Abstract Body (Do not enter title and authors here): Background: Myosin heads are the contractile units of the heart. Genetic variants in sarcomeric proteins, such as titin (TTN) or myosin (MYH7), can alter the function and/or the availability of myosin heads forming force-generating cross-bridges, and are associated with dilated cardiomyopathy (DCM). To date, no targeted therapies have been approved to address this pathophysiology in patients with DCM. Danicamtiv (DANI) is a novel investigational direct cardiac myosin activator capable of enhancing the function/availability of cardiac motors with the potential to address the underlying dysfunction of these patients. Here the role of DANI in the setting of myosin dysfunction mimicking DCM was evaluated.

Methods: DANI was studied in systems of increasing complexity ranging from the cardiac myosin sub-fragment 1 (cS1, the catalytic unit of myosin) to in vivo. ATPase rates of recombinant human cS1, and pig LV myofibrillar (cMF) preparations were studied in vitro. In skinned pig LV fibers, biomechanical relationships (force and Ca2+-sensitivity) were evaluated, while echocardiographic/hemodynamic indices were assessed in healthy rats and pigs. The responses to DANI were assessed under control conditions and in the setting of myosin dysfunction, resulting from either DCM-pathogenic MYH7 mutations (in recombinant cS1), or via pharmacological cardiac myosin inhibition (MYHi: CK-274 or CK-586) in vitro/in vivo.

Results: DANI enhanced the ATPase cycling rate of cS1 in wild-type and five DCM MYH7 variants (AC50, WT: 3.8 µM vs. F764L: 3.2µM). Induced MYH7 dysfunction, markedly depressed cMF ATPase rates (-47±2 to -23±1% vs CTRL, P< 0.05) and force generation (Fmax: 21±1 vs. 34±2 mN/mm2 in CTRL, P<0.05), resulting in depressed systolic cardiac performance in vivo. At clinically relevant concentrations, DANI corrected the induced sarcomere/myofilament depression, restoring Ca2+-dependent ATPase rates (0.12 ± 0.01 vs. 0.08 ± 0.01 in MYHi), Ca2+-sensitivity and peak force (29±1 mN/mm2), leading to increased EF, LV dP/dtmax, and cardiac output in vivo (all P<0.05).

Conclusion: Danicamtiv can directly improve the function of cardiac myosin in the setting of (genetic or induced) dysfunction, increasing the number of myosin heads forming productive cross-bridges, thus increasing/restoring force generation and systolic performance in vivo. These observations, if confirmed in humans, could suggest a potential novel mechanism-based targeted therapy for a subset of patients with DCM