Single-molecule mechanics and kinetics of cardiac myosin interacting with regulated thin filaments.

The cardiac cycle is a tightly regulated process wherein the heart generates force to pump blood to the body during systole and then relaxes during diastole. Disruption of this finely tuned cycle can lead to a range of diseases including cardiomyopathies and heart failure. Cardiac contraction is driven by the molecular motor myosin, which pulls regulated thin filaments in a calcium-dependent manner. In some muscle and nonmuscle myosins, regulatory proteins on actin tune the kinetics, mechanics, and load dependence of the myosin working stroke; however, it is not well understood whether or how thin-filament regulatory proteins tune the mechanics of the cardiac myosin motor. To address this critical gap in knowledge, we used single-molecule techniques to measure the kinetics and mechanics of the substeps of the cardiac myosin working stroke in the presence and absence of thin filament regulatory proteins. We found that regulatory proteins gate the calcium-dependent interactions between myosin and the thin filament. At physiologically relevant ATP concentrations, cardiac myosin's mechanics and unloaded kinetics are not affected by thin-filament regulatory proteins. We also measured the load-dependent kinetics of cardiac myosin at physiologically relevant ATP concentrations using an isometric optical clamp, and we found that thin-filament regulatory proteins do not affect either the identity or magnitude of myosin's primary load-dependent transition. Interestingly, at low ATP concentrations at both saturating and physiologically relevant subsaturating calcium concentrations, thin-filament regulatory proteins have a small effect on actomyosin dissociation kinetics, suggesting a mechanism beyond simple steric blocking. These results have important implications for the modeling of cardiac physiology and diseases.