Effect of acute low oxygen exposure on the proliferation rate, viability and gene expression of C2C12 myoblasts in vitro

Abstract

INTRODUCTIONChanges in the oxygen concentration of cellular microenvironments play a significant role in regulating cell function during muscle regeneration. Generally, most in-vitro cell culture experiments have been carried out in atmospheric conditions with 21% O2, which compared to the actual micro-environment of mature skeletal muscle of between 1% and 10% pO2 is extremely hyperoxic (Li et al, 2007). Culturing skeletal muscle cells in vitro within their typical physiologically hypoxic environment in situ (2-10% pO2) has been shown to increase proliferation rate, reduce apoptosis and increase multiple MRF gene expressions, compared to culturing in a normoxic environment (21% O2). However, chronic exposure (>24 hr) to a semi-severe hypoxic environment ([≤]5% O2) can lead to a decrease in cell proliferation and differentiation (Chakravarthy et al, 2001). The effects of acute hypoxic exposure (24 h) has limited research and could be important in understanding the effects of hypoxia on skeletal muscle during brief exposures such as those observed within intermittent hypoxic training programmes. The purpose of this work was to examine the role of acute hypoxia (24 h) on C2C12 proliferation and relevant gene expression in 2D culture. METHODSC2C12 mouse myoblast cells were seeded into six well plates. The cells were maintained in DMEM with 20% FCS. C2C12 myoblasts were either exposed to 21% or 5% O2. The effects of acute hypoxic exposure (24hours) at different time points during the proliferative phase of myogenesis, rather than chronic exposure, on cell proliferation, cellular viability and myogenic regulatory factor gene expression was examined. At 24, 48, 72 and 96 hours RNA was extracted using the Trizol(R) method and mRNA expression of myogenic regulatory factors, myoD, myf5 and myogenin were detected using the 2-{Delta}{Delta}CT method. Cell counts and cell viability were also quantified RESULTSNo significant difference was found between cells cultured in normoxic conditions (21%) and those that were exposed to low oxygen conditions for 24hours at various time points over a 96 hour culture period, with regards to proliferation rate, cell viability, and myogenic regulatory gene expression (Myf5, MyoD and Myogenin). DISCUSSIONThe effect of acute low oxygen exposure lasting 24hours appears to not be insufficient in having an effect on the proliferation rate, viability or transcription factor expression of C2C12 cells during the proliferative phase of myogenesis.