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.