Cardiac contraction occurs as a cooperative response to the release of Ca2+ from the sarcoplasmic reticulum. The molecular basis for the cooperativity of this response will be investigated using purified bovine cardiac and rabbit skeletal muscle proteins, specifically, troponin, tropomyosin, actin, any myosin, This proteins will be reconstituted with and without modifications designed to clarify the mechanism of the cooperative effect of Ca2+ on the heart. The following interrelated protein properties and/or structural features will be studied. (1) The relationships of actin-myosin binding and troponin-Ca2+ binding to cooperative activation of myosin MgATPase rate. (2) The N-terminal region of troponin T is highly regulated by alternative mRNA processing. The function of this region will be investigated by a variety of techniques, with particular emphasis on its interactions with other thin filament proteins and its effect on thin filament cooperativity. (3) Developmental and, if they occur, pathophysiological changes in mammalian cardiac troponin T isoform expression will be studied by the Western blot technique. Regulation of these isoforms may influence the response of the cardiac cell to the Ca2+ concentration. Considered together, these experiments will advance the understanding of the complex response of striated muscle to Ca2+. In the long term, insights such as these may clarify the mechanism of regulation of cardiac contractility and the molecular basis for altered cardiac performance.
