Hirata Hiromi, Watanabe Takaki, Hatakeyama Jun, Sprague Shawn M, Saint-Amant Louis, Nagashima Ayako, Cui Wilson W, Zhou Weibin, Kuwada John Y
Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
Development. 2007 Aug;134(15):2771-81. doi: 10.1242/dev.004531. Epub 2007 Jun 27.
Wild-type zebrafish embryos swim away in response to tactile stimulation. By contrast, relatively relaxed mutants swim slowly due to weak contractions of trunk muscles. Electrophysiological recordings from muscle showed that output from the CNS was normal in mutants, suggesting a defect in the muscle. Calcium imaging revealed that Ca(2+) transients were reduced in mutant fast muscle. Immunostaining demonstrated that ryanodine and dihydropyridine receptors, which are responsible for Ca(2+) release following membrane depolarization, were severely reduced at transverse-tubule/sarcoplasmic reticulum junctions in mutant fast muscle. Thus, slow swimming is caused by weak muscle contractions due to impaired excitation-contraction coupling. Indeed, most of the ryanodine receptor 1b (ryr1b) mRNA in mutants carried a nonsense mutation that was generated by aberrant splicing due to a DNA insertion in an intron of the ryr1b gene, leading to a hypomorphic condition in relatively relaxed mutants. RYR1 mutations in humans lead to a congenital myopathy, multi-minicore disease (MmD), which is defined by amorphous cores in muscle. Electron micrographs showed minicore structures in mutant fast muscles. Furthermore, following the introduction of antisense morpholino oligonucleotides that restored the normal splicing of ryr1b, swimming was recovered in mutants. These findings suggest that zebrafish relatively relaxed mutants may be useful for understanding the development and physiology of MmD.
野生型斑马鱼胚胎会对触觉刺激做出反应并游走。相比之下，相对松弛的突变体由于躯干肌肉收缩无力而游动缓慢。对肌肉进行的电生理记录显示，突变体中中枢神经系统的输出是正常的，这表明肌肉存在缺陷。钙成像显示，突变体快肌中的Ca(2+)瞬变减少。免疫染色表明，负责膜去极化后Ca(2+)释放的兰尼碱受体和二氢吡啶受体在突变体快肌的横管/肌浆网连接处严重减少。因此，游动缓慢是由于兴奋 - 收缩偶联受损导致肌肉收缩无力所致。事实上，突变体中大多数兰尼碱受体1b（ryr1b）mRNA携带一个无义突变，该突变是由于ryr1b基因内含子中的DNA插入导致异常剪接而产生的，从而导致相对松弛的突变体出现功能减退的情况。人类中的RYR1突变会导致一种先天性肌病——多微小核病（MmD），其特征是肌肉中出现无定形核。电子显微镜照片显示突变体快肌中有微小核结构。此外，在引入能恢复ryr1b正常剪接的反义吗啉代寡核苷酸后，突变体的游动能力得以恢复。这些发现表明，斑马鱼相对松弛的突变体可能有助于理解MmD的发育和生理学。
