Skeletal Muscle

BackgroundPathogenic mitochondrial (mt)DNA variants cause neuromuscular disease with highly variable severity and phenotypic presentation, the reason for which is poorly understood. Cells are thought to tolerate the presence of pathogenic mtDNA variants up to a threshold proportion with little or no functional consequence, developing significant respiratory complex defects above this threshold. We developed a robust method to identify deficient muscle fibres, applied it to biopsies from 17 patients carrying the common m.3243A>G variant and examined the relationship between respiratory deficiency and m.3243A>G level in hundreds of single skeletal muscle fibres. We hypothesised that single-cell between-patient differences may explain the vast clinical heterogeneity of mtDNA disease. ResultsImmunohistochemical measurements of respiratory complexes I and IV and unsupervised machine learning identified muscle fibres with respiratory deficiency; the pattern of deficiency and proportion of deficient fibres (range 0-64%) varies between patients. Tissue homogenate m.3243A>G level is a poor surrogate for the broad and complex distributions of m.3243A>G level in single cells from individual patients. Estimated thresholds do not differ between patients, but sections with narrow m.3243A>G distributions have a lower proportion of deficient fibres. ConclusionsInter-individual differences in respiratory complex deficiency in muscle fibres from patients with m.3243A>G are more complex than previously thought and may be driven by differential segregation and expansion of mtDNA molecules. Our quantitative observations could constrain the range of feasible mechanisms responsible for phenotypic diversity in mitochondrial disease.

Manual quantitation of skeletal muscle myonuclear number, spatial orientation, and morphology is time-consuming and subject to error and bias. To overcome these limitations, we developed and validated a semi-automated, quantitative, and reproducible image-analysis pipeline. The workflow combines FIJI-based preprocessing with custom Python scripts to process immunohistological images of individual muscle fibers, enabling high-resolution and scalable quantification of nuclei. Analyses incorporate morphometric parameters including nuclear position, shape, and three-dimensional orientation, as well as centroid-to-skeleton distance and nearest-neighbor relationships to capture spatial patterns of myonuclear organization along the fiber. Outputs include per-fiber and biopsy-level summaries integrated with Imaris metrics. This semi-automated approach provides a robust and efficient platform for high-throughput analysis of myonuclear number and structural features across large single fiber datasets.

BackgroundDuchenne muscular dystrophy (DMD) is a devastating disease manifested in skeletal muscle by repetitious myonecrosis and regeneration. Because the regenerative process is closely linked to the cumulative severity of muscle damage, which is variably distributed within and between muscle groups, accurately quantifying muscle regeneration has remained a significant challenge. MethodsMyofibers are delineated by immunostaining for laminin, and subsequent image analysis employed to generate a masked outline precisely within each myofiber boundary. Morphometric parameters including minimal Ferets diameter, cross-sectional area, and circularity were measured for each myofiber. In addition, the number of Pax7-expressing satellite cells were quantified. To evaluate regenerative activity, newly formed myofibers were identified by immunostaining for expression of embryonic myosin heavy chain (eMHC). Necrotic myofibers were enumerated by immunofluorescent detection of immunoglobulin G (IgG) infiltration. The Regenerative Index (RI) was calculated as the number of regenerating (eMHC+) myofibers divided by the number of necrotic (IgG+) myofibers. Determination of RI was performed on muscle biopsies from 10 boys with DMD and 3 non-DMD controls of similar age. ResultsA trend toward an increasing minimal Ferets diameter, cross-sectional area and circularity was observed with increasing age in DMD boys, with circularity showing the strongest trend. Furthermore, compared to DMD boys 7- to 8-years old, the boys 9- to 11-years old had significantly increased myofiber circularity. Pax7-expressing cells were significantly elevated in DMD boys compared to control boys of similar ages, without any observation of age-related changes. Notably, the Regenerative Index in DMD boys exhibited a pronounced decline between 7-11 years of age, and a significant inverse correlation between RI and age was observed. ConclusionsUsing eMHC and IgG immunostaining to calculate RI accurately assesses regeneration despite the variation in histopathologic severity between biopsies. This methodology demonstrated a significant negative correlation between RI and age of DMD boys from 7 to 11 years of age.

This study aims to use spatial transcriptomics to characterize the cell-type-specific expression profile associated with the microscopic features observed in Wooden Breast myopathy. 1 cm3 muscle sample was dissected from the cranial part of the right pectoralis major muscle from three randomly sampled broiler chickens at 23 days post-hatch and processed with Visium Spatial Gene Expression kits (10X Genomics), followed by high-resolution imaging and sequencing on the Illumina Nextseq 2000 system. WB classification was based on histopathologic features identified. Sequence reads were aligned to the chicken reference genome (Galgal6) and mapped to histological images. Unsupervised K-means clustering and Seurat integrative analysis differentiated histologic features and their specific gene expression pattern, including lipid laden macrophages (LLM), unaffected myofibers, myositis and vasculature. In particular, LLM exhibited reprogramming of lipid metabolism with up-regulated lipid transporters and genes in peroxisome proliferator-activated receptors pathway, possibly through CD36-mediated signaling. Moreover, overexpression of fatty acid binding protein 5 could enhance fatty acid uptake in adjacent veins. In myositic regions, increased expression of cathepsins may play a role in muscle homeostasis and repair by mediating lysosomal activity and apoptosis. A better knowledge of different cell-type interactions at early stages of WB is essential in developing a comprehensive understanding.

The pathology in Duchenne muscular dystrophy (DMD) is characterized by degenerating muscle fibers, inflammation, fibro-fatty infiltrate, and edema, and these pathological processes replace normal healthy muscle tissue. The mdx mouse model is one of the most commonly used preclinical models to study DMD. Mounting evidence has emerged illustrating that muscle disease progression varies considerably in mdx mice, with inter-animal differences as well as intra-muscular differences in pathology in individual mdx mice. This variation is important to consider when conducting assessments of drug efficacy and in longitudinal studies. Magnetic resonance imaging (MRI) is a non-invasive method that can be used qualitatively or quantitatively to measure muscle disease progression in the clinic and in preclinical models. Although MR imaging is highly sensitive, image acquisition and analysis can be time intensive. The purpose of this study was to develop a semi-automated muscle segmentation and quantitation pipeline that can quickly and accurately estimate muscle disease severity in mice. Herein, we show that the newly developed segmentation tool accurately divides muscle. We show that measures of skew and interdecile range based on segmentation sufficiently estimate muscle disease severity in healthy wildtype and diseased mdx mice. Moreover, the semi-automated pipeline reduced analysis time by nearly 10-fold. Use of this rapid, non-invasive, semi-automated MR imaging and analysis pipeline has the potential to transform preclinical studies, allowing for pre-screening of dystrophic mice prior to study enrollment to ensure more uniform muscle disease pathology across treatment groups, improving study outcomes.

AO_SCPLOWBSTRACTC_SCPLOWMitochondrial myopathies are rare genetic disorders characterized by muscle weakness and exercise intolerance. Currently, no effective treatment exists for these myopathies. Interestingly, the pharmacological cyclophilin inhibitor cyclosporine A (CsA) extended lifespan and prevented loss of force and mitochondrial Ca2+ overload in muscle fibers in the skeletal muscle-specific Tfam knockout mouse model of lethal mitochondrial myopathy (Tfam KO). The unaffected expression of proteins involved in mitochondrial energy metabolism suggests that these improvements occurred without improvement in metabolism. In this study, we aimed at investigating the effects of four weeks of CsA administration on in vivo contractile function and mitochondrial energy production in Tfam KO mice. The treatment started before the terminal phase with severe muscle weakness and weight loss. Our results show that CsA treatment delayed progression into the terminal disease phase. This occurred without any obvious positive effects on mitochondrial energy production at rest or during fatigue induced by repeated contractions. In conclusion, cyclophilin inhibitors may have the potential of counteracting devastating muscle weakness in patients with mitochondrial myopathies most probably by preventing deleterious effects triggered by excessive mitochondrial Ca2+ uptake rather than by improving mitochondrial energy production.

Volumetric muscle loss (VML) injuries result in chronic fibrosis, inflammation, and persistent functional deficits. Fibro-adipogenic progenitor (FAP) cells are a heterogeneous, muscle-resident stromal cell population that play a crucial role in muscle regeneration, but also contribute to fibrosis in muscle disease. The role of FAPs in VML is not well established and may be critical target to ensure functional muscle regeneration after VML. We utilized a VML model in the mouse quadriceps to study the location, secretome, surface marker distribution, gene expression, and single-cell transcriptional profile of FAPs after VML. After VML, a subpopulation of FAPs highly expressed {beta}1-integrin and were elevated in the post-VML muscle tissue; these FAPs had increased fibrotic gene expression and increased myofibroblast differentiation potential. Transforming growth factor-{beta}1 (TGF-{beta}1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) were identified as secreted proteins from VML derived FAPs that produced both pro-fibrotic and anti-myogenic signaling. These data establish an aberrant FAP sub-population that are elevated in VML injury and provides novel targets for future scarless muscle regeneration in VML.

Skeletal muscle biopsy commonly used for light microscopic, electron microscopic and biochemical and transcriptional evaluation remains the gold standard for establishing the etiology of a myopathy. While most myopathies exhibit one or more phenotypes, early stages or several metabolic myopathies often exhibit normal muscle morphology, making diagnosis difficult. In such cases where standard staining techniques fail to offer definitive diagnostic information, a combination of expensive and time-consuming electron microscopy and biochemical testing is required to provide definitive diagnosis. As a step toward overcoming these limitations in diagnostic pathology of skeletal muscle tissue, here we report the application of parameter estimation machine learning approaches on immunofluorescent images of human skeletal muscle tissue acquired using fluorescent microscopy. The machine learning morphometric approach enables the recognition of fine cellular changes in skeletal muscle tissue, allowing determination of skeletal muscle remodeling as a consequence of immobilization.

Joint hyper-resistance is a common symptom in neurological disorders. It has both neural and nonneural origins, but it has been challenging to distinguish different origins based on clinical tests alone. Combining instrumented tests with parameter identification based on a neuromechanical model may allow us to dissociate the different origins of joint hyper-resistance in individual patients. However, this requires that the model captures the underlying mechanisms. Here, we propose a neuromechanical model that, in contrast to previously proposed models, accounts for muscle shortrange stiffness and its interaction with muscle tone and reflex activity. We collected knee angle trajectories during the pendulum test in 15 children with cerebral palsy (CP) and 5 typically developing children. We did the test in two conditions - hold and pre-movement - that have been shown to alter knee movement. We modeled the lower leg as an inverted pendulum actuated by two antagonistic Hill-type muscles extended with SRS. Reflex activity was modeled as delayed, linear feedback from muscle force. We estimated neural and non-neural parameters by optimizing the fit between simulated and measured knee angle trajectories during the hold condition. The model could fit a wide range of knee angle trajectories in the hold condition. The model with personalized parameters predicted the effect of pre-movement demonstrating that the model captured the underlying mechanism and subject-specific deficits. Our model thus allows us to determine subject-specific origins of joint hyper-resistance and thereby opens perspectives for improved diagnosis and consequently treatment selection in children with spastic CP.

Bethlem myopathy (BM) is an incurable muscle disease characterized by joint contractures and muscle weakness worsening with age. BM results from mutations in genes encoding one of the three chains of collagen VI (ColVI), a component of the skeletal muscle extracellular matrix produced by interstitial fibroblasts. A still unresolved issue in BM is how alteration in ColVI present outside muscle fibers induces dysfunction within muscle fibers. In the present study, we explored properties of excitation-contraction coupling on isolated fast skeletal muscle fibers from one-year old zebrafish col6a1{Delta}ex14 harboring an exon-skipping mutation (col6a1{Delta}ex14) that is the most frequently found in BM patients. Col6a1{Delta}ex14 fish muscle exhibited age-dependent progressive loss of ColVI deposition, accompanied with defects in basement membrane organization and ColVI intracellular accumulation. Muscle action potentials were found to be unchanged in col6a1{Delta}ex14 fish as compared to wild-type. The density of charge movements produced by depolarization-induced activation of dihydropyridine receptors (DHPRs), that control sarcoplasmic reticulum (SR) Ca2+ release, was found to be reduced and their voltage-dependence shifted toward negative potentials in col6a1{Delta}ex14 fish. Concomitantly, the voltage dependence of depolarization-evoked intracellular Ca2+ transients was also shifted toward negative voltages, promoting in this way an elevated pathogenic SR Ca2+ leak at resting membrane potentials, as evidenced by higher density of Ca2+ sparks at rest, larger SR Ca2+ release in response to long-duration depolarizations between -70 and -40 mV and reduced twitch muscle force and swimming performance. Finally, clusters of 1S subunits of DHPR were found to be mis-localized in mutant fibers t-tubules. These data suggest that Col6 deficiency in BM leads to an alteration of the DHPR function that contributes to promote a pathogenic SR Ca2+ leak. DHPR could represent the still elusive link that allows altered myomatrix to transduce pathogenic signals within muscle.

Skeletal muscle is a structurally organized and functionally diverse organ composed of heterogeneous myofiber types and supporting non-myocyte populations that act in concert to generate force, regulate metabolism, and maintain systemic homeostasis. Myopathies occur in many different diseases, but the mechanisms that drive these muscle pathologies are still largely unknown, partly because conventional approaches cannot link histopathological features to molecular states at single-fiber resolution. To address this challenge, we brought histopathology and spatial transcriptomics together by applying high-resolution Seq-Scope technology to a rodent model of mTORC1 hyperactivation, which produces diverse pathological alterations within individual myofibers. Cross-sections from extensor digitorum longus (EDL) and soleus (SOL), two muscles with distinct fiber-type compositions, were profiled to determine how transcriptome changes are linked to histopathological outcomes. Our analyses reveal that mTORC1 hyperactivation elicits distinct, fiber-type-dependent pathological programs. Type I and IIa fibers, abundant in SOL but scarce in EDL, were largely resistant to mTORC1-induced pathology, exhibiting only minimal morphological alterations and no fiber type-specific responses beyond those commonly observed throughout the tissue. In contrast, type IIx fibers, shared between both muscles, diverged into opposing fates: in SOL, they underwent abnormal enlargement driven by sustained growth signaling, cytoskeletal remodeling, and impaired proteostasis with defective autophagy; whereas in EDL, they developed basophilia characterized by lipid-supported respiration fueling excessive ribonucleotide synthesis and RNA accumulation. Within the same muscle, type IIb fibers displayed striking heterogeneity with discrete transcriptional states encompassing canonical stress responses, oxidative metabolic activation, and developmental reprogramming. In parallel, non-myocytic populations, including activated macrophages and fibroblasts, accumulated preferentially in SOL, forming a fibrotic microenvironment supporting inflammation, tissue remodeling and hypertrophy. Taken together, these findings reveal that sustained mTORC1 signaling disrupts muscle homeostasis through distinct metabolic and structural routes, directly linking histopathological phenotypes to their molecular states at single-fiber resolution.

SummaryWhile manual quantification is still considered the gold standard for skeletal muscle histological analysis, it is time-consuming and prone to investigator bias. We assembled an automated image analysis pipeline, FiNuTyper (Fiber and Nucleus Typer), from recently developed deep learning-based image segmentation methods, optimized for unbiased evaluation of fresh and postmortem human skeletal muscle. We validated and utilized SERCA1 and SERCA2 as type-specific myonucleus and myofiber markers. Parameters including myonuclei per fiber, myonuclear domain, central myonuclei per fiber, and grouped myofiber ratio were determined in a fiber type-specific manner, revealing a large degree of gender- and muscle-related heterogeneity. Our platform was also tested on pathological muscle tissue (ALS) and adapted for the detection of other resident cell types (leukocytes, satellite cells, capillary endothelium). In summary, we present an automated image analysis tool for the simultaneous quantification of myofiber and myonuclear types, to characterize the composition of healthy and diseased human skeletal muscle. HighlightsO_LIA deep learning-based automated platform for skeletal muscle microscopic analysis C_LIO_LIHigh-fidelity identification and characterization of myonuclei and myofibers C_LIO_LIValidation of SERCA1 and SERCA2 as markers for myofiber and myonuclear subtypes C_LIO_LICharacterization of healthy and pathological human skeletal muscle tissue features C_LIO_LIAdaptations provided for studies on other resident cell types like satellite cells C_LI eTOC BlurbAn automated platform for unbiased analysis of skeletal muscle immunohistochemical images, focusing on type-specific myofiber-myonucleus relationships, facilitating high-throughput studies of healthy and diseased tissues.

BackgroundIn vertebrates, skeletal muscle grows postnatally through different strategies. While mammals predominantly rely on fiber hypertrophy after birth, many teleost fish retain the unique ability to generate new fibers via hyperplasia well into juvenile stages. The molecular mechanisms governing the transition between hyperplastic-hypertrophic and hypertrophic growth modes in fish muscle remain poorly understood. ResultsWe generated a single-cell transcriptomic atlas of muscle-derived cells from juvenile Oncorhynchus mykiss (rainbow trout) at five growth stages. Fifteen tissue resident cell populations were identified, including eight myogenic subpopulations spanning from quiescent stem cells to terminally differentiating myocytes. Two distinct transcriptional trajectories were uncovered thanks to RNA velocity analysis: one present only during hyperplastic growth and another maintained throughout growth, indicating specialization of satellite cells toward hyperplasia or hypertrophy. Comparative analyses with human single-cell atlases indicate that subpopulations specifically related to hyperplasia and hypertrophy are conserved, depending on stage (fetal or adult). Strikingly, we identified a population of pax7+/pdgfr+ cells, indicating plasticity toward fibroblastic lineage and associating these cells with hypertrophic growth. Furthermore, both intrinsic changes in muscle stem cells and extrinsic remodeling of the extracellular matrix accompanied the decline of hyperplasia, highlighting dynamic crosstalk between myogenic and mesenchymal compartments. ConclusionsOur findings reveal the existence of two transcriptionally distinct muscle stem cell fates that underlie hyperplastic versus hypertrophic growth in fish. The identification of a tissue-resident pax7+/pdgfr+ subpopulation provides new insights into muscle stem cell plasticity and niche remodeling. This work establishes a comparative framework to explore the regulation of postnatal muscle growth across vertebrates.

Spinal Muscular Atrophy (SMA) is due to a deficit in SMN protein encoded by the SMN1 gene. SMN-targeted disease modifying treatments have greatly improved the clinical outcomes of this neuromuscular disease. However, uncertainties remain regarding their long-term efficacy and non-neuronal tissue involvement in disease progression. We found that SMA type II patient muscles display a reduced number of quiescent PAX7+ Muscle Stem Cells (MuSC). In SMA mice, we showed that SMN is an important regulator of myogenic progenitor fate during early postnatal growth. In Pax7 Cre-driven conditional knockout mouse models, we demonstrated that high levels of SMN are required to ensure the maintenance of the quiescent MuSC pool in adult muscle. We further established that depletion of SMN-deficient MuSC yielded neuromuscular junctions remodeling followed by a non-cell autonomous loss of motor neurons in the long term. Overall, our findings demonstrate that MuSC are a crucial therapeutic target for SMA treatment. HIGHLIGHTSO_LISMN regulates myogenic lineage progression and quiescent MuSC pool establishment during postnatal growth C_LIO_LIBoth Smn alleles are necessary for the survival of quiescent MuSC in adult muscle C_LIO_LIDepletion of SMN-deficient MuSC leads to NMJ remodeling and non-cell autonomous loss of MN C_LI

Lack of dystrophin is the genetic basis for the Duchenne muscular dystrophy (DMD). However, disease severity varies between patients, based on specific genetic modifiers. D2-mdx is a model for severe DMD that exhibits exacerbated muscle degeneration and failure to regenerate even in the juvenile stage of the disease. We show that poor regeneration of juvenile D2-mdx muscles is associated with enhanced inflammatory response to muscle damage that fails to resolve efficiently and supports excessive accumulation of fibroadipogenic progenitors (FAPs). Unexpectedly, the extent of damage and degeneration of juvenile D2-mdx muscle is reduced in adults and is associated with the restoration of the inflammatory and FAP responses to muscle injury. These improvements enhance myogenesis in the adult D2-mdx muscle, reaching levels comparable to the milder (B10-mdx) mouse model of DMD. Ex vivo co-culture of healthy satellite cells (SCs) with the juvenile D2-mdx FAPs reduced their fusion efficacy and in vivo glucocorticoid treatment of juvenile D2 mouse improved muscle regeneration. Our findings indicate that aberrant stromal cell response contributes to poor myogenesis and greater muscle degeneration in dystrophic juvenile D2-mdx muscles and reversal of this reduces pathology in adult D2-mdx mouse muscle, identifying these as therapeutic targets to treat dystrophic DMD muscles.

Transmission electron microscopy (TEM) is the gold standard for assessing subcellular glycogen localization in skeletal muscle fibres, but conventional manual analysis is extremely time-consuming and limits large-scale studies. Here, we developed and validated a deep learning-based semantic segmentation approach to automate quantification of glycogen particles across defined subcellular compartments in human skeletal muscle. Skeletal muscle biopsies were obtained from seven healthy men under conditions of normal, depleted, and supercompensated glycogen content. TEM images were acquired from myofibrillar and subsarcolemmal regions and manually annotated to train two complementary attention U-Net models: a region model identifying subcellular structures (intermyofibrillar space, intramyofibrillar regions including A-band, I-band and Z-disc, and mitochondria) and a glycogen model detecting individual glycogen particles. Combining the two models enabled estimation of compartment-specific glycogen areal densities. Model performance was evaluated against manual point-counting. At the fibre level, estimates based on 10-12 images per region achieved biases below 15% and coefficient of variation below 26% for all compartments. Importantly, model-derived total glycogen volume density showed strong concordance with biochemically determined muscle glycogen content across biopsies. In conclusion, this validated semantic segmentation workflow provides a robust, objective, and highly time-efficient tool for quantifying subcellular glycogen distribution in skeletal muscle. The model substantially reduces analysis time and enables high-throughput investigations of compartmentalized glycogen metabolism, with model weights and code made openly available.

Therapeutic strategies for Duchenne Muscular Dystrophy (DMD) will likely require complementary approaches. One possibility is to explore genetic modifiers that improve muscle regeneration and function. The beneficial effects of the overexpression of Jagged-1 were described in escaper golden retriever muscular dystrophy (GRMD) dogs that had a near-normal life and validated in dystrophin-deficient zebrafish (1). To clarify the underlying biology of JAG1 overexpression in dystrophic muscles, we generated a transgenic mouse (mdx5cv-JAG1) model that lacks dystrophin and overexpresses human JAG1 in striated muscles. Skeletal muscles from mdx5cv-JAG1 and mdx5cv mice were studied at one, four, and twelve-month time points. JAG1 expression in mdx5cv-JAG1 increased by three to five times compared to mdx5cv. Consequently, mdx5cv-JAG1 muscles were significantly bigger and stronger than dystrophic controls, along with an increased number of myofibers. Proteomics data show increased dysferlin in mdx5cv-JAG1 muscles and an association of Nsd1 with the phenotype. Our data supports the positive effect of JAG1 overexpression in dystrophic muscles. Significance StatementDuchenne Muscular Dystrophy (DMD) patients present a progressive decline in motor function. DMD is caused by mutations in the DMD gene that lead to the absence of dystrophin - an essential component of muscle cells. However, dystrophin-deficient dogs overexpressing JAG1 had a normal lifespan with remarkable motor function. In this study, we increased expression of human JAG1 in mouse skeletal muscles lacking dystrophin to explore mechanisms responsible for these benefits. Our observations show that overexpression of JAG1 counterbalances the lack of dystrophin by generating bigger and stronger muscles as the mouse ages. Moreover, our proteomics dataset suggests a role of dysferlin in the phenotype. Therefore, our study supports the exploration of JAG1 in pre-clinical models.

Skeletal muscle function is inferred from the spatial arrangement of myofiber architecture and the molecular and metabolic features of myofibers. Features of myofiber types can be distinguished by the expression of myosin heavy chain (MyHC) isoforms, indicating contraction properties. In most studies, a local sampling, typically obtained from the median part of the muscle, is used to represent the whole muscle. It remains largely unknown to what extent this local sampling represents the entire muscle. Here we studied myofiber architecture over the entire wild type mouse tibialis anterior muscle, using a high-throughput procedure combining automatic imaging and image processing analyses. We reconstructed myofiber architecture from consecutive cross-sections stained for laminin and MyHC isoforms. The data showed a marked variation in myofiber geometric features, as well as MyHC expression and the distribution of neuromuscular junctions, and suggest that muscle regions with distinct properties can be defined along the entire muscle. We show that in these muscle regions myofiber geometric properties align with biological function and propose that future studies on muscle alterations in pathological or physiological conditions should consider the entire muscle.

Mutations in the DMD gene lead to Duchenne muscular dystrophy, a severe X-linked neuromuscular disorder that manifests itself as young boys acquire motor functions. DMD is typically diagnosed at 2 to 4 years of age, but the absence of dystrophin negatively impacts muscle structure and function before overt symptoms appear in patients, which poses a serious challenge in the optimization of standards of care. In this report, we investigated the early consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Here, dystrophin deficiency was linked to marked dysregulations of cell junction protein families involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether, this work demonstrates that in vitro, dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development, which should be considered in future therapeutic strategies for DMD.

Mitochondria are essential for regulation of the metabolic state of skeletal muscle, making their structure and function crucial for muscle performance. Myosin VI (MVI), an unconventional minus-end-directed motor, is expressed in skeletal muscle and myogenic cells. To explore its role in mitochondrial function and muscle metabolism, we used MVI knockout mice (Snells waltzer, SV) and their heterozygous littermates. We analyzed muscle samples from newborn (P0) and adult mice (3- and 12-month-old) and found that both MVI mRNA and protein levels were highest in newborn muscles and decreased with age. MVI expression also varied by muscle type, being highest in the slow-twitch soleus muscle (SOL) of adult mice. Loss of MVI had the most significant effects on SOL, which contains the highest number of mitochondria compared to fast-twitch muscles. MVI loss resulted in reduced respiratory capacity and ATP production in myogenic cells, indicating impaired mitochondrial function. Furthermore, MVI deficiency caused a shift from glycolytic to oxidative fiber types, especially in SOL. We also observed increased phospho-AMPK levels in MVI-KO SOL across all time points, along with downregulation of the mTOR pathway and upregulation of proteins involved in lipolysis. These findings highlight MVI as a novel regulator of metabolic processes in skeletal muscle.