We propose to clarify the role of myosin phosphorylation by studying its effect on the actomyosin interaction in permeable fibers, in myofibrils, in reconstituted contractile systems and in the biochemistry of purified proteins in solution. Reversible phosphorylation of one subunit of myosin occurs in vivo in skeletal and cardiac muscles and several experimental results suggest that it is an important control mechanism that modulates the contraction of these muscles. However this conclusion is controversial and many details of the action of myosin phosphorylation remain unknow. Our recent work has indicated that myosin phosphorylation decreases the rate of cycling of crossbridges in both skeletal and cardiac muscle thus making the muscle more efficient at maintaining tension. Results found in some studies of living muscle are in agreement with our conclusions, while other results have appeared to be in conflict. To determine the role of myosin phosphorylation we will phophorylate myosin in situ in permeable fibers, where the levels of myosin phosphorylation, the activation by Ca++, and the concentration of substrates can all be independently controlled. We will then measure the mechanics and energetics of contracting fibers. In addition we will measure the effect of myosin phosphorylation on the actomyosin interaction in solution, in reconstituted motile systems and in myofibrils. The results of these experiments will help define which steps in the actomyosin interaction are affected by phosphorylation in solution and in organized filament arrays. The above studies will be extended to cardiac fibers and proteins. Synthesis of results from the diverse experiments proposed will lead to a better understanding of how myosin phosphorylation modulates the contractile interaction. There is increasing evidence that myosin phosphorylation modulates cross-bridge kinetics and that it plays a significant role in determining the characteristics of fiber contraction. Understanding this process will aid in unraveling the complex physiological responses of living skeletal and cardiac muscles, and may eventually lead to therapeutic interventions with alter cardiac function by manipulating the level of myosin phosphorylation.
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