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However, in general, they do not show the functional excitation\u2010contraction coupling or the striated sarcomere arrangement typical of mature myofibers. Epigenetic modifications have been shown to play a key role in regulating the progressional changes in transcription necessary for muscle differentiation. In this study, we demonstrate that treatment of murine C2C12 mesenchymal progenitor cells with 10 \u03bcM of the DNA methylation inhibitor 5\u2010azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the up\u2010regulation of muscle genes at the myoblast stage, while at later stages nearly 50% of the 5AC\u2010treated myotubes displayed a mature, well\u2010defined sarcomere organization, as well as spontaneous contractions that coincided with action potentials and intracellular calcium transients. Both the percentage of striated myotubes and their contractile activity could be inhibited by 20 nM TTX, 10 \u03bcM ryanodine, and 100 \u03bcM nifedipine, suggesting that action potential\u2010induced calcium transients are responsible for these characteristics. Our data suggest that genomic demethylation induced by 5AC overcomes an epigenetic barrier that prevents untreated C2C12 myotubes from reaching full maturity.\u2014Hupkes, M., Jonsson, M. K. B., Scheenen, W. J., van Rotterdam, W., Sotoca, A. M., van Someren, E. P., van der Heyden, M. A. G., van Veen, T. A., van Ravestein\u2010van Os, R. I., Bauerschmidt, S., Piek, E., Ypey, D. L., van Zoelen, E. J., Dechering, K. J. Epigenetics: DNA demethylation promotes skeletal myotube maturation.\n                    <jats:italic>FASEB J.<\/jats:italic>\n                    25, 3861\u20133872 (2011).\n                    <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"http:\/\/www.fasebj.org\">www.fasebj.org<\/jats:ext-link>\n                  <\/jats:p>","DOI":"10.1096\/fj.11-186122","type":"journal-article","created":{"date-parts":[[2011,7,28]],"date-time":"2011-07-28T00:11:52Z","timestamp":1311811912000},"page":"3861-3872","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":58,"title":["Epigenetics: DNA demethylation promotes skeletal myotube maturation"],"prefix":"10.1096","volume":"25","author":[{"given":"Marlinda","family":"Hupkes","sequence":"first","affiliation":[{"name":"Department of Cell and Applied Biology Nijmegen Centre for Molecular Life Sciences"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Malin K. 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G.","family":"van der Heyden","sequence":"additional","affiliation":[{"name":"Department of Medical Physiology, Division of Heart and Lungs University Medical Center Utrecht  Utrecht The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toon A.","family":"van Veen","sequence":"additional","affiliation":[{"name":"Department of Medical Physiology, Division of Heart and Lungs University Medical Center Utrecht  Utrecht The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roselinde I.","family":"van Ravestein\u2010van Os","sequence":"additional","affiliation":[{"name":"Merck Research Laboratories  Oss The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susanne","family":"Bauerschmidt","sequence":"additional","affiliation":[{"name":"Merck Research Laboratories  Oss The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ester","family":"Piek","sequence":"additional","affiliation":[{"name":"Department of Cell and Applied Biology Nijmegen Centre for Molecular Life Sciences"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dirk L.","family":"Ypey","sequence":"additional","affiliation":[{"name":"Department of Cardiology Leiden University Medical Centre  Leiden The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Everardus J.","family":"van Zoelen","sequence":"additional","affiliation":[{"name":"Department of Cell and Applied Biology Nijmegen Centre for Molecular Life Sciences"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koen J.","family":"Dechering","sequence":"additional","affiliation":[{"name":"Department of Cell and Applied Biology Nijmegen Centre for Molecular Life Sciences"},{"name":"Merck Research Laboratories  Oss The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2011,7,27]]},"reference":[{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tcb.2009.03.002"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.4161\/epi.4.8.10258"},{"key":"e_1_2_6_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.gde.2006.08.008"},{"key":"e_1_2_6_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0959-437X(00)00224-0"},{"key":"e_1_2_6_6_1","doi-asserted-by":"publisher","DOI":"10.1002\/jcp.20489"},{"key":"e_1_2_6_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2005.12.008"},{"key":"e_1_2_6_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/ng1990"},{"key":"e_1_2_6_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(79)90317-9"},{"key":"e_1_2_6_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(18)42351-4"},{"key":"e_1_2_6_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(87)90585-X"},{"key":"e_1_2_6_12_1","doi-asserted-by":"publisher","DOI":"10.1242\/dev.121.3.637"},{"key":"e_1_2_6_13_1","doi-asserted-by":"publisher","DOI":"10.1006\/dbio.1998.9126"},{"key":"e_1_2_6_14_1","doi-asserted-by":"publisher","DOI":"10.1006\/dbio.1996.0180"},{"key":"e_1_2_6_15_1","doi-asserted-by":"publisher","DOI":"10.4161\/cc.9.19.13193"},{"key":"e_1_2_6_16_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M008234200"},{"key":"e_1_2_6_17_1","doi-asserted-by":"publisher","DOI":"10.1128\/MCB.00050-10"},{"key":"e_1_2_6_18_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/29.21.4452"},{"key":"e_1_2_6_19_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/24.14.2718"},{"key":"e_1_2_6_20_1","first-page":"551","article-title":"Expression of an alternative Dnmt1 isoform during muscle differentiation","volume":"11","author":"Aguirre\u2010Arteta A. 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