Theodore M. Brody - US grants

The overall objective of this project is to understand the mechanisms by which aging pathological conditions or drug interactions alter the sensitivity of heart muscle to therapeutic and toxic effects of the digitalis glycosides. This is important because the digitalis glycosides are still widely used despite their narrow margin of safety, and Ca2 overload is a potential mechanism for cardiotoxicity of digitalis and also many agents and pathological conditions. Understanding the mechanisms by which cellular Ca2 overload causes myocardial dysfunctions and also regulation of sarcolemmal sodium pump under physiological and pathological conditions are essential for understanding of basic biology of the heart muscle. During the proposed project period, following studies will be performed: (1) The hypothesis that reduced Na pump capacity accounts for reduced tolerance of senescent heart to digitalis will be examined. Our previous data suggest that leak Na influx is increased and the number of active Na pumping sites is decreased in the myocardium of senescent Fischer 344 rats. Therefore, passive fluxes of Na, K and Ca2 across the sarcolemma, reserve capacity of the Na pump and possible causes of changes in the number of active Na pump units observed in senescent heart will be examined primarily in myocytes obtained from rat, guinea pig or dog heart. (2) The hypothesis that Ca2 overload of sarcoplasmic reticulum and subsequent uncontrolled Ca2 release are responsible for all phases of digitalis toxicity (contracture and reduction of developed tension, after contractions and oscillatory afterpotentials) will be critically evaluated in atrial muscle and cardiac Purkinje fibers using Ca2 entry blockers, Na/Ca2 exchange inhibitor and ryanodine. (3) Recently, the possible regulation of Na,K-ATPase by Ca2, H and inorganic phosphate has been suggested. Therefore, effects of changes in intracellular concentrations of these ions, ischemia and hypoxia and myocardial hypertrophy on the sarcolemmal Na pump and on glycoside binding to Na,K-ATPase will be examined using viable myocytes. Underlying hypothesis for this part of the project is that Ca2 regulates the Na pump under pathological conditions. Seemingly diverse aims of the proposed project converge into the testing of a unifying hypothesis that myocardial Na pump and its glycoside sensitivity are regulated by Na, K and Ca2 under physiological or pathological conditions, and to assess the biological impact of sodium pump inhibition on myocardial functions or dysfunctions.

The overall objective of this project is to understand the mechanisms of the positive inotropic and toxic actions of the cardiac glycosides. It is now clear that Na pump inhibition is a cause of the positive inotropic and arrhythmogenic effects of the cardiac glycosides; however, the steps, which link Na pump inhibition to the inotropic effect, are not clear. In addition, whether Na,K-ATPase inhibition is the sole mechanism for the positive inotropic effect is unknown. Proposed studies are to evaluate if an enhancement of a putative "phasic" increase in [Na+]i which may occur during membrane depolarization at the inner surface of the sarcolemma is responsible for the enhancement of intracellular Ca2+ transients. Alternatively, a slight "tonic" increase in [Na+]i which is observed during the diastolic phase of the cardiac cycle may enhance the Ca2+ transient which occurs during membrane depolarization. Because the proposed mechanism which links Na pump inhibition to an enhancement of intracellular Ca2+ transients is the Na/Ca exchange reaction, the direction and magnitude of this reaction will be examined in isolated myocytes obtained from guinea pig and rat heart under conditions in which normal Na+, Ca2+ and electrical gradients are maintained. 13-Propylberberine, an agent which is claimed to inhibit adenylate cyclase in other types of tissues, eliminates the positive inotropic effect of ouabain. Thus, we will examine the mechanism by which this effect is brought about. This will define a critical step required for the expression of the inotropic effect. The possible presence of a second "digitalis receptor" which is unrelated to Na,K-ATPase and might have been overlooked because of the lack of appropriate ligands which are required for glycoside binding in cell fragment preparations will be examined using myocytes. Additionally, we will test the hypothesis that Ca2+ pools responsible for the positive inotropic effect of the cardiac glycosides are separate from those involved in Ca2+ overload and digitalis toxicity. Finally, the possible presence of an endogenous ligand for glycoside binding sites on Na,K-ATPase will be examined. These studies are designed to fill the missing links in the present scheme representing our understanding of the positive inotropic and toxic effects of the cardiac glycosides. Because the glycoside produces its positive inotropic effect by increasing the apparent efficiency of excitation-contraction coupling, understanding the mechanism of action of the glycoside will shed light on the coupling mechanism in the cardiac muscle.