Abstract
We clarified in our previous study that hypoxic training promotes angiogenesis in skeletal muscle, but the mechanism of angiogenesis in skeletal muscle remains unknown. In this study, we investigated the influence of differences in hypoxia exposure on angiogenesis in skeletal muscles at differing ages and metabolic characteristics at which the production of reactive oxygen species and nitric oxide may differ. Ten-week-old (young) and 20-month-old (old) mice were separated into control (N), continuous hypoxia (H), and intermittent hypoxia (IH) groups. The H group was exposed to 16% O2 hypoxia for 5 days and the IH group was exposed to 16% O2 hypoxia at one-hour intervals during the light period for 5 days. After completion of hypoxia exposure, the soleus and gastrocnemius muscles were immediately excised, and mRNA expression of angiogenesis- and satellite cell-related genes was investigated using real-time RT-PCR. In addition, muscle fiber type composition, muscle fiber area, number of satellite cells, and capillary density were measured immunohistochemically. In the young soleus muscle, the muscle fiber area was decreased in the H group, and mRNA expression of satellite cell activation-related MyoD, MHCe, and BDNF was significantly increased. On the other hand, in the old soleus muscle, nNOS and VEGF-A mRNA expression, and the capillary density were significantly increased in the H group. In the superficial portion of the gastrocnemius, mRNA expression of FGF2, an angiogenic factor secreted by satellite cells, was significantly increased in the young IH group. In addition, a positive correlation between VEGF-A mRNA expression and nNOS mRNA expression in the soleus muscle and eNOS mRNA expression in the superficial portion of the gastrocnemius was noted. These data demonstrated that age, hypoxia exposure method and muscle metabolic characteristics are related, which results in significant differences in angiogenesis.
Highlights
The muscle weights of young mice were lower in the H [soleus muscle (SOL): 13.3±0.3 g, gastrocnemius muscle (GA): 173.6±17.4 g] and intermittent hypoxia (IH) [SOL: 13.4±0.6 g, GA: 173.7±5.2 g] groups
No change was observed in the muscle weights in all groups of old mice [(N group) SOL: 16.8±1.8 g, GA: 177.3±16.0 g, (H group) SOL: 17.0±0.6 g, GA: 178.2±3.9 g, (IH group) SOL: 15.9±1.1 g, GA: 197.6±16.1 g)]
In the young muscle, mRNA expression of a gene involved in proteolysis [17], Atrogin1, was significantly increased due to the reduction of the muscle fiber area in the H group compared with that in the IH group, and mRNA expression of myostatin, which is involved in the promotion of proteolysis and inhibition of muscle formation [18], was significantly increased in the IH group
Summary
A hypoxic environment [1] and exercise stimulation [2] are factors that increase reactive oxygen species (ROS), and hypoxic training combining these may markedly increase ROS [3]. Increases in ROS and reactive nitrogen species (RNS) may be necessary to acquire beneficial effects of exercise through hormesis effects of ROS and RNS on mitochondrial biosynthesis, angiogenesis and satellite cell (SC) activation [4, 5]. We previously demonstrated the beneficial effects of hypoxic training using thoroughbreds, but the capillary density was evaluated only in the gluteus medius muscle, which is a glycolytic metabolism-dominant fast twitch muscle [4]. Intermittent exposure to hypoxia has been combined with training to improve angiogenesis and endurance [9], it is unclear which method of hypoxic exposure is more effective
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