HIF-1α Negatively Regulates Irisin Expression Which Involves in Muscle Atrophy Induced by Hypoxia

Abstract

Exposure to high altitude environment leads to skeletal muscle atrophy. As a hormone secreted by skeletal muscles after exercise, irisin contributes to promoting muscle regeneration and ameliorating skeletal muscle atrophy, but its role in hypoxia-induced skeletal muscle atrophy is still unclear. Our results showed that 4 w of hypoxia exposure significantly reduced body weight and gastrocnemius muscle mass of mice, as well as grip strength and the duration time of treadmill exercise. Hypoxic treatment increased HIF-1α expression and decreased both the circulation level of irisin and its precursor protein FNDC5 expression in skeletal muscle. In in vitro, CoCl₂-induced chemical hypoxia and 1% O₂ ambient hypoxia both reduced FNDC5, along with the increase in HIF-1α. Moreover, the decline in the area and diameter of myotubes caused by hypoxia were rescued by inhibiting HIF-1α via YC-1. Collectively, our research indicated that FNDC5/irisin was negatively regulated by HIF-1α and could participate in the regulation of muscle atrophy caused by hypoxia.

Keywords: FNDC5; HIF-1α; hypoxia; irisin; muscle atrophy.

Figure 1

Hypoxia reduced the lean weight and fat weight of mice. Representative morphology (left) and dual X-ray digital images (right) of mice after 4 w of hypoxic treatment (A). DEXA results showed that body weight (B), lean weight (C), and fat weight (D) were significantly reduced after 14 d and 28 d of hypoxia. Fat in tissue (E), bone area (F), and BMD (bone mineral density) (G) were not affected in the whole experiment, while BMC (Bone mineral content) and (H), bone volume (I) decreased only at 14 d of hypoxia, and returned to normal levels at 28 d. Values represented means ± SD. * p < 0.05, ** p < 0.01. n = 5–6.

Figure 2

Four weeks of hypoxic exposure induced muscle atrophy of mice. The grip strength (A) of mice (n ≥ 8) and run duration time (B) in the treadmill experiment were significantly reduced after 4 w of hypoxia (n = 4–6). The weight of gastrocnemius (Gas) and quadriceps (Quad) muscles were reduced but not soleus (Sol) and triceps (Tric), of which were all normalized to the tibia length (C, n ≥ 6). (D) shows the representative images of H&E staining of Gas muscle. Scale bar, 50 μm. (n = 6). Hypoxia significantly decreased the average CSA (the cross-sectional area from the mid-belly of Gas muscle) and diameter (Feret’s diameter) in Gas muscles (E,F) and affected the area and diameter distribution of the myofibers (G,H, n = 6). The expression of myogenic factors Mrf4 and MyoG significantly reduced in Gas muscles after 4 w of hypoxia (I, n = 5). Values represented means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Hypoxia reduced the expression of FNDC5/irisin in mice. Hypoxic treatment significantly increased HIF-1α expression (A,B) and reduced FNDC5 expression (C) in Gas muscles (n = 4). Irisin concentration in plasma (D) of mice was reduced in hypoxia (n = 8–10), and the mRNA level of FNDC5 (E) and ADAM10 (F) in Gas muscle were not changed (n = 5). PGC-1α expression was not changed both in protein (G,H, n = 3) and mRNA level (I, n = 5). Myostatin concentration in both plasma (J, n = 5) and quadriceps muscle (K, n = 4) was not affected by hypoxia. Values represented means ± SD. * p < 0.05, ** p < 0.01.

Figure 4

Inhibition of HIF-1α induced by CoCl₂ increased FNDC5 expression in C2C12 myotubes (A), CoCl₂ treatment all significantly increased the expression of HIF-1α (B,C), meanwhile decreased FNDC5 expression (D) and did not affect PGC-1α expression (E, n = 3). The expression of FNDC5 negatively correlated with HIF-1α, r > −0.5 represented the negative relationship (E). 50 μM of YC-1 treatment abrogated the increase in HIF-1α (F) and the decrease in FNDC5 (G) induced by CoCl₂ (n = 3). PGC-1α expression was not affected by both CoCl₂ (H) and YC-1 (I). Values represented means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ^# p < 0.05.

Figure 5

Inhibition of HIF-1α in 1% O₂ ambient hypoxia increased FNDC5 expression, and myotube formation in C2C12 myotubes HIF-1α was increased significantly in hypoxia for 12h (A), which was rescued by YC-1 (B); meanwhile, YC-1 reversed the decrease in FNDC5 (C, n = 3). The expression of PGC-1α was not affected by both hypoxia and YC-1 (D). Hypoxia significantly reduced the area and diameter of C2C12 myotubes, and which were rescued by YC-1 treatment (E–G), while myotube fusion index was not influenced (H, n = 13). Values represented means ± SD. * p < 0.05, *** p < 0.001. Green, MyHC; blue, Hoechst. Scale bar, 50 µm.