Beneficial effects of resistance training on both mild and severe mouse dystrophic muscle function as a preclinical option for Duchenne muscular dystrophy.

Mechanical overloading (OVL) resulting from the ablation of muscle agonists, a supra-physiological model of resistance training, reduces skeletal muscle fragility, i.e. the immediate maximal force drop following lengthening contractions, and increases maximal force production, in mdx mice, a murine model of Duchene muscular dystrophy (DMD). Here, we further analyzed these beneficial effects of OVL by determining whether they were blocked by cyclosporin, an inhibitor of the calcineurin pathway, and whether there were also observed in the D2-mdx mice, a more severe murine DMD model. We found that cyclosporin did not block the beneficial effect of 1-month OVL on plantaris muscle fragility in mdx mice, nor did it limit the increases in maximal force and muscle weight (an index of hypertrophy). Fragility and maximal force were also ameliorated by OVL in the plantaris muscle of D2-mdx mice. In addition, OVL increased the expression of utrophin, cytoplamic γ-actin, MyoD, and p-Akt in the D2-mdx mice, proteins playing an important role in fragility, maximal force gain and muscle growth. In conclusion, OVL reduced fragility and increased maximal force in the more frequently used mild mdx model but also in D2-mdx mice, a severe model of DMD, closer to human physiopathology. Moreover, these beneficial effects of OVL did not seem to be related to the activation of the calcineurin pathway. Thus, this preclinical study suggests that resistance training could have a potential benefit in the improvement of the quality of life of DMD patients.

Valproic acid reduces muscle susceptibility to contraction-induced functional loss but increases weakness in two murine models of Duchenne muscular dystrophy.

Skeletal muscles in animal models of Duchenne muscular dystrophy (DMD) are more susceptible to contraction-induced functional loss, which is not related to fatigue. Valproic acid (VPA) reportedly improves serological and histological markers of damage in dystrophin-deficient murine muscle. Here, we tested whether VPA would reduce the susceptibility to contraction-induced functional loss in two murine DMD models. Adult female mdx (mild) and D2-mdx (severe) DMD murine models were administered VPA (240 mg/kg) or saline for 7 days. Some VPA-treated mdx mice also performed voluntary running in a wheel, which is known to reduce the susceptibility to contraction-induced functional loss; that is, isometric force drop following eccentric contractions. In situ muscle function was assessed before, during and after eccentric contractions. Muscle utrophin and desmin expression were also evaluated using immunoblotting. Interestingly, VPA reduced the isometric force drop following eccentric contractions in both murine models, without change in the relative eccentric maximal force and in the expression of utrophin and desmin. VPA for 7 days combined with voluntary running had no additive effect compared to VPA alone. Furthermore, VPA reduced the absolute isometric maximal force before eccentric contractions in both murine models. The results of our study indicated that VPA in both murine DMD models reduced the susceptibility to contraction-induced functional loss but increased muscle weakness.

Dystrophin Restoration after Adeno-Associated Virus U7-Mediated Dmd Exon Skipping Is Modulated by Muscular Exercise in the Severe D2-Mdx Duchenne Muscular Dystrophy Murine Model.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease caused by Dmd mutations, resulting in the absence of dystrophin in skeletal muscle, and a greater susceptibility to damage during contraction (exercise). The current study evaluated whether voluntary exercise impacts a Dmd exon skipping and muscle physiology in a severe DMD murine model. D2-mdx mice were intramuscularly injected with an adeno-associated virus (AAV) U7 snRNA to correct Dmd reading frame, and allowed to voluntary run on a wheel for 1 month. Voluntary running did not induce muscle fiber regeneration, as indicated by the percentage of centronucleated fibers, Myh3 and Myh4 expression, and maximal force production, and thus possibly did not compromise the gene therapy approach. Voluntary running did not impact the number of viral genomes and the expression of U7 and Dmd 1 month after injection of AAV-U7 injected just before exercise initiation, but reduced the amount of dystrophin in dystrophin-expressing fibers from 80% to 65% of the muscle cross-sectional area. In conclusion, voluntary running did not induce muscle damage and had no drastic detrimental effect on the AAV gene therapy exon skipping approach in a severe murine DMD model. Moreover, these results suggest considering exercise as an additional element in the design and conception of future therapeutic approaches for DMD.

The cell polarity protein Vangl2 in the muscle shapes the neuromuscular synapse by binding to and regulating the tyrosine kinase MuSK.

The development of the neuromuscular junction (NMJ) requires dynamic trans-synaptic coordination orchestrated by secreted factors, including Wnt family morphogens. To investigate how these synaptic cues in NMJ development are transduced, particularly in the regulation of acetylcholine receptor (AChR) accumulation in the postsynaptic membrane, we explored the function of Van Gogh-like protein 2 (Vangl2), a core component of Wnt planar cell polarity signaling. We found that conditional, muscle-specific ablation of Vangl2 in mice reproduced the NMJ differentiation defects seen in mice with global Vangl2 deletion. These alterations persisted into adulthood and led to NMJ disassembly, impaired neurotransmission, and deficits in motor function. Vangl2 and the muscle-specific receptor tyrosine kinase MuSK were functionally associated in Wnt signaling in the muscle. Vangl2 bound to and promoted the signaling activity of MuSK in response to Wnt11. The loss of Vangl2 impaired RhoA activation in cultured mouse myotubes and caused dispersed, rather than clustered, organization of AChRs at the postsynaptic or muscle cell side of NMJs in vivo. Our results identify Vangl2 as a key player of the core complex of molecules shaping neuromuscular synapses and thus shed light on the molecular mechanisms underlying NMJ assembly.

The beneficial effect of chronic muscular exercise on muscle fragility is increased by Prox1 gene transfer in dystrophic mdx muscle.

PURPOSE: Greater muscle fragility is thought to cause the exhaustion of the muscle stem cells during successive degeneration/repair cycles, leading to muscle wasting and weakness in Duchenne muscular dystrophy. Chronic voluntary exercise can partially reduce the susceptibility to contraction induced-muscle damage, i.e., muscle fragility, as shown by a reduced immediate maximal force drop following lengthening contractions, in the dystrophic mdx mice. Here, we studied the effect of Prospero-related homeobox factor 1 gene (Prox1) transfer (overexpression) using an AAV on fragility in chronically exercised mdx mice, because Prox1 promotes slower type fibres in healthy mice and slower fibres are less fragile in mdx muscle. METHODS: Both tibialis anterior muscles of the same mdx mouse received the transfer of Prox1 and PBS and the mice performed voluntary running into a wheel during 1 month. We also performed Prox1 transfer in sedentary mdx mice. In situ maximal force production of the muscle in response to nerve stimulation was assessed before, during and after 10 lengthening contractions. Molecular muscle parameters were also evaluated. RESULTS: Interestingly, Prox1 transfer reduced the isometric force drop following lengthening contractions in exercised mdx mice (p < 0.05 to 0.01), but not in sedentary mdx mice. It also increased the muscle expression of Myh7 (p < 0.001), MHC-2x (p < 0.01) and Trpc1 (p < 0.01), whereas it reduced that one of Myh4 (p < 0.001) and MHC-2b (p < 0.01) in exercised mdx mice. Moreover, Prox1 transfer decreased the absolute maximal isometric force (p < 0.01), but not the specific maximal isometric force, before lengthening contraction in exercised (p < 0.01) and sedentary mdx mice. CONCLUSION: Our results indicate that Prox1 transfer increased the beneficial effect of chronic exercise on muscle fragility in mdx mice, but reduced absolute maximal force. Thus, the potential clinical benefit of the transfer of Prox1 into exercised dystrophic muscle can merit further investigation.

Benefits of therapy by dynamin-2-mutant-specific silencing are maintained with time in a mouse model of dominant centronuclear myopathy.

Dominant dynamin 2 (DNM2) mutations are responsible for the autosomal dominant centronuclear myopathy (AD-CNM), a rare progressive neuromuscular disorder ranging from severe neonatal to mild adult forms. We previously demonstrated that mutant-specific RNA interference is an efficient therapeutic strategy to rescue the muscle phenotype at the onset of the symptoms in the AD-CNM knockin-Dnm2 R465W/+ mouse model. Our objective was to evaluate the long-term benefit of the treatment along with the disease time course. We demonstrate here that the complete rescue of the muscle phenotype is maintained for at least 1 year after a single injection of adeno-associated virus expressing the mutant-specific short hairpin RNA (shRNA). This was achieved by a maintained reduction of the mutant Dnm2 transcript. Moreover, this long-term study uncovers a pathological accumulation of DNM2 protein occurring with age in the mouse model and prevented by the treatment. Conversely, a physiological DNM2 protein decrease with age was observed in muscles from wild-type mice. Therefore, this study highlights a new potential pathophysiological mechanism linked to mutant protein accumulation and underlines the importance of DNM2 protein expression level for proper muscle function. Overall, these results strengthen the allele-specific silencing approach as a robust, safe, and efficient therapy for AD-CNM.

Reversal of RNA toxicity in myotonic dystrophy via a decoy RNA-binding protein with high affinity for expanded CUG repeats.

Myotonic dystrophy type 1 (DM1) is an RNA-dominant disease whose pathogenesis stems from the functional loss of muscleblind-like RNA-binding proteins (RBPs), which causes the formation of alternative-splicing defects. The loss of functional muscleblind-like protein 1 (MBNL1) results from its nuclear sequestration by mutant transcripts containing pathogenic expanded CUG repeats (CUGexp). Here we show that an RBP engineered to act as a decoy for CUGexp reverses the toxicity of the mutant transcripts. In vitro, the binding of the RBP decoy to CUGexp in immortalized muscle cells derived from a patient with DM1 released sequestered endogenous MBNL1 from nuclear RNA foci, restored MBNL1 activity, and corrected the transcriptomic signature of DM1. In mice with DM1, the local or systemic delivery of the RBP decoy via an adeno-associated virus into the animals' skeletal muscle led to the long-lasting correction of the splicing defects and to ameliorated disease pathology. Our findings support the development of decoy RBPs with high binding affinities for expanded RNA repeats as a therapeutic strategy for myotonic dystrophies.

Alteration of skeletal and cardiac muscles function in DBA/2J mdx mice background: a focus on high intensity interval training.

Duchenne muscular dystrophy (DMD) is a recessive hereditary myopathy due to deficiency of functional dystrophin. Current therapeutic interventions need more investigation to slow down the progression of skeletal and cardiac muscle weakness. In humans, there is a lack of an adapted training program. In animals, the murine Mdx model with a DBA/2J background (D2-mdx) was recently suggested to present pathological features closer to that of humans. In this study, we characterized skeletal and cardiac muscle functions in males and females D2-mdx mice compared to control groups. We also evaluated the impact of high intensity interval training (HIIT) in these muscles in females and males. HIIT was performed 5 times per week during a month on a motorized treadmill. Specific maximal isometric force production and weakness were measured in the tibialis anterior muscle (TA). Sedentary male and female D2-mdx mice produced lower absolute and specific maximal force compared to control mice. Dystrophic mice showed a decline of force generation during repetitive stimulation compared to controls. This reduction was greater for male D2-mdx mice than females. Furthermore, trained D2-mdx males showed an improvement in force generation after the fifth lengthening contraction compared to sedentary D2-mdx males. Moreover, echocardiography measures revealed a decrease in left ventricular end-diastolic volume, left ventricular ejection volume and left ventricular end-diastolic diameter in sedentary male and female D2-mdx mice. Overall, our results showed a serious muscle function alteration in female and male D2-mdx mice compared to controls. HIIT may delay force loss especially in male D2-mdx mice.

The non-muscle ADF/cofilin-1 controls sarcomeric actin filament integrity and force production in striated muscle laminopathies.

Cofilins are important for the regulation of the actin cytoskeleton, sarcomere organization, and force production. The role of cofilin-1, the non-muscle-specific isoform, in muscle function remains unclear. Mutations in LMNA encoding A-type lamins, intermediate filament proteins of the nuclear envelope, cause autosomal Emery-Dreifuss muscular dystrophy (EDMD). Here, we report increased cofilin-1 expression in LMNA mutant muscle cells caused by the inability of proteasome degradation, suggesting a protective role by ERK1/2. It is known that phosphorylated ERK1/2 directly binds to and catalyzes phosphorylation of the actin-depolymerizing factor cofilin-1 on Thr25. In vivo ectopic expression of cofilin-1, as well as its phosphorylated form on Thr25, impairs sarcomere structure and force generation. These findings present a mechanism that provides insight into the molecular pathogenesis of muscular dystrophies caused by LMNA mutations.

Alternative splicing of clathrin heavy chain contributes to the switch from coated pits to plaques.

Clathrin function directly derives from its coat structure, and while endocytosis is mediated by clathrin-coated pits, large plaques contribute to cell adhesion. Here, we show that the alternative splicing of a single exon of the clathrin heavy chain gene (CLTC exon 31) helps determine the clathrin coat organization. Direct genetic control was demonstrated by forced CLTC exon 31 skipping in muscle cells that reverses the plasma membrane content from clathrin plaques to pits and by promoting exon inclusion that stimulated flat plaque assembly. Interestingly, mis-splicing of CLTC exon 31 found in the severe congenital form of myotonic dystrophy was associated with reduced plaques in patient myotubes. Moreover, forced exclusion of this exon in WT mice muscle induced structural disorganization and reduced force, highlighting the contribution of this splicing event for the maintenance of tissue homeostasis. This genetic control on clathrin assembly should influence the way we consider how plasticity in clathrin-coated structures is involved in muscle development and maintenance.

Desmin prevents muscle wasting, exaggerated weakness and fragility, and fatigue in dystrophic mdx mouse.

KEY POINTS: Desmin, similar to dystrophin, is associated with costameric structures bridging sarcomeres to the extracellular matrix. Deletion of the desmin gene in mdx mice [double knockout (DKO) mice] induces marked muscle weakness and fatigue resistance compared to mdx mice. Muscle fragility (higher susceptibility to contraction-induced injury) was also aggravated in DKO mice compared to mdx mice. By contrast to mdx mice, the DKO mice did not undergo muscle hypertrophy. Desmin cDNA transfer with adeno-associated virus in newborn mdx mice reduced muscle weakness. Overall, desmin plays important and beneficial roles in muscle wasting, performance and fragility in dystrophic muscle. ABSTRACT: Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease caused by dystrophin deficiency. Desmin, similar to dystrophin, is associated with costameric structures bridging sarcomeres to the extracellular matrix that contributes to muscle function. In the present study, we attempted to provide further insight into the roles of desmin, for which the expression is increased in the muscle from the mouse mdx DMD model. We show that a deletion of the desmin gene (Des) in mdx mice [double knockout (DKO) mice, mdx:desmin-/-] induces a marked muscle weakness; namely, a reduced absolute maximal force production and increased fatigue compared to that in mdx mice. Fragility (i.e. higher susceptibility to contraction-induced injury) was also aggravated in DKO mice compared to mdx mice, despite the promotion of supposedly less fragile muscle fibres in DKO mice, and this worsening of fragility was related to a decreased muscle excitability. Moreover, in contrast to mdx mice, the DKO mice did not undergo muscle hypertrophy, as indicated by smaller and fewer fibres, with a reduced percentage of centronucleated fibres, potentially explaining the severe muscle weakness. Notably, Desmin cDNA transfer with adeno-associated virus in newborn mdx mice improved specific maximal force normalized to muscle weight. Overall, desmin plays important and beneficial roles in muscle wasting, performance and fragility in dystrophic mdx mice, which differ, at least in part, from those observed in healthy muscle.

Combined Treatment with Peptide-Conjugated Phosphorodiamidate Morpholino Oligomer-PPMO and AAV-U7 Rescues the Severe DMD Phenotype in Mice.

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by an absence of the dystrophin protein, which is essential for muscle fiber integrity. Among the developed therapeutic strategies for DMD, the exon-skipping approach corrects the frameshift and partially restores dystrophin expression. It could be achieved through the use of antisense sequences, such as peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) or the small nuclear RNA-U7 carried by an adeno-associated virus (AAV) vector. AAV-based gene therapy approaches have potential for use in DMD treatment but are subject to a major limitation: loss of the AAV genome, necessitating readministration of the vector, which is not currently possible, due to the immunogenicity of the capsid. The PPMO approach requires repeated administrations and results in only weak cardiac dystrophin expression. Here, we evaluated a combination of PPMO- and AAV-based therapy in a mouse model of severe DMD. Striking benefits of this combined therapy were observed in striated muscles, with marked improvements in heart and diaphragm structure and function, with unrivalled extent of survival, opening novel therapeutic perspectives for patients.

An embryonic CaVß1 isoform promotes muscle mass maintenance via GDF5 signaling in adult mouse.

Deciphering the mechanisms that govern skeletal muscle plasticity is essential to understand its pathophysiological processes, including age-related sarcopenia. The voltage-gated calcium channel CaV1.1 has a central role in excitation-contraction coupling (ECC), raising the possibility that it may also initiate the adaptive response to changes during muscle activity. Here, we revealed the existence of a gene transcription switch of the CaV1.1 ß subunit (CaVß1) that is dependent on the innervation state of the muscle in mice. In a mouse model of sciatic denervation, we showed increased expression of an embryonic isoform of the subunit that we called CaVß1E. CaVß1E boosts downstream growth differentiation factor 5 (GDF5) signaling to counteract muscle loss after denervation in mice. We further reported that aged mouse muscle expressed lower quantity of CaVß1E compared with young muscle, displaying an altered GDF5-dependent response to denervation. Conversely, CaVß1E overexpression improved mass wasting in aging muscle in mice by increasing GDF5 expression. We also identified the human CaVß1E analogous and show a correlation between CaVß1E expression in human muscles and age-related muscle mass decline. These results suggest that strategies targeting CaVß1E or GDF5 might be effective in reducing muscle mass loss in aging.

Improvement of Dystrophic Muscle Fragility by Short-Term Voluntary Exercise through Activation of Calcineurin Pathway in mdx Mice.

Dystrophin deficiency in mdx mice, a model for Duchenne muscular dystrophy, leads to muscle weakness revealed by a reduced specific maximal force as well as fragility (ie, higher susceptibility to contraction-induced injury, as shown by a greater force decrease after lengthening contractions). Both symptoms could be improved with dystrophin restoration-based therapies and long-term (months) voluntary exercise. Herein, we evaluated the effect of short-term (1-week) voluntary wheel running. We found that running improved fragility of tibialis anterior muscle (TA), but not plantaris muscle, independently of utrophin up-regulation, without affecting weakness. Moreover, TA muscle excitability was also preserved by running, as shown by compound muscle action potential measurements after lengthening contractions. Of interest, the calcineurin inhibitor cyclosporin A prevented the effect of running on both muscle fragility and excitability. Cyclosporin also prevented the running-induced changes in expression of genes involved in excitability (Scn4a and Cacna1s) and slower contractile phenotype (Myh2 and Tnni1) in TA muscle. In conclusion, short-term voluntary exercise improves TA muscle fragility in mdx mice, without worsening weakness. Its effect was related to preserved excitability, calcineurin pathway activation, and changes in the program of genes involved in excitability and slower contractile phenotype. Thus, remediation of muscle fragility of Duchenne muscular dystrophy patients through appropriate exercise training deserves to be explored in more detail.

Gonad-related factors promote muscle performance gain during postnatal development in male and female mice.

To better define the role of male and female gonad-related factors (MGRF, presumably testosterone, and FGRF, presumably estradiol, respectively) on mouse hindlimb skeletal muscle contractile performance/function gain during postnatal development, we analyzed the effect of castration initiated before puberty in male and female mice. We found that muscle absolute and specific (normalized to muscle weight) maximal forces were decreased in 6-mo-old male and female castrated mice compared with age- and sex-matched intact mice, without alteration in neuromuscular transmission. Moreover, castration decreased absolute and specific maximal powers, another important aspect of muscle performance, in 6-mo-old males, but not in females. Absolute maximal force was similarly reduced by castration in 3-mo-old muscle fiber androgen receptor (AR)-deficient and wild-type male mice, indicating that the effect of MGRF was muscle fiber AR independent. Castration reduced the muscle weight gain in 3-mo mice of both sexes and in 6-mo females but not in males. We also found that bone morphogenetic protein signaling through Smad1/5/9 was not altered by castration in atrophic muscle of 3-mo-old mice of both sexes. Moreover, castration decreased the sexual dimorphism regarding muscle performance. Together, these results demonstrated that in the long term, MGRF and FGRF promote muscle performance gain in mice during postnatal development, independently of muscle growth in males, largely via improving muscle contractile quality (force and power normalized), and that MGFR and FGRF also contribute to sexual dimorphism. However, the mechanisms underlying MGFR and FGRF actions remain to be determined.