Abstract
Background Connexin43 (Cx43) dephosphorylation contributes profoundly to arrhythmias and infarction in ischemia/reperfusion (IR) heart, however, its causative role and underlying mechanism in cardiomyocyte damage are unknown. We aim to identify Cx43 specific phosphorylation site(s) that causes cardiomyocyte apoptosis and contributes to IR-induced myocardium injury.
Methods and Results Rats (11/22) with left coronary artery ligation followed by reperfusion had ventricular tachyarrhythmia compared with Sham rats (0/20). Immunohistochemistry/immunoblotting showed a marked infarcted area, increased levels of factor associated suicide (Fas)/Fas-associating protein with a novel death domain (FADD) and caspase-8/3, and reduced Cx43 phosphorylation at Ser279/282 (pS279/282, 0.48 versus 1.0, P=0.02), compared to Sham ventricles. Neonatal rat ventricular myocytes (NRVMs) that were exposed to anoxia/reoxygenation (AR) showed similar results as IR rats, which could be prevented by the treatment with a phosphatase inhibitor. NRVMs mutated at S282 with alanine (S282A) displayed cell death, activation of caspase-8/3 and Fas/FADD, and uncoupled Ca2+ transients compared to wild-type Cx43 (Cx43-wt) cells. Importantly, homozygous animals with Cx43 S282A mutation died prenatally. In addition, rats that received S282A virus intramyocardially also showed ventricular arrhythmias (8/22 versus 1/13, P<0.05), a reduced ejection fraction (P<0.01, 60.3% versus 76.1%) and robust TUNEL-positive cells with activated caspase-8/3 and Fas/FADD compared to Cx43-wt rats. Moreover, significant activation of p38 mitogen-activated protein kinase (MAPK) was detected in the ischemic cell and rat models, and increased Cx43/p38 MAPK interaction was found in S282A transfected cardiomyocytes. SB239063, a p38 MAPK inhibitor, suppressed S282A-induced activation of Fas/FADD and cell apoptosis in NRVMs.
Conclusion Our findings reveal deficient S282 phosphorylation as a trigger in Cx43-mediated cardiomyocyte death, offering a novel mechanism and potential target in protecting heart from IR injury.
