Abstract
In skeletal muscle, chronic oxygen depletion induces a disturbance leading to muscle atrophy. Mechanical stress (physical exercise) and nutritional supplement therapy are commonly used against loss of muscle mass and undernutrition in hypoxia, while oxygenation therapy is preferentially used to counteract muscle fatigue and exercise intolerance. However, the impact of oxygenation on skeletal muscle cells remains poorly understood, in particular on signalling pathways regulating protein balance. Thus, we investigated the effects of each separated treatment (mechanical stress, nutritional supplementation and oxygenation therapy) on intracellular pathways involved in protein synthesis and degradation that are imbalanced in skeletal muscle cells atrophy resulting from hypoxia. Myotubes under hypoxia were treated by electrical stimulation, amino acids supplement or oxygenation period. Signalling pathways involved in protein synthesis (PI3K-Akt-mTOR) and degradation (FoxO1 and FoxO3a) were investigated, so as autophagy, ubiquitin–proteasome system and myotube morphology. Electrical stimulation and oxygenation treatment resulted in higher myotube diameter, myogenic fusion index and myotubes density until 48 h post-treatment compared to untreated hypoxic myotubes. Both treatments also induced inhibition of FoxO3a and decreased activity of ubiquitin–proteasome system; however, their impact on protein synthesis pathway was specific for each one. Indeed, electrical stimulation impacted upstream proteins to mTOR (i.e., Akt) while oxygenation treatment activated downstream targets of mTOR (i.e., 4E-BP1 and P70S6K). In contrast, amino acid supplementation had very few effects on myotube morphology nor on protein homeostasis. This study demonstrated that electrical stimulation or oxygenation period are two effective treatments to fight against hypoxia-induced muscle atrophy, acting through different molecular adaptations.
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References
Arsham AM, Howell JJ, Simon MC (2003) A novel hypoxia-inducible factor-independent hypoxic response regulating mammalian target of rapamycin and its targets. J Bio Chem 278(32):29655–29660. https://doi.org/10.1074/jbc.M212770200
Baptista IL, Silvestre JG, Silva WJ, Labeit S, Moriscot AS (2017) FoxO3a suppression and VPS34 activity are essential to anti-atrophic effects of leucine in skeletal muscle. Cell Tiss Res 369(2):81–394. https://doi.org/10.1007/s00441-017-2614-z
Bensaid S, Fabre C, Fourneau J, Cieniewski-Bernard C (2019) Impact of different methods of induction of cellular hypoxia: focus on protein homeostasis signaling pathways and morphology of C2C12 skeletal muscle cells differentiated into myotubes. J Physiol Biochem 1–11 https://doi.org/10.1007/s13105-019-00687-3
Costes F, Gosker H, Feasson L, Desgeorges M, Kelders M, Castells J, Freyssenet D (2015) Impaired exercise training-induced muscle fiber hypertrophy and Akt/mTOR pathway activation in hypoxemic patients with COPD. J Appl Physiol 118(8):1040–1049. https://doi.org/10.1152/japplphysiol.00557.2014
Dal Negro RW, Aquilani R, Bertacco S, Boschi F, Micheletto C, Tognella S (2016) Comprehensive effects of supplemented essential amino acids in patients with severe COPD and sarcopenia. Monal Arch Chest Dis 73(1). https://doi.org/10.4081/monaldi.2010.310
Di Carlo A, De Mori R, Martelli F, Pompilio G, Capogrossi MC, Germani A (2004) Hypoxia inhibits myogenic differentiation through accelerated MyoD degradation. J Bio Chem 279(16):16332–16338. https://doi.org/10.1074/jbc.M313931200
Duan Y, Zeng L, Li F, Wang W, Li Y, Guo Q, Yin Y (2017) Effect of branched-chain amino acid ratio on the proliferation, differentiation, and expression levels of key regulators involved in protein metabolism of myocytes. Nutrition 36:8–16. https://doi.org/10.1016/j.nut.2016.10.016
Emtner M, Porszasz J, Burns M, Somfay A, Casaburi R (2003) Benefits of supplemental oxygen in exercise training in nonhypoxemic chronic obstructive pulmonary disease patients. Am J Respir Crit Care Med 168(9):1034–1042. https://doi.org/10.1164/rccm.200212-1525OC
Jewell JL, Russell RC, Guan KL (2013) Amino acid signalling upstream of mTOR. Nat Rev Molec Cell bio 14(3). https://doi.org/10.1038/nrm3522
Kent BD, Mitchell PD, McNicholas WT (2011) Hypoxemia in patients with COPD: cause, effects, and disease progression. Int J Chron Obstruct Pulmon Dis 6:199. https://doi.org/10.2147/COPD.S10611
Krieger JW (2010) Single vs. multiple sets of resistance exercise for muscle hypertrophy: a meta-analysis. J Stren Condition Res 24(4):1150–1159. https://doi.org/10.2147/COPD.S10611
Langen RCJ, Gosker HR, Remels AHV, Schols AMWJ (2013) Triggers and mechanisms of skeletal muscle wasting in chronic obstructive pulmonary disease. Int J Biochem Cell Biol 45(10):2245–2256. https://doi.org/10.1016/j.biocel.2013.06.015
Launay T, Hagström L, Lottin-Divoux S, Marchant D, Quidu P, Favret F, Beaudry M (2010) Blunting effect of hypoxia on the proliferation and differentiation of human primary and rat L6 myoblasts is not counteracted by Epo. Cell Prolif 43(1):1–8. https://doi.org/10.1111/j.1365-2184.2009.00648.x
Levett DZ, Radford EJ, Menassa DA, Graber EF, Morash AJ, Hoppeler H, Grocott MP (2012) Acclimatization of skeletal muscle mitochondria to high-altitude hypoxia during an ascent of Everest. FASEB J 26(4):1431–1441. https://doi.org/10.1096/fj.11-197772
Maltais F, Simon M, Jobin J, Desmeules M, Sullivan MJ, BÉlanger M, Leblanc P (2001) Effects of oxygen on lower limb blood flow and O2 uptake during exercise in COPD. Med Sci Sports Exerc 33(6):916–922. https://doi.org/10.1097/00005768-200106000-00010
Maltais F, Decramer M, Casaburi R, Barreiro E, Burelle Y, Debigare R, Gosker HR (2014) An official American Thoracic Society/European Respiratory Society statement: update on limb muscle dysfunction in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 189(9):e15–e62. https://doi.org/10.1164/rccm.201402-0373ST
Martin NR, Aguilar-Agon K, Robinson GP, Player DJ, Turner MC, Myers SD, Lewis MP (2017) Hypoxia impairs muscle function and reduces myotube size in tissue engineered skeletal muscle. J Cell Biochem 118(9):2599–2605. https://doi.org/10.1002/jcb.25982
Miyatake S, Bilan PJ, Pillon NJ, Klip A (2015) Contracting C2C12 myotubes release CCL2 in an NF-κB-dependent manner to induce monocyte chemoattraction. Am J Physio-Endocrin Metabol 310(2):E160–E170. https://doi.org/10.1152/ajpendo.00325.2015
Nakai N, Kawano F, Nakata K (2015) Mechanical stretch activates mammalian target of rapamycin and AMP-activated protein kinase pathways in skeletal muscle cells. Molec Cell Biochem 406(1):285–292. https://doi.org/10.1007/s11010-015-2446-7
Nedachi T, Fujita H, Kanzaki M (2008) Contractile C2C12 myotube model for studying exercise-inducible responses in skeletal muscle. Am J Physio-Endocrin Metabol 295(5):E1191–E1204. https://doi.org/10.1152/ajpendo.90280.2008
Paddon-Jones D, Rasmussen BB (2009) Dietary protein recommendations and the prevention of sarcopenia: protein, amino acid metabolism and therapy. Curr Opin Clin Nutr Metab Care 12(1):86. https://doi.org/10.1097/MCO.0b013e32831cef8b
Ren K, Crouzier T, Roy C, Picart C (2008) Polyelectrolyte multilayer films of controlled stiffness modulate myoblast cell differentiation. Adv Func Mater 18(9):1378–1389. https://doi.org/10.1002/adfm.200701297
Ren H, Accili D, Duan C (2010) Hypoxia converts the myogenic action of insulin-like growth factors into mitogenic action by differentially regulating multiple signaling pathways. Proc Natl Acad Sci 107(13):5857–5862. https://doi.org/10.1073/pnas.0909570107
Reyes de la Cruz H, Aguilar R, Sánchez de Jiménez E (2004) Functional characterization of a maize ribosomal S6 protein kinase (ZmS6K), a plant ortholog of metazoan p70S6K. Biochemistry 43(2):533–539. https://doi.org/10.1021/bi035222z
Sandri M (2013) Protein breakdown in muscle wasting: role of autophagy-lysosome and ubiquitin-proteasome. Int J Biochem Cell Biol 45(10):2121–2129. https://doi.org/10.1016/j.biocel.2013.04.023
Sakushima K, Yoshikawa M, Osaki T, Miyamoto N, Hashimoto T (2020) Moderate hypoxia promotes skeletal muscle cell growth and hypertrophy in C2C12 cells. Biochem Biophys Res Commun 525(4):921–927. https://doi.org/10.1016/j.bbrc.2020.02.152
Schneider A, Younis RH, Gutkind JS (2008) Hypoxia-induced energy stress inhibits the mTOR pathway by activating an AMPK/REDD1 signaling axis in head and neck squamous cell carcinoma. Neoplasia 10(11):1295–1302. https://doi.org/10.1593/neo.08586
Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, Glass DJ (2004) The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors. Mol Cell 14(3):395–403. https://doi.org/10.1016/S1097-2765(04)00211-4
Tan CY, Hagen T (2013) Post-translational regulation of mTOR complex 1 in hypoxia and reoxygenation. Cell Signal 25(5):1235–1244. https://doi.org/10.1016/j.cellsig.2013.02.012
Tarum J, Folkesson M, Atherton PJ, Kadi F (2017) Electrical pulse stimulation: an in vitro exercise model for the induction of human skeletal muscle cell hypertrophy. A proof‐of‐concept study. Experimental Physiology 102(11):1405–1413. https://doi.org/10.1113/EP086581
Vermeeren MA, Schols AM, Wouters EF (1997) Effects of an acute exacerbation on nutritional and metabolic profile of patients with COPD. Eur Respir J 10(10):2264–2269. https://doi.org/10.1183/09031936.97.10102264
Veliça P, Bunce CM (2011) A quick, simple and unbiased method to quantify C2C12 myogenic differentiation. Muscle Ner 44(3):366–370. https://doi.org/10.1002/mus.22056
Vivodtzev I, Debigaré R, Gagnon P, Mainguy V, Saey D, Dubé A, Maltais F (2012) Functional and muscular effects of neuromuscular electrical stimulation in patients with severe COPD: a randomized clinical trial. Chest 141(3):716–725. https://doi.org/10.1378/chest.11-0839
Wang C, Liu W, Liu Z, Chen L, Liu X, Kuang S (2015) Hypoxia inhibits myogenic differentiation through p53 protein-dependent induction of Bhlhe40 protein. J Bio Chem 290(50):29707–29716. https://doi.org/10.1074/jbc.M115.688671
Zheng R, Huang S, Zhu J, Lin W, Xu H, Zheng X (2019) Leucine attenuates muscle atrophy and autophagosome formation by activating PI3K/AKT/mTOR signaling pathway in rotator cuff tears. Cell and Tissue Research 378(1):113–125. https://doi.org/10.1007/s00441-019-03021-x
Acknowledgements
We thank Elsa Heyman for statistical analysis assistance, Charlotte Claeyssen for technical assistance and Amir Yahya Rajaei for writing assistance.
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This work was supported by the French Ministry for Research and Tertiary Education. Samir Bensaid is a recipient from the Lille University Hospital Center.
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Conceived and designed the research: Bensaid S. and Cieniewski-Bernard C, Performed experiments: Bensaid S. and Cieniewski-Bernard C, Analysed data and interpreted results of experiments: Bensaid S, Fabre C, Pawlak-Chaouch M, Cieniewski-Bernard C, Edited and revised the article: Bensaid S, Fabre C, Cieniewski-Bernard C.
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Bensaid, S., Fabre, C., Pawlak-Chaouch, M. et al. Oxygen supplementation to limit hypoxia-induced muscle atrophy in C2C12 myotubes: comparison with amino acid supplement and electrical stimulation. Cell Tissue Res 387, 287–301 (2022). https://doi.org/10.1007/s00441-021-03492-x
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DOI: https://doi.org/10.1007/s00441-021-03492-x