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Exercise-induced muscle damage (EIMD) is characterized by a severe and prolonged decline in force-generating capacity. However, the precise cellular mechanisms underlying the observed long-lasting decline in force-generating capacity associated with EIMD are still unclear. We investigated in vivo force generation and ex vivo Ca 2+ -activated force generation, Ca 2+ sensitivity, and myofiber Ca 2+ handling systems (SR and t-tubules) in human biceps brachii before and 2, 48, and 96 h after eccentrically muscle-damaging contractions and in non-exercised control arm. The force-generating capacity declined by 50 ± 13% 3 h after exercise and was still not recovered after 96 h. The force-Ca relationship of skinned myofibers revealed an impaired maximal Ca 2+ -activated force in MHC I-fibers, but not MHC II-fibers 48 h after exercise. Further, Ca 2+ sensitivity was increased in MHC II-fibers, which was reversed after incubation with a strong reductant. There was a biphasic increase in SERCA sulfonylation, and a parallel reduction in the SR Ca 2+ uptake rate, with no effects on SR vesicle leak or SR vesicle Ca 2+ release rate. T-tubules showed a progressive increase in the density of longitudinal tubules by 96 h after exercise. In conclusion, MHC II-fiber Ca 2+ sensitivity was increased 48 h after exercise, attributed to changes in the REDOX status. 96 h after exercise SR vesicle Ca 2+ uptake was impaired, and an increased number of longitudinal tubules were observed. These alterations may contribute to the impaired force generation evident at the late stage of recovery., (© 2025 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Background: COVID-19 has been associated with both respiratory (diaphragm) and non-respiratory (limb) muscle atrophy. It is unclear if SARS-CoV-2 infection of skeletal muscle plays a role in these changes. This study sought to: 1) determine if cells comprising skeletal muscle tissue, particularly myofibres, express the molecular components required for SARS-CoV-2 infection; 2) assess the capacity for direct SARS-CoV-2 infection and its impact on atrophy pathway genes in myogenic cells; and 3) in an animal model of COVID-19, examine the relationship between viral infection of skeletal muscle and myofibre atrophy within the diaphragm and limb muscles., Methods: We used in silico bioinformatics analysis of published human single cell RNA-seq datasets, as well as direct qPCR examination of human myotubes and diaphragm biopsies, to assess expression of key genes involved in SARS-CoV-2 cellular entry. In Vitro, we determined the ability of SARS-CoV-2 to directly infect myogenic cells and employed qPCR to assess the impact on muscle atrophy pathway genes (ubiquitin-proteasome, autophagy). In vivo, the diaphragm and quadriceps of Roborovski hamsters with SARS-CoV-2 respiratory infection were examined at day 3 post-inoculation to evaluate the relationship between atrophy pathway and SARS-CoV-2 transcripts by qPCR, as well as histological measurements of myofibre morphology., Results: Angiotensin converting enzyme 2 (ACE2), the primary receptor for SARS-CoV-2, as well as cooperating proteases (furin, cathepsins B and L), are expressed by myofibres. ACE2 expression was increased 5-fold (p = 0.01) in the diaphragms of mechanically ventilated human subjects compared to controls. In Vitro, a time-dependent increase of SARS-CoV-2 transcript levels was observed in myotubes directly exposed to the virus (p = 0.002). This was associated with downregulation of the ubiquitin ligase MuRF1 (by 64%, p = 0.002) and the autophagy gene LC3B (by 31%, p = 0.009). In contrast, in vivo infection led to upregulation of MuRF1 in quadriceps (23-fold, p = 0.0007) and autophagy genes in both quadriceps (5.2-fold for Gabarapl1, p = 0.03; 7-fold for p62, p = 0.0002) and diaphragm (2.2-fold for Gabarapl1, p = 0.03; 2.3-fold for p62, p = 0.057). In infected hamsters the diaphragm lacked viral transcripts but exhibited atrophy (48% decrease in myofibre area; p = 0.02), whereas the quadriceps lacked myofibre atrophy despite elevated viral transcripts in the muscle., Conclusions: Although myogenic cells express the genes required for SARS-CoV-2 entry and can be directly infected, there was no evident relationship between viral transcript levels and manifestations of atrophy, either in vitro or in vivo. Our results do not support direct myofibre infection by SARS-CoV-2 as a likely cause of atrophy in COVID-19., (© 2025 The Author(s). Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)

In this study, we used an ex vivo-in vitro model to assess the effect of feeding older (50-70 yr) adults a casein protein hydrolysate (CPH) compared with nonbioactive nonessential amino acid (NEAA) supplement on muscle protein synthesis (MPS) and markers of muscle protein breakdown (MPB). As a secondary objective, to assess any attenuation with aging, we compared the anabolic response to CPH-fed serum from older and young adults. Serum from seven healthy older and seven young men following overnight fast and 60-min postprandial ingestion of CPH or NEAA (0.33 g·kg -1 body mass) was used to condition C2C12 myotube media. Analysis by two-way ANOVA of the fed relative to fasted MPS response revealed a main effect for protein type in pmTOR ( P = 0.009), p70S6K ( P = 0.031), p4E-BP1 ( P = 0.047), and MPS ( P = 0.041) with a greater response to CPH-fed serum, and interaction effects (age × protein) between young and old serum for pmTOR ( P = 0.009) and p70S6K ( P = 0.016). In addition, significant changes in myotube diameter ( P = 0.049), atrogin-1 ( P = 0.004), and MuRF-1 ( P = 0.012) in response to CPH-fed compared with fasted serum were observed with no differences between young and old serum. In conclusion, this study demonstrated that CPH-fed serum from both young and older (50-70 yr) adults can stimulate MPS and muscle growth and can suppress biomarkers of muscle protein breakdown processes. NEW & NOTEWORTHY This study extended previously developed coculture models and found that treating skeletal muscle cells with ex vivo human serum following feeding with a casein protein hydrolysate resulted in greater protein signaling, muscle protein synthesis, muscle growth, and lower expression of genes related to muscle protein breakdown compared with feeding with a nonessential amino acid control. These findings were similar using serum from young and older adults.

Background: Muscle diseases are serious challenges to human health. Prokineticin receptor 1 (PROKR1) has emerged as a potential target to improve muscle function through increasing oxidative muscle fibres, but there are no clinically applicable synthetic PROKR1 agonists., Methods: Drugs with biological properties of prokineticin 2 (PK2) were discovered through connectivity map (CMap) analysis. Their effects on PROKR1 were evaluated using molecular docking, PROKR1 signalling and competitive binding assays. Pregnant dams were fed diets containing varying celecoxib concentrations (0, 500, 1000 and 1500 ppm) from gestation day 5 through weaning. Offspring were given high-fat diets (HFD) from weaning until 20 weeks old, and body composition, insulin resistance, energy expenditure, exercise performance and histological analysis of muscle tissues were evaluated., Results: Celecoxib, with a connectivity score of 64.19 to PK2 and a docking score of -9.0 to PROKR1, selectively activated Gs signalling at 4 μM of EC 50 and increased NR4A2 protein levels by 1.6-fold (p < 0.01) in PROKR1-overexpressing cells. It competitively inhibited PK2 binding to PROKR1 and reduced cAMP accumulation. In murine and human myotubes, celecoxib increased Prokr1 protein levels by 1.8-fold (p < 0.05), pCreb by 1.5-fold (p < 0.05) and Nr4a2 by 1.3-fold (p < 0.05). It also elevated Myh7 index by 2.2-fold (p < 0.0001), mitochondrial content by 1.6-fold (p < 0.001) and fatty acid oxidation (FAO) activity by 4.1-fold (p < 0.05). Offspring exposed to celecoxib during pre- and postnatal muscle development exhibited activated Prokr1 signalling, enhanced oxidative muscle fibre formation and improved muscle phenotype despite HFD. At weaning, both male and female offspring showed dose-dependent increases in lean mass (> 9.35%, p < 0.001) and grip strength (< 18.0%, p < 0.01). At 12 weeks old, mice displayed a dose-dependent decrease in weight loss (> 13.3%, p < 0.05), increased lean mass (> 16.2%, p < 0.05), improved insulin resistance (> 70.4%, p < 0.0001), energy expenditure (> 173%, p < 0.0001) and grip strength (> 23.5%, p < 0.001). Celecoxib also increased Myh7-positive muscle fibre composition (> 10.8%, p < 0.05) and mitochondrial mass (> 32.8%, p < 0.05) in the gastrocnemius and soleus muscles, accompanied by significant Prokr1 signalling activation. These effects persisted in both male and female mice at 20 weeks old., Conclusions: Celecoxib shows promise as a PROKR1 agonist and clinically applicable exercise mimetic for the treatment of muscular disorders., (© 2025 The Author(s). Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)

1. In the following experiment meat quality traits of a Gushi-Anka F2 resource population were measured, and their heritability estimated. Intramuscular fat (IMF) had medium heritability (0.35) but leg muscle fibre density (LMD), leg muscle fibre diameter (LMF), breast muscle fibre density (BMD), fresh fat content (FFA), and absolute dry fat content (AFC) had low heritability (0-0.2). The IMF presented the most important genetic additive effect among the poultry meat quality-related traits studied.2. The phenotypic data of meat quality traits in the Gushi-Anka F2 resource population were combined with genotyping by sequencing (GBS) data to obtain genotype data. Six meat quality traits in 734 birds were analysed by GWAS. Based on these variants, 83 significant (-log 10 (p) > 4.42) single nucleotide polymorphisms and four quantitative trait loci (QTL) regions corresponding to 175 genes were identified. Further linkage disequilibrium (LD) analysis was conducted on chromosome 13 (Chr13) and chromosome 27 (Chr27) QTL regions.3. Based on the transcriptome data and GWAS results, 12 shared genes - ITGB3 , DNAJC27 , ETV4 , C7orf50 , FKBP1B , G3BP1 , IGF2BP1 , KCNH6 , LOC416263 , SCARA5 , SMIM5 and TBL1XR1 were identified as candidate genes influencing muscle fibre and fat traits.

Collective cell migration is critical for morphogenesis, homeostasis, and wound healing. Migrating mesenchymal cells form tissues that shape the body's organs. We developed a powerful model, exploring how Drosophila nascent myotubes migrate onto the testis during pupal development, forming the muscles ensheathing it and creating its characteristic spiral shape. To define genes regulating this, we used RNA sequencing (RNA-seq) to identify genes expressed in myotubes during migration. Using this dataset, we curated a list of 131 ligands, receptors, and cytoskeletal regulators, including all Rho/Ras/Rap1 regulators, as candidates. We then expressed 279 short hairpin RNAs (shRNAs) targeting these genes and examined adult testes. We identified 29 genes with diverse roles in morphogenesis. Some have phenotypes consistent with defective migration, while others alter testis shape in different ways, revealing the underlying logic of testis morphogenesis. We followed up on the Rho-family GEF dPix in detail. dPix knockdown drastically reduced migration and thus muscle coverage. Our data suggest different isoforms of dPix play distinct roles in this process and reveal a role for its partner Git. We also explored whether dPix regulates Cdc42 activity or cell adhesion. Our RNA-seq dataset and genetic analysis provide an important resource for the community to explore cell migration and organ morphogenesis., Competing Interests: Conflicts of interests: The authors declare no financial conflict of interest.

Skeletal muscle generates superoxide during contractions, which is converted to hydrogen peroxide (H 2 O 2 ). H 2 O 2 has been proposed to activate signalling pathways and transcription factors that regulate adaptive responses to exercise, but the concentration required to oxidize and activate key redox-sensitive signalling proteins in vitro is much higher than the typical intracellular levels seen in muscle after exercise. We hypothesized that 2-Cys-peroxiredoxins (PRDX), which rapidly oxidize in the presence of physiological concentrations of H 2 O 2 , serve as intermediary signalling molecules and play a crucial role in activating adaptive pathways following muscle contractions. This study has examined the human muscle myotube responses to contractile activity, or exposure to low extracellular concentrations (2.5-5 μM) of H 2 O 2 and whether knock down of muscle PRDX2 alters the differential gene expression (DEG) that results from these stresses. Exposure of human skeletal muscle myotubes to a 15 min period of aerobic electrically stimulated isometric contractions or 5 μM H 2 O 2 induced substantial changes in DEG with modification of many genes associated with adaptations of skeletal muscle to contractile activity. Common DEG in these conditions included upregulation of genes associated with increased mitochondrial oxidative phosphorylation, including COX1, COX2, COX3 and ATP6. In myotubes with PRDX2 knock down (94 % decrease in PRDX2 mRNA), the upregulation of genes associated with increased mitochondrial oxidative phosphorylation was abolished following contractile activity or exposure to H 2 O 2 . These data indicate that a common effect of contractile activity and exposure to "physiological" levels of H 2 O 2 in human myotubes is to increase the expression of multiple genes associated with increased mitochondrial oxidative phosphorylation. Furthermore, these effects were abolished in PRDX2 knock down myotubes indicating that adaptations to upregulate multiple genes related to increased mitochondrial capacity in human muscle myotubes in response to exercise is both redox regulated and requires PRDX2 as an essential mediator of the effects of H 2 O 2 ., Competing Interests: Declaration of competing interests None of the authors have any competing financial interests relevant to this work., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Plasma membrane repair (PMR) restores membrane integrity of cells, preventing cell death in vital organs, and has been studied extensively in skeletal muscle. Dysferlin, a sarcolemmal Ca 2+ -binding protein, plays a crucial role in PMR in skeletal muscle. Previous studies have suggested that PMR uses membrane trafficking and membrane fusion, similar to neurotransmission. Soluble N -ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion in neurotransmission with the help of synaptotagmin, a crucial Ca 2+ -binding protein. Interestingly, dysferlin shares structural similarity with synaptotagmin and was shown to promote SNARE-mediated membrane fusion in a liposome-based assay. However, whether dysferlin facilitates SNARE-mediated membrane fusion in PMR in muscle cells remains unclear. In this study, we aimed to test if SNARE-mediated PMR requires dysferlin in muscle cells with pharmacological and genetic approaches. TAT-NSF700, which disrupts the disassembly of SNARE complexes, was used to disrupt functions of SNAREs in muscle cells. We found that human-induced pluripotent stem cells-derived cardiomyocytes (hiPS-CMs) treated with TAT-NSF700 showed a higher loss of membrane integrity after repetitive mechanical strains. Moreover, laser-wounded mouse flexor digitorum brevis (FDB) fibers treated with TAT-NSF700 showed an increased Ca 2+ influx, but a decreased FM1-43 uptake, which depends on dynamin-regulated endocytosis as we previously showed in FDB fibers. Importantly, overexpression of STX4-mCitrine or eGFP-SNAP23 decreased Ca 2+ influx in laser-wounded FDB fibers. Furthermore, overexpression of STX4-mCitrine also decreased Ca 2+ influx in laser-wounded dysferlin-deficient FDB fibers. Overall, these results suggest that disassembly of SNARE complexes is required for efficient PMR and STX4-enhanced PMR does not require dysferlin in skeletal muscle. NEW & NOTEWORTHY Dysferlin, a crucial Ca 2+ -binding protein in plasma membrane repair (PMR), shares homology with synaptotagmin, which binds Ca 2+ and regulates SNARE-mediated vesicle fusion in neurons. Dysferlin was thus hypothesized to function as synaptotagmin in PMR. We demonstrate here that the activity of SNAREs is important for PMR, and overexpression of STX4 enhances PMR in both intact and dysferlin-deficient skeletal muscle. These data suggest that SNARE-mediated PMR may be independent of dysferlin in skeletal muscle.

The positive role of melatonin in obesity control and skeletal muscle (SKM) preservation is well known. We recently showed that melatonin improves vastus lateralis muscle (VL) fiber oxidative phenotype. However, fiber type characterization, mitochondrial function, and molecular mechanisms that underlie VL fiber switching by melatonin are still undefined. Our study aims to investigate whether melatonin induces fiber switching by NRF2/RCAN/MEF2 pathway activation and mitochondrial oxidative metabolism modulation in the VL of both sex Zücker diabetic fatty (ZDF) rats. 5-Weeks-old male and female ZDF rats (N = 16) and their age-matched lean littermates (ZL) were subdivided into two subgroups: control (C) and orally treated with melatonin (M) (10 mg/kg/day) for 12 weeks. Interestingly, melatonin increased oxidative fibers amounts (Types I and IIa) counteracting the decreased levels found in the VL of obese-diabetic rats, and upregulated NRF2, calcineurin and MEF2 expression. Melatonin also restored the mitochondrial oxidative capacity increasing the respiratory control ratio (RCR) in both sex and phenotype rats through the reduction of the proton leak component of respiration (state 4). Melatonin also improved the VL mitochondrial phosphorylation coefficient and modulated the total oxygen consumption by enhancing complex I, III and IV activity, and fatty acid oxidation (FAO) in both sex obese-diabetic rats, decreasing in male and increasing in female the complex II oxygen consumption. These findings suggest that melatonin treatment induces fiber switching in SKM improving mitochondrial functionality by NRF2/RCAN/MEF2 pathway activation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondria are metabolic hubs that govern skeletal muscle health. Although exercise has been established as a powerful inducer of quality control processes that ultimately enhance mitochondrial function, there are currently limited pharmaceutical interventions available that emulate exercise-induced mitochondrial adaptations. To investigate a novel candidate for this role, we examined sulforaphane (SFN), a naturally occurring compound found in cruciferous vegetables. SFN has been documented as a potent antioxidant inducer through its activation of the nuclear factor erythroid 2-related factor 2 (Nrf-2) antioxidant response pathway. However, its effects on muscle health have been underexplored. To investigate the interplay between chronic exercise and SFN, C2C12 myotubes were electrically stimulated to model chronic contractile activity (CCA) in the presence or absence of SFN. SFN promoted Nrf-2 nuclear translocation, enhanced mitochondrial respiration, and upregulated key antioxidant proteins including catalase and glutathione reductase. These adaptations were accompanied by reductions in cellular and mitochondrial reactive oxygen species (ROS) emission. Signaling toward biogenesis was enhanced, demonstrated by increases in mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α nuclear translocation, PGC-1α promoter activity, mitochondrial content, and organelle branching, suggestive of a larger, more interconnected mitochondrial pool. These mitochondrial adaptations were accompanied by an increase in lysosomal proteins, suggesting coordinated regulation. There was no difference in mitochondrial and antioxidant-related proteins between CCA and non-CCA SFN-treated cells. Our data suggest that SFN activates signaling cascades that are common to those produced by contractile activity, indicating that SFN-centered therapeutic strategies may improve the mitochondrial phenotype in skeletal muscle. NEW & NOTEWORTHY Nrf-2 is a transcription factor that has been implicated in mitigating oxidative stress and regulating mitochondrial homeostasis. However, limited research has demonstrated how Nrf-2-mediated adaptations compare with those produced by exercise. To investigate this, we treated myotubes with Sulforaphane, a well-established Nrf-2 activator, and combined this with stimulation-induced chronic contractile activity to model exercise training. Our work is the first to establish that sulforaphane mimics training-induced mitochondrial adaptations, including enhancements in respiration, biogenesis, and dynamics.

The EP4 (prostaglandin E2) receptor plays a crucial role in myogenesis and skeletal muscle regeneration, yet its involvement in regulating insulin-dependent metabolic pathways is not well characterised. Our research investigates the expression of EP4 in rat skeletal L6 myotubes and its impact on insulin signalling. We found that activation of EP4 by selective agonists disrupts insulin signalling and insulin-stimulated glucose uptake. This impairment is associated with enhanced pro-inflammatory NF-κB signalling, a process that can be attenuated by EP4 antagonists. Importantly, EP4 antagonism also reduces NF-κB activation induced by palmitate and the associated reduction in insulin signalling, an effect not replicated by antagonists of EP1, EP2, or EP3 receptors. These observations indicate that the EP4 receptor is a modulator of insulin action and that it contributes to fatty-acid-induced insulin resistance in skeletal muscle cells. Our findings suggest that EP4 could be a potential therapeutic target for managing insulin resistance., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Professor Hari Hundal reports financial support was provided by Diabetes UK. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Environmental factors such as physical activity induce epigenetic modifications, with exercise-responsive DNA methylation changes occurring in skeletal muscle. To determine the skeletal muscle DNA methylation signature of endurance swim training, we used whole-genome methylated DNA immunoprecipitation (MeDIP) sequencing., Methods: We utilized endurance-trained rats, cultured L6 myotubes, and human skeletal muscle cells, employing MeDIP sequencing, gene silencing, and palmitate oxidation assays. Additional methods included promoter luciferase assays, fluorescence microscopy, and RNA/DNA analysis to investigate exercise-induced molecular changes., Results: Gene set enrichment analysis (GSEA) of differentially methylated promoter regions identified an enrichment of four gene sets, including those linked to lipid metabolic processes, with hypermethylated or hypomethylated promoter regions in skeletal muscle of exercise-trained rats. Bisulfite sequencing confirmed hypomethylation of CpGs in the Serhl2 (Serine Hydrolase Like 2) transcription start site in exercise-trained rats. Serhl2 gene expression was upregulated in both exercise-trained rats and an "exercise-in-a-dish" model of L6 myotubes subjected to electrical pulse stimulation (EPS). Serhl2 promoter activity was regulated by methylation and EPS. A Nr4a binding motif in the Serhl2 promoter, when deleted, reduced promoter activity and sensitivity to methylation in L6 myotubes. Silencing Serhl2 in L6 myotubes reduced intracellular lipid oxidation and triacylglycerol synthesis in response to EPS., Conclusions: Exercise-training enhances intracellular lipid metabolism and phenotypic changes in skeletal muscle through epigenomic modifications on Serhl2. Hypomethylation of the Serhl2 promoter influences Nr4a transcription factor binding, promoter activity, and gene expression, linking exercise-induced epigenomic regulation of Serhl2 to lipid oxidation and triacylglycerol synthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The skeletal muscle interstitial space is the extracellular region around myofibers and mediates cross-talk between resident cell types. We applied a proteomic workflow to characterize the human skeletal muscle interstitial fluid proteome at rest and in response to exercise. Following exhaustive exercise, markers of skeletal muscle damage accumulate in the interstitial space followed by the appearance of immune cell-derived proteins. Among the proteins up-regulated after exercise, we identified cathelicidin-related antimicrobial peptide (CAMP) as a bioactive molecule regulating muscle fiber development. Treatment with the bioactive peptide derivative of CAMP (LL-37) resulted in the growth of larger C2C12 skeletal muscle myotubes. Phosphoproteomics revealed that LL-37 activated pathways central to muscle growth and proliferation, including phosphatidylinositol 3-kinase, AKT serine/threonine kinase 1, mitogen-activated protein kinases, and mammalian target of rapamycin. Our findings provide a proof of concept that the interstitial fluid proteome is quantifiable via microdialysis sampling in vivo. These data highlight the importance of cellular communication in the adaptive response to exercise.

The present study was conducted on specific skeletal muscles of six weaned male kids from each of the Angora, Hair, Honamlı, and Kilis goat breeds. The relationships between the expression of myogenic factor 5 (Myf5) and myogenic factor 6 (Myf6) genes and muscle fibre characteristics were analysed. Muscle samples from the longissimus dorsi (LD) and semitendinosus (ST) were collected from six 90-day-old weaned male kids of each breed. Muscle fiber characteristics were assessed through histochemical staining, while expression levels of Myf5 and Myf6 genes were quantified using real-time PCR. Total RNA content in the LD and ST muscles was significantly higher (p < 0.05) in Honamlı kids compared to those of the other breeds. Similarly, the expression of Myf5 gene in Honamlı kids was significantly higher (p < 0.05) than that observed in kids from the other breeds in LD muscle. Conversely, in the ST muscle, Hair kids exhibited a significantly lower expression of Myf5 (p < 0.05) when compared to both Honamlı and Kilis kids Additionally, Kilis kids demonstrated a significant reduced expression of Myf6 gene (p < 0.05) relative to the other breeds. The highest expression levels of the Myf6 gene (p < 0.05) were detected in the ST muscle of Honamlı and Angora kids, significantly surpassing those observed in Hair and Kilis kids. Moreover, significant correlations (p < 0.05) were observed among Myf5 and Myf6 gene expression levels and various muscle fiber characteristics differing across breeds. The results of this study underscore the pivotal role of these myogenic regulatory factors in muscle development, offering insights into the molecular mechanism driving breed-specific muscle growth. This association between gene expression and muscle phenotype could have profound implications for targeted breeding programs aimed at optimizing muscle traits in livestock species., Competing Interests: Declarations. Ethics approval: The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to. The experimental procedures were approved by the Local Animal Care and Ethics Committee of Kırşehir Ahi Evran University, Kırşehir, Turkey (approval number; 021013.04.1), and ensuring compliance with EC Directive 86/609/EEC for animal experiments. Conflict of interest: None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (© 2025. The Author(s).)

This study aims to assess the effects of aquatic training (AT) and its combination with photobiomodulation (PBM) on cytokine synthesis and plantar muscle morphology during compensatory hypertrophy (H) in Wistar rats. H was induced by bilateral ablation of synergistic muscles, and PBM using a laser (780 nm). AT involved 60 min sessions, 5 times/week, for 7 and 14 days. Muscle weight relative to body weight did not differ significantly between groups. TNF-α synthesis levels decreased in the H + AT + PBM group at 7 days compared to H Control. IL-6 and IL-1β levels showed no significant changes. Cross-sectional area (CSA) was higher in H + AT + PBM at 7 days and 14 days compared to H + AT and H Control. Fiber diameter increased in H + AT and H + AT + PBM compared to H at 14 days. AT with PBM decreased TNF-α expression, increased CSA and fiber diameter, whereas AT alone increased fiber diameter. In conclusion, the combination of photobiomodulation (PBM) therapy and aquatic training (AT) effectively reduced TNF-α levels while increasing muscle fiber diameter and cross-sectional area (CSA). Furthermore, AT alone demonstrated the capacity to maintain fiber diameter., Competing Interests: Declarations. Ethical approval: This study received approval form the local animal research ethics committee – Nove de Julho University (certificate number: 1664030522). Conflict of interest: The authors declare no conflict of interest., (© 2025. The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature.)

Duchenne muscular dystrophy (DMD) is a severe genetic disorder characterized by progressive muscle degeneration, primarily affecting young males. In this study, we investigated arginine-modified hydroxyapatite nanoparticles (R-HAp) as a novel non-viral vector for DMD gene therapy, particularly for delivering the large 18.8 kb dystrophin gene. Addressing the limitations of traditional adeno-associated viral vectors, R-HAp demonstrated efficient binding and delivery of the dystrophin plasmid to DMD patient-derived skeletal muscle cells. Using confocal imaging and RT-PCR analysis, our results showed effective gene delivery and expression in both mouse myotubes and patient-derived cells, with sustained expression evident up to 5 days post transfection. The patient-derived myotubes also showed dystrophin protein production 7 days post transfection. These findings suggest R-HAp nanoparticles as a promising and cost-effective alternative for DMD treatment, highlighting their potential for overcoming current gene therapy challenges.

Spinal muscular atrophy (SMA) is characterized by low levels of the ubiquitously expressed Survival Motor Neuron (SMN) protein, leading to progressive muscle weakness and atrophy. Skeletal muscle satellite cells play a crucial role in muscle fiber maintenance, repair, and remodelling. While the effects of SMN depletion in muscle are well documented, its precise role in satellite cell function remains largely unclear. Using the Smn2B/- mouse model, we investigated SMN-depleted satellite cell biology through single fiber culture studies. Myofibers from Smn2B/- mice were smaller in size, shorter in length, had reduced myonuclear domain size, and reduced sub-synaptic myonuclear clusters-all suggesting impaired muscle function and integrity. These changes were accompanied by a reduction in the number of myonuclei in myofibers from Smn2B/- mice across all disease stages examined. Although the number of satellite cells in myofibers was significantly reduced, those remaining retained their capacity for myogenic activation and proliferation. These findings support the idea that a dysregulated myogenic process could be occurring as early in muscle stem cells during muscle formation and maturation in SMA. Targeting those pathways could offer additional options for combinatorial therapies for SMA., (© The Author(s) 2024. Published by Oxford University Press.)

Background/aims: MOTS-c belongs to a group of mitochondrial peptides involved in metabolic processes in the body. This peptide has garnered increasing attention since its discovery in 2015 because of its potential to ameliorate metabolic parameters in animals with diabetes or insulin resistance. MOTS-c is involved in muscle metabolism; however, little is known about its role in fiber differentiation., Methods: We conducted a study to explore the effect of MOTS-c on cellular processes using the C2C12 and L6 cell lines, representing different metabolic types of muscle fibers. The research methods were real-time PCR, Western blot, and lipid accumulation measurement., Results: Notably, our investigations revealed that MOTS-c increased the survival of C2C12 cells at doses of 10 and 100 nM (p<0.01) and stimulated the phosphorylation of extracellular signal-regulated kinase within 5 min of incubation (p<0.05). Remarkably, these effects were not observed in L6 cells; however, both cell lines showed a reduced rate of proliferation. Furthermore, MOTS-c promotes the differentiation of C2C12 cells by increasing the expression of muscle regulatory factors, but it does not produce such an effect in L6 cells. Additionally, cells were treated with physiological concentrations of free fatty acids and MOTS-c, unveiling an augmentation in lipid accumulation observed in L6 cells and a decrease in lipid accumulation in C2C12 cells., Conclusion: In conclusion, our findings have suggested a diverse response to MOTS-c depending on the type of muscle fibers, particularly in the domains of survival, cell differentiation, and lipid accumulation., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Nuclear growth differentiation factor 15 (GDF15) reduces the binding of the mothers' against decapentaplegic homolog (SMAD) complex to its DNA-binding elements. However, the stimuli that control this process are unknown. Here, we examined whether saturated fatty acids (FA), particularly palmitate, regulate nuclear GDF15 levels and the activation of the SMAD3 pathway in human skeletal myotubes and mouse skeletal muscle, where most insulin-stimulated glucose use occurs in the whole organism. Human LHCN-M2 myotubes and skeletal muscle from wild-type and Gdf15 -/- mice fed a standard (STD) or a high-fat (HFD) diet were subjected to a series of studies to investigate the involvement of lipids in nuclear GDF15 levels and the activation of the SMAD3 pathway. The saturated FA palmitate, but not the monounsaturated FA oleate, increased the expression of GDF15 in human myotubes and, unexpectedly, decreased its nuclear levels. This reduction was prevented by the nuclear export inhibitor leptomycin B. The decrease in nuclear GDF15 levels caused by palmitate was accompanied by increases in SMAD3 protein levels and in the expression of its target gene SERPINE1, which encodes plasminogen activator inhibitor 1 (PAI-1). HFD-fed Gdf15 -/- mice displayed aggravated glucose intolerance compared to HFD-fed WT mice, with increased levels of SMAD3 and PAI-1 in the skeletal muscle. The increased PAI-1 levels in the skeletal muscle of HFD-fed Gdf15 -/- mice were accompanied by a reduction in one of its targets, hepatocyte growth factor (HGF)α, a cytokine involved in glucose metabolism. Interestingly, PAI-1 acts as a ligand of signal transducer and activator of transcription 3 (STAT3) and the phosphorylation of this transcription factor was exacerbated in HFD-fed Gdf15 -/- mice compared to HFD-fed WT mice. At the same time, the protein levels of insulin receptor substrate 1 (IRS-1) were reduced. These findings uncover a potential novel mechanism through which palmitate induces the SMAD3-PAI-1 pathway to promote insulin resistance in skeletal muscle by reducing nuclear GDF15 levels., Competing Interests: Declarations. Ethical approval and consent to participate: All procedures were approved. Animal experiments were approved by the Bioethics Committee of the University of Barcelona, as stated in Law 5/21 July 1995 passed by the Generalitat de Catalunya and were following the guidelines of the National Institute of Health. Consent for publication: All authors have read the manuscript and agree with submission for publication. Conflict of interests: The authors declared no conflicts of interest., (© 2025. The Author(s).)

Quiescent skeletal muscle satellite cells (SCs) located on myofibers activate in response to muscle injury to regenerate muscle; however, identifying the role of specific matrix signals on SC behavior in vivo is difficult. Therefore, we developed a viscoelastic hydrogel with tunable properties to encapsulate myofibers while maintaining stem cell niche polarity and SC-myofiber interactions to investigate how matrix signals, including viscoelasticity and the integrin-binding ligand arginyl-glycyl-aspartic acid (RGD), influence SC behavior during muscle regeneration. Viscoelastic hydrogels support myofiber culture while preserving SC stemness for up to 72 hours post-encapsulation, minimizing myofiber hypercontraction and SC hyperproliferation compared to Matrigel. Pax7 is continuously expressed in SCs on myofibers embedded in hydrogels with higher stress relaxation while SCs differentiate when embedded in elastic hydrogels. Increasing RGD concentrations activates SCs and translocates YAP/TAZ to the nucleus as revealed by photo-expansion microscopy. Deleting YAP/TAZ abrogates RGD-mediated activation of SCs, and thus, YAP/TAZ mediates RGD ligand-induced SC activation and subsequent proliferation. STATEMENT OF SIGNIFICANCE: Satellite cells (SCs) are responsible for muscle maintenance and regeneration, but how the extracellular matrix regulates SC function is less understood and would benefit from new biomaterial models that can recapitulate the complexity of SC niche in vitro. Upon isolation of myofibers, SCs exit quiescence, becoming activated. To circumvent this issue, we developed a viscoelastic hydrogel for encapsulating myofibers, which maintains SC quiescence and limits differentiation, allowing the study of RGD effects. We showed that increasing RGD concentration promotes activation and suppresses differentiation. Finally, to allow high resolution imaging for resolving the subcellular localization of YAP/TAZ transcriptional co-activators, we applied photo-expansion microscopy and gel-to-gel transfer techniques to quantify YAP/TAZ nuclear-cytoplasmic ratio, revealing that RGD-mediated activation relies on YAP/TAZ nuclear translocation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

The myotendinous junction (MTJ) is a weak link in the musculoskeletal system. Here, we isolated the tips of single myofibres from healthy (non-injured) human hamstring muscles for confocal microscopy (n=6) and undertook RNAscope in situ hybridisation (n=6) to gain insight into the profiles of cells and myonuclei in this region, in a fibre type manner. A marked presence of mononuclear cells was observed coating the myofibre tips (confirmed by serial block face scanning electron microscopy and cryosection immunofluorescence), with higher numbers for type I (median 29; range 16-63) than type II (16; 9-23) myofibres (P<0.05). The number of these cells expressing COL22A1 was comparable between fibre types. Myonuclear number and density gradually increased from the myofibre proper towards the tip for both fibre types (P<0.05). COL22A1 was expressed by similar proportions of myonuclei in type I (median 26%; range 13-56) and type II (19%; 3-67) myofibre tips. 70% of the COL22A1-positive nuclei in the MTJ region were myonuclei, and the remaining 30% were MTJ cells. This insight refines our fundamental understanding of the human MTJ at the cell and structural levels., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2025. Published by The Company of Biologists.)

Background: In intensive care units, sepsis-related muscle atrophy is a severe complication of numerous diseases, yet the underlying mechanism and potential therapeutic options remain elusive. Recent research has identified melatonin as a promising candidate for attenuating organ dysfunction triggered by sepsis., Methods: We used in vitro and in vivo models to simulate sepsis, C2C12 myotubes were treated with LPS, and the mice underwent cecal ligation and puncture (CLP) surgery. Following a pretreatment regimen involving melatonin and the AKT inhibitor MK-2206 2HCl, we analyzed changes in p-Akt and MuRF1 protein levels, fiber cross-sectional areas, and myotube diameters. The analyses included RNA sequencing, Western blotting, qRT-PCR, and immunofluorescence staining., Results: Activation of the PI3K/Akt pathway in skeletal muscle occurred 24 h post-CLP surgery in mice. This was accompanied by upregulated MuRF1 expression and reduced muscle fiber cross-sectional area, which culminated in muscle atrophy. However, these detrimental effects were attenuated when the mice were pretreated with melatonin via intraperitoneal injection for seven consecutive days. Similarly, LPS treatment of C2C12 myotubes activated the PI3K/Akt pathway, elevated MuRF1 expression, and markedly reduced myotube diameter after 48 h, leading to muscle atrophy. Pretreatment of C2C12 myotubes with melatonin 24 h in advance mitigated these adverse effects. However, cotreatment of C2C12 myotubes with melatonin and MK-2206 2HCl attenuated the beneficial effects of melatonin., Conclusion: Melatonin can attenuate sepsis-induced muscle atrophy by regulating the PI3K/Akt pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cultured meat (CM) is derived from the in vitro myogenesis of muscle satellite (stem) cells (MSCs) and offers a promising alternative protein source. However, the development of a cost-effective media formulation that promotes cell growth has yet to be achieved. In this study, laxogenin (LAX) and 5-alpha-hydroxy-laxogenin (5HLAX) were computationally screened against myostatin (MSTN), a negative regulator of muscle mass, because of their antioxidant properties and dual roles as MSTN inhibitors and enhancers of myogenesis regulatory factors. In silico analysis showed LXG and 5HLXG bound to MSTN with binding free energies of -7.90 and -8.50 kcal/mol, respectively. At a concentration of 10 nM, LAX and 5HLAX effectively inhibited the mRNA and protein expressions of MSTN, promoted myogenesis, and enhanced myotube formation and maturation. In addition, by acting as agonists of ROS downregulating factors, they exhibited antioxidative effects. This study shows that supplementation with LAX or 5HLAX at 10 nM in CM production improves texture, quality, and nutritional value. We believe this study fills a research gap on media development for myotube formation and maturation, which are important factors for large-scale in vitro CM production that improve product quality, nutritional value, and efficacy.

Hibernating brown bears, due to a drastic reduction in metabolic rate, show only moderate muscle wasting. Here, we evaluate if ATPase activity of resting skeletal muscle myosin can contribute to this energy sparing. By analyzing single muscle fibers taken from the same bears, either during hibernation or in summer, we find that fibers from hibernating bears have a mild decline in force production and a significant reduction in ATPase activity. Single fiber proteomics, western blotting, and immunohistochemical analyses reveal major remodeling of the mitochondrial proteome during hibernation. Furthermore, using bioinformatical approaches and western blotting we find that phosphorylated myosin light chain, a known stimulator of basal myosin ATPase activity, is decreased in hibernating and disused muscles. These results suggest that skeletal muscle limits energy loss by reducing myosin ATPase activity, indicating a possible role for myosin ATPase activity modulation in multiple muscle wasting conditions., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

While total RNA concentrations putatively represent ribosome content, there is a need to homologize various quantification approaches. Thus, total RNA concentrations ([RNA]) provided through UV-Vis spectroscopy (UV), fluorometry-only (Fluor), and fluorometry-based microfluidic chip electrophoresis (MFGE) were examined in C2C12 myotubes and mouse skeletal muscle to determine if values aligned with [18S + 28S rRNA] (i.e., criterion ribosome metric). A novel antibody cocktail (termed RiboAb) was also tested and compared to [18S + 28S rRNA] in these models. In myotubes, 24-h IGF-1 treatments increased [18S + 28S rRNA] (~2.0-fold) and [RNA] based on UV (~1.9-fold), Fluor (~2.3 fold), and MFGE (~2.1-fold). In C57BL/6 mice, 10 days of mechanical overload (MOV) elevated plantaris [18S + 28S rRNA] (~1.7-fold) and [RNA] according to UV (~1.5-fold), Fluor (~1.6-fold), and MFGE (~1.8-fold). Myotube and mouse plantaris RiboAb levels were significantly higher with IGF-1 treatments and MOV, respectively, versus controls (1.3-fold and 1.7-fold, respectively), and values correlated with [18S + 28S rRNA] (r = 0.637 and r = 0.853, respectively, p ≤ 0.005). UV, Fluor, and MFGE [RNA] are seemingly valid surrogates of cell/tissue ribosome content, although each method has advantages (e.g., ease of use) and disadvantages (e.g., magnitudes of bias) discussed herein. Finally, the RiboAb cocktail may also represent ribosome content, although this should be further explored in other models., (© 2025 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Glucosamine (GlcN) is one of the dietary supplements used in the treatment of osteoarthritis. Endogenously, GlcN is synthesized from glucose through the hexosamine pathway. In addition to ameliorating arthritis, several biological functions of GlcN have been reported, including insulin resistance in skeletal muscle. However, the regulatory role of GlcN in skeletal muscle development is not clear. We therefore investigated the effect of GlcN on myoblast proliferation, differentiation, and myotube development and their underlying mechanisms in C2C12 cells. Myoblast proliferation was measured by MTT assay. The expressions of MyoD, myogenin (MyoG), and myosin heavy chain (MyHC) were identified as determinants of myoblast differentiation. Expressions of atrogin-1 and muscle RING-finger protein-1 (MuRF-1) were identified as markers of myotube atrophy. The results show that treatment with GlcN significantly reduced myoblast proliferation and phosphorylation of Stat3 and S6K. These findings suggest that GlcN can inhibit growth of myoblasts through inhibiting phosphorylation of Stat3 and S6K. In addition, GlcN significantly suppressed the expression of MyoD, MyoG, and MyHC, as well as myotube formation. Pretreatment of C2C12 myoblast cells with ER stress inhibitors significantly blocked GlcN-inhibited MyHC expression and myotube formation. It can be concluded that GlcN suppressed myogenic differentiation via a pathway that involved ER stress. Moreover, GlcN decreased myotube diameter and expression of MyHC, as well as increased MuRF-1 in C2C12 myotubes. Meanwhile, GlcN also reduced the expressions of phosphorylated Akt and mTOR were stimulated after GlcN treatment in C2C12 myotubes. Thus, GlcN induced skeletal muscle atrophy by inhibiting the protein synthesis pathway. Chronic GlcN infusion also caused skeletal muscle atrophy in mice. In conclusion, GlcN regulated important stages of skeletal muscle development through different signaling pathways., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The present study assessed whether single-leg daily blood flow restriction (BFR) treatment attenuates the decline in muscle fiber size, capillarization, and satellite cell (SC) content during 2 wk of bed rest in healthy, young men. Twelve healthy, young men (age: 24 ± 3 yr; BMI: 23.7 ± 3.1 kg/m 2 ) were subjected to 2 wk of bed rest, during which one leg was exposed to three times daily 5 min of BFR, whereas the contralateral leg received sham treatment [control (CON)]. Muscle biopsies were obtained from the m. vastus lateralis from both the BFR and CON legs before and immediately after 2 wk of bed rest. Types I and II muscle fiber size, myonuclear content, capillarization, and SC content were assessed by immunohistochemistry. No significant decline in either type I or type II muscle fiber size was observed following bed rest, with no differences between the CON and BFR legs ( P > 0.05). Type I muscle fiber capillary density increased in response to bed rest in both legs ( P < 0.05), whereas other muscle fiber capillarization measures remained unaltered. SC content decreased in both type I (from 7.4 ± 3.2 to 5.9 ± 2.7 per 100 fibers) and type II (from 7.2 ± 3.4 to 6.5 ± 3.2 per 100 fibers) muscle fibers (main effect of time P = 0.018), with no significant differences between the BFR and CON legs ( P > 0.05). In conclusion, 2 wk of bed rest has no effect on muscle capillarization and decreases the SC content, and daily BFR treatment does not affect skeletal muscle fiber size and SC content in healthy, young men. NEW & NOTEWORTHY We recently reported that the application of daily blood flow restriction (BFR) treatment does not preserve muscle mass or strength and does not modulate daily muscle protein synthesis rates during 2 wk of bed rest. Here, we show that 2 wk of bed rest resulted in a decrease in satellite cell (SC) content. In addition, the BFR treatment did not affect muscle fiber size, capillarization, and SC content during 2 wk of bed rest.

Defect in membrane repair contributes to the development of muscular dystrophies such as limb girdle muscular dystrophy (LGMD) type R2 or R12. Nevertheless, many other muscular dystrophies may also result from a defect in this process. Identifying these pathologies requires the development of specific methods to inflict sarcolemma damage on a large number of cells and rapidly analyze their response. We adapted a protocol hitherto used to study the behavior of cancer cells to mechanical constraint. This method is based on forcing the passage of cells through a thin needle, which induces shear stress. Due to size considerations, this method requires working with mononuclear muscle cells instead of myotubes or muscle fibers. Although functional sarcolemma repair was thought to be restricted to myotubes and muscle fibers, we show here that 24h-differentiated myoblasts express a complete machinery capable of addressing membrane damage. At this stage, muscle cells do not yet form myotubes, revealing that the membrane repair machinery is set up early throughout the differentiation process. When submitted to the shear-stress assay, these cells were observed to repair membrane damage in a Ca 2+ -dependent manner, as previously reported. We show that this technique is able to identify the absence of membrane resealing in muscle cells from patient suffering from LGMDR2. The proposed technique provides therefore a suitable method for identifying cellular dysregulations in membrane repair of dystrophic human muscle cells., (© 2024 The Author(s). Journal of Cellular Biochemistry published by Wiley Periodicals LLC.)

The lactate shuttle concept has revolutionized our understanding and study of metabolism in physiology, biochemistry, intermediary metabolism, nutrition, and medicine. Seminal findings of the mitochondrial lactate oxidation complex (mLOC) elucidated the architectural structure of its components. Here, we report that the mitochondrial pyruvate carrier (mPC) is an additional member of the mLOC in mouse muscle and C2C12 myoblasts and myotubes. Immunoblots, mass spectrometry, and co-immunoprecipitation experiments of mitochondrial preparations revealed abundant amounts of mitochondrial lactate dehydrogenase (mLDH), monocarboxylate transporter (mMCT), basigin (CD147), cytochrome oxidase (COx), and pyruvate carriers 1 and 2 (mPC1 and 2). In addition, using confocal laser scanning microscopy (CLSM) and in situ proximity ligation, we also demonstrated planar and three-dimensional (3-D) colocalization of pyruvate and lactate transporters with COx in fixed mouse skeletal muscle sections and C2C12 myoblasts and myotubes skeletal muscle sections, mouse muscle and C2C12 myoblasts and myotubes myotubes, and C2C12 myoblasts. This work serves as a landmark for configuring the final pathway of carbohydrate oxidation. NEW & NOTEWORTHY We expand on knowledge of the architectural design of the mitochondrial lactate oxidation complex (mLOC); key members are: mitochondrial lactate dehydrogenase (mLDH), monocarboxylate transporter 1 (mMCT1), cytochrome oxidase (COx), basigin scaffolding protein (CD147), and the mitochondrial pyruvate carrier (mPC). The mLOC is key in creating the lower end of the concentration gradient for disposal of lactate and pyruvate.

Aims: Redox signaling plays a key role in skeletal muscle remodeling induced by exercise and prolonged inactivity, but it is unclear which oxidant triggers myofiber hypertrophy due to the lack of strategies to precisely regulate individual oxidants in vivo . In this study, we used tetrathiomolybdate (TM) to dissociate the link between superoxide (O 2 •- ) and hydrogen peroxide and thereby to specifically explore the role of O 2 •- in muscle hypertrophy in C2C12 cells and mice. Results: TM can linearly regulate intracellular O 2 •- levels by inhibition of superoxide dismutase 1 (SOD1). A 70% increase in O 2 •- levels in C2C12 myoblast cells and mice is necessary and sufficient for triggering hypertrophy of differentiated myotubes and can enhance exercise performance by more than 50% in mice. SOD1 knockout blocks TM-induced O 2 •- increments and thereby prevents hypertrophy, whereas SOD1 restoration rescues all these effects. Scavenging O 2 •- with antioxidants abolishes TM-induced hypertrophy and the enhancement of exercise performance, whereas the restoration of O 2 •- levels with a O 2 •- generator promotes muscle hypertrophy independent of SOD1 activity. Innovation and Conclusion: These findings suggest that O 2 •- is an endogenous initiator of myofiber hypertrophy and that TM may be used to treat muscle wasting diseases. Our work not only suggests a novel druggable mechanism to increase muscle mass but also provides a tool for precisely regulating O 2 •- levels in vivo . Antioxid. Redox Signal. 42, 1-15.

Estrogen is an essential hormone for the development and functional activities of reproductive organs. Recent studies showed that estrogen signaling is also an important regulator of lipid and glucose metabolism in a number of tissues, but the molecular mechanism is not fully understood. We report here that estrogen is a stimulator of brain-derived neurotrophic factor (BDNF) synthesis in the skeletal muscle. Estradiol (E2), but not testosterone, induces a dose- and time-dependent BDNF production in cultured myotubes. Estrogen depletion in ovariectomized mice significantly reduced Bdnf expression in the glycolytic myofibers, which could be rescued after E2 administration. Mechanistically, E2 stimulation triggered the tethering of estrogen receptor (ER) α, but not ERβ, to the estrogen-responsive element on promoter VI of the Bdnf gene in skeletal muscle. When Bdnf production was inhibited by shRNA in C2C12 myotubes, E2-induced mitochondria activation and pyruvate dehydrogenase kinase 4 expressions were jeopardized. Collectively, our results demonstrate that BDNF is an underrecognized effector of estrogen in regulating mitochondrial activity and fuel metabolism in the skeletal muscle., (© 2024 Wiley Periodicals LLC.)

Muscle disuse has rapid and debilitating effects on muscle mass and overall health, making it an important issue from both scientific and clinical perspectives. However, the myocellular adaptations to muscle disuse are not yet fully understood, particularly those related to the myonuclear permanence hypothesis. Therefore, in this study, we assessed fiber size, number of myonuclei, satellite cells, and capillaries in human gastrocnemius muscle after a period of immobilization following an Achilles tendon rupture. Six physically active patients (5 males/1 female, 43 ± 15 yr) were recruited to participate after sustaining an acute unilateral Achilles tendon rupture. Muscle biopsies were obtained from the lateral part of the gastrocnemius before and after 6 wk of immobilization using a plaster cast and orthosis. Muscle fiber characteristics were analyzed in tissue cross-sections and isolated single fibers using immunofluorescence and high-resolution microscopy. Immobilization did not change muscle fiber type composition nor cross-sectional area of type I or type II fibers, but muscle fiber volume tended to decline by 13% ( P = 0.077). After immobilization, the volume per myonucleus was significantly reduced by 20% ( P = 0.008). Myonuclei were not lost in response to immobilization but tended to increase in single fibers and type II fibers. No significant changes were observed for satellite cells or capillaries. Myonuclei were not lost in the gastrocnemius muscle after a prolonged period of immobilization, which may provide support to the myonuclear permanence hypothesis in human muscle. Capillaries remained stable throughout the immobilization period, whereas the response was variable for satellite cells, particularly in type II fibers. NEW & NOTEWORTHY The impact of prolonged immobilization on muscle fiber characteristics is difficult to study in humans and therefore remains poorly understood. We analyzed cross-sections and single fibers from gastrocnemius before and after 6 wk of immobilization due to an Achilles tendon rupture. Our data suggest that myonuclei are not lost in response to such stimuli, thus lending support to the hypothesis of myonuclear permanency in human muscle.

Cancer cachexia affects up to 80% of patients with cancer and results in reduced quality of life and survival. We previously demonstrated that the transcriptional repressor Forkhead box P1 (FoxP1) is upregulated in the skeletal muscle of cachectic mice and people with cancer, and when overexpressed in skeletal muscle, it is sufficient to induce pathological features characteristic of cachexia. However, the role of myofiber-derived FoxP1 in both normal muscle physiology and cancer-induced muscle wasting remains largely unexplored. To address this gap, we generated a conditional mouse line with myofiber-specific ablation of FoxP1 (FoxP1 SkmKO ) and found that in cancer-free mice, deletion of FoxP1 in skeletal myofibers resulted in increased myofiber size in both males and females, with a significant increase in muscle mass in males. In response to murine KPC pancreatic tumor burden, we found that myofiber-derived FoxP1 mediates cancer-induced muscle wasting and diaphragm muscle weakness in male but not female mice. In summary, our findings identify myofiber-specific FoxP1 as a negative regulator of skeletal muscle with sex-specific differences in the context of cancer. NEW & NOTEWORTHY Here we identify myofiber-derived FoxP1 as a negative regulator of skeletal muscle with sex-specific effects in cancer. Under cancer-free conditions, FoxP1 knockout increased myofiber size in male and female mice. However, in response to pancreatic cancer, FoxP1 myofiber-specific deletion attenuated muscle wasting and weakness in males but not females. This highlights the need to consider sexual dimorphism in cancer-induced muscle pathologies and provides evidence suggesting that targeting FoxP1 could help mitigate these effects in males.

Maintaining healthy skeletal tissue is essential for overall well-being and quality of life. Skeletal muscle plays a key role in this process, yet models for studying its detailed function are limited. While RNA-binding motif protein 20 (RBM20) is primarily associated with dilated cardiomyopathy (DCM), its role in skeletal muscle remains largely unexplored. This study investigates RBM20 function in skeletal muscle using an RBM20 exon 9 deletion pig model (RBM20E9D). The deletion of exon 9 resulted in loosely arranged muscle fibers, large inter-fiber gaps, and irregular organization, leading to impaired muscle growth and development. Analysis of skeletal muscle satellite cells revealed significantly reduced proliferation, diminished myotube formation in vitro, and disrupted sarcomere structure due to exon 9 deletion. Given the critical role of satellite cell proliferation and differentiation in muscle repair, RBM20E9D pigs offer a novel model for studying the mechanisms underlying skeletal muscle injury, repair, and growth., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Obesity is associated with a series of skeletal muscle impairments and dysfunctions, which are characterized by metabolic disturbances and muscle atrophy. Luteolin is a phenolic phytochemical with broad pharmacological activities. The present study aimed to evaluate the protective effects of Luteolin on muscle function and explore the potential mechanisms in high-fat diet (HFD)-induced obese rats and palmitic acid (PA)-treated C2C12 myotubes. Male Sprague-Dawley (SD) rats were fed with a control diet or HFD and orally administrated 0.5% sodium carboxymethyl cellulose (vehicle) or Luteolin (25, 50, and 100 mg/kg, respectively) for 12 weeks. The results showed that Luteolin ameliorated HFD-induced body weight gain, glucose intolerance and hyperlipidemia. Luteolin also alleviated muscle atrophy, decreased ectopic lipid deposition and prompted muscle-fiber-type conversion in the skeletal muscle. Meanwhile, we observed an evident improvement in mitochondrial quality control and respiratory capacity, accompanied by reduced oxidative stress. Mechanistic studies indicated that AMPK/SIRT1/PGC-1α signaling pathway plays a key role in the protective effects of Luteolin on skeletal muscle in the obese states, which was further verified by using specific inhibitors of AMPK and SIRT1. Moreover, the mRNA expression levels of markers in brown adipocyte formation were significantly up-regulated post Luteolin supplementation in different adipose depots. Taken together, these results revealed that Luteolin supplementation might be a promising strategy to prevent obesity-induced loss of mass and biological dysfunctions of skeletal muscle., Competing Interests: Declaration of competing interests Y. Zhang, C. Luo, P. Huang, Y. Cheng, Y. Ma, J. Gao, H. Ding, no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Muscle and adipose tissue (AT) are in mutual interaction through the integration of endocrine and biochemical signals, thus regulating whole-body function and physiology. Besides a traditional view of endocrine relationships that imply the release of cytokines and growth factors, it is becoming increasingly clear that a metabolic network involving metabolites as signal molecules also exists between the two tissues. By elevating the number and functionality of mitochondria, a key role in muscle metabolism is played by the master regulator of mitochondrial biogenesis peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α), that induces a fiber type shift from glycolytic to oxidative myofibers. As a consequence, the upregulation of muscle respiratory rate might affect metabolite production and consumption. However, the underlying mechanisms have not yet been fully elucidated. Here, we used a muscle-specific PGC-1α overexpressing mouse model (MCK-PGC-1α) to analyze the metabolite secretion profile of serum and culture medium recovered from MCK-PGC-1α muscle fibers by NMR. We revealed modified levels of different metabolites that might be ascribed to the metabolic activation of the skeletal muscle fibers. Notably, the dysregulated levels of these metabolites affected adipocyte differentiation, as well as the browning process in vitro and in vivo. Interestingly such effect was exacerbated in the subcutaneous WAT, while only barely present in the visceral WAT. Our data confirm a prominent role of PGC-1α as a trigger of mitochondrial function in skeletal muscle and propose a novel function of this master regulator gene in modulating the metabolite production in turn affecting the activation of WAT and its conversion toward the browning., (© 2024 The Author(s). Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Purpose: to investigate the early consequences of type 1 diabetes (T1D) on the neural strategies of muscle force production., Methods: motor unit (MU) activity was recorded from the vastus lateralis muscle with High-Density surface Electromyography during isometric knee extension at 20 and 40% of maximum voluntary contraction (MVC) in 8 T1D (4 males, 4 females, 30.5 ± 3.6 years) and 8 matched control (4 males, 4 females, 27.3 ± 5.9 years) participants. Muscle biopsies were also collected from vastus lateralis for fiber type analysis, including myosin heavy chain (MyHC) isoform content via protein and mRNA expression., Results: MVC was comparable between groups as well as MU conduction velocity, action potentials' amplitude and proportions of MyHC protein isoforms. Nonetheless, MU discharge rate, relative derecruitment thresholds and mRNA expression of MyHC isoform I were lower in T1D., Conclusions: young people with uncomplicated T1D present a different neural control of muscle force production. Furthermore, differences are detectable non-invasively in absence of any functional manifestation (i.e., force production and fiber type distribution). These novel findings suggest that T1D has early consequences on the neuromuscular system and highlights the necessity of a better characterization of neural control in this population., Competing Interests: Declarations. Conflic of interest: None., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Eccentric contractions (ECC) induce excessive intracellular calcium ion (Ca 2+ ) accumulation and muscle structural damage in localized regions of the muscle fibers. In this investigation, we present the novel hypothesis that the ryanodine receptor (RyR) plays a central role in evoking a Ca 2+ dynamics profile that is markedly distinguishable from other muscle adaptive responses., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American College of Sports Medicine.)

The micronutrient vitamin C is essential for the maintenance of skeletal muscle health and homeostasis. The pro-myogenic effects of vitamin C have long been attributed to its role as a general antioxidant agent, as well as its role in collagen matrix synthesis and carnitine biosynthesis. Here, we show that vitamin C also functions as an epigenetic compound, facilitating chromatin landscape transitions during myogenesis through its activity as an enzymatic cofactor for histone H3 and DNA demethylation. Utilizing C2C12 myoblast cells to investigate the epigenetic effects of vitamin C on myogenesis, we observe that treatment of cells with vitamin C decreases global H3K9 methylation and increases 5-hmC levels. Furthermore, vitamin C treatment enhances myoblast marker gene expression and myotube formation during differentiation. We identify KDM7A as a histone lysine demethylase markedly upregulated during myogenesis. Accordingly, knockdown of Kdm7a prevents the pro-myogenic effects of vitamin C. Chromatin immunoprecipitation analysis showed that KDM7A occupies the promoter region of the myogenic transcription factor MyoD1 where it facilitates histone demethylation. We also confirm that the methylcytosine dioxygenases TET1 and TET2 are required for myogenic differentiation and that their loss blunts stimulation of myogenesis by vitamin C. In conclusion, our data suggest that an epigenetic mode of action plays a major role in the myogenic effects of vitamin C., (© 2024 Wiley Periodicals LLC.)

Engineering biointerfaces with nanoscale clustering of integrin-binding cell adhesive peptides is critical for promoting receptor redistribution into signaling complexes. Skeletal muscle cells are exquisitely sensitive to integrin-mediated signaling, yet biomaterials supporting myogenesis through control of the density and nanodistribution of ligands have not been developed. Here, materials are developed with tailorable cell adhesive ligands distribution at the interface by independently controlling their global and local density to enhance myogenesis, by promoting myoblast growth and myotube formation. To this end, RGD-functionalized low-fouling polymer surfaces with global ligand densities (G) from 0-7 µg peptide/mg polymer and average local ligand densities (L) from 1-6.3 ligands/cluster, are generated and characterized. Cell studies demonstrate improvements in cell adhesion, spreading, growth, and myotube formation up to a density of 7 µg peptide/mg polymer with 4 ligands/cluster. Optimizing ligand density and distribution also promotes early myofiber maturation, identified by increased MF20 marker protein expression and sarcomere-forming myotubes. At higher ligand densities, these cell properties are decreased, indicating that ligand multivalency is a critical parameter for tailoring cell-material interactions, to a certain threshold. The findings provide new insights for designing next-generation biomaterials and hold promise for improved engineering of skeletal muscle., (© 2024 Wiley‐VCH GmbH.)

Myogenic differentiation plays a fundamental role in myogenesis during development and in muscle regeneration. Sequential expression of myogenic regulatory factors (MRFs) including myogenin in the progenitor cells triggers the expression of effector proteins such as myosin heavy chain (MHC), leading to the terminal muscle differentiation. Although we have a snapshot-like understanding of molecules at each stage of the differentiation, how these molecules are interrelated in the continuum of myogenic differentiation remains poorly understood. In this study, we analyzed the dynamics of the minimal Myogenin promoter activity in live myoblasts. With the development of a new co-expression analysis method, we were able to reveal in detail the relationship between this Myogenin promoter activity and the expression of endogenous myogenin or MHC, as differentiation markers. Consequently, we found that our visualization system of myogenic differentiation is suitable for monitoring the transition from myoblasts to myotubes, in which the Myogenin promoter activity quantitatively represents the degree of myogenic differentiation. Thus, this system allows simultaneous observation of the degree of myoblast differentiation in relation to other molecules, which would contribute to deepening our understanding of myogenic differentiation as a continuous process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Lumbar disc herniation (LDH) is a common degenerative condition causing low back pain (LBP) due to nerve compression. Previous studies show conflicting findings regarding the multifidus (MF) muscle's microscopic changes in LDH patients. So, this study aimed to compare the affected MF to the adjacent MF on the ipsilateral and contralateral sides in LDH patients and examined correlations with clinical features of LBP. Four muscle biopsies were collected from each of 30 surgical participants. Immunohistochemistry was performed on tissue sections and imaged with an epifluorescence microscope. Data was analysed using a two-way ANOVA for muscle fibre cross-sectional area, perimeter, diameter, and composition, while pathological fibres were analysed using a one-way ANOVA. Pearson's correlation was employed to examine MF microscopy associations with clinical features. Results revealed no significant differences between the affected MF and MF from other sites, though significantly more pathological fibres were present in the affected MF (p < 0.05). A weak but significant negative correlation was found between type I fibres and LBP clinical features, though no such correlations were observed for type IIA fibres. In conclusion, LDH primarily impacts the pathological status of the MF rather than fibre phenotype or size, and severity of clinical features is associated with the size of type I fibres., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The fiber type transition of skeletal muscle is an intricate and essential physiological process in the body, significantly influencing both the function and metabolism of skeletal muscle. This phenomenon is not only affected by external environmental changes but also intricately regulated by internal physiological mechanisms. Therefore, exploring the physiological process of muscle fiber type transition holds considerable significance for the treatment of human neuromuscular disorders and the improvement of meat quality in livestock and poultry. It has been discovered that the cytokines secreted by skeletal muscle, i . e ., myokines, play a role in the fiber type transition of skeletal muscle. Myokines mainly act on skeletal muscle in autocrine and paracrine forms to participate in signal transduction and regulate the fiber type transition of skeletal muscle. This paper reviews the functional differences among various muscle fiber types, expounds the effects and mechanisms of myokines in regulating the transition processes of these fiber types, and prospects the future research directions in this field. This review is expected to provide theoretical support for enhancing the meat quality of livestock and poultry and treating skeletal muscle-related diseases.

Injured or atrophied adult skeletal muscles are regenerated through terminal differentiation of satellite cells to form multinucleated muscle fibers. Transplantation of satellite cells or cultured myoblasts has been used to improve skeletal muscle regeneration. Some of the limitations observed result from the limited number of available satellite cells that can be harvested and the efficiency of fusion of cultured myoblasts with mature muscle fibers (i.e., terminal differentiation) upon transplantation. However, the possible use of immature myotubes in the middle of the terminal differentiation process instead of satellite cells or cultured myoblasts has not been thoroughly investigated. Herein, myoblasts (Mb) or immature myotubes on differentiation day 2 (D2 immature myotubes) or 3 (D3 immature myotubes) were transferred to plates containing D2 or D3 immature myotubes as host cells. The transferred Mb/immature myotubes on the plates were further co-differentiated with host immature myotubes into mature myotubes in six conditions: Mb-to-D2, D2-to-D2, D3-to-D2, Mb-to-D3, D2-to-D3, and D3-to-D3. Among these six co-differentiation conditions, the D2-to-D3 co-differentiation condition exhibited the most characteristic myotube appearance and the greatest availability of Ca 2+ for skeletal muscle contraction. Compared with non-co-differentiated control myotubes, D2-to-D3 co-differentiated myotubes presented increased MyoD and myosin heavy chain II (MyHC II) expression and increased myotube width, accompanied by parallel and swirling alignment. These increases correlated with functional increases in both electrically induced intracellular Ca 2+ release and extracellular Ca 2+ entry due to the increased expression of ryanodine receptor 1 (RyR1), sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 1a (SERCA1a), and stromal interaction molecule 1 (STIM1). These increases were not detected in any of the other co-differentiation conditions. These results suggest that in vitro-cultured D2-to-D3 co-differentiated mature myotubes could be a good alternative source of satellite cells or cultured myoblasts for skeletal muscle regeneration.

Background: Muscle stem cells (MuSCs) undergo numerous state transitions throughout life, which are critical for supporting normal muscle growth and regeneration. Epigenetic modifications in skeletal muscle play a significant role in influencing the niche and cellular states of MuSCs. Mixed-lineage leukemia 4 (Mll4) is a histone methyltransferase critical for activating the transcription of various target genes and is highly expressed in skeletal muscle. This raises the question of whether Mll4 has a regulatory function in modulating the state transitions of MuSCs, warranting further investigation., Methods: To assess if myofiber-expressed Mll4, a histone methyltransferase, contributes to the maintenance of MuSCs, we crossed MCK Cre/+ or HSA MerCreMer/+ mice to Mll4 f/f mice to generate myofiber-specific Mll4-deleted mice. Investigations were conducted using 8-week-old and 4-week-old MCK Cre/+ ;Mll4 f/f mice, and adult HSA MerCreMer/+ ;Mll4 f/f mice between the ages of 3 months and 6 months., Results: During postnatal myogenesis, Mll4 deleted muscles were observed with increased number of cycling MuSCs that proceeded to a differentiation state, leading to MuSC deprivation. This phenomenon occurred independently of gender. When Mll4 was ablated in adult muscles using the inducible method, adult MuSCs lost their quiescence and differentiated into myoblasts, also causing the depletion of MuSCs. Such roles of Mll4 in myofibers coincided with decreased expression levels of distinct Notch ligands: Jag1 and Dll1 in pubertal and Jag2 and Dll4 in adult muscles., Conclusions: Our study suggests that Mll4 is crucial for maintaining MuSCs in both pubertal and adult muscles, which may be accomplished through the modulation of distinct Notch ligand expressions in myofibers. These findings offer new insights into the role of myofiber-expressed Mll4 as a master regulator of MuSCs, highlighting its significance not only in developmental myogenesis but also in adult muscle, irrespective of sex., Competing Interests: Declarations. Ethics approval and consent to participate: The care and treatment of animals in this study were approved by the Institutional Animal Care and Use Committee (IACUC) protocols (SNU-240103-3) of Seoul National University. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

We outline a protocol to visualize all mouse lower hindlimb skeletal muscles simultaneously. We describe procedures for orientating the whole lower hindlimb in gum tragacanth prior to freezing, simplifying the proceeding experimental steps, and enhancing the comprehensiveness of characterizations. We then detail steps for quantifying muscle fiber size and fiber type characteristics in a single cryosection using histochemistry and immunofluorescence. This protocol can be applied to histological and (immuno)histochemical evaluations such as muscle regeneration, fibrosis, enzymatic activity, and glycogen content., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Insulin resistance and type 2 diabetes impair cellular regeneration in multiple tissues including skeletal muscle. The molecular basis for this impairment is largely unknown. Glucose uptake via glucose transporter GLUT4 is impaired in insulin resistance. In healthy muscle, acute injury stimulates glucose uptake. Whether decreased glucose uptake via GLUT4 impairs muscle regeneration is presently unknown. The goal of this study was to determine whether GLUT4 regulates muscle glucose uptake and/or regeneration following acute injury., Methods: Tibialis anterior and extensor digitorum longus muscles from wild-type, control, or muscle-specific GLUT4 knockout (mG4KO) mice were injected with the myotoxin barium chloride to induce muscle injury. After 3, 5, 7, 10, 14, or 21 days (in wild-type mice), or after 7 or 14 days (in control & mG4KO) mice, muscles were isolated to examine [ 3 H]-2-deoxyglucose uptake, GLUT4 levels, extracellular fluid space, fibrosis, myofiber cross-sectional area, and myofiber centralized nuclei., Results: In wild-type mice, muscle glucose uptake was increased 3, 5, 7, and 10 days post-injury. There was a rapid decrease in GLUT4 protein levels that were restored to baseline at 5-7 days post-injury, followed by a super-compensation at 10-21 days. In mG4KO mice, there were no differences in muscle glucose uptake, extracellular fluid space, muscle fibrosis, myofiber cross-sectional areas, or percentage of centrally nucleated myofibers at 7 days post-injury. In contrast, at 14 days injured muscles from mG4KO mice exhibited decreased glucose uptake, muscle weight, myofiber cross sectional areas, and centrally nucleated myofibers, with no change in extracellular fluid space or fibrosis., Conclusions: Collectively, these findings demonstrate that glucose uptake via GLUT4 regulates skeletal myofiber regeneration following acute injury., Competing Interests: Declarations. Ethical approval: Procedures were performed in accordance with the Indiana University School of Medicine Institutional Animal Care and Use Committee (IACUC, protocol# 21053 approved on 17 May 2021 and protocol# 21118 approved on 31 August 2021), and the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Skeletal muscle cells (myofibers) require multiple nuclei to support a cytoplasmic volume that is larger than other mononuclear cell types. It is dogmatic that mammalian resident myonuclei rely on stem cells (specifically satellite cells) for adding new DNA to muscle fibers to facilitate cytoplasmic expansion that occurs during muscle growth. In this review, we discuss the relationship between cell size and supporting genetic material. We present evidence that myonuclei may undergo DNA synthesis as a strategy to increase genetic material in myofibers independent from satellite cells. We then describe the details of our experiments that demonstrated that mammalian myonuclei can replicate DNA in vivo. Finally, we present our findings in the context of expanding knowledge about myonuclear heterogeneity, myonuclear mobility and shape. We also address why myonuclear replication is potentially important and provide future directions for remaining unknowns. Myonuclear DNA replication, coupled with new discoveries about myonuclear transcription, morphology, and behavior in response to stress, may provide opportunities to leverage previously unappreciated skeletal muscle biological processes for therapeutic targets that support muscle mass, function, and plasticity., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: The increased degradation of the insulin receptor β subunit (InsRβ) in lysosomes contributes to the development of insulin resistance and type 2 diabetes mellitus. Endoplasmic reticulum (ER) stress contributes to insulin resistance through several mechanisms, including the reduction of InsRβ levels. Here, we examined how peroxisome proliferator-activated receptor (PPAR)β/δ regulates InsRβ levels in mouse skeletal muscle and C2C12 myotubes exposed to the ER stressor tunicamycin., Methods: Wild-type (WT) and Ppard -/- mice, WT mice treated with vehicle or the PPARβ/δ agonist GW501516, and C2C12 myotubes treated with the ER stressor tunicamycin or different activators or inhibitors were used., Results: Ppard -/- mice displayed reduced InsRβ protein levels in their skeletal muscle compared to wild-type (WT) mice, while the PPARβ/δ agonist GW501516 increased its levels in WT mice. Co-incubation of tunicamycin-exposed C2C12 myotubes with GW501516 partially reversed the decrease in InsRβ protein levels, attenuating both ER stress and the increase in lysosomal activity. In addition, the protein levels of the tyrosine kinase ephrin receptor B4 (EphB4), which binds to the InsRβ and facilitates its endocytosis and degradation in lysosomes, were increased in the skeletal muscle of Ppard -/- mice, with GW501516 reducing its levels in the skeletal muscle of WT mice., Conclusions: Overall, these findings reveal that PPARβ/δ activation increases InsRβ levels by alleviating ER stress and lysosomal degradation., Competing Interests: Declarations. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The conserved Runt-related (RUNX) transcription factor family are master regulators of developmental and regenerative processes. Runx1 and Runx2 are expressed in satellite cells (SCs) and in skeletal myotubes. Here, we examined the role of Runx1 in mouse satellite cells to determine the role of Runx1 during muscle differentiation. Conditional deletion of Runx1 in adult SCs negatively impacted self-renewal and impaired skeletal muscle maintenance even though Runx2 expression persisted. Runx1 deletion in C2C12 cells (which retain Runx2 expression) identified unique molecular functions of Runx1 that could not be compensated for by Runx2. The reduced myoblast fusion in vitro caused by Runx1 loss was due in part to ectopic expression of Mef2c, a target repressed by Runx1. Structure-function analysis demonstrated that the ETS-interacting MID/EID region of Runx1, absent from Runx2, is essential for Runx1 myoblast function and for Etv4 binding. Analysis of ChIP-seq datasets from Runx1 (T cells, muscle)- versus Runx2 (preosteoblasts)-dependent tissues identified a composite ETS:RUNX motif enriched in Runx1-dependent tissues. The ETS:RUNX composite motif was enriched in peaks open exclusively in ATAC-seq datasets from wild-type cells compared to ATAC peaks unique to Runx1 knockout cells. Thus, engagement of a set of targets by the RUNX1/ETS complex define the non-redundant functions of Runx1 in mouse muscle precursor cells., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)