NMR STUDIES OF ISCHEMIC INJURY IN THE PERFUSED HEART

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The uniquely diagnostic and noninvasive advantages of NMR spectroscopy will be employed to investigate the regulation of transarcolemmal cation gradients during ischemia and reperfusion, and their role in cell damage and recovery, in the perfused isovolumic rate heart model. Intracellular Na+ will be continuously monitored with 23Na NMR using the recently developed 23Na NMR shift reagent Tm(DOTP)5-. This unique reagent not only provides excellent resolution of the intracellular Na+ resonance, but is compatible with acquisition of high quality 31P spectra. Taking advantage of this, a specially designed NMR probe will be used to collect interleaved 23Na and 31P NMR spectra on the same preparation. High energy phosphates and intra- and extracellular pH will be monitored from the 31P NMR spectra, using the chemical shifts of inorganic phosphate and phenylphosphate, an extracellular pH marker. Additionally, free intracellular Ca2+ will be measured with 19F NMR by using loading the fluorinated Ca2+ indicator, 5F- BAPTA. These methodologies will ultimately be combined; Na+ and Ca2+ will be measured in the same preparation for the first time by NMR. The specific aims of this project are to determine the mechanisms which alter the sarcolemmal Na+ and Ca2+ gradients during ischemia and reperfusion, and to determine how these two gradients are coupled together, and to the energy supply. The relation of cation homeostasis to functional recovery will be investigated. The importance of glycolytic energy production in maintaining cation gradients during ischemia and reperfusion will be investigated. Two different models of ischemia will be employed, both low- flow and zero-flow global ischemia. The role of Na+/H+ exchange will investigated directly by using the specific inhibitor ethylisopropylamiloride. These issues will be addressed in the hypertensive animal model, the Spontaneously Hypertensive Rat, using age- matched Wistar-Kyoto rats as controls, to investigate the sensitivity of the hypertrophied heart to ischemia. The role of Ca2+ as a mediator of cell damage could be elucidated, leading to a better understanding of myocardial ischemia with regard to possible treatments.