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Actin and myosin light chain binding sites on human cardiac myosin heavy chain fragments synthesized by E. Coli
J Mol Cell Cardiol 21 {Supplement
III) (1989)
19 ACTIN AND MYOSIN LIGHT CHAIN BINDING SITES ON IiUMAN CARDIAC MYOSIN HEAVY CHAIN FRAGMENTS SYNTHESIZED BY E. COLI. P.Eldin, B.Comillon, A.M.Cathiard, M.Le cunff, M.Anoal, J.O.C.LCger, D.Mornet, H.P.Vosberg* and J.J.Ltger. Institut National de la Sante et de la Recherche MCdicale, INSERM U300, Fat. Pharmacie, Montpellier, France, *Max Planck Institute, Heidelberg, Federal Republic of Germany To investigate the structural basis of the functional differences between tcardiac isomyosins, fragments of human cardiac cDNAs coding for myosin I3heavy chain and venaicular light chain 1 (a gift from P.Gunning) were amplified by PCR, inserted in the coding portion of the l3-galactosidase gene and expressed in E.Coli. The different synthesized protein fragments were detected by monoclonal or polyclonal antibodies specific for the particular myosin light and heavy chain domains being studied. Certain of these antibodies, called “functional” antibodies, positively or negatively influence myosin functions, such as ATPase activity, actin binding, and myosin light-heavy chain interactions. Several synthetic myosin heavy chain fragments either in solution or blotted on a solid support, interacted with myosin light chains (or fragments) and with actin. Using the differential reactivities of some monoclonal antibodies with free light chains and native myosins, several light chain domains interacting with the myosin heavy chains were also identified. Dissection, isolation and reconstitution of some of the multiple functions within the cardiac myosin molecule are therefore possible using this approach combining molecular genetics snd hybridoma technology. The next steps will involve a comparison with cardic myosin subunits, as soon as the corresponding cDNA becomes available, mutagenesis studies involving putative “functional” amino acids, and injection of chimeric constructs into cardiac cells.
20 CONTRACTILE DYSFUNCTIONAND IMF'AIRED SR CALCIUM UPTAKE IN HEARTS EXPOSED TO OXIDANT. D.W.Eley, J.M.Eley, H.Fliss and B.Rorecky, University of Ottawa, Ottawa, Canada Isolated perfused rat hearts develop elevated coronary vascular resistance. increased diastolic pressure and reduced pulse pressure upon perfusion with 100 UM hypochlorous acid (HOCI). Subsequent perfusion with 1mM dithiothreitol (DTT), a disulfide reducing
agent, partially restored mechanical performance. We i;I+vestigated the possibility that this ‘contracture’ is linked to impaired cytosolif+ Ca transport due to the partial inactivation of the sarcoplasmic reticulum (SR) Ca ATPase by the oxidation of protein sulfhydryls. s isolated from HOC1 perfused (1 hour) tar hearts shtyed marked depression of sbRoevhes~?’ ATPase activity (121 2 39 "moles Pi/mg/min) and Ca uptake (10.7t5.6 nmoles/mn/min) as compared with control (595t74 and 45.623.6 respecrively). Vesicles isolated“from oxidized hearts subseque$ly treated with DTT (j0 minu&) showed a significant (~~0.05) recovery of both Ca ATPase activity (415f46) and ,Ca uptake (38.3'5.2). The 100 kD CaLT ATPase molecule was then labelled wi@ ‘*C Iodoacetamide (IAA) which sel5r+tively binds to free SH residues and counted for c-IAA binding. The decrease in Ca ATPase activity coincided with an increased level of oxidized thiols following HOC1 perfusion, both of which were reversed after D$$ administration. We conclude rhat HOC1 acts intracellularly to iqqctivate rhe Ca ATPase protein through thiol oxidation leading to a disrupted Ca homeostasis and impaired myocardia@echanics, and that restoration of protein thiol levels with DTT can restore both Ca ATPase and contractile function to these hearts.
21REAPPEARANCE OF@-SMOOTH MUSCLE ACTIN ISOFORM IN CULTURED ADULT RAT CARDIOMYOCYTES. M.E.Eppenberger, V.Kurer, I.Vollenweider, H.M.Eppenberger. Dept.of Cell Biology,ETH,8093 Zurich,Switzerland Adult rat cardiomyocytes in culture undergo extensive morphological changes accompanied by degradation and regeneration of cytoskeleton and contractile apparatus.In the flattened out cardiomyocytes new sarcomeres are preferentially formed in the center of the cells.The formation of myofibrils throughout the cell might be accomplished using actin stress fibers as scaffold (Eppenberger et al. Dev.Biol.l30,1,1988).o(smooth muscle(sm) actin,one of the muscle actin isoforms,is accumulated in a fraction of the regenerating cardiomyocytes.Usinq an anti-ti-smactin antibody(prov.by G.Gabbiani,Univ.of Geneva) we could demonstrate by immunofluorescence that newly isolated cardiomyocytes do not express 6<-sm-actin,whereas after 7 days in culture 13% of the cells expressksm-actin,after 9 days 44% and after 2 weeks 55%.The protein is mainly localized within stressfiber-like structures,but appears only weakly in the sarcomeres.This result was confirmed by immunoblot.Freshly isolated adult cardiomyocytes,which have ceased to expressm-sm-actin thus are capable of reexpressing thei%-sm-actin gene once they are in culture reminding of what is found in fetal rat heart. (SNF grant no. 3.497-86) S.8
III) (1989)
19 ACTIN AND MYOSIN LIGHT CHAIN BINDING SITES ON IiUMAN CARDIAC MYOSIN HEAVY CHAIN FRAGMENTS SYNTHESIZED BY E. COLI. P.Eldin, B.Comillon, A.M.Cathiard, M.Le cunff, M.Anoal, J.O.C.LCger, D.Mornet, H.P.Vosberg* and J.J.Ltger. Institut National de la Sante et de la Recherche MCdicale, INSERM U300, Fat. Pharmacie, Montpellier, France, *Max Planck Institute, Heidelberg, Federal Republic of Germany To investigate the structural basis of the functional differences between tcardiac isomyosins, fragments of human cardiac cDNAs coding for myosin I3heavy chain and venaicular light chain 1 (a gift from P.Gunning) were amplified by PCR, inserted in the coding portion of the l3-galactosidase gene and expressed in E.Coli. The different synthesized protein fragments were detected by monoclonal or polyclonal antibodies specific for the particular myosin light and heavy chain domains being studied. Certain of these antibodies, called “functional” antibodies, positively or negatively influence myosin functions, such as ATPase activity, actin binding, and myosin light-heavy chain interactions. Several synthetic myosin heavy chain fragments either in solution or blotted on a solid support, interacted with myosin light chains (or fragments) and with actin. Using the differential reactivities of some monoclonal antibodies with free light chains and native myosins, several light chain domains interacting with the myosin heavy chains were also identified. Dissection, isolation and reconstitution of some of the multiple functions within the cardiac myosin molecule are therefore possible using this approach combining molecular genetics snd hybridoma technology. The next steps will involve a comparison with cardic myosin subunits, as soon as the corresponding cDNA becomes available, mutagenesis studies involving putative “functional” amino acids, and injection of chimeric constructs into cardiac cells.
20 CONTRACTILE DYSFUNCTIONAND IMF'AIRED SR CALCIUM UPTAKE IN HEARTS EXPOSED TO OXIDANT. D.W.Eley, J.M.Eley, H.Fliss and B.Rorecky, University of Ottawa, Ottawa, Canada Isolated perfused rat hearts develop elevated coronary vascular resistance. increased diastolic pressure and reduced pulse pressure upon perfusion with 100 UM hypochlorous acid (HOCI). Subsequent perfusion with 1mM dithiothreitol (DTT), a disulfide reducing
agent, partially restored mechanical performance. We i;I+vestigated the possibility that this ‘contracture’ is linked to impaired cytosolif+ Ca transport due to the partial inactivation of the sarcoplasmic reticulum (SR) Ca ATPase by the oxidation of protein sulfhydryls. s isolated from HOC1 perfused (1 hour) tar hearts shtyed marked depression of sbRoevhes~?’ ATPase activity (121 2 39 "moles Pi/mg/min) and Ca uptake (10.7t5.6 nmoles/mn/min) as compared with control (595t74 and 45.623.6 respecrively). Vesicles isolated“from oxidized hearts subseque$ly treated with DTT (j0 minu&) showed a significant (~~0.05) recovery of both Ca ATPase activity (415f46) and ,Ca uptake (38.3'5.2). The 100 kD CaLT ATPase molecule was then labelled wi@ ‘*C Iodoacetamide (IAA) which sel5r+tively binds to free SH residues and counted for c-IAA binding. The decrease in Ca ATPase activity coincided with an increased level of oxidized thiols following HOC1 perfusion, both of which were reversed after D$$ administration. We conclude rhat HOC1 acts intracellularly to iqqctivate rhe Ca ATPase protein through thiol oxidation leading to a disrupted Ca homeostasis and impaired myocardia@echanics, and that restoration of protein thiol levels with DTT can restore both Ca ATPase and contractile function to these hearts.
21REAPPEARANCE OF@-SMOOTH MUSCLE ACTIN ISOFORM IN CULTURED ADULT RAT CARDIOMYOCYTES. M.E.Eppenberger, V.Kurer, I.Vollenweider, H.M.Eppenberger. Dept.of Cell Biology,ETH,8093 Zurich,Switzerland Adult rat cardiomyocytes in culture undergo extensive morphological changes accompanied by degradation and regeneration of cytoskeleton and contractile apparatus.In the flattened out cardiomyocytes new sarcomeres are preferentially formed in the center of the cells.The formation of myofibrils throughout the cell might be accomplished using actin stress fibers as scaffold (Eppenberger et al. Dev.Biol.l30,1,1988).o(smooth muscle(sm) actin,one of the muscle actin isoforms,is accumulated in a fraction of the regenerating cardiomyocytes.Usinq an anti-ti-smactin antibody(prov.by G.Gabbiani,Univ.of Geneva) we could demonstrate by immunofluorescence that newly isolated cardiomyocytes do not express 6<-sm-actin,whereas after 7 days in culture 13% of the cells expressksm-actin,after 9 days 44% and after 2 weeks 55%.The protein is mainly localized within stressfiber-like structures,but appears only weakly in the sarcomeres.This result was confirmed by immunoblot.Freshly isolated adult cardiomyocytes,which have ceased to expressm-sm-actin thus are capable of reexpressing thei%-sm-actin gene once they are in culture reminding of what is found in fetal rat heart. (SNF grant no. 3.497-86) S.8