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In this study, we focus on the molecular mechanisms associated with the A57G (Ala57-to-Gly57) mutation in myosin essential light chains (ELCs), found to cause hypertrophic cardiomyopathy (HCM) in humans and in mice. Specifically, we studied the effects of A57G on the super-relaxed (SRX) state of myosin that may contribute to the hypercontractile cross-bridge behavior and ultimately lead to pathological cardiac remodeling in transgenic Tg-A57G mice. The disease model was compared to Tg-WT mice, expressing the wild-type human ventricular ELC, and analyzed against Tg-Δ43 mice, expressing the N-terminally truncated ELC, whose hearts hypertrophy with time but do not show any abnormalities in cardiac morphology or function. Our data suggest a new role for the N terminus of cardiac ELC (N-ELC) in modulation of myosin cross-bridge function in the healthy as well as in HCM myocardium. The lack of N-ELC in Tg-Δ43 mice was found to significantly stabilize the SRX state of myosin and increase the number of myosin heads occupying a low-energy state. In agreement, Δ43 hearts showed significantly decreased ATP utilization and low actin-activated myosin ATPase compared with A57G and WT hearts. The hypercontractile activity of A57G-ELC cross-bridges was manifested by the inhibition of the SRX state, increased number of myosin heads available for interaction with actin, and higher ATPase activity. Fiber mechanics studies, echocardiography examination, and assessment of fibrosis confirmed the development of two distinct forms of cardiac remodeling in these two ELC mouse models, with pathological cardiac hypertrophy in Tg-A57G, and near physiologic cardiac growth in Tg-Δ43 animals., (© 2020 Federation of European Biochemical Societies.)

Myosin regulatory light chain (RLC) phosphorylation is important for cardiac muscle mechanics/function as well as for the Ca 2+ -troponin/tropomyosin regulation of muscle contraction. This study focuses on the arginine to glutamine (R58Q) substitution in the human ventricular RLC (MYL2 gene), linked to malignant hypertrophic cardiomyopathy in humans and causing severe functional abnormalities in transgenic (Tg) R58Q mice, including inhibition of cardiac RLC phosphorylation. Using a phosphomimic recombinant RLC variant where Ser-15 at the phosphorylation site was substituted with aspartic acid (S15D) and placed in the background of R58Q, we aimed to assess whether we could rescue/mitigate R58Q-induced structural/functional abnormalities in vitro. We show rescue of several R58Q-exerted adverse phenotypes in S15D-R58Q-reconstituted porcine cardiac muscle preparations. A low level of maximal isometric force observed for R58Q- versus WT-reconstituted fibers was restored by S15D-R58Q. Significant beneficial effects were also observed on the V max of actin-activated myosin ATPase activity in S15D-R58Q versus R58Q-reconstituted myosin, along with its binding to fluorescently labeled actin. We also report that R58Q promotes the OFF state of myosin, both in reconstituted porcine fibers and in Tg mouse papillary muscles, thereby stabilizing the super-relaxed state (SRX) of myosin, characterized by a very low ATP turnover rate. Experiments in S15D-R58Q-reconstituted porcine fibers showed a mild destabilization of the SRX state, suggesting an S15D-mediated shift in disordered-relaxed (DRX)↔SRX equilibrium toward the DRX state of myosin. Our study shows that S15D-phosphomimic can be used as a potential rescue strategy to abrogate/alleviate the RLC mutation-induced phenotypes and is a likely candidate for therapeutic intervention in HCM patients., (© 2018 Federation of European Biochemical Societies.)

Aims: The E143K (Glu → Lys) mutation in the myosin essential light chain has been associated with restrictive cardiomyopathy (RCM) in humans, but the mechanisms that underlie the development of defective cardiac function are unknown. Using transgenic E143K-RCM mice, we sought to determine the molecular and cellular triggers of E143K-induced heart remodelling., Methods and Results: The E143K-induced abnormalities in cardiac function and morphology observed by echocardiography and invasive haemodynamics were paralleled by augmented active and passive tension measured in skinned papillary muscle fibres compared with wild-type (WT)-generated force. In vitro, E143K-myosin had increased duty ratio and binding affinity to actin compared with WT-myosin, increased actin-activated ATPase activity and slower rates of ATP-dependent dissociation of the acto-myosin complex, indicating an E143K-induced myosin hypercontractility. E143K was also observed to reduce the level of myosin regulatory light chain phosphorylation while that of troponin-I remained unchanged. Small-angle X-ray diffraction data showed a decrease in the filament lattice spacing (d1,0) with no changes in the equatorial reflections intensity ratios (I1,1/I1,0) in E143K vs. WT skinned papillary muscles. The hearts of mutant-mice demonstrated ultrastructural defects and fibrosis that progressively worsened in senescent animals and these changes were hypothesized to contribute to diastolic disturbance and to mild systolic dysfunction. Gene expression profiles of E143K-hearts supported the histopathology results and showed an upregulation of stress-response and collagen genes. Finally, proteomic analysis evidenced RCM-dependent metabolic adaptations and higher energy demands in E143K vs. WT hearts., Conclusions: As a result of the E143K-induced myosin hypercontractility, the hearts of RCM mice model exhibited cardiac dysfunction, stiff ventricles and physiological, morphologic, and metabolic remodelling consistent with the development of RCM. Future efforts should be directed toward normalization of myosin motor function and the use of myosin-specific therapeutics to avert the hypercontractile state of E143K-myosin and prevent pathological cardiac remodelling., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions please email: journals.permissions@oup.com.)

We have investigated the physiology and mechanical profiles of skinned papillary muscle fibers from transgenic mice expressing the N47K mutation in the myosin regulatory light chain (RLC), shown to cause hypertrophic cardiomyopathy in humans. The results were compared with wild-type (WT) mice, both expressing the human ventricular RLC. Rate constants of a cross-bridge (XB) cycle were deduced from tension transients induced by sinusoidal length changes during maximal Ca 2+ activation, and were studied as a function of MgATP, MgADP, and Pi concentrations. N47K mutant showed slower XB cycles but higher actin-activated ATPase activity compared with WT. Consequently, N47K exhibited larger tension than WT. K 0 (ADP association constant) and K 4 (equilibrium constant of force generation) were larger in N47K, and K 1 (ATP association constant) was slightly larger in N47K vs. WT, demonstrating stronger nucleotide binding and force generation abilities of the mutant, but no changes in rigor acto-myosin binding were observed. Tension per XB was similar among groups, but N47K exhibited more XB distribution in the attached state. Larger values of tension and higher ATPase in N47K suggested that more cross-bridges participated in tension production in the mutant myocardium compared with WT. In vivo analysis of heart function, performed in ~ 12.5-month-old mice by echocardiography and invasive hemodynamics, demonstrated a significant decrease in dP/dt max -end-diastolic volume relationship, indicating a depression of ventricular contractility in N47K mice. Our findings suggest that the N47K mutation exerts its action through direct alterations of myosin motor function that ultimately result in pathological hypertrophic remodeling in N47K hearts., (© 2017 Federation of European Biochemical Societies.)

Using microarray and bioinformatics, we examined the gene expression profiles in transgenic mouse hearts expressing mutations in the myosin regulatory light chain shown to cause hypertrophic cardiomyopathy (HCM). We focused on two malignant RLC-mutations, Arginine 58→Glutamine (R58Q) and Aspartic Acid 166 → Valine (D166V), and one benign, Lysine 104 → Glutamic Acid (K104E)-mutation. Datasets of differentially expressed genes for each of three mutants were compared to those observed in wild-type (WT) hearts. The changes in the mutant vs. WT samples were shown as fold-change (FC), with stringency FC ≥ 2. Based on the gene profiles, we have identified the major signaling pathways that underlie the R58Q-, D166V- and K104E-HCM phenotypes. The correlations between different genotypes were also studied using network-based algorithms. Genes with strong correlations were clustered into one group and the central gene networks were identified for each HCM mutant. The overall gene expression patterns in all mutants were distinct from the WT profiles. Both malignant mutations shared certain classes of genes that were up or downregulated, but most similarities were noted between D166V and K104E mice, with R58Q hearts showing a distinct gene expression pattern. Our data suggest that all three HCM mice lead to cardiomyopathy in a mutation-specific manner and thus develop HCM through diverse mechanisms., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We discuss here the potential mechanisms of action associated with hypertrophic (HCM) or dilated (DCM) cardiomyopathy causing mutations in the myosin regulatory (RLC) and essential (ELC) light chains. Specifically, we focus on four HCM mutations: RLC-A13T, RLC-K104E, ELC-A57G and ELC-M173V, and one DCM RLC-D94A mutation shown by population studies to cause different cardiomyopathy phenotypes in humans. Our studies indicate that RLC and ELC mutations lead to heart disease through different mechanisms with RLC mutations triggering alterations of the secondary structure of the RLC which further affect the structure and function of the lever arm domain and impose changes in the cross bridge cycling rates and myosin force generation ability. The ELC mutations exert their detrimental effects through changes in the interaction of the N-terminus of ELC with actin altering the cross talk between the thick and thin filaments and ultimately resulting in an altered force-pCa relationship. We also discuss the effect of mutations on myosin light chain phosphorylation. Exogenous myosin light chain phosphorylation and/or pseudo-phosphorylation were explored as potential rescue tools to treat hypertrophy-related cardiac phenotypes.

In this study we aimed to provide an in-depth proteomic analysis of differentially expressed proteins in the hearts of transgenic mouse models of pathological and physiological cardiac hypertrophy using tandem mass tag labeling and liquid chromatography tandem mass spectrometry. The Δ43 mouse model, expressing the 43-amino-acid N-terminally truncated myosin essential light chain (ELC) served as a tool to study the mechanisms of physiological cardiac remodeling, while the pathological hypertrophy was investigated in A57G (Alanine 57 → Glycine) ELC mice. The results showed that 30 proteins were differentially expressed in Δ43 versus A57G hearts as determined by multiple pair comparisons of the mutant versus wild-type (WT) samples with P < 0.05. The A57G hearts showed differential expression of nine mitochondrial proteins involved in metabolic processes compared to four proteins for ∆43 hearts when both mutants were compared to WT hearts. Comparisons between ∆43 and A57G hearts showed an upregulation of three metabolically important mitochondrial proteins but downregulation of nine proteins in ∆43 hearts. The physiological model of cardiac hypertrophy (∆43) showed no changes in the levels of Ca(2+)-binding proteins relative to WT, while the pathologic model (A57G) showed the upregulation of three Ca(2+)-binding proteins, including sarcalumenin. Unique differences in chaperone and fatty acid metabolism proteins were also observed in Δ43 versus A57G hearts. The proteomics data support the results from functional studies performed previously on both animal models of cardiac hypertrophy and suggest that the A57G- and not ∆43- mediated alterations in fatty acid metabolism and Ca(2+) homeostasis may contribute to pathological cardiac remodeling in A57G hearts.

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., (© 2015 FEBS.)

Familial hypertrophic cardiomyopathy (HCM) is associated with mutations in sarcomeric proteins, including the myosin regulatory light chain (RLC). Here we studied the impact of three HCM mutations located in the NH2 terminus of the RLC on the molecular mechanism of β-myosin heavy chain (MHC) cross-bridge mechanics using the in vitro motility assay. To generate mutant β-myosin, native RLC was depleted from porcine cardiac MHC and reconstituted with mutant (A13T, F18L, and E22K) or wild-type (WT) human cardiac RLC. We characterized the mutant myosin force and motion generation capability in the presence of a frictional load. Compared with WT, all three mutants exhibited reductions in maximal actin filament velocity when tested under low or no frictional load. The actin-activated ATPase showed no significant difference between WT and HCM-mutant-reconstituted myosins. The decrease in velocity has been attributed to a significantly increased duty cycle, as was measured by the dependence of actin sliding velocity on myosin surface density, for all three mutant myosins. These results demonstrate a mutation-induced alteration in acto-myosin interactions that may contribute to the pathogenesis of HCM., (Copyright © 2014 the American Physiological Society.)

We have examined, for the first time, the effects of the familial hypertrophic cardiomyopathy (HCM)-associated Lys104Glu mutation in the myosin regulatory light chain (RLC). Transgenic mice expressing the Lys104Glu substitution (Tg-MUT) were generated and the results were compared to Tg-WT (wild-type human ventricular RLC) mice. Echocardiography with pulse wave Doppler in 6month-old Tg-MUT showed early signs of diastolic disturbance with significantly reduced E/A transmitral velocities ratio. Invasive hemodynamics in 6month-old Tg-MUT mice also demonstrated a borderline significant prolonged isovolumic relaxation time (Tau) and a tendency for slower rate of pressure decline, suggesting alterations in diastolic function in Tg-MUT. Six month-old mutant animals had no LV hypertrophy; however, at >13months they displayed significant hypertrophy and fibrosis. In skinned papillary muscles from 5 to 6month-old mice a mutation induced reduction in maximal tension and slower muscle relaxation rates were observed. Mutated cross-bridges showed increased rates of binding to the thin filaments and a faster rate of the power stroke. In addition, ~2-fold lower level of RLC phosphorylation was observed in the mutant compared to Tg-WT. In line with the higher mitochondrial content seen in Tg-MUT hearts, the MUT-myosin ATPase activity was significantly higher than WT-myosin, indicating increased energy consumption. In the in vitro motility assay, MUT-myosin produced higher actin sliding velocity under zero load, but the velocity drastically decreased with applied load in the MUT vs. WT myosin. Our results suggest that diastolic disturbance (impaired muscle relaxation, lower E/A) and inefficiency of energy use (reduced contractile force and faster ATP consumption) may underlie the Lys104Glu-mediated HCM phenotype., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The functional consequences of the familial hypertrophic cardiomyopathy A57G (alanine-to-glycine) mutation in the myosin ventricular essential light chain (ELC) were assessed in vitro and in vivo using previously generated transgenic (Tg) mice expressing A57G-ELC mutant vs. wild-type (WT) of human cardiac ELC and in recombinant A57G- or WT-protein-exchanged porcine cardiac muscle strips. Compared with the Tg-WT, there was a significant increase in the Ca²⁺ sensitivity of force (ΔpCa₅₀ ≅ 0.1) and an ~1.3-fold decrease in maximal force per cross section of muscle observed in the mutant preparations. In addition, a significant increase in passive tension in response to stretch was monitored in Tg-A57G vs. Tg-WT strips indicating a mutation-induced myocardial stiffness. Consistently, the hearts of Tg-A57G mice demonstrated a high level of fibrosis and hypertrophy manifested by increased heart weight-to-body weight ratios and a decreased number of nuclei indicating an increase in the two-dimensional size of Tg-A57G vs. Tg-WT myocytes. Echocardiography examination showed a phenotype of eccentric hypertrophy in Tg-A57G mice, enhanced left ventricular (LV) cavity dimension without changes in LV posterior/anterior wall thickness. Invasive hemodynamics data revealed significantly increased end-systolic elastance, defined by the slope of the pressure-volume relationship, indicating a mutation-induced increase in cardiac contractility. Our results suggest that the A57G allele causes disease by means of a discrete modulation of myofilament function, increased Ca²⁺ sensitivity, and decreased maximal tension followed by compensatory hypertrophy and enhanced contractility. These and other contributing factors such as increased myocardial stiffness and fibrosis most likely activate cardiomyopathic signaling pathways leading to pathologic cardiac remodeling.

Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in sarcomeric proteins including the myosin regulatory light chain (RLC). Two such FHC mutations, R58Q and N47K, located near the cationic binding site of the RLC, have been identified from population studies. To examine the molecular basis for the observed phenotypes, we exchanged endogenous RLC from native porcine cardiac myosin with recombinant human ventricular wild type (WT) or FHC mutant RLC and examined the ability of the reconstituted myosin to propel actin filament sliding using the in vitro motility assay. We find that, whereas the mutant myosins are indistinguishable from the controls (WT or native myosin) under unloaded conditions, both R58Q- and N47K-exchanged myosins show reductions in force and power output compared with WT or native myosin. We also show that the changes in loaded kinetics are a result of mutation-induced loss of myosin strain sensitivity of ADP affinity. We propose that the R58Q and N47K mutations alter the mechanical properties of the myosin neck region, leading to altered load-dependent kinetics that may explain the observed mutant-induced FHC phenotypes.

Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca(2+)-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (tau(1)) of myosin heads from thin filaments, 2) rebinding time (tau(2)) of the cross bridges to actin, and 3) dissociation time (tau(3)) of ADP from the active site of myosin. tau(1) was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. tau(2) was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. tau(3) was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. tau(1) was statistically greater in Tg-m than in controls. tau(2) was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. tau(3) was the same in Tg-m and controls. To determine whether the difference in tau(1) was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in tau(1) was probably caused by myofibrillar disarray. The decrease in tau(2) of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice.

Clinical studies have revealed that mutations in the ventricular myosin regulatory light chain (RLC) lead to the development of familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray and sudden cardiac death. While mutations in other contractile proteins have been studied widely by others, there is no report elucidating the mechanism(s) associated with FHC-linked RLC mutations. In this study, we have assessed the functional consequences of two RLC mutations, R58Q and N47K, in transgenic mice. Clinical phenotypes associated with these mutations included inter-ventricular hypertrophy, abnormal ECG findings and the R58Q mutation caused multiple cases of premature sudden cardiac death. Simultaneous measurements of the ATPase and force in transgenic skinned papillary muscle fibers from mutated versus control mice showed an increase in the Ca(2+) sensitivity of ATPase and steady-state force only in R58Q fibers. The calculated energy cost or rate of dissociation of force generating myosin cross-bridges (ATPase/force ratio) plotted as a function of activation state was the same in all groups of fibers. Both mutations caused prolonged [Ca(2+)] transients in electrically stimulated intact papillary muscles; however, the R58Q mutation also resulted in a significantly prolonged force transient. Our results suggest that the phenotypes of FHC observed in patients harboring these RLC mutations correlate with the extent of physiological changes monitored in transgenic fibers. Cardiac hypertrophy observed in patients is most likely caused by the activation of compensatory mechanisms ensuing from higher workloads due to incomplete relaxation as evidenced by prolonged [Ca(2+)] transients for both N47K and R58Q fibers. Furthermore, the poor prognosis of the R58Q patients may be associated with more severe diastolic dysfunction due to the slower off-rate of Ca(2+) from troponin C leading to longer force and [Ca(2+)] transients and increased Ca(2+) sensitivity of ATPase and force.

We have studied the physiological effects of the troponin T (TnT) F110I and R278C mutations associated with familial hypertrophic cardiomyopathy (FHC) in humans. Three to four-month-old transgenic (Tg) mice expressing F110I-TnT and R278C-TnT did not develop significant hypertrophy or ventricular fibrosis even after chronic exercise challenge. The F110I mutation impaired acute exercise tolerance, whereas R278C did not. Skinned papillary muscle fibers from transgenic mice expressing F110I-TnT demonstrated increased Ca(2+) sensitivity of force and ATPase activity, and likewise an increased Ca(2+) sensitivity of force was observed in F110I-TnT-reconstituted human cardiac muscle preparations. In contrast, no changes in force or the ATPase-pCa dependencies were observed in transgenic R278C fibers or in human fibers reconstituted with the R278C-TnT mutant. The maximal level of force development was dramatically decreased in both transgenic mice. However, the maximal ATPase was not different (R278C-TnT) or only slightly less (F110I-TnT) than that of non-Tg and WT-Tg littermates. Consequently, their ratios of ATPase/force (energy cost) at all Ca(2+) concentrations were dramatically higher compared with non-Tg and WT-Tg fibers. This increase in energy cost most likely results from a decrease in force per myosin cross-bridge, because forcing all cross-bridges into the force generating state by substitution of MgADP for MgATP in maximum contracting solutions resulted in the same increase in maximal force (15%) in all transgenic and non-transgenic preparations. The combination of increased Ca(2+) sensitivity and energy cost in the F110I hearts may be responsible for the greater severity of this phenotype compared with the R278C mutation.