Skeletal Muscle

Manual analysis of skeletal muscle cross-sections is time-consuming and subject to error and user bias. To overcome these limitations, we developed and validated a semi-automated, quantitative, and reproducible image-analysis pipeline specifically tailored to quantify Pax7+ satellite cells, myonuclei, and cross-sectional area by fiber type. The workflow combines FIJI/ImageJ-based image preprocessing with CellProfiler, Cellpose, and a custom Python script to process and analyze immunohistological images of muscle tissue cross-sections. Outcomes include Pax7+ satellite cells and myonuclei quantified per fiber by fiber type, along with cross-sectional area, perimeter, and fiber type classification. This semi-automated approach provides a robust and efficient platform for high-throughput analysis of muscle tissue cross-sections from large datasets. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/729866v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@a3401dorg.highwire.dtl.DTLVardef@1c63145org.highwire.dtl.DTLVardef@ccbf76org.highwire.dtl.DTLVardef@2e0da0_HPS_FORMAT_FIGEXP M_FIG C_FIG

A hallmark of damaged skeletal muscle fibers is displaced myonuclei that are no longer peripherally positioned. Displaced myonuclei are dogmatically thought to be derived exclusively from muscle stem cell (satellite cell) fusion. Using a surgical resection muscle injury model and in vivo recombination-independent resident myonuclear labeling, we detail the prevalence, time course, and origin of displaced myonuclei in response to a non-chemically-mediated muscle trauma. We found that: 1) non-satellite cell-derived (resident) displaced myonuclei emerge seven days after surgical injury in similar proportion to exogenous (satellite cell-derived) displaced myonuclei in intact muscle fibers, with a biased prevalence in myosin heavy chain IIB muscle fibers, 2) muscle fibers with multiple ([≥]2) displaced resident myonuclei was an unexpected but noteworthy feature of muscle fibers 7 days after injury, 3) embryonic myosin-expressing fibers at seven days post-surgery expectedly contain predominantly satellite-cell derived displaced myonuclei, but a subset have displaced resident myonuclei, and 4) satellite cell numbers in intact muscle do not increase until 7 days post-surgery. These data may help inform whether to target satellite cell-initiated processes, myonuclear-initiated processes, or both to facilitate muscle fiber injury repair. This information could lead to more effective therapeutic strategies for treating muscle trauma.

BackgroundCancer cachexia (CC) is characterized by skeletal muscle atrophy and reduced strength, partly linked to dysfunction of muscle stem cells (MuSCs) and alterations in their niche. Although exercise may mitigate muscle loss, its effects in CC remain debated and its feasibility is often limited in advanced patients. Neuromuscular electrical stimulation (NMES) offers a promising alternative, by promoting MuSC proliferation and fusion, increasing muscle size and macrophage content in healthy muscle. This study investigated whether NMES, initiated at tumor onset, could improve MuSC regulation and its niche while limiting muscle atrophy and weakness in a tumor-bearing mouse model. MethodsTen-week-old male BALB/c mice were subcutaneously injected with C26 tumor cells or PBS. Tumor-bearing mice were divided into NMES-treated (C26 NMES) and non-stimulated controls (C26). NMES consisted of six sessions (two series of three consecutive daily sessions separated by one rest day), starting seven days post-inoculation when tumors became visible. Each session was delivered at a submaximal intensity corresponding to 15% of maximal strength. Muscle mass, myofiber size, strength and cellular composition were assessed. ResultsMuscle mass was decreased by 13% in C26 mice as compared to PBS controls, while C26 NMES mice showed a [~]7% improvement over C26 mice. Mean myofiber size decreased similarly in both tumor-bearing groups as compared to PBS controls (-12-14%). However, NMES reduced the proportion of small myofibers (400-600 {micro}m{superscript 2}) as compared to C26 mice. Maximal torque loss was less severe in C26 NMES mice (-28%) than in C26 mice (-34%). As compared with PBS mice, C26 mice exhibited increased MuSC proliferation (+97%) but reduced differentiation (-61%), as indicated by fewer myogenin-positive cells. NMES normalized MuSC proliferation, restored myogenin-positive cell number, and enhanced MuSC fusion, reflected by an increased number of PCM1-positive myonuclei (+8-11%). NMES also modulated inflammation, reducing neutrophils (-42%) and increasing macrophages (+35%), through the proliferation of CD169-positive resident macrophages (+106%). In vitro, macrophages exposed to C26 muscle extracts showed elevated pro-inflammatory markers (COX2 and TNF-; +21% and +16%) as compared to PBS controls. This effect was abolished with extracts from C26 NMES muscles. Additionally, C26 extracts reduced the expression of anti-inflammatory markers by macrophages (CD206 and IL-10; -23%), whereas NMES restored their levels to those of controls. ConclusionNMES-induced mild contractile activity is an effective stimulus for preserving muscle strength and mass, improving MuSC regulation, and modulating muscle inflammation in a mouse model of CC.

Increased mechanical loading induces skeletal muscle growth and, at the ultrastructural level, promotes myofibrillogenesis and the radial growth of myofibrils. However, the mechanisms regulating these ultrastructural adaptations are not known. Here, we sought to determine whether the mechanistic target of rapamycin complex 1 (mTORC1) regulates these processes. To accomplish this, muscle-specific, tamoxifen-inducible raptor knockout (iRAmKO) mice were used to inhibit signaling through mTORC1, and growth was induced with a model of chronic mechanical overload (MOV). Using a next-generation fluorescence imaging pipeline for ultrastructural analyses, we found that mTORC1 is a critical regulator of the myofibrillogenesis and radial growth of myofibrils that occur in response to MOV. Together with other recent advances in the field, we propose a model in which mTORC1 acts as a gatekeeper that permits the retention, rather than the synthesis, of proteins that drive the ultrastructural adaptations.

Skeletal muscle satellite cells, residing between the myofiber plasma membrane and the surrounding basement membrane, maintain and repair skeletal muscle throughout life. Typically quiescent, satellite cells can transition into a reversible alert state (GAlert) that primes them for rapid activation to maintain or repair muscle. From GAlert, SCs can either re-enter quiescence or commit to the cell cycle, expand, and differentiate to fuse with existing regenerating myofibers. Exit from quiescence requires extensive post-transcriptional remodeling, including changes in RNA processing and RNA-binding protein activity. We show that TDP-43, an RNA binding protein, is essential for SC maintenance and muscle repair. Conditional deletion of TDP-43 in SCs caused a consistent and progressive loss of GAlert SCs even in uninjured muscle, leading to depletion of the SC pool. TDP-43 haploinsufficiency was sufficient to impair SC maintenance, indicating that both alleles are required. Integrative analysis suggests that TDP-43 supports expression of stress response-associated transcripts during the quiescent-to-GAlert transition, and that failure to mount this response contributes to SC apoptosis. Thus, we identified TDP-43 as a critical regulator of satellite cell survival as satellite cells activate and establish a TDP-43 requirement for maintaining and repairing skeletal muscle.

Resistance training lasting a few weeks increases maximal force mainly through neural adaptations that enhance the drive from the nervous system to muscle. While these adaptations have been well documented at the motor unit (MU) level during submaximal force contractions, the mechanisms underlying force increases during maximal voluntary contractions are poorly understood. This is due to a classic technical limitation in tracking MUs longitudinally during maximal force tasks. Here, we solved this technical challenge, enabling the investigation of MU adaptations during MVCs in both the trained and untrained limbs following unilateral resistance training. High-density surface electromyography was recorded from the biceps brachii of both limbs before and after a 4-week unilateral resistance-training intervention, and the same MUs were longitudinally tracked across sessions during MVCs by concatenation of three MVC trials of ~5-s each.Unilateral training increased maximal force in the trained limb (+16%) and induced strength transfer to the untrained limb (+8%). In both limbs, maximal contractions after training were characterized by greater EMG amplitude, faster muscle-fiber conduction velocity, and higher MU discharge rates, indicating enhanced neural drive to the motoneuron pool. These adaptations were strongly associated with improvements in maximal force (R2 > 0.7 for all). Importantly, longitudinal MU tracking revealed a non-uniform adaptation across the MU pool: MUs with higher baseline conduction velocity, indicative of higher recruitment threshold, exhibited the largest pre-post increases in discharge rate, whereas lower-threshold units showed smaller changes. Collectively, these findings demonstrate that gains in maximal force and their transfer to the untrained limb are primarily mediated by enhanced rate coding of higher-threshold MUs during MVCs.

Congenital myasthenic syndromes (CMS) are rare inherited diseases of the neuromuscular junction (NMJ). There are 40 identified CMS genes, but many patients go without genetic diagnosis, which suggests new genes have yet to be discovered and characterised. Here, we describe an optogenetic approach to study fatigable muscle weakness and NMJ function in larval zebrafish to facilitate screening approaches for uncovering novel CMS genes. Using blue-light illumination of spinal motoneurons that express channelrhodopsin-2 (ChR2) to induce muscle contraction, we measure motor defects at the behavioural, synaptic, and genetic level through a novel behavioural assay, standard whole-cell electrophysiology of individual muscle fibers and a customized NMJ gene panel. We employ this approach in synaptotagmin-2 (syt2) morphant zebrafish, an identified CMS gene model, to validate its usefulness. Our customized optogenetic behavioural assay successfully demonstrates reduced, fatigable, locomotor response during repeated activation of spinal motoneurons. Whole-cell electrophysiology recordings of optogenetically-elicited endplate currents in muscle fibers reveal similarities to altered properties of NMJ function in syt2 morphants reported in other studies using the standard paired motoneuron-muscle electrophysiology technique. Finally, we develop a genetic panel of CMS and NMJ-related genes to characterize the expression landscape of syt2 morphants to elucidate potential pathomechanisms and novel therapeutic targets. We propose that this three-tiered approach successfully links behaviour, synaptic motor function, and genetic expression and can be used as a tool in the screening of novel genes associated with CMS.

Duchenne muscular dystrophy (DMD) is the most common, lethal X-linked neuromuscular disorder of childhood and is caused by mutations in the Dmd gene that disrupt dystrophin expression. Although adeno-associated virus-mediated gene therapies hold tremendous promise for DMD treatment, their clinical applications have been limited by dose-dependent vector and genome-level toxicities. Here, we developed and tested a single-vector adenine base editing strategy as a potentially safer genome editing approach to recode the pathogenic nonsense mutation into a benign missense mutation in mdx4cvDMD mouse model. Delivered using a muscle-tropic adeno-associated virus (MyoAAV) at a clinically-feasible dose (4E13 VG/kg), this strategy enabled detectable molecular recoding of the mdx4cv mutation in mice ranging in age from 3 days to 6 months. Yet, the overall efficiency and therapeutic impact of in vivo base editing with this system was highest in mice treated at the juvenile stage, with animals administered MyoAAV vectors at 3 weeks of age showing robust recovery of dystrophin expression and significant improvement in muscle contractile properties only one month later. Notably, introduction of adenine base editors either earlier in development, in neonatal mice, or later, in adulthood, yielded substantially lower editing efficiencies, particularly in muscle satellite cells whose editing is essential to ensure durable rescue of dystrophin expression in growing and regenerating muscle. Taken together, these results demonstrate the therapeutic potential of single-vector adenine base editing for DMD and underscore the importance of recipient age and disease stage in achieving optimal treatment outcomes for this and other genetic muscle disorders.

Caveolinopathies caused by CAV3 mutations present with heterogeneous clinical phenotypes ranging from asymptomatic hyperCKemia to limb-girdle-type muscular dystrophy. Although prior imaging studies have described commonly affected muscles, structured modeling of muscle involvement patterns in caveolinopathy has not been established. We analyzed whole-body skeletal muscle computed tomography imaging in eight patients with pathogenic or likely pathogenic CAV3 variants, comprising 14 imaging study samples. Fat infiltration across 43 muscles was graded using modified Mercuri scores. Computational multivariate analysis,including principal component analysis, clustering, and pseudotime modeling,was applied to characterize severity staging and distribution patterns. A statistically supported, stage-dependent continuum of muscle involvement was identified. Most samples demonstrated a distributed limb-girdle-predominant pattern with coordinated progression across muscle clusters. In contrast, one patient (three samples in longitudinal series) exhibited a compartment-restricted thigh-dominant pattern characterized by early posterior and medial thigh involvement. Rectus femoris showed consistent stage-dependent progression, while greater medial gastrocnemius involvement was associated with advanced severity. None of the patients exhibited clinical evidence of rippling muscle disease. These findings suggest that integrating semi-quantitative imaging with computational modeling may provide an objective framework for characterizing muscle involvement patterns in CAV3-related myopathy.

BackgroundSkeletal muscle represents around 40% of total human body weight and exhibits remarkable plasticity. It can hypertrophy, atrophy, or regenerate in response to changes in activity, nutrient availability, or injury. The main component of striated muscle, the myofiber, is a post-mitotic, multinucleated cell that contains the muscles contractile unit, the sarcomere. The myonuclei within these fibers are specialized and differ in terms of gene expression and localization. Adult muscles also contain various other cell types, including adult muscle stem cells (MuSCs), macrophages, fibro-adipogenic progenitors (FAPs), and endothelial cells. MuSCs are central to muscle plasticity, and are capable of activation, proliferation, differentiation, and fusion to form new myofibers during regeneration, or to fuse with existing myofibers during hypertrophy. Muscle hypertrophy and myofibers enlargement involve increased protein synthesis and reduced protein degradation, as well as myonuclear accretion following satellite cell activation. Multiple signaling pathways, such as the mTOR pathway and the RhoA/SRF mechanotransduction pathway, are involved in these processes. MethodsWe performed single-nucleus RNA sequencing (snRNA-seq) on plantaris muscles of adult mice, comparing samples 7 days after hypertrophy induction (overload, 7OV) to non-hypertrophied controls (Ctl). RNAscope experiments on isolated myofibers identified the heterogeneity of myonuclei along the myofiber. ResultsSnRNA-seq analysis revealed a previously unknown population of myonuclei (UM). UM-Ctl, which is present only in the Ctl condition, and UM-7OV, only in the 7OV condition. These myonuclei are localised at the tips of myofibres. Furthermore, we determined that UM-7OV are not newly fused myonuclei from activated satellite cells. Trajectory analyses suggest that UM-Ctl transition into UM-7OV during hypertrophy, returning to a near-basal homeostatic state after 21 days of overload (21OV). Gene expression analysis showed that UM-Ctl and UM-7OV have distinct gene expression profiles compared to other myonuclei and respond differently to hypertrophy. ConclusionOur findings suggest the existence of a specific population of myonuclei with unique localization and gene expression profiles, which play distinct roles at baseline and during hypertrophy. These results highlight the differential properties of myonuclei in the myofiber and their potential specific functions in muscle homeostasis and adaptation.

Histopathological evaluation of skeletal muscle biopsies relies on subjective, semi-quantitative assessment with no standardized grading system. We developed a four-tissue deep learning segmentation pipeline using Cellpose-SAM for myofiber instance segmentation, a pixel classifier for fat infiltration, and watershed detection for nuclei. We applied this pipeline to 478 H&E whole-slide images from two independent cohorts: HuashanMuscle (n = 79; China; myotonic dystrophy type 1 [DM1], n = 28; limb-girdle muscular dystrophy type R1 [LGMDR1, calpainopathy], n = 12; type R2 [LGMDR2, dysferlinopathy], n = 22; controls, n = 17) and GTEx (n = 399; United States; three-level myopathy spectrum). Thirty-seven unique morphometric features were extracted per sample. Nuclear centralization index (NCI) and fiber size variability coefficient (fiber CV) discriminated myopathy from controls (p = 1.3E-05, rank-biserial r = 0.69; and p = 2.9E-04, r = 0.58, respectively). DM1 showed the highest NCI (median 0.121), consistent with its centronuclear pathology, and NCI correlated with CTG repeat count (Spearman rho = 0.46, p = 0.042, n = 20). In the GTEx cohort, both biomarkers exhibited significant dose-response trends across the myopathy spectrum (Jonckheere-Terpstra p < E-04). The MyoPath Score, a logistic regression composite of seven pathology indicators trained on GTEx, achieved AUC = 0.788 (LOO-CV 0.735) and transferred to the independent HuashanMuscle cohort with AUC = 0.873 without retraining. Segmentation achieved Dice coefficients of 0.92 (myofiber), 0.95 (fat), 0.87 (nucleus), and 0.88 (connective tissue), with intraclass correlation coefficients exceeding 0.88. NCI and fiber CV provide objective, reproducible quantitative biomarkers for skeletal muscle pathology severity assessment with potential as standardized grading criteria and clinical trial endpoints.

This study examined how lower-body lengthened partial (LP) versus full range of motion (FULL) resistance training affects acute post-exercise signaling, chronic hypertrophy, and cellular adaptations of the vastus lateralis (VL) muscle in resistance-trained men. Eight males (22{+/-}1 years old, 5.6{+/-}1.4 years training) completed a crossover study whereby VL biopsies were collected pre-exercise and 0, 3, and 24 hours following LP and FULL leg extension bouts for transcriptomic and anabolic signaling analyses (Experiment 1). Another 16 males (26{+/-}5 years old; 8.0{+/-}4.9 years training) completed an 8-week, twice-weekly lower-body intervention using a within-subject design (Experiment 2). One leg was assigned to FULL and the contralateral leg to LP training across three exercises (leg press, leg extension, and lying leg curl). Pre- and post-intervention outcomes included VL muscle cross-sectional area (mCSA) summed across five equidistant MRI-derived transverse slices and mid-thigh VL biopsy outcomes. As a secondary outcome, other hip and thigh muscles from Experiment 2 MRI scans were assessed. Condition x Time interactions for all outcomes were assessed using linear mixed-effects models. In Experiment 1, both conditions produced similar time-dependent changes in the VL transcriptome and anabolic (mTORC1 and Hippo) signaling, but minimal between-protocol interactions. In Experiment 2, VL summed mCSA significantly increased over time (mean change: 9.3 cm{superscript 2}, 95% CI [6.8, 11.8], P<0.001), but there was no clear evidence of differential change between protocols (LP-FULL change: -1.4 cm{superscript 2}, 95% CI [-6.1, 3.8], P=0.640). Additionally, no significant interactions existed for type I fiber CSA (P=0.476), type II fiber CSA (P=0.350), type I fiber myonuclei (P=0.813), type II fiber myonuclei (P=0.589), type I and II satellite cell number (P=0.102 and P=0.797, respectively), or total RNA content (P=0.537). Despite these null VL-centric findings, secondary Experiment 2 analyses provided some evidence that whole hamstring hypertrophy was greater following LP versus FULL (LP-FULL change: 3.9 cm{superscript 2}, 95% CI [-0.2, 7.9], P=0.058). In conclusion, 8 weeks of LP and FULL resistance training broadly elicit similar acute and chronic VL responses in previously trained men, though secondary hamstring findings suggest that differential responses may depend on exercises included in the resistance training program.

Neuromuscular contractures arising from neonatal brachial plexus injuries (NBPI) are highly disabling and currently incurable. We previously showed that contractures involve impaired longitudinal growth of denervated muscles, a defect mediated through myostatin (MSTN) signaling, a potent negative regulator of muscle size. However, MSTN-mediated contractures occur independent of canonical signaling pathways, including SMAD 2/3 and AKT/mTOR. Through a mouse model of NBPI, our present study extended these findings by revealing pharmacologic inhibition of JNK signaling, a noncanonical pathway downstream of MSTN, partially rescues contractures without restoring muscle length. Rather, JNK activation upregulates myofiber expression of the target gene Lmna, which encodes the nuclear envelope proteins Lamin A and Lamin C that are vital for nuclear stability, resulting in pervasive myonuclear displacement. These results suggest that other factors contribute to contracture pathology beyond deficits in longitudinal muscle growth. Further, while JNK inhibition does not restore length of denervated muscles, it impedes size and mass of normally innervated neonatal muscles, suggesting a requirement of JNK signaling for neonatal muscle growth. Our collective findings thereby establish new mechanistic insights into the molecular basis of aberrant muscle growth and neuromuscular contracture formation, potentially leading to novel targets for muscle restorative strategies and medical contracture prevention.

Duchenne muscular dystrophy (DMD) presents a critical therapeutic gap in adolescent patients, where extensive fibro-fatty muscle replacement and depletion of the regenerative niche render existing interventions insufficient. Prostaglandin E2 signaling through the EP4 receptor stimulates bone and muscle regeneration and repair, but systemic off-target effects have limited the clinical translation of EP4 agonism in diseases such as DMD. We therefore evaluated irodanoprost (IROD), a bone-targeted prodrug of an EP4-selective agonist, in a DMD rat model, comparing early- and late-intervention cohorts. In adolescent rats, 8 weeks of treatment reduced body weight deficit by 41.4% and restored hindlimb muscle mass and maximum tetanic force to wild-type levels. IROD dose-dependently inhibited fibro-adipogenic progenitor differentiation into -SMA myofibroblasts, facilitating active resolution of established fibrosis below pre-treatment baseline. This was accompanied by re-activation of a synchronized regenerative program marked by clustered eMHC fibers, restoring the total myofiber pool to wild-type levels. A strong linear correlation between intramuscular fat reduction and fibrosis resolution suggests that MRI-based fat imaging may serve as a non-invasive surrogate for monitoring anti-fibrotic efficacy. The efficacy of IROD is likely aided by the fact that while it selectively distributes to bone in healthy animals, we observed markedly enhanced accumulation of the drug in dystrophic muscle. These findings establish IROD as a pathology-targeted approach capable of resolving the fibro-fatty niche and restoring regenerative capacity in advanced DMD. SummaryPathology-targeted EP4 agonism resolves established fibrosis and restores myofiber regeneration in a rat model of adolescent Duchenne dystrophy. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/728674v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@141d591org.highwire.dtl.DTLVardef@12c4723org.highwire.dtl.DTLVardef@1f26514org.highwire.dtl.DTLVardef@ca146e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Laminin-2 (gene: LAMA2) is a key protein in the basement membrane of muscle and Schwann cells. A complete lack of this protein results in LAMA2-related congenital muscular dystrophy (LAMA2-RD), a severe muscle disease characterized by progressive muscle weakness, respiratory insufficiency, failure to thrive and shortened life span. One key signature of this disease is early onset of fibrosis coupled with poor muscle growth. We previously showed that TGF-{beta} and its activator, integrin-V, are elevated in dystrophic fibers of DyW mice, a mouse model of LAMA2- RD. Other than activating TGF-{beta}, integrin-V is also known to facilitate the transdifferentiation of various cell types to myofibroblasts. In this study we present evidence for transcriptional dysregulation of genes driving myofibroblast transdifferentiation and extracellular matrix (ECM) remodelling during the early development of DyW mice that is also reflected in muscle biopsies from young LAMA2-RD patients. We hypothesize that the early ECM remodelling, seen in both DyW mice and LAMA2-RD children, may explain the congenital onset of fibrosis with poor muscle growth seen in the disease.

IntroductionThe quality of preclinical studies for rare diseases, such as Duchenne muscular dystrophy (DMD), relies on the availability of comprehensive natural disease history data. In addition to the classic BL10-mdx mouse, in recent years, the D2-mdx model has increasingly been used as an alternative model due to its reportedly more severely impaired phenotype. To improve our understanding of disease progression in these two DMD models, we conducted a comprehensive natural history study. Materials and MethodsThis involved a cross-sectional analysis of key in vivo and ex vivo outcome measures performed in two independent laboratories, using the same study setup in compliance with TREAT-NMD Standard Operating Procedures (SOPs), while also taking advantage of site-specific expertise. Globally, largely comparable results were obtained across the two study sites. ResultsBody composition showed pronounced differences between the strains, with BL10-mdx mice displaying a hypertrophic and D2-mdx mice displaying an atrophic phenotype. Dystrophic mice of each strain exhibited significant alterations of disease-relevant indices related to muscle functionality and integrity, mostly worsening with age, in comparison to their wildtypes. Cardiac function was affected earlier and more severely in D2-mdx mice. DiscussionNotably, for some parameters, genetic-background related differences were observed, emphasizing the need to include control groups with matching genetic backgrounds in experimental designs. ConclusionsCollectively, our natural history study provides benchmark data for these two mdx mouse strains to guide model selection for preclinical DMD studies, allowing accurate data interpretation. HighlightsO_LIDistinct body composition phenotypes: BL10-mdx mice exhibit pseudohypertrophy while D2-mdx mice display pronounced atrophy. C_LIO_LIEarlier cardiac dysfunction in D2-mdx: D2-mdx mice develop reduced ejection fraction and stroke volume from 28 weeks, while BL10-mdx only at 52 weeks. C_LIO_LIGenetic background-dependent variations: Intrinsic deficits in wildtype D2 mice demonstrate that genetic background influences outcome measures independent of dystrophic pathology. C_LIO_LIComparable ex vivo muscle physiology: Despite divergent in vivo phenotypes, isolated muscle contractile parameters show similar impairment in both dystrophic models. C_LIO_LIMulti-site standardized validation: Cross-sectional study at two independent laboratories following harmonized TREAT-NMD Standard Operating Procedures. C_LI

Eya3 and Eya4 are two Eya genes expressed in adult myogenic stem cells, where they may act as SIX cofactors. We analyzed muscle regeneration in single and compound Eya3 and satellite cell-specific Eya4 mutant mice. A kinetic analysis of muscle regeneration after Notexin injury of the Tibialis Anterior revealed no major phenotype at 4, 14, and 30 days after injury in terms of PAX7+ cell number and myofiber cross-sectional area in Eya3 mutants, while all parameters were decreased in Eya4 mutants and further worsened in Eya3/Eya4 double mutants, in which we also observed a modification of the myofiber phenotype at 30 days after injury. Satellite cells were cultured ex vivo and Eya4 deletion was induced by Ad-Cre-mediated recombination. While single Eya3 mutant cells showed normal proliferation and differentiation, double mutant cells exhibited normal proliferation but failed to fuse. Analysis of their transcriptome revealed that the expression of Myomixer, Follistatin, and Noggin was severely downregulated specifically in double mutant cells, explaining their fusion deficiency. To gain a better understanding of the involvement of Eya genes during embryonic development and the genesis of PAX7+ myogenic stem cells, we analyzed Eya1 / ;Eya2 / , Eya3 / , Eya4 / , and Eya3 / ;Eya4 / E18.5 mutant fetuses at the limb and craniofacial levels. In Eya1 / ;Eya2 / fetuses, we confirmed the absence of distal limb muscles and observed reduced craniofacial muscles. In Eya3 / ;Eya4 / fetuses, craniofacial myogenesis appeared preserved and PAX7+ myogenic stem cells were present. BackgroundThe Eyes absent (Eya) genes encode transcriptional co-activators and phosphatases that function within the PAX-SIX-EYA-DACH (PSED) regulatory network. In skeletal muscle, EYA proteins cooperate with SIX homeoproteins to control myogenic gene expression during both embryonic development and adult regeneration. While Eya1 and Eya2 are predominantly expressed in embryonic myogenic progenitors and Eya3 and Eya4 are the dominant paralogs in adult satellite cells (SC), the specific and redundant contributions of individual family members to myogenesis remain poorly characterized. MethodsWe analyzed compound Eya mutant mice during adult Tibialis anterior muscle regeneration and during embryogenesis. We complemented this analysis by performing ex vivo myogenic stem cell cultures from compound Eya mutants and examining their fusion capacity. ResultsAnalysis of muscle regeneration following Notexin injury revealed that Eya2 and Eya3 single mutants display no major regenerative deficit. In contrast, satellite cell-specific deletion of Eya4 (Eya4sc/sc) caused a transient impairment of early regeneration, with reduced numbers of smaller regenerating MYH3+ (embryonic myosin heavy chain) myofibers and a transient decrease in SC number at 4 days post-injury (dpi). Compound Eya3-/-;Eya4sc/scdouble mutants showed a more severe and persistent phenotype, with decreased myofiber cross-sectional area, reduced myonuclear accretion, accumulation of PAX7+ cells associated with regenerated myofibers, and altered fiber-type composition at 14 and 30 dpi. Ex vivo analysis of double mutant SCs revealed a specific and complete blockade of myogenic fusion without defects in proliferation or MYOD expression. Transcriptomic analysis identified severe downregulation of Myomixer, Noggin, and Follistatin in differentiating Eya3-/-;Eya4-/- SCs. Open-access SIX1 and SIX4 ChIP-seq publicly available data confirmed direct binding at the Myomixer, Noggin, and Follistatin loci, supporting a direct SIX-EYA transcriptional mechanism. In parallel, embryonic analysis demonstrated that Eya1-/-;Eya2-/-E18.5 fetuses lack distal limb musculature and display severe craniofacial muscle hypoplasia, while in Eya3-/-;Eya4-/-fetuses limb and craniofacial musculature developed with no detectable defects. ConclusionsThese results reveal distinct temporal requirements for EYA proteins in skeletal muscle: EYA1 and EYA2 are essential SIX cofactors for embryonic myogenic fate acquisition in hypaxial and craniofacial progenitors, while EYA3 and EYA4 act redundantly in adult satellite cells to enable myogenic fusion by maintaining BMP antagonist expression and Myomixer activation downstream of the SIX-EYA transcriptional complex.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a mitochondrial lipid storage myopathy characterized by impaired fatty acid {beta}-oxidation, mitochondrial dysfunction, and progressive neuromuscular and cardiac disease. MADD is most commonly caused by pathogenic variants in electron transfer flavoprotein dehydrogenase (ETFDH), which encodes electron transfer flavoprotein-ubiquinone oxidoreductase (Etf-QO), a critical redox enzyme that transfers electrons from acyl-CoA dehydrogenases to the mitochondrial electron transport chain. Defective Etf-QO activity disrupts electron flow, promotes reactive oxygen species (ROS) production, and impairs cellular energy metabolism, linking abnormal lipid oxidation to oxidative stress-mediated tissue damage. To investigate the role of redox imbalance in MADD pathogenesis, we generated CRISPR/Cas9 knock-in Drosophila melanogaster models carrying patient-relevant Etf-QO missense mutations (L127R, S296C, and L399F; corresponding to human L138R, S307C, and L409F) within conserved FAD- and ubiquinone-binding domains. Mutant flies developed progressive locomotor impairment, reduced muscle performance, and marked lipid droplet accumulation in skeletal muscle, cardiac tissue, and fat bodies, indicating systemic defects in mitochondrial lipid utilization. Cardiac analyses demonstrated reduced fractional shortening, prolonged heart period, and increased arrhythmia index, consistent with metabolic cardiomyopathy associated with mitochondrial oxidative stress. In vivo respirometry revealed significantly decreased oxygen consumption, reflecting impaired oxidative phosphorylation. At the molecular level, mutant flies exhibited elevated ROS levels and ATP depletion, accompanied by increased expression of AMPK, PGC-1, and Tfam, suggesting activation of energy stress signaling and compensatory mitochondrial biogenesis. Importantly, endurance exercise significantly improved locomotor and cardiac function while reducing lipid accumulation and oxidative stress. Together, these findings establish a redox-centered in vivo model of MADD and identify oxidative stress as a major driver of disease pathology and a potential therapeutic target.

Pathogenic RYR1 variants are associated with a set of rare neuromuscular disorders termed RYR1-related disorders (RYR1-RD). Clinical manifestations of RYR1-RD include proximal/axial muscle weakness, delayed motor milestones, impaired mobility, muscle pain, and fatigue. Muscle-specific microRNAs (miRNAs) are mostly expressed in muscle tissue and can be detected peripherally in plasma. Using a digital detection system, here we identified and quantified differential amounts of miRNAs in six adult (four monoallelic and two biallelic) RYR1-RD patient plasma samples compared to controls. Overall, 51 differentially expressed miRNAs were identified and hsa-miR-4454+hsa-miR-7975, in particular, was significantly overexpressed relative to controls (+ 39-fold, P=0.00285). Exploration of these differentially expressed miRNAs warrant further investigation as potential biomarkers of RYR1-RD.

Macroglossia is a clinically significant feature of Beckwith-Wiedemann syndrome (BWS), but the cellular basis of tongue overgrowth remains poorly defined. Here, using pediatric tongue specimens from molecularly defined BWS subtypes and age-matched nonBWS controls, we show that BWS macroglossia is characterized by skeletal muscle fiber hypertrophy rather than increased fiber number. This phenotype is not explained by expansion or increased proliferation of satellite cells in situ, and prospectively isolated tongue satellite cells do not exhibit enhanced proliferation under growth conditions in vitro. Instead, BWS progenitors adopt distinct differentiation-associated regulatory states. IC2 loss of methylation cells sustain proliferative activity during differentiation and form enlarged myotubes, consistent with a cell-autonomous hypertrophic program. In contrast, pUPD11 cells display activation of NOTCH signaling and progenitor-associated programs, together with attenuated progression toward terminal myogenic differentiation. These findings identify skeletal muscle hypertrophy as a core tissue-level feature of BWS macroglossia and reveal that epigenetically defined BWS subtypes engage divergent myogenic programs that converge on a shared hypertrophic tissue phenotype. Together, these data define subtype-specific myogenic states in a rare human disease tissue and provide a framework for understanding how distinct epigenetic changes can produce a common overgrowth phenotype. HighlightsO_LIBWS macroglossia is associated with skeletal muscle fiber hypertrophy, not fiber hyperplasia C_LIO_LITongue satellite cell abundance and proliferation are not increased in situ in BWS C_LIO_LIIC2 loss of methylation cells sustain proliferation during differentiation and form enlarged myotubes C_LIO_LIpUPD11 cells show enhanced NOTCH signaling and a constrained myogenic state C_LI In briefTichy et al. show that Beckwith-Wiedemann syndrome macroglossia is driven by skeletal muscle hypertrophy and that distinct BWS molecular subtypes engage different myogenic regulatory programs. IC2 loss of methylation cells sustain proliferation during differentiation, whereas pUPD11 cells exhibit NOTCH-associated restraint of myogenic progression.