During the tenure of the Physician Investigator award I will explore experimentally the hypothesis that cardiac cells derived from dog heart tissue with experimentally-induced congestive cardiomyopathy differ from normal heart cells in that they contain cytoskeletal protein components unable to assemble into myofibrils and that these events may lead to the formation of functionally deficient sarcomeres. For this study I plan to use viable cardiomyocytes dissociated from embryonic heart and from normal and diseased dog heart muscle. I will use microinjection procedures to observe in vivo and in situ in the cytoplasm of myocytes the modes and rates of diffusion and assembly of fluorescein-conjugated myofibril proteins. In addition, I will attempt to establish in myocytes derived from diseased tissue the mode by which fluorescinated protein compounds are either reinserted into myofibrils (renewal) or are utilized to build completely new myofibril bundles (replacement). For these goals I would use the fluorescent photobleaching technique. For ultrastructural studies I will acquire expertise in the newest gold-labeling techniques and try to establish the location and site of assembly of microinjected antigens and their antibodies using well-characterized antibodies and marker-conjugated immunoglobulins. My long-term objectives as I train as an experimental pathologist are to understand how--in humans--derangements in myofibril/sarcomere assembly contribute to the mosaic of cellular events that lead to cardiac muscle disease.
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