Identification of a muscle-specific isoform of VMA21 as a potent actor in X-linked myopathy with excessive autophagy pathogenesis.

Defective lysosomal acidification is responsible for a large range of multi-systemic disorders associated with impaired autophagy. Diseases caused by mutations in the VMA21 gene stand as exceptions, specifically affecting skeletal muscle (X-linked Myopathy with Excessive Autophagy, XMEA) or liver (Congenital Disorder of Glycosylation). VMA21 chaperones vacuolar (v-) ATPase assembly, which is ubiquitously required for proper lysosomal acidification. The reason VMA21 deficiencies affect specific, but divergent tissues remains unknown. Here, we show that VMA21 encodes a yet-unreported long protein isoform, in addition to the previously described short isoform, which we name VMA21-120 and VMA21-101, respectively. In contrast to the ubiquitous pattern of VMA21-101, VMA21-120 was predominantly expressed in skeletal muscle, and rapidly up-regulated upon differentiation of mouse and human muscle precursors. Accordingly, VMA21-120 accumulated during development, regeneration and denervation of mouse skeletal muscle. In contrast, neither induction nor blockade of autophagy, in vitro and in vivo, strongly affected VMA21 isoform expression. Interestingly, VMA21-101 and VMA21-120 both localized to the sarcoplasmic reticulum of muscle cells, and interacted with the v-ATPase. While VMA21 deficiency impairs autophagy, VMA21-101 or VMA21-120 overexpression had limited impact on autophagic flux in muscle cells. Importantly, XMEA-associated mutations lead to both VMA21-101 deficiency and loss of VMA21-120 expression. These results provide important insights into the clinical diversity of VMA21-related diseases and uncover a muscle-specific VMA21 isoform that potently contributes to XMEA pathogenesis.

Cellular and Genomic Features of Muscle Differentiation from Isogenic Fibroblasts and Myoblasts.

The ability to recapitulate muscle differentiation in vitro enables the exploration of mechanisms underlying myogenesis and muscle diseases. However, obtaining myoblasts from patients with neuromuscular diseases or from healthy subjects poses ethical and procedural challenges that limit such investigations. An alternative consists in converting skin fibroblasts into myogenic cells by forcing the expression of the myogenic regulator MYOD. Here, we directly compared cellular phenotype, transcriptome, and nuclear lamina-associated domains (LADs) in myo-converted human fibroblasts and myotubes differentiated from myoblasts. We used isogenic cells from a 16-year-old donor, ruling out, for the first time to our knowledge, genetic factors as a source of variations between the two myogenic models. We show that myo-conversion of fibroblasts upregulates genes controlling myogenic pathways leading to multinucleated cells expressing muscle cell markers. However, myotubes are more advanced in myogenesis than myo-converted fibroblasts at the phenotypic and transcriptomic levels. While most LADs are shared between the two cell types, each also displays unique domains of lamin A/C interactions. Furthermore, myotube-specific LADs are more gene-rich and less heterochromatic than shared LADs or LADs unique to myo-converted fibroblasts, and they uniquely sequester developmental genes. Thus, myo-converted fibroblasts and myotubes retain cell type-specific features of radial and functional genome organization. Our results favor a view of myo-converted fibroblasts as a practical model to investigate the phenotypic and genomic properties of muscle cell differentiation in normal and pathological contexts, but also highlight current limitations in using fibroblasts as a source of myogenic cells.

Prime editing strategies to mediate exon skipping in DMD gene.

Duchenne muscular dystrophy is a rare and lethal hereditary disease responsible for progressive muscle wasting due to mutations in the DMD gene. We used the CRISPR-Cas9 Prime editing technology to develop different strategies to correct frameshift mutations in DMD gene carrying the deletion of exon 52 or exons 45 to 52. With optimized epegRNAs, we were able to induce the specific substitution of the GT nucleotides of the splice donor site of exon 53 in up to 32% of HEK293T cells and 28% of patient myoblasts. We also achieved up to 44% and 29% deletion of the G nucleotide of the GT splice site of exon 53, as well as inserted 17% and 5.5% GGG between the GT splice donor site of exon 51 in HEK293T cells and human myoblasts, respectively. The modification of the splice donor site for exon 51 and exon 53 provoke their skipping and allowed exon 50 to connect to exon 53 and allowed exon 44 to connect to exon 54, respectively. These corrections restored the expression of dystrophin as demonstrated by western blot. Thus, Prime editing was used to induce specific substitutions, insertions and deletions in the splice donor sites for exons 51 and 53 to correct the frameshift mutations in DMD gene carrying deletions of exon 52 and exons 45 to 52, respectively.

High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cells.

Genome editing based on dual CRISPR-Cas9 complexes (multiplexes) permits removing specific genomic sequences in living cells leveraging research on functional genomics and genetic therapies. Delivering the required large and multicomponent reagents in a synchronous and stoichiometric manner remains, however, challenging. Moreover, uncoordinated activity of independently acting CRISPR-Cas9 multiplexes increases the complexity of genome editing outcomes. Here, we investigate the potential of fostering precise multiplexing genome editing using high-capacity adenovector particles (AdVPs) for the delivery of Cas9 ortholog fusion constructs alone (forced Cas9 heterodimers) or together with their cognate guide RNAs (forced CRISPR-Cas9 heterodimers). We demonstrate that the efficiency and accuracy of targeted chromosomal DNA deletions achieved by single AdVPs encoding forced CRISPR-Cas9 heterodimers is superior to that obtained when the various components are delivered separately. Finally, all-in-one AdVP delivery of forced CRISPR-Cas9 heterodimers triggers robust DMD exon 51 splice site excision resulting in reading frame restoration and selection-free detection of dystrophin in muscle cells derived from Duchenne muscular dystrophy patients. In conclusion, AdVPs promote precise multiplexing genome editing through the integrated delivery of forced CRISPR-Cas9 heterodimer components, which, in comparison with split conventional CRISPR-Cas9 multiplexes, engage target sequences in a more coordinated fashion.

Aberrant Adenosine Triphosphate Release and Impairment of P2Y2-Mediated Signaling in Sarcoglycanopathies.

Sarcoglycanopathies, limb-girdle muscular dystrophies (LGMD) caused by genetic loss-of-function of the membrane proteins sarcoglycans (SGs), are characterized by progressive degeneration of skeletal muscle. In these disorders, muscle necrosis is associated with immune-mediated damage, whose triggering and perpetuating molecular mechanisms are not fully elucidated yet. Extracellular adenosine triphosphate (eATP) seems to represent a crucial factor, with eATP activating purinergic receptors. Indeed, in vivo blockade of the eATP/P2X7 purinergic pathway ameliorated muscle disease progression. P2X7 inhibition improved the dystrophic process by restraining the activity of P2X7 receptors on immune cells. Whether P2X7 blockade can display a direct action on muscle cells is not known yet. In this study, we investigated eATP effects in primary cultures of myoblasts isolated from patients with LGMDR3 (α-sarcoglycanopathy) and in immortalized cells isolated from a patient with LGMDR5 (γ-sarcoglycanopathy). Our results demonstrated that, owing to a reduced ecto-ATPase activity and/or an enhanced release of ATP, patient cells are exposed to increased juxtamembrane concentrations of eATP and display a higher susceptivity to eATP signals. The purinoceptor P2Y2, which proved to be overexpressed in patient cells, was identified as a pivotal receptor responsible for the enhanced ATP-induced or UTP-induced Ca2+ increase in affected myoblasts. Moreover, P2Y2 stimulation in LDMDR3 muscle cells induced chemotaxis of immune cells and release of interleukin-8. In conclusion, a higher eATP concentration and sensitivity in primary human muscle cells carrying different α-SG or γ-SG loss-of-function mutations indicate that eATP/P2Y2 is an enhanced signaling axis in cells from patients with α-/γ-sarcoglycanopathy. Understanding the basis of the innate immune-mediated damage associated with the dystrophic process may be critical in overcoming the immunologic hurdles associated with emerging gene therapies for these disorders.

Endosomal lipid signaling reshapes the endoplasmic reticulum to control mitochondrial function.

Cells respond to fluctuating nutrient supply by adaptive changes in organelle dynamics and in metabolism. How such changes are orchestrated on a cell-wide scale is unknown. We show that endosomal signaling lipid turnover by MTM1, a phosphatidylinositol 3-phosphate [PI(3)P] 3-phosphatase mutated in X-linked centronuclear myopathy in humans, controls mitochondrial morphology and function by reshaping the endoplasmic reticulum (ER). Starvation-induced endosomal recruitment of MTM1 impairs PI(3)P-dependent contact formation between tubular ER membranes and early endosomes, resulting in the conversion of ER tubules into sheets, the inhibition of mitochondrial fission, and sustained oxidative metabolism. Our results unravel an important role for early endosomal lipid signaling in controlling ER shape and, thereby, mitochondrial form and function to enable cells to adapt to fluctuating nutrient environments.

Prime editing optimized RTT permits the correction of the c.8713C>T mutation in DMD gene.

Duchenne muscular dystrophy is a severe debilitating genetic disease caused by different mutations in the DMD gene leading to the absence of dystrophin protein under the sarcolemma. We used CRISPR-Cas9 prime editing technology for correction of the c.8713C>T mutation in the DMD gene and tested different variations of reverse transcription template (RTT) sequences. We increased by 3.8-fold the editing percentage of the target nucleotide located at +13. A modification of the protospacer adjacent motif sequence (located at +6) and a silent mutation (located at +9) were also simultaneously added to the target sequence modification. We observed significant differences in editing efficiency in interconversion of different nucleotides and the distance between the target, the nicking site, and the additional mutations. We achieved 22% modifications in myoblasts of a DMD patient, which led to dystrophin expression detected by western blot in the myotubes that they formed. RTT optimization permitted us to improve the prime editing of a point mutation located at +13 nucleotides from the nick site to restore dystrophin protein.

Quantum Dot-Based Screening Identifies F3 Peptide and Reveals Cell Surface Nucleolin as a Therapeutic Target for Rhabdomyosarcoma.

Active drug delivery by tumor-targeting peptides is a promising approach to improve existing therapies for rhabdomyosarcoma (RMS), by increasing the therapeutic effect and decreasing the systemic toxicity, e.g., by drug-loaded peptide-targeted nanoparticles. Here, we tested 20 different tumor-targeting peptides for their ability to bind to two RMS cell lines, Rh30 and RD, using quantum dots Streptavidin and biotin-peptides conjugates as a model for nanoparticles. Four peptides revealed a very strong binding to RMS cells: NCAM-1-targeting NTP peptide, nucleolin-targeting F3 peptide, and two Furin-targeting peptides, TmR and shTmR. F3 peptide showed the strongest binding to all RMS cell lines tested, low binding to normal control myoblasts and fibroblasts, and efficient internalization into RMS cells demonstrated by the cytoplasmic delivery of the Saporin toxin. The expression of the nucleophosphoprotein nucleolin, the target of F3, on the surface of RMS cell lines was validated by competition with the natural ligand lactoferrin, by colocalization with the nucleolin-binding aptamer AS1411, and by the marked sensitivity of RMS cell lines to the growth inhibitory nucleolin-binding N6L pseudopeptide. Taken together, our results indicate that nucleolin-targeting by F3 peptide represents a potential therapeutic approach for RMS.

Brush-Induced Orientation of Collagen Fibers in Layer-by-Layer Nanofilms: A Simple Method for the Development of Human Muscle Fibers.

The engineering of skeletal muscle tissue, a highly organized structure of myotubes, is promising for the treatment of muscle injuries and muscle diseases, for replacement, or for pharmacology research. Muscle tissue development involves differentiation of myoblasts into myotubes with parallel orientation, to ultimately form aligned myofibers, which is challenging to achieve on flat surfaces. In this work, we designed hydrogen-bonded tannic acid/collagen layer-by-layer (TA/COL LbL) nanofilms using a simple brushing method to address this issue. In comparison to films obtained by dipping, brushed TA/COL films showed oriented COL fibers of 60 nm diameter along the brushing direction. Built at acidic pH due to COL solubility, TA/COL films released TA in physiological conditions with a minor loss of thickness. After characterization of COL fibers' orientation, human myoblasts (C25CL48) were seeded on the oriented TA/COL film, ended by COL. After 12 days in a differentiation medium without any other supplement, human myoblasts were able to align on brushed TA/COL films and to differentiate into long aligned myotubes (from hundreds of µm up to 1.7 mm length) thanks to two distinct properties: (i) the orientation of COL fibers guiding myoblasts' alignment and (ii) the TA release favoring the differentiation. This simple and potent brushing process allows the development of anisotropic tissues in vitro which can be used for studies of drug discovery and screening or the replacement of damaged tissue.

Development of versatile allele-specific siRNAs able to silence all the dominant dynamin 2 mutations.

Dominant centronuclear myopathy (CNM) is a rare form of congenital myopathy associated with a wide clinical spectrum, from severe neonatal to milder adult forms. There is no available treatment for this disease due to heterozygous mutations in the DNM2 gene encoding Dynamin 2 (DNM2). Dominant DNM2 mutations also cause rare forms of Charcot-Marie-Tooth disease and hereditary spastic paraplegia, and deleterious DNM2 overexpression was noticed in several diseases. The proof of concept for therapy by allele-specific RNA interference devoted to silence the mutated mRNA without affecting the normal allele was previously achieved in a mouse model and patient-derived cells, both expressing the most frequent DNM2 mutation in CNM. In order to have versatile small interfering RNAs (siRNAs) usable regardless of the mutation, we have developed allele-specific siRNAs against two non-pathogenic single-nucleotide polymorphisms (SNPs) frequently heterozygous in the population. In addition, allele-specific siRNAs against the p.S619L DNM2 mutation, a mutation frequently associated with severe neonatal cases, were developed. The beneficial effects of these new siRNAs are reported for a panel of defects occurring in patient-derived cell lines. The development of these new molecules allows targeting the large majority of the patients harboring DNM2 mutations or overexpression by only a few siRNAs.

Inactivation of Sirt6 ameliorates muscular dystrophy in mdx mice by releasing suppression of utrophin expression.

The NAD+-dependent SIRT1-7 family of protein deacetylases plays a vital role in various molecular pathways related to stress response, DNA repair, aging and metabolism. Increased activity of individual sirtuins often exerts beneficial effects in pathophysiological conditions whereas reduced activity is usually associated with disease conditions. Here, we demonstrate that SIRT6 deacetylates H3K56ac in myofibers to suppress expression of utrophin, a dystrophin-related protein stabilizing the sarcolemma in absence of dystrophin. Inactivation of Sirt6 in dystrophin-deficient mdx mice reduced damage of myofibers, ameliorated dystrophic muscle pathology, and improved muscle function, leading to attenuated activation of muscle stem cells (MuSCs). ChIP-seq and locus-specific recruitment of SIRT6 using a CRISPR-dCas9/gRNA approach revealed that SIRT6 is critical for removal of H3K56ac at the Downstream utrophin Enhancer (DUE), which is indispensable for utrophin expression. We conclude that epigenetic manipulation of utrophin expression is a promising approach for the treatment of Duchenne Muscular Dystrophy (DMD).

Moxifloxacin rescues SMA phenotypes in patient-derived cells and animal model.

Spinal muscular atrophy (SMA) is a genetic disease resulting in the loss of α-motoneurons followed by muscle atrophy. It is caused by knock-out mutations in the survival of motor neuron 1 (SMN1) gene, which has an unaffected, but due to preferential exon 7 skipping, only partially functional human-specific SMN2 copy. We previously described a Drosophila-based screening of FDA-approved drugs that led us to discover moxifloxacin. We showed its positive effect on the SMN2 exon 7 splicing in SMA patient-derived skin cells and its ability to increase the SMN protein level. Here, we focus on moxifloxacin's therapeutic potential in additional SMA cellular and animal models. We demonstrate that moxifloxacin rescues the SMA-related molecular and phenotypical defects in muscle cells and motoneurons by improving the SMN2 splicing. The consequent increase of SMN levels was higher than in case of risdiplam, a potent exon 7 splicing modifier, and exceeded the threshold necessary for a survival improvement. We also demonstrate that daily subcutaneous injections of moxifloxacin in a severe SMA murine model reduces its characteristic neuroinflammation and increases the SMN levels in various tissues, leading to improved motor skills and extended lifespan. We show that moxifloxacin, originally used as an antibiotic, can be potentially repositioned for the SMA treatment.

Targeting the Ubiquitin-Proteasome System in Limb-Girdle Muscular Dystrophy With CAPN3 Mutations.

LGMDR1 is caused by mutations in the CAPN3 gene that encodes calpain 3 (CAPN3), a non-lysosomal cysteine protease necessary for proper muscle function. Our previous findings show that CAPN3 deficiency leads to reduced SERCA levels through increased protein degradation. This work investigates the potential contribution of the ubiquitin-proteasome pathway to increased SERCA degradation in LGMDR1. Consistent with our previous results, we observed that CAPN3-deficient human myotubes exhibit reduced SERCA protein levels and high cytosolic calcium concentration. Treatment with the proteasome inhibitor bortezomib (Velcade) increased SERCA2 protein levels and normalized intracellular calcium levels in CAPN3-deficient myotubes. Moreover, bortezomib was able to recover mutated CAPN3 protein in a patient carrying R289W and R546L missense mutations. We found that CAPN3 knockout mice (C3KO) presented SERCA deficits in skeletal muscle in the early stages of the disease, prior to the manifestation of muscle deficits. However, treatment with bortezomib (0.8 mg/kg every 72 h) for 3 weeks did not rescue SERCA levels. No change in muscle proteasome activity was observed in bortezomib-treated animals, suggesting that higher bortezomib doses are needed to rescue SERCA levels in this model. Overall, our results lay the foundation for exploring inhibition of the ubiquitin-proteasome as a new therapeutic target to treat LGMDR1 patients. Moreover, patients carrying missense mutations in CAPN3 and presumably other genes may benefit from proteasome inhibition by rescuing mutant protein levels. Further studies in suitable models will be necessary to demonstrate the therapeutic efficacy of proteasome inhibition for different missense mutations.

Interplay between mitochondrial reactive oxygen species, oxidative stress and hypoxic adaptation in facioscapulohumeral muscular dystrophy: Metabolic stress as potential therapeutic target.

Facioscapulohumeral muscular dystrophy (FSHD) is characterised by descending skeletal muscle weakness and wasting. FSHD is caused by mis-expression of the transcription factor DUX4, which is linked to oxidative stress, a condition especially detrimental to skeletal muscle with its high metabolic activity and energy demands. Oxidative damage characterises FSHD and recent work suggests metabolic dysfunction and perturbed hypoxia signalling as novel pathomechanisms. However, redox biology of FSHD remains poorly understood, and integrating the complex dynamics of DUX4-induced metabolic changes is lacking. Here we pinpoint the kinetic involvement of altered mitochondrial ROS metabolism and impaired mitochondrial function in aetiology of oxidative stress in FSHD. Transcriptomic analysis in FSHD muscle biopsies reveals strong enrichment for pathways involved in mitochondrial complex I assembly, nitrogen metabolism, oxidative stress response and hypoxia signalling. We found elevated mitochondrial ROS (mitoROS) levels correlate with increases in steady-state mitochondrial membrane potential in FSHD myogenic cells. DUX4 triggers mitochondrial membrane polarisation prior to oxidative stress generation and apoptosis through mitoROS, and affects mitochondrial health through lipid peroxidation. We identify complex I as the primary target for DUX4-induced mitochondrial dysfunction, with strong correlation between complex I-linked respiration and cellular oxygenation/hypoxia signalling activity in environmental hypoxia. Thus, FSHD myogenesis is uniquely susceptible to hypoxia-induced oxidative stress as a consequence of metabolic mis-adaptation. Importantly, mitochondria-targeted antioxidants rescue FSHD pathology more effectively than conventional antioxidants, highlighting the central involvement of disturbed mitochondrial ROS metabolism. This work provides a pathomechanistic model by which DUX4-induced changes in oxidative metabolism impair muscle function in FSHD, amplified when metabolic adaptation to varying O2 tension is required.

Synthesis and Exon-Skipping Properties of a 3'-Ursodeoxycholic Acid-Conjugated Oligonucleotide Targeting DMD Pre-mRNA: Pre-Synthetic versus Post-Synthetic Approach.

Steric blocking antisense oligonucleotides (ASO) are promising tools for splice modulation such as exon-skipping, although their therapeutic effect may be compromised by insufficient delivery. To address this issue, we investigated the synthesis of a 20-mer 2'-OMe PS oligonucleotide conjugated at 3'-end with ursodeoxycholic acid (UDCA) involved in the targeting of human DMD exon 51, by exploiting both a pre-synthetic and a solution phase approach. The two approaches have been compared. Both strategies successfully provided the desired ASO 51 3'-UDC in good yield and purity. It should be pointed out that the pre-synthetic approach insured better yields and proved to be more cost-effective. The exon skipping efficiency of the conjugated oligonucleotide was evaluated in myogenic cell lines and compared to that of unconjugated one: a better performance was determined for ASO 51 3'-UDC with an average 9.5-fold increase with respect to ASO 51.

Myogenin controls via AKAP6 non-centrosomal microtubule-organizing center formation at the nuclear envelope.

Non-centrosomal microtubule-organizing centers (MTOCs) are pivotal for the function of multiple cell types, but the processes initiating their formation are unknown. Here, we find that the transcription factor myogenin is required in murine myoblasts for the localization of MTOC proteins to the nuclear envelope. Moreover, myogenin is sufficient in fibroblasts for nuclear envelope MTOC (NE-MTOC) formation and centrosome attenuation. Bioinformatics combined with loss- and gain-of-function experiments identified induction of AKAP6 expression as one central mechanism for myogenin-mediated NE-MTOC formation. Promoter studies indicate that myogenin preferentially induces the transcription of muscle- and NE-MTOC-specific isoforms of Akap6 and Syne1, which encodes nesprin-1α, the NE-MTOC anchor protein in muscle cells. Overexpression of AKAP6ß and nesprin-1α was sufficient to recruit endogenous MTOC proteins to the nuclear envelope of myoblasts in the absence of myogenin. Taken together, our results illuminate how mammals transcriptionally control the switch from a centrosomal MTOC to an NE-MTOC and identify AKAP6 as a novel NE-MTOC component in muscle cells.

Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMDJ) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.

Nitric Oxide (NO) and Duchenne Muscular Dystrophy: NO Way to Go?

The discordance between pre-clinical success and clinical failure of treatment options for Duchenne Muscular Dystrophy (DMD) is significant. The termination of clinical trials investigating the phosphodiesterase inhibitors, sildenafil and tadalafil (which prolong the second messenger molecule of nitric oxide (NO) signaling), are prime examples of this. Both attenuated key dystrophic features in the mdx mouse model of DMD yet failed to modulate primary outcomes in clinical settings. We have previously attempted to modulate NO signaling via chronic nitrate supplementation of the mdx mouse but failed to demonstrate beneficial modulation of key dystrophic features (i.e., metabolism). Instead, we observed increased muscle damage and nitrosative stress which exacerbated MD. Here, we highlight that acute nitrite treatment of human DMD myoblasts is also detrimental and suggest strategies for moving forward with NO replacement therapy in DMD.

BETs inhibition attenuates oxidative stress and preserves muscle integrity in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) affects 1 in 3500 live male births. To date, there is no effective cure for DMD, and the identification of novel molecular targets involved in disease progression is important to design more effective treatments and therapies to alleviate DMD symptoms. Here, we show that protein levels of the Bromodomain and extra-terminal domain (BET) protein BRD4 are significantly increased in the muscle of the mouse model of DMD, the mdx mouse, and that pharmacological inhibition of the BET proteins has a beneficial outcome, tempering oxidative stress and muscle damage. Alterations in reactive oxygen species (ROS) metabolism are an early event in DMD onset and they are tightly linked to inflammation, fibrosis, and necrosis in skeletal muscle. By restoring ROS metabolism, BET inhibition ameliorates these hallmarks of the dystrophic muscle, translating to a beneficial effect on muscle function. BRD4 direct association to chromatin regulatory regions of the NADPH oxidase subunits increases in the mdx muscle and JQ1 administration reduces BRD4 and BRD2 recruitment at these regions. JQ1 treatment reduces NADPH subunit transcript levels in mdx muscles, isolated myofibers and DMD immortalized myoblasts. Our data highlight novel functions of the BET proteins in dystrophic skeletal muscle and suggest that BET inhibitors may ameliorate the pathophysiology of DMD.

A Novel Bioengineered Functional Motor Unit Platform to Study Neuromuscular Interaction.

BACKGROUND: In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. In vivo studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered in vitro MUs. OBJECTIVE: to develop and characterise a functional MU model in vitro, permitting the analysis of MU development and function. METHODS: an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists. RESULTS: blocking acetylcholine receptors with α-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional. CONCLUSION: a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions.