Molecular effects of the myosin activator omecamtiv mecarbil on contractile properties of skinned myocardium lacking cardiac myosin binding protein-C

Abstract

Decreased expression of cardiac myosin binding protein-C (cMyBP-C) in the myocardium is thought to be a contributing factor to hypertrophic cardiomyopathy in humans, and the initial molecular defect is likely abnormal cross-bridge (XB) function which leads to impaired force generation, decreased contractile performance, and hypertrophy in vivo. The myosin activator omecamtiv mecarbil (OM) is a pharmacological drug that specifically targets the myosin XB and recent evidence suggests that OM induces a significant decrease in vitro motility velocity and an increase in the XB duty cycle. Thus, the molecular effects of OM maybe beneficial in improving contractile function in skinned myocardium lacking cMyBP-C because absence of cMyBP-C in the sarcomere accelerates XB kinetics and enhances XB turnover rate, which presumably reduces contractile efficiency. Therefore, parameters of XB function were measured in skinned myocardium lacking cMyBP-C prior to and following OM incubation. We measured ktr, the rate of force redevelopment as an index of XB transition from both the weakly- to strongly-bound state and from the strongly- to weakly-bound states and performed stretch activation experiments to measure the rates of XB detachment (krel) and XB recruitment (kdf) in detergent-skinned ventricular preparations isolated from hearts of wild-type (WT) and cMyBP-C knockout (KO) mice. Samples from donor human hearts were also used to assess the effects of OM in cardiac muscle expressing a slow β-myosin heavy chain (β-MHC). Incubation of skinned myocardium with OM produced large enhancements in steady-state force generation which were most pronounced at low levels of [Ca2+] activations, suggesting that OM cooperatively recruits additional XB’s into force generating states. Despite a large increase in steady-state force generation following OM incubation, parallel accelerations in XB kinetics as measured by ktr were not observed, and there was a significant OM-induced decrease in krel which was more pronounced in the KO skinned myocardium compared to WT skinned myocardium (58% in WT vs. 76% in KO at pCa 6.1), such that baseline differences in krel between KO and WT skinned myocardium were no longer apparent following OM-incubation. A significant decrease in the kdf was also observed following OM incubation in all groups, which may be related to the increase in the number of cooperatively recruited XB’s at low Ca2+-activations which slows the overall rate of force generation. Our results indicate that OM may be a useful pharmacological approach to normalize hypercontractile XB kinetics in myocardium with decreased cMyBP-C expression due to its molecular effects on XB behavior.

Impact of the Myosin Modulator Mavacamten on Force Generation and Cross‐Bridge Behavior in a Murine Model of Hypercontractility

“…When the myocardial preparations attained a steady‐state force, they were subjected to a sudden 2% stretch of their initial muscle length (ML), held at the new ML for 8 s, and were then returned back to their initial ML. The key features of the cardiac muscle stretch activation responses have been described earlier33, 37, 39 (Figure 4A). Briefly, a sudden 2% stretch in ML evokes an instantaneous spike in the force response (P1) because of a sudden strain of elastic elements within the strongly bound XBs (Phase 1).…”

“…Force‐ pC a relationships were constructed by plotting normalized forces generated against a range of pC a to assess pC a 50 in WT and KO myocardial preparations 37, 38, 40. Detergent‐skinned myocardial preparations were exposed to a 2‐minute incubation with either 0.5 or 1.0 μmol/L Myk461 to assess the impact of Myk461 on force‐ pC a relationships in (A) WT and (B) KO myocardium.…”

“…Forces were subsequently measured on the same preparations using the same range of pC a solutions following a 2‐minute incubation with Myk461. The net decrease in force generation following Myk461 incubation at each pC a was calculated and expressed as % decrease in force from the pre‐incubation baseline force in (A) WT and (B) KO preparations as done earlier 37, 40. Incubation with Myk461 significantly decreased the myocardial force generation at all the concentrations of Myk461 tested and at all the pC a's tested in the WT and KO groups, but the Myk461‐induced force depression is attenuated as the level of Ca 2+ activation increases.…”

“…Detergent‐skinned multicellular ventricular preparations were attached between a motor arm (312C; Aurora Scientific Inc., Aurora, Ontario, Canada) and a force transducer (403A; Aurora Scientific Inc.), as described previously 37, 38, 39, 40. Changes in the motor arm position and force transducer signals were sampled at 2000 Hz using a custom‐built sarcomere length control software 41.…”

“…Changes in the motor arm position and force transducer signals were sampled at 2000 Hz using a custom‐built sarcomere length control software 41. For all mechanical measurements, the sarcomere length of the ventricular preparations was set to 2.1 μm 33, 37, 38, 40. Force‐pCa relationships were determined by measuring the forces generated by the skinned myocardial preparations in a range of pCa solutions that yield submaximal to maximal forces.…”