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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL071781-02
Application #
6790648
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Buxton, Denis B
Project Start
2003-09-01
Project End
2007-08-31
Budget Start
2004-09-01
Budget End
2005-08-31
Support Year
2
Fiscal Year
2004
Total Cost
$219,000
Indirect Cost
Name
University of Tennessee Health Science Center
Department
Pharmacology
Type
Schools of Medicine
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
Thangavel, Muthusamy; Liu, Xiaoqiu; Sun, Shu Qiang et al. (2009) The C1 and C2 domains target human type 6 adenylyl cyclase to lipid rafts and caveolae. Cell Signal 21:301-8
Liu, Xiaoqiu; Thangavel, Muthusamy; Sun, Shu Qiang et al. (2008) Adenylyl cyclase type 6 overexpression selectively enhances beta-adrenergic and prostacyclin receptor-mediated inhibition of cardiac fibroblast function because of colocalization in lipid rafts. Naunyn Schmiedebergs Arch Pharmacol 377:359-69
Liu, Xiaoqiu; Sun, Shu Qiang; Hassid, Aviv et al. (2006) cAMP inhibits transforming growth factor-beta-stimulated collagen synthesis via inhibition of extracellular signal-regulated kinase 1/2 and Smad signaling in cardiac fibroblasts. Mol Pharmacol 70:1992-2003
Insel, Paul A; Head, Brian P; Ostrom, Rennolds S et al. (2005) Caveolae and lipid rafts: G protein-coupled receptor signaling microdomains in cardiac myocytes. Ann N Y Acad Sci 1047:166-72
Liu, Xiaoqiu; Sun, Shu Qiang; Ostrom, Rennolds S (2005) Fibrotic lung fibroblasts show blunted inhibition by cAMP due to deficient cAMP response element-binding protein phosphorylation. J Pharmacol Exp Ther 315:678-87
Ostrom, Rennolds S; Bundey, Richard A; Insel, Paul A (2004) Nitric oxide inhibition of adenylyl cyclase type 6 activity is dependent upon lipid rafts and caveolin signaling complexes. J Biol Chem 279:19846-53
Ostrom, Rennolds S; Insel, Paul A (2004) The evolving role of lipid rafts and caveolae in G protein-coupled receptor signaling: implications for molecular pharmacology. Br J Pharmacol 143:235-45