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

Abstract

BackgroundMutations 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.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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-016-0096-6) contains supplementary material, which is available to authorized users.

Defects in G-Actin Incorporation into Filaments in Myoblasts Derived from Dysferlinopathy Patients Are Restored by Dysferlin C2 Domains

“…As compared with C25 myoblasts, the expression of dysferlin was reduced by 68%, 87%, 88%, and 83% in DYSF2, DYSF3, AB320, and ER myoblasts, respectively. These results agree with the expected dysferlin expression in these myoblasts [31,32,36,37] and validate these cell lines as in vitro models of dysferlinopathy. We first analyzed the relative expression of dysferlin in the cell lines DYSF2, DYSF3, AB320, and ER and compared it with the expression of the protein in C25 control myoblasts.…”

“…Four different cell lines of immortalized myoblasts were derived from skeletal muscle biopsies from dysferlinopathy patients. These cell lines named DYSF2 (also called 107), DYSF3 (also called 379), AB320, and ER myoblasts were previously characterized [31,32]. Table 1 describes the origin of each cell line, including the mutations carried by donors.…”

“…We also performed experiments in RCMH myoblasts stably transfected with shRNA for dysferlin, a strategy to knockdown dysferlin expression previously reported by our group [32]. The RCMH cell line was established from a quadriceps biopsy of a healthy human [41], and it has been previously characterized [41,42].…”

Connexins and Pannexins in Bone and Skeletal Muscle

“…In agreement with this statement, depletion or reduction of CD4+ or CD8+ T cells [94] or neutrophils [95] in mdx mice reduces the severity of the dystropathology. In addition, mdx myofibers were recently shown to express de novo three connexins (39, 43 and 45) [96]. Accordingly, myofibers of DMD or BMD patients were found to express connexins 45.…”

“…In agreement with a local inflammatory response and the role of connexin hemichannels, it was recently demonstrated that connexin hemichannels also participate in LGMDs. In immortalized myotubes derived from patients harboring dysferlin mutations, it was found that connexin hemichannels are still expressed in mature myotubes and are responsible of an increase (~10%) of basal cytoplasmic Ca 2+ levels, suggesting that these hemichannels could mediate the posterior muscle atrophy and adult myofibers death [96]. …”