Activation of PKC‐ε occurs upstream of mitoK ATP channel activation in the mechanism of diazoxide‐induced cardioprotection

Abstract

In this study, we examined which isoforms of protein kinase C (PKC) family are modulated by diazoxide (DZX) and evaluated their roles in hypoxia-induced apoptotic cell death mediated via mitochondrial death pathway. We further investigated the interrelationship between PKC isoforms and the mitochondrial ATP-sensitive potassium (mitoKATP) channel in the mechanism of cardioprotection by DZX in rat heart-derived H9c2 cell lines and neonatal rat cardiomyocytes. Treatment with 100 μM DZX induced isoform-specific translocation of PKC-ε from the cytosolic to the mitochondrial fraction in both H9c2 and neonatal cardiomyocytes. A specific blockade of PKC-ε translocation by εV1-2 peptide abrogated the anti-apoptotic effect of DZX, suggesting an essential role of PKC-ε in cardioprotection. DZX-induced flavoprotein oxidation was completely inhibited by 10 μM εV1-2, and DZX-induced translocation of PKC-ε remained unaltered after treatment with 100 μM 5-hydroxydecanoate (5-HD). A direct PKC activation by phorbol ester mimicked flavoprotein-oxidizing effect of DZX, and this effect was completely blocked by εV1-2 or 5-HD. During hypoxia, the treatment with 100 μM DZX prevented the increase in mitochondrial Ca2+, mitochondrial depolarization and cytochrome c release induced by hypoxia, and all these effects of DZX were almost completely blocked by εV1-2 as well as 5-HD. In summary, these results suggest that DZX induces translocation of PKC-ε as an upstream signaling molecule for the mitoKATP channel, leading to cardioprotection through inhibition of mitochondrial death pathway.