Quantification of hormone-induced atrophy of large myotubes from C2C12 and L6 cells: atrophy-inducible and atrophy-resistant C2C12 myotubes

2006 ◽  
Vol 290 (2) ◽  
pp. C650-C659 ◽  
Author(s):  
Karim R. Sultan ◽  
Birgit Henkel ◽  
Maarten Terlou ◽  
Henk P. Haagsman
Keyword(s):  
Muscle Atrophy ◽  
Horse Serum ◽  
Skeletal Muscle Atrophy ◽  
C2c12 Myotubes ◽  
L6 Cells ◽  
L6 Myotubes ◽  
Ideal System ◽  
Key Factor ◽  
Set Up

Myofiber atrophy is the final outcome of muscle wasting induced by catabolic factors such as glucocorticoids and thyroid hormones. We set up an in vitro system to define the catabolic reaction based on myotube atrophy. Both mouse C2C12 and rat L6 cells were used. C2C12 myotube formation was improved by replacing horse serum with the serum substitute Ultroser G. A new method was developed to quantify size changes of large (0.5–1 mm) myotubes only, excluding remaining myoblasts and small myotubes. Dexamethasone reduced myotube size by 30% in L6 but not in C2C12 myotubes. Expression of the glucocorticoid receptor was twofold higher in L6 myotubes than in C2C12 myotubes. In both cell lines, 3,3′,5-triiodo-l-thyronine (T3) did not induce a significant size reduction. Expression of the major T3 receptor (T3Rβ1) was higher in L6 myotubes. We investigated whether the changes in myotube size are related to changes in atrogin-1 expression, as this enzyme is thought to be a key factor in the initiation of muscle atrophy. Dexamethasone induced a twofold increase of atrogin-1 mRNA; again, only L6 myotubes were susceptible. Interestingly, atrogin-1 expression in Ultroser G-fused C2C12 myotubes was lower than that in horse serum-fused myotubes. Furthermore, dexamethasone treatment increased atrogin-1 expression only in horse serum-fused myotubes but not in Ultroser G-fused myotubes. Ultroser G-induced fusion may result in atrophy-resistant C2C12 myotubes. Therefore, C2C12 myotubes offer an ideal system in which to study skeletal muscle atrophy because, depending on differentiation conditions, C2C12 cells produce atrophy-inducible and atrophy-resistant myotubes.

scholarly journals Trimetazidine attenuates dexamethasone-induced muscle atrophy via inhibiting NLRP3/GSDMD pathway-mediated pyroptosis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Li Wang ◽  
Xin-Feng Jiao ◽  
Cheng Wu ◽  
Xiao-Qing Li ◽  
Hui-Xian Sun ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Atrophy ◽  
Muscle Performance ◽  
Skeletal Muscle Atrophy ◽  
C2c12 Myotubes ◽  
High Dose ◽  
Protective Effects ◽  
Akt Inhibitor ◽  
Caspase 1

AbstractSkeletal muscle atrophy is one of the major side effects of high dose or sustained usage of glucocorticoids. Pyroptosis is a novel form of pro-inflammatory programmed cell death that may contribute to skeletal muscle injury. Trimetazidine, a well-known anti-anginal agent, can improve skeletal muscle performance both in humans and mice. We here showed that dexamethasone-induced atrophy, as evidenced by the increase of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) expression, and the decrease of myotube diameter in C2C12 myotubes. Dexamethasone also induced pyroptosis, indicated by upregulated pyroptosis-related protein NLR family pyrin domain containing 3 (NLRP3), Caspase-1, and gasdermin-D (GSDMD). Knockdown of NLRP3 or GSDMD attenuated dexamethasone-induced myotube pyroptosis and atrophy. Trimetazidine treatment ameliorated dexamethasone-induced muscle pyroptosis and atrophy both in vivo and in vitro. Activation of NLRP3 using LPS and ATP not only increased the cleavage and activation of Caspase-1 and GSDMD, but also increased the expression levels of atrophy markers MuRF1 and Atrogin-1 in trimetazidine-treated C2C12 myotubes. Mechanically, dexamethasone inhibited the phosphorylation of PI3K/AKT/FoxO3a, which could be attenuated by trimetazidine. Conversely, co-treatment with a PI3K/AKT inhibitor, picropodophyllin, remarkably increased the expression of NLRP3 and reversed the protective effects of trimetazidine against dexamethasone-induced C2C12 myotube pyroptosis and atrophy. Taken together, our study suggests that NLRP3/GSDMD-mediated pyroptosis might be a novel mechanism for dexamethasone-induced skeletal muscle atrophy. Trimetazidine might be developed as a potential therapeutic agent for the treatment of dexamethasone-induced muscle atrophy.

scholarly journals Trimetazidine Attenuates Dexamethasone-Induced Muscle Atrophy via Inhibiting NLRP3/GSDMD Pathway-Mediated Pyroptosis

2020 ◽  
Author(s):  
Li Wang ◽  
Ming-Qing He ◽  
Xi-Yu Shen ◽  
Kang-Zhen Zhang ◽  
Can Zhao ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Atrophy ◽  
Ring Finger ◽  
Muscle Performance ◽  
Skeletal Muscle Atrophy ◽  
C2c12 Myotubes ◽  
High Dose ◽  
Therapeutic Compound

Skeletal muscle atrophy is one of the major side effects of high dose or sustained usage of glucocorticoids. Pyroptosis is a novel form of pro-inflammatory programmed cell death that may contribute to skeletal muscle injury. Trimetazidine, a well-known anti-anginal agent, can also improve skeletal muscle performance both in human and mice. We here showed that dexamethasone induced atrophy, evidenced by the increase of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) expression , and the decrease of myotube diameter in C2C12 myotubes. Dexamethasone also induced pyroptosis, indicated by upregulated pyroptosis-related protein NLRP3, Caspase-1 and GSDMD. Knockdown of NLRP3 or GSDMD attenuated dexamethasone-induced myotube pyroptosis and atrophy. Trimetazidine administration ameliorated dexamethasone-induced muscle atrophy both in vivo and in vitro. Moreover, trimetazidine improved exercise tolerance, as evidenced by increased running distance and running time, as well as increased skeletal muscle mass in dexamethasone-treated mice. Mechanically, trimetazidine could reverse dexamethasone-induced activation of pyroptosis both in C2C12 myotubes and in mice. Taken together, our present study demonstrated that NLRP3/GSDMD pathway-mediated pyroptosis was involved in dexamethasone-induced skeletal muscle atrophy. Trimetazidine could partially alleviate dexamethasone-induced skeletal muscle atrophy, and increase the diameter of C2C12 myotubes via inhibiting pyroptosis. Thus, trimetazidine might be a potential therapeutic compound for the prevention of muscle atrophy in glucocorticoid-treated patients.

scholarly journals BMSC-Derived Exosomes Inhibit Dexamethasone-Induced Muscle Atrophy via the miR-486-5p/FoxO1 Axis

2021 ◽  
Vol 12 ◽  
Author(s):  
Ziyi Li ◽  
Chang Liu ◽  
Shilun Li ◽  
Ting Li ◽  
Yukun Li ◽  
...  
Keyword(s):  
Muscle Atrophy ◽  
Nuclear Translocation ◽  
Animal Experiments ◽  
Public Health Problem ◽  
Protective Role ◽  
Skeletal Muscle Atrophy ◽  
C2c12 Myotubes ◽  
Mechanism Analysis

Sarcopenia, characterized by reduced muscle function as well as muscle mass, has been a public health problem with increasing prevalence. It might result from aging, injury, hormone imbalance and other catabolic conditions. Recently, exosomes were considered to regulate muscle regeneration and protein synthesis. In order to confirm the effect of BMSC-derived exosomes (BMSC-Exos) on muscle, dexamethasone-induced muscle atrophy was built both in vitro and in vivo. In the present research, BMSC-Exos attenuated the decrease of myotube diameter induced by dexamethasone, indicating that BMSC-Exos played a protective role in skeletal muscle atrophy. Further mechanism analysis exhibited that the content of miR-486-5p in C2C12 myotubes was up-regulated after treated with BMSC-Exos. Meanwhile, BMSC-Exos markedly downregulated the nuclear translocation of FoxO1, which plays an important role in muscle differentiation and atrophy. Importantly, the miR-486-5p inhibitor reversed the decreased expression of FoxO1 induced by BMSC-Exos. In animal experiments, BMSC-Exos inhibited dexamethasone-induced muscle atrophy, and miR-486-5p inhibitor reversed the protective effect of BMSC-Exos. These results indicating that BMSC-derived exosomes inhibit dexamethasone-induced muscle atrophy via miR486-5p/Foxo1 Axis.

scholarly journals Identification of Withania somnifera-Silybum marianum-Trigonella foenum-graecum Formulation as a Nutritional Supplement to Contrast Muscle Atrophy and Sarcopenia

Nutrients ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 49
Author(s):  
Laura Salvadori ◽  
Manuela Mandrone ◽  
Tommaso Manenti ◽  
Catia Ercolani ◽  
Luca Cornioli ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Muscle Mass ◽  
Muscle Atrophy ◽  
Withania Somnifera ◽  
Myoblast Differentiation ◽  
C2c12 Myotubes ◽  
Silybum Marianum ◽  
Trigonella Foenum Graecum ◽  
Age Related

Background: Muscle atrophy, i.e., the loss of skeletal muscle mass and function, is an unresolved problem associated with aging (sarcopenia) and several pathological conditions. The imbalance between myofibrillary protein breakdown (especially the adult isoforms of myosin heavy chain, MyHC) and synthesis, and the reduction of muscle regenerative potential are main causes of muscle atrophy. Methods: Starting from one-hundred dried hydroalcoholic extracts of medical plants, we identified those able to contrast the reduction of C2C12 myotube diameter in well-characterized in vitro models mimicking muscle atrophy associated to inflammatory states, glucocorticoid treatment or nutrient deprivation. Based on their ability to rescue type II MyHC (MyHC-II) expression in atrophying conditions, six extracts with different phytochemical profiles were selected, mixed in groups of three, and tested on atrophic myotubes. The molecular mechanism underpinning the effects of the most efficacious formulation, and its efficacy on myotubes obtained from muscle biopsies of young and sarcopenic subjects were also investigated. Results: We identified WST (Withania somnifera, Silybum marianum, Trigonella foenum-graecum) formulation as extremely efficacious in protecting C2C12 myotubes against MyHC-II degradation by stimulating Akt (protein kinase B)-dependent protein synthesis and p38 MAPK (p38 mitogen-activated protein kinase)/myogenin-dependent myoblast differentiation. WST sustains trophism in C2C12 and young myotubes, and rescues the size, developmental MyHC expression and myoblast fusion in sarcopenic myotubes. Conclusion: WST strongly counteracts muscle atrophy associated to different conditions in vitro. The future validation in vivo of our results might lead to the use of WST as a food supplement to sustain muscle mass in diffuse atrophying conditions, and to reverse the age-related functional decline of human muscles, thus improving people quality of life and reducing social and health-care costs.

Temperature as a key factor for successful inoculation of soybean with Bradyrhizobium spp. under cool growing conditions in Belgium

2018 ◽  
Vol 156 (4) ◽  
pp. 493-503 ◽  
Author(s):  
J. Pannecoucque ◽  
S. Goormachtigh ◽  
J. Ceusters ◽  
J. Debode ◽  
C. Van Waes ◽  
...  
Keyword(s):  
Field Trials ◽  
Protein Yield ◽  
Control Treatment ◽  
Rhizobial Symbiosis ◽  
Key Factor ◽  
Growing Conditions ◽  
Set Up ◽  
Biological Nitrogen ◽  
Intermediate Performance

AbstractBacterial inoculation of soybean seeds to improve biological nitrogen fixation is a well-known practice to achieve higher seed and protein yield with reduced fertilization. The optimal inoculation strategy in temperate regions is unknown because soybeans are rarely cultivated under colder growing conditions. The aim of the present work was to determine the most suitable inoculation strategy for soybean cultivation in Belgium. Field trials were set up with four Bradyrhizobium inoculants (HiStick, Force 48, Biodoz and Optimize) at two locations over 2 years (2014–2015) and compared with a non-inoculated control treatment. In addition, HiStick was tested at three doses and Optimize at two time periods prior to sowing. Under Belgian conditions, all inoculants were effective in establishing rhizobial symbiosis, resulting in increased yield, protein content, protein yield and thousand-grain weight compared with the non-inoculated control. A single dose of HiStick was sufficient to establish symbiosis. Pre-inoculation with Optimize 2 weeks before sowing gave an intermediate performance for most parameters between the non-inoculated control treatment and inoculation with Optimize 24 h prior to sowing. Among the four products tested, Biodoz seemed the best product for inoculation under cool growing conditions. Based on the atpD gene, the bacterial strain of Biodoz showed complete similarity with Bradyrhizobium diazoefficiens, while strains of other products were identified as Bradyrhizobium japonicum. In vitro growing capacity of the Biodoz strain at 8 °C was higher compared with the other strains. Better cold adaptation of the Biodoz strain might be a possible explanation for the better performance of Biodoz in Belgium.

scholarly journals Protective Effect of Pyropia yezoensis Peptide on Dexamethasone-Induced Myotube Atrophy in C2C12 Myotubes

Marine Drugs ◽  
2019 ◽  
Vol 17 (5) ◽  
pp. 284 ◽  
Author(s):  
Min-Kyeong Lee ◽  
Jeong-Wook Choi ◽  
Youn Hee Choi ◽  
Taek-Jeong Nam
Keyword(s):  
Skeletal Muscle ◽  
Growth Factor ◽  
Signaling Pathway ◽  
Muscle Atrophy ◽  
Skeletal Muscle Atrophy ◽  
C2c12 Myotubes ◽  
Protective Effects ◽  
Factor I ◽  
Igf I ◽  
Foxo Signaling Pathway

Dexamethasone (DEX), a synthetic glucocorticoid, causes skeletal muscle atrophy. This study examined the protective effects of Pyropia yezoensis peptide (PYP15) against DEX-induced myotube atrophy and its association with insulin-like growth factor-I (IGF-I) and the Akt/mammalian target of rapamycin (mTOR)-forkhead box O (FoxO) signaling pathway. To elucidate the molecular mechanisms underlying the effects of PYP15 on DEX-induced myotube atrophy, C2C12 myotubes were treated for 24 h with 100 μM DEX in the presence or absence of 500 ng/mL PYP15. Cell viability assays revealed no PYP15 toxicity in C2C12 myotubes. PYP15 activated the insulin-like growth factor-I receptor (IGF-IR) and Akt-mTORC1 signaling pathway in DEX-induced myotube atrophy. In addition, PYP15 markedly downregulated the nuclear translocation of transcription factors FoxO1 and FoxO3a, and inhibited 20S proteasome activity. Furthermore, PYP15 inhibited the autophagy-lysosomal pathway in DEX-stimulated myotube atrophy. Our findings suggest that PYP15 treatment protected against myotube atrophy by regulating IGF-I and the Akt-mTORC1-FoxO signaling pathway in skeletal muscle. Therefore, PYP15 treatment appears to exert protective effects against skeletal muscle atrophy.

scholarly journals Mechanical loading of tissue engineered skeletal muscle prevents dexamethasone induced myotube atrophy

2020 ◽  
Author(s):  
Kathryn W. Aguilar-Agon ◽  
Andrew J. Capel ◽  
Jacob W. Fleming ◽  
Darren J. Player ◽  
Neil R. W. Martin ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Mechanical Loading ◽  
Muscle Atrophy ◽  
Mechanical Load ◽  
3D Models ◽  
Skeletal Muscle Atrophy ◽  
Treatment Modalities ◽  
Murine Cell Line

Abstract Skeletal muscle atrophy as a consequence of acute and chronic illness, immobilisation, muscular dystrophies and aging, leads to severe muscle weakness, inactivity and increased mortality. Mechanical loading is thought to be the primary driver for skeletal muscle hypertrophy, however the extent to which mechanical loading can offset muscle catabolism has not been thoroughly explored. In vitro 3D-models of skeletal muscle provide a controllable, high throughput environment and mitigating many of the ethical and methodological constraints present during in vivo experimentation. This work aimed to determine if mechanical loading would offset dexamethasone (DEX) induced skeletal muscle atrophy, in muscle engineered using the C2C12 murine cell line. Mechanical loading successfully offset myotube atrophy and functional degeneration associated with DEX regardless of whether the loading occurred before or after 24 h of DEX treatment. Furthermore, mechanical load prevented increases in MuRF-1 and MAFbx mRNA expression, critical regulators of muscle atrophy. Overall, we demonstrate the application of tissue engineered muscle to study skeletal muscle health and disease, offering great potential for future use to better understand treatment modalities for skeletal muscle atrophy.

scholarly journals Preclinical Evaluation of a Food-Derived Functional Ingredient to Address Skeletal Muscle Atrophy

Nutrients ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2274
Author(s):  
Roi Cal ◽  
Heidi Davis ◽  
Alish Kerr ◽  
Audrey Wall ◽  
Brendan Molloy ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Atrophy ◽  
Soleus Muscle ◽  
Murine Model ◽  
The Body ◽  
Skeletal Muscle Atrophy ◽  
Type I ◽  
Disuse Atrophy ◽  
Tnf Α

Skeletal muscle is the metabolic powerhouse of the body, however, dysregulation of the mechanisms involved in skeletal muscle mass maintenance can have devastating effects leading to many metabolic and physiological diseases. The lack of effective solutions makes finding a validated nutritional intervention an urgent unmet medical need. In vitro testing in murine skeletal muscle cells and human macrophages was carried out to determine the effect of a hydrolysate derived from vicia faba (PeptiStrong: NPN_1) against phosphorylated S6, atrophy gene expression, and tumour necrosis factor alpha (TNF-α) secretion, respectively. Finally, the efficacy of NPN_1 on attenuating muscle waste in vivo was assessed in an atrophy murine model. Treatment of NPN_1 significantly increased the phosphorylation of S6, downregulated muscle atrophy related genes, and reduced lipopolysaccharide-induced TNF-α release in vitro. In a disuse atrophy murine model, following 18 days of NPN_1 treatment, mice exhibited a significant attenuation of muscle loss in the soleus muscle and increased the integrated expression of Type I and Type IIa fibres. At the RNA level, a significant upregulation of protein synthesis-related genes was observed in the soleus muscle following NPN_1 treatment. In vitro and preclinical results suggest that NPN_1 is an effective bioactive ingredient with great potential to prolong muscle health.

scholarly journals Potential Therapeutic Role of L-Carnitine in Skeletal Muscle Oxidative Stress and Atrophy Conditions

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Anna Montesano ◽  
Pamela Senesi ◽  
Livio Luzi ◽  
Stefano Benedini ◽  
Ileana Terruzzi
Keyword(s):  
Oxidative Stress ◽  
Skeletal Muscle ◽  
Morphological Changes ◽  
Ubiquitin Proteasome System ◽  
Skeletal Muscle Atrophy ◽  
C2c12 Myotubes ◽  
Mitochondrial Activity ◽  
Potential Therapeutic Role ◽  
Essential Nutrient

The targeting of nutraceutical treatment to skeletal muscle damage is an emerging area of research, driven by the need for new therapies for a range of muscle-associated diseases. L-Carnitine (CARN) is an essential nutrient and plays a key role in mitochondrialβ-oxidation and in the ubiquitin-proteasome system regulation. As a dietary supplement to improve athletic performance, CARN has been studied for its potential to enhanceβ-oxidation. However, CARN effects on myogenesis, mitochondrial activity, and hypertrophy process are not completely elucidated. Thisin vitrostudy aims to investigate CARN role on skeletal muscle remodeling, differentiation process, and myotubes formation. We analyzed muscle differentiation and morphological features in C2C12 myoblasts exposed to 5 mM CARN. Our results showed that CARN was able to accelerate C2C12 myotubes formation and induce morphological changes, characterizing the start of hypertrophy process. In addition, CARN improved AKT activation and downstream cellular signaling pathways involved in skeletal muscle atrophy process prevention. Also, CARN positively regulated the pathways involved in oxidative stress defense. In this work, we provide an interesting novel mechanism of the potential therapeutic use of CARN to treat pathological conditions characterized by skeletal muscle morphological and functional impairment, oxidative stress production, and atrophy process in aging.

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