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.
Highlights
Skeletal muscle satellite cells are ubiquitous mononuclear cells, classically identified by their location between the fibre plasmalemma and basal lamina [1]
There were some variations in the efficiency of proliferation and fusion; proliferation of satellite cells was clearly increased when cells were grown in 6% O2 compared with cells grown in 20% O2 (Fig. 1)
Myoblasts grown in 20% O2 showed a significant increase in DHE (Fig. 3A) and DCF (Fig. 3B) fluorescence at each time point compared with myoblasts grown in 6% O2
Summary
Skeletal muscle satellite cells are ubiquitous mononuclear cells, classically identified by their location between the fibre plasmalemma and basal lamina [1]. When skeletal muscle damage occurs, satellite cells within the muscle bulk are activated and proliferate to become myogenic precursor cells (mpc's otherwise known as myoblasts; [2]). Regeneration of skeletal muscle in vivo involves similar processes to those occurring during myogenesis and can be studied in wellcharacterised cell culture models. The environmental O2 concentration used for satellite cell cultivation in vitro is almost always 20%, whereas normal adult skeletal muscle tissue O2 levels are significantly lower, potentially between 1.8 and 10.5% [3,4]. Environmental oxygen concentration has been previously shown to modify satellite cell behaviour [3] in a process that has been linked to reactive oxygen species (ROS) generation [5]. The pattern of generation of specific ROS in skeletal muscle cells during the processes of myogenesis under different oxygen concentrations is currently unknown
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