This project's long-term goal is to define the mechanisms by which altered Ca2+ homeostasis contributes to hypoxic injury in cardiac muscle, and to identify beneficial pharmacological mechanisms that can target these mechanisms. Therefore these studies are aimed at obtaining basic information that will also be relevant to myocardial ischemia/reperfusion. The project will test a new hypothesis focused on the cytoprotective mechanisms of diazoxide, an activator of mitochondrial ATP-dependent K+ channels (KATP). Specifically, it is proposed that diazoxide inhibits the reactivation of the sarcolemmal Na+/Ca2+ exchanger (NCX) during reoxygenation, and that this protects against Ca2+ overload and many types of Ca2+-induced injury in cardiac myocytes. ? This central hypothesis will be tested through four specific aims. 1) Determine the mechanism by which the reactive oxygen species (ROS) generated during hypoxia/reoxygenation stimulate NCX activity. 2) Determine whether diazoxide inhibits NCX activity during reoxygenation by decreasing the generation of ROS. 3) Determine whether diazoxide exerts its cytoprotective effects during hypoxia by modulating cytosolic and intra-mitochondrial [Ca2+]. 4) Test the hypothesis that diazoxide can selectively inhibit arrhythmogenic transient inward currents when they are induced by Ca2+ overload and NCX activation during hypoxia/reoxygenation. ? These aims will be investigated in hypoxic guinea-pig and rat cardiac myocytes. Ca2+ homeostasis and NCX activity will be studied using cell imaging, immunocytochemical, and electrophysiological methods. The role of mitochondrial KATP channels will be studied using pharmacological methods and confocal imaging. Two particular strengths of the proposal are that it hypothesizes a novel link between activation of mitochondrial KATP and Ca2+ signaling based on this lab's previous studies, and that our extensive experience studying the NCX should facilitate a definitive test of the hypothesis. ? ?