Cardiomyopathy is a degenerative disease of the myocardium without obvious systemic etiology. Although therapeutic intervention may offer symptomatic relief, the only successful treatment for cardiomyopathy is cardiac transplantation. Idiopathic dilated cardiomyopathy (DCM) accounts for 87% of diagnosed cardiomyopathies in the United States. Although both systolic and diastolic dysfunction accompany DCM, their underlying cause remains to be established. Indirect evidence suggests that altered Ca2+ cycling by the myocardial sarcoplasmic reticulum (SR) contributes to the functional impairment associated with DCM, but prior to the project described in this proposal, a direct measurement of the Ca2+ content of the SR from human hearts has not been obtained. The goal of this study is to directly measure Ca2+ storage, release, and re-uptake by the human cardiac SR in situ in cardiac muscle from failing (DCM) and non-failing human hearts and to correlate these measurements with parameters of isometric contraction and relaxation in the same muscles. Methods used include a unique combination of functional studies on human trabecular muscles and measurements of Ca2+ within the SR of the same muscles at fixed time points during the contractile cycle by electron probe microanalysis (EPMA). In addition to measuring SR Ca2+, EPMA on rapidly frozen human muscles allows quantification of Ca2+ in the mitochondria and at the level of the myofilaments (A-band) within each cell and provides simultaneous data on other elements of physiological interest (Na, Mg, P, S, Cl and K) within each of these subcellular areas. In order to ensure that functional parameters of contraction and relaxation measured in isolated trabecular muscles are representative of systolic and diastolic cardiac function in vivo, hemodynamic indices of cardiac function prior to transplantation or organ harvest will be obtained from each patient. Correlations will be drawn for each heart between the following parameters: 1) in vivo measures of cardiac function 2) in vitro parameters of muscle contraction and 3) Ca2+ in the SR at fixed time points during the cardiac cycle. This project proposes a direct method for correlating human cardiac muscle function with intracellular calcium homeostasis and provides the first direct test of the hypothesis that defects in SR Ca2+ cycling may be associated with the functional alterations observed in human DCM.
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