Omecamtiv Mecarbil Enhances Actin Affinity and Slows Force Production in Human β-Cardiac Myosin

Abstract

The small molecule drug Omecamtiv Mecarbil (OM) specifically targets cardiac myosin and is known to enhance cardiac muscle performance, while it is unclear how it impacts the motor properties of human cardiac muscle myosin. We expressed and purified human beta-cardiac myosin subfragment 1 (M2B-S1) containing a C-terminal Avi tag. We demonstrate that the maximum actin-activated ATPase activity of M2B-S1 is slowed 4-fold in the presence of 10 µM OM, while the actin concentration at which the ATPase activity is one-half maximal was reduced dramatically (32-fold). The ATPase activity was less ionic strength dependent in the presence of OM. The C-terminal Avi tag of M2B-S1 was biotinylated in vitro, which allowed specific attachment to the motility surface with streptavidin. We observed that the actin sliding velocity measured in the in vitro motility assay was 100-fold reduced in the presence of 10 µM OM. Increasing the ionic strength attenuated the inhibition of sliding velocity, which suggests the inhibition may be due to drag forces from myosin heads in the weak binding states. It has been proposed that OM slows the strain dependent ADP release step, which would dramatically increase duty ratio. However, we did not observe a significant increase in duty ratio in the presence of OM as measured by examining the sliding velocity as a function of actin filament length. Experiments with permeabilized human myocardium demonstrate that OM increases calcium sensitivity and slows force generation (K-TR) in a concentration-dependent manner but does not change maximally-activated force. Our results suggest that OM dramatically enhances the affinity for actin but slows the production of force. The enhanced recruitment of myosin crossbridges may cooperatively activate the thin filament which shifts the calcium sensitivity in human myocardium.