Regulatory light chain mutations associated with cardiomyopathy affect myosin mechanics and kinetics

The myosin regulatory light chain (RLC) wraps around the alpha-helical neck region of myosin. This neck region has been proposed to act as a lever arm, amplifying small conformational changes in the myosin head to generate motion. The RLC serves an important structural role, supporting the myosin neck region and a modulatory role, tuning the kinetics of the actin myosin interaction. Given the importance of the RLC, it is not surprising that mutations of the RLC can lead to familial hypertrophic cardiomyopathy (FHC), the leading cause of sudden cardiac death in people under 30. Population studies identified two FHC mutations located near the cationic binding site of the RLC, R58Q and N47K. Although these mutations are close in sequence, they differ in clinical presentation and prognosis, with R58Q showing a more severe phenotype. We examined the molecular based changes in myosin that are responsible for the disease phenotype by purifying myosin from transgenic mouse hearts expressing mutant myosins and examining actin filament sliding using the in vitro motility assay. We found that both R58Q and N47K show reductions in force compared to the wild type that could result in compensatory hypertrophy. Furthermore, we observed a higher ATPase rate and an increased activation at submaximal calcium levels for the R58Q myosin that could lead to decreased efficiency and incomplete cardiac relaxation, potentially explaining the more severe phenotype for the R58Q mutation.     

Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice

Myosin light chain kinase (MLCK)-dependent phosphorylation of the regulatory light chain (RLC) of cardiac myosin is known to play a beneficial role in heart disease, but the idea of a phosphorylation-mediated reversal of a hypertrophic cardiomyopathy (HCM) phenotype is novel. Our previous studies on transgenic (Tg) HCM-RLC mice revealed that the D166V (Aspartate166 →Valine) mutation-induced changes in heart morphology and function coincided with largely reduced RLC phosphorylation in situ. We hypothesized that the introduction of a constitutively phosphorylated Serine15 (S15D) into the hearts of D166V mice would prevent the development of a deleterious HCM phenotype. In support of this notion, MLCK-induced phosphorylation of D166V-mutated hearts was found to rescue some of their abnormal contractile properties. Tg-S15D-D166V mice were generated with the human cardiac RLC-S15D-D166V construct substituted for mouse cardiac RLC and were subjected to functional, structural, and morphological assessments. The results were compared with Tg-WT and Tg-D166V mice expressing the human ventricular RLC-WT or its D166V mutant, respectively. Echocardiography and invasive hemodynamic studies demonstrated significant improvements of intact heart function in S15D-D166V mice compared with D166V, with the systolic and diastolic indices reaching those monitored in WT mice. A largely reduced maximal tension and abnormally high myofilament Ca²⁺ sensitivity observed in D166V-mutated hearts were reversed in S15D-D166V mice. Low-angle X-ray diffraction study revealed that altered myofilament structures present in HCM-D166V mice were mitigated in S15D-D166V rescue mice. Our collective results suggest that expression of pseudophosphorylated RLC in the hearts of HCM mice is sufficient to prevent the development of the pathological HCM phenotype.Hypertrophic cardiomyopathy (HCM) is a complex and heterogeneous disorder with extensive diversity in the course of the disease, age of onset, severity of symptoms, and risk for sudden cardiac death (SCD) (1). HCM is the most common cause of SCD among young people, particularly in athletes (2). The characteristic pathologic features of HCM include cardiac hypertrophy with disproportionate involvement of the ventricular septum and extensive disorganization of the myocyte structure and myocardial fibrosis (3). At present, there is no cure for HCM, and it is now becoming evident that any effective therapy must target the underlying mechanisms involved in the pathogenesis of the disease.The D166V (aspartic acid replaced by valine) mutation in the ventricular myosin regulatory light chain (RLC) was reported by Richard et al. to cause HCM and SCD (4). Our extensive study of the mechanisms underlying the D166V phenotype revealed that the severity of mutation-induced effects in transgenic (Tg) mice correlated with reduced in situ RLC phosphorylation (5, 6). Tg-D166V papillary muscle preparations demonstrated a significantly lower contractile force and abnormally increased myofilament Ca²⁺ sensitivity compared with Tg-WT, expressing a nonmutated human ventricular RLC (5). Likewise, single molecule detection applied to fluorescently labeled cardiac myofibrils from Tg-D166V mice showed slower rates of cross-bridge cycling, and, as in Kerrick et al. (5), these functional abnormalities were paralleled by a low level of RLC phosphorylation compared with Tg-WT hearts (7).Cardiac myosin RLC is a major regulatory subunit of muscle myosin and a modulator of the troponin and Ca²⁺-controlled regulation of muscle contraction (8). It is localized at the head-rod junction of the myosin heavy chain (MHC), and, in addition to the N-terminal Ca²⁺-Mg²⁺ binding site, it also contains the myosin light chain kinase (MLCK)-specific phosphorylatable Serine15. Phosphorylation of Ser15 has been recognized to play an important role in cardiac muscle contraction under normal and disease conditions (9). Significantly reduced RLC phosphorylation was reported in patients with heart failure (10, 11) and observed in animal models of cardiac disease (5, 12–14). Attenuation of RLC phosphorylation in cardiac MLCK knockout mice led to ventricular myocyte hypertrophy, with histological evidence of necrosis and fibrosis, and to mild dilated cardiomyopathy (15, 16). These results and our decade-long investigation of the RLC mutant-induced pathology of the heart suggest that RLC phosphorylation may have an important physiological role in the heart and serve as a rescue tool to mitigate detrimental disease phenotypes.We first addressed this hypothesis in vitro and pursued the MLCK-induced phosphorylation studies on Tg-D166V mice (6), followed by the use of the pseudophosphorylation mimetic proteins exchanged in porcine myosin or skinned muscle fibers (17). Results from both lines of investigation showed that phosphorylation of RLC could counterbalance the adverse contractile effects of an HCM-causing mutation in vitro. In this report, we aimed to test the idea of pseudophosphorylation-induced prevention of a deleterious phenotype in HCM mice. Transgenic S15D-D166V mice were generated expressing the pseudophosphorylated Ser15 (S15D) in the background of the disease-causing D166V mutation. Functional, structural, and morphological assessments were conducted on Tg-S15D-D166V mice, and the results were compared with those of previously generated Tg-D166V (5) and Tg-WT (18) mice. Our findings indicate that myosin phosphorylation may have an important translational application and be used to prevent the development of a severe RLC mutant-induced HCM phenotype.