Endothelial cell Nrf2-KO attenuates endothelial function and skeletal muscle antioxidant capacity

INTRODUCTION: Endothelial cells line the inner surface of blood vessels and play a major role in modulating blood flow and gas exchange. Endothelial dysfunction is thought to be a contributor to cardiovascular disease development, and it is well-accepted that excessive reactive oxygen species (harmful molecules) likely contribute to endothelial dysfunction. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is considered the master regulator of cellular protection in response to elevated reactive oxygen species. Therefore, Nrf2 may be a potential therapeutic target to protect against endothelial dysfunction. However, the roles of endothelial cell-specific Nrf2 on endothelial function are not known. The purpose of this study was to investigate the impacts of endothelial cell-specific Nrf2 deletion on vascular function (endothelium-dependent and endothelium-independent vasodilation) and skeletal muscle antioxidant status. METHODS: Leg arteries were harvested from 6-mo old C57BL/6 mice (WT, n = 6) and endothelial cell-specific Nrf2-knockout mice (Tie2-Cre-Nrf2 floxed-KO, n = 6). Endothelium-dependent vasodilation was assessed in response to flow (30 uL·min-1) and acetylcholine (ACh, 10-7-10-3 M) with and without Nω-Nitro-L-arginine methyl ester (L-NAME), and endothelium-independent vasodilation was assessed with sodium nitroprusside (SNP, 10-9-10-4 M) using videomicroscopy. Skeletal muscle antioxidant protein expression for glutathione peroxidase-1 (GPX-1) and catalase (CAT) was assessed by immunoblotting. RESULTS: Endothelium-dependent vasodilation was lower in Nrf2-KO compared to WT induced by flow (WT: 34.8±2.9%, Nrf2-KO: 20.7±3.7%, P-3M, WT: 68.3±8.2%, Nrf2-KO: 44.5±7.1%, PP-3 M, 19.1±4.4%, PP=0.28) or ACh (10-3 M, 37.7±7.0%, P = 0.16). Endothelium-independent vasodilation was not different (SNP 10-4 M, WT: 92.7±3.6%, Nrf2-KO: 81.9± 0.2%, P=0.157). In addition, GPX-1 was lower in Nrf2-KO mice (WT: 0.47±0.06, Nrf2-KO: 0.001±0.003, PP=0.08). CONCLUSIONS: Endothelial cell Nrf2 may play a key role in endothelial-mediated vasodilatory function. The nitric oxide synthase inhibitor L-NAME attenuated endothelial-mediated vasodilation in WT but not in endothelial cell Nrf2-KO. Furthermore, endothelial cell Nrf2 may play a role in skeletal muscle antioxidant homeostasis, which suggests potential systemic implications of endothelial cell Nrf2 deletion. These results collectively suggest that the endothelial cell Nrf2 system is linked to endothelial dysfunction and changes in the skeletal muscle redox environment, likely through nitric oxide- and oxidative stress-related mechanisms

Functional, proteomic and bioinformatic analyses of Nrf2- and Keap1- null skeletal muscle

Key points Nrf2 is a master regulator of endogenous cellular defences, governing the expression of more than 200 cytoprotective proteins, including a panel of antioxidant enzymes. Nrf2 plays an important role in redox haemostasis of skeletal muscle in response to the increased generation of reactive oxygen species during contraction. Employing skeletal muscle-specific transgenic mouse models with unbiased-omic approaches, we uncovered new target proteins, downstream pathways and molecular networks of Nrf2 in skeletal muscle following Nrf2 or Keap1 deletion. Based on the findings, we proposed a two-way model to understand Nrf2 function: a tonic effect through a Keap1-independent mechanism under basal conditions and an induced effect through a Keap1-dependent mechanism in response to oxidative and other stresses. Although Nrf2 has been recognized as a master regulator of cytoprotection, its functional significance remains to be completely defined. We hypothesized that proteomic/bioinformatic analyses from Nrf2-deficient or overexpressed skeletal muscle tissues will provide a broader spectrum of Nrf2 targets and downstream pathways than are currently known. To this end, we created two transgenic mouse models; the iMS-Nrf2flox/flox and iMS-Keap1flox/flox, employing which we demonstrated that selective deletion of skeletal muscle Nrf2 or Keap1 separately impaired or improved skeletal muscle function. Mass spectrometry revealed that Nrf2-KO changed expression of 114 proteins while Keap1-KO changed expression of 117 proteins with 10 proteins in common between the groups. Gene ontology analysis suggested that Nrf2 KO-changed proteins are involved in metabolism of oxidoreduction coenzymes, purine ribonucleoside triphosphate, ATP and propanoate, which are considered as the basal function of Nrf2, while Keap1 KO-changed proteins are involved in cellular detoxification, NADP metabolism, glutathione metabolism and the electron transport chain, which belong to the induced effect of Nrf2. Canonical pathway analysis suggested that Keap1-KO activated four pathways, whereas Nrf2-KO did not. Ingenuity pathway analysis further revealed that Nrf2-KO and Keap1-KO impacted different signal proteins and functions. Finally, we validated the proteomic and bioinformatics data by analysing glutathione metabolism and mitochondrial function. In conclusion, we found that Nrf2-targeted proteins are assigned to two groups: one mediates the tonic effects evoked by a low level of Nrf2 at basal condition; the other is responsible for the inducible effects evoked by a surge of Nrf2 that is dependent on a Keap1 mechanism

Quantitative Proteomics Identifies Novel Nrf2-Mediated Adaptative Signaling Pathways in Skeletal Muscle Following Exercise Training

Exercise training (ExT) improves skeletal muscle health via multiple adaptative pathways. Nrf2 is a principal antioxidant transcription factor responsible for maintaining intracellular redox homeostasis. In this study, we hypothesized that Nrf2 is essential for adaptative responses to ExT and thus beneficial for muscle. Experiments were carried out on male wild type (WT) and iMS-Nrf2flox/flox inducible muscle-specific Nrf2 (KO) mice, which were randomly assigned to serve as sedentary controls (Sed) or underwent 3 weeks of treadmill ExT thus generating four groups: WT-Sed, WT-ExT, KO-Sed, and KO-ExT groups. Mice were examined for exercise performance and in situ tibialis anterior (TA) contractility, followed by mass spectrometry-based proteomics and bioinformatics to identify differentially expressed proteins and signaling pathways. We found that maximal running distance was significantly longer in the WT-ExT group compared to the WT-Sed group, whereas this capacity was impaired in KO-ExT mice. Force generation and fatigue tolerance of the TA were enhanced in WT-ExT, but reduced in KO-ExT, compared to Sed controls. Proteomic analysis further revealed that ExT upregulated 576 proteins in WT but downregulated 207 proteins in KO mice. These proteins represent pathways in redox homeostasis, mitochondrial respiration, and proteomic adaptation of muscle to ExT. In summary, our data suggest a critical role of Nrf2 in the beneficial effects of SkM and adaptation to ExT