The major goal of this proposal is to uncover in skeletal and cardiac muscle the time course, and to study ultrastructural changes attending the spatial displacements of, all elements of physiological relevance (notably Ca) during the process of excitation-contraction coupling (ECC). Knowledge of that time course, and of its ultrastructural substrata, is crucial to an understanding of kinetic aspects of ECC and for striking comparative, dynamic in vivo and in vitro structure-function correlations in muscle. There are 4 specific aims: 1. to determine the time of calcium release, its spatial distribution (50 nm resolution) and ultrastructural manifestations at various time intervals (fractions of ms) after electrical stimulation and pharmacologic interventions, 2. to improve spatial resolution and X-ray sensitivity of data acquisition, 3. to apply established methodology and improvements to the study of cardiac muscle and, 4. to continue to study the 3-D geometry of cardiac muscle. The proposal is unique in that all elements, especially calcium, are measured directly in single, demonstrably living cells that, prior to quick- freezing, have remained untouched by invasive procedures. Briefly, isolated, single intact frog skeletal muscle cells are cryofixed at high rates which keeps all elements """"""""frozen"""""""" in their respective anatomic positions at various predetermined points in time. Cryosections are cut, freeze-dried, and examined by electron probe X-ray microanalysis. The quantitative measurements are made (a) with static raster probes and, (b) by quantitative digital elemental X-ray imaging. In the former, specific regions are measured in succession; in the latter, a very large part of a cell is scanned in tot by a matrix of pixels, each containing precise quantitative data on all the elements. Freeze-etch replicas, and thin sections from embedded, freeze-substituted parts of the same muscle fiber adjacent to the collection are also planned. The proposed research will provide new, otherwise inaccessible quantitative physiological and structural data of dynamic intracellular events in vivo from which to gauge and derive mechanisms of skeletal and cardiac muscle function, both in health and disease. Beyond, the methodology will be suitable to expand our abilities to investigate many important aspects of fast physiological events in vivo at an ultrastructural level in other biological models, and to explore, directly, in vivo diffusion rates of various elements in certain single cell preparations. Concerning the final aim, the 3-D geometry of cardiac cell bundles is crucial to understanding electrical current distribution in normal and abnormal cardiac rhythms. The study requires 3-D reconstruction of many serial sections from various heart muscle cell bundles.
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