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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., Funding Agencies:Ministere de I'Enseignement Superieur et de la Recherche Association Francaise contre les Myopathies, 13955

Aim: To determine whether hypoxia influences the phenotypic adaptation of skeletal muscle induced by mechanical overload. Methods: Plantaris muscles of female rats were submitted to mechanical overload following synergist ablation. After 3 days of overload, rats were exposed to either hypobaric hypoxia (equivalent to 5500 m) or normoxia. Muscles were collected after 5, 12 and 56 days of overload (i.e. after 3, 9 and 53 days of hypoxia). We determined the myosin heavy chain (MHC) distribution, mRNA levels of myocyte-enriched calcineurin-integrating protein 1 (MCIP1) to indirectly assess calcineurin activity, the changes in oxidative capacity from the activities of citrate synthase (CS) and cytochrome c oxidase (COX), and the expression of regulators involved in mitochondrial biogenesis (Pgc-1α, NRF1 and Tfam) and degradation (BNIP-3). Results: Hypoxia did not alter the fast-to-slow MHC shift and the increase in calcineurin activity induced by overload; it only transiently slowed down the overload-induced transition in MHC isoforms. Hypoxia similarly decreased CS and COX activities in overloaded and control muscles. Nuclear respiratory factor 1 (NRF1) and transcription factor A (Tfam) mRNA and BNIP-3 protein were not influenced by hypoxia in overloaded muscles, whereas Pgc-1α mRNA and protein contents did not correlate with changes in oxidative capacity. Conclusion: Hypoxia is not a critical stimulus to modulate the fast-to-slow MHC transition associated with overload. Thus, the impairment of the fast-to-slow fibre shift often observed during post-natal development in hypoxia could be explained by the lower voluntary locomotor activity associated with hypoxia. Hypoxia alters mitochondrial oxidative capacity, but this adaptive response is similar in overloaded and control muscles., Funding Agency:Ministere de I'Enseignement Superieur et de la Recherche

Hypoxia induces a loss of skeletal muscle mass, but the signaling pathways and molecular mechanisms involved remain poorly understood. We hypothesized that hypoxia could impair skeletal muscle hypertrophy induced by functional overload (Ov). To test this hypothesis, plantaris muscles were overloaded during 5, 12, and 56 days in female rats exposed to hypobaric hypoxia (5,500 m), and then, we examined the responses of specific signaling pathways involved in protein synthesis (Akt/mTOR) and breakdown (atrogenes). Hypoxia minimized the Ov-induced hypertrophy at days 5 and 12 but did not affect the hypertrophic response measured at day 56. Hypoxia early reduced the phosphorylation levels of mTOR and its downstream targets P70(S6K) and rpS6, but it did not affect the phosphorylation levels of Akt and 4E-BP1, in Ov muscles. The role played by specific inhibitors of mTOR, such as AMPK and hypoxia-induced factors (i.e., REDD1 and BNIP-3) was studied. REDD1 protein levels were reduced by overload and were not affected by hypoxia in Ov muscles, whereas AMPK was not activated by hypoxia. Although hypoxia significantly increased BNIP-3 mRNA levels at day 5, protein levels remained unaffected. The mRNA levels of the two atrogenes MURF1 and MAFbx were early increased by hypoxia in Ov muscles. In conclusion, hypoxia induced a transient alteration of muscle growth in this hypertrophic model, at least partly due to a specific impairment of the mTOR/P70(S6K) pathway, independently of Akt, by an undefined mechanism, and increased transcript levels for MURF1 and MAFbx that could contribute to stimulate the proteasomal proteolysis., Funding Agency:Ministere de I'Enseignement Superieur et de la Recherche

The aim of this study was to examine the effect of ambient hypoxia on the main intracellular pathways involved in muscle regeneration. Left soleus muscles of female rats were degenerated by notexin injection before exposure to either normoxia (N) or ambient hypoxia (H) (10% O2) during 3, 7, 14 and 28 days (d). The expected muscle-mass loss of injured muscles was higher in H than in N rats at d3 and d7, whereas the recovery of muscle mass was similar in H and N rats at d28. The mammalian target of rapamycin (mTOR) activity, assessed from both eIF-4E binding protein (4E-BP1) and P70S6K phosphorylation, was markedly increased during the early period of regeneration, but remained two-fold lower in H than in N groups at d3. The hypoxia-induced alteration of mTOR activity, independently of Akt, was associated with an activation of AMP-activated kinase (AMPK) at d3. In contrast, REDD1, another negative regulator of mTOR, was markedly activated by H at d14 and d28 in intact muscles, but was blunted during the first days of regeneration (d3–7), independently of H. Taken together, we show for the first time, that hypoxia enhances the muscle-mass loss after extensive injury. This could be due to a specific impairment of mTOR activation during muscle regeneration, independently of Akt, at least partly related to AMPK activation, without detectable effect of REDD1.

Aim: The aim of the present study was to examine the effects of hypoxic endurance training on both aerobic performance and mitochondrial changes within plantaris muscle, independently of hematopoietic modifications. Methods: Four groups of female rats were constituted either sedentary (S) or trained (T), in either hypoxia (H) or normoxia (N). H conditions corresponded to 14% O2 and the training program to 5 running sessions/week for 5 weeks. Duration and intensity reached progressively 75Õ up to 80% of individual maximal aerobic running velocity (MAV) in either H or N. Performances of each rat were analysed through MAV values and time to exhaustion at 65% MAV (T65). Mitochondrial oxidative capacities (Vmax) for pyruvate (pyr), palmitoyl-carnitine (PC) and palmitoyl-CoA (PCoA) were measured in plantaris skinned fibers. Citrate synthase (CS) and HAD activities were also measured. Results: MAV increased in both TN and TH rats (respectively +52%, +39%, P<0.001) without difference between H and N, whereas hypoxia specifically increased T65 (+ 39%, P<0.05) independently of training effect. The training-induced increase in CS activity (P<0.001) was more marked in TN than in TH group (+39% vs +26%, P<0.001) whereas HAD activity rose similarly in TN and TH (respectively +83%, +64%, P<0.05). Physical training increased Vmaxpyr only in N rats (+30%, P<0.001), while VmaxPCoA decreased in hypoxia (P<0.05) without change in VmaxPC. This suggests that LCFA transport by CPT-1 was limiting in hypoxia. As expected, training improved creatine kinase efficiency in N rats (+80%, P<0.005), but no change was shown in H rats. Conclusion: Regarding the modest changes in mitochondrial function, it is likely that other factors contribute to explain the improvement of physical performance after an endurance training in hypoxia.

Quantifying target mRNA using real-time quantitative reverse transcription-polymerase chain reaction requires an accurate normalization method. Determination of normalization factors (NFs) based on validated reference genes according to their relative stability is currently the best standard method in most usual situations. This method controls for technical errors, but its physiological relevance requires constant NF values for a fixed weight of tissue. In the functional overload model, the increase in the total RNA concentration must be considered in determining the NF values. Here, we pointed out a limitation of the classical geNorm-derived normalization. geNorm software selected reference genes despite that the NF values extensively varied under experiment. Only the NF values calculated from four intentionally selected genes were constant between groups. However, a normalization based on these genes is questionable. Indeed, three out of four genes belong to the same functional class (negative regulator of muscle mass), and their use is physiological nonsense in a hypertrophic model. Thus, we proposed guidelines for optimizing target mRNA normalization and quantification, useful in models of muscle mass modulation. In our study, the normalization method by multiple reference genes was not appropriate to compare target mRNA levels between overloaded and control muscles. A solution should be to use an absolute quantification of target mRNAs per unit weight of tissue, without any internal normalization. Even if the technical variations will stay present as a part of the intergroup variations, leading to less statistical power, we consider this method acceptable because it will not generate misleading results.