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

Luis A. Cea; Carlos Puebla; Bruno A. Cisterna; Rosalba Escamilla; Aníbal A. Vargas; Marina Frank; Paloma Martínez-Montero; Carmen Prior; Jesús Molano; Isabel Esteban-Rodríguez; Ignacio Pascual; Pía Gallano; Gustavo Lorenzo; Héctor Pian; Luis C. Barrio; Klaus Willecke; Juan C. Sáez

doi:10.1007/s00018-016-2132-2

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

Luis A. Cea, Carlos Puebla, Bruno A. Cisterna, Rosalba Escamilla, Aníbal A. Vargas, Marina Frank, Paloma Martínez-Montero, Carmen Prior, Jesús Molano, Isabel Esteban-Rodríguez, Ignacio Pascual, Pía Gallano, Gustavo Lorenzo, Héctor Pian, Luis C. Barrio, Klaus Willecke, Juan C. Sáez

Medicine School

Research output: Contribution to journal › Article › peer-review

37 Citations (Scopus)

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 Cx43^fl/flCx45^fl/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 Cx43^fl/flCx45^fl/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 Cx43^fl/flCx45^fl/fl:Myo-Cre mice was significantly better than that of control mdx Cx43^fl/flCx45^fl/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.

Original language	English
Pages (from-to)	2583-2599
Number of pages	17
Journal	Cellular and Molecular Life Sciences
Volume	73
Issue number	13
DOIs	https://doi.org/10.1007/s00018-016-2132-2
Publication status	Published - 1 Jul 2016

Keywords

Cell death
Connexons
Evans blue uptake
NF-κB
P2X receptors
Pannexin1

ASJC Scopus subject areas

Molecular Medicine
Molecular Biology
Pharmacology
Cellular and Molecular Neuroscience
Cell Biology

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10.1007/s00018-016-2132-2

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Cea, L. A., Puebla, C., Cisterna, B. A., Escamilla, R., Vargas, A. A., Frank, M., Martínez-Montero, P., Prior, C., Molano, J., Esteban-Rodríguez, I., Pascual, I., Gallano, P., Lorenzo, G., Pian, H., Barrio, L. C., Willecke, K., & Sáez, J. C. (2016). Fast skeletal myofibers of mdx mouse, model of Duchenne muscular dystrophy, express connexin hemichannels that lead to apoptosis. Cellular and Molecular Life Sciences, 73(13), 2583-2599. https://doi.org/10.1007/s00018-016-2132-2

@article{02a47438e6be4da6a87f9d72b017c3e1,

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

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.",

keywords = "Cell death, Connexons, Evans blue uptake, NF-κB, P2X receptors, Pannexin1",

author = "Cea, {Luis A.} and Carlos Puebla and Cisterna, {Bruno A.} and Rosalba Escamilla and Vargas, {An{\'i}bal A.} and Marina Frank and Paloma Mart{\'i}nez-Montero and Carmen Prior and Jes{\'u}s Molano and Isabel Esteban-Rodr{\'i}guez and Ignacio Pascual and P{\'i}a Gallano and Gustavo Lorenzo and H{\'e}ctor Pian and Barrio, {Luis C.} and Klaus Willecke and S{\'a}ez, {Juan C.}",

note = "Funding Information: We thank Ms. Teresa Vergara and Ms. Paola Fern{\'a}ndez 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 Inserci{\'o}n en la Academia 79140023 (to LAC); Fondo Nacional de Desarrollo Cient{\'i}fico y Tecnol{\'o}gico (FONDECYT): Grant 3130662 (to CP); 1150291 (to JCS), ICM-Econom{\'i}a P09-022-F Centro Interdisciplinario de Neurociencias de Valpara{\'i}so (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. Publisher Copyright: {\textcopyright} 2016, Springer International Publishing.",

year = "2016",

month = jul,

day = "1",

doi = "10.1007/s00018-016-2132-2",

language = "English",

volume = "73",

pages = "2583--2599",

journal = "Cellular and Molecular Life Sciences",

issn = "1420-682X",

publisher = "Birkhauser Verlag Basel",

number = "13",

}

Cea, LA, Puebla, C, Cisterna, BA, Escamilla, R, Vargas, AA, Frank, M, Martínez-Montero, P, Prior, C, Molano, J, Esteban-Rodríguez, I, Pascual, I, Gallano, P, Lorenzo, G, Pian, H, Barrio, LC, Willecke, K & Sáez, JC 2016, 'Fast skeletal myofibers of mdx mouse, model of Duchenne muscular dystrophy, express connexin hemichannels that lead to apoptosis', Cellular and Molecular Life Sciences, vol. 73, no. 13, pp. 2583-2599. https://doi.org/10.1007/s00018-016-2132-2

TY - JOUR

T1 - Fast skeletal myofibers of mdx mouse, model of Duchenne muscular dystrophy, express connexin hemichannels that lead to apoptosis

AU - Cea, Luis A.

AU - Puebla, Carlos

AU - Cisterna, Bruno A.

AU - Escamilla, Rosalba

AU - Vargas, Aníbal A.

AU - Frank, Marina

AU - Martínez-Montero, Paloma

AU - Prior, Carmen

AU - Molano, Jesús

AU - Esteban-Rodríguez, Isabel

AU - Pascual, Ignacio

AU - Gallano, Pía

AU - Lorenzo, Gustavo

AU - Pian, Héctor

AU - Barrio, Luis C.

AU - Willecke, Klaus

AU - Sáez, Juan C.

N1 - Funding Information: 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. Publisher Copyright: © 2016, Springer International Publishing.

PY - 2016/7/1

Y1 - 2016/7/1

N2 - 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.

AB - 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.

KW - Cell death

KW - Connexons

KW - Evans blue uptake

KW - NF-κB

KW - P2X receptors

KW - Pannexin1

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U2 - 10.1007/s00018-016-2132-2

DO - 10.1007/s00018-016-2132-2

M3 - Article

C2 - 26803842

AN - SCOPUS:84955323343

SN - 1420-682X

VL - 73

SP - 2583

EP - 2599

JO - Cellular and Molecular Life Sciences

JF - Cellular and Molecular Life Sciences

IS - 13

ER -

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

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