The HCM-Linked Ala13thr Mutation in the Cardiac Myosin Regulatory Light Chain Increases Isometric Force Production

Abstract

The myosin regulatory light chain (RLC) is attached to the α-helical neck region of the myosin head, the so called lever arm, which connects the catalytic and actin binding domains with the thick filament backbone thus participating in the transmission of external forces to the myosin active site. It is understandable that mutations in the RLC associated with hypertrophic cardiomyopathy (HCM) may lead to alterations in force generation affecting cardiac muscle performance. Here, we studied the physiological consequences of an Alanine to Threonine (A13T) mutation in the N-domain of myosin RLC, found in population studies to cause HCM with a specific disease phenotype characterized by mid-ventricular obstruction. We observed an A13T-induced 30-50% increase in maximal force measured in skinned cardiac muscle fibers from transgenic Tg-A13T mice compared to control, Tg-WT and non-Tg littermates. Furthermore, a mutation-mediated 1.3-fold decrease in Vmax and a 1.5-fold increase in Km were observed in the actin-activated myosin ATPase activity compared with myosin from the healthy controls. The binding of Tg-myosin to pyrene-actin was similar for all groups of mice. No changes in the maximal myofibrillar ATPase or in the Ca2+-sensitivity were noted. The same was true for the force-pCa relationship and the mutation did not introduce any alterations in the Ca2+-sensitivity of force development. Gross morphological evaluation revealed enlarged inter-ventricular septa and left ventricles in the hearts from Tg-A13T mice, a phenotype observed in patients harboring the A13T mutation. Our results indicate that the A13T mutation may result in a hypertrophic response through abnormally increased force that may exceed the tolerance of a healthy myocardium. A decreased rate of cross-bridge turnover further demonstrates inadequate energy generation in Tg-A13T mice adding to impaired sarcomeric function. Supported by NIH-HL071778 (DSC).