The mechanisms responsible for switching muscle on and off and regulating its force and shortening will be studied. Current theories indicate that contraction is initiated by the binding of calcium to the regulatory protein complex, troponin and tropomyosin, and on the thin filament. The binding of calcium of troponin is thought to produce a movement of tropomyosin in such a fashion that charged sites on the actin and myosin are exposed and permitted to interact with myosin, resulting strong binding of the myosin head to the actin filament. This binding then initiates the force production and shortening in a fashion involving the use of ATP. The amount of force produced is thought to be dependent on the number of actin sites exposed while the unloaded shortening velocity is independent of the extent of thin filament activation. By combining measurement of the rate of shortening of single regulated thin filaments and the force exerted on single regulated thin filaments by myosin under a variety of conditions with site directed mutagenesis of the proteins involved in regulation, we plan to directly test hypotheses concerning: how many actin sites need to be switched on to activate contraction; how tropomyosin blocks the interaction surfaces of actin and myosin; whether mechanisms currently believed to modulate activation of the actin and myosin in the test tube can produce alterations in actomyosin s mechanical behavior; and whether the weak and strong binding sites on actin and myosin determine how much force or how fast the muscle shortens. Using the same techniques we will use mutant regulatory proteins known to be associated with hypertrophic cardiomyopathy to determine how these mutations alter cardiac muscle s ability to develop force and shorten.
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