Cardiomyopathy Mutations In The Converter Domain Of Human Beta-Cardiac Myosin Impairs Mechanochemistry In The Presence And Absence Of Load

Missense mutations in human β-cardiac myosin are associated with inherited cardiomyopathies, a leading cause of heart failure worldwide. The mechanisms for how mutations alter myosinu0027s motor performance are still unclear. We expressed and purified human beta-cardiac myosin subfragment 1 (M2β-S1) containing point mutations that are associated with hypertrophic (R723G) or dilated (F764L) cardiomyopathy. We demonstrate that both the R723G and F764L mutations slow down the maximum actin-activated ATPase activity (20-30%). Direct measurements of the ADP release rate constant in the presence of actin using mant labeled ADP demonstrate R723G has a 35% faster ADP release while F764L has a 20% slower ADP release compared to wild-type. Since the actin-activated ATPase is reduced in both mutants, it suggests a slower actin-activated phosphate release rate constant or slower transition between actomyosin.ADP states. The in vitro motility assay demonstrates relatively small differences compared to wild-type, with a 5-10% higher actin sliding velocity for R723G and 5-10% slower velocity for F764L. We also examined the sliding velocity as a function of ADP concentration, which provides information about the strain sensitivity of the mutants. We found that both mutants were less sensitive to ADP inhibition of in vitro actin sliding velocity suggesting reduced strain sensitivity. Therefore, we propose that both the hypertrophic R723G mutation and dilated F764L mutation have slower ATPase kinetics in the absence of load, while the mechanochemistry in the presence of load indicates a reduced ability to adapt to higher loads.