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Fast skeletal myofibers of mdx mouse, model of Duchenne muscular dystrophy, express connexin hemichannels that lead to apoptosis

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Abstract

Skeletal muscles of patients with Duchenne muscular dystrophy (DMD) show numerous alterations including inflammation, apoptosis, and necrosis of myofibers. However, the molecular mechanism that explains these changes remains largely unknown. Here, the involvement of hemichannels formed by connexins (Cx HCs) was evaluated in skeletal muscle of mdx mouse model of DMD. Fast myofibers of mdx mice were found to express three connexins (39, 43 and 45) and high sarcolemma permeability, which was absent in myofibers of mdx Cx43fl/flCx45fl/fl:Myo-Cre mice (deficient in skeletal muscle Cx43/Cx45 expression). These myofibers did not show elevated basal intracellular free Ca2+ levels, immunoreactivity to phosphorylated p65 (active NF-κB), eNOS and annexin V/active Caspase 3 (marker of apoptosis) but presented dystrophin immunoreactivity. Moreover, muscles of mdx Cx43fl/flCx45fl/fl:Myo-Cre mice exhibited partial decrease of necrotic features (big cells and high creatine kinase levels). Accordingly, these muscles showed similar macrophage infiltration as control mdx muscles. Nonetheless, the hanging test performance of mdx Cx43fl/flCx45fl/fl:Myo-Cre mice was significantly better than that of control mdx Cx43fl/flCx45fl/fl mice. All three Cxs found in skeletal muscles of mdx mice were also detected in fast myofibers of biopsy specimens from patients with muscular dystrophy. Thus, reduction of Cx expression and/or function of Cx HCs may be potential therapeutic approaches to abrogate myofiber apoptosis in DMD.

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Acknowledgments

We thank Ms. Teresa Vergara and Ms. Paola Fernández for their technical support. We also thank members of the Spanish families, whose participation made this study possible. This work was partially supported by CONICYT/PAI Proyecto de Inserción en la Academia 79140023 (to LAC); Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT): Grant 3130662 (to CP); 1150291 (to JCS), ICM-Economía P09-022-F Centro Interdisciplinario de Neurociencias de Valparaíso (to JCS), grants from the Spanish Ministry of Economy and Competitiveness (Consolider CSD2008-00005 and BFU2013-33821) and the Community of Madrid (Neurotec-P2010/BMD-2460) (to LCB). The research stay of LAC and CP in the Bonn laboratory was supported by a grant of CONICYT and the German Academic Exchange Service (to JCS and KW). Additional work in the Bonn laboratory was funded by the German Research Foundation (Wi 270/33.1 and SFB 645, B2) to KW.

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  1. Luis A. Cea

    Present address: Present: Program of Anatomy and Developmental Biology, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago, Chile

Authors and Affiliations

  1. Departamento de Fisiología, Pontificia Universidad Católica de Chile, Av. Libertador Bernardo O’Higgins 340, Santiago, Chile

    Luis A. Cea, Carlos Puebla, Bruno A. Cisterna, Rosalba Escamilla, Aníbal A. Vargas & Juan C. Sáez

  2. Centro Interdisciplinario de Neurociencias de Valparaíso, Valparaíso, Chile

    Carlos Puebla, Bruno A. Cisterna, Rosalba Escamilla & Juan C. Sáez

  3. Division of Molecular Genetics, Life and Medical Sciences Institute, University of Bonn, 53115, Bonn, Germany

    Marina Frank & Klaus Willecke

  4. Unidad de Genética Molecular-INGEMM, Hospital Universitario La Paz-IdIPAZ, Madrid, Spain

    Paloma Martínez-Montero, Carmen Prior & Jesús Molano

  5. Servicio de Anatomía Patológica, Hospital Universitario La Paz-IdIPAZ, Madrid, Spain

    Isabel Esteban-Rodríguez

  6. Servicio de Neuropediatría, Hospital Universitario La Paz-IdIPAZ, Madrid, Spain

    Ignacio Pascual

  7. Servicio de Genética, Hospital Santa Creu i Sant Pablo-CIBERER, Barcelona, Spain

    Pía Gallano

  8. Servicio de Pediatria, “Ramón y Cajal” Hospital-IRYCIS, Madrid, Spain

    Gustavo Lorenzo

  9. Servicio de Anatomía Patológica, “Ramón y Cajal” Hospital-IRYCIS, Madrid, Spain

    Héctor Pian

  10. Unidad de Neurología Experimental, “Ramón y Cajal” Hospital-IRYCIS, Madrid, Spain

    Luis C. Barrio

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The authors certify that the experiments comply with the current laws of Chile, where the experiments were performed. All protocols were approved by the Bioethics Committee of the Pontificia Universidad Católica de Chile (Protocol No. 176) in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All efforts were made to minimize animal suffering, reduce the number of animals used, and alternatives to in vivo techniques, if available.

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Cea, L.A., Puebla, C., Cisterna, B.A. et al. Fast skeletal myofibers of mdx mouse, model of Duchenne muscular dystrophy, express connexin hemichannels that lead to apoptosis. Cell. Mol. Life Sci. 73, 2583–2599 (2016). https://doi.org/10.1007/s00018-016-2132-2

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