Expert Panel Curation of 31 Genes in Relation to Limb Girdle Muscular Dystrophy.

Introduction Limb girdle muscular dystrophies (LGMDs) are a group of genetically heterogeneous autosomal conditions with some degree of phenotypic homogeneity. LGMD is defined as having onset >2 years of age with progressive proximal weakness, elevated serum creatine kinase levels and dystrophic features on muscle biopsy. Advances in massively parallel sequencing have led to a surge in genes linked to LGMD. Methods The ClinGen Muscular Dystrophies and Myopathies gene curation expert panel (MDM GCEP, formerly Limb Girdle Muscular Dystrophy GCEP) convened to evaluate the strength of evidence supporting gene-disease relationships (GDR) using the ClinGen gene-disease clinical validity framework to evaluate 31 genes implicated in LGMD. Results The GDR was exclusively LGMD for 17 genes, whereas an additional 14 genes were related to a broader phenotype encompassing congenital weakness. Four genes (CAPN3, COL6A1, COL6A2, COL6A3) were split into two separate disease entities, based on each displaying both dominant and recessive inheritance patterns, resulting in curation of 35 GDRs. Of these, 30 (86%) were classified as Definitive, 4 (11%) as Moderate and 1 (3%) as Limited. Two genes, POMGNT1 and DAG1, though definitively related to myopathy, currently have insufficient evidence to support a relationship specifically with LGMD. Conclusions The expert-reviewed assertions on the clinical validity of genes implicated in LGMDs form an invaluable resource for clinicians and molecular geneticists. We encourage the global neuromuscular community to publish case-level data that help clarify disputed or novel LGMD associations.

The super-healing MRL strain promotes muscle growth in muscular dystrophy through a regenerative extracellular matrix.

The Murphy Roths Large (MRL) mouse strain has "super-healing" properties that enhance recovery from injury. In mice, the DBA/2J strain intensifies many aspects of muscular dystrophy, so we evaluated the ability of the MRL strain to suppress muscular dystrophy in the Sgcg-null mouse model of limb girdle muscular dystrophy. A comparative analysis of Sgcg-null mice in the DBA/2J versus MRL strains showed greater myofiber regeneration, with reduced structural degradation of muscle in the MRL strain. Transcriptomic profiling of dystrophic muscle indicated strain-dependent expression of extracellular matrix (ECM) and TGF-ß signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized myoscaffolds. Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-ß1 and TGF-ß3 throughout the matrix. Dystrophic myoscaffolds from the MRL background, but not the DBA/2J background, were enriched in myokines like IGF-1 and IL-6. C2C12 myoblasts seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J muscles showed the MRL background induced greater myoblast differentiation compared with dystrophic DBA/2J myoscaffolds. Thus, the MRL background imparts its effect through a highly regenerative ECM, which is active even in muscular dystrophy.

Successes and challenges in clinical gene therapy.

Despite the ups and downs in the field over three decades, the science of gene therapy has continued to advance and provide enduring treatments for increasing number of diseases. There are active clinical trials approaching a variety of inherited and acquired disorders of different organ systems. Approaches include ex vivo modification of hematologic stem cells (HSC), T lymphocytes and other immune cells, as well as in vivo delivery of genes or gene editing reagents to the relevant target cells by either local or systemic administration. In this article, we highlight success and ongoing challenges in three areas of high activity in gene therapy: inherited blood cell diseases by targeting hematopoietic stem cells, malignant disorders using immune effector cells genetically modified with chimeric antigen receptors, and ophthalmologic, neurologic, and coagulation disorders using in vivo administration of adeno-associated virus (AAV) vectors. In recent years, there have been true cures for many of these diseases, with sustained clinical benefit that exceed those from other medical approaches. Each of these treatments faces ongoing challenges, namely their high one-time costs and the complexity of manufacturing the therapeutic agents, which are biological viruses and cell products, at pharmacologic standards of quality and consistency. New models of reimbursement are needed to make these innovative treatments widely available to patients in need.

Innate and adaptive AAV-mediated immune responses in a mouse model of Duchenne muscular dystrophy.

High systemic doses of adeno-associated viruses (AAVs) have been associated with immune-related serious adverse events (SAEs). Although AAV was well tolerated in preclinical models, SAEs were observed in clinical trials, indicating the need for improved preclinical models to understand AAV-induced immune responses. Here, we show that mice dual-dosed with AAV9 at 4-week intervals better recapitulate aspects of human immunity to AAV. In the model, anti-AAV9 immunoglobulin G (IgGs) increased in a linear fashion between the first and second AAV administrations. Complement activation was only observed in the presence of high levels of both AAV and anti-AAV IgG. Myeloid-derived pro-inflammatory cytokines were significantly induced in the same pattern as complement activation, suggesting that myeloid cell activation to AAV may rely on the presence of both AAV and anti-AAV IgG complexes. Single-cell RNA sequencing of peripheral blood mononuclear cells confirmed that activated monocytes were a primary source of pro-inflammatory cytokines and chemokines, which were significantly increased after a second AAV9 exposure. The same activated monocyte clusters expressed both Fcγ and complement receptors, suggesting that anti-AAV-mediated activation of myeloid cells through Fcγ receptors and/or complement receptors is one mechanism by which anti-AAV antigen complexes may prime antigen-presenting cells and amplify downstream immunity.

Nanoparticles systemically biodistribute to regenerating skeletal muscle in DMD.

Skeletal muscle disease severity can often progress asymmetrically across muscle groups and heterogeneously within tissues. An example is Duchenne Muscular Dystrophy (DMD) in which lack of dystrophin results in devastating skeletal muscle wasting in some muscles whereas others are spared or undergo hypertrophy. An efficient, non-invasive approach to identify sites of asymmetry and degenerative lesions could enable better patient monitoring and therapeutic targeting of disease. In this study, we utilized a versatile intravenously injectable mesoporous silica nanoparticle (MSNP) based nanocarrier system to explore mechanisms of biodistribution in skeletal muscle of mdx mouse models of DMD including wildtype, dystrophic, and severely dystrophic mice. Moreover, MSNPs could be imaged in live mice and whole muscle tissues enabling investigation of how biodistribution is altered by different types of muscle pathology such as inflammation or fibrosis. We found MSNPs were tenfold more likely to aggregate within select mdx muscles relative to wild type, such as gastrocnemius and quadriceps. This was accompanied by decreased biodistribution in off-target organs. We found the greatest factor affecting preferential delivery was the regenerative state of the dystrophic skeletal muscle with the highest MSNP abundance coinciding with the regions showing the highest level of embryonic myosin staining and intramuscular macrophage uptake. To demonstrate, muscle regeneration regulated MSNP distribution, we experimentally induced regeneration using barium chloride which resulted in a threefold increase of intravenously injected MSNPs to sites of regeneration 7 days after injury. These discoveries provide the first evidence that nanoparticles have selective biodistribution to skeletal muscle in DMD to areas of active regeneration and that nanoparticles could enable diagnostic and selective drug delivery in DMD skeletal muscle.

Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis.

Macrophages are essential for skeletal muscle homeostasis, but how their dysregulation contributes to the development of fibrosis in muscle disease remains unclear. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six clusters and unexpectedly found that none corresponded to traditional definitions of M1 or M2 macrophages. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 (gal-3) and osteopontin (Spp1). Spatial transcriptomics, computational inferences of intercellular communication, and in vitro assays indicated that macrophage-derived Spp1 regulates stromal progenitor differentiation. Gal-3+ macrophages were chronically activated in dystrophic muscle, and adoptive transfer assays showed that the gal-3+ phenotype was the dominant molecular program induced within the dystrophic milieu. Gal-3+ macrophages were also elevated in multiple human myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining their transcriptional programs and reveal Spp1 as a major regulator of macrophage and stromal progenitor interactions.

Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis.

The monocytic/macrophage system is essential for skeletal muscle homeostasis, but its dysregulation contributes to the pathogenesis of muscle degenerative disorders. Despite our increasing knowledge of the role of macrophages in degenerative disease, it still remains unclear how macrophages contribute to muscle fibrosis. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six novel clusters. Unexpectedly, none corresponded to traditional definitions of M1 or M2 macrophage activation. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 and spp1. Spatial transcriptomics and computational inferences of intercellular communication indicated that spp1 regulates stromal progenitor and macrophage interactions during muscular dystrophy. Galectin-3 + macrophages were chronically activated in dystrophic muscle and adoptive transfer assays showed that the galectin-3 + phenotype was the dominant molecular program induced within the dystrophic milieu. Histological examination of human muscle biopsies revealed that galectin-3 + macrophages were also elevated in multiple myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining the transcriptional programs induced in muscle macrophages, and reveal spp1 as a major regulator of macrophage and stromal progenitor interactions.

Single cell sequencing maps skeletal muscle cellular diversity as disease severity increases in dystrophic mouse models.

Duchenne muscular dystrophy (DMD) is caused by out-of-frame mutations in the DMD gene resulting in the absence of a functional dystrophin protein, leading to a devastating and progressive lethal muscle-wasting disease. Little is known about cellular heterogeneity as disease severity increases. Advances in single-cell RNA sequencing (scRNA-seq) enabled us to explore skeletal muscle-resident cell populations in healthy, dystrophic, and severely dystrophic mouse models. We found increased frequencies of activated fibroblasts, fibro-adipogenic progenitor cells, and pro-inflammatory macrophages in dystrophic gastrocnemius muscles and an upregulation of extracellular matrix genes on endothelial cells in dystrophic and severely dystrophic muscles. We observed a pronounced risk of clotting, especially in the severely dystrophic mice with increased expression of plasminogen activator inhibitor-1 in endothelial cells, indicating endothelial cell impairment as disease severity increases. This work extends our understanding of the severe nature of DMD which should be considered when developing single or combinatorial approaches for DMD.

Molecular and cellular basis of genetically inherited skeletal muscle disorders.

Neuromuscular disorders comprise a diverse group of human inborn diseases that arise from defects in the structure and/or function of the muscle tissue - encompassing the muscle cells (myofibres) themselves and their extracellular matrix - or muscle fibre innervation. Since the identification in 1987 of the first genetic lesion associated with a neuromuscular disorder - mutations in dystrophin as an underlying cause of Duchenne muscular dystrophy - the field has made tremendous progress in understanding the genetic basis of these diseases, with pathogenic variants in more than 500 genes now identified as underlying causes of neuromuscular disorders. The subset of neuromuscular disorders that affect skeletal muscle are referred to as myopathies or muscular dystrophies, and are due to variants in genes encoding muscle proteins. Many of these proteins provide structural stability to the myofibres or function in regulating sarcolemmal integrity, whereas others are involved in protein turnover, intracellular trafficking, calcium handling and electrical excitability - processes that ensure myofibre resistance to stress and their primary activity in muscle contraction. In this Review, we discuss how defects in muscle proteins give rise to muscle dysfunction, and ultimately to disease, with a focus on pathologies that are most common, best understood and that provide the most insight into muscle biology.

A Small-Molecule Approach to Restore a Slow-Oxidative Phenotype and Defective CaMKIIß Signaling in Limb Girdle Muscular Dystrophy.

Mutations in CAPN3 cause limb girdle muscular dystrophy R1 (LGMDR1, formerly LGMD2A) and lead to progressive and debilitating muscle wasting. Calpain 3 deficiency is associated with impaired CaMKIIß signaling and blunted transcriptional programs that encode the slow-oxidative muscle phenotype. We conducted a high-throughput screen on a target of CaMKII (Myl2) to identify compounds to override this signaling defect; 4 were tested in vivo in the Capn3 knockout (C3KO) model of LGMDR1. The leading compound, AMBMP, showed good exposure and was able to reverse the LGMDR1 phenotype in vivo, including improved oxidative properties, increased slow fiber size, and enhanced exercise performance. AMBMP also activated CaMKIIß signaling, but it did not alter other pathways known to be associated with muscle growth. Thus, AMBMP treatment activates CaMKII and metabolically reprograms skeletal muscle toward a slow muscle phenotype. These proof-of-concept studies lend support for an approach to the development of therapeutics for LGMDR1.

Genome Editing-Mediated Utrophin Upregulation in Duchenne Muscular Dystrophy Stem Cells.

Utrophin upregulation is considered a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). A number of microRNAs (miRNAs) post-transcriptionally regulate utrophin expression by binding their cognate sites in the 3' UTR. Previously we have shown that miRNA: UTRN repression can be alleviated using miRNA let-7c site blocking oligonucleotides (SBOs) to achieve utrophin upregulation and functional improvement in mdx mice. Here, we used CRISPR/Cas9-mediated genome editing to delete five miRNA binding sites (miR-150, miR-296-5p, miR-133b, let-7c, miR-196b) clustered in a 500 bp inhibitory miRNA target region (IMTR) within the UTRN 3' UTR, for achieving higher expression of endogenous utrophin. Deleting the UTRN IMTR in DMD patient-derived human induced pluripotent stem cells (DMD-hiPSCs) resulted in ca. 2-fold higher levels of utrophin protein. Differentiation of the UTRN edited DMD-hiPSCs (UTRNΔIMTR) by MyoD overexpression resulted in increased sarcolemmal α-sarcoglycan staining consistent with improved dystrophin glycoprotein complex (DGC) restoration. These results demonstrate that CRISPR/Cas9-based UTRN genome editing offers a novel utrophin upregulation therapeutic strategy applicable to all DMD patients, irrespective of the dystrophin mutation status.

Myostatin inhibition promotes fast fibre hypertrophy but causes loss of AMP-activated protein kinase signalling and poor exercise tolerance in a model of limb-girdle muscular dystrophy R1/2A.

KEY POINTS: Limb-girdle muscular dystrophy R1 (LGMD R1) is caused by mutations in the CAPN3 gene and is characterized by progressive muscle loss, impaired mitochondrial function and reductions in the slow oxidative gene expression programme. Myostatin is a negative regulator of muscle growth, and its inhibition improves the phenotype in several muscle wasting disorders. The effect of genetic and pharmacological inhibition of myostatin signalling on the disease phenotype in a mouse model of LGMD R1 (CAPN3 knockout mouse-C3KO) was studied. Inhibition of myostatin signalling in C3KO muscles resulted in significant muscle hypertrophy; however, there were no improvements in muscle strength and exacerbation of exercise intolerance concomitant with further reduction of muscle oxidative capacity was observed. Inhibition of myostatin signalling is unlikely to be a valid therapeutic strategy for LGMD R1. ABSTRACT: Limb-girdle muscular dystrophy R1 (LGMD R1) is caused by mutations in the CAPN3 gene and is characterized by progressive muscle loss, impaired mitochondrial function and reductions in the slow oxidative gene expression programme. There are currently no therapies available to patients. We sought to determine if induction of muscle growth, through myostatin inhibition, represents a viable therapeutic strategy for this disease. Myostatin is a negative regulator of muscle growth, and its inhibition improves the phenotype in several muscle wasting disorders. However, the effect of myostatin depends on the genetic and pathophysiological context and may not be efficacious in all contexts. We found that genetic inhibition of myostatin through overexpression of follistatin (an endogenous inhibitor of myostatin) in our LGMD R1 model (C3KO) resulted in 1.5- to 2-fold increase of muscle mass for the majority of limb muscles. However, muscle strength was not improved and exercise intolerance was exacerbated. Pharmacological inhibition of myostatin, using an anti-myostatin antibody, resulted in statistically significant increases in muscle mass; however, functional testing did not reveal changes in muscle strength nor endurance in treated C3KO mice. Histochemical and biochemical evaluation of follistatin overexpressing mice revealed a reduction in the percentage of oxidative fibres and decreased activation of AMP-activated protein kinase signalling in transgenics compared to C3KO muscles. Our data suggest that muscle hypertrophy, induced by myostatin inhibition, leads to loss of oxidative capacity, which further compromises metabolically impaired C3KO muscles and thus is unlikely to be a valid strategy for treatment of LGMD R1.

CRISPR for Neuromuscular Disorders: Gene Editing and Beyond.

This is a review describing advances in CRISPR/Cas-mediated therapies for neuromuscular disorders (NMDs). We explore both CRISPR-mediated editing and dead Cas approaches as potential therapeutic strategies for multiple NMDs. Last, therapeutic considerations, including delivery and off-target effects, are also discussed.

Spp1 (osteopontin) promotes TGFß processing in fibroblasts of dystrophin-deficient muscles through matrix metalloproteinases.

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. Prior work has shown that DMD progression can vary, depending on the genetic makeup of the patient. Several modifier alleles have been identified including LTBP4 and SPP1. We previously showed that Spp1 exacerbates the DMD phenotype in the mdx mouse model by promoting fibrosis and by skewing macrophage polarization. Here, we studied the mechanisms involved in Spp1's promotion of fibrosis by using both isolated fibroblasts and genetically modified mice. We found that Spp1 upregulates collagen expression in mdx fibroblasts by enhancing TGFß signaling. Spp1's effects on TGFß signaling are through induction of MMP9 expression. MMP9 is a protease that can release active TGFß ligand from its latent complex. In support for activation of this pathway in our model, we showed that treatment of mdx fibroblasts with MMP9 inhibitor led to accumulation of the TGFß latent complex, decreased levels of active TGFß and reduced collagen expression. Correspondingly, we found reduced active TGFß in Spp1-/-mdxB10 and Mmp9-/-mdxB10 muscles in vivo. Taken together with previous observations of reduced fibrosis in both models, these data suggest that Spp1 acts upstream of TGFß to promote fibrosis in mdx muscles. We found that in the context of constitutively upregulated TGFß signaling (such as in the mdxD2 model), ablation of Spp1 has very little effect on fibrosis. Finally, we performed proof-of-concept studies showing that postnatal pharmacological inhibition of Spp1 reduces fibrosis and improves muscle function in mdx mice.

The molecular tweezer CLR01 inhibits aberrant superoxide dismutase 1 (SOD1) self-assembly in vitro and in the G93A-SOD1 mouse model of ALS.

Mutations in superoxide dismutase 1 (SOD1) cause 15-20% of familial amyotrophic lateral sclerosis (fALS) cases. The resulting amino acid substitutions destabilize SOD1's protein structure, leading to its self-assembly into neurotoxic oligomers and aggregates, a process hypothesized to cause the characteristic motor-neuron degeneration in affected individuals. Currently, effective disease-modifying therapy is not available for ALS. Molecular tweezers prevent formation of toxic protein assemblies, yet their protective action has not been tested previously on SOD1 or in the context of ALS. Here, we tested the molecular tweezer CLR01-a broad-spectrum inhibitor of the self-assembly and toxicity of amyloid proteins-as a potential therapeutic agent for ALS. Using recombinant WT and mutant SOD1, we found that CLR01 inhibited the aggregation of all tested SOD1 forms in vitro Next, we examined whether CLR01 could prevent the formation of misfolded SOD1 in the G93A-SOD1 mouse model of ALS and whether such inhibition would have a beneficial therapeutic effect. CLR01 treatment decreased misfolded SOD1 in the spinal cord significantly. However, these histological findings did not correlate with improvement of the disease phenotype. A small, dose-dependent decrease in disease duration was found in CLR01-treated mice, relative to vehicle-treated animals, yet motor function did not improve in any of the treatment groups. These results demonstrate that CLR01 can inhibit SOD1 misfolding and aggregation both in vitro and in vivo, but raise the question whether such inhibition is sufficient for achieving a therapeutic effect. Additional studies in other less aggressive ALS models may be needed to determine the therapeutic potential of this approach.

Development of self-assembled multi-arm polyrotaxanes nanocarriers for systemic plasmid delivery in vivo.

Polyrotaxane (PRX) is a promising supramolecular carrier for gene delivery. Classic PRX exhibits a linear structure in which the amine-functionalized α-cyclodextrin (CD) is threaded along the entire polyethylene glycol (PEG) backbone. While promising in vitro, the absence of free PEG moieties after CD threading compromised the in vivo implementation, due to the unfavorable pharmacokinetics (PK) and biodistribution profile. Herein, we developed a multi-arm PRX nanocarrier platform, which has been designed for protective nucleic acid encapsulation, augmented biodistribution and PK, and suitable for intravenous (IV) administration. A key design was to introduce cationic CD rings onto a multi-arm PEG backbone in a spatially selective fashion. The optimal structural design was obtained through iterative rounds of experimentation to determine the appropriate type and density of cationic charge on CD ring, the degree of PEGylation, the size and structure of polymer backbone, etc. This allowed us to effectively deliver large size reporter and therapeutic plasmids in cancer mouse models. Post IV injection, we demonstrated that our multi-arm polymer design significantly enhanced circulatory half-life and PK profile compared to the linear PRX. We continued to use the multi-arm PRX to formulate a therapeutic plasmid encoding an immunomodulatory cytokine, IL-12. When tested in a colon cancer syngeneic mouse model with same background, the IL-12 plasmid was protected by the multi-arm PRX and delivered through the tail vein to the tumor site, leading to a significant tumor inhibition effect. Moreover, our delivery system was devoid of major systemic toxicity.

Repurposing Dantrolene for Long-Term Combination Therapy to Potentiate Antisense-Mediated DMD Exon Skipping in the mdx Mouse.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin, which is essential to muscle health. DMD "exon skipping" uses anti-sense oligo-nucleotides (AONs) to force specific exon exclusion during mRNA processing to restore reading frame and rescue of partially functional dystrophin protein. Although exon-skipping drugs in humans show promise, levels of rescued dystrophin protein remain suboptimal. We previously identified dantrolene as a skip booster when combined with AON in human DMD cultures and short-term mdx dystrophic mouse studies. Here, we assess the effect of dantrolene/AON combination on DMD exon-23 skipping over long-term mdx treatment under conditions that better approximate potential human dosing. To evaluate the dantrolene/AON combination treatment effect on dystrophin induction, we assayed three AON doses, with and without oral dantrolene, to assess multiple outcomes across different muscles. Meta-analyses of the results of statistical tests from both the quadriceps and diaphragm assessing contributions of dantrolene beyond AON, across all AON treatment groups, provide strong evidence that dantrolene modestly boosts exon skipping and dystrophin rescue while reducing muscle pathology in mdx mice (p < 0.0087). These findings support a trial of combination dantrolene/AON to increase exon-skipping efficacy and highlight the value of combinatorial approaches and Food and Drug Administration (FDA) drug re-purposing for discovery of unsuspected therapeutic application and rapid translation.

Calpain 3 and CaMKIIß signaling are required to induce HSP70 necessary for adaptive muscle growth after atrophy.

Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3) is a calcium dependent protease residing in the myofibrillar, cytosolic and triad fractions of skeletal muscle. At the triad, it colocalizes with calcium calmodulin kinase IIß (CaMKIIß). CAPN3 knock out mice (C3KO) show reduced triad integrity and blunted CaMKIIß signaling, which correlates with impaired transcriptional activation of myofibrillar and oxidative metabolism genes in response to running exercise. These data suggest a role for CAPN3 and CaMKIIß in gene regulation that takes place during adaptation to endurance exercise. To assess whether CAPN3- CaMKIIß signaling influences skeletal muscle remodeling in other contexts, we subjected C3KO and wild type mice to hindlimb unloading and reloading and assessed CaMKIIß signaling and gene expression by RNA-sequencing. After induced atrophy followed by 4 days of reloading, both CaMKIIß activation and expression of inflammatory and cellular stress genes were increased. C3KO muscles failed to activate CaMKIIß signaling, did not activate the same pattern of gene expression and demonstrated impaired growth at 4 days of reloading. Moreover, C3KO muscles failed to activate inducible HSP70, which was previously shown to be indispensible for the inflammatory response needed to promote muscle recovery. Likewise, C3KO showed diminished immune cell infiltration and decreased expression of pro-myogenic genes. These data support a role for CaMKIIß signaling in induction of HSP70 and promotion of the inflammatory response during muscle growth and remodeling that occurs after atrophy, suggesting that CaMKIIß regulates remodeling in multiple contexts: endurance exercise and growth after atrophy.

ERBB3 and NGFR mark a distinct skeletal muscle progenitor cell in human development and hPSCs.

Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenicity of hPSC-SMPCs relative to human fetal or adult satellite cells remains unclear. We observed that hPSC-SMPCs derived by directed differentiation are less functional in vitro and in vivo compared to human satellite cells. Using RNA sequencing, we found that the cell surface receptors ERBB3 and NGFR demarcate myogenic populations, including PAX7 progenitors in human fetal development and hPSC-SMPCs. We demonstrated that hPSC skeletal muscle is immature, but inhibition of transforming growth factor-ß signalling during differentiation improved fusion efficiency, ultrastructural organization and the expression of adult myosins. This enrichment and maturation strategy restored dystrophin in hundreds of dystrophin-deficient myofibres after engraftment of CRISPR-Cas9-corrected Duchenne muscular dystrophy human induced pluripotent stem cell-SMPCs. The work provides an in-depth characterization of human myogenesis, and identifies candidates that improve the in vivo myogenic potential of hPSC-SMPCs to levels that are equal to directly isolated human fetal muscle cells.

Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury.

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor ß (TGFß) pathway, osteopontin encoded by the SPP1 gene and latent TGFß binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFß and TGFß-associated pathways. We identified that increased TGFß resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFß and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy.