The proposed program is an integrated study of cellular and molecular processes that regulate contraction and kinetics of contraction of mammalian myocardium, objectives are to determine mechanisms of Ca2+ delivery during excitation and contraction, mechanisms of regulation of mechanical properties, mechanisms of action of intracellular messengers, and mechanisms of the protective effects of adenosine on cardiac function during ischemia. The program involves six sub-projects and four cores. Sub-project 1 will use immunolocalization techniques, gene targeting and electrophysiological methods to study the function of the beta subunit of the dihydropyridine-sensitive Ca2+ channel, sub-project 2 will study regulation of the Ca2+ release channel of sarcoplasmic reticulum. Channel activation and inactivation will be investigated by studying channel function in lipid bi-layers and using toxins from scorpion and gila monster venoms as probes. Sub-project 3 will focus on mechanisms by which adenosine reduces or prevents contractile dysfunction normally associated with post-ischemic, stunned myocardium. An in vivo porcine model of stunning will be used to characterize the effects of adenosine on myocardial function in the working heart, Ca2+ movements in the intact cell, and regulation of cross-bridge interaction. Sub-project 4 will examine mechanisms of regulation of mechanical properties of single skinned myocytes, and will emphasize selective extraction or alteration of proteins thought to be mediators of regulation. Sub-project 5 will investigate the roles of second messengers in modulating contraction in living cardiac myocytes. Mechanisms of action of alpha adrenergic agonists will be investigated by applying putative second messengers, by intracellular release of second messengers from chemically caged precursors, and by modifying activity of selected kinases. Sub-project 6 will use physical chemical, physiological and molecular biological techniques to investigate contributions of the elastic protein titin to tension in passive and actively contracting myocardium. Characterization of titin phosphorylation will explore potentially important mechanisms for regulating mechanical properties. Scientific cores on protein biochemistry and antibody production, caged compounds, and molecular biology provide support to the sub-projects and will facilitate development of new research directions. Our uniquely complementary approaches will yield new information concerning regulation of myocyte function under normal and ischemic conditions, as well as proteins and second messengers that mediate regulation.
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