Targeted Inhibition Of Mitochondrial Calpain Reduces Myocardial Ischemia/reperfusion Injury

Objective:To determine whether and how targeted inhibition of mitochondrial calpain prevents myocardial ischemia-reperfusion(I/R)injury.Methods:Cultured H9c2 cells were infected with adenoviral vectors containing mitochondrial-targeted calpastatin(Ad-mt CAST),ATP synthase ? subunit(ATP5A1,Ad-ATP5A1)or beta-gal(Ad-gal).Twenty-four hours after infection,the cells were subjected to hypoxia for 24 hours followed by rexygenation for another 24 hours(H/R).For global I/R injury,isolated hearts were perfused and subjected to ischemia(stopping perfusion)for 60 minutes followed by reperfusion for 30 minutes.For in vivo I/R,myocardial ischemia was achieved by left coronary artery ligation for 30 minutes and the perfusion was re-constructed by releasing the suture.For both global and in vivo I/R,both transgenic mice with over-expression of mitochondrial-targeted calpastatin(Tg-mt CAST)and their wild-type(WT)littermates were randomly allocated into the following four groups: WT control group,WT I/R group,Tg-mt CAST control group and Tg-mt CAST I/R group.Apoptosis,cell viability,necrosis,ATP5A1 protein levels,mitochondrial calpain-1 protein levels,mitochondrial reactive oxygen species(ROS)generation,ATP synthase activity and myocardial function were determined.Results:In cultured H9c2 cardiomyocytes,H/R increased the protein levels of calpain-1 and ROS generation in mitochondria,induced apoptosis as determined by caspase-3 activation and DNA fragmentation,and decreased cell viability.These adverse effects of H/R were attenuated by over-expression of mitochondrial-targeted calpastatin.Mechanistically,the elevation of calpain-1 in mitochondria was associated with a reduction of ATP5A1 protein and ATP synthase activity in H/R-stimulated cardiomyocytes,which could be reversed by up-regulation of mitochondrial-targeted calpastatin.Furthermore,over-expression of ATP5A1 prevented apoptosis and increased cell viability in H/R-treated cardiomyocytes.In mouse I/R model,Tg-mt CAST/t TA mice exhibited less cell death in hearts and improved myocardial function 7 days post I/R as compared to their wild-type littermates.This protective effect of over-expression of mitochondrial-targeted calpastatin was also demonstrated in perfused whole hearts subjected to global I/R.Conclusions:We have provided the evidence that selective over-expression of mitochondrial-targeted calpastatin reduces myocardial I/R injury at least in part by preventing ATP5A1 activity and ROS generation.Thus,targeted inhibition of mitochondrial-targeted calpain may be a new strategy to attentuate I/R injury in hearts.