Protein kinase C (PKC) has been implicated in a number of cardiac functions, including regulation of the strength and rate of contraction and protection from hypoxia-induced cell death. We found that there are at least six different PKC isozymes in neonatal heart. These isozymes share long stretches of sequence homologies. In addition, each isozyme has unique sequences that are quite well conserved in evolution. It is therefore expected that individual PKC isozymes play specific roles in cardiac function and that the isozyme-unique sequences mediate this specificity. Our research has focused on identifying PKC isozyme-selective inhibitors. We have capitalized on observations that individual isozymes translocate to different subcellular sites following activation. We suggested that this isozyme-selective subcellular localization reflects the association of individual isozymes with their selective anchoring proteins, and that inhibition of isozyme translocation selectively inhibits the function mediated by each isozyme. To this end, we have designed translocation inhibitors that interfere with the association of each PKC isozyme and its selective anchoring protein. Recently, we have also identified activators of individual isozymes. We now have selective inhibitors for all the isozymes present in heart and selective activators for some of them. With the exception of one inhibitor, all were developed in our laboratory. In this proposal, we plan to apply these novel tools to identify the role of different PKC isozymes in cardiac functions that are altered in specific disease states. We will test a new working hypothesis that individual PKC isozymes have opposing effects in cardiac function. If correct, it may explain the current controversy in the field. Using isolated adult cells, intact heart and whole animal studies, we plan to determine the role of PKC isozymes in the response of cardiac myocytes and fibroblasts to hypoxia (AIM A) and in the regulation of chronotrophy (AIM B). We will also determine the role of PKC isozymes in the regulation of proliferation of cardiac fibroblasts (AIM C). Our published and preliminary data demonstrate the feasibility of these studies as well as our substantial progress in sorting out these complex events. Our overall goal is to relate these findings to human disease; identification of the individual PKC isozyme whose activity is required and sufficient to induce the tested cardiac function may reveal novel targets for therapy of heart diseases.
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