Myosin with hypertrophic cardiomyopathy mutation M493I demonstrates altered crossbridge kinetics and force sensitivity despite normal working stroke

Familial hypertrophic cardiomyopathy (HCM) is a genetic disorder and prominent cause of sudden cardiac death. Among sarcomeric proteins, HCM-causing mutations have been identified in the MYH7 gene, encoding beta-cardiac myosin, but the etiologic mechanisms for HCM are unknown. Recent biochemical and biophysical studies of cardiac myosin containing different HCM mutations have shown either activation or inhibition of myosin activity. A leading model is that HCM-causing mutations may disrupt the super-relaxed state of myosin, causing hypercontractility of the sarcomere. Thus, mapping the mechanochemical effects of mutations on myosin function is essential to understand the disease. Here, we use a combination of bulk-phase kinetics and single-molecule optical trap studies to determine the kinetics and mechanics of the severe HCM-causing mutation M493I. In in vitro gliding filament assays, M493I-myosin reduced actin gliding velocity by 70% relative to WT, from 1.6 μm/s to 0.45 μm/s. Transient kinetics measurements revealed that actin-bound M493I-myosin has a significantly decreased the rate of ADP release (13 s−1 vs. 70 s−1 for WT), and slightly increased rate of ATP binding (5.5 μM−1s−1 vs. 4.5 μM−1s−1 for WT). In the optical trap the M493I mutation does not substantially affect the length of the myosin’s working stroke, which has two steps as in WT myosin. The distance parameter, a measure of the force-dependent enhancement of actomyosin-ADP lifetime, was calculated to be 0.33 nm for M493I-myosin, compared to 0.8 nm for WT, indicating that the detachment rate of M493I-myosin is less sensitive to force than WT. Finally, with careful attention to molecular alignment and stage stability, preliminary experiments suggest the kinetics of actin reattachment are accelerated by the M493I mutation. Together, these data demonstrate kinetic and mechanical changes in beta-cardiac myosin bearing the mutation M493I.