Effects of Ischemia on Cardiomyocyte Connexin-43 Distribution and Phosphorylation Studied in in vivo and in vitro Models

  • Stéphane Tanguy
  • Madhumathy Jeyaraman
  • Bradley W. Doble
  • Zhisheng Jiang
  • Robert R. Fandrich
  • Elissavet Kardami
Part of the Progress in Experimental Cardiology book series (PREC, volume 10)


The gap junction protein connexin-43 (Cx43) exists mainly in the phosphory-lated state in the normal heart. We have investigated short-term effects of ischemia on cardiac Cx43 phosphorylation and distribution, in four models: global ischemia of the ex vivo perfused heart, left ventricular ischemia induced by irreversible coronary ligation in vivo, simulated ischemia on isolated adult myocyte pellets, and neonatal cardiomyocytes incubated in a hypoxia chamber. Antibody AB. 13-800 that recognizes specifically the 41kDa nonphospho-rylated form of cardiac Cx43 labeled intercalated discs (ICDs) in myocytes from perfused rat hearts subjected to 30min global ischemia; also in myocytes at the infarct border 6 hours post-infarction. Ischemia induced a sharp increase in the 41 kDa Cx43 from perfused hearts, isolated adult myocyte pellets and neonatal myocyte cultures subjected to hypoxia. The protein phosphatase type 1/2A inhibitors okadaic acid and calyculin A, tested in the in vitro models, decreased ischemia-induced Cx43 dephosphorylation. The 41 kDa Cx43 was present in both Triton-soluble as well as Triton-insoluble (enriched in ICDs) cardiac membrane fractions, assessed by western blotting. We conclude that ischemia causes dephosphorylation of car-diomyocyte Cx43 in vivo as well as in vitro, and that this phenomenon occurs irrespectively of stage (neonatal or adult) or presence of cell contact. Cx43 dephosphorylation occurs at ICDs, is mediated at least in part by PPl/2-type phosphatases, and would be expected to affect GJ function and contribute to ischemia-induced conductance and contractile changes.

Key words

Ischemia models gap junctions phosphorylation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Kumar NM, Gilula NB. 1996. Gap junction communication channel. Cell 84:381–388.PubMedCrossRefGoogle Scholar
  2. 2.
    Beyer EC. 1993. Gap junctions. Int Rev Cytol: 1–37.Google Scholar
  3. 3.
    Saffitz JE, Yamada KA. 2000. Closing the gap in understanding the regulation of intercellular communication. Cardiovasc Res 45:807–809.PubMedCrossRefGoogle Scholar
  4. 4.
    Hossain MZ, Boynton AL. 2000. Regulation of Cx43 gap junctions: the gatekeeper and the password. Sci STKE 54:E1.Google Scholar
  5. 5.
    Lampe PD, Lau AF. 2000. Regulation of gap junctions by phosphorylation of connexins. Arch Biochem Biophys 384:205–215.PubMedCrossRefGoogle Scholar
  6. 6.
    Lau AF, Hatch-Pigott V, Crow DS. 1991. Evidence that heart connexin43 is a phosphoprotein. J Mol Cell Cardiol 23(6):659–663.PubMedCrossRefGoogle Scholar
  7. 7.
    Huang XD, Sandusky GE, Zipes DP. 1999. Heterogeneous loss of connexin43 protein in ischemic dog hearts. J Cardiovasc Electrophysiol 10(1):79–91.PubMedCrossRefGoogle Scholar
  8. 8.
    Kaprielian RR, Gunning M, Dupont E, Sheppard MN, Rothery SM, Underwood R, Pennell DJ, Fox K, Pepper J, Poole-Wilson PA, Severs NJ. 1998. Downregulation of immunedetectable con-nexin43 and decreased gap junction size in the pathogenesis of chronic hibernation in the human left ventricle. Circulation 97:651–660.PubMedCrossRefGoogle Scholar
  9. 9.
    Dupont E, Matsushita T, Kaba RA, Vozzi C, Coppen SR, Khan N, Kaprielian R, Yacoub MH, Severs NJ. 2001. Altered connexin expression in human congestive heart failure. J Mol Cell Cardiol 33:359–371.PubMedCrossRefGoogle Scholar
  10. 10.
    Gutstein DE, Morley GE, Vaidya D, Liu F, Chen FL, Stuhlmann H, Fishman GI. 2001. Heterogeneous expression of Gap junction channels in the heart leads to conduction defects and ventricular dysfunction. Circulation 104:1194–1199.PubMedCrossRefGoogle Scholar
  11. 11.
    Gutstein DE, Morley GE, Tamaddon H, Vaidya D, Schneider MD, Chen J, Chien, KR Stuhlmann H, Fishman GI. 2001. Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43. Circ Res 88:333–339.PubMedCrossRefGoogle Scholar
  12. 12.
    Curtis MJ, Macleod BA, Tabrizchi R, Walker MJ. 1986. An improved perfusion apparatus for small animal hearts. J Pharmacol Methods 15:87–94.PubMedCrossRefGoogle Scholar
  13. 13.
    Beardslee MA, Lerner DL, Tadros PN, Laing JG, Beyer EC, Yamada KA, Kleber AG, Schuessler RB, Saffitz JE. 2000. Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia. Circ Res 87:656–662.PubMedCrossRefGoogle Scholar
  14. 14.
    Jiang ZS, Padua RR, Ju H, Doble BW, Jin Y, Hao J, Cattini PA, Dixon IM, Kardami E. 2002. Acute protection of ischemic heart by FGF-2: involvement of FGF-2 receptors and protein kinase C. Am J Physiol Heart Circ Physiol 282:H1071–H1080.PubMedGoogle Scholar
  15. 15.
    Padua RR, Merle PL, Doble BW, Yu CH, Zahradka P, Pierce GN, Panagia V, Kardarm E. 1998. FGF-2-induced negative inotropism and cardioprotection are inhibited by chelerythrine: involvement of sarcolemmal calcium-independent protein kinase C. J Mol Cell Cardiol 30:2695–2709.PubMedCrossRefGoogle Scholar
  16. 16.
    Armstrong SC, Ganote CE. 1992. Effects of the protein phosphatase inhibitors okadaic acid and calyculin A on metabolically inhibited and ischaemic isolated myocytes. J Mol Cell Cardiol 24:869–884.PubMedCrossRefGoogle Scholar
  17. 17.
    Doble BW, Chen Y, Bosc DG, Litchfield DW, Kardarm E. 1996. Fibroblast growth factor-2 decreases metabolic coupling and stimulates phosphorylation as well as masking of connexin43 epitopes in cardiac myocytes. Circ Res 79:647–658.PubMedCrossRefGoogle Scholar
  18. 18.
    Doble BW, Ping P, Kardami E. 2000. The epsilon subtype of protein kinase C is required for car-diomyocyte connexin-43 phosphorylation. Circ Res 86(3):293–301.PubMedCrossRefGoogle Scholar
  19. 19.
    Zhao J, Renner O, Wightman L, Sugden PH, Stewart L, Miller AD, Latchman DS, Marber MS. 1998. The expression of constitutively active isotypes of protein kinase C to investigate preconditioning. J Biol Chem 273:23072–23079.PubMedCrossRefGoogle Scholar
  20. 20.
    Nagy JI, Li WE, Roy C, Doble BW, Gilchrist JS, Kardami E, Hertzberg EL. 1997. Selective monoclonal antibody recognition and cellular localization of an unphosphorylated form of connexin43. Exp Cell Res 236:127–136.PubMedCrossRefGoogle Scholar
  21. 21.
    Beardslee MA, Laing JG, Beyer EC, Saffitz JE. 1998. Rapid turnover of connexin43 in the adult rat heart. Circ Res 83:629–635.PubMedCrossRefGoogle Scholar
  22. 22.
    Jeyaraman M, Tanguy S, Fandrich RR, Lukas A, Kardami E. 2002. Ischemia—induced dephosphory-lation of cardiomyocyte connexin-43 is reduced by okadaic acid and caliculin A but not fostriecin. Mol Cell Biochem In press.Google Scholar
  23. 23.
    Manjunath CK, Page E. 1986. Rat heart gap junctions as disulfide-bonded connexon multimers: their depolymerization and solubilization in deoxycholate. J Membr Biol 90:43–57.PubMedCrossRefGoogle Scholar
  24. 24.
    Musil LS, Goodenough DA. 1991. Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques. J Cell Biol 115:1357–1374.PubMedCrossRefGoogle Scholar
  25. 25.
    Moreno AP, Saez JC, Fishman GI, Spray DC. 1994. Human connexin43 gap junction channels. Regulation of unitary conductances by phosphorylation. Circ Res 74:1050–1057.PubMedCrossRefGoogle Scholar
  26. 26.
    Kim DY, Kam Y, Koo SK, Joe CO. 1999. Gating connexin43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation. J Biol Chem 274:5581–5587.PubMedCrossRefGoogle Scholar
  27. 27.
    Ruiz-Meana M, Garcia-Dorado D, Lane S, Pina P, Inserte J, Mirabet M, Soler-Soler J. 2001. Persistence of gap junction communication during myocardial ischemia. Am J Physiol Heart Circ Physiol 280:H2563–H2571.PubMedGoogle Scholar
  28. 28.
    Li WE, Ochalski PA, Hertzberg EL, Nagy JI. 1998. Immunorecognition, ultrastructure and phosphorylation status of astrocytic gap junctions and connexin43 in rat brain after cerebral focal ischaemia. Eur J Neurosci 10:2444–2463.PubMedCrossRefGoogle Scholar
  29. 29.
    Duthe F, Plaisance I, Sarrouilhe D, Herve JC. 2001. Endogenous protein phosphatase 1 runs down gap junctional communication of rat ventricular myocytes. Am J Physiol Cell Physiol 281: C1648–C1656.PubMedGoogle Scholar
  30. 30.
    Kwak BR, Jongsma HJ. 1996. Regulation of cardiac gap junction channel permeability and conductance by several phosphorylating conditions. Mol Cell Biochem 157:93–99.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2004

Authors and Affiliations

  • Stéphane Tanguy
    • 1
  • Madhumathy Jeyaraman
    • 1
  • Bradley W. Doble
    • 1
  • Zhisheng Jiang
    • 1
  • Robert R. Fandrich
    • 1
  • Elissavet Kardami
    • 1
  1. 1.Institute of Cardiovascular Sciences, Departments of Human Anatomy and Cell Sciences and PhysiologyUniversity of ManitobaWinnipegCanada

Personalised recommendations