The Mechanisms of Cobaltous Chloride-Induced Hypoxia on Muscle Atrophy / 中国运动医学杂志

ABSTRACT

Objective To study the regulating mechanisms of cobaltous chloride (CoCl2)induced hypoxia on muscle atrophy.Methods The mouse skeletal C2C12 myotube line was used as the cell model and divided into a control group,a CoCl2 group,a 3-Methyladenine(3MA)group and a CoCl2+3MA group.The control group was not given any treatment,the CoCl2 group was treated with 200 μM CoCl2 to induce hypoxia,and the 3MA group or CoCl2+3MA group was treated with 5 mM 3MA in the absence or presence of 200 μM CoCl2.The Giemsa stain was conducted to observe the morphology of myotubes.The multifunctional microplate reader was utilized to quantify the reactive oxygen species (ROS)expression.The autophagosome was observed by using the transmission electron microscopy.The mRNA and protein expression of hypoxia-inducible factor-1α(HIF-1α),Bcl2/adenovirus E1B19kDa interacting protein 3(BNIP3),microtubule associated protein1 light chain 3(LC3)were detected using the quantitative real time polymerase chain reaction (QRT-PCR)and Western blotting (WB).Moreover,3-Methyladenine(3MA)was used to suppress autophagy and the expression of muscle atrophy F-box(MAF-bx)was detected.Results More spindly ring-shaped myotubes were formed in the control group,while CoCl2 induced myotubes atrophy and rupture.The expression of ROS increased significantly in the CoCl2 group compared with the control group(t=-4.965,P=0.008).Transmission electron microscopy results showed autophagosome formation induced by CoCl2,and significant improvement in the HIF-1α,BNIP3 and LC3 in the CoCl2 group compared with the control group(P＜0.05).The protein expression of MAFbx decreased when co-cultured with CoCl2 and 3MA(F=18.246,P=0.001).Conclusions The skeletal muscle atrophy caused by CoCl2 induced hypoxia may be associated with autophagy promoted by the HIF-1α/BNIP3 signal pathway,and inhibition of hypoxia induced autophagy can partly reduce the atrophy in skeletal C2C12 myotubes.