verbatim): To design new means of therapeutic interventions in heart disease requires a detailed understanding of the complex interactions between the thin filament and contractile proteins involved in the cooperative activation of cardiac muscle. Biochemical, structural and cellular mechanics studies indicate that activation mechanisms differ between cardiac and skeletal muscle. However, discerning the exact mechanisms by which cooperative activation occurs has been difficult because a technique has not been available to separate local events from events occurring along the thin filament. We have developed a simple but powerful approach to do this, using recombinant technology and skinned muscle techniques. This approach allows the cooperative cross-bridge binding in single regulatory units (RUs) to be studied separately from cross-bridge interactions that activate neighboring RUs along the thin filament. Our preliminary data suggest that strong cross-bridges can increase the interactions between neighboring RUs, and substantially increases cooperative activation of skeletal muscle, but there is minimal interaction between neighboring RUs in cardiac muscle. Furthermore, even with maximal Ca2+ activation, the cardiac thin filament may not become fully activated. In cardiac and skeletal muscle we will determine (1) if functional RU size differs from the structural A7TmTn in the absence of nearest-neighbor interactions, (2) the role of strong cross-bridges in establishing this minimum RU size, (3) the role of strong cross-bridge binding in individual RUs on cooperative activation of near-neighbor RUs, (4) the role of cooperative activation within individual RUs and between near-neighbor RUs on the rate of force development, and (5) the role of thin filament proteins in establishing RU size and on nearest-neighbor cooperative interactions.
These Aims will be studied by measuring force and kinetics of force development, varying the number of functioning RUs, using different isoforms of Tn and TnC (or TnI, TnT), and with the use of inhibitors and augmentors of strong cross-bridge binding. These studies will provide an exciting new insight into regulation of contraction in both skeletal and cardiac muscle.
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