Impact of Dilated Cardiomyopathy Mutation and Small Molecule Regulator on Human Beta-Cardiac Myosin

Abstract

Missense mutations in human β-cardiac myosin are associated with inherited cardiomyopathies. The mechanisms for how mutations alter myosin's motor performance are still unclear. We expressed and purified human beta-cardiac myosin subfragment 1 (M2β-S1) containing a point mutation in the converter domain (F764L) that is associated with dilated cardiomyopathy (DCM) and compared it to wild-type (WT) M2β-S1. We demonstrate that the F764L mutation slows down maximum actin-activated ATPase activity (20-30%). Direct measurements of ADP release in the presence of actin using mant labeled ADP demonstrate the F764L mutant has a 20% slower ADP release rate constant. The in vitro motility assay demonstrates a 10-15% slower actin sliding velocity for F764L. Monitoring ATP binding and hydrolysis with intrinsic tryptophan fluorescence demonstrates that the F764L mutation causes a 2-fold increase in K0.5 without changing the maximal rate. We also examined the impact of the heart failure drug Omecamtiv Mecarbil (OM) on the F764L mutant. The steady-state actin-activated ATPase results suggest that the F764L mutant shares a similar OM binding affinity with WT. Interestingly, the drug does not reduce the maximum ATPase activity and actin sliding velocity as much as it does with WT. We observed no difference in the ionic strength dependence or density dependence of in vitro motility in WT and F764L in the presence and absence of OM. Our previous results suggested OM dramatically slows down the ADP isomerization step in WT M2β-S1, making it the rate-limiting step. Our results demonstrate OM does not slow the ADP isomerization step as much in F764L, but this step is still rate-limiting. Therefore, we propose that OM will increase the myosin duty ratio in the F764L mutant enough to enhance isomeric force while only moderately slowing shortening velocity.