The long range goals of the proposed research are to elucidate the molecular mechanisms by which catecholamine stimulation enhances contractility and hence the work output of the mammalian heart. The hypotheses to be tested are that both alpha1- and beta-adrenergic receptors are coupled to known second messenger pathways involving cyclic AMP, inositol 1,4,5-triphosphate, calcium and diacylglycerol, which mediate direct modifications (e.e. phosphorylation) of the contractile apparatus and its associated calcium regulatory system. Previous investigations have concentrated on adrenergic mechanisms related to excitability of cardiac cell membranes. The proposed experiments make use of mechanical measurements of single isolated cardiac muscle experiments make use of mechanical measurements of single isolated cardiac muscle cells (myocytes) in conjunction with a novel photochemical technique for generating ATP, calcium, inositol 1,4,5-triphosphate and cyclic AMP rapidly and uniformly within the cell. This permits rigorous control of second messenger concentrations in skinned and living myocytes as well as time-resolved measurements of cross-bridge cycling and the activation of contraction by calcium in skinned myocytes. Analysis of single myocytes by gel electrophoresis will be used to identify specific phosphorylations of contractile and regulatory proteins and to characterize the isozyme composition of contractile proteins. The principal objectives of the proposed research are to identify the rate limiting steps of contraction in a well-defined cardiac muscle preparation and to understand mechanisms of adrenergic regulation of these steps. Information obtained from the proposed measurements may lead to therapeutic approaches for enhancing myocardial contractility during heart failure and will provide a basis for understanding changes in cardiac performance in chronic disease conditions such as hypertension, diabetes and hypothyroidism.
