Mature Muscle Cells Research Articles

Human induced pluripotent stem cell models for the study and treatment of Duchenne and Becker muscular dystrophies.

Duchenne and Becker muscular dystrophies are the most common muscle diseases and are both currently incurable. They are caused by mutations in the dystrophin gene, which lead to the absence or reduction/truncation of the encoded protein, with progressive muscle degeneration that clinically manifests in muscle weakness, cardiac and respiratory involvement and early death. The limits of animal models to exactly reproduce human muscle disease and to predict clinically relevant treatment effects has prompted the development of more accurate in vitro skeletal muscle models. However, the challenge of effectively obtaining mature skeletal muscle cells or satellite stem cells as primary cultures has hampered the development of in vitro models. Here, we discuss the recently developed technologies that enable the differentiation of skeletal muscle from human induced pluripotent stem cells (iPSCs) of Duchenne and Becker patients. These systems recapitulate key disease features including inflammation and scarce regenerative myogenic capacity that are partially rescued by genetic and pharmacological therapies and can provide a useful platform to study and realize future therapeutic treatments. Implementation of this model also takes advantage of the developing genome editing field, which is a promising approach not only for correcting dystrophin, but also for modulating the underlying mechanisms of skeletal muscle development, regeneration and disease. These data prove the possibility of creating an accurate Duchenne and Becker in vitro model starting from iPSCs, to be used for pathogenetic studies and for drug screening to identify strategies capable of stopping or reversing muscular dystrophinopathies and other muscle diseases.

Altered Integrins Expression of Patients Affected by Cryptorchidism

Objectives: The study aimed to investigate the expression of the integrin isoforms α7A and β1A, expressed by myogenic precursor cells, and α7B and β1D, expressed by mature muscle cells in the cremaster of patients affected by an undescended testis. Methods: Fifteen samples of cremaster were obtained from patients undergoing surgery for an undescended testis. Thirty control specimens of cremaster were harvested from patients with congenital hydrocele or inguinal hernia. Immunofluorescent analysis was carried out using anti-α7A, β1A, α7B, and β1D integrin antibodies. Sections were observed using confocal laser scanning microscopy. Results: As compared with controls, a significant loss of a α7B (p = 0.0355) and β1D (p = 0.0069) integrins and a higher expression of α7A (p = 0.0003) and β1A (p = 0.0150) was detected in the cremaster of patients affected by an undescended testis. Conclusions: Our data document a critical alteration of the cytoskeleton of cremasteric smooth muscle cells in patients with an undescended testis. This might explain the altered function in smooth muscle cells in cremaster implied during testicular descent. We therefore speculate that the postnatal splicing of α7A to α7B and of β1A to β1D integrins is delayed. This could account for the common clinical scenario of spontaneous descent of the testes in the first months of life.

Supplementation with IL-6 and Muscle Cell Culture Conditioned Media Enhances Myogenic Differentiation of Adipose Tissue-Derived Stem Cells through STAT3 Activation.

Mature skeletal muscle cells cannot be expanded in culture systems. Therefore, it is difficult to construct an in vitro model for muscle diseases. To establish an efficient protocol for myogenic differentiation of human adipose tissue-derived stem cells (hADSCs), we investigated whether addition of IL-6 and/or myocyte-conditioned media (CM) to conventional differentiation media can shorten the differentiation period. hADSCs were differentiated to myocytes using the conventional protocol or modified with the addition of 25 pg/mL IL-6 and/or C2C12 CM (25% v/v). The expression of MyoD and myogenine mRNA was significantly higher at 5–6 days after differentiation using the modified protocol than with the conventional protocol. mRNA and protein expression of myosin heavy chain, a marker of myotubes, was significantly upregulated at 28 and 42 days of differentiation using the modified protocol, and the level achieved after a 4-week differentiation period was similar to that achieved at 6 weeks using the conventional protocol. The expression of p-STAT3 was significantly increased when the modified protocol was used. Similarly, addition of colivelin, a STAT3 activator, instead of IL-6 and C2C12 CM, promoted the myogenic differentiation of ADSCs. The modified protocol improved differentiation efficiency and reduced the time required for differentiation of myocytes. It might be helpful to save cost and time when preparing myocytes for cell therapies and drug discovery.

Senescence in Myoblasts; Role in Age‐Related Muscle Dysfunction

Loss of skeletal muscle mass and function are critical factors in the development of frailty although the mechanisms by which this loss occurs are poorly understood. The number of senescent cells increases with age in rodents (Krishnamurthy et al., 2004) and primates (Kreiling et al., 2011) and it is proposed that the presence of these cells plays a role in age‐related muscle dysfunction. We hypothesise that resident satellite cells become senescent and exert detrimental effects on mature muscle fibres via the senescence associated secretory phenotype molecules (SASP).Initial studies have investigated whether a C2C12 mouse muscle myoblast cell line could be induced into senescence, whether key senescence associated molecules (such as p16, p21, and p53) are upregulated and determined the nature of the SASP.C2C12 myoblasts were treated for 24 hours with the topoisomerase II inhibitor, Etoposide, to cause DNA damage. Myoblasts were examined for up to 5 days following treatment for cell viability, Senescence Associated β‐Galactosidase (SAβ‐Gal) activity, immunocytochemistry and western blot analysis for p16, p21, and p53.Treated myoblasts exhibited a significantly altered morphology consistent with a senescence phenotype, increased SAβ‐Gal activity, and cyclin‐dependent kinase inhibitor levels compared with untreated cells.These data suggest that myoblasts can be induced into a senescent phenotype. Further work will characterise the SASP of the cells and determine the effect on mature myotubes co‐cultured with senescent myoblasts.Support or Funding InformationWork generously funded by the British Society for Research into Ageing and the Rosetrees Trust.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recapitulating early development of mouse musculoskeletal precursors of the paraxial mesoderm in vitro.

Body skeletal muscles derive from the paraxial mesoderm, which forms in the posterior region of the embryo. Using microarrays, we characterize novel mouse presomitic mesoderm (PSM) markers and show that, unlike the abrupt transcriptome reorganization of the PSM, neural tube differentiation is accompanied by progressive transcriptome changes. The early paraxial mesoderm differentiation stages can be efficiently recapitulated in vitro using mouse and human pluripotent stem cells. While Wnt activation alone can induce posterior PSM markers, acquisition of a committed PSM fate and efficient differentiation into anterior PSM Pax3+ identity further requires BMP inhibition to prevent progenitors from drifting to a lateral plate mesoderm fate. When transplanted into injured adult muscle, these precursors generated large numbers of immature muscle fibers. Furthermore, exposing these mouse PSM-like cells to a brief FGF inhibition step followed by culture in horse serum-containing medium allows efficient recapitulation of the myogenic program to generate myotubes and associated Pax7+ cells. This protocol results in improved in vitro differentiation and maturation of mouse muscle fibers over serum-free protocols and enables the study of myogenic cell fusion and satellite cell differentiation.

Protein arginine methyltransferase expression and activity during myogenesis.

Despite the emerging importance of protein arginine methyltransferases (PRMTs) in regulating skeletal muscle plasticity, PRMT biology during muscle development is complex and not completely understood. Therefore, our purpose was to investigate PRMT1, -4, and -5 expression and function in skeletal muscle cells during the phenotypic remodeling elicited by myogenesis. C2C12 muscle cell maturation, assessed during the myoblast (MB) stage, and during days 1, 3, 5, and 7 of differentiation, was employed as an in vitro model of myogenesis. We observed PRMT-specific patterns of expression and activity during myogenesis. PRMT4 and -5 gene expression was unchanged, while PRMT1 mRNA and protein content were significantly induced. Cellular monomethylarginines (MMAs) and symmetric dimethylarginines (SDMAs), indicative of global and type II PRMT activities, respectively, remained steady during development, while type I PRMT activity indicator asymmetric dimethylarginines (ADMAs) increased through myogenesis. Histone 4 arginine 3 (H4R3) and H3R17 contents were elevated coincident with the myonuclear accumulation of PRMT1 and -4. Collectively, this suggests that PRMTs are methyl donors throughout myogenesis and demonstrate specificity for their protein targets. Cells were then treated with TC-E 5003 (TC-E), a selective inhibitor of PRMT1 in order to specifically examine the enzymes role during myogenic differentiation. TC-E treated cells exhibited decrements in muscle differentiation, which were consistent with attenuated mitochondrial biogenesis and respiratory function. In summary, the present study increases our understanding of PRMT1, -4, and -5 biology during the plasticity of skeletal muscle development. Our results provide evidence for a role of PRMT1, via a mitochondrially mediated mechanism, in driving the muscle differentiation program.

Vascular Smooth Muscle Cell Maturation Stage and Ki-67 Index are Diagnostic Biomarkers for Pathologic Grade of Ovarian Teratoma.

Although immature teratoma of the ovary is a rare disease, its pathologic grading, especially between low-grade (grade 1) and high-grade (grade 2 or 3) immature teratomas, is important for optimal therapy and prognosis. This grading, however, is currently solely dependent on quantitation of neuroepithelial components as judged by subjective assessments. As we have recently successfully studied the maturation of vascular smooth muscle cells (SMCs) in other organs using an h-caldesmon to α-smooth muscle actin (α-SMA) ratio, we decided to use this ratio to investigate a potential link between teratoma grade and SMC maturation, in combination with Ki-67 index. Sixteen immature teratomas along with 5 mature teratomas of the ovary were studied and stained with antibodies to CD31, α-SMA, h-caldesmon, and Ki-67. The number of vascular SMCs calculated using the α-SMA/CD31 ratio did not differ between teratoma grades (except between grade 0 and 3), whereas the number of mature vascular SMCs calculated using the h-caldesmon/CD31 ratio and maturation state calculated using the h-caldesmon/α-SMA ratio reduced significantly as teratoma grade progressed from 0 to 3. Furthermore, these parameters were significantly lower in high-grade than in low-grade immature teratomas (P<0.05). Ki-67 labeling index, regardless of germ cell layer, also significantly increased with teratoma grade (P<0.05). These results suggest that not only the number of neuroepithelial elements but also vascular immaturity and proliferating cell counts are biomarkers for ovarian teratoma grading. Thus, assessment of the maturity of vascular SMCs may serve as a valuable diagnostic tool for assessing teratoma maturity.

Identification and functional characterization of muscle satellite cells in Drosophila.

Work on genetic model systems such as Drosophila and mouse has shown that the fundamental mechanisms of myogenesis are remarkably similar in vertebrates and invertebrates. Strikingly, however, satellite cells, the adult muscle stem cells that are essential for the regeneration of damaged muscles in vertebrates, have not been reported in invertebrates. In this study, we show that lineal descendants of muscle stem cells are present in adult muscle of Drosophila as small, unfused cells observed at the surface and in close proximity to the mature muscle fibers. Normally quiescent, following muscle fiber injury, we show that these cells express Zfh1 and engage in Notch-Delta-dependent proliferative activity and generate lineal descendant populations, which fuse with the injured muscle fiber. In view of strikingly similar morphological and functional features, we consider these novel cells to be the Drosophila equivalent of vertebrate muscle satellite cells.

NI.O.23 - Sh3kbp1 involvement during skeletal muscle fibers formation: a new candidate for centronuclear myopathies

Centronuclear myopathies (CNM) are a group of congenital myopathies characterized by skeletal muscle weakness, fatigability and atrophy. In affected muscles, myonuclei are abnormally located at the center of mature muscle fibers since they never migrate to the cell periphery as in healthy muscles. Initially considered as a consequence of the pathology, myonuclear mispositioning has recently emerged as one of the possible causes of muscle functionality defects observed in CNM. So far, the entire molecular machinery responsible for myonuclear positioning during myofibers formation is not known but recent clues involve cytoskeleton proteins. To discover new actors of myonuclear positioning, we performed a siRNA screen on potential cytoskeleton regulators and identified Sh3kbp1 as a new key regulator protein. Sh3kbp1 is ubiquitously expressed, but is up regulated upon muscle differentiation in vitro and specifically reactivated during in vivo muscle regeneration in mice. In in vitro conditions (C2C12 and primary muscle cells) and in mouse muscle in vivo (Tibialis anterior), Sh3kbp1 accumulates progressively both around myonuclei and at triads. Sh3kbp1 knockdown using siRNA in primary myoblasts or stable shRNA C2C12 cells reveals that the protein is not essential for the activation of myogenesis, but is required for normal myoblasts fusion and muscle fibers maturation. Indeed Sh3kbp1 downregulation increases fusion capacity and induces defects in T-tubules establishment and myonuclear positioning, which are typical features of CNM. Additionally, we demonstrated that Sh3kbp1 is interacting with Dynamin2 and is upregulated in a Dynamin2 mouse model of CNM. Further experiments are currently on-going to elucidate the involvement of the two partners in the CNM phenotype. Altogether our results show that Sh3kbp1 is a new key regulator of both T-tubules maturation and myonuclear positioning, and identify Sh3kbp1 as a central player to better understand CNM physiopathology.

Use of a neonatal rat system as a bioincubator to generate adult-like mature cardiomyocytes from human and mouse pluripotent stem cells.

Pluripotent stem cells (PSCs), including induced PSCs, hold great potential for personalized disease modeling, drug testing and cell-based therapeutics. However, cells differentiated from PSCs remain immature in a dish, and thus there are serious caveats to their use in modeling adult-onset diseases such as cardiomyopathies and Alzheimer's disease. By taking advantage of knowledge gained about mammalian development and from bioinformatics analyses, we recently developed a neonatal rat system that enables maturation of PSC-derived cardiomyocytes into cardiomyocytes analogous to those seen in adult animals. Here we describe a detailed protocol that describes how to initiate the in vitro differentiation of mouse and human PSCs into cardiac progenitor cells, followed by intramyocardial delivery of the progenitor cells into neonatal rat hearts, in vivo incubation and analysis. The entire process takes ∼6 weeks, and the resulting cardiomyocytes can be analyzed for morphology, function and gene expression. The neonatal system provides a valuable tool for understanding the maturation and pathogenesis of adult human heart muscle cells, and this concept may be expanded to maturing other PSC-derived cell types, including those containing mutations that lead to the development of diseases in the adult.

Tissue-specific extracellular matrix promotes myogenic differentiation of human muscle progenitor cells on gelatin and heparin conjugated alginate hydrogels.

Alginate based biomaterials are commonly used in tissue engineering and regenerative medicine field, however, the inefficient sequestration of growth factors restricted its utilization. In this study, a novel alginate based substrates was produced covalently modified with gelatin and heparin, in order to capture more effective cytokines and proteins in the culture milieu, keep homeostasis for cell survival and tissue regeneration with growth factor sequestration and long-term release capacities. Combining with skeletal muscle derived ECM, the modified Alginate-Gelatin-Heparin gel could most effectively mimic the tissue specific microenvironment to support skeletal muscle progenitor cells proliferation, differentiation and myotube formation. Additionally, the economical and practical features will make it more promising in high-throughput application for regenerative medicine research.

Direct reprogramming of fibroblasts into skeletal muscle progenitor cells by transcription factors enriched in undifferentiated subpopulation of satellite cells

Satellite cells comprise a functionally heterogeneous population of stem cells in skeletal muscle. Separation of an undifferentiated subpopulation and elucidation of its molecular background are necessary to identify the reprogramming factors to induce skeletal muscle progenitor cells. In this study, we found that intracellular esterase activity distinguishes a subpopulation of cultured satellite cells with high stemness using esterase-sensitive cell staining reagent, calcein-AM. Gene expression analysis of this subpopulation revealed that defined combinations of transcription factors (Pax3, Mef2b, and Pitx1 or Pax7, Mef2b, and Pitx1 in embryonic fibroblasts, and Pax7, Mef2b and MyoD in adult fibroblasts) reprogrammed fibroblasts into skeletal muscle progenitor cells. These reprogrammed cells formed Dystrophin-positive mature muscle fibers when transplanted into a mouse model of Duchenne muscular dystrophy. These results highlight the new marker for heterogenous population of cultured satellite cells, potential therapeutic approaches and cell sources for degenerative muscle diseases.

Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells

Dynamin-2 is a ubiquitously expressed GTP-ase that mediates membrane remodeling. Recent findings indicate that dynamin-2 also regulates actin dynamics. Mutations in dynamin-2 cause dominant centronuclear myopathy (CNM), a congenital myopathy characterized by progressive weakness and atrophy of skeletal muscles. However, the muscle-specific roles of dynamin-2 affected by these mutations remain elusive. Here we show that, in muscle cells, the GTP-ase activity of dynamin-2 is involved in de novo actin polymerization as well as in actin-mediated trafficking of the glucose transporter GLUT4. Expression of dynamin-2 constructs carrying CNM-linked mutations disrupted the formation of new actin filaments as well as the stimulus-induced translocation of GLUT4 to the plasma membrane. Similarly, mature muscle fibers isolated from heterozygous knock-in mice that harbor the dynamin-2 mutation p.R465W, an animal model of CNM, exhibited altered actin organization, reduced actin polymerization and impaired insulin-induced translocation of GLUT4 to the sarcolemma. Moreover, GLUT4 displayed aberrant perinuclear accumulation in biopsies from CNM patients carrying dynamin-2 mutations, further suggesting trafficking defects. These results suggest that dynamin-2 is a key regulator of actin dynamics and GLUT4 trafficking in muscle cells. Our findings also support a model in which impairment of actin-dependent trafficking contributes to the pathological mechanism in dynamin-2-associated CNM.

Polyurethane acrylates as effective substrates for sustained in vitro culture of human myotubes.

We developed polyurethane acrylates (PUA) to modulate the human skeletal muscle cell growth and maturation in vitro by controlling surface chemistry, morphology and tuning material's stiffness. PUA was able to maintain muscle cell viability for over a month without any detectable signs of material degradation. The best performing PUA prevented premature cell detachment from the substrate which often hampered long-term muscle cell studies. It also supported muscle cell maturation up to the late stages of differentiation. The significance of these findings lies in the possibility to advance studies on muscle cell biology, disease and therapy by using human muscle cells instead of relying on the widely used animal-based in vitro models.

TEAD transcription factors are required for normal primary myoblast differentiation in vitro and muscle regeneration in vivo.

The TEAD family of transcription factors (TEAD1-4) bind the MCAT element in the regulatory elements of both growth promoting and myogenic differentiation genes. Defining TEAD transcription factor function in myogenesis has proved elusive due to overlapping expression of family members and their functional redundancy. We show that silencing of either Tead1, Tead2 or Tead4 did not effect primary myoblast (PM) differentiation, but that their simultaneous knockdown strongly impaired differentiation. In contrast, Tead1 or Tead4 silencing impaired C2C12 differentiation showing their different contributions in PMs and C2C12 cells. Chromatin immunoprecipitation identified enhancers associated with myogenic genes bound by combinations of Tead4, Myod1 or Myog. Tead4 regulated distinct gene sets in C2C12 cells and PMs involving both activation of the myogenic program and repression of growth and signaling pathways. ChIP-seq from mature mouse muscle fibres in vivo identified a set of highly transcribed muscle cell-identity genes and sites bound by Tead1 and Tead4. Although inactivation of Tead4 in mature muscle fibres caused no obvious phenotype under normal conditions, notexin-induced muscle regeneration was delayed in Tead4 mutants suggesting an important role in myogenic differentiation in vivo. By combining knockdown in cell models in vitro with Tead4 inactivation in muscle in vivo, we provide the first comprehensive description of the specific and redundant roles of Tead factors in myogenic differentiation.

Myogenic Maturation by Optical-Training in Cultured Skeletal Muscle Cells.

Optogenetic techniques are powerful tools for manipulating biological processes in identified cells using light under high temporal and spatial resolutions. Here, we describe an optogenetic training strategy to promote morphological maturation and functional development of skeletal muscle cells in vitro. Optical stimulation with a rhythmical frequency facilitates specific structural alignment of sarcomeric proteins. Optical stimulation also depolarizes the membrane potential, and induces contractile responses in synchrony with the given pattern of light pulses. These results suggest that optogenetic techniques can be employed to manipulate activity-dependent processes during myogenic development and control contraction of photosensitive skeletal muscle cells with high temporal and special precision.

Isolation, in-vitro expansion, and characterization of human muscle satellite cells from the rectus abdominis muscle

Introduction: Satellite cells are a resident population of stem cells beneath the basal lamina of mature skeletal muscle fibers. Their capacity to regenerate muscle makes them a potentially ideal source for human cell therapy with respect to muscle-related diseases such as urinary and fecal incontinence, and others. In this study, we describe a protocol to isolate, expand in-vitro, and characterize human muscle satellite cells from the rectus abdominis muscle. Materials and Methods: Muscle biopsies from human donors were harvested, digested using collagenase type II, and then plated on extracellular matrix-coated plates. Results: Immunocytochemistry revealed that satellite cells on day 8 were 70–80% Pax7 positive; in contrast, cells expanded until day 12 showed 50–75% positivity for Pax7. The real-time polymerase chain reaction for day 8 culture indicated four-fold increase in Pax3 and Pax7 gene expression, four-fold increase in MyoD gene expression, and five-fold increase in Myf5 gene expression. Conclusion: These findings suggest that satellite cells can be cultured until day 8 for translational purposes. The protocol described here is modest, operational, and reproducible and involves only basic cell culture equipment.

Human Satellite Cell Isolation and Xenotransplantation.

Satellite cells are mononucleated cells of the skeletal muscle lineage that exist beneath the basal lamina juxtaposed to the sarcolemma of skeletal muscle fibers. It is widely accepted that satellite cells mediate skeletal muscle regeneration. Within the satellite cell pool of adult muscle are skeletal muscle stem cells (MuSCs), also called satellite stem cells, which fulfill criteria of tissue stem cells: They proliferate and their progeny either occupies the adult MuSC niche during self-renewal or differentiates to regenerate mature muscle fibers. Here, we describe robust methods for the isolation of enriched populations of human satellite cells containing MuSCs from fresh human muscle, utilizing mechanical and enzymatic dissociation and purification by fluorescence-activated cell sorting. We also describe a process for xenotransplantation of human satellite cells into mouse muscle by injection into irradiated, immunodeficient, mouse leg muscle with concurrent notexin or bupivacaine muscle injury to increase engraftment efficiency. The engraftment of human MuSCs and the formation of human muscle can then be analyzed by histological and immunofluorescence staining, or subjected to in vivo experimentation.

Isolation, Culture, and Differentiation of Fibro/Adipogenic Progenitors (FAPs) from Skeletal Muscle.

Fibro/Adipogenic Progenitors (FAPs) are a multipotent progenitor population resident in skeletal muscle. During development and regeneration, FAPs provide trophic support to myogenic progenitors that is required for muscle fiber maturation and specification. FAPs also represent a major cellular source of fibrosis in degenerative disease states, highlighting them as a potential cellular target for anti-fibrotic muscle therapies. Effective and reproducible methods to isolate and culture highly purified FAP populations are therefore critical to further understand their biology. Here, we describe a fluorescent activated cell sorting (FACS) based protocol to isolate CD31-/CD45-/Integrin-α7-/Sca1+ FAPs from murine skeletal muscle including details of tissue collection and enzymatic muscle digestion. We also incorporate optimized methods of expanding and differentiated FAPs in vitro. Together, this protocol provides a complete workflow to study skeletal muscle derived FAPs and compliments downstream analytical, drug screening, and disease modeling applications.

Muscle-specific deletion of SOCS3 increases the early inflammatory response but does not affect regeneration after myotoxic injury.

BackgroundMuscles of old animals are injured more easily and regenerate poorly, attributed in part to increased levels of circulating pro-inflammatory cytokines. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling cascade is a key mediator of inflammatory cytokine action, and signaling via this pathway is increased in muscles with aging. As a negative regulator of JAK/STAT signaling, a key mediator of myogenic proliferation and differentiation, altered expression of suppressor of cytokine signaling (SOCS3) is likely to have important consequences for muscle regeneration. To model this scenario, we investigated the effect of SOCS3 deletion within mature muscle fibers on injury and repair. We tested the hypothesis that reduced SOCS3 function would alter the inflammatory response and impair muscle regeneration after myotoxic injury.MethodsMice with a specific deletion of SOCS3 within mature skeletal muscle fibers were used to assess the effect of SOCS3 deletion on muscle injury and repair. Twelve-week-old or 24-month-old SOCS3 muscle-specific knockout (SOCS3 MKO) mice and littermate controls were either left uninjured or injured with a single injection of notexin (10 μg/ml) into the right tibialis anterior (TA) muscle. At 1, 2, 3, 5, 7, or 14 days post-injury, the right TA muscle was excised and subjected to histological, western immunoblotting, and gene expression analyses. Force production and fatigue were assessed in uninjured muscles and at 7 days post-notexin injury.ResultsIn uninjured muscles, SOCS3 deletion decreased force production during fatigue but had no effect on the gross or histological appearance of the TA muscles. After notexin injury, deletion of SOCS3 increased STAT3 phosphorylation at day 1 and increased the mRNA expression of the inflammatory cytokine TNF-α, and the inflammatory cell markers F4/80 and CD68 at day 2. Gene expression analysis of the regeneration markers Pax7, MyoD, and Myogenin indicated SOCS3 deletion had no effect on the progression of muscle repair after notexin injury. Inflammation and regeneration were also unchanged in the muscles of 24-month-old SOCS3 MKO mice compared with control.ConclusionsLoss of SOCS3 expression in mature muscle fibers increased the inflammatory response to myotoxic injury but did not impair muscle regeneration in either adult or old mice. Therefore, reduced SOCS3 expression in muscle fibers is unlikely to underlie impaired muscle regeneration. Further investigation into the role of SOCS3 in other cell types involved in muscle repair is warranted.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0108-4) contains supplementary material, which is available to authorized users.