PP62 - Manipulation of Environmental Oxygen Modifies Reactive Oxygen Species Generation during Myogenesis

Abstract

Regulated changes in ROS formation are important in myogenesis. Both excessive formation and reduction in ROS affect muscle differentiation in a negative way. Cultured cells are typically grown in 20% O 2 , but this is not physiological for a number of cell types, including skeletal muscle. The aim was to examine the generation of ROS in cultured skeletal muscle cells under more physiological conditions (6% O 2 ) and determine whether this affects muscle myogenesis. Primary muscle cells were cultured in 35mm gelatin coated tissue culture plates in DMEM containing 20% (v/v) FCS. To induce myotube formation the medium was replaced with DMEM containing 2% horse serum. Cultures were grown in 20% or 6% O 2 environments throughout myogenesis and ROS were monitored at different stages of myogenesis using dihydroethidium (DHE) and other fluorescent probes. Data demonstrate that proliferation of satellite cells was increased when cells were grown in 6% compared with 20% O 2 . Myoblasts grown in 20% O 2 showed an increase in DHE oxidation compared with myoblasts grown in 6% O 2 (1764.1±141.9 vs 1003.7±124.6 relative fluorescence units [RFU]). Myotubes differentiated in 20% O 2 also showed an increase in DHE oxidation compared with myotubes grown in 6% O 2 (2337.6±155.5 vs 1712.9±110.0 RFU). These data indicate that the oxidation of DHE (reflecting superoxide activity) in resting skeletal muscle myoblasts and myotubes is influenced by changes in environmental oxygen concentration and associated with inhibited myogenesis. Supported by Research into Ageing/AgeUK. Supported by Research into Ageing/AgeUK .