The absence of dysferlin induces the expression of functional connexin-based hemichannels in human myotubes.
The absence of dysferlin induces the expression of functional connexin-based hemichannels in human myotubes.
复制标题
DOI：
10.1186/s12860-016-0096-6
复制
发表时间：
2016-05-24
期刊：
影响因子：
--
通讯作者：
Caviedes P
中科院分区：
文献类型：
--
作者：
Cea LA;Bevilacqua JA;Arriagada C;Cárdenas AM;Bigot A;Mouly V;Sáez JC;Caviedes P
编码 Dysferlin 的基因突变会导致隐性常染色体性肌营养不良症，称为 Dysferlin 病。这些突变会引起骨骼肌的多种变化，包括炎症、膜通透性增加和细胞死亡。尽管事实上铁蛋白病的病因是已知的，但解释上述改变的机制仍然难以捉摸。因此，我们现在评估了基于连接蛋白的半通道在肌肉异常疾病的病理生理学中的潜在参与。 5 名智利铁蛋白病患者的人体三角肌活检显示存在肌肉连接蛋白（Cx40.1、Cx43 和 Cx45）。在来自永生化成肌细胞的人肌管中也观察到了这些连接蛋白的存在，这些肌管源自其他具有突变型 Dysferlin 的患者。除了上述连接蛋白之外，这些肌管还表达基于功能性连接蛋白的半通道，通过乙锭摄取测定进行评估，这与从正常人肌肉细胞系 RCMH 获得的肌管不同。通过用特异于 Dysferlin 的小发夹 RNA (RCMH-sh Dysferlin) 处理 RCMH 细胞系，在 Dysferlin 敲低模型中重现了这种反应。此外，在表达突变 Dysferlin 的肌管中检测到 P2X7 受体和瞬时受体电位通道 TRPV2（另一种 Ca2+ 通透通道）的存在，并且在后者的肌管中发现静息细胞内 Ca2+ 水平升高，而在分子 D4（一种选择性 Cx HCs 抑制剂）存在下，该水平又降低至对照水平。数据表明，由 Dysferlin 突变或下调引起的 Dysferlin 缺乏可促进 Cx HC 的表达。然后，从头表达 Cx HC 会导致细胞内游离 Ca2+ 水平失调，这可能是与 Dysferlin 突变相关的肌肉损伤的基础。这种机制可能构成铁蛋白病的潜在治疗靶点。本文的在线版本 (doi:10.1186/s12860-016-0096-6) 包含补充材料，可供授权用户使用。
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. 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 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. 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 Ca2+ levels, which could underlie muscular damage associated to dysferlin mutations. This mechanism could constitute a potential therapeutical target in dysferlinopathies. The online version of this article (doi:10.1186/s12860-016-0096-6) contains supplementary material, which is available to authorized users.