Department of Physiology, VU University Medical Centre, Amsterdam, The Netherlands.
Brain. 2013 Jun;136(Pt 6):1718-31. doi: 10.1093/brain/awt113. Epub 2013 May 28.
Nebulin--a giant sarcomeric protein--plays a pivotal role in skeletal muscle contractility by specifying thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy, the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which Neb exon 55 is deleted (Neb(ΔExon55)) to replicate a founder mutation seen frequently in patients with nemaline myopathy with Ashkenazi Jewish heritage. Neb(ΔExon55) mice are born close to Mendelian ratios, but show growth retardation after birth. Electron microscopy studies show nemaline bodies--a hallmark feature of nemaline myopathy--in muscle fibres from Neb(ΔExon55) mice. Western blotting studies with nebulin-specific antibodies reveal reduced nebulin levels in muscle from Neb(ΔExon55) mice, and immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is significantly reduced. In line with reduced thin filament length, the maximal force generating capacity of permeabilized muscle fibres and single myofibrils is reduced in Neb(ΔExon55) mice with a more pronounced reduction at longer sarcomere lengths. Finally, in Neb(ΔExon55) mice the regulation of contraction is impaired, as evidenced by marked changes in crossbridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. A novel drug that facilitates calcium binding to the thin filament significantly augmented the calcium sensitivity of submaximal force to levels that exceed those observed in untreated control muscle. In conclusion, we have characterized the first nebulin-based nemaline myopathy model, which recapitulates important features of the phenotype observed in patients harbouring this particular mutation, and which has severe muscle weakness caused by thin filament dysfunction.
肌联蛋白——一种巨大的肌节蛋白——通过确定细肌丝的长度和功能，在骨骼肌收缩中发挥关键作用。尽管编码肌联蛋白（NEB）的基因突变是导致肌无力型肌营养不良症（最常见的非营养不良性先天性肌病）的常见原因，但 NEB 基因突变导致肌肉无力的机制在很大程度上仍不清楚。为了更好地理解这些机制，我们构建了一种小鼠模型，其中肌联蛋白外显子 55 缺失（Neb(ΔExon55)），以复制在具有阿什肯纳兹犹太血统的肌无力型肌营养不良症患者中经常发现的一个起始突变。Neb(ΔExon55) 小鼠的出生比例接近孟德尔比例，但出生后生长迟缓。电子显微镜研究显示，Neb(ΔExon55) 小鼠的肌肉纤维中存在杆状体——一种肌无力型肌营养不良症的标志性特征。用肌联蛋白特异性抗体进行的 Western blot 研究显示，Neb(ΔExon55) 小鼠的肌肉中肌联蛋白水平降低，用原肌球蛋白抗体和鬼笔环肽进行的免疫荧光共聚焦显微镜研究显示，细肌丝长度显著缩短。与细肌丝长度缩短一致，Neb(ΔExon55) 小鼠的肌纤维通透性和单个肌原纤维的最大产生力能力降低，在较长的肌节长度下降低更为明显。最后，在 Neb(ΔExon55) 小鼠中，收缩的调节受损，这表现在横桥循环动力学的显著变化以及产生力的钙敏感性降低。一种新型药物可促进钙与细肌丝结合，可显著增加亚最大力的钙敏感性，使其超过未处理对照肌肉的水平。总之，我们描述了第一个基于肌联蛋白的杆状体肌营养不良症模型，该模型重现了携带该特定突变的患者中观察到的表型的重要特征，并且由于细肌丝功能障碍导致严重的肌肉无力。
