Despite the key role of intracellular Ca (Ca) in the pathogenesis of ischemic and reperfusion injury to the myocardium and coronary vasculature, the effects of metabolic inhibition and other components of the ischemic environment on the complex interaction between the multiple cellular processes responsible for regulating Ca is not well-understood. The objectives of this continuation proposal are to further characterize the subcellular mechanisms by which metabolic inhibition and components of the ischemic environment alter Ca regulation in ventricular myocytes and vascular endothelial cells. In ventricular myocytes, Ca, (using fura-2), membrane current and voltage and cell shortening will be monitored simultaneously under whole cell patch clamp conditions during exposure to combined or selective inhibition of glycolysis and oxidative metabolism, and to various components of the selective inhibition of glycolysis and oxidative metabolism, and to various components of the ischemic environment. Using appropriate pharmacologic interventions and ionic substitutions, the effects of these interventions and ionic substitutions, the effects of these interventions on individual components of cardiac excitation-contraction coupling will be characterized in detail. Studies will also be performed in giant excised membrane patches to assess Na-Ca exchange and Na-K pump function, and in permeabilized myocytes to test whether glycolysis plays a preferential role in supporting SR function. In vascular endothelial cell monolayers, Ca (Using fura-2 imaging), membrane potential and membrane resistance will be monitored simultaneously under whole cell patch clamp conditions to characterize: 1) cell signalling pathways which couple receptor binding of endothelium-dependent vasodilators (e.g. bradykinin, ACh, histamine, etc.) to increases in Ca associated with release of EDRF, ii) the effects of combined and selective metabolic inhibition, components of ischemia and cytokines on these cell signalling pathways, and iii) the effects of these interventions on cell-to-cell propagation of Ca wavers in response to locally-applied endothelium-dependent vasodilator agonists. Experiments in single endothelial cells using whole cell voltage clamp and single channel recordings will be used to elucidate underlying ionic currents involved. The proposed experiments offer a uniquely comprehensive approach to understanding how metabolic factor alter Ca regulation in myocardium and vascular endothelium.
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