*3.4. TGF-*β *Induces Col1a1 Expression via Ctgf and Fgf-2 in Myoblasts*

To investigate whether TGF-β induces *Col1a1* expression in C2C12 myoblasts directly or via the autocrine expression of *Ctgf* or *Fgf-2*, the effects of TGF-β treatment on *Col1a1* expression were studied in the presence of siRNA targeting *Ctgf* or *Fgf-2*. At all time points, treatment with siRNA reduced *Ctgf* or *Fgf-2* mRNA expression levels compared to levels of control siRNA treatment by >90% and >80%, respectively (Figure 4j,m). At 48 h of TGF-β treatment the induction of *Col1a1* mRNA expression was substantially lower (approximately 50%) in the presence of siRNA targeting either *Ctgf* or *Fgf-2* compared to controls (Figure 4k,n), suggesting that *Col1a1* mRNA expression is at least in part regulated by TGF-β dependent *Ctgf* and *Fgf-2* expression. In addition, after 3 h of TGF-β treatment, *Ctgf* knockdown did not affect *Fgf-2* mRNA expression, although after 48 h *Fgf-2* mRNA expression was significantly lower (approximately 70%) in the presence of siRNA targeting *Ctgf*, compared to controls (Figure 4l). *Ctgf* mRNA expression was significantly lower (>55%) in the presence of siRNA against *Fgf-2* compared to controls at all time points (Figure 4o).

#### *3.5. TGF-*β *Has a Larger E*ff*ect on Muscle Di*ff*erentiation and Fibrosis than Myostatin*

Due to the functional and mechanistic similarities between TGF-β and myostatin, the effects of myostatin and TGF-β on C2C12 and primary myoblasts were studied. C2C12 and primary myoblasts, as well as myotubes, were treated with different doses of myostatin or TGF-β. Although a higher concentration of myostatin was needed compared to that of TGF-β in C2C12 and primary myoblasts, as well as myotubes, both proteins induced the translocation of SMAD2 to the nucleus. Figure 5a shows that in primary myotubes and undifferentiated myoblasts, 1 h of 10 ng/mL TGF-β or 300 ng/mL myostatin treatment resulted in the nuclear translocation of SMAD2. Little effect was observed for 0.01 ng/mL TGF-β or 10 ng/mL myostatin. Both of these ligands have a molecular weight of 25 kDa. In primary myoblasts, the comparison of effects of 3 and 48 h myostatin or TGF-β treatment on myogenic and fibrotic gene expression levels showed that after 48 h TGF-β reduced *Myh3* expression by approximately twofold compared to controls, while myostatin did not affect *Myh3* expression (Figure 5b,c). Furthermore, although in primary myoblasts after 3 h of treatment both myostatin and TGF-β significantly enhanced *Ctgf* mRNA expression levels, TGF-β increased *Ctgf* expression levels by 2.2-fold, while myostatin increased *Ctgf* expression levels only by 1.6-fold (Figure 5d,e). These results indicate that TGF-β has a stronger effect on fibrotic and myogenic gene expression levels than myostatin.

**Figure 4.** TGF-β affects fibrotic gene expression levels in myoblasts and myotubes in a time-dependent matter. (**a**–**h**) mRNA expression levels of (**a**) *Ctgf* in myoblasts, (**b**) *Ctgf* in myotubes, (**c**) *Fgf-2* in myoblasts, (**d**) *Fgf-2* in myotubes, (**e**) *Col1a1* in myoblasts, (**f**) *Col1a1* in myotubes, (**g**) *Nox4* in myoblasts, (**h**) *Nox4* in myotubes. (**a–d**) In myoblasts and myotubes, expression levels of *Ctgf* and *Fgf-2* were acutely induced by TGF-β. (**e–h**) *Col1a1* and *Nox4* expression levels were gradually induced by TGF-β. *Gapdh* served as housekeeping gene; data were normalized to values of control cells at 0 h; \* indicates significant difference at *p* < 0.05; *n* = 3 experiments per condition (**a**–**h**). (**i**) *Col1a1* mRNA expression levels in myotubes were approximately 10-fold higher compared to those in myoblasts. *Gapdh* served as housekeeping gene; data were normalized to expression values in myoblasts. \* *p* indicates significant difference at <0.05; *n* = 6 experiments per condition. (**j**) At all time points after siRNA treatment, *Ctgf* expression was knocked down by >90%. (**k**) *Col1a1* expression was reduced in the presence of siRNA targeting *Ctgf*. (**l**) After 3 h of TGF-β treatment, *Fgf-2* expression increased independent of *Ctgf*. After 48 h of TGF-β treatment, in the presence of siRNA targeting *Ctgf, Fgf-2* expression was significantly reduced. **m** At all time points after siRNA treatment, *Fgf-2* expression was knocked down by >81%. Both *Col1a1* (**n**) and *Ctgf* (**o**) expression levels were significantly reduced in the presence of siRNA targeting *Fgf-2* compared to those of control siRNA condition. *Gapdh* served as housekeeping gene; data were normalized to values of control cells at 0 h; \* indicates significant difference at *p* < 0.05; *n* = 6 experiments per condition (**j–o**).

**Figure 5.** TGF-β has a larger effect on myoblasts compared to myostatin. (**a**) Primary cells were induced to differentiate for 2 days and subsequently treated with TGF-β (0.01 or 10 ng/mL) or myostatin (10 or 300 ng/mL). Myotubes were stained for MHC (green) and nuclei were stained using DAPI (blue). SMAD2 is visible in red. The scale indicates 100 μm. After TGF-β or myostatin treatment, SMAD2 was translocated to the nucleus in both myotubes and undifferentiated myoblasts compared to controls. (**b**,**c**) In primary mouse myoblasts, expression levels of *Myh3* mRNA were reduced after 48 h TGF-β treatment compared to those in untreated cells (**b**), while no differences were observed for myostatin (MSTN) (**c**). (**d**,**e**) Expression levels of *Ctgf* were increased after 3 h TGF-β (**d**) or myostatin treatment (**e**) compared to those in untreated cells, although TGF-β had a larger effect. (**f**,**g**) Treatment with specific siRNAs reduced levels of *Acvr1b* (**f**) or *Tgfbr1* (**g**). (**h**,**i**) Knockdown of *Acvr1b* or *Tgfbr1* did not affect *Myod* (**h**) or *Myog* (**i**) expression levels. (**j**,**k**) *Tgfbr1* knockdown slightly reduced *Ctgf* (**j**) and *Col1a1* (**k**) expression levels, while *Acvr1b* knockdown had little effect. The combined knockdown of *Tgfbr1* and *Acvr1b* did significantly reduce *Ctgf* and *Col1a1* expression levels. *Gapdh* served as housekeeping gene; error bars indicate standard error of the mean; \* indicates significant difference at *p* < 0,05; *n* = 3 experiments per condition; data were normalized to values of control cells at 0 h. (**l**,**m**) There is a significant correlation between *Tgfbr1* expression level and *Ctgf* and *Col1a1* expression levels (**l**), while no such correlations were found between *Acvr1b* expression levels and *Ctgf* or *Col1a1* expression levels (**m**).

#### *3.6. Tgfbr1 Levels Correlate with Ctgf and Col1a1 Expression Levels*

To further examine the effects of myostatin and TGF-β, type I receptors *Acvr1b* and *Tgfbr1* were individually or simultaneously blocked in myoblasts using specific siRNAs. C2C12 myoblasts were treated with siRNA against *Acvr1b* or *Tgfbr1* for 24 h in growth medium and were additionally treated with siRNA for 48 h in differentiation medium. *Acvr1b* and *Tgfbr1* siRNA reduced receptor mRNA levels by >60% and >50%, respectively, without affecting expression of the other receptor (Figure 5f,g). No significant effects of siRNA treatment on *Myod* or *Myog* expression levels were observed (Figure 5h,i). In line with these results, receptor blocking during differentiation using chemical blocker Ly364947 did not affect fusion or differentiation index nor myotube thickness after 3 days of differentiation (Figure 6a–e). In addition, Ly364947 treatment did not affect *Myh3* expression levels after 48 h of differentiation. However, when C2C12 myoblasts were simultaneously treated with TGF-β and Ly364749, *Myh3* mRNA expression levels were significantly increased compared to those in TGF-β treated cells and similar to those in control cells. In line with observations in primary myoblasts, in C2C12 cells myostatin treatment had no significant effect on *Myh3* expression (Figure 6f). In addition, when the receptors were blocked with Ly364947 during proliferation for 24 h and subsequent differentiation for 2 days, *Myh3* expression was significantly increased (Figure 6g). Knockdown of *Acvr1b* did not significantly affect levels of *Ctgf* and *Col1a1* mRNA, whereas *Tgfbr1* knockdown reduced expression levels of *Ctgf* and *Col1a1* mRNA. Combined knockdown of *Acvr1b* and *Tgfbr1* did not reduce *Ctgf* or *Col1a1* mRNA levels significantly further than *Tgfbr1* knockdown (Figure 5j,k). In addition, *Tgfbr1* mRNA expression levels significantly correlated with both *Ctgf* and *Col1a1* mRNA expression levels (Figure 5l,m). Taken together, these results indicate that TGF-β signalling via *Tgfbr1* has a stronger effect on muscle fibrosis compared to myostatin.

**Figure 6.** Effects of type I receptor blocking on myoblast differentiation is time-dependent. (**a**,**b**) C2C12 cells were induced to differentiate in control medium (**a**) or in medium supplemented with the TGF-β receptor inhibitor Ly364947 (**b**). Myotubes were stained for MHC (green) and nuclei were stained using DAPI (blue). Scale indicates 100 μm. (**c**) Fusion index, defined as number of myotubes ≥ 2 nuclei/total number of nuclei and (**d**) differentiation index defined as number of nuclei within MHC+ myotubes/total number of nuclei were not significantly affected by Ly364947 treatment. Error bars indicate standard error of the mean; \* indicates significant difference at *p* < 0.05; *n* = 4 experiments per condition. (**e**) Myotube thickness was not affected by Ly36447 treatment, compared to control condition. (**f**) 48 h of Ly364947 treatment did not affect *Myh3* expression levels in differentiating C2C12 myoblasts. *Myh3* expression levels were significantly increased in cells simultaneously treated with TGF-β and Ly364947 compared to those of cells treated with TGF-β and similar to those of untreated cells. Myostatin did not significantly affect *Myh3* expression levels. (**g**) *Myh3* expression levels were significantly increased when C2C12 myoblasts were treated with Ly364947 during proliferation for 24 h and subsequent culture in differentiation medium for 48 h.

#### **4. Discussion**

The aim of this study was to assess the time-dependent effects of TGF-β signalling on gene expression in myoblasts and myotubes and compare the effects of TGF-β and myostatin signalling in myoblasts. Here we show that in vitro TGF-β treatment inhibits the expression of a subset of myogenic genes in both myoblasts and myotubes, but does not affect myotube thickness. Most importantly, our results show that TGF-β regulates the expression of fibrotic genes in both myoblasts and myotubes in a time-dependent manner. TGF-β regulates *Col1a1* mRNA expression at least in part via *Ctgf* and *Fgf-2* and, in addition, *Ctgf* and *Fgf-2* are also required to induce the expression of each other. Moreover, our results show a more prominent role for TGF-β in SMAD signalling, as well as myogenic and fibrotic gene expression in comparison to myostatin.

#### *4.1. TGF-*β *A*ff*ects Myogenic Gene Expression in Both Myoblasts and Myotubes*

TGF-β is known for its inhibitory effect on myoblast differentiation in vitro through inhibition of MyoD [14,32]. As expected, TGF-β inhibited myoblast differentiation and myogenic gene expression. Also, in myotubes, a reduction in *Myh3* expression was observed after 24 and 48 h of TGF-β treatment. Embryonic myosin heavy chain (eMHC), which is encoded by *Myh3*, is normally only expressed during embryonic/fetal and neonatal development, but is transiently re-expressed during muscle regeneration. The loss of eMHC in adult muscle in vivo has been shown to change MHC isoform expression, while in vitro *Myh3* knockdown may result in reduced fusion index and a reduced number of reserve cells, which suggests that loss of *Myh3* results in the early differentiation of MuSCs, depleting the MuSC pool [33]. Together, these results suggest that long-term TGF-β expression in muscle fibres after injury or in chronic disease may impede proper regeneration through repression of *Myh3* expression.

Additionally, TGF-β has been known from previous studies to interfere with MyoD function via two different mechanisms. First, TGF-β-induced SMAD3 can directly interact with MyoD. Second, TGF-β/SMAD3 interferes with the interaction between MyoD and myocyte enhancer factor 2 (MEF2), which is required for the expression of many myogenic genes [14,32]. Here, we show that TGF-β induces *Id1* expression acutely and transiently in both myoblasts and myotubes. *Id1* is known to inhibit myoblast differentiation by interfering with the formation of MyoD/E complexes, which are required for MyoD function [27]. Our data suggest that the upregulation of *Id1* mRNA may be another mechanism through which TGF-β interferes with MyoD function.

#### *4.2. TGF-*β *Does Not A*ff*ect Myotube Size In Vitro*

TGF-β overexpression within mouse muscle has been shown to result in the stimulation of atrogin-1 expression and atrophy in vivo [17,18]. To investigate whether this increase in atrogin-1 expression was a direct or indirect effect of TGF-β, time-dependent effects of TGF-β on E3 ligase mRNA expression were studied. In contrast to what has been shown in vivo, C2C12 myotubes did not show evidence for any effect of TGF-β on muscle atrophy. TGF-β treatment resulted in a reduction in *Atrogin-1* and *Murf-1* mRNA expression, rather than an increase. Moreover, an increase in both *Atrogin-1* and *Murf-1* expression was observed during differentiation, which suggests that the observed TGF-β-induced effects on E3 ligase mRNA expression were likely related to inhibition of differentiation. In both myoblasts and myotubes, expression levels of the ligase *Musa1* were transiently increased. Furthermore, TGF-β did not affect Akt or ERK1/2 phosphorylation nor myotube size. Together, these data indicate that in C2C12 myotubes, TGF-β does not directly contribute to atrophy. However, in vivo long term overexpression of TGF-β may lead to a reduction in muscle fibre size [17,18]. Based on our data, this observed in vivo TGF-β overexpression-induced atrophy is possibly mediated via *Musa1* rather than by elevated *Murf-1* or *Atrogin-1* expression levels. Furthermore, we show that TGF-β stimulates *Nox4* and *Id1* mRNA expression. These genes have been implied to play a role in muscle atrophy [34,35]. TGF-β has been shown to induce caspase 3 expression and DNA fragmentation in C2C12 cells [36]. As such, myonuclear apoptosis and loss of muscle stem cells induced by TGF-β may

contribute to muscle atrophy as well. The role of TGF-β in the regulation of muscle fibre size requires further investigation.

#### *4.3. TGF-*β *Contributes to Fibrosis by Stimulation of Fibrotic Gene Expression in Myoblasts and Myotubes*

Our data show that both myoblasts and myotubes express various pro-fibrotic genes and TGF-β stimulates the expression of these genes in a time-dependent manner. This suggests that, in addition to its effect on fibroblasts, TGF-β likely also contributes to muscle fibrosis through effects on myoblasts and muscle fibres. The stimulatory effects of TGF-β on *Col1a1* mRNA expression in myotubes were relatively small compared to those in myoblasts. Nevertheless, myotubes may contribute substantially to collagen type I production. Basal expression levels of *Col1a1* in myotubes were approximately 10-fold higher than in myoblasts. Moreover, MuSCs comprise approximately 2%–5% of the myonuclei within mature muscle [37] and the number of fibroblasts is roughly 10-fold lower than the number of myonuclei [38,39]. Therefore, it is conceivable that within mature skeletal muscle, differentiating myoblasts and muscle fibres contribute substantially to the production of collagen type I.

Collagen type I is found in the endo-, peri- and epimysium surrounding muscle fibre [40,41]. Collagen fibres reinforce the ECM surrounding muscle fibres, which is essential in providing a niche for MuSCs, giving structure to the muscle and is even crucial for proper muscle function [42–44]. It is conceivable that during myogenesis and muscle regeneration, muscle fibres will secrete collagen type I to contribute to the deposition of connective tissue that provides a scaffold for the regenerating parts of the muscle fibre. However, chronic high expression of TGF-β in skeletal muscle may contribute to muscle fibrosis via the continuous elevated expression of collagen. In muscular dystrophies and aged muscle, TGF-β expression in damaged areas of the muscle may cause excessive collagen deposition. This may result in locally enhanced stiffness along the muscle fibre, which may cause strain distributions along the length of the muscle fibre. As a consequence, muscle fibres are likely to become susceptible to further injuries. In addition, excessive collagen deposition will result in enhanced stiffness of the muscle stem cell niche and likely alter MuSC mechanosensitivity, which may reduce myoblast differentiation and thus impair muscle regeneration capacity [44–47].

Besides pro-fibrotic growth factors and ECM genes, TGF-β also induced the expression of *Nox4*. *Nox4* expression is induced by TGF-β within various cell types such as endothelial cells or lung mesenchymal cells [31,48]. *Nox4* is part of an enzyme family which catalyses the reduction of oxygen into reactive oxygen species (ROS). In lung fibrosis, *Nox4*-dependent H2O2 generation is required for TGF-β mediated myofibroblast differentiation and ECM production [31]. Furthermore, *Nox4* is a known source for oxidative stress in many tissues and in chronic kidney disease both *Nox4* and oxidative damage markers are increased in muscle [49]. Therefore, we suggest that prolonged TGF-β expression in muscle wasting disorders may contribute to oxidative damage via *Nox4* upregulation.

### *4.4. TGF-*β *Induces Col1a1 Expression via Autocrine Ctgf and Fgf-2 Signalling*

In lung fibrosis, TGF-β is known to induce collagen 1 expression via CTGF [50–52]. This, in combination with the observed expression patterns for *Ctgf*, *Fgf-2* and *Col1a1* in our myoblasts and myotubes, raised the question regarding whether in muscle cells TGF-β directly induced *Col1a1* expression or indirectly via enhancement of expression of these growth factors. *Ctgf* and *Fgf-2* were significantly knocked down using siRNA. After 48 h of TGF-β treatment, *Col1a1* mRNa expression levels were significantly reduced when *Ctgf* or *Fgf-2* was knocked down. This suggests that *Col1a1* expression is at least in part dependent on both *Ctgf* and *Fgf-2* expression in an autocrine manner. In corneal endothelial cells and human vertebral bone marrow stem cells, FGF-2 has been implied to stimulate collagen production [53,54], while in muscle FGF-2 is best known to stimulate MuSC activation and proliferation [55,56]. In this study, we show for the first time that in C2C12 muscle cells *Fgf-2* is required for TGF-β induced *Col1a1* mRNA expression.

Our results show that after 3 h of TGF-β treatment, *Ctgf* knockdown did not significantly affect *Fgf-2* expression; however, *Fgf-2* expression was significantly reduced after 48 h of TGF-β treatment in the presence of siRNA against *Ctgf*. These data suggest that TGF-β acutely induces *Fgf-2* expression independently of changes in *Ctgf* expression, though chronic expression of *Fgf-2* depends on *Ctgf* expression levels. *Ctgf* expression was shown to depend on *Fgf-2* levels both acutely and chronically. To the best of our knowledge, this interaction has not been reported before. See Figure 7 for a schematic of the proposed mechanism for TGF-β induced regulation of *Ctgf*, *Fgf-2* and *Col1a1.* We suggest that TGF-β stimulates *Col1a1* expression largely via the autocrine and paracrine signalling of *Ctgf* and *Fgf-2* and that *Ctgf* and *Fgf-2* may regulate the expression of each other via a positive feedback loop.

**Figure 7.** Schematic illustration of a proposed mechanism of how TGF-β regulates *Col1a1* mRNA expression. TGF-β binds to its receptors and activates downstream SMAD2/3 signalling. Subsequently, R-SMAD complexes translocate into the nucleus to regulate mRNA expression of growth factors such as *Ctgf* and *Fgf-2*. CTGF and FGF-2 proteins are secreted by the muscle cell and subsequently induce *Col1a1* expression via autocrine or paracrine signalling. Furthermore, expression levels of *Fgf-2* and *Ctgf* are dependent on each other.

#### *4.5. TGF-*β *Has a More Pronounced E*ff*ect than Myostatin on Myoblast Di*ff*erentiation and Fibrotic Gene Expression*

Because of overlap in functional implications and mechanistic similarities between TGF-β and myostatin signalling in myoblasts, we compared the effects of both growth factors on myogenic and fibrotic gene expression. In order to induce downstream activation of SMAD2 signalling, a higher concentration of myostatin was required compared to TGF-β. Furthermore, in myoblasts, TGF-β had a larger effect on *Myh3* and *Ctgf* expression compared to myostatin. To further compare effects of TGF-β and myostatin signalling on myoblasts, these ligands were inhibited by using siRNA against their type I receptors. TGF-β is best known to signal via the TGF-β type I receptor TGFR-1 [24]. In epithelial cells, it has been shown that myostatin can signal via TGFR-1, as well as via ACTR-1B [23]. In mouse myoblasts, myostatin has been shown to signal mainly via ACTR-1B and not via TGFR-1, while in mouse fibroblasts myostatin signals mainly via ACTR-1B [25]. Together, these studies suggest that the knockdown of *Tgfbr1* mainly inhibits TGF-β signalling, while *Acvr1b* knockdown inhibits myostatin signalling. Here, we show in C2C12 myoblasts that *Ctgf* and *Col1a1* mRNA levels correlate with *Tgfbr1* mRNA expression levels, but not with *Acvr1b* expression levels. Moreover, no synergistic effects on the expression of pro-fibrotic genes were observed for combined receptor knockdown. Together, these data indicate that in muscle cells TGF-β has a more pronounced effect on fibrosis than myostatin and that pro-fibrotic gene expression in muscle is mainly mediated via *Tgfbr1*, and not via *Acvr1b*.

*Acvr1b* and *Tgfbr1* inhibition using siRNA did not affect the expression of myogenic genes. Furthermore, we showed that receptor blocking during differentiation using chemical blocker Ly364947 did not affect the differentiation or fusion index after 3 days, nor the expression of *Myh3* after 2 days. However, in cells treated with both TGF-β and Ly364947, *Myh3* expression levels were similar to those of control cells, which indicates that Ly364947 cancels out the negative effect of TGF-β on *Myh3* expression levels. In addition, when *Acvr1b* and *Tgfbr1* receptors were blocked by Ly364947 during proliferation for 24 h and subsequent differentiation for 2 days, *Myh3* expression levels were significantly increased compared to those of controls. This indicates that the negative effects of TGF-β on myoblast differentiation were cancelled by *Tgfbr1* blocking. The role of *Acvr1b* in myoblast differentiation cannot be concluded based on these results.

Under differentiation conditions, receptor blocking does not further enhance the expression of myogenic genes, which indicates that effects of TGF-β and possibly myostatin on differentiation are dose-dependent and time-dependent. The serum levels (i.e., growth factors such as TGF-β) are relatively low in the differentiation medium compared to the levels in growth medium. This suggests that low concentrations of TGF-β have a minor effect on myogenic gene expression and myoblast differentiation. Note that there is a difference between the chemical blocker Ly364947 and the siRNAs targeting *Acvr1b* and *Tgfbr1* in interference with type I receptor function. While Ly364947 blocks TGF-β signalling within one hour, as demonstrated in Figure 1, siRNAs interfere with the translation of the target mRNA, which may result in a delayed knockdown of type I receptors (Figure 5). This may explain why the presence of siRNA in growth medium did not affect myogenic gene expression. Based on our results, it seems that myoblast differentiation is less sensitive to myostatin signalling than to TGF-β signalling.

#### *4.6. Implications in Therapeutic Treatments*

Altogether, our results demonstrate that TGF-β signalling has an inhibitory effect on myoblast differentiation and contributes substantially to fibrosis. Therefore, the TGF-β pathway proves to be an interesting potential therapeutic target for treatment of muscular dystrophies. The inhibition of the TGF-β pathway may relieve and attenuate progressive muscle pathology characterized by severe fibrosis and loss of muscle mass. However, taking into account that TGF-β affects various cellular processes throughout the body, generic inhibition of the protein may have serious consequences. Our data show that TGF-β inhibits differentiation and induces fibrosis directly via its receptor in myoblasts and differentiated myotubes. This indicates that the inhibition of TGF-β exclusively within

muscle tissue may be an effective approach to improve muscle regeneration in muscular dystrophy. Furthermore, our data demonstrate that TGF-β has a larger effect on differentiation and fibrosis than myostatin. Moreover, *Tgfbr1*, but not *Acvr1b* inhibition, significantly reduced *Ctgf* and *Col1a1* mRNA expression levels, while simultaneous receptor knockdown did not reduce expression levels even further. This suggests that solely blocking *Tgfbr1* and concomitant inhibition of TGF-β signalling may be sufficient to reduce fibrosis in muscular dystrophy. However, when in pathological conditions, both the inhibition of fibrosis and improved regeneration are required, thus simultaneous blocking of the *Tgfbr1* and *Acvr1b* receptor may be desirable. It has been shown that both myostatin and activins signal via *Acvr1b* and that these ligands synergistically inhibit regulation of muscle size [57,58]. Thus, simultaneous targeting of *Tgfbr1* and *Acvr1b* in vivo may still have a synergistic effect on overall muscle function improvement.
