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. 2018 Jul 10;9(7):776.
doi: 10.1038/s41419-018-0792-6.

Nintedanib Decreases Muscle Fibrosis and Improves Muscle Function in a Murine Model of Dystrophinopathy

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Free PMC article

Nintedanib Decreases Muscle Fibrosis and Improves Muscle Function in a Murine Model of Dystrophinopathy

Patricia Piñol-Jurado et al. Cell Death Dis. .
Free PMC article

Abstract

Duchenne muscle dystrophy (DMD) is a genetic disorder characterized by progressive skeletal muscle weakness. Dystrophin deficiency induces instability of the sarcolemma during muscle contraction that leads to muscle necrosis and replacement of muscle by fibro-adipose tissue. Several therapies have been developed to counteract the fibrotic process. We report the effects of nintedanib, a tyrosine kinase inhibitor, in the mdx murine model of DMD. Nintedanib reduced proliferation and migration of human fibroblasts in vitro and decreased the expression of fibrotic genes such as COL1A1, COL3A1, FN1, TGFB1, and PDGFA. We treated seven mdx mice with 60 mg/kg/day nintedanib for 1 month. Electrophysiological studies showed an increase in the amplitude of the motor action potentials and an improvement of the morphology of motor unit potentials in the animals treated. Histological studies demonstrated a significant reduction of the fibrotic areas present in the skeletal muscles. Analysis of mRNA expression from muscles of treated mice showed a reduction in Col1a1, Col3a1, Tgfb1, and Pdgfa. Western blot showed a reduction in the expression of collagen I in skeletal muscles. In conclusion, nintedanib reduced the fibrotic process in a murine model of dystrophinopathy after 1 month of treatment, suggesting its potential use as a therapeutic drug in DMD patients.

Conflict of interest statement

Patricia Piñol-Jurado, Xavier Suárez-Calvet, Esther Fernández, Eduard Gallardo, Natalia de la Oliva, Anna Martínez-Muriana, Pedro Gómez-Gálvez, Luis M. Escudero, María Pérez-Peiró, Noemi de Luna, Xavier Navarro, and Isabel Illa have not any relevant conflict of interest. Lutz Wollin is an employee of Boehringer-Ingelheim the inventor of nintedanib used in this study. Jordi Díaz-Manera has received a research grant from Boehringer-Ingelheim.

Figures

Fig. 1
Fig. 1. Effect of nintedanib on fibroblasts from healthy human muscle in vitro.
Fibroblasts proliferation was analyzed after 2, 4, and 6 days of culture (a–d). We cultured fibroblast with increasing concentrations of nintedanib in a culture containing 10% fetal bovine serum (FBS) and we observed decreased fibroblast proliferation after 4 and 6 days in a dose-dependent manner (a). Fibroblasts were cultured in the presence of platelet-derived growth factor AA (PDGF-AA, 10 ng/mL) (b), basic fibroblast growth factor (FGFb, 10 ng/mL),(c) or vascular endothelial growth factor A (VEGF-A, 50 ng/mL) (d) with 1% FBS with or without nintedanib 0.4 μM. Only PDGF-AA increased cell proliferation, an effect that was reversed with the addition of nintedanib to the culture. Fibroblast migration was analyzed using a scratch assay. Nintedanid treatment at 0.4 μM reverted the promigratory effect of PDGF-AA, FGFb, VEGF, and CTGF (e). Representative images of this assay are shown in (f). Nintedanib treatment at 0.4 μM (blue bars) reverted the effect of PDGF-AA in the expression of ADAM metallopeptidase domain 17 (ADAM-17), tissue inhibitor of metalloproteinase 1 (TIMP-1) and tissue inhibitor of metalloproteinase 2 (TIMP-2) (g). Nintedanib treatment at 0.4 μM (blue bars) produced a statistically significant reduction of collagen type I alpha 1 chain (COL1A1), collagen type III alpha 1 chain (COL3A1), fibronectin 1 (FN1), platelet-derived growth factor A (PDGFA), connective tissue growth factor (CTGF), transforming growth factor beta 1 (TGFβ1) expression compared with control samples (black bars) analyzed by qPCR (h). Data are expressed as means ± SD. N = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.005
Fig. 2
Fig. 2. Motor nerve conduction tests and electromyographic analysis of WT, mdx, and nintedanib-treated mdx mice after 4 weeks of treatment.
a–c Motor nerve conduction test showed that nintedanib administration preserved the compound muscle action potential (CMAP) i`n the tibialis anterior (a), gastrocnemius (b), and plantar (c) muscles. d, e Electromyography (EMG) of tibialis anterior. AUC quantification of electromyography (EMG) responses of the TA muscle after application of Von Frey filaments 5.47 to the ipsilateral hind paw (d). Percentage of MUAPs from each type that were activated (e) and EMG showed a lower proportion of small pathologic motor unit action potentials (MUAPs) in nintedanib-treated mdx mice. f–h Representative recordings of EMG bursts after nociceptive stimuli in WT (f), mdx (g), and nintedanib-treated mdx mice (h). Note that mdx animals showed lower amplitude and shorter duration MUAPs compared with WT and nintedanib-treated mdx mice. Data are expressed as means ± SD. Genetic background mouse strain C57BL (WT); n = 5, mdx mice (mdx), n = 5; nintedanib-treated mdx mice (mdx + Ninte.), n = 7. Data are expressed as means ± SD. *P < 0.05, **P < 0.01, **** p < 0.0001
Fig. 3
Fig. 3. Histological analysis of muscle samples of WT, mdx and nintedanib-treated mdx mice.
Hematoxylin and eosin (H&E) staining of muscle samples obtained (a–i). Representative examples of cross-sectional muscle fibers within quadriceps (a–c), diaphragm (d–f), and tibialis anterior (g–i) of WT, mdx and nintedanib-treated mdx mice. Schematic representation of the fiber mean area in quadriceps (j), diaphragm (k), and tibialis anterior (l) showed no differences on the size of the muscle fibers. Analysis of the proportion of central nucleated fibers in quadriceps (m), diaphragm (n), and tibialis anterior (o) did not present significant differences after 1 month of treatment with nintedanib (60 mg/kg). Data are expressed as means ± SD. Genetic background mouse strain C57BL (WT); n = 5, mdx mice (mdx), n = 5; nintedanib-treated mdx mice (mdx + Ninte), n = 7. Data are expressed as means ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001. Scale bar = 100 µm
Fig. 4
Fig. 4. Nintedanib reduced the number of regenerative muscle fibers in mdx mice.
Immunostaining of eMHC and quantification of positive fibers in muscle samples of WT, mdxand nintedanib-treated mdx mice (a-l). The number of eMHC-positive fibers (green) is reduced in quadriceps, diaphragm and tibialis anterior in mdx mice treated with nintedanib. Background in red is shown to localize the positivity within the muscle. Data are expressed as means ± SD. *P < 0.05 and **P < 0.01. Scale bar = 100 µm
Fig. 5
Fig. 5. Nintedanib reduced skeletal muscle fibrosis in mdx mice.
Representative pictures of immunofluorescence staining for collagen VI in quadriceps (a–c), diaphragm (d–f), and tibialis anterior (g–i) of WT, mdx, and nintedanib-treated mdx mice. Endomysial collagen deposition increased in all mdx muscles compared with WT group and decreased in nintedanib-treated mdx muscles, significantly in quadriceps (j) and diaphragm (k) but not in tibialis anterior (l) compared with non-treated mdx mice. Data are expressed as means ± SD. Genetic background mouse strain C57BL (WT); n = 5, mdx mice (mdx), n = 5; nintedanib-treated mdx mice (mdx + Ninte), n = 7. *P < 0.05. Scale bar of quadriceps and tibialis anterior = 200 µm. Scale bar of diaphragm = 100 µm.
Fig. 6
Fig. 6. RT-qPCR studying mRNA expression of fibrosis-related genes.
Collagen type I alpha 1 chain (Col1a1), Collagen type III alpha 1 chain (Col3a1), Fibronectin 1 (Fn1), Platelet-derived growth factor A (Pdgfa), Platelet-derived growth factor B (Pdgfb), Connective tissue growth factor (Ctgf), Transforming growth factor beta 1 (Tgfβ1), and Adhesion G protein-coupled receptor E1 (Adgre1) showed changes in relative abundance following nintedanib in quadriceps (a–h), diaphragm (i–p), and tibialis anterior (q–y). Col1a1, Col3a1, Pdgfa, Tgfb1, and Adgre1 gene expression was increased in mdx mice compared with WT. Col1a1, Col3a1, Pdgfa, Pdgfb, Tgfb1, and Adgre1 expression was reduced in all muscles analyzed from nintedanib-treated mdx mice compared with mdx mice. In contrast, Ctgf expression was increased in muscles from nintedanib-treated mdx mice compared with mdx mice. Data are expressed as means ± SD. Genetic background mouse strain C57BL (WT); n = 5, mdx mice (mdx), n = 5; nintedanib-treated mdx mice (mdx + Ninte), n = 7. *P < 0.05, **P < 0.01, ***P < 0.005
Fig. 7
Fig. 7. Nintedanib decreased collagen I expression in mdx muscles.
Western blotting of Collagen I in quadriceps (a), diaphragm (b), and tibialis anterior (c) showed a reduction in the protein expression in diaphragm (e) and tibialis anterior (f) but not in quadriceps (d) of nintedanib-treated mdx mice compared with mdx mice by quantification of Collagen I normalized to α-tubulin levels. Data are expressed as means ± SD. Genetic background mouse strain C57BL (WT); n = 4, mdx mice (mdx), n = 4; nintedanib-treated mdx mice (mdx + Ninte), n = 4. *P ≤ 0.05 and **P ≤ 0.01

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