Omecamtiv Mecarbil Enhances the Duty Ratio of Human β-Cardiac Myosin Resulting in Increased Calcium Sensitivity and Slowed Force Development in Cardiac Muscle

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 motility assay. The ionic strength dependence of 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 () 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.

Dose-Dependent Effects of the Myosin Activator Omecamtiv Mecarbil on Cross-Bridge Behavior and Force Generation in Failing Human Myocardium

“…Consistent with previous reports 17 , we observed reduced steady-state maximal force generation and increased pCa 50 in HF myocardium when compared to donor myocardium (Table 1; Figure 3A). However, unlike previous studies 11, 25 , OM incubation did not enhance the pCa 50 (Table 1). The cause of this discrepancy is unclear, but may be related to a basal temperature-dependent sensitization of thin filaments which acts to decrease the impact of OM on pCa 50 (15°C 25 and 22°C 11 in earlier studies vs. 24°C in the present study).…”

“…However, unlike previous studies 11, 25 , OM incubation did not enhance the pCa 50 (Table 1). The cause of this discrepancy is unclear, but may be related to a basal temperature-dependent sensitization of thin filaments which acts to decrease the impact of OM on pCa 50 (15°C 25 and 22°C 11 in earlier studies vs. 24°C in the present study). Results from stretch activation experiments revealed a slowed rate of XB detachment (i.e., k rel , Table 2) in HF myocardium, which acts to prolong XB duty ratio and increase force generation at low Ca 2+ -activations.…”

“…11, 13, 35 As shown earlier 11 , we observed that OM-induced force enhancements were primarily confined to sub-maximal Ca 2+ -activations and that force enhancements gradually decline with increasing Ca 2+ levels (Figure 2). As reported earlier 11 , OM treatment also reduced the cooperativity of force generation, n H (Table 1) which may be related to the fact that at low [Ca 2+ ], thin filament activation relies more on XB-mediated cooperative XB recruitment as most of the regulatory units (RU) are in the off state.…”

“…Data were analyzed with a linear mixed (LM) effect model 25 with an unstructured covariance matrix across the conditions and across the groups. The R package lme4 was used to implement the LM analysis.…”

A szív pozitív inotróp támogatása a miozin-aktivátor hatású omecamtiv mecarbil segítségével

“…A legfrissebb adatok szerint az OM a fenti esetben inkább csökkentené a miozin ATPáz aktivitását, mintsem fokozná azt. Tehát egyáltalán nem zárható ki, hogy az OM a P i -disszociá-ció ütemének fokozásán kívül mást is tesz az aktin-miozin ciklussal (46)(47)(48). In vitro motilitási vizsgálatokban is megerősítették, hogy OM hatására csökken az aktin fi lamentumok miozin-függő elmozdulásának sebessé-ge (38,46).…”