Phosphorylation site at serine 282 of connexin 43 has a regulatory effect on survival and apoptosis of cardiomyocytes

OBJECTIVE To find out the specific serine phosphorylation site of connexin 43 which involved in regulating cardiomyocytes survival and apoptosis and its underlying mechanism. METHODS Ca;transients was performed in NRVMs transfected with connxin 43 mutants（S282A, S279A） and NRVMs exposed to anoxia and reoxygenation（A/R） and AR cells treated with cantharidinate（CA）. Lucifer Yellow Uptake Assay was detected in S282A NRVMs. Cell deaths were analyzed in all cell models. Assessment of caspase 8, 9 and 3 activity by ELISA kits, P38 and Fas/FADD expression by Western blotting, connexin 43-P38 physical interaction by Coimmunoprecipitation were carried out in both cell models and mouse models including S282A+/-mice, Ischemia/reperfusion rats and rats injected with S282A adenovirus.Echocardiography and electrocardiograph, evaluations of histological analysis and immunostaining of ventricular structurewere conducted on mouse models to monitor heart functions. RESULTS Deficiency in phosphorylation of S282 in NRVMs transfected with connxin 43 mutants（S282A, S279A） and AR cells induced cardiomyocyte death and blockaded intercellular coupling. Markedly activated caspase 3, 8 and increased Fas/FADD expression were found in S282A and AR cells. But CA, an inhibitor of PP2A-A which regulates Cx43 serine dephosphorylation, restored decreased phosphorylation of S282 and completely inhibited FAS/FADD apoptotic pathway in A/R cells. Cx43 S282A;mice, ischemia/reperfusion rats and rats injected with S282A adenovirus with S282 dephosphorylation exhibited arrhythmias and dispersed myocardial damage including disarranged myocardium,fibrosis, apoptosis of cardiomyocytes, and impaired cardiac output. CONCLUSION Deficiency in serine 282 phosphorylation is linked to ventricular arrhythmias and also triggers cardiomyocyte death via activation of P38 MAPK and Fas/FADD pathway.