Catch a Tiny Fish by the Tail.
Catch a Tiny Fish by the Tail.
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抓住尾巴的小鱼。
DOI：
10.1016/j.bpj.2020.07.010
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发表时间：
2020
影响因子：
3.4
通讯作者：
Granzier,HenkL
中科院分区：
文献类型：
--
作者：
Granzier,HenkL
动物模型系统对于深入了解肌肉功能和疾病的分子机制至关重要。然而，很少有脊椎动物动物模型适合于快速遗传操作，具有可以容易地可视化的快速发展，并且可以用于高通量治疗测试。一个例外是斑马鱼（Danio rerio），这是Mead等人研究的动物模型。（一）.斑马鱼与哺乳动物具有高度的序列和结构保守性，诱导靶向突变的快速方法使斑马鱼成为研究基因功能和人类疾病的有吸引力的模型（2）。肌肉组织的结构完整性可以通过利用斑马鱼在发育早期是透明的这一事实来非侵入性地评估。此外，可以使用双折射测定，其中斑马鱼被放置在两个偏振光滤光器之间，并且斑马鱼的高度组织化的骨骼肌使它们在黑暗背景中变得明亮（3）。肌病的斑马鱼经常表现出双折射的减少，反映了肌节的紊乱。此外，斑马鱼在发育早期对药物具有渗透性，双折射可用于高通量药物发现筛选，以寻找改变肌节结构的化合物（2）。然而，长期以来一直缺乏准确反映肌节力学的详细功能测定。在早期的工作，窦等。(4)研究人员开发了一种技术，用于表征几天大的斑马鱼幼鱼尾部的肌肉组织。该年龄段的肌纤维长度为100 mm，排列成肌节，肌节由肌间隔（纤维附着的结缔组织片）划分（图1）。连续连接的肌隔膜构成了尾巴的大部分。尾部的一大段可以在一端连接到力传感器，在另一端连接到控制器（以控制长度）。通过将尾巴置于两个电极之间的生理溶液中，可以刺激尾部肌肉并记录收缩反应（图1）。中轴骨骼直到受精后1周才成熟，这使得肌纤维可以拉伸，并且可以测量力，而不会受到骨骼系统的严重限制。此外，幼虫的透明度使得肌节图案能够被分析。可以将化合物添加到浴中，然后扩散到肌肉组织中（例如肌球蛋白抑制剂），并研究它们对肌肉功能的影响（2）。虽然这种方法已经成功地揭示了完整的尾部肌肉的收缩特性的相关见解，这些特性如何准确地反映肌节的力学和最终的收缩蛋白还没有很好地建立。这就是Warshaw小组的研究（Mead et al. (1))取得了重大进展。通过测量肌节的长度以及肌节在抽搐和强直收缩时的长度，Mead等。(1)示出了夹紧段的中心区域缩短了15%，而夹紧端附近的段被拉伸。这可能是由于当研究节段的末端连接到力和长度传感器时发生的损坏，这是通过弹簧加载夹具发生的。众所周知，内部缩短发生在其他肌肉类型中，这些肌肉类型在其末端被夹紧，甚至存在于通过其骨附件（5）保持的鱼类完整肌肉中，内部缩短可能是由于肌腱的拉伸。内部缩短如何影响力的产生是不确定的。为了防止内部缩短，作者使用了前馈长度。
Animal model systems have been essential for gaining an in-depth understanding of molecular mechanisms underlying muscle function and disease. However, few vertebrate animal models are amenable to fast genetic manipulation, have a rapid development that can be easily visualized, and can be used for high-throughput therapeutic testing. An exception is the zebrafish (Danio rerio), the animal model studied by Mead et al.(1). Zebrafish has a high sequence and structural conservation with mammals, and rapid methods for inducing targeted mutations make the zebrafish an attractive model, both to study gene function and human disease (2). The structural integrity of the musculature can be evaluated noninvasively by taking advantage of the fact that early in development, zebrafish are transparent. Furthermore, birefringence assays can be used in which zebrafish are placed between two polarized light filters, and the highly organized skeletal muscle of zebrafish makes them bright amid a dark background (3). Myopathic zebrafish often display a reduction in birefringence, reflecting the disorganization of sarcomeres. Additionally, zebrafish are permeable to drugs early during development, and birefringence can be used in high-throughput drug discovery screens to search for compounds that alter sarcomere structure (2). Detailed functional assays that accurately reflect sarcomere mechanics, however, had been lacking for a long time. In earlier work, Dou et al.(4) developed techniques for characterizing the musculature of the tail of zebrafish larvae that was a few days old. Muscle fibers are 100 mm in length at that age and are arranged in myotomes that are demarcated by myosepta, sheets of connective tissue to which fibers attach (Fig. 1). Serially linked myosepta make up the majority of the tail. A large segment of the tail can be attached to a force transducer at one end and a servomotor (to control length) at the other end. By placing the tail in a physiological solution between two electrodes, the tail muscle can be stimulated and the contractile responses recorded (Fig. 1). The axial skeleton does not mature until 1 week postfertilization, which allows the muscle fibers to be stretched and force to be measured without serious limitations from the skeletal system. In addition, the transparency of the larvae enables the sarcomere pattern to be analyzed. Compounds can be added to the bath that then diffuse into the musculature (eg, myosin inhibitors) and their effect on muscle function studied (2). Although this approach has been successful in revealing relevant insights in the contractile properties of the intact tail muscle, how accurately these properties reflect the mechanics of the sarcomere and ultimately that of the contractile proteins is not well established. This is where the study by the Warshaw group (Mead et al.(1)) makes important advances. By measuring the length of the myotomes as well as that of the sarcomeres during twitch and tetanic contractions, Mead et al.(1) show that the central region of the clamped segment shortens up to 15% at the expense of the segments near the clamped ends that are stretched. This is likely due to the damage that occurs when the ends of the studied segment are attached to the force and length transducers, which occurs via spring-loaded clamps. Internal shortening is well known to take place in other muscle types that are clamped at their ends and is even present in fish intact muscles held via their bony attachments (5) with internal shortening likely due to the stretch of the tendons. How internal shortening affects force generation is uncertain. To prevent internal shortening, the authors used a feedforward length …