The applicant proposes to study the b-adrenergic receptor-stimulatory GTP binding protein-adenylyl cyclase (AC) pathway in heart failure. b-adrenergic agonist stimulation increases intracellular cAMP in cardiac myocytes, activating protein kinase A which augments contractile function. In heart failure, despite increased sympathetic drive, cardiac function remains depressed. It has been established that altered b-receptor mediated signalling occurs in heart failure; changes include a reduction in myocardial b-receptor-stimulated cAMP production and decreased b1- receptor number. Nevertheless, there are two areas, potentially quite important in the pathogenesis of heart failure, that have received little attention: 1) the importance of AC content and catalytic function during the development of heart failure; and 2) the role of GTP binding receptor kinase (GRK) in myocardial b-receptor desensitization and the temporal regulation of their expression in the failing heart. The applicant proposes first to determine the alterations in AC isoforms and G protein receptor kinase expression during the development of heart failure. These studies will provide information regarding the specific abnormalities underlying b-receptor desensitization in heart failure, and lay the foundation for subsequent studies designed to determine mechanisms for these changes. The applicant will examine the role of AC isoform and GRK in b-receptor mediated signalling by altering their gene expression in cardiac myocytes in vitro and in vivo. Using an animal model of dilated systolic heart failure, the applicant will isolate cardiac myocytes from the left ventricle, and apply biochemical and molecular approaches to define and altered adrenergic signalling. The proposed studies will define the manner in which elements of b-receptor signalling are altered during the development of heart failure and will establish the importance of these elements in regulating b-receptor mediated cAMP responsiveness in cardiac myocytes. The applicant proposes to study the mechanism for altered myocardial b-receptor signalling transduction in heart failure by examining the regulatory mechanisms of two of the key elements of the b-receptor signalling pathway, AC and GRK. The applicant will combine molecular biology and integrated physiology approaches to define the roles of AC and GRK for b-receptor desensitization in vitro and in vivo.
