The long range goals of the proposed research are a) to establish, at the molecular level, how the energy available from the hydrolysis of ATP in cardiac muscle is utilized by the contractile proteins actin and myosin to perform external work and b) to determine how this mechanism is altered in different physiological and pathophysiological states of the heart. Previously, the elementary steps of the actin and myosin ATPase system in cardiac muscle have only been characterized in biochemical studies using isolated proteins. The objective of this proposal is to test the hypothesis that the interaction of actin and myosin (and hence the rates and equilibrium constants of the chemical steps in the cross-bridge cycle) are modified by the steric and mechanical constraints present within the filament structure of permeable cardiac muscle fibers. The proposed experiments make use of a novel method in the study of muscle kinetics. The general approach will be to first induce rigor tension in chemically skinned ventricular trabeculae from the guinea-pig; equilibrate the tissue with caged-ATP a biologically inactive, photosensitive precursor of ATP either in the presence or absence of calcium; and initiate ATP hydrolysis by laser- induced release of ATP. The actin and myosin interaction will be monitored by: 1) analyzing the mechanical responses of the tissue following the liberation of ATP, and 2) measuring the chemical changes that occur during the actomyosin-nucleotide interaction by terminating the ATP hydrolysis by rapid freezing.
The specific aims are to obtain estimates of the rate of: 1) ATP-induced cross-bridge detachment, 2) the """"""""phosphate burst"""""""", 3) ADP dissociation form the cross-bridge and the equilibrium constant for the hydrolysis step. This information will facilitate the interpretation of mechanical studies on cardiac muscle contraction; contribute to the general understanding of the mechanochemistry of this process; and clarify the underlying causes of alterations produced by physiologic stimuli and pathological changes.
