Disrupted membrane structure and intracellular Ca²⁺ signaling in adult skeletal muscle with acute knockdown of Bin1.
Disrupted membrane structure and intracellular Ca²⁺ signaling in adult skeletal muscle with acute knockdown of Bin1.
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DOI：
10.1371/journal.pone.0025740
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Ma J
中科院分区：
文献类型：
--
作者：
Tjondrokoesoemo A;Park KH;Ferrante C;Komazaki S;Lesniak S;Brotto M;Ko JK;Zhou J;Weisleder N;Ma J
骨骼肌中有效的细胞内 Ca2+ ([Ca2+]i) 稳态需要完整的三联连接复合体，该复合体由质膜的 T 管内陷和肌浆网末端池组成。 Bin1 由一个专门的 BAR 结构域组成，该结构域与骨骼肌中的 t 管发育相关，并参与将二氢吡啶受体 (DHPR) 束缚到 t 管。在这里，我们证明 Bin1 对于成人骨骼肌中的 Ca2+ 稳态很重要。由于小鼠体内Bin1的全身性消融会导致出生后致死，因此采用体内电穿孔介导的转染方法递送RFP标记的质粒，该质粒产生靶向Bin1的短发夹（sh）RNA（shRNA-Bin1），以研究Bin1敲低对成年小鼠FDB骨骼肌的影响。在确认内源性 Bin1 表达减少后，我们发现 shRNA-Bin1 肌肉表现出肿胀的 t 管结构，表明 Bin1 是维持成人骨骼肌完整膜结构所必需的。 Bin1 表达减少导致 t 管结构破坏，这与细胞内 Ca2+ 释放的改变有关。 shRNA-Bin1 的分离单肌纤维中电压诱导的 Ca2+ 释放表明，与 shRNA 对照相比，Ca2+ 电流和 SR Ca2+ 瞬变的平均幅度均降低，表明 DHPR 和兰尼定受体 1 之间的耦合受损。shRNA-Bin1 中渗透应激诱导的 Ca2+ 火花的平均频率降低，表明 DHPR 激活受损。 ShRNA-Bin1 纤维还表现出 Ca2+ 火花幅度降低，这是由于 shRNA-Bin1 纤维中总 Ca2+ 储存减少。 Bin1 的人类突变与中心核肌病相关，Bin1 的 SH3 结构域对于骨骼肌中的肌节蛋白组织很重要。我们的研究表明 Bin1 在维持成人骨骼肌完整 T 管结构和 ([Ca2+]i) 稳态中的重要性，可以为 Bin1 在骨骼肌收缩性和肌病病理学中的潜在作用提供机制见解。
Efficient intracellular Ca2+ ([Ca2+]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca2+ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short –hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca2+ release. Voltage-induced Ca2+ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca2+ current and SR Ca2+ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca2+ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca2+ sparks' amplitude that was attributed to decreased total Ca2+ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca2+]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy.
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