Wang Xueqian, Chong Mei, Wang Xin, Wang Hongkui, Zhang Jie, Xu Hui, Zhang Jingjing, Liu Dong
Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Qixiu Road 19, 226001, Nantong, China,
In Vitro Cell Dev Biol Anim. 2015 Mar;51(3):211-7. doi: 10.1007/s11626-014-9836-0. Epub 2014 Nov 18.
Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes encoding different nonmuscle myosin heavy chains including NM II-A, NM II-B, and NM II-C. Blebbistatin is a small molecule that has been shown to be a relatively specific inhibitor of NM II. Blocking the function of NM II by blebbistatin induces zebrafish embryo cardia bifida at a dose-dependent manner. In situ hybridization analysis with ventricular marker ventricular myosin heavy chain (vmhc) and atrial marker atrial myosin heavy chain (amhc) showed each of the heart contained both distinct atria and ventricle. However, the cardia bifida embryos had highly variable distance between two separate ventricles. We also provided evidence that time window from 12 to 20 h post fertilization (hpf) is necessary and sufficient for cardia bifida formation caused by blebbistatin treatment. Expression of spinster homolog 2 (spns2) was decreased in blebbistatin-treated embryos, suggesting the cardia bifida phenotype caused by NM II inhibition was relevant to precardiac mesoderm migration defects. Through in situ hybridization analysis, we showed that foxa1 was expressed in endoderm of blebbistatin-treated embryos at 24-hpf stage, suggesting the endoderm formation is normal in cardia bifida embryos caused by blebbistatin treatment. In addition, we demonstrated that blebbistatin treatment resulted in morphology alteration of zebrafish cardiomyocytes in vivo and neonatal mouse cardiomyocytes in vitro.
非肌肉肌球蛋白II（NM II）是由三个基因编码的多亚基蛋白质产物的名称，这些基因编码不同的非肌肉肌球蛋白重链，包括NM II - A、NM II - B和NM II - C。blebbistatin是一种小分子，已被证明是NM II的相对特异性抑制剂。用blebbistatin阻断NM II的功能会以剂量依赖的方式诱导斑马鱼胚胎心脏双裂。用心室标记物心室肌球蛋白重链（vmhc）和心房标记物心房肌球蛋白重链（amhc）进行原位杂交分析表明，每个心脏都包含不同的心房和心室。然而，心脏双裂胚胎的两个独立心室之间的距离差异很大。我们还提供了证据，即受精后12至20小时（hpf）的时间窗口对于blebbistatin处理引起的心脏双裂形成是必要且充分的。在blebbistatin处理的胚胎中，spinster同源物2（spns2）的表达降低，这表明由NM II抑制引起的心脏双裂表型与心前中胚层迁移缺陷有关。通过原位杂交分析，我们发现foxa1在24 hpf阶段的blebbistatin处理胚胎的内胚层中表达，这表明在blebbistatin处理引起的心脏双裂胚胎中内胚层形成正常。此外，我们证明blebbistatin处理导致斑马鱼体内心肌细胞和体外新生小鼠心肌细胞的形态改变。
