The long range goals of the proposed research are to elucidate molecular mechanisms by which agonists coupled to phosphoinositide hydrolysis influence cross-bridge function in the mammalian heart. Two general approaches are proposed: 1) dissection of intact signalling pathways to identify differences in second messenger cascades activated by different agonists, and 2) evaluation of the effects of protein kinases on cross- bridge kinetics in skinned myocytes. Initial experiments are designed to characterize intracellular biochemical changes that occur on exposure of cardiac myocytes to receptor agonists, particularly those that stimulate phosphoinositide hydrolysis. Elevation of specific lipids, translocation of protein kinase C isoforms and phosphorylation of contractile proteins will be assessed in response to agonists that cause unique changes in the twitch amplitude and twitch time course of electrically paced ventricular myocytes. Effects of selective protein kinase inhibitors on physiological responses to agonists will also determined. Protein kinase C will then be activated within living myocytes in a controlled way by photorelease of diacylglycerols. The time course and diacylglycerol dose dependence of inotropy and myofilament protein phosphorylation will be established. Emphasis will be placed on the potential for certain species of diacyglcyerol to selectively activate protein kinase C isoforms either directly or as a result of diacylglycerol metabolism. Skinned myocytes will be treated with protein kinases A, c-alpha, C-alpha or myosin light chain kinase, then several active cross-bridge processes will be measured including activation and relaxation rates, and the kinetics of dissociation of Pi and ADP from the cross-bridge. Results from these experiments should provide unique insights into the roles of putative second messengers, protein kinases, and their target proteins in mediating the effects of surface receptor stimulation on cross-bridge kinetics. Thus, this project directly addresses specific objective 2, 3, and 4 of the overall program.
Showing the most recent 10 out of 103 publications
