Effects of Ischemia On Cardiomyocyte Connexin-43 Distribution and Phosphorylation Studied in in Vivo and in Vitro Models

摘要

Summary. 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 cardiomyocyte Cx43 in vivo as well as in vitro, and that this phenomenon occurs irrespectively of stage (neonatal or adult) or presence of cell contact.