BMP antagonists enhance myogenic differentiation and ameliorate the dystrophic phenotype in a DMD mouse model

Abstract

Duchenne Muscular Dystrophy (DMD) is an X-linked lethal muscle wasting disease characterized by muscle fiber degeneration and necrosis. The progressive pathology of DMD can be explained by an insufficient regenerative response resulting in fibrosis and adipose tissue formation. BMPs are known to inhibit myogenic differentiation and in a previous study we found an increased expression of a BMP family member BMP4 in DMD myoblasts. The aim of the current study was therefore to investigate whether inhibition of BMP signaling could be beneficial for myoblast differentiation and muscle regeneration processes in a DMD context. All tested BMP inhibitors, Noggin, dorsomorphin and LDN-193189, were able to accelerate and enhance myogenic differentiation. However, dorsomorphin repressed both BMP and TGFβ signaling and was found to be toxic to primary myoblast cell cultures. In contrast, Noggin was found to be a potent and selective BMP inhibitor and was therefore tested in vivo in a DMD mouse model. Local adenoviral-mediated overexpression of Noggin in muscle resulted in an increased expression of the myogenic regulatory genes Myog and Myod1 and improved muscle histology. In conclusion, our results suggest that repression of BMP signaling may constitute an attractive adjunctive therapy for DMD patients.

Fig. 1. Endogenous BMP signaling during myogenic differentiation

(A) Myogenic differentiation of C2C12 cells. Immunofluorescent staining of myogenic marker desmin in red and differentiation marker Myosin Heavy Chain (MyHC) in green. After 3 days of differentiation in low serum medium, formation of myotubes was observed. (B) Western blot analysis of pSmad1/5/8 and Myog levels on C2C12 protein lysates at different time points of differentiation. αTubulin is shown as a loading control in all samples. (C) qRT-PCR analysis of Bmp4 expression on C2C12 cDNA at different time points of differentiation. Fold expression relative to PM is shown and error bars indicate s.d. of triplicate experiments. *P<0.05. PM, proliferation medium; DM, differentiation medium.

Fig. 2. Effect of BMP inhibitors on BMP and TGFβ signaling

(A) Luciferase reporter assay with the BRE-luc reporter showing the activity of endogenous BMP signaling in transfected C2C12 cells treated with or without BMP antagonists Noggin (NOG; 200ng/ml), LDN-193189 (LDN; 120nM) and dorsomorphin (DMPN; 4μM).(B) Luciferase reporter assay with BRE-luc showing the activity of BMP4-induced signaling in transfected C2C12 cells treated with or without different concentrations of BMP antagonists (C) Luciferase reporter assay with the CAGA-luc reporter showing the activity of TGFβ-induced signaling in transfected C2C12 cells treated with or without different concentrations of BMP antagonists. Error bars indicate s.d. of triplicate samples and the luciferase activity relative to the control is shown in each graph; *P<0.01.

Fig. 3. Effect of BMP inhibitors on myogenic differentiation

(A) Immunofluorescent staining of desmin and MyHC showing the effect of Noggin (NOG; 200ng/ml), LDN-193189 (LDN; 120nM) and dorsomorphin (DMPN; 4 μM) on myogenic differentiation of C2C12 cells. Experiment was performed in duplo and representative pictures are shown. (B) Quantification of immunofluorescent staining in (A) showing the differentiation index and fusion index of C2C12 cells treated with or without the different BMP antagonists. Error bars indicate the s.d. of 10 areas measured in duplo. *P<0.01 (C) Western blot analysis of C2C12 protein lysates showing the pSmad1/5/8 levels and Myogenin levels after treatment with the different compounds after 1 day and 2 days of differentiation. αTubulin is shown as a loading control in all samples. (D) Immunofluorescent staining of Myog (in green) and desmin (myogenic cells; in red) of C2C12 cells differentiated for 3 days and 6 days with or without Noggin (200ng/ml). Top row shows merged pictures with DAPI (in blue) staining the total nuclei in the field. Representative pictures are shown from experiments performed in duplo. The graph on the right shows the quantification of the percentage of Myog⁺ nuclei compared to the total amount of nuclei. Error bars indicate the s.d. of 10 areas measured in duplo. *P<0.01

Fig. 4. Effect of BMP inhibitors on myogenic differentiation of primary human myoblasts

Immunofluorescent staining of human primary myoblasts differentiated for 7 days with our without Noggin (NOG; 200ng/ml), LDN-193189 (LDN; 120nM) and dorsomorphin (DMPN; 4 μM). Staining shows desmin (myogenic cells; in red) and MyHC (differentiated myotubes; in green). Experiment was performed in duplo and representative pictures are shown. Error bars indicate s.d. of 10 measured areas in duplo; *P<0.01

Fig. 5. Adenoviral-mediated overexpression of Noggin in dystrophic muscle

(A) The left panel shows qRT-PCR analysis of control (n=5) and ad-Noggin (n=5) gastrocnemius cDNA with primers specific for human Noggin. The right panel shows a representative gel with RT-PCR product amplified with human Noggin specific primers. The amplified products of all ad-Noggin samples were isolated, sequenced and identified as human Noggin. (B) qRT-PCR analysis of myogenic, inflammation and fibrosis marker gene expression in gastrocnemius samples of control (n=5) and ad-Noggin (n=5). Error bars represents s.d. *P<0.01 (C) Quantitative analysis of fibrotic/necrotic areas as determined with H&E staining and the percentage of eMyHC+ fibers of control (n=5) and ad-Noggin (n=5) muscle sections. Error bars represents s.d. (D) Representative pictures of H&E stainings of control muscle (NaCl and LacZ) and contralateral ad-Noggin injected muscle. Scale bar = 100 μm