This project is dedicated to the interdisciplinary development of analytical electron microscopic and other structural approaches and their application to the determination of calcium-dependent and independent mechanisms of regulation in vascular smooth and cardiac muscle. A major goal is to perfect the sensitivity and spatial resolution of energy- filtered transmission electron microscopy (STEM-EELS) to a previously un- attained (2-4 nm) level suitable for sensitive compositional imaging of subcellular domains. STEM-EELS and X-ray mapping with an improved solid- state detector will be used to determine the functionally important, binding of calcium to membranes of cardiac and vascular smooth muscle, the sub-mitochondrial distribution of calcium and the composition of (putative) Ca- and Na buffering domains beneath the plasma membrane. The hypotheses, that the calcium content of mitochondria varies depending on their cellular location relative to the plasma membrane or the sarcoplasmic reticulum, will be quantitatively tested with electron probe microanalysis (EPMA). Manganese (Mn), used to quench the fluorescence of calcium-sensitive fluorophores used for measuring intracellular free calcium, will be quantitatively localize to assess the contribution of Mn accumulation by mitochondria and sarcoplasmic reticulum to fluorescent signals. The distribution of aluminum and fluoride, used to activate G- proteins will be determined in order to ascertain whether AIF/4 directly interacts and co translocates with monomeric GTP-binding proteins in cells or only interacts with trimeric G-proteins. Fluorescence confocal microscopy will be used to related to function the stimulus-dependent distribution of telokin, Rho and Rho-associated proteins and smooth muscle myosin phosphatase in vascular smooth muscle. Health-related aspects of the research include the role of calcium binding to cardiac gap junctions in ventricular fibrillation, the most common cause of sudden cardiac death, and the importance of abnormalities of vascular and bronchial smooth muscle regulation in diseases such as high blood pressure and asthma. The STEM-EELS methodology developed for mapping,a t 2-4 nm resolution, biologically important elements, such as calcium, will be widely applicable to a broad range of problems related to normal and diseased cardiovascular function and to cellular physiology and pathology of nearly every biological system.
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