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 halfmaximal ATPase was reduced dramatically (30-fold).WefindOM does not change the overall actin affinity. Transient kinetic experiments suggest that there are two kinetic pathways in the presence ofOM.The dominant pathway results in a slow transition between actomyosinADP 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.OMcausesanincrease in duty ratioexaminedin 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 thatOM increases the myosin duty ratio, which results in enhanced calcium sensitivity but slower force development in human myocardium.
|Number of pages||11|
|Journal||Journal of Biological Chemistry|
|State||Published - Mar 3 2017|
Bibliographical notePublisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology