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Normal myosin ATPase activity in the volume overload hypertrophied right ventricle
ABSTRACTS
INHIBITION OF MYOCARDIAL B-ADRENERGIC RECEPTOR (MBAR) BINDING BY A DIGITALIS GLYCOSIDE Ohun H. Sethna, MD, Todd A. Goodglick, William E. Shell, MD, and Michael H. Burnam, MD; Cedars Sinai Medical Center Los Angeles, California. The interphase between drug receptor binding and the f i nal biological response is a poorly understood phenomenon. I t is conceivable that drugs acting on presumably d i f f e r ent receptors but having a common final biological response (increased myocardial c o n t r a c t i l i t y ) may share a portion of this interphase pathway. Canine L.V. microsomes prepared by ultracentrifugation were s e r i a l l y didiluted (0.8-1.6 mgms protein/ml) with buffer (0.075 Tris, 0.025 M MgCl2, pH" 7.6), and assayed for MBAR binding by incubation a~ 37°C for 5 mins with lOnM (-) 3H dihydroalprenolol (DHA), 25-50 Ci/m mole, followed by a 0.45_~ millipore f i l t r a t i o n . Preincubation with ouabain 10-6 M at 37°C for 5 mins resulted in a s i g n i f i c a n t decrease in DHA binding at each protein concentration as follows: Protein Untreated Pretreated mgms/ml MBAR binding* MBAR binding* 0.8 1.0 1.2 1.4 1 6 "
11.2 14.7 17.6 19.3 23.8
+ 2.1 _+ 2.5 + 2.0 _+ 2.7 ± 4.3
5.7 7.6 9.0 12.2 14.2
_+ 0.7 ± 0.8 _+ 1.7 +_ 2.3 +•2.7
p
N=6 N=6 N=6 N=6 N=6 *c p.m. x 10-3
This is the f i r s t dembnstration of a modulation by a d i g i t a l i s glycoside of MBAR binding, thus offering the glycoside as a potential probe for further exploration of MBAR binding and coupling mechanisms.
PRI~IARY CHANGES IN MITOCHONDRIAL RESPIRATION RESULTING IN ALTERED CALClUH TRANSPORT IN ISCHEMIC HEART Louis A. Sordahl, Ph.D., FACC, University of Texas Med~cal~ Branch~, Gaiveston, Texas
STIMULATION BY GROWTH HORMONE (GH) OF MYOCARDIAL THYMIDINE KINASE: DEPENDENCE OF MICROTUBULE ASSEMBLY Constantinos J. Limas, M.D., University of Minnesota School of Medicine, Minneapolis, Minnesota 55455. The ability of myocardial cells to synthesize DNA and undergo mitosis shows an age-dependent decline during postnatal development. In order to obtain more information about the regulatory mechanisms involved, we have studied 3H-thymidine incorporation into and thymidine kinase activity of isolated cardiac myocytes from hypophysectomized rats treated with growth hormone (GH). A single injection of growth hormone (0.5 unit) to 3-week old rats resulted in increased 3H-dTR incorporation (118 ± 0.42 dpm/~g DNA vs. 63.7 ± 0.12 in controls, p<0.Ol) and thymidine kinase activity (117 ± 6.3 vs. 63.5 ± 3.1 pmoles [3H]dTMP/mg DNA/20 minutes). There was a 12-hour lag before significant stimulation of DNA synthesis occurred in response to GH and the peak was reached in 48 hours. Concomitant administration of actinomycin D (8 pg) or cycloheximide (0.8 mg) ~ith GH prevented the subsequent increase in DNA synthesis but did not affect thymidine kinase activity; daunomycin (lO0 ~g), on the other hand, inhibited both DNA synthesis and thymidine kinase stimulation. Vinblastine and colchicine [but not lumicolchicine] showed a dose-dependent inhibition of thymidine kinase and DNA synthesis stimulation by growth hormone. This inhibitory effect was limited to the first 8-10 hours from GH administration. Despite thymidine kinase activity in the myocardium of 6-month old rats comparable to that of 3-week old animals, no stimulation of enzymatic activity orDNA synthesis occurred inresponse to GH injection. These results indicate an age-dependent induction of myocardial thymidine kinase activity after proliferative stimuli; this induction depends on protein synthesis in the pre-replicative phase and is sensitive to inhibitors of microtubule assembly.
NORMAL MYOSIN ATPase ACTIVITY IN THE VOLUME OVERLOAD HYPERTROPHIED RIGHT VENTRICLE Rita A. Ca reyPhD, FACC, Gangaiah Natarajan MBBS, FACC, Alfred A. Bove MD, PhD, FACC, Richard L. Coulson PhD, James F. Spann MD, FACC, Temple Univ. Philadelphia, PA. ~ . _
A number of early changes in mitochondrial energy-linked functions have been reported in ischemic heart. The role of altered mitochondrial function in subsequent myocardial cell death during ischemia is not known. Mitochondria from ischemic dog hearts (XH) were isolated I0 min to 1 hr following l e f t circumflex coronary l i g a t i o n . ~litochondria from the non-ischemic area of the same heart served as controls (CM). Hitochondrial respirations was measured polarographically and calcium uptake by dualbeam spectrophotometry. Decreased XM respiratory rates were observed 15 min after the onset of ischemia. XM respiration continued to decrease throughout the f i r s t hour following ischemic i n s u l t . Parallel decreases in the rates of respiration-supported calcium uptake by XM were also observed. XM exhibited rapid, premature release of accumulated calcium. The i n a b i l i t y of XM to retain accumulated calcium was associated with loss of intramitochondrial adenine nucleotides. Addition of small amounts of adenine nucleotides (lOnmoles/mg) to XM resulted in retention of accumulated calcium similar to CM. Spectrophotometric measurements revealed rapid oxidation of internal NADH during calcium uptake by XM. Adenine nucleotide addition prevented this rapid oxidation and premature calcium loss in XH. The data suggest tha~ alterations in the "energy poise" (reduced-steady State) of the mitochondrial electron chain results in the early and rapid turnover of calcium. This rapid and s i g n i f i c a n t loss of calcium from the intramitochondrial compartment may contribute to the early loss of c o n t r a c t i l i t y of ischemic myocardium.
Although it is clear that chronic pressure overload (CPO) leads to hypertrophy (H), depressed mechanical function (MF) and reduced myosin ATPase activity (M-ATPase), it is not known if the lowered M-ATPase results from the hypertrophic process per s~ or if CPO is required for the depressed M-ATPase. Further, a causal relationship between lowered M-ATPase and weakened MF in CPO has not been established. Chronic volume overload (CVO) on the myocardium leading to H equivalent to that in CPO allows the effects of pressure overload to be separated from the effects of H and provides insite into the association between M-ATPase and MF. Large atrial septal defects (ASD) were produced with a transvenous biopsy catheter in 6 adult cats and resulted in 62~o hypertrophy, normal (p>.O5) RV papillary muscle velocity of shortening at .5 g/mm 2 load (C:l.Ol+.O5 l/s, ASD=].02+.I6 I/s) peak isometric force develop--ment (C=6.53+.30 g-/mm2, ASD=6.57+ 51 g/mm2). M-ATPase was determined in 3 activating media and enzymatic Vma x was normal (p>.05) in the ASD group: •
M-ATPase (pmol es Pi/min'mg) Stimulating Medium Act in K-EDTA Ca++
C O. 20+.02 l. 67~. 05 O. 4 I¥. 03
ASD 0.21+. 03 I. 69¥. 07 O. 38¥. 02
It was concluded that CPO is required for depressed MATPase. The concept that depressed M-ATPase is causally related to depressed MF in CP0 is supported.
February 1979
The American Journal of CARDIOLOGY
Volume 43
393
INHIBITION OF MYOCARDIAL B-ADRENERGIC RECEPTOR (MBAR) BINDING BY A DIGITALIS GLYCOSIDE Ohun H. Sethna, MD, Todd A. Goodglick, William E. Shell, MD, and Michael H. Burnam, MD; Cedars Sinai Medical Center Los Angeles, California. The interphase between drug receptor binding and the f i nal biological response is a poorly understood phenomenon. I t is conceivable that drugs acting on presumably d i f f e r ent receptors but having a common final biological response (increased myocardial c o n t r a c t i l i t y ) may share a portion of this interphase pathway. Canine L.V. microsomes prepared by ultracentrifugation were s e r i a l l y didiluted (0.8-1.6 mgms protein/ml) with buffer (0.075 Tris, 0.025 M MgCl2, pH" 7.6), and assayed for MBAR binding by incubation a~ 37°C for 5 mins with lOnM (-) 3H dihydroalprenolol (DHA), 25-50 Ci/m mole, followed by a 0.45_~ millipore f i l t r a t i o n . Preincubation with ouabain 10-6 M at 37°C for 5 mins resulted in a s i g n i f i c a n t decrease in DHA binding at each protein concentration as follows: Protein Untreated Pretreated mgms/ml MBAR binding* MBAR binding* 0.8 1.0 1.2 1.4 1 6 "
11.2 14.7 17.6 19.3 23.8
+ 2.1 _+ 2.5 + 2.0 _+ 2.7 ± 4.3
5.7 7.6 9.0 12.2 14.2
_+ 0.7 ± 0.8 _+ 1.7 +_ 2.3 +•2.7
p
N=6 N=6 N=6 N=6 N=6 *c p.m. x 10-3
This is the f i r s t dembnstration of a modulation by a d i g i t a l i s glycoside of MBAR binding, thus offering the glycoside as a potential probe for further exploration of MBAR binding and coupling mechanisms.
PRI~IARY CHANGES IN MITOCHONDRIAL RESPIRATION RESULTING IN ALTERED CALClUH TRANSPORT IN ISCHEMIC HEART Louis A. Sordahl, Ph.D., FACC, University of Texas Med~cal~ Branch~, Gaiveston, Texas
STIMULATION BY GROWTH HORMONE (GH) OF MYOCARDIAL THYMIDINE KINASE: DEPENDENCE OF MICROTUBULE ASSEMBLY Constantinos J. Limas, M.D., University of Minnesota School of Medicine, Minneapolis, Minnesota 55455. The ability of myocardial cells to synthesize DNA and undergo mitosis shows an age-dependent decline during postnatal development. In order to obtain more information about the regulatory mechanisms involved, we have studied 3H-thymidine incorporation into and thymidine kinase activity of isolated cardiac myocytes from hypophysectomized rats treated with growth hormone (GH). A single injection of growth hormone (0.5 unit) to 3-week old rats resulted in increased 3H-dTR incorporation (118 ± 0.42 dpm/~g DNA vs. 63.7 ± 0.12 in controls, p<0.Ol) and thymidine kinase activity (117 ± 6.3 vs. 63.5 ± 3.1 pmoles [3H]dTMP/mg DNA/20 minutes). There was a 12-hour lag before significant stimulation of DNA synthesis occurred in response to GH and the peak was reached in 48 hours. Concomitant administration of actinomycin D (8 pg) or cycloheximide (0.8 mg) ~ith GH prevented the subsequent increase in DNA synthesis but did not affect thymidine kinase activity; daunomycin (lO0 ~g), on the other hand, inhibited both DNA synthesis and thymidine kinase stimulation. Vinblastine and colchicine [but not lumicolchicine] showed a dose-dependent inhibition of thymidine kinase and DNA synthesis stimulation by growth hormone. This inhibitory effect was limited to the first 8-10 hours from GH administration. Despite thymidine kinase activity in the myocardium of 6-month old rats comparable to that of 3-week old animals, no stimulation of enzymatic activity orDNA synthesis occurred inresponse to GH injection. These results indicate an age-dependent induction of myocardial thymidine kinase activity after proliferative stimuli; this induction depends on protein synthesis in the pre-replicative phase and is sensitive to inhibitors of microtubule assembly.
NORMAL MYOSIN ATPase ACTIVITY IN THE VOLUME OVERLOAD HYPERTROPHIED RIGHT VENTRICLE Rita A. Ca reyPhD, FACC, Gangaiah Natarajan MBBS, FACC, Alfred A. Bove MD, PhD, FACC, Richard L. Coulson PhD, James F. Spann MD, FACC, Temple Univ. Philadelphia, PA. ~ . _
A number of early changes in mitochondrial energy-linked functions have been reported in ischemic heart. The role of altered mitochondrial function in subsequent myocardial cell death during ischemia is not known. Mitochondria from ischemic dog hearts (XH) were isolated I0 min to 1 hr following l e f t circumflex coronary l i g a t i o n . ~litochondria from the non-ischemic area of the same heart served as controls (CM). Hitochondrial respirations was measured polarographically and calcium uptake by dualbeam spectrophotometry. Decreased XM respiratory rates were observed 15 min after the onset of ischemia. XM respiration continued to decrease throughout the f i r s t hour following ischemic i n s u l t . Parallel decreases in the rates of respiration-supported calcium uptake by XM were also observed. XM exhibited rapid, premature release of accumulated calcium. The i n a b i l i t y of XM to retain accumulated calcium was associated with loss of intramitochondrial adenine nucleotides. Addition of small amounts of adenine nucleotides (lOnmoles/mg) to XM resulted in retention of accumulated calcium similar to CM. Spectrophotometric measurements revealed rapid oxidation of internal NADH during calcium uptake by XM. Adenine nucleotide addition prevented this rapid oxidation and premature calcium loss in XH. The data suggest tha~ alterations in the "energy poise" (reduced-steady State) of the mitochondrial electron chain results in the early and rapid turnover of calcium. This rapid and s i g n i f i c a n t loss of calcium from the intramitochondrial compartment may contribute to the early loss of c o n t r a c t i l i t y of ischemic myocardium.
Although it is clear that chronic pressure overload (CPO) leads to hypertrophy (H), depressed mechanical function (MF) and reduced myosin ATPase activity (M-ATPase), it is not known if the lowered M-ATPase results from the hypertrophic process per s~ or if CPO is required for the depressed M-ATPase. Further, a causal relationship between lowered M-ATPase and weakened MF in CPO has not been established. Chronic volume overload (CVO) on the myocardium leading to H equivalent to that in CPO allows the effects of pressure overload to be separated from the effects of H and provides insite into the association between M-ATPase and MF. Large atrial septal defects (ASD) were produced with a transvenous biopsy catheter in 6 adult cats and resulted in 62~o hypertrophy, normal (p>.O5) RV papillary muscle velocity of shortening at .5 g/mm 2 load (C:l.Ol+.O5 l/s, ASD=].02+.I6 I/s) peak isometric force develop--ment (C=6.53+.30 g-/mm2, ASD=6.57+ 51 g/mm2). M-ATPase was determined in 3 activating media and enzymatic Vma x was normal (p>.05) in the ASD group: •
M-ATPase (pmol es Pi/min'mg) Stimulating Medium Act in K-EDTA Ca++
C O. 20+.02 l. 67~. 05 O. 4 I¥. 03
ASD 0.21+. 03 I. 69¥. 07 O. 38¥. 02
It was concluded that CPO is required for depressed MATPase. The concept that depressed M-ATPase is causally related to depressed MF in CP0 is supported.
February 1979
The American Journal of CARDIOLOGY
Volume 43
393