Monday 3 July 1995 abstracts Mo1-Mo188

Monday 3 July 1995 Abstracts Mol-Mo188 Developmental cardiology Coronary circulation Smooth muscle, endothelium Contractile proteins Cardiac mechanics Cardiomyopathies, diabetes

Mol-Mo42 Mo43-Mo66 Mo67-Mo120 Mo121-Mo139 Mo140-Mo148 Mo149-Mo188

A MODEL FOR CARDIAC HYPERPLASIA. Mo001 Maurice J.B. van den Hoff, Ronald H. Lekanne dit Deprez, Wout H. Lamers & Antoon F.M. Moorman. Department of Anatomy and Embryology, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

Treatment of pregnant rats with an inhibitor of prostaglandin synthesis results in closure of the ductus arteriosus in utero. Initially, a pressure ovedoad is created in the right ventricle, because the blood can no longer drain on the descending aorta. In response to this overload, the fetal heart is expected to increase its contractile potential by increasing the number of myocytes. This fetal hyperplastic response contrasts the observed hypertrophic response upon pressure overload in adults and provides hence an interesting model to compare the differences. Near-term pregnant rats were injected with Indomethacin for various time periods and 2 hours before sacrifice bromodeoxyuridine (BrdU) was injected to monitor DNA-synthesis. The obtained fetal hearts were processed for immuno-histochemical staining and for determination of total DNA, RNA and protein content. One day of treatment did not result in changes in DNA, RNA and protein content, whereas after two days all three parameters had almost doubled. Immunohistochemical showed an increase in the number of BrdU-containing nuclei, slightly prior to the observed increase in DNA content of the stressed hearts. These results indicate a balanced growth of the fetal heart upon pressure overload.

EXPRESSION OF LAMININ VARIANT mRNAMO003 DURING NORMAL AND PATHOLOGICAL CARDIAC GROWTH IN THE RAT

Farhadian F, P. Olivi6ro, F. Marotte, L. Rappaport, J.L. Samuel. U 127,1NSERM, IFR Circulation, 75010 Paris

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Laminin, (LAM) a major component of basal lamina, is said to play a critical role in cell growth, and differentiation. The prototype molecule is an heterotrimer consisting of A or M, S or B 1 and B2 chains. The expressioa of LAM isoforms is tissue specific and developmentally regulated. In the striated and smooth muscle cells of adult mammals, the specific LAM heterotrimers are B 1/M/B2 and S/A/B2, respectively. In order to determine the pattern of expression of LAM isovariants during physiological and pathological cardiac growth of the rat, we analysed the cardiac level of mRNAs encoding respectively BI, B2, M and S-LAM chains using Northern- and dot-blots during post natal development and hypertension-induced hypertrophy (Goldblatt, lclip, 2 kidneys). At birth, the levels of all LAM mRNAs were high. In 25 d-old rat, BI, B2 and M-LAM m R N A had decreased 3 fold, then remained constant until 7 month whereas those of S - L A M decreased only in 2-3 month-old rat. In Goldblatt rat heart, B 1 m R N A amount did not vary whereas that of M-LAM d ~ d (-50%) as early as lweek after surgery and remained stable a~rwards. The S-LAM m R N A level decreased (-50%) only 4 weeks after surgery. In conclusion, it emerged that i) all the mRNA encoding a laminin subunit are highly expressed during physiological growth, ii) the muscle S and M - L A M mRNA are down-regulated during pathological growth.

CHANGES IN THE RNA CONTENT DURING RATMo002 CARDIAC DEVELOPMENT. Maurice J.B. van den Hoff, Ronald H. Lekanne dit Deprez, Marisol Monteiro, Wout H. Lamers & Antoon F.M. Moorman. Department of Anatomy and Embryology, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands. It is common knowledge that in situ hybridisation signals from fetal hearts are relatively high compared to Northern blot signals. Changes in RNA content during development could account for these differences. To identify these changes, a developmental profile was made to determine total DNA (diphenylamine (DPA) colorimetric assay), total RNA (orcinol assay) and total protein (BCA assay; Pierce). The DNA content was found to decrease almost 5-fold in post-natal live (adult level: 2.0 lag/mg). The RNA content was found to decreased almost 3-fold after birth (adult level: 2.0 ~g/mg). The protein content gradually increased 3-fold between ED13 and adolescence (130 t.tg/mg). The 5-fold change in the RNA to protein ratio suggests that during development post-transcriptional in addition to transcriptional control is occurring. The important implication of this study is that when e.g. the expression level of a message is similar in fetal and adult life based on Northern blot analysis, in fact its level has 3-fold decreased, which fully explains the relatively low in situ hybridisation signals in adult hearts.

Mo004 CHOLINE ACETYLTRANSFERASE EXPRESSION PATTERN DURING RAT HEART DEVELOPMENT D. Franco, P.A.J. de Boer, A.F.M. Moorman & W.H. Lamers. Dept. of Anat. & Embryol. AMC. UvA. Amsterdam. Acetylcholinesterase is present in the downstream part of the embryonic heart and maybe involved in the mobilisation of the calcium. To verify whether other components of the acetylcholine biosynthetic pathway are present in the embryonic heart, we localised choline acetyltransferase (CHAT) mRNA by in situ hybridisation. Rat embryos, ranging from 13ED to 20ED, were studied. Adult rat spinal cord served as positive control and [~-MHC mRNA was used to visualise the myocardium. The f'ust expression of ChAT was observed at 15ED in neural tissue covering the interatrial septum. Subsequently this area expands to the sinus horns in such a way that it comes to surround the pulmonary veins at ED18. The observed data fits with the position of the cardiac ganglia. ChAT mRNA is, therefore, not present where acetylcholinesterase activity has been localized. The implications may be that the function of the acetylcholinesterase, in the embryonic heart, in not related to the hydrolysis of the acetylcholine, but exert its action in another way to mobilise calcium.

COORDINATEEXPRESSIONOF PH0SPH0,I~tBAN (PLB)Mo005 AND SARCOPLASMICRETICULUM(SR) Ca2r-ATPase DURINGMURINEMYOCARDIALDEVELOPMENT. Judy M. Harrer, Kobra Haghighi, Hae Won Kim, Evangelia G. Kranias. Univ. Cincinnati, Cincinnati, OH and *UI San Univ., Seoul, Korea

POST-NATAL CHANGES IN TRANSFORMING MO006 GROWTH FACTOR BETA ISOFORM EXPRESSION IN THE RAT HEART Robert Haworth x.z, Gavin Brooks I. Metin Avkiran I. Derrick Chilton 2 & Peter Cummins 2. 1Cardiovascular Research. Rayne Institute. St. Thomas' Hospital, London, SEI 7EH, UK and -~Department of Physiology. U. Birmingham. UK.

To elucidate the role of the relaxing system in the developing heart, mice at different ages of development were analyzed for cardiac SR Ca2+-ATPase and PLB expression levels as well as SR function. The mRNA and protein levels of both SR Ca2+-ATPase and PLB were 40% of adult levels (100%) at birth and they increased in parallel to near adult levels by day 15 of development. SR 45Ca2+-uptake assays were performed to determine the functional consequences of the coordinate expression of PLB and Ca2+-ATPase in the developing hearts. Analysis of the data indicated that over the course of development: 1) the increase in maximal rates of Ca2+-uptake correlated with the developmental up-regulation of SR Ca2+-ATPase protein; and 2) the affinity of the SR Ca2+-uptake for Ca2+ remained constant (6.54+.01 pCa Units). In parallel studies, PLB-deficient hearts, which have an increased Ca2+ affinity for SR Ca2+-uptake (7+.04 pCa units), were also analyzed during development. An increase in the rates of SR Ca2+-uptake, without alterations in Ca2+ affinity, were observed during development similar to wild-type hearts. These findings suggest that phospholamban and SR Ca2+-ATPase expression are coordinately regulated in the developing mouse heart to maintain a constant affinity of the transport system for Ca2+.

Translornting growth factor beta (TGF-[3) has been implicated in post-natal cardiac development, but little is known about isolorm expression during this period. Isoform-specific antibodies were raised against synthetic peplides based on the sequence of the latency-associaled protein (LAP) for each of the three human TGF-13 isoforms. These antibodies were used to determine the myocardial levels of LAP and precursor TGF-I~ proteins during postnatal development (at 2, 5.7, I0. 11. 14. 21 and 28 days of age) in the rat. At 2 days. TGF-I3j was expressed mainly as cleaved LAP. A progressive shift from TGF-13j LAP to full lenglh precursor was observed with increasing age, such that LAP was undeteclable at 28 days• In contrast, TGF-[~ was present predominantly as the full length precursor in 2 day old myocardium, wilh an age-dependent shift from precursor protein to LAP between 2 and 28 days. TGF-[3.~ was detected only as LAP Ihroughoul the period studied. However. a four-fold increase in the expression of TGF-133 protein was observed between 2 and 28 days. Fractionation sludies indicated that TGF-[~2 and TGF-133 were associaled with the cardiac myocyle, whereas TGF-I3t was not. The substantial changes in myocardial TGF-[3 protein isoform expression that occur during the first 28 days of life in Ihe rat supI~rt a role for these proteins in post-natal cardiac developnlent.

MYOSIN LIGHT CHAIN 2V GENE Mo007 EXPRESSION DOES NOT DEMARCATE THE A N A T O M I C A L B O U N D A R I E S O F THE RAT EMBRYONIC VENTRICLE Gerry T.M. Wagenaar, Marry W.M. Markman, Wouter

ISOLATION OF A DEVELOPMENTALLYMo008 EXPRESSED AVIAN GLYPICAN H O M O L O G U E IN HEART Shi Niu, Kaoru Akimoto, Parker Antin, Irwin Flink & Eugene Morkin, Univ. of Arizona, Tucson, Arizona, USA.

H. Lamers and Antoon F.M. Moorman. Department of Anatomy and Embryology, University of Amsterdam, The Netherlands. During enibryonic development the single heart tube develops into a four chambered pump with atria and ventricles. Ventricular myosin light chain (MLC) 2V has been reported to specify ventricular chamber formation. To study very early ventricle formation we performed an in situ hybridization and immunohistochemical study. At embryonic day (ED) 10, when atrium and ventricle can be clearly recognized morphologically. MLC2V can not be detected in the heart tube in contrast to atrial and ventricle myosin heavy chain. From ED 11 onward MLC2V is mainly present in the ventricle. Remarkably, the staining intensity of mRNA and protein extends into atrium and outflow tract (OFT), indicating expression in the atrio-ventricular canal and under rim of the atrium, and the OFT. Interestingly, structures derived from the inflow tract (IF'I3, e.g. myoeardium surrounding the pulmonary vein and carat vein, also express MLC2V. So MLC2V expression is predominant in the developing ventricle, but it can not be used to delineate the process of ventricular formation.

Heparan sulfate proteoglycans (HSPG) modulate specific growth factor-receptor interactions and mediate interactions of cell surface with enzymes and structural proteins. The core proteins of H S P G have been classified into syndecan-like or glypican-like families depending on their mechanism of attachment to cell membranes. In this study, we isolated a eDNA encoding a protein with 60% overall identity to the human and rat glypicans. Within the protein, however, 6 stretches of 13-36 residues were identical to the mammalian proteins. Sequence analysis showed that the protein was a precursor of a phosphatidylinositola n c h o r e d cell m e m b r a n e protein and c o n t a i n e d consensus sequences for heparan sulfate attachment sites, indicating that this e D N A encodes an avian h o m o l o g u e of m a m m a l i a n glypicans. U s i n g the antisense message as a probe in whole mount-in-situ hybridizations, expression of the transcript was found to be developmentally regulated. In day 3 or 4 chick hearts, the message was highly expressed in outflow tract and AV junction areas, suggesting that the encoded protein may be involved in AV canal formation during cardiac development.

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PUTATIVE MEMBRANE FATTY ACID TRANSLOCASE Mo009 (CD36) AND HEART-TYPE FATTY ACID-BINDING PROTEIN ARE CO-EXPRESSED IN RAT MUSCLES F.A. Van Nieuwenhoven1, C.P.H.J. Verstijnen1, N.A. Abumrad2, P.H.M. WillemsenI, G.J.J.M. Van Eyst, G.J. Van Der Vusse1 and J.F.C. Glatz1.

tCardiovascular Research Institute Maastfieht [CARIM), University of Limburg, NL and ZOept. of Physiology and Biophysics, State University of New York at Stony Brool; U.S.A. A 88-kDa membraneprotein (FAT) with high homology to CD36 has recently been implicated in the transmembrane transport of long-chain fatty acids (FA). Expression of FAT was studied in rat heart during development, in oxidative and glycolytic skeletal muscles and in various cell-types isolated from adult rat heart. Expression of FAT was comparedto that of the cytoplasmic fatty acid carrier, heart-type fatty acid-binding protein (H-FABP) to determine whether co-expressioncould be demonstrated.FAT and H-FABP mRNAs showed a similar muscle tissue distribution. The highest expression was found in heart tissue, expression in oxidative skeletal muscle was lower and glycolytic skeletal muscle showed the lowest expression level. In isolated cardiac cells mRNAs of both proteins were detected in cardiomyocytes,but not in endothelial or fibrablast-like cells. During heart development from the day before birth to day 70, mRNA levels for both proteins increased about 5-fold. In conclusion, expression of FAT and H-FABP in muscle tissues and cell-types with high FA-metabolism and the upregulation during heart developmentsupport the notion that these proteins play an important role in muscle FAmetabolism.

MOLECULAR ANALYSIS OF CARDIAC PROGENITOR MoO 10 CELL SPECIFICATIONAND DIFFERENTIATION. M.A.Q.Siddiqui, S. Goswami, S. Ghatpande, S. M.A. Baig, S.Carleton, Department of Anatomy & Cell Biology, SUNY Health Science Center at Brooklyn, Brooklyn, NY 11203 USA In order to understand the mechanisms underlying the establishment of the cardiac muscle cell lineage, we have analyzed the early heart development by monitodng the onset of the cardiac muscle specific gene expression and the appearance of early DNA-binding regulatory protein factors. We observed that the first discernable transcript of cardiac myosin light chain (MLC2) and alpha-actin genes appear immediately after the cardiogenic fate assignment stage (stage 4) in the early evian embryos. The functional expression of the cardiac specific transcription factor (BBF-1), a member of the myocyte enhancer factor (MEF-2) family implicated in the cardiogenic specification, precedes the cardiogenic fate assignment. A recombinant plasmid pGCBI was isolated by screening a cDNA library from mRNA isolated from the heart-forming region of stage 6 chicken blastoderm using DNA primers containing MEF-2 conserved sequence, pG-CBI was able to transactivate the expression of the cardiac specific MLC2 promoter (pMLC371CAT) in non-permissive fibroblast C3H10T112 cells. In-situ hybridization studies indicated that the pGCBI specific mRNA were localized in the cardiogenic mesodermal area in early embryonic stages. These results, thus, demonstrate the potential of pG-CB1 as a regulator of cardiogenic lineage determination.

Study supported by the Netherlands Heart Foundation

THYROID HORMONE STATUS AFFECTS CARDIAC Mo011 Na*-Ca=* EXCHANGER mRNA LEVELS IN IMMATURE RABBIT VENTRICLES Scott R. Boerth and Michael Artman. University of South Alabama College of Medicine, Mobile, AL and New York University School of Medicine, NY, NY, USA Previous studies in adult rabbits demonstrate that cardiac Na*-Ca2. exchanger steady-state mRNA levels are increased by hypothyroidism and decreased by hyperthyroidism. In developing rabbits, expression and functional activity of the sarcolemmal Na*-Ca2. exchanger are high at birth and decline to adult levels by 2-3 weeks of age. This transition occurs in temporal relation with the postnatal increase in thyroid hormone levels. Therefore, in order to determine if the postnatal surge in thyroid hormone mediates the downregulation of the cardiac Na*-Caz* exchanger, exchanger steady-state mRNA was measured using poly(A)* RNA isolated from ventricles of control and hypothyroid immature rabbits. The normal postnatal thyroid surge was blocked by administering PTU to the mothers from gestational day 25 through 3 weeks postpartum. Exchanger mRNA levels were quantitated at 3 weeks of age by Northern slot blot analysis. Results (mean + SD) are tabulated below: Group (litters) SerumT4(pgldl) mRNA(%control) Control (4) 9.4 + 1.0 100 + 24 Hypothyroid (3) 2.2 + 0.4 205 + 30 Thus, blocking the postnatal surge in thyroid hormone attenuates the normal postnatal decline in cardiac Na÷-Ca2. exchanger steady-state mRNA levels. These results support the concept that thyroid hormone is important in regulating expression of the Na*-Ca=* exchanger during pednatal cardiac maturation.

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IN VITRO REGULATION OF SERCA AND MoO12 PLB EXPRESSION BY ADRENERGIC AGONISTS Dylan G. Wynne & Kenneth R. Boheler. Dept of Cardiothoracic Surgery, NHLI, London SW3 6LY, UK. To determine the effects of adrenergic (Adr) agonists on the expression of the SR CaATPase (SERCA) and its regulator protein phospholamban (PLB), rat neonatal cardiac myocytes were cultured in defined serum-free media (n>6). After addition of 4pM norepinepherine (NE), isoproterenol (ISO) or phenylepherine (PHE) for 5560hrs, the mRNA expression of ANF, SERCA and PLB were determined. Values normalised to 18S rRNA are expressed as a ratio of ascorbate (ASC) control. Relative ratios ofisomRNA's of actin (skeletal (S) and cardiac (C)) and MHC (ct and [~) were determined by 'hot' RT-PCR. ASC NE ISO PHE SIC actin 0.19_+0.01 0.47_+0.07* 0.37_+0.05* 0.42_+0.07* ~/czMI-IC 0.61_+0.12 0.14_+0.06" 0.35_+0.10 0.28_+0.12" ANF 2.80_+0.66* 1.77_+0.57 3.10_+0.88" SERCA 0.65_+0.09 0.57_+0.15" 0.654-0.07* PLB * p<0.05 0.79_-_+0.18 0.84_+0.25 0.72_+0.09* Adr agonists increase the levels of ANF, S-actin and BMHC transcripts. The decrease in SERCA is due to ct- and p-Adr effects. The reduction in PLB expression may be mediated primarily by ct-Adr. These data have been partially confirmed through the use of specific inhibitors, and suggest a role of Adr stimulation in the downregulation of SERCA and PLB with cardiac hypertrophy.

EXPRESSION OF A HYPERPOLARIZATION- Mo013 ACTIVATED INWARD CURRENT FROM THE EMBRYONIC CHICK HEART IN XENOPUS OOCYTES Andrew A. Munk & Alvin Shrier. Department of Physiology, McGill University, Montr6al, Qu6bec, Canada, H3G IY6. We have investigated a hyperpolarization-activated inward current in single cells dissociated from embryonic chick hearts, with the whole-cell patch clamp technique, and in Xenopus laevis oocytes microinjected with poly-A" mRNA isolated from the embryonic chick heart, with the two-microelectrode voltage clamp method. In isolated embryonic chick heart cells a slow time-dependent current (It) was activated in the range between -90 to -120 mV during hyperpolarizing steps from a holding potential of -50 mV and had a reversal potential between -30 and -10 inV. In oocytes injected with poly-A* mRNA, a similar slow time-dependent inward current was activated in response to hyperpolarizing voltage-clamp pulses from a holding potential of-50 or -40 mV. As in the chick cells, this current was found to activate for steps between -90 and -120 mV and had a reversal potential between -30 and -20 mV. This inward current was not observed in oocytes that had not been injected. These results suggest that the expressed current in the injected oocytes is the (It) current observed in the isolated chick heart cells. These findings indicate that the embryonic chick heart may be a good source of mRNA for the expression and cloning of this channel. Supported by the Heart and Stroke Foundation and the vlRC o f Canada. Figure I: Currents elicited during A B succesive 10 mV hyperpolarizing pulses from a holding potential of-40 mV in an uninjected oocyte 500ms¢¢ (A) and in an oocyte injected with poly-A" mRNA (B).

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EXCITATION-CONTRACTION COUPLING IN CARDIAC DYSMORPHOGENESlS Tony L. Creazzo & Marco A. P. Brotto. Medical College of Georgia, Augusta GA USA.

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Persistent truncus arteriosus (PTA), a neural crest related heart defect, is among the most lethal of the congenital heart anomalies. Bilateral ablation of the cardiac neural crest in chick embryos (neural crest from mid-otic placode to caudal somite 3) leads to aortic arch. anomalies and, later, to failure of septation of the cardiac outflow tract or PTA. In this study, we found that the peak of the Ca2+transients in fura-2 loaded myocytes were depressed in hearts with PTA, at days 11 and 15 of incubation, and not observable above background noise. Treatment with isoproterenol (ISO) restored peak L-type Ca current (ica.L) to the level found in normal hearts (=-2.9pNcm 2 at +10mV without ISO;joatch clamp) but could only partially restore the Ca z* transient (300+40nM in PTA with ISO vs 465+22nM in normal hearts with no ISO). Ca2. release from the sarcoplasmic reticulum (SR) stimulated by caffeine was significantly reduced (1249±186 vs 533±73nM) and greatly slowed. The results suggest impaired SR Ca2* release in addition to reduced iCa.L. Interestingly, decreased iCa,Lwas detected at day 3 in neural crest ablated embryos (-6.4 vs -9.7pNcm" in normal~,and further decreased by day 5 (-3.1 vs -10.1pNcm'). These decreases were observed during and after aortic arch formation but before outflow septation (days 5-7). Note that ica.L is normally much larger at the earlier ages. Supported by NIH HL36059.

IS DEPRESSED VENTRICULAR CONTRACTILITYMo014 IN AN EXPERIMENTAL HEART DEFECT DUE TO ELEVATED MICROTUBULE CONTENT? R.E. Godt, T.M. Nosek, J.C. Chu & W.A. Lutin'. Depts. of Physiology & Endo. and Pediatrics'; Med. College of Georgia; Augusta, GA 30912, USA. Ablation of cardiac neural crest at stage 9 produces a structural heart defect (persistent truncus arteriosus, PTA) in chicks. PTA is associated with decreased myocardial contractility, as indicated by decreased left ventricular ejection fraction determined echocardiographically (Lutin et al. C/in. Res., in press) at embryonic days 12-18. Using pressure-overloaded right ventricle of cat, Tsutsui et al. (Science 260:682, 1993) found reduced isotonic shortening of myocytes was associated with a doubling of microtubule content, and suggested that excessive microtubules caused the reduced shortening. To examine whether increased microtubule content could explain the decreased ventricular contractility in chick hearts showing PTA, we compared the content of ~tubulin in left ventricle from hearts whose left ventricular volume ejection fraction had been determined previously with in ovo echocardiography. In preliminary studies at embryonic days 16-17, we found no difference in left ventricular content of [3-tubulin between control hearts and those showing PTA, even though the PTA hearts had a significantly decreased ejection fraction. These observations do not support a role for elevated microtubules in the depression of ventficular contractility in this heart defect. The mechanism for these changes remains to be elucidated. (Support: NIH HL36059)

INTERMEDIATE FILAMENT VIMENTIN AND DESMIN Mo016 IN THE HUMAN FETAL HEART Ho-dirk Kim Dept of Histology, College of Medicine, Chung-Ang Univ, Seou1156-756,Korea Intermediatefilament vimentin (Vim) plays an important role in maintainingthe stability of mesenchymal cells and desmin (Des) provides support for contractile machinary in muscle cells. In this study, localization and development of Vim and Des in the human fetal heart aged from 9 to 28 wks gestation (n=38)were investigated by Western blotting 0NB) and immunofluorescencetechnique with monoclonal antibodies (MAb) against Vim and Des. Cardiac needle biopsy samples from 1-yr-old infant was used for the control. By WB analyses, 54-kDa Vim and 53-kDa Des bands appeared in all heart tissues but Vim intensity was progressively decreasedwith fetal age while Des increased. Vim appeared in the mesenchymal tissue including fibroblasts and surroundings of blood vessels. In part, some cardiac myocytes showed weakly positive reaction with MAb against Vim in 914 wks gestation.Vim-positive area, however, was progressively diminished with fetal age. Vim appeared only in the connective tissue and coverings of the 1-yr-old heart. Des appearedonly in the cardiocytes. In the earlier period (10-14 weeks gestation) Des was localized along the cell membrane and/orZ-lines with regular intervals and later (2528 wks gestation) structurally well integrated, however, its network was incomplete. Only 1-yr-old cardiac myocytes revealed highly developed and structurally integrated Des lattice.

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CONA-BmlD~IGPROTONSIN SERUMPROMOTECARDIAC MoO1 MESENCHYMAL~ &IGRAT]ONWITH RGD DEPENOENTMAMUER. Hlroshl Sumldal, Harukazu Nakamura2, Tatsuhiro Matsu01 & MIneo Yasuda3 1DIv. of Anat., Sanyo Women's College, Hatsukalchl, 2Dept. of Molec. Neuroblol., Inst. of Develop., Aging & Cancer, T o h o k u Univ., Sendal, 3Dept. of Anat., Hlroshlma Univ. Sch. Med., HIroshlma, Japan. The developing heart at an early phase is composed of the endocardium, cardiac jelly and myocardium. Cardiac jelly is a substrate rich in extracellular matrix and cardiac mesenchymal (CM) cells. CM cells migrate in the cardiac jelly of two main cushions of the developing heart, the conotruncal and atrioventricular cushions, toward the myocardium. Migration of CM cells is certainly an important step for the formation of the intracardiac septa. Migration of CM cells can be mimicked in culture system. Although migration relating molecules have not been identified, we have found that Concanavalin A (Con A)-binding proteins (BPs) in chicken serum promote migration of chick CM cells in vitro. Collagen type-I did not significantly enhance migration promotion activity of these proteins. The GRGDSP synthetic peptide, a cell attachment fragment in fibronectin, inhibited Con A-BPs dependent migration of CM ceils at 500 to 1000 ~.M. Western blotting, however, revealed that fibronectin was not one of the major components of Con A-BPs. These results suggest that Con A-BPs directly interact with CM cells, not mediated by collagen type-I, and promote migration of CM cells padially with an RGD dependent manner.

CARDIAC NEURAL CREST ABLATION (CNCA) Mo019 DECREASES CALCIUM UPTAKE AND RELEASE BY THE CARDIAC SARCOPLASMIC RETICULUM Thomas M. Nosek, Rosalvo T,H. Fogac...a, Paula L. Allee, Marco A.P. Brotto, and Robert E. Godt. Medical College of Georgia, Augusta, GA 30912-3000 L~SA CNCA at embryonic stage 9 produces outflow tract anomalies in chick heart by embryonic days E10-E11 (Kirby & Waldo Circ. 82:332, 1990). Leatherbury et al. (Circ. 81:1047, 1990) found that CNCA results in decreased myocardial contractility at stage 18. We found that at E15, CNCA decreases twitch force of intact left ventdcular trabeculae and maximum Ca z+ activated force of Triton-skinned trabeculae (Fogac..a et al., J.CelI.Biochem. 17D:211, 1993). In the present study, we found that Ca 2+ sensitivity of the contractile apparatus is unchanged at E15 by CNCA. However,.. CNCA dramatically reduces the magnitude of the Ca" transient measured in Fura-2/AM loaded intact trabeculae. Saponin-skinned trabeculae were used to investigate the role of SR function in this response (see Zhu & Nosek, Am.J.Physiol. 261:H620, 1991 for technical details). CNCA has no effect on the half-time of Ca z+_uptake by the SR. It does, however, decrease the Ca z÷ sensitivity of both Ca =+ uptake and calciuminduced calcium release (CICR) by the SR at E15. Therefore, the decrease in cardiac function associated with CNCA results from both decreased function of the contractile apparatus and a reduced Ca =* transient. Depressed Ca=+-sensitivity of SR Ca =+ loading and CICR account for the decreased Ca =+ transient. (Supported by NIH grant HL 36059.)

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THE ATRIOVENTRICULAR VALVES. Mo018 NORMAL DEVELOPMENT TO UNDERSTAND ABNORMALITIES. Petra W. Oosthoek, Ant6nio J. Macedo, Lambertus J. Wisse, Rob E. Poelmann, Addana C. Gittenberger-de Groot, Arnold C.G. Wenink. Dept of Anatomy, University of Leiden, NL. We studied the atrioventricular (AV) region in normal embryonic human (5 through 20 weeks of development) and chicken hearts (Hamburger Hamilton stage 24 through hatching). We used immunohistochemistry for both species to differentiate the myocardium, AV-cushions and AV-sulcus, the regions that might contribute to the adult valves and chords. Three-dimensional reconstructions were made of successive stages. Human heads were also examined in toto under a dissection microscope. In addition gross studies of heads with congenital malformations of AV-valves were performed, such as Ebstein's malformation, congenital dysplasia and parachute valves. During development AVcushions are surrounding the lumen of both the left and the right AV--canal. Delamination of myocardium from the free walls of the ventricles and from the interventricular septum "lifts" the cushions including a layer of myocardium. As a result relatively thick valve-like leaflets become visible composed of two layers: myocardium at the ventricular side and cushion tissue at the atrial side. These primitive leaflets loose their myocardial layer except at the site of the future papillary muscles. The papillary muscles in human get lheir adult location at 16 weeks of development. In chicken a completely m6Jscular valve leaflet in the adult right ventricle represents the persistence of the myocardial layer. The developmental processes suggest that in Ebstein's malformation there has been incomplete "lifting". In congenital dysplasia persistence of cushion tissue is the pathogenetic mechanism, whereas the parachute is an example of abnormal papillary muscle formation.

RESPONSES OF ANOXIC-REOXYGENATED Mo020 EMBRYONIC HEART TO pH CHANGES Meiltz Alexandre, Kucera Pavel, Raddatz Eric Institute of Physiology, University of Lausanne, CH-1005 We investigated the mechanisms underlying the response of spontaneously contracting hearts from 4-day-old chick embryos to alterations of pH and oxygen availability. Under normoxia, acidosis (10 mM bicarbonate/CO2 buffer, pH 6.5) induced 1) negative chrono- (30%) and dromotropic (25%) effects, 2) transient decrease of contraction velocity (40%), and 3) chaotic and periodic bursts of contractions (incidence 35%). Return to pH 7.4 suppressed arrhythmias within 1-5 seconds and restored normal activity. Further alkalinisation (pH 8.2) had slight positive chrono-, dromo- and inotropic effects (10%). In bicarbonate-free buffer (i.e. phosphates), acidosis induced positive chrono- (17%) and negative dromotropic (20%) effects and provoked also arrhythmias (incidence 50%). The same effects were observed at pH 7.4 in bicarbonate by blocking anion exchange (DIDS 100gM) but not by blocking Na+/H + exchange (HMA lgM). During anoxia and reoxygenation in bicarbonate buffer, acidosis provoked arrhythmias and prolonged the reoxygenation-induced cardiac arrest. At pH 7.4, DIDS or bicarbonate-free buffer had positive chronotropic effect during anoxia and were arrhythmogenic during anoxia and reoxygenation. However, at pH 7.4 HMA had no effect. These findings indicate that intracellular pH regulation in the anoxic-reoxygenated embryonic myocardium depends predominantly on bicarbonate availability and transport. Supported by the SNSF Grant 31-37668.93

SARCOPLASMIC RETICULUM FUNCTION Mo021 OF NEONATAL PIGLET MYOCYTES. Charlene M. Hohl, Beth Livingston, James Hensley & Ruth A. AItschuld. Dept of Med Biochem, Ohio State University, Columbus, Ohio, USA. The increased susceptibility of neonatal hearts to ischemia is often attributed to increased sensitivity to abnormal levels of [Ca2+]i due to an immature sarcoplasmic reticulum (SR). Many of these studies have been performed on neonatal rats and rabbits which are much less developed at birth than larger mammals. Accordingly, SR function was characterized in ventricular cardiac myocytes isolated from newborn (< 20 hr) piglets. Vmax and K0.5 for 45Ca2+ uptake by the SR of digitonin-lysed myocytes were 279 + 18 nmol/min/mg and 0.68 + 0.08 /~M, respectively. In the presence of H-89 (to inhibit phosphorylation of phospholamban) Vmax was reduced by 30% and Kd increased to 1.28 ~M. In the presence of 1 /~M isoproterenol peak amplitude of electrically stimulated fura-2 Ca transients was increased by 465 ::1:85 % and T0.5 decreased from 1.67 to 0.94 sec. [3H]-Ryanodine binding studies yielded a Bmax of 195 fmol/mg myocyte protein and a Kd of 1.4 nM. Ca transients were completely abolished by superfusion of cells with 2 nM ryanodine. Raising extracellular Ca (0.5 to 5raM) increased peak amplitude and decreased T0. 5 of Ca transients. Corresponding effects of [Ca] o on changes in cell length were also recorded. The results suggest that SR function of newborn piglet myocytes is relatively mature.

Supported by AHA and HL36240.

TRANSIENT INOTROPIC RESPONSE TO Mo022 LOW EXTRACELLULAR SODIUM IN PERFUSED PERINATAL RAT HEART Ivana O~t~dalov~i, Franti~ek Kol~f* and Bohuslav O~t~dal. Institute of Physiology, Academy of Sciences of the Czech Republic, Czech Republic. Detailed information on the development of contractile function d u r i n g the early postnatal period in rats is, as yet, lacking. Contractile performance of the isolated perfused rat heart and its inotropic response to low extraceIlular sodium (LES) and ryanodine was, therefore, studied in rat hearts on prenatal day 20 and postnatal days 1, 2, 4 and 7. The effect of LES on the developed force (DF) changes significantly day by day: whereas a p e r s i s t i n g increase was recorded in the prenatal hearts, this increase was only t r a n s i e n t on postnatal day 1 and 2. Starting from day 4, the signs of a triphasic response, typical for adult h e a r t s , appeared (an initial increase of DF, followed by decrease of DF and a rise of r e s t i n g force, and finally a delayed increase of DF); this t r e n d was more pronounced on day 7. The negative inotropic effect of ryanodine was observed already prenatally; however, pretreatment with ryanodine abolished the day by day ohanges in the response to LES. Our data show that inotropio response to LES is a g e - d e p e n d e n t and changes rapidly d u r i n g the first week of life.

THE COMPARISON OF THE EXCITATIONMo023 CONTRACTION COUPLING IN VENTRICULAR MYOCARDIUM OF NEWBORN AND ADULT CATS. Pavel PuEelik and Milan Stengl. Dept. of Physiology, Charles University Medical faculty, Plze~, Cz.

PEPTIDE-CONTAINING NEURONS AND SYNAPTIC Mo024 VESICLE MARKERS IN THE DEVELOPING RAT

The excitation-contraction coupling (ECC) in the immature (newborn or fetal) myocardium exhibits substantial differences in the regulation of contraction and relaxation processes, compared with the adult ones. The underdeveloped sarcoplasmic reticulum (SR) in neonatal hearts causes that contraction cycle is more directly regulated by the transsarcolemmal fluxes of Ca (sarcolemmal type of ECC). The experiments were performed on the right ventricular papillary muscles of the newborn and adult cats. The action potential (AP) and force of isometric contraction (MG) were measured simultaneously. The following results were acquired. 1) While the adult myocardium exhibited pronounced postextrasystolic potentiation, this property was not found in the newborns. 2) The period of rest evokes age difference in the course of the post-rest contraction. In newborn cats the time of the reaching of maximum of postrest MG was extended as a function of period of inactivity. This parameter was unaffected in the adult cats. 3) The changes of extracellular Ca caused more pronounced inotropic responses in the newborn myocardium than in the adult one. 4) The negative inotropic effect of verapamil was considerably higher in the newborns. Our results suggest that ability of SR to accumulate and to release Ca maturates during the early postnatal ontogeny and therefore the sarcolemmal (immature) type of ECC converts to the reticular (adult) type of ECC. This process causes that the internal cycle of the calcium turnover become more effective.

The presence and distribution of peptide-containing neurons in the rat heart during early postnatal development are investigated by indirect immunocytochemical technique. Antibodies against neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) were applied to the whole mount stretch preparations of the rat right atda from hearts of newborn, 10, 18, and 40 days old animals. Compadson with other known vesicle membrane components p38 (synaptophysin) and SV2 in nerve axons and terminals was done in double immunoincubation studies. Immunofluoresce was documented with epi-fluorescence and confocal laser scanning microscopy in order to improve resolution in the thick whole-mount of the tissue, and to allow also some more information on the subcellular distdbution of CGRP in comparison to the other markers. NPY-like immunoreactivity (LI) and CGRP-LI are present within the right atria of rats from birth. The bundles of fibres entedng the right atrium along the supedor caval vein seem to be extdnsic in odgin. The postnatal changes are basically quantitative in that of the density and the number of vadcosities. With increasing age, the intensity of fluorescence increased, while the basic pattern of innervation was essentially unchanged. CGRP was not colocalised in the same preparations with either SV2 or p38 antigens, which are present in small synaptic vesicles of the classical autonomic innervation, supporting NANC origin of CGRP.

RIGHT ATRIUM Jana Slavikov~, Annica Dahlstr0m. Dept. of Physiol., Med. Faculty, Charles Univ., Plze6, Czech Republic, Inst. of Neurobiol., Univ. of GOteborg, Sweden.

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MYOCARDIAL ISCHEMIC TOLERANCE: DIFFERENCES BETWEEN NEONATAL AND ADULT RATS

Mo025

Narcisa Tribulova, Ludmila Okruhlicova, Barbara ZiegelhSffer & Jan Slezak. Inst Heart Res, SAS, Bratislava, Slovak Republic.

The age-related response of the myocardium to 20 min of normothermic global ischemia was evaluated ultrastructurally and cytochemically using neonatal (0-4 days old ), transitional (8d), weanlings (22d) and adult (90 days old) rat hearts. Structural developmental changes previously described were accompanied by cytochemical maturation of cardiomyocytes. The activity of 5"nucleotidase was observed predominantly in capillary endothelium, and later on also on the sareolemma of myocytes. The activity of G-6-phosphatase (marker of SR) was low in newborn hearts and frequency of positive sites increased with age. 20 min of global ischemia did not induce any structural or cytoehemical alterations in the newborn hearts. Mild, ischemia-related structural alterations but not cytochemical were observed on the 4th day. The ischemic injury was more pronounced in 2nd postnatal week and characterized by depletion of glycogen, oedematous mitoehondria, hypercontractions of myofibrills and intracellular oedema. The enzyme activities were reduced. By 22nd day all cardiomyocytes were fully differentiated and resembled adult ones. lschemia-related structural changes were evident and enzyme activities reduced. These results demonstrate increased tolerance of the neonatal heart to ischemic injury.

In different phases of development of the heart, there have been reported contraversal data concerning the sensitivity of cardiac tissue against ischcmia. In the present study the capability of developing heart to tolerate 1 h lasting global ischemia (37oc) was investigated in rat in respect to preservation of activities of I 1 enzymes in the time interval between the first postnatal day and the maturity. The study involved histochemical detection and quantification by computer image analysis activities of dehydrogenases (13HBDH, G-6-PDH, LDH, SDH), cytochromoxidase, ATPases, glycogen phosphorylase, 5-nucleotidase, acetylcholinesterase and alkaline phosphatase (AP) in the myocardial tissue as well as AP and diaminopeptidase IV in cardiac vessels. Between the first and 8th postnatal day the enzyme activities investigated in ischemic hearts revealed no significant alterations in comparison to those in nonischemic controls. It was concluded that from the point of view of preservation of enzyme activities the hearts of neonatal and suckling rats are less sensitive to ischemia than those of weanlings (22 days old rats) and adult animals.

AGE-RELATED DIFFERENCESIN SUPEROXIDEAND HYDROGEN PEROXIDE PRODUCTION IN ISOLATED GUINEA PIG HEARTS DURING REPERFUSION

FREE-RADICAL SCAVENGERS IN CARDIAC Mo028 MEMBRANES; QUALITATIVE AND QUANTITATIVE CHANGES DURING POSTNATAL DEVELOPMENT Alexander Lebedevj, V~iclav Pelouch2, Natalija UtkinaJ, Nina

Mo027

Richard Southwodh, Frank J. Kelly, Michael J. Shattock and David J. Hearse. Cardiovascular Research, The Rayne institute, St. Thomas' Hospital, London, U.K. The immature myocardium is known to be more resistant to ischaemic injury than the mature myocardium. This study determined whether cardiac maturity influences reperfusioninduced free radical production. Heads (n=6/grp) from adult and 3 day premature (caesarean section) guinea pigs were perfused in the Langendoflf mode for 10min, made globally ischaemic for 20rain (37°C) and reperfused with 10mM succinylated ferricytochrome c (cyt c) for 20min. Coronary effluent was collected in lmin aliquots throughout reperfusion and superoxide (O2") reduction and hydrogen peroxide oxidation (H20,i of cyt c was estimated spectrophotometrically (550nm). Additional groups were reperfused with cyt c with either SOD (lx105 lull), catalase (lx10 e lUll) or both, with or without allopurinol (2raM). Superoxide production(nmol/min/g wet wt) Age/ treatment

-SOD

+SOD

Immature Mature Mature + allopurinol

23.0 :!: 7.8 ' 82.8 + 14.0 8.34- 3.4 *

3.6 + 5.6 + 0 + 0.3 '

2.8 + 2.4

* = Different from mature .SOD; += different from immature .SOD p
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HISTOCHEMISTRY OF THE DEVELOPING Mo026 RAT HEART: THE INFLUENCE OF GLOBAL ISCHEMIA IN VITRO Barbara Ziegelhoeffer, Narcisa Tribulova, Jan Slezak. Inst Heart Res, Slovak Acad Sci, Bratislava, Slovak Republic.

Differences in H202 production mirrored those of 02" (data not shown). Mature hearts produced 4 times more O2~ than immature heads after the same duration of ischaemia - the majority via xanthine oxidase. Thus, developmental changes in reperfusion-induced oxidant stress on may either contribute to, or reflect, age-related differences in ischaemic tolerance.

Prokazova~, Bohuslav O~t'/tdaP. ~ExptlCardiology Res Ctr, Moscow, Russia, :Inst or Physiology, Acad Sci, Prague, Czech Republic, JPacific Inst of Bioorganic Chemistry, Vladivostok, Russia.

The purpose of this study was a) to analyze the postnatal development of cardiac lipid-soluble antioxidants and b) to compare the effect of chronic hypoxia (hypobaric chamber, stepwise up to 7000 m, 8 h a day, total of 24 exposures) on the free radical scavengers (FRS) in Wistar male rats acclimatized either from birth or in adulthood. Vitamin E, active FRS and total FRS in cardiac lipids were assessed in myocardial n-hexane extracts by fluorescence measurement, l,l-diphenyl-2-picrylhydrazyl free radical test, TLC and HLPC methods. Total (maximal) antiradical activity was determined in the same extracts after treatment with borohydride, taking into account reversible losses of antiradical activity during oxidations of quinols. The total pool of FRS decreased by 300%, whereas the proportion of active FRS increased by 150% during postnatal development.Vitamin E represents only 12% of active FRS in newborns but 70-90% in adult animals; the complementary part is formed by coenzyme Q in both developmental periods. Chronic hypo,,da was without any effect on the level of total FRS and vitamin E in animals acclimatized from birth, on the other hand, acclimatization in adulthood induced a significant decrease in the concentration of vitamin E. It may be assumed that the composition of the active pool of FRS changes significantly during development. Chronic hypoxia decreased the concentration of vitamin E only in adult animals.

INCREASED EXPOSURE TO CHRONIC HYPOXEMIA M o 0 2 9 FROM BIRTH DECREASES RECOVERY OF RIGHT AND LEFT VENTRICULAR FUNCTION AFTER GLOBAL ISCHEMIA Brad Schubert, Gordon N. Olinger & John E. Baker. Department of Cardiothoracic Surgery, Medical College of Wisconsin, Milwaukee, WI, USA In order to establish the relationship of chronic hypoxemia to recovery of ventdcular function after ischemia, we assessed the relative tolerance to global ischemia of the right ventricle (RV) and left ventricle (LV) from immature rabbits chronically hypoxemic (H) from birth (FIO 2 = 0.09, PaO 2 = 34 + 11 mm Hg, SaO 2 = 63 _+ 3%) versus normoxemic (N) rabbits (FIO 2 = 0.21, PaO 2 = 92 + 4 mm Hg, SaO 2 = 94 + 2%) in two age groups (7-10 days old (DO), and 14-18 DO). After 30 min of aerobic perfusion with bicarbonate buffer in the Langendorff mode with water-filled latex balloons placed in both ventricles, all hearts underwent 3 min of perfusion with St. Thomas' II cardioplegic solution at 39°C. Warm (39=C) global ischemia followed. Thirty-five min of reperfusion then began after intraventricular LV pressure increased by 20 mm Hg above baseline ischemic values to equalize the ischemic insult in all hearts. In the H 7-10 DO group, percent recovery of right ventricular developed pressure (RVDP) and left ventricular developed pressure (LVDP) were greater than in the N 7-10 DO group (86 + 8 vs 71 + 2, p < 0.05 and 79 + 10 vs 64 + 10, p < 0.05, respectively). However in the 14-18 DO group there was no difference in percent recovery of RVDP and LVDP between H and N groups (55 _+7 vs 56 + 10, p > 0.05, and 55 + 5 vs 52 + 8, p > 0.05, respectively). We conclude that increased duration of exposure of developing rabbit heart to chronic hypoxemia decreases tolerance of both RV and LV to ischemia in comparison to normoxemic controls.

PHOSPHOLIPID COMPOSITION MO031 AND OXIDATIVE CAPACITY IN PRESSURE-OVERLOADED RAT MYOCARDIUM Olga N o v & k o v ~ I , Libor Mrnka I , V~clav P e l o u c h 2- M a r t i n Kalous 2, Franti~ek N o v ~ k 3. IDept of Animal Physiol, 3Dept of B~ochemistry, Fac of Sci, Charles U n i v , ~ I n s t of P h y s i o l o g y , C z e c h A c a d e m y of Sci, Prague

ALTERED

PressuWe-overload was induced by abdominal c o n s t r i c t i o n in 2 days-old male W i s t a r rats. Sixty days after surgery rats were killed. H o m o g e n a t e and m i t o c h o n d r i a l fraction (mito) from left v e n t i c l e s (LV) were prepared. Phospholipids and cytochrome oxidase a c t i v i t y were d e t e r m i n e d . P r o p o r t i o n of phosphatidylcholine(PC), Phosphatidylethanolamine, diphosphatidylglycerol (DPG) d e c r e a s e d and P h o s p h a t i d y l s e r i n e (PS), Sphingomyelin(SM) increased in homogenate, p r o p o r t i o n of PC increased and PS, SM d e c r e a s e d in mito from LV of b a n d e d rats. Decrease in DPG c o n c e n t r a t i o n (by 25%) correlated with lower c y t o c h r o m e oxidase in homogenate whereas no d i f f e r e n c e in D P G , c y t o c h r o me oxidase was observed between mito from banded and control rats. This suggests that lower DPG and oxidative c a p a c i t y is due to lower c o n c e n t r a t i o n of m i t o c h o n d r i a in LV of banded rats.

P H O S P H O L I P I D C O M P O S I T I O N IN T H E Mo030 RAT H E A R T EXPOSED TO H Y P O X I A DURING EARLY P O S T N A T A L DEVELOPMENT Franti~ek Nov~k I , Olga Nov~kov~ 2 , David Smik 2 , V~clav Pelouch 3 , E v a ~vrzick~4.1Dept of Biochemistry, ADept of P h y s i o l o g y , F a c of Sciences, Angiol. 4Lab. of First Medical Fac., Charles Univ., ~Institute of Physiology, Czech Academy of Sci, Prague

The Male Wistar rats four-days old were exposed to intermitent high a l t i t u d e hypoxia (IHA) in barochamber (7000 m, 5 days a week, 8 hrs a day, 24 expositions). Alterations in fatty acyl (FA) composition of phospholipids (PLP) in hypertrophied right and left ventricles of hypoxic animals were observed: C18:0, C24:4n-6, C22:5n-3, C22:6n-3 increased whereas C18:1n-9 and C18:2n-6 decreases in phosphatidylcholine, C18:0, C22:5n-3, C22:6n-3 increased and 18:2n-6 decreased in phosphatidylethanolamine. The increase in p r o p o r t i o n of saturated FA and d e c r e a s e in C18:2n-6 was found in cardiolipin in right ventricles only. No change in PLP concentration was found in ventricles after IHA. 30 days after returning into normoxia hypertrophy still persisted but FA composition r e t u r n e d to control values.

PROTEIN CHANGES IN THE RAT HEART Mo032 EXPOSED TO PRESSURE OVERLOAD FROM BIRTH Jan (~ernohorsk~, Vficlav Pelouch, Bohuslav O~t'fidal, Marie Milerovfi. Inst of Physiology, Acad Sci, Prague, Czech Republic. A gradual pressure overload was induced in 2- and 6-day-old rats (A2, A6) by abdominal aorta banding (to 0.25 and 0.45 nun respectively). Quantitative changes of collagenous and non-collagenous proteins were estimated by the measurement of protein and hydroxyproline concentration in individual protein fractions, qualitative changes by using SDS-PAGE and UV spectrophotometry (220-240 nm). Left ventricular (LV) enlargement was found in both A2 and A6 animals, fight ventricular (RV) enlargement, however, in the A2 group only. Whereas the concentration of soluble collagenous proteins in the LV was already elevated 30 days after surgery, higher concentrations of insoluble collagenous proteins were found 60 days after aorta banding to the detriment of metabolic proteins; these changes were less pronounced in A6 animals. Protein remodelling of the RV in both A2 and A6 groups was characterized by lower concentrations of soluble collagenous proteins. The proportion of major collagen types changed significantly in the LV of A2 and A6 animals: the amount of collagen HI and V increased, whereas the amount of collagen I decreased. Furthermore, qualitative remodelling of myosin light chains was observed in hypertrophied LV of the A2 group. The above results suggest that aorta banding induced during the early postnatal period is accompanied by remodelling of both collagenous and non--eoUagenous proteins which depends on the onset of the pressure overload. A85

Mo033 INFLUENCE OF THYROID HORMONE ON POSTNATAL ULTRASTRUCTURAL DEVELOPMENT OF RAT ATRIAL AND VENTRICULAR MYOCYTES 'Daniela Jarkovsk~i, 2Franti~ek Kolsi[ & 2Bohuslav O~t'sidal. ~Department of Histology and Embryology, Charles University, ~Institute of Physiology, Academy of Sciences of the Czech Republic, Prague. Newborn male Wistar rats were rendered hyperthyroid by daily injections of L-triiodothyronine (10 p.g/100 g b.w.) or hypothyroid by adding 6-n-propyl-2-thiouracil to the drinking water to mothers from day 2 to days 7 or 14 of postnatal life. Compared to the euthyroid group, the hyperthyroid state resulted in an acceleration of the development of sarcoplasmic reticulum (SR) and T-tubules in cardiac myocytes. The most striking feature of atrial myocytes was the presence of a very rich network of the SR_ In the ventricular myocytes, the duplicated T-tubules were quite common and formed large junctional areas with Z-tubules and SR terminal cistemae. Mitochondria were enlarged with densely packed cristae. Hypothyroidism generally resulted in opposite changes: T-tubules were extremely rare and the sparse profiles of SR formed couplings largely with the peripheral sarcolemma. Structural maturation of mitochondria and the contractile apparatus was retarded. The observed changes are consistent with the ~4ew that thyroid hormones are essential for the normal postnatal development of the heart, in particular for the maturation of intracellular membrane structures involved in excitation-contraction coupling of adult myocytes.

S L AND S R C a 2 + - A T P a s e I S O F O R M S IN Mo035 DEVELOPING HUMAN HEART Angel Zarain-Herzberg, Juan Santiago-Garcfa* & Jaime M a s - O l i v a * Div. of C a r d i o v a s c u l a r Sciences, St. Boniface Res. Ctre., Winnipeg, Canada & *Dept. Bioenergdtica, Inst. de Fisiologia Celular, Univ. Nacional Aut6noma de Mdxico. In this study we have investigated the expression of the m R N A encoding the various P M C A and S E R C A proteins in fetal and adult human heart and placenta by the reverse-transcriptase-polymerase-chain-reaction (RTPCR) and eDNA cloning. W e have found that PMCA1 and P M C A 4 genes were expressed in 8-, 12- and 20week fetal heart and in adult heart. PMCA2 gene was expressed at low levels in adult heart but was not detected in fetal heart. PMCA3 m R N A was not detected in the heart nor placenta. In contrast, the m R N A encoding SERCA2a, S E R C A 2 b and SERCA3 were expressed in all cardiac developmental stages. Multiple alternatively spliced m R N A transcripts which differ at splice site A and B/C of the P M C A I , P M C A 2 and P M C A 4 genes were detected in the h u m a n heart. Interestingly, a novel tissue specific isoform of the PMCA4 gene was detected in both fetal and adult human heart but not in placenta that accounts for about 30% of the total P M C A 4 m R N A isoform expression. D N A sequence analysis of this novel isoform revealed that corresponds to the equivalent of the P M C A I d isoform and accordingly we have named it PMCA4d.

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RECIPROCAL CHANGES IN SR Ca TRANSPORT ANDMo034 Na/Ca EXCHANGE IN THE IMMATURE RAT HEART DUE TO ALTERED THYROID STATUS Roland Vetter & Monika Kott. Max Delbr0ck Center for Molecular Medicine, Berlin-Buch, Germany. Reverse changes in sarcolemmal Na/Ca exchanger (NaCaX) and sarcoplasmic reticulum (SR) Ca ATPase (SERCA2a) function contribute to developmental changes in cardiac Ca handling in rat heart. Because thyroid hormones are involved in heart maturation, we examined the effect of thyroid status on Ca transport by SERCA2a and NaCaX of 21-day old rats made hypo- or hyperthyroid by treatment with either 0.05% 6-n-propyl-2-thiouracil (PTU; in drinking water given to nursing mothers) or L-triiodothyronine (T3, 10 or 2.5 pg/d/lO0 g BW, s.c.) from day 2 postpartum (n=6/group). Oxalate-facilitated SR 4SCa uptake and Na-dependent 46Ca transport rates were measured in cardiac membranes. In hyperthyroidism, SR Ca uptake was increased (p <0.01) by 116% (19.5 + 1.7 vs. 9.0 ± 1.1 nmolCa/mg protein/min in euthyroid controls) and NaCaX was reduced ( p<0. 01 ) by 50% (0.089 -+ 0.009 vs. 0.179 ± 0.011 nmol Ca/mg protein/s in controls). By contrast, hypothyroidism resulted in a decrease ( p<0. 01) in SR Ca uptake to 41% and an increase ( p<0. 01) in NaCaX of 70% compared to control values. These alterations were reversed by Ta-treatment. Values of NaCaX and SR Ca uptake were found to be inversely related to each other ( r = - 0 . 8 9 , p < 0 . 0 0 0 1 } . Although the mechanism underlying the observed alteration in NaCaX remains to be elucidated, the results suggest that thyroid hormones are not only a major determinant of phenotypic SERCA2a expression but appear to be also involved in the postnatal decline of NaCaX in the rat myocardium.

MORPHOLOGICAL CHANGES IN HUMAN SINUS Mo036 NODE DURING GROWTH AND AGEING. POST-MORTEM MORPHOMETRIC ANALYSIS. M. Lasiak. S. Grajek, M. Pyda. I Clinic of Cardiology, ul Dluga I/2 Po~rmA, Poland. The aim of the study was the estimation of morphological changes of the sinus node (SN) in healthy subjects during g~owth and ageing. The subjects of the study consisted of 21 autopsy hearts taken ~om patients ranging in age from 4 months to 79 yearn (mean 37.2+22.9), with no history of heart sickness, died of non cardiac disease. On necropsy the hearts were substantiallynormal. The histologicalslices were taken perpendicularly to the long axis of the node's artery.The volume of the node was estimated by planimetry of the serialsections. On the basis of the point-counling method, a morphomettic analysis was performed to calculate the density of the nodal cells, relative percentage of the cells, connective and fatty tissues, and capillaries. Also, the actual volume of the cells and connective tissue was calculated. Besides, the mean diameter of typical nodal P cells, and their total number in the SN was determined. The volume of the node increases until adulthood because of the hyper~ophy and hyperplasia of its cells,and the expansion of the fibrous tissue,whereas the density and volume ratioof the nodal cells decrease, and the volume ratio of the fibrous tissue increases. During ageing, the volume of the node decreases, mostly because of atrophy of the nodal cells in peripheral areas, with resultant replacement of fatty tissue. The number of the cells starts to reduce from the age of about 30. During ageing, the actual volume of the connective tissue does not increase, however the atrophy of the ceils gives a false impression of fibrosis. The volume percentage of fatty tissue and capillaries does not significantly change during growth an ageing. Conclusions: Until adulthood, the actual volume of SN cells and connective tissue increases, but the more significant expansion of connective tissue gives a false impression of involution of the nodal calls. Atrophy of that calls, however, occur during ageing, which in turn is the reason of apparent fibrosis, while the actual volume of the fibrous tissue is constant.

HG2+-DEFICIENCY AND IMPAIRED SENSITIVITY Mo037 OF ADENYLATE CYCLASE (AC) OF AGING HEART C.Clb*,C.Pignatti,B.Tantini and C.M.Caldarera.*Inst. Biochemistry,University of Parma and Dept.Biochemistry,University of Bologna, I t a l y . Basal a c t i v i t y of cardiac AC progressively declines with the age of the r a t . Compared to AC from youn9 (I month) hearts, AC from aged (24 months) ones e x h i b i t lower responsiveness in v i t r o to e i t h e r stimulatory (isoproterenol, forskolin or stable GTP analogs) or i n h i b i t o r y (polyamines, morphine) effectors. However a net

recovery of responsiveness of "aged" AC is observable by increasing M92+ in the assay mixture, thus suggestin9 t h a t the requirement of the ion f o r optimal f u n c t i o n a l i t y of AC system ~ocreases with the age. The p o s s i b i l i t y that Hgz* improves the c a p a b i l i t y of the catal y t i c moiety to bind-activated G proteins (Gs or Gi), very l i k e l y l i m i t i n g in AC system of aged hearts, is supported by the fact that the responsiveness in v i t r o of AC is strongly i n creased a f t e r a d a i l y administration f o r one week of MgSO4 to aged rats. The same treatment had only a s l i g h t e f f e c t on AC from young hearts. Q u a l i t a t i v e s i m i l a r results have been obtained with cultured myocytes isolated from rat or chick embryo. Supported by a grant from CNR,Progetto F i n a l i zzato "Invecchiamento",Sottoprogetto"Gerontologia".

T R O P O N I N T IN THE C O R D B L O O D - M A R K E R Mo039 OF M Y O C A R D I A L INJU RY A F T E R TOCOLYSlS

Zden6k Kok~tein, Michaela Adamcov61, Vladimlr PALI(~KA2, Department of Pediatrics Faculty of Medicine, 1Department of Physiology Faculty of Medicine, 2Institute for Clinical Biochemistry and Diagnosis, Hradec Kr61ov~, Czech republic The study has been designed to evaluate the diagnostic efficiency of cardiac troponin T (TnT) in neonates ~xposed in utero to tocolytic therapy. First part of our study confirmed that almost no myocardial troponin T is present in the cord blood in healthy term infants (0.06 +0.04 ug/I). Then TnT concentration was measured in the cord blood of 51 neonates after tocolysis. The first group (acute tocolysis) was divided into subgroups determined by the duration of treatment. Troponin T levels were in physiologica! range in all of the neonates after 12 hours of therapy. All of the newborns had TnT concentration in the pathological range after 3 days of treatment. The second group (preventive tocolysis) was divided into subgroups determined by the interval between the end of therapy and delivery. TnT concentration decreased depending on the interval between withdrawal of treatment and delivery. The results of this study shows that the used tocolytic therapy may have side effect on the fetal myocardium. The finding of elevated TnT concentration in the cord blood corresponds with the kinetics of TnT and peaks about the 3rd day of the treatment.

A G E - R E L A T E D C H A N G E S IN THE CON-Mo038 T R A C T I L E R E S P O N S E TO ET-1 AND THE R E C E P T O R IN T H E RAT CARDIAC MUSCLE Yumi Katano, Akira Ishihata & Masao Endoh. Dcpt of Pharmacol, Yamagata University School of Medicine, Yamagata, Japan. Endothelin (ET) receptors are widely distributed in the cardiac muscle and ET elicits a positive inotmpic effect (PIE) on mammalian cardiac muscles. We reported that the E-T-l-induced PIE decreased with age in isolated perfused rat heart. We examined to elucidate the mechanisms of age-related changes in the ET-l-induced PIE and the binding sites in rat isolated ventricular muscles. Methods: Right ventricular papillary muscles isolated fi-om 2-monthold (too) and 24-mo male Fisher 344 rats were electrically driven at 1 Hz (37°C). The [125I] ET-1 binding assay was carried out in membrane fi'action derived fi'om rat ventricle. Results: ET-1 elicited a PIE in 2-too raLs; the PIE of ET1 was markedly decreased in 24-mo rats. Relaxation time (RT) of isometric contractions in 2-mo rats was significantly briefer than that in 24-mo rats. The PIE of ET-1 was markedly attenuated by cyclopiazonic acid (C'PA; 3x10 "6 M) in 2-too, but not in 24-mo rats. These findings suggest that the decreased function of SR to uptake and release Ca 2+ may be an important factor to contribute to the age-related changes in RT and the ET-l-induced PIE. The density of [12sI] ET-1 binding site was significantly higher in 24-mo than in 2-mo rats; the affinity was unaltered with aging. These results suggest that the diminished cardiac response to ET-1 in aged rats may partly be due to uncoupling of E-T-1 receptors to subsequent signal transduction processes.

DNA-BASED CLINICAL DISSECTION OF Mo040 PFHBI, A CARDIAC BUNDLE BRANCH DISORDER. P Brink, A Christoffels, A Ferreira, V Corfield. Univ Stellenbosch and Tygerberg Hosp, S A.

Progressive familial heart block I (PFHBI), an autosomal dominantly inherited disease, has been mapped to chromosome 19q13.3-13.4. Closely linked markers allow characterisation of the clinical spectrum and determination of whether it is specific to the ventricular conduction system. Disease-associated haplotypes were constructed using markers from DM to Rras in the PFHBI pedigrees (154 living, 59 affected). Individuals displaying genotype/phenotype inconsistencies were clinically re-evaluated. Twelve individuals were identified in 4 categories, viz., i. gene carrier/clinically unaffected (4), ii. carrier/ambiguous phenotype (2), iii. noncarrier/clinically affected (1), iv. noncarrier/ambiguous phenotype (5). Importantly, selective disease of the SA or AV nodes was not associated with carrier status. One carrier (category ii) died of a dilated cardiomyopathy. We conclude that results indicate that PFHBI is a disease of the ventricular conduction system. The single case of dilated cardiomyopathy may be co-incidental. Alternatively, PFHBI may be a generalisad cardiomyopathy, which like myotonic dystrophy, a chromosomally adjacent muscle and systemic disorder, manifests clinically with emphasis on the bundle branch system.

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T I ~ U E ~ N B I B I ~ Z A T I O N IN OHII=DRENMo041 A ~ E R NON-RJ~EUMA~IC MI~OCAP~DITIS

IC~AmmSZS/ Elena M Levitska~a, Galina D Dorofeeva MedloaiUniversity, Donetsk, UKRAINE Previous our studies demonstrated that autosensibility pla~s significant role in recovery period of non-rheumatic myocarditis / N I ~ . We have now Invesb~ gated the immune processes in 78 children with slow & chronic NRM courses after 9-12 months from disease, using the tests blasttransformation of lymphocytes /BT~v ~ and local hemolysis of immunocompeten~ antibod~ plaque-form~n~ cells /APFC/, stimulated by myocardial antigen. 37pts with later NRM courses took standard therapy /group 1/; Alpts had therapy with immunocorrectors /galascorbine,aloes,eleutherococcus/. Results~ n BTL APFC Control 36 3,q~1,5 q,2 ~ 0,08 Group I q9,4~2,0 9,7 ~ 1,0 Group 2 qO,O~q,8 3,8 ~ 0,8 P o. 05 o. off This data indicate the role of autosensibilization in pathogenesis of chronic Nl~ courses and effect of immunocorrective therapy in catamnesis.

ESTIMATION OF CORONARY BLOOD FLOW BYMo043 THERMOGRAPHY IN OPEN CHEST CONDITIONS Naama Gordon, Shmuel Rispler, Samuel Sideman, Rona Shoffi & Rafael Beyar. The Heart System Research Center, Technion-IIT, Haifa, Israel.

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Background: Thermography has been previously suggested as an imaging method during open heart surgery. We hypothesized that coronary blood flow could be estimated from the dynamics of the thermographic images during cold saline injection in the aortic root. Methods: In nine open chest dogs the heart was exposed and imaged by an Agama thermographic camera. Coronary flow was measured by a Transit time flowmeter. Images were acquired for 30 s alter aortic injection of 20 ml of cold saline. Several flow levels were achieved in each dog by obtaining a reactive hyperemic response to transient coronary occlusions. A temperature washout curve, characterized by a rapid initial cooling followed by gradual rewarming was obtained over an edge detected arterial segment. Results: The thermographic image during cold saline injections allows for direct visualization of the arterial tree in all cases. The following correlations were obtained with flow: exponential coefficient of the ascending part of the temperature curve (r=0.67, p<0.005), the peak temperature difference (r=0.7, p<0.001), the slope of the descending part of the response curve (r=0.64, p<0.002), and the area under the curve (r=0.55, p<0.02). Conclusions: Thermographic imaging of the coronary tree provides a tool 'for quantification of coronary blood flow and myocardial perfusion.

HE~ODYNA~ICS IN CHILDREN OF Mo042 DIFFERENT AGES SUFFERING FROM POSTNATAL NON-RHEUMATIC MYOCARDITIS G.D.Dorofeyevs, V.H.Zabirov & S.I.Tsctashov. Medical University. Donetsk, Ukraine. Age peculiarities of child's growing heart and of its regu]etion ere of pronounced eppesrance under myocardium affections. Activity of blood cholinestereses es well ss hemodynemics have been investigated (the latter - with the method of di]ution of the dye-stuff Evens' blue) in 109 children (I group - 48 st the age from 6 months up to 3 years, end II group 61 from 10 up to 15 years) suffering from acute non-rheumatic myocsrditis (NRM). Reliable fell of the scetylcholinesterase activity (I,042+0,029. normal 1,247+__0,015, p~O,O5)-and rise of the butyrilcholinestersse one (0,744±O,O31, normal 0,612±0,O06, p~0,05) sre revealed being of the most appearance in children under 3 years. Hemodynamics is characterized with the blood flow moderetion (by 22,2% in I group, and by 4,5% in II group) beat volume diminution (by 42,6% in I group and by 10,5% in II group, p~O,05), minute volume diminution (by 26,2% in I group and by 8,1% in II group, p
THE RELATIONSHIP BETWEEN THE DYNAMICSMo044 OF THE EPICARDIAL CORONARY FLOW AND VENTRICULAR HEMODYNAMICS Dan Manor, Samuel Sideman, Rona Shofti & Rafael Beyar. Dept. of Biomed. Eng., Technion-IIT, Haifa, Israel. Hemodynamic events were studied in a series of 19 dogs at sinus rhythm, where left anterior descending coronary flow measured by transit time and LV and aortic pressures by tip manometers. The following parameters were defined: early systolic flow decrease Fes; mid-systolic flow increase Fms; and late systolic flow increase Fis. The flow dynamics during constant coronary perfusion pressure conditions were studied in six dogs. During baseline conditions Fee did not correlate with either (dP/dt)max or the length of the Isovolumic contraction, whereas F ~ depended strongly on the magnitude of Pao early systolic increase (r=0.71, p<0.005) and FIs correlated with vantricular relaxation time. Constant perfuslon conditions abolished the Fie component. The isovolumic relaxation flow increase rate (dF/dt)max was found to be inversely related to the relaxation time. Therefore, with the natural variability between dogs the relationship between early systolic flow dynamics and LV pressure buildup is weak and probably masked by variability in heart rate, isovolumic contraction time and aortic pressure. However, mid-systolic coronary flow events are more consistently determined by the aortic pressure waveform, and late systolic events by ventricular relaxation dynamics.

EPICARDIAL CAPACITANCE IS A MAJOR M0045 DETERMINANT OF CORONARY DYNAMICS Alan S. Lader, James E. McNamee, & Francis L. Abel. Dept of Physiology, Univ of South Carolina, Columbia SC, USA. Models of the coronary circulation utilize capacitance as a parameter in predicting coronary flow. However, the anatomical distribution of the capacitance is controversial. We used computer simulation to evaluate the relative influence of capacitance location and magnitude on input flow. A three capacitor electrical analog model containing epicardial(C,~), intramural(C,,,), and venous(C~) capacitances was used along with four resistances of constant value (R,p,=50, R,,=150, R,=175, Rout=25 PRU) arranged as three low pass filters in series. Capacitances values were varied in order to evaluate the importance of each. Simulation was done on a microcomputer with Maple V software. The response of the model to a pressure impulse was evaluated; following the impulse, flow was retrograde and decayed exponentially.The decay was comprised of three exponents. The contribution due to Cv was always less than 1%. Therefore, a two exponential function was sufficient to model the flow. The dominant exponent was associated with the epicardial capacitance. However, when C,p,was of the same order of magnitude as C,m,the exponent associated with C,, became apparent (>5% of the calculated flow). Using a single exponential to approximate the flow, C~ was distinguishable from C,p, only when Cep, < 0.1xC,m. We have observed changes in C~ in animal experiments using pressure impulses, therefore, this model suggests that C,. cannot be greater than ten times C°p~.(Supported by a grant from the American Heart Association, Inc.)

GREGG's PHENOMENON I N R A T A N D Mo046 GUINEA PIG HEARTS Andrei N. Khatkcvich & Valeri I. Kapelko.

Cardiology Research Centre, Moscow, Russia The contractile responses of rat and guinea pig isovolumic hearts to a rise in coronary perfusion pressure in the range 60-120 mmHg (Gregg's phenomenon) were compared. Left ventricular CLV) systolic pressure was lower in guinea pig hearts than in rat hearts at low coronary perfusion pressure and increased markedly less at a higher perfusion pressure, despite higher increment in coronary flow. The rise i,1 LV systolic pressure in guinea pig hearts was entirely due to an increased LV end-diastolic pressure while LV developed pressure did not rise. The wet weight of the hearts m situ was similar in both species, but after perfusion the guinea pig hearts gained significantly more fluid than the rat hearts (65% of the initial heart weight compared to 37%). The group of rat hearts perfused with a low external Ca2+ concentration developed a similar LV pressure to the guinea pig hearts and gained a similar amount of fluid (63%), but Gregg's phenomenon was the same as in rat hearts peffused with normal Ca 2+ concentration. The results suggest that the weak Gregg's phenomenon in guinea pig hearts can be attributed to factors other than myocardial edema and a lower LV systolic pressure.

C O R O N A R Y O C C L U S I O N F O R ONE Mo047 HOUR D E P R E S S E S ANTEGRADE AND C O L L A T E R A L BLOOD F L O W AFTER R E P E R F U S I O N Belcher PR 1, Kingsbury M 2, Etherington p3, Winlove p3, Sheridan D 2. Noble MIM ]. Chafing Cross & Westminster Medical School l, St Mary's Hospital Medical School2, Imperial College3, London

ROLLER PUMPS, BUT NOT CENTRIFUGAL PUMPS, Mo048 ACTIVATE NEUTROPHIL5 DURINGCARDIOPULMONARYBYPASS (CPB) Uday H. Trivedi, Marion G. Macey, Des A. McCarthy, K. Alun Brown, Graham E. Venn & David J. Chambers. Cardiac Surgical Research and Dept of Immunology, The Rayne Institute, St Thomas' Hospital, London, UK.

Coronary ha~modynamics were studied pre and post 1 hours octlusion of the circumflex artery in 8 anau~sthetised open-chest dogs. SIN-1 at 10 p.g/kg/min had no effect on pressure or flow but bradykinin at 30 ~tg/kg/min decreased coronary pressure (p<0.05) and increased flow (p<0.05); only pressure fell after ischu~mia (p<0.05). The hyperasmic response to transient coronary occlusion significantly decreased after repel'fusion (50i-_19 vs. 95+_2.1 ml/min; p<0.005). The distal coronary pressure/flow ratio rose linearly with time between 10 and 70 seconds (all r 2 >0.95) after release of occlusions, at the same rate before and after the ischa=mic period. The mean y-intercept of this relation was 0.50-1-0.30 vs. 1.57:L-0.69 mmHg.min/ml (p<0.01) after repeffusion, indicating higher distal resistance. At zero time (no flow) the pressure in the arteries (PZF) averaged 30-Y_5mmHg before and after reperfusion. This was considered to bc due to collateral blood flow, which was calculated from the resistance (y-intercept) and PzF. This fell from 58+9-8 to 28+18 ml/min after 1 hour of isch~mia (p<0.005). Conclusion: The depressed level of reflow following coronary occlusion is related to downsu'cam resistance changes which arc in part endotheliallymediated.

Neutrophil activation during CPB may lead to reperfusion injury. A comparative study was undertaken for evidence of neutrophil activation in 18 patients randomly allocated to centrifugal (Bio-pump) pumps (n=9) or to conventional r o l l e r pumps (n=9) during CPB. Flow cytometric measurement of the expression of surface adhesion molecules and the high a f f i n i t y Fc receptor (CD64) was used as an indicator of neutrophil activation and results presented as the mean fluorescence intensity (MFI). In relation to values taken immediately before CPB, neutrophils f r o m r o l l e r pump samples obtained after lh of CPB exhibited an increased expression of CDIIb (mean MFI 91±37 v 36±9; p=O.O07) and CD64 (mean MFI 292±73 v 122±78; p=O.O01). The expression of CDI|a/CDI8, CDIIc/CD18 and L-selectin was not modified. None of the above markers were significantly increased on neutrophils from centrifugal pump samples. We conclude that CPB patients on r o l l e r pumps are at risk to neutrophil activation and possible tissue damage.

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EFFECT OF CORONARY ANGIOPLASTY ON Mo049 AUTONOMIC CARDIAC CONTROL Ugo Limbruno, Giancarlo Strata, Roberto Baglini, Domenica Di Santo, Giovanni Amoroso, Riccardo Zucchi, Anna Sonia Petronio, Alberto Balbarini, Mario Mariani. Institute of Cardiology. University of Pisa. Italy. We used the spectrum analysis of heart rate (HR) variability to assess the modifications of sympatho-vagal balance associated with angioplasty-induced myocardial ischemia. We studied 23 pts with left ,anterior descending (LAD) and 16 pts with right coronary (RC) proximal non occlusive stenosis.Spectrum analysis of HR variability was performed in basal conditions and during each balloon inflation. ECG recordings were digitized and analyzed by an autoregressive model to obtain the power spectrum of HR variability. The high-frequency (HF) band (0.18-0.35 Hz) and the low-frequency (LF) band (0.05-0.18 Hz) of power spectrum were considered indexes of vagal activity and sympathetic activity with vagal modulation respectively. Results: the first balloon inflation in LAD induced a significant increase of the LF band (+27+7n.u., p<0,01) and LF/HF ratio (+132+_59%, p<0,01). During the following inflations such modifications were markedly blunted and not significantly different from basal values. On the contrary, balloon inflation in RC induced a significant increase in the HF component (+21+8 n.u., p<0.05) and decrease in the LF/HF ratio (-55+28%. p
L O N G T E R M I N C R E A S E IN P R E L O A D COM- M0051 P R O M I S E S THE R E S P O N S I V E N E S S OF C O R O N A RY AND SYSTEMIC ARTERIES DIFFERENTLY.

Anna Hol4cyov~, M~ria Gerov~,Franti~ek Kristek, Ala Klasov~, *Fedor ~imko. Inst. of Norm. and Pathol. Physiology, Slovak Acad. of Sci.& *Dept. of Pathol. Physiology, Fac. of Medicine, Comenius University, Bratislava, Slovakia. Increase in preload was induced by insufficiency of aortic valves in rabbit and verified by heart/body weight. After 4 months,the contractions of coronary rings to acetylcholine (ACh), 5-HT and KCl decreased significantly to 22%, 17% and 21% of the resp. control values. An increase in both extracellular matrix (EM) and EM/muscle cell ratio in the media underlied the compromised function. In contrast, maximum contraction of aorta and renal artery to noradrenaline was unchanged and that to KCl even potentiated. In these vessels, attenuation of relaxation to ACh was observed. Morphology revealed intimal thickenings and local reduplications of internal elastic lamina. The compromised contraction in coronary artery ensues very probably from the additional deformation due to alteration of ventricular volume. A90

STENTS BUT NOT BALLOON ANGIOPLASTY MO050 INDUCE CHRONIC NEOINTIMAL PERMEABILITY Heleen MM van Beusekom, Sjoerd H Hofma, Deirdre MC Whelan, Pieter D Verdouw, VV~llem J van der Giessen. Thoraxcenter, Erasmus University Rotterdam, The Netherlands. We have shown earlier that porcine coronary arteries exhibit an increased permeability for 1 kDa molecules (angiotensins, Evans Blue) during several weeks after stent implantation. Our objectives were to study the extent and specificity of endothelial permeability using different stents and compare this to balloon angioplasty at 2 (immature endothelium) to 12 weeks (mature endothelium) after intervention. Implantation of Palmaz-Schatz (n=20), Wiktor stents (n=12) or balloon angioplasty (n=12) was performed in porcine coronary artedes under quantitative angiographic guidance to prevent oversizing. At follow-up the animals were catheterized for control angiography and Evans Blue infusion (EB). To assess endothelial leakage, 500 ml of EB in saline (1 kDa) was infused either directly into the treated coronary arteries in vivo to assess for permeability to small molecules (1 kDa), or given intravenously to assess for permeability to the Evans-Blue-Albumin (EBA) complex (70 kDa). After the dye-exclusion test, the coronary arteries were pressure-fixed for macro- and microscopic analysis. Macroscopical analysis revealed penetration of both EB and EBA into the vessel wall up to 12 weeks after stenting. After balloon angioplasty permeability to either EB or EBA was not observed. Microscopical analysis revealed that all treated coronary segments were completely endothelialized.This study indicates that stenting but not balloon angioplasty decreases long-term endothelial integrity in porcine coronary arteries, enabling the passage of large molecules into the vessel wall. These results seem to give an explanation for our observation of increased lipid accumulation in stented human venous bypass grafts, and may be related to the increased neointimal build-up (late loss) after clinical coronary stenting.

COMPARISON OF CONTRACTILE RESPONSES Mo052 TO POLYMORPHONUCLEAR LEUKOCYTES IN RABBIT AORTA AND CORONARY ARTERIES. Joanne L. Hart, Christopher G. Sobey & Owen L. Woodman, Dept of Pharmacology, Univ of Melbourne, Parkville, Australia. Polymorphonuclear leukocytes (PMNL) are known to release several vasoconstrictor substances. In this study we have compared the vasoconstrictor effects of products from rabbit PMNL in large and small arteries. PMNL were isolated from rabbit blood and suspended at 5x107/ml. The conditioned medium (CM) from this suspension, containing stable PMNL derived products was collected by centrifugation. CM (3-4431.1.1 per 10 ml organ bath) was then added cumulatively to isolated aortic tings, or as a bolus (300El) to isolated Krebs-perfused (30mi/min) rabbit hearts. CM caused a further increase in contraction of pre-contracted aortic tings (0.6 + 0.1g, n=21). This contraction was abolished by haemoglobin (Hb, 10gM), attenuated by N-nitroL-arginine (NOLA, 301aM) but unaffected by superoxide dismutase (30 U/ml). The factor causing this contraction was found to be >30 kDa, and was destroyed by incubation at 90"C for 10 min. CM caused a constriction of the coronary vascular bed (32 + 6 mmHg), which was unaffected by the presence of Hb (61a.M), NOLA (10gM), indomethaein (31aM), phentolamine (101aM) or the endothelin receptor antagonist bosentan (10RM). These data suggest that PMNL may release multiple vasoconstrictor factors, one which acts in the aorta by inhibiting the action of NO and another which acts in the coronary microeirculation through a mechanism yet to be determined.

EFFECt OF NIFEDIPINE ON ACIDOSIS-INDUCED Mo053 CORONARY CONSTRICTION IN THE RAT HEART. Darren A. Wilson & Brian Woodward. Pharmac. Dept, Univ. O f Bath, Bath, Avon, BA2 7AY, U.K. Metabolic acidosis(MA) is an early event in myocardial ischaemia but its effect on the coronary circulation is not clear. 5min perfusion(10ml.min q) of isolated rat hearts with MA(pH6.8)increased coronary perfusion pressure (CPP) from 79±8 to 156 +9 mmHg (p<0.01 n=4). Perfusing with 15mM [K]o to arrest cardiac contractility or pacing at 8Hz showed the increase in CPP to be separate from the myocardial depressant effects of MA. The response to acidosis was independent of flow rate(5-20 ml.min ~) and it was not affected by L-NOARG (100p.M) or indomethacin (10p.M) ruling out a role for nitric oxide inhibition or production of vasoconstrictor cyclooxygenase products. In contrast nifedipine(lnM- 100nM) inhibited the MA-induced increase in CPP. Therefore this data suggests that MA is probably activating L-type calcium channels in the coronary vessels of the isolated rat heart. This contrasts to the finding that acidosis inhibits L-type calcium channels in isolated smooth muscle and cardiac cells.

THE RESPONSE OF ADENOSINE RECEPTORS IN Mo055 THE CORONARY RESISTANCE VESSELS TO AGONIST STIMULATION IS BIPHASIC Fiona Harden, Glenn Harrison, Lind.say Jordan, Roger Willis. Rotary Centre for Cardiovascular Research, Griffith University, Nathan, 4tl t, Australia.

A biphasic vasodilatory response to adenosine and 5'-Nethylcarboxamidoadenosine (NECA) was observed in the coronary vasculature of potassium-arrested porfused rat hearts. The dose-response data for both agonists was best fitted to a two-receptor model. For adenosine, negative log ED~ovalues of 8.06 ± 0.11 (mean + SEM) and 5.22 + 0.13 were obtained and represented 31.2% and 68.8% of the receptors respectively. In the presence of 5 pM 8-phenyltheophylline (8-P'I'), the vasodilatory response to adenosine was also best fated to a two-site receptor model and negative leg ED~ values of 7.03 ± 0.22 and 5.39 ± 0.16 were obtained. The proportion of receptors observed in the presence of 8-PT was 26.3% for the high potency site and 73.7% for the low potency-site. For NECA, negative log EO~ values of 9.57± 0.12 and 6.84 ± 0.23 representing 48% and 52% of the receptors were found. In contrast, ATP produced a monophasic response and yielded a negative leg EDso value of 8.75 ± 0.08. These results support previous evidence of adenosine receptor heterogeneity in vascular tissue.

C A P I L L A R Y D E N S I T Y AND Mo054 P E R F O R M A N C E IN B R A D Y C A R D I A L L Y PACED INFARCTED PIG HEARTS Margaret D. Brown l, Michael K. Davies 2, Olga Hudlicka 3 & Paul Townsend 4, School of Sport and Exercise Sciences 1, Dept.s of Cardiovascular Medicine 2, Physiology 3 and Biomedical Sciences 4, University of Birmingham, Birmingham BI5 2"VF, UK. Long-term electrically induced bradycardia stimulated capillary growth and increased stroke work in rabbit and pig hearts. Its effect was studied in pigs with myocardial infarction caused by introduction of a copper coil into the left anterior descending coronary artery. Heart rates in farm pigs instrumented with Medtronic~R~dual chamber pacemakers were decreased by atrial-atrial pacing (P) from 112+4bpm to 60-80bpm. After 4-6 weeks, under isofluorane anaesthesia, stroke volume and stroke work index (calculated from cardiac output measured by thermodilution, heart rate and mean blood pressure) were similar at rest. During dobutamine challenge (151ag/kg/min) both indices were higher in P infarcted than infarcted animals (SWI 34.3+1.1 Joules/beat/100g heart weight vs 27.6+ 3.2 respectively, p<0.05). The heart/body weight was similar in both groups, but marginally lighter left ventricle in proportion to heart weight in infarcted pigs was corrected by P. Capillary density assessed in lectin stained frozen sections, was decreased in the border zone (940!'_ 110 cap/mm 2 vs 1451+65 in the remaining left ventricle); P increased it in both regions (to 1138+128 and 1650i-_181 respectively, p<0.05). Since coronary sinus flow measured by thermodilution was similar in both groups, the improvement of SWI by P may be explained by more homogeneous perfusion of the enlarged capillary bed.

ANGIOTENSIN-CONVERTING E N Z Y M E M0056 INHIBITORS POTENTIATE THE C O R O N A R Y VASODILATOR EFFECT OF BRADYK.ININ IN THE INTACT HEART.

Peter B Arming, Richard M Grocott-Mason, Malcolm J Lewis, Ajay M Shah. Cardiovascular Sciences Research Group, University of Wales College of Medicine, Cardiff, CF4 4XN. Angiotensin-converting enzyme (ACE) converts angiotansin I to anginteusin II and degrades the vasodilator peptida bradyldnln (BK). BK activates B2 receptors on endothelial cells to cause the release of several factors, eg nitric oxide (NO), prosteglandins. Increased B2 receptor activation may thus be involved in the mechanism of action of ACE inhibitorS (ACEi). In this study, we examined the effects of captopril (CAP, l~M) and fisinopril (LIS, lldvD, both alone and in combination with exogenous BK (0.1 and 1.0nM), on coronary flow (CF) in isolated ejecting guinea-pig hearts (constant perfusion pressure & heart rate; Kreb's buffer;, 37°C; lizM indomethacin). CF was measured from timed collections of effluent from the pulmonary artery. At the concentration chosen, neither CAP (n=9) nor LIS (n--A) alone had any effect on CF (-0.4 + 2.0%, +1.0 + 0.6% respectively; p = NS). BK (lnM) caused a significant increase in CF (+15.5:1:.3%,p<0.05 e.f control) whereas BK (0.1nM) had no effect (-0.7:L-0.5%,p=NS). In the presence of either CAP or LIS, ]~K-in,4.e~l incm~-¢ in CF were significantlyautnnented (Table~. CAP/BK L I S / B K CAP/BK LIS/BK Coronary O.lnM O.lmM lnM lnM Flow 9.1.+.2.8"k 9.8+3.0"k 66+8.89rOt 25.5:~.5.9"k Maximal % change (+ s.e) in coronary flow, n > 6; -kp<0.01 c.f BK alone; ¢tp<0.01 c.f LIS/BK lnM.

These date show that although CAP and LIS alone have no effect on CF, they both increase the vasodilatur effects of BK. Since the effects of the two ACEi on CF are similar with 0.1nM BK, but differ with lnM BK, involvement of the sulfllydryl group of CAP does not appear to be essential for potentiation of the vesodflator response to BK, but may account for the greater effect of CAP with hi~her concentrationsof BK.

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OPPOSING EFFECTS OF AGING ON ADENOSINE AIMo057 AND A2 MEDIATED RESPONSES IN RAT HEART John Headrick. James Cook University, Townsville 4811 Australia. A~ and A2-mediated responses (bradycardla and vasodilation) were compared in perfused heads from young adult (4-6 months) and aged (18-20 months) rats. Basal heart rate fell from 308 + 5 to 244 + 12 beats/rain, and MVO2 fell from 95 + 5 to 78 + 4 p.I O2/min/g during aging. Coronary flow was not significantly reduced (11.5 + 0.8 to 10.7 + 0.7 ml/min/g). Basal endogenous adenosine levels, indexed by venous or cardiac microdialysate concentrations, increased significantly (- 2-fold) with aging. Al-mediated bradycaKlia was 10-fold more sensitive in aged heads with a pEC50 for adenosine of 4.87 + 0.13 in aged compared with 3.88 + 0.10 in young adult hearts (p<0.005). Conversely, A2 dependent vasodilation was much less sensitive in aged heads with a pEC50 for adenosinedependent vasodilation of 4.46 + 0.10 in aged compared with 6.10 + 0.09 in young adult heads (p<0.005). Isolated aodic rings from aged animals were also less sensitive to adenosine (pEC50 = 3.05 + 0.10) compared with aoflic rings from young adult animals (pEC50 = 3.78 ± 0.09). Age related changes in cardiovascular AI and A2 responses were not secondary to differences in adenosine uptake and/or metabolism. These data indicate that Al-mediated responses become increasingly sensitive with aging whereas A2-mediated responses becomes less sensitive. These opposing changes in A1 and A2 responses may undedie functional and metabolic changes in aging myocardium.

CROSS-TALK OF ENDOGENOUS VASODILATORS Mo059 IN THE REGULATION OF BASAL CORONARY FLOW Michal M~lczewski & Andrzej Berqsewicz. Dept Clin Physiol, Med Ctr Postgrad Education, Warsaw, Poland Interaction of NO, K^w channels (CK^~) and adenosine (Ado) in the regulation o[ basal coronary t'Iow (CF) was studied. In Langendorff perfused rat hearts, the inhibition of NO synthesis (L-NOARG, 10~M), CK^-re (glyburide, Glyb, 600 nM) and Ado receptors (8-PT, 10 I~M) resulted in a drop in CF by 44%, 29% and 0%, resp. In L-NOARG treated hearts, a 3.5-fold and 2.7fold rise in the effluent lactate and Ado concentration occurred, resp. 8-PT and GIFb caused a further 26% and 24% drop in CF in those hearts. Glyb produced rightward shift of concentrationeffect curves for pinacidil- (PIN, CK^~ opener), Ado-, and SIN1- (NO donor) induced increase in CF (Tab). In the presence of Table 1. ED~ for drug-induced increase in CF

A92

PIN (nlVD

Ado (nM)

Untreated

170.5"'5.9

547--.36

SIN (pM) 1.31--.0.12

Olyb

1511--.1 0 1 .

4750--+190o

4.43__.0.5.

L-NOARG Ado

484-+24.

5724"41

65.5"4"12. 321+-45. L-NOARG, the PIN and Ado curves were shifted either to the right or were not changed. In the presence of Ado (150 nM, to mimic Ado status during L-NOARG treatment), the PIN and Ado curves showed leftward shift. Thus, in rat heart, basal vasodilation is maintained by NO and CK^w, but not by Ado. Although mainly CK^~ activation accounts for Ado-induced rise in CF, it is NO (and other factors?), rather than Ado, which regulates the basal activity of vascular CK^~. Although, in NO deficient heart, Ado partially compensates for the absence of the NO-induced vasodilation, vascular responsiveness to stimulation of CK^~ and Ado receptors is impaired.

MEASUREMENT OF INTERSTITIAL ADENOSINE IN Mo058 CRYSTALLOID-PERFUSED RAT HEART BY MICRODIALYSIS. Glenn Harrison, Lindsay Jordan, Roger Willis. Rotary Centre for Cardiovascular Research, Griffith University, Nathan,41 t t, Australia. Adenosine is a coronary vasodilator. In the potassiumarrested crystalloid-perfused rat head, we have shown that the dilation produced by infusion of adenosine into the coronary vessels is biphasic. This response was best explained by the presence of two adenosine receptors with negative EDso values of 8.06 and 5.22. Other work with paced, perfused rat hearts has shown only a monophasic dilatory response to adenosine. These different observations may be explained by differing levels of endogenous adenosine in beating and arrested heads. In the present expedments, we have used microdialysis and HPLC to measure interstitial adenosine. After equilibration of the microdialysis fibre in the wall of the left ventdcle, interstitial adenosine was found to be 0.52+0.07p.M. This value reduces 4-fold to 0.12±0.06pM dudng 30 minutes of potassium arrest. A complex relationship would be expected between infused adenosine, its concentration in the region of the target receptors in the coronary resistance vessels, and its interstitial level. Nevertheless, the present results suggest that one reason why a high potency adenosine receptor has not been detected in the vasculature of the beating heart is that endogenous adenosine levels are high enough to mask receptor function.

CROSSTALKBETWEEN NITROGLYCERINE MO060 AND P R O S T A G L A N D I N S C A U S E C O R O N A R Y CONSTRICTION. S.A.Gupte, T.Okada and R.Ochi. Dept. of Physiol., Juntendo Univ. Sch. Med., Tokyo, Japan. In isolated perfused rat hearts, superoxide [O21 significantly elevated coronary perfusion pressure [CPP] between 15 to 20 min. 02" was washed out at the end of 15 min (n=14) or 20 min (n=14). Upon washout of 02" at 15 min, CPP transiently decreased (from 67+5 to 59+6 mmHg, n=7, 13<0.05, at 2 and 4min, respectively) and then elevated (to 70±8 mmHg, n=7, p
ROLE OF HISTAMINE AND NITRIC OXIDE(NO) INMo061 CORONARY AUTOREGULTION (CA) Hllosav H. KostlC & Milan R. Petronljevid Institute of Physlology, Faculty of Hedlclne, 34000 Kragujevac, FR Yugoslavia The aim of the present study was to evaluate the roles of histamine and NO in CA. Isolated hearts of rats were perfused with constant pressure (30-110 cm H20) according to Langendorff technique. Coronary flow (CFI, release of endogenous histamine and release of NO (as NO2-) were measured under control conditions and during perfusion with selective antagonists of all three types of histamine receptors, as well as, with L-NAHE and D-NAME. Heasurements of CF revealed an autoregulatory interval of 50-80 cm H20. During autoregulation great changes of histamine release occur showing a_n inverse relationship with CF: at 50 cm Ha0 release of histamine vras about 70 higher than at 80 cm H20. In contrast, release of NO slightly increases during autoregulation parelelling CF. Histamine-receptors' blockade reduces CF (especially bellow 60 cm H20) and decreases autoregulation shifting it to the left (30-50 cm H20). L-NAME, but not D-NAHE, decreases CF (especially above 80 cm H20) and widens autoreglatory interval (40-I00 cm H20). We concluded that (I) histamine mediates CA, mainly at lower perfuslon pressure, while (2l NO modulates CA, particularly at higher coronary pressure values.

NO-Donors Elicit Vasodilation in an Extra-/ Mo063 Intra-cellular Redox Potential Dependent M a n n e r M. Skatchkov, O. Sommer, L. Larina*, A. Vanin*, E. Bassenge. Inst. of Applied. Physiology, Univ. Freiburg, Germany, *Inst. of Chemical Physics, Moscow, Russia NO donors are important modulators on vascular tone. The dependency of their activity on the extra/ intracellular .redox state is not known. We compared nitroprusside (SNP) and dinitrosyl-iron-cysteine (DNIC) for their capacity to both stimulate guanylyl cyclase (sGC) by release of cGMP and to induce vasodilation. Experiments were carried out in perfused isolated guinea-pig heart preparations. NO-release in vitro was monitored by infrared-spectroscopy (SNP at 1930 cm -1, DNIC at 1730 cm -l) in transparent electrochemical cell (EC) in the beam of IR )ectrometer Bruker IFS 25. DNIC; SNP; Total amount infused in heart 0.05/tM 10 FM 2.1 nM/g 1.8 nM/g NO content in heart, ESR data 238+_11 212_+12 cGMP release increased, [%] Coronary flow increase, [%] 62+6 45+5 Start of NO release in EC, at re- -0.22 V -0.12V ducin~ ootential applied to EC vs AgC1 vs AgC1 Conclusions: NO-release from DNIC, is more effective intracellularly in the vicinity of sGC due to its specific redox-potential which is well suited to that one maintained inside the myocyte. Thus premature extracellular inactivation of NO is avoided when DNIC is used, but not when SNP is applied.

NO AND IMPAIRMENT OF K^TI,CHANNEL INDUCEDMo062 VASODILATION IN REPERFUSED RAT HEART Andrzej Berqsewicz,Micha! M~lczewski& Michat Rabijewski Medical Ctr Postgraduate Education, Warsaw, Poland Brief myocardial ischemia followed by reperfasion results in disturbances of endothelial function and vascular responsiveness. Here the vascular responsiveness to stimulation of K^Techannels (CK^Te) in post-ischemic hearts was studied and compared to that in the hearts with the inhibited NO synthesis (L-NMMA, 50 I~M). In Langendorff perfused rat hearts subjected to 20 rain of global ischemia, 30 rain reperfusion resulted in a recovery of coronary flow to 87%. While in the control hearts, L-NMMA treatment resulted in 49% drop in CF, in the reperfused hearts, this drop amounted to only 31%, suggesting an impairment of NO pathway in those hearts. Both reperfused and L-NMMA treated hearts showed a 3-fold rightward shift of a concentrationresponse curve for pinacidil- (PIN, CK^rr opener) induced rise in CF, while a leftward shift of the PIN curve occurred in SIN-I(NO donor, 0.8 I~M) perfused hearts (Fig). Glybenclamide (CK^avantagonist, 600 nM) produced a 9-fold rightward shift of the PIN curve and only a 3.5-fold rightward shift of the curve for ,~ ~ ~,~ ~.~.~ ~ jSIN-l-induced I ~/J" ~72".~-~-~I ./"~ increase in CV

/

| HI /

/

~d &r ./ ~ff ff [ // // /

| [ /

1(Fig).

It

is

J .] concluded that fl/~ o c_o~t¢ Jin rat heart, the _ / j ~ c,y~. activity of vas-

o~.2_~" -7

~ , ~,~,7..~ o s,s-~ lpositively regu-s -5 -7 -s -s -, lated by NO. [Pinocid;I l log U iS'"-TI ,09~ Therefore, the CKaar-dependent vasodilation may be impaired in the hearts with depressed NO pathway, including the reperfused hearts.

ADRIAMYCIN MAY IMPAIR VASCULAR SMOOTH Mo064 MUSCLE RELAXATION VIA A NITRIC OXIDERELATED MECHANISM. Maurizio Cappelli-Bigazzi, "Giuseppe Ambrosio, Carmine Battagfia, Antonello D'Andrea, Massimo Chiadelio. Divisions of Cardiology, University of Naples and "Perugia, Italy. The free radical-dependent cardiac toxicity of addamycin (ADR) is well known. Recently we observed that ADR is also able to affect vascular function, reducing endothelium-dependent relaxation. Moreover, endothelium-independent relaxation to nitroglycerin (NTG) was also impaired. Therefore, we wanted to investigate the mechanism through which ADR induces Impairment of vascular smooth muscle function. In isolated rings of rabbit thoracic aorta preconstrioted with 1 laM phenylephdne, maximal relaxation to NTG was decreased after 1 h incubation with ADR in a dose-dependent manner. In 6 experiments relaxation to NTG was 103_+3%in control dngs, and was reduced to 975:5% (p=NS), 85+8 (p=NS) and 77+8% (p<.05) in presence of 30 pM, 90 M and 150 laM ADR. As a consequence, ED~oincreased from -7.93-+0.11 Log Mol NTG in controls to -7.81_+0.11-(p=NS),-7.204_-0.15(p<.05) and -6.99-+0.25Log Mol NTG (p<.01)with increasing ooncentrations of ADR. Similar results were observed with two other endotheliumindependent vasodilators, SIN-1 (which activates guanylate cyclase) and isoproterenol (which activates adenylate cyclase). However, the inhibitory effect of AD R on NTG relaxationwas much less pronounced if endothelium was absent. In fact, in denuded rings control maximalrelaxation to NTG was 101+ 3 " and after 150 p.M ADR it was 89t_4% (n=6 ; P=NS), with no significant changes in ED~. Furthermore, when nitric oxide synthesis in rings with endotfieliumwasinhibitedby300 p.M L-NAME, maximal relaxation to NTG was not significantly affected, being 100+_2%before and 85_+4%after 150 l.tM ADR incubation in=6; p--NS). Thus, ADR may acutely impair smooth muscle relaxation. This impairment is dependent on the presence of normal endothelium. Formation of toxic peroxynitrite, via interaction of nitric oxide with ADR-formed free radicals, might mediate this phenomenon.

A93

INHIBmON OF ENDOTHELIUM-DEPENDENT Mo065 RELAXATION BY CERULOPLASMIN, A CIRCULATING PROTEIN WHICH BINDS NITRIC OXIDE. Maurizio CappellI-Bigazzi, #Giovanni Musci, Carmine Battaglia, Massimo Chiariello, @LIlia Calabrese, *Giuseppe Ambrosio. Div. of Cardiology, Univ. of Naples and *PenJgia; #C.N.R. Center of Molecular Biology, @Dept. Biochemical Sciences, Univ. of Rome 'La Sapienza', Italy. It is known that native ceruloplasmin (Cp), a copper-containing plasma protein, can bind nitric oxide (NO). Since endotheliumdependent vascular relaxation has been mainly attdbuted to the release of NO, or a NO related compound, we investigated whether pudfied Cp inhibits endothelium-dependent relaxation (R) in rings of rabbit thoracic aorta in organ chambers. Concentration response curves to the endothelium-dependent agent acetylcholina (Ach) in rings pre-constricted with PGF2a were obtained before and after incubation with different concentrations of native Cp (f ,3 and 10 ~M). Control rings were incubated with 10 ~ heat-inactivated Cp (I-Cp), or 10 ~M copper-free Cp (Apo-Cp). Finally, the effets of 10 p.M Cp on endothelium-independent relaxation to nitroglycerin (N) were evaluated, n = 6 in all the experiments. ° = P < 0.05. Cont Cplp.M Cp31~M C p l 0 ~ M I-Cp Apo-Cp %R Ach 92.t:5 80+4 60+6 40-~_7 82+6 81+4 %R N 100+2 . . . . . . . . . . 94+3 ........... The effects of Cp on endothelium-dependent relaxation were reversible upon wash-out. In conclusion, ceruloplasmin significantly blunts endotheliumdependent relaxation in vitro, without affecting smooth muscle function at concentrations similar to those present in plasma. Thus, our data suggests a potential role for circulating ceruloplasmin in modulating endothelial function.

ANGIOTENSIN II M O D U L A T E S THE GROWTH M0067 OF CULTURED C O R O N A R Y ENDOTHELIAL C E L L S VIA THE AT2 RECEPTOR Sabine Bernotat-Danieiowski, Werner Mederski, Dieter Dorsch, Mathias Osswald, Norbert Beier & Pierre SchelUng, Cardiovasc. Res., E. Merck, 64271 Darmstadt, FRG.

A94

The renin-angiotensin system plays an important role in blood pressure homeostasis, water and electrolyte balance. More recently the effector peptide angiotensin II (All) has been shown to exhibit trophic effects on vascular smooth muscle cells (VSMC) via stimulation of ATl-receptors. Much less is known about regulation of cell growth by All in endothelial cells which in contrast to VSMC express both ATl-and AT2receptors. We studied (1) the receptor subtype distribution in cultured endothelial cells from rat coronary arteries (RCEC) and (2) the influence of All on the proliferation measured by 3H-thymidine uptake with/without specific and mixed AT1 and AT2-antagonists. Binding assays revealed a distribution of ATl/AT2-receptors of 80:20. bFGF-induced proliferation of RCEC was inhibited by All with a maximal effect of 40 % at 10"7M. Inhibition was completely abolished by the selective AT2-receptor antagonist L-159586 at subnanomolar concentrations. In contrast Losartan, a selective ATl-receptor antagonist had no effect on All induced growth inhibition. Effects of mixed AT1/AT2-antagonists (L-159689, EMD84391, EMD84938) were attenuated as compared to those of AT2antagonists pointing to a potential interaction between both receptor subtypes. In conclusion these results show that All inhibits the proliferation of RCEC stimulated by bFGF. Inhibition is not affected by ATl-antagonists, but is completely reversed by AT2-blockade. As a consequence All might attenuate the repair of the endothelial layer in injured vessels and inhibit neoangiogenesis via AT2-receptor stimulation.

CHANGES OF ERYTROCYTIC MEMBRANE IN PATIENTSMo066 WITH ISHEMIC HEART DISEASE UNDER THE INFLUEN'E OF LASER IRRADIATION Aleksander P.VasllJev, Nlna StrelRova, Dmltrty Teffenberg. Institute of clinical & preventive cardiology, Tyumen, Russia.

In 24 patients with angina Pactorisof Ig-W functional class there were studient the structural changes in erytrocytic membrane under the influence often daily procedures ofintmvenous blood laser irradiation (IBLI). In the middle of the IBLI course there was observed the reliable increase of phospbolipase A2 (PL-A2) activity by 127.7%, membrane cholesterol (CL) level by 27.1%, tendency to rualonic dialdehyde (MDA) rise as well as levering of phosphatidilethenolamine (PEA) level from 0.21 I_+0.01 to 0.205_+0.005 mkg/l Ph (p<0.01) and phosphatidilcholine (PC) from 0.383_+0.015 to 0.333_+0.02 mkg/l Ph (p<0.00l) and tendency to lysalecithin growlh. At the same time there was revealed the decrease of outlet fraction by 11.4%, maximum filling speed by 13.7% and maximum evacuating speed by 19.8% (p<0.05). By the end of the IBLI treatment there was regist~ed the restoration of level of MDA and CS as phospholipid membrane content and decrease of FL-A2 activity. There was also observed the rise of the total outlet fraction, speed of filling and evacuating of the heart which topped the initial valies by 6.2%, 19.4% and 22.2%, correspondialy (p<0.05). One month after finishing the irradiation course there was revealed the rise of the content of PEA by 6.7%, PC by 23.6% and growth of phospholipida/cholesterol ratio from 1.4_+0.3 to 2.2_+0.2 (p<0.05) in erythrocytes. Positive changes in biomembrane structure were associated with the increase of outlet fraction and effort tolerance by 26.4% (p<0.01). These changes remained up to 4-6 months. Thus, it may by assumed thet IBLI has a s¢cssor influence accompanying by modification in cell membrane, changes in cell metabolism and lowering of activity values. As compensate antistrass mechanisms start to work and laser influence through photoaccepter apparatus begins to realize there occurs the restoration of lipid structure of membrane, rise of functional heart activity and Patients tolerance to exercise.

TWO DISTINCT PATHWAYS OF HYPOXIA-SENSINGMo068 FOR THE INDUCTION OF VASCULAR ENDOTHELIAL GROWTH FACTOR mRNAIN THE HEART Etsuo Hashimoto, Kumie Kage, Teruhiko Ogita, Takashi Nakaoka, and "~'uji Kira. University of Tokyo, *Showa General Hospital, Tokyo, Japan We previously reported that transient ischemia applied on perfused rat hearts selectively induced vascular endothelial growth factor (VEGF) mRNA as an angiogenic factor. In this report, we studied the role of adenosine and the responsive cell varieties for the ischemic induction of VEGF. Ischemia for 5, 15 and 30 minutes applied on perfused hearts enhanced adenosine concentration detected in the perfusate up to 711 (10.8), 5173 (304) and 5350 (27.1), respectively, in comparison with the control of 61 (2.0) (mean (SD), nM). Adenosine (10 .8 to 10-SM) used in non-ischemic perfusion dose-dependently enhanced VEGF mRNA expression with the maximum of 2.8 fold of the control. The enhancement was reduced by about 60% with the use of adenosine deaminase, non-selective and A2 selective receptor antagonists. In order to define the cell varieties to respond to hypoxia and adenosine, cardiac myocytes and non-cardiac myocytes from neonatal rats were separately cultured. Hypoxia of about 40 mmHg enhanced VEGF mRNA expression both in cardiac myocytes and non-cardiac myocytes in 2 hours. In contrast, adenosine up to 10-s M elevated the mRNA only in non-cardiac myocytes. These findings suggest that endogenous adenosine produced by hypoxia and ischemia enhances VEGF mRNA in non-cardiac myocytes through A2-receptor and that another hypoxia sensing mechanism may exist in cardiac myocytes for the induction of VEGF mRNA.

CORONARY ENDOTHELIAL DENUDATION LIMITS MO069 REACTIVE HYPEREMIA: ROLE OF PLATELET EMBOLIZATION Jos(~ A Barrab(~s, David Garcia-Dorado, Miguel A Gonz-51ez, Amparo Garcfa, Marisol Ruiz-Meana, Jos(~ Blanco, Yolanda Puigfel, J Soler Soler. Hospital General Vail d'Hebron, Barcelona, Spain. The influence of coronary endothelial injury on reactive hyperemia was investigated in 16 pigs submitted to 30 rain of occlusion of the LAD preceded or not by catheter induced endothelial denudation of this artery (CED). The day before, platelets had been labelled ex vivo with 99mTc-HMPAO, and reinjected. Coronary blood flow was measured at the occlusion site (electromagnetic flowmeter) during 5 h of reperfusion, and specific radioactivity was measured in samples from the area at risk and expressed as a percent of activity in control myocardium. Animals did not present myocardial infarction (tryphenyltetrazolium reaction). The hyperemic response was blunted in the CED group (coronary blood flow, ml/min): Baseline CED 29±1 No C E D 31±4 MANOVA: CED: p =

5minR 15minR 30minR 43±6 35±6 27±3 69±12 52±4 46±5 0.02; Time: p = 0.04.

1 hR 32±2 39±4

5 hR 26±4 35±3

Platelet content was increased in the area at risk without between group differences (specific activity 197 + 71% and 235 + 7 7 % respectively in CED and controls, p = NS). Thus, coronary endothelial injury limits hyperemic response and platelet microembolization does not contribute to this effect.

Mo071 ENDOTHELIAL CELLS OF MYOCARDIAL CAPILLARIE.S SWELL AS A RESULT OF REOXYGENATION, NOT HYPOXIA. John L. Donnelly & Barbara J. Ward, Dept of Anatomy, Queen Mary & Westfield College, London, UK. Qualitative .studies of the endothelial response to hypoxia report a swelling (ie. intracellular oedema) effect. This was assessed quantitatively in capillaries from isolated rat hearts perfused with hypoxic medium for 30 or 60 minutes, or 30 minutes followed by oxygenated" medium for 15 minutes. Areas and perimeters of digitised capillary profiles were computed, corrected for obliqueness of section and compared statistically. Contrary to the" literature no endothelial swelling was found after 30 or 60 minutes of hypoxia, as compared with controls. Overall capillary cross-sectional area was significantlyreduced in both cases, but no change was found in endothelial cell area (although an increase in cell thickness gave the impression of swelling). Hypoxia followed by reoxygenation, however, did result in an increased cell area concomitant with a reduction in overall capillary area and exacerbating reduction of the luminal area. Implications of a localised control of blood flow are discussed.

SUBCELLULAR LOCALIZATION OF Mo070 ADHESION MOLECULES IN HUMAN ENDOTHELIAL CELLS Dimitri Scholz, Bruno Devaux, Bernd P6tsch*, Bettina Kropp* & Jutta Sehaper. Max-Planck-Institute and *Kerckhoff-Cllnic, Bad Nauheim, Germany The aim of this study was to follow the different stages of synthesis and transfer of cell adhesion molecules (AM) E-Selectin, ICAM-1 and VCAM-1 after IL-11~ stimulation of HUVEC. Methods were immunohistochemistry using monoclonal antibodies, confocal microscopy and immunogold electron microscopy (EM). PECAM-1 is expressed in all and ICAM-1 in 30% of the cells under control conditions. Synthesis of E-Selectin, VCAM-1 and ICAM-1 begins 1.5h after IL-stimulation in the perinuclear rough endoplasmic reticulum (pn), achieves a maximum at 2.5-4h, decreases between 4-8h and returns to zero at 24h, but the proteins persist longer. From the pn the proteins are transferred to the cell membrane (m) and exocytosed, maintaining the binding with the m to ensure function. After 2h of stimulation, ESelectin and VCAM-1 positive granula (but never ICAM1) are observed, become larger till 6h and persist in some cells fill 72h. After 2.5h diffuse fluorescence occurs indicating labeling of m confirmed by EM. The maximal labeling for E-Selectin is at 4-6h, for VCAM-1 at 8-12h, for ICAM-1 at 24h. After 24h after IL, numerous cellular processes labeled strongly for ICAM-1 appear. For ICAM-1, we assume a direct connection between a synthetic site and m (cytoskeleton?). Conclusion: an active synthesis of all AM occurs after stimulation indicating the necessity of all AM for final cell adhesion. Cellular transport, however, is different for the different AM.

FREE RADICALSCAVENGERSANDANTIOXIDANTMo072 REDUCE REPERFUSION-INDUCEDENDOTHELIAL CELL SWELLINGAND MEMBRANEBLEBBING Barbara J. Ward & Mark Scoote, Dept of Anatomy, Queen Mary & Westfield College, London, UK. Reperfusion after ischaemia causes swelling of capillary endothelial cells and blebbing of the luminal membrane, both of which may contribute to the "noreflow" effect. We have used the isolated rat heart rendered globally ischaemic and reperfused with KrebsHenseleit medium to quantify these changes both before and after the addition of either superoxide dismutase and catalase or the antioxidant trolox. Parameters measured from a digitised image of each capillary include whole capillary area, luminal area, endothelial cell area, the outer and inner perimeters and the proportion of luminal area occupied by membrane blebs. The measurements showed that endothelial cell crosssectional area was increased on reperfusion compared with the pre-ischaemic period (we have previously shown that ischaemia alone does not cause endothelial cell swelling) with a concomitant decrease in overall capillary size. This was ameliorated by both scavengers and trolox, as was the degree of luminal membrane blebbing which was more effectively reduced by the trolox than by the scavengers. We conclude that both free radical scavengers and antioxidant minimize structural changes to the cardiac microvasculature on reperfusion after ischaemia. A95

TISSUE PLASMINOGEN ACTIVATOR (t-PA) Mo073 AS A MARKER OF ENDOTHELIAL INJURY Guro Valen, Anders Owall, Elsa Eriksson, Anders Kallner, Bo Risberg, & Jade Vaage. Karolinska Hospital, Stockholm, and Ostra Hospital, Gothenburg, Sweden. t-PA is a possible plasmatic marker of endothelial injury. Release of t-PA antigen (Ag) and activity, creatine kinase (CK-MB), and troponin-T in artedal and coronary sinus (CS) blood was investigated in 14 patients scheduled for coronary bypass surgery (cardiopulmonary bypass 83+5 min, aortic crossclamping (AXC) 42:1:4 min) (mean+SEM). CK-MB increased more in CS than arterial blood (18+_2 vs 14+_2 mg/I, p<0.002, 20 min after AXC), as did troponin-T (max 1.01+0.2 vs. 0.7+0.1 mg/1 60 min after AXC, p<0.002), t-PA Ag and t-PA activity also increased more in the CS (37+6 vs 31+5 ng/ml, p<0.009, 5 min after AXC, and 321+108 vs 244+87 pmol/I, p<0.02, 10 min after). CK-MB and troponin-T were higher in patients with the longest time of AXC. Increase of t-PA Ag and activity was independent of time of AXC. In conclusion, CK-MB, troponin-T, and t-PA were released from the heart after cardioplegia. Release of CK-MB and troponin-T increased with increasing AXC, but t-PA did not. t-PA is not a suitable plasmatic marker of graded endothelial injury in the coronary circulation.

ALTERATION OF F-ACTIN DISTRIBUTION IN Mo075 ENDOCARDIAL ENDOTHEUUM INDUCED BY RAPID VENTRICULAR PACING Luc J. Andries, Grzegorz L Kaluza, Stanislas U. Sys, Dirk L Brutsaert, Laboratory of Physiology, University of Antwerp, Belgium. The role of cardiac endothelium in tachycardiainduced heart failure is at present unknown. We investigated the morphology of endocardial endothelium (EE) in rabbits subjected to left ventdcular pacing with increasing rate (250-440 bpm). After hemodynamic measurements in vivo, left ventricles of control and dilated hearts were processed for en face confocal microscopy. In the apex of control hearts, F-actin staining in EE was restricted to the peripheral actin band. In the apex of dilated hearts, large zones of EE contained many actin filament bundles which often seemed to cross over several EE cells. Peripheral actin bands of EE cells could not be discerned or were broad and consisted of disconnected actin filaments. In endocardial interstitial tissue subjacent to EE with a modified cytoskeleton, more actin-rich interstitial cells were observed than in control hearts. However, in the outflow tract and elsewhere along the base of the heart, endocardial interstitial cells and EE cells had a normal appearance. The observed structural differences between endocardium of paced and control hearts may result from changes of mechanical stress in the heart wall. Transendothelial permeability and other physiological properties may be affected in EE cells with an altered cytoskeleton. A96

I N C R E A S E D P R O T E O S Y N T H E S I S OF T H E Mo074 E N D O C A R D I A L E N D O T H E L I A L C E L L S AND T H E CARDIOMYOCYTES OF THE RAT LEFT VENTRICLE I N D U C E D BY O N E DAY L A S T I N G H Y P E R T E N S I O N Marcel Hork~, Pavel Horsk~, Franti~lek Kol/t~ and Vladimir Z,nojU. Dept of Pathological PhysiololD', Medical faculty, M a s s a r y k University, 60000 Brno, C Z

We detected minute changes in the proteosynthesis of endocardial endothelial cells and cardiomyocytes in h)1~ertensive rats. AgNOR (argyrophilic nucleolar organizer region) - the most prominent feature of the interphase nucleus, involved in the ribosome biogenesis-was used to mark proteosynthetically active cells in the stenosis of the rat abdominal aorta. Ten female rats underwent surgeD' and the mbp in the aortic arch was 178 + 25 mmHg. Ten controls showed mbp in the aortic arch 104 + I 1 mm Hg. The data were analyzed by multiple t- test, see the Table. We found that one day lasting hypertension activated the endoeardial endothelium and the cardiomyocytes of the left ventricle. We concluded that the activation of the endothelium ~as a direct response to the blood pressure elevation and secondarily the synthetic reaction of the cardiomyocytes was coupled to endocardial endothelium. Our morphological findings supported the hypothesis encountering the existence of humoral intracavitary regulation mediated by a variety of peptides produced by activated endoeardium. Moreover the endoeardium is certainly involved in the pathogenesis of cardiovascular diseases, e.g. hypertension. Nucleolarnumberaftertwentyfourhours lastingstcnosis Controls Experimental Animals endocardid

1.49 ± 0.12

2.46 ± 0.33

tmdothelium cardiomyoc~,les 2.92 ± 0.07 3.65 ± 0.22 All data arc pre~-mtedas arithmeticalmean± staadarddeviation.

COMPARISON OF A NEGATIVE INOTROPIC Mo076 E N D O T H E L I A L CELL (EC) F A C T O R W I T H BDM IN INTACT CARDIAC MYOCYTES. Chris B. Pepper, Malcolm J. Lewis & Ajay M. Shah, Cardiovascular Sciences Research Group, University of Wales College of Medicine, Cardiff, UK Both the superfusing effluent of cultured porcine aortic EC and coronary effluent of isolated rat hearts contain an unidentified factor(s) that inhibits m y o c y t e contraction with tittle change in Ca 2+ transient. W e have compared the effect of this effluent with that of the Ca 2+ 'desensitiser' butanedione monoxime (BDM, l m M ) on the steady-state relationship between [Ca2+]i (fura-2 fluorescence ratio-R) and tetanic shortening (S) in intact rat ventricular cardiac myocytes tetanised (10Hz) in the presence of the sarcoplasmic reticulum (SR) Ca 2+ATPase inhibitor, thapsigargin (0.2pM, 10rnins). Application of effluent caused an increase in resting cell length (Ld) (126.5+4.4 to 128.1+4.71am, I>=0.02, n=10 myocytes), and a reduction in S (25.2_+_5.1%, p=0.001), but no associated change in diastolic R (+4.0-J:2.8%, p=ns) or tetanic R amplitude (-0.8+1.8%, p=ns) was observed. BDM (lm_M) also caused an increase in Ld (122.8+4.31am to 123.4+4.31.tm, n=8, p=0.004), and a reduction in S (-18.8%, p=0.003) without significant change in diastolic R (-0.8i-0.8%, p=ns) or tetanie R amplitude (-6.4:P_2.2%, p=0.1). These results suggest that EC effluent and BDM have similar actions on myofdament Ca2+ response, although the underlying mechanisms remain to be elucidated.

ADENINE REUTILISATION IN RAT Mo077 ENDOTHELIUM Ryszard T. Smolenski, Marek Zych & Zdzislaw Kochan, Department of Biochemistry, Medical University of Gdansk, Poland Adenine (ADE) reutilisation is important pathway of adenylate pool regeneration, but unlike with adenosine, only few studies addressed the question of regulation of this pathway in heart cells. Supply of ribose (R) greatly accelerates ADE incorporation into ATP in cardiomyocytes. Inorganic phosphate (Pi) exerts also stimulating effect in many cell types. We have evaluated the effect of R and Pi on the rate of adenine reutilisation in rat endothelium. Microvascular endothelial cells were cultured from the rat heart and used for experiment after 2-3 passages. 14C ADE was added for 5 min at 10 pM concentration followed by determination of its incorporation into ATP in cell extracts using HPLC with in line radiodetection. Control rate was 8.2 pmol/min/flask and little change was observed in the presence of 5 mM R (9.2 pmol/min/flask). High Pi in the medium (6.2 mM) exerted some, but still minor effect (11.5 pmol/min/flask). Combined effect of R and Pi produced the most significant increase (12.9 pmol/min/flask), but still small in comparison to the other cell types. In conclusion, endothelial adenine reutilisation is relatively irresponsive to metabolic stimulation, suggesting that supply of pyrophosphoribosylphosphate, a co-substrate of adenine reutilisation is .sufficient, contrasting cardiomyocytes

Combination of Kca channel and Kv channel blockers completely Inhibits relaxation by EDHF (endothellum derived hyperpolarizlng factor).

Mo079

Yoshihito Nakashlrna,Yasumasa Fukami, Masanori Shinoda, Yukio Told, Kenji Okumura, Takayuki Ito, Internal Medicine 2, Nagoya Univ. School of Medicine The aim of our study is to clarify which K channels are responsible for endothelium derived hyperpolarizing factor (EDHF) induced relaxation in canine coronary arteries. We investigated the effects of various K channel blockers on endothelium dependent relaxation in response to acetylcholine (10/zM) in isolated canine coronary rings precontracted with prostaglandin F2a(2uM) in the presence of Nconitro-L-arginine methyl ester (L-NAME); an inhibitor of nitric oxide synthase, and indomethacin; an inhibitor of cycloox"ygenase, because residual relaxation resistant to these 2 inhibitors is considered as relaxation by EDHF. Extracellu]ar K at 30ram completely inhibited the residual relaxatlon.A non selective K channel blocker; tetrabutylamrnonium (3raM) completely Inhibited the residual relaxation .An ATP-sensitive K channel blocker; glibenclamide (2/z M), did not inhibit the residual relaxation. In contrast Ca-activated K (Kca) channel blockers; charybdotox:in (ChTX) (i00nM) and tetraethylammonium (10raM), and a voltage-dependent K (Kv) channel blocker; 4-arninopyridine (4-AP) (imM), partly inhibited the relaxation (76.2%, 56.1%, and 46.9%of maximum relaxation, respectively). A combination of ChTX and 4-AP completely inhibited the residual relaxation. This suggests that EDHF may exert its relaxing action on canine coronary arteries through two types of K channels, i.e. Kca and Kv channels.

MEMBRANE CONDUCTANCE OF RESTING M0078 ENDOCARDIAL ENDOTHELIAL CELLS Paul F. Fransen, Marc J.M. Demolder, David M. Van Bedaf and Stanislas U. Sys. Laboratory of Physiology, University of Antwerp (RUCA), Antwerp, Belgium.

The resting membrane potential of endocardial endothelial (EE) cells (-45 to -65 mY) is dependent on the extrecellular K+-concentretion, but is always more depolarized than the equilibrium potential for K+-ions (-85 mV) (Laskey et aL, 1990; Fransen et al., 1995; Manabe et al., 1995). Therefore, the membrane of these cells must be permeable to other ions than K* alone. In the present study, membrane permeability of non-stimulated cultured EE cells from the porcine right ventricle to K+-, Na+-, Ca2+- and cr-ions was investigated. The conventional whole-cell mode of the patch-clamp technique was used to construct currant-voltage (I-V) relations in different ionic conditions. In baseline, the bathing solution was a normal Tyrode's solution, while the pipette contained (in mmol/I): 30 KCI, 100 KAsp. 1 CaCI2, 5 MgCI2, 10 HEPES, 10 EGTA, pH 7.2, 37 °C. The main membrane current in non-stimulated EE cells was the inwardly rectifying K+-current. Removal of Na+ or Ca2+ from the bathing solution had no or only a slight depolarizing (5 to 10 mV) effect on the zere-current potential of the I-V relations.When, however, the extra- or intracellular Cr-concentration was changed, the zero-current potential followed the shift of the reversal potential for CI-ions and an outwardly rectifying CI-current ~:ould be identified. Results indicated that the membrane of non-stimulatedEE cells was permeable to K*- and cr-ions. Therefore, it is suggested that Cl'-ions, in addition to K+-ions, can affect the release of endothelium-derivedmediators.

HUMAN MITRAL VALVE ENDOTHELIAL Mo080 CELLS POSSESS CALCIUM-ACTIVATED K+-CHANNELS. Herbert M. Himmel. Dept. of Pharmacology, University of Essen, Essen, Germany. Endothelial cells (EC) respond to hormonal signals and shear stress with the generation of mediators (e.g. NO) that modulate smooth muscle tone. The initial response of EC involves elevation of [Ca2+], the magnitude and duration of which is influenced by membrane potential and currents such as Ca z+activated K+-currents (IK(Ca)). Human mitral valve EC were isolated and cultured, and membrane currents were measured by means of the patchclamp technique (cell-attached mode, 22°C, bath and pipette solution with 140 mM K+). Under control conditions, two types of channels were observed: an inward rectifier-like channels in 2 of 11 EC with a conductance of 68 pS, and K(Ca)-channels in 7 of 11 EC with a conductance of 220+19 pS. When cells were exposed to stimuli in the bath such as bradykinin (BK, 1 IJM), ATP (100pM) or BHQ (2,5-di-(tert-butyl)benzohydroquinone, 301JM), K(Ca)-channel activity increased dramatically after a delay of 10-30 s. The open probability in the potential range of +60 to +100 mV increased from 0.05 to 0.92, irrespective of the agonist applied. Thus the increased channel activity is due to the intracellular second messenger Ca ;'+, because an increase in [Ca :'+] is common to BK, ATP and BHQ. In conclus on, mitral valve EC could serve as a model for studying human endothelial cells. A97

BASELINE SYSTOLIC CYCLE LENGTH PREDICTSMo081 MYOCARDIAL INOTROPIC RESPONSIVENESS TO ENDOTHELIN-I. Gilles W De Keulenaer, Stanislas U Sys, Dirk L Brutsaert. Laboratory of Physiology. University of Antwerp, Belgium As myocardial inotropic responsiveness to endothelin-1 (Et) depends on the functional state of cardiac endothelium (CE), which modulates systolic cycle length, we analyzed the relation between inotropic responsiveness to Et and baseline systolic cycle length (baseline time to half isometric relaxation; bTHR) in isolated cat cardiac muscle (35°C; 1.25 mM [Ca2+]o). The inotropic response to Et (10"°-107M) was strongly correlated with bTHR (n=17, left panel). In contrast, the response to [Ca~*]o (7.5 mM) was poorly correlated with bTHR (n=26, right panel). Dimethyl amiloride, inhibiting Na*/H + exchange, markedly reduced the response to Et.

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Iennart Faijser, John Pernow, G~m~ur Ahlburg & Jan M. ~ . Dqpt of ~ n i c a l Physiology, ~nge _~P~_~ ital, D ~ t of Cardiology, Karolinska ~=pital and Dept of Pharmacology, Karolinska Institute, ~ , S~n.

Accordingly, baseline cardiac isometric twitch duration predicted responsiveness to Et (which influences [Ca2+]~sensitivity through Na÷/H÷ exchange), but not to [Ca2"]o. Baseline cardiac twitch duration may, therefore, reflect functional state of CE and [Ca2"],-sensitivity recruitable inotropic reserve.

Endctbelin-1 (L-T-l) i s a powerful vasoconstrictor. Also the precursor peptide, big ET-I, produces vasoconstriction. Effects of i.v. infusions of EP-I and big ET-I on myocardial blood flow and metabolism were studied in healthy men using catheters in the bradlial artery and the coronary sinus for m ~ u r e m e n t of mean arterial pressure (MAP) and coronary sinus blood flow (CSBF; thermodilution) and oxygen, lactate, and glucose content. Both ET-I and big EP-I increased MAP and reduced heart rate by 15%. CSBF and CS oxygen saturation d~v~ased by 25% m e i n t e ~ m y ~ oxyg'en u p t a k e unaltered. Myocardial lactate uptake increased. Big ET-I but not ET-I also myocardial glucose uptake. It is concluded that ET-I and big ED-I produce pronouncea____ and similar coronary vasoconstriction. does not lead to anaezr~ic m e t a b o l ~ . On the oontrary lactate metabolism, and in the case of big EP-I also glucose metabolism may be stimulated.

METABOLISM OF ENDOTHELIN-1 0ET-1) MO083 AND BIG ET-1 IN RAT lVlYOCARDIUM. l.J. Zeitlin, A. Garjani, C.L. Wainwright & A.K. Pitt. Dept of Physiology & Pharmacology, University of Strathclyde, Glasgow, Scotland, U.K.

PLASMA ENDOTHELIN-1 AND ENDOGENOUS Mo084 DIGOXIN-LIKE FACTOR IN HYPERTENSIVE PATIENTS WITH ACUTE CEREBROVASCULARDISEASE Qiang Zeng,Dogn Ning Wei,Ying Xian Fan & Xia dun Ys. Institute of Geriatrics, Chinese Great Wall Hospital & PLA 309 Hospital, Beijing, P.R. China

We have recently reported that in rats, ET-1 exacerbates and ETA receptor blockade diminishes myocardial ischaemiarelated arrhythndas (Garjani et al, 1995, I Cardiovasc. Pharmacol., In Press). The processing of big ET-1 and ET-1 in the myocardium remains to be characterized. The ventricnlar myocardiums of male Sprague Dawley rats were homogenized in phosphate buffer (O.01M, pH7.2, O.1 Triton X100). Degradation of the endothelin peptides (8 nmole/ml) was assessed by incubation with the extracts (1:20 dil.), boiling, and drying supematants under N 2. Mature ET-I and big ET-1 were measured using RP-HPLC (C18, acetonitrile 15-70%, 0.09% 1TA). Degradation of ET-1 by rat myocardium was optimal at acid pH (
The aim of p r e s e n t study was to measur plasma l e v e l s of immunoreactive e n d o t h e l i n ( i r - E T - l ) and endogenous d i g o x i n - l i k e f c a t o r (EDLF) in e s s e n t i a l hypertension(EH) p a t i e n t s with acute c e r b r o v a s c u l a r d i s e a s e (ACVD). E n d o t h e l i n and Gigoxin radioimmunoassays were used to determine plasma i r - E T - I and EDLF l e v e l s in 31 EH p a t i e n t s w i t h ACVD and 35 c o n t r o l s u b j e c t s . R e s u l t s showed t h a t plasma ir-ET-1 and EDLF l e v e l s in EH p a t i e n t s with ACVD were s i g n i g i c a n t l y higher than those in normal s u b j e c t s r e s p e c t i v e l y ( 4 . 8 0 ± 0 . 1 6 p g / m l VS 3 . 6 2 ± 0 . 0 8 pg/ml P<0.001; 288.15± 30.60 pg/ml VS 151.67± 18.23 pg/ml P<0.01). Plasma ir-ET-1 l e v e l s in p a t i e n t s with d i f f e r e n t kind of ACVD were s i g n i g i c a n t correlation between ir-ET-1 and EDLF (r=-0.4720, P<0.05), but t h e r e was no s i g n i f i c a n t c o r r e l a t i o n between i r - E T - I and blood p r e s s u r e ( r = - 0 . 1 6 9 2 , P<0.05). Conclusions. The results suggest t h a t the e l e v a t e d plasma ir-ET-1 and EDLF l e v e l s in EH p a t i e n t s with ACVl). There was a s i g n i f i c a n t correlation between i r - E T - I c o n c e n t r a t i o n and EDLF l e v e l . ET and EDLF may be important h o m e o s t a t i c f a c t o r s in human.

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INOTROPIC EFFECTS OF THE ENDOTHELINB-Mo085 RECEPTOR-AGONtST IRL 1620 IN VIVO Martin E. Beyer, Gtinther Slesak & Hans Martin Hoffmeister. Medical Dept. III, University of Tiibingen, Germany In vivo studies could not confirm the positive inotropy of endothelin-1 (ET-I) described in in vitro experiments due to its strong vasoconstriction with consecutive cardiodepression. As ETa-receptors seem to be involved in ET-induced inotropy we studied the effects of the highly specific ETa-agonist IRL 1620 on hemodynamics and inotropy. We investigated in open-chest rats the dose-dependent effects of 7 min i.v. IRL 1620 (0.4, 1.0, 2.0, 4.0 nmol/kg vs. NaCI) on the intact circulation and on isovolumic left ventricular pressure generating capacity (isovol. LVSP, isovol, dp/dtmax as indexes of contractility). To study IRL 1620-effects on energy metabolism myocardial high-energy phosphates (energy charge as ischemia-index: EC = [ATP+ ~AADP]/[ATP+ADP+AMP]) were measured. IRL 1620 [nmol/kg] NaCI 0.4 1.0 2.0 4.0 TPR 132+3 # 128+3 # 163+8 # 179+7 # 97+1 isovol. LVSP 102+2" 102+1" 107+2 # 108+1 # 95+1 isovol, dp/dtmax 112+4 # 113+2 # 122+4 N 117+3 # 99+2 Means+SEM in % of preinfusion values, *p<0.05, #p<0.01 After initial vasodilation IRL 1620 causes a dose-dependent vasoconstriction less pronounced compared to ET-1. EC is reduced by 1 nmol/kg ET-1 (0.73+0.03 # vs. NaCI: 0.84-1- 0.02) and not influenced by 2 nmol/kg IRL 1620 (0.85+0.02). .CONCLUSION: Selective activation of ETa-receptors by IRL 1620 produces (in contrast to unselective activation of ET A- and ETa-receptors by ET-1) no vasoconstriction-induced myocardial ischemia and thus causes a positive inotropy.

A C U T E E F F E C T OF C A P T O P R I L A N D A N T I ENDOTHELIN ANTIBODY ON BLOOD PRESSURE

MO087

IN S P O N T A N E O U S L Y H Y P E R T E N S I V E R A T S L.P.Zhang,Y.X.Fan,Q.Zeng and X . J . Y u . l n s t l t u t e of Geriatrics,General H o s p i t a l of P L A , B e l j i n g , C h l n a Endothelin (ET) is a powerful vasoconstrictor peptide produced in the endothelium of blood vessels. When ET is i n j e c t e d into i n t a c t a n i m a l , | i r s t r e s p o n s e is a transient "quickly decrease of blood pressure. Depressor response to ET is followed by a sustained and longlasting c o n s t r i c t o r effect of vascular smooth muscle. So ET may be play important role in pathophysiology of h y p e r t e n s i o n . In our study 24 spontaneously hypertensive rats(SHR) were divided into three groups: control, captopril(1 mg/kg IV)treatment group and antiET a n t i b o d y ( 1 : 5 0 0 0 50 ul IV )treatmen.t group. We found that captopril caused a sign=ficant decrease of blood pressure throughout the observation period but anti-ET antibody did not. Captopril and anti- ET body induced a significant decrease of angiotension ] l a n d ET blood levels c o m p a r e d with control group. Aortic immunostaining of ET in captopril treatment group was decreased compared with control and anti- ET antibody t r e a t m e n t group. This s t u d y s u j e s t s that ET and A l l play ~mportant roles in r e g u l a t i o n of blood p r e s s u r e in SHR C i r c u l a t i n g ET l e v e l s do not r e f l e c t the l o c a l vascular production of the peptide local ET levels is a better parameter in modulating vascular resistance Anti- ET a n t i b o d y =s no u s e f u l in d e p r e s s u r e .

Mo086 EFFECTS OF HYPOXIAAND TAURINEON ET-t mRNA EXPRESSION AND RELEASEOF ET-I, AT-II AND EDF FROM CULTUREDBOVINE PULMONARYARTERIAL ENDOTHELIALCELLS(PAEC)AND DOT BLOT HYBRIDIZATIONWERE STUDIED Yu Zhonghs ,Sun Binwong, Qlan Gulsheng, U JIcheng, Chesg HangweI,Lui Xlaofian. Department of Resplratow Diseases, Beirptg MUhry General Hospital& Center of Respiratory Diseases,XJnqiao Hospital, The Third Military Medical University & Department of Pathophy~oiogy,ThoThird M~ary Medical University(Chine) EndothsFm-I can cause comraction of pulmonary vascu~atura and produce pulmonary hypertension.In recent years,several authors indicated that taurine could protect several Idnds of tissue cells from injury was effective in treating related diseases.But it remains unclear whether that taurine has the same effect on diseases of respirntoW system.Our results suggest that: 1.PAECcuJuradunder enoxlafor 3. 12 and 24 hour resulted in an increase In ET-I mRNA expression.Itreached the peak at the end of 24 hour.By adding taurine to the culture madlum,ET-I mRNA expression was kfnJbited. 2. PAEC c~tured under 0%02 and 3%02 induced increases in ET-I release at the end of 12. 24 and 48 hour.The increased release was inh~ited by tam'ins. 3. The release of AT-II from PAEC cultured under 0%02 and 21%O2 was enhanced by ET-I at the end of 12. 24 and 48 hour,white taurine inhibited the effect of ET-I. 4. the release of EDF from PAEC cultured under 0%02 increased at the end of 24,48and 72 hour, taut'me inhibited it.Positive correlation e,d~s between ET-I end EDF releases hem PAEC under 0%O2condition. It ts concluded: 1. Expressionof ET-t mRNA and release of ET-I from PAEC increased under hypoxla. 2. Tatatne can it.'bit the hypo~dainduced expression of ET-lmRNA of PAECandreduce the release of ET-I from PAEC. 3. Taurine inhibits the release of AT-II from PAEC either under 21%O2 or under 0%02. 4. "latrine inhibits the Increasedrelease of EDF from PAECunder hypoxla.

FOREARM ENDOTHELIUM-DEPENDENT VASOMo088 DILATION IS NOT SELECTIVELY IMPAIRED IN HEART FAILURE DUE TO V A L V U L A R HEART DISEASE M o t o y u k i Nakamura, Hiroaki Yoshida, Naoshi Arakawa, T s u t o m u Funakoshi, M a k o t o C h i b a , K a t s u h i k o Hiramori.

Iwate Medical University, Morioka, Japan Recent reports have suggested that, in patients (pts) with congestive heart failure (CHF-), endothelium-dependent peripheral vasodilation (EDV) mediated by acethycholine (ACH) is impaired, but endothelium-independent vasodilation mediated by nitroprusside (SNP) is preserved. This suggests that EDV is selectively depressed in CHF. However, a classical paper demonstrated that nitrate-mediated forearm vasodilation is depressed in CHF due to valvular heart disease (R. Zelis et el. JCI 1968;47:960). To examine whether endothelial dysfunction in peripheral vascular bed is consistently impaired in CHF, forearm blood flow (FBF; ml/min/100ml) following intra-artedal graded infusion of ACH (0.75-4.51ag/min/100ml) and SNP (0.05-0.31~g/min/100ml) were measured by forearm plethysmography in 52 pts with CHF due to valvular heart disease and matched normal controls (n=29). The following data were obtained: rest FBF

ACH-induced SNP-induced peak FBF peak FBF Control (n=29) 2.8 _+0.2 14.7 _+1.6 13.0 + 0.8 NYHA1 (n=9) 2.3+0.3 13.6+2.0 13.2_+1.4 NYHA 2 (n=24) 2.0 _+0.2 8.6 4-1.4"* 8.5 _+0.8** NYHA 3-4 (n=l 9) 1.8 _+0.2* 7.7 __.1.4"* 7.0 _+0.7** *p<0.05 **p<0.001 vs. control group (rnean_+SEM) We conclude that the selective endothelial dysfunction is not a common phenomenon in the peripheral vascular bed in CHF.

A99

P R O S T A G L A N D I N E1 AND P R O S T A N I T Mo089 DILATE RAT AORTA WITH DIFFERENT MECHANISMS V l a d i m i r N. S e r e b r y a k o v & Stanislav Z a k h a r e n k o I n s t i t u t e of E x p e r i m e n t a l Cardiology, Cardiology R e s e a r c h C e n t e r of Russia, 3 r d C h e r e p k o v s k a y a str. 15A, 121552 Moscow, Russia.

SIGNALLING PATHWAYS IN Mo090 PROSTAG LANDIN-MEDIATED V A S C U L A R SMOOTH MUSCLE CELL GROWTH. Lorraine Yau and Peter Zahradka. Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre; Departments of Physiology and Medicine, University of Manitoba, Winnipeg, Canada.

The effects ofprostaglandin E l (PGE1) and prostanit (1,3-dinitroglycerol ether o f PGE1) on membrane currents were investigated using the patch-clamp technique in fresh-isolated and cultured rat aortic smooth muscle cells. Mechanical responses o f rat aortic rings induced by P G E 1 and prostanit under isometric conditions were studied as well. PGE 1 (100 nM) and prostanit (100 nM) produced an increase in whole-cell outward currents at positive holding potentials. These currents were blocked by internal Cs + and are likely due to increased Ca2+--activated K+ permeability. In the symmetrical K + solution in configuration inside-out P G E 1 was ineffective, but prostanit and dinitrogiycerol increased K + permeability through the cell membrane. Both P G E 1 (100 riM) and prostanit (100 n_M) produced dilation o f aortic rings in normal physiological solution, but dilation caused by prostanit was sicnifieantly longer than that caused by PGE I. These results suggest that PGE I caused dilation o f aorta via intraceIlular messenger(s), presnm,bly cAMP. Prostanit augment K + permeability directly, and this mechanism could be responsa"ole for the prolonged dilation o f aorta in comparison with the brief effect o f PGE 1.

Prostaglandins (PG) have recently been s h o w n to stimulate the g r o w t h of vascular smooth muscle cells (SMC). To characterize this process, w e examined potential signalling p a t h w a y s that may couple PG's to both rRNA and DNA synthesis. Mitogen-associated protein kinase (MAPK) activation was assessed using an in gel assay following stimulation of SMC w i t h PGE2, PGF2a and PGI 2 (prostacyclin). Both PGE 2 and PGF 2_ induced MAPK activity w h i c h peaked at minutes and returned to basal levels after 20 minutes. Interestingly, PGI 2 stimulated MAPK activity, but it was unable to stimulate either rRNA or DNA synthesis. Thus, MAPK activation may not be strictly coupled to g r o w t h events. Addition of phosphatidylinositol-3-kinase inhibitors and the mono-ADP-ribosylation inhibitor rneta-iodobenzylguanidine i n h i b i t e d the stimulation of rRNA and DNA synthesis by PGE 2 and PGF2w These data indicate that nontraditional signal transduction p a t h w a y s are associated with the stimulation of g r o w t h in SMC by prostaglandins. Supported by the MRC Group in Experimental Cardiology and an MRC studentship to LY.

OBLIGATORY ROLE OF ENDOTHELIUM IN MO091 HYPOXIA-INDUCED PGI 2 RELEASE IN ISOLATED RABBIT HEART Nabil Nakhostine, REginaldNadeau, & Daniel Lamontagne, Centre de recherche, HSpital du Sacr~-Coeur de Montreal, Faculties of Pharmacy and Medicine, Universit~ de Mont~al, Canada.

TREATMENT WITH NG-MONOMETHYL-LMo092 ARGININE INCREASES BLOOD PRESSURE AND IMPROVES SURVIVAL IN A PIG MODEL OF ABDOMINAL SEPSIS Oystein A. Strand, Karl E. Giercksky & Knut A. Kirkebeen. Inst. Exp. Med. Res. and Dep. of Infect. Dis., Ullev~l Hospital, and Dep. of Surg. Onc., National Cancer Hosp., Oslo, Norway.

The mechanism underlying the release of prostaeyclin (PGI2) was studied in isolated saline perfused rabbit hearts under constant flow conditions. The contribution of vascular endothelium, and the role of calcium influx through L-type Ca**channels in hypoxia-induced release of PGI2, was investigated. Reduction in the perfusion solution PO2 (from 476 + 13 to 127 + 24 mmHg) in the presence of glucose (5 mlV0 and pyruvate (2 raM) caused a significant release of 6-keto..PGF==, the stable metabolite of PGI2 (from 3.0 + 0.4 to 7.3 + 2.0 pmol mitt"~ g.t, p<0.05, n=12). Endothelium impairment by electrolysis abolished almost completely the vasodilation induced by serotonin and aeetylcholine, without affecting that of papaverine. Basal release of 6-keto-PGF,, was significantly enhanced after electrolysis. However, hypoxia-induced release of 6-keto-PGF==was completely abolished. Verapamil (I 0"~ M) blocked completely the release of 6-keto-PGF==during hypoxia. Similarly, hypoxia in 15 mM KCl-arrested hearts was not accompaniedby 6-keto-PGFt=release.Furthermore, the use of diclofenac,a cyclooxygenaseinhibitor, during hypoxia in arrested hearts did not affect the coronary vasodilation while 8phenyltheophyllinehalvedthe CPP variations.Our results suggest that endothelialcells are the major site of PGIa synthesisduring hypoxia.Myocardialcontractilityis somehowinvolvedin hypoxiainduced PGIz release,since no releasewas observedin arrested hearts. The effect of verapamilin reducing the hypoxia-induced PGI2 releaseis mostprobablythroughits negativeinotropicaction. AIO0

Immunological and inflammatory stimuli have been shown to induce an isoenzyme of nitric oxide (NO) synthase in endothelium, vascular smooth muscle cells, macrofages and other tissues. NO produced in pathophysiological amounts by this enzyme has been proposed to be of importance for the hypotension and cardiovascular hyporeactivity in septic shock. In this study we tested if continuous treatment with the inhibitor of NO synthesis, N%nonomethyl-L-arginine (LNMMA), improves hemodynamics and survival in a model of abdominal sepsis. Three groups of anesthetized, open-chest pigs were included. Group A (n=6) was monitored for 9 hours after completion of surgery (Time-control). In group B (n=9) 0.5 gram faeces from the caecum/kg body weight was administered inlraperitoneally, and the pigs were monitored for 9 hours. In group C (n=6) the animals were treated as in group 2, but a continuous intravenous infusion of LNMMA (10 mg/kg/hour) was given from 3 hours after faeces was administered. In group A all the pigs survived. In group B only 1 pig survived for 9 hours. The other 8 pigs died after a mean survival time of 6 hours and 20 min after a time with hypotension and tachycardia. In contrast, in the group treated with LNMMA 5 of the 6 pigs survived for 9 hours, and 1 pig died after 7 hours and 10 rain (difference in mortality between group B and C: p<0.01). In group C LNMMA increased mean arterial blood pressure by =15 mmHg, and this effect lasted throughout the experiment. Importantly, coronary blood flow was not reduced by LNMMA. In conclusion, early and continuous treatment with LNMMA increases blood pressure and improves survival in pig model of abdominal sepsis.

CONTRIBUTION OF NITRIC OXIDE TO BRADYKININMo093 AND DES -ARGg-BRADYKININ-INDUcED VASODILATATION IN ANAESTHETIZED DOGS. Daniel Lamontagne, Nabil Nakhostine, R6jean Couture & R~ginald Nadeau. Research Center, H6pital du Sacr~-Coeur de Montreal, Faculties of Pharmacy and Medicine, Universit6 de Montreal, Canada.

THE NITRIC OXIDE SYNTHASE INHIBITOR Mo094 DIPHENYLENEIODONIUM RELAXES VASCULAR SMOOTH MUSCLE AND DECREASES CYTOSOLIC pH Jeffrey M Dodd-o, Howard S Silverman, Gemin Zheng, Edward G Lakatta, Roy C Ziegelstein. Cardiology Div, Johns Hopkins Univ & Gerontology Research Ctr, NIA, NIH, Baltimore, MD, USA

The present study was performed to investigate the mechanism responsible for the hypotensive effect of bradykinin (BK) and des-Argg-bradykinin (DBK) in anaesthetized dogs. Bolus intravenous (iv) or intra-arterial (ia) injections of 0.6 pg/kg BK produced identical transient hypotensions (from 110 + 6 to 66 ± 6mmHg, or -41 ± 5% for both iv and ia). The ia injection of 0.6 pg/kg DBK induced a weaker hypotension compared to its iv injection (-27 + 2 vs -39 + 3%, p<0.05). The hypotension was accompanied by increases in heart rate, maximum left ventricular dP/dt, and aortic blood flow. The positive inotropic and chronotropic effects of BK and DBK were practically abolished by 1 mg/kg propranolol. In contrast, the increase in aortic blood flow was only slightly attenuated. Inhibition of NO synthase with NGnitro-L-arginine (L-NNA) induced an increase in arterial pressure (from 106 + 5 to 141 + 6mmHg, p<0.05) and halved the vasodilatation induced by 10 pg/kg acetylcholine iv (-38 + 8 vs -19 ± 7%, p<0.05) without affecting that of sodium nitroprusside. The hypotension induced by BK, iv and ia, and DBK iv was only slightly reduced. In contrast, the hypotensive effect of DBK ia was markedly reduced by L-NNA (-27 + 2 vs -13 ± 3%, p<0.05). In conclusion, the hypotensive effect of BK and DBK results from a peripheral vasodilatation. The contribution of NO in this vasodilatation is substantial for DBK when administered ia, but limited for BK and DBK iv.

Systemic administration of different nitric oxide synthase (NOS) inhibitors produces distinct blood pressure responses in rats. The pressor response to N6-nitro-L-arginine is sustained, while that induced by diphenyleneiodonium (DPI) is transient. To examine the mechanism of this transient response, isolated PGF=,,preconstricted endothelium-containing (E+) or denuded (E-) rat aortic rings were exposed to 5pM DPI in 37°C Krebs buffer. Following an initial transient contraction (39+13%, n=12) which was abolished by N%nitro-L-Arg methyl ester pretreatment (1 mM, n=7), a sustained dilation occurred which was greaterat 15 min in E- (-92+14%, n=8) than in E+ rings (-28% + 19%, n=11, p=0.02). In SNARF-Ioaded cultured rat aortic vascular smooth muscle cells (VSMC), 51~M DPI (23°C, HEPES buffer, pH=7.40) reversibly decreased pH~ from 7.01+0.02 to 6.90+_0.02 (n=5, P<0.02). This was not affected by prior NaVH÷ exchange inhibition with ethylisopropylamilodde(n=3), suggesting that DPI does not decrease pH= by inhibition of this exchanger. No effect of DPI on intracellular Ca2" was seen in indo-1 loaded cultured VSMC. This effect of DPI on VSMC pH~ may decrease myofilament Ca2. sensitivity and thereby contribute to the Erelaxation of aortic rings and the shortened presser response observed during systemic administration.

NITRIC OXIDE SYNTHASE ACTIVITY FOLLOWING M0095 BALLOON ANGIOPLASTY. Patrick Black, Adrian P Banning, Malcolm J Lewis. Cardiovascular Sciences Research Group, University of Wales College of Medicine, Cardiff, UK.

TACHYPHYLAXlSTO VASOPRESSlN: Mo096 ENDOTHELIUM- DEPENDENTAND NITRIC OXIDE MEDIATED Esther Millette, Eric Dumont & Daniel Lamontagne, Facult~ de pharmacie, Universit6 de Montreal, Montreal, Canada.

Re-endothelialisation has a fundamental role in limiting neo-intimel hyperplasia and platelet adhesion following balloon angioplasty. Nitric oxide (NO) may be responsiblefor regulating theseprocesses. We investigated the time course of recovery of functional endothelium (E) in the pig carotid artery. Pigs underwent bilateral angioplasty and were sacrificed at either 30 rnirt~([r"~), 24hra (n=t), 48hrs (n=4), 7 days (n=6) or 21 days (n=6). Mm3)hological analysis was performed using stained sections. DBA lectin (L) antibody provided a marker of sWactural E (porcine arterial E does not contain vWF antigen) NADPH diaphorase (which co-localisus with NO synthase) a marker of functional E. Expression of profifereting cell nuclear antigen (PCNA) indicated active cell division. Scanning (SEM) and transmission (TEM) electron microscopy examined cell morphology and platelet deposition. Results were categorised as either minimal injury (M) or deep injury (D) ( r u p ~ of the internal elastic lamina) intimal area was expressed in nun----2-'andcoverage by eedothelinm or platelets as trace (+/-), non-confluent (++) or confluent 30 rni~q 1 days 2 days 7 days 21 days

The present study was performed to characterize the tachyphylaxis of rat aortas to vasopressin. Isometric tension generated by rat thoracic aorta sliced in 4 mm rings, was recorded. Tension generated by intact rings increased with cumulative additions of vasopressin up to 10 nM (1.51 + 0.15 g). After this concentration, most rings lost their tension and relaxed to 1.09 + 0.17 g (p < 0.001) despite further addition of vasopressin to 100nM This desensitization was not observed in endothelium-denuded rings (from 2.87 + 0.12 to 2.68 ± 0.17 g, NS). No desensitization to phenylephrine was observed in intact as well as endothelium-denuded rings. Pretreatment of intact aortic rings with the cyclo-oxygenase inhibitor, diclofenac (lpM), did not prevent the desensitization to vasopressin. In contrast, NO synthase inhibition with N6nitro-L-arginine (30pM) resulted in an attenuated desensitization to vasopressin in intact rings (from 2.33 ± 0.77 to 1.93 + 0.48 g, NS). To confirm the involvement of NO, endothelium-denuded rings were pretreated with sodium nitroprusside (SNP). At a concentration of 10nM, SNP induced a desensitization, to vasoprerLsin (from 1.02 ± 0.21 to 0.38 + 0.15 g) as well as phenylephrine (from 1.88 + 0.14 to 1.14 :t: 0.07 g). However, pretreatment of endotheliumdenuded rings with 8-bromo-cGMP (100pM) reduced maximum contraction to vasopressin (2.57 + 0.17 vs 1.68 + 0.42 g for control and treated rings, respectively) without producing any desensitization. The results suggest that the endothelium-dependent tachyphylaxis to vasopressin is mediated by NO, but independent of cGMP.

M

D

M

D

M

D

A 0 PCN 0 DIAP ~ctin

0 0 -

0 0

0 0

0 0

0 0

+++

+

+++

+

+++

/~dPt

+++

M

D

M

D

0.1 0.24 0.1 0.53 7.4 16.5 2.3 4.7 +++ +/- +++ +++ ++ +/- +++ +++ -

+Io

-

Abbreviations for above table: A, intimal erea(mm '); PCN, PCNA Index; DIAP, Diaphorase; AdPt, Adherent Plaiters. NO synthase activity is detectable 7 days following balloon injury. Endogenous NO production may be responsible for limiting platelet adhesion and n e o - ~ growth.

A101

POLAROGRAPHIC ~ OF Mo097 NTrRIC OXIDE I/~I.I~.ASED BY "NO-DONOR" COMPOUNDS IN AQUEOUS SOLUTIONS J~[nos Pataricza, +Botond Penke, *G6bor E. Balogh & Julius Gy. Papp. Dept. of Pharmacology, +Dept. of Medical Chemistry, Albert Szent-GySrgyi Medical University, and *Inst. of Biochem., Centr. Biol. Res., Szeged, Hungary Nitric oxide releasing capacity of sodium nitroprusside (SNP), nitroglycerine (NTG) and nicorandil (NIC) was investigated by the recently developed polarographic sensor, ISO-NO 0/¢PI, Mauer, Germany). In the absence of biological materials, all the three substances liberated free nitric oxide radical (NO) concentration-dependently in 2 ml samples of Krebs-Hensaleit (KI-I) solution or that of a reducing medium (sodium iodide in strong acid) at 37°C. In KH solution, SNP produced 1.5+0.7 tam (mcan+S.E.M.) NO per 1 ~tM of the compound (n=7), and no detectable NO was obtained from NTG (up to 140 p.M) or from NIC (up to 6.4 raM). In the presence of 5 mM L-eysteine, an SH-group donor, NTG produced 3.3-1-1.9 nM NO per 1 #M of the substance in KH solution (n=5). NIC did not release NO in KH solution even in the presence of L-eysteine, and 1 mM of the drug converted to only 1.1+0.9 nM NO in reducing medium (n=4). In conclusion, 1) SNP spontaneously liberates NO,and 2)NTG releases biologically active amounts of NO in the absence of specific enzymes. 3) NIC is a weak "NO-donor" and the mechanism by which it releases NO may differ from that of SNP and NTG. This work was

supported by the Hungarian National Scientific Foundation (OTKA 772848).

EXERCISE TRAINING AND BASAL ENDOTHELIAL Mo099 NITRIC OXIDE PRODUCTION Bronwyn A Kingwell, Garry L Jennings, Anthony M Dart Alfred and Baker Medical Unit, Baker Medical Research Institute, Melbourne, Australia.

Moderate aerobic exercise is now widely accepted as contributing to a reduction in cardiovascular morbidity and mortality. Enhanced endothelium-dependent vasorelaxation is a possible mechanism underlying these cardiovascular benefits. Nine healthy male volunteers (mean age _+_ SD; 24.3 + 4.4 yrs) performed, in a randomised order, both 4 weeks of cycling for 30 minutes at 65% of maximum capacity, 3 times per week and 4 weeks of sedentary activity. Training increased both maximum work capacity (mean _-h SEM; 228 _:L 18 vs 255 _+_ 14W; p=O.O1) and maximum oxygen consumption (46 .:h 3 vs 50 _+_ 3 ml/kg/min; pffi0.01). Forearm venous occlusion ptethysmography was used to study the forearm blood flow response to intra-arterial administration of the nitric oxide synthase inhibitor NGmonomethyl L-arginine (L-NMMA 4 ~tmol/min). After training L-NMMA reduced forearm blood flow by 1.16 + 0.31 ml/lOOml (p=O.001) whereas the reduction was not significant after the sedentary intervention (0.67 _+_ 0.34 ml/1OOml; p=O.34). In addition, forearm oxygen consumption was reduced by a greater amount after LNMMA in the trained compared with the sedentary state (44.2 _-L 12.1 vs 5.2 + 15.7 lal/1OOml/min; p=O.O1). These data suggest that basal release of endothelium derived nitric oxide is increased with short term exercise training and that this effect may contribute to the cardiovascular protective effects of exercise. A102

ENDOTHEUAL CYCUC GMP PRODUCTION Mo098 IS AN INDEX FOR "ACTIVE" NITRIC OXIDE Gabriele Wiemer, Brian Pierchala*, Bernward A. Sch01kens & Tadeus Malinski*. Hoechst AG, Frankfurt/M., Germany; *Oakland University, Rochester, Michigan, USA

We investigated whether the reported enhanced accumulation of endothelial cyclic GMP by bradykinin (BK) or the angiotensin converling enzyme inhibitor ramiprilat (RT) reflects enhanced nitric oxide (NO) generation and release. Using a selective porphyrinic intracellular microsensor for NO its release was detected on the cell surface of single primary cultured bovine aorlic endothelial cells. Maximal NO release was achieved by BK and RT at 5 x 10-8 M and 10-7 M, respectively. The time course of NO release by BK showed a rapid increase rate (22.5 +1.6 nM/sec) and was transient within 15 min (decrease rate 0.60t-0.05 nM/sec). In contrast the increase rate of NO release by RT was delayed (0.34_+0.03nM/sec) and reached a plateau level, which remained stable for at least 14 rain. The effects of both compounds were prevented by the kinin receptor antagonist icatibant These results are in agreement with data for intracellular cyclic GMP accumulation in the same cells. Thus, endothelial cyclic GMP content can be considered as an index for that amount of released NO which is not intracellularly inactivated (e.g. reactions with iron, thiols, superoxide anions and other free radicals) and most likely reaches adjacent target cells.

CORRELATION BETWEEN INDUCTION OF Me100 MYO-CARDIAL iNOS mRNA, ENZYME ACTIVITY AND PLASMA NO PRODUCTS DURING ENDOTOXAEMIA Alan N. Bateson, Olga M. Jakiwczyk & Richard Schulz. Departments of Pharmacology and Pediatrics, University of Alberta, Edmonton, Alberta, Canada. We have previously shown that endotoxin (ETOX) causes an increase in the activity of inducible Ca2*-independent NO synthase (iNOS) in the myocardium which contributes to depression of cardiac contractile function. We have now determined the time course of induction of myocardial iNOS mRNA, iNOS activity, and nitrite/nitrate levels in plasma of ETOX-treated (4 mg/kg, i.p.) rats. iNOS mRNA was detected at 3, 6, and 12 hr after ETOX treatment (maximum at 3 hr) by Northern blot analysis, but not in saline-injected controls (6 hr, n=6). iNOS activity was not detectable in saline-injected controls, but rose to 0.76+0.31, 1.01+0.28, 4.68+1.07, 1.84+0.66 and 0.32+0.08 pmol/min/mg protein following 0.5, 3, 6, 12, and 24 hr ETOX treatment, respectively (n=5-6). Ca2+-dependent NO synthase activity was observed (2.66+0.57 pmol/min/mg protein) in control rats, however, this did not change during endotoxaemia. The level of total plasma nitrite/nitrate in control rats was 21_+_2ixM; this rose following ETOX injection to 23+2, 47+8, 436+86, 768_+_212 and 439+73 I.tM at 0.5, 3, 6, 12 and 24 hr, respectively (n=4-6). Therefore, during endotoxaemia there is a rapid induction of myocardial iNOS activity which is a result of increased iNOS gene expression. Selective block of iNOS expression, or its activity, may prevent detrimental effects of excess NO in the heart during endotoxaemia.

E N D O T O X I N - A N D C Y T O K I N E S - E F F E C T S ONM0101 NITRIC O X I D E P R O D U C T I O N IN CARDIOMYOCYTES S I G N I F I C A N C E FOR cONTRACTILE D E P R E S S I O N Ursula Mfiller-Werdan, Ralph Fuchs, Josef Stadler*, Karl Werdan. Dept. of Med. I, K l i n i k u m Groflhadern, U n i v e r s i ty of Munich, Germany, and * Dept. of Surgery, Technical University of Munich, Germany. -

It is an open q u e s t i o n to which extent tumor n e c r o s i s factor a (TNF)-induced c a r d i o d e p r e s s i o n in sepsis is m e d i a t e d by i n d u c t i o n of n i t r i c o x i d e (NO) synthase. N i t r i t e [nmol/mg p r o t e i n x 24h] was d e t e r m i n e d as a m e a s u r e of NO after a 24-h-incubation-period with TNF or I n t e r l e u k i n - i (IL-I) or endotoxin (LPS) (x ± SD, n=3) : Control TNFa TNFa IL-I LPS 10U/ml 100U/ml 20U/ml 10#g/ml 30,5 25,7 20,2 36,8 139,0 ±5,8 ±3,3 ±2,4 ±4,5 ±15,0 L P S - i n d u c e d NO p r o d u c t i o n is completely s u p p r e s s e d by L - N M M A (0.5 mM) or dexamethasone (0.i ~M) . 24-hi n c u b a t i o n w i t h LPS a t t e n u a t e s catec h o l a m i n e i n o t r o p y , a r g u i n g f o r an NO-dependent cardiodepression. TNF depresses catecholamine i n o t r o p y in the absence of enhanced NO production.

TISSUE SPECIFIC REGULATION OF Mo102 ANGIOTENSINOGEN Conrad Sernia, Tang-Sheng Zeng & David Kerr. Department of Physiology and Pharmacology, University of Queensland, Australia. All the angiotensin peptides are derived from the glycoprotein, angiotensinogen (Ao). Its expression in tissues other than the liver (the source of plasma Ao), is likely to directly affect the activity of local reninangiotensin systems. In this study we show that dexamethasone (DEX) and cAMP differentially regulate Ao expression and secretion in hepatoma (H4), glioma (C6), pituitary and glial cells. Using a direct radioimmunoassay for Ao and quantitative RTPCR for its mRNA, H4 and pituitary cells showed marked increases in both Ao secretion and mRNA in response to DEX (5-7 fold) while glial cells responded weakly with a 30-50% increase and C6 with a 60% decrease, cAMP alone stimulated secretion from H4 and pituitary cells by 1-2 fold but had no effect in C6 and glial cells. However, in the presence of both DE)( (llaM) and cAMP (lmM) marked 40 fold increases were seen in pituitary cells, 10 fold in H4 cells and 6-8 fold in gila. In C6 cells, cAMP prevented the inhibition by DEX. Actions of cAMP could be mimicked by 13-adrenoceptor agonists, suggesting involvement of catecholamines. In conclusion, cAMP and glucocorticoids, both alone and in synergy, regulate angiotensinogen, and consequently the production of angiotensins, in a tissue-specific manner.

BOTH AT 1 AND AT2RECEPTORS MEDIATE Me103 SMOOTH MUSCLE ~;ELL GROWTH BY ANGIOTENSIN II Laura Saward & Peter Zahradka. Div Cardiovasc Sci, Dept of Physiology, University of Manitoba, Winnipeg, Canada.

AT1 and AT2 receptor subtypes Mo104 in AII dependant vascular remodeling Sabri A, P Poitevin§, B.Levy§, L Rappaport, JL Samu-J. U127 and §U141 IFR Circulation, 75010 paris, France

Vascular smooth muscle cells (SMCs) express at least t w o distinct Agll receptor subtypes, AT 1 and AT2; however, the role of the AT 2 receptor remains poorly defined. To determine more precisely the relationship between angiotensin receptor subtypes and the c~llular processes coupled to growth, a cell culture model based on SMC migration from porcine coronary artery explants was studied. Immunofluorescent microscopy was used to examine Agll stimulation of PCNA, an early S phase marker. Agll stimulated PCNA expression with a distinct nuclear localization which was blocked by both AT 1 (Losartan) or AT 2 (PD1 23319) antagonists. Similarily, Agll stimulated DNA synthesis 200% as measured by 3H-thymidina and BrdU incorporation which could be effectively blocked by both the AT 1 or AT 2 antagonists. To distinguish between a hypertrophic or hyparplastic response, the MTT cell count assay was used. Agll resulted in increased cell number over 72 hours which was blocked by both AT 1 or AT 2 antagonists. Binding studies with 1251_ Agll demonstrated that both AT 1 and AT 2 receptors were present on these cells. These results present conclusive evidence that the AT 2 receptor is coupled to growth in vascular SMCs and suggests that growth stimulation by Agll requires activation of both AT 1 and AT 2 receptor pathways. Supported by MRC Group in Exp Cardiology & MRC Studantship to L.S.

The relative importance of angiotensin II (AID receptor subtypes 1 and 2 on vascular remodeling was analysed. Wistar rats were treated for 23 days with either solvant or AII (120 ng/kg/min),+ DUP753 (10 mg/kg/day) or PD123319 (30 mg/kg/day), respectively AT1 or AT2 receptor antagonists. Myocardial sections were immunolabelled with antibodies directed against extracellular matrix [cellular fibronectin, c-FN] or contractile [e-smooth muscle actin (ct-SM actin) and non muscle myosin (NMM)] proteins. The sizes of the medial coronary arteries were determined by morphometric analysis using a-SM actin labelling. The expression of c-FN and NMM indicated an immature ~e of smooth muscle cells. Control &ll AII+ DUP AII+ PD BP

168

(mml-Ig) +4 Media 2.3 xl0-3~tm2 -t-0.2 c-FN NMM

210

+8" 3.3 +_0.2== ++ ++

DUP 172

172

+_16 +7 2.6 3.2 + 0 . 2 +0.3*

PD 225

180

+15" 2.3 +_0.2

+_4 2.4 +_0.2

++ ++

*:p
A r i a ANGIOTENSIN RECEPTOR LOCALISATION Mo105 IN RAT CARDIOMYOCYTES REVEALED BY IMMUNOCYTOCHEMICAL TECHNIQUES. Wolfgang Schulze*, Michael L.X. Fu °, Ake Hjalmarson°, Johan Hoebeke°. *Max-Delbmck-Centre, Cardiology, Berlin, Germany; °Wallenberg Lab., Univ. of GOteborg, Sweden

MODULATION OF G-PROTEIN EXPRESSION MO106 AND ASSOCIATED FUNCTIONS BY ANGIOTENSION II IN VASCULAR SMOOTH MUSCLE CELLS Anuradha Palaparti & Madhu B. Anand-Srivastava. Dept. of Physiology, Univ. of Montreal, Quebec, Canada H3C 3J7.

Polyclonal anti-peptide antibodies were produced against a synthetic peptide corresponding to the second extracellular loop of the rat ATIa angiotensin receptor in rabbit. Affinity purified antibodies were used to localize ATIa angiotensin receptors in rat heart tissue and in cultivated neonatal cardiomyocytes and noncardiomyocytes. Immunofluorescence and postembedding gold-labelling indicated three main sites of antigenic reactions. In cardiomyocytes: along sarcolemma, including T-tubuli membranes, at intercalated discs and at the heterochromatin of nuclei. In non-cardiomyocytes : at the transluminal side of endothelial cell membranes, at the surface of the smooth muscle cells and at the heterochromatin of fibroblasts and endothelial nuclei. These results demonstrated the specific localization of ATla angiotensin receptors using anti-peptide antibodies against the second extracellular loop of the rat ATIa angiotensin receptor at the subcellular and cellular level in rat heart.

In the present studies, we have investigated the effect of angiotensin II (AID on G-protein expression and functions in vascular smooth muscle cells (VSMC) (A10). AH treatment of the VSMC enhanced levels of Gi protein as well as mRNA as determined by immunoblot and Northern blot analysis, however the inhibition of adenylyl cyclase by AII and C-ANF4.m were completely attenuated. On the other hand, AH had no effect on the levels of Gs but inhibited the stimulatory effects of GTPyS, isoproterenol and NECA on adenylyl cyclase activity by 44, 28 and 40% respectiviely without affecting forskolin and NaFstimulated adenylyl cyclase activities. Staurosporine (a protein kinase C inhibitor) prevented AII-mediated attenuation of AII and C-ANF~_~ responsiveness to adenylyl cyclase inhibition and decreased Gi levels. These results indicate that AH alters Gi expression which is not associated with Gi-mediated inhibition of adenylyl cyclase. It can be suggested that PKC sigrmlllng pathway may be implicated in the observed A/I-mediated alterations.(Supported by grants from Medical Research Council of Canada and Quebec Heart Foundation).

ENCAPSULATION OF VASOACTIVE INTESTINAL Mo107 PEPTIDE INTO LIPOSOMES POTENTIATES ITS VASORELAXANT EFFECTS IN VIVO

VANADATE-INDUCED CONTRACTION AND PHOSPHORYLATION PREGNANCY.

Hideyuki Suzuki, Sudhir Paul, Yasuko Noda & israel Rubinstein. Universities of Illinois & Nebraska, Chicago & Omaha, USA. Vasoactive intestinal peptide (VIP) induces vasodilation in the peripheral microcirculation. The purpose of this study was to determine whether encapsulation of VIP into liposomes potentiated its vasorelaxant effects in situ. Using intravital microscopy, 'we found that suffusion of native human VIP (0.05, 0.1 and 1.0 nmol) for 7 min induced a significant, concentration-dependent vasodilation in the hamster cheek pouch (8.6_+1.4%, 12.7_+3.1% & 14.4_+0.6% increase from baseline, respectively; n=11, p<0.05). Arteriolar diameter returned to baseline within 1-2 min after suffusion was stopped. By contrast, encapsulation of VIP (0.05, 0.1 and 1.0 nmol) into liposomes was associated with significant potentiation of its vasoralaxant effects in comparison to VIP alone (26.1_+5.5%, 38.1_+6.5%, & 34.1-+2.9% increase from baseline, respectively; n=11; p<0.05). Furthermore, arteriolar diameter returned to baseline 10-16 min after suffusion was stopped. Suffusion of empty liposomes had no significant effects on arteriolar diameter. Suffusion of VIP with and without liposomes had no significant effects on systemic arterial blood pressure and heart rate. We conclude that encapsulation of VIP into liposomes potentiates and prolongs its vasorelaxant effects in the peripheral microcirculation in situ. A104

SMOOTH

MUSCLE Mo108

PROTEIN TYROSINE IS IMPAIRED IN

Jean St-Louis, BenoR Sicotte, Eric Breton and Ashok K. Srivastava. Centres de recherche/H6pital Ste-Justine and H6teI-Dieu de Montr6al, University of Montreal, Montrdal, Canada. The present study was undertaken to characterize the contractile effects of vanadate on thoracic aorta rings from virgin and term-pregnant rats. Vanadate caused concentration-dependent contraction in rat aortic rings with an EC5o of 0.10 mM, which were equivalent to the ones measured with 1 p.M phenylephrine. The effects of vanadate was not affected by Indomethacin (up to 10 p.M), an inhibitor of prostanoid cyclooxygenase, but was blocked by staurosporine, an inhibitor of protein kinases. Furthermore, vanadate-mediated contraction was associated with tyrosyl phosphorylation of at least 5 proteins having apparent molecular masses of 40, 50, 60, 75 and 85 kDa. The tyrosyl phosphorylation of these proteins was diminished in aorta of pregnant rats. Correspondingly, vanadate elicited a significant decrease in contractile responses in aorta of pregnant as compared to virgin rats. These results suggest that: 1) the contractile effect of vanadate on rat aorta is independent of endogenous prostanoids and may be mediated by protein kinase C and/or protein tyrosine kinase dependent pathway, and 2)the contractile response to vanadate on the rat aorta is impaired during pregnancy. (Supported by grants from BIOPEDIA Inc., FRSQ and QHSF).

Mo109 PRESSURE DEPENDANCE AND INTRINSIC EFFECTS OF SMOOTH MUSCLE ACTIVATION ON THE AORTIC WALL MECHANICS IN CONSCIOUS DOGS. Juan G. Barra, Ricardo L. Armentano, Jaime Levenson*, Alain Simon* & Ricardo H. Pichel. Favaloro Foundation, Buenos Aires, Argentina and *Hbpital Broussais, Paris, France

RELATIVE EFFICACY OF REDUCTION OF Mol 10 CALCIUM INFLUX BY BLOCKADE OF CALCIUM CHANNELS OR ACTIVATION OF POTASSIUM CHANNELS IN VASCULAR TISSUE (BLOOD VESSELS) Klfira Csete, Ott6 I-Idla, Z o l t ~ Vezek~nyi and Julius Gy. Papp.Department of Pharmacology, Albert SzentGy6rgyi Medical University, Szeged, Hungary

Recently we demonstrated that aortic smooth muscle (SM) activation increases blood pressure with local vasoconstriction altering elastic, viscous and inertial behaviors of the aortic wall in conscious dogs. To elucidate if this alterations are due to high peripheral resistance or to the aortic SM itself (local vasoconstriction) we measured aortic pressure and diameter in 8 chronically instrumented dogs during control (CTL), under activation of SM (SMA, phenylephrine, 5 pg/Kg/min) and during high resistance control state (HRCS, transient distal aortic cuff occlusion). Although good correlation existed between mean pressure (MP, mmHg) and elastic (E, 10e dyn/cm 2, r=0.81), viscous (q, 104 dyn.s/cm 2, r=0.72) and inertial (M, dyn.s2/cm 2, ==0.85) moduli in CTL and SMA, no changes were found in and M between CTL and HRCS, indicating that SMA produces genuine increases only in ~1 and M Conversely, E is highly pressure dependent due to the concomitant predominance of collagen fibers in the resistance to stretch in HRCS. MP MD E q M CTL 100±8 16.5+1.4 5.0±1.6 4.3+1.5 41+14 SMA 156+11.17.0±1.3. 10.6+1.3. 7.8+1.1. 91+23. HRCS 154±13. 17.6±1.4.1- 14.2±1.4.1- 5.4±2.01- 36±17t*p<0.01. vs CTL; 1-p<0.03, vs SMA. (MD: mean diameter, mm). The presence of SM in the aorta improves the elastic behavior with respect to HRCS (even though there is energy loss mainly due to viscosity augmentation) increasing aortic compliance mediated by isobaric vasoconstriction.

The action of the calcium channel modulating group of drugs has proved to be of interest in recent years. The aim of our present work was to compare the effect of drugs which resemble each other in reducing calcium influx. Verapamil (VERAP). Diltiazem (DILT) and Nifedipine (NIF) as calcium antagonists were compared to cromakalim (CROM3, a potassium channel activator, both in vitro and in vivo. In isolated rin~s of rabbit aorta depolarized by.50 mM K + all three Ca z~" antagonists antagonized the Ca z + induced contractions in very small concentrations, and NIF was the most effective (ICs0 values: VERAP:0.215:0.05, DILT:l.01+0.34, NIF:0.DI5+0.002, means+SEM in p.M). CROM was effective in very high concentrations only (1904+491 #M). In conscious rabbits NIF as well as CROM, when given in the same dose (0.3mg/kg i.v.), had a significant arterial blood pressure decreasing effect (NIF:from 111+6 to 79+2, CROM: from 90+4 to 58+5 in mmHg) accompanied by some tachycardia and a marked improvement in the reduced cortical blood flow (NIF:from 56+10 to 86+17, CROM: from 465:5 to 84+11 in ml/min/100g tissue, t?<0.001 n=20) measured after unilateral carotid occlnslon by hydrogen polarography. Our results suggest that major differences exist in the in vi~r~ and in wvo vasodilator profiles of CROM and Ca'~T antagonists, and for the treatment of hypertension and impaired cerebral circulation and ischemia not only NIF, but also CROM might be of considerable use.

POLYPEPTIDE GROWTH FACTORS AND HYPOXIA M0111 SYNERGISE IN THE EXPRESSION OF VEGF mRNA George T.Stavri, Jorge D.Erusalimsky & John F.Martin. Dept of Cardiovascular Science, King's College School of Medicine and Dentistry, London, UK.

OVEREXPRESSION OF CALRETICULINMo112 D O E S N O T A L T E R I N T R A C E L L U L A R FREE C A L C I U M LEVEL IN S M O O T H M U S C L E CELLS Nasrin Mesaeli*, Amy Tse #, Fred Tse #, and Marek M i c h a l a k * . Departments of * B i o c h e m i s t r y and #Pharmacology, University of Alberta, Edmonton, Alberta, Canada.

Hypoxia of the arterial wall has been implicated in the pathogenesis of atherosclerosis. This study investigated the effect of hypoxia in cultured vascular smooth muscle cells (VSMC) by examining the expression of the proto-oncogene c-myc and the gene for Vascular Endothelial Growth Factor (VEGF). VSMC cultures were incubated in humidified hypoxic chambers and levels of mRNA for c-mycand VEGF were measured by Northern blotting. Hypoxla had no effect on the level of c-myc mRNA expression. In m.arked contrast, steady-state levels of VEGF mRNA were markedly elevated in response to hypoxla in a time-dependent and oxygen concentration-dependent manner. The polypeptide growth factors PDGF-BB, bFGF hnd TGF-~ at 21% 02 each stimulated a modest dose-dependent increase in the expression of VEGF mRNA. The same concentrations of these growth factors at a submaximal level of hypoxia (2.5% 02) resulted in a striking increase in the level of VEGF that was greater than the expected additive effect of these stimuli alone. The c-mycrespone to these growth factors was similar under normoxio and hypoxic conditions. These results s .uggest that hypoxia is not a direct proliferative signal for VSMC but hypoxia is a potent stimulus for the expression of VEGF. Physicochemical stimuli such as hypoxia and polypeptide growth factors may act in concert on VSMC within the arterial wall to promote atherogenesis.

Calreticulin (CRT) and calsequestdn (CSQ) are the major C a 2÷ binding proteins present in the lumen of sarco/endoplasmic reticulum membranes. Using immunological techniques CRT was detected both in cultured aortic smooth muscle (Ato) and tissue extracts and was further localized to the sarcoplasmic reticulum compartement. In contrast, we did not found any immunologically detectable CSQ in these smooth muscle preparations. We concluded that CRT is a major Ca 2÷ binding protein of the sarcoplasmie mticulum membranes in the rat aortic smooth muscle cells. To study a role of CRT in Ca 2÷ homeostasis in smooth muscle cells, Ate ceils were transfected with pRe/CMVCRT vector encoding CRT. Ca 2÷ indicator indo-1 AM was then used to measure the changes in intracellular free Ca 2÷. Overexpression of CRT did not produce any changes in the basal free Ca 2÷ concentration. Addition of vasopressin (100 I.tM) resulted in an increase in free Ca 2÷ concentrations in control, mock-transfected and CRT overexpressing cells, however, no differences in peak Ca 2÷ levels was observed. We conclude that increased expression of CRT in Ato cells does not modulate significantly free cytosolic Ca2÷ concentration. supported by the Heart and Stroke Foundation of Alberta A105

COMPARISON OF THE CHARACTERISTIC AND D E N - M o 1 1 3 SITY OF DOPAMINE-1 RECEPTORS IN D I ~ E R E N T ARTERXES USING [ 3 H ] S C ~ 3 3 9 0 BINDING R.R.Zhao, X.H.Jin & W.Z.Wang. Dept of Physiology, S h a r ~ Medical College, Taiyuan, China By using radioreceptor binding techniques, a comparative study was performed on the property and density of DA-1 receptors in different vascular systems. [511]SCH23390 was specifically bound to membranes from rabbit renal,mesenteric and pulmonary, but not femoral, arteries . The binding was saturable and in a manner consistent with the labeling of DA-1 receptor.The Kd~alue was similar in all three arteries,indicating a homogenous binding sites with a single class of high affinity. In competitive tests, selective DA-1 receptor antagonist inhibited the binding much more potently than DA-2 receptor antagonist, indicating pharmacologically characteristic of DA-1 receptors.The Bmax value, however, differed considerably among these arteries, with the value being the largest in the renal artery and smallest in the pttlmoru~ry a r t e r y . These findings are indicative of the e ~ s t e n c e of DA-1 receptor site with identical property but diverse density in different vascular beds, which underlies the relative functional importance of the receptors i n regulating local blood flow in distinct vessels.

Regulation of Peripheral Venous Tone in Severe Tricuspid Regurgitation Festinese S, Ciavarella GM, "Calcagnini G jr, Calcagnini G.

MO115

Clinical PathophysiologF. School of Medicine and *Department of Information System Sciences. "La Sapienza" University of Rome. Alrial mechanoreecptors respond to changes in volume end pressure within the atrial chambers and elicit reflex changes in neural activity that modulate the cardiovascular function. Aim of the research was to assess the relationship between right atrial mechanoreceptors stimulation and regulation of venous peripheral circulation. Method: the study group included 15 patients (6 males, 9 females, average 51 years - range 23-71) with SevereTricuspidRegurgitation (STR) measured by EchocardioDoppler examination (CWmaximum regurgitation veloeity>3.6m/s).Tbe study group was compared with a sex and age matched controlgroup (N) with no or trivialuicospidregurgitation(CW maximum regurgitationvelocity < 2.0

m/s). Exclusion criteriawere: congenitalheart disease,heart failure, pericardialdisease,cardiacarrhythnua,arterialperipheral disease,venous peripheral disease, neuro-vascular compression syndrome, thyroid dysfunction.In conditionof no pharmacologicaltreatmenteach patientwas submitted to mercury slrain-gaugeplethysmographyat 20-25-30-40-60 mmHg venous occlusion;strain-gaugewas applied at maximum right forearm diameter at zere phiebostaticlevel (right atrium). Each examinationwas performed after 10' of rest(forearmabduction < 30*; physiologicalbreath;temperature:24-26"C).We calculatedthe following plethysmugraphicparametara:Venous Tone (VT), Venous Distensibility (VD) at 60-40mmHg occlusionsand Venous Pressure(VP) estimatedby a linearregressionon 20-25-30-40-60MVIV (Maximal Venous incrernental Volume) points. The tests were pcM'ormedby two operatorsin single blind. The T-test forunpaireddatawas pcri'orm.edforstatistical analysis.Results: On)up N(16) STR(15)

VT (mmHg/ml) VD (ml]mmHg) VP (mmHg) 29+ 9 0.050+0.015 10.9+5 41 -+ 19 0.030 :t: 0.012 11.5 + 8 p < .05 p < .05 n.s. Conclusion: our data show in STR group a significant increase of V'f and a symmetrically decrease of VD, without changesof VP. The observed phenomenon can be explainedas consequenceof a raising in constrictor sympatheticdrive on venous wall probably related to stimulation of right atrial mechanureceptors. A106

Mo114 EFFECT OF AORTIC SMOOTH MUSCLE ACTIVATION ON ARTERIAL HYDRAULIC LOW PASS FILTERING PERFORMANCE IN CONSCIOUS DOGS. Ricardo L. Armentano, Juan G. Barra, Jaime Levenson °, Alair Simon* & Ricardo H. Pichel. Favaloro Foundation, Buenos Aires, Argentina and *H6pital Broussais, Paris, France Hydraulic low pass filtering performance (LPFP) can be evaluated by deriving two corner frequencies (fcl and fc2, Hz) from a second order system used to fully characterize aortic wall mechanics. Elastic (E, 106 dyn/cm2), viscous (rh 104 dyn.s/cm2), and inertial (M, dyn.s2/cm2) moduli might be affected by aortic smooth muscle activation (SMA), but these effects could be masked both by increased mean pressure (MP, mmHg) as result of peripheral vasoconstriction and by use of anesthetized preparations. To reveal the true changes, we measured aortic pressure (microtransducer) and diameter (sonomicrometry) in 8 conscious dogs during control (CTL, MP=100+8, mean diameter (MD, mm)=16.5+1.4), under SMA (phenylephrine, 5 pg/Kglmin, MP=156+11., MD=17.0+1.3') and during high resistance CTL state by transient vessel occlusion (HRCS, MP=154+13., MD=17.6+1.4.1.). E q M fcl fc2 CTL 5.0±1.6 4.3+1.5 41+14 23+5 167+98 SMA 10.6+1.3" 7.8+1.1" 91±23' 29+8' 115±50 HRCS14.2+1.4*t 5.4±2.0t" 36¢171" 63±24'1" 198+571" *:p<0.01, vs CTL; "i':p<0.03, vs SMA. SMA alters wall mechanics and LPFP. In all cases fc2 governed by M is distant from fcl governed by rh indicating that the aortic wall is essencially viscoelastic. Despite the increment in fcl by SMA, this was 70+14% lower than under HRCS suggesting that aortic smooth muscle exerts a beneficial effect maintaining good LPFP during systemic vasoconstriction.

BEHAVIOUR OF VENOUS PERIPHERAL CIRCULATION MO116 IN ESSENTIAL HYPERTENSION F e s t i n e s e S, C i a v a r e l l a G M , F e r r u c c i A, C a l c a g n i n i G.

Clinical Pathophysiology "La Sapienza" University of Rome. Venous PeripheralCircointion(VPC) have not been thoroughlystudiedin EssentialHypertension(H), despitetheirrelevancein clinicsand therapy. The aim of thisstudywas to assessthe regulationof VPC in I-LMethod: the studygroupincluded30 patients(12 males,18 females;,age average60 years - range 32-73;BMI average23.9 Kg/m2 - range 18.9-25.2)with H (PAD > 90 mmHg );thisgroupwas comparedwith sex,age,BMI matched normotensive control group (N). Exclusion criteriawere: secondary hypertension, congenital heart disease, heart failure, perieardial disease, cardiac arrhythmia, valvular disease, arterial peripheral disease, venous peripheral disease, thyroid dysfunction. In condition of no pharmacological treatment (no less than 10 days) each patient was submflted to mercury strain-gauge plethysmography 0FSG) at 20-25-30-40-60 mmI-Ig venous occlusions; strain-gauge was applied at maximum calf diameter at zero phlebostatic level (fight atrium); each examirmtion was performed after 10' of rest, physiological breath, knee flexion about 20", tamperamre:24-26*. We calculated (getting arithmetical average on two legs) following parameters: Venous Tone (VT), Venous Distensibility(VD), time of Total Emptying (tTE) and Venous Pressure O/P) estimated by linear regression on 20-25-30-40-60 Maximal Venous Incremental Volume points (MVIV). The PSGs were performed by an operator in single blind. The T-test for m mired data was ~erformed for statistical anal rsis. Results: Group VT VD tTE VP mmHg/ml ml/mmHg see. mmHg N (15) avr-~d 27-+5 .042-+.010 89-+59 7+7 H (30) avr-+sd 35+3 .0345:.015 36-+17 6_+6 N vs H p <.016 <.026 <.001 n.s. Conclusion: the H group shows significant increase in VT and, symmetrically, decrease in VD and tTE, without difference in VP. The data can be expression of increased sympathetic drive on peripheral veins;

however the higherstandarddeviationof VT in H group (12.95 vs 5.25) seems to indicate the presence of subgroups.

METABOLIC PROFILE OF THE DISTAL END OF Mol 17 THE INTERNAL MAMMARY ARTERY (IMA) : IMPLICATIONS IN CORONARY A R T E R Y SURGERY.

p. Nataf, P. Hadjiisky, M. Peuchmaurd, A. Pavle, Ch. Cabrol & I. G a n d j b a k h c h , Hopital Piti6 PARIS FRANCE. Metabolic studies of the distal segment of the IMA were undertaken in order to evaluate mllabiltty of Its use as anastomotlc site for bypass grafting with regard to its local susceptibility to develop intimal changes. Twenty distal segments of IMA were harvested from patients who had undergone myocardial mvasculadzation. Histological examinations indicated an elastomuscular stmctum (13 Pts), a muscular structure (6 Pts) and an elastic structure (1Pt). Intimal proliferative changes were observed in 18/20 IMP.. Luminal narrowing was never significant. There was no histological evidence of athemsclemtic plaque. The enzyme-histochemical profile of the proliferating smooth muscle cells ('Synthetic SMC') found in the intimal thickening differed from normal contractile SMC on the media by the loss of myosin and mitochondrial ATPases, plasma membrane 5' nucleotidase, mederately decreased aerobic dehydrogenase and increased lactate dehydrogenase activity and ribonucleoprotein-linked pymninophilla. Lysosomal betaglucumnidase and sulfatase were strongly active. This enzyme behavior is unfavorable to contractile function and favorable to cell proliferation and lipid accumulation, two events strongly involved in the atherogenic process. Selection of the anastomotic site in the distal segment of the IMA is not recommended because of the potentially increased risk of cellular proliferation after surgery.

ACUTE IMPAIRMENT OF VASCULAR

MOl 19

FUNCTION BY ADRIAMYCIN: POSSIBLE ROLE OF OXYGEN RADICAL FORMATION. Maurizio Cappelli-Bigazzi, Carmine Battaglia, Antonello D'Andrea, *Giuseppa Ambmsio. Divisions of Cardiology, University of Naples and *Perugia, Italy. Adriamicyn (ADR) is an antitumoral agent, whose clinical usefulness is limited by its dose-dependentcardiactoxicity, probably due to oxygen radical formation. We wanted to investigate if ADR may also impair vascular relaxation. In isolated rings of rabbit thoracic aorta preconstricted with 1 I.zMphenylephrine, maximal relaxation to the endothelium-dependentvasodilatoracatylcholine was reduced from 85,6+4.1% of phenylephrine constriction in control dngs to 75.2.t.4.2 % (p=NS) after 1 h incubation with 30 I.tM ADR, and to 43.75.-6.7 % (n=6; p<.05) and to 22.5_+5.0 % (n=6; p<.01) after 1 h incubation with 90 I.tMand 150 I.zMADR, respectively. Relaxationto acatylcholine was significantly improved when ADR was incubated in presence of SOD (150 U/ml) plus catalase (112 U/ml). In presence of the two scavengers, in fact, ADR incubation caused only aslight impairmentof maximal acetylcholine relaxation (63.5 _+4.0%), as compared to 73.4+~.5 % in normal rings also exposed to SOD plus catalase (n=6; p=NS). Finally, relaxation to two different endothelium-independent agents, nitmglycarin and SIN-1, was also impaired by ADR. This effect was less pronounced as compared to that observed on acetylcholine relaxation.Maximal relaxation to nitroglycerin and SIN-1 was, in fact, 102.9.+_2.8and 116.8+3.5 % in control rings, respectively, and was reduced to 77.2t:7.8 (n=6; p<.05) and 76.3_+5.6%(n=6; p<.05), respectively, after 1 h incubation with 1501.tM ADR. In conclusion, addamycin significantly affects vascular relaxation in a dose-dependent manner. This effect seems to be mainly due to the inhibition of endothelial function, but it is accompaniedby impairmentofvascularsmooth muscle relaxation. This vasculartoxicityby adriamycin is probably due, at least in part, to oxygenradical production, since Ach relaxationcan be prevented by SOD plus catalase.

MECHANICAL STRAIN INDUCES MITOGENESIS IN Mo118 CORONARY ARTERY SMOOTH MUSCLE CELLS. Peter J. Little, Krishnankutty Sudhir, Garry L. Jennings, Rodney J. Dilley & Alex Bobik. Baker Medical Research Institute, Alfred Hospital, Prahran 3181, Australia Vascular smooth muscle cells (SMC) are subjected to mechanical strain both naturally and in pathophysiological environments such as that following saphenous vein grafting into the coronary circulation. We investigated the effects of cyclical strain in inducing a mitogenic response in coronary artery SMC derived, by explant, from ovine left anterior descending (LAD) coronary arteries. Cells were grown on flexible supports and subjected to strain (60 cycles/min) using a vacuum actuated strain device. Proliferative response was evaluated by the incorporation of 3H-thymidine into DNA. Cell membrane integrity was evaluated with the nuclear dyes, ethidium bromide and bisbenzimide. Strain induced an increase in 3H-thymidine incorporation with a peak occurring at 18 hours, identical to the time to peak effect in response to basic-fibroblast growth factor (b-FGF) and platelet derived growth factor. Supernatant from stretched cells induced a 2.01 fold increase in 3H-thymidine incorporation in unstretched cells. The proliferative response to cyclical mechanical strain was abolished in the presence of monoclonal antibodies to b-FGF. Bioassay of supematants from stretched cells showed a time dependent appearance of TGF-131, which is a weak inhibitor of mitogenicity in these cells. Dye exclusion studies showed no increased level of cell membrane damage in cells stretched for 24 h. We conclude that mechanical strain activates a proliferative response in LAD smooth muscle cells; the mechanism appears to be due to the acute release of growth factor(s), possibly b-FGF.

ION IMPLANTATION IMPROVES ADHESION M0120 OF HUMAN VASCULAR SMOOTH MUSCLE CELLS TO POLYMERIC SUPPORTS IN CULTURE Lucie Ba~ikov~i, Vfra Lisf, Vfclav gvor~ik I, Vladimir Rybka 1, Viadimir Hnatowiez 2 & Vladislav Mare~, Inst. o f Physiology, Acad. Sci. o f the Czech Republic, Prague, 1Dept. o f Solid State Engineering, Inst. o f Chemical Technology, Prague, 2Inst. o f Nuclear Physics, Aead. Sci. o f the Czech Republic, Rez, Czech Republic. Polymers are often used for construction o f the artificial vascular grafts. One o f the limitations o f the use o f this technology is the high hydrofobicity o f these molecules resulting in low adhesivity and limited growth o f vascular cells on the polymer constructed supports. In this tissue culture study, we have evaluated the adhesion o f human aortic smooth muscle cells (passage 8, Dulbecco minimal essential medium with 10% o f fetal calf serum, inoculation density o f 15000 cells per cm 2) on the thin polystyrene supports modified with F + ions (doses from 1012 to 1015 ions per em 2, energy o f 150 keV). It was found that 18 hours after starting cultures, the densities o f adhering cells on the ion modified growth supports were higher (8200+500 to 16300:t: 1200 cells per cm 2) than on unmodified surface o f the same polymers (1200+200 cells per cm2)i The highest cell densities were achieved when the fluorine ions were implanted at the doses o f 1013 and 10 Is ions per cm 2. The increased cell adhesion at the dose o f 1013 ions per cm2 was probably due to slightly increased surface polarity (polar component o f surface free energy ysP about I0 mJ m-2). At the dose of 1015 ions per cm ~, it was more likely due to a simultaneous carbonization o f the polymer. A107

EXPRESSION OF THE RAT CARDIAC ALPHA-MYOSIN M0121 HEAVY CHAIN GENE IN HEART IS CONTROLLED BY A STRONG NEGATIVE REGULATORY ~t.~M.EN'r. Madhu Gupta, Radovan Zak* and Mahcsh P. Gupta °. Heart Institute for Children, Christ Hospital Medi~l Center, Oak Lawn, 11, *Department of Medicine, University of Chicago, Chicago, IL

Expression of cardiac =-MX-IC gene is regulated at transcriptional level by hormonal and developmental stimuli. Although, several positive cis-regulatory elements have been i.:lcntifie.d in the promoter region, cardiac muscle-specifice.xpresstonof this gene remains largely unknown. We analy'zcdthe in viva ¢xprcasionof=MHC gene promoter encompassing- 600 to +429 bp sequences relative to its transcription start site. Different progressive 5' and 3' deletion mutan= of this region were cloned upstream of a chloramph~nicol acetyl transferase (CAT) reporter gene. The chimeric constructs were directly injected into adult rat heart muscle and CAT activity determined alter 72 hours. Our analysis showed presence of a 40 bp long negative regulatory clement (NP,E) downstream of the transcription start site. Deletion of NRE resulted in 15-20 fold induction in the ¢xprossion of --600 =MHC-JCAT reporter construct. However, activity o[ the minimum =-MHC promoter/reporter construct containing -70 to +32 sequences remained unaffected by the prnsenco or absence of NRE, suggesting that factors binding to NRE must diminish the role of positive regulatory element present within the -600 to -70 bp region.

Gel shift analysis with NRE revealed presence of three DNAprotein complexeswith the nuclear extract prepared from neonatal rat heart and sol 8 skeletal musclecells as well as from non-muscle cells, Hep G2 and JEG. Howcver, the pattern of DNA-protcin complex formation seen with muscle cell nuclear extract was different than that observedwith non-muscleceils. This suggestsa different regulatory role for th.:se transcription factors in =-M/-[C generegulationin muscle and non-musclecells.

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CARDIAC AND SKELETAL TROPONIN I AS MARKERS OF I~OCARDIAL INFARCTION AND MUSCLE INJURY D. Rama, I. Margaritis, P. Marconnet, A. Orsetti, C. Larue, B. Pau, C. Calzolari. Sanofi Recherche, Montpellier, France. Troponin I ( ~ ) is the inhibitory protein of the Troponin complex that regulates the interaction of actin and myosin in striated muscles. The skeletal isoform (sTnI) has a b~l of 18.5 ~dga; the cardiac £soform (eTnI) has an extra 30 residues on the N-terminus resulting in a M~ of 22.6 kDa. By l~mphocyte hgbridisation, we obtained high affinity monoelonal antibodies (NAbs).. ~ t y - f i v e were specifically directed against the cardiac form while 15 recognized the skeletal form as well. Screening of all Mabe made by pairs (one blAb coated on NUNC Maxi sorb immunotube, the other one c~ Horse Radish Pero=idase (BRP) conjugate) was used to construct the antigenic map. This epitopic map allo~ed to identify relevant pairs of ~lbs capable of measuring TnI concentrations. Two op.timal pairs of ~ b s were selected to determ'~ne both c~aZ and sTnI concentrations by using 2 independent immunoenzb~atio assays (]EMA). The combination of both assays is proposed to quantify the cardiac muscle damage after myocardial infarction and to quantify muscle injur H during and after exercise in various athlete populations. A108

Pathological Significance of Gene Mutation in cardiac B-Myosin H e a v y Chain in a Family with S u d d e n Death

Mo122

Masayoshi Kate, Yoshiyuki Sakaki¶, Kenji Takazawa', Akinori Kimura#, Wee Sea Shin, Teruhiko Toyo-oka The 2nd Dept. Int.Med., ¶Human Genome Center, Inst. Med. Sci., Univ. of Tokyo, "Tokyo Med. Coll., and #Tokyo Med. Dent. Coll., Japan We have screened 75 patients with hypertrophic cardiomyopathy (HCM) to detect mutation of cardiac 8-Myosin Heavy Chain (cBMHC) gene by Orphan Peak Analysis (Y. Sakaki, Genomics 1993) and identified novel missense mutation in exon 20 at codon 731 from Pro to Leu in a family with cardiomyopathy and sudden death in three generations. To verify pathological significance of mutated sequence, we developed "peptide competition assay". After constructing two peptides; NPAAILEGQF corresponding to mutated sequence found in this family and NPAAIPEGQF (authentic sequence), we measured isometric tension of glycerinetreated porcine ventricular fibers. At pCa 8.0, both peptides had no effect. At pCa 5.6, authentic peptide increased the tension, suggesting the cooperativity of myosin heads, whereas at pCa 4.3, mutated peptide attenuated the tension, suggesting that mutated myosin interacts with actin more potently than authentic myosin. We also prepared a mutated peptide (NE-IVVGLYQK) observed in exon 16 of HCM family without sudden death and a peptide with authentic sequence. At any pCa, these peptides had no effect on the tension, indicating that ceMHC mutated at this point has no direct action on the myocardial contractility. Mutated cBMHC which we found might effect on actomyosin interaction and cause sudden death. Thus, this newly developed assay is useful for predicting the patient's prognosis.

ALTERED CONTROL OF TROPONIN T GENEMo124 AND INCREASED ACTIVITY OF MEKRATIN, A PROTEASE, IN DILATED C A R D I O M Y O P A T H Y . Sarkis S. Margossian, Page A.W. Anderson, Paul Norton, James B. Caulfield, Peter D. Chantler and Henry S. Slayter. Albany Med Col., Duke Univ Med Ctre, Univ. of Alabama, Royal Vet Col and Dana-Farber Cancer Inst. We have shown a decrease in myosin regulatory light chain (LC2) content and a dysfunction in Calciumregulation in the human heart during idiopathic dilated cardiomyopathy (IDC), to be related to activation of a protease cleaving myosin LC2 and TnT. The ability of TnC to bind calcium properly is being investigated. Western blots revealed that the smaller T2 isoform of TaT rather than the larger T1 isoform, was often prevalent in IDC. In several other individual cases, only T1 isoform was present. Moreover, no "1-2 isotype could be detected in the myofibrils isolated from whole tissue in which T2 was also absent, suggesting that T2 is a distinct gene product and is not produced by proteolysis of T1 during myofibrillar preparation (PAW Anderson et al., Circ Res 1995). In parallel studies concentrating on the characterization of the protease, we have observed three proteolytic protein bands in the original extract. Of these only one could hyrolyze LC2 as was the case with the protease isolated from the skeletal muscle of myopathic hamsters. The human heart protease, identified as mekratin (Armenian for scissors) constitutes a very small fraction of the tissue; less than a milligram of mekratin could be obtained from 40g of tissue. Activation or synthesis of mekratin along with the presence of T2 isoform (resulting from alternate splicing of the primary transcript), are two major changes in the the IDC cardiac tissue. Whether or not the presence of T2 implies an inherited element in the etiology of IDC is under investigation.

EFFECT OF LEVOSIMENDAN VIA TROPONIN C IS Mo125 CONTROLLED BY INTRACELLULAR CALCIUM LOAD AND CALCIUM INFLUX Helmo Halkala, Jouko Levljokl & Inge-Brltt Llnd6n, Orlon.Farmos, Orlon Research, Espoo, Finland Levosimendan acts on troponln C In a calcium dependent manner thereby increasing the calcium sensitivity of cardiac muscle. We have now investigated the calcium dependency of the positive inotropic effect of levoslmendan. The cellular calcium load was changed by verapamil, lidocaine and digoxin. In addition, the calcium influx was adjusted by using different stimulus strengths, 20% above threshold (study A) and two-fold threshold (study B), in pacing (1 Hz, 37"C) of the guinea-pig papillary muscle. Milrinone was used as reference compound. In study A and B levosimendan (0.03-30 laM) increased twitch tension maximally by 47% and 129%, respectively. In study B lidocaine (100 ktM) and verapamil (3 I~M) alone decreased twitch tension by 55% and 60%, respectively. Lidocaine and verapamil noncompetitively antagonised (-59% and -57%, respectively) the effect of levosimendan. The effect of milrinone was stimulus strength independent and it was not antagonised by lidocaine. In study A and B dlgoxin (0.1 ktM) increased twitch tension by 11 and 17%, respectively. DIgoxin reduced the stimulus sensitivity of the muscle. In study A digoxin potentiated (+129%) but in study B it non-competitively antagonised (-45%) the effect of levosimendan (1 /aM). In contrast, In study B digoxin potentiated the effect of mtlrinone (10 p.M) by 79%. The results indicate that levosimendan does not compensate the lowered intracellular calcium load either indirectly via sodium current or directly via calcium current. However, the antagonism found with digoxtn indicates that the inotropy by levosimendan is very sensitive to changes in calcium influx.

OXYGEN FREE RADICALS AND CARDIAC Mo127 MYOFIBRILS. FACTS AND ARTEFACTS Ren6e Ventura-Clapier, Hassane Mekhfi, Vladimir Veksler. CJF INSERM 92-11. Universit(~ Paris-Sud, 92 296 Ch~tenay-Malabry FRANCE. Reactive oxygen species (ROS) have been reported to act on myofibrillar proteins and to alter cardiac myofibrillar function. However, their precise mode and site of action are still unknown. The effects of xanthine oxidase/xanthine system (XO/X) and of hydrogen peroxide (H202) on myofibrillar mechanical properties have been studied in the presence and in the absence of phosphocreatine (PCr) in Triton X-100 treated cardiac fibers. We found that the reported decrease in maximal calcium activated force induced by xanthine oxidase was prevented by the proteolytic inhibitor PMSF and could thus be attributed to a high contaminating proteolytic activity. After 25 rain incubation of the fibers with either XO/X system (30 mU.ml'l/30 gM + PMSF) or H202 (2.5 raM), a large increase in calcium sensitivity was observed in the absence of added MgATP, when MgATP was provided by myofibrillar creatine kinase with PCr and ADP. After incubation with XO/X or H20 ~ the MgATP concentration at which half maximal' rigor tension developed was shifted towards higher MgATP concentrations in the presence of PCr. However, when added MgATP was the only source of energy, no change in calcium sensitivity or rigor tension was observed. These results suggest that ROS do not induce profound deterioration of the fibers but inactivate bound creatine kinase, inducing a decrease in local ATP/ADP ratio.

SARCOMERE LENGTH, INTERFILAMENT Mo126 SPACING, AND MYOFILAMENT Ca 2+ SENSrI'IV1TY IN CARDIAC MUSCLE Franklin Fuchs and Yi-Peng Wang. Dept. Cell Biology and Physiology, University of Pittsburgh Sch. Mad., Pittsburgh, PA 15261, USA. The Ca 2+ sensitivity of skinned cardiac muscle is increased by either an increase in sarcomere length (Hibberd and Jewell, 1982) or by osmotic compression of the filament lattice (Harrison, et al, 1988). Since increase in sarcomere length is associated with a reduction in interfilament spacing it is not evident which factor is the primary determinant of Ca 2+ sensitivity. An analysis of Ca 2+ sensitivity in relation to length and spacing was carried out with skinned bovine cardiac muscle. At sarcomere length 1.7 I.tm addition of 5% Dextran T-500 increased Ca2+ sensitivity to the same extent as extension to samomere length 2.3 I.tm (ApCa0.5 = 0.25). Both interventions reduced fiber bundle width by -13%. At sarcomere length 2.0 laxn Dextran had a lesser effect (ApCa0.5 = 0.15) and at 2.3 I.tm it had essentially no effect on Ca2+ sensitivity. A comparison of force-pCa curves in relation to both sarcomere length and muscle width showed that pCa0.5 values were more closely correlated with interfilament spacing than with sarcomere length. The length dependence of force in cardiac muscle (Frank-Starlingrelation) may be mainly based on length-dependent changes in actin-myosin separation. Supported by NIH grant AR10551.

EFFECTS O F Rp-8-Br-cGMPS AND Rp-8Mo128 Br-cAMPS ON THE CHANGES IN Ca 3+ SENSITIVITY BY CYCLIC NULEOTIDES Tomie Kawada, Mikio Nakazawa and Shoichi Imai. Dept of Pharmacology, Niigata University School of Medicine, Niigata 951, Japan. The participation of activation of protein kinase G (Gkinase) or protein kinase A (A-kinase) in changes in Caz÷ sensitivity of contractile element (CE) by agents producing increases in cyclic GMP (cGMP) or cyclic AMP (cAMP) was studied using rat mesenteric arterial smooth muscle preparations skinned with a-toxin (30 /~g/ml) in the presence of A23187 (10/zM). A rightward shift of the concentration-response curve for Ca2+ denoting the decrease in Ca z+ sensitivity of CE was observed with cGMP, 8-Br-cGMP, cAMP and 8-BrcAMP. The shift by 8-Br-cGMP (107M) was counteracted completely by a specific inhibitor of activation of G-kinase, Rp-8-Br-cGMPS (Rp-8-Br guanosine-3',5'-cyclic monophosphorothioate.). A specific inhibitor of activation of A-kinase, Rp-8-BrcAMPS (Rp-8-Br adenosine-3',5'-cyelic monophosphorothioate) which itself produced a left-ward shift of the concentration-response curve counteracted most of the shift by 8-Br-cAMP (10 .6 M). These results indicate the involvement of activation of G- and A-kinases in the decrease in Ca 2+ sensitivity of CE produed by respective cyclic nucleotides.

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SELENITE-INDUCED INCREASE IN FORCE Mo129 OF RAT SKINNED CARDIOMYOCYTES Catherine Vannier, Hugues Chevassus, *Belma Turan & Guy Vassort. INSERM U-390, Montpellier France, *Biophysics Ankara, Turkey. Selenite (Se032-) has been shown in some tissues to cause cell dysfunction that was attributed to oxidative stress. Contractile activity was previously measured at 35* C on rat papillary muscles stimulated at 0.1 Hz. Na2SeO3 (lmM) induced a small but significant (20%) increase m force that was followed by a gradual decline, down to 30% of control value after 20 rain exposure• This decrease was simultaneous to an augmentation of resting force. Parallel experiments with Fura-2 loaded cells indicate that at l mM selenite rapidly decreased Ca transients while resting Cai was enhanced (Turan & Desilets, 1995). In this work, the effects of selenite were investigated on the properties of the contractile proteins of a rat single ventricular cell, after its chemical skinning by Triton X100. Under control conditions, the tension/pCa curve could be fitted by an Hill equation with pCa50 = 5.79 + 0.01 and nn = 2.93 + 0.0l (mean + SEM, n=5). The presence of 1 mM selenite increased force developed at intermediate and optimal Ca concentrations. The pCa50 and nrl values were then 5.87 -k 0.0l and 2.28 + 0.01 respectively. This effect was sustained and not reversible on washing the selenite; however, it could be reversed by the disulfide reducing agent dithiothreitol (DTT, 0.3 mM). Consequently the transient positive inotropic effect could be imputed to a Ca sensitisation of the contractile proteins due to their oxidation by selenite ions.

DIFFERENTIAL REGULATION OF MO131 CALMODULIN-DEPENDENT CYCLIC NUCLEOTIDEPHOSPHODIES~ERASE 1SOZYMES Rajendra K. Sharma, Department of Pathology, Royal University Hospital, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interactions which occur between the cyclic nucleotide and Ca2* second messenger systems. CaMPDE exists in different isozymic forms which exhibit distinct molecular and/or catalytic properties. Immunological, kinetic and regulatory characterization have revealed subtle differences among these isozymes. The 63 kDa CaMPDE is kinetically distinct from the other three isozymes. This isozyme is also further distinguished from other isozymes by the ginsenosides inhibition study. Ginsenosides are found to be potent inhibitiors of brain 60 kDa, heart and lung CaMPDE isozymes but not of the brain 63 kDa isozyme. The three other members, brain 60 kDa CaMPDE, heart CaMPDE anti hmg CaMPDE, show differential activation by cahnodtdin and Ca 2+. These observations are consistent with the notion that differential regulalion hy calmodulin and kinetics inhibition Ca 2+, is an important flmction of these isozymes which provide fine-ttming mechanisms for calmodulin action. AllO

STUDIES ON PROTEIN CHANGES IN Mo130 DILATED CARDIOMYOPATHY USING TWO-DIMENSIONAL ELECTROPHORESIS Joseph M. Corbett, Colin H. Wheeler, Howard H. Why', Peter J. Richardson*, Magdi H. Yacoub & Michael J Dunn. Heart Science Centre Harefield Hospital, Middx, UK and *Dept of Cardiology, Kings College Hospital, Denmark Hill, London. Dilated cardiomyopathy (DCM) is a disease of unknown aetiology. A variety of pathological mechanisms and predisposing factors have been implicated including autoimmune disorders, viral infections and exposure to several toxic agents. Irrespective of the primary cause it is expected that alterations in protein expression will result, leading to heart failure. We have investigated protein changes in DCM using two-dimensional electrophoresis (2-DE) which separates proteins on the basis of two independent properties, charge and molecular mass, on a single gel. Protein expression profiles from DCM myocardial tissue samples (n=47) have been combined in a computerised 2-D human heart protein database which currently contains in excess of 1,200 proteins. Significant decreases (p<0.05) in the expression of 10 proteins have been detected in DCM compared to non-DCM heart failure material using this database. 2-D analysis of isolated myocytes and cultured fibroblast, smooth muscle, endothelial and mesothelial cells has confirmed that the protein changes occur in myocyte contractile proteins. Protein characterisation using N-terminal and internal microsequencing, MALDI MS and Western immunoprobing has identified the proteins as myosin light chain 2 and desmin, major components of the sarcomere.

INFLUENCE OF THE CALCIUM-SENSITIZER

Mo132

EMD 57033 ON SYSTOLIC AND DIASTOLIC PROPERTIES OF HUMAN LEFT VENTRICULAR INTACT AND SKINNED MUSCLE PREPARATIONS

Thorsten Rut, Katja Pioch, Wemer Nickl, Jens L0demann, Burkert Pieske, Gerd Hasenfuss, Hanj6rg Just, Christian Holubarsch, Innere Medizin Ill, Universit&t Freiburg, F.R.G. By means of experiments using skinned and intact muscle preparations obtained from explanted human left ventricles the pharmacological mode of action of EMD 57033 (10.5 M) and its influence on systolic and diastolic tension were studied. Results: ECso of skinned fiber preparations is shifted significantly (n=8; p<0.01) from 5.76+0.06 to 6.16+_0.08 by EMD (10s M). (2) EMD increases peak systolic tension from 8.0-+-1.9to 12.6+_2.1 mN/mm 2 (n=l 0; p<0.01 ; experimental temperature 37°C, 30 beats/min). The 95% relaxation time is increased from 334+_24 to 783_+80 msec (n=10; p<0.01). (3) The extent of shortening of the intact preparation increases in propation to the increase in isometric force induced by EMD. (4) When increasing stimulation rate (30,60,90,120,150,180 beats/min) diastolic tone is observed with EMD due to impaired relaxation. Resting tension increases from 7.3+_0.8 to 17.0+-1.8 mN/mm 2. Conclusion: EMD 57033 increases force development and shortening capability in human cardiac preparations due to calcium sensitization. These positive inotropic effects are associated with impaired relaxation and thereby - diastolic contracture.

Mo 133 THYROXINE (T3) AND PIOGLITAZONE INDUCES CARDIAC HYPERTROPHY VIA DIFFERENT MECHANISMS Norman S. Kato,* Jing-Liang Wang,* Mehmet E. Ergun,* Woemer P. Meehan. °* *Dept of Surgery, UCLA Medical Center, Los Angeles, CA. **USC Medical Center, Diabetes, Los Angeles, CA. Thyroxine (T3) is the only currently known drug to induce cardiac hypertrophy. We have shown that the thiazolidinedione, pioglitazone, also causes cardiac hyperthrophy in rats. Prior studies in expedmental animals have shown that T3 alters contractile machinery of myocardium by differential expression of myosin isoforms. We have recently isolated the total proteins from left ventricles of normal, hypothyroid, pioglitazone treated, and T3 injected rat hearts by use of phenol/sucrose buffer method. By comparison of the protein profiles resolved on high resolution two dimensional PAGE gel electrophoresis, we have found that myosin isoform expression is not the only cause for induced cardiac hyperthrophy. Regulatory proteins are also involved in the processes. Additionally, analysis of the gels reveal that the mechanism responsible for induced cardiac hypertrophy is different for T3 and pioglitazone. The precise differences are under investigation.

EFFECT OF SHORTENING VELOCITY ON Mo135 CROSSBRIDGE KINETICS AND ATPase ACTIVITY Amir Landesberg, Rafael Beyar & Samuel Sideman. Technion-IIT, Haifa, Israel. This study describes a model for the regulation of mechanical activity in the intact cardiac muscle. A simple linear dependence of the rate of crossbridge (Xb) turnover from the force generating (strong) to the nonforce generating (weak) conformation on Xb strain rate is suggested, thus explaining a wide spectrum of phenomena. Analytical solutions for Hill's equation of the force-velocity relationship and for the phenomena of unloaded shortening velocity and force deficit are provided. The dependence of the shortening velocity on calcium concentration, sarcomere length, internal load and rate of Xb cycling are described. Moreover, the model explains the linear stiffness-force relation, as well as the relation between ATPase activity and the average force obtained during length perturbation. At high activation levels, ATPase activity during shortening increases over the isometric ATPase activity, which relates to the reported positive effect of ejection on force generation. However, at low activation, ATPase activity decreases below the isometric value. The Fenn effect is explained through the acceleration of the rate of Xb turnover from the strong to the weak conformation. The Fenn effect in cardiac muscle is diminished since the sarcomere is not maximally activated. The effect of the shortening velocity on force generation, energy turnover and Xb cycling can be understood by this simple model.

LOCALIZATION OF HSP25 IN THE I-BAND Mo134 OF CARDIAC MYOFIBRILS OF ADULT RAT Gudrun Lutsch, Roland Vetter, Joachim Stahl & Rainer Benndorf. Max Delbr{Jck Center for Molecular Medicine, Berlin-Buch, Germany. The mammalian heart is an organ in which the small heat shock protein HSP25 is constitutively expressed at elevated levels compared to other organs. Previous investigations of our group showed a drastic differentiation-dependent reduction of the HSP25 level when comparing heart tissue from embryos, neonatal and adult rats by immunoblotting and immunoelectron microscopy ~. Here we show by immunofluorescence microscopy of semithin cryo-sections that HSP25 is located in the I-bands of cardiomyocytes as deduced from its colocalization with actin. Furthermore, diffuse cytoplasmic staining occurs in endothelial and smooth muscle cells. The homologous ctB-crystallin, which is not subjected to developmental regulation in heart ~, has a similar location as HSP25 in cardiomyocytes but was not detected in endothelial and smooth muscle cells. The high abundance of HSP25 in immature cardiomyocytes and its location in the I-band of myofibrils of differentiated cardiomyocytes suggest an important role of this stress protein in the assembly of myofibrils. Furthermore, it may be involved in the organization of the microfilament system in other cell types as can be concluded from in vitro experiments2. Ref: 1. Lutsch, Get al., J Mol Cell Cardio126: CVI (Abstr), 1994; 2. Benndorf, R et al., J Biol Chem 269: 20780, 1994.

EFFECT OFISOPROTERENOLONPROTEIN Mo136 PHOSPHORYLATIONIN HEARTSDEFICIENTIN PHOSPHOLAMBAN E. Kiss, I. Edes*, W, Luo & E.G. Kranias; Univ. of Cincinnati, Cincinnati, OH, USA and *Medical School of Debrecen, Hungary Phosphorylation of phospholamban (PLB), troponin I, Cprotein and phospholemman has been suggested to mediate the 13-adrenergic inotropic and lusitrcpic effects of the heart. To delineate the relative contdbution of these phosphoproteins in the myocardial p-responses, phosphorylation studies were performed using intact, beating heart preparations of wild-type (WT) and phospholamban-knockout (PLB-KO) mice. Langendorff perfusion of the hearts revealed significant increases in the basal rates of left ventdcular +dP/dt (155%) and dP/dt (170%) in PLB-KO compared to WT (100%) hearts. Isoproterenol (Iso) perfusion (0.15 p.M) was associated with a gradual increase in +dP/dt and -d.Pldt in the WT hearts while there were no changes in the PLB-KO hearts. Examination of the basal and Isostimulated tissue cAMP levels revealed no significant differences between the WT and PLB-KO hearts. Isostimulation of WT hearts was associated with significant increases in the degree of phosphorylation of all phosphoproteins (PLB: 7.6x; troponin I: 7.1x; C-protein: 1.9x; phospholemman: 5.3x) compared to basal (1:<) values). In PLB-KO hearts, Iso-stimulation was associated with similar increases in phosphorylation of troponin I (7.4x), C-protein (2x) and phospholemman (5.6x). These findings indicate that the lack of Isomediated changes in the contractile parameters of PLBKO hearts is not due to lack of increases in cAMP levels or the degree of phosphorylation of regulatory phosphoproteins in myofibdls and sarcolemma.

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CARDIAC MUSCLE CELLS AS TARGETS FOR Mo137 THE ACTION OF ADENOVIRAL E1A PROTEINS Lorrie A. Kirshenbaum & Michael D. Schneider, Molecular Cardiology Unit, Baylor College of Medicine, Houston, Texas Though molecular events governing cell-cycle exit in cardiac muscle are cryptic, potential clues are available from skeletal muscle and other model systems. Mitogens control the cell cycle in part through cyclin-dependent protein kinases, which alter the phosphorylation or expression of so-called 'pocket" proteins including the retinoblastoma gene product, Rb, p107, end p130. To disrupt "pocket" protein function in ventricular myocytes and test for potential impact upon cardiac differentiation and growth, we expressed a series of 12S EIA proteins in neonatal ventricular myocytes, using adenoviral gene transfer. All 4 12S E1A proteins were efficiently expressed and appropriately targeted to the nucleus. In the absence of a second adenoviral gene, E1B, EIA was cytotoxic, with features typical of apoptosis. In the presence of E1B, E1A preferentially inhibited transcription of cardiac-restricted c¢-actin promoters, and reactivated DNA synthesis in cardiac myocytes, without cell death. DNA synthesis (BrdU incorporation) was accompanied by a parallel increase in DNA content (Feulgen stain). Cells infected with a substitution that abrogates known functions of both the amino-terminus and conserved region 2 (R2G,C124G) were indistinguishable from uninfected cells. Mutations of the amino-terminus alone (R2G), or conserved region 2 alone (Y47H,C124G), demonstrate that these respective "pocket" protein- and p300-binding domains each suffice, in the absence of the other, for transcriptional repression and re-entry into S phase.

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EJECTION IMPROVES CONTRACTILE PERFORMANCE Mo138 INDEPENDENT OF END-DIASTOLIC LENGTH HISTORY Matthew W. Watkins, Akihiro Higashiyama, Martin M. LeWinter, University of Vermont, Burlington, VT, USA. The positive effect of ejection on contractile performance compared to isovolumic contraction at the same end-systolic volume has been attributed to greater length-dependentactivation. However, there have been reports of an ejection related increase in contractility which is independentof end-diastolic volume (EDV). To further characterize the latter effect, we studied isolated, rbcperfused rabbit hearts at constant EDV during is0volumic contraction (ISO), volume reduction beginning at end-systole (VR), and ejection (EF .34 + .08) beginning at mid-syst01e(EJ). Both ejection modes led to an increase in contractile function, which required > 50 beats to attain steady-state. Comparedto IS0, VR (N - 7) increased LV pressure by 15% [92.2 + 23.7 (± SD) vs 105.9 ± 27.6 mmHg] and maximum dPIdt by 17%, all p <0.01, while EJ (n-6) increased developed pressure 10% compared to the first beat of ejection (61.0 ± 10.9 vs 67.4 + 11.7 mmHg, p<0.01}. In separate hearts (n-8), 2,3-butanedione monoxime (BDM) was used to estimate nonmechanicalV02 (V02 for ECC and basal metabolism). Compared to IS0, VR ejection increased n0nmechanicalV0 z 26% (0.0248 ± 0.0021 vs 0.0312 ± 0.0022 ml 02"beatt'lO0g~, p<0.Ol), consistent with an increase in total calcium cycled per beat. In conclusion, ejection is associated with a slowly appearing increase in contractile performance which is independent of length-dependent activation at EDV. This "shortening activation" may represent a positive feedback between myefilament shortening and calcium cycled per beat.

GENOMIC ORGANIZATIONAND MUTATIONAL Me139 ANALYSISOF THE CELLULAR NUCLEIC ACID BINDING PROTEIN (CNBP) GENRE Irwin L. Flink & Eugene Morkin, University Heart Center, University of Arizona College of Medicine, Tucson, AZ 85724, U.S.A.

PRELOAD AND AFTERLOAD INDEPENDENT Mo140 SENSITIVITY OF SINGLE BEAT INOTROPIC STATE INDEXES Elena C. Lascano, Jorge A. Negroni, Alberto J. Crottogini, H~ctor del Valle & Ricardo H. Pichel. Favaloro Foundation, Buenos Aires, Argentina

The human CNBP gene locus is about 6.9 kbp in length. The gene consists of five exons, four of which contain coding information for two alternatively spliced products, CNBPt~ and 6. CNBPot (Mr 19,463) and 13 (Mr 18,742) proteins are encoded by exons 2 through 4. Both forms contain seven zinc finger domains. CNBP13 lacks seven amino acids in the linker region between the first two zinc fingers because of the use of an alternative 5' donor site within exon 2. The sixth zinc finger is the only finger domain that is not completely encoded within a single exon. The 5' untranslated region contains 850 bp and is interrupted by intron 1. The 3' untranslated region, ending at the polyadenylation signal, is 859 bp long and is contained within exon 5. One A l u - repeat was identified within intron 1. CNBPt~ and 13 differentially regulate the 13myosin heavy chain (13-MHC) gene by interacting with a repressor element (RE). E. coli expressed protein containing a His to Asn site-specific mutation of the first finger does not interact with RE. These results suggest that this finger may be essential for DNA binding and functional activity.

Inotropic state indexes are preload and afterload dependent. The purpose of this work was to determine the inotropic sensitivity of single beat indexes independently of errors introduced by preload lend-diastolic volume (EDV)] and afterload [conductance (G)=l/peripheral resistance] using a mathematical model. Eleven slightly anesthetized blocked (atropine, propranolol, enalaprilat) chronically instrumented [volume (V, micrQc~stals) and ventricular and aortic pressures (P, microtransducers)] dogs were used. At normal, decreased (aortic occlusion) and increased (sodium nitroprusside) G , P-V data of stable beats at 5 decreasing EDV (vena cava occlusion) were measured in control and increased inotropic state (amrinone, .65 mg/min). Each index was fitted to a second order surface as a function of EDV and G and the errors due to preload and afterload were calculated using a 10% change in EDV and G. The indexes' relative inotropic increase free of EDV and G errors showed that the maximum velocity of circumferential fiber shortening (Vcf, 22.8 +15.6%), maximum dP/dt (P'm, 19.5+_.26.8%), P'm/IP (IP:P at P'm, 29.1_+22.9%) and P'm/EDV (38.9.+.25.9%) were equally able to evaluate inotropic state, whereas ejection fraction (5_+ 13%), stroke work [SW] (-46+_20.5%), SW/EDV (-17.7:1:2.4.7%) and maximum PN (-6.1+_22.4%) were significantly lower than any of them (P<.05) and consequently less adequate for this purpose.

THE BASIS FOR THE PRESSURE-VOLUME Mo141 RELATIONSHIP AND ENERGY TURNOVER Amir Landesberg, Rafael Beyar & Samuel Sideman. Technion-IIT, Haifa, Israel.

Recent observations have shown the Important effects of ejection dynamics on the end-systolic pressure-volume relationship (ESPVR). We have tested the effect of loading conditions on the ESPVR using our understanding of the intracellular control of crossbridge cycling. Based on biochemical studies, two main feedback mechanisms were described: 1) Cooperativity, in which the affinity of troponin for calcium depends on the number of cycling crossbridges. 2) Mechanical feedback whereby the filament sliding velocity accelerates the rate of crossbridge weakening. These intracellular mechanisms describe cardiac muscle fiber characteristics (the force-length and the force-velocity relationship) and provide the molecular basis for left ventricular function as described by the time varying elastance model, in the pressure-volume plane. Moreover, the study explains the positive effect of ejection and the excessive end-systolic pressure (ESP) in an ejecting beat over the isovolumic beat at a small ejection fraction; at increased activation levels, which depend on the cooperativity mechanism, sarcomere shortening increases the ATPase activity, through the mechanical feedback. For a large ejection fraction (EF>0.3) the ESP of the ejecting beat approaches the isovolumic ESP. The ESPVR and the energy turnover depend on the balance between the intracellular mechanisms i.e. the cooperativity and the negative mechanical feedback.

MECHANICAL AND MORPHOMo143 METRIC DIFFERENCES BETWEEN ATRIAL AND VENTRICULAR MYOCYTES T Onodera', B Heileson, JM Capasso, AM Gerdes. Jikei U n i v School Med, Tokyo, Japan', U South Dakota, Vermillion, SD

Recent data suggest that increased myocyte length/width ratio (L/W; e.g. t from approx. 8 to 11) and d e p r e s s e d myocyte mechanics (e.g. $ velocities of shortening [Vs] and r e - l e n g t h e n i n g [VR] ) are associated with heart failure. Compared to v e n t r i c u l a r myocytes, atrial myocytes from normal guinea pigs also have a high L/W. To determine if mechanical p r o p e r t i e s are related to cell shape, V s an~ V a were examined in isolated atrial and ventricular myocytes from guinea pigs (n=8). V s was 266% greater in atrial (476±36 ~m/sec) vs. ventricular (179±63) myocytes. V R was 277% greater in atrial (498±62) vs. v e n t r i c u l a r (180±65) myocytes. Atrial m y o c y t e L/W was 13.4±1.0 while v e n t r i c u l a r myocyte L/W was 8.4±0.8. Unlike failing ventricular myocytes, the high L/W of normal atrial myocytes is associated with enhanced, rather than depressed, cell mechanics. These mechanical properties may be related to regional differences in myosin isoforms.

PAPILLARY MUSCLES SPLIT IN THE Mo142 PRESENCE OF BDM HAVE NORMAL ENERGETIC AND MECHANICAL CHARACTERISTICS Helen Kiriazis & Colin L. Gibbs. Dept of Physiology, Monash University, Clayton 3168, AUSTRALIA. A number of studies have used 2,3-butanedione monoxime (BDM) to avoid myocardial damage when cutting small muscle preparations from large hearts. The present study investigates the effects of BDMdissection and muscle cross sectional area (CSA) reduction. Using adult rat hearts three muscle groups were obtained: whole left ventricular papillary muscles (Group 1, Grl) and papillary muscles split longitudinally in the presence of 30 mM BDM, removing approximately 10 % (Gr2) or 40-50 % (Gr3) of the muscle (5 animals in each group). Isolated muscle preparations were studied at 27 °C and stimulated at 1/6 Hz. Grl and Gr2 muscle preparations had comparable CSAs: 0.90_+0.11 versus 0.80_+0.12 mm2 (mean_+SEM). Stress (force/CSA), work and energy use were similar for these two groups implying that BDM-dissection did not alter muscle characteristics. The thinner Gr3 muscles (0.54-0.05 mm=), however, showed an enhanced mechanical performance. This outcome was in accordance with literature data documenting a negative correlation between stress and CSA. The results suggest that BDM-split and intact papillary muscles of similar CSA have comparable energetic and mechanical properties.

THE EFFECT OF EXERCISE-INDUCED Mo144 TACHYCARDIA ON LEFT VENTRICULAR EFFICIENCY OF ENERGY TRANSFER IN EXERCISING DOGS Alberto J. Crottogini, Juan G. Barra, Peter Willshaw & Ricardo H. Pichel. Basic Sciences Research Institute, Favaloro Foundation, Buenos Aires, Argentina. The area circumscribed by the left ventricular (LV) end-systolic (ES) and end-diastolic pressure (P)-volume (V) relationship (R) and the systolic trajectory of the PV loop is termed PV a r e a (PVA) and represents total mechanical energy of contraction. The ratio of stroke work (SW) to PVA (TransPVA) represents the efficiency of the LV in transferring mechanical energy to SW. Exercise has been shown to increase TransPVA. We determined on which mechanical bases this occurs and examined whether the increase in TransPVA is due to the force-frequency effect resulting from exercise-induced tachycardia. LW (sonomicrometry) and LVP were measured in 6 dogs during standing (PRE-EX), treadmill exercise (EX) and following EX but paced at the heart rate observed during EX (TACHY). The ESPVR under each condition was generated by vena caval occlusion and its slope (Ees) and intercept (V0) calculated, as were PVA, SW and TransPVA. EX increased TransPVA from 0.62+0.06 to 0.72+0.07 (p<0.05), by means of significantly increased SW at constant PVA. Both Ees and V0 increased during EX. This resulted in crossover of the PRE-EX and EX ESPVRs at around 100 mm Hg, allowing the increase in TransPVA to occur at constant PVA. TACHY had no effect on Ees, V0 or TransPVA. Conclusion: in exercising dogs, the increase in TransPVA occurs through an increase in SW with no change in PVA. This effect in independent of the tachycardia associated with exercise.

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ELASTIC RECOIL IN ISOLATED MYOCYTES Mo145 Hideki Okayama, Hideo Kawakami, Yuji Shigematsu, Mareomi Hamada, Kunio Hiwada. Second Dept of Internal Medicine, Ehime University, Japan. Early diastolic ventricular filling is an active suction of blood depending on elastic recoil of the ventricle. However, the origin of this recoiling force (i.e. extracellular or intmcellular) has not been entirely clarified. To elucidate this issue we examined the contractile function of the isolated myocyte which is independent of extraceilular matrix or chamber geometry. Peak velocity of shortening (PVS: am/see), peak velocity of relengthening (PVR: g.m/see) and twitch amplitude (AMP:. lam) were analyzed by a video system. These parameters were normalized by each resting cell length [Norm',dized PVS (NPVS)= PVS/resting cell length, Normalized PVR (NPVR) =PVR/resting cell length, extent of shortening (ES) =AMP/resting cell length]. Peak velocity of shortening was similar to that of relengthening (mean PVS ( N P V S ) = 2 ~ (1.61:t-0.02), mean PVR (NPVR) =247i-4 (1.56-,A3.02); No. of cell~108). PVS (NPVS) correlated well with PVR (NPVR) (r=0.93 (0.91)). There was a positive correlation between A M P and PVR (r=0.68, p---0.000l). Similarly, there was a positive correlation between ES and NPVR (r---0.60, p= 0.13001). Minimum cell length had an inverse correlation with NPVR (r=0.43, p---0.0001). These data suggest that peak velocity of relengthening in the myocytes increased in accordance with the extent of cellular shortening and that the recoiling force exists in shortend myocyte itself

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CO~I-TRASTiN-G LEFT VENT-I~I(2ULAR Mo146 R.~e~LAXrXN'T EFFECT OF CAPTOPRIL AND LISINOPRIL IN THE INTACT HEART. Peter B Annlng, Richard M Grocott-Mason, Malcolm J Lewis, Ajay M Shah. Cardiovascular Sciences Research Group, University of Wales College of Medicine, Cardiff, CF4 4XN. Angiotensin-converting enzyme (ACE) converts angioteusin I to angioteusin II and also catalyses the breakdown of bradykinin (BK). BK activates B2 receptors on endothelial cells to release nitric oxide (NO), and may be involved in the mechanism of action of ACE inhibitors (ACEi). We have previously shown that exogenous BK (>lnM) acting on B2 receptors modulates left ventricular (LV) relaxation, in part via an NO-dependent mechanism and that captopril (CAP) mimics this effect. We examined the effects of the ACEi CAP (ttdV[) and lisinopril (LIS, liuM) both alone and with a threshold concentration of exogenous BK (0.1aM) on LV performance in isolated ejecting guinea-pig hearts (constant loading & heart rate; Kreb's buffer; 37"C). Haemoglobin (Hb, llaM) was used as an NO-scavenger in some experiments. LV pressure (P) and dP/dt were measured using a 2F Millar catheter inserted into the LV cavity. LV relaxation was assessed by a mono-exponential time constant (TE), as described previously. BK (0.InM) had minimal effect on TE, peak LVP or dP/dt (n=6). CAP alone caused a large decrease in TE (-15.0 :t: 1.2% at 16rain; n=9; p<0.01), whereas LIS had no effect (-4.4:1: 2.5% at 16rain; n--4; p=NS). The fall in T E with CAP was inhibited by Hb (-1.68 5: 1.2%, at 16rain; n=5; p<0.01 c.f CAP alone). Peak LVP and dP/dt were unchanged. In the presence of CAP but not LIS, BK induced a greater fall in TE (21 -I- 1.5%, after 16min; n ~ ; p<0.01, c.f CAP alone or BK alone). There were no significant changes in peak LVP or dP/dt c.f CAP alone.

These date demonstrate that the LV relaxant effect of CAP both alone and in the presence of exogenous BK is not mimicked by LIS, suggesting the involvement of the sulfhydryl group of CAP in this reslx)nse. Also, this effect of CAP is mediated by an NO-dependent mechanism.

AFTERLOAD DEPENDENCE OF INOTROPIC MO147 INDEXES DERIVED FROM PRELOAD MANEUVERS Jorge A. Negroni, Elena C. Lascano, Alberto J. Crottogini, Carlos A. Perazzo & Ricardo H. Pichel. Favaloro Foundation, Buenos Aires, Argentina.

AUTOMATED SYSTEM FOR CARDIAC Mo148 ULTRASOUND IMAGE ANALYSIS / C a r m A ) Bal~izs Asztalos t'2 & Roger J. Willis t. Rotary Centre for Cardiovascular Research, Brisbane, Australia; 2Technical University of Budapest, Hungary

In intact animals it is difficult to independently control preload from alterload to evaluate the afterload dependence of indexes derived from preload maneuvers. We analized, by means of a mathematical model, the affedoad [conductance (G)= l/peripheral resistance] dependence of indexes dedved from preload maneuvers [decrease in end-diastolic volume (EDV)] and their inotropic response at a fixed G in closed chest dogs. Eleven slightly anesthetized, blocked, chronically instrumented [volume (V, microcrystals) and ventricular and aortic pressures (P, microtransducers)] dogs were used. At normal, decreased (aortic occlusion) and increased (sodium nitroprusside) G, P-V data of stable beats at 5 decreasing EDV (vena cava occlusion) were measured in control (C) and increased inotropic state [amdnone, .65 mg/min (1+)]. In each loading condition, stroke work (SW) and maximum dP/dt (P'm) were fitted to a second order surface as a function of EDV and G and end-systolic P asl a function of end-systolic V and G. End-systolic P-V (Em), SW= EDV (Mw) and P'm-EDV (E'm) relations were calculated as thel derivative with respect to EDV of the fitted surface in thd reference point (mean G and EDV of each animal and inotropiQ condition). The inotropic response was Em (5.7+2.7 (C)' 8.7:1:3.7 (I+) P<.001), Mw (63.3+17.6 (C), 90.6:1:14.4 (1+)! P<.005) and E'm (30.8:1:22.3 (C), 71.3+47.8 (1+). A 50°/=1 decrease in G produced an increase of 52+30% in Em (P<.001} and 361:1:823% in E'm, and an 11:t:25% decrease in Mw. We conclude that Em is the index most affected by afferload but together with Mw is very sensitive to I+ in these conditions.

Ultrasound echocardiography is a widely used clinical technique, but the images obtained using current technology, are still processed manually with semiautomated methods. In contrast to this, the newly developed system works in an automated way, first obtaining a series of long and short axes views of the heart synchronised by the ECG in real time, then processing them off-line. After detection of the internal edges of the left ventricle, the system determines the short/long axes areas, diameters, calculates the volume of the left ventricle frame by frame and, based on this, the ejection fraction for each cardiac cycle. The validation processes were carried out as a part of a larger study which focused on the role of the endogenous hormone, adenosine, in regulating cardiac efficiency in anaesthetised sheep with elevated cardiac work loads. After a right thorocomy was performed, the heart was imaged directly from its surface. Image analysis was carded out by a fully automated and an operator assisted edge tracking method. Quantitatively, the results indicate that the manual and automatic boundary tracking method both give relative area changes of the left ventricle minor axis, throughout the cardiac cycle. Based on agar casts of the left ventricle in end-diastole, the volume correlation was found to be good with r=0.93.

C H A N G E S IN P H O S P H O L I P I D Mo149 C O M P O S I T I O N DURING D E V E L O P M E N T O F C A R D I O M Y O P A T H Y IN U M - X 7.1 H A M S T E R Alba Vecchini, Luciano Binaglia, Paolo Di Nardo t, Marilena Minieri t, Vincenzo Panagia 2 & Naranjan S. Dhalla 2. Institute of Biochemistry, Univ. of Perugia and ~Dept. oflnternal Medicine, Univ. o f R o m a "Tor Vergata", Italy, and 2Div. of Cardiovascular Sciences, Univ. of Manitoba, Winnipeg, Canada. Since phospholipids play a crucial role in membrane function, the lipid composition of the heart of UM-X 7. I cardiomyopathic hamster was analyzed and compared with that of Syrian Golden Hamster (SGH). The left ventricle of both SGH and UM-X 7.1 hamsters exhibited less phospholipids in comparison with the right ventricle. The phospholipid content of the ventricles decreased with age (20 days - 5 months) in UM-X 7.1 ; such a decrease was mainly due to depletion o f choline and ethanolamine phospholipid pools. Cardiolipin content augmented with age in both ventricles o f healthy animals, whereas it decreased significantly in UM-X 7.1. The percent content ofoleic acid increased with age in SGH ventricles, whereas it was depressed in UM-X 7.1. The percentage of arachidonic acid decreased during the same age interval in SGH, but did not change significantly in cardiomyopathic animals. These alterations may contribute to the membrane dysfunction in the UM-X 7.1 strain o f genetic cardiomyopathic hamster.

Mo151 ENDOTHELIAL DYSFUNCTION AND ENHANCE1] CONTRACTILITY TO ANGIOTENSIN II IN YOUNG CARDIOMYOPATHIC SYRIAN HAMSTERS Crespo MJ, Escobales N, and Altieri Pl. Departments of Physiology and Medicine, University of Puerto Rico Medical School, San Juan, P.R., U.S.A. 00936-5067 Endothelial dysfunction has been shown to be a common characteristic of congestive heart failure (CHF) in both patients and animal models with this condition, however, whether the dysfunction precedes or is secondary to CHF is unknown. To evaluate this point, we evaluate the vasodilator response to acetylcholine (10-8 to 10.4 M) in norepinephrine ( 10-7 M) precontracted aortic rings from 2-months old, cardiomyopathic hamsters (Bit-TO-2) that had not yet developed heart failure. BitFIB hamsters (age matched) were used as controls. L-NAME (ImM) was used to inhibit the relaxation of aortic rings by the acetylcholine-inducedproduction of nitric oxide.ln Controlanimals, acetylcholine (10-5 M) induced full relaxation by a mechanism sensitive to L-NAME. In contrast, in cardiomyophatic hamsters the maximal relaxation induced by acetylcholine was 48+10% (n=8) of that observed in control animals, supporting the presence of endothelial dysfunction in this animal model of CHF. Concentration-responsecurves for acetylcholine yielded an IC50 of 0.7 _+ 0.05 IIM in Bit-TO-2 hamsters and 1.5 + 0.2 ~M in control animals (P<0.002, n=5). We also evaluated the effect of angiotensin II (100 aM) on the contraction of aortic rings from this animals. The contractile response to angiotensin II is 50% greater (P<0.001) in Bio-TO-2 (1.63 + 0.1 g) than in control animals (1.08 + 0.01 g), suggesting a state of increasing contractility in animals that have not yet developed CHF. These results indicate that in this animal model, the endothelial dysfunction precedes the clinical manifestation of CHF. Furthermore, the results indicate that in this animal model, the endothelial dysfunction precedes the clinical manifestation of CHF. Supported by Merck, Sharp and Dohme Co. and NIH-GM-RR 08224.

EFFECTS OF TCV-116 AND AMLODIPINE ON THE CARDIAC REMODEUNG AND DYSFUNCTION IN CARDIOMYOPATHIC HAMSTERS

ao150

Masashi Watanabe,Hitoshi Okada, Hisao Onozuka, Satoshi Yamada, Taisei Mikami, Hideaki Kawaguchi and Akira Kitabatake Department of CardiovascularMedicine, Hokkaido University School of Medicine, Sapporo,Japan. Reoentlythere are several reports about roles of angiotensin converting enzyme inhibitors in cardiac remodeling and their beneficial effects on congestiveheart failure (CHF) Calcium(Ca) handling in myocyte is disturbed in CHF. This study examined the effects of long-term treatmentswith angiotensin II subtype 1 receplor antagonist, TCV-116 (T)and a Ca antagonist,Amlodipine (A) on the morphologicalchanges in extracellularmatrix and progressive LV dysfunction in cardiomyopathichamsters (B1053.58). Five-week.oldB1053.58 hamsters were randomized to T (10mg/kg/day po) therapy, to A(10mg/kg/daypo) therapy or fo no therapy (C)group. Each group was treated for 15weeks. In A group, ult rasonoscopic echocardiogram revealedan increase in LV percent fractional shortening (%FS) (35.2 +--5.0 versus C:20.8 ± 4.0, P=0.001),whereasT showed no significant improvement in %FS (23.7±4.2).Each heart was fixed with 10% formalin and embedded in paraffin. Hearts were sectioned horizontaly with serial sections and stained with Gomori's aldehyde-lucshinwith Masson Goldner method. Fibrous tissue to total myocardial volume ratio calculatedby serial sections had a stronger tendency toward a reducuction in A (11.4± 1.7, P--0.08) than in T group (12.4 ± 1.6%)comparedwith C group (13.7± 1.6%).Theseresults indicate that long term treatment with A has beneficialeffect on cardiac dysfunction and may prevent the progressiveremodeling, whereas failure of T therapy may be relatedto the period of treatment, the loading dose or susceptibilityof receptor.

DISRUPTION OF CELL-CELL ADHESION INMo152 HERED1TARY CARDI 0MYOPATHY HAMSTER Nobuhiko Shibata, Yasushl Fujio, Fumiko HondaYamada, Naoyuki Sato, Hiroko Funai, A k i r e Wade= The Center for A d u l t Diseases, Osaka, Japan. In dilated cardiomyopathy arrangement of cardiomyocytes is disturbed pathologically. In the present study, we investigated on cell-cell adhesion system in hereditary cardiomyopathy hamster ( B i t 14.6). Light microscopic observation revealed t h a t intercalated disks were less prominent in B i t 14.6 than normal. Electron microscopy showed reduction in submembranous density in B i t 14.6. By immunofluorescence microscopy using the a n t i - A - C A M (adherens junction-specific cell adhesion molecule), we examined the localization of A • CAM, which plays an important role in cellular arrangements by regulating cell-cell adhesion in intercalated disk. In contrast to normal, fluorescence is weak in intensity and vague in border in B i t 14.6. By Westernblotting, we measured content of A.CAM in heart. In B i t 14.6, the content of A.CAM is 40% of that in normal. In a study how the content of A - C A M reduces in the cardiomyopathy we c l a r i f i e d 1) A . C A M was proteolized in m i l l i - m o l a r calcium ion, 2) proteolysis of A . C A M was inhibited by protease inhibitors and 3) m-calcium-activated neutral protease was localized with A - C A M in intercalated disks. Thus, we suppose A . CAM is proteolyzed in response to calcium overload in heart, resulting in disarrangement of cardiomyocytes in dilated cardiomyopathy.

AII5

MYOCAFd)IAL FIBERDISA~AYINHYPERTROPHIC CARDIOMYOPATIrf, MYOC~]DITISA~DII~TEDCARDIOMYOPATHY

Me1 s3

CELL CYCLE OF MYOCYTESINDILATEDMols4 CARDIOMYOPATHY AND MYOCARDITIS

ATSUSIII TAI~ED& Hldeo A. Baba*t, Nobuakira Takeda, Sachio [await and

Atanshi Takeda', Yukihiro Takeuchi, ^kihiko Sakata, ~buaktra Takeda" and

Ryozo Okada*.Dept. of Int.Med. Jlkei Uni.. Jnntendo Uni.*.Tokyo,

Idakoto Nagano'. Dept. of Int. Ik~cl.". Patho.. hoto llosp., J ikei Uni.. Tokyo.

Dept. of Pathology.Aachen Uni.t*,Geraany To evaluate tbe DNA synthesis of i~,ocytes in dilated cardio,o'opathy(D~) Disorganized ~ocardial fiber, so - called m'ofiber disarray in hyper-

and chronic(persistent}~ocarditis(MC), the zyocardial cell' sDNA contents

trophic cardiotvopatby0teM)

were seasured in autopsied hearts fros DCM.MC and non- cardiac diseases

Is characteristic and although not specific,

i t is also observed sicroscopically in normal hearts, dilated cardio~o-

(Control) with the fluorescence- activated cell sorter(F^C~).

pathy(DC~) and ,ffocarditis(I/,C).To

evaluate the structural differences of

ventrleular free walls of thirty hearts were obtained vlthin six hours

the fiber disarray among HQCl)Q/ and I,IC, the disorganized cardiac lyocyts

after death:DQd group(n=lO, male(Id):female(F) = 8 : 2. nean 66.0 Y),MC

were investigntednorphologically in detail with a colputer. The auto-

group(nffilO.M : F = 4 : 6, aean 62.6 Y)and control group (n=lO.M : F = 8

psied hearts were used froi patients with HCM,DCM and Mc:gc~l(nf4.mean 48

The left

2,mean 61.2 Y). The nuclei lere taken out from the tissue by iledley

Y), DCM(n=8.mean 69Y) and MC(n=5.mean 56Y).40 serial section(5 # m)s

nethed and were stained with Propidius iedide(PI) by Yindelov eethed.

were made from the paraffin block in each case and PkS or Azan stain in

The lyocardial cell cycles were estinated by using these stained nuclei. D CM

regular nethed. 200 u g n i f i e d picture of the nonparallel arrangements of

MC

Con

t r o 1

cardiac euscle c e l l s fro~ theeach slide was traced out on a sheet of tracing paper and then these serial tracing pictures were reconstructed

G 0 - 1 phase %

for throe--dimensional visualization with the Till systeu in 8ATOCsystem

S phase %

engineering eolpany.Cowplieated multiplex ~'ofiber netcork was observed

GZM phase g

really among cardiac lyocytes in HCK Side to side connections among cardiac ~ecytes became loose in MC and end to end connections among

S G 2 M phase %

81.7±3.9

80.6±8.4

7.7+3.2*

9.7-1-2.9.

10.6+3.4

18.3__.3.9

77.8__.4.1 12.6±3. Z

9.7+6.9

9.7±3.4

19.4±8.4

22.3+_.4.1

# p< 0.05 VS control

lean±SD

hypertrophic or atrophic nyocytes were couparatively predoninant in DQL We consider that the fiber disarray depends on the laay-sided cross-

S phase van decreasing significantly in DQd and MC groups compared with

linked l u l t i p l e x network in HCMor the progress of perilyocardial and

control. Polyploid..vwas detected in DCldgroup. We suspect

i n t e r s t i t i a l fibrosis in MC and DCK

v i t a l i t y of ,o'ocytes in DC~ and ~

THE EXPRESSION OF HEAT SHOCK PROTEINS Mo155 60 AND 90 IN PATIENTS WITH DILATED CARDIOMYOPATHY Najma Latif, Michael J. Dunn, Marlene L. Rose, Patricia M.

DOXORUBICIN CARDIOMYOPATHY IS Mo156 N O T M E D I A T E D B Y M O D U L A T I O N S IN PHOSPHOLAMBAN GENE EXPRESSION Mahmood Alam, Mikhail Vaynblat, Mirza M A Baig, Gale Grijalva, M a r i e Chiavarelli, G e e t a Ramdev, Joseph N. C u n n i n g h a m , Richard A Stein, M A Q Siddiqui, SUNY / HSC, Brooklyn, NY USA

Taylor and Magdi H. Yaeoub. National Heart and Lung Institute, Heart Science Centre, Harefield Hospital, Harefield,

Middlesex, UK

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In a study of anti-heart antibodies using SDS-PAGE and Western blotting, a significantly greater frequency of antibodies directed against the 60 and 90 kDa heat shock proteins (hsp)s were detected in the sera of patients with dilated eardiomyopathy (DCM) than in those with ischaemic heart disease (IHD). Circulating antibodies were detected in 44/52 (85%) and 20148 (42%) of patients with DCM and IHD respectively against hsp60, and in 27152 (52%) and 7/48 (14.5%) of patients with DCM and IHD respectively against hsp90. No normal individuals showed reactivity of this specificity. We have now investigated whether the level of expression of hsp60 and hsp90 is upregulated in the rnyocardium of patients with DCM. Pieces of explanted heart from 30 patients with DCM and biopsies from 30 normal heart transplant donors were used to quantitate the expression of these hsp's using SDS-PAGE followed by Western immunoblotting. The blots were probed with monoclonal antibodies specifically reactive with these hsp's and the results were quantitated using laser densitometry. The results showed a 5-fold increase in the expression of hsp60 in the DCM hearts (mean OD 135.26) compared with donors (mean OD 26.51) (0=0.0037), and a 3.5-fold increase in the expression of hspg0 in the DCM hearts (mean OD 27.1) compared with donors (mean OD 7.4) (0=0.0003). The distribution of hsp60 in the myocardium was investigated by immunocytochemistry and results showed increased staining of the myocardiurn, endothelium and connective tissue. Our results have demonstrated not only the production of autoantibodies reactive with hsp60 and hsp90, but also an upregulatinn in the expression of these proteins in the myocardium of patiants with DCM.

that the

i s f a i r l y weaker than the control.

Molecular basis of dilated cardiomyopathy (CMP) remains unknown, so as the doxorubicin (Dxr) CMP. Changes in sarcoplasmic reticulum (SR) dependent Ca++ transport have been implicated. Quantitative changes in mRNA level of Phospholamban (PLB) were investigated by PCR. Intracoronary Dxr (0.25 mg/kg) was given weekly for 4 weeks into the LAD coronary artery of four dogs. Two dogs served as a control. Cardiac function was evaluated by 2 - D echocardiography weekly. Open myocardial biopsies were obtained from anterior left ventricular wall at 16 weeks. RT-PCR assay was performed using PLB specific primers on equal amounts of quantitated eDNA made from total RNA extracted from bioptic tissues. Primers were end labeled with g a m m a 32p-ATP for quantitation. Beta Actin primers were used as positive control. Results are expressed as mean +SD. No difference was found in the m R N A level of PLB in controls compared with Dxr treated left ventricles (1712+218 versus 1791+288). Progressive fall in ejection fraction was documented by echocardiography (from 57+11 to 41+5%). In conclusion, PLB gene transcription did not change in this model of Dxrinduced CMP. Our data supports the recent report of unaltered PLB protein levels in human dilated CMP.

OF DOXORUBICIN ON CARDIOMYOCYTES M0157 FROM ADULT RAT AND GUINEA PIG HEARTS Hans-G. Olbrich, Andreas Rupp, Harald Klepzig, Ernst Mutschler 1 . Abt. f. Kardiologie, Zentrum der Inneren Medizin and 1Pharmakol. Inst. f. Naturwissenschaftler, Universit~it Frankfurt/M, Germany. EFFECT

Doxorubicin (DX) modifies intracellular Ca2 + homeostasis. Therefore, cardiotoxicity of DX may depend on the relative importance of various Ca 2 + pools in regulating myocardial Ca 2 + homeostasis in different species. We examined the effect of DX on cardiac myocytes from rat - Ca2 + homeostasis being predominantly regulated by the sarcoplasmic reticulum (SR) - and guinea pig - Ca 2 + homeostasis being mainly regulated by the sarcolemma (SL). Freshly isolated MYO from hearts of adult Wistar rats or Hartley guinea pigs (n = 6 preparations) were incubated with 350 pM DX and paced by electrical field stimulation (1 Hz). The number of rod shaped MYO - divided in responding ,.d ,,,p,~ ¢,,,, (x). ,,t ,.~ ,,,p,~ cc,,,(x).~o~.,,p:9 (rhythmically contracting) and non responding cells - was 4O evaluated at the be20 ginning and after 15 0 and 30 rain of pacing. tespoP~de,$ (Z). rot ¢esponaecs (~¢}. Qu;neo p;g Survival of rod shaped MYO and number of responding MYO was considerably more reduced in MYO from rats than from guinea pigs (Fig. DX: black, control: white}. In conclusion, impairment of the SR might be of more importance for the cardio.toxicity of DX than its influence of the SL.

IMPLANTATION OF A CARDIAC SUPPORT SYSTEM LEADS TO A LOSS OF THE ANTI-BI-

Mo159

ADRENOCEPTOR AUTOANTIBODIES (AAB) IN PATIENTSwrrH DILATED CARDIOIvIYOPATHY (DCIvl) Gerd Wallukat, Johannes Mfiller*, Hans Peter Luther & Roland I-Ietzer*. Max Delbrtick Centre for Molecular Medicine, Berlin, *German Heart Institute, Berlin, Germany. Recently it was shown that in the sera of patients with DCM and myocarditis AAB were present that might play a role in the development of these diseases. In the present study we investigated the A.AB in DCM patients with an implanted cardiac support system. In 18 patients with endstage DCM (LVEF < 15%) a left ventricular or biventricular assist system was implanted. In 83.3% of the cases we observed a high level of AAB that we previously had found to induce in our bioassay "cultured neonatal rat heart myocytes" a positive ohronotropic effect realized via the Bl-adrenoceptor. The A ~ . were measured in all patients before implantation and once weekly thereafter. Patients without AAB (16.7%) showed no functional improvement over an observations period of 12 weeks while in the other patients the AAB disappeared completely during the postoperative course in parallel to an inproven~nt in all of these patients in LVEF (from mean of 15% to 65%; only to 40% for those with distinct fibrosis). A coneurrant reduction in heart rate was observed in conjunction with the disapperance of the AA.B. This is remarkable as the AAB have a positive ehronotropic effecL Why the AAB disppear remains unexplained. Improved coronary circulation and a reduetiun in afterload of the left ventricle may remove the presence of the antigen necessary for the development and maintenance of autoantibodies.

HYPOTHESIS OF SYMPATHETIC PARTICIPATION Mo158 IN COBALT CARDIOMYOPATHY: EVIDENCE FOR COBALT INDIRECT SYMPATHOMIMETIC ACTION Sergey F.Dugin, Lab. of Experimental Pharmacology, Cardiology Research Center, Moscow, Russia Studies were performed on conscious, chronically catheterized male Wistar rats to investigate the effect of cobalt sulfate infusion (mean rate 0.195+0.011 mg/kg/min) on arterial pressure and heart function. Cobalt administration increased heart rate by 30.4+2.8 beats/min (P
MULTINUCLEATED GIANT CELLS UNDERGOM0160 A PO P T O S IS IN EXPERIMENTAL AUTOIMMUNE MYOCARDITIS

Keisuke Suzuki, Tohru Izumi, Yusuke Ogawa, Takayuki Inomata, and Akira Shibata. First Dept. of Internal Medicine, Niigata University School of Medicine, Niigata, Japan. Giant cell myocarditis is well known a rapidly fatal disease in human. But, characteristics of the giant cell, especially its function and life cycle, have remained unclear. The present study aims to clarify the problems using an animal model of autoimmune myocarditis induced by cardiac myosin fragments. Multinucleated giant cells frequently occurred in the inflammatory loci, being intermingled with a number of EDl-positive inflammatory macrophages. The giant cells reacted with EDI antibody but not with ED2, which indicated no surface antigen of resident macrophages. Fine structures of giant cells closely resembled those of inflammatory macrophages. The giant cells containing less than 5 nuclei were apt to be rich in phagosomes, whereas those with more over nuclei were filled with many lipid droplets but few small phagosomes in the cytoplasm. With hematoxylineosin staining, it was disclosed that some giant cells were presenting apoptotic bodies which corresponded to condensed ehromatin. Such multinueleated giant cells were also positively stained with the TUNEL method. Ultrastructurally, their nuclei showed a condensed chromatin as homogenous dense masses. Consequently, this observation is the first report of apoptosis of giant cells shown in myocarditis. Through these observations, multinucleated giant cells seemed to be formed by fusion of inflammatory macrophages, and die by apoptosis. The phagocytofic activity, which characterizes the inflammatory macrophages, is presumed to be loosing during the maturation process of the cells.

Al17

C A R D I A C D E N D R I T I C C E L L AND Mo161 A U T O I M M U N E M Y O C A R D I T I S IN R A T Tohru Izumi, Keisuke Suzuki, Yusuke Ogawa, Yuji Ohkura, Takayuki Inomata, Akira Shibata. First Department of Internal Medicine, Niigata University School of Medicine, Niigata 951, Japan

PORCINE CARDIAC MYOSIN EPITOPES THAT INDUCE EXPERIMENTAL AUTO{MMUNE MYOCARDITIS

Mo162

Takeyuki Inornata, Haruo Hanawe, Makoto Kodema & Tohru IzumL let Dept Int Med, Niigata Unlv, Nligeta, Japan. An autoimmune mechanism has been found to be responsible for the pathogenesis of myocarditis and dilated cardiomyopathy.

Cardiac dendritic cells must be working as antigen presenters in autoimmune myocarditis. In addition to it, concerning the non-muscle cells, some multipotentialities have been emerged in cardiocytology. Thus, the present purpose was to study on experimental autoimmune myocarditis with a special reference to dendritic cells. Male Lewis rats, which were immunized with cardiac myosin, were sacrificed on day 0, 14, 17, 21 and 40. The samples were investigated ultrastructurally and immunohistologically. Even in the non-immunized state, small numbers of dendritic cell were found among the myofibers. The whole figures were very slender likely perieytes around blood capillaries, which were quite different from those in the activated phase: rich organelles in the cytoplasm and invaginated cell membranes. Obviously, at the initiation, the dendritic cells damaged cardiocytes as targets, invading into the sarcoplasma. At the peak, the dendritic cells were distributed at the inflammatory periphery, probably working as immune-mediators. They were also dispersed among the intact myofibers. In the healing state, the cells seemed to contributed to post-myocarditic reconstruction.

Al18

Our established experimental autoimmune myocarditis is a unique model in which active myocarditis is elicited in Lewis rats by immunization with human, porcine, or rat cardiac myosin. We investigated the localization of myocarditogenic epitopes using porcine cardiac myosin. Subfragment-1 (S-1) and the rod were obtained by c¢-chymotryptic digestion. The rod was further fragmented using cyanogen bromide cleavage.

Three

subfragments of S-1 were prepared by tryptic digestion.

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Lewis rats immunized with the rod exhibited severe myocarditis, and the immunization of the cyanogen bromide cleaved peptide equivalent to human B-cardiac myosin heavy chain RDCB9 (residues 1070-1165) induced moderate myocarditis. Although none of the rats immunized with the whole S-1 exhibited any myocardial lesions, moderate inflammatory cardiac lesions were detectable in rats immunized with the tryptic digests of S-1. Our results indicate that the myocarditogenic epitopes in Lewis rats are located in the RDCB9 (1070-1165) of the cardiac myosinrod, and that a cryptic minor epitope may reside in S-1.

Mo164

Mo 163 IDENTIFICATION OF A VARIED ANTIGENIC MARKER IN RHEUMATIC FEVER / RHEUMATIC HEART DISEASE BY MONOCLONAL ANTIBODY : A FOLLOW UP STUDY Kumar Dinender, Sukhbir Kaur, Harinder Ball 1 and Nirrnal K Ganguly. Dept. of Experimental Medicine and Biotechnology and Cardiology1, Postgraduate Institute, Chandigarh, INDIA.

DIFFERENT ETIOLOGY OF CONGESTIVE CARDIOMYOPATHY IN A DANISH AND AN ESKIMOIC POPULATION.

Various studies have shown the reactivity of B-cell alloantigen of RF/RHD patients with different monoclonal antibodies (MAb's). But these antibodies have shown only 59.1 & 62% RF/RHD patients positive in Indian population as compared to 12-15% of normal controls. An attempt was made to develop a MAb against B-cell alloantigen in Indian RF/RHD patients to see the presence of alloantigen marker tn normal controls (20), RF/RHD (30) and patients with recurrence of Rheumatic Activity (RRA) (20). Further follow up of the same patients after three months was conducted to see whether the expression of marker increases or decreases on the B-cell of patients on penicillin treatment. Three MAb's (PG-DS/12A, PG-DB/13A, PG-DB/20A) have been developed and used for the study. The sample was analysed on LYSYS II software of FacScan. It was found that these antibodies could detect the presence of marker in 100%, 97%, 97% of RHD and 100% (with all antibodies) of RRA and 8.3% of normal controls. After three months follow up % positive patients were 90%, 96.6%, 96.6% of RHD and 95%, 90%, 95% of RRA patients with three different MAb's. Interestingly, the percentage of cells stained were less for RRA after three months follow up (Data not shown). The present study suggests that there might be presence of a varied or additonal genetic marker in RF/RHD patients which plays a major role in complexity of the disease. Another important feature of follow up study has shown that during activated stage of disease B-cell might play a role with a high frequency as compared to Non-activated stage.

An echocardiographic survey has been c o n d u c t e d o n G r e e n l a n d in o r d e r t o s t u d y h e a r t d i s e a s e in t h e arctic. A c o n g e s t i v e c a r d i o m y o p a t h y was d e s c r i b e d with a m a s s i v e r e d u c t i o n in ejection fraction. Values b e l o w t e n w a s n o t u n c o m m o n . In all t h e s e p a t i e n t s toxic s e r u m Mercury levels were f o u n d , the Selenium levels being normal. In an equal s i z e d D a n i s h p o p u l a t i o n o f 35000 p e o p l e (Morso c o m m u n i t y at the w e s t c o a s t in Denmark) congestive cardiomyopathy could likewise be o b s e r v e d , in quite m a n y p a t i e n t s . S e r u m M e r c u r y levels w e r e h o w e v e r always within reference values. C o r o n a r y v a s c u l a r h i s t o r y with typical i s c h e m i c e v e n t s c o u l d be d o c u m e n t e d in all b u t o n e in t h e d a n i s h p o p u l a t i o n in c o n t r a s t t o the eskimoic population, where coronary heart disease is u n k n o w n .

Inger Asmussen Denmark

7900-Nykobing

M

Mo165

INGER ASMUSSEN MD DK-7900 NYKE~BING M DENMARK

ALTERED SARCOPLASMIC RETICULUM Ca 2+- Mo166 ATPase FUNCTION AND EXPRESSION IN DIABETIC BB RAT HEARTS. Philippe Jourdon, Brigitte Berton, Dominique LagadicGossmann, Karine Le Prigent, Anne-Mane Lompr6. ERS 100 CNRS, U 274 INSERM, URA 1131 CNRS, Universit6 Paris Xl, Orsay, France.

Echocardiography has been performed in two equal sized districts (35000people) none of which had had previous service by a cardiologist. Rheumatic heart disease was well represented in both locations. On Greenland valvular heart disease could be documented even in adolescence with slight mitral valve involvement. In young women this would give rize to serious trouble during pregnancy with heart failure. In Denmark the valvular defects were found in the more mature population: 40 years and up, but also here the symtomps should have made doctors refer the patients to a cardiologist years earlier.The major clinical picture was that of cerebral emboli and fainting spels. Cardiologists of today focus on coronary vascular disease and seems to have forgotten classical cardiology as the university centres are localized in well serviced areaes, where diseases of yeasterday are nonexisting..

Most of the experimental studies concerning the effects of diabetes on cardiac function have been carded out in chemically-induced diabetic animal models. Bio-Breeding (BB) rat develops a syndrom similar to human type 1 insulin-dependent diabetes mellitus. We investigated the alterations both of the mechanical activity and of the sarcoplasmic function (SR) in this animal model. In papillary muscles isolated from spontaneously diabetic BB rat hearts the time course of the contractile activity is modified when compared to that of control WB rats: the time to peak force and the time for half relaxation are significantly increased (by 23% and 22%, respectively). These results suggested an abnormal calcium handling in BB rat hearts. The function of the SR Ca2+-ATPase was assessed by measuring the rate of oxalate-stimulated Ca uptake and the level of Ca2+-ATPase mRNA was quantified by slot blot analysis. Our results showed that the rate of the SR Calcium uptake was significantly decreased in BB rats (by 28%) without any significant modification of the apparent Km for calcium, which was associated with a decrease in Ca2+-ATPase mRNA level.

DECREASED TRANSIENT OUTWARD Mo167 POTASSIUM CURRENT IN DIABETIC BB RAT VENTRICULAR MYOCYTES Philippe Jourdon and Danielle Feuvray. ERS 100 CNRS, Physiologie Cellulaire, Universit6 Paris ×1, Orsay, France.

DIABETIC STATE REDUCES ISCHEMIC K* LOSSM0168 AND H* EFFLUX IN ISOLATED RAT HEARTS Ryuko Anzawa, Fumio Tanaka, Fumiko Suzuki, Satoshi Takeda, Shingo Seki, Masayuki Tanignchi, Seibu Mochizuki. Department of Medicine, Aoto Hospital, Jikci University School of Medicine, Tokyo, Japan.

The spontaneously diabetic Bio-Breeding rat (BB) seems to be a suitable model for investigating the factors involved in the development of diabetic cardiomyopathy. BB rats have to be daily treated with insulin such that ketonuria is prevented whereas hyperglycemia is maintained. Ventricular cardiac muscle from such rats exhibit a lengthened action potential (AP) in comparison with AP recorded from cardiac muscle of control WB rats. This occurs without any significant change in the membrane resting potential. Membrane potassium currents (IK) were recorded in enzymatically dissociated ventricular cells by using the whole-cell patch clamp technique. There was no difference in membrane capacitance between WB and BB cells.The amplitude of the outward potassium current was significantly reduced in BB rats. This resulted from a decrease in the transient outward K current (Ito), determined as the 4aminopyridine sensitive component of IK. The voltagedependent properties and the steady-state inactivation of Ito were not significantly modified.

We previously demonstrated in the diabetic rat heart perfused without exogenous subs~ate a mechanism that reduces the loss of potassium and lactate production from the myocardium during ischemia that might reduce its susceptibility to such ischemia and improve functional recovery during reperfusion. Our present objective was to further clarify the relationship between ventdcular function, lactate and proton cfflux, and K" loss during ischemia and reperfusion. Hearts from control and diabetic rats were pcrfused by isolated, working heart preparation with 10rain ischemia followed by 20min reperfusion. Diabetic rat hearts demonstrated an decreased susceptibility to ischemia and an improved functional recovery during reperfusion in the presence of 22raM glucose. Proton production during isehemia was lower in diabetic vs. control hearts. There was no significant difference between groups in lactate efflux at 10-min ischemia. Loss of K* from the diabetic myocardium was significantly suppressed during ischemia vs. controls. Results suggest that an increased susceptibility to ischemia was due, at least in part, to an increased myocardial loss of K*. Reduction in loss of K ÷ in the diabetic heart may be related to the slowed activity in ATP-sensitive K÷ channels, which may result from the decreased rate of inlracellular pH during ischemia in the diabetic heart.

C L A S S I C A L C A R D I O L O G Y IS S T I L L O V E R L O O K E D IN 1995. D A T A F R O M DENMARK AND GREENLAND o

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THE EFFECT OF GLIBENCLAMIDE ON Mo169 PERFUNDED HEALTHY AND STREPTOZOTOCIN DIABETIC RAT HEARTS Krzysztof t.angowski, *Janina Szewczyk, **Joanna Szulc, Btefan Angielski. Dept of Clinical Biochemistry, *Dept of Internal Medicine, **Dept of Cardiology Medical University of Gda~sk, PL. The effect of glibenclamide on the electrical, mechanical function and ischaemic and post ischaemic injury of rat hearts in Langendorff-perfused system was examined. Four groups were studied ( n>6/group ) :-ischaemic and non ischaemic, healthy and streptozotocin diabetic rat hearts.Comparad results from four examined group show that glibenclamide ( in concentration - dependent manner ) decreased coronary outflow ( by 24 and 30% at glibenclamide concentrations 5 and 25 p.M respectlvly; p<0.005 ), increased the release of cell enzymes ( CK,LDH ) and lactate (p<0.01). Glibenclamide increased severity of arrhythmias dudng reperfusion. In case of perfused diabetic rat hearts, there was observed significant decrease in coronary outflow ( by 63 and 93% at glibenclamide concentrations 5 and 25p.M respectivly; t p<0.005 ), increase in cell enzymes and lactate release p<0.01 ) and extension of arrhythmias, as compared to both, 'control and healthy rats. Conclusion: Our results suggest that the blockade of ATPIsensitive potassium channels by glibenclaroide intensify the injury and arrhythmias of rat hearts. These negative effects of glibenclamide were much more profound in streptozotocin diabetes groups.

The @im of, t h i ~ stucLy was t o analyse ne n a e m o o y n a m l c e T T e c t s o f t w o ~OW dai,]y dose sulphgny]urea " cp,mpou.nds ~ g/~oenciam~oe ano {u~meplrloe]n /u metaDoi i cal]y healthy(H) and 62 al loxan-diabetic(D) (al loxan t,etrabyCrate, 51~ p m p l / k g ) , rabbit@. /no 1nT/uence OT S u , / p n o n y / u r e a s in 0.2-2-10 pmol/kg ooses iv, was examined on mean arterial blood pressure,. . cardiac outpu,t an~ myocaroiai Torce, unoer,pentooarbita L anesthesia. ~ilmepirioe i ncreaseo blood pressure (15H ~ b D , t=19.32, p
EFFECT OF HYPOGLYCAEMIC SULPHONYL- MO171 UREA COMPOUNDS ON THE MEMBRANE CHANNELS OF THE HEART r Pog~tsa,Zs6fia M . K o l t a i , E r z ~ d b e t ocsis, Il~iK6 P6sa, matlonai Institute of Cardiology, Budapest,

ROLE OF PARASYMPATHETHIC ACTIVITY MO172 IN THE ARRHYTHMOGENIC EFFECT OF SULPFONYLUREAS ldik6 Pgsa, Maria Z s 6 f i a Koltai, ~oor Pogatsa National Institute of cardiology, Budapest, Hungary

The effects of d i f f e r e n t A T P - s e n s i t i v e p p ~ B s ~ i u m channel b l o c k e r ~ulphonvl~-=~ orugs (0 01-1000 mmoq/l] were ~n% e s t i g a t e ~ 6n'~tro~ha~thin- pnd r~perTUS~On.lnoucQ9 arrny~qmlas ~n r a o D l t s ano ra~s. ~iloenciam~oe tv~m: y=zox+ 339; o<0.01; VF: v=53x+761; D<0.01) a0d g]imepiride L~EB: y = 1 0 x + 5 0 8 ; ~ p<
Lo~ #nd high d a i ] y . ~ o s e hypoglyc@em~c su/pnonyiureas lnr/uenc~ oppositely the arrhythmog~nesis o~ the he~rt~ ~#oce ,to elucloat.~ the oacK~rouna o~ ~O&~ pnenomeno,n, zge acute e f f e c t s OT OiTTerenz s u l p n o n y l u r e a s were compareo on. h e @ r t r a t e and. blood . p r e s s u r e a l z e r a z i o n s inoucee oy zne e i e c t r l c a l s t i m u l a t i oD ( I - 1 6 .~z) o f the vagal nerve wiznou~ a ~ n o r a c o ~ o m y in 42 young, mongrel dogs. T h e br@dycerdiac e f f e c ~ prgvoKeg oy vagai ~8Fimu~a~lqn 8coved t o De ennanceo o ~ i ~ o e n c l a m i o @ tTrom 65% t o 45%,. p
~abo

A120

OPPOSITE CARDIOVASCULAR EFFECTS OF Mo170 GLIBENCLAMIDE AND GLIMEPIRIDE IN HEALHTY AND DIABETIC RABBITS Erzsebet Kocsis, l l d i k 6 P 6 s a , M&ria Z s 6 f i a Koltai" G ~ b o r Pog~tsa National Ins'ti t u t e of Cardiology, Budapest

~

pH i REGULATION IN RELATION TO POST- MO173 ISCHEMIC RECOVERY OF VENTRICULAR FUNCTION IN DIABETIC RAT HEARTS Nassirah Khandoudi, *Monique Bernard, *Patrick Cozzone & Danielle Feuvray. Physiologie Cellulaire, Universit(~ Paris Xl-Orsay; *CRMBM, Marseille, France.

EFFECTS OF pHo AND Ca 2+ ON Na+ / H + MO174 EXCHANGE IN DIABETIC RAT VENTRICULAR MYOCYTES Karine Le Prigent & Dominique Lagadic-Gossmann. Lahoratoire de Physiologie Cellulaire, ERS CNRS 100, Bat. 443, Universit6 Paris-XI, 91405 Orsay cedex, France.

A marked decrease in the activity of the amiloride-sensitive Na+/H + exchanger has been demonstrated in hearts from STZ-induced diabetic rats. The aim of this study was to investigate the contribution of other specific sarcolemmal transport mechanisms to pH i recovery upon reperfusion in STZ-induced diabetic rat hearts and their relation to recovery of ventricular function, lsovolumic rat hearts were submitted to a zero-flow ischemic period of 28 min at 37"C and then reperfused for 28 min. The time course of pH i decline during ischemia and of recovery on reperfusion was followed by means of 31p-labeled NMR. An HCO3-dependent mechanism contributed to pH i recovery after ischemia in the diabetic rat hearts. Even when the Na+/H + exchanger was blocked by amiloride in nominally HCO3free solution, a rapid rise in pH i occurred during the first 3 min of reperfusion. The early rise in pH i was reduced by external lactate and inhibited by o~-cyano-4hydroxycinnamate. This suggested that a coupled H+-lactate efflux may be a major mechanism for acid extrusion in the initial stage of reperfusion. The observation of a higher functional recovery on reperfusion in diabetic hearts is in accordance with previous studies using HCO 3- buffer. However, this study shows that a good recovery of function occurred even more rapidly in diabetic hearts receiving HEPES-buffered solution than in those receiving HCO3buffered solution. This suggests that the HCO3--dependent mechanism of regulation may be depressed in diabetic rat hearts.

We have previously shown that diabetes is associated with a decrease in Na+/H + exchange activity in rat cardiac papillary muscle (Pfliigers Archiv, 1988, 412: 613). The present work has been carried out in order to elucidate the factors responsible for such an alteration. In this aim, we have studied effects of extracellular pH (pHo) and Ca2+ on Na+/H + exchange in ventricular myocytes isolated from streptozotocin-induced diabetic rat hearts, lntracellular pH (PHi) was recorded using carboxy-SNARF-l. The NH4 + (10mM)-prepulse method was used to induce an acid load in order to activate Na+/H + exchange in HEPES-buffered Tyrode. Whereas diabetes did not change intracellular buffering power (13i), it significantly decreased acid efflux through Na+/H + exchange (Jen=4.9l + 0.4, n=32, normal v e r s u s 3.7 + 0.24 mequiv/l/min, n=41, diabetic, at pH i 6.85; P<0.02). Upon decreases of pH o, acid efflux similarly decreased in normal and diabetic cells (by about 80% at pH i 6.85, in the range 8.0-6.8). Whereas Na+/H + exchange was unaffected by Ca2+o-removal, pretreatment of diabetic cells with BAPTA-AM (25/~M; CaZ+-chelator) resulted in a decrease by 54% of acid efflux. Interestingly, this decrease was even more marked in normal cells (by 64%). In conclusion, this study suggests that the diabetesrelated decrease in intracellular calcium may play an important role in altering Na+/H + exchange activity in diabetic ventricular myocytes.

MINI-GLUCAGON POTENTIATESCALCIUM Me175 MOBILIZATION BY GLUCAGON IN HEART CELLS. Catherine Pavoine, Anne Sauvadet and Fran(;oise Pecker. INSERM. Unit~ 99, HSpital Henri Mondor, 94010 Cr~teil, France.

COMPARATIVE STUDY ON INOTROPIC Mo176 RESPONSE OF M Y O C A R D I U M IN IDDM A N D NIDDM Tam&s B&ny~sz Univ. Med. School of Debrecen, Debrecen Hungary

It has been recently shown that the processing of glucagon into its C-terminal (19-29) fragment, mini-glucagon, by the cardiac cells was essential for the contractile positive inotropic effect of the hormone. However, the mechanisms underlyir]g the effects of mini-glucagon remained undetermined. In the present study, we assessed the effects of mini-glucagon on Ca2+ homeostasis in embryonic chick ventricular myocytes. Short term applications of 0.1 nM mini-glucagonmarkedly increasedthe accumulationof [45Ca] into intracellular compartments, resistant to digitor~in lysis. Ca2+ accumulation into the sarcoplasmic store was further attested by the following experimental evidences obtained from Fura-2 imaging studies: mini-glucagon potentiated ryanodine-induced Ca2+ mobilization and evoked a rise in cytosolic [Ca2+] when it was applied on cells pretreated with 1 I~M thapsigargin, the specific inhibitor of the sarcoplasmic Ca2+ pump. Glucagon alone produced a small Ca2+ mobilization into the cytosol which was considerably potentiated by mini-glucagon. This was interpreted as the ability of glucagon to release Ca2+ accumulated into intracellular stores upon mini-glucagon action. The action of glucagonwas mimicked by 8-Bromo-cyclicAMP, and blocked by verapamil, suggesting that it relied on the activation of L-type Ca2+ channels, via phosphorylation.We conclude that the combined actions of mini-glucagon and glucagon on Ca2+ accumulation into sarcoplasmic stores, and Ca2+ release from the same stores mediated by cyclic AMP are likely to support the positive inotropic effect of glucagon on heart contraction.

In spite of the great energy invested into the diabetes research, results are fairly contradictive in respect of contractility or inotropy of the heart. Developed tension of myocardium is reported increased, decreased or even unaltered by different authors. Besides, representation of the t w o main form of diabetes (IDDM and NIDDM) are disproportional in todays research. Significant amount of information have been collected in respect of IDDM, whereas our knowledge is more limited in regard of NIDDM. Considering gaps listed above we felt obliged to take steps to cease discre-pancies and hiatus. Using streptozotocin treated rat as experimental model, we compared the contractility of trabecular muscle from left ventricle in IDDM and NIDDM animals to age matched controls. We applied paired pulse stimulation and catecholamine as inotropy model, to compare the inotropism of the t w o type of diabetes. We found that, intervallum dependency of paired pulse induced inotropy is different in the t w o form of diabetes. Isoproterenol and norepinephrine sensitivity of the IDDM and NIDDM myocardium was found to be decreased, but catecholamine resistance of old control rats must be taken into consideration when drawing conclusions. A121

A L T E R A T I O N IN M I C R O S O M A L Mo177 MEMBRANOUS PHOSPHOLIPIDS AND F U N C T I O N IN D I A B E T I C R A T H E A R T Yoshihisa Arata, Toshikuni Yanagishita, Yumiko Kuwabara, Eiichi Geshi, Noburu Konno, Takashi Katagiri The 3rd Dept. of Internal Medicine, SHOWA University School of Medicine, Tokyo, JAPAN

RESPONSES TO PHOSPHATIDIC ACID Mo178 AND RATE OF PACING IN DIABETIC HEART

Diabetes mellitus causes disorder in glyco, lipid metabolism. Alteration in components of membranous phospholipids may affect on activities in membrane bound emzymes. So we studied the relationship between altered microsomal membrane phospholipids and Ca uptake of sarcoplasmic reticulum (SR) and Na+-K + ATPase activity in sarcolemma (SL), as an indicator of microsomal function in streptozotocin (ST-Z) induced diabetic rats. Light microsomal fraction was obtained by centrifuge method, and phospholipids were extracted by modified method of Folch. phospholipids were separated with two dimensional thin layer chromatography. Phospholipids composing fatty acids were analysed with gas chromatography. Ca uptake of SR were measured with '=s Ca in millipore technic in isolated permeabilized cardiomyocytes. Na+-K + ATPase activity was also measured spectrophotometoricaly and oubain binding sites of SL was assayed with 3H-oubain in millipore technic. Contents of total phospholipids in microsome were more in DM rats from 8 weeks after STZ administration. In each component of phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE) were increased in DM rats. In percentage of fatty acids, C20:4 was decreased significantly. Ca uptake of SR was decreased to 53% of control in diabetic rat. Activity of Na+-K + ATPase was decreased to 86% and oubain binding sites were also decreased in DM rat. These results suggest that alteration of microsomal membranous phospholipids occured in diabetic rat hearl causes the disturbance of function in SR and SL, and leads to diabetic cardiomyopathy.

To investigate the mechanisms of cardiac dysfunction in diabetic rats, the effects of phosphatidic acid (PA) and rate of pacing on the speed of contraction and relaxation (±dP/dt) as well as left ventricular end diastolic pressure (LVEDP) were studied on the isolated rat heart preparation. PA (10"~ 10.5 M) produced concentration-dependent increases of±dP/dt in control groups. However, these responses were attenuated in diabetic rats (P < 0.05, compared to control groups). In another series of experiments, the change of ±dP/dt demonstrated negative staircase pattern when the stimulating frequency was increased. But the drop of these parameters was more steep in diabetic rats. In addition, LVEDP was augmented in parallel with the increases of rate of pacing in diabetic rats while no significant elevation was observed in control groups. These data suggest that in diabetic heart (i) either the level of PA or its regulatory role on intracellular free Ca2' is abnormal, and (ii) the time required for [Ca2]i to associate and disassociate with contractile proteins is prolonged. (Supported by the Canadian Diabetes Association.)

NON-DIABETIC THAN DIABETIC HEARTS Mo179 WITH G L Y B U R I D E A R E M O R E V U L N E R A B L E T O H Y P O P E R F U S I O N WITH N O R E P I N E P H R I N E Makle Hlguchl, Kanako Miyagl, Susumu Kayo* & Matao Sakanashi. Dept of Pharmacol & *Res Lab Center, Sch of Med, Univ of the Ryukyus, Okinawa, Japan.

OXYGEN RADICALS IN DIABETESMo180 INDUCED E N D O T H E L I A L DYSFUNCTION.

The effects of glyburide (a specific K^~ channel inhibitor) on cardiac dysfunctions during hypoperfusion with norepinephrine (NE) were examined in 6-week streptozotocin-induced diabetic and non-diabetic rat hearts. The Langendorff hearts with a balloon in the left ventricle (LV) were used. Glyburide was infused for 15 min before as well as during hypoperfusion (2 ml/mini g heart weight) with NE. The hypoperfusion with NE caused marked elevations in the LV diastolic tension and pressure in diabetic hearts, and only slight elevations in non-diabetic hearts. Glybudde accelerated the starts and the degrees of the elevations in both heart groups. At 30-rain and 60-rain hypoperfusion with NE in non-diabetic and diabetic hearts, respectively, decreases in subendocardial ATP, energy charge, creatine phosphate/inorganic phosphate ratio, phosphorylation potential and glycogen content, and an increase in subendocardial lactate were more marked in the glyburide-treated hearts. The concentration of glyburide required to induce the harmful effects in non-diabetic hearts was about one-tenth of that in diabetic hearts. Levcromakalim (a selective K* channel opener) restored these aggravated dysfunctions to the glybudde-nontreated hypoperfusion with NE levels. It was concluded that K^~ channel blockade accelerated an increase in the LV stiffness and abnormal myocardial energy metabolism during hypoperfusion with NE in both heart groups, and non-diabetic hearts rather than diabetic hearts suffered from the harmful effects.

A122

Yan-Jun Xu, Mike Botsford & Naranjan S. Dhalla. Division of Cardiovascular Sciences, St. Boniface Hospital Research Centre, University of

Manitoba, Winnipeg, Canada.

Galen M. Pieper and Ching-San Lai. Dept. of Transplant Surgery & Biophysics Research Institute, Medical College of Wisconsin, Milwaukee USA. Nitric oxide (NO) is believed to mediate the phenomenon known as endothelium-dependentrelaxation (EDR). In this study, we investigated a possible mechanism for defective EDR in diabetes. NO-mediated EDR was assessed ex vivo in thoracic aortic tings taken from 8-wk streptozotocin-induced diabetic and age-matched control rats. In both control and diabetic rings, relaxation to acetylcholine (but not nitroglycerin, NTG) was completely blocked by removal of the endothelium or by inhibiting NO synthase using 100 laM of either NG-nitroL-arginine or NG-nitro-L-arginine methyl ester. Maximum EDR was also blocked by nearly 50 % using the water soluble, nitric oxide spin-trapping agent, Nmethyl-D-glucaminedithiocarbarnate-Fe 2+. EDR to acetylcholine was impaired while endotheliumindependent relaxation to NTG was unaltered by diabetes. In vitro addition of either 20 U/ml superoxide dismutase or 100 U/ml catalase did not alter EDR to acetylcholine in control or diabetic rings. In contrast, incubation with both scavenger enzymes augmented EDR in diabetic but not control tings. Administration of both scavengers did not alter relaxation to NTG. Thus, enhanced production of both superoxide anion radicals and H202 may limit agonist-stimulated NO activity arising from diabetic endothelium and contribute to endothelial dysfunction.

CARDIO-VASCULAR R E A C T I O N S T O M0181 ACUTE MYOCARDIAL ISCHEMIA IN ALLOXANEDIABETIC DOGS Alexander P. Nescheret, Irene V. Shepelenko, Nadezhda V. Okhrimenko, Irene V. Gonchar & Anastas I. Khomazjuk .Lab. Neurohormonal Control of Circulation, Inst. of Endocrinology & Metabolism, Kiev, Ukraine The investigations of peculiarities of the disturbances of cardiac function and systemic circulation in acute myocardial ischemia(MI) were performed on 36 dogs with alloxane diabetes (D) using catheterization and extracorporal perfusion of coronary artery (CA) without openning the chest. MI was induced by the cessation of left circumflex CA perfusion on 60 s. In D with mild hyperglycemia (7-12 mmol/I) during MI the degree of myocardial contractility reduction and bradycardia, arterial blood pressure (ABP) and peripheral vessels resistance (PVR) lowering in comparison with those in healthy dogs was inconstant, that is augmanted or diminished, but the same reactions to intracoronary epinephrine injection (IEI) were increased. In severe D (hyperglycemia 12-25 mmol/I) myocardial contractility lowering, bradycardia, PVR and ABP reduction were markedly decreased both in the reactions to MI and IEl.ln MI cardiac contractility, ABP and PVR reduced on 81%; 55.5% and 51.3 %, resp., in comparison with those in healthy dogs. The period of halfrestoration of CA resistance in the area of MI during CA reperfusion was more prolonged. It is suggested that in severe D the heart sensitivity to ischemic and catecholamines injuries are decreased.

Mo183 Detection of Insulin Resistance in the Myoeardium of Patients with Coronary Artery Disease by Positron Emission Tomography Giovanni Paternostro, Norma VS Marinho, Eleuterio Ferrannini* and Paolo G Camici. MRC Clinical Sciences Centre, RPMS, Hammersmith Hospital, London, UK and CNR Institute of Clinical Physiology*, Pisa, Italy. Insulin resistance is known to be associatedwith coronaryartery disease (CAD). However,it is not clear whetherthe myocardiumof patients with CAD is resistant to insulin action. We studied 14 male patients, aged 53+8 years, with angiographically proven CAD and chronic regional wall motion abnormalities,assessed by radionuclideventriculography.None of the patientshad diabetesor arterial hypertension.Regional myocardial glucose utilization was measured with positron emission tomography (PET) and 18Ffluorodeoxyglucose (FDG) during euglycemic-hyperinsulinemic clamp. Myocardial glucose utilization was measured in regions with normal wall motion subtended by angiographically normal coronary arteries. Results in patients were compared with those obtained in a group of 7 normal male subjects aged 49-'-~.5years. Fasting plasma glucose was 5.0+-0.9 mM in normal subjects and 6.1+0.9 mM in patients (1)<0.05). During clamp mean plasma glucose was 6.3+1.0 mM in normal subjects and 5.5+0.7 mM in patients (.p=NS). Whole body glucose utilization,calculatedduring clamp, was 6.1+_2.4 mg/(kg min) in normal subjects and 2.7+1.1 mg/(kg rain) in patients (p<0.001). Myocardial glucose utilization was 0.68+0.08 lamol/min/g tissue in the normal subjects and 0.41+_0.13 in patients (p<0.001). In conclusion, myocardial regions with normal wall motion subtended by angiographically normal coronary arteries are insulin resistant in nondiabetic patients with whole body insulin resistanceand CAD elsewhere.

IMPAIRED ENDOTHELIAL DEPENDENT RE-Mot82 LAXATION IN CANINE DIABETIC CORONARIES: DEFECT IN GENERATION OF NO? Zs6fla M. Koltai, E r z s ~ b e t K o e s i s , lldik6 P 6 s a , P e t e r R 6 s e n , Gfibor Pogfitsa. Nat. I n s t . C a r d iol., Budapest, Hungary and Diabetes Res. Institute, Dfisseldorf, Germany. Diabetic c o r o n a r y blood flow is k n o w n not to b e d i f f e r e n t from t h a t o f t h e h e a l t h y . H o w e v e r , i t s s e n s i t i v i t y to v a s o d i l a t o r s is m a r k e d l y dimin i s h e d . To s t u d y t h e m e c h a n i s m s u n d e r l y i n g the impaired endothelial dependent relaxation ( E D R ) , e f f e c t of a e e t h y l e h o l i n e ( A e h 2.25-36 p m o l / k g ) was m e a s u r e d on c o r o n a r y blood flow in 6 h e a l t h y ( C ) a n d 6 d i a b e t i e (D) d o g s . A d d i t i o n a l l y t h e i n f l u e n c e o f Ach ( 0 . l - 1 0 jamol/l) a n d t h a t o f L - a r g i n i n e (A lmmol/l) on t h e r e l e ase o f eGMP b y i s o l a t e d c o r o n a r y r i n g s , as p a r a m e t e r for t h e g e n e r a t i o n o f NO was d e t e r m i n e d . The i n c r e a s e o f c o r o n a r y b l o o d flow d u e to Ach was s i g n i f i c a n t l y (p<0.01) d e p r e s s e d in D. The b a s a l r e l e a s e o f cGMP b y D c o r o n a r i e s was l e s s (1.3+0.3 fmol,]h~g) t h a n b y C o n e s ( 2 . 2 + 0 . 4 f m o l / l ~ g ) _ Ach s t i m u l a t e d t h e r e l e a s e o f eGMP d o s e - d e p e n d e n t l y , h o w e v e r , i t s amount m a x i mally r e l e a s e d was r e d u c e d (p<0.05) in D (D: 1.86+0.2, C : 3 . 5 4 + 0 . 5 f m o l l m g ) . Similarly t h e d o s e - e f f i c a c y curSres w e r e d i f f e r e n t ( C : y = 0 . 0 5 . l o g x + 3 . 2 , r=0.9245 ; D : y = 0 . 0 1 l o g x + l . 69, r=0. 8990) L - a r g i n i n e i n d u c e d cGMP e l e v a t i o n o n l y i n C (179+10%), b u t n o t in a r t e r i e s drom D d o g s . T h u s - r e d u o e d g e n e r a t i o n o f NO c o n t r i b u t e s to t h e i m p a i r e d EDR o b s e r v e d i n D c o r o n a r i e s .

DIABETES AND PRECONDITIONING IN Me184 ISOLATED RAT HEART A. Tosaki & D.K. Das. Univ. Conn. Health Ctr., Farmington, CT, USA. The preconditioning (PC) phenomenon has been extensively studied in healthy myocardium, but such a study has not been previously done in sick hearts. We studied the incidence of reperfusion-induced ventricular fibrillation (VF), and tachycardia (VT), heart function, maldistrihution of tissue Na÷, K÷, Ca2+, and Mg2÷ in isolated hearts from rats with streptozotocin-induced diabetes. Following 2, 4, 6, and 8 weeks of diabetes, hearts were isolated and subjected to 30 min global ischemia (ISA) followed by reperfusinn (RE). In the 2-week diabetic group the incidence of VF and VT were reduced from their non-diabetic age-matched control values of 100% and 100% to 42% (p<0.05) and 42% (p<0.05), respectively. Such a reduction in VF and VT was not observed with progressive diabetes. In the 2-week diabetics, the reduction in VF and V'I" was reflected in the improvement of postisehemic function, the reduction of ISA/RE-induced Na÷ and Ca2÷ gains, and the prevention in K÷ and Mg2÷ loss. This diabetes-induced initial protection was not seen in the 4and 6-week diabetics, and a deterioration of postischemic function was observed in the 8-week diabetics. Four cycles of PC, each consisting of 5 rain ISA followed by I0 min RE, failed to reduce the incidence of VF and VT, improve cardiac function, and prevent ISA/RE-induced ion shifts in diabolics. In the early phase of diabetes the heart is more resistant to ISA/RE than the nondiabetic heart. PC does not afford a protection against a prolonged period of ISA in diabetics, indicating that PC may be a "healthy heart phenomenon".

A123

O X I D A T I V E STRESS ADAPTATION DOES Mo185 NOT INCREASE CELLULAR TOLERANCE TO I S C H E M I A IN DIABETIC RAT HEARTS

Nilanjana Maulik, Arpad Tosaki, Richard M. Engelman, Gerald A. Cordis, James A. Schley and Dipak K. Das, University of Connecticut School of Medicine, Farminglon, CT, USA. Adaptation to oxidative stress has been found to increase cellular tolerance to myocardial ischemia. In this study we examined whether the same oxidative stress adaptation could also protect the hearts of diabetic animals from the ischemic injury. Oxidative stress was applied to both normal and diabetic rats by injecting them with lipid A (0.5 mg/kg) which exerted oxidative stress within l hr of treatment as evidenced by enhanced malonaldehyde formation. The oxidative stress disappeared progressively with time in concert with the appearance of the induction of glutathione (GSH) and antioxidative enzymes that included SOD, catalase, GSH peroxidase and GSH reductase. After 24 hr, the amount of oxidative stress and antioxidant enzyme levels were significantly lower and higher, respectively, in the normal animals compared to those at the baseline levels. Corroborating these results, lipid A provided protection against myocardial ischemic injury as evidenced by significantly improved postischemic recovery of left ventricular functions.In contrast, in the diabetic hearts oxidative stress continued to exist even after 24 hours of lipid A treatment; antioxidant enzyme levels were not stimulated over the baseline levels, and post-ischemic myocardial functions were not improved. The results of our study suggest that while oxidative stress adaptation can enhance the cellular tolerance to ischemia in normal hearts presumably by enhancing intracellular antioxidants, the same adaptive protective mechanism is absent in diabetic hearts. EXPRESSION AND LOCALIZATION OF C-MYC Mo187 PROTEIN IN CARDIOMYOPATHIC HAMSTER HEARTS Naomasa Kawaguchi, Noboru Fujitani*, Hiroshi Kimura*, Akira Wada** & Shunzo Onishi. School of Allied Health Sciences, Osaka Univ., Osaka, *Dept. of Legal Med., Kurume Univ. , Fukuoka, and * * C e n t e r for A d u l t Diseases, Osaka, Osaka, Japan An immunohistochemical investigation was performed to examine the expression of c-Myc protein in the heart of cardiomyopathic UM-X7.1 hamsters(UM). Male UM hamsters at 30 days of age and age- and sexmatched normal BIO-RB hamsters(CT) were used in this study. Immunohistochemical staining was performed on frozen sections by avidin-biotin complex method, using monoclonal antibody against the c-Myc protein. The heart weight and the heart weight to body weight ratio did not differ between UM and CT. Light microscopically, hypertrophy of myocytes or inflammatory cell infiltration were not observed in the heart of UM. In the heart of both hamsters, immunoreactive c-Myc protein was found in the nuclei of myocytes and nonmuscle cells. However, the number of c-Myc-positive cells was markedly increased in the heart of UM as compared with that of CT. These results indicate that increase of c-Myc protein may relate to pathogenesis of the hereditary cardiomyopathy.

A124

EXPRESSION OF B-ADRENERGIC RECEPTORMo186 K I N A S E W A S I N C R E A S E D IN H E A R T S OF C A R D I O M Y O P A T H I C SYRIAN HAMSTERS Ichiro Yoshida, Kazushi Urasawa, Taisei Mlkami, Hideaki Kawaguchi & Akira Kitabatake. Dept of Cardiovascular Medicine, Hokkaido University, Sapporo, Japan. In chronic heart failure, B-adrenegic receptor (BAR) response to its agonist is markedly decreased. Desensitization of Badrenergic receptors is thought to be a mechanism of such reduced responsiveness. Recently an increased expression of B-adrenegic receptor kinase (BARK) was demonstrated in the hearts of CHF patients, suggesting that the enhanced expression ol BARK might be involved in the desensitization of BAR in failing hearts. We have theretore investigated messenger RNA (mRNA) expression of BARK in cardiomyopathic Syrian hamsters (BIO 53.58). Total RNA of hearts were extracted from 20 week-old BIO 53.58, and from age-matched control hamsters, F1B. Total RNA were reverse transcripted into complementary DNA (cDNA) in vitro, then semi quantitative polymerase chain reaction was performed with primers synthesized against rat BARK cDNA. In Hamster hearts, BARK1 was mainly expressed. BARK1 mRNA level in BIO 53.58 was increased more than twofold compared with that in F1B. These results suggest that an altered expression of BARK1 might play an important role even in the BAR desensitiztion in the hearts of cardiomyopathic Syrian hamsters.

EFFECT OF LONG-TERM ACE-INHIBITOR THERAPY IN HYPERTROPHIC CARDIOMYOPATHY (HCM)

Mo188

Andreas Hartmann,AngelaPiitz, ROdigerHopf, Dept. of Cardiology, University Medical Center, and Division of Internal Medicine, KrankenhausSachsenhausen,Frankfurt, Germany ACE-inhibitors (ACI) reduce left ventricular hypertrophy in patients with hypertension. There is experimental evidence that ACI may reduce nonhypertensive left ventricu]ar hypertrophy. It was the goal of this study to investigate the effect of ACI therapy on left ventriculsr dimensions and exercise capacity in patients with HCM. 26 patients (P) with HCM were divided into two groups matched for age, sex and severity of disease. 13 P (group I: 2 f, 11 m, age 59 ± 3.7 years) were treated with Ca-channel or beta-blockers. 13 P (group I1:2 f, 11 m, age 62 ± 2.6 years) were additionally treated with ACI {coptopril 37,5 mg/d, enolapril 5 -10 mg/d). M-mode, 2-D end Doppler echocardiogrephy as well as exercise tests were performed at the beginning (BEGIN) end at the end (END) of the observation period (group 143 ± 3 months, group I1:44 ± 7 months). Septum and left ventriculor posterior well thickness (Sep, PWmm), left atrial and left ventricular end diastolic diameter (LA, Edd- mm), intraventriculor gradient (gra • mmHg), duration (dor - min.) and level (watt) of exercise were documented(Mean ± SEMI.

Groupl i Sep JPW JLA BEGIN 20±1 13±1 46+3 END 18+1 12+1 47+3 Groupll Sap JPW JLA BEGIN 19±2 13±1 45±2 END 17±2 11±1 47±2

]Edd 47+2 47+3 JEdd 50±2 50±2

]gra 32±5 42+12 Jgra 20+6 23±9

Idur 5±1 5±1 Idur 5±1 5±1

I watt 98+9 94+8 Jwatt 96±9 98±9

Conclusion: There were no significant differences in left ventricular

dimensions and exercise capacity between treated and untreated patients. In contrast to hypertensiveleft ventricular hypertrophy, non-hypertensiveHCM is not reducedby ACID

Tuesday 4 July 1995 Abstracts Tu1-Tu182 Excitation-contraction coupling Potassium channels Preconditioning Atherosclerosis, thrombosis

Tul-Tu47 Tu48-Tu70 Tu 71-Tu 162 Tu163-Tu182

A DISCRETE MODEL OF THE BEAT TO Tu001 BEAT C O N T R O L O F MECHANICAL A C r l V I T Y IN C A R D I A C C E L L Jh'i Simurda, Milena Simurdova, Pavd Braven~ & IGeorges Christ~, Dept of Physiology, Faculty of Medicine, Masaryk University, Brno, CZ, and tINSERM U121, Bron, F. A quantitative model has been designed that incorporates negative feedback modulation of sarcolemmal calcium transport (via Ca channels and Na/Ca exchange) by Ca2+ released from intracellular stores during excitation. A signalflow diagram was constructed. Signals are represented by sequences of the amounts of ions (Ca2+, Na+, K+) transported during each cycle between compartments. The Na/Ca exchange current was expressed as INoco = l~oco, + IN,,cQf The component 1Noco., reflects slow changes of activities aca and a/~a and depends on the Na/K exchange pump. 1/~,,cofiS the fast component related to the Ca2+ transient. Each contraction is calculated from a sequence of foregoing amplitudes of contractions and intervals using recurrent formulae. Our aim was to find the simplest formulation with minimum variable parameters necessary to reflect the observed rate-dependent variations of contraction (i.e. mechanical restitution and potentiation, steady state rate dependence, postrest potentiation or decay and alternans). In order to fit the known differences b~ween rat and rabbit rate dependence of contractility, the variables of the model related to sarcoplasmic reticulum (SR) function had to be changed. This finding is in full agreement with structural and functional differences of SR in both species. The model suggests an important role ofl:voc,, s in most of the simulated rate dependent features.

THE EXCITATION-CONTRACTION COUPLING Tu003 IN S T R O N T I U M T R E A T E D RABBIT V E N T R I C U L A R MUSCLE Milan ,~tengl, Pavel PuEelik. Dept. of Physiol., Med. Faculty, Charles Univ., Plzefi, Czech Republic. The purpose of our study was to divide the excitationcontraction coupling with the aid of strontium into particular events. The experiments were performed on the adult rabbit right ventricular papillary muscles. Action potentials (AP) and isometric contractions were simultaneously recorded. Replacing 90 - 97% of the calcium (Ca2.) in the bathing solution with strontium (Sr2") produced prolonged AP accompanied by biphasic contraction. These phenomenons are frequency sensitive, they are emphasized at low frequencies. The first (phasic) component disappeared, when the extracallular solution contained ryanodine. Nifedipine preferentially eliminated the late (tonic) component of the biphasic contraction. If the rest dependent shortening of AP did not occur in the control solution (caused probably by low intensity of It0), the triphasic contraction in Sr" solution appeared. The new, third component of the contraction was incorporated among the first and second components. 4aminopyridine (wall known blocker of I~) leads to emphasizing of the third component. We conclude that the first component is caused by intracellular release of activator cation from the sercoplasmic reticulum; the second component is the result of Sr~÷entry across the sercolemma, probably by way of I=. The third inserted component of the contraction is closely related to I=; it increases with decrease of Ira. Our results suggest that this component is probably caused by the Na/Ca (resp. Na/Sr) exchange working in the reverse mode.

A126

THE G E O M E T R Y OF QUASI SALTATORYTu002 EXCITATION - C O N T R A C T I O N C O U P L I N G Joachim R. Sommer, Teresa High, and Isaiah Taylor. Dept. Pathology, Veterans Administration and Duke University Medical Centers, Durham, NC 27705/10. Activation of contraction in most striated muscle cells proceeds inward along transverse tubules (TT), and is consummated in a one-step calcium release (CR) from JSRs at couplings. For global contraction of bird hearts, absence of TT calls for a different vectorial, transverse, inward travel of the basic calcium-release mechanism initiated at couplings, such as, e.g., in a two-step, quasi saltatory propagation of calcium-induced CR along vicinal CR channels of EJSRs pervading Z-discs. What are the interJSR and inter-EJSR distances of mouse and finch cardiac myocytes? Preliminary results: in contrast to the straightforward, large transverse inter-JSR (i.e.between couplingsmouse) distances (~ 0.5->1.0 I.tm), inter-EJSR (finch) distances as estimated in transverse section planes through tubules of the unique, highly convoluted avian EJSR Z-retes (mesh-width < 0.4 I.tm) are only between << 0.1 (majority) - 0.4 I.tm long, thus, allowing to relegate a unique, if still hypothetical, mechanism for effective global cardiac contraction in the absence of TT to but differences in geometry, not basic properties, between EJSR and mammalian JSR of couplings. By comparison, corbular SR, an infrequent EJSR derivative, especially in Purkyn6 cells, is an atavism. (Support: NIH-HL12486 and VA Res.Service)

FORCE-FREQUENCY RELATIONSHIP Tu004 IN M A M M A L I A N CARDIAC MUSCLES P~ter P. N~n~si, Pdter Szigligeti, AndrOs Varr6", Csaba Pankucsi Dept. of Physiol., Univ. Med. School of Debrecen and Dept. of Pharmacol., SZAOTE Med. Univ., Szeged °, Hungary The effect of action potential duration (APD) and elevated intracellular Na activity was studied on the force-frequency relationship in isolated rabbit, guinea pig and rat papillary muscle preparations. Shortening of guinea pig and rabbit APD ( 1 5 0 - 2 0 0 ms), using the KCATP) channel activator lemakalim (15 pM), to values characteristic of rat (20-40 ms) markedly reduced the force of contraction and converted the positive staircase into negative staircase at lower pacing frequencies. This conversion was greatly enhanced in the presence of acetylstrophanthidin (0.2-1 pM), an inhibitor of the Na-K pump. Prolongation of rat APD w i t h 10 mM 4-AP + 2 mM TEA (inhibitors of K channels) increased contractile force and abolished the negative staircase. It is concluded that both APD and intracellular Na activity are major determinants of the forcefrequency relationship in mammalian myocardium.

IS THE MYOCARDIAL Na+ PUMP Tu005 ACTIVATED BY DIRECT PHOSPHORYLATION? John H. Bothwell, KJeran Clarke and George K. Radda. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. In skeletal muscle increases in the Na + pump activity occur by direct phosphorylation, thus increasing the rate of K + transport into the cell. We have used the K + congener 87Rb to determine whether the same direct phosphorylation occurs in heart, quantifying changes in Na + pump activity by NMR. The [3-adrenergic agonist, isoproterenol, increased the rate of 87Rb uptake in the isolated, perfused rat heart, but failed to do so in a KCIarrested heart in which Ca 2+ channels were closed. 87Rb uptake changed linearly with heart function, as shown below. This implies that direct phosphorylation of the Na + pump did not occur in heart, and that [3-adrcnergic agonists caused an increase in intracellular Ca 2+, which in turn raised intracellular Na + via increased Na+/Ca 2+ exchange. The rise in intracellular Na + then stimulated the Na+ pump activity. 0.8 0.4

,.v= 0.0

oo

0 40 80 Rate Pressure Product (mmHg/min x 10-3)

INTERACTIONS OF THE ENANTIOMERS OF Tu007 THE NA-CHANNEL MODULATOR BDF9148 IN GUINEA-PIG AND HUMAN VENTRICULAR MYOCYTES. Herbert M. Himmel & Ursula Ravens. Dept.of Pharmacology, University of Essen, Essen, Germany. The positive inotropic action of S-BDF9148 (4-[3'(l"-benzhydryl-azetidine-3"-oxy)-2'-hydroxypropoxy]-lH-indole-2-carbonitrile) is due to inhibition of inactivation of Na+-current INa; the enantiomer R-BDF is inactive. A possible interaction of R- and S-BDF on INa was investigated in whole-cell voltage-clamped ventricular myocytes of guineapig (GP) and human (HU) heart (30°C, 20 mM [Na+]o, 20 ms-pulses from -100 to -30 mV, 0.2 Hz). In GP, S-BDF (IlJM, n=8) increased peak-INa from -14+_1 to -18+_1 pA/pF, while R-BDF (3pM, n=9) decreased peak-INa from -15+_1 to -11+_1 pNpF; these effects were partially reversible after prolonged washout. INa-inactivation was described by the sum of 2 exponentials; the fraction of the slow phase (time constant ~ 31 ms) increased from 8% (control) to 74% (S-BDF). S-BDF-induced ( l p M ) current measured after 20 ms was reversibly diminished by R-BDF (3pM)in GP (n=6) and in HtU (n=2). On the other hand, S-BDF (3pM) in the presence of R-BDF ( l p M ) still increases current in both GP (n=5) and HU (n=2) albeit to a smaller extent. Thus interactions of the two enantiomers have to be accounted for when racemic BDF is used for inotropic stimulation.

HUMAN MUSCLE NA,K-ATPase CONCENTRATIONTu006 AND EXERCISE INDUCED PLASMA-K CHANGES EFFECTS OF TRAININGAND DIGITALIZATION Henning Bundgaard, Thomas A. Schmidt, Keld Kjeldsen. The Heart Centre, Rigshospitalet,UniversityofCopenhagen,Denmark Na, K-ATPase mediated skeletal muscle K-uptake counteracts exercise induced K-release. Moderate training reduces in htnnans rnaximttm exercise plasma-K (p-K) from 6.15:0.2 to 5.6_/-0.2 mmol/I (mean__~EM), i.e. 8.2% (p<0.05) without changes in muscle Na, K-ATPase concentation and increases total blood volmr~ by up to 12.3% (p<0.01). If this had been taken into account the peak p-K reduction might had been abolished. Sprint training reduces in htunans maximmn exercise p-K by 19% (p<0.05) corrected for volume changes. This occurs concomitantly with an increase in muscle Na, K-ATPase concentration by 16% (p<0.02). In heart failure patients increases in exercise p-K of 0.5:!:0.1 mmol/1 (p<0.01) and veno-arterial p-K differences by 50% (p<0.05) were observed at equal work rates after digitalization when values were totally corrected for volume redistribution. The results show that changes in functional skeletal muscle Na, K-ATPase concentation, i.e. increase during training or reduction e.g. by inhibition with digoxin affect extrarenal potassium handling during exercise. Corrections for volume redistribution are crucial for the interpretation of p-K fluctuations.

BDF 9198, A NEW Na+-CHANNEL ACTIVATING Tu008 INOTROPIC AGENT WITH HIGHER IN VITRO AND IN VIVO POTENCY AS BDF 9148 AND D P I 201-106 A. Raap, H-J. Mest, B. Armah, W. Grott & W. Blechacz Unlike cAMP dependent positive inotropic agents, sodium channel activators like BDF 9148 (4[-(1-diphenyl-methylazetidine-3-oxy)-2-hydroxy-propoxy]-l-H-2-carbonitril)exert a sustained effectiveness in failing human myocardium (Schwinger et al., J Mol Cell Cardiol, 1991, 23, 460) and hence could stand a real chance to replace digoxin. BDF 9198 (4[3(1-diphenyl-methyl-azetidine-3-oxy)-2-hydroxy-propylamin]1H-indol-2-carbonitril) was nearly 10-100 fold more potent in increasing contractility as BDF 9148 and its diphenyl-methylpiperazinyl analogue DPI 201-106. In guinea-pig papillary muscles the ECS0"s of increasing force of contraction for BDF 9198, BDF 9148 and DPI were 0.01, 0.8 and 1.0 pM, respectively. At these concentrations myocardial cAMP was not elevated. Equieffective concentrations of 0.01 pM BDF 9198 and lpM BDF 9148 and DPI prolonged APDg0 by 51 + 5.01, 42 + 5.7 and 32 + 10.2 ms above baseline values of 188 ± 11.8, 186 + 11.3 and 178.5 + 11.8 ms, respectively. The effects on force and action potential were contributedto the S(-) enantiomer, while the R(+) was without effect. BDF 9198 enhanced dp/dtmax in pithed rats and conscious rabbits with EC50"s of 0.01 and 0.3 mg/kg, respectively, and was10 fold more potent than BDF 9148 and DPI. In conscious cats 30 rag/animal of 9198 p.o. enhanceddp/dtmax 2 h p.a. by 79J:30 % while 100 mg BDF 9148 revealed 58:1:24%at comparable baselines of 3000-3200 mmHg/s. The effect on heart rate was poor or absent in all models. In conclusion, beside a higher potency which was 1-2 orders of magnitude compared with BDF 9148 and DPI, data in cats suggest a batter bioavailibility. Dept. of Pharmacology, Beiersdorr-Lilly GmbH,20253Hamburg

A127

UNIFORM DISTRIBUTION OF Na + AND Ca2+Tu009 CHANNELS IN THE SARCOLEMMA OF FROG CARDIAC MYOCYTES Jonas Jurevicius & Rodoiphe Fischmeister. INSERM CJF 92-11, Univ. Paris-XI, Facuit~ de Pharmacle, 92296 Chhtenay-Malabry, France

FUNCTIONAL UPREGULATION OF DIHYDRO- Tu010 PYRIDINE RECEPTORS IN CARDIAC MUSCLE. Gladys E. Chiappe de Cingolani, Susana M. Mosca and Horacio E. Cingolani. Centro de Investigaciones Cardiovasculares. Facultad de Cs. Mddicas. (1900) La Plata, Argentina.

We used a double-barrelled perfusion of single cells to examine whether Na + and L-type Ca 2+ channels were uniformly distributed on the cell surface. To do this, the cell was sealed at both ends to two patch-clamp pipettes and whole-cell recording condition was established for both electrodes. Na + and Ca2+ currents (INa and Ica) were simultaneously recorded every 8 s at -30 and 0 mV, respectively, from a holding potential of -80 inV. The cell was positioned transversally at the mouth of two adjacent capillaries separated by a thin wall. One part of the cell was superfused with a control solution (S1) while the other part was exposed to a solution ($2) containing 300 nM tetrodotoxin and 1 #M nifedipine, to fully block INa and Ica, respectively. As the cell was progressively moved laterally from S1 to $2, Ica and INa were progressively decreased. The reduction in INa appeared proportional to the reduction in Ic~, suggesting a similar distribution of Na + and Ca2"F-channels on the cell surface. Moreover, the reduction in current amplitude was proportional to the amount of cell length exposed to $2. This suggests that the density of Na + and Ca2+ channels was identical at any place on the cell surface. We conclude that Na + and Ca 2+ channels are uniformly distributed on the sarcolemmal membrane of frog ventricular myocytes.

Experiments were designed to investigate if the increased density of cardiac DHP receptors induced by pretreatment with nifedipine are functional active Ca 2÷ channels. New Zealand White rabbits were treated with 20mg/day nifedipine for 25 days. In isovolumically perfused hearts at constant coronary flow and heart rate, left ventricular developed pressure (LVDP) and its first derivative (dP/dt) were monitored. Basal contractility after isolation of the hearts was higher in nifedipine-pretreated rabbits. Doseresponse to nifedipine showed that the decrease in contractility elicited by this DHP was less in pretreated animals. The depression of contractile performance in response to nifedipine is shifted about 0.5 log units toward higher concentrations (less sensitivity to the drug) when the animals were pretreated with this compound. ED = 1.09 + 0.09 x 10 -7 (control) and 1.55 + 0.17 x 10"~M nifedipine (pretreated) (P < 0.05). Up regulation of DHP receptors in cardiac muscle membranes from nifedipinepretreated rabbits was detected by specific binding at the DHP receptor with [methyl-3H]PN 200-110. Bmax = 269 + 38 (control) and 429 + 46 fmol/mg protein (pretreated) (P < 0.05). The enhanced basal contractility and the lesser sensitivity to nifedipine in hearts from nifedipine-pretreated rabbits are evidences of functional upregulation of L-type Ca 2+ channels.

SINGLE CHANNEL INVESTIGATION Tu011 OF THE RATE-DEPENDENT D~CREASE OF THE CARDIAC L-TYPE Ca 2+ CURRENT. Stephen O. McMorn, Simon M. Harrison, Wei-Jin Zang & Mark R. Boyatt. Dept. of Physiology, University of Leeds, Leeds, UK On increasing the rate of stimulation from 0.5 to 3 Hz, the L-type Caz+ channel current (with Ca 2+, Ba2+or Na + as the charge carrier) declines progressively over -16 s as a result of ultra-slow voltage-dependent inactivation (USI; Boyett et aL, 1994, Pfliigers Archiv 428, 39-50). In the present study, we have investigated whether USI occurs in rat ventricular myocytes at the single channel level in cell attached patches at 37°C. The membrane was clamped from -40 to 0 mV for 200 ms at 0.5 I-Iz and 3 Hz. At 0.5 Hz, single channel currents (110 mM Ba 2+ and II~M Bay K 8644) had a unitary amplitude of 2.4 + 0.06 pA and an NP o of 0.56 + 0.17 (mean + SEM, n=5). In the same patches at 3 Hz, unitary amplitude was 2.4 + 0.09 pA and an NP o of 0.47 + 0.13. These data show that at 3 Hz, NP o does not fall and so cannot explain the reduction in current magnitude seen in whole cell experiments. The reason for this is unclear but may reflect the different conditions employed in whole ceU and cell-attached experiments (e.g. ['Ba2+] or presence of Bay K 8644).

Supported by the BHF and the Wellcome Trust. A128

ELECTROPHYSIOLOGICAL EFFECTS OF TU012 FLECAINIDE IN RABBIT SINOATRIAL NODE Tomoakl Saeki, Yusaku Sakakibara, Kenjl Suzuki, Kazuhiro Yajlma & Takao Fujlnami. 3rd Dept of Medicine, Nagoya City University, Nagoya, Japan. Electrophysiological effects of flecainide on spontaneous action potentials and membrane currents were studied using small preparations (0.2x0.2x0.1mm) of rabbit sinoatrial node. Flecainide (1,3,10pM) progressively decreased action potential amplitude, maximal rate of depolarization and spontaneous firing frequency, and prolonged action potential duration and diastolic interval in a concentration-dependent manner. At 10pM, spontaneous activity ceased in 4 of 7 preparations. In voltage clamp using double microelectrodes (n=5), 10pM flecainide reduced Ca2÷ current (Ica) from -40 to 0 mV (27%), delayed rectifying K÷ current (IK) tail from +10 to -60 mV (59%), and hyperpolarization-activatad inward current at 90 mV (8'/o). 10pM flecainide shifted steady-state inactivation curve for Ica toward negative potentials by 4mV without significant change in slope factor. 10pM flecainide caused marked use-dependent block of Ica in a frequencydependant manner. 10pM fiecainide retarded recovery of Ica from inactivation (time constant=2s) at -40 mV. 10pM fiecainide shifted steady-state activation curve for I K toward negative potentials by 6mY without significant change in slope factor. These results suggest that (1) flecainide exerts negative chronotropic action by decreasing conductance of all membrane currents in rabbit sinoatrial node, (2) the drug has high affinity to inactivated Ca 2÷ channels and activated K÷ channels, and (3) since recovery kinetics of flecainide from Ca2÷ channel inactivation was slow, the drug showed marked use-dependent block of Ica.

ELECTROPHYSIOLOGICAL EFFECTS OF BQ-123 Tu013 ON VENTRICULAR CARDIOMYOCYTES ISOLATED FROM RABBIT MYOCARDIUM Elizabeth Kelso, Barbara McDermott, "Paul Spiers, Bernard Silke, & "Norman Scholfield. Dept. Therapeutics & Pharmacology and "School of Biomedical Sciences, The Queen's Universityof Belfast, Northern Ireland.

P O T E N T I A T I O N O F L - T Y P E Ca Tu01 C H A N N E L C U R R E N T S BY V A S O P R E S S I N IN G U I N E A P I G V E N T R I C U L A R M Y O C Y T E S Masayasu Hiraoka, Shetuan Zheng & Yuji Hirano Dept. Cardiovasc. Dis., Med. Res. Inst., Tokyo Medical and Dental University, Tokyo 113, Japan

BQ-123 is extensively used as a selective antagonist of ETA receptors. In this study, the effect of BQ-123 per se on action potentials (APs) and L-type Ca2+ currents were examined in ventricular cardiomyocytesisolated from adult, male, New Zealand White rabbits (2.5-3Kg). Cells were porfused with a solution of composition (mM): NaCI 137, KCI 5.4, CaCI2 3.0, MgCI2 1.2, HEPES 10, glucose 10 (pH 7.4, 35°C). APs were elicited by the application of depolarizing pulses of 6-12ms duration in current clamp mode using an Axopatch ID patch-clamp amplifier. L-type Ca2+ currents were recorded using a whole cell patch-clamp technique and electrodes filled with (mM): K-gluconate 120. KCI 20, MgCI2 2, HEPES 5, and EGTA 5, GTP 0.1 creatine phosphate 2.5 (pH 7.2). Following stabilization, control readings were made from a holding potential of -40mV, and voltage steps of 120ms duration were applied every Is. Durations of AP at 90% repolarization (APD) and peak Ca2+ current values were expressed as mean±standard error. Data were analysed using the paired t-test. BQ-123 produced an increase (p<0.005) in APD at a concentration of 10"SM (278±49ms) from a control duration of 240q-39ms (n=ll). At 10"6M, BQ-123 increased (p<0.005) the L-type Ca2+ current to -2.76-i-0.3nA from a control peak amplitude of 2.45"0.28nA (n=9). The increases in both APD and L-typo Ca2. current were reversed upon washout (239±41ms and 2.32±0.31 nA, respectively)and were not different from the control values in the absence of BQ-123. These results indicate that BQ123, at the concentration generally used to block vascular responses, exerts a positive effect per se in ventricular cardiomyocytes. The mechanism of action of BQ-123, therefore, is not confined to ET receptor antagonism.

Arginine-vasopressin plays important roles for cardiovascular regulation, but its direct effect on cardiac ion channels has not been clarified. We studied effects of vasopressin on L - t y p e Ca channel currents in guinea pig ventricular myocytes. Unitary Ca channel currents were recorded by the c e l l attached patch configuration with Ba 2÷ as the charge carrier. Bath application of 100 nM vasopressin increased channel open probability (NPo) by a factor of 2.91---1.43 (n=15) due to increased numbers of channel openings and increased open times without changing the single channel conductance. Vasopressin-induced increase in NPo was suppressed by O P C - 2 1 2 6 8 , a blocker of V t receptor, but not by O P C - 3 1 2 6 0 , a V 2 blocker. The potentiation by vasopressin was also suppressed by 100 p.M H7, but not by 1 p.M H89. The results indicate that vasopressin potentiates cardiac L - t y p e Ca channel currents via V 1 receptor activation.

COMPARATIVE EFFECTS OF EHNA, A PDE2Tu015 INHIBITOR, ON Ca CURRENT IN HUMAN ATRIAL AND RAT VENTRICULAR MYOCYTES Mich~le Rivet-Bastide, Agnb.s Benardeau*, St6phane Hatem*, Jean-Jacques Mercadier* & Rodolphe Fischmeister. I N S E R M C J F 92-11, Fac. Pharmacie, Ch~itenay-Malabry, & *CNRS URA 1159, Hop. Marie-Lannelongue, Le Plessis Robinson, France EHNA (erythro-9-[2-hydroxy-3-nonyl]-adenine) is a well-known inhibitor of adenosine deaminase (ADA). Recently EHNA has also been found to be a selective inhibitor of the cardiac cGMP-stimulated phosphodiesterase (PDE2). In frog myocytes, EHNA reversed the inhibitory effect of cGMP on cAMPelevated calcium current (Ic,) but had no effect on basal Ica. Here, we have tested the effect of EHNA on Ica in freshly isolated atrial human myocytes. Extracellular application of 0.1, 1, 10 ~M o f EHNA induced an increase in the amplitude of basal Ica (116 + 30% at 1 #M) without any modification in the current-voltage or inactivation curves. The stimulatory effect of EHNA on ICa was not mimicked by the application of 2-deoxycoformycin (3 tzM), another ADA inhibitor, nor by the addition of adenosine (1 /.tM). Thus, the stimulatory effect of EHNA on Ica was not due to ADA inhibition. In isolated rat ventricular myocytes, EHNA (1-10 ~M) did not modify basal ICa. Our results suggest that PDE2 is involved in the control of basal Ca current amplitude in human atrial myocytes, but not in rat ventricular myocytes. (Supported by Association Franfaise contre les Myopathies, F#ddration Franfaise de Cardiologie, and Glaxo).

S P E C I F I C I N T E R A C T I O N O F F E N D I L I N E Tu016 I S O M E R S & BAY K 8644 ON L - T Y P E Ca z+ CHANNELS. Tripathi, O., Meera, P., & Schreibmayer, W.', Physiology Division, Central Drug Research Institute, Luclmow, India; "Department of Medical Physics & Biophysics, Graz University, Graz, Austria. Mechanism underlying the conversion of agonist property of Bay K 8644 (BK) into antagonistic one by fendiline (F), I2 a Ca2 + channel blocker', was investigated by studying interactions between isomers of F and BK on L-channel current (I_) in guinea pig ventricular myocytes using whole-cell"apateh-clamp technique. Both (+) and (-)F blocked I_a with IC50 of 3.1+1.1 and 10.1+0.9 /.tM respectivet:~. Resting and inactivated channel block and slowed repriming caused this effect. BK failed to overcome block by (+) and (-)F and induced inhibition of I,.. in theft presence as reported for ( + ) F I. 10 #M (-)F, reduced gca from 14+4 pS to 4.2+4 pS but E,,~ of activation was unaltered from -2.9+2.6 mV to -2.7-i:2.8 mV. 1 #M BK in presence of (-)F shifted these values to 2.6+0.7 pS and -10.7+5.1 mV. The E,. offoo was shifted from-19.4+4.7 mV to -29.4+4.5 mV ~y (-)F and to -38.7+5 mV by lp.M BK plus (-)E 7"-Repriming was prolonged from 0.17+0.37s to 2+0.8s by (-)F and to 434.8-1-167.8s by BK plus (-)E Similar effects were seen with (+)E These findings and reports that F competes for DHP binding sites 3 suggest novel sites of action of F isomers and specific cooperativity between them and BK causing reversal of agonistie action of the latter. I. Schreibmayer, W. et al., Br. J. Pharmacol. 106, 151(1992); 2. Tripathi, O. et al., Br. J. Pharmacol., 108+ 865(1993); 3. Glossmann, H. et al., TIPS, 3, 431(1982)

A129

EFFECT OF ANTIDEPRESSANTS ON CATu017 CURRENT IN RAT VENTRICULAR MYOCYTES Igor MinaroviE, Ivan Zahradnik & Alexandra Zahradnfkovd. Inst Molec Physiol & Genetics SAS, Bratislava, SIovak Republic.

MUSCARINIC INHIBITION OF Ca CURRENT IS Tu018 ENTIRELY DUE TO ADENYLYL CYCLASE INHIBITION IN FROG CARDIAC MYOCYTES

Antidepressants display harmful side effects on cardiovascular system. Their interaction with Ltype calcium channels of enzymatically isolated rat ventricular myocytes was studied using patchclamp technique in whole-cell configuration. Cacurrents were recorded on 70 ms voltage pulses applied from holding potential of -50 mV to different potentials or to 0 mV repetitively at 3 Hz. The peak amplitude of Ca-current was found to be decreased in dose dependent manner by clomipramine, amitriptyline, maprotiline, citalopram and dibenzepin with ICso values of 9.7, 22.9, 23.1, 60.7 and 378 (in mmol/I), respectively. Kinetics of Ca-current was not affected. No shift in I-V relationship was observed. Phosphorylation of the channels had no effect on ICso. Potency of antidepressants correlates with their hydrophobicity index. At their therapeutic doses, clomipramine, amitriptyline and maprotiline can significantly decrease Ca-current during action potential. This effect can contribute to cardiotoxicity of structuraly related drugs.

Muscarinic receptor-induced inhibition of adenylyl cyclase (AC) is considered to be a major mechanism in the inhibition of 13-adrenergic stimulated Ca current (Ica) by acetylcholine (ACh). However, ACh has been found to activate a number of other processes, such as phosphodiesterase, phosphatase, and NO-synthase activities, and cGMP synthesis. To examine to which extent these mechanisms participate in the regulation of Ica by ACh, we used a double-barrelled perfusion of single frog ventricular myocytes. Half of the myocyte was superfused with a control solution (SI) while the other part of thecell was exposed to a Ca-free (nominal) solution ($2) containing either 10 t~M isoprenaline (ISO) or 3 0 / z M forskolin (FSK). ISO or FSK induced an increase in Ica of 360 + 80% (mean + s.e.m., n = 7 ) and 534 + 21% (n=4) over basal level. This increase reflected the distant effect of ISO and FSK since the only Ca channels generating a current were those not exposed to the drugs, i.e. in the region of the cell where AC was not stimulated. Addition of 1-3 #M ACh to $2 reduced the stimulatory effect of ISO and FSK to 20 + 4% (n=7) and 66 + 27% (n=4) increase over basal level, respectively. However, addition of up to 10 ~M ACh to S1 had no significant effect on Ica. These results rule out the participation of any additional mechanism besides AC inhibition in the muscarinic inhibition of Ica in frog ventricular cells.

EFFECTS OFDIFFERENTRECEPTOR AGONISTS Tu019 THAT ACCRLRRATEPI HYDROLYSIS ON THE M~4BRANECmtRENT IN RABBIT ~RTRICOLARMYOCYTEB MasaoEndoh, Hideyuki Morita & Junko Kimura.

EFFECTS OF CLONIDINE ON THE SLOW INWARD CALCIUM CURRENT AND TENSION

Dept of Pharmacology, Yamagata, Japan

Yamagata University,

Activation of angiotensin (ANG), endothelin (ET) and alpha-i receptors are coupled to a c c e l e r a t i o n of p h o s p h o i n o s i t i d e (PI) hydrolysis in cardiac muscle. We examined the effects of respective receptor agonists on action potential (AP) and membrane currents in rabbit single ventricular cardiomyocytes to elucidate the role of PI hydrolysis in the regulation. ET-I, ET-3 and ANG II elicited a biphasic alteration of AP duration: a transient shortening and subsequent long-lasting prolongation. ANG II caused a slowly developing activation of CIcurrent, but ET-I did not activate the CIcurrent. ET-I produced likewise a biphasic change, i.e., a transient decrease followed by a long-lasting increase in IC_. ANG II increased Ica but to a less extefi~ than ET. Phenylephrlne (PE + B-blocker) caused a monophasic prolongation of AP duration, did not affect ICa, and had a weak effect on CIcurrent. PE and methoxsmine suppressed IKI, but ANG II transiently activated IKI current. Thus, the modulation of membrane currents induced by agonists that accelerate PI hydrolysis cannot b e interpreted solely by its metabolic products. A130

Rodolphe

Fischmeister

&

Jonas

Jurevicius.

I N S E R M C J F 92-11, Univ. Paris-XI, Facult~ de Pharmacie, 92296 Ch~tenay-Malabry, France

Tu020

OF FROG ATRIAL FIBERS Elena A.Alexandrova & Alexander K.Filippov. Dept of Physiology, Volgograd State University, Russia. Cardiac beta-adrenoceptors of frogs are predominantly beta-2 receptors, while the existence of alpha-2 adrenoceptors in anlphibian heart remains unclear. We have investigated the action of alpha-2 selective agonist clonidine on electrophysiological and mechanical activity of the frog myocardial fibers. Experiments were perfomed on frog atrial trabeculae using the double sucrose-gap technique. Tension was recorded by the optical method. Clonidine (0.l-t0 p.M) dose-dependently decreased the action potential duration, the height of plateau and twitch tension. Corresponding decreases in the peak of tension, rate of tension development, thne to peak tension and rate of relaxation were noted. Under voltage clamp clonidh~e inlfibited the amplitude of slow inward current (Isi) and Isi-dependent phasic tension in a concentrationdependent manner. These effects reached of maxinml level after 3-5 rain exposure to clonidine and were reversible upon washing in normal Rh~ger. In addition, clonidine decreased the time-independent outward current. The alpha-2 antagonist yohimbine (lp.M) did not influence on the inhibitory effects of clonidine. These results indicate that clonidine produces the decrease in transmembrane currents and tension in frog atrium which are not due to action on alpha-2 adrenoceptors. Moreover, these data support to the view that the existence of alpha-adrenoceptors in a frog heart unlykely.

Tu021 EFFECT O F V ~ % A P A M I L C ~ Ca =+ ]2qFLUX AND

Cv'~-Rz~

R~LZCACZCU

CU

CULCUR~

~ HEART c ~ , T -q ~ WTI'H C v ' ~ Yang Ying-Zhen, Guo Qi, Per~Yian-Qing, ChenHaoZtm, Gu Quan-Ba:L & Z t m o - J i a n - X L - ~ . Shanghai I n s t i t u t e o f C a r d i o v a s c u l a r Diseases. S ~ , PRC. The effect of varapamil (Ver) on Ca2. influx across the myocardial plasma membrane and CV~-RNA replication of cultured neonatal rat heart cells infected with CVB3 was investigated. It was found that heart cells infected for 48 h, the rapid Ca2. influx couldn't be inhibited by Ver (i0 pmol/L). But the alow Ca2÷ influx could be done significantly (P<0.01). However, when cultured heart cells treated with Ver and infected.with CV~ at the same time for 72 h, the reflection peak area (RPA) and hybridizationindex (HI) of CVB3-RNAinsupernatant and suspension are significant higher than that infected with control group (P<0.05). These phenomena suggest that in the early stage the increase of Ca2. influx of cultured heart cells could be inh/bited by some calcium antagonist e .g. Ver. On the other hand, Ver might accelerated viral replication in myocardium. Thus, though Ver could be good for decreasing the secondary Ca2÷ damages and improving the myocardial electric activity, it isn't a good choice for therapy in acute stage of virus infection with cardiac symptoms.

DIGOXIN-LIKE SUBSTANCE INVOLVEMENT IN Tu022 Ca++ TRANSMEMBRANE FLOW: A HYPOTHESIS FOR DIGOXlN POSITIVE INOTROPIC ACTIVITY William Vergoni, Anna Iori, Patrizia Zanoni, Vincenzo Franco, Velmore Davoli. Public Health Service, Arcispedale S. Maria Nuova, Reggio Emilia, Italy. Our aim was to focus on Digoxin-Like Immunoreactive Factors (DLIF) biological activity. DLIF is detected with RIA methods for Digoxin in various sick and healthy patients. We found the following evidences: 1.There is a significant inverse correlation between DLIF and Ca++ in newborns (r = -0,40, p<0,0001, N = 158). It is well known that newborn hypocalcemia is not modified by supporting diet with Ca++ and that heart fibre Ca++ increases to normalise fibre function .2. In dialysed patients there is an unusual failure in significant blood Ca++ increase if DLIF is detectable.It must be underlined that Ca++ dose is the same and cannot belost in any way .3.The significant difference in DLIF detectability in CAE channel blocker in treated vs. untreated patients (30•36 vs 5•59, X2 test, p<0,001). Our data could only suggest DLIF involvement in transmembrane cellular Ca++ flow. But given reports showing an inverse correlation between heart function depression and DLIF increase, an important question comes immediately to mind: does DLIF mimic Digoxin or viceversa?The hypothesis that Digoxin positive inotropic activity could be related to interference in transmembrane Ca++ flow, increasing intracellular concentration, is something that will be the subject of further research over the years.

ALTERED [Ca2+]i REGULATION IN BEATING Tu023 CARDIAC MYOCYTE FROM SPONTANEOUSLY HYPERTENSIVE RATS (SHR) Monique Dufilho, Elisabeth Millanvoye, Marianne Freyss, Marie-Aude Devynck. Dept of pharmacology, CHU Necker, Paris, France

INTRACELLULAR SYSTOUC AND DIASTOUC Tu024 [Ca2.] AFTER LENGTH CHANGES IN ISOLATED RAT VENTRICULAR TRABECULAE. Jonathan C. Kentish & Antonl Wrzosek. Dept of Pharmacology, U.M.D.S., St. Thomas' Campus, London 8E1 7EH. U.K.

The genetic hypertension of the Okamoto-Aoki rat is associated with early cardiac hypertrophy. The intracelluiar Ca 2+ concentration ([Ca2+]i) which directly controls cell growth and contractility, was investigated in cardiac myocytes from 3-4 day-old SHR and their normotensive controls (WKY). The ratio of heart weight to body weight was already increased in SHR (p<0.01). Myocytes were separated from fibroblasts by differential Elating and cultured on glass coverslips (40 for each strain). [Ca;'+]i was determined with fura-2. The spontaneous beating frequency was similarly decreased in fura-2 loaded myocytes of the two strains. [Ca2+]i was higher in non beating myocytes from SHR than in those from WKY (p<0.01). In synchronously beating cells, we observed rythmic and transient [Ca2+]i variations, minimal and maximal values o! which were higher in SHR than in WKY (p= 0.02 and 0.004 respectively). Elevation of the external Ca ;'+ concentration to 3.6 mM increased minimal and maximal [Ca2+]i values in WKY only, suppressing the differences between the two strains. In the absence of Ca 2+ influx, the transient [Ca2+]i variations were suppressed and the resting value was higher in SHR than in WKY myocytes (p<0.05). The Ca 2+ channel blocker isradipine dose-dependently decreased [Ca2+]i in SHR only but maximal [Ca2+]i values still remained higher in SHR than in WKY myocytes. These results indicate activation of Ca 2+ influx, alteration of Ca 2+ transport mechanisms in intracellular organelles and enhanced.sensitivity to i~r~dlnine in SHR cardiac myocytes

An Increase In length of cardiac muscle produces an Immediate potentiation of twitch force, followed by a further Increase of force over the next 10 - 20 mln. The delayed Increase In force Is at least partly due to an Increase In the Intracalluiar Ca2+ transient, but its underlying mechanIsm Is unknown. To see whether the Increase In systolic [Ca2+] was the result of a raised diastolic [Ca2+], so enhancing SR Ca2+ loading, we imposed length changes on Isolated rat ventricuiar trabeculae, in which the calls were iontophoresed with fura-2 (K salt), to measure Intracalluiar [Ca2+]. In Tyrodea with 1-3 mM Caz+ (30°C), Increasing length from 90% to 100% of the optimal length Increased delayed force by 49.1 + 6.0% (mean + SE, n=8), and the 340/380 nm fluorescence ratio (oc [Ca2+j) dudng systole by 15.5% (from 1.10 + 0.10 to 1.27 + 0.11, p<0.02). However, there was no significant In diastolic [Ca2+] change (from 0.39 + 0.01 to 0.40 + 0.02), and this remained true even after the SR had been Inhibited with 1 ~ ryanodlne + 30 pM cyciopiazonic acid. These results suggest that the systolic changes In Ca2+ are not secondary to diastolic changes In [CaZ+L but may be due to a mechanIsm that occum during systole. In addition, a comparlscn of the force-[Ca2~ relationships produced by length changes and by other means suggested that all of delayed force response could be explained by the modest Increase In systolic [Ca2'~J: there was no evidence for a change in myofibdllar Ca2+ sensitivity. Supported by The Wellcome Trust A131

CHEMICAL DERIVATION OF RYANODINE SEPATu025 RATES ITS FUNCTIONAL EFFECT TO ACTIVATE T~J~ CALCIUM RELEASE C ~ (CRC) FROM ITS DEACTIVATION.

Henry R. Besch, Jr., Keshore R. Bldasee, Lisa Meritt, William Welch*, John L. Sutko*, and Luc Ruest ^, Dept Pharmacol and Toxicol, Indiana U Sch Med, Indpls, IN, *U Nevada-Rend and ^U Sherbrooke, Quebec, Can. The heptacycllc dlterpene, ryanodlne (Ry), produces dual functional effects on Intracellular CRCs, activating them at low (nM) concentrations and deactivating them at uM concentrations, functionally closing the CRC.s to Ion flux. Certain Cl0-Oeq derivatives of Ry effect a further separation of ECS0aet from ECS0deact values, but because shifts are not proportional to changes In receptor affinity, differences In Intrinsic activity among such derivatives could not be clearly elucidated. Derivation at > one site, however, permits partial deconvolution of contributions of the Involved functional groups to the Intrinsic activity of the molecules. For example, expansion of the C3 substituent fve-membered pyrrole ring on Ry to a slxmembered pyridine moiety as In 3-O-(pyrldyl-3-carbonyl) ryanodol (RPC}, reduces affinity by 150-fold, without changing ECS0aet, but while shifting the ECS0deact an additional 6-fold rightward (ECS0deact = 1500~tM for RPC as compared to 280~tM for Ry). Esterlflcatlon of the secondary OH at the C10 position of RPC with ILalanine or guanldlno-proplonate, returned the affinity of these ryanolds essentially to that of the parent Ry (IC50 = 25nM and 20nM, respectively), but effected little or no increase In their ability to activate the CRC. Like the same CI0 derivatives of Ry and DehyRy, these compounds retain no ability to deactivate the CRC. These data substantiate that although the C3 pyrrole-2-carbonyl substituent is very Important for Its high affinity binding to the CRC, It plays a much lesser role In the channel activating effects of ryanolds. Supported In part by the Show'alter Trust.

PROLONGED EXPOSURE TO HIGH Tu027 A M B I E N T T E M P E R A T U R E A U G M E N T S PRESSURE GENERATION BY T H E HEART. Horowitz M., Novakova MI., Gross H., Meiri U. and Hasin y1. Div. of Physiology and lcardiology, The Hebrew University, Jerusalem, Israel. This study was aimed to follow the dynamics o f augmented pressure generation (P) acquired with heat acclimation (AC) and to unravel the mechanisms responsible for this phenomenon. Isolated hearts and myocytes of rats acclimated for 0 (C), 2, 5 and 30 days (d) at 34oC were studied in parallel. In the isolated heart P was significanfly elevated upon 2d and 30d of heat acclimation whereas 5d AC hearts were similar to C hearts. P augmentation was associated with a better responsiveness to extracellular [Ca2+]. Administration of ryanodine abolished this phenomenon, suggesting the involvement o f the sarcoplasmic reticulum in the phenomenon observed. In AC isolated myocytes (2d, 30d), both Ca +2 transients and A S M (amplitude systolic motion) were more pronounced than in C hearts. Nevertheless, response sensitivity in both C and AC myocytes to extracellular [Ca 2+] was similar. This may suggest that P augmentation in AC hearts is due to alteration in Ca 2+ recruitment and not due to altered Ca 2+myosin sensitivity. ASMs in the presence o f verapamil and restitution curves in the absence and presence of ryanodine indicate that upon initiation o f AC (2d), the sarcolemma also contributes to increased Ca 2+. A132

CALCIUM RELEASE FROM SR IN CONTROL AND Tu026 CARDIOMYOPATHIC HUMAN HEARTS. F. Rannou, Y. Belikova, C. Sainte Beuve and D. Charlemagne. U127 INSERM, Hopital Lariboisi~re, 75010 Paris, France. Abnormal intracellular calcium handling has mainly been associated with an alteration of the gene expression of the sarcoplasmic reticulum (SR) proteins. We have shown that during human heart failure, there is a 2-fold increase in the number of cardiac ryanodine receptor (RyR-2). To determine whether this increase is correlated to an increase in active calcium release channels, we studied calcium release from microsomal preparations of control (n=5) and CM (n= 8) human hearts. The vesicles were passively loaded with 45 Ca2+ washed and total calcium release was elicited by ultra-rapid filtration (25 to 200 msec) with buffer containing 10 IJM free Ca2+ and 5 mM ATP. The passive efflux of calcium was performed under inhibitory conditions (10 IJ_Mof free Ca2+, 10 mM of MgCI2 and 50 laM ruthenium red) is very low (12% after 50 msec and 10 % after 200 msec). The labeled Ca2+ remaining in the vesicles was counted. Loading reached equilibrium after 120 min, and remained stable for .several hours. Active release represented the difference between total and passive efflux and fit to a single exponential + constant curve. We have measured the loading capacity of the vesicles (368+56 and 361+_64nM/mg in C and CM hearts, respectively), the rate of active release (0.074 + 0.008 and 0.06 _+0.007 nmol/ms in C and CM hearts respectively), the total active release (98 +_ 18 and 75 + I0 nmol/mg protein in C and CM) and the number of RyR-2 (178 + 27 and 343 + 31 fmol/mg protein in C and CM hearts, respectively; p<0.001). l-fence, the rate of release of a fully activated channel, and the calcium release per mg protein were similar in C and CM human hearts despite the two-fold increase in the RyR-2 number in CM. The discrepancy between the increased number of RyR-2 and the unchanged calcium release in CM hearts compared to C hearts suggested that the activation procedure of the calcium release channel (by the way of associated proteins such as FKBP 12 fimaily?)is different during heart failure.

CHARACTERIZATION OF THE N A - C A Tu028 EXCHANGE IN HUMAN ATRIAL MYOCYTES A. Btnardeau, S. N. Hatem, L. Mac~, P. Dervanian, J'.J. Mercadier, E. Coraboeuf. University of Paris XI - CNRS URA 1159, Hfpital Marie Lannelongue, Le Plessis Robinson, France

The role the Na-Ca exchange current in the electrical activity of human atrial myocytes was studied in whole-cell patch-clamped ceils. Following the activation of ICa by a short (20 msec) test pulse to +20 mV, membrane repolarization to -80 m V activated a tail current (Itl) of -1.35 + 0.08 pA/pF, (n=21), which was suppressed by substitution of [Na+]o by Li+ and by caffeine, indicating that Itl was essentially generated by the Na-Ca exchange. Two types of action potentials (AP) we observed in human atrial cells: type A characterized by a large initial peak with a narrow early plateau and a late plateau phase of low amplitude, and type B characterized by an initial peak and a prolonged plateau phase. On both AP types, [Na+]o substitution by Li+ induced a marked shortening in the AP duration measured at 0 and -50 mV. Caffeine (5 mM) shortened the late part of the plateau of the two AP types whereas it lengthened the initial plateau phase of type A and reduced the duration of the plateau of type B. There was no evidence for a direct effect of isoproterenol on Na-Ca exchange current. The suppression of the delayed after depolarizations (DAD) upon caffeine exposure and during substitution of [Na+]o by Li indicated that, in human atrial myoeytes, DAD are essentially generated by the Na-Ca exchange. These results indicate that INa-Ca is a prominent current in human atrial cells which participates significantly in the control of the late part of the AP and in the development of triggered activities.

T H E F U N C T I O N I N G AND T H E R O L E O F Ca 2+ E X C H A N G E IN C A R D I O M Y O C I T E S . Kucherenko S.N., Bevza A.V. Kurskii M.D. Institute of Biochemistry, Kiev, Ukraine, 252030.

Tu029

Suspension of vesicles is obtained from plasma membrane (PM) of pig heart's cardiomyocites. Cations transport was registered as radioactive label counting. [Ca2+]i and [H+]i were determined using fluorescent probes. Ca2+/H+ exchanger (CH) is found in PM. It was activated only by transported cations' gradient changing on PM.Kinetic constants and exchanging rates were estimated for exchanging cations. For extracellular Ca2+-binding side Km=3.6+-0.5 raM, Vmax= 12.1-+1.1 nmol/106 cells per 10 see, for intracellular one Kin= 463+59 riM. For extracellular H+-binding side Km=138t54 riM, for inracellular one is 29L-6 riM. Extracellular side expressed negative cooperation effect with KHm -1.5. CH is low affinity, low capacity system.It makes electroneutral, 2H + coupled with 1Ca 2+ transport, and may express Ca 2+ leakage through PM and act as intracellular Ca 2+ feeder despite to Na+/Ca 2+ exchanger. Some antagonists action on CH activity studying showed it may be a structure differed from known Ca 2+- and H+-transporting molecules. CH's Ca 2+- and H+-transporting activities were changed by the same proportion.Na+/Ca 2+ exchanger's Ca2+-ATPase's and CH activities comparison predicted that the last may act as a tool for providing that [Ca2+]i is needed for C-type proteinkinase activation, especially in non-excitable cells.

ABNORMAL Na+/Ca2+ EXCHANGER ACTIVITY IN Tu031 TRANSGENIC MICE WHICH OVEREXPRESS ANNEXIN VI Guojie Song, Gerald W. Dora If, *Ann-Marie E. GunteskiHamblin, Darryl L. Kirkpatrick, *John R. Dedman, Richard A. Walsh. Divisionof Cardiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, U.S.A. We recently demonstrated decreased intracellularcalcium levels and contrgctility in cardiomyocytes of transgenic mice with cardiac specific overexpression of annexin Vl (TG). To investigate the mechanism for this effect, enzymatically disaggregated cardiomyocytes were field stimulated and superfused with incremental concentrations of dichlorobenzamil (DCB), an inhibitor of the Na÷/Ca2÷ exchanger. Cell mechanics were quantitated with video edge detection and calcium kinetics with FURA-2 AM. DCB caused dose-dependent decreases in myocyte shortening and relengthening as compared to controls (C). Results of FURA-2 studies (340/380 nm ratio) include: IDCBI

0 pM

I Idd

5 pld

|0 e.tM

20 Old

Ca 1+ Baseline C

1.202+_.0.113

TG

0.692+-0.021'*

1.108+_0.111 0.885+-0.068++

0.682+-0.030"" 0,638+-0.026°*

O12+ Amplitude C 1.764+-0.233

1.460+-0.109+

TG

1.150+_0,204 0.691+0.102 ++

1.240+-0.173'

0.708+-0.051++ 0.543+--0"048++

0.594+_.0.032

0.852+-0,141"H" 0-393-+0.070++ 0,439+-0.101++

0,541+-0.047+ 0-181+-0'062++ 0,174+-0.030++

C group n~6; TG group, n-6. Values are showed as M+_~E. • P<0.05, " ' P<0.01 competed with control; + P<0.05, ++ P<0.01 compared with 0 JAMDCB

We conclude that (I) in normal mouse eardiomyoeytes the Na+ICa2÷ exchanger promotes cytosolic Ca2÷ influx and (2)

overexpression of annexin VI inhibits the ability of the Na÷/Ca2, exchanger to modulate basal cytosolic calcium levels.

OF 'II-E ~I'I(]N' ~ Oiq ~ CA=* Tu030 I:~M3VAL MB[][-¥,NISM8 IN ~ ~ Mrr~rl~-q Etsu.lm Tanaka & Satoshi Kurihara. Dept of l ~ i o l , qhe Jikei Univ Seh of Mad, Tokyu 105, Japan In ,,~,,A]ian cardiac muscles, three ~ mechanisms are k n m m to r~move intraeellular Ca" from myupla~a: sarcoplasmie reticulum ( ~ ) , Na*-Ca" exchanger (1~) and mitochnndria (Mr). We investigated the effect of the action potential (AP) on the Ca'* rm~val

mechanisms in f e r r e t vontricular muscles. For this purpose, we used aequorin and measured the decay time (I)T) of the light signal. Ca" was released by e l e c t r i c a l st~n,,lation (0.07 Hz) (]~) at 19.'0 and by

rapid cooling (temperature ~ s rapidly lowered from 30 to 12"C) (1~3). qhe following ~nh~bitore v~re used to identify the Ca'* removal mer}mn~n ~hic.h influenced by AP: for SR, t e r t - b u t y l h ~

( T ~ ) ; for b-'X, NiC1, ; for Mr, ruthenium red 0 ~ ) . q]ae DT ~ s _me_A_mared in the presence of one or two inhibitors of Ca'* removal mechanisms. At 12"C, ER markedly prolonged the DT with 1~2. qhe DT with ES vas prolonged by TI~ or NiC1,. In the presence of both TB~ and NiCl, (Ca" was removed by Mr), the ~ with ES a r t 1~2 vms almost the same. 0n the other hand, in the presence of both NiCI, and I~ (Ca'÷ ~ removed by S~), the Dr with E8 was shorter than that with I~. A similar result vms observed in the presence of both T ~ and RR (Ca'* ~ x-~x~ovedby ~ ) . q]aese results ~st that ~ and ]~ more effectively function to relxDve Ca'* vJn~n the AP i s generated and that the

Ca,. removal by Mr i s not influenced by AP.

SERINE 38 PHOSPHORYLATION IN THE Ca '+- Tu032 ATPase STIMULATES Ca z+ PUMPING IN CARDIAC SARCOPLASMIC RETICULUM (SR) Njanoor Narayanan & A n d e Xu. Dept of Physiology, The University of Western Ontario, London, Canada. Our previous studies have demonstrated phosphorylation of the cardiac and slow-twitch muscle isoform (SERCA 2) but not the fast-twitch muscle isoform (SERCA 1) of the SR CaZ÷-ATPase (at S e r ~ by a membrane-associated, CaZ*/ calmodulin-dependent protein kinase (CaM kinase). Analysis of the functional consequence of CaZ+-ATPase phosphorylation in native SR membranes, however, is complicated by the concurrent, CaM kinase mediated phosphorylation of the SR protein phospholamban (PLN) which in turn stimulates Ca z÷ sequestration by the Ca z+ATPase. In the present study, we utilized a PLN monoclonal antibody (PLNab) known to block PLN phosphorylation, to determine the effect of CaZ*-ATPase pbosphorylation on Ca z* pumping in cardiac SR. Treatment of SR with PLNab resulted in an increase in V,~ for Ca 2+ transport as well as decrease in I ~ for Ca z÷. When the PLNab-treated SR was incubated in the presence of Ca z÷ and calmodulin, the CaZ+-ATPase but not PLN" underwent phosphorylation by the endogenous CaM kinase. The phosphorylation of Ca2*-ATPasc was accompanied by further increase in V ~ for Ca z+ transport with no further change in K~ for Ca z+. Thus, direct phosphorylation of the CaZ÷-ATPase results in stimulation of Ca z÷ pumping in cardiac SR. (Supported by MRC of Canada and H.S.F.O.) A133

;.; ; LC/'S OFTHAPSIGARGINON CONTRACTIONIN Tu033 GUINEA,PIG VENI~CULAR MYOCYTES. S.M. Bryant and G. Hart. Deptarlrnant of Cardiovascular Medicine,U ~ of Oxford,Oxfa'd, OX3 9DU. UK.

Cell shonatag of gtinea-~ myoctas is p~narily depandant on calcium re/easedfrom the SR but, ~le is known about other possible sources of calcium. We have looked at the effect of inhibiting SR function on contraction of single gtinea-pig myocytes. Thapsigarg~ fiG) (lpM) aboftshed caffeine-induced c~aract3xe and reduced contraction associatedwith an action potential by 52% (-16.4+1 to -8.1 + 1/xn, p<0.001, n = l 1). -time to peak shortering was prokr~ed (185 + 9 to 279 + 13rns p<0.001) and mean rate of rise of corYaactionwas decreased (91±6 to 29±3/.~3.s"l p
THAPSIGARGIN INHIBITS EFFECT OF NATu034 AND HIGH [Ca2*]o ON KINETICS BUT NOT ON AMPLITUDE OF CARDIAC CONTRACTION. Robert Janiak and Bohdan Lewartowski. Department of Clinical Physiology, Medical Center of Postgraduate Education, Warsaw, Poland. We investigated influence of thapsigargin (TG), a selective blocker of the Ca2'-ATPase of the sarcoplasmic reticulum (SR) on the effects of noradrenaline (NA) and 5.0mM ICa2'l, in the single myocytes of guinea- pig heart. The fluorescence of Indo-1 AM and cell shortening were measured. In control part of experiment both NA (I(YglVl) and high ICa='lo increased the rate of rise and the amplitude of Ca2'transients. The amplitude of shortening, and negative and positive dL/dt were also greatly increased. 2x10"7 M TG did not affect the amplitudes of Ca2' transients and shortening but greatly decreased the rate of their rise and negative dL/dt. In the cells pretreated with TG, NA or high [Ca2'lo increased the amplitudes of Ca2'transients and contractions like in the normal cells but they did not alter their kinetics. These results support our hypothesis (Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H 1829-H1839. 1994) that the main function of the SR in guinea- pig cardiomyocytes is control of the kinetics of contraction.

charr~ and reversedNa÷/Ca÷e x p e r t .

AGING AND SARCOPLASMIC RETICULUM Tu035 FUNCTION IN GUINEA-PIG CARDIOMYOCYTES Kerry Davia and Sian E. Harding, National Heart and Lung Institute, Dovehouse St., London SW3 6LY, UK. Aging may be a contributory factor in the development of heart failure. We have investigated whether changes in SR function were age related. Left ventricular myocytes were enzymatically isolated from young (Y, <4 weeks), mature (M, 4-8 weeks) and senescent (S, >lyr) guinea-pigs. Myocytes were field stimulated at 0.5 Hz, 32°C in Krebs Henseleit solution (4-6mM Ca2+). There was a significant difference in time to 50% relaxation (s) between the three groups (0.13 ± 0.01, n=10, Y; 0.12 + 0.01, n=50, M; 0.17 ± 0.01, n=15, S; P<0.01 ANOVA). Pairwise comparison showed significant differences between M and S only. Addition of II~M thapsigargin prolonged time-to peak contraction in myocytes from Y (0.20 ± 0.01 n=7 to 0.28 ± 0.01, P<0.01) and M (0.22 ± 0.01 to 0.25 ± 0.01, n=13, P<0.05). No significant effect of thapsigargin was noted with myocytes from S. To investigate SR function further we compared the amplitude of the first beat following 3 mins rest (B 1) to that of the steady state contraction (SS). Myocytes from M showed more pronounced decay in BI/SS than S (57 ± 4%, M, n=38 vs 74 ± 8%, S, n=15, P<0.05). Thapsigargin reduced the post-rest decay in myocytes from M (43 ± 7% vs 60 ± 5%, n=9, P<0.02) but had little effect on S. In conclusion, the slowed relaxation, reduced response to thapsigargin and flattened post rest beats seen in human heart failure also occur in senescent guinea-pigs. A134

PHOSPHOLAMBANGENEDOSAGEEFFECTSIN THE Tu036 MURINEHEART. Wusheng Luo*, Beata M. Wolskat Ingrid L. Grupp*, Jay Slack, Judy M. Harrer, Kobra Hagh=ghl , R. John Solarot and Evangelia G. Kranias* *Univ. Cincinnati College of Medicine, Cincinnati, OH and tUniv. Illinois, Chicago, IL *

t

i

.

.t

Phospholamban (PLB) is a regulatory phosphoprotein in the heart. The PLB gene was targeted in embryonic stem cells and mutant mice heterozygous (+/-) and homozygous (-/-) for the targeted allele were generated. The -/- mice expressed no PLB, while the +/- mice expressed reduced PLB mRNA and protein (40+5%) levels compared to wild-type mice (+/+). The reduction in PLB mRNA levels was not associated with any alterations in either the number of the PLB transcripts or their relative abundance in +/- hearts compared to +/+ hearts (45% for the 2.8 kb, 8% for the 1.5 kb and 47% for the 0.7 kb mRNA). To assess the PLB gene dosage effects, hearts from +/+, +/- and -/mice were studied in parallel at the subcellular, cellular and intact organ levels. There was a linear correlation between the relative levels of PLB in the 3 groups and the: a) affinity of the Ca2+-ATPase for Ca2* in SR; b) rates of relaxation in isolated cardiomyocytes, and c) rates of relaxation in intact-beating hearts. These findings indicate that PLB is a major regulator of myocyte mechanical function and cardiac contractile parameters through its regulatory effects on the SR Ca2*-ATPase activity in the mammalian heart.

REGULATION OF P H O S P H O L A M B A N (PLB) E X P R E S S I O N IN INTACT NEONATAL RAT HEART CELLS

Tu037

Sabine Bartel, Michael Bader, Birgitt Stein*, Stefanie Gsell* Peter Karczewski, Joachim Neumann*, Hasso Scholz*, Ernst-Georg Krause Max Delbrtick Centre for Molecular Medicine, Berlin; *Abt. Allg. Pharmakologie, Universit~itsKrankenhaus E p p e n d o r f , Hamburg, F R G Phospholamban is the regulator of the Ca2+-ATPase in cardiac sarcoplasmic reticulum, and it has been suggested to be an important determinant in the inotropic and lusitropic responses o f the heart to 13adrenergic stimulation. In the present studies we demonstrate that long term exposure (2 days) of neonatal rat heart cells (HC) to the cAMP-elevating agents isoproterenol (I, 0.1-10 laM), forskolin (0.1-10 laM) or 8Br-cAMP (100 laM) induced a concentration dependent reduction in PLB content detected by quantitative immunoblot analysis. Propranolol (0.5 taM) attenuated the I-induced reduction in PLB content. Conclusion: Chronic stimulation o f intact HC by c A M P elevating agents reduced the PLB content and might therefore induce relevant alterations in contractile activity.

THE CARDIAC PHOSPHOLAMBAN KINASE Tu038 IS A CAM KINASE TYPE I1 DINSTINCT ISOFORM. Leonidas G. Bahas, Peter Karczewski & Ernst-G. Krause. Max Delbrtick Centre for Molecular Medicine, Berlin, Germany Pho~pholamban (PLB) the regulator of the Ca2*-ATPasein cardiac sarenplasmic reticulum (SR) is the key phosphoprotein in mediating the heart's contractile responses to 8-adrenergic agonists. PLB is phosphorylated by an endogenous SR Ca2*/CaM-dependentprotein kinase (PLB-kinaso) which we .set out to characterize in the present study. In isolated cardiac SR vesicles the specific inhibitor of the C;dvl kinase I], peptide CamK [I-(289-302), inhibited strongly the phe~phorylation of PLB (IC5o 71aM). In addition Zn2., which is known to inhibit the brain CaM kinase I1, and the general inhibitor of the CaM kinase family, KN-62, both inhibited the Ca2*/Calmodulindependent phosphory'lation of PLB. Following fractionation of cardiac SR proteins by SDS-PAGE and in situ renaturation a doublet of ~ 54 kDa was detected which was also recognized by a mAb specific for the ct- subunit of the brain CaM kinase II. An important feature of the PLB-kinase is the regulation of its activity through autophosphorylation. Initial rapid autophosphorylation converts a completely Ca~'*/CaM (conu'ol value 100%) dependent enzyme to a partially Ca2*/CaM independent (26% of control). At this state of incomplete autopho~'phorylationthe kinase is still partially sensitive to Ca"*/CaMand in its presence the majority of maximal activity can be obtained (87%). If Ca2* is removed with EGTA autophosphorylation continues and results in an enzyme which is in~nsifive to Ca2*/CaM (20%). Dephosphorylation by protein Phosphatase 2A converts the kinase to Ca2*/CalVl~nsitive and the majority of the maximal activity is regained (55%). Based on the above functional and structural similarities,with the CaM kinase U, we propose that the cardiac SR PLB-kinase is a dinstinct isoform of the CaM kinase [I subclass, with the potential to become the target of inotropic modulators.

DIFFERENTIALTENSIONDEVELOPMENTIS ASSOCIATED Tu039 WITH THE RELATIVEABUNDANCEOF PHOSPHOLAMBAN AND SERCA2 mltNAs IN MURINEATRIUMAND VENTRICLE Kimberly L. Koss, Sathivel Ponniah, Ingrid L. Grupp, and Evangelia G. Kranias Univ. Cincinnati College of Medicine; Cincinnati, OH 45267

INCREASING [Ca] oENHANCES Tu040 PHOSPHOLAMBAN PHOSPHORYLATION AND MYOCARDIAL RELAXATION WITHOUT CHANGES IN cAMP. Leticia Vittone, Cecilia Mundifia-Weflenmann, Gladys Chiappe de Cingolani, Alieia Mattiazzi. Centre de Investigaeiones Cardiovasculares, 1900 La Plata, Argentina.

Phospholamban (PLB), the phosphoprotein regulator of the cardiac sarcoplasmic reticulum (CSR) Ca2+ATPase, is functionally important in modulating cardiac contraction and relaxation. Transcriptional analyses of PLB relative to o~-myosinheavy chain (ct-MHC) revealed a 3-fold higher PLB mRNA abundance in the ventricle compared to the atrium. However, the relative mRNA level of CSR Ca2+-ATPase (SERCA2/ct-MHC) in the ventricle was 80% of that in the atrium. Consequently, the relative ratio of PLB/SERCA2 was 4.2fold lower in atrium than in ventricle. This lower atrial transcript ratio of PLB/SERCA2 was associated with significantly shortened times to peak-tension (TPT) for atrium (27.36+0.82 msec) compared to ventricle (44.60+2.55 msec), assessed in superfused cardiac tissue preparations recorded at maximum lengthtension. The atrial muscles also exhibited significantly shortened times to half-relaxation of tension (RT½) (17.40+0.71 msec) compared to ventricular musc~s (30.58_+2.04 msec). ~adrenergic stimulation was associated with further shortening of TPT and RT½ in both atrial and ventricular muscles. Therefore, the relative abundances of PLB/~-MHC and SERCA2/¢MHC may be important determinants of the contractile parameters in atrial and ventricular muscles.

Increases in [Ca]o (f[Ca]o) enhanced phospholamban (PHL) phosphorylation only when cAMP is elevated. In preliminary experiments we found that f[Ca] o also enhance PHL phosphorylation in the presence of the phosphatase inhibitor okadaic acid (OA). The present experiments performed in rat hearts perfused with a2p~, studied whether this enhanced PHL phosphorylation occurs without changes in cAMP and correlates with a positive lusitropic effect. Sarcoplasmic reticulum (SR) was isolated and PHL was analyzed by SDS-PAGE, autoradiography and liquid scintillation counting. 1'[Ca]o from 1.35 to 3.85 mM increased maximal rate of contraction (+i-) from 129 + 17 to 218 + 20 g/sec (n=12, P<0.05) without changes in either relaxation or PHL phosphorylation. With 0.1 #M OA the same increase in [Ca]o produced a similar inotropic effect and an increase in PHL phosphorylation from 49 +_6 to 83 + 14 pmol a2p/mg SR prot (n=8, P<0.05) which was associated with a positive lusitropic effect (decrease in the ratio between +l" and the maximal rate of relaxation (-1") from 1.47 + 0.04 to 1.31 + 0.03 (n=12, P<0.05)). Neither the f[Ca] o nor OA altered cAMP levels. It is suggested: 1) f[Ca] o in the presence of OA produced a Ca-calmodulin-dependent (CaCM) PHL phosphorylation and 2) inhibition of phosphatases is necessary to unmask the Ca-CM PHL phosphorylation and its role on myocardial relaxation.

A135

EFFECTS OF ACIDOSIS ON PHOSPHORYLATION Tu041 OF PHOSPHOLAMBAN IN RAT CARDIAC MUSCLE Cecilia Mundifia-Weilenmann, Leticia Vittone, Horacio E. Cingolani and Clive Orchard*. Centro de Investigaciones Cardiovasculares, 1900 La Plata, Argentina and *Dept. of Physiology, University of Leeds, Leeds LS2 9NQ, U.K.

EFFECTS OF LINOLEIC ACID HYDROPEROXIDE ON CARDIAC CONTRACTILITY AND MEMBRANE ION TRANSPORT

Since acidosis inhibits the sarcoplasmic reticulum (SR) Ca2÷-ATPase of cardiac muscle we have investigated whether this acid-base changes alters phospholamban (PHL) phosphorylation. Isolated rat hearts were per'fused with Ringer solution (pH 7.4) containing 32pi for 30 rain and then for 3 min with Ringer solution either at pH 7.4 or 6.8 in the absence and presence of 3 x 10-8M isoproterenol (ISO). SR membranes were isolated by differential centrifugation and PHL was separated using SDS-PAGE. In the absence of ISO, acidosis had no effect on PHL phosphorylation. In the presence of ISO, acidosis significantly increased PHL phosphorylation from 136.5 + 14.3 to 210.1 + 28.7 pmol 32p/mg prot SR (n=9, P<0.05) and decreased time to half relaxation from 43 + 2 to 36 + 1 msec (n=9, P<0.05). Acidosis did not change basal or ISO-induced cAMP levels. The activity of the SR-associated PHL phosphatase (PP1), measured in membranes isolated from control hearts decreased by 56.5 + 12.4 % (n=4, P<0.05) when pH decreased from 7.3 to 6.5. We conclude that: 1) the inhibition of the Ca2*-ATPase caused by acidosis is not mediated by dephosphorylation of phospholamban and 2) the increase in PHL phosphorylation produced by acidosis in the presence of ISO could be explained by the inhibition of PP1.

The aim of the present study was to investigate the effects of llpoxygenase derivatives of llnolelc acid (LA) on cardiac contractility and ion transport mechanisms In cardiac membranes. 13(S)-Hydroperoxlde of LA was synthesized using immobilized soybean llpoxygenase. Perfuslon of isolated Langendorff heart wlth 20 ~M of HPODE decreased systolic pressure, dp/dtmax, dp/dtmln and increased diastolic pressure. Coronary vessel resistance was not changed significantly. Using highly purified isolated sarcolemma we have found that HPODE at the concentration above 10 ~uM enhanced passive 45Ca permeability. The compound (0.I-I0 ~M) also stimulated Na-Ca exchange activity in a concentratlon-dependent manner. Sarcolemmal Na,K-ATPase was inhibited by HPODE with IC50 of 18 pM and complete loss in activity was achieved at 100 p/~. 13(S)-Hydroxlde, a reduced form of HPODE, was slgnlflcantly less potent In activation of Na-Ca exchange and inhibition of Na,K-ATPase. These data imply that HPODE can be an effective modulator of cardiac contractility, affecting basic properties of plasma membrane, and that hydroperoxy functional group is a prerequisite.

LYSOPHOSPHATIDYLCHOLINE ACTIVATES Tu043 NON-SELECTIVE CATION CURRENT IN GUINEA PIG VENTRICULAR MYOCYTES

POSITIVE INOTROPIC EFFECT OF THYROTROPIN-RELEASlNG HORMONE ON ISOLATED RAT HEARTS Robin Socci, Ralph C. Kolbeck & I.~szl6 G. M6szAros, Medical College of Georgia, Dept. of Physiology and Endocrinology, Augusta, GA, USA

Katsuakl Maglshl, Yasushl Ablko. D e p t of Pharmacology, Asahlkawa Medical College, Japan.

Effects of lysophosphatidylcholine (LPC) on nonselective currents (NSC) were studied by whole-cell patch clamp technique with ramp pulse in isolated guinea pig ventricular myocytes using normal Tyrode solution (mM) containing Na+ 143, K÷ 5.4, Ca 2. 1, Mg 2. 1 or 5 and Ba 2. 2. The I-V relationship exhibited a straight line, having the slope conductance of 0.87 ± 0.10 nS. LPC (10 pM) increased NSC and also increased the slope conductance greatly without changing the straight line relationship. Even when the external solution was changed to a NaCI (150 mM) solution, a CaCI2 (90 mM) solution, or an N-methyi-Dglucamin (140 mM) solution, LPC increased the NSC. The increase in NSC induced by LPC was still observed after the treatment with lmM 4,4'dinitrostilbene-2,2'-disulfonic acid disodium salt (DNDS: a CI" channel blocker), and even after the extra and intra cellular CI" concentration was reduced to 6.7 mM and 4.0 mM, respectively. These findings suggest that LPC increases influx of cation but not CI-, leading to a decrease of the membrane potential, which may produce severe arrythmia in the heart.

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Tu042

Gennady A.Cherednichenko, Ludmlla I.Kvochlna, A l e x e l A.Motbenko, Sergey A.Ohly. Institute of Physiology, Klev, Ukraine.

The effect of thyrotropin releasing hormone (TRH) on the contractility of electrically stimulated (at 240-280 BPM) and perfused Isolated rat heads were investigated. TRH at up to 1 pmol/L concentrations was found to enhance the rate of both cardiac

contraction and relaxation, which effect of the hormone culminated in a 25-30% increase of peak contractile force. At higher concentrations, the positive inotrepic effect of TRH, although still significant, was reduced, which was apparently associated with a TRH-induced decrease of cardiac relaxation rate, a secondary effect of the hormone. Since TRH is expressed in the heart, the above findings suggest that TRH

i :]lllllill o

OT 10

/

20

30

Time (rain)

TRH

Fig. t. Effect of TRH on cardiac

contractileforce

might play a role in a direct autocdne modulation of cardiac contractility, in addition to its indirect role through the central nervous system.

Tu044

MECHANISM OF THE POSITIVE INOTROPICTu045 EFFECTS OF THE ACTIVE METABOLITE OF PIMOBENDAN ON FERRET MYOCARDIUM Kimiaki Komukai, Makoto Kawai & Satoshi Kurihara Dept of Physiology, The Jikei University School of Medicine, Tokyo 105, JAPAN The phosphodiesterase inhibitor, pimobendan, shows an inotropic effect. However, orally-administered pimobendan is changed to UD-CG 212 by hepatic demethylation. We investigated the effects of UD-CG 212 on intracellular Ca2÷ transients and contraction in aequorin-injected ferret papillary muscles and on pCa-tension relation in ferret skinned trabeeulae. With an extracellular Caz+ concentration ([Ca2*]o) of 2 mM, UD-CG 212 increased the peak of Ca2+ transients without a significant increase in tension. Developed tension reached 40-50 mN/mm2 before application of UD-CG 212, suggesting that tension is near maximal level of the pCatension relation. With a [Ca2+]o of 0.5 raM, UD-CG 212 (10-s-10-4 M) potentiated the contraction accompanying an increase in the peak of Ca2+ transients. When UD-CG 212 concentration was increased from 10.7 M to 10.6 M, the increase in tension was larger than that in Ca2÷ transients. We treated the preparation with ryanodine and repetitively stimulated the preparation to produce tetanic contraction. Then, we measured the pCa-tension relation at a steady state in intact papillary muscle. However, UD-CG 212 (10.6 M) did not alter the pCa-tension relation. UD-CG 212 did not increase the Caz+ sensitivity of Triton-X-treated skinned trabeeulae. We conclude that UD-CG 212 potentiates contraction via an increase in Ca2+ transients.

SIGMA RECEPTOR LIGANDS MODULATE Tu046 CONTRACTILITY IN CARDIAC MYOCYTES FROM NEONATAL AND ADULT RATS. *Catherine Ela, ^Marie Novakova, ^Shmuel Raz, Ojacob Barg, °Zvi Vogel, ^Yonathan Hasin & *Yael Eilam. *Hebrew University-Hadassah Medical School, Jerusalem; ^Hadassah Hospital, Jerusalem; °Weizman Institute, Rehovot. israel.

Sigma receptors are present in mammalian brain and also in immune, endocrine and other peripheral tissues. A diverse array of functions have been suggested for o receptors but their exact physiological role is not yet elucidated. We have characterized specific binding sites for t~ ligands on cardiac myocytes from neonatal and adult rats. Exposure to nanomolar concentrations of prototypic o ligands such as (+)-pentazocine or haloperidol, induced marked effects on contractility, [Ca2+]i-transients , Ca 2+ fluxes and beating rates. The pattern of response to o ligands of cardiomyocytes from adult rats was different from that observed previously in cardiomyocytes from neonatal rats (Ela et a/., J. Pharmacol. Exp. Therap. 269:1300-1309, 1994). In adu t rat cardiomyocytes o ligands induced marked positive inotropic effects, reaching 148% of control level. The response of neonatal cardiomyocytes to o ligands became similar to that of the aduR, after desensitization of a putative o receptor subtype by preincubation with ~ ligand. Isolated perfused rat hearts were also sensitive to o ligands. Prototypic o ligands, at nanomolar concentrations, induced a marked increase in force of contraction and a decrease in beating rates. Our results suggest that o receptors may have important regulatory functions in the myocardium.

CYTOPROTECTION WITH BDM: ARE THE Tu047 ELECTROPHYSIOLOGICAL EFFECTS REVERSIBLE? Franz Metzger, Erich Wettwer, Gregory J. Amos & Ursula Ravens. Department of Pharmacology, University of Essen, Essen, Germany.

CARDIOPROTECTIVE EFFECTS OF PGE 0 Tu048 ARE PREVENTED BY GLIBENCLAMIDE: ROLE OF ATP-SENSITIVE POTASSIUM CHANNELS. E. Hide, J. Piper, Ip. Ney, C. Thiemermann & J.IL Vane. The William Harvey Research Institute, St. Bartholomew's Hospital Medical College, Charterhouse Sq., London, U.IL

2,3-Butanedionemonoxime (BDM) is commonly used for myocardial ceil protection, despite the fact that it also affects electrophysiological parameters. Our aim was to determine whether these effects are reversible. Action potentials (AP) were measured in guinea pig ventricular myocytes either isolated and stored in BDM (20 mM) containing solutions (A) or isolated without BDM and then exposed to BDM for 5 min (B). Human atrial myocytes were isolated and stored with (C), or without (D) BDM. Ito and Ica were investigated. In A, APD90 in BDM was 431+30 ms, 806+35 after I min and 646+40 ms after 5 min wash--out. AP amplitude increased from 112+3 mV to 115+3 mV, resting membrane potential changed from -62+1 mV to -65+1 mV. In B APD90 decreased from 620+59 ms (control) to 384+16 ms (20mM BDM) and increased to 606+49 ms after wash-out. The current density of Ito at +60 mV was 16.3+2.5 pA/pF after washout in C, and 15.8_+0.7 in D. The current density of Ica at +5 mV in BDM was 4.0_-+0.7 pA/pF, 5.0:t:0.8 pA/pF after 1 min and 3.4_+0.4 pA/pF after 5 min wash-out, comparable to 3.1z'--0.2 pA/pF in D. Conclusion: Since the effects of BDM are quickly reversible, they should not limit the agents use for the protection of myocytes during isolation for electrophysiological investigations.

The PGE 1 metabolite 13, I4-dihydro PGE I (or PGE0) causes vasodilatation, inhibition of platelet and neutrophil function and reduces infarct size (IS) in a rabbit model of myocardial ischaemia (MI) and reperfusion (REP). Here we use an identical model to investigate whether (i) a lh pretreatment with PGE0 reduces IS, and (ii) whether this effect is blocked by the ATP-sensitiv¢ K+ channel blocker Glibenclamide (Glib). Anaasthetised NZW rabbits were tracheotomised and ventilated with room air. After thoracotomy, the LAL coronary artery was occluded for 60 rain followed by 120 min of repeffusion. PGE0 (l btg kg'lmin "1) was infused i.v. for 60 rain and stopped 15 min prior to LAL occlusion. Glib (0.3 mg kg-l; i.v.) was administered l0 min prior to PGE0 pretreatment. Area at risk (AR) and IS were determined by Evans blue and NBT staining. AR was not significantly different between groups. PGE0 caused a reduction in IS from 59-J:6% (control, n--9) to 42-+-2%* (*p<0.05, n = 6). Glib prevented the cardioproteetive effect of PGE 0, thus returning IS to control levels (52:1:3%*; *p<0.05 when compared to PGE0 treated group; n=8). Thus pretreatment with PGE0 prior to LAL occlusion reduces IS through a mechanism that may be related to the activation of ATP-sensitive potassium channels. This work was supported by a project grant from I Schwarz Pharma AG; EH is the recipient of a BHF grant.

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A138

PROTECTION AFFORDED BY K*-CHANNEL Tu049 OPENERS IN THE CARDIOMYOPATHIC HAMSTERS UM-X7.1. Ga~tan Jasmin and Libuse Proschek, Department of Pathology, Universit~ de Montreal, Montreal, Canada.

ELECTROPHYSIOLOGICAL EFFECTS OF ATRIAL Tu050 NATRIURETIC PEPTIDE (ANP) Valeria Kecskemeti, R. Marko and K. Kelemen. Dept of Pharmacology, Semmelweis University of Medicine, Budapest, Hungary

ATP-sensitive K'-channels have been recognized to play an important role in such pathological conditions as ischemia or metabolic stress in heart under which they are activated. Moreover, K'-channel openers have been shown to prevent the Ca** overload in ischemic head. The aim of this study was to investigate whether in vivo treatments with cromakalim (CR) or PZ-400N reduce the necrotizing process in cardiomyopathic hearts. Young 28+1 days old cardiomyopathic hamsters UM-X7.1 were injected with CR and PZ-400N dissolved in DMSO (5%) and EtOH (4%) respectively in a twice daily dose of 2.5 mg/kg for 4 consecutive weeks. The necrotic changes were assessed microscopically in the head, the diaphragm and the tongue. Both K" openers were well tolerated despite of slight reduction in body weight and induction of cardiac hypertrophy. Necrotic lesions were significantly (p<0.001) reduced by CR and PZ-400N. CR however, prove to be a more potent cardioprotector than PZ-400N as evidenced by the sole presence of myolytic foci without calcification. The severity of the skeletal muscle degenerative process was also modestly, but significantly (p<).05) reduced. Thus the present data provide evidence that K° openers are potent cardiopmtectors presumably by maintenance of the membrane resting potential together with improvement of Ca÷~ homeostasis. These K" openers therefore may find interest in the management of primary cardiomyopathies.

The effects of ANPs (Atdopeptin III, frog ANP) on transmembrane action potential (AP) of electrically driven left audcle and right ventdcular papillary muscle of guineapig and on the ionic currents of frog atdal fibers, using double sucrose gap voltage clamp technique were studied. Atdopeptin III (1-100 nM) caused a slight, but significant hyperpolarizing effect in either in atrial or in ventdcular muscles of guinea-pig. It decreased the height of the overshoot (OS) and the plateau phase in both preparations. It had no effect on the maximum rate of rise of depoladzation phase (Vmax) of ventdcular AP but slightly diminished the atdal one. Its effect on the repoladzation phase in the two types of muscle is different. While the duration of atdal AP remained unchanged the repoladzation phase was dosedependently shortened in ventdcular fiber. The K + channel blocker 4-aminopyddine (1 mM) prevented the effect of ANP on the duration of ventdcular AP. In depoladzed (by 25 mM K Tyrode) fibers, when fast Na+-channels were inactivated, ANP was not able to generate slow Ca 2+ dependent APs and it prevented epinephrine-induced slow APs, too. Frog ANP(IO0 nM) decreased the slow component of the inward current in frog atrial fibres and it was not able to restore the electromechanical activity of K+ (27 mM)-arrested frog head. These results suggest the possibility that ANP decreases Ca2+infiux via the slow inward current, but the action of ANP on K+current can not be excluded either. This work was supported by OTKA 1077 and ETT 3-286.

IONIC BASIS OF ACTION POTENTIAL Tu051 PROLONGATION IN MYOCYTES FROM DILATED VENTRICLES OF SYRIAN HAMSTERS Dominique Thuringer, Edith Deroubaix, Alain Coulombe, Nathalie Aubailly, Edouard Coraboeuf & Jean-Jacques Mercadier. CNRS URA 1159, Le Plessis Robinson, France

DEPRESSEDTRANSIENT OUTWARD CURRENT

IN SYRIAN HAMSTERS DURING AGING AND DILATED CARDIOMYOPATHY Dominique Thuringer, Alain Coulombe, Edith Deroubaix, Edouard Coraboeuf & Jean-Jacques Mercadier. CNRS URA 1159, Le Plessis Robinson, France

To determine the electrophysiological alterations associated with cardiac dilation, we compared action potentials (AP; recorded with standard microelectrodes from isolated hearts) and ionic currents (recorded from ventricular myocytes using the whole-cell patch-clamp technique) in 120 day-old cardiomyopathic (MS200) and age-matched control (CHF148) Syrian hamsters. In hearts isolated from MS200, the AP duration was prolonged and the plateau phase was markedly increased compared to CHFI48. In myocytes, membrane capacitance was not higher in MS200 than in CHFI48, indicating the absence of cell hypertrophy in dilated ventricles. The ICa,L density was significantly reduced in MS200 and the voltage dependence of both activation and inactivation was shifted towards positive potentials. The steady-state current-voltage relations between -120 and +60 mV were superimposable in MS200 and CHF148, in agreement with the observation that the resting potential did not differ between strains. The voltage-dependent outward current was composed of both transient (Itol) and sustained (Iss) components. The Itol density was strongly depressed in MS200 compared to CHF148, whereas Iss was not significantly different in the two strains. Prolongation of AP duration in dilated heart is due in part to depression of Itol. (Study supported by a grant from Procter & Gamble Pharmaceuticals)

The aim was to investigate alterations in the Ca 2+independent transient outward current (Itol) using the whole-cell patch-clamp technique, in normal hamsters (CHF148) and in a model of dilated cardiomyopathy (MS200) with no initial hypertrophy. Since cardiac dilation is a time-dependent process, ventricular cells were studied at different ages of 60, 90, 120 and 180 days. In CHF148, Itol density increased from 60 to 120 days then decreased whereas, in MS200, its value increased slowly until 180 days. The Ito 1 density was drastically reduced, at 90 and 120 days, in MS200 compared to CHF148 whereas, at 180 days, Itol density was the same in both strains. The conductancevoltage and steady-state inactivation relationships were shifted towards positive potentials in 180-day-old CHF148 compared to younger animals, and in 90- and 120-day-old MS200 compared to CHF148. The overall recovery process of Itol from inactivation was markedly slower in 180 than in 120 day-old CHF148 and in MS200 than in CHF148. Depression of Itol contributes to the increase in the action potential duration in MS200 and this effect is more marked at higher frequencies. Alterations of Ito 1 in dilated hearts resemble those in normal senescent hearts. (Study supported by a grant from Procter & Gamble Pharmaceuticals)

Tu052

PHARMACOLOGICAL MODULATION OF Tu053 THE TRANSIENT OUTWARD CURRENT Ito IN HUMAN VENTRICULAR IMIYOCYTES. Herbert M. Himmel, Qi Li, Erich Wettwer, Franz Metzger, Ursula Ravens. Dept. of Pharmacology, University of Essen, Essen, Germany. Early repolarization of action potential in human ventricular cardiomyocytes is mainly determined by Ito. We tested interactions with Ito of antiarrhythtalc drugs with class-Ill action: E-4031 (N-[4-[[1-[2(6-methyl-2-pyridinyl)-ethyl]-4-piperidinyl]-carbonyl]-phenyl]-methanesulfonamide), d-sotalol (SOT), quinidine (QU). In voltage-clamped human ventricular myocytes (22°C, 0.2 Hz, V h -80 mV), Ito was elicited by 300ms-clamp steps (range -30 to +60 mV); Na + current was.,~repulse-inactivated (from V~ -80 to -40 mV), Ca z current was inhibited by C'c~2+ (100 IJM). Neither E-4031 (10t.tM, n=5) nor SOT (100tJM, n=5) had an effect on current amplitude, voltage-dependence of activation and steadystate inactivation; the inactivation time course was slightly accelerated. QU (101.tM) reduced peak-lto at +60 mV from 5.9+1.0 pA/pF to 5.1_+0.8 pA/pF, while the current at the end of the clamp step (lateIto ) was unaffected. The inactivation time constants at +60 mV were 59_+3 ms (control) and 21_+2 ms (QU). The fractional block of Ito at +60 mV developed with a time constant of 9.5+1.5 ms. Our results are consistent with an open channel block of Ito by QU. Thus, some antiarrhythmic drugs with class-Ill action (e.g. QU) affect Ito in ventricular myocytes from human hearts, whereas E-4031 .and SOT hardly influence this current.

I O N I C M E C H A N I S M OF EARLY AFTER-

Tu055

D E P O L A R I Z A T I O N INDUCED BY L O W K + A N D Cs + I N M O U S E V E N T R I C U L A R M Y O C Y T E Ying-Ying Zhou, Xue-Mei Hao, Jing-Song Fan & Tai-Feng Liu College of Life Sciences, Peking University, Beijing, PRC. Our previous work demonstrates that early afterdepolarization (EAD) is considerably easy to be induced in mouse myocardium. To explore the underlying mechanism we studied the change of ionic currents and action potential (AP) in single myocytes isolated from mouse ventricles after the treatment of perfusion of Tyrede's solution with 3 mM K+ plus 3 mM Cs+ by the whole-cell patch clamp technique. Both transient outward current and delayed outward current did not vary significantly in treatment vs. control (1:'>0.05). Interestingly, the inward-rectifying potassium current (IKI) of mouse ventricuiar myocyte differed from those of other species in that its current-voltage (I-V) curve showed no negative slope, i.e., the slope in the membrane potential range between --40 and -70 mV was virtually fiat and remained at a low current level (59 ± 39pA). Under treatment, IKI in the above region nearly decreased to zero. Then in the same potential range EAD eccured and the I-V curve displayed a net inward current (-37 ± 5SPA) which could be blocked by 20 ¢ M TTX and 10 p M nifedipine. Furthermore, we examined the contribution of ionic currents to EAD with the EAD clump for the first time and found that in the repolarizing phase of AP under treatment, the outward currents decreased before the inward currents increased which parallel to the occurrence of EAD. Thus, the distinctive characteristic of IKl in mouse ventricuiar myocyte may relate to the high susceptility to EAD in mouse myocardium. The inhibition of IKi seems to be a prerequisite for the occurrence orEAD in this experiment.

OPEN CHANNEL BLOCK BY TEDISAMIL Tu054 OF TRANSIENT OUTWARD CURRENT (Ito) IN HUMAN VENTRICULAR MYOCYTES. Erich Wettwer, Herbert M Himmel, Qi Li, Gregory J Amos & Ursula Ravens. Dept of Pharmacology, University of Essen, Essen, Germany. In ventricular myocytes of the human heart, I,n determines the early phase of repolarization d~ the epicardial action potential. We have studied the effects of the new class-Ill antiarrhythmic agent tedisamil (TED) on Ito in whole-cell clamped human ventricular myocytes enzymatically isolated from explanted hearts. Ito was elicited with 300 ms clamp steps between -30 and +60 mV (holding potential -80 mV, 22°C). TED concentration-dependently reduced only peak-lto: with 101JM from 10.0+ 2.6 pA/pF to 6.7_+1.5 pA/pF at +60 mV (n=6). The time constant x of apparent Ito-inactivation was acce erated from 65.5_+4.5 ms (control) to 20.3+3.7 ms (10 IJM TED) with an IC50 value of 4.4 IJM. From the onset of the fractional block at 3, 5 and 10 pM, we determined the rate constants for the Ito-TED interact on k 1 9.106 M-is -1 k 1 23 s-1 Kn 2.6 IJM. TED block developed only with channeq activation, showed use dependence and recovered completely with a biphasic time course within 120 s (5 pM). The effects of TED on Ito could be described with a four state kinetic model assuming an irreversible closed-open transition and subsequent equilibrium of inactivated and TED-bound channel states. Therefore, in human ventricular cardiomyocytes, TED behaves as an open channel blocker of Ito.

COMPARISON OF POTASSIUM CURRENTS IN Tu056 ISOLATED MOUSE A T R I A L AND V E N T R I C U L A R MYOCYTES Ying-Ying Zhou, Xue-Mei Hao, Jing-Song Fan & Tai-Feng Liu College of Life Sciences, Peking University, Beijing, PRC.

Though the action potential configurations of mouse atrial and ventricular myocytes are very similar, the incidence and the types of early aflerdepolarization (EAD) are quite different between them in our previous experiments assuming the heterogenoas in potassium channels of mouse atrial and ventricuiar myocytes. Using both whnle-cell and single channel patch-clump techniques, we compared the inward-rectifying potassium current (IKl), the transient outward current (It,,) and the delayed outward current (Ig) between these two kinds of cells. IK1 was relatively larger in ventricle (31 + 8pA/pF, 120mV) than in atrium (20+ 7pA/pF) and showed fast inactivation upon hyperpolarization in more ventricle cells (7/21) than in atrial cells (3/18), however, the difference between the conductances oflK! in ventricle (39+ 7nS) and atrium (34 ± 9nS) was not significant. Ito in ventricle (72 ± 18pA/pF, +60mV) was relatively little compared with that in atrium (83 + 19pA/pF), but the activation process were almost identical between them. Ig was very small (1.2 + 0.3pA/pF, tail current after depolarized to 30mV for 4s) and only present in some ventricle myocytes (11/29) while there could not detect any Ig in atrium. We conclude that there exist some, though not very striking differences between the potassium currents in mouse atrial and ventricular myocytes. Since the membrane resistance is considerably high in the plateau potential range, a small change in currents can cause a large alteration in membrane potential which may lead to the generation orEAD.

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Tu057 THE CARDIOPROTECTIVE EFFECT OF ENANTIOMERS OF ClCLETANINE INVOLVES A GLIBENCLAMIDE SENSITIVE MECHANISM IN ISOLATED RAT HEARTS Tem6s Caont, Csaba Csonka, P6ter Ferdinandy, Zolt6n SzilvSsey," Matyas Koltai,# Marie-T. Droy-Lefaix,' Thierry Tarrade.* Dept Biochem, "lst Dept Medicine, Szent-Gy6rgyi Univ., Szeged, Hungary; #lPSEN-Beaufour, Paris, France.

EFFECT OF BERTOSAMIL ON THE Tu058 ATP DEPENDENT POTASSIUM CHANNELS IrOn Krassti, .*Dieter Ziegler and Julius Gy. Papp. Department of Pharmacology, Albert SzentGytirgyi Medical University, Szeged, Hungary, *Department of Pharmacology, Kali-Chemie Pharma, Hannover, Germany

The anti-ischemic effect of cicletanine racemate was shown to involve opening of ATP-sensitive K*-channels (KATp). In the present study, interaction of the KArp blocker glibenclamide with enantimers of cicletanine was studied on ischemic myocardial function and lactate-dehydrogenase (LDH) release in isolated working rat hearts subjected to 10min coronary artery occlusion (CO) (n = 10 in each group). The most effective concentration of cicletanine[-] (BN50418, 3x10 "s M) increased aortic flow (AF) from its control value of 20.3 + 1.16 to 30.3 + 2.6 mL/min (p < 0.01 ), decreased left ventricular end-diastolic pressure (LVEDP) from 1 . 8 1 ± 0 . 0 5 to 0.97:1:0.08 kPa (p
In earlier experiments bertosamil was shown to lengthen cardiac action potential duration (APD). It was however not clear whether the drug was capable of inhibiting the ATP dependent potassium channels (KATP) which are known to be activated in m2}66xirdial ischaemia, thereby shortening APD. To study this question, in the present experiments, transmembrane potentials were recorded from canine Purkinje fibres using standard microelectrode technique. Shortening of APD via specific activation of KATP channels was achieved by applying 5 #M pinac~dil, and after about 30 min washout period when the evoked APD shortening (-50% at APD90) recovered, bertosamil (1, 3 and 10 pM), or glibenclamide (1 /zM), a selective blocker of KATp, was added to the organ bath. Application of 5"~M pinacidil resulted in significantly less APD shortening in the presence of 1 /zM Bertosamil (-13% at APD90) than in the absence of this drug. As glibenclamlde (0%), 3 and 10 #M bertosamil prevented almost completely the pinacidil induced APD shortening (-7, -4% at APD9o). From these findings it is concluded that Bertosamfl inhibits not only the Ito, as reported earlier, but also the ATP dependent potassium current.

TEDISAMIL IS MORE POTENT TO I N H H } I T Tu059 IK(ATP) THAN GLIBENCLAMIDE IN CARDIAC MUSCLE Mikl6s N~meth, AndrOs Varr6, Julius Gy. Papp. Department of Pharmacology Albert Szent-

Gy~rgyi Medical University Szeged, Hungary

THE EFFECTS OF CHRONIC AMIODARONETu060 TREATMENT ON THE CARDIAC CALCIUM AND POTASSIUM CHANNELS A n d r ~ Varrt, IAszl6 Virdg and Julius Gy. Papp. Department of Pharmacology, Albert SzentGy~rgyi Medical University, Szeged, Hungary

The ATP dependent potassium current flKATP) plays an important pathophysiological role during ischemia. Therefore, drugs influencing this current may be useful in the treatment of ischemic heart disease. In this study the effects of tedisamil flED), a new antiischemic/bradycardie agent, and those of glibenelamide (GLI) were examined on IKATP in dog ventrieular muscle applying standard microelectrode technique. IKATP was activated by adding I0 #M pinacidil (PIN) which caused significant shortening in the action potential duration (AID). Prevention or attenuation of the PIN induced APD shortening (p<0.05) by the tested drugs were considered as inhibition of IKATP. In group 1. PIN shortened APD by 39.6+1.8% (n=22, p<0.001). After washout of PIN GLI was administered in different concentrations. Then, PIN was added again. GLI prevented the PIN evoked APD shortening with an EC50 value of 410 nM. In group 2. PIN shortened APD by 37.5+3.3% (n=20, p<0.001). With similar protocol TED prevented the PIN evoked APD shortening with an EC50 value of 97 nM which is considerably lower than that of GLI. These data suggest that in the heart TED is a very powerful blocker of IKATP. This action may play an important role in the well documented beneficial effect of TED during cardiac ischemia. Supported by OTKA T 676.

The effects of the chronic amiodarone treatment (AM) were studied on transmembrane ionic currents in single rabbit ventricular myocytes using the wholecell configuration of the patch clamp technique at 37°C. Rabbits (n=5-5) were treated i.p. either with AM (50 mg/kg/day) or with the solvent for 3-4 weeks. Measuring ECG parameters, AM but not the solvent increased QTe (from 134-t-2 ms to 167-1-9 ms, n=5, p<0.05). Myocytes isolated from rabbits treated chronically with AM showed no significant differences in current densities and the kinetical behavior of Ikl and L-type ICa compared to those of the solvent treated animals. Chronic AM treatment however, significantly reduced the current densities of IK from 1.284+0.085 pA/pF at +30 mV (solvent, n=15) to 0.687+0.093 pA/pF (AM, n=16, p<0.001). The density of Ito at +60 mV was also reduced after chronic AM treatment from 10.24+0.94 pA/pF n=17 to 7.383+0.88 .pA/pF (n=17, p<0.05) without an alteration m its inactivation kinetics. We assume that the repolarization lengthening effect of AM treatment is, at least partly, due to the reduced number of IK and Ito channels, while the use-dependent inhibition of ICa observed earlier is presumably due to direct drug interaction with the L-type calcium channels. This work was supported by the OTKA T 676 grant.

EFFECT OF DISOPYRAMIDE ON Tu061 POTASSIUM CURRENTS IN ISOLATED RABBIT CARDIAC MYOCYTES L~iszl6 Virfig, Andr~ls Varr6 and Julius Gy Papp. Department of Pharmacology, Albert SzentGyiJrgyi Medical University, Szeged, Hungary. The effects of I0~M Disopyramide on transient outward (Ito), delayed rectifier (IK) and inward rectifier (IKI) currents were studied in isolated single rabbit ventricular myocytes applying the whole-cell confi.guration of the patch-clamp technique.The experiments were carried out at 35°C. The Ito was activated by depolarizing voltage pulses from holding potential of-90 mV. Disopyramide at 50 mV markedly decreased the integral of the Ito (57.8-4-4.2 %, n=7), while the amplitude of the current was less reduced by the drug (78.5+3.8 %, n=6). This is consistent with the observation that Disopyramide accelerates inactivation of Ito. The fast time constants were 6.284-0.18 ms in control conditions and 3.88+0.27 ms (n=9, p<0.05) after application of Disopyramide. The slow time constant was unaffected by the drug. IK was activated by depolarizing voltage steps (range -10 to 30 mV) from holding potential o f - 4 0 inV. Disopyramide decreased the IK tail current from control value of 139.54-10.9 pA to 30.74-3.2 pA (n=ll, p<0.05) at 30 mY. The IKI was measured by a series of 300 ms depolarizing or hyperpolarizing voltage step (range 140 to 50 mV) from holding potential of-90 mV. This current was not influenced by Disopyramide. It is concluded that the repolarization lengthening effect of Disopyramide is due to the depression of IK and Ito. This work was supported by OTKA F6328 grant.

ELECTROPHYSIOLOGIC EFFECTS OF A TU063 NEW K+-CHANNEL BLOCKER, GLG-V13, IN GUINEA PIG Csaba Lengyel*, Leif Carlsson**, Tam,is V~konyi*, Tarmts Fazekas*, Benjamin J. Scherlag#, Ralph Lazzara# *Ist Dept of Medicine, Szent-Gyrrgyi Medical University, Szeged, Hungary; **Dept of Cardiovasc Pharmacology, Astra H~issle, Mrlndal, Sweden; #Dept of Medicine, OUHSC, Oklahoma City, OK, USA GLG-V-13, a new K+-channel (Ikr) blocker was examined in vivo in order to "~haracterize its electrophysiological properties using a guinea pig screening model. The variables measured were the prolongation of the epicardial LV-MAP (at 75% repolarization, MAPD7~), the AV-conduction time (AV-time) and the venfi'icular activation time (QRS interval or the rise time of the MAP). All these parameters were recorded at a constant atrial pacing rate close to the intrinsic HR (R.R interval: 258+10.5 ms). Cumulatively increasing doses of GLG-V-13 were administered i.v. and dose-response curves were constructed. When studied in a dose interval (1 nmol/kg --, 1 tzmol/kg), GLG-V-13 lengthened the MAPDTs (13<0.05 at doses > 10 nmol/kg), AV-time (p<0,ffS" at doses > 2 nmol/kg) and RR interval (p<0.05 at doses > 50 nmol/kg) in a dose-dependent manner. At the highest dose (1 /.tmol/kg) these variables were increased by 30%, 13% and 23%, respectively. No effects were noted on the ventricular conduction time. Conclusion: GLG-V-13 is a novel pure K-r-channel blocker lacking Class I action.

R E G I O N A L D I F F E R E N C E S IN A C h - A C T I V A T E D T u 0 6 2 K ÷ C U R R E N T IN C A N I N E V E N T R I C L E Z h a o - K a n g Yang & Mark R. Boyett. Dept of P h y s i o l o g y , University of Leeds, Leeds, UK. We have previously shown that ACh has dose-dependent effects on both contraction and action potential duration in epicardial cells, but not endocardial cells, from the canine ventricle (Yang et al., 1995, J. Physiol., in press). The mechanism underlying these effects has been investigated in 13 epicardial cells and 10 endocardial cells. Cells were stimulated with 200 ms voltage clamp pulses from -80 to 0 mV at 1 Hz (37°C). In epicardial cells, on application of 10"SM ACh, there was a rapid outward shift in current at 0 mV and during continued application of ACh the current partly recovered towards control. These characteristic changes were absent in endocardial cells. The mean amplitude of the AChactivated current at 0 mV early during an exposure to ACh was 70+8 pA and 10+_2 pA in epicardial and endocardial cells, respectively. In the presence of 2 mM Ba2., the ACh-activated outward current in epicardial cells was abolished, which suggests that it is a K* current. We conclude that ACh-activated K* current is present in epicardial cells (but not in endocardial cells) and this is responsible for the parallel changes in action potential duration and contraction in this cell type. Supported by the Royal Society

EFFECTS OF ZATEBRADINE IN RABBIT SINO-ATRIAL TU064 NODE AND ATRIO-VENTRICULAR NODE CELLS Yusaku Sakakibara, Tomoaki Saekl, Kenjl Suzuki, Kazuhiro Yajima & Takao Fujinaml. Third Department of Medicine, Nagoya City University Medical School, Nagoya Japan Zatebradine (Zb) is a newly developed bradycardic agent which blocks hyperpolarization activated inward current (Ih). We studied electrophysiologic effects of Zb in rabbit sinoatrial node (SAN) and atrio-ventricular node (AVN) cells using small preparations (0.2xO.2x0.1 mm). Membrane potentials were recorded by conventional glass microelectrodes filled with 3 M KCI. Voltage clamp experiments were conducted by double-microelectrode techniques. 1 pM Zb showed a negative chronotropic effect in both SAN and AVN cells. 1 pM Zb significantly prolonged diastolic interval from 173 + 18 msec to 1 8 4 ± 2 3 msec in SAN cells (n=10) and from 194 ± 12 msec to 205 ± 15 msec (n = 10) in AVN cells. 1/JM Zb also prolonged action potential duration from 1 6 3 ± 6 msec to 176 ± 6 msec in SAN cells (n = 10) and from 148 ± 8 msec to 1 5 9 ± 8 msec in AVN cells (n=lO). However, Zb showed no significant changes in maximal diastolic potential, rate of diastolic depolarization, action potential amplitude and maximal rate of depolarization either in SAN or AVN cells. Voltage clamp experiments revealed that Zb did not alter Ca=* current and delayed rectifying K + current (IK) either in SAN or AVN cells. Ih was recorded by hyperpolarizing the membrane from -60 mV to -90 mV for 3 sec immediately after an initial hyperpolarization from -40 mV to -60 mV for 1 sec to fully deactivate IK. Zb reduced Ih by 12 ± 3 % in SAN cells (n=6) and 11 ± 3 % in AVN cells (n=6). These results suggests that Zb exerts a negative chronotropic effect by blocking Ih in both SAN and AVN cells.

AI41

EFFECT OF GLYBURIDE ON MYOCARDIAL Tu065 METABOLISM IN THE ISCHEMIC DOG HEARTS Masahiko Kamigaki, A.N. Ehsanul Hoque, Kazuo Ichihara & YasushiAbiko. Dept of Pharmacology, Asahikawa Medical Coll©g¢, Asahikawa, Japan.

Both pancreatic B-cells and myocardial cells possess ATP scnstive K channel (KArl,). Glyburidc appears to be highly spcctific for the KATe. Myocardial ischcmia leads to activation of the KATedue to reduction of the tissue ATP level during ischcmia. The activation of KATemay reduce Ca2÷ influx through the voltage-dependant Ca2* channel and hence decrease myocardial contractility. This would lead to preservation of the ATP level in the ischcmic tissue, resulting in attenuation of ischcmic damage. Accordingly, the prescnse of glyburide, a selective blocker of the KATe, may worsen the ischcmic myocardial metabolic derangement further. We examined therefore the effect of glyburide on myocardial energy and carbohydrate metabolism in dog hearts whose left anterior descending coronary artery was occluded for 15 min. Glyburidc (1 mg/kg, injected i.v. 10 rain before the occulusion) accelerated the ischcmia-induecd decrease in the tissue level of ATP and also accelerated the ischemia-induccd increase in the tissue lactate level compared to ischcmic control. In addition, a continuous infusion of glucose (3 mg/kg/min) that was performed to prevent mild hypoglycemia induced by glyburidc, did not ameliorate the deleterious effects of glyburidc. These results indicate that glyburidc enhances the ischcmic myocardial metabolic derangement, and that the activation of the KATe may play a role as an endogenous cardioproctivc mechanism during ischcmia.

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ENHANCED ISUS BUT NOT ITO1 IN H U M A N T u 0 6 6 A TRIAL M YOCYTES D URING S E R U MSTIMULATED GROWTH S. N. Hatem, A. Btnardeau, C. RiJcker-Martin, E. Coraboeuf, J.J. Mercadier. Universit6 de Paris XI - CNRS URA 1159, Htpital Marie Lannelongue, Le Plessis Robinson, France In human atrial myocytes, the transient outward current is composed of two components, Itol and Isus, carried by different potassium channels, the expression of which is differentially regulated in rat heart during development and hypertrophy. We examined whether, in human atrial myocytes, Itol and Isus undergo differential evolution during serum-induced myocyte growth in vitro. Currents were recorded in whole-cell patch-clamped myocytes. After one week of culture (Clt), the density of Itol did not differ from that in freshly dissociated myocytes (C) (4.38+0.8 pA/pF vs 3.71_+0.6 pA/pF), while that of Isus was more than 4-fold higher (9.76+2.1 pA/pF vs 2.21_+0.29 pA/pF). In Clt, following inactivation of Itol by a prepulse, sustained depolarization elicited a rapidly activating outward current (10.22.+.1.18 pA/pF), with a threshold at -30 mV, and activation times of 39.2+2.1 msec at -10 mV and 9.8_+0.7 msec at +40 mV. The apparent reversal potential of the tail current was -73.5+_3.2 mV, indicating high K + selectivity. Isus was highly sensitive to 4-AP (55.4+4.4% inhibition in 50 mM 4AP). D600 suppressed Isus (47.9-&-_4.1 inhibition in 10 p.M D600) but not Itol. These results suggest that Isus in cultured myocytes is similar to the Kvl.5 cloned channel current and that the expression of the channels carrying Isus and Itol is differentially regulated in the human heart.

ACTION POTENTIAL PROLONGATION IN SINGLE T u 0 6 7 RABBIT VENTRICULAR MYOCY'rES DURING HYPOXIA BY ITO DEPRESSION Antoni C.G. van Ginneken, Arie O. Verkerk, Marieke W. Veldkamp & Lennart N. Bouman. Dept of Physiology, University of Amsterdam, the Netherlands.

INFARCT LIMITATION BY PROTEIN KINASE Tu068 C (PKC) IS ATTENUATED BY BLOCKADE OF ATP-SENSITIVE POTASSIUM (KATp) CHANNELS D.M. Van Winkle, K. Kuzume, K. Dote, and R.A. Wolff. Dept. of Anesthesiology, Oregon Health Sci. Univ., and Research Service, V A Medical Center, Portland, OR.

Effects of hypoxia were studied on isolated rabbit ventricular cells. In a chamber on the stage of an inverted microscope the cells were superfused with a HEPES-buffered Tyrode solution of 33-34 °C which was gassed either by 02 or N2. Electrical access to the cell interior was obtained by the perforated patch technique, using amphotericin B. Action potentials were elicited by 5 ms, 1 Hz current pulses of 1.5x threshold via the patch electrode. On switching to hypoxia by means of N2gassed Tyrode, we observed a decrease in action potential duration (APD), which was thought to be caused by opening of IK,ATP channels. However, in 9 out of 14 cells the decrease was preceded by an initial increase in APD of 13g:I:41%(:I:SD). Steady-state outward current and peak Ca++-current (IcaL), did not change during the APD increase. Phase-plane plots'indicated that the change predominantly occurred in the early plateau phase, suggesting a role for the transient outward current ITO in the increase in APD. ITO was found to be present in 50-60% of the cells. When present, ITO gradually disappeared during superfusion of N2-gassed solution, with a time course similar to that of the observed increase in APD. These results suggest that the hypoxia-induced initial increase in APD is caused by a decrease in ITO. It is likely that in the border zone of ischemic cardiac tissue due to the gradient in pO 2 cells with an increased APD as well as a decreased APD are present, which results in a potentially arrhythmogenic dispersion of the refractory period. The finding that the initial increase in APD is not observed in all cells is suggestive for epi-/endocardial differences.

We investigated the role(s) of PKC and KATp channels in ischemic preconditioning (IP). Isolated buffer perfused rabbit hearts u n d e r w e n t 4 0 min regional ischemia (RI) and 2 hours reperfusion (REP). IP w a s elicited by 5 min RI, f o l l o w e d by 10 min REP. PKC w a s activated w i t h oleyl acetyl glycerol (OAG, 15 pmoles/min) or 4-1~phorbol 12-myristate 13-acetate (PMA, 0.015 nmoles/min) and blocked w i t h p o l y m y x i n B (PB, 4 0 pM). KATP channels were blocked w i t h 5-hydroxydecanoate (5HD, 3 0 0 pM); this dose abolished 10 p M cromakalim-induced infarct limitation. Infarct size w a s assessed w i t h tetrazolium, and is expressed b e l o w as a percentage of the risk zone. GROUP IS ± SEM n No IP 28 + 3 % 18 IP 11 ± 2 % § 13 PMA 12 ± 3 % § 11 OAG 14 + 5 % 9 PB 31 ± 5 % 12 PB + IP 23 ± 4 % 15 5HD 26 ± 9 % 7 5HD + PMA 28 ± 4 % 11 We conclude that preconditioning involves activation of PKC w i t h subsequent opening of KATp channels.

NITROGLYCERINE ACTIVATES AN Tu069 IBERIOTOXIN-SENSITIVE POTASSIUM CHANNEL IN HUMAN SAPHENOUS VEIN J6zsef H6hn, +Janos Pataricza, *G~bor K. T6th, .~ddm Balogh & +Julius Gy. Papp. Dept. of Surgery, +Dept. of Pharmacology and *Dept of Medical Chemistry, Albert Szent-Gy6rgyi Medical University, Szeged, Hungary Recendy, synthetic iberiotoxin 0BTX), the known most selective inhibitor of large conductance calcium activated potassium channel (BKCa), has been shown to be an efficient pharmacological tool for studying the functional role of BKCa in coronary artery (Pataricza et al., J. Mol. Cell. Cardiol., 26:Ahstr.336., 1994). In the present study, the possible involvement of BKCa in the venodilator mechanism of nitroglycerine (NTG) was investigated using isolated human saphenous veins. Isometric contractions of venous rings (5 nun in width) were induced by 0.125 #M 5hydroxytryptamine and cumulative concentrations of NTG (22-330 #M) were applied in the presence and absence of 90 nM IBTX (preincuhation: 30 rain). Concentration-relaxation curve for NTG was significantly shifted to the right by IBTX (ICs0 (NTG)= 14.9+7.9 p.M, IC50 (NTG+IBTX)= 55.3+5.3 #M, mean+S.E.M., p<0.01, n=5). In conclusion, an iberiotoxin-sensitive potassium channel,

BKCa is involved in the venodilator mechanism of NTG in vitro, and this effect may be mediated by nitric oxide in human saphenous vein. This work was supported by the

Hungarian National Scientific Foundation (OTKA T12848).

THE DEVELOPMENT OF NECROSES IN Tu071 ISCtlEMIC PRECONDITIONED MYOCARDIUM Jochen Rose, Rainer Schulz, Heiner Post, Gerd Heusch. Department of Pathophysiology, University of Essen, FRG Ischemic Preconditioning refers to the reduction in infarct size (IS) during a prolonged ischemic period by one or more preceding short episodes of ischemia and reperfusion. Nevertheless, even in ischemic preconditioned myocardium some necroses may develop. We tested in 8 anesQ I--1: NON-INrARCTED,I : INFARCTED(%) thetized swine whether 0,.-, Oo the necrotic areas in g~)m.2 .12 ischemic preconditioned myocardium are ~E <\0.9 characterized by a greater flow reduction 6 than the surviving myocardium. The left 3 anterior descending m coronary artery was f'~ D 0.0 CONIROL 5 MIN REDUCTION cannulated and hypoISCHEMIA(%CONTROl.) perfused for 90 rain. Ischemic preconditioning was induced by a cycle of 10 min ischemia and 15 min reperfusion. IS was determined after 2 his of reperfusion by TTC-staining. Subendocardial blood flow (microspheres) was determined in the infarcted and non-infarcted myocardium under control conditions and after 5 rain of the prolonged ischemic period (*: p<0.05 vs NONINFARCTED, t-test). Our data indicate that the infarcted myocardium was subjected to a greater flow deficit than the noninfarcted tissue.

nl

li

BOTH OPENING AND CLOSING OF ATPTu070 SENSITIVE POTASSIUM CHANNELS PROTECT AGAINST LETHAL REPERFUSION ARRHYTHMIAS IN ANAESTHETIZED RATS Istvdn Baczk6, Istvfin Leprfin, Andrfis Varr6, Julius Gy. Papp. Department of Pharmacology, Albert SzentGySrgyi Medical University, Szeged, Hungary Controversial data have been published concerning the effects of agents acting on ATP-sensitive potassium channels (KAT P) on reperfusion induced arrhythmias. In the present experiments the influence of a KAT P opener, pinacidil, and the effect of glibenclamide, a KATp inhibitor, were investigated on the mmdence of reperfusion induced arrhythmias. In anaesthetized, artificially ventilated Sprague-Dawley CFY rats myocardial ischaemia was produced by the ligation of the left main coronary artery for 6 min. The release of the ligature was followed by reperfusion induced arrhythmias within 20 seconds. Pinacidil given in a dose of O.1 mg/kg intravenously significantly decreased the incidence of ventricular fibrillation during reperfusion (37 % vs. 100 % in controls). Moreover, 2 1 % of the animals survived without developing any arrhythmia. This dose of pinacidil, as well as 5 mg/kg glibenclamide, given intraperitoneally, significantly improved the survival rate (68 % and 81 % vs. 9% in controls, respectively). Pinacidil, in a larger dose (0.3 mg/kg) did not protect against reperfusion arrhythmias in anaesthetized rats but permanently decreased the mean arterial blood pressure (78+7.4 vs. 107+5.8 mmHg in controls). Our conclusion is that both activating and inhibiting KAT P may offer beneficial effects against reperfusion induced arrhythmias in anaesthetized rats. This work was supported by the Hungarian National Research Fund (OTKA No. T 5270).

DOTEMPORALELECTROPHYSIOLOGICALANDCOLLATERALTu072 FLOW CHANGES MIRROR PRECONDITIONING-INDUCED INFARCTSIZELIMITATIONINTHEPIG? Michael J Shattock, Clive S Lawson, David J Hearse, and James M Downey*. Cardiovascular Research, St Thomas' Hospital, London & *Univ. of South Alabama, USA. Temporal changes in S-T segment elevation during repetitive short (<2min) balloon inflations during angioplasty have been cited as evidence for preconditioning in man. However, these changes may reflect recruitment of collaterals. This study characterised temporal changes in the S-T segment and collateral flow, in the preconditioned pig heart. Open-chested pigs were subjected to 2 cycles of 8min LAD occlusion and 8min reperfusion prior to 60rain ischemia and 2h reperfusion. Two epicardial ECGs were continuously recorded from the ischemic zone and one ECG from the normal zone. Flow was measured using Xenon washout. Infarct (IS) and risk zone (RZ) sizes were assessed after repeffusion in a subset of 6 pigs and confirmed profound protection with preconditioning (IS/RZ=14+10% vs 42_~3%in controls, P<0.05). S-T segment elevationwas smaller early (0-4 min) in the 2nd or 3rd ischemic cycles than in the 1st (integrated S-T elevation was 11.7+3.0, 9.5+_2.9and 6.5_+l.9*mV.minrespectively:*P<0.05). However, this difference was lost later during ischemia. Collateral flow during the 3 ischemic cycles was 4.8+3.7, 5.8+_2.3 and 5.6+_2.9% (n=5/grp, ns) respectively.Thus, in the absence of significant changes in collateral flow, S-T segment changes provide a sensitiveindex of preconditioning but only during the first few minutes of occlusion. S-T segment changes dudng short (1-2rain) repeatedangioplastyocclusions may, therefore, be indicativeof preconditioning in man. A143

LESS ST SEGMENT ELEVATION DURING Tu073 SUBSEQUENT CORONARY OCCLUSIONS INDICATES PRECONDITIONING IN RABBIT Michael V. Cohen, Xi-Ming Yang, I. Raymond Fletcher, & lames M. Downey. Univ. So. Alabama, Mobile, AL ST elevation diminishes with subsequent balloon inflations in clinical angioplasty. However, whether this is evidence of preconditioning (PC) or merely recruitment of collaterals is unclear. Open-chest rabbits which have negligible collateral flow were subjected to 2 cycles of 5 min coronary occlusion and I0 min reperfusion prior to 30 rain ischemia and 3h repeffusion. Epicardial ECGs from ischemic and normal zones were continuously recorded. Infarct size was measured with tetmzolium. ST segment elevation after 5 rain occlusion averaged 7.8__. 1.5my for the second and 6.84-1.3mv for the third occlusion and both were significantly less than that during the first occlusion (9.3 4-1.3mv) (p < 0.005). 8-(p-sulfophcnyl)theophylline (SPT, 7.5mg/kg), an adenosine receptor antagonist, injected 5 rain prior to each occlusion abolished the reduction of ST segment elevation seen during second and third occlusions. SPT also blocked the limitation of infarct size typically observed following ischemic PC (32.8 4-1.7 % infarction of risk zone). ST segment changes during the second I0 rain reperfusion returned to baseline much more quickly than after the first occlusion (p < 0.05). Curiously this hastened ST segment recovery was not blocked by SPT. Hence lessening of the magnitude of ST segment elevation during later coronary occlusions does indeed reflect the onset of PC. Because the effect was blocked by SPT, the mechanism is likely to be the same as for myocardial salvage.

CARDIOPROTECTIVE EFFECTS OF ISCHEMIC PRECONDITIONING ARE NOT DETERMINED BY THE ISOHEMIC TEMPERATURE.

Tu075

J.S. Juggi, A. Prehash, and G. Telahoun. Department of Physiology, Faculty of Medicine, Kuwait University, Kuwait. The effect of ischamic temperature during preconditioning on myocardial protection was evaluated in the globally ischemicreperfuaed rat heart. Preconditioning at 34"C (PC)or at 10"C (HPC) was achieved by two episodes of 5 mln ischemia and 10 mln reperfusion (34"C) respectively and the hearts were studied in three groups: Group A: PC followed by global ischemia at 34"C (I) for 60 min; Group B: HPC+I; and Group C: HPC+HI (hypothermic global ischemia at 10"C for 60 min). Therafter, all the hearts were reperfused at 34"C for 30 min when recovery in left ventricular contractility and coronary dynamics was recorded and compared (Table). % Post-lschemic Recover Prn~ CI SS LVEDP CF CVR PC+I 68 100 55 71 62 173 HPC+I 77 99 58 115 60 170 HPC+HI 72 102 81 70 74 146 Clffi contractility Index, SS = segment shortening, CF ffi coronary flow, CVR = coronary vascular resistance. Except for a significant improvement in SS with HPC+HI, there were no other signircant differences between PC+I, HPC+I and HPC+HI in the post-ischemic recovery of contractility and coronary dynamics. These results indicate that ischemic temperature during preconditioning does not determine the cardioprotectiva effect of preconditioning. (Supported by Kuwait University research grants MY021, and MLY-045-1 .)

A144

IS STUNNING AN IMPORTANT COMPONENT OF Tu074 PRECONDITIONING? Anna Cargnoni, C l a u d i o Ceconi, Palmira Bernocchi, Evasio Pasini, Salvatore Curello, Roberto Ferrari. Fondazione Clinica del Lavoro, Centre S. Maugeri, Gussago, Brescia; Cattedra di C a r d i o l o g i a , Universit~ di Brescia, Italy. The mechanisms of preconditioning (P) -mediated cardiac protection are not known and an ATP-spadng effect of P has been suggested. ATP-spadng, in turn, could be consequence of an incomplete recovery of ventdcular function or stunning after the brief P pedods of ischaemia. We tested this hypothesis in rat hearts subjected to different P protocols: Group 1 (PI), two 2 min of ischaemia followed by two 5 rain of reperfusion (rap); Group 2 (P2) identical to group 1 with the exception that the second reperfusion was prolonged to 20 min to allow a complete recovery of systolic pressure (SP). After these procedures, all hearts were made ischaemic for 30 min and then reperfused. Mechanical function, CPK release and tissue content of ATP and CP were determined. The data obtained are the following: sP before % of SP total CPK CP ATP ischaemia recovery release mmol/gdw mmol/gdw rnrnHg after rap. mU/min219ww C 77.7+7.0 56.9~7.6 54905±5734 16.8±2.4 3.9~0.5 PI 60.4±4.1" 75.7±6.1" 40064+7128 31.4±1.8" 6.2±0.8° P2 82.8±5.4 59.7±12.8 32043±4974 17.8±2.7 3.9~0.5 CP=creatine phosphate * p< 0.05 vs C (control) Thus P resulted in cardiac protection only when the prolonged ischaemia was started before a complete contractile recovery. It is likely that stunning plays an important role by sparing ATP.

INTRMSCHESOCPRECONDITIONING:INCREASED Tu076 TOLERANCETO SUSTAINEDLow-FLOW ISCHEMIA BY A BRIEF EPISODE OF NO-FLOW ISCHEM~, WrrHotrr INTERMI'ITI~NTREPERFUSION Rainer Schulz, Heiner Post, Samir Sakka, David R. Wallbridge, G. Heusch. Department of Pathophysiology, University of Essen, FRG Recent data from isolated buffer-perfused rabbit hearts pointed to a link between ischemic preconditioning (IP) and myocardial hibernation (MID in that an initial stimulus of no-flow ischemia (I) was required to permit the development of MH during subsequent sustained low-flow I. In the in situ pig heart we therefore investigated whether a brief episode of no-flow I enhances the myocardial tolerance to subsequent sustained low-flow I. By blocking ATP-dependent potassium channels (K^rp) we attempted to further determine whether such increased tolerance to I is related to IP or MH, since blockade of K^~ with glibenclamide ((3) abolishes the cardio,protection achieved by IP but not that by MH. In 8 enflursne-an'esthetized pigs (group 1), inflow into the cannulated LAD was reduced to achieve a 90% reduction in anterior myocardial function (sonomicrometry) for 90 rain. In 15 pigs (group 2), l0 rain no-flow I preceded 80 rain sustained I at an identical blood flow reduction as in pigs of group 1. In 8 additional pigs (group 3), G was administered prior to the l0 min no-flow I. In all pigs, infarct size (IS, % of area at risk) was determined by TTC-staining following 120 min reperfusion, mean+SD, *:P<0.05 vs. GROUP 1, ona-way .ANOVA IS ENDO in T r c + GROUP 1 13.2+9.8 0.11+0.04 GROUP 2 6.8+6.0* 0.06+0.02* GROUP 3 16.7+8.3 0.10+0.06 In group 2, subendocardial blood flow (microspheres, ENDO, ml/min/g) of tissue which remained viable (TTC+) was lower than that in groups 1 and 3. Thus, a brief episode of no-flow I without intermittent reperfusion increases the tolerance to sustained lowflow I. The cardioprotective effect is mediated by activation of K^Tp, and therefore relates to IP rather than to MH.

PRECONDITIONINGAGAINSTCONTRACTILEDYSFUNCTION TU077 is ABOLISHEDINTHEPRESENCEOF RESIDUALFLOWIN THEISOLATED BLOODPERFUSEDRAT HEART.

Alison C. Cave, Adam S. Silverman, Cad S. Apstein. Boston University, Boston, MA. We have previously demonstrated that preconditioning (PC) increases functional recovery after prolonged zero-flow ischemia (Isc) in the rat heart, but does not protect when O2 deprivation is accompanied by a high level of perfusion (hypoxia). Since most clinical ischemla is low flow, not zero flow, we wished to determine whether PC could protect against a prolonged period of low flow ischemla at a clinical level of ischemic perfusion. Thus, functional recovery after 30 rain of repedusion (Rep) was assessed in Isolated blood pedused hearts subjected to one of four protocols (n=6/group): (A) 25 min of zero flow Isc without PC, (B) 25 rain of zero flow Isc preceded by 3 cycles of PC (PC=5min Isc + 5rain Rep), (C) 90 rain of low flow Isc at 10% of baseline coronary flow (0.32_+0.01 ml/min/g wet wt.) without PC, (D) 90 rain of low flow Isc preceded by 3 x PC. Groups A and C were subjected to 30 rain more pre-ischaemic pedusion than groups B and D so that total pedusion time was equal in all groups. Contractile function was assessed throughout via a LV balloon; LVEDP was set initally Io 10mmHg. PC provided significant protection following 25 rain of zero flow Ischemla (recovery of developed pressure (DP) was 67:t:6% vs 3i+12% in preconditioned (B) and control (A) groups respectively;, p<0.05) but, no protect/on from PC was apparent either during or following low flow ischemla (recovery of DP was 40-2:8 vs 37+7% in control (C) and preconditioned (D) respectively). In further experiments, inclusion of adenosine (100p.M) during the period of low flow ischemia could not restore PC (recovery of DP was 36+8 vs 26+7% in control and preconditioned groups respectively). In conclusion, ischemic PC cannot protect against contractile dysfunction when a clinically relevant level of ischemic pedusion remains during the prolonged insult.

CHARACTERIZATION OF DIFFER Tu079 ENTIAL EXPRESSED GENES FOLLOWING SHORT CORONARY OCCLUSIONS Ralph Kniill, Ren~ Zimmermann, Klaus Berwing*,

Margarete Arras, Wolfgang Schaper. Max-PlanckInstitute, Kerckhoff-Clinic*, Bad Nauheim, Germany. Short coronary occlusions mainly cause long lasting but reversible myocardial dysfunctions as well as ischemic preconditioning. In order to identify differential expressed genes, "probably responsible for these reactions, we employed differential display reverse transcribed polymerase chain reaction (=DDRT-PCR) on postischemic pig myocardium subjected to 2 cycles of 10 minutes of coronary occlusion followed by 30 minutes of reperfusion. Total RNA, isolated from the LAD (=experimental area) as well as from the circumflex area (=control area), was reverse transcribed by the use of 12 different downstream poly A primers and the resulted eDNA was amplified by the use of additional 26 upstream primers. Fragments showing differential ex-pression after gelelectrophoresis and autoradiography were reamplified, cloned and sequenced (600 clones expected). One of these clones, a mitochondrial creatine kinase, revealed differential expression by northern blotting. Some clones showed homologies to vitronectin-receptors (58%), proteinphosphatases (62%), lactate dehydrogenases (57%), interferon receptors (52%), vitamin D binding proteins (61%) or retinoblastoma sus-ceptibility genes (53%). About 30% of the fragments represent transcripts of unknown genes. We conclude that DDRT-PCR is applicable on myocardial tissue and that dysfunctions following short term ischemia might be the result of unexpectedly complex molecular reacti6as._

TIME COURSE OF PACINGTu078 INDUCED PRECONDITIONING IN DOGS. Karoly Kaszala, Agnes Vegh, James R Parran*, Julius Gy Papp, Department of Pharmacology, Albert Szent-Gyorgyi Medical University, Szeged, Hungary, *University of Strathclyde, Glasgow, UK We have shown (Cardiovasc Res 1991, 25, 1051) that marked suppression of ventricular arrhythmias during myocardial ischaemia is achieved following short periods of cardiac pacing. Cardiac pacing also protects against arrhythmias when performed 24 h before the ischaemic stress (Br J Pharmac 1994, 113, 1081). In the present study we examined whether antiarrhythmic protection exists I, 6, 48 and 72 h after pacing. Cardiac pacing was performed (4 x 5 rain, 220 beats rain"1) through the fight ventricle, using a bipolar pacing electrode, in pentobarbitune anaesthetised spontaneously ventilated dogs. The animals were subjected to a 25 rain occlusion-reperfusion of the LAD, 5 rain (n = 11, group I), I h (n = 8, group 2), 6 h (n = 8, group3), 24 h (n = 10, group 4), 48 h (n = 9, group 5) and 72 h (n = g, group 6) after pacing. The incidence of arrhythmias, as occlusion-induced ventricular premature beats and ventricular fibrillation, and survival from the occlusion-perfusion insult were as follows: Group I: 202 + 69; 0%; 55%, Group 2:247 + 114; 75*/,,; 13./o, Group 3:180 + 26; 62.5%; 12.5%, Group 4:77 :t: 32; 10%; 60%, Group 5:79 + 34; 56%; 22%, Group 6:98 + 34; 50%; 12.5%. We can conclude, that 4 x 5 rain pacing is sufficient to precondition the heart. 1 h after pacing the protection is lost but reappears by 24 h and is markedly diminished after 48 and 72 h. The time course of the protection achieved by pacing-induced preconditioning is similar to that brought about by short periods of coronary occlusion. Supported by OTKA, British Council, OMFB and the European Commission (ERB CT 924009).

ISCHEMIC PRECONDITIONING ATTENUATES To080 INCREASES IN [H+]i AND [Ca2+]i DURING PROLONGED ISCHEMIA IN ISOLATED RAT HEARTS Hiroyuki Okumura, Satoshi Takeda*, Kohji Tamura*, Takeki Ohashi**, Shingo Seki, Masayuki Taniguchi, Seibu Mochizuki. Department of Medicine Jikei University School of Medicine Tokyo, *Yamanashi Medical University and **Osaka University Medical School, Osaka, Japan. Although the mechanisms of ischemic preconditioning (IP) could related to ionic alternations, especially [Ca2*]i, there is few direct evidence to support this hypothesis. The aim of present study is to test this hypothesis in the intact heart. We studied isolated perfused rat hearts loaded with BCECF and Fura-2 which are the fluorescence indicators of pHi and [CaZ*]i, respectively. In IP group (n=18), hearts were preconditioned with two 5-rain episodes of ischemia separated by 5-rain of repeffusion. Control hearts (n=6) received no preconditoning. All hearts received 20-min of whole heart ischemia and 20-rain reperfusion. Percentile recovery of LVP in controls was 72.3+6.5%, whereas IP revealed significant recovery (p
DENSITY OF L-TYPE CALCIUM CHANNELS AFTER Tu081 60 MIN ISCHEMIA IN NORMAL AND ISCHEMICALLY PRECONDITIONED MYOCARDIUM Knut A. Kirkebeen, Mimi Stokke, P~II A. Naess, Else M. Hagelin, Arnfinn Ilebekk & Odd Brers. Inst. Expr. Med. Res. and Div. Clin. Pharmacol. Toxieol., Ullev~l Hospital, Oslo, Norway. L-type calcium channels (LCC) regulate sareolemmal calcium ion (Ca2.) influx. Ischemic preconditioning (IP) might be explained by reduced cellular Ca"* influx and cytosolic Ca~ overload resulting in delayed cell injury during subsequent Ionglasting ischemia, We tested if IP is associated with reduced density or altered state of LCC in sarcolemma or cytosoI after 60 min ischemia. The density and the dissociation constant of (+)-[3H]isradipine binding to LCC in membranes and homogenates in IP (two I0 rain LAD occlusions, 30 rain reperfusion) (n=7) and non-IP (n=7) porcine hearts were compared. Biopsies were obtained after 60 min LAD occlusion from iscbemic regions and control regions supplied by the circumflex artery. The density of (+)-[3H]isTadipine binding sites in membranes was reduced by 23.3+ 10.5 (Mean+SEM) % (p<0.05) in non-IP hearts and by 20.3+ 7.5 % (p<0.05) in IP hearts. In homogenates it was reduced by 35.5±5.5 % (p<0.05) in non-IP hearts and by 21.3±4.5 % (p<0.05) in IP hearts. The reductions in the two groups and the reductions in membranes and homogenates were not statistically different. The dissociation constant of (+)-[3H]isradipine binding to LCC was not altered in either group. In conclusion, 60 rain ischemia reduces the density of (+)-[~H]isradipine binding sites in membranes and homogenates by 20-35 %. This shows that the reduction in density of binding sites is not caused by redistribution of LCC to cytosol. IP does not affect the decline in binding sites. Thus, the protective effect of IP does not involve changes in density or state of LCC that can be detected by (+)-[3H]isradipine binding.

THE EFFECT OF MULTIPLE REPETITIVE PRETU083 C O N D I T I O N I N G IN A C O L L A T E R A L D E F I C I E N T S P E C I E S

Efstathios K.Iliodromitis,Costes Papadopoulos,Zenon S. Kyriakides, Ioannis Paraskevaidis,Constantina Flessa,Nikolaos Soudas,Dimitrios Th. Kremestinos, Onassls Cardiac Surgery Center, Athens Greece Few cycles of short ischemia and reperfusion confer protection to the vulnerable ischemic myocardium. This study was designed to determine in a collateral deficient species: a. Whether multiple repetitive periods of 5 min ischemla and 10 min reperfusion (5 iscfl0 rap) are protective and b. The effect of an additional 5 rain ischemic preconditioning (PC) given 60 min after the last short ischemia and 10 mln prior to the sustained ischemia (SI). 28 rabbits were divided in 4 groups and were: Group A (n=7) 6 times to 5 isc/10 rap and immediately after to 45 mln SI. Elapsed time from the first PC to SI was 90 min. Group B (n=7) 6 times to 5 isc/10 rap and 60 min after the last PC to 45 min SI. Elapsed time from the first PC to SI was 140 rain. Group C (n=8) six times to 5 isc/10 rap but 60 rain after the last PC to another 5 lsc/10 rap and immediately after to 45 min SI. Group D (n=6) was finally subjected six times to 5 iscfl0 rap without any SI. All animals were subjected to a final 120 min raperfusion, infarct size (I) was measured using tetrazollum staining, risk zone (R) was delineated with the aid of fluorescent particles and I/R ratio was expressed in percent. Groups A B C D % I/R 40.4+ 7.2 38.1i-6.6 11.4_+.2.2 0±0 p values: C vs (A,B) <0.01. A vs B = NS. The results indicate that multiple repetitive PC are no protective in collateral deficient species. Afforded protection depends on the elapsed time between the first PC and the SI. An additional PC given immediately pdor to the SI restores the protection. Myocardial infarction does not appear after the introduction of multiple PC only.

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PRECONDITIONING OF PERFUSED RAT HEART Tu082 INHIBITS REPERFUSION-INDUCED RELEASE OF INOSITOL( 1,4,5)TRISPHOSPHATE. Karen E. Anderson and Elizabeth A. Woodcock, Baker Medical Research Institute, Australia. Preconditioning the heart by brief episodes of ischemia and reperfusion can be cardioprotectivc to injury from subsequent sustained ischemia and reperfusion. Such protective effects include the reduction of repeffusion-induced arrhythmias. We have previously reported a rapid rise in the second messenger inositol( 1,4,5)tr/sphosphate (Ins(l,4,5)P3) with post-ischemic reperfusion (Anderson et al., 1995 Circ Res, in press), which has been implicated in the generation of reperfusion-induced arrhythmias. Studies were performed in isolated perfused rat hearts to determine if preconditioning could reduce the reperfusion-induced rise in Ins(l,4,5)P3. 3H-labeled hearts underwent 20 rain global ischemia with or without 2 min reperfusion. This was proceeded by preconditioning of 2 or 3 cycles of 2 or 5 rain ischemia separated by 5 or 10 min reperfusion, or by dme matched control perfusions. Two min reperfusion following 20 rain ischemia in dme matched controls caused an increase of [3H]Ins(l,4,5)P3 from 20 + 3 cpm/mg protein to 52 ± 5 cpm/mg protein (mean + SEM, n--4, p<0.01). Preconditioning of three cycles of 5 min ischemia and 5 rain reperfusion inhibited the 2 rain reperfusion-induced rise in [3H]Ins(l,4,5)P3 (26 + 4 cpnYmg protein p<0.01 rcladve to 20 min ischemia), however protocols that involved either fewer ischemic cycles or shorter ischemic periods were ineffective. Preconditioning did not effect myocardial ATP levels or norepinephrine release with sustained ischemia and reperfusion. Inhibition of the reperfusioninduced rise in Ins(1,4,5)P3 may provide an explanation for the inhibitory effects of preconditioning on reperfusion-induced arrhythmias.

Tu084 ALTERATIONS IN CIRCULATING CYOLIC-GUANOSINE MONOPHOePHATE DURING SHORT AND LONG ISCHEMIA IN PRECONDITIONING

Efstathios K. Iliodromitis, Costes Papadopoulos, Manolis Markianos, Ioannis Paraskevaidis, Zenon S. Kyriakides and Dim±trios Th. Kremastinos. Onassls Cardiac Surgery Canter, Athens, Greece. The aim of this study was to evaluate the alterations in circulating cyclic-guanosine monophosphate (c-GMP)in preconditioning (PC). 16 rabbits were divided in 2 groups (Gp) and in-viva hearts were preconditioned either with lmin (GpA) or with 5rain (GpB) ischemia (isc), followed by 10min reperfusion (rep).Protection was determined subjecting both groups to 30rain regional isc followed by 2 hours rep. 7 blood samples were drawn from every animal for c-GMP assessment;A or B was used for the samples of GpA or GpB respectively: A1 and Bl,baseline values,A2 end of the 1st rain isc,B2 end of the 5th min isc,A3 and B3 1 rain before long isc,A4 and B4 the 15th rain,A5 and B5 the 30th min of long isc,A6 and B6 the 1st min rep,A7 and B7 the 1st hour of rep.Results are expressed in pmol/ml, risk zone (R) was defined by the use of fluorescent particles, infarct area (I) by tetrazoltum and %IIR ratio was calculated by planimetry. All results are in mean values +ISEM. %I/R: GpA (n=8) 29.9 4. 2.1 GpB (n=8) 12.2 ± 1.4 p<0.001 A1 A2 A3 A4 A5 A6 A7 44.9+6 43.3+4 36.7+4 43.9+4 51.2+7 43.1+8 41.7+7 B1 B2 B3 B4 B5 B6 B7 47.0+4 52.2+6 48.0+9 60.5+4 60.44-4 52.74-6 38.74-4 p: (B1) vs (B4, B5) <.05. (A4) vs (B4) <.05. Circulating c-GMP increases only in the classically preconditioned animals during the long isc (GpB). Furthermore, this group has significantly higher circulating o.GMPlevels in comparison to GpA after 15 rain of isc. c-GMPmight be involved in the mechanism of PC.

INDOMETHACINIMPROVESIN-VIVO THE BENEFICIAL TU085 EFFECTOF HEAT SHOCKPROTEINSIN RABBIT HEARTe Efstathios K. lliodromltis, Costes Papadopoulos, Nikolaos Sourlas, Zenon S. Kyriakides, Constantina Flessa, Dimitrios Th. Kremastinos. Onass/s C..ard/acSurgery Center, Athens, Greece. Whole body heat stress limits the infarct size after 45 min ischemia in-vitro but not in-vivo. Protection of the heat stressed rabbit hearts has been observed in vivo when they were exposed to 30 min Ischemia only. We investigatedthe effect of indomethacin (IND), in an attempt to determine if the inflammation negates in-vivo any benefit obtained by the heat shock proteins. Twenty-four rabbits were divided in 3 groups of 8 animals each. After anesthesia, in group (Gp) A body temperature raised to 42°C for 15 min; in Gp B the same protocol but with the addition of IND (15 mg/kg iv for 60 min, starting 30 min before fever). Gp C served as control and was subjected for 15 rain in sham heat. All the animals recovered for 24 h, then re-anesthetizedand subjected to 30 min ischemia and 2 h reperfusion. In Gp B, additional IND (15 mg/kg) was administered from the beginning to the end of experiment. Infarcted (I) and risk (R) areas were delineatedwith the aid of tetrazolium staining and Zn-Cd fluorescent particles and their ratio was expressedin percent

(%I/R).

Gp A Gp B Gp C %I/R 33:L-5.2 "16.1+4.4 48.9+4.0 p: BvsC<0.001, BvsA and AvsC<0.05. This study shows that IND augments the beneficial effect of heat shock proteins in-vivo. Inflammation is involved in the heat stressed ischemic heart decreasingthe favourableoutcome in-vivo.

OXYGEN RADICALS MAY INDUCE Tu087 PRECONDITIONING IN ISOLATED HEARTS. Isabella Tritto, Annalisa Scognamiglio, Pietro P. Ella, Davide D'Andrea, Nicola Eramo, Ciro De Simone, Annamada Esposito, Plinio Cirillo, Massimo Chiariello, Giuseppe Ambrosio*. Divisions of Cardiology, Univ. of Naples and Perugia*, Italy. Oxygen radical (OR) scavengers have been shown to prevent development of preconditioning, suggesting that this phenomenon may be partly mediated by ORs. The present study was designed to evaluate whether exposure to ORs per se, in the absence of ischemia, would reproduce the beneficial effects of ischem.ic preconditioning on infarct size. A first group of isolated rabbit hearts was subjected to 30 min of regional ischemia, induced by coronary artery occlusion, followed by 2.5 hs of reflow (Controls; n=8). Another group was initially exposed for 5 rain to a low flux of ORs (5 nmoles/min/ml) generated by infusing purine/xanthine oxidase (P/XO); this dose had minimal hemodynamic effects. After 15 rain of washout also this group underwent 30 min of coronary artery occlusion, followed by 2.5 hrs of reflow (P/XO; n=7). Risk region (RR) was delineated by injecting fluorescent particles and it was similar in Controls (56+4% of left ventricle) and in the P/XO group (58+3% of left ventricle). In contrast, infarct size (by triphenyltetrazolium staining) was significantly reduced in the P/XO group (13+4% of RR vs 49-~_17%of RR in Controls; p<0.05). Thus, our data show that a brief exposure to low concentrations of oxygen radicals may precondition the heart toward a subsequent period of ischemia, and suggest that the beneficial effects of preconditioning induced by short ischemic episodes might be partly due to oxygen radical generation.

RELEASE OF (GLYCO)PROTEINS DURING Tu086 ISCHEMIC PRECONDITIONING OF DOG MYOCARDIUM. Jfin Styk, Albert Breier, Narcisa Tribulov~i, Ivan Gabauer, J=in Slez~k & Dezider Pancza, Inst Heart Res Bratislava, SK Multiple brief episodes of ischemia followed by reperfusion render the myocardium more resistant to subsequent severe ischemia. This phenomenon has been known as ischemic preconditioning. The precise mechanisms remain rather unclear. Release of (glyco)proteins from the dog myccardium to the blood serum during experimental preconditioning procedure was tested in the present study. Blood serum samples from the control and after short-term ischemia and reperfusion intervals (tree times repeated) were precipitated with 50% ammonium sulfate and centrifuged (3000xg). Supematant was additionally precipitated by 80% ammonium sulfate and pellet was resolved in PBS. Glyeoproteins were estimated using concanavalin A (Con-A) and mouse polyclonal anti-Con-A antibody by ELISA and Western bloting. Significant increase of Con-A reactive glycoproteins was observed after precondition procedure applied. Western blot analysis of described blood serum fraction revealed the several new protein's bands after third occlusion and reperfusion period in comparison with the control. Some of them could be stained by Con A and anti-Con-Antibody. The same blood fraction when applied intravenously exerts a eardio-protective effect against ischemia in nonpreconditioned dog myoeardium.

THE INFLUENCE OF AGE AND HORMONAL STATUS Tu088 ON MYOCARDIAL ISCHAEMIC PRECONDITIONING. Rachael A. Humphreys, Kathleen A Kane & James R Parratt, Dept of Physiology & Pharmacology, University of Strathclyde, Glasgow, UK. Previous studies of myocardial ischaemic preconditioning (pc) have been carried out in young healthy male animals. The aim of this study was to investigate whether age or sex influenced the antiarrhythmic effect of pc. Three groups of Sprague-Dawley rats were studied: group 1 = 3 month old males (300-350 g), group 2 = 3 month old females (200-270 g) and group 3 = 16 month old males (800-1300 g). Rats were anaesthetised with pentobarbitone sodium (60 mg kg-1 i.p.) and the left coronary artery occluded for either 30 min (control) or 3 min followed by 10 min reperfusion before the permanent 30 min occlusion (pc). In group 1 animals, pc had a marked antiarrhythmic effect reducing significantly the number of ectopic beats from 2074:1:206 (n=10) to 490+139 (n=8) and the % incidence of ventricular fibrillation (VF) from 40 to 0%. Female controls exhibited fewer ectopic beats on occlusion than males (1130+287 [n=8] vs 2074+206) and had no sustained VF (0 vs 40%). Pc protocol led to a further reduction in the number of ectopic beats in females to 154+48. Control aged rats had an 80% incidence of VF on occlusion (n=10) and this was markedly attenuated by pc (to 17%). In these rats, pc also reduced the number of ectopic beats from 1797+100 to 300+93. Heart rate and arterial blood pressures, prior to occlusion, were not significantly different between the 3 groups with the exception that arterial blood pressure was lower in female than male controls (76:t:5 vs 101+7 mmHg). It is concluded that neither age nor hormonal status modifies the antiarrhythmic effect of pc in anaesthetized rats.

A147

Tu089 MYOCARDIAL PROTECTION BY TRANSIENT RENAL ISCHEMIA IN RATS IS TEMPERATURE DEPENDENT Ben CG Gho, "Regien G Schoemaker & Pieter D Verdouw. Exp. Cardiology, Thoraxcenter, "Dept. of Pharmacology, Erasmus University Rotterdam, Rotterdam, The Netherlands

T a t y a n a O x m a n , Natalia A v a z o v , Ella Elazar, Rodica Klein, L i a n a Horowitz, Michael Arad, B a b e t h R a b i n o w i t z . Heart I n s t i t u t e , T e l - H a s h o m e r , Israel.

Ischemic preconditioning is usually acquired by transient ischemic stress preceding the infarction in the same area. Our previous study showed that "remote" myocardial protection can be obtained by transient renal artery occlusion, however, data were scattered. Therefore, we studied whether temperature modulates the cardioprotective effect of transient renal ischemia. Anesthetized rats were assigned to two study groups, one in which the body temperature was kept "normal" between 35"C and 36°C (NT) and in the other it was kept "low" between 30°C and 31 °C (LT). In both groups, the animals underwent a transient 15 min left renal artery occlusion and 10 rain reperfusion ("fRO) before a 60 min left anterior descending coronary artery occlusion (CO) followed by 3 hours of reperfusion (NT-TRO, n=7 and LT-TRO, n=9). Control animals underwent an abdominal sham operation 25 min before the CO (NT-Con, n=6 and LT-Con, n=6). The area at risk (AR as % of left ventricular mass) was delineated with Trypan Blue (negative staining) and the infarct area (IA) was negatively stained with NitroBlue-Tetrezollum. In NT-Con and NT-TRO the AR were not different (28 + 5% and 32 :t: 8%, respectively) neither was the developed INAR in NT-Con and NT-TRO (72 ± 4% and 69 + 3%, respectively). In LT-Con and LT-TRO the AR were not different (39 + 4% and 35 + 3%, respectively), whereas the developed INAR was significant limited in LT-TRO (49 + 6% compared to 69 + 3% in LT-Con, p = 0.02). The INAR in NT-Con and LT-Con were not different, indicating that the lower bodytemperature itself did not limit INAR. We conclude that bodytemperature modulates the cardioprotective effect of transient renal artery occlusion in rats.

P r e c o n d i t i o n i n g (PC] is classically i n d u c e d by s i n g l e or repetitive brief e p i s o d e s of regional i s c h e m i a (RI] in t h e heart. T o d e t e r m i n e if a cardioprotective effect c a n be o b t a i n e d by ext.racardiac i s c h e m i e PC we i n v e s t i g a t e d t h e a n t i a r r h y t h m i c effect d u r i n g RI a n d r e p e r f u s i o n [R) in 3 g r o u p s of S p r a g u e - D o w l e y L a n g e n d o r f f p e r f u s e d rat h e a r t s : I - control (N:18) 30 m i n RI + R , II - PC [N:12] b y s i n g l e 5 m i n RI + 15 rain R followed by 30 rain RI + R a n d III PC [N:20] by 10 rain limb i s c h e m i a ILl] + 10 rain R, p r o d u c e d in vivo by t o u r n i q u e t at lower limb, followed b y 30 rain RI of t h e h e a r t + R. T a e h y a r r h y t h m i a at R [RTA] w a s defined a s v e n t r i c u l a r t a e h y c a r d i a or fibrillation of at least 5 s e c d u r a t i o n . RTA, t h e release of p r o s t a c y c l i n [PGI 2] a n d n o r e p i n e p h r i n e (NE] in t h e c o r o n a r y effluent were c o m p a r e d b e t w e e n t h e 3 g r o u p s . Results: R T A w a s r e d u c e d in g r o u p s II a n d III (1/12 a n d 2/20 v s 12/18 in I, p < 0 , 0 2 ] . In g r o u p Ill t h e b a s a l release of PGI 2 a n d NE w a s h i g h e r t h a n I (p < 0.05). R e l e a s e of PGI 2 a n d NE d u r i n g R h a d n o s i g n i f i c a n t difference b e t w e e n g r o u p s . C o n c l u s i o n s : Brief i s c h e m i a of a n e x t r e m i t y p r o t e c t s t h e h e a r t w i t h regional i s c h e m i a a g a i n s t r e p e r f u s i o n t a c h y a r r h y t h m i a s . T h i s a p p e a r s to be a p r e c o n d i t i o n i n g effect w h i c h m o s t p r o b a b l y h a s a different m e c h a n i s m of a c t i o n t h a n brief i s c h e m i a in situ.

M E T A B O L I C E V I D E N C E OF Tu091 P R E C O N D I T I O N I N G IN H U M A N S DURING P T C A Kameljit K. Kalsi, Nigel S.Jep.son, Charles D. Ilsley, nne-Marie L. S e y m o u r . u e p t s o f Caroio-Thoracic Surgery, (NHLI) 8; Caroiology, Harefield, Middx. UK.

E N E R G E T I C S OF I S C H E M I C P R E C O N D I T I O N E D T u 0 9 2 DOG MYOCARDIUM Oleg I. Pisarenko, Olga V. Tskitishvily, Irina M. Studneva & Larisa I. Serebryakova. Cardiology Research Center, Moscow, Russia

Evidence for p r e c o n d i t i o n i n g in humans remains controversial. The threshold for ischaemic injury is increased (based on angina and ECG changes) following repeated coronary occlusions at PTCA. T h e aim of this study was to measure the extent of iscnaemia by monitodnjg the release of Hypoxanthine (Hx, an !noex of iscnaemia) into the blood ano myocaroiai . energy metaoohte revels in . oiopsy specimens our!ng angioplasty. 14 male.patients Lage 53.6+2.2 years) unoergoing PTCA OT isoJateo prox=mai left anterior descending (LAD) artery stenoses (>70%) were studied. LAD collateral filling was absent and LV function normal. PTCA comprised of three balloon inflations; 1) 90 secs, 2) .90 secs 3) 1.80-300 secs with 5 minutes of reperfusion cetween eacn inflation. Paireo artedo-venous (A-V) blood samples were taken before PTCA and after each deflation. Endomyocaroial biopsies were taken from the LAD perfusion area immediate!y before and after the first and third balloon inflations. Hx, ATP and total adenine nucleotides ('rAN) content were analyzed by HPLC. (+ SEM) A - V differences of Hx pre-PTCA 1 2

This study was designed to assess effects of myocardial preconditioning (PC) on energy metabolism of dog heart in situ during regional ischemia and subsequent reperfusion. The control group was subjected to 40-min occlusion of the left anterior descending coronary artery (LAD) followed by 1hr reperfusion. The PC group received 5-rain occlusion of LAD followed by 10-rain reperfusion prior to the same period of sustained regional ischemia and reperfusion. Group 3 was treated additionally by 8-p-sulphophenyl theophylline (SPT, 5 mg/kg iv over 30 s) 20 min before PC. Interstitial (IS) lactate (Lac) and creatine (Cr) were sampled by microdialysis probes implanted in the control and risk area of the left ventricle. Biopsies from both areas were taken for measurement of ATP, ADP, AMP, phosphocreatine (PCr), Cr and Lac. In the control area, tissue and interstitial metabolite contents did not change in all groups throughout the experiment. PC significantly reduced ATP, ADP and PCr content and slightly increased AMP and Cr level in the risk area before ischemia. By the end of ischemia, Lac, Cr and AMP accumulation and degree of ATP, ADP and PCr depletion were markedly reduced by PC. After reperfusion, the PC group showed a significantly better recovery of PCr, lesser loss of Cr and reduction of myocardial Lac compared to the control one. In the PC group, an increase in IS Lac and Cr for the first period of ischemia/reperfusion was accompanied by a reduction of Lac and Cr release from myocytes during the sustained ischemia and raperfusion compared to these indices in the control. SPT pretreatment attenuated PC influence on the energy state of ischemic and reperfused myocardium and significantly increased IS Cr and reduced IS Lac accumulation for the first period of ischemia/reperfusion. Thus, PC reduces energy-waste during ischemia which, in turn, promotes repletion of PCr stores during reperfusion. These effects are mediated in part via adenosine receptors.

10.03,0.2 I-2 s,2, 31-1

TAN

I-2 474-0 I

prel

Biopsies(pmol/g d r y wt) post1 %A p r e 3 post3

%A

22.14-2

14.523

-27.2

-34.5

17.122 12.54-2

Ischaemia is less severe following the long 3rd balloon inflation,oudng a,ngioplasty inoicating that the human myocaroium can De preconditioned during PTCA. A148

IN V l V O B R I E F L I M B I S C H E M I A Tu090 PROTECTS ISOLATED RAT HEARTS AGAINST REPERFUSION TACHYARRHYTHMIAS

ENHANCEMENT OF GLYCOLYSIS DURING AN EARLY Tu093 PHASE OF REPERFUSION AS A POSSIBLE MECHANISM FOR PRECONDITIONING IN ISOLATED RAT HEARTS Ken-lchJ Yabe, Yoshlhlsa Nasa, Mamoru Sato & Satoshl Takeo. Dept of Pharmacology, Tokyo University of Pharmacy and Life Science, Hachloji, Japan.

h p o s s i b l e mechanism f o r p r e c o n d i t i o n i n g (PC) o f i s o l a t e d r a t h e a r t s was e x a m i n e d . Rat h e a r t s were s u b j e c t e d t o 5-min I s c h e m i c PC b e f o r e 4 0 - m l n l s c h e m l a , f o l l o w e d by 30-min reperfuslon. Rate p r e s s u r e p r o d u c t ( h e a r t r a t e x left ventrlcular developed pressure), a measure of cardiac contractile f o r c e , was significantly I m p r o v e d by PC. The r a t i o o f [fructose 1,6-dIphosphate]/ [glucose 6-phosphate]+[fructose 6-phosphate] and t h e p h o s p h o f r u c t o k l n a s e a c t i v i t y a t t h e end o f l s c h e m i a and 5 - m i n o f r e p e r f u s i o n were h i g h e r i n t h e PC h e a r t t h a n in u n p r e c o n d l t i o n e d (unPC) h e a r t . PC e n h a n c e d t h e r e c o v e r y o f t i s s u e h i g h - e n e r g y p h o s p h a t e s (ATP and c r e a t i n e p h o s p h a t e ; HEP) a t 30-mln r e p e r f u s l o n . The m i t o c h o n d r i a l oxygen c o n s u m p t i o n r a t e o f t h e PC h e a r t was h i g h e r a t t h e end o f l s c h e m i a , 5-mln and 30-mln o f r e p e r f u s l o n t h a n t h e unPC h e a r t . The r e s u l t s s u g g e s t t h a t PC e n h a n c e s r e s t o r a t i o n o f t i s s u e HEP upon r e p e r f u s l o n which may be due t o acceleration o f g l y c o l y s l s In t h e EmbdenMeyerhof pathway and/or preservation of the m l t o c h o n d r l a l a b l l l t y t o g e n e r a t e HEP.

ENERGY M E T A B O L I S M I M B A L A N C E DURINGTu095 THE ISCHEMIC PRECONDITIONING PHASE CONTRIBUTES TO C A R D I O P R O T E C T I O N . Anne Garnier, Nieole Lavanehy. Lab. Bio~nerg~tique, Univ. J. Fourier, Grenoble, France. The kinetics of alterations in HEP were studied in isolated rat hearts during single and multiple i s c h e m i c preconditioning (IPC) using 31-P NMR spectroscopy. Aortically perfused hearts were subjected to a 25 min sustained ischemia and a 30 min reperfusion. The protocols' designed for IPC used a basic pattern of 3 rain ischemia + 6 rain reflow, increasing the 0ast) reflow period from 6 to 12 min. Efficient IPC (improved function on reperfusion) was associated during ischemia with a reduction in ATP degradation, in intracellular acidosis and a maintainance of a residual pool o f PCr. Analysis of the IPC phase showed that each short ischemia was followed by a vasodilation (40-60% increase), accompanied by a clear PCr overshoot (115-125% o f initial values), accounting for processes that consumed all the eytosolic free Pi (Pi undershoot). Thus, the energy producing reactions were brung out of their initial equilibrium. The P e r overshoot remained up to the onset of the sustained ischemia in the efficient models, whereas it has practically vanished in the unefficient ones. In efficacious multiple IPC, despite the lengthening o f the last reperfusion period, the PCr overshoot was still present, as if the memory o f Cr overphosphorylation was also lengthened by the multiple IPC. We suggest that a single IPC cycle induced, through cycling o f 0 2 deprivation and delivery, a time-dependent positive imbalance in the mitochondrial oxphnsphorylation reactions and that the benefit for the heart was exhibited only when the prolonged ischemia was imposed under such conditions. In addition, the duration of such a positive desequilibrium appeared reinforced by the multiple IPC.

EFFECT OF ISCHAEMIC PRECONDITIONING ON Tu094 MYOCARDIAL ISCHAEMIAIREPERFUSION INDUCED ARRHY'rHMIAS IN RATS FED SUNFLOWER SEED OIL RICH DIET Istv~in Lepr~n, IstvSn Baczk6, Julius Gy. Papp. Department of Pharmacology, Albert Szent-GySrgyi Medical University, Szaged, Hungary

The present experiments were devoted to investigate whether protection by ischaemic preconditioning could be elicited after a polyunsaturated fatty acid (PUFA) rich diet. Spragua-Dawley CFY rats were fed a PUFA-diet (10 % sunflower seed, SSO) or pork fat as a saturated fatty acid source (SF) for 8 weeks. At the end of the feeding period animals were anaesthetized by pentobarbitone (60 mg/kg i.p.) and after opening the chest wall myocardial ischaemia was produced by ligation of the left main coronary artery for 6 min that followed by reperfusion for 5 min. SSO diet significantly decreased the incidence of ventricular fibrillation (VF, 33% vs. 82%) and increased the survival rate (SR, 67% vs. 27%) during reperfusion after 6 min ischaamia. Preconditioning was consisted of a twominutes coronary artery ligation that followed by 5 min reperfusion. During this period arrhythmias were rarely occurred. Preconditioning significantly decreased the incidence of raperfusion induced arrhythmias in SF fed animals (VF=29%, SR=71%) and 14 % of the animals survived without developing any arrhythmias. Preconditioning in SSO fed rats somewhat further improved the chance to survive ischaemia/reperfusion induced arrhythmias (VF=23%, SR=85%). We may conclude that SSO diet alone decrease the incidence of ischaemia/reperfusion induced arrhythmias in rats. A single 2 min ischaemic preconditioning produced similar degree of protection and using this in SSO fed animals could further improve the protective effect.

Supported by OTKA No. T 5270.

IS PRECONDITIONING LINKED TO GLYCOGEN Tu096 DEPLETION?

Linda M. King, Lionel I I. Opie Heart ResearchUnit, UCT Medical School,Cape Town, South Africa Preconditioning(PC) hastens the time to onset (TOC) and peak of contractur¢, but improvesrecovery,of function in an isolated perfused rat heart. The brief ischacmicepisode depletes glycogen stores. We tested whetherglycogendepletioncould explain the effects or PC. Methods: The isolatedLangendorffrat heart, with a LV balloon, was perfused with glucose I Imbt, acetate 5mlVl, or glucose I ImM+insulin,to alter glycogenlevels (umol glucoseYgwet wt) prior to 30min total global ischacmia. PC hearts were made totally ischucmic for 5min followed by 5min rcperfusionprior to sustained ischacmia. Time to onset of conlracture (TOC - rain), peak contracture and recoveryof developedpressure (Day P) after 20rain repcrfusion (both cx'p~'~,,=das % prcischaemicdeveloped pressure) were measured(n=13-16). ~cetate ~lucose ~lucose + ~s-ain Fennel Pc IConLrol IPC Icon~ol pc Glyc [7.2i'0.9# p.l:k0.8*#lI0,61"0.7~.41"0.6 *116.2+1.4# [9.1"20.7*#

TOC p.6~.3#p.~.l'#11,.7~0.7F.8:~l.l'll4.1~.9 F.6~0.8"

P~

191-8~4.9II13.4~3.2~.I-+-3.S152.7~.0 13g.I~.6 pI.7~.6

~vPIS.2.+.3.0 III.7~6.5 122.5+7.014Z6+Z3111.6~.3 IIS.6+S.S * p<0.05vscontrol;# p<0.05vsglucose A decreaseinglycogenreducedTOC and increasedpeckconUacture. An increasedglycogenhad oppositeeffects.In preconditionedhearta, TOC was also reduced,and peak contractureincreasedexcept in higll glycogen hearts. PC improved recovery in glucose hearts, but not when glycogenwas altered. Conclusions: The effect of PC on worseningcontractummay be due to glycogen depletion. PC is ineffective in protecting against insufficient glycogen(lack of ATP production) or excess glycogen (excess lactate production). The protective effects of PC are not linked to glycogendepletion.

A149

PRECONDITIONING AND INTERMITTENT Tu097 ISCHEMIA IN AN ISOLATED RAT HEART MODEL. A P-31 NMR STUDY. R. Sharony*, A. Olivsen**, E. Berman**, J.B. Bonnan, G. Merin, G. Uretzky*, H. Schwalb. *Cardiothoracic Surg. Carmel Hosp., Haifa & Lunenfeld Cardiac Surg. Res. Center & **HBR Center, Hadassah Univ. Hosp., Jerusalem, Israel. The effect of myocardial preconditioning (PCO) on intermittent cross clamping, a method used in cardiac surgery, was tested in Iwo groups of isolated rat hearts. Gr A (n=9) was exposed to PCO prior to 3 ischemia-reperfusion cycles (10 and 15 rain, respectively) and then to Krebs-Henseleit 30 rain reperfusian. Or B (n=12) was exposed to the same protocol but without PCO. All studies were carded out inside the NMR. allowing real time monitoring of hemodynamics and high energy phosphates. During the ischemic intervals, no significant difference was observed between the high-energy phosphates in both groups. On the other hand, during the reperfusion stages % recovery of ATP in gr A were higher than in gr B (68+72%, 74-1-8.9%, 74+10.5% vs 52+8.7%, 59:t:13%, 63+12%, respectively; M.-i:SD, p<0.05). The near-zero phosphocreatine (Pcr) levels during the ischemic intervals were regenerated to supranormal values during repoffusion. The Pcr increase in gr A was less pronounced than in gr B (122+16%, 117.+.22%, 120+19% vs [40+19%, 133:i:18%. 135+14.5%, respectively, p<0.05). Higher recovery of the left ventricniar developed pressure was observed in gr A during the reperfusion stages (80.5+8.8%, 86+70/0, 88+4.5% vs 63+18%, 68:1: 19%, 67+19%, respectively, p<0.025). We conclude that PCO imparts metabolic and hemodynamic protection to the intermittent isehemic isolated rat hearL

PRECONDITIONING PREVENTS VASCULAR Tu098 DYSFUNCTION INDUCED BY ISCHAEMIA AND REPERFUSION IN RAT HINDQUARTERS Kit E. Loke & Owen L. Woodman. Dept of Pharmacology, University of Melbourne, Victoria, Australia. Preconditioning with brief periods of ischaemia and reperfusion (I/R) has been shown in the myocardium to reduce infarct size caused by a prolonged ischaemic period. In this study we investigated whether I/R impairs vasodilator responses to exogenous and neuronally released ACh (Loke et al, 1994). We also examined whether preconditioning protects against the I/R-induced loss of vasodilator function. The abdominal aortae of anaesthetized rats were caanulated for hindquarters perfusion with Krebs bicarbonate solution. Rats were subjected to sham 2h aortic occlusion and 15rain Krebs reperfnsion (Group 1), 2h aortic occlusion and 15min Krebs reperfusion (Group 2) or similar to Group 2 but preceded by 5rain aortic occlusion and 10min blood reperfusion (is preconditioning, Group 3). In control rats nerve stimulation, ACh and the endothelium-independent vasodilator sodium nitroprussids (SNP), caused frequency- or dose- dependent decreases in perfusion pressure, indicating vnsodilatation. I/R significantly attenuated vnsodilator responses to both nerve stimulation (I/R: 65.+.5% of control) and ACh (I/R: 84+3% of control) but not SNP (I/R: 101+6% of control). Preconditioning prevented the l/R-induced decrease in vasodilator responses to nerve stimulation (preconditioned: 98+6% of control, P<.001 vs l/R) and ACh (preconditioned: 103+9% of control, P<.001 vs I/R). Thus I/R impairs endotheliumdependent but not endothelium-independent vasodilatation and that impairment was prevented by preconditioning. Lokc KE et al (1994) Br J Pharmacol, 112, 630-634.

ISCHAEMIC PRECONDITIONING PROTECTS Tu099 ENDOTHELIAL FUNCTION AGAINST ISCHAEMIC INJURY IN THE ISOLATED RAT HEART. Jean-Francois Bouchard & Daniel Lamontagne, Facult~ de pharmacie, Universit~ de Montreal, Montreal, Quebec, Canada. Ischaemic preconditioning is a phenomenon whereby a brief or quite of few brief periods of ischaemia render the myocardium resistant to a subsequent more severe ischaemic injury. The aim of the present study was to assess whether ischaemic preconditioning can preserve endothelial function from a subsequent ischaemic insult in resistance coronary arteries of the rat. Isolated Langendorff rat hearts, perfused under constant flow conditions, were exposed to 30 min of partial ischaemia (flow rate 1 ml/min) followed by 20 min of reperfusion. After this period of reperfusion, coronaries were precontracted with U-46619 0.1 pM. Endothelial function was measured by the vasodilatation produced by serotonin 10 pM (5-HT). Coronary smooth muscle function was evaluated with 3 pM sodium nitroprusside (SNP). In control group (without ischaemia or preconditioning), infusion of 5-HT and SNP induced marked vasodilatation, as reflected by the reduction in coronary perfusion pressure of 25 + 3, and 30 + 4 % respectively. Ischaemia significantly reduced the vesodilatation to 5-HT (10 + 5 %), whereas that of SNP was unaffected. Preconditioning with 5 min ischaemia and 10 min reperfusion before ischaemia reestablished the vasodilatation produced by 5-HT (31 + 3 %). The vasodilatation to SNP was still unchanged. These observations suggest that ischaemic preconditioning affords protection to endothelial function in resistance coronary arteries of the rat. A150

ISCHEMIA-REPERFUSION IMPAIRS HYPOXIC Tul00 CORONARY VASODILATION: PROTECTION BY ISCHEMIC PRECONDITIONING Eliana Giannella, Hans-Christian Mochmann and Roberto Levi. Dept. of Pharmacology, Cornell University Medical College, New York, NY, USA. We tested the hypothesis that ischemic preconditioning (PC) prevents the impairment of hypoxic coronary vasodiiation (HCVD) caused by myocardial ischemia and reperfusion. HCVD was elicited in isolated guinea-pig hearts by a 1-min perfusion with Ringer's solution equilibrated with 100% N=. HCVD (i.e., a =70% increase in coronary flow, ml/min) was associated with an increase in adenosine (ADO) overflow from 3.9±0.5 (basal pre-hypoxic) to a maximum of 19.2±2.7 nmol/min/g (1st min of reoxygenation). Following 30-min global ischemia and 20-min reperfusion, HCVD was decreased by =60% (from 3.4±0.3 to 1.4±0.5 ml/min; n=10, p<.05), and ADO overflow was markedly reduced (from 19.2±2.7 to 3.5±0.5 nmol/min/g; n=9; p<0.05). PC (i.e., three cycles of 5-min global ischemia + 5-min reperfusion) prevented the impairment of HCVD (PC: 3.3±0.3 ml/min, n=5, p<.05 vs. 1.4±0.5 ml/min ischemia/reperfusion) and restored =50% of ADO overflow (11.4±5.9 nmol/min/g, p<.05 vs. prehypoxic or reoxygenated). The protective effect of PC was mimicked by three 5-min perfusion cycles with the adenosineA I receptor (A1R) agonist Ne-cyclopentyladenosine (CPA; 100 nM)(2.6±0.6 ml/min; n=5; N.S. vs. control) and prevented by the A1R antagonist N-0861 (5 pM)(1.4±0.7 ml/min; n=4; p<.05 vs. control). In contrast, the AaR agonist NS-2-(4-aminophenyl) ethyladenosine (APNEA; 65 nM) had no protective effect. Our data indicate that PC restores HCVD by a process which involves A~R and is accompanied by an increase in ADO overflow. (USPHS grants HL34215/46403)

NITRIC OXIDE IS INVOLVED IN PACING-INDUCED TU101 PRECONDITIONING IN ISOLATED WORKING RAT HEARTS P6ter Ferdinandy, Zolt6n Szilv6ssy', Csaba Csonka, Tam6s Csont, Erzs6bet Nagy", Matyas Koltai', L6szl6 Dux. Dept Biochem, "lst Dept Medicine, "'Central Laboratory, SzentGy6rgyi Univ., Szeged, Hungary; IPSEN-Beaufour, Paris, France',

THE ANTI-ISCHEMIC EFFECT OF NITROGLYCERIN TU102 INVOLVES A DIRECT MYOCARDIAL MECHANISM IN ISOLATED WORKING RAT HEARTS P6tar Ferdinandy, Zolt8n Szilv~say," TamSs Csont, Csaba

The objective of the present study was to investigate the involvement of nitric oxide (NO) in the mechanism of ventricular overdrive pacing (VOP)-induced preconditioning. Isolated working rat hearts ( n = 8 in each groups) were subjected to 10-min coronary artery occlusion (test ischamia) after three intermittent periods of VOP (10 Hz) of 5-rain duration. In the non-preconditioned group, coronary occlusion [CO] decreased aortic flow [AF] from 46.6 ± 2.4 to 1 3 . 9 ± 1 . 7 mL/min and increased left ventricular enddiastolic pressure [LVEDP] from 0.53 ± 0 . 0 5 to 2 . 0 0 ± 0 . 0 7 kPa. CO resulted in a relative lactate dehydrogenase (LDH) release of 199 ± 52 mU/min/g wet weight. Preceding VOP significantly increased AF to 25.3 ± 2.0 mL/min (p <0.001) and decreased LVEDP to 1.38 ± O. 1 kPa (p < 0.001 ) and LDH release to 3 9 + 1 4 mU/min/g (p<0.05) after CO. In tissue samples of the left ventricle, pretreatment with 1 mglkg NGnitro-L-arginine (LNNA) completely eliminated the specific spectra of NO-Diethyl-dithio-carbamate complex assessed with electron spin resonance spectroscopy. The same concentration of LNNA inhibited the effect of VOP-induced preconditioning (AF: 17.3 ± 1.3 mL/min; LVEDP:1.89 + 0.05 kPa; LDH release:132 ± 2 6 mU/min/g). The combination of 1 mg/L LNNA with 200 mg/L L-Arg did not significantly affect VOP-induced protection (AF: 2 7 . 2 + 1 . 7 mL/min; LVEDP:1.47±O.O9 kPa; LDH release:103±43 mU/min/g). We conclude, that a NO-mediated mechanism may be involved in VOP-induced preconditioning in isolated working rat hearts. (Supported by grants from OTKA F6396, E6277; MHB 709/94; Hungarian Space Research Office, Budapest; IPSEN-Beaufour, Paris)

The present study was devoted to investigate whether the anti-ischemic effect of nitroglycerin (NTG) involves a direct myocardial action of the drug. Rats were treated with 100 mg/kg nitroglycerin and corresponding vehicle subcutaneously 3 times a day for 3 days to induce vascular tolerance to NTG. Hearts isolated from tolerant and nontolerant rats were perfusad in a working mode, and subjected to 10-min coronary occlusion followed by 3 rain reperfusion in the presence of 10 .7 M NTG or its solvent, respectively (n = 7 in each group). In the nontolerant, solvent treated group, coronary occlusion (CO) decreased aortic flow (AF) from the preischemic value 47 ± 1.7 to 22.4 ± 1.3 mL/min, and increased left ventricular end-diastolic pressure (LVEDP) from 0 . 5 8 ± 0 . 0 8 to 1.86±0.O7 kPa. CO resulted in a relative lactate dehydrogenase (LDH) release of 203 ± 58 mU/min/g. NTG at a concentration of 10 "~ M did not affect preischemic myocardial function and coronary vascular resistance, however, it increased ischemic AF to 3 5 . 5 + 1 . 2 mL/min (p<0.05), and decreased LVEDP to 1 . 4 2 ± 0 . 0 5 kPa (p<0.01) and LDH release to 30:t:20 mU/min/g (p
IMPAIRMENT OF cGMP-SlGNALING CORRELATES Tu103 WITH THE LOSS OF PRECONDITIONING IN RABBITS WITH VASCULAR TOLERANCE TO NITROGLYCERIN Zolt~n Szilv6ssy, P6ter Ferdinandy," Ildik6 Jakab, Istv6n Nagy, M~ty~s Koltai.'" 1st Dept. Medicine, "Dept. Biochem. Albert Szent-Gyorgyi Univ. Szeged, Hungary; "'IPSEN-

PRECONDITIONING WITH I~-ADRENERGIC TU104 STIMULATION IN CHOLESTEROL-FED CONSCIOUS RABBITS Zolt6n Szilv~tssy, P6ter Ferdinandy,° Istv6n Nagy, Sarolta Karcsu, Matyas Koltai.'" 1st Dept. of Medicine, "Dept. Biochem. Albert Szent-Gy6rgyi Univ. Szeged, Hungary; "'lPSEN-Baaufour, Paris, France.

Beaufour, Paris, France. We explored if experimental vascular tolerance to nitroglycerin interferes with the protection conferred by RPinduced preconditioning. Right ventricular electrode and left ventriculqr polyethylene catheter-instrumented conscious rabbits were subjected to ventricular RP (500 bpm over 10 minutes). The resulting intracavital ST-segment elevation (2,2±0.3 mV), increase in left ventricular end-diastolic pressure (LVEDP): from resting 3 . 9 + 0 . 5 to postpacing 19.4± 2.5 mm Hg (p <0.001 ), and reduction of ventricular effective refractory period (VERP): from prepacing 103 + 3.2 to postpacing 8 0 + 4 . 2 (p<0.001) were determined, The same rabbits were anesthesized and the RP-induced changes in cardiac cGMP content determined by radioimmunoassay were measured (resting: 0 . 0 4 9 + 0 . 0 0 7 9 , postpacing: 0.099 + 0 . 0 1 5 pmol/mg wet wt; p < 0 . 0 0 1 ) . ST-segment elevation, postpacing LVEDP-increase and VERP-shortening were reduced to 1 . 4 + 0 . 3 mV (p <0.001 ), 1 0 , 6 + 2 . 0 mmHg (p<0.01), and 9 3 + 5 . 5 (p
Caonka, Matyas Koltai,# L/iszl6 Dux. Dept Biochem, "lst Dapt Medicine, Szent-Gy6rgyi Univ., Szeged, Hungary; 'IPSENBeaufour, Paris, France.

The aim of the study was to assess the ability of isoprenaline (ISO) to precondition the in situ heart of cholesterol-fed rabbits. Right vantricular electrode and left ventricular polyethylene catheter-instrumented conscious rabbits were subjected to ventricular RP (500 bpm over 10 minutes). The resulting intracavital ST-segment elevation and increase in left ventricular end-diastolic pressure (LVEDP) were measured. Both variables were significantly reduced when a preceding 5-min RP was applied with an interpacing period of 30 minutes to induce a preconditioning (PC) effect. The same protection was seen when ISO (2pg/kg) was infused intravenously 30 min prior to a single 10-min RP. The same animals were then fed cholesterolenriched diet over 8 weeks. Exposure to dietary cholesterol resulted in the loss of RP-induced PC effect on both parameters, nevertheless, ISO was found to significantly reduce both postpacing ST-segment elevation (Table) and LVEDP-increasa from 2 9 , 2 + 5 . 5 (without ISO} to 1 4 . 3 ± 2 . 9 mm Hg (p
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Without PC Preconditioned ISO, 2 pg/kg .

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2.2 ± 0.34 1.3 + 0.22" 1.4±0.19" .

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2.9 + 0.33 + 1.8 ±0.29" .

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+ :cholesterol v$ normal (p
Conclusion: Hypercholastarolaamia blocks RP-inducad PC, nevertheless, PC with beta adrenergic stimulation confer protection even in hyparcholesterolaemia. (Supported by grants from OTKA F6396, and IPSEN-Beaufour)

AIS1

A= RECEPTOR MEDIATES THE CARDIOPROTECTIVE Tu105 EFFECT OF ADENOSINE IN ISOLATED RABBIT HEART. Claudio Ceconi, Anna Cargnoni, Anna Mazzoletti, A n t o n e l l a B o r a s o , S a l v a t o r e Curello, R o b e r t o Ferrad. F o n d a z i o n e Clinica del L a v o r o , C e n t r o S. Maugeri, G u s s a g o , Brescia; C a t t e d r a di C a r d i o l o g i a , Universitzt di Brescia, Italy.

The cardioprotective effect of adenosine (Ado) can be mediated by vascular A2 or cardiac A1 receptors. We have investigated the role of both receptors in reducing ischaemic and reperfusion damage of isolated rabbit heart by using: 1) unselective A1 and A2agonists: Ado (10"s and 10"4 M) and NECA (10-7 and 5x10 "s M); 2) a selective AI agonist: CCPA (10"6 M) and 3) selective A2a agonists: CGS 21680 (10"s and 10"s M) and 2-hexynylNECA (10.7 M). All drugs were delivered 10 min before ischaemia (45min; 0ml/min) and during reperfusion (30min; 22ml/min). The heads were paced at 180 b/min and perfused at costant flow in order to eliminate chronotroplc and vasodilator effects. Ado, when delivered at 104 M, exerted a protective effect in terms of a greater recovery in systolic pressure (36% vs 20% in control group; p<0.01), a reduced increase in diastolic pressure dudng reperfusion (46.7+7.1 vs 67.6:1:5.0 mmHg; p<0.05), a lower CPK release (654.8:1:85.4 vs 1558.8:1:218.6 mUlmin/gww; p<0.01) and a better maintenance of mitochondrial function and of energy phosphates content. A similar protection was also observed with A2a (CGS 21680, 2-hexynyI-NECA) but not with A1 selective agonist (CCPA). Thus, "in vitro', the cardioprotective effect of Ado is mediated by A2 receptors.

ISCHEMIC PRECONDITIONING (PC) AND Tu107 ADENOSINE P.JEIJF_ASEIN ISO][.&'I'F_,D PAT HEART Norbert If.. Urba~ski & Andrzej Ber~sewicz. Dept Clin Physiol, Med Ctr Postgrad Education, Warsaw, Poland Adenosine (Ado) receptor stimulation is implicated in the mechanism of PC induced cardioprotection in many species, excluding rat. In dog heart, PC was reported to increase Ado release during the final reperfusion period, a potentially protective effect. Adenosine release in rat heart subjected to PC has not been studied. Reperfusion induced arrhythmias and the release of Ado (and other purines) was studied in Langendorff perfused rat hearts subjected to either I0 min regional or I0 global ischemla, resp, followed by I0 rain reperfusion. The incidence of the reperfusion induced sustained VF was reduced from 68%, in control hearts, to 8%, in PC group (5 rain global isch + 5 min rep prior to I0 rain isch). While SIN-I (NO donor, I0 I~M), given for 5 mln and washed for another 5 min prior to the 10 min ischemin, was able to mimic the effect of PC on the arrhythmJas, Ado, 10 tiM, was not effective. In PC group, upon the first reperfusion, the release of Ado was greatly increased and it declined during 5-rain of reperfusion to a basal level. Quantitatively similar profile of Ado release occurred during Ado washout period in the hearts "preconditioned" with Ado. Basal Ado release was not affected by SIN-1. The profiles of Ado release during the reperfusion which followed 10 rain ischemla did not differ among the experimental groups. In conclusion: In rat heart, PC episode is accompanied by an increased release of Ado, an effect not fikely to mediate the PC inducedprotection. Ado metabolism during ischemia/reperfusion is not affecte~i by p c in rat heart. It is NO- but not Ado-induced mechanism which, may be responsible for the PC induced protection in rat. A152

PRECONDITIONING OF MYOCYTES: Tu106 DOSE/RESPONSE EVIDENCE FOR T W O (Az a n d A s ) A D E N O S I N E R E C E P T O R S Stephen C. Armstrong & Charles Ganote. Dept of Pathology, East Tenn State Univ, Johnson City TN, USA. Rabbit cardiomyocytes were preincubated (10') with log doses (lnM to 100p.M) adenosine (ADO) or (lnM to 10p.M) APNEA or CCPA, with or without 1 or 10tam of the selective AzR antagonist DPCPX. After 30' post-incubation (no drug), cells were ischemically pelleted and sampled at intervals to 180 minutes, assessing viability after 85mOsMol swelling.

o7

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ADO (fig A) produced a broad smooth D/R curve. CCPA (fig B) produced a biphasic curve with a plateau between 10-1000 nM. DPCPX blocked the early agonist low dose effects, reducing protection by approximately 40%. APNEA (not shown) produced a steep monophasic curve displaced to the left of the ADO curve by about one log unit. Agonist and agonist]antagunist curves closely resembled calculated two receptor binding curves (plotted on figures without symbols) assuming a low and a high affinity receptor with binding affinities corresponding to Az and A3 receptors. The results are consistent with induction of preconditioning being due to two additive adenosine receptors, with approximatly 40% being high (Az) and 60% being low (A3) affinity receptors.

A D E N O S I N E CONSERVES OXYGEN IN

Tu108 NORADRENALINE-STIMULATED SHEEP HEART IN VIVO lindsay Jordan, Glenn Harrison, David McGiffin, Roger Willis. Rotary Centre for Cardiovascular Research, Griffith University, Nathan, 4111, Australia. We have proposed that adenosine modulates head function dudng increased cardiac work to conserve oxygen. In the present experiments sheep (n=32) were anaesthetised with thiopentone, and noradrenaline (NA) was infused intravenously to increase cardiac work in two steps (NA1 and NA2) from the initial level (I). In 17 sheep, the effect of endogenous adenosine was blocked by 8-phenyitheophylline (8-P'I'). Once steady conditions were reached at each level of cardiac work and dudng a recovery pedod (R, open symbols), MVO2 and cardiac work were measured. Cardiovascular function of sheep in each group was matched initially, but work level increases due to NA were higher in sheep treated with 8-PT than in untreated sheep. MVO2 was significantly higher (ANOVA, P<0.05) in 8-PT treated sheep (squares) than untreated sheep (circles). i i i 2~ 20-

10.

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Adenosine is involved in cell volume

Tul09

regulation in myocardial preconditioning Nadir Askenasy, Avi Koller and Gil Navon, School of Chemistry, Tel Aviv University, Israel Previously we have reported a cellular shrink induced by short ischemia/reperfusion (I/R) episodes and excessive swelling during sustained ischemia. The aim of this study is to determine whether adenosine Al receptors and ATP-sensitive K channels are involved in cell volume regulation during myocardial preconditioning (P). Cell volumes were measured in the perfused and }~chemic rat heart using ~H NMR of water and Co NMR of the extracellular marker cobalticyanide. Adenosine (20 or 200 paM) induced a transient cell swelling (2.74i-0.08 from an initial value of 2.504_0.08 ml/gdw), followed by cellular shrink to 2.28:L'0.07 ml/gdw after 30 min of perfusion. During ischemia, the ceils swelled by 0.32 rrd/gdw in the presence of adenosine, while hearts preconditioned by 3 UR episodes (5/5 rain) swelled by 0.70-k0.12 ml/gdw. The characteristic pattern of cell volumes in P-hearts was abolished by the adenosine receptor inhibitor 8-phenyl teophylline (10 ~.M), but was not affected by the ATP-sensitive K channel inhibitor glibenclamide (10 ~JVl). It is concluded: a) Al adenosine receptors are involved in cell volume regulation in the rat heart; b) adenosine influences cell volume dynamics during the preconditioning phase and during sustained ischemia; c) involvement of ATP-sensitive K channels is not supported.

ISCHEMIC PRECONDITIONING -VIEW OF Tu111 ADENOSINE RECEPTOR AND K CHANNELHaruhlko Ishloka, EIIchl Geshl, Takashl Kataglrl. 3rd Dept. of Internal Medicine, Showa Univ. School of Med.,Tokyo 142, Japan.

The aim of this study is to investigate the mechanism of preconditioning on ischemic cellular injury in the canine head of LAD occlusion for 60 rain (I) with two times of initial ischemia for 5 rain and reperfusion for 10 rain (PC). 7.5rng/kg of 8-phenyitheophilline (adenosine receptor blocker)(8PT),20p. g/kg/min of Nicorandil (K channel opener) (N) and 0.3mg/kg of glybenclamide(K channel blocker) (G) were given prior to PC, After continuous records of hemodynamics, segment shortening (SS), and regional myocardialblood flow (MBF)I biochemical analyses of SR and Mt were performed. At the end of 60min reperfusion SS of ischemic area reduced tO -30% of the previous level in I, 8PT and G groups, but these reserved to 35% in PC and 55% in N groups, respectively. MBFwas 70% of the control in all groups. Ca-ATPaseactivity of sarcoplasmicreticulum (SR) was higher in PC and N than Ii 8PT and G. SDS gel electrophoresis of SR showed well maintain of major ATPase protein in PC and N group. Botll respiratoryand DNP-ATPaseactivitiesof Mt were also higher irl PC and N. These results indicatedthat activationof adenosine receptor and followed opening of K channel were initial factor~ of t he mechanismsof ischemicpreconditioning.

MECHANISM OF ISCHEMIC PRECONDITI-Tu110 ONING -ROLE OF POTASSIUM CHANNELEiichi Geshi, Haruhiko Ishioka, Katsumichi lijima, Takuya Watanabe, Toshiki lwata, Hirohisa Arata, Takashi Katagiri. 3rd Dept. of Int. Med., Showa Univ. School of Med., Tokyo, Japan. Numerous mechanisms have been discussed concerning to the effect of ischemic preconditioning (IP). The aim of this study is to clarify the role of potassium channel on the IP. In the canine hearts of 60 min ischemia and 60 min reperfusion, prior IP of 5 min ischemia with 10 min interval were performed twice. The 20ug/kg/min of Nicorandil (potassium channel opener with nitrate effect: N), SG209 (pure potassium channel opener: S) or vehicle (V) were given under the continuous records of hemodynamics and segment shortening (SS), then biochemical and ultrastructural analyses were performed. %SS diminished to -20% of control (C) in the without IP (I), this maintained to 40% in V. This value more recovered to 55% in N, however that of S indicated 38% which was similar to V. Ca-ATPase activity of sarcoplasmic reticulum showed 11.0 umoles Pi/mg/hr in C. This decreased to 6.7 in I, however those in V, N and S were 9.5, 10.5 and 8.9, respectively. State III activity of mitochondda and ultrastructural changes were also more maintained in N than in S and in V. From these results, we speculate that the potassium channel is not a initial factor of the mechanism of ischemic preconditioning.

PROTECTING THE MYOCARDIUM AGAINST Tu112 ISCHAEMIA USING HEAT STRESS AND PRECONDITIONING David P Jenkins, Marko Kerac,Justin S Zaman, & Derek M Yellon. The Hatter Institute, UniversityCollege London Hospitals, London,UK.

It is known that the myocardium has endogenous defences against ischaemic damage. Prior stress, such as heat stress (HS) or ischaemic preconditioning(PC) can protect the heart against a more severe ischaemic insult. However, the protectionof HS and PC has not been directly comparedin the same model, and it is not yet known whether this protectionis additive. In this study, isolated buffer-perfused rabbit hearts were subjected to 20 min globalischaemiaat 37oc followedby 2 hours reperfusion. Rabbits, heat stressed (HS) to 42oC under anaesthesia 24 hrs prior to the ischaemic insult were compared with sham-treated non-HS animals. Hearts preconditioned (PC) with 5 min ischaemia and 10 min reperfusion were compared with controls. Infarct volume was measured by tetrazolium staining and expressed as a percentageof the left ventricularvolume. Results are mean ± SEM, n = 8 per group

IContro,l PC ShamI

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31+6 13+3 24±5 9+4 * 4±1"# • p < 0.05 v respectivecontrols, # p = ns v HS or PC; ANOVA In this model of global ischaemia,HS and PC resulted in equal protection when used alone. There was no significan! additionalprotectionby using both HS and PC together.

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PRECONDITIONING: PROTEIN KINASE C AND THE Tu113 KA-rp CHANNEL IN THE ISOLATED RABBIT HEART David P Jenkins, Marko Kerac, & Derek M Yellon. The Hatter Institute, University College London Hospitals, London, UK. In rabbit hearts activation of protein kinase C (PKC) is known to be an essential step in the mechanism of ischaemic preconditioning (PC). In other species there is evidence for the involvement of the KATP channel and it has been suggested that it may be the 'end-effector' of PC. We investigated the relationship between PKC and the KATP channel, using the PKC activator 1,2-dioctanoyl-sn-glycerol (DOG) and the KATP channel blocker glibenclamide (Glib). Isolated buffer-perfused rabbit hearts were subjected to 25 min global ischaemia at 37oC, and reperfused for 2 hours. A PC group was pretreated with 5 min ischaemia and 10 min reperfusion. Further groups were treated with DOG (30 IJM); PC in the presence of Glib (1JJM), and DOG in the presence of Glib. Infarction was determined by tetrazolium staining and expressed as a percentage of the left ventricular volume. Results: mean ± SEM. Control PC DOG PC+Glib DOG+Glib 35.3+8 13.1 ± 7 * 7.3±3# 17.8±7' 16.4+3 * n=10 n=6 n=6 n=8 n=8 "p < 0.05, # p < 0.01 v Control; ANOVA These results demonstrate that both PC and treatment with DOG give significant myocardial protection which is attenuated b y Glib, but not abolished. Therefore, the KATP channel may be involved in PC in the rabbit, but is not the sole 'end effector'.

A154

CLASS HI AGENT DOFETILIDE DOES NOT Tul 14 ABOLISH ISCHEMIC PRECONDITIONING. Jens Munch-Ellingsen, Einar Bugge and Kirsti Yla'ehus, Department of Medical Physiology, University of Troms¢, Norway. Ischemic preconditioning (IP) is a powerful mechanism for cardioprotection. Increased K+ conductance (gO early during the sustained ischemic episode has been proposed as an important event for cardioprotection caused by IP. Most but not all in vitro studies of K+ channel blocking class III agents have shown a loss of class III activity during ischemia/hypoxia. We have investigated if dofetilide a class HI antiarrhytmic agent could abolish IP in an in situ rabbit heart infarct model. Controls received 30 min of regional ischemia followed by 180 min reperfusion and IP group 5 min of regional ischemia and 10 min of reperfusion before the 30 min ischemia. Dofetilide group received 20 ~g.kg"t during the first minutes of the first reperfusion periode. Results: % infarction (mean + SEM) of the risk zone in controls was 39.06 ±7.30, in IP 4.55 ± 2.05* and in dofetilide 10.47 :L-4.39' (* pg 0.05 vs control). Conclusion: In an in situ rabbit heart model, dofetilide did not abolish the beneficial effect of ischemic preconditioning on infarct size.

VENTRICULAR PACING PROTECTS MYOCARDlUM BYTu115 NON-ISCHEMIC ACTIVATION OF K*A-mCHANNELS Ben CG Gho, Monique MG Koning, Edk van Klaarwater, Rend LJ Opstal, Dirk J Duncker & Pieter D Verdouw. Thoraxcenter, Erasmus University Rotterdam, Rotterdam, The Netherlands

THE ATP-SENSITIVEPOTASSIUMCHANNEL: ITS ROLE IN Tu116 ISCHAEMIC PRECONDITIONINGIN HUMANATRIALTRABECULA M. Elsya Speechly-Dlck, Charles W. Pattison, Wilt B. Pugsley, Gary J. Grover', Derek M. Yellon,The Hatter Instlt., DepL of Cardiology,Univ. CollegeLondonHospitals,London,U.K. Bristol-MyersSquibb*,USA.

We studied whether ventdcular pacing at 200 bpm (VP) protects against myocardial infarction induced by a 60 min coronary artery occlusion (6Omin CAO) in open-chest pigs and determined if ischemia and activation of K*^rp channels play a role in the protection by VP. Infarct size (IS) was expressed as a percent of the area at risk (AR). AR was not different between the 5 study groups (33:1:1% (mean+SEM) of left ventricular (LV) mass, n=38). In 10 control animals IS after 60min CAO was 85:~..2%. 10 min of VP followed by 15 min of normal sinus rhythm (NSR) prior to the 6Omin CAO was not protective (IS=83+3%, n=4). Extending the VP period to 30 min followed by 15rain NSR resulted in limitation of IS (72+9%, P
The ATP-dependent potassium channel (KATP) has been implicated in ischaemic preconditioning (IP). Aim: To establish whether the protection of IP in humans involves KATP activation. Methods: A human atrial model was used in which IP and the role of protein kinase C in IP have been shown. Trabecula were supertused, paced at 1Hz, and subjected to simulated ischaemia (SI) of 90min of substratefree hypoxia paced at 3Hz, followed by 2hr of reoxygenation at 1Hz. IP was 3min substrate-tree hypoxia at 3Hz and 7min reoxygenation. We used a selective KATPopener, cromakalim (Ck, 30urn), and blocker glibenclamide (Gb, 1urn). The endpoint was post-ischaemic recovery of function as a percentage of baseline (%R). There were six groups: i)Control (C): SI (n=6); ii)Preconditioned (PC): IP then SI (n=6); iii)Ck: 5min Ck prior to SI (n=6); iv)Ck+Gb: as Group Ck in the presence of Gb (n=4); v)PC+Gb: as Group PC in the presence of Gb (n=4); and vi)C+Gb: as Group C with 10min Gb prior to SI (n=4). Results: ,group means with SEM

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65.1" 53.6* 5.5 2.9 . . . Pratreatment with IP or the KATP opener, Ck, results in significant protection against SI (*=p<0.01 v C). This protection is abolished by KATP blockade. Gb alone gave a similar %R to Control. C o n c l u s i o n : Activation of KATP results in similar protection against ischaemia as IP and this effect is lost with KATP blockade. This study supports the hypothesis that KATP may be involved in ischaemic preconditioning in human myocardium.

IS THE KA1.PCHANNELTHE EFFECTORBY WHICH Tu 117 PRECONDITIONINGPROTECTS? Mihaela M Mocanu °, David P Jenkins, and Derek M Yellon. The Hatter Institute, University College London Hospitals, London, UK and *Victor Babes Inst, Bucharest, Romania.

DOSE- AND PERFUSION MEDIUM-DEPENDENCY OF Tul 18 THE PRECONDMONING-INDUCED ACCELERATION OF ISCHAEMIC CONTRACTURE Kyriacos G. Kolocassides, Manuel Galihanes, David J. Hearse. The Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK.

It is known that activation and translocation of Protein Kinase C (PKC) is central to the myocardial protection observed in rabbit hearts following ischemic preconditioning (PC). It has also been shown that opening of K,~ channels results in myocardial protection and it has been suggested that these channels may be the "end effector" of preconditioning in some species. To investigate the relationship between PKC activation and K,~ channel opening, a specific activator of PKC, 1,2-dioctanoyl-sn-glycerol (DOG), and a blocker of the K,~, channel, glibenclamide (Glib), were used. Isolated buffer perfused rabbit hearts were subjected to 45 mins regional ischemia at 37°C and repedused Ior 2 hours (controls). The PC group was pretreated with 5 mins regional ischemia and 10 mins reperfusion. Another group underwent PC with the same protocol in the presence of Glib. Additional hearts were treated with DOG (30uM) as well as DOG in the prescence of Glib (luM). The risk (R) and infarct (I) areas were determined following administration of fluorescent particles and tetrazolium staining respectively. Results are expressed as percent infarct/risk zone(I/R). Mean ± SEM of 5-6 animals per group. P<0.05 for all groups vs control. Control PC DOG PC+GLIB DOG+GLIR 46.7+9.6 13.6±1.5 17.0±3.0 14.2+2.4 11.0+1.5 These results suggest that PC with either ischemia or the PKC activator, DOG, are highly protective to the myocardium. We were unable however to prevent this protection using the K,.r, channel blocker glibenclamide. Thus it appears that in this rabbit model of regional myocardial ischemia, the K,~,, channel may not be the only end effector through which PC protection occurs.

We investigated the effect of (i) varying the dose (frequency) of preconditioning ischaemlaand (ii) the compositionof the perfusion medium, on the ability of preconditioning (PC) to accelerate ischaemic contracture (IC) in the rat head. Isolated heads (6/ group), with an intraventricular balloon, were aerobicallyperfussd with either whole blood from a support rat or buffer. They were then subjected to zero flow global ischaemia (37°C) for 20min. The groups were: (i) controls (C) with unprotected ischaemla, (ii), (iii) and (iv) PC with 1,2 and 3 cycles of 3min ischaemia/3min reperfusion prior to ischaemia. In buffer-perfused heads, 1 cycle of PC was without effect on IC (time-to-75%lC C=14.7±0.2 v PC(1 )=15.1 ±1.0min).Two and three cycles of PC accelerated IC considerably (time-to-75%lC PC(2)=11.4±1.0 and PC(3)=10.6±1.4min). In no instant was peak contractura elevated. Bycontrast, in the blood-perfusion groups peakcontracture was significantly increased in a frequency dependent manner as was time-to-75%lC (PC(1)=6.0+0.2, PC(2)=5.6±0.2 and PC(3)--4.8+0.2 v C=14.5±0.4min). The origins of the inter-preparation differences are unknown, but may relate to differing vulnerabilities to ischaemia-induced ATP depletion and acidosis.

OPPOSING EFFECTS OF PRECONDmONING AND Tu119 CAFIDIOPLEGIA ON ISCHAEMIC CONTRACTURE AND A'FP DEPLETION IN THE BLOOD-PERFUSED RAT HEART Kyriacos G. Kolocassides, Manuel Galihanes, David J. Hearse. The Rayna Institute, St Thomas' Hospital, London SE1 7EH, UK. In vivo dog heart studies have suggested that preconditioning (PC) delays the ischaemia-induced depletion of ATP. However, usingthe bufter-perfused rat head, we have previously shown that PC accelerates ATP depletion and ischaemic contracture whereas cardioplagla (CP) delays both these events. We investigated whether this also occurs in the blood-perfused rat head. Isolated heads were aerobically perfused with blood from a support rat and subjected to zero flow global ischaemia (37"C) for periods up to 35min (n=5 heads/group). The heads were frozen and taken for metabolic analysis. Ischaemic contractura was assessed with an isovolumic intraventricular balloon. The study groups were: (i) control (C) with unprotected ischaemia, (ii) PC (two cycles of 3min ischaemla/3mm reperfusion) and 0ii) cardioplegia (St Thomas') prior to ischaemia. Preconditioning accelerated whilst cardioplagia delayed ischaemic contracture (IC, time-to-peak contracture PC=8.1+0.3min and CP=25.1+0.2mm v C=15.6+0.3min P<0.05). The ischaemlainduced decline in ATP was delayed by cardioplegia but accelerated by preconditioning(e.g, after 9min ischaemia ATP was: C=8.1 ¢0.4, PC=4.1 +0.6 and C P=I 7.2±1.0p mol/g dry wt, P<0.05). In a parallelstudy PC and CP protected post-ischaemiccontractilefunctiontoasimilarextent.Thus, intheblood-andcrysta o aperfused rat heart PC accelerates ischaemic contracture and - depletion of ATP. In m'~(~ "''"1~ --~TpIl°° ~ contrast, CP slows ~-=o, ischaemic contrac~=,s ~ \ ~ ? - =" " e ' " -'8~ ~ ~ ~ tureandATPdeple-

THE PROTEIN IGNASEC INHIBITORPOLYMYXINB BLOCKSTu 120 THE FUNCTIONAl.PROTECTIONOF PRECONDmONING BUT NOTTHE A ~ ' i ' I O N OF ISCHAEMIC CONTRACTURE Kyrlacos G. Koiocassides, Manuel GaliRanes, David J. Hearse. The Rayne Institute, St Thomas' Hospital, London SE1, UK.

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We have previously shown that preconditioning (PC) exacerbates ischaemic contracture despite improving post-ischaemic contractile function. We investigated whether both of these contrasting effects can be blocked by the protein kinase C (PKC) inhibitor polymyxin B (501~mol/L).Four groups of buffer-perfused heads (n--6/group) were used: (i) controls (C) with unprotected ischaemla, (ii) PC by 3min ischaemla/3min reperfusion + 5min ischaemia/Smin reperfusion, (iii) controls with polymyxin B (C+POL) infused for 9min prior to ischaemia and (iv) PC with polymyxin B (PC+POL) infused prior to and during preconditioning. Heads were subjected to 35min of global ischaemla (37°C) followed by 40min of reperfusion. Left ventricular developed pressure (LVD P) and ischaemic contracture were assessed with an intraventricular balloon. PC accelerated ischaemic contracture (time-to-50% peak contracture PC=7.7+l.0min v C=10.8+0.6min, P<0.05). Polymyxin B had no significant effect on this accelaration (6.9!-_0.8min),but in control heads it delayed contracture (14.6-J:1.7min, P<0.05). LVDP was improved by PC from 23+7% in controls to 67+5% (P<0.05). With polymyxin B, PC failed to improve the recovery of LVDP (C+POL=26!-_8% v PC+POL=34+5%, P=NS). Thus, polymyxin B was able to abolish the protection of post-isshaemic contractile function afforded by preconditioning but was without effect on the preconditioninginduced exacerbation of ischaemic contracture. Whilst our resuits support the concept that PKC may be involved in some aspect of the preconditioning mechanism, other PKC-independent mechanisms might also be operative.

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A155

CAN ISCHEMIC PRECONDITIONINGTu121 COMPENSATE F O R CARDIOPLEGIA WHEN DELIVERY IS IMPAIRED? M. Galifianes, A.N.A. Wilson, D.J. Hearse. The Rayne Institute, St Thomas' Hospital, London SE1, U.K.

PRECONDITIONING AND TEMPORAL ISCHEMIA-Tu122 INDUCED CHANGES IN PROTEIN KINASE C DELTA EXPRESSIONAND DISTRIBUTION M. GaUfianes,F.J. Sutherland, G. Brooks, D.J. Hearse.The Rayne Institute, St Thomas' Hospital, LondonSE1, U.K.

We have previously shown that preconditioning (P) and cardioplegia (C) afford similar pro.tection against injury during ischemia and reperfusion but that their e/rects are not additive. However, it is not known whether the same applies in thepresence ofstenotic artery lesions, where the delivery of C is likely to be severely reduced. We have therefore investigated the protective effect of C vs P in the presence of reduced delivery of C. Isolated rat hearts (paced at 400bpm) were perfused (Langendorff) aerobicallyfor 20rain with bicarbonate buffer. They were madeglobally ischemic (370C) for 40rain and reperfused for 40min (37°C). LV function was assessed with an intraventricular balloon. Seven groups of hearts (lg wet wt) were studied (n=8/ group): (i) control unprotected ischemia (I), (ii) 2.0ml of C, (iii) L0ml of C, (iv) 0.5ml of C, (v) 0.2ml of C, (vi) two cycles (3+5rain) of P, and (vii) P + 1.0ml of C. The postischemic recovery of LVDP was 33-*3% in the I group; it was improved by 2.0ml of C (59-*5%; p<0.05) but aoses ot 1.0, 0.5-or 0.2ml of C were ineffective (37+_5%, 37.*5% and 32+6%). P alone and the combination of P + 1.0ml of C afforded similar protection (57±3% and 58.*3%; p<0.05 vs 13. LVEDP increased to 57±3mmHg in the I group; it was decreased by 2.0ml of C (40.*5mmHg; p<0.05), but not lay 1.0, 0.5 or 0.2ml of C (54-*4, 55-*3 and 59±4mmHg). Again, P alone (39-*ImmHg; p.<0.05) orP + 1.0ml ofC (37±2mmHg; p<0.05) afforded similar protection to the 2.0ml of C group but greater protection than any of the groups with reduced delivery of C. In conclusion, in the presence of reduced delivery of C (<2.0ml/g myocardium) P alone (or in combination with C) is more protective than C alone.

It has been postulated that the mechanism underlying ischemic preconditioning involves protein kinase C (PKC) activation. The present study was designed to characterize the effect of ischemia, with and without preconditioning on the amount and distribution (cytosolic vs membrane) of the delta isoenzyme of PKC. Isolated rat hearts, pedused at 37°C with buffer and paced at 400bpm, were subjected to 20rain aerobic'pedusion followed or not by ischemic preconditioning (3min ischemia+3min repertusion plus 5min ischemia+5min reperfusion). The hearts in both groups were then made globally ischemic for 5, 10, 20, 30 and 40min (n=at least 5/group) after which LV tissue was taken and assayed for the expression of PKC5 protein and its distribution between membraneand cytosoliccompartments.Dudngischemia,cytosolic PKC5 protein expression increased to 225~49% of control after 10min and to 453+68% after 20min before falling to within control levels with longer durations of ischemia (p<0.05). In contrast, the expression of membrane bound PKC5 fell to 24+6% of control after only 5rain ischemia and then increased to 184+66% after 20min ischemia before falling again with longer durations of ischemla. In the preconditioned hearts similar biphasic patterns of PKCSexpressionwere observed for both cytosolicand membrane PKC. In conclusion, although PKC5 expression could be shown to change during ischemia, no evidence for net movement of PKC6 from the cytosolic to the membrane fraction (consistent with PKC activation) could be found suggesting that activation of PKC5 is not required for preconditioning.

CALCIUM PRECONDITIONINGMEDIATES PROTECTION AGAINST ISCHEMIA VIA ACTIVATION OF PROTEIN KINASE C

RAMIPRILAT PRECONDITIONS THE FREE Tu124 RADICAL-DAMAGED AORTIC ENDOTHELIUM Xiu Chen, Li-Ying Liu & Chao-Min Cai Dept of Pharmacol, Hunan Medical University, Changsha, China.

Tu123

Muhammad Ashraf, Hiroshi Miyawaki, Xiaobo Zhou, Xiaolin Zhai. Dept. of Pathology, University of Cincinnati, Cincinnati, Ohio, U.S.A. To test the hypothesis that calcium preconditioning (CPC) stimulates protein kinase C (PKC) which is responsible for protection against ischemia (I), isolated rat hearts were subjected to multiple brief periods of Ca2"depletion (lmin) and repletion (5min) (CPC). In control I gp, hearts were subjected to 40min global ischemia and 30min reperfusion (R) (n=5). In CPH gp, CPC hearts were exposed to I&R (n=5). In PKI gp, Chelerythrine (25gg/min), a PKC inhibitor was given during CPC prior to I&R (n=5). During CPC phase, LV dP/dt was increased by 30%. CPC significantly (p<.01) restored LV dP/dt compared with control (91+4 vs 35+7%). Tissue AT P (19.3+0.2p.mol/gr.dr.wt. in normal non I hearts) was significantly preserved in CPH (13.7+0.4, vs 6.6+1.2 I/R control and 9.4+1.2 PKI) (p<.01). Sixty percent reduction il LDH release and minimal morphological changes were observed in CPH. In PKI, the protection was lost. Membrane fraction of PKC was significantly increased in CPH (27.5+_3.1%) compared with control (6.7+2.1%) and PKI (7.5+1.1%) (p<.01). These results suggest that brief Ca2÷ depletion and repletion causes stimulation of PKC perhaps due to increase in intracellular Ca2. and PKC participates h different signaling pathways leading to reduction of ischemic injury. This work was supported by NIH grant HL23597. A156

The mechanism of myocardial ischemic preconditioning has attributed to protein kinase C (PKC) activation, which can be induced by some pharmacologic agents. Thus we investigate if Ramiprilat (Ram) can serve as a pharmacologic preconditioning (PC) agent to protect aortic endothelium (EC) against free radical damage. The EC of rabbit aortic ring was injured in tissue bath by exposing for 10 rain to 2.5 I~M I,l Diphenyl-2-picryl-hydrazyl(DPPH), a stable free radical. The ACh-induced relaxation was reduced from 70 +_5.5% to 19 _+4.9% immediately after l 0 min DPPH incubation, and it recovered to 36.5 + 8.3, 50.3 +_ 6.5 and 56.0 +_5.2 %, 30, 60 and 90 rain respectively after washing out of DPPH. In Ram PC group, the aortic rings were preincubated with 5 I~M Ram for 20 rain. After washing out of Ram, the aortic rings were exposed to DPPH. It shows that 5 I~M Ram PC preserved the AChinduced relaxation to 43.7 +_8.4, 65.3 + 3.0, 69.5 + 3.7, 71.8 + 3.0 % by 0, 30, 60 and 90 rain after washing out of DPPH respectively (n=6, all p<0.05 vs DPPH group). PC effect of Ram 15 I~M was similar to that of 5 p.M. As a control, l0 laM bradykinin PC for 20 min also preserved ACh-induced relaxation injured by DPPH. In addition, PC effect of Ram was not significantly altered by 10 pM H7, a PKC antagonist. Conclusion: Ram acts as a pharmacologic preconditioning agent against free radicalinjured rabbit aortic endothelium and this protection is probably not mediated by PKC activation as in the myocardial preconditioning.

PROTEIN KINASE C-e IS DOWNREGULATEDBY Tu125 ISCHAEMIC PRECONDITIONING IN THE CANINE MYOCARDIUM Susan Wilson,Tanya Ravingerova,KazushigeSakai*, Agnes Vegh, Nigel Pyne, James Parran, Dept Physiology & Pharmacology, Universityof Strathclyde, Glasgow,UK.

ISCHEMIC PRECONDITIONING(IP) IN ISOLATED RABBIT TU126 HEART: EFFECTON PROTEIN KINASE C, EXTRACELLULAR Ca2+, INFARCTSIZE AND LEFT VENTRICULAR (LVI FUNCTION Ho-dirk Kim, Hyun Kim, Bong-jin Rah Dept of Histology, College of Medicine, Chung-Ang Univ, Seou1156-756, Korea

Protein kinase C (PKC) seems to play a pivotal role in ischaemie preconditioning(PC) in rat and rabbit hearts. This study aimed to clarify the role of the Ca2+-independent PKC-e isoform in preconditioning in the dog. Tissue samples were taken from ischaemic areas of the myocardium of dogs which has been preconditioned by 2 x 5 rain coronary artery (GA) occlusions. Membrane and high-speed supernatant fractions were isolated by centrifugation of homogenates and proteins separated by SDSPAGE. Western blots were probed with an antibody which specifically identifiedPKC-e and immuno-crossreaetivityvisualized using chemiluminescence. An immunoreactive protein band correspondingto PKC-E (7lkDa) disappeared from the high-speed supernatant fractions containing the cytosol and also from samples taken from dogs subjected to a sustained period of CA occlusion followingpreconditioning. There was no correspondingincrease in the levels of PKC-e in either the membrane or nuclear fractions, although these fractions did contain the PKC-e isoform in unstimulated myocardium. No decrease in PKC-e levels was observed in supernatant fractions of samples taken from nonischaemic areas of the same hearts. No consistentdecrease in PKClevels was observed in ischaemic areas 24 h after PC by rapid cardiac pacing. These observations may suggest that PKC-E is downregulated during short-term PC. It remains to be determined whether PC offers cardioprotectionas a result of the downregulation of a possible pro-arrhythmogeniceffect of PKC-e. Supported by the British Council, the EEC (ERB CT 924009) and *Chugai Pharmaceuticals.

Protein kinase C (PKC) which is activated by Ca2+ has been implicated in the cardioprotective effect of IP in the rat heart, however, controversies still remain. The_present study was perfomed to assess whether PKC and Ca 2+ are related to IP effect in isolated rabbit heart. Hearts were subjected to 60min sustained ischemia (I) and 120-min reperfusion (R) with IP (4 cycles of 5-min I and 5-min R) (IP heart, n=13) or without IP (control, C heart, n=12). LV functional parameters (LVFP), coronar,/ flow (CF), creatine kinase (CK) leakage, extracellular Ca;'+ and frequency of arrhythmias (VF and VPC) were measured during 12g-rain R. After experiments, each 5 hearts were randomly selected for determination of PKC activity and infarcted area/LV area (IA/LVA). There was no significant difference in the LVFP, CK leakage, CF, and frequency of VF and VPC between the C and the IP hearts. However, LV end-diastolic pressure (higher in the C hearts during the first 30 min of R, p<0.01), Ca 2+ (lower in the C hearts during the first 60 rain of R, p<0.05), and IA/LVA (mean(%):l:SEM, 51.7+9.5 in the C vs 36.1+4.8 in the IP hearts, p<0.05) were significantly different. Cytosolic PKC was lower (29.37_+.2.01 pmol/mg protein/rain vs 62.88:1:8.33 in the C hearts, p<0.004), while membrane PKC was higher (4.77:1:1.47 in the C vs 17.43+4.00 in the IP hearts, p<0.O2) in the IP hearts. These results indicate that: 1) IP does not attenuate postischemic LV dysfunction, however, it has an infarct-limiting effect, and 2) this cardioprotective effect of IP may be related to PKC activation and Ca ;'+ homeostasis.

THE LONG ISCHEMIA IS THE CRITICAL Tu127 TIME FOR KINASE ACTIVITY DURING ISCHEMIC PRECONDITIONING Xi-Ming Yang, J. Raymond Fletcher, James M. Downey, & Michael V. Cohen. University of South Alabama, Mobile, AL We have proposed that protein kinasc C (PKC) activation is necessary for protection in ischemic preconditioning (PC). However, the timing of this critical activation has been inferred from experiments with PKC antagonists in an in situ rabbit model in which it was difficult to carefully control both the concentration of drug perfusing the myocardium and the duration of drug exposure. Consequently we used an in vitro rabbit heart preparation to determine more precisely the timing of required kinasc activity. The PKC blocker staurosporine (STA) was used because it washes out quickly and does not block PKC's translocation, but rather only its kinasc activity. In control hearts a 30 min regional coronary occlusion followed by 2 hr reperfusion resulted in 31.4+1.5% infarction of the risk area. PC with 5 rain global ischemia + 10 rain repcrfusion reduced infarction to 11.4 +2.3 % (p < 0.01 vs control). STA (100nM) starting 5 min before and ending 5 min after PC ischemia did not block protection (14.1_+1.7% infarction, p < 0 . 0 1 vs control). However, STA starting 5 min before and continuing for 10 min into the long ischemia totally blocked the protection of PC (33.7_+2.6% infarction, pNS vs control). These observations demonstrate that kinase activity is required only at the beginning of the long ischemia, but not during the PC ischemia, and further support the translocation theory of ischemic PC.

MECHANISMSINVOLVEDIN CARDIOPROTECTION. "1"U128 E. Murphy, W. Chen, S. Gabel, and C. Steenbergen. LMB, NIEHS, Research Triangle Park, NC and Dept. of Pathology, Duke University, Durham, NC, USA. It is well established that ischernic preconditioning (PC) protects the heart during subsequent more sustained periods of ischemia. We have investigated several pathways which appear to be involved in ischemic preconditioning in Langendorff perfused rat hearts. We find that lipoxygenase metabolism appears to be involved in PC since addition of the lipoxygenase inhibitors NDGA and ETYA during preconditioning, block the protective effects of preconditioning. We further find that 12HETE, the stable end-product of lipoxygenase metabolism, is made during the preconditioning protocol. We also find that redox state appears to be involved in preconditioning. Addition of the antioxidant and glutathione precursor, N-acetyl-cysteine (NAC) during the preconditioning protocol is found to block the protective effects of preconditioning. In addition, consistent with the results of other investigators, we find that the endogenous PKC activator, DOG, can mimic preconditioning, and that the PKC inhibitor, ehelerythrine (CH) blocks the protection afford by preconditioning when it is added during the preconditioning protocol. Recovery of LVDP (%initial) after 20 rain I & 20 rain R Control 40-2_12 PC 91+11 PC + NAC 22.~_3 PC + NDGA 17+ 8 PC + CH 54+4 DOG 81+6 A157

ENDOTHI~LIN-1REDUCESIMPROVED SYSTOLIC TU129 RECOVERY IN PRECONDITIONEDRAT HEARTS. Josephine Ho & Morris Karmazyn. Dept of Pbarmacol & Toxicol, Univ of Western Ontario, London, Ont, Canada. As levels of endothelin-1 (ET-1) are ele.yated in the ischemic heart we determined whether the peptide (0.5, 5 or 10 nM) modulates the protective effect of ischemic preconditioning (IPC) in isolated rat hearts. ET-I was added 5 min before each 5 min IPC episode after which the hearts were made ischemic for 45 min followed by 30 rain of reperfusion in the absence of ET-1. IPC hearts demonstrated significantly improved recovery of left ventricular systolic pressure (I..VSP), reduced LV end-diastolic pressure (LVEDP) and faster time to recovery (TTR) after reperfusion. At all 3 concentrations, ET-1 produced a significant attenuation of LVSP recovery whereas the beneficial effects of IPC on LVEDP and TTR were unaffected. The effects of 5 and 10 nM ET-1 but not the lowest concentration were associated with a significant decrease in tissue ATP contents. As ET-1 stimulates Na+/H + exchange (NHE) and as the antiporter mediates reperfusion injury we tested the potential modulatory effect of the NHE inhibitor methylisobutylamiloride (MIA). However, MIA was without influence either on the cardiac response to IPC or the effects of ET-I. In contrast, the ET^ receptor antagonist BQ-123 totally prevented both the functional and metabolic effects of ET-I on IPC hearts. Our results demonstrate that ET-1 diminishes the protective effects of IPC on systolic recovery through a mechanism involving ET^ receptor activation and a reduction in ATP content but unrelated to NHE activation.

CARDIOPROTECTIVE EFFECTS OF ENDOTHELIAL Tu131 VERSUS NEURONAL NITRIC OXIDE IN RABBIT Ravinder Pabla & Michael J. Curtis, Kings Coll., London, U.K. Nitric oxide (NO) is an endogenous protectant against repeffusion-induced ventricular fibrillation (VF). In rat hearts, the tissue source of this NO is neuronal (protection blocked by the neuronal-selective NO synthase blocker, 7-nitro indazole; 7-NI). The aim of the present study was to investigate the tissue source of cardioprotective NO in rabbit hearts by use of L-NAME (a tissue non-selective NO synthase inhibitor) and 7-NI and direct measurement of NO levels in the coronary effluent during the first rain of reperfusion (chemiluminescence). Male New Zealand White rabbit hearts (n=50) were perfused (Langendorff) with modified Krebs buffer (K+ 4,0 mM; Ca2. 2.8 mM) at 37°C & pH 7.4, randomised to contain nothing (control), vehicle (0.1% ethanol), 100 ~M L-NAME, 100 I~M L-NAME plus 10 mM Larginine, or 10 IIM 7-NI (n=10/group). Following 60 min regional ischemia, reperfusion-inducedVF incidence was zero in controls and 50% in hearts perfused with L-NAME (p < 0.05). L-NAME reduced NO levels from 4929 + 893 to 2505 _+ 483 pmol/min/g (p < 0.05) and reduced coronary flow. The pro-fibrillatory effect of L-NAME was attenuated by Larginine co-perfusion (20% incidence) and NO levels were increased (6430 + 330 pmol/min/g). 7-NI had no effect on VF incidence (20% vs 20% in vehicle hearts), NO levels (4469 _+ 777 vs. 3779 + 824 pmol/min/g) or coronary flow. In conclusion, endogenous NO appears to be cardioprotective in rabbit hearts, but the tissue source is not neuronal. A158

THE PROTECTIVE EFFECT OF ISCHEMICTu130 PRECONDITIONING IS ABOLISHED BY A SULFHYDRYL REDUCING AGENT Riccardo Zucchi, G o n g y u a n Yu, Simonetta Testoni, Ugo Limbruno, Giovanni Ronca, Mario Mariani. University of Pisa, Italy We investigated whether changes in the redox state of sulfhydryl groups may play a role in ischemic preconditioning. Isolated working rat hearts were preconditioned with three 3-rain periods of global ischemia, each followed by 3 min of retrograde reperfusion, and then subjected to 30 rnin of global ischemia and 120 min of retrograde reperfusion. In parallel experiments, 1 mM dithiothreitol (DTT), a sulfhydryl reducing agent, was included in the perfusion buffer during the preconditioning procedure (while it was omitted during the final 120 rain of reperfusion). In control experiments preconditioning was replaced by 18 rain of aerobic perfusion, performed either in the absence or in the presence of DTT. Myocardial injury was evaluated on the basis of triphenyltetrazolium chloride (TTC) staining and of intracellular enzyme (CK and LDH) release. In the absence of DTT, ischemic preconditioning determined a significant decrease in tissue injury (TTC negative tissue 9+_3% vs 24_+2%, P<0.01). In the presence of DTT, no significant difference in tissue injury was observed in preconditioned vs control hearts (average value of TTC negative tissue: 28+-6%). Similar results were obtained with regard to CK and LDH release. We conclude that the development of myocardial protection after ischemic preconditioning might require sulfhydryl oxidation.

CYTOKINES INDUCE NITRIC OXIDE SYNTHASE TU132 ACTIVITY IN CULTURED NEONATAL RAT CARDIAC MYOCYTES. L. Max Buja, Andrea Karoly, Roger J. Bick, Dachun Wang, Anne LeMaistre & Jeanie B. McMillin. Dept. of Pathology, UTHSC, Houston, Texas. Nitric oxide (NO), synthesized from L-arginine by nitric oxide synthase (NOS), may have an important role in some diseases. The constituative form (cNOS) is calcium calmodulin (CaC) dependent and the inducible form (iNOS) is CaC independent, but may be induced by lipopolysaccharides (LPS) and cytokines. Neonatal rat cardiac myocytes served as control, or were treated individually or in combination with IL-1/Y, F-IFN, IL-4 and IL-10 (1Ong/ml), up to 72 hours. Total RNA was isolated, and amplified by reverse transcription PCR and iNOS primers. IL-I# increased iNOS expression in a time-dependent manner, up to 48 hours. IL-I~'+ F-IFN increased iNOS expression above levels obtained with cytokines alone, peaking at 18 hours and IL-4 and IL-10, combined with IL-I~', also increased iNOS. Findings were confirmed by Southern Blot analysis. Expression of iNOS was associated with increased nitrite levels, an index of NO. N-mono-methyI-L-arginine (LNMMA), a NOS substrate analogue, did not reduce the iNOS mRNA levels, but did completely block the cytokine induced nitrite production (with IFN +1L-1.8, 25nmol/mg, +LNMMA, 7.5 nmol/m;. Control, 7.5=t:1.7 nmol nitrite/mg). Neonatal rat cardiac myocytes respond to cytokine treatment with increased iNOS and NO production, the cytokines demonstrating different time courses of action, iNOS, therefore, may be induced in cardiac tissue and lead to autocrine effects on cardiac function.

Tu133

Activation of Protein Kinase C Mimics lschemic P r e c o n d i t i o n i t i o n g t h r o u g h A u g m e n t a t i o n of

Ecto-5'-nucleotidase Activity Koichi Node, Masafumi Kitakaze, Kazuo Komamura, Tetsuo Minamino, Michihiko Tada, Michitoshi Inoue, Masatsugu Hori, Takenobu Kamada, The First Dept. of Med., Osaka Univ., Osaka, Japan. The activation of protein kinase C mediates the infarct size(IS)-limiting effect of ischemic preconditioning (IP). Since activation of ecto-5'nucleotidase is reported to mediate IP, we tested whether exposures to PMA (413-phorbol 12myristate 13-acetate) activate ecto-5'-nucleotidase and mimic IP in the dogs. PMA (0.5pmol/kg/min) was administered four times for 5 rain separated by a 5 min interval (PMA preconditioning). Then the coronary artery was occluded for 90 min followed by 6 h of reperfusion. Ecto-5'-nucleotidase activity was increased after PMA administration (83.5+8.3 vs. 38.2+4.5 nmol/mg/min in the control group). Infarct size normalized by risk area were smaller than the control group (18.4+5.4 vs 40.1+3.9 %), although no difference existed in endomyocardial collateral flow during ischemia. The IS-limiting effect of PMA preconditioning was attenuated by AOPCP, an inhibitor of ecto-5'-nucleotidase (IS: 32.8+9.6%). Thus we conclude that activation of ecto-5'nucleotidase due to the PKC activation is primarily responsible for the IS-limiting effect of ischemic preconditioning.

Tu135 a ] - A d r e n o c e p t o r Activity Regulates Release o f Nitric Oxide From Ischemic Myocardium in Dogs Koichi Node, Masafumi Kitakaze, *Hiroaki Kosaka, Kazuo Komamura, Tetsuo Minamino, Michihiko Tada, Michitoshi Inoue, Masatsugu Hori, Takenobu Kamada, The First Dept. of Med., *The Dept. of Physiol. I, Osaka Univ., Osaka, Japan. We examined whether Ctl-adrenoceptor activity plays a key role in the release of nitric oxide (NO) from ischemic myocardium. In 32 open-chest dogs, the left anterior descending coronary artery was perfused with blood from the left carotid artery. In the untreated condition, the coronary arteriovenous differences of nitrate+nitrite (end-products of NO) concentration (AV(NO)D) was increased 10 minutes after the onset of hypoperfusion (13.6+2.41.tM vs. 3.2+l.ll.tM in the baseline). Antagonists of 13- or a2adrenoceptors did not attenuated release of NO during hypoperfusion (10.7+1.9 and 11.8:t:2.61.tM, respectively). In contrast, intracoronary infusions of prazosin (4 I.tg/kg/min) and phentlamine (9 I.tg/kg/min) during coronary hypoperfusion attenuated the release of NO (5.7+1.3 and 6.4+1.11xM). The extents of decreases in fractional shortening (FS) and lactate extraction ratio (LER) were comparable between the untreated and al-adrenoceptor attenuation (FS: -1.8+1.2 vs 3.8+1.4 % and LER: -34.6+6.7 vs -52.3+9.7 %). We concluded that al-adrenoceptor activity largely contributes the mechanisms whereby NO is released in the ischemic myocardium.

Tu 134

Evidence

for

Nitric

Oxide Production from Human Ischemic Hearts Koichi Node, Masafumi Kitakaze, Hiroshi Yokoyama, Katsuji Imai, Kazuo Komamura, Tetsuo Minamino, *Hiroaki Kosaka, Hideyuki Sato, Michihiko Tada, Michitoshi Inoue, Masatsugu Hori, Takenobu Kamada. The First Dept. of Med., The First Dept. of Physiol., Osaka Univ., Osaka, Japan. There is no clear and firm evidence to support the idea that nitric oxide is produced in the human ischemic heart. To test this idea, we thus measured the coronary arteriovenous differences o f n i t r a t e + n i t r i t e ( e n d - p r o d u c t s o f NO) concentration (AV(NO)D). Both AV(NO)D and lactate extraction ratio (LER) were measured in the coronary sinus blood and arterial blood before and during the cycling exercise. In the patients with coronary artery diseases (% stenosis >90% in the LAD, LCX or RCA), A V ( N O ) D was increased from 2.2+1.8 to 10.7+4.1 ~tM and LER decreased during the exercise. In the control group without coronary aretry diseases, neither AV(NO)D nor LER was changed before and during exercise. These results suggest that nitric oxide is produced during myocardial ischemia in man. DUALBLOCKADEOF THE CYCLO-OXYGENASE Tu 136 AND L-ARGININE-NITRICOXIDEPATHWAYSPREVENTS THE ANTIARRHYTHMICEFFECTOF PRECONDITIONING Adriann Kis, Agnes Vegh, Julius Gy Papp, *James R Parratt. Departments of Pharmacology, Albert Szent-Gyorgyi Medical University,Szeged, *Universityof Strathelyde,Glasgow, UK.

The mechanismof the antiarrhythmiceffectof preconditioning(PC) involvesthe generationof nilzicoxide(NO)and prostacyclin(PG[2), since separate blockadeof the cyclo-oxygenaseand L-arginine-NO pathways attenuates this protection. We have now examined whetherblockadeof bothpathwaysis able to abolish'completelythis marked cardioprotection. In anaesthetized dogsPC was inducedby two 5 rain occlusionsof the leftanteriordescending(LAD)coronary artery, followed,20 rain later, by a 25 rain occlusionof that same artery. Meclofenamate(M; 2 mgkg"I, iv) and L-NAME(5 rngkgl,ic) were given either before the PC procedure or after PC but before the long occlusion. PC markedly reduced the severity of arrhythmias(VPBs:72+_21v 429+68in controls;P<0.05,%VT;21% v 95%; VT episodes:0.7+0.4 v 7.6:k2.1;P<0.05;%VF 0% v 44%; P<0.05), and increased survival (47% v 0%). However, it was difficult to preconditiondogs in the presence of M and L-NAME; 77% of the dogs died during the PC procedure and the remaining dogs fibrillated duringprolongedocclusion. WhenM and L-NAME were givenbeforethe longocclusion,the protectionwas attenuated but still present (VPBs: 118+74;VT episodes;2.6.+_.2.2,%VT:50%, %VF: 17%). The resultsshow that NO and PGI2 generatedduring the PC procedure contribute to the antiarrhythmic effect of PC. Furthermore, dual blockadeof the cyclo-oxygenaseand L-arginineNO pathways prevents the protection more effectively than inhibition of either pathwayalone. Supportedby OTKA(1"-5725), British Council, OMFBand the European Commission(ERB CT 924009). A159

BRADYKININ AND ISCHEMIC PRECONDITIONING IN FUNCTIONAL

Tu137

PROTECTION OF THE ISOLATED R A T HEART. Joel Starkopf, Kirsti Ytrehus. Dept. of Medical Physiology Univ. of Troms~ Norway and Dept of Biochemistry, University of Tartu, Estonia. We compared preischemic treatment with bradykinin (BK, 0.1p.M for 10min) and ischemic preconditioning (IP, 5min ischemia 5min reperfusion) using bufferperfused rat heads subjected to 30min global ischemia and 30min reperfusion. Two additional groups of heads received BK-antagonist Arg[Hyp-Thi-Phe]BK (Sigma) l t t M either with BK (BK+) or PC (PC+). This dose abolished BK induced vasodilatation. Recovery of left ventricular (LV) function and coronary flow (CF) was: qroups (n) LVdP/dt,~,(%) CF(%) control(11) 26.2:1:4.3 68.3+4.5 IP(11) 50.~.9" 87.8+10.3* BK(11) 49.9i-5.0" 77.3+4.2* IP+(7) 59.7+7.9* 99.1+8.8" BK+(5) 66.1:L-5.3" 75.45:2.8* * p<0.05 vs control In conclusion, preischemic BK was as effective as IP in improving post-ischemic function. The BKantagonist did not reduce protection.

CAPTOPRIL POTENTIATES INFARCT SIZETu139 LIMITING EFFECT OF PRECONDITIONING VIA BRADYKININ-2 RECEPTOR ACTIVATION. Takayuki Miki,Tetsuji Mlura, Nobuyuki Ura, KazuakiShimamoto, Osamu llmura. 2nd Dept of Int Med, Sapporo Med Univ,Japan The role of bradykinln (BK) and its origin in preconditioning (PC) remainsunclear. To examine these issues, we assessed the effects of captopril (Cap), a Idninase IIinhibitor, on PC and the arterialplasma BK level. Coronaryarteryof the rabbitwas occluded for 30 min and reperfused for 3 hr. Infarctsize (IS) and area at risk (AR)were determined by tetrazoliumand fluorescent padicles, respectively,and IS was expressed as % of AR (IS/AR). Adedal blood was sampled under baseline conditions and immediatelybefore the 30 rain ischemlafor redioimmunoassayof BK. In the controls, IS/ARwas 43.7±2.8[SE]%, and PC with 2 min-ischemiaslightly limited IS/AR to 34.5 i3.3%. Cap (1mg/kg) alone did not modify IS (IS/AR= 41.7+5.1%), but combination of Cap and 2 rain-PC significantly limited IS/ARto 20.2 ±3.0%. This enhancement of PC by Cap was prevented when 2 p.g/kg of Hoe140, aspecific B2 receptor antagonist, wasgiven before the PC (IS/AR=38.5 +5.1%). Hoe140 alone did not modify IS (IS/AR=41.2:1:5.7%). The heart rate and double products were not significantly different in all groups under baseline condition and during ischemia, though Cap tended to lower blood pressure by about 10 mmHg. PlasmaBK was similarin the control, Cap and Cap plus PC groups under baseline conditions (3.8:1:1.0, 6.3:1:1.9 and 5.2:1:1.7 pg/ml, respectively), and immediately before ischemta (4.14-1.2, 6.2:1:1.9 and 4.2.-1:0.4pg/ml). These results suggest that BK endogenously produced in the heart during PC contributes to IS limitationvia B2 receptors. A160

BRADYKININ CAN PROTECT AGAINST Tu138 INFARCTION BUT IS NOT THE MEDIATOR OF ISCHAEMIC PRECONDITIONING IN THE RAT HEART Einar Bugge and Kirsti Ytrehus, Department of Medical Physiology, Univeslty of Troms~, Norway We tested the hypothesis that prelsehaemic Ireatment with bradykinin (BK) can protect against infarction in an isolated rat heart model of regional ischaemia and mperfuston. We also wanted to clarify if stimulation of BK receptors alone is the mechanism behind isehaemic preconditioning in the rat heart. Buffeaperfused rat hearts were subjected to 30 rain of regional lsehaemla and 120 minutes of reperfuslon. The risk zone was determined by fluorescent particles and Infarct size was detemained by T I C staining.Treatment with BK (0.5 jaM, 3 cycles of 5 rain treatment separated by 5 rain of washou0 prior to lsehaemla significantly reduced infarct size In per cent of the risk zone from 41.8 ± 3.6% In the untreated control group to 9.6 ± 1.3% (p<0.001), a protection similar to the protection afforded by lschaemic preconditioning (IP) (8.4 ± 2.0%). The BK-antagonist HOE 140 reversed the protective effect of BK (42.5 ± 3.8%), but did not influence the effect of IP (6.3 ± 0.9%). Chelerythdne (2 jaM), a protein idnase C inhibitor, reversed the infarct-size reducing effect of bradyldnin (30.0 ± 2.8%) We conclude that pretmatment with BK can protect against Infarction in an isolated rat heart model of regional ischaemia, and that this protection, like IP, is dependent on activation of protein kinase C. Stimulation of BK receptors alone does not, however, mediate IP in the rat.

P R E C O N D I T I O N I N G BY D R U G S ? Tu140 W H E T H E R or O R ]~ A D R E N E R G I C S Y S T E M IS IN THE BACKGROUND OF ILOPROST INDUCED D E L A Y E D AND PROLONGED CARDIOPROTECTIVE ADAPTATION TO I S C H A E M I C STRESS ? Istv~in Kovanecz, Julius Gy. Papp, Lhszl6 Szekeres, Department of Pharmacology, Albert Szent-Gy0rgyi Medical University, Szeged, Hungary It was shown by us earlier that a single dose of ILOPROST afforded a dose- and time-dependent protection against the consequences of global myocardial ischaemia induced by intravenous injection of ISOPROTERENOL, a beta-adrenergic agonist. In the present study we tested PHENYLEPHRINE, an alphaadrenergic agonist, in a dose of 16 lxg/kg under the same circumstances. For comparison lsoproterenol (2 Itg/kg) was used. Both series of experiments were undertaken in conscious rabbits with implanted left ventricular catheter and right ventricular endocardiai electrode. Changes in heart rate, left ventricular end-systolic pressure and in ST segment of the endocardial ECG induced by Phenylephrine were not affected, but when evoked by Isoproterenol they were significantly reduced 24, 48 and 72h after administration of I0 ltg/kg iloprost. Presumably, the beta-adrenergic system plays a more important role than the alpha-adrenergic mechanism in the Iioprnst induced delayed and prolonged cardioprotective adaptation to ischaemic stress.

ISCHEMIC PRECONDITIONING: INTER ACTION WITH Tu141 ANTI-ADRENERGIC INTERVENTIONS. Johan Moolman, Sonia Genade, Erna Tromp and Amanda Lochner. Dept. of Medical Physiology, Univ. of Stellenbosch, Rep. South Africa.

PROTECTIVE MECHANISMS 1. IP MODULATES CATECHOLAMINE RELEASE

The interaction of preconditioning and anti-adrenergic interventions with regard to metabolism (cAMP and energy utilization) [EU]) and functional recovery was examined in the isolated rat heart subjected to 25 rain global ischemia. Six groups were studied: Nonpreconditioned and preconditioned hearts of normal (Non-PC and PC) and reserpinated rats (Non-PC Res and PC Res), and 7Non-PC and PC hearts perfused with propranolo110" M (Non-PC Prop and PC Prop).

AIM: This study was designed to examine whether ischaemic preconditioning (IP) may modulate liberation of noradrenaline (NA) and/or cotransmitters from the nerve plexus. METHODS: Ischaemia was produced by coronary artery occlusion in anesthetised open-chest pigs. Serial drill biopsies were obtained from left ventricular myocardium during and following ischaemia and were immediately frozen in liquid nitrogen. The neuronal NA content was measured, using fluorescence microscopy (glyoxylic acid staining) and computer aided image analysis. RESULTS: A brief (> 10 min) period of myocardial ischemia that resulted in preconditioning caused substantial reduction (approx. 25%) of the synaptic NA content of the affected myocardium. The release of NA stopped instantly upon reperfusion but the synaptic NA stores were not readily replenished (30 rain tested). In hearts preconditioned by ischaemia, there was no early liberation of NA during subsequent coronary occlusions, but there was delayed liberation of NA, with nearly complete lack of fluorescence at 90 min ischaemia. CONCLUSION: The present data indicate that IP reduces the amount of neuronal NA and/or cotransmitters (ATP?), like cardiac sympathetic denervation and delays the release of neurotransmitters during a subsequent ischaemic episode. By limiting the activity of adenylyl cyclase, this may allow the myocardium to withstand better a subsequent ischaemic challenge (see our ancillary report on IP and reduced inhibition of PDEs).

Results:

cAMP* EU • " No reperfusion

Aorta flow # Reperfusion

Non-PC PC Non-PC Prop PC Prop Non-PC Res PC Res

654±30 403±65 a 4 0 7 ± 16 a 3 7 6 ± 13 a 403±18 a 400+23 a

4.2±2.4 16.3±1.7 a 19.8±4.0 a 18.6± 1.9 a 13.3+ 1.2 a 18.6±2.5 a

2.27 1.88 1.48 1.39 1.94 1.74

a = p < 0 . 0 0 1 vs. Non-PC, *p moles/g wet weight, • * =pmoles/g wet weight/min, # = ml/min Conclusion: Non-PC and PC with anti-adrenergic interventions have similar effects on cAMP and postischemic recovery as PC alone, despite varying effects on energy utilization.

PROTECTIVE MECHANISMS 2. Tu143 IP ATTENUATES ISCHAEMIA INDUCED PDE INHIBITION Thomas Podzuweit, Sylvia Thomas, Kiaus Binz & Antje Miiller. Max Planck Institute, Bad Nauheim, FRG. Background: Regional myocardial ischaemia caused

inhibition of cyclic nucleotide phosphodiesterase (PDE) activities in pig myocardium that was readily reversed by reperfusion (TP et al., JMCC 26, 486, 1994). AIM: It was exarrlined whether the beneficial effects of ischaemic preconditioning lIP) may be mediated by attenuation of PDE inhibition during a subsequent ischaemic episode. METHODS: Ischaemia was produced by coronary artery occlusion in anesthetised open-chest pigs. Serial drill biopsies were obtained from left ventricular myocardium during and following ischaemia and were immediately frozen in liquid nitrogen. PDE assay: Specimens were processed and incubated for 1 rain at 25°(2 with c[3H]AMP or c[3I-I]GMP. Mixtures were analysed by reversed phase HPLC with radiochemical detection of labeled nueleotides. RESULTS: A brief (> 10 rain) period of myocardial ischaemia that resulted in preconditioning caused substantial Cz 40%) inhibition of total PDE activity of the affected myocardium. The inhibition of PDEs was readily reversed by reperfusion. In hearts preconditioned by ischaemia-reperfusion (15 min each), there was no significant inhibition of PDEs during subsequent ischaemic episodes (15 min tested). CONCLUSION: The present data indicate that IP may attenuate the inhibition of myocardial PDE activities during subsequent ischaemic episodes, thereby limiting the accumulation of both, cAMP and cGMP (see our ancillary report on synaptie cameholamine release).

Tu142

Thomas Podzuweit, Antje Winkelmann, Antje Miiller & Achim Vogt. Max-Planek-Institute, Bad Nauheim, FRG.

AN INHIBITOR OF ADENYLATE Tu144 CYCLASE BUT NOT BETA-BLOCKADE PROTECTS ISCHEMIC PIG HEARTS Achim Vogt, I-Iiroshl Ando, Doroth~e Weihraueh, Margarete Arras, Albrecht Elsisser, Thomas Podzuweit, and Wolfgang Schaper, Max-Planck-Institute, Bad Nauheim, Germany

Development and size of myocardial infarction depends on the adrenergic influence on the ischemie myocardial region. Contrary to expectation, beta-blockade in studies investigating experimental myocardial infarction did not clearly show a decrease in infarct size. Since prolonged ischemie episodes are known to modify the betaadrenergic receptor - adenylate cyclase system and may thus limit the effect of beta-blockade, we investigated whether direct inhibition of adenylate cyclase may protect ischemic hearts by circumventing said alterations. In chloralose-anesthetized open-chest pigs, myocardial infarction was evoked by a 60 rain LAD-occlusion (CO) followed by 120 min reperfusion. In five of six animals, intramyocardial microinfusion (IM) of the direct adenylate cyclasc inhibitorSQ22536 (lmM) for 10 min prior to CO exerted local protection of the isehemic myocardium against infarction (p<0.01). The beta-blocker atenolol (0.1 mM), tested in three animals, and infusions of Krebs-Henseleit-buffer (four animals) were without protective effect. We conclude that direct inhibition of adenylate eyclase protects ischemic hearts from infarction. The lacking effect of beta-blockers may be due to the altered coupling of the beta adrenergie receptor to its second-messenger system in sustained isehemie episodes. A161

I N S U L I N - L I K E G R O W T H F A C T O R II Tu145 AND INSULIN PROTECT ISCHEMIC MYOCARDIUM Achim Vogt, Hiroshi Ando, Doroth~e Weihrauch, Margarete Arras, Albrecht EIs~.~ser and Wolfgang Schaper, Max-Planck-Institute, Bad Nauheim, Germany Insulin-like growth factor II (IGF-II) is a polypeptide that exerts its metabolic actions via the insulin receptor (a tyrosine-kinase) and its mitogenic activity via the IGF-II receptor. Its increased expression (mRNA) in myocardium subjected to ischemic preconditioning (IP) by short-term coronary occlusions was discovered in our laboratory. To investigate IGF-II's cardioprotecdve role, we applied human recombinant IGF-II (0.25 I.tg/ml) by means of intramyocardial microinfusion rIM) for 60 min prior to a 60 min LAD-occlusion and 120 min reperfusion. Myocardial infarction (IA/RA) was significantly decreased by IGF-II treatment (from 66.6+1.7% to 50.95:2.0%; p<0.0l), whereas IM of Krebs-Henseleitbuffer did not show any protective effect. A comparable degree of cardioprotection was observed after IM of an equipotent concentration of recombinant human insulin (0.02 IE/ml; 64.6+2.5% to 56.4+1.4%; p<0.05). To investigate the receptor involved, we coinfused either IGF-II or insulin together with lavendustin A, an inhibitor of protein tyrosine kinases. Since lavendustin A prevented the observed cardioprotection, it is likely to be mediated by the insulin-receptor. In conclusion, IGF-II, "the heart's own insulin", is cardioprotective during ischemic stress comparable to IP. Its observed actions are likely to be mediated by the insulin receptor, and may lead to increased glucose uptake.

A162

DURATION OF THE 'SECOND WINDOW Tu146 OF PROTECTION' FOLLOWING ISCHAEMIC PRECONDITIONING IN THE RABBIT Gary F Baxter, Fastone M Goma & Derek M Yellon. The Hatter Institute for Cardiovascular Studies, University College London Medical School, UK Previously we have reported that a 'second window of protection' is present 24 h after ischaemic preconditioning (PC) in the rabbit. We have now undertaken a study to examine the duration of this delayed myocardial protection. Male New Zealand White rabbits underwent a midline stemotomy. The myocardium was preconditioned by four 5 rain circumflex occlusions while control animals had a sham operation. The animals were allowed to recover for 24, 48, 72 or 96 h before re-occluding the artery for 30 rain. After 2 h reperfusion the heart was excised and risk volume (R) and infarct volume (I) were determined by fluorescent microspheres and tetrazolium staining respectively. Risk volume was similar in all experimental groups (0.8-1.0 cm3). Infarct size is expressed as % I/R (mean + sere of 5-8 animals).

~

30

• control

20

[] PC

I0 0

* p<0.01

24h 48h 72h 96h These results suggest that the 'second window of protection' after PC extends between 24 and 72 h in this species. The magnitude of the delayed protection appears to increase beyond 24 h although the effect is lost by 96 h.

DELAYED ANTIARRHYTHMIC EFFECT OF Tu147 PRETREATMENT WITH NOREPINEPHRINE IN RATS: THE ROLE OF NalK ATPase Tanya Ravingerova, Wu Song*, Attila Ziegelhoeffer & Jim Parratt*. Inst Heart Res, Bratislava, SIovakia, *Strathclyde University, Glasgow, Scotland

ANTIARRHYTHMIC EFFECT OF ACUTE Tu148 STIMULATION OF RAT HEART WITH EXOGENOUS NOREPINEPHRINE (NE): THE ROLE OF NalK ATPase Tanya Ravingerova, Attila ZiegelhOffer & Jim Parratt* Inst Heart Res, Slovak Acad Sci, Bratislava, Slovakia, *Strathclyde Univ, Glasgow, Scotland.

Although a delayed phase of myocardial protection after ischaemic preconditioning (IP) has been demonstrated in different models, its molecular basis remains poorly elucidated. Norepinephdne (NE) has been recently implicated as a trigger of IP in rats. Our objectives were to test whether:. (i) exogenous NE can mimick the late protective effect of IP in anaesthetized rats and (ii) to investigate the role of Na/K ATPase in this mechanism. Rats were pretreated with NE (lmg/kg i.p.) or saline 24h prior to induction of regional ischaemia in vivo (occlusion of LAD, 30min). Heart rate and blood pressure did not differ between the groups before or dudng ischaemia. In the NE-pretreated group, a total number of VPBs was reduced to 654 from 1662 in controls (p<0.05). All rats survived and only 11% exhibited VF, as compared to 22% mortality and 67% incidence of VF in controls (p<0.05). Although incidence of VT was not affected by NE, its duration was shorter. Antiarrhythmic effect of NE pretreatment was accompanied by an enhanced activity of Na/K ATPase in the heart sarcolemmal fraction (109.7% of initial value vs 83% in ischaemia, p<0.01). Concl.: (i) NE induces late protection against arrhythmias in vivo, and (ii) the protective mechanism may involve stimulation of Na/K ATPase.

Exogenous NE has been found to mimick ischaemic preconditioning (IP) and to afford functional protection in rats and to reduce infarct size in rabbits. There is an indirect evidence that during ischaemia endogenous NE stimulates Na/K ATPase resulting in hyperpolarization of the cell membrane. In this study we tested the hypothesis that protection against arrhythmias induced by exogenous NE can be attributed to the changes in the activity of Na/K ATPase. For this reason, isolated Langendorff-perfused rat hearts were subjected to 30 min regional ischaemia. NE (50 nmol) was applied at a rate of 10ml/min for 5 min with a 10 min recovery prior to ischaemia. Enzyme activities were estimated in the sarcolemmal fraction of control and NE-treated hearts. In the control group, VF and VT developed in 67% and 100% of hearts. In the NE-treated group, VT occured only in 17% of hearts and VF was totally abolished (p<0.01). However, no significant differences in the activity of Na/K ATPase between the groups were observed. Concl.: transient adrenergic stimulation by exogenous NE affords protection against arrhythmias in isolated rat heart. This IP-like antiarrhythmic effect seems to require a different mechanism than immediate stimulation of the activity of Na/K ATPase

DELAYED PROTECTION IN RABBIT HEART Tu149 FOLLOWING 1SCHAEMIC PRECONDITIONING IS ASSOCIATED WITH MODULATION OF HSP27 AND SUPEROXIDE DISMUTASE AT 24 HOURS Richard J. Heads, Gary F. Baxter, David S. Latchman & Derek M. Yellon. The Hatter Institute, University College London, UK. The mechanism of adenosine (A1) receptor-dependent delayed protection 24 hours after i s c h a e m i c preconditioning in rabbit myocardium is unknown. Here we report molecular characterisation of this phenomenon. Rabbit hearts were preconditioned with four 5 min coronary occlusions separated by I0 min reperfusion (4xPC) or given a bolus of 0.1 m g k g -t 2chlorocyclopentyl adenosine (CCPA) or saline in place of 4xPC. After 24h, hearts were frozen in N2(1) prior to analysis. Western blotting with specific antibodies did not detect significant changes in the expression of the stress proteins HSP70i or HSP90 compared to a two-fold elevation 24h following whole body heat stress (HS). However, we observed a decrease in immuno-reactivity towards the low molecular weight stress protein HSP27 24h following 4xPC or CCPA treatment compared to sham or saline controls, possibly consistent with posttranslational phosphorylation of HSP27. Manganese (Mn) and copper/zinc (Cu/Zn) superoxide dismutase (SOD) activities were determined by activity gel analysis as shown below: Results are mean +-SEM, *p <-_0.05 SOD HS Sham 4xPC Saline CCPA Mn 3.2+--0.1 1.9+--0.3 5.1+1.5 2.+6.t-0.l 3.6~-0.2" Cu/Zn 1.2+_0.05 0.9+_0.3 1.8+_0.7 2.0~-0 2.5+--0.1* Therefore, both modulation of HSP27 and increased superoxide dismutase activity may be involved in delayed protection 24 h after preconditioning in rabbit myocardium.

DIFFERENTIAL PROTECTION OF PRIMARY RAT Tu151 CARDIOC;YTES BY TRANSFECTION OF SPECIFIC HEAT STRESS PROTEINS Debbie VE Cumming, R i c h a r d J Heads, David S Latchman and Derek M Yellon. The Hatter Institute, University College London Hospitals, London, UK.

The contribution of specific stress proteins to cellular protection has to date only been observed in cell types that are not fully representative of mammalian myocardium. The aim of this study was to examine the individual contribution of specific heat stress p r o t e i n s in primary rat cardiocytes to any protection observed following lethal heat stress or simulated lethal

ischaemia, cDNAs for hsp70i, hsp90, hsp60 or a control plasmid were cotransfected with a detectable marker, alkaline phosphatase, into primary rat cardiocytes. Survival assays were performed after a subsequent lethal stress. Expression of transfected hsp70i was found to increase survival following a subsequent lethal stress by 2.45 fold (p<0.01) and against a subsequent lethal ischaemic stress by 2.71 fold (p<0.01). Transfection of the hsp90 gene improved survival against subsequent heat by 2.55 fold (p<0.05) but failed to have any effect on survival against subsequent lethal ischaemia. Transfection of the hsp60 gene offered no protection against either stress. This study identifies specific differences in the ability of three members of the stress protein family to confer protection against a subsequent lethal heat stress and subsequent simulated ischaemia in primary rat cardiocytes.

ADENOVIRUS MEDIATED GENE Tu150 T R A N S F E R O F A H E A T S H O C K PROTEIN70 (HSP70i) P R O T E C T S A G A I N S T I S C H E M I A Ruben Mestril, Frank Giordano & Wolfgang H. Dillmann UCSD Medical Center, San Diego, CA, USA Our laboratory has recently demonstrated that overexpression of a hsp70i in a rat myogenic cell line confers protection against simulated ischemia. We have developped and shown that overexpression of a rat hsp70i in the cardiomyocytes of a transgenic mouse line protects against ischemia/reperfusion injury. We have presently inserted the hsp70i gene in an adenoviral vector and show that we are able to transfer and achieve overexpression of this protein in neonatal cardiomyocytes and a rat myogenic cell line. We have also found that cells infected with the adenoviral-hsp70i are rendered more tolerant to simulated ischemia as compared to cells infected with an adenoviral-Lac Z control construct.We conclude that our findings demonstrate the feasibility of using adenoviral vectors to overexpress hsp70i in cardiomyocytes and the efficacy of this approach for providing protection against myocardial ischemia.

SPECIFIC INDUCTION OF HSP72 BY HERBIMYCIN-A Tu152 PROTECTS CARDIOMYOCYTES FROM A SUBSEQUENT STRESS Susie D. Morris, Debbie V.E. Cumming, David S. Latchman,Derek M. Yegon. The Hatter Institute, University College London Hospilals, London, UK Heat shock protein (HSP) induction by stressful stimuli such as heat and ischaemia is known to protect cardiac calls from a subsequent severe stress. The ability to directly increase the level of HSPs via a pharmacalogicalmeans has yet to be demonstrated in Ihe heart, however in non-cardiac cells the tyrosine kinase inhibitor Hetbimycin-A has been shown to induce HSP72. We examined whether Herbimycin-A and another tyrosine kinase inhibitor, Genistein, could induce HSP72 in primary cultures of rat neonatal cardiomyocyles; and whether these treatments could improve survival on exposure to severe stress. Primary cardiomyocytes were incubated with Herbimycin-A (50p.M) or Genislein (501.tM). HSP induction was measured 16-20 hours later by Western blotting. Cell survival lollowing subsequent lethal heat stress (47*C for 1 hour) or simulaled ischaemia was assessed using Irypan blue exclusion and an LDH assay. Our results indicate that Herbimycin-A induces HSP72 but not HSPg0 or 60, and Genistein has no efiecl on HSP levels. Moreover HSP72 induction correlated with the ability of Herbimycin-A to protect cells against lethal simulated ischaemia, whereas Genistein had no protective effects. Cell death in Herbimycin-A treated cells subjected to lethal ischaemia was 50.6% (+ 5.6), compared to 68.1% (+ 1.9) in controls, (p<0.001, n=10). Cell death in the Genistein group was 69.8% (:1:t.8), with no significant protection. Comparable levels of protection by Herbimycin-A against severe heat stress were also obtained. Thus Herbimycin-A specifically induces HSP72 in primary cardiomyocytes, and protects against subsequent lethal stress. The lack of effect of Genistein on both HSP72 induction and protection, suggests that Herbimycin-A may well act via a tyrosinekinaseindepedentmechanism. ElucidalJonof thesemechanisms may lead to slrategies for targeted hsp72 induction and cardiac protection in clinical practice.

A163

CARDIAC AND SKELETAL MUSCLE HEAT SHOCK Tu153 PROTEINS IN HYPERTROPHY AND HYPERTENSION. Giuseppina Gala, Laura Comini, Evasio Pasini, Laura Agnoletti, Massimo Benigno, Salvatore Curello, Roberto Ferrari. Fundazione Clinica del Lavoro, Centre S. Maugeri, Gussago, Brescia, Cattedra dl Cardiologia, Universit~t di Brescia, Italy.

INDUCTION AND P H O S P H O R Y L A T I O N STATE Tu154 OF HEAT S H O C K P R O T E I N HSP25/27 IN T H E RAT M Y O C A R D I U M Brigitte Hoch, Rainer Bermdorf, Gudrun Lutsch, Wolfgang Schlegel, Sabine Bartel, Ernst-Georg Krause, Gerd Wallukat & Peter Karczewski. Max Delbriick Cenlre for Molecular Medicine, Berlin, Germany.

To elucidate the relationship between hypertension and hypertrophy in the induction of heat shock protein (lISP 72), we measured HSP72 synthesis in cardiac and gracilis muscles of normotensive (WKY) and hypertensive (SHR) rats of different ages subjected to hypertermia (42"C+0.5 for 15 rain). We divided the animals as follows: (a) no-shocked age-matched control: (b) 2 months WKY and SHR with developing cardiac hypertrophy; (c) 18 months WKY and SHR with fully developed chronic cardiac hypertrophy. HSP72 protein was investigated by SDS-Page and Western Blot analysis, expressed as % (mean _+ SE) of a commercial standard. Our data show that: 1) the heart of young SHR responds to heat shock more than their normotensive age-matched control (298.8+24.7% vs 88.3+8.5%, p<0.001); 2) there is no significant difference between normo- and hypertensive heart of 18 months rats after exposure to hypertermia (43.6+5.3% vs 65.3+10.4%); 3) the gracilis muscle shows a basal spontaneous HSP72 synthesis in both SHR (71.4+10.8%) and WKY (40.6+11.7%) animals; 4) there is a significant increase in HSP72 synthesis in the gracilis muscle of young SHR with respect to WKY (186.2+18.7% vs 115.8+9.9%, p<0.02); 5) this pattern is maintained also during aging (171.9+17.3% vs 95.2+10.5%, p<0.01). These data suggest that hypertension results in increased synthesis of HSP72 both in cardiac and gracilis muscle in responce to heat shock. This abnormal response is mitigated by aging in the heart but not in the gracilis muscle. HSP 72 synthesis is independent from hypertrophy.

Heat stress induced cardiac protection against subsequent ischemia and reperfusion injuries has found considerable interest in the last years. Several authors suggest that this cardioprotective effect is linked to the expression of heat shock protein HSP70. Here we report on induction and phosphorylation o f HSP25/27, another m e m b e r of the heat shock protein family, which has been implicated in the organization of microfilaments. The expression o f the hsp25127 gene under heat stress was examined in Langendorff hearts and in isolated neonatal and adult cardiomyocytes by Western blotting. In perfused hearts the HSP25/27 was found to be induced by heat stress at 42°C for 45 min. The phosphorylation state o f HSP25/27 was analysed by 2D-PAGE. Isolated cardiomyocytes and intact perfused hearts contain mainly non-phosphorylated and monophosphorylated isoforms and a minor amount of the biphosphorylated isoform. In neonatal cardiomyocytes HSP25/27 was one of the major phosphoproteins. The distribution o f HSP25/27 was analysed by immunofluorescence microscopy in order to characterize the effect o f heat shock on distribution of HSP25/27 and organization o f microfilaments in isolated cardiomyocytes.

PHARMACOLOGIC 'PRECONDITIONING' WITH Tu 1 5 5 MONOPHOSPHORYL LIPID A IN THE RABBIT HEART IS NOT MEDIATED BY THE SYNTHESIS OF A 70-KILO DALTON HEAT SHOCK PROTEIN Rakesh (2. Kukreja, Kazu-ichl Yoshida, Joshua B. Shlpley, Mark DeloreJeo, Yong-Zhen, Qlan, Mohammed Maaleh, and Gary T. Elliott*. Medical College of Virginia, Richmond VA, USA * RIBI ImmunoChem R~eareh Ine, Hamilton, MT USA.

PROTECTIVE EFFECT OF THERMAL STRESS A G A I N S T CRYOPROTECTANT TOXICITY ON AORTIC RINGS. C. Poussin, M. Joyeux, C. Ribuot, J. Verdetti

Monophosphoryl lipid A (MI,A) represents a novel class of pharmacologic agent capable of producing myocardial protection, in this study,the effect ofMLA pre-treatment was evaluated in an in vivo model of regional isehex~a/reperfusion in rabbits. Animals (n=9 for each group) were treated with MLA (35 ]:~-'~¢';~,,.,l ug/kg IV or equivalent vehicle in controls) 12 and 24 hr prior to ischemia/reperfusion. AT 12 or 24 hr, hearts were subjected to 30 min of regional ischemia (via ligation of the LAD) ...,., . . . . followed by four hours of repeffusion. At the end of reperfusion, infarct size was determined by tetrazolium staining. No significant improvement in hemodynamics was seen in treated animals, and CK and LDH release were slightly increased over controls. Infarct size, however, was significantlyreduced in the MLA treated animals. Interestingly, western blot analysis did not demonstrate the synthesis of Heat Shock Protein 70 (lISP) in MLA treated animals, though the inducible form of this protein has been proposed to have a role in mynoardial protection. We conclude that MLA has a powerful anti-infarct effect in the isehemic rabbit heart, which does not appear to be mediated by synthesis of liSP. A164

Univ. J. Fourier-F38041

GRENOBLE

Tu156

- France

The goal of cold-preservation and cryo-praservation is to preserve organs for transplantation. The extent to which an organ recovers from cold ischemia is organs and species specific depending on cold storage time, cold and cryopreservation techniques, presence or not of cryoprotectant in the preservative solution. I,2 propanediol (1,2 Pd) is a potential cryoprotectant since it permeates well through cell membrane and has a strong glass-ice forming tendency. However, its toxicity increases rapidly beyond 3 M. The purpose of this study was to determine whether prior (2A-h) heat stress (42°C-20min) could protect vascular cells against 1,2 Pd toxicity. We compared the reactive properties of isolated rat aortic rings from non-thermal stressed non-l'2Pd perfused (GO to 1,2 Pd-perfused (60 min-3,3M) ((31I) or thermal-stressed 1,2 Pd perfused (GIll) groups. The results first show that thermal stress induces the vascular synthesis of heat shock proteins (lISP) 70 and 27, and second that the relaxant effect of acetylcholine on noradrenaline-precontracted aortic rings from stressed rats is well-conserved after 1,2 Pd perfusion, since the relaxation is 43% for thermal stressed rats (GILD, whereas it is only 15% for GII and 58% for GI. Likewise, the nitroprusside-induced relaxation of KCl-precontmeted aortic rings from stressed rats increases up to 62% after 1,2 Pd treatment (Gill), compared to the 32% for GII and 17% for GI. Conclusion : acute heat stress protects vascular cells against 1,2 Pd toxicity. This protective effect could be correlated to the enhancement of HSP synthesis.

This work has been supported by grants of MRE and R~gion Rh~ne -Alpes.

INCREASED TOLERANCE TO ISCHEMIA BY Tu157 ADAPTING TILE, HEART TO OXIDATIVE STRESS INDUCED BY VP-16.

Nllanjana Maulll~ A/pad Tosald, Rlcharrl M. Engelman,ValprianE. logan, and D/pakK. Das, Univ. of Connect~utSchoolof Medldne,Farmlngton,

CT, and Univ. of Pittsburg, Pittsburg, PA, USA

The antitumor activity of VP-I6 (etoposide) is believed to be mediated by the transient formation of VP-16 phenoxy.l radical in the course of its oxidative/reductive conversmns. We used this property of VP-16 to adapt the heart to oxidative stress by injecting the rats with VP16 (0.1-10mg/kg). Induction of oxidative stress was confirmed by enhanced malonaldehyde formation in heart. After 24 hr, rats were sacrificed and isolated working rat hearts were prepared. Ischemia was induced for 30 rain followed by 30 min of reperfusion. VP-16 (0.1 and 1 mg/kg) did not exert any effects on the heart rate and coronary flow before and ischemia. Significant improvement in aortic flow, left ventricular pressure (LVP) and the maximum first derivative of LVP (LVdp/dt) was noticed during, post-ischemic repeffusion. Corroborating with the improved post-ischemic ventricular functional recovery, decreased oxidative stress and enhanced intracellular antioxidants enzymes and increased glutathione content were observed in the VP-16 treated rats. At 10 mg/kg, VP-16 was found to be arrhythmogenic and caused loss of intracellular ascorbate and thiol. The results of this study, thus, suggest that hearts can be adapted to oxidative stress with a lower dose of VP-16 that can render the heart more tolerant to ischemia / reperfusion injury.

H Y P E R T H E R M A L I M M E R S I O N IS B E ' I ' r E R Tu159 T O L E R A T E D T H A N E X E R C I S E T E S T IN C A D PATIENTS Juraj Celko, J~-n Luk&~ B r a h o V o h n o u t , D e n n i s Valent, Jdn L i e t a v a S l o v a k Spa, T r e n ~ i a n s k e Teplice M e d i c a l F a c u l t y of C o m e n i u s University, B r a t i s l a v a Slovak Republik Balneologists fear to indicate hyperthermal immersion (HI) to CAD patients,but the same patients are taking hot bath without any control at home.We verified the hemo dynamic response comparing standard maximal syrup tom-limited exercise test ( E l ) with HI in 40"C water (terminated by increase of core temperature about 2 "C) in 21 middle aged men ( 60.3 <45-72 yrs > ) with CAD. Thoracic electric bioimpedance, ECG and BP were monitored to estimate the cardiac load in the both tests. Subjective tolerance was compared by Borg scale. Results: HI had Iongerduration ( 2 2 . 2 vs. 12.1 min; p<0.003), but exhibit less ischemic episodes(1 vs. 5; p<0.001), less complex ventricular arrhythmias (2 vs. 4; NS) and peak SBP was substantially lower (147.0 vs. 213.6 mmHg; p<0.004 ). Peak hemodynamic response was lower in HI than in ET : cardiac index was 4.2 vs. 6.4 Vmin/m 2 (p< 0.001), index of contrac tility was 0.038 vs. 0.060 l / s e c (p<0.001) and heart rate was 105.8 vs. 126.9 bpm (p<0.02). Borg scale suggests better subjective tolerance of HI than ET: 7.6 vs. 10.8 (NS). We conclude that HI can be safely indicated also to CAD patients.

DIFFERENCE IN HEAT-INDUCED SYNTHESIS O F T u l 5 8 NITRIC OXIDE IN THE HEART AND OTHER ORGANS OF RATS OF DIFFERENT GENETIC LINES Igor Yu.Malyshev, *Anatoly F.Vanin, "Vasak D.Mikoyan, Ludmila N.Kubrina, Elena V.Malysheva, & Eugenia B.Manukhina. Inst of Gen Pathol & Pathophysiology, Inst of Chem Physics, Moscow, Russia Heat shock (HS) is a systemic disorder which can be even fatal. Death induced by HS is associated with myocardial injury, vascular collapse, endotoxemia and hemorrhage. These complications are characterized by potentiated nitric oxide (NO) production. Previously we have shown that resistance of animals to HS depends on the genetic line: the death rate of August rats from HS was 6 times as high as in Wistar rats. This suggests that genetic lines may differ by the extent of activated NO synthesis in response to HS. Our aim was to compare the NO generation response to HS in Wistar and August. HS was produced by heating of rats to 41°C. NO was measures in the liver, kidneys, spleen, heart, intestine and the brain by EPR spectra. In the liver, heart and spleen, the NO content was significantly higher in August than in Wistar. HS increased the NO content in both Wistar and August. However, the increase in NO was considerably greater in August than in Wistar. For instance, in HS-exposed August, the NO content was increased 10 times in the heart, 15 times in the kidney, 6 times in the spleen, 3 times in the liver, 3 times in the intestine, and 2 times in the brain as compared to HS-exposed Wistar. Thus the less resistant to HS August line displayed much more pronounced activation of NO synthesis in response to HS than the more resistant to HS Wistar line. We conclude that genetic mechanisms determining the organismic resistance to HS are related with mechanisms of NO production.

THE INTERLINES DIFFERENCE-IN INDUCEDTH~-Tu16o MOTOLERANCEFOR RATS ISOLATEDHEAHTS~ Alexander Zamotri,nsky.Nt,kolay Lart.onov. Igor

Malushev~ Inst. Cardiology. Tomsk. Russia. Adaptation to repeated restraint stresses(MS) results In increase of rats isolated hearts stabl,llty~ The difference l,n formation of AHSinduced hearts thermotolerance for rats Wistar and August lines were studted~ Heat stroke was modelea for Isolated hearts by increase of temerature of perfuston solutLon to d2C: 15 mtn,

phen~

contraction function of hearts and crea-

tin k inase(CK) release from ones sere compared for 4 groups (n~6/group} of rats~ , Wlstar [ Auqus~ leontd adaptJ l contr, l adaptedd Contractur~0.8 ] 0.3~O. llO.9±O, l lO.8*O.I +Contraction[ l, [ l ampli~tude~m~O,4 I I.OiO. II O,410.Z [ 0,5~0,I +CK{mUlmin/g)E95 [* ZO~15 [310160 l Z75150

• p< O~OI compared with control, ÷ p< O.OI between lines . for adapted rats~ Unabtlity of August rats to develop of AHS-Induced thermotolerance Is associated wlth lack of induction of hap 70 proteins synthesis in their heartsJ In contrast. AHS induce In Wis tar rats both the hap 70 synthesis and the heart tolerance formatlon~ ~e conclude that: I~the hap 70 synthesis have a c r i t i c a l role In formation of ~ e r m o t o l e r a n c e and Z. there are the line-dependent d i f f e r e n c e s tn a b i l i t y to form the heart p r o t e c t i v e reaction for rats, A165

BENEFICIAL EFFECTS OF HEAT ACCLIMATION Tu161 DURING ISCHEMIA AND REPERFUSION: METABOLIC ADAPTATIONS Eynan M l, Knubovets T 3, Meiri U I, Navon G 3, Hasin y2, Horowitz M t. Physiology I & Cardiology2, Hebrew Univ. Med. Sch. & Physical Chemistry 3 TeI-Aviv Univ. Israel Previous studies showed that: heat acclimation provides protection during ischemia and reperfusion. Decreased number of acclimated hearts (AC) developing ischemic contracture (IC) with delayed onset compared to norrnothermic hearts (C) was associated with conservation of ATP during the ischemia. Upon reperfusion, recovery of both systolic and diastolic functions was markedly better in these AC hearts. Since glycolysis is the primary source of ATP during ischemia, our present study was conducted to elucidate it's role in the adaptive responses observed. Experiments were carried out on isolated C and 1 month acclimated (34°C) rats's hearts. Measurements of." 1. glycogen conoentration 2. Effects of the duration of glycolysis inhibition (with iodoacetate for 15 and 30 min) on the development of 1C during total ischemia and its recovery upon reperfusion 3. Lactate (LA) production using 13C NMR spectroscopy were performed. Glycogen concentration was 1.5 fold greater in AC than in C hearts. Longer time was required to inhibit glycolysis in AC compared to C hearts (30 and 15 min respectively). 13C glucose incorporation to glycogen in AC hearts exceeded that of C hearts. LA production during ischemia is slower but ultimately reached higher levels in AC than in C hearts. Those results indicate that in the AC hearts higher glycogen levels and slower but prolonged glycolysis contribute to ATP conservation leading to a better recovery of the AC heart upon rel~erfusion.

Cardioprotective effects el adaplation to environmental lactors are determined by increased aclivity of stress-limiting syslems including that ot prostaglandins (PC), and also by enhanced resistance of the myocardium itself to injuries. Adaptation to exercise exert.,-; more pronounced cardioproteclivo ellects against infarction and reperlusion than adaptation to hypoxia. The aim was to compare the activity el PG system and of myocardial delence mechanism in these types el adaptation. One group of rats (HR) was adapted to hypoxia in a hypobaric altichamber; another group (TR) was trained to swimming. Plasma PC, catecholamines (CA), and corticostefone (CS) were radioassayed. In HR, PGE, PGI2 and PGE/PGF2,, were increased by 78, 60 and 40% respectively as compared to the control; PGI2/-I'XA2 remained unchanged. In TR, PGE2, PGI2 were increased by 54 and 65% respectively; PGI2./TXA2 was unchanged. After acute stress, PG levels were signilicanlly higher while CA and CS release was lesser in HR and TR than in stress-exposed controls. Thus both adaptations activale the PG system and decrease the stress reaction. However protective effects of these adaplalions on the isolated head in total ischemia and reperlu.~ion were strikingly dissimilar. The ischemic and repeHusion contracture, creatine kinase release and arrhythmias were 3-12 times lesser in TR than in control. Adaptation to hypoxia lacked these protective elfects. This suggests that dillerenlial cardioproleclive eflects el exercise training and adaptation to hypoxia observed in vivo are due Io the difference in local cytoprotective mechanism in the hearl.

AN3]ARRHYTHMIC EFFECTS OF ACAJTE Tu103 HYPERCHOLESTEROLAEMIA Linda A Shaw & Susan J Coker, Pharmacology & Therapeutics, University of Liverpool, UK.

ACUTE HYPERCHOLESTEROLAEMIAAND Tu164 PLATELET AGGREGATION Linda A Shaw & Susan J Coker, Pharmacology & Therapeutics, University of Liverpool, UK.

To study the effects o f acute hypercholesterolaemia on

It has been reported that after only 3 days on a cholesterol enriched diet, platelet responsiveness is increased in rabbits. We have studied whether feeding a diet enriched with 5~; cholesterol and 1% taurocholate to rats for 7-10 days altered platelet aggregation. The ability of 5-HT and the TxA2 mimetic U46619 (U4) to potentiate platelet aggregation induced by ADP (10TM) or collagen (1 #g ml") was measured in heparinized whole blood (n=5 or 6 per group).

arrhythmias associated with myocardial ischaemia, male Wistar rats and NZW rabbits were fed cholesterolenriched diets for 7-10 days, then anaesthetized and prepared for coronary artery occlusion. In rats (n=12 per group), 5~ cholesterol with 1~ taurocholate in drinking water increased plasma cholesterol from 0.35-+0.03 (in controls receiving normal diet and water) to 2.85+0.40mM (P
A166

PROTECTION OF THE HEART BY ADAPTATIONS TU162 TO HYPOXIA AND EXERCISE Maya G. Pshennikova, Bella Ao Kuznetsova, Peter A. Prodius, Maria V. Shimkovich, Felix Z. Meerson & Igor Yu. Malyshev. Inst. of General. Pathology & Pathophysiology, Moscow 125315, Russia

Platelet Aggregation (fl) Norm Chol

ADP +5-HT 1.0__.0.5 6.4___1.0 0.8_+0.4 4.8+_1.5

+U4 +5-HT&U4 15.5___2.8 17.9-t-1.0 10.3_+2.7 18.3_+1.7

Norm Chol

Collagen +5-HT 6.7___1.4 12.9-+1.0 4.8-+0.9 11.4-+2.1

+U4 +5-HT&U4 17.3-+0.6 15.0___1.8 12.6+_1.7 20.7-t-3.8

Neither 5-HT nor U4 alone caused aggregation, but both substances potentiated responses to low concentrations oFADP or collagen. The responses of platelets from rats with acute hypercholesterolaemia were not increased when compared to those of platelets from rats fed a normal diet. Similarly, in further studies in rabbits fed a 2~o cholesterol enriched diet for 7 days we found no changes in platelet responses to ADP or collagen.

IMMUNOCYTOCHEMICAL STUDY OF THE VASCULAR Tu165 COMPOSITION OF CAROTID ATHEROSCLEROTIC PLAQUES Jos~ Milei, Graciela Fernandez Alonso, Ricardo Beigelman & Juan C. Parodi. Cardiopsis, Buenos Aires, ARGENTINA. Although carotzd plaque fissure has been demonstrated as the main mechanism leading to intraplaque hemorrhage (IPH) and/or thrombosis, a number of IPH do not present a connection with the lumen. The goal of this stud¥ was to characterize the vascular components of 68 surgically excised carotid endarterectomies to get information about the pathogenesis of IPH. CD3I (PCAM) and C D 3 4 monoclonal antibodies were used for cell zmmunophenotyping. 14/68 cases presented IPH; serial sections showed that intimal surfaces were clean and a continuous, row of endothelial cells (anti CD31 and anti CD34 +) was observed. Highly vascularized lipid cores, with neoformed vessels stained with CD34 and weakly with CD31 were found. T-lymphocytes were found in contact to neoformed vessels, and in some cases disrupting the endotheliums. The deeper layers of the pia~ues also showed neoformed vessels and old hemorrhages as shown by hemosiderin-laden macrophages. This suggest that because of its vascularized constitution, in 20 % of cases, IPH might originate from the rupture of neoformed vessels.

Increased Intramural Expression of

Tu167

P l a s m i n o g e n a c t i v a t o r I n h i b i t o r t y p e - 1 (PAl-l) after Balloon Injury Hirofumi Sawa, Toshiyuki Kudo, Hideaki Kawaguchi, "Satoshi Fujii, "Burton E Sobel, Akira Kitabatake:Hokkaido Univ., ,Sapporo, Japan; *Univ., Vermont Burlington, VT, USA PAl-l, the primary physiological inhibitor of endogenous plasminogen activators, is present within atherosclerotic vessels. To in'vestigate the relationship between expression of PAl-1 and neointimal information after angioplasty, PAl-1 gene and protein expression were assayed sequentially in rabbit carotid arteries subjected to balloon injury with the use of Northern blotting, in situ hybridization, immunostaining. In uninjured vessels, PAl-1 mRNA was not detectable. However, injury was followed within 3 hrs by increase in PAl-1 mRNA of 5.9 fold compared with that in contralateral control carotid arteries. PAIl mRNA was detectable by in situ hybridization early after injury first in adventitia; after 24 hrs it was particularly prominent in the media. From 1 to 4 weeks after injury it was consistently detectable and was localized in neointimal vascular smooth muscle and endothelial cells at a time when neointimal thickening was marked. Cells of both types exhibited PAl-1 protein detected immunohistochemically. In vessels maintained in organ culture after balloon injury in vivo, sustained increases in PAl-1 activity appeared in conditioned media as well. Conclusions: Balloon injury stimulating angioplasty in patients induces intramural expression of PAl-1 in vascular smooth muscle and endothelial cells. The decreased cell surface fibrinolytic activity likely to result from the incresed PAl-1 expression may initiate or exacerbate mural thrombosis. Accordingly, excessive stimulation with clot-associated mitogens may stimulate vascular smooth muscle cell proliferation.

CAROTID PLAQUE RUPTURE. Tu166 AN IMMUNOCYTOCHE~4ICAL APPROACH. J0s~ Milei, Graoiela Fernandez Al0ns0, AndreaBar0ne, Ricard0 Beigelman,Juan C.Par0di, Cardi0psis,Buenos Aires.

The exact mechanists causinq carotid plaque rupture are yet not known. The goal of this study was to analyse the cellular componentsof 68 surqically excised carotid endarterectomiesin order to obtain informationabout their role in the pathoqenesis o( plaque rupture, the cell populations were characterizedwith cell specific monoclonal antibodies (Bndothelialcells: CO31, CD34;T-ly~phooytes:CD45 RO; nacrophages : CD6B; B lymphocytes:CD20; nusclecells: HHF35. Streptavidinbiotin peroxidase and streptuvidin biotin alkalin phosphatasewere used and unique and double immunostainings. Twenty-fivespecimens presented plaque rupture with intraplaquehemorrhaqe (4/68) plus thrombosis (15/68) or only thrombosis(16/68). The fibrous caps at the site of the ruptureshowed loss of the endothelial lining. In the remaininq surfaces, a continuousnon damaqed row of endothelial cells (anti CD3 and anti CD34+) was observed. The collagenousfibrous cap fissure showed inflammatory components oonsistinqof 2/3 of nacrophages [CD68+), i/3 Tlymphocytes and scarce B-lymphccytesand mast cells. A close interaction between macrophaqesand capillariesand =acrophaqes and T-lymphooyteswere cononly observed. In the lipid cores, T-lymphocyteswere found to be in close contact to neorf0rmed vessels, and in somecases disrupting the end0theliums. Mononuclearinfiltrates(CD68 and CD45+) were observed half of the cases in the peripheryand 'shoulders'of the plaques, surroundinqneoformed vessels. These data suqqest that inflanmation throughenzymaticdeqradation of the cap miqht cause weakiniqand rupture of the fibrous plaque.

GENETIC RISK FACTORS IN PATIENTS WITHTu168 CORONARY OBSTRUCTION: AN ASSOCIATION STUDY CONCERNING ACE AND LRP Katrin Handschug, Gerhard Miiller, Wilhelm Teichmann, Christiane Giiiser, University of Halle, Germany Investigating the importance of genetic factors in the atherosclerotic risk constellation we studied the Angiotensin Converting Enzyme (ACE) D/I and the Low Density Lipoprotein Receptor Related Protein (LRP) A1/A2 (tetranucleotide length (TFrC),) polymorphism of 70 patients (coronary stenosis of more than 70%) from the Central German region elected for percutaneous transluminnl coronary angioplasty (PTCA). The allele frequencies of the PTCA patients were compared with those of healthy blood donors of the same geographical region. The ACE D/I ratios of all the patients (D/I: 0.58/0.42) did not differ significantly from the control group (D/I: 0.59/0.41). But in consideration of the multifamiliai coronary affliction the 26 high risk patients had higher D allele frequencies (0.63) than the remaining patients (0.55)(p<0.01). The LRP polymorphism (A1/A2:91bp/87bp) ratio was 0.67/0.33 in the PTCA group and 0.58/0.42 in the controls (odds ratio 1.47). We found a heterozygosity index of 0.42 in the patients and 0.63 in the control group. Our results suggest that first the ACE polymorphism should only be included in risk assessments in combination with high mnltifamilial afflictions and second the LRP polymorphism showing different allele frequencies in patients with coronary obstructions and controls and should be taken into account in the investigation of individual risk constellation for atherosclerosis development. A167

PLATELET STIMULATED PROLIFERATION Tu169 OF CORONARY SMOOTH MUSCLE CELLS Christina Unterberg, Thomas Meyer, Arnd B. Buchwald Dept. of Cardiology, University Clinic, G6ttingen, Germany Human coronary smooth muscle cells (SMC) from atherectomized restenotic lesions and porcine SMC 4 weeks after coronary angioplasty were cultivated to passage 3-7. Human (H-PL) and porcine (P-PL) platelets were isolated from citrate-anticoagulated blood (60 ml, platelet recovery 20 ± 2 %). Proliferation (PROL) (3H-thymidin incorporation) following 24 hrs stimulation with 10 % fetal calf serum (FCS) was used as 100 % reference. Both H-PLA and P-PLA stimulated PROL of SMC in a concentration dependent manner with a maximum of 73 ± 2 1 % (H-PL, 5x104/I.tl) and 69 + 16 % (P-PL, 22.5x104/I.tl) of FCS values (mean + SD). Similar results were obtained with PL lysed by sonication. Parallel experiments using the growth factors PDGF-BB, bFGF, EGF and ILGF-I alone or in combination at their maximal effective concentrations showed significantly less stimulation (p < 0.05, ANOVA). PDGF-BB at 50 ng/ml was the most potent stimulating individual growth factor. Results: I. It. III. IV. V. (PROLPDGF- PDGFPDGF-BB PDGF-BB PL % FCSBB BB +bFGF+ +bFGF values, +bFGF EGF + EGF mean+SD) +IGF- 1 H-SMC 17±6 22±12 22±8 43+13# 73±21" P-SMC 16±9 2 3 ± 1 7 21±11 41±14# 69+16' #: p < 0.05 vs. I, II and IIl. *: p < 0.05 vs I, 11, 111 and IV.

Conclusion: SMC - PROL in vitro is significantly more stimulated by platelets than by growth factors suggested to cause SMC proliferation after coronary angioplasty in vivo. This might have implications for anticoagulation during coronary angioplasty.

THE EFFECT OF R-HIRUDIN IN A MODEL Tu171 OF RESTENOSIS. Patrick Hadoke, Cherry Wainwright, Roger Wadsworth, Keith Butler*, June Giddings*, University of Strathclyde, Glasgow, UK; *Ciba Pharmaceuticals,Horsham, UK. This study assessed the effects of acute and chronic r-hirudin therapy on the consequences of angioplasty of the left subclavian artery in NZW rabbits following 1 month of 1% cholesterol feeding, r-Hirudin (0.5 - 0.6 mg kg-1 h-1) was administered via subcutaneous minipumps for either 24 hours (n = 8) or 28 days (n = 6), starting immediately after angioplasty; controls (n = 10) received saline. Four weeks after angioplasty the rabbits were sacrificed and both right (control) and left (angioplastied) subclavian arteries were removed for morphological(Table) and functional analysis. Lumen(ram 2) Intima(mm 2) PN M Control R 53.5+9.8 4.8+9.8 14±6 58+30 L 47.2+9.0 15.0+1.8"* 34+16 118+51" Acute R 60.5+9.2 1.8+1.3 3+2 28+16 L 41.8±4.1"* 23.5+4.6** 35+12" 80+96* ChronicR 60 6+9.4 0.3+0.3 1+1 0 L 35.3+6.7** 28.5+5.6"* 55±15" 141+44" PN = Proliferatingnuclei; M = macrophages.No in intima. *P < 0.05; **P < 0.01 vs right artery. In controls, the maximum relaxation to carbachol in left subclavian artery rings precontracted with 5-HT (llaM) was reduced 10.4 + 6.6% compared to 20.6 :t: 9.1% in right artery rings; responses to calcimycin and SIN-1 were unaffected by angioplasty. Neither acute nor chronic r-hirudin treatment altered this reduced responsiveness to carbachol. These results show that, in this model of restenosis following angioplasty, r-htrudin does not prevent either the development of neointimaor functional abnormalitiesof the endothelium. A168

S u p p r e s s i o n of V a s c u l a r S m o o t h Muscle Tu170 Cell Proliferation by an A n t i s e n s e O l i g o n u c l e o t i d e a g a i n s t P D G F Receptor Hiroyuki Sugiki, kazushi Urasawa, Hideaki Kawaguchi, Akira Kitabatake,Departmentof CardiovascularMedicine, Hokkaido University School of Medicine,Sapporo,Japan Although the pathogenesis involved in coronary restenosis after Percutaneous transluminal coronary angioplasty (PTCA) is not fully understood, multiple growth factors promoting vascular smooth muscle cell (VSMC) proliferation and migration are likely to play a pivotal role in this process. The purpose of this study is to suppress the platelet-derived growth factor (PDGF) - induced VSMC growth by antisense oligonucleotides against PDGF ~ receptor mRNA. Rat cultured VSMCs (A10) were incubated with antisense oligonucleotide against PDGF ~ receptor mRNA (AS) for 3days, then exposed to 10ng/ml PDGF for 24 hours. Subsequently, DNA synthesis was assessed by 3H-thymidine incorporation into the cells. The DNA synthesis of A10 cells induced by PDGF-AB and-BB were significantly inhibited by 1/z M AS (44% and 49%, respectively). PDGFindeced DNA synthesis was inhibited dose dependently by AS. A quantitative RT-PCR method showed that the expression of c~ receptor message was significnatly suppressed in the cells treated by 1/z M AS. In this study, antisense oligonucleotide targetting rat PDGFe receptor effectively suppressed the expression of the receptor, and inhibited PDGF- induced DNA systhesis in rat VSMCs. In addition, these results provide an important impetus to initiating in vivo studies to determine the feasibility of antisense strategies in the prevention of coronarv restenosis.

The A T 1 receptor antagonist Losartan Tu172 inhibits neointimal proliferation in an in vitro model of restenosis. PoKee Cheung, David P. Wilson, & Peter Zahradka. Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre; Departments of Physiology and Medicine, University of Manitoba, Winnipeg, Canada. The efficacy of an in vitro model for the study of angiotensin involvement in restenosis was examined. Early post-mortem (30 min) pig hearts u n d e r w e n t balloon angioplasty in the left anterior descending coronary artery, f o l l o w e d by coronary vessel culture for 14 days. With this in vitro model, angioplasty produced a greater than 4 0 0 % increase in neointimal proliferation vs. non injured controls. Immuno-staining w i t h Proliferating Cell Nuclear Antigen (PCNA) provided no evidence of smooth muscle cell division in the medial layer. Presentation of the angiotensin II A T 1 receptor blocker Losartan reduced neointimal proliferation in a concentration dependent manner. Losartan at 1 0 7, 1 0 8 and 10" D~ M decreased neointimal proliferation by 4 9 . 6 % , 65% and 7 0 % , respectively, Interestingly, the ACE inhibitor captopril (lO'eM) was unable to significantly reduce neointimal proliferation. This data suggests an ACE independent p a t h w a y may be involved in the local generation of All, [Funding was provided by the Thorlakson Foundation (to PKC} and the MRC Group in Experimental Cardiology (to PZ)]

METALLOPROTEINASE INHIBITORS Tu173 PREVENT NEOINTIMAL PROLIFERATION IN AN I N VITRO MODEL OF RESTENOSlS. Peter Zahradka1'2, Daniel E. Levy3 & David P. Wilson1'2. 1Divisionof CardiovascularSciences,St. BonifaceResearchCentre, Winnipeg, MB, 2Dept. Physiology, University of Manitoba, Winnipeg, MB and "Dept. MedicinalChemistry, Glycorned, Inc., Alameda CA. Metalloproteinases are associated with cell growth and migration, and thus may contribute to the development of vascular disease. The activation of smooth muscle cell growth following injury, for example, is mediated by a cascade of proteolytic activities which convert latent growth factors to their active forms (eg. TGF-~). To examine the potential involvement of metalloproteinases in vascular tissues, specific rnetalloproteinase inhibitors were evaluated in a porcine coronary artery organ culture model. Balloon angioplasty was performed on the left anterior descending coronary artery, and vessel segments were cultured for up to 14 days. Injured vessels had a significant increase (418%) in neointimal proliferation compared with non-injured controls. Administration of GM6001, an effective inhibitor of gelatinase, at concentrations of 10"" M or higher, prevented restenosis. A similar result was obtained with GM1489, a water-soluble analogue of GM6001. These results suggest that changes in smooth muscle proliferative potential are directly coupled to the activation or release of metalloproteinase activities from damaged smooth muscle cells. This investigation was supported by the MRC Group in Experimental Cardiology.

CORONARY VASOREACTIVITY IN THE DOG Tu174 PR Belcher, AJ Drake-Holland, I Vergroesen, MIM Noble. AUCVM, Chariag Cross & Westminster Medical School, W6 8RF

LOW DOSES OF ACETYLSALICYLIC ACID IN ANTIAGG~EGATION Tu175 THERAPY OF ATHEROSCLEROSIS AND ITS COMPLICATIONS Martin Gajdo~, Viera Spustov~ & Rastislav Dz~rik. Clinic of Phamacotherapy, Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic

INCREASED RESISTANCE TO ISCHEMIC Tu176 INJURY IN ISOLATED PERFUSED HEARTS OF ATHEROSCLEROTIC RABBITS. Bruno Le Grand, Bruno Vi~, Philippe Faur~, AnneDominique Degryse, Pierre Mouillard, Gareth W. John. Division of Cardiovascular Diseases, Centre de Recherche Pierre Fabre, Castres, France.

The optimal dose of ASA in antiaggregation therapy is able to inhibit the production of platelet thromboxane more than 95 t. The aim of this study was to evaluate, if the effect of daily dose 30 mg •of ASA is sufficient to ihibit TxB2 production and to compare the effect of initial doses 30 and 200 mg of ASA . METHODS: 30 mg of ASA was administered to the first group of 16 patients with atherosclerosis (age 45-63, male 8). 200 mg of ASA was administered to the second group of 16 patients with atheroslerosis (age 40-74, male 7). The second group followed up with the administration of 30 mg daily dose of ANA. In the intervals of 2 hours after administration of initial doses and 1 and 3 months after 30 mg daily dose of ASA was native blood collected and 1 hour production of TxB2 was measured by RIA method in serum. The serum was reached after 1 hour blood incubation near 37 °C and i0 minutes centrifugation. ~HULTH: The production of TxB9 after one-dose of 30 mg of ASA in the first group was decreased from 269.3±25.9 ng/ml to 151.4~ 16.8ng/ml (-42t). The production of TxB2 after one.dose of 200 mg of ASA in the second group was decreased from 303.4!37.1 nglml to 9.1± 0.i nglml (-98%). The decrease of TxB2 production after 1 month of 30 mg ANA administration was to 6.6±2.7 ng/ml (-97|} and after 3 months of 30 mg ASA administration to 23.6±10.4 ng/ml (-93t). CONCLUSIONS: We recommend to administer the initial dose 200 mg of ASA, which inhibits the production of TxH sufficiently and immedietly and follow up with daily dose 230 mg of ASA in antiaggregation therapy and prevention.

Blood flow in the critically stenosed coronary artery with endothelial damage declines with accumulating platelet thrombosis. After embolisation of the thrombus, flow is restored, but falls agm.'n cyclically. Distal coronary pressure behaves similarly but on a different time scale. In the open-chest pentobarbitone anesthetized dog, after embolization, flow increased more rapidly than pressure (reactive vasodilatation) in a first period of low distal resistance. In the next period, pressure rose while flow fell, (increasing distal pressure/flow [DP/F]). In the third period, flow and pressure declined together (constant DP/F), due to thrombus growth. We observed transient complete occlusions of the critically stenosed artery, with (n=6) and without (n=4) thrombus growth. Stenosis resistance was equal in both groups before and after the second period of 65+36s (meard:SD) Distal coronary DP/F'was lower without thrombosis (p<0.005), but rose similarly (p<0.05) during the second period, albeit to a lower level (p<0.025). It is concluded that a condition analagous to reactive hypermmia persists with a critical proximal coronary stenosis. At any given time after release of occlusion, DP/F was higher in the presence than in the absence of thrombus, suggesting thrombus induced distal vasoconstriction.

In isolated, Langendorff-perfused hearts from rabbits fed a 2% cholesterol diet (CFR) for 6 weeks (n=23), and age-matched controls fed standard chow (n=12), we studied baseline cardiac hemodynamics, lipid accumulation in sections of coronaries and left ventricular (LV) tissue, and the susceptibility of these hearts to 30 min global ischernia and 90 min reperfusion. Baseline HR (147±6 vs 173a:6 bpm), LVDP (64~-5 vs 95:J:3 mmHg) and CF (13±I vs 24±2 ml/min/g) were all significantly lower in hearts from CFR than in controls, respectively (p<0.01). Atheromatous lesions in coronary vessels and significant interstitial lipid accumulation in LV sections were observed in hearts from CFR. Although large differences in several baseline hemodynamic parameters in hearts from CFR and controls were evident before ischemia, no statistically significant differences were discernible in these parameters between the two groups from 60 min reperfusion onwards (pfNS). In conclusion, hearts from CFR exhibited markedly improved recovery upon reperfusion compared to controls, strongly suggesting increased myocardial resistance to ischemic injury.

A169

Tu177 THE ARACHIDONATE CASCADE OF H U M A N PLATELETS Zs6fia Mezei, B61a Kis, Arp=~d Gecse, Gyula Telegdy. Department of Pathophysiology, Albert Szent-Gy6rgyi Medical University, Szeged, Hungary The involvement of platelets in the pathophysiology of human diseases is well known, but its background is not clearly defined. The aim o f the present in vitro experiments was to investigate the arachidonate (AA) cascade of isolated human platelets o f different blood groups, and to compare it to that of the rat. Platelets were incubated with 1-14C-arachidonic acid in Parker medium 199 ( p H = 7 . 4 , 37 °C, 10 min). The A A metabolites were extracted with ethyl-acetate and after separation by OPTLC, were quantitatively determined by liquid scintillation counter. In the platelets o f human blood group 'A' the quantiw of cyclooxygenase (CO) products and TXB 2, as well as the ratio of vasoconstrictor to vasodilator CO metabolites were positively correlated, while the ratio of lipoxygenase (LO) to CO products was inversely correlated to the decrease of platelat count in the incubation media. In human platelets, isolated from blood group '0', there was significantly higher LO and lower CO activity than in platelets of blood group 'B'. This difference was mainly due to the lower amount of TXB 2 synthesis in blood group '0' platelets. Human platelets (10 ° cells/ml) synthesized less LO products and more TXB 2, as compared to rat platelets (10 o cells/ml). We may conclude that isolated human platelets are useful tools to investigate the AA cascade. Human platelets may have a different profile in the A A metabolism compared to animals. During such in vitro investigations one should pay attention to the differences in the A A cascade in platelets from different blood groups.

Supported by grants from OTKA (7"-6084, T-2683, T/3 1354), by the Ministry of Social and Welfare of Hungary (7"-11 549/93) and FEFA (1008/I).

Tu179

Microviseosity of Platelet Membrans Depends on Ratio Intra/Extraeellular t=a~T B. Fink, K. Schrfir*, E. Bassenge. Inst. of Appl. Physiology, Univ. Freiburg, *Inst. of Pharmacology, Ufiiv. Dfisseldorf, Germany

Platelet activity (PA) and microviscosity of platelet membranes (MV)are closely interconnected. We studied a potential influence of changes in intra/extracellular concentrations of [Cai] on MV. Methods: Significant differences of fluorescence light (FL) emitted from DPH and from FURA-2 provide an unique chance for simultaneous analyses both of MV and of [Cal] in platelets in the same sample. Polarization's amsofropy of the steady-state FL emitted from DPH at 430 nm was used to analyze MV and FL from FURA-2 at 505 nrn for [Cai]. Tile influence of extracellular Ca 2+ [Ca~j on MV was studied in 10 mM HEPES-buffers at p H 7 . 4 containing 20 #M or .1 m:M [Cae] both in the unstimulated and in the stimulated state using ionomycin (Io 5, /I/L) or thrombin ( r h .01, 0.1, 0.5 U/ml). [Ca¢] = 20/~M [Cad = 1 mM [CaJ]/nM "q/Poise [Call/raM rl/Poise Control 4,517:K),027 115+8 2,3484.0,013 122+12 a[Cai] /'~l A[cai] rh 0.01 0,0404.0,006 19+10 0,048+0,009 464.5 l'h 0.5 0,719i~0,018 9554-60 0,449+0,010 3112_+362 [o 5 0,039+0,009 1314.22 0,0454.0,009 28+9 [o 50 0,186i-0,028 888+60 0,2244.0,037 1208+9-36 or the ratio Bns: Decrea causes significant chart I

A170

V[V and thus

:ts e n -

hanc.cd PA. Both Th atd Io are potent,,st~ulators ot p_latelets in KEPES buffer containing z u ~ t~a~l. Concomitant chanRes in PA can be attributed to the 2 fold increase i n M'V. Changes in MV by modifying [Cae.] or the ratio [Cae]/[Cai] are a promising procedure tor tUture studies on enhanced PA.

Upregulation of Platelet Activity by ChronicallyTu178 SuppIied NO-Donors N. Fink, B. Fink, E. Bassenge. Inst. of Applied Physiology, University of Freiburg, Germany Platelet activity (PA) was analyzed during long term supplementation of exogenous NO using NO-donors such as isosorbide dinitrate (ISDN, of 20 #g/kg/min) or a recently synthesized compound with a furoxan-like structure CAS-1609 (0.5 mg/kg twice daily per os). Methods: Washed, nonstimulated platelets of dogs were tested daily before, during, and after continuous five day nitrovasodilator admifiistration. Changes in membrane microviscosity ( M V ) a n d thrombin stimulated intracellular Ca2+-levels (fluorescence techniques) as well as cGMP levels were monitored. Resulfs: MV, reflecting receptor externalization, increased progressively up to 141 +5 % (ISDN) and up to 119 _+4 % (CAS) on the fifth day of treatment compared to controls obtained before infusions were started. Simultaneously the thrombin induced increase in [Cai] rose progresswely to 257 +36 % (ISDN) on fifth day, but only to 109 +14 % using CAS. There was a progressive drop i n c G M P levels from 1.5 _+0.1 down to 0.55 _+0.10 p-M/ml on the fifth day (CAS) and from 1.44 _+0.20 to 0.40 _+0.10pM/ml (ISDN.) at aplatelet concentration of 80.000f/A. Concluston: Chronic administration of ISDN causes an upregulation of PA. This increase is due to the drop in cGMP-synthesis. As a result of higher thrombin-induced stimulation ot platelets, there is an increase both in [Cai] and in MV. The chronic administration of CAS elicits a still more pronounced suppression of cGMP-synthesis, which is surprisingly not paralleled by a greater increase in thrombin-induced [Ca i] and in MV. A possible explanation may be that the increase in [Cai] is inhibited by CAS. The results may indicate a more favourable action of CAS in clinical practice with regard to PA.

PLATELET LIPID DISORDER IN THE PATIENTS TU180 WITH ARTERIAL HYPERTENSION CAUSING BY CHRONIC PYELONEPHRITIS Lyudmila I. Gapon, Nina Ju. Saveljeva. Dept of Arterial Hypertension, Institute of Clinic & Preventive Cardiology, Tyumen, Russia. The aim of the study was to investigate the mechanisms of lipid disorders in platelet membranes in pts with arterial hypertension/All I causing chronic pyelonephdtis. Cholesterol content, quantitative contents of main phospholipid, cholesterol/phospholipid ratio and lipid peroxydes products /dien conjugates, malonic dialdehyde and shift bases/were examined in 20 healthy subjects/Grl/, 23 pts with essential hypertension/Gr2/, 18 pts with chronic pyelonephritis without AH/Gr3/and 18 ptswith All causing chronic pyelonephritis /Gr4L Blood pressure levels were same in 10ts with Gr2 and Gr4. Cholesterol content had tendency to increasing in pts with Gr4 in comparison with Gr2 and Gr3. Phospholipid profile was changed in pts Gr4 due to the decreasing of lightoxidative fraction /phosphatidylcholine level consists 0,200+0,42 mmol/I in pts Gr4 in comparison with pts Gr3 ,406+0,4 mmol/I, p<0,01, Gr2 - 0,516+0,12 mmol/I, p<0,05 and Grl - 0,420+0,05 mmol/I, p<0,05/ and the accumulation of hardoxidative product Ilysophasphatidylcholine had tendency to increasing/. Cholesterol/phospholipid ratio was significantly higher in pts Gr4/4,6+0,6/ in comparison Grl /2,6+_0,2, p
METOPROLOL AND CAPTOPR1L TREATMENTTu181 ON PATHOLOGICAL FINDINGS AT DEATH IN RUNNING CARDIOMYOPATHIC HAMSTERS Suzanne Desjardins, Michael J. Cauchy & Colin G. Rousseaux*, Health Canada and * Global Tox International Consultants Inc., Ottawa, Canada.

CARDIAC STRUCTURAL ALTERATION ANDTu182 THROMBOTIC TENDENCY IN SPONTANEOUSLY HYPERCHOLESTEROLEMIC RAT Emma Riva, Giuseppina Luvara, Giuseppe Andreoni, Cristina M. D'adamo, I.JciaJacoviello. Istituto Ricerche Farmacologiche "Mario Negd", Milan and Santa Maria Imbaro, Italy.

We recently showed that voluntary running decreased survival in cardiomyopathic (CM) hamsters. Running performance before death was improved by treatment with low doses of combined metoprolol (M) and captopril (C). This report describes histopathological findings in these passive (P) and running (R) CM hamsters. At the age of 60 days, CM hamsters (CHFI47) were provided (n=48 R), or not (n=38 P), with a wheel in their cage. Starting at the age of 200-210 days, hamsters received either no treatment (PNT n= I 1; RNT n= 16) or were treated with M lmg/kg/day (PM n=9; RM n=17), C 2mg/kg/day (PC n=8; RC n=7) or M Img/kg/day and C 2mg/kg/day (PMC n = I0; RMC n=8) in their drinking water. At spontaneous death, heart, lungs and liver were assessed histologically for severity of cardiac failure. There was no statistically significant treatment effect tbr P hamsters. For R hamsters, MC reduced both the incidence (0% vs 67, 78, 91% for RC, RM, RNT) and the size of atrial thrombi (0+0 vs 1.64 + 0.28 for RNT; Dunnett's test) as well as the severity of hepatic congestion (0.38+0.26 vs 2.31+0.33). C alone also reduced hepatic congestion scores (0.86+0.4). These results suggest that low doses of combined M and C may have an effect on thrombus tbrmation and that this may, in part, be related to the better exercise pertbrmance in treated CM hamsters.

Hypercholesterolemia is a risk factor for coronary artery disease. Rats are resistant to acquired and experimentallyinduced atherosclerosis. A special line of rats with high (FHC) and one with low (FNC) serum cholesterol have been obtained. We have investigated alterations in (i) cardiac structure and (ii) vascular function in relation to cholesterol levels. Blood samples were collected and the heart removed for collagen determination (as % area of histological section) by image analysis. Thrombotic tendency was measured as occlusion time of a polythene cannula inserted into the abdominal aorta. Triglycerides, body and heart weight, and mean arterial pressure were similar in all rats. Ventdcular collagen content, serum cholesterol levels, and factor VII significantly increased in FHC rats, whereas occlusion time shortened (Table 1). Table 1

Collagen Cholesterol Factor Occlusion (%) (mg/dl) VII (%) time (h) FNC (n= 7) 4.7±0.3 95±8 208±25 171±29 FHC (n= 9) 6.0±0.3§ 162±9§ 325±18§ 95±6" § p< 0.Ol, "p<0.o3 A linear correlation (p< o.o5) was found between serum cholesterol and ventricular collagen. These results show that hypercholesterolemia is associated with cardiac structural and vascular alteration independent of the atherosclerotic process.

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Thursday 6 July 1995 Abstracts Thl-Th169 Cardiac metabolism Arrhythmias Ischemic injury Cardioplegia Cardiac transplantation

Th1-Th66 Th67-Th100 Th101-Th136 Th137-Th160 Th161-Th169

The effect of ischemic glycolysis on cell Th001 volumes - multinuclear NMR studies of rat hearts Antonio Vivi, Maria Tassini, Nadir Askenasy and Gil Navon, School of Chemistry, Tel Aviv University Accumulation of metabolites generated by ischemic glycolysis and degradation of high energy phosphates are two factors which increase cytosolic osmolarity during myocardial ischemia. The influence of these factors on cell volumes in the non-perfused ischemic rat heart was assessed by ~H, 59Co and 3Sp NMR spectroscopy. In one set of experiments, preischemic metabolic inhibitions were used to deplete the high energy phosphates prior to ischemia: substitution of 11 mM glucose by 2-deoxyglucose (2DG), administration of 200 /aM iodoacetate, and combination of 11 mM 2DG with 1 mM cyanide. When high energy phosphates were depleted in the perfused hearts, cell volumes were stable during a subsequent period of ischemia. Hearts treated with these protocols failed to resume function at reperfusion. In a second set of experiments, ischemic glycolysis was altered either by reduction of lactate production by 1 mM dichloroacetate (DCA) or partial depletion of glycogen by 11 mM 2-deoxyglucose and 5 mM pyruvate (2DG+pyr). In these protocols, the functional recovery at reperfusion was superior to that of the controls. During ischemia the ceils swelled by 0.26 and 0.14 ml/gdw with 2DG+pyr and DCA, respectively compared to 0.38 rnl/gdw in controls). It is concluded that during reversible ischemia, inorganic phosphate released from the high energy phosphates and lactate generated by ischemic glycolysis, contribute to ischemic cell swelling.

LACTATE AND FATTY ACIDS AS SOURCES Th003 OF ATP PRODUCTION IN THE HEART Brett O. SchiJnekess, Gary D. Lopaschuk, University of Alberta, Edmonton, Canada. Fatty acid oxidation is generally considered the major source of energy in the heart, although a number of studies have suggested that lactate oxidation can be a major contributor to ATP production. In this study we used isolated working rat hearts to directly determine the relationship between lactate and fatty acid oxidation. Low or high concentrations of lactate (0.5 or 8.0 mM) were added to hearts perfused with buffer containing 5.5 mM glucose, and either low or high palmitate (0.4 or 1.2 raM). Rates of glycolysis and glucose oxidation, or lactate and palmitate oxidation was determined by radiolabelling the perfusate with [5-3H/UJ4C]-glucose, or [U-~4C]-lactate and [9,10-3H]-palmitate, respectively. In the presence of low lactate and low palmitate, lactate oxidation provided 17% of the ATP production, palmitate oxidation 68%, glucose oxidation 10% and glycolysis 5%. High lactate concentrations increased the contribution of lactate oxidation to 37% of the total ATP production. High lactate and high palmitate resulted in lactate oxidation contributing only 13% of the total ATP production, with palmitate oxidation contributing 81%. This data demonstrates that under near physiological conditions of lactate (0.5 mM) and fatty acids (0.4 mM) the preferred energy substrate of the heart is fatty acids. At high levels of lactate such as can be observed during exercise, lactate oxidation becomes a significant source of ATP production but only if fatty acid levels are low.

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Glycolysis increases and aerobic metabolism Th002 decreases cell volumes in the perfused rat heart Maria Tassini, Antonio Vivi, Nadir Askenasy and Gil Navon, School of Chemistry, Tel Aviv University, Israel The effect of energy metabolism on cell volumes was monitored in the perfused rat heart, and with a series of metabolic inhibitions. Cell ~lumes were monitored by IH NMR of water and Co NMR of cobalticyanide, in parallel with ° ' P NMR spectra. Substitution of glucose with 11 mM 2-deoxyglucose (2DG) induced a triphasic volume timecourse: an initial swelling (from 2.50L--0.08 to 2.68_+0.09 ml/gdw) during the f'urst 6 min, a cellular shrink to 2.43_-t-0.10 ml/gdw during the next 8 min and a second period of swelling to 2.57_+0.08 ml/gdw. This time course was not caused by accumulation of 2DG-6P, as it was observed also when iodoacetate (200 IxM) was applied. Perfusion of the hearts with 11 mM 2DG and 5 mM pyruvate induced a gradual increase in cell volumes to 2.64:L-0.09 ml/gdw after 20 rain, while perfusion of the hearts with i mM cyanide induced a transient cellular shrink followed by swelling (2.45_-t-0.10 and 2.62_+0.11 rnl/gdw at 9 and 20 min, respectively). Cell volumes of hearts perfused with 11 mM 2DG and 1 mM cyanide resembled those of cyanide treated hearts. The cell volume dynamics were not related to the concentrations of either ATP, inorganic phosphate or phosphomonoeters as measured by31P spectroscopy~ It is concluded that in the perfused heart, glycolysis acts toward reduction of the cell volumes, while aerobic metabolism increase the cellular water contents.

LACTATE INDUCED POOR REPERFUSION ThO04 RECOVERY: VENTRICULAR FIBRILLATION OR LACK OF GLYCOLYSIS. Jacques van Rooyen* & Lionel H Opie. MRC Heart Research Unit, Univ. Cape Town & Univ Stellenbosch*, South Africa. Hypothesis: Glycolytic ATP may benefit the ischaemic myocardium. Lactate (L) inhibits glycolysis and may impair reperfusion recovery. L may also aggravate reperfusion ventricular fibdllation (VF), caused by cytosolic Ca 2÷ overload. Our aim was to examine the mechanism of the adverse effects of L by addition of glucose (G) and/or insulin (I) in ischaemia and/or defibrillation. Methods: Isolated working rat hearts (n=6) were perfused at a fixed ischaemic coronary flow of 0.2 ml/min/g for 30 rain. L (10mM) or L + G (11raM) or L + I (lmU/ml) or L + G + I were used as ischaemic substrates with only G during reperfusion. Reperfusion cardiac output (RCO) recovery (%) and VF were determined. Tissue ATP at the end of ischaemia, L production (mmol/g dry weight) and G uptake (IGU)(mmol/min/g) during ischaemia were measured. To defibrillate (DF), hearts were briefly immersed in cold KrebsHenseleit solution. Subst RCO ATP IGU L VF L 7.5 1.52 No G 47.07 515 DF L+G 71 9.80 0.51 94.26 015 L+I 58 5.65 No G 73.94 6/6 DF L+G+I 93 9.61 0.84 113.62 0/5 Results: In L hearts RCO was low despite successful DF. However, addition of G abolished VF and improved RCO (P<0.05). Addition of I improved RCO (p<0.05), but left VF unchanged. Conclusions: The adverse effects of L were independent of VF and fully removed by G + I. Impaired RCO with L alone may be related to lack of glycolytic flux.

HPLC ASSAY OF LACTATE, PYRUVATE AND

ThO05

TCA CYCLE COMPONENTS IN HEART TISSUE Paolo Pedersini, Claudio Ceconi, Massimo Benigno, Palmira Bernocehi, Antonella Boraso, Roberto Ferrari. Fondazione Cllnica del Lavoro "Centro S. Maugeri", Gussago, Brescia; Cattedra di Cardiologia, Universita' di Brescia, Italy.

Whilst HPLC quantification of CP and purine nucleotides is routinarely, that of lactate, pyruvate and TCA cycle intermediates require tedious and often unreliable enzymatic assays. We coupled a reversedphase HPLC method for purine and pyridine nuclcetides separation, to a strong cation-exchange chromatography. HCIO4 tissue extract was injected in the reversed-phase system (C18 column equilibrated with 0 . 1 M KH2PO4, 5ndv[ tetrabutylammonium hydrogen sulphate and a gradient of acetonitrile to a concentration of 25%). In series with detection at 260 nm for quantification of CP, ATP, ADP, AMP the eluate was timely collected in definite fractions and immediately reinjected in the ion-exchangesystem (Aminex HPX-87H column using 0.004M H2SO4 as mobile phase) for a further analytes separation. Effluent was monitored at 210 nm. The method was applied to aerobic and ischaemic rabbit heart and to liver and skeletal muscle for comparison. Results are expressed in nmol/mgdwt. Tissue Pyruvate Malate Sueeinate Lactate Aerob.Heart(n=6) Isch.Heart(n=6) hmer Leg(n=6) Liver(n=6)

< 15 94:t:2 15084-307 2594-41

<0.05 0,554-0.09 <0.05 2.144-0.07

<0.20 5.784-0.27 0.264-0.03 0.474-0.20

4.6:t:1.7 76.g4-20.7 45.44-9.5 6.0"M).I

We conclude that the coupled chromatographic techniques are useful to asses analytical intermediates of energetic metabolism. The disadvantages of high sample dilution are balanced by the optimal peaks separation and high "signal to noise" ratio.

M Y O C A R D I A L FLUX O F C R E A T I N E Th007 KINASE IN P H O S P H O C R E A T I N E D E P L E T E D P E R F U S E D R A T H E A R T . V. Stepanov, P.Mateo, B. Gillet*, J.-C. Beloeil*, J.A. Hoerter. *RMN Biol., ICSN Gif s/Yvette & Cardiol. Cellul. Mol., CJF9211 INSERM, Univ. ParisXI, Chatenay-Malabry, France Myocardial Creatine Kinase (CK) isozymes participate in the production, utilisation and cytosolic transfer of energy. To check CK kinetics, we compared the forward CK flux measured by p31 NMR in acetate perfused rat hearts in control ( C ) and in an experimental model of adenylate depletion by deoxyglucose (DG) which results in sustained contractility and ATP synthesis despite a marked reduction in phosphocreatine content [PCr]. A steady state with 50% [PCr] was achieved by transient infusion of 2DG. The apparent rate constant of CK kfor increased from 0.51_+0.0Is -1 in C (n=5) to 1.03_+0.08 in DG (n=5, p<0.001). Thus CK flux remained similar in C (13.7+1.5 raM.see-I) and DG (14.8+0.3) in agreement with sustained contractility and ATP synthesis. By contrast when a similar PCr decrease was achieved by moderate hypoxia (25-30% O2), contractility decreased. A marked decrease in kfor led to a 75% reduction in CK flux. In conclusion we show that cardiac CK flux can not be unequivocally predicted from the in vitro CK kinetics and cytosolic content of CK substrate and products. This may be related to isozymes compartmentation of CK isozymes and their substrates and/or to flux visibility.

PYRUVATE DEHYDROGENASE (PDH) Th006 ACTIVITY AND MALONYL CoA LEVELS IN NORMAL AND ISCHEMIC SWINE MYOCARDIUM. W.C. Stanley, L.A. Hernandez, D.A. Spires, J. Bringas, S. Wallace, and J.G. McCormack. Syntex Discovery Research, Pale Alto, USA, and Edinburgh, Scotland, UK. The purpose of this study was to assess the effects of dichloroacetate (DCA) on myocardial malonyl CoA levels and free fatty acid (FFA) uptake during ischemia. Experiments were performed in anesthetized open-chest swine under nonischemic conditions, and with a 60% reduction in left anterior descending coronary artery (LAD) flow (n=8/group). Myocardial needle biopsies for PDH activity were taken aRer 57 min of intracoronary saline or DCA (1 mM in LAD blood) under aerobic conditions, and after 37 min of ischemia. Myocardial malonyl CoA and acetyl CoA levels were measured after 40 minutes of LAD ischemia. DCA treatment under aerobic conditions increased the amount of active dephosphorylated PDH to 88% of total activity compared to 55% with saline treatment (P<0.01). Ischemia did not significantly change PDH activation state in either group. Acetyl CoA and malonyl-CoA contents were significantly elevated in ischemic DCA-treated myocardium compared to saline-treated ischemic myocardium. DCA treatment significantly lowered rates of myocardial FFA uptake under both aerobic and ischemic conditions, but did not effect glucose uptake or lactate exchange. FFA uptake was negatively correlated to malonyI-CoA levels (r'=--0.68), and it is proposed that this is due to malonyl CoA inhibition of carnitine palmityl transferase I.

PROTECTION OF M~TABOLICALLY P O I S O N E D C A R D I O M Y O C Y T E S BY

ThO08

A PEPTIDE

Philip S. Duffy & Peter H. Cobbold. Dept of Human Anatomy and Cell Biology, University of Liverpool, UK. Rigor-activation of myosin-ATPase induces shortening of isolated, metabolically-poisoned cardiomyocytes and a simultaneous dramatic fall in cytoplasmic ATP, and a consequent rise in cytoplasmic free Ca2+. Here we show that a Troponin-I mimetic pcptide (Gly 137 - Arg 148), when loaded into isolated cardiomyocytes, prevents for at least 1.5 hours' of cyanide and 2-deoxyglucose exposure, both the entry of cells into rigor and appreciable rises in free calcium, which was monitored with fura-2. Thereai~r free Ca rises slowly to micromolar levels. In contrast, control cells loaded with inactive peptides, enter rigor after 5 to 20 minutes, and free calcium rapidly rises through micromolar levels 10 minutes later. Removal of cyanide and 2-deoxyglucose to simulate the 'oxygen paradox' causes a sudden rounding-up and large free calcium fluctuations in control cells, but peptideprotected cells show no shape change and flee calcium returns to close to healthy cell levels, even afar 2 hours' poisoning. The peptide studies highlight the inhibition of rigor-activation of the mynsin-ATPase as a key targe~ for, ultimately, protecting the myocardlum from ischaemic damage.

AI75

MATHEMATICAL MODELING OF ThO09 D I F F U S I O N A L E N E R G Y T R A N S F E R IN CYCLIC C O N T R A C T I O N S O F H E A R T M U S C L E Mayis K.Aliev & Valdur A.Saks. Cardiology Research Center, Moscow, Russia & Institute of Chemical and Biologi-

eal Physics, Tallinn, Estonia To gain some insight into controversial matter of phasic character of energy restoration in cyclically contracting heart cells, we have simulated the diff~sional exchange of ATE ADP, Cr, PCr and Pl between mitochondria and myofibrils during each contraction cycle. The path for plane diffusion (1, 0.1 and 0.2 um in myofibrils, cytoplasm and mitochondria, respectively) was considered along the radii of organellae. The mitochondrial comparmaent included: l) matrix with ATP-synthase enzyme working at P. and ADP concentrations, that exceeded those for mtermembrane space by 10- and about 25-fold, respectively; 2) intermembrane space (0. I urn) with mitochondrial isoform of creatine kinase (CK), coupled to adenine nucleotide translocase; 3) highly permeable mitochondrial outer membrane, (0.1 um). Myofibrillar compartment and cytoplasm were taken as the spaces with randomly distributed myofibrillar isoform of CK; during contraction the myofibrils hydrolyze ATP with predicted kinetics. With maximal activities of CK and ATP-synthase, corresponding to those for rat heart muscle, it was possible to model the steady energy exchange even at maximal work output with O: consumption of 33 mmol/min/kg wwt. The energy restoration was clearly phasic: in the first half of entire cycle the system has taken away the ADP burst (from 11 to 130 uM) in myoplasm at the expense of PCr decrease by 0.24 mM from initial level of 17.76 mM. The PCr, Cr and P. levels were restored gradually by the end of the cycle at th~ expense of mitochondrial activation mainly by ADP from mitochondrial CK. Both CK isoforms are functioning out of equilibrium. •

I

Ca2+-INDUCED FORMATION OF CONTACT Th011 SITES IN RAT HEART MITOCHONDRIA: ROLE OF MITOCHONDRIAL CREATINE KINASE (mCK) Attila Ziegelhoeffer, Annette Bakker*, Willem Jacob* Tanya Ravlngerova, Narcisa Tribulova, Jan Slezak. Inst Heart Res, Slovak Acad Sci, Bratislava, Slovakia,

*Dept of Med, Univ of Antwerp, Antwerp, Belgium. Mitochonch'ial contact sites (MCS) are well established as dynamic structures created by fusion of the inner and outer mitoehondrial membranes. Stimulation of the intermediary metabolism in the myoeardium results in an increase of the number of MCS. Since high activity of roCK demonstrated eytochemieally has been localized in MCS, this was utilized for their better visualization. We have recently reported that a perfusion (Langendorff) of the isolated rat heart with increasing concentrations of Ca may also enhance the formation of MCS. It was already hypothesized that the high incidence of mCK in the MCS results from the changes in oligomeric structure of the membrane bound enzyme by forming oetamers (from originally dimers) coupled with a molecule of ATP-ADP transloeasc. However, little information was available about the relation between the Ca-induced local changes in mCK activity in the MCS and the bulk activity and properties of the roCK estimated bioehemieally. Our results revealed, that acoumulation of roCK in MCS is accompanied by a considerable decrease in the bulk activity of roCK in conjunction with a slight depression in the kinetic properties of the enzyme. A176

D I F F E R E N T C H E M I C A L S T A T E OFTh010 INTRACELLULAR ATP AMONG RAT BRAIN, HEART AND SKELETAL MUSCLE TISSUES Eiichi Takayama, *Motoaki Bessho, Fumitaka Ohsuzu, Shigeki Yanagida, *Tetsuya Nakamura, and Haruo Nakamura, Dept of Medicine 1, National Defence Medical College, and *Eisai Research Laboratories, Tokyo, Japan ATP and creatine phosphate (CP) were extracted separately from rat brain and skeletal muscle tissues using two chemical methods, i•e•, conventional direct perchloric acid (0.4M) extraction and stepwise extraction using 70-100% alcohol and perchloric acid (0.4M) solutions sequentially. The ATP and CP values for the two extraction methods were measured bioluminescently and data were compared among the brain, skeletal muscle and heart (previously determined). ATP in the brain was barely extracted by an 85% or higher concentration of ethanol and this finding was commonly seen in the other two tissues. The extraction ratio of ATP in the brain when extracted with 70% ethanol was 78+6% of the total (mean+SD, n=5) and the value was higher than those observed in the heart (68+5%, n=5) and skeletal muscle (61+5%, n=5). In contrast to ATP, the extraction ratio of CP when extracted with 70% ethanol was nearly the same in these three tissues. As in the other two tissues, the total CP content in the brain was higher when it was extracted using the stepwise method than when extracted by perchloric acid solution alone. These results suggest that intracellular ATP exists in different chemical states among brain, heart and skeletal muscle; however, intracellular CP exists in a similar chemical state in these three tissues.

Ca 2÷ STIMULATES BOTH RESPIRATORY AND Th012 PHOSPHORYLATION SUBSYSTEMS IN HEART lV[ITOCHONDRIA Vida Mildaziene, Rasa Baniene, Zita Nauciene, Vilmante Borutaite, Ausra Marcinkeviciute, Adolfas Toleikis, *Guy C. Brown. Kaunas Medical Academy, Kaunas, Lithuania, *Dept. of Biochemistry, Cambridge University, UK Intramitochondrial free Ca 2+ concentration appears to be the most likely candidate for an additional metabolic regulator to supplement ADP, ATP and phosphate. In heart mitochondria, the 2-oxoglutarate dehydrogenase is the most important target of direct activation by Ca z+. The evidence suggesting Ca 2+ effects on mitochondrial function at several other sites between NADH oxidation and ADP phosphorylation is relatively poor. We studied the effect of extramitochondrial Ca z+ concentration (5 rum versus 1 IxM) on mitochondrial respiration with 1 mM 2oxoglutrate using the "top-down" approach of Metabolic Control Analysis. The rate of respiration was stimulated nearly twice by Ca z+. The results showed that the most significantly Ca z+ activated the block of reactions generating protonmotive force - 2-oxoghtarate dehydrogenase plus respiratory chain. The proton leak across the inner mitochondrial membrane was not altered by Ca 2+. At the same time the phosphorylation subsystem was clearly stimulated by the increase in Ca z+ concentration. Our previous experiments have shown, that Ca z+ has no effect on adenine nucleotide translocator. Thus, ATP synthase and/ or phosphate carrier are the possible sites of Ca z+ action•

ASSESSMENT OF MITOCHONDRIAL RESPI-Th013 RATION AND EFFECT OF ISCHEMIA IN SAPONIN-SKINNED CARDIAC FIBERS Adolfas Toleikis, Daiva Krasauskait@, Sonata Trumbeckait6, Arvydas Dagys. Kaunas Medical Academy, Kaunas, Lithuania Saponin-skinned fibers (F) of the rabbit and rat heart were used to study the effect of total ischemia (I; 0.5, 1.O, 1.5h) on the respiration of mitochondria (M) with pyruvate & malate and succinate. I-induced changes of respiration of F with pyruvate $ malate ( - and $ ADP) were similar though less pronounced when compared to isol~ ted M. These parameters with succinate were not different from control; cytochrome c significantly stimulated State 5 respiration which increased above control level (4~%) what had never been observed with isolated M. Experiments with F homogenates showed clearly that the respiration rate of M in control F is not maximal, probably, due to sub-optimal permeability of cellular membranes what increases during I. Preparation of control F with saponin & collagenase enhances succinate oxidation several times with the concomitant decrease of apparent K for ADP. m

ThO14 ANTIOXIDATIVE AND PROTECTIVE EFFECT OF AN ALPHA-TOCOPHEROL ANALOGUE IN SMOKE MITOCHONDRIAL CARD1OMYOPATHY Anna Gvoz@ikovfi, Jarmila Kucharski Ivona Herichovfi, Snircov& Ol'ga Uli6ni Viliam Bada, JAn Gvoz@ik Pharmacobiochernical Laboratory. of Medical Faculty, Komenskn) University, Bratislava, Slovak Republic Prolonged cigarette smoking produces free oxygen radicals, decreases the level of alpha tocopherol in mitochondria of the heart muscle.The substances with antioxidative effect can diminish toxic effect of smoking. The aim of this study was to investigate the effect of an analogue of alpha-tocopherol (MDL 73,404) on the bioenergetics of heart muscle and liver mitochondria in three weeks lasting passive smoking of rabbits.The prolonged effect of MDL 73,404 (in dose 3rng/kg b.w., last 5 days before sacrifice) improved bioenergetics of the heart muscle and liver mitochondria (state 3,OPIL ADP:O), when NAD-substrates were used (glutamate,pyruvate). MDL 73,404 decreased peroxidation of lipids in the plasma, increased the activity of catalase in the heart muscle mitochondria.These results show on one of the possible mechanisms of protective and antioxidative effect of IVlDL 73,404 in the mentioned smoke mitochondrial eardiomyopathy. (MDL 73,404 was "kindlyprovided by the Marion Merrell Dow Research Institute, Strasbourgh, Cedex, France).

Th015 ON THE BENEFICIAL EFFECT OF AN ALPHATOCOPHEROL ANALOGUE ON THE HEART BIOENERGETICS AFTER CHRONIC ETHANOL APPLICATION Snircov~, Jarmila Kucharsk,~ Ivona Herichovi J~n Gvozdj&k, Viliam Bada, Anna GvozdjAkowi Pharmacobiochemical Laboratory. of Medical Faculty Komensk%' University, Bratislava, Slovak Republic

Th016 ENDOGENOUS COENZYME Qto AND ALPHATOCOPHEROL IN SMOKE MITOCHONDRIAL CARDIOMYOPATHY. THE EFFECT OF ANTIOXIDATIVE THERAPY. Jarmila Kucharsk£, Anna Gvozdjhkov~, Ivona Herichov~ MAria Snircov~, J~n Gvozdj~k, Viliam Bada, Pharmacobiochemical Laboratory of Medical Faculty, Komensky University, Bratislava, Slovak Republic

Prolonged effect of MDL 73,404 (an analogue of alphatocopherol) had a beneficial effect on the heart muscle bioenergetics (in dose 3 mg/kg b.w., 5 days). It increased the level of the ATP, ADP, total AdN, hypoxantine in the heart muscle. In previous our studies we proved, that 7 weeks lasting drinking of 20% ethanol provoked alcoholic mitochondrial cardiomyopathy.In this study we proved, that levels of ADP and energy charge were statistically decreased after chronic ethanol administration. The levels of ATP, AMP, total AdN were decreased, but not statistically. Chronic effect of alcohol and simultaneous effect of MDL 73,404 (last 5 days before sacrifice) increased the levels of ATP, ADP and total AdN, participated on the levels their metabolites ( adenosine, inosine, hypoxantine, xantine, uric acid). These results show that analogue of alpha-tocopherol MDL 73,404 had beneficial effect on heart muscle bioenergetics as well as in alcoholic cardiomyopathy of the rats.(MDL 73,404 was kindly provided by the Marion Me, reU Dow Research Institute, Strasbourgh, Cedex, France).

Prolonged cigarette smoking induces metabolic injury of heart muscle mitochondria characterised as smoke mitochondrial cardiomyopathy. Smoking provokes free oxygen radicals production and therefore endogeous antioxidants may participate in protecting against lipoperoxidation and preventing damage of mitochondrial function. The aim of this work was to study the effect of prolonged smoking on the alpha-tocopherol and CoQ, 0 levels in reduced and oxidized form. Three weeks lasting passive smoking of rabbits provoked significant decrease of alpha-tocopherol level in myocardial mitochondria and marg.sign, in blood. COQto,+d was practically not detecteable in myocardial mitochondria isolated from smoking rabbits. No changes were in CoQ t0.~ levels. Antioxidative therapy with alphatocopherol analogue MDL 73,404 improved the levels of alpha-tocopheroi and CoQm,j in mitochondria to controls. The results indicate involvement of endogeous antioxidants in development of smoking mitochondrial cardiomyopathy and possibilities of antioxidative therapy. (MDL 73,404 was kindly provided by the Marion Merrell Dow Research Institute, Strasbourg Cedex, France)

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EXTENSIVE FRAGMENTATION OF Th017 M I T O C H O N D R I A L DNA D E T E C T E D USING A NOVEL TOTAL DETECTION SYSTEM Mika Hayakawa, Kazumi Katsumata & Takayuki Ozawa. Department of Biomedical Chemistry, Faculty of Medicine, University of Nagoya, Nagoya 466, Japan A total detection system, using 180 kinds of primer pairs covering all regions of mitochondrial DNA circle, was newly devised to detect all deletions in mitochondrial DNA without exception. This system was applied to the survey of the heart mitochondrial DNA from 3 human cadavers (a patient with mitochondrial cardiomyopathy and two controls). The patient, having a germ-line synmutation in transfer RNAAsp gene, died of heart failure at age 19. The control-1 subject was a 28 year-old woman who could be regarded as a syn" mutation negative control, as having a similar genotype to the patient except for the syn- mutation, and the control-2 was a 24 year-old man. In the patient's specimen, extensive fragmentation of mitochondrial DNA was demonstrated: 235 types of deletions, accounted for 87% of the total, were detected, including 97 types of "minicircles" lacking the two replication origins. On the other side, 67 or 50 types of deleted genome, accounting for 31 or 27% of the total, were respectively detected in the control-1 or -2 subjects. Moreover, in the patient's heart, the accumulation of oxygen damage, corresponding to that of 78-year-old normal specimen, was demonstrated. Therefore, it could be concluded that the syn" mutation in the patient's mitochondrial D N A triggered oxygen damage that promoted massive accumulation of DNA fragments.

DOES ISCHEMIA-INDUCED FIFo-ATPASE Th019 INHIBITION OCCUR IN FAST-BEAT RODENT HEARTS? Kad V. Ylitalo, Klaus H. Vuorinen, Keijo J. Peuhkudnen, Antti Ala-R~mi, M.J. Pekka Raatikainen & Ilmo E. Hassinen. Depts of Medical Biochemistry and Internal Medicine, University of Oulu, Oulu, Finland The mitochonddal ATP synthase (F1Fo-ATPase) is regulated by an inhibitor protein (IF1). Reversible inhibition by IF1 occurs upon disappearance of the mitochondrial membrane potential. We have shown that a reversible inhibition of F1Fo-ATPase occurs in isolated rat hearts during a short ischemia and that this inhibition persists over an intermittent reperfusion preceding a longer sustained ischemia. Because of discrepant views of the significance of IF 1 in the hearts of small rodents, we wanted to corroborate a method for the measurement of ex vivo activity of F1Fo-ATPase. For the observation of.the ischemiainduced inhibition it was necessary mmmlmlze the time used for cell fractionation to 30 s. The observed inhibition was dependent on pH of the suspension medium. At pH 6.5 the inhibition was 63% after 20-min ischemia, whereas at pH 8.0 the inhibition was only 17%. In Ca2+-free medium at pH 6.5 and 0*C the F1Fo-ATPase activity and its inhibited state were stable for 1 h. The results indicate that contrary to earlier assumptions a reversible metabolic F1Fo-ATPase activity modulation can be observed in the rat heart. This is in accord with a role of IF1 in preventing ATP wastage by mitochondria in ischemia. The phenomenon may be related to increased tissue viability after reperfusion. A178

Th018 A PHENOMENON OF " R E S P I R A T O R Y CONTROL' BY TRYPSIN IN SKINNED FIBERSe MITOCHONDRIAL CYTOSKELETON INTERACTIONS? K. Anflous*, R. Ventura-Clapier*, J.L.Samuel**,L.Rappaport,** M.Toompuu, T. Tiivel, Saks V.A.. Laboratory of Bioenergetics, Inst. Biol. Chem. Phys., Tallinn, Estonia, **INSERM Unit 127, Paris, and *INSERM CJF 92-11, Chatenay-Malabry, France. In skinned cardiac fibers the respiration rate in the presence of 100 uM ADP is low due to its retarded intracellular diffusion. We found that under these conditions respiration rate is increased by a factor of 2.5 by 0.5 mg/ml trypsin. Inhibition of the proteolytic activity by leupeptin eliminated the effect which was of kinetic nature due to a shift of Km for ADP from 300 uM to 40 uM at constant Vmax. Trypsin had no effect on properties of isolated mitochondria. It was proposed that in intact cells permeability of the mitochondrial outer membrane porin channels for ADP could be regulated by some cellular proteins which may be connected to cytoskeleton. For identification of these factors, microtubular network of cells was dissociated by colchicine or stabilized by taxol, using the immunohistochemical methods for control. This procedure did not change the Km for ADP thus indicating the participation of other cytoplasmic (cytoskeletal) factors than the microtubular system itself in the control of respiration.

Th020 RESPIRATION BY CONTROL OF CELLULAR MEMBRANE AND MITOCHONDRIAL OUTER RELATED STRUCTURES of Bioenergetics, V.A. Saks, Laboratories University of Joseph Fourier, Grenoble, France, Biological Physics, and Institute of Chemical and Tallinn, Estonia. Cellular regulation of respiration was studied using skinned cardiac fibers and permeabilized Cells where mitochondria are localized in natural surrounding but the cell membrane is dissolved. Apparent Km value for ADP in regulation of respiration was 295+ 35 I.tM, in isolated mitochondria - 17+1 I.tM. Activation by creatine of ~;reatine kinase (CK) reaction in mitochondria reduced Km to 85+5 RM, and this effect was lost in ~25 mM KCt which dissociates CK from membrane. Removal of myofibrillar proteins by 800 mM KCI 0r inhibition of myokinase reaction did not Change the value for ADP but mitochondrial 8welling reduced this value to 32+5 I.tM. Most remarkably, short treatment of fibers or ceils by trypsin, 0.125 mg/ml, reduced the value to 82+22 M. Very similar effects were seen in heart omogenates. Thus,ADP diffusion in cells in vivo is retarded due to low permeability of mitochondriai outer membrane for ADP that is controlled by YtOplasmic protein factors which are lost during olation of mitochondria, and mitochondrial respiration is regulated in vivo most probably with participation of periferial kinases as signal ~amplifiers.

~

CARDIAC PLASMA MEMBRANE Ca2+/Mg2+ Th021 ATPase: MICROHETEROGENITY AND MICROSEQUENCING ANALYSIS Subburaj Kannan, Vijayan Elimban, Ken S. Dhalla & Naranjan S. Dhalla. Division of Cardiovascular Sciences, St. Boniface Hospital Research Centre, University of Manitoba, Winnipeg, Canada. Cardiac plasma membrane Ca-"/Mg-" ATPase (Ecto ATPase) is a membrane bound enzyme which requires millimolar concentrationofeither Ca2' or Mg:' for maximal hydrolysis of ATP. In this study we investigated the protein structure by matrix assisted laser desorption and ionization mass spectroscopy (MALDI-MS) and by microsequencing. EctoATPase was purified from the rat heart sarcolemmal vesicles to homogeneityand analyzx~lfor its purity by HPLC. By using four different matrix support systems, i) 2,5,dihydro benzoic acid (DHB); ii) [2-(4-hydroxy phenylaxol)l-benzoic acid (HABA); iii) 3,5, dimethoxy-4-hydroxy cinnamic acid (SINAPINIC); iv) alpha-cyano-4-hydroxy cinnamic acid (ALPHA), the analysis was performed on a linear TOF spec MALDI mass spectrometer. In all four matrix combinations we observed multicomponent species indicative of microheterogenity. Further, a partial amino acid sequence of the ecto-ATPase by tryptic digestion and data bank analysis showed that the rat cardiac ecto ATPase exhibits sequence homologywith the cell adhesion molecule. It is suggested that rat cardiac sarcolemmal ecto ATPase is made up of multi componentswith microheterogcnity in its molecular structure and it may be a cell adhesion molecule. (Supported by MRC Group in Experimental Cardiology).

DISTRIBUTION OF ct SUBUNIT ISOFORMS T h 0 2 3 OF NA,K-ATPase IN W O R K I N G , CONDUCTING AND TISSUE CULTURED CARDIOMYOCYTES. I M M U N O F L U O R E S C E N C E STUDY. Jan Slezak, Wolfgang Schulze, Ludmila Okruhlicova & Narcisa Tribulova, Inst Heart Res, SAS Bratislava & Max Delbruck Center Berlin The exact localization of the site specific distribution of Na,K-ATPase isoforms and their subunits in specialized compartments of the myocyte is necessary prerequisite for better understanding their functional significance. Polyclonal and monoclonal antibodies to c~ 1, 2 and 3 subunit isoforms were used for accurate determination of localization of Na, K-ATPase immunoreactive sites. Immunofluorescence data were compared with cytochemical and immunocytochemical ones.The results indicate that differential localization of high and low affinity binding sites bound to different localizations of morphologically specialized structures may account for the differences in sensitivity to cardiac glycosides. Ouabain-resistant Na, K-ATPase cytochemically localized in junctional sarcoplasmic reticulum is probably ctl isoenzyme which is directly involved in modulation of action potential-ionic fluxes of Na and K and participate in the translation of the action potential at the sarcolemma into Ca release, mediated through junctional processes containing calcium release channels.

ELECTROPHILIC REAGENTS AS INHIBITORS T h 0 2 2 OF THE HEART SARCOLEMMAL (Na/K)-ATPase. Albert Breier, Attila Ziegelhoeffer & Tania Stankovi[ov~l, Inst Mol Physiol and Genet & lnst Heart Res. Slovak Acad Sci. Bratislava, Sk. The effect of electrophilic substances: p-bromophenylisothiocyanate (PBITC); fluoresceinisothiocyanate (FITC); [4-isothiocyanatophenyl-(6-thioureidohexyl)-earbamoylmethyl]-ATP (ATPITC); 2,4,6-trinitrobezenesulfonic acid (TNBS); I-(5-nitro-2-furyl)-2-phenylsulfonyl-2-furylcarbonyl ethylene (FE I); l-(5-phenylsulfonyl-2-furyl)-2-phenylsulfonyl -2-furylcarbonyl ethylene (FE2) and 1-(5-phenylsulfonyl-2furyl)-2-phenylsulfonyl-2-tienocarbonyl ethylene (FE3) on the sarcolemmal (Na/K)-ATPase isolated from guinea-pig hearts was studied. FITC and PBITC were found to inhibit competitively the activation of (Na/K)-ATPase by ATP. Being for the enzyme inhibitor and substrate at the same time ATPITC does not offered clear kinetic behavior. These data indicated that isothiocyanates may interact predominantly in the ATP-binding site of the enzyme molecule. In contrary to isothiocyanates, TNBS and FEI both manifested affinities to the potassium binding site of the (Na/K)-ATPase molecule (FE2 and FE3 were ineffective). More specific was the effect of FEI that showed clearly competitive inhibition of potassium-stimulation of the enzyme activity. FE1 exerted also an ouabain-like effect on the mechanical activity of isolated perfused guinea-pig heart.

THE STRUCTURE AND FUNCTION OF SARCOLEMMAL CARDIOMYOCYTES AND THEIR BASIC PROPERTIES AT HYPERLIPIDEMIA

Th024

L.S.Mkhytarian, I.N.Yevstratova, N.N.Odova, A.G.Korotkoruchko, N.N.Fedun, T.F.Drobot'ko. Institute of Cardiology, Kiev, Ukraine. The role of hypercholesterolemia (HCh) in deterioration of fundamental cardiomyocyte sarcolemmal (SL) properties receptor and barrier functions, ion transport was studied at early Ht stages. HCh was modulated by feeding rabbits with cholesterol (Ch) - 0,5 g per kg body mass - during 2 months. By the end of this period SL content of Cfl in

experimental animals rose averagely by 17 % compared to control. Simultaneously lipid peroxidation (LP) in SL was enhanced, accumulation of primary and end LP products occurred (respective increase by 20 % and 3 1 % compared to control), membrane vesicular chemoluminescence both spontaneous and induced was intensified. Their permeability for 45Ca 2 + estimated by velocity of passive transfer through LS, appeared 18 % higher than in control. Remarkable is the significant Na+,K+-ATPase cataly2ti+c activity decrease and retention of the initial Na+-Ca exchange rate. At HCh the content of main structural components - sialic acids and hexose amines in the glycocalex - the superficial SL layer - decrease by 17 % and 15 % respectively, the membrane Ca 2 + binding

capacity being lowered by 25 % compared to control. Investigation results of betaadrenergic receptor sensitivity and betaadrenoreceptors density on the SL assessed with radioligand allow to conclude that the myocardial SL in rabbits with HCh has a higher quantity of Hdihydroalprenolol binding sites compared to control.

A179

MODULATION OF CARDIAC MEMBRANE LIPIDS Th025 AND AGING AFFECT ACTIVATION OF PDH AND COUPLING OF OXIDATIVE PHOSPHORYLATION Salvatore Pepe, Naotaka Tsuchiya, Edward G. Lakatta and Richard G. Hansford. Laboratory of Cardiovascular Science GerontologyResearchCenter NIA NIH BaltimoreMaryland USA

DECREASED MYOCARDIAL MALONYL COA Th026 WITH DOBUTAMINE-INDUCED WORK Jennifer L. Hall, Gary D. Lopaschuk, Amy Barr, Robert.D. Pizzurro, C. Dawn Hamilton, and William C. Stanley. Syntex Discovery Research, Palo Alto, CA and University of Alberta, Edmonton, Alberta.

High saturated animal fat consumption (SAT) alters membrane lipid composition and exacerbates ageassociated arrhythmias and decreases the efficiency of oxidative phosphorylation in the rat heart in situ, whereas dietary fish oil (o-3 PUFA; FO) attenuates arrhythmogenesis & improves O 2 utilization efficiency. We measured the relative activation of pyruvate dehydrogenase (PDHa/PDHt,,a)) in freeze-clamped ventricles, previously crystalline perfused and paced at 3Hz, of 6 and 24 m o o " Wistar rats after 6 weeks feeding on SAT or FO regimens. At 6mo this fraction was slightly higher in SAT than FO (11.1% +0.4 vs 7.9%_5:0.8, p<0.05) but at 24mo this increased to 21%+2 in SAT and 13%+1 in FO (p<0.05). As PDHa/PDHt== is Ca2*-dependant, this is consistent with increased Ca 2÷ cycling across mitochondrial membranes of 24mo hearts. In isolated mitochondria from 24mo SAT hearts relative to 24mo FO hearts, state IV oxidation rates (-ADP,-Ca 2÷) were increased (pyruvate, 25%_+5; palmitoyl carnitine, 23%_+5;p<0.05). Thus oxidative metabolism is less coupled in 24mo SAT heart mitochondria, consistent with lowered thermodynamic efficiency seen in this model in situ.

Malonyl CoA (MCoA) inhibits carnidne palmitoyl transferase 1, thereby limiting the transport of fatty acyl CoA's across the mitochondrial membrane. We assessed MCoA levels and free fatty acid uptake (FFAup) in 8 yucatan micro-pigs before, during and following a dobutamine-induced (15 Iag/kg/min i.v.) increased work state. FFAup was calculated as the product of arterial minus coronary venous FFA and mean blood flow. MCoA levels were measured in needle biopsies by I-IPLC. All animals received glucose and intralipid throughout the protocol. Dobutamine resulted in increases of 106% and 126% in rate pressure product (RPP) and MVO~, respectively. MCoA fell 68% with dobutamine, concomitant with a 239% increase in FFAup. Contzol Dobutanine Recovery RPP 10420-+705 21501_+724* 10267__.1181 MCoA 6.43_+0.62 2.04_+.77* 5.72_+0.23 FFAup 0.14-+0.02 0.46_+0.18" 0.11-+0.05 (*significant from control and recovery, P < 0.05). In summary, MCoA decreased and FFAup increased with dobutamine-induced increased work. This suggests that MCoA could play an important role in the mechanism of matching energy supply and demand in the heart.

ASPARTATE AND 2-OXOGLUTARATE Th027 IMPROVE! FUNCTIONAL AND METABOLIC RE COVERY OF POSTHYPOXIC ISOLATED RAT HEART Oleg I. Pisarenko, lrina M. Studneva, Olga V.

ADP AND H+ MODULATE RIGOR IN CARDIOMYOCYTES

Korchazhkina & Valentin S. Shulzhenko. Cardiology Research Centre, Moscow, Russia

A180

The hypothesis that stimulation of cytosol aspartate aminotransferase by increasing substrate availability might improve the energy state of posthypoxic myocardium was tested in isolated working rat hearts perfused with glucose. The hearts were subjected to 20-min anoxic perfusion followed by 30-min reoxygenation without additives or in the presence of 3.5 mM aspartate (ASP) plus 3.5 mM 2-oxoglutarate (2-OG). Metabolite contents and lactate dehydrogenese (LD) release were assayed by enzymatic methods. By the end of anoxia, the supplemented group showed significantly lesser decrease of phosphocreatine (PCr) and creatine (Cr), augmented formation of lactate (Lac) and pyruvate (Pyr), and markedly reduced LD release. Reoxygenation with ASP and 2-OG reduced an increase in the initial LD release and decreased myocardial Lac and perfusate Lac/Pyr ratio to near the normal values. An increased content of Asp and 2-OG in reoxygenated hearts was combined with a higher level of myocardial glutamate, lesser loss of Cr and a better repletion of ATP and PCr stores. These metabolic effects were associated with ehanced recovery of cardiac functon. Thus, facilitation of anaerobic glycolysis due to promoting NADH reoxidation by malate dehydrogenase coupled to asparate aminotransferase, and improvement of the malate-aspartate shuttle operaton during reoxygenation are important components of cardioprotection afforded by ASP and 2-OG.

Th028

Mary Stapleton & Ashley AIIshire Department of Pharmacology & Therapeutics, University College, Cork, Ireland. Ischaemic contracture of the myocardium appears to be a rigor process (no actively cycling crossbridges) yet is associatedwith millimolarATP whereas in vitro rigor occurs at 10-100 fold lower concentrations. Rat ventricular myocytes permeabilized with digitonin were used to study the rigor reaction, rigor taken as cell shortening in the absence of calcium at 37°C. Rigor was half maximal (50% rods shortened) at pMgATP 4.0 (100~M) and complete at pMgATP 4.747 (171.LM). Inorganic phosphate (Pi; 30 raM), lactate (30 mM) or moderate acidosis (pH 6.8) had no effect but rigor was attenuatedat pH 6.2 (no shortening at MgATP > 56~M). Converselyall cells went into rigor at pMgATP 3.0 and 4.0 (pH 7.1) when equimolar MgADP was present. Lowering pH at pMgATP = pMgADP = 3.0 (1 mM) attenuated the process (n=4): pH %rods

7.1 2_+1

6.8 55+9

6.5 81 _+13

6.2 92_+8

Thus of metabolites accumulating in ischaemia MgADP promotes rigor, H+ inhibits it and Pi or lactate have no effect. Elevated MgADP may cause ischaemic contracture at mM ATP if it precedessevere acidosis.

[Supported by The/rish Heart Foundation)

DO INTRACELLULAR ATP AND ADP Th029 LEVELS SUBSTANTIALLY CHANGE IN EARLY MYOCARDIAL ISCHAEMIA ? Fumitaka Ohsuzu, *Motoaki Bessho, Eiichi Takayama, Katsumi Hayashi, Shigeki Yanagida, Nobuhiro Sakata, *Tetsuya Nakamura, and Haruo Nakamura, Dept of Medicine 1, National Defence Medical College, and *Eisai Research Laboratories, Tokyo, Japan Changes in ATP and ADP levels during early (up to 300s) global ischaemia were determined in Langendorff perfused rat hearts using two chemical extraction methods, i.e., conventional direct perchloric acid (0.4 M) extraction and stepwise extraction using 80% alcohol and perchloric acid (0.4 M) solutions sequentially. No difference in the ATP content of normally-perfused tissue was found using the two chemical extractIon methods. In the stepwise extraction, the ATP level hardly changed for up to 40s of ischaemia then gradually decreased; it significantly decreased only after 300s of ischaemia. In the direct perchloric a c i d extraction, the ATP level decreased after the onset of ischaemia and this decrease became significant after 90s of ischaemia. In accordance with this ATP change, ADP in these tissues increased more rapidly when it was extracted directly by perchloric acid solution. The results indicated that the changes in ATP and ADP with progression of ischaemia are smaller when both compounds are extracted using a stepwise method than when they are extracted by direct perchloric acid extraction. These results suggest that ATP and ADP change their intracellular chemical state and/or the susceptibility of ATP to perchloric acid increases with progression of ischaemia in rat cardiac myocytes.

REOXYGENATION FOLLOWING C H R O N I C Th031 HYPOXIA INCREASES MYOCYTE GLYCOGEN STORES AND TOLERANCE TO SEVERE HYPOXIA

Howard S Silverm--. Chris J Ocampo, Shao-kui Wei. Div of Cardiology,Johns Hopkins Univ, Baltimore, MD, USA. Adult r a t cardiac myooytes were cultured for 48 h o u r s in normoxic (N), 21% O = / 5 % CO~ or moderately hypoxic (H), 1% O = / 5 % CO 2 conditions followed b y reoxygenation (reox) for 10 mln (H48h-O21Om), a n d 3 h o u r s (H48h-O23h) or no reox (H48h) to . . . . ;-~ the effect of chronic hypoxia +/- reox

on toleranceto acute severe hypoxia. Myocyte viabilityw a s ~m~ffected by H. Culture m e d i a glucose fell f~om 5.6 to 4.7~0.1 in N a n d 3.5~).3 m M in H 4 8 h (p<.005 v s IN), while lactate increased to 0.7~).1 in N a n d 3 . 0 ~ . 5 m M in H 4 8 h (p<.001 vs N). Cellular glycogen w a s n o t depleted by H (68.8~6.7 nmoles glucosehng protein i n N v s 94.4~q.7 i n H 4 8 h , n = l l each, paNS) and w a s increased by reox following chronic H (190.3±45.2 in H48h-O=3h, p<.05 v s IN). During a subsequent exposure to severe hypoxia (.003% O=), t h e time to ATP-dapletion rigor cont~mcture w a s increased i n cells cultured in H a n d f u r t h e r ~ d b y reox following H (see table). Myocyte recovery following severe hypoxia w a s enhanced in H a n d H + reox groups even w h e n normAi;74~c] for t h e prolonged time to ATP-dep]etion rigor. _N H48h H48h-O a H48h-Os 10m 3h TIME TO RIGOR 27.6~6.3 42.2±1.2 51.3+1.7 71.3:~.5

(rain) (n=54) (n=69) (n=68) (n=97) Thus, chronic hypoxia enhances myooyte to]eranoo to subsequent severe hypoxia. A n intervenln~ period of O2 r e a a m ; ~ o n increases glycogen stores a n d m a r k e d l y delays later hypoxic energy depletio~

Th030 CYTOPLASMIC ATP CHANGES IN METABOLICALLY POISONED MYOCYTES MONITORED BY FIREFLY LUCIFERASE AT CONSTANT pH Isidro Allue & Peter H. Cobbold. Dept of Human Anatomy and Cell Biology, Univ Liverpool, UK

In order to test the hypothesis that rigor-mediated contracture of hypoxic cardiomyocytes coincides with a sudden fall in cytoplasmic ATP levels caused by rigor complex activation of myosin $1-ATPases; single, isolated cardiomyocytes were microinjected with the bioluminescent enz3n'nefirefly luciferase during metabolic inhibition. This enzyme in the presence of luciferin, O2, Mg2+ and ATP produces light, which can be used to monitor cytoplasmic ATP. Luciferase light emission is highly dependent on pH. Unfortunately, intracellular pH (pHi) varies greatly during metabolic inhibition. Measurementswith the fluorescence dye BCECF (2',7'-bis(2-Carboxyethyl)-5(6)carboxyfluorescein) showed that after addition of 2mM cyanide, 5mM 2-deoxyglucose, there is an initial increase in pHI to around pH 7.46, followed by a longer acidosis until the cell shortens at pH c. 6.94. To avoid pH-dependent artefacts in the luciferase signal, the cells were superfused with a modified bicarbonate-buffered Kreb's saline with 92mM NaHCO3 and equilibrated at pH 7.4 with 20% CO2/80% 02. This, clamped the pHi during metabolic inhibition. Simultaneous measurements of cell length and luciferase signal free of pH-dependent artefacts, show a sudden drop of the luciferase signal to background levels coincident with myocyte shortening. Calibration of the luciferase signal in terms of [ATP] indicate a cytosolic [ATP] of c.l.5mM as the threshold for the onset of cell contracture. We conclude that shortening of a cardiomyocyte is a key event in cell deterioration, involving a cytosolic ATP drop from millimolar to micromolar levels.

REPETITIVE STRETCHING DURING MYOCARDIAL T h 0 3 2 LOW-FLOW ISCHEMIA IMPAIRS CONTRACTILE FUNCTION IN ISOLATED PIGLET HEARTS Jon Offstad, Knut A. Kirkebeen, Hiichen Sommerschfld, Arnflnn Ilebekk, S. Evans Downing, InsL for Exp. Med. Res., Univ. of Oslo, Oslo, Norway and Yale Univ. School of Med., New Haven, USA. Infarction in pigs appears after 20 rain of no-flow segmental ischcmia in vivo. In contrast, much longer time of global no-flow ischemia is needed to produce necrosis in isolated hearts. We tested the hypotheses that repetitive stretching of the ischemic segment in vivo may explain this difference and that blockade of stretch activated channels (SAC) by Gadolinium (Gd) during repetitive myocardial stretching (RMS) attenuatesstretch-induced cell injury. Isolated piglet hearls (2-10 d) were perfused with Krebs-Henseleit buffer enriched with red blood cells. A system was constructed to induce RMS using a balloon in the left ventricle. Functional and metabolic recovery were compared in hearts exposed to 2 h of low flow ischemis (l 0O% of control) with or without RMS (pressure 120 mmHg during 1/3 of the cycle, and 0 mmHg during the remainingphase, frequency 150/min), followed by I h ofrepeffusiun. Non RMS hearts were exposed to saline (Isch, n=g) or Gd 25 ( ~ n=5) and RMS hearts were exposed to saline (Puls, n=l 0) or C,-d (C-dpuls,n=l 2). During low flow LVSP stabilized in the non RMS hearts, but a further decrease combined with signs of increased anaerobic metabolism were seen in RMS hearts. The table shows LVSP and ATP during repeffuaica. Isch Gdisch Puls Gdpuls LVSP (% of control) 774-4 74=1:2 46--1:4"* S 1"4-3** ATP (t~mol/gdw) 2.64.0.4 2.2--t:0.3 1.5:t:0.3" 1.64.0.2" (vs Isch, *P<0.05,**P<0.0 ] ). We conclude that RMS during low-flow ischemia severely reduces postischemic functional recovery and levels of ATP, probably due to increased energy d=uend during ischemia. Blockade of SAC by C-d (25 Idv0 does not attenuate the alterations induced by R_MS. A181

Th033 CHRONIC HYPOXIA IMPROVES CARDIAC METABOLIC RESPONSE TO B STIMULATION Vall~rle Novel-Chata, Jean Aussedat, Valdur Saks & Andr6 Rossl. Lab. Bloanerg6tlqua. Unlverslt6 J. Fourier, Grenoble, France. The aim of this study was to check whether the response of the energy metabolism of isolated rat hearts to increased energy demand was altered in hearts from animals chronically adapted to normobaric hypoxia (3 weeks, FO 2 = 10%). We used 31-P NMR spectroscopy to monitore the dynamics of the changes in Pi, PCr and ATP contents, and evaluated in parallel oxygen consumption and metabolite contents. Hearts were pertused under constant pressure (74 mmHg) with a medium containing glucose 9 mM + pyruvate 2 mM. They were subjected to increase in calcium concentration (from 0.5 to 2.5 mM) and to isoprenaline stimulation (10 -6 M). When compared to that of control animals, the hearts from hypoxic animals presented the following features : (i) for an unchanged ATP content, the myocardial concentrations of PCr and Cr were decreased. (ii) the inotrepic response was larger while the oxygen consumption was unaltered. (iii) under maximal inotropic effect by isoprenaline, in both groups a reduction of PCr and ATP contents and an increase in Pi concentration were observed, but subsequent pedusion without isoprenaline resulted, in the hearts from hypoxic animals, in a significant overshoot of PCr, which was not observed in the control group. These alterations of mechanical and metabolic responses during and after 13 stimulation in hearts from chronically hypoxic animals are assumed to indicate more efficient energy conversion than in normoxic hearts.

IMPAIRMENT OF MYOCARDIAL HIGH ENERGY Th035 PHOSPHATE METABOLISM IN AORTIC VALVE DISEASE ASSESSED BY alp MR SPECTROSCOPY V(t Herynek, Milan Hdjek, Dana K0rkov& Josef Kautzner*, MR Unit, Institute for Clinical and Expedmantal Medicine and "Department of Medicine II, Charles University Medical School I, Prague, Czech Rep. Depressed level of myocardial high energy phosphates (HEP) have been demonstrated In various cardiac diseases. To assess whether HEP abnormalities may help in early detection of cardiac failure in aorttc valve disease (AVE)),ECG-tdggered 1D ISIS MR spectroscopy technique using a 1.5 Tesia Siemens Magnetom with 10 cm surface coil and correction for blood contamination was employed in 18 male AVD patients (mean age 49): 7 with aortic stenosis (la), 5 with aortic regurgitation (113)and 6 with combined disorder (Ic), and in a group of 12 age matched healthy volunteers (11). Significant decrease in PCr/ATP ratios characterised the group of

patients compared to control population. However, neither these ratios differentiated among heamodynamicallywell defined subgroups of patients nor correlated with the severity of haamodynamic impairment.

°'°l'l'l'l

PCr/(xATP PCr~ATP

0.86i-().21 1.05:[-0.28 1.00-ZO.82 1.74$-0.51" 1.30-ZO.83 1 . 5 ~ . 5 4 1.20-ZO.61 2.01 :L-0.30"

"p<0.05 The principal component analysisapplied to the whole set of heamodynamic, echocardiographic and spectroscopic indices distinguished between groups la and Ib, based on the parameters clustered in the I st main component containing also PCr/I}ATP ratio (13<0.014).Group Ic was different only from group lb (13<0.045) because of heamodynamic similaritywith group la. In conclusion, evaluationof resting HEP levels does not appear to provide additional clinically relevant Informationbeyond that obtained from echocardiographyor basic heamodynamic data.

SIMULTANEOUS ACQUISITION OF NMR SPECTRA Th034 AND RADIOTRACERUPTAKE IN THE PERFUSED RAT HEART. Morilyn Buchanan,Paul K Marsden, Alison C. Cave, PamelaB. Garrick. RadiologicalSciences,UMDS,London,UK. NMR spectroscopyand radiotracer uptake measurementsore recognised as complementarymethodsfur the assessmentof the bioenergeticstatus of tissues. Correlationof data from separatestudies, however,is subject to potential errors; the ideal solution is simultaneousdata acquisitionin a single experiment. We havenow built e "y-photondetector that both fits in end functions in our 9.4T, 8cm bore, vertical magnet. Here we present the world's first, SIMULTANEOUSLYACQUIRED, 18FDGuptake end 31p NMR data from e SINGLE,isolatedrat heart, perfusedunder normoxicand hypoxic conditions. The figure shows continuousmFDG uptake, together with NMR spectra acquiredat the markedtimes. Thesedata confirm that hypoxia, as expected, causes a reversibleincrease in the leFOG uptake and a reversibledecreasein the phosphocreatine:P=ratio. We have thus shown that it is 31p NMR Spectra possible to combine 18FDG Uptake PerI ATP these two, powerful non-invesive techniques into o single analytical method, DREAM (Dual acquisition of , 80 120 Radioactive Emission Time(mini And Magnetic ppm resonance). Not only does this have enormous experimental potential but, clinically, the combinationof PET sensitivity with MRI resolutioncould be invaluable.

HYPERTHYROIDISM AFFECTS MYOCARDIAL Th036 GLUCOSE UPTAKE AND INSULIN SENSITIVITY James C.A. Hopkins, Kieran Clarke & George K. Radda Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Peripheral insulin resistance and impaired glucose tolerance are observed in the hyperthyroid state. Hyperthyroidism increases glucose oxidation and insulin stamulated glucose disposal in skeletal muscle. In hyperthyroidheart the contribution of glycolysis to ATP synthesis is increased. In this study we examine basal and insulin stimulated glucose uptake in the isolated perfused heart from normal and hyperthyroid rats. Myocardial glucose uptake rates were measured using 2deoxyglucose (2DG) as a marker of glucose and 31p NMR spectroscopy to monitor 2-deoxyglucose-6phosphate accumulation. Hyperthyroidism was induced by injection of T3one week before heart per'fusion. The basal rate of 2DG uptake in the normal heart was 0.54 mM/min, but 37% lower in the hyperthyroid state (0.34 mM/min). Insulin increased 2DG uptake 4-fold in both normal and hyperthyroid hearts (2.22 & 1.74 mM/min respectively). Decreased basal glucose uptake and normal insulin sensitivity is consistent with reported decreased GLUT1 and normal GLUT4 levels m the hyperthyroid heart. 60

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KINETIC CONTROL OF MITOCHONDRIAL Th037 RESPIRATION IN THE IN SlTU RAT HEART. John Headrick 1, Geoffrey Dobson 1, Lindsay Jordan z, Roger Willis 3. 1. James Cook University, Townsville 4811 Australia. 2. Rotary Centre for Cardiovascular Research, Griffith University, Nathan 4111, Australia.

Th038 E F F E C T O F D - I 3 - H Y D R O X Y B U T Y R A T E ON F A T T Y ACID AND G L U C O S E O X I D A T I O N IN T H E I S O L A T E D P E R F U S E D R A T H E A R T John R. Forder, David A. Schulman & John C. Chatham. Johns Hopkins University, Baltimore, Maryland, USA.

The role of ADP in control of mitochondrial respiration was studied in in situ rat heart. Under control conditions, the NMR determined phosphocreatinine/ATP ratio (PCr/ATP) was 2.30 + 0.05, pH was 7.35, free [Mg2'] was 0.57mM and inorganic phosphate (Pi) was estimated to be 0.83 mM The in situ rat myocardium is highly energised as indicated by [ATP]/[ADP] [Pi], dGATP, free [ADP] and [AMP] values of 700 + 78 mM~, -63.93 + 0.33 Kj/mol, 18 + 3 i.tM and 47 ± 19 nM, respectively. 0.9 Fg/min/kg epinephrine increased the rate-pressure product by 105%. [ATP] was stable (97% baseline), [Pcr] declined by 25% and [Pi] increased by 1.00 mM. Cytosolic pH and [Mg2"] were calculated to be 7.27 + 0.01 and 0.64 ± 0.05 mM, respectively. The PCr/ATP ratio, [ATP]/[ADP] [Pi] and dGATP declined to 1.83 + 0.13 I~M, 108 ± 15 mM"1 and -59.56 ± 0.49 Kj/mol respectively. Free [ADP] and [AMP] increased to 55 ± 10 p.M and 495 ± 183 nM respectively. Analysis of data yielded an in vivo Km for ADP of 37 ± 8 p.M and supports the proposal that ADP is important in kinetic control of myocardial respiration in the in situ rat head. Experimental data from larger species indicates that kinetic control may =scale" with body mass, larger animals displaying lower energy states and higher [ADP]. If the Km for respiration is constant across species (at ~ 35 p.M), the results suggest that ADP is of regulatory importance in small but not larger mammals.

The purpose of this study was to examine the effect of the ketone body D-I~-hydroxybutyrate (13-HB) upon the relative utilization of fatty acid and carbohydrate fuels in the isolated, isovolumic rat heart preparation (Langendorff). Hearts were perfused with KrebsHenseleit buffer containing uniformly labeled 13Cpalmitate (0.5 mM) and 1-t3C-glucose (5 mM) plus insulin (0.05 mU/mL) in the presence of increasing concentrations of I3-HB (0-5 mM). At the end of each experiment, hearts were freeze-clamped, extracted with perchloric acid, and analyzed by high resolution 13CNMR spectroscopy. Isotopomer distribution analysis was performed to determine the relative contributions of substrates to TCA cycle turnover. The lowest concentration of I~-HB (0.2 mM) caused a significant inhibition of glucose oxidation, from 37+6% (in the absence of ~-HB) to 16+_.3% (p<0.01) without affecting fatty acid oxidation. At higher concentrations of 13-HB (0.6-5 mM), glucose oxidation was not further reduced, but fatty acid oxidation was inhibited in a concentrationdependent manner (p<0.01). Anaplerosis was calculated to be 19+4%, and was not altered by I~-HB. Neither the initial contractile function (as assessed by rate,pressure product), nor the stability of the preparation was affected by increased fI-HB concentration. This work has important implications regarding the modulatory role of ketone bodies upon myocardial metabolism and function.

THE EFFECT OF ADENOSINE ON CELLULAR Th039 ENERGETICS AND PURINE PRODUCTION IN THE POSTISCHEMIC RAT HEART Ryszard T Smolenski, Jay R Jayacumar, Anne-Marie L Seymour & Magdi H Yacoub, Dept Cardiothoracic Surgery, Natl Heart & Lung Inst, Harefield Hospital, UK

Th040 In Vivo Depletion of Free Thiols Does Not Account for Nitroglycerin-Induced Tolerance, but ThiolNitrate Interactions May Exist. A, Hai-Yehi~ 1 and L. Z. Benet2, School of Pharmacy, The Hebrew University of Jerusalem, Israel I and The University of California, San Francisco, USA2. The in vivo role of the endogenous thiols, ~utathionc (GSI-I) and cysteinc (O/s) in nitroglycerin (G'rN) activity and tolerance has bex:omcincreasingly controversial. We investigated the role of these thiols in an in vivo rat model. Rats were divided into treatment groups including controls, GTN-tolerant animals and rats after thiol depletion or supplementation. Tissue and plasma

A brief period of ischemia (I) can cause increases in adenosine (ADO) and total pudne catabolite release from the heart. However, if repeated periods of I are induced, this release is markedly reduced whilst supply of ADO during reperfusion restores this deficit. We evaluated the relationship between changes in intracellular high energy phosphates and pudne production in Langendorff perfused rat hearts with or without ADO. Hearts were subjected to (A) 1' (min) I at 40' of perfusion, 10' I after 50' and 1' I after 85' while in (B) 30 pM ADO was added for 15 min of reperfusion after 10' I at 50'. % changes in ATP, phosphocreatine (PCr) and intracellular inorganic phosphate (Pi) were measured using 31P-NMR. ATP and release of purine catabolites into the coronary effluent (Pur) were determined by HPLC. without ADO (A) with ADO (]3) PCr Pi ATP Put PCr Pi ATP Put initial = I00% initial = I00% l'lat40' 64 163 90 I00 52 159 92 100 prior l'Iat 85' 146 108 69 138 106 74 l'lat85' 77 192 66 12 64 166 82 37 ATP at end 14.8 nmol/g dry wt 16.4 nmol/g dry wt ADO infusion increases release of purines by elevating adenine nucleotide pool rather than by alterations of cellular energetics.

cGMP

and thiol concentradons were measured. Lower basal

and GTN-induccd cGMP levels were found in vascular tissue from thiol-deplctcd rats. However, thiol depletion was not associated with tolerance devcloprnent in control rats and did

not significantly enhance tolerance in thiol depleted ram. Thiol repletion did not restore the response in OTN-tolcrant rats. These findings suggest that depletion of free thiols does

not

underlie GTN tolerance.Thiol repletion in depleted-tolerantrats or supplementation in control tolerantrats did not reverse GTN tolerance. However, a depletion of sullhydryl =croups from sources other than free GSH and cysteine may be involved in GTN action and tolerance.

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ATRIAL MITOCHONDRIAL DNA DELETION IN Th041 OPEN HEART SURGERY

PATIENTS.

A.Giaccari, P.Mikus °, G.Coll °, C.M uscari, C.Stefanelli A.Pierangeli°,C.M.Caldarera,C.Guamieri.Department of Biochemistry and Heart Surgery°,University of Bologna, Italy. Mitochondrial DNA (mitDNA) mutations or deletions have been detected in degenerative diseases and accumulated in a variety of aging tissues.The consequence of increased frequency of mitDNA deletions in heart tissue may be cardiomyopathy. We investigated the presence of the common 4977 nucleotide pair mtDNA deletion in atrial tissue of patients undergoing open heart surgery for severe coronary artery disease or valve replacement. The biopsies were taken before and after surgery. The total DNA was prepared by SDS-proteinase K digestion. The presence of mtDNA deletion was estimated by PCR analysis separating the amplified segment on agarose gel and capillary electrophoresis. Preliminary results indicate that all patients examined (n=22) had the mtDNA 4977 deletion identified by a single amplified fragment of 469 bp. In some patients, the amplified fragment was also present in lymphocytes withdrawn before the surgical operation,differently from normal subject. Further studies will ascertain if the mitDNA deletion found in these patients is caused by the diseases,or is a manifestation of the aging.

DOES INHALATION ANESTHETIC NEGATE ~ Th043 CARDIOPROTECTIVE EFFECTS OF D-RIBOSE? Jack Wdlan, Michael Belangcr,and Carin Witlnich DcpLof Physiology,Universityof Toronto,Toronto,Canada D-ribose significantly augments myocardial adenine nuclantide content, enhancing restitution of preischemin ATP content and functional recovery. To date, howev~, acute studies of D-ribose have not investigated its effects when administered in the presence of inhalation anesthetics, especially those used clinically. To determine whether D-ribose pretreatment concurrent with inhalation anesthesia protected hems against ischemic insult, adult Sprague-Dawley rats were anesthetized with halothane and received either A) Saline or B) D-ribose (100 mg/kg) twice in 24 hours, varying only the route of adminislration between intravenous (IV) and inlrmnuscular(IM). Additional 8roups were similarly pretreated in the absenceofhalotlmn~ H e n were excised and myocardial tolerance to global ischemla assessed using time to ischemin conU'actm'e(TIC). Initiation of contracture (TICo), the onset of irreversible damage, was measured as a 2 mmHg pressure rise in a LV balloon. HALOTHANE AWAKE Iv IM IV IM SALINE 15.05=~-0.3415.0S~'0.34 14.10~1.30 14.41:~0.77 (n=5) (n-s) (n=8) (n=lr) D-RIBOSE

IV

IM

Iv

IM

11.954-~.02 5.93:~0,61"~ 17.71~.0,62*16.64~-0.74* Cn=3) Cn'6) Cn'II) (n=13) •rlco (mira) Mean~EM *p<0.05v=.=,,~,uv,;at=oomrol #p<0.05vs. IV Compmisonof halothene and awake saline pretreated rats showed that halothan¢did not alter TICo (p=0.8). D-ribose significantly prolonged TICo in both IVand IM awake groups (25.6% and 15.5%, respectively). However, D-n'bose with halothane decreased TICo by 20.6% and 60.6% in the IV and IM groups, respectively. Hence, D-ribose, in the presence of halothane, is detrimental to myocardial ischemic tolerance. A184

P R O T E C T I O N OF THE H E A R T IN PATIENTS Th042 UNDERGOING CORONARY SURGERY: THE EFFECT OF L-CARNITINE. HEMODYNAMIC STUDY Ivan Gabauer, Roman Zahorec, Ivan Pechan, Viliam Fischer & Katarina Kanalikova. Inst Heart Res, SAS, Inst Cardiovasc Diseases, Bratislava, Slovak Republik. L-camitine has been found to play an important role in cardiac metabolism. Its effect on ischemia-reperfusion injury has been evaluated in patients with low left ventricular ejection fraction (below 40%), randomly selected for coronary artery bypass grafting. 10 patients were given an infusion of L-camitine immediately before the surgery, and then 12 and 24 h postoperatively. 10 patients with placebo served as a control. The following functional and hemodynamic variables were estimated: restoration of the sinus rhythm, number of defibrillations, occurrence of the peri and postoperative arrhytbmias, mean arterial pressure, pulmonary wedge pressure, cardiac index, systemic resistance index and left ventricular systolic work index. The protective effect of L-eamitine was manifested by increased occurrence of spontaneous restoration of sinus rhythm, by decreased incidence o f ventricular fibrillations after aortic deelamping, by lesser number o f defibrillations and by decreased incidence of peri and postoperative arrbythmias. Values o f estimated hemodynamie variables immediately after surgery were significantly increased in camitine group as compared to control group.

SUBSTRATE AVAILABILITY AND Th044 M Y O C A R D I A L C A L C I U M HOMEOSTASIS Ellen Aasum & Terje S. Larsen. Dept of Medical Physiology, Univ. of Troms~, Norway In the present study we examined the possibility that substrate availability can influence myocardial calcium uptake and ventricular function in hearts exposed to hypothermia and rewarming. Thus, 3 groups of Langendorff pertused guinea pig hearts, given either 11.1 mM glucose (G), glucose + 1.2 mM palmitate (GP) or glucose + palmitate + 5 mM pyruvate (GPP) were exposed to 40 min hypothermic perfusion (15 °C), followed by 30 min rewarming at 37 oC. Calcium content ([Ca2*]tol=) was measured by means of the "SCa~*technique. All perfuslon groups showed marked elevations in [Ca2*]t== at 40 min hypothermia, compared to prehypothermic values (9.3-12.2 vs. 0.6-0.7 pmol • g dry wt'l ), which were associated with pronounced elevations in diastolic tension. Following rewarming, GP-perfused hearts showed higher [Ca2÷]1=.,and less recovery of developed pressure (LVDP, % of prehypothermic value) than Gperfused and GPP-perfused hearts ([Ca2÷]t,~,,:6.0 ± 1.3 vs. 2.0 ± 0.6 and 3.8 ± 0.5 pmol • g dry wt " ; LVDP: 27 ± 8 vs. 62 ± 3 and 62 ± 8%). In separate experiments we found that palmitate oxidation (3H-palmitate)was more than 40% lower in GPP-perfused hearts as compared to GP-perfused hearts. We conclude that the presence of fatty acids impair recovery of calcium homeostasis and ventrlcular function following hypothermia. These effects can be overcome by addition of pyruvate, probably as a consequence of reduced fatty acid oxidation.

ROLES OF ENERGY SUBSTRATES, Ca 2÷, AND Th045 NO IN MYOCARDIAL DEPRESSION. Kassim Abou-Chehade & Richard Schulz. Depts. Pediatrics and Pharmacology, Univ. of Alberta, Edmonton, Canada. We have shown that bacterial endotoxin or proinflammatory cytokines depress cardiac mechanical function due to the expression of inducible NO synthase (iNOS) activity in the heart. We now measured the effects of energy substrate, extraeellular Ca 2., and cycloheximide (Cx) on cardiac mechanical function and expression of iNOS. Isolated working rat hearts paced at 300 bpm were perfused with recirculating Krebs-Henseleit buffer (>lng/ml endotoxin) containing: a) 1.25mM Ca2*/5mM pyruvate, b) 1.25mM Ca2*/0.SmM palmitate in the presence or absence of 10l.tM Cx or, c) 2.5mM Ca2"/0.8mM palmitate. Cardiac work (cardiac output x peak systolic pressure) was measured as an index of cardiac function. At 1.25mM Caz÷, 2hr perfusion with either palmitate or pyruvate did not significantly alter the loss of cardiac work (47.7+8.2%, n=9 vs. 24.7+14.2%, n=5, respectively). Doubling of buffer Ca 2÷ in palmitate-perfused hearts reduced the loss of cardiac work at 2 hr measured at preloads _>13.5mmHg (n=9, p<0.05). The loss of cardiac work after 2 hr perfusion with 1.25raM Ca2÷/palmitate was abolished by Cx (n=6, p<0.01). Cx prevented the increase in [NOS activity in tissue homogenates of 1.25mM Ca2÷/palmitate-perfused hearts (0.05+0.04 vs. 0.60+0.09 pmol/min/mg protein, p<0.001). We conclude that the loss of cardiac function: a) cannot be due to the lack of fatty acid, the major energy substrate in the heart, b) is reduced by extracellular Ca2÷ and, c)'is abolished by Cx by preventing an increase in iNOS activity.

Th047 THE RELATION BETWEEN INSULIN RESISTANCE ANI~ THE FAITY ACID COMPOSITION IN RAT MYOCARDIUM Hldeo Matsul, KenJl Okumura, Yuklo Tokl & Takayukl Ito. Internal Medlclne 2, Nagoya Unlverslty, Nagoya, Japan. The aim of this study was to investigate the relation between insulin resistance and myocardial lipid metabolism. We analyzed lipid composition in the myocardium from male Sprague-Dawley rats fed high fructose (40%) diet, which is known to cause insulin resistance and hypertension in rats, for14 days. High fructose diet elevated blood pressure and the levels of plasma triglyceride, free-fatty acid, LDL and VLDL concentrations, but myocardial cholesterol, cholesteryl ester, triglyceride, and phospholipid contents remained unchanged. However, the percentage of Fnonounsaturated fatty acids (MUFAs) in triglyceride fraction was increased by 44% (p<0.01) and that of polyunsaturated fatty acids (PUFAs) was decreased by 36% (p<0.01). In the PUFAs, n-3 fatty acids and n-6 fatty acids were decreased by 28%(p<0.05) and 37%(I)<0.01), respectively. We also had similar results in phospholipid fraction, which plays Important roles in various metabolic events as a major membrane component. These alterations of fatty acid compos tion in myocardium were similar to those in skeletal muscle and plasma LDL, which are major sites of insulin action. The addition of 1% probucol to high fructose diet decreased plasma lipid concentrations and myocardial trig yceride content, but failed to reduce insul n secret on and normalize the fatty acid composition of each lipid. These results suggest that insulin resistance may be associated with alteration of fatty acid composition, especially increase of MUFAs and decrease of PUFAs in myocardium.

CLONAZEPAM INHIBITS MITOCHONDRIAL Th046 Ca'-* EFFLUX IN SINGLE CARDIOMYOCYTES. Elinor J. Griffiths, M. C. Haigney, C. Oeampo, & H. S. Silverman. Division of Cardiology, Johns Hopkins University, Baltimore, MD 21205. Increases in mitochondrial [Ca'-'] ([Ca2"],") levels stimulate ATP production by activating dehydrogenases of the citric acid cycle. In isolated mitochondria, clonazepam (CZ) c a n maintain [Ca2+],, by inhibiting Na ÷ dependent Ca2+ efflux. Here we show that clonazepam can inhibit the decline in [Ca:*],. in intact, single rat myocytes following cellular Ca -'~ loading with 50nM norepinephrine (NE). Myocytes loaded with the fluorescent Ca2÷ indicator indo-l/am were superfused with Mn'-+ which quenches the cytosolic Ca2÷ signal leaving a signal due to [Ca2*],, only. Myocytes were exposed to NE alone or NE plus 100uM CZ for 6rain followed by washout of NE. Values of [Ca'-'],. (riM) for NE (n=5) and NE + CZ (n=4) were: 59+0.5 vs 58+0.4 (pre-treatment), 606+ 159 vs 1075+ 174 (peak), 69+7 vs 584+84 (10min washout). Thus CZ increases the peak [Ca~-+],, and greatly delays the return to pre-stimulation levels. Lower [CZ], 50, 25 and 5uM, gave progressively less inhibition. Exposure of myoeytes to CZ alone (10min) prolonged the action potential (15.3 +2.1 vs 54.9+7.2reset for 50% decay, n=5) and increased contractility (extent of shortening 9+2.3 vs 13.7+1.1%, n=5) but had only minimal effects on cytosolic Ca~-÷. Thus the major effect of CZ appears to be an inhibition of mitochondrial Na*/Ca:* exchange, but there are other, as yet undefined, effects on contractility and membrane potential.

EFFECT OF RETINOIDS ON PLASMA AND Th048 MYOCARDIAL LIPID PROFILES IN RATS E.Tvrzick~, M.Jir~skov~, K.Filipov~, O.Nov~kov~, A.Z~k. ist Faculty of Medicine and Faculty of Science, Charles University, Prague, Czech Rep.

We have investigated the effect on rat l i p i d metabolism of two retinoids, Tigason (T) and Roaccutane (R), which are used for long-time therapy in dermatologic practice. Male Wistar rats ( i n i t i a l body mass 448.3 ± 25.8 g) received 0.5 mg of T or R daily for a period of 6 weeks. Lipid classes were separated by thin-layer chromatography. Fatty acid (FA) profiles were determined by capillary gas chromatography. The ratio of the polar/neutral l i p i d in myocardial tissue was decreased in the T group. Increase of polyunsaturated FA anddecrease of saturated FA was observed in the R group. No significant changes in individual plasma l i p i d levels were observed. In cholesteryl esters, increased content of polyunsaturated FA was found in the R group and that of the stearic acid in the T group. In phosphatidylcholine, increased content of palmitic acid was observed in the T group; decreased content of oleic and increased one of vaccenic acid were found in the R group. Changes in the l i p i d composition of plasma and myocardial tissue implicate different actions of both drugs on the enzyme system of l i p i d metabolism.

A185

DIHOMOGAMMA-LINOLENIC A C I D (DGLA) Th049 METABOLISM IN S P O N T A N E O U S L Y H Y P E R T E N S I V E RATS (SHR). G r a c i e l a E. H u r t a d o de C a t a l f o , I r m a N.T. de G6mez D- mm. I n s t i t u t e de I n v e s t i g a c i o n e s B i o q u ~ m i c a s de La Plata, A r g e n t i n a .

In the development of hypertension in SHR a role has been attributed to alterations in lipid metabolism. Considering that DGLA and arachidonic acid (AA) are the immediate precursors of eicosanoids with hypotansive effects, we studied the activity levels of enzymes directly related with polyunsaturated fatty acid metabolism in SHR compared to normotensive controls (WKY). The dihomogamma-linolenylCoA formation, and the AA biosynthesis at the &5 desaturation step were measured in the micresomal fraction of liver, testis and kidney using labeled DGLA as substrata in both enzymatic assays. In kidney microsomes from VVKY basal experimental conditions and the kinetic properties (Km and Vmax) of the long-chain fatty acyI-CoA (LCFA) synthetase were determined: 45 pM and 12.5 nmol/min/mg protein for DGLA. SHR exhibited higher levels in the LCFA synthetase activity in liver than WKY (114.7_+2.8 %, P<0.02). Delta 5 desaturation activity in SHR was depressed in liver (76.6_+2.5 %, P<0.01) and testis (83.9_+4.0 %, P<0.05) compared to WKY. The testicular and kidney thioesterification of DGLA were also significantly depressed in SHR compared to controls. The data obtained suggest that in SHR lesser amounts of dihomogammalinolenyI-CoA and lower levels of AA would be available for the formation of prostaglandins and/or other eicosanoids which are important regulators of blood pressure.

METABOLIC RESPONSE TO ISCHEMIA IN Th051 CHRONICALLY INFARCTED RAT HEARTS; EFFECTS OF LONG-TERM CAPTOPRIL Ed A.J. Kalkman, Pramod R. Saxena, Regien G. Schoemaker, Dept of Pharmacol, Erasmus University, Rotterdam, The Netherlands Captopril (Cap) has been shown to improve mechanical and biochemical parameters of the heart after myocardial infarction (MI). We studied the effects of chronic Cap on cardiac metabolic response to additional ischemia in MI hearts. Hearts from rats, treated with Cap from 3-8 weeks after MI were isolated and perfused. Maximal vasodilation to exogenous adenosine was determined, and the release of ATP catabolites (purines) and lactate into the coronary effluent was measured after 30 minutes of low-flow ischemia. Maximal coronary flow (corrected for heart weight) was reduced in MI hearts (16.7:1:1.2 v 25.0:1:2.0 ml/min.g) and restored by Cap (24.3 5:1.5 ml/min.g). Release of both purines and lactate was significantly lower in MI compared to sham hearts (purines 6 . 9 + 0 . 5 v 12.0:1:1.0 nmol/min.g; lactate 0.63 + 0.05 v 0.90 5: O. 15 pmollmin.g). Cap did not affect purine release, but further reduced lactate release (0.345:0.05 pl/min.g). These data indicate that chronically infarcted rat hearts are less sensitive to ischamia, since both ATPbreakdown and lactate production were reduced. Cap restored decreased maximal coronary flow in MI hearts. In addition, Cap further decreased lactate production during ischemia, suggesting alterations toiwards a more aerobic ATP-production.

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REGULATION OF ACETYL CoA Th050 CARBOXYLASE (ACC) IN DIABETES Jim Gamble*, Lee A. Witters and Gary D. Lopaschuk. Edmonton, Canada and Hanover, U.S.A. Abnormal lipid metabolism contributes to the high incidence of cardiovascular complications associated with diabetes. A key enzyme involved in lipid metabolism is ACC, which is both the rate limiting enzyme for fatty acid biosynthesis and is important in the regulation of fatty acid oxidation. In this study we determined if changes in ACC expression or activity contributed to the alterations in lipid metabolism seen in diabetes. Tissues were harvested from control or diabetic rats (injected with 110 mg/kg streptozotocin 48 hrs prior to study). Western blots revealed the presence of both a 280 kDa ACC (important in fatty acid oxidation) and a 265 kDa ACC (important in lipogenesis) in heart and liver. Skeletal muscle (SM) and white adipose tissue (WAT) contained exclusively the 280 kDa and 265 kDa ACC, respectively. ACC-265 protein expression in diabetic WAT declined to undetectable levels, which was accompanied by a 87% decrease in ACC activity (from 363.2+133.2 to 48.2:1:15.0 pmol malonyl CoAJmin/mg). Although ACC protein levels in heart remained unchanged, a significant decrease in ACC activity was observed (162.8+2.1 versus 94.9+7.0 in control and diabetic rats, respectively). In liver both isozymes decreased (to 79% and 62% of ACC-280 and ACC-265 levels seen in control rats, respectively). This was accompanied by a 56% decrease in ACC activity (from 70.7:t:5.8 to 31.0+_5.8 pmol malonyl CoA/min/mg). No change in either ACC expression or activity was observed in SM, The decrease in ACC-265 protein expression and activity in lipogenic tissues may contribute to the decrease in fatty acid biosynthesis seen during acute ketotic diabetes. Decreased ACC activity, but not protein level, may contribute to the overeliance of the heart on fatty acid oxidation in the diabetic.

E F F I C I E N C Y O F O X Y G E N U T I L I Z A T I O N Th052 IS A L T E R E D IN I N F A R C T E D R A T H E A R T S R. Tian, P. Gaudron, S. Neubauer, K. Hu, G. Ertl. Medizinische Universatetsklinik, Wurzburg, Germany The relationship of performance and energy consumption in chronically infarcted heart has not been clarified though structural and biochemical remodeling were found. We measured oxygen consumption (MVO2) during stepwise increases in left ventrieular filling volume, isoproterenol infusion or high frequency pacing in isolated isovolumic buffer-perfused rat hearts 8 weeks after left coronary artery ligation or sham-operation. Systolic pressure-volume area (PVA) and rate-pressure product (RPP) were used to assess total mechanical energy consumed for each beat and ventricular performance, respectively. Structural dilatation and reduced performance of infarcted hearts were indicated by substantial rightward shift of pressure-volume curves and a reduced maximal developed pressure of the left ventricle (80 + 5 vs. 119 + 4 mmHg, p<0.05). The slope of the MVO2-PVA relation was lower in the infarcted compared to the control groups (1.02 + 0.16 vs. 1.44 + 0.10 10-5mlO2/ mI-Ig/ml, p<0.05), reflecting an increased efficiency of chemomechanical energy transduction in surviving myoeardium. However, RPP/MVO 2 of infarcted hearts was significantly reduced (315+30 vs. 505+38 p.mol/mmHg, p<0.05). Thus, efficiency of myocardial contractile machinery was increased in infarcted hearts while efficiency of left ventricular performance was reduced, possibly due to unfavorable geometry resulting from chamber dilatation.

Th053 PARTITIONING OF MYOCARDIAL ENERGY DEMAND IN REGIONAL MYOCARDIUM IN VlVO Serge AlP Trines, Rob Krams, Cees J Slager, Loe Kie Soei, Sjoerd H Hofma & Pieter D Verdouw. Experimental Cardiology, Thoraxcenter, Erasmus University Rotterdam, The Netherlands Myocardial oxygen is not only used for generation of work, but also for Ca"-cycling and basal metabolic rate. The contribution of each of these components for regional myocardium has sofar only been studied in isolated hearts. We have evaluated a new method to obtain the contribution of each of these three components in vivo for regional myocardium. In 9 open thorax pigs, regional area shortening was measured with two sets of perpendicular crystals and wall tension calculated from left ventricular (LV) diameter and LV pressure. Preload variations were applied to calculate E,, and the area of tension area (TAA) from regional tension area relationships. Subsequently heart rate was varied to evaluate the 3 weighing factors a,13and V in two physiological oxygen demand models: MVO2=a.TAA+I3.E°,+ y, where y is basal metabolic rate (BMR;model 1) and MVO2=a.TAA+I3.E,,=+ y*(I/HR) (model II). The first model assumes a constant BMR per beat, while the second model assumes a constant BMR per minute. The results are: a* 10"4 13"10.2 y r2 model I model I1

0.62 ± 0.20 0.14+0.20

-0.24 + 0.14 0.3+0,06

0.09 + 0.03 0.92 9.11 + 1.42 0.99

Model II appeared superior over model I (F-test, p<0.05;higher value and I~>0). As a consequence, in regional myocardium in vivc under resting conditions, MVO 2 is approximately partitioned 25% for contractility, 25% for generation of work and 50% for Basal Metabolic Rate. The high contribution of BMR in vivo is unexpected and implies that in further studies this factor has to be taken into account.

DIABETIC-LIKE BEHAVIOUR OF THE RAT HEART I N T h 0 5 5 EXPERIMENTAL RENAL INSUFFICIENCY Hana Vavfinkovfi, Milada Tutterovfi, Milada Bohdaneckd, Ludmila Kazdov~ Inst.Clin.Exp.Medicine, Prague, Czech Republic Chronic renal insufficiency (CR[) in man is often associated with cardiac dysfunction of unknown etiology. We studied this problem in female rats of an inbred Wistar line, with CRI induced by 5/6 nephrectomy. The metabolic and functional status of the heart and its resistance to ischemia were determined.at week 10 since nephrectomy. At the start and end of the experiment, blood pressure (BP) was measured using plethysmography. Before decapitation, the oral glucose tolerance test (OGTI') was performed in control (C) and nephrectomized (N) rats. Mechanical parameters of the heart before ischemia and at the end of 30-rain reperfusion after 20-minute ischemia were measured in the isolated rat heart perfused according to Neely. ATP and glycogen (G) contents were determined in the heart frozen with liquid nitrogen. Results: At the end of experiments, BP significantly rose in N from 1 1 2 + 1 . 5 to 1 4 4 + 2 . 8 mmHg, while it remained unaltered in C. Left ventdcular weight was higher by 43% in N than in C. Right ventdcuiar weight did not differ. Coronary flow (CF), aortic flow (AF), and stroke volume (SV) were lower in N rats after calculation to 1 g of weight (CF -30%, AF -10%, SV -14%). By contrast, cardiac function recovery after 20-minute ischemia was significantly higher in N than in C. Expressed in % of function before ischaemia: N vs C = AF 90.2 + 2.3 vs 85.7+10.0, p < 0.005; SV 97.4:]:2.7 vs 68.7+5.6, p < 0.001). Myocardial G conltent was higher in N than in C (98+4.6 vs 5 9 + 5 . 7 ~mol.g- d.w., p < 0.001). N rats had also an impairment of glucose tolerance, increased cardiac resistance to ischemia and higher post-ischemic G have also been reported in diabetic perfused rat hearts. Our findings show that CRI leads to changes in heart function and metabolism similar to those characteristic for the diabetic heart. This research was supported by a grant IGA MZ CR reg. Nr. 2023-3

Th054 REDUCED MYOCARDIAL EFFICIENCY IN STUNNED MYOCARDIUM CAN BE EXPLAINED BY A DIFFERENT PARTITIONING OF MYOCARDIAL OXYGEN DEMAND Rob Krams, Serge AlP Trines, Loe Kie Soei, Cees J Slager, Jos MJ Lamers & Pieter D Verdouw. Experimental Cardiology, Thoraxcenter, Erasmus University Rotterdam, The Netherlands Cardiac dysfunction after brief periods of ischemia is accompanied by relatively high oxygen consumption (MVO2) leading to a reduced mechanical efficiency (external work over MVO2 ). MVO2 is not only determined by the generation of work, but also by the energy necessary for maintenance of contractility or excitation-contraction coupling (EC-coupling) and basal metabolic rate (BMR). We determined how myocardial sfunning,induced by two cycles of 10 min of occlusion and 30 min of reperfusion in 5 open thorax pigs, affected the contribution of these components to MVO 2. Area shortening was calculated from two perpendicular sets of ultrasonic crystals placed in the mesomyocardium and wall tension from Left Ventricular (LV) diameter and LV pressure. Preload changes were induced to calculate contractility (E==) and total work (tension area area or TAA) from regional tension-area relationships. Secondly we changed heart rate in 6 steps, before and after stunning in each animal to determine the 3 unknowns of the oxygen demand model: MVO2= a.TAA + {3E,,=+ y(I/HR). Regional En decreased, signifying the reduced EC-coupling, from 6.1 to 3.5 mmHg/ml. Regional TAA decreased from 758 to 495 mmHg.ml. The a, 13and y were: a*10 "~ 13.10-2 V r= Baseline 0.22+0.18 0.35+0.08 8.03±1.19 0.99 Stunning 0.85 + 0.28 1.00 + 0.20 2.87 ± 1.54 0.99 In conclusion, the disproportional increased oxygen cost for generation of work (a) and maintenance of contractility (13),reflecting a less efficient EC-coupling, may explain the reduced mechanical efficiency during stunning.

BIOCHEMICALCHARACTERISATIONOF THE ,Th056 BIREFRINGENCEASSAY Sally Darracott-Cankovic, Stephen Large, Terence Kealey. Dept of Clinical Biochemistry, Addenbrooke's Hospital and Transplant Unit, Papworth Hospital, Cambridge UK

THE

Quantitative birefringence microscopy (QBM) has been used as a predictor of donor heart viability, but its mechanism has not been elucidated. We speculated that QBM reflected rigor following a fall in ATP and ADP content with ischaemia. We have, therefore, correlated changes in adenine nucleotides, creatine phosphate (CP) and glycogen (Glyc) during ischaemia with QBM in 75 rat hearts excised and stored at 37oc for up to 6 hrs (results=mean_+SEM gmoles/gram wet weight). Time CP 0 3.1

Glyc 224

ADP AMP QBM 1.22 0.25 1.55 _+0.2 _+38 _+0.25 _+0.15 +_0.05 +0.04

5min 0.65 81 _+0.06 _+7

ATP 4.29

3.54 1.90 0.73 1.15 _+0.19 _+0.13 _+0.08 _+0.03

30rain 0.13 5_+3 0.51 0.6 1.37 0.93 +0.01 -+0.08 _-_+-0.06_+0.07 _+0.02 6hr

0.03

0.11

0.43

0.69

-+O.Ol _+O.Ol -+O.Ol _+0.Ol The fall in QBM over 6 hours warm ischaemia closely follows the fall in high energy phosphate and glycogen content, and it confirms that the fall in QBM does indeed reflect the onset of rigor. A187

ELONGATION FACTOR 2 - ITS REGULATI-Th057 ON IN CARDIOMYOCTES BY METABOLIC SIGNALS RELEVANT FOR SEPSIS AND SHOCK Ursula Mfiller-Werdan, Christian Seliger, Christopher Reithmann, Heinz Rupp*, Karl Werdan. D e p t . o f Med. I, Klinikum Gro~hadern, University of Munich, Germany, and * Molecular Cardiology, Klinikum, University of Marburg, Germany. C e l l u l a r p r o t e i n s y n t h e s i s is d e p e n d ent on the activity of ribosomal elongation factor 2 (EF-2) . The influence of metabolic signals and mediators with pathogenetic relevance for septic cardiomyopathy on E F - 2 w a s documented in neonatal rat cardiomyocytes: Pseudomonas Exotoxin A by inactivating EF-2 inhibits the synthesis of ~-adrenoceptor molecules and myosin isoenzyme Vl, yielding an attenuation of catecholamine inotropy. Energy depletion with deoxyglucose ( 2 . 5 m M ; 48h) o r h y p o x i a (6h) r e d u c e s the content of EF-2 (to 7 6 % / 6 2 % ) . Triiodothyronine (0.03~M;48h) enhances E F - 2 (126%). B y c o n t r a s t , a n i n c r e a s e d glucose oxidation resulting from a reduced fatty acid oxidation by etomox i r ( 0 . 0 1 m M ; 4 8 h) r a i s e d E F - 2 t o 152%. These data argue also for metabolic i n f l u e n c e s o n E F - 2 in s e p s i s .

ROLE OF PROTEIN KINASE C IN c¢I-ADRENERGIC Th059 ACTIVATION OF SARCOLEMMAL Na+/H + EXCHANGE Masahiro Yasutake, William A. Coetzee & Metin Avkiran. The Rayne Institute, St Thomas' Hospital, London, U.K.

Stimulation of c¢l-adrenoceptors (AR) increases sarcolemmal Na+/H + exchanger (NHE) activity. We have studied the role of protein kinase C (PKC) in this phenomenon in adult rat ventricular myocytes, using an activator (phorbol-12-myristate-13-acatate [PMA]) and 2 inhibitors (chelerythrine [CH] or GF 109203X [GF]) of PKC. Cells (n=4-6) in HCO3--free medium were loaded with the fluorescent pH indicator carboxy-SNARF-1 and subjected to intracellular acidification by 2 consecutive transient (2-3 min) exposures to NH4CI (20 mM). Acid efflux (JH), used as an index of NHE activity, was estimated during recovery from acidosis following both NH4CI pulses (the second of which was in the presence of the c¢I-AR agonist phenylephrine [PH E] or PMA, with or without pretreatment with CH or GF) and the change in JH at pHi 6.9 (AJH) determined. AJH was 0.3+0.3 mM/min in control cells; PHE (100 pM) and PMA (100 nM) significanty increased this to 5.6:1:0.6"and 4.9-/:1.1° mM/min (°p<0.05 vs. control). The inactive stereoisorner of PMA, 4¢¢-PMA, had no effect. CH and GF reversed the increase in AJH in response to PHE and PMA in a dose-dependent manner, although the inhibitory effect of GF on PHE was not significant (Figure). Thus, PKC appears to be involved in (Zl-AR-mediated activation of NHE, although other (GF-insensitive) pathways may also contribute.

_

A188

0 10 100 GF (nM)

0 0.5 5 CH (~M)

0 10 100 GF (nM)

0 0.5 5 CH (F,M)

OF GUANINE (Gu) A N D C.UANINE Th058 NUCLEOTIDES (GuRN) IN PRIMARY RAT CARDIOMYOCYTE CULTURES Chaim Shirin, Yechezkel Sidi, ¥ael Bromberg, Oded Sperling and Esther Zoref-Shani. Dept of Clinical Biochemistry, Sackler Faculty of Medicine, Tel-Aviv University, Israel. I¢~T/LI]OLISM

The metabolic fate of labeled Gu and of prelabeled GuRN was studied in cultured rat cardiomyocytes in order to clarify the metabolic fate of Gu and GuRN. In accordance with the 3 to 4-fold higher activity of guanase in comparison to hypoxanthine-guanine PRTase, the rate of Gu deamination to xanthine exceeded that of Gu incorporation into nucleotides by 13.2-fold. The label from Gu that incorporated into nucleotides, was found mainly (81%) in GuRN, but also in IMP and adenine nucleotides (AdRN). The prelabeled GuRN lost 43 % of the label in 4 h, reflecting mainly degradation to xanthine (and uric acid) and synthesis of nucleic acids. 8-Aminoguanosine caused an almost total block of the production of xanthine, associated with a marked accumulation of label in guanosine and inosine. The results indicate that: t. Gu is a poor substrate for salvage synthesis of GuRN, and its contribution to the homeostasis of AdRN is negligible. 2. GMP is degraded to xanthine (and uric acid) through guanosine and through IMP. 3. The cardiomyocytes contain the activity of GMP reductase and of the enzymes converting IMP to GMP (guanine nucleotide cycle).

EFFECTS OF LACTATE ON RELAXATION Th060 MECHANISMS IN GUINEA-PIG CARDIAC MYOCYTES. Cesare M.N. Terracciano & Kenneth T. MacLeod. Dept. Cardiac Med, NHLI, London, SW3 6LY, U.K. The effects of intracellular acidosis produced by extracellular application of 20 mM lactate (l.,ac) on Ca extrusion mechanisms were studied in isolated ventrieular myocytes. Rapid cooling of cardiac tissue to 0*C in < ls brings about SR Ca release. Rewarming activates SR Ca ATPase and Na/Ca exchange and the cell relaxes. After 5 min of Lac superfusion indo-1 fluorescence recovery during rewarming was slower than control (C) (t50 in Lac was 78.5+6.3% of C, n= 17). In the presence of caffeine, rewarming from a RCC elicited in Lac was slower than C suggesting that Na/Ca exchange is affected by Lac (t50 in Lac was 73.1+7.1% of C, n=8). After 5 min in Lac rewarming in absence of Na. and Ca, again produced a slower decline of Indo-I fluorescence suggesting that the SR Ca ATPase is also affected (t50 in Lac was 42.4+ 10% of C, n= 12). When a second cooling period (RCC.~ is elicited, the size of contracture depends on the amount of Ca taken up by the SR during the relaxation of the first RCC (RCC,). In C RCC2/RCC, -- 0.60 4- 0.03; in Lae RCC2/RCC, = 0.38 40.05 (n=14). In the absence of Na. and Ca., RCCJRCC~ was unchanged (C = 0.954 _+ 0.017, Lae = 0.959 40.026, n =" 10) so the minor contribution of mitochondria and sarcolemmal Ca ATPase to cytosolic Ca extrusion was unchanged. In Lae the relative role of the SR Ca ATPase and Na/Ca exchange are - 38% and 58% respectively.

THE EFFECT OF HIGH GLYCOGEN ON THE ISCHAEMIC Th061 MYOCARDIUM. A CONTROVERSY RESOLVED? Heather R. Cross, Kieran Clarke, Lionel H. Opie, George K. Radda Dept of Biochemistry, University of Oxford, Oxford OXl 3QU, UK

Glycogenolysis may be beneficial during ischaemia by supplying ATP or detrimental by increasing intracellular lactate and H ÷. In this study, rat heart glycogen was elevated (.~ly) before 32 min low flow ischaemia +_glucose (iG) and 32 mm reperfusion plus glucose, aW NMR spectra were acquired sequentially throughout. Other hearts were reperfused early or in the presence of HOE 694 (a Na+/H ÷ exchange inhibitor). Functional recovery was highest in .G hearts, a condition in which contracture did not occur. In the absence of glucose during ischaemia, contracture was later and recovery lower in high glycogen hearts (--G.gly). Contracture onset occurred at the point of depletion of glycogen and cessation of glycolysis, followed by a rapid decrease in ATP and intraceUular pH. Functional recovery in +gly hearts was increased by reperfusing prior to contracture or adding HOE 694 during reperfusion. We conclude that, until glycogen depletion and contracture onset, high glycogen prevented myocardial injury. After glycogen depletion, the higher [H ÷] caused damage by increasing Na+/H ÷ exchange during reperfusion. Thus the paradoxical findings of past studies may be explained by the existence or absence of ischaemic glycogen depletion under different experimental conditions. Functional Contracture

+G -(3

-G+gly -G+gly, early rep -G+gly,+HOE694

recovery(%) onset (min) 88+35 --

52 + 7*t 14:1:5*¢ 89 + 3¢ 57 + 5~

5+1) 13 + 2 15 + 1

End i schaemia IATPImM

pH 6.60 + 0,05 6.87+ 0.02~ 6.61 + 0.03 6.83:1:0.05"~ 6.56 + 0.04

6.6 + 0.25 3.2 5: 0.3* 2.5 + 0.6° 7.5 + 0.2"I 2.2 + 0.8"

W A S H O U T O F CN D U R I N G M E T A B O L I C Th062 CONTRACTURE LEADS TO A RAPID OLIGOMYCIN-SENSITIVE ALKALOSIS Matthew K. Lancaster & Simon M Harrison, Dept. of Physiology, University of Leeds, Leeds, U.K. Changes in cell length, intracellular Ca 2+ (Ca2+i) and pH (pHi) were determined in rat ventricular myocytes during and following complete metabolic inhibition (MI; 5 mM deoxyglucose, 2 mM NaCN, 1 mM iodoacetate. After 4-5 min of MI, Ca2+i transients and contractions were abolished and the cell entered a contracture. Returning to normal Tyrode during the development of the contracture led to a rapid fall of resting Ca2+i, a rapid alkalosis and the relaxation of the contracture. This was followed by a gradual recovery of evoked contractions. When oligomycin (a blocker of the mitochondrial F1,F 0ATPase) was present throughout IVlI and washout, removal of CN did not relax the contracture, Ca2+i did not fall and pH i did not change. These results suggest that upon removal of CN, the rapid changes in PHi, Ca2+i and cell length result from the rephosphorylation of ADP via the mitochonddal FI,Fo-ATPase (ADP+Pi+H+--*ATP+H20). This reaction, which consumes protons, represents a mechanism which may induce the rapid alkalosis seen on removal of CN.

Supported by The Physiological Society.

DISSOCIATION BETWEEN RECOVERY OF Th063 MYOCARDIAL INTRACELLULAR pH (pH i) AND DEVELOPED FORCE (DF) AFTER HYPERCAPNIA. N~stor G. P~rez, Bernardo Alvarez, Marfa C. Camili6n de Hurtado, Horacio E. Cingolani. Centro de Investigaciones Cardiovasculares, 1900 La Plata, Argentina. After hypercapnia, the force of contraction of cardiac muscle initially decreases and then recovers. The decrease "is believed to be due to a decrease in responsiveness of the contractile proteins to Ca**. Since the intracellular acidosis activates the Na*/H* exchanger, a recovery of pH~ takes place. The recovery Of pHt would restore the changes in myofilament sensitivity and might explain the recovery in force. However, the activation of Na*/H ÷ exchanger increases intracellular Na ÷ that would result in an increase in intracellular Ca *÷, through the Na*/Ca** exchange. Li* can drive the Na*/H + exchanger, but not the Na*/Ca *÷ exchanger. In experiments in isometrically contracting cat papillary muscles (0.2 Hz), Na* was replaced by Li* and hypercapnia at constant extracellular pH was induced, pH~ decreased by 0.12 + 0.01 pH units and DF by 52.55 + 13.94 % (P<0.05). In spite of the full recovery in pH i, no recovery in contractility . was detected. In separate experiments in Ringer solution with 148 mM Na*, the recovery of DF was 28.1 + 1.5 % (P<0.05), and intracellular Na ÷ measured by liquid sodium-sensitive microelectrodes, showed an increase of 61.1 + 23.8 % (P<0.05). Inhibition of SR by ryanodine reduced the recovery of contractility without altering the recovery in pHt. We conclude that the recovery of contractility following hypercapnia is not due to the

Wellcome Trust and

The

M Y O C A R D I A L I N T R A C E L L U L A R pH (pHi) Th064 REGULATION IN SPONTANEOUSLY HYPERTENSIVE

RATS

(SHR).

Maria C. C a m i l i t n de H u r t a d o , B e r n a r d o Alvarez, N~stor G. P~rez & H o r a c i o E. Cingolani. Centro de I n v e s t i g a c i o n e s Cardiovasculares, 1900 La Plata, Argentina. The regulation of pH i in the hypertrophic myocardium from SHR was investigated in isometrically contracting (0.2 Hz) papillary muscles loaded with BCECF-AM. In nominally HCOz'-free media, in which the solely operative pH i regulatory mechanism is the Na*IH* exchanger, pH i was more alkaline in SHR (7.23 + 0.03, n=7) than in normotensive age-matched Wistar Kyoto rats (WKY) (7.10 + 0.03, n=6, P<0.05) indicating an enhanced Na*/H ÷ exchange activity in the myocardium of SHR. SITS inhibition (0.1 mM) did not decrease pH i in SHR (7.26 + 0.03 and 7.28 + 0.03 before and after SITS, respectively) ruling out HCOs"-dependent mechanism(s) as a possible reason for the difference in pH I, Initial velocity of pH i recovery (dpHi/dtt) from acidosis was faster in SHR than in WKY (0.055 + 0.0016 vs 0.014 + 0.0005, respectively, P< 0.05, measured at pH i 6.99). In the physiological CO~HCOs--buffer myocardial pH i of SHR (7.15 + 0.03) was not different from WKY (7.11 + 0.05). Na ÷independent HCO 3" efflux after an alkaline load was larger in myocardium from SHR than in WKY, suggesting that the Na÷-independent CIo'IHCOs'I exchanger is compensating the increased activity of Na÷/H * exchange in the hypertrophied myocardium of SHR. A189

EFFECTS O F $20787 ON PHi-REGULATING Th065 MECHANISMS IN THE RAT ISOLATED VENTRICULAR MYOCYTE D. Lagadic--Gossmarm, K. Le Prigent, *E. Scalbert & D. Feuvray.. Lab. Physiologie Cellulaire, Univ. Paris XI,

Orsay; IRIS, Courbevow, France. It is now well recoguized that pH i regulation in the heart relies on the activity of three membrane mechanisms: the Na+/H + antiport and a Na +, HCO3---dependent system, both activated following an acid load, and the CI-/HCO 3exchange, activated by an alkaline load. Whereas several specific inhibitors of Na+/H + antiport exist, it remains as yet difficult to distinguish between both HCO3--dependent mechanisms, especially around the steady-state, because of the lack of specific inhibitors. In order to find out such an inhibitor, we have tested the effects of $20787 on pH iregulation in the rat isolated ventricular myocyte. Intraeellular pH was recorded using the fluorescent probe earboxy-SNARF-l. The NH4CI (10mM)-prepulse method was used to induce an acid load in order to activate the dual acid extrusion sytem, whereas CI-/HCO 3- exchange was activated using the acetate (40mM)-prepulse method. Our results show that: i)-a high dose (5.10"°M) of $20787 did not change intraeellular buffering power, I~i; ii)-the dual acid extrusion system was unaffected by $20787, in the concentration range of 10-]L10-6M; iii)-$20787 partially inhibited (by about 50%) the activity of CI/I-ICO3- exchange in a dose-dependent manner, with an IC5o of 8.8.10-t°M. This inhibitory action of $20787 did not change the steady-state pH i . In conclusion, this study demonstrates that $20787 is a highly specific, partial inhibitor of CI'/I-ICO3- exchange in cardiac muscle.

W e studied the effect of thyroid state on p ~ in rat ventrieular myocytes and Langendorff-perfused rat hearts. To study changes in pHi in single cells, cardiac myoeytes isolated from hyperthyroid, euthyroid and hypothyroid animals were loaded with B C E C F / A M and fluorescence ratio 490/450 as a function of p h i was calculated. There was no change in BCECF fluorescence ratio between cells isolated from hypothyroid and euthyroid animals. However, a decrease in 490/450 ratio was observed in myoeytes isolated from hyperthyroid animals compared to hypothyroid (3.79 _ 0.17 ( n = 2 9 ) vs. 4.32 + 0.04 ( n = 3 0 ) respectively). In the perfused heart, pHi was measured from chemical shift of the exogenous 3~p N M R pI-I indicator 2-deoxy-D-glucose-6-phosphate. The pHi in hyperthyroid hearts was 0.14 units lower compared to hypothyroid animals. These results agreed with the direction of changes observed in BCECF loaded myoeytes. W e conclude that thyroid state changes cardiac contractility not only by an effect on Ca1.2, but also by an effect on pHi.

A P O P T O S I S AND A R R t t Y T H M I A Th067 Valeria G.Tsyplenkova, Nina N.Beskrovnova, Elena Z. Golukhova, Leo A.Bokeriya. Cardiology Research Center

VENTRICULAR ACTION POTENTIAL DURATION Th068 DURING ISCHAEMIA ON ENDOCARDIUM IN HUMANS AT LONG AND SHORT INTERVALS: RELEVANCE TO AN ARRHYTHMOGENIC MECHANISM

and Bakulev Institute of Cardiovascular surgery, Moscow, Russia Human subendocardium of 5 patients with different ventricle arrhythmias was excised from arrhythmogenic areas of the heart during the operation of cryodestruction and examined by light and electron microscopy. Light microscopy revealed focal fibrosis and development of fatty tissue in interstitium, degeneration of myoeytes and narrowed small vessels. In the majority of cardinmyocytes electron microscopy showed dilation of the sareoplasmie retieulum, lipid and lypofuscin granules accumulation in cytoplasm, mitoehondrial alterations. The hypercontrac. tion of myofibriis and dehiscence of the intercalated discs were often seen in arrhythmogenic areas. In some myocytes the mitoehondria were abnormally abundant with high electron density of matrix, remarkably pleomorphie and smaller than in majority of cardiomyoeytes. The typical signs of different stages of apoptosis (so called programmed cell death) were seen in both arrhythmogenie and non-arrhythmogenic areas of three patients. Apoptosis takes place during embriogenesis, in the course of normal tissue turnover, but it is unusual for high differentiated cardiac muscle. What causes apoptosis to begin is poorly understood, especially for the heart. Apoptnsis progression once it begins may proceed with astonishing speed, going from intact state to cell death within minutes. So the apoptosis may be an explanation of eloctrical instability of myocardium and lead to arrhytlunias.

A190

EFFECTS OF THYROID STATE ON Th066 I N T R A C E L L U L A R p H (pH0 I N R A T H E A R T S . B e a t a M . W o l s k a , A k i r a O m a c h i & R . J o h n Solaro. Dept. of Physiol. & Biophysics, U n i v . of Illinois, Chicago, USA.

Peter Taggart, Peter Sutton, Mark Boyett, Max Lab, Howard Swanton. Middlesex Hospital, Charing Cross Hospital, London and University of Leeds, UK.

Differences in action potential duration (APD) and activation time (AT) across an ischaemic border may initiate arrhythmia by local current flow. Monophasic action potentials were recorded in 14 patients (22 sites) from the right ventricular septum during angioplasty of a mid left anterior descending coronary artery lesion. A test pulse sequence with shorter and longer coupling intervals was incorporated. Under control conditions test beats with the shortest coupling interval had the shortest APDs and test beats with the longer coupling interval had a longer APD. This resulted in a rising curvilinear relationship between increasing APD and increasing diastofic interval. During ischaemia the APD dependence on cycle length was minimized thereby flattening the curve: eg, at the shortest interval shortening of APD at control 16.7 + 1.7ms compared to ischaemia 0.14 + 1.2ms, P < 0.0001 and at the long intervals shortening at control 5.5+2.0ms compared to ischaemla 0.5=1=1.0ms; P<0.05. Using best fit curves AT delay to fulfil experimental criteria for local current flow was eg, for a 40ms shortening of the steady state APD (cycle length 600ms) the AT delay required for a premature beat of cycle length 500ms to reexcite was 19.6ms: and for a premature beat with cycle length 400ms the AT delay was 1.4ms. We conclude 1) Ischaemia rapidly diminishes the cycle length dependence of APD in the human heart, 2) AT delay for a premature beat to generate a voltage gradient sufficient to reexcite across a border zone is minimised at short coupling intervals and maximised at long coupling intervals.

CYTOSKELETON MODIFIERS MODULATETh069 CARDIAC SODIUM CHANNEL GATING Victor A. Maltsev, Hani N. Sabbah, Sidney Goidstein, and Albertas I. Undrovinas. Henry Ford Heart & Vascular Institute, Detroit, Michigan, USA. The membrane environment including the attached cytoskeleton may influence ion channel function. In the present study we tested if the cytoskeleton can affect cardiac Na channel gating. We studied availability (hoo) of Na channel in rat ventricular cardiomyocytes at 2224 °C. The half-point (Via) of h.o defined as the voltage at which 50% of channels are available was monitored in the whole-cell patch-clamp configuration. In control conditions, V j,2 shifted towards hyperpolarizing potentials with a velocity of 0.37+0.20 mV/min (mean+SD, n=27). The shift was significantly faster (0.74+0.38 mV/min, n=10, p<0.01) when the cytoskeleton was disrupted by cytochalasin-D and colchicine (both 20 p.M). In contrast, with the cytoskeleton stabilizers phalloidin and taxol (100 p.M) the shift was slower (0.21+0.18 mV/min, n=13; p<0.01). These data suggest that cardiac Na channel gating is regulated by the membrane attached cytoskeleton. Any pathological factors (e.g. ischemia) that can damage the cytoskeleton may result in decreased myocardial excitability due to a reduction ifi the pool of available Na channels.

MULTIELECTRODE ENDOCARDIAL MAPPING Th071 DURING VENTRICULAR TACHYCARDIA IN SWINE Dan Ohad, Adam P. Fitzpatrick, Michael F. Smith, James Wayne, Zvi Vered, Zeev Rotstein, Thomas Cordis, David K. Swanson, Michael D. Lesh, Melvin M. Scheinman, Arnold J. Greenspon, Michael Eldar. Neufeld Cardiac Research Institute, Tel Aviv Univ., Israel, Dept. of Medicine, Univ. of California, San Francisco and Thomas Jefferson Univ. Hospital, Philadelphia, USA, EPT, Sunnyvale, CA, USA. Identification of critical areas within the ventdcular tachycardia (VT) circuit is a prerequisite for catheter ablation. Currently, mapping dudng VT, usually performed with a standard catheter carrying a small number of electrodes, is difficult ,time consuming and limited to long and hemodynamically stable tachycardias. A multyelectrode mapping catheter (MC) carrying 64 electrodes was deployed in the left ventricle of pigs which had undergone closed chest induction of myocardial infarction. Pacing at 3 mA, conceivably denoting good electrode-tissue contact, was successful in 78% of the basket catheter electrodes. Hemodynamic and echocardiographic measurements did not reveal any significant interference with myocardial or valvular function dudng or following catheter deployment. In 28 pigs, 118 episodes of VT were induced via right ventdcular stimulation and 81 were mapped and analyzed. VT mapping was rapid and required several beats and <10 seconds to complete. Presystolic potentials, a possible target for ablation, were identified in 58% of the VTs mapped. Pathologic examination revealed only minor valvular and endocardial catheter induced lesions immediately after mapping and none a month later. _Conclusion. The MC enables rapid and safe transcutaneous endocardial mapping of VT in swine. Exploration of the clinical potential of the MC seems warranted.

A ROLE OF CI-/HCO3- EXCHANGER FOR Th070 THE DEVELOPMENT OF ISCHEMIA-REPERFUSION INDUCED ARRHYTHMIAS Shingo Seki, Tatsuyuki Onodera, Satoshi Takeda, Masayuki Taniguchi, Seibu Mochizuki. Department of Medicine, Aoto Hospital, Jikei University School of Medicine, Tokyo, Japan. The roles of Na+/H÷ exchanger and CI-/HCO3- exchanger for the development of reperfusion induced ventricular arrhythmias were investigated in isolated, perfused rat heart. The hearts were perfused by the working heart mode with Krebs Henseleit bicarbonate buffer 0CHB) and whole heart ischemia was induced by one-way ball valve with 330 bpm pacing. Ischemic peffusion was continued for 15 rain followed by 20 min of reperfusion. 1.0raM amiloride (AM) treatment with KHB during ischemia decreased the incidence of reperfusion induced ventricular fibrillation (VF) from 100% to 0% (N=7). AM plus 0.1mM 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid (SITS), inhibitor of CI-/HCO3- exchanger with KHB during ischemia was less effective than AM treatment only, decreasing the incidence to 57% (n=7). When the hearts were perfused with HEPES buffer, AM treatment showed no effect on the prevention of reperfusion induced VF (100%, n=7). These findings suggest that CI-/HCO3- exchanger may be involved in the genesis of reperfusion induced arrhythmias as well as Na*/H+ exchanger.

HEART RATE VARIABILITY CHANGES IN

Th072 PATIENTS WITH ATRIAL FLUTTER Rt3ta V a l ( ~ l u l y t e , RQto S t n k e v l ( ~ l e n e , K a u n a s Medical Academy, Kaunas, Lithuania 27 pts with atdal flutter (AF) were examinated. The 500 RR intervals were recorded in the lying position (LP), dudng orthostatic test and in the standing position (SP). Heart rate (HR), it's vadability, HR responses to orthostatic test was evaluated using computedsed system. HR power spectral analysis was performed also. RESULTS: The pts with AF were divided into 3 groups: I 7pts with the regular AF in the LP and in the SP; II - 11 pts with AF in LP and with atdal fibdllation in the SP; Ifi - 9 pts with atrial fibdllaUon and atdoventdcular block i the LP and SP. The high frequency power spectrum was charactedstic in the LP and SP and very reduced HR response to orthostasis for the first group; the high frequency power spectrum in LP and not any expressed power spectrum waves in SP. More significant HR response to orthostasis was charactedstic for the second group; III 17 very often atdoventdcular block were in the LP and reduced atdoventdcular block in the SP. The high frequency power spectrum in both positions and reduced reaction to orthostatic test was charactedstic for the 3-th group. CONCLUSIONS: 1 - the high freqt~ency power spectrum was charactedstic for regular atdal flutter. 2 - for 40.7% pts the atrial fibrillation changes to atdal flutter in the standing position influenced by increased sympathic system affect;

A191

LONG-TERM PROTECTION AGAINST Th073 R E P E R F U S I O N TACHYA.RRHYTIIMIAS WITH L A C T O B A C I L L U S CULTIVATION COMPONENTS Tatyana Oxman, Babeth Rabinowitz. Heart Institute, Sheba Medical Center, Israel.

ACETYLCHOLINE INDUCED INOTROPIC Th074 RESPONSES IN HUMAN CARDIAC TISSUE Regien G. Schoemaker, Xiao Y. Du, and Pramod R. Saxena. Dept of Pharmacol, Erasmus Univ Rotterdam, The Netherlands.

Single intravenous injection of nontoxic doses of lactobacillus cultivation components (LCC) [author's "know-how"] were used in anesthetized male Sprague Dowley rats. Rectal temperature was measured before and during 30 rain after LCC injection [fluctuation was + I °C.). One, 7 or 21 days after injection the rats were anesthetized, the hearts excised and retrogradely Langendorff perfused. Fifteen rain global (G] or 30 rain regional ischemia (RI} were followed by 15 rain reperfusion. GI was produced by full cessation of perfusion, RI by occlusion of the left coronary artery. Two control groups for GI and RI were performed: 1) GI or RI without any pretreatment; 2] GI or RI after 7 days of i.v. placebo in anesthetized rats. Reperfusion tachyarrhythmia (RTA] was diagnosed as ventricular tachyeardia or fibrillation of at least 5 second duration. Results of RTA, in all hearts are shown: RI GI Day 1 7 21 1 7 21 LCC I / I 0 " 3/27* 5/29* 3/16" 7/38* 6/24* Placebo 11/17 7/10 Control 12/18 10/16 *p<0.02 vs Control and Placebo. Conclusion: LCC possesses long-term protective effect against reperfusion tachyarrhythmias in isolated rat hearts.

Acetylcholine (Ach) elicit complex inotropic responses in human cardiac tissue, including a biphasic (negative followed by positive) inotropic response in atrial, and a positive inotropic effect in ventricular tissue. The aim of this study was to characterize the receptors involved in these responses. Effects of Ach on isometric tension developed by electrically stimulated (1Hz) right atrial and left ventricular trabeculae from non-diseased human donor hearts, were evaluated in absence or presence of relatively selective antagonists (pirenzepine, M1; AFDX 1 16, M2; HHSID, M3(M1)). The negative inotropic effect in atrial, as well as the positive inotropic effect in ventricular tissue were best antagonized by AFDX 1 16 (changes in pD 2 values from 6 . 8 + 0 . 2 to 5 . 9 + 0 . 2 at 1 pM AFDX 116, n = 6 in atrial, and from 5 . 8 + 0 . 2 to 3.9±0.1 at 10 pM AFDX 1 16, n = 7 in ventricular tissue). The positive inotropic response in atrial tissue was not affected by AFDX 116 or HHSID, but depressed by pirenzepine 10 pM (from 7 8 ± 1 1 to 4 ± 3 % at 1 mM Ach). The M 1 agonist McN-A-343 could mimic the positive inotropic effects of Ach in atrial tissue, and the effect was antagonized by pirenzepine. Results indicate that the negative inotropic effect in atrial and the positive inotropic effect in ventricular tissue are mediated by M 2 receptors, though coupled to different pathways, whereas the positive inotropic effect in atrial tissue is mediated by M 1 receptors.

MAPPING AND MICROELECTRODE STUDYING Th075 OF ACETYLCHOLINE-IN DUCED TACHYARRHYTHMIAS IN THE RABBIT RIGHT ATRIUM Tania M. Vinogradova, Aleksey V. Zaitsev, Tania N. Yuzuk, Aleksandr Yu. Kaliadin, Leonid V. Rosenshtraukh. Lab of Heart Electrophysiology, Cardiology Resarch Center, Moscow, Russia.

THE NOVEL CLASS III ANTIARRHYTHMIC Th076 AMONG DICYCLOHEXYLAMIDES OF AMINOCARBONIC ACIDS N.V. Kaverina, V.V. Lyskovtsev & S.F. Sokolov. Institute o f Pharmaco]ogyRAMS. Cardiological Scientific Center RAMS, Moscow, ~,ussia

It was shown that in dogs acetyicholine (ACh) induces atrial tachyarrhythmias. The objectives of the present study were to test whether ACh (10-4 M) can induce spontaneous tachyarrhythmias in the preparation of rabbit right atdum and to make insight into the mechanisms underlying these arrhythmias. From total of 54 infusions 35 induced different types of tachyarrhythmia. We used 64-channel mapping set-up to determine the localization of ACh-induced tachyarrhythmia. The mapping demonstrated uni-, sometimes multifocal activation patterns with only few cases of transition to the reentrant pattern. The focal sites were clustered in the sinus node (SN) region and between inferior vena cava (IVC) and crista terminalis. Intracellular recordings in these areas revealed that ACh induced marked heterogeneity in activity of cells with slow diastolic depolarization. The action potential (AP) duration of some fibers reduced to <10% of the control value while AP duration of the others remained unchanged. Under ACh some of cells stopped generating AP, namely, the AP amplitude fell below 30% of the control value, though the preparation retained regular or irregular rhythm. Focal patterns observed during tachycardias may be a result of micro-reentry induced by nonuniform refractoriness and excitability in the SN and IVC regions. A192

In studies of the Institute of Pharmacology RAMS performed jointly with AWD (Germany) tSere were synthesised and investigated the new compounds, aricyclohexylamides of aminicarbonic acids with a pronounced antiarrhythmic mode of action. Programmed myocardium stimulation was used in anesthetized dogstaken f o r testing 3-5 days after the experimental myocardial infarction. The compound AWD 160-275 at doses of 1.0, 2.5 and 5.0 mg/kg i.v. was found to prolonc~ ventricular repolarisation, to increase, not less intensively than sotalol, the e f f e c t i v e atrium and ventricular refractory periods and demonstrated a high antiarrhythmic and antifibrillatory activity. Unlike sotalol, AWD 160-275 does not inhibit the function of the sinus node and produces a slight e f f e c t on conduction rate in A-V node. The reported properties evidently identify AWD 160-275 as class III antiarrhythmic compound.

NIBENTAN IS A NEW TYPICAL CLASS III Th077 ANTIARRHYTHMIC AGENT. Alexei Ed.Tararak, Irina N.Merkulova, Mikhail Ya.Ruda. Cardiology Research Centre, Moscow, Russia. Tbe effects of Nibentan (1 phenyl-l-(paranitrobenzoilamine)-5diethylaminopentan hydrocloride) a new class II1 antiarrhyflmfic agent (AA) on ECG parameters were evaluated in 68 patients (pts) with arrhythnfias (age 48,9±8,29 yrs, 58 men). 49 pts had organic heart diseases (NYHA 0-I). The dosage schedule of Nibentan (N) included 5-min boluses (1,2 or 3) injected i.v. at 15 min interval for achievement of antiarrhythmic effect or loading of proposed dose (0,125-0,5 mg/kg). Results: N showed some actiotts that were evident for class Ill AAs (see table): 1) N prolonged significant QTc interval but had no effect on P, PQ and QRS duration in normal sinus cycle length (SCL) because the drug increased repolarization phase of action potential duration (APD) by potassium-channel blockade and did not i,lfluence on depolarization. Appearance of additional "U" waves in 95% of pts also related to this action and possibly reflected early afterdepolarization. 2) N- induced reverse rate dependence of QTc prolongation was particularly evident in beats occurring after compensatory pauses.3) N increased SCL,probably, by lengthening of sinus node APD. 4) N produced rate dependent appearance of aberrant intraventricular conduction due to different APD prolongation of distal parts of conduction system and myocardium. This effect was found in 100% of pts with supraveqtricular arrhytlunias (n=38). P, ms PQ, ms QRS,ms QT(U)c,ms SCL,ms Before N 109+17 169_+.21 9 0 + 1 1 433_+.24 769+956 60'after N 112+17 169+-21 9 1 + 1 3 557:t23" 968+_218" M+SD *-p<0.0001 vs baseline level Conclusion: N in doses of 0.125-0.5 mg/kg i.e. showed clinical effects which were evident for actions of class III antiarrhythmic agents.

DOES A M I O D A R O N E HAVE A FREQUENCY- Th079 DEPENDENT POSITIVE INOTROPY IN VIVO? Martin E. Beyer, Hans Martin Hoffmeister & Ludger Seipel. Medical Dept. Ill, University of Tiibingen, Germany The positive inotropic effect of class-III antiarrhythmic drugs in vitro is caused by prolongation of the action potential duration (APD). As a frequency-dependence of the APD-prulongation ("reverse use-dependence") was described for class-III drugs a hea~ rate reduction after administration of these drugs should have a positive effect on myocardial contractility. We examined the hemodynamic and inotropic effects of amiodarune (10 and 20 mg/kg i.e.) without and with bradycardia in thoracotomized rats vs. saline controls. Heart rate reduction was caused by vagal stimulation (reduction about 50%). Additionally to measurements in the intact circulation isovolumic registrations (isovol. LVSP and isovol. @/&max) were performed to determine myocardial contractility. spontaneous heart rate vagal stimulation 10mg/kg 20mg/kg N a C I 20mg/kg NaCI isovol. LVSP 934-2 88+1" 98+1 80+5* 100+2 isovol, dp/dtmax 814-3" 734-3* 944-3 544-5* 824-4 AOPme~ 764-5* 674-4* 964-2 584-4 684-4 CO 904-7 754-7 934-8 574-6 68+4 Means 4- SEM in % of preinfusion values, *p<0.01 CONCLUSTON: The prolongation of the APD by the class-Ill antiarrhythmic drug amiodarone does not cause in vivo a positive inotropic effect as the cardiodepressive properties of the drug (e.g. sodium- and calcium-channel blockade) are stronger. A reverse use-dependence of the inotropism of this class-HI antiarrhythmie drug is not detectable in vivo.

DOFETILIDE - A NEW CLASS-III ANTITh078 ARRHYTHMIC DRUG WITH POSITIVE INOTROPIC EFFECTS IN VIVO? Martin E. Beyer, Hans Martin Hoffmeister & Ludger Seipel. Medical Dept. llI, University of Ttibingen, Germany Prolongation of the action potential duration by class-Ill antiarrhythmic drugs causes in vitro a positive inotropic effect. Our previous in vivo studies could not show such a positive inotropy of the class-Ill drugs amiodarone and D-sotalol. Such a positive inotropy would be an advantage to other antiarrhythmic drugs (e.g. class-I drugs) with cardiodepressive properties. The dose-dependent effects of the new class-Ill antiarrhythmic drug dofetilide (20, 40, 80/~g/kg i.v. vs. NaCI-controls) were examined in the previously described in vivo model (openchest rats). Besides measurements in the intact circulation the isovolumic left ventricular pressure generating capacity (isovol. LVSP, isovol, dp/dtmax) was determined for quantification of myocardial contractility. dofetilide NaCI 20p.g/kg 40/Lg/kg 80#g/kg heart rate 102+4 107+4 99+6 99__+ 1 cardiac output 1174-6" 123+5 N 110+4 100+6 isovol. LVSP 99+ 1 1004-2 974-2 984- 1 isovol, dp/dtma x 994-2 105_+5" 974-3 93+2 Means+SEM in % of preinfusion values, *p<0.05, Yp<0.01 CONCLUSION: The new class-Ill antiarrhythmic drug dofetilide has no cardiodepressive effect in vivo. 40 ,ug/kg dofetilide even elevates significantly cardiac output and isovol, dp/dtma x. Especially in patients with reduced left ventricular function this may be an advantage to other antiarrhythmic drugs.

SL 87.0495, A NEW Ca~* ANTAGONIST WITH Th080 MINOR EFFECTS ON AV NODAL CONDUCTION Alaln Grosset, Josette Sabatler, Alain Villeneuve, Etienne Gautier, Thierry Pouyet, Pierre Lain~ & Stephen E. O'Connor, Synth~labo Recherche, 31 avenue P.V. Couturier B.P. 110, 92225 Bagneux Cedex, France. SL 87.0495 is a new calcium channel blocker unrelated structurally to the dihydropyridines, phenylalkylamines and benzothiazepines which acts at the benzothiazepine recognition site (Abst. EPHAR'95, Milan). We have compared the cardiovascular profiles of SL 87.0495 and diltiazem in anaesthetised and conscious dogs. Anaesthetised open-chest dogs were instrumented to monitor haemodynamic parameters and the ECG (PR interval as index of atrio-ventricular [AV] conduction). Diltiazem and SL 87.0495 were given as 30 sac i.e. ascending doses (0.01 to 1.0 mg/kg) at 20 min intervals. Both compounds dose-dependentlyloweredthe heart rate, arterial and left ventricular pressures, with concomitant increases in aortic blood flow and stroke volume. SL 87.0495 was as potent as diltiazem to decrease the coronary vascular resistance, ~ = at 0.3 mg/kg: -61 + 2 % and -60 + 4 % raspectively. At this dose total peripheral resistances decreased by -52 + 4 % and -37 4. 4 % respectively. PR intervals at 0.3 mg/kg were unchanged by SL 87.0495 (96 + 6 ms, initial value: 85 + 4 ms) and increased (p<0.05) with diltiazem (142 + 20 ms, initial value: 85 4- 7 ms). Diltiazem (1 mg/kg i.v.) induced second-dogree AV block in this model, these arrhythmias were not elicited by high doses of SL 87.0495. Coronary vasodilation without slowing of AV conduction after SL 87.0495 (doses as high as 1 mg/kg i.v. and 6 mg/kg p.o.) was confirmed in conscious dogs. Hence, SL 87.0495 is better tolerated on AV conduction than diltiazem (typos I and II AV block) with potentially similar anti-anginal properties.

A193

EFFECT OF LEVOSIMENDAN ON ATRIAL Th081 AND VENTRICULAR IMPULSE GENERATION IN DOGS WITH ARTIFICIAL ATRIO-VENTRICULAR BLOCK l~va Udvary, Julius Gy. Papp, Agnes V6gh. Department of Pharmacology, Albert Szent-Gy6rgyi Medical University, Szeged, Hungary Levosimendan is a novel calcium sensitizer inodilator with some PDE III inhibitory activity. It was also found to reduce severe ventricular arrhythmias in anaesthetized dogs subjected to coronary artery occlusion. In order to analyse the possible antiarrhythmic action of levosimendan, the present study was designed to investigate the effects of the drug on the cardiac nomotopic and heterotopic impulse generation following artificial atrio-ventricular (AV) block in anaesthetized, thoracotomized dogs. Blockade of the AV conduction was induced by injection of 40 % formaldehyde around the AV node. Changes in atrial rate were recorded by means of a bipolar electrode clamped on the right auricle. Standard limb lead If ECG was used for recording alterations in ventricular rate. Blockade of the AV nodal conduction resulted in a significant decrease in the arterial blood pressure (28 %), a slight reduction in the atrial (6 %) and a marked decrease in the ventricular (59 %) rate. Increasing doses of levosimendan (from 0.03 to 0.3 ~mol kg1) elevated the atrial rate dose-dependently (15 %, 28 % and 39 %), whereas the ventricular rate and rhythm remained virtually unchanged. It is concluded that although levosimendan possesses some PDE Ill inhibitory activity which could result in a dangerous enhancement of the ventricular ectopic impulse generation, doses of the drug which produce positive inotropic effect do not exhibit ventrieular arrhythmogenic potential.

K~rmCTS OF TAICATOXINON 'IIBEELECTROTh083 PHYSIOLOGICAL, MECHANICAL ANDBIOCHEMICAL C H A R A u w ~ S T I C S OF ISOLATED RATCARDIOMYOCYTES ElisabethFantini, PierreAthias, Regine Tirosh I & Ari6 Pinson I - D~p. d'Electrophysiologie, Laboratoirede Physiologie, Facult~ deM~dicine, Dijon, FRANCE and 1Laboratory for Myocardial Research, The Hebrew gniversity-Hadassah Medical school, Jerusalem, ISRAEL.

A194

TaiCatoxin (TCX), a complex toxin isolated from Taipen snake venom, is believed to specifically block the voltage-dependent cardiac calcium channels. The aim of this study was to investigate the effect of TCX on a broad range of heart muscle cell functions and the related biochemical modifications. TCX decreased the amplitude of the action potential, mainly by lowering the plateau. The action potential duration and the contraction amplitude were also decreased. Although TCX had a minor overall chronic effect, this peptide toxin evoked severe, transient arrhythmias and induced sustained alteration of the intercellular electrical coupling. These effects are consistent with specific blockade of the L-type, voltage-dependent calcium channels. TCX also exhibits phospholipase A 2 activity, leading to release of lysophospholipid and FFA (acyl CoA and acyl carnitine), which have detrimental effects on cellular integrity and function.

CONTROL OF VENTRICULAR RATE Th082 D U R I N G A T R I A L F L U T T E R BY V E R A P A M I L , AMIODARONE, DIGOXIN AND DILTIAZEM H e l m u t A. T r i t t h a r t , Ulrike S t a r k , G e r h a r d S t a r k , Karin Kasper, Ingrid Schwarzl. Department of M e d i c a l P h y s i c s a n d Biophysics, G r a z , Austria. A lot o f substances, prolonging AV-nodal conduction and refractoriness were used to control ventricular rate during atrial flutter. In a newly developed atrial flutter model on Langendorff perfused guinea-pig hearts, we studied the influence o f verapamil (0.01 p.M), amiodarone (10p.M), digoxin (0.6nM) and diltiazem (0.03p.M) on a frequency histogram o f the ventricular cycle length (VCL) during rapid atrial pacing ( S l - S l = 5 0 m s ) . The shape o f the histogram during this pacing cycle length, resembles very well the histogram during spontaneous atrial flutter. All four drugs induced a comparable depression o f AV-nodal conduction during spontaneous sinus rate. They also reduced significantly the mean, minimal and maximal V C L in the atrial flutter model. But the overall prolongation o f V C L was less pronounced by dilitazem compared to the other substances, which corresponds to its short time constant (x). Therefore a short time constant, which characterises a fast association and dissociation kinetic o f a c o m p o u n d to the calcium channel, results also in a reduced m e m o r y effect from one AV-nodal activity to the other.

DOES SINGLE LEAD QT INTERVAL REFLECT LOCALTh084 ENDOCARDIAL REPOLARISATION 1N MAN? Suzanna MC Ilardman, Peter Taggart, Peter MI Sutton, Martin R Cowie, J Malcobn Walker.

University College London Hospitals & Medical School, UK. Measurement of QT interval dispersion provides a non invasive measure of global dispersion of repolarisation. However, the extent to which QT interval in a single precordial lead represents local repolarisation has not been explored. We therefore compared precordial QT intervals with endocardial action potential durations (APD) recorded from five patients following diagnostic cardiac catheterisation. Monophasic action potentials (MAPs) were recorded from a sequence of left ventricular endocardial sites spanning apex to base, during right atrial pacing. A roving procordial ECG lead was positioned as close as possible to each endocardial MAP recording site to allow QT measurement from the same area of myocardium. APD was measured at 100% repolarisation. APD & QT interval were corrected for cycle length (after Bazett) and normalised to mean values for each patient. The dispersion of APD between recordings sites was 60.3 + 5.8ms and of QT interval 46.3 + 5.9ms (mean + SEM). The regional variability in APD was mirrored by local QT measurements. ~ 1 ~ 1 ° ° 1,~ •° ••5• •e ~,

r2= 0.93, p < 0.0001 95 90

95

10o 105 Iio APDc % The strong correlation obtained between precordial QT interval and endocardial action potential duration recorded from corresponding left ventricular regions suggests QT interval in a single precordial lead does reflect local repolarisation. These findings may have clinical application in the management of arrhythmias.

THE INFLUENCE OF DC C A R D I O V E R S I O N Th085 ON SELECTED FUNCTIONS OF P O L Y M O R P H O NUCLEAR N E U T R O P H I L S (PMN) - M O D I F I C A T I O N BY DEXTRAN INFUSION

Yuliusz Smielecki, Andrzej Wykretowicz, Henryk Wysockl, Andrzej Minc~kowski, Mirosla~v Kazmierczak; DepL of Intensive Therapy, University School of Medicine, Poznan, Poland

CONTRIBUTION OF MECHANO-ELECTRICAI. FEEDBACK TO RESPIRATORY SINUS ARRHYTHMIA

Th086

Hax J.Lab, Zuzana Vespalcov&, Arun Holden. Dept. of P h y s i o l o g y , Charing Cross and Westminster Medical School, St. Dunstan's Rd., W6 8RF, London, U.K. R e s p i r a t o r y sinus a r r h y t h m i a ( h e a r t changes related to respiration)

rate has

Hydrogen peroxide (I-I202) and superoxide anion (02 ) have arrhythmogenic effects and these both toxic oxygen metabolites play a major role in endothelial cell injury. PMN activation is accompanied by the increased production of thcsc toxic oxygen mctabelites. PMN activation leeds also to cell aggregation and microembolization. It was hypothetised that DC cardioversion can cause PMN activation. To test this hypothesis we evaluated the influence of DC cardioversion in patients with atrial flutter or fibrillation on P/VIN autoaggregation as well as unstimulated and stimulated superoxide anion production. We also evaluated plasma hydrogen peroxide and malonyldialdehyde (IvlDA) levels. Blood was obtained from 20 patients before and in 10 minute after DC cardioversion. The obtained data indicate that PMN obtained after eerdioversion showed increased autoaggregation as well as unstimulated and stimulated superoxide anion production. Also hydrogen peroxide and MDA plasma levels increased after DC cardioversion. In the second group of 6 patients we infused 250 ml of dextran 40 solution just before DC cardioversion. Resets obtained in these patients showed that dextran 40 diminished PMN aggregation and Of production while H:O2 and MDA plasma levels did not changed after DC cardioversion. Obtained results indicate that PMN activation during cardioversion may constitute an additional mechanism responsible for complications observed during this procedure and that dextran 40 infusion may reduce these negative events.

traditionally been attributed to autonomic nerve activity on the cardiac pacemaker. We suggest that there is an intrinsic mechanism, mechano-electrical feedback, independent of autonomic reflexes also involved. We measured changes in the heart rate respiration cycle in artificially ventilated, deeply anaesthetised pigs. We used serial correlograms, deviation from local mean and average frequency method to relate the changes in the heart rate to the ventilation cycle. Two observations support our hypothesis: a) sinus arrhythmia was detected in hearts with complete autonomic blockade, and b) the periodic changes in the heart rate disappear when the sinoatrial node is fixed to suppress mechanical changes of the tissue evoked by respiration. We suggest that mechano-electrieal feedback due to cyclic stretch of the pacemaker cells during respiration contributes to respiratory sinus arrhythmia.

ANTIARRHYTHMIC AND ELECTROPHYSIOTh087 LOGICAL EFFECTS OF CERULOPLASMIN IN ISOLATED RAT HEARTS. Roxana Atanasiu, Marie-Josde Dumoulin, Ramez Chahine, Mircea Ao Mateescu and Rdginald Nadeau. HSpital du Sacr6-Coeur de Montrdal and Universitd de Montrdal, Montrdal, QC, Canada

CARDIOVASCULAR CHANGES DURING SUDDEN Th088 ASCENT TO THE MIDDLE HIGH ALTITUDE IN MEN OVER FIFI'Y YEARS Stafan Kujanik, Mad~n Sninc~k, Karolina Galajdov&, Kadn Rackov~, Eva Zubajov~ - ~vecov~. Dept of Physiology, Faculty of Medicine, ~af~dk University, Dept of Internal Medicine, Military Hospital, Ko~,ice,SIovakia.

The ability of ceruloplasmin (CP), an important serum antioxidant, to reduce the incidence of reperfusioninduced arrhythmias has been investigated. Isolated rat hearts were submitted to 1 5 min of regional ischemia and 10 min of reperfusion. Ischemic hearts were treated with CP at different concentrations, 5 min before reperfusion. As negative controls we used heatdenatured CP and albumin. The incidence of ventricular fibrillation was significantly decreased by CP in a concentration-dependent manner. Heat-denatured CP and albumin had no anti-arrhythmic effects. Furthermore, we checked whether CP can alter left ventricular effective refractory periods (ERP) and conduction time (CT) during ventricular pacing. The ERP and CT were measured in control hearts and in hearts treated for 1 5 min with different concentrations of CP, SOD, catalase, deferoxamine and albumin. Ceruloplasmin increased significantly the ERP in a concentration-dependent manner and did not alter CT. SOD, catalase, deferoxamine and albumin had no effect on ERP and CT. The CP-induced prolongation of ERP might explain its antiarrhythmic effects at reperfusion, in isolated rat hearts.

In 20 healthy men (50-64 years) permanently living at the sea level under 8 0 0 metres the occurence of supraventdcular (SVPB), ventdcular (VEB) extrasystoles, systolic, diastolic blood pressures, and heart rate was studied during cable cabin transportation by Holter ECG monitoring (Memoport C Hellige). Comparisons were performed at stations 898 m, 1752 m, 2632 m in both directions. Our records show that values of the blood pressure and pulse rate were within normal limits during the whole period of transportation. Both SVPB and VEB show a sudden increase with increasing sea level and decrease to initial values during the descent compared with values in 898 m.The highest values were observed at the top station (VEB were 6 times more frequent, SVPB not present at 898 m were at the top 7 times more frequent than at 1752 m). The results demonstrate that healthy men in higher dacennia show the intensive increase in SVPB and VEB during the sudden ascent to the middle high altitude but the blood pressure and heart rate do not exceed the normal limits. Although number of extrasystoles is still within normal limits related to that age, a similar sudden ascent in persons with other cardiovascular risks (stress, hypoxia, alterations in blood mineral levels, etc) may be dangerous. A195

SUPPRESSION OF REPERFUSION Th089 ARRHYTHMIAS BY INHIBITION OF RELEASE OF INS(1,4,5)P 3 Xiao-Jun Du, Karen E. Anderson, Alexander N. Jacobsen, Anthony M. Dart & Elizabeth A. Woodcock Baker Medical Research Institute, Melbourne, Australia.

DIMETHYLAMILORIDE FACILITATES Th090 VENTRICULAR FIBRILLATION IN AN IN-SITU PORCINE MODEL OF REGIONAL MYOCARDIAL ISCHEMIA William T. Smith, Hua Yang, Barbara J. M011er-Borer, Connie L. Engle, Timothy A. Johnson, John J. Lemasters & Wayne E. Cascio. Depts of Medicine and Cell Biology, University of North Carolina, Chapel Hill, NC, USA.

Reperfusion following myocardial ischaemia causes a rapid and transient release of inositol(1,4,5)trisphosphate (Ins(l,4,5)P3) (Anderson et al., Circ Res 76: 261-268, 1995). The current study examined anti-arrhythmic effects of compounds which inhibit release of Ins(l,4,5)P 3. Ventricular tachycardia (VT) and fibrillation (VF) as well as Ins(1,4,5)P 3 content were measured during early reperfusion following 20 min ischaemia, in perfused rat hearts. A number of different compounds including the aminoglycosides neomycin (0.5-5 mM), streptomycin (0.015-1.5 raM), gentamicin (0.015-1.5 mM), the polyamine spermine (5 mM), prazosin (1 IxM) and pertussis toxin (25 mg/Kg IV 48 h pretreatment) were effective in inhibiting the reperfusion-induced release of Ins(l,4,5)P 3 and reperfusion arrhythmias. There were strong correlations between Ins(l,4,5)P 3 content at 2 min reperfusion and both %VT (r=0.71) and %VF (r=0.83). In addition, intravenous gentamicin (2.5-10 mg/Kg/min) suppressed the onset of arrhythmias under ischaemic and reperfusion conditions in vivo. We conclude that agents which inhibit Ins(l,4,5)P 3 release are anti-arrhythmic and form a new class of anti-arrhythmic agent.

Ventricular activation time (AT) and arrhythmias were measured during 23 short (8 min) LAD artery occlusions in 5 pig hearts. Blood and drugs were infused via a carotid to LAD shunt. VF occurred in 2/14 control (C) ischemic episodes, 6/6 with dimethyl amiloride (DMA, 50pM), and 0/3 treated with DMA and Verapamil (8pg/ml, DMA+V). Compared to C, DMA increased AT more and facilitated VF during ischemia. In order to study the mechanism of VF, changes of conduction velocity (e), passive cable-like properties, action potential characteristics and extracellular K (Ke) were measured in arterially perfused rabbit papillary muscles during ischemia without drug (n=7), with DMA (501JM, n=9), and with DMA+V (1uM, n=2). Intracellular and extracellular voltages recorded during subthreshold and threshold current pulses were used to determine intracellular resistance and e. Ke was measured with a K-selective electrode. During the first 12 min of ischemia, in 7 control experiments e <10 cm/s was not observed prior to block. By contrast, e slowed more rapidly to <10 cm/s (P=0.01) by12 min in 7/9 with DMA. Onset of slow conduction was not attributed to differences in Ke or coupling. V prevented DMA facilitated slow conduction during ischemia in the rabbit. Suppression of DMA facilitated VF and slow conduction by V suggests that the mechanism of this DMA induced VF is related to the slow inward current.

T H E H Y P O X E M I C T E S T E V A L U A T E D Th091 BY LATE P O T E N T I A L S AND H E A R T RATE VARIABILITY M. Janota, J. Bytegnik, M. Vrhna, V. Stan~k Department o f Cardiology, Institute for Clinical and Experimental Medicine, Prague, Czech Republic

I N C R E A S E D V A R I A B I L I T Y O F REPOLARIZATION INTERVALS IN PATIENTS WITH S P O N T A N E O U S

The hypoxemic test (HX), producing changes in the 12-Iead ecg and precordial body surface map, is rel~arded as an alternative to exercise testing m the iaiagnosis of myocardial ischemia. We used HX for late potenlaals (LP) evaluation to assess excitation conduction in the heart as well as for heart rate variability (RR variability) determination to assess me degree ot stress and neurovegetative readiness. Our patient group included 22 subjects with ischemic heart disease and an g~ga pectoris with LP determination. (Group I), and _8 patients with RR variability determination (Group II). HX was performed by inhaling a mixture of 10% O2 in 90% N2 flora a gas bottle through a mouthpiece for 5 minutes. HX-LP did not provide consistent results. HX-RR variability m the group with a mean square successive difference of the RR vfiriability (MSSD) lower than 500 ms - hence also an ificreased risk for arrhythmias - led to a significant decrease in the RR variability. compared to the low-risk group. We recommen~l to use HX-RR variability hi clinical practice when determinin"g We risk of stratified patients and selecting future merapy. A196

Th092

VENTRICULARTACHYCARDIA Oal Bayam, Shlomo A. Ben-Baim, Malka Yahalom, 1 T a m a r Harel, Guy Dori, Nathan Roguin, I & Kelley P. A n d e r s o n 2 for the ESVEM Investigators. B r u c e Rappaport Faculty of M e d i c i n e , T e c h n i o n , Haifa, Israel; IICCU and Heart Inst., Western Galilee Regional Hospital, Nahariya, Israel; 2Univ. of Utah, Salt Lake City, UT, USA. Cardiac repolarization duration is dependent on cycle length (RR) and autonomic nervous system tone. We studied 20 patients with repeated episodes of spontaneous monomorphic ventricular tachycardia (VT), i0 post-myocardial infarction (MI) patients without documented VT, and I0 healthy control subjects. We analyzed the Holter recordings prior to the VT when appropriate. The power of RR spectra in VT and post-MI patients and the control group was not significantly different: 78.81±21.17 (SD), 55.91±13.99 and 42.43±7.73 ms2/Hz, respectively. However, the total power of RT spectra was significantly increased in VT patients: 3.42±0.67 vs. 0.22±0.55 ms2/Hz (VT vs. post-MI, p<0.0001). There was no difference between the post-MI patients and the control group: 0.22±0.55 vs. 0.61±0.23 ms2/Hz. It is suggested that RT interval variability measurements with power spectral techniques might help to discriminate those patients who are prone to VT.

TIME COURSE OF THE USE-DEPENDENT DECAY Th093 OF EFFICACY OF D,L-SOTALOL AT HIGHER HEART RATE. Juraj Melichercik, Wolfgang Schoels, Thorsten Beyer, Thomas Hilbel, Mark W. F. Schweizer, Patricia Kraft, Johannes Brachmann. Dept. of Cardiology, University of Heidelberg, Germany. Use-dependency is a well known parameter that affects the efficacy of antiarrhythmic drugs (AAD). Little is known about the time course of change of the rate-dependent changes in efficacy of AAD. To assess this parameter, monophasic action potential duration (MAPD) was recorded during constant pacing (steady state) at a cycle length (CL) of 700 ms and after abrupt changes in CI to 550, 400, 330ms in 7 Pts undergoing routine electrophysiologic study before and after d,l-sotalol (1,Smg/kg iv.) Results are expressed as mean_+_SD.A MAPD = % change in MAPD at 90% of repolarisation after d,i-sotalol measured at the 1 to 200 beats after step increase in heart rate. EEL AMAPD(%) AMAPD(%) AMAPD(%) (ms) PCL 700 ms 1 beat 20 beats 700-550 15t2-~4r4 15r?:t:4r7 15~1+4tl 700-400 15,1+4fl 14,8+5,8 14,0+_2,5 700-330 15,4:1:4,1 12,8+4,1 12,6:t2,7 50 beats 100 beats 200 beats 700-550 14r9-J:3,5 1 3 , 7 ~ 3 t 7 12fl-~3fl 700-400 13rS'x2,1 lltl:f.2.1 9t2.+.2,2 700-330 llt6:L2,0 8~6+1t~ 6~0"/:1t6 Conclusions : D,I-sotalol exhibits favorable time-dependent effect on the MAPD prolongation in the right ventricle after abrupt increase in heart rates. This might preserve its efficacy in ventricular tachyarrhythmias even at higher rates.

DISYS®DATA ACQUISITION SOFTWARE IN

Th095

CARDIAC CELLULAR ELECTROPHYSlOLOGY

F.Bart~k, L.Bolek and T.Mr~zekt. Dept. of Physiology and Biophysics, Med. Faculty, Charles Univ., Plzefi, MERLIN Ltd. t, Prague, Czech Republic,. The new computer data acquisition system for application in cardiac cellular electrephysiology is described. The system is based on the DISYS® software (MERLIN Ltd.) running under WindowsTM and usinf, new virtual driver technology for real time data acquisition. The conventional 486DX2 personal computer equiped with low cost PCL812PG A/D interface card (ADVANTECH Ltd.) is sufficient hardware. The A/D card is conected with a newly developed laboratory interface unit to exclude problems with its connections with other laboratory devices and to prevent electrical ovedoading of the computer circuits. Important features of this user friendly and powerfull laboratory system are as follows: • Real time signal processing and display. • Unlimited length of signal data files (up to the storage capacity of the hard disk). • Modular design. • Customization using Plug-in Librades • Experiment automation by Scripts ;. Export-import capabilities for sharing experimental data with other Windows applications.

P R O A R R H Y T H M I C EFFECT O F GLG-V-13 Th094 IN A RABBIT M O D E L O F THE ACQUIRED LONG QT SYNDROME T a m ~ Fazekas a, Leif Carlsson b, Benjamin J. Scherlag c, Csaba Lengyel a, A n d r ~ Varr6 d, Julius Gy~ Papp ~l, Ralph Lazzara c. a l s t Dept. of Medicine, aDept, of Pharmacology, ~zent-GySrgyi Medical University, Szeged/Hungary; "Cardiovascular Pharmacology, Astra H~ssle, M61ndal/Sweden; CDept. of Medicine, The University of Oklahoma, Oklahoma City, OK/USA The proarrhythmic potential of a novel potassium channel (Ikr) blocking antiarrhythmic compound GLG-V-13 was studied in an animal model of the acquired long QT syndrome. Anaesthetized rabbits (n=8) were given a concomitant infusion of the al-agonist methoxamine and GLG-V-13. The latter compound produced a significant lengthening of the QTU interval and reduction in heart rate. It increased the QTUe interval from 125+3.0 ms to 161+6.2 ms (p<0.001) after a cumulative dose of 0.84 /Jmol/kg, caused ventricular extrasystoles in all eight rabbits at a dose of 3.9+1.13/~mol/kg, and in five of the animals the premature beats turned into "torsades de pointes" VT at a dose of 5.2+3.24 /~mol/kg. Compared to almokalant and dofetilide, GLG-V-13 showed a tendency towards a lower, but still marked proarrhythmic potential. The proarrhythmic feature of GLG-V-13 was also obvious from our recent observations made in isolated canine Purkinje fibers stimulated at a low rate (0.2 Hz). Here again, in the presence of GLG-V-13 (18.5 gmol/I), early after-depolarizations developed. It is concluded that, irrespective of the chemical nature of the compound applied, blockade of the cardiac Ikr potassium channel is usually associated with proarrhythmic effect.

CARDIOANAPHYLAXIS IN TRICHINELLOSIS Th096 Nazarene Paolocci*, Slawomir Michalak, Marco Bettini*, Isabel Castillo Romera, Franen Magni* & Fabrizio Bruschi. lstitato di Patologia Generale, *Cattedra di Fisiologia Umana, Universit~ degli Studi di Pemgia, Pemgia, Italy Myocarditis and arrhythmias during trichinellosis are far from being clarified. We studied histological modifications in the myocardium of rats orally infected with 3.000 Trichinella spiralis muscle larvae, with particular reference to the presence and state of mast cells, the degranulation products of which, such as PAF, can induce arrhythmias in normal animals. Mast cells were evaluated for distribution (parivascular or not) and degranulation in heart sections stained with toluidine blue at 21 and 50 days of infection (d.i.). A second set of hearts was Langandorff pcffused to evaluate the response to a single bolus (100 pM) of PAT, given after 50 minutes of parfusion. Coronary flow (CF), ESLVP and ECG were monitored. No difference in the distribution of mast cells between infected animals and controls was observed, however mast cells were much more dogranulatod in 21d.i. than in 50d.i. or control animals (67, 23 and 20% respectively). PAl; injection during parfusion induced a negative inotropic effect in 21d.i. animals (up to -60%), whereas PAT increased ESLVP in control and 50d.i. groups. CF decreased during stabilization in 21 d.i. group to decrease further after the PAF bolus. This occurred in controls and 50d.i hearts, too, but at 21d.i. not only perivascular mast cell degranulation was more important but also irreversible arrhythmias are more frequent and blood and myocar~al eosinophil levels reach the peak. In conclusion the rhythm disturbances observod also in human trichinellosis could be due to a cardioanaphylasis phenomenon. This work was in part supported by the Italian MURST.

A197

LONG QT SYNDROME: PHENOTYPIC AND Th097 GENOTYPIC DIAGNOSIS Paacale Gulcheney, Erlc Dauase, Isabelle DenJoy*, Mohammed Bennaceur, Mlchel Komjade, Ketty Schwartz, Phlllppe Coumel*

INSERM UR153, Groupe hospitalier Piti~-Salp~tri~re " Service de Cardiologie, H6pital Lariboisi~re, Paris, France Long QT syndrome (LQTS) is an autosomal dominant disease leading to syncopes and sudden death. While the diagnosis is obvious in patients with the most characteristic presentation, it is difficult in asymptomatic subjects with minor QT abnormalities on the ECG who are nevertheless at risk of sudden cardiac arrest. Three genetic loci have been identified on chromosomes 1lp15.5 (LQT1), 7q35-36 (LQT2) and 3p21-24 (LQT3). We attempted to refine diagnostic criteria by using the recent tools provided by molecular genetics to determine in French families the disease gene carders. The affected subjects were defined according to the following criteria: QTc> 460ms; or QTc > 440ms associated with bradycardia or abnormal T wave; or syncopes under stress with family history. The disease locus was on chromosome 11 for eight families, but the phenotypes were variable. Asymptomatic carriers were found in all families. On the basis of the genetic data, we found that the mean computed daytime QTc value determined from Holter recordings gave a better evaluation of QTc prolongation than ECG, and was a powerful indicator of the disease. We therefore propose genotyping combined with daytime QTc evaluation as new tools to diagnose LQT syndrome for which effective treatment exists.

TREATMENT OF VENTRICULAR Th099 ARRHYTHMIAS BASED ON THE EVALUATION OF MYOCARDIAL DYSFUNCTION MECHANISMS. O.I.Zharinov. Cardiology Clinics, Medical University, Lviv, Ukraine

A198

The aim oft.he investigationwas to find clinical and hemodynamic predictors ofthe antiarrhythmic efficacy ofatenolol and tiraeisine in the patients with ventricular arrhythmias (VA) and congestive heart failure (CHF). The study included 51 patient with VA and CI-IF, among them 25 with CAD, 19 with arterial hypertension, 7 with congestive eazdiomyopathy. The methods of investigation were Holter ECG monitoring, ChestX-ray and Echoeardiography, with Doppleranalysis of transmittal flow. Antia_rrhythmictherapy was initialized after the failure of conventional drugs (ACE-inhibitors and diuretics)to suppress VA. Atenolol (A) was effective in 17 cases (33.3%), tLracisine (3") - in 21 (41.1°/o). The most importer predictors ofatenolol antiatrhythmic and hemodynamic effect were tachycardia, arterial hypertension and combination of lung congestion and ejection fraction > 40% (high possibility of diastolic dysfunction). On the contrary, no important predictors ofantiarthythmic effect of tiracisine were found; moreover, this drug had unfavourable hemodynamic impact. Thus, the treatment of VA in CHF with beta-blockers must be based on the evaluation of myocardial dysfunction mechanisms. Tachycardia, arterial hypertension and diastolic dysfunction seem to be the most important signs to prexiict antiarrhythmic and hemodynsmic response to atcnolol.

Arrhythmlas in the elderly patients w i t h Th098 hypertension and left ventricular h y p e r t r o p h y XY.Li, ZY.Zhen, GQ.Chen,T.Liu,XCh Jiang. Chinese PLA General Hospital, Beijing , 100853, P.R.China In order to observe the arrhythmlau In the patients over 60 years old with essential hypertension ~ and left ventricular Hypertrophy (LVIO, 196 patients were derided into two groups according to the left ventrlcular mass Index (U/MI) on eohooardiogram:LVH or non LVH (IqLVIO, nod into three subgroups: without angina peotoris(1), with unstable angina peotorls fill, with myocardial infarction(Ill). The analysis for the Holter results in both groups showed that the VPeu/34hr In L~-I ( 1796± 1732} were significantly more than those In NLVH (1163±1026, p<0.06), but no significant difference was round between the two groups In SVPCs/24hr. The mean heart rate in the 34 hr In LVIt 173.3± 10. 9 bents/min) was higher than that In NLVH (63.3± 9. 3 boats/min, p<0.003), and the mean lowest heart rate in 34 hr In LVH (01.6±3.6 beats/mln) was also higher than that in NLVH(48. 9 ± 6 . 0 boato/mln, p<0. 05). The eomparlsiou among the subgroups in the LVtt showed that the VPOs/34hr in group Ill (4114±3690} wore much more than those In grpup II (1499±3310, p<0. 06) and group 1 (1284±2766, p<0.01), but there were no significant differences among the subgroupu in the NLVII.The positive relationships were showed by the regression analysis between VPCo/24hr sod the LV ejection fraction (r=O. 3671, pC0. 02) and LVMI(r=O. 7004, p<0. 001). h conclusion, the increased WCs/24hr In the group of elderly hypertension patients with LVll were related to the degree of LVK besides, to the severity of accompanied coronary artery disease, the decreased LV function and increased activity of the sympathetic nervous system.

ARRTHYTHMOGENESIS, ACUTE MYOCAR- Thl00 DIAL INFARCTION AND ANTIARRHYTHMICS. Kryzhanovsky S., Vititnova M., Lyskovtsev V. and Kaverina N. Institute of Pharmacology RAMS, Moscow, Russia The present was undertaken with the purpose to conduct a systemic study on the effects exerted by the antiarrhythmic drugs of different classes according to Vaughan Williams's classification on arrhythm0genesis promotion during the acute experimental myocardial infarction. To achieve this goal we have designed a unique model for -the assessment of arrhythmogenesis development features by daily Holter's monitorinq in doqs (n= 135) with the acute myocardial inTarct['on. The following antiarrhythmic drugs were subjected to a comparative examination: Ethmosine (class I, subclass IA), Lidocain (class I, subclass IB), Ethacizin (class I, subclass/C), Propranolol (class II), Sotalol (class I11), Verapamil (class IV) and Neoton (IA and IV classes combination). The experiments showed that the largest changes in arrhythmogenesis are caused by Lidocain, Verapamil and Neoton; all the drugs prevent the advancement of ectopic activity. I-or instance, at arrhythmogenesis peak Lidocain reduce the total number of ectopic constrictions by mean 32.7%, Verapamil by 54.1% and Neoton by 51.9%. The latter two drugs do not only cause a statistically siqnificant decrease in total ectopic constrictions, bSt almost entirely eliminate from arrhythmogenesis structure the "malignant" (that is inducing the fibrillation) cardiac arrhythmia.

Mechanisms involved in cell volume regulation Thl01 during myocardial ischemia Nadir Askenasy, Ruth Panigel and Gil Navon, School of Chemistry, Tel Aviv University, Israel Cellular swelling during myocardial ischemia is generally considered to be caused by an increase in cytosolic osmolarity. Using an NMR method for continuous measurement of cell volumes in the perfused and ischemic rat heart, several ischemic events were assigned volume-related activities: 1) Inhibition of sodium influx via the Na/l-I antiport, the Na/K/2C1 cotransport and the Na+ channels reduced the extent of cellular water accumulation, while inhibition of the Na/K ATPase pumps did not cause an excessive swelling. 2) Reduction of cytosolic free calcium concentration either by inhibition of the Caz+ channels, the Na/Ca exchange or magnesium cardioplegia, abolished the ischemic cell swelling. 3) Anaerobic generation of lactate and release of inorganic phosphate due to high energy phosphates degradation promote cellular swelling during ischemia. 4) The presence of ATP at the onset of ischemia has a permissive effect on cellular water accumulation. In the absence of high energy phosphates, cells shrink during ischemia, similar to the behavior observed in hearts treated with toxic doses of ouabain. 5) Adenosine AI receptors modulate cell volume dynamics in hearts preconditioned to ischemia. It is therefore evident that in addition to the direct effect of cellular osmolarity on volumes, calcium ion homeostasis and receptoriN systems are also involved in cell volume regulation in the ischemic myocardium.

Th 103 DIRECT DAMAGING EFFECTS OF LIVER ISCHEMIAREPERFUSION (I/R) ON THE ISOLATED HEART. Edith Hochhauser, Avi Weinbroum, Zen Mhagna, Valery Rudick, Bemardo Vidne. Cardiac Res Lab Beilinson Israel Cardiovascular dysfunction frequently occurs after major vascular surgery or liver transplantation. Our hypothesis was that substances released from I/R organs may directly cause cardiac dysfunction. Method: Male rat livers and hearts were separately perfused with oxygenated modified Krebs-Henseleit solution (KH) via portal vein or retrogradely (modified Langendorff prep) at 100 mmHg pressure, respectively. Temperature was kept at 37+ 0.5°C. After stabilization, livers were perfused with KH (CON, n=5) or made globally ischemic (I/R, n=6) for 2h. Followed inseries liver+heart re-perfusion:liver effluent was directed into the heart for 15 min; the heart was then separately re-circulated with accumulated liver+heart effluent for 45 min. Results: Baseline +dP/dTmax in CON and I/R hearts was similar. In I/R hearts it then decreased significantly (fig). CPK significantly increased in I/R hearts staring at 1 (12:1:5 vs 21+12 [mean+SD]) throuhout 60 min (12t-6 vs 165+32) mU.ml-~. Conclusions: I/R Myocarclial Contmcti~y liver causes direct 3~0 T "P(O~ ~ Comrol myocardial injury. This remote cardiac ~ • . dysfunction model may prove useful to better understand the mechanism(s) leading to decreased T~,me(m.) auring le-pe'hi~l cardiac dysfunction.

POSSIBLE METABOLIC MEDIATORS OFThl02 ISCHEMIA-INDUCEDACUTE CONTRACTILE FAILURE M. Galifianes, T. Smolenski, J.C. Kentish*, D.J. Hearse. *U.M.D.S. and St Thomas' Hospital, London SE1, U.K.

The mechanism(s) underlyingthe abrupt contractile failure that occurs immediately following the onset of ischemia remains controversial.To investigatetile possibleroleof ischemia-induced metabolic changes, isolated rat hearts, perfused at 37°C with bufferand pacedat 400bpm, weresubjectedto globalischemiafor 300s. At varioustimes between 1 and 300s they were subjected to electronically-controlled high speed freeze clamping and metaboliteanalysis(,n=8/time-~int).The profilesforthe declineof LVDP,phosphorylatJonpotent=al(PP) and NAD/NADH ratiowere essentmllysupenmposible(Figure).Of the PPcomponents([ATP]t'. [ADP][Pi]) only Pi exhibited changes that were the reciprocalof contractilefailure. ATP and ADP changed little over this p.eriod.A possiblelink between Pi increaseand acutecontractilefadurewas investigated in skinned fibers (n=3), in which Pi was increased from 4.0 to 14.0raM 2 0 0 "~ PI (comparableto that seen during the first 300s of ischemia).This induceda 50% decline in force (c.f. 0 100% declineof LVDP in whole hearts) whilst a decrease in the NAD/ NADH ratio from 52 to 4 lequivalent to the 300s Jschemia) had no effect. In conclusion Pichanges induced by ischemiamay J LVDP NADI~AOH~ play a significant role in 0J , . . . . . determining the rate of Os 5s lOs 15s 20.' acute contractilefailure.

Th104 ON THE CAUSES OF ISCHEMIC DAMAGE OF OXIDATIVE PHOSPHORYLATION IN HEART MITOCHONDRIA Vilmante Borutaite, Ramune Morkuniene, Aiste Budriunaite, Rasa Baniene, Vida Milda~iene, Adolfas Toleikis, xGuy C. Brown. Kaunas Medical Academy, Kaunas, Lithuania; XUniversity of Cambridge,UK Using the top-down approach of Metabolic Control Analysis we have shown that ischemia causes inhibition of respiratory chain and phosphorylation system, and increased proton leak. The addition of cytochrome c to ischemic mitochondria restores the activity of the respiratory chain and phosphorylation system to that of normal but does not cause the activity of the proton leak to return to normal. Thus, inhibition of the respiratory chain and the phosphorylation system is due to loss of cyt.c from mitochondria. A defect in a respiratory chain after 45 min ischemia is unrelated to loss of cyt.c. The proton leak in ischemic mitochondria decreases after incubation of mitochondria with albumin. However even after that the proton leak in ischemic mitochondria is greater than in normal. This shows that the increase in proton leak is only partially related to the accumulation of unesterified fatty acids in myocardium during ischemia.

A199

MECHANISM OF THE INHIBITORY Th105 A C T I O N O F O X Y R A D I C A L S ON C A R D I A C S A R C O L E M M A L Na+-K + A T P A S E Qiming Shao, Taku Matsubara & Naranjan S. Dhalla. Div. of Cardiovasc. Sciences, St. Boniface Res. Ctr., Univ. of Manitoba, Winnipeg, Canada.

SUPEROXIDE ANION INDUCESATP DEPLETION ThlO6 BEFORE PEROXIDATIVE DAMAGEIN RAT HEART. Nazareno Paolocci, Fabrlzio Bruschi, Marco Bettini, Stefania Banditelli*, Maria Vittoria Boeri', Alessandro Riva°, Franco Magni & Renata Barsacehi *°. General Pathology Institute. Perugia University,*Physiologyand Biochemistry Department. Pisa Universityand °Clinical Physiology Institute. CNR. Pisa. Italy.

Although different oxyradical generating systems are known to depress the cardiac Na'-K ~ ATPase activity, the mechanisms for this effect are not clear. In this study we employed xanthine plus xanthine oxidase and low concentrations ofH20 z plus Fe 2+ for the generation of superoxide and hydroxyl radicals, respectively. Exposure of porcine cardiac sarcolemma to either superoxide radicals or hydroxyl radicals decreased the Na+-K + ATPase activity as well as SH group content and increased the lipid peroxidation (MDA content). Superoxide-induced decrease in Na'-K ~ ATPase and increase in MDA content were prevented partially whereas the depression in SH group content was prevented completely. Likewise, mannitol prevented hydroxyl radical-induced decrease in Na+-K + ATPase and increase in MDA content partially while complete prevention was observed with respect to changes in SH group content. These results suggest that changes in lipid peroxidation due to superoxide and hydroxyl radicals play an important role in depression of the cardiac sarcolemmal Na+-K + ATPase. (Supported by MRC Group in Experimental Cardiology).

Superoxide anion (SA) is released by inftammatot3' cells in man), different situations including myocardial tissue. We studied changes in heart rate (F). ECG. ESLVP. coronet3, flow (CF), ultra-weak chemilunlinescence emission (as lipid pcroxidation marker), the levels of 3 major antioxidant systems (ascorbate. vit.E and GSH) and energetic nucleotide levels in isolated perfused rat heart after SA generation in the perfusion fluid by electrolysis. After 30' of stabilization, a 5mA current was applied for 30"or 60" to 37°C warmed solution and the results obtained are reported below. 30' stabiliz. 3' after SA 30' aller SA 30"electr. CF ml/min 11 +0.9 -49% -74% F beat/min 326 +15 -14% -38% ESLVP 63.3 :t:11 -34% -58%

ROLE OF POLYMORPHONUCLEAR LEUKOCYTES IN Th107 CARDIOVASCULAR DEPRESSION AND CELLULAR INJURY IN HEMORRHAGIC SHOCK AND REINFUSION. Rakesh Kapoor and Kailash Prasad. Department of Physiology, College of Medicine, University of Saskatchewan,. Saskatoon, Canada.

A200

We investigated th~ role of polymorphonuclear leukocytes (PMNLs) in cardiac depression and cytotoxiclty during hemorrhagic shock and relnfuslon ( H S ) . The dogs were assigned to 4 groups: I (sham), 4 hrs duration; II, 2 hrs of shock followed by relnfusion for 2 hrs; III, shock and relnfusion in neutrophils depleted with immune serum; IV, same as III but pretreated with non-lmmune serum. Cardiac function and contractility decreased during shock while plasma creatine klnase (CK), CKMB and lactate increased. Relnfuslon tended to bring hemodynamlc 9arameters and plasma lactate towards control values while OFR producing activity of PMNLs, plasma CK, and CK-MB increased further. Cardiac malondialdehyde and superoxldedismutase activity were higher while left ventricular chemiluminescence was lower in group II as compared to group I. Despite the increase in the antioxidant reserve and antioxldant enzymes, there was oxidative damage. PMNL depletion attenuated the deleterious effects of HS on the hemodynamic and biochemical parameters. The changes in group IV were similar to those in group II. These results suggest that PMNLs contribute to eytotoxiclty during HS.

60"elect r.

CF ml/min F beaVmin ESLVP

18 :t3 3(175+75 68 :tl

-36% - 10% -44%

-(~% - 15% -59%

Anti-oxidant s)'stems were not affected: only after 6 pulses of 60" electrolysis vit.E was slightly decreased. The energy cell content decreased already after I poise of 30"electrolysis(60% of controls). Chemiluminescence started to increase only after 3 pulses of 60" and stabilized at vet3' low values after 6 pulses. Our dala show that ATP depletion induced by SA is not related to a lipid-poroxidative damage. In conclusion myocardial fimetional changes observed are dne to mechanism which is currently under investigation.

C a l c i u m accumulation promotes cellular Thl08 swelling d u r i n g myocardial ischemia Avi Koner, Nadir Askenasy and Gil Navon, School of Chemistry, Tel Aviv University, Israel Intracellular accumulation of calcium during myocardial ischemia is considered as one of the injury-promoting factors. The effect of intraeeUular calcium concentration on cell volumes was studied in perfused rat hearts: a) inhibition of Ca z* channels with verapamil (10 I.tM), b) inhibition of the Na/Ca exchange with bepri~lil (10 gM), c) modulation of sarcoplasmatic Ca 2. release with magnesium cardio,plegia (16 mM). Cell volumes were measured using.[ "H NMR of water and "'Co NMR spectroscopy o f cobalticyanide, which was used as an extraeellular marker. Administration of either verapamil oi" bepridil to the perfused heart did not induce changes in cell volumes (10 min). During ischemia, inhibition of the channels, the Na/Ca exchange and magnesium arrest reduced the extent of cellular swelling to 0.08, 0.18 and 0.12 ml/gdw, respectively (compared to 0.38 ml/gdw in controls). At reperfusion, cell volumes recovered their pre-ischemic values in verapamil, bepridil and magnesium treated hearts, while volumes were higher by 7% in controls. It is concluded that calcium influx during isehemia promotes cellular swelling. Blockade of intracellular calcium influx during isehemia stabilize cellular volumes, although this ion does not contribute significantly to the increase in cytosolic osmolarity. The mechanism which couples calcium with the cell volume remains to be determined.

AN INCREASE OF Ca2+ CYCLING ACROSS THE Th109 MITOCHONDRIAL MEMBRANE CAUSES AN UNCOUPLING OF RESPIRATION ON CALCIUM PARADOX Kazunobu Ban, Reg A.Chapman, Dept of Physiohigy, Bristol University, Bristol, UK.

A markedfall in energy rich phosphatesand increasedglucose utilization are features of the calcium paradox of the heart. In isolatedguinea-pigventricularmyocytessubjectedto Ca2+ ovcrload, a collapseof mitochondrialmembranepotential (Win) could be the resultof increasedCa2. cycling acrossthe mitochondrialmembrane and would be expected to uncouple respiration from ATP production. Myocytes,were preloadedby 51.dvlJC-1, subjectedto Ca2+ overloadand the ratio of JC-I fluorescence(excitedat 490nm and measuredat 530 and 590rim) was monitored.On exposureto 3 ~vI ruthenium red (R.R.), 3pzM CGP-37157 (CGP) or 0.2p.M cyclosporinA (CyA). the changein JC-I fluorescenceratio on Ca2. overload,indicativeof a depolarizationof Win, was either delayed. slowed and reduced or failed to develop. As R.IL inhibits Ca2+ influx via the uniport in cardiacmitochondriawhile CGP and CyA inhibit the Na+/Ca2+ antiport, these results implicate Ca2"~ cycling across the mitochondrial membrane in the fall in ~Fm- As intraccllular ATP is complcxed by Mg2+. measurements of intracellularfrec Mg2. can indicate changesin ATP. On calcium depletion, [Mg2+]i, as measuredby the mag-fura2 ratio (excitedat 350 and 380nm and measured at 510rim), was reduced. On subsequentCa2+ overload,the mag-fura2 ratio increasedin two phases;the first was smaller and coincidentwith hypercontracture and was probablydue to the increased[Ca2+]i. Exposureto R.R., CGP and CyA reducedor further delayed the second rise in the mag-fura2ratio. The bettermaintenanceof c¢tlular ATP when Ca2+ cycling acrossthe mitochondrialmembraneis blocked indicatesthe importanceof oxidative phosphorylationin meeting the increased energ)'demandprovokedby Ca2+ overload.

EFFECTS OF HYPOXIA ON THE FORWARD Thl 11 AND REVERSE MODES OF Na*/Ca 2. EXCHANGER Fumiko Suzuki, Satoshi Takeda, Masayuki Taniguchi, Seibu Mochizuki. Department of Medicine, Aoto Hospital, Jikei University School of Medicine, Tokyo, Japan. The pathophysiological roles of the Na÷/Ca 2. exchange system are still controversial so that the contribution of this system to Ca2. regulation during ischemia or hypoxia remains uncertain. The aim was to investigate whether the forward and reverse modes of Na÷/Ca 2. exchange are inhibited dtldng hypoxia in the isolated, perfused rat heart. [Ca2*]i was monitored using ratio of Fura 2 fluorescence intensities at emission wavelengths of 340-and 380-nm. The hearts from SD rats were perfused by Langendorff method with HEPES buffer containing 5.5mM glucose. A latex balloon was inserted into the left ventricle to measure LV pressure. The reverse mode of Na~/Ca 2. exchange was estimated by the complete replacement of extraeellular Na* by tris-aminomethane. The forward mode of this exchange was estimated by a decrease in[Ca2*]i after the administration of caffeine to the perfusate. When the hearts were switched to hypoxia, there was a decrease in the rate of rise and magnitude of the [CaZ*]i increase compared with aerobic conditions. The rate of decay in [Ca2*]i after the addition of caffeine was significantly reduced during hypoxia. In addition, this rate of decay in [Ca2*]i was significantly attenuated when Ni2. was present. These results indicate both efflux and influx of Ca2. through the sarcolemma are inhibited during hypoxia, however the mechanism underlying the increase in [Ca2*]i seems to occur by the delay of expelling [Ca2*]i since this exchanger normally represents the major efflux pathway for Ca2* in the heart.

CALCIUM OVERLOAD INDUCED BY LYSO-Thl 10 PHOSPHATIDYLCHOLINE IN ISOLATED RAT CARDIAC MYOCYTES Hiroko Hashizume & Yasushi Abiko. Department of Pharmacology, A s a h i k a w a Medical College, Asahikawa, Japan. Lysophosphatidylcholine (LPC) is one of the amphiphilic metabolites of membrane phospholipid, and accumulates in the ischemic myocardium. Effect of e x o g e n o u s LPC on the intracellular calcium concentration ([Ca2+]i) was studied in isolated rat cardiac myocytes. The addition of LPC (15 pM) increased [Ca2*]i and produced Ca2. overload in the cells. The increase of [Ca2+]i was dependent on the extracellular Ca2* concentration. Verapamil (200/JM) did not attenuate the LPC-induced Ca2+ overload. Simultaneous treatment with ryanodine (10 pM) and thapsigargin (200 nM) attenuated the LPC-induced Ca2+ overload. Manoalide (10 pM), aphospholipase (PL) A2 inhibitor, also attenuated the LPC-induced Ca2* overload. U73122 (10 pM), a PLC inhibitor, did not attenuate it, however. LPC has chemical structure similar to platelet-activating factor (PAF). WEB 2086 (10 pM), a PAF inhibitor, did not attenuate the LPCinduced Ca2. overload. These results indicate that 1) LPC produces Ca2+ overload due to both Ca2+ entry (not via the voltage-dependent Ca 2+ channel) and Ca2+ release from intracellular Ca2. stores; 2) the increase of [Ca2+]j induced by LPC would activate PLA2, and it may in turn produce further accumulation of lysophospho I ipids including LPC, and 3) LPC which accumulates in the ischemic myocardium would accelerate the ischemic damage because of Ca2* overload in the cells.

EFFECTS OF HOE 642 ON pH i AND Ca i IN Th112 ISOLATED RAT CARDIOMYOCYTES Ulrich Rul~, Heinz Gtigelein, Wolfgang Scholz, Udo Albus, Hans-J. Lang, Andreas Weichert & Bernward A. Sch61kens. Hoechst AG, PGU Cardiovascular Agents, H 821, D-65926 Frankfurt/Main, Germany Isolated cardiomyocytes of adult rat were dual loaded with the fluorescent dyes fura-2 and BCECF. Under anoxic conditions the intracellular pH (PHi) acidified by 0.4 pH units until rigor contraction of the cell occurred, lntracellular calcium (Cai) started to rise to a constant level (>1 pM) when cell length was reduced by rigor shortening. The rise in Ca i was always preceded by the fall in pH i. After reoxygenation, all cells hypercontracted and pH i returned to its resting level within 10-20 rain. Ca i immediately started to decrease and reached its resting level within 10 min. During this period slow Ca i oscillations superimposed by fast ones were observed. When 1 pM of the specific NHE-1 inhibitor HOE 642 was present during begin of the experiment, the PHi-recovery was abolished, but no obvious differences in the Ca i signals could be observed. However, in the presence of HOE 642 hypercontracture after reoxygenation was not observed in 2 out of 6 cells. In conclusion, HOE 642 has no obvious effect on the Cai-overioad during anoxia and on the decline of Ca i during reoxygenation, but seems to protect cells in the latter phase, probably by keeping pH i at a low level. A201

Th113 THE NEUTROPHIL DERIVED OXIDANT HYPOCHLOROUS ACID REDUCES THE L-TYPE CALCIUM CURRENT IN ISOLATED VENTRICULAR CARDIOMYOCYTES Stefan Hammerschmidt, Hans Wahn, Friedarike yon zur Milhlcn. BcrudD. Gonska. University G6ttingen, Dept of Cardiology Gtttingen, Germany Disturbances of the cellular calcium homeostasis due to oxidative stress are regarded as important factors in the genesis of reperfusion-induced arrhythmias. This study investigates the effect of the oxidant hypochlorous acid (HOCI) on L-type (high voltage activated) calcium current (ICa,L). The whole-cell recording configuration of the patch-clamp technique was used in single hamster vcntricular myocytcs. The recordings (holding potential -80 mV) were started 5 min after patch rupture. The ICa,L was recorded at a frequency of 0.5 Hz over 12.5 min. Voltage-current relationships were obtained at the beginning and the end of the registration. HOCI (0.04 mmol]l) was applied externally to the cells beginning one minute after the start of the registration. A control group of cells was not exposed to HOCI. The cells were dialyscd with different internal solutions: EGTA (raM) A T P (raM)

control 0.5 0

l 0.5 0

II 5.0 0

m 0.5 1.0

lV 5.0 1.0

The tableshows the ICa,L after 12.5 rain (in % of the current at 0 rain, n=5/group).Theapparentrevcrsalpotentialdid not change. time (min) control I* [I ** m ** IV ** 12.5 99+4.9 38+6.3 51:15.7 52:.+.5.2 64+4.8 • : p<0.01 vs. control, **: p<0.01 vs control and vs. l A higher intracellular calcium buffer capacity and intracellular addition of ATP prevents the HOCl-induced decrease of ICa,L. Therefore this effect may be explained rather by a calcinm-dependent inactivation of the channels due to an increased intracellular calcium concentration caused by inactivation of calcium transporting membrane proteins or by reduced energy supply than by a direct inactivation of the channel proteins due to oxidative alteration. A decrease of ICa.L may contribute to the shortening of the action potential that is observed during rcperfusion.

THE R O L E O F THE Na÷-CHANNEL IN Th115 THE A C C U M U L A T I O N OF I N T R A C E L L U L A R Na ÷ D U R I N G M Y O C A R D I A L ISCHEMIA Jan G. van Emous, Marcel G.J. Nederhoff, Tom J.C. Ruigrok & Cees J.A. van Echteld. Heart Lung Institute, University Hospital, Utrecht, The Netherlands. The mechanisms of intracellular [Na'] ([Na*]~ increase during ischemia (I) are still a matter of debate. To examine the role of the Na*-channel, 200 p.M lidocaine (L) was included in the perfusate for 5 min prior to 30 rain of global I in isolated rat hearts at 37 °C. Z~Naand sip NMR were used to monitor [Na']~ and high energy phosphates, resp. During perfusion with L, [Na÷]~ declined from 10.2±1.3 mM (mean±SD; n=6 in all groups) to 8.7±1.9 mM, and the rate pressure product (RPP) decreased by 50%. During I [Na']~ rose to 15.9±2.4 mM. In control (C) hearts [Na*]i rose from 10.9±1.3 to 26.2±2.9 mM during I. Additional experiments with low extracellular [Ca2"] showed that this beneficial effect of L could not be explained by the decrease in RPP prior to I. At 20 min of I, pH~ in Chearts was 5.76±0.04 and in L-hearts 6.06±0.07, but after 30 min of I pH~ was 5.78±0.05 and 5.73±0.04, resp. Furthermore, L attenuated the decline of ATP during I and delayed the time to onset of contracture. Within 5 min of reperfusion (R) developed pressure (DP) recovered completely in L-hearts. At 30 min of R in C-hearts DP recovery was 17.2±16.2% and enddiastolio pressure was 80.7_,12.0 cmH20 compared to 29.6±15.6 cmH20 in L-hearts. During R [Na*]~ declined to 13.2±2.5 mM in L-hearts and 18.3±1.5 mM in C-hearts. PCr and ATP recovered significantly better in L-hearts. Conclusion: Na*-influx dunng I occurs, at least partly, via the Na*-channels, and blocking this channel during I improves postischemic functional and metabolic recovery. A202

EFFECT OF I S C H E M I A ON C A R D I A C DIDHYDROPYRIDINE RECEPTORS

Th114

Riccardo Zucchi, Simonetta Testoni, Gongyuan Yu, Ugo Limbruno, Giovanni Ronca, Mario Mariani. University of Pisa, Italy We investigated the effect of ischemia on cardiac dihydropyridine receptors, which correspond to L-type sarcolemmal calcium channels. Isolated working rat hearts were perfused aerobically for 10 min, and then subjected to 20 min of global ischemia. Control hearts were perfused aerobically for 30 rain. At the end of the perfusion, the ventricles were homogenized and a microsomal fraction was prepared. 3 H - P N 2 0 0 . 1 1 0 binding was measured in the crude homogenate and in the microsomal fraction. In the homogenate obtained from control hearts, the Kd and Bmax averaged 0.23_+0.05 nM and 84_+4 fmol/mg of proteins respectively. Ischemia did not produce any significant change in these variables. On the contrary, in the microsomal fraction ischemia caused a significant reduction in the Bmax (115_+15 vs 190_+34 fmol/mg, P<0.05), without any change in the Kd. In this fraction, the yield of PN200-110 binding sites was 4.7_+0.6% in the control condition and 2.8_+0.5% after ischemia (P<0.05). The yield of other sarcolemmal markers such as quinuclidinyl benzilate and ouabain binding sites was not reduced in the microsomal fraction obtained from ischemic hearts. In conclusion, the total number of dihydropyridine receptors was unchanged after ischemia, although their distribution after tissue Iractionation was slightly modified, suggesting receptor redistribution between different subcellular pools.

Th116 ALT]~RATIONS IN CYTOSOLIC MAGNESIUM ACTIVITY IN ISOLATED RAT VENTRICULAR MYOCYTES IN RESPONSE TO HYPOXIA AND ADRENERGIC ACTIVATION Mark M. Gnilagher & Asltlcy Allshire. Dept of Clinical Pharmacology & Therapeutics, University College Cork, Ireland. Magnesium concentration in plasma alters significantly in the hours following myocardial infarction and intravenous administration of raagnesium sulphate is used in the treatment of acute MI in some cantres. It has been shown that cytoselic levels of magnesimn increase during ischaemia in intact hearts and in isolated cardiac myocytes. It is known that adrancrgic activation induces an effiux of magnesium from the myocardium in a beta receptor mediated manner. The interaction of these factors was studied using the fluorescent probe Mag-Fura 2 to quantifycytosolicmagnesium in fleshlyisolatedratventricular myocytes paced at 60 contractions/ min, attachedto a glasscover slip and supeffnsedwith H E P E S bufferedcxtmcellularmedium containing either0.6 or 1.2 mmol/L M g S O 4. HypoxJa was simulated by use of N a C N at 2 mmol/L and 2..danxyglucoseat 2.5 mmol/L. Each cellwas studiedfor 100see under physiologicalconditions,then exposed 1o the cyanide solutionfor 300see followedby 300see of superfusionwith physiologicalextracalIulurfluid.Adrenergic stimulationwas achieved by inclusionof adrenalineat 0.1mmol/L in the extracallularracdium from at least5 mins before the experiment untilthe end ofthc expe1"imanL73 cells were studied, 19 in 0.6 mmol/L Mg solution without adrenaline, 16 in 0.6 mmol/L Mg with adrenaline, 20 in 1.2 mmol/L Mg without adrenaline and 18 in 1.2 mmol/L Mg with adrenaline. We observed a consistent rise in cytoselic magnesium activity, typically beginning 100 see after onset of exposore to cyanide and 2-denxyglucose. This was followed in most cases by ccssationof cellcontractionwithin 30 sec.Magnesium activityfellfollowing washout of cyanide and 2-deoxyglucose. The changes in magnesium activity were not altered by alteration of extracollular magnesium concealtration, or by inclusion of adrenaline in the extracellular fluid.

CALCIUM-INDEPENDENT CO-RELEASE OF NOR- Th11 EPINEPHRINE AND NEUROPEPTIDE Y DURING MYOCARDIAL ISCHEMIA Markua Haase, Caraten Kr0ger, Armin Haunstetter & Wolfgang K0bler. Department of Cardiology, University of Heidelberg, Germany The effect of global myocardial ischemia (stop flow for 5-40 min) on spontaneous release of endogenous norepinephrine (NE; determined by HPLC) and of the sympathetic cotransmitter neuropeptide Y (NPY; determined by RIA) was investigated in the isolated perfused guinea-pig heart. During the first 10 min of myocardial ischemia no spontaneous release of NE and NPY was detectable. However, 15 rain of myocardial sichemia were accompanied by a marked spontaneous release of both NE (182¢36 pmol/g) and NPY (205¢30 fmol/g). The spontaneous release of both transmitter was further enhanced in parallel by longer periods of global myocardial ischemia (20-40 min). Consistent with a nonexocytotic release mechanism, the ischemia-induced corelease of both NE and NPY was calcium-independent. Furthermore, the ischemia-induced co-release of both NE and NPY was suppressed by blockade of the neuronal catecholamine uptake carrier (uptake-I; e.g by 300 nmol/I desipramine or 300 nmol/I nisoxetine) and by inhibition of sodium/proton exchange (e.g. by 10 pmol/I ethylisopropylamilorid). In conclusion, global myocardial ischemia of > 15 min is accompanied by a spontaneous calciumindependent co-release of both NE and NPY. Its suppression by uptake-1 blockade and by inhibition of sodium/proton exchange is consistent with a regulated release mechanism,

cAMP - PHOSPHODIESTERASES OF RAT Thl 18 CARDIOVASCULAR SYSTEM CYTOCHEMICAL STUDY Ludmila Okruhlicova, Narcis Tribulova, Anita Eckly" & Jan Slezak. Inst Heart Res, Bratislava, S R , ULP Fac Pharmacie, IIIkirch, F r a n c e , . It is known that cyclic AMP (cAMP) modulates the contraction and relaxation of cardiovascular system. Its synthesis by adenylate cyclase and the inactivation by phosphodiesterase (PDE) are, therefore, one of the main regulatory steps in the transfer of signal. The cyto-chemical localization of cAMP-dependent PDE was studied in the rat myocardium. 40 ~tm thick slices from perfusion fixed rat heart were incubated in the medium with cAMP as the substrate and Pb ions as the capture metal of the reaction product. After the incubation in the basic medium the specific precipitate of cAMP-PDEs was localized on the sarcolemma (SL) of cardiomyocytes. In addition, it was localized on the plasmalemma (PL) of endothelial cells of capillaries and small coronary arteries. The same distribution of the precipitate was detected in the presence of selective inhibitor of PDE IV (50 p.M rolipram). However, the precipitate was less intensive. After the inhibition of PDE III with the specific inhibitor (100 laM SK&F94120) the intensity of precipitate on the SL was weaker. The results demonstrate cytochemical distribution of PDE IV on SL and PL and PDE III on SL only. These results correlate with biochemical data.

DELETION POLYMORPHISM IN THE ACE GENE Thl 19 IN ANGIOGRAPHICALLY PROVEN CORONARY HEART DISEASE. Justyna Bigda, Tomasz Nylk, Darek Cie~wierz, Pawel Skarzyfiski, Ryszard Pawlowski, Roman Hauser, Andrzej Rynkiewicz. I Dept.Cardiology, Dept. Forensic Medicine, Medical University of Gdafisk, Poland.

DEPOLARIZATION PATTEPd~ OF BODY Th120 SURFACE POTENTIAL ~ P S IN CORONARY ARTERY DISEASE

The DD genotype of an insertion/deletion polymorphism of the anglotensin I converting enzyme (ACE) gene, associated with higher ACE activity has been reported as a risk factor for myocardial infarction and sudden cardiac death. The aim of the present study was to estimate the potential association between ACE polymorphism and coronary heart disease (CHD). The polymerase chain reaction was carried out to identify I/D polymorphism with standard procedures. 107 patients with CHD and 77 healthy subjects were studied. CHD was angiographically confirmed. The frequency of DD genotype 35% was nonsignificanfly higher in CHD group in comparison to 27% in the controls. The H genotype was found in 18% of patients with CHD and in 22% of controls. Among males with CHD DD genotype was present in 41% of patients and in 27% of healthy males (borderline significance). Moreover frequency of D allele occurence was higher among males with CHD (0.62) in comparison to healthy males (0.52). In older patients with CHD (age > 60 years ) DD was less frequent ( 33% ) than in younger group of patients ( age < 60 years ) (48 % ). The rate of DD genotype in subjects with single vessel or multiple vessel CHD was almost identical. Conclusion: The ACE DD genotype is a potential risk factor for the coronary heart disease.

The aim of the study was to assess if depolarization body surface potential maps (BPS~) were able to evaluate the site, size and severity of chronic ischaemic damages. The B S P ~ w e r e obtained from 84 patients sufferin~ from coronary artery disease confirmed by ccronarography, with at least 75% occlusion of at least one coronary artery. According to the site of single occlusion or a combination of sites of multople occlusions, the patients were divided into 6 subgroups. The reference standard consisted of 28 normal subjects. The depolarization maps can distinguish patients with coronary artery disease from healthy persons with a sensitivity of 92% and a specificity of 63% in the case of iscareal maps from the first half of depolarization and 52% and 75%, respectively, in the case of the iscintegral map from the whole depolarization.

O.Kittnar, J.Slav~ek, ~.V~vrov~, A.Dchnalov~ Inst. of Physiology, Ist ~ed. Faculty, Charles University, Prague, Czech Rep.

A203

ESTIMATION OF PEROXIDE PLASMA LEVEL Th121 IN PATIENTS EVALUATED FOR CORONARY HEART DISEASE BY DIPYRIDAMOLE INFUSION FOLLOWED BY SPECT.

Miroslaw Kazmierczak, Henryk Wysocki, Andrzej Wykretowicz, Andrzej Mincz~kowskl, Juliusz Smieleeki ; Dept.

of Intensive Therapy,

University

School

of

Medicine, Poznan, Poland Myocardial ischemia leads to increased formation of toxic oxygen radicals. These substances exert deleterious effects on myocardial cells, contributing to reperfusion injury and arrhythmia generation. However, considerably less information was obtained concerning toxic oxygen species generation during transient ischemia. The purpose of our study was to estimate hydrogen peroxide (H~O2)plasma levels in patients subjected to short lasting ischemia induced by dipyridamolc stress test, applied for diagnosis of coronary artery disease. Evaluation was performed with the use of ~I'c-SestaMIBI followed by SPECT. Blood for studies was obtained from peripheral veins of 53 patients (37 men and 16 women), mean age 49a:11 yrs. Plasma hydrogen peroxide level was estimated by spectrophotometric method just before dipyridamole challenge and after drug infusion. Hydrogen peroxide plasma level in patients with negative SPECT test was 23.5 4- 3.0 mmolq (mean 4- SEM) and 21.04-2.9 after dipyridamole infusion (p--0.474). H202 plasma concentration in patients with positive SPECT was 30.5:i:4.6 and increased after dipyridamole challenge up to 50.34-5.4 (p=0.004). Further analysis revealed that observed difference can not be attributed to previous myocardial infarction. Even transient myocardial ischemia can induce generation of toxic oxygen derivatives.

TRANSIENT REDUCTION OF BAROREFLEX Th123 SENSITIVITY AFTER MYOCARDIAL INFARCTION Carsten Krflger, Armin Kalenka, Markus Hanss, Armin Haunstetter, Mark Schweizer, Christoph Maier & Wolfgang K0bler Department of Cardiology, University of Heidelberg, Germany The baroraflex control of the R-R-interval ('baroreflex sensitivity', BS) was investigated in conscious rats 3, 28 and 56 days after myocardial infarction (MI; induced by left coronary artery ligation; n=23-31) or sham-operation (S; n=14-19). Tile BS was determined by linear regression analysis of R-R-interval changes vs. changes of mean artedal pressure in response to i.v. bolus injections of nitroprusside (NIT; 0.2-2.0 I~J/kg) or methoxamine (MET; 2.0-20.0 I.Lg/kg). Consistent with moderate heart failure, LVEDP was increased 3, 28 and 56 days after MI (MI: 11+1, 12=1:1and 14+1, S: 6i-0, 6+0 and 7i'0 mmHg; means + SEM; p<0.05 each). A twofold increase of the plasma concentration of ANF was found after MI, while both plasma catacholamines and basal heart rate (HR) remained unchanged. Three and 28 days after MI, a marked decrease of BS was observed (Mh 1.0-J:0A and 1.li-0.1, S: 1.4i-0.1 and 1.6:1:0.2ms/mmHg; p<0.05 each), whereas 56 days after MI BS was normalized (Mh 1.51-0.1, S: 1.4:1:0.2 ms/mmHg; n. s.). Subanalysis revealed that the attenuation of BS was due to a decrease of reflex bradycardia (RB; induced by MET), whereas reflex taohycardia (induced by NIT) was intact. Pretreatment with atropine (0.5 mg/kg) did not cause an additional impairment of RB 3 and 28 days after MI, but markedly reduced RB 56 days after MI and in all S-groups. As compared to S, the increase of basal HR induced by atropine was less pronounced 3 and 28 days but not 56 days after MI. These data indicate that both RB and control of basal HR are impaired in rats with moderate head failure after MI. This may be due to a decreased basel tone and a reduced reflectory activity of the vagus. These changes occur eady but are no longer observed 56 days after MI, despite persisting heart failure.

CLINICAL SIGNIFICANCE OF VITAMIN 0 T h 1 2 2 TO AMELIORATE LIPID PEROXIDATION DAMAGE IN THE PREVENTION AND TREATMENT OF CORONARY ARTERY DISEASE. Rong Ye-Zhi,Lu Bao-Jing, Zhu Xiang-Yang Research ~ab for Myocardial Disease, Xin-Hua Hospital, Shanghai, CHINA 200092 The activities of superoxlde dismutase (SOD),glutathlone peroxidase(GSH-PX) in blood and the content of melondialdehyde(MDA) in plasma of 30 patients (male 17,femal 13) with coronary artery dlsease(CAD) were determined in this study. It was shown that SOD and GSH-PX activities were decreased and the content of MDA was increased in the patients comparatiye with the healthy controls(P
ELEVATED LEVELS OF PLASMA GASTRIN Th124 AND VASOACTIVE INTESTINAL PEPTIDE IN ACUTE MYOCARDIAL INFARCTION. +Mariann Gy6ngy6si, + + +Tamds Sdri, + + +J6zsef Kaszaki, + +J6zsef N~meth, + +Tamds Vdrkonyi, +Mikl6s Csan~idy. +2nd and + + l s t Dept. Int. Mad., + + + D e p t . of Exp. Surgery,A. Szent-Gy6rgyi Med. Univ.,Szeged,Hungary The plasma level of vasoactive intestinal peptide (VIP) and gas.trm ((3) are raised by adrenergic stimulation. The aim of this study was to check whether these two gastrointestinal peptides are elevated in acute myocardial infarction (AMI). Experiments were carried out on 6 mongrel dogs (22.6+5.4 kg) anesthetized with pentobarbital sodium. ECG, the heart rate and the arterial blood pressure was monitored. The left circumflex (LCX) coronary artery was prepared, and the pneumatic occluder was located 1 cm towards the bifurcation of the left anterior descending coronary, artery and LCX. After 30 minutes preconditioning, the coronary artery was occluded, and blood samples were collected 1, 5, 10, 30 and 60 minutes, and 2, 3, 4, 5 and 6 hours after the coronary occlusion. Plasma levels of G and VIP were determined by RIA assay. The plasma G concentration gradually increased in dogs following acute coronary occlusion, and the change became significant compared with the control value in the third hour (10.44-4.2 vs 6.14-3.4 fmol/ml, p=0.042). The plasma VIP concentration in dogs with AMI was significantly elevated in the first hour after the acute coronary occlusion (5.744-1.28 vs 3.524-1.66 fmol/ml, p=0.036). In conclusion, the slightly but significantly elevated plasma VIP concentration in the early phase of AMI may have a possible protective role in acute myocardial ischemia. The reason for and the role of the increased plasma G level in the third hour after the onset of AMI need further explanation.

CHANGES IN ACTIVITY OF TRANSMEMBRANE Th125 lONE TRANSPORT OF LEUKOCYTE MEMBRANES IN PATHOGENESlS OF MYOCARDIAL INFARCTION Kremneva L.V., Abaturova O.V. Institute of Clinical and Preventive Cardiology, "rjumen, Russia In 70 pts with acute myocardial infarction during the first 24 hours of disease progression there was studied ferment activity of transmembrano transport of iones of Na +- K+- and Ca 2+ -ATPase of the whole polymorphonuclear leukocytes (PMNL), content of Ca and K in PMNL, cell adhesion on glass and level of zymozanstimulated chemiluminescence (Ho). There was revealed decrease of Na +- K +- and Ca 2+- ATPase activity (0.18+0.01 and 0.71+0.06 mkmol Ph hour 106 cl., correspondingly, P<0.05), lowering of K level (1.21+0.14 mmol 108 cl. P<0.05) and Ca accumulation in cells (0.066+0.001 mmol 108 cl., P<0.05). There was also observed rise of Ho level (0.85+0.5 105 r.u. 108 cl.)as well as cell ability to adhesion (0.338+0.019 r.u. 108 cl., P<0.05). There was determined correlation between Na +- K +- ATPase activity and cell adhesion which was equal to r---0.68, Ca 2+- ATPase and adhesion r=-0.48. Thus, lowering of ferment activity of transmembrane lone transport in pts within the acute period of myocardial infarction is associated with Ca accumulation in cells that causes the rise of PMNL functional activity as increase of adhesion and cell secretion of active oxygen forms. Manifestation of oppression of transport ATPase activity, changes in electrolyte cell homeostasis and their functional activity may be criteria of inauspicious disease processing.

MB FRACTION OF CUMULATIVE CREATINE Th127 K I N A S E C O R R E L A T E S WITH INSULIN S E C R E T I O N IN P A T I E N T S WITH M Y O C A R D I A L I N F A R C T I O N . Hlroyukl Matsui, Hldekazu Hashlmoto*, Sachio Mitanl, Yuklo Tokl, Kenj| Okumura, Takayukl Ito, A k i h l k o Fukushlma**, HItoshl Kanayama**, Internal M e d i c i n e 2, Nagoya Univ. School of Medicine, *Aichi Pref. Coll. of Nursing & Health, Nagoya, **Internal Medicine, Kamo Hosp., Toyota, Japan. To test whether insulin is a regulatory factor of myocardial MB creatine kinase (CK) content, we investigated the correlation between the ability of insulin secretion and the MB fraction of cumulative CK released in plasma in 18 patients who underwent successful direct angioplasty within 10 h of the onset of their first acute myocardial infarction. Exclusion criteria were age > 75 y, heart failure, obesity, multivessel d i s e a s e and diabetes. Cumulative MB CK divided by that of total CK was defined as MB%. Two weeks or more after the onset, 75 g oral glucose tolerance test with serial determination of plasma glucose and insulin (0, .5, 1, 2, 3h) was done and catecholamines and echoic LV mass were measured. MB% significantly correlated with insulinogenic index (r = .56, p = .02), insulin area (r = .59, p = .02), insulin area / glucose area (r = .63, p = .007) and urinary adrenaline (r = - .54, p = .03). Age, BMI, infarct size, glucose metabolism and LV mass were not significant univariate predictors of MB%. Multivariate analysis showed that the ability of insulin secretion contributed to MB% more than catecholamines and that insulin area I glucose area was the strongest independent predictor of MB% (t = 3.01, p = .02). Thus, MB fraction of cumulative CK released in plasma, indicative of myocardial MB CK distribuUon, significantly related to the ability of insulin secretion in subjects without overt insulin resistance. Regulation by Insulin of myocardial MB CK was suggested.

In-vlvo sequence of left ventrlcular depolarization Th126 does not change the Infarct size In rabbit hearts Zenon S. Kyrlakides, Efstathios K. Iliodromitls, Costas Papadopoulos, Nikolaos Sourlas, Dimitrios Th. KremasUnos. Onass/s Cardiac Center, Ath/nal, Greece. We examined the hypothesis that altered left ventricular depolarization sequence may augment the infarct size. Twenty-one New Zealand male white rabbits were anesthetised and ventilated. The chest was opened via a median thoracotomy and two electrodes were placed on the right atrium and ventricle. After that the rabbits were randomized to atrial (n=7) and atrial ventricular (AV) sequential pacing (n=7) whereas 7 rabbits sewed as controls without pacing. The pacing rate was 20 beats/min higher than the sinus rate. Blood pressure and ECG were recorded continously. During AV pacing the AV delay was decreased to prevent fusion beats. After 1 rain of pacing the left coronary artery was occluded by snare. After 30 min the snare was released and pacing was stopped. After 120 rain of reperfusion the experiment was terminated. Hearts were removed and mounted on a Langerdoff apparatus via the aorta and perfused retrogradely with normal saline. When blood was removed from the coronary arteries, the coronary ligature was retightened at the same site and Zn-Cd fluorescent particles (10-20 u) were infused to delinate the normal and risk areas. Infarct size was measured using tetrazolium staining and the infarcted and risk area were planimetered. All results were expressed in cm 3 and the ratio of the infarcted to risk area in percent (%I/R). Atrial pacing AV pacing Controls p %I/R 43.9-J:5.4 38.3+6.4 4 1 . 4 + 6 . 4 >.05 In conclusion, altered left ventricular depolarization sequence in rabbit hearts does not increase the infarct size in vivo.

THE DISTRIBUTION OF INSULIN-LIKE GROWTH T h 1 2 8 FACTOR-I IN RELATION TO EXPERIMENTAL MYOCARDIAL INFARCTS Linda Maxwell, John B Gavin, Barbara Johnston, Peter D Gluckman. Department of Pathology and Research Centre for Developmental Medicine and Biology, University of Auckland, New Zealand. IGF-I is expressed in normal myocaxdium, increases the contractitility of neonatal cardiac myocytes, and can induce hypertrophy in cultured myocytcs. This study determined the relative distributionof IGF-I in myocardium within, distant from, and adjacent to regional infarctsproduced by ligation (6hr - 20d) of the leftdescending coronary artery in male Wistar rats (200-300g). IGF-I was identified in transverse 6pm sections through the ventricles using a polyclonal IGF-1 primary antibody, biotinylated rabbit IgG secondary antibody and horseradish peroxidase. It was located within myocyte sarcoplasm and in the cytoplasm of neutrophil leukocytes and macrophages. The intensity of staining of left ventricular ntyocardinm distant from infarcts was 337% higher after 6hr and 382% higher after 24hr of ligation than corresponding regions of sham-operated hearts. The layer of viable myocytes immediately adjacent to infarcts was 504% more intensely stained after 6hr and 617% after 24hr than for sham operated myocardinm. The elevated levels declined toward control levels by 4 days of infarction, but myocytes immediately adjacent to the margin were still significantly above control (170%) at 20d. This spatial and chronological pattern of expression of IGF-I is consistent with a role for IGF-1 in the healing of myocardial infarcts.

A205

FISH OIL MODULATION OF MYOCARDIAL Th129 INFARCTION AND LEUKOCYTE FUNCTION Peter L McLennan, Mahinda Y Abeywardena & Thelma M Bridle. CSlRO Div Human Nutrition, Adelaide, Australia. Several studies have reported that fish oil feeding limits infarct size in animal models. This study sought to extend these observations to examine the effects of diet and reperfusion on leukocyte function and infarction. Rats were fed for 12w on a control diet (C) or a diet containing 6% fish oil (FO). Infarcts were produced by coronary artery occlusion either permanently (PERM) or with reperfusion (REP) after 30min and examined after 4h and 24h. Infarct size determined by tetrazolium chloride staining and computerised area measurement was significantly reduced by early reperfusion (PERM: 4h: C 3 7 + 4 % ventricular area; FO 3 8 + 5 % ; 24h: C 3 6 + 6 % ; FO 31:I:6%)(REP: 4h: C 1 8 + 6 % ; FO 1 8 + 5 % ; 24h: C 1 6 + 4 % ; FO 1 7 + 5 % ) with no significant dietary differences. Polymorphonuclear leukocyte (PMNL) infiltration into non-infarcted ventricle was increased with REP in 4h hearts. At 24h all hearts showed greater remote PMNL infiltration than at 4h. At 24h, PMNL infiltration into small ( < 3 0 % ) infarcts was decreased by FO with no difference in large infarcts. In vitro stimulated PMNL chemotaxis was reduced by FO. This study could show no acute reduction in infarct size with dietary fish oil. Effects on PMNL behaviour suggest that effects on chronic, healed infarcts may be worthy of investigation.

A206

PROPHYLAXIS OF VENTRICULAR ARRHYTHMIAS Th130 IN ACUTE MYOCARDIAL INFARCTION BY INTRAMUSCULAR ADMINISTRATION OF LIDOCAINE

Grlgore Lupescu, *Constantln Mlrclolu, *Mlheela Ionescu, Grlgore Lupescu Jr. Tg.Jlu Hospital, Tg.Jlu and *Army Center for Medical Research, Bucharest, Romanla Systematic prevention of cardiac arrhythmias in acute myocardial infarction (AMI) is a logic therapeutical measure meant to prevent the onset of primaryventricularfibrillation and"undeserved deaths" occurring to "hearts too good to die". According to an original therapeutical scheme, the authors administered mean doses of 200 mg i.m. lidocaine at 6 hours interval, with a supplementary dose at lh 30 min. after the first administration, during hospitalization (except for contraindications). A comparative study included two groups of (Q wave) AMI patients: 87 treated conventionally during the 1971-1975 period and 721 patients treated also by i.m. lidocaine during the 1976-1994 period.The intrahospital mortality rate fell significantly from 20.7*/, to 9.6% (p<0.05), especially by spectacular diminution (p<0.001) of "surprise deaths".The need of i.v. lidocaine diminished significantly.The local i.m. anaesthetic injection is virtually painless and has no adverse effects in the mentioned doses.The favourable results may be explained:(1 ) by the accumulation of lidocaine following repetition of doses(in 17 patients without heart failure the mean plasma level of lidocaine, measured just before the next dose, once daily for the first 5 days running, was 1.88,SEM+0.85 gg/ml), (2) by the need of smaller doses in AMI patients and (3) by reducing the risk of malign degeneration of arrhythmias: the coupled premature beats decreased in number significantly(p < 0.01) in comparison with the isolated.In conclusion,by this method, systematic prophylaxis of ventricular arrhythmias in AMI patients during the hospitalization period lead to a significant decrease of mortality rate.

DOSE-DEPENDENT REDUCTION IN MYO- Th131 CARDIAL INFARCT SIZE IN RABBITS BY THE NA+/H+-EXCHANGE INHIBITOR HOE 642 Wolfgang Linz, Wolfgang Jung, Udo Albus, Andreas Weichert, Woifgang Scholz & Bemward A. SchOlkens. Hoechst PGU CVA, D-65926 Frankfurt/Main, Germany

ETHYLISOPROPYLAMILORIDE REDUCES Th132 INFARCT SIZE IN THE RABBIT HEART IN VIVO. A ROLE OF Na+/H+ EXCHANGE Einar Bugge, Jens Munch-Ellingsen and Kirsti

Myocardial ischemia decreases intracellular pH and leads to an activation of Na+/H+ exchange with consequential increase in intracellular Na + and Ca++. To interrupt this process we used the Na÷/I-I+ exchange inhibitor HOE 642 in rabbits with acute myocardial infarction. Rabbits (n:28) were randomized in 4 groups: saline vehicle, HOE 642: 0.01, 0.l and 0.3 mg/kg, and subjected to a 30-min left main coronary artery occlusion followed by 2-h reper~sion. HOE 642 was given as a bolus intravenously l0 min before occlusion. After reperfusion, myocardial infarct size was determined by tetrazolium staining and expressed as a percentage of area at risk. HOE 642 led to a dose-dependent reduction (p<0.05) in infarct size by 38%, 55% and 81% respectively, whereas area at risk did not differ in between the groups. Sustained decrease in blood pressure (15-20 mmHg) and a significant reduction in lef~ ventricular dP/dtma x in the control group throughout occlusion and reperfusion was observed. In treated groups these changes were dosedependently diminished. Our results show that inhibition ofNa+/I-I+ exchange with HOE 642 is cardioprotective by reducing infarct size in rabbits in a dose dependent manner.

Inhibition of Na÷/I-I÷ exchange with amiloride analogues has been shown to offer functional protection and reduce infarct size in isolated hearts. The aim of the present study was to clarify if pre- or postischaemic treatment with ethylisopropylamiloride (EIPA), a selective Na÷/I-I÷ exchange inhibitor, could reduce infarct size in an in vivo rabbit model of regional ischaemia and reperfusion. Anaesthetized, open chest rabbits were subjected to 30 min of regional ischaemia and 180 min of reperfusion. The risk zone was determined by fluorescent particles, and infarct size was determined by TTC staining. Preischaemic treatment with EIPA (0.65 mg/kg) significantly reduced infarct size from 45.8 _+3.5% of the risk zone in the control group to 10.6 __ 3.1% (p<0.01). EIPA-treatment during the first part of the reperfusion period did not reduce infarct size compared to controls (41.9 + 3.5%). We conclude that EIPA, when administered prior to ischaemia, reduces infarct size in the rabbit heart in vivo, a protection most likely due to inhibition of Na÷/I-I÷ exchange.

Ytrehus, Department of Medical Physiology, University of Tromse, Norway

Th 133 MEANING OF THE INITIAL PERIOD OF MODERATE ISCHEMIA FOR THE FLOW/FUNCTION BALANCE IN HYPOPERFUSED MYOCARDIUM Hans Martin Hoffmeister, Markus Str6bele, Andrea B/ifiler, Silke Kazmaier, Martin E. Beyer, Ludger Seipel, Medical Department III, University of Tiibingen, Germany.

A match between the reduction in coronary flow and function prohibits the development of a myocardial necrosis ("hibernation"). Using isovolumetric, beating isolated rat hearts we investigated the meaning of the initiation period of hypoperfusionfor this delicate balance. In group A the period of ischemia was started with 10 min of "no-flow" ischemia followed by a hypoperfusion for 3h with 15% of the preischemic coronary flow. In group B the coronary flow was gradually decreased during 5 min to a reduction in coronary flow of 15% of the control values (for 3h). Data were compared to controls (group C). Histologicallyno irreversible damage was observed in the hearts of both groups. Data after 3h of hypoperfusion(n= 20/group): group A group B group C isovol. LVP (mmHg) 42 + 2* 40 + 2* 91 + 3 TAN (pmol/g) 1.9 + 0.1" 2.1 4- 0.1" 4.6 +0.2 (Means + SEM,*=p<0.05; ~AN= ATP+ADP +AMP) Reduction in contractile function and decrease in high energy phospates during hypoperfusion were comparable in the two groups after 3h of hypoperfusion. Conclusion: For the development of a coronary flow/ contractile function balance in hypoperfused myocardium the conditionsof initiation (gradual reduction or no-flow ischemia with consecutivehypoperfusion)are not of importance.

Chronic Myocardial Hibernation: an adaptive mechanism to isehemia. A. Bakker, M. Borgers', W. Jacob

Th 134

University of Antwerp, Belgium • Janssen Res. Foundation, Beerse, Belgium

Chronic hibernating myocardium refers to the presence of a myocardial dysfunction, associated with a severely reduced coronary blood flow. Ultrastructurally these ceils are transformed to a non-functional 'surviving' state since they are chamcterised by the absence of contractile material and the presence of many 'mini'mitochondria. We quantified the surface density of mitochondrial contact sites, since these sites are sensitive markers for the energy state of the cell and for the intracellular [Ca2÷]. In biopsies from patients 8 months after coronary bypass surgery who showed markedly improved wall motion, the surface density of contact sites was evaluated. The surface density of contact sites show in hibernating zones (0.27 + 0.05) is significantly lower than in the normal zones (0.357 + 0.005). In conclusion we may state that our data strenghtens the idea that chronic hibernation is a low demand-low supply situation, characterised by a low intracellular [Ca2*].

"HIBERNATING MYOCARDIUM": AN Th135 INCOMPLETE ADAPTATION Albrecht EIs~sser, Wolf-Peter Kliivekorn, Klaus-Detlef Miiller, Rudolf Strasser, Achim Vogt, Martin Schlepper, Jutta Schaper, Kerckhoff-Clinic and Max-PlanckInstitute, Bad Nauheim, Germany

KINETICS OF ADAPTATION OF ISOLATED Th136 RAT HEARTS TO ISCHEMIA AND HYPOXEM/K. Giampiero Merati, Sonia Allibardi, Silvia Sommaruga, Stefania Casalini and Michele Samaja. University of Milano and Scientific Institute H San Raffaele, ITALY.

The definition of "hibernating myocardiurn" (I-IM)implies impaired myocardial function caused by a chronically reduced coronary blood flow. After restoration of adequate per,fusion cardiac function will recover. This adaptive mechanism supposedly preserves the myocardial structure. The aim of our study was to test this hypothesis by morphological analysis. HM was diagnosed by dobutamine echocardiography, thaUium-201 SPET myocardial scintigraphy and radionuclide ventriculography. In 36 patients biopsies were taken from regions of HM during coronary bypass surgery and examined by electron microscopy as well as immun-histochemistry. We observed intra- and extraceUular changes: Intracellular alterations include loss of myofilaments, disorganization of the cytoskeleton, changes in size and shape of nuclei and storage of glycogen. The extracellular space is enlarged and shows an increase in adhesive and structural proteins as well as in number of fibroblasts and macrophages. The morphological changes are indicative of a loss of contractile function, of cellular integrity and of a synthesis of fibrotic material. The final stage of this degeneration is cell death and scar tissue. In conclusion: The adaptive mechanism as proposed for HM is incapable to preserve the structural integrity and cannot prevent further degeneration of the tissue. A completly functional recovery of HM areas after restoration of an adequate perfusion is therefore not possible.

We measured the time course of adaptation of isolated perfused rat hearts to low-flow ischemia CLFI, n=13) or hypoxemia (H, n--12) matched for O2 supply, 10% o f in vivo. A computer-assisted system was developed to monitor performance (5 s time resolution) and to control the apparatus. In LFI, the fall of peak systolic pressure was monophasic (-320-Y_.27mmHg/s, meard:SE). In I-I,the fall was similar to LFI during the first 20 s (-271+_24 mmHg/s, p=NS) being followed by a short bout of activity and a slower monophasie decrease. The change of PvO2 was faster in H than in LFI (p<0.05). Whereas net lactate release in LFI reached a steady value within 120 s (1.3+0.2 p.moles/min), in H it overshooted up to 7 Ixmoles/min -120 s after beginning the 02 shortage and then stabilized at 4.4+0.4 I.tmoles/min (p<0.0001). The overshoot corresponded to the delayed adaptation of performance in H hearts. It appears that anaerobic ATP production provided the bulk of the needed metabolic energy during the first 2 rain of LFI but not during H and longer LFI. This approach may provide clues to assess the metabolic pathways of regulation of myocardial performance at the onset of acute 02 shortage. Supported by BTBS Target Project, CNR, Roma, Italy. A207

Morphological, Tracer and Biochem.Th137 Examinations on Canine Heart post HTK Cardioplegia and following ischaemia. G. Mogllevskl, A. Schmiedl, J. Richter, M.M. Gebhard and H.J. Bretschneider , Dept. of Anatomy and Dept of Physiology, University of G@ttingen, Germany

This study examined the degree of ultrastructural maintenance after cardioplegia with modified HTK (histidin tryptophan ketoglutarat) solution and following hypothermia (5 o C). As morphological functions ware examined the cell and mitochondrial volumes and the permeability of membranes by adding the extracellular tracer Lanthan high energy phosphates were determined enzymatically. After 240 min ischaemle there was no significant volume increase, Lanthan could not found intracellular by ESI (electron spectroscopy imaging) and EELS (electron energy loss spectroscopy), ATP average 5,5 p.mol/gww. After 600 rain ischaemie increased the cell volume. Lanthan was found in the myofibrils by EELS and on the outer mitochondrial membran and ATP average 4,2 p.mol/gww, After 1320 min the mitochondrial matrix was damaged irreversible. Lanthan occured now in granular form in the myoflbrils, ATP average 2,5 pmol/gww. The presents of intracellular Lanthan during the reversible ischeamic damaging phase points to disturbances of the ional exchange process and/or changes of membrane permeability.

_&208

MYOCARDIAL METABOLISM DURING Th139 INTERMrTTElqT WARM CARDIOPLEGIA Evasio Pasini, Lucia Torecca, Giuseppe Coletti, Massimo Benigno, Ottavio Alfieri, Roberto Ferrari, Fondazione Clinica del Lavoro, Centro S. Maugeri, Gussago, Brescia; II Divisions Cardiochirurgia, Spedali Civili, Brescia Italy. Intermittent warm blood cardioplegia (IWBC) has been proposed to protect the myocardium during open heart surgery. No data, however, are available on the effects of IWBC on the metabolism of the heart. The aim of this study was to evaluate functional and metabolic changes of isolated and blood perfused rabbit hearts exposed to perfusion sequence of IWBC. After a stabilisation pedod a cardioplegic arrest was inducedfor 5' by intracoronaryadministrationof 25 mEq/l KCL. Then coronary flow was abolished for 10'. Thereafter coronary flow was rainstarted for 5' in the presence of high KCL. This sequence was repeated three times. Finally the hearts were reperfused for 30' in absence of KCL. This resulted in a prompt recovery of developed pressure (100% after 31. Quiescence caused a reduction of myocardial VOz (from 4.9¢-0.8 to 0.8i.0.07 ml/min/100g; p<0.001). KCL also induced an increase of coronary perfuslon pressure (from 50+3 to 135+8 mmHg; p<0.01) suggesting depoladsation of coronary smooth muscle. Reperfusion after no flow was associated with a transient washout of lactate (from 26:t:3 to 290+15 pgr/min/gww; p<0.01) followed by a return to pre-ischemic value. Myocardial content of ATP and CP (17.5:1:0.6 and 32.3:u?..4 pmol/gdw, respectively) after IWBC was within the normal aerobic value. These data suggest that:l) IWBC allows a prompt recovery of myocardial function without stunning. 2) Dudng each period of no flow anaerobic metabolism take place, but myocardial content of ATP and CP is maintained after IWBC. 3) KCL depoladses the coronary artery of isolated heart.

THE CHALLENGE OF THE DIASTOLIC CARDIOPLEGY:Th138 THE CARDIAC ACTIVITY UNDER SYSTOLIC ARREST. Otoni M. Gomes;Dalton R. Weigl;FAbio I.Pedroso HEART SERVICE-HSFA/UFRG MEDICAL SCHOOL-BRAZIL

To analyse the cardiac activity under deep(20 ° C) hypothermic systolic arrest 24 dogs were st~ died:Group 1:8 hearts (control)removed soon a~ far anesthesia and thoracothomy;Group If:8 hear ts submitted to normothermic anoxic arrest (60-min);Group Ill:8 hearts arrested by cold blood coronary perfusion and kept ischemic during 60 min, with the temperature preserved by cold p~ ricardium saline perfusion.PARARETERS STUDIED: ATP concentrations,fine structure alterations including endothelial cell pinocytotic activity mitochondrial respirometry(alpha-ketoglutaric , succinic,beta-hydroxybutyric,glutamic and malic deshydrogenase activies-Warburg Method).RESULTS ATP-Group 1,1.210;Group II,O.439;Group III,1.3 50~mol/g. ENZYRES: The enzymatic activity depr~ ssion was not statistically different between groups II and Ill (p>O,O5).ELECTRON MICROSCOPY STUDY:Group II~85.5% of normal and 14.5% of ~tered mitochondria;Group III,82.3% normal and 17.7% altered mitochondria(p > O,05).PINOCYTOSIS Group I,I0.16 vesicles/~ of endothelial membrane ;Group II,g.68 and Group III,7.60 ves/~membrane. Differences between groups II and Ill were not statistically significant(pp O,05)oIt is concluded that the cold cardiac systolic a~ rest is a high energetic (ATP) ischemic state with litlle impairing effects on the diastolic cell activity.

MYOCARDIAL SUBSTRATE OXIDATION Th140 FOLLOWING WARM CARDIOPLEGIA Terje S. Larsen, Thomas V. Andreasen, Terje K. Steigen, Oivind Irtun, Dag S~rlie. University of Troms~, Norway

In this study we investigated the recovery of oxidative metabolism during the first hour following cardioplegic cardiac arrest (2 h continuous warm blood cardioplegia, 120 ml. min") in pigs subjected to cardiopulmonary by-pass. Thus, we measured rates of oxidation of glucose (~4Cglucose) and oleate (aH-oleate) before and 15, 30 and 60 min following cardioplegia. Arterial and coronary sinus blood samples were taken simultaneously to evaluate myocardial substrata uptake. Sixty min after cardioplegia uptake and oxidation of oleate had recovered to 61 ± 26 and 25 ± 7%, respectively, of the values obtained before cardioplegia. The corresponding numbers for glucose were 78 ± 44 and 60 ± 12%. At 15 rain following cardioplegia myocardial uptake of lactate was essentially zero, but at 60 min it had recovered to 60 ± 28% of pre-cardioplegic values. These data indicate that warm cardioplegia leads to stunning of oxidative metabolism, which may be related to the functional stunning observed after cardioplegic arrest of the heart.

s+p NMR EVALUATION OF ENERGY METABOLISM Th141 IN THE RAT HEART DURING CARDIOPLEGIC ARREST Ryszard T Smolenski, Hitoshi Ogino, Jay R Jayacumar, Magdi H Yacoub, Anne-Made L Seymour. Dept Cardiothorac Surgery, Natl Heart & Lung Inst, Harefield Hospital, UK The profile of metabolic changes in the ischemic heart following repeated infusions of cardioplegic solution is not clear. The changes in myocardialATP, phosphocreatine(PCr) and intracellular inorganic phosphate (Pi) were followed by ~P NMR in an isovolumic preparation of Langendorff per/used rat heart. After 40 min of per/usion, St. Thomas' Hospital No.1 cardioplegiawas infused for 4 min at 250C. Hearts were then maintained at 25"C under ischemic conditions for 2 hours with infusions of cardioplegic solution every 30 min. There was an initial increase of 16 % in PCr concentration and total depletion of Pi during the first infusion of cardioplegic fluid. After infusion, there was a gradual decrease in PCr with a rate of 7% per min concomitant with accumulation of Pi. PCr was rapidly restored after each infusion of cardioplegic solution while Pi completely disappeared. However, depletion of PCr was faster (12% and 15% per min) after the second and the third infusion and increases in Pi during the ischemic intervals were more pronounced (119% and 188% of the first increase after the second and the third cardioplegia, respectively).ATP concentrationdecreased dudng cardioplegic arrest at the rate of 0.8% per min and cardioplegic infusions exerted little effect here. Our results demonstrate that transient infusions of cardioplegic solution are sufficient to restore energy equilibrium in the heart and that more frequent infusions of cardioplegic solution in the later phase of ischemia may be beneficialfor preservationof the heart during surgery.

Effects of T r a n s i e n t Acidic R e p e r f u s i o n on Th142 the Level of High Energy P h o s p h a t e s Tetsunori Kawase,Fumio Yamamoto,HidekazuHirai,Masao Hirano, Yutaka Sakakibara,Yositsugu Kito,YasunaruKawashima.Dept.of CardiovascularSurgery,National CardiovascularCenter,Osaka,Japan. The purpose of this isolated working rat hearl study is to investigate the protective ability and mechanism of Ihe transient acidic reperfusion(TAR).(Study1)After measuringpreischemiecardiac function and infusion of St.Thomas' solution,hearts were subjected to 35rain.of normothermic ischemia,followed by Langendofff(L) reperfusion with either pH7.5 or 6.8 for 3rain.Then hearts were ctmvened to working(W) perfusion to measure the postischemie cardiac function and to express as the percent of preisehemie control value.A1 the end,hearls were freeze-clamped for measurementof high energy phosphates (HEP).(Study 2)Basic time protocol was the same as study-I .After 3rain.of L-reperfusion with either pH7.5 or 6.8, hearts were freeze-clamped to measure HEP. Energy charge(EC) and adenine nucleotide pool(ANP) were calculated for the evaluation of tissue energy condition.(Results)Aeidic reperfusion improved postischemie functional recovery, although there was no significant improvement in HEP both at the end of 3min.of L-reperfasion and W-perfusion.Our results suggested that TAR is beneficial to improve poslischemic functional recovery although its mechanism effects related to TAR.

W~LS~Ot e x

C.P

ADO

EC

A.NP

pH6.8

53.5

11.4

2.89

0.33

28.1

0.03

0.88

1.4.6

pH7.5

38.7

1.2.1

3.07

0.32

30.0

0.03

0.88

IStudyI % A o F "

kTP

ADP

~MP

t5.4

Study2

ATP

ADP

AMP

~

ADO

EC

ANP

~H6.8

12.8

3.78

t.62

!3.4

0.95

0.80

18.2 18.7

REPERFUSION RESPONSE A N D A T P Th143 SUFFICIENCY CORRELATE WITH RAPID POSTISCHAEMIA RECOVERY OF HEART OUTPUT Ralph J F Houston, Berend Oeseburg, Arend Heerschap & Stefan H Skotnicki. Faculty of Medical Sciences, University of Nijmegen, The Netherlands. Fax + 3 1 80 5 4 0 5 3 5 .

PROTECTION OF THE HEART IN PATIENTS Th144 UNDERGOING CORONARY SURGERY: THE EFFECT OF L-CARNITINE. BIOCHEMICAL STUDY Ivan Pechan, Ivan Gabaner, Roman Zahorec, Vifiam Fischer, Katarina Kanalikeva, Helena Minarova, Viera Rendekova, Lukac Halcak & Terezia Kalnovicova. Inst Cardiovasc Dis, Inst Heart Res, SAS, Inst Biochem, Med Fac, Comenius Univ, Bratislava, Siovakia

In this project we have developed and are using an ejecting, b'lood-perfused isolated rat heart model for the haemodynamic and 31p NMR assessment of cardioprotective regimes, especially normothermic intermittent blood cardioplegia. Isovolumic models do not predict the ability of the heart to support a physiological workload postoperatively. In pilot w o r k w e determined the response of the unprotected heart to normothermic ischaemic insults of varying duration. As this heart receives a generous oxygen supply, coronary f l o w autoregulation functions. We noted that a postischaemic ATP level around 8 0 % of starting value combined with a reperfusion response of a rapid overshoot of coronary f l o w to 2-3 times its original value then decay to baseline led to rapid recovery of left ventricular p o w e r output (flow x pressure): approximately 7 5 % of initial value within 10 minutes. This response could predict the need for post-operative patient support. We acknowledge Dutch Heart Foundation grant 902-19-11 5.

L-camitine is a compound with important role in fatty acid transport processes. The protective effect of eamitine was studied in patients with progressive isehemic heart disease (NYHA m - I V ) with low left ventricular ejection fraction undergoing aorto-eoronary bypass surgery during operation and in the early postoperative periods. The following biochemical variables were estimated: variables o f energy and nucleotide metabolism (lactate, pyruvate, inorganic phosphate, purine nueleotide degradation products) and some representatives of cell antioxidant status (malondialdehyde, reduced glutathione, superoxide dismutase, glutathione peroxidase). The protective effect o f L-earnitine was manifested especially by decreased malondialdehyde and lactate levels and by stabilization o f glutathione level.

A209

A210

CLINICAL COMPARISON BETWEEN CRYSTALLOIDANDTh145 BLOOD ST THOMAS' CARDIOPLEGIC SOLUTION No 1. Mohamed F Ibrahim, Graham E Venn, Christopher P Young, David J Chambers. Cardiac Surgical Research & Cardiothoracic Surgery, St Thomas' Hospital, London, UK.

MECHANISMS OF ACTION OF UW AND ST TItOMAS' Th 146 SOLUTION: IMPORTANCE OF IONIC COMPOSITION Franklin L. Rosenfeldt, Robert A. J. Conyers, Paula Jablonski, Lesley Langley, Stephen M. Richards, Gregory Self, Vernon Marshall. Baker Institute and Monash Medical Centre, Melbourne Australia

Increasingly, cardiac surgeons are using blood, rather than crystalloid, cardioplegia to protect the ischaemic myocardium. Few studies, however, have compared similar solutions with blood as the only variable. We studied 50 patients undergoing coronary artery surgery who were randomly allocated to receive either St Thomas' No 1 (ST1) crystalloid cardioplegia (n=25) or blood-based (BST1) solution (n=25). We determined myocardial function (thermodilution) and post-ischaemic arrhythmias. Preoperative haemodynamic data were similar in both groups of patients, as were aortic cross clamp (38+_2vs 43+_2)and bypass (76+5 vs 84+4) durations (ST1 vs BST1, respectively). Defibrillation was significantly (p
Although University of Wisconsin solution (UW) is in limited clinical use for heart transplantation, there are concerns about its efficacy and high K + concentration. Using a modified (starchfree) variant of UW (MUW) we studied: 1) Recovery of function with UW compared with espartate-containing St Thomas' solution; 2) The effect of reduction in K + in UW and the addition of Ca2+ or aspartate; 3) The effect of elevation of K + in St Thomas' solution to the level in UW. Isolated rat hearts underwent 7 h of arrest at I'C using MUW with or without 20 mol/L aspartate, or using aspartate-containing St Thomas' solution. The results showed that functional recovery with MUW, 51.8+2.5%, was superior to aspartate-containing St Thomas' solution, 37.15=4.3 % (p<0.01), as was rapidity of arrest and preservation of adenine nucleotides. Addition of aspartate to MUW had no effect. During 6 h of arrest, lowering the K + in MUW from 125 mmol/L to 20 mmol/L reduced functional recovery from 59.9 :t: 4.2% to 42.3 + 4.3% (p<0.01). The addition of 1 mmol/L Ca 2+ had no effect. Elevation of K + in St Thomas' solution produced more rapid arrest but did not improve recovery. Conclusions: 1) The protective effect of starch-free UW is greater (+15%) than aspartata-em'iched St Thomas' solution. 2) Reduction of K + in MUW decreases its protective effect by about 30% to a lever comparable with that of St Thomas' solution; the addition of either Ca2+ or aspartate produces no improvement. 3) The efficacy of MUW cannot be matched in St Thomas' solution either by raising the K + level or by adding aspartate.

MORE SENSITIVE CLINICAL MARKERS FOR Th147 MYOCARDIAL P R O T E C T I O N - W I T H OXYGENATED OR NON-OXYGENATED ST. T H O M A S ' SOLUTION Yutaka Sakakibara, Fumio Yamamoto, Masao Hirano, Tetsunori Kawase, Hidekazu Hirai, Yositsugu Kitoh, Yasunaru Kawashima. Dept. o f Cardiovascular Surgery, National Cardiovascular Center, Osaka, Japan

THE EFFECTS OF AMILORIDE AND NI2"IN Th148 CARDIOPLEGIC SOLUTION OR REPERFUSATE UPON MYOCARDIAL PROTECTION Hidekazu Hirai, Fumio Yamamoto, Takumi Ishikawa, Yutaka Sakaldbara, Masao Hirano,Tetsunori Kawase, Yositsugu Kitoh, Yasunaru Kawashima. Dept. of Cardiovascular Surgery, National CardiovascularCenter, Osaka, Japan

T h e changes o f T r o p o n i n - T ( T n - T ) , myosin light chain (MLC) and CPK were investigated in this clinical study related to protective ability o f the oxygenated(O) and non -oxygenated(NO) St.Thomas' solution(STS). (Patients) 46 pts operated under extracorporeal circulation (ECC) divided into 4 groups: Group A: I0 pts o f aortic valve replacement, Group B: 12 of coronary bypass grafting (CAG), Group C: 12 o f M A Z E operation (NO-STS was used in Group A, B and C) , Group D: 12 of CAG with O-STS. (Methods) Blood samples were obtained at the induction o f anesthesia, before aortic cross-clamp (ACC) on, and at 10,30 min.,l,2,3,8,12,16,22,28,40,52 hours after ACC off. (Results) In all groups,Tn-T level reached their peaks at between 120-180rain.after ACC off. T h e leakage pattern o f CPK was similar to T n - T although that o f M L C in each g r o u p was not uniform. Peak CPK and T n - T levels in group C were significantly higher than those in other groups.And there was no significant difference in CPK and T n - T levels between g r o u p B and D. In g r o u p A, peak CPK and T n - T value were corelated to ACC tin'ie. In g r o u p B and D, T n - T was corelated to ACC time. (In conclusion) 1) T n - T might be most useful a m o n g the markers used in this study.2) Maze operation m i g h t show the different pattern o f these markers leakage influenced by atrium damage.3) O-STS did not imporved myocardial protective ability.

This isolated working rat heart study was desi~nedto investigate whether the addition of amiloride(Am) or Ni to cardioplegic solution or repeffusate can improve postischemic functional recovery and to know how Na-H and Na-Ca exchange systems contribute the development of this injury. (Methods) Hearts were subjected to 20 min. of working(W) perfusion and then to 3 rain of cardioplegic infusion with the St. Thomas' solutiou(STS) followed by 37 rain. of global ischemia at 37°C. And then hearts were subjected to 20 rain. ofLangendofff(L)reperfusion followedby 20 min of W repeffusion. During pre- and post-W period cardiac function were mcasuredand expressed asthe percent of preischemic control value. Several concenlrations of Am and/or Ni2" were added to either STS or reperfusate. (Results) The addition of Am to STS did not improve postischemic recovery of function at any concentration (0.2 to 1.0rely0 although its addition to reperfusate did improve these indices at the concentration of 0.5 mM. The addition of Ni to reperfusate also improved them at the concentration of 0.1 and 0.5 IJM. And the addition of both Am and Ni ~ at optimal concentration to reperfusate did improve these indices ,however the improvement obtained from the addition of both Am and Ni to reperfuasatowas almost the same level to that of Am alone. (In conclusion) Protective ability of Am such as Na-H and/or Na-Ca exchange inhibition might not operate during ischemia when addedto STS although it became dominant during reperfusion with Na-Ca exchangeinhibition.

THE EFFECTS OF GLUCOSE UPON POST Th149 ISCHEMIC FUNCTIONAL RECOVERY IN ISOLATED NEONATAL RABBIT HEART Takumi Ishikawa*, Fumio Yamamoto, Shigefumi Suehiro*, Hiroaki Kinoshita*, Toshikatsu Yagihara, Yasunaru Kawashima. Dept of Cardiovascular SurgeD,, National Cardiovascular Center. *; Second Dept of Surgery, Osaka City University Medical School, Osaka, Japan. The major ~fference in metabolism between mature and immature heart is that immature hearts are more dependent on glycolysis for energy charge- The aim of this isolated working heart study is to investigate the effects of glucose in isdaemic immature mbbit(3-day old) heart upon functional recovery and enzyme leakageAfter measuring pre.ischemic function, hearts were subjected to 360 minutes of ischemia at 20 *C after infusion of either K.rebs-Hensdeit bicarbonate buffer(KHBB), glucose free KHBB (KHBB(-)), St.Thomas' solution(STS), or glucose (llmM) added STS(STS(+)). In multidose cardople~c infusion study, STS or STS(+)was reinfused every 60 min. Hearts were then aerobically reperfused in Langendorff mode for 15 min. Subsequently, function was measured and expressed as percent of the preischemie control value. :mearmSEM, n=6, *;p<0.05 v.s. other groups Cardioplegia %AoF(%) CKleakage(IU/dry H.W.) KHBB 64.5+ I 1.4 143.9±41.2 KHBB(-) 57.0:v-I-.0 138.4±8.9 STS 64.7+2.9 146.9±18.1 STS(+) 62.5±3. t 141.3±31.8 STS muld 62.1+_3.4 153.5±22.7 STS(+) multi 86.3:1:3.5" 111.4±22.2 These data demonstrated that glucose ad~don to cartioplegia in single infusion dd not improve protective ability of them although glucose addition to STS in multi-infusion did improve it.

CREATINE-STIMULATED R E S P I R A T I O N Th151 OF S K I N N E D F I B E R S AS S E N S I T I V E T E S T FOR INTRAOPERATIONAL HEART PROTECTION. Clinical studies. Sulling, T., Ratsep I, Toompuu M., Utno L., Saks V.A. Centre for C a r d i o v a s c u l a r s u r g e r y , I n s t i t u t e o f Chemical and Biological Physics, Tallinn, Estonia.

The purpose of the work was to study the question i f determination of the mitochondrial respiration rates in small skinned cardiac fibers is as effective test for myocardial intraoperative protection in 10atients as it is in well-defined experiments. In the group of 6 patients with ischemic heart disease bperated for coronary bypass biopsy samples (5-7 rag) were taken from left ventricle and atrium before, during intraoperational ischemia and after Operations. Duration of ischemia was 98-120 min, land c a r d i o p l e g i c protection with St. Thomas Solution was used in combination with hypothcrmia. In skinned fibers from biopsy ~amples the respiration rate at 0.1 mM ADP was increased by 20 mM creatine in average by 99 %, at the end of ischemic period the degree of i~timulation was decreased to about 27 %. However, I~fter 20 min of recovery after perfusion gtimulation by creatine increased again to 78 %. The results show very significant changes in the p a r a m e t e r studied d u r i n g b y p a s s o p e r a t i o n and OOd recovery at rvperfusion, that being dicativ¢ o f reversibility o f ischemic injury and ~fficiency of cardioplegic intraoperative ftrrtt~t, r ..........

EFFICACY OF INTRACELLULAR CARDIOPLEGIA

Th150 DEPENDS ON HIGHVOLUMEDELIVERY Paul A. Human,DieterH. Bochm*,Mike Worthington,JermiSutherland, Ulrich yon OppeU Dpt. of Cardio Thoracic Surgery,UCT Medical School, Cape Town *DpL of Cardio Thoracic Surgery,Universityof Munich Bretschneidets HTK-4 e,,,u'dioplegie solution (HI'K) has an intmecllular electrolyte equivalentcomposition,with its recommendedadministrationas a singledose on induction of cardioplegic arrest. We evaluated the efficacy of delivering HTK as either standard multidose infusions or only as single high volume dose infused over a period of seven to eight minutes on induction to ensure equilibration of component ions within the interstitial space. As a control we used the extracellnlar electrolyte equivalent St Thomas No 2 cardioplegie solution. Clutcma baboons were supported on cardiopuimena=7 bypass during three hours of hypothermic cardioplegic arrest. Animals were divided into the followingthree groups:In the £u'st group, hearts were arrested according to a multidose regimen(15 ml/kg, 80 mmI-Igmaintained with half hourly 100 ml maintenance infusions)using HTK (HTK-MD). In the second group, hearts were arrested according to a single high volume induction dose regimen (100 ml/kg, 50 mmHG)only using I--rrK (HI'K-HV). In the third group, hearts were arrested end maintaIned with St. Thomas'Hospital cardioplegie solutionNo. 2 using the multidose regimen(ST2). Precardioplegic isehaemic fibrillation fur 20 min was employed to simulate clinically diseased hearts. Postischaernic recovery is shownas a percentof praisehaemicvalues(+ SEM): CPS

n

CI

SVI

SWI

HTK-MD ? ~ . 3 + &4 TL7 _+&7 63.4+106 H'I'K-ItV "/ IW2,0+ $.6' 10l.$ ~"J.0• 121.6+_9.2| ~'s test v= SLY:" F
MW1 61.?+112 129,6+ 1 4 ~

dPldi I~i3+_ 7,7 1253 + 4.8~

|412 +_~A n $ 3 ~- IZI

I-IaemodyusmJcrecoveryof hearts protectedwith HTK was markedly improved when the solution was administered in the high volume mode. This finding emphasizesthe importance of selecting the appropriate mode of delivery where comparing solutions of differing basic design. The intracellular HTK-4, therefore, offered superior performance to the extnicellular ST2 pmvidod the appropriate administration regimen was employed.

Th 152 CREATINE STIMULATED RESPIRATION OF SKINNED CARDIAC FIBERS AS A SENSITIVE TEST FOR EARLY ISCHEMIC DAMAGE DURING HEART PRESERVATION.

Laurence Kay, Zoya Daneshrad,Valdur Saks & Andr6 Rossl. Lab. Bio6nergdtique, Universit6 J. Fourier, Grenoble, France. The skinned fiber technique was used to analyse the alterations of mltochondrial respiration following hypothermlc isolated rat head preservation and global normothermlc ischemia. The experimental protocol of respiration rate determination included a cytochrome c test to check the intactness of the outer mitochonddal membrane, kinetics of regulation by ADP, and creatine test for assessment of the functional coupling between creatine kinase (CK) and adenine nucleotide translocase (ANT). The experiments revealed that one hour normothermic ischemia or 15h preservation by immersion of the head In cardloplegic solution at 4°C, induced outer membrane mitochondrial damage and a loss of the functional coupling of CK and ANT. Preservation of the heads under low-flow pedusion of cardioplegic solution (0.3mL/min), at 4"C for 15 hours, resulted in preservation of coupled CK reaction and intactness of outer mitochonddal membrane. The changes in creatine stimulation of the respiration correlated well with left ventdcular developed presslon on repeduslon and free inorganic phosphate concentration (NMR determined) at the end of preservation. It is concluded that the most sensitive indicators of eady ischemic damage to mltochondria are the functionally coupled CK reaction and intactness of the outer membrane. Determination of these parameters may be a simple and rapide test for cardiac protection.

t ;/~n

A211

RESTING MEMBRANE POTENTIAL IN THE COLD Th153 STORED RAT HEART: POLARISED vs. DEPOLARISED ARREST Andrew K Snabaitis, Michael J Shattock, David J Chambers. Cardiac Surgical Research, St Thomas' Hospital, London, UK. Hypothermia and hyperkalaemic cardioplegic solutions are currently used for donor heart preservation. Hyperkalaemiainduced depolarisationof the resting membrane potential (Ern) may predispose the heart to Na+ and Ca2+ loading, exacerbating injury and limiting the safe storage duration. The aims of this study were to establish whether (i) reversible sodium channel blockade with tetrodotoxin (TTX) could induce and maintain po/arised arrest, and (ii) this confers significant protection. Isolated working rat hearts (n=8/gp) were aerobically perfused (37°C) with KrebsHenseleit (KH) buffer (20rain) and control pre-ischaemic aortic flow (AF) measured. Hearts were arrested with a 2ml infusion (20°C) of either KH (control), KH+high K+ (16mM) or KH+TTX (221~M) and the heart surface slowly superfused with an identical solution at 7.5°C during 5h storage. Polarised arrest was confirmed during storage by measuring Em via conventional 3M KCl-fiUed intracellular microelectrodes inserted into the left-ventricular free wall (see Figure). Recovery of AF (% of control) was determined during working mode (20rain) following Langendorff -40 KHB+K+(16mM) reperfusion (15min), and was higher in the TI'X gp (48+3%) than in the high K + (37+4%) KHB(control) and control (28+8%; p
RAMIPRILAT DURING REPERFUSION Th155 Bruno Podesser, Martin Zegner, Gerda Koci, Seth HallstrOm, Gregor Wollenek. Dept. of CardioThoracic Surgery, General Hospital Vienna, Austria ,In an experimental study adult white Elco-rabbits were treated with ramiprilat (ram) (2.58x10-6 mol/I) during reperfusion after 60 minutes of cardiac arrest ,on an isolated blood-perfused "working heart" model. The aim was to determine whether known antiischemic effects of ramiprilat on high-energy phosphates (HEP) can be obtained also, if offered only during reperfusion. ~myocardial biopsies (,umol/g wet weight/HPLC). HEP ram(9) control (9) p-value ATP 2.659-J:0.19 1.941+0.399 n.s. ADP 0.64+0.076 0.703+0.081 n.s. AMP 0.089-J:0.025 0.172:1:0.073 n.s. GTP 0.147+0.01 0.102:1:0.02 0.05 cGMP 0.049-J:0.03 0.03+0.005 0.01

These results indicate that GTP and cGMP, the substance responsible for vascular smooth muscle relaxation, are significantly higher in the ramiprilal group. Consequently it can be speculated that reperfusion-induced injuries such as low coronary flow followed by arrhythmias might be attenuated. Further studies on hemodynamics during reperfusion under ramprilat will have to follow. A212

EPICARDIAL VENTRICULAR ACTION POTENTIAL Th 154 REPOLARISATION DURING GLOBAL ISCHAEMIA IN HUMANS Peter Sutton, Wilfred Pugsley, Mark Boyett, Max Lab, Peter Taggart. Middlesex Hospital, Charing Cross Hospital, London and University of Leeds, UK. Experimental studies recording action potentials during simulated ischaemia have shown a sequence characterised by a rapid early repolarisation followed by subsequent decay of the plateau in contrast tu uniform shortening on endocardium. We recorded monophasic action potentials (MAP) from the left veturicular epicardium in 8 patients (14 sites) following the initiation of cardiopalmonary bypass. Novmothermia was maintained and epicardial temperature monitored. Right ventricular pacing was employed and beats of shorter cycle length interpolated. Global ischaemia was created by aortic cross-clamping for two 3 minute periods separated by 2 minutes reflow. After 3 minutes of ischaemia action potential duration at 30% repolarisation (APD~) shortened by 39(13)ms (mean(SEM)) from control (P < 0.01) and at 100% repolarisation (APDtoo) shortened by 16(5)ms (P<0.02); after 6 minutes of ischaemia APD30 shortened by 67(22)ms (P<0.02), and APD~00 shortened by 30(10)ms (P<0.02). Repolarisation shape changes typical of experimental epicardial preparations were frequently observed resulting in a progressive decrease in the ratio APD~o/APD~o0(P<0.02 after 3 minutes and P<0.01 after 6 minutes). We conclude: MAPs recorded from the human epicardium during global ischaemia showed a progressive decrease in APD and the ratio APD~/APD~co. Repolarisation shape changes for steady state beats correspond closely with experimental studies but contrast for premature beats. These findings may relate to differences in the dominance of currents during repriming in the ischaemic human heart compared to animal species.

H A L O T H A N E AND POSTTh156 CARDIOPLEGIC RECOVERY Amanda Lochner, Grant Trollip, S. Gcnade. MRC Exporimcntal Biology Programmc; Dept. Medical Physiology and Biochemistry, Univcrsity of Stcllcnbosch, Republic of South Africa. Halothane has been shown to be a powerful cardioprotectant during normothermic cardioplegic arrest and reperfusion(t). Its mechanism of action may involve the voltage depeedent Ca'* channels and/or Na*/H * exchanger. The aim of this study was to compare halothane's effects on post-cardioplegic recovery with those of nifedipin¢, amiloride and HOE 694. Our hypothesis was that if the effect of the combination of halothanc and a drug was similar to those when administered separately, they may have similar mechanisms of action. Conversely, if the effects of the combination of halothane and the drug exce~ those when administered separately, they may have different mechanisms of action. Methods: Isolated working rat hearts were subjected to 45 rain normothermic cardioplegic arrest followed by reperfusion. Halothane (1.5%) was administered during cardioplegia. Nifedipine (10 .7 M), amiloride (0.1 raM) and HOE 694 (10 "~M) were administered in the perfusate. Results: Halothane, nifedipinc, amiloride and HOE 694, when administered separately, significantly improved functional recovery. Combination of halothane and nifedipine did not further improve recovery, while halothane plus amiloride caused complete mechanical failure upon reparfusion. Simultaneous administration of halothane and HOE 694 significantly improved recovery. Conclusion: Halothane probably mediates its protective effects via the voltage-dependent Caz* channels. (1) Coetzee AR et al. Anest Analg 1991; 73:711 - 719.

THE COMBINED EFFECTSOF PROTAMINESULFATETh157 AND ISOFLURANEON THE ISOLATEDRAT HEART RECOVERING FROMCARDIOPLEGIC ARREST Edith Hochhauser, Pinchas Halpern, Victor Zolotarski, & Bernado Vidne. Cardiac Res.Lab. FMRC, Beilinson Campus, Petah Tikva, Israel. Protamine sulfate (P) is currently used for reversal of heparin anticoagulation. Isoflurane (IS) is used as an alternative to narcotic anesthesia. The purpose of this study was to compare the combined effects of P and IS on the isolated rat heart with (+) and without (-) cardioplegic ischemia. Methods: The isolated rat hearts were perfused with Krebs Henseleit (KH) 30 min: thereafter with KH, IS 1.5%, P 10#g/ml or IS+P for 15 min; and 60 min KH. We also subjected the hearts to cardioplegic (KCI 16 meq/L) ischemica (ISC) 30"C 30 min. Then the effect of the drugs was tested. For results of drug effects on +dP/dtm (dP) & CF (% of base line values) see table"lSi~low(Mean±SD,n=8-10): Control P IS IS+P dP CF dP CF dP CF dP CF 91±13 98±3 40±19 51±24 78±8 105±14 39±8 72±19 + 98±19 98±9 20±7 47±1082±9 108±9 45±6 70±15 Conclusion: IS caused a small depression of c o n t r a c t i l i t y which reverted to baseline. P in ISC and non-ISC hearts, caused a marked decrease in c o n t r a c t i l i t y and CF which did not revert to baseline even after 60 min of KH reperfusion. IS with P had a protective effect on the degree of depression of c o n t r a c t i l i t y and CF of the ischemic hearts. -

MECHANISM OF THE CARDIOPROTECTIVE ACTION T h 1 5 0 OF OROTIC ACID Stephen M. Richards, Robert A.J. Conyers, Jane L. Fisher, Franklin L. Rosenfaldt. Baker Institute, Melbourne, Australia Background: Orntic acid (OA) protects the recently infarcted heart against ischaemic damage in dogs and rats. It is thought that OA corrects a relative deficiency of pyrimidine nucleotides in the surviving, hypertrophying myocardium, but the bulk of OA is taken up by the liver and < 1% reaches the heart. Aim: To investigate the metabolism and mechanism of OA protection of infarcted hearts. Methods: Normal rats were given 100mg/kg OA and liver, plasma and cardiac pyrimidine levels monitored. In a second study coronary artery ligation was followed by 30mg/kg/d OA for 2d and either OA metabolites assayed or hearts perfused and subjected to 30rain of cardioplegie arrest and ~recovery of function (ill) assess~. Results: While in normal hearts a single dose of OA raised in turn, liver, plasma and heart pyrimidine levels (heart :EUTP peaked 121% at 4h), in OA-treated, infarcted hearts pyrimidine nuclentide and RNA levels in surviving myocardium did not differ significantly from infarcted controls (p=0.83; p=0.90 respectively). %Recov fn Pre-ATP Pro-TAN

Sham 62.2+4.0 21.7+0.8 30.3+0.8

Infarct 26.4+9.1"* 14.7+0.7" 22.4+1.1"

Infarct + OA 56.6+5.5 20.7+0.5 29.1+0.6

*Infarct vs Sham: p<0.05; **Infarct vs Sham: p<0.01. Preischaemic ATP and total adenine nueleotides (TAN) were significantly reduced in infarcted hearts, while OA treatment prevented the reduction. Conclusions: OA improves function by preventing the depletion of TAN, rather than correcting a relative deficiency of pyrimidines, so that OA-treated, infarcted hearts enter ischaemia with higher energy reserves.

MAGNESIUM ATTENUATES THE DETRIMENTAL Th158 EFFECT OF HIGH POTASSIUM IN CARDIOPLEGIA Ganghong Tian, Bo Xiang, Christopher Lindsay, Hooman R. Ghomeshi and Roxanne Deslauriers. Inst. for Biodiagnostics, National Research Council Canada, Winnipeg, Canada. This study was to determine whether magnesium (Mg ++) can attenuate the detrimental effects produced by high concentrations of potassium (K +) in cardioplegia. After 15 rain perfusion with K-H solution, isolated rat hearts were arrested with a solution containing 22 mmol/L K*, 1.2 mmol/L Mg ++ for 10 mins. The K~" concentration was subsequently raised to 45 mmol/L with either 16 mmol/L Mg ~ (group I) or 1.2 mmol/L Mg ++ (group 11). Hearts were then maintained in the solution for 25 rains. The temperature of the hearts was 37°C. 3~p NMR spectroscopy was used to monitor the changes in energy phosphates, and intracellular pH(pH0. We found that the hearts in group I showed a significantly slower decrease in ATP, PCr and pHi with a correspondingly slower increase in inorganic phosphate (Pi) during the 45 mmol/L K ÷ cardioplegia period compared to the hearts in group II. During reperfusion, the recovery of contractile function (rate-pressure product, 91% vs. 28%), ATP (60% vs. 22%), PCr (90% vs. 42%), Pi (110% vs. 250%) pHi (7.12 vs. 6.93 pH unit) and coronary flow (16 ml/min vs. 10 ml/min) was significantly improved in group I compared to group II. Moreover, myocardial contracture was also significantly delayed and attenuated by 16 mmol/L Mg H. The results indicate that Mg ~+ can attenuate some detrimental effects of high K +. This is postulated to be related to its reduction of Ca ~ influx which can be activated by high [K÷].

THE

EFFICIENT

GENE

INTO MYOCARDIUM

TRANSFECTIONTh160

BY CORONARY

INFUSION OF HVJ-LIPOSOME. Yoshiki Sawa, Yasuhisa Shimazaki, Hong-Zhi Bai, Ken Suzuki, Yasnfumi Kaneda* and Hikarn Matsndn. 1st Dept. of Surgery, Inst. of Mol. and Cell. Biol.*, Osaka Univ., Osaka, Japan To improve in-vivo gone transfer into myocardium, a gone transfer method of I-IVJCdemagglutinating virus of Japan)-liposome can be alternative. In this study, in-rive gone Iransfection of FITC(F) labeled oligonuclentide(ODN) was examIned using HVJliposomeCH group) or only lipesome(L group). After the harvest of donor rat hearts arrested by cardioplegia, coronary artery was infused with lipesome-gene complex through aortic cannula during cardioplegic arrest, transplanted(anoxic time: 30min.) into the abdomen of the recipient rat of the same strain and sacrificed after 3 days of transplantation. All hearts showed no rejection at the time of sacrifice. H group showed apparent F staining in the nuclei of myocytes and coronary endothelial cells in the whole layer of myocardium as compared to L group which showed very low F staining. Intensity of F was significantly higher in H group(939±l12 F.I.) than L group(166±TgF.L). These results demonstrated that donor hearts were transfecled with F-ODN in the whole layer of myocardium by coronary infusion of HVJ-liposome during cardioplegic arrest at the time of harvest. Our method appears to be a novel in-rive gone transfer technique for the heart, and may provide a new tool for researches and therapies of cardiovascular disease. A213

LEFT HEART BYPASS IN THE PIG WITH PERCUTANEOUS CANULA: BRIDGE FOR TRANSPLANTATION B.H.WaIDoth. V. Mehan, R. Rogulenko, G. Vucic, D. Mettler, B. Meier Thoracic and Cardiovascular Surgery, Cardiology and Experimental Surgery, University of Berne, 3 0 1 0 Berne, Switzerland

Th161

A rapid and efficient circulatory support system may save a patient in cardiogenic shock. Left heart bypass with percutaneous and transseptal placement of the aspiration canula simplifies the circuit and eliminates the need for an oxygenator. We assessed this preclinical set-up in 5 anaesthetized pigs using a centrifugal pump w i t h a 14 F arterial catheter and a 16 F left atrial aspiration line. Animals were supported for t w o hours at a mean flow of 3.1 liters (3,680 RPM), a mean hematocrit of 2 9 % and low heparinisation (ACT double baseline). Hemodynamic and laboratory samples were taken at baseline (A), 10 minutes (B), one hour (C) and 2 hours (D) on support. Results (mean values): TimoPufse A 94 B 74 C 74 D 82

Pressures (mm fig) A.Sysl A.MeanA.DlasLVED CO 90 69 57 4 6. I 79 68 6~3 I 1 6,5 78 65 60 12 6.3 71 59 54 16 6.9

V~/hen the pump was turned on (0-3 litrest there was usually a decrease in heart rate, systolic pressure and left ventricular pressure, w i t h unchanged cardiac output (non failing model). Potassium increased from 3.9 - 4.2 mmol/I and plasma haemoglobin from 6.0 - 18.2 mg/dl. Thrombocytes decreased from 187155 lOS/1. In conclusion, this preclinical support study has demonstrated the feasibility of an efficient (3 Iitres/minute) short time left heart bypass support with percutaneously implantable canula.

CEREBRAL PERFORMANCE LATE AFTER ACCIDENTAL OR INDUCED DEEP HYPOTHERMIA AND CIRCULATORY ARREST

Th 163

B.H. Walooth. B.N. Walpoth-Aslan, H. Mattle, G. Schroth, B. Radanov, A. Fischer, L. v. Segesser and U. Althaus Thoracic and Cardiovascular Surgery, Neurology, Neuroradiology and Psychiatry, Universities of Bern, Lausanne and Zurich Switzerland Late sequels with special regard to cerebral lunciion have not been reported yet Ior patients rewarmed by cardiopulmonary bypass (CPB) following accidental deep hypolhermia (ADH) with circulatory arrest (CA). In a comparative study 15 patients (8 males. 7 lemales, age 11-51 years) after CPB rawarming for ADH with CA were assessed (group A) and matched to 15 patients (13 males, 2 females, age 29-6g years) submitted to induced hypothermic CA during cardiac surgery (group B). The mean follow-up for patients of both groups was 6.5 and 5.5 years respectively. Cardiac arrest was signilicanfly longer in group A (mean 2.5 hours vs. 0.6 hours in group B; p < 0.02) bur mean age was higher in group B (51,9 vs. 32.0 years; ns). The degree of maximal hypothermia was similar in both groups (mean 22.1°C in group A vs. 20.1°C in group B). The specific tests show the following results (number el patients): Test Neurological status

Borderline Group B Group A 9 5

Pathologic Group A (peripheral lesions in 2) 1 0

GroupB 0

8 0 i EEG 6 1 6 Doppler flow 1 measurements 6 2 (1 Poly5 Brain MRi with 1 trauma) volumetric analysis 4 1 ,2" Neuropsycholooical test 2 conclusions: All survivors of accidental hypofhermia feel healthy and are free of symptoms st follow-up. Patients of group A show significantly less alterations in the doppler flow measurements (p < 0,01) and in brain MRi (p < 0,003) than patients of group B (age and pathology related). These results confirm that resuscitative measures are very promising for ADH patients even alter prolonged cimulatory arrest.

A214

ASSESSMENTOF TROPONIN-TFOR DETECTIONOF CARDIAC REJECTION Th162 B.H. Walpolh. A. Tschopp, E. Peheim.T. Schaffnerand U. Althetls Thoracic and Cardiovascular Surgery, Clinical Chemistry and Patholnay, University of Berne, 3010 Berne. Switzerland Non Inveslvo detection of rejection after heart transplanlation is slill unsolved. We assessed a new myocardial protein serum assay, Troponln-T (TNT) which shows a high sensitivity f87%f and specificity (92%1 in myocardial iachemla. Fortyfour cardiac heterotopic rat transplnnts were perlomled using isografts (LEW-LEWI or allografts (DA-LEWI with end wlthoul Immunosuppression, One week after transplantation blood samples were drawn for an,~lysisof cfPatlne phospho.kinsse (CK}, CK-MB and TNT and compared wBh histologlc grndlng el rejection according to Sillfngham (0 =none, 3 =severe] and Iho amount of viable myocardlum. Results (mean$;'p < 0,05;# p< 0,05 vs grade O): Grade CK IU/L) CKMB (U/L) TNT (ug/L) 0 (n=ff 70 3.5 O,1 I (n = 20) 682 8,1 0,5 II (n =9) 578 I 9,1 2,0 Ig In=9] 305 58 # 3,3 # With increasing degree of rejection TNT shows a gr.'lduol ar,d significant (p <0,05) elevation in all reJection gradings when compared Io he,'trts wilhout reiection. In addition the differences were atalisticelly siglliticnnl between rejection grade none to mild end mild to moderate. Furthermore a signiflcont correlation (p < 0,0tl exists between TNT and the degree of rejeclion yielding the best correlation factor of 0,66 when compared to the other serum enzymes. The amount of viable myocerdiumis decreasedwith the degree uf rejection: 0 = 92 %, I = 84 %, II = 77 % and III = 5S % (p < 0.05 III vs O. I.IIL The correlation factor between viable myocardium and rejcetion is only 0,46 (P < 0,03); thtlS we feel that TNT measures other cellular clmngns besides myocytolysls, especially in mild relectlon. In conclusion, Troponin.T may be used as an early and sensitive serum marker for detection of cerdlac rntoclion even before severe myocylolysis becomes evident. However these findings should be confirmed by humanstudies,

R E C O V E R Y O F C A R D I A C FUNCTION A F T E R TRANSPLANTATION

IS

RELATED

TO

THE

Th164 METABOLIC

S T A T E O F T H E C A R D I O P L E G I C H U M A N DONOR HEART. J. a n n e v a n D o b b e n b u r g h , J a a p R. L a h p o r , S t e p h e n R. Woolley, Nlcolaae de Jonge, Corinne Kl6pplng &Cees J.A. v a n E c h t e l d . H e a r t L u n g Institute, University Hospital, Utrecht, The Netherlands. A reliable and rapid test of myocardial viability during cold cardioplegic preservation could be of great value. In this study the m e t a b o l i c condition of 25 h u m a n donor hearts was evaluated prior to transplantation(HT'X) by a 6 min alp MRS scan at 1 . 5 T and O°C; phosphocreatine(PCr)/ATP, inorganic p h o s p h a t e ( P i ) / A T P , p h o s p h o m o n o e s t e r s ( P M E ' ) / A T P ratios and p H w e r e calculated a n d extrapolated to values at the onset of reperfusion(R), using linear regression of the pooled data. Right heart h e m o d y n a m i c s w e r e m e a s u r e d with a S w a n - G a n z catheter during the first hours and 1 w e e k after R. No significant correlation(C) w a s f o u n d between the cardiac index(CI) at the first hours after H T X and extrapolated P C r / A T P , Pi/ATP, PMF_.JATP and pH, possibly d u e to general anesthesia, inotropic support, cardiac stimulation and stunning. H o w e v e r , 1 w e e k after H ' I X significant C between CI and e x t r a p o l a t e d P C r / A T P and P M E / A T P (r=0.49, p=0.01 and r=-0.45, p=O.02, resp.) w a s found. In contrast, extrapolated P i / A T P a n d p H s h o w e d poor C with CI (r=-0,10, p>0.5 a n d r=0.31, p>0.1, rasp.). C b e t w e e n CI and P C r / A T P m a y be related to creatine loss during early R and slow replenishment. C b e t w e e n CI and P M E / A T P is less clear. T h e a b s e n c e o f C b e t w e e n CI and P i / A T P and pH m a y be due to w a s h o u t or redistribution during 1 w e e k of R. In conclusion, alp M R S m a y be used as a m e a s u r e of functional viability of the cardioplegic h u m a n d o n o r heart.

HISTOLOGICAL CHANGES OF Th165 CARDIAC NERVES AFTER HETEROTOPIC HEART TRANSPLANTATION IN R A T S Yoshihiro Kurisu, Yuichiro.Mastuura, Taijiro Sueda, Kazumasa Orihashi, Hiroki Kajihara* & Yoshio Kate**. 1st Dept of Surgery, Inst of Health Sciences*, Hiroshima Univ Sch of Medicine, Miyoshi Medical Association Hosp.**, Japan. Distribution of cardiac nerve fibers after heart transplantation remains unclear. In this study, both catecholamine- and peptide-containing nerve fibers were examined immuno-histochemically in normal and denervated conditions using syngeneic heterotopic heart transplantation model. The immunohistochemical staining was performed by the indirect method with the following polyclonal antibody; anti-neuron specific enolase antibody against the neuron, anti-tyrosine hydroxylase antibody against the catecholamine-containing neuron, anti-calcitonin gene- related peptide (CGRP) antibody and anti-neuropeptide Y (NPY) antibody. In the transplanted hearts, nerve fibers gathered like a bundle and axonal varicosities were unclear. Reinnervation waq not observed. Catecholamine-containing nerve fibers decreased. But, after 24 weeks, a few were observed only in the atria. CGRP-containing nerve fibers decreased only slightly. Almost all were retained. NPY~ containing nerve fibers disappeared completely. It w a s suggested that the transplanted heart remains under hypoadrenergic status for at least 24 weeks aftet transplantation and that CGRP-containing neurons might be dominant in the transplanted hearts of rats.

Th167 TIME COURSE OF ENDOTHELIAL FUNCTION IN CONDUIT CORONARY ARTERIES AND IN THE MICROCIRCULATiON AFTER CARDIAC TRANSPLANTATION Andreas Hartmann, Nora Marzilli, Michael Weis, Hans-Georg. 01brich, Wolfram Burger, Peter Satter, Depts. of Cardiology and Cardiothoracic Surgery, University Medical Center, Frankfurt, FRG It has been assumed that endothelial (END) dysfunction after cardiac transplantation (TX) may progress into transplant vasculopathy,(TVP). We investigated changes of END function in conduit coronary arteries and in the microcirculationduringthe longterm course after TX in 3 groups of patients (P): Group I: 17 P up to 2 years after TX; group I1:11 P 2 to 4 years after TX; group II1:16 P more than 4 years after TX. Changes of conduit coronary artery diameters (DIA) were investigated after END-dependent and ENDindependentstimulation with acetylcholine(A 50 and 100 pg i.c.) and nitrates (GTN 0.3 mg i.c.). Coronaryflow changes(FLOW)were determined END-dependent (ACh 50 and 100 pg i.c.) and ENDindependent (dipyridamole 0.56 mglkg i.v.[DIP]) utilizing an 8 F Judkins-styleDopplercatheter in the left main coronary artery. Results:(% changes, mean + SD) group I group II group III DIA FLOW DIA FLOW DIA FLOW ACh50 -15 + 217 + -9 + 113 + -12 + 108+ AChl00 -16 + 236 + -19 + 77 + -16 + 109 + GTN 14 + 14 + 12 + DIP 296 + 63 +* 113 +* (+p < 0.05 vs.baseline*p < 0.05 vs. group I) A constant vasosonstrictor response to ACh in conduit coronary arteries indicated that END-dysfunctionafter TX is independentof the time course. END-dysfunction in these vessels may not be a precursor of TVP. END-dependent and independent flow reserves decreased 2 years after TX and remained constant thereafter.

Th166 CHAMBER AND MYOCARDIAL ELASTIC PROPERTIES OF HETEROTOPICALLY TRANSPLANTED RAT HEART. 'Franti~ek Papou~ek, 'Franti~ek Kolfi[', =Cathy MacNaughton, =Borivoj Korecky. '[nst of Physiol, Acad Sci., Prague, Czech Republic and 'Dept of Physiol, Univ of Ottawa, Canada. The relationship between chamber stiffness and intrinsic elastic properties of myocardial tissue was studied in atrophic left ventricles of inbred Lewis rats after heterotopic heart-lung transplantation. This was evaluated from pressure-volume (P-V) and stress-strain (a-e) curves in the isolated perfused hearts under isovolumetric conditions. The decrease of the ventricular mass to 43 % within 28 days after surgery was associated with opposite changes in chamber and myocardial stiffness (dP/dV and daME respectively)" whereas the constant of chamber stiffness ct (dP/dV= oLP+const.) increased to 215%, the constant of myocardial stiffness k (da/d8 = ktr+const.) decreased to 76% of corresponding recipient values. Following normalization of volume differences dV to instantaneous cavity volume V, an approximately linear relationship between normalized chamber and myocardial stiffness, expressed as dP/(dV/V)~ const. (da/d~), was observed. The constant of normalized chamber stiffness 7 (dP/(dV/V) = 7P+const.) decreased after transplantation similarly as the constant k to 77%. Relationship -/= 0.24k - 1.07 with a correlation coefficient r = 0.96 was proved between these constants. It may be concluded that the chamber stiffness at any point of P-V curve is directly proportional to myocardial stiffness and indirectly related to chamber volume under our conditions. Moreover, the constant 7 derived simply from the course of dP/(dV~10 may serve to indicate changes of myocardial elastic properties instead of the conventional constant k, which requires further mathematical transformations.

XENOREACTIVE NA~ANTIBODIES Th168 (XNA) - C A R D I O D E P R E S S A N T EFFECTS IN C A R D I O M Y O C Y T E S OF N E O N A T A L RATS (RCM) Ursula M~ller-Werdan, B e r n d Koidl*, Arnd Autenrieth, Dieter Klein**, Karl Werdan, Claus Hammer**. Dept. of Med. I and **Institute for S u r g i c a l R e search, K l i n i k u m Gro~hadern, University of Munich, Germany, and *Institute for M e d i c a l P h y s i c s and B i o p h y s i c s , U n i v e r i s t y of Graz, Austria. In x e n o t r a n s p l a n t a t i o n XNA, immunoglobulins rather n o n - s p e c i f i c a l l y recognizing several antigens, are supposed to t r i g g e r the h y p e r a c u t e r e j e c t i o n and d y s f u n c t i o n of the donor organ. In s p o n t a n e o u s l y beating RCM, XNA present in t h e s e r u m of man, p i g a n d o t h e r species, induce a s t e r e o t y p e p a t t e r n of altered c o n t r a c t i l i t y with a temporary standstill of b e a t i n g for a c o u p l e of m i n u t e s , which could be p r e v e n t e d by the a b s o r p t i o n of XNA. H a v i n g s p o n t a n e o u s l y resumed contract i l i t y , t h e c e l l s of t h e s y n c y t i a l m o n o l a y e r c o n t i n u e to b e a t a s y n c h r o n o u s l y for 12 hours after the addition of the X N A - c o n t a i n i n g serum. In adult g u i n e a pig v e n t r i c u l a r c e l l s - u n d e r the same conditions - the main elect r o p h y s i o l o g i c a l e f f e c t of the s e r u m was an e n h a n c e d excitation threshold.

A215

THE VIABILITY OF CARDIAC MYOCYTES TRANS-Th169 PLANTED INTO HEART. Thomas Nagano, Tomoko Tsuchiya, Atsusht Takeda*, M i t s u t o s h i Kato* and Makoto Nagano*. Department o f Surgery and Organ T r a n s p l a n t a t i o n , The I n s t i t u t e of Medical Science, The U n i v e r s i t y of Tokyo. J i k e i U n i v e r s i t y School of Medicine*.Tokyo. The decrease of c a r d i a c myocytes a f t e r myoc a r d i a l i n f a r c t i o n and cardiomyopathy plays an i m p o r t a n t r o l e f o r c a r d i a c performance. This study was performed t o examine the v i a b i l i t y o f t r a n s p l a n t e d c a r d i a c myocytes i n t o in vivo hearts.The used animals were hamsters. Cardiac myocytes used for transplantation were isolated from embryonic and neonatal hamster cardiac myocytes and then cultured for 24 hrs. The c u l t u r e d c e l l s 5x10 /0.1ml were t r a n s p l a n t e d i n t o the v e n t r i c u l a r f r e e wall of a d u l t S y r i a n hamsters.Control animals t r e a t e d by an i n j e c t i o n of s o l u t i o n ( O . l m l ) f o r c e l l suspension. As tmmunosuppressive t r e a t m e n t , c y c l o s p o r t n A was used during the observ a t i o n p e r i o d a f t e r t r a n s p l a n t a t i o n . Thece11 viability o f the t r a n s p l a n t e d myooytes was examined immunohistochemtcally. C e l l i n f i l t r a t i o n in the t r a n s p l a n t e d area as sign o f tmmunorejeotton could not be observed. Someof these c a r d i a c myocytes t r a n s p l a n t e d i n t o the h e a r t s u r v i v e d . But a t t h i s time, i t can not be e s t a b l i s h e d whether they develop hyperp l a s i a and h y p e r t r o p h i a or not.

A216

Friday 7 July 1995 Abstracts Fr1-Fr187 Hypertension Cardiac hypertrophy Heart failure Catecholamines Ischemia and reperfusion Varia

Frl-Fr15 Fr16-Fr59 Fr60-Fr94 Fr95-Fr116 Fr117-Fr170 Frl 71-Fr 187

NEW TRANSGENIC ANIMALS IN Fr001 HYPERTENSION RESEARCH liidenori Urata,Slgfrid Hoffmann,HikaruNishimura, Martin Paul, Detlev Ganten, Max-Delbrfick-Centrefor Molecular Medicine (MDC), Robert-R6ssle-Strasse 10, 13122 Berlin-Buch, Germany

The genetic defects causing primary hypertension are still unknown, however, various regulatory systems have been identified and studied for their roles in regulation of cardiovascular function. The use of molecular biologic techniques has demonstrated that these regulatory systems not only act via the circulation as hormones but that the genes of the components of these systems are ex'pressed in cardiovascular organs where they may have important, local functions. This seems to be the case for e.g. the renin-angiotensin system, the endothelins, nitric oxide, and the catecholamines. It is, however, still not clear how each component of these systems can contribute to pathophysiological changes observed in cardiovascular diseases in vivo. To address this problem, we have developed several independent transgenic rat lines harboring target genes such as mouse renin 2, human renin, haman angiotensinogen, endothelin 2, chymase, and angiotensin II subtype 1 receptor (ATt-R). These transgenic models are useful not only to examine roles of each gene in a systematic fashion and in various pathophysiological conditions in vivo but also to study detailed mechanisms of gene expression. Recently, we have developed transgenic rat harboring human chymase gene [TGR(h-Chymase)193], which showed normal grox~ethand blood pressure with a slight cardiac hypertrophy. In addition, another transgenic line expressing human AT,-R specifically targeted in the heart ITGR(hANF-hATcR)I also showed a cardiac hypertrophy without any changes in blood pressure. Those results obtained from newly developed transgenic rats indicate that, independent of the effects on blood pressure, direct angiotensinergic input to the heart appears to be an important determinant in e.g. development of cardiac hypertrophy.

EFFECTS OF HIGH CALCIUM INTAKE ON HEART Fr003 FUNCTION IN SPONTANEOUSLY HYPERTENSIVE RATS Milada Tutterovd, Hana Vav~inkovd, Ludmila Kazdovd, Antonin Vr~na, Ji[i Heller. Inst.Clin.Exp.Medicine, Prague, Czech Republic Epidemiological studies have demonstrated an inveme relation between Ca intake and blood pressure (BP). Some clinical observations and experimental studies of hypertension have shown that a high calcium (Ca) intake reduces BP. This effect was tested in spontaneously hypertensive lines and normotensive controls selected from a single parental pair of outbred Wistar rats in our Institute (Prague Hypertensive and Normotensive Rats: Heller et al. Clin. Exp. Hypertens.; 15:807,1993). The effect of a high Ca intake on functional and metabolic parameters of the heart was also tested. Normotensive and hypertensive young, 6-week-old rats (PNR-Y and PHR-Y) and adult 24-week-old rats (PNR-A and PHR-A) .,drank water or 1.5% (w/v) of Ca chloride for 3-4 weeks. High Ca intake decreased BP by 17 mmHg in PHR-Y and by 21 mmHg in PHR-A without affecting BP in PNR. No effect was observed on heart dry weight and ATP or glycogen content in any group. There were no differences in total serum and ionized Ca in PHR and PNR drinking water. On the other hand, increased Ca intake led to increased serum total and ionized Ca levels in PHR-Y. In the isolated heart, perfused according to Neely, the values of its mechanical parameters were not changed by hypertension in PHR-Y and PHR-A. High Ca intake led to a reduction in coronary flow by 30%, aortic flow by 30-40%, and stroke volume by 25-28% in young rats of both lines. High Ca intake decreased BP in PHR-Y, raised serum Ca levels and had an adverse effect on cardiac output. High Ca intake in PHR-A decreased BP while leaving dynamic heart parameters unaltered; only tolerance to ischemia was reduced. The results indicate that the decreased BP after Ca supplementation, observed in PHR, was associated with cardiac function impairment in young rats and a lower resistance to ischemia in adult rats.

A218

GENE THERAPY FOR HYPERFr002 TENSIVE CORONARY VESSELS. Pedro C u e v a s , V i v e n c i o Barrios*, Diana Reimers, G u i i l e r m o G i m 6 n e z - G a l l e g o * * . Depts. H i s t o l o g y and C a r d i o l o g y * . Hospital R a m 6 n y Cajal. C S I C * * . Madrid. Spain. Acidic and basic fibroblast growth factors (aFGF and b F G F ) are hypotensive and angiogenic peptides participating in coronary blood flow and collateral vessel growth after myocardial ischemia. Moreover, a progressive decrease of endothelial F G F expression is associated to progression of hypertension in SHR, suggesting a decrease capacity for heart collateral vessel growth and endothelial vasomotor disburbances in the hypertensive heart. Thus, exogenously administered FGFs could be effective to correct F G F endothelial cells deficiency, directly transfecting endothelial cells of the coronary vessels with genes encoding FGFs. Human aFGF recombinant gene was incorporated in the p M A M a F G F expression plasmid, packaged into cationic liposomes and systemically administered in 18 month old SHR ( n = 6 ) . Control SHR received plasmid p M A M n e o ( n = 6 ) . Animals were sacrificed 3 months later and expression of vector encoding aFGF was determined in coronary vessels using anti-aFGF immunohistochemistry. Transfected coronary vessels showed high expression of aFGF protein whereas control non-transfected animals did not. These data suggest that a F G F gene systemic lipofection appears as a safe and efficient procedure to correct the deficient aFGF expression in hypertensive coronary vasculature.

THE INFI/JENCE OF CALCIUM CHANNELS B ~ S FRO04 ON THE LIPIDS METABOLISM Costas L.Petrogiannopoulos,Antonis K.Zaharof, John G.Panagopoulos,Lambros G.Lambropoulos and John P. Poulikakos.Dpt of Medicine, Hellenic Red Cross Hospital. The aim of the study was to investigate the effect of calcium channel blockers on the lipids metobolism in hypertensive patients with dylsipidemia. Matterial and methods:The study included 35 dyslepidemic patients with mild hypertension(25 men and i0 women age 49-57years).All the patients started one step diet for the dyslipidemia while the/were receiving nifedipine (Adalat)20-40mg daily for one year. Every two months lipids control was doing . Results:During the 1 year treatment with nifedipine the levels of TC, reduced from281_+9mg% before therapy to 223_+7mg% after, the levels of LDL-C and TGS from 216+--4 to 143_+9n~% and from 160+7 to 99_+12mg% respectively while the levels of HDL-C increased slightly from 44+5 to 47+6 mg% . COnclusions:The one year antihypertensive therapy of dyslipidemic subjects with calcium channel , blockers(nifedipine)is quite effective as concern as the blood pressure while does not influence unfavorable the levels of ser~n lipids.

DO CALCIUM ANTAGONIST AFFECT VENOUS FRO05 PERIPE~RAL CIRCULATION IN HYPERTENSION ? Festinese S, Messa F, Ferruccl A, Calcagninl G. Chnical Pathophystology, "Lo Sapienza" Untversstyof Rome. Ankle swelling, by chronic calcium antagonists (Ca) therapy in hypertensive patients, is a side effect of considerable interest. The genesis is actually poorly understood but it seems to be dependent on peripheral circulation derangement. Aim of this study was to assess the hypothetical effects of calcium antagonists on regulation of peripheral veins in hypertensive patients. Method: the study group included 43 hypertensive patients in calcium antagonists therapy, Nifedipine, Amlodipine, Felodipine, Lacidipine (Ca group) 20 males, 23 females, age average 49 years - range 39-71, BIvfi average 23,2 Kg/m2 - range 19,9-24,8; this group was compared with 27 hypertensive patients sex, age, BMI matched fi'ces of pharmacological therapy (H group). Exclusion criteria were: secondary hypertension, congenital heart disease, heart failure, pcricardial disease, cardiac arrhythmia, valvular disease, arterial peripheral disease, venous peripheral disease, thyroid disfunction. Each patient was submitted to Mercury Strain..Gauge Plethismography (PSG), strnin-gangu was applied at maximum calf diameter at zero pldebustatic level (right atrium); each examination was performed after 10' of rest, physiological breath, knee flexion about 20° , temperature 24-26 ° . We calculated (getting arithmetical average on two legs) following plethysmographic parameters: Venous Tone (VT), Venous Distensibility(VD), time of Total Emptying (tTE) and Venous Pressure (VP) estimated by linear regression on 20-25-3040-60 MVIV ( Maximal Venous Incremental Volume) points. The PSG were performed by an operator in single blind. The T-test for unpaired data was :~¢rformedfor statistical anal sis. R~ults:

Group

VT

~/D

tTE

VP

H (27) avr_+sd34 +13 .033 _+.012 34 _+16 6 -+6 Ca(43) avr-+sd 36+12.032_+.011 33_+19 4_+4 H vs Ca p n.s. n.s. n.s. n.s. Conclusion: Our datashow that the chronic calcium-antagonists therapy in hypertensive patients does not medifie the regulation of peripheral veins and therefore the ankle swelling, in this kind of treatment, is not dependent by venous disfunction.

NATRIURETIC PEPTIDE PRODUCTION IN Fr007 HYPERTENSIVE RATS WITH OR WITHOUT CARDIAC HYPERTROPHY T. Ogawa,*W. Linz, M. Stevenson, B.G. Bruneau, M.L.K. de Bold, J.H. Chen, H. Eid, *B. A. Sch61kens and A. J. de Bold. Heart Institute, Ottawa, Canada and *Hoechst AG, Frankfurt/Main, Germany Cardiac natduretic peptide (NP=ANF and BNP) production was studied in the aortic banded, hypertensive rat after prevention or reversal of cardiac hypertrophy by treatment with low (10 pg/kg=LR) or high (1 mg/kg=HR) dose Ramipdl. Treatments started at the beginning or after 6 wks of aortic banding and lasted 6 wks. In aortic banded, non treated rats, blood pressure, heart weight (HW)/body weight (BVV) ratio, left and nght ventdcular (LV, RV) 6MHC mRNA levels (LV, RV), NP mRNA levels (LV), NP content(LV) and plasma NP were significantly higher than those in sham operated rats. In both the regression and prevention experiments atdal MHC isoform was c( and did not change nor did NP mRNA levels or NP content. In HR rats all parameters were similar to sham operated rats. The LR rats were hypertensive but HW/BW ratio was normal. ~MHC mRNA levels remained at the same levels as that of aortic banded, non treated rats. NP mRNA levels and NP content (LV) and NP plasma concentration were partially higher than controls but lower than banded animals. It is concluded that: 1. HR treatment normalizes ventdcular NP production but regression or prevention of hypertrophy alone (LR) only padly does so suggesting a relationship to both BP and hypertrophy. 2. ACE inhibition does not affect atrial NP production. 3. LR treatment reverses anatomical hypertrophy but does not revert the increased 13MHC expression suggesting that these are independently regulated processes.

MAGNESIUM IN HYPERTENSION: P H Y S I O L O G I C A L B A S I S FOR PREVENTION

FRO06

Victoria Kolomiets, Elena Bobrova Medical university, Donetsk, Ukraine We studied magnesium (ME) homeostasis i n hypertension. We examined 40 normotensive ~]d 54 hypertensive persons (HP) and 1200 workers'population. Using peroral ME load we revealed a decreased intestinal ME ab sorption in HP caused a deorease~ vitamin 1,25(OH)~D3 production and a decreased intestine sensitivity to vitamin's action. The result is an increased parathyroid gland activity confirmed increased plasma immunoreactive papathormone level. Calcium a]ta~onists were more effective in HP having ME homeostasis alterations. 0,25 I~]DI/kE b. w. ME oxide daily i n t a k e d u r i n ~ 3 m o n t h s decreased parathormon plasn~ level and decreased effective daily dose of calcium antagonists. The vegetabl e s and fruits fortified foods int-

ake in equivalent ME content during i year was accompanied by a decrease of hypertension incident of 1.9 ~ (p
FRO08 ALPHA-1 BLOCKING AGENT, DOXAZOSINPREVENTS LVH INDEPENDENTLY OF BLOOD PRESSURE LOWERING EFFECT IN SPONTANEOUSLY HYPERTENSIVE RAT Hitoshi Matsuoke, Hitoshi Hasegawe, Ken Ueki, Toshifumi Nemoto, Osamu Yasuda, Kaoru Shimeda, Koichi Kawamura, Hirotake Masuda, Mamoru Miura. Second Dept of

Intern Med, Akita Univ, Akita, Japan. We investigated the effects of doxazosin on LVH and myocardial fibrosis in in vivo rat model. Spontaneously hypertensive rats of 16 weeks (w) of age were divided into three group(Gr)s. In Gr DI and D2, low (0.3 mg/kg/day) or moderate (0.9 mg/kg/day) dose of doxazosin was administered for 20 weeks respectively. Rats were served as nontreated in Gr C and Wistar-Kyoto rats were bred as normal blood pressure control in Gr W. Systolic blood pressure (BP), heart rate (FIR) and body weight (BW) were measured periodicallly. At 36 w of age, perfusion fixation was performed retrogradely from the abdominal aorta, the heart was excised, weighed and examined histologically. Percent ratio of the fibrotic area (%FR) of each slice was determined. BP was around 190 mmHg in Gr C. In Gr DI, BP was similar as in Gr C, although BP reduced significandy in Gr D2 during the u'eatment period. Left ventricular weight (LV)/BW ratio decreased significandy in both Gr D1 and Gr D2. Myocardial fibrosis was significantlyprevented in Gr D1 and D2. Gr C Gr DI Gr D2 Gr W BP at 16 w (mmHg) 188-+5 183i-4 180"/:6 117-+-3"** BP at 36 w (mmHg) 192+.3 186.+.2 16hV.2** 128:i:3"** LV/BW (rag/g) 3.84+0.07 3.60i'0.08" 3.57-+0.05** 3.02-+0.02*** %FR(%) 2.7+0.6 1.2i-0.3" 0.3-+0.0** 1,0i4).l* p<0.05 **p
In conclusion, alpha-I blocking agent, doxazosin prevents LVH and myocardial fibrosis. Our results suggest that alpha-I blocking mechanism operates independently of BP reduction in in vivo rat model.

A219

AUTOANTIBODIES AGAINST THE Ul- Fr009 ADRENOCEPTOR IN PATIENTS WITH PRIMARY HYPERTENSION Hans Peter Luther, Volker Homuth, Gerd Wallukat. Max DelbrOck Centre for Moleculare Medicine Berlln-Buch, Germany.

Previously, it was reported that autoantibodies against the ctladrenoceptor were detected in 3 of 20 patients with malignant hypertension (Fu et al, 1994 Lancet: 334:1660-63). We tested affinity-chrematography purified antibodies against the cd-adrenoceptor in the system of cultured neonatal heart ceil. These antibodies were able to mimic the effect of agonist stimulation in a concentration dependent manner as demonstrated by a positive chrontropic effect on cultured neonatal cardiomyocytes. The oil-antagonist Prazosin was able to block the stimulatory effect, whereas the pl-antagonist Propranoiol had no effect. To evaluate the incidence of these autoantibodies we investigated seras of 60 patients with primary hypertension and 20 control subjects. Serum from 30 (50 %) hypertensive patients and 2 (10%) of controls were shown to be positive.To localize the epitope of the antibodies, aliquots of the IgG fractions were incubated with peptides of the three extracellular loops of the ctl-adrenoceptor. It was shown that the peptides of either the first (75%) or the second extracellular loop (25%) were able to neutralize the effect of the antibodies, suggesting to be the binding sites. The peptide of the third extracellular loop had no effect. In the present study we report evidence for the presence of autoantibodies against the ctl-adrenoceptor in 50% of seras from patients with pdmary hypertension. Because the autoantibodies have pharmacological activity in vitro they might play a role in elevation of peripheral resistance in the pathogenensis of primary hypertension.

Ventricular myocytes produce atrial natriuretic peptide (ANP) prenatally or at pathological states. Using immunohistochemical techniques we mapped ANP containing cells across the ventricular wall of SHRs subjected to antihypertensive therapy, comparing them with untreated SHRs and Wistar rats (WR). In WRs, ANP was localized to subendocardial and Purkinje cells, whereas in untreated SHRs, the majority of ventricular myocytes showed ANPimmunoreactivity. Treatments with hydralazine (HZ), captopril (CP), or nifedipine (NF), previously shown by us to markedly reduce ANP mRNA, eliminated ANP-immunoreactivity from most myocytes, irrespective of variations in the resulting blood pressure or hypertrophy, but with HZ being less effective than CP and NF. Myocytes adjacent to fibrotic lesions, predominantly found in subendocardium, subepicardium and papillary muscles, retained ANP-immunoreactivity. Purkinje cells showed strong ANP-immunoreactivity, similar to WR. We conclude that in treated SHRs, ANPimmunoreactivity of myocytes on the border of fibrous tissue and cells which express ANP in the normal heart, resist the suppressive effect(s) of antihypertensive drugs.

EXPERIMENTAL NO-DEFICIENT HYPER-Fr011 TENSION: PATHOPHYSIOLOGICAL CHANGES IN THE M Y O C A R D I U M O~ga P e c h ~ o v ~ , ipavol Bab~l & Iveta Bern~tov~. Inst. of Norm. and Pathol. hysiol., Slovak Academy of Sciences, Dept. of Pathol., Faculty of Medicine Comenius Univ.,Bratislava, Slovak Rep.

CARDIAC FIBROSIS DEVELOPMENT IN Fr012 ALDOSTERONE-SALT HYPERTENSIVE RATS. Valerie Robert, Jean-Sebastien Silvestre, Thierry Dakhli, Bernard Swynghedauw, Dani~le Charlemagne, Claude Delcayre.

~

In the study, changes of physiological and morphological parameters were investigated in rats during 4 week oral administration of NO-synthase inhibitor: L-NAME. The 20 and 40mg/kg/ day doses significantly increased systolic pressure by 34% and 30% resp. after the first week. The heart rate decreased by 15% and 20% respectively. These changes persisted during the following three weeks. Histological investigation of rat hearts after 4 weeks of L-NAME administration, especially with the 40mg/kg/day dose, showed large areas of fibrosis unpresent in control hearts. These findings imply that the inhibition of NO-synthase leads to hypertension development connected with myocardial fibrosis reflecting reparative process after ischemic injury. These fenomena represent consequences of impaired arterial relaxation by L-NAME. A220

IMMUNOLOCALIZATIONOF ANP IN THE HEARTOF F~)IO

SHR FOLLOWING ANTIHYPERTENSIVETREATMENTS Kessler-lcekson G*, Kaganovsky E*, Barhum Y*, Schaper J#, & Schaper W#. *FMRC, Petah-Tikva, Israel, & #MPI, Bad-Nauheim, FRG.

INSERM U 127, HoSpital Lariboisi~re, F-75010 Paris. Chronic treatment by aldosterone-salt in rats results in arterial hypertension, left ventricle hypertrophy and biventricular fibrosis. To gain further insight into the mechanism of aldo-induced fibrosis, we studied in parallel protein and mRNA accumulation of collagens I and III over a 60 day-long period in both ventricles. After 3 and 7 days of treatment, systolic blood pressure was normal and no histological or biochemical change was reen in the hearts. At day 15, systolic blood pressure was raised (4.40%) and left veatricular hypertrophy reached 15%. Cardiac examination after eosin-hemalun staining or immunolabelling using anti-collagen I and III antibodies showed no structural alterations. A 83% increase of type III procollagen mRNA levels was found in the right ventricles. By contrast at 30 and 60 days a progressive cardiac fibrosis with an infiltration of fibroblasts and macrophages were readily evident and were accompagnied by a consistent and sustained elevation of biventricular levels of both collagen mRNAs.To test the hypothesis that the myocyte necrosis observed at 60 days in this model might arise from ionic disturbances via aldosterone effect on the cardiac Na/K-ATPase (NKA), a well known renal target of aldosterone, the major cardiac NKA isoforms genes were studied at early time of treatment. Whereas at day 1, ctl and 131 NKA subunit mRNA levels were elevated in the kidney, neither these cardiac transcripts nor the specific ~2 isoform were altered by 1, 3 and 15 days of treatment. These results indicate that the accumulation of collagen mRNAs appears before collagen deposition and is a sustained event. These modifications in collagen metabolism are late and are not preceded by changes in cardiac NKA gene expression precluding a direct control of cardiac collagen synthesis and NKA by aldosterone.

Fr013 An e x t r a c e l l u l a r d o m a i n on a n g l o t e n s l n II r e c e p t o r s - d i r e c t e d a u t o i m m u n e r e s p o n s e in patients w i t h m a l i g n a n t h y p e r t e n s i o n Michael L. X. Fu, Hans Herlitz, °Wolfgang Schulze °Gerd Wallukat, Elisabet Hilme, Thomas Hedner, Johan Hoebeke, Ake Hjalmarson. Wallenberg Laboratory, University of Gfteborg, Sweden; o MDC, Berlin, Germany Peptides corresponding to the sequences of the second extracellular loops of the human angiotensin II receptors was used. Sera were tested from patients with malignant essential hypertension (MEH, n= 15), malignant secondary hypertension (MSH, n=11) and from normotensive healthy blood donors who served as controls (n=33). Sera from patients with MEH and MSH and from control had autoantibodies against the angiotensin II receptor peptide in 13%, 45% and 11% of cases respectively. Autoantibodies against the angiotensin II receptors were able to specifically localize the angiotensin II r e c e p t o r s a l o n g the s a r c o l a m m a and at heterochromatin of nuclei of rat cardiomyocytes. The autoantibodies displayed pharmacological activity as shown by an increase in heart beating frequency of neonatal cultured rat cardiomyocytes. These results demonstrate autoantibodies against a functional epitoe on second extracellular loop of human angiotensin II receptors in a subgroup of sera of patients with malignant hypertension.

M Y O C A R D I A L NA,K- AND CA-ATPASE FRO15 IN S P O N T A N E O U S L Y H Y P E R T E N S I V E RATS. J.S. Larsen, T . A . S c h m i d t & K. Kjeldsen, The Heart Centre, Rigshospitalet, University of Copenhagen, Denmark. Spontaneously hypertensive rats (SHR) of 1I, 20, 32 and 80 weeks of age were studied with age-matched Wistar Kyoto Rats (~NKY) as controls (n = 6 in each group). Furthermore SHR and WKY rats at the age of 27 weeks were injected i.p. 30 mg × kg" body weight captopril once daily for a period of 5 weeks. Na+,K +- and Ca2+-ATPase was quantified using K +- and Ca2+-dependent p-nitrophenyl phosphatase (pNPPase) assay, respectively. The heart to body weight ratio of SHR rats was 10%*, 23%*, 41%* and 75%* (* ffi p < 0.05) higher than of age-matched WKY rats. No significant differences in myocardial protein content between the two age-matched groups could be detected. SHR myocardial Na+,K+-ATPase concentration tended to be 21% and 3% higher at I1 and 20 weeks of age compared to age-matched WKY rats, but was significantly lower at 32 and 80 weeks of age, i.e. - 1 5 % * and -42%*. The Ca2+-ATPase concentration of SHR rats was 22%, -18%*, - 6 % and - 2 7 % * compared to values of agematched WKY rats. Captopril treatment of SHR and WKY rats for 5 weeks had no significant effect on the above given parameters compared to non-treated age-matched control animals, respectively.

RELATIONSHIP BETWEEN LEFT VENTRICULAR PERFORMANCE Fr014 AND CELL PARAMETERS IN PATIENTS WITH BOTH ESSENTIAL HYPERTENSIONAND CORONARY ARTERY DI8EASE

Marina J. Shafer, Vadim A. Kuznetsov, Inna R. Kirshenbaum, Elena P. Gultyaeva, Lyudmila I. Gapon. Institute of Clinical and Preventive Cardiology, Tjumen, Russia. To assess the correlation between some cell parameters and left ventdcular (LV) performance erythrocytes Na+ - K+ - ATP- ase, erythrocytes and platelets Ca++ - ATP - ase, Ca content and parameters of LV performance (and diastolic and systolic diameters, ejection fraction, stroke volume and cardiac output) determined by M-mode guided by 2D echo were studied in 10 patients with mild to moderate essential hypertension combined with chronic stable angina pectods. The negative correlations between erythrocytes Na+ - K+ ATP- ase and LV end systolic diameter (,"=-0.70, 1>=0.036), erythrocytes Ca++ - ATP - asa and cardiac output (r=-0.70, p=0.033), erythrocytes Ca++ - ATP - ase and stroke volume (r=-0,65, p=0.050) were revealed using univadate regression analysis. With stepwise regression analysis 50% of the cardiac output and 42% of stroke volume variation was connected with erythrocytes Ca++ - ATP - ase. Erythrocytes Na+ - K+ - ATP - ase explained 48% of end systolic diameter variation and 39% of ejection fraction vadation. Thus there is the relationship between cell transport ATPase and LV performance in patients with combination of essential hypertension and coronary artery disease. Erylhrocytes Na+ - K+ - ATP- ase and Ca++ - ATP - ase can be predictors of LV performance.

CARDIAC CALCIUM CHANNELS AND Fr016 CATION TRANSPORT ATPASES AFTER AORTIC CONSTRICTION IN NEWBORN RATS Lei Zheng, Maurice Wibo, °Frantisek Kolar & Thdophile Godfraind. Dept of Physiology and Pharmacology, Catholic University of Louvain, B-1200 Brussels, Belgium, and "Institute of Physiology, Academy of Sciences of the Czech Republic, CZ-142 20 Prague, Czech Republic. Cardiac enlargement due to gradual pressure overload was induced by aortic constriction in 2-day-old Wistar rats. At 90 days, the functional performance of the left ventricle (LV) was assessed by acute load test (ligation of ascending aorta) in open-chest anesthetized animals. Two sub-groups, designated compensated and dacompensated hypertrophy (CH and DH) were distinguished on the basis of right ventricle weight, which was increased by less than 30% in CH and by at least 100% in DH. The LV weight (including saplum) was increased by 65% in CH and 88% in DH. The functional reserve of LV was significantly impaired in DH but not in CH. In total particulate fractions prepared from hypertrophied LV, 131-adrenoceptors ([3H]CG P-12177 binding) were markedly down-regulated (CH, - 33%; DH, -45%), but the levels (pmol/ g tissue) of L-type voltagedependent Ca channels ([3H]isradipina binding) and sarcoplasmic raticutum (SR) Ca channels ([3H]ryanodina binding) were only slightly decreased (- 10-20%). The SR Ca-ATPase activity per g tissue was reduced by 28% in the same fractions, while the Na,K-ATPase activity insensitive to 3 [aM ouabain (low-affinity (11 isoform) was reduced by 27% in CH and 38% in DH. The only distinguishing feature of DH fractions vs. CH was the quasi-disappearance of high-affinity [3H]ouabain binding sites (Kd 100-200 nM) attributable to the (12 isoform of Na,K-ATPase (CH, - 48%; DH, - 83%); the small very-high-affinity component (Kd 10 nM) attributable to the (13 isoform was not modified significantly after aortic constriction.

A221

DISTRIBUTION OF SARCOPLASMIC Fr017 RETICULUM CA:z+ PUMPS AND RELEASE CHANNELS IN RAT CARDIAC OVERLOAD. Marielle Anger#, Anne-Made Lompr6#, Fran~oise Marotte* & Jane-Lyse Samuel*. #URA-CNRS 1131, Orsay, France. *INSERM U127, Paris, France. Expression of the genes coding for the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA): i.e. SERCA 2a and SERCA 2b, and for Ca2+ relcasc channels (i.e. ryanodine receptor, RYR, and IP3 receptor, IP3R) was studied in early (24 hours, 5 days) and late (one-month) stages of cardiac overload induced in rat by thoracic aortic stenosis (AS). Quantification by slot-blot hybridization indicated that, SERCA 2a and RyR mRNA levels were unchanged 24 hours after AS, whereas 5 days and one-month after AS, they were drastically lower than in sham. One-month after AS, the decrease in SERCA 2a mRNA level was greater than that in RyR mRNA level (60% vs 32%, respectively). In situ hybridization indicated that 24 hours after AS, SERCA 2a and RyR mRNAs were homogeneously distributed in cardiomyocytes, whereas one-month after AS, the distribution of SERCA 2a and RyR mRNAs became heterogeneous. In both shams and AS, SERCA 2b and IP3R mRNAs were mainly expressed in the vessels and at low level in the myocytes. In conclusion, the decrease in accumulation of SERCA 2a and RyR mRNAs in compensated cardiac hypertrophy was accompanied by a heterogeneous distribution; Some myocytes no more expressed SERCA 2a and RyR mRNAs whereas others expressed normal levels of these mRNAs.

Augmented Expression of ANP and BNP Fr019 Genes in Hypertrophied Rat Heart. Correlation with Expression of the Ca-ATPase Gene. Hideo Kawakami, Hideki Okayama, Yuji Shigematsu, Mareomi Hamada, Kunio Hiwada. 2nd Dept of Internal Medicine, Ehime University, Ehime, Japan. We assessed the changes of AN'P, BNP and sarcoplasmic reticulum Ca-ATPase (SRCA) genes expression associated with progression and regression of cardiac hypertrophy in renovascular hypertentsive rats (RHR). Renovascular hypertension was made by constriction of left renal artery using male Wistar rats and were studied 6 or 10 weeks after the procedure. Regression of cardiac hypertrophy was induced by nephrectomy 6 weeks after constriction and was maintained for 4 weeks (RHR-NEP.). ANP, BNP and SRCA gent expressions in the left ventricles were analyzed by Northem blotting. The mean density scores for the sham group were set at 1.0. SBP LVweight (mmH~ (m~/~/ ANP BNP SRCA Sham-6wks 134a:7 1.88±0.1 1.00 1.00 1.00 RHR-6wks 220e23" 2.99:1:0.5" 2.37* 1.59" 0.72* Sham-10wks 140±8 1.76e0.1 t.00 t.00 1.00 RHR-10wks 236el6t 3.40+03t 4.20t 1.80t 0.69t RI-IR-NEP

146~8

1.834-0.1

0,98

I.I0

0.96

*p<0,01 vs Sham-6wks.tp<0.01 vs Sham-10wksand RHR-.NEP. The levels of ANP and BNP mRNAs were correlated negatively with those of SRCA mRNA and positively with LVweight. These data suggest that ANP and BNP may play a crucial role in a compensatory mechanism in the myocardial hypertrophy. A222

EFFECT OF CARDIAC HYPERTROPHYAND Fr018 REGRESSION ON SARCOPLASMIC RETICULUM GENE EXPRESSION AND FUNCTION Samuel Y. Boateng, Michael J. Dunn, Anne-Marie L. Seymour, Magdi H. Yacoub & Kenneth R. Boheler. Dept of Cardiothoracic Surgery, NHLI, London UK. Cardiac hypertrophy (CH) is associated with changes in calcium handling. The effect of regression (R) following CH on cardiac sarcoplasmic reticulum (SR) CaATPase (SERC) gene expression and function was examined. CH was induced in the rat by banding of the abdominal aorta for 5 weeks and regression by means of daily oral administration of an angiotensin converting enzyme (ACE) inhibitor ramipril (50pg/Kg body wt/day) for 4 weeks. SERC activity was assessed by oxalate stimulated calcium transport into the SR. Western blotting of cardiac ACE showed no significant alteration with CH or with R. Cardiac atrial natriuretic factor (ANF) levels were found to be elevated at the mRNA and protein level (20 fold n=6) with CH. In regression, ANF mRNA decreased to normal, but remained 6 fold hicjher at the protein level (n=5).

nmol/min/m~ 21.3 ± 2 . 8 14.64-3.8 19.484"2.5 Western blotting showed that CaATPase levels were not significantly altered in CH or R, but phospholamban decreased in CH (20% n=6) and returned to normal in R. These changes suggest that ramipril can reverse changes in cardiac gene expression induced by CH without producing complete regression of cardiac size.

ROLE OF ANGIOTENSIN II RECEPTOR SUB Fr020 TYPES IN CARDIAC HYPERTROPHY IN SPONTANEOUSLYHYPERTENSIVERATS. Naoki Makino, Masahiro Sugano, Tomoji Hata, Takasi Yanaga, Beppu Japan Our purpose was to evaluate whether the early treatment of spontaneously hypertensive rats (SHR) with the Ang.II receptor type 1 (AT1) or type II (AT2) blockades could permanently alter the course on left ventricule (LV) hypertrophy. The both doses of agents were administrated into SHR and aged from 5 to 15 weeks and compared with untreated SHR and the Wister Kyoto rats (WKY).Arterial blood pressure was significantly decreased in AT1 treated SHR compared with untreated SHR. The left ventricular collagen concentration and the ACE activity were also significantly decreased in AT1 -treated SHR. The Scatchard analysis for [lZSl]-Ang II receptor binding which showed the increased densities in in AT1-treated SHR was significantly decreased, but not in AT2-treated SHR. Using by the reverse transcriptass polymerase chain reaction, we examined mRNA expression for the ACE, AT1 and AT2 in rat hearts. In LV of SHR, enhanced mRNA expressions for ACE and AT1 were found. These observations were inhibited only by AT1 blockade. These results indicates that AT1 receptor but not AT2 receptor in SHR has a crucial role in the development of the pressure-overload cardiac hypertrophy.

IN UTERO EXPRESSION OF CARDIAC Fr021 ANGIOTENSIN I1 R E C E P T O R S IN CARDIOMYOPATHIC HAMSTERS Chantal Lambert, Yolette Massillon & Sylvain Meloche. Department of Pharmacology, Universit~ de Montreal and Centre de Recherche, H6teI-Dieu de Montreal, Quebec, Canada.

We have recently reported that the development of ventricular hypertrophy in cardiomyopathic hamsters is associated with up-regulation of angiotensin II (AII) AT~ receptors suggesting that AII plays an important role. The aim was to study the cardiac expression of AII receptors in utero. Ventricles of fetal (El6) cardiomyopathic (CHF 146) and inbred control hamsters (Canadian Hybrid Farms, Nova Scotia) were dissected and membranes were prepared for binding assays. Competition binding studies were performed using [~2~ISar~,IleS]AIIas non-selective radioligand in the presence of either [Sar~,IleS]AII or AT t and AT2 selective antagonists losartan (DuP 753) and PD123319, respectively. No significant difference were observed between the two groups in the total expression and in the density of AT) receptors. In contrast with the results obtained in adult hearts, significant and similar concentrations of AT2 receptors were expressed in both groups of animals. Our results demonstrate that the upregulation of All AT~ receptors retrieved in the cardiomyopathic hamsters is not genetically transmitted.

SUPPRESSION OF CARDIAC ANGlO-FrO23 TENSINOGEN mRNA IN SHR TREATED WITH ANTIHYPERTENSIVE DRUGS Toshiyuki Kudou, Hirofumi Sawa, Hiroshi Okamoto, Hideaki Kawaguchi, Akira Kitabatake. Dept of Cardiovasular Medicine, Hokkaido University. Sapporo, Japan

It is well known thal systemic hypertension induces cardiachypertrophy, ~mdit can be prevented by antihypertensive drugs. But its mechanism is still unclear. Our study is designed to investigate the relationship of LVH and cardiac angiotensinogen (ATN) mRNA expression by using spontaneous hypertensive rat (SHR). Method: SHR fed normal diet or antihypertensive drugs (Captopril 30mg/kg/day, Bunazosin 10mg/kg/day, Nifedipine 20mg/kg/day, Atenolo120mg/kg/day, Thiazide 5mg/kg/day) containing diet during 11W to 15W. In 15 weeks SHR and normal diet Wistar Kyoto rat 0NKY) were checked body weight, total heart weight, left vantric~lar weight. ATN mRNA was extracted by chomczynski's method and quantified by ribonuclease protection assay. Result: All antihypertensive drugs could prevent the increase of blood pressure and the development of left ventricular hypertrophy of SHR. Left venlricular ATN mRNA expression of SHR was 2-fold higher than WKY. And all antihypertensive drugs could prevent left vanlrioular ATN mRNA expression. There was little differences between the drugs on the suppressive effects on ATN mRNA expression. Conclusion: It is suggested that mechanism of LVH which is induced by systemic hypertension is related with cardiac ATN mRNA expression. And it can be modulated by antihypertensive drug treatments.

RELATIONSHIPS BETWEEN ANGIOTENSIN-Fr022 CONVERTING-ENZYME GENE POLYMORPHISM AND HYPERTROPHIC CARDIOMYOPATHY Keiji Yoneya, Hiroshi Okamoto, Hideaki Kawaguchi, Akira Kitabatake. Dept of cardiovascular Medicine, Hokkaidou University, Sapporo, Japan. (Purpose) To elucidate the relation between angiotensin converting e n z y m e ( A C E ) gene and left ventricular hypertrophy, we investigated the ACE gone insertion/deletion polymorphism in hypertrophic cardiomyopathy(HCM) patients and their relalives.(Methods) The subjects were 80 Japanese HCM patients diagnosed in our hospital by using echo cardiography and their healthy 88 relatives. Detection of the ACE gone genotype was performed using PCR method as Rigat's.(Results) The ACE II, ID, DD genotype were present in 69, 69, and 30 subjects respectively in total, and the allele D frequency was 0.38. In HCM patients group and normal heart(control) group, the allele D frequencies were 0.42 and 0.35 for each, and the genotype showed significant dilference for each groups. Further, comparison of the ACE genotype between familial HCM(FHCM) and sporadic HCM(SHCM) did not show significant difference, but the allele D frequency seemed higher in SHCMs. Other comparison between SHCMs and controls showed significant difference, on the other hand the another comparison between FHCMs and controls did not.(Conclusion) In conclusion, we deduced that the ACE gone was related to the left ventricular hypertrophy in HCM patients.

CHRONIC HYPOXIA REGULATES a t SUB- Fr024 TYPE mRNAs AND INHIBITS aI-STIMULATED HYPERTROPHY AND SIGNALING. Hong-Tai Li, Carlin S. Long, D. Gregg Rokosh, Norman Y. Honbo, Joel S. Karliner. VA Medical Center and University of California, San Francisco In neonatal rat cardiac myocytes, we studied the effects of chronic hypoxia (I % 02 for 72 hr) on i) aladrenoceptor-mediated hypertrophy and signaling; and ii) a I density, subtypes and mRNAs. Chronic hypoxia inhibited norephinephrine (IslE) stimulated hypertrophy (25% decrease in new protein synthesis, n= 14, p<.01), al-AR transduced phosphoinositide hydrolysis was similarly inhibited (n=8, p < . 0 l ) . Chronic hypoxia differentially regulated a I subtypes (cqA,aiQ defined by 5-methylurapadil displacement, and subtype mRNAs ((giA/D,0~iS,G/-iC) by RNAs¢ protection.Hypoxia for 72 hr coordinately downregulated both the pharmacologically defined at^ density and alc mRNA. During norrnoxia, NE increased the pharmacologically defined al density and alc mRNA level, but hypoxia for 72 hr prevented the.so changes, Thus, chronic hypoxia inhibits cq-mediated hypertrophy and signaling, which may be related to decreases in both the pharmacologically defined oq density and the alc mRNA level. A223

HUMORAL AND HEMODYNAMIC Fr025 REGULATION OF NATRIURETIC P E P T I D E S IN C A R D I A C H Y P E R T R O P H Y Tatsuya Iso, Masashl Arai, Tadashi Suzuki, K a z u h i k o Murata, C a r d i o l o g y Dept, G u n m a U n i v S c h o o l of M e d , M a e b a s h i , G u n m a , J a p a n It has been demonstrated that the peptide level and genc expression of atrial and brain natriuretic peptide(ANP and BNP) are increased in pressure overload hypertrophic hearts. To determine whether pure mechanical or humoral factors secondary to hemodynamic load contribute the increase of natriuretic factors, we compared the level of these peptides in heterotopically isografted hearts, which is completely free from mechanical load, with or without systemic hypertension due to aortic banding in host rats. ANP BIffP peptide m R N A peptide mRNA in situ 329 ""51 1.00-,-0.2 17.8-,-2.1 1.0 ± 0.l sham transplant 408 "29* 2.05 __.0.3' 32.3--3.6* 1.91---0.3~ serum 296 ± 97 17.3±2.4 in situ 530 -,-78 # 2.36 ± 0.5# 41.6-,-5.7# 3.22 ±0.3~ pressure overload !transplant 492 -,-.58 2.89±0.2# 53.2±8.7# 2.35±0.5 serum 250 ± 60 20.4--.4.1 *in situ vs transplant p
(pghng protein) semm(pghnl)

The level of these peptides were elevated in transplanted hearts than in host hearts, and were highest in transplanted hearts of pressure overload group. These data suggest that natriuretic peptides in hearts are regulated by both enhancement and withdrawal of mechanical load, and by humoml factors secondary to hemodynamic load.

EFFECT OF R56865 ON PHENYLEPHRINE- AND Fr027 ENDOTHELIN-I-INDUCED HYPERTROPHY OF NEONATAL RAT VENTRICULAR CARDIOMYOCYTES Josde A.A.M. Claes, Johan E. de Vries, Ger J. van der Vusse, Robert S, Reneman & Marc van Bilsen, Dept of Physiology, University of Limburg, Maastricht, The Netherlands.

The contribution of alterations in intracellular Ca2*-homeos tasis to the development of cardiac hypertrophy is still incompletely understood. To gain more insight into the role of Ca2÷ in this process isolated neonatal rat ventricular cardiomyocytes were stimulated with phenylephrine (PE, 10.5 M) and endothelin-1 (ET-1, 10o M) in the absence or presence of R56865 (10"a-10"e M), a putative Ca2*-overload inhibitor. 48 Hours of stimulation with PE resulted in an increase in cell size and a marked induction of ANF mRNA and upregulation of MLC-2 mRNA levels. In PE-stimulated cells co-administration of R56865 inhibited the increase in cell size and both ANF- and MLC-2-expression in a dosedependent manner. At 10"e M R56865 prevented the induction of ANF-expression. Cardiomyocytes transfected with a plasmid carrying the luciferase gene under the control of the ANF-promoter underwent the same protocol as described above. Induction of ANF-promoter activity by PE was abolished by 10-7 M R56865. In ET-l-stimulated cells a reduction of ANF-promoter activity was observed with 10e M R56865. These results indicate that R56865 inhibits the development of cellular hypertrophy, suggesting that alterations in Ca2*-homeostasis may contribute to the development of myocardial hypertrophy.

A224

NATRIUREIIC PElrrIDE C - ~ EXPRE~ION IN Fr026 "IHE AORTOVEFIOCAVALFIS'IUI.A RAT HF_ART Lesley A. Brown, Richard A.D. Rutherford~, Derek J. Nunez, John Whartonl, Martin R. Wilkins. Clinical Phanmcology and Histochemistry~, Royal Postgraduate Medical School, London, UK. Chang~ in the cardiac expression of the 3 natriuretic peptide receptors (NPR-A, NPR-B and NPR-C) may be an important mechanism for regulating the local effects of the peptides in the heart. Previously, we have demonstrated a reduction in mRNA transcript expression of NPR-C (the putative "clearance" receptor) in the hypertrophied anrtovenocaval (AV)-fistula rat heart. To investigate whether the NPR-C transcript changes axe represented at the receptor protein level, we have performed western blotting and binding studies for NPR-C in hypertrophied 35d AV-fistula and 35d sham-operated con~ol rat hearts. NPR-C binding was assessed using in vitro autoradiography with 101aMfrozen sections of heart and [lZI]rANPl.n ± C-ANP,~ (to define total and non-NPR-C binding). Western blot analysis was performed using 20-50pg of atrial and venlricular membranes, a rabbit anti-rat NPR-C antibody (supplied by D.LOwe, Genetech) and the ECL detection system (Amersham). High density [t~I]rANPv~ binding was localised to the ventricular endoeardial endothelium in both control and AV-fistula rat hearts. Co-incubation with an excess (lla/Vl) of C-ANP,~ reduced the endoeardial binding below the level of detection. The distribution and density of [mI]rANP~.~binding sites was significantly lower in AV-fistula rat heaxts than in controls (p<0.05, n=6). lmmunoblotting produced a single band (~120kDa) in both atrial and ventricular membranes which was reduced to --60kDa with mercaptoethanol. Quantification of autoradiographs by optical densitometery suggested a 20-50 % non-significant reduction in NPR-C protein in AV-fistula compared to control hearts. Fhe data suggest a reduction in NPR-C protein accompanies hypertrophy of the AV-fistula rat heart but that there is variability in the response.

REDUCTION OF THE C M H 4 LOCUS Fr028 IN F A M I L I A L H Y P E R T R O P H I C C A R D I O M Y O P A T H Y BY FINE H A P L O T Y P E A N A L Y S I S L. Carrier 1, J. Bercovici 1, G. Bonne 1, P. Richard 1, N. Lautid 1, M. Komajda2, & K. Schwartz 1. IlNSERM UR153,GroupeHospltalierPilldSalpl~ldbra. Paris, France. 2Groups des CardLomyopathies de la Socl6ffl Fran~aise de Cardlologie. Pads. France,

Familial hypertrophic cardiomyopathy (FHC) is a cardiac disease transmitted as an autosomal dominant trait. Four disease loci have been reported on chromosomes 14q11-q12 (CMH1), lq3 (CMH2), 15q2 (CMH3), and 11p13-q13 (CMH4). The genes implicated for three out of these four loci encode sarcomeric proteins. To precise the location of the morbid gene in the CMH4 locus, we examined the segregation of highly informative microsatellite markers in three unrelated french families with FHC. Methods. Families 714, 717, and 728 were diagnosed by M mode and 2D echocardiography, and by echocardiogram. Highly informative microsatellite markers, obtained from the G+n+thon map, were genotyped by multiplex PCR procedure, and the haplotypes were analysed. Results. A segregation between the disease gene and a particular haplotype obtained with several markers from the CMH4 locus was found for each family. Family 717 shows linkage on 23 cM. Two unrelated and affected individuals from the families 714 and 728 were recombinant for 2 markers that suggests a reduction of the linkage interval to 9 cM. Furthermore, the haplotype analyses on CMH1, CMH2 and CMH3 show affected recombinants for each of the families suggesting no linkage to the other FHC loci. Conclusion. We found no linkage to CMH1, CMH2, and CMH3 in three french families with FHC. The fine haplotype analysis with highly informative markers suggest linkage between the disease gene of these three families and the CMH4 locus, and permit to reduce the linkage interval from 23 to 9cM.

X-linked dilated cardiomyopathy associated Fr029 with skipping of the dystrophin exon 29 Wolfgang M. Franz,*Ralf Herrmann,#Marion Cremer, Eckart Griinig, Thomas Scheffold, *Thomas Voit, Hugo A. Katus, Inhere Medizin IIl, #Humangenetik, Univ. of Heidelberg,*Kinderklinik, Univ. of Diisseldorf, FRG.

SPECTRUM OF I~MHC MUTATIONS IN HCM IN Fr030 RSA AND PROGNOSTIC ASSOCIATIONS Johanna Moolman, Bert Posen, Paul Brink, Valerie Corfield. US/MRC Centre for Mol. and Cell. Biology, Tygerberg, RSA.

We report on the genetic linkage analysis and characterization of a novel dystrophin defect in a family with dilated cardiomyopathy. Two brothers, the elder requiring heart transplantation, and a maternal cousin presented elevated creatine kinase levels, increased right ventricular diameters and ECG abnormalities. All had exertional cramping myalgia. Immunofluorescence against the dystrophin protein showed preserved, but reduced staining of the sarcolemmal membranes of heart and skeletal muscle. Westernblot analyses revealed a dystrophin molecule, which was quantitatively reduced by 80 %. However, the monoclonal dys-1 antibody, directed against the mid rod region (1181 aa -1388 aa), did neither react with heart nor skeletal muscle protein. Dystrophin mRNA of muscle and heart tissue was reverse transcribed and amplified by PCR using 18 subsets of primers. The PCR-products containing codon 1275- 1591 revealed a band of reduced size. SSCPanalysis confirmed an abnormal banding pattern. Subcloning and sequencing of the dys-1 epitope revealed a point mutation in exon 29 (1251 aa - 1300 aa) leading to a stop mutation in all affected individuals and carriers. In addition, fragments missing the 50 codons of exon 29 were identified. We conclude, that an alternative splicing of exon 29 has occured as a secondary effect of the stop mutation. The mechanism leading to heart muscle damage may either be due to a quantitative or qualitative change of the dystrophin protein or by a combination of both.

Cardiac ~ myosin heavy chain gene (I~MHC) defects cause about 20% of familial hypertrophic cardiomyopathy (HCM). A correlation between HCM prognosis and specific 13MHCmutations have been reported. This study investigated the prevalence of HCM-causing r~MHC mutations and prognostic association in South Africa. Mutation screening of r~MHC in 50 unrelated HCM patients by PCR-SSCP and restriction enzyme-based DNA assays identified the following mutations (prevalence in brackets): R249Q (1); R403W (1) novel A797T (6). The prognosis associated with the R249Q and R403W mutations was good, the poor prognosis associated with the prevalent A797T mutation may be due to a gene dosage effect. Knowledge of the full spectrum of 13MHC mutations and their associated clinical features against different ethnic backgrounds may elucidate the molecular mechanisms of HCM and lead to better understanding of genotype/phenotype correlations. This in turn will facilitate patient management and counselling.

IMPAIRED COUPLINGBETWEENPALMITOYL- Fr031 CoA SYNTHETASE AND CPTo IN THE MITOCHONDRIA OF HYPERTROPHIED RAT HEARTS. Brunold Ch., E1Alaoui-TalibiZ. and Moravec J. Dept de Physiologie,UCB Lyon I, 69622 Villeurbanne, France.

ACE INHIBITION INCREASES SURVIVAL IN Fr032 RATS WITH LEFT VENTRICULAR HYPERTROPHY AND MYOCARDIAL INFARCTION Wolfgang Linz, Gabriele Wiemer, Hans-Ludwig Schmidts, Wolfgang Ulmer & BernwardSch01kens Hoechst AG, D-65926 Frankfurt/Main, Germany

In our previous work on chronicallyvolume-overloadedrat heartswe demons~atedthatthe developmentof cardiac hypertrophy results in an impaired long-chainfattyacid u"tflization. In this work we studied the oxidation of I- 14(3 palmitate an.d of its CoA and camitine esters by the mitochondriafrom volume-overloadedhearts. The ez~¢mes kinetics of palmitoyl-CoAsynthetase and carnitinepalmitoylh-ansfcrase (CPTo) were also analyzed in order to assess the functionalproperties of these two enzymes in conditions characterized by high carnifineconcentrations.Our results suggest that the rate of conversionof labelledpalmitate into acid soluble products by the mRochondriafrom volumeoverloaded hearts is ~imnificantly.slowed down even in presence of saturable carnitineconcentrations.The rate of palmitoylcarniaueoxidafionis, on the other hand, quite comparable to thatof mitochondriafrom controlhearts. The kinetic studies of palmitoyl-CoAsynthetase and CPTo do not revealany significant difference betweenthe two groups tested. However, the conversionof exogenous palmitate to paimitoylcamitineis significantlyslowed down, atleast at high substrate concentrations(above 1O0 uM). The decrease of palmitate utilization,as observed in mechanicallyoverlonded hearts, may therefore result from analtered cooperation of the above two enzymes supposed to form a functionalcomplex at the level of the outer mitochondrial membrane.

In rats with left ventricular hypertrophy (LVH) induced by aortic constriction (AC) and subsequent myocardial infarction (MI, n=25) by occlusion of the left coronary artery the effects of ACE inhibition with ramipril (IRA, 1 mg/kg/day via the drinking water, n=26) on survival and remodeling were investigated during 6 weeks. Respective groups (sham AC, n=10; AC, n=12; AC + sham MI, n=20; sham MI, n= 21; MI, n=21; MI + RA, n=20) served as controls. Results: RA led to a significant reduction in mortality rate (17 deaths out of 25 rats in the AC+MI group versus 11 deaths out of 26 rats in the AC+MI+RA group = -26 %). Furthermore, LVH and MI size and hydroxyproline content were reduced. In the isolated hearts of RA treated rats cardiac function and metabolism was improved. Conclusion: Rats with LVH undergoing MI experience increased mortality which was reduced by RA. In isolated hearts of these animals marked deteriorations are taking place. ACE inhibition with RA improved these deteriorations and attenuated the sequelae of remodeling. A225

EFFECTS OF MODULATIONS OF ANGIOTENSIN ON Fr033 REACTIVE CARDIAC HYPERTROPHY AFTER MYOCARDIAL INFARCTION IN RATS Tomoji Hata, Naoki Makino, Masahiro Sugano, Takashi Yanaga. Department of Bioclimatlogy and Medicine, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan

PREVENTIVE EFFECTS OF ACE INHIBITOR IN FRO34 HYPERTROPHIED VENTRICULAR MYOCYTES ISOLATED FROM SPONTANEOUSLY HYPERTENSIVE RAT Takuya Watanabe, Toshikuni Yanagishlta, Noburu Konno EIIchl Geshl & Takashl Kataglrl. 3rd Dept. of Internal Medicine, Shown Univ. School of Med. ,Tokyo, Japan

To elucidate the role of angiotensin in the reactive hypertrophy after myocardial infarction (MI), we evaluated the effects of an angiotensin converting enzyme (ACE) inhibitor, enalapril, and an angiotensin (Ang) II receptor blockade, E-4177 (AT1 blocker), after MI in rats. MI were induced by the ligation of left coronary artery in rats, thereafter, oral drug therapy was initiated and compared with untreated MI rats at 4, 8 and 12 weeks later. Hearts were divided into 2 sections as an infamted scar and a viable tissue. Since scar weights were not altered in the study and the left ventricular weight to body weight ratio was significantly higher in MI rats, the reactive hypertrophy was obvious in the viable region. Collagen content, ACE activity and Ang II receptor density were increased in scar and viable tissue, while Ang receptor mRNA expression determined by RT-PCR method was also increased in the viable tissue compared with the respective region of healthy rats. The administration of enalapril (30 mg/kg/day) or E-4177 (10 mg/kglday) showed the marked reduction of the reactive hypertrophy with the improvement in contractility and significant decreases of collagen content and ACE activity. Enhanced expression of Ang receptor mRNA was markedly restored in viable tissues by both drugs. Ang II receptor densities significantly reduced by E-4177 in viable tissue, but not by enalapril. These results suggest ACE inhibitor and AT1 blocker have beneficial effects against the development of reactive hypertrophy in MI and these effects may not be dependent only on AT1 receptor-mediated mechanisms.

The effects of Imidapril, an ACE inhibitor, and diltiazem, a calcium channel blockade on functional end structural changes of myocardium were assessed in spontaneously hypertensive rats (SHR). Imidaprll (2.5mg/kg/day), dlltiazem (30mglkg/day), or vehicle were given by gavage from10 week-old. Single myocytes were Isolated enzymatically from the left ventricles of three SHR groups and Wister-Kyoto (WKY) control in 18 week-old. Enzyme activitiesof sarcoplasmicreticulum (SR) and salcolenma (SL) and the respiratory control index (RCI) of mitochondrla were measured with single myocytes, and cardiac hypertrophy and fibrosis were histmetrically observed. In 10 week-old SHR without treatment several significant enzymatic changes and cardiac hypertrophy and fibrosis were already observed compared with age-matched WKY. Imidapril reduced heart to body weight ratio and regressedcardiac hypertrophyand fibrosis significanUy more than diltiazem corn ~ared with vehicle in SHR groups. :aATP ase I Ca ATPase Na-KATP RCI of of SR ( * ) of SL ( * ) aseofSL(* "nitochondria 10W-WKY 60.8±4.51 5.8±2.0 6.6±4.0 4.0±0.7 10W-SHR 46.9±9.3) 5.1±2.3 15.0±1.9 3.3±1.3 'i8~;k;~;( ~;~/T:;i"EI~E~:Z-~ ..... ~E~~;T~ ...... ~;~~i:i ...... IMIDAPRIL 54.2+9.9[ 8.6+4.3 25.1+13..( 3.1±0.9

ANGIOTENSIN RECEPTORS DURING Fr035 CARDIOVASCULAR REMODELLING Lindsay Brown & Conrad Semia, Department of Physiology and Pharmacology, The University of Queensland, Australia.

THE EFFECT OF LOSARTAN ON CARDIAC FRO36 REMODELING FOLLOWING EXPERIMENTAL INFARCTION IN RATS. Tomas Sladek, Jarmila Sladkova, FrantisekKolar, Frantisek Papousek, NicholasCicutti, Borivoj Korecky & Karel Rakusan. Dept. Physiol., Fac. ~d., Univ. Ottawa, Ottawa, Canada

Angiotensin II (AT) produces cardiovascular responses such as vasoconstriction, salt retention through aldosterone release and thirst but the most important effects are myocardial fibrosis, and hyper~ophy and hyperplasia of the heart and blood vessels; all responses are effected by specific AT receptors. Discase-ioduced changes in AT receptor density may contribute to the cardiovascular remodelling. Thus, we have determined AT receptor densities by radioligand binding studies using mIsaP,ileLAT in heart and kidney membranes from rats with thyroid dysfunction (triiodothyronine or meLhimazole-treated), in deoxycorticesteroneacetate (IX)C.A)-salt hypertensive rats and in spontaneous hypertensive rats (SHR, and normotensive Wigar-Kyoto (WKY) controls). Hyperthyroid and hypertensive rats showed cardiac hypertrophy; renal hyperffophy resulted fi'om hyperthyroidism and DOC_A-salt hypertension. Left veatricular AT receptor density increased from 10.9:1=3.6 (control) to 24.8:1-,.2.2 (hyperthyroid rat) and 205.3 fmol/mg protein (hypothyroid rats) with selective increases in AT2 receptor subtype density;, from 8.9-J:I.9 (WKY) to 14.1Y_2.2 fmol/mg protein (SHR); but did not change in IX)CA-salt hypertensive rats. Renal AT receptor density increased from 516:1=77 (control) to 903_+.275 (hyperthyroid rat) and 71.5-~9 fmol/mg protein (hypothyroid rat); from 531.+.80 (WKY) to 852.t:156 (SHR); IX)CA-salt rats showed no changes in renal AT receptor density. Thus, increases in AT receptor density may contribute to the cardiovascular remodelling in thyroid dysfunction and genetic hypertension in rats although not in DOCA-salt hypertension, a low AT model, where cardiac and renal hypertrophy occurred without alteration of AT receptor density.

A226

I

O,L ,AZE.

VEHICLE 40.5+'1.4 / 4.0+].4 1 2.5+1.1 (* : p moles Pi/cell/hr) Thus,these results suggest that imidapril may prevent more effectively than diltiazem against the progressive detriment failing to hypertensive heart disease.

Hemodynmics and merphometric changes in a surviving heart tissue has been investigated 3 weeks following coronary artery ligation in rats with and without treatment with AIl receptor antagonist Iosartan (15 mg/kg/day). Ihe therapy decreased (p
ALTERATION OF MYOCARDIAL COLLAGEN IN T H E FRO37 VOLUME-OVERLOAD RATS AND EFFECT OF C A P T O P R I L O N IT G I V E N IN D I F F E R E N T T I M E Zhiquan Liu, Qiming Feng, Dingyi Yang The C V Medicine of Xian Med. Univ., Xian 710061, PR China To determine effect of captopril on myocardial content and its component in volume-overload hearts, an aorta-cava] shunt was established in 51 rats, divided into 4 groups at random. Two grps were given captopril orally (f~ng/kg/day) in their drinking water immediately and after one month post the A-V fistula, respectively, the rest of two groups as controls. After total 48-49 days observations, with left ventricular cavity and weight increasing in the fistula group, the collagen concent, tended to be decreased, collagen type I/III

ratio markedly decreased,

compared with those in the sham, 4.7+_2.0 vs 8.8±4.8, P
THE EFFECTS OF BDF 9148 ON THE LEFT Fr039 VENTRICLE ARE REDUCED IN HYPERTENSION-INDUCED HYPERTROPHY. Sheila A. Doggrell + Vinita Nand, Department of Pharmacology, University of Auckland, New Zealand B D F 9148 prolongs the opening of sodium channels. W e have investigated the effects of B D F 9148 on the action potentials and contractility of the leftventricles from 14 and 21 week old (wk) Wistar Kyotu (WKY) normotensive rats and Spontaneously Hypertensive ,rats(SHRs). At 21, but not 14 wk, the SHRs had developed left ventricular hypertrophy with the weights of the W K Y rat and S H R left ventricles being 352 mg and 438 mg, respectively. B D F 9148 prolonged the action potentials and augmented the contractile responses. Thus on the 14 wk W K Y rat left ventricle, B D F 9148 at I0"v, 3x10 "~, I0"s and 3xl0"SM prolonged the APDso by 8, 14, 22 and 18 msec, and the APD~o by 14, 20, 42 and 36 msec and augmented the contractile responses by 45, 93, 122 and I01%, respectively. B D F 9148 had similar effects on the action potentials and contractilityof leftventricles from 14 wk SHRs and 21 wk W K Y rats. The duration of action potentialswas increased in the hypertrophical leftventricles from 21 wk SHRs; the APD~o and ~o values were 21 and 59 msec, respectively. The ability of B D F 9148 to prolong the action potentials and augment the contractile force was reduced in the hypertrophied left ventricle. Thus on the 21 wk S H R left ventricle, B D F 9148 at I0"v,3x10 "7, 10"~and 3x10"6M prolonged the APDso by 4, 10, 14 and I I msec, the APDgo by 8, 15, 32 and 26 msec and augmented the force by 30, 63, 88 and 77%, respectively. In summary our study has shown that B D F 9148 prolongs action potentials and augments force and that these effects are reduced in the hypertrophied leftventricle of the 21 wk SHR. (Supported by the Auckland Medical Research Foundation).

METABOLICALLY-INDUCED CARDIAC HYPER- Fr038 TROPHY IS ASSOCIATED WITH IMPROVED SR Caz• TRANSPORT Heinz Rupp', Wolfgang Schulze & Roland Vetter. "Mol Cardiol Lab, Univ Marburg; Max Delbr~ck Ctr Mol Med, Berlin-Buch, Germany. To characterize sarcoplasmic reticulum (SR) Ca2. handling in metabolically-induced cardiac hypertrophy, Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were treated for 5 wks with a high dose of enantiomeric etomoxir (Eto, 15 mg/kg/d), which partially inhibits fatty acid utilization at the level of carnitine palmitoyltransferase-1 (CPT-1) and causes an increased glycolytic flux. To bypass CPT-1 inhibition, Eto-treated rats were fed a medium-chain fatty acid (MCFA) diet. The Eto-treatment resulted in a proportionate growth of the heart which could partially (WKY) or completely (SHR) be prevented by the MCFA diet. This diet also reduced (p<0.05) the etomoxir-induced increase in myosin VI. Etomoxir increased (p<0.05) the rate of SR Ca2. uptake in left ventricular homogenates of both strains in the presence and absence of the Ca2" release channel inhibitor ruthenium red. Electronmicroscopic inspection revealed no obvious intergroup differences in the ultrastructure of SR membrane network and myofibrils. Thus, a sustained pharmacologically induced reduction in fatty acid oxidation and the subsequent increased glucose oxidation may induce (at high dosis) biventricular growth as well as qualitative and quantitative molecular alterations of cellular organelles such as myofibrils and SR which seem to be independent of the pressure-load of the ventricles. A metabolic modulation of cardiac substrate utilization could therefore be a beneficial approach to improve the altered SR Ca2" transport function in a diseased myocardium.

REGRESSION OF HYPERTROPHY IMPROVES Fr040 SUSCEPTIBILITY TO ISCHAEMIC INJURY. Nabeela S.Bhutta, John F.Unitt*, Magdi H.Yaceub and Anne-Marie L.Seymour, Dept. Cardio-Thoracic Surgery, N.H.L.I. at Harefield Hospital, *Dept. Biochemistry, Fisons Pharmaceuticals, Loughborough, UK. Hypertrophy (HT) increases the susceptibility of the heart to ischaemic injury. It is unclear however, if regression (R) of HT can improve this state. We have investigated energy metabolism during total global ischaemia (TGI) and reperfusion in HT, R and control (C) isolated rat hearts using 31P-NMR. HT was induced by ligation of the abdominal aorta. R was caused by daily administration of the ACE inhibitor, ramipdl, 5 weeks post-surgery for 4 weeks. Banding caused a 23% HT(p<0.001), R caused a 35% reduction in the extent of HT and a recovery of the decrease in phosphocreatine (PCr) content (7.2+0.3 HT(n=4) (p<0.05 compared to C), 7.5+0.3 R(n=5), 8.5+0.2 C(n=4), pmoles/g wet wt ). Following TGI, the rate of high energy phosphate use and the accumulation of intracellular inorganic phosphate (Pi) were more marked in HT than in R or C. Time to onset of contracture was increased in HT (12.1+0.5min HT vs 6.2+0.2min C). R reduced this time by 13%(n=4). Metabolic changes in TGI and reperfusion were less severe in R, indicating that regression has a beneficial effect on the susceptibility of the hypertrophied heart to TGI. A227

HYPERTROPHIC EFFECTS OF CGRP & AMYLIN ON FRO41 ADULT R~T VENTRICULAR C~RDIOMYOCYT~ZS David Bell , Klaus-Dieter Sehliite~ , Xi-Juan Zhou~ ,Barbara J.McDermottl, H.Miehael Piper2 . Dept.Therap.& Pharmacol., The Queen's University of Belfast, Northern lreland~ UKl, Physiol. lustitut 1, Universitat Diisseldorf, Germany '~. It was addressed whether CGRP and amylin exert hypertrophic effects in day-1 cultures of cardiomyocytes, isolated from ventricles of adult rats and maintained in serum-free medium. Fetal Calf Serum (FCS,10% v/v), employed as a positive control, increased incorporation of/-[t'tC] phenylalanine into cellular protein, total content of cellular RNA and total mass of cellular protein significantly. CGRP and amylin also increased each of these parameters significantlyin a concentration-dependent manner (maximum responses: 100 pM and 10 aM, respectively). The selective antagonist at CGRPl-receptors, CG,RPs.37 (100 nM), inhibited significantlythe incorporation of/-[1'~C] phenylalanine into cellular protein in response to CGRP and amylin. The selective inhibitor of protein kinase C (PKC), Bisindolylmalemide (BIM) (5 //M), reduced significantly the incorporation of I-[14C] phenylalanine into cellular protein in response to phenylephrine (1 #M), employed as a positive control, but not to insulin (1 unit/ml), employed as a negative control. BIM (5/~M) reduced significantly the responses to FCS (10% v/v), amylin (10 nM) and CGRP (10 pM), but not to CGRP (100 pM). Phenylephrine (1 #M) increased significantly the specific activity of the cytosolic enzyme, creatine kinase (CK), but not of lactate dehydrogenase (LDH). Significant induction of CK, but not LDH, was also stimulated by CGRP and amylin (maximum responses: 100 pM and I00 nM, respectively). In conclusion, CGRP and amylin exert hypertrophic effects directly on ventricolar cardiomyoeytes from the hearts of adult rats in vitro. These effects are:(1) due to de novo protein synthesis since total content of cellular RNA and incorporation of l-[14C] phenylalanine into cellular protein were increased;(2) mediated by CGRP~ receptors at which amylin binds with lower potency;(3) recreated, at least partly, by activation of PKC;(4) may be associated with a fetal shift in gene expression, characterized by selective induction of CK.

HYPERTROPHIC C A R D I O M Y O P A T H Y : Fr043 TEMPERATURE EFFECTS ON SS C P ANALYSIS OF AFFECTED GENES Pascal McKeown, Bernd Weist, Alessandra Trojani, Hans-Peter Vosberg, Max-PlanckInstitute, Bad N a u h e i m , Germany A well accepted technique for the detection of point mutations in DNA sequences is based on single strand conformation polymorphism (SSCP). The method involves electrophoretic separation of PCR amplified and denatured DNA fragments. The migration pattern depends on the different secondary structures adopted by the single stranded DNAs, which in turn relate to the nucleotide sequence of the molecules; point mutations may be capable of changing the migration of one or both strands. Conditions influencing separation of the bands include fragment length, base composition, buffer and gel conditions, and temperature. We systematically checked the influence of temperature on SSCP analysis of 7 hypertrophic cardiomyopathy related point mutations in two different genes : the I~-myosin heavy chain gene and the tx-tropomyosin gene. We installed a temperature gradient ranging from 25°C to 5°C perpendicular to the migration direction of the DNA samples, which were loaded on non denaturing gradient gels ( 5-20% ). Under SSCP conditions we applied mutation carrying DNA fragments (135bp-296bp) next to wildtype sequences over the total range of the gradient. Our results show that temperature has marked effects on the migration pattern of single stranded DNA molecules with a high variability among different fragments. The choice of optimal temperature conditions is essential to the success of SSCP analysis. A228

EXPRESSION OF PROLIFERATING CELL FRO42 NUCLEAR ANTIGEN IN THE MYOCARDIUM OF HYPERTENSIVE RATS Tohru Arino, Makoto Nagai, Masahito Tsuchiya, Nobuakira Takeda, Seibu Mochizuki. Department of Medicine, Aoto Hospital, Jikei University School of Medicine, Tokyo, Japan. The aim of this study is to investigate whether DNA synthesis is accelerated and an expression of proliferating cell nuclear antigen (PCNA), cell cycle associated protein, can be observed in adult myocardial cells, although it is generally accepted that mitosis and cytokinetic cease to occur in myocardiac cells several days after birth. Six-week-old male Wistar rats were made hypertensive by constriction of one renal artery (Goldblatt). Age-matched sham operated Wistar rats served as controls. Hearts were excised at 5, 6, 8 and 10 weeks after the operation. DNA synthesis was examined using incorporation of bromodeoxyuridine (BrdU) into DNA and the expression of PCNA was evaluated using the monoclonal antibody to PCNA. DNA synthesis and expression of PCNA were not observed in myocardial cells in control rats. On the other hand, DNA synthesis was stimulated in Goldblatt rats after 5 weeks, and PCNA was observed in more than 30% of myocardial cell nuclei in Goldblatt rats at 5, 6, 8 and 10 weeks after the operation. Moreover, the expression of PCNA decreased in Goldblatt rats of which blood pressure was normalized by nephrectomy. In conclusion, the rate of myocardial DNA synthesis was accelerated during cardiac hypertrophy due to pressure overload, indicating that the similar regulation system as normal mitosis is involved in the adult myocardiac cells.

MUTATION ANALYSIS O F F A M I L I A L FRO44 HYPERTROPHIC CARDIOMYOPATHY Bernd Weist, Pascal McKeown, Alessandra Trojani, Hans-Peter Vosberg, Max-PlanckInstitute, Bad Nauheim, G e r m a n y Familial hypertrophic cardiomyopathy (FHC) is an inherited primary disease of the heart showing variable clinical features and prognosis. Dominant mutations in three different genes have been reported as causes of the disease. These genes code for proteins involved in contractile functions (B-myosin heavy chain, cardiac troponin T, and ot-tropomyosin). Hence, the disease may be described as a sarcomeric disorder. It is not known whether mutations in non-sarcomeric components can also cause FHC. Most of the known mutations have been found in the B-myosin heavy chain gene. Over 30 different missense mutations have been described, 'all of them located in the head domain or in the head-rod junction of the protein. We are screening families and sporadic cases (by SSCP analysis) for mutations in known FHC-genes in order to evaluate the prevalence of distinct non-allelic alterations and the contribution of separate genotypes to their respective phenotypes. We report on two families with mutations in the B-myosin heavy chain gene. Their corresponding amino acid exchanges are Arg-Gln in position 143 (exon 5) and ValMet in position 606 (exon 16), respectively. These positions suggest that ATP binding and the interaction with actin filaments may be affected by these changes.

CONTRACTILE CHARACTERISTICS OF FRO45 MYOCYTES ISOLATED F R O M T H E HEARTS OF AORTIC-BANDED RABBITS. Ruby U. Naqvi, David Tweedie, Kenneth T. MacLeod. Dept. Cardiac Medicine, NHLI, London, SW3 6LY, U.K. Cardiac myocyte hypertrophy was produced by constriction of the ascending aorta. Three banded animals together with weight-matched controls were kept for four weeks before removal of the heart. In banded animals, blood pressure was slightly but not significantly elevated (systolic BP=Ill.0+I1 vs 89.9+13 mmHg; diastolic BP=78.11+5.1 vs 71.7+11 mmHg) but cell area was increased (3236+144 vs 2066+259 p.m2, p < 0 . 0 5 ) . Left ventricular myocytes enzymatically isolated and loaded with indo-I were superfused with Tyrode solution (2 mM Ca, 22"C, 0.5 Hz). Time-to-peak CVI'P) and 90% relaxation (R90) of the twitch and its indo-I Ca transient were prolonged;

TTP (s)

R90 (s)

CL

Ca

CL

Ca

control

0.603

0.376

0.648

0.664

banded

0.841

0.444

0.923

0.869

p value < 0.001 <0.001 <0.001 <0.001 on potent was pr (APD90=0.747_0.05s, n = 2 7 vs 0.581_+0.058s, n=24, p < 0 . 0 5 ) and peak Ca current density (It0 was greater (3.1 +0.3 nA/pF vs 1.7 +0.2 nA/pF, p < 0.05).

CARDIAC PROTEIN KINASES IN VENTRICULAR HYPERTROPHY INDUCED BY MYOCARDIAL INFARCTION

FRO47

Lavandero S, Slinchez MI, Corbalin R, Sapag-Hagar M, Mel,~ndez J, Godoy L Fac. Ciencias Quimicas y Farmec,~uticas, U Chile &

Hesp. Clinico PU Cat61ica Chile, Santiago, CHILE. VenU'icularhypertrophy(VII) is the main compensatorycellular response to cardiomyocytesloss in myocardial infarction (MI) but its biochemical mechanism is still unknown. We have studied the activities of cardiac cyclic AMP dependant protein kinase (PICA) and the Ca2+ and phospholipid dependent protein kinase (PKC) in the MI elicited by leR coronary arte/y ligation in male rats. Sham animals were used as controls. MI was evaluated by ECG and enzyme profile (LDH, CKMB). VII was assessed by the relative vent.r'Jcular~ (RVM). PKA and PKC activities by phosphorylationof histune with [32P]-ATP at 7, 28 and 56d post MI. Resultsare: Day Group n

BW

7

213i-6 346i-4" 216:f.5 308i-6 289:/:10"320+13'* 322i-9 276i-6 390~:13 290+7* 380+13 268i-6

28 56

MI C ]VII C MI C

5 6 7 5 8 8

RVM

PKA (UV) 151-4 6+I 38:1:6 32+7 43:£4 31i'6

PKC (UV) 1.9-'~.4 1.2i-,0.2 1.9i-0.2" 1.3i'0.2 2.7:~.4" 1.7:t0.3

n--n° animals, BW=bedy weight:, RVM--mg ventricles xl00g/BW); UV= unit (nmol/min)per ventricles;*<0.05;**p<0.0l vs control. We conclude that only PKC changed in VII induced by MI with a 58% increase at 28 and 56 days. Results are similar to those obtained in the pressureoverloadVH. FONDECYT92-0738

SIZE OF ISOLATED VENTRICULAR CARDIOMYOCYTES (CMC) I N D I S E A S E D HUMAN MYOCARDIUM

Isaiah D. Pathology, Israel.

F~46

Shperling. D e p a r t m e n t of Kaplan Hospital, Rehovot,

A l k a l i n e CMC isolation was applied to 15 adult p a t i e n t s who died from heart and lung diseases in the C a r d i o l o g i c a l Center of Armenia. The volume (V) of 1383 CMC was calculated from c y l i n d e r V formula by direct m e a s u r i n g of the m a x i m u m length and average t h i c k n e s s of each cell. In the left ventricle (mean weight 307±35 g) the V of mono, bi and t r i n u c l e a ~ e ~ CMC was 130±6, 218±12, 274±58xi0 ~m respectively, in the right one (mean w e i g h t 311~±9 g) 143±6, 217±16, 285±76xi0 ~m . Our data suggest that the degree of CMC h y p e r t r o p h y depends on their nuclear class and the length a u g m e n t a t i o n of bi and t r i n u c l e a t e d CMC is greater. As the CMC have a cylindrical form the comparative stability of the thickness confirms the metabolic e f f i c i e n c y of these CMC because in such conditions the d i f f u s i o n track from e x t r a c e l l u l a r space into the cell increases slightly. A l k a l i n e CMC isolation enables m y o c a r d i a l biopsy investigation.

REGRESSION OF ACROMEGALIC HEART F~48 DISEASE AFTER SUCCESSFUL TREATMENT J. H r a d e c , J. K r ~ l , T. J a n o t a , M. K r ~ek, V. H ~ n a & J. Marek. Dept. Medici-

ne 3, Charles University, Prague, CZ. This study was designed to prove rev e r s i b i l i t y of acromegalic heart disease after successful treatment of acromegaly. Three groups of patients (pts) were followed: A: pts with permanent normalization of plasma growth hormone (GH) levels after pituitary s u r g e r y (n=11), B: pts with continuing GH h y p e r s e c r e t i o n after failed surgery who were treated by long-acting somatostatin analogue lanreotide (n=13), C: pts after surgery with later relapse of GH hypersecretion (n=6). Left ventricular size, systolic function, d i a s t o l i c filling and hypertrophy were e v a l u a t e d using echocardiography. Both left ventricular diastolic volume (LVDV) and mass index (LVMI) d e c r e a s e d in groups A and B (p<0.05, p<0.01 in both). On the contrary, both LVDV and LVMI increased in group C (p<0.001, p<0.01). Nor ejection fraction or diastolic filling changed s i g n i f i c a n t l y in any group. In conclusion, effective treatment of acromegaly, or surgical or pharmacological, leads to regression of acromegalic heart disease. A229

Monika Eppenberger-Eberhardt*, Marc Y. Donath+, Marcus C. Schaub° and Hans M. Eppenberger*,*lnst.of Cell Biology, ETH, CH-8093 Z=3rich, +Dept. of Medicine, University Hospital, CH-8091 ZQrich, °Inst. of Pharmacology, University of Z0rich, CH-8057 ZOdch, Switzerland

CORONARY MICROVASCULATURE IN NEOFr050 NATAL HYPO- AND HYPERTHYROID RATS. CHANGES IN MORPHOMETRY AND CARDIAC FUNCTION AFTER RETURN TO EUTHYROIDISM. M. I. Heron, F. Kolar*, F. Papousek*, and K. Rakusan. Dept. of Physiology, Univ. of Ottawa, Ottawa, Canada. *Inst. of Physiology, Czech Academy of Sciences, Prague, Czech Republic.

Ventricular adult rat cardiomyocytes (ARC) in Iongterm culture after a period of degeneration, followed by regeneration do not only exhibit contraction (Review: H.M. Eppenberger et al. Trends Cardiovasc.Med. 1994; 4: 187193) but they also resume hormonal activity. The peptide hormone atrial natriuretic factor (ANF) which in adult heart in vivo is only expressed in the atrium, but no more in the ventricle is, concomitant to the regeneration of the myofibrils, reexpressed in vitro in cultured ventricular ARC (Eppenberger-Eberhardtet al. JMCC 1993; 25: 753-757). By immunofluorescent studies using polyclonal antibodies against 0¢-ANFit is shown that ANF is on the one hand stored in ARC partly in ANF granules, on the other hand it is secreted into the medium. Storage as well as secretion can be influenced by growth factors: Under bFGF treatment both storage and secretion of ANF are increased whereas IGF I brings about a decrease of storage and secretion of the hormone. By Northern blots it can be shown that this up- and downregulationresp. is regulated on the transcriptional level. We hypothesize that control ARC and ARC under bFGF treatment, the latter leading to a further increase of the cell size (Harder et al. 1995; submitted to JMCC), resemble in vitro a state we find in vivo in hypertrophy, where ANF is upregulated. IGF I, though, which promotes differentiation leads concomitant to an enhanced myofibril regeneration (Donath et al. PNAS 1994; 91: 1686-90) to a downregulation of ANF expression, resembling the state found in the differentiatedadult ventricle in vivo.

Microcirculatory networks were studied in newborn male rats given 28 days of tdiodothyronine (HYPER) or propylthiouracil (HYPO). Persistence of changes were examined in adult rats (80 days old), after cessation of treatment and re-establishment of euthyroidism. Neonatal hyperthyroidism induced cardiac hypertrophy; neonatal hypothyroidism induced slower cardiac growth with possible myocyte atrophy. Capillary density in neonatal HYPER was comparable to control (CON) indicating commensurate capillary growth; arteriolar density was greater despite cardiac hypertrophy. Neonatal HYPO had higher capillary density reflecting lower ventricular mass, but lower than expected arteriolar density. After treatment, ventricular mass increased in corrected HYPER and HYPO, but remained below CON. Capillary and artedolar density returned to CON values in both corrected groups. Hemodynamic measurements from an identical parallel study, showed that corrected HYPER (80 day old) had higher developed, maximum, and enddiastolic pressures, (dP/dt)max and (dP/dt)min, in both ventricles compared to CON. Corrected HYPO had similar ventricular hemodynamics to CON. Our data suggest neonatal hyperthyroidism induces compensatory capillary growth, sizeable arteriolar growth, and lasting ventdcular hemodynamic changes. Neonatal hypothyroidism induces slower artedolar growth.

A CYSTEINE PROTEASE INVOLVED IN Fr051 ISOPROTERENOL (ISO) INDUCED CARDIAC HYPERTROPHY Gavin D. Arthur, Douglas Priestley and Angelo N, Belcastro. School of Rehabilitation Sciences, University of Bdtish Columbia, Vancouver, B.C., Canada, V6T 2B5.

CLENBUTEROL INDUCES PHYSIOLOGICAL Fr052 HYPERTROPHY IN LATISSIMUS DORSI MUSCLE AND HEART. Mario Petrou, Simon Windeatt, Anne-Marie L.Seymour, Kenneth R.Boheler & Magdi H.Yacoub.Dept Cardiothoracic Surgery, National Heart & Lung Institute, London U.K.

During the development of cardiac hypertrophy a number of morphological changes occur which are reminiscent of the actions of a calcium activated nonlysosomal cysteine protease, calpain. The purpose of this study was to determine, if calpain, a cysteine protease was involved in the increased cardiac function observed during ISO induced hypertrophy. Rats (N=40) were distributed to 4 groups; control (C); ISO; protease inhibitor (E64c) and E64c+1SO. Three days post injection (1 mg/kg;s.c.), body and ventdcular mass were recorded, left ventricular function assessed and hearts assayed for cAMP and calpain-like activity. The hypertrophic index increased for I S O v s C (p<0.05), while E64c+1SO induced an intermediate response. CyclicAMP levels rose from 103.5 + 2.6 (C) to 128.7 + 4.0 pmol/g (ISO) (p<0.05), E64c+1SO prevented the rise in cAMP (90.9 + 2.7 pmovg; compared to ISO (p<0.05). Left ventdcular pressure development (+dP/dt) and maximum left ventdcular pressure were greater with ISO compared to C (p<0.05), E64c+1SO attenuated the ISO increases, which were similar to C after 3 days (p>0.05). Calpain-like activities increased from 27.26 + 4.64 to 39.54 + 4.96 U/g for ISO) with E64c+1SO minimizing the calpain-like activity (24.64 41.41 U/g). It is concluded that the cysteine protease, calpain is implicated in ISO induced cardiac hypertrophy, perhaps by altedng cardiac function. The [cAMP] datq suggests that its site of action occurs proximal to cAMP forma~on in the heart. Supported by Heart & Stroke Fdn of B.C. & Yukon

Skeletal muscle assistance of the circulation of patients in endstage heart failure requires electrical training of the latissimus dorsi muscle (LDM) to produce fatigue resistance.This process of stimulation and the post-mobilisation atrophy which occurs, result in a profound loss in peak power generated. The 13-2adrenoceptor agonist, clenbuterol (Clen), was used to investigate its potential to induce hypertrophy (H) selectively in skeletal muscle, particularly in LDM, independent of adverse effects on cardiac muscle. Rats received 2p.g/g BW/day Clen subcutaneously for either 5 weeks (Group A) or 3 weeks (Group B). Controls (C) received received 0.5ml saline daily for the same periods of time.The LDM to tibia length ratio was increased by 29% in A (n=12) and 20% in B (n=8) which were both significantly greater than C (n=21) (p<0.01). Measurements of metabolic enzyme activities in LDM showed a marked increase in glycolysis in A consistent with the degree of H (PFK - 17.9+ 0.4p.mol/min/gwet wt, A n=5; 14.8+ 0.5 C, n=4,p<0.01). RNA analyses and RT-PCR amplifications indicate that Clen hearts express elevated levels of mRNA for atdal natriuretic factor (ANF) without a concomitant increase in skeletal ~-actin and 13-myosinheavy chain, consistent with a physiological form of cardiac hypertrophy. Clen induces significant physiological hypertrophy in the LDM associated with specific changes in cardiac gene expression.These findings could have important implications for the use of skeletal muscle assistance of the failing heart.

THE EXPRESSION OF ATRIAL NATRIURETIC FACTOR F r 0 4 9 IN VENTRICULAR ADULT RAT CARDIOMYOCYTES IN CULTURE IS INFLUENCED BY IGF I AND bFGF.

A230

pYRIMIDINE METABOLISM IN THE Fr053 HYPERTHYROID RAT HEART Ryszard T. Smolenski, Piotr Jagodzinski, Magdi H. Yacoub & Anne-Marie L. Seymour, Dept Cardiothoracic Surgery, National Heart & Lung Institute, Harefield Hospital, UK. Hyperthyroidism produces changes in metabolism and morphology of the heart which may affect myocardial nucleotide pathways. Such alterations include impaired energy metabolism, increased protein synthesis and a decrease in the cell surface/cell volume ratio due to hypertrophy. In this study rats were treated for one week with thyroxine (T4) which resulted in a 40% increase in heart weight/body weight ratio. Purine and pyrimidine nucleotide contents were evaluated in extracts of hearts frozen in situ. Uridine phosphorylase was measured in heart homogenates. There was a 42% increase in CTP/ATP ratio and 32% increase in UTP/ATP ratio as a result of T 4 induced hypertrophy. The activity of uridine phosphorylase was 0.165 (+0.020, n=4) pmol/min/g wet wt in control heart and increased to 0.283 (+0.026, n=4) pmol/min/g wet wt in the heart of T 4 treated animals (p<0.02). As uridine phosphorylase is predominantly located in the endothelium our results suggest that, in hyperthyroidism capillary density increases in parallel with the hypertophy. In conclusion: hyperthyroidism is associated with increases in pyrimidine nucleotide content and metabolism.

INTERLEUKIN-1 DIFFERENTIALLY Fr055 REGULATES CARDIAC CELL GROWTH IN CULTURE Carlin S. Long, James Palmer, and W e n d y Hartogensis Cardiology Section, San Francisco VA Medical Center Interleukin-1 0 L - l ) , is primarily known as a mediator of inflammation. However, it also plays many other diverse physiologic roles including the stimulation and inhibition of both ceils in culture and the interstitial and parenchymal cells of a number of organs including the heart. In heart, IL-1 expression has traditionally been reported in situations of myocardial injury such as occurs during transplant rejection and congestive heart failure. For this reason, all of the effects of IL-1 have been presumed to be deleterious. Using a ceil culture model which allows both the muscle cells (myocytes) and nonmuscle ceils (fibroblasts) to be evaluated separately, we have found that IL-1 induces both cardiac myocyte h y p e r t r o p h y (1.385:0.04-fold o v e r c o n t r o l , n=5, p<0.001) a n d re-initiates m y o c y t e D N A synthesis (increase in myocyte ceil numbers of 1.24_+0.04, n=3, p< 0.01). In stark contrast, IL-1 exerts a potent antiproliferative effect on cardiac fibroblasts (0.19-k0.03-fold vs control, n=5, p~0.001), a result that extends to the inhibition of mitogen-stimulated (serum) proliferation. To our knowledge this is the first report concerning the differential effects of I D 1 on myocardial ceil growth in culture and, given the expression of IL-1 in heart during stress, underscores the importance of investigating the complex nature of the intracardiae cell-ceil interactions that occur in the heart.

EFFECT OF T E T R A N D R I N E O N CARDIAC MASS Fr054 H A E M O D Y N A M I C S A N D BINDING O F (all) PN2oo-~oIN DOCA-SALT HYPERTENSIVE LV HYPERTROPHIED RA2S

M.R.Rao, Y.Xu Dept of C.ardovascularPl'ermacology, Nan/ragMeclcal U~versity.NANJING,210029CHINA The effect of regression of left ventricular hypertrophy (LVH) on ventricular haemodynamics, coronary flow, LV developed pressure (DP), LV compliance, LV stiffness constant and dihydropyridine (['H] PNaoo--,xo) binding to heart, hipocampus and thalamus membranes were studied in DOCA- salt rats (at 18 weeks after hypertension) treated with Tetrandrine (TCt) (Tet 40rag-" qd for 9 weeks). Cardiac haemodynamioa was determined through working heart. LV compliance was measured with LV volume-- pressure curves. LV haemodynamics tended to be depressed in DOCA -salt hypertensive rats with cardiac hypertrophy. After treatment with Tet, ventricular performance (I,VH CF 8.82+ 0.9 to 11.58+ 1.12 ml.na i n - ' , LVH CO 35.45± 2.4 to 47.7± 2.8 nal.mln-') LV-to body weight ratio (LVH 3. 9± 0.1 to 2.5± 0.2 nag.g-a), LV stiffness (LVH 8.64± 0.6 to 4.6± 0.3) and LV compliance returned to control level. The B m ~ of dihydrop3h'idine binding sites in LVH, hipocampus and thalamus increased by 106.6, 57.0 and 138. 0%. All parameters returned to normal after 'let treatment.

EARLY EXPRESSION OF C-MYC IN Fr056 MYOCYT ES CAUSES HYPE RT ROPHY IN V O L U M E - O V E R L O A D RAT HEART. Satoshi T a k e t a n i , Yoshiki Sawa, Yasuhisa Shimazaki, Kazuhiro T a n i g u c h i , Shunzo Onishi, Naomasa Kawagnchi, Noboru Fujitanf**, Hikaru Matsuda. 1st Dept of Surg , School of Allied Health Science*, Osaka Univ., Osaka, Japan, Dept of Forensic Saga, Medical School, Saga, Japan** Proto-oncogene has been implicated in the palhogenesis of hyperlrophy in the hearl. However, its important role especially in volume-overload hypertrophy in the heart is undelermined. To investigate the localization and its role of c-myc in volume-overload hypertrophy, expression of MYC protein was immunuhistochemically investigated in the rat hearts following volume overload by A-V shunt belween abdominal aorta and inferior caval venous. The volume-overload(V) hearts showed significantly greater size of myocytes and heavier weight than did control hearts after 2 weeks of A-V shunt. Expression of MYC protein in the nuclei of myocytes of V hearts peaked at day 2 after A-V shunt and then, this expression gradually decreased. On the other hand, number of fibroblasts and MYCexpression in nuclei of fibroblasts increased after 2 weeks of A-V shunt. These data demonstrated that MYC protein was expressed in myocytes in the early phase of volume overload associated wilh the increase of myocyte size and heart weight as compared with those expressed in fibrobrasts. This suggests that early expression of cmy c in myocytes may cause cardiac hypertrophy rather than those in fibroblasts in the early phase of volume-overload heart. A231

CHANGES OF MYOCARDIAL ANTIOXIDANT STATUS F~57 FOLLOWING CHRONIC INTOXICATION WITH CADMIUM Radoslav V. ~Ikl~, Branka Ognjanovl~, Andra~ ~taJn & Milosav M. Kostie, Faculty of Sciences and Faculty of Medicine, Univeresity of I~'aEu-

J e v a c , 34000 KraguJevac, FR Y u g o s l a v i a Chronic exposure of male Wistar alblno rats to dietary intake of cadmium (15 mg CdCl2/d/kg) leads to signlflcant reductlon of total body

mass (-33±3 Z) and ETowth r a t e (-63±7 %), d e c r e a s e o f r e s t i n g 02 c o n s u m p t i o n (-28±2 Z) and, among o t h e r s , d e v e l o p m e n t o f c a r d i o v a s o u l a r and h a e m a t o l o E i c a l c h a n g e s . Myocardial hyp e r t r o p h y , c h a r a c t e r i z e d by a c c u m u l a t i o n o f cadmium (1.66±0.01 ~E/g wt) a n d l o w e r p r o t e a n content (4.2 v s 5.5 mE, p<0.02) is found In cadmium-tpeated rats. Activitles of all myocardial antioxldant enzymes (SOD, catalase, GSH-Px, GST), as well as plasma concentrations of ascorbate and tocopherol, are significantly Increased in treated animals. Particularly, the specfflc actlvlty of SOD showed remarkable Increase of 197±8.3 Z (p<0.00S), indicating an Important role of superoxide anion radical in cadmlum-lnduced cardiotoxlcity. Simultaneous ingestion of Na-selenlte (7 ~g/d/kE) prevents cardiac hypertrophy, reduces cadmium accumulatlon in the heart and partlaly corrects protein defficiency. Activities of all myocardial antioxidant enzymes, except catalase, were also significantly increased in selenite-cotreated rats.

CLmEBR~d. OA8 EXCHANOE IN "NORMAL"SUBJECTS Fr059 AND IN PATl~rr8 WITH ~ HYPERTROPHIC CARDIQMIOPATHY OeorgyYa,Oebei, AlexandrO.Kmglov, Ludmllmm,Ooloatmtovm, Nexandr ~ I ~ i N V , VmkaT ~ UUdn, Vlotor F- BqdstJev, Hloolsy O.lgnmov*, 8wgey O~uvorov. 8gat= 6ol,Contmr. InM~uto of B~nl~ Pmblwn~ "'Medicine for you=Coqxxxtton, Rumd=

The mUwlmtzsUm dM= ~ m JS =ubJe~k fNm o( oenSmmmum ~homw

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peUem ~ h 0re.P) we Blood ImmplN Yam tlklm from i.fem(xdll (i) i~d •k~ ~dd(nS) mndR~RX)2 (mm~X sOm,~lm evelumd. The sumnvuy O ~ prmum (SGP) (PE=PO2+PCO2) In

hypemn~lvehypemm~ cemnmyoNmy(HHC

(PEa+PEvJ)m m c~cullkl In mm Hg., The meen mira/el p r o e m in m ~ p J x ) ) , u u ~ o n ~ m (pep), m . = ~ (p=S), vJuoulmtm(pvJ)- - vmn - - ~ mD (HR) ~ ~ (Hb)

weremmmm~ PO2 I 2il6,72~0,1 vi sT,m~0,s • till,li(~_~Ol v~ 4A,trr~,l

PCO2 PE SO2 pH $7,30~2,3 N, IL~0,1 7,391_~0,004N 4~,W~_,. ~ , ~ , S U,~L*O,Z 7,SSS_*0,00S ~ll,,~li~l,li I~,(i1_~,6 7~41_e4,0~IHHC 4il,01_4~,0 ~lll,:tS~.§ 71~17_*t,1 7,N4~0,011

pAO pAP I~S lWJ HR 286,5~._1,7 1 4 ~ 2 9,4~_12 (~7~1~ 76,6fI,2

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The 10~OWlll0 R11~ ~illolgd~k~ ~ the 0fo4J~ ale rlo(~l; i) the diffemn~lnADba, mm Ihe gmul~; b)lhecommon ohan~mrln Immmbral venous ~ dMa. S~o In venous bl~xl oubloMngfromthebrs~iecons~mtvAh 1=O2 Inwtmd blood Inflo~ng k~ Ule b ~ n ~ b~h gmu~L erndk~ t~mebe~o~, the "norm~ vlue o~m~k~ ~ eq/to0,e3+0,~mmHg taproom l ~ Ilmlte el~cl Ill dlmlC4Mlblk~ wore pmeented blfonl. In HHC i k l _.~j_mJto 0,693_+3,4 mmH0, I.e. II b ~ ~lh "normer.

A232

A NEW SURGICAL THERAPY FOR PATIENTS WITH Fr058 INOPERABLE CORONARY ARTERY STENOSIS. M. Beyer, U. Beyer, T. Scheunert, S. Mierdl, A. Hannekum Experimentally it was shown, that the grafting of a free skeletal muscle flap onto the heart produces a functional myocardial revascularization. Therefore clinical application of this new surgery was performed. Methods: 5 male patients, aged over 65 with diffuse and peripheral coronary artery stenosis and suffering from severe angina pectoris, unterwent the intervention in May 1993 (i), April 1994 (2, 3), June 1994 (4) and August !994 (5). The ECG showed in all patients ischemic ST-segment changes at 50 to 80 Watt in the bicylce ergometry. Thus, a free skeletal muscle flap (musculus latissimus dorsi) was grafted onto the anterior wall of the heart. The flap artery was implanted into the aorta, venous flow was directed into the right atrium. Results: All patients had no more anginal episodes except in the first postoperative weeks. Ischemic ST-segment changes at bicycle ergometry were no more evident. Angiographically, a visible myocardial revascularization was lacking. The quality of life of all patients was i00 % improved. Therefore, the grafti~9 of a free skeletal muscle flap onto the heart should be performed on patients with diffuse and peripherical coronary artery stenosis, who cannot undergo direct coronary artery surgery.

COLLAGEN GENE EXPRESSION, MYOCARDIAL Fr060 STIFFNESS, AND HEART FAILURE IN THE SPONTANEOUSLY HYPERTENSIVE RAT Chester H. Conrad, Marvin O. Boluyt, Wesley W. Brooks, Oscar H.L. Bing. VA Medical Center & Boston University, Boston MA and GRC, NIA, Baltimore MD Myocardial stiffness, fibrosis, and hydroxyproline are increased in the spontaneously hypertensive rat (SHR) with heart failure (SHR-F), relative to age-matched (18-24 mo) non-failing SHRs (SHR-NF), and non-hypertensive controls (WKY). We studied collagen gene expression in LV myocardium from SHR-F, SHR-NF, and WKY. Central segment myocardial stiffness (kcs) was measured in isolated LV papillary muscles using passive stretches. Procollagen GI(I) (C1) and al(lll) (C3) mRNA (expressed relative to WKY) vs. kcs are shown below. OWC+

i°!o/+1 J "

l"

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~.0

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!

,.o ooo

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o.ol . . . . . =0 30 4o .so 6o 7o MYOCARDIASTI L FFNESS ~¢.)

Myocardial stiffness (kcs) was correlated with C1 (r=0.93, p<0.001) and C3 (r=0.92, p<0.001) in the SHR; there was no significant correlation in the WKY group. These data suggest that heart failure in the SHR is associated with upregulation of collagen gene expression, active fibrosis, and increased stiffness.

Genotype and phenotype or severe Fr061 mitochondrial cardiomyopathy Takayuki Ozawa, Kazumi Katsumata, Mika Hayakawa, Masashi Tanaka & Satoru Sugiyama Dept of Biomed Chemi, Faculty of Medicine, University of Nagoya, Nagoya, Japan Some of cardiomyopathies appear to be caused by mutations in mitochondrial DNA (mtDNA), thus, being regarded as a mitochondrial disease.

Comprehensive

analyses of mitochondrial (mt) DNA of a 7-year-old girl who underwent heart transplantation because of severe cardiomyopathy

revealed

three germ

line point

mutations, each one in the 12S rRNA gene, in the cytochrome oxidase subunit 1 (CO1) gene, and in the cytochrome b gene. As her somatic mutation, extensive

ABNORMAL MITOCHONDRIAL RESPIRATION IN Fr062 FAILED HUMAN AND DOG MYOCARDIUM. Victor G. Sharov, Hani N. Sabbah, Jane M. Cook, Norman Silverman, Michael Lesch & Sidney Goldstein. Henry Ford Heart & Vascular Institute, Detroit, Michigan, USA. Abnormalities of mitochondria (MIT) exist in the failing heart and include hyperplasia, reduced organdie size and structural injury. We examined MIT respiration in LV tissue (30 mg) obtained from explanted hearts of patients with ischemic cardiomyopathy (ICM,n=8) and idiopathic dilated cardiomyopathy (IDC,n=13) and in normal dogs (NL,n=8) and dogs with heart failure produced by intracoronary microembolizatious (HF,n=I 1). MIT respiration in the presence of the substrates (V-SUB) glutamate and malate and state-3 respiration (V-ADP, after addition of I mM ADP) was measured with an oxygraph and Clark electrode using saponin-skinned fiber bundles. Respiration rate was calculated in ngatoms of oxygergmirg 3rotein.

fragmentation of mtDNA associated with 212 kinds of deletions was detected in contrast to 5 kinds in an agematched negative control. A recipient's positive control, having almost

the

same

base-substitutions

and

mutations with the recipient except one in the CO1 gene, also developed severe cardiomyopathy, and died at age 20. The close relation between phenotype and mtDNA genotype provides basis of our understanding to cell death and premature ageing.

FRO63 PECULIARITIES OF ACUTE CARDIAC PUMP FAILURE CAUSED BY VARIOUS ENERGY SUPPLY LIMITATIONS Vladimir L. Lakomkin, Valeri I. Kapelko, Olga V. Korchazhkina, Irina M. Studneva & Oleg I. Pisarenko. Cardiol. Res. Centre. Moscow, Russia

The contractile function of the isolated rat heart and high energy phosphate content were evaluated under conditions of depressed energy supply caused by disturbances either in mitochondrial ATP production or ATP-phosphocreatine transformation. Amytal (0.3 mM), an inhibitor of mitochondrial respiration, or iodoacetamide (/AA, 0.1 mM) reducing in this dose creatine kinase activity to 16% of the initial level, were used, respectively. Myocardial ATP content remained unaffected in both groups and PCr content decreased to 37% only in amytaltreated group. Very similar alterations in cardiac pump function during volume load were observed in both groups; maximal cardiac output was significantly less by 30%, cardiac pressurevolume work by 38-40%, left ventricular (LV) systolic pressure by 24-29%, and LV +dP/dt by 36-39%. In contrast, the extent of a fall in LV distensibility was different, a curve relating LV filling volume and end-diastolic pressure was shifted up and to the left much more prominently after /AA treatment. The difference was corresponded to decreased LV filling pressure area after /AA treatment. Heart rate slightly increased at volume load both in control and /AA-treated groups while a definite decrease was observed in amytal-treated group, heart rate in the latter group at maximal load was 24% less than in two other groups. Results indicate that a decreased myocardial distensibility is a dominating feature in the acute cardiac pump failure caused by an inhibition of myocardial creatine kinase.

V-SUB

NL Dog

HF Dog

ICM

IDC

31+2

18±3

21±2

15±2"

V-ADP 46±4 ~ 19±3 22--t:4 14±2' [*=P<0.05 ICM vs. IDC; :I:=P<0.001 V-SUB vs. V-ADP] Addition of ADP increased MIT respiration rate in NL dogs but not in HF dogs or ICM and IDC patients. Furthermore, V-SUB and V-ADP were lower in IDC compared to ICM. Conclusion: MIT state-3 respiration is abnormal in failed human and dog hearts. In failed human hearts, this abnormality is even more profound in IDC compared to ICM.

IN(UDENCEOFDNASTRANDBREAKSINMYOCYTESAC1JT~Y Fr064 AFTERM Y ~ [ A L I]~ARCTION AND ~ ]-EAR'E FAILUREINHUMANS Federico Quaint, Costanza Lagrasla, Roberta Maestri, Domenico

Corradi, Elena Cigola, Edgardo Bonacina, Giorgio Olivetti, Piero

Anversa, Depl of Pathology, Hospital of Parma and Milan-ltaly and NY Medical College, NY USA

After coronary artery occlusion, the magnitude of myocyte loss exceeding the growth reactive processes of the myocardium may dictate the onset and the development of intractable congestive heart failure. Although ischemic in origin, myocyte loss may occur as a result of mechanical myocyte death since diastolic stress is maximally elevated under this condition and the relationships between blood flow hemodynamic and necrotic injury have not been clearly defined. Mechanical myocyte death is included in the category of programmed cell death or apoptosis. A trademark of apoptosis is the internucleosomal DNA fragmentation in otherwise morphologically normal myocytes. DNA strand breaks in myocytes were detected in situ by biotinylated deoxyuridyl triphosphate (dUTP) labeling, using exogenous terminal deoxynucleotidyl transferase, in hearts obtained from 10 patients who died within 2 to 5 days after a large myocardial ,nfarction. On a qualitative basis, positive myocyte nuclei were mostly observed in areas of myocardium lacking inflammatory infiltrates and in the subendocardium. Quantitative estimation of the incidence of this phenomenon showed that in the region bordering the necrotic area 62 +_13% myocyte nuclei exhibited DNA strand breaks, whereas, in a 13 mm2 area lateral to infarction, 30 + 26 % myocyte nuclei were found to be positively stained. In the region remote from infarction, scattered myocytes (0,009 + 0,001%) showing positive labeling were observed. In conclusion, acute myocardial infarction in humans is associated with the induction of DNA strand breaks in myocytes suggesting that apoptotic myocyte cell death may participate to the loss of contractile mass leading to acute ventricular dilation and failure.

A233

Ca2+ATPase AND RYR-2 GENE EXPRESSION IN THE FRO65 EARLY PHASE OF HEART FAILURE P. Trouvt, I. Coquard, F. Rannou, L. Soufir, F. Cart6 and D. Charlemagne INSERM U127, Hopital Lariboisi~re, 75010 Paris, France. Among the mechanisms that contribute to the transition phase from compensated cardiac hypertrophy to heart failure, we hypothesized that an alteration of the sareoplasmic retieulum (SR) proteins might be involved and lead to alterations in calcium handling, and in turn, to arrhythmias. To test this hypothesis, the occurrence of spontaneous arrhythmias was explored by Holier monitoring and the mRNA levels of Ca2+ATPase and ryanodine receptor (RyR-2) were studied in rats, 3, 6 and 12 months after abdominal aortic stenosis and compared to age-matched controls, mRNA levels from left ventricles (LV) were quantified by ribonuclense protection assays, Northern and slot blot analysis. After 3 and 6 months of stenosis, the animals exhibited 20 to 30% of hypertrophy. Ca2+ATPase and RyR-2 mRNA levels were unchanged, confirming previous results obtained in mild hypertrophy. After 12 months of aortic stenosis, a similar hypertrophy of the LV (24,57% +: 2,51; p<0.05) was observed, whereas the weight of right ventricles, lungs, and livers remained unchanged, suggesting that heart failure did not yet occur. In these 12 month groups, the Ca2+ATPase and RyR-2 mRNA levels relative to GAPDH (RPA) or 18S rRNA were significantly decreased by 57% and 46% respectively in hypertrophied LV, compared to age-matched controls. Moreover, Holter monitoring on 4 animals from the 12 month group showed severe characterized arrhythmias (ventricular extrasystoles and ventricular tachycardia) and conduction troubles (AVB) which were not observed in age-matched controls. The decrease of the mRNA levels of the main SR proteins suggest that these proteins might be involved in intracellular calcium handling abnormalities and oeeurence of arrhythmias. These alterations might be one of the first event leading to heart failure.

ENDOTHELIN RECEPTOR BLOCKADE IMPROVES FRO67 CENTRAL HEMODYNAMICS IN DOGS WITH CHRONIC HEART FAILURE. Sidney Goldstein, ttlsashi Shimoyama, Mitsuhiro Tanimura, Serguei Shevlyagin, Steven Borzak & Haul N. Sabbah, Henry Ford Heart & Vase. Inst., Detroit, Michigan, USA. Plasma enclothelin (El3 levels are increased in heart failure ffIF) and may contribute to the vasoconstriction seen in this disease state. We examined the hemodynamic effects of intravenous Bosentan (BOS) (10 mg/kg), a non-peptide, competitive El"I receptor blocker, in 8 dogs with I-IF produced by intracoronary mieroemboliT~tions. Measurements wea'e made at baseline and at 30 and 60 rain after BOS. BOS had no significant effect of heart rate or blood pressure, but improved LV end-diastolic pressure (I.,VEDP, mmHg) peak LV +dP/dt (mmHg/see), cardiac output (CO, l/rain), systemic vascular resistance (SVR, dynes-see-era"s) and LV fractional shortenin ~ (LVFS, %).

LVEDP +dP/dt

Baseline

30 min

60 min

19-J:3

14~3 *

12-+2 *

2420-~-150*

2364:t:191 *

3.47~0.42 *

3.69-~-0.54"

1788-+-85

CO

2.83i0.31

SVR

3489-~247

LVFS

31~3

3002-+-356 38-+-2*

2757:1:356* 38.+.2 *

"*=P<0.05 Baseline vs. 30 and 60 rain] Conclusion: Intravenous BOS, an ET~ receptor blocker, improves overall LV function in dogs with chronicHF. A234

EFFECT OF BETA-RECEPTOR DOWN-REGULATION FRO66 ON SARCOPLASMIC RETICULUM CALCIUM HANDLING IN RABBIT CARDIACTRABECULAE Martin A Denvir, *Nalll G MacFarlane, *David J Miller & Stuart M Cobbe, Department of Medical Cardiology and *Institute of Physiology, Glasgow University, Glasgow, UK. We have previously reported enhanced sarcoplasmic reticultan (SR) calcium loading in a rabbit coronary ligation model of heart failure. Alterations in beta-receptor populations could explain this finding. We therefore examined SR calcium loading and myofilament force production using Saponin- and Triton-treated right ventricular trabeculae from rabbits 7 days after isoprenaline infusion by subcutaneous mini-osmotic pump (100y.g/kg/hour, IMP, n=10). Saiine-infused rabbits (SMP, n=7) were used as controls. Invasive haemodynamic measurements of heart rate, left ventricular pressures and cardiac output in response to bolus injections of isoprenaline were made at the end of the 7 day infusion. IMP rabbits demonstrated a diminished peak heart rate response (336.7+8.1 vs 368.6.'!:6.3 beats/rain, P<0.05) and a reduced cardiac output response (22.9+2.3 vs 26.9+...2.1% increase) to isoprenaline suggestive of betareceptor down-regulation. Trabeeulae were mounted for isometric tension measurement and the amplitude of the caffeine induced contmcture used as a measure of SR Ca 2+ content. There was no significant difference between groups in the amplitude of peak caffeine-induced contracture (34.3+10.6 vs 32.6+6.2%Cmax; P=0.65). There was also no difference in the conditions ([Caz+] and time) at which the onset of spontaneous tension oscillations occurred. There was no difference in myofilument maximum force production between groups (4.76+:3.55 vs 5.21+3.88gwt/mm 2) although there was a slight difference in myofilament calcium sensitivity (pCa50-5.51+0.07 vs 5.61_+0.05; P=0.053). These results suggest that enhanced cardiac SR calcium loading in the rabbit coronary ligation model was not directly related to adrenoreceptor down-regulation.

C Y C L O H E X I M I D E (Cx) ABOLISHES LOSS IN FRO68 CARDIAC EFFICIENCY IN ISOLATED H E A R T S Richard Schulz and Donna Panas. Depts. Pediatrics and Pharmacology, University o f Alberta, Edmonton, Canada Endotoxin and pro-inflammatory cytokines cause a delayed depression in myocardial contractile function by the expression of inducible NO synthase (iNOS) in the isolated heart. As NO inhibits mitochondrial respiration, we determined whether the cardiac depression may be due to an inhibition of MVO 2 and energy metabolism. Isolated working rat hearts paced at 290 bpm were perfused with modified Krebs' buffer (>Ing/ml endotoxin) containing 1 l m M glucose, 5 mM pyruvate and either [U-14C] pyruvate or [U-"C/5-3H] glucose, for the measurement o f pyruvate oxidation or, glucose oxidation and glycolysis, respectively. TCA cycle activity was determined by measuring the rate o f acetyl CoA entering the cycle derived from pyruvate oxidation (which comprised over 90% o f TCA cycle acetyl CoA). Cardiac work remained constant for the first hour and then progressively declined to 39.2+6.6% o f initial values by 2hr (n=12, p<0.05). Cx (I0 l.tM) prevented the loss in cardiac work at 2hr (83.3+4.6%, n=16, p<0.0001). Despite the loss in cardiac work, MVO z and TCA cycle activity remained constant between control and Cx groups. Cardiac efficiency (ratio o f cardiac work/MVO2) dropped by 55% in control hearts but did not change over 2 hr perfusion in Cx-treated hearts. Mitochondrial function is not compromised in failing hearts, rather, ATP is not being efficiently utilized for contractile work. Cx prevents the loss in efficiency possibly by inhibition o f iNOS expression.

PROGNOSTIC IMPLICATIONS OF PLASMA Fr06g ATRIAL NATRIURETIC PEPTIDE CONCENTRACTION IN PATIENTS WITH ACUTE MYOCARDIAL NFARCTION Aleksander G6recki, Grzegorz Opolskl, Andrzej Januszewicz*, Bo~enna Wocial*, Krzysztof Slomka, Mariusz Lapifiski*, Hanna lgnatowska-~witalska*. Dept. of Cardiology and Dept. of Hypertension and Angiology * Medical Academy in Warsaw, Poland. The aim of the study was to assess whether plasma concentration of atrial natriuretic peptide (ANP) might predict the prognosis of acute myocardial infarction (AM]). We studied 40 pts with first AMI up to 6 hrs onset the first symptoms. Plasma ANF' concentrations were determined at 0, 4, 8, 16, 24, 48 and 72 hrs post admission. Patients were classified into 2 groups according to ANP level (21 pts with high ANP > 28 finol/ml - group I, 18 pts with low ANP < 28 fmol/ml - group II). The mean period of follow-up was 36 months (range 24 - 48 months). The ANP 0 in pts with complications of AM] (CI-IF, arrhythmias, reinfarction, death) was significantly higher than in pts without complications (61.9 9-a:114.2 vs 13.8 9-a:13.1 fmol/ml). In group I of pts we observed significantly more frequently new CI-IF, second AMI and cardiac death during follow-up period: group I

group I!

p value

8

1

< 0.05

New CHF Second AMI

8

1

< 0.05

Cardiac death

6

0

< 0.05

The data suggest that high ANP 0 concentration is unfavourable predicting prognosis of AMI. The plasma ANP level is a useful non-invasive prognostic marker of identifying those at high risk of CHF, second AMI and cardiac death among pts after AM].

THE INFLUENCE

OF EXTRA-THRESHOLD

Fr071

ELECTRICAL STIMULATION ON CONTRACTILE ACTIVITY OF BIOPTATS OF HUMAN

MYOCARDIUM IN ISCHEMIC HEART DISEASE S.A.Afanasyev,A.V.Lebedev,A.M.Chernyavsky,

PACING-INDUCED HEART FAILURE REDUCES Fr070 TISSUE Mg CONTENT AND DEPRESSES MYOCARDIAL [Mg2+]~ IN DOGS MCP Haigney, BS Silver, SK Wei, R Tunin, D Kass, S Kalh, EJ Griffiths and HS Silverman. Uniformed Servic~ University, Bethesda, MD and Johns Hopkins University, Baltimore, MD USA Reduction of total cellular Mg ([Mg]~) in hearts of patients with CHF was shown at autopsy and explantation, but it is unknown if Mg loss is due to diuretic use, concurrent disease, or it"CHF causes Mg depletion primarily. It is also not known if the reduction in [Mg]i diminishes cardiac ionized Mg ([Mg2*]i). [Mg:+]~ represents < 10% of [MgL but is more physiologically significant. To test the hypothesis that CHF results in a loss of tissue Mg in the absence of confounding drugs or diseases, we obtained smears of sublingual epithelial cells from normal dogs (n=4) and dogs exposed to 4 weeks of ventricular pacing at 250 bpm (n = 4), the latter manifesting bi-ventricular failure. IMg]~ was measured using energy dispersive X-radiation. Cardiac myocytes were isolated from 4 dogs (3 CHF, 1 normal), loaded with the fluorescent probe Mg-indo to measure [Mg~-+]i,and studied in HEPES-based buffer (20"C, 1.2raM MgCI,). Sublingual cells had lower [Mg]~ in paced dogs (34.8mEq/L_+0.8 vs 38.7mEq/L+0.5, p < 0.05). The fluorescence ratio indexing [Mg2~l~ was reduced in paced animals (0.57+0.01, n=49 cells) compared with the ,ormal dog (0.79 +0.03, n = 18 cells, p < 0.001). Thus short periods of CHF result in a loss of tissue Mg and a decrease in myocyte [Mg-'*]~ which may have important consequences.

EFFECTS OF ACE-I}THIBITOR ENALAPRIT, FRO72 ON NATRIURETIC HOR~ONE IN CONGESTIVE HEART FAILURE Oleg I.Shushlyapin,Vladlmir D.Babadzhan&Larisa A.Lapshina State ~edical University,Khar'kov,U~raine

I.V.Ponomarenko. Institute of Cardiology,Tomsk,Russia The work is performed on biopsy of human heart (37-55 years). The influence of extra-threshold electrical stimulation on inotropic abilities of human myocardium in ischemic heart disease, on the content of peroxide oxidation products and the state of hydrophobic antioxidant system in cardiac muscle was studied, Heart bioptat (trabeculas extracted from atrium) of patients with IHD of III and IV functional classes (NIOA) were used. Patients with WPW syndrome Without ischemia sewed as controls In isometric regime muscle preparations of IHD patients heart were found to increase (p<0,05) inotropic effect in extrathreshold increase of stimulating electrical impulse.The increase in relaxation developed by muscle (mean 84 % vs threshold values), the rate of its rise and fall is demonstrated. Bioptats of WPW patients do not exhibit such change. The peculiarities of contractile activity of myocardium in IHD correlated with reliable (p<0,05) accumulations of bioptats of peroxide oxidation products in hydrophobic fraction. IHD patients of severe functional classes demonstrated the accumulation of trien conjugates.The accumulation of peroxide oxidation products correlated with the inhibition of endogenous antioxidant defence of myocardium Extra-threshold stimulation in IHD possibly eliminates disorganization of membrane structures of cardiomyocytes favouring coordinated functioning their ion-transporting of systems.

The aid of the present study was to assess interaction of the natriuretic hormone and indices of peripheral hemocirculation in succesful treatment of 86 patients with congestive heart failure(CH~) of II-III classes NYNA by ACE-inhybitcr enalapril. The results of the investigation given in the table Result s Treatment Before After C cmmon peripheral vascular_resistance (d. s.dm-D/m2) 2289+I 70 1 837+I 5~ Natriuretic hormone of plasma (ink-tool/l) 1 06 +--II 71 +_8* *P <0.05 These results indicate that the reduction of initially high concentration of natriuretic hormone by the influence of enalapril can serve as a marker of improved peripheral vascular resistance in congestive heart failure A235

SYNTHESIS OF E X T R A C E L L U L A R MATRIX PROTEINS IN F A I L I N G HUMAN M Y O C A R D I U M OF PATIENTS W I T H DILATED CARDIOMYOPATHY Hanke Mollnau, Brigitte Miinkel, Jutta Schaper, Max-Pianck-Institute, Bad Nauheim, Germany

Fr073

As p a r t of our s e a r c h for the molecular b a s i s of h e a r t failure (HF) we studied t h e composition of t h e extracellular m a t r i x (ECM) in 9 p a t i e n t s with H F due to dilated c a r d i o m y o p a t h y (DCM). Tissue obtained during t r a n s p l a n t a t i o n surgery w a s studied using immunohistochemistry and eonfocal microscopy (CM). Monoclonal antibodies were used a g a i n s t the E C M proteins : fibronectin (F), laminin (L), chondroitinsulfate (CS), t e n a s c i n (T), v i m e n t i n (V), collagen (C) I, III, IV, VI, and C D 6 8 for m a c r o p h a g e s . Sirius-red staining showed a distinct fibrosis. All ECM proteins, b u t especially C VI, were increased in amount. CM showed a close s p a t i a l relationship b e t w e e n myocytes (myo) and C VI. Dedifferentiated myo and t h e i r debris as detected by L were numerous. The n u m b e r of cells labeled with V or CD 68 was high i n addition to the occurrence of T and F indicating injury and formation of new s e a r tissue. Older s c a r s contained m a i n l y all C b u t no F or T. Conclusion: Dedifferentiation of myo a c c o m p a riled b y s e q u e s t r a t i o n of cell f r a g m e n t s plus development of fibrosis occur simultaneously. The increased a m o u n t of ECM m a y trigger apoptesis t h e r e b y e s t a b l i s h i n g a circulus vitiosus leading to continuous deterioration of the cardiac tissue.

CELL ADHESION M O L E C U L E S IN ADVANCED HUMAN H E A R T DISEASE. Bruno Devaux, Dimitri Scholz & Jutta Schaper. Max-Planck-Institute, Bad Nauheim, Germany

Fr075

The expression of cell adhesion molecules was studied in severely diseased hearths with myocarditis (M, n=6) and dilated cardiomyopathy (DCM, n=4). Tissue was obtained at transplantation. Control samples (C, n=3) were from patients undergoing mitral valve replacement. ICAM-1, PECAM-1, VCAM-1 and E-SELECTIN were studied using specific antibodies and confocal microscopy. R e s u l t s : Capillary density (cap d) determined by PECAM was significantly increased in DCM. E-SELECTIN and VCAM were absent. ICAM and PECAM see table. Electron microscopy showed ICAM only at the luminal side of cap. Values for M equaled C because only non-affected myocardium was counted. In necrotic areas with mononuclear infiltrates (inf) cap d was high, and ICAM labeling exceeded PECAM. CD 68 revealed the presence of ICAM labeled macrophages. Myocytes bordering inf were also positive for ICAM. Group n patients PECAM-1 ICAM-1 I/PE M 6 985 + 79 973 5:77 0.95 DCM 4 11205:86* 11505:105" 1.03" C 3 694 + 88 605 + 70 0.87 m + SEM, n cap/ram2, * p<0,05 C vs DCM. Conclusion: in DCM, an increase in cap d occurs as adaptative angiogenetic response to hypertrophy. Chronic inflammation is present in DCM. Inflammation is focally and dramatically enhanced in inf of acute M. A236

EXPRESSION OF CELL ADHESION MOLECULES IN SEVERELY I S C H E M I C HUMAN HEARTS Bruno Devaux, Dimitri Scholz & Jutta Schaper. Max-Planck-Institute, Bad Nauheim, Germany

Fr074

We studied the expression of cell adhesion molecules and capillary density (cap dens) in non-infarcted myocardium (NI) from 3 failing human hearts with chronic severe ischemic heart disease (IHD) and multiple infarcts obtained at transplantation surgery. Controls (C, n=3) were from patients undergoing mitral valve replacement. ICAM-1, PECAM-1, VCAM-1 and E-SELECTIN were studied using specific antibodies and confocal microscopy. Results: PECAM-1 was present in all vessels and therefore used to determine cap dens, see table. The tissues were negative for E-SELECTIN, and VCAM indicating the absence of an acute inflammatory response. Almost all vessels in NI expressed ICAM-1 and the significant increase of the ratio ICAM/PECAM indicates a process of chronic inflammation ( see table). Group n patients PECAM-1 ICAM-I I/PE I 3 1076 5: 49* 1075 +70" 0.94* C 3 694 + 88 605 + 70 0.87 m 5: SEM, n of capillaries / mm 2, * p<0,05 C vs I. Conclusion: In human hearts with severe IHD and multiple infarcts the NI is afflicted by the sequels of the disease. NI shows adaptative hypertrophy and an increased angiogenic response accompanied by chronic inflammation but is incapable to prevent heart failure, probably because only small vessels respond.

a- AND ~-ADRENOCEPTOR MEDIATED Fr076 INOTROPIC EFFECTS IN FALLING HUMAN VENTRICULAR MYOCARDIUM Tor Skomedal 1, Kjell Borthne 1, Jan-BJorn Osnes 1 and Halfdan Aass =. ~)Department of Pharmacology, University of Oslo, and 2)Medical Department B, Rikshospitalet, Oslo, Norway. In failing human hearts the role of an a-adrenoceptor (a-AR) mediated inotropic component is still a matter of discussion as only a small inotropic effect of phenylephrine has been found. We studied the adrenergic inotropic components in explanted human hearts stimulated by noradrenaline. Strips from ventricular trabeculae were mounted in organ baths (37 ° C, paced at 1 Hz). a-AR and [3adrenoceptor (13-AR)mediated inotropic effects were studied separately in the presence of appropriate receptor blockers. The inotropic responses to a-AR and 13-AR stimulation, respectively, were of comparable size but varied markedly between different preparations, a-AR stimulation prolonged and 13-AR stimulation shortened time to peak tension. The ventricular muscle preparations from explanted human hearts thus responded unexpectedly well to a-AR stimulation compared to I~-AR stimulation. These findings indicate a functional role of the a-AR mediated inotropic component also during heart failure.

RECOVERY OF RESPONSIVENESS OF FAILING F~77 HEARTS TO BETA-ADRENOCEPTOR STIMULATION BY TREATMENT WITH ACE INHIBITORS Atsushl Sanbe, Satoshi Takeo, Norio Takagi & Ken-ichJ Yabe. Dept of Pharmacology, Tokyo University of Pharmacy and Life Science, Hachlojl, Japan. R e s p o n s i v e n e s s of c a r d i a c f u n c t i o n to b e t a a d r e n o c e p t o r s t i m u l a t i o n has been r e c o g n i z e d to be d e c r e a s e d in c h r o n i c h e a r t f a l l u r e of a n i m a l s and humans. The p r e s e n t s t u d y was u n d e r t a k e n to d e t e r m l n e whether a n g i o t e n s l n I c o n v e r t l n g enzyme (ACE) i n h i b i t o r s , c a p t o p r l l , e n a l a p r i 1 and t r a n d o l a p r i l , r e s t o r e the reduced r e s p o n s i v e n e s s of c a r d i a c o u t p u t to b e t a - a d r e n o c e p t o r s t l m u l a t l o n in r a t s with c h r o n i c h e a r t f a i l u r e (CHF) a f t e r m y o c a r d i a l i n f a r c t i o n . CHF in r a t s was developed 12 weeks a f t e r l e f t c o r o n a r y a r t e r y l i g a t i o n . Treatment wlth 3 to i0 mg/kg/day ACE inhibitors from the 2nd to 12th weeks resulted in an appreciable recovery of the reduced responsiveness of cardiac output to beta-stimulants, the decreased Bmax of beta-adrenoceptor blnding and the decreased cardiac norepinephrlne content of the CHF animal. The r e s u l t s suggest that t h l s recovery of cardiac function of the CilF animal by ACE inhibitors is partly a t t r i b u t e d to prevention of down-regulation caused by the excessive stimulation of beta-

adrenoceptor.

EFFECTS OF ACE INHIBITION, BETA-BLOCKADE Fr078 AND DIGITALIS ON MYOCYTE HYPERTROPHY .4aND INTERSTITIAL FIBROSIS IN MYOCARDIUM OF DOGS WITH MODERATE HEART FAILURE. Hani N. Sabbah, Victor G. Sharov, Michael Lesch & Sidney Goldstein. Henry Ford Heart & Vascular Institute, Detroit, Michigan, USA. Cardiocyte hypertrophy (CH) and accumulation of interstitial collagen (IC) occur in the failing heart and are thought to contribute to the progression of LV dysfunction. We examined the effects of long-term (3 months) monotherpay with the ACE inhibitor enalapril (ENA, n=7), the beta-blocker metoprolol (MET, n=7) and the digitalis preparation digoxin (DIG, n=7) on CH and IC in dogs with moderate heart failure (HF) produced by intracoronary microembolizations. Seven untreated HF dogs (CON) and 7 normal (NL) dogs were used for comparison. Cryostat LV sections were prepared and stained with a collagen III antibody. CH was defined on the basis of average myocyte cross-sectional area (MCSA, i.tm2) and IC volume fi'action was calculated as the percent of total surface area occupied by collagen u ' uter-based videodensitometr

MCSA

NL

CON

ENA

MET

DIG

616

924:1:

711 *

979:1:

922:1:

IC(%)

3.5 11.5::1: 3.9* 10.8::1: 9.2:1: [:I:=P<0.05 vs. NL; *=P<0.05 vs. CON] Conclusion: Among the 3 prototypical drugs used in treating HF, only ACE inhibition with ENA attenuated the development of CH and the accumulation of IC.

EFFECTS OF 3,5-DIIODOTHYROPROPIONIC ACID Fr079 (DITPA) ON RAT AND RABBIT MODELS OF HEART FAILURE AFTER MYOCARDIAL INFARCTION. Eugene Morkin, Gregory Pennock, Thomas E. Raya & Steven Goldman, Univ. of Arizona, Tucson, Arizona, USA.

DIRECT COMPARISON OF THE INTRAFr080 CELLULAR CA=*-INDICATORS AEQUORIN ARD FURA-2 IN ISOLATED HUMAN MYOCARDIUM Burkert Pieske, Albrecht Schmidt, J5rg Schattmann, Christian Holubarsch, Hanjbrg Just, Gerd Hasenfuss. Medizinische Klinik III, Universit&t Freiburg, F.R.G.

An agent that improves LV performance by non-cyclic AMP-mediated mechanisms would be valuable in the therapy of congestive heart failure (CHF). Thyroid hormone is known to increase cardiac performance by a combination of effects on the heart and peripheral circulation. Accordingly, the efficacy of DITPA, a thyroid analogue with low metabolic activity, was studied in rat and rabbit postinfarction models of CHF. In the rat model, we compared treatment with captopril to a combination of DITPA and captopril beginning 3 wks. after infarction. Both captopril and captopril/DITPA reduced LV end-diastolic pressure (LVEDP), but the addition of DITPA improved resting cardiac index and shifted the cardiac function curve upward and to the left, indicative of enhanced performance. In a rabbit postinfarction model, DITPA decreased LVEDP and improved LV relaxation without changing heart rate or myosin isoenzyme composition. Although LV diameter increased after infarction, there were no effects of DITPA on LV remodeling. Thus DITPA may be a useful adjunct to other measures in the therapy of CHF.

It is uncertain whether the different Ca2÷ indicators Aequorin and FURA-2 reveal comparable results. We investigated the influence of stimulation frequency (30180 beats per minute; 37"C) on contractile behaviour and intracellular Ca2+ transients of isolated ventricular muscle strips obtained from 10 end-stage failing human hearts. Muscle strips from each heart were loaded in parallel with either Aequorin or FURA-2 AM. Results: Stimulation frequency 30/min 90/min 180/min Twitch tension FURA-2 100 120~ 92~:9 (Amplitude) Aequorin 100 1 3 1 + 1 9 101:L21 Light signal FURA-2 100 103+3 65-2:4 (Amplitude) Aequonn 100 117¢15 98.t:20 RT~ FURA-2 348±23 305±16 211¢14 Aequorin 340+~8 298+91 182~8 RL m FURA 596+37" 404±13" 246+7' Aequorin 154+-28 149+-28 102:1:12 Diastolic FURA 0 2:t:4 28:t:13 Tension (/,%) Aequorin 0 3+13 27:1:23 Diastolic FURA 0 33+4' 86-'~0' Light (/,%) Aequorin 0 -1±4 ('p
FURA-2 or Aequorin did not influence contractile behaviour. However, FURA-2 Ca 2÷ transients are prolonged. This results in fusion of FURA-2 signals at higher stimulation rates with an increase in the diastolic signal without contractile counterpart. This difference may be due to different Ca"÷ binding kinetics of the compounds. A237

INFLUENCE OF RYANODINE ON FORCE OF Fr081 CONTRACTION, FORCE-FREQUENCY-RELATION AND INTRACELLULAR CA=*-TRANSIENTS IN ISOLATED HUMAN AND RAT MYOCARDIUM B. Pieske, J. Schattmann, A. Schmidt, H. Just, Ch. Holubarsch, G. Hasenfuss. Medizinische Klinik III, Universit&t Freiburg, F.R.G. Ryanodine (RYA) inhibits the function of the sarcoplasmic reticulum. We investigated the influence of RYA (lpM) on isometric force of contraction ~FOC), force-frequency-relation (FFR 30-180 beats per minute) and intracellular Ca =" transients (aequorin method) in isolated ventricular muscle strip preparations from 8 end-stage failing human hearts and 5 nonfailing rat heads (37"C). Results: Human rnyocardium: Without RYA, the FFR was slightly positive up to a stimulation frequency of 90/min (increase in FOC to 137+_28% of the basal value at 30/roin), and then declined at higher stimulation frequencies. There was a parallel change in aecluorin light signals. Addition of RYA (at 60/roin) decreased FOC and light signal by 51+-12% and 79+17% respectively, p<0.05. However, under the influence of RYA, the FFR became posit ve: ncrease in FOC to maximal 213+-32% at 120/min, p< 0.05 vs. pre-RYA. Again, the aequorin light signal run parallel to force. Total duration of the isometric twitch was significantly prolonged under RYA. Rat myocardiuro: As already described for rat myocardiuro, the force-frequency was steeply negative before RYA (At 180/min: force 59__.5%, light 52+_5% of the basal values at 30/min p<0.05). With RYA (at 60/min), force decreased by 95+_12% and aequorin light signal by 91-+13% (p<0.05). However after RYA, the force-frequency and aequorin lightfrequency became positive: increase in FOC to 141---18% and aequorin light to 112-+21% at 180/rain as compared to 30/min, p<0.05 vs. pre-RYA. Conclusion: With RYA, the negative force-frequency and aequorin light frequency relation in failing human myocardium and in nonfailingrat myocardiuro b,ecome positive. This may be due to inhibition of sarcopmsroic reticuluro function with consecutive direct activation of contractile proteins by transsamolammal Ca2. influx.

INFLUENCE OF THAPSIGARGIN AND Fr083 ISOPROTERENOL ON FORCE-FREQURNCYRELATION AND POST-REST-POTENTIATION IN ISOLATED HUMAN MYOCARDIUM Burkert Pieske, Christiane Schwan, Christian Holubarsch, Hanj6rg Just, Gerd Hasenfuss. Medizinische Klinik III, Universit&t Freiburg, Germany

Excitation-contraction coupling is altered in end-stage failing human myocardium. We investigated the influence of Thapsigargin (THAPSI), an inhibitor of sarcoplasmic reticulum (SR) Ca 2+ uptake, and Isoproterenol (ISO), which stimulates SR Ca 2+ uptake, on force-frequency-relation (FFR) and post-rest-potentiation of isometric force (PRP). Experiments were performed in isolated, isometrically contracting ventricular muscle strips (n=42) from 19 endstage failing human hearts. (37"C; FFR: 30-180 beats per minute; PRP: Rest intervals from 2-240 s).Results: 1. FFR: without intervention, optimal stimulation frequency was 48+12min "1 with an increase in force of contraction (FOC) by 14+9%. ISO (3x10 a) partially normalized the inverse FFR: increase in optimal stimulation frequency to 91+19 rain"1, maximal increase in FOC 47_+16%, p<0.05. THAPSI (10 s) worsened the FFR: decrease in optimal stimulation frequency to 32-+4 min "1, significant reduction in frequencydependent FOC (at 180 m i n ,1 34_+12oYoof basal value at 30 minl). 2. PRP: without intervention, the optimal rest interval was 30 sec with an increase in FOC of the first beat upon restimulaUon by 35+10%. After ISO, the optimal rest interval was 40 s and FOC increased by 74_+17%, p< 0.05 vs. predrug value. After THAPSl, the optimal rest interval was 5 s and FOC increased by only 14-+9%. Conclusion: In failing human myocardium, stimulation of Ca 2÷ uptake partially normalizes FFR and PRP, whereas inhibition of Caz÷ uptake further worsenes FFR and KFB.

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INFLUENCE OF EXPERIMENTAL TEMPERATURE Fr082 ON ISOMETRIC FORCE, FORCE-FREQUENCY-RELATrOR AND INTRACELLULAR CA 2. TRANSIENTS IN HUMAN MYOCARDIUM Burkert Pieske, Rena Bornemann, Sophia Tsipizidou, Bodo Kretschroann, Gerd Hasenfuss. Medizinische Klinik III, Universit&t Freiburg, Germany Discrepant find!ngs of previous experiments in human myocardium may be due to different experimental conditions. We therefore investigated the effects of different temperatures (37 vs. 30"C) on force generation and intracellular Ca2~ transients (aequorin method) in ventricular muscle strip preparations from nonfailing (n=5) and end-stage failing (n=12) human heads. Experimental protocol: 1) Influence of a stepwise reduction of temperature from 39 to 29"C on the isometric twitch; 2) Force-frequency-relation (FFR: 0.253.0Hz) in aequorin-loaded preparations at 37"C followed by FFR at 30"C. Results: Reducing bath temperature increased isometric force of contraction to maximally 212~21% in nonfailing and 227_+21% in end-stage failing myocardium at 30 vs 3TC (p<0.05). The total duration of the twitch was prolonged by 36_+8% and 38+7%, respectively (p<0.05). The decline of the amplitude of the isometric twitch with increasing stimulation rates (inverse FFR) was significantly steeper at 30"C as compared to 3TC in the failing myocardiuro. This resulted from a pronounced increase in diastolic tension with higher stimulation rates at 30°C. Lowering bathing temperature from 37°C to 30 "C (1Hz) resulted in a reduction of the amplitude of the aequorin light signal by 58+_23% (p<0.05) and a prolongation of its time course (RLso, 63+_8ms at 1Hz, 3TC and 126+9 ms at 1Hz, 30"C, p<0.05). A second light component, L2, was observed under hypothermic conditions in most preparations. In conclusion, experimental temperature crucially influences contractile force, twitch configuration and force-frequency behaviour as well as the amplitude and the time parameters of the aequorin light signal in isolated human myocardium.

BENEFICIAL INOTROPIC EFFECT OF Fr084 EMD57033 IN DILATED HEART FAILURE *JA Lee, AJ Drake-Holland, J W Hynd, SB Clarke, MIM Noble, *Department of Pathology, University of Sheffield & AUCVM, C h a r i n g Cross & Westminster Medical School, London, UK. EMD57033 is a drug which enhances the sensitivity of the contractile proteins to calcium, and may thus be of benefit in the failing heart. We studied 6 dogs in dilated heart failure, induced by right ventricular pacing at 250 beats/min for 6 weeks. Evidence of heart failure was determined clinically and by measurement of the left ventricular (LV) dimensions by transoesophageal echocardiography. Haemo-dynamic measurements of LV pressure, maximum rate of rise of LV pressure (LVdP/dt), cardiac output, mean arterial pressure, heart rate, pulmonary artery and wedge pressures, were made during infusion of solvent, EMD57033 (0.6mg.kg-l.min-1), and the PDE inhibitor EMD57439 (0.03mg.kg-l.min-l). In each dog studied there was an improvement in left ventricular haemodynamics and dimension with both EMD57033 and EMD57439. The % increase in LVdP/dt was 57 and 44%, and cardiac output 79 and 81% respectively, but the % decreases in systemic vascular resistance were larger with EMD57439 (58%) compared to 38% for EMD57033, reflecting the greater vasodilator effect of the PDE inhibitor. We have confirmed that the positive inotropic effects of EMD57033 are p.resent in this model of heart failure and zmprove haemodynamics.

REDUCED LEFT VENTRICULAR Na,K-ATPase FRO85 CONCENTRATION IN SYMPTOMATIC PATIENTS WITH CHRONIC AORTIC VALVUI.AR DISEASE Thomas A. Schmidt, Henning Bundgaard, Jim S. Larsen, Henrik Arendrup, Keld Kjeldsen. Copenhagen University Heart Center, Rigshospitalet, Denmark. The mechanism for sudden death related to aortic stenosis (AS) ( - 2 0 % ) and chronic aortic regurgitation (AR) is unknown. Left ventricular (LV) Na,K-ATPase is of importance for development of arrhythmias, Na-, K-, Ca-handling and digoxin treatment. Thus, we evaluated whether AS and AR were associated with regulation of LV Na,K-ATPase. In symptomatic, non digitalized patients (means _+ SEM; n = 5) with AS and A R 65 -+ 5 and 52 -+ 4 years old (p > 0.05) cardiac catheterization was performed. During aortic valve replacement vital LV biopsies were obtained for 3H-ouabain binding to intact samples. AS and AR patients had an ejection fraction of 46 -+ 5 and 52 -+ 7% (p > 0.50) respectively, and a LV 3H-ouabain binding site concentration of 286 _+ 35 and 430 +- 26 pmol/g wet wt., i.e. a reduction of 61 and 41% (p < 0.05), respectively compared to normal LV Na,KATPase concentration. In patients with AS an inverse relationship was found between Na,K-ATPase concentration and the systolic aortic gradient (r = -0.97, p = 0.007, n = 5). Thus, there appears to be a correlation between severity of AS and LV Na,K-ATPase concentration. Reduced Na,K-ATPase concentration in aortic valvular disease may be detrimental to cardiac ion homeostasis including digoxin tolerance and could be arrhythmogenic.

SYSTOLIC BLOOD PRESSURE AND CONGESTIVE HEART FAILURE IN ESSENTIAL HYPERTENSION M Crlppa, G DamlanI, E BonI, L Corda, R Farlello DptlnternalMedlclne, SpedallCIvllI, B~scla, ltaly

Fr087

Systolic blood pressure (BP) is more of a determinant of risk than is the diastolic in developing congestive heart failure (CHF). The high pressures generated during systole put an immediate, direct burden on the heart and the resistance arterioles. The aim of the present study was the evaluation of the systolic BP values from a 24-h ABPM (A&D TM 2420) in 421 uncomplicated untreated essential hypertensive patients (stage 1 & 2, JNC V), divided into 2 groups: .qroup I, from 26 to 45 years (mean 37), including 85 males (M, mean age 37) and 61 females (F, mean age 39); qroup I1, from 46 to 65 years, including 144 males (mean age 54) and 131 females (mean age 54). Our data seem to suggest that: 1) group II of older males have higher systolic BP, markedly during the night-time (in comparison with younger males in group I); 2) group II of older females always have higher systolic BP values during the night-time, the daytime and the 24-h period as a whole (in comparison with younger females in group I); 3) younger males have higher systolic BP values only during the daytime, as compared to younger females while during the night-time the systolic BP profile is similar; 4) the systolic BP values in older males and older females are similar during the morning, evening and night-time periods, while during the early afternoon there is a significant fall in systolic BP in females (this last finding seems to be a constant difference in both younger and older females, compared with males); 5) older hypertensive patients, both males and females, have a lesser systolic BP fall during night-time. Thus, the increased risk of developing CHF in elderly hypertensives could be related to the higher levels of systolic BP during both day and night-time.

COMBINATION OF SYSTOLIC AND DIASTOFRO86 LIC DYSFUNCTION: DIAGNOSTIC POSSIBILITIES JiK Toman, Lenka Spinarovfi, Jaroslav Meluzin, Miroslav Sou}ek~ Marek Richter. Ist Dept. Med., Masaryk University, Brno, Czech Republic In many cases, systolic and diastolic dysfunction appear simultaneously. While the former one may be easily detected from the global ejection fraction or from segmental left ventricular (LV) disturbances, an evaluation of the latter one is much more difficult. Only invasive examination yields precise information. Nevertheless, there are several noninvasive means that may be useful for clinical practice. We exammed 62 pts with chronic heart failure (CHF), all with significant systolic dysfunction (ejection fraction lower than 40%), NYHA H-Ill. 21 of them had coronary heart disease, 41 dilated cardiomyupathy. Right heart catheterization esttmated filling LV pressure. From echocardiography we assessed volumes, ejection fraction, isovolumic relaxation time, filling period (RR intervalduration of mitral regurgitation) and both peak rates of posterior wall relaxation and of LV dimension increase. Examinations were performed at rest and in stress. Our complex approach showed that patients may be principially divided into two groups: the first with a marked diastolic dysfunction (60-70% ofpts) and the second without it. However, a spectrum with small differencies exists among pts. The therapy of systolic dysfunction is well known now. A specific treatment of diastolic dysfunction is not still established. Differentiation of them may help to this aim.

CARDIOMYOCYTE LOSS BY APOPTOSIS IN DOGS Fr088 WITH CHRONIC HEART FAILURE. Victor G. Sharov, Hani N. Sabbah, Michael Lesch & Sidney Goldstein. Henry Ford Heart & Vascular Institute, Detroit, Michigan, USA. Progressive deterioration of LV function in heart failure (HF) is often attributed to ongoing loss of viable myocytes. We examined the possibility that myocyte loss in HF may be due, in part, to apoptosis: an active process of gene-directed cellular self-

desta'uction. This process differs from necrosis in that myocyte loss occurs in the absence of membrane rupture. Immunohistochemical studies were performed in LV tissue obtained from 10 dogs with chronic HF produced by intracomnary mierocmbolizations. The presence of nuclear DNA fragments (DNAf), a marker of apoptosis, was assessed in frozen sections using the Oneor ApopTag fluorescein kit. Sections were double stained with striated muscle antimyosin antibody to identify apoptotic bodies ofcardiocyte origin. In 10 transmural LV sections obtained from the 10 HF dogs, a total of 393 bodies showed evidenceof DNAf. Of these, 43 (I 1%) contained myosin thus confimaing their cardiocyte origin. Among all apoptotic bodies identified as being of cardioeyte origin, 95% were localized to LV regions bordering old infarctions. Conclusions: 1) Cardiocyte loss by apoptosis occurs in LV myocardium of dogs with chronic heart failure. 2) Apoptotic bodies of cardioeyte origin are primarily localizedto regions bordering old infarctions. 3) Ongoing loss of viable myocyte by apoptosis may be a factor leading to the progression of LV dysfunction which characterizes the heart failure state. A239

TUMOR NECROSIS FACTOR RECEPTORS IN Fr089 CHRONIC HEART FAILURE Tiziana Baehetti, Roberta Confortini, Cristina Opasich, Angelo Corti, Giuseppe Cassani, Roberto Ferrari. Fondazione Clinica del Lavoro "Centro S Maugeri", Gussago, Brescia, and Heart Failure Unit, Montescano, Pavia, Italy; Cattedra di Cardiologia, Spedali Civili, Brescia; Tecnogen S.c.p.A., Piana di Monte Verna, Caserta, Italy Tumor necrosis factor alpha ('I'NF-a) increases in patients with severe chronic heart failure (CHF). Two naturally occurring modulators of TNF-ct activity, sTNF-RI and sTNF-RII, have been identified in human serum. This study was undertaken to examine sTNF-Rs and bioaetive and antigenic TNF-a in 37 patients with varying degrees of CHF, compared to that of 26 normal subjects. Antigenic TNF.-a rose (from 14.3+7.08 to 33.54-13.1 pg/mL, p<0.001) in patients with severe CI--IF(NYHA class IV). sTNF-Rs were also increased (sTNF-RI from 1.174-0.43 to 4.43±2.14 ng/mL and sTNF-RII from 2.24-0.44 to 7.55±2.28 ng/mL, p<0.001). Cytolytic TNF-a was undetectable (<100 pg/mL). Addition of 625 pg/mL recombinant human TNF-a (r-hTNF-a, corresponding in the bioassay to 60% of the lethal dose) to the serum of normal subjects resulted in an increase of the expected cytotoxicity (from 625 to 12904-411 pg/mL, p<0.001). Addition of the same dose of r-hTNF.-a to the serum of patients with mild to moderate CHF (NYHA classes I1 and lII) increased the cytotuxicity from 625 to 8774-132 pg/mL, p<0.001. In four patients with severe CHF, the expected eytotoxicity was inhibited whereas it was reduced from 625 to 263±198 pg/m.L, p<0.001 in the remaining eight patients. Ten patients died within one month of entry into the study. The), had the highest level of sTNF-RII (8.18--1=1.92 ng/mL). In conclusion, TNF..ct and beth sTNF-Rs increase in patients with severe CI--[F. The increased level of sTNF-RII independently correlate with poor short-term prognosis.

IMPAIRED ENDOTHELIUM-DEPENDENT VASODILA- Fr091 TION: RELATIONSHIP TO HYPEREMIC CALF BLOOD FLOW AND EXERCISE CAPACITY IN HEART FAILURE Makoto Chiba, Motoyuki Nakamura, Motoko Ishikawa, Kenji Ueshima, Kohya Hashimoto, Katsuhlko Hlramori.

Iwate Medical University, Morioka, Japan This study determined the peripheral endothelium-dependent vasodilatory response to acetylcholine (ACH) and the endothelium-independent vasodilatory response to sodium nitroprusside (SNP) in 19 patients (pts) with chronic heart failure (CHF") and 8 normal controls. These peripheral blood flow responses were compared with hyperemic calf blood flow (CBF) changes after maximum leg exercise (EX) and with 5-min femoral occlusion (OC). The peripheral blood flow response to forearm intra-artedal infusion of ACH and SNP, and reactive hyperemic CBF changes were measured by plethysmography. All peripheral blood flow responses were reduced in pts with CHF (p<0.05). Reduction of ACHmediated changes in peripheral blood flow was correlated with EX-induced CBF responses (r=0.51, p<0.05), but not with OC-induced CBF responses (r=0.02, N.S.). SNPmediated change.s were not correlated with any reactive hyperemic blood flow responses (EX; r=0.27, N.S.: OC; r=0.11, N.S.). When the pts were divided into two subgroups based on the median EX-induced CBF changes, the subgroup with the lower CBF response showed a reduction in EX capacity (anaerobic threshold: 11.8_+0.6 vs 14.6-+1.0 mllkg/min, p<0.05). These findings suggest that endothelial dysfunction is related to decrease in EX-induced skeletal blood flow and EX capacity in pts with CHF. A240

ALTERED IN VIVO K I N E T I C S OF A T R I A L F r 0 9 0 NATRIURETIC PEPTIDE (ANP) IN PATIENTS WITH HEART FAILURE. Giorgio Iervasi, *Frantisek Vitek, Aldo Clerico, Sergio Berti, Alessandro Pilo, Andrea Biagini, Luigi Donato. CNR Institute of Clinical Physiology, and *Institute of Byophysics, Charles Unicersity, Prague, Czech Republic. To investigate whether differences in the renewal and distribution of ANP may play a role in the pathogenesis and evolution of heart failure in idiopathic dilated eardiomyopathy (IDC), a tracer method was used to study ANP kinetics in the steady state condition in I0 normal subjects and in 13 patients with IDC with different degrees of hemodynamic dysfunction at variable sodium intake. 'Z~l labeled ANP was bolus injected and an HPLC was used to purify the labeled hormone in venous plasma samples collected up to 50 min after injection; the main ANP kinetic parameters were then derived from the disappearance curve of the labeled hormone. Patients with IDC showed a gradual reduction in the total distribution volume, mainly due to a contraction of the peripheral distribution spaces in the early phases of the disease, and to a reduction of both the initial distribution volume and the peripheral spaces in the late phases of the disease (from 20.5+4.5 L/m2 to 12.9+7.2 L/m2, p<0.0279). Moreover, the ANP production rate, which was in the normal range (120.0+-104.7 ng/min/m2) in the early (NYHA class < II) stages (99.8+_52.3 ng/min/m2), increased more than 3 times in patients with more severe (NYHA class >II) myocardial involvement (378.9+_189.1 ng/min/m 2, p<0.0055). Different relationships between the metabolic clearance rate (MCR) values and daily sodium excretion were observed in patients (r=0.837, p<0.0001) and in controls (r=0.962, p<0.0001). The significantly (p<0.02) different linear regression coefficients indicate that, on average, for each mmol rise in sodium excretion the ANP MCR increased by 17 ml/min/m2 in the patients, i.e. an increase of about 2 fold with respect to the controls. Our study demonstrates a markedly altered peripheral distribution and degradation of ANP in patients with IDC, even those in the early phase of the disease and plasma ANP levels in the normalrange.

THE CAPILLARIES OY COR PULMONALE F~92 Alexander ~ . Z ~ r i t s k y . Dept of Pathology,Centr Reg Hosp, Taldom,Russia. The study was aimed at quantitative assessment of capillaries (C) of the right ventricular myocardium in comensation (n=7) and decompensation n=7) stages of cot pulmonale (CP) in chronic obstructive lung diseases. The control group was composed of 6 forensic-medicine cases of people of corresponding age and sex who died a violent death without pulmonary and cardiac diseases. We determined volume density of C in IO fields of vision of 3 myocardial zones under magnification x4OO by means of Avtandilov ocular grid according to randomized choice trial using a "point" method. Histostereometric analysis showed that in all myocardial zones the C density significantly decreases as decompensation of CP develops. It is as early as in the compensated stage that reduction of C net in each myocardial zones is reveal (p~O.OOI). Hence, the reduction of C induces relative hypoxia of the right ventricle tissues, and a decrease in C density should be considered as a micrometric diagnostic sign of CP decompensation, in spite of the fact that it is observed in the compensated stage.

p

B I V E N T R I C U L A R G E O M E T R Y A L T E R A - Fr093 TIONS UNDERLYING HEART FAILURE IN P R I M A R Y P U L M O N A R Y H Y P E R T E N S I O N Victoria A. KhomazJuk, Lldla F. Konoplyova, Taras D. Nikula. Dept of Internal Medicine, Ukrainian State Medical University, Kiev, Ukraine. To investigate the role of geometrical interdependency between the right and left ventricles in development of heart failure 44 patients with primary pulmonary hypertension aged 16-48 years were prospectively followed for 3 years. Geometrical and functional parameters of both ventricles were measured by means of echocardiography and compared with clinical data. Progression of heart failure was associated with leftward displacement of the interventricular septum and alterations of the shape of both ventricles. Diastolic diameter of the right ventricle (DDRV) was increasing progressively, end diastolic diameter of the left ventricle (EDD LV) was diminishing. The ratio of DDRV to EDD LV correlated positively with mean pulmonary arterial pressure (r=0.30, p<0.05) and right ventricular wall thickness in diastole (r = 0.45, p<0.001). An inverse correlation existed between the DDRV / EDD LV ratio and LV stroke volume (r=-0.63, p<0.001), cardiac output (r= -0.51, p< 0.001 ). An analogous correlation was observed between the DDRV/EDD LV ratio and the degree of systolic thickening of interventricular septum (r= -0.48, p<0.001). We suggested that alterations of biventricular geometry must be considered in pathophysiologic analysis of heart failure in primary pulmonary hypertension.

TWO INBRED RAT STRAINS AS A N I M A L F~95 MODELS FOR STUDYING ISOPRENALINE I N D U C E D M Y O C A R D I A L L E S I O N S (ISOML) Sixtus Hynie, M i l a n ~am~nek, M i r o s l a v Mr&z. Inst. of P h a r m a c o l . , i s t Fac. of Med., Charles Univ., Kardiocentrum, Univ. Hosp. Motol, Prague, CR. Two inbred rat strains w h i c h differ in ISOML r e s u l t e d from 37 g e n e r a t i o n s of selective breeding. M e c h a n i s m s of this d ~ f f e r e n c e were s t u d i e d on the receptor-adenylyl cyclase complex (AC) level. Rats s e n s i t i v e to ISOML have a lower c o n t e n t of myocardial ~cogen and h i g h e r i n c o r p o r a t i o n of ~Hg into d a m a g e d m y o c a r d i u m as compared to rats r e s i s t a n t to ISO. These two rat strains also d i f f e r in their behavior, immunological reactivity, sensitivity to stress induced organ lesions and in vitro liver b i o t r a n s formation of drugs. Both strains develop m y o c a r d i a l h y p e r t r o p h y after 5 daily doses of ISO. The t r e a t m e n t resulted in a r e d u c e d s e n s i t i v i t y of AC to ISO, however, no d i f f e r e n c e was observed between both strains. In conclusion, d i f f e r e n c e s in the sensitivity of the m y o c a r d i u m to ISO lay behind the r e c e p t o r - A C complex. Supported by G r a n t s IGA MZ C R No. Z 2040-3 and G A C R No. 307/95/0643.

ADRIAIVIYCIN-INDUCED CONGESTIVE HEART FRO94 FAILURE IN RABBITS. Zsuasanna Rozsa, Tamas Forster*, Dirk Thorm~ihlen** Zsuzsa Szorenyi, Julius Gy. Papp Dept. of Pharmacology, Dept. of Medicine* Szent-Gy0rgyi University, Szeged, Hungary and Dept. of Pharmacology, Kali-Chemie, Hannover, Germany** Aims: in this study we analysed the changes of the systemic and cardiac hemodynamics and electrocardiogram (ECG) during the development of heart failure in adriamycininduced cardiomyopathy in rabbits. Methods: adriamycin was administered intravenously in a dose of 1 mg.kg"! twice weekly for 8 weeks in 8 rabbits. At the beginning and on the 4th and on the 8th week paired studies were performed in 20 control rabbits. Noninvasive measurement of left ventrieular function was carried out by echocardiography (ECHO) at weeks 0 and 4. ECG was recorded from the chest lead trace at weeks 0 and 8. In anaesthetized animals systemic arterial pressure (MAP), heart rate (HR) and left ventricular pressures and their derivates (LVSP, LVEDP, +dP/dtmax, -dP/dtraax) were measured at weeks 0 and 8. Results: on the 4th week adriamycin treatments resulted in a significant decrease of left ventricular ejection fraction concomitantly with a dilation of the left ventricular cavity as determined from ECHO. Adriamycin-induced ECG changes were characterized by a prolongation of the QT interval, a widening of the QRS complex and a flattening of the T-wave. On the 8th week, the MAP was lower and the HE was higher in adriamycin treated animals vs control. In addition, adriamycin treatments resulted in a marked deterioration of the left ventrieular function namely, a significant increase in LVEDP and a considerable decrease in +dP/dt compared to the controls. These changes were accompanied by a significant increase in the heart weight / body weight ratio. Conclusion: these findings show that we have succeeded in developing a reliable experimental model for the study of the adriamycin-induced heart failure in rabbits. Our model is suitable for comparative analysis of the effects of potential cardiotonic agents.

THYROID CONTROL OVER ATRIAL CONTRACFr096 TILITY AT THE LEVEL OF PHOSPHODIESTERASES Enn K. Seppet, Allen Kaasik, Urmo Braun & Jorma J. Ohisalo. Dept of Pathophysiology, University of Tartu, Estonia & Dept Medical Chemistry, University of Helsinki, Finland. The present study was undertaken to reveal whether hypothyroidism modulates atrial contractility via changes in cAMP-phosphodiesterase (PDE) activity and isozyme profile. The rats were made hypothyroid by addition of 0.08% 6-n-propyl-2-thiouracil in their drinking water for 3 weeks. Analysis of PDE isozyme activities using isozyme-specific inhibitors (rolipram and SK&F 94836) showed that total particulate PDE activity was lowered by 30% in hypothyroid atria, and that it was due to diminished type IV PDE activity. The positive inotropic effects of isozymespecific inhibitors of PDE and isoproterenol (ISO) on atrial contractility (dP/dtm.x) were also studied. The effect of rolipram, (20 I~M), relative to the cumulative effect of rolipram and SK&F 94836 (20 I~M), was lowered from 7 6 + 9 % in normal atria to 2 2 + 9 % in hypothyroid atria. The dose-response curve fc)r ISO was shifted to the right under hypothyroidism (EC50% 2.6+0.9 nM and 54+22 nM, respectively). At the same time the maximal increment in contractility was much larger in hypothyroid than in euthyroid atria (1400% and 200%, respectively, as compared to control level before ISO addition). We suggest that the decreased particulate type IV PDE activity may be a reason for increased contractile responsiveness to ISO in hypothyroid atria. A241

A242

ALTERATION OF CARDIAC axF~97 ADRENOCEPTOR SUBTYPES IN HYPERTHYROIDRATS Chide Han, Geng-sheng Yu, Kai-ming Xu, Peng Qu & Er-dan Dong. Institute of Vascular Medicine, Beijing Medical University, Beijing 100083, CHINA

Tnl phosphorylation does not Increase the rate Fr098 of relaxation following photolysis of diazo-2 in skinned trabeculae from guinea-pig. EC Johns, SJ Simnett, IP Mulligan', CCAshley. University. Laboratory of Physiology, University of Oxford. OX1 3PT 'Dept. Cardiology, J.R.Hospital, Oxford OX3 9DU UK.

The alteration of cardiac axadrenoceptor (a,-AR) and its subtypes in hyperthyroid rats was studied. The results showed that in hyperthyroidism the cardiac a x - A R d e n s i t y (B~=) reduced from control of 51.6~6.0 to 40.9±3.7 fmol/mg (p<0.01). The high affinity sites for 5-MU (axA-AR) were reduced (from 12.0 to 4.4 fmol/mg), while the low affinity sites for 5-MU (ax,- plus a,,-AR) were not changed (from 39.6 to 36.5 fmol/mg). The results of RT-PCR showed that steady state levels of mRNA for axA- and ax,-ARwere declined, while that for ax,-AR was raised. The phenylephrine induced maximal contractions of isolated left atria of hyperthyroid rats were reduced (p<0.05). Thus we conclude that in hyperthyroid rats the total amount of cardiac ax-AR are reduced, which accounts for the decreased positive inotropic response induced by a~-AR agonists. The change of a,-AR is subtype selective, with ax,- and a~,-AR reduced and ax,-AR increased.

In vivo two of the effects of beta-adrenergic stimulation am phosphorylation of cardiac Tnl and an increase in the rate ot~ relaxation of the heart. In vitro, cardiac Tnl can bo phosphorylated by protein kinase A (PKA). We have used the technique of laser flash photolysis of the calcium~ chelator diazo-2 to investigate the effect of phosphorylation of Tnl on the relaxation rate of skinned (1% v/v Triton X, 100; 45 minutes) trabeculae from the guinea-pig at 12°C.Thel fibres were phosphorylated by PKA for 30 minutes at 30°C~ pH 6.8; this resulted in a decrease in calcium sensitivity (thel pCas0 decreased by ~0.15pCa units). The tin of relaxation was 62.2 _ 7.7 msec (mean _+SD; n = 6) in untreated fibre~t and 56.3 _+2.7 msec (n = 4) in PKA treated fibres. Double exponential curve fits of the average curves showed no significant difference between the rate constants of the twd cases. From this we conclude that TnI phosphorylation ha~t no effect on the rate of relaxation in skinned trabeculae from guinea-pig following diazo-2 photolysis. The increase irl relaxation rate seen in vivo during adrenergic stimulatiorl may be due to the phosphorylation of phospholamban whictl results in the acceleration of calcium uptake into the S.R.

MECHANISM OFTHE NEGATIVE LUSITROPIC EFFECT Fr099 OF al-ADRENERGIC STIMULATION (uI-AS). Martin Vila-Petroff, N~stor G. P~rez, Bernardo Alvarez, Horacio E. Cingolani, Alicia Mattiazzi. Centro de Investigaciones Cardiovasculares, 1900 La Plata, Argentina.

a~-ADRENERGIC STIMULATION OF NATRIURETIC Fr100 PEPTIDE SECRETION AND GENE EXPRESSION IN ISOLATED RIGHT ATRIA. Benoit G. Bruneau, Leonardo A. Piazza, and Adolfo J. de Bold. University of Ottawa Heart Institute at the Ottawa Civic Hospital, Ottawa, ON, Canada

The mechanism of the negative lusitropic effect of .1-AS is not clear. Experiments were performed in chemically skinned trabeculae (ST) and papillary muscles (PM) from the right ventricles of cats to explore this issue. Perfusion of EGTA-ST with 10 pM phenylephrine (Phe) in the presence of 1,uM atenolol (Phe+A)(n=8), failed to affect both Ca myofilament (CAM) sensitivity and maximal tension. Similar results were obtained in a-toxin-ST (n=4) and triton Xl00-ST pre-treated with Phe+A (n=6). In PM contracting isometrically at 0.2 Hz, Phe+A increased maximal rate of rise of tension (+T) by2 132 + 23% (P< 0.05, n=8, control= 12.3 + 4g/mm /sec), and half relaxation time (tl/2) by 30 + 10 % (p<0.05, n=8, control= 235 _+ 32msec). Intracellular pH (pH i) (BCECF fluorescence) increased by 0.18 + 0.05 pH units (P<0.05, n=4). In the presence of 10 ~uM of EIPA, an inhibitor of the Na÷-H * exchanger, the increase in +1- produced by Phe+A was reduced to 54 + 9 % (P
To elucidate the mechanisms of stimulation of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) secretion and synthesis in rat atrial cardiocytes, we have studied the effects of a~-adrenergic stimulation on isolated right atria. The early response gene Egr-1 was also studied as a potential marker of transcdplJonalactivation following a ~-adrenergic stimulation. Isolated right atria from rat were stimulated for 4, 6, or 8 h by the acadrenergic agonist phenylephrine (PE) at 10, 50, or 100 pM. As measured by RIA, PE stimulated the secretion of immunoreactive Or)ANF after 30 min. IrANF secretion peaked at 90 min, remained significantly elevated for 4, 6, and 8 h with 10, 50, and 100 pM PE, respectively. PE (50 & 100 pM) stimulated irBNP secretion after 15 min; irBNP secretion remained above basal levels throughout the period of stimulation. Northern blot analysis of atrial mRNA revealed that 50 pM PE for6 h caused a 49% and 135% increase in ANF and BNP mRNA levels, respectively. BNP mRNA levels remained increased by 77% after 8 hours. Egr-1 mRNA levels were increased by 88% after 4 h, 167% after 6 h, and 40% after 8h of treatment. We conclude that al-adrenergic receptor stimulation may play an important role in the regulation of natriuretic peptide secretion and production in atrial cardiocytes. The increased expression of Egr-1 suggests it may be involved in the modulation of the atrial cardiocyte phenob/pe in response to a~-adrenergic stimulation.

¢xI-ADRENOCEPTOR MEDIATED Fr101 I N C R E A S E I N K + E F F L U X IS INHIBITED BY BUMETANIDE. Geir O. Andersen, Mette Enger, Tor Skomedal and Jan -B. Osnes. Dept of Pharmacology, University of Oslo, Norway. Recently, increased 86Rb+ efflux, as a measure of K + movement, was reported after Ctl-adrenoceptor (AR) stimulation in rat hearts. The aim of the presented study was to study the effect of bumetanide, a blocker of the Na/K/2CI cotransport, on the ct I-AR mediated K+efflux. Methods: Spontaneously beating, isolated rat hearts were perfused retrogradely with a constant flow, at 3 I*C. Timolol (10 -6 mol/l) was used to block iB-AR. After a loading period with 86Rb+, washout started. After 25 rain. wash-out, the hearts were stimulated with 3x10 -5 mol/l phenylephrine in the presence or absence of 5x10 "5 mol/l bumetanide. Results: The 86Rb+ efflux was increased after a l - A R stimulation. Average increase in 86Rb+ efflux was 21.4% (:i:2.9). When bumetanide was present during the stimulation, the increase was 12.3% (±1.7). Bumetanide significantly (p=0.016) reduced phenylephrine mediated increase in 86Rb+ efflux by 43%. Conclusion: a t - A R stimulation increases a bumetanide sensitive K + efflux. This is, to our knowledge, the first report indicating a linkage between Ctl-AR stimulation and the Na/K/2CI cotransport in the heart.

POST SYNAPTIC OPIOID PEPTIDE (OP) Fr103 AND ADRENERGIC (AD) SIGNALLING INTERACTIONS IN INTACT RAT HEART Salvatore Pepe, Rui-PingXiao and Edward G. Lakatta Lab. of CardiovascularScience GRC NIA NIH BaltimoreMD USA This study sought evidence for a post-synaptic interaction of OP and AD receptor stimulation in isolated, isovolumic hearts of ~' Sprague-Dawley rats pretreated, with 6-hydroxy-dopamine to destroy cardiac nerve endings. Norepinephrine (NE) 5x10TM, increased left ventricular systolic pressure (LVP) to 200% of control values. When the 5-OP receptor agonist, leucine-enkephalin (LE) 10aM, was delivered in the presence of NE, LVP decreased to 66% of control values. When naloxone 10SM was added with LE and NE, the effect of LE was blocked. In contrast, LE failed to inhibit the positive inotropic effects of an equipotent concentration of either forskolin (adenylate cyclase activator) or the cAMP analogue, CPT-cAMP (180% and 178% of control LVP respectively). In hearts treated with pertussis toxin (251ag/kg Lp.) for 24hr, the anti-NE effect of LE was abolished. Similar effects were observed in isolated cardiac myocytes. These results indicate that in both single cells and the intact rat heart 8-OP receptor stimulation specifically renders negative feedback modulation of the AD signal transduction pathway via a pertussis toxinsensitive G protein, thus altering cardiac excitationcontraction coupling processes.

ADRENOCEPTOR.MEDIATED INCREASE IN Frl02 POTASSIUM-UPTAKE OF CARDIOMYOCYTES: EVIDENCE FOR CROSS-TALK H.~vard Viko. Jan-Biotin Osnes and Tor Skomedal. Dep. of Pharmacology. University of Oslo. Oslo. Norway. The aim of the present study was to investigate the effect of eq- and [~-adrennceptor stimulation, alone and in combination, on K'-uplake in ventricular ardiomyocytes fmm adult rat bean. The cellular uptake of the K"-analogue ~'Rb" was measured after 15 rain in the prescence of tzt-, ~or combined adrenoceptor stimulation. At 15 rain both ct]-, 13-and combined adrenoceptor stimulation dose-dependently increased the ~'Rb'-uplake, with an equal potency (pD2=6,77+0,35; 6,62+0,31 and 6,73:t:0,27 respectively) for all three stimulation types. The maximal values for the increased St'Rb*-uptake was 20-J:35%; 21+3% and 31+3% above basal level respectively. The value for combined adrenoceptor stimulation was significantly higher than the values for the separate types, but significandy lower than their (calculated) sum. The mutual auenuation between a t- and ~-adrenoceptor stimulation when applied simultaneously, may be due to a common limiting step for the final effects. Alternatively mutual inhibitory regulations of the two signal transduction systems may occur, i.e. a cross-talk may excist between ctaand ~-adrenoceptor mediated regulation of K'-fluxes in cardiomyocytes.

EFFECT OF IS-ADRENERGIC STIMULATION Fr104 ON CALCIUM-INDUCED CALCIUM-RELEASE IN RAT VENTRICULAR MYOCYTES M. Hussain & C.H. Orchard, Department of

Physiology, Universityof Leeds, Leeds, LS2 9NQ, U.K. Perforated patch clamp experiments were conducted to investigate the role of the L-type calcium current (/Ca) in triggering the release of Ca 2+ from the sarcoplasmic reticulum (SR) during 13-adrenergic stimulation. Membrane potential was depolarised for 300 ms from 40 mV to different test potentials at 0.5 Hz. Under control conditions, both ICa and the Ca 2+ transient (monitored using fura-2) showed a bell shaped voltage dependence: both parameters reached a peak at ---4-10 mV and declined at more positive potentials (n=6). A plot of the amplitude of the Ca 2+ transient vs. the amplitude of ICa was linear with a correlation coefficient of ~0.8. Isoprenaline (lgM) caused a simultaneous increase in the amplitude of Ica and the Ca2+ transient (n=5). However, the voltage-dependence of the Ca 2+ transient was no longer bell-shaped: submaximal ICa now elicited a maximal Ca 2+ transient. These data suggest that an increase in the Ca 2+ content of the SR, rather than the increase in Ica, is the major determinant of the positive inotropic effect of 13adrenergic stimulation. Supportedby the BtIF A243

I N F L U E N C E O F H Y P O C H L O R O U S A C I D ON Fr105 CARDIAC I~-ADRENOCEPTOR-ADENYLYL CYCLASE SYSTEM Sujata Persad, Jasvinder Kaila & Naranjan S. Dhalla. Division of Cardiovascular Sciences, St. Boniface Hospital Research Centre, University of Manitoba, Winnipeg, Canada. Although hypochlorous acid (HOCI) is known to produce heart dysfunction, virtually nothing is known about its action on 13-adrenergic receptor-adenylyl cyclase system. In this study we examined the effects of HOCI on the rat heart 13/- and 132-adrenergic receptors and adenylyl cyclase activity in the absence or presence of isoproterenol. Treatment of heart membranes with HOCI was found to markedly depress the isoproterenolinduced activation of adenylyl cyclase; this effect was completely prevented by the addition of methionine, a well known antioxidant. The affinities of both 13t and 132 adrenoceptors as well as the density of 13t adrenoceptors were depressed without any changes in the density of 132-receptors upon treating heart membranes with HOCI. HOCI also decreased the basal adenylyl cyclase activity as well as stimulation by NaF, forskolin and Gpp(NH)p. These results indicate that defects in signal transduction due to oxidative stress may involve both adrenoceptors and adenylyl cyclase enzyme in the myocardium (Supported by MRC Group in Experimental Cardiology).

I3-ADRENERGIC RECEPTOR-MEDIATED CHANGES F r l 0 7 IN SUBCELLULAR LOCALIZATION OF G PROTEIN [~ SUBUNITS IN PERFUSED RAT HEARTS Ken Kageyama, Takeshi Murakami, Hideaki Kawaguchi & Akire Kitabatake. Dept of Cardiovasc. Med., Hokkaido Univ. Sch. of Med., Sapporo, Japan.

G proteins consist of three subunits, termed a, 13,and ~'. Recently, it has been recognized that the J3ysubunits play active roles .Therefore, we investigated the effects of 13-adrenergic receptor stimulation on steady-state level of G protein [~subunits (GI3) in the rat heart. Rat heads were per/used by Langendorff's technique with or without isoproterenol (ISO), epinephrine (EPI), and ISO with propranolol (PROP). Immunoblotting using isoformspecific antisera against G protein 13subunits revealed that the rat heart contains at least three G protein 13subunits. The level of G~3 in the cytosol dramatically decreased in the presence of ISO or EPI. On the other hand, the level of GJ~3in membrane fractions significantly increased in the presence of ISO or EPI. The levels of G[3= and G~2 did not change in the presence of ISO or EPI in cytosol and membrane fractions. ISO with PROP did not change the level of G~3 in cytosol and membrane fractions.Taken together, J3-adranoceptor agonist might induce translocation of isoform-speciflc GI3.~from the cytosol to the membrane.

MODULATION OF THE 13-ADRENERGIC Fr106 RESPONSE OF CULTURED CARDIOMYOCYTES BY THE LIPOXYGENASE PRODUCT (15S)-HETE Oerd Wallukat, Hartlnut Ktlhn*, Rosemarie Morwinsld & Albert Wollenberger. Max Delbrtick Centre for Molecular Medicine, Berlin, *Humboldt University, Berlin, Germany. Arachidonic acid and its 15-1ipoxygenase metabolite (15S)hydroxy-(5,8,11,13)-eicosatetraenoic acid (15S-HETE) modulates the B-adrenergic response of cultured neonatal rat cardiomyocytes as indicated by a shill of the isoprenaline doseresponse curve to the left by 4 order of magnitude. The induction of the R-adrenergic supersensitivity was enandoselective for the S-isomer and could be detected at concentratioas as low as lff ~2 M. (11S)-I-IETEdid also exhibit this effect, whereas other mono, di, and trihydroxyeicmanoids as well as the 15- lipoxygenase products of ll,14-eicosadinoic acid and two eicosatrienoic acid isomers and of 5,8,11,14,17- eicosapentaenoic acid were ineffective. Immunohist~hemical studies indicated the expression of a 15-1ipoxygenase in cardiomyocytes and in heart mast cells. The inductiom of the supersensitivity was accompanied by a selective incorporation of (15S)-HETE into the cellular phosphatidylinositol pool In contrast, (12S)-HETE, which did not induce B-adrenergic supersensitivity, was incorporated preferently into phosphatidylcholine. Calphostin C, an inhibitor of protein kinase C, blocked the induction of the supersensitivity by (15S)-HETE. These data suggest that the induction of the B-adrenergic supersensitivity by arachidonic acid is mediated by a 15-1ipoxygenaseand protein kinase C involving pathway.

NOREPHINEPHRINE REDUCES NITRIC OXIDE Fr108 PRODUCTION WITHOUT AFFECTING NO SYNTHASE SYNTHESIS IN HUMAN ENDOTHELIAL CELLS. Tiziana Bachetti, Laura Comini, Paolo Pedersini, Laura Agnoletfi, Marcella Bellet, Giuseppina Gala, Salvatore Curello, Roberto Ferrari. Fondazione Clinica del Lavoro, Centro "S. Maugeri", Gnssago e Cattedra di Cardiologia, Brescia, Italy. It has been suggested that endothelium function and nitric oxide (NO) production are impaired in congestive heart failure as a result of chronic adrenergic activation. We measured NO production and constitutive NO synthase synthesis (cNOS) in human umbilical vein endothelial cells (HUVEC) incubated for 7 days in the presence of 1 ng/mL of norepinephrine (NE) and 10.4 M ascorbic acid as antioxidant. NO production was monitored by a spectrophotometric ox'yhemoglobinassay, cNOS was determined by SDS-PAGE and Western Blot Analysis. Release of NO was measured before and after addition of 1O"~ M bradikinin (BK) and 10.4 M ionomycin (IONO). The results are: Basal Control,n---6 N E (I ng/mL), n=6

NO released(nM) BK IONO

421+_34
572_+39
5424-50
The synthesis ofcNOS was unchanged after NE treatment. We conclude that: chronic exposure of HUVEC to NE results in a blunted release of NO and does not affect the synthesis of eNOS.

A244

NEUROMODULATION OF ELECTRICAL ACTIVITY Fr109 IN ADULT CULTURED CARDIOMYOCYTES IN PRESENCE OF NITRIC OXIDE Magda Horackova, Drew Armour; Dept. of Physiology & Biophysics, Faculty of Medicine, Dalhousie University, Halifax, N.S. Canada

LONG-TERM CULTURES OF ADULT Fr110 CARDIOMYOCYTES WITH PERIPHERAL CARDIAC NEURONS: IMMUNOHISTOCHEMICAL PROPERTIES Magda Horackova, J. Drew Armour, Roger P. Croll, Dept. of Physiology & Biophysics, Faculty of Medicine, Dalhousie University, Halifax, N.S. Canada

To determine whether nitric oxide (NO) modifies cardiomyocytes directly or indirectly via peripheral autonomic neurons, the effects of NO were studied in long-term (3-6 weeks) cultures of adult guinea-pig ventricular myocytes alone as well as in cocultures with cardiac neurons. The beating frequency of ventricular myocytes cocultured with intrinsic cardiac neurons (M-intrinsic) or stellate ganglion neurons (M-stellate) increased by 20-30% following administration of the NO donor SNAP; this effect was abolished by the guanylate cyclase inhibitor LY 83583. The beating frequency of noninnervated myocyte cultures was not affected by SNAP. The precursor of NO, L-arginine also increased the beating rate (-20%) of M-intrinsic cocultures, not affecting that of M-stellate cocultures or noninnervated myocyte cultures. Augmentor effects induced by SNAP were no longer elicited in the presence of TTX, and were unaffected by .8-adrenergic or muscarinic receptor blockade. It is concluded that (1) NO-sensitive neurons are present in stellate and intrinsic cardiac ganglia and these neurons increase the beating rate of cardiomyocytes in the presence of NO; (2) more NO-synthesizing neurons are present in Mintrinsic than M-stellate cocultures as L-arginine increased the beating frequency of myocytes significantly only in Mintrinsic cocultures. (3) The beating rate of noninnervated myocyte cultures is not directly affected by NO.

Adult guinea-pig ventricular myocytes and neurons from stellate or intrinsic cardiac ganglia cultured together or separately for 2-10 weeks were morphologically investigated by means of a variety of markers and specific antibodies. Examination of cultured myocytes by phalloidin-rhodamine (for F-actin) and by antibodies raised against myomesin revealed that myofibrillar striations in rod-shaped myocytes remained present while those cells changed from cylindrical to a flattened form as they established intercellular contacts. Microtubular networks, identified by e-tubulin DM1A antibody, were arrayed longitudinally among the myofibdls. Histochemically identified adult peripheral autonomic neurons cultured alone or with myocytes had a variety of shapes. Both populations of neurons exhibited staining for markers of nine different neurochemicals; tyrosine hydroxylase, acetylcholinesterase, choline acetyltransferase, neuropeptide Y, vasoactive intestinal peptide, calcitonin gene-related peptide, bradykinin, oxytocin, and NADPH-diaphorase. This study demonstrates that (1) the structural properties of the cultured myocytes resemble those of myocytes in vivo and (2) the cultured intracardiac and extracardiac neurons are associated with a number of neurochemicals, as was indicated by our earlier functional studies. This model provides a valuable tool for investigations of the neuronal regulatory mechanisms of cardiac function.

INTRAMURAL NEURONS OF THE RAT HEART Frl 11 AS SEEN IN SCANNING ELECTRON MICROSCOPE. M. Moravec, J. Moravec, Dept de Physiologie, UCB Lyon Io 69622 Villeurbarme, France. According to our previous work in the optical and transmission electron microscopes, the intramural nerve plexus of the interatrial septum contains several types of ganglion ceils. In this work rite morphology of the intracardiac neurons was assessed by use of scanning electron microscopy. Our results con£trm the three dimensional reconstruction of the intramural nerve plexus and correlate reasonablyenough with our immunohistochemical data. Some of the pictures cord'trm the presence of both unipolar and multipolar neurons all along the nerve fibres associated with different structures of the atrioventricularjunction. The unipolar neurons (35 um in diameter) occur isolated in the atrioventricular node, proximally to the emergence of the right bundle branch. In contrast, large muldpolar neurons (90 u m in diameter) are associated with the varicose fibres of the perinodal glomeruli. In the vicinity of these latter structures we found numerous terminal varicosifies (3 to 15 u m in diameter). Some of which are closely associated with the muscle ceils of the surrounding specialized tissue. The typical spindle-like structures characterized by several whorls of their coiled endings are more difficult to be preserved. Apart from nerve fibres innervating their axonal hillocks, the cell bodies of the neurons observed present only scarce synapfic buttons which may underlie their functional autonomy. (This work has been done at the CMAB of the University of Burgundy, Dijon, France).

STUDY OF ASYNCHRONOUS RESPONSE OF THE Frl 12 LEFT AND RIGHT CARDIAC PUMPS UNDER ACUTE HYPOXIA* Zhao-Nian Zhou, Lei-Min Gu, Feng Yuan, Yun Xiao

Shanghai Institute of Physiology, Academia Sinica, Shanghai Previous experiments proved that mechanical response of the left ventricle did not show a simultaneous change with the right one in vivo and in vitro under acute hypoxia. The present work tries to investigate the effects of calcium channel blockers and the changes of catecholamine content in contact with the asynchronous response of left and right cardiac pumps under acute hypoxia. The study was carded out in anesthetized and thoracotomized adult dogs under artificial respiration. Effects of three calcium channel blockers (nifedipline, diltiazem and verapamil) on the mechanics of the left and right cardiac pumps were observed. After treatment with this blockers, pressor responses on LVP by acute hypoxia disappeared. Decrease in pressor response of RVP and Ppa was also observed in acute hypoxic. Changes of the eatecholamine (NA, A and DA) content in left and right myocardium when asynchronous mechanics of loft and fight pumps occurred were measured by HPLC. The content of these substances in left myoeardium increased. In right myocardium, except the A content, NA and DA increased significantly. The results suggested that: (1) the effects of calcium channel blockers on the performance of the left and fight ventricles were different~ It may due to the different degree of the dependence on calcium in left and right myoeardiura. (2) Heterogeneity of eateeholamines distribution in left and right myoeardium may play a part role in the asynchronous response of the left and right cardiac pumps under acute hypoxia. *The project supported by NSFC. A2~5

SINGLE COMPONENTS OF ISCHEMIA AND Fr113 CARDIAC NORADRENALINE RELEASE Armin Haunsletter, Markus Haass, Carsten Kr0ger, Babette Schulze Icking & Wolfgang K0bler. Dept. Cardiology, University of Heidelberg, Heidelberg, Germany.

The influence of global myocardial ischemia and its single components on noradrenaline release and its modulation by the muscarinic agonisl carbachol was studied. Release of endogenous noradrenaline from the in situ perfused guinea pig heart (n=5-19) was induced by two electrical stimulations (Sl, $2) of the left stellate ganglion allowing for interventions belween $1 and $2. Under control conditions $1 and S2 resulted in an equivalent noradrenaline release ($2/$1=0.98+0.05) that was reduced by carbachol (10 laM) to 0.41+_.0.07(P<0.05). Ten min of stopflow ischemia resulted in an inhibition of noradrenaline release to 0.14_+0.01 (P<0.05). In addition, presynaptic modulation by carbachol was lost. Under anoxic (pO2<5mmHg) and hyperkalaemic conditions (16mM) noradrenaline release was reduced to 0.76_+0.08 (P<0.06) and 0.43+0.14 (P<0.05), respectively. However, an additional inhibitory effect of carbachol was still preserved during both anoxic (0.27-+0.05; P<0.05 vs. anoxia) and hyperkalaemic perfusion (0.04+0.02; P<0.05 vs. 16 mM K+). Acidification to a pH value of 6.0 reduced stimulated noradrenaline overflow to 0.37_+0.09 (P<0.05), whereas moderate acidosis had no inhibitory effect (pH 6.8: 0.82_+0.13; P not significant). However, moderate acidosis completely suppressed muscarinic modulation of noradrenaline release (0.70+0.12; P not significant vs. pH 6.8). Inhibition of stimulated cardiac noradrenaline release by ischemia could be attributed to the combined action of anoxia, acidosis and hyperkalaemia. In contrast, muscarinic modulation of noradrenaline release was only affected by acidosis.

Frl 1 5 ROLE OF Ca ~ IN ENERGY DEPLETION-INDUCED NORADRENALINE (NA) RELEASE IN PERFUSED HEART AND INCUBATED BRAIN SYNAPTOSOME. Xiao-Jun Du, Alex Bobik, Murray D Esler, Anthony M Dart. Baker Medical Research Institute, Melbourne, Australia. Ischaemia or energy depletion in the heart induces NA release which has been considered as a Ca2÷-independent, carriermediated release. We studied the role of Ca 2÷ in such NA release in perfused rat heart and incubated brain synaptosomes with energy depleted by either anoxic and glucose-free perfusion or metabolic inhibitors (MI, rotenone 10 laM and iodoacetate 1 raM). NA levels were analysed with HPLC. At 1.85 mM Ca 2+ 40-min anoxia or MI induced marked NA release in both preparations starting at 10 min. The neuronal uptake1 inhibitor desipramine (0.3 pM) and a Ca 2÷ entry blocker Ni z÷ (1 raM) inhibited NA release by 70-80% in both preparations. N-type channel blockers Cd 2 (0.1 mM) showed a small inhibition on anoxic NA release in hearts, and both Cd z÷ (0.1 mlV0 or m-conotoxin (50 nM) had no effect on MIinduced NA release in synaptosomes. In hearts perfused with Ca 2+ free buffer, an intracellular Ca 2÷ chelator BAPTA AM(20 gM) reduced NA release by 25%. In synaptosomes incubated with 1.85 mM Ca2+ and MI, BAPTA'¢M (2, 20 and 100 laM) reduced Ml-induced NA release in a dose-dependent manner with 80% suppression at 100 laM. Conclusion: 1) Energy depletion-induced NA release is a CaZ+-mediated process. 2) Brain synaptosome is a useful model for mechanistic studies on NA release in simulated ischaemic conditions. A246

OVEREXPRESSION OF 132-ADRENOCEPTORS IN Frl 1 4 TRANSGENIC MICE CHANGES CARDIAC 13ADRENERGIC ACTIVITY X.iao-Jun Du, Elizabeth Vincan, Anthony Dart, Ca.rmelo Milano, Elizabeth Woodcock, Robert Lefkowitz. Baker Medical Research Institute, Melbourne, Australia and Dept of Medicine, Duke University Medical Center, USA. Transgenic mice have been created with 200 fold overexpression of human 132-adrenoceptors in the heart (Milano et al, Science 1994;264:582). We studied cardiac function at baseline and during adrenergic stimulation by isoprenaiine infusion or sympathetic nerve stimulation in transgenics (+132) versus their controls (-132). The model was an in situ perfused (coronary flow of 2.2 ml/min), innervated heart with the left ventricle cannulated to measure dp/dtm~ and heart rate (HR). Basal HR and dp/dtm.~ were 20--40% higher in +132 than -132 hearts. Electrical stimulation of the left stellate ganglion (2-8 Hz) or infusion of isoprenaiine (10"l°-10-5 M) markedly increased HR and dp/dtmax in -I]2 hearts. In +132hearts, there was no response in HR and dp/dtrr~ during isoprenaline infusion. In contrast, the-~132hearts retained HR response to nerve stimulation and there was a small increase in dp/dt,~. Atenolol (13t-antagonist, 3 p.M) abolished the response to nerve stimulation in -13zhearts whilst ICI 118551 (132-antagohist, 3 I.tM) abolished the response in +132hearts. Basal HR and dp/dtm.u were decreased by ICI I 18551 only in +112hearts. Thus, overexpression of cardiac 132-adrenoceptors modifies 13adrenergic activity but the responses to endogenous and exogenous adrenergic stimulation are affected differently.

OF H Y P O X E M I A ON A D E N Y L Y L Fr116 CYCLASE A C T I V I T Y IN A T R I A AND V E N T R I C L E S OF C H I L D R E N W I T H CONGENITAL HEART DISEASE Milan ~am~nek, B o h u s l a v Hu~in, V~ra Klenerov~, Sixtus Hynie. K a r d i o c e n t rum, Univ. Hosp. Motol and ist Fac. of Med., Charles Univ., Prague, CR. EFFECTS

Our previous study has shown that human heart is able to adapt to hypoxemia by c h a n g i n g the a c t i v i t y of energy s u p p l y i n g enzymes. T h e r e was a d i f f e r e n c e in a d a p t a t i o n of atria and ventricles. A total of 29 samples of right atrial or v e n t r i c u l a r tissue were obtained during heart surgery from 21 c h i l d r e n 2 to 15 years old with congenital heart disease. Adenylyl cyclase level (AC) and its response to i s o p r e n a l i n e , glucagon, Gpp/NH/p and forskolin (F) were m e a s u r e d using 32p-a-ATP. s i g n i f i c a n tly higher basal and F s t i m u l a t e d A C a c t i v i t y was found in atria of hypoxemic children (H) as c o m p a r e d to normoxemic (N) patients. Basal as well as F s t i m u l a t e d r i g h t v e n t r i c u lar AC a c t i v i t y in H did not d i f f e r from right atrial activity. In conclusion, AC a c t i v i t y and its r e s p o n s e to s t i m u l a t i o n is h i g h e r in H than in N children.

COHPARATIVEEVALUATIONOF VARIOUSANTIOXIDANTS Fr117 ON ISOLATEDAND ISCHEMIZED RAT HEARTS. Marianna V. Bilenko, AlexanderA. Morgunov. Inst. of Biomedical Chemistry RAMS, Moscow, Russia. Anti-ischemic and toxic effects of lipid-soluble- BHT, and water-soluble antioxidants (AO)-K-Phenosane (KPh), 1-hydroxy 2,2,6,6,-tetramethylpiperidine (HTMP), Se-Methionine (Se-Met), and AO enzyme- h-SOD, were studied on 276 Wistar rat hearts which were perfused with Krebs-Henseleit medium25 min before and reperfused 40 min after their total 30 min ischemia at 37oC (stop-flow Langendorff-Fallen model). All water-soluble AO were added to the mediumprior and after ischemia. BHT was added to the medium inside monolayer liposomes from egg phospholipids either before or after ischemia. Effect of AO was estimated by their influence on the contractile function (Pmax, +dP/dtmax, -dP/dtmax, EDP) and coronary flow (CF) of the heart. I t was shown that all studied AO had dose-dependent anti-ischemic action on myocardium contractile function, while BHT, KPh and HTMP increased also pre- and postischemic CF. Protective effects of Se-Met and COD on ischemized heart have already appeared in the early reperfusion period, while the effects of BHT, KPh, and HTMPwere the most significant only in the late reperfusion period. The approximately equal protective effect of studied AO on ischemized heart was found in the optimal concentrations which were increasing in the row: Se-Met (10-9 M) < BHT (10-6 M) < HTMPand KPh (10-5 M). SOD was the most effective at the dose 30000 U/L. In higher doses AO produced a moderate (KPh - 10-4 M, SOD - 60000 U/L), or significant (BHT - 10-4 M) toxic action on ischemized myocardium.

STOBADINE AND TROLOX-C AFFECT THE Fr119 REPERFUSION-INDUCED DYSRHYTHMIAS Peter Kovfics, Vlado Knezl*, Ivo Jurfinek*, Tania StankoviEovfi, Pavel .~vec, Comenius Univ, Fac Pharm, Dept Pharmacol, *lnst Exp Pharmacol SAS, Bratislava, Slovak Republic We investigated the effects of antioxidant Trolox-C and stobadine on reperfusion-induced dysrhythmias. On isolated perfused rat hearts, Trolox-C (0.1mM) and stobadine (0.01mM) decreased incidence and duration of reperfusioninduced dysrhythmias and prevented the contractile dysfunction observed in ischemicreperfusion damaged hearts. In stobadinepretreated hearts the decrease of malondialdehyde and conjugated dienes (CD) levels, 9 and 4 times respectively, was found with respect to control group. However, in Trolox-Cpretreated hearts only CD levels were decreased (3 times). Concentrations of cAMP and cGMP were maintained near the control levels in drugspretreated hearts. These results suggest that both compounds prevent peroxidation of cardiac lipids, imbalance between cyclic nucleotides, and in this way protect myocardium against reperfusioninduced dysrhythmias.

Sensitization

of postischemic

reperfused

Fr118

isolated rat heart by copper and protection by stable radical. Galina Zeltcer, Eduard Berenshtein, Amram Samuni & Mordechai Chevion. Hebrew University, School of Medicine, Jerusalem, Israel. The sensitizing effect of isolated rat heart by copper following global ischemia and the protection by stable radical 4-OH-2,2,6,6 tetramethyl-piperidin-l-yloxy ( T E M P O L ) h a v e been studied. Hearts were equilibrated with Krebs-Henseleit buffer for 10rain and subjected to 18min o f normothermic global ischemia. Most of the heart function, impaired upon ischemia, was restored following 20min reperfusion, as demonstrated by the hemodynamic parameters: v e n t r i c u l a r d e v e l o p e d pressure (79%), rate of contractility (78%) and relaxation (80%), heart rate 87%, enddiastolic pressure (12mmHg), and work i n d e x (70%). T E M P O L , w h e n g i v e n d u r i n g reperfusion provided no protection. W h e n 40p.M Cu(histidine)2 was included in the perfusate before, during, and following ischemia, the heart injury was more extensive and work index was restored only to 17% of the preischemic value, demonstrating the sensitizing effect of copper. Cu-(nitrilotriacetate)2 was more potent as 101.tM complex caused similar sensitization. The inclusion during reperfusion of 1001.tM TEMPOL, which oxidizes reduced metal ions, a b o l i s h e d the c o p p e r - i n d u c e d s e n s i t i z a t i o n . Surprisingly, the presence of T E M P O L in the perfusate during preischemic normoxia impaired heart function, even in the absence of copper.

TRANSIENT MYOCARDIAL ISCHEMIA STIMULATES Frt 20 pOLYMORPHONUCLEAR NEUTROPHILS (PMN).

przemyslaw Guzik,Andrzej Wykrftowic~MiroslawKabnierczak Henryk Wysockifrom Dep. of Intensive Therapy, Institute of llnternal Medicine, University School of Med., Poznad, Poland 'It is established that a number of stimuli for neutrophils ~MN) are released during myocardial infarction and prolonged ischemia. However, there is no data explaining the impact of transient myocardial ischemia (TMI) on release ofPMN stimul. The aim of this study was to determine whether TMI could affect PMN function. TMI was induced during positive dipyridumole stress test evaluated by 99"Tc-SestaMIBI followed by SPECT. Samples of plasma were obtained from patients just before and 5-8 minutes l~ncerdipyridamole injection (0.84 mg/kg). Aliqouts of plasma were ubated with PIvIN isolated from healthy donors. PMN function lwas estimated by superoxide anion (05") production and expressed hs 02" pmol/2.5 x l0 s PMN/30 rain. In the group without TMI (4 females, 3 males, 52 + 13 yrs) 02" production by PMN incubated ,with plasma obtained after dipyridamole infusion 070 + 54.8) was significantly lower than caused by plasma collected before drug administration (293.2 + 66.2: p = 0.004). In contrast there was no significant difference in the 05" release by PMN incubated with plasma obtained after (288.3 + 79.2) and before (288.7 + 74.7) dipyridamole injection in patients with TMI (2 females, 6 males; ;53 + 10 yrs). 02" release by PMN incubated with plasma taken after drug administration was significantly higher in the group with TMI than in patients without transient ischemia (p = 0.015). In patients without TMI a decrease in 02" release by PMN observed t~er drug infusion was caused by antioxidant and/or neutrophil |nltibiting properties of dipyridamole. A lack of similar decrease in patients with TMI may indicate that substances released during transient myocardial ischemia stimulated neutrophils to produce 0 2"and to abolish dipyridamole scavenging and inhibiting effect. A247

EBSELEN FOR MYOCARDIAL Fr121 PROTECTION IN DOGS DURING ISCHEMIA AND REPERFUSION Shiro Hoshida, Masashi Nishida, Nobushige Yamashita, Junsuke Igarashi, Masatsugu Hori,

Takenobu

Kamada,

Tsunehiko

Kuzuya,

Michihiko Tada, 1st Dept. of Med. Univ. School of Med., Suita, JAPAN

Osaka

We investigated the infarct-limiting effect of a selenoorganic compound, ebselen (2-phenyl-l, 2benzisoselenazol-3[2H]-one), in a canine coronary artery occlusion (90 min)-reperfusion (5 h) model of myocardial infarction. Ebselen was administered 1 h before coronary artery occlusion (50 mg/kg po). When we examined the reladon between infarct size and plasma ebselen level, infarct size in dogs with plasma ebselen level>5/zM before reperfusion was significantly smaller (20.8+3.1%) (P<0.05) than that in dogs with plasma ebselen level < 5/z M(34.5+7.0%) or in the control dogs (39.1+4.9%). This infarct limitation produced by ebselen treatment was associated with an increase in reduced glutathione content and a reduction in myeloperoxidase activity in the ischemic myocardium. No differences between the control and treated groups were found in hemodynamic parameters or regional myocardial blood flow in the course of the experiment. The findings of this study demonstarate that ebselen effectively reduced the myocardial ischemia-reperfusion injury associated with preservation of the glutathione redox state and a reduction in neutrophil infiltration into the ischemic myocardium.

EFFECT OF ACE-I (ALACEPRIL) ON STUNNED Fr123 MYOCARDIUM Katsumlchl Iljlma, HIrohlsa Arata, Haruhlko Ishloka HIdehiko AIba, Kazuhiko Umetsu, EIichl Geshl, Takashl Kataglrl. 3rd Dept. of Internal Medicine, Showa Univ. School of Med., Tokyo, Japan.

We studied the mechanism of stunned myocardium with special standpoint of the relation o1 free radicals using ACE-I (alacepdl). In the anesthetized dog, 15 min o1 LAD occlusion and 60 rain reperfusion were performed with continuous records of segment shortening (%SS) and hemodynamics, then biochemical analyses of sarcoplasmic reticulum (SR) and mitochondda (Mt) were performed. Alacepdl(A) (10mg/kg/hr) or saline (S) were given intravenously before the ischemia. Changes of hemodynamics were not indicated significant differences between two groups, however %SS more recovered in A group (78% of control) than in S group (58%). Ca-ATPase of SR showed higher in A group (85%) than in S group (78%). State III respiratory and DNP-ATPase activities of Mt also indicated higher in A group (89% and 830) than in S group (77% and 75%). SDS gel electrophoresis of SR showed no significant changes in both groups. Free radical generation is considered one of the main causes of the stunned myocardium. These results indicated that ACE°I improved regional contractility and energy metabolism of stunned myocardium caused by scavenging of the free radical.

A248

SUPEROXIDE DISMUTASE IS INEFFECTIVE Fr122 AGAINST REPERFUSION INJURY IN CATALASE INHIBITED RAT HEART

Toshiki Iwata, Noburu Konno, Toshikuni Yanagishita, Eiichi Geshi & Takashi Katagid. The Third Department of Internal Medicine, Showa University School of Medicine, Tokyo,Japan The sequential univalent reduction of oxygen gives rise to very reactive intermediats, and these radicals may initiate to cause the cellular injury. In myocyte, the superoxide anion is dismutated to hydrogen peroxide by superoxide dismutasa (SOD), and hydrogen peroxide catalytically redused to H~) mainly by cataiase, but it is uncertain the relation between SOD and cat°lass. Therefore in this study we used 3-amino1,2,4°tdazole (AT-Z) as the inhibitor of cat°lass and investigate the effect of endogenous catalase to the action of SOD as an antioxidant for heart tissues. We adopted the perfused rat head by working head model, and the sarcoplasmic reticulum (SR) was extracted by cantdfugation, it was resulted that on the ATZ treated heart, SOD cured the contractile function only 80% of the control. The incidences of reperfusion arrhythmia (especially prolonged ventdcular fibrillation) was increased in the A'I'Z group, and more increased significantly by SOD treatment during reperfusion. In A'rz treatment group the Ca2*-ATPase activity of SR during reperfusion was decreased significantly to 9.52/J. moles Pi/mg protein/hr (control group level is 13.1), and more over decreased in ATZ+SOD group. The content of major ATPase protein of SR were decreased by ATZosoD treatment, correlated with the changes in the ATPase activity. The results indicated that SOD has not the protective effects against reperfusion injury without endogenous or exogenous catalase which has the ability to reduce hydrogen peroxide catalytically in myocytes.

PHOLASIN:A NOVELPHOTOPROTEINFOR THE DETECTION Fr124 OF FREE RADICAL PRODUCTION DURING ISCHEMIA AND REPERFUSIONIN THE ISOLATEDRAT HEART

Michael J Shattock, Jonathan IA Miller, Richard Southworth, Jan Knight*, David J Hearse. Cardiovascular Research, St Thomas' Hosp, London & *Knight Scientific Ltd, Plymouth, UK. Free radicals and oxidant stress have been implicated in ischemia/reperfusion injury in the heart. Detection and quantification of radicals has relied on indirect methods such as electron spin resonance and/or the measurement of radical adducts - techniques with limited temporal resolution. In a series of preliminary experiments, we have used the radicalsensitive photoprotein, Pholasin® (isolated from the marine bivalve Pholas dactylus), in the isolated rat heart. In initial experiments, A-V consumption of Pholasin was measured in rat hearts subjected to 15rain ischemia and 8min reperfusion. A peak in Pholasin consumption was measured after 2-5rain reperfusion retuming to control levels after ~6rain. In a second series of experiments, light emission was measured directly using a photomultiplier tube placed adjacent to the leftventricle. A large peak in radical-inducad luminescence on reperfusion was seen that correlated with the hyperdynamic phase of contractility (see figure). LVDP~ _ , (_m m H g )~ , A sharp peak in radical production ' - ] - I~l~ during early ischemia was also o.i ~ . . _ _ / ~ apparent. This technique requires L.uminescence ~k further characterisation but may provide an important new tool for measuring, in real time, free Ischemia radical production during ischemia and repeffusion.

C A R D I A C A C T I O N S OF T H E R A D I C A L Fr125 S C A V E N G E R WR2721: AN E P R AND N M R STUDY Margit Pissarek. Reincr Hascloff, Thomas Keller,ingolf E. Blasig & Ernst-Ccorg Krausc. INSERM, Chatcnay-Malabr3', France; Inst. Mol. Pharm. & Ma×-Delbrueck-Centrc,Berlin, Germany The radical scavenger properties of S-2 (3 aminopropyl-amino) ethylphosphorothioic acid (WR2721) were investigated in a radical generating system in vitro as well as in isolated rat hearts subjected to 30 rain ischaemia and 30 rain reperfusion with the electron paramagnctic resonance (EPR) spin trap technique. For these experiments the spin trap o~-phenyI-N-tertbutylnitrone (3raM) was added to the perfusate before and following ischacmia.The action of WR2721 on high-energy phosphates was observed by means of phosphorus-3 t nuclear magnetic resonance (NMR,) spectroscopy. WP-,.2721 (0. l raM) reduced the vascular concentration of spin trap adducts in isolated hearts by up to 78% (p< 0.05) between 5 and 12.5 rain of rcpcrfusion. The left ventricular enddiastolic pressure was reduced in the rcperfused hearts from 42 + 8 mmHg in the absence of WR2721 to 9+6 mrnI-Ig in the presence of the compound (p <0.02). Simultaneously, the developed pressure remained unaffected. The NMK spectroscopically assessed creatinc phosphate levels returned in the post-ischaemic hearts following application of WR2.72I to 78+5% of the preischaemic values but only to 41+ 5% in untreated controls (p<0.05).In contrast, the contents of adenosine triphosphatc were unchanged.The data confirm an effective reduction in the exposure of the rcpcrfused hearts to endogenously released frea radicals by WP,2721 as well as a partial preservation of highenergy phosphates and suggest an improvement of the postischaemic cardiac performance possibly due to an int'tuence on its diastolic component.

Fr127 Effect of c=:{e'c~ine A1 receptor agonlst R-PtA on nitric oxide production and mRNA of nitric oxk:le synthase Tn rnyocard'~ ~ a h rabblts XY.U,HG2Heng,LI_~,YP.'I'en,Yt-2h~.Departmentof CaddooY, G i ~ PLA Gen~ Hosp~l, Being, 100853, pJ~.CHre The aim of this study was to test the effect of adenosine At receptor agonist R-PIA on endogenous nitric oxide products (NOP) and the expression of nitric oxide syntbane 040S) geaa is myocardial iscbanmia and reperfusion In anaesthetized rabbits. gyocardial iacb~amia was induced by ligation of LAD artery for 10 min followed by 90 mln of reperfasion (group 1, n=~. R-PIA ~.3 mg/k~ was infused i.v. for 5 mln starting 15 min before LADocclusion (group It, n=~. A selective adenosine AI receptor antagonist DPCPX (1.0 mg/k~ was infused i.v. for 5 miu starting t6 min before R-PIA infusion (group II[, n=5). The blood levels of NOP (nitrite) were determined by ameliorated Greian metbod at baseline, 5,10 min after drug infusions and LAD occlusion, 5,10, ~O, 60, 90 min after reperfusion. The expression of 1 ~ gene in iscbaamie area of myocnrdiamwas studied by Northern Blot in the three groups. I ~ T S The levels of NOP at 6 min after R-PIA infusion rapidly increased to the double of baseline (1. t5±0.28 • to ]. 16~0. O ug, p<0.05) and continued to be bigber than baseline during ischuemla and reperfusion accompanied by decreases of heart rate for 27.6% and blood pressure for 20%. All the effects above were abolished by infusion of DPCPX previously. The results of Horthern Blot showed that the ml~ expression of ~ increased in Group l[ compared with those In Group I and II!. CO~LUSI~ The R-PIA Infusion increased the blood level of NOP and attenuated its decrease during iecbaemia and the early stage of reperfusion, the mechanism of which is most probably due to the stimulation of adenosine AI receptor and the increase of NOS.

Effects o f myocardial ischaemia and Fr126 reperfuslon on nitric o x i d e production in rabbits XY.Li,HG.2~-~.~g,YP.T~,IV~h.Y'ng,P.Mao.~ of carci01ogy, Chinese PLA Gene~ Hospital, Bei/~g, 100853, p.R.Q~na In order to investigate the effects of myocardial lscbaemia and reperfus{on on the blood level of endogenousnitric oxide products fifteen anaesthetized rabbits were randomly divided into three gronps:l, subjected to 10-min occlusion of LAD followed by 90-min reperfusion, (n=5), II. received two 10-min occlusion of LAD interposed by 10-min reperfusion and finally followed by 90-min reperfusion, (n=5), II[. received two 10-min occlusion as in grpup It followed by 60-min occlusion and 90-min reperfusion, (n=5).Tbe brood levels of NOP (nitrite) were determined by ameliorated Greieo method at baseline, 5, 10, 30 =in of occlusion and 5, 10, 30, 60, 90 min of reperfusion. The resstts: I~3P (ul~O l It lit baseline Oce. 5 rain 10 rain Rep. 5 rain Last 10min 30min 60 rain 90 mlo

1.42-1-0.41 0.78:f:0.17" O. 8l:f:O. 18" 0.97±0.19' 0.95=1:0.33 1.71:i:0.36" l. 87:i:0. 26'*

1.24~: 0. 48 0.83:t:0.19'

2. 34:[:0. 3 1 "

1.64:t: O. 3"2' '

O.70+0. 27" 0.74~0.41' 0. 78~:0. 44 0. 89:i:0. 49 1.20±0.26

1.39~: 0. 20 0.80-t-0.31,' 0.78"t"0.35" 0.59-t-0.20,, O. 57~:0. 15" 0. 54±0. 18" 0.53:t:0. l I " 0. 54~0. 14~'

* P<0.0fi ,, P<0.01 Comparedwith baseline In each group. In conclusion, the blood level of NOP decreased rapidly after n brief episode of iucbaemia and could not return to the normal immediately after rcpcrfusiou. The longer the iscbuomia, the slower the NOP rose again. Preconditioning by 10-min ischaemin could not improve the return of NOP level (group II).

CALCIUM MOBILIZATION BY INTRACELLULAR pH Fr128 IN CULTURED AORTIC ENDOTHELIAL CELLS Hiroshi Watanabe, Hireyasu Kakizawa, Reiko Takahashi and Hideharu Hayashi. Dept of Internal Medicine III, Hamamatsu University School of Medicine, Hamamatsu 431-31, Japan. The purpose of this study was to determine the interrelationship between cytosolic pH (pHi) and cytosolic calcium concentration ([Ca2+]i) in cultured porcine aortic endothe}ial cells. Changes n pHi and [Ca2*]i were measured with the pHsensitive probe 2',7'-bis(carboxyethyl)-5,6-carboxyf uorescein, and the Ca2+ sensitive fluorescent probe fura-2. The basal pHi of endothelial monolayers was 7.25 + 0.03 (mean±SD) and the value of [Ca2*]i was 45 --. 7 nM. in a modified .Tyrode solution. Intracellular acidification, wnicn was inouceo e=iner by addition of propionate (April = -0.26 + 0.04), removal of NH4CI (April = -0.28:L 0.05),or changing from a 5 to a 30 % CO2-HCO3" solution (tLpHi = -0.33 + 0.05), did not affect [Ca2*]i. On the other hand, recovery from intracellular acidification increased [Ca2*]i to 264:1:29 nM, or to 362 + 48 nM either by removal of propionate (April = +0.29 + 0.04), or returning from a 30 to a 5 % CO2-HCO3" solution (ApHi= +0.37 ± 0.06), respectively. In addition, NH4CI-induced alkalinization (pHi from 7.25 + 0.03 to 7.63 + 0.06) increased [Ca2*]i to 334:1:34 nM. Elevation of [Ca2*]i caused by the removal of propionate was completely inhibited in the absence of extracellular Ca2.. NH4Cl-induced increase in [Ca2*]i was partly inhibited in the absence of extracellular Ca2. and completely blocked by the combination with bradykininpretreatment These results showed that not decreasing phi, but increasing pHi, therefore, caused significant increase in [Ca2*]i. In addition, different mechanisms seem to be involved in this calcium mobilization during the recovery from intracellular acidification and intracellular alkalinization. A249

T, L-CALCIUM CHANNEL BLOCKER RO 40-5967 Fr129 MAINTAINS MODERATE NEGATIVE INOTROPY BOTH ON NORMAL AND POSTISCHEMIC MYOCARDIUM David Simper, David J. Chambers. Cardiac Surgical Research, Rayne Inst., St Thomas' Hospital, London, UK We have previously shown a protective effect of R0 40-5967 on ischemic myocardium in paced Langendorff rat hearts subjected to 15min ischemia and 30 min reperfusion. As the inherent negative inotropy of calcium antagonists is more pronounced in failing and ischemically injured hearts, we investigatedthe protectiveand negative inotropic effects of Ro 40-5967 in rat hearts subjected to 40 min global ischemia and 60 rain reperfusion. Drug (3x10-7 M, n=6/group) was administered as a pretreatment (30 min) before ischemia (P), pretreatment and during 5, 30 or 60 min of reperfusion (groups P+5, P+30, P+60) or during reperfusion only (R). Pretreatment increased the preischemic coronary flow by 13% (p=0.0001) and decreased the developed pressure (LVP) by 12% (p=0.0001), delayed onset of ischemiccontracture by 1.8 rain (p=0.000t) but had no effect on recovery of LVP during reperfusion (% preischemicvalue) P: 61+7, P+5: 47_+9, P+30: 50-&_6,P+60: 50+4, all NS compared to untreated controls (C): 55+6. In R, recovery of LVP was slightly reduced: 39+5%, NS, at the end of reperfusion, by 15% compared to C. In this model of extended ischemia and reperfusion, Ro 405967 delayed ischemic contracture and did not depress ventricular function in ischemically injured and failing myocardium. In contrast to other calcium antagonists, Ro 405967 may be more useful in the treatment of patients with chronic myocardialinfarction and compromised LV function.

Na+/H + EXCHANGER INHIBITION DURING ISCHEMIA, REPERFUSION OR BOTH?

Fr131

Yasuyuki Shimada & Metin Avkiren The Rayne Institute, St Thomas' Hospital, London, U.K.

There is controversyover whethert he cardioprotectiveeffectsof Na+/H+ exchanger inhibitors are exerted primarily during ischaemla or during subsequent reperfusion, possibly due to inter-study differences in experimental conditions. We studied the impact of perfusate buffer composition on the protection affordedby Na+/H+ exchangerinhibitionduring ischaemlaversus reperfusion. Isolated rat hearts (n=8/group) were perfused with bicarbonate- or HEPES-buffered medium and subjected to 20 min of global ischaemlaand 45 min of reperfusion,with transient (5 min) infusion of one of two structurally distinct Na+/H+ exchanger inhibitors, dimethylamiloride (DMA) or HOE694 (110pM), immediatelybefore ischaemla,during initial reperfusion, or during both periods. With bicarbonate medium, neither drug improvedthepost-ischaemicrecoveryofleftventriculardeveioped pressure (LVDP) when given only during reperfusion.However, they tendedto improverecoverywhen present during ischaemia, with HOE694 exhibiting greater efficacy than DMA. In contrast, with HEPES medium, both DMA and HOE694 significantly improved recovery of LVDP (from 34+5 % in controls to 56-J:3" and 71+8%*; °p
EFFECT OF PILSICAINIDE HCI ON ISCHEMIA Fr130 REPERFUSION ARRHYTHMIAS:POSSIBLE ROLE OF INTRACELLULAR Na*AND Ca 2+ DURING ISCHEMIA Masayuki Taniguchi, Fumiko Suzuki, Ryuko Anzawa, Kuni Miyoshi, Shingo Seki, Seibu Mochizuki. Division of Cardiovascular Research, Department of Medicine, Aoto Hospital, Jikei University School of Medicine, Tokyo, Japan. We previously demonstrated that pilsicainide HCI (Class Ic) significantly prevented mperfusion-induced ventricular arrhythmias. The aim of the study was to further evaluate the possible role of intracellular Na+([Na*]i) and Ca2'([Ca2*]i) during ischemia with and without an addition of pilsicainide. The isolated rat hearts were perfused by Langendorff mode, and were loaded with 100p.l of SBFI/AM or 50ul of Fura 2 as [Na*]i and [Ca2'l i respectively. Whole hearts ischemia was induced for 20 min followed by 20 min reperfusion. For fluorescence recordings, fiber optic cable connected to lamp was attached to anterior epicardial surface of the left ventricle. Fluorescence emissions of [Na*]i and [Ca2*]i indicators were collected with another fiber optic system. Ruorescence of Na + and Ca 2+ was obtained from the ratio of emission during excitation. [Na*]i quickly began to rise in early phase of ischemia and gradually rose during 20 min ischemia and that of Ca 2. showed 3 peaks during ischemia in control group. On the other hand, pilsicainide HCI significantly inhibited both the rise of [Na*]i in early phase and second peak of [Ca2*]i during ischemia. These results suggested that prevention of reperfusion arrhythmias by pilsieainide was due, at least in part, to the inhibition of the [Na*]i and [Ca2*]i accumulation during ischemia.

ENERGY METABOLISM OF MITOCHONDRIA ISOLATED Fr132 FROM POST-ISCHEMIC MYOCARDIUM L. Demalson, I. Chaudron, D. Moreau (I) L. Martine and A. Grynberg. INRA, Unit~ de Nutrition Lipidique, Dijon and (i) Facult~ de M~decine, Dijon The effect of ischemia and reper~sion on the energy metabolism was evaluated in isolated cardiac mitochondria. Rat hearts were perfused according to the working mode in normoxia for 15 minutes and were then subjected to a local 20 minute ischemia by ligation of the main coronary artery and 30 minute reperfusion. The aortic and coronary flows and electrocardiogram were continuously monitored. At the end of reperfusion, the mitochondria were extracted from the healthy (H) and reperfused (R) zones. The mitochondrial oxidation uptake was measured with palmitoylcarnitine as substrate in 2 buffers differing in their free calcium (fCa) concentration (0 or 0.34 BM). The rate of energy production was also determined in the same conditions. After ischemia, the coronary flow recovered partially, but the aortic flow remained low because of severe arrhythmia and stunning. The mitochondrial state Ill respiration rate was lower in R zone than in H zone, whatever the tea concentration. This was associated with an enhanced rate of energy production, outlining a better metabolic efficiency. In the fCa free buffer, the mitochondrial metabolic efficiency was high. Conversely, the presence of fCa decreased noticeably the oxygen cost of energy synthesis. This last phenomenon was associated with AMP production, maintaining elevated both ADP level and oxygen uptake. These results showed that, despite of the ischemia-induced cell injuries, the energy capacities of isolated mitochondria remained elevated. Conversely, in the whole cell, the ionic conditions might contribute to reduce the mitochondrial oxygen cost of energy synthesis. This was observed in vitro in the present study when the fCa concentration was high. Such disorders could bepartly responsible for the reduced contractile function observed during reperfusion.

BENEFICIAL EFFECTS OF VANADATE ON Fr133 ISCHEMIA-REPERFUSION Satoshi Takeda, Kanu R. Shah, Seibu Mochizuki & Naranjan S. Dhalla. Division of Cardiovascular Sciences, St. Boniface Hospital Research Centre, University of Manitoba, Winnipeg, Canada. Earlier we have shown that vanadate at low concentrations serves as an antioxidant. Since ischemiareperfusion injury in the myocardium is associated with an increased level of oxidative stress, this study was undertaken to examine the effects of vanadate by making the isolated hearts ischemic for 30 min and then perfusing with normal medium for a period of 30 min in the absence or presence of different concentrations of vanadate. The left ventricular developed pressure, rate of contraction and rate of relaxation were depressed by 35 to 40 % upon perfusing the ischemic hearts with normal medium. However, these abnormalities were prevented upon treatment of hearts with 1 to 4 uM vanadate. The sarcoplasmic reticulum (SR) isolated from the ischemic-reperfused hearts showed a marked depression in the ATP-dependent Ca2'-uptake activity; this defect was prevented by the addition ofvanadate in the perfusion medium. These results demonstrate beneficial effects ofvanadate on cardiac dysfunction and changes in SR Ca2'-transport due to ischemiareperfusion injury. (Supported by MRC Group in Experimental Cardiology.)

CALCIUM ANTAGONISM REPERFUSION INJURY

IN ISCHEMIA Fr'135

Ingeborg M. Kober, T. Br011, PauI-G. Spieckermann. Institute of Medical Physiology, University of Vienna, Austria Inhibition of excessive Ca 2+ entry may represent an effective means of ensuring myocardial protection during ischemia and reperfusion. Our aim was to examine if supplementation of gallopamil to Bretschneider's HistidineTryptophane-Ketoglutarate (HTK) cardioplegic solution influences heart preservation significantly. Isolated working rat hearts (n=5"in each group) were flushed for 5 rain. with either 1. Krebs-Henseleit buffer (KHB) or 2. commercial HTK solution or 3. HTK+gallopamil (501~g/L). After 45 rain. of total global ischemia at 25°C, the hearts were reperfused in the Langendorff mode for 15 min., and then made to work for 45 min.. The addition of gallopamil to HTK resulted in a significantly (p<0.05) higher coronary flow of 109.8±7.9% of the preischemic value in HTK+gallopamil treated hearts compared to 82.5±10.6% after storage in commercial HTK. The cardiac output of KHB stored control hearts recovered to 86.2±14.1%, and was 93.8_+15.8% for HTK, and 93.1_+12.2% for HTK+gallopamil preserved hearts. In gallopamil treated hearts, there was a significant (p<0.01) increase in the recovery of left ventricular pressure generation to 124.9_+16.7% of the preischemic value compared to 92.3_+14.5% in hearts of the HTK group. Trends were seen for the following parameters: diastolic pressure, oxygen consumption, edema formation and myocardial high-energy phosphates. The results of this study suggest that the elimination of Ca2+ from cardioplegic solution might be beneficial when applied under a hypothermic condition. The negative inotropic action of calcium antagonists may be overcome by addition of extra Ca2+ after the early repeffusion period.

CALCIUM, MITOCHONDRIAL INJURY AND Fr134 CONTRACTURE OF ANOXIC/REPERFUSED ADULT CARDIOMYOCYTES. lan S. Harper¶, Donna R. Trollinger, Samuel A. Tesfai and John J. Lemasters. ¶Experimental Biology, MRC, Tygerberg, South Africa, and Dept Cell Biology & Anatomy, University of North Carolina at Chapel Hill, NC, USA. The relationship between intracellular Ca2+, mitochondrial injury, contracture and cell death in adult cardiomyocytes is under investigation. Isolated cardiomyocytes, loaded with various fluorescent dyes, were examined by confocal microscopy at 37°C. During anoxia (either at pH 6.2 or pH 7.4) myocytes shortened to a square form by 15-30 min without loss of mitochondrial Aq" or an increase in cytosolic Ca2+. Subsequently, Aq' dissipated, Ca2* rose and the cells hypercontracted (rounded up). Mitochondrial Ca2+ increased after cytosolic Ca2* and hypercontracture during the anoxic period. Reperfusion at pH 7.4 after 120 min caused a transient increase in AW, but mitochondria did not fully repolarise and soon loaded with Ca2+. In some, but not all, cells this was followed by leakage of PI across the sarcolemma, indicating cell death. BDM during anoxia delayed cell shortening, but subsequent mitochondrial depolarisation occurred and was followed by increased Ca2+ and hypercontracture as before. We conclude that mitochondrial depolarisation occurs prior to marked increases in cytosolic and apparently also mitochondrial Ca2+, and leads ultimately to hypercontracture, but not necessarily to cell death.

E F F E C T S O F H Y P O X I A AND CN- ON Fr136 (+)-[3H]ISRADIPINE B I N D I N G IN HEART CELLS Mimi Stokke, Else M. Hagelin & Odd BrCrs, Clin. Chem. Dept., Ullev~ University Hospital, Oslo, Norway. Sixty rain ischemia decreases the binding density of (+)[3H]isradipine to L-type Ca 2+ channels (LCC) in heart membranes and homogenates. We investigated if 2 hrs hypoxia (incubation in glucose-free 30 mM K + buffer and N2 gass) in the absence or presence of I mM CN- affected (+)-[3H]isradipine binding in 48 hrs cultured adult rat ventricular heart cells (HC). Such incubations of HC have been shown to reduce intracellular ATP by 90%. Control HC were incubated for 2 hrs in glucose-buffer, 02 gass. (+)-[3H]isradipine was added concomitantly during (group 1) or after 2 hrs incubation (group 2). The table shows the effects on 1 nM (+)-[3H]isradipine binding: Cells/% bindin~ ~roup I $roup 2 Control 100 100 Hypoxia 90+9 82+21 Hypoxia and CN- 117+23 127+27 The binding was not significantly altered by hypoxia in the absence or presence of CN- compared to control in either group (mean+SEM, n=4). The difference in controls in the groups was not significant. In conclusion, 2 hrs hypoxia with or without CN- had no effect on (+)-[3H]isradipine binding in HC in either group. Thus, longlasting ischemia, reducing intracellular ATP, has different effects on LCC in HC and in tissue from in vivo ischemie heart regions. A251

EFFECTS OF THROMBOXANEA, RECEPTOR Frl37 ANTAGONIST SQ29548 ON MYOCARDIAL METABOLISM AND INFARCT SIZE IN DOGS. Shiva D.Seth,Uma Singh, Subhash C,Manchanda*, Departments of Pharmacology & *Cardiology, All India Institute of Medical Sciences, New Delhi-110029, India. In the present study, 22 anesthetized open chest dogs were subjected to left anterior descending coronary artery occlusion for 24h. menty minutes post-occlusion the dogs were given i.v. saline (0.9% NaCl) or the thromboxane A, (TXA,) antagonist SQ2954S (0.2mg/ kg+O.2rq/kg/h i.v. for 4h. Administraticn of SQ29548 (n=lO) resulted in significant (~~0.01) reduction of the myocardial infarct size 24h post-occlusion and a concomitant improvement of the cardiac metabolism when ampared to the control (saline-treated) group (n=12). Myocardial ATP & CP levels and activity of myocardial enzymes CPK & LDH which had been depleted on account of 24h ischemia were significantly restored by this agent, alongwith lowering of the mycardial lactate levels. Further, myocardial TXB, levels were lowered and 6 keto prostaglandin F,dlpha levels were enhanced in the drugtreated

group

as

cunpared

to

the

control

group. Hence, TXA, receptor blockade may have potential as a mode of theraw for ischemiainduced myocardial injury and modulate the actions of cyclic endoperoxides and TXA2

MODIFICATION OF THE CYCLOOXYGENASE Frl39 PATHWAY DURING REPERFUSION AFFECTS PLASMA-MEDIATED NEUTROPHIL STIMULATION Tomasz Siminiak”, David Flu& Julian F. Dye, Desmond J. Sheridan, (*) Academy of Medicine, Poznan, Poland and St. Mary’s Hospital Medical School, London, UK

Leakage Profile of Purine Nucleotides during Reperfusion in an Isolated Working Rat Heart Model. Yoshiaki Fukuhiro, Seiichi Mochizuki’, Takashi Fujiwara, Tatsuki Katsumura. Dept of Thoracic and Cardiovascular Surgery and *Medical Engineering, Kawasaki Medical School, Kurashiki, Japan.

Frl38

The purpose of this sludy is IO evaluate the characreristics of Icakage profile of each purinc nucleotide such as adenosinc(AD0). inosine(lN0) and hypoxanthine(HYP) during reperfusion and to search the relationship with the rccovcry profile of coronary flow in an isolated working(W) rat heart model. (Methods) Hearts were subiected 10 5 min. of LaneendorfHL) and to 20 min. of W-DC& . sion followed by 30 min. if ischemia at 37°C after 3 min. of infusion with St.Thomas’cardioplegic solution(STS). They were subjcclcd 10 L-repcrfusion for i0 min. followed by 20 min; of Wrepcrfusion. During pre-and posl-ischcmic W-periods, cardiac function such as aortic and coronary flow, heart rate and aortic pressure were measured and expressed as the percent of preischcmic value. Coronary effluent during pre-and post-ischemic L-reperfusion was collecrcd with 5 min. of intervnl for the measuremenl of ADO, IN0 andHYP with usinghigh performance liquid chromnlography. (Rcsulls) Normolhermic ischemia with STS offered the heart damage at the level of almost 50% recovery of function. ADO, IN0 and HYP were not detected during L-rcpcrfusion. Peak concentration of ADO was observed at 5 min. after L-reperfusion with the level of 41.6k2.1 nmolcs/min/g. dry.wt. In contrast with ADO, peak concenrrarion of IN0 and HYP were observed at onset of L-rcpcrfusion with the level of 1335+183 and 734il49 nmoles/minlg.dry.wt. Recovery profile of coronary flow was very similar to leakaec orofile of ADO with the maximum recovery level of 66.8&l .<%.‘These data demonstrated that the leakage profile of ADO was different to IN0 and HYP during L-repcrfusion in an isolated working rat heart model.

SOLUBLE ADHESION MOLECULES ICAMFrl40 AND E-SELECTIN RELiEASE DURING MYOCARDIAL INFARCTION AND CORONARY ANGIOPLASTY Tomasz Siminiak*, Robin M. Egdell, Julian F. Dye, Daniel J.O’Gorman, David Hackett, M. Shahi, Desmond J. Sheridan, (*) Academy of Medicine, Poznan, Poland and St.Mary’s Hospital Medical School, London, UK

Pharmacological interventions which modify cyclooxygenase

pathwayhavebeen shownto exert an effect on accumulation of polymorphonuclearneutrophils (PMN) in ischeamicrepefised myocardium. We investigated whether modification of the cyclooxygenasepathway af%cts plasma- mediated PMN stimulation during repetision. Rabbits underwent 40 min of coronary occlusion followed by 90 min of reperfusion and were treated with saline (n=6), TX receptor blocker (GR3219B; lmg/kg; n=6), TX synthetase antagonist (UK38485; Smglkg; n=6) or aspirin (Aspisol; SOmglkg; n=6). Plasma samples were taken pm-trealment and at 30 and 90 min. after repefision and incubated with washed control PMN. TheexpressionofCD18antigen,a commonp chainof PMN intergins was measured by flow cytometry with the use of a specific monoclonal antibody and phycoerythrin labelled F(ab’), fragments. Percent of CD18 expression by PMN incubated with plasma taken beforeocclusion (*p
Modulationofthe inflammatoryreactionby endothelialcells involves expression of adhesion molecules. To investigate whether myocardialischemiaresults in release of soluble adhesion molecules, we collected peripheral venous plasma samples from 14 consecutive patients with acute myocardial infarction (AMI) at the time of admission, 6 hours, 1 and 5 days later. Additionally, coronary sinus (CS) and femoral artery (FA) samples from 7 patients undergoing LAD coronary angioplasty were drawn before (A) and after (B) the first balloon inflation. Soluble ICAM- (intercellular adhesion molecule) and E-selectin (ELAM-I) levels were measured by ELISA. Mean&SEM, *p
EVIDENCE FOR PLASMA-MEDIATED Fr141 STIMULATION OF NEUTROPHIL ADHESION MOLECULES DURING CORONARY ANGIOPLASTY Tomasz Siminiak*, Daniel J. O'Gorman, Manjit Shahi, David Hackett, Desmond J. Sheridan. *Academy of Medicine~ Poznan, Poland & St. Mary's Hospital, London, UK Activation of polymorphonuclear neutrophils (PMN) in ischaemic/reperfused myocardium is known to result in an increase of the extent of tissue injury. Increased PMN adhesion leads to plugging of coronary capillaries, is involved in the "no-reflow" phenomenon and decreases coronary collateral flow. The purpose of the present study was to verify the possibility of plasma- mediated PMN activation during coronary angioplasty (PTCA). Plasma samples were taken from coronary sinus ostium area (CS), femoral artery (Ar) and femoral vein (Ve) in 12 patients with stable angina pectoris undergoing PTCA of proximal LAD before the procedure fl), immediatelyafter the first balloon deflation (II) and at the end of the procedure-5min aRer last inflation (III). Samples were incubated with washed PMN obtained from healthy donors and the expression of CDI8 adhesion molecule (common chain of integrins) was measured by flow cytometry using specificantibodies. Mean valuesd:SEM,*p<0.05 vs (I) CS (I):41.2+6.6 (II):49.9+7.9' (11I):45.0+7.1 Ar (I):54.2-4:9.7 (1I):62.6+10.8" ('111):54.6+8.7 Ve 0):43.2--1:5.l (II):46.2--L:8.1 (III):45.1±7.2 Conclusion: brief ischaemia during PTCA leads to release of soluble stimuli leading to activation of neutrophils and increased expression of integrin adhesion molecules.

IMPORTANCE OF THE DEGREE AND DURATION OF FLOW Fr143 REDUCTION FOR PRECONDITIONING BY A PARTIAL CORONARY OCCLUSION WITHOUT REPERFUSION. Dirk J Duncker, Monique MG Koning, Erik van Klaarwater, Pister D Verdouw. Thoraxcenter, Erasmus University Rotterdam, Thb Netherlands. We recently reported that a partial coronary occlusion (PCO) immediately preceding a sustained coronary artery occlusion limited infarct size. Now we studied whether (i) the protection against infarction exerted by PC's depends on the severity and(or) duration of the flow reduction and (il) the protection afforded by the PC's varies in the different myocarolal layers. Left ventricular area at risk (AR) and infarct area (IA) were determined for the endo- and epicardial half of the taft ventricle. In control pigs that underwent a 60 min total coronary artery occlusion (TCO) followed by 120 min of reperfusion (Rep) there were highly linear relations between IA and AR in the endocardium (r=0.98, p<0.001) and epicardium (r=0.97, p<0.001). Two groups of pigs were subjected to either a 30 or 90 min 70% reduction in coronary blood flow (FR) immediately preceding the 60 min TCO and 120 min Rep, without intervening reperfusion. A 30 min 70% FR decreased infarct size to the same degree in the endo- and epicardial half (both p<0.01). A 90 min 70% FR resulted in greater protection in epi- than in the endocardium (both p<0.01), likely because the 90 min 70% FR alone (without 60 min TCO) already caused some infarction which was larger in the endothan in the epicardium (p<0.01). Infarct size in pigs subjected to either a 30 or 90 min 30% FR prior to the 60 min TCO was identical to that in control pigs. In conclusion, 70% but not 30% FR protected against infarction produced by subsequent 60 min TCO. Trensmurel distribution of infarct size limitation by a 70% FR depended on the duration, as a 30 min 70% FR produced similar decreases in infarct size in the endo- and epicardium, while a 90 min 70% FR preferentially limited epicardial infarct size. This suggest that perfusion abnormalities immediately preceding a coronary occlusion in patients can modulate infarct size.

HYPERDYNAMIC CONTRACTILE RESPONSE DURING Fr142 EARLY REPERFUSION IN THE ISOLATED BLOODPERFUSED PAPILLARY MUSCLE Jonathan IA Miller and Michael J Shattock. Cardiovascular Research, St Thomas' Hospital, London. There is increasing evidence for the role of calcium overload in contractile and electrical dysfunction during early reperfusion. We have characterised functional recovery, during the early seconds of reperfusion, in the vascularly bloodperfused rabbit papillary muscle. Right-ventricular muscles were isolated with a portion of the septum, the septal artery cannulated and perfused with bovine washed red cells (Hct 35%) in a modified Krebs-Henseliet buffer. Muscles were stimulated at 1.5Hz, impaled with conventional (3M KCI-filled) microelectrodes for action potential recording, and isometric tension measured. Muscles were subjected to 10, 20, 30, or 40rain zero-flow ischaemia in a N2 atmosphere (35°C) and then reperfused. A hyperdynamic phase of contraction was observed during early reperfusion which was most pronounced in muscles reperfused after 20rain ischaemia (see figure). Developed tension then fell before recovering to achieve a steady level after 40-60 rain reperfusion of 87.5_+12, 52.8_+11, 49.8_+3 or 38+_2% respectively. This hyperdynamic phase in early reperfusion may be obscured in other models of ischaemia where contractility is I¢ Ischaemia more difficult to assess. This model, in which action potential and metabolic changes have also been characterised, may contribute to the understanding of cellular injury 5mn during early reperfusion.

LACK OF A ROLE FOR OSMOTIC SHOCK Fr144 IN LETHAL ISCHEMIA/REPERFUSION INJURY TO ADULT CARDIAC MYOCYTES Donna R. Trollinger, Hisayuki Ohata, Wayne E. Cascio* and John J. Lemasters. Depts. of Cell Biology and *Medicine, University of North Carolina, Chapel Hill, NC 27599-7090, U.S.A. Accumulation of metabolites increases tissue osmolarity during myocardial ischemia. Relative osmotic shock has been proposed to cause osmotic lysis and cell killing during reperfusion (Eur Heart J 4(Suppl. H), 123-137, 1983). To test this hypothesis, adult rabbit cardiac myocytes were exposed to 5 mM NaCN and 20 mM 2-deoxyglucose at pH 6.2 to mimic the ATP depletion and acidosis of ischemia. Cell death was determined by propidiurn iodide labeling, and changes in cell shape were observed by bright.field microscopy. After 30 minutes, myocytes began to shorten. More than 50% of cells were hypercontracted after 45 minutes. Hypotonic shock was imposed by decreasing osmolarity to 150 mOs/kg after 45-120 rain. Cell swelling and bleb formation occurred within minutes, but cell killing was not accelerated. Similarly, when cells were exposed to hypertonic (450 mOs) buffer during NaCN, subsequent hypotonic shock did not precipitate cell killing. These results suggest that osmotic shock does not cause cell death to cultured cardiac myocytes during ischemia/reperfusion injury. A253

EFFECT OF DOBUTAMINE ON Fr145 HEMODYNAMICS AND ENERGY METABOLISM IN STUNNED AND NORMAL MYOCARDIUM Kazuhiko Umetsu, Eiichi Geshi, Takashi Katagiri, Louis A. Sordahl*. Third Dept.of Int. Med.,Showa Univ. Tokyo,Japan. *Univ.of Texas Medical Branch,Galvestou,Tx Impairment of energy metabolism is not considered to be a causal factor in stunned heart (SH) since positive inotropic stimulation restores cardiac contractility to control level. Simultaneous measurements of cardiac function (ECG, LVP, CBF, % segment-shortening[%SS]) and 31P-magnetic resonance spectra were obtained in regionally stunned canine hearts (15 rain isehemia-30 rain reperfusion) with subsequent dobutamine (DOB) infusion at 3 doses (2.5, 5.0 & 7.5ug/Kg/min X 15 rain) and compared to normal control (NH). SH exhibited significantly depressed %SS (-22.8-1-57.8% of control) and elevated PCr/ATP ratios (2.8_3-~.55) following 30 rain reperfusion compared to control (%SS=100%; PCr/ATP=I.74_40.24). DOB produced dose-dependent increases in rate-pressure product in both NH and SH. DOB increased %SS in a dose-dependent manner in SH (69, 130 & 148% of control),but had little effect on NH at all three do6ages. PCr/ATP ratios did not change in either Nil or SH during DOB treatment. However, 30 rain post-DOB treatment the PCr/ATP ratio in SH had increased to 3.44-~_.36 due to further increases in PCr and decreases in ATP. These results show significantly different hemodynamic and energy metabolism responses to DOB stimulation in NH and SH. DOB treatment of SH enhances the supranormal levels of PCr and increases ATP loss beyond that produced by isehemia-reperfusion suggesting a significant shift in the energy transfer kinetics of the stunned hearts.

EFFECT OF NUTRITIONAL STATE ON SUBSTRATE Fr147 METABOLISM AND CONTRACTILE FUNCTION IN NORMAL AND POSTISCHEMIC MYOCARDIUM Gh. Montessuit, I. Papageorgiou, I. Tardy & R. Lerch. Cardiology Center, University Hospital, Geneva, Switzerland

The pattern of substrate utilization may influence postischemic myocardial injury. To study the effect of nutritional state, independently of substrate availability, on substrate metabolism and contractile function, hearts isolated from fed and fasted rats were perfused retrogradely with erythrocyteenriched buffer containing 0.4 mM palmitate, 8 mM glucose and 175 p.U/ml insulin. Under control conditions hearts from fasted animals exhibited lower glucose oxidation (33 + 2 (SEM) nmol/min/g wet weight) as compared to hearts from fed animals (84 + 12 nmol/min/g, p=O.O05). Conversely, palmitate oxidation was significantly higher after fasting (59 + 6 vs. 22 + 2 nmoVmin/g, p=O.O003). Hearts subjected to 40 minutes of no flow ischemia exhibited 15 minutes after the onset of reperfusion marked stimulation of glucose oxidation without a difference between hearts from fasted and fed animal (113 + 9 vs 117:1:12 nmol/g/min, respectively). However, oxidation of palmitate remained significantly higher after fasting (47 + 5 vs. 28 + 4 nmol/min/g, p=0.018). The nutdtional state did not influence left ventricular (LV) diastolic contracture during ischemia and the time to onset of contracture. However, 45 minutes after the onset of reperfusion, hearts from fasted animals exhibited significantly lower LV diastolic pressure (50 5:4 vs. 85 + 19 mm Hg, p=O.O08) and higher LV systolic pressure development (33 + 6 vs. 1 2 + 6 mm Hg, p=0.027). Conclusion: 1) substrate selection in myocardium is influenced by the nutritional state independently of circulating substrate levels; 2) glucose oxidation is activated during reperfusion irrespective of the nutdtional state; 3) postischemic myocardial recovery is improved in the fasted compared to the fed state. A2 54

"CROSS-TALK" B E T W E E N SECOND Fr146 MESSENGERS DURING M Y O C A R D I A L ISCHAEMIA AND REPERFUSION A m a n d a Lochner, Ronelle Mouton, Erna Tromp. MRC Experimental Biology Programme; Dept. Medical Physiology and Biochemistry, University of Stellenbosch, Republic of South Africa. Concomitant stimulation of both at- and 8-adrencrgic signalling pathways during ischaemia-rcpcrfusion could potentially lead to intracellular Ca 2. ovcrload. Since inhibitory "cross-talk" occurs during normoxia (l), thc aim of this study was to evaluate the interaction between these two pathways during different periods of ischaemia and rcpcrfusion. Methods: Isolated rat hearts were retrogradely pcrfused for 20 min before (i) 5 - 20 rain global ischaemia, (ii) 20 mln global ischacmia plus 1 - l0 min reperfusion. Hearts were perfused in the absence or presence of prazosin (3 x 10"?M) or propranolol (10-7 M). Resull.5: Increasing periods of ischaemia caused a progressive decrease and increase in tissue inositoltrisphosphate (InsP3) and cAMP levels respectively. Reperfusion caused a timedependent elevation in lnsPj levels and reduced cAMP. Alpha-l-blockade caused a significant increase in cAMP levels during both ischacmia and reperfusion, indicating inhibitory "cross-talk". Conclusions: Evidence was obtained for inhibitory "cross-talk" during both ischacmia and reperfusion which may .serve to protect the myocardium against excessive Ca z÷ overloading.

1. Guse A Met al 1991; J Mol Cell Cardiol t23:1375 - 1382.

gitochondrial enlargement during reperfusion Fr148 a f t e r global c a r d i a c ischemia Kajihara, H., Takagi, A., & Nakayama, H., Dept. of P a t h . , l n s t . Health S c i . , Hiroshima Univ. Sch. Med., Hiroshima, Japan

Enlarged or giant mitochondria are f r e q u e n t l y observed in the myocardial c e l l in various pathologic conditions. There is, however, l i t t l e information about morphogenesis of mitoehondrial enlargement. In this study, 28 pairs of adult dogs, weighing 8-15 kg, were used. The heart of one member of each pair was removed, subjected to global ischemia of 30, 60, 90, 120 or 150 min, and then reperfused with whole blood of the partner dog. Restoration of heart beat was observed in 100~ of 30 and 60 min ischemia, in 57~ of 90 min ischemia, and in 33% of 120 min ischemia. But r e s t o r a t i o n was not seen in the h e a r t s of 150 min ischemia. In u l t r a s t r u c t u r a l s t u d i e s , f u s i o n of n e i g h b o r i n g p a i r s of m i t o c h o n d r i a was f r e q u e n t l y observed in myocardial c e l l s of h e a r t s with 60 min ischemia. A f t e r 90 to 120 min ischemia, as a r e s u l t of m i t o c h o n d r i a l f u s i o n , l a r g e e l o n g a t e d m i t o c h o n d r i a were u s u a l l y observed in i n t e r m y o f i b r i l l a r spaces. In the h e a r t s of 150 min isehemia, the m i t o c h o n d r i a were s e v e r e l y i n j u r e d and f u s i o n was not found. These r e s u l t s d e s c r i b e d above i n d i c a t e t h a t e n larged or g i a n t m i t o c h o n d r i a were formed mainly by f u s i o n of o r d i n a r y mitochondria.

SWELLING, HYPERCONTACTURE AND MECHANICAL Fr149 FRAGILITY IN REOXYGENATED CARDIOMYOCYTES. Marisol Ruiz-Meana, David Garcfa-Dorado, Miguel A. Gonz~lez, Jos6 A. Barrab~s, J. Soler-Soler, Hospital General Vail d'Hebron, Barcelona, Spain. Cell viability was assessed (Trypan blue test) in freshly isolated, adult rat myocytes submitted to 60 min of metabolic inhibition (M.I., NaCN 2 mM), and then to reoxygenation with either normoosmotic (312 mOsm) or hypoosmotic (80 mOsm) medium. Rreoxygenation induced hypercontracture in 85 + 2% of cells in both groups, but 74 + 8% of cells undergoing normoosmotic reoxygenation remained viable vs 26 + 4% with hypoosmotic reoxygenation. Sarcolemmal rupture did not occur when hypercontracture was prevented with 2,3 BDM 30 mM during hypoosmotic reoxygenation. In cells not previously submitted to M.I., hypercontracture induced by exposure to hypoosmotic, high Ca2 + buffer (Ca2 + 20mM) did not result in sarcolemmal rupture (viability = 99%). Delaying osmotic stress 20 or 40 rain after the onset of reoxygenation did not result (p=NS) in improved viability (19.3 + 3.9 and 34.9 + 1.3% respectively). When hypercontracture was delayed with 2,3 BDM during the first 20 or 40 rain of reoxygenation, less cells hypercontracted upon exposure to BDM free-hypoosmotic medium (58 + 7 and 12 + 2% respectively), but the % of hypercontracted cells remaining viable did not increase (25 and 27%, respectively). Conclusions: 1) Swelling and hypercontracture may cooperate to disrupt the sarcolemma of mechanically fragile myocytes during reoxygenation 2) Fragility persists for at least 40 min after restoration of metabolic activity.

INDUCED ACTIVITY OF THE SKELETAL Fr151 c¢-ACTIN P R O M O T E R IN I S C H E M I C / REPERFUSED MYOCARDlUM. Patricla McDonald, MarUn N. Hlcks 1, Keith A. Webster2, Andrew Wylie, Stuart M. Cobbe 1, Nanette H. Bishopric 2 and Howard Prentice. Department of Genetics and Department of Medicine and Therapeutics, University of Glasgow, 1Depertment of Medical Cardiology, Royal Infirmary, Glasgow, UK and 2Division of Life Sciences,

SRI International, Menlo Park, CA, USA. DNA transfer into the myocardium by direct DNA injection has been valuable in assessing tissue specific and inducible promoter activities and has been extended to measurements of foreign gene expression in ischemic/ reperfused (IR) myocardium. The immediate-early genes c-los and c-jun and the skeletal (z-actin (sAct) gene were introduced in neonatal and adult cardiac myocytes in response to either adrenergic mediated hypertrophy or hypoxia in vitro. In extension of these studies we employed direct DNA injection into the myocardium to assess the effects of transient ischemia on sAct promoter activation, sAct promoter constructs were injected into rat heart in vivo after a 15 minute coronary ligation and raperfusion. Our data indicated that the full length sAct promoter -2000SA was activated 3.5 fold at 7 days after ischemia/raperfusion by comparison to levels obtained from sham operated control animals. By contrast the truncated construct -1282SA was not activated under these conditions. The induction of full length sAct promoter activity probably represents a transcriptional induction mediated by cardiac remodelling in response to ischemia. Thus the domain from -2000 to -1282 mediates long term transcriptional activation of the sAct gene in IR myocardium.

EFFECT OF 2,3- BUTANEDIONEMONOXIME (BDM) ON Fr150 ISCHEMIC CONTRACTURE(IC) AND REPERFUSIONINJURY Hiroshi Hasegawa, Masato Tani, Ken Shinmura, Yoshirou Nakamura. Dept of Geriatric Med, Kelo Univ., Tokyo,Japan IC deterioratesmyocardialdamage during ischemia/repedusion. IC is related to crossbridge formation and/or rigor- bond formation. We investigated whether IC could be attenuated by BDM, a reversible desensitizer of contractile protein to Ca", resulting in improvement of recovery of function after reperfusion. [Methods] Isolated rat hearts were subjected to 30 min of Iowflow ischemia (0.3 ml/min) and 30 rain of reperfusion. In the BDM group K-H buffer containing5 mM of BDM was used during ischemia while the buffer without the agent was used in the control group. Left ventricular (LV) pressure, dP/dt, and energy metabolltes and myocardial Ca" uptake were compared between the groups. [Results] There were no difference in myocardial levels of high energy phosphates (HEP) after ischemia between the groups. However, the rise in LV diastolic pressure during ischemia was significantly less in the BDM group (11.6 vs. 28.6 mmHg). Although there were no significant difference in recoveries of indices of LV systolic function between the groups after reperfusion, peak negative dP/dt recovered better (54.0 vs. 81.0%), the rise in LV end- diastolic pressure was reduced (25.5 vs. 38.6 mmHg), and myocardial "Ca" uptake were reduced (6.5 vs. 1.3 IJmol/g dwt), and recovery of HEP was improved (ATP: 26.4 vs. 14.3, CP:49.0 vs. 36.6 ijmol/g dwt) in the BDM group after reperfusion. [Conclusions] Our results indicated that IC was reduced by BDM without any changes in HEP after ischemla, suggesting that IC is caused, at least in part, by the crossbridge formation due to rise in [Ca"]. We concluded that reduction of the sensitivity of contractile protein to Ca" could attenuate myocardial damage during ischemia- reperfusion.

EVIDENCE FOR A PROTECTIVE EFFECT OF VASOACTIVE INTESTINAL PEPTIDE (VIP) UPON

Fr152

ISCHEMIC AND REPERFUSED RAT HEART John E. Baker, E. Jack Baker, *Sami I. Said & Bjarne K, Semb. Medical College of Wisconsin, Milwaukee, WI, USA; *University of Illinois at Chicago, Chicago, IL, USA We recently showed that VIP protects the lung against oxidant injury. We have now assessed whether VIP can protect the rat heart from ischemic/reperfusion injury. Isolated rat hearts (n=6/group) were peffused isovolumically in the Langendorff mode with bicarbonate buffer and left ventdcular developed pressure (LVDP), heart rate (HR) and coronary flow rate (CFR) were recorded during steady-state conditions. Heads were perfused for 3 min with VIP (1,7,10 and 50 I~g/ml) prior to 17 min global ischemia at 37°C, followed by 60 min aerobic reperfusion in the absence of the peptide. Recovery (mean + SD) of LVDP, HR and CFR in heads not treated with VIP was 60 + 4%, 88 + 9% and 61 _+4%, respectively, of pre-ischemic values. In VIP-treated heads recovery of LVDP improved to a maximum of 78 +_3% during early (10-40 min) reperfusion with 7-50 I~g VIP/ml (p < 0.05) but then declined to control values by 60 rain. Recovery of CFR was consistently (p < 0.05) better (90 + 3%) throughout the entire reperfusion period ,with VIP treatment (7-50 p.g/ml). Post-ischemic creatine kinase leakage in control heads (52 + 5 IU/g) and recovery of HR were unaffected by VIP. We conclude that VIP exerts a protective effect on functional recovery of isolated rat heads following ischemia.

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F O R M A T I O N OF 8 - H Y D R O X Y G U A N I N E IN C E L L U L A R DNA F R O M I S C H E M I A -REPERFUSED HEART OF RAT M y u n g - s u k Kim, Ho-jin You, J o n g - w a n Myung-hee Chung, Dept. of Pharmaeol. National Univ. Coll. of Med. Seoul, Korea

Fr153

Park, Seoul

As an index of oxidative damage of myocardial DNA, formation of 8-hydroxyguanine moiety has been studied in c e l l u l a r DNA extracted from the isolated, ischemia-reperfused

hearts of rats.

The amount of

8-hydroxyguanine(8-OH-Gua) was measured in the enxyaatically digested DNA by using HPLC-ECD method. The isolated hearts perfused by Lengendorff's method were exposed to 30 rain or 60 rain global ischemia followed by 30 rain reporfusion with the oxygenated VJ'ebs-Henseleit solution. The 8-OH-Gua contents of DNA isolated from the ischemia-reporfused hearts were three to five times higher then those from the normoxically perfused hearts(3.12~3.75 8-OH-Gua rasidue/lO s dGua). The oxygen free radical scavengers, SOD end mannitol which were administered before ischamia end throughout reperfusion period caused a decrease in the amount of 8-OH-Gua formed. Treatment of rats with a xanthine oxidase i n h i b i t o r , allopurinol also caused a decrease in the formation of 8-OH-Gua. These results indicate that 8-OH-Gua is formed in c e l l u l a r DNA of the ischemia-reperfused hearts and suggest that oxidative damage of myocardial DNA mediated by oxygen free radicals is produced during the process of ischemia and reperfusion in the hearts.

ARRHYTHMOGENICACTION OF THROMBIN Fr155 DURING MYOCARDIALREPERFUSION. AlexanderN. Jacobsen, Kim A. Lambert, Xiao-JunDu, Elizabeth A. Woodcock, AnthonyM. Dart. Baker Medical ResearchInstitute, Melbourne,Australia

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Myocardial reperfusion causes arrhythmias, especially ventricular tachycardia (VI') and fibrillation (VF), associated with a rapid, transient release of inositol(1,4,5) trisphosphate (Ins(1,4,5)P3) (Anderson, Dart, Woodcock Circ.Res. 76: 261-268, 1995). The current study investigated the direct pro-arrhythmic effects of thrombin (10 lU/min) during early reperfusion. VT and VF durations were measured following 20 rain regional ischaemia in catecholamine depleted perfused rat hearts (n=34). Thrombin was arrhythmogenic (VF time 138 sec vs. 6 sec in reserpinised control, p
CONTRACTILE RESERVE OF POSTISCHEMICFr154 MYOCARDIUM: COMPARISON OF DIFFERENT STIMULI Hans Martin Hoffmeister, Markus Str6bele, Martin E. Beyer, Ludger Seipel, Medical Department III, University of Ttibingen, Germany. Stunned myocardium is characterized by a prolonged reversible contractile dysfunction, but can be stimulated by positive inotropic stimuli. Using isovolumetrically beating isolated rat hearts we investigated the max. contractile reserve during different pharmacological stimuli (dopamine, Ca2.) and compared the effects on stunned myocardium (after 30min. ischemia) and on non ischemic myocardium. The hearts were stimulated with increasing doses until the maximum contractile response (contractile reserve) was obtained. Prior to the stimulation LVP and dp/dtmax were reduced to 80% of control data in the stunned left ventricles (n=8/group). LVP dp/dtmax coronary flow noniseh, dopamine 173+8% 251+19% 142+11% nonisch. Ca z+ 164+7% 183+ 8% 112+ 7% postiseh, dopamine 114+4%* 154+13%* 113+13% postisch. Ca 2+ 120+5%* 141+ 9%* 99+13% (means + SEM in % of preischemic data; * = p < 0 . 0 5 ) Stunned myocardium has a reduced contractile reserve and cannot be stimulated to the same amount as non-ischemic myocardium independenly of the stimulus used. Conclusion: The contractile reserve of stunned myoeardium is considerably reduced indicating the direct injury of the contractile apparatus. This reduction in contractile response is independent from the mode of action of the pharmacological stimulus used and thus not a receptor mediated phenomenon.

EFFECT OF PLA2 INHIBITORS ON POST-ISCHEMIC Fr156 RECOVERY IN ISOLATED RAT HEARTS. Gulzar S. Sandhu, Althea C. Burrier & Jay K. Thakkar. Research Dept., Pharmaceutical DIvslon, CIBA-GEIGY Corp., Summit, NJ, USA. Isolated hearts were perfused with Krebs Henseleit Buffer (KHB) for30 minutes, then with KHB+ qulnacrlne or KHB+ (E)-6-(Idodomethylene)tetrahydro-3-(1-naphthalenyl)-2Hpyran-2-one (Iodoenol lactone) for 15 minutes, and subjected to normothermlc Ischemla for 30 minutes. FlnaUy, hearts were reperfused for 60 minutes (with respective agent In treated groups). Coronary effluent samples were collected before and after Ischemia for determination of creatine klnase (CK) release from hearts. Sroup Qulnacrlne Iodoenol lactone +dP/dt CK +dP/dt CK % of PI units/mln % of PI units/rain Vehicle 56.21 0.88 50.9 0.74 3.1 pM 50.8 0.17" 1.0 p M 54.77 1.06 28.2* 0.00" 3.0 p M 67.62* 0.77 20.4* 0.00" 10.0 p M 82.37* 0.44* PI, pre-lschemlc; *, p < 0.05 vs Vehicle Qulnecrlne enhanced post-ischemlc contractile function and amellorated CK release during reperfuslon In a dose dependent manner. Iodoenol lactone completely blocked CK release, but compromised post-lschemlc contractile function. The effect of Iodoenol lactone on CK release was not due to direct inhibition of CK or interference with the assay. These data suggest that the mechanism of action of the two agents is different, and while Iodoenol lactone might have membrane protective effects, it does not have antl-lschemlc properties.

THE ROLE OF LIPOXYGENASE PATHWAY IN Fr157 PATHOGENESIS OF CARDIAC INJURIES A ~ X d l A.Molbenko, Gennady A.Cherednichenko, Vera N.Kotsuruba, Galina I.Marchenko, Vladimir I.Azarov, Victor M.Brovkovlch, LesJa V.Tumanovskaya. Inst. of Physiology, Kiev, Ukraine. The aim of the present study was to determine the role of llpoxygenase (LO) activation in development of Ischemla-reperfusion (IR) and immune (ID) damages of the heart. We elaborated a new model of IR reproduced by double catheterlsation of left coronary artery in anestheti~ zed dogs. ID was produced by intracoronary inJection of anticardlac antibody. We observed progressive elevation of LTC4-LTE4 level in coronary blood during IR and ID; in case of ID these changes correlated with coronaroconstrlctor reaction. Preliminary inhibition of LO by llnoleylhydroxamic acid (3 mg/kg), by soluble form of quercetin (10 mg/kg) or by membranostabillzer phosphocreatlne (500 mg/kg) prevented immunogenlc coronaroconstrlction, increase in sarcolemmal permeability for Ca ions and hypercontraction of myoflbrills. Inhibition of LO in IR attenuated the increase in blood LTC4-LTE4 level but did not decrease elevated level of induced chemiluminescence ~n blood from ischemlc region. LO inhibition also largely prevented decrease of retrograde collateral flow and decreased infarct size. We suggest that activation of LO pathway plays an important role in pathogenesis of cardiac injuries.

Fr159 NITRIC OXIDE SYNTHASE INHIBITION ATTENUATES ISCHAEMIC AND REPERFUSION INJURY IN THE ISOLATED PERFUSED RAT HEART MODEL. Eugene du Toit, Jody Miyashiro, Joy McCarthy and Lionel OpieMRC Heart Research Unit, Univ.CapeTown, S.Afdca.

Hypothesis: We hypo!hesise that: 1) elevated cAMP levels contribute to Ca2 ovedoad and worsen ischaemic and reperfusion injury, and 2) that stimulation of the NOcGMP pathway may be protective and decrease these forms of myocardial injury. Methods: To investigate the effect of manipulation of the cAMP a n d NO-cGMP pathways on the sevedty of injury, we used the isolated rat heart model with a LV balloon. Heads were perfused dudng 25 min low-flow ischaemia (0.2 ml/min) with a ~agonist dobutamine (Dob), or one of two nitdc oxide synthase inhibitors, N*-nitre-L-arginine (LNA) or N~-nitre-Larginine methyl ester (L-NAME) or membrane permeable 8-Bromo-cGMP. Percentage ischaemic contracture (% of pre-ischaemic developed pressure) and the reperfusion rate-pressure product (RPP) was monitored. Results: (* p<0.05)(n=5-10) Treatment % Contracture % RPP Recover]/ Control 46.5:P.2.8 28.0+4.6 Dob (10 -= M) 65.3.+.2.5 * 19.4+5.9 * LNA (104 M) 24.2_+6.8 * 74.1+13.2 * cGMP (104 M) 49.2:1:3,2 28.6:L-6.9 L-NAME data are inconclusive. Conclusions: These data show that ~-agonism exacerbates and NO-cGMP pathway inhibition attenuates ischaamic and reparfusion injury. Exogenous cGMP administration has no effect on ischaemic or reperfusion injury. These findings argue against a protective role for the NO-cGMP pathway in ischaemic and reperfusion injury.

INVERSE REGULATION OF ACETYLCHOLINESTERASE Fr158 ACTIVITY AND ITS mRNA LEVEL IN CHOMNERGIC JUNCTIONS OF ISCHEMIC/REPERFUSED RAT HEARTS. Ern6 Z6dor, P6ter Ferdinandy, Csaba Csonka, Tam/~s Csont, L/=szl6 Dux. Dept Biochem, Szent-GyOrgyi Univ., Szeged, Hungary. The aim of this study was to investigate the histochemical activity of acetylcholinesterase (ACHE) and its mRNA level in the ventricles of normal and ischemic rat hearts. Isolated working rat hearts (n=4 in each group) were subjected to 30 min global ischemia followed by 10 min reperfusion. Slices of the ventricles were than stained histochemically for AChE activity, and in situ hybridization were performed with the 5' end Eco RI fragment of the Torpedo californica AChE gene showing extensive homology with the rat AChE cDNA. The AChE mRNA was accumulated in the presynaptic nerve endings and right beneath the postsynaptic membrane of myocytes. This pattern partially overlapped the histochemical AChE activity along the neural fibers. Ischemia/reperfusion decreased the number of AChE positive fibers from the control 88.5+10.4 to 30.9.+6.1 signal/transversal section (p
AN NMR STUDY OF HIGH K PROTECTION AGAINST Fr160 ISCHEMIA-REPERFUSlON INJURY IN THE RAT HEART "Shang-Gong Chen, John C Docherty & Roxanne Deslauriers, National Research Council Canada, Winnipeg, Canada and "Fuwai Hospital, Beijing, China Isolated perfused rat heads were subjected to 40 min total global ischemia at 37°C followed by 40 min raperfusion. Left ventricular end diastolic pressure (LVEDP) and developed pressure (LVP) were recorded and the intracellular pH, Pi, PCr and ATP contents were monitored by =P NMR. In the control group (n--4) activity ceased after 2-3 min ischemia and after 15 min the LVEDP rose, reaching a peak of 70-100 mm Hg which was maintained following reperfusion. Intracellular pH (pH~ of the heart decreased rapidly from 7.2 to 5.75 and remained low throughout ischemia, pH= recovered within 2.5 min following initiation of repeffusion. PCr and ATP declined rapidly and did not recover upon reperfusion. In the test groups, after administration of 5 ml of 4.7 mM W (n = 5), 16 mM K+ (n = 5) or 22 mM K+ (n = 5) in Krebs-Henseleit buffer (no O=, no glucose) as a bolus at 5 min ischemia, pH~ rose and remained above pH 6.75 until reperfusion. Mechanical activity resumed soon after reperfusion with a decrease in LVEDP to levels lower than observed in the reperfused controls. Recovery of the product of HR x LVP in the 22 mM IC group was 80% in contrast to the 0%, 20% and 20% observed in the control, 4.7 mM K+and 16 mM K* groups respectively. The enhanced recovery of function observed in the treated groups appears to be due to a "flushing" effect leading to a partial reversal of the ischemic intracellular acidosis and an energy sparing effect. In addition to this flushing effect, the 22 mM K° treatment conferred additional, and more profound, cardioprotection by other mechanisms, possibly related to improved ionic homeostasis, particularly Na° and Ca=*.

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EFFECTS OF TRANDOLAPRILAT, AN ACTIVE FORM Fr161 OF ACE INHIBITOR TRANDOLAPRIL, ON ISCIIEMIA AND REPERFUSION INJURY OF ISOLATED RAT IIEARTS Satoshl Takeo, Toru Kamlyama, Aya Takezono & Klmlko Sakal. Dept of Pharmacology, Tokyo University of Pharmacy and Life Science,

t l a c h i o j i , Japan. The e f f e c t s of t r a n d o l a p r l l a t , an a c t i v e form of ACE i n h i b i t o r t r a n d o l a p r i l , on ischemia and r e p e r f u s l o n i n j u r y were examined. Rat hearts were s u b j e c t e d to 25-mln lschemla and subsequent 60-mln r e p e r f u s i o n . Ischemia and r e p e r f u s l o n r e s u l t e d In l i t t l e recovery of c a r d i a c c o n t r a c t i l e f o r c e , r e l e a s e of c r e a t i n e klnase and ATP m e t a b o l i t e s into the p e r f u s a t e and d e p l e t i o n of t i s s u e high-energy phosphates. Treatment of the heart with 30 to 100 ~mol/L t r a n d o l a p r i l a t during 10-min of the prelschemlc period r e s u l t e d In an enhancement of the postischemlc c o n t r a c t i l e recovery and suppression of the r e l e a s e of c r e a t i n e kinase and ATP m e t a b o l i t e s during r e p e r f u s i o n . This was a s s o c i a t e d with an a p p r e c i a b l e r e s t o r a t i o n of t i s s u e ATP and c r e a t i n e phosphate of the reperfused h e a r t . The f u n c t i o n a l improvement of the t r a n d o l a p r i l a t - t r e a t e d heart was v i r t u a l l y abolished by treatment with flOE140, a bradyklnln a n t a g o n i s t . The r e s u l t s suggest t h a t b r a d y k l n i n - l i k e a c t i o n plays a r o l e in the enhancement of postischemic c o n t r a c t i l e recovery by t r a n d o l a p r i l a t .

INTERMrI-I'ENTGLOBALISCHEMiA-REPERFUSION Frl 63 IS DELETERIOUSTO CORONARYVASCULATURE WHILE IT PRESERVESTHE CONTRACTILE FUNCTION OF PERFUSEDRATHEART. J.S. Juggi, and A. Prahash,Departmentof Physiology,Facultyof Medicine,KuwaitUniversity,Kuwait(A. Gull). With the increasingawarenessabout tha cardioprotectiverole of ischemicpreconditioning,it was of interest to re-evaluatethe role of intermittentglobal ischemia-reperlusionand to compare it with ischemic preconditioningand hypotharmiccardioplegia.Isolated aorta perfused rat hearts were divided into four groups. GroupA: Ischamia (ISI, 34"C, 60 min); Group B: Ischemic-preconditioning (PC, 2 cycles of 5 min global ischemia 34"C and 10 min raperfusion,34"C)followedby hypothermicischemia(HI, 10"C, 60 rain); GroupC: Intermittentglobal ischemia-repertusion(IT-ISI, 6 cycles of 10 min ischemia,34'C and 10 rain reperfusion,34"C); Group D: Hypothermiccardioplegia (CPL+HI; 'Plagisol', 2 rain pertusion followed by HI). Post-ischemic recovery in left ventricular (LV) contractility (Pmax; CI, contractility index; SS, segment shortening) and coronarydynamics, (CF, coronarytlow; CVR, coronaryvascular resistance;MA, mogenicautoregulation), was compared(table). Prna~ ISl 39 PC+HI 68 IT-ISI 85 CPL+HI 92

%Post-lschemicRecovery CI SS LVEDP CF CVR 31 19 747 51 209 100 55 71 62 173 100 69 100 58 173 100 108 102 83 129

MA 55 78 89 192

As compared to ISI, post-ischemicrecoveryin LV contractilityand -.coronarydynamicswas significantlybetter with PC+HI, IT-ISI and CPL+HI. There were no significant differences in recovery between PC+HI and IT-ISI groups. Global recoveryin contractile functionbetweenIT-ISIand CPL+HIwas not different,but CPL+HI offered better protection to coronary vascular dynamics. These results indicate near identical preservation with PC+HI and IT-ISI and a superior preservation with CPL+HI. Supported by KuwaitUniversityresearchgrant MY021.

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EFFECTS OF ATENOLOL AND PACING ON Fr162 PERFORMANCE AND METABOLISM OF THE ISCHEMIC-REPERFUSED RAT HEART. Sonia Allibardi, Silvia Sommaruga, Stefania Casalini, Giampiero Merati, Gabdele Fragasso, Michele Samaja, and Sergio L. Chierchia. Scientific Institute H San Raffaele, University of Milano, ITALY. We assessed the effects of atenolol and pacing on recovery after low-flow ischaemia (I) of isolated hearts. Rat hearts were perfused with Krebs-Henseleit medium at costant coronary pressure (CP = 100 mmHg) and exposed to I for 30 min (CP=10 mmHg) followed by reperfusion for 20 rain. Hearts were freeze-clamped at end of reperfusion to determine high-energy phosphates (HEP) and their metabolites by HPLC methods. Three groups were considered: C (controls, n=17); A (2.5 lag/l atenolol in the medium, n=lJ); P (as C but with electrical pacing during I to assess the effect of energy demand in the absence of ~-blocker, n=lJ). During I, heart rate (HR.) and the systolic pressure x HR product (PSPxHR) were higher in A and P than in C (p<0.02). During recovery, PSPxHE and HEP were higher in C than in A and P (p<0.05). We conclude that atenolol and pacing depress recovery after I in this model likely by increasing HR and energy demand during I and suppressing the protective effects of downregulation during I. The reason of higher HR in A remains unclear. Supported by BTBS Target Project, CNE, Roma, Italy.

THE SPECIFIC BRADYCARDIC AGENTS Fr164 AND ISCHEMIC MYOCARDlUM. G.G. Chichkanov, I.B. Tsorin & G.Yu. Kirsanova. Institute o f Pharmacology RAMS, Moscow, Russia In benzimidazole series there were found the novel compounds SM-266 and SM-345, at a dose of 1-2 mg/kg i.v. they are able to cause a pronouncecl bradycardia and do not actually change the arterial pressure and heart contractility. Electro-physiological studies showed that these compounds belong to the novel group of drugs - "specific bradycardic agents"-, and since the induced bradycardia is associated with the direct action of the compounds on sinoatrial node cells and slow down the prolonged diastolic depolarisation. The compounds produce the significant antiischemic action. They decrease ST segment elevation on epicardial electrogram during the 5-rain occlusion o f coronary a r t e r y in cats and makes haemodynamics parameters steady during 20- and 40-rnin occlusion with 30- ani:l 60-rain reperfusion of coronary artery accordingly. In the 40-rain occlusion and 60-min reperfusion o f coronary a r t e r y in cats the compound SM-345 was }'ound to increase ATP concentration in myocardium o f the ischemic area. The compounds under study exert a substantial antiarrhythmic effect. They were found e f f e c t i v e in models o f adrenaline and chloridecalcium arrhythmias, Harris model and produced a n t i f i b r i l l a t o r y action in rats with occlusion and reperfusion of coronary artery. Thus it can be assumed that the compounds found show strong promises f o r further studies.

MEASUREMENTOFELEMENTCHANGESAND RECOVERY Fr165 OF FUNCTION AFTER ISCHEMIA/REPERFUSION. Clare Marchant, Alice Warley 1, David J Chambers. CardiacSurgicalResearch,The RayneInstitute,1PhysiologyDept, UMDS, St Thomas' Hospital, London, UIC Disturbance of ionic homeostasis may contribute to ischemia/ reperfusion injury in the heart. X-ray microanalysis (XRMA) can determine element concentration changes within the cell and subcellular organelles and relate these to structural and functional observations. In this study, isolated rat hearts (n=6/group) were aerobically perfused at 37°C for 20 min and left ventricular developed pressure (LVD P) measured; hypothermic (20°C) global ischemia (I) was induced for 30, 60, 90, 120, 180,240 or 360 rain and hearts reperfused for 30 rain. LVDP (expressed as percent of pre-ischemic values) recovered to 75+_2, 74+3, 65+5, 69!-_5, 71+3, 43+_2and 12_~3%after the respective ischemic durations. Consequently, further hearts (n=5/group) were subjected to ischemia for 15, 60, 90,180,210,240 or 360 min, freeze-clamped at the end of 30 min reperfusion and analysed by XRMA for [Na] and [Ca] (expressed as mmol/kg dry wt) in "whole cell" (W), cytoplasm (C) and mitochondria (M) areas. [Na] was significantly (p<0.05) increased at 180 min (I) to 335,208 and 191 from 121, 105 and 112 at 90 min (I) in W, C and M, respectively, and further increased to 309 and 285 (C and M) at 210 min (I). W [Ca] was constant up to 180 min (I) when it increased from 4.8 to 12.0; subsequently, M and C [Ca] increased from 3.1 to 8.8 (p<0.05) and from 4.8 to 7.9, respectively, at 210 rain (I). In conclusion, a rapid, but relatively late, rise in [Na] was associated with a similar [Ca] rise; these element changes in the ischemic/reperfused heart appeared to influence functional recovery.

STUNNED MYOCARDIUM IS NOT CAUSED BY CHANGES OF THE EXTRACELLULAR MATRIX PROTEINS

Fr167

Udo Sprenget, Cornelia Delvatle, Margarete Arras, Stefan Sack & Jutta Schaper. Max Planck Institute, Bad Nauheim, Germany The him of this study was to to

test the hypothesis t h a t stunned myocardium is based on disturbances of the ECM (Zhao et al, 1987) and to examir~e the effect of ischemia (isch) on extraceUular matrix proteins (ECM). Samples from stunned pig myocardium (SPM, 2x10' isch, 30' or 240' reperfusion) and normal pig hearts, (NPM) were removed and frozen in liquid nitrogen. H u m a n myocardium (H'M) was used as control for species differences, because of the presence of increased ECM and as a model with more severe in vitro isch. Left ventricular samples from explanted h u m a n hearts (HI-I) with dilated cardiomyopathy were incubated in PBS-buffer at 20°C, removed at 0, 15', 20', 60', 120', and 180' and frozen for immunohietochemistry. C r y o s t a t sections were prepared from SPM and the various samples of HM and NPM and stained with specific antibodies against the interstitial proteins fibronectin, lnmlnin, collagen I, III, IV and VI. NPM and SPM showed a similar, distribution and localization pattern of ECM at' all time intervals. I-IM lacked changes due to isch in the distribution pattern of ECM. It is concluded 1. the ECM proteins are not sensitive to isch and 2. stunned myocardium is not based on disturbance of extracellular matrix.

B E N E F I C I A L E F F E C T OF T E I - 2 3 0 6 IN Fr166 S T U N N E D M Y O C A R D I U M OF D O G S Masatoshi Kanoh, Mildo Ota, Naold Hase, Yasuhide Uelime, Himhumi Tanabe, MamonJ Kiyold, Shim Hoshida 1 & Tsunehiko Kuzuya 2 Pharmaceutical Development Research Laboratories, Tei~n Insllute for Bion'wdical Research, Tokyo, Japan and The First Department of medicine 1 and Depmtment of Pathophysiology 2, Osaka University School of Medicine, Osaka, Japan. .

Myocardial stunning shows reversible but persisted contractile dysfunction. In animal models, some anti-oxidative agents are known to attenuate stunning as long as administered before coronary occlusion. In open-chest dogs undergoing a 15-rnin LAD ligation followed by 4-hr reperfusion, we studied the effect of y-glutamylcysteine ethyl ester (TEI-2306), which is a derivative of glutathione biosynthesis intermediate and can penetrate into cells in its intact form. TEI-2306 was intravenously infused from 5 min after the LAD ligation for 30 rain with a total dose of 100 mg/kg. Regional myocardial function was assessed as thickening fraction (TF) in the severe ischernic dogs ( <0.20 ml/min/g of ischemic region flow ). Control group exhibited little recovery of contractile function in the ischemic region after reperfusion. In contrast, TEI-2306-lreated group showed significant improvement of TF after 3 hr of reperfusion. During coronary occlusion, TF was similar between the two groups. It is indicated that TEI-2306 is effective in scavenging oxygen radicals and replenishing intracellular GSH, thereby enhancing the recovery of myocardial contractile function during reperfusion, even when administered after the occlusion.

ASPIRIN DOES NOT PREVENT THE Fr168 ATTENUATION OF MYOCARDIAL STUNNING BY THE ACE-INHIBITOR RAMIPRILAT Samir G. Sakka, Thomas Ehring, Jochen .Rose., Andreas Skyschally and Gerd Heusch. Dept. of Patlaoplaysiology, Umversity of Essen Medical School, FRG. The attenuation of myocardial stunning by the ACEinhibitor ramiprilat (R) ns mediated by a signal cas,cade of bradykinin and prostaglandins an(/ preventeo oy cvclooxygenase-inhibttion with indometliacin. In the clinical setting of ischemia/reperfusion, however, the cyclooxygenase inhibitor aspirih (A) is widely used to prevent platelet aggregation (PA). The present study therefore investigated whether or not A, ifi a dose sufficient to inhibit-PA, interferes with the attenuation of myocardial stunning by R. Eight dogs served as controls. Fifteen dogs received either "I (n--T) or 10 mg(.(kg.day~ (n=-8) A p. o. for 1 week. Both dosages of b/ inlilbited ADP-induced PA by 73+13% and. b y , 68~25°A, respectively_. The dogs were then anesthetized and sqejected to 15 min leffcircumtlex coronary artery occlusion and subsequent 4 h reperfusion. All dogs received R .(20 ~gLk_g i.v.)" 0rior to occlusion. Posterior mvocaroiaa blood flow (PMBF, colored microspheres) and, systolic wall thickening (PWT% sonomicrometry) were measured during-control, at ]0 min occlusion and at 4 h reperfusion. Mean aortic pressure was k.ept constan,t by an intraaortic balloon. -Heart rate did not change throughout the protocol. PMBF was not different between the 3 ~roups at ~e differe.nt time points of the p.rotocol. PWTr/* was not different between the 3 groups iluring control and coronary occlusion. At 4 h reperfusion PWT% had recovered to 55+20% of the control value '-(R only) to 47±23% (low dose A+R), and to 50-~15% (higli i:loseA+R). . . . . . In coni:lusion, the attenuation oI myocaroiaa stunning bvthe ACE-inh~itor R is not prevented b y A in a dose v~hich js nevertheless sufficient to inhibit PA. A259

EFFECTS OF NIFEDIPINE ON MYOCARDIAL Fr169 STUNNING: ACUTE VERSUS CHRONIC INTERVENTION. Susana M. Mosca, Abel E. Moreyra, Martin Carriquiriborde, Horacio E. Cingolani. Centre de Investigaciones Cardiovasculares, 1900 La Plata, Argentina. Acute infusion of nifedipine (N) is known to prevent myocardial stunning. Since chronic administration of this compound elicited up-regulation of myocardial dihydropyridine receptors in rabbits (Am.J.Physiol.267: H1222-H1226, 1994) and this effect would decrease the response to N, we tested the hypothesis that the prevention of stunning would be blunted after chronic treatment with N. After 15 min of ischemia and 30 min of reperfusion, left ventricular developed pressure (LVDP) and +dP/dtma x in isovolumically perfused rabbit hearts stabilized at 45 +2% and 48+2% of preischemic values, respectively (control group). In the presence of 15 nM of N in the perfusate (group I) or when the rabbits were pretreated with only one single oral dose (20mg)l-4h before isolation (group II), values of LVDP and +dP/dtma x at the end of the reperfusion period were 67+2% and 74+3% (group I) and 63+6% and 66+6% (group II) respectively (P<0.05 compared to control group). When the rabbits were chronically treated with 20 mg N for 50 days (group liD, LVDP and +dP/dtmax stabilized at 50+4% and' 59+5% respectively (NS compared to control). Since stunning is prevented by either infusion of N in the perfusate or a single oral dose, whereas it is not after chronic treatment, we suggest that the up-regulation of L-channels may be the mechanism involved in this phenomenon. Binding studies with 3H-PN 200110, a diminished response to N and an enhanced contractile basal state in pretreated animals, support this position.

ACE inhibitors i.v. reduce post-ischemic myocardial stunning. In the present study, ACE inhibitors were infused i.c. during repetitive occlusion (ROC as in angioplasty) of a stenosed coronary artery. In anesthetized pigs, aortic pressure (AoP), heart rate (HR), stroke volume (SV), left descending ('LAD) and circumflex (LCX) coronary artery blood flow (Q), and myocardial systolic shortening (by sonomicrometry) were monitored. LAD was stenosed (40 rain, distal myocardial power MP reduced to 20%) followed by ROC (5x occlusion lmin, perfusion lmin), and reperfused (90rain). Into the LAD, hydroxy-ethyl-starch (lIES) was infused throughout the experiment (0.5ml/min, n=9), or replaced by lisinopril (LISI 8p.g/min/kg, n=7), or captopril (CAP 171.tg/min/kg, n=8) from 5min before to 5min after ROC. The following mean differences [reperfusion - pre-stenosis] were observed (*p<0.05): A% Q-LAD -LCX MP-LAD -LCX AoP HR SV HES +21" +26* -35" +43* - 3 +26* -12" LISI - 2 - 10 -47" + 9* -12" +17" -12" CAP +33* +30* - 40* +31" - 7 ' +22* - 5 LAD hypoperfusion+ROC caused stunning of LAD-area and a compensatory increase of power of non-ischemic area. LISI or CAP i.c. did neither improve post-ischemic Q as compared to HES nor ameliorate myocardial stunning. This disappointing result is possibly due to the limited time of i.c. infusion, and/or the regional (non-systemic) application of the drugs.

James W. Gaubatz & Susan Rooks. Department of Biochemistry & Molecular Biology, University of South Alabama, Mobile, AL 36688 USA

Fr172 MECHANISMS OF THE HEART AND CIRCULATORY SYSTEM INSUFFICIENT IN ENDOTOXlC SHOCK Ludmlla I. Kvochlna. Dpt Exp. Cardlology, Bogomolets Inst. Physlology, Klev, Ukralne

Cooked food mutagens are heterocyclic amines (HCAs) formed during the ordinary cooking Of meats and fish. Activated HCAs will form DNA adducts and are mutagenic, cytotoxic, and carcinogenic. Recent work has indicated that some HCAs preferentially damage DNA in heart cells. To obtain detailed information about the ability of cardiac cells to sustain function when exposed to HCAs, we have studied the 2 most abundant HCAs produced in cooked meats. Cultured rat neonatal myocytes and non-myocytes were exposed to varying concentraions of N-hydroxylated HCAs for an interval, then cell viability, lactate dehydrogenase leakage, and DNA adduct levels were measured. Cardiac myocytes were significantl y m o r e sensitive than non-myocytes to the cytotoxic, cell-damaging effects of N-0H-HCAs. Myocytes treated with N-OH-HCAs had 3- times more DNA adducts than non-myocytes. Thus, HCAs appear to be more genotoxic to myocytes than to non-myocytes, and this genotoxicity might be the mechanism responsible for the observed cardiotoxicity. (Supported by American Heart Association, Alabama

Relative contribution of cardiac and vascular components, alterations of vascular reactions and heart activities in early period of endotoxic shock (ES), the role of arachidonic acid metabolites (AAM) in the development of that alterations have been investigated in experiments on the dogs and on the isolated heads. There were 3 series of experiments on the mongrel dogs: with lntracoronary administration of endotoxin E.coli (E); with pretreatment of indomethacin (In) (inhibitor of cyclooxygenase) before E; with pretreatment of linoleate-hydroxamatlc acid (LHA) (inhibitor of lipoxygenase), - and 4 series of experiments on the isolated perfused guinea pig hearts performed with perfusion by E or self blood incubating with E. In one series of experiments guinea pigs were preliminary conditioning in vivo with prodigiosin (an activator of REScells) 24 h before cutting a heart. It was determined In the experiments on the mongrel dogs that a different masons have a main significance in the different periods of ES: a blood pool In peripheral capacitance vessels, that Is likely due to PG 12 effect on the vessels; the effect of AAM on the myocardium and peripheral blood vessels. Administration of E increased the plasma content of prostanoids: Tx A2 and PG 12. In and LHA prevent the development of the ES. Preliminary conditioning in vivo guinea pigs caused a significant alterations in the heart activity and in the resistance of the coronary vessels of the isolated heads.

CELL ~ C I T Y

ASSOCIATED W1a~

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O0uK~u F O O D ~

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EFFECT OF INTRACORONARY ACE INHIBITORSFr170 ON POST-ISCHEMIC MYOCARDIAL POWER. Hubert Schad, Werner Heimisch, Nikolaus Mendler & Rudolf Blasini*, Deutsches Herzzentrum & *I. Medizinische Klinik der Technischen Universit~t Mfinchen, FRG

PHARMACOLOGICAL MODULATION OF THE Fr173 DAMAGE INDUCED TO GUINEA PIG VENTRICULAR MYOCYTES BY CYTOTOXlC T LYMPHOCYTES (CTL) IBella Felzen, 2Gideon Berke, 3Phyllis Gardner & IOfer Binah. IRappaport Inst., Technion, Haifa, Israel, 2Weizmann Inst., Rehovot, Israel, Zstanford Univ., Stanford, CA, USA To evaluate possible new immunosuppressive modalities, we studied the pharmaco-modulatlon of CTL-induced damage to ventdcular myocytes (VM). We utilized patch clamp techniques to record membrane potential and currents from a Con A-treated VM conjugated with peritoneal exudate CTL (PEL), as well as in free PEL. In VM conjugated with PEL for 60 min (drug-free experiments), resting potential (Rp) was reduced from -80.9t-0.5 to -72.5±1.5 mV, action potential amplitude (APA), from 134.9±1.9 to 104.2t-6.2 mV, APDso from 731.7+57.8 to 195.3±58.0 ms, and the delayed outward K* current (Ik) (at Vm=+40 mV) was increased from 0.5£-0.07 to 1.18±0.14 nA. While 2xl0"eM verapamil was ineffective, 3 mM COCI2 (Co) and 10 nM margatoxin (Mgx) prevented CTL-induced damage, expressed as myocyte contructure, reduction in Rp, APA and APDso (only Mgx), as well as CTL-induced increase in Ik. We then determined whether this protective activity was due to effects of Co and Mgx on Ik in VM and/or in PEL. Co and Mgx did not significantly affect Ik in VM, but in PEL, decreased Ik by 80%. We suggest that inhibition of ik in PEL, probably required for activation and delivery of lethal hit, is responsible for the protection provided by Co and Mgx against VM damage induced by CTL.

REGULATION OF CONSTITUTIVE NO Fr175 SYNTHASE IN CARDIAC MYOCYTES BY CONTRACTILE ACTIVITY David M K.aye, Laurent B¢lhassen, Jean-Luc Balligand, Ralph A Kelly,Thomas W Smith. Cardiovascular Division,Brigham and Women's Hospital, Boston, MA, USA A constitutivelyactive isoform of nitricoxide synthasc (ecNOS; N®S l[I) is present in adult rat venu'icular myocytes (ARVM), and is important in mediating some of the effectsof muscadnic and adrenergic agonists on myocyte contractileactivity.W e examined the effects of 24hr continual electricalfieldstimulation on N O S Ill gen¢ regulation in A R V M . Control, non-paced A R V M showed a 2 to 3 fold increase in both N O S m R N A and protein content by northern and western blotting studies respectively, over paced ventricular myocytes (which remained similar to those in freshly isolated cells). Similarly, NOS III enzymatic activity (3H Arg to 3H Citmlline conversion) increased 2 fold compared to paced myoeytes. These data indicate that constitutive NOS expression and actvlty is regulated by contractile activity. The mechanism by which this occurs is, however, unknown.

CYTOKINES INCREASE NEONATAL CARDIAC Fr174 MYOCYTE CALCIUM TRANSIENTS. ARE NITRIC OXIDE AND CYCLIC NUCLEOTIDES INVOLVED IN THESE CHANGES ? Roger J. Bick, David E. Wood, Andrea Karoly, Dachun Wang, Anne LeMaistre, Jennie B. McMillin and L. Max Buja. Dept. of Pathology, UTHSC, Houston, Texas Neonatal rat cardiac myocytes were treated with a number of cytokines for varying times. Systolic and diastolic calcium were measured by fluorescence microspectroscopy and scanning laser confocal microscopy (SLCM). With each of the cytokines, tumor necrosis factor (TNF), interleukin 1-alpha (IL-1G), I-beta (IL-.8) and gamma-interferon (y-IFN), systolic calcium levels were raised above basal levels. With y-IFN, the calcium levels were raised abruptly and dramatically (325 ± 50nmol vs 1550 ± 500nmol), and cells would not respond to pacing, but beat rapidly for 2 to 18 hours (115±20 bpm vs 425+77 bpm). Inclusion of, Imethyl arginine (LMNNA), allowed the cells to respond to pacing. Inclusion of LMNNA in myocytes treated with TNF, IL-1,8 and IL-la caused only minor changes in systolic calcium, but in IFN treated myocytes, the systolic calcium was reduced from 1550 ± 500 nmol to 600 ± 180 nmol. A major point of interest, was that co-treatment of myocytes with IFN & IL-I~', caused the cells to respond to pacing. Cyclic nucleotides in acutely (2hr) treated myocytes showed no major changes, except with TNF, where there was a significant rise in cyclic guanosine monophosphate (cGMP) (30pmol/mg/min for control to 65 for TNF treated). Stimulation with isoproterenol and forskolin caused no significant increase in cGMP levels, except in the case of TNF, where isoproterenol caused a 25 ± 5% increase over basal levels.

ARE PHYSIOLOGICAL-INDUCED CHANGES IN Fr176 MUSCLE MYELOPEROXIDASE AND CALPAIN-LIKE ACTIVITIES CORRELATED? Angelo N. Belcastro, Tim Booker and Daniel Raj. School of Rehabilitation Sciences, University of Bdtish Columbia, Vancouver, B.C., Canada, V6T 2B5. Physiological and pathological sUmuli have been reported to induce an inflammatory response in which neutrophil infiltration into muscle tissues is accompanied by changesl in tissue protease activity. Although qualitative evidence exists for this neutrophil--protease interaction, a direct quantitative assessment of this interaction is lacking. The purpose of this study was to determine the extent to which myeloparoxldase (MPO) (a neutrophil enzyme] and the calcium-activated neutral nonlysosomal proteasej calpaln, changed over a period of increased contractil~ activity for rat skeletal and cardiac muscles. Following 1 hr of run exercise (25m/min), rat hearts and plantaris musde.~ were assayed for MPO and calpaln-like activities (N=IO) and compared to control values. Ca/pain-like activities ranged from 2.79 to 58.9 U/g for all samples and conditions, while MPO ranged from 0.03 to 4.88 U/g. Correlation coefficients (r) within heart and plantads data set were 0.98 and 0.68, respectively. With all data (i.e r across tissues and conditions) the r was 0.83 (p
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BAROREFLEX SENSITIVITY DURING Fr177 M E N T A L L O A D AND I N D U C E D E M O T I O N Natasa Honzikova, Ivana Rybkova+& Jan M. Honzik*. Dept of Physiol and + Center of Cardiovas Sur and Transplantation, Masaryk Univ, *Dept of Comp Sci and Eng, Technical Unix,, Bmo, Czech Republic. Wc compared baroreflex sensitivity (BRS) after light mental load (ML) and light induced emotion (E). Nine subjects of both sexes (19-22 years) were tested in each trial. ML: discrimination between three tones of different pitch; E: instructed imagination of hostile environment. Blood pressure was evaluated from continuous noninvasive 3 rain records at rest and during ML or E. Power spectra of pulse intervals (I), systolic (SP) and diastolic pressure (DP) were calculated. BRS was determined by spectral analysis of SP and I fluctuations at 0.1 Hz. During ML, SP and DP significantly increased (p<0.01) and SD of I and SP decreased (p<0.05, p<0.01 respectively). These changes were not associated with unambiguous changes of BRS. During E, the only significant change was a decrease of BRS @<0.01). In both trials, the change of BRS did not correlate with mean I. We conclude that light / e l l increased sympathetic and decreased parasympathetic tone, contrary to light E which decreased reflex parasympathetic regulation of the heart.

R E V E R S I B L E P E R M E A B I L I S A T I O N OF CARDIOMYOCYTES TO LOAD MEMBRANEIMPERMEANT SOLUTES

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Claudia Fuchsbauer& Ashley AIIshire, Department of Pharmacology& Therapeutics, Universly College Cork, Cork, Ireland. Lysosphingolipid sphingosylphosphorylchollne (SPC) added exogenously inserts into the plasmalemma of cells rendering it leaky. We used SPC to transiently permeabilise cardiomyocytes, in order to introduce foreign molecules into the cytosol. Membrane permeabilisation is then reversed by absorbing the SPC with fattyacid free bovine serum albumin (BSA). Rat cardiomyocytes (2x10s cells/ml) are washed with a Ca2+-free Ringer solution containing 300gM EGTA and transferred to an 'intracellular' type medium (ICM) at 37°C containing lmM-ATP and 15mM-phosphocreatine. SPC is added at 12.5 I.tgJmlfor 5 min, together with the solute to be loaded into the cells. At this stage trypan blue (TB) can enter 93+7% of SPC-treated cardiomyocytes (n=27) versus 4+3% of untreated cells (n=l 1). BSA (1% w/v) is then added for 2 rain, after which cells again exclude TB. 'Resealed' cells are transferred to fresh ICM lacking SPC/BSA and Incubated for 30 mtn twice, then harvested into nominally Ca-free Ringer. Finally Ca2+ is broughtstepwise to 1mM in the medium over 30 min, after which 40+10% (n=l 1) of the initial population of rods is recovered. Of these, 55+_22% (n=8) are found to have been loaded with the membrane-impermeant fluorescent marker lucifer yellow. Cells appear morphologically normal (maintain rod shape and clear sarcomere pattern) for at least several hours after the process. Thus reversible permeabilisation with SPC is straightforward, rapid, inexpensive and cell membrane-specific.

[Supportedby EOLASThe Irish Science& TechnologyAgency] A262

PURIFICATION AND CHARACTERIZATION OF Fr178 RECOMBINANT PHOSPHOLIPASE C-,81 EXPRESSED IN E. eo# Johanna T.A. Meij and Elliott M. Ross, Dept. of Pharmacology, University of Texas Southwestern Med. Ctr., Dallas TX, U.S.A.

Reconstitution into phospholipid vesicles of the purified components of receptor-G protein signaling pathways provides detailed insight into the mechanistics of their interactions. To obtain active recombinant receptors and G protein subunits, posttranslational modification and therefore, eukaryotic expression is required. Presumably, however, this does not apply to phospholipase C (PLC). Thus, the PLC-~'I coding region, preceded by 6 His codons to allow Ni+-affinity purification, was cloned into the bacterial expression vector pQE60. Several bacterial strains transformed with the plasmid pQEPLC were submitted to various induction conditions. Expression was screened by SOSPAGE, anti-PLC Western blotting, and by assay of PLC activity stimulated by GTPyS.preactivated aq. Optimum results were obtained with SG13OO9[pREP4] cells when induced at ODsoo-1.2 with 0.1 mM IPTG for 2h at 30°C. 2 M NaCI extraction of cell lysate, followed by Ni*NTA chromatography, and anion and cation exchange FPLC, yielded a single protein peak of lOO.fold purified aq-stimulated PLC activity. A major (>90%) 150 kDa band and 3 bands in the 120-135 kDa range were visible both by SDS-PAGEIsilver stain and Western blotting. These findings showed that, although bacterially expressed PLC-/5'I is proteolysed, purified Gq-responsive protein can be obtained that can also act as GTPase accelerating protein (GAP) for Gq in a reconstituted assay. As an approach to examine the physiological function of GAP activity, we have aimed to construct PLC.,81 mutants lacking GAP activity (GAP-PLC-,81) by site-directed mutagenesis in the extended Y-domain of PLC-,81 (C-terminus). The above expression and purification procedure supplies a rapid method to screen for GAP-PLC-,81.

ANGIOTENSIN II STIMULATES EXPRESSION Fr180 OF TISSUE PLASMINOGEN ACTIVATOR AND ITS IHIBITOR IN CULTURED CARDIAC FIBROBLASTS Hari S. Sharma, Han A. van Heugten, Miriam A. Goedbloed, Pieter D. Verdouw and Jos M.J. Lamers, Cardiovascular Research h~titute (COEUR), Erasmus University Rotterdam, The Netherlands Angiotensin II (ANG II) has been shown to be a mitogenic peptide for the cardiac fibroblasts in vitro, the intracellular mediators of its action are however, unknown. Tissue type plasminogen activator (t-PA) and its physiological inhibitor (PAl-l) contribute to a number of biological processes, such as fibrinolysis, matrix degradation and tissue remodeling. We examined the mitogenic action of ANG II on the expression pattern of t-PA and PAI-1 in cultured cardiac fibroblasts. Fibroblasts were isolated by enzymatic dissociation of cardiac tissue from 0-3 days old rat pups and were grown for a week in Dulbecco's modified Eagle's medium containing 5% each horse and fetal calf serum. Cells were passaged and seeded at a density of 3-5x103 cells/cm2 and were serum deprived for 24 hrs prior to incubate with [Sarl]-ANG II for 30 min, 1, 2, 4, 8, 12, 24, and 48 hrs. Total cellular RNA was extracted and processed for Northern hybridization using eDNA probes encoding t-PA and PAI-I. ANG II induced steady state mRNA levels of t-PA and PAI-I as early as 1 hr, reaching maximal at 12 hrs and thereafter the expression declined but remained elevated as compared to untreated cells. DNA and protein biosynthesis was increased after 24 hrs of ANG II incubation and this effect was further aggravated by serum. We conclude that the induced expression of t-PA and PAI-1 in cardiac fibroblasts by ANG II indicate for a stimulated plasminogen/plasmin system which may contribute to extra cellular matrix modulation and tissue remodeling during e.g. development of cardiac hypertrophy.

Gene Transfer in vivo into Rat Fr181 Myocardium by HVJ-Liposome Method Hiroyuki Kawaguchi, Wee Soo Shin, Yuepeng Wang, Wei Dong Yang, Teruhiko Toyo-oka The 2nd Dept. Int. Med., Univ. Tokyo, Japan Gene therapy for myocardial disease needs simple and efficient transfection. We prepared liposomes which include CMV promoter to improve transfection for 13-galactosidase (13-gal) gene expression vector. To make the gene transfer to nucleus more effectively, the plasmid DNA was mixed with high mobility group-1 (HMG-1) protein which forms a complex with DNA and the liposomes were coated with HVJ (Hemagglutinating virus of Japan). This HVJ-liposome complex was directly injected into anterior wall of left ventricle of rats under guidance of echocardiography. Most striking findings compared with adenovirus-mediated transfection were that 1) 13-gal staining was confined to a portion between two intercalated disks in individual myocardial cells, making it possible to contrast morphology of adjacent transfected and nontransfected parts, 2) HVJ vector induced much less local inflammation, and 3) the expression peaked on thelOth day and remained high level on the 14th day. Our results suggest that percutaneous direct gene transfer by HVJ-liposome into cardiomyocyte provide a useful technique for gene transfer.

MAP-KINASE (MAPK) ACTIVATION IN ATRIAL

MYOCYTES (AM) - INTERACTION WITH THE ANP SECRETORY SYSTEM. Anton F Doubell & Carine Page. Dept of Internal

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Medicine, University of StellenboscH, South Africa.

MAPK is one of a family of kinases, activated in response, to stimuli acting throug,h receptor tyrosine Kinases and G protem-coupma receptors. It's activation results from phosphorylation, on both Ser/Thr and / y r residues ana it is thus thought to funct on as an integration point tor oifferent siqnam, since tyrosine- ano ~.~proteincoup,led ~.inases are import.ant signalling mecnan sms in AM, integration ot these systems by MAPK coud be mDortant in their function. The reported role of M.APK during se.cretion in chromaffin cells raises the ,qossibihty that .MAPK may, ne invoIveo in regum(ing the secretion OX atrial.natriuretic peptide(ANP) from AM. We therefore characterized MAPK in cultured AM, studied it.s .act vat on by ANP secretagogues and measured the secretion of ANP as a runction or MAPK activity. Th.e presenqe of MAPK in AM was confirmed,by isolatinQ it throu~n ion exchanQe cnromatoqrapny. Stimulation of AM with PMA, ET-I_bFGF and thrombin resulted in the activation of MAPK. The concomitant secretion of ANP was measure.d, w t h PMA, ET.-1 and thrombin resulting m sign f cant st mu ation of ANP levels. In response to these st m u , both MAPK activity an,d A N P secret on were mpaired by the selective innioition of signals upstrea.m of MAPK. Dissociation of the two responses =n some instances (e.g. ET.-1 stimuaton n the Dresence or cnorera toxm~ indicated crosstalk between different signalling

THYROID HORMONE-INDUCED NUCLEAR Fr182 PROTEIN IN CARDIAC FIBROBLASTS BINDS TO THE AP-! CONSENSUS SEQUENCE. Mahboubeh Eghbali-Webb, James Douglas & David C. Ward. Depts of Anesthesiology, Genetics & Molec. Biophys and Biochemistry, Yale School of Medicine, New Haven, Connecticut, USA. Thyroid hormones inhibit collagen type I gene expression in the heart and in cardiac fibroblasts. Previously, we showed the binding of triiodothyronine (T.~)-induced nuclear proteins in cardiac fibroblasts to DNA sequences on pro al(l) collagen promoter, proximal to the transcription initiation site. In this study, using a gel retardation assay we showed that T3-induced nuclear proteins in cardiac fibroblasts bind to a ~[P]-end-labelled double stranded oligonucleotide corresponding to the AP1 transcription factor binding site. This binding was competed completely by molar excess of unlabelled AP-I oligonucleotide. On the other hand, the binding of T3induced nuclear factors in cardiac fibroblasts to a 3-'[PIend-labelled 340 bp fragment of pro al(I) collagen promoter (-225/+ 115) was competed in a dosedependent manner, by molar excess of unlabelled AP-I oligonucleotide but not by an SP-1 oligonucleotide. These data conform with previous results on T3-induced expression of mRNAs for c-fos and c-jun, components of AP-1, in cardiac fibroblasts and further suggest a role for this transcription factor in thyroid hormone-mediated regulation of collagen type I gene expression in cardiac fibroblasts.

IDENTIFICATION OF DOMAINS ON G PROTEIN -~2 SUBUNIT FOR ASSOCIATION WITH I~1 SUBUNIT BY DOMINANT-NEGATIVE ASSAY

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Takeshi Murakami,Ken Kageyama,Hideaki Kawaguchi& Akira Kitabatake. Dept. of CardiovascularMedicine, Hokkaido Univ. Sch. of Med., Sapporo,Japan. It is now clear that many effectors of cardiac signal transductions are regulatedby G protein 97 subunits. Five 13and at least six Y subunits can not form all the possible pairs. To study moleculardeterminantson Y subunits to form specific [37pairs we made three different deletion mutants of ~ at conserved regionsamong several Y subunits. Three internal deletions are at amino acids 16-23 (VEQLKMEA), 27-32 (RIKVSK) and 47-50 (EDPL). We used dominant-negativeassay in which a deleted T2 was coexpressedwith wild-type~2 along with B1 in transiently transfected COS cells. The RIKVSK mutant showed the most negativeeffect on expressionof [31. EDPLshowed less negative effect. In conclusion, multipledomains of •~mightbe responsiblefor associationwith 91 and the RIKVSK region on y2 has a key role in 13-¥interaction.

~mI~APK is present in AM, is activated by d verse st mu ani:l s nked to the proce.ss of AN,,P secretion. This coupling is complex with crosstaIK ~etween dif.feren.t secona messenger systems oetermining the ultimate secretory response. A263

FUNCTIONAL I S C H E M I C P R E C O N D I T I O N I N G D O E S F r l 8 5 N O T P R O T E C T AGAINST R E P E R F U S I O N . I N D U C E D F I B R I L L A T I O N IN A D J A C E N T M Y O C A R D I A L REGIONS

Roberl M. Lust, You Su Sun, David T. Rock, W. Randolph Chitwood, Jr. East Carolina University School of Medicine, Greenville, NC. Ischemic preconditioning (IPC) has been demonstrated to locally reduce infarct size and limit repcrfusion arrhythmias. Extension of IPC benefits to adjacent, "unconditioned" myocardial regions is unresolved. In the last year, 42 animals received two venous bypass grafts, one to the proximal LAD, and one to the proximal circumflex (CFX), placed without bypass, using local occlusion of the target artery. Proximal aortic anastomoses were constructed f'wst to reduce ischemic time. Average anastomotic time (ischemic time) was 13.7 3.5 minutes. The incidence of reperfusion-induced fibrillation after opening the first graft was 40.4% (17/42). Stable reperfusion ,was allowed for 29.4 + 6.1 minutes and then the second coronary anastomosis was performed. Thus, this model was functionally equivalent of a 15 rain occlusion, 30 rain repcrfusion IPC paradigm. The incidence of reperfusion-fibrillation after opening the second lanastomosis (occlusion time, 12.9 + 4.2 minutes) was 50% (21]42). Thus, the IPC paradigm in one region did not attenuate repcrfusion fibrillation following subsequent, brief ischemia-reperfusion in adjacent myocardium. H o w e v e r , the local IPC effect was quite pronounced. 9 technical failures required re-occlusion of the native artery to reconstruct the anastomosis (LAD, 5/42; CFX, 4/42), with similar occlusion times, but the incidence of repcrfusion fibrillation was decreased significantly (1/9, 11.1%, p < 0.05 vs 1st occlusion). Therefore, within the same region, IPC did attenuate reperfusioninduced fibrillation, but these effects were localized, and did not extend to adjacent myocardium. These data demonstrate precise ifunctional l o c a l i z a t i o n of I P C - i n d u e e d c a r d i o - p r o t e c t i o n from ~rhythmia, without extension to adjacent tissue.

EFFECTS OF DIETARY EICOSAPENTAENOIC AND Vr187 DOCOSAHEXAENOIC ACIDS ON HEART FUNCTION. A. Grynberg, J.P. Sergiel, L. Martine D. Readerstorff "~ and L. Demaison. INRA, Unit6 de Nutrition Lipidique, Dijon, France and ~t) Hoffmann La Roche, CH 4002 Bale, Suisse. The influence of pure dietary eicosapentaenoic acid (EPA) and docosahesaenoic acid (DHA) on heart physiology was investigated before and after ischemia. Rats were fed for 6 weeks a diet enriched with either sunflower seed oil (6.75%) + EPA ethyl ester (0.75%; EPA group), or sunflower seed oil (6.75%) ÷ DPA ethyl ester (0.75%; DHA group) or 7.5% of sunflower seed oil (SSO group). The hearts were peffused according to the working mode. Eight hearts of each group were maintained in normoxic conditions for 65 minutes. Ten other hearts were perfused in normoxia for 15 minutes and then subjected to a 17 minute global zero-flow ischemia and reperfusion. The aortic and coronary flows, aortic pressure and electrocardiogram were continuously monitored. The n-3 PUFA rich diets noticeably decreased the n-6/n-3 PUFA ratio of cardiac phospholipids. The membrane DHA content in the EPA group was lower than in the DHA group. Conversely. the amounts in EPA and DPA (22:5 n-3) were higher in the EPA group. These modifications altered the cardiac function in normoxic conditions. Dietary EPA increased the aortic flow although the coronary flow was enhanced in the SSO group. The cardiac rate was reduced in the DHA group. On the other hand, the aortic pressure recovery after ischemia was improved in the EPA group as compared to the other groups. This was due to a better coronary flow recovery. However, the aortic flow recovery was not significantly different. These results indicate that pure dietary n-3 PUFA altered the phospholipid fatty acid composition. These changes modified the cardiac function before and after ischemia. The improved mechanical activity recovery in the EPA group might be related to an effect of either EPA or DPA on the coronary bed. A264

LOW FLOW ISCHAEM]A ABOLISHES THE PROTECTIVE EFFECT OF PRECONDITIONING ON STUNNING Linda M. King, Lionel H, Opie Heart Research Unit, UCT Medical School, Cape Town, South Africa

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Preconditioning (PC) has been shown to protect against infarct size, arrhythmias and stunning. Most studies have tested the effect of PC on total ~vcre ischaemia. We tested thc effect of PC with low residual coronary flow on isclmemic contracture and stunning. Methods: We used the isolated Langcndorff pcrfused rat heart, with LV balloon. PC hearts were made totally ischaernic for 5rain followed by 5min reperfusion prior to 30rain sustained global ischacmia, either zero flow, or low flow (0.2ml/min/g wet wt). Time to onset of contractnre (TOC - rain), peak contracture and recovery of developed pressure (Dev P) after 20rain rcperfusion (both expressed as % preischaemic developed pressure) were measured (n = 13-16) Zero flow IZaro flow 0.2ml/min 0.2ml/min Control IPC TOC 11.66i0.73 [7.81±1.09 * 111.96±1.03 * [10A6±1.53 Peak 44.13±3.84 52.70~1.95 36.96:~5.55 ]31.59±3.75 ** DevP 22.54±7.03 47.59~7.31 * 55.06+8.58 47.45±11.78 • p<0.05 vs Control zero flow, ** p<0.05 vs PC zero flow PC reduced TO(2 and increased peak contracture but improved recovery after zero flow ischacmia. With low flow, TOC was delayed, and peak contracture reduced in PC hearts. There was no difference in recovery of function compared to zero flow PC hearts, or to control low flow hearts. Conclusions: Low flow ischaemia abolished the protective effect of PC on stunning, although positive effects on contracture were observed, conmL,ry to results obtained under zero flow conditions. Low flow improves the metabolic status of the heart, as observed by improved recovery, whereas with PC hearts, there may Ix: washout of a protective substanco, possibly adenosine, which modulates metabolism in the absence of flow. I

IControl

[PC

Invited Lectures Abstracts L1-L59

POSTNATALDEVELOPMENTOFTHE MAMMALIAN HEART: GROWTH OF CARDIAC MYOCYTES AND INTERSTITIAL CELLS

LO1

Piero Anversa, Wei Cheng, Jan Kajstura, Krzysztof Reiss & Edmund H. Sonnenblick. Department. of Medicine, New York Medical College, Valhalla, N.Y. To determine

whether

the growth

of myocytes

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~02

Controversies on acetylcholine-induced increases or decreases in coronary blood flow arise from obvious species differences, the role of the endothelium in mediating vascular smooth muscle responses and the marked negative chronotropic and inotropic effects of acetylcholine. In man, there appears to be a predominant dilation of intact epicardial coronary arteries and a constriction of atherosclerotic segments. However, at present there is no evidence for a vagal initiation of myocardial ischemia. Coronary vascular beta receptors mediate dilation, but appear to be functionally insignificant during sympathetic activation. The beta-adrenergic mechanisms contributing to myocardial ischemia are indirect, mediated by a tachycardia-related redistribution of blood flow away from the ischemic myocardium. Alpha receptors mediating epicardial coronary artery constriction in experimental studies appear not to be responsible for the initiation of ischemia in patients with angina at rest. However, alpha adrenergic constriction of coronary resistance vessels resulting in the precipitation of poststenotic myocardial ischemia was demonstrated in experimental studies and recently confirmed in patients with effort angina. Non-adrenergic, noncholinergic neurotransminers exist; however, their role in regulating coronary blood flow remains entirely unclear.

and cardiac

tibroblasts during development is mod;lated by the IGF-1 recentor (IGF-1Rf. the exoression of IGF-I. IGF-2 and IGF:lR was dete&ed in’myocytes and fibriblasts from fetal and postnatal hearts. The expression of proliferating cell nuclear antigen (PCNA) and DNA polymerase CLwas also evaluated in combination with the estimation of DNA replication. Myocardial development was characterized by a progressive decrease in the expression of late growth related genes in myocytes and fibroblasfs which was particularly evident at 21 days after bii. The expression of IGF-2 in myocytes decreased at birth, whereas the attenuation in IGF-1 mRNA became apparent a few days later. The induction of IGF- 1R at the message and protein levels decreased by 11 days. Moreover, DNA synthesis in myocytes was sharply reduced at 21 days after birth. In comparison with fetal hearts, at day 21 postnatally, IGF-1R mRNA in fibroblasts was reduced by 77% and IGF-2 by 70%. Moreover, IGF-IR recepior protein decreased bv 48% at 2 1 days. Bromodeoxvnridine labeling decreased by 88% at &is time. In co&lusion, the IGF-1R receptor system appears to play a major role in the regulation of myocyte and fibroblast growth in the heart & @.

RAPID GROWTH OF THE NEONATAL HEART Cathy J. Beiniich, Howard E. Morgan. Weis Center for Research, Geisinger Clinic, Danville, PA

NEURONAL CONTROL OF CORONARY BLOOD FLOW Dietrich Baumgart and Gerd Heusch. Dept. of Patiophysiology, University of Essen, FRG.

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During the first two weeks of life the IefI ventricular free wall of the neonatal pig heart grows rapidly. This rapid growth is restrained by pretreating the piglet with the angiotensin converting enzyme inhibitor, enalapril maleate. The purpose of this study was to determine whether the rapid growth of the neonatal pig heart was the result of myocyte hyperplasia, hypertrophy or a combination of hyperplasia and hypertrophy. From birth to 14d of age, the mass of the left ventricular free wall (LVFW) increased 4.3 fold while the right ventricular free wall (RVNV) increased 1.75 fold. During the same period, the cellular volume of myocytes from the LVFW increased 3.43 fold while myocytes from the RVFW increased in volume 1.62 fold. The number of RVFW myocytes did not change during the first 2 weeks of life while the number of LVFW myocytes Myocytes from both ventricles were increased 26%. approximately 90% mononuclear from birth to 4-5 days of age. By 14 d of age, 67% of LVFW myocytes and 53% of RVFW myocytes were multinucleated. When piglets were treated with enalapril for 2 weeks, the LVFW mass was reduced by 24% and was accounted for by a reduction in myocyte volume. Enalapril treatment did not alter the number of myocytes in either the LVFW or RVFW as compared to hearts from untreated piglets. After 14 days of enalapril treatment, the percentage of multinucleated cells was reduced in the LVFW and unchanged in the RVFW as compared to hearts from untreated piglets. These results indicate that cellular hypertrophy accounts in large part for the rapid growth of the LVFW of the neonatal pig heart and that enalapril reduced normal growth of the left ventricle by reducing left ventricular myocyte hypertrophy.

REGULATION EXPRESSION

OF CARDIAC GENE DURING CARDIAC GROWTH.

Kenneth R. Boheler Dept. of Cardiothoracic NHLI, London SW3 6LY U.K.

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Surgery,

Two of the primary tinctions of the heart are to contract and relax, processes that are regulated by the coordinated interactions

of numerous

proteins.

The

expression

of these

proteins can be controlled at transcriptional, posttranscriptional or translational levels of regulation. By analysing the expression of the contractile proteins, actin and myosin heavy chain, and those responsible for sarcoplasmic reticulum mediated Ca uptake in both the in viva and in vilro settings, we have been able to delineate responsible for their regulation. some of the pathways This talk will focus on our recent results involving regulation by a, fil and p2 adrenergic agonists with growth and hypertrophy and the effects of angiotensin converting enzyme inhibitors on the reversal of cardiac hypertrophy. Finally, the changes in gene expression due to these interventions will be discussed in terms of in vitro transcription reactions to determine precisely what level of control the changes in RNA accumulations are due.

HUMORAL REGULATIONOF CARDIAC FIBROBLAST L05 FUNCTION Christian G. Brilla & Heinz Rupp. Molecular Cardiol. Lab, DIv. of Cardiology, Univ. of Marburg, Germany.

PRECONDITIONING POTENTIATES M O L E C U L A R S I G N A L I N G FOR MYOC A R D I A L A D A P T A T I O N T O ISCHEMIA

In hypertensiveheart disease (HHD) or congestive heart failure (CHF), myocardial fibrosis is associated with an activated reninangiotensin-aldosteronesystem (RAAS). Cardiac fibroblasts (Fb) express mRNAs for types I and III collagens, the major fibrillar collagens in the heart, and for matrix metalloproteinase 1 (MMP1), the key enzyme for interstitial collagen degradation. Therefore, adult human Fb were cultured to ascertain whether the RAAS effector hormones angiotensin I1 (All) or aldosterooe (ALDO) directly stimulate collagen synthesis (CollSyn) or inhibit MMF1 production. CollSyn,determined by 3H-proline incorporation, and MMP1 activity, determined by degradation of 14Ccollagen,were measured under serum-free conditions in confluent Fb after 24 hr incubationwith All or ALDO over a wide range of concentrations (i0 "I1 -10-6M). In addition, CollSynwas measured after incubation with the mineralocorticoid, deoxycorticosterone (DOC), or the prostaglandin, PGF-~. CollSyn, normalized per total protein synthesis,increased significantlyin a dose-dependent manner after incubation with either mineralocorticoid hormone, ALDO or DOC, or after incubation with All compared with untreated control cells. In contrast, CollSyn was significantly decreased with PGE2 treatment. This increase in CollSynin Allor mineralocorticoid-stimulatedFb could be abolished by All type 1 or mineralocorticoid receptor antagonists, respectively. In addition, All significantlydecreased MMP1 activity while PGF-~ increased it; mineralocorticoidhormones had no effect on collagen degradation. These findings suggest a direct interaction between ALDO, DOC, All, PGF-.2 and F'b in regulating the collagen matrix in HHD or CHF, where circulating R.AASor tissue renin-angiotensin systems are activated.

While ischemic preconditioning renders the heart tolerant to ischemia, its effects worns out after several hours. The results of our study implicate that preconditioning potentiates the intracellular signaling leading to the stimulation of both phospholipase C and phospholipase D thereby genarating the second messengers, diacylglycerol and inositol triphosphate, and resulting the translocation and activation of protein C kinase. Preconditioning also leads to the induction for the expression of a number of protooncogenes and stressrelated genes including the mRNAs for heat shock proteins and antioxidant enzymes within minutes to hour. The mRNAs of superoxide dismutase (SOD) and catalase as well as HSP 70 have been found to be translated into the corresponding proteins within hours of preconditioning as evidenced by the enhanced antixidant enzyme activities and increased content of heat shock proteins. The preconditioned hearts remained tolerant to ischemia even after 24 hours. It may be speculated from our results that activation of protein C kinase exerts a negative feedback that downregulates the early effects of ischemic preconditioning. However, the activation of protein C kinase and diacyl glycerol leads to the gene expression at the transcription level which is likely to play a role in the myocardial adaptation to ischemia. Thus, protein C kinase/diacylglycerol may serve as the signaling link between the early preconditioning and ultimate adaptation process.

THE PARTICIPATION OF NITRIC OXIDE (NO) L07 IN THE REGULATION OF CORONARY FLOW Rail J. Domenech and Pilar Macho. Department of Pathophysiology. Faculty of Medicine, University of Chile. Numerous agents produce coronary endothelial disfunctlon characterized by a decrease in the endothellum mediated vasodUatory response. However these findings do not necessarily mean that the endothellum mediates in the regulation of coronary flow through a cross - talk with the cardiac myocyte. Thus, experiments in dogs reveal: 1.- NO does not seem to be essential for the adaptation of coronary flow to increments in the oxygen consumption of the heart since NO synthesis inhibition does not modify the slope of the linear regression between myocardial oxygen consumption and coronary flow; 2.- It is uncertain that NO is essential in the autoregulation of coronary flow since the autoregulatory gain during the plateau response of flow versus coronary perfusion pressure was not changed after NO synthesis inhibition; 3.- NO synthesis inhibition produces a very small redistribution of flow towards the subendocardium; 4.- Conversely, the Inhibition of NO synthesis produces a decrease in coronary reactive hyperamia of about 40% in the dog and of about 100% in Isolated perfused guinea pig hearts. However it cannot be discarded that this participation of NO ls due to its release by the sudden increase in shear stress on the endothelium at the time of reopening the vessel rather than to a signal from the hypoxic myocardial cell. Therefore, in regard to NO, these results support a probably cross - talk between the endothellum and the myocardial cell In the regulation of coronary flow during ischemla but the talk does not appear to be obligatory for the physiological regulation of coronary flow. (Grants FONDECY'r N a 1940296 and DTI, U. of Chile N= 322g).

L06

Dipak K. Das, University of Connecticut School of Medicine, Farmington, CT 06030,USA

TGF-fl LIGAND & RECEFIDREXPRESSIONVARI- L08 ATIONS DURING POSTNATAL HFART DEVELOPMENT MODIFIED BY AORTIC CONSTRICTION. Gary L. Engelmanna, Patricia Grutkoski a, Frautisek Kolarb & Bohuslav Ostadal b. aDept, of Med., Loyola Univ. Chicago, Maywood, IL, bAcad. Sci. of the Czech Rep., Prague, Czech. Republic Postnatal heart development is characterized by rapid changes in myocyte proliferative growth & cellular hypertrophy. In concert with these changes, extracellular matrix (ECM) deposition & expansion of the capillary network are ongoing. A regulatory component of these processes may be the transforming growth factor-beta (TGF-J3) family of ligands & receptors. Although postnatal myocyte proliferation is limited, acute abdominal aortic constriction (AC) on day 2 may extend their normal proliferative potential. We evaluated transcript levels for TGF-8 & TGF-8-reeeptors (TGF-SR) in regional ventrieular samples from normal & AC animals. A late-fetal-to-early postnatal "upregulation" i n transcript levels for TGF-~ ligands & Type II TGF-SR was found in normal pups. Ventricular samples from animals obtained 1-8 days post-AC showed that TGF-8 & Type I & II TGF-/3R expression is reduced in the 1-day AC group. Depending on the ventricular region, by 3- & 8-days post-AC expression of these same genes was increased relative to controls. In accord with biochemical & morphological studies suggesting a n extended period of postnatal myocyte proliferation had occurred in AC neonates, our data supports the postulate that the TGF-13 family is involved in norreal regulatory control of postnatal myocyto growth.

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GROWTH FACTOR INFLUENCE A N D L09 HYPERTROPHIC B E H A V I O U R OF A D U L T CARDIOMYOCYTESIN CULTURE.

Hans M. Eppenbergerand MarcusC. Schaub* Institute of Cell Biology, ETH, CH-8093 Z0rich, *Institute of Pharmacology, University of ZOrich, CH-8057 Z0rich, Switzerland Adult rat cardiomyocytes do not undergo cell divisions and remain immobile when kept in cell culture. New cell-cell contacts are made by growing in size only, leading to an e n l a r g e m e n t of the cardiomyocytes similar to that observed in adaptive growth during cardiac hypertrophy. The cells have been shown to undergo a considerable change in morphology and to express fetal cytoskeletal and myofibrillar proteins. Here we report on the effects of basic fibroblast growth factor (bFGF) and insulinlike growth factor (IGF-I) during these changes. While IGF-I accelerates myofibril reassembly and sarcomeric growth and is a c c o m p a n i e d by a considerable protein synthesis, b F G F inhibits sarcomer formation but induces a significant increase in size of the cells. A comparable effect o b s e r v e d for IGF-I is d e m o n s t r a t e d for cardiomyocytes grown in a medium conditioned through 12 day old cardiac cells. While IGF-I and conditioned medium produce a more differentiated cardiomyocyte, bFGF seems to keep the cells in a less differentiated state.

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MYOCARDIALGLUCOSETRANSPORTREGULATION L10 BY CATECHOLAMINESAND ALTERNATIVESUBSTRATES Yvan Fischer, Institute of Physiology, Aachen, FRG Glucose metabolism is essential to myocardial function, and its impairment in pathological conditions such as Diabetes mellitus may contribute to heart failure. Glucose transport into heart muscle cells is the first and one of the key steps controlling the utilization of the sugar by the myocardium. Cardiomyocyte glucose transport is subject to acute stimulation by insulin, contractile activity, catecholamines, and to counterregulation by alternative substrates of energy metabolism. The stimulating action of catecholamines,which occurs via ~l-adrenergic receptors, is not mediated by second messenger systems such as cAMP, cGMP, diacylglycerol or calcium release, but involves, at least in part, a product of oxidative monoamine degradation. Catecholamines act, like insulin, via a recruitment of glucose transporters to the plasma membrane. However, based on several findings, distinct mechanisms must be postulated; for instance, the extent of transport stimulation by catecholamines is not altered in cells from diabetic animals, while the effect of insulin is largely impaired. Counterr~.gulationof glucose transport is exerted by a variety of alternative substrates (lactate, pyruvate, ketone bodies, etc.), in contrast, the aminoacids alanine or valine, and inhibitors of aminotransferase reactions, are stimulatory. Alterations in glucose transport correlate with anaplerotic or cataplerotic properties of these agents, respectively, as indicated by corresponding changes in the cellular malate level. Transport inhibition eventually occurs through a decrease of the amount of glucose transporters in the plasma membrane. In conclusion, catecholamines and altemative substrates appear to regulate cardiomyocyte glucose transport, in addition to their well-known effects on glycolysis and pyruvate oxidation.

T I T I N A N D N E B U L I N IN S K E L E T A L L1 AND CARDIAC MUSCLE. Dieter FOrst, Wolfgang Obermann & Klaus Weber, Dept of Biochemistry, Max Planck Institute for biophysical Chemistry, D-37077 GSttingen, Germany.

CELL SIGNAL MODULATIONIN CORONARYARTERY L12 DISEASE : A CLINICAL AND EXPERIMENTALSTUDY. Nirmal K Ganguly, Veena Dhawan, Sudhir Gupta, Manjit Singh. Dept. of Experimental Medicine & Biotechnology, PostgraduateInstitute, Chandigarh,India.

The contractile machinery of the vertebrate striated muscle myofibril is held together by an endosarcomeric cytoskeleton that is comprised of titin and several associated proteins. In the A band titin interacts with thick filaments at least via Cprotein, M-protein and myomesin. Molecular cloning revealed that all these proteins, like titin, are intracellular members of the immunoglobulin superfamily of proteins, where each protein is characterized by a specific repeat pattern. Human myomesin and M-protein display unique N-terminal domains, followed by 12 repeats in the order 2-2-1-1-1-1-1-2-2-2-2-2, where 1 and 2 correspond to fibronectin type III- and immunoglobulin cll-like domains, respectively. Over this region both polypeptides share 50% sequence identity (Vinkemeier et al., 1993). We have recently succeeded in purifying bovine skeletal muscle myomesin. This allowed for a biochemical characterization of the polypeptide. The protein reacts with myomesin-specific monoclonal antibodies as well as polyclonal antibodies directed against internal peptides derived from the human cDNA. Analytical uitracentrifugation, circular dichroism and electron microscopy confirmed the strong similarity to M-protein predicted from sequence comparisons. In dot-blot overlay-assays purified myomesin specifically bound to myosin-rods and LMM. Limited proteolysis revealed that this myosin-binding site seems to be located in the first two immunoglobulindomains of myomesin.

Maintenanceof cellular homeostasisand control of the local environmentare critical factors involved in the prevention of the disease. Various clinical and experimental studies have been designed by us to elucidate the role of some of the key modulators/triggers in exacerbation and amelioration of myocardial and vascular damage. Our data shows that inflammatory cells are extremely important in mediating myocardial injury and these processes could be modulated through several k-~y messengrs like calcium, ECGF and prostaglandins.The cardiac cells exposed to OFR generated artifically showed a marked decrease in cellular utilization of glucose alongwith a significant decrease in calcium uptake. A direct relationship of neutrophil OFR production with the extent of myocardial ischemia in patients of MI has been shown. In another elaborate study endotoxin was shown by activating TH1 and TH2 cells and production of cytokines which by activating macrophages led to the secretion of nitric oxide and OFR causing cell damage. Studies aiming to prevent the damage were designed and revealed that the use of an antiinflammatory agent indomethacin in rhesus monkeys provided a significant protective effect against atherosclerosis by modulating arachidonate pathway. Captopril and an iron chelating agent desferrioxamine inhibited the myocardial infarct size in vivo whereas nifedipine and other scavengers and antioxidants showed the ability to reduce Lhe myocardial damage.

ENDOTHELIAL CONTROL OF CORONARY FLOW L13 IN PERFUSED GUINEA PIG HEART Ryszard J. Gryglewski, S. Chlopicki & P. Niezabitowski, Department of Pharmacology, dagiellonian University Medical College,Cracow,Poland. In perfused isolated guinea pig hearts reactive hyperaemia (RH) was induced by occlusion of coronary flow for periods ranging from 1-60 se~ RH was hampered by 100-60% in ~he presence of an inhibitor of NO synthase, N -nitro-f.-arginine (i00 ~H) and to a lesser extent ( up to 37~ by an antagonist of adenosine receptors, 8pbenyl-theophylline (I0 @M). An inhibitor of PGH synthase, indomethacin (5 ~M) did not affect R}]. During RH the heart generated prostacyclin, nitric oxide and adenosine as indicated by the appearance of 6-keto-PGF]. cyclic GMP, urete, inosine, hypoxanthine and xanthine in the perfusate. Out of these factors only NO and adenosine were responsible for R}I. ~I0 was responsible for Rll which was evoked by shortterm (I-i0 sec) coronary occlusion whereas concurred efforts of NO and adenosine were required to maintain R|| that followed longer (20-60 sec) periods of interruption of coronary inflow. Thus, in the investigated system ~ and adenosine but not prostacyclin can be considered as mediators of myocardial reactive hyperaemia.

DELAYED AFrERDEPOLARIZATIONS L15 Masayasu Hiraoka, Seiko Kawano & Akinori Kuruma. Dept. Cardiovasc. Dis., Med. Res. Inst., Tokyo Medical and Dental University, Tokyo 113, Japan Delayed afterdepolarizations (DADs) are a unique form of abnormal impulse formation, which initiate triggered arrhythmias. DADs are seen as damped membrane oscillations following the end of preceding action potentials. Their amplitude and mode of appearance of triggered arrhythrnias are dependent on preceding excitations. DADs are mostly observed in the conditions of Ca2*-overload and are formed by the transient inward current (I-n) during cyclic release of Ca2÷ from SR in the Ca2*-overloaded conditions. I~ is believed to be carried by Na ÷ through the nonselective cation channels or the Na+-Ca2÷ exchange mechanism. When ventricular myoeytes from guinea pig or rabbits were exposed to the low K ÷ (0-1 raM), high Ca2÷ (3.6 raM) solution, all the myocytes (n=45) easily developed DADs in current clamp mode and I.n in response to voltage clamp pulses. At this stage, we could not determine the reversal potential of I~ in most preparations, except 3 rabbit and 1 guinea pig myocytes which showed the reversal potential between 0 and -10 inV. These results suggest that the ionic mechanism for I.n is mainly caused by the Na*-Ca z÷ exchange mechanism but the non-selective cation channels may also be responsible in certain stage.

[Na+]l AND [Ca2*]i D U R I N G E N E R G Y D E P L E T I O N L14 A N D R E P L E T I O N IN M Y O C Y T E S : R O L E OF Na+/H ÷ E X C H A N G E AND Na+/Ca 2+ E X C H A N G E Hideharu Hayashi, Hiroshi Satoh', Hideki Katoh*, Hiroshi Watanabe', Hajime Terada°. Photon Med. Res. Ctr, "Internal Medicine III, Harnamatsu Univ. Sch. of Med, Harnamatsu,Japan. To study the dynamics of [Na+]i and [Ca2+]i, guinea pig ventricular myocytes were dual-loaded with 5 ~M SBFI/AM and 10 IJM fluo-3/AM. During the first 20 min of energy depletion (ED: 3.3 mM amytal and 5 pM CCCP in the absence of glucose), [Na+]i increased from 6.2+0.5 mM to 18.6_+1.6 rnM (M_+SE; p<0.01), whereas [Ca2+]i was low. In the following 30 rnin, 94 % of the cells developed contracture, and [Ca2+]i began to increase after cells had contracted (167_+14% of the control, 50rain). The increase in [Na+]i during ED was not affected by 10 v.M TTX, but was suppressed by 1 pM hexamethylene amiloride (HMA). The application of glucose from the start of ED prevented both the increase in [Na+]i and cell contracture. The levels of [Ca2+]i when cells contracted or hypercontracted were much lower than those during 500 pM strophanthidin perfusion, whereas [Na+]i increased further. However, the addition of 10 rnM glucose after 20 rain of ED (energy repletion: ER) led to a dramatic increase in [Ca2+]i to 442_+72% of the control (50 rnin, p
CARDIAC

ANGIOGENESIS

L16

Olga Hudlicka 1, & Margaret D. Brown 2. Depts of Physiology t and Sport and Exercise Sciences 2, University of Birmingham, Birmingham B15 2TT, UK. Growth of vessels in the heart during development of collateral circulation after constriction of coronary arteries is induced mainly by various growth factors. Under physiological circumstances - in endurance training and during long-term bradycardia - it is initiated by physical factors (increased capillary wall tension or stretch). While training stimulates mainly growth of larger vessels, bradycardia (brought about by electrical pacing in rabbits and pigs and by a bradycardic drug alinidine in rats) stimulates growth of capillaries without accompanying growth of larger vessels. Physiological consequences of increased capillary supply were more homogeneous distribution of flow (demonstrated by more uniform ratio between capillary permeabilitysurface area product in rabbits and by increased oxygen extraction in rabbits and pigs) and by improved maximal cardiac work in control (rabbit, pig) and hypertrophic (rabbit) hearts.

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INTRACELLULAR Ca a÷ GRADIENTS IN [_17 S M O O T H MUSCLE CELLS: MEASUREMENT AND POSSIBLE PHYSIOLOGICAL RELEVANCE Gerrit Isenberg, Department of Physiology, Martin-Luther University, 06097 Halle, Germany In comparison to global [CaZ*]o [Ca2+] at inner side of the plasma membrane increases at a faster rate to a higher peak, declines earlier and faster, and reprimes along a faster time course. Upon depolarization, this dissociation is measured in both visceral (urinary bladder) and vascular (coronary artery) smooth muscle cells, comparing fluorescence of Ksindol ([Ca2*]~) with ratiometric data from the membrane binding dye KsFIP18 or with currents through Ca 2+ activated BK + channels. 3-D digital imaging reveals Ca 2+ gradients from the sarcolemma to the center of the cell during the initial 500 ms of the depolarization, later, the Ca 2* gradient reverses the direction. The spatial arrangement of DHPreceptors in the plasma membrane closely to the ryanodine-receptors in the peripheral sarcoplasmic reticulum (SK) suggest that Ca 2+ induced Ca z+ release can start a Ca z+ wave propagating through urinary bladder cells. In coronary myocytes, however, depolarization is unable to inkiate a propagating Ca z+ wave. Ca z+ waves and large Ca z+ increments induced by agonists (e.g. acetylcholine) is shown to usually start at the central SR, provided the SR Ca z+ load is high. It is discussed that InsP3 induced Caz+ release originates predominantly at the central SR.

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REGULATION OF ENERGY GENERATION IN L19 THE MYOCARDIUM Helmut Kammermeier, Dept. of Physiology, Med. Fat., RWTH, Panweisstr. 30, D-52057 Aachen Metabolic pathways and other processes taking place in living organisms are implicit also pathways of flow of energy along f e e energy gradients, where a part of the energy available is also utilized to create energy gradients. This is brought at optimum in the generation of ATP, where a high percentage of energy available from subtrate oxidation or glycolysis is preserved as energy source for various energy dependent processes as ion pumping contractile activity and numerous steps of chemical synthesis. In contrast to technical processes in which energy transformation can take place in a sliding scale of efficiency, processes of ATP synthesis and utilization take place in stoiohiometric ratios. Even if integer ratios are not met in some cases owing to mixed processes or slipping and/or leaking, the processes per se take place in fixed ratios (e.g. Na-K-ATPase 3:2:1, SE-Ca-ATPase 2:1, SL Ca-ATPase l:l, Na-Caexchange 3:1, ATPase-erossbridge cycle 1:1 (persumed)). These ratios are determined by thermodynamic limits in most cases i.e. for example with physiological concentration and voltage gradients higher ratios are not possible owing to the limited energy available from ATP-hydrolysis. Recent findings on rather low (<1 raM) cytosolic inorganic phosphate contn'oute new aspects to the kinetics and thermodynamics of energy transformation and its regulation.

MYOCARDIAL REPERFUSION INJURY: PROTECTIVE L18 ROLE OF ISCHEMIC PRECONDITIONING. J. S. Juggi, Department of Physiology, Faculty of Medicine, Kuwait University, P.O. Box 24923, 13110 Safat, Kuwait, (A. Guff). Inspire of the best efforts to curtail the reperfusion injury during open-heart surgery, variable degree of residual ischemic damage still persists. The role of ischemic preconditioning in limiting global reperfusion injury has gained recent attention. However the mechanism and extent of this protection is not clear although some inroads have been made in relation to regional ischemia and infarction. In order to ascertain the role of ischemic preconditioning in limiting the reperfusion injury of the globally ischemic heart employing surgically relevant conditions of hypothermia, isolated aorta perfused rat hearts were exposed to different protocols of preconditioned global ischemia. Preconditioning (PC, 2 cycles of 5 min ischemia and 10 rain reperfusion, 34°C) significantly improved the ischemia (60 rain, 34=C)-reperfusion (30 rain, 34°C) recovery in left ventricular global and regional contractility and coronary flow. This recovery was not affected by the preconditioning temperature (34°C or 10°C) or the temperature during sustained ischemia (34 ° or 10°C). Cardioprotection by PC was found to be mediated by stimulation of the endogenous nitric oxide synthesis. Unprotected or protected (PC) hypothermic ischemia (10°C, 60 rain) - repedusion (34°C, 30 rain) offered nearly identical protection except that regional contractile function was better preserved with unprotected hypothermic ischemia. Hypothermic cardioplegia (Plegisol) with or without PC offered nearly identical recovery in global and regional contractile function and coronary flow and additive protective effect of PC was not observed. There was a dissociation of recovery in contractile function and coronary flow after intermittent global ischemia-reperfusion (IT-ISI, 6 cycles of 10 min ischemia and 10 rain reperfusion, 34°C). IT-ISI produced stunning of the coronary vasculature. It was concluded from these studies that ischemic preconditioning and intermittent ischemia-reperfusion significantly reduced the reperfusion injury but the beneficial effect of these interventions was not superior to the conventional cardioprotective technique of hypothermic cardioplegia. (Supported by Kuwait University Research Grant MY021.)

TOWARD THE GENE THERAPY OF CARDIOVASCULAR DISEASES Yasufumi Kaneda 1, Yoshlkl Sawa 2, Yoshikazu Yonemltsu 3 & Ryuichi Morlshlta 4" Inst. MoI.Cell BiolJ, Dept. of Surgery 2, Dept. of Geriatrics 4, Osaka Univ., Dept. of Surgery 3, Kyushu Univ.

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Gene therapy has arrested attentions for a novel therapeutic strategy and, also in cardiovascular field, gene therapy will provide us with new possibilities to treat various disorders. We have developed efficient gene delivery system based on liposome, virus mediated cell fusion, and nuclear targeting. Finally, we developed protein-liposome delivery system, in which DNA and nuclear protein are entrapped in liposomes having fusion proteins derived from HVJ (Sendal virus) on the surface. Recently, using this system, we showed the possibility to treat the restenosis after angioplasty by transfer of antisense oligonucleotides (ODN), double -strand oligo and c-NOS DNA. Then, we attempted to introduce DNA into myocytes from the coronary artery. During cardiac transplantation, FITC-ODN was introduced into more than 50 % of myocytes of rat heart through the infusion of the vector into the coronary artery. Using this ex vivo system, TGF-beta DNA was introduced and expressed in the rat heart without any injury to the myocytes. The transplanted heart expressing TGF-beta recovered coronary flow and LVDP, and reduced neutrophil adherence. These results suggest that our gene transfer system may be useful for myocardial protection.

QUANTITATIVE AND MOLECULAR BIOLOGICAL L21 CHANGES OF ADP/ATP CARRIER PROTEIN IN CARDIOMYOPATHIC HAMSTERS Mitsutoshi Kato, Nobuaklra Takeda, Selbu Mochizuki, and Makoto Nagano. Dept. of Internal Medicine, Aoto Hospital, Jlkel Univ., Tokyo Japan. ADP/ATP carrier protein (AAC) is an integral protein of the inner mltochondrial membrane that is involved In the exchange of cytoplasmic and intramitochondrialADP and ATP. The myocardial AAC contents was studied in J-2-N cardlomyopathic hamsters. These hamsters develop cardiac lesions which resemble human dilated cardlomyopathy. The myocardial AAC content was found to be significantly decreased in J-2-N. For molecular biological analysis, hamster AAC cDNA was cloned by the plaque hybridization method. This AAC cDNA hybridized specifically with AAC mRNA, allowing RNA dot-blot hybridization to be performed. The highest AAC mRNA level was observed in control hamsters followed by J-2-N with mild myocardial damage, J-2-N with severe myocardial damage, and Bio 14.6 hamster. T h e s e results s u g g e s t that a reduced myocardial AAC content may contribute to the pathogenesis of cardiomyopathy and that a decrease of AAC mRNA may explain the abnormal AAC level in J-2-N cardiomyopathic hamsters.

SARCOPLASMIC RETICULUM: ITS ROLE L23 IN EXCITATION-CONTRACTION COUPLING AND OUTWARD C a 2' T R A N S P O R T Bohdan Lewartowski and Robert Janiak, Dept. of Clinical Physiology, Med. Centre of Postgraduate Education,Warsaw,Poland. We reinvestigated the role of sarcoplasmic reticulum (SR) in excitation-contraction coupling of single, enzymatically isolated myocytes of guinea-pig (G-P) heart. Voltage clamping, free [Ca"]i recording by Indo1AM fluorescence and recording of contractions were used. We found that compounds that block Ca2+ flux through the SR by inhibiting SR Ca2*uptake (thapsigargin, TG) or by blocking the SR Ca~ release channels (1.0 mM ryanodine, Ry), did not affect the amplitude of Ca 2. transients or contractions but decreasd the rate of their rise and decay. TG did not affect the Ca2~ current. 1.0 #M Ry, which increases the Ca '~ flux through SR by opening the Ca2~ release channels decreased the amplitude of Ca~* transients and contractions and stimulated Na/Ca exchange. The effects of 1.0 #M Ry were reversed or blocked by TG and 1.0 mM Ry or by block of Na/Ca exchange. We conclude that the main function of SR in G-P myocytes is control of kinetics of contraction by handling of Ca~÷ derived from extra-SR sources. Control of the rate of Caa÷ flux through the pathway: sarcoplasm-SR-Na/Ca exchange may be an important way of operation of the system.

PHENOTYPE OF THE RELAXINGSYSTEM DURING CARDIACDEVELOPMENT

L22

Evangelia G. Kranias, Judy M. Harrer, Grazyna Szymanska, Jay P. Slack and Ingrid L. Grupp, Univ. Cincinnati, Cincinnati, OH 45267 Developmental changes in sarcoplasmic reticulum (SR) function, which may reflect alterations in the rates of myocardial relaxation, were assessed in mouse and rabbit hearts at different stages post-birth. In mouse hearts, the Ca2+-ATPase and phospholamban (PLB) protein levels were 40% of adult levels at birth and expression of both proteins increased in parallel during development. The upregulation of Ca2+-ATPase expression was associated with ,ncreases in the maximal rates of Ca2÷-uptake by SR, while the coordinate regulation of PLB and Ca2+ATPase expression was associated with maintenance of the affinity of the SR Ca2+-ATPase for Ca 2+ throughout development. In rabbit hearts, a similar coordinate regulation of the expression levels of SR Ca2*-ATPase and PLB as well as maintenance of the pump's Ca 2+ affinity were observed during development. Perfusion of developing rabbit hearts with isoproterenol was associated with significant increases in the rates of myocardial relaxation and increases in the Ca2+-affinity of the SR Ca2+-ATPase. At maximal isoproterenol, the Ca2+-affinity was similar between the different age groups, due to complete phosphorylation of PLB. These findings indicate that the coordinate regulation of the expression levels of SR Ca2÷-ATPase and PLB during cardiac development may be critical in regulating SR function and myocardial relaxation.

LEFT VENTRICULARMECHANOENERGETICSDURING L24 MILD PRESSURE0VERLOAD:RELATIONSHIPTO CONTRACTILE PROTEINALTERATIONS Martin M. LeWinter, Zeng-Yi Chen, Akihiro Higashiyama, Matthew W. Watkins, StephenBell, David Maughan,University of Vermont, Burlington,VT, USA Contractile protein isoform switches appear to be common in heart failure. It is unclearwhether this phenomenonis related to severedec0mpensationor representsan early manifestation of the responseto hemodynamicstress. To clarify this issue, we studiedleft ventricular(LV}mechanoenergetics(isolated,rbcperfused, isovolumicallycontracting heart) and myosin and troponin T (TnT, SDS-PAGE)isoforms in a rabbit modelof mild pressure overload(P0) produced by 12 weeks of cellophane wrap of the kidneys. LV to bodyweight ratio was 28% greater in P0(N-6) than shamcontrols(n-6,p<.001}. Therewere no significantmechanoenergeticdifferencesbetweenPO and shams, including contractian duration, relaxationrate, normalizedEmax (an index of contractility), and contractile efficiency and economy(inverseslopesaf V02-pressurevolumeareaand stress time integralrelations). AverageV3 isomyosincontentwas 80% in shams and > 95% in PO, while the % af total TnT accountedfor by the TnT2 isofarmwas 10 and 14, respectively (pooled data). We conclude that mild P0 causes modest contractile protein isoform shifts in the absenceof detectable alterations in LV mechanoenergeticperformance. Thus, in this model changesin contractileprotein phenatypeare not related solely to severedecompensation.

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REGULATORY MECHANISM OF THE L25 INTERACTION BETWEEN GLUCOSE AND F A T T Y A C I D M E T A B O L I S M Gary D. Lopaschuk, U. of Alberta, Edmonton, Canada The majority of energy production in the heart is derived from the oxidation of fatty acids, with the other important sources of energy being the oxidation of carbohydrates and to a lesser extent ATP production from glycolysis. While the mechanisms by which fatty acids inhibit carbohydrate oxidation have been well established, much less in known as to how carbohydrates regulate fatty acid oxidation in the heart. While an increase in intramitochondrial acetyl CoA derived from carbohydrate oxidation (via the pyruvate dehydrogenase complex) can down regulate 13-oxidation of fatty acids, it is not clear how fatty acid acyl group entry into the mitochondria is decreased when carbohydrate oxidation increases. Our studies have focused on the involvement of acetyl CoA carboxylase (ACC) in this process. While it has been known for some time that malonyl CoA exists in heart tissue, and that it is a potent inhibitor of carnitine palmitoyltransferase 1, it has only recently been demonstrated that a 280 kDa isoenzyme of ACC exists in the heart that is the source of malonyl CoA. We have provided evidence that heart ACC, via the production of malonyl CoA, is a key regulator of fatty acid oxidation. We have also shown that ACC represents a key enzyme in a feedback loop that decreases acyl CoA transport into the mitochondria when carbohydrate oxidation rates are increased. ACC represents a novel and potentially important site for pharmacological intervention in situations involving abnormal fatty acid metabolism.

THE ROLE OF ADENOSINE AND A D E N O S I N E RECEPTOR SUBTYPES IN P R E C O N D I T I O N I N G Tetsuji Miura, Katsuo Suzuki, Takayuki Miki, Osamu limura. 2nd Dept of Int Med, Sapporo Med Univ, Japan

L27

Previous studies from our laboratory indicated that an elevation of the interstitial adenosine (ADO) level and activation of ADOA1 receptors during preconditioning (PC) ischemia contribute to infarct size (IS) limitation by PC. In the present studies, modulators of the ADO level and effectors of ADO receptors in the mechanism of PC were investigated. In the first series of experiments, the contribution of fl-adrenoceptors to ADO production during PC was tested. PC significantly limited IS, but this protection was not detected in reserpinized rabbits and those pretreated with atenolol. An infusion of isoproterenol restored the PC effect in reserpinized rabbits, which was abolished by 8-phenyltheophylline. In the second series of experiments, to test the role of ecto-5'-nucleotidase (ecto-5'N) in PC, PC effect was assessed when 0.75~1.50 mg/kg/min of a, f]-methyleneadenosine 5'-dtphosphate (AOPCP), an inhibitor of ecto-5'N, was infused into the left atrium. The efficacy of AOPCP on ecto-5'N in vivo was confirmed by attenuated inotropic response to an AMP injection. However, IS limitation by PC was not blocked by the AOPCP treatment. These results suggest that sympathetic nerves may be an important modulator of ADO level during PC, but ADO production by ecto-5'N activity is probably not necessary for triggering PC in the rabbit. In the third series of experiments, it was found that IS limitation by PIA, an Al-receptor agonist, was abolished by polymyxin B and staurosporine. This finding suggests acrucial role of protein kinase C in the PC mechanism downstream to Al-receptor activation.

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MODULATION OF CALCIUM CHANNELS BY ct-ADRENOCEPTORS IN VASCULAR MYOCYTES Jean Mironneau, Physiopathologie et Pharmacologie vasculaire, URA CNRS 1489, Universit6 Bordeaux II, Bordeaux, France

L26

The purpose of this study was to investigate the contribution of both oh- and cq-adrenoceptor subtypes in inducing stimulation of voltage-operated calcium channels in single myocytes from rat portal vein. Norepinephrine, phenylephrine, clonidine and ox'ymetazoline stimulated the calcium channel current by a similar extent. Using subtype-selective antagonists we showed that both ohA- and cz.~-adrenoceptors modulated calcium channels through two distinct transduction pathways. ~x~Aadrenoceptors coupled with a pertussis toxin-insensitive Gprotein, whereas ¢~-adrenoceptors coupled with a pertussis toxin-sensitive G-protein. Portal vein myocytes expressed Gproteins that were recognized by anti-czq/Cql,(zi~.2,eq3- and oto2antibodies. Using antibodies directed against the cx-subnnits of G-proteins and phosphatidyl-inositols, we demonstrated that : (1) cqA-adrenoceptors activated a Gq/G~-protein to stimulate phosphatidylinositol hydrolysis leading to release of calcium from intracellular stores and enhancement of calcium channel current ; (2) ct.,.A-adrcnoceptors activated a Gij.2-protein but did not induce any calcium release from intracellular stores. Calcium channel current was stimulated and the coupling involved activation of protein kinase C. These results suggest that two distinct G-proteins, Gq/Glj and Gil-2, coupled to ~Z~A-and o~z~adreneceptors regulate calcium influx flu'ough voltage-operated calcium channels by two different transduction pathways leading to activation of protein kinase C and subsequent phosphorylation of calcium channels.

MYOCARDIAL ION REGULATION DURING L28 ISCHEMIA AND HYPOXIA Seibu Mochizuki, Satoshi Takeda. Fumiko Suzuki, Masayuki Taniguchi. Department of Medicine, Aoto Hospital, Jikei University School of Medicine, Tokyo, Japan. Complex changes in ionic movement through the sarcolemma occur after ischemia or hypoxia, and may be restored or aggravated after reperfusion or reoxygenation. The earl!est change is a loss of K'and Mg÷ and significant changes in ion content, increases in [Ca-~*]i,Hi" and [Na*]i are also observed. However, the interactions between these ions are not fully understood. In this study, the effect of iscbemia or hypoxia on intracellular and extracellular ionic changes was investigated in isolated perfused rat heart. [Ca:+]i, pHi and [Na+]i were measured using fluorescence indicators, Fura-2. BCECF and SBFI, respectively. Cytoplasmic K ÷ and H* are lost during ischemia, possibly causing the functional deterioration in reperfusion. Both glibenclamide and amilori.'de increase the functional recovery during reperfusion. During hypoxia [Ca-~*]i increases in a biphasic pattern while pHi decreases rapidly from 7.2 to 6.8 at 3 min and 6.6 at I0 rain after hypoxia starts. [Na*]i increases gradually during hypoxia. Both the forward and reverse modes of the Na*/Caz" exchanger were markedly inhibited during hypoxia and recovered slowly on reoxygenation. Na÷ channel blocker partially prevented an increase in [Ca~'°]i during hypoxia. These data collectively indicate the complex spectrum of ionic changes that occur during both ischemia/hypoxia and reperfusion/reoxgenation. They include: I ) intracellular acidosis, 2) increases in K* and H* loss caused by the inhibition of Na/K ATPase and the opening of Al"P-sensitive K channel, 3) an increase in [Na°]i via Na"/H" exchanger and Na ÷ channel, and 4) an increase in [Ca-'÷]i via the inhibition of the Na+/Ca2÷exchanger.

ALTERATIONS IN CYTOSOLIC FREE MAGNESIUM DURING M Y O C A R D I A L ISCHEMIA. E. Murphy. LMB, NIEHS, Research Traingle Park, NC, USA.

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Recent development of new fluorescent and NMR sensitive indicators for measuring cytosolic free magnesium (Mgi) have advanced our understanding of magnesium homeostasis. Under normal conditions Mg i is regulated far from electrochemical equilibrium; although the tranport processes responsible for Mg i regulation are still debated. The fluorescent magnesium indicator, furaptra, has been used to investigate the regulation of Mgi. We have also used the NMR sensitive magnesium indicators which are fluorine labelled derivatives of o-aminophenol -N,N,O-triacetate (FAPTRA) to measure changes in Mgi during global myocardial ischemia in Langendorff perfused rat hearts. Hearts were loaded intracellularly with FAPTRA by perfusion with Krebs-Henseleit buffer containing the acetoxymethyl ester of the indicator. Basal Mgi concentrations averaged 0.85 _+0.1 mM (n=9). Between 10 to 15 minutes of ischemia, Mgi rose nearly 3-fold to 2.1+0.4 mM. This increase occurred over the same time course as the decrease in ATP. After 20 minutes of reperfusion with oxygenated Krebs-Henseleit buffer, Mgi declined to 1.5+0.5 mM.

LIMITED CAPACITYFORWORKINGOVERLOAD IN THE L30 V3 MYOSIN PREDOMINANTRAT HEART. Makoto Nagano, Makoto M i y a i r i , OsamuKawashima, Jikei University School of Medicine, Tokyo/Japan In smal mammalians, a l t e r a t i o n of cardiac cond i t i o n as well as cardiac overload, diabetic and hypothyroidic status induce a change in the composition of v e n t r i c u l a r myosin isoenzymes.The cardiac function of hypertrophied hearts with overloaded work or heart with negative energy balances are regulated economically by the isomyosin V3 predominant c o n t r a c t i l e protein. The V3 predominant v e n t r i c l e shows aslower contraction - r e l a x a t i o n and a decreased Vmax. The sliding v e l o c i t y of myosin V3 on the actin cables in v i t r o is slower than t h a t of myosin VI. Fromt h i s standpoint, we examined the limited capacity f o r working overload of the V3 myosin predominant r a t heart. The used animals: Diabetic, hypothyroid and age-mached Wistar rats. Cardiac overloads were performed using a t r i a l pacing a t the heartlung-preparation of these r a t s . Pacing frequence was performed a t a pacing interval from 180 msec u n t i l 120 msec.Changes of myosin isoenzyme, blood

g l u c o s e and serum T3 l e v e l were measured. The i n i t i a l heart r a t e s in both pathological animals were lower than in t h a t of c o n t r o l . When the pacing rate goes up, cardiac output(CO) goes down and central venous pressure(CVP) up. The pacing rate of the cross point(RCP) between the CVP- and CO-curves was calculated. RCPwas s i g n i f i c a n t l y lower in the myosin V3 predominant heart than in the control heart. A s i g n i f i c a n t l y negative r e l a tionship between RCP and myosin V3 content was o b s e r v e d . These r e s u l t s suggest that t h e V3 p r e -

dominant h e a r t i s i n t o l e r a n t

f o r high frequency

heart rate.

FUNCTION OF ION CHANNELS IN MODULATION L31 OF Ca =" SIGNALING IN HUMAN ENDOTHELIAL CELLS B.Nilius, G.Droogmans, F.Viana, J. Eggermont. KU Leuven, Campus Gasthuisberg, Department of Molecular

Cellbiology, Laboratoryof Physiology, B-3000 LEUVEN, Belgium Endothelial cells plays an important role in the control of a variety of biological functions, such as the tone of vascular smooth muscle cells, maintenance of blood fluidity, inflammatop/and immunological processes. Most of these processes (e.g. NO release) depend on changes in the intracellular Ca2° concentration ( [Ca2*]i ). An increase in [Ca2"]~ occurs via Ca2" -release and transmembrane Ca= entry. Depletion of intracellular Ca 2" stores activates a tiny Ca2" entry (Ca2. release activated Ca2" currents, CRAC (1)) which depends on the driving force, and is in the range of 1-3 pA/cell. The same Ca2" entry can be also activated by mechanical stimulation of endothelial cells. This entry is efficiently modulated via changes in the driving force by at least two other types of channels: a) a Ca2" dependent K channel, b) a CI channel. Co-activation of these channels clamps the membrane potential at a negative potential and stabilizes the inwardly driving force for CRAC. The CI channel can sense changes in the cell configuration, as well as mechanical forces, such as shear stress and cell swelling, and could thus act as a mechano-sensor in endothelial cells. A possible candidate for this channel or a regulator of this mechanically induced CI conductance is the 235 amino acid protein Inn (2), which has been detected and cloned in human endothelial cells. Gating, permeation properties and pharmacological modulation of this channel will be described in more detail. (1) Hoth M, Penner R. Nature 355, 353-3576, 1992 (2) Paulmichl M. et aL Nature 356, 238-241, 1992

The Guanine Nucleotide-Binding Regulatory Protein (G protein) in A c u t e I s c h e m i c Myocardium

L32

Mitsumasa Ohyanagl, Tadaakl Iwasaki. First Dept of Internal Medicine, Hyogo College of Medicine, Nishinomlya, Japan

It has been reported that the function of the stimulatory guanine-binding regulatory protein (G protein) in myocardium is decreased with acute ischemia. However, it is unclear whether this decrease is due to transcriptional or post-transcriptional changes. To explore alterations in mRNA of G proteins (Gs and Gi) in ischemic hearts, we measured the levels of mRNA for Gs a and Gi a in isehemie and nonischemic myocardium by in situ hybridization using a radioisotope imaging system. We compared these mRNA levels in ischemic and nonischemic myoeardium with Northern blot analysis and the protein levels of G proteins by Western blot analysis. One and three hours of ischemia resulted in a decrease in mRNA of Gs and Gi, accompanied by a decrease in the level of these proteins as measured by Western blot. These results were confirmed by the activity of adenylate eyelase. These results suggest that decreased levels of mRNA and protein for G proteins may underlie the impaired function of the receptor - G protein - adenylate eyelase system in ischemic myoeardium. A273

REGULATION OF THE SARCOLEMMAL L33 P H O S P H O I N O S I T I D E P A T H W A Y BY THYROID HORMONE Vincenzo Panagia & Nasrin Mesaeli. Division of Cardiovascular Sciences, St. Boniface G.H. Research Centre, Univ. of Manitoba, Winnipeg, Canada. We examined the effects of different plasma levels of thyroid hormone on the rat heart sarcolemmal (SL) phosphoinositide pathway. Hyperthyroidism was induced by daily injection of thyroxine (T,, 50 lag/100g b.w.) for 7 days. Two hypothyroid groups were examined: thyroidectomized and propylthiouracil (PTU, 0.05% in drinking water for 7 weeks) treated rats. The latter group was injected with T, for 14 days while kept on PTU to reverse hypothyroidism. Both SL phosphoinositide kinases were upregulated in hyperthyroid hearts and unaffected by hypothyroidism. SL phospholipase C (PL C) activity was measured using 3H-phosphatidylinositol 4,5-bisphosphate as a substrate, and was found to be significantly higher than controls in both PTU-treated and thyroidectomized rats. This enzyme activity was normalized upon reversal of hypothyroidism and was decreased in hyperthyroid SL. The observed PL C changes, which paralleled thyroid hormone-induced changes in heart ctt/13j adrenoceptor ratio (NaunynSchmied. Arch. Pharmacol. 334:275, 1986), indicate a regulatory role of thyroid hormone on SL PL C. (Supported by MRC Group in Exptl. Cardiol.)

NORMAL AND ABNORMAL DEVELOPMENT OF L35 THE HUMAN AND ANIMAL HEART - ORGAN LEVEL ANALYSIS. Tomas Pexieder, Institut d'Histologie et d'Embryologie, Universite de Lausanne, Lausanne, Switzerland

Understanding of prenatal organogenesis requires a multilevel analytical approach. Each level needs adequate methodology. The microdissection and scanning electron microscopy provide details about morphology of the developing heart for efficient studies of cellular, molecular and functional aspects. Using chick, mouse, rat, dog and human embryos we have focused on the morphogenesis of the outflow tract (conotruncos)disproving many of the textbook myths like "transfer" of the aorta into the left ventricle. Our understanding was helped by experimental models such as fetal mouse trisomies, Keeshond dogs, trimethadione treated rats, retinoic acid exposed mice or more recently transgenic mice with either TGF 3 or RXR knocked out. Combining the use of immunohistochemistry (PDGF, ESI30,GP23, Cx37, carbonic anbydrase) with in situ hybridization, PCR and computer aided 3D reconstruction we try to identify the positional information guiding the conotruncus ridges development. Their form, size and location are modified in experimental situations mentioned, and are involved in pathogenesis of most frequent cardiovascular malformations. The retinoic acid and RXR knockout experiments open an entirely new field: development of myocardial architecture and its regulation.

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PROTEIN REMODELLING OF THE NORMAL L34 AND MALFORMED FETAL HEART. V/tclav Pelouch1, Tomas Pexieder2, Marie IVlilerovtP,Bohuslav Ott'~daP, IInst of Physiology, Acad Sci, Prague, Czech Republic, 2Inst of Histology and Embryology, Lausanne,

Switzerland. There is almost no evidence available that cardiac malformations are associated with altered protein composition. To study this question, an intraperitoneal injection of 20 mg/kg retinoic acid (RA) - a potent cardiovascular teratogen - was given to pregnant mice on the 8th embryonic day (ed). The quantitative and qualitative remodelling of collagenous (CP) and noncollagenous (NCP) cardiac proteins was analyzed during the late fetal period. It has been shm~ that application of the oily vehicle results in an increase of total protein (TP) of the fetal hearts; this increase is mainly due to a higher NCP concentration. RA significantly decreased the amount of TP in both morphologically normal and malfomaed hearts (transposition of great vessels - TGA and double outlet of right ventricle - DORV). This change was significantly smaller on ed 19, namely in the TGA group. NCP (fractions of metabolic and contractile proteins) were more affected by RA than CP (composed of proteinaceous material of pepsin-soluble and -insoluble extracellular matrix) on both ed 15 and 19. The concentration of contractile proteins was significantly higher in the malformed heart as compared with the RA-treated non-damaged myoeardium. Our results suggest that surgeons who are reconstructing cardiac anatomy should be aware that they are dealing with a biochemically abnormal tissue. This might explain some of the failures or late problems in children who had surgery for congenital heart disease.

A FORGO'CI'EN M E M B R A N E SYSTEM: L36 P R O T E I N S IN T H E CELL NUCLEI Grant N. Pierce, Michael P. Czubryt, James S.C. Gilchrist & Brain Ramjiawan. Div. Cardiovasc. Sci., St. Boniface Res. Ctr., Winnipeg, Canada In comparison to work devoted to the manipulation of DNA within the cell, relatively little attention has been given to the nuclear membrane which encapsulates the cell's genetic material. Our work on nuclear proteins has focussed on 2 types: Ca2+ binding proteins (CaBP) & a nucleotide triphosphatase (NTPase). The NTPase has been suggested to be associated with the nuclear pore complex & provides energy for the nuclear export of mRNA into the cytoplasm. The NTPase activity is sensitive to membrane cholesterol. It is stimulated by in vitro cholesterol incorporation into the nuclear membrane & inhibited by cholesterol oxidation. Elevated nuclear NTPase activity & membrane cholesterol have been identified in animals with high circulating lipid levels. We have also identified several CaBPs in the nuclei which partition either into the membrane or the nucleoplasm. Their function is unknown but they may modulate the effects of Ca2+ on cell growth. In summary, initial work on the NTPase and the CaBPs has been exciting and clearly justifies further study of these & other nuclear membrane proteins. (Supported by the Medical Research Council of Canada).

CRITICAL STEPS IN THE R E C O V E R Y OF L37 CATION CONTROL IN THE R E O X Y G E N A T E D CARDIOMYOCYTE. H.M. Piper, Yu.V. Ladilov, B. Siegmund. Physiologisches Institut, Justus-Liebig-Universitat, Giessen, Germany. In isolated v e n t r i c u l a r cardiac myocytes (adult rat), the changes in i n t r a c e l l u l a r ion homeostasis during anoxia (pH o 6.4) and reoxygenation (pH o 7.4) were analysed. Cells were incubated in anoxic media at pH 6.4 until pCa i ~ 5, pH4 6.5 and cytosolic Na + 50 mM was reac~ed. Upon reoxygenation m e d i u m pH was changed to 7.4, to activate Na+~H + exchange. Cytosolic Ca ~* and Na t ~eturned to control within 10 min. Ca 2+ recovery d e p e n d e d on (i) s e q u e s t r a t i o n into sarcoplasmic r e t i c u l u m and [ii) extrusion via sarcolemmal Na+/Ca 2+ exchange. Prerequisite was sufficient activation of Na+-K + pump. pH i r e n o r m a l i s e d (pH i 7.2) within i0 min. Activation of Na+/H + exchange impaired r e c o v e r y 9f Ca 2+ control only if activity of Na ~K + pump is critically low. Prolongation of cytosolic acidosis is beneficial primarily because acidosis protects against reoxygenation-induzce~+ h y p e r c o n t r a c t u r e Ca and cytosolic oscillations.

POSTNATAL DEVELOPMENT OF THE L39 MAMMALIAN HEART: GROWTH OF THE VASCULAR STRUCTURES. Karel Rakusan and Marcia I. Heron, Dept. of Physiology, Fac. of Medicine, University of Ottawa, Ottawa, ON, Canada.

The early postnatal period of development in the mammalian heart is characterized by a sizeable proliferation of capillaries and artedoles - larger vessels are alread~ formed before birth. This is accompanied by the maturation of the vascular walls with the establishment of more definite morphological and biochemical features around the weaning pedod. Knowledge of normal vascular growth and development should serve as a basis for understanding the agerelated differences in response to vadous growth pathological stimuli. For instance, comparison of neonatal and adult cardiac hypertrophy induced by pressure overload revealed vascular proliferation in the former and yet minimal vascular growth in the latter situation. In addition, a study examining the effect of hypo- and hyperthyroidism in rats demonstrated that ~)oth interventions affected coronary capillary and artedolar growth. Surprisingly, responses of arterioles were more pronounced than those of capillaries. Thus, developmental stage and the type of stimulus determine the cardiac adaptive response. Increasing age of the organism is characterized by decreasing plasticity of myocardial tissue.

THE ROLE OF PROTEIN KINASE C A N D G - P R O T E I N S IN I S C H A E M I A AND PRECONDITIONING.

L38

N. J. PYNE University o f Strathclyde, Department o f Physiology and Pharmacology, 204 George St, Glasgow, Scotland, G1 1XW. During sustained periods of coronary occlusion adenylyl cyclase activity is acutely sensitised, a response which becomes refractory as a consequence of preconditioning. This may be a consequence of a rapid adaptive modulation by protein ldnase C. Since adenylyl cyclase is regulated by stimulatory (Gs) and inhibitory G-proteins (Gi), much attention has been placed upon alterations in the functioning of these proteins. The presentation will review possible mechanisms of G-protein and adenylyl cyclase modulation by protein kinasc C. Preconditioning appears to elicits an alteration the conformation state of the G-proteins, as evidenced from ADPribosylation studies. Under conditions of linear toxin-catalysed ADP-ribosylation, the susceptibility of Gi to pertussis toxincatalysed ADP-ribosylation is increased, whilst the susceptibility el Gs to cholera toxin is reduced. We propose that preconditioning may remove a functional pro-arrhythmic protein kinase C-dependent regulation of the Gproteins. In this context, we will also demonstrate that the calcium. independent protein Idnase Cs isoform is rapidly removed from the cytoplasm of the myocarduim during preconditioning. Whilsl this isoform is localised in isolated membranes and the nuclear fraction, its rapid removal from the cytoplasm is not associated with an increased level in these cell fractions. We suggest that protein kinase CE is rapidly down-regulated and therefore, may be linked to the refractory changes in adenylyl cyclase responsiveness. The potential relationship between adenylyl cyclase and protein kinase C may have important physiological implications in allowing cardioprotection.

NOVEL ASPECTS OF THE REGULATION L40 OF VOLTAGE-SENSITIVE K-CHANNELS Jochen ROper and Olaf Pongs , Zentrum fQr Molekulare Neurobiologie, Haus 42, Martinistr. 62, D-20246 Hamburg, Germany.

Several distinct classes of potassium channel genes are expressed in heart and involved in different aspects of repolarization of the cardiac action potential (AP). Several members of the voltage-gated Shaker-like K channels (Kv) are expressed in cardiac tissue coding for either rapidly inactivating (Kvl.4) and delayed-rectifier type K channels (e.g. Kvl.5, Kvl.2). Biophysical properties of Kv channels can be dramatically altered by coexpression of KvSsubunits. The rapidly inactivating ( I t o ) K v l . 4 channels play an important role in early repolarisation setting the plateau potential and thereby determining AP duration. Patch-clamp studies on heterologously expressed Kvl.4 in HEK 293 cells showed that the fast inactivation which is thought to be mediated by a N-terminal ball and chain mechanism is modulated by protein kinase activity and also affected by the metabolic parameters like the redox state or intracellular ATP levels. In addition, the gating of Kvl.4 is modulated by a rise in intracellular Ca2+. These new mechanisms of statedependent modulation of fast inactivating Kv channels add further aspects to the already puzzling complexities of multiple channel genes, heteromerization, and novel 8-subunits of Kv channels. Factors which are likely to contribute to the fine tuning of cardiac repolarization.

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CROSSBRIDGES IN CALCIUM SENSITIVITY L41 MODULATION Johann C. Rfiegg and Anders Arner*. Dept of Physiology II, Univ. of Heidelberg, Germany, and Dept of Physiology & Biophysics, Univ. of Lund, Sweden.

EFFECTS OF CPT-1 INHIBITION ON MYOCYTE L42 GENE EXPRESSION IN OVERLOADED HEARTS Heinz Rupp, Christian G. Brilla" , Bernhard Maisch" & Marian Turcanf'. University of T0bingen, "University of Marburg, FRG & "'University of Bratislava, SVR.

During contraction crossbridges attach and detach from thin filaments with rate constants fapp and gapp., respectively. According to Brenner (P~/~.S 85, 3265, 1988), calcium activation increases fapp, but the ratio of fanm (at maximal Ca2+ activation) and gapp also deter~nri'nes the calcium responsiveness or the "Ca2+ sensitivity. Accordingly, we found that a novel cardiotonic drug (EMD 53998) increased fapp. thereby increasing the Ca2+ responsiveness of skinned cardiac muscle fibres suspended in ATP-salt solution. The drug affects crossbridges directly, since it also alters contractility in skinned fibres that are depleted from a functional regulatory protein system (by extracting Tnl with a vanadate extraction procedure). The novel cardiotonic drugs EMD 53998 (and its enantiomer 57033) may therefore be considered as crossbridge agonists. By affecting the rate of crossbridge attachment at a given Ca 2+ concentration such drugs antagonize as it were the effects of inorganic phosphate that is released during strong crossbridge attachment as well as mimicking the effect of myosin light chain phosphorylation. The latter also increases fapp. thereby increasing calcium sensitivity.

In the failing heart, gene expression of various cell types is affected in a detrimental manner. Fibroblasts are stimulated by angiotensin II and aldosterone, resulting in an adversely remodeled extracellular matrix. Because interstitial heart disease is most probably preceded by neuroendocrine activation due to inadequate myocyte performance (impaired protein phenotype), efforts are needed to pharmacologically modulate cardiocyte gene expression. The carnitine palmitoyltransferase (CPT-1) inhibitor, etomoxir (Eto), emerged as lead compound which raises glucose oxidation, the density of SR Ca 2+ pumps and myosin Vl (AJP 1994;267:H2091-2099)° In overloaded (supracoronary aortic constriction; AC) rat ventricles, the reduction in V1 (26% vs 50% sham) was partially (P
CONTRACTILE PROTEINS IN THE CARDIOMYOCYTE Marcus C. Schaub, and Hans M. Eppenberger* Institute of Pharmacology, University of Zurich, CH-8057 Zurich, and *Institute of Cell Biology, ETH, CH-8093 Zurich, Switzerland

L43

The contractile apparatus of the cardiomyocyte is akin to that of skeletal muscles. A number of differences on the molecular level though allows for more subtle regulation of contraction involving both the contractile proteins and the calcium handling devices. In rodents the isoform expression changes under cardiac overload from co-myosin heavy chain (cz-MHC) to the more economically working 13-MHC. In man 13-MHC is evailing in ventricles. Mutations in critical regions of I~HC have been found in familial hypertrophic cardiomyopathy which affect contractile function in vitro. We have described the appearance of the atrial/fetal myosin light chain-1 (ALC1) under mechanical overload. This ALC1 increases the calciumsensitivity of skinned papillary muscle fibers from hypertrophic human ventricles. Under chronic ischemic conditions ~-smooth muscle actin (¢z-SMA) accumulates in ventricular myocytes together with disturbances of the myofibrillar structures (Ausma et al., Cardiovasc. Pathol., in press). We observed strong upregulation of c¢-SMA in adult rat cardiomyocytes in long-term culture under treatment with fibroblast growth factor (bFGF). Accumulation of c¢-SMA seems to inhibit myofibrUlar assembly and growth. ALC1 and ~-SMA are transiently expressed in ventricular tissue during development. Their re-appearance under overload and/or paracrine cell mediators represents in part a recourse to the fetal expression program in order to meet changed demands.

~

A276

AI2PI{A-~kDRE~GIC RECEP'~RS IN THE HEART AND

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MESSENGER SYSTEMS Hasso Scholz, Joachim Neumann, Thomas Eschenhagen & eirgitt Stein. Pharmakologisches Institut, Universit~ts-Krankenhaus Eppendorf, 20246 Hamburg, FRG. In the failing heart the beta-adrenergic positive inotropic effect (PIE) is attenuated, and alphaadrenoceptor (A)-mediated inotropism, which involves an increase in IP3, may gain importance under these conditions. The number of cardiac alphaadrenoceptors has been shown to be enhanced in endstage myocardial failure. However, previous studies with phenylephrine (Ph) as an A agonist revealed only small PIEs of A-stimulation. Therefore, the effect of noradrenaline (NA) as an A agonist was compared to that of Ph. Trabeculae were obtained from the hearts of patients undergoing cardiac transplantation due to idiopathic dilated cardiomyopathy. HA (i00 ~J4 in the presence of i0 ~M propranolol) increased force of contraction by about 90% of the predrug value. Subsequently applied carbachol (I0 ~M) had no negative inotropic effect indicating the absence of a cAMP-dependent effect. T h e A 1 antagonist prazosin (i ~M) and the A 1 A antagonist WB 4101 blocked the PIE of NA wheras the A 1 a antagonist chlorethylclonidine was nearly ineffective. The PIE of Ph (i00 ~M) was much smaller than that of NA. Thus, in the failing human h e a r t the magnitude of the A l-mediated PIE apparently is agonist-dependent and A 1-mediated effects of the physiological agonist NA might be important in order to sustain contractility under conditions of impaired beta-sdrenergic stimulation. (Supported by the Deutsche Forschungsgemeinschaft)

NA/H EXCHANGE IN ESSENTIAL HYPERL45 TENSION Winfried Siffert, Institut fOr Pharmakologie, Universit~itsklinikum, Hufelandstr.55, D-45122 Essen, Germany

OVERVIEW: QUANTITATIVE MODELS L46 OF THE BEAT TO BEAT CONTROL OF CARDIAC CONTRACTION Jiri ~imurda, Dept of Physiology, Faculty of Medicine, Masaryk University, Brno, CZ.

An enhancement of Na/H exchange (NHE) activity has been determined in approximately 50% of all patients with essential hypertension (EH). This enhancement is not only determined in blood cells, but also in skeletal muscle (Rosskopf et al., Hypertension 21:607-617, 1993). It was hypothesized that the accelerated pH i regulation could contribute to abnormal proliferation processes in EH, e.g. media hypertrophy in resistance vessels and left ventricular hypertrophy. We found that enhanced NHE activity persisted in immortalized cells from patients with EH even after prolonged cell culture (Rosskopf et al., J.Clin. Invest. 92: 2553-2559, 1993) which suggests that this abnormality is under genetic control. However, an overexpression of the NHE or a mutation in its gene could be ruled out. Interestingly, cells from patients with EH proliferate distinctly faster than those from normotensive controls, and this enhanced proliferation is not a consequence of enhanced NHE activity. Recent findings suggest that the enhanced NHE activity results from an enhanced signal transduction via pertussis toxin-sensitive G proteins, which also leads to an increased formation of inositol trisphosphate and increased Ca2+ mobilisation.

The early quantitative models of the beat to beat control of cardiac contraction were mostly based on formal mathematical descriptions of largely unrecognized mechanisms. With increasing knowledge the models have been gradualy identified with concrete physical and chemical processes. The present quantitative models are supported by increasing evidence related to particular steps in the sequence of events between excitation and contraction. Partial quantitative models have utilized the experimental data concerning the Ca2+ fluxes via membrane channels and Na/Ca exchange, diffusion of Ca2+ between various compartments, binding to intraceflular ligands and the interactions of Ca z+ with sarcoplasmic retieulum and mitochondria. However, the internal movements of Ca2+ are not yet fully understood leaving space for alternative interpretations and modelling. Recently, the partial models have been suceesfully integrated into quantitative models of the entire system of the beat to beat control of cardiac contraction. In addition, such models must reflect negative feedbacks regulating the level of available Ca 2+ and the balance in movements of all other ions involved. Own results of computer modelling including different variants of intemal Ca2+ movements and respecting the above premises will be presented.

PROPRANOLOL PROTECTION AGAINST L47 ISCHEMIC INJURY: AN ANTIOXIDANT EFFECI" P.K. Singal, N. Singlh C.K. Seneviratne, C. Rigatto and .B. Kowaluk. Div. of Cardiovasc. Sci., St, Boniface Gea. Hosp. Res. Ctr., Dept. of Physiol., Univ. of Manitoba, Winnipeg, Canada. Effects of propranolol (PROP), a non-specific/3-adrenergic blocking drug, on ischemia-reperfusion (I-R) hJjury ill isolated perfused hearts were studied in relation to changes ha myocardial autioxidants. Rats were treated with 10 mg/kg/day PROP (i.p.) for 1 and 3 weeks and the hearts were analyzed for contractile function and autioxidaut enzyme activities as well as mRNAs. Hearts from PROP treated rats were better protected against injury due to 60 m of global I followed by 40 m R. Catalase (CAT) and glutathione peroxidase (GSHIXx) activities were increased in the treated group as compared to ~utrols, whereas superoxide dismu tase (SOD) activity showed no dlange. Total RNA was isolated and probed with human GSHPx, CAT and SOD cDNAs nick-translated to a specific activity of I08 dpnffug DNA. Mouse 28s r~osomal RNA was used as a control for RNA loading. PROP treatment did not change mRNA abundance for any of the enzymes. In in vitro studies, addition of PROP (1 rag/L) in the perfusion medium did not cause any change in antioxidant enzyme activities but it offered protection against I-R iujury. Use of atenolol (65 rag/L), a cardiosclective fl-blocker, in in vitro studies offered no protection agahlst I-R injury. It is suggested that protection by a chronic treatment with PROP may involve increased antioxidaut enzyme activities without any change in mRNA expression. Protection under acute conditions may involve direct antioxidaut effect of the drug rather than ,0-blockade or antioxidant enzyme chauges. (Supported by the Medical Research Council of Canada).

THE ROLE OF I-I202 IN SEQUENCE OF L48 EVENTS LEADING TO ISCHEMIA-REPERFUSION INJURY OF THE HEART. Jan Slezak, Inst Heart Res, Bratislava, SIovak Republic Deteriorative effect of oxygen radicals is accompanied with the accumulation of 1-1202from different sources generated by critically but reversibly injured ischemie cardiac myocytes. Cytochemieal as well as biochemical assessment of I-hO2 in the early period of postischemic reperfusion revealed up to sixfold increase in I-h02 concentation in the 2 min of reperfusion period. I-I202 accumulation at the end of 30 rain ischemia or in the 8 min of reperfusion was about threefold of the preischcmic level. The intensity of cytoehemically demonstrated precipitate of cerium perhydroxide indicate that in the reperfused heart the highest accumulation of l-hO2 was localized in the glycocalyx of myocytes and on the abluminal surface of endothelial cells of the vessels. A lower amount was found randomly distributed throughout the myocytes, mitochondria and endothelial cells. Attempts to find the most important source of H202 failed to discriminate any of the specific oxidases assessed. The effect of I-~02 is mediated through its influence on the membrane proteins involved in maintenance of ion homeostasis in cardiac ceils resulting in the uncontrolled Ca influx. The second effect of 1-1202 is associated with damage to the endothelial cells affecting blood flow and related functions. Conclusion: sudden transitional depression in myocardial contractility detected in the early reperfusion period can be caused by an increase in l'hO2 concentration affecting the structures responsible for ion homeostasis resulting in the enhanced Ca influx. A277

DESENSITIZATION MECHANISM AT THE CONTRACTILE L49 PROTEINS IN THE STUNNED MYOCARDIUM L.K. Soei, K. Bezstarosti, J.M.J. Lamers, R. Krams & P. D. Verdouw. Experimental Cardiology, Thoraxcenter, and Department of Biochemistry, Erasmus University, Rotterdam, The Netherlands

Two mechanisms have been proposed as underlying myocardial stunning: A decrease in activator Ca2. or a reduced sensitivity of the myofibrils (MF) to Ca2÷. In our stunning model of 2 consecutive sequences of 10 rain ischemia and 30 min occlusion we established that the expression of SR Ca2÷pump protein and the SR Ca2÷ pump activity were not reduced refuting the first mechanism. Subsequently we studied the effects of the specific Caz÷ sensitizer EMD 60263 on stunned myocardium in situ. Stunning reduced segment shortening and external work to 50% (16+1% and 183:1:23 mmHg.mm at baseline, respectively) and mechanical efficiency (EW/MVOz) to 60% of baseline. EMD 60263 dose-dependently restored these contractile variables of stunned myocardium to baseline values, also in the presence of (:{- and adrenoceptor blockade. Ca2. sensitivity of the SR and MF Ca=*ATPase was measured in vitro in control and stunned myocardium, but no differences were observed. We used the specific inhibitor of the SR Ca2÷ATPase, thapsigargin, to differentiate between the 2 types of ATPase. EMD 60263 caused in vitro a leftward shift of the Ca2÷ activator curve of control MF Ca2*ATPase, but not of the SR Ca2÷ATPase. In conclusion, the EMD 60263 effects in vivo and in vitro suggest that a decreased sensitivity of MF ATPase to Ca2÷ plays an important role in the prolonged contractile dysfunction in stunned myocardium. Which cellular signal(s) are responsible for these changes in the MF properties is presently unknown. (Supported by grant 92.308 of The Netherlands Heart Foundation)

CATECHOLAMINES,POSSIBLE PREVENTIVE THERAPY?

AGENTS

FOR

L51

L~szI6 Szekeres.Instituteof Pharmacology, A.Szent-Gy6rgyiMedical Univ.Szeged,HUNGARY We described earlier the delayed cardiac adaptation to stress, protecting the heart from harmful consequences of a more severe stress.Adaptation was induced by drugs such as PgI2 or its stable analogs (Basic Res. Cardiol.86:215-221,1991) or by repeated brief ischaemic periods (rapid pacing; Cardiovasc. Res.27:593-596,1993) .A common trait of these interventions is an activation of the adenylcyc lase/cAMP pathway. Hence we tested in instrt.nented,conscious rabbits whether ~-adrenergic agonists might evoke delayed cardiac adaptation to stress. A preventive application of this principle in therapy requires that the dose inducing adaptation should be wel I tolerated and reproducible. 2 ug/kg i.v. isoprenaline(ISO) meets these requirements.A single or a few injections repeated at i0 min intervals were not sufficient to induce adaptation. However, i0 doses given in a similar way proved to be successful. Thus changes due to cardiac loading by 2 ug/kg ISO+I6 ug/kg phenylephrine i.v., such as a rise in ST segment and in the heart rate were markedly reduced 24 and 48 h after treatment with i0 ISO doses.Conclusion: Fractionated administration of low dose Oagonists is promising for preventive therapy.

A278

L50 MUTAGENESIS AND TRANSGENIC APPROACHES TOWARD UNDERSTANDING REGULATION OF HEART MYOFILAMENTS BY TROPONIN-TROPOMYOSIN. R. John Solaro, Kimberly Palmiter, Yoshimi Kitada, Mariappan Muthuchamy, David Wieczorek. Colleges of Medicine, Univ. Illinois Chicago, Chicago, IL 60612 and Univ of Cincinnati, 45267.

The myofilament response to Ca 2+ ultimately involves an altered state of tropomyosin (Tm). By use of a cardiac specific promoter, we have generated transgenic mice, which express muscle specific B-Tm in the cardiac compartment. The/3Tm incorporates into the myofilaments in stoichiometric amounts and the amount of a-Tm, the native form, is reduced. Compared to preparations from non-transgenic (NTG) controls, the pCa-force relation of Triton X-100 extracted bundles of fibers from transgenic (TG) mouse hearts demonstrated a small but significant increased sensitivity to Ca 2+. When extracted with saponin and treated with cAMP/protein kinase A desensitization of NTG was greater than TG myofibrils. Reconstituted preparations containing a deletion mutant of TnI demonstrated that it is TnI that is responsible for this desensitization. Our results indicate that the isoform of Tm may be an important determinant of control of myofilaments.

MUTATIONS OF MITOCHONDRIAL DNA L52 IN CARDIOVASCULAR DISORDERS Nobuakira Takeda, Yuusaku Hayashi, Satoko Nomura, Takaaki Iwai & Akira Tanamura. Department of Internal Medicine, Aoto Hospital, Jikei University School of Medicine, Tokyo, Japan. Myocardial mitochondria play an important role in cardiac function with their energy-supplying system. Mitochondrial DNA mutations have recently been found in patients with idiopathic cardiomyopathy. These mutations in mitochondrial genes related to the electron transport system can lead to deteriorated energy production and myocardial dysfunction. We have investigated whether myocardial mitochondrial DNA mutations exist in secondary cardiac lesions. Human left ventricular myocardium obtained at autopsy was used. Myocardial DNA was extracted and mutations of mitochondrial DNA were detected using the polymerase chain reaction (PCR). To ensure that amplified DNA fragments were not obtained by misannealing of the primers to an unexpected site, primer shift PCR was also carried out. A 7.4 kb deletion between the D-loop and ATPase 6 gene of mitochondrial DNA was found in myocardium from patients with myocardial infarction and diabetes mellitus, as well as patients treated with adriamycin. These mutations might be induced by free radicals produced in these pathological conditions.

~ I - A D R E N E R G I C AND P 2 - P U R I N E R G I C S T I M U L A T I O N S IN C O N T R O L AND ISCHEMIC CARDIAC CELLS Guy Vassort & Michel Puc~at. I N S E R M U-390, Montpellier, France.

L53

aradrenergic and P2-purinergic stimulations share a number of similarities in their effects as well as salient differences in their signal transduction pathways. Both induce positive inotropism without significant change in the time course of contraction. This is related in part to an alkalosis following the activation of the Na-H antiport by both neurohormonal stimulations and to either a Ca-sensitization of contractile apparatus by phenylephrine or to an increase in Ca current by ATP. Both agonists increase phosphatidylinositol turnover; however, only ATP markedly enhances resting Cai and Ca transients. Membrane depolarization most often occurs with both agonists. Indeed, phenylephrine reduces the K conductance while ATP opens a CI- and a non specific cationic conductances; the latter results from a transient acidosis following the activation of the CI/HCO 3 exchanger on sudden ATP application in the micromolar range. Evidence has been obtained to implicate al-adrenoceptors in the arrhythmias that occur during coronary artery occlusion and/or reperfusion. Under this pathological situation, the enhanced al-adrenergic responsiveness is associated with an increase in ~]-receptors and IP3 production. Adenosine and ATP were shown most generally to reduce arrhythmias; however, this might result from an adenosine-induced K conductance increase. Acute ATP application on cells triggers automaticity and favors EAD. Furthermore Na current inactivation as well as Ca and H increases, which reduce gap junction communication might impair cell to cell conduction.

D E V E L O P M E N T A L C H A N G E S IN C A R D I A C CALCIUM REGULATION M a t t i V o r n a n e n . D e p a r t m e n t of Biology, University of Joensuu, Finland

L55

The contraction of mammalian myocardium is dependent on both transsarcolemmal Ca 2* influx and intracellular Ca 2÷ release from the sarcoplasmic reticulum (SR). In the adult myocardium SR provides the main source of contractile Ca 2+, while several lines of evidence suggest that immature heart relies largely on transsarcolemmal Ca 2* influx to support contraction. In the rat rapid changes in heart rate and contraction duration occur immediately after birth and functional studies on right ventricular preparations suggest that a shift from sarcolemma-dominated contractile regulation to SR-dominated one occurs during the third postnatal week in this species. Peak amplitude of L-type Ca z* current increases strongly and steadily during the postnatal growth but changes in current density are relatively small. Volume of rat ventricular myocytes increases approximately 24-fold (from 1.21 x 103 to 27.96 x 103 # m 3) but surface area only 8-fold (from 1.13 x 103 to 8.87 x 103/zm 2) from birth to adulthood. Due to the higher surface to volume ratio of immature myocytes Ca 2* influx through Ca 2÷ channels probably contributes a much larger part of contractile Ca 2+ in neonatal myocardium than in the adult rat heart.

BRADYKININ, A TRIGGER FOR DELAYED L54 PACING-INDUCED PRECONDITIONING. Agnes Vegh, K Kaszala, J Gy Papp, J R. Parratt*, Department of Pharmacology, Albert Szent-Gyorgyi Medical School, Szeged, Hungary and *Department of Physiology and Pharmacology, Unviersity of Strathclyde, Glasgow.

We have recently demonstrated a role for bradykinin in the antiarrhythmic effects of'classical' preconditioning in dogs (Br J Pharmac 1994, 113, 1167). The purpose of the present studies was to determine whether this peptide plays any role in the delayed cardioprotection afforded by cardiac pacing i.e. 20 h after the preconditioning stimulus (J Physiol 1994, 480, 89P). Dogs were paced (220 beats rain-1 for 4 x 5 rain periods). Either the left anterior descending coronary artery (LAD) was occluded 5 min after pacing or the dogs were allowed to recover from the anaesthetic and 20 h later reanaesthetised and subjected to LAD occlusion. The B2 antagonist icatibant (HOE-140) was given (300 tag kg"1 iv) before the pacing procedure. Ventricular fibrillation (VF) occurred in all the dogs given icatibant before pacing when the LAD was occluded 5 rain later (ef 0/11 in the paced dogs not given icatibant (P < 0.01) and 6 out of 14 in control, non-paced dogs). When the LAD was occluded 20 h after icatibant in paced dogs the incidence of VF was 60% (cf 10% without icatibant; P < 0.05) and 63% in the controls (pacing electrode in right ventricle but not paced). These results suggest that early bradykinin release during the pacing procedure is involved as a trigger in the delayed protective effects of cardiac pacing. Supported by OTKA, British Council, OMFB and the European Commission (ERB CT 924009)

EFFECTS OF ANTIOXIDANTS AND NEUROPEPTIDE BLOCKADE ON CARDIOMYOPATHY DUE TO MAGNESIUM DEFICIENCY William B. Weglicki, lu T.Mak, Jay H. Kramer, Marie M. Cassidy, Richard E. Stafford, Benjamin F. Dickens, Tammy Wagner, Terry M. Phillips. Departments of Medicine and Physiology, GWUMC, Washington, D.C. 20037 Animals placed on a magnesium deficient diet develop over a period of weeks cardiovascular lesions which result ultimately In focal necrosis of the myocardium. In studying the early pathobiological mechanisms leading to cardiomyopathy we have identified lipid peroxidation and oxidation of myocardial proteins dudng the second and third weeks of magnesium deficiency. Inflammatory cytokines ILL1, IL6, and TNF~] are elevated both in the plasma and tissues during the third week of deficiency. Treatment with antioxidants [probucol, vitamin E] diminished lesion formation but not the cy~okine elevations. Recently, we have identified neuropeptide elevations during the first week of magnesium deficiency and have utilized neuropeptide receptor blocking agents in this model. The results indicate that lesion formation is diminished, cytokine responses are blunted and tissue neuropeptide changes are prevented by neurogenic peptide receptor blockade. These findings support a neurogenic inflammation meohanism leading to excess free radical formation as a significant pathological pathway contributing to the cardiomyopathy of magnesium deficiency. Supported by NIH Grants HL49232, and HL36418.

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Can ATP-sensitive K + C h a n n e l s C o n t r i b u t e 157 to Extracellular K + A c c u m u l a t i o n in M y o c a r d i a l Ischemia. Arthur A.M. Wilde, Patrick Henry*, Denis Escande*, Michiel J. Janse. Dept. of Clinical and Experimental Cardiology, Academic Medical Centre, University of Amsterdam (NL). *Laboratoire de Physiologie Cellulaire, URA CNRS 1121, Universit~ Paris XI, Orsay (FR) We examined the possible role of ATP-sensitive K + channels (IKATP) in causing extracellular K + accumulation during acute global ischemia in Langendorff perfused rabbit hearts by on line assessment of [K+]o with K + sensitive mini-electrodes inserted in the LV wall. In quiescent hearts neither blocking (3 I.tM glibenclamide) nor activating IKATP (50 I.tM cromakalim) altered the time course of changes in [K+]o. This demonstrates that IKATP is not involved in cellular K + loss when the membrane potential remains at its resting level. In nomaoxic beating hearts IKATP activation produced a transient increase in [K+]o, demonstrating that IKATP activation in itself is sufficient to increase K + efflux. A computer model of guinea-pig cardiac electrical activity (Oxsoft) was used to explain the mechanism by which IKATP activation may lead to enhanced K + efflux. The model predicts that activation of IKATP will lead to an abbreviated action potential (AP) which will in turn increase the total amount of inward charges entering the cell during the course of the AP, because the Ca-current is not yet inactivated and the increased driving force will lead to enhanced Ca influx. To produce abbreviated repolarization, the amount of positive charges leaving the cell necessarily also increases. We conclude that IKATP activation can lead to cellular K + loss in beating but not in quiescent hearts.

REGULATION OF ENERGY CONSUMPTION IN THE MYOCARDIUM: ADAPTATION OF ENZYMES TO HYPOXIA OR TO ISCHEMIA Attila Ziegelhoeffer. Inst Heart Res, SIovak Aead Sciences, Bratislava, Slovak Republic. Myocardial energcties is characterized by a tendency to establish a state of dynamic equlibrium characterized by constant ratios between ATP, ADP AMP and the total of adenine nucleotides. This may be reached by transitory balance between the processes of energy production and energy utilization in the heart. However, many mechanisms involved in lowering energy demands in hearts adapted to iscbemia or hypoxia to meet their lowered capacity of energy production and delivery, still have to be elucidated. Principles are proposed that may enable the modelling of the balanced energetic state in myoeardium preconditioned by iscbemia or high altitude hypoxia. These principles are based on thermodynamics and involve a more economic handling of ATP by the ATPase systems. It is demonstrated, that sarcolemmal ATPases can adapt to lowered availability of ATP in cardiac tissue by decreasing their values of Vmax and increasing their affinity for ATE The known less effective utilization of energy preserved in ATP by ATPases in iscbemia may be in some cases compensated by adaptation of their activation energy (the anaerobiosis-indueed elevation of Ea may be missing). Catecholamines, prostacyelin and some shock proteins may also be involved in modulation of cardiac ATPases in iscbemia. A280

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CYTOKINES AND COMPLEMENT /58 ACTIVATION FOLLOWING MYOCARDIAL ISCHAEMIA AND REPERFUSION Frances M Williams, Claire L Ivey, Paul D Collins, Peter J Jose & Timothy J Williams. Dept of Applied Pharmacology, NHLI, Dovehouse Street, London, UK. A characteristic feature of infarcted myocardium is the presence of infiltrated neutrophils and these cells have been implicated in reperfusion injury. Extravascular accumulation of neutrophils at a site of inflammation depends on the generation of chemoattractant mediators and the expression of adhesion molecules by the leukocytes and endothelial cells. Inhibitors of complement activation are reported to reduce myocardial neutrophil accumulation, indicating a role for the chemoattractant, C5a. Both C5a and the cytokine IL-8, another potent neutrophil chemoattractant, have been detected in ischaemic/reperfused rabbit myocardium. However, the kinetics of generation of these mediators differs. C5a appears within minutes of reperfusion whilst substantial amounts of IL-8 are only detected 4.5 h later. Depletion of circulating neutrophils results in almost complete suppression of IL-8 generation whilst the concentration of C5a is not significantly affected. These results indicate that there is a sequential generation of mediators during reperfusion. Initially C5a is generated, followed subsequently by IL-8 possibly generated by infiltrating neutrophils. This work was supported by the BHF and the Wellcome Trust.

ABSTRACT INDEX

AUTHOR

ABSTRACT NUMBER

Aass H Aasum E Abaturova OV Abel FL Abeywardena MY Abiko Y Abou-Chehade K Abumrad NA Adamcova M Afanasyev SA Agnoletti L Ahlborg G Aiba H Akimoto K Alam M Ala-Rfimi A Albus U Alexandrova EA Alfieri O Aliev MK Allee PL Allibardi S AIIshire A Allue I Alonso GF Althaus U AItieri Pl Altschuld RA Alvarez B Ambrosio G Amoroso G Amos GJ Anand-Srivastava MB Andersen GO Anderson KE Anderson KP Anderson PAW Ando H Andreasen "IV Andreoni G Andries LJ Anflous K Anger M Angielski S Anning PB Antin P Anversa P Anzawa R Apstein CS Arad M Arai M Arakawa N Arata H Arata Y Arendrup H Arino T Armah B Armentano RL Armour JD

Fr076 Th044 Th125 Mo045 Thl2O Tu043, Tu065, Thl l0 Th045 Mo009 Mo039 Fr071 Tu153, Frl08 Mo082 Fr123 Mo008 Mo156 Th019 Thll2, Thl31 Tu020 Th139 Th009 Mo019 Th136, Fr162 Th028, Th116, Fr179 Th030 Tu165, Tu166 Th 162, Th163 MoI51 Mo021 Th063, Th064, Fr099 Mo064,Mo065, Mo119, Tu087 Mo049 Tu047, Tu054 Mo106 Frl01 Tu082, Th089 Th092 Mo124 Tu 144, Tu145 Thl40 Tu182 Mo075 Th018 Fr017 Mo169 Mo056, Mo 146 Mo008 L01, Fr064 Mo168, Frl30 Tu077 Tu090 Fr025 Mo088 Tu110, Fr123 Mo177 Fr085 Fr042 Tu008 Mo109, Mo114 Frl09, F r l l 0

Armstrong SC Amer A Arras M Arthur GD Artman M Ashley CC Ashraf M Askenasy N Asmussen I Asztalos B Atanasiu g Athias P Aubailly N Aussedat J Autenrieth A Avazov N Avkiran M Azarov Vl Baba HA Babadzhan VD Babal P Bacakova L Bachetti T Baczko I Bada V Bader M Bagdatjev VE Baglini R Bai HZ Baig MMA Baig SMA Baker EJ Baker JE Bakker A Balbarini A Bali H Balligand JL Balogh A Balogh GE Baltas LG Ban K Banditelli S Baniene R Banning AP Banyasz T Barg J Barhum Y Barone A Barr A Barra JG Barrabes JA Barrios V Barsacchi R Bartak F Barrel S BassengeE B/issler A Bate,son AN

Tu 106 L41 Tu079, Tu144, Tu145, Fr167 Fr051 Mo011 Fr098 Tu 123 Tul09, Th001, Th002, Thl01, Thl08 Mo164, Mo165 Mo148 Th087 Th083 Tu051 Th033 Th168 Tu090 Mo006, Th059, Frl31 Fr157 Mo153 Fr072 Fr011 Mo120 Fr089, Frl08 Tu070, Tu094 Th014, Th015, Th016 Tu037 Fr059 Mo049 Thl60 Mo156 Mo010 Fr152 Mo029, Fr152 Th01 I, Th 134 Mo049 Mo163 Fr175 Tu069 Mo097 Tu038 Thl09 Thl06 Th012, Thl04 Mo095 Mo176 Tu046 Fr010 Tu166 Th026 Mol09,Mo114, Mo144 Mo069, Fr149 Fr002 Thl06 Th095 Tu037, Tu 154 Mo063, Tu 178, Tu179 Th133 Mol00

A281

Battaglia C Baumgart D Baxter GF Beier N Beigelman R Beinlich CJ Belanger M Belcastro AN Belcher PR Belhassen L Belikova Y Bell D Bell S Bellet M Boloeil JC Benardeau A Benet LZ Benigno M Bennaceur M Benndorf R Ben-Haim SA Bercovici J Berenshtein E Beresewicz A Berke G Berman E Bernard M Bemardeau A Bematova I Bemocchi P Bemotat-Danielowski S Berti S Berton B Berwing K Besch Jr HR Beskrovnova NN Bessho M Bettini M Beuve CS Bevza AV Beyar R Beyer M Beyer ME

A282

Beyer T Beyer U Bezstarosti K Bhutta NS Biagini A Bick RJ Bidasee KR Bigda J Bilenko MV Binaglia L Binah O Bing OI-IL Bingyong S Binz K Bishopric Nit Black P Blanco J Blasig IE Blasini R Blechacz W

Mo064,Mo065, Mo I 19 L02 Tu146, Tu149 Mo067 Tu165, Tu166 L03 Th043 Fr051, Fr176 Mo047, Tu 174 Fr 175 Tu026 Fr041 L24 Frl08 Th007 Tu028, Tu066 Th040 Tu153, Th005, Th139 Th097 Mo134, Tu154 Th092 FRO28 Frl 18 Mo059,Mo062, Tu 107 Fr173 Tu097 Mo173 Tu015 Fr011 Tu074, Th005 Mo067 Fr090 Mo166 Tu079 Tu025 Th067 Th010, Th029 Th096, Thl06 Tu026 Tu029 Mo043,Mo044, Mo135, Mo141 Fr058 Mo085, Th078, Th079, Th133, Fr154 Th093 Fr058 IA9 Fr040 Fr090 TuI32, Fr174 Tu025 ThlI9 Fr117 Mo149 FrI73 Fr060 Mo086 Tu143 Frl51 Mo095 Mo069 Fr125 FrI70 Tu008

Boateng SY Bobik A Bobrova E Boehm DH Boeri MV Boerth SR Bohdanecka M Boheler KR Bokefiya LA Bolek L Boluyt MO Bonacina E Boni E Bonne G Booker T Boraso A Borgers M Borman JB Bomemann R Borthne K Borutaite V Borzak S Bothwell JH Botsford M Bouchard JF Bouman LN Boyett M Boyett MR Brachmann J Braun U Braveny P Breier A Breton E Bretschneider HJ Bridle TM Brilla CG Bringas J Brink P Bromberg Y Brooks G Brooks WW Brors O Brotto MAP Brovkovich VM Brown GC Brown KA Brown L Brown LA Brown MD Briill T Bnmeau BG Brunold C Bruschi F Brutsaert DL Bryant SM Buchanan M Buchwald AB Budriunaite A Bugge E Buja LM Bundgaard H Burger W Burrier AC Butler K Bytesnik J

Fr018 Mo118, Frll5 Fr006 Thl50 Thl06 MoO I 1 Th055 L04, Mo012, Fr018, Fr052 Th067 Th095 Fr060 Fr064 FRO87 Fr028 Frl76 Tu 105, Th005 Th134 Tu097 Fr082 Fr076 Th012, Thl04 Fr067 Tu005 Mo178 Tu099 Tu067 Th068, Th154 Tu011, Tu062 Th093 Fr096 Tu001 Tu086, Th022 Mo108 Th137 Th129 L05, L42 Th006 Mo040, Fr030 Th058 Mo006, Tu 122 Fr060 Tu081, Fr136 Mo015, Mo019 Fr157 Th012, Thl04 Mo048 Fr035 Fr026 LI6, Mo054 Fr135 Fr007, Frl00 Fr031 Th096, Th 106 Mo075, Mo081 Tu033 Th034 Tu169 Thl04 Tu114, Tu138, Th132 Tu132, Fr174 Tu006, Fr085 Th167 Fr156 Tul71 Th091

Cabrol C Cai CM Calabrese L Calcagnini G Calcagnini jr G Caldarera CM Calzolari C Camici PG Camilion de Hurtado MC Capasso JM Cappelli-Bignzzi M Cargnoni A Carleton S Carlsson L Carre F Carrier L Carfiquiriborde M Casalini S Cascio WE Cassani G Cassidy MM Cauchy MJ Caulfield JB Cave AC Ceconi C Celko J Cemohorsky J Chahine R Chambers DJ Chantler PD Chapman RA Charlemagne D Chatham JC Chaudron I Chen GQ Chen HZ Chen JH Chen SG Chen W Chen X Chen ZY Cheng W Cherednicheriko GA Chemyavsky AM Cheung P Chevassus H Chevion M Chiappe de Cingolani GE Chiariello M Chiavarelli M Chiba M Chiehkanov GG Chierehia SL Chilton D Chitwood WR Chlopieki S Christe G Christoffels A Chu JC Chung M Ciavarella GM Cicutti N Ciecwierz D Cigola E

Mol 17 Tu 124 Mo065 Mo115,Mo116, Fr005 Mo115 Mo037, Th041 Mo123 Mo 183 Th063, Th064 Mo143 Mo064,Mo065, Mo119 Tu074, Tu 105 Mo010 Tu063, Th094 Fr065 Fr028 Fr169 Th136, Fr162 Th090, Fr144 Fr089 L56 Tul81 Mo124 Tu077, Th034 Tu074, Tu 105, Th005 Tu159 Mo032 Th087 Mo048, Th145, Th153, Fr129, Fr165 Mo124 Thl09 Tu026, Fr012, Fr065 Th038 Fr132 Th098 Tu021 Fr007 Frl60 Tu128 Tu124 L24 L01 Tu042, Fr157 Fr071 Tu172 Mo129 Fr 118 Tu010, Tu040 Mo064,Mo065, Tu087 Mo156 Mo088, Fr091 Fr164 Fr162 Mo006 Fr185 L13 Tu001 Mo040 Mo014 Fr153 Mo115, Moll6 Fr036 Thll9 Fr064

Cingolani HE Cirillo P Claes JAAM Clarke K Clarke SB Clerico A CIo C Cobbe SM Cobbold PH Coetzee WA Cohen MV Coker SJ Coletti G Coli G Collins PD Comini L Confortini R Conrad CH Conyers RAJ Cook JM Coquard I Coraboeuf E Corbalan R Corbett JM Corda L Cordis GA Cordis T Corfield V Corradi D Corti A Coulombe A Coumel P Couture R Cowie MR Cozzone P Creazzo TL Cremer M Crespo MJ Crippa M Croft RP Cross HR. Crottogini M Csanady M Csete K Csonka C Csont T Cuevas P Cumming DVE Cummins P Cunningham.IN Curello S Curtis MJ Czubryt MP D Adamo CM D Andrea A D Andrea D Dagys A Dahlstr~m A Dakhli T Damiani G Daneshmd Z Darracott-CankovicS Dart AM

Tu010, Tu041, Th063, Th064, Fr099, Fr169 Tu087 Fr027 Tu005, Th036, Th061 Fr084 Fr090 Mo037 FRO66, Frl51 Th008, Th030 Th059 Tu073, Tu127 Tu163, Tu164 Th139 Th041 L58 Tu153, Frl08 Fr089 Fr060 Th146, Th159 Fr062 Fr065 Tu028, Tu051, Tu052, Tu066 Fr047 Mo130 Fr087 Mo185 Th071 Mo040, Fr030 Fr064 Fr089 Tu051. Tu052 Th097 Mo093 Th084 Mo173 Mo015 Fr029 Mo151 Fr087 Fr110 Th061 Mo140,Mo144, Mo147 Th124 Mo110 Tu057, Tul01, 'Tul02, Fr158 Tu057, Tul01, Tul02, Fr158 Fr002 Tul51, Tu152 Mo006 Mo156 Tu074, Tul05, Tu153, Frl08 Tul31 L36 Tu182 Mo064, Mo119 Tu087 Th013 Mo024 Fr012 Fr087 Th152 Th056 Mo099, Th089, Fr114, Fr115, Fr155

_4,283

Das DK Dasaev AN Dausse E Davia K Davies MK Davoli V De KeulenaerGW De Simone C de Boer PAJ de Bold AJ de Bold MLK de Jonge N de "CriesJE D~iman JR Degryse AD del Valle H Detaeayre C Deloreseo M Delvalle C Demaison L Demolder MJM Denjoy 1 Denvir MA Deroubaix E Dervanian P Desjardins S Deslauriers R Devaux B Devynek MA Dhalla KS Dhalla NS

A284

Dhawan V Di Nardo P Di Santo D Dickens BF Dilley RJ Dillmann WH Dinender K Dobson G Doeherty JC Doddo JM DoggrellSA Dohnalova A Domenech RJ Donath M Y Donato L Dong E Donnelly JL Doff G Dom II GW Dorof~yeva GD Dorsch D Dote K Doubell AF Douglas J Downey JM Downing SE Drake-Holland AJ Drobotko TF Droogmans G Droy-Lefaix MT Du XJ Du XY du ToitE

L06, Mo184, Mo185, Tu157 Fr059 Th097 Tu035 Mo054 Tu022 Mo081 Tu087 Mo004 Fr007, Frl00 Fr007 Th164 Fr027 Tu031 Tu176 Mo140 Fr012 Tu155 Fr167 Fr132, Fr187 Mo078 Th097 Fr066 Tu051, Tu052 Tu028 TulSl Th158, Frl60 Mo070, Fr074,Fr075 Tu023 Th021 Mo 149,Mo178, Th021, Th 105, Frl05, Fr133 LI2 Mo149 Mo049 L56 Mo118 Tul50 Mo163 Th037 Frl60 Mo094 Fr039 Thl20 L07 Fr049 Fr090 Fr097 Mo071 Th092 Tu031 Mo041, Mo042 Mo067 Tu068 Fr183 Fr182 Tu072, Tu073, Tu 127 Th032 Tu174, Fr084 Th024 L31 Tu057 Th089, Fr114,Fr115,Fr155 Th074 Fr159

Duffy PS Dufilho M Dugin SF Dumont E Dumoulin MJ Duneker DJ Durra MJ Dux L Dye JF Dzurik R Eckly A Edes I Egdell RaM Eggermont J Eghbali-Webb M Ehring T Eid H Eilam Y El Alaoui-Talibi Z Ela C Elazar E Eldar DM Ella PP Elimban V Elliott GT EIs/isser A Endoh M Engelman RM Engelmann GL Enger M Engle CL Eppenberger HM Eppenberger-Eberh.-,rdt M Eramo N Ergun ME Eriksson E Ertl G Erusalimsky JD Escande D Eschenhagen T Escobales N Esler MD Esposito A Etherington P Eynan M Fan JS Fan YX Fantmi E Farhadian F Fariello R Faure P Fazokas T Fedun NN Felzan B Feng Q Ferdinandy P Ferrannini E Ferrari R FerreiraA Ferrueei A Festinese S Feuvray D Filipova K

Th008 Tu023 Mo158 Mo096 Th087 Tu115, Fr143 Mo130,Mo155, Fr018 Tul01, Tul02, Fr158 FrI39, Frl40 TuI75 ThIl8 Mo136 Frl40 L31 Fr182 Fr168 FRO07 Tu046 Fr031 Tu046 Tu090 Th071 Tu087 Th021 Tul55 Tu144, Tu 145, Thl35 Mo038, Tu019 Mo185, Tul57 L08 Frl01 Th090 L09, L43, Fr049 Fr049 Tu087 Mo133 Mo073 Th052 Moll 1 L57 L44 Mo151 Fr115 Tu087 Mo047 Tul61 Tu055, Tu056 Mo084, Mo087 Th083 Mo003 Fr087 Tu176 Tu063, Th094 Th024 Fr173 Fr037 Tu057, Tul01, Tul02, Tul03, Tul04, Fr158 Mo183 Tu074, Tul05, Tu153, Th005, Th139, Fr089, Frl08 Mo040 Mo116, Fr005 Mo115,Mo116, Fr005 Mo167,Mo173, Th065 Th048

Filippov AK Fink B Fink N Fischer A Fischer V Fischer Y Fischmeister R Fisher JL Fitzpatrick AP Flessa C Fletcher JR Flink IL Fluck D Fogaca RTH Forder JR Forster T Fragasso G France D France V Fransen PF Franz WM Freyss M Fu MLX Fuchs F Fuchs R Fuchsbauer C Fujii S Fujinami T Fujio Y Fujitani N Fujiware T Fukami Y Fukuhiro Y Fukushima A Funai H Funakoshi T Fiirs't D Gabauer I Gabel S Gala G Gajdos M Galajdova K Galinanes IV[. Gallagher MM Gamble J Gandjbakhch I Ganguly NK Ganote C Ganten D Gapon LI Garcia A Gareia-Dorado D Gardner P Garjani A Garliek PB Gamier A Gaubatz J W Gaudron P GautierE Gavin JB Gebel GY Gebhard MM Geese A Genade S

Tu020 Tu178, Tu179 Tu 178 Th163 Th042, Th 144 L10 Tu009, Tu015, Tu018 Th159 Th071 Tu083, Tu085 Tu073, Tu127 Me008, Mo139 Fr139 Me019 Th038 FRO94 Fr162 Me004 Tu022 Mo078 FRO29 Tu023 Mo105, FRO13 Mo126 Mol01 Fr179 Tu167 Tu012, Tu064 Mo152 Mo187, Fr056 Fr138 Mo079 Fr138 Th127 Mo152 Mo088 LII Tu086, Th042, Th144 Tu128 Tu153, Frl08 Tu175 Th088 Tu118, Tu119, Tul20, Tul21, Tu122, Thl02 Thll6 Th050 Mo117 L12, Mo163 Tul06 Fr001 Tul80, Fr014 Mo069 Mo069, Fr149 Fr173 Mo083 Th034 Tu095 Frl71 Th052 Th080 Th128 FRO59 Th137 Tu177 Tul41, Th156

Gerdes AM Gerova M Geshi E Ghatpande S Gho BCG Ghomeshi HR Giaccari A Giannella E Gibbs CL Giddings J Giercksky KE Gilchrist JSC Gillet B Gimenez-Gallego G Giordano F Gittenberger-de Greet AC Gl~iser C Glatz JFC Gluckman PD Godfraind T Godoy I Godt RE Goedbloed MA G6gelein H Goldman S Goldstein S Golostenova LM Golukhova EZ Goma FM Gomes OM Gomez Dumm INT Goncbar IV Gonska BD Gonzalez MA Gordon N Gorecki A Goswami S

Grajek S Greenspon AJ Griffiths EJ Grijalva G Grocott-Mason RaM Gross H Grosset A Grott W Grover GJ Grfinig E Grupp IL Gmtkoski P Gryglewski KI Grynberg A Gsell S Gu LM Gu QB Guamieri C Guieheney P Guisheng Q Gultyaeva EP Gunteski-Hamblin A/VIE Guo Q Gupta M Gupta MP Gupta S

Mo143 Me051 Mo177, Tu110, Tul 11, Fr034, Fr122, Fr123, Fr145 Me010 Tu089, Tu115 Th158 Th041 Tul00 Mo142 Tul71 Mo092 L36 Th007 Fr002 Tul50 Me018 Tul68 Me009 Th128 Fr016 Fr047 Me014, Me019 Frl80 Thll2 Fr079 Th069, Fr062, Fr067, Fr078, Fr088 Fr059 Th067 Tu146 Th138 Th049 Mol81 Thll3 Mo069, Frl49 Mo043 Fr069 Mo010 Mo036 Th071 Th046, Fr070 Mo156 Mo056, Mo146 Tu027 Th080 Tu008 Tull6 Fr029 L22, Tu036, Tu039 L08 L13 Fr132, Frl87 Tu037 Frll2 Tu021 Th041 Th097 Mo086 FrOl4 Tu031 Tu021 Mol21 Mol21 Ll2

A285

Gupte SA Guzik P Gvozdjak J Gvozdjakova A Gy6ngy6si M Haass M Hackett D Hadjiisky P Hadoke P Hagelin EM Haghighi K Haigney MC Haikala H Hajek M Haj-Yehia A Hala 0 Halcak L Hall JL Hallsltr6m S Halpem P Hamada M Hamilton CD Hammer C Hammerschmidt S Han C Hana V Hanawa H Handsehug K Hangwei C Hannekum A Hansford RLG Hao XM Harden F Harding SE Hardman SMC Hard T Harper IS Hatter JM Harrison G Harrison SM HartG Hart JL Hartmann A Hartogensis W Hase N Hasegawa H Haseloff R

Hasenfuss G

A286

Hashimoto E Hashimoto H Hashimoto K Hashizume H Hasin Y Hassinen IE Ham T Hatem SN Haunstetter A Hauser R Haworth R Hayakawa M Hayam G Hayashi H Hayashi K Hayashi Y

Mo060 Frl20 Th014, Th015, Th016 Th014, Th015, Th016 Th124 Th117, Th123, Fr113 Frl40, Frl41 Mol 17 Tul71 Tu081, Fr136 Mo005, Tu036 Th046, Fr070 Mo125 Th035 Th040 Mo110 Th144 Th026 Th155 Th157 Mo145, Fr019 Th026 Th168 Thll3 Fr097 Fr048 Mo162 Tu 168 Mo086 Fr058 Th025 Tu055, Tu056 Mo055 Tu035 Th084 Th092 Fr134 L22, Mo005, Tu036 Mo055,Mo058, Tu 108 Tu011, Th062 Tu033 Mo052 Mo188, Th167 Fr055 Fr166 Fr008, Frl50 Fr125 Mo132, Fr080, Fr081, Fr082, Fr083 Mo068 Th127 Fr091 Thll0 Tu027, Tu046, Tul61 Th019 Fr020, Fr033 Tu015, Tu028, Tu066 Th117, Th123, Fr113 Thll9 Mo006 Th017, Fr061 Th092 L14, Fr128 Th029 L52

Headrick J Heads RJ Hearse DJ

Hedner T Heerschap A Heileson B Heimisch W Heller J Henry P Hensley J Herichova I Herlitz H Hemandez LA Heron MI Herrmann R Herynek V Hetzer R Heusch G Hicks MN Hide E Higashiyama A High T Higuchi M Hilbel T Hilme E Himmel HM Hirai H Hiramori K Hirano M Hirano Y Hiraoka M Hiwada K Hjalmarson A Hnatowicz V Ho J Hoeh B Hochhauser E Hoebeke J Hoerter JA Hoffmann S Hoffmeister HM Hofma SH Hohl CM H6hn J Holden A Holecyova A Holubarsch C Homuth V Honbo NY Honda-Yamada F Honzik JM Honzikova N Hopf R Hopkins JCA Hoque ANE Horaekova M Hori M Horky M Horowitz L Horowitz M Horsky P Hoshida S

Mo057, Th037 Tu149, Tul51 Mo027, Tu072, Tu118, Tu119, Tul20, Tul21, Tu122, Thl02, Fr124 Fr013 Th143 Mo143 Frl70 Fr003 L57 Mo021 Th014, Th015, Th016 Fr013 Th006 L39, Fr050 Fr029 Th035 Mo159 L02, Tu071,Tu076, Fr168 Frl51 Tu048 L24, Mo 138 Tu002 Mol79 Th093 Fr013 Mo080, Tu007, Tu053, Tu054 Th142, Th 147, Th148 Mo088, Fr091 Th142, Th147, Th148 Tu014 L15, Tu014 Mo145, Fr019 Mo105, Fr013 Mo120 Tu129 Tu154 Thl03, Th157 Mo105, Fr013 ThO07

Fr001 Mo085, Th078, Th079, Th133, Fr154 Mo050, Th053 Mo02 I Tu069 Th086 Mo051 Mo132, Fr080, Fr081, Fr083 Fr009 Fr024 Mo152 Fr177 Fr177 Mol88 Th036 Tu065 Frl09, Fr110 Tu133, Tu134, Tu135, Frl21 Mo074 Tu090 Tu027, Tul61 Mo074 Frl21, Fr166

Houston RJF Hradec J Hu K Hucin B Hudlicka O Human PA Humphreys RA Hurtado de Catalfo GE Hussain M Hynd JW Hynie S lbrahim MF Ichihara K lervasi G Igarashi J lgnatov NG lgnatowska-Switalska H lijima K limura O llebekk A Iliodromitis EK llsley CD Imai K Imai S Inoescu M Inomata T lnoue M loft A lrtun R Isenberg G lshihata A lshikawa M Ishikawa T Ishioka H Iso T Ito T Ivey CL lwai T lwasaki T lwata T Izumi T Jablonski P Jacob W Jacobsen AN Jacoviello L Jagodzinski P Jakab I Jakiwczyk OM Janiak R Jano~ M Janota T Janse MJ Januszewicz A Jarkovska D Jasmin G Jayacumar JR Jenkins DP Jennings GL Jepson NS Jiang XC Jicheng L Jin XJ Jiraskova M John GW Johns EC

Th143 Fr048 Th052 Fr116 L 16, Mo054 Thl50 Tu088 Th049 Frl04 Fr084 Fr095, Fr116 Th145 Tu065 Fr090 Frl21 Fr059 Fr069 Tu110, Fr123 L27, Tu139 Tu08 I, Th032 Tu083, Tu084, Tu085, Th126 Tu091 Tu 134 Mo128 Thl30 Mol60,Mol61, Mo162 Tu133, Tu134, Tu135 Tu022 Thl40 LI7 Mo038 Fr091 Th148, Th149 Tu110, Tul 1 I, Fr123 Fr025 Mo079, Th047, Th127 L58 L52 L32 Tu110, Fr122 Mol60,Mol61, Mo162 Th146 Th011, Th134 Th089, Fr155 Tu182 Fr053 Tul03 Mol00 L23, Tu034 Th091 Fr048 L57 Fr069 Mo033 Tu049 Th039, Thl41 Tu112, Tu113, Tu117 Mo099, Mo118 Tu091 Th098 Mo086 Mo113 Th048 Tu176 Fr098

Johnson TA Johnston B Jordan L Jose PJ Jourdon P Joyeux M Juggi JS Jung W Juranek I Jurevicius J Just H Kaasik A Kagan VE Kaganovsky E Kage K Kageyama K Kaijser L Kaila J Kajihara H Kajstura J Kakizawa H Kalb S Kalenka A Kaliadin AY Kalkman EAJ Kallner A Kalnovicova T Kalous M Kalsi KK Kaluza GL Kamada T Kamigaki M Kamiyama T KammermeierH Kanalikova K KanayamaH Kane KA Kaneda Y Kannan S Kanoh M Kapelko VI Kapoor R Karcsu S Karezewski P Karliner JS Karmazyn M Karoly A Kasper K Kass D Kaszaki J Kaszala K Katagiri T Katano Y Kato M Kato NS Kato Y Katoh H

Katsumata K Katsumura T Katus HA Kaur S Kautzner J Kaverina NV Kawada T

Th090 Th128 Mo055,Mo058, Tu 108, Th037 L58 Mo166, Mo167 Tu156 L18, Tu075, Fr163 Thl31 Fr119 Tu009, Tu018 Mo132, Fr080,Fr081, Fr083 Fr096 Tu157 Fr010 Mo068 Frl07, Fr184 Mo082 Frl05 Th165, Fr148 L01 Fr128 Fr070 Th123 Th075 Th051 Mo073 Th144 Mo031 Tu091 Mo075 Tu133, Tu134, Tu135, Frl21 Tu065 Frl61 L19 Th042, Th 144 Th127 Tu088 L20, Thl60 Th021 Fr166 Mo046, Fr063 Thl07 Tul04 Tu037, Tu038, Tu 154 Fr024 Tu129 Tu132, Fr174 Th082 Fr070 Th124 L54, Tu078 Mo177, Tul I0, Tul 1I, Fr034, Fr122, Fr123, Fr145 Mo038 L21, Mo122, Th169 Mo133 Th165 LI4 Th017, Fr061 Fr138 Fr029 Mo163 Th035 Th076, Thl00 Mo!28

A287

Kawaguche H Kawaguchi H Kawaguchi N Kawai M Kawai S Kawakami H Kawamura K Kawano S Kawase T Kawashima O Kawashima Y Kay L Kaye DM Kayo S Kazdova L Kazmaier S Kazmierczak M Kealey T Kecskemeti V Kelemen K Keller T Kelly FJ Kelly RA Kelso E Kentish JC Kerac M Kerr D Kessler-lcekson G Khandoudi N Khatkevich AN Khomazjuk AI Khomazjuk VA Kim H Kim HW Kim M Kimura A Kimura H Kimura J King LM Kingsbury M Kingwell BA Kinoshita H Kira Y Kiriazis H Kirkeboen KA Kirk-patrick DL Kirsanova GY Kirshenhaum IR Kirshenbaum LA Kis A Kis B Kiss E Kitabatake A

A288

Kitacla Y Kitakaze M Kito Y Kitoh Y Kitmar 0 Kiyoki M Kjeldsen K Klasova A Klein D Klein R

Mo150 Mo186, Tu167, Tul70, Fr022, Fr023, Frl07, Frl81, Fr184 Mol87, Fr056 Tu045 Mo153 Mo145, Fr019 Fr008 L15 Th142, Th147, Th148 L30 Th 142, Th147, Th 148, Th149 Thl52 Fr175 Mol79 Th055, Fr003 Th133 Th085, Thl21, Frl20 Th056 Tu050 Tu050 Fr125 Mo027 Fr175 Tu013 Tu024, Th 102 Tull2, Tu113 Mo102 Fr010 Mol73 Mo046 MolS1 Fr093 Mo016, Tu126, Tu126 Mo005 Fr153 Mo122 Mo187 Tu019 Tu096, Fr186 Mo047 Mo099 Th149 Mo068 Mo142 Mo092, Tu081, Th032 Tu03 I Fr164 FrOI4 Mo137 Tu136 Tu177 Mo136 Mo150,Mo186, Tu167, Tul70, Fr022, Fr023, Frl07, Fr184 L50 Tu133, Tu134, Tu135 Th142 Th147, Th148 Thl20 Fr166 Tu006, Fr015, Fr085 Mo051 Th168 Tu090

Klenerova V Klepzig H Kl6pping C Klovekom WP Knezl V Knight J KnOll R Knubovets T Kober IM Kochan Z

Koci G Kocsis E Kodama M Koidl B Kokstem Z Kolar F Kolbeck RC Koller A Kolocassides KG Kolomiets V Koltai M Koltai MZ Komajda M Komamura K Komukai K Koning MMG Konno N Konoplyova LF Korehazhkina OV Korecky B Korotkoruchko AG Kosaka H Koss KL Kostic MM Kotsuruba VN Kott M Kovacs P Kovanecz 1 Kowaluk B Kraft P Kral J Kramer JH Krams R Kranias EG Krasauskaite D Krassoi I Krause EG Kremastinos DT Kremneva LV Kretschmann B Kristek F Kropp B Krsek M Krfiger C Kruglov AG Kryzhanovsky S Kfibler W Kubrina LN Kucera P Kucharska J Kucherenko SN

Fr116 Mol57 Th164 Th135 Frl 1O Fr124 Tu079 Tul61 Fr135 Mo077 Th155 Mo170,Mo171, Mol82 Mo162 Th168 Mo039 L08, Mo022, Mo033,Mo074, Th 166, Fr016, Fr036, Fr050 Tu044 Tu 109, Th 108 Tu118, Tu119, Tul20 Fr006 Tu057, Tu I 0 I, Tu 102, Tu 103, Tu ! 04 Mo170,MolTl, Mo172, Mo182 Th097, Fr028 Tu133, Tu 134, Tu135 Tu045 Tu115, Fr143 Mol77, Fr034, Fr122 Fr093 Th027, Fr063 Th 166, Fr036 Th024 Tu134, Tu135 Tu039 Mo061, Fr057 Fr157 Mo034 Fr119 Tu 140 L47 Th093 Fr048 L56 L49, Th053, Th054 L22, Mo005, Mol36, Tu036, Tu039 Th013 Tu058 Tu037, Tu038, Tu154, Fr125 Tu083, Tu084, Tu085, Th 126 Th125 Fr082 Mo051 Mo070 Fr048 Th117, Th123, Fr113 Fr059 ThlO0 Th117, Th 123, Fr113 Tu158 Mo020 Th014, Th015, Th016 Tu029

Kudo T Kudou T Kiihn H Kujanik S Kukreja RC Kurihara S Kurisu Y Kurkova D Kurskii MD Kuruma A Kuwabara Y Ku~metsov VA Kuznetsova BA Kuzume K Kuzuya T Kvochina LI Kyriakides ZS Lab M Lader AS Ladilov YV Lagadic-Gossmann D Lagrasta C Lahpor JR Lai CS Lainee P Lakatta EG Lakomkin VL Lambert C Lambert KA Lambropoulos LG Lamers JMJ Lamers WH Lamontagne D Lancaster MK Landesberg A Lang HJ Langley L Langowski K Lapinski M Lapshina LA Large S Larina L Larionov N Larsen JS Larsen TS Lame C Lascano EC Latchman DS LatifN Lautie N Lavanchy N Lavandero S Lawson CS Lazzara R Le Grand B Le Prigent K Lebedev A Lebedev AV Lee JA Ler'kowitz R Lekanne dit Deprez RH LeMaistre A Lemasters JJ Lengyel C

Tu167 Fr023 Frl06 Th088 Tu155 Tu030, Tu045 Th165 Th035 Tu029 LI5 Mol77 FrO 14 Tu 162 Tu068 Frl21, Fr166 Tu042, Fr172 Tu083, Tu084, Tu085, Th 126 Th068, Th 154, "1-11086 Mo045 L37 Mo166, Mo174, Th065 Fr064 Th164 Mo180 Th080 Mo094, Th025, Frl03 Fr063 FrO2 I Fr155 Fr004 L49, Th054, Frl80 Mo001, Mo002, Mo004, Mo007 Mo091,Mo093, Mo096, Tu099 Th062 Mo135, Mol41 Thll2 Th146 Mo169 Fr069 Fr072 Th056 Mo063 Tu 160 Fr015, Fr085 Th044, Th 140 Mo123 Mo140, Mo147 Tu 149, Tul51, Tu152 Mo155 Fr028 Tu095 Fr047 Tu072 Tu063, Th094 Tu 176 Mo166,Mo174, Th065 Mo028 Fr071 Fr084 Fr114 Mo00 I, Mo002 Tu132, Fr174 Th090, Fr134, Fr144 Tu063, Th094

Lepran 1 Lerch R Lesch M Lesh MD Lesiak M Levenson J Levi R Levijoki J Levitskaya EM Levy B Levy DE Lewartowski B LeWinter MM Lewis MJ Li HT Li Q Li XY Lietava J Limbruno U Linden IB Lindsay C Linz W Lisa V Little PJ Liu LL Liu LY Liu T Liu TF Liu Z Livingston B Lochner A Loke KE Lompre AM Long CS Lopaschuk GD Lu BJ Liidemann J Lukac J Lundberg JM Luo W Lupescu G Lupescu Jr G Lust RM Luther HP Lutin WA Lutsch G Luvar G Lyskovtsev VV Maaieh M Mace L Macedo A Macey MG Machanda SC Macho P MacLeod KT MacNaughton C Maczewski M Maestri R Magishi K Magni F Maier C Maisch B Mak IT Makino N Malinski T

Tu070, Tu0O4 Fr147 Fr062, FRO78, Fr088 Th071 Mo036 Mo109, Mo114 Tu 100 Mo125 Mo04 I Mo104 Tu 173 L23, Tu034 L24, Mo138 Mo056,Mo076, Mo095,Mo 146 Fr024 Tu053, Tu054 Th098, Fr126, Fr127 Tu159 Mo049, Tul30, Th114 Mo125 Th158 Thl31, Fr007, Fr032 Mo120 Mo118 Fr127 Tu124 Th098 Tu055, Tu056 Fr037 Mo02 I Tul41, Th156, Fr146 Tu098 Mo166, Fr017 Fr024, Fr055 L25, Th003, Th026, Th050 Th122 Mo132 Tu159 Mo082 Mol36, Tu036 Thl30 Thl30 Fr185 Mo159, Fr009 Mo014 Mo134, Tu154 Tu182 Th076, Th 100 Tu155 Tu028 Mo018 Mo048 Fr137 L07 Th060, Fr045 Th166 Mo059, Mo062 Fr064 Tu043 Th096, Thl06 Th123 L42 L56 Fr020, Fr033 Mo098

A289

A290

Maltsev VA Malushev I Malyshev IY Malysheva EV Manor D Manukhina EB Mao P Marchant C Marchenko G! Marcinkeviciute A Marconnet P Marek J Mares V Margaritis I Margossian SS Mariani M Marinho NVS Markianos M Markman MWM Marko R Marotte F Marsden PK Marshall V Martin JF Martine L Marzilli N Massillon Y Mastuura Y Masuda H Mas-Oliva J Mateescu MA Mateo P Matsubara T Matsuda H Matsui H Matsuo T Matsuoka H Mattiazzi A MaRie H Maughan D Maulik N Maxwell L Maz.zoletti A McCarthy DA McCarthy J McCormack JG McDermott BJ McDonald P McFarlane NG McGiffin D McKeown P McLennan PL MeMillin JB McMom SO MeNamee JE Mederski W Meehan WP Meera P Meerson FZ Mehan V Meier B Meij JTA Meiltz A Meifi U Mekhfi H

Th069 Tul60 Tu158, Tu162 Tu158 Mo044 Tu158 Fr126 Fr165 Fr157 Th012 Mol23 Fr048 Mo120 Mo123 Mo124 Mo049, Tu 130, Th114 Mo183 Tu084 Mo007 Tu050 Mo003, FrO 17 Th034 Th146 Molll Fr132 Th167 Fr02 I Th165 Fr008 Mo035 Th087 Th007 Thl05 Th 160, Fr056 Th047, Th127 Mo017 Fr008 Tu040, Fr099 Th163 L24 Mo185, Tu157 Th128 Tu 105 Mo048 Fr159 Th006 Tu013, Fr041 Frl51 Fro66 Tul08 Fro43, Fr044 Th129 Tu132, Fr174 Tu011 Mo045 Mo067 Mo133 Tu016 Tu 162 Thl61 Thl61 Fr178 Mo020 Tu027, Tul61 Mo127

Melendez J Melichercik J Meloche S Meluzin J Mendler N Merati G Mercadier JJ Merin G Meritt L Merkulova IN Mesaeli N Messa F Mest HJ Mestril R Meszaros LG Mettler D Metzger F Meyer T Mezei Z Mhagna Z Michalak M Michalak S Mierdl S Mikami T Miki T Mikoyan VD Mikus P Milano C Mildaziene V Milei J Milerova M Millanvoye E Miller DJ Miller JIA Millette E Minamino T Minarova H Minarovic 1 Minczykowski A Minieri M Mircioiu C Mironneau J Mitani S Miura M Miura T Miyagi K Miyairi M Miyashiro J Miyawaki H Miyoshi K Mkhytarian LS Mocanu MM Mochizuki S

Mochmann HC Mogilevski G Moibenko AA Mollnau H -Monteiro M Montessuit C Moolman J Moorman AFM

Fro47 Th093 Fro21 Fro86 Fr 170 Th136, Fr162 Tu015, Tu028, Tu05 I, Tu052, Tu066 Tu097 Tu025 Th077 L33, Mo112 Fro05 Tu008 Tul50 Tu044 Thl61 Tu047, Tu053 Tu169 Tu177 Thl03 Mo112 Th096 Fro58 Mo150, Mol86 L27, Tu139 Tu158 Th041 Fr114 Th012, Thl04 Tu165, Tu166 L34, Mo032 Tu023 Fr066 Fr124, Fr142 Mo096 Tu133, Tu134, Tu135 Th144 Tu017 Th085, Thl21 Mol49 Thl30 L26 Th127 Fro08 L27, Tu 139 Mo179 L30 Fr159 Tu 123 Frl30 Th024 Tu117 L21, L28, Mo168, Tu080, Th070, Thl I 1, Fr042, Frl30, Fr133, Fr138 Tul00 Th137 Tu042, Fr157 Fro73 Mo002 Fr147 Tul41, Fr030 Mo001, Mo002, Mo004, Mo007

Moravec J Moravec M Moreau D Moreyra AE Morgan HE Morgunov AA Morishita R Morita H Morkin E Morkuniene R Morris SD Morwinski R Mosca SM Mouillard P Mouton R Mraz M Mrazek T Mmka L MSller A Miiller G Miiller J MSller KD Miiller-Borer BJ Miiller-Werdan U Mulligan IP Munch-Ellingsen J Mundina-Weilenmann C Munk AA Miinkel B Murakami T Murata K Murphy E Muscari C Musci G Muthuchamy M Mutschler E Nadeau R Naess PA Nagai M Nagano M Nagano T Nagy E Nagy I Nakamura H Nakamura M Nakamura T Nakamura Y Nakaoka T Nakashima Y Nakayama H Nakazawa M Nakhostine N Nanasi PP Nand V Naqvi RU Narayanan N Nasa Y Natal P Nauciene Z Navon G Nederhoff MGJ Negroni JA Nemeth J Nemeth M

Fr031, F r i l l Frl I 1 Fr132 Fr169 L03 Frl 17 L20 Tu019 Mo008, Mo139, Fr079 Thl04 Tu152 Fr106 Tu010, Frl6q Tu176 Fr146 Fr095 Th095 Mo031 Tu142, Tu143 Tu168 Mo159 Th135 Th090

Mol01, Th057, Th168 Fr098 Tu114, Th132 Tu040, Tu041 Mo013 Fr073 Frl07, Fr184 Fr025 L29, Tu128 Th041 Mo065 L50 Mo157 Mo091, Mo093, Th087 Tu081 Fr042 L21, L30, Mol54, Th169 Th169 Tul01 Tu 103, Tu 104 Mo017, Th010, Th029 Mo088, Fr091 Th010, Th029 Frl50 Mo068 Mo079 Fr148 Mo128 Mo091, Mo093 Tu004 Fr039 Fr045 Tu032 Tu093 Mo117 Th012 Tul09, Tul61, Th001, Th002, Thl01, Thl08 Thll5 Mo140, Mo147 Th124 Tu059

Nemoto T Nescheret AP Neubauer S Neumann J Ney P Nickl W Niezabitowski P Nikula TD Nilius B Nishida M Nishimura H Niu S Noble MIM Noda Y Node K Nomum S Norton P Nosek TM Novak F Novakova M Novakova O NoveI-Chate V Nunez DJ Nylk T 0 Connor SE O Gorman DJ Obermann W Ocampo CJ Ochi R Oeseburg B Offstad J Ogawa T Ogawa Y Ogh~o H Ogita T Ognjanovic B Ohad D Ohashi T Ohata H Ohisalo JJ Ohiy SA Ohkura Y Ohsuzu F Ohyanagi M Okada H Okada R Okada T Okamoto H Okayama H Okhrimenko NV Okruhlicova L Okumura H Okumura K Olbrich HG Olinger GN Olivetti G Oliviero P Olivson A Omachi A Onishi S Onodera T Onozuka H Oosthoek PW " Opasich C Opie L H

Fr008 Mo181 Th052 L44, Tu037 Tu048 Mo132 L13 Fr093 L31 Frl21 Fr001 Mo008 Mo047, Tu 174, Fr084 Mo107 Tu133, Tu134, Tu135 L52 Mo124 Mo014, Mo019 Mo030, Mo031 Tu027, Tu046 Mo030, Mo031, Th048 Th033 Fr026 Thll9 Th080 Frl40, Frl41 LI1 Th031, Th046 Mo060 Th143 Th032 Fr007 Mol60, Mo161 Thl41 Mo068 Fr057 Th071 Tu080 Fr144 Fr096 Tu042 Mo161 Th010, Th029 L32 Mo150 Mo153 Mo060 Fr022, Fr023 Mo145, Fr019 Mo181

Mo025, Th023, Th118 Tu080 Mo079, Th047, Th127 Mo 157, Th 167 Mo029 Fr064 Mo003 Tu097 Th066 Mo187, Fr056 Mo143, Th070 Mo150 Mo018

Fr089 Tu096, Th004, Th061, Fr 159, Fr186

A291

Opolski G Opstal RLJ Orchard C Orchard CH Orihashi K Orlova biN Orsetti A Osnes JB Osswald M Ostadal B Ostadalova I OtaM Owall A Oxmann T Ozawa T Pabla R Page C Palaparti A Palicka V Palmer J Palmiter K Panagia V Panagopoulos JG Panas D Pancza D Panigel R Pankucsi C Paolocci N Papadopoulos C Papageorgiou I Papousek F Papp JG

Paraskevaidis I Park J Parodi JC Parratt JR Pasini E Pataricza J Patemostro G Pattison CW Pau B Paul M Paul S Pavie A Pavoine C Pawlowski R Pechan I Pechanova O Pecker F Pedersini P Pedroso FI Peheim E Pelouch V

A292

Peng YQ Penke B Pennock G Pepe S Pepper C B Perazzo C A

Fr069 Tu115 Tu041 Fr 104 Th165 Th024 Mo123 Fr076, Frl01, Frl02 Mo067 L08, L34, Mo022, Mo028, Mo032, Mo033 Mo022 Fr166 Mo073 Tu090, Th073 Th017, Fr061 Tul31 Fr183 Mo106 Mo039 Fr055 L50 L33, Mo149 Fr004 Fr068 Tu086 Thl01 Tu004 "111096, Th 106 Tu083, Tu084, Tu085, Th 126 Fr147 Th 166, Fr036, Fr050 L54, Mo097, Mo110, Tu058, Tu059, Tu060, Tu061, Tu069, Tu070, Tu078, Tu094, Tu 136, Tul40, Th081, Th094, Fr094 Tu083, Tu084 Fr153 Tu165, Tu166 L54, Tu078, Tu088, Tu125, Tu 136, Tu 147, Tu148 Tu074, Tu153, Th139 Mo097, Tu069 Mol83 Tull6 Mo123 Fr001 Mol07 Moll7 Mo175 Thll9 Th042, Th144 Fr011 Mo175 Th005, Frl08 Th138 Th162 L34, Mo028, Me030, Me03 I, Mo032 Tu021 Mo097 Fr079 Th025, Frl03 Mo076 Mo147

Perez NG Pemow J Persad S Petrogiannopoulos CL Petronijevic MR Petronio AS Petrou M Peuchmaurd M Peuhkurmen KJ Pexieder T Phillips TM Piazza LA Pichel RH Pieper GM Pierangeli A Pierce GN Pierchala B Pieske B Pignatti C Pile A Pinson A Pioch K Piper J Piper HM Pisarenko e l Pissarek M Pitt AR Pizzurro RD Podesser B Podzuweit T Poelmann RE Pogatsa G Pongs O Ponniah S Ponomarenko IV Posa ! Posen B Post H Potevin P P6tzseh B Poulikakos JP Poussin C Pouyet T Prahash A Prasad K Prentice H Priestley D Prodius PA Prokazova N Proschek L Pshennikova MG Puceat M Pucelik P Pugsley WB Pugsley W Puigfel Y PfitzA Pyda M Pyne NJ Qian YZ Qu P

Th063, Th064, Fr09q Mo082 Frl05 Fr004 Me061 Mo049 FRO52 Mo117 Th010 L34, L35 L56 Frl00 Mo109, Mo114, Mo140, Mo144, Mo147 Mo180 Th04 l L36 Mo098 Mo132, FRO80, Fr08], Fr082, FRO83 Mo037 Fr0qO Th083 Mo132 Tu048 L37, Fr041 Tu092, Th027, Fr063 Fr125 Mo083 Th026 Th155 Tu 142, Tu 143, Tu144 Me018 Mo170, Mo171, Mol72, Mol82 L40 Tu039 Fr07 I Mo170, Mo171, Mol72, Mo182 Fr030 Tu071, Tu076 Mo104 Me070 Fr004 Tu156 Th080 Tu075, Fr163 Thl07 Frl51 Fr051 Tu162 Mo028 Tu049 Tu162 L53 Mo023, Tu003 Tu 116 Th154 Mo069 Mo188 Mo036 L38, Tu125 Tu155 Fr097

Quaini F Raap A Raatikainen MJP Rabijewski M Rabinowitz B Rackova K Radanov B Radda GK Raddatz E gah B gaj D Rakusan K Rama D Ramdev G Ramijawan B Rannou F Rao MR Rappaport L Ratsep I Ravens U Ravingerova T Raya TE Raz S Readerstorff LM D Reimers D Reiss K Reithmannn C Rendekova V Reneman RS Ribuot C Richard P Richards SM Richardson PJ Richter M Richter J Rigatto C Risberg B Rispler S Riva E Riva A givet-Bastide M Robert V Rock DT Roguin N Rogulenko R Rokosh DG Romera IC Ronca G Rong YZ Rooks S R6per J Rose J Rose ML R6s~ P Rosenfeldt FL Rosenshtraukh LV Ross EM Rossi A Rotstem Z Rousseaux CG Rozsa Z Rubinstein 1 Riicker-Martin C Ruda MY Rudiek V

Fr064 Tu008 Th019 Mo062 Tu090, Th073 Th088 Th163 Tu005, Th036, Th061 Mo020 Tu126 Fr176 L39, Fr036, Fr050 Mol23 Mo156 L36 Tu026, Fr065 Fr054 Mo003, Mo 104, Th018 Thl51 Tu007, Tu047, Tu053, Tu054 Tu125, Tu147, Tu148, Th01 I Fr079 Tu046 Fr187 Fr002 L01 Th057 Th144 Fr027 Tu 156 Fr028 Th 146, Th 159 Mo130 Fr086 Th137 L47 Mo073 Mo043 Tu182 Thl06 Tu015 Fr012 Fr185 Th092 Thl61 Fr024 Th096 Tu 130, Th114 Th122 Frl71 L40 Tu071, Fr168 Mo155 Mo182 Th 146, Th159 Th075 Fr178 Th033, Th 152 Th071 TulSl Fr094 Mo107 Tu066 Th077 Thl03

Rfiegg JC Ruest L Ruf T Ruigrok TJC Ruiz M Ruiz-Meana M Rupp H Rupp A Russ U Rutherford RAD Rybka V Rybkova I Rynkiewicz A Sabatier J Sabbah FIN Sabri A Sack S Saeki T Said SI Sakai K Sakaki Y Sakakibara Y Sakanashi M Sakata N Sakata A Sakka SG Saks V Samaja M Samanek M Samini A Samuel JL Sanbe A Sanchez MI Sandhu GS Santiago-Garcia J Sapag-Hagar M Sari T Sato H Sato N Sato M Satoh H Satter P Sauvadet A Saveljeva NJ Sawa H Sawa Y Saward L Saxena PR Scalbert E Schad H Schaffiaer T Schaper W Schaper J Schattmann J Schaub MC Scheffold T Scheinman MM Schelling P Scherlag BJ " Scheunert T Schlegel W

L41 Tu025 Mol32 Thll5 Mo069 Fr149 L05, L42, Th057, Fr038 Mo157 Thll2 Fr026 Mo120 Fr177 Thll9 Th080 Th069, Fr062, Fr067, Fr078, Fr088 Mo104 Fr167 Tu012, Tu064 Fr152 Tu125, Frl61 Mol22 Tu012, Tu064, Th 142, Th 147, Th148 Mo179 Th029 Mo154 Tu176, Fr168 Th009, Th018, Th020, Th033, Thl51, Th152 Th136, Fr162 Fr095, Fr116 Fr118 Mo003, Mo104, Th018, Fr017 Fr077 Fr047 Fr156 Mo035 Fr047 Th124 Tu134 Mo152 Tu093 L14 Th167 Mo175 Tul80 Tu 167, Fr023 L20, Th 160, Fr056 Mo103 Th05 I, Th074 Th065 Frl70 Th162 Tu079, Tu144, Tu145, Fr010 Mo070, Th135, Fr010, Fr073, Fr074, Fr075, Fr167 Fr080, Fr081 L09, I.A3, FRO49 Fr029 Th071 Mo067 Tu063, Th094 Fr058 Tu154

A293

Schlepper M Schley JA Schliiter KD Schmidt A Schmidt TA Schmidts HL Schmiedl A Schneider MD Schoels W Schoemaker RG Scholfield N Seh61kens BA Scholz W Seholz H Scholz D Sch6nekess BO Schreibmayer W Schr6r K Schroth G Schubert B Schulman DA Schulz R

SchulzeW Schulze-lckingB Schwalb H Schwan C Schwartz K Schwarzl I Schweizer MWF Schweizer M Seognamiglio A Scoote M Seipel L Seki S Self G Seliger C Semb BK Senevirame CK Seppet EK Serebryakov VN Serebryakova LI Sergiel JP Semia C Seth SD Seymour AML

Sharer MJ Shah AM Shah KR Shahi M Shao Q Sharma Sharma HS Sharony R Sharov V G Shattoek

Shaw LA Sh~elenko IV Sheridan DJ Shevlyagin S Shibata A A294

Shibata N

Th135 Mo185 Fr041 Fr080, Fr081 Tu006, Fr015, Fr085 Fr032 Th137 Mo137 Th093 Tu089, Th051, Th074 Tu013 Mo098, Th112, Thl31, Fr007, Fr032 Th112, Thl31 IA4, Tu037 Me070, Fr074, Fr075 Th003 Tu016 Tu179 Th163 Mo029 Th038 Mol00, Tu071, Tu076, Th045, Fr068 Mo105, Th023, Fr013, FRO38 Fr113 Tu097 Fr083 Th097, Fr028 Th082 Th093 Thl23 Tu087 Mo072 Th07$, Th079, Th133, Fr154 Mo168, Tu080, Th070, Frl30 Th146 Th057 Fr152 IA7 Fr096 Mo089 Tu092 Fr187 Mo102, Fr035 Fr137 Tu091, Th039, Thl41, Fr018, Fr040, Fr052, Fr053 Fr014 Mo056, Mo076, Mo146 Fr133 Frl40, Frl41 Thl05 Mo131 FrlS0 Tu097 Fr062, Fr078, Fr088 Mo027, Tu072, Th 153, Fr 124, Fr142 Tu163, Tu164 MolSl Mo047, Fr139, Frl40, Frl41 Fr067 Mol60, Mo161 Mo152

Shigematsu Y Shimada K Shimada Y Shimamoto K Shimazaki Y Shimkovich MV Shimoyama H Shin WS Shinmura K Shinoda M Shipley JB Shifin C Shofti R Shperling ID Shrier A Shulzhenko VS Shushlyapin Ol Sicotte B Siddiqui MAQ Sideman S Sidi Y Siegmund B Siffert W Silke B Silver BS Silverman AS Silverman N Silverman HS Silvestre JS Siminiak T Simko F Simnett SJ Simon A Simper D Simurda J Simurdova M Singal PK

Singh M Singh U Singh N Sinkeviciene R Skarzynski P Skatchkov M Skomedal T Skotnicki SH Skyschally A Slack JP Slack J Sladek T Sladkova J Slager CJ Slavicek J Slavikova J Slayter HS Slesak G Slezak J Slomka K Smielecki J Smik D Smith MF Smith WT Smith TW Smolenski T

Mo145, Fr019 Fr008 Frl31 Tu139 Th 160, Fr056 Tu 162 Fr067 Mol22, Frl81 Frl50 Mo079 Tu155 Th058 Mo043, Mo044 Fr046 MoO 13 Th027 Fr072 Mol08 Me010, Mol56 Mo043, Mo044, Mol35, Mol41 Th058 L37 L45 Tu013 Fr070 Tu077 Fr062 Mo094, Th031, Th046, Fr070 Fr012 Fr139, Frl40, Frl41 Me051 Fr098 Mol09, Mo114 Fr129 L46, Tu001 Tu001 L47 LI2 Fr137 L47 Th072 Thll9 Mo063 Fr076, Frl01, Frl02 Th143 Fr168 L22 Tu036 Fr036 Fr036 Th053, Th054 Thl20 Mo024 Mo124 Mo085 L48, Mo025, Mo026, Tu086, Th011, Th023, Th118 Fr069 Th085, Thl21 Me030 Th071 Th090 Fr 175 Thl02

Smolenski RT Snabaitis AK Snineak M Snircova M Sobel BE Sobey CG Socci R Soei LK Sokolov SF Solaro RJ Soler JS Soler-Soler J Sommaruga S Sommer O Sommer JR Sommersehild H Song G Song W Sonnenblick EH Sordahl LA Sorlie D Soucek M Soufir L Sourias N Sourlas N Southworth R Speechly-Dick ME Sperling O Spieekermann PG Spiers P Spinarova L Spires DA Sprengel U Spustova V Srivastava AK Stadler J Stafford RE Stahl J Stajn A Stanek V Stankovicova T Stanley WC Stapleton M Stark U Stark G Starkopf J Stavri GT Steenbergen C Stefanelli C Steigen TK Stein RA Stein B Stengl M Stepanov V Stevenson M Stokke M Strand RA Strasser R Strata G Streltzova N Str6bele M StudnevaIM Styk J St-Louis J Sudhir K

Mo077, Th039, Thl41, FRO53 Th153 Th088 Th014, Th015, Th016 Tu167 Mo052 Tu044 L49, Th053, Th054 Th076 L50, Tu036, Th066 Mo06O Fr149 Th136, Fr162 Mo063 Tu002 Th032 Tu03 I Tu147 L01 Fr145 Thl40 Fr086 Fr065 Th126 Tu083, Tu085 Mo027, Fr124 Tu116 Th058 Fr135 Tu013 Fr086 Th006 Fr167 Tu175 Mol08 Mol01 L56 Mo134 Fr057 Th091 Th022, Fr119 Th006, Th026 Th028 Th082 Th082 Tu137 Moll l Tu128 Th041 Thl40 Mo156 L44, Tu037 Mo023, Tu003 Th007 Fr007 Tu081, Fr136 Mo092 Th135 Mo049 Mo066 Th133, Fr154 Tu092, Th027, Fr063 Tu086 Mo108 Mo118

Sueda T Suehiro S Sugano M Sugiki H Sugiyama S Sulling T Sumida H Sun YS Sutherland J Sutherland FJ Sutko JL Sutton P Sutton PMI Suvorov SG Suzuki K Suzuki T Suzuki H Suzuki F Svec P Svoreik V Swanson DK Swanton H Swynghedauw B Sys SU Szekeres L Szewczyk J Szigligeti P Szilvassy Z Szorenyi Z Szule J Szymanska G Tactashov S1 Tada M Taggart P Takagi A Takagi N Takahashi R Takayama E Takazawa K Takeda S Takeda A Takeda N Takeo S Taketani S Takeuchi Y Takezono A Tamura K Tanabe H Tanaka M Tanaka F Tanaka E Tanamura A Tani M Taniguehi K Taniguehi M Tanimura M Tantini B Tararak AE Tardy [ Tarrade T

Th165 Th149 Fr020, Fr033 Tu 170 Fr061 Thl51 Mo017 Fr185 Thl50 Tu122 Tu025 Th068, Th154 Th084 Fr059 L27, Mol60, Mol61, Tu012, Tu064, Th 160 Fr025 Mol07 L28, Mol68, Thl 11, Frl30 Fr119 Mol20 Th071 Th068 Fr012 Mo075, Mo078, Mo081 L51, Tul40 Mo169 Tu004 Tu057, Tul01, Tul02, Tul03, Tu 104 Fr094 Mo169 L22 Mo042 Tu133, Tu134, Tu135, Frl21 Th068, Th084, Th154 Fr148 Fr077 Fr128 Th010, Th029 Mo122 L28, Mo168, Tu080, Th070, Thl 1 I, Fr133 Mo153, Mo154, Th169 L21, L52, Mo153, Mo154, Fr042 Tu093, Fr077, Frl61 Fr056 Mo154 Frl61 Tu080 Fr166 Fr061 Mo168 Tu030 L52 Fr150 Fr056 L28, Mo168, Tu080, Th070, Th111, Frl30 Fr067 Mo037 Th077 Fr147 Tu057

A295

Tassini M Taylor PM Taylor I Toffenborg D Teichmann W Telahoun G Telegdy G Terada H TerraccianoC M N TesfaiSA TestoniS Thakkar JK Thiemermann C Thomas S Thorm~len D Thuringer D Tian G Tian R Tian YP Tiivel T Tirosh R Toki Y Toloikis A "roman J Toompuu M Toraeea L Tosaki A Toth GK Townsend P Toyo-oka T Tdbulova N TrinesSAIP Tdpathi 0 TritthartH A TrittoI TrivodiU H TrojaniA TroUingerD R Trollip G Tromp E Trouve P Trumbeekaite S Tsehopp A Tse A Tse F

Tsipizidou S Tskitishvily OV

A296

Tsorin IB Tsuchiya T Tsuehiya M Tsuehiya N Tsyplenkova VG Tumanovskaya L V Tunin R Turan B Tureani M Tutterova M Tvrzicka E Tweedie D Udvary E Uejima Y Ueki K Uoshima K Ulicna O

Th001, Th002 Mo155 Tu002 Mo066 Tu168 Tu075 Tu177 LI4 Th060 Fr134 Tul30, Th114 Fr156 Tu048 Tu143 Fr094 Tu05 l, Tu052 Th158 Th052 Fr126, Fr127 Th018 Th083 Mo079, Th047, Th127 Th012, Th013, Thl04 Fr086 Th018, Thl51 Th139 Mo184, Mo185, Tu157 Tu069 Mo054 Mo122, Frl81 Mo025, Mo026, Tu086, Th011, Th023, Th I 18 Th053, Th054 Tu016 Th082 Tu087 Mo048 Fr043, Fr044 Fr134, Fr144 Th156 Tul41, Fr146 Fr065 Th013 Th162 Mo112 Mo112 Fr082 Tu092 Fr164 Th169 Fr042 Th025 Th067 FrI57 Fr070 Mo129 L42 Th055, Fr003 Mo030, Th048 Fr045 Th081 Fr166 Fr008 Fr091 Th014

Ulmer W Umetsu K Undrovinas AI Unitt JF Unterberg C Ura N Urasawa K Urata H Urbanski NK Uretzky G Utkin VN Utkina N Umo L Vaage J Vaiciulyte R Valen G Valent D van Bedaf DM van Beusekom HMM van Bilsen M van den Hoff MJB van der Giessen WJ van der Vusse GJ van Dobbenburgh JO van Echteld CJA van Emous JG van Eys GJJM van Gmneken ACG van Heugten HA van Klaarwater E van Nieuwenhoven FA van Rooyen J van Winkle DM Vane JR Vanin A Vanin AF Vannier C Varkonyi T Varro A Vasiljev AP Vassort G Vavrinkova H Vavrova M Vaynblat M Vecchmi A Vegh A Veksler V Vetdkamp MW Verm GE Ventura-Clapier R Verdetti J Verdouw PD Verecl Z Vergoni W Vergroesen I Verkerk AO Verstijnen CPHJ Vespalcova Z Vetter R Vezekenyi Z Viana F Vidne B

Fr032 Fr123, Fr145 Th069 Fr040 Tu 169 Tu139 Mo186, Tul70 Fr001 Tul07 Tu097 Fr059 Mo028 Thl51 Mo073 Th072 Mo073 Tu159 Mo078 Mo050 Fr027 Mo001, Mo002 Mo050 Mo009, Fr027 Th164 Th115, Th164 Thll5 Mo009 Tu067 Frl80 Tu115, Fr143 Mo009

Th004 Tu068 Tu048 Mo063 Tu158 Mo129 Tu063, Th 124 Tu004, Tu059, Tu060, Tu061, Tu070, Th094 Mo066 L53, Mo129 Th055, Fr003 Thl20 Mol56 Mo149 L54, Tu078, Tu125, Tu136, Th081 Mo127 Tu067 Mo048, Th145 Mo127, Th018 Tu156 L49, Mo050, Tu089, Tu I 15, Th053, Th054, Fr143, Frl80 Th071 Tu022 Tu 174 Tu067 Mo009 Th086 Mo034, Mol34, Fr038 Mo110 L31 Thl03, Th157

Vie B Viko H Vila-Petroff M Villeneuve A Vincan E Vinogradova TM Virag L Vitek F Vititnova M Vittone L Vivi A Vogel Z Vogt A Vohnout B Volt T yon Oppell U von Segessser L von zur Miihlen F Vomanen M Vosberg HP Vrana M Vrana A Vucic G Vuorinen KH Wada A Wadsworth R Wagenaar GTM Wagner T Wahn H Wainwright CL Walker JM Wallace S Wallbridge DR Wallen J Wallukat G Walpoth BH Walpoth-Aslan BN Walsh RA Wang D Wang YP Wang Y Wang J L Wang WZ Ward BJ Ward DC Warley A Watanabe H Watanabe T Watanabe M Watkins MW Wayne J Weber K Webster KA Weglicki WB Wei SK Wei DN Weichert A Weigl DR Weihrauch D Weinbroum A Weis M Weist B Welch W Wenink ACG

Tu176 Fr102 Fr099 Th080 Fr114 Th075 Tu060, Tu061 Fr090 Th 100 Tu040, Tu041 "1"11001,"1"11002 Tu046 Tu142, Tu144, Tu145, Th135 Tu159 Fr029 Th 150 Th 163 Th113 L55 Fr043, Fr044 Th091 Fr003 Thl61 Th019 Mo 152, Mo 187 Tu 171 Mo007 L56 Th I 13 Mo083, Tu 171 Th084 Th006 Tu076 Th043 Mo159, Tu154, Fr009, Fr013, Fr 106 Thl61, Th162, Th163 Th 163 Tu031 Tu 132, Fr 174 Mo 126 FrlSl Mo 133 Mo113 Mo071, Mo072 Fr182 Fr165 LI4, Fr128 Tu110, Fr034 Mo 150 L24, Mo138 Th071 L11 Frl51 L56 Th03 I, Fr070 Mo084 Th112, Thl31 Th 138 Tu144, Tu145 Th 103 Th167 Fr043, Fr044 Tu025 Mo018

Werdan K Wettwer E Wharton J Wheeler CH Whelan DMC Why HH Wibo M Wieczorek D Wiemer G Wilde AAM Wilkins MR Willemsen PHM Williams TJ Williams FM Willis R Willis RJ Willshaw P Wilson S Wilson DP Wilson ANA Wilson DA Windeatt S Winkelmann A Winlove P Wisse LJ Witters LA Wittnich C Wocial B Wolff RA Wollenberger A Wollenek G Wolska BM Wood DE Woodcock EA Woodman OL Woodward B Woolley SR Worthington M Wrzosek A Wykretowicz A Wylie A Wynne DG Wysocki H Xiang B Xiao RP Xiao Y Xiaolian L Xu K Xu Y Xu A Xu YJ Yabe K Yacoub MH

Yagihara T Yahalom M Yajima K Yamada S Yamamoto F Yamashita N Yanaga T Yanagida S Yanagishita T Yang YZ

Mol01, Th057, Th168 Tu047, Tu053, Tu054 Fr026 Mo 130 Mo050 Mo130 FrO 16 L50 Mo098, Fr032 L57 Fr026 Mo009 L58 L58 Mo055, Mo058, Tul08, Th037 Mo 148 Mo 144 Tu125 Tu 172, Tu 173 Tu 12 I Mo053 Fr052 Tu142 Mo047 MoO 18 Th050 Th043 Fr069 Tu068 Fr 106 Th 155 Tu036, Th066 Fr174 Tu082, Th089, Fr114, Fr155 Mo052, Tu098 Mo053 Th164 Th 150 Tu024 Th085, Th 121, Fr 120 Fr 151 Mo012 Th085, Th 12 I, Fr 120 Th158 Frl03 Fr112 Mo086 Fr097 Fr054 Tu032 Mo178 Tu093, Fr077 Mo130, Mo155, Th039, Thl41, Fr018, Fr040, Fr052, Fr053 Th149 Th092 Tu012, Tu064 Mo 150 Th142, Th147, Th148, Th149 Frl 21 Fr020, Fr033 Th010, Th029 Mo177, Fr034, Fr122 Tu021

A297

Yang H Yang XM Yang ZK Yang WD Yang D Yasuda M Yasuda 0 Yasutake M Yau L Yellon DM Yevstratova IN Ying MZ YlitaloKV Yokoyama H Yonemitsu Y Yoneya K Yoshida H Yoshida I Yoshida K You H Young CP Ytrehus K YuXJ YuG Yuan F Yuzuk TN Zabirov VII Zador E Zaharof AK Zahkharenko S Zahor~ R Zahradka P Zahradnik I Zahradnikova A Zaitsev A V Zak A Zak R Zaman JS Zamotrinsky A

A298

Th090 Tu073, Tu 127 Tu062 Frl81 Fr037 Mo017 Fr008 Th059 Mo090 Tu112, Tu113, Tu116, Tu117, Tu146, Tu149, Tul51, Tu152 Th024 Fr126 Th019 Tu134 L20 Fr022 Mo088 Mo186 Tu155 Fr153 Th145 Tu114, Tu137, Tu138, Th132 Mo084, Mo087 Tul30, Th114, Fr097 Frll2 Th075 Mo042 Fr158 Fr004 Mo089 Th042, Th 144 Mo090, Mo103, Tu172, Tu173 Tu017 Tu017 Th075 Th048 Mo121 Tull2 Tul60

Zang WJ Zanoni P Zarain-Herzberg A Zegner M Zoitlin IJ Zeltcer G Zeng Q Zeng TS Zhai X Zhang LP Zhao JX Zhao RR Zhao YL Zharinov Ol Zhen ZY Zheng L Zheng G Zheng HG Zheng S Zhonghe Y Zhou ZN Zhou X Zhou XJ Zhou YY Zhu XY Ziegelh6ffer A Ziegelh6ffer B Ziegelstein RC Ziegler ID Zikic RV Zimmermann R Znojil V Zolotarski V Zoref-Shani E Zubajova-Svecova E Zubritsky AN Zueehi R Zyeh M

Tu011 Tu022 Mo035 Th155 Mo083 Fr118 Mo084, Mo087 Mo 102 Tu123 Mo087 Tu021 Mo113 Fr127 Th099 Th098 Fr016 Mo094 Fr126, Fr127 Tu014 Mo086 Frll2 Tu123 Fr041 Tu055, Tu056 Th122 L59, Tu147, Tu148, Th011 Th022 Mo025, Mo026 Mo094 Tu058 Fr057 Tu079 Mo074 Th157 Th058 Th088 Fr092 Mo049, Tul30, Th114 Mo077