Elsevier

Life Sciences

Volume 248, 1 May 2020, 117465
Life Sciences

FGF6 enhances muscle regeneration after nerve injury by relying on ERK1/2 mechanism

https://doi.org/10.1016/j.lfs.2020.117465Get rights and content

Abstract

Background

Severe peripheral nerve injury leads to skeletal muscle atrophy and impaired limb function that is not sufficiently improved by existing treatments. Fibroblast growth factor 6 (FGF6) is involved in tissue regeneration and is dysregulated in denervated rat muscles. However, the way that FGF6 affects skeletal muscle repair after peripheral nerve injury has not been fully elucidated.

Methods

In this study, we investigated the role of FGF6 in the regeneration of denervated muscles using myoblast cells and an in vivo model of peripheral nerve injury.

Results

FGF6 promoted the viability and migration of C2C12 and primary myoblasts in a dose-dependent manner through FGFR1-mediated upregulation of cyclin D1. Low concentrations of FGF6 promoted myoblast differentiation through FGFR4-mediated activation of ERK1/2, which upregulated expression of MyHC, MyoD, and myogenin. FGFR-1, FGFR4, MyoD, and myogenin were not upregulated when FGF6 expression was inhibited in myoblasts by shRNA-mediated knockdown. Injection of FGF6 into denervated rat muscles enhanced the MyHC-IIb muscle fiber phenotype and prevented muscular atrophy.

Conclusion

These findings indicate that FGF6 reduces skeletal muscle atrophy by relying on the ERK1/2 mechanism and enhances the conversion of slow muscle to fast muscle fibers, thereby promoting functional recovery of regenerated skeletal muscle after innervation.

Introduction

Severe peripheral nerve injury leads to denervation of skeletal muscles, which causes pathological changes including muscular atrophy. In resting muscle, satellite cells are quiescent. Following muscle injury, satellite cells are activated and become proliferating myoblasts that either self-renew or differentiate to form new muscle fibers or repair existing fibers [1]. However, when the muscle is denervated, functional recovery is significantly impaired, even after nerve connections are reestablished [2]. Existing treatments do not sufficiently improve muscle function. Therefore, understanding the mechanisms underlying the regeneration of denervated muscle is important for the development of more effective treatments.

Fibroblast growth factor (FGF) regulates various biological responses [3] via FGF receptors (FGFRs). The FGF family comprises 10 members, but only Fibroblast growth factor 6 (FGF6) expression is restricted to cells of the myogenic lineage [4]. After skeletal muscle injury, wild-type mice up-regulated FGF6 and completely restored the experimentally damaged skeletal muscle. In contrast, FGF6 (−/−) mutant mice showed severe regeneration defects, including fibrosis and myotube degeneration. [5,6]. Regenerative capacity can be restored by the injection of recombinant FGF6 [7]. These findings implicate FGF6 in the regeneration of denervated muscle.

The ability of muscle tissue to grow or regenerate after injury depends on the activation and proliferation of satellite cells. These small mononuclear cells are located under the basal layer of the muscle fibers and are mainly present in resting muscles [8]. However, during growth or muscle injury, satellite cells begin mitotic activity, show programmed proliferation and differentiation, and then express muscle-specific proteins [9,10]. These processes are tightly adjusted by Myogenic determining factor (MyoD), myogenin, and Myogenic regulatory factor 5 (myf5) [11]. FGF6 signaling is transduced predominantly through Fibroblast growth factor receptor 1 (FGFR1) and Fibroblast growth factor receptor 4 (FGFR4) [12] and is believed to have dual functions during myogenesis, depending on which receptor is used. During early stages of regeneration, high concentrations of FGF6 promote myogenic precursor proliferation through FGFR1 [13]. At later stages, lower concentrations of FGF6 activate differentiation pathways through FGFR4 [14].

In this study, we studied the protective effect of FGF6 on skeletal muscle and the effect of FGF6 on the proliferation and differentiation of C2C12 myoblasts in the rat sciatic nerve injury model. Our results show that FGF6 can protect the injured sciatic nerve and promote the viability, migration and differentiation of C2C12 myoblasts. These beneficial effects of FGF6 are achieved by activating specific components of MAPK signaling pathway in a concentration dependent manner, and FGF6 can convert slow muscle fibers into fast muscle fibers to prevent muscle atrophy.

Section snippets

Ethical statement

All animal experiments were approved by the Animal Research Committee of Shanghai Tenth People's Hospital Affiliated to Tongji University and were conducted in accordance with established international guiding principles for Animal Research.

Rat model of sciatic nerve injury

Male Sprague-Dawley rats weighing 250 to 350 g were obtained from Shanghai Laboratory Animal Center (Shanghai, China), and housed in a facility with a 12:12 h light/dark cycle at a controlled temperature and humidity with free access to food and water.

FGF6 promotes the viability, migration, and differentiation of C2C12 myoblast cells

FGF6 increased the proliferation (Fig. 1A) and migration (Fig. 1B) of C2C12 myoblast cells in a dose-dependent manner. To monitor the differentiation of C2C12 cells, we analyzed the expression of MyHC and FGFR4 by immunostaining. We selected a minimum of P values <0.05 and < 0.001 for subsequent testing. A low concentration of FGF6 (0.8 nmol/ml) increased MyHC and FGFR4 expression in C2C12 cells (Fig. 1C, middle panel), indicating enhanced myoblast differentiation. In contrast, a higher

Discussion

In this study, we investigated the mechanisms underlying FGF6-mediated muscle recovery after denervation. The functional recovery of the reinnervated muscle after low and high doses of FGF6 was indicated by the sciatica functional index. Treatment with high concentrations of recombinant FGF6 after nerve injury increased myoblast cell proliferation, in agreement with previous findings [14]. FGFR1 and cyclin D1 expression was significantly upregulated during this time, suggesting that enhanced

Conclusion

In conclusion, the present study has shown that FGF6 controls myoblast precursor proliferation and differentiation via ERK1/2-dependent signaling pathways. Furthermore, treatment with recombinant FGF6 reduces muscle atrophy after denervation by promoting the MyHC-IIb fiber phenotype in fast muscles. These findings suggest that FGF6 may promote the maintenance of muscle mass following reinnervation.

Abbreviations

    FGF

    Fibroblast growth factor

    FGFRs

    Fibroblast growth factor receptors

    FGF6

    Fibroblast growth factor 6

    MyoD

    Myogenic determining factor

    myf5

    Myogenic regulatory factor 5

    FGFR 1

    Fibroblast growth factor receptors 1

    FGFR 4

    Fibroblast growth factor receptors 4

    SFI

    Sciatic function index

    DMEM

    collagenase/Dulbecco's modified Eagle's medium

    FBS

    Fetal bovine serum

    CEE

    Chick embryo extract

    bFGF

    Basic fibroblast growth factor

    H&E

    Hematoxylin and eosin

    CSAs

    Calculate cross-sectional areas

    MyHC

    Myosin heavy chain

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 81401851) and the Program for Young Excellent Talents in Tongji University (No. 2016kj056).

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Consent for publication

Not applicable.

Authors' contributions

Planned experiments: SX, PW; performed experiments: QC, GW, MZ, HG, CX, QZ, BC; analyzed data: QC, GW; contributed reagents: PW; wrote the paper: QC, GW, PW.

Declaration of competing interest

The authors confirm that there are no conflicts of interest.

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    These authors contributed equally to this work.

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