2004 ISHR North American Section Meeting

Journal of Molecular and Cellular Cardiology 36 (2004) 609–640 www.elsevier.com/locate/yjmcc

Abstracts from the 26 Annual Scientific Sessions, North American Section of the International Society for Heart Research th

Bench to Bedside and Back: Exploring New Paradigms - A Multinational Perspective of Cardiovascular Research in North America 2-5 May, 2004 Westin Regina Resort, Cancun, Mexico doi:10.1016/j.yjmcc.2004.02.008

2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

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2004 ISHR North American Section Meeting Abstract N° 1 Effects of fixed-dose combination trandolapril-verapamil in stage 2 uncontrolled hypertensive patients Alberto Rubio, Adalberto Arceo, Jose Lozano, German Vargas, Leticia Rodriguez, Luz Ramos. Hospital General de Ticomán, México D.F. About 70% of hypertensive patients will need more than one drug for their control. It has been proposed that a better blood pressure control will be achieved with fixed-dose combination formulations. Methods. – 40 hypertensive patients with blood pressure > 160/100 mm Hg in spite of more than 6 month of drug treatment, self-measured their blood pressure (SMBP) during 3 days with a validated equipment; later on, they received the fixed-dose combination of trandolapril-verapamil (2/180 mg) for 12 weeks, when a second SMBP was performed. The antihypertensive control achieved and its behavior during the day was evaluated. The statistical analysis was performed using ANOVA. Results. – 39 patients controlled their blood pressure (10 men and 29 women age 59 ± 9) from 184/100 to 132/77 mm Hg all day long (p < 0.001), and one man did not respond. One patient suffer headache and one more referred constipation, no one stopped the drug. Conclusion. – Fixed dose combination of trandolaprilverapamil seems to be an effective and safe option for the management of stage 2 hypertensive patients uncontrolled with monotherapy. Abstract N° 2 In vivo NADPH oxidase inhibition restores endothelial function in preproinsulin gene mutant Ins2Akita diabetic mice Xiang-Qun Yang, Alex F. Chen. Departments of Pharmacology and Neurology and the Neuroscience Program, Michigan State University, East Lansing USA Oxidative stress causes endothelial dysfunction and atherogenesis in diabetes. We tested the hypothesis that NADPH oxidase-derived superoxide (O2–) induces endothelial dysfunction in Ins2Akita diabetic mice, with a spontaneous autosomal preproinsulin gene mutation. Compared to agematched normal mice, a 4-month high fat dieting significantly increased plasma lipid peroxidation and arterial O2– levels and reduced NO synthase (NOS) cofactor tetrahydrobiopterin (BH4) levels in diabetic mice, resulting in impaired endothelium-dependent relaxation in carotid arteries (91.8 ± 3.3 vs. 74.9 ± 3.7 %, control vs. diabetes, n = 8, p < 0.001). Vascular cellular adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) levels were also markedly elevated. In vivo treat-

ment of Ins2Akita mice with the selective NADPH oxidase inhibitor apocynin (4 mg/kg/d) for 3 mo not only significantly reduced plasma lipid peroxidation and arterial VCAM-1, MCP-1 and O2– levels, but also reversed the BH4 deficiency and restored impaired endothelium-dependent relaxation (92.0 ± 3.4%, n = 5, p < 0.001). These results indicate that NADPH oxidase is a key source for O2–-induced endothelial dysfunction in preproinsulin gene mutant Ins2Akita diabetic mice. Abstract N° 3 Expression and activity of aromatase in rat coronary microvascular endothelial cells in culture *Alfredo Sierra-Ramı´rez; **Tomas Morato; *Ivan Rubio; *Rafael Campos; *Claudia Calzada, *Guillermo Ceballos. *Laboratorio Multidisciplinario, Seccio´n de Posgrado, E.S.M, I.P.N. **U.A.M-I, México. Email: [email protected] In this work we explored the participation of aromatase enzyme (P450AROM, the product of the CYP19 gene) on testosterone-induced [Ca2+]i kinetics on rat male and female coronary micro-vascular endothelial cells (CEMC) in culture. We also explored the expression and catalytic activity of aromatase by immunoblot, immunocytochemical and in vitro enzymatic assays. Our results showed that testosterone induced-effects were of nongenomic origin and phospholipase C activity is involved in the androgen induced increase on [Ca2+]i, those effects can be blocked by highly selective inhibitors; amino-glutethimide and 4OH-androstenedione. Immuno-cytochemical assays and immunoblots were positive for aromatase. We also found that in vitro enzymatic assays using [1B-3H]Androstendione as substrate result in the formation of tritiated water (an indirect proof of aromatization). Either, male or female CEMC of rats are able of aromatize androgens to estrogens. Abstract N° 4 Toluene inhibits Na+ currents from cardiac and skeletal muscle transfected into Xenopus laevis oocytes Marcia Y. Gauthereau 1, Silvia L. Cruz 1, Gerardo J. Orta *, Lourdes Millan *, Eduardo M. Salinas-Stefanon *. 1 Department of Pharmacobiology, Cinvestav, IPN, Mexico, D.F. * Institute of Physiology, BUAP, Puebla, Mexico Sodium channels are responsible for membrane depolarizations and the initial phase of action potential. Inhibition of these channels can result in cardiac arrythmias and conduction disturbances. In this work we tested the hypothesis that the abused solvent toluene inhibits cardiac and skeletal muscle sodium channels. Sodium channels (Nav 1.5, and Nav 1.4, alfa and beta1 subunits) were transfected into Xenopus laevis oocytes. Sodium currents (INa+) were elicited by step depo-

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2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

larizations from a holding potential of -110 mV to various test voltages. Toluene inhibited Nav1.5 INa+ in a concentration-dependent manner with an IC50 of 274 µM; while the Nav 1.4 INa+ have a IC50 of 2.61mM. The inhibition was complete, voltage-independent and slowly reversible. Toluene had no effect on the reversal potential of sodium or the steady-state inactivation. The slow time recovery constant from inactivation of INa+ decreased with toluene, while the fast time recovery constant remained unchanged. Blockade of INa + by toluene was use-dependent and frequencydependent. Data using a post-rest stimulation protocol suggested that toluene does not bind to closed sodium channels. The blocking effect of INa+ by toluene, enhanced by channel use and opening frequency might be responsible, at least in part, of the cardiac arrythmias and conduction disturbance in skeletal muscle that have been related to toluene exposure. 1 30571M CONACyT and *VIEP-BUAP II77G01. Abstract N° 5 Clortalidone (ClorT) inhibits potassium currents in Guinea-pig myocytes Cludia Mancilla 1, Angelica Lopez *, Eduardo M. Salinas-Stefanon 1. Instituto de Fisiología-BUAP Puebla, México ClorT is a tiazidic diuretic used communly as first choice in hipertensive cardiac disease. ALLHAT study shows that the drug have a protective effect on cardiac function; the electrophysiological effects of ClorT are poorly understood. This work studies the direct actions of the drug on potassium cardiac currents in isolated cardiac myocytes. We used perforated patch-clamp whole cell technic in order to explore the effects of Clort on the repolarization currents (Ik1, Ito and Ik ). Stimulation and recording protocols were made using a pClamp software. Standard, external and internal solutions were used; all the experiments were performed at 37 °C, pH 7.40. ClorT prolong the cardiac action potential repolarization by a 46% without changes in RP or dV/dt. ClorT (30 µM) have an inhibitory effect, 48% ± 2.6 on delay rectified potassium current, mainly at expenses of the rapid component (Ikr), it is dose-dependent, with a IC50 of 32.4 µM and a Hill number of 1.02. The drug has a blocking effect on Ito current ~23% ± 3.2 the inward rectified potassium current. This effect may be responsible at least in part of the protective effect on cardiac function. 1 Supported by BUAP-VIEP # II77G01 and *CONACyT fellowship 176941. Abstract N° 6 Differential effects of primaquine on sodium channels isoforms Gerardo J.Orta *, Lourdes Millan, Eduardo M. Salinas-Stefanon 1. Instituto de Fisiología, BUAP, Puebla, México Voltage-gated Na+ channels underlie rapid conduction in heart and skeletal muscle. We studied the effects of prima-

quine (PQ) on human cardiac (Nav 1.5) and rat skeletal muscle (Nav 1.4) sodium channels expressed in Xenopus oocytes. At 20 µM, PQ caused a use dependent inhibition of Nav 1.5 Na + current (INa+) by 50% after 20 pulses at 1 Hz. It was necessary 60 µM PQ to decreased Nav 1.4 INa+ by 50%. In control condition, recovery from inactivation occurred with two times constants in Nav 1.5 channels: a fast time constant (sf) of 5.9 ms and slow time constant (ss) of 57.3 ms. After PQ (20 µM) sf was 16.2 ms and ss 104.0 ms. Recovery from inactivation time constant for Nav 1.4 channels were 1.9 ms and 52.7 ms in control conditions and 1.8 ms and 66.4 ms after the exposure to PQ (60 µM). Steady-state inactivation curves had a V1/2 = – 75.6 mV and k = – 6.4 for Nav 1.5 in control conditions and – 71.6 mV and – 6.8 after exposure to PQ. In Nav 1.4 channels V1/2 = – 62 mV and k = – 5.0 in control conditions and – 66.4 mV and – 6.9 after exposure PQ. Here we showed that the primaquina is most effective at blocking cardiac than skeletal muscle sodium channels isoforms. * CONACYT fellowship 122184. 1 BUAP-VIEP II77G01. Abstract N° 7 The role of reactive oxygen species and apoptosis in anthracycline-induced myocyte death Richard S. Vander Heide, Thomas L’Ecuyer. Depts. of Pathology and Pediatrics, Wayne State Univ. and John D. Dingell VAMC, Detroit, MI 48201 Anthracyclines (AC) are anti-tumor antibiotics with significant activity against solid and hematologic malignancies. One problem preventing more widespread use has been the development of cardiac toxicity. Experimental evidence supports oxidant stress-induced apoptosis as an important mediator of AC-induced cardiotoxicity. We developed a cell culture model system using the H9C2 cardiac cell line exhibiting controlled overexpression of the antioxidant glutathione transferase (GST). Treatment with the AC doxorubicin (DOX) produced both oncosis, manifested by an increase in the number of cells staining positive for trypan blue, and apoptosis, indicated by the presence of positive deoxynucleotidyl transferase (TUNEL) staining. In both cases, ACinduced increase in fluorescence with carboxy-dichlorofluorescein diacetate (DFDA) demonstrated the presence of high levels of reactive oxygen species (ROS) within one hour of exposure. The DOX-induced increase in ROS was reduced to control levels by maximal GST-overexpression. Coincident with this elimination of oxidative stress, there was a reduction in DOX-induced cell death. Inhibition of mitochondrial permeability transition or caspase 9 reduced DOXinduced cell death while caspase 8 inhibition (marker of Fas pathway) had no effect on cell death. We conclude that ROS appear early and play a role in DOX-induced cell death and that the mitochondrial pathway of apoptotic cell death appears more important than the extrinsic pathway in causing apoptotic cell death in this model system.

2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

Abstract N° 9 Length dependence of activation in mice expressing troponin I with non-phosphorylatable PKA sites Julian E. Stelzer, Jitandrakumar R. Patel, Daniel P. Fitzsimons, Jeffery W. Walker, Richard L. Moss. Dept. of Physiology, University of Wisconsin, Madison, Wisconsin, USA Phosphorylation of troponin I (TnI) by Protein Kinase A (PKA) has been proposed to have an important role in modulating the Ca2+ sensitivity of force (pCa50) in myocardium. We examined the role of PKA phosphorylation on length dependence of pCa50 in a transgenic murine model that has non-phosphorylatable PKA sites (TG). The pCa50 of skinned ventricular myocardial preparations from wild-type (WT) and TG mice was measured at short (1.90 µm) and long (2.35 µm) sarcomere lengths (SL). Lengthening the SL increased the pCa50 in both WT and TG preparations, the change in pCa50 was not significantly different between WT and TG mice (D pCa50 = 0.12 ± 0.01 units, respectively). These data suggest that PKA mediated phosphorylation of TnI does not play a role in length dependent modulation of Ca2+ sensitivity of force in murine myocardium. Abstract N° 11 Anti-apoptotic ARC Blocks Bax Activation Åsa B. Gustafsson, Joseph G. Tsai, Susan E. Logue, Michael T. Crow, Roberta A. Gottlieb. The Scripps Research Institute and Johns Hopkins University School of Medicine Myocardial ischemia/reperfusion (I/R) is associated with apoptosis. The Apoptosis Repressor with caspase recruitment domain (ARC) is a protein that is highly expressed in heart and protects against ischemia/reperfusion (I/R) injury. In this study, we show that transduction of TAT-ARCL31F, a mutant of ARC in the CARD domain, did not reduce CK release and infarct size after I/R. TAT-ARCL31F also failed to protect against hydrogen peroxide-mediated cell death in H9c2 cells, suggesting that the CARD domain is important in mediating ARC’s protective effects. ARC co-immunoprecipitated with Bax. TAT-ARC, but not TAT-ARCL31F, prevented Bax activation and cytochrome c release in peroxide-treated H9c2 cells. TAT-ARC was also effective in blocking cytochrome c release after ischemia and reperfusion, whereas TAT-ARCL31F had no effect on cytochrome c release. In addition, recombinant ARC protein abrogated Bax-induced cytochrome c release from isolated mitochondria. This suggests that ARC can protect against cell death by interfering with activation of the mitochondrial death pathway through the interaction with Bax, preventing mitochondrial dysfunction and release of pro-apoptotic factors. Abstract N° 12 Inhibition of protein phosphatase protects hearts from ischemia-reperfusion injury Monika Skrzypiec *, Meltem Sariahmetoglu, Jolanta Sawicka, Grzegorz Sawicki, Richard Schulz. * Medical

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Univ. Wroclaw, Poland; Dept. of Pharmacology, Univ. of Alberta, Canada Matrix metalloproteinase-2 (MMP-2) contributes to cardiac dysfunction resulting from ischemia-reperfusion (I/R) injury. MMP inhibitors protect the heart from I/R injury. The latest evidence suggests that phosphorylation is a novel mechanism in regulating MMP-2 activity in addition to its activation by proteolytic cleavage, oxidative stress, or inhibition with natural protein inhibitors (TIMPs). Dephosphorylation of MMP-2 with protein phosphatase enhances its activity. We used isolated rat hearts perfused with Krebs buffer at 37°C at constant pressure and subjected them to 20 min global ischemia and 30 min reperfusion. Serine/threonine phosphatase and MMP-2 activities were measured in samples from aerobically perfused hearts (control) or those subjected to I/R +/- the serine/threonine phosphatase inhibitor okadaic acid (n = 3–5 hearts/group). Treatment of MMP-2 preparations with alkaline phosphatase increases its activity by at least twofold. With 10 nM okadaic acid, total phosphatase activity in homogenates from hearts subjected to I/R was inhibited by approximately 36%. Okadaic acid (100 nM) protected hearts from I/R injury (72 ± 5 vs. 53 ± 5% of baseline rate pressure product, p < 0.05). These results suggests that inhibiting the dephosphorylation of MMP-2 could be a novel strategy to protect hearts from I/R injury.

Abstract N° 13 Postconditioning protects isolated hearts through PI3K signaling Xi-Ming Yang, Lin Cui, Thomas Krieg, James M. Downey, Michael V. Cohen. University of South Alabama, Mobile, AL. Multiple short coronary occlusions immediately after prolonged myocardial ischemia protect in situ rabbit hearts through ERK activation, nitric oxide production, and opening of mitochondrial KATP channels. We determined if postconditioning also protects buffer-perfused, isolated rabbit hearts. Infarct size was measured with tetrazolium staining. Control hearts undergoing 30 min of regional ischemia and 2 h of reperfusion infarcted 31.5 ± 2.4% of the risk zone. Four postconditioning cycles of 30-sec global ischemia/30-sec reperfusion starting 30 sec after release of the index coronary occlusion decreased infarction to 22.7 ± 2.4% of the risk zone (p < 0.05). Wortmannin (100 nM), a PI3K antagonist, infused for 20 min starting 5 min before reperfusion, blocked postconditioning’s protection (35.5 ± 2.1% infarction). Western blotting showed that Akt phosphorylation, a reporter for PI3K activity, fell during ischemia with partial rebound at 10 min following reflow in control hearts. The increase was greater after postconditioning. Thus, postconditioning’s protection is not dependent on circulating blood factors or cells, and its anti-infarct effect requires PI3K activation.

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2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

Abstract N° 14 A novel long-chain free fatty acid export pathway in heart mitochondria Lamar K. Gerber, Bruce Aronow, Mohammed A. Matlib. Department of Pharmacology, Cell Biophysics, and Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45267 Microarray analysis was used to identify genes in fatty acid (FA) metabolism that are altered in streptozotocin (STZ)-induced diabetic rat hearts. The results show increased expression of key genes in long-chain FA mitochondrial transport and oxidation. There are similar increases in mitochondrial thioesterase-1 (MTE-1) and uncoupling protein-3 (UCP3). The roles of these two proteins in FA oxidation are unknown. These proteins may constitute a novel FA export pathway in which MTE-1 synthesizes longchain free FA from FA-CoA in the matrix and UCP3 exports the free FA across inner mitochondrial membrane. We propose this system is up regulated to prevent feedback inhibition of â-oxidation in mitochondria of STZ-induced diabetic rat hearts. To test this hypothesis, synthesis and export of free palmitic acid during oxidation of palmitoyl-carnitine in mitochondria of normal and STZ-induced diabetic rat hearts were determined. Synthesis and export of free palmitic acid increased in diabetic rat hearts. The rate of synthesis of free palmitic acid accompanies increases in rate of MTE-1 activity and MTE-1 protein levels. The rate of free palmitic acid export accompanies UCP3 protein level. This study is the first to demonstrate existence of a novel long-chain free FA synthesis and export system in heart mitochondria. Abstract N° 15 Expression of sarcoglycan complex in vascular tissue from human umbilical cord Ramírez-Sánchez I. §, Rosas V.H. §, Ceballos R.G. *, Salamanca G.F. §, Coral-Vazquez R. §. § U.I.G.H. Centro Médico Nacional Siglo XXI. * E.S.M-IPN The sarcoglycan complex (SGC) is composed of a,b,g, d,e,z SG and sarcospan (SPN). Mutations in a,b,g and d, SG lead to limb-girdle muscular dystrophies, ocationaly assosiated with dilated cardiomyopaty, which is presumably caused by the loss of SGC in the coronary microvasculature. This suggests a great importance of SGC in the vascular phisiology. To know more about the expression of the SGC in the vascular tissue, we analyzed the expression of transcripts and proteins of the SGC in endothelium (E) and smooth muscle (SM) from vein and artery of human umbilical cord. The results show the transcripts expression of a,g,b,d,e SG and SPN in the SM. In the case of a SG, to our best knowledge, this is the first time that its transcript is detected in SM. In contrast, by immunofluorescence assays, in the same tissue, there was only evident expression of b,d,e SG and SPN. On other hand, in E we detected the transcripts expression of b,d,e SG and SPN; while to protein level it was only detected e-SG and SPN. Taken together all the results, it may be

suggested a posttranscriptional down regulation process to a and g SG in SM, and to b and d SGs in E. Abstract N° 16 Cytoprotection through modulation of succinate dehydrogenase activity Petras Dzeja, Arturo Valverde, Peter Bast, Andre Terzic. Mayo Clinic, Rochester, MN, USA Succinate dehydrogenase (SDH) has been implicated in the respiratory burst and generation of reactive oxygen species at reoxygenation after ischemia. Here, two murine strains with inherited differences in myocardial SDH activity were tested for their response to ischemia/reperfusion. After 30 min ischemia, hearts from 129T2SvEmsJ mice displayed 20% lower SDH activity and reduced oxidative stress, along with a 28% greater functional recovery and preserved cellular nucleotide pool, compared to C57Bl/6 mice. Reduction of oxidative stress on reperfusion was also induced by 3-nitropropionic acid, an inhibitor of SDH, and by diazoxide, a potassium channel opener. Diazoxide, within its cardioprotective concentration range, reduced SDH activity and diminished the generation of reactive oxygen species (ROS). In an SDH-deficient cell line, with increased stress resistance, diazoxide had little effect on flavoprotein oxidation and ROS generation. Thus, reduced cellular oxidative injury can be achieved by modulation of mitochondrial dehydrogenase activity. Abstract N° 17 ATP-loaded liposomes protect mechanical functions of myocardium during and after global ischemia in isolated rat heart Tatyana S. Levchenko, Daya D. Verma, Eugene A. Bernstein, Vladimir P. Torchilin. Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, 02115 USA ATP-loaded liposomes have been used as a bioenergetic supplement for protecting mechanical function during ischemia-reperfusion in the isolated rat heart. ATP-loaded liposomes were prepared from egg PC, Ch, DSPE-PEG2000, and DOTAP (2.3:1:0.016:0.09 molar ratio) by freezingthawing, passing through polycarbonate membranes, and dialysis. Isolated Langendorff buffer-perfused hearts were used. Sprague-Dawley rats were anesthetized intraperitoneally with sodium pentobarbital. Hearts were perfused through the aorta to the coronary arteries with oxygenated Krebs-Henseleit (KH) buffer pH 7.4 at coronary perfusion pressure of 80 mmHg. Thebesian drainage was removed via an apical drain placed through the left ventricular apex, and a cannula was secured into the pulmonary artery for coronary venous drainage. Left ventricular developed pressure (LVDP) was measured via the cateter inserted through the left atrial incision into the LV. After 25 min of global ischemia hearts were reperfused for 30 min and contractility was measured. The LVDP at the end of reperfusion in the liposo-

2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

mal ATP group recovered significantly better than in the KH buffer, plain liposomes, and ATP in KH buffer control groups. At the end of reperfusion, LVEDP (left ventricular end diastolic pressure) was significantly reduced in the liposomal ATP group as compared to the KH buffer, plain liposomes, and ATP in KH buffer control groups. Cardioprotection with free ATP was totally eliminated after incubating the free ATP with ATPase, while the protective effect of the liposomal ATP remained unchanged. Liposomal ATP delivery effectively protects the severely ischemic isolated heart by supporting its systolic and diastolic functions. Abstract N° 18 Nitric oxide mediates apoptosis in acute myocarditis induced by a Mexican strain of trypanosoma cruzi Maria L. Loredo 1,2, Luis C. Mita-Alban 1, Victor M. Monteon 1, Julian C. Escobar 2, Zu-Xi Yu 2. 1 Instituto Nacional de Cardiología, Mexico D.F. 2 NHLBI, NIH, Bethesda, MD, USA Trypanosoma cruzi strains of low virulence cause cardiomyopathy. To explore the mechanisms of acute myocardial injury mediated by nitric oxide (NO) in this disease, Balb/c mice (n = 25) were infected with a low virulence strain (Mexican, Ninoa), and treated with or without L-NMMA (inhibitor of iNOS). At day 25, Apoptotic Index (AI:number of apoptotic nuclei per mm 2, identified by TUNEL assay) for myocytes and nonmyocytes was significantly higher in the infected group (n = 4) vs the non-infected group (n = 4, p = 0.004). In addition AI was lower in the infected, L-NMMA treated group (n = 4) than in the infected, untreated group (n = 4, p = 0.01). The inflammatory and vascular reactivities were moderate and showed no differences in both groups. Immunolabeling for iNOS and nitrotyrosine, performed to evaluate NO production, revealed less intense reactivity and fewer positive cells in the infected, treated group compared with the infected, untreated group. In conclusion, apoptosis, as well as inflammation and vascular reactivity contribute to myocardial damage in this model of acute Chagasic cardiomyopathy. Apoptosis was significantly reduced by inhibition of NO products suggesting that is a NO mediated phenomenon, in contrast to inflammatory and vascular reactivities, which were unaffected. Abstract N° 19 Effect of the caveolin-1 scaffold peptide and E2 on Ang ii-induced [Ca+2]i in vascular smooth muscle cells Enrique Méndez B., * Javier Sánchez G., Pedro López S., Guillermo Ceballos R. * Escuela Médico Militar. SEPI-Escuela Superior de Medicina-I PN. México, D. F. We analized the effects of caveolin-1 scaffold peptide on angiotensin II-induced intracellular calcium kinetics and the coexpression of caveolin-1, AT1-AT2 receptors and estrogen receptor (alpha) in VSMC from HMIAMC. Angiotensin II through AT1 and AT2 receptors activation induce several effects in the vasculature, like vasoconstriction and synthesis

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of vasodilator factors. Caveolae, have been related with several signalling molecules, i. e, G-proteins, PI3K-Akt. Caveolin-1 develop a scaffold zone in caveolae related to “preassembled signalling complexes”. Using Human Vascular Smooth Muscle in culture we assay the effects of the scaffold peptide of caveolin-1 on intra-cellular calcium kinetics induced by angiotensin II and, the coexpression of AT1, AT2 receptor types, estrogenic receptor by immunofluorescence and immuno-blots. Our results showed that increase intracellular calcium kinetics induced by angiotensin II was inhibit by the scaffold peptide of caveolin-1 and AT1, caveolin-1 and estrogenic receptor colocalized. We found AT2 immunoexpression in these cells and AT2, AT1, caveolin-1 and estrogenic receptor colocalized. Abstract N° 20 Changes in the expression of AT1 and AT2 receptors in huvec from preeclamptics: role of oxidative/nitrosative damage Cecilia Fernández del Valle 1, María Elena López 1, Alfredo Sierra-Ramírez 1, José Antonio Chimal 2, Guillermo Ceballos 1. 1 Escuela Superior de Medicina, I.P.N. 2 Hospital de la Mujer SSA, México, D.F. [email protected] Preeclampsia occurs after 20th week of gestation features hypertension, edema and proteinury; the etiology is unknown. Women that develop preeclampsia show an increased vascular reactivity to angiotensin II (AII). Thus has been suggested that the local RAS is involved in the pathogenesis. Placenta of preeclamptic women shows an increased activity of ACE and an enhanced release of renin, prorenin, and angiotensinogen. The signaling mechanisms of AII are linked to free radicals. Peroxinitrite is a potent oxidant and nitrating of proteins, modifying their function. For inmunochemistry methods, we found an increased expression of AT1 and AT2 in HUVEC of preeclamptics, and a higher oxidative/nitrosative damage (Indirectly measured by 3Ntyrosin concentration and directly by Electronic Paramagnetic Resonance). This work was supported by IPN and CONACYT 31423-M and 634998-N Grants. Abstract N° 21 ECE-1-mediated signaling in ventricular myocyte contractility and apoptosis in sepsis Akanksha Gupta, Nicholas S. Aberle II, Jun Ren, Avadhesh C. Sharma Dept. Pharm. Sci., NDSU, Fargo, ND, & Div. of Pharm. Sci., Univ. Wyoming, Laramie, WY We tested the hypothesis that endothelin-converting enzyme-1 (ECE-1) modulation would alter sepsis-induced adult rat ventricular myocyte (ARVM) contractile dysfunction and apoptosis along with upregulation of p38-MAPK. ARVM were isolated from septic or sham rat hearts and treated with FR901533 (FR, 1, 10 and 50 µM) in presence and absence of ET-1 precursor bigET-1 (100nM). FR produced a significant time-dependent elevation of caspase-3 activity in septic ARVM, an effect accentuated by bigET-1.

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2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

FR-induced downregulation of p38-MAPK phosphorylation in sepsis was reversed by bigET-1. Although septic ARVM demonstrated an up-regulation of ERK, it was significantly depressed in sham-ARVM. BigET-1 significantly increased the cell length in sham and septic ARVM. In septic ARVM, FR decreased the cell length, an effect that was reversed by bigET-1. These data demonstrated that ECE-1 inhibition downregulated p38-MAPK phosphorylation and upregulated caspase-3 activity in septic ARVM. We conclude that ECE-1 plays a crucial role in development of ARVM dysfunction via p38-MAPK signaling and apoptosis during polymicrobial sepsis. (Funded by NIH & AHA) Abstract N° 22 E2 inhibits contractile effects of phenylephrine associated with the release of [Ca2+]i Carlos Castillo, Guillermo Ceballos, Javier Larios, Cleva Villanueva, Roberto Medina, Jorge López, Enrique Méndez, Enrique F. Castillo. SEPI-Escuela Superior de Medicina-IPN., México, D.F. In rat aortic rings incubated in Ca2+-free solution, we evaluated the ability of E2 to affect phenylephrine (phen)induced contraction, and increase in resting tone (IRT) associated with capacitative Ca2+ entry. Applied 5 min before phen (10-6M), E2 (10-9-10-7M) inhibited both the pheninduced contraction and IRT. Neither cycloheximide (106M) nor tamoxifen (10-6M) modified the effects of E2. E2(10-4M) also blocked the contractile response to serotonin but not to caffeine. In addition, E2 (10-4M) inhibited contractile responses to cyclopiazonic acid (10-5M) associated with capacitative Ca2+ influx through on-L-type Ca2+ channels. Finally, E2 inhibited the increases in intracellular free Ca2+ elicited by the addition of Ca2+ (after pretreatment with phen) in cultured rat aorta smooth muscle cells incubated in Ca2+-free solution. In conclusion, E2, interferes with intracellular-Ca2+ dependent contractile effects mediated by alpha1- adrenergic- and 5-HT2 serotonergic receptors and inhibits capacitative Ca2+ influx, through both L-type and non L-type Ca2+ channels. Such effects are nongenomic and non-mediated by the intracellular estrogenic-receptor. Abstract N° 23 The alpha-adrenoceptors related capacitative Ca2+ influx in rat aorta María C. Castillo, *Rafael Villalobos, Cleva Villanueva, Enrique F. Castillo, Carlos Castillo. Escuela Superior de Medicina-IPN and *CINVESTAV-IPN. México, D.F. We analized the role of alpha-adrenoceptors in mediating the capacitative Ca2+ influx in rat aorta, using toracic and abdominal aortic rings from the male rat, with and without endothelium, incubated in Ca2+-free solution. We compare the contractil effect of phenilephryne, oximetazolyne and UK-14304, and we found that the all of them exert a contractil effect in rings without endhotelium, but the rings with

endothelium with UK-14304 didn´t responde, a diference with the anothers two agonist. Also we analyzed the Ca2+ capacitative inward induced for the three agonist, in control rings and with adrenergic antagonist, like prazosine, rawolscine, phenoxybenzamine; until this moment we found that phenilephryne induce capacitative Ca2+ influx due to alpha1-adrenoceptor activation because the effect was inhibit in presence of prazosine; and seems to be that the effect of oximetazolyne and UK-14304 is mediating for alpha1 and 2-adrenoceptors because the effect was inhibit with phenoxybenzamine. Also we study the temporal effect and we saw that rings with endothelium have a different temporal effect that rings without endothelium this effect can be caused by NO or/and some prostanoid.

Abstract N° 26 PPARgamma agonists, Ciglitazone and GW7845, induce apoptosis in cultured rat neonatal cardiomyocytes independent of PPARgamma Qianhong Qin, Lihong Cheng, Guoliang Ding, David Woods, Qinglin Yang. Cardiovascular Research Institute, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta GA 30310 PPARgamma agonists of the thiazolidinedione (TZD) class have been shown to inhibit cardiac hypertrophy. However, these ligands may exert potential apoptotic effect on a variety of cells. To test the hypothesis that PPARgamma ligands is also a proapoptotic factor in cultured cardiomyocytes, we treated cultured rat neonatal cardiomyocytes with various doses of Ciglitazone and GW7845 (a highly selective synthesized PPARgamma ligand). Apoptotic cardiomyocytes detected by TUNEL assay were significantly increased in both ligand treatment groups in a dose dependent manner. On the other hand, GW9662, a PPARgamma-selective antogonist, did not block the apoptotic effects induced by both Ciglitazone and GW8475. In addition, adenoviral overexpression of PPARgamma in cardiomyocytes did not induce significant cardiomyocytes apoptosis. We conclude that PPARgamma ligands induced cardiomyocytes apoptosis in a dose dependent manner and this effect is independent of PPARgamma receptor.

Abstract N° 27 Calcium binding to cardiac troponin c assessed by caging the regulatory binding site Karen L. Reece, Jeffery W. Walker, Gerard J.D. Marriott, Richard L. Moss. Department of Physiology, University of Wisconsin Medical School, Madison, WI, 53706 The direct effect of Ca2+ binding on the regulation of myocardial force has not been determined because fluorescent reporters of Ca2+ binding are sensitive to other factors. To address this problem, a caged Ca2+ binding site has been

2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

engineered as a tool for assessing Ca2+ binding to cTnC and its role in thin filament activation. A photolabile group, 4,5dimethoxy-2-nitrobenzyl bromide [DMNBB, Aldrich], has been incorporated into the regulatory Ca2+ binding loop of cTnC, preventing Ca2+ from binding and “caging” the protein. Incorporation of caged-cTnC into skinned myocardium will allow the kinetics of Ca2+ binding to cTnC to be recorded as changes in fluorescence of a Ca2+ sensitive fluorophore (e.g., Fluo-3) while simultaneously measuring force development. Incorporation of the cage into the binding loop of cTnC has been verified both by absorbance at 350 nm and MALDI-TOF mass spectrometry. The cage can be subsequently removed by irradiation with UV light as shown by absorption spectroscopy. Abstract N° 28 Pro-oxidant activation of PKC d prevents pyruvate dehydrogenase reactivation during cardiac reperfusion Eric N. Churchill 1, Christopher L. Murriel 2, Daria Mochly-Rosen 2, Luke I. Szweda 1. 1 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH USA. 2 Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA USA The mitochondrial multienzyme complex, pyruvate dehydrogenase (PDH), exhibits diminished activity during cardiac ischemia/reperfusion (I/R). Augmentation of pyruvate oxidation through pharmacological stimulation of PDH improves contractile recovery during reperfusion. We sought to find processes that control inactivation and reactivation of PDH during I/R. Cardiac I/R is associated with increased generation of mitochondrial derived superoxide and hydrogen peroxide (H2O2) and translocation of PKC d to the mitochondria. Using a Langendorf model of I/R and a specific peptide inhibitor of PKCd we found that inhibition of PKC d translocation to the mitochondria during reperfusion resulted in dephosphorylation and activation of PDH. Infusion of a pharmacological activator of PKC d into perfused control hearts resulted in translocation of PKC d to the mitochondria and loss in PDH activity. Similarly, infusion of the pro-oxidant H2O2 resulted in a loss in PDH activity that was prevented when PKC d translocation was blocked. These results indicate that pro-oxidant dependent activation and translocation of PKC d to the mitochondria during reperfusion maintains PDH in a phosphorylated and inactive state. Abstract N° 29 Lipopolysaccharide mediated vasorelaxation: role of matrix metalloproteinases and the endothelium Jonathan Cena, Richard Schulz. Departments of Pharmacology and Pediatrics University of Alberta, Edmonton, Canada Recent evidence implicates the role of matrix metalloproteinases (MMPs) in vascular contractile dysfunction during sepsis. The contribution of the endothelium to vascular chan-

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ges observed in models of sepsis is unknown. Our lab has shown that inhibition of MMPs is beneficial in models of sepsis. We investigate the role of the endothelium in lipopolysaccharide (LPS) induced vascular dysfunction. Aortic rings from normal rats either with or without endothelium were mounted into organ baths in Krebs Henseleit buffer (37°C, 95% O2:5% CO2). The endothelium was carefully removed in some rings by light rubbing. Rings were contracted with phenylephrine (750 nM) and the LPS (300 ng/mL) mediated relaxation was observed over 8 hr in the presence or absence of the MMP inhibitor doxycycline (30 microM). Control rings relaxed greater than endothelium denuded rings (to 30.1 ± 3.5% and 56.2 ± 3.2% of the initial contractile response to phenylephrine, respectively; p < 0.01). Doxycycline inhibited the vasorelaxation seen over 8 hours; however, this was not different in normal (62.3 ± 3.5%) or denuded rings (62.6 ± 9.8%). This study demonstrates the contribution of the endothelium in LPS mediated vasorelaxation. The effects of MMP inhibition appear to be independent of the vascular endothelium.

Abstract N° 30 Cytoskeletal protein alterations in myocardial ischemia/reperfusion injury: potential role of matrix metalloproteinases Miranda M. Sung, Christina G. Schulz, Jolanta Sawicka, Grzegorz Sawicki, Richard Schulz. Departments of Pharmacology and Pediatrics, University of Alberta, Edmonton, Canada We have previously reported that MMP-2 is co-localized with troponin I (TnI) of the sarcomere, where degradation of TnI by MMP-2 contributes to acute myocardial ischemiareperfusion (I/R) injury. We tested the hypothesis that MMP-2 may also degrade cytoskeletal proteins to contribute to the contractile dysfunction after I/R. In vitro degradation assays of purified alpha-actinin, desmin and spectrin by purified MMP-2 showed that alpha-actinin and desmin, but not spectrin are susceptible to proteolytic cleavage after 1-2 hr incubation at 37ºC (n = 3). Isolated rat hearts were perfused aerobically for 75 min or subjected to 20-min global, no-flow ischemia followed by 30-min reperfusion in the presence or absence of MMP inhibitors, doxycycline or o-phenanthroline (100 microM each). Immunoblotting of heart homogenates demonstrated a twofold increase in alpha-actinin and more than sixfold increase in desmin following I/R (p < 0.05 vs. controls; n = 5-6/group). Both MMP inhibitors normalized alpha actinin levels to that of the controls. In contrast, o-phenanthroline, but not doxycycline, normalized desmin levels. The data suggests that MMPs could mediate, by an unknown mechanism, the increased alpha-actinin and desmin levels seen following I/R. This mechanism could involve increased biosynthesis, decreased degradation and/or posttranslational modifications of the protein.

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Abstract N° 31 Inhibition of dephosphorylation downregulates MMP-2 activity in vivo M. Sariahmetoglu, H. Leon, J. Sawicka, G. Sawicki, R. Schulz. Depts. of Pharmacology and Pediatrics, University of Alberta, Edmonton, Canada We have shown that matrix metalloproteinase-2 (MMP-2) is activated in hearts subjected to ischemia-reperfusion injury and that a unique intracellular proteolytic action of MMP-2 contributes to the acute cardiac mechanical dysfunction by cleavage of troponin I. MMPs can be directly activated by oxidative stress or by proteolytic removal of the propeptide domain, however, other post-translational mechanisms controlling MMP activity are not well understood. We hypothesized that the activity of MMP-2 could also be affected by phosphorylation. Ten possible phosphorylation sites on human MMP-2 for each of serine or threonine and nine for tyrosine were identified using NetPhos 2.0. Recombinant MMP-2 or that partially purified from conditioned media of HT1080 cells were examined. Immunoprecipitation of conditioned media demonstrated that MMP-2 is phosphorylated at threonine, serine, and tyrosine residues. Dephosphorylation of MMP-2 resulted in significantly increased gelatinolytic activity for both recombinant and partially purified MMP-2. Cells treated with the protein phosphatase inhibitor okadaic acid significantly decreased MMP-2 activities in the media in a concentration-dependent manner. These findings show that MMP-2 is phosphorylated and its phosphorylation status determines its activity. Abstract N° 32 Ca-insensitive phospholipase A2 activity inhibits the Ca paradox Katherine T. Potter, Robert A. Haworth. Dept. Surgery, Univ. Wisconsin, Madison, USA Ca restoration following a period of zero Ca perfusion of hearts causes massive Ca uptake and enzyme release, a phenomenon called the Ca paradox. Inhibition of Ca-sensitive phospholipase A2 (cPLA2), was reported to inhibit this enzyme release. Inhibition of the Ca-insensitive phospholipase A2 (iPLA2) with bromoenol lactone (BEL), on the other hand, was reported to protect hearts from ischemia-reperfusion injury, while AACOCF3, an inhibitor of both cPLA2 and iPLA2, did not protect. We therefore investigated the role of iPLA2 in the Ca paradox. Rat hearts were perfused for 15 min with Krebs-Henseleit medium containing 2.5 mM Ca, for 5 min -Ca +0.1 mM EGTA, then again with the +Ca medium. Reperfusion caused release of lactic dehydrogenase (LDH), 16.9 ± 5.5% of total in the first 3 min. When 10µM BEL was included throughout, the enzyme release was 43.6 ± 5.1% (p < 0.005, n = 3 in each group). These results show that while cPLA2 activity may promote the Ca paradox, iPLA2 activity protects against the Ca paradox. This suggests that iPLA2 and cPLA2 may play an opposite signaling role in the Ca paradox, and not necessarily a direct membrane-destructive role.

Abstract N° 33 Inhibition of intracellular MMP-2 protects from ONOO– -induced mechanical dysfunction in single cardiac myocytes Hernando León 1, Istvan Baczko 2, Peter E. Light 2, Gregorz Sawicki 2, Richard Schulz 1,2. Departments of Pediatrics 1 and Pharmacology 2, Cardiovascular Research Group, University of Alberta, Canada. The potent oxidant peroxynitrite (ONOO– ) induces mechanical dysfunction in the heart through activation of matrix metalloproteinase-2 (MMP-2). This may be an effect independent of MMPs actions on the extracellular matrix. Therefore, the purpose of this study was to study the effects of ONOO– on contractile function at the level of the single cardiac myocyte. Methods. – Freshly isolated rat ventricular myocytes were continuously superfused at 21°C with K-H solution and paced at 0.5 Hz. Contractility was measured using a video edge-detector. Active ONOO– or decomposed ONOO– (300 mM) were superfused over 40 min to evaluate the contraction stop time (CST). Results. – Myocytes subjected to 300 mM ONOO– had shorter CST than decomposed ONOO– (14.9 + 1.7 vs. 29.6 + 4.3 min, n = 6; p < 0.05). The MMP inhibitor doxycycline (100 mM) significantly delayed the CST induced by ONOO– (22.1 + 3.3 vs. 15.1 + 2.2 min for ONOO–, n = 3; p < 0.05). Conclusions. – This is the first demonstration that inhibition of intracellular MMP-2 protects myocytes from peroxynitrite-induced cardiac dysfunction independent of an action of MMPs on the extracellular matrix. Abstract N° 36 Signals mediating the high molecular weight FGF-2induced chromatin disruption and cell death Xin Ma, Cheryl Hirst, Farah Sheikh, *Peter Claus, Robert R Fandrich, *Claudia Grothe, Peter A Cattini and Elissavet Kardami. Institute of Cardiovascular Sciences, University of Manitoba, Winnipeg, Manitoba. Canada, and *Hannover Medical School, Hannover, Germany Previously we showed that overexpression of the stressinduced, nuclear CUG-initiated (hi) FGF-2 in cardiomyocytes induces chromatin compaction, leading to cell death presenting apoptotic features. We have now addressed the role of casein kinase 2 (CK2), ERK1/2, PKC e, and intracellular FGF-2 receptor (FGFR1) in mediating the effects of hiFGF-2. Chromatin disruption was prevented/ decreased by overexpression of dominant negative forms of FGFR1 and MEK1, pharmacological inhibition of ERK1/2 or CK2, by overexpressing a mutant, non-nuclear hi-FGF-2, or a mutant hi-FGF-2 unable to activate CK2. In contrast, the phenotype was not prevented by neutralizing anti-FGF-2 antibodies (blocking action of all extracellular FGF-2), exogenous addition of the 18 kDa FGF-2, or overexpression of dominant negative PKC e. Similar results were obtained for cardiomyocytes and HEK293 cells. We conclude that the hi-FGF-

2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

2-induced cell death phenotype follows an intracrine-nuclear pathway that requires activity of FGFR1, ERK1/2 and CK2. Abstract N° 37 Is moderate drinking associated with alcoholic behavior? K.A. Ammar, R. Rodeheffer, M.B. Shapiro, R. Morse. Olmsted Medical Center and Mayo Clinic, MN, USA Objectives. – Despite the documented cardiovascular (CV) protective effect of moderate alcohol intake, clinicians are reluctant to prescribe alcohol. We hypothesized that alcoholism is associated with a higher number of drinks per day, beyond the CV benefit range. Identifying a cut-off point in terms of number of drinks per day, beyond which one has a high risk of developing alcoholic behavior, would decrease the reluctance in medical community against alcohol prescription. Methods. – In a randomly selected, populationbased sample of 2042 adults, age >= 45, alcohol quantity consumed was assessed by a self-administered questionnaire, and alcoholic behavior by using Self-Administered Alcoholism Screening Test (SAAST). The prevalence of coronary, peripheral or cerebrovascular disease was determined by ECG and systematic medical record review. Results. – Identified alcoholics (A) were 439, nonalcoholic problem drinkers (NAPD) were 1256, 160 were careful alcohol users (CAU), 166 were abstainers and 341 had vascular disease. The amount of alcohol consumed was similar (<= 2 drinks/day) in 86% of A, 93% of NAPD and 94% of CAU (p > 0.05). In multivariate analyses, alcohol intake was associated with reduced odds ratio of vascular disease in A (0.4; CI 0.3 to 0.7) and in NAPD (0.5; CI 0.3 to 0.7) but not in CAU (OR 1.9; CI 0.9 to 3.8), as compared to abstainers. Conclusion. – There may be no safe cut-off point, since most alcohol consumers drink similar quantities, within the CV benefit range, not withstanding the tendency of alcoholics to underreport. Abstract N° 38 The influence of alcohol intake on ventricular function K.A. Ammar, M.M. Redfield, R.J. Rodeheffer, S.J. Jacobsen. Mayo Clinic, Rochester, MN, USA Background. – Moderate alcohol consumption has been shown to be associated with reduced incidence of congestive heart failure (CHF) in the community. However, it is not known if alcohol protects against systolic dysfunction (SD) or diastolic dysfunction (DD). Methods. – In a randomly selected, population-based sample of 2042 adults, age >= 45, we assessed alcohol quantity by a self-administered questionnaire. Diastolic Dysfunction (DD) and Systolic Dysfunction (SD) were assessed by M-mode and 2D Doppler echcoardiography. Results. – 46 cases of CHF, 244 with Coronary Artery Disease (CAD), 186 abstainers, 316 past alcohol users (PAU)

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and 1501 current alcohol users (CAU) were identified. 1-7 drinks per week was associated with reduced odds of CHF (OR 0.4; CI 0.1, 1.0; p 0.06). SD (ejection fraction) was not associated with alcohol intake.DD was progressively more prevalent in abstainers vs. PAU vs. CAU (50% vs. 39% vs 35%; p 0.002). A J-shaped curve was observed in odds of exposure to alcohol quantitiy and DD, with the lowest odds seen in the 1-7 drinks per week, as compared to abstainers (OR 0.5;CI 0.4,0.8). CAU were less likely to have diastolic dysfunction (OR 0.55;CI 0.4, 0.8; p < 0.001) as compared to abstainers, a relationship which persisted in bivariate stratified analysis with diabetes, hypertension, smoking and BMI, but disappeared whenadjusted for age. CAU were less likely to have CAD (OR 0.5; CI 0.4,0.8). Conclusion. – The protective effect of moderate alcohol intake may be due to prevention of coronary disease leading to reduced prevalence of diastolic dysfunction. Abstract N° 39 Hyperthyrodism in hamsters reverses fetal gene expression while producing chf phenotype James A. Kuzman, Tracy A. Thomas, Kathryn A. Vogelsang, Brent E. Anderson, Suleman Said, A. Martin Gerdes. Univ South Dakota, Sioux Falls, SD Recent evidence suggests that thyroid dysfunction may be an important component of CHF but potential toxicity has not been well investigated. Hyperthyroidism (T) was induced in 3 mo old normal male F1B (C) and dilated cardiomyopathic (CM) hamsters. After 2 mo treatment, hemodynamics, echos, and isolated myocyte dimensions were collected. T in C and CM hamsters produced a similar increase in heart weight/body weight (~35%) but very different phenotypes. Compared to C, TC hamsters showed significant ventricular dilatation (↑ LVIDd and LVIDs), ↓ +dP/dT, ↓ -dP/dT, and a selective ↑ in myocyte length (e.g. CHF) despite ↑ $-myosin and ↓ b-myosin. Compared to C, CM hamsters had ↑ LVIDd, ↓ ejection fraction, ↓ +dP/dT, ↓ -dP/dT, and severe myocardial fibronecrosis (e.g. CHF). TCM hamsters showed a similar reversal of the fetal gene program but no further ventricular dilatation or further deterioration of ventricular function. Thus, T was tolerated better in CM than C hamsters. Abstract N° 40 Insulin deficiency causes free radical generation, DNA damage, and apoptosis Viktor Pastukh, Craig Ricci, Stephen W. Schaffer. University of South Alabama, College of Medicine, Department of Pharmacology, Mobile, Alabama, USA Diabetes is associated with major changes in mitochondrial metabolism, including defects in oxidative phosphorylation. Since type I diabetes is characterized by the lack of insulin, we hypothesized that insulin withdrawal could be an important mediator of the mitochondrial defects observed in diabetes. To test this hypothesis, cardiomyocytes were incubated in insulin-free medium. Removal of insulin caused an

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increase in iNOS levels, as well as a rise in reactive oxygen species (ROS). Inhibition of iNOS with aminoguanidine, or ROS with tiron, partially prevented mtDNA damage, cytochrome c release, and cell death associated with insulin deficiency. The scavengers also partially prevented the drop in the levels of subunit ND5 of complex 1 and cytochrome c oxidase associated with insulin withdrawal. Since cardiomyopathies are known to arise in hearts showing abnormalities in mitochondrial DNA and electron transport, we propose that insulin deficiency plays an important role in their development in the diabetic individual. Abstract N° 41 Hyperglycemia preconditions the cardiomyocyte against hypoxia Craig Ricci, Viktoriya Solodushko, Stephen W. Schaffer. University of South Alabama, College of Medicine, Department of Pharmacology, Mobile, Alabama, USA Many animal studies have reported that diabetes protects the heart against ischemia. The mitochondrial permeability transition pore (mPTP) may be involved since its inhibition blocks hypoxia-induced cell death. We hypothesized that hyperglycemia (HG) can precondition the heart by upregulating cardioprotective factors, such as p-Akt, Bcl-2, and PKC-e, which regulate the mPTP. Thus, cardiomyocytes cultured in insulin-free medium and either 5 mM or 25 mM (HG) glucose were subjected to chemical hypoxia. HG reduced hypoxia-induced cell death, and HG cells exhibited elevated levels of p-Akt, Bcl-2, and activated PKC-epsilon. Chelerythrine, a PKC inhibitor, or dom/neg Akt, reversed preconditioning. Interestingly, HG caused increased cell death during hypoxia in cells cultured in medium containing insulin. These cells also showed decreased levels of p-Akt, Bcl-2, and activated PKC-e. Our data suggest that the dominant effect of HG is to induce insulin resistance, which is detrimental to the cell during hypoxia. However, HG alone preconditions the cell by mimicking some of insulin’s favorable effects, including upregulating p-Akt, Bcl-2, and PKC-e, promoting mPTP closure. Abstract N° 42 The effect of anesthesia on echocardiographic left ventricular structure and function in rats Adam B. Stein, Roberto Bolli, Paul Thomas, Marcus F. Stoddard, Greg Hunt, Buddhadeb Dawn. From the Institute of Molecular Cardiology, University of Louisville, and the Jewish Hospital Heart and Lung Institute, Louisville, KY The use of anesthetic agents during echocardiographic studies may potentially alter left ventricular (LV) structural and functional parameters. We compared echocardiographic parameters in male Fischer 344 (F344) rats anesthetized with pentobarbital (PB, 25 mg/kg i.p.) (group I, n = 6) and inhaled isoflurane (ISF, 1.0%) (group II, n = 6). The body weight and temperature during the study were comparable between the two groups, while the heart rate was higher in group I (491 ±

15 bpm vs. 356 ± 9 bpm in group II, p < 0.0001). Rats in group I exhibited greater LV ejection fraction (81 ± 1.5% vs. 76 ± 1.3% in group II, p < 0.05), LV fractional shortening (58 ± 2% vs. 48 ± 1% in group II, p < 0.05), LV fractional area change (66 ± 2% vs. 59 ± 1% in group II, p < 0.01), and the velocity of circumferential fiber shortening corrected for heart rate (3.7 ± 0.2 cir/s vs. 2.8 ± 0.1 cir/s in group II, p < 0.01) as compared with group II. Rats in group I also exhibited smaller LV anatomical parameters, including LV end-diastolic diameter (5.6 ± 0.2 mm vs. 6.7 ± 0.1 mm in group II, p < 0.001), LV area in diastole (25 ± 1.8 mm2 vs. 36 ± 1.4 mm 2 in group II, p < 0.001), and LV end-diastolic volume (160 ± 16 m l vs. 236 ± 9 m l in group II, p < 0.001) as compared with group II. The cardiac output was similar between two groups despite a greater stroke volume in group II (189 ± 5 m l vs. 129 ± 9 m l in group I, p < 0.01). We conclude that in F344 rats, PB anesthesia is associated with less depression of cardiac contractility and LV dilatation as compared with ISF anesthesia. Abstract N° 43 MMP-dependent degradation of myosine light chain in isolated rat hearts subjected to ischemia-reperfusion injury Grzegorz Sawicki 1, Hernando Leon 1, Jolanta Sawicka 1, Meltem Sariahmetoglu 1, Danuta Szczesna-Cordary 2, Richard Schulz 1. 1 University of Alberta, Canada. 2 University of Miami, USA Matrix metalloproteinase-2 (MMP-2) contributes to cardiac dysfunction resulting from ischemia-reperfusion (I/R) injury. MMP-2 not only remodels the extracellular matrix but also acts at the intracellular level by degradation of troponin-I. We used a proteomics approach to search for other possible targets of MMP-2. Isolated rat hearts were subjected to 20 min ischemia and 30 min reperfusion. The impaired recovery of mechanical function of the heart was attenuated by MMP inhibitors (100 µM of phenanthroline and 100 µM of doxycycline). 2-D electrophoresis was performed for protein analysis using samples from aerobically perfused hearts (control) or those subjected to I/R injury in the presence or absence of MMP inhibitors (n = 6 in each group). Quantitative analysis showed two low molecular weight proteins whose levels were significantly increased upon I/R injury and normalized to control heart levels by MMP inhibitors. Mass spectrometry analysis identified that both proteins are fragments of myosin light chain 1. Our results demonstrate the usefulness of the proteomics approach in identifying possible new targets for MMP-2 which may contribute to contractile dysfunction of the heart. Abstract N° 44 A cardiac or cardiac-like milieu, rather than lengthy exposure to 5-azacytidine, is enough to initiate rat bone marrow stromal cell cardiomyogenic differentiation Weixin Liu a,d, Jian Song a,b, Yu Wan c, Guodong Pan a, Yu Liu a, Bangchang Cheng d, Xichang Chen a, Jiang Chang e.

2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640 a

Faculty of Anatomy and Embryology. b Center for Research in Structural Biology. c Dept. of Physiology. d Dept. of Thoracic-Cardiovascular Surgery of Renmin Hospital, Wuhan University School of Medicine, Wuhan, Hubei, P.R. China. e Dept. of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA Objectives. – Previously we have reported that rat marrow stromal cells (MSCs) cannot be significantly induced into cardiomyocytes by 5-azacytidine. In the present study, we further investigated this induction by testing whether long term exposure to 5-azacytidine could expand MSCs’ cardiac lineage. In addition, coculture of MSCs with cardiomycytes was conducted to explore the mechanisms involved in this differentiation. Finally, the cardiomyogenic potency of MSCs was investigated using in vivo transplantation. Methods. – A stable MSC cell line with the reporter gene ECFP (enhanced cyan fluorescent protein), under ventricular myosin light chain 2 promoter, was established. The stable MSCs were exposed for up to thirty days to 5-azacytine treatments (single/repeat) at varying concentrations (3, 5 and 10 µM). In coculture experiments, the primary MSCs were cocultured with rat neonatal cardiomyocytes for periods up to sixteen days. For the transplantation, primary MSCs were directly injected into the border region of infarcted myocardium and expression of cardiac $/bMHC and Tn I was analyzed. To evaluate cell-cell interaction, FRAP (fluorescence recovery after photobleaching) assays were performed. Results. – No ECFP expression was detected in any of the thirthy-day experiments among all of the groups. In contrast, spontaneously contracting MSCs were first observed on day one under coculture with cardiomyocytes. $/bMHC and Tn I were also observed on day one, day four and reached the highest levels on day eight, which were 2.92 ± 0.76% and 1.96 ± 0.62%, respectively (both p < 0.0001 compared to MSCs alone). FRAP assays indicated that there were direct intercellular communications between MSCs and adjacent cardiomyocytes. In vivo transplantation revealed the expression of $/bMHC and Tn I MSCs cells beginning at the third week and lasting for three weeks. Conclusions. – We questioned the novel effects of 5-azacytidine on the cardiomyogenics of MSCs. Direct intercellular communications between MSCs and cardiomyocytes and establishment of a cardiac microenvironment are necessary and sufficient for MSCs to differentiate into cardiomyocytes both in vitro and in vivo.

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was examined in an in vitro ischemic model, utilizing adult rabbit cardiomyocytes. Akt and GSK-3 phosphorylation in the cytosolic fraction were determined by immuno-blotting for Akt p-Ser473 and GSK-3$/b p-Ser21/9. Akt and GSK3$/b were observed at high levels in oxygenated (O2) and 15 min ischemic (Isch), control (Ctrl) and APNEA (APN) cells. Akt p-Ser473 and GSK-3$/b p-Ser21/9 were minimal in O2/Ctrl cells. Akt p-Ser473 was increased 3.0 ± 0.7 fold in O2/APN cells (p < 0.003, vs O2/Ctrl); 2.4 ± 0.3 fold in Isch/Ctrl cells (p < 0.001, vs O2/Ctrl) and 5.7 ± 1.2 fold (vs O2/Ctrl) in Isch/APN cells (p < 0.005, vs Isch/Ctrl). GSK-3 a/b p-Ser21/9 was increased 2.1 ± 0.3/4.1 ± 0.9 fold in O2/APN cells (p < 0.01/0.007 vs O2/Ctrl) and 2.6 ± 0.5/2.0 ± 0.3 fold in Isch/Ctrl cells (p < 0.02/0.05 vs O2/Ctrl). Akt p-Ser473 and GSK-3$/b p-Ser21/9 were attenuated in Isch/Ctrl cells (p < 0.004 /0.002/0.02) by the PI-3K inhibitor, Ly294002 (10 m M). The cardioprotection provided by pharmacologic preconditioning might be mediated by activation of the Akt pathway. Abstract N° 46 NADPH and BH4 partially restore coronary flow in rat hearts subjected to global ischemia and reperfusion Cristian Dumitrescu, Yong Xia, Arturo J. Cardounel, Jay L. Zweier, Davis Heart and Lung Research. Institute, The Ohio State University, Columbus, Ohio The overall goal of this study was to establish the role of eNOS substrates and cofactors in restoring vasoreactivity in hearts subjected to different ischemia times followed by reperfusion. Hearts were perfused in Langendorff mode and allowed a 15-20 minute equilibration period after cannulation. Histamine (10 µM) was than infused through a side arm of the perfusion apparatus and vascular relaxation determined by measuring changes in coronary flow (CF). After CF returned to baseline, we initiated global ischemia for 30, 45 or 60 minutes, followed by 30 minute reperfusion. We found that CF decreased by 19 ± 3.2, 30 ± 3.6 and 50 ± 2.6 % respectively when compared with baseline preischemic CF. NADPH (175 µM) together with BH4 (50 µM) infusion restored vasoreactivity to 77 ± 4, 60 ± 10 and 59 ± 13 % of the histamine response before ischemia. HPLC experiments showed a severe reduction of BH4 and NADPH levels on reperfusion. Arginine (1mM) and calmodulin infusion had no significant effect on postischemic CF after 30minute reperfusion.

Abstract N° 45 Pharmacologic preconditioning with apnea enhances the phosphorylation of AKT and GSK-3 in adult cardiomyocytes Stephen Armstrong. Dept. of Pathology, College of Medicine, East Tenn State Univ; Johnson City, TN

Abstract N° 47 Role of akt signaling in mitochondrial survival pathway triggered by hypoxic preconditioning Takamichi Uchiyama, Nilanjana Maulik, Dipak K. Das. Cardiovascular Research Center, University of Connecticut School of Medicine

The enhancement of Akt and glycogen synthase kinase-3 (GSK-3) phosphorylation by pharmacologic preconditioning with the A1/A3 adenosine receptor agonist, APNEA (1 µM),

The intracellular signaling pathways that control ischemia/reperfusion-induced cardiomyocyte apoptosis in heart have not fully defined. In this study, we investigated

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whether Akt signaling has a role in the anti-apoptotic pathways of preconditioning against hypoxia/reoxygenation (H/R) in adult cardiomyocytes. Primary culture of adult rat ventricular myocytes (ARVMs) were subjected to hypoxic preconditioning by exposing the cells for 10 min hypoxia followed by 30 min of reoxygenation (PC). Nonpreconditioned and PC myocytes were subjected to 90 min of hypoxia followed by 120 min reoxygenation. Hypoxic-PC protected the myocytes from subsequent H/R injury as evidenced by decreased apoptosis and LDH release and increased cell viability. H/R-induced cytochrome c release and activation of caspase-3 and -9 were blocked by PC. This protective effect was inhibited by treating the cells with LY294002 (50 mM), a PI3-kinase inhibitor, for 10 min before and during PC. PC also induced phosphorylaton of Akt and BAD. Protein levels of Bcl-2 in mitochondria were maintained in PC. ARVMs were infected with either a control adenovirus (Adeno lac-Z), an adenovirus expressing dominant-negative Akt, or an adenovirus expressing constitutively active Akt. Ectopic overexprssion of constitutively active Akt protected ARVMs from apoptosis induced by hypoxia/reoxygenation compared with Adeno lac-Z. In contrast, dominat negative Akt overexpression abolished the anti-apoptotic effect of PC. Our data demonstrated that in adult cardiomyocytes, the anti-apoptotic effect of PC against H/R requires Akt signaling leading to phosphorylation of BAD, inhibition of cytochrome c release and prevention of caspase activation. Abstract N° 48 Myocyte dysfunction during ventricular remodeling induced by chronic volume overload in rats Yanfeng Ding, Ruijiao Zou, James A. Stewart Jr., Gregory L. Brower, Joseph S. Janicki, Juming Zhong. Auburn University, Auburn, AL 36849 Previous results from our group demonstrated a progressive ventricular hypertrophy, dilatation, and contractile depression in response to chronic volume overload induced by an infrarenal aorto-caval fistula in rat. Whether this decompensation was related to myocyte dysfunction has not been determined. The present study evaluated ventricular myocyte function in rats at 5- and 10-weeks post fistula. Pulmonary edema occurred in rats at 10 wk post fistula. Although the cell size of myocytes from both fistula groups were significantly larger than controls, resting sarcomere lengths and intracellular Ca 2+ concentrations ([Ca2+]i) were not different among the three groups of myocytes. When myocytes were electrically stimulated at 0.5 Hz, sarcomere shortening and peak [Ca2+]i at 5-wk post fistula were not significantly different from that of control, but were significantly reduced at 10-wk. Corresponding to the reduced peak [Ca2+]i, the whole-cell peak Ca2+ current (ICa) density was reduced in myocytes from 10 wk post-fistula hearts. Bath application of isoproterenol increased cell shortening, peak [Ca2+]i, and ICa density in all three groups of myocytes, but failed to fully compensate for the functional deficiency in

myocytes at 10 wk post-fistula. These data indicate that in this model of chronic volume overload, ventricular myocyte contraction is depressed due to altered intracellular Ca2+ homeostasis and reduced Ca2+ channel activity during the development of heart failure. Abstract N° 49 Ischemia-induced ventricular arrhythmias in rats in vivo Arvinder K. Dhalla, Wei-Qun Wang, Luiz Belardinelli. Dept of Drug Research and Pharmacological Sciences, CV Therapeutics, Inc. Palo Alto, CA 94304 Ranolazine (RAN) has been shown to be cardioprotective against ischemia-reperfusion injury. Recent in-vitro studies show that RAN has anti-arrhythmic effects; however, they are yet to be evaluated in-vivo. The objective of the present study was to determine the effect of RAN on ischemiainduced ventricular arrhythmias (ventricular tachycardia; VT ventricular fibrillation; VF). Anesthetized male rats were artificially ventilated with room air and were subjected to in vivo left anterior descending coronary artery ligation. RAN treatment (10 mg/kg, iv bolus followed by 9.6 mg/kg/hr iv infusion) significantly (p < 0.01) decreased the severity of arrhythmias (arrhythmia score 5.1 ± 0.5; control vs. 3 ± 0.5; RAN) even though the ischemic area was not different in two groups (control; 39 ± 2% vs. RAN; 40 ± 1%). Number of VT episodes (29 ± 8 vs.10 ± 6) and duration (124 ± 30 vs. 40 ± 18 sec) was also less (p < 0.01) in RAN group. RAN treatment significantly (p < 0.01) reduced the incidence (50% vs. 13%), and duration (41 ± 23 vs.2 ± 0.3 sec) of VF episodes as compared to control group. Mortality was significantly reduced in RAN group (0%) as compared to control (30%). In conclusion, RAN reduced the severity of ischemia-induced ventricular arrhythmias in anesthetized rats. Abstract N° 50 CVT-4325, A new fatty acid oxidation inhibitor, inhibits long-chain enzyme(s) of fatty acid beta oxidation Jeffrey J. McVeigh, Paige E. Ivey, Brent K. Blackburn, Luiz Belardinelli, Heather Fraser. CV Therapeutics, Inc., CA A shift in myocardial energy substrate utilization from fatty acids to glucose may be desirable, particularly during times of limited oxygen availability. CVT-4325 inhibits fatty acid oxidation and increase glucose oxidation in rat isolated working hearts. To determine at what level CVT-4325 inhibits fatty acid oxidation in rat hearts, the oxidation of the long chain fatty acids, palmitate and oleate, the medium chain fatty acid, octanoate, and glucose were measured. CVT-4325 did not affect cardiac work or rate-pressure product in the presence of palmitate (C16), oleate (C18:1) or octanoate (C8). CVT-4325 inhibited the oxidation of palmitate and oleate by 57 ± 2% (P < 0.05) and 58 ± 3% (P < 0.05), and concomitantly increased glucose oxidation by 182 ± 20% (P < 0.05) and 252 ± 25% (P < 0.05), respectively. Furthermore, CVT-4325 increased the rate of glycolysis in hearts

2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

perfused with palmitate by 100 ± 14%, (P < 0.05). In contrast, in hearts perfused with the medium chain fatty acid, octanoate, CVT-4325 had no effect on fatty acid oxidation, glucose oxidation or on glycolysis. In summary, CVT-4325 induces a metabolic shift in myocardial substrate preference in isolated hearts that is likely mediated by the inhibition of long chain enzymes (e.g. 3-ketoacyl CoA thiolase, Acyl-CoA dehydrogenase, or enoyl-CoA hydratase) of mitochondrial b-oxidation. Abstract N° 51 Modulation of cardiac metabolism and serum lipids by tecadenoson Heather Fraser 1, Roger W Brownsey 2, Jerzy E Kulpa 2, Luiz Belardinelli 1. 1 CV Therapeutics, Inc., Palo Alto, CA. 2 University of British Columbia, Vancouver, Canada Tecadenoson (TECA), an A1 adenosine receptor agonist, lowers serum non-esterified free fatty acid (NEFA) levels at doses at least 5-fold lower than those that cause bradycardia. This study investigated whether lowering serum NEFA levels leads to changes in myocardial biochemical markers of lipid metabolism. Rats were instrumented with telemetry transmitters for recording heart rate and with catheters for delivery of drugs and blood sampling. TECA (20 µg/kg) or vehicle (saline) was administered i.p.. After 1hr or 2hr, blood samples were drawn and the heart frozen in situ and stored at -80°C. TECA had no effect on heart rate or blood pressure. In contrast, tecadenoson caused a 2.7-fold decrease in serum NEFA, a 67% decrease in triglyceride levels and a 46% decrease in the ratio between myocardial acetyl-CoA and CoASH, an index of the shift towards greater oxidation of glucose and a less oxidation of fatty acid. These data demonstrate that the systemic administration of an A1 adenosine receptor agonist lowers serum lipid concentrations and induces significant changes in cardiac energy metabolism. This may offer a new approach to modulate myocardial energy metabolism and protect the heart from the deleterious effects of free fatty acids on myocardial function and efficiency. Abstract N° 52 Identification of the PKC-epsilon binding domain on Connexin-43 Xitong Dang, Elissavet Kardami. Department of Human Anatomy and Cell Sciences, University of Manitoba, Winnipeg, Manitoba, Canada The epsilon-subtype of protein kinase C (PKCe) is a central mediator of cardioprotection. One of its targets is the cardiac gap junction protein connexin-43. This interaction may be important for cardioprotection. To selectively perturb the Cx43 - PKCe interaction it would be important to identify the binding Cx43 domain. To this end we produced cDNAs coding for different regions of the N-terminal (NT) or the C-terminal (CT) of Cx43. These cDNAs were co-transfected with a cDNA coding for PKCe in the HEK293 cell line that

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does not express Cx43. Subsequently, cell extracts were immunoprecipitated with anti- PKC e antibodies, or with antibodies recognizing Cx43 fragments (or their ’tags’); and immunoprecipitated proteins were probed for Cx43 or for PKCe respectively. CT-[228-382]- and CT-[243-382]-Cx43, displayed weak or absent interaction with PKC e, respectively. NT-[1-242]-Cx43 interacted strongly while NT-[1228]-Cx43, showed weak interaction. CT-[207-382]-Cx43 displayed the highest potency for interaction with PKC e. Our data indicated that the PKC e binding domain was located within res.207-242. We created an adenoviral vector expressing [207-242]-Cx43, and found that, when expressed in cardiomyocyte cultures, it co-localized with PKCe, indicative of interaction. It remains to be seen if an excess of this fragment will prevent the interaction of Cx43 and PKCe in situ. Abstract N° 53 Extracellular or nuclear 21-25 kda FGF-2 causes myocyte hypertrophy or apoptosis Zhi-Sheng Jiang, Xin Ma, Cheryl Hirst, Robert R Fandrich, Elissavet Kardami. Institute of Cardiovascular Sciences, University of Manitoba, Winnipeg, Manitoba. Canada We have examined the effects of the stress-induced 21-25 kDa (hi-) FGF-2 isoforms on cardiomyocytes, in vitro and in vivo. To simulate the auto- or paracrine -mode of action, the hi-FGF-2 protein was added to cardiomyocytes in vivo and in vitro. Intracrine-mode of action was achieved by overexpressing hi-FGF-2 in vitro; neutralizing antibodies blocked the action of exported FGF-2. Administration of hi-FGF-2 to the ischemic rat heart after irreversible coronary occlusion resulted in both acute as well as long term cardioprotection from tissue loss and contractile dysfunction (similar to the 18 kDa FGF-2). Despite decreased infarcts the hi-FGF-2 group presented substantial cardiomyocyte hypertrophy 4-8 weeks later. Added hi-FGF-2 induced a 40% myocyte size increase in vitro. When overexpressed, hi-FGF-2 localized mainly to the nucleus of cardiomyocytes resulting in cell death with apoptotic features (DNA ladder, nuclear phenotype); cell death was dose and time-dependent and required nuclear localization and interaction with intracellular FGFR1. We conclude that increases in extracellular hi-FGF-2 (added or ’secreted’) would contribute to myocardial hypertrophy as well as cardioprotection, but prolonged upregulation of endogenous nuclear hi-FGF-2 would contribute to cell death and a maladaptive cardiac phenotype. Abstract N° 54 Myotrophin-induced cardiac hypertrophy is influenced by PKC-mediated activation of both ERK and NF-jB signaling pathways Sudhiranjan Gupta, Dave Young, Subha Sen. Department of Molecular Cardiology, Cleveland Clinic Foundation, Cleveland, Ohio-44195 Multiple intracellular signaling pathways have been shown to regulate the hypertrophic growth of myocytes. Our

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previous studies suggested that protein kinase C (PKC) activation contributes to hypertrophy in vitro. PKC can modulate many intracellular signaling cascades, e.g., Raf, Ras, mitogen-activated kinase (MAPK) and IKK. We have shown previously that myotrophin-induced hypertrophic growth was mediated through PKC associated with activation of IKKb. However, whether PKC and MAPK play a role during progression of hypertrophy and its transition to heart failure has not been determined. The present study elucidated the activation of the extracellular signal regulated kinase (ERK) cascade and PKC in regulating cardiac hypertrophy in vivo. MAPK and PKC activation and translocation of various isoforms of PKC were characterized in the hearts of transgenic (Tg) mice, in which hypertrophy was initiated at 4 weeks of age and progressed to heart failure in 9 months as a result of cardiac-specific overexpression of myotrophin, a candidate gene for myocardial hypertrophy. Tg mouse hearts demonstrated activation of ERK cascade via Raf, but not JNK, p38 MAPK and Ras, during the progression of hypertrophy. Tg mice also showed an increased activation of total PKC activity, which reached a maximum by 12 weeks of age. Furthermore, we observed translocation of PKC-$, -b, -d, -k and -e but not -c, -i and -θ in Tg mouse hearts during the progression and transition phases of hypertrophy. To further confirm our in vivo observations, neonatal cultured myocytes were treated with myotrophin, and MAPK signaling cascades were examined. Data from experiments using a pharmacological inhibitor of ERK (PD98059) and a dominantnegative (DN) mutant of ERK, Raf and Ras suggest that myotrophin-induced expression of hypertrophic genes (ANF, c-myc and c-fos) and protein synthesis were partially inhibited by PD98059 and overexpression of DN MEK. Furthermore, overexpression of DN MEK1 and PDTC (an NF-jB-inhibitor) inhibit myotrophin-induced NF-jB transcriptional activity and translocation of NF-jB into the nucleus. Taken together, these results suggest that translocation of PKC and activation of both ERK and NF-∏B pathways are necessary for myotrophin-induced myocytes growth. Abstract N° 55 Insulin induces a repertoire of genes during ischemia in children with tetralogy of fallot Cathy Boscarino, John Coles, Jason Goncalves, Xiaojing Dai, Igor Konstantinov, Vivek Rao, Glen Van Arsdell. Cardiovascular Surgery Research, Hospital for Sick Children, Toronto, Canada Myocardial damage due to heart surgery remains the number one cause for the mortality and morbidity in pediatrics. Insulin has been shown to protect the heart from ischemia and reperfusion. Our institution has previously demonstrated a significant increase in survival in heart transplant patients with insulin enhanced cardioplegia (IHC). However, the effect of IHC in a pediatric heart with a severe and common heart defect during surgery has yet to be elucidated. The latter has been addressed in a randomized placebo controlled trial in which patients with tetralogy of Fallot were

randomly chosen to receive placebo cardioplegia or 10UI insulin prior to ischemia. To date, a gene repertoire from the RV at end ischemia in 6 patients (n = 4 placebo; age: 7 mos and n = 2 IHC; age: 11 mos) has been analyzed using a U133A Affymetrix platform. Significance was set to.05 with a fold change greater than 2.0 performed with an RMA. Of the 44 significant genes, PGK, SH2D and ITGA4 suggest a potential protective role to the pediatric heart during ischemia. Abstract N° 57 Sex differences in expression of beta-adrenergic receptors and Ca2+-handling proteins in rat heart ventricle Sang Hui Chu, Karen Sutherland, Jenny Beck, Jill Kowalski, Paul Goldspink & Dorie Schwertz. Univ. of Illinois, Colleges of Nursing and Medicine. Chicago, IL. Gender has been implicated as an important factor in the development of cardiovascular diseases. Beta-adrenergic receptors and Ca2+-handling proteins have been shown to be altered in heart failure. Yet, it is unknown if these proteins differ by sex in the healthy heart. To determine whether there are sex differences in ventricular beta-adrenergic receptors and cardiac Ca2+-handling proteins, the levels of beta1- and beta2-adrenergic receptors, L-type Ca2+ channel, SERCA2, phospholamban and the Na+-Ca2+ exchanger were examined using Western blot analysis in age-matched male (n = 12) and female (n = 12) Sprague-Dawley rats. The mRNA level of L-type Ca2+ channel and beta1-adrenergic receptor was also determined using real time RT-PCR normalized to abundance of GAPDH mRNA. Results demonstrate that female ventricle has significantly higher levels of L-type Ca2+ channel and Na+-Ca2+ exchange protein than males. No differences were found in the level of beta-adrenergic receptors, SERCA, and phospholamban. Female ventricle has significantly less L-type Ca2+ channel mRNA compared to males, but no difference in beta1-adrenergic receptor mRNA was observed. The results reveal gender differences in expression of cardiac Ca2+-handling proteins. These differences may contribute to observed gender differences in cardiac function and development of cardiovascular diseases. Abstract N° 58 Abnormal cardiac wall motion and matrix metallopoteinase activity Ricardo Garcia, Kristian Brown, Richard Pavelec, Katrina Go, James Covell, Francisco Villarreal. Department of Medicine, University of California at San Diego Activation of matrix metalloproteinases (MMPs) in the heart is known to facilitate cardiac remodeling and progression to failure. We hypothesized that regional dyskinetic wall motion of the left ventricle would stimulate activation of MMPs. Abnormal wall motion at a target site on the anterior lateral wall of the left ventricle was induced by pacing atrial

2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

and ventricular sites of five open-chest anesthetized dogs. Changes in shortening at the left ventricular (LV) pacing site and at a remote site at the anterior base of the left ventricle were monitored with piezoelectric crystals. Simultaneous atrial-ventricular pacing resulted in abnormal wall motion at the LV pacing site yielding early shortening and late systolic lengthening, while shortening at the remote site was essentially unaffected. Global myocardial MMP activity showed a seven-fold increase in substrate cleavage at the LV pacing site relative to the remote site. Gelatin zymography revealed increases of fifty-fold in 92kDa MMP-9 activity and ten-fold in 84 kDa MMP-9 at the LV pacing site relative to the remote site, whereas MMP-2 activity was unaffected. Abnormal wall motion was associated with a two-fold increase in collagen degradation and with increases in plasmin activity and inflammatory infiltrate relative to the remote site. Results indicate that regional dyskinesis by epicardial activation is sufficient to stimulate significant MMP activity in the heart, suggesting that abnormal wall motion is a major stimulus for MMP activation. Abstract N° 59 Anti-apoptotic properties of d-Propranolol (d-Prop) I.T. Mak, A.Torres-Duarte, Y. Su, W.B. Weglicki. George Washington Univ. Med. Ctr. Dept. Biochem., Mol. Biol. Div. of Exp. Med. Washington DC, 20037 When confluent (>90%) bovine arterial endothelial cells (ECs) were treated with staurosporine (STS, 0.5 µg/ml); caspase 3 activity (by fluorometric assay) was enhanced 243 ± 22% (p < 0.01) at 2 hrs and pronounced DNA laddering occurred between 3-5 hrs. Cell survival (by MTT) was reduced to 33.5% ± 3 (P < 0.001) of control at 20 hrs. Pretreatment of EC with d-Prop provided dose-dependent (1-10 µM) inhibition of these indices; at 5 µM, caspase 3 activation was suppressed 51 ± 4.9%, (p < 0.01), DNA laddering was substantially diminished, and cell survival was restored back to 67 ± 6.4% of control. In separate experiments, atenolol, a water soluble b -blocker (up to 10 µM), was not effective (< 10% effect), whereas Trolox (5 µM) was only partially effective (≤ 25% effect). However, the Fe-chelator 2,2’dipyridyl, provided potent protection against caspase 3 activation, DNA laddering, and cell survival comparable to d-Prop. STS alone elevated the GSSG/GSH ratio significantly from 4.2% (control) to 31.5 ± 8% within 3 hrs; d-Prop and dipyridyl (5 µM), but not atenolol effectively preserved the GSSG/GSH ratio to < 10% (p < 0.05). It is suggested that cytosolic chelatable Fe participated in the apoptotic cascade and that d-Prop prevented Fe-promoted GSH oxidation, caspase 3 activation and subsequent apoptosis. Abstract N° 60 Cardiac expression of FGF-16 David P. Sontag, Peter A. Cattini. Department of Physiology, University of Manitoba, Winnipeg, Canada The heparin-binding fibroblast growth factor (FGF) family consists of 23 known members, many of which have

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been implicated in cellular processes such as cell chemotaxis, division and differentiation. We sought to test the ability of 3 FGF-16 antibodies to detect protein via western blot and/or immumofluorescence. Sources of FGF-16 tested included 1) recombinant protein, 2) 293 cells transiently transfected with an FGF-16 cDNA expression vector or GFPFGF-16 tagged expression vector 3) adult mouse heart and 4) cultures of rat neonatal myocytes. Western blots containing heart and cell culture samples revealed band sizes similar to recombinant protein (24 kD) as well as a larger band (28 kD). Treatment of samples from 293 cells and neonatal rat myocytes with gylcosylase enzyme revealed the larger band to be a glycosylated form of FGF-16. Neonatal myocytes transfected with FGF-16 or GFP-FGF-16 expression constructs were detectable through immunofluorescence, however, endogenous FGF-16 could not be detected. Immunofluorescence of cryosections from adult mouse heart revealed signal in myocytes that decreased with antibody preabsorbtion. Abstract N° 61 FGF-2 autoregulation in cardiac myocytes S.K. Jimenez, E. Kardami, P.A. Cattini. Dept of Physiology, University of Manitoba, Canada Fibroblast growth factor-2 (FGF-2) is a cardioprotective molecule shown to be released in the heart on a beat-to-beat basis. Although reported to be autoregulated in other systems (including astroglial cells and endothelial cells), very little information is available on FGF-2 gene regulation in the cardiac myocyte. We show evidence for the autoregulation of FGF-2 in cardiac myocytes at the transcriptional level, directly in vitro, and indirectly in vivo. Direct addition of FGF-2 to culture media significantly increased (2.5-fold) transfected FGF-2 promoter activity in cardiac myocytes. Increased FGF-2 release in transgenic mouse hearts through b -agonist stimulation resulted in significant 1.8- and 3.3-fold increases in FGF-2 promoter activity. Overexpression studies showed the involvement of the transcription factors Egr-1 and/or Sp1 in FGF-2 gene regulation while DNA binding studies confirm their binding within the GC-rich sequences of the proximal FGF-2 promoter region. However, mutation of binding sites for both factors within this GC-rich region did not abrogate the increased activity detected after direct treatment with FGF-2, although basal levels are significantly decreased. Our results suggest that Egr-1 and Sp1 are involved in FGF-2 gene regulation in cardiac myocytes, although their role in FGF-2 autoregulation at the transcriptional level in cardiac myocytes cannot be considered rigorously established. Abstract N° 62 The role of frnk in the development of neointimal hyperplasia after VASCULAR injury Steven J. Engman, Allen M. Samarel. Cardiovascular Institute, Loyola University Medical Center, Maywood, Illinois

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Focal adhesion kinase (FAK) plays a central role in cell migration and proliferation. FAK related non-kinase (FRNK), the autonomously expressed C-terminal portion of FAK, is an endogenous inhibitor of FAK signaling. In this study, we used immuno-histochemistry and Western blotting to investigate the role of FAK and FRNK in neointimal hyperplasia following rat carotid artery balloon injury (BI). FAK and FRNK expression were increased after BI, as determined by Western blotting with an antibody that detects both FAK and FRNK. FRNK expression was localized to vascular smooth muscle cells within the neointima and the adjacent media, as determined by immunohistochemistry with an antibody that is selective for FRNK. FRNK expression was maximal, on a per cell basis, at 1 wk after BI, and expression decreased as the neointimal volume increased. FRNK expression also correlated with a decrease in FAK autophosphorylation at Y397. The expression pattern of FRNK in the early stages of neointimal development suggests that rather than inhibiting the development of neointimal tissue, FRNK may function in the initiation of smooth muscle migration during neointimal formation. Abstract N° 63 FAK-dependent akt activation in neonatal rat ventricular myocytes Maria C. Heidkamp, Kalpana Vijayan, Allen M. Samarel. The Cardiovascular Institute, Loyola University Medical Center, Maywood, Illinois Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase involved in adhesion- and growth factordependent signal transduction. We previously showed that adenoviral overexpression of GFP- FRNK (a dominantnegative inhibitor of FAK signaling) induces cell detachment and apoptosis, termed anoikis. The PI3K-PDK1-AKT pathway in cardiomyocytes can prevent apoptosis in response to a variety of noxious stimuli. However, the role of AKT in cardiomyocyte anoikis is not known. We therefore investigated the FAK-dependence of basal, and growth-factor stimulated AKT phosphorylation (pAKT) in GFP-FRNK overexpressing NRVM stimulated with either endothelin-1 (ET) or insulin-like growth factor-1 (IGF1). As determined by Western blotting, both ET and IGF1 induced FAK tyrosine autophosphorylation. The PI3K inhibitor LY294002 inhibited both adhesion-dependent and growth factor-stimulated pAKT. GFP-FRNK reduced basal pAKT, and also suppressed ET-induced pAKT. Activation of AKT by IGF1, however, was not inhibited by GFP-FRNK overexpression. We conclude that the anti-apoptotic signals generated through the AKT pathway by the hypertrophic agonists ET and IGF1 are differentially dependent on signaling through FAK. Abstract N° 64 Capsaicin receptors role in nitric oxide release induced by coronary flow in the guinea pig heart J. Carlos Torres 1, Leonardo del Valle 1, Fermín A. Tenorio 1 , Amalia González 1, Gustavo Pastelín 1, Jorge Suárez 2.

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Department of Pharmacology. National Institute of Cardiology ′Ignacio Chávez′, Mexico City, Mexico. 2 Departament of Medicine. University of California, San Diego, USA Introduction. – Nitric oxide (NO) release in the heart is determinated by many factors, being shear stress one of the most important. In earlier works, we demonstrated that coronary flow induces NO release through stretch-activated nonselective cation channels (SACs) activation promotes an increase in cytosolic calcium, followed by activation of endothelial nitric oxide synthase (eNOS). Although SACs have not been neither cloned or purified, at the Genomic Bank recently appeared a report in which a DNA sequence has been proposed to be similar to the vanilloid receptor (VR1) sequence. Our aim is to explore if the SACs and the VR1 receptor are the same structure. Methods. – Guinea pigs hearts were obtained and exposed according to Langendorff. Krebs solution was used as perfusion liquid and perfusion flow was varied (5, 10, 15, 20 and 25 mL/min) with the presence or absence of 10 m M capsaicin, 1 m M capsazepine, 100mM L-NAME and 3 m M gadolinium (III) chloride. NO release was estimated by the oxyhemoglobin method. Under these conditions, left intraventricular pressure, perfusion pressure, and coronary vascular resistance were recorded. Results. – Capsaicin produced a positive inotropic effect and 42%, 39% and 35% in-fold of NO release at 10, 15 and 20 mL/min flows, respectively, which were blocked by gadolinium. Capsazepine induced a negative inotropic effect and decrease of the NO release was observed in 30%, 42%, and 44% in range of 10, 15 and 20 mL/min respectively compared to control. These results are indicative that VR1 receptor is involved in the NO release. Abstract N° 67 Role of ERK and p38 in cardioprotection induced by overexpression of fibroblast growth factor 2 in the heart Stacey L. House, Gilbert Newman, Thomas Doetschman, Jo El J. Schultz. Department of Pharmacology, University of Cincinnati College of Medicine, Cincinnati, OH We have previously shown that cardiac-specific overexpression of fibroblast growth factor 2 (FGF2 Tg) results in increased recovery of contractile function and decreased infarct size following ischemia-reperfusion (IR) injury. The elucidation of the signaling mechanisms mediating this cardioprotection is vital to the development of FGF2 as a therapeutic strategy. Mitogen activated protein kinase signaling has been shown to be downstream of FGF2 and has been implicated to be important in other models of cardioprotection. Treatment of FGF2 Tg and wildtype (Wt) hearts with UO126 (2.5 µM), an inhibitor of the ERK signaling pathway, significantly reduced recovery of contractile function and increased infarct size following global low-flow IR injury in FGF2 Tg hearts but not Wt hearts (p < 0.05). In contrast,

2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

treatment of FGF2 Tg and Wt hearts with SB203580 (2 µM), an inhibitor of p38, did not abrogate FGF2-induced cardioprotection from post-ischemic contractile dysfunction and myocardial infarction. Instead, inhibition of p38 resulted in increased recovery of contractile function and decreased infarct size in Wt hearts (p < 0.05) but did not alter these parameters in FGF2 Tg hearts. Western analysis of ERK and p38 activation reveals signaling alterations in FGF2 Tg and Wt hearts throughout IR injury. These data suggest that activation of ERK but not p38 is necessary for FGF2-induced cardioprotection. Abstract N° 68 OXI/Nitrosative stress and bone loss in aldosteronism Vikram S. Chhokar, Syamal K. Bhattacharya, Robert A. Ahokas, Antonio Martinez, Arnold E. Postlethwaite, Yao Sun, Karl T. Weber, Univ. Tennessee Health Science Center, Memphis, TN Congestive heart failure (CHF) is accompanied by a systemic illness with oxi/nitrosative stress and wasting of lean tissue, fat and bone, where the role of renin-angiotensinaldosterone system effector hormones is unclear. Herein we hypothesized aldosterone (ALDO)/salt treatment (ALDOST) has adverse systemic effects. In uninephrectomized, male rats receiving standard chow, 1% NaCl and ALDO (0.75 µg/h) for 4-6 wks, we monitored plasma $1-antiproteinase ($1-AP) activity (µmol/L), an inverse correlate of oxi/nitrosative stress. We examined tibia for: bone mineral density (BMD, g/cm2) and content (BMC, g) by dual-energy x-ray densitometry; and Ca2+ and Mg2+ concentrations (µg/mg fat-free dry bone) by atomic absorption spectroscopy. Unoperated, untreated, age-/gender-matched rats served as controls. ALDOST led to reduced (p < 0.01) plasma $1-AP activity at wk 1 and which persisted thereafter. At 4 and 6 wks ALDOST, a respective reduction (p < 0.001) in BMD 14% and 53% (0.135 ± 0.002 and 0.074 ± 0.003 vs. 0.156 ± 0.002) and BMC of 25% and 71% (0.376 ± 0.017 and 0.147 ± 0.040 vs. 0.502 ± 0.010). At wk 6, a 47% and 49% fall (p < 0.001) in Ca2+ and Mg2+ (115 ± 6 vs. 216 ± 3 and 2.22 ± 0.16 vs. 4.38 ± 0.06) concentrations was found. Thus, ALDOST in rats is accompanied by systemic evidence of oxi/nitrosative stress and a progressive, marked loss of bone mineral composition. The aldosteronism seen in CHF may contribute to the oxidative stress and bone loss that appears in such patients.

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fibroblasts (CF). We wished to determine the effects of HG on CF protein and collagen synthesis, and the role played by angiotensin II (Ang II) in these responses. We cultured rat CF in either normal (NG, 5.5 mM) or HG media (25 mM) and assessed changes CF functions. Results indicate that HG CF synthesized more protein and collagen, that the effects were not due to changes in osmotic pressure, and that Ang II did not potentiate these effects on HG CF. Losartan pretreatment blocked the HG- or Ang II-induced increases in both protein and collagen synthesis. Vitamin E pre-treatment blocked HG effects on total protein synthesis only. HG decreased total MMP activity. This was reversed by pre-treatment with losartan, but not vitamin E. HG increased MMP-2 activity. AT1 mRNA levels were comparable between NG and HG groups. Results suggest that HG promotes fibrosis by increasing CF protein and collagen synthesis and decreasing MMP activity. Abstract N° 70 Differential phosphorylation of MARCKS by PKC e and PKC d in neonatal and adult rat ventricular myocytes Maria C. Heidkamp, Erika L. Szotek, Leanne L. Cribbs, Allen M. Samarel. The Cardiovascular Institute, Loyola University Medical Center, Maywood, Illinois MARCKS is a PKC substrate that has been implicated in the regulation of cell spreading, stress fiber formation, and focal adhesion assembly in nonmuscle cells. We show that MARCKS is expressed in cultured neonatal and adult rat ventricular myocytes (NRVM and ARVM, respectively) by immunocytochemistry and Western blotting. Endogenous MARCKS expression was higher in NRVM as compared to ARVM, but adenoviral overexpression of GFP-tagged MARCKS occurred in both. In NRVM, endothelin-1 (ET), phenylephrine, and angiotensin II caused rapid MARCKS phosphorylation, with ET inducing the greatest response. ET also rapidly phosphorylated (p) MARCKS in ARVM. Since ET activates both PKCe and PKCd, but not PKCa, NRVM and ARVM were infected with adenoviruses encoding constitutively active (ca) PKCe and PKCd. caPKCe but not caPKCd increased pMARCKS in both ARVM and NRVM. In addition, overexpression of dominant-negative PKCe reduced ET-stimulated pMARCKS in NRVM. We conclude that MARCKS is expressed in cardiomyocytes, and phosphorylated by PKCe.

Abstract N° 69 Relationship between high glucose and angiotensin II in cardiac fibroblasts Juan Asbun, Ana M. Manso, Francisco J. Villarreal. Dept. of Medicine UC San Diego, USA

Abstract N° 71 Endothelial activation and a proinflammatory vascular phenotype in aldosteronism Vikram S. Chhokar, Mohammad F. Kiani, Yao Sun, Karl T. Weber. Univ. Tennessee Health Science Center, Memphis, TN

Diabetic patients can develop a cardiomyopathy characterized by myocardial fibrosis. Whereas exposure of cultured kidney and skin fibroblasts to high glucose (HG) is known to increase collagen synthesis, little is known about cardiac

Chronic elevations in plasma aldosterone (ALDO), inappropriate for dietary Na+ intake, are accompanied by a proinflammatory vascular phenotype that includes intramural coronary arteries. Lesions, which appear at ≥ 4 wks ALDO/salt

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treatment (ALDOST), include monocytes/macrophages and lymphocytes that invade the perivascular space of involved vessels. Prior to tissue invasion, the transcriptome of peripheral blood mononuclear cells (PBMC) is activated to include a differential (> 2-fold) expression of genes for intercellular adhesion molecule (ICAM)-1 and various chemoattractant chemokines. A role for endothelial cell activation in the homing of activated PBMC to these vascular sites is uncertain. Accordingly, we examined the coronary vasculature using: ICAM-1 antibody-specific and IgG-nonspecific, labeled microsphere binding; and by immunohistochemistry and in situ hybridization for ICAM-1 and monocyte chemoattractant protein (MCP)-1 expression. Uninephrectomized rats received ALDO (0.75 µg/h) and a 1% NaCl diet for 4-6 wks. Unoperated, untreated, age-/gender-matched rats served as controls. We found: a progressive increase in endothelial cell activation, expressed by upregulated adhesion of ICAM-1 labeled microsphere binding at wks 4, 5 and 6 ALDOST (24.3 ± 2.3, 50.0 ± 15.5, and 188.7 ± 44.7 spheres/hpf, respectively), a response also seen with IgG binding (35.3 ± 2.7, 109.7 ± 22.7, and 216.0 ± 41.3 spheres/hpf, respectively). Upregulated expression (mRNA and protein) of ICAM-1 and MCP-1 within the coronary vasculature, where monocytes and lymphocytes first appeared at wk 4. Thus, in rats receiving ALDOST an activation of the endothelium, involving ICAM-1 and other adhesion molecules, appears at sites where PBMC have homed to the intramural coronary vasculature. Whether this is a requisite to the proinflammatory vascular phenotype remains uncertain.

Abstract N° 72 The I593R HERG mutation in long QT syndrome activates the unfolded protein response Steve H. Keller UCSD, San Diego, CA Hereditary long QT syndrome is a life-threatening arrhythmia associated with mutations in potassium and sodium channels that coordinate the action potential. We demonstrate activation of an endoplasmic reticulum (ER) stress response by the mutant I593R HERG K+ channel identified in long QT syndrome. I593R HERG is trafficking impaired, forms Triton insoluble aggregates, and its expression elicits cell rounding, phenomena consistent with generation of cellular stress. Expression of I593R HERG activates the unfolded protein response pathway, and separately, NF-∏B signaling. ATF6, the activating transcription factor of the unfolded protein response pathway, is processed into the active transcription factor in cells expressing I593R HERG. The ER chaperones/calcium binding proteins Grp78, Grp94 and calreticulin are also elevated in the cells expressing I593R HERG. Increases of ER chaperones suggest changes in calcium handling, which potentially impacts contraction and heartbeat rhythm independent of attenuated IKr.

Abstract N° 73 Doxycycline flanks the zinc-binding domains of matrilysin: a putative mode of inhibition by a broad-spectrum MMP inhibitor Ricardo A. Garcia, Dennis Pantazatos, Virgil L. Woods Jr., Francisco J. Villarreal. Department of Medicine, University of California at San Diego Matrix metalloproteinases (MMPs) play an essential role in normal and pathological collagen matrix degradation. Here we report on the effects of a broad-spectrum MMP inhibitor, Doxycycline, on the structure of the MMP matrilysin. Deuterium exchange mass spectrometry studies on matrilysin reveal two putative Doxycycline-binding sites (residues 145-155; residues 231-243) of similar affinity that flank the zinc-binding domains of the enzyme. Examination of the X-ray crystal structure of matrilysin shows that the Doxycycline-binding site at residues 231-243 is positioned within the active site cleft adjacent to the catalytic zinc atom. Comparisons of drug-bound versus drug-free forms of matrilysin show discrete changes in deuterium exchange suggesting that drug binding induces alterations in structure. In addition, fluorescence-quenching studies of matrilysin shown minor changes in conformation induced by doxycycline. In the absence of doxycycline, tryptophan fluorescence is completely accessible to quencher, whereas in the presence of Doxycycline, accessibility decreases by approximately 8%. These results suggest a mode of MMP inhibition by doxycycline that could involve interactions with the catalytic zinc atom. Abstract N° 74 Adenosine receptor signaling in cardiac fibroblasts Sara Epperson1,2, Francisco Villarreal 1, Laurence Brunton 1,2. Dept. of Medicine 1 and Pharmacology, 2 UC, San Diego Adenosine (ADO) inhibits the deposition of collagen in rat myocardium. The stimulation of ADO A2 receptors (A2 R) in rat cardiac fibroblasts (CFs) inhibits functions associated with collagen deposition such as proliferation and proteinand collagen synthesis.#i However, the signaling mechanisms by which ADO inhibits these functions are currently unknown. To identify these mechanisms, we have studied adenosine receptor (AR) expression, G-protein coupling and second messenger production in adult rat CFs. Standard RT-PCR indicates the presence of mRNA for all four ARs. Treatment of CFs with 2-Cl-ADO and NECA does not stimulate inositol phosphate accumulation, indicating that CFs lack an AR that signals via the Gq-PLC pathway. 2-Cl-ADO and NECA stimulate cAMP accumulation in a time- and concentration-dependent manner, indicating that an AR is present that couples to Gs. We do not observe any coupling of ARs to the Gi -pertussis toxin (Ptx)-sensitive pathway, as Ptx does not alter cAMP accumulation in response to 2-Cl-ADO or NECA; furthermore, 2-Cl-ADO and NECA activate MAPK in a Ptx-insensitive manner. Thus, our data indicate that ADO is unable to activate Gq-PLC and

2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

Gi-Ptx-sensitive pathways, but does activate the Gs-adenylyl cyclase pathway in CFs. These data are consistent with mediation of the anti-fibrotic effects of ADO by the A2b R. Ongoing experiments are directed to determining the intracellular signaling pathways by which ADO inhibits collagen production. Abstract N° 75 Functional effects of enhancing or silencing adenosine A2B receptors in cardiac fibroblasts Yinhong Chen, Francisco Villarreal. Department of Medicine, University of California at San Diego Heart failure can be accompanied by fibrosis, which contributes to the loss of pump function. The adenosine receptor family (A1, A2a, A2b and A3) has been proposed to be important in the regulation of cell functions such as collagen synthesis. Pharmacological evidence suggests the involvement of the A2 (A2a and A2b) subtypes in inhibiting cell functions involved in fibrosis. We wished to determine the effects of over- (adenovirus mediated) and underexpression (siRNA mediated) of A2a or A2b receptors (R) on cardiac fibroblast (CF) collagen and protein synthesis. Results indicate that CF express all adenosine receptor subtypes. Treatment of CF with 2-chloroadenosine inhibits CF protein and collagen synthesis in a dose dependent manner. The use of A2R agonists, which were capable of inhibiting CF protein and collagen synthesis, did not clarify the contributions derived from A2aR vs. A2bR. A 2-fold enhancement of A2bR by recombinant adenovirus infection yielded 40% and 20% decreases in adenosine-induced collagen and protein synthesis vs. control virus infected CF. siRNA mediated silencing of CF A2bR yielded increases in collagen and protein synthesis when treated with adenosine analogs. However, A2aR siRNA silencing did not affect CF protein and collagen synthesis. In conclusion, we demonstrate that A2bR rather than A2aR mainly mediate the in vitro regulation of CF protein and collagen synthesis. Abstract N° 76 Synthesis and evaluation of new chelators for use as MMP inhibitors using cardiac cell cultures David Puerta, Jana Lewis, Michael Griffin, Ricardo Garcia, Francisco Villarreal, Seth Cohen. Depts. of Chemistry and Medicine, University of California at San Diego Matrix metalloproteinases (MMPs) are hydrolytic enzymes involved in the breakdown of connective tissue. MMP activity is associated with a number of illnesses including arthritis, cancer, and cardiovascular disease. Compounds that can inhibit MMP activity may prove to be useful therapeutic agents. The MMP active site contains a Zn(II) ion bound to three histidine ligands with open coordination sites for substrate binding. Our research program is focused on developing novel MMP inhibitors and exploring the binding modes of known inhibitors. Using hydrotris(pyrazolyl)borate (Tp) model complexes the interaction of MMP inhibitors with the

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enzyme has been reproduced. This strategy has revealed several chelators that may be promising compounds for MMP inhibition. These lead compounds were evaluated in an MMP activity assay that utilizes a fluorescent peptide substrate. The results of these assays indicate that the newly identified ligands have activities ranging from 3- to 600-fold more potent than the hydroxamic acid ligands used in most clinically tested MMP inhibitors. These activities and lack of evidence for cytotoxicity were validated using cultures of rat cardiac fibroblasts. We propose that these new chelators represent a significant step toward producing improved, nextgeneration MMP inhibitors. Abstract N° 77 Reduction of infarct size by doxycycline: a role for plasmin inhibition Michael O. Griffin, Francisco J. Villarreal. Department of Medicine, University of California at San Diego Reperfusion of ischemic myocardium leads to the upregulation of several extracellular matrix (ECM)-degrading proteases. The purpose of this study was to determine whether doxycycline (DOX), a matrix metalloproteinase (MMP) inhibitor, would reduce infarct size following ischemiareperfusion injury and to identify potential mechanisms for its effects. Results showed that DOX treatment reduced infarct size by ~37% in the rat. DOX did not reduce acute inflammation (ie, neutrophil infiltration) but did attenuate increases in 92 kDa MMP-9 and plasmin levels observed in the infarct region. To investigate the role of these proteases in mediating myocardial injury, we utilized cultures of neonatal rat ventricular myocytes (NRVMs). NRVMs stimulated with physiological levels of plasminogen generated the active plasmin form of the protease in a dose-dependent manner. Stimulation with plasminogen (or plasmin), but not active MMP-9, caused cell detachment and death (ie, anoikis). Plasminogen stimulation did not activate endogenous MMPs, and co-treatment with GM6001 did not preserve cell attachment suggesting MMP activity was not responsible for cell detachment. Interestingly, co-treatment with DOX preserved NRVM attachment and viability, and DOX inhibited plasmin activity in culture with an apparent IC50 of ~18 m g/ml. However, inhibition of plasmin was an indirect effect, as DOX displayed no dose-dependent or time-dependent inhibition of plasmin in an in vitro test. These results suggest a novel role for DOX in preserving cardiomyocyte cell-matrix interactions by indirectly inhibiting plasmin activity. Abstract N° 78 Stretch induced activation of myocardial matrix metalloproteinases Ricardo Garcia, Sheila George, Francisco Villarreal, Jeffrey Omens. Departments of Medicine and Bioengineering, University of California, San Diego Abnormal myocardial stretch may stimulate cardiac remodeling via activation of extracellular matrix remodeling

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enzymes called matrix metalloproteinases (MMPs). We hypothesized that cardiomyocytes (CM) produce latent MMPs, which are amenable to activation by stretch. For this purpose, CM were plated onto collagen type I coated silastic membranes assembled in equibiaxial stretch devices. CM were allowed to grow for 24 h using 10% serum containing culture media, then replaced with low serum (0.5%) media for 72 h. CM were subjected to different degrees of static stretch of 0%, 4%, 8% at time intervals spanning up to 48 hours. Analyses of MMP activity by gelatin zymography indicate that stretching CM gives rise to a time-dependent increase in MMP-2 activity, and not MMP-9. MMP-2 activity increases with increasing stretch, where 8% shows the highest comparative levels of activity. Assessment of global MMP activity using fluorescent peptide probes showed increases at 4%, but not 8%, suggesting that some isoforms may be downregulated by high levels of stretch. Parallel studies with isolated fibroblasts also showed stretch-dependent increases in MMP-2 activity, where the highest activity levels were observed at 8% stretch. Profiling of specific MMP activities are currently underway and will shed light on the specific roles of CM-derived MMPs during stretch-induced MMP activation. Abstract N° 79 Activation of adenosine A1/A3 receptors protects cardiomyocytes by enhancing cell volume regulation Roberto J. Diaz, Julia Warden, Paul Salamone, Krish Parameswaran, Alina Hinek, Peter H. Backx, Gregory J. Wilson. Hospital for Sick Children, Toronto, and University of Toronto, Canada We have shown that regulatory volume decrease can be enhanced by activating adenosine A1/A3 receptors. Thus, we hypothesize that activation of those receptors enhances volume regulation during ischemia. Cultured (48 hours) rabbit ventricular myocytes were subjected to either 60-min simulated ischemia (SI, severe hypoxia and metabolic inhibition)/60-min simulated reperfusion (SR, reoxygenation) to assess myocyte nercrosis (trypan blue staining) or 30-min SI to assess cell swelling (% volume change by confocal microscopy). Myocytes were preconditioned with N6-2-(4-aminophenyl)ethyladenosine (APNEA, 1µM) given for 10 min, then washed out (10 min) before SI/SR. APNEA significantly reduced myocyte % necrosis after the long index SI/SR (APNEA 24.5 ± 3.2% (mean ± SEM) vs control+DMSO 41.9 ± 2.9%, p < 0.0001, n = 6 hearts) and prevented ischemic cell swelling (APNEA – 5.20 ± 5.0% vs control+DMSO 30.7 ± 9.1%, p < 0.001, n = 22-28 cells/group). Cl– channel inhibition with indanyloxyacetic acid 94 (IAA94, 50 µM) abolished protection by APNEA against necrosis (APNEA + IAA94 45.2 ± 1.6% vs APNEA, p < 0.0001) and ischemic cell swelling (APNEA + IAA94 22.3 ± 9.3% vs APNEA, p < 0.001). Control or baseline myocytes were not affected by drug vehicles. These data suggest that activation of the adenosine A1/A3 receptor pa-

thway triggers a potent cell volume regulatory mechanism that confers protection against necrosis in cardiomyocytes. Abstract N° 80 Effect of dietary saturated fat on ischemia reperfusion injury in hypertension and glucose intolerance Mahmood S. Mozaffari, Champa Patel, Claudia Ballas. Dept. of Oral Biology, MCG Georgia School of Dentistry, Augusta, Georgia, 30912 Impaired glucose tolerance, alone or in combination with hypertension, is associated with lower myocardial infarct size following ischemia-reperfusion injury. We tested the hypothesis that chronic (e.g., 18 weeks) consumption of a saturated fat diet increases susceptibility to ischemia reperfusion injury in hypertensive (H) and hypertensive-glucose intolerant (HGI) rats. The fat-fed H group displayed significantly higher body weight than the H group or the fat-fed HGI group. While the fat-fed H group displayed mild glucose intolerance, the fat-fed HGI group was markedly glucose intolerant. The fat-fed HGI rats displayed marked reduction in myocardial contractility and relaxation but higher enddiastolic pressure compared to either the H or the fat-fed H groups. However, infract size was similar among the 3 experimental groups. In conclusion, dietary excess of saturated fat causes reduction of myocardial performance in the HGI rat. Further, the treatment abrogates the previously reported differential in infarct size between H and HGI rats that were fed a basal fat diet thereby indicating greater susceptibility to ischemia-reperfusion-induced cell death in the fat-fed HGI rat. Abstract N° 81 Rapid onset of heart failure in male SHHF rats induced by high salt diet and thoracic aortic banding Sylvia. A, McCune, Thomas K. Pope, Jody M, Kronlage, Kathy L. Schreiber, Richard J. Gorczynski. Myogen, Inc., Westminster, CO The SHHF (Spontaneously Hypertensive Heart Failure) rat model, a genetic animal model of heart failure (HF), shows a large number of biochemical, physiologic and gene alterations in common with humans in HF. SHHF rats develop hypertension early and overt HF by 16-20 mo, Since all of the SHHF are programmed to go into HF, we wanted to see if we could accelerate the onset of HF by superimposing a cardiac insult, aortic banding and/or a high salt diet. Four groups (n = 5) of 7 wk lean SHHF males A) Sham; B) Pressure overload induced by banding of the thoracic aorta (TAB); C) 8% NaCl (HS) and D) TAB+HS were followed until a group developed HF. Only group D developed overt HF after 4 wks with significantly enlarged hearts and lungs, edema, peritoneal fluid, atrial thrombi, ascites, increased % of LV beta myosin and hemodynamic changes of decreased LV ± dPdt and increased LVEDP compared to the other 3 groups. Heart weights compared to the control group A (0.85 ± 0.03 g) were all significantly hypertrophied in order

2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

of D (1.5 + 0.04 g)> B (1.3 ± 0.06 g) > C (1.15 + 0.04 g). The combination of HS and TAB induced a rapid onset of HF in young SHHF male rats with similar changes that have been observed in older SHHF with natural onset of HF. Abstract N° 82 Pyrogallol impairs cardiac contractile function via a P38 map kinase-dependent pathway: antagonism of herbal medicines Jun Ren, Bonnie H. Zhao, Lucy B. Esberg. Div. of Pharm. Sci., Univ. Wyoming, Laramie, WY 82071, USA Oxygen free radicals contribute to the pathogenesis of myocardial dysfunction. This study was to examine the role of the superoxide generator pyrogallol on cardiac function and intervention with herbal medicines anisodamine and tetramethylpyrazine (TMP). Mechanical properties were evaluated in rat ventricular myocytes using an IonOptix system including peak shortening (PS), time-to-PS (TPS), timeto-90% relengthening (TR90) and maximal velocity of shortening/relengthening (± dL/dt). A 10-minute exposure of pyrogallol (0-10–2 M) did not affect cardiac contractile mechanics. However, longer duration of pyrogallol exposure (1, 3 and 6 hrs) significantly shortened resting cell length, reduced PS and ± dL/dt, and prolonged TPS and TR90 in time- and concentration-dependent manners. The pyrogallol (10–4 M at 6 hr incubation)-induced mechanical defects were prevented by the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 (1 µM) and superoxide dismutase (SOD, 500 U/ml). Interestingly, incubation of herbal antioxidants anisodamine (10–5 M) and TMP (10–5 M) effectively attenuated pyrogallol-induced cardiac defects with the exception of PS being unaffected by TMP. Our data demonstrate a direct inhibitory effect of pyrogallol on cardiac contraction probably through a superoxide and p38 MAP kinase-dependent manner. Antioxidant medicines anisodamine and TMP may be useful in the treatment of oxygen free radical-induced myocardial dysfunction (supported by NIH and ADA). Abstract N° 83 Magnesium deficiency stimulates angiogenesis in the rat heart M. Isabel Tejero-Taldo, Joanna J. Chmielinska, William B. Weglicki. Division of Experimental Medicine. The George Washington University Medical Center, Washington, D.C. New vessel formation in adult tissues is dependent upon local VEGF release. Substance P (SF) has been linked to iNOS up regulation, which can induce VEGF expression. We previously reported that Mg deficiency (MgD) resulted in SP release, and increased VEGF and RECA (rat endothelial cell marker) presence in the rat heart. We investigated the proangiogenic effects of MgD in the rat heart by examining the VEGF signaling pathway. Rats were fed either food with normal or low Mg (9% of the USDA-recommended) for 1, 2, or 3 weeks, when hearts were removed and frozen in liquid

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N2. When compared with diet controls, western blot analysis of heart tissue showed significant increase of iNOS protein at 1 week in MgD, reaching 2.5-fold at week 2 (p < 0.01). VEGF was 2-fold higher at week 1, and continued elevated through week 3 (p < 0.01). Furthermore, Flk-land Flt-1 (VEGF receptors) protein levels were significantly elevated at 1 week (1.5 and 1.75-fold respectively, p < 0.05), with Flk-1 remaining elevated through week 3 (p < 0.05). eNOS, essential in VEGF signaling, was also significantly elevated at week 1 (p < 0.05). In conclusion, severe MgD results in significant up regulation of the VEGF-signaliag pathway in the rat heart, and could result in new vessel formation. Supported byNIH/HL-62282. Abstract N° 84 p38-mitogen-activated protein kinase (MAPK) inhibition improves cardiac function during acute myocardial injury Zhihe Li, Thomas-Toan Tran, Jing Ying Ma, Gilbert O’Yong, Ann Kapoun, Sarvajit Chakravarty, Sundeep Dugar, George Schreiner, Andy Protter. Dept. of Pharmacology, Scios Inc., Fremont, CA p38 MAPK is activated during myocardial injury. However, the role of activated p38 MAPK on cardiac function during the injury, especially acute myocardial injury, is not fully understood. We have investigated the cardioprotective effects of p38 MAPK in a rat model of acute myocardial injury induced by isoproterenol (ISO, 20 mg/kg/day, s.c.). A synthetic p38 MAPK inhibitor, SD-282 (40 mg/kg, p.o.) remarkably improved ISO-induced deterioration of cardiac function indicated by increases in ejection fraction, cardiac output, stroke volume and cardiac index (all p < 0.01). ISOinduced expression of COX-2, collagen I and III, and fibronectin genes was significantly abolished by SD-282 (all p < 0.05). SD-282 also significantly reduced ISO-induced myocardial injury as judged by the reduction of myocardial necrosis (p < 0.05). Data suggest that p38 MAPK may be involved in the pathogenesis of cardiac dysfunction in acute myocardial injury. Inhibition of p38 MAPK may improve cardiac function and protect myocardium from ischemic/hypoxic injury that occurs during ischemic heart disease. Abstract N° 85 Rat tissue iron content is differentially altered by AZT and Mg deficiency (MgD) Jay H. Kramer, Shabnam Dadgar, Jonathon Hall, I. Tong Mak, William B. Weglicki. Dept. Biochem. & Mol. Biol., George Washington Univ., Washington D.C. Oxidative toxicity of AZT may be related to its influence on prooxidant metal tissue distribution, and may be further aggravated by MgD, which frequently co-exists with HIV infection. Iron (Fe) content in cardiac (C), hepatic (H) and small intestinal (S) tissues were assessed in rats on Mgsufficient (MgS = 100% RDA) or MgD (20 % RDA; 50%

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lower plasma Mg) diets for 6 wks ± AZT (daily 0.5 mg/ml, drinking water). Acid-digested tissues were assessed for Fe by atomic absorption and plasma for oxidative (conjugated diene [CD] levels) and gross tissue (LDH activity) injury. AZT alone caused a 20% decline in C-Fe, had no effect on S-Fe, but increased H-Fe 95%; MgD alone increased C-Fe 84%, H-Fe 212%, and S-Fe 243%; AZT + MgD increased C-Fe 11%, H-Fe 72% and decreased S-Fe 13%. Plasma CD and LDH were markedly higher in the combined group (66 & 41% increases vs MgS), compared to AZT (29 & 17%) and MgD alone (44 & 11%). The data suggest that AZT and MgD each can differentially alter Fe levels in select tissues. Enhanced oxidative stress with AZT + MgD may relate to liberation of tissue Fe; this could underlie the previously observed cardiac intolerance to postischemic oxidative stress.

Abstract N° 86 Assesment of KATP activation in isolated mouse hearts using 87RB magnetic resonance spectroscopy Olga Jilkina, Bo Xiang, Bozena Kuzio, John Rendell, Valery V. Kupriyanov. Institute for Biodiagnostics, NRC, Winnipeg, Canada We previously used 87Rb-MRS to study regulation of KATP channels in isolated rat hearts. In this work, we applied the same method to study K+ fluxes in mice. Hearts (~ 160 mg) of male CD-1 mice were perfused in a Langendorff mode with Krebs Henseleit buffer (KHB). NMR experiments were performed using a Bruker AM-360 spectrometer. The hearts were perfused with KHB-Rb containing 50% of K+ substituted with Rb+. Rb+ accumulation in the hearts resulted in an increase in 87Rb peak. Rb+ efflux was initiated by switching to Rb+-free KHB. The rates for Rb+ efflux were derived using monoexponential fits. Rb+ efflux was stimulated directly with a KATP opener, P-1075, or indirectly with 2,4-dintrophenol (DNP) via a decrease in ATP/ADP (see Table). Table Rate constants of Rb+ efflux (× 10–3, min–1), n = 3-6 Groups Rats Mice

Control 39.6 ± 0.4 51.1 ± 5.1

P-1075, 5 µM 92.7 ± 3.3 72.0 ± 5.9

DNP, 50 µM 65.4 ± 1.4 69.8 ± 7.5

The higher heart rate of spontaneously beating mouse hearts in comparison to that of rat hearts (~ 370 vs. ~ 260 beats/min) indicates that mouse cardiac K+ voltage-gated channels open more frequently, which is probably the factor responsible for faster kinetics of Rb+ efflux under baseline conditions. Less significant stimulation of KATP with P-1075 and DNP in mice, could be explained by species differences in 1) surface density of KATP, 2) sensitivities of the channels to the ligands.

Abstract N° 87 Thermal assessment of cardiac ischemia in vivo Darren Manley, Stephen Nighswander-Rempel, Bo Xiang, Valery Kupriyanov. Institute for Biodiagnostics, NRC, Winnipeg, Manitoba, Canada Thermal imaging can identify areas of increased or decreased blood flow as either an increase or reduction in surface temperature, respectively. We have acquired thermal image sequences of in vivo porcine hearts (open-chest model) during normal perfusion, partial (50 and 20% flow) and complete occlusion of left anterior descending (LAD) artery, and reperfusion, with and without pharmacological stimulation. We show that reduction of blood flow through the LAD bed produces an immediate reduction in epicardial temperature. Infusion of the adrenergic agonist dobutamine (10 µg/min/kg) increases blood flow producing temperature increases during normal perfusion and reperfusion (0.7 ± 0.1 °C) and 20% and 50% regional flow (1.7 ± 0.2 °C and 2.1 ± 0.2 °C, respectively). We show there is a non-linear dependence of cardiac surface temperature on blood flow in vivo (see table), and that thermal imaging may be a valuable tool for assessing sites of potential ischaemic injury. LAD flow, % DT

0 (n = 5) – 0.87

20 (n = 3) – 0.93

50 (n = 2) – 0.46

100 (n = 5) 0

>200 (n = 5) 0.85

Abstract N° 88 Near-infrared spectral imaging of cardiac ischemia in vivo Darren Manley, Stephen Nighswander-Rempel, Bo Xrang, Valery Kupriyanov. Institute for Biodiagnostics, NRC, Winnipeg, Manitoba, Canada Spectroscopic images were acquired (650 to 1050 nm) from in vivo porcine hearts. Images where acquired during normal perfusion, partial (50 and 80%) and total occlusion of left anterior descending (LAD) artery, and reperfusion, with and without pharmacological stimulation. The images reveal a decrease in oxygen saturation levels proportional to changes in LAD flow in the ischaemic regions (see Table), relative to normally-perfused control regions. Dobutamine infusion (10µg/min/kg) increases blood flow yiefding increases in oxygen saturation ranging from 11-12% during normal perfusion and reperfusion to 60% during 20% regional flow (45% during 50% regional flow). Partial LAD occlusion also produces a change in indocyanine green (bolus injection) kinetics causing a shift of peak position and a decrease in peak height and washout kinetics, relative to normal perfusion and reperfusion. These data confirm that oxygenation and flow can be mapped in cardiac tissue in vivo by nearinfrared spectroscopic imaging. LAD flow, % O2 Sat., %

0 0

20 20.7

50 27.0

100 73.3

> 200 85.1

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Abstract N° 89 Time course of changes in heart function in pressure and volume overloaded rat hearts Elliott Cantor, Andrea Babick, Zainisha Vasanji, Rishi Seth, Naranjan S. Dhalla, Thomas Netticadan. Institute of Cardiovascular Sciences, University of Manitoba, Winnipeg, Manitoba, Canada When the heart is subjected to chronic pressure overload (PO) or volume overload (VO), it responds with a compensatory mechanism known as hypertrophy. Cardiac hypertrophy may be beneficial to the stressed myocardium, but sustained hypertrophy leads to heart failure. Using echocardiographic measurements, we examined a time course of changes in heart function during the development of hypertrophy and heart failure. After surgical induction of PO and VO, heart function was assessed at five time intervals. Our results show that both PO and VO induced concentric and eccentric hypertrophy respectively, leading to heart failure. Systolic dysfunction occurred early in VO hearts in comparison to PO hearts, as evident from a significantly reduced fractional shortening and ejection fraction. (This study was supported by the Heart and Stroke Foundation of Manitoba, Canadian Institutes of Health Research & Manitoba Health Research Council).

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The combination of high glucose and high insulin (HG/HI) has been shown to protect against cardiac ischemic injury. One proposed mechanism is due to increased glucose oxidation and decreased fatty acid oxidation. However, the impact of HG/HI on cardiac metabolism following ischemia/reperfusion has not been reported. Therefore, we investigated effect of HG/HI on cardiac metabolism following repsrtusion. Using a physiological substrate mixture, hearts were subjected to 30 min of low flow ischemia (LFI, 0.3 mls/min) followed by 60 min reperfusion and divided into three groups: 1) glucose 30 mM and insulin 1000 µU/ml (HG/HI), 2) 5mM and 1000 µU/ml (HI), 3) 30mM and 50 µU/ml (HG). HG/HI attenuated the increase in end diastolic pressure during LFI; 6 ± 2 mmHg compared to 60 ± 6 and 75 ± 5 in HG and HI respectively p < 0.01 and improved % recovery of rate pressure product at the end of reperfusion; 86 ± 2 compared to 33 ± 10 and 41 ± 6 in HG and HI p < 0.01. Surprisingly, the relative oxidation of the different substrates at the end of reperfusion was not significantly different among groups as shown in Fig. This data suggests that the protective effect of HG/HI may be not due to increased glucose oxidation during reperfusion.

Abstract N° 90 Alterations in cardiac function in sucrose-fed rats Zainisha Vasanji, Satyajeet Rathi, Elliott Cantor, Andrea Babick, Xing-Hai Yao, Naranjan S. Dhalla, Thomas Netticadan. Institute of Cardiovascular Sciences, University of Manitoba, Winnipeg, Manitoba, Canada Diabetes mellitus may cause a specific type of cardiomyopathy independent of cardiovascular disease. The objective of this study was to determine whether a diet-induced model of type II diabetes caused cardiomyopathy. Cardiac function was studied in control and sucrose-fed rats at 2.5, 5 and 10 weeks. Plasma insulin, glucose and triglyceride levels were significantly higher in sucrose-fed animals at all time points studied, while plasma cholesterol was significantly elevated only after 10 weeks. Changes in cardiac structure in sucrosefed rats were evident after 10 weeks as the internal ventricular septal wall thickness at systole was significantly decreased and the left ventricular internal dimensions at systole and diastole were significantly increased. Cardiac dysfunction was also apparent after 10 weeks as fractional shortening and ejection fraction were significantly reduced. These results suggest that sucrose-fed rats developed a type II diabetes-induced cardiomyopathy after 10 weeks. (This study was supported by the Canadian Diabetes Association). Abstract N° 91 Ischemic cardiac protection of high glucose/high insulin is not due to increased glucose oxidation Peipei Wang, Steven G Lloyd, John C Chatham. Div. of Cardiovas. Disease, Dept. Med. Univ. Alabama at Birmingham, Birmingham, AL 35294-4470

Abstract N° 92 Mechanisms underlying cardiac contractile impairment in dilated cardiomyopathy Andrea Babick, Elliott Cantor, John Babick, Nobuakira Takeda, Thomas Netticadan, Naranjan S. Dhalla. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and University of Manitoba, Winnipeg, Canada Dilated cardiomyopathy (DCM) is characterized by impaired cardiac contractile function; however, the underlying mechanisms remain unclear. In this study, we examined cardiac performance and SR function in the J2N-k cardiomyopathic hamster (CM). Significant decreases in ejection fraction (EF), fractional shortening (%FS), cardiac output (CO) and heart rate were associated with a reduction in SR Ca2+-uptake activity and a decrease in the expression of the SR Ca2+ ATPase (SERCA2a) and cAMP-dependent protein kinase (PKA)-mediated phospholamban (PLB) phospho-

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rylation at ser-16 in CM as compared to J2N-n control hamster. Depressed PLB phosphorylation was associated with a reduction in the activity of the SR associated PKA and elevated protein phosphatase activity in CM. The results suggest that an impairment of SERCA2a function leads to cardiac contractile dysfunction in DCM. (Supported by the CIHR Group in Experimental Cardiology). Abstract N° 93 Modification of SR gene expression in heart failure by blockade of RAS Xiaobing Guo, Jingwei Wang, Donald Chapman, Naranjan S Dhalla. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and University of Manitoba, Winnipeg, Canada Although depression of the sarcoplasmic reticular (SR) Ca -regulating proteins are known to be associated with congestive heart failure (CHF), the mechanisms are poorly understood. In view of the increased activity of reninangiotensin system (RAS) in heart failure, the effects of enalapril, an angiotensin converting enzyme inhibitor, and losartan, a AT1 receptor antagonist, were studied on SR Ca2+-regulating protein gene expression in the left ventricle (LV) and right ventricle (RV) at 8 weeks after myocardial infarction (MI) due to coronary artery ligation. Hemodynamic assessment revealed depression in + dP/dt and -dP/dt as well as elevation in LVEDP. Gene expression of SR Ca2+pump ATPase (SERCA2), ryanodine receptor (RYR), and phospholamban (PLB) are decreased in LV; only PLB mRNA was decreased in RV. Blockade of RAS partially prevented the remodeling of SR in both LV and RV in failing heart (Supported by CIHR Group in Experimental Cardiology). 2+

Abstract N° 94 Prevention of myofibrillar remodeling in failing heart by blockade of RAS Xiaobing Guo, Jingwei Wang, Donald Chapman, Naranjan S Dhalla. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and University of Manitoba, Winnipeg, Canada In this study, congestive heart failure was induced in rats by coronary artery ligation for 3 weeks and then the animals were treated with an angiotensin converting enzyme inhibitor, enalapril (10 mg/kg/day; orally) and/or losartan, an AT1 receptor antagonist (20 mg/kg/day; orally) for 5 weeks. Hemodynamic assessment revealed depression in + dP/dt and – dP/dt as well as elevation in LVEDP. Gene expression of $-myosin heavy chain ($-MHC) mRNA was decreased and b-myosin heavy chain (b-MHC) mRNA was increased in both left ventricle (LV) and right ventricle (RV). However, myosin light chain (MLC) and b-actin were different in each of the groups. Although enalapril and losartan partially attenuated all the changes in hemodynamic parameters, and myosin isoform gene expression, the combined therapy of

enalapril and losartan did not show any additional benefit. The results suggest that the therapeutic effects of blockade of RAS in CHF may be partly related to prevention of changes in cardiac myofibril remodeling (Supported by CIHR Group in Experimental Cardiology). Abstract N° 95 Effects of b-blockers on cardiac function and remodeling in heart failure in rats Jarmila Machackova, Vijayau Elimban, Naranjan S. Dhalla. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and University of Manitoba, Winnipeg, Canada Although the beneficial effects of different b-blockers on mortality and heart function have been reported in humans with heart failure, their mechanisms at molecular level are not clear. Therefore, we examined the effects of b-blockers on changes in myofibrillar ATPase as well as myosin heavy chain (MHC) in an animal model of heart failure. Heart failure in rats was induced by occluding the coronary artery and 3 weeks later the animals were treated daily with and without 20 mg/kg atenolol or propranolol for 5 weeks. The animals were assessed for left ventricular (LV) function and myofibrillar ATPase activities (Mg2+ and Ca2+ stimulated) as well as $- and b-MHC isoform contents. The infarcted hearts exhibited depression in LV function and myofibrillar Ca2+ ATPase activity, unlike that in the right ventricle, compared with controls. MHC $-isoform contents were decreased whereas those of MHC b-isoform were increased in the LV due to myocardial infarction. Although atenotol or propranolol did not completely prevent LV dysfunction, these drugs partially normalized LV myofibrillar ATPase activities and MHC isoforms contents (Supported by CIHR Group in Experimental Cardiology). Abstract N° 96 Antioxidants and arrhyhmias: role of oxidatrve metabolites of catecholamines Rajat Sethi, Vijayan Elimban, Ken S. Dhalla, Naranjan S. Dhalla. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and University of Manitoba, Winnipeg, Canada Since oxidation products of catecholamines play an important role in the genesis of arrhythmias, this study was designed to investigate, whether a reduction in oxidative metabolites by antioxidants is responsible for their beneficial effect on catecholamine induced arrhythmias. Rats were treated with or without different antioxidants such as vitamins A (30 mg/kg), E (20 mg/kg), C (100 mg/kg) and N-acetylcysteine (NAC, 200mg/kg) two days before epinephrine (32 µg/kg) injection. Plasma levels of malondialdehyde (MDA) in nmoles/L were 1.54 ± 0.1, 1.43 ± 0.08, 1.16 ± 0.09, 1.30 ± 0.07, 1.32 ± 0.12, whereas the oxidative metabolites of catecholamines in ng/ml were 0.25 ± 0.02, 0.06 ± 0.003, 0.2 ± 0.02, 0.3 ± 0.02 in control, Vit C, Vit E, Vit A and NAC

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treated animals, respectively. Epinephrine induced arrhythmias were significantly attenuated by antioxidant treatments. These results support the view that the epinephrine induced arrhythmias may be mediated by oxidation products of catecholamine. (Supported by CIHR Group in Experimental Cardiology). Abstract N° 97 Altered phospholipase C isozymes in heart failure due to volume overload Melissa R. Dent, Naranjan S. Dhalla, Paramjit S. Tappia. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and University of Manitoba, Canada The arteriovenous (AV) shunt model of volume overload in the rat results in the development of heart failure, a response that may be mediated by phospholipase C (PLC). This study examined the changes in PLC isozyme (b1, c1, d1) gene expression, protein content and activities in heart failure induced by AV shunt in Sprague-Dawley rats by the needle technique. The reduced +dP/dt and -dP/dt as well as elevated LVEDP demonstrated the occurrence of heart failure at 8 and 16 weeks after the induction of the shunt. At 8 weeks after the induction of volume overload, PLC b1 activity was elevated but the activities of PLC c1 and d1 isozymes were significantly decreased. At 16 weeks of heart failure, PLC b1 activity was elevated to a lesser extent but PLC c1 and d1 activities were further reduced. These changes were associated with altered mRNA and immunoprotein expression. In addition a decreased SL DAG amount was observed. It is suggested that heart failure is associated with a depressed PLC c1 and d1 mediated signal transduction. [Supported by the Manitoba Health Research Council]. Abstract N° 98 Cardiac failure induced by maternal undernutrttion Kuljeet K. Ckeema, Melissa R. Dent, Nina Aroutiounova and Paramjit S. Tappia. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and University of Manitoba, Winnipeg, Canada We examined if heart failure has its origins during fetal cardiac development. A rat model in which pregnant dams were fed diets containing either 180 (normal) or 90 g (low) casein/kg diet during pregnancy. The ejection fraction (EF) of the low protein (LP) exposed pups was severely depressed in the first 2 weeks of life, followed by a progressive recovery of the EF of the offspring by 40 weeks of age. The left ventricular (LV) internal diameters were increased between 24 hrs and 84 days of age in the LP exposed group. Although, between 3 days and 2 weeks of age the LV wall of the hearts of the LP group were thinner, a progressive increase in the LV wall thickness was seen. Early increases in the atrial natriuretic factor and phospholipase C isozymes mRNA levels occurred in the LP group. At 40 weeks of age although the EF was normal, a 2-fold elevation in the LV end diastolic pres-

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sure, a reduced mean arterial pressure and cardiac output as well as decreased ± dp/dtmax were observed. Exposure of the developing fetus to maternal undernutrition may program heart failure in offspring in later life. [Supported by the MMSF]. Abstract N° 99 Changes in cardiac function and beta adrenoceptor depend upon the type and stage of heart failure Rajat Sethi, Harjot K. Saini, Naranjan S. Dhalla. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and University of Manitoba, Winnipeg, Canada Although cardiac remodeling has been shown to be responsible for heart failure, its relation with subcellular changes needs to be examined. Since cardiac function and betaadrenoceptor are either upregulated or down regulated in the failing heart, this study was designed to investigate if these changes are dependent upon cardiac remodeling. Heart failure in rats was induced by aortocaval shunt (AV shunt), myocardial infarction (MI) or pressure overload (PO) and changes in cardiac remodeling, cardiac function and beta adrenoceptors were studied after 4 and 24 weeks of inducing heart failure. Cardiac remodeling, demonstrated by increased heart weight was evident in all types and at all stages of heart failure. On the other hand, changes in cardiac function and beta adrenoceptor were dependent on the type and stage of heart failure; however, attenuation of these parameters was always evident at the later stages. These results thus support the view that changes in cardiac function and beta adrenoceptor are dependent upon the type and stage of heart failure and not on cardiac remodeling. (Supported by CIHR Group in Experimental Cardiology). Abstract N° 100 Endogenous nitric oxide formation accounts for the extent of recovery during ischemia reperfusion injury Punam K. Chohan, Vijayan Elimban, Dasliang Prajapati, Naranjan S. Dhalla. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and University of Manitoba, Winnipeg, Canada In order to investigate the role of endothelial integrity and the release of nitric oxide on the magnitude of ischemia reperfusion (I/R) injury, isolated rat hearts were either perfused at constant pressure or at constant flow. Hearts were exposed to global ischemia for different periods followed by reperfusion for 60 min. Our results show that longer duration of ischemia was required to elicit same changes in contractile parameters in the hearts perfused at constant pressure as compared to constant flow. Treatment with L-Arginine (3 mM), a precursor of NO, improved the recovery at constant flow. Treatment with L-NAME (100 µM), an inhibitor of NO, further depressed the recovery in cardiac function at constant pressure. These results suggest that hearts were more sensitive to IR changes during constant flow than constant pres-

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sure and the recovery depends on their ability to produce NO (Supported by the CIHR Group in Experimental Cardiology). Abstract N° 101 Sarcoplasmic reticulum function in pressure overload-induced cardiac hypertrophy Xing-Hai Yao, Elliott Cantor, Zainisha Vasanji, Jason Greville, Thomas Netticadan. Institute of Cardiovascular Sciences, University of Manitoba, Winnipeg, Manitoba, Canada When the heart is subjected to chronic pressure overload (PO), it counters with a compensatory hypertrophic response. Hypertrophy may be beneficial to the stressed myocardium; however, sustained hypertrophy leads to heart failure. To determine whether PO-induced hypertrophy results in cardiac contractile dysfunction, we examined cardiac performance as well as sarcoplasmic reticulum (SR) function and regulation. Eight weeks of PO induced concentric left ventricular hypertrophy. Although systolic function was not abnormal in PO hearts, SR Ca2+-uptake was significantly reduced. This attenuation was consistent with an increase in the ratio of phospholamban (PLB) to SR Ca2+-ATPase as well as a decrease in the phosphorylation of PLB. The reduction in PLB phosphorylation could be associated with an elevation in the SR-associated protein phosphatase activity. These results suggest that 8-week PO induced alterations in SR calcium handling without systolic dysfunction. (This study was supported by the Canadian Institutes of Health (Research and Manitoba Health Research Council). Abstract N° 103 Prolactins with actions on angiogenesis and inflammation Carmen Clapp, Yazmin Macotela, Ana M. Corbacho, Carmen González, Jose C. Rivera, Jorge Aranda, Pavel Montes de Oca, Gonzalo Martinez de la Escalera. Neurobiology institute, National University of Mexico, 76230 Queretaro, QRO, Mexico Angiogenesis is associated with tissue repair after injury and inflammation and is an important mechanism underlying human diseases such as cancer and heart disease. Prolactin hormones and cytokines regulate angiogenesis and inflammation by modulating the functions of endothelial cells. The cleaved fragment of prolactin (16K-prolactin) is a potent inhibitor of angiogenesis in vivo and in vitro. Inhibiting endothelial cell proliferation and protease activation. These effects may involve autocrine mechanisms since endothelial cells from different vessels and species produce and secrete prolactin isoforms. Furthermore, prolactin molecules can regulate inflammation by affecting other cell types within the vicinity of the capillary network. Treatment of leukocytes with full-length prolactin stimulates their adhesion to vascular endothelium. Moreover, prolactin and 16K-prolactin can function on fibroblasts to have dichotomous effects on nitric

oxide synthesis via inducible nitric oxide synthase (iNOS). While 16K-prolactin acts as a pro-inflammatory cytokine inducing the expression of iNOS in resting fibroblasts, prolactin acts as an anti-inflammatory factor, attenuating cytokine-induced iNOS expression. These findings reveal a new repertoire of prolactin actions in the vasculature and in the inflammatory reaction, and open a new perspective in the functional link between angiogenesis, vascular function and inflammation. (Supported by Conacyt 36041 -N, and UNAM IN227502 and PUIS).

Abstract N° 104 16K-prolactin inhibits nitric oxide production and calcium mobilization in endothelial cells Carmen Gonzalez 1, Ana M Corbacho 2,Celina Garcia 1, Macotela Y. 1, Jason P. Eiserich2, Morales V 1, Diaz M 1, Gonzalo Martinez de la Escalera 1, Carmen Clapp 1. 1 Neurobiology Institute, National University of Mexico, Queretaro, Qro. Mexico; Department of Internal Medicine. University of California, Davis, CA Activation of endothelial nitric oxide synthase (eNOS) lies downstream from and mediates the effect of important angiogenic, vasopermeability, and vasorelaxation factors, including vascular endothelial growth factor (VEGF), bradykinin (BK), and acetylcholine (Ach). We have found that the N-terminal 16 KDa fragment of prolactin (16K-PRL) inhibits the activation of eNOS induced by the three vasoactive substances as determined by the citrulline assay in endothelial cells from different species and blood vessels. Because the activity of eNOS is calcium-dependent, we hypothesized that interference with the mobilization of intracellular calcium is one of the mechanisms underlying inhibition of eNOS by 16K-PRL. Consistent with this hypothesis, 16KPRL inhibited the transient increase in intracellular calcium evoked by BK in endothelial cells as determined by fura2AM, a fluorescent marker for free calcium. Inhibition of eNOS could mediate putative effects of 16K-PRL on vasorelaxation. We found that 16K-PRL blocks vasodilation of rabbit aortic segments induced by acetylcholine, indicating that it interferes with the endothelial NO signal to the smooth muscle. In contrast, vascular relaxation responses to sodium nitroprusside (endothelium-independent relaxation) were similar in treated and untreated segments, indicating that 16K PRL does not interfere with the smooth muscle response to NO. Altogether, these findings suggest that 16K PRL is a potential regulatory factor of vascular tone and further suggest that 16K-PRL inhibition of endothelial-NO derived signal could mediate some of the known anti-angiogenesis effects of this hormonal fragment. (Supported by Conacyt 36041-N and UNAM-IN227502 and -PUIS).

2004 ISHR North American Section Meeting / Journal of Molecular and Cellular Cardiology 36 (2004) 609–640

Abstract N° 105 Postconditioning: a new link in nature’s armor against ischemia-reperfusion injury Jakob Vinten-Johansen, Zhi-Qing Zhao, Faraz Kerendi, Michael E. Halkos, He-Ying Sun, Joel S. Corvera, Robert A. Guyton. Cardiothoracic Research Laboratory, Emory University School of Medicine, Atlanta, Georgia USA Ischemic preconditioning (IPC) is the most potent endogenous cardioprotective strategy identified. We have recently reported (Am J Physiol Heart Circ Physiol 285: H579-H588, 2003) in a canine model of 60 minutes LAD occlusion and 3 hours of reperfusion that 3 brief (30 seconds each) cycles of reperfusion and ischemia interposed during the immediate onset of reperfusion (i.e. postconditioning, PostC) reduced infarct size to the same extent as IPC. 3 cycles of PostC was as effective as 6 cycles. PostC also reduced the incidence of apoptosis in AAR myocardium, arrhythmias, reduced coronary artery endothelial injury and P-selectin expression, and neutrophil accumulation in AAR. PostC attenuated the generation of reactive oxygen species in AAR comparable to IPC. Further studies in rats and rabbits suggest that the first moments of reperfusion during PostC are critical in its cardioprotection. PostC can be observed in cell culture, suggesting that inhibition of inflammatory cell processes is not the sole mechanism. Further studies implicate activation of adenosinergic receptors, release of nitric oxide, activation of KATP channels, and intracellular signaling pathways (MAP kinases) as mechanisms of PostC. The results suggest that PostC is a new endogenous cardioprotective phenomenon in attenuating reperfusion injury. Application of PostC at reperfusion may lend to more rapid clinical adoption for adjunct treatment during percutaneous coronary interventions. Abstract N° 106 Expression analysis of the human calsequestrin-2 gene in cardiocytes Angel Zarain-Herzberg, Raúl Juárez-Rubí. Dept. Bioquímica y Biología Molecular, Facultad de Medicina UNAM, México, D.F., 04510 Calsequestrin (CSQ2) is the main calcium binding protein located inside the sarcoplasmic reticulum and participates in the contraction/relaxation cycle of cardiac muscle. Analysis of CSQ2 mRNA expression in normal human heart revealed that exists 1.5 to 2-fold higher amount in left atrium and ventricle compared to the light cavities. In order to understand the transcriptional regulation of the hnman CSQ2 gene in the cardiomyocytes, a 3276 bp genomic DNA fragment was cloned in pGL3-basic. The resulting hCSQ2p-Luc construct contains 3103 bp of 5’-flanking sequence and 173 bp of 5’-nontranslated sequence. Three additional hCSQ2p-Luc constructs were generated containing 1097, 582 and 290 bp of 5’-regulatory region. To analyse the transcriptional activity of the hCSQ2p-Luc constructs, transient transfection studies were performed in primary neonatal rat cardiomyocytes in culture. The -290 and -582 bp constructs showed

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basal transcriptional activity, this region contains one TATAbox, 3 CAAT-boxcs, 3 MEF2 binding sites and 5 E boxes. The -1.1 kb and -3.1 kb constructs exhibited 3- to 4-fold higher transcriptional activities compared to the -582 bp construct. The -582 bp to -3.1 kb region contains consensus sites for MyoD, MEF2, NFAT and AP1 suggesting that these factors increase the transcriptional activity of the hCSQ2 gene in cardiocytes.

Abstract N° 107 dPKC mediates reperfusion-induced cytochrome c release, BAD stability, Akt activation, and apoptosis 1 Christopher L. Murriel, 2Eric Churchill, 1Koichi Inagaki, 2 Luke Szweda, and 1Daria Mochly-Rosen. 1Dept. of Molecular Pharmacology, Stanford University, Stanford, CA and 2Dept. of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH. Introduction: Increased dPKC translocation during ischemia and reperfusion is involved in myocardial cell apoptosis. Here, we determined if dPKC activity modulates apoptotis proteins during this process. Methods: We subjected adult male Wistar rat hearts, ex vivo, to 30 min of no-flow global ischemia followed by treatment for the first 15 min of reperfusion with or without 500 nM of Tat protein-derived conjugated dPKC inhibitor, Tat-dV1-1. Reperfusion was stopped or continued for 60 or 120 min. dPKC activation and changes in apoptosis-related proteins were determined by Western blot analysis. Results: Ischemia and reperfusion induced an increase in both dPKC mitochondria translocation and cytochrome c release, vs perfused controls. Tat-dV1-1 treatment during reperfusion inhibited mitochondrial dPKC translocation and cytochrome c release (p ≤ 0.02). Ischemia and reperfusion also enhanced caspase 3 activation, PARP cleavage, and DNA fragmentation–all inhibited by Tat-dV1-1. Ischemia and reperfusion induced a significant increase (> 60 %; p ≤ 0.05) in pro-apoptotic BAD levels and decreases in antiapoptotic Bcl-2 (< 40 %; p ≤ 0.03) and Bcl-xL (< 40 %; p ≤ 0.01). Importantly, inhibiting dPKC translocation with Tat-dV1-1 during reperfusion inhibited the increase in BAD protein only, but not decreases in Bcl-2 or Bcl-xL. In addition, we observed a decrease in the phosphorylation of prosurvival Akt protein in response to ischemia and reperfusion. However, this decrease was inhibited with Tat-dV1-1 (< 50 %; p ≤ 0.005). Conclusion: dPKC activity during ischemia and reperfusion results in myocardial apoptosis by decreasing mitochondrial stability and Akt activity, while increasing the levels of pro-apoptotic BAD. These data illustrate the molecular basis for the cardioprotective effect of the dPKC inhibitor recently reported in a porcine model of acute myocardial infarction, in vivo, (Inagaki, K., et al. Circulation. 108, 2304-7, 2003).

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Abstract 108 Effect of ascorbic acid on lipidic profil and insulin sensitivity in healthy Obese volunteers Cardona G, Pascoe S, Totsuka S, Miranda A & Garcia L. Cardiovascular Investigation Center, CUCS, University of Guadalajara The Obesity represents a worldwide health problem and asociated with diabetes mellitus, hypertension, dyslipidemia and atherosclerotic cardiovascular disease as part of multifaceted syndrome of insuline resistence (IRS) is a major cause of morbi-mortality. Over the past several years significant progress has been made in our understanding of IRS. Several studies have shown that oxidative stress plays an important role in the pathophysiological events of IRS. In view of these data, we considered it important to investigate the effect of ascorbic acid a potent antioxidant on lipidic profil and insulin sensitivity in healthy obese volunteers. Methods: Rabdomized single center, double-blind study was done in 16 healthy volunteers with BMI between 30 y 40 kg/m@ with stable body weight for at least 3 months before start the study, They were divided in two parallel groups: 8 received 1 gr of ascorbic acid everyday for four weeks and 8 received placebo, all them were investigated about relevant medical history and their current medical condition and blood samples were taken before and after the medication to laboratory test included: creatinine, glucose, uric acid, cholesterol, triglycerides, HDL, LDL. VLDL. Insulin sensitivity was assessed by hyperglycemic hyperinsulinemic clamp technique. Results and conclusions: Ascorbic acid orally administrated in obese volunteers not show significant differences in lipidic profil and insulin sensitivity, perhaps a more long period is necessary to reflect significant differences. Keywords: Antioxidants, Free Radicals, Endocrine abnormalities.

Abstract N° 109 Adenosine-mediated relaxation in A1 knockout mouse aorta Huda Tawfik, P.J. Oldenburg, and S. Jamal Mustafa. Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, NC 27858 Adenosine has been shown to be an important regulator of vascular tone through the activation of four subtypes of adenosine receptors (AR). The A2A and A2B AR have been shown to be involved in the relaxation of vascular smooth muscle. However, the roles of the A1 and A3 AR in the regulation of vascular tone are not substantiated. The aim of this study was to determine the role of A1AR in the relaxation of mouse aorta. Isolated aortic rings from A1AR knockout (KO) and wild-type (WT) mice were precontracted with phenylephrine (1 µM). Concentration response curves for adenosine receptor agonists, NECA (nonselective), CGS21680 (A2A selective), and CCPA (A1 selective) were obtained to determine aortic relaxation. At low doses (0.01-1 µM), NECA caused a contraction in WT aortic rings. NECA and CGS-21680 produced increased maximal relaxation in KO compared to WT aortic rings. The EC50 values were shifted from 9.86 ± 2.26 to 0.55 ± 1.46 µM for NECA and from 9.90 ± 3.12 to 0.64 ± 1.78 µM for CGS-21680. Selective activation of A1AR with CCPA produced significant contraction (45% ± 4.7) in WT aortic rings. These data suggest that A1AR cause contraction of the vascular smooth muscle and negatively modulate the vascular tone in conjunction with other AR subtypes. (Supported by HL-27339)

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Abstract Author Index Ammar, K. Afzal : 37, 38 Armstrong, Stephen : 45 Asbun, Juan : 69 Babick, Andrea : 92 Bolaina, Enrique Mendez : 19 Boscarino, Cathy : 55 Buddhadeb, Dawn : 42 Cantor, Elliott : 89, 101 Castillo, Maria del Carmen : 23 Cena, Jonathan : 29 Chang, Jiang : 44 Chapman, Donald : 93, 94 Chatham, John C : 91 Chen, Yinghong : 75 Churchill, Eric N : 28 Clapp, Carmen : 103 Dent, Melissa : 97 Dhalla, Arvinder : 49 Dhalla, Ken : 96 Diaz, Roberto J : 79 Dumitrescu, Cristian : 46 Dzeja, Petras : 16 Elimban, Vijayan : 100

Engman, Steven : 62 Epperson, Sara : 74 Fernandez del Valle, Cecilia : 20 Fraser, Heather : 50, 51 Garcia, Leonel : 108 Garcia, Ricardo : 58, 73, 78 Gauthereau, Marcia Y : 4 Gerber, Lamar : 14 Gonzalez, Carmen : 104 Gottlieb, Roberta A : 11 Gupta, Akanksha : 21 Gupta, Sudhiranjan : 54 Haworth, Robert A : 32 Heidkamp, Maria C : 70 Henkel, Carlos Castillo : 22 House, Stacey L : 67 Jiang, Zhi-Sheng : 53 Jilkina, Olga : 86 Jimenez, Sarah K : 61 Kardami,, Elissavet : 36, 52 Keller, Steve : 72 Kramer, Jay H : 85

Kuzman, James A : 39 Levchenko, Tatyana S : 17 Li, Zhihe : 84 Loredo, Maria L : 18 Machackova, Jarmila : 95 Mak, I. Tong : 59 Mancilla, Cludia : 5 Manley, Darren : 87, 88 McCune, Sylvia A : 81 Millan, Lourdes : 6 Mozaffari, Mahmood S : 80 Murriel, Christopher L : 107 Narvaez, Juan Carlos Torres : 64 Pastukh, Viktor : 40 Ramirez, Jose Alfredo Sierra : 3 Reece, Karen : 27 Ren, Jun : 82 Ricci, Craig : 41 Rubio, Alberto : 1 Samarel, Allen : 63

Sanchez, Israel Ramirez : 15 Schulz, Richard : 12, 31, 33, 43 Schwertz, Dorie W : 57 Sethi,Rajat : 99 Sontag, David P : 60 Stelzer, Julian E : 9 Sung, Miranda M : 30 Tappia, Pram : 98 Tawfik, Huda : 109 Tejero-Taldo, Isabel : 83 Uchiyama, Takamichi : 47 Vander Heide, Richard S : 7 Vasanji, Zainisha : 90 Villarreal, Francisco J : 76, 77 Vinten-Johansen, Jakob : 105 Weber, Karl T : 68, 71 Yang, Qinglin : 26 Yang, Xiang-Qun : 2 Yang, Xi-Ming : 13 Zarain-Herzberg, Angel : 106 Zhong, Juming : 48

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Abstract Subject Index 5-azacytidine : 44 Adenosine : 51 Adenosine Receptors : 79 Adhesion Molecules : 71 Akt : 45 Alcohol : 37, 38 Aldosterone : 68, 71 Anesthetics : 42 Angiogenesis : 83, 103, 104 Anthracyclines : 7 Antiarrhythmics : 49 Antioxidants : 82, 96, 108 Apoptosis : 7, 11, 26, 47, 59, 107 Arrhythmias : 72, 96 ATP : 17 b-Adrenergic Receptors : 57 b-Adrenoceptor : 99 b-Blockers : 95 Bone Marrow Stromal Cells : 44 Calcium : 19, 22, 23, 68, 104, 106 Calcium Channel : 19, 22, 23, 64 Calcium Handling : 92, 101 Calcium Handling Proteins : 57 Calcium Paradox : 32 Cardiac Function : 90, 99 Cardiac Hypertrophy : 53, 54 Cardiac Remodeling : 99 Cardiology : 55 Cardiomyocytes : 26, 79 Cardiomyopathies : 39, 40, 44, 48, 90, 92 Cardioplegia : 55 Cardioprotection : 53, 67, 105 Cardiovascular Disease : 37 Caveolae : 19 Caveolin-1 : 19 Cell Culture : 20, 22, 59, 60, 61, 76, 77 Cell Death : 36, 40, 41, 63, 77 Cell Volume Regulation : 79

Chloride Channels : 79 Compliance : 1 Connective Tissue : 58, 69, 74, 75, 76, 78 Contractile Proteins : 30, 33, 43 Coronary Flow : 46, 87, 88 Cytoskeleton : 15, 62, 63, 70 Diabetes Mellitus : 90 Differentiation : 44 Dinucleotide Phosphate : 46 Drug Interactions : 73 Drug Toxicity : 85 Echocardiography : 42, 89, 90, 98 Endocrine Abnormalities : 108 Endothelial Nitric Oxide Synthase (eNOS) : 46 Endothelium : 20, 29, 64, 71, 100, 103, 104 Energetics : 86 Enzymes : 78 Enzymology : 12, 43 ERK : 54 Fatty Acids : 14, 50, 51 Fischer 344 : 42 Free Radicals : 20, 33, 82, 108 GAP Junctions : 44, 52 Gelatinases : 31 Gender : 57 Gene Expression : 14, 54, 61, 84, 106 Glucose Intolerance : 80 Glycogen Synthase Kinase : 45 Growth Factors : 36, 53 Heart : 57 Heart Failure : 38, 39, 40, 48, 54, 58, 81, 89, 93, 94, 95, 97, 98, 101 Hyperplasia : 62

Hypertension : 1, 20, 80, 81 Hypertrophy : 70, 81, 89, 98, 101 Hypoxia : 41, 47 Imaging : 87, 88 Infarction : 67, 77 Infarct Size : 80, 105 Inflammation : 103 Inotropy : 64 Ion Channel : 72 Iron : 85 Ischemia : 12, 17, 30, 41, 43, 45, 46, 49, 55, 67, 79, 87, 88, 91, 100, 107 Ischemia/ Reperfusion : 11, 30, 46, 77, 91, 100, 105, 107 Isoforms : 95 Isolated Rat Heart : 17 Liposomes : 17 Magnesium : 68, 83 Magnesium Deficiency : 85 Matrix : 29, 31 Mechanics : 58 Metabolism : 14, 50, 51, 55, 91 Metalloproteinases : 29, 31 Mitochondria : 7, 11 Molecular Biology : 15, 106 Myocardial : 55, 83 Myocardial Preservation : 84 Myocytes : 33, 39, 47, 48, 60, 61, 77, 82 Myofibrillar ATPase : 95 Myofibrillar Remodeling : 94 Myosin Heavy Chain : 95 Myotrophin : 54 NFkB : 54 Nicotinamide Adenine : 46 Nitric Oxide : 100 Nuclear Magnetic Resonance : 86 Nutrition : 98

Oxidative Metabolites : 96 Oxygen-Derived Free Radicals : 7 Pharmacology : 1, 22, 23, 49, 50, 69, 73, 74, 75, 76, 84 Phospholipase A2 : 32 Phospholipase C : 97 Phosphorylation : 31 Potassium : 86 PPARgamma : 26 Preconditioning : 47 Prevention : 37 Propranolol : 59 Protection : 12, 55, 77, 84 Protein Kinases : 52, 62, 63, 70, 84 Proteins : 73 Protein Synthesis : 60, 72 Raf : 54 Receptors : 19, 20, 22, 23, 36, 64, 74, 75 Reduced Tetrasodium Salt (NADPH) : 46 Renin Angiotensin System : 93, 94 Reoxygenation : 47 Sarcolema : 97 Sarcolemmal : 15 Sarcoplasmic Reticulum : 92, 93, 101, 106 Saturated Fat Diet : 80 Sepsis : 29 Sex Differences : 57 Signal Transduction : 54, 97, 98 Smooth Muscle : 19, 22, 62 Stem Cells : 44 Structure-Function : 52 Systolic Dysfunction : 89 Tetrahydrobiopterin (BH4) : 46 Tissue Culture : 69, 78 Vasoactive Drugs : 23 Ventricular Function : 38, 88 Volume Overload : 97