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Background: Decreased ryanodine receptor type 1 (RyR1) protein levels are a well-described feature of recessive RYR1-related myopathies. The aim of the present study was twofold: (1) to determine whether RyR1 content is also decreased in other myopathies and (2) to investigate the mechanisms by which decreased RyR1 protein triggers muscular disorders., Methods: We used publicly available datasets, muscles from human inflammatory and mitochondrial myopathies, an inducible muscle-specific RYR1 recessive mouse model and RyR1 knockdown in C2C12 muscle cells to measure RyR1 content and endoplasmic reticulum (ER) stress markers. Proteomics, lipidomics, molecular biology and transmission electron microscopy approaches were used to decipher the alterations associated with the reduction of RyR1 protein levels., Results: RYR1 transcripts were reduced in muscle samples of patients suffering from necrotizing myopathy (P = 0.026), inclusion body myopathy (P = 0.003), polymyositis (P < 0.001) and juvenile dermatomyositis (P < 0.001) and in muscle samples of myotonic dystrophy type 2 (P < 0.001), presymptomatic (P < 0.001) and symptomatic (P < 0.001) Duchenne muscular dystrophy, Becker muscular dystrophy (P = 0.004) and limb-girdle muscular dystrophy type 2A (P = 0.004). RyR1 protein content was also significantly decreased in inflammatory myopathy (-75%, P < 0.001) and mitochondrial myopathy (-71%, P < 0.001) muscles. Proteomics data showed that depletion of RyR1 protein in C2C12 myoblasts leads to myotubes recapitulating the common molecular alterations observed in myopathies. Mechanistically, RyR1 protein depletion reduces ER-mitochondria contact length (-26%, P < 0.001), Ca 2+ transfer to mitochondria (-48%, P = 0.002) and the mitophagy gene Parkinson protein 2 transcripts (P = 0.037) and induces mitochondrial accumulation (+99%, P = 0.005) and dysfunction (P < 0.001). This was associated to the accumulation of deleterious sphingolipid species. Our data showed increased levels of the ER stress marker chaperone-binding protein/glucose regulated protein 78, GRP78-Bip, in RyR1 knockdown myotubes (+45%, P = 0.046), in mouse RyR1 recessive muscles (+58%, P = 0.001) and in human inflammatory (+96%, P = 0.006) and mitochondrial (+64%, P = 0.049) myopathy muscles. This was accompanied by increased protein levels of the pro-apoptotic protein CCAAT-enhancer-binding protein homologous protein, CHOP-DDIT3, in RyR1 knockdown myotubes (+27%, P < 0.001), mouse RyR1 recessive muscles (+63%, P = 0.009), human inflammatory (+50%, P = 0.038) and mitochondrial (+51%, P = 0.035) myopathy muscles. In publicly available datasets, the decrease in RYR1 content in myopathies was also associated to increased ER stress markers and RYR1 transcript levels are inversely correlated with ER stress markers in the control population., Conclusions: Decreased RyR1 is commonly observed in myopathies and associated to ER stress in vitro, in mouse muscle and in human myopathy muscles, suggesting a potent role of RyR1 depletion-induced ER stress in the pathogenesis of myopathies., (© 2023 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)

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 analyze the effects of common MYH7 and MYH2 mutations in the light meromyosin (LMM) region of myosin. Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in silico modeling showed that myosin coiled coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients and fluorescent ATP analog 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, myofiber mechanics experiments to investigate contractile function showed that myofiber contractility was not affected. These findings indicate that the structural remodeling 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.

Aim: Conditions related to mutations in the gene encoding the skeletal muscle ryanodine receptor 1 (RYR1) are genetic muscle disorders and include congenital myopathies with permanent weakness, as well as episodic phenotypes such as rhabdomyolysis/myalgia. Although RYR1 dysfunction is the primary mechanism in RYR1-related disorders, other downstream pathogenic events are less well understood and may include a secondary remodeling of major contractile proteins. Hence, in the present study, we aimed to investigate whether congenital myopathy-related RYR1 mutations alter the regulation of the most abundant contractile protein, myosin., Methods: We used skeletal muscle tissues from five patients with RYR1-related congenital myopathy and compared those with five controls and five patients with RYR1-related rhabdomyolysis/myalgia. We then defined post-translational modifications on myosin heavy chains (MyHCs) using LC/MS. In parallel, we determined myosin relaxed states using Mant-ATP chase experiments and performed molecular dynamics (MD) simulations., Results: LC/MS revealed two additional phosphorylations (Thr1309-P and Ser1362-P) and one acetylation (Lys1410-Ac) on the β/slow MyHC of patients with congenital myopathy. This method also identified six acetylations that were lacking on MyHC type IIa of these patients (Lys35-Ac, Lys663-Ac, Lys763-Ac, Lys1171-Ac, Lys1360-Ac, and Lys1733-Ac). MD simulations suggest that modifying myosin Ser1362 impacts the protein structure and dynamics. Finally, Mant-ATP chase experiments showed a faster ATP turnover time of myosin heads in the disordered-relaxed conformation., Conclusions: Altogether, our results suggest that RYR1 mutations have secondary negative consequences on myosin structure and function, likely contributing to the congenital myopathic phenotype., (© 2023 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Objective: The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca 2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle., Methods: A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes., Results: STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue., Conclusions: We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Dilated cardiomyopathy (DCM) is a naturally occurring heart failure condition in humans and dogs, notably characterized by a reduced contractility and ejection fraction. As the identification of its underlying cellular and molecular mechanisms remain incomplete, the aim of the present study was to assess whether the molecular motor myosin and its known relaxed conformational states are altered in DCM. For that, we dissected and skinned thin cardiac strips from left ventricle obtained from six DCM Doberman Pinschers and six nonfailing (NF) controls. We then used a combination of Mant-ATP chase experiments and X-ray diffraction to assess both energetic and structural changes of myosin. Using the Mant-ATP chase protocol, we observed that in DCM dogs, the amount of myosin molecules in the ATP-conserving conformational state, also known as superrelaxed (SRX), is significantly increased when compared with NF dogs. This alteration can be rescued by applying EMD-57033, a small molecule activating myosin. Conversely, with X-ray diffraction, we found that in DCM dogs, there is a higher proportion of myosin heads in the vicinity of actin when compared with NF dogs (1,0 to 1,1 intensity ratio). Hence, we observed an uncoupling between energetic (Mant-ATP chase) and structural (X-ray diffraction) data. Taken together, these results may indicate that in the heart of Doberman Pinschers with DCM, myosin molecules are potentially stuck in a nonsequestered but ATP-conserving SRX state, that can be counterbalanced by EMD-57033 demonstrating the potential for a myosin-centered pharmacological treatment of DCM. NEW & NOTEWORTHY The key finding of the present study is that, in left ventricles of dogs with a naturally occurring dilated cardiomyopathy, relaxed myosin molecules favor a nonsequestered superrelaxed state potentially impairing sarcomeric contractility. This alteration is rescuable by applying a small molecule activating myosin known as EMD-57033.

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., (© 2023 Lewis et al.)

Specific force (SF) has been shown to be reduced in some but not all studies of human aging using chemically skinned single muscle fibers. This may be due, in part, not only to the health status/physical activity levels of different older cohorts, but also from methodological differences in studying skinned fibers. The aim of the present study was to compare SF in fibers from older hip fracture patients (HFP), healthy master cyclists (MC), and healthy nontrained young adults (YA) using two different activating solutions. Quadriceps muscle samples and 316 fibers were obtained from HFPs (74.6 ± 4 years, n = 5), MCs (74.8 ± 1, n = 5), and YA (25.5 ± 2, n = 6). Fibers were activated (pCa 4.5, 15°C) in solutions containing either 60 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid pH buffer (TES) or 20 mM imidazole. SF was determined by normalizing force to fiber cross-sectional area (CSA) assuming either an elliptical or circular shape and to fiber myosin heavy chain content. Activation in TES resulted in significantly higher MHC-I SF in all groups and YA MHC-IIA fibers, irrespective of normalization method. Although there were no differences in SF between the participant groups, the ratio of SF between the TES and imidazole solutions was lower in HFPs compared with YAs (MHC-I P < 0.05; MHC-IIA P = 0.055). Activating solution composition, as opposed to donor characteristics, had a more notable effect on single fiber SF. However, this two-solution approach revealed an age-related difference in sensitivity in HFPs, which was not shown in MCs. This suggests further novel approaches may be required to probe age/activity-related differences in muscle contractile quality. NEW & NOTEWORTHY Whether specific force (SF) decreases with advancing age in human single skeletal muscle fibers is uncertain. Equivocal published findings may be due to the different physical activity levels of the elderly cohorts studied and/or different chemical solutions used to measure force. We compared single fiber SF between young adults, elderly cyclists, and hip fracture patients (HFP) using two solutions. The solution used significantly affected force and revealed a difference in sensitivity of HFP muscle fibers.

Slow myosin heavy chain 1 (Smyhc1) is the major sarcomeric myosin driving early contraction by slow skeletal muscle fibres in zebrafish. New mutant alleles lacking a functional smyhc1 gene move poorly, but recover motility as the later-formed fast muscle fibres of the segmental myotomes mature, and are adult viable. By motility analysis and inhibiting fast muscle contraction pharmacologically, we show that a slow muscle motility defect persists in mutants until about 1 month of age. Breeding onto a genetic background marking slow muscle fibres with EGFP revealed that mutant slow fibres undergo terminal differentiation, migration and fibre formation indistinguishable from wild type but fail to generate large myofibrils and maintain cellular orientation and attachments. In mutants, initial myofibrillar structures with 1.67 ​μm periodic actin bands fail to mature into the 1.96 ​μm sarcomeres observed in wild type, despite the presence of alternative myosin heavy chain molecules. The poorly-contractile mutant slow muscle cells generate numerous cytoplasmic organelles, but fail to grow and bundle myofibrils or to increase in cytoplasmic volume despite passive movements imposed by fast muscle. The data show that both slow myofibril maturation and cellular volume increase depend on the function of a specific myosin isoform and suggest that appropriate force production regulates muscle fibre growth., Competing Interests: Declaration of competing interest All authors declare they have no financial or competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The druggability of small-molecule binding sites can be significantly affected by protein motions and conformational changes. Ligand binding, protein dynamics and protein function have been shown to be closely interconnected in myosins. The breakthrough discovery of omecamtiv mecarbil (OM) has led to an increased interest in small molecules that can target myosin and modulate its function for therapeutic purposes (myosin modulators). In this work, we use a combination of computational methods, including steered molecular dynamics, umbrella sampling and binding pocket tracking tools, to follow the evolution of the OM binding site during the recovery stroke transition of human β-cardiac myosin. We found that steering two internal coordinates of the motor domain can recapture the main features of the transition and in particular the rearrangements of the binding site, which shows significant changes in size, shape and composition. Possible intermediate conformations were also identified, in remarkable agreement with experimental findings. The differences in the binding site properties observed along the transition can be exploited for the future development of conformation-selective myosin modulators., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Akter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Skeletal muscle is the most abundant component of the mature mammalian phenotype. Designed to generate contractile force and movement, skeletal muscle is crucial for organism health, function and development. One of the great interests for muscle biologists is in understanding how skeletal muscle adapts during periods of stress and stimuli, such as disease, disuse and ageing. To this end, genomic-based experimental and analytical approaches offer one of the most powerful approaches for comprehensively mapping the molecular paradigms that regulate skeletal muscle. With the power, applicability, and robustness of 'omic' technologies continually being developed, we are now in a position to investigate these molecular mechanisms in skeletal muscle to an unprecedented level of accuracy and precision, heralding the dawn of a new era of functional genomics in the field of muscle physiology., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Congenital myopathies are a vast group of genetic muscle diseases. Among the causes are mutations in the MYH2 gene resulting in truncated type IIa myosin heavy chains (MyHCs). The precise cellular and molecular mechanisms by which these mutations induce skeletal muscle symptoms remain obscure. Hence, in the present study, we aimed to explore whether such genetic defects would alter the presence as well as the post-translational modifications of MyHCs and the functionality of myosin molecules. For this, we dissected muscle fibers from four myopathic patients with MYH2 truncating mutations and from five human healthy controls. We then assessed 1 ) MyHCs presence/post-translational modifications using LC/MS; 2 ) relaxed myosin conformation and concomitant ATP consumption with a loaded Mant-ATP chase setup; 3 ) myosin activation with an unloaded in vitro motility assay; and 4 ) cellular force production with a myofiber mechanical setup. Interestingly, the type IIa MyHC with one additional acetylated lysine (Lys35-Ac) was present in the patients. This was accompanied by 1 ) a higher ATP demand of myosin heads in the disordered-relaxed conformation; 2 ) faster actomyosin kinetics; and 3 ) reduced muscle fiber force. Overall, our findings indicate that MYH2 truncating mutations impact myosin presence/functionality in human adult mature myofibers by disrupting the ATPase activity and actomyosin complex. These are likely important molecular pathological disturbances leading to the myopathic phenotype in patients.

Background: Chronic mTORC1 activation in skeletal muscle is linked with age-associated loss of muscle mass and strength, known as sarcopenia. Genetic activation of mTORC1 by conditionally ablating mTORC1 upstream inhibitor TSC1 in skeletal muscle accelerates sarcopenia development in adult mice. Conversely, genetic suppression of mTORC1 downstream effectors of protein synthesis delays sarcopenia in natural aging mice. mTORC1 promotes protein synthesis by activating ribosomal protein S6 kinases (S6Ks) and inhibiting eIF4E-binding proteins (4EBPs). Whole-body knockout of S6K1 or muscle-specific over-expression of a 4EBP1 mutant transgene (4EBP1mt), which is resistant to mTORC1-mediated inhibition, ameliorates muscle loss with age and preserves muscle function by enhancing mitochondria activities, despite both transgenic mice showing retarded muscle growth at a young age. Why repression of mTORC1-mediated protein synthesis can mitigate progressive muscle atrophy and dysfunction with age remains unclear., Methods: Mice with myofiber-specific knockout of TSC1 (TSC1mKO), in which mTORC1 is hyperactivated in fully differentiated myofibers, were used as a mouse model of sarcopenia. To elucidate the role of mTORC1-mediated protein synthesis in regulating muscle mass and physiology, we bred the 4EBP1mt transgene or S6k1 floxed mice into the TSC1mKO mouse background to generate 4EBP1mt-TSC1mKO or S6K1-TSC1mKO mice, respectively. Functional and molecular analyses were performed to assess their role in sarcopenia development., Results: Here, we show that 4EBP1mt-TSC1mKO, but not S6K1-TSC1mKO, preserved muscle mass (36.7% increase compared with TSC1mKO, P < 0.001) and strength (36.8% increase compared with TSC1mKO, P < 0.01) at the level of control mice. Mechanistically, 4EBP1 activation suppressed aberrant protein synthesis (two-fold reduction compared with TSC1mKO, P < 0.05) and restored autophagy flux without relieving mTORC1-mediated inhibition of ULK1, an upstream activator of autophagosome initiation. We discovered a previously unidentified phenotype of lysosomal failure in TSC1mKO mouse muscle, in which the lysosomal defect was also conserved in the naturally aged mouse muscle, whereas 4EBP1 activation enhanced lysosomal protease activities to compensate for impaired autophagy induced by mTORC1 hyperactivity. Consequently, 4EBP1 activation relieved oxidative stress to prevent toxic aggregate accumulation (0.5-fold reduction compared with TSC1mKO, P < 0.05) in muscle and restored mitochondrial homeostasis and function., Conclusions: We identify 4EBP1 as a communication hub coordinating protein synthesis and degradation to protect proteostasis, revealing therapeutic potential for activating lysosomal degradation to mitigate sarcopenia., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

The development of small molecule myosin modulators has seen an increased effort in recent years due to their possible use in the treatment of cardiac and skeletal myopathies. Omecamtiv mecarbil (OM) is the first-in-class cardiac myotrope and the first to enter clinical trials. Its selectivity toward slow/beta-cardiac myosin lies at the heart of its function; however, little is known about the underlying reasons for selectivity to this isoform as opposed to other closely related ones such as fast-type skeletal myosins. In this work, we compared the structure and dynamics of the OM binding site in cardiac and in fasttype IIa skeletal myosin to identify possible reasons for OM selectivity. We found that the different shape, size, and composition of the binding pocket in skeletal myosin directly affects the binding mode and related affinity of OM, which is potentially a result of weaker interactions and less optimal molecular recognition. Moreover, we identified a side pocket adjacent to the OM binding site that shows increased accessibility in skeletal myosin compared with the cardiac isoform. These findings could pave the way to the development of skeletal-selective compounds that can target this region of the protein and potentially be used to treat congenital myopathies where muscle weakness is related to myosin loss of function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

After years of intense research using structural, biological, and biochemical experimental procedures, it is clear that myosin molecules are essential for striated muscle contraction. However, this is just the tip of the iceberg of their function. Interestingly, it has been shown recently that these molecules (especially myosin heavy chains) are also crucial for cardiac and skeletal muscle resting state. In the present review, we first overview myosin heavy chain biochemical states and how they influence the consumption of ATP. We then detail how neighboring partner proteins including myosin light chains and myosin binding protein C intervene in such processes, modulating the ATP demand in health and disease. Finally, we present current experimental drugs targeting myosin ATP consumption and how they can treat muscle diseases.

Nemaline myopathy (NM) is one of the most common non-dystrophic genetic muscle disorders. NM is often associated with mutations in the NEB gene. Even though the exact NEB-NM pathophysiological mechanisms remain unclear, histological analyses of patients' muscle biopsies often reveal unexplained accumulation of glycogen and abnormally shaped mitochondria. Hence, the aim of the present study was to define the exact molecular and cellular cascade of events that would lead to potential changes in muscle energetics in NEB-NM. For that, we applied a wide range of biophysical and cell biology assays on skeletal muscle fibres from NM patients as well as untargeted proteomics analyses on isolated myofibres from a muscle-specific nebulin-deficient mouse model. Unexpectedly, we found that the myosin stabilizing conformational state, known as super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting muscle fibres from NEB-NM patients when compared with controls or with other forms of genetic/rare, acquired NM. This destabilization of the myosin super-relaxed state had dynamic consequences as we observed a remodeling of the metabolic proteome in muscle fibres from nebulin-deficient mice. Altogether, our findings explain some of the hitherto obscure hallmarks of NM, including the appearance of abnormal energy proteins and suggest potential beneficial effects of drugs targeting myosin activity/conformations for NEB-NM., (© 2022. The Author(s).)

Introduction: Nemaline myopathies (NM) represent a group of clinically and genetically heterogeneous congenital muscle disorders with the common denominator of nemaline rods on muscle biopsy. NEB and ACTA1 are the most common causative genes. Currently, available treatments are supportive., Areas Covered: We explored experimental treatments for NM, identifying at least eleven mainly pre-clinical approaches utilizing murine and/or human muscle cells. These approaches target either i) the causative gene or associated genes implicated in the same pathway; ii) pathophysiologically relevant biochemical mechanisms such as calcium/myosin regulation of muscle contraction; iii) myogenesis; iv) other therapies that improve or optimize muscle function more generally; v) and/or combinations of the above. The scope and efficiency of these attempts is diverse, ranging from gene-specific effects to those widely applicable to all NM-associated genes., Expert Opinion: The wide range of experimental therapies currently under consideration for NM is promising. Potential translation into clinical use requires consideration of additional factors such as the potential muscle type specificity as well as the possibility of gene expression remodeling. Challenges in clinical translation include the rarity and heterogeneity of genotypes, phenotypes, and disease trajectories, as well as the lack of longitudinal natural history data and validated outcomes and biomarkers.

The transcriptional demands of skeletal muscle fibres are high and require hundreds of nuclei (myonuclei) to produce specialised contractile machinery and multiple mitochondria along their length. Each myonucleus spatially regulates gene expression in a finite volume of cytoplasm, termed the myonuclear domain (MND), which positively correlates with fibre cross-sectional area (CSA). Endurance training triggers adaptive responses in skeletal muscle, including myonuclear accretion, decreased MND sizes and increased expression of the transcription co-activator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Previous work has shown that overexpression of PGC-1α in skeletal muscle regulates mitochondrial biogenesis, myonuclear accretion and MND volume. However, whether PGC-1α is critical for these processes in adaptation to endurance training remained unclear. To test this, we evaluated myonuclear distribution and organisation in endurance-trained wild-type mice and mice lacking PGC-1α in skeletal muscle (PGC-1α mKO). Here, we show a differential myonuclear accretion response to endurance training that is governed by PGC-1α and is dependent on muscle fibre size. The positive relationship of MND size and muscle fibre CSA trended towards a stronger correlation in PGC-1a mKO versus control after endurance training, suggesting that myonuclear accretion was slightly affected with increasing fibre CSA in PGC-1α mKO. However, in larger fibres, the relationship between MND and CSA was significantly altered in trained versus sedentary PGC-1α mKO, suggesting that PGC-1α is critical for myonuclear accretion in these fibres. Accordingly, there was a negative correlation between the nuclear number and CSA, suggesting that in larger fibres myonuclear numbers fail to scale with CSA. Our findings suggest that PGC-1α is an important contributor to myonuclear accretion following moderate-intensity endurance training. This may contribute to the adaptive response to endurance training by enabling a sufficient rate of transcription of genes required for mitochondrial biogenesis., (© 2021 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Myosinopathies are defined as a group of muscle disorders characterized by mutations in genes encoding myosin heavy chains. Their exact molecular and cellular mechanisms remain unclear. In the present study, we have focused our attention on a MYH1 -related E321G amino acid substitution within the head region of the type IIx skeletal myosin heavy chain, associated with clinical signs of atrophy, inflammation and/or profound rhabdomyolysis, known as equine myosin heavy chain myopathy. We performed Mant-ATP chase experiments together with force measurements on isolated IIx myofibres from control horses ( MYH1 E321G-/- ) and Quarter Horses homozygous ( MYH1 E321G+/+ ) or heterozygous ( MYH1 E321G+/- ) for the E321G mutation. The single residue replacement did not affect the relaxed conformations of myosin molecules. Nevertheless, it significantly increased its active behaviour as proven by the higher maximal force production and Ca 2+ sensitivity for MYH1 E321G+/+ in comparison with MYH1 E321G+/- and MYH1 E321G-/- horses. Altogether, these findings indicate that, in the presence of the E321G mutation, a molecular and cellular hyper-contractile phenotype occurs which could contribute to the development of the myosin heavy chain myopathy.

Chemically skinned fibres allow the study of human muscle contractile function in vitro. A particularly important parameter is specific force (SF), that is, maximal isometric force divided by cross-sectional area, representing contractile quality. Although SF varies substantially between studies, the magnitude and cause of this variability remains puzzling. Here, we aimed to summarize and explore the cause of variability in SF between studies. A systematic search was conducted in Medline, Embase and Web of Science databases in June 2020, yielding 137 data sets from 61 publications which studied healthy, young adults. Five-fold differences in mean SF data were observed. Adjustments to the reported data for key methodological differences allowed between-study comparisons to be made. However, adjustment for fibre shape, swelling and sarcomere length failed to significantly reduce SF variance (I 2 = 96%). Interestingly, grouping papers based on shared authorship did reveal consistency within research groups. In addition, lower SF was found to be associated with higher phosphocreatine concentrations in the fibre activating solution and with Triton X-100 being used as a skinning agent. Although the analysis showed variance across the literature, the ratio of SF in single fibres containing myosin heavy chain isoforms IIA or I was found to be consistent across research groups. In conclusion, whilst the skinned fibre technique is reliable for studying in vitro force generation of single fibres, the composition of the solution used to activate fibres, which differs between research groups, is likely to heavily influence SF values., (© 2021 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Background: Myofibrillar myopathy (MFM) of unknown aetiology has recently been identified in Warmblood (WB) horses. In humans, 16 genes have been implicated in various MFM-like disorders., Objectives: To identify variants in 16 MFM candidate genes and compare allele frequencies of all variants between MFM WB and non-MFM WB and coding variants with moderate or severe predicted effects in MFM WB with publicly available data of other breeds. To compare differential gene expression and muscle fibre contractile force between MFM and non-MFM WB., Study Design: Case-control., Animals: 8 MFM WB, 8 non-MFM WB, 33 other WB, 32 Thoroughbreds, 80 Quarter Horses and 77 horses of other breeds in public databases., Methods: Variants were called within transcripts of 16 candidate genes using gluteal muscle mRNA sequences aligned to EquCab3.0 and allele frequencies compared by Fisher's exact test among MFM WB, non-MFM WB and public sequences across breeds. Candidate gene differential expression was determined between MFM and non-MFM WB by fitting a negative binomial generalised log-linear model per gene (false discovery rate <0.05). The maximal isometric force/cross-sectional area generated by isolated membrane-permeabilised muscle fibres was determined., Results: None of the 426 variants identified in 16 candidate genes were associated with MFM including 26 missense variants. Breed-specific differences existed in allele frequencies. Candidate gene differential expression and muscle fibre-specific force did not differ between MFM WB (143.1 ± 34.7 kPa) and non-MFM WB (140.2 ± 43.7 kPa) (P = .8)., Main Limitations: RNA-seq-only assays transcripts expressed in skeletal muscle. Other possible candidate genes were not evaluated., Conclusions: Evidence for association of variants with a disease is essential because coding sequence variants are common in the equine genome. Variants identified in MFM candidate genes, including two coding variants offered as commercial MFM equine genetic tests, did not associate with the WB MFM phenotype., (© 2020 EVJ Ltd.)

Mammalian cells exhibit remarkable diversity in cell size, but the factors that regulate establishment and maintenance of these sizes remain poorly understood. This is especially true for skeletal muscle, comprised of syncytial myofibers that each accrue hundreds of nuclei during development. Here, we directly explore the assumed causal relationship between multinucleation and establishment of normal size through titration of myonuclear numbers during mouse neonatal development. Three independent mouse models, where myonuclear numbers were reduced by 75, 55, or 25%, led to the discovery that myonuclei possess a reserve capacity to support larger functional cytoplasmic volumes in developing myofibers. Surprisingly, the results revealed an inverse relationship between nuclei numbers and reserve capacity. We propose that as myonuclear numbers increase, the range of transcriptional return on a per nuclear basis in myofibers diminishes, which accounts for both the absolute reliance developing myofibers have on nuclear accrual to establish size, and the limits of adaptability in adult skeletal muscle.

X-linked myotubular myopathy (XLMTM) is a life-threatening skeletal muscle disease caused by mutations in the MTM1 gene. XLMTM fibres display a population of nuclei mispositioned in the centre. In the present study, we aimed to explore whether positioning and overall distribution of nuclei affects cellular organization and contractile function, thereby contributing to muscle weakness in this disease. We also assessed whether gene therapy alters nuclear arrangement and function. We used tissue from human patients and animal models, including XLMTM dogs that had received increasing doses of recombinant AAV8 vector restoring MTM1 expression (rAAV8-cMTM1). We then used single isolated muscle fibres to analyze nuclear organization and contractile function. In addition to the expected mislocalization of nuclei in the centre of muscle fibres, a novel form of nuclear mispositioning was observed: irregular spacing between those located at the fibre periphery, and an overall increased number of nuclei, leading to dramatically smaller and inconsistent myonuclear domains. Nuclear mislocalization was associated with decreases in global nuclear synthetic activity, contractile protein content and intrinsic myofilament force production. A contractile deficit originating at the myofilaments, rather than mechanical interference by centrally positioned nuclei, was supported by experiments in regenerated mouse muscle. Systemic administration of rAAV8-cMTM1 at doses higher than 2.5 × 10 13  vg kg -1 allowed a full rescue of all these cellular defects in XLMTM dogs. Altogether, these findings identify previously unrecognized pathological mechanisms in human and animal XLMTM, associated with myonuclear defects and contractile filament function. These defects can be reversed by gene therapy restoring MTM1 expression in dogs with XLMTM.

Classical dynamins are large GTPases regulating membrane and cytoskeleton dynamics, and they are linked to different pathological conditions ranging from neuromuscular diseases to encephalopathy and cancer. Dominant dynamin 2 (DNM2) mutations lead to either mild adult onset or severe autosomal dominant centronuclear myopathy (ADCNM). Our objectives were to better understand the pathomechanism of severe ADCNM and test a potential therapy. Here, we created the Dnm2SL/+ mouse line harboring the common S619L mutation found in patients with severe ADCNM and impairing the conformational switch regulating dynamin self-assembly and membrane remodeling. The Dnm2SL/+ mouse faithfully reproduces severe ADCNM hallmarks with early impaired muscle function and force, together with myofiber hypotrophy. It revealed swollen mitochondria lacking cristae as the main ultrastructural defect and potential cause of the disease. Patient analysis confirmed this structural hallmark. In addition, DNM2 reduction with antisense oligonucleotides after disease onset efficiently reverted locomotor and force defects after only 3 weeks of treatment. Most histological defects including mitochondria alteration were partially or fully rescued. Overall, this study highlights an efficient approach to revert the severe form of dynamin-related centronuclear myopathy. These data also reveal that the dynamin conformational switch is key for muscle function and should be targeted for future therapeutic developments.

Skeletal muscle weakness is an important determinant of age-related declines in independence and quality of life but its causes remain unclear. Accelerated ageing syndromes such as Hutchinson-Gilford Progerin Syndrome, caused by mutations in genes encoding nuclear envelope proteins, have been extensively studied to aid our understanding of the normal biological ageing process. Like several other pathologies associated with genetic defects to nuclear envelope proteins including Emery-Dreifuss muscular dystrophy, Limb-Girdle muscular dystrophy and congenital muscular dystrophy, these disorders can lead to severe muscle dysfunction. Here, we first describe the structure and function of nuclear envelope proteins, and then review the mechanisms by which mutations in genes encoding nuclear envelope proteins induce premature ageing diseases and muscle pathologies. In doing so, we highlight the potential importance of such genes in processes leading to skeletal muscle weakness in old age., (© 2020 The Author(s).)

Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, Neb Y2303H, Y935X , has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, Neb Y2303H,Y935X mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications.

Skeletal muscle fibres are large, elongated multinucleated cells. Each nucleus within a myofibre is responsible for generating gene products for a finite volume of cytoplasm-the myonuclear domain (MND). Variation in MND sizes during atrophy, hypertrophy and disease states, are common. The factors that contribute to definitive MND sizes are not yet fully understood. Previous work has shown that peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1-α) modulates MND volume, presumably to support increased biogenesis of mitochondria. The transcriptional co-regulator peroxisome proliferator-activated receptor gamma coactivator 1β (PGC1-β) is a homologue of PGC1-α with overlapping functions. To investigate the role of this protein in MND size regulation, we studied a mouse skeletal muscle specific knockout (cKO). Myofibres were isolated from the fast twitch extensor digitorum longus (EDL) muscle, membrane-permeabilised and analysed in 3 dimensions using confocal microscopy. PGC1-β ablation resulted in no significant difference in MND size between cKO and wild type (WT) mice, however, subtle differences in nuclear morphology were observed. To determine whether these nuclear shape changes were associated with alterations in global transcriptional activity, acetyl histone H3 immunostaining was carried out. We found there was no significant difference in nuclear fluorescence intensity between the two genotypes. Overall, the results suggest that PGC-1α and PGC-1β play different roles in regulating nuclear organisation in skeletal muscle; however, further work is required to pinpoint their exact functions.

Nemaline myopathy (NM) is a skeletal muscle disorder caused by mutations in genes that are generally involved in muscle contraction, in particular those related to the structure and/or regulation of the thin filament. Many pathogenic aspects of this disease remain largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mouse models and patients with NM: irregular spacing and morphology of nuclei; disrupted nuclear envelope; altered chromatin arrangement; and disorganisation of the cortical cytoskeleton. Impairments in contractility are the primary cause of these nuclear defects. We also establish the role of microtubule organisation in determining nuclear morphology, a phenomenon which is likely to contribute to nuclear alterations in this disease. Our results overlap with findings in diseases caused directly by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to explain some of the hallmarks of NM, including contractile filament disarray, altered mechanical properties and broad transcriptional alterations.

Myopathies are notably associated with mutations in genes encoding proteins known to be essential for the force production of skeletal muscle fibers, such as skeletal alpha-actin. The exact molecular mechanisms by which these specific defects induce myopathic phenotypes remain unclear. Hence, in the present study, to better understand actin dysfunction, we conducted a molecular dynamic simulation together with ex vivo experiments of the specific muscle disease-causing actin mutation, D286G located in the actin-actin interface. Our computational study showed that D286G impairs the flexural rigidity of actin filaments. However, upon activation, D286G did not have any direct consequences on actin filament extension. Hence, D286G may alter the structure of actin filaments but, when expressed together with normal actin molecules, it may only have minor effects on the ex vivo mechanics of actin filaments upon skeletal muscle fiber contraction.

The pyruvate dehydrogenase complex (PDC) converts pyruvate to acetyl-CoA and is an important control point for carbohydrate (CHO) oxidation. However, the importance of the PDC and CHO oxidation to muscle metabolism and exercise performance, particularly during prolonged or high-intensity exercise, has not been fully defined especially in mature skeletal muscle. To this end, we determined whether skeletal muscle-specific loss of pyruvate dehydrogenase alpha 1 ( Pdha1), which is a critical subunit of the PDC, impacts resting energy metabolism, exercise performance, or metabolic adaptation to high-fat diet (HFD) feeding. For this, we generated a tamoxifen (TMX)-inducible Pdha1 knockout (PDHmKO) mouse, in which PDC activity is temporally and specifically ablated in adult skeletal muscle. We assessed energy expenditure, ex vivo muscle contractile performance, and endurance exercise capacity in PDHmKO mice and wild-type (WT) littermates. Additionally, we studied glucose homeostasis and insulin sensitivity in muscle after 12 wk of HFD feeding. TMX administration largely ablated PDHα in skeletal muscle of adult PDHmKO mice but did not impact energy expenditure, muscle contractile function, or low-intensity exercise performance. Additionally, there were no differences in muscle insulin sensitivity or body composition in PDHmKO mice fed a control or HFD, as compared with WT mice. However, exercise capacity during high-intensity exercise was severely impaired in PDHmKO mice, in parallel with a large increase in plasma lactate concentration. In conclusion, although skeletal muscle PDC is not a major contributor to resting energy expenditure or long-duration, low-intensity exercise performance, it is necessary for optimal performance during high-intensity exercise.

Centronuclear myopathies (CNM) are a group of severe muscle diseases for which no effective therapy is currently available. We have previously shown that reduction of the large GTPase DNM2 in a mouse model of the X-linked form, due to loss of myotubularin phosphatase MTM1, prevents the development of the skeletal muscle pathophysiology. As DNM2 is mutated in autosomal dominant forms, here we tested whether DNM2 reduction can rescue DNM2 -related CNM in a knock-in mouse harboring the p.R465W mutation ( Dnm2 RW/+ ) and displaying a mild CNM phenotype similar to patients with the same mutation. A single intramuscular injection of adeno-associated virus-shRNA targeting Dnm2 resulted in reduction in protein levels 5 wk post injection, with a corresponding improvement in muscle mass and fiber size distribution, as well as an improvement in histopathological CNM features. To establish a systemic treatment, weekly i.p. injections of antisense oligonucleotides targeting Dnm2 were administered to Dnm2 RW/+ mice for 5 wk. While muscle mass, histopathology, and muscle ultrastructure were perturbed in Dnm2 RW/+ mice compared with wild-type mice, these features were indistinguishable from wild-type mice after reducing DNM2. Therefore, DNM2 knockdown via two different strategies can efficiently correct the myopathy due to DNM2 mutations, and it provides a common therapeutic strategy for several forms of centronuclear myopathy. Furthermore, we provide an example of treating a dominant disease by targeting both alleles, suggesting that this strategy may be applied to other dominant diseases., Competing Interests: Conflict of interest statement: H.T., J.L., and B.S.C. are inventors of a patent on targeting DNM2 for the treatment of centronuclear myopathies. J.L. and B.S.C. are scientific advisors for Dynacure.

Physiological and premature aging are frequently associated with an accumulation of prelamin A, a precursor of lamin A, in the nuclear envelope of various cell types. Here, we aimed to underpin the hitherto unknown mechanisms by which prelamin A alters myonuclear organization and muscle fiber function. By experimentally studying membrane-permeabilized myofibers from various transgenic mouse lines, our results indicate that, in the presence of prelamin A, the abundance of nuclei and myosin content is markedly reduced within muscle fibers. This leads to a concept by which the remaining myonuclei are very distant from each other and are pushed to function beyond their maximum cytoplasmic capacity, ultimately inducing muscle fiber weakness.

Skeletal muscle fibers are giant multinucleated cells wherein individual nuclei govern the protein synthesis in a finite volume of cytoplasm; this is termed the myonuclear domain (MND). The factors that control MND size remain to be defined. In the present study, we studied the contribution of the NAD + -dependent deacetylase, sirtuin 1 (SIRT1), to the regulation of nuclear number and MND size. For this, we isolated myofibers from mice with tissue-specific inactivation (mKO) or inducible overexpression (imOX) of SIRT1 and analyzed the 3D organisation of myonuclei. In imOX mice, the number of nuclei was increased whilst the average MND size was decreased as compared to littermate controls. Our findings were the opposite in mKO mice. Muscle stem cell (satellite cell) numbers were reduced in mKO muscles, a possible explanation for the lower density of myonuclei in these mice; however, no change was observed in imOX mice, suggesting that other factors might also be involved, such as the functional regulation of stem cells/muscle precursors. Interestingly, however, the changes in the MND volume did not impact the force-generating capacity of muscle fibers. Taken together, our results demonstrate that SIRT1 is a key regulator of MND sizes, although the underlying molecular mechanisms and the cause-effect relationship between MND and muscle function remain to be fully defined., (© 2018 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.)

The congenital myopathies are a group of early-onset, non-dystrophic neuromuscular conditions with characteristic muscle biopsy findings, variable severity and a stable or slowly progressive course. Pronounced weakness in axial and proximal muscle groups is a common feature, and involvement of extraocular, cardiorespiratory and/or distal muscles can implicate specific genetic defects. Central core disease (CCD), multi-minicore disease (MmD), centronuclear myopathy (CNM) and nemaline myopathy were among the first congenital myopathies to be reported, and they still represent the main diagnostic categories. However, these entities seem to belong to a much wider phenotypic spectrum. To date, congenital myopathies have been attributed to mutations in over 20 genes, which encode proteins implicated in skeletal muscle Ca 2+ homeostasis, excitation-contraction coupling, thin-thick filament assembly and interactions, and other mechanisms. RYR1 mutations are the most frequent genetic cause, and CCD and MmD are the most common subgroups. Next-generation sequencing has vastly improved mutation detection and has enabled the identification of novel genetic backgrounds. At present, management of congenital myopathies is largely supportive, although new therapeutic approaches are reaching the clinical trial stage.

Nemaline myopathy (NM) is a heterogeneous congenital skeletal muscle disease with cytoplasmic rod-like structures (nemaline bodies) in muscle tissue. While weakness in NM is related to contractile abnormalities, myofiber smallness is an additional abnormality in NM that may be treatable. We evaluated the effects of mRK35 (a myostatin inhibitor developed by Pfizer) treatment in the TgACTA1D286G mouse model of NM. mRK35 induced skeletal muscle growth that led to significant increases in animal bodyweight, forelimb grip strength and muscle fiber force, although it should be noted that animal weight and forelimb grip strength in untreated TgACTA1D286G mice was not different from controls. Treatment was also associated with an increase in the number of tubular aggregates found in skeletal muscle. These findings suggest that myostatin inhibition may be useful in promoting muscle growth and strength in Acta1-mutant muscle, while also further establishing the relationship between low levels of myostatin and tubular aggregate formation., (© The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Muscle fibres are multinucleated cells, with each nucleus controlling the protein synthesis in a finite volume of cytoplasm termed the myonuclear domain (MND). What determines MND size remains unclear. In the present study, we aimed to test the hypothesis that the level of expression of the transcriptional coactivator PGC-1α and subsequent activation of the mitochondrial biogenesis are major contributors. Hence, we used two transgenic mouse models with varying expression of PGC-1α in skeletal muscles. We isolated myofibres from the fast twitch extensor digitorum longus (EDL) and slow twitch diaphragm muscles. We then membrane-permeabilised them and analysed the 3D spatial arrangements of myonuclei. In EDL muscles, when PGC-1α is over-expressed, MND volume decreases; whereas, when PGC-1α is lacking, no change occurs. In the diaphragm, no clear difference was noted. This indicates that PGC-1α and the related mitochondrial biogenesis programme are determinants of MND size. PGC-1α may facilitate the addition of new myonuclei in order to reach MND volumes that can support an increased mitochondrial density. J. Cell. Physiol. 232: 1270-1274, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The congenital myopathies - including Central Core Disease (CCD), Multi-minicore Disease (MmD), Centronuclear Myopathy (CNM), Nemaline Myopathy (NM) and Congenital Fibre Type Disproportion (CFTD) - are a genetically heterogeneous group of early-onset neuromuscular conditions characterized by distinct histopathological features, and associated with a substantial individual and societal disease burden. Appropriate supportive management has substantially improved patient morbidity and mortality but there is currently no cure. Recent years have seen an exponential increase in the genetic and molecular understanding of these conditions, leading to the identification of underlying defects in proteins involved in calcium homeostasis and excitation-contraction coupling, thick/thin filament assembly and function, redox regulation, membrane trafficking and/or autophagic pathways. Based on these findings, specific therapies are currently being developed, or are already approaching the clinical trial stage. Despite undeniable progress, therapy development faces considerable challenges, considering the rarity and diversity of specific conditions, and the size and complexity of some of the genes and proteins involved. The present review will summarize the key genetic, histopathological and clinical features of specific congenital myopathies, and outline therapies already available or currently being developed in the context of known pathogenic mechanisms. The relevance of newly discovered molecular mechanisms and novel gene editing strategies for future therapy development will be discussed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The dysfunction in a number of inherited cardiac and skeletal myopathies is primarily due to an altered ability of myofilaments to generate force and motion. Despite this crucial knowledge, there are, currently, no effective therapeutic interventions for these diseases. In this short review, we discuss recent findings giving strong evidence that genetically or pharmacologically modulating one of the myofilament proteins, myosin, could alleviate the muscle pathology. This should constitute a research and clinical priority., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomers are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. These phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The greater susceptibility to contraction-induced skeletal muscle injury (fragility) is an important dystrophic feature and tool for testing preclinic dystrophin-based therapies for Duchenne muscular dystrophy. However, how these therapies reduce the muscle fragility is not clear., Methods: To address this question, we first determined the event(s) of the excitation-contraction cycle which is/are altered following lengthening (eccentric) contractions in the mdx muscle., Results: We found that the immediate force drop following lengthening contractions, a widely used measure of muscle fragility, was associated with reduced muscle excitability. Moreover, the force drop can be mimicked by an experimental reduction in muscle excitation of uninjured muscle. Furthermore, the force drop was not related to major neuromuscular transmission failure, excitation-contraction uncoupling, and myofibrillar impairment. Secondly, and importantly, the re-expression of functional truncated dystrophin in the muscle of mdx mice using an exon skipping strategy partially prevented the reductions in both force drop and muscle excitability following lengthening contractions., Conclusion: We demonstrated for the first time that (i) the increased susceptibility to contraction-induced muscle injury in mdx mice is mainly attributable to reduced muscle excitability; (ii) dystrophin-based therapy improves fragility of the dystrophic skeletal muscle by preventing reduction in muscle excitability.

Objective: Nemaline myopathy, one of the most common congenital myopathies, is associated with mutations in various genes including ACTA1. This disease is also characterized by various forms/degrees of muscle weakness, with most cases being severe and resulting in death in infancy. Recent findings have provided valuable insight into the underlying pathophysiological mechanisms. Mutations in ACTA1 directly disrupt binding interactions between actin and myosin, and consequently the intrinsic force-generating capacity of muscle fibers. ACTA1 mutations are also associated with variations in myofiber size, the mechanisms of which have been unclear. In the present study, we sought to test the hypotheses that the compromised functional and morphological attributes of skeletal muscles bearing ACTA1 mutations (1) would be directly due to the inefficient actomyosin complex and (2) could be restored by manipulating myosin expression., Methods: We used a knockin mouse model expressing the ACTA1 His40Tyr actin mutation found in human patients. We then performed in vivo intramuscular injections of recombinant adeno-associated viral vectors harboring a myosin transgene known to facilitate muscle contraction., Results: We observed that in the presence of the transgene, the intrinsic force-generating capacity was restored and myofiber size was normal., Interpretation: This demonstrates a direct link between disrupted attachment of myosin molecules to actin monomers and muscle fiber atrophy. These data also suggest that further therapeutic interventions should primarily target myosin dysfunction to alleviate the pathology of ACTA1-related nemaline myopathy. Ann Neurol 2016;79:717-725., (© 2016 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.)

The sarcomeric tropomodulin (Tmod) isoforms Tmod1 and Tmod4 cap thin filament pointed ends and functionally interact with the leiomodin (Lmod) isoforms Lmod2 and Lmod3 to control myofibril organization, thin filament lengths, and actomyosin crossbridge formation in skeletal muscle fibers. Here, we show that Tmod4 is more abundant than Tmod1 at both the transcript and protein level in a variety of muscle types, but the relative abundances of sarcomeric Tmods are muscle specific. We then generate Tmod4(-/-) mice, which exhibit normal thin filament lengths, myofibril organization, and skeletal muscle contractile function owing to compensatory upregulation of Tmod1, together with an Lmod isoform switch wherein Lmod3 is downregulated and Lmod2 is upregulated. However, RNAi depletion of Tmod1 from either wild-type or Tmod4(-/-) muscle fibers leads to thin filament elongation by ∼15%. Thus, Tmod1 per se, rather than total sarcomeric Tmod levels, controls thin filament lengths in mouse skeletal muscle, whereas Tmod4 appears to be dispensable for thin filament length regulation. These findings identify Tmod1 as the key direct regulator of thin filament length in skeletal muscle, in both adult muscle homeostasis and in developmentally compensated contexts., (© 2015. Published by The Company of Biologists Ltd.)

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.

In humans, mutant skeletal muscle α-actin proteins are associated with contractile dysfunction, skeletal muscle weakness and a wide range of primarily skeletal muscle diseases. Despite this knowledge, the exact molecular mechanisms triggering the contractile dysfunction remain unknown. Here, we aimed to unravel these. Hence, we used a transgenic mouse model expressing a well-described D286G mutant skeletal muscle α-actin protein and recapitulating the human condition of contractile deregulation and severe skeletal muscle weakness. We then recorded and analyzed the small-angle X-ray diffraction patterns of isolated membrane-permeabilized myofibers. Results showed that upon addition of Ca(2+), the intensity changes of the second (1/19 nm(-1)) and sixth (1/5.9 nm(-1)) actin layer lines and of the first myosin meridional reflection (1/14.3 nm(-1)) were disrupted when the thin-thick filament overlap was optimal (sarcomere length of 2.5-2.6 μm). However these reflections were normal when the thin and thick filaments were not interacting (sarcomere length>3.6 μm). These findings demonstrate, for the first time, that the replacement of just one amino acid in the skeletal muscle α-actin protein partly prevents actin conformational changes during activation, disrupting the strong binding of myosin molecules. This leads to a limited myosin-related tropomyosin movement over the thin filaments, further affecting the amount of cross-bridges, explaining the contractile dysfunction., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Skeletal muscle is one of the most dynamic and plastic tissues of the human body. In humans, skeletal muscle comprises approximately 40% of total body weight and contains 50-75% of all body proteins. In general, muscle mass depends on the balance between protein synthesis and degradation and both processes are sensitive to factors such as nutritional status, hormonal balance, physical activity/exercise, and injury or disease, among others. In this review, we discuss the various domains of muscle structure and function including its cytoskeletal architecture, excitation-contraction coupling, energy metabolism, and force and power generation. We will limit the discussion to human skeletal muscle and emphasize recent scientific literature on single muscle fibers.

Nemaline myopathy, the most common congenital myopathy, is characterized by mutations in genes encoding myofilament proteins such as skeletal α-actin. These mutations are thought to ultimately lead to skeletal muscle weakness. Interestingly, some of the mutations appear to be more potent in males than in females. The underlying mechanisms remain obscure but may be related to sex-specific differences in the myofilament function of both limb and respiratory muscles. To verify this, in the present study, we used skeletal muscles (tibialis anterior and diaphragm) from a transgenic mouse model harbouring the His40Tyr amino acid substitution in skeletal α-actin. In this animal model, 60% of males die by 13weeks of age (the underlying causes of death are obscure but probably due to respiratory insufficiency) whereas females have a normal lifespan. By recording and analysing the mechanics of membrane-permeabilized myofibres, we only observed sex-related differences in the tibialis anterior muscles. Indeed, the concomitant deficits in maximal steady-state isometric force and stiffness of myofibres were less exacerbated in transgenic females than in males, potentially explaining the lower potency in limb muscles. However, the absence of sex-difference in the diaphragm muscles was rather unexpected and suggests that myofilament dysfunction does not solely underlie the sexually dimorphic phenotypes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In skeletal muscle, thick and thin filaments are arranged in a myofibrillar lattice. Tropomodulin 1 (Tmod1) is a pointed-end capping and tropomyosin-binding protein that controls thin-filament assembly, stability, and lengths. It remains unknown whether Tmods have other functional roles, such as regulating muscle contractility. To investigate this, we recorded and analyzed the mechanical properties and X-ray diffraction patterns of single membrane-permeabilized skeletal muscle fibers from mice lacking Tmod1. Results show that absence of Tmod1 and its replacement by Tmod3 and Tmod4 may impair initial tropomyosin movement over actin subunits during thin-filament activation, thus reducing both the fraction of actomyosin crossbridges in the strongly bound state (-29%) and fiber force-generating capacity (-31%). Therefore, Tmods are novel regulators of actomyosin crossbridge formation and muscle contractility, and future investigations and models of skeletal muscle force production must incorporate Tmods.

Multiple congenital myopathies, including nemaline myopathy, can arise due to mutations in the ACTA1 gene encoding skeletal muscle α-actin. The main characteristics of ACTA1 null mutations (absence of skeletal muscle α-actin) are generalized skeletal muscle weakness and premature death. A mouse model (ACTC(Co)/KO) mimicking these conditions has successfully been rescued by transgenic over-expression of cardiac α-actin in skeletal muscles using the ACTC gene. Nevertheless, myofibres from ACTC(Co)/KO animals generate less force than normal myofibres (-20 to 25%). To understand the underlying mechanisms, here we have undertaken a detailed functional study of myofibres from ACTC(Co)/KO rodents. Mechanical and X-ray diffraction pattern analyses of single membrane-permeabilized myofibres showed, upon maximal Ca(2+) activation and under rigor conditions, lower stiffness and disrupted actin-layer line reflections in ACTC(Co)/KO when compared with age-matched wild-types. These results demonstrate that in ACTC(Co)/KO myofibres, the presence of cardiac α-actin instead of skeletal muscle α-actin alters actin conformational changes upon activation. This later finely modulates the strain of individual actomyosin interactions and overall lowers myofibre force production. Taken together, the present findings provide novel primordial information about actin isoforms, their functional differences and have to be considered when designing gene therapies for ACTA1-based congenital myopathies.

Human tropomyosin mutations deregulate skeletal muscle contraction at the cellular level. One key feature is the slowing of the kinetics of force development. The aim of the present study was to characterize the potential underlying molecular mechanisms by recording and analyzing the X-ray diffraction patterns of human membrane-permeabilized muscle cells expressing a particular β-tropomyosin mutation (E41K). During resting conditions, the d1,0 lattice spacing, Δ1,0 and I1,1 to I1,0 ratio were not different from control values. These results suggest that, in presence of the E41K β-tropomyosin mutation, the myofilament lattice geometry is well maintained and therefore may not have any detrimental influence on the contraction mechanisms and thus, on the rate of force generation.