title: Manipulation of environmental oxygen modifies reactive oxygen and nitrogen species generation during myogenesis.
abstract: Regulated changes in reactive oxygen and nitrogen species (RONS) activities are important in maintaining the normal sequence and development of myogenesis. Both excessive formation and reduction in RONS have been shown to affect muscle differentiation in a negative way. Cultured cells are typically grown in 20% O2 but this is not an appropriate physiological concentration for a number of cell types, including skeletal muscle. The aim was to examine the generation of RONS in cultured skeletal muscle cells under a physiological oxygen concentration condition (6% O2) and determine the effect on muscle myogenesis. Primary mouse satellite cells were grown in 20% or 6% O2 environments and RONS activity was measured at different stages of myogenesis by real-time fluorescent microscopy using fluorescent probes with different specificities i.e. dihydroethidium (DHE), 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) and 5-(and-6)-chloromethyl-2',7' -dichlorodihydrofluorescein diacetate (CM-DCFH-DA). Data demonstrate that satellite cell proliferation increased when cells were grown in 6% O2 compared with 20% O2. Myoblasts grown in 20% O2 showed an increase in DCF fluorescence and DHE oxidation compared with myoblasts grown at 6% O2. Myotubes grown in 20% O2 also showed an increase in DCF and DAF-FM fluorescence and DHE oxidation compared with myotubes grown in 6% O2. The catalase and MnSOD contents were also increased in myoblasts and myotubes that were maintained in 20% O2 compared with myoblasts and myotubes grown in 6% O2. These data indicate that intracellular RONS activities in myoblasts and myotubes at rest are influenced by changes in environmental oxygen concentration and that the increased ROS may influence myogenesis in a negative manner.
authors.affiliation: MRC-Arthritis Research UK Centre for Integrated Research into Musculoskeletal Ageing, Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool L7 8TX, UK.
authors.forename: Rachel
authors.initials: R
authors.lastname: McCormick
authors.affiliation: MRC-Arthritis Research UK Centre for Integrated Research into Musculoskeletal Ageing, Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool L7 8TX, UK.
authors.forename: Timothy
authors.initials: T
authors.lastname: Pearson
authors.affiliation: MRC-Arthritis Research UK Centre for Integrated Research into Musculoskeletal Ageing, Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool L7 8TX, UK. Electronic address: vasilaki@liverpool.ac.uk.
authors.forename: Aphrodite
authors.initials: A
authors.lastname: Vasilaki
mesh_terms: D000818:Animals; D002374:Catalase / Q000737:chemistry / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D024510:Muscle Development / Q000235:genetics*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D018482:Muscle, Skeletal / Q000254:growth & development / Q000378:metabolism*; D032446:Myoblasts / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D061251:Primary Cell Culture; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D032496:Satellite Cells, Skeletal Muscle / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism
chemical_list: C503301:4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; C029569:diacetyldichlorofluorescein; D002374:Catalase; D013482:Superoxide Dismutase; D009584:Nitrogen; D010100:Oxygen
keywords: Antioxidant defence enzymes; Low oxygen concentration; Reactive oxygen and nitrogen species; Skeletal muscle
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