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Updated: Dec 9, 2025
Skeletal Muscle Gender Dimorphism from Proteomics
Published on: December 14, 2011
Functional, proteomic and bioinformatic analyses of Nrf2- and Keap1- null skeletal muscle
Lie Gao1, Vikas Kumar2, Neetha Nanoth Vellichirammal3
1Department of Cellular & Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, USA.
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.
Abstract:
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.
Insights
Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates cellular defenses and skeletal muscle redox homeostasis. This study reveals Nrf2
Area of Science:
- Cellular Biology
- Molecular Biology
- Physiology
Background:
- Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular defense mechanisms, controlling over 200 cytoprotective proteins, including antioxidant enzymes.
- Nrf2 plays a critical role in maintaining skeletal muscle redox homeostasis, particularly in response to reactive oxygen species generated during muscle contraction.
- The precise functional significance and complete spectrum of Nrf2 targets and downstream pathways in skeletal muscle remain incompletely defined.
Purpose of the Study:
- To comprehensively identify novel Nrf2 target proteins, downstream pathways, and molecular networks within skeletal muscle.
- To elucidate the distinct roles of Nrf2 and its inhibitor Keap1 in regulating skeletal muscle function and cellular defense mechanisms.
- To propose a refined model for understanding the dual mode of Nrf2 action in skeletal muscle.
Main Methods:
- Creation and utilization of skeletal muscle-specific transgenic mouse models (iMS-Nrf2flox/flox and iMS-Keap1flox/flox) for Nrf2 and Keap1 deletion.
- Application of unbiased omics approaches, including mass spectrometry, to analyze proteomic changes in Nrf2-deficient and Keap1-deficient skeletal muscle.
- In-depth bioinformatics analyses (Gene Ontology, canonical pathway, and Ingenuity Pathway Analysis) to interpret proteomic data and validate findings through glutathione metabolism and mitochondrial function assays.
Main Results:
- Selective deletion of skeletal muscle Nrf2 or Keap1 resulted in distinct alterations in skeletal muscle function.
- Mass spectrometry identified significant changes in protein expression: 114 proteins in Nrf2-KO and 117 proteins in Keap1-KO models, with 10 common proteins.
- Gene Ontology analysis indicated that Nrf2-KO affected proteins involved in basal functions (e.g., oxidoreduction, ATP metabolism), while Keap1-KO affected proteins related to induced effects (e.g., detoxification, glutathione metabolism, electron transport chain).
Conclusions:
- Nrf2 exerts both tonic effects via a Keap1-independent mechanism under basal conditions and inducible effects via a Keap1-dependent mechanism under stress.
- The study proposes a two-way model for Nrf2 function, differentiating between basal and stress-induced regulatory roles in skeletal muscle.
- Proteomic and bioinformatic analyses successfully identified distinct sets of Nrf2-targeted proteins mediating these tonic and inducible functions, impacting cellular defense and metabolism.
