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.

AMPKα2 is a skeletal muscle stem cell intrinsic regulator of myonuclear accretion.

Due to the post-mitotic nature of skeletal muscle fibers, adult muscle maintenance relies on dedicated muscle stem cells (MuSCs). In most physiological contexts, MuSCs support myofiber homeostasis by contributing to myonuclear accretion, which requires a coordination of cell-type specific events between the myofiber and MuSCs. Here, we addressed the role of the kinase AMPKα2 in the coordination of these events supporting myonuclear accretion. We demonstrate that AMPKα2 deletion impairs skeletal muscle regeneration. Through in vitro assessments of MuSC myogenic fate and EdU-based cell tracing, we reveal a MuSC-specific role of AMPKα2 in the regulation of myonuclear accretion, which is mediated by phosphorylation of the non-metabolic substrate BAIAP2. Similar cell tracing in vivo shows that AMPKα2 knockout mice have a lower rate of myonuclear accretion during regeneration, and that MuSC-specific AMPKα2 deletion decreases myonuclear accretion in response to myofiber contraction. Together, this demonstrates that AMPKα2 is a MuSC-intrinsic regulator of myonuclear accretion.

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.

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.

Absence of Desmin Results in Impaired Adaptive Response to Mechanical Overloading of Skeletal Muscle.

Background: Desmin is a muscle-specific protein belonging to the intermediate filament family. Desmin mutations are linked to skeletal muscle defects, including inherited myopathies with severe clinical manifestations. The aim of this study was to examine the role of desmin in skeletal muscle remodeling and performance gain induced by muscle mechanical overloading which mimics resistance training. Methods: Plantaris muscles were overloaded by surgical ablation of gastrocnemius and soleus muscles. The functional response of plantaris muscle to mechanical overloading in desmin-deficient mice (DesKO, n = 32) was compared to that of control mice (n = 36) after 7-days or 1-month overloading. To elucidate the molecular mechanisms implicated in the observed partial adaptive response of DesKO muscle, we examined the expression levels of genes involved in muscle growth, myogenesis, inflammation and oxidative energetic metabolism. Moreover, ultrastructure and the proteolysis pathway were explored. Results: Contrary to control, absolute maximal force did not increase in DesKO muscle following 1-month mechanical overloading. Fatigue resistance was also less increased in DesKO as compared to control muscle. Despite impaired functional adaptive response of DesKO mice to mechanical overloading, muscle weight and the number of oxidative MHC2a-positive fibers per cross-section similarly increased in both genotypes after 1-month overloading. However, mechanical overloading-elicited remodeling failed to activate a normal myogenic program after 7-days overloading, resulting in proportionally reduced activation and differentiation of muscle stem cells. Ultrastructural analysis of the plantaris muscle after 1-month overloading revealed muscle fiber damage in DesKO, as indicated by the loss of sarcomere integrity and mitochondrial abnormalities. Moreover, the observed accumulation of autophagosomes and lysosomes in DesKO muscle fibers could indicate a blockage of autophagy. To address this issue, two main proteolysis pathways, the ubiquitin-proteasome system and autophagy, were explored in DesKO and control muscle. Our results suggested an alteration of proteolysis pathways in DesKO muscle in response to mechanical overloading. Conclusion: Taken together, our results show that mechanical overloading increases the negative impact of the lack of desmin on myofibril organization and mitochondria. Furthermore, our results suggest that under these conditions, the repairing activity of autophagy is disturbed. Consequently, force generation is not improved despite muscle growth, suggesting that desmin is required for a complete response to resistance training in skeletal muscle.

Myod1 and GR coordinate myofiber-specific transcriptional enhancers.

Skeletal muscle is a dynamic tissue the size of which can be remodeled through the concerted actions of various cues. Here, we investigated the skeletal muscle transcriptional program and identified key tissue-specific regulatory genetic elements. Our results show that Myod1 is bound to numerous skeletal muscle enhancers in collaboration with the glucocorticoid receptor (GR) to control gene expression. Remarkably, transcriptional activation controlled by these factors occurs through direct contacts with the promoter region of target genes, via the CpG-bound transcription factor Nrf1, and the formation of Ctcf-anchored chromatin loops, in a myofiber-specific manner. Moreover, we demonstrate that GR negatively controls muscle mass and strength in mice by down-regulating anabolic pathways. Taken together, our data establish Myod1, GR and Nrf1 as key players of muscle-specific enhancer-promoter communication that orchestrate myofiber size regulation.

Differential physiological roles for BIN1 isoforms in skeletal muscle development, function and regeneration.

Skeletal muscle development and regeneration are tightly regulated processes. How the intracellular organization of muscle fibers is achieved during these steps is unclear. Here, we focus on the cellular and physiological roles of amphiphysin 2 (BIN1), a membrane remodeling protein mutated in both congenital and adult centronuclear myopathies (CNM), that is ubiquitously expressed and has skeletal muscle-specific isoforms. We created and characterized constitutive muscle-specific and inducible Bin1 homozygous and heterozygous knockout mice targeting either ubiquitous or muscle-specific isoforms. Constitutive Bin1-deficient mice died at birth from lack of feeding due to a skeletal muscle defect. T-tubules and other organelles were misplaced and altered, supporting a general early role for BIN1 in intracellular organization, in addition to membrane remodeling. Although restricted deletion of Bin1 in unchallenged adult muscles had no impact, the forced switch from the muscle-specific isoforms to the ubiquitous isoforms through deletion of the in-frame muscle-specific exon delayed muscle regeneration. Thus, ubiquitous BIN1 function is necessary for muscle development and function, whereas its muscle-specific isoforms fine tune muscle regeneration in adulthood, supporting that BIN1 CNM with congenital onset are due to developmental defects, whereas later onset may be due to regeneration defects.

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.

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.

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.

Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice.

Antisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including myotonic dystrophy (DM1). Thus, ASO strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nucleus-retained mutant DMPK (DM1 protein kinase) transcripts containing CUG expansions (CUGexps). However, systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6a-conjugated morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison with unconjugated PMO and other ASO strategies. Thus, low-dose treatment with Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease transcriptome. Moreover, treated DM1 patient-derived muscle cells showed that Pip6a-PMO-CAG specifically targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and consequently MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces long-lasting correction with high efficacy of DM1-associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide conjugates for systemic corrective therapy in DM1.

Functional muscle recovery following dystrophin and myostatin exon splice modulation in aged mdx mice.

Duchenne muscular dystrophy (DMD) is a rare genetic disease affecting 1 in 3500-5000 newborn boys. It is due to mutations in the DMD gene with a consequent lack of dystrophin protein that leads to deterioration of myofibres and their replacement with fibro-adipogenic tissue. Out-of-frame mutations in the DMD gene can be modified by using antisense oligonucleotides (AONs) to promote skipping of specific exons such that the reading frame is restored and the resulting protein produced, though truncated, is functional. We have shown that AONs can also be used to knock down myostatin, a negative regulator of muscle growth and differentiation, through disruption of the transcript reading frame, and thereby enhance muscle strength. In young mdx mice, combined dystrophin and myostatin exon skipping therapy greatly improved DMD pathology, compared to the single dystrophin skipping approach. Here we show that in aged (>15-month-old) mdx mice, when the pathology is significantly more severe and more similar to the one observed in DMD patients, the effect of the combined therapy is slightly attenuated but still beneficial in improving the disease phenotype. These results confirm the beneficial outcome of the combination approach and support its translation into DMD clinical trials.

Effects of the selective inhibition of proteasome caspase-like activity by CLi a derivative of nor-cerpegin in dystrophic mdx mice.

Previous studies have shown that proteasome inhibition can have beneficial effects in dystrophic mouse models. In this study, we have investigated the effects of a new selective proteasome inhibitor, CLi, a strong caspase-like inhibitor of the 20S proteasome, on skeletal and cardiac muscle functions of mdx mice. In the first series of experiments, five-month-old male mdx mice (n = 34) were treated with 2 different doses (20 and 100 µg/kg) of CLi and in the second series of experiments, five-month-old female mdx (n = 19) and wild-type (n = 24) mice were treated with 20 µg/kg CLi and Velcade (1 mg/kg) for 1-month. All animals were treadmill exercised twice a week to worsen the dystrophic features. In the first series of experiments, our results demonstrated that 20 µg/kg CLi did not significantly increase absolute and specific maximal forces in skeletal muscle from male mdx mice. Moreover, the higher susceptibility to contraction induced skeletal muscle injury was worsened by 100 µg/kg CLi since the force drop following lengthening contractions was increased with this high dose. Furthermore, we found no differences in the mRNA levels of the molecular markers implicated in dystrophic features. Concerning cardiac function, CLi had no effect on left ventricular function since ejection and shortening fractions were unchanged in male mdx mice. Similarly, CLi did not modify the expression of genes implicated in cardiac remodeling. In the second series of experiments, our results demonstrated an improvement in absolute and specific maximal forces by CLi, whereas Velcade only increased specific maximal force in female mdx mice. In addition, exercise tolerance was not improved by CLi. Taken together, our results show that CLi treatment can only improve maximal force production in exercised female mdx mice without affecting either exercice tolerance capacity or cardiac function. In conclusion, selective inhibition of caspase-like activity of proteasome with CLi has no compelling beneficial effect in dystrophic mdx mice.

Aged Nicotinamide Riboside Kinase 2 Deficient Mice Present an Altered Response to Endurance Exercise Training.

Background: Skeletal muscle aging is marked by the development of a sarcopenic phenotype, a global decline of muscle energetic capacities, and an intolerance to exercise. Among the metabolic disorders involved in this syndrome, NAD metabolism was shown to be altered in skeletalmuscle, with an important role for the NAMPT enzyme recycling the nicotinamide precursor. An alternative pathway for NAD biosynthesis has been described for the nicotinamide riboside vitamin B3 precursor used by the NMRK kinases, including the striated muscle-specific NMRK2. Aim: With this study, our goal is to explore the ability of 16-month-old Nmrk2 -/- mice to perform endurance exercise and study the consequences on muscle adaptation to exercise. Methods: 10 control and 6 Nmrk2 -/- mice were used and randomly assigned to sedentary and treadmill endurance training groups. After 9 weeks of training, heart and skeletal muscle samples were harvested and used for gene expression analysis, NAD levels measurements and immunohistochemistry staining. Results: Endurance training triggered a reduction in the expression of Cpt1b and AcadL genes involved in fatty acid catabolism in the heart of Nmrk2 -/- mice, not in control mice. NAD levels were not altered in heart or skeletal muscle, nor at baseline neither after exercise training in any group. Myh7 gene encoding for the slow MHC-I was more strongly induced by exercise in Nmrk2 -/- mice than in controls. Moreover, IL-15 expression levels is higher in Nmrk2 -/- mice skeletal muscle at baseline compared to controls. No fiber type switch was observed in plantaris after exercise, but fast fibers diameter was reduced in aged control mice, not in Nmrk2 -/- mice. No fiber type switch or diameter modification was observed in soleus muscle. Conclusion: In this study, we demonstrated for the first time a phenotype in old Nmrk2 -/- mice in response to endurance exercise training. Although NMRK2 seems to be predominantly dispensable to maintain global NAD levels in heart and skeletal muscle, we demonstrated a maladaptive metabolic response to exercise in cardiac and skeletal muscle, showing that NMRK2 has a specific and restricted role in NAD signaling compared to the NAMPT pathway.

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.

Allele-specific silencing therapy for Dynamin 2-related dominant centronuclear myopathy.

Rapid advances in allele-specific silencing by RNA interference established a strategy of choice to cure dominant inherited diseases by targeting mutant alleles. We used this strategy for autosomal-dominant centronuclear myopathy (CNM), a rare neuromuscular disorder without available treatment due to heterozygous mutations in the DNM2 gene encoding Dynamin 2. Allele-specific siRNA sequences were developed in order to specifically knock down the human and murine DNM2-mRNA harbouring the p.R465W mutation without affecting the wild-type allele. Functional restoration was achieved in muscle from a knock-in mouse model and in patient-derived fibroblasts, both expressing the most frequently encountered mutation in patients. Restoring either muscle force in a CNM mouse model or DNM2 function in patient-derived cells is an essential breakthrough towards future gene-based therapy for dominant centronuclear myopathy.