Effects of Ischemia on Cardiomyocyte Connexin-43 Distribution and Phosphorylation Studied in in vivo and in vitro Models
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 wordsIschemia models gap junctions phosphorylation
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- 2.Beyer EC. 1993. Gap junctions. Int Rev Cytol: 1–37.Google Scholar
- 4.Hossain MZ, Boynton AL. 2000. Regulation of Cx43 gap junctions: the gatekeeper and the password. Sci STKE 54:E1.Google Scholar
- 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
- 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
- 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