2.1 Animals and ethics statement
The study was approved by the Animal Ethics Committee at Beijing Sport University. 40 male Sprague-Dawley rats (8 weeks of age) were housed at 22–25℃and 12-h light-dark cycles, with food and water available ad libitum. The animals were divided randomly into four groups and weighed once every day: control group (C group, n = 10), resistance training group (R group, n = 10), hypoxia group (H group, n = 10) and hypoxia resistance training group (HR group, n = 10).
2.2 Hypoxic intervention and resistance training (RT)
The rats in the H and HR groups lived in normobaric hypoxia condition that simulated the environment at a height of 4000m (12.4%O2). The C and R groups acted as controls and were housed in a normal oxygen environment. The rats in the R group trained in a normal environment while the HR group trained in a hypoxic room. We designed and made a 1.2m ladder with a 1 cm grid, which was inclined at 85° against the wall during training. The R and HR groups pre-trained for a week to familiarize them with the climbing ladder. Weights were placed in a Corning tube, with small iron balls and a hook, attached to the tail root of the rat by a rubber belt (Fig. 1B). The rats were trained to climb from the bottom to the top in 10s by gentle stimulation on their tails. The initial load was 50% of their body weight and was increased 10% every other day throughout the 4 weeks of the training period.
Table 1
Training load of R and HR groups
Day
|
R group load
|
HR group load
|
1
|
121 g
|
115 g
|
3
|
153 g
|
145 g
|
5
|
188 g
|
177 g
|
7
|
217 g
|
196 g
|
9
|
257 g
|
234 g
|
11
|
294 g
|
272 g
|
13
|
323 g
|
310 g
|
15
|
376 g
|
353 g
|
17 ~ 28
|
422–485 g
|
395–442 g
|
2.3 Body composition, muscle wet weight and cross-sectional area (CSA)
Dual energy x-ray absorptiometry (DEXA) was used to measure the body composition of the rats. 48 h after the last training session, the animals were anesthetized with 3% pentobarbital sodium (3 ml/kg body weight). Parameters measured in the test included body weight, lean body mass, fat mass and the percentage of lean body mass. The rats were sacrificed by drawing blood from the abdominal aorta after the DEXA test. The extensor digitorum longus (EDL), soleus, gastrocnemius and bicipital muscle of forelimb were isolated quickly and weighed, followed by fixation of the muscle in 4% paraformaldehyde overnight at 4 ℃. The tissue specimens were stained with hematoxylin and eosin (HE) according to standard protocols and the CSA of each muscle analyzed using Image J software.
2.4 Cell culture experiment
Rat L6 skeletal muscle cells were purchased from ScienCell library. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco. The cells were cultured at 37℃ in high glucose DMEM (10% FBS, penicillin 100 U/ml, and streptomycin 0.1 mg/ml ) at 5% CO2. When the cells had grown to a confluence of 80–90%, the DMEM was supplemented with 2% equine serum, followed by incubation for a further 7–8 days to induce the cells to differentiate into myotubes. The differentiated myotubes were then transferred to a hypoxia chamber (Smarter 118 hypoxia system) maintained at 1% O2, 5% CO2 and 94% N2 for a further 6 h. 1, 3-Dicaffeoylquinic acid is an effective activator of the PI3K/Akt pathway, was added to the medium of the Akt-Act group and regulated to a final concentration at 50µM/L.
2.5 Immunohistochemistry and imaging
Paraffin sections of the skeletal muscle were dewaxed and repaired using citric acid antigen repair buffer. Fluorescence quenching agent was then added dropwise onto the tissue section. After washing in PBS, the sections were incubated overnight in FoxO1-S256 antibody (1:100 dilution, Cell Signaling Technology, Boston, USA). The sections were then incubated with fluorophore-conjugated secondary antibody for 1 h in a dark room. The nucleus was stained with DAPI. L6 cells were grown on glass coverslips and fixed in 4% paraformaldehyde for 15 min, followed by washing in PBS and incubation in goat serum for 30 min. The next steps were similar to those used for the tissue sections. The images were captured by laser scanning confocal microscopy and merged with NIS-Elements D image analysis software.
2.6 Western Blotting
EDL and arm biceps muscles from at least six rats tissue were dissected and frozen in a -80℃ ultra cold storage freezer. Total protein was abstracted using RIPA lysis buffer (Thermo Fisher Scientific, Massachusetts, USA) supplemented with protease inhibitor and phosphatase inhibitor (Roche, Basel, Switzerland). 30 mg of tissue was crushed using beads suspended in in homogenizer system (Eppendorf, Hamburg Germany) in 300 µl of RIPA buffer at 4℃ for 5 min. The samples were then centrifuged (12000 rpm, 10 min,4℃) and the supernatant isolated. L6 myotubes grown in six wells plates were washed with ice DPBS, followed by drop-wise addition of RIPA buffer into the wells and then left to stand for five minutes. The cell fraction was scraped from the bottom of plate situated in an ice bath and then centrifuged at 12000 rpm at 4 ℃ for 10 min. The protein concentration was measured using a bicinchonininc acid (BCA) kit (Thermo Fisher Scientific). A 20 µg aliquot of protein of each sample was subjected to SDS-PAGE (4–12% Bis-Tris gel, Thermo Fisher Scientific) and then transferred onto PVDF membrane (Thermo Fisher Scientific). The tailored membranes were then immunoblotted with primary antibodies: PI3K, (1:1000 dilution, Proteintech, Chicago, USA ); Akt (1:1000 dilution, Abcam, Cambridge, UK); FoxO1 (1:500 dilution, Abcam); FoxO1 (S256) (1:1000 dilution, Cell Signaling Technology); MuRF1 (1:2000 dilution, Abcam); Atrogin-1 (1:1000 dilution, Abcam); β-Actin (1:1000, Cell Signaling Technology), and Tubulin (1:1000, Cell Signaling Technology).
2.7 Antibodies and instruments
PI3K P110 (Alpha) antibody (20583-1-AP) was purchased from Proteintech. Anti-pan-AKT antibody (ab8805), anti-FOXO1 antibody (ab52857), anti-Fbx32 antibody (ab74023), anti-MuRF1 antibody (ab172479) and anti-fast myosin skeletal heavy chain antibody (ab91506) were purchased from Abcam. Phospho-FoxO1(Ser256) antibody (#9461), β-actin (#4970) and α-tubulin antibody (#2144) were purchased from Cell Signaling Technology. A resistance training ladder was designed and made by our group (Patent number: 201521113887.1).1, 3-dicaffeoylquinic acid was used as the Akt activator and was purchased from MCE (New Jersey, USA).
2.8 Statistical analysis
Images of the immunohistochemistry sections were analyzed using Image-Pro Plus software. The CSA analyses were carried out using Image. J. All values were expressed as mean ± SD. One- or two-way analysis of variance (ANOVA) was used to examine the effects, in the various experiments. All statistical analyses were performed using SPSS 19.0. A P value < 0.05 was considered statistically significant.