Differential Responses of Myogenic C2C12 Cells to Hypoxia between Growth and Muscle-Induction Phases: Growth, Differentiation and Motility

Abstract

Abstract.[Introduction] Local hypoxia plays a favorable role in muscle regeneration. [Subjects and Methods] The effect of hypoxia on cell growth, differentiation, and motility was examined in differentiating and growing C2C12 mouse myoblasts cells, respectively.[Results] Hypoxia induced growth suppression in the growth phase, but the suppression diminished in the differentiation phase. Hypoxia inhibited the expression of differentiation marker proteins, accompanying MyoD mRNA suppression. Expression of HIF-1α protein was induced only in the induction phase, regardless of oxygen concentration. Hypoxia did not alter motile activity in the growing phase, but augmented the motile property in the differentiation phase. Expression of autocrine motility factor mRNA was augmented under hypoxic conditions. [Conclusion] In differentiating cells, HIF-1α induced by myogenic differentiation may compensate for the cell growth suppression due to hypoxia, and support the motility augmentation. Hypoxia may shift differentiating cells into the growth phase which provides the ability to translocate to an appropriate area.

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

“…The design study aimed to establish whether acute exposure is effective at eliciting a change in proliferative capacity or MRF expression and if so then at which stage of the proliferative phase of myogenesis should it be used. Sato et al (2011) looked at the effect of culturing C2C12's continuously for 122hours at 2%, 5% and 10% oxygen levels. Interestingly they found that growth rate of the cells in the hypoxic environments was the same as cells under normal conditions for upto 2 days after plating before growth rate increased (Sato et al, 2011).…”

“…Sato et al (2011) looked at the effect of culturing C2C12's continuously for 122hours at 2%, 5% and 10% oxygen levels. Interestingly they found that growth rate of the cells in the hypoxic environments was the same as cells under normal conditions for upto 2 days after plating before growth rate increased (Sato et al, 2011). Lui et al (2012) found that the initial 48hours of culturing primary murine myoblasts in 1% pO2 affected myoblast fate decision by promoting self-renewal and inhibiting differentiation, but overall cell proliferation was not affected.…”

“…Nevertheless, the results of this study with that of recent literature, do tend to indicate 24hours of exposure to a low, physiological oxygen environment may not be an adequate exposure time to induce an effect on cell proliferation rate. It is still unclear whether the hypoxic condition used in the present study reflects the drop in intramyocellular oxygen pressure induced by exercise, muscular damage or hypoxic exposure (Sato et al, 2011;Hoppeler et al, 2003), however when using manipulation of O2 concentrations in improving muscle self renewal for damage repair or treating regenerative disease, it may be possible that exposure times must be longer than 24hours to be effective.…”

“…Aswell as chronic low oxygen exposure being known to increase expression of all these transcription factors, short term exposure has been shown to upregulate the myogenic regulatory factors Myf5 and MyoD when at 6% O2 (Csete et al, 2001). When culturing cells in more extreme hypoxic conditions (2 -0.1% pO2), there appears to be a down regulation of Myf5 and MyoD expression (Sato et al, 2011;Koning et al, 2011, Lui et al 2012. As no research has yet looked at the effects acute exposure at 5% O2 has on these specific MRF's, it may be possible that 5% O2 is the cut off point between which hypoxia either starts having an up-regulatory or down-regulatory effect on Myf5 and MyoD and expression.…”

“…When the pO2 is dropped below 2% within the microenvironment, also known as pathological hypoxia, the expression of differentiation markers (MyoD, Myf5, and Myogenin) begin to be inhibited, along with the inhibition of quiescient satellite cell activation and of multinucleated myotube formation (Roy et al, 2003). Furthermore there is a notable reduction in satellite cell proliferation and increased apoptosis (Sato et al, 2011;Di Carlo et al, 2004). The inhibition of myogenic differentiation is found to be through accelerated MyoD degredation by the ubiquitin-proteasome pathway (DiCarlo et al, 2004).…”