Sympathetic control of cardiac myosin heavy chain gene expression

  • Mahesh P. Gupta
  • Madhu Gupta
  • Evelyn Dizon
  • Radovan Zak
Part of the Developments in Molecular and Cellular Biochemistry book series (DMCB, volume 17)


Several neuroendocrine factors have been shown to influence the muscle phenotype. Various physiological reports have suggested the role of adrenergic nervous system for cardiac myosin heavy chain (MHC) expression. We have used cultured fetal rat heart myocytes to investigate the role of cAMP on the α- and β-MHC gene expression. In low density cultures, addition of 1 mM 8 Br cAMP resulted in up regulation of α-MHC and down regulation of β-MHC mRNA. This antithetic effect of cAMP depends on the basal expression of both MHC transcripts. In transient transfection analysis employing a series of α-MHC gene promoter/reporter constructs, we identified a 13 bp E-box M-CAT hybrid motif (EM element) which conferred a basal muscle specific and cAMP-inducible expression of the α-MHC gene. Data obtained from the mobility gel-shift analysis indicated that one of the factor(s) binding to the EM element is related to troponin T M-CAT binding factor (TEF-1). To test whether the protein binding to this sequence could be a substrate for cAMP-dependent phosphorylation, the cardiac nuclear proteins were preincubated in a kinase reaction buffer either with a catalytic subunit of PKA (CatPKA) or with cAMP, and binding activity of proteins to the EM element was evaluated by mobility gel shift assay. In a concentration dependent manner, a twofold increase in the intensity of the retarded band was observed. Furthermore, at 100 units of CatPKA, an additional band of faster mobility was observed which was not present either when phosphorylated nuclear extract was incubated with alkaline phosphatase or when ATP was absent in kinase reaction buffer. These results strongly suggest that factor(s) binding to the EM element is a substrate for cAMP dependent phosphorylation.

Key words

cardiac myocytes myosin heavy chain gene cAMP phosphorylation 


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  1. 1.
    Bandman E: Contractile protein isoforms in muscle development. Dev Biol 154: 273–283, 1992PubMedCrossRefGoogle Scholar
  2. 2.
    Shimizu N, Camoretti-Mercada B, Jakovcic S, Zak R: RNA transcription in heart muscle. In: H.A. Fozzard, E. Haber, R.B. Jennings, A.M. Katz, H.E. Morgan (eds). The Heart and Cardiovascular System, 2nd ed., Raven Press, Ltd, New York, 1992, pp 1525–1538Google Scholar
  3. 3.
    Bugaisky LB, Gupta M, Gupta MP, Zak R: In: Cellular and molecular mechanisms of cardiac hypertrophy, In: H.A. Fozzard, E. Haber, R.B. Jennings, A.M. Katz, H.E. Morgan (eds). The Heart and Cardiovascular System, 2nd edn, Raven Press, New York, 1992, pp 1621–1640Google Scholar
  4. 4.
    Emerson CP, Bernstein SI: Molecular genetics of myosin. Annu Rev Biochem 56: 695–726, 1987PubMedCrossRefGoogle Scholar
  5. 5.
    Mahdavi V, Strehler EE, Periasamy M, Wieczovek DF, Izumo S, Nadal-Ginard B: Cardiac myosin heavy chain genes are organized in tandem. Proc Natl Acad Sci USA 81:2626–2630, 1984PubMedCrossRefGoogle Scholar
  6. 6.
    Chizzonite RA, Zak R: Regulation of myosin isoenzyme composition in fetal and neonatal rat ventricle by endogeneous thyroid hormones. J Biol Chem 259: 12628–12632, 1984PubMedGoogle Scholar
  7. 6.
    Swynghedauw B: Developmental and functional adaptation of con-contractile proteins in cardiac and skeletal muscle. Physiol Rev 66: 710–771, 1986PubMedGoogle Scholar
  8. 7.
    Pagani ED, Julian FJ: Rabbit papillary muscle myosin isozymes and the velocity of papillary muscle shortening. Circ Res 54: 586–594, 1984PubMedGoogle Scholar
  9. 8.
    Lyons GE, Schiaffino S, Sasson D, Barton P, Buckingham M: Developmental regulation of myosin gene expression in mouse cardiac muscle. J Cell Biol 111: 2427–2436, 1990PubMedCrossRefGoogle Scholar
  10. 9.
    Lompre AM, Mercadier JG, Wisnewsky C, Bouveret P, Pantaloni D, Albis D, Schwartz K: Species and age-dependent changes in the relative amounts of cardiac myosin isoenzymes in mammals. Dev Biol 84: 286–290, 1981PubMedCrossRefGoogle Scholar
  11. 11.
    Izumo S, Nadal-Ginard B, Mahdavi V: All members of the MHC multigene family respond to thyroid hormone in a highly tissue-specific manner. Science 231: 597–600, 1986PubMedCrossRefGoogle Scholar
  12. 12.
    Izumo S, Mahdavi V: Thyroid hormone receptor a isoforms generated by alternative splicing differentially activate myosin heavy chain gene transcription. Nature 334: 539–542, 1988PubMedCrossRefGoogle Scholar
  13. 13.
    Buttrick, PM, Malhotra A, Factor S, Geener D, Scheuer J: Effects of chronic dobutamine administration on hearts of normal and hypertensive rats. Circ Res 63: 173–181, 1988PubMedGoogle Scholar
  14. 14.
    Dowell RT: Myocardial contractile function and myofibrillar adenosine triphosphatase activity in chemically sympathectomized rats. Circ Res 39: 683–689, 1976PubMedGoogle Scholar
  15. 15.
    Kawana M, Ischizuka N, Taira A, Kimata S, Hosoda S: Effects of cardiac sympathetic activity on myosin isozymes of rabbit heart. Circulation 80 (Suppl II): 462, 1989Google Scholar
  16. 16.
    Advani SV, Malhotra A, Liang D, Geener DL, Buttrick PM, Scheuer J: Swimming attenuates the shift in myosin isoenzymes in the rat heterotopic cardiac isograft. Circulation 80 (Suppl II):297, 1989Google Scholar
  17. 17.
    Winegrad S, McClellan G, Wiesberg A, Lin LE, Weindling S Horowitz A: Beta adrenergic regulation of cardiac myosin. Can J Physiol Pharmacol 65: 606–609, 1986CrossRefGoogle Scholar
  18. 18.
    Karin M: Complexities of gene regulation by cAMP. Trends Genet 5: 65–66, 1989PubMedCrossRefGoogle Scholar
  19. 19.
    Roesler WJ, Vanderbark GR, Hanson RW: Cyclic AMP and the induction of eukaryotic gene transcription. J Biol Chem 263: 9063–9066, 1988PubMedGoogle Scholar
  20. 20.
    Nag AC, Cheng M: Expression of myosin isoenzymes in cardiac muscle cells in culture. Biochem J 221: 21–26, 1984PubMedGoogle Scholar
  21. 21.
    Clark WA, Chizzonite RA, Everett AW, Rabinowitz M, Zak R: Species correlations between cardiac isomyosins: A comparison of electrophoretic and immunological properties. J Biol Chem 257: 5449–5454, 1982PubMedGoogle Scholar
  22. 22.
    Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5298, 1979PubMedCrossRefGoogle Scholar
  23. 23.
    Higuchi R: Recombinant PCR. In: M.A. Iunis, D.H. Gelfand, J.J. Sninsky, T.J. White (eds). PCR Protocols: A Guide to Methods and Applications, Academic Press, New York, pp 177–183, 1990Google Scholar
  24. 24.
    Olsen DB, Eckstein F: High-efficiency oligonucleotide-directed plasmid mutagenesis. Proc Natl Acad Sci USA 87: 1451–1455, 1990PubMedCrossRefGoogle Scholar
  25. 25.
    Dignam JD, Lebovitz RM, Roeder RG: Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acid Res 11: 1475–1489, 1983PubMedCrossRefGoogle Scholar
  26. 26.
    Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH: Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci 83: 6682–6686, 1986PubMedCrossRefGoogle Scholar
  27. 27.
    Moss JB, McQuinn TC, Schwartz RJ. The avian cardiac a-actin promoter is regulated through a pair of complex elements composed of E boxes and serum response elements that bind both positive and negative acting factors. J Biol Chem 269: 12731–12740, 1994PubMedGoogle Scholar
  28. 28.
    Winter B, Braun T, Arnold HH: cAMP-dependent protein kinase represses myogenic differentiation and the activity of the muscle-specific helix-loop-helix transcription factors myf-5 and myoD. J Biol Chem 268: 9869–9878, 1993PubMedGoogle Scholar
  29. 29.
    Ostman-Smith I: Prevention of exercise-induced cardiac hypertrophy in rats by chemical sympathectomy. Neurosci 1: 497–507, 1976CrossRefGoogle Scholar
  30. 30.
    Womble JR, Larson DF, Copeland JG, Brown BR, Maddox MK, Russel DH: Adrenal medulla denervation prevents stress-induced epinephrine plasma elevation and cardiac hypertrophy. Life Sci 27: 2417–2420, 1980PubMedCrossRefGoogle Scholar
  31. 31.
    Sartorelli V, Kedes L: Myocardial activation of the human cardiac α-actin promoter by helix-loop-helix proteins. Proc Natl Acad Sci USA 89: 4047–4051, 1992PubMedCrossRefGoogle Scholar
  32. 32.
    Mar JH, Ordahl CP: M-CAT binding factor, a novel transcription factor governing muscle-specific transcription. Mol Cell Biol 12: 619–630, 1990Google Scholar
  33. 33.
    Gupta MP, Gupta M, Zak R: An E-box/M-CAT hybrid motif and cognate binding protein(s) regulate the basal muscle-specific and cAMP-inducible expression of the rat cardiac a-myosin heavy chain gene. J Biol Chem 269: 29677–29687, 1994PubMedGoogle Scholar
  34. 34.
    Shimizu N, Smith G, Izumo S: Both a ubiquitous factor mTEF-1 and a distinct muscle-specific factor bind to the M-CAT motif of the myosin heavy chain S gene. Nucleic Acid Res 21: 4103–4110, 1993PubMedCrossRefGoogle Scholar
  35. 35.
    Ishiji T, Lack MJ, Parkkinen S, Anderson RD, Hangen TH, Crippe TP, Xiao JH, Davidson I, Chambon P, Turek LP: Transcription enhancer factor (TEF-1) and its cell-specific co-activator activates human papillomavirus E6 and E7 oncogene transcription in keratinocytes and cervical carcinoma. EMBO J 11: 2271–2281, 1992PubMedGoogle Scholar
  36. 36.
    Baeuerle PA, Baltimore D: IkB: A specific inhibitor of the NFkB transcription factor. Science 242: 540–546, 1988PubMedCrossRefGoogle Scholar
  37. 37.
    Morikoshi M, Kai T, Kin Y-S, Green MR, Roeder: Transcription factor ATF interacts with the TATA factor to facilitate establishment of a preinitiation complex. Cell 54: 1033–1042, 1988CrossRefGoogle Scholar
  38. 38.
    Corsi PS, Sisson JC, Verma EM: Trascriptional auto-regulation of the proto-oncogene fos. Nature 334: 314–319, 1988CrossRefGoogle Scholar
  39. 39.
    Pagani ED, Julian FJ: Rabbit papillary muscle myosin isozymes and the velocity of papillary muscle shortening. Circ Res 54: 586–594, 1984PubMedGoogle Scholar
  40. 40.
    Pope B, Hoh JFY, Weeds A: The ATPase activities of rat cardiac myosin isoenzymes. FEBS Lett 118: 205–208, 1980PubMedCrossRefGoogle Scholar
  41. 41.
    Winegrad S, McClellan G, Tucker M, Lin L-E: Cyclic AMP regulates myosin isoenzymes in mammalian cardiac muscle. J Gen Physiol 81: 749–765, 1983PubMedCrossRefGoogle Scholar
  42. 42.
    Jacob R: Chronic reactions of myocardium at the myofibrillar level. Reflections on ‘adaptation’ and ‘disease’ based on the biology of long-term cardiac overload. In: Jacob R et al (ed.). Cardiac Adaptation of Hemodynamic Overload, Training and Stress. Steinkopff Verlag, Darmstadt pp 3–24, 1983Google Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Mahesh P. Gupta
    • 1
  • Madhu Gupta
    • 2
  • Evelyn Dizon
    • 1
  • Radovan Zak
    • 1
    • 3
    • 4
  1. 1.Department of Medicine, MC5105University of ChicagoChicagoUSA
  2. 2.The Heart Institute for ChildrenThe Christ Hospital Medical CenterOak lawnUSA
  3. 3.Department of Physiology and PharmacologyUniversity of ChicagoChicagoUSA
  4. 4.Department of Organismal Biology and AnatomyUniversity of ChicagoChicagoUSA

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