We will study the contraction of permeable muscle fibers under a variety of conditions that alter cross-bridge function. The long range goal of this research is two fold. 1. to understand the kinetics of the cross- bridge cycle and 2. to understand how the cross-bridge cycle is altered when muscles undergo heavy activity leading to fatigue. The interactions of the contractile proteins in solution are now relatively well known, and a central goal of the field is to understand how this interaction is altered when the proteins interact in the intact fibers. To help answer this question we will measure the mechanical responses of fibers activated by one of a series of nucleotides modified at either the base or the ribose moiety. This data will be correlated with the solution biochemistry of the actomyosin interaction measured by Dr. Howard White. These studies will also identify nucleotides that inhibit the cycle at specific transitions, producing altered states of muscle fibers which would be good candidates for structural studies. When skeletal or cardiac muscles undergo sustained activity, a number of changes occur in their mechanics and energetics. The proposed research will investigate two mechanisms that are involved in these changes: the reversible phosphorylation of a subunit of the myosin head, and the inhibition of contraction by the buildup of the products of ATP hydrolysis. Previous work by our laboratory and by others has now established that the products of ATP hydrolysis, ADP, phosphate and protons, inhibit cross- bridge function. This work has been largely carried out at low temperatures (5-15oC) and we will extend it to more physiological temperatures where preliminary results show that rather different results are obtained. 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 during and following intense activity. We have shown that phosphorylation increases the tension obtained in partially activated fibers, and several experiments will explore the steps in the contractile cycle that are altered by phosphorylation. All of the experiments proposed above will provide information on the kinetics and energetics of cross-bridge action while some will also directly address the mechanism of fatigue. Understanding these processes will aid in unraveling the complex physiological responses of living skeletal and cardiac muscles and may eventually lead to the development of new therapeutic interventions to alter cardiac function.
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