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
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
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DOI：
10.1007/s13105-019-00687-3
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发表时间：
2019-08-01
影响因子：
3.4
通讯作者：
Cieniewski-Bernard, Caroline
中科院分区：
文献类型：
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作者：
Bensaid, Samir;Fabre, Claudine;Cieniewski-Bernard, Caroline
缺氧，发生在几种病理，对骨骼肌，特别是蛋白质稳态有害的影响。不同的缺氧诱导方法常用于细胞模型中，以研究缺氧应激后肌肉功能的变化。然而，关于低氧诱导方法学尚未明确建立共识。我们的目的是比较缺氧与化学诱导的缺氧，使用氯化钴（CoCl 2）或去铁胺（DFO）C2 C12肌管，无论是在缺氧室培养在4%的氧气水平或处理与CoCl 2或DFO。对于每种缺氧诱导方法，我们确定了它们对肌细胞形态以及合成和降解途径的关键信号蛋白的表达或活化状态的影响。无论采用何种缺氧诱导方法，HIF-1 α的表达均增加。肌管直径和蛋白质含量下降专门为C2 C12肌管提交生理性缺氧（4%O-2）或处理与氯化钴。结果与调节合成途径的关键蛋白（Akt、GSK 3-β和P70 S6 K）的低磷酸化相关。同样，FoxO 1的磷酸化减少，自噬相关的LC 3-II在4%O-2和CoCl 2条件下过表达。我们的研究结果表明，在体外缺氧和使用模拟剂，如氯化钴，不像DFO，诱导类似的反应，肌管形态和萎缩/肥大标志物。因此，生理性缺氧或其人工诱导使用氯化钴可用于精细地了解骨骼肌细胞的分子变化，并评估新的治疗缺氧相关的肌肉疾病。
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-1 alpha increased whatever the method of hypoxia induction. Myotube diameter and protein content decreased exclusively for C2C12 myotubes submitted to physiological hypoxia (4% O-2) or treated with CoCl2. Results were correlated with a hypophosphorylation of key proteins regulated synthesis pathway (Akt, GSK3-beta and P70S6K). Similarly, the phosphorylation of FoxO1 decreased and the autophagy-related LC3-II was overexpressed with 4% O-2 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.