Growth differentiation factor 11 induces skeletal muscle atrophy via a STAT3-dependent mechanism in pulmonary arterial hypertension

Increased serum concentrations of GDF11 in PAH patients

As seen in Fig. 1A, serum concentration of GDF11 increased significantly in PAH patients (543.1 ± 197.2 pg/ml) compared to healthy controls (92.9 ± 37.8 pg/ml). We also detected the serum concentration of MSTN and Activin A, the molecules closely related to GDF11, and transduced SMAD2/3 activation and downstream transcriptional responses [14]. Although the expression of MSTN and Activin A was higher in PAH patients, only circulating GDF11 levels in PAH patients had the most significantly increased. Therefore, we supposed that GDF11 may play a key role in patients with PAH.

Fig. 1figure 1

MCT-induced PAH caused body weight loss and muscle atrophy. A Circulating GDF11 levels in PAH patients and health control (n=8). B RVSP of rats 4 weeks after MCT-treated, C body weight, and D the weight of gastrocnemius muscle, soleus muscle, tibialis anterior, and Extensor Digitorum Longus normalized per body weight (BW). E The weight of gastrocnemius muscle. F Representative images of EDL, TA, Sol, GM, and the cross-sectional areas of approximately 250 myofibers per group were determined. Scale bar represents 25 μm. G The distribution of myofiber cross-sectional area. n = 8 rats/group. Data are shown as mean ± SEM. Versus vehicle control, *P < 0.05, **P < 0.01, ***P < 0.001

MCT induces a skeletal muscle wasting phenotype in a PAH rat model

All MCT rats developed PAH as indicated by the increase in RVSP (Fig. 1B). Loss of weight in MCT rats was observed 4 weeks after MCT injection (17% reduction; Fig. 1C). Although the MCT rat tibialis anterior (TA), gastrocnemius muscle (GM), and soleus (SOL) exhibited muscle loss, only the GM atrophy index (muscle weight/total weight) was reduced which was accompanied by a significant decrease of CSA (Fig. 1D–G). Therefore, further research was limited to the GM.

GDF11 levels are accumulated in serum and lung in MCT rats

Serum GDF11 levels were raised in MCT rats when compared with controls (Fig. 2A). GDF11 protein levels were also elevated remarkably in the lung of MCT rats compared with controls (Fig. 2B). In the MCT model, immunohistochemical staining for GDF11 showed that the expression of GDF11 is higher in the pulmonary arteries which are concentrated in the endothelial cells in MCT-treated rats than in the control group (Fig. 2C). In addition, the protein levels of Trim63, Fbx32, and Foxo1, which are important factors in the ubiquitin-proteasome pathway, were raised significantly in GM of MCT rats when compared with controls (Fig. 2D).

Fig. 2figure 2

GDF11 levels are accumulated in serum and lung in MCT rats. A GDF11 was measured by ELISA in the serum from rats. B GDF11 expression in lung was detected by Western blot, and GAPDH served as a loading control. C Representative immunohistochemistry of lung sections showing pulmonary arteries stained for GDF11 or CD31 in rats, scale bar is 20 μm. D Western blot analysis of trim63, fbx32, and foxo1 was assessed by Western blot, and protein expression levels were quantified by densitometry. Data are shown as mean ± SEM. Versus vehicle control, *P < 0.05, **P < 0.01, ***P < 0.001, n = 8 rats/group

In vitro model of PAH-derived GDF11 is sufficient to induce the myotube atrophy

To explore the signaling pathways which contribute to muscle wasting in PAH, we utilized a PAEC-induced myotube atrophy model. The myotube was from the differentiation of C2C12. We collected CM from PAEC which was under hypoxia culture for 24h. The myotubes were treated with Hypo-CM (20%, 50%) for 48 h, and then fixed for the measurement of myotube diameter (Fig. 3A). Hypo-CM induced a decrease in myotube diameter which was accompanied by an increase of GDF11 level in Hypo-CM (Fig. 3B, C).

Fig. 3figure 3

The GDF11 is involved in myotube atrophy induced by CM from PAEC. A Schematic drawing depicting the generation of CM by hypoxia-culture of PAEC, then myotube was stimulated with CM for 48 h. B Concentrations of GDF11 (pg/mL) in 50% Norm-CM, 20% or 50% Hypo-CM. C Bright-field images of C2C12-derived myotubes treated with either 50% Norm-CM, 20% or 50% Hypo-CM from PAEC, and myotube diameter for conditions represented in the panel. Scale bar is 50 μm. D Myotubes were transfected with GDF11 siRNA or NC siRNA and 50% Norm-CM or 50% Hypo-CM. Protein levels were examined by immunoblotting. E Bright-field images of myotubes treated with 50% Hypo-CM with GDF11 antibody or isotype control, and myotube diameter for conditions represented in the panel. Scale bar is 50μm. F Immunoblots of trim63, fbx32, and foxo1 using lysates from myotubes treated with 50% Norm-CM or 50% Hypo-CM with GDF11 antibody or isotype control; and protein expression levels were quantified by densitometry. Values are presented as average ± SEM. Versus vehicle control, *P < 0.05, **P < 0.01, ***P < 0.001; versus Hypo-CM control, #P < 0.05, ##P < 0.01, ###P < 0.001; n=3

To verify whether Trim63, Fbx32, and Foxo1 was a downstream target of GDF11 in myotubes, GDF11 was silenced with specific siRNA. We found that loss of GDF11 down-regulated Trim63, Fbx32, and Foxo1 expression in Hypo-CM treated myotubes (Fig. 3D).

Next, the myotube diameter was measured in response to anti-GDF11 (neutralizing GDF11 antibody) or isotype control. Notably, anti-GDF11 could alleviate the myotube atrophy induced by 50% Hypo-CM significantly (Fig. 3E). Furthermore, Western blotting of treated myotubes demonstrated inhibition of proteolysis by anti-GDF11 showed as the reduction of Trim63, Fbx32, and Foxo1 levels (Fig. 3F). These results demonstrate that GDF11 released from in vitro model of PAH induce significant myotube atrophy.

GDF11 acts via STAT3, SOCS3, and iNOS to induce proteolysis in muscle atrophy in vitro

To further investigate intracellular signaling pathways activated in myotube in response to GDF11, we transfected C2C12 with NF-κB, ERK, Smad, or STAT3 dependent luciferase reporter plasmids. After differentiation of myotubes, the myotubes were subsequently treated with rGDF11 for 48 h. We selected these transcription factors for which were potential targets and have been implicated in skeletal muscle atrophy [1921]. Neither NF-κB nor ERK-dependent reporter activity changes in response to rGDF11 treatment (Fig. 4A, B). However, rGDF11 induced activation of the Smad reporter (Fig. 4C). rGDF11 also significantly increased STAT3 reporter activity in a dose-dependent manner (Fig. 4D). Based on these findings, we next measured the activation of Smad2/3 and STAT3 in myotubes treated with rGDF11. rGDF11 treatment induced a significant increase in phospho/total STAT3 and the effect was concentration-dependent, especially at the dose of 50 and 100 ng/ml (Fig. 4E). However, we had not found a significant increase in phospho/total Smad2/3 levels (Fig. 4E).

Fig. 4figure 4

GDF11 acts via STAT3, SOCS3, and iNOS to induce proteolysis in muscle wasting in vitro. AD NF-κB, ERK, Smad, or STAT3 dependent luciferase reporters in C2C12 myotubes treated with rGDF11 with the dose ranging from 0 to 100 ng/ml. E Representative Western blots of target proteins (iNOS, phosphorylation and total STAT3, phosphorylation and total Smad2/3, socs3) and loading control (GAPDH) from myotubes treated with rGDF11 with the dose ranging from 0 to 100 ng/ml for 48 h. F NO levels were measured in supernatant from the myotubes described in the panel. G Total protein content of rGDF11-treated myotubes. H Representative western blotting images of ubiquitin from myotubes. I Protein expression of trim63, fbx32, and foxo1 in myotubes treated with rGDF11 with the dose ranging from 0 to 100 ng/ml. GAPDH was used as an internal control. J Bright-field images of myotubes treated with rGDF11, with or without the 26S ribosome inhibitor MG-132 (10 μM) for 48 h; diameter of myotubes for conditions represented in the panel. Scale bar is 50μm. Data presented as mean ± SEM. Versus vehicle control, *P < 0.05, **P < 0.01, ***P < 0.001; versus rGDF11 control, #P < 0.05, ##P < 0.01, ###P < 0.001; n=3

We next measured protein levels of iNOS and socs3 since both would be increased in response to activation of STAT3 in atrophic myotubes in other conditions [26, 27]. Levels of iNOS and socs3 were significantly increased at 48h of rGDF11 treatment with a corresponding increase in the production of NO, which was paralleled by an increase in phospho/total STAT3 (Fig. 4E, F), suggesting they were the potential target of STAT3 which was activated by GDF11.

Correspondingly, we detected reduced protein content in myotubes treated with rGDF11 (Fig. 4G). The ubiquitin-proteasome system (UPS) and Foxo1could regulate muscle degradation via the E3 ubiquitin ligases, Trim63 and Fbx32 [28]. In our study, the levels of UPS were increased at 48h of rGDF11 treatment with a corresponding increase in the expression of Trim63, Fbx32, and Foxo1 dose-dependently (Fig. 4H, I). Based on these findings, we suppose that the exposure of myotubes to GDF11 activates STAT3 pathways implicated in skeletal muscle wasting in PAH.

We set out to determine if GDF11 induce myotube wasting via proteolytic effects. A 26S proteasome inhibitor, MG-132, was added to myotubes. We detected that MG-132 prevented GDF11-induced reductions in myotube diameter (Fig. 4J). These findings suggest that GDF11 has direct proteolytic effects on atrophic myotube via STAT3, SOCS3, and iNOS.

STAT3 inhibition abrogates myotube atrophy treated by GDF11

Results showed that Stattic, a STAT3 inhibitor, reversed myotube atrophy induced by GDF11, as assessed by myotube diameter (Fig. 5A). Next, we observed that Stattic completely inhibited iNOS and socs3 expression in myotube treated with rGDF11 (Fig. 5B). We detected that total protein level inhibition by GDF11 was reversed by Stattic (Fig. 5C). NO production and UPS levels were also dependently decreased by Stattic (Fig. 5D, E). In addition, GDF11-induced expression of Trim63, Fbx32, and Foxo1 protein was drastically inhibited by Stattic (Fig. 5F). These data indicate that Stattic has a therapeutic effect on myotube wasting induced by GDF11.

Fig. 5figure 5

Blocking STAT3 activation with Stattic, a STAT3 inhibitor, prevents GDF11 mediated atrophy in vitro. A Bright-field images of myotubes treated with rGDF11 (50ng/ml), with or without STAT3 inhibitor Stattic for 48 h. Scale bars = 50 μm. The fiber widths were measured and calculated (right panel). B Myotubes treated with rGDF11 then with Stattic for 48h were used for Western blot analysis with antibodies against iNOS, pY-STAT3, total STAT3, socs3, and GAPDH. C Total protein content of rGDF11-treated myotubes, with or without STAT3 inhibitor Stattic for 48 h. D NO levels were measured in supernatant from the myotubes described in the panel. E Representative western blotting images of ubiquitin from myotubes. F Protein expression of trim63, fbx32, and foxo1 in myotubes treated with rGDF11 then with Stattic for 48h. G Representative Western blots of phosphorylation and total STAT3 from myotubes treated with rGDF11, siALK5, or AcvRIIb. Data presented as mean ± SEM. Versus vehicle control, *P < 0.05, **P < 0.01, ***P < 0.001; versus rGDF11 control, #P < 0.05, ##P < 0.01, ###P < 0.001; n=3

Canonical signal transduction begins with GDF11 binding to its type II serine/threonine kinase receptor (ACVR2B) which then recruits and activates type I serine/threonine kinase receptors (ALK5) [29]. Results showed that knocked down ALK5 and ACVR2B suppressed the phosphorylation of STAT3. Therefore, we think GDF11 mediated the STAT3 signal which is dependent of ACVR2B/ALK5.

STAT3 inhibition improves muscle wasting in the MCT-treated rats

We next evaluated the therapeutic effect of STAT3 inhibition in an MCT-treated model of PAH (Fig. 6A). STAT3 inhibition had no significant effect on RVSP (Fig. 6B).

Fig. 6figure 6

STAT3 inhibition prevents muscle atrophy in the MCT rat. A SD rats were treated with MCT or saline on day 1, with or without intraperitoneal injection of Stattic daily for 2 weeks since day 14. The effects of Stattic on the main features of PAH model were examined, including B RVSP, C body weight, and D gastrocnemius muscles mass. E Cross-sections cut from the gastrocnemius muscle and stained with wheat germ agglutinin (blue). Scale bar is 25μm, n=3. F The cross-sectional areas of approximately 250 myofibers per group were determined and the distribution of myofiber cross-sectional area. Data are shown as mean ± SEM. Versus vehicle control, *P < 0.05, **P < 0.01, ***P < 0.001; versus MCT control, #P < 0.05, ##P < 0.01, ###P < 0.001; n=8 rats

Furthermore, Stattic administration distinctly improved chief features of muscle wasting, with significant prevention of the loss of body weight, GM weight with a corresponding prevention of the decrease of CSA (Fig. 6C–F).

STAT3 is a regulator of leptin signaling, which can affect food intake. However, MCT-treated rats in our study ate significantly less than their control counterparts (354g vs 422g, respectively). There was no difference in average food intake per animal between the MCT and MCT/Stattic-treated rats (354g vs 357g, respectively). This, along with the above data, suggests that STAT3, as a down-stream mediator of GDF-11, has a pro-atrophic effect on the skeletal muscle that is independent of its role as an appetite suppressant.

STAT3 inhibition improves muscle wasting through inhibition proteolysis

Results showed that Stattic specifically suppressed the phosphorylation of STAT3 and expression of its down-stream target gene iNOS and socs3 in GM of MCT-treated rats (Fig. 7A). Consistent with cellular data, Stattic administration also inhibited the expression of Trim63, Fbx32, and Foxo1 in gastrocnemius muscles of MCT-treated rats as shown by western-blot (Fig. 7B), which further confirmed that STAT3 inhibition improves muscle wasting through inhibition proteolysis.

Fig. 7figure 7

Pathway of STAT3 inhibition in improvement muscle atrophy in the PAH model. A, B The expression of indicated proteins in gastrocnemius muscles was detected by western blot. The band intensities were quantified and total STAT3 or GAPDH was used as control. C Model depicting how STAT3 promotes GDF11-induced muscle wasting. The GDF11 binds to ACVR2B/ALK5 and then activates STAT3 via phosphorylation. Following p-STAT3 translocates to the nucleus and upregulates the expression of iNOS and socs3, leading to the activation of the ubiquitin-proteasome pathway and iNOS/NO pathway, which in turn promotes muscle wasting. Data are shown as mean ± SEM. Versus vehicle control, *P < 0.05, **P < 0.01, ***P < 0.001; versus MCT control, #P < 0.05, ##P < 0.01, ###P < 0.001; n=3 rats

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