The goal of this research is to determine the mechanisms of reversible and irreversible injury of cultured chick and mouse embryonic ventricular cells resulting from ATP depletion. Specifically we will investigate the interaction of ATP depletion (produced by cyanide or hypoxia combined with zero glucose-2 deoxyglucose) and sarcolemmal injury produced by either oxygen- free radicals (derived from xanthine-xanthine oxidase, or activated neutrophils), or exposure to lysophosphatidyl choline. We will study the importance of alterations in (Ca2+)i homeostasis (assessed by the fluorescent Ca2+ probe Indo-1, 45Ca fluxes, and Ca content) in these interactions; and the effects of drugs which alter Ca2+ influx into the cell via the slow Ca2+ channel, and via Na-Ca exchange on cell injury and dysfunction. The role of altered K+ homeostasis in ATP depletion injury, and the effects of drugs which alter K permeability or transport, (42K flux, Na and K contents) will also be investigated. In these experiments, contractility of isolated myocytes is quantitated using a motion detector system, and cell injury will be assessed by LDH release. Cells will be studied during ATP depletion for up to 6 hours, in the presence and absence of a diffusion limited aqueous extra-cellular volume; and after 24 hours to two weeks of recovery in serum- free culture medium. It is anticipated that the proposed experiments will test the hypotheses that ATP depletion combined with exposure to oxygen free radicals or to lysophospholipids, produces more cell injury than exposure to either stress alone; that impairment of Ca2+ and K+ homeostasis is involved; and that the degree of cell injury and dysfunction during ATP depletion and during recovery can be ameliorated by drugs or interventions which decrease K+ depletion, Ca2+-overload, and/or sarcolemmal injury.
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