Mitochondrial PKC-ε deficiency promotes I/R-mediated myocardial injury via GSK3β-dependent mitochondrial permeability transition pore opening.

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)-induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase-2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross-clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2 ) and wild-type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin-related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N-acetylcysteine (NAC) or PKC-δ shRNA treatment on glycogen synthase kinase-3β (GSK-3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2 mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC-ε translocation was lower in ALDH2 mice than in WT mice, and PKC-δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre-treatment under I/R injury. In addition, PKC-ε inhibition caused activation of caspase9, caspase3 and Drp1Ser in response to I/R stress. Importantly, expression of phosphorylated GSK-3β (inactive form) was lower in ALDH2 mice than in WT mice, and both were increased by NAC pre-treatment. I/R-induced mitochondrial translocation of GSK-3β was inhibited by PKC-δ shRNA or NAC pre-treatment. In addition, mitochondrial membrane potential (∆Ψ ) was reduced in ALDH2 mice after I/R, which was partly reversed by the GSK-3β inhibitor (SB216763) or PKC-δ shRNA. Collectively, our data provide the evidence that abnormal PKC-ε/PKC-δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK-3β-dependent mPTP opening, which results in mitochondrial injury-triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2 mice following I/R stress.