The objective of this proposal is to study the ionic derangements of acute myocardial ischemia -- extracellular K+ accumulation, and intracellular calcium overload -- and to explore the role which these abnormalities play in the electrophysiological manifestations of ischemia. Fluorescent indicators will be used throughout the proposal to monitor membrane potential, cytosolic calcium, and extracellular K+ in rabbit hearts during normal beating and ischemia. Apparatus will be designed to permit simultaneous acquisition of pairs of signals from the same region of the heart. Accumulation of extracellular K+, for example, will be compared with changes in the resting and action potential recorded with the potentiometric dye, di-4-ANEPPS. Particular emphasis will be placed on determining whether the pattern of intracellular calcium alternans is concordant with alternans of action potential duration during ischemia, and whether the concordance is preserved when two different regions of myocardium exhibit alternans that is out of phase. This determination is important in showing whether calcium alternans plays a causal role in the genesis of membrane potential changes that lead to ventricular fibrillation. A second major focus of the project will be the deliberate modulation of [Ca++]i in intact hearts using the photolabile calcium chelator Nitr-5. Flash photolysis of Nitr-5 will be performed before and during ischemia to determine whether the ability of the myocytes to sequester calcium is impaired. Photolysis during ischemia may provoke calcium alternans, and attempts will be made to provoke alternans under several conditions which favor its development. Finally, confocal laser scanning microscopy will be used to obtain fluorescent [Ca++]i images from intact hearts before and during ischemia. Scanning will be gated to avoid movement artifact. Partial images can be acquired rapidly by reducing the number of raster lines scanned. This will permit the acquisition of temporarily resolved calcium transients in small regions. Major objectives are to determine whether the [Ca++]i increase during ischemia originates in the cytoplasm of cardiac myocytes, or whether other sources such as endothelial cells and mitochondria are important. These observations are an important step in understanding the early [Ca++]i increase that has been observed in the intact ischemic heart.
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