Proteomic and Functional Analyses of Keap1‐Nrf2 Pathway in Skeletal Muscle

Abstract

The Nrf2/Keap1 pathway plays a critical role in maintaining redox homeostasis. To determine its significance in skeletal muscle, we created two novel transgenic mice, iMS-Nrf2flox/flox and iMS-Keap1flox/ flox, which allowed us to knock out (KO) Nrf2 or Keap1 selectively in skeletal muscle by ip tamoxifen (Tam). Employing these two models, we analyzed exercise capacity and soleus (Sol) muscle contractility and protein profiles after deleting Nrf2 or Keap1. Experiments were performed in 12 adult male mice treated with Tam or vehicle (Veh) to generate Nrf2-WT, Nrf2-KO, Keap1-WT, and Keap1-KO, 3 mice in each group. Twenty weeks after treatment, mice were tested for treadmill exercise endurance (6 m/min x 6 min warm up, followed by an increase of 3 m/min every 3 min until exhaustion). In situ muscle fatigue was tested on the left Sol in anesthetized mice using an intermittent tetanic stimulation (2.5V, 50Hz, 1 s per 3 s x 20 min). The right Sol was used for proteomic analysis by a Thermo Orbitrap Fusion Lumos Tribrid mass spectrometry. We found that running distance was significantly reduced in Nrf2-KO (234 ± 92 vs 548 ± 58 m, P < 0.05) and was increased in Keap1-KO (1067 ± 163 vs 576 ± 51 m, P < 0.05). Sol fatigue development was faster and greater in Nrf2-KO than WT, whereas no significant fatigue was observed in Keap1-KO. Proteomic data showed that Nrf2 KO significantly upregulated 6 proteins and downregulated 4 proteins. The downregulated proteins include NADH dehydrogenase (ubiquinone), epoxide hydrolase, and immunoglobulin-κ, suggesting impaired antioxidant defense and immune function in the muscle with reduced Nrf2. On the other hand, Keap1 KO significantly changed 105 proteins (P ≤ 0.05 vs Keap1-WT), among which 98 were upregulated, including Nrf2 and its targets involved in anti-oxidation, detoxification, anti-inflammation, and metabolism. For example, sulfiredoxin-1, immunoglobulin-κ, NAD(P)H dehydrogenase (ubiquinone), carbonyl reductase, pirin, and GSTA2 were increased at 34, 21, 17, 12, 11, 10 fold, respectively suggesting an enhancement in cytoprotective mechanisms when Keap1 was deleted. In addition to these Nrf2-targets, Keap1 KO also altered muscular structural and contractile proteins. Titin, a large and abundant protein of striated muscle was upregulated. L-type calcium channel, responsible for excitation-contraction coupling was downregulated. Arp2/3 complex, a major component of the actin cytoskeleton was upregulated. Keratin, fibrous structural proteins were upregulated. In conclusion, the functional data suggest that the Nrf2/Keap1 system plays a critical role in skeletal muscle contractility. The proteomics data further suggest that, at least in the Sol, the Nrf2/Keap1 pathway not only exerts cytoprotection but also modulates muscular structure and function. Keap1 KO upregulated more proteins than were downregulated by Nrf2 KO (98 vs 4), confirming the consensus that the Nrf2/Keap1 system exerts effects primarily in the stress state where Keap1 is inactivated, which can be observed during strenuous exercise and may be important in skeletal myopathies. Support or Funding Information Supported by NIH P01-HL62222 and UNMC MSPCF 33-1209-0400. (A) Maximal running distance indicating exercise capacity, *P < 0.05;(B) Representative courses of force generation showing Sol muscle resistance to fatigue;(C) A representative volcano plot illustrating fold change (x-axis) and statistical significance distribution (y-axis) of the proteomic data set of Sol-Keap1-WT vs Sol-Keap1-KO. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.