A large body of literature exists that characterizes the elementary steps of the actin-myosin ATPase cycle in skeletal muscle using both isolated contractile proteins and muscle fibers. In contrast, the steps in the ATPase cycle of cardiac muscle have been studied almost exclusively using isolated contractile proteins; a re lost. Only recently, have studies begun to examine the individual steps of the cross-bridge cycle in cardiac muscle using laser photolysis of caged compounds. These experiments have shown that there are significant differences in cardiac and skeletal muscle in the mechanical transients elicited by the photolytic release of ATP. The long range goal of our research is first to elucidate the precise mechanism by which the chemical energy available from the hydrolysis of ATP is transformed into mechanical work by the heart and then how this mechanism is altered in different physiological and pathological states. The specific objective in this proposal is to test the hypothesis that separate steps in the cross-bridge cycle regulate force production, steady- state cycling, and shortening velocity. We will use laser photolysis of caged compounds, including caged fluorescent analogs of ATP and ADP to initiate or perturb specific steps in the cross-bridge cycle in chemically skinned trabeculae of the guinea-pig. This technique allows the delivery of known concentrations of ligand to the cross-bridges without diffusional delays. The effects of these perturbations on the various steps in the cycle will be monitored as changes in force, stiffness, nucleotide content, and fluorescence.
The specific aims that will be addressed to test this hypothesis are: 1) To determine if the non steady-state kinetics of the """"""""phosphate burst"""""""" or the ATP hydrolysis step in cardiac muscle is sufficiently slow to control the rate of force development. Laser photolysis of caged tritiated ATP in conjunction with rapid freezing will be used to initiate and arrest ATP hydrolysis; 2) To study the kinetics of Ca2- induced force development using a novel form of caged Ca2- to determine if the rate of force development is Ca2- dependent and which cross-bridge state is responsible for force development; 3) To determine the mechanism by which cross-bridge attachment to the thin filament promotes further attachment in the absence of Ca2-; 4) To study the transient kinetics of the phosphate release steps in the cycle using caged P; and 5) To determine if the rate of ADP release from the cross-bridge limits shortening velocity and exhibits strain dependence. The information from such studies will facilitate the interpretation of mechanical studies of the heart; contribute to the general understanding of the mechanochemistry of the cross-bridge cycle; and clarify some of the causes for the changes in cardiac contractility associated with heart disease.
