Zhiqun Zhou

Novel familial dilated cardiomyopathy mutation in MYL2 affects the structure and function of myosin regulatory light chain

Dilated cardiomyopathy (DCM) is a disease of the myocardium characterized by left ventricular dilatation and diminished contractile function. Here we describe a novel DCM mutation in the myosin regulatory light chain (RLC), in which aspartic acid at position 94 is replaced by alanine (D94A). The mutation was identified by exome sequencing of three adult first‐degree relatives who met formal criteria for idiopathic DCM. To obtain insight into the functional significance of this pathogenic MYL2 variant, we cloned and purified the human ventricular RLC wild‐type (WT) and D94A mutant proteins, and performed in vitro experiments using RLC‐mutant or WT‐reconstituted porcine cardiac preparations. The mutation induced a reduction in the α‐helical content of the RLC, and imposed intra‐molecular rearrangements. The phosphorylation of RLC by Ca2+/calmodulin‐activated myosin light chain kinase was not affected by D94A. The mutation was seen to impair binding of RLC to the myosin heavy chain, and its incorporation into RLC‐depleted porcine myosin. The actin‐activated ATPase activity of mutant‐reconstituted porcine cardiac myosin was significantly higher compared with ATPase of wild‐type. No changes in the myofibrillar ATPase–pCa relationship were observed in wild‐type‐ or D94A‐reconstituted preparations. Measurements of contractile force showed a slightly reduced maximal tension per cross‐section of muscle, with no change in the calcium sensitivity of force in D94A‐reconstituted skinned porcine papillary muscle strips compared with wild‐type. Our data indicate that subtle structural rearrangements in the RLC molecule, followed by its impaired interaction with the myosin heavy chain, may trigger functional abnormalities contributing to the DCM phenotype.

Sarcomeric perturbations of myosin motors lead to dilated cardiomyopathy in genetically modified MYL2 mice

Significance Dilated cardiomyopathy (DCM) is a progressive heart disease with no current cure, often culminating in heart transplantation. Transgenic D94A (aspartic acid-to-alanine) mice carrying a novel DCM-causative mutation in the MYL2 gene, encoding the cardiac myosin regulatory light chain, were created and investigated by echocardiography and invasive hemodynamic and molecular structural and functional assessments. Our data show that hypocontractile myosin motors and structural perturbations at the level of sarcomeres trigger aberrant functional remodeling in D94A hearts and the development of DCM, which closely follows the clinical phenotype. Left ventricular chamber dilation and decreased ejection fraction, observed in D94A hearts, were indicative of systolic dysfunction, a hallmark of DCM. Our study suggests that MYL2 may be considered a therapeutic target for dilated cardiomyopathy. Dilated cardiomyopathy (DCM) is a devastating heart disease that affects about 1 million people in the United States, but the underlying mechanisms remain poorly understood. In this study, we aimed to determine the biomechanical and structural causes of DCM in transgenic mice carrying a novel mutation in the MYL2 gene, encoding the cardiac myosin regulatory light chain. Transgenic D94A (aspartic acid-to-alanine) mice were created and investigated by echocardiography and invasive hemodynamic and molecular structural and functional assessments. Consistent with the DCM phenotype, a significant reduction of the ejection fraction (EF) was observed in ∼5- and ∼12-mo-old male and female D94A lines compared with respective WT controls. Younger male D94A mice showed a more pronounced left ventricular (LV) chamber dilation compared with female counterparts, but both sexes of D94A lines developed DCM by 12 mo of age. The hypocontractile activity of D94A myosin motors resulted in the rightward shift of the force–pCa dependence and decreased actin-activated myosin ATPase activity. Consistent with a decreased Ca2+ sensitivity of contractile force, a small-angle X-ray diffraction study, performed in D94A fibers at submaximal Ca2+ concentrations, revealed repositioning of the D94A cross-bridge mass toward the thick-filament backbone supporting the hypocontractile state of D94A myosin motors. Our data suggest that structural perturbations at the level of sarcomeres result in aberrant cardiomyocyte cytoarchitecture and lead to LV chamber dilation and decreased EF, manifesting in systolic dysfunction of D94A hearts. The D94A-induced development of DCM in mice closely follows the clinical phenotype and suggests that MYL2 may serve as a new therapeutic target for dilated cardiomyopathy.

Molecular and Functional Effects of a Splice Site Mutation in the MYL2 Gene Associated with Cardioskeletal Myopathy and Early Cardiac Death in Infants

The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1+∕+-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure. In this report we have investigated the molecular mechanism and functional consequences associated with the IVS6-1 mutation using recombinant human cardiac IVS6-1 and wild-type (WT) RLC proteins. Recombinant proteins were reconstituted into RLC-depleted porcine cardiac muscle preparations and subjected to enzymatic and functional assays. IVS6-1-RLC showed decreased binding to the myosin heavy chain (MHC) compared with WT, and IVS6-1-reconstituted myosin displayed reduced binding to actin in rigor. The IVS6-1 myosin demonstrated a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT. In stopped-flow experiments, IVS6-1 myosin showed slower kinetics of the ATP induced dissociation of the acto-myosin complex and a significantly reduced slope of the kobs-[MgATP] relationship compared to WT. In skinned porcine cardiac muscles, RLC-depleted and IVS6-1 reconstituted muscle strips displayed a significant decrease in maximal contractile force and a significantly increased Ca2+ sensitivity, both hallmarks of hypertrophic cardiomyopathy-associated mutations in MYL2. Our results showed that the amino-acid changes in IVS6-1 were sufficient to impose significant conformational alterations in the RLC protein and trigger a series of abnormal protein-protein interactions in the cardiac muscle sarcomere. Notably, the mutation disrupted the RLC-MHC interaction and the steady-state and kinetics of the acto-myosin interaction. Specifically, slower myosin cross-bridge turnover rates and slower second-order MgATP binding rates of acto-myosin interactions were observed in IVS6-1 vs. WT reconstituted cardiac preparations. Our in vitro results suggest that when placed in vivo, IVS6-1 may lead to cardiomyopathy and early death of homozygous infants by severely compromising the ability of myosin to develop contractile force and maintain normal systolic and diastolic cardiac function.