bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2025–08–31
fifty papers selected by
Anna Vainshtein, Craft Science Inc.



  1. Exp Physiol. 2025 Aug 25.
      MicroRNAs (miRNAs) are key regulators of cellular processes, including mitochondrial function and energy metabolism. This study explores the regulation of miR-494 in skeletal muscle and circulation, investigating its response to exercise training and an acute exercise bout, its association with metabolic disorders, and the effects of electrical pulse stimulation (EPS). In addition, it validates the gene targets and physiological role of miR-494 using gain- and loss-of-function studies in primary human skeletal muscle cells. We demonstrate that miR-494 levels in both skeletal muscle and circulation are influenced by long-term exercise training, which induces adaptive changes, but remain unaffected by an acute bout of exercise. EPS does not alter miR-494 levels in cultured primary human skeletal muscle cells. Moreover, muscle miR-494 levels remain unchanged under various metabolic challenges, including obesity and type 2 diabetes. Genetic manipulation of miR-494 in primary human skeletal muscle cells modulates mitochondrial biogenesis and function, as well as lipid metabolism, through targeting PGC1A and SIRT1. Injection of a miR-494 inhibitor into skeletal muscle of mice supports the role of miR-494 in regulating Pgc1α mRNA, suggesting potential therapeutic implications. These findings highlight miR-494 as a significant modulator of mitochondrial dynamics and energy metabolism in skeletal muscle.
    Keywords:  exercise; metabolism; microR‐494; mitochondria; skeletal muscle; type 2 diabetes
    DOI:  https://doi.org/10.1113/EP092977
  2. Muscles. 2025 Jul 31. pii: 26. [Epub ahead of print]4(3):
      Skeletal muscle aging and related diseases are characterized by progressive loss of muscle mass, strength, and metabolic function. Central to these processes is mitochondrial dysfunction, which impairs energy metabolism, redox homeostasis, and proteostasis. In addition, non-mitochondrial factors such as muscle stem cell exhaustion, neuromuscular junction remodeling, and chronic inflammation also contribute significantly to muscle degeneration. This review integrates recent advances in understanding mitochondrial and non-mitochondrial mechanisms underlying muscle aging and disease. Additionally, we discuss emerging therapeutic approaches targeting these pathways to preserve muscle health and promote healthy aging.
    Keywords:  inflammation; mitochondrial dysfunction; muscle aging; muscle stem cells; neuromuscular junction; therapy
    DOI:  https://doi.org/10.3390/muscles4030026
  3. Cell Prolif. 2025 Aug 24. e70120
      Sarcopenia profoundly impacts the quality of life and longevity in elderly populations. Notably, alterations in thyroid hormone (TH) levels during ageing are intricately linked to the development of sarcopenia. In skeletal muscle, the primary action of TH is mediated through the thyroid hormone receptor alpha (TRα). Emerging evidence suggests that decreased TRα expression may precipitate mitochondrial dysfunction in ageing skeletal muscle tissues. Yet, the precise mechanisms and the potential causative role of TRα deficiency in sarcopenia are not fully understood. This study suggests that TRα may regulate mitochondrial calcium (Ca2+) transport across membranes by targeting the inositol 1,4,5-trisphosphate receptor 1 (IP3R1), as evidenced by ChIP-seq and RNA-seq analyses. Experiments using naturally aged mice, skeletal muscle-specific TRα knockout (SKT) mice, and C2C12 myoblasts were conducted to investigate this process further. Findings include increased IP3R1, mitochondria-associated endoplasmic reticulum membranes (MAM), and mitochondrial Ca2+ in aged skeletal muscle. Additionally, SKT mice exhibited smaller muscle fibres, increased IP3R1 and MAM, and mitochondrial dysfunction. ChIP-qPCR and TRα manipulation in C2C12 cells showed that TRα negatively regulates IP3R1 transcription. Moreover, TRα knockdown cells exhibited increased Ca2+ transfer in MAM and mitochondrial dysfunction, which was ameliorated by the IP3R1 inhibitor 2-aminoethoxydiphenyl borate. Reintroduction of TRα improved IP3R1-mediated mitochondrial Ca2+ overload in aged cells. Our findings uncover a novel mechanism by which TRα deficiency induces mitochondrial Ca2+ overload through IP3R1-mediated Ca2+ transfer in MAM, exacerbating skeletal muscle atrophy during ageing. The TRα/IP3R1 pathway in MAM Ca2+ transfer presents a potential therapeutic target for sarcopenia.
    Keywords:  IP3R1; MAM; mitochondrial Ca2+ overload; sarcopenia; senescence; thyroid hormone receptor α
    DOI:  https://doi.org/10.1111/cpr.70120
  4. Nat Commun. 2025 Aug 25. 16(1): 7671
      Mitochondrial dynamics enable cellular adaptation to fluctuations in energy demand, such as those imposed on skeletal muscle by exercise, metabolic disorders, or aging. Here, we report a novel pathway that modulates mitochondria dynamics in skeletal muscle involving the scaffolding protein ankyrin-B. Rare variants in ankyrin-B, encoded by ANK2, increase risk for cardio-metabolic syndrome in humans and mice. We show that mice selectively lacking skeletal muscle ankyrin-B have reduced endurance exercise capacity without alterations in muscle strength or systemic glucose regulation. Muscle fibers in these mice have increased oxidative stress, reduced fatty acid oxidation, and enlarged and hyperconnected mitochondria. We found that ankyrin-B interacts with and is required for efficient mitochondria recruitment of fission modulators and sarcoplasmic reticulum-mitochondria coupling. Thus, we conclude that ankyrin-B enables substrate adaptability and bioenergetic homeostasis under energetic stress, and exercise capacity by promoting efficient mitochondrial fission in skeletal muscle.
    DOI:  https://doi.org/10.1038/s41467-025-62977-3
  5. Genes (Basel). 2025 Aug 11. pii: 948. [Epub ahead of print]16(8):
      Sarcopenia, the progressive loss of skeletal muscle mass and function with age, significantly contributes to frailty and mortality in older adults. Notably, muscles do not age uniformly-some retain structure and strength well into old age. This review explores the mechanisms underlying differential resistance to muscle aging, with a focus on sarcopenia-resistant muscles. We analyzed current literature across molecular biology, genetics, and physiology to identify key regulators of muscle preservation during aging. Special attention was given to muscle fiber types, mitochondrial function, neuromuscular junctions, and satellite cell activity. Muscles dominated by slow-twitch (type I) fibers-such as the soleus, diaphragm, and extraocular muscles-demonstrate enhanced resistance to sarcopenia. This resilience is linked to sustained oxidative metabolism, high mitochondrial density, robust antioxidant defenses, and preserved regenerative capacity. Key molecular pathways include mTOR, PGC-1α, and SIRT1/6, while genetic variants in ACTN3, MSTN, and FOXO3 contribute to interindividual differences. In contrast, fast-twitch muscles are more vulnerable due to lower oxidative capacity and satellite cell depletion. Unique innervation patterns and neurotrophic support further protect muscles like extraocular muscles from age-related atrophy. Resistance to sarcopenia is driven by a complex interplay of intrinsic and extrinsic factors. Understanding why specific muscles age more slowly provides insights into muscle resilience and suggests novel strategies for targeted prevention and therapy. Expanding research beyond traditionally studied muscles is essential to develop comprehensive interventions to preserve mobility and independence in aging populations.
    Keywords:  muscle atrophy; sarcopenia; skeletal muscle aging
    DOI:  https://doi.org/10.3390/genes16080948
  6. Nat Commun. 2025 Aug 22. 16(1): 7858
      Myogenesis in amniotes occurs in two waves. Primary myotubes express slow myosin (often with fast myosin) and likely act as scaffolds for secondary myotubes, which express only fast myosin. The embryonic origins and relationships of these lineages, and their connection to satellite cells, remain unknown. Here, we combine a TCF-LEF/β-catenin signaling reporter with precise in vivo electroporation in avian embryos to trace limb muscle progenitors from early migration to fetal stages. We identify two distinct progenitor populations that coexist from the onset: reporter-positive cells give rise exclusively to primary myotubes, while reporter-negative cells generate secondary myotubes and satellite cells. We also reveal a previously unrecognized role for TCF-LEF/β-catenin signaling in spatially organizing the primary lineage via Cxcr4-mediated control of myoblast migration. These findings redefine the developmental origin of myogenic lineages, resolve a longstanding question in muscle biology, and provide a molecular framework for investigating how muscle fiber diversity emerges and how distinct lineages contribute to the functional specialization of skeletal muscle.
    DOI:  https://doi.org/10.1038/s41467-025-61767-1
  7. Int J Mol Sci. 2025 Aug 21. pii: 8098. [Epub ahead of print]26(16):
      Monocytes/macrophages promote the repair of acutely injured muscle while contributing to dystrophic changes in chronically injured muscle in Duchenne muscular dystrophy (DMD) patients and animal models including mdx and mdx5cv mice. To elucidate the molecular mechanisms underlying this functional difference, we compared the transcriptomes of intramuscular monocytes/macrophages from wild-typed (WT) uninjured muscles, WT acutely injured muscles, and mdx5cv dystrophic muscles, using single cell-based RNA sequencing (scRNA-seq) analysis. Our study identified multiple transcriptomically diverse monocyte/macrophage subclusters, which appear to be induced by the intramuscular microenvironment. They expressed feature genes differentially involved in muscle inflammation, regeneration, and extracellular matrix (ECM) remodeling, but none of them conform to strict M1 or M2 activation. The Gpnmb+Spp1+ macrophage subcluster, an injury-associated subcluster that features the signature genes of reported scar-associated macrophages (SAMs) involved in ECM remodeling and fibrosis, is present transiently in acutely injured muscle and persistently in chronically injured dystrophic muscle, along with the persistence of monocytes. Our findings suggest that the persistent monocyte/macrophage infiltration and activation induced by continuous injury may underlie the pathogenic roles of macrophages in mdx5cv muscles. Controlling muscle injury and subsequent macrophage infiltration and activation may be important to the treatment of DMD.
    Keywords:  inflammation; macrophages; muscle injury; muscular dystrophy
    DOI:  https://doi.org/10.3390/ijms26168098
  8. Physiol Rep. 2025 Aug;13(16): e70519
      Running promotes skeletal muscle remodeling through metabolic and inflammatory signaling pathways, though the extent to which these responses are sex-dependent remains unclear. We profiled cytokine responses in quadriceps lysates from sedentary, voluntary wheel-running (VWR), and muscle-specific TFEB-overexpressing (cTFEB;HSACre) male and female mice. Cytokine analysis revealed 40 differentially expressed factors associated with exercise and/or TFEB overexpression, many exhibiting sex-dimorphic patterns. In males, VWR increased interleukins (IL-1α, IL-1β, IL-2, IL-5, and IL-17) and chemokines (e.g., MCP-1, CCL5, and CXCL9), including components of TNF signaling (e.g., TNFα, sTNFR1/2, and Fas ligand). These elevations were partially recapitulated by TFEB overexpression in sedentary males. In contrast, female VWR muscle showed limited changes, with significant differences restricted to IL-3, IL-3Rb, IL-13, and CXCL16. These findings demonstrate sex-specific cytokine responses to endurance-like stimuli and suggest a broader or more prolonged inflammatory remodeling profile in male skeletal muscle. Moreover, muscle-specific TFEB overexpression reproduced similar endurance-induced cytokine changes, particularly in males, highlighting TFEB as a partial molecular mimic of exercise-associated inflammatory signaling in skeletal muscle. Together, our data underscore the importance of sex as a biological variable in exercise-induced cytokine remodeling and support the utility of TFEB overexpression as a platform for prioritizing exercise-associated phenotypes.
    Keywords:  cytokines; exercise; running; sex‐differences
    DOI:  https://doi.org/10.14814/phy2.70519
  9. J Gen Physiol. 2025 Sep 01. pii: e202513866. [Epub ahead of print]157(5):
      JGP study (Woods et al. https://doi.org/10.1085/jgp.202413679) suggests that stretch activation of fast-contracting skeletal muscle fibers might increase muscle endurance by boosting force production during fatigue.
    DOI:  https://doi.org/10.1085/jgp.202513866
  10. Nat Aging. 2025 Aug 20.
      Aging is associated with a progressive decline in tissue function and regenerative capacity, partly due to genomic instability, one of the hallmarks of aging1,2. Genomic instability encompasses DNA damage and the accumulation of somatic mutations in post-zygotic cells, yet the specific impact of these mutations on age-related tissue dysfunction remains poorly understood. To address this, we developed a mouse model in which genomic instability was induced specifically in muscle progenitor cells3 through targeted deletion of the Msh2 (ref. 4) and Blm5 genes. This allowed us to assess how elevated DNA damage and somatic mutations, from single-nucleotide variants (SNVs) to structural variants, affect muscle regeneration following injury. These mice exhibited impaired muscle regeneration, characterized by smaller muscle fibers, reduced muscle mass gain and decreased grip strength. Importantly, similar muscle deficits were observed in a second mouse model where somatic mutations were elevated with less substantial DNA damage. These findings provide evidence that the accumulation of somatic mutations can potentially compromise the function of somatic cells, contributing to the aging phenotype in skeletal muscle.
    DOI:  https://doi.org/10.1038/s43587-025-00941-y
  11. Int J Mol Sci. 2025 Aug 19. pii: 7986. [Epub ahead of print]26(16):
      Muscle wasting and weakness are critical clinical problems that limit mobility and independence, reduce health span, and increase the risk of physical disability. The molecular basis for this has not been fully determined. Klotho expression is downregulated in conditions associated with muscle wasting, including aging, chronic kidney disease, and myopathy. The objective of this study was to investigate a mechanistic role for Klotho in regulating muscle wasting and weakness. Body weight, lean mass, muscle mass, and myofiber caliber were reduced in Klotho-deficient mice. In the tibialis anterior muscle of Klotho-null mice, type IIa myofibers were resistant to changes in size, and muscle composition differed with a higher concentration of type IIb fibers to the detriment of type IIx fibers. Glycolytic GPDH enzymatic activity also increased. Klotho-deficient mice showed impaired muscle contractility, with reduced twitch force, torque, and contraction-relaxation rates. RNA sequencing revealed upregulation of synaptic and fetal sarcomeric genes, prompting us to examine muscle innervation. Klotho deficiency led to neuromuscular junction remodeling, myofiber denervation, and functional motor unit loss. Loss of motor units correlated with absolute torque. Collectively, these findings reveal a novel mechanism through which systemic Klotho deficiency disrupts muscle synapses and motor unit connectivity, potentially contributing to muscle wasting and weakness.
    Keywords:  Klotho; motor unit; skeletal muscle; wasting
    DOI:  https://doi.org/10.3390/ijms26167986
  12. J Physiol. 2025 Aug 22.
      
    Keywords:  oxidative stress; passive dehydration; protein synthesis; resistance exercise; skeletal muscle
    DOI:  https://doi.org/10.1113/JP289527
  13. Proc Natl Acad Sci U S A. 2025 Aug 26. 122(34): e2500449122
      Type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum in skeletal muscle. In excitation-contraction (E-C) coupling, RyR1 opens by depolarization of transverse tubule membrane via physical interaction with dihydropyridine receptor, which is referred to as depolarization-induced Ca2+ release (DICR). RyR1 can also be gated via Ca2+-induced Ca2+ release (CICR), in which binding of Ca2+ directly opens the channel. Thus, RyR1 has two Ca2+ release modes; DICR and CICR, but the physiological role of CICR has been a matter of debate: whether CICR can amplify Ca2+ signals in E-C coupling. To address this issue, we created a mouse model carrying a mutation in the Ca2+-binding site in RyR1 (RyR1-E3896A), which selectively inhibits CICR. Surprisingly, the homozygous RyR1-E3896A mice show no appreciable changes in E-C coupling, ex vivo muscle contraction, in vivo muscle performance, or muscle fiber type. Gain-of-function mutations in RyR1 cause malignant hyperthermia (MH), which is a lethal disease triggered by inhalational anesthetics. The E3896A mutation conferred resistance to isoflurane-induced MH episodes and severe heat stroke triggered by environmental heat stress. Our data suggest that RyR1-mediated CICR plays a negligible role in E-C coupling of normal skeletal muscle but may increase the risk for muscle diseases when excessively activated.
    Keywords:  calcium release channel; calcium-induced calcium release; excitation–contraction coupling; ryanodine receptor; skeletal muscle
    DOI:  https://doi.org/10.1073/pnas.2500449122
  14. J Adv Res. 2025 Aug 18. pii: S2090-1232(25)00636-8. [Epub ahead of print]
       INTRODUCTION: Skeletal muscle function is profoundly challenged under high-altitude environments, where hypobaric hypoxia disrupts structural integrity and impairs physiological function. However, few animal studies have examined the impact of hypobaric hypoxia on skeletal muscle and molecular basis. While exercise training holds promise for alleviating hypoxia-induced muscle dysfunction, the understanding of its protective mechanisms remains limited.
    OBJECTIVES: We aimed to investigate chronic hypobaric hypoxia-induced myotube atrophy and mitochondrial dysfunction in mouse models and C2C12 cells, and develop a combined exercise strategy (preconditioning and hypoxic training) to mitigate hypoxia-related muscle pathology.
    METHODS: A mouse chronic hypobaric hypoxia model (45-day exposure, 6,000 m equivalent) combined with in vitro C2C12 myotube hypoxia simulations was employed. Muscle atrophy, mitochondrial ultrastructure, and molecular pathways were analyzed via histology, proteomics, and functional assays. Exercise interventions included preconditioning (9-week treadmill training) followed by voluntary wheel running under hypobaric hypoxia.
    RESULTS: Chronic hypobaric hypoxia induced pronounced skeletal muscle dysfunction and mitochondrial structural disorganization. However, exercise preconditioning combined with hypoxic training attenuated these hypoxia-induced impairments. Both hypoxic skeletal muscles in vivo and C2C12 cells in vitro exhibited significant Sirt1 downregulation. Notably, overexpression of Sirt1 or treatment with exercise mimetics partially reversed hypoxia-induced myotube atrophy and mitochondrial dysfunction through the PGC-1α/FoxO3a signaling pathway-a mechanism shared with exercise interventions.
    CONCLUSION: This study uncovers exercise as a potent inducer of hypoxia resilience through Sirt1-dependent mitochondrial repair and multicellular crosstalk (vascular-endothelial-satellite cell axis). Our "train-before-you-climb" approach could transform how we prepare for high-altitude living, offering a drug-free way to keep muscles strong where the air is thin.
    Keywords:  Exercise; Hypoxia; Mitochondria; Sirt1; Skeletal muscle dysfunction
    DOI:  https://doi.org/10.1016/j.jare.2025.08.022
  15. Cells. 2025 Aug 18. pii: 1277. [Epub ahead of print]14(16):
      Type 2 diabetes (T2D), a growing global health concern, is closely linked to obesity and sedentary behavior. Central to its development are insulin resistance and impaired glucose metabolism in peripheral tissues, particularly skeletal muscle, which plays a key role in energy expenditure, glucose uptake, and insulin sensitivity. Notably, increased accumulation of lipid metabolites in skeletal muscle is observed both in endurance exercise-associated with improved insulin sensitivity-and in high-fat diets that induce insulin resistance. The review examines the contrasting metabolic adaptations of skeletal muscle to these opposing conditions and highlights the key signaling molecules involved. The focus then shifts to the role of the stress kinase p38α mitogen-activated protein kinase (MAPK) in skeletal muscle adaptation to overnutrition and endurance exercise. p38α enhances mitochondrial oxidative capacity and regulates nutrient utilization, both critical for maintaining metabolic homeostasis. During exercise, it cooperates with AMP-activated protein kinase (AMPK) to boost glucose uptake and fatty acid oxidation, key mechanisms for improving insulin sensitivity. The co-activation of p38α and AMPK in skeletal muscle emerges as a promising therapeutic avenue to combat insulin resistance and T2D. The review explores strategies for selectively enhancing p38α activity in skeletal muscle. In conclusion, it advocates a comprehensive approach to T2D prevention and treatment, combining established caloric intake-reducing therapies, such as GLP-1 receptor agonists, with interventions aimed at increasing energy expenditure via activation of p38α and AMPK signaling pathways.
    Keywords:  AMPK; insulin resistance; mitochondrial metabolism; p38α MAPK; skeletal muscle
    DOI:  https://doi.org/10.3390/cells14161277
  16. Autophagy. 2025 Sep 01. 1-18
      The neuromuscular junction (NMJ) is essential for transmitting neural stimulus to muscles, triggering muscle contraction. Mitochondria are enriched in NMJ to support the energy needs required for neuromuscular function and stability. Thus, maintaining mitochondrial homeostasis through the clearance of damaged mitochondria, a process known as mitophagy, is vital for preserving neuromuscular health. Here, we highlight the crucial role of muscle PRMT1 in maintaining NMJ and mitochondrial homeostasis via mitophagy regulation. PRMT1 is distinctively expressed in myofibers, accumulating in the postsynaptic area, with its levels upregulated in denervated muscles. PRMT1-ablated muscles displayed disrupted NMJs and an accumulation of abnormal mitochondria, accompanied by increased mitochondrial oxidative stress. Additionally, prmt1 depletion in muscles specifically impaired TBK1 (TANK binding kinase 1)-OPTN (optineurin)-mediated mitophagy. Overall, our findings suggest that PRMT1 plays a critical role in maintaining NMJ and mitochondrial health by regulating selective mitophagy through TBK1-OPTN.Abbreviations: ADMA: asymmetric arginine dimethylation; BTX: α-bungarotoxin; EDL: extensor digitorum longus; FDB: flexor digitorum brevis; GAS: gastrocnemius; NMJ: Neuromuscular junction; Mko: mice with muscle-specific prmt1 ablation; MTOR: mechanistic target of rapamycin kinase; OPTN: optineurin; PRMT1: protein arginine methyltransferase 1; SA: sodium arsenate; SNI: sciatic nerve crush injury; Sol: soleus; SQSTM1/p62: sequestosome 1; TBK1: TANK binding kinase 1; TOMM20: translocase of outer mitochondrial membrane 20; TA: tibialis anterior; VDAC1: voltage dependent anion channel 1.
    Keywords:  Mitophagy; PRMT1; TBK1; neuromuscular junction; skeletal muscle
    DOI:  https://doi.org/10.1080/15548627.2025.2551477
  17. Extracell Vesicles Circ Nucl Acids. 2025 ;6(2): 324-327
      A recent study from Liu et al. described the role of skeletal muscle-derived extracellular vesicles in promoting liver fibrosis as an outcome of chronic overtraining in mice. Here, we highlight this work and discuss its implications within the fields of exercise physiology and inter-organ communication.
    Keywords:  Extracellular vesicles; exercise physiology; inter-organ communication; liver fibrosis; overtraining; skeletal muscle
    DOI:  https://doi.org/10.20517/evcna.2025.36
  18. J Physiol. 2025 Aug 22.
      Fibro/adipogenic progenitors (FAPs) are muscle-resident stem cells essential for muscle regeneration because of their ability to differentiate into adipocytes and fibroblasts. This differentiation contributes to tissue remodelling and is implicated in the accumulation of fat and fibrotic tissue seen in neuromuscular, cardiovascular and degenerative diseases. FAPs also interact with other muscle cells and modulate inflammation, playing a central role in muscle degeneration across various disease contexts. This review summarises current knowledge on FAP adipogenic differentiation in muscle degeneration and regeneration, with a focus on cardiovascular and neuromuscular diseases, which share common features of impaired muscle remodelling. We discuss established methods for culturing, maintaining, and differentiating FAPs in vitro to support future research. Additionally, we examine FAP subpopulations, key signalling pathways and pharmacological agents influencing FAP differentiation into adipocytes. Understanding these mechanisms offers promising avenues for developing therapeutic strategies to restore muscle homeostasis and slow down pathological muscle remodelling.
    Keywords:  FAPs; adipogenesis; fibro/adipogenic progenitors; fibrosis; muscle; remodelling
    DOI:  https://doi.org/10.1113/JP288924
  19. Biomolecules. 2025 Jul 24. pii: 1072. [Epub ahead of print]15(8):
      Prolonged high-dose administration of synthetic glucocorticoids (GCs) leads to limb muscle atrophy and weakness, yet its underlying mechanisms remain incompletely understood. Muscle fibers and muscle satellite cells (MSCs) are essential for skeletal muscle development and associated pathologies. This study demonstrates that dexamethasone (Dex) induced MSC-derived extracellular vesicles (EVs) impair myogenesis in muscle fiber-like cells (MFLCs) via inducible nitric oxide synthase (iNOS) suppression. High-throughput sequencing revealed a marked upregulation of miR-335-5p in MSC-derived EVs following Dex treatment. Mechanistically, EV miR-335-5p targeted MAPK11, leading to iNOS downregulation and subsequent UPS activation in MFLCs, which directly promoted muscle protein degradation. Collectively, our findings identify the EV miR-335-5p/MAPK11/iNOS axis as a critical mediator of GC-induced muscle atrophy, offering novel insights into therapeutic strategies targeting EV-mediated signaling in muscle wasting disorders.
    Keywords:  extracellular vesicles; glucocorticoids; inducible nitric oxide synthase; miRNA; muscle satellite cells
    DOI:  https://doi.org/10.3390/biom15081072
  20. Front Cell Dev Biol. 2025 ;13 1614732
      This review explores the multifaceted impact of physical exercise on FoxO signaling pathways, which play a central role in cellular homeostasis, stress response, metabolism, and longevity. Exercise influences FoxO proteins-particularly FoxO1, FoxO3, FoxO4, and FoxO6-through diverse mechanisms, including phosphorylation, acetylation, and ubiquitination, determining their localization, transcriptional activity, and stability. Regular exercise modulates FoxO signaling by activating pathways like PI3K/AKT, AMPK, SIRT1, and IGF-1, promoting cellular resilience against oxidative stress, apoptosis, and metabolic dysfunction. The review highlights how exercise-induced modulation of FoxO pathways contributes to improved insulin sensitivity, muscle hypertrophy, cardiovascular health, neuroprotection, and reduced risks of chronic diseases, including metabolic syndrome, neurodegeneration, cardiovascular disease, and cancer. Additionally, it addresses the role of exercise in preventing muscle atrophy under various conditions, such as pharmacological interventions, aging, disease, and dietary factors. By enhancing FoxO signaling, exercise promotes anabolic processes, mitochondrial function, autophagy, and antioxidant defenses. Understanding the intricate relationship between exercise and FoxO pathways offers insights into developing therapeutic strategies to mitigate disease progression.
    Keywords:  FoxO signaling pathways; complex; left; metabolic regulation; physical exercise; skeletal muscle
    DOI:  https://doi.org/10.3389/fcell.2025.1614732
  21. Dev Biol. 2025 Aug 19. pii: S0012-1606(25)00233-7. [Epub ahead of print]527 308-317
      Liquid-liquid phase separation (LLPS) is a crucial biophysical process that enables dynamic compartmentalization within cells, gaining prominence in recent research on cellular organization. However, its implications for skeletal muscle-a vital tissue for movement and metabolic regulation-remain largely uncharted. This review highlights recent insights into LLPS's roles in other tissues while examining its potential functions in skeletal muscle development, regeneration, and disease. By synthesizing existing knowledge, we propose that exploring LLPS in skeletal muscle could uncover fundamental aspects of muscle physiology and offer innovative therapeutic avenues for muscle-related disorders. A deeper grasp of LLPS may thus transform our understanding and treatment of skeletal muscle pathologies.
    Keywords:  Development; Diseases; LLPS; Skeletal muscle
    DOI:  https://doi.org/10.1016/j.ydbio.2025.08.016
  22. Trends Mol Med. 2025 Aug 21. pii: S1471-4914(25)00172-8. [Epub ahead of print]
      The muscle regeneration niche comprises various cell types, including muscle stem cells (MuSCs; also termed satellite cells), immune cells, and stromal cells, all of which have crucial roles in the regeneration process. Intracellular metabolic reprogramming during injury responses is closely linked to the functional activities of these cells, thus necessitating a comprehensive understanding for developing targeted metabolic interventions that promote regeneration. Recent studies have suggested the existence of a more intricate network, involving cell-cell metabolic crosstalk and even cross-organ regulation, which underpins muscle regeneration. In addition, aging and diseases that disrupt overall metabolic homeostasis contribute to muscle dysfunction, due, in part, to metabolic disorders in the regeneration niche. In this review, we provide a comprehensive overview of the metabolic profile within the muscle regeneration niche and highlight potential interventions to reprogram metabolism to improve regenerative capacity.
    Keywords:  inflammation; metabolic intervention; metabolism; muscle pathology; regeneration niche; stem cell
    DOI:  https://doi.org/10.1016/j.molmed.2025.07.006
  23. Antioxidants (Basel). 2025 Jul 25. pii: 911. [Epub ahead of print]14(8):
      Sarcopenia, the progressive loss of muscle mass, strength, and regenerative capacity with age, is driven by interconnected processes such as oxidative stress, chronic inflammation, mitochondrial dysfunction, and reduced activity of muscle stem cells. As the population ages, nutritional strategies that target these mechanisms are becoming increasingly important. This review focuses on nicotinamide (vitamin B3) and pyridoxine (vitamin B6), two essential micronutrients found in functional foods, which play complementary roles in redox regulation, immune balance, and muscle repair. Nicotinamide supports nicotinamide adenine dinucleotide (NAD+) metabolism, boosts mitochondrial function, and activates sirtuin pathways involved in autophagy and stem cell maintenance. Pyridoxine, via its active form pyridoxal 5'-phosphate (PLP), is key to amino acid metabolism, antioxidant defense, and the regulation of inflammatory cytokines. We summarize how these vitamins influence major molecular pathways such as Sirtuin1 (SIRT1), protein kinase B (AKT)/mechanistic target of rapamycin (mTOR), Nuclear factor-κB (NF-κB), and Nrf2, contributing to improved myogenic differentiation and protection of the aging muscle environment. We also highlight emerging preclinical and clinical data, including studies suggesting possible synergy between B3 and B6. Finally, we discuss how biomarkers such as PLP, nicotinamide mononucleotide (NMN), and C-reactive protein (CRP) may support the development of personalized nutrition strategies using these vitamins. Safe, accessible, and mechanistically grounded, nicotinamide and pyridoxine offer promising tools for sarcopenia prevention and healthy aging.
    Keywords:  inflammaging; muscle regeneration; muscle stem cells; nicotinamide; nicotinamide adenine dinucleotide (NAD+) metabolism; nutritional intervention; pyridoxine; sarcopenia
    DOI:  https://doi.org/10.3390/antiox14080911
  24. Cells. 2025 Aug 10. pii: 1234. [Epub ahead of print]14(16):
      Fibro-adipogenic progenitor cells (FAPs) support muscle tissue homeostasis, regulate muscle growth, injury repair, and fibrosis, and activate muscle progenitor cell differentiation to promote regeneration. We aimed to investigate the effects of co-culturing FAPs with muscle satellite cells (MuSCs) on myogenic differentiation. Proteomic profiling of co-culture supernatants identified significant DCX, IMP2A, NUDT16L1, SLC38A2, and IL-6 upregulation. Comparative transcriptomics of mono-cultured versus co-cultured MuSCs revealed differential expression of oxidative stress-related genes (HMOX1, ALOX5, GSTM3, TRPM2, PADI1, and CTSL). Pathway enrichment analyses highlighted cell cycle regulation, TNF signaling, and ferroptosis. Gene ontology analysis of MuSCs indicated significant gene enrichment in myosin-related components. Combined transcriptomic and proteomic analyses demonstrated HO-1 downregulation at the transcriptional and translational levels, with altered pathways being predominantly related to myosin filament, muscle system process, and muscle contraction cellular components. HO-1 knockdown reduced intracellular iron accumulation in MuSCs, suppressing iron-dependent autophagy. This alleviated oxidative stress and promoted myogenic differentiation. Exogenous IL-6 (0.1 ng/mL) downregulated HO-1 expression, initiating an identical regulatory cascade, while HO-1 overexpression reversed the IL-6-mediated reduction in the expression of the autophagy markers LC3 and ATG5, suppressing myogenic enhancement. This establishes the co-culture-induced IL-6/HO-1 axis as a core regulator of iron-dependent oxidative stress and autophagy during myogenic differentiation.
    Keywords:  fibro-adipogenic progenitor cells (FAPs); heme oxygenase-1 (HO-1); interleukin-6 (IL-6); muscle satellite cells (MuSCs); myogenic differentiation; oxidative stress
    DOI:  https://doi.org/10.3390/cells14161234
  25. Life (Basel). 2025 Aug 07. pii: 1259. [Epub ahead of print]15(8):
      Heme, an essential prosthetic group involved in mitochondrial respiration and transcriptional regulation, is synthesized via the rate-limiting enzyme 5-aminolevulinic acid synthase (ALAS). Utilizing heterozygous mouse models for ALAS1 and ALAS2, our studies have revealed diverse systemic consequences of chronic heme deficiency. ALAS1-heterozygous (ALAS1+/-) mice develop metabolic dysfunction characterized by insulin resistance, glucose intolerance, and abnormal glycogen accumulation, linked mechanistically to reduced AMP-activated protein kinase (AMPK) signaling. These mice also exhibit pronounced mitochondrial dysfunction, impaired autophagy, and accelerated aging phenotypes, including sarcopenia and metabolic decline, highlighting heme's role as a critical metabolic regulator. Additionally, ALAS2 heterozygosity (ALAS2+/-) leads to impaired erythropoiesis, resulting in anemia and ineffective iron utilization. Importantly, supplementation with the heme precursor 5-aminolevulinic acid (5-ALA) significantly mitigates ALAS1+/- phenotypes, restoring metabolic function, mitochondrial health, autophagy, and immune competence. This review encapsulates key findings from our group's research together with advances made by multiple research groups over the past decade, collectively establishing heme homeostasis as a central regulator of systemic physiology and highlighting the therapeutic potential of 5-ALA in treating heme-deficient pathologies.
    Keywords:  5-aminolevulinic acid; aging; autophagy; glucose intolerance; insulin resistance; mitochondria; multi-organ; native immunity; sarcopenia; skeletal muscle
    DOI:  https://doi.org/10.3390/life15081259
  26. Free Radic Biol Med. 2025 Aug 20. pii: S0891-5849(25)00931-1. [Epub ahead of print]240 373-383
       BACKGROUND: High-fat diet (HFD)-induced sarcopenic obesity can lead to reductions in muscle fiber diameter, enhanced protein degradation, and various forms of cell death. Exercise training has been shown to alleviate HFD-induced muscle atrophy. However, the underlying mechanism remains unclear. Stimulator of interferon genes (STING) is involved in ferroptosis and various forms of muscle atrophy. This study aimed to investigate the role of STING in exercise training against HFD-induced skeletal muscle atrophy.
    METHODS: In vivo, HFD-fed mice were subjected to exercise training and were intraperitoneally injected with the STING agonist diABZI or selective STING inhibitor C-176 for 8 weeks. In vitro, the differentiated C2C12 myotubes were treated with palmitic acid (PA), followed by interventions with Ferrostatin-1 (Fer-1), Erastin, diABZI or C-176. Grip strength test, body composition analysis, serum assay, histology analysis, dihydroethidium staining, transmission electron microscopy, myosin heavy chain staining, mitochondrial membrane potential, Western blot, and real-time quantitative PCR were performed.
    RESULTS: In vivo, exercise training significantly reduced the mRNA and protein expression of STING and ameliorated skeletal muscle atrophy and lipid peroxidation associated ferroptosis in HFD-fed mice. The STING agonist diABZI blunted the alleviative effect of exercise training in HFD-induced skeletal muscle atrophy and ferroptosis. The selective STING inhibitor C-176 and exercise training synergistically alleviated HFD-induced skeletal muscle atrophy and ferroptosis. In vitro, the ferroptosis inhibitor Fer-1 partially rescued PA-triggered C2C12 myotubes atrophy and ferroptosis, whereas the ferroptosis activator Erastin aggravated myotubes atrophy and ferroptosis. diABZI exacerbated PA-induced C2C12 myotubes atrophy and ferroptosis. Erastin impaired the ameliorative effect of C-176 in PA-induced C2C12 myotubes atrophy and ferroptosis.
    CONCLUSIONS: Exercise training effectively suppressed HFD-mediated upregulation of STING in skeletal muscle. STING is a response factor for the alleviative effect of exercise training in HFD-induced skeletal muscle atrophy and ferroptosis.
    Keywords:  Exercise training; Ferroptosis; High-fat diet; STING; Skeletal muscle atrophy
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2025.08.043
  27. Genes (Basel). 2025 Aug 18. pii: 968. [Epub ahead of print]16(8):
      Mechanotransduction, also referred to as mechano-signal transduction, is a biophysical process wherein cells perceive and respond to mechanical stimuli by converting them into biochemical signals that initiate specific cellular responses. This mechanism is fundamental to the development and growth, and proper functioning of mechanically active tissues, such as the diaphragm-a respiratory muscle vital for breathing in mammals. In vivo, the diaphragm is subjected to transdiaphragmatic pressure, and therefore, its muscle fibers are subjected to mechanical forces not only in the direction of the muscle fibers but also in the direction transverse to the fibers. Previous research conducted in our laboratory uncovered that stretching the diaphragm in either the longitudinal or transverse direction activates distinct mechanotransduction pathways. This indicates that signaling pathways in the diaphragm muscle are regulated in an anisotropic manner. In this review paper, we discussed the underlying mechanisms that regulate the anisotropic signaling pathways in the diaphragmatic muscle, emphasizing the mechanical role of cytoskeletal proteins in this context. Furthermore, we explored the regulatory mechanisms governing mechanosensitive gene transcription, including microRNAs (mechanomiRs), within the diaphragm muscle. Finally, we examined potential links between anisotropic signaling in the diaphragm muscle and various skeletal muscle disorders.
    Keywords:  anisotropic gene regulation; cytoskeletal proteins; mechanical stretch; mechanomiRs; mechanotransduction; skeletal muscle
    DOI:  https://doi.org/10.3390/genes16080968
  28. Skelet Muscle. 2025 Aug 22. 15(1): 22
       BACKGROUND: Pathogenic variants in RYR1 cause a spectrum of rare congenital myopathies associated with intracellular calcium dysregulation. Glutathione redox imbalance has been reported in several Ryr1 disease model systems and clinical studies. NAD+ and NADP are essential cofactors in cellular metabolism and redox homeostasis. NAD+ deficiency has been associated with skeletal muscle bioenergetic deficits in mitochondrial myopathy and sarcopenia.
    METHODS: Using a new colorimetric assay and large control dataset (n = 299), we assessed redox balance (glutathione, NAD+, and NADP) in whole blood from 28 RYR1-RM affected individuals (NCT02362425). Analyses were expanded to human skeletal muscle (n = 4), primary myotube cultures (n = 5), and whole blood and skeletal muscle specimens from Ryr1 Y524S mice. The in vitro effects of nicotinamide riboside (NR) on cellular NAD+ content and mitochondrial respirometry were also tested.
    RESULTS: At baseline, a majority of affected individuals exhibited systemic NAD+ deficiency (19/28 [68%] < 21 µM) and increased NADPH concentrations (22/26 [85%] > 1.6 µM). When compared to controls, decreased NAD+/NADH and NADP/NADPH ratios were observed in 9/28 and 23/26 individuals, respectively. In patient-derived myotube cultures (n = 5), NR appeared to increase cellular NAD+ concentrations in a dose and time-dependent manner at 72-h only and favorably modified maximal respiration and ATP production. Average whole blood GSH/GSSG ratio was comparable between groups, and redox imbalance was not observed in Ryr1 Y524S specimens.
    CONCLUSIONS: NAD+ and NADP dyshomeostasis was identified in a subset of RYR1-RM affected individuals. Further experiments are warranted to confirm if NAD+ repletion could be an attractive therapeutic approach given the favorable outcomes reported in other neuromuscular disorders.
    Keywords:   RYR1 ; Congenital myopathy; Glutathione; NAD+ ; NADP; Oxidative stress
    DOI:  https://doi.org/10.1186/s13395-025-00390-6
  29. Int Immunopharmacol. 2025 Aug 23. pii: S1567-5769(25)01374-8. [Epub ahead of print]164 115383
       OBJECTIVE: Skeletal muscle is crucial for glucose metabolism, but diabetes impairs this function, leading to muscle atrophy. Although the mutation of BOLA Family Member 3 (BOLA3) resulted in disease Multiple Mitochondrial Dysfunctions Syndrome, the role of which in diabetic muscle atrophy is unclear.
    METHODS: Firstly, datasets were downloaded and assessed from skeletal muscle tissue with diabetic individuals in GEO database and key genes were screened using weighted gene co-expression network analysis (WGCNA) and machine learning algorithms. Subsequently, external validation datasets were applied to screen and verify specific genes. We also used qRT-PCR, Western Blot, H&E staining and MitoSOX in vitro and vivo experiments. Finally, molecular docking was employed to predict potential therapeutic drugs.
    RESULTS: BOLA3 and LPIN1, were screened by WGCNA, machine learning and external datasets verification. Among these, only BOLA3 emerged positively correlated with ferroptosis marker. Notably, muscle atrophy and ferroptosis phenotypes were observed in diabetic mice with reduced BOLA3 levels. Moreover, BOLA3 knockdown in C2C12 cells confirmed its association with both ferroptosis and muscle atrophy, with a surge in mitochondrial ROS emerging as a critical factor. At last, based on the protein structure of BOLA3, handelin showed the highest binding affinity to BOLA3.
    CONCLUSION: BOLA3 is a potential biomarker and therapeutic target for diabetic skeletal muscle atrophy. Handelin may represent a promising therapeutic candidate for modulating BOLA3 function in diabetes.
    Keywords:  BOLA3; Diabetic skeletal muscle atrophy; Ferroptosis
    DOI:  https://doi.org/10.1016/j.intimp.2025.115383
  30. J Muscle Res Cell Motil. 2025 Aug 20.
      Marfan syndrome is an autosomal dominant multisystemic connective tissue disorder caused by mutations in the FBN1 gene. Although clinical changes in the cardiovascular, ocular, and skeletal systems have been described in detail in Marfan syndrome patients, investigations about skeletal muscle alterations are still incipient. This systematic review describes cellular, molecular, and functional changes in skeletal muscles of patients and mice with Marfan syndrome. Study selection (from EMBASE, MEDLINE, and Web of Science databases), data extraction, and quality appraisal were performed by two independent reviewers. A total of 2634 articles were identified; 26 were included in the analysis based on the selection criteria. The risk of bias was evaluated using the Critical Appraisal Skills Programme and Joanna Briggs Institute Critical Appraisal tool for human studies and the Systematic Review Centre for Laboratory Animal Experimentation RoB tool for animal studies. The findings indicate that skeletal muscle alterations in Marfan syndrome are characterized by fibrosis, reduced muscle mass and myofiber size, compromised muscle regeneration, and impaired muscle function. Future studies are warranted to investigate the mechanisms involved in the development of this muscle phenotype to help develop effective strategies to improve skeletal muscle function and the quality of life of individuals with Marfan syndrome.
    Keywords:  Animal model; Marfan syndrome; Patients; Skeletal muscle alterations
    DOI:  https://doi.org/10.1007/s10974-025-09706-x
  31. Pharmaceuticals (Basel). 2025 Aug 19. pii: 1222. [Epub ahead of print]18(8):
      Regular physical activity induces a dynamic crosstalk between skeletal muscle and adipose tissue, modulating the key molecular pathways that underlie metabolic flexibility, mitochondrial function, and inflammation. This review highlights the role of myokines and adipokines-particularly IL-6, irisin, leptin, and adiponectin-in orchestrating muscle-adipose tissue communication during exercise. Exercise stimulates AMPK, PGC-1α, and SIRT1 signaling, promoting mitochondrial biogenesis, fatty acid oxidation, and autophagy, while also regulating muscle hypertrophy through the PI3K/Akt/mTOR and Wnt/β-catenin pathways. Simultaneously, adipose-derived factors like leptin and adiponectin modulate skeletal muscle metabolism via JAK/STAT3 and AdipoR1-mediated AMPK activation. Additionally, emerging exercise mimetics such as the mitochondrial-derived peptide MOTS-c and myostatin inhibitors are highlighted for their roles in increasing muscle mass, the browning of white adipose tissue, and improving systemic metabolic function. The review also addresses the role of anti-inflammatory compounds, including omega-3 polyunsaturated fatty acids and low-dose aspirin, in mitigating NF-κB and IL-6 signaling to protect mitochondrial health. The resulting metabolic flexibility, defined as the ability to efficiently switch between lipid and glucose oxidation, is enhanced through repeated exercise, counteracting age- and disease-related mitochondrial and functional decline. Together, these adaptations demonstrate the importance of inter-tissue signaling in maintaining energy homeostasis and preventing sarcopenia, obesity, and insulin resistance. Finally, here we propose a stratified treatment algorithm based on common age-related comorbidities, offering a framework for precision-based interventions that may offer a promising strategy to preserve metabolic plasticity and delay the age-associated decline in cardiometabolic health.
    Keywords:  IL-6; adipokines; adiponectin; irisin; leptin; metabolic flexibility; mitochondrial biogenesis; myokines; skeletal muscle–adipose tissue crosstalk
    DOI:  https://doi.org/10.3390/ph18081222
  32. Front Vet Sci. 2025 ;12 1636652
      
    Keywords:  environment; genetics; muscle; skeletal muscle development; skeleton
    DOI:  https://doi.org/10.3389/fvets.2025.1636652
  33. Sci Adv. 2025 Aug 29. 11(35): eadw1059
      Androgens act through androgen receptor (AR) to maintain muscle mass. Evidence suggests that this pathway is influenced by "the gene for speed," ACTN3 (α-actinin-3). Given that one in five people lack α-actinin-3, it is possible that they may respond to androgens differently. Here, we show that α-actinin-3 deficiency decreases AR in muscles of mice and humans (in males and females) and that AR positively correlates with α-actinin-3 expression in a dosage-dependent manner. α-Actinin-3 deficiency exacerbates gastrocnemius mass loss with androgen deprivation in male mice and stunts the muscle growth response to dihydrotestosterone in female mice at the onset of puberty. This is mediated by differential activation of pathways regulating amino acid metabolism, intracellular transport, autophagy, mitochondrial activity, MAPK, and calcineurin signaling, likely driven by seven key genes that are both androgen sensitive and α-actinin-3-dependent in expression. Our results highlight a role for ACTN3 as a regulator of muscle mass and a genetic modifier of androgen action in skeletal muscle.
    DOI:  https://doi.org/10.1126/sciadv.adw1059
  34. Am J Physiol Endocrinol Metab. 2025 Aug 26.
      The mitochondrial tRNA-derived fragment mt-tRF-LeuTAA couples mitochondrial metabolism to insulin secretion. While its role in pancreatic β-cell function is well established, its broader impact on multi-organ glucose homeostasis remains unclear. In insulin target tissues, the presence, regulation, and mechanism of action of mt-tRF-LeuTAA are entirely unexplored. This study addresses this gap by investigating the impact of diet, nutritional status and diabetes on mt-tRF-LeuTAA regulation and by assessing its role in insulin sensitivity. We examined mt-tRF-LeuTAA levels in different insulin target tissues, including skeletal muscle, liver, and epididymal white adipose tissue, of rodents under physiological and pathological conditions. In skeletal muscle myotubes, we combined subcellular fractionation, antisense oligonucleotide-mediated knockdown and glucose uptake assays to determine mt-tRF-LeuTAA's mitochondrial localization and its influence on insulin sensitivity. mt-tRF-LeuTAA levels in mouse skeletal muscle decreased twofold in response to fasting. In myotubes, this tRNA fragment was enriched in mitochondria, and its downregulation enhanced glucose uptake. While the levels of mt-tRF-LeuTAA remained unchanged in insulin target tissues of diabetic mice, we observed a skeletal muscle-specific downregulation of mt-tRF-LeuTAA in young adult rats exhibiting insulin hypersensitivity. This study identifies mt-tRF-LeuTAA as a candidate regulator of skeletal muscle insulin response. By modulating both insulin secretion and action, mt-tRF-LeuTAA appears to play a notable role in systemic metabolic control, and may represent a promising target for diabetes treatment.
    Keywords:  Diabetes susceptibility; Mitochondrially-encoded tRNAs; Muscle glucose uptake; Nutritional status; mt-tRNA-derived fragments
    DOI:  https://doi.org/10.1152/ajpendo.00284.2025
  35. Biomaterials. 2025 Aug 21. pii: S0142-9612(25)00564-2. [Epub ahead of print]326 123645
      Skeletal muscle regeneration mediated by muscle satellite cells (MuSCs) is supported by the specific vascular niche containing endothelial cells, pericytes, and mesenchymal stem cells. Volumetric muscle loss (VML) severely disrupts the vascular niche and impairs the ability of MuSCs to regenerate functional skeletal muscle. Until now, it remains a great challenge to reconstruct the vascular niche for muscle regeneration. Here, we successfully developed a specific 3D induction strategy based on collagen matrix and pro-angiogenic culture system to fabricate a multifunctional human pre-vascularized microtissue (h-VM) in vitro. The h-VM featured a robust vascular network formed by endothelial cells, with pericytes and mesenchymal stem cells accompanying. Under a specific induction medium, the h-VM could differentiate into adipogenic and osteogenic tissues, indicating a strong stemness within the microtissue. Of note, the h-VM significantly boosted the myogenic differentiation of MuSCs through paracrine signaling. Transplantation of the h-VM into the volumetric muscle defect not only facilitated early vessel integration with the host vasculature but also maintained muscle structural stability and provided sustained mechanical support to the defect site. Mechanistically, the h-VM effectively induced MuSCs differentiation and myofiber formation, and M2 macrophage polarization, thereby mitigating muscle fibrosis and atrophy post-injury, ultimately achieving structural and functional angio-myogenesis. Our results highlight that the h-VM can effectively re-establish the vascular regenerative microenvironment, which presents an innovative therapeutic approach to promote functional skeletal muscle regeneration following volumetric muscle loss.
    Keywords:  Angio-myogenesis; Collagen matrix; Muscle regeneration; Pre-vascularized microtissue; Tissue engineering; Vascular niche
    DOI:  https://doi.org/10.1016/j.biomaterials.2025.123645
  36. Brain. 2025 Aug 25. pii: awaf260. [Epub ahead of print]
      Inclusion body myositis (IBM) is a progressive muscle disorder characterized by inflammation and degeneration with altered proteostasis. To better understand the interrelationship between these two features, we aimed at establishing a novel preclinical mouse model. First, we used quantitative PCR to determine expression of pro-inflammatory chemo- and cytokines including lymphotoxin (LT)-signaling pathway components in human skeletal muscle tissue diagnosed with myositis. Based on these results we generated a mouse model that we analyzed at the histological, ultrastructural, transcriptional, biochemical, and behavioral level. Lastly, we subjected this model to anti-inflammatory treatments. After confirming and extending previous data on activation of lymphotoxin (LT)-signaling in human myositis, we generated distinct transgenic mouse lines co-expressing LTα and -β in skeletal muscle fibers. Transgenic mice displayed chronic myositis accompanied by dysregulated proteostasis, including an altered autophagolysosomal pathway that initially shows signs of activation and later exhaustion and decreased flux. To enhance the latter, we genetically impaired autophagy in skeletal muscle cells. Autophagy impairment alone induced a pro-inflammatory transcriptional state, but no obvious cellular inflammation. However, the combination of LT-driven myositis with autophagy impairment induced the full spectrum of characteristic molecular and pathological features of IBM in skeletal muscle, including protein inclusions with typical ultrastructural morphology and mild mitochondrial pathology. Our attempts to treat the pathology by subjecting these mice to corticosteroids or anti-Thy1.2 antibodies mirrored recent treatment failures in humans, i.e., none of these treatments resulted in significant clinical improvement of motor performance or the transcriptional profile of muscle pathology. In summary, these data provide evidence that inflammation and autophagy disruption play a synergistic role in the development of IBM-like muscular pathology. Furthermore, once established, IBM-like pathology in these mice, as in human IBM patients cannot be reverted or prevented from progression by conventional means of immunosuppression. We expect that this novel mouse model will help to identify future treatment modalities for IBM.
    Keywords:  NF-κB signaling; autophagy; inclusion body myositis; lymphotoxin; lymphotoxin signaling; myositis
    DOI:  https://doi.org/10.1093/brain/awaf260
  37. Int J Mol Sci. 2025 Aug 19. pii: 8011. [Epub ahead of print]26(16):
      Cancer-associated cachexia is a multifaceted wasting syndrome characterized by progressive loss of skeletal muscle mass, systemic inflammation, and metabolic dysfunction and is particularly prevalent in gastrointestinal cancers. Physical activity has emerged as a promising non-pharmacological intervention capable of attenuating key drivers of cachexia. Exercise modulates inflammatory signaling (e.g., IL-6/STAT3 and TNF-α/NF-κB), enhances anabolic pathways (e.g., IGF-1/Akt/mTOR), and preserves lean body mass and functional capacity. Exercise-induced signaling molecules, known as exerkines, are key mediators of these benefits, which are released during physical activity and act in an autocrine, paracrine, and endocrine manner. However, many of these molecules also exhibit context-dependent effects. While they exert protective, anti-inflammatory, or anabolic actions when transiently elevated after exercise, the same molecules may contribute to cachexia pathogenesis when chronically secreted by tumors or in systemic disease states. The biological effects of a given factor depend on its origin, timing, concentration, and physiological milieu. This review presents recent evidence from clinical and experimental studies to elucidate how physical activity and exerkines may be harnessed to mitigate cancer cachexia, with particular emphasis on gastrointestinal malignancies and their unique metabolic challenges.
    Keywords:  cancer cachexia; context-dependent signaling; exerkines; gastrointestinal cancers; metabolic dysfunction; physical activity; skeletal muscle; systemic inflammation
    DOI:  https://doi.org/10.3390/ijms26168011
  38. Muscles. 2025 Aug 19. pii: 35. [Epub ahead of print]4(3):
      Baicalin is a natural flavonoid that has anti-apoptotic and anti-inflammatory effects. It shows some beneficial effects on muscle atrophy. However, its effects on age-related muscle atrophy are poorly understood. In this paper, we investigated whether baicalin exerts protective effect against skeletal muscle atrophy and its underlying mechanisms in aged mice using the grip strength test, histological analysis, and Western blots. Baicalin increased total muscle mass and strength in aged mice. Consistently, the cross-sectional area of quadriceps (QD) muscle significantly increased in both baicalin-administrated groups. Moreover, baicalin induced a shift in muscle fiber size distribution toward large fibers in both groups of mice. Expression levels of muscle atrophic factors, such as myostatin (MSTN) and atrogin-1, as well as pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), were elevated in aged mice, but these increases were reduced by baicalin. While mitochondrial fission regulator, dynamin-related protein 1 (DRP-1), and apoptosis-related protein (apoptotic protease activating factor 1 (Apaf-1)) expressions were higher in aged mice than young mice, and their expression were downregulated following baicalin administration. The comprehensive results of this study suggest that baicalin provides beneficial effects on the treatment of sarcopenia not only by suppressing muscle atrophic factor expression and inflammation but also attenuating DRP-1-mediated mitochondrial fission and apoptosis.
    Keywords:  apoptosis; baicalin; inflammation; mitochondria fission; muscle atrophy; sarcopenia
    DOI:  https://doi.org/10.3390/muscles4030035
  39. Curr Issues Mol Biol. 2025 Jul 23. pii: 583. [Epub ahead of print]47(8):
      Resistance exercise can enhance or preserve muscle mass and/or strength. Modifying factors are secreted following resistance exercise. Biomarkers like cytokines and extracellular vesicles, especially small extracellular vesicles, are released into the circulation and play an important role in cell-to-cell and inter-tissue communications. There is increasing evidence that physical activity itself promotes the release of extracellular vesicles into the bloodstream, suggesting the importance of vesicles in mediating systemic adaptations following exercise. Extracellular vesicles contain proteins, nucleic acids like miRNAs, and other molecules targeting different cell types and tissues of distant organs. Therefore, extracellular vesicles and encapsulated miRNAs are fine tuners of protein synthesis and are important in the adaptation after resistance training. However, there is a lack of strong data supporting the precise mechanisms of these processes. In this literature review, we collected publications related to miRNA and extracellular vesicle profile changes induced by resistance exercise. To the best of our knowledge, the changes in human extracellular vesicle and microRNA profiles following resistance exercise have not been reviewed yet. We aimed to assess the shortcomings and difficulties characterizing this research area, to summarize the existing results to date, and to propose possible solutions that could help standardize the implementation of future investigations.
    Keywords:  exosome; intercellular communication; microvesicle; physical activity; resistance training; signaling
    DOI:  https://doi.org/10.3390/cimb47080583
  40. Skelet Muscle. 2025 Aug 25. 15(1): 24
      Double Homeobox 4 (DUX4) is a potent transcription factor encoded by a retrogene mapped in D4Z4 repeated elements on chromosome 4q35. DUX4 has emerged as pivotal in the pathomechanisms of facioscapulohumeral muscular dystrophy (FSHD), a relatively common hereditary muscle wasting condition, although classified as a rare disease. DUX4 contributes to zygote genome activation before its expression is repressed in most somatic tissues through epigenetic mechanisms, including DNA methylation and chromatin modifications. In FSHD, inappropriate activation of DUX4 expression is driven by a complex interplay of genomic and epigenetic alterations. The ectopic presence of DUX4 in skeletal muscle cells activates genes, viral elements and pathways that are typical of very early embryonic development, disturbing cell function and ultimately contributing to muscle weakness and wasting. This review first traces the history of DUX4, from the FSHD genetic linkage studies in the early 1990s, through to identification and characterization of the DUX4 gene in 1999. We then discuss the seminal studies that showed how and why DUX4 is expressed in FSHD and the effects of this ectopic expression in muscle, notably cellular toxicity. Other pathological roles of DUX4, such as participation in cancer and viral infection, are also highlighted. Maintenance of DUX4 in the genome was explained by discovery of the function of DUX4 in zygotic genome activation to institute the totipotent cells of the embryo. Thus, we encompass the gradual transition of DUX4 over the past 25 years from being considered a pseudogene in "junk DNA" to becoming central to understanding the molecular pathogenesis of FSHD and the primary focus for FSHD therapeutics.
    Keywords:  D4Z4; DUX4; FSHD; Facioscapulohumeral muscular dystrophy; Muscle; Pathology
    DOI:  https://doi.org/10.1186/s13395-025-00388-0
  41. Int J Biol Macromol. 2025 Aug 23. pii: S0141-8130(25)07642-1. [Epub ahead of print]322(Pt 4): 147085
      During muscle stretch, an elastic or "passive" force develops, primarily determined by the giant protein titin, which forms the third filament system of the muscle sarcomere. The magnitude of this passive force depends largely on the elasticity of titin, governed by the structure of its polypeptide chain. In addition to its structural role, post-translational modifications-particularly phosphorylation by protein kinases-have been suggested to modulate sarcomeric passive force. Mechanical studies on single myofibrils have shown that different kinases can alter passive tension in opposing directions, highlighting phosphorylation as a critical regulatory mechanism. However, the direct contribution of titin phosphorylation to these effects has not been investigated at the single-molecule level. To address this, we conducted single-molecule experiments on individual titin molecules isolated from the m. longissimus dorsi skeletal muscle of rabbit. Phosphoprotein gel staining revealed that the native molecules were highly phosphorylated. To manipulate phosphorylation status, the molecules were treated with λ-protein phosphatase, thereby generating a hypophosphorylated state. Atomic force microscopy of surface-bound titin filaments revealed a marked conformational change in response to dephosphorylation: the C-terminal region of titin collapsed into a compact, coiled structure. These findings suggest that phosphorylation influences titin's nanomechanical structure and may play a direct role in regulating passive muscle tension.
    Keywords:  Atomic force microscopy; Phosphorylation; Posttranslational modification; Skeletal muscle; Titin; λ-protein phosphatase
    DOI:  https://doi.org/10.1016/j.ijbiomac.2025.147085
  42. Clin Exp Med. 2025 Aug 27. 25(1): 306
      Exercise has long been recognized for its systemic health benefits, including modulation of the immune system. Contemporary scientific inquiry has increasingly turned toward understanding the regulatory effects of exercise on immune cell dynamics within muscle tissue, highlighting their potential role in facilitating tissue repair and modulating chronic disease pathways. Following acute bouts of exercise, especially those involving eccentric or high-intensity contractions, muscle fibers experience micro-damage that triggers a well-orchestrated immune response. This phenomenon entails a coordinated, time-sensitive accumulation of immune effector cells-namely neutrophils, macrophages, and T lymphocytes-within compromised muscle tissue. Through the release of immunoregulatory and regenerative mediators like cytokines and growth factors, these cells actively participate in coordinating tissue repair by eliminating cellular debris and resolving inflammation.Macrophage polarization from a pro-inflammatory (M1) to an anti-inflammatory (M2) phenotype is particularly crucial in coordinating effective muscle repair and preventing fibrosis. However, dysregulation of this immune response, such as persistent inflammation or impaired immune cell transition, can hinder regeneration and contribute to the pathogenesis of chronic conditions like sarcopenia, insulin resistance, and muscular dystrophies. Moreover, in chronic disease states, immune cell infiltration into muscle may become maladaptive, exacerbating tissue damage and metabolic dysfunction.Regular moderate-intensity exercise appears to modulate this immune infiltration in a way that enhances repair mechanisms while reducing chronic inflammation, highlighting a potential therapeutic avenue for managing muscle-related pathologies. In-depth insight into the molecular and cellular crosstalk between physical activity and immune cell regulation in muscle tissue forms the basis for crafting specialized therapeutic strategies aimed at facilitating muscle regeneration and limiting the development of chronic pathological conditions. Through a detailed evaluation of exercise-elicited immune dynamics, this review underscores the dichotomous functions of immune cell infiltration in supporting muscle regeneration and in contributing to strategies for chronic disease prevention and management.
    Keywords:  Exercise; Immune cell infiltration; Muscle; Repair
    DOI:  https://doi.org/10.1007/s10238-025-01852-3
  43. Am J Physiol Cell Physiol. 2025 Aug 25.
      Uncoupling protein 3 (UCP3), a member of the mitochondrial solute carrier family, shares high homology with both UCP1 and UCP2. Its exact functional role has been elusive since its discovery, with previous studies primarily focusing on studying UCP3 function in differentiated skeletal muscle myotubes or whole animal models because basal levels of UCP3 protein are low in undifferentiated myoblasts. In the present study, we demonstrate that UCP3 plays a role in modulating energy and redox stress related pathways in undifferentiated muscle myoblasts. Although low, UCP3 mRNA and protein levels were detectable in WT myoblasts. Both whole-body UCP3 knockout (wKO) and conditional UCP3 knockout (cKO) myoblasts displayed increased activation of AMPK (pAMPK) and elevated levels of PPARδ/β and GLUT4 proteins compared to wild type (WT) myoblasts. This altered energy signaling was further associated with UCP3 KO myoblasts exhibiting impaired insulin-stimulated glucose uptake, while WT cells and UCP3 KO cells expressing WT UCP3 were sensitive to insulin stimulation. Moroever, UCP3 KO myoblasts had an accumulation of fatty acids and upregulation of downstream PPARδ target genes in UCP3 KO cells. Lastly, UCP3 KO myoblasts were found to be more sensitive to oxidative stress and hypoxia, due in part to a decrease in the GSH/GSSG ratio compared to WT myoblasts. Collectively, these findings demonstrate that UCP3 is a key modulator of energy sensing and oxidative stress in undifferentiated skeletal muscle myoblasts.
    Keywords:  Energy signaling; Metabolism; Myoblast; Redox signaling; Uncoupling Protein 3
    DOI:  https://doi.org/10.1152/ajpcell.00366.2025
  44. Int J Mol Sci. 2025 Aug 15. pii: 7875. [Epub ahead of print]26(16):
      Osteosarcopenia is a widespread geriatric condition resulting from the coexistence of osteoporosis and sarcopenia, where the connection between bone and muscle is, in part, driven by bone-muscle crosstalk. Given the close, reciprocal influence of muscle on nerve, and vice versa, it is not surprising that there are corresponding aging changes in the biochemistry and morphology of the neuromuscular junction (NMJ). Indeed, degeneration of motor neurons and progressive disruption of the neuromuscular connectivity were observed in old age. Extracellular vesicles (EVs) derived from human amniotic fluid stem cells (hAFSC), exhibiting antioxidant properties, which can also explain their anti-aging and cytoprotective effects, can be considered as potential treatment for age-related diseases. To study cell interactions under both healthy and pathological conditions occurring in musculo-skeletal apparatus, we developed a three-culture system exploiting the use of well-known transwell supports. This system allows both myotubes and neurons, eventually treated with EVs, and osteoblasts, induced to osteoporosis, to interact physically and biochemically. Collectively, this method allowed us to understand how the modifications induced in osteoblasts during bone disorders trigger a cascade of detrimental effects in the muscle and neuron parts. Moreover, we demonstrated the efficacy of hAFSC-EVs in preventing NMJ dysfunction, muscle atrophy, and osteoblast impairment.
    Keywords:  NMJ; bone; muscle; neurons; osteosarcopenia
    DOI:  https://doi.org/10.3390/ijms26167875
  45. J Adv Res. 2025 Aug 18. pii: S2090-1232(25)00643-5. [Epub ahead of print]
       INTRODUCTION: Cachexia is a lethal syndrome with massive muscle wasting that occurs in 60% of colon cancer patients. Several studies in humans and mice show that males are more prone than females to muscle atrophy caused by colorectal cancer. Understanding whether muscle atrophy precedes or follows other organ alterations may unravel the sex-specific drivers of cachexia.
    OBJECTIVES: In two mouse models of colon cancer, we explored when cachexia affects multiple organs in both sexes and their sex development, if/how sex hormone may affect in vitro the inflammatory state of colon adenocarcinoma C26, HCT116 and human primary colorectal cancer cells and in vivo the progression of cachexia in C26-carriers.
    METHODS: We compared tumor growth and cachexia-related responses in C26 males and females and C57BL/6J-ApcMin/+ mice of both sexes. C26, HCT116 and human primary colorectal cancer cells exposed to 17β-estradiol or the antagonist fulvestrant were analysed for Il-6 expression and secretion. β-estradiol 3-benzoate was given to C26 males.
    RESULTS: In both models, cancer-bearing males display more/earlier muscle wasting than females. Muscle proteasome activity is enhanced only in cachectic males and only when they were sexually mature. During cachexia hypogonadism appears earlier in males than females of both models. Tumor-bearing females as long as they are "cycling" are more preserved from cachexia than males. Circulating levels of IL-6 increased more in C26 males than C26 females and spleens were bigger in C26 males also displaying more atrophic and inflamed muscles. 17β-estradiol halved the expression of Il-6 and abrogated the secretion of IL-6 from C26 cells, while fulvestrant surprisingly highly enhanced Il-6 expression, supporting an anti-inflammatory effect of estrogens directly on C26 cells. Similar data were obtained from human (primary) colorectal cancer cells. In vivo β-estradiol 3-benzoate prevented some signs of cachexia in C26 carriers.
    CONCLUSION: Overall, we herein report a novel direct role of 17β-estradiol on colon cancer cells explaining why multiple tissues from males display more signs of cachexia than females.
    Keywords:  Cancer cachexia; Colon cancer; Mouse models; Sex difference; Sex hormones
    DOI:  https://doi.org/10.1016/j.jare.2025.08.028
  46. JVS Vasc Sci. 2025 ;6 100294
       Objective: In people with peripheral artery disease (PAD), the Telmisartan Plus Exercise to Improve Functioning in Peripheral Artery Disease (TELEX) randomized clinical trial tested whether telmisartan (TEL), with or without exercise, significantly improved 6-minute walk distance at 6-month follow-up, compared with placebo (PLA). This study investigated the effects of TEL on exploratory muscle biopsy outcomes of muscle cellular characteristics (myofiber size, satellite cell content, capillary density, extracellular matrix, and collagen area) and molecular characteristics (cell-specific transcriptomics) in people undergoing supervised exercise in the TELEX Trial.
    Methods: Baseline and 6-month follow-up muscle biopsies were obtained from 13 participants with PAD in the TELEX trial randomized to exercise + TEL (n = 6) or exercise + PLA (n = 7). Immunohistochemistry was used to measure muscle cellular characteristics, and the GeoMx digital spatial profiling system was used for transcriptomic analyses of alpha-smooth muscle actin (α-SMA)-positive and α-SMA-negative cells (primarily myofibers).
    Results: Compared with exercise + PLA, exercise + TEL increased mean myofiber cross-sectional area (+2175 μm2; 95% confidence interval, -266 to 4615; P = .04) and the number of satellite cells associated with type II myofibers (+17; 95% confidence interval, -1 to 35; P = .03). In α-SMA-negative cells, exercise + TEL upregulated peroxisome proliferator-activated receptor gamma activation-related pathways, including nitric oxide-cyclic guanosine monophosphate-protein kinase G signaling (P = .008), and fatty acid oxidation (P = .011). Exercise + TEL also reduced myostatin expression relative to exercise + PLA in α-SMA-negative cells (Log2fold-change = -1.24; false discovery rate = 0.010).
    Conclusions: TEL may influence the effects of exercise on muscle in individuals with PAD by reducing myostatin expression, increasing myofiber size, and increasing activation of peroxisome proliferator-activated receptor gamma. Further study is needed to confirm these findings.
    Keywords:  Alpha-smooth muscle actin; Cell deconvolution; Digital spatial profiling; Myostatin; Spatial transcriptomics
    DOI:  https://doi.org/10.1016/j.jvssci.2025.100294
  47. Free Radic Biol Med. 2025 Aug 21. pii: S0891-5849(25)00932-3. [Epub ahead of print]240 284-295
      Mechanisms of skeletal muscle fatigue are commonly studied under isometric conditions, which exclude muscle shortening and limit physiological relevance. We developed a novel in vitro protocol to examine isotonic fatigue using afterload contractions that permits the study of additional active (velocity, power, work) and passive (stiffness, energy loss) mechanical properties of muscle. During the development of this protocol, we examined the impact of shortening load during afterload contractions on the development of fatigue, and observed a relationship where fatigue onset is more rapid and severe with larger shortening loads (30 % vs. 45 % vs. 60 % maximal isometric force). We then applied this protocol to investigate the contribution of NAD(P)H Oxidase 2 (Nox2) to fatigue development and recovery. Nox2 was deleted from skeletal muscle using the Cre-LoxP system (skmNox2KO), while Cre-negative littermates were used as controls. Knockout of Nox2 attenuated the decline in power and increased total isotonic work performed during repeated contractions compared to controls. Recovery kinetics of power, work, and isometric force were similar between groups. Passive mechanical properties-including stiffness and energy loss- increased with fatigue but were unaffected by Nox2 deletion. These findings highlight the importance of incorporating isotonic contractions to uncover fatigue mechanisms and suggest that Nox2, and presumably reactive oxygen species, contributes to the decline in muscle power during repetitive shortening contractions.
    Keywords:  Contraction; Diaphragm; Fatigue; Isotonic; NADPH oxidase; Power
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2025.08.045
  48. Skelet Muscle. 2025 Aug 25. 15(1): 23
       BACKGROUND: The development of functional muscles in Drosophila melanogaster relies on precise spatial and temporal transcriptional control, orchestrated by complex gene regulatory networks. Central to this regulation are cis-regulatory modules (CRMs), which integrate inputs from transcription factors to fine-tune gene expression during myogenesis. In this study, we investigate the transcriptional regulation of the LIM-homeodomain transcription factor Tup (Tailup/Islet-1), a key regulator of dorsal muscle development.
    METHODS: Using a combination of CRISPR-Cas9-mediated deletion and transcriptional analyses, we examined the role of multiple CRMs in regulating tup expression.
    RESULTS: We demonstrate that tup expression is controlled by multiple CRMs that function redundantly to maintain robust tup transcription in dorsal muscles. These mesodermal tup CRMs act sequentially and differentially during the development of dorsal muscles and other tissues, including heart cells and alary muscles. We show that activity of the two late-acting CRMs govern late-phase tup expression through positive autoregulation, whereas an early enhancer initiates transcription independently. Deletion of both late-acting CRMs results in muscle identity shifts and defective muscle patterning. Detailed morphological analyses reveal muscle misalignments at intersegmental borders.
    CONCLUSIONS: Our findings underscore the importance of CRM-mediated autoregulation and redundancy in ensuring robust and precise tup expression during muscle development. These results provide insights into how multiple CRMs coordinate gene regulation to ensure proper muscle identity and function.
    Keywords:  Enhancers; Multiple CRMs; Muscle identity; Muscle patterning; Myogenesis; Transcriptional regulation
    DOI:  https://doi.org/10.1186/s13395-025-00392-4
  49. EMBO Rep. 2025 Aug 26.
      Terminal cell differentiation is often associated with permanent withdrawal from proliferation, termed the postmitotic state. Though widespread among vertebrates and determinant for their biology, the molecular underpinnings of this state are poorly understood. Postmitotic skeletal muscle myotubes can be induced to reenter the cell cycle; however, they generally die as a result of their inability to complete DNA replication. Here, we explore the causes of such incompetence. Genomic hybridization of newly synthesized DNA shows that the replicative failure does not concern specific genomic regions, but can stochastically affect any of them. Myoblast and myotube nuclei are incubated in replicative Xenopus egg extract, which provides a full DNA replication machinery. While myoblast nuclei attain complete DNA replication, those from myotubes, even in these conditions, duplicate less than half of their genomes, strongly indicating that the structure of myotube chromatin obstructs DNA replication. Furthermore, disassembling and disorganizing chromatin with a strong salt treatment does not modify the replicative differences between the two types of nuclei, suggesting that they are rooted in the core structure of chromatin.
    Keywords:  Cell Cycle; Chromatin; Histones; Terminal Differentiation; Xenopus
    DOI:  https://doi.org/10.1038/s44319-025-00554-x