title: Epigenetics: DNA demethylation promotes skeletal myotube maturation.
abstract: Mesenchymal progenitor cells can be differentiated in vitro into myotubes that exhibit many characteristic features of primary mammalian skeletal muscle fibers. However, in general, they do not show the functional excitation-contraction 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 μM of the DNA methylation inhibitor 5-azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the up-regulation of muscle genes at the myoblast stage, while at later stages nearly 50% of the 5AC-treated myotubes displayed a mature, well-defined 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 μM ryanodine, and 100 μM nifedipine, suggesting that action potential-induced 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.
authors.affiliation: Department of Cell and Applied Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands. m.hupkes@science.ru.nl
authors.forename: Marlinda
authors.initials: M
authors.lastname: Hupkes
authors.forename: Malin K B
authors.initials: MK
authors.lastname: Jonsson
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Scheenen
authors.forename: Walter
authors.initials: W
authors.lastname: van Rotterdam
authors.forename: Ana M
authors.initials: AM
authors.lastname: Sotoca
authors.forename: Eugene P
authors.initials: EP
authors.lastname: van Someren
authors.forename: Marcel A G
authors.initials: MA
authors.lastname: van der Heyden
authors.forename: Toon A
authors.initials: TA
authors.lastname: van Veen
authors.forename: Roselinde I
authors.initials: RI
authors.lastname: van Ravestein-van Os
authors.forename: Susanne
authors.initials: S
authors.lastname: Bauerschmidt
authors.forename: Ester
authors.initials: E
authors.lastname: Piek
authors.forename: Dirk L
authors.initials: DL
authors.lastname: Ypey
authors.forename: Everardus J
authors.initials: EJ
authors.lastname: van Zoelen
authors.forename: Koen J
authors.initials: KJ
authors.lastname: Dechering
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D020013:Calcium Signaling / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D019175:DNA Methylation / Q000187:drug effects / Q000502:physiology*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D044127:Epigenesis, Genetic; D020869:Gene Expression Profiling; D059630:Mesenchymal Stem Cells; D051379:Mice; D009119:Muscle Contraction / Q000187:drug effects; D024510:Muscle Development / Q000187:drug effects*; D018485:Muscle Fibers, Skeletal; D018482:Muscle, Skeletal / Q000166:cytology*; D009543:Nifedipine / Q000494:pharmacology; D012433:Ryanodine / Q000494:pharmacology; D012518:Sarcomeres / Q000187:drug effects / Q000648:ultrastructure; D013779:Tetrodotoxin / Q000494:pharmacology
chemical_list: D002614:Chelating Agents; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D012433:Ryanodine; D013779:Tetrodotoxin; D004533:Egtazic Acid; D009543:Nifedipine; D001374:Azacitidine