Date of Award
Master of Science in Biology
Hypoxic injury in skeletal muscle occurs in response to musculoskeletal traumas, diseases and following reconstructive and transplantation surgeries. However, the molecular mechanisms responsible for hypoxic injury in skeletal myoblasts have yet to be fully investigated. Understanding of these molecular mechanisms would identify potential therapeutic targets. Herein, we have determined that treatment with cobalt chloride (CoCl2, a hypoxia mimicking agent) first leads to an inhibition of proliferation followed by a decrease in the number of viable myoblasts over time. FACS analysis revealed a marked increase in G2/M arrest after 24 hours of treatment. We next assessed PARP cleavage, a well-known hallmark of apoptosis, and detected cleavage after 48 hours, suggesting apoptosis as a mechanism contributing to myoblast death. Since we have recently determined that PUMA (p53 up-regulated modulator of apoptosis) plays a critical role in the apoptosis associated with skeletal myoblast differentiation, we assessed the level of PUMA expression in response to CoCl2 treatment and documented an increase in PUMA protein and mRNA. We did find an increase in p53 expression suggesting that this transcription factor is responsible for the increased expression of PUMA. Moreover, this increase in PUMA expression occurs under condition of eIF2a phosphorylation, which implies the possibility of IRES (Internal Ribosome Entry Site)-mediated regulation of PUMA expression under hypoxic conditions. Indeed, actinomycin D induced abrogation of the increase in PUMA mRNA did not prevent the increase in PUMA protein. In skeletal myoblasts, the processes of differentiation and apoptosis are coordinately regulated during myogenesis and muscle regeneration and this coordinated regulation is mediated by the transcription factor MyoD. Thus, we next assessed the effect of CoCl2 treatment on MyoD expression and documented decreased levels of MyoD protein and mRNA. Although we have also determined that the decrease in mRNA was not a consequence of a shortened half-life suggesting an effect on SRF, the transcription factor controlling MyoD expression, we were unable to detect a change in SRF expression as a consequence of CoCl2 treatment. Future experiments will assess the activation of SRF.
Zhao, Shuai, "Effects of Hypoxic Conditions on Skeletal Myoblasts" (2016). ETD Archive. 939.