The magnitude of developed force during muscle contraction is dependent upon the number of strong interactions between actin and myosin. These interactions in striated muscle are modulated by a Ca(2+)-regulated molecular switch that determines the level of Ca(2+) saturation in troponin (Tn) and a potentiated state than involves a feedback control by force-generating crossbridges. The switching and potentiating mechanisms involve interactions among the three Tn subunits and interactions of Tn with tropomyosin (Tm). Although a description of the regulatory process cannot be formulated with detail in the absence of a three-dimensional structure of the protein system, it is possible to gain significant insights into the molecular interactions that are important in the regulation of activation of cardiac myofilaments. This has been made possible based on recent progress in the determination of the structure of individual proteins and elucidation of the arrangement of myofilament proteins. The present research addresses two broad issues that are related to both the switching and potentiating mechanisms and draws upon recently published fluorescence studies from this laboratory, a continued collaboration to allow entry into a new direction, and collaborative modeling studies of protein assemblies. This project has three specific aims: (1) comparison of the kinetic mechanisms by which activator Ca(2+) binds to cardiac and fast skeletal Tn and determination of the kinetics of Ca(2+)-induced opening of the regulatory domain in both isoforms, (2) topography mapping of the cardiac Tn complex, and (3) investigation of the mechanism of fiber length dependence of Ca(2+) activation of tension and Ca(2+)-induced conformational changes of the regulatory proteins that occur during isometric tension development. These studies will require the use of specific mutant proteins. The kinetic studies will be carried out by stopped-flow fluorometry. Topography mapping will be accomplished using fluorescence resonance energy transfer (FRET); both standard heterotransfer between different fluorophores and homotransfer between identical fluorophores will be used. The new direction in Aim 3 will use skinned muscle fibers and involve simultaneous measurement of tension and fluorescence signals arising from myofilament proteins which are exchanged into the filaments. Results from these studies are expected to enhance our understanding of the mechanisms that modulate changes in cardiac myofilament response to Ca(2+) and provide insights into intrinsic mechanisms of the control of the heart by Starling's law. Since many cardiac abnormalities appear to involve changes in myofilament response to Ca(2+) and altered structures of regulatory proteins, results from this project may provide insights into the molecular events that can contribute to abnormalities.
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