Patient Myoblasts Research Articles

Functional benefit of CRISPR-Cas9-induced allele deletion for RYR1 dominant mutation

More than 700 pathogenic or probably pathogenic variations have been identified in the RYR1 gene causing various myopathies collectively known as "RYR1-related myopathies”. There is no treatment for these myopathies, and gene therapy stands out as one of the most promising approaches. In the context of a dominant form of Central Core Disease due to a RYR1 mutation, we aimed at showing the functional benefit of inactivating specifically the mutated RYR1 allele by guiding CRISPR/Cas9 cleavages onto frequent single nucleotide polymorphisms (SNPs) segregating on the same chromosome. Whole-genome sequencing was used to pinpoint SNPs localized on the mutant RYR1 allele and identified specific CRISPR/Cas9 guide-RNAs. Lentiviruses encoding these guide-RNAs and the SpCas9 nuclease were used to transduce immortalized patient myoblasts, inducing the specific deletion of the mutant RYR1 allele. The efficiency of the deletion was assessed at DNA and RNA levels, and at the functional level after monitoring calcium release induced by the stimulation of the RyR1-channel. This study provides in-cellulo proof of concept regarding the benefits of mutant RYR1 allele deletion, in the case of a dominant RYR1 mutation, from both a molecular and functional perspective, and could apply potentially to 20% of all patients with a RYR1 mutation.

Enhancing Sarcolemmal Membrane Repair Using a Novel Protein Therapeutic Approach can Improve Membrane Integrity in Duchenne Muscular Dystrophy Cells and Mouse Models

Duchenne Muscular Dystrophy (DMD) is a fatal neuromuscular disease involving progressive cardiac and skeletal myocyte cell death and subsequent muscle degeneration. DMD and Becker muscular dystrophy (BMD) result from mutations in the dystrophin gene that compromise the structural integrity of the sarcolemma. While there has been important recent progress in the treatment of DMD, the currently available therapeutics have significant limitations. Thus, novel therapies that address the compromised sarcolemmal membrane integrity are an unmet medical need. Tripartite motif protein 72, or mitsugumin 53 (TRIM72/MG53), facilitates the sarcolemma repair response after disruption of the membrane. Loss of TRIM72/MG53 function in mice leads to a progressive myopathy. Exogenous delivery of recombinant human MG53 protein (rhMG53) can increase sarcolemmal membrane repair in many different cell types and ameliorate pathology in models of DMD and multiple limb girdle muscular dystrophies. Despite the promise of rhMG53 as a therapeutic invention the development of this protein to treat DMD has been complicated by findings showing that high levels of the protein correlate with metabolic dysfunction in animal models and some human patients. Deletion analysis of the major domains of the rhMG53 protein allowed us to design an improved version of rhMG53 called MyoTRIM. MyoTRIM contains amino acid replacements to eliminate metabolic effects and improve solubility while maintaining membrane repair effects. One set of modifications inactivate the E3 ligase activity of the protein, which revealed this function is dispensable for subcellular localization of the protein. TRIM72/MG53 is reported to mediate this effect by binding phosphatidylserine (PS) at membrane injury sites, so we demonstrate that MyoTRIM can bind PS-coated beads to illustrate that carboxy-terminal domains are required for effcient PS binding rather than the E3 ligase function. External application of MyoTRIM can increase membrane repair capacity in a variety of cell-based assays using DMD and BMD patient myoblasts and myotubes. We also establish that MyoTRIM can increase membrane repair and prevent eccentric contraction induced injury in a dystrophin deficient mouse model. Taken together, these results provide mechanistic insight into rhMG53 function and indicate that MyoTRIM can recapitulate the therapeutic effects on sarcolemmal membrane repair while eliminating E3 ligase activity and associated side effects. This work was supported by the Offce of the Assistant Secretary of Defense for Health Affairs through the Duchenne Muscular Dystrophy Research Program (DMDRP) under Translational Research Award HT9425-23-1-0940. The opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differentiation shifts from a reversible to an irreversible heterochromatin state at the DM1 locus

Epigenetic defects caused by hereditary or de novo mutations are implicated in various human diseases. It remains uncertain whether correcting the underlying mutation can reverse these defects in patient cells. Here we show by the analysis of myotonic dystrophy type 1 (DM1)-related locus that in mutant human embryonic stem cells (hESCs), DNA methylation and H3K9me3 enrichments are completely abolished by repeat excision (CTG2000 expansion), whereas in patient myoblasts (CTG2600 expansion), repeat deletion fails to do so. This distinction between undifferentiated and differentiated cells arises during cell differentiation, and can be reversed by reprogramming of gene-edited myoblasts. We demonstrate that abnormal methylation in DM1 is distinctively maintained in the undifferentiated state by the activity of the de novo DNMTs (DNMT3b in tandem with DNMT3a). Overall, the findings highlight a crucial difference in heterochromatin maintenance between undifferentiated (sequence-dependent) and differentiated (sequence-independent) cells, thus underscoring the role of differentiation as a locking mechanism for repressive epigenetic modifications at the DM1 locus.

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.

Dystrophin Expressing Chimeric (DEC) Cell Therapy for Duchenne Muscular Dystrophy: A First-in-Human Study with Minimum 6 Months Follow-up.

Duchenne Muscular Dystrophy (DMD) is a X-linked progressive lethal muscle wasting disease for which there is no cure. We present first-in-human study assessing safety and efficacy of novel Dystrophin Expressing Chimeric (DEC) cell therapy created by fusion of patient myoblasts with myoblasts of normal donor origin. We report here on safety and functional outcomes of the first 3DMD patients. No study related adverse events (AE) and no serious adverse events (SAE) were observed up to 14months after systemic-intraosseous administration of DEC01. Ambulatory patients showed improvements in functional tests (6-Minute Walk Test (6MWT), North Star Ambulatory Assessment (NSAA)) and both, ambulatory and non-ambulatory in PUL, strength and fatigue resistance which correlated with improvement of Electromyography (EMG) parameters. DEC01 therapy does not require immunosuppression, involves no risks of off target mutations, is not dependent upon the causative mutation and is therefore a universal therapy that does not use viral vectors and therefore can be readministered, if needed. This study was approved by the Bioethics Committee (approvalNo. 46/2019). Mechanism of action of the Dystrophin Expressing Chimeric Cell (DEC) cells created via ex vivo fusion of human myoblast from normal and DMD-affected donors. Following systemic-intraosseous administration, DEC engraft and fuse with the myoblasts of DMD patients, deliver dystrophin and improve muscle strength and function. (Created with BioRender.com).

P.146 Characterising the molecular consequences of LMNA-related congenital muscular dystrophy in patient myoblasts

P.146 Characterising the molecular consequences of LMNA-related congenital muscular dystrophy in patient myoblasts

Key Facilitator Proteins that Mediate Sarcolemma Membrane Repair have Potential as Therapeutics for Muscle Disease and Injury

A common aspect of many diseases and traumatic injuries affecting skeletal muscle is the necrotic death of muscle fibers. Necrotic death of muscle fibers involves the breakdown of the sarcolemmal membrane that can be exacerbated by increased fragility of the membrane or by compromised endogenous sarcolemmal membrane repair processes. Increasing the efficacy of these repair mechanisms could act as a therapeutic approach for several muscular diseases and injuries. Limb‐girdle muscular dystrophy 2B (LGMD2B), a subtype of LGMD, is an autosomal recessive neuromuscular disorder caused by mutations in the DYSF gene that encodes dysferlin, a protein that is vital in membrane repair. The dysferlin protein has been previously shown to facilitate membrane repair in skeletal muscle and knockout mice for dysferlin develop progressive muscular dystrophy. When dysferlin is absent or inactivated through mutation, membrane damage that cannot be repaired accumulates until muscle fibers undergo necrosis that eventually overwhelms the regenerative capacity of the muscle. This causes skeletal muscle deterioration in LGMD2B patients, particularly in the pelvic and shoulder girdle muscles. There is currently no cure for LGMD2B and treatments that stimulate the membrane repair process in muscle fibers have been greatly overlooked and underutilized as a potential therapeutic approach. This project investigates a novel membrane repair signaling cascade in skeletal cells by utilizing SC79, an Akt activator, to increase membrane repair capacity in skeletal muscle. We hypothesize that activation of the phosphoinositide‐3 kinase (PI3K)/Akt1 signaling axis regulates membrane repair in cultured muscle cells and mouse tissue. Based on our data, we find that SC79 injection leads to decreased sarcolemmal membrane injury during treadmill exercises where injection of SC79 decreases the entry of Evans blue dye into muscle fibers. We determined that various doses of SC79 can increase membrane repair in cultured muscle cells and Bla/J dysferlin deficient mice. In these studies, multi‐photon infrared laser microscopy was used to damage the cell membrane of myoblasts, transdifferentiated from skin fibroblasts isolated from LGMD2B patients, in the presence of FM4‐64 dye, a lipophilic dye that fluoresces when it enters the cells and binds to the phospholipids of the cell membrane and intracellular organelles. The extent of localized FM4‐64 dye fluorescence provides a measurement of how well the membrane is repaired. We found SC79 could increase membrane repair in these human patient myoblasts in a dose dependent fashion. This assay was also used on isolated complete muscles from the Bla/J dysferlin deficient mice, where SC79 also increased membrane repair responses. We conclude that activation of the PI3K/Akt1 signaling axis increases membrane repair in dysferlin deficient skeletal muscle. This project explores a novel signaling cascade controlling membrane repair that could be leveraged to develop new therapies for muscle disease and injury.

Transiently expressed CRISPR/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications

Among the mutations arising in the DMD gene and causing Duchenne Muscular Dystrophy (DMD), 10–15% are multi-exon duplications. There are no current therapeutic approaches with the ability to excise large multi-exon duplications, leaving this patient cohort without mutation-specific treatment. Using CRISPR/Cas9 could provide a valid alternative to achieve targeted excision of genomic duplications of any size. Here we show that the expression of a single CRISPR/Cas9 nuclease targeting a genomic region within a DMD duplication can restore the production of wild-type dystrophin in vitro. We assessed the extent of dystrophin repair following both constitutive and transient nuclease expression by either transducing DMD patient-derived myoblasts with integrating lentiviral vectors or electroporating them with CRISPR/Cas9 expressing plasmids. Comparing genomic, transcript and protein data, we observed that both continuous and transient nuclease expression resulted in approximately 50% dystrophin protein restoration in treated myoblasts. Our data demonstrate that a high transient expression profile of Cas9 circumvents its requirement of continuous expression within the cell for targeting DMD duplications. This proof-of-concept study therefore helps progress towards a clinically relevant gene editing strategy for in vivo dystrophin restoration, by highlighting important considerations for optimizing future therapeutic approaches.

Inhibition of Postn Rescues Myogenesis Defects in Myotonic Dystrophy Type 1 Myoblast Model.

Myotonic dystrophy type 1 (DM1) is an inherited neuromuscular disease caused by expanded CTG repeats in the 3′ untranslated region (3′UTR) of the DMPK gene. The myogenesis process is defective in DM1, which is closely associated with progressive muscle weakness and wasting. Despite many proposed explanations for the myogenesis defects in DM1, the underlying mechanism and the involvement of the extracellular microenvironment remained unknown. Here, we constructed a DM1 myoblast cell model and reproduced the myogenesis defects. By RNA sequencing (RNA-seq), we discovered that periostin (Postn) was the most significantly upregulated gene in DM1 myogenesis compared with normal controls. This difference in Postn was confirmed by real-time quantitative PCR (RT-qPCR) and western blotting. Moreover, Postn was found to be significantly upregulated in skeletal muscle and myoblasts of DM1 patients. Next, we knocked down Postn using a short hairpin RNA (shRNA) in DM1 myoblast cells and found that the myogenesis defects in the DM1 group were successfully rescued, as evidenced by increases in the myotube area, the fusion index, and the expression of myogenesis regulatory genes. Similarly, Postn knockdown in normal myoblast cells enhanced myogenesis. As POSTN is a secreted protein, we treated the DM1 myoblast cells with a POSTN-neutralizing antibody and found that DM1 myogenesis defects were successfully rescued by POSTN neutralization. We also tested the myogenic ability of myoblasts in the skeletal muscle injury mouse model and found that Postn knockdown improved the myogenic ability of DM1 myoblasts. The activity of the TGF-β/Smad3 pathway was upregulated during DM1 myogenesis but repressed when inhibiting Postn with a Postn shRNA or a POSTN-neutralizing antibody, which suggested that the TGF-β/Smad3 pathway might mediate the function of Postn in DM1 myogenesis. These results suggest that Postn is a potential therapeutical target for the treatment of myogenesis defects in DM1.

Nanopattern surface improves cultured human myotube maturation

BackgroundIn vitro maturation of human primary myoblasts using 2D culture remains a challenging process and leads to immature fibers with poor internal organization and function. This would however represent a valuable system to study muscle physiology or pathophysiology from patient myoblasts, at a single-cell level.MethodsHuman primary myoblasts were cultured on 800-nm wide striated surface between two layers of Matrigel, and in a media supplemented with an inhibitor of TGFβ receptor. Gene expression, immunofluorescence, and Ca2+ measurements upon electrical stimulations were performed at various time points during maturation to assess the organization and function of the myotubes.ResultsWe show that after 10 days in culture, myotubes display numerous functional acetylcholine receptor clusters and express the adult isoforms of myosin heavy chain and dihydropyridine receptor. In addition, the myotubes are internally well organized with striations of α-actinin and STIM1, and occasionally ryanodine receptor 1. We also demonstrate that the myotubes present robust Ca2+ responses to repetitive electrical stimulations.ConclusionThe present method describes a fast and efficient system to obtain well matured and functional myotubes in 2D culture allowing thorough analysis of single-cell Ca2+ signals.

Drug Repurposing Patent Applications October-December 2020.

Drug Repurposing Patent Applications October-December 2020.

Estradiol differentially regulates DUX4, β-catenin and PAX3/PAX7 in primary myoblasts of facioscapulohumeral muscular dystrophy patients

Abstract Objectives There is a clinical variability and heterogeneity among Facioscapulohumeral Muscular Dystrophy (FSHD) patients. Escalation after menopause in women, early onset in men suggests that estrogen might be a protective factor on the course of FSHD. In spite of few molecular studies supporting the protective role of estrogen in FSHD in vitro, there is no study revealing the effect of estradiol on the protein levels of DUX4, β-catenin and PAX3/PAX7. In present study, we investigated the effect of estradiol treatment on the expressions of DUX4, β-catenin and PAX3/PAX7 protein levels. Materials and Methods Primary myoblasts of 63 and 71 years old (63yM/71yM) males; 47 years old (47yF) female FSHD patients were used. Cells were processed under these conditions; (i) untreated, (ii) 10 nM-30 min estradiol and (iii) 10 nM-4 h estradiol treated. The expression of DUX4, PAX3/PAX7 and β-catenin were examined by western-blotting. Results Expression of DUX4 significantly downregulated after 4 h treatment of estradiol while PAX3/PAX7 56 kDa variant expression upregulated in 71yM cells. β-catenin and PAX3 expression was variable among the samples. Conclusion Our results suggest that estrogen might be a palliative treatment option via downregulation of DUX4 protein in DUX4 expressing FSHD patients.

Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5

Sporadic inclusion body myositis (sIBM) is the most common idiopathic inflammatory myopathy, and several reports have suggested that mitochondrial abnormalities are involved in its etiology. We recruited 9 sIBM patients and found significant histological changes and an elevation of growth differential factor 15 (GDF15), a marker of mitochondrial disease, strongly suggesting the involvement of mitochondrial dysfunction. Bioenergetic analysis of sIBM patient myoblasts revealed impaired mitochondrial function. Decreased ATP production, reduced mitochondrial size and reduced mitochondrial dynamics were also observed in sIBM myoblasts. Cell vulnerability to oxidative stress also suggested the existence of mitochondrial dysfunction. Mitochonic acid-5 (MA-5) increased the cellular ATP level, reduced mitochondrial ROS, and provided protection against sIBM myoblast death. MA-5 also improved the survival of sIBM skin fibroblasts as well as mitochondrial morphology and dynamics in these cells. The reduction in the gene expression levels of Opa1 and Drp1 was also reversed by MA-5, suggesting the modification of the fusion/fission process. These data suggest that MA-5 may provide an alternative therapeutic strategy for treating not only mitochondrial diseases but also sIBM.

Palmitate but Not Oleate Exerts a Negative Effect on Oxygen Utilization in Myoblasts of Patients with the m.3243A>G Mutation: A Pilot Study

It is known that exposure to excess saturated fatty acids, especially palmitate, can trigger cellular stress responses interpreted as lipotoxicity. The effect of excessive free fatty acids on oxidative phosphorylation capacity in myoblasts of patients with the m.3243A>G mutation was evaluated with the mitochondrial (Mito) stress test using a Seahorse XF96 analyzer. ß-oxidation, measured with the Seahorse XF96 analyzer, was similar in patients and controls, and reduced in both patients and controls at 40 °C compared to 37 °C. Mito stress test in the absence of fatty acids showed lower values in patients compared to controls. The mitochondrial activity and ATP production rates were significantly reduced in presence of palmitate, but not of oleate in patients, showing a negative effect of excessive palmitate on mitochondrial function in patients. Diabetes mellitus is a frequent symptom in patients with m.3243A>G mutation. It can be speculated that the negative effect of palmitate on mitochondrial function might be related to diacylglycerols (DAG) and ceramides (CER) mediated insulin resistance. This might contribute to the elevated risk for diabetes mellitus in m.3243A>G patients.

The Effect of Resveratrol on Mitochondrial Function in Myoblasts of Patients with the Common m.3243A>G Mutation.

Mitochondrial function is essential for ATP-supply, especially in response to different cellular stressors. Increased mitochondrial biogenesis resulting from caloric restriction (CR) has been reported. Resveratrol (RSV) is believed to mimic the physiological effects of CR mainly via a sirtuin (SIRT) 1-dependent pathway. The effect of RSV on the physiological function of mitochondrial respiratory complexes was evaluated using a Seahorse XF96. Myoblasts of five patients harboring the m.3243A>G mutation and five controls were analyzed. The relative expression of several genes involved in mitochondrial biogenesis was evaluated for a better understanding of the coherent mechanisms. Additionally, media-dependent effects of nutritional compounds and hormonal restrictions (R) on myoblasts from patients and controls in the presence or absence of RSV were investigated. Culturing of myoblasts under these conditions led to an upregulation of almost all the investigated genes compared to normal nutrition. Under normal conditions, there was no positive effect of RSV on mitochondrial respiration in patients and controls. However, under restricted conditions, the respiratory factors measured by Seahorse were improved in the presence of RSV. Further studies are necessary to clarify the involved mechanisms and elucidate the controversial effects of resveratrol on SIRT1 and SIRT3 expression.

Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei.

FSHD is characterized by the misexpression of DUX4 in skeletal muscle. Although DUX4 upregulation is thought to be the pathogenic cause of FSHD, DUX4 is lowly expressed in patient samples, and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. To better understand the native expression profile of DUX4 and its targets, we performed bulk RNA-seq on a 6-day differentiation time-course in primary FSHD2 patient myoblasts. We identify a set of 54 genes upregulated in FSHD2 cells, termed FSHD-induced genes. Using single-cell and single-nucleus RNA-seq on myoblasts and differentiated myotubes, respectively, we captured, for the first time, DUX4 expressed at the single-nucleus level in a native state. We identified two populations of FSHD myotube nuclei based on low or high enrichment of DUX4 and FSHD-induced genes (“FSHD-Lo” and “FSHD Hi”, respectively). FSHD-Hi myotube nuclei coexpress multiple DUX4 target genes including DUXA, LEUTX and ZSCAN4, and also upregulate cell cycle-related genes with significant enrichment of E2F target genes and p53 signaling activation. We found more FSHD-Hi nuclei than DUX4-positive nuclei, and confirmed with in situ RNA/protein detection that DUX4 transcribed in only one or two nuclei is sufficient for DUX4 protein to activate target genes across multiple nuclei within the same myotube. DUXA (the DUX4 paralog) is more widely expressed than DUX4, and depletion of DUXA suppressed the expression of LEUTX and ZSCAN4 in late, but not early, differentiation. The results suggest that the DUXA can take over the role of DUX4 to maintain target gene expression. These results provide a possible explanation as to why it is easier to detect DUX4 target genes than DUX4 itself in patient cells and raise the possibility of a self-sustaining network of gene dysregulation triggered by the limited DUX4 expression.

G-quadruplex ligands mediate downregulation of DUX4 expression.

Abnormal DUX4 expression in skeletal muscles plays a key role in facioscapulohumeral muscular dystrophy (FSHD) pathogenesis, although the molecular mechanisms regulating DUX4 expression are not fully defined. Using bioinformatic analysis of the genomic DUX4 locus, we have identified a number of putative G-quadruplexes (GQs) forming sequences. Their presence was confirmed in synthetic oligonucleotiode sequences derived from the enhancer, promoter and transcript of DUX4 through circular dichroism and nuclear magnetic resonance analysis. We further examined the binding affinity of a naturally occurring GQ stabilizing compound, berberine, to these non-canonical genetic structures using UV–Vis and fluorescence spectroscopy. Subsequent in vitro study in FSHD patient myoblasts indicated that berberine treatment reduced DUX4 expression and also expression of genes normally switched on by DUX4. Further investigation in a mouse model overexpressing exogenous DUX4 confirmed the therapeutic effects of berberine in downregulating DUX4 protein expression, inhibiting muscle fibrosis, and consequently rescuing muscle function. Our data demonstrate for the first time that GQs are present in the DUX4 locus and that the GQ interactive ligand reduces DUX4 expression suggesting potential role of GQs in FSHD pathogenesis. Our work provides the basis of a novel therapeutic strategy for the treatment of FSHD.

Sporadic Inclusion Body Myositis Underling Mitochondrial Dysfunction Ameliorated by Mitochondrial Homing Drug, MA-5

Background: Sporadic inclusion body myositis (sIBM) is the most common idiopathic inflammatory myopathy, and mitochondrial abnormalities may be involved. Methods: We recruited 9 sIBM patients and we examined mitochondrial functions with bioenergetic and examinations and dynamics of patient myoblasts with measuring a mitochondrial disease marker growth differential factor 15 (GDF15). We also examined the effect of a new candidate drug, Mitochondria acid-5 (MA-5). Findings: Bioenergetic analysis of sIBM patient myoblasts revealed damaged mitochondrial function with decreased ATP, mitochondrial size and impaired mitochondrial dynamics and cell vulnerability. MA-5 increased the ATP level and reduced mitochondrial ROS, following the protection against cell death. The reduced levels of Opa1 and Drp1 showed the impairment of mitochondrial fusion/fission process and that MA-5 ameliorated. Skin fibroblasts from sIBM patients can be used for diagnosis. Interpretation: GDF15 and MA-5 could provide both a new maker and therapeutic strategy for sIBM patients. Funding Statement: These works were supported in part by National grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (18H02822), Translational Research Network Program (C50) from Japan Agency for Medical Research and Development (AMED). MEXT and AMED, JAPAN. Declaration of Interests: The authors state: None. Ethics Approval Statement: Myoblasts and fibroblasts obtained by muscle and skin biopsy of sIBM patients were collected in Tohoku University Hospital under the approval of the Ethical Committee of Tohoku University.

Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells

Autosomal recessive mutations in Anoctamin 5 (ANO5/TMEM16E), a member of the transmembrane 16 (TMEM16) family of Ca2+-activated ion channels and phospholipid scramblases, cause adult-onset muscular dystrophies (limb girdle muscular dystrophy 2L (LGMD2L) and Miyoshi Muscular Dystrophy (MMD3). However, the molecular role of ANO5 is unclear and ANO5 knockout mouse models show conflicting requirements of ANO5 in muscle. To study the role of ANO5 in human muscle cells we generated a myoblast line from a MMD3-patient carrying the c.2272C>T mutation, which we find causes the mutant protein to be degraded. The patient myoblasts exhibit normal myogenesis, but are compromised in their plasma membrane repair (PMR) ability. The repair deficit is linked to the poor ability of the endoplasmic reticulum (ER) to clear cytosolic Ca2+ increase caused by focal plasma membrane injury. Expression of wild-type ANO5 or pharmacological prevention of injury-triggered cytosolic Ca2+ overload enable injured patient muscle cells to repair. A homology model of ANO5 shows that several of the known LGMD2L/MMD3 patient mutations line the transmembrane region of the protein implicated in its channel activity. These results point to a role of cytosolic Ca2+ homeostasis in PMR, indicate a role for ANO5 in ER-mediated cytosolic Ca2+ uptake and identify normalization of cytosolic Ca2+ homeostasis as a potential therapeutic approach to treat muscular dystrophies caused by ANO5 deficit.

Dynamic transcriptomic analysis reveals suppression of PGC1α/ERRα drives perturbed myogenesis in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy, linked to epigenetic derepression of D4Z4 repeats on chromosome 4q, leading to ectopic DUX4 expression. FSHD patient myoblasts have defective myogenic differentiation, forming smaller myotubes with reduced myosin content. However, molecular mechanisms driving such disrupted myogenesis in FSHD are poorly understood. We performed high-throughput morphological analysis describing FSHD and control myogenesis, revealing altered myogenic differentiation results in hypotrophic myotubes. Employing polynomial models and an empirical Bayes approach, we established eight critical time points during which human healthy and FSHD myogenesis differ. RNA-sequencing at these eight nodal time points in triplicate, provided temporal depth for a multivariate regression analysis, allowing assessment of interaction between progression of differentiation and FSHD disease status. Importantly, the unique size and structure of our data permitted identification of many novel FSHD pathomechanisms undetectable by previous approaches. For further analysis here, we selected pathways that control mitochondria: of interest considering known alterations in mitochondrial structure and function in FSHD muscle, and sensitivity of FSHD cells to oxidative stress. Notably, we identified suppression of mitochondrial biogenesis, in particular via peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), the cofactor and activator of oestrogen-related receptor α (ERRα). PGC1α knock-down caused hypotrophic myotubes to form from control myoblasts. Known ERRα agonists and safe food supplements biochanin A, daidzein or genistein, each rescued the hypotrophic FSHD myotube phenotype. Together our work describes transcriptomic changes in high resolution that occur during myogenesis in FSHD ex vivo, identifying suppression of the PGC1α-ERRα axis leading to perturbed myogenic differentiation, which can effectively be rescued by readily available food supplements.