Skeletal Muscle Myosin Research Articles

Sex differences in skeletal muscle size, function, and myosin heavy chain isoform expression during post-injury regeneration in mice.

Skeletal muscle regeneration is an essential process to restore muscle function after injury and is influenced by various factors. Despite the known importance of sex hormones in muscle regeneration, whether and what sex difference exists in this process is still unclear. In this study, we provide evidence for a clear sex difference in muscle regeneration in mice. At 7 and 14 days after barium chloride-induced muscle injury, female mice showed a faster recovery of muscle fiber size than males. Consistently, muscle force in female mice was restored faster than in males after injury, and this functional difference was maintained at 14 months of age when regenerative capacity declined. Myosin heavy chain isoform profiling and fatigability test revealed dynamic remodeling of myosin heavy chain isoform expression including a type IIB to IIA/X MHC transition and reduced fatigability in regenerated muscles compared to uninjured muscles. A significant sex difference was detected in myosin heavy chain IIX content, although this did not lead to different fatigability. Together, our results suggest that sex is an important determinant of the recovery of regenerating skeletal muscle and is partially involved in the remodeling of myosin heavy chain isoforms during muscle regeneration.

Alternative relay regulates the adenosine triphosphatase activity of Locusta migratoria striated muscle myosin.

Locust (Locusta migratoria) has a single striated muscle myosin heavy chain (Mhc) gene, which contains 5 clusters of alternative exclusive exons and 1 differently included penultimate exon. The alternative exons of Mhc gene encode 4 distinct regions in the myosin motor domain, that is, the N-terminal SH3-like domain, one lip of the nucleotide-binding pocket, the relay, and the converter. Here, we investigated the role of the alternative regions on the motor function of locust muscle myosin. Using Sf9-baculovirus protein expression system, we expressed and purified 5 isoforms of the locust muscle myosin heavy meromyosin (HMM), including the major isoform in the thorax dorsal longitudinal flight muscle (FL1) and 4 isoforms expressed in the abdominal intersegmental muscle (AB1 to AB4). Among these 5 HMMs, FL1-HMM displayed the highest level of actin-activated adenosine triphosphatase (ATPase) activity (hereafter referred as ATPase activity). To identify the alternative region(s) responsible for the elevated ATPase activity of FL1-HMM, we produced a number of chimeras of FL1-HMM and AB4-HMM. Substitution with the relay of AB4-HMM (encoded by exon-14c) substantially decreased the ATPase activity of FL1-HMM, and conversely, the relay of FL1-HMM (encoded by exon-14a) enhanced the ATPase activity of AB4-HMM. Mutagenesis showed that the exon-14a-encoded residues Gly474 and Asn509 are responsible for the elevated ATPase activity of FL1-HMM. Those results indicate that the alternative relay encoded by exon-14a/c play a key role in regulating the ATPase activity of FL1-HMM and AB4-HMM.

Variants in ACTC1 underlie distal arthrogryposis accompanied by congenital heart defects.

Contraction of the human sarcomere is the result of interactions between myosin cross-bridges and actin filaments. Pathogenic variants in genes such as MYH7, TPM1, and TNNI3 that encode parts of the cardiac sarcomere cause muscle diseases that affect the heart, such as dilated cardiomyopathy and hypertrophic cardiomyopathy. In contrast, pathogenic variants in homologous genes such as MYH2, TPM2, and TNNI2 that encode parts of the skeletal muscle sarcomere cause muscle diseases affecting skeletal muscle, such as distal arthrogryposis (DA) syndromes and skeletal myopathies. To date, there have been few reports of genes (e.g., MYH7) encoding sarcomeric proteins in which the same pathogenic variant affects skeletal and cardiac muscle. Moreover, none of the known genes underlying DA have been found to contain pathogenic variants that also cause cardiac abnormalities. We report five families with DA because of heterozygous missense variants in the gene actin, alpha, cardiac muscle 1 (ACTC1). ACTC1 encodes a highly conserved actin that binds to myosin in cardiac and skeletal muscle. Pathogenic variants in ACTC1 have been found previously to underlie atrial septal defect, dilated cardiomyopathy, hypertrophic cardiomyopathy, and left ventricular noncompaction. Our discovery delineates a new DA condition because of variants in ACTC1 and suggests that some functions of ACTC1 are shared in cardiac and skeletal muscle.

Effects of Atmospheric Ammonia on Skeletal Muscle Growth in Broilers

Ammonia, one of the most polluted gases in poultry houses, has always been an urgent problem to solve. Exposure to ammonia can threaten the respiratory tract, induce inflammation, and decrease growth performance. To date, there are few studies investigating the effects of ammonia on skeletal muscle growth. In this experiment, a total of 144 broilers were randomly divided into two groups, and 0 ppm and 35 ppm atmospheric ammonia were administered in the chambers. The trial lasted for 21 days. The breast muscle, thigh muscle, dressed weight, and serum biochemical indexes were measured. The skeletal muscle fibre morphology was observed using light microscopy, and the expressions of genes associated with skeletal muscle development and myosin heavy chain genes were assessed. After 7 days of ammonia exposure, the broilers’ weight in the ammonia group decreased. On the 21st day of the experiment, in the ammonia group, the breast muscle weight, thigh muscle weight, and dressed weight decreased, the blood urea nitrogen content increased, skeletal muscle fibre diameter shortened, the expression of myostatin increased, and the expression of myosin heavy chain-FWM and myosin heavy chain-FRM decreased significantly. This article suggests that 35 ppm atmospheric ammonia seriously affects the skeletal muscle gain rate of broilers, and the myostatin pathway could be a potential regulation of the growth rate of muscle fibre under ammonia exposure.

Physical activity impacts resting skeletal muscle myosin conformation and lowers its ATP consumption.

It has recently been established that myosin, the molecular motor protein, is able to exist in two conformations in relaxed skeletal muscle. These conformations are known as the super-relaxed (SRX) and disordered-relaxed (DRX) states and are finely balanced to optimize ATP consumption and skeletal muscle metabolism. Indeed, SRX myosins are thought to have a 5- to 10-fold reduction in ATP turnover compared with DRX myosins. Here, we investigated whether chronic physical activity in humans would be associated with changes in the proportions of SRX and DRX skeletal myosins. For that, we isolated muscle fibers from young men of various physical activity levels (sedentary, moderately physically active, endurance-trained, and strength-trained athletes) and ran a loaded Mant-ATP chase protocol. We observed that in moderately physically active individuals, the amount of myosin molecules in the SRX state in type II muscle fibers was significantly greater than in age-matched sedentary individuals. In parallel, we did not find any difference in the proportions of SRX and DRX myosins in myofibers between highly endurance- and strength-trained athletes. We did however observe changes in their ATP turnover time. Altogether, these results indicate that physical activity level and training type can influence the resting skeletal muscle myosin dynamics. Our findings also emphasize that environmental stimuli such as exercise have the potential to rewire the molecular metabolism of human skeletal muscle through myosin.

Modulating fast skeletal muscle contraction protects skeletal muscle in animal models of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by absence of the protein dystrophin, which acts as a structural link between the basal lamina and contractile machinery to stabilize muscle membranes from mechanical stress. In DMD, mechanical stress leads to exaggerated membrane injury and fiber breakdown, with fast fibers being the most susceptible to damage. A major contributor to this injury is muscle contraction, controlled by the motor protein myosin. However, the relationship between how muscle contraction and fast muscle fiber damage contribute to the pathophysiology of DMD has not been well characterized. We explored the role of fast skeletal muscle contraction in DMD with a novel, selective, orally active inhibitor of fast skeletal muscle myosin, EDG-5506. Surprisingly, even modest decreases of contraction (<15%) were sufficient to protect skeletal muscles in dystrophic mdx mice from stress injury. Longer-term treatment also decreased muscle fibrosis in key disease-implicated tissues. Importantly, therapeutic levels of myosin inhibition with EDG-5506 did not detrimentally affect strength or coordination. Finally, in dystrophic dogs, EDG-5506 reversibly reduced circulating muscle injury biomarkers and increased habitual activity. This unexpected biology may represent an important alternative treatment strategy for Duchenne and related myopathies.

Myosin light chain of shark fast skeletal muscle exhibits intrinsic urea-resistibility

Marine elasmobranch fish contain urea, a protein denaturant, in their bodies. The urea-trimethylamine N-oxide (TMAO) counteraction mechanism contributes to urea-resistibility, where TMAO compensates for protein denaturation by urea. However, previous studies revealed that shark fast skeletal muscle myosin exhibits native activity at physiological urea concentrations in the absence of TMAO, suggesting that shark myosin has urea-resistibility. In this study, we compared the urea-resistibility of myosin alkali light chains (A1-LC and A2-LC) from banded houndshark and carp by examining the α-helical content at various urea concentrations. The α-helical content of carp myosin A1-LC and A2-LC gradually decreased as urea concentrations increased to 2 M. In contrast, the α-helical content of banded houndshark A1-LC increased between 0 and 0.5 M urea, and the α-helical content of A2-LC remained constant until 0.5 M urea. We determined the full-length sequences of the banded houndshark myosin light chains (A1-LC, A2-LC and DTNB-LC). Hydrophilicity analysis revealed that the N-terminal region (residues 28–34) of A1-LC from banded houndshark is more hydrophilic than the corresponding region of A1-LC from carp. These findings support the notion that shark myosin exhibits urea-resistibility independent of the urea-TMAO counteraction mechanism.

Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays

Myosin expression and purification is important for mechanistic insights into normal function and mutation induced changes. The latter is particularly important for striated muscle myosin II where mutations cause several debilitating diseases. However, the heavy chain of this myosin is challenging to express and the standard protocol, using C2C12 cells, relies on viral infection. This is time and work intensive and associated with infrastructural demands and biological hazards, limiting widespread use and hampering fast generation of a wide range of mutations. We here develop a virus-free method to overcome these challenges. We use this system to transfect C2C12 cells with the motor domain of the human cardiac myosin heavy chain. After optimizing cell transfection, cultivation and harvesting conditions, we functionally characterized the expressed protein, co-purified with murine essential and regulatory light chains. The gliding velocity (1.5–1.7 µm/s; 25 °C) in the in vitro motility assay as well as maximum actin activated catalytic activity (kcat; 8–9 s−1) and actin concentration for half maximal activity (KATPase; 70–80 µM) were similar to those found previously using virus based infection. The results should allow new types of studies, e.g., screening of a wide range of mutations to be selected for further characterization.

Structural changes in myosin affect chemo-mechanical properties of the myosin-actin interaction.

Mutations in the genes encoding striated muscle myosins lead to a variety of diseases including hypertrophic (HCM) and dilated (DCM) cardiomyopathies (MYH7) and distal arthrogryposis syndromes (MYH3 and MYH8). We have used a muscle myoblast cell line to express and purify recombinant human beta-cardiac myosin and human embryonic skeletal muscle myosins, comparing the WT versions of these proteins with an array of disease-causing variants. HCM-causing mutations in the beta-cardiac motor domain often destabilize a folded back, autoinhibited state of myosin, leading to an increase in the number of heads available within the sarcomere to interact with actin and produce force, which in turn results in the hypercontractility characteristic of HCM. Destabilization is seen for mutations that alter residues involved in the regions of myosin important for forming the intramolecular contacts in the folded back state. However, mutations affecting residues remote from these interfaces can also increase the number of available heads by destabilizing the pre-power stroke state of myosin that enables formation of the folded back state. HCM mutations that affect only myosin motor function without affecting the stability of the folded back state are less common. In contrast, we have studied a variety of DCM-causing mutations and found that most DCM-mutant myosins exhibit decreases in one or more aspects of motor function, including actin-activated ATPase activity and in vitro gliding velocity. Current efforts focus on correlating the effects of distal arthrogryposis syndrome-causing mutations on MYH3 biomechanical function and contrasting the effects of these mutations with paralogous mutations in MYH7.

Effects of blebbistatin on force production, actin motility and the force-velocity relation produced by myosin II filaments and myofibrils.

Blebbistatin is a myosin II-specific inhibitor that is often used to study the relation between force generation and Pi release. The goal of this study was to investigate the effects of blebbistatin on force production and the actin motility while interacting with arrays of myosin motors. We used myosin filaments isolated from mammalian smooth muscles, rabbit psoas and the anterior byssus retractor (ABRM) muscles extracted from fresh Mytilus edulis in different experiments. We also used whole myofibrils isolated from the rabbit psoas. The force during myosin-actin interactions was measured with a system of micro-fabricated cantilevers, and the sliding velocity of actin was measured with a standard in-vitro motility assay. The force-velocity relation was measured in myofibrils with atomic force cantilevers. The force produced by the myosin filaments upon interaction with actin decreased with increasing concentrations of blebbistatin (5 μM-12.5 μM). The force produced by smooth and skeletal muscle myosin filaments were 95.93 ± 12.05 pN/μm before blebbistatin treatment, 71.45 ± 5.94 pN/μm with 5 μm blebbistatin, 62.05 ± 9.44 pN/μm with 7.5 μM blebbistatin, 38.28 ± 8.77 pN/μm with 10 μm blebbistatin and dropped to zero with 12.5 μM blebbistatin. All these differences were significantly different (p<0.0001). Sliding velocity of actin filaments also decreased consistently with increasing concentrations of blebbistatin. Before treatment, filaments slid over HMM at 3.68 ± 0.06 μm/s, decreasing to 2.30 ± 0.056 μm/s with 1 μM, 1.27 ± 0.043 μm/s with 5 μM, and 1.44 ± 0.032 μm/s with 30 μM. The maximal velocity decrease was observed at 5 μM of blebbistatin. These differences were significantly different (P<0.0001). There was a downward shift in the force-velocity relation in myofibrils treated with blebbistatin. These results suggest that blebbistatin regulates the mechanics of myosin motors working collectively in different experimental preparations.

Embryonic myosin mutations alter the mechanics and maturation of HiPSC derived skeletal muscle.

Distal arthrogryposis (DA) is a skeletal muscle disorder characterized by joint contractures predominantly localized in the distal extremities. DA associated syndromes like Freeman-Sheldon syndrome (FSS) are linked to mutations in the MYH3 gene that encodes the embryonic skeletal muscle myosin. To study the mutation and its effect on developing muscle, we generated human induced pluripotent stem cell (hiPSC) lines bearing T178I or R672C MYH3 mutations. hiPSC were differentiated into skeletal muscle and evaluated for differences in the maturation of the contractile unit and its functional performance. Preliminary myofibril mechanics measurements showed T178I myotubes generated significantly greater specific force compared to control myofibrils. Diseased myofibrils also expressed significantly slower rates of relaxation and prolonged thin filament deactivation compared to control myofibrils. Myofibril preparations were also used to compare ATP binding rates across conditions. Preliminary results showed homozygous R672C myofibrils have faster ATP binding rates compared to heterozygous and control preparations. Mutations in MYH3 were also associated with alterations in myosin isoform content, with homozygous R672C having reduced MYH3 levels in day 7 cells. Molecular dynamics simulations of R672C and T178I mutations in the post rigor state showed greater separation between the beta-sheet and SH-helix than in controls. Mutation also disrupted local salt bridges and hydrogen bond formation in some residues that help stabilize the nucleotide binding pocket and converter-connected helix. Interrupted structural communication between the nucleotide binding pocket and surrounding functional regions may underly the impaired relaxation phenotype. Ongoing studies will further characterize the effect of mutation on actin-myosin binding, crossbridge cycling kinetics, the maturation of skeletal myotubes over time, and myosin isoform switching dynamics.

Semi-automated technique for bovine skeletal muscle fiber cross-sectional area and myosin heavy chain determination.

Myosin heavy chain (MyHC) type and muscle fiber size are informative but time-consuming variables of interest for livestock growth, muscle biology, and meat science. The objective of this study was to validate a semi-automated protocol for determining MyHC type and size of muscle fibers. Muscle fibers obtained from the longissimus and semitendinosus of fed beef carcasses were embedded and frozen within 45 min of harvest. Immunohistochemistry was used to distinguish MyHC type I, IIA, and IIX proteins, dystrophin, and nuclei in transverse sections of frozen muscle samples. Stained muscle cross sections were imaged and analyzed using two workflows: 1) Nikon workflow which used Nikon Eclipse inverted microscope and NIS Elements software and 2) Cytation5 workflow consisting of Agilent BioTek Cytation5 imaging reader and Gen5 software. With the Cytation5 workflow, approximately six times more muscle fibers were evaluated compared to the Nikon workflow within both the longissimus (P < 0.01; 768 vs. 129 fibers evaluated) and semitendinosus (P < 0.01; 593 vs. 96 fibers evaluated). Combined imaging and analysis took approximately 1 h per sample with the Nikon workflow and 10 min with the Cytation5 workflow. When muscle fibers were evaluated by the objective thresholds of the Cytation5 workflow, a greater proportion of fibers were classified as glycolytic MyHC types, regardless of muscle (P < 0.01). Overall mean myofiber cross-sectional area was 14% smaller (P < 0.01; 3,248 vs. 3,780) when determined by Cytation5 workflow than when determined by Nikon workflow. Regardless, Pearson correlation of mean muscle fiber cross-sectional areas determined by Nikon and Cytation5 workflows was 0.73 (P < 0.01). In both workflows cross-sectional area of MyHC type I fibers was the smallest and area of MyHC type IIX fibers was the largest. These results validated the Cytation5 workflow as an efficient and biologically relevant tool to expedite data capture of muscle fiber characteristics while using objective thresholds for muscle fiber classification.

Simultaneous loss of skeletal muscle myosin heavy chain IIx and IIb causes severe skeletal muscle hypoplasia in postnatal mice.

The skeletal muscle myosin heavy chain (MyHC) is a fundamental component of the sarcomere structure and muscle contraction. Two of the three adult fast MyHCs, MyHC-IIx and MyHC-IIb, are encoded by Myh1 and Myh4, respectively. However, skeletal muscle disorders have not yet been linked to these genes in humans. MyHC-IIb is barely detectable in human skeletal muscles. Thus, to characterize the molecular function of skeletal muscle MyHCs in humans, investigation of the effect of simultaneous loss of MyHC-IIb and other MyHCs on skeletal muscle in mice is essential. Here, we generated double knockout (dKO) mice with simultaneous loss of adult fast MyHCs by introducing nonsense frameshift mutations into the Myh1 and Myh4 genes. The dKO mice appeared normal after birth and until 2 weeks of age but showed severe skeletal muscle hypoplasia after 2 weeks. In 3-week-old dKO mice, increased expression of other skeletal muscle MyHCs, such as MyHC-I, MyHC-IIa, MyHC-neo, and MyHC-emb, was observed. However, these expressions were not sufficient to compensate for the loss of MyHC-IIb and MyHC-IIx. Moreover, the aberrant sarcomere structure with altered expression of sarcomere components was observed in dKO mice. Our findings imply that the simultaneous loss of MyHC-IIb and MyHC-IIx is substantially detrimental to postnatal skeletal muscle function and will contribute to elucidating the molecular mechanisms of skeletal muscle wasting disorders caused by the loss of skeletal muscle MyHCs.

Isoform selectivities of novel 4-hydroxycoumarin imines as inhibitors of myosin II

Muscle myosin inhibition could be used to treat many medical conditions involving hypercontractile states, including muscle spasticity, chronic musculoskeletal pain, and hypertrophic cardiomyopathy. A series of 13 advanced analogs of 3-(N-butylethanimidoyl)ethyl)-4-hydroxy-2H-chromen-2-one (BHC) were synthesized to explore extended imine nitrogen side chains and compare aldimines vs. ketimines. None of the new analogs inhibit nonmuscle myosin in a cytokinesis assay. ATPase structure-activity relationships reveal that selectivity for cardiac vs. skeletal myosin can be tuned with subtle structural changes. None of the compounds inhibited smooth muscle myosin II. Docking the compounds to homology models of cardiac and skeletal myosin II gave rationales for the effects of side arm length on inhibition selectivity and for cardiac vs. skeletal myosin. Properties including solubility, stability and toxicity, suggest that certain BHC analogs may be useful as candidates for preclinical studies or as lead compounds for advanced candidates for drugs with cardiac or skeletal muscle myosin selectivity.

Pseudo-phosphorylation of essential light chains affects the functioning of skeletal muscle myosin

The work aimed to investigate how the phosphorylation of the myosin essential light chain of fast skeletal myosin (LC1) affects the functional properties of the myosin molecule. Using mass-spectrometry, we revealed phosphorylated peptides of LC1 in myosin from different fast skeletal muscles. Mutations S193D and T65D that mimic natural phosphorylation of LC1 were produced, and their effects on functional properties of the entire myosin molecule and isolated myosin head (S1) were studied. We have shown that T65D mutation drastically decreased the sliding velocity of thin filaments in an in vitro motility assay and strongly increased the duration of actin-myosin interaction in optical trap experiments. These effects of T65D mutation in LC1 observed only with the whole myosin but not with S1 were prevented by double T65D/S193D mutation. The T65D and T65D/S193D mutations increased actin-activated ATPase activity of S1 and decreased ADP affinity for the actin-S1 complex. The results indicate that pseudo-phosphorylation of LC1 differently affects the properties of the whole myosin molecule and its isolated head. Also, the results show that phosphorylation of LC1 of skeletal myosin could be one more mechanism of regulation of actin-myosin interaction that needs further investigation.

Actin-binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II.

We report a case in which sub-stoichiometric binding of an actin-binding protein has profound structural and functional consequences, providing an insight into the fundamental properties of actin regulation. Rng2 is an IQGAP contained in contractile rings in the fission yeast Schizosaccharomyces pombe Here, we used high-speed atomic force microscopy and electron microscopy and found that sub-stoichiometric binding of the calponin-homology actin-binding domain of Rng2 (Rng2CHD) induces global structural changes in skeletal muscle actin filaments, including shortening of the filament helical pitch. Sub-stoichiometric binding of Rng2CHD also reduced the affinity between actin filaments and muscle myosin II carrying ADP and strongly inhibited the motility of actin filaments on myosin II in vitro. On skeletal muscle myosin II-coated surfaces, Rng2CHD stopped the actin movements at a binding ratio of 11%. Rng2CHD also inhibited actin movements on myosin II of the amoeba Dictyostelium, but in this case, by detaching actin filaments from myosin II-coated surfaces. Thus, sparsely bound Rng2CHD induces apparently cooperative structural changes in actin filaments and inhibits force generation by actomyosin II.

Insect Sf9 cells are suitable for functional expression of insect, but not vertebrate, striated muscle myosin

Insect Sf9 cells are widely used for producing recombinant proteins, including myosin. It is expected that the protein folding machinery in Sf9 cells can meet the requirement for the proper folding of exogenous myosin. Of interest is that not all class II myosins are expressed functionally in Sf9 cells. Among vertebrate class II myosins, non-muscle myosin and smooth muscle myosin, but not striated muscle myosin, are functionally expressed in Sf9 cells, presumably due to lacking vertebrate striated muscle myosin-specific chaperone Unc45b in Sf9 cells. Insects only express a generic myosin-specific chaperone Unc45, which is expected to be responsible for the folding of all insect myosins, including striated muscle myosin. This rationale promotes us to investigate the folding of recombinant insect striated muscle myosins in Sf9 cells. We expressed the heavy meromyosin version of the striated muscle myosins from three insect species (Locusta migratoria, Drosophila melanogaster and Plutella xylostella) in Sf9 cells. Similar to vertebrate smooth muscle myosin, but unlike vertebrate striated muscle myosin, the insect striated muscle myosin expressed in Sf9 cells are soluble. The purified recombinant insect striated muscle myosins display normal myosin functions, including ATP-dependent actin interaction, actin-activated ATPase activity, and in vitro actin-gliding activity, indicating that Sf9 cells are suitable for expressing insect striated muscle myosin. We therefore conclude that, unlike vertebrate striated muscle myosin requiring striated muscle-specific chaperones (such as Unc45b) for its folding, insect striated muscle myosin can be properly folded by the generic protein folding machinery in insect cells.

P.124 EDG-5506 targets fast skeletal myosin and reduces muscle damage biomarkers in a phase 1 trial in Becker muscular dystrophy (BMD)

To assess safety, pharmacokinetics (PK), pharmacodynamics and biomarkers of muscle damage in healthy adults and adults with BMD. Fast (Type II) muscle fibers are affected early and disproportionately in dystrophinopathies. EDG-5506 targets fast skeletal muscle myosin, and in animal models of Duchenne muscular dystrophy (DMD), decreased muscle damage biomarkers and fibrosis, while increasing muscle strength and habitual activity. Ascending EDG-5506 doses (5-40 mg daily) were administered orally for 14 days in healthy adults who underwent assessments of safety, plasma and muscle PK, and biomarkers. Seven ambulatory adults with BMD were enrolled to assess safety, PK and effects of EDG-5506 or placebo in the setting of dystrophic muscle after 14 days. EDG-5506 was well-tolerated at all doses in healthy adults. Most common adverse events were somnolence and dizziness, which were mild and transient. Plasma and muscle exposures exceeded exposures that had demonstrated positive effects in animal models of DMD. In healthy adults and in participants with BMD, muscle concentrations substantially exceeded those in plasma, consistent with high affinity of EDG-5506 for fast fiber myosin. Muscle strength was maintained in all cohorts. In BMD participants, EDG-5506 was well-tolerated and significantly reduced creatine kinase, myoglobin, and fast skeletal muscle troponin I (TNNI2). In a plasma proteomic panel (SOMAscan), proteins associated with muscle damage and muscle metabolism were elevated in participants with BMD when compared to healthy controls. This panel of proteins decreased significantly with EDG-5506, particularly for those proteins that become elevated in response to exercise. EDG-5506 demonstrated favorable safety and PK profiles at dose levels that resulted in robust reductions in muscle damage biomarkers in adults with BMD. By targeting fast skeletal muscle myosin to protect dystrophic muscle, EDG-5506 has potential to be a novel disease-modifying approach in DMD and BMD regardless of mutation type. Phase 2 studies in BMD and DMD are planned.

Molecular-level evidence of force maintenance by smooth muscle myosin during LC20 dephosphorylation.

Smooth muscle (SM) is found in most hollow organs of the body. Phasic SM, as found in the gut, contracts to propel content, whereas tonic SM, as found in most blood vessels, maintains tension. This force maintenance is referred to as the latch state and occurs at low levels of myosin activation (myosin light chain [LC20] phosphorylation). Molecular mechanisms have been proposed to explain the latch state but have been studied only at the whole-muscle level because of technological limitations. In the current study, an assay chamber was devised to allow injection of myosin light chain phosphatase (MLCP) during laser trap and in vitro motility assays, without creating bulk flow, to reproduce latch state conditions at the molecular level. Using the laser trap in a single-beam mode, an actin filament was brought in contact with several myosin molecules on a pedestal. Myosin pulled on the actin filament until a plateau force was reached, at which point, MLCP was injected. Force maintenance was observed during LC20 dephosphorylation, the level of which was assessed in a parallel in vitro motility assay performed in the same conditions. Force was maintained longer for myosin purified from tonic SM than from phasic SM. These data support the longstanding dogma of strong bonds caused by dephosphorylated, noncycling cross-bridges. Furthermore, MLCP injection in an in vitro motility mixture assay performed with SM and skeletal muscle myosin suggests that the maintenance of these strong bonds is possible only if no energy is provided by surrounding actively cycling myosin molecules.

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.