Metabolic adaptation of murine skeletal muscle to nicotinamide nucleotide transhydrogenase and to hypoxia

Skeletal muscle is a metabolically active tissue reliant on both anaerobic and aerobic pathways, with mitochondrial oxidative phosphorylation (OXPHOS) playing a key role in energy production and redox balance. Nicotinamide nucleotide transhydrogenase (NNT), a mitochondrial enzyme, generates NADPH using the proton motive force and contributes to antioxidant defense. A spontaneous NNT dysfunction mutation in C57BL/6J mice, a commonly used strain in biomedical research, complicates interpretation of studies due to background genetic differences with other substrains. To overcome this, a congenic NNT-deficient mouse model was developed to investigate the role of NNT in skeletal muscle metabolism, particularly in the gastrocnemius muscle of young mice, under both normoxic and hypoxic (12% O₂ for 6 hours) conditions.

This thesis first examined the molecular response of gastrocnemius muscle to acute hypoxia in adult and prepubertal male mice with functional NNT. In both age groups, hypoxia activated the forkhead box-O1 (FOXO1) signaling pathway. In adult mice on a high-fat diet, hypoxia-induced genes are associated with muscle fiber-type switching, differentiation, and neuromuscular junction (NMJ) denervation. Similarly, in prepubertal mice, FOXO1 activation led to increased protein degradation and decreased protein synthesis via suppressed mammalian target of rapamycin complex 1 (mTORC1) activity.

Next, using 24-day-old male mice from the congenic NNT-deficient model (NntDELTA) and wild-type controls (Nnt-wt), the impact of NNT dysfunction under normoxia was assessed. Transcriptomic analysis revealed impaired mitochondrial energy metabolism, particularly a downregulation of OXPHOS-related genes, though this was not reflected at the protein or functional levels. Genes involved in the TCA cycle were unaffected, but those associated with fatty acid transport were downregulated. Mitochondrial antioxidant gene expression remained stable, but increased superoxide dismutase 1 (SOD1) activity in NntDELTA mice indicated a compensatory response to oxidative stress.

Finally, the interaction between NNT dysfunction and acute hypoxia was investigated. While both Nntwt and NntDELTA mice mounted a transcriptional response to hypoxia, the response in NntDELTA mice was markedly blunted, affecting only 2.7% of the transcriptome. Key pathways including mTORC1 signaling and glucocorticoid receptor (GR)-FOXO1 activation were also suppressed in NntDELTA mice. Evidence suggested that constitutive GR-FOXO1 activation under normoxic conditions in NntDELTA mice may impair hypoxia-induced adaptive responses, disturb proteostasis, and increase vulnerability to NMJ denervation.

In conclusion, this work elucidates the role of NNT in regulating skeletal muscle metabolism and stress responses, highlighting its importance in maintaining mitochondrial function and redox balance, particularly under hypoxic conditions. The findings underscore the limitations of using C57BL/6J mice without considering NNT status and advocate for careful selection of mouse models in muscle biology research. Future studies should extend to aged and female mice to broaden the understanding of NNT’s physiological relevance.