Ouyang Shi, Qin Wu-Ming, Niu Yu-Juan, Ding Yong-He, Deng Yun
State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Normal University, Changsha, China.
Laboratory of Zebrafish Genetics, College of Life Sciences, Hunan Normal University, Changsha, China.
Front Cardiovasc Med. 2022 Apr 5;9:839166. doi: 10.3389/fcvm.2022.839166. eCollection 2022.
Drug exposure during gestation or in prematurely born children represents a significant risk to congenital heart disease (CHD). Amantadine is an antiviral agent also effective in the treatment of Parkinson's disease. However, while its potential side effects associated with tetralogy of fallot (ToF) and birth defects were implicated, its underlying etiologic mechanisms of action remain unknown. Here, we report teratogenic effects of amantadine drug during early cardiogenesis through developing a novel zebrafish () knock-in (KI) animal model and explore the underlying mechanisms.
Homologous recombination (HR) pathway triggered by CRISPR/Cas9 system was utilized to generate an enhanced green fluorescent protein (EGFP) KI zebrafish animal model. Dynamic fluorescence imaging coupled with a whole-mount hybridization (WISH) assay was employed to compare the spatial and temporal expression patterns of the EGFP reporter in the KI animal model with the KI-targeted endogenous gene. Heart morphology and EGFP expression dynamics in the KI animal models were monitored to assess cardiac side effects of different doses of amantadine hydrochloride. Expression of key genes required for myocardium differentiation and left-right (LR) asymmetry was analyzed using WISH and quantitative reverse transcription-PCR (RT-PCR).
A novel EGFP KI line targeted at the ventricular myosin heavy chain ( gene locus was successfully generated, in which EGFP reporter could faithfully recapitulate the endogenous expression dynamics of the ventricle chamber-specific expression of the gene. Amantadine drug treatment-induced ectopic expression of gene in the atrium and caused cardiac-looping or LR asymmetry defects to dose-dependently during early cardiogenesis, concomitant with dramatically reduced expression levels of key genes required for myocardium differentiation and LR asymmetry.
We generated a novel zebrafish KI animal model in which EGFP reports the ventricle chamber-specific expression of gene dynamics that is useful to effectively assess drug safety on the cardiac morphology . Specifically, this study identified teratogenic effects of amantadine drug during early cardiogenesis dose dependent, which could be likely conveyed by inhibiting expression of key genes required for cardiac myocardium differentiation and LR asymmetry.
孕期或早产儿接触药物是先天性心脏病（CHD）的重大风险因素。金刚烷胺是一种抗病毒药物，也可有效治疗帕金森病。然而，尽管其与法洛四联症（ToF）和出生缺陷相关的潜在副作用已被提及，但其潜在的病因作用机制仍不清楚。在此，我们通过建立一种新型斑马鱼基因敲入（KI）动物模型，报告金刚烷胺药物在心脏早期发育过程中的致畸作用，并探索其潜在机制。
利用CRISPR/Cas9系统触发的同源重组（HR）途径，构建一种增强型绿色荧光蛋白（EGFP）敲入斑马鱼动物模型。采用动态荧光成像结合全胚胎原位杂交（WISH）分析，比较KI动物模型中EGFP报告基因与KI靶向的内源性基因的时空表达模式。监测KI动物模型中心脏形态和EGFP表达动态，以评估不同剂量盐酸金刚烷胺的心脏副作用。使用WISH和定量逆转录聚合酶链反应（RT-PCR）分析心肌分化和左右（LR）不对称所需关键基因的表达。
成功构建了一种靶向心室肌球蛋白重链（基因位点的新型EGFP KI品系，其中EGFP报告基因能够忠实地重现基因在心室腔特异性表达的内源性表达动态。金刚烷胺药物治疗在心脏早期发育过程中，剂量依赖性地诱导基因在心房中的异位表达，并导致心脏环化或LR不对称缺陷，同时显著降低心肌分化和LR不对称所需关键基因的表达水平。
我们构建了一种新型斑马鱼KI动物模型，其中EGFP报告基因可报告基因在心室腔的特异性表达动态，这有助于有效评估药物对心脏形态的安全性。具体而言，本研究确定了金刚烷胺药物在心脏早期发育过程中的剂量依赖性致畸作用，这可能是通过抑制心肌分化和LR不对称所需关键基因的表达来实现的。
