Intracellular Ca overload is an important pathogenetic factor contributing to cardiac dysfunction and irreversible damage in ischemic and reperfused cardiac muscle. Although it is now well-documented that intracellular free Ca rises during early ischemia and combined inhibition of aerobic and anaerobic metabolism, the complex interaction between the multiple cellular processes responsible for regulating intracellular Ca has made it difficult to identify which processes are specifically affected by metabolic inhibition and lead to the Ca overload state. The objective of this proposal is to study the effects of both selective and combined inhibition of aerobic and anaerobic metabolism on intracellular Ca regulation and excitation-contraction coupling in isolated ventricular myocytes in order to address the following issues: i). the mechanisms causing failure of excitation-contraction coupling during metabolic inhibition, in particular relationships between diastolic and systolic intracellular Ca levels and the development of contracture and depressed contraction, ii). the relationship between these functional changes and cellular high energy phosphates levels, iii). the effects of metabolic inhibition on individual cellular processes involved in the regulation of intracellular Ca (the T and L components of the Ca current, the sarcoplasmic reticulum, Na-Ca exchange, the sarcolemmal Ca pump and mitochondria), using ionic and pharmacologic manipulations to isolate the various processes, and iv). the effects of various sequelae of ischemia (acidosis, lactate accumulation, extracellular K accumulation, lysophosphoglyceride and fatty acid ester accumulation, and extracellular ATP release) on intracellular Ca, contractile function and membrane current and voltage. The studies will be performed on voltage-clamped ventricular myocytes using FURA-2 and INDO-1 to measure intracellular Ca, a video motion detector to monitor cell length and contraction, and a rapid extracellular solution exchange device to facilitate ionic and pharmacologic interventions. Experiments in which membrane potential is uncontrolled and the intracellular environment minimally altered (to study """"""""natural history"""""""") will complement experiments in voltage-clamped internally dialyzed myocytes (to isolate pathophysiologic mechanisms). This comprehensive approach will enhance our understanding of the pathophysiology of abnormal intracellular Ca regulation and contractile failure during metabolic inhibition and may provide insights relevant to the search for rational approaches towards preventing Ca overload in ischemic myocardium.
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