Protein kinase Cε mediates salutary effects on electrical coupling induced by ischemic preconditioning

BACKGROUND Ischemic preconditioning delays the onset of electrical uncoupling and prevents toss of the primary ventricular gap junction protein connexin 43 (Cx43) from gap junctions during subsequent ischemia. OBJECTIVE To test the hypothesis that these effects are mediated by protein kinase C epsilon (PKC epsilon), we studied isolated Langendorff-perfused hearts from mice with homozygous germline deletion of PKCe (PKC epsilon-K0). METHODS Cx43 phosphorylation and distribution were measured by quantitative immunoblotting and confocal microscopy. Changes in electrical coupling were monitored using the 4-electrode technique to measure whole-tissue resistivity. RESULTS The amount of Cx43 located in gap junctions, measured by confocat microscopy under basal conditions, was significantly greater in PKC epsilon-K0 hearts compared with wild-type, but total Cx43 content measured by immunobtotting was not different. These unanticipated results indicate that PKC epsilon regulates subcellular distribution of Cx43 under normal conditions. Preconditioning prevented toss of Cx43 from gap junctions during ischemia in wildtype but not PKC epsilon-K0 hearts. Specific activation of PKC epsilon, but not PKC delta, also prevented ischemia-induced loss of Cx43 from gap junctions. Preconditioning delayed the onset of uncoupling in wild-type but hastened uncoupling in PKC epsilon-K0) hearts. Cx43 phosphorylation at the PKC site Ser368 increased 5-fold after ischemia in wild-type hearts, and surprisingly, by nearly 10-fold in PKC epsilon-K0 hearts. Preconditioning prevented phosphorylation of Cx43 in gap junction plaques at Ser368 in wild-type but not PKC epsilon-K0 hearts. CONCLUSION Taken together, these results indicate that PKCe plays a critical role in preconditioning to preserve Cx43 signal in gap junctions and delay electrical uncoupling during ischemia.