Journal of Physiology and Biochemistry

, Volume 75, Issue 3, pp 367–377 | Cite as

Impact of different methods of induction of cellular hypoxia: focus on protein homeostasis signaling pathways and morphology of C2C12 skeletal muscle cells differentiated into myotubes

  • Samir Bensaid
  • Claudine Fabre
  • Julie Fourneau
  • Caroline Cieniewski-BernardEmail author
Original Article


Hypoxia, occurring in several pathologies, has deleterious effects on skeletal muscle, in particular on protein homeostasis. Different induction methods of hypoxia are commonly used in cellular models to investigate the alterations of muscular function consecutive to hypoxic stress. However, a consensus is not clearly established concerning hypoxia induction methodology. Our aim was to compare oxygen deprivation with chemically induced hypoxia using cobalt chloride (CoCl2) or desferrioxamine (DFO) on C2C12 myotubes which were either cultured in hypoxia chamber at an oxygen level of 4% or treated with CoCl2 or DFO. For each method of hypoxia induction, we determined their impact on muscle cell morphology and on expression or activation status of key signaling proteins of synthesis and degradation pathways. The expression of HIF-1α increased whatever the method of hypoxia induction. Myotube diameter and protein content decreased exclusively for C2C12 myotubes submitted to physiological hypoxia (4% O2) or treated with CoCl2. Results were correlated with a hypophosphorylation of key proteins regulated synthesis pathway (Akt, GSK3-β and P70S6K). Similarly, the phosphorylation of FoxO1 decreased and the autophagy-related LC3-II was overexpressed with 4% O2 and CoCl2 conditions. Our results demonstrated that in vitro oxygen deprivation and the use of mimetic agent such as CoCl2, unlike DFO, induced similar responses on myotube morphology and atrophy/hypertrophy markers. Thus, physiological hypoxia or its artificial induction using CoCl2 can be used to understand finely the molecular changes in skeletal muscle cells and to evaluate new therapeutics for hypoxia-related muscle disorders.


Hypoxia Protein homeostasis Myotube Atrophy Cobalt chloride Desferrioxamine 



We thank Amandine Girard and Matthias Lambert for their technical assistance.

Author contributions

Samir Bensaid: Conceived, designed and performed the experiments, analysed the data and wrote the paper; Julie Fourneau: contributed to acquisition and analysis of the data; Claudine Fabre: conceived the experiments and wrote the paper; Caroline Cieniewski-Bernard: conceived and designed the experiments, performed the experiments, analysed the data and wrote the paper.

Compliance with ethical standards

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Informed consent

Not concerned.


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Copyright information

© University of Navarra 2019

Authors and Affiliations

  1. 1.Team Physical Activity, Muscle, HealthUniversity Lille - EA 7369 – URePSSSLilleFrance
  2. 2.Research PoleCHU LilleLilleFrance

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