bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2026–11–01
forty-four papers selected by
Anna Vainshtein, Craft Science Inc.
  1. Chromatin Immunoprecipitation Followed by PCR (ChIP-PCR) from Prospectively Isolated Murine Muscle Stem Cells.
  2. Automated Bioinformatic Analysis of Skeletal Muscle Immunofluorescence for the Quantification of Muscle Stem Cells.
  3. Co-cultures of Macrophages with Muscle Stem Cells or Fibroadipogenic Precursor Cells from Skeletal Muscle.
  4. Beyond Mitochondrial Fission: Emerging Roles of Drp1 in Skeletal Muscle Physiology and Disease.
  5. Measurement of Oxygen Consumption Rate of Primary Mouse Skeletal Muscle Stem Cells and Their Progeny on the Seahorse Analyzer.
  6. Modeling Skeletal Muscle Stem Cell Fate In Vitro.
  7. Ex Vivo Analysis of Muscle Stem Cells on Single EDL Myofibers, from Quiescence to Self-Renewal.
  8. Aquaporin-4 Orchestrates Muscle Cell Differentiation and Satellite Cell Preservation: A Novel Target in Age-Related Muscle Decline.
  9. Binding of a Novel 11β-Hydroxysteroid to the Apelin Receptor (APLNR) and Its Possible Role in Regulating Skeletal Muscle Health in Mice.
  10. Automated Quantitative Analysis of Myogenesis in Isolated Mouse and Rat Muscle Stem Cells.
  11. Neuromuscular Junction Degeneration in Skeletal Muscle Atrophy: Disease-Specific Mechanisms, Evidence-Informed Stratification, and Therapeutic Windows.
  12. Midlife time-restricted feeding preserves skeletal muscle insulin responsiveness and function during aging.
  13. Rapsyn overexpression improves neuromuscular junction and skeletal muscle function after tourniquet-induced mouse hindlimb ischemia-reperfusion injuries.
  14. Skeletal Muscle Aging Is a Significant Contributor to Systemic Aging.
  15. Flow Cytometry Analysis of Mitochondrial Function in Muscle Stem Cells.
  16. An Ex Vivo Cross-Grafting and Live-Imaging Assay to Analyze Myofiber Niche Impact on Muscle Stem Cells.
  17. Annexins cooperate with Myomaker to promote myoblast fusion.
  18. Isolation of Mouse, Rat, and Human Muscle Stem Cells by Fluorescence-Activated Cell Sorting Based on α7-Integrin.
  19. M4-like Macrophages Drive Skeletal Muscle Fibrosis and Suppress Myogenesis via the MMP12/SPP1/NF-κB Axis.
  20. Non-steady-state modelling reveals reduced skeletal muscle protein synthesis as the primary driver of disuse-atrophy in young, healthy females.
  21. An Ion-Releasing Bioactive Glass Accelerates Skeletal Muscle Regeneration and Functional Recovery Following Acute Injury.
  22. Low-Input RNA-Sequencing of In Situ Fixed Adult Murine Muscle Stem Cells.
  23. Loss of LanC-like proteins delays post-injury regeneration of aging skeletal muscles.
  24. Proteomic ageing clocks: methods, mechanisms and the muscle connection.
  25. Bioinformatic Workflow for the Analysis of Single-Nucleus RNA-Sequencing Data from Skeletal Muscle.
  26. Biological Accessibility and Analytical Recovery in Myofibrillar Protein Loss During Skeletal Muscle Atrophy.
  27. NEMUCO: The In Vitro 4D NMJ as an Innovative Powerful Tool to Study Nerve and Muscle Cell-Cell Communication.
  28. The peripheral neuromuscular continuum: Histological remodeling induced by physical training.
  29. Intracellular calcium handling and not contraction type determines task-dependent skeletal muscle fatigue.
  30. Niche-reprogramming CAR T therapy improves skeletal muscle regeneration and function in preclinical murine models of Duchenne muscular dystrophy.
  31. The annexin A1 mimetic peptide Ac2-26 promotes inflammation-resolution and stimulates skeletal muscle regeneration in the mdx mouse model of Duchenne muscular dystrophy.
  32. Disruption of the Muscle-Brain Axis in Ageing: Role of Myokines in Sarcopenia and Neurodegeneration.
  33. Tissue Clearing and Immunostaining of Mouse and Human Skeletal Muscles for In Situ Analysis of Muscle Stem Cell Spatial Organization.
  34. A Cross-Condition Single-Cell Atlas Defines Skeletal Muscle Adaptation and Its Predictive Limits.
  35. Sex-dimorphic DNA lesion accumulation drives a PDK4 metabolic checkpoint in skeletal muscle.
  36. Characterization of sex-dependent skeletal muscle alterations in the 3xTg Alzheimer's disease mouse model.
  37. Mdfi overexpression promotes oxidative muscle fiber formation without affecting muscle development in mature skeletal muscles.
  38. Progesterone does not modulate myofibrillar protein synthesis at rest or following acute resistance exercise in premenopausal females.
  39. RNA-Based Biological Age Clocks Track Chronological Age in Skeletal Muscle but Show Limited Discrimination of Sarcopenia.
  40. Adipocyte lipolysis is a determinant of skeletal muscle size during fasting and feeding.
  41. Preserved anabolic skeletal muscle capacity in response to exercise and protein intake after critical illness.
  42. Directed Differentiation of Human Pluripotent Stem Cells into Skeletal Muscle Progenitor Cells.
  43. Population-scale single-nucleus multi-omic profiling of skeletal muscle reveals extensive context-specific genetic regulation.
  44. Bazi Bushen Attenuates Skeletal Muscle Decline Associated With Oestrogen Deficiency and Modulates FGFR1/ERK Signalling.
  1. Methods Mol Biol. 2027 ;3093 271-280
    Chromatin Immunoprecipitation Followed by PCR (ChIP-PCR) from Prospectively Isolated Murine Muscle Stem Cells.
    Marie-Claude Sincennes.
      Muscle stem cells are essential for skeletal muscle regeneration, but their scarcity makes epigenetic studies technically and financially challenging. Here, we describe a robust chromatin immunoprecipitation (ChIP) protocol compatible with freshly isolated muscle stem cells using as few as 25,000 cells. This method avoids sample pooling, reduces animal use, and enables reliable analysis of chromatin modifications and transcription factor binding.
    Keywords:  Chromatin immunoprecipitation; Epigenetics; Gene expression; Histone marks; Muscle stem cells; Myogenesis; Satellite cell; Skeletal muscle; Transcription
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_19
  2. Methods Mol Biol. 2027 ;3093 49-62
    Automated Bioinformatic Analysis of Skeletal Muscle Immunofluorescence for the Quantification of Muscle Stem Cells.
    Rémi Kurdian, Martina Canatelli-Mallat, Daniel Stockholm, Colin Crist, Alicia Mayeuf-Louchart.
      Muscle stem cells (MuSCs) are essential for skeletal muscle regeneration. They express the transcription factor PAX7 and reside beneath the basal lamina of muscle fibers in a quiescent state. Disruption in MuSCs quiescence or activation impairs regenerative capacity, as observed in myopathies and during ageing. Consequently, the accurate quantification of PAX7-expressing MuSCs constitutes a key readout in myology research. Here, we present a complete and standardized workflow enabling the automated quantification of MuSCs immunolabeled with anti-PAX7 antibodies by immunofluorescence on skeletal muscle sections. This methodology comprises three main steps: cardiotoxin-induced muscle injury followed by muscle dissection, muscle freezing, cryosectioning, and immunofluorescence staining with subsequent MuSCs quantification using MuscleJ2, a freely available high-content analysis Fiji plugin that we previously developed. This automated pipeline ensures reproducible, unbiased, and high-throughput assessment of MuSCs abundance, providing a robust tool for studies investigating muscle homeostasis, pathology, and regeneration.
    Keywords:  Bioinformatic tool; Histology; Muscle stem cell (MuSC); MuscleJ2; PAX7; Regeneration; Satellite cells; Skeletal muscle
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_5
  3. Methods Mol Biol. 2027 ;3093 19-33
    Co-cultures of Macrophages with Muscle Stem Cells or Fibroadipogenic Precursor Cells from Skeletal Muscle.
    Stéphanie Gobert, Orane Mercier, Gaëtan Juban.
      Skeletal muscle is capable of complete regeneration after an acute injury thanks to the properties of muscle stem cells (MuSCs), which can activate, proliferate, differentiate, and fuse to replace the damaged myofibers. However, this regeneration process involves the interaction of several cell types, including fibroadipogenic precursor cells (FAPs) that reorganize the extracellular matrix and macrophages (MPs), which modulate MuSC and FAP behavior. Here, we present a protocol to isolate MuSCs, FAPs, and MPs from skeletal muscle and assess the regulation of MuSC and FAP function by MPs through a short-term co-culture system, which allows to preserve key in vivo characteristics of the isolated cells.
    Keywords:  Co-cultures; Differentiation; Fibroadipogenic precursor cell; Macrophage; Muscle stem cell; Proliferation; Skeletal muscle
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_3
  4. Am J Physiol Cell Physiol. 2026 Oct 29.
    Beyond Mitochondrial Fission: Emerging Roles of Drp1 in Skeletal Muscle Physiology and Disease.
    Kai Zou, Nicolas J Berger, Zhenqi Zhou, Benjamin A Kugler.
      Dynamin-related protein 1 (Drp1) is best recognized as the primary regulator of mitochondrial fission. However, emerging evidence indicates that Drp1 is a multifaceted regulator of skeletal muscle physiology whose functions extend well beyond its canonical role in mitochondrial fission. Recent studies have identified novel roles of Drp1 in regulating substrate utilization, calcium homeostasis, reactive oxygen species signaling, respiratory complex assembly, and mitochondrial-to-nuclear signaling, including mechanisms independent of its canonical fission activity. Importantly, Drp1 regulation in skeletal muscle is highly context-dependent, as both excessive and insufficient Drp1 activity can disrupt skeletal muscle homeostasis. For example, excessive Drp1 activation and mitochondrial fragmentation are associated with insulin resistance, type 2 diabetes, and neuromuscular diseases, whereas severe or prolonged Drp1 deficiency impairs mitochondrial quality control, bioenergetics, induce muscle atrophy, and compromise the exercise training adaptations. Therefore, the biological consequences of Drp1 modulation are highly context dependent and influenced by the metabolic and physiological state of skeletal muscle. In this review, we summarize the molecular regulation and emerging functions of Drp1 in skeletal muscle, with particular emphasis on fission-independent mechanisms and Drp1 dysregulation in metabolic and neuromuscular diseases. We further discuss pharmacological and non-pharmacological strategies for modulating Drp1 signaling and highlight emerging approaches for skeletal muscle-specific targeting. Collectively, current evidence supports a paradigm in which skeletal muscle Drp1 functions as a multifaceted regulator of mitochondrial and skeletal muscle homeostasis, highlighting the therapeutic potential of modulating Drp1 activity to optimize mitochondrial and muscle health across different physiological and pathological contexts.
    Keywords:  DYNAMIN-RELATED PROTEIN 1; MITOCHONDRIA DYNAMICS; SKELETAL MUSCLE
    DOI:  https://doi.org/10.1152/ajpcell.90065.2026
  5. Methods Mol Biol. 2027 ;3093 185-194
    Measurement of Oxygen Consumption Rate of Primary Mouse Skeletal Muscle Stem Cells and Their Progeny on the Seahorse Analyzer.
    Audrey Saugues, Lucie Ancel, Ha My Ly, Lola E R Lessard, Rémi Mounier, Anita Kneppers.
      To establish mature myofibers with a complex mitochondrial network, skeletal muscle stem cells (MuSCs) need to undergo large metabolic alterations. This goes beyond an increase in mitochondrial abundance during myogenesis and includes mitochondrial morphological adaptations by fusion and fission. Recent insights into the epigenetic, transcriptomic, and cell signaling alterations supported by mitochondrially generated intermediates and end products demonstrate how mitochondrial metabolism dictates MuSC behavior. The measurement of mitochondrial oxidative phosphorylation (OXPHOS), as an indicator of mitochondrial function under stressed states, therefore gives crucial insight into metabolic alterations that drive MuSC function. In this chapter, we describe the measurement of the oxygen consumption rate of primary mouse skeletal muscle stem cells and their progeny on the Seahorse analyzer.
    Keywords:  Cellular respiration; Metabolism; Muscle stem cells; Myotubes; Oxygen consumption rate; Seahorse technology; Skeletal muscle
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_14
  6. Methods Mol Biol. 2027 ;3093 115-125
    Modeling Skeletal Muscle Stem Cell Fate In Vitro.
    Muriel Sébastien, Emilie Christin, Vincent Gache.
      Skeletal muscle stem cells (MuSCs) are necessary for muscle development and regeneration. Upon external cues, they activate from their quiescent state to self-renew and/or differentiate. The study of MuSCs is challenging as they tend to differentiate when isolated from their microenvironment. Here, we describe an in vitro model that mimics a skeletal muscle environment, allowing MuSC preservation together with myofiber maturation.
    Keywords:  In vitro; Matrigel; MuSCs; Myofibers; Niche modeling; Pax7; Skeletal muscle
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_10
  7. Methods Mol Biol. 2027 ;3093 77-90
    Ex Vivo Analysis of Muscle Stem Cells on Single EDL Myofibers, from Quiescence to Self-Renewal.
    Ludivine Rotard, Hsiao-Fan Lo, Anna Rausch De Traubenberg, Robert S Krauss, Caroline E Brun.
      Skeletal muscle regeneration relies on normally quiescent muscle stem cells that, upon tissue injury, activate, proliferate, and either differentiate to repair the myofibers or self-renew to reconstitute the stem cell pool. Quiescence regulation, myogenic lineage progression or self-renewal are tightly controlled by intrinsic factors and extrinsic signals released from the muscle stem cell microenvironment. Here, we provide a method for isolating individual extensor digitorum longus myofibers and visualizing muscle stem cells in their quiescent state and during their progression into the myogenic lineage.
    Keywords:  Cytoskeleton; Immunostaining; Muscle stem cell; Myofiber culture; Myofiber isolation; Quiescence; Skeletal muscle; Stem cell fate
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_7
  8. J Cachexia Sarcopenia Muscle. 2026 Dec;pii: e70412. [Epub ahead of print]17(6):
    Aquaporin-4 Orchestrates Muscle Cell Differentiation and Satellite Cell Preservation: A Novel Target in Age-Related Muscle Decline.
    María D Girón, Manuel Manzano, Suzette L Pereira, Antonio Reinoso, Chiara Burgio, Nora Montero-Martin, Guillermo Jiménez-Jiménez, Josep M Argilés, Ricardo Rueda, José M López-Pedrosa, Rafael Salto.
       BACKGROUND: Impaired muscle regeneration is a key contributor to age-related muscle decline, yet the molecular mechanisms linking muscle water homeostasis to regenerative failure remain incompletely understood. Aquaporin-4 (AQP4), a major skeletal muscle water channel, may have functions beyond water transport that are relevant to muscle maintenance and repair. We investigated whether altered AQP4 expression contributes to age-related defects in skeletal muscle regeneration and whether AQP4 may represent a targetable pathway for muscle dysfunction associated with ageing.
    METHODS: Skeletal muscle from young adult (25-week; n = 9) and aged (85-week; n = 8) rats was analysed for extracellular and intracellular water content by bioimpedance. AQP4, AQP1 and myogenic markers were measured by Western blot. Satellite cells isolated from skeletal muscle were assessed for proliferation, differentiation and expression of differentiation markers. To define the mechanistic role of AQP4 in myogenesis, CRISPR-generated AQP4-silenced L6 rat myoblasts were evaluated for proliferation, differentiation, senescence and Akt/PKB signalling. Rescue experiments were performed by restoring AQP4 expression in silenced cells. Data are reported as mean ± SEM.
    RESULTS: Compared with young rats, aged rats showed an altered intracellular water-to-lean mass ratio (0.94 vs. 0.62, p < 0.05), a 33% reduction in AQP4 expression (p < 0.05) and reduced muscle strength (3.1 vs. 2.6, p < 0.05), with AQP4 levels correlating with muscle performance. Satellite cells from aged muscle exhibited 29% lower AQP4 expression and 39% lower Pax7 expression (both p < 0.05), together with reduced myogenic differentiation (30% decrease, p < 0.05). In L6 myoblasts, AQP4 silencing reduced proliferation (20% decrease, p < 0.05), impaired myotube differentiation (50% decrease, p < 0.05), increased senescence (30% increase, p < 0.05) and decreased Akt/PKB signalling by 42% (p < 0.05). AQP4 silencing was also associated with decreased dystrophin localization at the plasma membrane (50%, p < 0.05). Re-expression of AQP4 reversed these abnormalities and restored the phenotype toward wild-type levels (all p < 0.05).
    CONCLUSIONS: Reduced AQP4 expression is associated with impaired muscle regeneration and functional decline in aged skeletal muscle. Experimental AQP4 loss disrupts myogenic signalling, membrane organization and increases progression to senescence, whereas AQP4 restoration rescues these defects. These findings identify AQP4 as a biologically plausible therapeutic target for strategies aimed at preserving or restoring muscle health and regenerative capacity in ageing-associated muscle dysfunction.
    Keywords:  AQP4; aquaporins; muscle proliferation and differentiation; sarcopenia; satellite cells; senescence; water transporters
    DOI:  https://doi.org/10.1002/jcsm.70412
  9. Int J Mol Sci. 2026 Oct 21. pii: 9379. [Epub ahead of print]27(20):
    Binding of a Novel 11β-Hydroxysteroid to the Apelin Receptor (APLNR) and Its Possible Role in Regulating Skeletal Muscle Health in Mice.
    Marcos González-Pérez, Citlali Gómez-Márquez, Miguel Ortiz-Flores, Marycarmen Godínez-Victoria, Nayelli Nájera, Guillermo Ceballos.
      Several molecules have been investigated for their potential to improve muscle health in models of muscle damage simulating sarcopenia, injury, or aging. Some small molecules induced beneficial effects on skeletal muscle by stimulating the apelin receptor (APLNR). This receptor has emerged as a promising pharmacological target for modulating muscle homeostasis. Using an in silico approach, we recently identified 11β-hydroxypregnenolone (11β-OHP5) as a potential skeletal muscle (SKM) modulator. We hypothesized that 11β-OHP5 interacts with and activates APLNR. This study employed several research approaches: (1) in silico molecular docking and molecular dynamics to explore the feasibility of 11β-OHP5 interacting with APLNR; (2) in vitro activation analysis to APLNR; (3) in vitro analysis C2C12 myoblast differentiation; (4) analysis of the molecule's presence in mouse skeletal muscle and its differential presence between young and older mice. The results show that 11β-OHP5, acting through APLNR, positively affects myoblast differentiation and may participate in the exercise-induced benefits in aging mice. We show that 11β-OHP5 interacts with APLNR, induces myogenic responses, is found in SKM; its concentration decreases with age, and exercise restores it. More work is necessary to understand the physiology of this possible endogenous molecule.
    Keywords:  11β-hydroxypregnenolone; apelin receptors; skeletal muscle
    DOI:  https://doi.org/10.3390/ijms27209379
  10. Methods Mol Biol. 2027 ;3093 127-152
    Automated Quantitative Analysis of Myogenesis in Isolated Mouse and Rat Muscle Stem Cells.
    Pauline Garcia, Zachary Gurlekian, Rebecca Desaulniers, Nicolas A Dumont.
      Understanding the mechanisms governing myogenesis is essential for studying skeletal muscle development, regeneration, and disease. Here, we present a detailed and automated protocol for the quantitative analysis of myogenesis in isolated muscle stem cells (MuSCs) from both mice and rats. Mouse MuSCs are isolated from skeletal muscle tissue using magnetic-activated cell sorting (MACS), while rat MuSCs are purified by fluorescence-activated cell sorting (FACS), ensuring high purity and viability of stem cell populations in both models. Following isolation, MuSCs are cultured under defined proliferative or differentiating conditions and subsequently stained by immunofluorescence for key myogenic markers. To ensure unbiased and high-quality analysis, automated quantitative analysis is done using QuPath, enabling objective measurement of proliferation (Pax7, Ki67), differentiation (Myogenin), and fusion indices (Myosin Heavy Chain). Overall, this method describes a robust and fast automated analysis of myogenesis on mouse and rat using automated quantification.
    Keywords:  Automated quantification; Image analysis; Immunofluorescence; Muscle stem cells; Myogenesis
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_11
  11. Int J Mol Sci. 2026 Oct 19. pii: 9264. [Epub ahead of print]27(20):
    Neuromuscular Junction Degeneration in Skeletal Muscle Atrophy: Disease-Specific Mechanisms, Evidence-Informed Stratification, and Therapeutic Windows.
    Shiming Li, Mengyu Liu, Peng Zhang, Xuemei Ma, Xiaoping Chen.
      Skeletal muscle atrophy involves not only the loss of muscle mass and fiber size, but also a significant decline in contractile performance. Force decline often occurs before visible fiber atrophy, suggesting that early defects in neuromuscular transmission might trigger or exacerbate the wasting process. Therefore, we argue that neuromuscular junction (NMJ) degeneration is not a universal, conserved pathway across all atrophy syndromes. Instead, it should be viewed as a stage- and disease-specific event, with its actual contribution varying based on specific evidence. To explore this, we compare NMJ maintenance and degeneration across a broad spectrum of conditions, ranging from primary neuromuscular diseases (like denervation and motor neuron disorders) to systemic states such as aging, cachexia, and metabolic or respiratory diseases. Based on this, we propose a framework to evaluate NMJ involvement by timing, lesion type, and reversibility, drawing on diverse evidence from morphological, electrophysiological, and biomarker studies. Crucially, this approach distinguishes primary synaptic disorders from systemic conditions where NMJ damage acts merely as a secondary modifier. Ultimately, aligning specific molecular lesions with practical intervention windows will help refine patient stratification and guide the design of more targeted NMJ therapies.
    Keywords:  agrin-MuSK signaling; evidence stratification; neuromuscular junction; skeletal muscle atrophy; therapeutic window; translational biomarkers
    DOI:  https://doi.org/10.3390/ijms27209264
  12. Am J Physiol Cell Physiol. 2026 Nov 01. 331(5): C1506-C1520
    Midlife time-restricted feeding preserves skeletal muscle insulin responsiveness and function during aging.
    Raz Ben-David, Nikita Sarvin, Eli Carmeli, Nitsan Dahan, Tomer Shlomi, Shenhav Shemer.
      Primary sarcopenia, the progressive loss of skeletal muscle mass and function during aging, is partly driven by anabolic resistance, a reduced ability of aged muscle to activate protein synthesis in response to nutrients and hormones. We compared 12-wk time-restricted feeding (TRF) and alternate-day fasting (ADF) interventions initiated at 3, 12, 15, or 18 mo of age in male C57BL/6J mice (analyzed at 6, 15, 18, or 21 mo, respectively). The effects of intermittent fasting on skeletal muscle were strongly age-dependent. Midlife (15 mo of age) TRF preserved lean mass, improved glucose tolerance, and increased insulin-stimulated glucose uptake in isolated muscle, whereas ADF induced broader metabolic remodeling but was associated with lean-tissue loss when initiated later in life. Muscle aging was accompanied by reduced membrane abundance of insulin receptor-plakoglobin-dystrophin glycoprotein complex (DGC) signaling assembly. However, midlife TRF was associated with greater abundance of these protein modules and enhanced postprandial AKT signaling. These findings identify midlife as a responsive window for nutritional timing interventions and link improved muscle insulin responsiveness and maintenance with diet-associated differences in sarcolemmal protein abundance and organization, although targeted perturbation studies are required to establish causality.NEW & NOTEWORTHY Midlife time-restricted feeding preserved lean mass, muscle performance, glucose tolerance, and insulin-stimulated glucose uptake in male mice. These physiological benefits were accompanied by greater membrane abundance of selected components of the insulin receptor-plakoglobin-DGC signaling assembly and enhanced postprandial AKT signaling. The data reveal an age-sensitive response to nutritional timing but do not establish a causal connection between preservation of this assembly and the physiological improvements.
    Keywords:  anabolic resistance; dystrophin glycoprotein complex; insulin signaling; sarcopenia; time-restricted feeding
    DOI:  https://doi.org/10.1152/ajpcell.00315.2026
  13. Front Bioeng Biotechnol. 2026 ;14 1906715
    Rapsyn overexpression improves neuromuscular junction and skeletal muscle function after tourniquet-induced mouse hindlimb ischemia-reperfusion injuries.
    Huiyin Tu, Emmanuel Adu-Agyekum, Aaron N Barksdale, Michael C Wadman, Yu-Long Li.
      Tourniquet-induced hindlimb ischemia-reperfusion (tourniquet/IR) results in the fragmentation of nicotinic acetylcholine receptors (nAChR) clusters in the neuromuscular junction (NMJ) and impairs neurally controlled skeletal muscle contraction. Rapsyn is a cytoplasmic nAChR-associated protein and participates in the nAChR assembly at the NMJ. Here, we observed whether the down-expression of rapsyn mediated the structural damage and functional impairment of the NMJ and skeletal muscle from tourniquet/IR injuries in mouse hindlimb. Unilateral hindlimb of C57BL/6 mice was subjected to 3 h of ischemia by placing a rubber band and followed by 6 weeks of reperfusion (tourniquet/IR). Ad-m-RAPSN was injected into the gastrocnemius muscle to increase rapsyn levels. LV-m-Rapsyn shRNA was used to knockdown rapsyn levels. After 6 weeks of sham treatment or tourniquet/IR, skeletal muscle contraction, endplate potentials (EPPs), and morphology of NMJs were assessed. Compared to sham mice, NMJs in the gastrocnemius muscle were significantly fragmented with low rapsyn expression, and the amplitude of EPPs and muscle contraction were also decreased in tourniquet/IR mice. Ad-m-RAPSN transfection into tourniquet mice increased rapsyn expression, reduced fragmented NMJs (38.2% ± 4.0%, compared to 74.7% in tourniquet/IR alone, P < 0.05), and raised EPP amplitudes and skeletal muscle contraction. Conversely, rapsyn shRNA-downregulated rapsyn expression in normal mice resulted in the NMJ fragmentation and neuromuscular dysfunction. These data suggest that tourniquet/IR decreases rapsyn expression in the skeletal muscle, which results in the nAChR cluster fragmentation and impairs skeletal muscle contractile function. Rapsyn overexpression could be a therapeutic strategy to improve the morphological and functional recovery of the NMJ as well as skeletal muscle contraction from tourniquet/IR injuries.
    Keywords:  ischemia/reperfusion; neuromuscular junction; rapsyn; skeletal muscle; tourniquet
    DOI:  https://doi.org/10.3389/fbioe.2026.1906715
  14. Biomolecules. 2026 Oct 20. pii: 1527. [Epub ahead of print]16(10):
    Skeletal Muscle Aging Is a Significant Contributor to Systemic Aging.
    Mei-Mei Li, Xinwei Liu, Yixuan Li, Han Li, Yuhao Wang, Kangli Qiu, Wen Kong.
      Skeletal muscle aging is a conserved biological process characterized by progressive declines in muscle mass, strength, regenerative capacity, and metabolic adaptability, contributing to impaired mobility, frailty and age-related diseases. Beyond its canonical role in locomotion, skeletal muscle functions as an endocrine and metabolic organ that actively regulates organismal aging through myokines, metabolites, extracellular vesicles, thermogenic activity, and dynamic communication with distant organs. In this review, we summarize the intrinsic mechanisms driving skeletal muscle aging, including protein turnover imbalance and anabolic resistance, myosteatosis, denervation and motor unit loss, and cellular composition remodeling. We also discuss how aging skeletal muscle influences systemic aging through bidirectional interactions with metabolic organs, brain, cardiovascular system, and bone via endocrine signaling and interorgan communication. Finally, we highlight emerging interventions targeting skeletal muscle aging, including exercise, nutritional strategies, pharmacological approaches, and other novel therapies. Understanding skeletal muscle as both a target and regulator of systemic aging provides a framework for developing strategies to promote healthy aging and reduce the burden of age-associated diseases.
    Keywords:  age-associated disease; aging; myokine; regeneration; skeletal muscle
    DOI:  https://doi.org/10.3390/biom16101527
  15. Methods Mol Biol. 2027 ;3093 167-184
    Flow Cytometry Analysis of Mitochondrial Function in Muscle Stem Cells.
    Lucia Acampora, Melania Murolo, Shahragim Tajbakhsh, Barbara Gayraud-Morel.
      Mitochondrial metabolism plays a central role in regulating muscle stem cell (MuSC) quiescence, activation, and differentiation, with dynamic metabolic transitions accompanying changes in cell fate and function. Flow cytometry-based analysis of mitochondrial function provides a rapid and accessible approach to characterize metabolic states at the single-cell level. Here, we present a detailed protocol to analyze mitochondrial activity, particularly by assessing mitochondrial membrane potential and mitochondrial mass in freshly isolated MuSCs and myoblasts using the fluorescent dyes TMRE and MitoTracker Deep Red in cytometry. This method is compatible with antibody-based identification of MuSCs and includes controls using mitochondrial uncouplers to validate ΔΨm-dependent staining. This protocol enables reliable discrimination between quiescent, activated, and differentiating muscle cells and can be applied to study physiological and pathological conditions in different contexts, including injury, aging, and diseases.
    Keywords:  Flow cytometry; Metabolism; MitoTracker; Mitochondria; Satellite cells; Skeletal muscle; Stem cells; TMRE
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_13
  16. Methods Mol Biol. 2027 ;3093 91-100
    An Ex Vivo Cross-Grafting and Live-Imaging Assay to Analyze Myofiber Niche Impact on Muscle Stem Cells.
    Liza Sarde, Shahragim Tajbakhsh, Brendan Evano.
      The isolation and culture of primary muscle fibers and their associated muscle stem cells (MuSCs) allow investigation of MuSC function in their myofiber niche, notably in disease conditions. However, this assay does not discriminate between stem cell-intrinsic and fiber-derived cues driving MuSC (dys)function. Here, we describe the development of an ex vivo cross-transplantation assay between healthy and mutant MuSCs and myofibers ex vivo, coupled with live imaging for the dynamic characterization of MuSC behavior. The progeny of up to 60 MuSCs can be analyzed per experiment, in four grafting conditions and dynamically over a period of 3 days. This approach allows assessment of the relative contribution of the myofiber niche to impaired MuSC functions in disease conditions, providing a framework to experimentally dissect niche-dependent mechanisms underlying muscle stem cell dysfunction.
    Keywords:  Differentiation; Grafting; Live imaging; Migration; Muscle stem cell; Muscular dystrophy; Myofiber; Proliferation; Satellite cell; Skeletal muscle
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_8
  17. Proc Natl Acad Sci U S A. 2026 Nov 03. 123(44): e2612167123
    Annexins cooperate with Myomaker to promote myoblast fusion.
    Zhisheng Xu, Key Tse, Ning Liu, Eric N Olson.
      Myoblast fusion is essential for skeletal muscle development and regeneration. Myomaker is a muscle-specific fusogen indispensable for this process. Cryoelectron microscopy analysis has shown that Myomaker dimerization is required for myoblast fusion and muscle formation. However, the precise molecular mechanisms of Myomaker function remain unclear. Notably, the short extracellular domain of Myomaker suggests the involvement of additional proteins that cooperate with Myomaker to facilitate membrane fusion. To identify potential interacting partners of Myomaker, we employed an APEX2-based proximity labeling approach in a synchronized myoblast fusion and differentiation system. By comparing results with a fusion-deficient mutant form of Myomaker, we identified several members of the Annexin family of calcium-dependent phospholipid-binding proteins as prominent candidates for interaction, particularly under prefusion conditions. Among these, Annexins A2, A5, and A6 were found to colocalize with Myomaker at the myoblast plasma membrane. Proximity ligation assays further confirmed the close association of these Annexins with Myomaker at the cell surface. Functional analyses demonstrated that these Annexins can promote heterologous cell-cell fusion between fibroblasts and myoblasts. While single knockdown of Annexin A2, A5, or A6 had minimal effects, the simultaneous knockdown of all three impaired myoblast fusion, indicating functional redundancy among them. Together, these findings indicate that Annexins A2, A5, and A6 cooperate with Myomaker to promote myoblast fusion, providing a mechanistic link between the fusogenic machinery and membrane dynamics during myoblast fusion.
    Keywords:  heterologous cell–cell fusion; myogenesis; proximity labeling; skeletal muscle
    DOI:  https://doi.org/10.1073/pnas.2612167123
  18. Methods Mol Biol. 2027 ;3093 11-17
    Isolation of Mouse, Rat, and Human Muscle Stem Cells by Fluorescence-Activated Cell Sorting Based on α7-Integrin.
    Anaïs Bleuzen, Ghislain Banos, Teoman Ozturk, Emeline Faraüs, Julien Mignot, Nathalie Didier.
      Muscle stem cells (MuSC) are necessary for skeletal muscle tissue homeostasis and repair following injury. These cells are therefore extensively studied and are considered promising candidates for cell-based therapies, rendering their purification a recurring technical requirement. For this purpose, adhesion molecules expressed on MuSC surface represent ideal positive selection markers, for magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS). Here, we describe a method for efficiently isolating MuSC from postnatal and adult muscles of mice, rats, and humans by FACS, using α7-integrin (ITGA7) as common selection marker. We provide a detailed protocol for the immunostaining of mononucleated cells derived from digested skeletal muscle and the gating strategies to obtain highly pure MuSC from the muscles of these three species.
    Keywords:  Fluorescence-activated cell sorting; Muscle stem cells; Skeletal muscle; α7-integrin
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_2
  19. Int J Mol Sci. 2026 Oct 21. pii: 9369. [Epub ahead of print]27(20):
    M4-like Macrophages Drive Skeletal Muscle Fibrosis and Suppress Myogenesis via the MMP12/SPP1/NF-κB Axis.
    Senwen Yao, Zhenyuan Lu, Guosheng Zhao, Yang Liu, Shan Wu, Jiaxuan Zhang, Renjie Dong, Yangming Hu, Ye Zhang, Yang Wang.
      Acute skeletal muscle repair depends on a finely tuned balance between myogenic regeneration and fibrotic remodeling. Macrophages are essential to this process, and specific subsets have been linked to fibrosis in some organs. The M4-like macrophage, a distinct polarization subset implicated in fibrotic diseases, has not been well characterized in the context of injured muscle, particularly regarding its impact on fibroblast activation and myogenic suppression. The study aims to explore the effects of M4-like macrophages in the repair of muscle injury and possible mechanism. We analyzed a murine single-cell RNA-sequencing dataset from cardiotoxin (CTX)-injured skeletal muscle to identify injury-expanded macrophage subsets and their molecular programs. Immunohistochemistry and immunofluorescence staining of muscle sections were used to assess CXCL4 expression and platelet (CD41+) infiltration in injured versus uninjured tissues. CXCL4-induced M4-like macrophages were established as an in vitro model to examine their effects on L929 fibroblasts and C2C12 myoblasts. The MMP12-SPP1 interaction was predicted by AlphaFold3 and validated by co-immunoprecipitation and immunofluorescence colocalization. Loss-of-function and rescue experiments targeting MMP12 and SPP1 were performed to determine their causal roles in fibroblast activation and myogenic suppression. Transcriptomic and biochemical analyses were used to define downstream macrophage-intrinsic NF-κB/TNF-α signaling. Finally, AAV-mediated macrophage-targeted SPP1 knockdown and pharmacological NF-κB inhibition were evaluated in the CTX injury model. Using a BALB/c mouse model of cardiotoxin (CTX)-induced acute skeletal muscle injury and a publicly available murine scRNA-seq dataset from CTX-injured skeletal muscle, we observed an injury-induced MMP12/SPP1-positive macrophage subset with an M4-like transcriptional signature. Mechanistically, co-immunoprecipitation assays and immunofluorescence colocalization supported an association between MMP12 and SPP1. MMP12 knockdown attenuated the pro-fibrotic and anti-myogenic effects of M4-like macrophages, whereas recombinant SPP1 largely restored these phenotypes. Furthermore, transcriptomic and biochemical analyses revealed that autocrine SPP1 activated NF-κB signaling in M4-like macrophages, and increased TNF-α secretion. Pharmacological inhibition of NF-κB signaling with QNZ reduced fibroblast activation and restored the expression of myogenic markers. However, QNZ is not TNF-α specific and may affect multiple cell types. In vivo, AAV-mediated macrophage-targeted SPP1 knockdown driven by the F4/80 promoter, as well as QNZ treatment in the CTX-induced muscle injury model, markedly decreased collagen deposition, enhanced myogenic regeneration, and improved functional recovery. These findings identify an MMP12/SPP1/NF-κB signaling pathway in M4-like macrophages, with TNF-α as a potential downstream mediator, that may contribute to an imbalance between regeneration and fibrosis and may represent a therapeutic target for enhancing skeletal muscle repair after acute injury.
    Keywords:  M4-like macrophage; MMP12; SPP1; fibrosis; skeletal muscle injury; skeletal muscle repair
    DOI:  https://doi.org/10.3390/ijms27209369
  20. J Physiol. 2026 Oct 27.
    Non-steady-state modelling reveals reduced skeletal muscle protein synthesis as the primary driver of disuse-atrophy in young, healthy females.
    Callum J Pufahl, Jonathan C Mcleod, Emily J Ferguson, Kristine Boileau, Brendan Ross, Thomas R Tripp, Jennifer A Wilkinson, Craig A Simpson, Brendon J Gurd, Chris McGlory.
      Skeletal muscle mass reflects the balance between muscle protein synthesis (MPS) and muscle protein breakdown (MPB). In healthy humans, a decline in MPS is recognized as the primary driver of muscle disuse-atrophy, with limited contribution from increased MPB. However, disuse-induced declines in protein pool size directly influence the calculation of muscle protein turnover. We examined how accounting for changes in protein pool size impacted MPS and MPB rates assessed using deuterium oxide in healthy humans. Fourteen females (21 ± 2 years of age; 24.5 ± 3.0 kg/m2) had one leg subjected to 10 days of unloading, whilst the contralateral leg underwent loading in the form of resistance exercise. Steady-state and non-steady-state modelling approaches were used to calculate MPS and MPB rates. In response to unloading, there was a significant decline in MRI-derived vastus lateralis volume, and thus non-steady-state equations were applied exclusively to this limb. Regardless of whether steady-state or non-steady-state approaches were applied, mixed MPS rates were significantly reduced in response to unloading, whilst mixed MPB rates remained unchanged. Notably, unloading elicited protein fraction-specific responses, whereby myofibrillar MPS rates declined significantly whilst sarcoplasmic MPS rates were unaltered. We also found that in response to loading, mixed, myofibrillar and sarcoplasmic MPS rates remained unchanged compared with basal rates. Our findings show that unilateral unloading in females induces muscle disuse-atrophy driven by reductions in MPS rates, rather than by elevations in MPB rates, a response that was protein fraction specific and preserved irrespective of whether steady-state or non-steady-state equations were used. KEY POINTS: In humans, short-term muscle disuse-atrophy is generally attributed to reduced muscle protein synthesis (MPS) rates, but integrated tracer approaches to measure protein turnover can be influenced by changes in protein pool size. We used deuterated water (2H2O) to measure rates of MPS and muscle protein breakdown (MPB) during 10 days of unilateral unloading in young, healthy females. Steady-state and non-steady-state equations were applied to account for changes in the protein pool size that occur with disuse-atrophy. Unloading reduced muscle size and MPS rates, whereas MPB rates were unchanged. Accounting for the change in the protein pool size using non-steady-state equations did not alter this conclusion. These findings refine our interpretation of the mechanisms driving muscle disuse-atrophy in healthy humans that might differ from more severe clinical or preclinical scenarios of muscle wasting.
    Keywords:  exercise; muscle atrophy; skeletal muscle; stable isotope
    DOI:  https://doi.org/10.1113/JP292118
  21. J Funct Biomater. 2026 Oct 03. pii: 500. [Epub ahead of print]17(10):
    An Ion-Releasing Bioactive Glass Accelerates Skeletal Muscle Regeneration and Functional Recovery Following Acute Injury.
    Jacob A Kendra, Jiwei Hao, Dillon R Harris, Rebekah L Blatt, Richard K Brow, Aaron B Morton.
      Bioactive glasses represent an emerging strategy for localized skeletal muscle repair. Here, we investigated whether an injectable borophosphate bioactive glass [cobalt oxide-time release ion matrix (CoO-TRIM)] can accelerate skeletal muscle regeneration and functional recovery following acute injury. Using a barium chloride (BaCl2) injury model, muscle function, myogenesis, angiogenesis, and inflammation were evaluated in mouse tibialis anterior (TA) and extensor digitorum longus (EDL) muscles at 8 and 14 days post-injury (dpi). Mice were randomly assigned to Untreated, Saline-treated, and CoO-TRIM-treated groups. CoO-TRIM accelerated the regenerative process, with increased myofiber size and restoration of TA contractile function to values not significantly different from Healthy muscle by 14 dpi compared to Injured Untreated and Saline-treated muscles. Vascular endothelial growth factor (VEGF) concentrations were elevated through 14 dpi following CoO-TRIM treatment and accompanied by enhanced muscle vascularity in both TA and EDL muscles. CoO-TRIM modulated the inflammatory response, with increased inflammatory signaling during active regeneration at 8 dpi, followed by an accelerated transition toward a pro-regenerative environment characterized by increased CD163+ M2 macrophages and sustained IL-10 signaling. In contrast, CoO-TRIM produced no detectable effects in healthy contralateral muscles. These findings support ion-releasing bioactive glass as a cell- and growth factor-free strategy for skeletal muscle repair.
    Keywords:  angiogenesis; bioactive glass; muscle injury; regenerative medicine; skeletal muscle
    DOI:  https://doi.org/10.3390/jfb17100500
  22. Methods Mol Biol. 2027 ;3093 211-226
    Low-Input RNA-Sequencing of In Situ Fixed Adult Murine Muscle Stem Cells.
    Indigo T C Chan, Wenxin Zhang, Gary J He, Tom H Cheung.
      Muscle stem cells (MuSCs) play a crucial role in skeletal muscle regeneration. Under homeostatic conditions, the majority of MuSCs remain quiescent, a reversible, nonproliferative state with minimal cell activity. Upon muscle injury, MuSCs rapidly activate, proliferate, differentiate, and form nascent muscle fibers. Notably, the tissue dissociation process in conventional cell isolation protocols disrupts the stem cell niche environment, exposes the MuSCs to extracellular stimuli, and swiftly changes the transcriptomic profiles of MuSCs, making it challenging to study the regulation of MuSC quiescence. In vivo fixation allows the preservation of MuSC quiescence for further analysis and profiling. This chapter will focus on the detailed downstream RNA-seq library preparation workflow for fixed MuSCs, including RNA purification and decrosslinking, cDNA synthesis, and tagmentation-based library construction.
    Keywords:  In situ fixation; Muscle stem cell; Quiescence; RNA-seq
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_16
  23. Am J Physiol Cell Physiol. 2026 Oct 20.
    Loss of LanC-like proteins delays post-injury regeneration of aging skeletal muscles.
    Adriana Reyes-Ordoñez, Tianhui Hina Zhou, Tarun C Rao, Pallob Barai, Wilfred A van der Donk, Jie Chen.
      The adult skeletal muscle regenerates robustly upon injury, but this regenerative capacity rapidly declines with age. In this study, we identify the lanthionine synthetase C-Like (LanCL) proteins, mammalian homologs of the bacterial peptide cyclase LanC, as positive regulators of muscle regeneration in middle-aged mice. In a barium chloride-induced injury model, we found the protein levels of LanCL1 and LanCL2 to increase during an early phase of regeneration in middle-aged (12-month-old) but not young adult (4-month-old) mice. Utilizing a mouse line lacking all three LanCL proteins (LanCL triple KO or LTKO), we examined a potential role of LanCL in injury-induced muscle regeneration. Consistent with an age-dependent function of LanCL, we observed a delayed regeneration of the tibialis anterior (TA) muscle after injury, as reflected by reduced regenerating myofiber sizes at day 7 after injury (D7AI) in middle-aged (but not young) LTKO compared to age-matched WT mice. The pool size of quiescent satellite cells (Pax7+) was comparable between 12-month-old LTKO and WT muscles without injury. However, the number of Pax7+ cells was significantly higher in regenerating LTKO muscles at both D3AI and D5AI, accompanied by reduced numbers of MyoD+ and MyoG+ cells. The percentage of proliferating Pax7+ cells was also higher in LTKO muscles at D5AI. Furthermore, we detected elevated expression of pro-inflammatory cytokines in regenerating LTKO muscles, while the number of macrophages was similar comparing LTKO and WT muscles. Taken together, our observations suggest that in aging muscles LanCLs are important for proper timing of inflammation resolution and regeneration upon injury.
    Keywords:  LanCL; aging; inflammatory response; muscle regeneration
    DOI:  https://doi.org/10.1152/ajpcell.00313.2026
  24. Genome Biol. 2026 Jul 30. pii: 327. [Epub ahead of print]27(1):
    Proteomic ageing clocks: methods, mechanisms and the muscle connection.
    Elizabeth G Reisman, Macsue Jacques, Robin Grolaux, Vadim N Gladyshev, Nir Eynon.
      Proteomic ageing clocks are emerging tools that estimate biological age from protein abundance patterns. Most models are developed using blood capturing inflammatory, metabolic, and extracellular proteins, including secreted signalling factors. Large-scale plasma studies now predict chronological age, disease risk and mortality, but muscle-related models remain indirect, relying on organ-enriched circulating proteins rather than biopsy-derived skeletal muscle clocks. We review current proteomic clock frameworks, computational approaches, platform considerations and validation strategies, highlighting the potential of muscle-based clocks to capture tissue and subcellular-specific ageing processes. We discuss future integration of spatial, single-cell, and multi-omic data to enhance mechanistic insight and translational relevance.
    Keywords:  Ageing biomarkers; Multi-omics; Proteomic clocks; Skeletal muscle
    DOI:  https://doi.org/10.1186/s13059-026-04220-w
  25. Methods Mol Biol. 2027 ;3093 237-267
    Bioinformatic Workflow for the Analysis of Single-Nucleus RNA-Sequencing Data from Skeletal Muscle.
    Chiara D'Ercole, Laura Virtanen, Lorenzo Giordani.
      Understanding cellular diversity within a tissue is essential to elucidate how distinct cell populations cooperate to maintain homeostasis, regenerate, and respond to aging or disease. Unlike bulk methods that measure averaged gene expression, single-cell RNA sequencing (scRNA-seq) enables high-resolution profiling of the transcriptomes of individual cells, providing a powerful tool for analyzing cellular heterogeneity. However, its application in skeletal muscle remains limited to the mononucleated cellular fraction, as standard single-cell approaches cannot efficiently capture muscle fibers due to their large and multinucleated nature. Single-nucleus RNA sequencing (snRNA-seq), which profiles individual nuclei rather than intact cells, offers a more suitable and technically robust approach, enabling comprehensive characterization of the transcriptional landscape across the entire tissue. In this chapter, we present a workflow for the bioinformatic analysis of snRNA-seq data generated from mouse skeletal muscle using the 10x Genomics Chromium platform. The chapter focuses on computational processing using R software and the Seurat package, including quality control, normalization, dimensionality reduction, clustering, and cell-type annotation. By applying this pipeline, researchers can identify distinct muscle-resident cell populations and explore the complexity of skeletal muscle. The described framework also illustrates how to perform downstream analyses such as differential gene expression and cell-cell communication analysis.
    Keywords:  Bioinformatics; Single-nucleus RNA-seq; Skeletal muscle
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_18
  26. Biomolecules. 2026 Sep 25. pii: 1398. [Epub ahead of print]16(10):
    Biological Accessibility and Analytical Recovery in Myofibrillar Protein Loss During Skeletal Muscle Atrophy.
    Thanh Huu Phan Ngo, Wan Lee.
      Myofibrillar loss during skeletal muscle atrophy is often described as selective. MuRF1 is considered to remove thick filament proteins preferentially, whereas thin filament proteins are assigned to other ligases or appear relatively spared. We reassess whether this pattern demonstrates molecular specificity or is partly explained by the state in which a protein is encountered and measured. Several observations motivate this analysis. Actin can be polyubiquitinated by MuRF1 even though loss of assembled thin filament proteins is not MuRF1-dependent in several models. Apparent preferential myosin loss varies with extraction chemistry, normalization, and the combination of catabolic stimuli with inactivity. KLHL40 and KLHL41 preserve major thin filament proteins, although the direct evidence comes mainly from development and myopathy. Turnover and ubiquitin-remnant proteomics quantify molecules recovered from defined fractions and do not, on their own, distinguish degradation from redistribution. We therefore distinguish two biological determinants from one observer-side analytical layer. Assembly-dependent exposure and stability or damage-state regulation influence biological availability. Extraction, fractionation, detectability, and normalization determine analytical recovery. Ubiquitination remains distinct from degradation because chain architecture, deubiquitination, extraction, unfolding, and productive disposal can still determine fate. This framework retains ligase specificity while defining the controls needed to establish preferential degradation in intact muscle.
    Keywords:  actin dynamics; atrophy; myofibril extraction; skeletal muscle; thin filament; ubiquitin–proteasome system
    DOI:  https://doi.org/10.3390/biom16101398
  27. Int J Mol Sci. 2026 Oct 09. pii: 8946. [Epub ahead of print]27(20):
    NEMUCO: The In Vitro 4D NMJ as an Innovative Powerful Tool to Study Nerve and Muscle Cell-Cell Communication.
    Katharina Block, Gaia Ziraldo, Gabor Trautmann, Sandra Furlan, Abhishek Singh, Sabine Grosser, Martina Gutsmann, Paola Lorenzon, Pompeo Volpe, Imre Vida, Dieter Blottner, Michele Salanova.
      Chronic muscle unloading, following denervation, aging, disease, or long-term bed rest, whether as a spaceflight analog or during an actual spaceflight mission, results in a decline in structure and function of the neuromuscular junction (NMJ). This decline leads to a decrease in muscle mass and impaired fine motor control. Similar changes are also observed in several neuromuscular diseases and myopathies' impaired movement control symptoms. Here, we report on a powerful 3D co-culture experimental model developing NMJ-like structures, as a novel in vitro platform for functional/regulatory studies during time (4D co-culture) for basic and translational research purposes. Murine NErve (NSC-34 motoneuron) and MUscle (C2C12 myoblast) cell CO-cultures (NEMUCO) were grown on either synthetic or biological three-dimensional (3D) scaffolds. Morphological analysis obtained through 3D reconstruction in proximity of the cell-cell contact revealed close apposition between NSC-34 motoneurons and C2C12 myotubes. The co-cultures were further analyzed using a significant array of molecular and cellular biology tools, based on NMJ-specific molecular marker expression combined with muscle- and nerve-cell-specific differentiation biomarkers. Specialized cell-cell contacts were present starting at day 2 of co-culture. Triple immunostaining indicated neurofilament-positive axonal nerve terminals approaching clustered α-bungarotoxin-positive nicotinic acetylcholine receptors (nAChRs) in desmin- or fast-type myosin heavy chain (fMyHC)-positive developing myotubes, representing the first signs of NMJ-like structure assembly in our in vitro system. It is noteworthy that co-cultured motoneurons showed a trend of increase in SNAP-25 transcription, one of the most important SNARE proteins, whereas co-cultured myotubes showed a slight increase in junctophilin and ryanodine receptor type 1 (RyR1) transcription, both critical proteins of the triadic junction of differentiated muscle fibers. In most respects, neuronal-myotube cell-cell contacts in our 3D co-cultures mimicked native pre- and postsynaptic NMJ microdomains. Neuronal-myotube co-cultures grown in 3D scaffolds represent a powerful tool for investigating the molecular mechanisms underlying NMJ adaptation and plasticity in muscle myopathies. Moreover, this approach could be adapted for customized miniaturized platforms designed for cellular neurobiology research under spaceflight conditions.
    Keywords:  muscle and nerve cell co-culture; muscle synaptogenesis in vitro; neuromuscular junction development
    DOI:  https://doi.org/10.3390/ijms27208946
  28. Histol Histopathol. 2026 Oct 26. 25161
    The peripheral neuromuscular continuum: Histological remodeling induced by physical training.
    Walter Krause Neto, Eliane Florencio Gama.
      Physical exercise induces structural adaptations that extend far beyond skeletal muscle, affecting virtually every component of the peripheral neuromuscular system. Although remodeling of peripheral nerves and the neuromuscular junction (NMJ) has been increasingly documented, current knowledge remains fragmented because researchers have largely studied these structures in isolation rather than as components of a continuous biological system. This review critically synthesizes evidence on histological, ultrastructural, and cellular adaptations induced by physical exercise throughout the peripheral neuromuscular system. It emphasizes remodeling of peripheral nerve fibers, Schwann cells, the blood-nerve barrier, the extracellular matrix, and the NMJ, as well as the cellular and molecular mechanisms that coordinate these responses. The review also examines how aging affects peripheral nerve and NMJ integrity, discusses current methodological limitations, and proposes an integrative framework that interprets exercise-induced adaptation as coordinated remodeling across the peripheral neuromuscular continuum. Collectively, the available evidence indicates that regular physical exercise preserves structural integrity throughout the neuromuscular system, providing a broader biological basis for its protective effects than skeletal muscle adaptations alone.
    DOI:  https://doi.org/10.14670/HH-25-161
  29. Am J Physiol Regul Integr Comp Physiol. 2026 Nov 01. 331(5): R743-R752
    Intracellular calcium handling and not contraction type determines task-dependent skeletal muscle fatigue.
    Nathaniel J Andrews, Daiki Watanabe, Håkan Westerblad, Arthur J Cheng.
      This study compared skeletal muscle fatigue induced by different contraction types to determine whether repeated shortening contractions cause greater fatigability via larger reductions in sarcoplasmic reticulum Ca2+ release than repeated isometric contractions. A secondary objective was to assess whether fatigability differs when initial shortening and isometric contraction forces are matched between tasks. Single intact flexor digitorum brevis fibers from female C57BL/6 mice were dissected and loaded with indo-1 AM to measure myoplasmic free [Ca2+] ([Ca2+]i). Fibers were assigned to one of three protocols: isometric, shortening, or force-matched isometric. Fatigue was induced by stimulating fibers with tetani (300 ms) every 2 s for 50 contractions. Isometric and shortening groups were stimulated at 70 Hz, with shortening velocity set to achieve peak power; the force-matched isometric group was stimulated at ∼31 Hz (25-45 Hz) to match the initial force of shortening contractions. Every 10th contraction during all fatigue protocols, force and [Ca2+]i were assessed with isometric as well as shortening contractions to compare fatigability across tasks using identical contraction types. When fatigued by 70 Hz tetani, shortening and isometric contractions produced similar declines in force, tetanic [Ca2+]i, and resting [Ca2+]i. However, when compared with force-matched isometric contractions, shortening contractions resulted in greater fatigue-induced force loss, larger reductions in tetanic [Ca2+]i, and greater increases in resting [Ca2+]i (P < 0.05). Contraction type-dependent differences in skeletal muscle fatigue are driven primarily by differences in intracellular Ca2+ handling rather than the contraction type as such.NEW & NOTEWORTHY We investigated contraction-type-dependent fatigue mechanisms during repeated shortening versus isometric contractions in mouse intact single muscle fibers. When stimulated with repeated 70 Hz tetani, fatigue-induced changes in force and myoplasmic free [Ca2+] ([Ca2+]i) were similar between contraction types. However, when the initial forces were matched by decreasing the frequency in isometric contraction, shortening contractions caused greater fatigue-induced reductions in tetanic [Ca2+]i and force than isometric contractions. These findings indicate that intracellular Ca2+ handling, rather than contraction type, plays a predominant role in contraction-type-dependent fatigue.
    Keywords:  contraction type-dependent fatigability; excitation-contraction coupling; sarcoplasmic reticulum calcium release; single muscle fiber; skeletal muscle fatigue
    DOI:  https://doi.org/10.1152/ajpregu.90070.2026
  30. Cell Stem Cell. 2026 Oct 29. pii: S1934-5909(26)00385-1. [Epub ahead of print]
    Niche-reprogramming CAR T therapy improves skeletal muscle regeneration and function in preclinical murine models of Duchenne muscular dystrophy.
    Hamayl Afzal, Tomer Rotstein, Debolina D Biswas, Xin Lin, Alastair Khodabukus, Wenzhe Luo, Yueyuan Xu, Emma C Frair, Yongwu Li, Jingjuan Chen, Shihuan Kuang, Kevin M Flanigan, Nenad Bursac, Mai K ElMallah, Charles A Gersbach, Yarui Diao.
      Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder with no cure. Emerging dystrophin-restoring gene therapies target mutant myofibers but face challenges in efficacy and toxicities, underscoring the need for new treatments. Current therapies do not address chronic inflammation and muscle fibrosis, key pathologies creating a pathological muscle microenvironment that impairs dystrophic muscle function and regeneration. Here, we identify urokinase plasminogen activator receptor (uPAR) as a shared surface marker on subsets of macrophages and fibro-adipogenic progenitors exhibiting strong pro-inflammatory and fibrogenic signatures in DMD. Notably, uPAR-directed chimeric antigen receptor (CAR) T cells selectively eliminate these pathogenic subpopulations, remodel the muscle microenvironment, and enhance dystrophic muscle regeneration and force production; they also improve diaphragm and respiratory function, addressing an unmet clinical need and a major cause of DMD mortality. These findings establish uPAR CAR T cells as a niche-remodeling regenerative immunotherapy that simultaneously addresses multiple DMD pathologies and improves dystrophic muscle regeneration and function.
    Keywords:  CAR T therapy; DMD; chronic inflammation; fibrosis; muscle microenvironment; muscle regeneration
    DOI:  https://doi.org/10.1016/j.stem.2026.10.004
  31. bioRxiv. 2026 Oct 06. pii: 2026.10.05.756885. [Epub ahead of print]
    The annexin A1 mimetic peptide Ac2-26 promotes inflammation-resolution and stimulates skeletal muscle regeneration in the mdx mouse model of Duchenne muscular dystrophy.
    Hamood Rehman, Xinyue Lu, Savannah M Nasrallah, Binayok Sharma, Sophie Lies, Shikha Adhikari, Steven S Welc, James F Markworth.
      Duchenne muscular dystrophy (DMD) is an X-linked disorder mainly affecting young boys characterized by repeated cycles of myofiber injury/regeneration and chronic muscle inflammation. Although glucocorticoids can slow disease progression, their potential long-term adverse effects highlight the need for new steroid-sparing therapies for DMD. Annexin A1 (ANXA1) is a glucocorticoid-induced pro-resolving protein that signals primarily through formyl peptide receptor 2 (FPR2). We investigated whether the ANXA1 mimetic peptide Ac2-26 could reduce inflammation, stimulate tissue regeneration, and improve skeletal muscle function in muscular dystrophy. 3-week-old dystrophin-deficient male mdx mice were randomized to receive daily intraperitoneal injections of vehicle control, deflazacort (1 mg/kg/day), or Ac2-26 (1 mg/kg/day) for a duration of 5-weeks. Body composition, skeletal muscle mass, contractile function, and tissue histopathology/immune-cell infiltration were assessed at 8-weeks of age. Direct effects on myogenesis were also examined in vitro using primary D2- mdx myoblasts. ANXA1 was expressed predominantly by myeloid cells rather than resident dystrophic muscle cells. Both deflazacort and Ac2-26 markedly reduced intramuscular inflammation and increased myofiber size. However, Ac2-26 produced broader anabolic and functional benefits, increasing body-weight gain, lean body mass, hindlimb muscle weights, and absolute contractile force. Ac2-26 treatment also directly promoted myogenesis of dystrophic myoblasts in vitro , while pharmacological FPR2 blockade impaired myotube development. Overall, Ac2-26 mimicked the anti-inflammatory effects of deflazacort while providing superior benefits for body weight and composition, skeletal muscle growth, myofiber regeneration, and absolute muscle strength. These findings support therapeutic targeting of the ANXA1-FPR2 axis as a potential safe and effective steroid-sparing immunomodulatory strategy for DMD.
    DOI:  https://doi.org/10.64898/2026.10.05.756885
  32. Cells. 2026 Oct 20. pii: 1910. [Epub ahead of print]15(20):
    Disruption of the Muscle-Brain Axis in Ageing: Role of Myokines in Sarcopenia and Neurodegeneration.
    Víctor Agustín Morales Molina, Cecibel Nicole Ludeña Salinas, Celina Andrade Vásquez, Andri Matos, Diego Aldeán Riofrío.
      This narrative review analyzes how myokines act as key links in communication between skeletal muscle and the brain, focusing on the impact of aging on this axis. Although the current literature links defects in this signaling pathway to sarcopenia, neurodegeneration, and cognitive decline, we still do not know exactly what role each molecule plays individually. Among the main gaps in our understanding are questions about how these substances are transported to the nervous system, what their specific receptors are, and what cause-and-effect relationship actually exists between losing muscle mass and developing neurological problems. In fact, in humans, it remains unclear whether myokines actually reach the brain in truly effective concentrations or whether their benefits are due more to peripheral metabolic, vascular, and inflammatory changes. Today, most of the mechanistic evidence comes from animal and cellular models, while observational studies predominate in humans. This is further complicated by uncertainties regarding the actual origin of these proteins in tissues, their passage through the blood-brain barrier, and their measurement in the blood, making it difficult to determine whether these proteins act as causes, consequences, or simply compensatory responses to aging and disease. On the other hand, although exercise remains the most robust tool for maintaining muscle health and protecting cognitive function, it is still unclear whether we could replicate those same benefits by isolating and stimulating specific myokines. The major challenge for the future is to develop human studies that combine standardized biomarkers with long-term monitoring of muscle function, cognition, and brain structure. Only then will we know whether this fascinating muscle-brain axis can move beyond being a promising hypothesis to become a real therapeutic target.
    Keywords:  ageing; myokines; neurodegeneration; sarcopenia; skeletal muscle–brain axis
    DOI:  https://doi.org/10.3390/cells15201910
  33. Methods Mol Biol. 2027 ;3093 37-48
    Tissue Clearing and Immunostaining of Mouse and Human Skeletal Muscles for In Situ Analysis of Muscle Stem Cell Spatial Organization.
    Marie Quétin, Julien Mignot, Juliette Berthier, Christelle Dubois, Nathalie Didier, Marianne Gervais.
      Skeletal muscle is essential for voluntary movement, postural maintenance, respiration, and thermoregulation. It consists of large, multinucleated myofibers and has strong regenerative capacity driven by resident muscle stem cells (MuSCs) located beneath the myofiber basal lamina. Traditional histological methods using longitudinal or cross sections have improved our understanding of muscle architecture, but their two-dimensional nature limits insight into the tissue's complex three-dimensional (3D) organization and cellular interactions. To better understand MuSC dynamics, 3D imaging of entire tissue volumes is necessary. However, deep tissue imaging remains challenging due to light scattering and autofluorescence within muscle tissue. While advances in microscopy and computational imaging have improved spatial resolution, optical tissue clearing has become essential to enhance tissue transparency. Yet, existing clearing methods, though effective, often cause tissue deformation or fluorescence loss, compromising data reliability. Moreover, in situ visualization of MuSCs frequently relies on fluorescent reporter models, which is incompatible with human tissues. This chapter introduces an optimized tissue-clearing protocol for skeletal muscle and MuSC visualization that preserves both morphology and fluorescence. Compatible with mouse and human samples, it enables high-resolution 3D imaging of MuSCs within their native microenvironment, providing a powerful tool to study muscle architecture, regeneration, and disease mechanisms.
    Keywords:  3D reconstruction; Muscle stem cells; Skeletal muscle; Tissue clearing
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_4
  34. Curr Issues Mol Biol. 2026 Oct 19. pii: 1080. [Epub ahead of print]48(10):
    A Cross-Condition Single-Cell Atlas Defines Skeletal Muscle Adaptation and Its Predictive Limits.
    Wanting Tong, Renyu Yang, Qi Yu, Chunxue Shao, Guang Yang, Ziheng Wang.
      Skeletal muscle adapts to diverse stresses through coordinated changes across myofibres and cellular niches, yet these responses are usually studied separately. We assembled a cross-species discovery atlas of 501,940 cells and nuclei from six public resources, comprising 2863 condition-time-state combinations defined at the declared analysis unit level, and evaluated it with two independent validation datasets. Injury produced the largest observed compositional displacement, followed by mechanical overload, whereas acute human endurance exercise and denervation showed smaller occupancy shifts despite substantial cell-type-specific molecular responses. Genetic lineage data identified distinct newly fused myonuclear programmes; the overloaded Myomaker-knockout versus sham comparison remained limited by loading, genetic background and batch differences. Spatial analyses identified local enrichment of a terminal Schwann cell expression module around neuromuscular junction regions, and paired human datasets provided design-sensitive translational support. Across 20 fixed model specifications with training-only predictor preprocessing, the baseline-plus-context control model showed small average improvements over composition-only and intervention permutation controls, with heterogeneous performance across study holdouts. Here, we show that an evidence-graded atlas can characterise observed adaptive remodelling alongside study-dependent baseline structure while defining the limits of prospective prediction based on coarse composition.
    Keywords:  cellular composition; muscle adaptation; predictive modelling; single-cell RNA sequencing; single-nucleus RNA sequencing; skeletal muscle; spatial transcriptomics
    DOI:  https://doi.org/10.3390/cimb48101080
  35. Res Sq. 2026 Oct 06. pii: rs.3.rs-10972321. [Epub ahead of print]
    Sex-dimorphic DNA lesion accumulation drives a PDK4 metabolic checkpoint in skeletal muscle.
    Aditi Gurkar, Sriravali Pulipaka, Akanksha Sachan, Zachary Clemens, Kritika Chaddha, Jagruti Kosaraju, Ulalume Hernández-Arciga, Zarifeh Rarani, Augustine George, Emily Madigan, Lillian Williams, Naghmeh Abbasi, Isabel Joyce, Bommana Reddy, Kritika Rao, Savita Khanna, Chandan Sen, Sruti Shiva, Andrey Parkhitko, Eric Goetzman, Michael Jurczak, Daniel Shiwarski, Natalie Gassman, Jishnu Das, Amrita Sahu.
      Skeletal muscle loss and functional decline are sexually dimorphic, with fast-twitch type II fibers disproportionately vulnerable to age-related atrophy. DNA damage accumulation is a hallmark of aging whose consequences in myofibers remain poorly understood. Using a type II fiber-specific DNA repair-deficient mouse, we show that the same genotoxic insult produces qualitatively different lesion landscapes by sex, oxidative damage in females and uracil misincorporation in males. Females exhibited greater atrophy pathway activation, paralleling clinical evidence that genotoxic stress disproportionately drives frailty in women. Single- nucleus RNA sequencing and metabolomics identified PDK4 as the key female-specific metabolic driver. Pharmacologic PDK4 inhibition restored oxidative metabolism and myotube health. Dietary α-ketoglutarate, replenishing TCA intermediates downstream of the PDK4 block, rescued contractile force and metabolic profiles in females. Human aging data confirmed conservation of this pathway, revealing a sex-specific mechanism linking DNA damage to metabolic dysfunction and identifying an accessible therapeutic strategy to combat female-predominant frailty.
    DOI:  https://doi.org/10.21203/rs.3.rs-10972321/v1
  36. Geroscience. 2026 Oct 28.
    Characterization of sex-dependent skeletal muscle alterations in the 3xTg Alzheimer's disease mouse model.
    Amy Creecy, David L Waning, Izza Khurram, Abdullahi Warsame, Rachel J Blosser, Sonali J Karnik, Jing Xu, Chiaki Yamada, Debra K Smith, Adrian L Oblak, Jill C Fehrenbacher, Alexandru Movila, Melissa A Kacena, Joshua R Huot.
      Alzheimer's disease (AD) is increasingly recognized as a systemic condition with effects beyond the central nervous system. Skeletal muscle dysfunction, including sarcopenia and reduced physical performance, is prevalent in AD patients, yet the underlying mechanisms remain poorly understood. In this study, we characterized skeletal muscle alterations in male and female 3xTg-AD mice at 3, 6, and 12 months of age and compared them to age- and sex-matched B6129 controls. We measured body composition, muscle function, and skeletal muscle parameters including fiber type distribution, cross-sectional area (CSA), and neural cell adhesion molecule (NCAM) expression. Our findings revealed early-onset and progressive impairments of skeletal muscle mass, morphology, and function in 3xTg-AD mice, with sex-specific differences. Male mice exhibited reduced lean mass, muscle weight, and torque generation as early as 3 months and decreased CSA of type IIB and IIX fibers and increased NCAM expression by 12 months, indicating a potential neuromuscular junction deficiency. Female mice in this study generally exhibited a milder phenotype. Together, these results identify skeletal muscle dysfunction as an early and sex-dependent feature of AD pathology in 3xTg-AD mice. Our study underscores the importance of considering systemic effects in AD and highlights skeletal muscle as a potential target for therapeutic intervention aimed at preserving mobility and quality of life in affected individuals.
    Keywords:  Alzheimer’s disease; Body composition; Denervation; Mouse model; Muscle function; Skeletal muscle
    DOI:  https://doi.org/10.1007/s11357-026-02569-1
  37. Cell Mol Life Sci. 2026 Aug 01. pii: 400. [Epub ahead of print]83(1):
    Mdfi overexpression promotes oxidative muscle fiber formation without affecting muscle development in mature skeletal muscles.
    Tingting Lu, Xulong Liu, Chao Wang, Bo Huang, Ziyuan Mo, Jiali Guo, Xuezhu Gong, Yifan Zhu, Xiong Tong, Ching Yuan Hu, Chong Wang.
      Skeletal muscles are composed of multinucleated fibers with distinct metabolic and contractile properties. Skeletal muscles support locomotion and systemic metabolism, and their dysfunction underlies the pathogenesis of muscular dystrophies. The MyoD family inhibitor (Mdfi) has been implicated in muscle biology, yet its function in muscle development and muscle fiber type conversion remains largely unclear. Here, we developed the first tamoxifen (TMX)-inducible, skeletal muscle-specific Mdfi overexpression mouse model using CRISPR/Cas9 and CreERT2 integration, enabling spatiotemporal control of Mdfi activity to mimic therapeutic modulation. This model revealed that Mdfi exerts dual-phase regulatory roles: suppressing early postnatal myogenesis while driving glycolytic-to-oxidative fiber switching in mature muscle. Mechanistically, elevated Mdfi activates the calcium signaling pathway to enhance mitochondrial oxidative phosphorylation, directly linking its function to improved muscle fatigue resistance. These Mdfi overexpression-induced phenotypic alterations exhibit stable transgenerational inheritance patterns. Our findings establish Mdfi as a key regulator of muscle plasticity and metabolic adaptation, providing a genetically tractable model for investigating muscle development and energy metabolism. Importantly, this study opens new avenues for understanding and potentially modulating fiber-type composition in muscular dystrophies and other disorders involving metabolic insufficiency, offering a conceptual framework for future therapeutic interventions aimed at improving muscle health.
    Keywords:  CRISPR/Cas9 technology; Calcium signaling pathway; CreERT2 system; Mdfi.; Transformation of skeletal muscle fiber types
    DOI:  https://doi.org/10.1007/s00018-026-06279-x
  38. J Physiol. 2026 Oct 23.
    Progesterone does not modulate myofibrillar protein synthesis at rest or following acute resistance exercise in premenopausal females.
    Alysha C D'Souza, Alison K Shea, Caroline B Dunlop, Matthew J Lees, Hafsa Ali, Dax Houtkamp, Joel L Prowting, James P Newbold, Stuart M Phillips.
      Progesterone has been proposed to modulate skeletal muscle protein turnover, although direct support for this hypothesis is lacking. We examined whether acute elevation of progesterone alters myofibrillar protein synthesis (MyoPS) at rest and following resistance exercise (RE) in naturally menstruating females. Twenty premenopausal females (21 ± 3 years of age) were randomised in a double-blind, placebo-controlled trial to receive oral micronized progesterone (400 mg) or matching placebo at -34 and -10 h before a stable isotope infusion on days 1-5 of their menstrual cycle. One leg performed RE while the contralateral leg remained rested, followed by ingestion of 30 g whey protein. MyoPS was quantified using the l-[ring-1 3C6]-phenylalanine precursor-product method across fasted-rested, fed-rested and fed-exercised conditions. Progesterone elevated serum concentrations well above the luteal-phase range (185 ± 105 vs. 1.0 ± 0.2 ng mL-1). Despite this, we detected no difference in MyoPS groups in any conditions, including the exercise-induced increment (treatment × leg interaction, P = 0.945). Both protein feeding (P = 0.00459) and RE (P < 0.001) independently stimulated MyoPS above resting fasted levels. Progesterone was associated with modestly lower total amino acid concentrations, but not essential amino acid or leucine specifically; postprandial kinetics did not differ between groups. Acute elevation of circulating progesterone above the luteal-phase range does not detectably modify the skeletal muscle anabolic response to RE or protein feeding in premenopausal females. These findings challenge the characterisation of progesterone as catabolic (or anabolic) to skeletal muscle and indicate that menstrual cycle phase-based training recommendations predicated on a progesterone-driven effect warrant critical re-evaluation. KEY POINTS: Progesterone has been proposed to exert catabolic effects on skeletal muscle, yet direct evidence in premenopausal adult females is absent. We examined whether pharmacological elevation of progesterone augments myofibrillar protein synthesis (MyoPS) at rest and following resistance exercise. Oral micronized progesterone administered at 34 and 10 h before testing successfully elevated serum progesterone to well above the luteal-phase range (185 ± 105 vs. 1.0 ± 0.2 ng mL-1) during the early follicular phase. Despite this, MyoPS did not differ between progesterone and placebo in fasted-rested, fed-rested or fed-exercised conditions. Resistance exercise robustly elevated MyoPS above the fed-rested conditions, confirming model sensitivity. Neither postprandial amino acid availability nor individual progesterone exposure was associated with MyoPS. These findings challenge the characterisation of progesterone as pro-catabolic/anti-anabolic to skeletal muscle and indicate that menstrual cycle phase-based training and nutrition recommendations predicated on progesterone-driven effects on skeletal muscle are not supported by direct mechanistic evidence.
    Keywords:  female; menstrual cycle; progesterone; protein synthesis; resistance exercise; skeletal muscle
    DOI:  https://doi.org/10.1113/JP292285
  39. Cell Biochem Biophys. 2026 Sep 21.
    RNA-Based Biological Age Clocks Track Chronological Age in Skeletal Muscle but Show Limited Discrimination of Sarcopenia.
    Sergio David Ledesma-Gallardo, Emma López-Espinosa, Alejandra Isabel Ortega-Meléndez, Eric Gustavo Ramírez-Salazar, Nelly Patiño, Berenice Rivera Paredez, Rafael Velázquez-Cruz, Alberto Hidalgo-Bravo, Rogelio Frank Jiménez-Ortega.
      Transcriptomic biological age clocks may track age-related transcriptional variation in skeletal muscle, but it remains unclear whether they detect sarcopenia-related molecular variation beyond chronological age. This multicohort study benchmarked directly implementable RNA-based age models in public human skeletal muscle RNA-seq datasets. GSE164471 was used as a healthy-aging reference, GSE111006/GSE111010 as a pooled discovery benchmark, GSE167186 as external validation, and GSE174106 as biological validation using appendicular lean mass trajectories. RNAAgeCalc skeletal muscle and across-tissue models were implemented for age estimation and sarcopenia discrimination. Gene coverage was used only as a preliminary technical-compatibility assessment, after which the published SkeletAge model was directly applied to GSE111006 and GSE111010 for independent chronological-age prediction and sarcopenia-discrimination analyses; GSE164471, which contributed to SkeletAge model development, was used only for an in-sample implementation check. Both RNAAgeCalc models showed high gene coverage. In GSE164471, the skeletal muscle model showed stronger chronological-age tracking than the across-tissue model (r = 0.604 vs. 0.266; MAE 15.2 vs. 16.7 years), but retained substantial absolute prediction error and positive mean bias (+ 10.4 years), indicating that stronger correlation did not imply adequate calibration. In the discovery benchmark, between-group differences in chronological age, transcriptomic age, and age-acceleration residuals were not statistically significant, with small-to-moderate standardized effect estimates and confidence intervals spanning zero. Logistic discrimination was influenced mainly by cohort origin, while transcriptomic aging measures provided limited independent discrimination. External validation showed broad age-group structure with substantial overlap between old healthy and sarcopenic participants, and no differences were detected across appendicular lean mass trajectories. Current RNAAgeCalc implementations therefore appear to track chronological-age-related transcriptomic structure more effectively than sarcopenia-related variation, although smaller disease-related effects cannot be excluded, and these models should not currently be considered standalone sarcopenia biomarkers.
    Keywords:  Biological aging; RNA-seq; Sarcopenia; Skeletal muscle; Transcriptomic age
    DOI:  https://doi.org/10.1007/s12013-026-02200-2
  40. Am J Physiol Endocrinol Metab. 2026 Nov 01. 331(5): E562-E571
    Adipocyte lipolysis is a determinant of skeletal muscle size during fasting and feeding.
    Daniel M Marko, Rhea V Vemula, Meghan O Conn, Nazli Robin, Angela M Schmidt, Irena A Rebalka, Jonathan D Schertzer.
      Intermittent fasting (IF) can lower fat mass, but also muscle mass. The mechanisms governing tissue partitioning during fasting remain unclear. During fasting, adipose-derived fatty acids from lipolysis and lipolysis-fueled hepatic ketogenesis maintain blood glucose and systemic energy homeostasis. We hypothesized that impaired lipolysis during fasting would shift substrate provision from adipose to muscle, increasing muscle catabolism during IF. We tested male adipocyte-specific adipose triglyceride lipase knockout (aKO) and littermate floxed-control mice during ad libitum feeding on a 10-wk 5:2 IF protocol consisting of two nonconsecutive 24-h fasts and 5 ad libitum feeding days per week. After 10 wk of IF, aKO mice had lower blood lipids and ketones and lost more lean mass and less fat mass than floxed-control mice when mice were fed a standard control diet and also retained more inguinal white adipose tissue and had lower hindlimb muscle mass when IF was compared with ad libitum feeding. In addition, aKO mice had larger adipocytes, smaller muscle fibers, and increased expression of the atrogene, Atrogin1. We then provided exogenous lipids by feeding an obesogenic high-fat diet (HFD). HFD-fed aKO mice had higher whole body fat oxidation and lower blood lipids and ketones than floxed-controls. Obese HFD-fed aKO mice still had smaller skeletal muscle myofibers and increased muscle atrogene expression despite smaller adipocytes during ad libitum feeding or IF. Therefore, adipocyte ATGL-mediated lipolysis is a determinant of skeletal muscle size, and lower lipolysis influences the balance of fat vs. muscle loss during fasting.NEW & NOTEWORTHY This study demonstrates that adipocyte ATGL is a critical regulator of adipose-muscle metabolic crosstalk. Limiting adipocyte lipolysis through adipose ATGL deletion reduced blood lipid and ketone fuel availability, thereby increasing adipose retention, decreasing skeletal muscle mass and fiber size, and increasing muscle atrogene expression, particularly during intermittent fasting. These findings support a model in which impaired adipocyte lipid mobilization shifts whole body energy demands away from adipose and toward skeletal muscle, promoting muscle catabolism during fasting.
    Keywords:  ATGL; intermittent fasting; lipolysis; obesity
    DOI:  https://doi.org/10.1152/ajpendo.00262.2026
  41. Crit Care. 2026 Oct 24. pii: 549. [Epub ahead of print]30(1):
    Preserved anabolic skeletal muscle capacity in response to exercise and protein intake after critical illness.
    Arabella Fischer-Hammerschmied, Nicolas Tardif, Mirko Mandić, Thomas Gustafsson, William Apro, Johan Helleberg, Mariam Amouzandeh, Jonathan Grip, Dag Seldén, Olav Rooyackers, Martin Sundström Rehal.
       BACKGROUND: Low muscle mass and persistent weakness are common after critical illness, but the mechanisms behind impaired recovery are incompletely understood. The aim of this study was to investigate the skeletal muscle anabolic response to combined resistance exercise and protein intake in intensive care unit (ICU) survivors, compared to matched controls without a history of critical illness. We hypothesised that residual anabolic resistance limits muscle regeneration after ICU stay.
    METHODS: Former patients were enrolled within 3 to 6 months of intensive care unit discharge. Muscle biopsies were obtained before and after resistance exercise using a flywheel leg extension ergometer, combined with ingestion of 24 g hydrolysed protein. Muscle protein fractional synthetic rate was determined from the incorporation of [ring-²H₅]phenylalanine into mixed-muscle protein during a primed continuous intravenous infusion. Anabolic and proteolytic pathways were measured using standard assays. Group-by-time differences were analysed using linear mixed models. The primary comparison was the between-group difference in pre-to-post change in muscle protein synthesis.
    RESULTS: Twenty ICU survivors (60 ± 12 years, 30% female, BMI 26.9 (IQR 22.9-28.7), SAPS 3 62.3 ± 15.8) and 20 age- and sex-matched controls were included. ICU survivors had significantly lower quadriceps muscle thickness (p = 0.02) and handgrip strength (p = 0.04) than controls. In ICU survivors, muscle protein synthesis from plasma precursor amino acids was higher at baseline than in controls (0.041%/hour vs. 0.030%/hour, p = 0.009), but increased less (Δ 0.031 vs. Δ 0.040%/hour, p = 0.04). Post-intervention muscle protein synthesis was similar between groups (0.071%/hour vs. 0.070%/hour, p = 0.70). At baseline, ICU survivors showed lower activation of the autophagy-lysosomal pathway. In both groups, the intervention increased anabolic signalling, decreased initiation of autophagy and increased parts of the ubiquitin-proteasome pathway.
    CONCLUSIONS: In ICU survivors with persistent muscle loss and weakness, the increase in muscle protein synthesis after combined resistance exercise and protein intake was smaller than in controls, in the context of a higher baseline fractional synthetic rate. Post-intervention muscle protein synthesis rates were similar between groups. These findings provide the first mechanistic support for structured exercise-nutrition rehabilitation to promote muscle recovery after critical illness.
    TRIAL REGISTRATION: This study was prospectively registered at ClinicalTrials.gov (NCT05261984), 2022-02-20.
    Keywords:  Anabolism; Critical illness; Muscle protein synthesis; Rehabilitation; Resistance training
    DOI:  https://doi.org/10.1186/s13054-026-06418-7
  42. Methods Mol Biol. 2027 ;3093 153-164
    Directed Differentiation of Human Pluripotent Stem Cells into Skeletal Muscle Progenitor Cells.
    Dieu-Huong Hoang, Nimisha Gupta, Sam Torres, Michael R Hicks, April D Pyle.
      Human pluripotent stem cells (hPSCs) hold the potential to differentiate into skeletal muscle progenitor cells (SMPCs), which provide a human model for muscle disease modeling, drug screening, and a promising cell source for future stem cell-based muscle therapies. Directed differentiation is a transgene-free approach to guide hPSCs through the developmental path and differentiate them into embryonic-like SMPCs. However, the directed differentiation of functional SMPCs from hPSCs can be challenging due to cell line variations leading to low efficiency and reproducibility. In this section, we introduce a robust protocol that has been validated in multiple hPSC lines, providing cues on how to ensure efficient SMPC differentiation routinely. We describe the directed differentiation and isolation of SMPCs, using well-characterized markers ERBB3 and NGFR. Previous work from our lab has generated transcriptomic profiles of hPSC-derived SMPCs and functionally characterized the differentiated SMPCs in vitro and in vivo using different mouse models, and in this chapter, we will provide a detailed method for generating SMPCs used in these and additional studies.
    Keywords:  Directed differentiation; Human pluripotent stem cells; Myotubes; PAX7; Skeletal muscle progenitor cells
    DOI:  https://doi.org/10.1007/978-1-0716-5643-3_12
  43. Nat Genet. 2026 Oct 28.
    Population-scale single-nucleus multi-omic profiling of skeletal muscle reveals extensive context-specific genetic regulation.
    Arushi Varshney, Nandini Manickam, Peter Orchard, Adelaide Tovar, Christa Ventresca, Zhenhao Zhang, Fan Feng, Joseph Mears, Michael R Erdos, Narisu Narisu, Kirsten Nishino, Vivek Rai, Heather M Stringham, Anne U Jackson, Tricia Tamsen, Chao Gao, Mao Yang, Olivia I Koues, Joshua D Welch, Charles F Burant, L Keoki Williams, Chris Jenkinson, Ralph A DeFronzo, Luke Norton, Jouko Saramies, Timo A Lakka, Markku Laakso, Jaakko Tuomilehto, Karen L Mohlke, Jacob O Kitzman, Heikki A Koistinen, Jie Liu, Michael Boehnke, Francis S Collins, Laura J Scott, Stephen C J Parker.
      Skeletal muscle tissue underlies several polygenic metabolic traits and diseases. Identifying their genetic mechanisms requires pinpointing cell types, regulatory elements, target genes and causal variants. Here we use genetic multiplexing to generate population-scale single-nucleus chromatin accessibility (single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq)) and transcriptome (single-nucleus RNA sequencing (snRNA-seq)) maps across 287 frozen human skeletal muscle biopsies. We identify 13 cell types and integrate genetic variation to discover >7,000 expression quantitative trait loci (eQTLs) and >100,000 chromatin accessibility QTLs (caQTLs) across cell types, including context-specific eQTL/caQTL with significant G × C interaction. We identify nearly 2,000 eGenes colocalized with caQTL and construct causal directional maps for chromatin accessibility and gene expression. Almost 3,500 genome-wide association study (GWAS) signals across 38 relevant traits colocalize with sn-eQTL/caQTL, most in a cell-specific manner. These signals typically colocalize with caQTL and not eQTL, highlighting the importance of population-scale chromatin profiling for GWAS functional studies. The results of this study illuminate the genetic regulatory architecture of human skeletal muscle.
    DOI:  https://doi.org/10.1038/s41588-026-02786-6
  44. J Cell Mol Med. 2026 Nov;pii: e71407. [Epub ahead of print]30(21):
    Bazi Bushen Attenuates Skeletal Muscle Decline Associated With Oestrogen Deficiency and Modulates FGFR1/ERK Signalling.
    Dezhi Huang, Na Xu, Qi Chu, Zhenhua Jia, Yan Wang, Jin Xu.
      Postmenopausal oestrogen deficiency is associated with skeletal muscle loss and functional decline, thereby increasing the risk of sarcopenia in women. Considering the safety risks linked to hormone replacement therapy (HRT), alternative strategies are needed to attenuate skeletal muscle decline after menopause. A total of 24 female C57BL/6J mice were randomly assigned to four groups (n = 6 per group): Sham-operated (SHAM), ovariectomized (OVX), OVX treated with estradiol valerate (OVX + E2) and OVX treated with Bazi Bushen (OVX + BZBS). Skeletal muscle mass and function were evaluated in four groups. RNA sequencing was applied to explore the molecular mechanisms and key signalling pathways underlying BZBS-induced muscle protection. We further examined the role of the FGFR1/ERK signalling pathway in mediating the effects of BZBS on myogenic differentiation in C2C12 myoblasts. The results showed that OVX mice had reduced grip strength and skeletal muscle mass, increased fat accumulation, reduced lean mass and impaired metabolic activity. These phenotypic alterations were accompanied by myofiber atrophy, increased collagen staining, endocrine perturbations and elevated expression of p16, p21, Atrogin-1 and MuRF1. BZBS ameliorated these abnormalities. RNA-seq suggested that FGF/MAPK signalling may be involved. In D-gal-treated C2C12 myoblasts, BZBS reduced Atrogin-1 and MuRF1 expression while restoring MYHC and MYOG expression. Mechanistically, BZBS suppressed FGFR1/ERK signalling activation and bFGF partially reversed the protective effects of BZBS. Taken together, these findings suggest that FGFR1/ERK signalling may contribute to the protective effects of BZBS. Our findings provide evidence for the beneficial effects of BZBS on skeletal muscle deterioration associated with oestrogen deficiency.
    Keywords:  Bazi Bushen; FGFR1/ERK signalling; oestrogen deficiency; skeletal muscle deterioration
    DOI:  https://doi.org/10.1111/jcmm.71407
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