It is becoming increasingly apparent that autocrine/paracrine factor mediated activation of cardiomyocyte G-protein coupled receptors may contribute in various ways to myocardial adaptation to hemodynamic stress. Recent in vitro and in vivo studies in our laboratories have demonstrated that signaling through Gq-coupled pathways can lead both to cardiac hypertrophy and to heart failure. The critical signal transducers of hypertrophy and heart failure mediated through Gq-coupled pathways are not known, but associations between hypertrophy/failure phenotypes and protein kinase C (PKC) activation suggest a role for this family of kinases. Cardiac tissue contains multiple different isoforms of PKC, each with its own unique pattern of stimulus recognition, subcellular translocation and substrate specificity. We therefore hypothesize that different cardiac PKC isoenzymes play distinct roles in cardiac developmental and adaptive responses. We will examine this hypothesis by selectively activating or inhibiting individual endogenous PKC isoforms through expression of peptides corresponding to their respective RACK binding or pseudo-PACK domains.
In Specific Aim #1, cultured immortalized HL-1 mouse atrial cardiomyocytes expressing different PKC isoform activating or inhibiting peptides will be studied to identify signal transduction pathways and areas of PKC isoform crosstalk.
In specific Aim #2 we will construct transgenic mice with cardiomyocyte- specific expression of PKC alpha/beta, delta or epsilon antagonist peptides to determine the consequences of loss of function for these endogenous PKC isoforms in pressure overload or genetic models of hypertrophy.
In Specific Aim #3 a gain of function approach will be employed by cardiomyocyte-specific expression of PKC alpha/beta, delta or epsilon activating peptides, for analysis of the biochemical, phenotypic and functional consequences of selective PKC isoform activation in the heart. Finally, in Specific Aim #4 we will measure the expression of Galphaq, phospholipase C and PKC isoforms in normal, non-failing hypertrophied, and failing human hearts. Together, these studies constitute the first detailed and comprehensive examination of PKC isoform signaling in the in vivo heart, and should contribute to a greater understanding of PKC activation in cardiac adaptation and maladaptation.
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