Although there is a clear relationship between the duration of myocardial ischemia and the extent of contractile failure and cell injury upon reperfusion, the cellular basis of these abnormalities is not well understood. Intracellular calcium (Ca2+) overload and abnormal intracellular Ca2+ handling are thought to underlie the contractile abnormalities and cell damage associated with reperfusion injury. However, the causes of altered Ca2+ homeostasis are unknown. One possible explanation is that potentially injurious compounds, ordinarily absent or at extremely low levels in the cellular environment under physiologic conditions, increase to toxic concentrations during ischemia and reperfusion. Three classes of compounds are of particular interest in this regard: oxygen derived free radicals, inflammatory cytokines, and amphiphiles. Agents from each of these classes have been found to accumulate in cardiac tissue during ischemia and reperfusion, and each has been implicated in the pathogenesis of abnormal Ca2+ handling and altered excitation-contraction coupling. The objective of this proposal is to investigate the mechanisms whereby these mediators of cardiac injury disrupt excitation-contraction coupling, with particular emphasis on the individual components of Ca2+ regulation. Experiments will be performed on patch clamped single ventricular myocytes from rabbits loaded with the Ca2+ sensitive fluorescent indicator FURA-2. Alterations in intracellular Ca2+ will be correlated with membrane current and voltage, as well as cell edge motion using a video motion detector. A rapid extracellular solution exchange device will facilitate ionic and pharmacologic interventions. The individual components of Ca2+ regulation to be studied in this fashion will include the Ca2+ current (both T and L components), sarcoplasmic reticulum, sodium-calcium exchanger, sarcolemmal Ca2+ ATPase, and mitochondria. Sodium-calcium exchange will also be studied using caged Ca2+ and, in other experiments, the giant excised patch technique in xenopus oocytes. We will also investigate the potential roles of Ca2+ leak channels in altered intracellular Ca2+ homeostasis. We believe these studies will improve our understanding of the cellular mechanisms of reperfusion injury and may also provide a basis for developing new strategies aimed at limiting contractile failure and irreversible injury following reperfusion in the setting of myocardial infarction.
