Human Ventricular Regulatory Light Chain Research Articles

Molecular basis of force-pCa relation in MYL2 cardiomyopathy mice: Role of the super-relaxed state of myosin

In this study, we investigated the role of the super-relaxed (SRX) state of myosin in the structure-function relationship of sarcomeres in the hearts of mouse models of cardiomyopathy-bearing mutations in the human ventricular regulatory light chain (RLC, MYL2 gene). Skinned papillary muscles from hypertrophic (HCM-D166V) and dilated (DCM-D94A) cardiomyopathy models were subjected to small-angle X-ray diffraction simultaneously with isometric force measurements to obtain the interfilament lattice spacing and equatorial intensity ratios (I11/I10) together with the force-pCa relationship over a full range of [Ca2+] and at a sarcomere length of 2.1 μm. In parallel, we studied the effect of mutations on the ATP-dependent myosin energetic states. Compared with wild-type (WT) and DCM-D94A mice, HCM-D166V significantly increased the Ca2+ sensitivity of force and left shifted the I11/I10-pCa relationship, indicating an apparent movement of HCM-D166V cross-bridges closer to actin-containing thin filaments, thereby allowing for their premature Ca2+ activation. The HCM-D166V model also disrupted the SRX state and promoted an SRX-to-DRX (super-relaxed to disordered relaxed) transition that correlated with an HCM-linked phenotype of hypercontractility. While this dysregulation of SRX ↔ DRX equilibrium was consistent with repositioning of myosin motors closer to the thin filaments and with increased force-pCa dependence for HCM-D166V, the DCM-D94A model favored the energy-conserving SRX state, but the structure/function-pCa data were similar to WT. Our results suggest that the mutation-induced redistribution of myosin energetic states is one of the key mechanisms contributing to the development of complex clinical phenotypes associated with human HCM-D166V and DCM-D94A mutations.

Cardiomyopathy mutations impact the actin-activated power stroke of human cardiac myosin

Cardiac muscle contraction is driven by the molecular motor myosin, which uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, although it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a fluorescence resonance energy transfer (FRET) strategy to investigate structural changes in the lever arm domain of human β-cardiac myosin subfragment 1 (M2β-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2β-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic measurements. We propose that the fast phase measured with our FRET probes represents the macroscopic rate constant associated with actin-activated rotation of the lever arm during the power stroke in M2β-S1. Our results demonstrated M2β-S1 has a slower actin-activated power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the actin-activated power stroke and phosphate release are consistent with a model in which the power stroke occurs before phosphate release and the two steps are tightly coupled. We suggest that the actin-activated power stroke is highly reversible but followed by a highly irreversible phosphate release step in the absence of load and free phosphate. We demonstrated that hypertrophic cardiomyopathy (R723G)- and dilated cardiomyopathy (F764L)-associated mutations both reduced actin activation of the power stroke in M2β-S1. We also demonstrate that both mutations alter in vitro actin gliding in the presence and absence of load. Thus, examining the structural kinetics of the power stroke in M2β-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction.

Impact of Regulatory Light Chain Mutation (K104E) on the Atpase and Motor Properties of Human Cardiac Myosin

Mutations in the cardiac myosin heavy chain (MYH7) as well as the essential (MYL3) and regulatory (MYL2) light chains are known to lead to cardiomyopathies with variable phenotypes, including hypertrophic (HCM) and dilated (DCM) cardiomyopathy. We investigated the impact of the HCM-associated regulatory light chain (RLC) mutation (K104E) on myosin ATPase kinetics and in vitro motility. We expressed and purified human cardiac myosin subfragment 1 (M2B-S1) and exchanged on the human ventricular RLC, with the mutant or WT RLC. A mouse model of K104E was found to cause cardiac hypertrophy, fibrosis, and diastolic dysfunction in older animals suggesting a slow development of HCM, while variable penetrance of the mutation in human populations suggest the impact of K104E may be subtle. We found that maximum actin-activated ATPase activity and the actin-dependence of ATPase were similar in K104E and WT. Actin gliding velocities in the in vitro motility assay were slightly increased in K104E. We found that the ADP release rate constant in the presence of actin also was slightly increased in K104E compared to WT. Our results suggest that HCM-associated RLC mutations may only have a minor impact on unloaded ATPase kinetics and in vitro actin gliding, which may suggest other factors could be altered by the mutation such as mechanical load and myosin regulation. Future experiments will address the impact of the mutation in mouse cardiac vs. human cardiac myosin as well as the impact of the mutation on load-dependent motility and the off state (super-relaxed state) of cardiac myosin.

Phosphomimetic-mediated invitro rescue of hypertrophic cardiomyopathy linked to R58Q mutation in myosin regulatory light chain.

Myosin regulatory light chain (RLC) phosphorylation is important for cardiac muscle mechanics/function as well as for the Ca2+ -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 invitro. 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 Vmax 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.

Cardiac contractility, motor function, and cross-bridge kinetics in N47K-RLC mutant mice.

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 Ca2+ 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. K0 (ADP association constant) and K4 (equilibrium constant of force generation) were larger in N47K, and K1 (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/dtmax -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.

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.

Hypertrophic cardiomyopathy associated Lys104Glu mutation in the myosin regulatory light chain causes diastolic disturbance in mice

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.

Familial Hypertrophic Cardiomyopathy-Linked Mutation (K104E) in the Myosin Regulatory Light Chain Affects Sarcomeric Structure and Function in Tg-Mice

Structural and functional effects of the familial hypertrophic cardiomyopathy (FHC)-linked mutation (Lysine 104-to-Glutamic Acid, K104E) in the human ventricular regulatory light chain (RLC) were studied in transgenic (Tg) mice. Small angle X-ray diffraction measurements on freshly skinned papillary muscle fibers demonstrated a mutation-induced increase in the interfilament lattice spacing at both short (2.3 µm) and long (2.5 µm) sarcomere lengths (SL) compared with Tg-WT. The intensity ratio (I1,1/I1,0) in WT fibers increased upon stretch (from SL=2.3 to SL=2.5 µm) indicating a shift in the cross-bridge mass distribution toward the thin filaments. In contrary, the K104E fibers demonstrated no change in I1,1/I1,0 on stretch. In line with X-ray data, K104E fibers developed higher passive tension at pCa 8 when consecutively stretched by 10%, 20%, 30% and 40% of their length compared to WT. Furthermore, the K104E mutation significantly decreased the endogenous RLC phosphorylation (∼2-fold) compared to WT myocardium. Histopathological changes included signs of hypertrophy and severe fibrotic lesions in Tg-K104E hearts. The higher mitochondrial content seen in the left ventricles of K104E mice by electron microscopy was supported by significantly increased myosin ATPase activity in Tg-K104E mice indicating higher energy demand and ATP consumption by the mutant hearts. Studies using single molecule detection demonstrated significantly decreased cross-bridge dissociation rates with lower duty cycle (indicative of lower force production) in Tg-K104E cardiac myofibrils compared to WT. Our results in Tg-K104E mice demonstrated cardiomyocyte hypertrophy and fibrosis, and significant alterations in sarcomere structure and function. Morphological abnormalities, increased passive stiffness and a slower cross bridge dissociation rate in Tg-K104E mice mirror the diastolic dysfunction phenotype observed in K104E positive patients. Supported by AHA12PRE12030412 (to WH), and NIH HL-071778 and HL-108343 (to DSC).

Myosin regulatory light chain mutation found in hypertrophic cardiomyopathy patients increases isometric force production in transgenic mice.

FHC (familial hypertrophic cardiomyopathy) is a heritable form of cardiac hypertrophy caused by mutations in genes encoding sarcomeric proteins. The present study focuses on the A13T mutation in the human ventricular myosin RLC (regulatory light chain) that is associated with a rare FHC variant defined by mid-ventricular obstruction and septal hypertrophy. We generated heart-specific Tg (transgenic) mice with ~10% of human A13T-RLC mutant replacing the endogenous mouse cardiac RLC. Histopathological examinations of longitudinal heart sections from Tg-A13T mice showed enlarged interventricular septa and profound fibrotic lesions compared with Tg-WT (wild-type), expressing the human ventricular RLC, or non-Tg mice. Functional studies revealed an abnormal A13T mutation-induced increase in isometric force production, no change in the force-pCa relationship and a decreased Vmax of the acto-myosin ATPase. In addition, a fluorescence-based assay showed a 3-fold lower binding affinity of the recombinant A13T mutant for the RLC-depleted porcine myosin compared with WT-RLC. These results suggest that the A13T mutation triggers a hypertrophic response through changes in cardiac sarcomere organization and myosin cross-bridge function leading to abnormal remodelling of the heart. The significant functional changes observed, despite a low level of A13T mutant incorporation into myofilaments, suggest a 'poison-peptide' mechanism of disease.

FHC-linked Mutations in the Myosin Regulatory Light Chain Interfere with RLC Phosphorylation in Transgenic Mice

Previous studies have shown that MLCK-phosphorylation of the ventricular regulatory light chain (RLC) increases myofilament Ca2+ sensitivity, maximal level of force and rate of tension development thereby enhancing the systolic function of the heart. We have investigated the effect of FHC (familial hypertrophic cardiomyopathy) mutations in the myosin RLC on its phosphorylation measured in rapidly frozen ventricular samples from transgenic (Tg) mice. We observe that the Aspartate to Valine substitution in Tg-D166V mice and Arginine to Glutamine in Tg-R58Q mice result in a decreased phosphorylation of RLC detected in left ventricular samples from Tg-mutant compared to Tg-WT mice expressing the human ventricular RLC. The level of RLC phosphorylation was determined by Western blotting utilizing human specific phospho-RLC antibodies (gift from Dr. N. Epstein, NIH). Our data from skinned Tg-D166V and Tg-R58Q papillary muscle fibers show that both RLC mutations lead to a decreased maximal level of force and to slower kinetics of force generating myosin cross-bridges compared to WT fibers. Studies in intact papillary muscle fibers show prolonged force transients for both D166V and R58Q mutants. In addition, the hearts of aging mutant mice demonstrate histopathological changes and frequent occurrences of fibrotic lesions. Clinical studies revealed that both D166V and R58Q mutations are associated with severe FHC phenotypes with multiple cases of sudden cardiac death. Our results suggest that phosphorylation of RLC plays an important role in regulating cardiac function and its deficit may contribute to malignant FHC phenotypes. Reduced RLC phosphorylation observed in the D166V and R58Q hearts correlates with our cellular findings and could be responsible for delayed force transients, slower cross-bridge kinetics and decreased force observed in Tg-D166V and Tg-R58Q papillary muscle fibers. Supported by NIH- HL071778 (D.S-C.).

The E22K mutation of myosin RLC that causes familial hypertrophic cardiomyopathy increases calcium sensitivity of force and ATPase in transgenic mice

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease caused by mutations in all of the major sarcomeric proteins, including the ventricular myosin regulatory light-chain (RLC). The E22K-RLC mutation has been associated with a rare variant of cardiac hypertrophy defined by mid-left ventricular obstruction due to papillary muscle hypertrophy. This mutation was later found to cause ventricular and septal hypertrophy. We have generated transgenic (Tg) mouse lines of myc-WT (wild type) and myc-E22K mutant of human ventricular RLC and have examined the functional consequences of this FHC mutation in skinned cardiac-muscle preparations. In longitudinal sections of whole mouse hearts stained with hematoxylin and eosin, the E22K-mutant hearts of 13-month-old animals showed signs of inter-ventricular septal hypertrophy and enlarged papillary muscles with no filament disarray. Echo examination did not reveal evidence of cardiac hypertrophy in Tg-E22K mice compared to Tg-WT or Non-Tg hearts. Physiological studies utilizing skinned cardiac-muscle preparations showed an increase by DeltapCa50>or=0.1 in Ca(2+) sensitivity of myofibrillar ATPase activity and force development in Tg-E22K mice compared with Tg-WT or Non-Tg littermates. Our results suggest that E22K-linked FHC is mediated through Ca(2+)-dependent events. The FHC-mediated structural perturbations in RLC that affect Ca(2+) binding properties of the mutated myocardium are responsible for triggering the abnormal function of the heart that in turn might initiate a hypertrophic process and lead to heart failure.