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The absence of dysferlin induces the expression of functional connexin-based hemichannels in human myotubes

Authors :
Christian Arriagada
Luis A. Cea
Vincent Mouly
Pablo Caviedes
Anne Bigot
Juan C. Sáez
Jorge A. Bevilacqua
Ana M. Cárdenas
Program of Anatomy and Developmental Biology
Universidad de Chile = University of Chile [Santiago] (UCHILE)-Institute of Biomedical Sciences
Departamento de Neurología y Neurocirugía [Santiago]
HCUCH-Instituto de Ciencias Biomédicas, Facultad de Medicina
Centro Interdisciplinario de Neurociencias de Valparaíso
Universidad de Valparaiso [Chile]
Centre de recherche en myologie
Université Pierre et Marie Curie - Paris 6 (UPMC)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Departamento de Fisiología
Pontificia Universidad Católica de Chile (UC)-Facultad de Ciencias Biologicas
Programa de Farmacología Molecular y Clínica
Instituto de Ciencias Biomedicas
HAL-UPMC, Gestionnaire
Source :
BMC Cell Biology, BMC Cell Biology, BioMed Central, 2016, 17 (S1), pp.15. ⟨10.1186/s12860-016-0096-6⟩, Bmc Cell Biology, Artículos CONICYT, CONICYT Chile, instacron:CONICYT, BMC Cell Biology, 2016, 17 (S1), pp.15. ⟨10.1186/s12860-016-0096-6⟩
Publication Year :
2016
Publisher :
BioMed Central, 2016.

Abstract

International audience; Background: Mutations in the gene encoding for dysferlin cause recessive autosomal muscular dystrophies called dysferlinopathies. These mutations induce several alterations in skeletal muscles, including, inflammation, increased membrane permeability and cell death. Despite the fact that the etiology of dysferlinopathies is known, the mechanism that explains the aforementioned alterations is still elusive. Therefore, we have now evaluated the potential involvement of connexin based hemichannels in the pathophysiology of dysferlinopathies. Results: Human deltoid muscle biopsies of 5 Chilean dysferlinopathy patients exhibited the presence of muscular connexins (Cx40.1, Cx43 and Cx45). The presence of these connexins was also observed in human myotubes derived from immortalized myoblasts derived from other patients with mutated forms of dysferlin. In addition to the aforementioned connexins, these myotubes expressed functional connexin based hemichannels, evaluated by ethidium uptake assays, as opposed to myotubes obtained from a normal human muscle cell line, RCMH. This response was reproduced in a knock-down model of dysferlin, by treating RCMH cell line with small hairpin RNA specific for dysferlin (RCMH-sh Dysferlin). Also, the presence of P2X 7 receptor and the transient receptor potential channel, TRPV2, another Ca 2+ permeable channels, was detected in the myotubes expressing mutated dysferlin, and an elevated resting intracellular Ca 2+ level was found in the latter myotubes, which was in turn reduced to control levels in the presence of the molecule D4, a selective Cx HCs inhibitor. Conclusions: The data suggests that dysferlin deficiency, caused by mutation or downregulation of dysferlin, promotes the expression of Cx HCs. Then, the de novo expression Cx HC causes a dysregulation of intracellular free Ca 2+ levels, which could underlie muscular damage associated to dysferlin mutations. This mechanism could constitute a potential therapeutical target in dysferlinopathies.ResultsHuman deltoid muscle biopsies of 5 Chilean dysferlinopathy patients exhibited the presence of muscular connexins (Cx40.1, Cx43 and Cx45). The presence of these connexins was also observed in human myotubes derived from immortalized myoblasts derived from other patients with mutated forms of dysferlin. In addition to the aforementioned connexins, these myotubes expressed functional connexin based hemichannels, evaluated by ethidium uptake assays, as opposed to myotubes obtained from a normal human muscle cell line, RCMH. This response was reproduced in a knock-down model of dysferlin, by treating RCMH cell line with small hairpin RNA specific for dysferlin (RCMH-sh Dysferlin). Also, the presence of P2X7 receptor and the transient receptor potential channel, TRPV2, another Ca2+ permeable channels, was detected in the myotubes expressing mutated dysferlin, and an elevated resting intracellular Ca2+ level was found in the latter myotubes, which was in turn reduced to control levels in the presence of the molecule D4, a selective Cx HCs inhibitor.ConclusionsThe data suggests that dysferlin deficiency, caused by mutation or downregulation of dysferlin, promotes the expression of Cx HCs. Then, the de novo expression Cx HC causes a dysregulation of intracellular free Ca2+ levels, which could underlie muscular damage associated to dysferlin mutations. This mechanism could constitute a potential therapeutical target in dysferlinopathies.

Details

Language :
English
ISSN :
14712121
Volume :
17
Issue :
Suppl 1
Database :
OpenAIRE
Journal :
BMC Cell Biology
Accession number :
edsair.doi.dedup.....a0f62ab177ab436c6d7fbe955fb5f2f4
Full Text :
https://doi.org/10.1186/s12860-016-0096-6⟩