[en] [en] BACKGROUND: Hypoxia is known to modify skeletal muscle biological functions and muscle regeneration. However, the mechanisms underlying the effects of hypoxia on human myoblast differentiation remain unclear. The hypoxic response pathway is of particular interest in patients with hereditary muscular dystrophies since many present respiratory impairment and muscle regeneration defects. For example, an altered hypoxia response characterizes the muscles of patients with facioscapulohumeral dystrophy (FSHD).
METHODS: We examined the impact of hypoxia on the differentiation of human immortalized myoblasts (LHCN-M2) cultured in normoxia (PO2: 21%) or hypoxia (PO2: 1%). Cells were grown in proliferation (myoblasts) or differentiation medium for 2 (myocytes) or 4 days (myotubes). We evaluated proliferation rate by EdU incorporation, used myogenin-positive nuclei as a differentiation marker for myocytes, and determined the fusion index and myosin heavy chain-positive area in myotubes. The contribution of HIF1α was studied by gain (CoCl2) and loss (siRNAs) of function experiments. We further examined hypoxia in LHCN-M2-iDUX4 myoblasts with inducible expression of DUX4, the transcription factor underlying FSHD pathology.
RESULTS: We found that the hypoxic response did not impact myoblast proliferation but activated precocious myogenic differentiation and that HIF1α was critical for this process. Hypoxia also enhanced the late differentiation of human myocytes, but in an HIF1α-independent manner. Interestingly, the impact of hypoxia on muscle cell proliferation was influenced by dexamethasone. In the FSHD pathological context, DUX4 suppressed HIF1α-mediated precocious muscle differentiation.
CONCLUSION: Hypoxia stimulates myogenic differentiation in healthy myoblasts, with HIF1α-dependent early steps. In FSHD, DUX4-HIF1α interplay indicates a novel mechanism by which DUX4 could interfere with HIF1α function in the myogenic program and therefore with FSHD muscle performance and regeneration.
Disciplines :
Biochemistry, biophysics & molecular biology Cardiovascular & respiratory systems
Author, co-author :
Nguyen, Thuy-Hang; Laboratory of Respiratory Physiology, Pathophysiology and Rehabilitation, Research Institute for Health Sciences and Technology, University of Mons, Mons, 7000, Belgium
Paprzycki, Lise ; Université de Mons - UMONS > Faculté de Médecine et de Pharmac > Service de Physiologie et réadaptation respiratoire
Legrand, Alexandre ; Université de Mons - UMONS > Faculté de Médecine et de Pharmac > Service de Physiologie et réadaptation respiratoire
Decleves, Anne-Emilie ; Université de Mons - UMONS > Faculté de Médecine et de Pharmac > Service de Biochimie métabolique et moléculaire
Heher, Philipp; Randall Centre for Cell and Molecular Biophysics, King's College London, Guy's Campus, London, SE1 1UL, UK
Limpens, Maelle; Laboratory of Respiratory Physiology, Pathophysiology and Rehabilitation, Research Institute for Health Sciences and Technology, University of Mons, Mons, 7000, Belgium
Belayew, Alexandra ; Université de Mons - UMONS > Faculté de Médecine et de Pharmac > Service du Doyen de la Faculté de Médecine et Pharmacie
Banerji, Christopher R S; Randall Centre for Cell and Molecular Biophysics, King's College London, Guy's Campus, London, SE1 1UL, UK ; The Alan Turing Institute, British Library, 96 Euston Rd, London, UK
Zammit, Peter S; Randall Centre for Cell and Molecular Biophysics, King's College London, Guy's Campus, London, SE1 1UL, UK
Tassin, Alexandra ; Université de Mons - UMONS > Faculté de Médecine et de Pharmac > Service de Physiologie et réadaptation respiratoire
Language :
English
Title :
Hypoxia enhances human myoblast differentiation: involvement of HIF1α and impact of DUX4, the FSHD causal gene.
M117 - Physiologie et réadaptation respiratoire M122 - Biochimie métabolique et moléculaire
Research institute :
R550 - Institut des Sciences et Technologies de la Santé
Funders :
Fonds De La Recherche Scientifique - FNRS ABMM - Association Belge contre les Maladies neuro-Musculaires Amis FSH Medical Research Council Austrian Science Fund FSHD Society Friends of FSH Research
Funding text :
THN and ML held a FRIA doctoral fellowship (FC 29703 and FC 47057, respectively) from the National Fund for Scientific Research (F.R.S – FNRS), Belgium. LP had a UMONS-UNAMUR doctoral fellowship. This scientific study was funded by the French non-profit organization AMIS FSH whose objective is to heal and support patients suffering from Facio Scapulo Humeral Dystrophy. THN also acknowledges funding from ABMM (Belgium). PH was mainly funded by the Medical Research Council (MR/P023215/1) and then by an Erwin Schroedinger post-doctoral fellowship awarded by the Austrian Science Fund (FWF, J4435-B), supported by Friends of FSH Research (Project 936270) and the FSHD Society (FSHD-Fall2020-3308289076) and currently the Medical Research Council (MR/S002472/1).
Semenza GL. HIF-1, O(2), and the 3 PHDs: how animal cells signal hypoxia to the nucleus. Cell. 2001;107:1–3.
Forcina L, Miano C, Pelosi L, Musaro A. An Overview about the Biology of Skeletal Muscle Satellite Cells. Curr Genomics. 2019;20:24–37.
Zammit PS. Function of the myogenic regulatory factors Myf5, MyoD, Myogenin and MRF4 in skeletal muscle, satellite cells and regenerative myogenesis. Semin Cell Dev Biol. 2017;72:19–32.
Nguyen T-H, Conotte S, Belayew A, Declèves A-E, Legrand A, Tassin A. Hypoxia and hypoxia-inducible factor signaling in muscular dystrophies: cause and consequences. Int J Mol Sci. 2021;22:7220.
Majmundar AJ, Lee DSM, Skuli N, Mesquita RC, Kim MN, Yodh AG, et al. HIF modulation of Wnt signaling regulates skeletal myogenesis in vivo. Dev Camb Engl. 2015;142:2405–12.
Cirillo F, Resmini G, Ghiroldi A, Piccoli M, Bergante S, Tettamanti G, et al. Activation of the hypoxia-inducible factor 1a promotes myogenesis through the noncanonical Wnt pathway, leading to hypertrophic myotubes. FASEB J. 2017;31:2146–56.
Jash S, Adhya S. Effects of transient hypoxia versus prolonged hypoxia on satellite cell proliferation and differentiation in vivo. Stem Cells Int. 2015;2015:e961307.
Sakushima K, Yoshikawa M, Osaki T, Miyamoto N, Hashimoto T. Moderate hypoxia promotes skeletal muscle cell growth and hypertrophy in C2C12 cells. Biochem Biophys Res Commun. 2020;525:921–7.
Koning M, Werker PMN, van Luyn MJA, Harmsen MC. Hypoxia promotes proliferation of human myogenic satellite cells: a potential benefactor in tissue engineering of skeletal muscle. Tissue Eng Part A. 2011;17:1747–58.
Launay T, Hagström L, Lottin-Divoux S, Marchant D, Quidu P, Favret F, et al. Blunting effect of hypoxia on the proliferation and differentiation of human primary and rat L6 myoblasts is not counteracted by Epo. Cell Prolif. 2010;43:1–8.
Yang X, Yang S, Wang C, Kuang S. The hypoxia-inducible factors HIF1α and HIF2α are dispensable for embryonic muscle development but essential for postnatal muscle regeneration. J Biol Chem. 2017;292:5981–91.
Niemi H, Honkonen K, Korpisalo P, Huusko J, Kansanen E, Merentie M, et al. HIF-1α and HIF-2α induce angiogenesis and improve muscle energy recovery. Eur J Clin Invest. 2014;44:989–99.
Olfert IM, Howlett RA, Wagner PD, Breen EC. Myocyte vascular endothelial growth factor is required for exercise-induced skeletal muscle angiogenesis. Am J Physiol Regul Integr Comp Physiol. 2010;299:R1059–1067.
Settelmeier S, Schreiber T, Mäki J, Byts N, Koivunen P, Myllyharju J, et al. Prolyl hydroxylase domain 2 reduction enhances skeletal muscle tissue regeneration after soft tissue trauma in mice. PLoS ONE. 2020;15:e0233261.
Banerji CRS, Knopp P, Moyle LA, Severini S, Orrell RW, Teschendorff AE, et al. β-Catenin is central to DUX4-driven network rewiring in facioscapulohumeral muscular dystrophy. J R Soc Interface. 2015;12:20140797.
Banerji CRS, Panamarova M, Hebaishi H, White RB, Relaix F, Severini S, et al. PAX7 target genes are globally repressed in facioscapulohumeral muscular dystrophy skeletal muscle. Nat Commun. 2017;8:2152.
Wang LH, Tawil R. Facioscapulohumeral dystrophy. Curr Neurol Neurosci Rep. 2016;16:66.
Dixit M, Ansseau E, Tassin A, Winokur S, Shi R, Qian H, et al. DUX4, a candidate gene of facioscapulohumeral muscular dystrophy, encodes a transcriptional activator of PITX1. Proc Natl Acad Sci U S A. 2007;104:18157–62.
Lemmers RJLF, van der Vliet PJ, Klooster R, Sacconi S, Camaño P, Dauwerse JG, et al. A unifying genetic model for facioscapulohumeral muscular dystrophy. Science. 2010;329:1650–3.
Geng LN, Yao Z, Snider L, Fong AP, Cech JN, Young JM, et al. DUX4 activates germline genes, retroelements and immune-mediators: implications for facioscapulohumeral dystrophy. Dev Cell. 2012;22:38–51.
Snider L, Geng LN, Lemmers RJLF, Kyba M, Ware CB, Nelson AM, et al. Facioscapulohumeral dystrophy: incomplete suppression of a retrotransposed gene. PLoS Genet. 2010;6:e1001181.
Vanderplanck C, Ansseau E, Charron S, Stricwant N, Tassin A, Laoudj-Chenivesse D, et al. The FSHD atrophic myotube phenotype is caused by DUX4 expression. PLoS One. 2011;6:e26820.
Lim KRQ, Nguyen Q, Yokota T. DUX4 Signalling in the Pathogenesis of Facioscapulohumeral Muscular Dystrophy. Int J Mol Sci. 2020;21:729.
Jagannathan S, Ogata Y, Gafken PR, Tapscott SJ, Bradley RK. Quantitative proteomics reveals key roles for post-transcriptional gene regulation in the molecular pathology of facioscapulohumeral muscular dystrophy. eLife. 2019;8:e41740.
Banerji CRS, Zammit PS. Pathomechanisms and biomarkers in facioscapulohumeral muscular dystrophy: roles of DUX4 and PAX7. EMBO Mol Med. 2021;13:e13695.
Tsumagari K, Chang S-C, Lacey M, Baribault C, Chittur SV, Sowden J, et al. Gene expression during normal and FSHD myogenesis. BMC Med Genomics. 2011;4:67.
Lek A, Zhang Y, Woodman KG, Huang S, DeSimone AM, Cohen J, et al. Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy. Sci Transl Med. 2020;12(536):eaay0271.
Nguyen T-H, Bouhmidi S, Paprzycki L, Legrand A, Declèves A-E, Heher P, et al. The DUX4-HIF1α axis in murine and human muscle cells: a link more complex than expected [Internet]. Preprints; 2022. [cited 2022 Dec 19]. Available from: https://www.preprints.org/manuscript/202211.0532/v2.
Barro M, Carnac G, Flavier S, Mercier J, Vassetzky Y, Laoudj-Chenivesse D. Myoblasts from affected and non-affected FSHD muscles exhibit morphological differentiation defects. J Cell Mol Med. 2010;14:275–89.
Banerji CRS, Panamarova M, Pruller J, Figeac N, Hebaishi H, Fidanis E, et al. Dynamic transcriptomic analysis reveals suppression of PGC1α/ERRα drives perturbed myogenesis in facioscapulohumeral muscular dystrophy. Hum Mol Genet. 2019;28:1244–59.
Banerji CRS, Henderson D, Tawil RN, Zammit PS. Skeletal muscle regeneration in facioscapulohumeral muscular dystrophy is correlated with pathological severity. Hum Mol Genet. 2020;29:2746–60.
Ganassi M, Muntoni F, Zammit PS. Defining and identifying satellite cell-opathies within muscular dystrophies and myopathies. Exp Cell Res. 2022;411:112906.
Ganassi M, Zammit PS. Involvement of muscle satellite cell dysfunction in neuromuscular disorders: expanding the portfolio of satellite cell-opathies. Eur J Transl Myol. 2022;32:10064.
Krom YD, Dumonceaux J, Mamchaoui K, den Hamer B, Mariot V, Negroni E, et al. Generation of isogenic D4Z4 contracted and noncontracted immortal muscle cell clones from a mosaic patient. Am J Pathol. 2012;181:1387–401.
Choi SH, Gearhart MD, Cui Z, Bosnakovski D, Kim M, Schennum N, et al. DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes. Nucleic Acids Res. 2016;44:5161–73.
Bosnakovski D, Gearhart MD, Toso EA, Ener ET, Choi SH, Kyba M. Low level DUX4 expression disrupts myogenesis through deregulation of myogenic gene expression. Sci Rep. 2018;8:16957.
Chaillou T, Lanner JT. Regulation of myogenesis and skeletal muscle regeneration: effects of oxygen levels on satellite cell activity. FASEB J Off Publ Fed Am Soc Exp Biol. 2016;30:3929–41.
Kook S-H, Son Y-O, Lee K-Y, Lee H-J, Chung W-T, Choi K-C, et al. Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD. Cell Biol Int. 2008;32:871–8.
Di Carlo A, De Mori R, Martelli F, Pompilio G, Capogrossi MC, Germani A. Hypoxia inhibits myogenic differentiation through accelerated MyoD degradation. J Biol Chem. 2004;279:16332–8.
Khanna S, Roy S, Maurer M, Ratan RR, Sen CK. Oxygen-sensitive reset of inducible HIF transactivation response: prolyl hydroxylases tune the biological normoxic setpoint. Free Radic Biol Med. 2006;40:2147–54.
Bylund-Fellenius AC, Walker PM, Elander A, Holm S, Holm J, Scherstén T. Energy metabolism in relation to oxygen partial pressure in human skeletal muscle during exercise. Biochem J. 1981;200:247–55.
Ikossi DG, Knudson MM, Morabito DJ, Cohen MJ, Wan JJ, Khaw L, et al. Continuous muscle tissue oxygenation in critically injured patients: a prospective observational study. J Trauma. 2006;61:780–8; discussion 788–790.
Boekstegers P, Riessen R, Seyde W. Oxygen partial pressure distribution within skeletal muscle: indicator of whole body oxygen delivery in patients? Adv Exp Med Biol. 1990;277:507–14.
Carreau A, Hafny-Rahbi BE, Matejuk A, Grillon C, Kieda C. Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J Cell Mol Med. 2011;15:1239–53.
Lin J-W, Huang Y-M, Chen Y-Q, Chuang T-Y, Lan T-Y, Liu Y-W, et al. Dexamethasone accelerates muscle regeneration by modulating kinesin-1-mediated focal adhesion signals. Cell Death Discov. 2021;7:1–16.
Elashry MI, Kinde M, Klymiuk MC, Eldaey A, Wenisch S, Arnhold S. The effect of hypoxia on myogenic differentiation and multipotency of the skeletal muscle-derived stem cells in mice. Stem Cell Res Ther. 2022;13:56.
Pirkmajer S, Filipovic D, Mars T, Mis K, Grubic Z. HIF-1alpha response to hypoxia is functionally separated from the glucocorticoid stress response in the in vitro regenerating human skeletal muscle. Am J Physiol Regul Integr Comp Physiol. 2010;299:R1693–1700.
Pandey SN, Khawaja H, Chen Y-W. Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts. Mol Basel Switz. 2015;20:8304–15.
Pircher T, Wackerhage H, Aszodi A, Kammerlander C, Böcker W, Saller MM. Hypoxic Signaling in Skeletal Muscle Maintenance and Regeneration: A Systematic Review. Front Physiol. 2021;12:684899.
Heher P, Ganassi M, Weidinger A, Engquist EN, Pruller J, Nguyen TH, et al. Interplay between mitochondrial reactive oxygen species, oxidative stress and hypoxic adaptation in facioscapulohumeral muscular dystrophy: Metabolic stress as potential therapeutic target. Redox Biol. 2022;51:102251.
Ganassi M, Figeac N, Reynaud M, Ortuste Quiroga HP, Zammit PS. Antagonism between DUX4 and DUX4c highlights a pathomechanism operating through β-catenin in facioscapulohumeral muscular dystrophy. Front Cell Dev Biol. 2022;10:802573.
Knopp P, Krom YD, Banerji CRS, Panamarova M, Moyle LA, den Hamer B, et al. DUX4 induces a transcriptome more characteristic of a less-differentiated cell state and inhibits myogenesis. J Cell Sci. 2016;129:3816–31.
Echeverri CJ, Beachy PA, Baum B, Boutros M, Buchholz F, Chanda SK, et al. Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat Methods. 2006;3:777–9.
Echeverri CJ, Perrimon N. High-throughput RNAi screening in cultured cells: a user’s guide. Nat Rev Genet. 2006;7:373–84.
Ganassi M, Badodi S, Wanders K, Zammit PS, Hughes SM. Myogenin is an essential regulator of adult myofibre growth and muscle stem cell homeostasis. eLife. 2020;9:e60445.
Agley CC, Velloso CP, Lazarus NR, Harridge SDR. An image analysis method for the precise selection and quantitation of fluorescently labeled cellular constituents. J Histochem Cytochem. 2012;60:428–38.
