Our studies in the past 20 years led to a better understanding of the molecular events contributing to cardiac injury by ischemia and reperfusion (I/R) and the role of protein kinase C (PKC) isozymes in this response. We showed that epsilon PKC activation protects from I/R-induced injury whereas activation of delta PKC increases this injury. Our work and recent discoveries by a number of other labs identified the importance of the mitochondria in the health of the myocardium. The mitochondria are critical for cardiac metabolism and energetics as well as for cell death through apoptosis and necrosis. Because we found a critical role for delta PKC-mediated mitochondrial regulation in I/R response, our current proposal is focused on elucidating the role of delta PKC in mitochondrial structure and functions following I/R and under basal conditions. Specifically, we plan Aim 1: Determine the role of delta PKC phosphorylation of the mitochondrial matrix enzyme, pyruvate dehydrogenase kinase, in inhibition of ATP regeneration Aim 2: Determine the role of delta PKC phosphorylation of dynamin-related protein 1 on mitochondrial fission Aim 3: Determine the role of delta PKC in elimination of damaged mitochondria Aim 4: Determine how delta PKC translocates onto the mitochondria, how delta PKC is imported into the mitochondrial matrix and whether I/R triggers activation of delta PKC that is different from hormone activation. Our hypothesis is that differen delta PKC substrates are critical at different stages of reperfusion injury. We previously showed that, when given right at reperfusion, a peptide inhibitor of all the phosphorylation events mediated by delta PKC that we have designed, reduced reperfusion injury by 50-70%, as measured in isolated cardiac myocytes, and in rat, mouse and pig models of myocardial infarction. However, what the role of each delta PKC substrate is in I/R and when its activity is critical is not known. Using a rational approach that we have developed, we will generate new separation-of-function inhibitors of delta PKC that will selectively inhibit phosphorylation of one substrate of delta PKC without affecting the phosphorylation of the others. These separation-of-function inhibitors of delta PKC will be used in isolated myocytes, in Langendorff preparations and in vivo, at reperfusion and during the first few weeks after MI to address the above aims. Focusing on the molecular events related to mitochondrial fate should elucidate fundamental mechanisms in cell biology, in general, and in the health of the myocardium, in particular. Further, this work will likely identify novel targets for therapeutic interventions for myocardial infarction patients.