Effects of Mini-Dystrophin on Dystrophin-Deficient, Human Skeletal Muscle-Derived Cells.

BACKGROUND: We are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells. AIMS: Our aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells. METHODS: Skeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3-R13 spectrin-like repeats (ΔR3R13) or hinge2 to spectrin-like repeats R23 (ΔH2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression. RESULTS: Cell proliferation was not affected in cells transduced with ΔR3R13, but was significantly increased in cells transduced with ΔH2R23. The fusion index of myotubes derived from both ΔR3R13- and ΔH2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ΔH2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs. CONCLUSIONS: Ectopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter.

Selective release of muscle-specific, extracellular microRNAs during myogenic differentiation.

MyomiRs are muscle-specific microRNAs (miRNAs) that regulate myoblast proliferation and differentiation. Extracellular myomiRs (ex-myomiRs) are highly enriched in the serum of Duchenne Muscular Dystrophy (DMD) patients and dystrophic mouse models and consequently have potential as disease biomarkers. The biological significance of miRNAs present in the extracellular space is not currently well understood. Here we demonstrate that ex-myomiR levels are elevated in perinatal muscle development, during the regenerative phase that follows exercise-induced myoinjury, and concomitant with myoblast differentiation in culture. Whereas ex-myomiRs are progressively and specifically released by differentiating human primary myoblasts and C2C12 cultures, chemical induction of apoptosis in C2C12 cells results in indiscriminate miRNA release. The selective release of myomiRs as a consequence of cellular differentiation argues against the idea that they are solely waste products of muscle breakdown, and suggests they may serve a biological function in specific physiological contexts. Ex-myomiRs in culture supernatant and serum are predominantly non-vesicular, and their release is independent of ceramide-mediated vesicle secretion. Furthermore, ex-myomiRs levels are reduced in aged dystrophic mice, likely as a consequence of chronic muscle wasting. In conclusion, we show that myomiR release accompanies periods of myogenic differentiation in cell culture and in vivo. Serum myomiR abundance is therefore a function of the regenerative/degenerative status of the muscle, overall muscle mass, and tissue expression levels. These findings have implications for the use of ex-myomiRs as biomarkers for DMD disease progression and monitoring response to therapy.

Loss-of-function mutations in SCN4A cause severe foetal hypokinesia or 'classical' congenital myopathy.

Congenital myopathies are a clinically and genetically heterogeneous group of muscle disorders characterized by congenital or early-onset hypotonia and muscle weakness, and specific pathological features on muscle biopsy. The phenotype ranges from foetal akinesia resulting in in utero or neonatal mortality, to milder disorders that are not life-limiting. Over the past decade, more than 20 new congenital myopathy genes have been identified. Most encode proteins involved in muscle contraction; however, mutations in ion channel-encoding genes are increasingly being recognized as a cause of this group of disorders. SCN4A encodes the α-subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4). This channel is essential for the generation and propagation of the muscle action potential crucial to muscle contraction. Dominant SCN4A gain-of-function mutations are a well-established cause of myotonia and periodic paralysis. Using whole exome sequencing, we identified homozygous or compound heterozygous SCN4A mutations in a cohort of 11 individuals from six unrelated kindreds with congenital myopathy. Affected members developed in utero- or neonatal-onset muscle weakness of variable severity. In seven cases, severe muscle weakness resulted in death during the third trimester or shortly after birth. The remaining four cases had marked congenital or neonatal-onset hypotonia and weakness associated with mild-to-moderate facial and neck weakness, significant neonatal-onset respiratory and swallowing difficulties and childhood-onset spinal deformities. All four surviving cohort members experienced clinical improvement in the first decade of life. Muscle biopsies showed myopathic features including fibre size variability, presence of fibrofatty tissue of varying severity, without specific structural abnormalities. Electrophysiology suggested a myopathic process, without myotonia. In vitro functional assessment in HEK293 cells of the impact of the identified SCN4A mutations showed loss-of-function of the mutant Nav1.4 channels. All, apart from one, of the mutations either caused fully non-functional channels, or resulted in a reduced channel activity. Each of the affected cases carried at least one full loss-of-function mutation. In five out of six families, a second loss-of-function mutation was present on the trans allele. These functional results provide convincing evidence for the pathogenicity of the identified mutations and suggest that different degrees of loss-of-function in mutant Nav1.4 channels are associated with attenuation of the skeletal muscle action potential amplitude to a level insufficient to support normal muscle function. The results demonstrate that recessive loss-of-function SCN4A mutations should be considered in patients with a congenital myopathy.

Extracellular microRNAs are dynamic non-vesicular biomarkers of muscle turnover.

Extracellular microRNAs (miRNAs) are promising biomarkers of the inherited muscle wasting condition Duchenne muscular dystrophy, as they allow non-invasive monitoring of either disease progression or response to therapy. In this study, serum miRNA profiling reveals a distinct extracellular miRNA signature in dystrophin-deficient mdx mice, which shows profound dose-responsive restoration following dystrophin rescue. Extracellular dystrophy-associated miRNAs (dystromiRs) show dynamic patterns of expression that mirror the progression of muscle pathology in mdx mice. Expression of the myogenic miRNA, miR-206 and the myogenic transcription factor myogenin in the tibialis anterior muscle were found to positively correlate with serum dystromiR levels, suggesting that extracellular miRNAs are indicators of the regenerative status of the musculature. Similarly, extracellular dystromiRs were elevated following experimentally-induced skeletal muscle injury and regeneration in non-dystrophic mice. Only a minority of serum dystromiRs were found in extracellular vesicles, whereas the majority were protected from serum nucleases by association with protein/lipoprotein complexes. In conclusion, extracellular miRNAs are dynamic indices of pathophysiological processes in skeletal muscle.

Donor satellite cell engraftment is significantly augmented when the host niche is preserved and endogenous satellite cells are incapacitated.

Stem cell transplantation is already in clinical practice for certain genetic diseases and is a promising therapy for dystrophic muscle. We used the mdx mouse model of Duchenne muscular dystrophy to investigate the effect of the host satellite cell niche on the contribution of donor muscle stem cells (satellite cells) to muscle regeneration. We found that incapacitation of the host satellite cells and preservation of the muscle niche promote donor satellite cell contribution to muscle regeneration and functional reconstitution of the satellite cell compartment. But, if the host niche is not promptly refilled, or is filled by competent host satellite cells, it becomes nonfunctional and donor engraftment is negligible. Application of this regimen to aged host muscles also promotes efficient regeneration from aged donor satellite cells. In contrast, if the niche is destroyed, yet host satellite cells remain proliferation-competent, donor-derived engraftment is trivial. Thus preservation of the satellite cell niche, concomitant with functional impairment of the majority of satellite cells within dystrophic human muscles, may improve the efficiency of stem cell therapy.

Defects in glycosylation impair satellite stem cell function and niche composition in the muscles of the dystrophic Large(myd) mouse.

The dystrophin-associated glycoprotein complex (DGC) is found at the muscle fiber sarcolemma and forms an essential structural link between the basal lamina and internal cytoskeleton. In a set of muscular dystrophies known as the dystroglycanopathies, hypoglycosylation of the DGC component α-dystroglycan results in reduced binding to basal lamina components, a loss in structural stability, and repeated cycles of muscle fiber degeneration and regeneration. The satellite cells are the key stem cells responsible for muscle repair and reside between the basal lamina and sarcolemma. In this study, we aimed to determine whether pathological changes associated with the dystroglycanopathies affect satellite cell function. In the Large(myd) mouse dystroglycanopathy model, satellite cells are present in significantly greater numbers but display reduced proliferation on their native muscle fibers in vitro, compared with wild type. However, when removed from their fiber, proliferation in culture is restored to that of wild type. Immunohistochemical analysis of Large(myd) muscle reveals alterations to the basal lamina and interstitium, including marked disorganization of laminin, upregulation of fibronectin and collagens. Proliferation and differentiation of wild-type satellite cells is impaired when cultured on substrates such as collagen and fibronectin, compared with laminins. When engrafted into irradiated tibialis anterior muscles of mdx-nude mice, wild-type satellite cells expanded on laminin contribute significantly more to muscle regeneration than those expanded on fibronectin. These results suggest that defects in α-dystroglycan glycosylation are associated with an alteration in the satellite cell niche, and that regenerative potential in the dystroglycanopathies may be perturbed.

Restoration of the dystrophin-associated glycoprotein complex after exon skipping therapy in Duchenne muscular dystrophy.

We previously conducted a proof of principle; dose escalation study in Duchenne muscular dystrophy (DMD) patients using the morpholino splice-switching oligonucleotide AVI-4658 (eteplirsen) that induces skipping of dystrophin exon 51 in patients with relevant deletions, restores the open reading frame and induces dystrophin protein expression after intramuscular (i.m.) injection. We now show that this dystrophin expression was accompanied by an elevated expression of α-sarcoglycan, ß-dystroglycan (BDG) and--in relevant cases--neuronal nitric oxide synthase (nNOS) at the sarcolemma, each of which is a component of a different subcomplex of the dystrophin-associated glycoprotein complex (DAPC). As expected, nNOS expression was relocalized to the sarcolemma in Duchenne patients in whom the dystrophin deletion left the nNOS-binding domain (exons 42-45) intact, whereas this did not occur in patients with deletions that involved this domain. Our results indicate that the novel internally deleted and shorter dystrophin induced by skipping exon 51 in patients with amenable deletions, can also restore the dystrophin-associated complex, further suggesting preserved functionality of the newly translated dystrophin.

Investigating the Impact of Delivery Routes for Exon Skipping Therapies in the CNS of DMD Mouse Models.

Nucleic acid-based therapies have demonstrated great potential for the treatment of monogenetic diseases, including neurologic disorders. To date, regulatory approval has been received for a dozen antisense oligonucleotides (ASOs); however, these chemistries cannot readily cross the blood-brain barrier when administered systemically. Therefore, an investigation of their potential effects within the central nervous system (CNS) requires local delivery. Here, we studied the brain distribution and exon-skipping efficacy of two ASO chemistries, PMO and tcDNA, when delivered to the cerebrospinal fluid (CSF) of mice carrying a deletion in exon 52 of the dystrophin gene, a model of Duchenne muscular dystrophy (DMD). Following intracerebroventricular (ICV) delivery (unilateral, bilateral, bolus vs. slow rate, repeated via cannula or very slow via osmotic pumps), ASO levels were quantified across brain regions and exon 51 skipping was evaluated, revealing that tcDNA treatment invariably generates comparable or more skipping relative to that with PMO, even when the PMO was administered at higher doses. We also performed intra-cisterna magna (ICM) delivery as an alternative route for CSF delivery and found a biased distribution of the ASOs towards posterior brain regions, including the cerebellum, hindbrain, and the cervical part of the spinal cord. Finally, we combined both ICV and ICM injection methods to assess the potential of an additive effect of this methodology in inducing efficient exon skipping across different brain regions. Our results provide useful insights into the local delivery and associated efficacy of ASOs in the CNS in mouse models of DMD. These findings pave the way for further ASO-based therapy application to the CNS for neurological disease.

Networking to Optimize Dmd exon 53 Skipping in the Brain of mdx52 Mouse Model.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that disrupt the open reading frame and thus prevent production of functional dystrophin proteins. Recent advances in DMD treatment, notably exon skipping and AAV gene therapy, have achieved some success aimed at alleviating the symptoms related to progressive muscle damage. However, they do not address the brain comorbidities associated with DMD, which remains a critical aspect of the disease. The mdx52 mouse model recapitulates one of the most frequent genetic pathogenic variants associated with brain involvement in DMD. Deletion of exon 52 impedes expression of two brain dystrophins, Dp427 and Dp140, expressed from distinct promoters. Interestingly, this mutation is eligible for exon skipping strategies aimed at excluding exon 51 or 53 from dystrophin mRNA. We previously showed that exon 51 skipping can restore partial expression of internally deleted yet functional Dp427 in the brain following intracerebroventricular (ICV) injection of antisense oligonucleotides (ASO). This was associated with a partial improvement of anxiety traits, unconditioned fear response, and Pavlovian fear learning and memory in the mdx52 mouse model. In the present study, we investigated in the same mouse model the skipping of exon 53 in order to restore expression of both Dp427 and Dp140. However, in contrast to exon 51, we found that exon 53 skipping was particularly difficult in mdx52 mice and a combination of multiple ASOs had to be used simultaneously to reach substantial levels of exon 53 skipping, regardless of their chemistry (tcDNA, PMO, or 2'MOE). Following ICV injection of a combination of ASO sequences, we measured up to 25% of exon 53 skipping in the hippocampus of treated mdx52 mice, but this did not elicit significant protein restoration. These findings indicate that skipping mouse dystrophin exon 53 is challenging. As such, it has not yet been possible to answer the pertinent question whether rescuing both Dp427 and Dp140 in the brain is imperative to more optimal treatment of neurological aspects of dystrophinopathy.

Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study.

BACKGROUND: We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with Duchenne muscular dystrophy. METHOD: We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duchenne muscular dystrophy aged 5-15 years with amenable deletions in DMD. Participants had a muscle biopsy before starting treatment and after 12 weekly intravenous infusions of AVI-4658. The primary study objective was to assess safety and tolerability of AVI-4658. The secondary objectives were pharmacokinetic properties and the ability of AVI-4658 to induce exon 51 skipping and dystrophin restoration by RT-PCR, immunohistochemistry, and immunoblotting. The study is registered, number NCT00844597. FINDINGS: 19 patients took part in the study. AVI-4658 was well tolerated with no drug-related serious adverse events. AVI-4658 induced exon 51 skipping in all cohorts and new dystrophin protein expression in a significant dose-dependent (p=0·0203), but variable, manner in boys from cohort 3 (dose 2 mg/kg) onwards. Seven patients responded to treatment, in whom mean dystrophin fluorescence intensity increased from 8·9% (95% CI 7·1-10·6) to 16·4% (10·8-22·0) of normal control after treatment (p=0·0287). The three patients with the greatest responses to treatment had 21%, 15%, and 55% dystrophin-positive fibres after treatment and these findings were confirmed with western blot, which showed an increase after treatment of protein levels from 2% to 18%, from 0·9% to 17%, and from 0% to 7·7% of normal muscle, respectively. The dystrophin-associated proteins α-sarcoglycan and neuronal nitric oxide synthase were also restored at the sarcolemma. Analysis of the inflammatory infiltrate indicated a reduction of cytotoxic T cells in the post-treatment muscle biopsies in the two high-dose cohorts. INTERPRETATION: The safety and biochemical efficacy that we present show the potential of AVI-4658 to become a disease-modifying drug for Duchenne muscular dystrophy. FUNDING: UK Medical Research Council; AVI BioPharma.

Dystrophin quantification and clinical correlations in Becker muscular dystrophy: implications for clinical trials.

Duchenne muscular dystrophy is caused by mutations in the DMD gene that disrupt the open reading frame and prevent the full translation of its protein product, dystrophin. Restoration of the open reading frame and dystrophin production can be achieved by exon skipping using antisense oligonucleotides targeted to splicing elements. This approach aims to transform the Duchenne muscular dystrophy phenotype to that of the milder disorder, Becker muscular dystrophy, typically caused by in-frame dystrophin deletions that allow the production of an internally deleted but partially functional dystrophin. There is ongoing debate regarding the functional properties of the different internally deleted dystrophins produced by exon skipping for different mutations; more insight would be valuable to improve and better predict the outcome of exon skipping clinical trials. To this end, we have characterized the clinical phenotype of 17 patients with Becker muscular dystrophy harbouring in-frame deletions relevant to on-going or planned exon skipping clinical trials for Duchenne muscular dystrophy and correlated it to the levels of dystrophin, and dystrophin-associated protein expression. The cohort of 17 patients, selected exclusively on the basis of their genotype, included 4 asymptomatic, 12 mild and 1 severe patient. All patients had dystrophin levels of >40% of control and significantly higher dystrophin (P = 0.013), ß-dystroglycan (P = 0.025) and neuronal nitric oxide synthase (P = 0.034) expression was observed in asymptomatic individuals versus symptomatic patients with Becker muscular dystrophy. Furthermore, grouping the patients by deletion, patients with Becker muscular dystrophy with deletions with an end-point of exon 51 (the skipping of which could rescue the largest group of Duchenne muscular dystrophy deletions) showed significantly higher dystrophin levels (P = 0.034) than those with deletions ending with exon 53. This is the first quantitative study on both dystrophin and dystrophin-associated protein expression in patients with Becker muscular dystrophy with deletions relevant for on-going exon skipping trials in Duchenne muscular dystrophy. Taken together, our results indicate that all varieties of internally deleted dystrophin assessed in this study have the functional capability to provide a substantial clinical benefit to patients with Duchenne muscular dystrophy.

CRISPR-mediated correction of skeletal muscle Ca2+ handling in a novel DMD patient-derived pluripotent stem cell model.

Mutations in the dystrophin gene cause the most common and currently incurable Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting. Although abnormal Ca2+ handling is a pathological feature of DMD, mechanisms underlying defective Ca2+ homeostasis remain unclear. Here we generate a novel DMD patient-derived pluripotent stem cell (PSC) model of skeletal muscle with an isogenic control using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated precise gene correction. Transcriptome analysis identifies dysregulated gene sets in the absence of dystrophin, including genes involved in Ca2+ handling, excitation-contraction coupling and muscle contraction. Specifically, analysis of intracellular Ca2+ transients and mathematical modeling of Ca2+ dynamics reveal significantly reduced cytosolic Ca2+ clearance rates in DMD-PSC derived myotubes. Pharmacological assays demonstrate Ca2+ flux in myotubes is determined by both intracellular and extracellular sources. DMD-PSC derived myotubes display significantly reduced velocity of contractility. Compared with a non-isogenic wildtype PSC line, these pathophysiological defects could be rescued by CRISPR-mediated precise gene correction. Our study provides new insights into abnormal Ca2+ homeostasis in DMD and suggests that Ca2+ signaling pathways amenable to pharmacological modulation are potential therapeutic targets. Importantly, we have established a human physiology-relevant in vitro model enabling rapid pre-clinical testing of potential therapies for DMD.

Muscle satellite cells are a functionally heterogeneous population in both somite-derived and branchiomeric muscles.

Skeletal muscles of body and limb are derived from somites, but most head muscles originate from cranial mesoderm. The resident stem cells of muscle are satellite cells, which have the same embryonic origin as the muscle in which they reside. Here, we analysed satellite cells with a different ontology, comparing those of the extensor digitorum longus (EDL) of the limb with satellite cells from the masseter of the head. Satellite cell-derived myoblasts from MAS and EDL muscles had distinct gene expression profiles and masseter cells usually proliferated more and differentiated later than those from EDL. When transplanted, however, masseter-derived satellite cells regenerated limb muscles as efficiently as those from EDL. Clonal analysis showed that functional properties differed markedly between satellite cells: ranging from clones that proliferated extensively and gave rise to both differentiated and self-renewed progeny, to others that divided minimally before differentiating completely. Generally, masseter-derived clones were larger and took longer to differentiate than those from EDL. This distribution in cell properties was preserved in both EDL-derived and masseter-derived satellite cells from old mice, although clones were generally less proliferative. Satellite cells, therefore, are a functionally heterogeneous population, with many occupants of the niche exhibiting stem cell characteristics in both somite-derived and branchiomeric muscles.

Direct effects of the pathogenic mutation on satellite cell function in muscular dystrophy.

Skeletal muscle is maintained and repaired by resident stem cells called muscle satellite cells, but there is a gradual failure of this process during the progressive skeletal muscle weakness and wasting that characterises muscular dystrophies. The pathogenic mutation causes muscle wasting, but in conditions including Duchenne muscular dystrophy, the mutant gene is not expressed in satellite cells, and so muscle maintenance/repair is not directly affected. The chronic muscle wasting, however, produces an increasingly hostile micro-environment in dystrophic muscle. This probably combines with excessive satellite cell use to eventually culminate in an indirect failure of satellite cell-mediated myofibre repair. By contrast, in disorders such as Emery-Dreifuss muscular dystrophy, the pathogenic mutation not only instigates muscle wasting, but could also directly compromise satellite cell function, leading to less effective muscle homeostasis. This may again combine with excessive use and a hostile environment to further compromise satellite cell performance. Whichever the mechanism, the ultimate consequence of perturbed satellite cell activity is a chronic failure of myofibre maintenance in dystrophic muscle. Here, we review whether the pathogenic mutation can directly contribute to satellite cell dysfunction in a number of muscular dystrophies.

The altered expression of neurofilament in mouse models and patients with spinal muscular atrophy.

OBJECTIVES: To investigate the levels of neurofilaments (NFs) in transgenic mice and patients with spinal muscular atrophy (SMA), and to evaluate their efficacy as a biomarker in SMA. METHODS: The levels of NF mRNA transcripts were measured by quantitative real-time PCR in spinal cord from SMA mice. Blood levels of NF heavy chain (NfH) from mice and patients were measured by an in-house ELISA method. The response of NFs to therapeutic intervention was analysed in severe SMA mice treated with morpholino antisense oligonucleotides. RESULTS: Significant changes in NF transcript and protein in spinal cord and protein levels in blood were detected in SMA mice with severe or mild phenotypes, at different time points. A decrease in blood levels of NfH after antisense oligonucleotide treatment was only transient in the mice, despite the persistent benefit on the disease phenotype. A drastic reduction of over 90% in blood levels of NfF was observed in both control and SMA mice during early postnatal development. In contrast, blood levels of NfH were found to be decreased in older SMA children with chronic disease progression. INTERPRETATION: Our results show that blood NfH levels are informative in indicating disease onset and response to antisense oligonucleotides treatment in SMA mice, and indicate their potential as a peripheral marker reflecting the pathological status in central nervous system. In older patients with chronic SMA, however, the lower NfH levels may limit their application as biomarker, highlighting the need to continue to pursue additional biomarkers for this group of patients.

Dystrophin involvement in peripheral circadian SRF signalling.

Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy. Dystrophin has an actin-binding domain, which binds and stabilises filamentous-(F)-actin, an integral component of the RhoA-actin-serum-response-factor-(SRF) pathway. This pathway plays a crucial role in circadian signalling, whereby the suprachiasmatic nucleus (SCN) transmits cues to peripheral tissues, activating SRF and transcription of clock-target genes. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised dystrophin loss causes circadian deficits. We show for the first time alterations in the RhoA-actin-SRF-signalling pathway, in dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios, altered MRTF levels, dysregulated core-clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from Duchenne patients harbouring an array of mutations. Furthermore, we show dystrophin is absent in the SCN of dystrophic mice which display disrupted circadian locomotor behaviour, indicative of disrupted SCN signalling. Therefore, dystrophin is an important component of the RhoA-actin-SRF pathway and novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.

High-Throughput Digital Image Analysis Reveals Distinct Patterns of Dystrophin Expression in Dystrophinopathy Patients.

Duchenne muscular dystrophy (DMD) is an incurable disease caused by out-of-frame DMD gene deletions while in frame deletions lead to the milder Becker muscular dystrophy (BMD). In the last decade several antisense oligonucleotides drugs have been developed to induce a partially functional internally deleted dystrophin, similar to that produced in BMD, and expected to ameliorate the disease course. The pattern of dystrophin expression and functionality in dystrophinopathy patients is variable due to multiple factors, such as molecular functionality of the dystrophin and its distribution. To benchmark the success of therapeutic intervention, a clear understanding of dystrophin expression patterns in dystrophinopathy patients is vital. Recently, several groups have used innovative techniques to quantify dystrophin in muscle biopsies of children but not in patients with milder BMD. This study reports on dystrophin expression using both Western blotting and an automated, high-throughput, image analysis platform in DMD, BMD, and intermediate DMD/BMD skeletal muscle biopsies. Our results found a significant correlation between Western blot and immunofluorescent quantification indicating consistency between the different methodologies. However, we identified significant inter- and intradisease heterogeneity of patterns of dystrophin expression in patients irrespective of the amount detected on blot, due to variability in both fluorescence intensity and dystrophin sarcolemmal circumference coverage. Our data highlight the heterogeneity of the pattern of dystrophin expression in BMD, which will assist the assessment of dystrophin restoration therapies.

Myostatin inhibition in combination with antisense oligonucleotide therapy improves outcomes in spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is caused by genetic defects in the survival motor neuron 1 (SMN1) gene that lead to SMN deficiency. Different SMN-restoring therapies substantially prolong survival and function in transgenic mice of SMA. However, these therapies do not entirely prevent muscle atrophy and restore function completely. To further improve the outcome, we explored the potential of a combinatorial therapy by modulating SMN production and muscle-enhancing approach as a novel therapeutic strategy for SMA. METHODS: The experiments were performed in a mouse model of severe SMA. A previously reported 25-mer morpholino antisense oligomer PMO25 was used to restore SMN expression. The adeno-associated virus-mediated expression of myostatin propeptide was used to block the myostatin pathway. Newborn SMA mice were treated with a single subcutaneous injection of 40 µg/g (therapeutic dose) or 10 µg/g (low-dose) PMO25 on its own or together with systemic delivery of a single dose of adeno-associated virus-mediated expression of myostatin propeptide. The multiple effects of myostatin inhibition on survival, skeletal muscle phenotype, motor function, neuromuscular junction maturation, and proprioceptive afferences were evaluated. RESULTS: We show that myostatin inhibition acts synergistically with SMN-restoring antisense therapy in SMA mice treated with the higher therapeutic dose PMO25 (40 µg/g), by increasing not only body weight (21% increase in male mice at Day 40), muscle mass (38% increase), and fibre size (35% increase in tibialis anterior muscle in 3 month female SMA mice), but also motor function and physical performance as measured in hanging wire test (two-fold increase in time score) and treadmill exercise test (two-fold increase in running distance). In SMA mice treated with low-dose PMO25 (10 µg/g), the early application of myostatin inhibition prolongs survival (40% increase), improves neuromuscular junction maturation (50% increase) and innervation (30% increase), and increases both the size of sensory neurons in dorsal root ganglia (60% increase) and the preservation of proprioceptive synapses in the spinal cord (30% increase). CONCLUSIONS: These data suggest that myostatin inhibition, in addition to the well-known effect on muscle mass, can also positively influence the sensory neural circuits that may enhance motor neurons function. While the availability of the antisense drug Spinraza for SMA and other SMN-enhancing therapies has provided unprecedented improvement in SMA patients, there are still unmet needs in these patients. Our study provides further rationale for considering myostatin inhibitors as a therapeutic intervention in SMA patients, in combination with SMN-restoring drugs.

Myogenic cell proliferation and generation of a reversible tumorigenic phenotype are triggered by preirradiation of the recipient site.

Environmental influences have profound yet reversible effects on the behavior of resident cells. Earlier data have indicated that the amount of muscle formed from implanted myogenic cells is greatly augmented by prior irradiation (18 Gy) of the host mouse muscle. Here we confirm this phenomenon, showing that it varies between host mouse strains. However, it is unclear whether it is due to secretion of proliferative factors or reduction of antiproliferative agents. To investigate this further, we have exploited the observation that the immortal myogenic C2 C12 cell line forms tumors far more rapidly in irradiated than in nonirradiated host muscle. We show that the effect of preirradiation on tumor formation is persistent and dose dependent. However, C2 C12 cells are not irreversibly compelled to form undifferentiated tumor cells by the irradiated muscle environment and are still capable of forming large amounts of muscle when reimplanted into a nonirradiated muscle. In a clonal analysis of this effect, we discovered that C2 C12 cells have a bimodal propensity to form tumors; some clones form no tumors even after extensive periods in irradiated graft sites, whereas others rapidly form extensive tumors. This illustrates the subtle interplay between the phenotype of implanted cells and the factors in the muscle environment.

Publisher Correction: Necroptosis mediates myofibre death in dystrophin-deficient mice.

The original version of this article contained an error in Fig. 3. In panel c, the labels 'mdx' and 'mdx Ripk3-/-' were inadvertently inverted. This has now been corrected in the PDF and HTML versions of the Article.