Single Molecule, Optical Trapping Studies Of Omecamtiv Mercarbil On Human Cardiac Myosin'S Force Production

The cardiac myosin small-molecule activator, omecamtiv mercarbil (OM), is in stage three clinical trials for the treatment of chronic heart failure, however the exact mechanism of its action is not settled. Initial reports suggested that OM increases the rate of phosphate release and the myosin duty ratio, resulting in increased force production due to an increased number of myosins strongly-bound to actin during contraction. Studies on muscle fibers and animal models corroborated these results as measured by force production and stroke volume, without changes in kinetics. However, in vitro motility assays published subsequently indicated that the sliding velocity is greatly reduced in the presence of OM in this experimental geometry. We utilized single molecule optical trapping techniques to study the step size and kinetics of actively cycling, recombinant human cardiac myosin under load in the presence of OM. We measured an increase in actin-attachment lifetime of myosin and a decrease in the length of the powerstroke from 6 nm to <1 nm in the presence of 10 µM OM. The decreased actomyosin dissociation rate and unitary displacement provide a mechanism for the inhibitory action of OM in the in vitro motility assay. Additionally, these results suggest that OM inhibits a kinetic step that occurs in between phosphate release and ADP release, as solution kinetic experiments indicate that OM does not slow the rates of ADP release or ATP-induced actomyosin dissociation. Single molecule optical trapping is providing a way to observe the entire cycle of myosin in the presence of OM and is thus helping to elucidate its therapeutic mechanism.