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.

Specific DMPK-promoter targeting by CRISPRi reverses myotonic dystrophy type 1-associated defects in patient muscle cells.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disease that originates from an expansion of CTG microsatellites in the 3' untranslated region of the DMPK gene, thus leading to the expression of transcripts containing expanded CUG repeats (CUGexp). The pathophysiology is explained by a toxic RNA gain of function where CUGexp RNAs form nuclear aggregates that sequester and alter the function of MBNL splicing factors, triggering splicing misregulation linked to the DM1 symptoms. There is currently no cure for DM1, and most therapeutic strategies aim at eliminating CUGexp-DMPK transcripts. Here, we investigate a DMPK-promoter silencing strategy using CRISPR interference as a new alternative approach. Different sgRNAs targeting the DMPK promoter are evaluated in DM1 patient muscle cells. The most effective guides allowed us to reduce the level of DMPK transcripts and CUGexp-RNA aggregates up to 80%. The CUGexp-DMPK repression corrects the overall transcriptome, including spliceopathy, and reverses a physiological parameter in DM1 muscle cells. Its action is specific and restricted to the DMPK gene, as confirmed by genome-wide expression analysis. Altogether, our findings highlight DMPK-promoter silencing by CRISPRi as a promising therapeutic approach for DM1.

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.

MBNL-dependent impaired development within the neuromuscular system in myotonic dystrophy type 1.

AIMS: Myotonic dystrophy type I (DM1) is one of the most frequent muscular dystrophies in adults. Although DM1 has long been considered mainly a muscle disorder, growing evidence suggests the involvement of peripheral nerves in the pathogenicity of DM1 raising the question of whether motoneurons (MNs) actively contribute to neuromuscular defects in DM1. METHODS: By using micropatterned 96-well plates as a coculture platform, we generated a functional neuromuscular model combining DM1 and muscleblind protein (MBNL) knock-out human-induced pluripotent stem cells-derived MNs and human healthy skeletal muscle cells. RESULTS: This approach led to the identification of presynaptic defects which affect the formation or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA-binding MBNL proteins, whose loss of function in vivo is associated with muscular defects associated with DM1. These experiments indicate that the functional defects associated with MNs can be directly attributed to MBNL family proteins. Comparative transcriptomic analyses also revealed specific neuronal-related processes regulated by these proteins that are commonly misregulated in DM1. CONCLUSIONS: Beyond the application to DM1, our approach to generating a robust and reliable human neuromuscular system should facilitate disease modelling studies and drug screening assays.

Identification of a CCG-Enriched Expanded Allele in Patients with Myotonic Dystrophy Type 1 Using Amplification-Free Long-Read Sequencing.

Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4000 CTG. The clinical variability depends on CTG repeat number, CNG repeat interruptions, and somatic mosaicism. Currently, none of these factors are simultaneously and accurately determined due to the limitations of gold standard methods used in clinical and research laboratories. An amplicon method for targeting the DMPK locus using single-molecule real-time sequencing was recently developed to accurately analyze expanded alleles. However, amplicon-based sequencing still depends on PCR, and the inherent bias toward preferential amplification of smaller repeats can be problematic in DM1. Thus, an amplification-free long-read sequencing method was developed by using CRISPR/Cas9 technology in DM1. This method was used to sequence the DMPK locus in patients with CTG repeat expansion ranging from 130 to >1000 CTG. We showed that elimination of PCR amplification improves the accuracy of measurement of inherited repeat number and somatic repeat variations, two key factors in DM1 severity and age at onset. For the first time, an expansion composed of >85% CCG repeats was identified by using this innovative method in a DM1 family with an atypical clinical profile. No-amplification targeted sequencing represents a promising method that can overcome research and diagnosis shortcomings, with translational implications for clinical and genetic counseling in DM1.

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 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.

Comprehensive transcriptome-wide analysis of spliceopathy correction of myotonic dystrophy using CRISPR-Cas9 in iPSCs-derived cardiomyocytes.

CTG repeat expansion (CTGexp) is associated with aberrant alternate splicing that contributes to cardiac dysfunction in myotonic dystrophy type 1 (DM1). Excision of this CTGexp repeat using CRISPR-Cas resulted in the disappearance of punctate ribonuclear foci in cardiomyocyte-like cells derived from DM1-induced pluripotent stem cells (iPSCs). This was associated with correction of the underlying spliceopathy as determined by RNA sequencing and alternate splicing analysis. Certain genes were of particular interest due to their role in cardiac development, maturation, and function (TPM4, CYP2J2, DMD, MBNL3, CACNA1H, ROCK2, ACTB) or their association with splicing (SMN2, GCFC2, MBNL3). Moreover, while comparing isogenic CRISPR-Cas9-corrected versus non-corrected DM1 cardiomyocytes, a prominent difference in the splicing pattern for a number of candidate genes was apparent pertaining to genes that are associated with cardiac function (TNNT, TNNT2, TTN, TPM1, SYNE1, CACNA1A, MTMR1, NEBL, TPM1), cellular signaling (NCOR2, CLIP1, LRRFIP2, CLASP1, CAMK2G), and other DM1-related genes (i.e., NUMA1, MBNL2, LDB3) in addition to the disease-causing DMPK gene itself. Subsequent validation using a selected gene subset, including MBNL1, MBNL2, INSR, ADD3, and CRTC2, further confirmed correction of the spliceopathy following CTGexp repeat excision. To our knowledge, the present study provides the first comprehensive unbiased transcriptome-wide analysis of the differential splicing landscape in DM1 patient-derived cardiac cells after excision of the CTGexp repeat using CRISPR-Cas9, showing reversal of the abnormal cardiac spliceopathy in DM1.

CRISPR gene editing in pluripotent stem cells reveals the function of MBNL proteins during human in vitro myogenesis.

Alternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly misregulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins.

A CRISPR-Cas13a Based Strategy That Tracks and Degrades Toxic RNA in Myotonic Dystrophy Type 1.

Cas13a, an effector of type VI CRISPR-Cas systems, is an RNA guided RNase with multiplexing and therapeutic potential. This study employs the Leptotrichia shahii (Lsh) Cas13a and a repeat-based CRISPR RNA (crRNA) to track and eliminate toxic RNA aggregates in myotonic dystrophy type 1 (DM1) - a neuromuscular disease caused by CTG expansion in the DMPK gene. We demonstrate that LshCas13a cleaves CUG repeat RNA in biochemical assays and reduces toxic RNA load in patient-derived myoblasts. As a result, LshCas13a reverses the characteristic adult-to-embryonic missplicing events in several key genes that contribute to DM1 phenotype. The deactivated LshCas13a can further be repurposed to track RNA-rich organelles within cells. Our data highlights the reprogrammability of LshCas13a and the possible use of Cas13a to target expanded repeat sequences in microsatellite expansion diseases.

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.

FISH Protocol for Myotonic Dystrophy Type 1 Cells.

Mutant DMPK transcripts containing expanded CUG repeats (CUGexp) are retained within the nucleus of myotonic dystrophy type 1 (DM1) cells as discrete foci. Nuclear CUGexp-RNA foci that sequester MBNL1 splicing factor represent a hallmark of this RNA dominant disease caused by the expression of expanded microsatellite repeats. Here we described fluorescent in situ hybridization (FISH) techniques to detect either RNA containing CUG expansion or DMPK transcripts in human DM1 or WT cells. In addition, we propose a combined FISH/immunofluorescence protocol to visualize the colocalization of MBNL1 with CUGexp-RNA foci in DM1 cells.

Cardiovascular manifestations of myotonic dystrophy.

Patients with myotonic dystrophy, the most common neuromuscular dystrophy in adults, have a high prevalence of arrhythmic complications with increased cardiovascular mortality and high risk for sudden death. Sudden death prevention is central and relies on annual follow-up and prophylactic permanent pacing in patients with conduction defects on electrocardiogram and/or infrahisian blocks on electrophysiological study. Implantable cardiac defibrillator therapy may be indicated in patients with ventricular tachyarrhythmia.

New recessive mutations in SYT2 causing severe presynaptic congenital myasthenic syndromes.

OBJECTIVE: To report the identification of 2 new homozygous recessive mutations in the synaptotagmin 2 (SYT2) gene as the genetic cause of severe and early presynaptic forms of congenital myasthenic syndromes (CMSs). METHODS: Next-generation sequencing identified new homozygous intronic and frameshift mutations in the SYT2 gene as a likely cause of presynaptic CMS. We describe the clinical and electromyographic patient phenotypes, perform ex vivo splicing analyses to characterize the effect of the intronic mutation on exon splicing, and analyze the functional impact of this variation at the neuromuscular junction (NMJ). RESULTS: The 2 infants presented a similar clinical phenotype evoking first a congenital myopathy characterized by muscle weakness and hypotonia. Next-generation sequencing allowed to the identification of 1 homozygous intronic mutation c.465+1G>A in patient 1 and another homozygous frameshift mutation c.328_331dup in patient 2, located respectively in the 5' splice donor site of SYT2 intron 4 and in exon 3. Functional studies of the intronic mutation validated the abolition of the splice donor site of exon 4 leading to its skipping. In-frame skipping of exon 4 that encodes part of the C2A calcium-binding domain of SYT2 is associated with a loss-of-function effect resulting in a decrease of neurotransmitter release and severe pre- and postsynaptic NMJ defects. CONCLUSIONS: This study identifies new homozygous recessive SYT2 mutations as the underlying cause of severe and early presynaptic form of CMS expanding the genetic spectrum of recessive SYT2-related CMS associated with defects in neurotransmitter release.

miR-7 Restores Phenotypes in Myotonic Dystrophy Muscle Cells by Repressing Hyperactivated Autophagy.

Unstable CTG expansions in the 3' UTR of the DMPK gene are responsible for myotonic dystrophy type 1 (DM1) condition. Muscle dysfunction is one of the main contributors to DM1 mortality and morbidity. Pathways by which mutant DMPK trigger muscle defects, however, are not fully understood. We previously reported that miR-7 was downregulated in a DM1 Drosophila model and in biopsies from patients. Here, using DM1 and normal muscle cells, we investigated whether miR-7 contributes to the muscle phenotype by studying the consequences of replenishing or blocking miR-7, respectively. Restoration of miR-7 with agomiR-7 was sufficient to rescue DM1 myoblast fusion defects and myotube growth. Conversely, oligonucleotide-mediated blocking of miR-7 in normal myoblasts led to fusion and myotube growth defects. miR-7 was found to regulate autophagy and the ubiquitin-proteasome system in human muscle cells. Thus, low levels of miR-7 promoted both processes, and high levels of miR-7 repressed them. Furthermore, we uncovered that the mechanism by which miR-7 improves atrophy-related phenotypes is independent of MBNL1, thus suggesting that miR-7 acts downstream or in parallel to MBNL1. Collectively, these results highlight an unknown function for miR-7 in muscle dysfunction through autophagy- and atrophy-related pathways and support that restoration of miR-7 levels is a candidate therapeutic target for counteracting muscle dysfunction in DM1.

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.