The long-term objective of the proposed research is to elucidate the mechanism of excitation-contraction coupling (ECC) in striated muscle with emphasis on frog skeletal and avian cardiac muscle. The proposal combines physiological approaches with high resolution electron microscopy, freeze- fracture, immunotopochemistry, morphometry and X-ray microanalysis. The proposal takes advantage of our ability to quickfreeze single intact skeletal muscle fibers at submillisecond intervals following electrical stimulation and to measure, essentially by non-invasive means, calcium depletion of internal stores directly (i.e. as chance in total [Ca]), and to investigate contemporaneous morphologic changes in the same fiber by high resolution EM, including thin sections following freeze-substitution as well as freeze-fracture replicas of the frozen-hydrate state of the cells at the time of freezing. Moreover, the proposal takes advantage of anomalous organelles discovered in this laboratory in avian and mammalian hearts that may shed light onto the mechanism by which an action potential is translated into calcium release (excitation-calcium release, ECR) because these organelles have no contact with the plasmalemma. Therefore, they cannot function by direct contact signal transduction from the plasmalemma but, rather, require a diffusible signal. This proposal promises to establish a time parameter of fundamental physiological importance for striated muscle: the time it takes between a stimulus and intracellular calcium release and, in addition, to uncover local morphological changes incident to that release. Specifically: (1) to test the hypothesis that calcium is released from the junctional SR (JSR)- during the first millisecond following electrical stimulation; by x-ray microanalysis. (2) to test the hypothesis that demonstrable morphological changes occur at the instant of calcium release at the JSR/plasmalemmal junction; by electron microscopy. (3) to identify ryanodine receptors on non-junctional SR in skeletal muscle, and to test the hypothesis that corbular SR is a calcium release organelle in mammalian hearts; by immunocytochemistry. (4) to establish the time course of developmental expression of ryanodine receptors in extended JSR (EJSR) in the avian heart; by immunocytochemistry. (5) to test the hypothesis that EJSR is a calcium release site; by x-ray microanalysis. (6) to obtain physiological data of calcium displacements in isolated avian cardiac cells to test specific aspects of our working hypothesis that the principal source of activating calcium is stored in the SR and is released through a diffusible messenger, e.g. calcium. Our results will describe physiological events in the intact system, the ultimate arbiter of biological function.
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