Cells respond to mechanical stress by rapid disassembly of caveolae.

The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that quickly accommodates sudden and acute mechanical stresses.

Myostatin in idiopathic inflammatory myopathies: Serum assessment and disease activity.

AIMS: In idiopathic inflammatory myopathies (IIM), disease activity is difficult to assess, and IIM may induce severe muscle damage, especially in immune-mediated necrotising myopathies (IMNM) and inclusion body myositis (IBM). We hypothesise that myostatin, a negative regulator of muscle mass, could be a new biomarker of disease activity and/or muscle damage. METHODS: Prospective assessment of myostatin protein level in 447 IIM serum samples (dermatomyositis [DM], n = 157; IBM, n = 72; IMNM, n = 125; and antisynthetase syndrome [ASyS], n = 93) and 59 healthy donors (HD) was performed by ELISA. A gene transcript analysis was also carried out on 18 IIM muscle biopsies and six controls to analyse myostatin and myostatin pathway-related gene expression. RESULTS: IIM patients had lower myostatin circulating protein levels and gene expression compared to HD (2379 [1490; 3678] pg/ml vs 4281 [3169; 5787] pg/ml; p < 0.0001 and log2FC = -1.83; p = 0.0005, respectively). Myostatin-related gene expression varied accordingly. Based on the Physician Global Assessment, inactive IIM patients showed higher myostatin levels than active ones. This was the case for all IIM subgroups, except IMNM where low myostatin levels were maintained (2186 [1235; 3815] vs 2349 [1518; 3922] pg/ml; p = 0.4). CONCLUSIONS: Myostatin protein and RNA levels are decreased in all IIM patients, and protein levels correlate with disease activity. Inactive ASyS and DM patients have higher myostatin levels than active patients. Myostatin could be a marker of disease activity in these subgroups. However, IMNM patients do not have significant increase in myostatin levels after disease remission. This may highlight a new pathological disease mechanism in IMNM patients.

Assessment of PABPN1 nuclear inclusions on a large cohort of patients and in a human xenograft model of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare muscle disease characterized by an onset of weakness in the pharyngeal and eyelid muscles. The disease is caused by the extension of a polyalanine tract in the Poly(A) Binding Protein Nuclear 1 (PABPN1) protein leading to the formation of intranuclear inclusions or aggregates in the muscle of OPMD patients. Despite numerous studies stressing the deleterious role of nuclear inclusions in cellular and animal OPMD models, their exact contribution to human disease is still unclear. In this study, we used a large and unique collection of human muscle biopsy samples to perform an in-depth analysis of PABPN1 aggregates in relation to age, genotype and muscle status with the final aim to improve our understanding of OPMD physiopathology. Here we demonstrate that age and genotype influence PABPN1 aggregates: the percentage of myonuclei containing PABPN1 aggregates increases with age and the chaperone HSP70 co-localize more frequently with PABPN1 aggregates with a larger polyalanine tract. In addition to the previously described PRMT1 and HSP70 co-factors, we identified new components of PABPN1 aggregates including GRP78/BiP, RPL24 and p62. We also observed that myonuclei containing aggregates are larger than myonuclei without. When comparing two muscles from the same patient, a similar amount of aggregates is observed in different muscles, except for the pharyngeal muscle where fewer aggregates are observed. This could be due to the peculiar nature of this muscle which has a low level of PAPBN1 and contains regenerating fibers. To confirm the fate of PABPN1 aggregates in a regenerating muscle, we generated a xenograft model by transplanting human OPMD muscle biopsy samples into the hindlimb of an immunodeficient mouse. Xenografts from subjects with OPMD displayed regeneration of human myofibers and PABPN1 aggregates were rapidly present-although to a lower extent-after muscle fiber regeneration. Our data obtained on human OPMD samples add support to the dual non-exclusive models in OPMD combining toxic PABPN1 intranuclear inclusions together with PABPN1 loss of function which altogether result in this late-onset and muscle selective disease.

Challenges in cell transplantation for muscular dystrophy.

For decades now, cell transplantation has been considered a possible therapeutic strategy for muscular dystrophy, but failures have largely outnumbered success or at least encouraging outcomes. In this review we will briefly recall the history of cell transplantation, discuss the peculiar features of skeletal muscle, and dystrophic skeletal muscle in particular, that make the procedure complicated and inefficient. As there are many recent and exhaustive reviews on the various myogenic cell types that have been or will be transplanted, we will only briefly describe them and refer the reader to these reviews. Finally, we will discuss possible strategies to overcome the hurdles that prevent biological efficacy and hence clinical success.

Pharmacological modulation of the ER stress response ameliorates oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare late onset genetic disease leading to ptosis, dysphagia and proximal limb muscles at later stages. A short abnormal (GCN) triplet expansion in the polyA-binding protein nuclear 1 (PABPN1) gene leads to PABPN1-containing aggregates in the muscles of OPMD patients. Here we demonstrate that treating mice with guanabenz acetate (GA), an FDA-approved antihypertensive drug, reduces the size and number of nuclear aggregates, improves muscle force, protects myofibers from the pathology-derived turnover and decreases fibrosis. GA targets various cell processes, including the unfolded protein response (UPR), which acts to attenuate endoplasmic reticulum (ER) stress. We demonstrate that GA increases both the phosphorylation of the eukaryotic translation initiation factor 2α subunit and the splicing of Xbp1, key components of the UPR. Altogether these data show that modulation of protein folding regulation is beneficial for OPMD and promote the further development of GA or its derivatives for treatment of OPMD in humans. Furthermore, they support the recent evidences that treating ER stress could be therapeutically relevant in other more common proteinopathies.

A promotive effect for halofuginone on membrane repair and synaptotagmin-7 levels in muscle cells of dysferlin-null mice.

In the absence of dysferlin, skeletal muscle cells fail to reseal properly after injury, resulting in slow progress of the dysferlinopathy muscular dystrophy (MD). Halofuginone, a leading agent in preventing fibrosis in MDs, was tested for its effects on membrane resealing post-injury. A hypo-osmotic shock assay on myotubes derived from wild-type (Wt) and dysferlin-null (dysf-/-) mice revealed that pre-treatment with halofuginone reduces the percentage of membrane-ruptured myotubes only in dysf-/- myotubes. In laser-induced injury of isolated myofibers, halofuginone decreased the amount of FM1-43 at the injury site of dysf-/- myofibers while having no effect on Wt myofibers. These results implicate halofuginone in ameliorating muscle-cell membrane integrity in dysf-/- mice. Halofuginone increased lysosome scattering across the cytosol of dysf-/- primary myoblasts, in a protein kinase/extracellular signal-regulated protein kinase and phosphoinositide 3 kinase/Akt-dependent manner, in agreement with an elevation in lysosomal exocytotic activity in these cells. A spatial- and age-dependent synaptotagmin-7 (Syt-7) expression pattern was shown in dysf-/- versus Wt mice, suggesting that these pattern alterations are related to the disease progress and that sytnaptotagmin-7 may be compensating for the lack of dysferlin at least with regard to membrane resealing post-injury. While halofuginone did not affect patch-repair-complex key proteins, it further enhanced Syt-7 levels and its spread across the cytosol in dysf-/- myofibers and muscle tissue, and increased its co-localization with lysosomes. Together, the data imply a novel role for halofuginone in membrane-resealing events with Syt-7 possibly taking part in these events.

Lamin-Related Congenital Muscular Dystrophy Alters Mechanical Signaling and Skeletal Muscle Growth.

Laminopathies are a clinically heterogeneous group of disorders caused by mutations in the LMNA gene, which encodes the nuclear envelope proteins lamins A and C. The most frequent diseases associated with LMNA mutations are characterized by skeletal and cardiac involvement, and include autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy type 1B, and LMNA-related congenital muscular dystrophy (LMNA-CMD). Although the exact pathophysiological mechanisms responsible for LMNA-CMD are not yet understood, severe contracture and muscle atrophy suggest that mutations may impair skeletal muscle growth. Using human muscle stem cells (MuSCs) carrying LMNA-CMD mutations, we observe impaired myogenic fusion with disorganized cadherin/ß catenin adhesion complexes. We show that skeletal muscle from Lmna-CMD mice is unable to hypertrophy in response to functional overload, due to defective fusion of activated MuSCs, defective protein synthesis and defective remodeling of the neuromuscular junction. Moreover, stretched myotubes and overloaded muscle fibers with LMNA-CMD mutations display aberrant mechanical regulation of the yes-associated protein (YAP). We also observe defects in MuSC activation and YAP signaling in muscle biopsies from LMNA-CMD patients. These phenotypes are not recapitulated in closely related but less severe EDMD models. In conclusion, combining studies in vitro, in vivo, and patient samples, we find that LMNA-CMD mutations interfere with mechanosignaling pathways in skeletal muscle, implicating A-type lamins in the regulation of skeletal muscle growth.

JAK inhibitor improves type I interferon induced damage: proof of concept in dermatomyositis.

Dermatomyositis is an acquired auto-immune disease characterized by skin lesions and muscle-specific pathological features such as perifascicular muscle fibre atrophy and vasculopathy. Dermatomyositis patients display an upregulation of type I interferon-inducible genes in muscle fibres, endothelial cells, skin and peripheral blood. However, the effect of type I interferon on muscle tissue has not yet been determined. Our aim was to study the pathogenicity of type I interferon in vitro and to evaluate the efficacy of the type I interferon pathway blockade for therapeutic purposes. The activation of type I interferon in differentiating myoblasts abolished myotube formation with reduced myogenin expression while in differentiated myotubes, we observed a reduction in surface area and an upregulation of atrophy-associated genes. In vitro endothelial cells exposure to type I interferon disrupted vascular network organization. All the pathogenic effects observed in vitro were abolished by ruxolitinib. Finally, four refractory dermatomyositis patients were treated with ruxolitinib and improvement ensued in skin lesions, muscle weakness and a reduced serum type I interferon levels and interferon-inducbile genes scores. We propose JAK inhibition as a mechanism-based treatment for dermatomyositis, a finding that is relevant for the design of future clinical trials targeting dermatomyositis.

Skeletal Muscle Regenerative Potential of Human MuStem Cells following Transplantation into Injured Mice Muscle.

After intra-arterial delivery in the dystrophic dog, allogeneic muscle-derived stem cells, termed MuStem cells, contribute to long-term stabilization of the clinical status and preservation of the muscle regenerative process. However, it remains unknown whether the human counterpart could be identified, considering recent demonstrations of divergent features between species for several somatic stem cells. Here, we report that MuStem cells reside in human skeletal muscle and display a long-term ability to proliferate, allowing generation of a clinically relevant amount of cells. Cultured human MuStem (hMuStem) cells do not express hematopoietic, endothelial, or myo-endothelial cell markers and reproducibly correspond to a population of early myogenic-committed progenitors with a perivascular/mesenchymal phenotypic signature, revealing a blood vessel wall origin. Importantly, they exhibit both myogenesis in vitro and skeletal muscle regeneration after intramuscular delivery into immunodeficient host mice. Together, our findings provide new insights supporting the notion that hMuStem cells could represent an interesting therapeutic candidate for dystrophic patients.

Pathogenic role of anti-signal recognition protein and anti-3-Hydroxy-3-methylglutaryl-CoA reductase antibodies in necrotizing myopathies: Myofiber atrophy and impairment of muscle regeneration in necrotizing autoimmune myopathies.

OBJECTIVE: Immune-mediated necrotizing myopathies (IMNM) may be associated with either anti-signal recognition protein (SRP) or anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) antibodies (Abs), and the titer of these Abs is correlated with disease activity. We investigated whether anti-SRP and anti-HMGCR Abs could be involved in muscle damage. METHODS: Muscle biopsies of patients were analyzed for atrophy and regeneration by measuring fiber size and by performing immunostaining of neonatal myosin heavy chain. To further understand the role of the Abs in the pathology, we performed muscle cell coculture with the Abs. Atrophy and regeneration were evaluated based on the myotube surface area as well as gene and cytokine profiles. RESULTS: In muscle biopsies of patients with anti-SRP+ and anti-HMGCR+ Abs, a large number of small fibers corresponding to both atrophic and regenerating fibers were observed. In vitro, anti-SRP and anti-HMGCR Abs induced muscle fiber atrophy and increased the transcription of MAFbx and TRIM63. In addition, the muscle fiber atrophy was associated with high levels of inflammatory cytokines: tumor necrosis factor, interleukin (IL)-6, and reactive oxygen species. In the presence of anti-SRP or anti-HMGCR Abs, mechanisms involved in muscle regeneration were also impaired due to a defect of myoblast fusion. This defect was associated with a decreased production of IL-4 and IL-13. The addition of IL-4 and/or IL-13 totally rescued fusion capacity. INTERPRETATION: These data show that molecular mechanisms of atrophy and regeneration are affected and contribute to loss of muscle function occurring in IMNM. This emphasizes the potential interest of targeted therapies addressing these mechanisms. Ann Neurol 2017;81:538-548.

Obestatin Increases the Regenerative Capacity of Human Myoblasts Transplanted Intramuscularly in an Immunodeficient Mouse Model.

Although cell-based therapy is considered a promising method aiming at treating different muscular disorders, little clinical benefit has been reported. One of major hurdles limiting the efficiency of myoblast transfer therapy is the poor survival of the transplanted cells. Any intervention upon the donor cells focused on enhancing in vivo survival, proliferation, and expansion is essential to improve the effectiveness of such therapies in regenerative medicine. In the present work, we investigated the potential role of obestatin, an autocrine peptide factor regulating skeletal muscle growth and repair, to improve the outcome of myoblast-based therapy by xenotransplanting primary human myoblasts into immunodeficient mice. The data proved that short in vivo obestatin treatment of primary human myoblasts not only enhances the efficiency of engraftment, but also facilitates an even distribution of myoblasts in the host muscle. Moreover, this treatment leads to a hypertrophic response of the human-derived regenerating myofibers. Taken together, the activation of the obestatin/GPR39 pathway resulted in an overall improvement of the efficacy of cell engraftment within the host's skeletal muscle. These data suggest considerable potential for future therapeutic applications and highlight the importance of combinatorial therapies.

Nuclear poly(A)-binding protein aggregates misplace a pre-mRNA outside of SC35 speckle causing its abnormal splicing.

A short abnormal polyalanine expansion in the polyadenylate-binding protein nuclear-1 (PABPN1) protein causes oculopharyngeal muscular dystrophy (OPMD). Mutated PABPN1 proteins accumulate as insoluble intranuclear aggregates in muscles of OPMD patients. While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice have been established, the molecular mechanisms which trigger pathological defects in OPMD and the role of aggregates remain to be determined. Using exon array, for the first time we have identified several splicing defects in OPMD. In particular, we have demonstrated a defect in the splicing regulation of the muscle-specific Troponin T3 (TNNT3) mutually exclusive exons 16 and 17 in OPMD samples compared to controls. This splicing defect is directly linked to the SC35 (SRSF2) splicing factor and to the presence of nuclear aggregates. As reported here, PABPN1 aggregates are able to trap TNNT3 pre-mRNA, driving it outside nuclear speckles, leading to an altered SC35-mediated splicing. This results in a decreased calcium sensitivity of muscle fibers, which could in turn plays a role in muscle pathology. We thus report a novel mechanism of alternative splicing deregulation that may play a role in various other diseases with nuclear inclusions or foci containing an RNA binding protein.

Mitochondrial dysfunction reveals the role of mRNA poly(A) tail regulation in oculopharyngeal muscular dystrophy pathogenesis.

Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.

KCC3 loss-of-function contributes to Andermann syndrome by inducing activity-dependent neuromuscular junction defects.

Loss-of-function mutations in the potassium-chloride cotransporter KCC3 lead to Andermann syndrome, a severe sensorimotor neuropathy characterized by areflexia, amyotrophy and locomotor abnormalities. The molecular events responsible for axonal loss remain poorly understood. Here, we establish that global or neuron-specific KCC3 loss-of-function in mice leads to early neuromuscular junction (NMJ) abnormalities and muscular atrophy that are consistent with the pre-synaptic neurotransmission defects observed in patients. KCC3 depletion does not modify chloride handling, but promotes an abnormal electrical activity among primary motoneurons and mislocalization of Na+/K+-ATPase α1 in spinal cord motoneurons. Moreover, the activity-targeting drug carbamazepine restores Na+/K+-ATPase α1 localization and reduces NMJ denervation in Slc12a6-/- mice. We here propose that abnormal motoneuron electrical activity contributes to the peripheral neuropathy observed in Andermann syndrome.

Muscle satellite cells are functionally impaired in myasthenia gravis: consequences on muscle regeneration.

Myasthenia gravis (MG) is a neuromuscular disease caused in most cases by anti-acetyl-choline receptor (AChR) autoantibodies that impair neuromuscular signal transmission and affect skeletal muscle homeostasis. Myogenesis is carried out by muscle stem cells called satellite cells (SCs). However, myogenesis in MG had never been explored. The aim of this study was to characterise the functional properties of myasthenic SCs as well as their abilities in muscle regeneration. SCs were isolated from muscle biopsies of MG patients and age-matched controls. We first showed that the number of Pax7+ SCs was increased in muscle sections from MG and its experimental autoimmune myasthenia gravis (EAMG) mouse model. Myoblasts isolated from MG muscles proliferate and differentiate more actively than myoblasts from control muscles. MyoD and MyoG were expressed at a higher level in MG myoblasts as well as in MG muscle biopsies compared to controls. We found that treatment of control myoblasts with MG sera or monoclonal anti-AChR antibodies increased the differentiation and MyoG mRNA expression compared to control sera. To investigate the functional ability of SCs from MG muscle to regenerate, we induced muscle regeneration using acute cardiotoxin injury in the EAMG mouse model. We observed a delay in maturation evidenced by a decrease in fibre size and MyoG mRNA expression as well as an increase in fibre number and embryonic myosin heavy-chain mRNA expression. These findings demonstrate for the first time the altered function of SCs from MG compared to control muscles. These alterations could be due to the anti-AChR antibodies via the modulation of myogenic markers resulting in muscle regeneration impairment. In conclusion, the autoimmune attack in MG appears to have unsuspected pathogenic effects on SCs and muscle regeneration, with potential consequences on myogenic signalling pathways, and subsequently on clinical outcome, especially in the case of muscle stress.

CellWhere: graphical display of interaction networks organized on subcellular localizations.

Given a query list of genes or proteins, CellWhere produces an interactive graphical display that mimics the structure of a cell, showing the local interaction network organized into subcellular locations. This user-friendly tool helps in the formulation of mechanistic hypotheses by enabling the experimental biologist to explore simultaneously two elements of functional context: (i) protein subcellular localization and (ii) protein-protein interactions or gene functional associations. Subcellular localization terms are obtained from public sources (the Gene Ontology and UniProt-together containing several thousand such terms) then mapped onto a smaller number of CellWhere localizations. These localizations include all major cell compartments, but the user may modify the mapping as desired. Protein-protein interaction listings, and their associated evidence strength scores, are obtained from the Mentha interactome server, or power-users may upload a pre-made network produced using some other interactomics tool. The Cytoscape.js JavaScript library is used in producing the graphical display. Importantly, for a protein that has been observed at multiple subcellular locations, users may prioritize the visual display of locations that are of special relevance to their research domain. CellWhere is at http://cellwhere-myology.rhcloud.com.

Nuclear protein spreading: implication for pathophysiology of neuromuscular diseases.

While transfer of a protein encoded by a single nucleus to nearby nuclei in multinucleated cells has been known for almost 25 years, the biological consequences for gain-of-function diseases have not been considered. Here, we have investigated nuclear protein spreading and its potential consequences in two of the three most prevalent neuromuscular diseases. By performing co-cultures between diseased or control human myoblasts and murine C2C12 myoblasts, we demonstrate that in facioscapulohumeral dystrophy, although the transcription of the toxic protein DUX4 occurs in only a limited number of nuclei, the resulting protein diffuses into nearby nuclei within the myotubes, thus spreading aberrant gene expression. In myotonic dystrophy type 1, we observed that in human-mouse heterokaryons, the expression of a mutated DMPK from human nuclei titrates splicing factors produced by neighboring nuclei, inducing the mis-splicing of several pre-mRNAs in murine nuclei. In both cases, the spreading of the pathological phenotypes from one nucleus to another is observed, highlighting an additional mechanism that contributes to the dissemination and worsening of the muscle pathogenesis. These results indicate that nuclear protein spreading may be an important component of pathophysiology of gain of function muscular diseases which should be taken into consideration in the design of new therapeutic approaches.

DUX4 and DUX4 downstream target genes are expressed in fetal FSHD muscles.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent adult muscular dystrophies. The common clinical signs usually appear during the second decade of life but when the first molecular dysregulations occur is still unknown. Our aim was to determine whether molecular dysregulations can be identified during FSHD fetal muscle development. We compared muscle biopsies derived from FSHD1 fetuses and the cells derived from some of these biopsies with biopsies and cells derived from control fetuses. We mainly focus on DUX4 isoform expression because the expression of DUX4 has been confirmed in both FSHD cells and biopsies by several laboratories. We measured DUX4 isoform expression by using qRT-PCR in fetal FSHD1 myotubes treated or not with an shRNA directed against DUX4 mRNA. We also analyzed DUX4 downstream target gene expression in myotubes and fetal or adult FSHD1 and control quadriceps biopsies. We show that both DUX4-FL isoforms are already expressed in FSHD1 myotubes. Interestingly, DUX4-FL expression level is much lower in trapezius than in quadriceps myotubes, which is confirmed by the level of expression of DUX4 downstream genes. We observed that TRIM43 and MBD3L2 are already overexpressed in FSHD1 fetal quadriceps biopsies, at similar levels to those observed in adult FSHD1 quadriceps biopsies. These results indicate that molecular markers of the disease are already expressed during fetal life, thus opening a new field of investigation for mechanisms leading to FSHD.

Mechanical Overloading Increases Maximal Force and Reduces Fragility in Hind Limb Skeletal Muscle from Mdx Mouse.

There is fear that mechanical overloading (OVL; ie, high-force contractions) accelerates Duchenne muscular dystrophy. Herein, we determined whether short-term OVL combined with wheel running, short-term OVL combined with irradiation, and long-term OVL are detrimental for hind limb mdx mouse muscle, a murine model of Duchene muscular dystrophy exhibiting milder dystrophic features. OVL was induced by the surgical ablation of the synergic muscles of the plantaris muscle, a fast muscle susceptible to contraction-induced muscle damage in mdx mice. We found that short-term OVL combined with wheel and long-term OVL did not worsen the deficit in specific maximal force (ie, absolute maximal force normalized to muscle size) and histological markers of muscle damage (percentage of regenerating fibers and fibrosis) in mdx mice. Moreover, long-term OVL did not increase the alteration in calcium homeostasis and did not deplete muscle cell progenitors expressing Pax 7 in mdx mice. Irradiation before short-term OVL, which is believed to inhibit muscle regeneration, was not more detrimental to mdx than control mice. Interestingly, short-term OVL combined with wheel and long-term OVL markedly improved the susceptibility to contraction-induced damage, increased absolute maximal force, induced hypertrophy, and promoted a slower, more oxidative phenotype. Together, these findings indicate that OVL is beneficial to mdx muscle, and muscle regeneration does not mask the potentially detrimental effect of OVL.

Correlation between low FAT1 expression and early affected muscle in facioscapulohumeral muscular dystrophy.

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is linked to either contraction of D4Z4 repeats on chromosome 4 or to mutations in the SMCHD1 gene, both of which result in the aberrant expression of the transcription factor DUX4. However, it is still difficult to correlate these genotypes with the phenotypes observed in patients. Because we have recently shown that mice with disrupted Fat1 functions exhibit FSHD-like phenotypes, we have investigated the expression of the human FAT1 gene in FSHD. METHODS: We first analyzed FAT1 expression in FSHD adult muscles and determined whether FAT1 expression was driven by DUX4. We next determined FAT1 expression levels in 64 muscles isolated from 16 control fetuses. These data were further complemented with analysis of Fat1 expression in developing mouse embryos. RESULTS: We demonstrated that FAT1 expression is independent of DUX4. Moreover, we observed that (1) in control fetal human biopsies or in developing mouse embryos, FAT1 is expressed at lower levels in muscles that are affected at early stages of FSHD progression than in muscles that are affected later or are nonaffected; and (2) in adult muscle biopsies, FAT1 expression is lower in FSHD muscles compared to control muscles. INTERPRETATION: We propose a revised model for FSHD in which FAT1 levels might play a role in determining which muscles will exhibit early and late disease onset, whereas DUX4 may worsen the muscle phenotype.