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

Abstract

Background: Omecamtiv mecarbil (OM) enhances systolic function in vivo by directly binding the myosin cross-bridges (XBs) in the sarcomere. However, the mechanistic details governing OM-induced modulation of XB behavior in failing human myocardium are unclear.

Methods and results: The effects of OM on steady state and dynamic XB behavior were measured in chemically skinned myocardial preparations isolated from human donor and heart failure (HF) left ventricle. HF myocardium exhibited impaired contractile function as evidenced by reduced maximal force, magnitude of XB recruitment (P_df), and a slowed rate of XB detachment (k_rel) at submaximal Ca²⁺ activations. Ca²⁺ sensitivity of force generation (pCa₅₀) was higher in HF myocardium when compared with donor myocardium, both prior to and after OM incubations. OM incubation (0.5 and 1.0 μmol/L) enhanced force generation at submaximal Ca²⁺ activations in a dose-dependent manner. Notably, OM induced a slowing in k_rel with 1.0 μmol/L OM but not with 0.5 μmol/L OM in HF myocardium. Additionally, OM exerted other differential effects on XB behavior in HF myocardium as evidenced by a greater enhancement in P_df and slowing in the time course of cooperative XB recruitment (T_rec), which collectively prolonged achievement of peak force development (T_pk), compared with donor myocardium.

Conclusions: Our findings demonstrate that OM augments force generation but also prolongs the time course of XB transitions to force-bearing states in remodeled HF myocardium, which may extend the systolic ejection time in vivo. Optimal OM dosing is critical for eliciting enhanced systolic function without excessive prolongation of systolic ejection time, which may compromise diastolic filling.

Keywords: heart failure; myocardium; omecamtiv mecarbil; sacromere; systolic function.

Figure 1. Phosphorylation of sarcomeric proteins and MHC expression in donor and HF myocardium

(A) Representative Coomassie stained (left) SDS gel and Pro-Q Diamond-stained (right) showing the expression and phosphorylation status of myofilament proteins in donor and HF samples. Cardiac samples isolated from 4 donor hearts and 4 failing hearts were used to analyze contractile protein expression and phosphorylation levels. (B) Quantification of phosphorylation of MyBP-C, TnT, and TnI in donor and HF samples. (C) Representative 5% Tris-HCl gel showing MHC isoform expression in donor and HF myocardium. Values are expressed as mean ± S.E.M. * P < 0.05.

Figure 2. Effect of OM on force enhancements at variable [Ca²⁺] activations in donor and HF myocardium

Baseline forces produced by the skinned myocardial preparations were first measured in Ca²⁺ solutions producing a range of submaximal forces. Forces were measured at baseline or following a 2-minute incubation with 0.5μM or 1.0μM OM. The net increase in force generation from the untreated baseline (pre-OM) following OM incubation was calculated and is expressed as % increase in force from baseline. Force enhancements were pronounced at submaximal Ca²⁺ activation but declined as the level of Ca²⁺ was increased. 12 skinned myocardial preparations (3 fibers each from 4 hearts) were used for both groups. *, significant force enhancement from baseline level in donor and HF myocardium following 1.0μM OM incubation; †, significant force enhancement from baseline level in donor and HF myocardium following 0.5μM OM incubation. Asterisks indicate P < 0.05.

Figure 3. Effect of OM on pCa₅₀ in donor and HF myocardium

Skinned myocardial preparations were exposed to a 2-minute incubation with 0.5 μM or 1.0μM OM. (A) Effect of OM on the force-pCa relationships in donor and HF myocardium. (B) Effect of OM on the force-pCa relationships in donor myocardium. (C) Effect of OM on the force-pCa relationships in HF myocardium. 12 skinned myocardial preparations (3 fibers each from 4 hearts) were used for both groups. *, pCa₅₀ is significantly higher in HF when compared to the donor myocardium. Asterisks indicate P < 0.05.

Figure 4. Representative stretch activation responses in donor and HF myocardium prior to and following OM incubations

Representative force responses are shown following a sudden 2% stretch in muscle length (ML) in isometrically-contracting donor (black traces), and HF (red traces) myocardial preparations prior to (A) and following incubation with 0.5μM OM (B) or 1.0μM OM (C). Expanded views are shown below panels A to C to demonstrate that k_rel is slower in HF myocardium compared to donor myocardium both prior to and following OM incubation. Insets below the expanded views depict the T_rec region of the trace. The highlighted parts of the stretch activation traces (blue colored regions) in the insets show that the time required for XB recruitment, T_rec, is prolonged post-OM incubations in the HF myocardium. Panel A highlights the important phases of the force transients and various stretch activation parameters that are measured from force responses to 2% stretch in ML (explained in methods section).

Figure 5. Effect of OM on the time required for XB recruitment (T_rec) and time required to achieve the peak of force development (T_pk) in donor and HF myocardium

The effect of OM was measured by incubating myocardial preparations in 0.5μM or 1.0μM OM. (A) T_rec prior to and following OM incubation in donor and HF myocardium. (B) T_pk prior to and following OM incubation in donor and HF myocardium. The supplement contains a description of how these parameters are measured. Values are expressed as mean ± S.E.M. 12 skinned myocardial preparations (3 fibers each from 4 hearts) were used for both groups. *, different vs. the corresponding pre-OM group; †, different vs. the corresponding donor group. Asterisks indicate P < 0.05.