The DUX4-HIF1α Axis in Murine and Human Muscle Cells: A Link More Complex Than Expected.

FacioScapuloHumeral muscular Dystrophy (FSHD) is one of the most prevalent inherited muscle disorders and is linked to the inappropriate expression of the DUX4 transcription factor in skeletal muscles. The deregulated molecular network causing FSHD muscle dysfunction and pathology is not well understood. It has been shown that the hypoxia response factor HIF1α is critically disturbed in FSHD and has a major role in DUX4-induced cell death. In this study, we further explored the relationship between DUX4 and HIF1α. We found that the DUX4 and HIF1α link differed according to the stage of myogenic differentiation and was conserved between human and mouse muscle. Furthermore, we found that HIF1α knockdown in a mouse model of DUX4 local expression exacerbated DUX4-mediated muscle fibrosis. Our data indicate that the suggested role of HIF1α in DUX4 toxicity is complex and that targeting HIF1α might be challenging in the context of FSHD therapeutic approaches.

Regional and bilateral MRI and gene signatures in facioscapulohumeral dystrophy: implications for clinical trial design and mechanisms of disease progression.

Identifying the aberrant expression of DUX4 in skeletal muscle as the cause of facioscapulohumeral dystrophy (FSHD) has led to rational therapeutic development and clinical trials. Several studies support the use of MRI characteristics and the expression of DUX4-regulated genes in muscle biopsies as biomarkers of FSHD disease activity and progression. We performed lower-extremity MRI and muscle biopsies in the mid-portion of the tibialis anterior (TA) muscles bilaterally in FSHD subjects and validated our prior reports of the strong association between MRI characteristics and expression of genes regulated by DUX4 and other gene categories associated with FSHD disease activity. We further show that measurements of normalized fat content in the entire TA muscle strongly predict molecular signatures in the mid-portion of the TA, indicating that regional biopsies can accurately measure progression in the whole muscle and providing a strong basis for inclusion of MRI and molecular biomarkers in clinical trial design. An unanticipated finding was the strong correlations of molecular signatures in the bilateral comparisons, including markers of B-cells and other immune cell populations, suggesting that a systemic immune cell infiltration of skeletal muscle might have a role in disease progression.

snRNA-seq analysis in multinucleated myogenic FSHD cells identifies heterogeneous FSHD transcriptome signatures associated with embryonic-like program activation and oxidative stress-induced apoptosis.

The sporadic nature of DUX4 expression in FSHD muscle challenges comparative transcriptome analyses between FSHD and control samples. A variety of DUX4 and FSHD-associated transcriptional changes have been identified, but bulk RNA-seq strategies prohibit comprehensive analysis of their spatiotemporal relation, interdependence and role in the disease process. In this study, we used single-nucleus RNA-sequencing of nuclei isolated from patient- and control-derived multinucleated primary myotubes to investigate the cellular heterogeneity in FSHD. Taking advantage of the increased resolution in snRNA-sequencing of fully differentiated myotubes, two distinct populations of DUX4-affected nuclei could be defined by their transcriptional profiles. Our data provides insights into the differences between these two populations and suggests heterogeneity in two well-known FSHD-associated transcriptional aberrations: increased oxidative stress and inhibition of myogenic differentiation. Additionally, we provide evidence that DUX4-affected nuclei share transcriptome features with early embryonic cells beyond the well-described cleavage stage, progressing into the 8-cell and blastocyst stages. Altogether, our data suggests that the FSHD transcriptional profile is defined by a mixture of individual and sometimes mutually exclusive DUX4-induced responses and cellular state-dependent downstream effects.

Autosomal dominant in cis D4Z4 repeat array duplication alleles in facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD) has a unique genetic aetiology resulting in partial chromatin relaxation of the D4Z4 macrosatellite repeat array on 4qter. This D4Z4 chromatin relaxation facilitates inappropriate expression of the transcription factor DUX4 in skeletal muscle. DUX4 is encoded by a retrogene that is embedded within the distal region of the D4Z4 repeat array. In the European population, the D4Z4 repeat array is usually organized in a single array that ranges between 8 and 100 units. D4Z4 chromatin relaxation and DUX4 derepression in FSHD is most often caused by repeat array contraction to 1-10 units (FSHD1) or by a digenic mechanism requiring pathogenic variants in a D4Z4 chromatin repressor like SMCHD1, combined with a repeat array between 8 and 20 units (FSHD2). With a prevalence of 1.5% in the European population, in cis duplications of the D4Z4 repeat array, where two adjacent D4Z4 arrays are interrupted by a spacer sequence, are relatively common but their relationship to FSHD is not well understood. In cis duplication alleles were shown to be pathogenic in FSHD2 patients; however, there is inconsistent evidence for the necessity of an SMCHD1 mutation for disease development. To explore the pathogenic nature of these alleles we compared in cis duplication alleles in FSHD patients with or without pathogenic SMCHD1 variant. For both groups we showed duplication-allele-specific DUX4 expression. We studied these alleles in detail using pulsed-field gel electrophoresis-based Southern blotting and molecular combing, emphasizing the challenges in the characterization of these rearrangements. Nanopore sequencing was instrumental to study the composition and methylation of the duplicated D4Z4 repeat arrays and to identify the breakpoints and the spacer sequence between the arrays. By comparing the composition of the D4Z4 repeat array of in cis duplication alleles in both groups, we found that specific combinations of proximal and distal repeat array sizes determine their pathogenicity. Supported by our algorithm to predict pathogenicity, diagnostic laboratories should now be furnished to accurately interpret these in cis D4Z4 repeat array duplications, alleles that can easily be missed in routine settings.

Hypoxia enhances human myoblast differentiation: involvement of HIF1α and impact of DUX4, the FSHD causal gene.

BACKGROUND: Hypoxia is known to modify skeletal muscle biological functions and muscle regeneration. However, the mechanisms underlying the effects of hypoxia on human myoblast differentiation remain unclear. The hypoxic response pathway is of particular interest in patients with hereditary muscular dystrophies since many present respiratory impairment and muscle regeneration defects. For example, an altered hypoxia response characterizes the muscles of patients with facioscapulohumeral dystrophy (FSHD). METHODS: We examined the impact of hypoxia on the differentiation of human immortalized myoblasts (LHCN-M2) cultured in normoxia (PO2: 21%) or hypoxia (PO2: 1%). Cells were grown in proliferation (myoblasts) or differentiation medium for 2 (myocytes) or 4 days (myotubes). We evaluated proliferation rate by EdU incorporation, used myogenin-positive nuclei as a differentiation marker for myocytes, and determined the fusion index and myosin heavy chain-positive area in myotubes. The contribution of HIF1α was studied by gain (CoCl2) and loss (siRNAs) of function experiments. We further examined hypoxia in LHCN-M2-iDUX4 myoblasts with inducible expression of DUX4, the transcription factor underlying FSHD pathology. RESULTS: We found that the hypoxic response did not impact myoblast proliferation but activated precocious myogenic differentiation and that HIF1α was critical for this process. Hypoxia also enhanced the late differentiation of human myocytes, but in an HIF1α-independent manner. Interestingly, the impact of hypoxia on muscle cell proliferation was influenced by dexamethasone. In the FSHD pathological context, DUX4 suppressed HIF1α-mediated precocious muscle differentiation. CONCLUSION: Hypoxia stimulates myogenic differentiation in healthy myoblasts, with HIF1α-dependent early steps. In FSHD, DUX4-HIF1α interplay indicates a novel mechanism by which DUX4 could interfere with HIF1α function in the myogenic program and therefore with FSHD muscle performance and regeneration.

Flavones provide resistance to DUX4-induced toxicity via an mTor-independent mechanism.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most common of the muscular dystrophies, affecting nearly 1 in 8000 individuals, and is a cause of profound disability. Genetically, FSHD is linked to the contraction and/or epigenetic de-repression of the D4Z4 repeat array on chromosome 4, thereby allowing expression of the DUX4 gene in skeletal muscle. If the DUX4 transcript incorporates a stabilizing polyadenylation site the myotoxic DUX4 protein will be synthesized, resulting in muscle wasting. The mechanism of toxicity remains unclear, as many DUX4-induced cytopathologies have been described, however cell death does primarily occur through caspase 3/7-dependent apoptosis. To date, most FSHD therapeutic development has focused on molecular methods targeting DUX4 expression or the DUX4 transcript, while therapies targeting processes downstream of DUX4 activity have received less attention. Several studies have demonstrated that inhibition of multiple signal transduction pathways can ameliorate DUX4-induced toxicity, and thus compounds targeting these pathways have the potential to be developed into FSHD therapeutics. To this end, we have screened a group of small molecules curated based on their reported activity in relevant pathways and/or structural relationships with known toxicity-modulating molecules. We have identified a panel of five compounds that function downstream of DUX4 activity to inhibit DUX4-induced toxicity. Unexpectedly, this effect was mediated through an mTor-independent mechanism that preserved expression of ULK1 and correlated with an increase in a marker of active cellular autophagy. This identifies these flavones as compounds of interest for therapeutic development, and potentially identifies the autophagy pathway as a target for therapeutics.

MATR3 is an endogenous inhibitor of DUX4 in FSHD muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common neuromuscular disorders and has no cure. Due to an unknown molecular mechanism, FSHD displays overlapping manifestations with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). FSHD is caused by aberrant gain of expression of the transcription factor double homeobox 4 (DUX4), which triggers a pro-apoptotic transcriptional program resulting in inhibition of myogenic differentiation and muscle wasting. Regulation of DUX4 activity is poorly known. We identify Matrin 3 (MATR3), whose mutation causes ALS and dominant distal myopathy, as a cellular factor controlling DUX4 expression and activity. MATR3 binds to the DUX4 DNA-binding domain and blocks DUX4-mediated gene expression, rescuing cell viability and myogenic differentiation of FSHD muscle cells, without affecting healthy muscle cells. Finally, we characterize a shorter MATR3 fragment that is necessary and sufficient to directly block DUX4-induced toxicity to the same extent as the full-length protein. Collectively, our data suggest MATR3 as a candidate for developing a treatment for FSHD.

The transcription factor DUX4 orchestrates translational reprogramming by broadly suppressing translation efficiency and promoting expression of DUX4-induced mRNAs.

Translational control is critical for cell fate transitions during development, lineage specification, and tumorigenesis. Here, we show that the transcription factor double homeobox protein 4 (DUX4), and its previously characterized transcriptional program, broadly regulates translation to change the cellular proteome. DUX4 is a key regulator of zygotic genome activation in human embryos, whereas misexpression of DUX4 causes facioscapulohumeral muscular dystrophy (FSHD) and is associated with MHC-I suppression and immune evasion in cancer. We report that translation initiation and elongation factors are disrupted downstream of DUX4 expression in human myoblasts. Genome-wide translation profiling identified mRNAs susceptible to DUX4-induced translation inhibition, including those encoding antigen presentation factors and muscle lineage proteins, while DUX4-induced mRNAs were robustly translated. Endogenous expression of DUX4 in human FSHD myotubes and cancer cell lines also correlated with reduced protein synthesis and MHC-I presentation. Our findings reveal that DUX4 orchestrates cell state conversion by suppressing the cellular proteome while maintaining translation of DUX4-induced mRNAs to promote an early developmental program.

Compromised nonsense-mediated RNA decay results in truncated RNA-binding protein production upon DUX4 expression.

Nonsense-mediated RNA decay (NMD) degrades transcripts carrying premature termination codons. NMD is thought to prevent the synthesis of toxic truncated proteins. However, whether loss of NMD results in widespread production of truncated proteins is unclear. A human genetic disease, facioscapulohumeral muscular dystrophy (FSHD), features acute inhibition of NMD upon expression of the disease-causing transcription factor, DUX4. Using a cell-based model of FSHD, we show production of truncated proteins from physiological NMD targets and find that RNA-binding proteins are enriched for aberrant truncations. The NMD isoform of one RNA-binding protein, SRSF3, is translated to produce a stable truncated protein, which is detected in FSHD patient-derived myotubes. Ectopic expression of truncated SRSF3 confers toxicity, and its downregulation is cytoprotective. Our results delineate the genome-scale impact of NMD loss. This widespread production of potentially deleterious truncated proteins has implications for FSHD biology as well as other genetic diseases where NMD is therapeutically modulated.

Influence of DUX4 Expression in Facioscapulohumeral Muscular Dystrophy and Possible Treatments.

Facioscapulohumeral muscular dystrophy (FSHD) represents the third most common form of muscular dystrophy and is characterized by muscle weakness and atrophy. FSHD is caused by the altered expression of the transcription factor double homeobox 4 (DUX4), which is involved in several significantly altered pathways required for myogenesis and muscle regeneration. While DUX4 is normally silenced in the majority of somatic tissues in healthy individuals, its epigenetic de-repression has been linked to FSHD, resulting in DUX4 aberrant expression and cytotoxicity in skeletal muscle cells. Understanding how DUX4 is regulated and functions could provide useful information not only to further understand FSHD pathogenesis, but also to develop therapeutic approaches for this disorder. Therefore, this review discusses the role of DUX4 in FSHD by examining the possible molecular mechanisms underlying the disease as well as novel pharmacological strategies targeting DUX4 aberrant expression.

Facioscapulohumeral Muscular Dystrophy is Associated With Altered Myoblast Proteome Dynamics.

Proteomic studies in facioscapulohumeral muscular dystrophy (FSHD) could offer new insight into disease mechanisms underpinned by post-transcriptional processes. We used stable isotope (deuterium oxide; D2O) labeling and peptide mass spectrometry to investigate the abundance and turnover rates of proteins in cultured muscle cells from two individuals affected by FSHD and their unaffected siblings (UASb). We measured the abundance of 4420 proteins and the turnover rate of 2324 proteins in each (n = 4) myoblast sample. FSHD myoblasts exhibited a greater abundance but slower turnover rate of subunits of mitochondrial respiratory complexes and mitochondrial ribosomal proteins, which may indicate an accumulation of "older" less viable mitochondrial proteins in myoblasts from individuals affected by FSHD. Treatment with a 2'-O-methoxyethyl modified antisense oligonucleotide targeting exon 3 of the double homeobox 4 (DUX4) transcript tended to reverse mitochondrial protein dysregulation in FSHD myoblasts, indicating the effect on mitochondrial proteins may be a DUX4-dependent mechanism. Our results highlight the importance of post-transcriptional processes and protein turnover in FSHD pathology and provide a resource for the FSHD research community to explore this burgeoning aspect of FSHD.

An in silico FSHD muscle fiber for modeling DUX4 dynamics and predicting the impact of therapy.

Facioscapulohumeral muscular dystrophy (FSHD) is an incurable myopathy linked to the over-expression of the myotoxic transcription factor DUX4. Targeting DUX4 is the leading therapeutic approach, however, it is only detectable in 0.1-3.8% of FSHD myonuclei. How rare DUX4 drives FSHD and the optimal anti-DUX4 strategy are unclear. We combine stochastic gene expression with compartment models of cell states, building a simulation of DUX4 expression and consequences in FSHD muscle fibers. Investigating iDUX4 myoblasts, scRNAseq, and snRNAseq of FSHD muscle we estimate parameters including DUX4 mRNA degradation, transcription and translation rates, and DUX4 target gene activation rates. Our model accurately recreates the distribution of DUX4 and targets gene-positive cells seen in scRNAseq of FSHD myocytes. Importantly, we show DUX4 drives significant cell death despite expression in only 0.8% of live cells. Comparing scRNAseq of unfused FSHD myocytes to snRNAseq of fused FSHD myonuclei, we find evidence of DUX4 protein syncytial diffusion and estimate its rate via genetic algorithms. We package our model into freely available tools, to rapidly investigate the consequences of anti-DUX4 therapy.

WDR5 is required for DUX4 expression and its pathological effects in FSHD muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.

Nutritional Status of Patients with Facioscapulohumeral Muscular Dystrophy.

In patients with facioscapulohumeral muscular dystrophy (FSHD), a rare genetic neuromuscular disease, reduced physical performance is associated with lower blood levels of vitamin C, zinc, selenium, and increased oxidative stress markers. Supplementation of vitamin C, vitamin E, zinc, and selenium improves the quadriceps' physical performance. Here, we compared the nutritional status of 74 women and 85 men with FSHD. Calorie intake was lower in women with FSHD than in men. Moreover, we assessed vitamin C, vitamin E, zinc, copper, and selenium intakes in diet and their concentrations in the plasma. Vitamin E, copper, and zinc intake were lower in women with FSHD than in men, whereas plasma vitamin C, copper levels, and copper/zinc ratio were higher in women with FSHD than in men. The dietary intake and plasma concentrations of the studied vitamins and minerals were not correlated in both sexes. A well-balanced and varied diet might not be enough in patients with FSHD to correct the observed vitamin/mineral deficiencies. A low energy intake is a risk factor for suboptimal intake of proteins, vitamins, and minerals that are important for protein synthesis and other metabolic pathways and that might contribute to progressive muscle mass loss. Antioxidant supplementation and higher protein intake seem necessary to confer protection against oxidative stress and skeletal muscle mass loss.

Human DUX4 and porcine DUXC activate similar early embryonic programs in pig muscle cells: implications for preclinical models of FSHD.

Human DUX4 and its mouse ortholog Dux are normally expressed in the early embryo-the 4-cell or 2-cell cleavage stage embryo, respectively-and activate a portion of the first wave of zygotic gene expression. DUX4 is epigenetically suppressed in nearly all somatic tissue, whereas facioscapulohumeral dystrophy (FSHD)-causing mutations result in its aberrant expression in skeletal muscle, transcriptional activation of the early embryonic program and subsequent muscle pathology. Although DUX4 and Dux both activate an early totipotent transcriptional program, divergence of their DNA binding domains limits the use of DUX4 expressed in mice as a preclinical model for FSHD. In this study, we identify the porcine DUXC messenger ribonucleic acid expressed in early development and show that both pig DUXC and human DUX4 robustly activate a highly similar early embryonic program in pig muscle cells. These results support further investigation of pig preclinical models for FSHD.

DUX4 double whammy: The transcription factor that causes a rare muscular dystrophy also kills the precursors of the human nose.

SMCHD1 mutations cause congenital arhinia (absent nose) and a muscular dystrophy called FSHD2. In FSHD2, loss of SMCHD1 repressive activity causes expression of double homeobox 4 (DUX4) in muscle tissue, where it is toxic. Studies of arhinia patients suggest a primary defect in nasal placode cells (human nose progenitors). Here, we show that upon SMCHD1 ablation, DUX4 becomes derepressed in H9 human embryonic stem cells (hESCs) as they differentiate toward a placode cell fate, triggering cell death. Arhinia and FSHD2 patient-derived induced pluripotent stem cells (iPSCs) express DUX4 when converted to placode cells and demonstrate variable degrees of cell death, suggesting an environmental disease modifier. HSV-1 may be one such modifier as herpesvirus infection amplifies DUX4 expression in SMCHD1 KO hESC and patient iPSC. These studies suggest that arhinia, like FSHD2, is due to compromised SMCHD1 repressive activity in a cell-specific context and provide evidence for an environmental modifier.

Gastrointestinal cancer occurs as extramuscular manifestation in FSHD1 patients.

Facioscapulohumeral dystrophy type1 (FSHD1) patients with a shortened D4Z4 repeat containing the DUX4 gene have a broad spectrum of clinical manifestations. In addition, high expression of DUX4 protein with an aberrant C terminus is frequently identified in B cell acute lymphoblastic leukemia. We investigated clinical manifestations in 31 FSHD1 patients and 30 non-affected individuals. Gastrointestinal cancers (gastric and colorectal cancers) increased after the age of 40 years and were more frequently observed in FSHD1 patients (n = 10) than in non-affected individuals (n = 2, p = 0.0217), though the incidence of cancers occurring in non-gastrointestinal tissues of FSHD1 patients was the same as that of non-affected individuals (p > 0.999). These comorbidities of FSHD1 patients were not associated with D4Z4 repeat number. Our results suggest that gastrointestinal cancers are among the extramuscular manifestations of adult FSHD1 patients, and do not depend on D4Z4 repeat number.

DUX4 expression activates JNK and p38 MAP kinases in myoblasts.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by misexpression of the DUX4 transcription factor in skeletal muscle that results in transcriptional alterations, abnormal phenotypes and cell death. To gain insight into the kinetics of DUX4-induced stresses, we activated DUX4 expression in myoblasts and performed longitudinal RNA sequencing paired with proteomics and phosphoproteomics. This analysis revealed changes in cellular physiology upon DUX4 activation, including DNA damage and altered mRNA splicing. Phosphoproteomic analysis uncovered rapid widespread changes in protein phosphorylation following DUX4 induction, indicating that alterations in kinase signaling might play a role in DUX4-mediated stress and cell death. Indeed, we demonstrate that two stress-responsive MAP kinase pathways, JNK and p38, are activated in response to DUX4 expression. Inhibition of each of these pathways ameliorated DUX4-mediated cell death in myoblasts. These findings uncover that the JNK pathway is involved in DUX4-mediated cell death and provide additional insights into the role of the p38 pathway, a clinical target for the treatment of FSHD.

Update on the Molecular Aspects and Methods Underlying the Complex Architecture of FSHD.

Despite the knowledge of the main mechanisms involved in facioscapulohumeral muscular dystrophy (FSHD), the high heterogeneity and variable penetrance of the disease complicate the diagnosis, characterization and genotype-phenotype correlation of patients and families, raising the need for further research and data. Thus, the present review provides an update of the main molecular aspects underlying the complex architecture of FSHD, including the genetic factors (related to D4Z4 repeated units and FSHD-associated genes), epigenetic elements (D4Z4 methylation status, non-coding RNAs and high-order chromatin interactions) and gene expression profiles (FSHD transcriptome signatures both at bulk tissue and single-cell level). In addition, the review will also describe the methods currently available for investigating the above-mentioned features and how the resulting data may be combined with artificial-intelligence-based pipelines, with the purpose of developing a multifunctional tool tailored to enhancing the knowledge of disease pathophysiology and progression and fostering the research for novel treatment strategies, as well as clinically useful biomarkers. In conclusion, the present review highlights how FSHD should be regarded as a disease characterized by a molecular spectrum of genetic and epigenetic factors, whose alteration plays a differential role in DUX4 repression and, subsequently, contributes to determining the FSHD phenotype.

Non-myogenic mesenchymal cells contribute to muscle degeneration in facioscapulohumeral muscular dystrophy patients.

Muscle-resident non-myogenic mesenchymal cells play key roles that drive successful tissue regeneration within the skeletal muscle stem cell niche. These cells have recently emerged as remarkable therapeutic targets for neuromuscular disorders, although to date they have been poorly investigated in facioscapulohumeral muscular dystrophy (FSHD). In this study, we characterised the non-myogenic mesenchymal stromal cell population in FSHD patients' muscles with signs of disease activity, identified by muscle magnetic resonance imaging (MRI), and compared them with those obtained from apparently normal muscles of FSHD patients and from muscles of healthy, age-matched controls. Our results showed that patient-derived cells displayed a distinctive expression pattern of mesenchymal markers, along with an impaired capacity to differentiate towards mature adipocytes in vitro, compared with control cells. We also demonstrated a significant expansion of non-myogenic mesenchymal cells (identified as CD201- or PDGFRA-expressing cells) in FSHD muscles with signs of disease activity, which correlated with the extent of intramuscular fibrosis. In addition, the accumulation of non-myogenic mesenchymal cells was higher in FSHD muscles that deteriorate more rapidly. Our results prompt a direct association between an accumulation, as well as an altered differentiation, of non-myogenic mesenchymal cells with muscle degeneration in FSHD patients. Elucidating the mechanisms and cellular interactions that are altered in the affected muscles of FSHD patients could be instrumental to clarify disease pathogenesis and identifying reliable novel therapeutic targets.