Effects of Cobalt Chloride, a Hypoxia-Mimetic Agent, on Autophagy and Atrophy in Skeletal C2C12 Myotubes

Abstract

Background: Hypoxia-induced autophagy and muscle wasting occur in several environmental and pathological conditions. However, the molecular mechanisms underlying the effects of the hypoxia-mimetic agent CoCl₂ on autophagy and muscle atrophy are still unclear.

Methods: C2C12 myotubes were exposed to increasing concentrations of CoCl₂ for 24 hours. Quantitative RT-PCR, Western blotting, and transmission electron microscopy were performed to confirm autophagy occurs. Autophagy proteins were measured to understand the molecule mechanisms. We also inhibited hypoxic autophagy and examined the changes in myogenin expression, myotubes formation, and apoptosis.

Results: Our results showed that CoCl₂-mimicked hypoxia upregulated the expression of the autophagy-related proteins LC3, HIF-1α, BNIP3, p-AMPKα, and beclin-1, whereas p62 and p-mTOR were downregulated. In addition, the autophagosome could be observed after CoCl₂ induction. The expression of the autophagy-related E3 ligase parkin and the muscle-specific ubiquitin ligase atrogin-1 was increased by CoCl₂. Inhibition of autophagy by 3MA increased myogenin expression and promoted myotubes formation and the percentage of cell death was decreased.

Conclusions: Our results confirmed that CoCl₂-mimicked hypoxia induced autophagy via the HIF-1α/BNIP3/beclin-1 and AMPK/mTOR pathways. Our results also revealed an important link between autophagy and muscle atrophy under hypoxia, which may help to develop new therapeutic strategies for muscle diseases.

Figure 1

CoCl₂ induced C2C12 myotubes autophagy. The C2C12 myotubes were incubated with different dilutions of CoCl₂ (10, 50, 100, or 200 μM) for 24 hours. QRT-PCR and Western blotting analysis were, respectively, used to determine the mRNA and protein levels of LC3 and p62 in C2C12 cells treated with CoCl₂ (a–d). The bands were quantified using Image J and tubulin was used as the internal control. ^∗P < 0.05 and ^∗∗P < 0.01 compared to control group. (e) Transmission electron microscopy images of C2C12 cells showing increased numbers of autophagosomes in the CoCl₂ (200 μM) group (E2) compared to the normal group (E1). Scale bar: 500 nm. The red arrows mean autophagosomes.

Figure 2

3MA and CQ inhibited autophagy induced by CoCl₂. Western blotting analysis revealed the protein level of LC3II/LC3Ι treated with 3MA (a) or CQ (b) in the presence and absence of CoCl₂ in C2C12 cells. The bands were quantified using Image J and the expression levels of LC3II/LC3Ι were normalized relative to tubulin. ^∗P < 0.05 and ^∗∗P < 0.01 compared to control group.

Figure 3

CoCl₂ induced autophagy via the HIF-1α/BNIP3/beclin-1 and AMPKα/mTOR pathways. Western blotting analysis revealed the protein levels of HIF-1α/BNIP3/beclin-1 (a) and p-AMPKα/AMPKα and p-mTOR/mTOR (b) in C2C12 cells treated with CoCl₂. The bands were quantified using Image J and the expression levels of proteins were normalized relative to tubulin. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 compared to control group.

Figure 4

CoCl₂ promoted C2C12 cells protein degradation. QRT-PCR and Western blotting were used to determine the mRNA and protein levels of parkin (a and b) and atrogin-1 (c and d) in C2C12 cells treated with CoCl₂. The bands were quantified using Image J and the expression levels of parkin and atrogin-1 were normalized relative to tubulin. ^∗P < 0.05 compared to control group.

Figure 5

Inhibition of autophagy induced by cobalt chloride promoted cell survival in C2C12 myotubes. (a) Western blotting result showed the protein level of C2C12 myotubes treated with CoCl₂ with/without 3MA. The bands were quantified by Image J and the expression level of myogenin was normalized relative to tubulin. (b) Giemsa staining images showing morphology changes in C2C12 cells. Pictures were taken at the same magnification (40x). Scale bar: 100 μm. (B1) Control group, (B2) 200 μM CoCl₂ group, (B3) 5 mM 3MA group, and (B4) 200 μM CoCl₂ + 5 mM 3MA group. (c, d) Flow cytometry for apoptosis stained with Annexin V-FITC on the x-axis and 7-ADD on the y-axis. ^∗∗P < 0.01 compared to control group; ^##P < 0.01 compared to CoCl₂ + 3MA group.