Skeletal Muscle

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.

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.

EMG-based muscle force predictions are often inaccurate following active muscle stretch or shortening because of residual force enhancement (rFE) or depression (rFD), respectively, which can alter the neural drive to a muscle. However, the extent of neural drive modulation due to rFE or rFD remains unknown, making it difficult to correct EMG-based force predictions. Therefore, seventeen participants performed dorsiflexion contractions at 20 and 40% of maximum voluntary torque (MVT) in three conditions: stretch-hold, shortening-hold, and fixed-end reference (REF) conditions. The ankle dorsiflexion torques and angles were matched using dynamometry to the REF condition over a 10-s steady state following a 1-s 25{degrees} stretch or shortening, during which we recorded and decomposed tibialis anterior individual motor unit action potentials from high-density surface EMG recordings to gain insights into neural drive. Normalized EMG amplitudes were 2% lower following stretch and 1 or 3% higher following shortening relative to REF at 20 versus 40% MVT (p[≤].008), respectively. Discharge rates (DRs) from 19 matched motor units per person on average obtained via DEMUSE and MUedit were similar (p=.871). Following stretch and shortening, DRs were [~]1 Hz lower (p[≤].004) and 0 (p=.966) to 1 Hz higher relative to REF (p=.003), respectively. More unique motor units were also detected following shortening versus REF and in REF versus following stretch. These findings indicate that to account for rFE or rFD, neural drive is respectively decreased or increased via reduced or additional motor unit recruitment and DR modulation, with a contraction-intensity specific discharge rate modulation following active shortening.

Spinal muscular atrophy (SMA) is characterized by motor neuron degeneration caused by deficiency of the survival motor neuron (SMN) protein. However, evidence increasingly supports broader systemic involvement. This study aimed to examine cardiac pathology in SMA patients and to investigate how reduced SMN levels impact cardiomyocyte homeostasis. We analyzed postmortem data from 14 SMA type I patients from the pre-treatment era, integrating gross anatomical, histopathological, and clinical findings. To investigate cardiomyocyte-intrinsic effects of SMN deficiency, healthy human cardiomyocytes were subjected to SMN knockdown and assessed using metabolic assays and transcriptomic profiling. Key findings were further investigated in vivo using the Smn2B/- mouse model of SMA. We found heterogeneous cardiac involvement in SMA patients, including cardiomegaly, variable fat deposition and interstitial fibrosis. SMN knockdown in human cardiomyocytes induced a metabolic shift and widespread transcriptional dysregulation, with pathway analyses identifying selective upregulation of PTEN signaling. Elevated PTEN protein levels were observed in a subset of human SMA hearts and in early postnatal hearts of Smn2B/- mice. Our results demonstrate that the heart remains a biologically relevant target of SMN deficiency and highlights cardiomyocyte-specific metabolic and PTEN signaling alterations as potential contributors to cardiac involvement in SMA.

DNA methylation is a key epigenetic mechanism influencing gene regulation and cellular identity. In skeletal muscle, methylation contributes to fiber-type specification, metabolic programming, and satellite cell function, with evidence of sex-specific differences. Here, we investigated whether spatial regionalization of gene expression along the proximal-distal axis of the tibialis anterior (TA) is mirrored by corresponding patterns of DNA methylation. Using MeDseq on TA sections from muscles previously analyzed by spatial transcriptomics, we profiled methylation across transcriptional start sites (TSS), gene bodies, and regulatory elements. Despite robust spatial differences in transcriptomes, methylation patterns were largely uniform along the proximal-distal axis, indicating that DNA methylation does not underlie regional gene expression in adult TA muscle. In contrast, sex emerged as the primary determinant of methylation variation. Male muscles exhibited widespread hypermethylation at TSS, gene-bodies and regulatory regions, corresponding with sex-specific transcriptional programs, including glycolytic fiber enrichment in males and oxidative fiber markers in females. Notably, chromatin- and methylation-associated regulators such as Setd7, Gsk3a, and Bmyc were upregulated in males, suggesting mechanisms linking transcriptional control to epigenetic state. These findings highlight that while spatial gene expression is transcriptionally driven, sex-specific epigenetic programs dominate adult skeletal muscle, underscoring the need to consider sex in multi-omic studies of muscle biology.

Force production relies on the interaction between neural control of spinal motoneurons and the mechanical properties of the muscles. Changes in muscle length provide a useful model for exploring this interaction; however, studies typically assess these properties in the same muscle undergoing length change. This study investigated how altering the length of one muscle influences motor unit discharge behavior of its synergists. Eighteen healthy participants performed submaximal isometric knee extensions with the hip joint positioned at 90{degrees} (shortened rectus femoris, RF) and 180{degrees} (lengthened RF). At each hip position, participants followed trapezoidal force profiles at 10% and 30% of maximal voluntary contraction (MVC), while high-density surface electromyography (HDsEMG) was recorded from the synergistic vastus medialis and vastus lateralis (VL). Motor unit spike trains were decomposed from HDsEMG, tracked across hip positions, and analyzed for mean discharge rate and coefficient of variation of interspike interval (CoV-ISI). Lengthening the RF led to increased discharge rates of vasti motor units at 10% MVC, but not 30% MVC, with no changes in CoV-ISI. To further explore these force-dependent changes in discharge rate, two sets of experiments were conducted. The first showed that the discharge rate at recruitment during ramp-up contractions increased with RF lengthening, but only for vasti units recruited below 20% MVC. In the second, electrically evoked twitch contractions in the vasti revealed reduced twitches at 180{degrees} during low-frequency, but not high-frequency stimulation. These findings collectively suggest that the force-dependent changes in the vasti motor unit discharge rates are likely driven by RF-length dependent changes in the vasti muscles contractile properties.

Unlike mammals and birds, where new muscle fiber formation (hyperplasia) ceases around birth, large and fast-growing fish such as rainbow trout undergo a spectacular post-hatching surge of hyperplasia, followed by a considerably delayed hyperplasia decline. This study investigated the roles of the satellite cells (SCs) and their niche in this decline by determining the number and the myogenic capacity of the muscle progenitors as well as the functionality of their direct tissue environment. Histological analysis revealed a significant decrease in hyperplasia (fibers <25 {micro}m) and SC numbers (Pax7+) between 10 g and 500 g trout. Transplantation experiments using muscle-derived cells (MDCs) from mlc2-GFP transgenic trout (10 g to 2 kg donors into 10 g to 2 kg recipients) demonstrated a marked decline in both intrinsic myogenic capacity and niche functionality as trout grow from 10 g to 500 g. Detailed analyses of GFP+ fibers produced after transplantation showed an enrichment of small-diameter GFP+ fibers in 10 g but not 100 g trout recipient muscles, showing a rapid impairment in niche ability to support hyperplasia. In addition, transplantation of MDCs from trout of different ages but the same weight, showed that increasing trout weight, but not aging, is associated with an impairment of the myogenic capacity of progenitors and their niche. Overall, these findings show that the muscle hyperplasia decline in trout is primarily driven by early impairment of the SC niche, followed by a reduction in their myogenic capacity and number, with weight gain playing a more critical role than aging.

IntroductionSuccessful reinnervation following peripheral nerve injury is highly variable, and the molecular programs underlying human muscle degeneration and recovery remain poorly defined. There is a critical need for high-resolution, spatially resolved gene expression data from human skeletal muscle obtained in clinically relevant settings. This study aimed to establish the feasibility of applying spatial transcriptomics to intra-operatively human muscle biopsies and to generate a framework for identifying gene expression signatures associated with reinnervation outcomes. MethodsTo validate the workflow, we collected biopsies intraoperatively from upper-extremity muscles during standard-of-care orthopaedic surgical procedures 5 months after traumatic brachial plexus injury. The flash-frozen biopsy was processed using the 10x Genomics Visium HD high-resolution platform. Quality metrics confirmed high RNA integrity and robust transcript detection at 8 {micro}m resolution. ResultsGenes involved in neuromuscular junction formation, degeneration, and regeneration were identified at subcellular resolution and showed fiber-type-specific expression patterns. Analyses were performed using complementary approaches in Seurat and Loupe Browser. ConclusionsTogether, these findings demonstrate the feasibility of spatial transcriptomics in human muscle, establish baseline gene-expression signatures, and provide a foundation for future studies aimed at identifying biomarkers associated with successful reinnervation and improved nerve-repair strategies.

Skeletal muscle hypertrophy results from the integrated regulation of anabolic and proteolytic processes in response to mechanical loading. Although increases in resistance training (RT) volume are used to increase mechanical stress, it remains uncertain whether large and abrupt volume progressions could exceed muscle adaptive capacity by disrupting the balance between anabolic and catabolic signaling. The present study investigated whether a large increase in weekly RT volume (+120%) leads to impaired hypertrophic outcomes and intracellular regulatory responses compared with a modest increase (+20%). Twenty-five resistance-trained men and women (18-35 years old) completed an 8-week randomized, single-blind, within-subject unilateral intervention. Each participant trained both legs twice weekly, with one leg assigned to the large (VOL120) and the contralateral leg to the modest (VOL20) weekly volume progressions relative to habitual training volume. Vastus lateralis muscle cross-sectional area (mCSA) was assessed by ultrasonography before and after training. Muscle biopsies were obtained at baseline, post-intervention, and 24 h after the last session to quantify muscle fiber cross-sectional area (fCSA), satellite cell myonuclear content, and anabolic/catabolic signaling markers. Both protocols induced increases in mCSA over time (p<0.001), with no protocol vs. time interaction. No significant effects were observed for fCSA nor satellite cell number or myonuclear content. Additionally, molecular responses related to translational regulation and protein degradation were largely similar between protocols. Collectively, these data indicate that a large, abrupt increase in weekly set volume does not impair hypertrophic adaptations or meaningfully alter the anabolic-catabolic signaling profile in resistance-trained individuals.

Optimized histological techniques are crucial for visualizing cellular morphology across zebrafish tissues. Here, we report a rapid and reliable hematoxylin and Oil Red O (H-ORO) staining protocol for frozen sections that can be completed in less than three minutes. Mayers hematoxylin is used for nuclear staining, followed by Oil Red O (ORO) to visualize lipid-rich structures such as the endomysium surrounding myofibers, white matter of the brain, and myelin layers of major axonal tracts. Importantly, our optimized H-ORO protocol preserves tissue integrity and minimizes artifacts such as myofiber shrinkage commonly observed with ethanol-based hematoxylin and eosin (H&E) staining in both frozen and paraffin sections.

BackgroundPeripheral nerve trauma denervates skeletal muscle resulting in paralysis and atrophy that is reversible if timely reinnervation occurs, due to its regenerative capacity. If reinnervation is delayed muscles regenerative ability is exhausted and resident fibroadipogenic progenitors (FAPs) differentiate into adipocytes and fibroblasts that replace muscle with non-contractile fibrotic tissue and fat, resulting in physical disability. Prostaglandin E2 (PGE2) inhibits adipogenesis and fibrosis in other tissues. We determined whether PGE2 could inhibit fibro-fatty degradation of long-term denervated muscle. MethodsWe utilized the rat tibial nerve transection model, denervating the gastrocnemius and selected a 5 week post-denervation time point to represent short-term muscle denervation injury (reversible with reinnervation), and 12 weeks to represent sustained, irreversible injury. Gastrocnemius FAPs were isolated via FACS and grown in culture to assess endogenous PGE2 production and the proliferative and differentiation response to exogenous PGE2. We evaluated transcript and protein expression of PGE2 synthesizing enzyme PTGS2, PGE2 degrading enzyme 15-PGDH and markers of proliferation, adipogenesis and fibrogenesis using RT-qPCR, immunofluorescence and SDS-PAGE/Western blotting. Paracrine impact of FAPs produced PGE2 was assessed by treating C2C12 myoblasts with FAPs conditioned media. ResultsTranscript expression of PTGS2 was increased and 15-PGDH decreased (4.37{+/-}2.63 and -3.06{+/-}0.85 fold change respectively, p<0.05) in 5 week, but not 12 week denervated gastrocnemius, consistent with increased PGE2 production in 5 week denervated muscle. Similarly, PTGS2 transcript levels were significantly increased (2.58{+/-}0.33 fold change, p<0.05) and 15-PGDH decreased (-5.24{+/-}3.19 fold change, p<0.05) in FAPs isolated from 5 week, but not 12 week denervated muscle, demonstrating that FAPs are a source of PGE2 in short-term denervated muscle. 16,16-dimethyl PGE2 did not impact naive FAPs in vitro proliferation, but significantly inhibited their differentiation as demonstrated by 88.9%, 82.3% and 94.2% decreases in FAPs expression of adipogenic marker perilipin-1, fibrogenic marker -smooth muscle actin (-SMA) and lipid content respectively, mediated via PGE2 binding to the FAPs EP4 receptor. FAPs isolated from 12 week denervated muscle demonstrated increased adipogenesis and fibrogenesis vs. naive FAPs (perilipin-1 and -SMA 7.93{+/-}2.96 and 2.00{+/-}0.33 fold increase respectively, p<0.05) and remained fully susceptible to PGE2 inhibition of fibro-adipogenic differentiation. Conditioned media from FAPs derived from 5 week, but not 12 week, denervated gastrocnemius stimulated C2C12 myoblast proliferation which was prevented by EP4 blockade. ConclusionsPGE2 is identified as a novel negative regulator of FAPs differentiation in traumatically denervated muscle, suggesting the therapeutic potential of PGE2 to prevent fibro-fatty degradation of long-term denervated muscle awaiting reinnervation.

Muscle mass significantly influences skeletal muscle behaviour, potentially explaining why traditional massless Hill-type models struggle to predict the forces generated by larger muscles during dynamic, submaximal contractions. However, the applicability of mass-enhanced Hill-type models in human locomotion remains unexplored. Here, we compared the predicted force from a 1D mass-enhanced Hill-type muscle model with a traditional 1D massless Hill-type muscle model across a range of experimentally measured human movements. Kinematic and electromyographic data were collected from twenty participants performing locomotor tasks and supplemented with existing cycling data. Muscle size was geometrically scaled by factors from 0.1 to 10, which causes lengths to be scaled proportionally, cross-sectional area and peak isometric force F0 with the square, and mass with the cube of the factor. Muscle tissue mass (inertia) and cadence increased the differences between mass-enhanced and massless predictions of force and power. At high cadence and the largest scale, the normalized root mean square difference between force traces reached 7% of F0, (averaged across muscles). However, differences between models were minimal (<1%) at human-sized scale 1. Real muscle additionally deforms in 3D, we still do not know the extent to which this extra dimensionality affects muscle forces for these human movements.

The dy3K/dy3K Lama2-/- mouse is a model for the severe form of LAMA2-related dystrophy and peripheral neuropathy (LAMA2-RD). In the dystrophic mice, a compensating laminin subunit, Lm4, that lacks polymerization and -dystroglycan-binding activity, replaces the missing Lm2 subunit. It was previously found that an 4-laminin can be modified with two small laminin-binding linker proteins, i.e. LNNd{Delta}G2 and miniagrin to facilitate polymerization and -dystroglycan binding respectively, to enable the key missing functions. Adeno-associated virus serotype 9 (AAV9) was used to deliver minigenes coding for the two proteins in dystrophic mice. AAV9-LNNd{Delta}G2 utilized a universal CBh promoter while AAV9-miniagrin utilized either the CBh promoter or muscle-specific SPc5-12 promoter. The phenotype in the dy3K/dy3K mice was evaluated following i.v. postnatal injection with either AAV9 -LNNd{Delta}G2 alone or in combination with AAV9- LNNd{Delta}G2 + AAV9- miniagrin. Double AAV treatment was found to substantially increase survival and ambulation, as well as increase forelimb grip-strength and improve muscle histology. Of note, the sciatic nerve amyelination characteristic of laminin 2-deficiency was prevented. While single treatment with LNNd{Delta}G2 was inferior to double treatment for muscle strength and survival, it corrected the radial sorting deficit equally, revealing that enablement of laminin polymerization is a sufficient requirement for myelination. HighlightsO_LIThe dy3K/dy3K (Lama2-/-) mouse, a model for severe LAMA2-related dystrophy, expresses laminin-411 that is unable to polymerize or bind to -dystroglycan (DG). C_LIO_LILNNd{Delta}G2 and miniagrin are laminin-411-binding proteins that enable polymerization and DG binding. C_LIO_LIAAV9 delivery of genes coding for LNNd{Delta}G2 and miniagrin ameliorated the dystrophic phenotype in muscle and nerve (survival, growth, mobility, and grip-strength, muscle and nerve histopathology). C_LIO_LISciatic nerve amyelination was prevented by LNNd{Delta}G2 alone. C_LI

BackgroundAging and disuse are two of the most clinically relevant conditions associated with the loss of skeletal muscle mass, yet the ultrastructural adaptations that drive these losses remain poorly defined. In particular, it is unclear whether radial atrophy of muscle fibers is driven by a reduction in the size of the existing myofibrils, and/or the loss of myofibrils. Accordingly, the objective of this study was to define the macro-to-ultrastructural adaptations that mediate aging- and disuse-induced loss of muscle mass. MethodsSkeletal muscle structure was assessed at the macroscopic, microscopic, and ultrastructural levels in humans and mice. In humans, magnetic resonance imaging was used to quantify knee extensor muscle volume and cross-sectional area (CSA) in young (19 - 40 years) and old (65 - 84 years) adults, and vastus lateralis biopsies were analyzed for microscopic and ultrastructural adaptations using immunohistochemistry and fluorescence imaging of myofibrils with image deconvolution (FIM-ID). Parallel studies were performed in young (4 months) and aged (24 months) mice, along with the use of unilateral hindlimb immobilization to model disuse. ResultsAging led to a robust loss of skeletal muscle mass that was mediated by coordinated macro-to-ultrastructural adaptations. In humans, aging reduced knee extensor muscle volume (34%, P < 0.005) and CSA (32%, P < 0.001) in a sex-independent manner, and these effects were associated with radial atrophy of SERCA1-positive fibers (23%, P < 0.05). Ultrastructural analyses revealed that the radial atrophy was driven by a reduction in the number of myofibrils per fiber (23%, P < 0.05) without changes in myofibril CSA. In mice, aging produced similar macro-to-ultrastructural adaptations in various flexor muscles; however, radial atrophy of the highly glycolytic/Type IIb fibers, which are not present in human limb muscles, was also associated with a decrease in the CSA of the myofibrils (9%, P < 0.005). We also determined that disuse led to radial atrophy of SERCA1-positive fibers (24%, P < 0.001), and this was mediated by a decrease in both the number (22%, P < 0.005) and size of the myofibrils (4%, P < 0.05). Notably, the results also revealed that the magnitude of the disuse-induced adaptations was significantly blunted with aging. ConclusionThis study identifies the loss of myofibrils as a central and conserved mediator of the radial atrophy of muscle fibers that occurs in response to disuse and aging, while also highlighting smaller context-dependent contributions that can arise from changes in myofibril size.

Quantitative histological analysis of skeletal muscle morphometry provides critical insights into muscle physiology but remains labor-intensive and technically demanding. While recent developments in machine-learning-based image segmentation techniques have facilitated large-scale tissue analysis, existing tools that automate muscle morphometry analysis are largely tailored to mammalian models, with limited applicability to teleosts. Moreover, there is a lack of effective tools for visualizing spatial organization and morphometric variability of teleost muscle fibers, a feature that is important for understanding hyperplastic muscle growth dynamics in teleosts. In this study, we show that cytoplasmic staining combined with deep learning-based cell segmentation offers a robust and accurate approach for automated muscle morphometry analysis in developing zebrafish. We also introduce a FIJI2 plugin, implemented in Jython, that streamlines both morphometric analysis and visualization. This tool accommodates shallow and deep learning-based segmentation techniques and incorporates novel quantification and visualization methods suited to teleost-specific muscle features, including mosaic hyperplasia dynamics. The plugin features an intuitive graphical user interface and is designed for flexibility, with minimal constraints regarding species, image quality, or staining protocol. Its modular architecture allows it to be used as a baseline for automated muscle morphometry analysis, while permitting integration with other tools and workflows.

Skeletal muscle dysfunction is a pervasive complication of critical illness that worsens survival and recovery, yet remains poorly explained by current clinical or molecular markers. To directly connect disease-associated molecular states to the contractile machinery, this study combined sequential functional, transcriptomic, and proteomic profiling of the same single human skeletal myofibers from critically ill patients in the intensive care unit with acquired weakness (ICU-AW) and controls. Despite marked donor-level heterogeneity, integrated analysis revealed a subtle yet conserved myofiber state enriched in ICU-AW, characterized by inflammatory and chemotactic gene programs, intracellular structural remodeling, and bioenergetic adaptation. Nineteen features were significantly altered at both RNA and protein levels from the same myofiber, linking an inflammatory transcriptional landscape to a proteomic shift toward mitochondrial and translational machinery and away from membrane-associated signaling. Functionally, fibers in this state displayed selectively disrupted myosin dynamics, evidenced by prolonged ATP turnover time of myosin heads in their super-relaxed conformation, implicating altered myosin energetics as a contributor to muscle dysfunction. These findings define a discrete, disease-associated myofiber state and establish an integrative single-fiber framework for connecting multi-omic heterogeneity to molecular motor function in complex human disease. Graphical AbstractSingle-fiber multi-omic and functional analysis reveals a stress-adapted myofiber state in ICU-AW. Specifically, for the present study, myofibers from ICU-AW donors and control donors were isolated and functionally profiled for myosin dynamics before being split for simultaneous transcriptomic and proteomic analysis. Integrated analysis then identified a reproducible fiber phenotype enriched in ICU-AW, characterized by inflammatory transcriptional signatures coordinated with mitochondrial proteomic remodeling and altered myosin super-relaxed state energetics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/706099v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@95715forg.highwire.dtl.DTLVardef@1462ff8org.highwire.dtl.DTLVardef@f74996org.highwire.dtl.DTLVardef@10055ec_HPS_FORMAT_FIGEXP M_FIG C_FIG

When running, metabolic cost increases as muscles are simultaneously fatigued. However, the contribution of an individual muscle group to fatigue-related increase in metabolic costs remains unclear. We investigated the metabolic consequence of running with local plantar flexor or knee extensor fatigue and associated neuromuscular control strategies. Recreational and experienced male runners (N=20) completed two sessions (one per muscle group), with each including two 10 min running bouts: without and with local fatigue ([~]20% reduction in peak joint torque). Net metabolic power and muscle activity (initial and final minutes) were determined. Metabolic power was unaffected by plantar flexor (p=0.367) or knee extensor (p=0.607) fatigue in both cohorts. Plantar flexor fatigue recovered during the fatigued run (p=0.033), while knee extensor fatigue only recovered for the recreational cohort (p=0.009; experienced: p=0.826). With plantar flexor fatigue, plantar flexor muscle activity was unchanged between runs (p[&ge;]0.312), however initial soleus activity was greater in the unfatigued than fatigued run for experienced runners (p=0.022), and initial medial gastrocnemius activity was greater in the unfatigued than fatigued run for the combined cohort (p=0.009). With knee extensor fatigue, knee extensor muscle activity was mostly lower in the unfatigued than fatigued run (p[&le;]0.009), except for final vastus lateralis activity, which was unchanged between runs (p=0.061). Therefore, muscle groups respond with different activation strategies when fatigued. Running with plantar flexor or knee extensor fatigue, at levels like those induced by prolonged running (10-42 km), does not increase metabolic power and thus, submaximal running energetics may be maintained despite local muscle fatigue. NEW & NOTEWORTHYWhile muscle fatigue is suggested to increase the metabolic cost of running, the individual contributions of key lower limb muscle groups have not been explored. We examined responses after fatigue of only the plantar flexors or the knee extensors. Results indicate that local fatigue did not affect the metabolic power of male runners for either fatigued muscle group. These findings enhance our understanding of running performance and the interaction between fundamental criteria dictating human locomotion.

BackgroundExercise-induced fatigue is a complex physiological phenomenon involving oxidative stress, inflammation, and metabolic disturbances. Ergothioneine (EGT), a naturally occurring amino acid with potent antioxidant properties, has garnered interest for its potential health benefits. This study aimed to evaluate the anti-fatigue effects of Gene III EGT in a mouse model of exhaustive exercise and to elucidate its underlying mechanisms. MethodsMale C57BL/6 mice were randomly divided into five groups: a control group (CTL), low-dose EGT (EGT-L, 10 mg/kg), medium-dose EGT (EGT-M, 30 mg/kg), high-dose EGT (EGT-H, 50 mg/kg), and a positive control group (Coenzyme Q10, 50 mg/kg). Mice were subjected to a 4-week treadmill training protocol, followed by an exhaustive running test. We measured exercise performance and collected blood and skeletal muscle samples at multiple time points to assess biochemical markers, inflammatory cytokines, antioxidant status, and key signaling proteins. ResultsGene III EGT supplementation, particularly at medium and high doses, significantly extended the time to exhaustion and running distance. Compared to the control group, EGT treatment significantly reduced post-exercise levels of lactic acid (LA), lactate dehydrogenase (LDH), and blood urea nitrogen (BUN). Furthermore, Gene III EGT suppressed the exercise-induced increase in pro-inflammatory cytokines, including IL-1{beta}, IL-6, and TNF-. The anti-fatigue effect of EGT was also associated with a reduction in malondialdehyde (MDA) and an increase in the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Mechanistically, EGT promoted the phosphorylation of AMP-activated protein kinase (AMPK) and the expression of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1) in skeletal muscle, while also increasing the Bcl-2/Bax ratio, suggesting enhanced mitochondrial biogenesis and reduced apoptosis. ConclusionsOur findings demonstrate that Gene III EGT effectively enhances exercise performance and alleviates fatigue. The underlying mechanisms involve the mitigation of oxidative stress and inflammation, as well as the activation of the AMPK/PGC-1 signaling pathway to promote mitochondrial function and cellular protection. These results highlight the potential of Gene III EGT as a nutritional supplement for combating exercise-induced fatigue.

Collagen organisation within the tumour microenvironment plays a critical role in tumour progression and has emerged as an important structural biomarker in cancer. Second Harmonic Generation (SHG) microscopy enables label-free visualisation and quantitative assessment of fibrillar collagen architecture; however, its high cost, specialised instrumentation, and limited field-of-view restrict routine clinical application. In this study, we evaluated whether collagen features quantified from digitally scanned Masson-Goldners Trichrome-stained histopathological sections can approximate measurements obtained from SHG microscopy. Formalin-fixed paraffin-embedded breast tumour tissues, including benign and invasive ductal carcinoma (IDC) samples with varying collagen content, were analysed using SHG microscopy and whole-slide brightfield imaging. Matched regions of interest were analysed using two independent digital image analysis approaches: a conventional ImageJ-based workflow (TWOMBLI) and a machine learning-based computational pipeline. Collagen structural parameters including collagen deposition area, fibre number, and alignment metrics were quantified and compared across imaging modalities using correlation analysis. SHG signals were consistently detected from trichrome-stained sections, confirming compatibility of SHG imaging. Quantitative comparison demonstrated significant concordance between SHG-derived collagen metrics and those obtained from digital image analysis pipelines, particularly for collagen area and fibre alignment. These findings demonstrate that computational analysis of routine histopathological images can capture key spatial features of collagen organisation comparable to SHG microscopy. Digital pathology-based collagen quantification therefore, represents a scalable and clinically accessible approach for assessing extracellular matrix architecture in tumour tissues.

This study examined how motoneuron activity influences transcription factor binding in mouse fast glycolytic Myh4+ muscle fibers. Single nucleus multiomics of innervated versus denervated tibialis anterior muscles revealed altered chromatin accessibility: SIX and c-MAF binding sites decreased while JUN, FOS, and RUNX1 sites increased in denervated Myh4+ myonuclei. c-MAF showed strong nuclear enrichment after 100 Hz stimulation and periods of increased motoneuron activity but was absent following denervation, establishing it as a primary readout of fast motoneuron firing. Genome-wide analysis demonstrated that c-MAF binding site spacing encodes functionally distinct muscle gene programs. Analysis of constitutive and inducible skeletal muscle-specific c-Maf mutants revealed that c-MAF loss caused region-specific MYH4+ fiber atrophy, MYH1/MYH2 fiber type shifts resembling ALS G93A mouse phenotypes, and progressive neuromuscular junction fragmentation with increased motoneuron terminal sprouting and ectopic reinnervation. These findings establish c-MAF as a critical mediator linking motoneuron activity to muscle gene regulation, fiber integrity, and neuromuscular junction maintenance in fast glycolytic fibers.