DESCRIPTION: (adapted from the abstract) The primary goal of this proposal is to make use of state-of-the-art equipment and techniques to elucidate the regulation of the formation and dissociation of force generating myosin cross-bridges by Ca2+ in both skeletal and cardiac muscle (striated muscle). The hypothesis to be tested is that the rate constants involved in both the formation and dissociation of the force-generating myosin cross-bridges are regulated during Ca2+ activation of contraction. This proposed research is therefore designed to study the kinetics of the myosin cross-bridge attachment and detachment as a functions of Ca2+ and inorganic phosphate concentration. This study will make use of skinned skeletal and cardiac cells from the rat and rabbit. In order to carry out these studies the principal investigator (PI) will measure muscle force, actomyosin ATPase activity, and fluorescence of both skeletal and cardiac troponin-C (TnC) labeled with a fluorophor which reports Ca2+ binding to TnC. The unique aspect of these studies is that the PI is able to measure actomyosin ATPase or TnC fluorescence simultaneously with force measurements. These measurements make it possible to study the effect of Ca2+ on the rate constants that control both the formation and dissociation of force generating, cross-bridges in striated muscle. Recent studies on fast-twitch skeletal muscle and preliminary studies on slow-twitch and heart muscle show that the rate of dissociation of the force generating myosin cross-bridge decreases. The significance of this research is that it will provide important new information regarding the kinetic parameters of myosin force generating cross-bridges which determine the range of Ca2+ concentrations which activate force, and control efficiency of striated muscle contraction. Since it is known that many factors such as ATP hydrolysis products (ADP, and inorganic phosphate), drugs (sulmazole, EMD 53998, pimobendan and adibendan), pH, and cardiac Tnl phosphorylation affect the Ca2+ activation and efficiency of contraction, their effects on the kinetics of myosin cross-bridge formation and dissociation will be determined and the results used to test the hypothesis stated above. Particular emphasis will be placed upon the effects of inorganic phosphate (Pi) as a tool for altering and studying myosin cross-bridge kinetics and consequently Ca2+ -sensitivity of contraction.