The main objective is to investigate the role of cardiac phosphoproteins in the modulation of cardiac function and excitability. These studies are proposed to test the hypotheses that cardiac phosphoproteins modulate cardiac function by mediating changes in calcium and potassium permeability as well as in the force and rate of contraction and that the phosphoproteins serve as the locus of interaction between beta-adrenergic and muscarinic cholinergic inputs to the heart. The proposed studies will use monolayer heart cells cultured from embryonic chick heart, which provide the advantage of permitting measurement at intervals as short as five seconds and hence the ability to obtain accurate time course analysis. The specific goals of these studies are to determine whether: 1) a subunit of the cardiac calcium channel becomes phosphorylated in response to isoproterenol and mediates the increase in calcium influx (measured as 45Ca++ influx rate) and contractility; 2) a close correlation exists between the time course of appearance of the phosphorylation of calcium channel subunit and other phosphoproteins (protein C and phospholamban) and that of development of the respective physiologic responses and whether a close correlation also exists between the time course of reversal of the phosphorylation of these proteins and that of reversal of the respective physiologic responses; 3) the muscarinic agonist carbachol antagonizes isoproterenol-stimulated physiologic responses by dephosphorylating the calcium channel subunit, phospholamban and protein C; 4) the coupling of muscarinic receptor to the inhibition of cAMP accumulation and the inhibition of calcium channel phosphorylation differs between the atria and the ventricle and whether the physiologic effects caused by the inhibition of cAMP formation differ between the atria and the ventricle; 5) one of the cardiac physiologic functions of protein kinase C is to modulate the physiologic responses to beta-adrenergic and muscarinic cholinergic agonists by phosphorylating its respective receptors or its respective GTP-binding proteins. These studies should enhance our understanding of the molecular basis of cholinergic-adrenergic interaction in modulating the inotropic and chronotropic state in the heart and should provide insights into the events important in predisposing to brady-and tachyarrhythmia.
