Abstract
The small molecule drug omecamtiv mecarbil (OM) specifically targets cardiac muscle myosin and is known to enhance cardiac muscle performance, yet its impact on human cardiac myosin motor function is unclear. We expressed and purified human β-cardiac myosin subfragment 1 (M2β-S1) containing a C-terminal Avi tag. We demonstrate that the maximum actin-activated ATPase activity of M2β-S1 is slowed more than 4-fold in the presence of OM, whereas the actin concentration required for half-maximal ATPase was reduced dramatically (30-fold). We find OM does not change the overall actin affinity. Transient kinetic experiments suggest that there are two kinetic pathways in the presence of OM. The dominant pathway results in a slow transition between actomyosin·ADP states and increases the time myosin is strongly bound to actin. However, OM also traps a population of myosin heads in a weak actin affinity state with slow product release. We demonstrate that OM can reduce the actin sliding velocity more than 100-fold in the in vitro motility assay. The ionic strength dependence of in vitro motility suggests the inhibition may be at least partially due to drag forces from weakly attached myosin heads. OM causes an increase in duty ratio examined in the motility assay. Experiments with permeabilized human myocardium demonstrate that OM increases calcium sensitivity and slows force development (ktr) in a concentration-dependent manner, whereas the maximally activated force is unchanged. We propose that OM increases the myosin duty ratio, which results in enhanced calcium sensitivity but slower force development in human myocardium.
Highlights
The drug can enhance the duty ratio of myosin heads that flux through the conserved ATPase pathway by increasing the period of time they are strongly bound to the actin filament during filament sliding
We complement this work with an investigation of the impact of OM on human cardiac muscle mechanics
Our results support a model that suggests OM slows ATPase cycle kinetics by slowing the transition between actomyosin1⁄7ADP states which results in an increase in duty ratio
Summary
SCHEME 1 and does not alter skeletal or smooth muscle myosin [6]. Muscle fiber studies that have examined the impact of OM are somewhat contradictory. A study on porcine cardiac myosin determined that OM alters the hydrolysis equilibrium constant to favor products and enhances the rate of actin-activated phosphate release, but it does not change the ADP release step [24]. These factors were predicted to enhance the number of myosin cross-bridges in the strongly bound state, which would explain the increased force in muscle fiber studies. It is important to directly measure the duty ratio, the fraction of the ATPase cycle myosin is bound to actin, in the in vitro motility assay to evaluate hypotheses about how OM dramatically slows sliding velocity. Based on measurements with permeabilized human cardiac muscle, we find that OM increases calcium sensitivity and slows force generation, but it does not change steady-state force at saturating calcium concentrations
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