MECHANISMS/CONSEQUENCES OF INTRACELLULAR CA OVERLOAD

University Of California Los Angeles, Los Angeles CA

Investigators

Linked publications & trials

Abstract

(Adapted from the Applicant's Abstract) During acute myocardial ischemia, ion imbalances such as extracellular K+ accumulation cause electrophysiologic abnormalities predisposing the heart to ventricular tachycardia, fibrillation and sudden death. Consistent with the theme of the SCOR, the major objective of Project 4 is to further probe the mechanisms of ion imbalance during acute myocardial ischemia and metabolic inhibition. Recently, the investigators presented evidence that hypoxic K+ loss is secondary to intracellular Na accumulation, which drives net K+ loss as a charge-balancing mechanism (Shivkumar et al., 1997). Since intracellular Na+ accumulation also contributes to reoxygenation and reperfusion injury by promoting intracellular Ca++ overload, this suggests that intracellular Na+ accumulation plays a central pathophysiological role in both ventricular arrhythmias and reperfusion injury. Understanding its pathogenesis is therefore of paramount importance. The investigators recently made the intriguing discovery that metabolic inhibition activates a novel pathway of Na+ influx by causing non-gap junctional connexin43 (Cx43) hemichannels to open (John et al., 1999). A major goal of the renewal is to evaluate the contribution of the connexin hemichannel pathway, in relation to conventional pathways, as a cause of increased Na+ influx during hypoxia and ischemia promoting K+ loss and intracellular Ca++ overload. The proposed studies will employ: intact ventricular muscle (using microelectrode, ion sensitive electrode, fluorescent dye uptake, radioisotopic and metabolic assay techniques), isolated ventricular myocytes (using patch clamp and fluorescent dye techniques), and cultured mammalian cells heterologously expressing various connexins (using patch clamp and fluorescent dye techniques, green fluorescent protein tagging and mutagenesis). Specific objectives are: 1). To determine whether ischemic, like hypoxic, K+ loss is driven by intracellular Na+ accumulation. 2). To determine whether intracellular Na+ accumulation during hypoxia results from increased Na+ influx or decreased Na+ efflux. 3). To determine whether functional connexin hemichannels are present in intact ventricular muscle, or whether they are an artifact of myocyte isolation. 4). To further document conditions under which connexin hemichannels are activated in HEK293 cells and isolated ventricular myocytes by metabolic inhibition, hypoxia or simulated ischemia. 5). To define the signaling pathway(s) by which metabolic inhibition activates connexin hemichannels, with special emphasis on signaling pathways involved in ischemic preconditioning. 6). To delineate the regions of the Cx43 protein which are responsible for hemichannel activation by metabolic inhibition. These studies will promote a better understanding of the pathogenesis of ventricular fibrillation and sudden cardiac death in its most common setting, acute myocardial ischemia, leading to novel therapeutic approaches.