title: Atmospheric oxygen tension slows myoblast proliferation via mitochondrial activation.
abstract: BACKGROUND
  Mitochondrial activity inhibits proliferation and is required for differentiation of myoblasts. Myoblast proliferation is also inhibited by the ~20% oxygen level used in standard tissue culture. We hypothesize that mitochondrial activity would be greater at hyperoxia (20% O(2)) relative to more physiological oxygen (5% O(2)).
  
  
  RESULTS
  Murine primary myoblasts from isolated myofibres and conditionally immortalized H-2K myoblasts were cultured at 5% and 20% oxygen. Proliferation, assayed by cell counts, EdU labeling, and CFSE dilution, was slower at 20% oxygen. Expression of MyoD in primary myoblasts was delayed at 20% oxygen, but myogenicity, as measured by fusion index, was slightly higher. FACS-based measurement of mitochondrial activity indicators and luminometric measurement of ATP levels revealed that mitochondria exhibited greater membrane potential and higher levels of Reactive Oxygen Species (ROS) at 20% oxygen with concomitant elevation of intracellular ATP. Mitochondrial mass was unaffected. Low concentrations of CCCP, a respiratory chain uncoupler, and Oligomycin A, an ATP synthase inhibitor, each increased the rate of myoblast proliferation. ROS were investigated as a potential mechanism of mitochondrial retrograde signaling, but scavenging of ROS levels by N-acetyl-cysteine (NAC) or α-Phenyl-N-tert-butylnitrone (PBN) did not rescue the suppressed rate of cell division in hyperoxic conditions, suggesting other pathways. Primary myoblasts from older mice showed a slower proliferation than those from younger adult mice at 20% oxygen but no difference at 5% oxygen.
  
  
  CONCLUSIONS
  These results implicate mitochondrial regulation as a mechanistic explanation for myoblast response to oxygen tension. The rescue of proliferation rate in myoblasts of aged mice by 5% oxygen suggests a major artefactual component to age-related decline of satellite cell proliferation in standard tissue culture at 20% oxygen. It lends weight to the idea that these age-related changes result at least in part from environmental factors rather than characteristics intrinsic to the satellite cell.
authors.affiliation: Research Center for Genetic Medicine, Children's National Medical Center, Washington, District of Columbia, United States of America.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Duguez
authors.forename: William J
authors.initials: WJ
authors.lastname: Duddy
authors.forename: Viola
authors.initials: V
authors.lastname: Gnocchi
authors.forename: James
authors.initials: J
authors.lastname: Bowe
authors.forename: Sherry
authors.initials: S
authors.lastname: Dadgar
authors.forename: Terence A
authors.initials: TA
authors.lastname: Partridge
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D018496:Hyperoxia / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008928:Mitochondria / Q000378:metabolism*; D017570:MyoD Protein / Q000378:metabolism; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000378:metabolism*; D051763:PAX7 Transcription Factor / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D017570:MyoD Protein; D051763:PAX7 Transcription Factor; C497322:Pax7 protein, mouse; D017382:Reactive Oxygen Species