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Published on: June 3, 2018
Reductive stress impairs myogenic differentiation
Namakkal S Rajasekaran1, Sandeep Balu Shelar2, Dean P Jones3
1Cardiac Aging & Redox Signaling Laboratory, Molecular and Cellular Pathology, Department of Pathology, Birmingham, AL, USA; Division of Cardiovascular Medicine, Department of Medicine, University of Utah School of Medicine, Salt Lake City, UT, USA; Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
Abstract:
Myo-satellite cells regenerate and differentiate into skeletal muscle (SM) after acute or chronic injury. Changes in the redox milieu towards the oxidative arm at the wound site are known to compromise SM regeneration. Recently, we reported that abrogation of Nrf2/antioxidant signaling promotes oxidative stress and impairs SM regeneration in C57/Bl6 mice. Here, we investigated whether the activation of intracellular Nrf2 signaling favors antioxidant transcription and promotes myoblast differentiation. Satellite cell-like C2C12 myoblasts were treated with sulforaphane (SF; 1.0 & 5.0 μM) to activate Nrf2/antioxidant signaling during proliferation and differentiation (i.e. formation of myotubes/myofibers). SF-mediated Nrf2 activation resulted in increased expression of Nrf2-antioxidants (e.g. GCLC and G6PD) and augmented the production of reduced glutathione (GSH) leading to a reductive redox state. Surprisingly, this resulted in significant inhibition of myoblast differentiation, as observed from morphological changes and reduced expression of MyoD, Pax7, and Myh2, due to reductive stress (RS). Furthermore, supplementation of N-acetyl-cysteine (NAC) or GSH-ester or genetic knock-down of Keap1 (using siRNA) also resulted in RS-driven inhibition of differentiation. Interestingly, withdrawing Nrf2 activation rescued differentiation potential and formation of myotubes/myofibers from C2C12 myoblasts. Thus, abrogation of physiological ROS signaling through over-activation of Nrf2 (i.e. RS) and developing RS hampers differentiation of muscle satellite cells.
Insights
Activating Nrf2 signaling to combat oxidative stress surprisingly inhibits skeletal muscle regeneration. Over-activation leads to reductive stress, impairing myoblast differentiation and muscle repair.
Area of Science:
- Muscle regeneration and cellular redox homeostasis.
- Molecular mechanisms of skeletal muscle differentiation.
- Role of Nrf2 signaling in cellular stress responses.
Background:
- Skeletal muscle regeneration relies on myo-satellite cells differentiating into muscle fibers.
- Oxidative stress at injury sites can impair muscle regeneration.
- Previous work showed Nrf2/antioxidant signaling abrogation impairs skeletal muscle regeneration.
Purpose of the Study:
- To investigate if activating intracellular Nrf2 signaling promotes antioxidant transcription and myoblast differentiation.
- To determine the effects of sulforaphane-induced Nrf2 activation on C2C12 myoblast differentiation.
Main Methods:
- C2C12 myoblasts were treated with sulforaphane (SF) to activate Nrf2 signaling.
- Gene expression of Nrf2-antioxidants (GCLC, G6PD) and differentiation markers (MyoD, Pax7, Myh2) were analyzed.
- Effects of N-acetyl-cysteine (NAC), GSH-ester, and Keap1 knockdown on differentiation were assessed.
Main Results:
- SF treatment increased Nrf2-antioxidant expression and reduced glutathione (GSH) production, creating a reductive redox state.
- This reductive state significantly inhibited myoblast differentiation, evidenced by morphological changes and reduced marker expression.
- NAC, GSH-ester, and Keap1 knockdown also induced reductive stress and inhibited differentiation.
Conclusions:
- Over-activation of Nrf2 signaling leads to reductive stress, which hampers skeletal muscle satellite cell differentiation.
- Disrupting physiological reactive oxygen species (ROS) signaling by inducing reductive stress impairs muscle regeneration.
- Modulating Nrf2 activation is crucial for effective muscle repair, as excessive activation is detrimental.
