Phosphorylation of the regulatory light chain of myosin (RLC) is altered in many forms of cardiac diseases, both inherited (HCM and DCM) and acquired (failure, infarction and diabetes). Phosphorylation of the RLC alters muscle mechanics by influencing calcium sensitivity, force, power and shortening velocity. Currently we do not understand how these effects on muscle are created at the molecular level of the cross-bridge. To this end, we isolated full-length cardiac myosin from porcine endocardium and altered its phosphorylation level in-vitro. We compare populations of myosins with complete (100%) RLC phosphorylation, intermediate phosphorylation (40% & 50%), and eradicated phosphorylation (0%). We could thereby correlate enzymatic and motility changes produced with RLC phosphorylation. We find that when myosin possesses 100% phosphorylated RLCs, the ATPase rate is accelerated and actin gliding velocities in putative unloaded and loaded (in presence of -actinin) conditions are accelerated. These results are consistent with the influence of RLC phosphorylation on the kinetics of the cross-bridge motor kinetics. RLC phosphorylation reduced the lifetime of actomyosin attachment as measured in the optical trap three-bead assay. These results suggest an important role for RLC phosphorylation in regulating the function of cardiac myosin. - The results are consistent with the hypothesis that reductions in normal levels of RLC phosphorylation may precipitate cardiovascular disorders and heart failure.
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