Hydrolysis of phosphatidylinositol 4,5 bisphosphate (PIP2) by phosphatidylinositol specific phospholipase C (PLC) is a central process in hormone regulated signal transduction. We, and others, recently identified a novel, multifunctional PLC, PLCe, regulated by both Ras related GTPases and heterotrimeric GTP binding proteins and thus, potentially downstream of both G protein coupled and growth factor receptors. Despite extensive knowledge of potential regulators of PLCe based on biochemical and overexpression experiments, the true pathways involving PLCe that function in vivo remain undefined. In the previous funding period we created a mouse model with targeted deletion of the PLCe gene. Our exciting central finding from analysis of the phenotype of these mice is that PLCe activity plays a critical role in the PAR signaling pathway in cardiac myocytes critical for supporting ionotropic responses to PAR stimulation. Analyzing this pathway in a true physiological context will allow us to understand signaling properties of this pathway relevant to human disease.
The specific aims are 1) Defining roles for a novel Epac/ PLCe pathway in cardiac function. The proposed experiments are designed to understand the significance of an additional pAR-dependent pathway in cardiac regulation. 2) Examination of the mechanisms for Epac/PLCe regulation of cardiac calcium induced calcium release. We have developed preliminary data supporting a mechanism where Epac-PLCe regulates Ca2+ transients by, at least in part, regulating activity of L-type Ca2+ channels through PKC dependent phosphorylation. 3) Roles for PLCe P Pz hydrolysis and RapGEF activities in cardiac myocytes. Our hypothesis is that PIP2 hydrolysis activity regulates Ca2+ transients but that the RapGEF activity may potentiate this pathway and feed into other critical cardiac signaling pathways that may also be relevant to other physiological systems. 4) Determining if PLCe expression directly in cardiac myocytes protects against development of cardiac hypertrophy. Our preliminary data indicate that treated PLCe -/- animals have increased sensitivity to chronic isoproterenol induced hypertrophy. Here we will measure development of hypertrophy in mice with cardiac specific deletion or overexpression of PLCe to determine if PLCe activity directly in myocytes is cardioprotective. These studies will elucidate a novel mechanism for action of a critical pathway in the cardiovascular system, the p-adrenergic system, p-adrenergic receptors regulate critical cardiac, vascular and pulmonary function. Elucidation of the functions and mechanisms of this novel Epac/PLCe pathway could lead to novel therapeutic strategies for heart failure, pulmonary disease and hypertension.
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