Angiotensin II receptor type 1 blockade decreases CTGF/CCN2-mediated damage and fibrosis in normal and dystrophic skeletal muscles

Abstract

Connective tissue growth factor (CTGF/CCN-2) is mainly involved in the induction of extracellular matrix (ECM) proteins. The levels of CTGF correlate with the degree and severity of fibrosis in many tissues, including dystrophic skeletal muscle. The CTGF overexpression in tibialis anterior skeletal muscle using an adenoviral vector reproduced many of the features observed in dystrophic muscles including muscle damage and regeneration, fibrotic response and decrease in the skeletal muscle strength. The renin-angiotensin system is involved in the genesis and progression of fibrotic diseases through its main fibrotic components angiotensin-II and its transducer receptor AT-1. The use of AT-1 receptor blockers (ARB) has been shown to decrease fibrosis. In this paper, we show the effect of AT-1 receptor blockade on CTGF-dependent biological activity in skeletal muscle cells as well as the response to CTGF overexpression in normal skeletal muscle. Our results show that in myoblasts ARB decreased CTGF-mediated increase of ECM protein levels, extracellular signal regulated kinases 1/2 (ERK-1/2) phosphorylation and stress fibres formation. In tibialis anterior muscle overexpressing CTGF using an adenovirus, ARB treatment decreased CTGF-mediated increase of ECM molecules, α-SMA and ERK-1/2 phosphorylation levels. Quite remarkable, ARB was able to prevent the loss of contractile force of tibialis anterior muscles overexpressing CTGF. Finally, we show that ARB decreased the levels of fibrotic proteins, CTGF and ERK-1/2 phosphorylation augmented in a dystrophic skeletal muscle from mdx mice. We propose that ARB is a novel pharmacological tool that can be used to decrease the fibrosis induced by CTGF in skeletal muscle associated with muscular dystrophies.

Fig 1

Blocking of AT-1 receptor decreases CTGF-dependent pro-fibrotic activity in skeletal muscle cells. (A) C2C12 myoblasts were pre-incubated for 1 hr with increasing concentrations of the ARB ZD-7155. Then, cells were incubated with 80 ng/ml of recombinant CTGF for 48 hrs. Fibronectin (FN) protein levels were analysed by Western blot in cell extracts. Tubulin levels are shown as loading control. Molecular weights are shown in kD. (B) Relative levels of FN normalized to tubulin. The graphic shown corresponds to densitometric analysis of Western blots shown in (A). Values correspond to the mean ± standard deviation of three independent experiments (*^,#,&P < 0.05). (C) C2C12 cells treated as described in (A). Western blot for phospho-ERK-1/2 and total ERK-1/2 levels are shown. FS: foetal serum bovine. Molecular weights are shown in kD. (D) The graphic shown corresponds to densitometric analysis of Western blots shown in (C). Values correspond to the mean ± standard deviation of three independent experiments (*^,#,&P < 0.05). (E) C2C12 cells were pre-incubated with losartan (10 μM) and incubated with 80 ng/ml of recombinant CTGF. Levels of fibronectin (FN) and phospho-ERK-1/2 were determined as in (A) and (C), respectively. Densitometric analysis of Western blots was performed [Fibronectin, fold of induction relative to control: 2.3 ± 0.5 (CTGF), 1.2 ± 0.3 (CTGF + Losartan), P < 0.05; Phospho-ERK-1/2, fold of induction relative to control: 1.9 ± 0.5 (CTGF), 1.1 ± 0.2 (CTGF + Losartan), P < 0.05]. (F) C2C12 myoblasts were treated as in (A) and processed for direct fluorescence using phalloidin coupled to fluorescein. Nuclei were labelled with Hoechst 33258. Bar corresponds to 50 μm.

Fig 2

Angiotensin II receptor type 1 blockade decreases CTGF-induced damage and collagen content in tibialis anterior muscle. Tibialis anterior (TA) of C57BL/10 mice systemically treated with vehicle or the ARB losartan were infected with control adenovirus (Ad-GFP) or with an adenovirus to overexpress CTGF (Ad-mCTGF). Three independent experiments were performed using two mice for each experimental condition. (A) Cryosections of TA muscles infected for 5 days were stained with haematoxylin and eosin. (B) Total content of collagen evaluated by Sirius red (SR) staining in cryosections of TA muscles infected for five days. Bar corresponds to 200 μm. (C) Quantification of total content of collagen stained by Sirius red (SR) using ImageJ software. Values correspond to the mean ± standard deviation (*^,#P < 0.05).

Fig 3

CTGF-induced ECM accumulation is decreased by blockade Angiotensin II receptor type 1 in skeletal muscle. Tibialis anterior (TA) of C57BL/10 mice systemically treated with vehicle or the ARB losartan were infected with control adenovirus (Ad-GFP) or with an adenovirus to overexpress CTGF (Ad-mCTGF). Levels of fibronectin (FN), collagen I (Col I) and collagen III (Col III) were detected by indirect immunofluorescence analysis in cryosections of TA muscles infected for five days. Bar corresponds to 200 μm. The images are representatives from three independent experiments performed using two mice for each experimental condition.

Fig 4

Tibialis anterior muscle fibrosis induced by CTGF is prevented by blocking Angiotensin II Receptor Type 1. (A) Protein levels of fibronectin (FN), collagen III (Col III), α-SMA and CTGF were detected by Western blot analysis in extracts obtained from TA muscles of C57BL/10 mice without treatment (Control) or systemically treated with vehicle or losartan five days post-infection with control (Ad-GFP) or CTGF-overexpressing (Ad-mCTGF) adenoviruses. Tubulin levels are shown as loading control. Molecular weights are shown in kD. (B) Quantification of data corresponding to densitometric analysis from Western blot. Values correspond to the mean ± standard deviation of three independent experiments performed using two mice for each experimental condition (*^,#,&P < 0.05).

Fig 5

CTGF-dependent skeletal muscle fibrosis is decreased by blockade AT-1 receptor through a mechanism that involves diminished ERK-1/2 phosphorylation. (A) Protein levels of phospho-ERK-1/2 and total ERK-1/2 were detected by Western blot analysis in extracts obtained from TA muscles of C57BL/10 mice systemically treated with vehicle or losartan 5 days post-infection with control (Ad-GFP) or CTGF-overexpressing (Ad-mCTGF) adenoviruses. GAPDH levels are shown as loading control. Molecular weights are shown in kD. (B) The graphic shown corresponds to densitometric analysis of Western blots shown in (A). Values correspond to the mean ± standard deviation of three independent experiments performed using two mice for each experimental condition (*P < 0.05).

Fig 6

Induction of TGF-β activity is consequence of the adenoviral infection, but not related to CTGF overexpression. (A) Detection of phospho-Smad-3 through IFI analysis in cryosections of tibialis anterior (TA) of C57BL/10 mice systemically treated with vehicle or ARB losartan for 5 days post-infection with control (Ad-GFP) or overexpressing CTGF (Ad-mCTGF). The pictures correspond to 40× (bar corresponds to 200 μm). The insert shown nuclei labelled with Hoechst 33258 (Bar correspond to 50 μm). (B) Phospho-Smad-3 positive nuclei quantification of cryosections of TA of C57BL/10 mice treated as in (A). Values correspond to the mean ± standard deviation from three independent experiments using two mice for each experimental condition (*^,#P < 0.05).

Fig 7

CTGF-mediated decrease in isometric force of skeletal muscle is prevented by blocking AT-1 receptor. Tibialis anterior (TA) from mdx mice injected with PBS or infected with Ad-GFP or Ad-mCTGF for five days and systemically treated with vehicle or losartan were compared for (A) isometric specific force (mN/mm²)-stimulation frequency (Hz) relationship and (B) tetanic-specific force measured at 80 Hz. In (B), the values are represented as percentage of specific isometric force generated by control muscle. In (A) and (B), the values corresponds to the mean ± standard deviation from three independent experiments using two mice for each experimental condition (*^,#P < 0.05).

Fig 8

Blockade of AT-1 receptor diminishes amount of fibrotic proteins, CTGF and ERK-1/2 phosphorylation in a dystrophic skeletal muscle. (A) Detection of fibronectin (FN), α-SMA, CTGF and ERK-1/2 phosphorylated levels were determinate by Western blot in TA muscles from normal (BL-10) and dystrophic mice (mdx) treated for 6 months with vehicle (water) or losartan as indicated in Material and methods. GAPDH levels are shown as loading control. Molecular weights are shown in kD. (B) Cryosections of TA muscles from normal (BL-10) and dystrophic mice (mdx) treated for six months with vehicle (water) or losartan as indicated in Material and methods were analysed for haematoxylin and eosin, Sirius red staining and fibronectin (FN) detected by indirect immunofluorescence analysis. Bar corresponds to 200 μm.