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Inhibition of the angiotensin-converting enzyme decreases skeletal muscle fibrosis in dystrophic mice by a diminution in the expression and activity of connective tissue growth factor (CTGF/CCN-2)

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Abstract

The renin-angiotensin system (RAS), through angiotensin II and the angiotensin-converting enzyme (ACE), is involved in the genesis and progression of fibrotic diseases characterized by the replacement of normal tissue by an accumulation of an extracellular matrix (ECM). Duchenne muscular dystrophy (DMD) presents fibrosis and a decrease in muscle strength produced by chronic damage. The mdx mouse is a murine model of DMD and develops the same characteristics as dystrophic patients when subjected to chronic exercise. The connective tissue growth factor (CTGF/CCN2) and transforming growth factor type beta (TGF-β), which are overexpressed in muscular dystrophies, play a major role in many progressive scarring conditions. We have tested the hypothesis that ACE inhibition decreases fibrosis in dystrophic skeletal muscle by treatment of mdx mice with the ACE inhibitor enalapril. Both sedentary and exercised mdx mice treated with enalapril showed improvement in gastrocnemius muscle strength explained by a reduction in both muscle damage and ECM accumulation. ACE inhibition decreased CTGF expression in sedentary or exercised mdx mice and diminished CTGF-induced pro-fibrotic activity in a model of CTGF overexpression by adenoviral infection. Enalapril did not have an effect on TGF-β1 expression or its signaling activity in sedentary or exercised dystrophic mice. Thus, ACE inhibition might improve muscle strength and decrease fibrosis by diminishing specifically CTGF expression and activity without affecting TGF-β1 signaling. Our data provide insights into the pathogenic events in dystrophic muscle. We propose ACE as a target for developing therapies for DMD and related diseases.

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Abbreviations

Ang-II:

Angiotensin II

ACE:

Angiotensin-converting enzyme

ARB:

Angiotensin II receptor type 1 blocker

AT-1 receptor:

Angiotensin II receptor type I

CTGF:

Connective tissue growth factor

DMD:

Duchenne muscular dystrophy

ECM:

Extracellular matrix

mdx :

DMD mouse model

RAS:

Renin angiotensin system

TGF-β:

Transforming growth factor beta

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Authors and Affiliations

  1. Laboratorio de Biología y Fisiopatología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas & Facultad de Medicina, Universidad Andres Bello, Santiago, Chile

    María Gabriela Morales, Yaneisi Vazquez & Claudio Cabello-Verrugio

  2. Laboratorio de Diferenciación Celular y Patología, Centro de Regulación Celular y Patología (CRCP), Centro de Regeneración y Envejecimiento (CARE), Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile

    María Gabriela Morales, Daniel Cabrera & Enrique Brandan

  3. Departamento de Fisiología, Centro de Regeneración y Envejecimiento (CARE), Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile

    Carlos Céspedes & Carlos P. Vio

  4. Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas & Facultad de Medicina, Universidad Andres Bello, Avenida Republica 239, 8370146, Santiago, Chile

    Claudio Cabello-Verrugio

Authors
  1. María Gabriela Morales
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  2. Daniel Cabrera
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  3. Carlos Céspedes
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  4. Carlos P. Vio
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  5. Yaneisi Vazquez
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  6. Enrique Brandan
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  7. Claudio Cabello-Verrugio
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Corresponding author

Correspondence to Claudio Cabello-Verrugio.

Additional information

This study was supported by research grants from Association-Francaise Contre Les Myopathies AFM 16670 (C.C.-V.); FONDECYT 1120380 (C.C.-V.), 11080212 (C.C.-V.), 1110426 (E.B.), 3130593 (M.G.M.); UNAB-DI-281-13/R (C.C.-V.); CARE PFB12/2007 (E.B., C.P.V.); CONICYT AT-24100047 (M.G.M.), AT-24090192 (D.C.) and Fundación Chilena para Biología Celular Proyecto MF-100 (E.B.).

The authors declare no conflicts of interest.

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Fig. S1

Improvement of skeletal muscle strength by enalapril in sedentary and exercised mdx mice. Tetanic specific force of normal, sedentary (a), or exercised (b) mdx mice treated with a vehicle (Vehicle), enalapril (Enal), or losartan (Los). Values are represented as percentages of specific isometric force (mN/mm2) shown by normal muscles (*,# P<0.05). (DOC 404 kb)

Fig. S2

Enalapril treatment does not change the number of central nuclei but decreases fibrosis in dystrophic skeletal muscle (Enal enalapril, Los losartan). a Central nuclei of the fibers were measured as indicated in Material and Methods. Values correspond to the means ± SD of three independent experiments with four mice for each experimental condition. b Fibrotic area (% of total) measured by using immunofluorescence for fibronectin (shown in Figure 4) as described in Materials and Methods. Values correspond to the means ± SD of three independent experiments with four mice for each experimental condition (*,**,#,## P<0.05). (DOC 468 kb)

Fig. S3

ACE inhibition decreases the angiotensin II signaling pathway in skeletal muscle that overexpress CTGF. Tibialis anterior (TA) of normal mice systemically treated with a vehicle (Veh), enalapril (Enal), or losartan (Los) were infected with a control adenovirus (Adv-Ctrl) or with an adenovirus to overexpress CTGF (Adv-CTGF). Phospho-p38 and total p38 protein levels were detected by Western blot analysis in extracts obtained from TA muscles. Molecular weights are shown in kDa. (DOC 389 kb)

Fig. S4

Extracellular signal-related kinase (ERK) phosphorylation is decreased by enalapril in sedentary and exercised mdx mice. a Protein levels of phospho-ERK and total ERK-1/2 were detected by Western blot analysis in extracts obtained from the gastrocnemius muscles of normal and of sedentary and exercised mdx mice treated with a vehicle or enalapril for 6 months. Molecular weight markers are shown in kDa. b Densitometric analysis of Western blots shown in a. Values correspond to means ± SD of three independent experiments with five mice for each experimental condition (*P<0.05). (DOC 552 kb)

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Morales, M.G., Cabrera, D., Céspedes, C. et al. Inhibition of the angiotensin-converting enzyme decreases skeletal muscle fibrosis in dystrophic mice by a diminution in the expression and activity of connective tissue growth factor (CTGF/CCN-2). Cell Tissue Res 353, 173–187 (2013). https://doi.org/10.1007/s00441-013-1642-6

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