Mutant Myosin Research Articles

Abstract Mo122: Investigating the Role of the Lysine Methyltransferase SMYD1 in Striated Muscle Motor Domain Folding

Properly coordinated folding and assembly of myosin into the sarcomere thick filament is critical for cardiac and skeletal muscle development, function, growth, and maintenance. The motor domain of myosin heavy chain (MHC) requires muscle-specific chaperones for proper folding, making it currently impossible to fold in non-muscle cells. Previous studies found that HSP90 and UNC45B are necessary, but not sufficient, for folding the motor domain, indicating other factors are involved. Defining the minimal set of factors for motor domain folding would allow for development of an improved expression system and accelerate the study of myosin mutations and myosin-targeted drug development. We hypothesize that the muscle-specific lysine methyltransferase SMYD1 contributes to MHC folding and stability. We found SMYD1 associates with UNC45B, HSP90, and MHC in the adult human heart. To study motor domain folding we generated a human MYH7 motor domain fused to GFP (MD::GFP). Transfection into COS-7 cells resulted in unfolded aggregates while adenoviral infection of C 2 C 12 myotubes leads to folded and functional MD::GFP. Interestingly, the folded MD::GFP contains lysine trimethylation, which may arise from the methyltransferase activity of SMYD1. When COS-7 cells were co-transfected with UNC45B and SMYD1, we observed increased total protein levels of non-functional MD::GFP without affecting RNA levels as assessed by RT-qPCR. This effect was not observed when MD::GFP was co-transfected with UNC45B or SMYD1 alone and is dependent on HSP90. We found that UNC45B and SMYD1 stabilized MD::GFP as determined by cycloheximide chase assays. We also found SMYD1 associates with MD::GFP, suggesting this stabilizing effect is due to a direct interaction. Interestingly, a catalytically inactive SMYD1 also increased MD::GFP protein levels in COS-7 cells, consistent with our observation that no significant changes in global protein methylation were observed in the presence of WT SMYD1. Our data suggests that SMYD1 stabilizes the motor domain of striated muscle myosin in conjunction with UNC45B and HSP90, in a manner that is independent of its methyltransferase activity. However, UNC45B and SMYD1 are insufficient for motor domain folding in a non-muscle cell line, suggesting that additional folding factors or proper stoichiometric ratios of chaperones/co-chaperones may be required.

Gene transfer of human cardiomyopathy β-MyHC mutant R403Q directly alters intact cardiac myocyte calcium homeostasis and causes hyper-contractility.

The R403Q mutation of human cardiac β-myosin heavy chain was the first missense mutation of a sarcomeric protein identified as being causal for hypertrophic cardiomyopathy (HCM), in humans. The direct effect of the R403Q mutant myosin on intracellular calcium homeostasis and contractility is not fully known. Here we have used in vitro gene transfer of the R403Q mutant human β-myosin to study its direct effects on single intact adult cardiac myocyte contractility and calcium homeostasis. In the first experiments, adult cardiac myocytes transduced with the R403Q mutant myosin recombinant viral vectors were compared to myocytes transduced with wild-type human β-myosin (wtMYH7). Efficiency of gene transfer was high in both groups (>98%) and the degree of stoichiometric myofilament incorporation of either the mutant or normal myosin was comparable at ∼40% in quiescent myocytes in primary culture. Sarcomere structure and cellular morphology were unaffected by R403Q myosin expression and myofilament incorporation. Functionally, in electrically paced cardiac myocytes, the R403Q mutant myosin caused a significant increase in intracellular calcium concentration and myocyte hyper-contractility. At the sub-cellular myofilament level, the mutant myosin increased the calcium sensitivity of steady state isometric tension development and increased isometric cross-bridge cycling kinetics. R403Q myocytes became arrhythmic after β-adrenergic stimulation with spontaneous calcium transients and contractions in between electrical stimuli. These results indicate that human R403Q mutant myosin directly alters myofilament function and intracellular calcium cycling. Elevated calcium levels may provide a trigger for the ensuing hypertrophy and susceptibility to arrhythmia that are characteristic of HCM.

Aficamten is a small-molecule cardiac myosin inhibitor designed to treat hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is an inherited disease of the sarcomere resulting in excessive cardiac contractility. The first-in-class cardiac myosin inhibitor, mavacamten, improves symptoms in obstructive HCM. Here we present aficamten, a selective small-molecule inhibitor of cardiac myosin that diminishes ATPase activity by strongly slowing phosphate release, stabilizing a weak actin-binding state. Binding to an allosteric site on the myosin catalytic domain distinct from mavacamten, aficamten prevents the conformational changes necessary to enter the strongly actin-bound force-generating state. In doing so, aficamten reduces the number of functional myosin heads driving sarcomere shortening. The crystal structure of aficamten bound to cardiac myosin in the pre-powerstroke state provides a basis for understanding its selectivity over smooth and fast skeletal muscle. Furthermore, in cardiac myocytes and in mice bearing the hypertrophic R403Q cardiac myosin mutation, aficamten reduces cardiac contractility. Our findings suggest aficamten holds promise as a therapy for HCM.

Dilated cardiomyopathy mutation in beta-cardiac myosin enhances actin activation of the power stroke and phosphate release.

Inherited mutations in human beta-cardiac myosin (M2β) can lead to severe forms of heart failure. The E525K mutation in M2β is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2β subfragment 1 (S1) we found that E525K enhances (threefold) the maximum steady-state actin-activated ATPase activity (k cat) and decreases (eightfold) the actin concentration at which ATPase is one-half maximal (K ATPase). We also found a twofold to fourfold increase in the actin-activated power stroke and phosphate release rate constants at 30 μM actin, which overall enhanced the duty ratio threefold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2β S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis.

Unveiling the Mechanism of Phenamacril Resistance in F. graminearum: Computational and Experimental Insights into the C423A Mutation in FgMyoI.

Phenamacril (PHA) is a highly selective fungicide for controlling fusarium head blight (FHB) mainly caused by F. graminearum and F. asiaticum. However, the C423A mutation in myosin I of F. graminearum (FgMyoI) leads to natural resistance to PHA. Here, based on the computational approaches and biochemical validation, we elucidate the atomic-level mechanism behind the natural resistance of F. graminearum to the fungicide PHA due to the C423A mutation in FgMyoI. The mutation leads to a rearrangement of pocket residues, resulting in increased size and flexibility of the binding pocket, which impairs the stable binding of PHA. MST experiments confirm that the mutant protein FgMyoIC423A exhibits significantly reduced affinity for PHA compared to wild-type FgMyoI and the nonresistant C423K mutant. This decreased binding affinity likely underlies the development of PHA resistance in F. graminearum. Conversely, the nonresistant C423K mutant retains sensitivity to PHA due to the introduction of a strong hydrogen bond donor, which facilitates stable binding of PHA in the pocket. These findings shed light on the molecular basis of PHA resistance and provide new directions for the creation of new myosin inhibitors.

Tail length and E525K dilated cardiomyopathy mutant alter human β-cardiac myosin super-relaxed state.

Dilated cardiomyopathy (DCM) is a condition characterized by impaired cardiac function, due to myocardial hypo-contractility, and is associated with point mutations in β-cardiac myosin, the molecular motor that powers cardiac contraction. Myocardial function can be modulated through sequestration of myosin motors into an auto-inhibited "super-relaxed" state (SRX), which may be further stabilized by a structural state known as the "interacting heads motif" (IHM). Here, we sought to determine whether hypo-contractility of DCM myocardium results from reduced function of individual myosin molecules or from decreased myosin availability to interact with actin due to increased IHM/SRX stabilization. We used an established DCM myosin mutation, E525K, and characterized the biochemical and mechanical activity of wild-type and mutant human β-cardiac myosin constructs that differed in the length of their coiled-coil tail, which dictates their ability to form the IHM/SRX state. We found that short-tailed myosin constructs exhibited low IHM/SRX content, elevated actin-activated ATPase activity, and fast velocities in unloaded motility assays. Conversely, longer-tailed constructs exhibited higher IHM/SRX content and reduced actomyosin ATPase and velocity. Our modeling suggests that reduced velocities may be attributed to IHM/SRX-dependent sequestration of myosin heads. Interestingly, longer-tailed E525K mutants showed no apparent impact on velocity or actomyosin ATPase at low ionic strength but stabilized IHM/SRX state at higher ionic strength. Therefore, the hypo-contractility observed in DCM may be attributable to reduced myosin head availability caused by enhanced IHM/SRX stability in E525K mutants.

Myosin-Catalyzed ATP Hydrolysis in the Presence of Disease-Causing Mutations: Mavacamten as a Way to Repair Mechanism.

Hypertrophic cardiomyopathy is one of the most common forms of genetic cardiomyopathy. Mavacamten is a first-in-class myosin modulator that was identified via activity screening on the wild type, and it is FDA-approved for the treatment of obstructive hypertrophic cardiomyopathy (HCM). The drug selectively binds to the cardiac β-myosin, inhibiting myosin function to decrease cardiac contractility. Though the drug is thought to affect multiple steps of the myosin cross-bridge cycle, its detailed mechanism of action is still under investigation. Individual steps in the overall cross-bridge cycle must be queried to elucidate the full mechanism of action. In this study, we utilize the rare-event method of transition path sampling to generate reactive trajectories to gain insights into the action of the drug on the dynamics and rate of the ATP hydrolysis step for human cardiac β-myosin. We study three known HCM causative myosin mutations: R453C, P710R, and R712L to observe the effect of the drug on the alterations caused by these mutations in the chemical step. Since the crystal structure of the drug-bound myosin was not available at the time of this work, we created a model of the drug-bound system utilizing a molecular docking approach. We find a significant effect of the drug in one case, where the actual mechanism of the reaction is altered from the wild type by mutation. The drug restores both the rate of hydrolysis to the wildtype level and the mechanism of the reaction. This is a way to check the effect of the drug on untested mutations.

A therapeutic leap: how myosin inhibitors moved from cardiac interventions to skeletal muscle myopathy solutions.

The myosin inhibitor mavacamten has transformed the management of obstructive hypertrophic cardiomyopathy (HCM) by targeting myosin ATPase activity to mitigate cardiac hypercontractility. This therapeutic mechanism has proven effective for patients with HCM independent of having a primary gene mutation in myosin. In this issue of the JCI, Buvoli et al. report that muscle hypercontractility is a mechanism of pathogenesis underlying muscle dysfunction in Laing distal myopathy, a disorder characterized by mutations altering the rod domain of β myosin heavy chain. The authors performed detailed physiological, molecular, and biomechanical analyses and demonstrated that myosin ATPase inhibition can correct a large extent of muscle abnormalities. The findings offer a therapeutic avenue for Laing distal myopathy and potentially other myopathies. This Commentary underscores the importance of reevaluating myosin activity's role across myopathies in general for the potential development of targeted myosin inhibitors to treat skeletal muscle disorders.

Sequences in the myosin A rod interact with UNC-89/obscurin and the zinc-finger protein UNC-98 during thick filament assembly and M-line formation in C. elegans striated muscle.

The M-line of striated muscle is a complex structure that anchors myosin-containing thick filaments and also participates in signaling and proteostasis. While the physical associations among many M-line components have been defined, the mechanism of thick filament attachment is not completely understood. In Caenorhabditis elegans, myosin A is essential for viability and forms the site of M-line attachment at the center of the filament, whereas myosin B forms the filament arms. Using a mutant myosin A that forms ectopic filaments, we examined interactions between myosin A and M-line proteins in intact muscle cells. Ectopic myosin A recruits the giant kinase UNC-89/obscurin, a presumed scaffolding protein, in an interaction that requires the zinc-finger protein UNC-98, but not UNC-82/NUAK, UNC-97/PINCH, or UNC-96. In myosin A mutants, UNC-89/obscurin patterning is highly defective in embryos and adults. A chimeric myosin containing 169 residues of the myosin A C-terminal rod, coincident with the UNC-98/ZnF binding site, is sufficient for colocalization of UNC-89/obscurin and UNC-98/ZnF in M-line structures whereas a myosin chimera lacking these residues colocalizes with UNC-89/obscurin in M-lines that lack UNC-98. Thus, at least two myosin A rod regions contribute independently to M-line organization. We hypothesize that these M-line-organizing functions correspond to the essential "filament initiation function" performed by this isoform.

Functional characterization of calmodulin-like proteins, CML13 and CML14, as novel light chains of Arabidopsis class VIII myosins.

Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck region to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a myosin VIII. Here, we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among CMLs tested for binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with red fluorescent protein-myosin fusion proteins containing IQ and tail domains of myosin VIIIs. In vitro actin motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains. Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant, myosin viii4KO. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.

Human Skeletal myopathy myosin mutations disrupt myosin head sequestration.

Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle, and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyse the effects of common MYH7 and MYH2 mutations in the light meromyosin region of myosin (LMM). Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in-silico modelling showed that myosin coiled-coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients, and Mant-ATP chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with X-ray diffraction measurements to estimate myosin head order we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofibre mechanics experiments to investigate contractile function showed myofibre contractility was not affected. These findings indicate that the structural remodelling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies.

Abstract 15515: SGLT2 Inhibitor Empagliflozin Increases Fatty Acid Oxidation, Coupling Between Glycolysis and Glucose Oxidation, and Decreases Left Ventricular Hypertrophy in Mice With Hypertrophic Cardiomyopathy Due to Myosin R403Q Mutation

Background: A metabolic switch from fatty acid oxidation (FAO) to glycolysis associates with LV hypertrophy (LVH). Glucagon signaling increases FAO in the heart and sodium-glucose co-transporter 2 inhibitors (SGLT2i) raise glucagon/insulin ratio (GIR) in patients. We have shown that SGLT2i reprogram cardiac metabolic genes favoring FAO, yet the mechanism is unknown. Hypothesis: SGLT2i increases GIR, thereby increasing FAO and glycolysis/glucose oxidation coupling (GGC), which in turn attenuates LVH and fibrosis in a murine model of HCM (R403Q myosin mutation). Methods: Mice 9 weeks of age were treated ± SGLT2i empagliflozin (EMPA, 0.15 mg/g chow). After 2 weeks, prior to LVH onset, myocardial transcriptomic analysis was performed. After 16 weeks, echocardiography, proteomics, fasted GIR, and histology were obtained. Glucose fluxes, energetics and 13 C substrate oxidation were measured in isolated beating hearts by 31 P and 13 C NMR spectroscopy. Results: R403Q mice had ↓GIR which was normalized by EMPA. Transcriptomics (2 weeks) and proteomics (16 weeks) confirmed downregulation of electron transport and FAO pathways, and upregulation of LVH and fibrosis pathways. At 16 weeks, R403Q hearts exhibited LVH, fibrosis, ↓diastolic function and ↓contractile reserve (rate pressure product increase between low and high workload). Metabolically, R403Q had ↑glucose uptake and ↓GGC with ↑lactate production; ↓FAO and impaired energetics (↓free energy of ATP hydrolysis, ΔG ~ATP , at both workloads). The HCM phenotype and all measures of metabolic dysfunction were partially or completely prevented by EMPA. Conclusion: R403Q hearts display marked metabolic reprogramming that appears early in the disease process and leads to energetic dysfunction. SGLT2i corrects GIR, attenuates metabolic reprogramming and the energetic, structural and functional features of HCM. SGLT2i and possibly other measures to modulate glucagon signaling hold therapeutic potential in HCM.

Structure of the Drosophila melanogaster Flight Muscle Myosin Filament at 4.7 Å Resolution Reveals New Details of Non-Myosin Proteins.

Striated muscle thick filaments are composed of myosin II and several non-myosin proteins which define the filament length and modify its function. Myosin II has a globular N-terminal motor domain comprising its catalytic and actin-binding activities and a long α-helical, coiled tail that forms the dense filament backbone. Myosin alone polymerizes into filaments of irregular length, but striated muscle thick filaments have defined lengths that, with thin filaments, define the sarcomere structure. The motor domain structure and function are well understood, but the myosin filament backbone is not. Here we report on the structure of the flight muscle thick filaments from Drosophila melanogaster at 4.7 Å resolution, which eliminates previous ambiguities in non-myosin densities. The full proximal S2 region is resolved, as are the connecting densities between the Ig domains of stretchin-klp. The proteins, flightin, and myofilin are resolved in sufficient detail to build an atomic model based on an AlphaFold prediction. Our results suggest a method by which flightin and myofilin cooperate to define the structure of the thick filament and explains a key myosin mutation that affects flightin incorporation. Drosophila is a genetic model organism for which our results can define strategies for functional testing.

Abstract EC.03: Distinct Molecular Signatures Of HCM- And DCM-causing Mutations At The Same Residue In The Transducer Of Cardiac Myosin

Mutations in cardiac myosin cause both hypertrophic (HCM) and dilated cardiomyopathy (DCM), and the clinical phenotype ultimately depends on the precise effect of the mutation on the function of myosin. Using recombinant human cardiac myosin, we studied the molecular effects of a novel Q222H mutation in MYH7 found in a patient with severe, early-onset DCM, and compared the effects to the Q222K mutation that was previously reported to cause HCM. The Q222 residue is located within the transducer of the head domain near the ATP binding pocket. The Q222H (DCM) mutant myosin exhibited significantly reduced steady-state actin-activated ATPase activity and a decreased rate of ATP binding in transient kinetic studies, while Q222K (HCM) mutant protein showed an increase in ATPase activity and ATP binding kinetics. In vitro motility assays showed 43% reduction and 32% increase in actin gliding velocity in Q222H and Q222K mutations, respectively. Finally, single nucleotide turnover assays revealed that the fraction of myosin with a slow ATP hydrolysis rate (super relaxed state, or SRX) thought to represent myosin in an autoinhibited state was significantly reduced in Q222K and increased in Q222H. Taken together, the Q222H/K mutations exerted contrasting effects on the motor properties of the cardiac myosin catalytic domain. Cellular characterization and molecular dynamic simulations of cardiac myosin to assess the structural impact of mutant residues are underway. These results highlight how subtle structural differences between cardiomyopathy-causing mutations can lead to significant differences in cardiac myosin motor activity and thus phenotypic expression of cardiomyopathy.

The Antiparallel Coiled-Coil Domain Allows Multiple Forward Step Sizes of Myosin X.

Myosin X forms an antiparallel dimer and moves processively on actin bundles. How the antiparallel dimer affects the stepping mechanism of myosin X remains elusive. Here, we generated several chimeras using domains of myosin V and X and performed single-molecule motility assays. We found that the chimera containing the motor domain from myosin V and the lever arm and antiparallel coiled-coil domain from myosin X has multiple forward step sizes and moves processively, similar to full-length myosin X. The chimera containing the motor domain and lever arm from myosin X and the parallel coiled-coil from myosin V takes steps of ∼40 nm at lower ATP concentrations but was nonprocessive at higher ATP concentrations. Furthermore, mutant myosin X with four mutations in the antiparallel coiled-coil domain failed to dimerize and was nonprocessive. These results imply that the antiparallel coiled-coil domain is necessary for multiple forward step sizes of myosin X.

Nonmuscle Myosin IIA Regulates the Precise Alignment of Hexagonal Eye Lens Epithelial Cells During Fiber Cell Formation and Differentiation.

Epithelial cells in the equatorial region of the ocular lens undergo a remarkable transition from randomly packed cells into precisely aligned and hexagon-shaped cells organized into meridional rows. We investigated the function of nonmuscle myosin IIA (encoded by Myh9) in regulating equatorial epithelial cell alignment to form meridional rows during secondary fiber cell morphogenesis. We used genetic knock-in mice to study a common human Myh9 mutation, E1841K, in the rod domain. The E1841K mutation disrupts bipolar filament assembly. Lens shape, clarity, and stiffness were evaluated, and Western blots were used to determine the level of normal and mutant myosins. Cryosections and lens whole mounts were stained and imaged by confocal microscopy to investigate cell shape and organization. We observed no obvious changes in lens size, shape, and biomechanical properties (stiffness and resilience) between the control and nonmuscle myosin IIA-E1841K mutant mice at 2 months of age. Surprisingly, we found misalignment and disorder of fiber cells in heterozygous and homozygous mutant lenses. Further analysis revealed misshapen equatorial epithelial cells that cause disorientation of the meridional rows before fiber cell differentiation in homozygous mutant lenses. Our data indicate that nonmuscle myosin IIA bipolar filament assembly is required for the precise alignment of the meridional rows at the lens equator and that the organization of lens fiber cells depends on the proper patterning of meridional row epithelial cells. These data also suggest that lens fiber cell organization and a hexagonal shape are not required for normal lens size, shape transparency, or biomechanical properties.

Integrating comparative modeling and accelerated simulations reveals conformational and energetic basis of actomyosin force generation

Muscle contraction is performed by arrays of contractile proteins in the sarcomere. Serious heart diseases, such as cardiomyopathy, can often be results of mutations in myosin and actin. Direct characterization of how small changes in the myosin-actin complex impact its force production remains challenging. Molecular dynamics (MD) simulations, although capable of studying protein structure-function relationships, are limited owing to the slow timescale of the myosin cycle as well as a lack of various intermediate structures for the actomyosin complex. Here, employing comparative modeling and enhanced sampling MD simulations, we show how the human cardiac myosin generates force during the mechanochemical cycle. Initial conformational ensembles for different myosin-actin states are learned from multiple structural templates with Rosetta. This enables us to efficiently sample the energy landscape of the system using Gaussian accelerated MD. Key myosin loop residues, whose substitutions are related to cardiomyopathy, are identified to form stable or metastable interactions with the actin surface. We find that the actin-binding cleft closure is allosterically coupled to the myosin motor core transitions and ATP-hydrolysis product release from the active site. Furthermore, a gate between switch I and switch II is suggested to control phosphate release at the prepowerstroke state. Our approach demonstrates the ability to link sequence and structural information to motor functions.

Dilated cardiomyopathy mutation (E525K) in human beta-cardiac myosin enhances actin-activated phosphate release but stabilizes the auto-inhibited super-relaxed state.

Mutations in beta-cardiac myosin (M2β) are a common cause of inherited cardiomyopathies including dilated (DCM) and hypertrophic cardiomyopathy (HCM). We investigated the impact of a DCM mutation (E525K) in the human M2β motor domain, which has been shown to stabilize both the interacting heads motif (IHM) and auto-inhibited super-relaxed (SRX) state via head-tail electrostatic interactions in 15 heptad heavy meromyosin (M2β HMM). These findings suggest E525K may reduce muscle force and power by triggering an increase in the IHM/SRX state. However, it is unclear how E525K impacts the intrinsic motor properties of M2β. Thus, we introduced the mutation into single-headed human cardiac myosin subfragment 1 (M2β-S1), unable to form the IHM, and examined its impact on the ATPase mechanism. We revealed that E525K induces a 2.8-fold increase in maximum steady-state actin-activated ATPase activity (kcat), a 6-fold decrease in the actin concentration at which ATPase is half maximal (KATPase), and a 3.4-fold increase in the fast phase of actin-activated phosphate release. Glutamate 525 is located in the conserved activation loop that is proposed to trigger phosphate release by interacting with the negatively charged N-terminus of actin. We propose the mutation may enhance the rate of rotation of the lower 50-kDa domain, movement of the relay helix/switch II, and lever arm rotation associated with the power stroke. Thus, we will directly examine the impact on the actin-activated power stroke utilizing a FRET approach that measures lever arm rotation. Our results suggest the E525K mutation triggers an increase in intrinsic motor properties, while its ability to stabilize the SRX/IHM state in the myosin dimer likely dominates the molecular mechanism that leads to decreased muscle force and power and DCM pathogenesis.

Molecular mechanics of E525K dilated cardiomyopathy mutant human cardiac myosin.

Dilated cardiomyopathy (DCM) is associated with point mutations in beta-cardiac myosin and characterized by cardiac hypo-contractility. Myocardial power generation can be tuned by modulation of the number of functionally available myosin motors through myosin's ability to adopt an autoinhibited “super relaxed” state (SRX). Therefore, hypo-contractility of DCM hearts may result from: 1) reduced function of individual myosin heads, and/or 2) decreased myosin availability through increased SRX stabilization. To define the molecular impact of an established DCM myosin mutation, E525K, we expressed both wildtype (WT) and E525K myosin in several GFP-tagged human cardiac myosin constructs. We then determined actin filament velocity versus myosin surface density (GFP fluorescence) in the in vitro motility assay (1 mM ATP, 25 mM KCl, 30oC). The SRX state is thought to be stabilized by intramolecular head-tail interactions, which is where the E525K mutation exists. Accordingly, myosin constructs incapable of these interactions, such as single headed (S1) and short-tailed, double-headed myosin (heavy meromyosin with 2-heptad tail) constructs generated similarly fast velocities (2298±298 nm/s and 2352±234 nm/s, respectively). At the same myosin density, longer-tailed, SRX-capable constructs generated progressively slower velocities: 961±379 nm/s with a 15-heptad tail and 635±195 nm/s with a 25-heptad tail. These SRX-capable myosin constructs required 4-fold more myosin surface density to generate velocity equivalent to non-SRX constructs. Therefore, we propose that the 15- and 25-heptad constructs adopt the SRX state with a 75% probability. Unexpectedly, the E525K had no apparent impact on velocity, suggesting that under the low salt, unloaded in vitro motility assay conditions, the E525K mutation does not alter ensemble motor function. However, power generation is dependent on both force (F) and velocity (V), so future measurements of F:V relationships may uncover the true impact of this DCM myosin mutation.

Molecular characterization of a novel MYH7 mutation Q222H in a patient with severe dilated cardiomyopathy.

Mutations in the cardiac myosin heavy chain gene (MYH7) are known to cause both hypertrophic and dilated cardiomyopathy (HCM and DCM), and the use of recombinant human cardiac myosin has enabled the study of the precise biochemical and biophysical effects of these mutations on myosin function at the molecular level. Here we report a case of a patient with severe, early-onset DCM who carries a previously unreported Q222H mutation in MYH7. This mutation affects a residue at the distal end of the ATP binding pocket of cardiac myosin. The Q222H mutant myosin exhibited significantly reduced steady-state actin-activated ATPase activity and a decreased rate of ATP binding in transient kinetic studies. It also showed a ∼50% reduction in actin gliding velocity in an in vitro motility assay. Single-nucleotide turnover assays were performed comparing Q222H mutant myosin 2-headed constructs with either a long S2 tail capable of forming a folded-back auto-inhibited state or a short S2 tail that cannot stably fold back and sequester heads. These studies revealed that the fraction of myosin with a slow ATP hydrolysis rate (super relaxed state, or SRX) thought to represent myosin in an autoinhibited state did not differ significantly between WT and the Q222H mutant myosins. Taken together, the Q222H mutation exerts substantial effects on the motor properties of the cardiac myosin catalytic domain, rather than affecting the availability of myosin heads in the sarcomere. Further transient kinetic assays are underway to delineate precise steps in the actin-myosin ATPase cycle affected by the mutation. Future work also includes comparing these effects with the Q222K mutation that has been reported to cause HCM, to gain further insights into the molecular basis for phenotypic determination of cardiomyopathy.