**Michèle M. G. Hillege, Ricardo A. Galli Caro, Carla O**ff**ringa, Gerard M. J. de Wit, Richard T. Jaspers \* and Willem M. H. Hoogaars**

Laboratory for Myology, Department of Human Movement Sciences, Faculty of Behavioural and Movement Sciences, Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, 1081 BT Amsterdam, The Netherlands; m.m.g.hillege@vu.nl (M.M.G.H.); r.a.galli@amsterdamumc.nl (R.A.G.C.); c.offringa@vu.nl (C.O.); g.m.j.de.wit@vu.nl (G.M.J.d.W.); w.m.h.hoogaars@umcg.nl (W.M.H.H.)

**\*** Correspondence: r.t.jaspers@vu.nl; Tel.: +31-(0)20-5988463

Received: 3 January 2020; Accepted: 3 February 2020; Published: 6 February 2020

**Abstract:** Transforming Growth Factor β (TGF-β) is involved in fibrosis as well as the regulation of muscle mass, and contributes to the progressive pathology of muscle wasting disorders. However, little is known regarding the time-dependent signalling of TGF-β in myoblasts and myotubes, as well as how TGF-β affects collagen type I expression and the phenotypes of these cells. Here, we assessed effects of TGF-β on gene expression in C2C12 myoblasts and myotubes after 1, 3, 9, 24 and 48 h treatment. In myoblasts, various myogenic genes were repressed after 9, 24 and 48 h, while in myotubes only a reduction in *Myh3* expression was observed. In both myoblasts and myotubes, TGF-β acutely induced the expression of a subset of genes involved in fibrosis, such as *Ctgf* and *Fgf-2,* which was subsequently followed by increased expression of *Col1a1*. Knockdown of *Ctgf* and *Fgf-2* resulted in a lower *Col1a1* expression level. Furthermore, the effects of TGF-β on myogenic and fibrotic gene expression were more pronounced than those of myostatin, and knockdown of TGF-β type I receptor *Tgfbr1*, but not receptor *Acvr1b,* resulted in a reduction in *Ctgf* and *Col1a1* expression. These results indicate that, during muscle regeneration, TGF-β induces fibrosis via *Tgfbr1* by stimulating the autocrine signalling of *Ctgf* and *Fgf-2.*

**Keywords:** *Acvr1b*; *Tgfbr1*; myostatin; *Col1a1*; skeletal muscle; fibrosis; myogenesis; atrophy
