During the first project period we have defined a detailed sequence of functional and ionic changes in single cardiac myocytes exposed to anoxia and reoxygenation, culminating in the oxygen paradox in cells which are reoxygenated after prolonged energy depletion. We have demonstrated marked abnormality of relaxation kinetics in myocytes reoxygenated after brief anoxia, and shown that this abnormality is at the myofilament level. We have developed a method for realtime measurement of mitochondrial free calcium in cardiac myocytes, and demonstrated that it rises during energy depletion and its level is predictive of cell survival at reoxygenation. During the next period we will concentrate on 3 areas: (1) We will pursue the mechanism of post- reoxygenation myofilament abnormality, looking particularly for phosphorylation of regulatory sites on myofilament proteins, the role of inorganic phosphate in the signaling pathway, and the possible participation of tyrosine kinases. (2) To define further the conditions predisposing to reoxygenation hypercontracture, we will (a) try to dissociate changes in cytosolic and mitochondrial calcium to determine which (if either) is causal and (b) Determine the mechanism of Na+ entry during rigor, looking particularly for evidence of involvement of voltage-dependent Na channels either vial small window currents activated by deplorization of the resting potential or by abnormal inactivation. (3) Because of marked species differences in the response to prolonged anoxia at the single myocyte level, we will isolate human ventricular myocytes from endomyocardial biopsies and explant hearts and characterize the response to anoxia/reoxygenation in these cells.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Cardiovascular and Pulmonary Research A Study Section (CVA)
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Johns Hopkins University
Internal Medicine/Medicine
Schools of Medicine
United States
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Silverman, H S; Wei, S; Haigney, M C et al. (1997) Myocyte adaptation to chronic hypoxia and development of tolerance to subsequent acute severe hypoxia. Circ Res 80:699-707
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Stern, M D (1996) ""Adaptive"" behavior of ligand-gated ion channels: constraints by thermodynamics. Biophys J 70:2100-9
Silverman, H S; Di Lisa, F; Hui, R C et al. (1994) Regulation of intracellular free Mg2+ and contraction in single adult mammalian cardiac myocytes. Am J Physiol 266:C222-33
Haigney, M C; Lakatta, E G; Stern, M D et al. (1994) Sodium channel blockade reduces hypoxic sodium loading and sodium-dependent calcium loading. Circulation 90:391-9
Silverman, H S (1993) Mitochondrial free calcium regulation in hypoxia and reoxygenation: relation to cellular injury. Basic Res Cardiol 88:483-94
Miyata, H; Lakatta, E G; Stern, M D et al. (1992) Relation of mitochondrial and cytosolic free calcium to cardiac myocyte recovery after exposure to anoxia. Circ Res 71:605-13
Silverman, H S; Stern, M D; Lakatta, E G (1992) Contrasting effects of anoxia and graded hypoxia on single cardiac myocyte function. Am J Cardiovasc Pathol 4:256-64
Ziegelstein, R C; Zweier, J L; Mellits, E D et al. (1992) Dimethylthiourea, an oxygen radical scavenger, protects isolated cardiac myocytes from hypoxic injury by inhibition of Na(+)-Ca2+ exchange and not by its antioxidant effects. Circ Res 70:804-11
Haigney, M C; Miyata, H; Lakatta, E G et al. (1992) Dependence of hypoxic cellular calcium loading on Na(+)-Ca2+ exchange. Circ Res 71:547-57

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