The Affect of Omecamtiv Mecarbilon the Phosphoate Dissociation and Motile Properties of the Recombinant Human β-Cardiac Heavymeromyosin

Abstract

Omecamtiv mecarbil (OM), a small-molecule increases cardiac contractility by directly activating cardiac myosin by accelerating phosphate (Pi) release (Malik et al., 2011). Here we analyzed the affect of OM on the mechanochemical cycle and motility of human β-cardiac heavymeromyosin expressed in a adenoviral/C2C12 muscle cell based system, and purified porcine ventricular myosin (PV-HMM). Double-mixing stopped-flow fluorescence and phosphate binding protein (MDCC-PBP) were used to determine the maximum rate (kmax) of phosphate release. Rates of 30s−1 and 35s−1 were obtained for human and porcine cardiac HMM respectively. Addition of 20 µM OM resulted in a 2-fold increase in the maximal rate of actin activated Pi release to kmax = 70 s−1 (AM-ADP-Pi _> AM-ADP + Pi) for both human and porcine cardiac HMM. The unloaded actin filament velocity measured in the in vitro motility assay was 0.76 ± 0.37 µm/sec for human β-cardiac HMM and 0.97 ± 0.23 µm/sec for PV-HMM. OM has two effects on the motor activity: 1) The actin filament velocity was significantly slower (∼ 16 -fold at saturation); 2) the filament motion became more persistent with long periods of uninterrupted movement. This suggests that OM recruits more crossbridges to participate in the movement of the actin filaments resulting in higher forces per filament, which overcomes the pinning defects that invariably trap actin filaments in normal motility assays. The increase in attached crossbridges/filament also slows the filament velocity. The motility data are consistent with the kinetic analysis indicating that OM accelerates Pi release without dramatically enhancing the rate of the ATPase hydrolysis. This produces a higher duty ratio in which the motor spends more of the ATPase cycle in tight binding states. Supported by AHA-BGIA to EF and AHA-GI to DAW.