The overall aim of the proposed research is to investigate the cellular mechanisms responsible for the systolic and diastolic abnormalities of contraction sen in myocardial hypertrophy and failure. Abnormalities in calcium homeostasis have previously been demonstrated in animal models of hypertrophy and failure and in cells isolated from failing human hearts. However, these studies fail to identify the individual cellular processes that underlie these abnormalities. The important question is what step(s) in the coupling of membrane excitation with subsequent contraction and relaxation is (are) altered in hypertrophy and failure. Within the theoretical framework of a quantitative scheme of calcium homeostasis, the various steps of excitation-contraction-relaxation coupling will be individually probed by current methods in voltage clamping (whole-cell & single-channel), intracellular fluorescent calcium (Ca2+) indicators, and the flash photolysis of caged compounds.
The specific aims of the proposed research include: 1.) Test the hypothesis that the influx of Ca2+ through the L-type Ca2+-channel required to """"""""trigger"""""""" the release of Ca2+ from the sarcoplasmic reticulum (SR) is altered in hypertrophy and failure. This will involve measurement of both hole-cell and unitary Ca2+-currents. 2.) Test the hypothesis that the grading of the SR calcium release b the L-type Ca2+-current is altered in hypertrophy and failure. This will involve the measurement of single-channel Ca2+-currents and the simultaneous measurement of whole-cell calcium currents, intracellular calcium transients, and cell shortening. 3) Test the general hypothesis that the handling of Ca2+ by the SR is altered in hypertrophy and failure. Specifically, 3.1) test the hypothesis that the systolic abnormalities of contraction result from abnormalities of Ca2=-efflux from the SR Ca2+- release channel, and 3.2) test the hypothesis that the diastolic or relaxation abnormalities seen in hypertrophy and failure are the result of impaired sequestration of Ca2+ by athe SR Ca2+=ATP-ase pump. 4.) Test the hypothesis that the Ca2+-binding ligands (including the contractile regulatory proteins troponin C and calmodulin) are altered in hypertrophy and failure.
These specific aims will be investigated in single isolated myocytes from animal models of cardiac hypertrophy and failure and from failing human heart. This research addresses the fundamental question regarding the precise cellular mechanism(s) responsible for the altered contractility characteristic of hypertrophy and failure in a quantitative way.
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