Block the function of nonmuscle myosin II by blebbistatin induces zebrafish embryo cardia bifida

Abstract

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.

MYH 3‐associated distal arthrogryposis zebrafish model is normalized with para‐aminoblebbistatin

“…Myosin ATPase inhibitors are being developed for the treatment of cardiomyopathy caused by similar mutations of cardiac myosin heavy chain genes (Green et al , 2016); therefore, we sought to determine whether drugs that directly inhibit function of myosin (Kovács et al , 2004) could reduce the adverse phenotypic effects of the s myhc1 R673H allele. Blebbistatin, a myosin ATPase inhibitor, which reduces actin–myosin affinity, was applied to dechorionated embryos at a concentration of 25 μM from 24 to 48 hpf (Wang et al , 2015). Blebbistatin prevented the development of notochord kinks in both smyhc1 R673H/+ and s myhc1 R673H/R673H embryos (Rauscher et al , 2018; Fig EV5B).…”