Effects of acute hypoxia exposure with different durations on activation of Nrf2-ARE pathway in mouse skeletal muscle

Abstract

Background: Hypoxia training enhances the endurance capacity of athletes. This response may in part be attributed to the hypoxia-induced increase in antioxidant capacity in skeletal muscles. Nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor which regulates the expression of genes via binding to the antioxidant-response element (ARE) of these genes, plays a crucial role in stimulating the body's defense system and potentially responds to hypoxia. Meanwhile, hypoxia-inducible factor-1α (HIF-1α) is an important player in protecting cells from hypoxic stress. The purpose of this study was to investigate the effects of acute hypoxia exposure with different durations on the activation of Nrf2-ARE pathway and a possible regulatory role of HIF-1α in these responses.

Methods: C57BL/6J mice were allocated into the non-hypoxia 0-hour, 6-hour, 24-hour, and 48-hour hypoxic exposure (11.2% oxygen) groups. The quadriceps femoris was collected immediately after hypoxia. Further, to investigate the possible role of HIF-1α, C2C12 myoblasts with HIF-1α knockdown by small interfering RNA (siRNA) and the inducible HIF-1α transgenic mice were employed.

Results: The results showed that 48-hour hypoxia exposure up-regulated protein expression of Nrf2, Nrf2/ARE binding activity and the transcription of antioxidative genes containing ARE (Sod1 and others) in mouse skeletal muscle. Moreover, HIF-1α siRNA group of C2C12 myoblasts showed a remarkable inhibition of Nrf2 protein expression and nuclear accumulation in hypoxia exposure for 72 hours compared with that in siRNA-Control group of the cells. In addition, HIF-1α transgenic mice gave higher Nrf2 protein expression, Nrf2/ARE binding activity and expressions of Nrf2-mediated antioxidative genes in their skeletal muscle, compared with those in the wild-type mice.

Conclusions: The findings suggested that the acute hypoxia exposure could trigger the activation of Nrf2-ARE pathway, with longer duration associated with higher responses, and HIF-1α expression might be involved in promoting the Nrf2-mediated antioxidant responses in skeletal muscle.

Fig 1

The changes in HIF-1α (A) and Nrf2 (B) total protein contents in mouse quadriceps femoris. Muscles were collected following acute exposure to hypoxia with different durations (n = 9 mice/group). Protein expression was measured by western blotting. The values are expressed as the means ± SEM. **p<0.01 or *p<0.05 vs. 0h group; #p<0.05 vs. 6h group.

Fig 2

The changes in the Nrf2-ARE binding activity (A) and the mRNA expressions of Nrf2-mediated antioxidative gene (B) in mouse quadriceps femoris. Muscles were collected following acute exposure to hypoxia with different durations (n = 9 mice/group). Nrf2-ARE binding activity was measured by trans-activation assay. The mRNA expressions were measured by RT-PCR. The values are expressed as the means ± SEM. **p<0.01 or *p<0.05 vs. 0h group; ##p<0.01 or #p<0.05 vs. 6h group; ††p<0.01 or †p<0.05 vs. 24h group.

Fig 3

Alterations of the protein expressions of HIF-1α at different time points after hypoxia exposure (A); siRNA-mediated knockdown of HIF-1α inhibited HIF-1α (B) and Nrf2 (C) protein contents in C2C12 cells. Anti-HIF-1αsiRNA (si-HIF-1α,) and its control siRNAs (siControl) were transiently transfected into C2C12 cells before HE. Cells were incubated in normoxia (21% O₂) or hypoxia (5% O₂) and protein content levels of relevant molecules were measured by western blotting. The values are expressed as the means ± SEM. **p<0.01 or *p<0.05 vs. HE or HE+siControl group; ##p<0.01 or #p<0.05 vs. normoxia group. HE = 72h hypoxia exposure at 5% O₂.

Fig 4. Effects of HE and HE+si HIF-1α on the nuclear distribution of Nrf2 in C2C12 cells.

Cells were incubated in normoxia (21% O₂) or HE (5% O₂) and then localization of Nrf2 was visualized with a fluorescence microscope after immunofluorescence staining with anti-Nrf2 antibody and rabbit anti-mouse IgG second antibody coupled to Alexa Fluor 555. The nuclei were counterstained with DAPI (blue). Images of Nrf2 staining (Orange red) and DAPI staining (blue) of the same area were merged together to locate the cells with nuclear Nrf2 accumulation. HE = 72h hypoxia exposure at 5% O₂.

Fig 5

The changes in HIF-1α (A) and Nrf2 (B) total protein contents between HIF-1α TG and WT mice groups. Mouse quadriceps femoris muscles were collected (n = 9 mice/group). Protein expression was measured by western blotting. The values are expressed as the means ± SEM. #p<0.05 vs. WT mice group.

Fig 6

The changes in Nrf2-ARE binding activity (A) and the mRNA expressions of Nrf2-mediated antioxidative genes (B) between HIF-1α TG and WT mice groups. Mouse quadriceps femoris muscle was collected (n = 9 mice/group). Nrf2-ARE binding activity was evaluated in the muscle nuclear extracts using a Trans AM Nrf2 transcription factor assay. The level of mRNA expression was measured by RT-PCR. The values are expressed as the means ± SEM. #p<0.05 or ##p<0.01 vs. WT mice group.