Cardiac Angiotensin: Hypertrophy and Failure

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DESCRIPTION (the applicant's description verbatim): The angiotensin II type 2 (AT2) receptor predominates in the left ventricle (LV) in hypertrophy and heart failure, and AT2 receptor activation suppresses growth and promotes apoptosis in vitro. This project will test the overall hypothesis that AT2 receptor signaling in vivo mediates anti-growth and pro-apoptotic effects in pressure overload hypertrophy. We will use transgenic mice with ventricular targeted overexpression of the AT2 receptor driven by the MLC2V promoter which are subjected to ascending aortic stenosis. Functional consequences will be studied by echocardiography and hemodynamic measurements, analysis of isolated myocyte contraction and intracellular ion regulation using fluorescence video microscopy, and confocal microscopy analysis of in situ cell morphology and apoptosis. Signaling pathways will be studied by immunohistochemistry and immunoblotting using antibodies to specific signaling molecules and identification of phosphorylation state. The Specific Aims are: Aim 1. To determine if AT2 receptor overexpression in transgenic mice suppresses in vivo hypertrophic growth in response to chronic systolic pressure overload from aortic stenosis. We predict that AT2 receptor overexpression severely depresses the development of LV hypertrophy, and promotes the rapid development of heart failure and premature death. Aim 2. To test the hypothesis that AT2 receptor overexpression promotes myocyte apoptosis in mice with pressure overload. Apoptosis will be identified by in situ Tunel and ligase assays using confocal microscopy, and complementary measurement of cytochrome c leakage to the cytosol. Aim 3. To determine if AT2 receptor overexpression in vivo modifies myocyte contractile function, and interferes with Ang II mediated inotropy. These experiments will employ measurements of contractility as well as intracellular pH and Ca2+ in isolated mouse myocytes, and test the hypothesis that AT2 receptor activation suppresses the coupling of Ang II with forward Na+-H+ exchange. Aim 4. To determine if AT2 receptor overexpression in vivo activates the kinin-cGMP pathway. In vitro studies suggest that this system contributes to AT2 receptor signaling, but its contribution, if any, in the adult heart is not understood. Measurements will be made of cGMP levels and kininogenase activation in LV tissues (and atrial tissues in which the transgene is minimally expressed). In addition, the functional effects of inhibition of this pathway on cardiac growth and hemodynamic performance will be tested in vivo in mice with AT2 overexpression, in presence and absence of pressure overload. These integrated molecular physiology studies, which examine in vivo and cellular cardiac physiology, will provide new insights regarding cardioprotective versus deleterious effects of AT2 receptor activation in hypertrophy and heart failure.