Abstract 4364366: Myosin modulator Aficamten inhibits force in cardiac muscle by altering myosin’s biochemical activity without changing thick filament structure

Abstract

Introduction: Inhibiting contractility by targeting cardiac myosin is an effective treatment for patients with hypertrophic cardiomyopathy (HCM). Aficamten (Afi), a second in class myosin inhibitor, improves hemodynamics and symptoms in HCM patients. While it is known that Afi inhibits force and cardiomyocyte contractility by stabilizing the weak pre-powerstroke conformation, measurements in organized sarcomeres are lacking. Goals: Determine effects of Aficamten on cardiac myosin structure and function and kinetics of contraction under load. Methods: Permeabilized porcine cardiac tissue and myofibrils were used for single-molecule imaging of ATP turn over, X-ray diffraction, and mechanical measurements. Engineered heart tissues (EHTs) from human induced pluripotent stem cell cardiomyocytes were used to evaluate force and contraction kinetics. Results: In contrast to Mavacamten (Mava), Afi does not structurally sequester myosin heads along the thick filament. Afi inhibits ATPase activity by shifting myosin heads from higher to slower ATPase, with the emergence of a super slow biochemical nucleotide turnover. This results in decreased maximal activated force and calcium sensitivity (ND=5.53 ± 0.02, Mava=5.40 ±0.03 and Afi=5.40±.03) without altering cross-bridge cycling to a similar extent as Mava. At myofibril level, both Afi and Mava significantly inhibited the maximally activated force (37.64±2.81 and 37.46±2.90 nN/μm 2 ) of the isolated porcine cardiac myofibrils compared to untreated controls (76.70±3.00 nN/μm 2 ). Afi significantly accelerated k REL,fast (21.9 ± 0.9 s -1 ) compared to Mava (14.8 ± 1.0 s -1 ) and to ND (11.3 ± 0.4 s -1 ). Both Afi and Mava significantly increased k REL,slow (1.10 ± 0.01 and 1.12 ± 0.06 s -1 ) compared to ND (0.40 ± 0.03 s -1 ). Both compounds also significantly decrease t REL,slow (45.1 ± 0.9 and 48.6 ± 3.3 ms) compared to ND (78.1 ± 1.8 ms). In EHTs, while Mavacamten and Aficamten inhibit cardiac twitch forces, Time to peak tension (TT P ) was significantly longer for Mava but not changed for Afi. Time to 50% relaxation (RT 50 ) and time to 90% relaxation (RT 90 ) were significantly shorter for Afi, while only RT 50 was shorter for Mava. Conclusion: We used a combination of biochemical and biomechanical assays to show that Aficamten inhibits myosin ATPase without appreciably altering myosin structure. This is different from Mavacamten that strongly affects both.