Characterization of the Cardiac Myosin Inhibitor CK-3773274: a Potential Therapeutic Approach for Hypertrophic Cardiomyopathy

Abstract

Hypercontractility of the cardiac sarcomere may be essential for the underling pathological hypertrophy and fibrosis in 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 (IC50:1.26 μM). CK-3773274 selectively targets cardiac myosin, as it reduced myosin ATPase activity in the absence of other sarcomere proteins, including actin, troponin, and tropomyosin. Importantly, CK-3773274 did not inhibit the ATPase activity of smooth muscle myosin. In transient kinetic studies, CK-3773274 substantially slowed the rate of actin-activated phosphate release, likely stabilizing myosin conformations that bind weakly to actin. Binding of CK-3773274 to cardiac myosin is mutually exclusive with the nonspecific myosin II inhibitor blebbistatin, suggesting they bind to the same or overlapping sites. Consistent with its biochemical phenotype, CK-3773274 (10 μM) reduced fractional shortening (FS) by 84% in electrically paced, isolated adult rat cardiomyocytes relative to control without any effect on the calcium (Ca2+) 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. Left ventricular dimensions and FS were determined by echocardiography at select time points over a 24-hour period. One hour after dose administration, CK-3773274 significantly reduced FS in a dose-related fashion by 20% to 70% relative to vehicle treatment without any changes to heart rate. 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.