LAUSR

Hypercontractility of the cardiac sarcomere appears to underlie pathological hypertrophy and fibrosis in select genetic hypertrophic cardiomyopathies. Here, we characterize the small molecule, CK-3773274, as a novel cardiac myosin inhibitor that decreases contractility in vitro and in vivo . In bovine cardiac myofibrils, CK-3773274 decreased myosin ATPase activity in a concentration-dependent fashion (IC 50 :1.26 μM). CK-3773274 specifically inhibited myosin activity, as it reduced myosin ATPase activity in a concentration-dependent manner in the absence of other sarcomere proteins, including actin, troponin, and tropomyosin. CK-3773274 (10 μM) reduced fractional shortening by 84% in electrically paced, isolated adult rat cardiomyocytes relative to control without any effect on the calcium transient. The effect of CK-3773274 on cardiac contractility in vivo was assessed in healthy male Sprague Dawley (SD) rats using single oral doses ranging from 0.5 to 4 mg/kg. Fractional shortening (FS) and left ventricular dimensions were determined by echocardiography at select time points over a 24-hour period. One hour after dose administration, CK-3773274 significantly reduced fractional shortening in a dose-related fashion by 20-70% relative to vehicle treatment (FS %: vehicle: 47.9± 1%; 0.5 mg/kg: 39 ± 2%; 4 mg/kg: 15 ± 4%; mean ±SEM, p<0.05 vehicle vs. all doses) without any changes to heart rate. Lastly, the effect of CK-3773274 was evaluated by echocardiography in healthy beagle dogs. Left ventricular ejection fraction (LVEF) was evaluated following single oral doses ranging from 0.75-3 mg/kg over a 48 hour period. 2 hours after dosing, CK-3773274 decreased LVEF in a dose-related fashion by approximately 15-50% relative to vehicle treatment (LVEF vehicle: 74.6 ± 3 %; 0.75 mg/kg: 62.5 ± 3%; 2 mg/kg: 44.9± 3%; 3 mg/kg: 36.8 ± 2%; mean ±SEM, p<0.05 vehicle vs. all doses). In conclusion, CK-3773274 is a novel, small molecule, cardiac myosin inhibitor that reduces cardiac contractility in vitro and in vivo . Cardiac myosin inhibition may be a viable approach to treat the underlying hypercontractility of the cardiac sarcomere in hypertrophic cardiomyopathies.