Genetic disruption of Smad7 impairs skeletal muscle growth and regeneration.

The transforming growth factor-β (TGF-β) family of growth factors plays an essential role in mediating cellular growth and differentiation. Myostatin is a muscle-specific member of the TGF-β superfamily and a negative regulator of muscle growth. Myostatin inhibitors are currently being pursued as therapeutic options for muscle disorders. Smad7 inhibits intracellular myostatin signalling via Smad2/3, and thus presents a means of regulating myostatin and potentiating muscle growth. We investigated the functional loss of Smad7 on muscle in vivo by examining muscle growth and differentiation in mice deficient in Smad7 (Smad7−/−). Smad7−/− mice showed reduced muscle mass, hypotrophy and hypoplasia of muscle fibres, as well as an increase in oxidative fibre types. Examination of muscle strength showed reduced force generation in vivo and ex vivo compared to wild-type controls. Analysis of muscle regeneration showed a delay in recovery, probably as a result of decreased activation, proliferation and differentiation of satellite cells, as confirmed in vitro. Additionally, myostatin expression was upregulated in Smad7−/− muscle. Our findings suggest that increased Smad2/3 signalling in the absence of Smad7 inhibition impedes muscle growth and regeneration. Taken together, our experiments demonstrate that Smad7 is an important mediator of muscle growth in vivo. Our studies enhance our understanding of in vivo TGF-β pathway modulation and suggest that Smad7 may be an important therapeutic target for muscle disorders. Key points Smad7 is an intracellular antagonist of transforming growth factor-β signalling pathways and modulates muscle growth in vivo. Loss of Smad7 results in decreased muscle mass, reduced force generation, fibre type switching from glycolytic towards oxidative type and delayed recovery from injury. Upregulated Smad2/3 signalling in Smad7−/− muscle results in reduced myoblast proliferation and differentiation. Smad7 is an important regulator of muscle growth and may be a potential intracellular therapeutic target for muscle disorders. Introduction The transforming growth factor-β (TGF-β) superfamily of growth factors exerts pleiotropic effects on tissue growth, cell proliferation and death (Burks & Cohn, 2011). Myostatin (growth/differentiation factor-8) is a member of the TGF-β superfamily and a key negative regulator of skeletal muscle growth (Lee, 2004). Mice carrying a homozygous myostatin-null mutation show dramatic increases in muscle mass arising from a combination of increased fibre size and fibre number (McPherron et al. 1997). Myostatin exerts numerous effects on muscles, including regulating myoblast proliferation and differentiation (Thomas et al. 2000; McCroskery et al. 2003; Trendelenburg et al. 2009), regulating myofibre number and size (McPherron et al. 1997; Amthor et al. 2007; Amthor et al. 2009), and regulating slow- and fast-twitch fibre type specification (Girgenrath et al. 2005; Hennebry et al. 2009). The canonical myostatin and TGF-β signalling pathways begin with ligand-dependent receptor activation and subsequent phosphorylation of receptor-mediated Smads (R-Smads): Smad2 and Smad3. Although TGF-β predominantly binds to TGF-β type II receptor, myostatin shows preferential binding affinity to the activin type IIB receptor (ActRIIB) (Lee & McPherron, 2001; Rebbapragada et al. 2003). Once engaged, the ActRIIB receptor heterodimerizes with activin receptor-like kinase-4 or -5, inducing a phosphorylation-dependent activation of the R-Smads (Rebbapragada et al. 2003). The activation of Smad2 and Smad3 induces oligomerization with the co-Smad, Smad4, leading to nuclear translocation and interaction with the context-specific transcription factors, including MyoD (Liu et al. 2001), as well as co-activators and co-repressors that modulate the expression of transcriptional targets. Activation of R-Smads is regulated by the inhibitory Smads: Smad6 and Smad7 (Hayashi et al. 1997; Imamura et al. 1997). Although Smad6 primarily functions within the bone morphogenic protein (TGF-β family member) pathway, Smad7 specifically inhibits TGF-β and myostatin signalling by competing with the R-Smads for binding with the type I receptor (Zhu et al. 2004). Thus, Smad7 inhibits Smad2/3 from activation and subsequent regulation of transcription. Because Smad7 inhibits the myostatin and TGF-β pathways, it may also play a role in modulating muscle mass. Over-expression of Smad7 in vitro accelerates myoblast differentiation, whereas inhibition of Smad7 expression by small interfering RNA results in deficient differentiation (Kollias et al. 2006). Mice carrying a genetic disruption or complete loss of Smad7 exhibit growth delays, abnormal development of immune tissues, and fibrosis in the kidney and lung (Li et al. 2006; Tojo et al. 2012; Liu et al. 2013). However, whether Smad7 plays a role in muscle growth in vivo has not been investigated previously. The aim of the present study was to examine the effects of Smad7 disruption in vivo on muscle growth and regeneration. We report that genetic reduction of Smad7 results in decreased muscle growth, decreased strength, delayed regeneration and alterations in fibre type composition. Our studies determine the mechanisms of myostatin inhibition in vivo and suggest a novel therapeutic target for muscle disorders.