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Muscle weakness and atrophy are clinical hallmarks of myotonic dystrophy type 1 (DM1). Muscle stem cells, which contribute to skeletal muscle growth and repair, are also affected in this disease. However, the molecular mechanisms leading to this defective activity and the impact on the disease severity are still elusive. Here, we explored through an unbiased approach the molecular signature leading to myogenic cell defects in DM1. Single cell RNAseq data revealed the presence of a specific subset of DM1 myogenic cells expressing a senescence signature, characterized by the high expression of genes related to senescence-associated secretory phenotype (SASP). This profile was confirmed using different senescence markers in vitro and in situ. Accumulation of intranuclear RNA foci in senescent cells, suggest that RNA-mediated toxicity contribute to senescence induction. High expression of IL-6, a prominent SASP cytokine, in the serum of DM1 patients was identified as a biomarker correlating with muscle weakness and functional capacity limitations. Drug screening revealed that the BCL-XL inhibitor (A1155463), a senolytic drug, can specifically target senescent DM1 myoblasts to induce their apoptosis and reduce their SASP. Removal of senescent cells re-established the myogenic function of the non-senescent DM1 myoblasts, which displayed improved proliferation and differentiation capacity in vitro; and enhanced engraftment following transplantation in vivo. Altogether this study presents a well-defined senescent molecular signature in DM1 untangling part of the pathological mechanisms observed in the disease; additionally, we demonstrate the therapeutic potential of targeting these defective cells with senolytics to restore myogenesis.

BCL-2-associated athanogene 3 (BAG3) is a co-chaperone to heat shock proteins important in degrading misfolded proteins through chaperone-assisted selective autophagy. The recurrent dominant BAG3-P209L mutation results in a severe childhood-onset myofibrillar myopathy (MFM) associated with progressive muscle weakness, cardiomyopathy, and respiratory failure. Because a homozygous knock-in (KI) strain for the mP215L mutation homologous to the human P209L mutation did not have a gross phenotype, compound heterozygote knockout (KO) and KI mP215L mice were generated to establish whether further reduction in BAG3 expression would lead to a phenotype. The KI/KO mice have a significant decrease in voluntary movement compared with wild-type and KI/KI mice in the open field starting at 7 months. The KI/KI and KI/KO mice both have significantly smaller muscle fiber cross-sectional area. However, only the KI/KO mice have clear skeletal muscle histologic changes in MFM. As in patient muscle, there are increased levels of BAG3-interacting proteins, such as p62, heat shock protein B8, and αB-crystallin. The KI/KO mP215L strain is the first murine model of BAG3 myopathy that resembles the human skeletal muscle pathologic features. The results support the hypothesis that the pathologic development of MFM requires a significant decrease in BAG3 protein level and not only a gain of function caused by the dominant missense mutation.

Lack of dystrophin causes muscle degeneration, which is exacerbated by chronic inflammation and reduced regenerative capacity of muscle stem cells in Duchenne Muscular Dystrophy (DMD). To date, glucocorticoids remain the gold standard for the treatment of DMD. These drugs are able to slow down the progression of the disease and increase lifespan by dampening the chronic and excessive inflammatory process; however, they also have numerous harmful side effects that hamper their therapeutic potential. Here, we investigated Resolvin-D2 as a new therapeutic alternative having the potential to target multiple key features contributing to the disease progression. Our in vitro findings showed that Resolvin-D2 promotes the switch of macrophages toward their anti-inflammatory phenotype and increases their secretion of pro-myogenic factors. Moreover, Resolvin-D2 directly targets myogenic cells and promotes their differentiation and the expansion of the pool of myogenic progenitor cells leading to increased myogenesis. These effects are ablated when the receptor Gpr18 is knocked-out, knocked-down, or blocked by the pharmacological antagonist O-1918. Using different mouse models of DMD, we showed that Resolvin-D2 targets both inflammation and myogenesis leading to enhanced muscle function compared to glucocorticoids. Overall, this preclinical study has identified a new therapeutic approach that is more potent than the gold-standard treatment for DMD., Glucocorticoids delay muscle wasting in Duchenne Muscular Dystrophy by reducing inflammation; but also have harmful side effects. Here, the authors show that Resolvin-D2 is more effective than glucocorticoids in mitigating muscular dystrophy in mouse models, due to its ability to dampen inflammation and target myogenic cells to improve muscle regeneration.

BCL-2-associated athanogene 3 (BAG3) is a co-chaperone to heat shock proteins important in degrading misfolded proteins through chaperone-assisted selective autophagy. The recurrent dominant BAG3-P209L mutation results in a severe childhood-onset myofibrillar myopathy (MFM) associated with progressive muscle weakness, cardiomyopathy, and respiratory failure. Because a homozygous knock-in (KI) strain for the mP215L mutation homologous to the human P209L mutation did not have a gross phenotype, compound heterozygote knockout (KO) and KI mP215L mice were generated to establish whether further reduction in BAG3 expression would lead to a phenotype. The KI/KO mice have a significant decrease in voluntary movement compared with wild-type and KI/KI mice in the open field starting at 7 months. The KI/KI and KI/KO mice both have significantly smaller muscle fiber cross-sectional area. However, only the KI/KO mice have clear skeletal muscle histologic changes in MFM. As in patient muscle, there are increased levels of BAG3-interacting proteins, such as p62, heat shock protein B8, and αB-crystallin. The KI/KO mP215L strain is the first murine model of BAG3 myopathy that resembles the human skeletal muscle pathologic features. The results support the hypothesis that the pathologic development of MFM requires a significant decrease in BAG3 protein level and not only a gain of function caused by the dominant missense mutation.

This study evaluated cellular and molecular effects of radicicol, a heat shock protein (HSP) inducer, on the regeneration of skeletal muscle injured by crotoxin, the main toxin isolated from Crotalus durissus terrificus venom. Regenerating muscles treated with radicicol had decreased NF-kB activation. Differentiating myoblasts treated with radicicol showed reduced number of NF-kB positive nuclei and increased fusion index. The results suggest that radicicol enhances regeneration of muscle by attenuating NF-kB activation and increasing myogenic differentiation.

Aims Previously, detection of ANO5 protein has been complicated by unspecific antibodies, most of which have not identified the correct protein. The aims of the study were to specify ANO5 protein expression in human skeletal muscle, and to investigate if the ANO5 protein levels are affected by different ANO5 mutations in anoctaminopathy patients. Methods Four different antibodies were tested for ANO5 specificity. A sample preparation method compatible with membrane proteins, combined with tissue fractionation was used to determine ANO5 expression in cell cultures expressing ANO5, in normal muscles and eight patient biopsies with six different ANO5 mutations in homozygous or compound heterozygous states, and in other dystrophies. Results Only one specific monoclonal N-terminal ANO5 antibody was efficient in detecting the protein, showing that ANO5 is expressed as a single 107 kD polypeptide in human skeletal muscle. The truncating mutations c.191dupA and c.1261C>T were found to abolish ANO5 expression, whereas the studied point mutations had variable effects, however, all the ANO5 mutations resulted in clearly reduced ANO5 expression in the patient muscle membrane fraction. Attempts to detect ANO5 using immunohistochemistry were not yet successful. Conclusions The data presented here indicate that the ANO5 protein expression is decreased in ANO5-mutated muscular dystrophy and that most of the non-truncating pathogenic ANO5 mutations likely destabilize the protein and cause its degradation. The method described here allows direct analysis of human ANO5 protein, which can be used in diagnostics, for evaluating the pathogenicity of the potentially harmful ANO5 variants of uncertain significance. This article is protected by copyright. All rights reserved.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS [MIM 270550]) is an early-onset neurodegenerative disorder caused by mutations in the SACS gene. Over 170 SACS mutations have been reported worldwide and are thought to cause loss of function of sacsin, a poorly characterized and massive 520 kDa protein. To establish an animal model and to examine the pathophysiological basis of ARSACS, we generated Sacs knockout (Sacs(-/-)) mice. Null animals displayed an abnormal gait with progressive motor, cerebellar and peripheral nerve dysfunctions highly reminiscent of ARSACS. These clinical features were accompanied by an early onset, progressive loss of cerebellar Purkinje cells followed by spinal motor neuron loss and peripheral neuropathy. Importantly, loss of sacsin function resulted in abnormal accumulation of non-phosphorylated neurofilament (NF) bundles in the somatodendritic regions of vulnerable neuronal populations, a phenotype also observed in an ARSACS brain. Moreover, motor neurons cultured from Sacs(-/-) embryos exhibited a similar NF rearrangement with significant reduction in mitochondrial motility and elongated mitochondria. The data points to alterations in the NF cytoskeleton and defects in mitochondrial dynamics as the underlying pathophysiological basis of ARSACS.

Skeletal muscles from old rats fail to completely regenerate following injury. This study investigated whether pharmacological stimulation of β2-adrenoceptors in aged muscles following injury could improve their regenerative capacity, focusing on myofiber size recovery. Young and aged rats were treated with a subcutaneous injection of β2-adrenergic agonist formoterol (2 μg/kg/d) up to 10 and 21 days after soleus muscle injury. Formoterol-treated muscles from old rats evaluated at 10 and 21 days postinjury showed reduced inflammation and connective tissue but a similar number of regenerating myofibers of greater caliber when compared with their injured controls. Formoterol minimized the decrease in tetanic force and increased protein synthesis and mammalian target of rapamycin phosphorylation in old muscles at 10 days postinjury. Our results suggest that formoterol improves structural and functional regenerative capacity of regenerating skeletal muscles from aged rats by increasing protein synthesis via mammalian target of rapamycin activation. Furthermore, formoterol may have therapeutic benefits in recovery following muscle damage in senescent individuals.

This work was undertaken to provide further insight into the role of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle regeneration, focusing on myofiber size recovery. Rats were treated or not with rapamycin, an mTORC1 inhibitor. Soleus muscles were then subjected to cryolesion and analyzed 1, 10, and 21 days later. A decrease in soleus myofiber cross-section area on post-cryolesion days 10 and 21 was accentuated by rapamycin, which was also effective in reducing protein synthesis in these freeze-injured muscles. The incidence of proliferating satellite cells during regeneration was unaltered by rapamycin, although immunolabeling for neonatal myosin heavy chain (MHC) was weaker in cryolesion+rapamycin muscles than in cryolesion-only muscles. In addition, the decline in tetanic contraction of freeze-injured muscles was accentuated by rapamycin. This study indicates that mTORC1 plays a key role in the recovery of muscle mass and the differentiation of regenerating myofibers, independently of necrosis and satellite cell proliferation mechanisms.

This work investigates the influence of heat shock proteins (HSPs) on necrosis and subsequent skeletal muscle regeneration induced by crotoxin (CTX), the major component of Crotalus durissus terrificus venom. Mice were treated with radicicol, a HSP inductor, followed by an intramuscular injection of CTX into the gastrocnemius muscle. Treated groups were sacrificed 1, 10 and 21 days after CTX injection. Muscle histological sections were stained with toluidine blue and assayed for acid phosphatase or immunostained with either neuronal cell adhesion molecule (NCAM) or neonatal myosin heavy chain (MHCn). Muscle samples were also submitted to Western blotting analysis. The results show that CTX alone and CTX combined with radicicol induced a similar degree of myofiber necrosis. CTX-injured muscles treated with radicicol had increased cross-sectional areas at 10 and 21 days post-lesion compared with untreated CTX-injured muscles. Additionally, radicicol significantly increased the number of NCAM-positive satellite cells in the gastrocnemius at one day post-CTX injury. CTX-injured muscles treated with radicicol contained more MHCn-positive regenerating myofibers compared with untreated CTX-injured muscles. These results suggest that HSPs contribute to the regeneration of myofibers damaged by CTX. Additionally, further studies should investigate the potential therapeutic effects of radicicol in skeletal muscles affected by Crotalus venom.

This work investigated the effect of gallium arsenide (GaAs) irradiation (power: 5 mW; intensity: 77.14 mW/cm(2), spot: 0.07 cm(2)) on regenerating skeletal muscles damaged by crotoxin (CTX). Male C57Bl6 mice were divided into six groups (n = 5 each): control, treated only with laser at doses of 1.5 J or 3 J, CTX-injured and, CTX-injured and treated with laser at doses of 1.5 J or 3 J. The injured groups received a CTX injection into the tibialis anterior (TA) muscle. After 3 days, TA muscles were submitted to GaAs irradiation at doses of 1.5 or 3 J (once a day, during 5 days) and were killed on the eighth day. Muscle histological sections were stained with hematoxylin and eosin (HE) in order to determine the myofiber cross-sectional area (CSA), the previously injured muscle area (PIMA) and the area density of connective tissue. The gene expression of MyoD and myogenin was detected by real-time PCR. GaAs laser at a dose of 3 J, but not 1.5 J, significantly increased the CSA of regenerating myofibers and reduced the PIMA and the area density of intramuscular connective tissue of CTX-injured muscles. MyoD gene expression increased in the injured group treated with GaAs laser at a dose of 1.5 J. The CTX-injured, 3-J GaAs laser-treated, and the CTX-injured and treated with 3-J laser groups showed an increase in myogenin gene expression when compared to the control group. Our results suggest that GaAs laser treatment at a dose of 3 J improves skeletal muscle regeneration by accelerating the recovery of myofiber mass.

Muscle mass and strength are variable traits in humans. Many French Canadians (FC) became international celebrities because of their exceptional strength. Though muscle hypertrophy has been associated in many mammals with myostatin mutations, to date only a single pediatric case has been reported in humans. Dominant pathological mutations in the CLCN1, SCN4A, CAV3, ATP2A1, LPIN1, LMNA and RYR1 gene have been known to lead to muscle hypertrophy in humans, in particular selective muscle hypertrophy in certain myopathies. These conditions are associated with either weakness or increased strength. We recruited a cohort of more than 115 cases belonging to more than 65 families of a heterogeneous dominant condition that we refer to as: “Strongman syndrome” (SM). In all families, the most affected case suffers from incapacitating myalgias with variable degree of muscle cramps, and they also have a personal and familial history of above average strength. On examination these patients have large well defined muscles, above average strength, non-electric prolonged muscle contractions and progressive weakness on repeated contractions. Exome sequencing of a large FC family identified a single segregating variant predicted to be damaging in the DCST2 gene shared also by two other FC families. The DCST2 gene is expressed in skeletal muscle and is predicted to code for a six transmembrane domains channel-like protein. To better characterize the herculean strength we assesses different biometric variables in a subset of cases with and without DCST2 mutations and investigated if myofiber hypertrophy is present. Results of quantifiable methods to evaluate the clinical and pathological phenotype of strongman cases will be presented, as well as functional data about DCST2. The uncovering of the function of DCST2 and other strongman genes will contribute to a better understanding of pathways important for muscle hypertrophy and strength in health and disease.

This work was undertaken to provide further insight into the role of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle regeneration, focusing on myofiber size recovery. Rats were treated or not with rapamycin, an mTORC1 inhibitor. Soleus muscles were then subjected to cryolesion and analyzed 1, 10, and 21 days later. A decrease in soleus myofiber cross-section area on post-cryolesion days 10 and 21 was accentuated by rapamycin, which was also effective in reducing protein synthesis in these freeze-injured muscles. The incidence of proliferating satellite cells during regeneration was unaltered by rapamycin, although immunolabeling for neonatal myosin heavy chain (MHC) was weaker in cryolesion+rapamycin muscles than in cryolesion-only muscles. In addition, the decline in tetanic contraction of freeze-injured muscles was accentuated by rapamycin. This study indicates that mTORC1 plays a key role in the recovery of muscle mass and the differentiation of regenerating myofibers, independently of necrosis and satellite cell proliferation mechanisms.