Adenylyl cyclase as a regulator of cardiac fibroblasts

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DESCRIPTION (provided by applicant): This application is designed to test the hypothesis that increased expression of adenylyl cyclase (AC, the enzyme that generates the second messenger, cAMP) in cardiac fibroblasts can limit cardiac fibrosis in heart disease. I and others have found that the expression level of AC limits the maximal generation of cAMP elicited by G protein-coupled receptor (GPCR) agonists. Therefore, increased expression of this enzyme enhances the response to hormones that regulate AC activity. Preliminary data demonstrate that agents which increase cellular levels of cAMP in cardiac fibroblasts decrease cell proliferation and collagen synthesis (two key cellular functions that contribute to cardiac fibrosis). Moreover, gene transfer of AC enhances this inhibition. The Aims of this proposal will characterize these effects in primary cultures of cardiac fibroblasts in which three different AC isoforms are overexpressed. The knowledge gained from cellular studies will then be extended to studies of an animal model of heart failure to determine if increased AC expression can attenuate the development of cardiac fibrosis and thereby improve cardiac function. However, my recent studies of cardiac myocytes and fibroblasts indicate that overexpression of a particular isoform of AC, AC6, increases the maximal cAMP generated by activation of beta-adrenergic receptors (betaAR) but does not enhance basal levels of cAMP or that stimulated by agents which activate other GPCR coupled to Gs (e.g. prostanoid receptors). In cardiac myocytes, this selective effect of AC6 overexpression is due to co-localization of betaAR and AC in caveolae - a microdomaln of the plasma membrane that excludes prostanoid receptors. Therefore, AC expression is highly compartmentalized in cardiac myocytes and co-localization of receptor and effector is essential for efficient signal transduction. In contrast, other studies I have conducted in vascular smooth muscle cells indicate that betaAR and AC are not highly compartmentalized in caveolae. Therefore, it is critical to characterize the co-localization of GPCR and AC in cardiac fibroblasts in order to define an AC isoform overexpression strategy that most efficaciously enhances the anti-fibrotic effect of endogenous hormones. The specific aims will assess the impact of overexpression of AC3, 4 and 6 on cAMP formation and extracellular matrix production/degradation, determine the functional coupling and compartmentation of endogenous hormonal receptors with these AC isoforms, and target AC transgene overexpression to cardiac fibroblasts of mice and assess if this treatment decreases cardiac fibrosis in an experimental model of heart failure.