Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest.

Abstract

OBJECTIVES: Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts. MATERIALS AND METHODS: Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot. RESULTS AND CONCLUSIONS: We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts.