Abstract Body: Clinical trials of cardiac myosin inhibitors, including mavacamten and aficamten, have been shown to induce cardiac remodeling and improve exercise capacity of patients with hypertrophic cardiomyopathy (HCM) with left ventricular outflow tract obstruction. A subset of patients with heart failure with preserved ejection fraction (HFpEF) have overlapping features with HCM including hypercontractility (EF >65%), thickened heart walls and impaired diastolic function. This similarity suggests myosin inhibitors could be therapeutic in treating HFpEF. Here, we describe the preclinical characterization of a novel cardiac myosin inhibitor, CK-4021586 (CK-586), which has a distinct mechanism of action from both mavacamten and aficamten. CK-586 is a partial inhibitor of cardiac myofibrillar ATPase activity (EC50: 2.9 µM, maximal biochemical inhibition ~50%) which requires the presence of two-headed heavy meromyosin (HMM) and the regulatory light chain. CK-586 potently inhibited sarcomere shortening in both rat and human adult ventricular cardiomyocytes, with IC50 values of 3.2 µM and 2.8 µM respectively, achieving over 80% inhibition at 10 µM. Importantly, even at high concentrations up to 20 µM, CK-586 did not affect calcium transients in rat myocytes. Similarly, CK-586 effectively normalized contractile function in engineered heart tissues derived from human iPSC-cardiomyocytes carrying the HCM-associated R403Q myosin mutation. At 2 µM, the compound restored contractile force and relaxation time (RT90) to near-normal levels, while showing >80% force inhibition at 10 µM. In the ZSF1 obese HFpEF rat model, 16 weeks of CK-586 treatment reduced fractional shortening (vehicle: 52.7 ± 2.9% vs. CK-586: 48.1 ± 2.6%) and as a measure of diastolic function, improved isovolumic relaxation time (vehicle: 27.0 ± 2.2 ms vs. CK-586: 23.3 ± 2.3 ms). ZSF1 obese rats treated with CK-586 also demonstrated approximately 50% lower interstitial fibrosis compared to untreated controls. In conclusion, CK-586 is a novel, small molecule, cardiac myosin inhibitor that reduces contractility in vitro and in vivo. These findings suggest that targeting cardiac myosin to reduce contractility could help reverse some of the structural changes associated with HFpEF and improve cardiac relaxation.