The molecular events that are activated during the response of the heart to reperfusion following ischemia have been the topic of much research. Our laboratory focused on the role for protein kinase C (PKC) in cardiac ischemia. We developed unique pharmacological tools, which provided new insights into the role of several pathways in cardiac reperfusion injury. Some involve more expected players, such as key metabolic enzymes or regulators of mitochondrial dynamics. Others identified unexpected players in the response to reperfusion injury. In this proposal, we describe our plans to investigate our recent finding that a component of the thin filament in the contractile apparatus, cardiac troponin I (cTnI), participates in the acute response to ischemia. A role for cTnI in regulating cardiac contractility is well described, but a role of cTnI in the acute response to ischemia is less expected. We showed that a brief and selective inhibition of ?PKC-mediated phosphorylation of cTnI at the onset of reperfusion is sufficient to greatly inhibit acute reperfusion injury in models of myocardial infarction. Our first hypothesis is that phosphorylated cTnI induces injurious response following ischemia, through a mechanism that may be independent of cTnI's role in contractility. The finding that mutations in cTnI result in severe cardiomyopathy and high incidence of sudden death in humans led to our second hypothesis, that at least some of the 74 mutations in cardiac cTnI that are associated with cardiomyopathy are the result of the novel role of cTnI in the response to ischemia that we discovered. These hypotheses are tested as follows: In the first aim, we will identify the signaling event that is transmitted by cTnI phosphorylation during reperfusion, thus contributing to cardiac injury. To this end, we will use live-cell imaging to determine the timing of cTnI-induced injury, a proteomic study to identify the cTnI interactome and a rational design of protein- protein interaction (PPI) inhibitors to confirm the role of the pathway identified by this study. In the second aim, we will determine if human cTnI mutations leading to CM affect the response to ischemia and reperfusion (IR). To this end, we will generate a structure-function relationship map of human cTnI using population-scale genetic variation data to identify mutation clusters on the cTnI surface that represent different functions of cTnI. We will determine the effect of representative mutations from each mutation cluster on the response to IR- induced injury in cardiac cell lines, adult cardiomyocytes and in transgenic mice. Finally, using iPSC-derived cardiomyocytes, we will determine if at least some of the human CM-associated cTnI mutations increase the sensitivity to ischemic insult by the molecular mechanism identified in Aim 1 and if the novel PPI inhibitors that we develop affect excessive reperfusion injury mediated by the mutations. Together, our work will characterize a new pathway by which reperfusion injury occurs, its potential role in cTnl-associated cardiomyopathy and possibly, a therapeutic approach to prevent or slow down cardiomyopathy in at least some of the patients with cTnI mutations.
Protein kinase C delta is a key mediator of cardiac injury following myocardial infarction. We focus on developing novel pharmacological agents using cardiac cell lines, transgenic mice, patient-derived cardiac cells and a large human genetic data set to identify the mechanism by which the thin filament component in the contractile apparatus, troponin I, contributes to cardiac injury following ischemia and how it may contribute to some genetic forms of cardiomyopathy.
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