Normobaric hypoxia shows enhanced FOXO1 signaling in obese mouse gastrocnemius muscle linked to metabolism and muscle structure and neuromuscular innervation

Abstract

Skeletal muscle relies on mitochondria for sustainable ATP production, which may be impacted by reduced oxygen availability (hypoxia). Compared with long-term hypoxia, the mechanistic in vivo response to acute hypoxia remains elusive. Therefore, we aimed to provide an integrated description of the Musculus gastrocnemius response to acute hypoxia. Fasted male C57BL/6JOlaHsd mice, fed a 40en% fat diet for six weeks, were exposed to 12% O₂ normobaric hypoxia or normoxia (20.9% O₂) for six hours (n = 12 per group). Whole-body energy metabolism and the transcriptome response of the M. gastrocnemius were analyzed and confirmed by acylcarnitine determination and Q-PCR. At the whole-body level, six hours of hypoxia reduced energy expenditure, increased blood glucose and tended to decreased the respiratory exchange ratio (RER). Whole-genome transcriptome analysis revealed upregulation of forkhead box-O (FOXO) signalling, including an increased expression of tribbles pseudokinase 3 (Trib3). Trib3 positively correlated with blood glucose levels. Upregulated carnitine palmitoyltransferase 1A negatively correlated with the RER, but the significantly increased in tissue C14-1, C16-0 and C18-1 acylcarnitines supported that β-oxidation was not regulated. The hypoxia-induced FOXO activation could also be connected to altered gene expression related to fiber-type switching, extracellular matrix remodeling, muscle differentiation and neuromuscular junction denervation. Our results suggest that a six-hour exposure of obese mice to 12% O₂ normobaric hypoxia impacts M. gastrocnemius via FOXO1, initiating alterations that may contribute to muscle remodeling of which denervation is novel and warrants further investigation. The findings support an early role of hypoxia in tissue alterations in hypoxia-associated conditions such as aging and obesity.

Keywords: FOXO; Hypoxia; Metabolism; Mitochondria; Neuromuscular junction; Skeletal muscle.

Fig. 1

Whole body effects of six hours exposure to normoxia (Norm) and mild hypoxia (Hypox; 12% O₂). (a) Body weight of the mice before exposure, (b) average levels of body energy expenditure and (c) RER during Norm or Hypox, (d) physical activity determined as total beam breaks, (e) blood glucose levels and (f) serum insulin levels directly after Norm or Hypox, n = 12 per group, n = 8 for physical activity, * p-value < 0.05, *** p-value < 0.001

Fig. 2

Volcano plot (a), confirmation (b, c) and cluster analysis (d) of differential expressed genes in M. gastrocnemius after Hypox (12% O₂) versus Norm. (a) All expressed genes with an Entrez annotation are displayed in a volcano plot based on their FC and t-test P-value. Dashed lines indicate P-value < 0.05 cutoff and FC = -1/1. Non-regulated, upregulated, and downregulated genes are labelled in grey, red, and blue respectively. Q-PCR analysis of (b) Depp1 and (c) Ttn. Values are represented as mean ± SD, n = 12 per group, ** P-value < 0.01, **** P-value < 0.0001. (d) Cluster analysis using gene ontology gene set enrichment using the cellular component GO-aspect (adjusted P-value < 0.05)

Fig. 3

Analysis of mitochondrial genes affected by six hours Hypox (12% O₂) versus Norm, (a) Gene set enrichment analysis of Hypox versus Norm using the MitoCarta 3.0 gene sets with adjusted P-value < 0.05 and (b) heatmap showing all significantly regulated genes under six hours hypoxia involved in mitochondrial metabolism using the MitoCarta 3.0 gene set, genes specifically involved in lipid metabolism are marked in bold. (c) Correlation between RER and Cpt1a expression (n = 24, combined Hypox and Norm)

Fig. 4

The effect of six hours hypoxia on the FOXO signaling pathway. (a) Heatmap showing all significantly regulated genes involved in the FOXO pathway in Hypox (12% O₂) versus Norm. (b) Schematic presentation of the FOXO pathway. (c) Correlation between blood glucose and Trib3 expression (n = 24, combined Hypox and Norm)

Fig. 5

The effect of six hour Hypox versus Norm on the muscle structure, ECM and NMJ. (a) SynGO sunburst of the genes involved in synapse based on the cellular components including child terms (b) Heatmap showing significantly regulated NMJ genes after hypoxia. (c) Heatmap showing significantly regulated genes under hypoxia based on the GO term contractile fiber and collagen genes. (d) Schematic presentation of regulated genes (bold in c) that are involved in the ECM and contractile fiber