L-Type Ca2+ Channel Function Is Linked to Dystrophin Expression in Mammalian Muscle
L-Type Ca2+ Channel Function Is Linked to Dystrophin Expression in Mammalian Muscle
复制标题
DOI：
10.1371/journal.pone.0001762
复制
发表时间：
2008-03-12
期刊：
影响因子：
3.7
通讯作者：
Rohrbach, Petra
中科院分区：
文献类型：
--
作者：
Friedrich, Oliver;von Wegner, Frederic;Rohrbach, Petra
背景：在营养不良的 mdx 骨骼肌中，发现了异常的 Ca2+ 稳态和纤维变性。杜氏肌营养不良症 (DMD) 模型中抗肌营养不良蛋白的缺失与离子通道特性的改变有关。 g。 L 型 Ca2+ 电流受损。在再生 mdx 肌肉中，“回复”纤维恢复肌营养不良蛋白的表达。它们涉及 DHPR-Ca2+- 通道的功能是难以捉摸的。方法和结果：我们开发了一种新型“原位”共聚焦免疫荧光和成像技术，首次允许定量亚细胞肌营养不良蛋白-DHPR 在个体非固定肌纤维中共定位。管状 DHPR 信号与源自肌球蛋白的二次谐波生成信号交替。抗肌营养不良蛋白-DHPR 共定位在 wt 纤维中很显着，但在大多数 mdx 纤维中减少。表达微型肌营养不良蛋白（MinD）的纤维成功恢复了共定位。有趣的是，在一些老化的 mdx 纤维中，共定位与 wt 纤维相似。大多数 mdx 纤维显示出非常弱的膜抗肌营养不良蛋白染色，并被归类为“mdx 样”。然而，一些 mdx 纤维具有强烈的“wt 样”肌营养不良蛋白信号，并被鉴定为“回复体”。分裂的 mdx 纤维大多是“mdx 样”，并且通常不是“回复体”。膜肌营养不良蛋白和 DHPR 共定位之间的相关性表明“回复体”中假定的联系已恢复。使用双微电极电压钳技术，Ca2+-电流幅度（imax）表现出非常相似的行为：大多数老年mdx纤维的幅度降低（仅在年轻的mdx小鼠中看到）和一些mdx纤维（很可能是“回复体”），其幅度与wt或MinD纤维相似。 Ca2+ 电流激活曲线在“wt-like”和“mdx-like”老化 mdx 纤维中相似，并不是电流幅度差异的原因。 imax 振幅在 MinD 纤维中完全恢复。结论：我们提供了 wt、MinD 和“回复”mdx 纤维中存在直接/间接 DHPR-肌营养不良蛋白相互作用的证据，但在其余 mdx 纤维中不存在。我们的成像技术能够可靠地检测单个孤立的“回复”纤维，这些纤维可用于后续的生理实验，以研究 DMD 的机制和治疗概念。
Background: In dystrophic mdx skeletal muscle, aberrant Ca2+ homeostasis and fibre degeneration are found. The absence of dystrophin in models of Duchenne muscular dystrophy (DMD) has been connected to altered ion channel properties e. g. impaired L-type Ca2+ currents. In regenerating mdx muscle, 'revertant' fibres restore dystrophin expression. Their functionality involving DHPR-Ca2+- channels is elusive.Methods and Results: We developed a novel 'in-situ' confocal immuno-fluorescence and imaging technique that allows, for the first time, quantitative subcellular dystrophin-DHPR colocalization in individual, non-fixed, muscle fibres. Tubular DHPR signals alternated with second harmonic generation signals originating from myosin. Dystrophin-DHPR colocalization was substantial in wt fibres, but diminished in most mdx fibres. Mini-dystrophin (MinD) expressing fibres successfully restored colocalization. Interestingly, in some aged mdx fibres, colocalization was similar to wt fibres. Most mdx fibres showed very weak membrane dystrophin staining and were classified 'mdx-like'. Some mdx fibres, however, had strong 'wt-like' dystrophin signals and were identified as 'revertants'. Split mdx fibres were mostly 'mdx-like' and are not generally 'revertants'. Correlations between membrane dystrophin and DHPR colocalization suggest a restored putative link in 'revertants'. Using the two-micro-electrode-voltage clamp technique, Ca2+- current amplitudes (imax) showed very similar behaviours: reduced amplitudes in most aged mdx fibres (as seen exclusively in young mdx mice) and a few mdx fibres, most likely 'revertants', with amplitudes similar to wt or MinD fibres. Ca2+ current activation curves were similar in 'wt-like' and 'mdx-like' aged mdx fibres and are not the cause for the differences in current amplitudes. imax amplitudes were fully restored in MinD fibres.Conclusions: We present evidence for a direct/indirect DHPR-dystrophin interaction present in wt, MinD and 'revertant' mdx fibres but absent in remaining mdx fibres. Our imaging technique reliably detects single isolated 'revertant' fibres that could be used for subsequent physiological experiments to study mechanisms and therapy concepts in DMD.