HYPOXIA AND THE PHOSPHOINOSITIDE PATHWAY IN THE HEART

Alpha(1)-adrenergic agonist activation of phosphoinositide hydrolysis has been linked to the modulation of myocardial cell contractile function as well as the regulation of growth in the neonatal heart. Recent studies indicate that ventricular myocardium contains at least two alpha(1)-receptor subtypes. The first specific aim of this proposal is to examine alpha(1)-receptor subtype specific activation of phosphoinositide hydrolysis in neonatal and adult cardiac myocytes. A characterization of alpha(1)-receptor subtype modulation of phosphoinositide hydrolysis and activation of protein kinase C will be achieved using several different biochemical techniques. Alpha(1)- receptor subtype modulation of intracellular calcium and pH will be analyzed using quantitative fluorescence microscopy with calcium and pH sensitive dyes. Simultaneous effects on cell motion will be determined using a video edge tracking system. Alpha(1)-receptor subtype actions to modulate action potential parameters will be assessed using electrophysiologic techniques. Alpha(1)-receptor subtype induction of a set of growth related genes which may play a permissive role in the hypertrophic response will be examined using molecular biological techniques. The second specific aim of this proposal is to delineate the cellular actions of thrombin, a coagulation factor which also stimulates phosphoinositide hydrolysis in cardiac myocytes. These studies are particularly significant in view of recent evidence that activation of phosphoinositide hydrolysis by alpha(1)-agonists and thrombin is associated with distinct effects on cytosolic calcium and contractile function. This result suggests that the relationship between activation of phosphoinositide hydrolysis and the pharmacologic or electrophysiologic response is not straightforward and that a comparison of the cellular actions of alpha(1)-agonists and thrombin is likely to provide insights into the biological actions of intracellular second messenger molecules generated via the phosphoinositide signaling pathway. Recent studies indicate that catecholamine actions at an alpha(1)-receptor subtype specifically linked to activation of phosphoinositide hydrolysis are enhanced in hypoxic ventricular myocytes. Thrombin-dependent phosphoinositide hydrolysis also is exaggerated in hypoxic ventricular myocytes. The third specific aim of this proposal is to use biochemical, electrophysiological and molecular techniques to determine the functional significance of the exaggerated phosphoinositide response in the hypoxic myocardium. Studies proposed in this application have far-reaching implications since myocardial cells may be exposed to high levels of catecholamines and/or thrombin during myocardial infarction. Enhanced catecholamine or thrombin responsiveness during acute ischemic coronary events may significantly impact on myocyte contractile function, arrhythmogenesis and survival.