Nrf2 deficiency induces skeletal muscle mitochondrial dysfunction: a proteomics/bioinformatics approach

Intracellular redox homeostasis is crucial for maintaining healthy skeletal muscle. Substantial redox imbalance due to excess reactive oxygen species (ROS) production is consequently associated with muscle fatigue, functional decline, metabolic dysfunction and even muscle wasting. The nuclear factor erythroid-derived 2 (Nrf2)/Kelch ECH-associated protein 1 (Keap1) complex is a redox-sensitive transcriptional regulatory system pivotal for the regulation of redox homeostasis, where the cytoplasmic repressor protein, Keap1, senses intracellular ROS and electrophiles, and the transcription factor, Nrf2, promotes antioxidant and other cytoprotective (e.g. anti-inflammation, metabolism) gene expression. In the resting state, Nrf2 is sequestered in the cytoplasm by Keap1 and targeted for proteasomal degradation and thus antioxidant expression remains low. However, in response to cellular stress, intracellular ROS levels increase and several Keap1 cystine residues become oxidised, facilitating the liberation of Nrf2 from Keap1. Nrf2 therein translocates to the nucleus where it binds to antioxidant response elements, in turn promoting the transcription of antioxidant enzyme genes. Targeted analytical research has shown that exercise stress induces Nrf2-mediated antioxidant expression in mouse skeletal muscle (Li et al. 2015) and, conversely, that Nrf2-deficient mice display higher intramyocellular ROS levels and attenuated antioxidant enzyme abundance (Miller et al. 2012). Consequently, impaired Nrf2/Keap1 function has been implicated in skeletal myopathy and disease and the Nrf2/Keap1 complex has been suggested as a promising therapeutic target against such pathologies. However, despite growing evidence for the Nrf2/Keap1 complex as a powerful intramuscular antioxidant system, the precise biological implications of Nrf2 in skeletal muscle tissue remain poorly defined. In a recent article published in The Journal of Physiology (Gao et al. 2020), the authors aimed to address this current research aperture by applying untargeted proteomic analysis to Nrf2-deficient or Nrf2-overexpressed mouse skeletal muscle tissue in order to identify a broad spectrum of novel targets and downstream pathways of Nrf2. For this purpose, two skeletal muscle-specific transgenic mouse models were produced: (i) iMS-Nrf2, a model for Nrf2 deficiency, and; (ii) iMS-Keap1, a model for Nrf2 overexpression. At 12 weeks of age, intraperitoneal injection of tamoxifen (or vehicle control) was administered for five consecutive days to activate Cre-recombination and thus induce selective Nrf2 (Nrf2-KO) or Keap1 (Keap1-KO) gene inactivation. At 32 weeks of age, mice were assigned to two cohorts for functional assessment and proteomic/bioinformatic analysis of skeletal muscle. Functional assessment included maximal exercise capacity via treadmill running, in situ muscle contractility under 2% isoflurane anaesthesia via tetanic stimulation, targeted protein content via immunoblotting, glutathione content via glutathione assays and mitochondrial function via high resolution respirometry and citrate synthase activity. Proteome-wide expression was quantified using mass spectrometry, with the resultant data being subjected to downstream proteomic/bioinformatic analyses. Gao et al. (2020) reported many interesting and novel findings. First, Nrf2-KO mice displayed significantly impaired maximal running speed, distance, duration and reduced muscle force-generating capacity (compared with vehicle, i.e. Nrf2-WT mice), whereas these performance parameters were improved in Keap1-KO mice, suggesting that Nrf2 has potent effects on skeletal muscle function. This is perhaps to be expected given the mechanisms of action of the Nrf2/Keap1 complex: specifically, that reduced Nrf2 (i.e. Nrf2-KO) leads to reduced antioxidant expression (either in the rested state and/or in response to exercise stress) and therein a redox imbalance, in turn negatively affecting muscle function. Second, differential protein-level analysis revealed 114 differentially expressed proteins in Nrf2-KO mouse muscle (11 upregulated, 103 downregulated) and 117 differentially expressed proteins in Keap1-KO mouse muscle (108 upregulated, nine downregulated), of which 10 proteins were found to be commonly regulated. Given this overall lack of commonality, the authors suggest that regulated proteins can be distinguished into two distinct categories: (i) upregulated proteins in Keap1-KO muscle are molecular targets activated by a high level of Nrf2, and; (ii) downregulated proteins in Nrf2-KO muscle are Nrf2 targets, which rely on basal Nrf2 activity. Downstream Gene Ontology enrichment analysis and Ingenuity Pathway Analysis (IPA) of differentially expressed proteins expectedly revealed that proteins upregulated by Keap1-KO were involved in well-known Nrf2 functions (e.g. detoxification, antioxidant defence). However, Nrf2-KO regulated proteins were found to be involved in (e.g.) purine ribonucleoside triphosphate metabolism, of which the implications are currently unclear. A particularly striking finding was that Nrf2-KO downregulated proteins were associated with many aspects of mitochondrial dysfunction, including abnormal mitochondrial morphology (COQ7), volume, length and coupling of the mitochondria (AK1), transmembrane potential of mitochondria (PHB and YWHAE), permeability transition of mitochondria (PPID) and mitochondrial complex I deficiency (NDUFS6) – implicating a role for, and candidate mechanistic targets of, Nrf2-related regulation of muscle mitochondrial health/ remodelling. Mitochondrial disturbance was subsequently confirmed at the functional level. Specifically, citrate synthase activity and electron transport chain (ETC) complex protein expression