Studies in this application are based upon the evidence that protease-activated receptors (PARs) exert profound effects on cardiac myocytes; responses to PARs include robust changes in calcium and the induction of a hypertrophic growth program. Our preliminary data indicate that cardiomyocytes express at least two PARs (PAR-1 and PAR-2) which possesses similar (but not identical) signaling properties and that PARs induces a form of hypertrophy that is morphologically (distinct from that elicited by the prototypical Gq-coupled alpha1-adrenergic receptors. The molecular underpinnings for distinct hypertrophic programs in response to PAR and alpha1-AR activation are not known, but differences in PAR- vs 1alpha-adrenergic receptor-G protein coupling and the activation of several important signaling molecules are identified in the preliminary data which could be contributory. Collectively, these results emphasize that pharmaceuticals targeted to PARs may have important cardiac consequences. This application will address general questions related to mechanisms for PAR action as well as specific questions regarding the expression and detailed coupling/functional properties of individual PARs in cardiomyocytes, an area of research that has largely been neglected.
The specific aims are (I) to investigate PAR linkage to downstream signaling pathways and growth responses; these studies will identify and distinguish the mechanisms for hypertrophic signaling by PARs vs. alpha1-adrenergic receptors, (II) to use newer molecular techniques to delineate the G protein-dependence of individual signaling pathways and define their contribution to hypertrophic signaling by PAR-1, and (III) to use PAR-1 knockout mice to define the functional role of PAR-1 in the integrated response in the intact mouse heart. By spanning cell culture to intact animal models (and employing a range of biochemical/molecular techniques, fluorescence microscopy with calcium-sensitive indicators, and PAR-1 knockout mice) the proposal will elucidate the mechanisms whereby proteases such as thrombin activate cardiomyocytes and resolve clinically relevant issues as whether drugs that are being developed to modulate signaling through PARs are likely to influence the heart under normal conditions or in the context of ischemic injury.
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