Resistance Training Alleviates Skeletal Muscle Atrophy in Rats Exposed to Hypoxia by inhibiting Autophagy Mediated by Acetyl-FoxO1
KEYWORDS 2 hypoxia, resistance training, skeletal muscle atrophy, autophagy, Acetyl-FoxO1 3 Abstract Background: Skeletal muscle atrophy induced by hypoxia could affect the physical fitness and training effect of the athletes in the rapid altitude, and also affect the production and life of the general public. Resistance training in a hypoxic environment could effectively alleviate the occurrence of muscular atrophy. Whether autophagy lysosomal pathway, as an important proteolysis pathway, is
... volved in this process, and whether FoxO1, the key gene of atrophy, plays a role by regulating autophagy is unclear. Methods: Male Sprague-Dawley (SD) rats were randomly divided into normoxic control group (group C), normoxic resistance-training group (group R), hypoxic control group (group H), and hypoxic resistance-training group (group HR). The H and HR groups were exposed to 12.4% oxygen for four weeks. The R and HR groups underwent incremental loaded training by climbing a ladder every other day for four weeks. Results: Compared to parameters in group H, resistance training increased lean body mass (LBM) and wet weight and decreased the expression of atrogin1 of the extensor digitorum longus (EDL) after four weeks ( P <0.05). Resistance training decreased the levels of FoxO1 and Ac-FoxO1 and the extent of their localization in the nucleus and cytoplasm, respectively ( P <0.05), as well as the LC3II/LC3I ratio, the integrated optical density (IOD) of LC3 and the levels of autophagy-related gene 7 (Atg7), and elevated the levels of sequestosome 1 (SQSTM1/p62) ( P <0.05). Most differentially expressed autophagy-related genes (ATGs) interacted with FoxO1, and the functions of these ATGs were mainly enriched in the early autophagy phase. Conclusions: Our findings demonstrate that resistance training lowers the levels of both nuclear FoxO1 and cytoplasmic Ac-FoxO1, as well as reduced autophagic flux in the EDL of rats exposed to hypoxia. Background Plateau/hypoxic training, plateau tourism, and hypoxic weight loss promote individuals to experience hypoxic environments, which induce a series of adverse effects, including skeletal muscle atrophy. Such reduction in the size and strength of muscles affects not only the general public health, but also 4 the competitive levels of athletes [1, 2] . Although appetite, food intake, and gastrointestinal function are attenuated during hypoxia, these changes occur only at the early stages of hypoxic stimulation and are not sufficient to induce skeletal muscle atrophy. Rather, this atrophy is attributed primarily to an alteration in protein metabolism, with the rate of protein degradation becoming approximately 3 times faster than synthesis [3, 4] . Resistance training is the most effective training method for promoting muscle hypertrophy and is therefore effective non-pharmaceutical treatment for various pathological or age-related muscle atrophies. However, it is unclear whether resistance training may also alleviate hypoxia-induced muscle atrophy. It is a fascinating possibility that resistance training might also attenuate hypoxia-induced muscle atrophy, thereby avoiding the use of sometimes illicit drugs. The autophagy-lysosome pathway (ALP) is an important pathway for protein degradation [5, 6] . The balance of autophagic flow may be disrupted by multiple factors-such as diet, environment, physical activity, systemic disease, and/or genetics-to induce muscle atrophy. Although insufficient autophagy can cause muscle atrophy, this effect results in a chronic loss and is more common in aging. However, excessive autophagy can cause a rapid loss in muscle mass due to the continued clearance of necessary cellular components. Whether and how autophagy is involved in hypoxiainduced muscle atrophy remains unclear. Exercise has been found to induce B-cell lymphoma-2 (Bcl-2) phosphorylation, providing a molecular regulatory link to autophagy. In studies of exercise-regulated autophagy, endurance exercise represents the most commonly investigated method. Earlier findings suggested that resistance training may not influence autophagy, but more recent observations indicating that such training attenuates age-related muscle atrophy have challenged this view. However, the role of resistance training in relieving autophagy in hypoxiainduced muscle atrophy remains an unanswered question. Forkhead box protein O1 (FoxO1), a member of the FoxOs family of transcription factor, is an important regulator of muscle mass  . FoxO1 transgenic mice exhibit decreased muscle mass and impaired muscular function  . Autophagic levels are directly regulated by FoxO1 to induce hypotrophy-induced muscle atrophy but are not changed in FoxO1 skeletal-muscle-specific knockout Animals and experimental design Twenty-eight male Sprague-Dawley (SD) rats, each weighing approximately 200 g, were kept at 25±2 ℃ on a 12 h day-night cycle in the Beijing Sports University SPF Animal Laboratory (Beijing, China) and provided with food and water ad-libitum. Three or four rats were housed in each cage, and these animals divided randomly into four groups of 7 each: normoxic control group (group C), normoxic resistance-training group (group R), hypoxic control group (group H), and hypoxic resistance-training group (group HR). All experiments were conducted with the pre-approval of the Animal Ethical Committee of Beijing Sport University (IACUC 2017009A) and in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). Hypoxia exposure and resistance training Groups C and R were placed in a normoxic environment; Groups H and HR were placed in a hypoxic chamber with a 12.4% oxygen concentration (simulated altitude of 4,000 m); Group R and HR performed incremental-load resistance-ladder training every other day, with pre-training for one week We would like to thank all of the members of our laboratory for their encouragement and assistance with this study. Ethical approval Animal care and experimental procedures were approved by the Animal Ethical Committee of Beijing Sport University and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.