Fifteen days of 3,200 m simulated hypoxia marginally regulates markers for protein synthesis and degradation in human skeletal muscle

Abstract

Chronic hypoxia leads to muscle atrophy. The molecular mechanisms responsible for this phenomenon are not well defined in vivo. We sought to determine how chronic hypoxia regulates molecular markers of protein synthesis and degradation in human skeletal muscle and whether these regulations were related to the regulation of the hypoxia-inducible factor (HIF) pathway. Eight young male subjects lived in a normobaric hypoxic hotel (FiO₂ 14.1%, 3,200 m) for 15 days in well-controlled conditions for nutrition and physical activity. Skeletal muscle biopsies were obtained in the musculus vastus lateralis before (PRE) and immediately after (POST) hypoxic exposure. Intramuscular hypoxia-inducible factor-1 alpha (HIF-1α) protein expression decreased (-49%, P=0.03), whereas hypoxia-inducible factor-2 alpha (HIF-2α) remained unaffected from PRE to POST hypoxic exposure. Also, downstream HIF-1α target genes VEGF-A (-66%, P=0.006) and BNIP3 (-24%, P=0.002) were downregulated, and a tendency was measured for neural precursor cell expressed, developmentally Nedd4 (-47%, P=0.07), suggesting lowered HIF-1α transcriptional activity after 15 days of exposure to environmental hypoxia. No difference was found on microtubule-associated protein 1 light chain 3 type II/I (LC3b-II/I) ratio, and P62 protein expression tended to increase (+45%, P=0.07) compared to PRE exposure levels, suggesting that autophagy was not modulated after chronic hypoxia. The mammalian target of rapamycin complex 1 pathway was not altered as Akt, mammalian target of rapamycin, S6 kinase 1, and 4E-binding protein 1 phosphorylation did not change between PRE and POST. Finally, myofiber cross-sectional area was unchanged between PRE and POST. In summary, our data indicate that moderate chronic hypoxia differentially regulates HIF-1α and HIF-2α, marginally affects markers of protein degradation, and does not modify markers of protein synthesis or myofiber cross-sectional area in human skeletal muscle.

Keywords: HIF-1α; autophagy; hypoxia; mTORC1.

Figure 1

Effect of chronic hypoxia on key markers of the HIF pathway. Notes: (A) HIF-1α, (B) HIF-2α, and (C) REDD1 protein content and (D) VEGF-A, (E) BNIP3, (F) Nedd4, (G) REDD1, (H) PLIN2, (I) HIF-1α, and (J) HIF-2α mRNA content in human skeletal muscle before (PRE) and after (POST) 15 days of exposure to environmental hypoxia. (K) Representative blots. Dashed lines are individual values, and solid lines are mean ± SEM (n=8). *P<0.05 vs PRE. Abbreviations: BNIP3, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3; HIF, hypoxia-inducible factor; HIF-1α, hypoxia-inducible factor-1 alpha; HIF-2α, hypoxia-inducible factor-2 alpha; Nedd4, neural precursor cell expressed, developmentally downregulated 4; PLIN2, perilipin 2; REDD1, regulated in development and DNA damage responses 1; SEM, standard error of mean; VEGF-A, vascular endothelial growth factor A.

Figure 2

Effect of chronic hypoxia on key markers of the Akt-mTORC1 pathway. Notes: (A) Phospho-Akt, (B) phospho-mTOR, (C) phospho-S6K1, (D) phospho-4E-BP1, (E) phospho-AMPK, (F) S6K1 activity, and (G) AMPK activity in human skeletal muscle before (PRE) and after (POST) 15 days of exposure to environmental hypoxia. (H) Correlation between delta (POST–PRE) AMPK and S6K1 activities. (I) Correlation between delta (POST–PRE) phospho-S6K1 and LC3b-II/I ratio. (J) Correlation between delta (POST–PRE) phospho-S6K1 and HIF-1α protein content. (K) Representative blots. Dashed lines are individual values, and solid lines are mean ± SEM (n=8). Abbreviations: 4E-BP1, 4E-binding protein 1; AMPK, AMP-activated protein kinase; HIF, hypoxia-inducible factor; HIF-1α, hypoxia-inducible factor-1 alpha; LC3b, microtubule-associated protein 1 light chain 3; mTORC1, mammalian target of rapamycin complex 1; S6K1, S6 kinase 1; SEM, standard error of mean.

Figure 3

Effect of chronic hypoxia on key markers of proteolytic pathways. Notes: (A) LC3b-II/I ratio, (B) LC3b-II and (C) LC3b-I protein content, (D) LC3b mRNA content, (E) P62 protein content, (F) P62 mRNA content, (G) Gabarapl1 and (H) Cathepsin L mRNA content, (I) phospho-FoxO1/3a, (J) MAFbx, (K) Psmb1, and (L) MuRF-1 mRNA content in human skeletal muscle before (PRE) and after (POST) 15 days of exposure to environmental hypoxia. (M) Representative blots. Dashed lines are individual values, and solid lines are mean ± SEM (n=8). *P<0.05 vs PRE. Abbreviations: FoxO1/3a, forkhead box 1/forkhead box O3a; Gabarapl1, gamma-aminobutyric acid receptor-associated protein-like 1; LC3b, microtubule-associated protein 1 light chain 3; MAFbx, muscle atrophy F box; MuRF-1, muscle RING-finger protein-1; Psmb1, proteasome subunit beta type 1; SEM, standard error of mean.

Figure 4

Effect of chronic hypoxia on LC3b and P62 migration in myofibers. Notes: (A–C) Autophagy-positive muscle cell immunostained with antibodies for LC3b (A), P62 (B), and merged (C). A representative image for the PRE biopsy is shown in (D–F), and for the POST biopsy in (G–I). The white arrows indicate punctae representative for LC3b and P62. Abbreviation: LC3b, microtubule-associated protein 1 light chain 3.

Figure 5

Effect of chronic hypoxia on key markers of satellite cell proliferation and differentiation. Notes: (A) Myogenin, (B) MyoD, (C) MRF4, (D) Myf-5, and (E) PCNA mRNA content in human skeletal muscle before (PRE) and after (POST) 15 days of exposure to environmental hypoxia. Dashed lines are individual values, and solid lines are mean ± SEM (n=8). *P<0.05 vs PRE. Abbreviations: MRF4, muscle regulatory factor 4; Myf-5, myogenic factor-5; MyoD, myogenic differentiation 1; PCNA, proliferating cell nuclear antigen; SEM, standard error of mean.

Figure 6

Effect of chronic hypoxia on muscle fiber cross-sectional area and distribution. Notes: (A) Cross-sectional area, (B) percentage of total area per fiber type, and (C) percentage of total count per fiber type. Representative immunofluorescent pictures (D) pre- and (E) posthypoxic exposure. Conjugated WGA was used to delineate the sarcolemma (yellow) of each myofiber. Type I fibers are stained in green type IIa in blue, and type IIx are nonstained fibers. Abbreviation: WGA, wheat germ agglutinin.