Heart development will be studied in normal and cardiac mutant axolotls and in normal and cardiomyopathic hamsters. Our objectives are to elucidate the sequence of events and mechanism(s) of myofibrillogenesis and to determine the roles of embryonic inductive interactions regulating and directing heart differentiation at the cellular and molecular levels. Developing heart cells will be examined by immunohistochemical methods to establish the appearance and precise locations of the various contractile/cytoskeletal proteins. Deep freeze etching methods in combination with immunoelectron microscopy will permit three-dimensional analyses of the protein distributions during myofibril formation. Two-dimensional gel electrophoresis studies and Western blots will aid in determining the early appearance and relative concentrations of the proteins in the differentiating cells. """"""""New"""""""" contractile/cytoskeletal proteins will be probed by preparing specific polyclonal and monoclonal antibodies against unknown proteins detected by two-dimensional gel electrophoresis. Micro-injection of fluorescently-tagged specific proteins into cultured heart cells will be employed to examine their incorporation into the myofibrils and/or cytoskeletal elements of the living differentiating cells. In addition, antibodies against the various proteins will be injected into cultured cells to help determine roles that specific proteins play in myofibril organization. Heart inductive processes which regulate normal myofibrillogenesis will be studied at the cellular and molecular levels. To accopmlish this, gene c in axolotls will be employed as a """"""""tool"""""""" in the studies. Since a diffusible RNA from normal anterior endoderm corrects the defect in cardiac mutant heart cells by promoting myofibrillogenesis, a variety of biochemical experiments will be performed to determine the mechanism(s) of this inductive relationship at the cellular and molecular levels. The use o these two unique genetic mutations should provide the biological """"""""tools"""""""" necessary for determining the sequence of events and mechanism(s) of normal myofibrillogenesis and heart induction. By understanding the basic mechanism(s) of how heart cells form into normal functional units, the various diseases which alter these processes can be understood, and, perhaps, reversed. In a broader sense, the proposed research may help to answer one of the major unsolved problems in modern biology: the control of gene expression during development.

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National Heart, Lung, and Blood Institute (NHLBI)
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Upstate Medical University
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