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Journal of Cachexia, Sarcopenia and... Dec 2022Muscle atrophy, leading to muscular dysfunction and weakness, is an adverse outcome of sustained period of glucocorticoids usage. However, the molecular mechanism...
BACKGROUND
Muscle atrophy, leading to muscular dysfunction and weakness, is an adverse outcome of sustained period of glucocorticoids usage. However, the molecular mechanism underlying this detrimental condition is currently unclear. Pyruvate dehydrogenase kinase 4 (PDK4), a central regulator of cellular energy metabolism, is highly expressed in skeletal muscle and has been implicated in the pathogenesis of several diseases. The current study was designed to investigated and delineate the role of PDK4 in the context of muscle atrophy, which could be identified as a potential therapeutic avenue to protect against dexamethasone-induced muscle wasting.
METHODS
The dexamethasone-induced muscle atrophy in C2C12 myotubes was evaluated at the molecular level by expression of key genes and proteins involved in myogenesis, using immunoblotting and qPCR analyses. Muscle dysfunction was studied in vivo in wild-type and PDK4 knockout mice treated with dexamethasone (25 mg/kg body weight, i.p., 10 days). Body weight, grip strength, muscle weight and muscle histology were assessed. The expression of myogenesis markers were analysed using qPCR, immunoblotting and immunoprecipitation. The study was extended to in vitro human skeletal muscle atrophy analysis.
RESULTS
Knockdown of PDK4 was found to prevent glucocorticoid-induced muscle atrophy and dysfunction in C2C12 myotubes, which was indicated by induction of myogenin (0.3271 ± 0.102 vs 2.163 ± 0.192, ****P < 0.0001) and myosin heavy chain (0.3901 ± 0.047 vs. 0.7222 ± 0.082, **P < 0.01) protein levels and reduction of muscle atrophy F-box (10.77 ± 2.674 vs. 1.518 ± 0.172, **P < 0.01) expression. In dexamethasone-induced muscle atrophy model, mice with genetic ablation of PDK4 revealed increased muscle strength (162.1 ± 22.75 vs. 200.1 ± 37.09 g, ***P < 0.001) and muscle fibres (54.20 ± 11.85% vs. 84.07 ± 28.41%, ****P < 0.0001). To explore the mechanism, we performed coimmunoprecipitation and liquid chromatography-mass spectrometry analysis and found that myogenin is novel substrate of PDK4. PDK4 phosphorylates myogenin at S43/T57 amino acid residues, which facilitates the recruitment of muscle atrophy F-box to myogenin and leads to its subsequent ubiquitination and degradation. Finally, overexpression of non-phosphorylatable myogenin mutant using intramuscular injection prevented dexamethasone-induced muscle atrophy and preserved muscle fibres.
CONCLUSIONS
We have demonstrated that PDK4 mediates dexamethasone-induced skeletal muscle atrophy. Mechanistically, PDK4 phosphorylates and degrades myogenin via recruitment of E3 ubiquitin ligase, muscle atrophy F-box. Rescue of muscle regeneration by genetic ablation of PDK4 or overexpression of non-phosphorylatable myogenin mutant indicates PDK4 as an amenable therapeutic target in muscle atrophy.
Topics: Animals; Humans; Mice; Body Weight; Dexamethasone; Glucocorticoids; Muscular Atrophy; Proteasome Endopeptidase Complex; Ubiquitin; Pyruvate Dehydrogenase Acetyl-Transferring Kinase
PubMed: 36259412
DOI: 10.1002/jcsm.13100 -
Biomedicine & Pharmacotherapy =... Dec 2019A high glucose level is usually considered to be the factor that induces tissue and cell dysfunction and damage, commonly known as "glucose toxicity".
BACKGROUND
A high glucose level is usually considered to be the factor that induces tissue and cell dysfunction and damage, commonly known as "glucose toxicity".
OBJECTIVE
This study aimed to explore the effects and the potential molecular mechanisms of high glucose on myoblast differentiation and insulin sensitivity.
MATERIALS AND METHODS
C2C12 cells were cultured in differentiation medium containing 25, 40, or 60 mM glucose for 1, 3, or 5 days. E-MHC positive area and GLUT4 fluorescence were evaluated through Immunofluorescence. The expression of Myf5, MyoD, myogenin were measured by performing western blot and qRT-PCR. The protein expression of GLUT4 on cell membrane and glucose uptake in C2C12 myotubes were measured through western blot and 2-NBDG assay. AKT activator SC79 and inhibitor MK2206 was utilized to reveal the important role of AKT signaling in myogenesis and insulin sensitivity inhibited by high glucose.
RESULTS
60 mM glucose inhibits myogenesis by decreasing the expression of MyoD and myogenin, and induces insulin resistance by reducing both basal and insulin-stimulated GLUT4 expressions and glucose uptakes. The influences of high glucose on myogenesis and IR was related to decreased AKT activation. SC79 rescued the inhibition of high glucose on myogenesis and attenuated IR. MK2206 inhibits the myogenic differentiation and induces IR.
CONCLUSION
The present study reveals that high glucose inhibited myogenisis accompanied by inducing IR, through AKT signaling inhibition, which may help to further research for resisting degenerative muscular diseases caused by glucose metabolism disorders.
Topics: Animals; Cell Differentiation; Cell Line; Cell Survival; Down-Regulation; Gene Expression Regulation; Glucose; Insulin Resistance; Mice; Muscle Development; Myoblasts; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt
PubMed: 31634780
DOI: 10.1016/j.biopha.2019.109498 -
Journal of Cachexia, Sarcopenia and... Aug 2023Sepsis-induced intensive care unit-acquired weakness (ICUAW) features profound muscle atrophy and attenuated muscle regeneration related to malfunctioning satellite...
BACKGROUND
Sepsis-induced intensive care unit-acquired weakness (ICUAW) features profound muscle atrophy and attenuated muscle regeneration related to malfunctioning satellite cells. Transforming growth factor beta (TGF-β) is involved in both processes. We uncovered an increased expression of the TGF-β receptor II (TβRII)-inhibitor SPRY domain-containing and SOCS-box protein 1 (SPSB1) in skeletal muscle of septic mice. We hypothesized that SPSB1-mediated inhibition of TβRII signalling impairs myogenic differentiation in response to inflammation.
METHODS
We performed gene expression analyses in skeletal muscle of cecal ligation and puncture- (CLP) and sham-operated mice, as well as vastus lateralis of critically ill and control patients. Pro-inflammatory cytokines and specific pathway inhibitors were used to quantitate Spsb1 expression in myocytes. Retroviral expression plasmids were used to investigate the effects of SPSB1 on TGF-β/TβRII signalling and myogenesis in primary and immortalized myoblasts and differentiated myotubes. For mechanistical analyses we used coimmunoprecipitation, ubiquitination, protein half-life, and protein synthesis assays. Differentiation and fusion indices were determined by immunocytochemistry, and differentiation factors were quantified by qRT-PCR and Western blot analyses.
RESULTS
SPSB1 expression was increased in skeletal muscle of ICUAW patients and septic mice. Tumour necrosis factor (TNF), interleukin-1β (IL-1β), and IL-6 increased the Spsb1 expression in C2C12 myotubes. TNF- and IL-1β-induced Spsb1 expression was mediated by NF-κB, whereas IL-6 increased the Spsb1 expression via the glycoprotein 130/JAK2/STAT3 pathway. All cytokines reduced myogenic differentiation. SPSB1 avidly interacted with TβRII, resulting in TβRII ubiquitination and destabilization. SPSB1 impaired TβRII-Akt-Myogenin signalling and diminished protein synthesis in myocytes. Overexpression of SPSB1 decreased the expression of early (Myog, Mymk, Mymx) and late (Myh1, 3, 7) differentiation-markers. As a result, myoblast fusion and myogenic differentiation were impaired. These effects were mediated by the SPRY- and SOCS-box domains of SPSB1. Co-expression of SPSB1 with Akt or Myogenin reversed the inhibitory effects of SPSB1 on protein synthesis and myogenic differentiation. Downregulation of Spsb1 by AAV9-mediated shRNA attenuated muscle weight loss and atrophy gene expression in skeletal muscle of septic mice.
CONCLUSIONS
Inflammatory cytokines via their respective signalling pathways cause an increase in SPSB1 expression in myocytes and attenuate myogenic differentiation. SPSB1-mediated inhibition of TβRII-Akt-Myogenin signalling and protein synthesis contributes to a disturbed myocyte homeostasis and myogenic differentiation that occurs during inflammation.
Topics: Animals; Mice; Cytokines; Inflammation; Interleukin-6; Muscle Development; Muscle, Skeletal; Myogenin; Proto-Oncogene Proteins c-akt; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha
PubMed: 37209006
DOI: 10.1002/jcsm.13252 -
Journal of Cachexia, Sarcopenia and... Dec 2021Sarcopenia and osteoporosis frequently co-occur in the elderly and have common pathophysiological determinants. Slit guidance ligand 3 (SLIT3) has been recently...
BACKGROUND
Sarcopenia and osteoporosis frequently co-occur in the elderly and have common pathophysiological determinants. Slit guidance ligand 3 (SLIT3) has been recently discovered as a novel therapeutic factor against osteoporosis, and a SLIT3 fragment containing the second leucine-rich repeat domain (LRRD2) had a therapeutic efficacy against osteoporosis. However, a role of SLIT3 in the skeletal muscle is unknown.
METHODS
Skeletal muscle mass, strength, and/or physical activity were evaluated in Slit3 , ovariectomized, and aged mice, based on the measurements of muscle weight and grip strength, Kondziella's inverted hanging test, and/or wheel-running test. Skeletal muscles were also histologically evaluated by haematoxylin and eosin staining and/or immunofluorescence. The ovariectomized and aged mice were intravenously injected with recombinant SLIT3 LRRD2 for 4 weeks. C2C12 cells were used to know cellular effects of SLIT3, such as in vitro myogenesis, fusion, cell viability, and proliferation, and also used to evaluate its molecular mechanisms by immunocytochemistry, immunoprecipitation, western blotting, real-time PCR, siRNA transfection, and receptor-ligand binding ELISA.
RESULTS
Slit3-deficient mice exhibited decreased skeletal muscle mass, muscle strength, and physical activity. The relative masses of gastrocnemius and soleus were lower in the Slit3 mice (0.580 ± 0.039% and 0.033 ± 0.003%, respectively) than those in the WT littermates (0.622 ± 0.043% and 0.038 ± 0.003%, respectively) (all, P < 0.05). Gastrocnemius of Slit3 mice showed the reduced number of Type I and Type IIa fibres (all, P < 0.05), but not of Type IIb and Type IIx fibres. SLIT3 activated β-catenin signalling by promoting its release from M-cadherin, thereby increasing myogenin expression to stimulate myoblast differentiation. In vitro experiments involving ROBO2 expression, knockdown, and interaction with SLIT3 indicated that ROBO2 functions as a SLIT3 receptor to aid myoblast differentiation. SLIT3 LRRD2 dissociated M-cadherin-bound β-catenin and up-regulated myogenin expression to increase myoblast differentiation, in a manner similar to full-length SLIT3. Systemic treatment with SLIT3 LRRD2 increased skeletal muscle mass in both ovariectomized and aged mice (all, P < 0.05). The relative masses of gastrocnemius and soleus were higher in the treated aged mice (0.548 ± 0.045% and 0.033 ± 0.005%, respectively) than in the untreated aged mice (0.508 ± 0.016% and 0.028 ± 0.003%, respectively) (all, P < 0.05). SLIT3 LRRD2 treatment increased the hanging duration of the aged mice by approximately 1.7-fold (P < 0.05).
CONCLUSIONS
SLIT3 plays a sarcoprotective role by activating β-catenin signalling. SLIT3 LRRD2 can potentially be used as a therapeutic agent against muscle loss.
Topics: Animals; Cell Differentiation; Membrane Proteins; Mice; Muscle Development; Muscle, Skeletal; Muscular Atrophy; RNA, Small Interfering; Receptors, Immunologic; Sarcopenia; Transfection
PubMed: 34423586
DOI: 10.1002/jcsm.12769 -
European Journal of Translational... Dec 2023Skeletal muscle possesses regenerative potential via satellite cells, compromised in muscular dystrophies leading to fibrosis and fat infiltration. Angiotensin II...
Skeletal muscle possesses regenerative potential via satellite cells, compromised in muscular dystrophies leading to fibrosis and fat infiltration. Angiotensin II (Ang-II) is commonly associated with pathological states. In contrast, Angiotensin (1-7) [Ang-(1-7)] counters Ang-II, acting via the Mas receptor. While Ang-II affects skeletal muscle regeneration, the influence of Ang-(1-7) remains to be elucidated. Therefore, this study aims to investigate the role of Ang-(1-7) in skeletal muscle regeneration. C2C12 cells were differentiated in the absence or presence of 10 nM of Ang-(1-7). The diameter of myotubes and protein levels of myogenin and myosin heavy chain (MHC) were determined. C57BL/6 WT male mice 16-18 weeks old) were randomly assigned to injury-vehicle, injury-Ang-(1-7), and control groups. Ang-(1-7) was administered via osmotic pumps, and muscle injury was induced by injecting barium chloride to assess muscle regeneration through histological analyses. Moreover, embryonic myosin (eMHC) and myogenin protein levels were evaluated. C2C12 myotubes incubated with Ang-(1-7) showed larger diameters than the untreated group and increased myogenin and MHC protein levels during differentiation. Ang-(1-7) administration enhances regeneration by promoting a larger diameter of new muscle fibers. Furthermore, higher numbers of eMHC (+) fibers were observed in the injured-Ang-(1-7), which also had a larger diameter. Moreover, eMHC and myogenin protein levels were elevated, supporting enhanced regeneration due to Ang-(1-7) administration. Ang-(1-7) effectively promotes differentiation in vitroand improves muscle regeneration in the context of injuries, with potential implications for treating muscle-related disorders.
PubMed: 38112612
DOI: 10.4081/ejtm.2023.12037 -
Journal of Animal Science May 2020Satellite cells are the myogenic stem and progenitor population found in skeletal muscle. These cells typically reside in a quiescent state until called upon to support... (Review)
Review
Satellite cells are the myogenic stem and progenitor population found in skeletal muscle. These cells typically reside in a quiescent state until called upon to support repair, regeneration, or muscle growth. The activities of satellite cells are orchestrated by systemic hormones, autocrine and paracrine growth factors, and the composition of the basal lamina of the muscle fiber. Several key intracellular signaling events are initiated in response to changes in the local environment causing exit from quiescence, proliferation, and differentiation. Signals emanating from Notch, wingless-type mouse mammary tumor virus integration site family members, and transforming growth factor-β proteins mediate the reversible exit from growth 0 phase while those initiated by members of the fibroblast growth factor and insulin-like growth factor families direct proliferation and differentiation. Many of these pathways impinge upon the myogenic regulatory factors (MRF), myogenic factor 5, myogenic differentiation factor D, myogenin and MRF4, and the lineage determinate, Paired box 7, to alter transcription and subsequent satellite cell decisions. In the recent past, insight into mouse transgenic models has led to a firm understanding of regulatory events that control satellite cell metabolism and myogenesis. Many of these niche-regulated functions offer subtle differences from their counterparts in livestock pointing to the existence of species-specific controls. The purpose of this review is to examine the mechanisms that mediate large animal satellite cell activity and their relationship to those present in rodents.
Topics: Animals; Cell Differentiation; Livestock; Mice; Muscle Development; Muscle Fibers, Skeletal; Muscle, Skeletal; Myogenic Regulatory Factor 5; Myogenic Regulatory Factors; Myogenin; Satellite Cells, Skeletal Muscle; Somatomedins
PubMed: 32175577
DOI: 10.1093/jas/skaa081 -
Animals : An Open Access Journal From... Dec 2020Growth rates in fish are largely dependent on genetic and environmental factors, of which the latter can be highly variable throughout development. For this reason,... (Review)
Review
Growth rates in fish are largely dependent on genetic and environmental factors, of which the latter can be highly variable throughout development. For this reason, muscle growth in fish is particularly dynamic as muscle structure and function can be altered by environmental conditions, a concept referred to as muscle plasticity. Myogenic regulatory factors (MRFs) like Myogenin, MyoD, and Pax7 control the myogenic mechanisms regulating quiescent muscle cell maintenance, proliferation, and differentiation, critical processes central for muscle plasticity. This review focuses on recent advancements in molecular mechanisms involving microRNAs (miRNAs) and DNA methylation that regulate the expression and activity of MRFs in fish. Findings provide overwhelming support that these mechanisms are significant regulators of muscle plasticity, particularly in response to environmental factors like temperature and nutritional challenges. Genetic variation in DNA methylation and miRNA expression also correlate with variation in body weight and growth, suggesting that genetic markers related to these mechanisms may be useful for genomic selection strategies. Collectively, this knowledge improves the understanding of mechanisms regulating muscle plasticity and can contribute to the development of husbandry and breeding strategies that improve growth performance and the ability of the fish to respond to environmental challenges.
PubMed: 33396941
DOI: 10.3390/ani11010061 -
Head and Neck Pathology Mar 2020Skeletal muscle tumors are classified into rhabdomyoma and embryonal, alveolar, spindle cell/sclerosing and pleomorphic rhabdomyosarcoma according to WHO classifications... (Review)
Review
Skeletal muscle tumors are classified into rhabdomyoma and embryonal, alveolar, spindle cell/sclerosing and pleomorphic rhabdomyosarcoma according to WHO classifications of tumors. These tumors arise mostly in the head and neck and, in childhood, represent the largest subset of soft tissue tumors. Although these skeletal muscle tumors show common immunoexpression of two myogenic regulatory factors, MyoD1 and myogenin, their molecular biological backgrounds are quite different. Therefore, treatment regimens vary a great deal depending on the histological subtype. Histopathologically, rhabdomyoma is characterized by well-demarcated lesions with no invasion of the surrounding tissue. Embryonal rhabdomyosarcoma is composed of primitive mesenchymal cells in various stages of myogenesis and shows heterogeneous nuclear staining for myogenin. Alveolar rhabdomyosarcoma, on the other hand, shows a proliferation of uniform primitive round cells arranged in alveolar patterns. The tumor cells at the periphery of alveolar structures adhere in a single layer to the fibrous septa. Diffuse and strong nuclear immunoexpression for myogenin is observed. In genetic backgrounds, almost all alveolar rhabdomyosarcomas contain a characteristic fusion gene such as PAX3/7-FOXO1. Spindle cell/sclerosing rhabdomyosarcoma is characterized by fascicularly arranged spindle-shaped cells or dense hyalinized collagenous matrix. NCOR2- or VGLL2-related gene fusions or MYOD1 (p.L122R) mutation is commonly recognized. Epithelioid rhabdomyosarcoma is a rare variant of rhabdomyosarcoma that shows a proliferation of epithelioid tumor cells having large vesicular nuclei, prominent nucleoli, and amphophilic to eosinophilic cytoplasm arranged in sheets. As these characteristic histological and molecular features are present in each subtype, it is possible to diagnose skeletal muscle tumors accurately.
Topics: Humans; Neoplasms, Muscle Tissue; Soft Tissue Neoplasms
PubMed: 31950473
DOI: 10.1007/s12105-019-01113-2 -
Nutrients Jan 2024Epicatechin is a polyphenol compound that promotes skeletal muscle differentiation and counteracts the pathways that participate in the degradation of proteins. Several... (Review)
Review
Epicatechin is a polyphenol compound that promotes skeletal muscle differentiation and counteracts the pathways that participate in the degradation of proteins. Several studies present contradictory results of treatment protocols and therapeutic effects. Therefore, the objective of this systematic review was to investigate the current literature showing the molecular mechanism and clinical protocol of epicatechin in muscle atrophy in humans, animals, and myoblast cell-line. The search was conducted in Embase, PubMed/MEDLINE, Cochrane Library, and Web of Science. The qualitative analysis demonstrated that there is a commonness of epicatechin inhibitory action in myostatin expression and atrogenes MAFbx, FOXO, and MuRF1. Epicatechin showed positive effects on follistatin and on the stimulation of factors related to the myogenic actions (MyoD, Myf5, and myogenin). Furthermore, the literature also showed that epicatechin can interfere with mitochondrias' biosynthesis in muscle fibers, stimulation of the signaling pathways of AKT/mTOR protein production, and amelioration of skeletal musculature performance, particularly when combined with physical exercise. Epicatechin can, for these reasons, exhibit clinical applicability due to the beneficial results under conditions that negatively affect the skeletal musculature. However, there is no protocol standardization or enough clinical evidence to draw more specific conclusions on its therapeutic implementation.
Topics: Animals; Humans; Catechin; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; MyoD Protein; TOR Serine-Threonine Kinases
PubMed: 38276564
DOI: 10.3390/nu16020326 -
Journal of Neuromuscular Diseases 2021The resident stem cell for skeletal muscle is the satellite cell. On the 50th anniversary of its discovery in 1961, we described the history of skeletal muscle research... (Review)
Review
The resident stem cell for skeletal muscle is the satellite cell. On the 50th anniversary of its discovery in 1961, we described the history of skeletal muscle research and the seminal findings made during the first 20 years in the life of the satellite cell (Scharner and Zammit 2011, doi: 10.1186/2044-5040-1-28). These studies established the satellite cell as the source of myoblasts for growth and regeneration of skeletal muscle. Now on the 60th anniversary, we highlight breakthroughs in the second phase of satellite cell research from 1980 to 2000. These include technical innovations such as isolation of primary satellite cells and viable muscle fibres complete with satellite cells in their niche, together with generation of many useful reagents including genetically modified organisms and antibodies still in use today. New methodologies were combined with description of endogenous satellite cells markers, notably Pax7. Discovery of the muscle regulatory factors Myf5, MyoD, myogenin, and MRF4 in the late 1980s revolutionized understanding of the control of both developmental and regerenative myogenesis. Emergence of genetic lineage markers facilitated identification of satellite cells in situ, and also empowered transplantation studies to examine satellite cell function. Finally, satellite cell heterogeneity and the supportive role of non-satellite cell types in muscle regeneration were described. These major advances in methodology and in understanding satellite cell biology provided further foundations for the dramatic escalation of work on muscle stem cells in the 21st century.
Topics: Animals; Cell Differentiation; History, 20th Century; History, 21st Century; Humans; Mice; Muscle Development; Muscle, Skeletal; Myogenic Regulatory Factors; Myogenin; PAX7 Transcription Factor; Satellite Cells, Skeletal Muscle
PubMed: 34459412
DOI: 10.3233/JND-210705