Role of miR-200c in Myogenic Differentiation Impairment via p66Shc: Implication in Skeletal Muscle Regeneration of Dystrophic mdx Mice.

Marco D’Agostino,Giulia Imeneo,Sara Sileno,Daisy Proietti,Chiara Salis,Luca Madaro,Lorenza Marchetti,Sara Beji,Alessio Torcinaro,Francesca De Santa,Alessandra Magenta,Maurizio C Capogrossi

doi:10.1155/2018/4814696

Abstract

Duchenne muscular dystrophy (DMD) is a genetic disease associated with mutations of Dystrophin gene that regulate myofiber integrity and muscle degeneration, characterized by oxidative stress increase. We previously published that reactive oxygen species (ROS) induce miR-200c that is responsible for apoptosis and senescence. Moreover, we demonstrated that miR-200c increases ROS production and phosphorylates p66Shc in Ser-36. p66Shc plays an important role in muscle differentiation; we previously showed that p66Shc−/− muscle satellite cells display lower oxidative stress levels and higher proliferation rate and differentiated faster than wild-type (wt) cells. Moreover, myogenic conversion, induced by MyoD overexpression, is more efficient in p66Shc−/− fibroblasts compared to wt cells. Herein, we report that miR-200c overexpression in cultured myoblasts impairs skeletal muscle differentiation. Further, its overexpression in differentiated myotubes decreases differentiation indexes. Moreover, anti-miR-200c treatment ameliorates myogenic differentiation. In keeping, we found that miR-200c and p66Shc Ser-36 phosphorylation increase in mdx muscles. In conclusion, miR-200c inhibits muscle differentiation, whereas its inhibition ameliorates differentiation and its expression levels are increased in mdx mice and in differentiated human myoblasts of DMD. Therefore, miR-200c might be responsible for muscle wasting and myotube loss, most probably via a p66Shc-dependent mechanism in a pathological disease such as DMD.

Highlights

We previously showed that oxidative stress inhibits myogenic differentiation [1] and in a model of oxidative stress such as acute hind limb ischemia, it was demonstrated that reactive oxygen species (ROS) production plays a causal role in tissue damage, leading to cell death by both apoptosis and necrosis [2].p66Shc adaptor protein is a redox enzyme implicated in mitochondrial ROS generation and translation of oxidative signals [3]
We demonstrated that miR-200c increases ROS production and phosphorylates p66Shc in Ser-36. p66Shc plays an important role in muscle differentiation; we previously showed that p66Shc−/− muscle satellite cells display lower oxidative stress levels and higher proliferation rate and differentiated faster than wild-type cells
C2C12 were cultured in growth medium ((GM) DMEM-GlutaMAX complemented with penicillin/streptomycin and 20% FBS)

Summary

Introduction

We previously showed that oxidative stress inhibits myogenic differentiation [1] and in a model of oxidative stress such as acute hind limb ischemia, it was demonstrated that reactive oxygen species (ROS) production plays a causal role in tissue damage, leading to cell death by both apoptosis and necrosis [2]. We wanted to dissect the role of miR-200c in muscle differentiation and to comprehend whether miR200c levels were modulated in muscle pathological diseases associated with oxidative stress increase, such as Duchenne muscular dystrophy (DMD) [11, 12]. In keeping with this hypothesis, in the paper of Greco et al, an interesting link between ischemia-, mdx-, and DMD-modulated miRNAs associated with apoptosis/myonecrosis was demonstrated [13]. We hypothesized a miR-200c role in muscle wasting and myotube loss via a p66Shc-dependent mechanism in DMD

Materials and Methods

Results

Discussion

Conclusion