Reductive stress impairs myogenic differentiation

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.

Keywords: Differentiation markers; Nrf2-signaling; Pro-oxidative setting; Reactive oxygen species (ROS); Reductive stress; Satellite cells; Skeletal muscle regeneration.

Graphical abstract

Fig. 1

Sulforaphane treatment establishes reductive stress in C2C12 myoblasts. (A) Glutathione levels and its redox (GSH/GSSG) ratio in the myoblasts following sulforaphane (SF) treatment was measured using enzymatic recycling assay. (B) qPCR based quantification of gene expression for some of the classical Nrf2-driven antioxidants (Gclc, Gclm, Nqo1, G6pd & Catalase) during proliferation (PM) and differentiation (day 1 & 5) phases. (C) Immunoblot analyses of antioxidant enzymes (i.e. GSR, GCLM, GCLC, NQO1, G6PD, Catalase, HO1 and SOD1/2) in proliferating and differentiating myoblasts on different time points (Differentiation on day 1 & 5). *p < 0.05, **p < 0.01 and ***p < 0.001, n = 3–4.

Fig. 2

Reductive stress impairs myoblast differentiation. C2C12 myoblasts were cultured in proliferation medium to 70–80% confluence, subjected to differentiation with or without SF (1.0 & 5.0 μM). (A) Bright field microscopy images of C2C12 myoblast during proliferation and differentiation phase under SF treatment (day 1 & 5). (B) Q-PCR based relative gene expression of myoblast differentiation markers (Myod1 and Myogenin) during myoblast proliferation and differentiation (day 1 & 5). (E) Immunoblots for differentiation markers (Myogenin and Myh2) during myoblast proliferation and differentiation (day1 & 5) phases. ***p < 0.001, n = 3–4.

Fig. 3

Direct augmentation of glutathione (i.e. NAC, GEE) or Keap1-silencing induce RS and inhibits myoblast differentiation. (A) NAC (1 and 3 mM) or (B) GEE treatment (1 and 5 mM) was performed in proliferating myoblast for 24 h and differentiating myoblasts until day-5. Total GSH and GSH/GSSG ratio in proliferating C2C12 cells treated with NAC/GEE, Bright field images of proliferating and differentiating myoblast (Magnification = 20X), immunoblot analysis of Myh2, and Myogenin in proliferating (PM) and differentiating (day 5) myoblast treated with NAC or GEE. *p < 0.05 and **p < 0.01, n = 3–4. (C) Immunofluorescence images of differentiating (day 5) myoblast after shRNA-mediated knockdown of Keap1 during proliferation phase. (B) GFP expressing myotubes were scored between 0-5 and the average of scoring were presented in bar graphs. (C) Western blots of markers of myoblast differentiation and antioxidants proteins in day 5 of differentiating myoblast after shRNA-mediated knockdown of Keap1. *p < 0.05, **p < 0.01 and ***p < 0.001, n = 3–4.

Fig. 4

Preventing RS (by withdrawing SF) rescues myoblast differentiation. Myoblasts were treated with SF (5 μM) during proliferation and differentiation phases until day 3. Removal of SF (washout) on day 3 by replenishing cells with fresh differentiation medium and allowed to differentiate until day 5. (A) Bright field images of proliferating and differentiating myoblast until day 5 under SF or rescued SF treatment. (B) Western blots of markers of myoblast differentiation and antioxidant proteins in proliferating and differentiating myoblasts. (C) Densitometry analysis of NQO1, myogenin, GCLC and MYH2 normalized with GAPDH was presented in bar graphs. *p < 0.05 and ***p < 0.001, n = 3–4.