Myosin Subfragment Research Articles

Homologous mutations in human β, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in β-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman-Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human β, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins but minimal effects in β myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing overall enzymatic (ATPase) cycle rate. In contrast, the only measured effect of R671C in β myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not β, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are a testament to myosin's highly allosteric nature.

New insights into the cross-linking between myosin and alkali-treated pea protein by transglutaminase under low ionic conditions: Contribution of legumin and vicilin fractions

This study investigated the cross-linking of myosin with pH12-shifted legumin (11SpH) or vicilin (7SpH), and the gel properties of composite gels, with or without transglutaminase, in 0.3 M NaCl. The free sulfhydryl content of 7SpH/11SpH increased significantly (P < 0.05). The covalent cross-linking degree between 7SpH/11SpH and myosin was enhanced, with the vicilin of approximately 45 kDa showing more pronounced cross-linking. SDS–PAGE analysis of myosin subfragments revealed that S1 and light meromyosin were the primary cross-linking regions for 11SpH, reducing the Ca2+-ATPase activity of the myosin head, while S2 was an alternative cross-linking region for 7SpH. The cross-linking of 7SpH/11SpH enhanced the thermal aggregation of S1. The cross-linking degree of tails followed this order: myosin > myosin+7SpH > myosin+11SpH. Thus, the use of 7SpH/11SpH and transglutaminase offers a new approach to improving texture in low-salt meat products.

Cooperative & competitive binding of anti-myosin tail antibodies revealed by super-resolution microscopy

The MF30 monoclonal antibody, which binds to the myosin subfragment-2 (S2), was found to increase the extent of myofibril shortening. Yet, previous observations found no effect of this antibody on actin sliding over myosin during in vitro motility assays with purified proteins in which myosin binding protein C (MyBPC) was absent. MF30 is hypothesized to enhance the availability of myosin heads (subfragment-1 or S1) to bind actin by destabilizing the myosin S2 coiled-coil and sterically blocking S2 from binding S1. The mechanism of action likely includes MF30's substantial size, thereby inhibiting S1 heads and MyBPC from binding S2. Hypothetically, MF30 should enhance the ON state of myosin, thereby increasing muscle contraction. Our findings indicate that MF30 binds preferentially to the unfolded heavy chains of S2, displaying positive cooperativity. However, the dose-response curve of MF30's enhancement of myofibril shortening did not suggest complex interactions with S2. Single, double, and triple-stained myofibrils with increasing amounts of antibodies against myosin rods indicate a possible competition with MyBPC. Additional assays revealed decreased fluorescence intensity at the C-zone (central zone in the sarcomere, where MyBPC is located), where MyBPC may inhibit MF30 binding. Another monoclonal antibody named MF20, which binds to the light meromyosin (LMM) without affecting myofibril contraction, showed less reduction in fluorescence intensity at the C-zone in expansion microscopy than MF30. Expansion microscopy images of myofibrils labeled with MF20 revealed labeling of the A-band (anisotropic band) and a slight reduction in the labeling at the C-zone. The staining pattern obtained from the expansion microscopy image was consistent with images from photolocalization microscopy which required the synthesis of unique photoactivatable quantum dots, and Zeiss Airyscan imaging as well as alternative expansion microscopy digestion methods. Consistent with the hypothesis that MF30 competes with MyBPC binding to S2, cardiac tissue from MyBPC knockout mice was stained more intensely, especially in the C-zone, by MF30 compared to the wild type.

Molecular Mechanisms of Deregulation of Muscle Contractility Caused by the R168H Mutation in TPM3 and Its Attenuation by Therapeutic Agents

The substitution for Arg168His (R168H) in γ-tropomyosin (TPM3 gene, Tpm3.12 isoform) is associated with congenital muscle fiber type disproportion (CFTD) and muscle weakness. It is still unclear what molecular mechanisms underlie the muscle dysfunction seen in CFTD. The aim of this work was to study the effect of the R168H mutation in Tpm3.12 on the critical conformational changes that myosin, actin, troponin, and tropomyosin undergo during the ATPase cycle. We used polarized fluorescence microscopy and ghost muscle fibers containing regulated thin filaments and myosin heads (myosin subfragment-1) modified with the 1,5-IAEDANS fluorescent probe. Analysis of the data obtained revealed that a sequential interdependent conformational-functional rearrangement of tropomyosin, actin and myosin heads takes place when modeling the ATPase cycle in the presence of wild-type tropomyosin. A multistep shift of the tropomyosin strands from the outer to the inner domain of actin occurs during the transition from weak to strong binding of myosin to actin. Each tropomyosin position determines the corresponding balance between switched-on and switched-off actin monomers and between the strongly and weakly bound myosin heads. At low Ca2+, the R168H mutation was shown to switch some extra actin monomers on and increase the persistence length of tropomyosin, demonstrating the freezing of the R168HTpm strands close to the open position and disruption of the regulatory function of troponin. Instead of reducing the formation of strong bonds between myosin heads and F-actin, troponin activated it. However, at high Ca2+, troponin decreased the amount of strongly bound myosin heads instead of promoting their formation. Abnormally high sensitivity of thin filaments to Ca2+, inhibition of muscle fiber relaxation due to the appearance of the myosin heads strongly associated with F-actin, and distinct activation of the contractile system at submaximal concentrations of Ca2+ can lead to muscle inefficiency and weakness. Modulators of troponin (tirasemtiv and epigallocatechin-3-gallate) and myosin (omecamtiv mecarbil and 2,3-butanedione monoxime) have been shown to more or less attenuate the negative effects of the tropomyosin R168H mutant. Tirasemtiv and epigallocatechin-3-gallate may be used to prevent muscle dysfunction.

Preclinical characterization of CK-4021586, a new class of cardiac myosin inhibitors for the treatment of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common monogenetic heart disease, with an estimated carrier prevalence of ∼1 in 500. Hypercontractility of the cardiac sarcomere is a common driver of the pathological hypertrophic cardiac remodeling that is a hallmark of the disease. Clinical trials of the selective cardiac myosin inhibitors mavacamten and aficamten have demonstrated that inhibition of the biochemical activity of cardiac myosin can induce cardiac remodeling and improve both patient symptoms and exercise capacity in HCM with left ventricular outflow tract obstruction (oHCM). The diverse genetic underpinnings of HCM suggest that it is possible that additional classes of cardiac myosin inhibitors with distinct biochemical mechanisms-of-action may also have a salutary impact on disease expression. While both mavacamten and aficamten bind to the single-headed motor domain of myosin (subfragment-1) and inhibit its ATPase activity, CK-4021586 (CK-586) is a new class of cardiac myosin inhibitor that inhibits the ATPase activity of two-headed heavy meromyosin (HMM) but not single-headed subfragment-1. CK-586 is a partial inhibitor of cardiac myofibrillar ATPase activity (EC50 2.9 µM, maximal inhibition ∼50%) that requires the regulatory light chain. Notably, fractional shortening of electrically-paced adult rat ventricular cardiomyocytes was inhibited almost completely (>80% at 5 µM) without alterations in the calcium (Ca2+) transient. In normal Sprague Dawley rats, CK-586 reduced cardiac fractional shortening in a dose and concentration-dependent manner. In conclusion, CK-586 is a novel, small molecule, cardiac myosin inhibitor that reduces cardiac contractility in vitro and in vivo. CK-586 has a biochemical mechanism of action distinct from both mavacamten and aficamten, providing an additional tool to decrease the number of functionally-available myosin heads and treat the cardiac hypercontractility that forms the pathologic basis of HCM.

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.

MyBP-C binds myosin S2 in mouse heart as revealed by super-resolution microscopy.

Supporting the hypothesis that myosin binding protein C (MyBP-C) binds to myosin subfragment-2 (S2) in the heart, a monoclonal antibody (MF30) to S2 was inhibited by the presence of MyBP-C in skinned mouse heart papillary muscle. Using a form of super-resolution microscopy called expansion microscopy, the relative positions of the fluorescent labels attached to a hydrogel could be expanded approximately four-fold in all three dimensions prior to imaging the sample with a Zeiss Airyscan super-resolution microscope. This muscle tissue was prepared for expansion microscopy by labeling with the fluorescently conjugated MF30 and embedding in a hydrogel, followed by crosslinking the fluorescent labels to the hydrogel and thoroughly digesting the proteins within the hydrogel. Images of these samples readily enable measurements of the relative fluorescent intensities of the P-, C-, and D-zones along thick filaments in the A-bands of the sarcomere. The A-bands from wild type mice showed low fluorescent intensities from MF30 in the C-zones relative to the P- and D-zones. Since MyBP-C resides in the C-zones of the thick filament, it was hypothesized that MyBP-C inhibited the MF30 binding in the C-zone. This hypothesis was tested by comparing tissues from MyBP-C knockout mice with the wild type mice using this super-resolution microscopy technique. The overall fluorescence intensity from MF30 was significantly greater in the tissue from MyBP-C knockout mice vs. wild type tissue. Furthermore, the MF30 fluorescence intensity in the C-zone was no longer inhibited in the MyBP-C knockout mice, relative to the D-zone in the wild type. These data support the hypothesis that MyBP-C binds myosin S2 and may compete with the MF30 binding to S2. (Supported by NIH/NHLBI R01HL149164.)

Properties of Cardiac Myosin with Cardiomyopathic Mutations in Essential Light Chains

The effects of cardiomyopathic mutations E56G, M149V, and E177G in the MYL3 gene encoding essential light chain of human ventricular myosin (ELCv), on the functional properties of cardiac myosin and its isolated head (myosin subfragment 1, S1) were investigated. Only the M149V mutation upregulated the actin-activated ATPase activity of S1. All mutations significantly increased the Ca2+-sensitivity of the sliding velocity of thin filaments on the surface with immobilized myosin in the in vitro motility assay, while mutations E56G and M149V (but not E177G) reduced the sliding velocity of regulated thin filaments and F-actin filaments almost twice. Therefore, despite the fact that all studied mutations in ELCv are involved in the development of hypertrophic cardiomyopathy, the mechanisms of their influence on the actin–myosin interaction are different.

Toxicity Assessment of an Anti-Cancer Drug of p-Toluene Sulfonamide in Zebrafish Larvae Based on Cardiovascular and Locomotion Activities.

p-Toluene sulfonamide (p-TSA), a small molecular drug with antineoplastic activity is widely gaining interest from researchers because of its pharmacological activities. In this study, we explored the potential cardio and neural toxicity of p-TSA in sublethal concentrations by using zebrafish as an in vivo animal model. Based on the acute toxicity assay, the 96hr LC50 was estimated as 204.3 ppm, suggesting the overall toxicity of p-TSA is relatively low in zebrafish larvae. For the cardiotoxicity test, we found that p-TSA caused only a minor alteration in treated larvae after no overall significant alterations were observed in cardiac rhythm and cardiac physiology parameters, as supported by the results from expression level measurements of several cardiac development marker genes. On the other hand, we found that acute p-TSA exposure significantly increased the larval locomotion activity during the photomotor test while prolonged exposure (4 days) reduced the locomotor startle reflex activities in zebrafish. In addition, a higher respiratory rate and blood flow velocity was also observed in the acutely treated fish groups compared to the untreated group. Finally, by molecular docking, we found that p-TSA has a moderate binding affinity to skeletal muscle myosin II subfragment 1 (S1), ATPase activity, actin- and Ca2+-stimulated myosin S1 ATPase, and v-type proton ATPase. These binding interactions between p-TSA and proteins offer insights into the potential molecular mechanism of action of p-TSA on observed altered responses toward photo and vibration stimuli and minor altered vascular performance in the zebrafish larvae.

Hypertrophic cardiomyopathy mutations in the pliant and light chain-binding regions of the lever arm of human β-cardiac myosin have divergent effects on myosin function.

Mutations in the lever arm of β-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy, a disease characterized by hypercontractility and eventual hypertrophy of the left ventricle. Here, we studied five such mutations: three in the pliant region of the lever arm (D778V, L781P, and S782N) and two in the light chain-binding region (A797T and F834L). We investigated their effects on both motor function and myosin subfragment 2 (S2) tail-based autoinhibition. The pliant region mutations had varying effects on the motor function of a myosin construct lacking the S2 tail: overall, D778V increased power output, L781P reduced power output, and S782N had little effect on power output, while all three reduced the external force sensitivity of the actin detachment rate. With a myosin containing the motor domain and the proximal S2 tail, the pliant region mutations also attenuated autoinhibition in the presence of filamentous actin but had no impact in the absence of actin. By contrast, the light chain-binding region mutations had little effect on motor activity but produced marked reductions in autoinhibition in both the presence and absence of actin. Thus, mutations in the lever arm of β-cardiac myosin have divergent allosteric effects on myosin function, depending on whether they are in the pliant or light chain-binding regions.

Study on nucleotide, myofibrillar protein biochemical properties and microstructure of freeze-dried scallop striated muscle during storage and rehydration

The biochemical properties and microstructural changes of freeze-dried Japanese scallop (Patinopecten yessoensis) striated muscle during room temperature storage and rehydration were investigated. The results showed that the content of ATP in freeze-dried scallop muscle remained stable with no significant difference (p > 0.05). However, ATP was rapidly decomposed and AMP accumulated within 1.5 min of rehydration, and HxR and Hx were gradually produced from AMP decomposition with the extension of rehydration time. Besides, the results of chymotryptic digestion patterns demonstrated that the rod of myosin was unstable after dehydration, reflecting lower salt solubility than that of frozen-thawed scallop. In contrast, the myosin subfragment-1 (S-1) was stable, as indicated by the constant of Ca2+-ATPase activity of freeze-dried scallops throughout the storage and rehydration (p > 0.05). Furthermore, the microstructural analysis revealed that the Z line of the freeze-dried scallop was broken after dehydration process. This study might be useful for developing high-quality dehydrated scallops in the future.

Nanosurfer assay dissects β-cardiac myosin and cardiac myosin-binding protein C interactions

Cardiac myosin-binding protein C (cMyBP-C) modulates cardiac contractility through putative interactions with the myosin S2 tail and/or the thin filament. The relative contribution of these binding-partner interactions to cMyBP-C modulatory function remains unclear. Hence, we developed a “nanosurfer” assay as a model system to interrogate these cMyBP-C binding-partner interactions. Synthetic thick filaments were generated using recombinant human β-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14- or 28-nm spacing, corresponding to the 14.3-nm myosin spacing in native thick filaments. The nanosurfer assay consists of DNA nanotubes added to the in vitro motility assay so that myosins on the motility surface effectively deliver thin filaments to the DNA nanotubes, enhancing thin filament gliding probability on the DNA nanotubes. Thin filament velocities on nanotubes with either 14- or 28-nm myosin spacing were no different. We then characterized the effects of cMyBP-C on thin filament motility by alternating HMM and cMyBP-C N-terminal fragments (C0–C2 or C1–C2) on nanotubes every 14 nm. Both C0–C2 and C1–C2 reduced thin filament velocity four- to sixfold relative to HMM alone. Similar inhibition occurred using the myosin S1 construct, which lacks the myosin S2 region proposed to interact with cMyBP-C, suggesting that the cMyBP-C N terminus must interact with other myosin head domains and/or actin to slow thin filament velocity. Thin filament velocity was unaffected by the C0–C1f fragment, which lacks the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A C0–C2 fragment with phospho-mimetic replacement in the M-domain showed markedly less inhibition of thin filament velocity compared with its phospho-null counterpart, highlighting the modulatory role of M-domain phosphorylation on cMyBP-C function. Therefore, the nanosurfer assay provides a platform to precisely manipulate spatially dependent cMyBP-C binding-partner interactions, shedding light on the molecular regulation of β-cardiac myosin contractility.

Mechanisms of the modulation of actin-myosin interactions by A1-type myosin light chains

BackgroundThe interaction of N-terminal extension of the myosin A1 essential light chain (A1 ELC) with actin is receiving increasing attention as a target in utilizing synthetic A1 ELC N-terminal-derived peptides in cardiac dysfunction therapy. MethodsTo elucidate the mechanism by which these peptides regulate actin-myosin interaction, here we have investigated their effects on the myosin subfragment 1 (S1)-induced polymerization of G-actin. ResultsThe MLCFpep and MLCSpep peptides spanning the 3–12 of A1 ELC sequences from fast and slow skeletal muscle, respectively, increased the rate of actin polymerization not only by S1(A2) but also the rate of S1(A1)-induced actin polymerization, suggesting that they did not interfere with the direct binding of A1 ELC with actin. The efficiency of actin polymerization in the presence of the N-terminal ELC peptides depended on their sequence. Substitution of aspartic acid for neutral asparagine at position 5 of MLCFpep dramatically enhanced its ability to stimulate S1-induced polymerization and enabled it to initiate polymerization of G-actin in the absence of S1. ConclusionsThese and other results presented in this work suggest that the modulation of myosin motor activity by N-terminal ELC peptides is exerted through a change in actin filament conformation rather than through blocking the A1 ELC-actin interaction. General significanceThe results imply the possibility of enhancing therapeutic effects of these peptides by modifications of their sequence.

The structure and function of the human cardiac actomyosin complex

We have solved high-resolution structures of the human cardiac actin-myosin complex and correlated our findings with kinetics data of the actomyosin ATPase. Our long-range goal is to compare the behavior of wild-type and mutant sarcomeric proteins on assembled thin filament complexes using human cardiac-specific proteins. Furthermore, we aim to dock small compounds into pockets associated with the thin filament-myosin interface as a means of mitigating effects of mutations. To date, human β-cardiac myosin subfragment 1 (M2β-S1, amino acids 1-843) was expressed with C-terminal Avi and FLAG tags in C2C12 cells.

Anti-S2 peptides and antibodies bind cooperatively to the myosin dimer with high affinity and stabilize the unfolded state of S2

Fluorescently labeled synthetic peptides (stabilizer and destabilizer) and antibodies (MF20, MF30, polyclonal), that target myosin subfragment-2 (S2) or light meromyosin (LMM), bound myosin, and all but polycolonal antibody caused a statistically significant change in the anisotropy. Their binding to myosin S2/LMM was confirmed when they labeled the A-band of the sarcomere in expansion microscopy images of rabbit skeletal myofibrils. The variations in the anisotropy readings depended on the mass that is small for the synthetic peptides, and large for the antibody and myosin.

Tetrasodium pyrophosphate ameliorates oxidative damage to the TGase-catalyzed gelation of actomyosins

The present study attempted to investigate the interactive roles of protein oxidation (0–20 mM H2O2) and tetrasodium pyrophosphate (TSPP) on the crosslinking efficiency of actomyosin mediated by transglutaminase (TGase). Oxidation at 0–20 mM H2O2 was not conducive to TGase-mediated crosslinking as indicated by the relative reduction of free amine consumption from 35.3% to 11.7%, and caused the principle crosslinking sites to progressively convert from myosin subfragment-1 (S1) to subfragment-1 (S2) as evidenced by electrophoresis. However, the binding of TSPP to myosin alleviated oxidation suppression to TGase-catalyzed crosslinking in varying degrees and retarded the migration of crosslinking site from S1 to S2. Moreover, oxidation (especially 20 mM H2O2) decreased the final (90 °C) elasticity index (EI) and water holding capacity of TGase-treated actomyosin gel, while TSPP intensified those of TGase-catalyzed actomyosin gel, indicating that TSPP had a positive effect on ameliorating the oxidative stress to TGase-catalyzed gelation of actomyosin.

Visualization of Actin Filaments Decorated with Smooth Muscle Myosin Subfragment 1 and Heavy Meromyosin by Cryoelectron Tomography

Visualization of Actin Filaments Decorated with Smooth Muscle Myosin Subfragment 1 and Heavy Meromyosin by Cryoelectron Tomography

Impact of regulatory light chain mutation K104E on the ATPase and motor properties of cardiac myosin.

Mutations in the cardiac myosin regulatory light chain (RLC, MYL2 gene) are known to cause inherited cardiomyopathies with variable phenotypes. In this study, we investigated the impact of a mutation in the RLC (K104E) that is associated with hypertrophic cardiomyopathy (HCM). Previously in a mouse model of K104E, older animals were found to develop cardiac hypertrophy, fibrosis, and diastolic dysfunction, suggesting a slow development of HCM. However, variable penetrance of the mutation in human populations suggests that the impact of K104E may be subtle. Therefore, we generated human cardiac myosin subfragment-1 (M2β-S1) and exchanged on either the wild type (WT) or K104E human ventricular RLC in order to assess the impact of the mutation on the mechanochemical properties of cardiac myosin. The maximum actin-activated ATPase activity and actin sliding velocities in the in vitro motility assay were similar in M2β-S1 WT and K104E, as were the detachment kinetic parameters, including the rate of ATP-induced dissociation and the ADP release rate constant. We also examined the mechanical performance of α-cardiac myosin extracted from transgenic (Tg) mice expressing human wild type RLC (Tg WT) or mutant RLC (Tg K104E). We found that α-cardiac myosin from Tg K104E animals demonstrated enhanced actin sliding velocities in the motility assay compared with its Tg WT counterpart. Furthermore, the degree of incorporation of the mutant RLC into α-cardiac myosin in the transgenic animals was significantly reduced compared with wild type. Therefore, we conclude that the impact of the K104E mutation depends on either the length or the isoform of the myosin heavy chain backbone and that the mutation may disrupt RLC interactions with the myosin lever arm domain.

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.

Synthetic thick filaments: A new avenue for better understanding the myosin super-relaxed state in healthy, diseased, and mavacamten-treated cardiac systems

A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low-ionic-strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filaments. Although these STFs have been extensively used for structural characterization, their functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin subfragments, heavy meromyosin (HMM) and myosin subfragment 1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and an increased ADP:ATP ratio, destabilize the SRX population. Furthermore, WT myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small-molecule inhibitor, mavacamten, showed that it is more effective in not only increasing myosin SRX population in STFs than in S1 or HMM but also in increasing myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens mavacamten’s ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, diseased, and therapeutic conditions.