Ambient hypoxia enhances the loss of muscle mass after extensive injury

Abstract

Hypoxia induces a loss of skeletal muscle mass and alters myogenesis in vitro, but whether it affects muscle regeneration in vivo following injury remains to be elucidated. We hypothesized that hypoxia would impair the recovery of muscle mass during regeneration. To test this hypothesis, the soleus muscle of female rats was injured by notexin and allowed to recover for 3, 7, 14, and 28 days under normoxia or hypobaric hypoxia (5,500 m) conditions. Hypoxia impaired the formation and growth of new myofibers and enhanced the loss of muscle mass during the first 7 days of regeneration, but did not affect the final recovery of muscle mass at 28 days. The impaired regeneration under hypoxic conditions was associated with a blunted activation of mechanical target of rapamycin (mTOR) signaling as assessed by p70(S6K) and 4E-BP1 phosphorylation that was independent of Akt activation. The decrease in mTOR activity with hypoxia was consistent with the increase in AMP-activated protein kinase activity, but not related to the change in regulated in development and DNA response 1 protein content. Hypoxia increased the mRNA levels of the atrogene muscle ring finger-1 after 7 days of regeneration, though muscle atrophy F box transcript levels remained unchanged. The increase in MyoD and myogenin mRNA expression with regeneration was attenuated at 7 days with hypoxia. In conclusion, our results support the notion that the enhanced loss of muscle mass observed after 1 week of regeneration under hypoxic conditions could mainly result from the impaired formation and growth of new fibers resulting from a reduction in protein synthesis and satellite cell activity.

Fig. 1

Cross sections of the soleus muscles stained with hematoxylin and eosin. Morphological data are presented in Table 2. All pictures are visualized at the same magnification (×20). Bars = 100 μm. Int-N, intact soleus muscle from normoxic rats; Int-H, intact soleus muscle from hypoxic rats; Reg-N, regenerated soleus muscle from normoxic rats; Int-H, regenerated soleus muscle from hypoxic rats

Fig. 2

Analysis of the Akt/mTOR signaling during muscle regeneration. Phosphorylated and total levels of Akt (A1 and A2, respectively), p70^S6K (B1 and B2, respectively), eukaryotic initiation factor-4E-BP1 (C1 and C2, respectively), and eEF2 (D1 and D2, respectively) during soleus regeneration from normoxic and hypoxic rats. The phosphorylated-to-total protein ratios were also quantified (Akt^Thr308/Akt1, A3; p70^{S6K Thr389}/p70^S6K, B3; 4E-BP1^Thr70/4E-BP1, C3; eEF2^Thr56/eEF2, D3). Immunoblot pictures are composites of original lanes from muscle samples that were derived at the same time and processed in parallel. Values are means±SE. Legends as in Fig. 1. The asterisk indicates significant difference from the respective Int group. The number sign indicates significant difference from the respective normoxic group. The dollar sign indicates significant difference from the previous time for the same group

Fig. 3

Analysis of the endogenous repressors of the mTOR signaling during muscle regeneration. Phosphorylated and total levels of AMPK (a and b, respectively) and the ratio of phosphorylated AMPK^Thr172 to AMPK (c). REDD1 protein levels (d). Immunoblot pictures are composites of original lanes from muscle samples that were derived at the same time and processed in parallel. Values are means±SE. Legends as in Fig. 1. The asterisk indicates significant difference from the respective Int group. The number sign indicates significant difference from the respective normoxic group. The dollar sign indicates significant difference from the previous time for the same group

Fig. 4

Protein levels of the myogenic regulatory factors MyoD (a) and myogenin (b) during muscle regeneration. Immunoblot pictures are composites of original lanes from muscle samples that were derived at the same time and processed in parallel. Values are means±SE. Legends as in Fig. 1. The asterisk indicates significant difference from the respective Int group. The number sign indicates significant difference from the respective normoxic group. The dollar sign indicates significant difference from the previous time for the same group

Fig. 5

mRNA levels of the muscle-specific ubiquitin ligases MAFbx (a) and MURF1 (b) during muscle regeneration. Values are means±SE. Legends as in Fig. 1. The asterisk indicates significant difference from the respective Int group. The number sign indicates significant difference from the respective normoxic group. The dollar sign indicates significant difference from the previous time for the same group