Amiodarone, an Anti-arrhythmia Drug, Inhibits Epithelial to Mesenchymal Transformation and Causes Cardiac Valve Defect during Zebrafish Embryogenesis

Amiodarone為一種第三型抗心律不整藥物，在臨床上可緩解多種不同類型的心律不整症狀。由於Amiodarone使用頻繁，因此希望了解若懷孕婦女不慎使用之後，藥物對胎兒發育的影響。斑馬魚胚胎透明，在研究胚胎發育方面有極大優勢，因此選用斑馬魚作為模式動物來研究Amiodarone對胚胎發育的影響。從10 hpf起，利用15 μM Amiodarone浸泡至72 hpf止，於72 hpf皆可發現到部分的胚胎具有血液逆流的性狀，而利用whole mount in situ hyridization觀察與瓣膜發育相關的基因可發現，標定瓣膜的基因versican具有異位性過量表現的現象，而另一個會表現在瓣膜的基因has2也具有此現象，因此確定瓣膜的發育在基因層次上的確受到Amiodarone的影響。此外，利用倍頻光學顯微術及雙光子螢光顯微術觀察心臟專一性標定遠紅外光的基因轉殖斑馬魚品系Tg (cmlc2:HcRFP)，可以活體直接觀察到浸泡15 μM Amiodarone的斑馬魚胚胎其心臟瓣膜無法正常發育，且利用石蠟切片及HE染色也可觀察到相同的結果，故Amiodarone的確會造成斑馬魚胚胎心臟瓣膜缺失。Epithelial to Mesenchymal Transformation (EMT) 為瓣膜發育的關鍵步驟之一，目的在於促使atrioventricular canal (AV canal) 處形狀固定的endocardial cell轉型成具移動能力的mesenchyme cell，進而遷移進入endocardium及myocardium間的cardiac jelly中堆積增生形成瓣膜。而cdh5為AV canal處endocarial cell進行EMT時表現量必須下降的基因，在浸泡藥物組觀察cdh5的表現可以發現，cdh5呈現過量表現的情況，顯示EMT的進行受到抑制，且注射1.6 ng cdh5 MO後，可救回Amiodarone所造成的瓣膜發育缺失。另外，在注射16 ng versican MO後，可以發現cdh5的表現量呈現下降的情況，且同樣可救回Amiodarone所造成的瓣膜發育缺失。綜合以上，我們發現Amiodarone會透過異位性過量表現versican造成cdh5的上升而抑制EMT的進行，進而阻斷瓣膜生成。Amiodarone, a type Ⅲ anti-arrhythmia drug, is commonly used to treat arrhythmia, including pregnant women. In order to develop an animal model for studying the effect of Amiodarone on embryonic development, we used zebrafish due to its transparent embryo which makes dynamical observation be possible. When zebrafish embryos were treated with 15 μM Amiodarone from 10 hour post-fertilization (hpf) to 72 hpf, blood regurgitation between ventricle and atrium was found. Whole mount in situ hybridization showed that versican and has2, molecular markers for cardiac valves were over-expressed ectopically at 72 hpf, suggesting that Amiodarone causes embryos to have cardiac valve defect. Moreover, VE-cadherin (cdh5), a marker downregulate during epithelial to mesenchymal transformation (EMT) which is an important process during cardiac valve formation at cardiac valve was also overexpression at cardiac valves, indicating that EMT during cardiac valve development was inhibited by treating Amiodarone. After knockdown of versican by injecting 16 ng versican-morpholino (MO), the expression of cdh5 was downregulated at 72 hpf, suggesting that versican modulates the cdh5 expression. Using harmonic optical microscopy and two-photon fluorescence microscopy, we observed in vivo that valves were not formed in the Amiodarone-treated embryos derived from zebrafish transgenic line Tg(cmlc2:HcRFP), whose RFP reporter was driven by heart-specific promoter cmlc2. Histochemical examination of embryos also strengthened the evidence that cardiac valve was defective due to Amiodarone treatment. Taken together, our findings conclude that Amiodarone overexpress versican to inhibit cardiac valve formation due to repression of EMT during embryonic cardiac valve development.目 錄文摘要………………………………………………………………………1文摘要………………………………………………………………………3言……………………………………………………………………………5驗材料與方法………………………………………………………………15果……………………………………………………………………………23論……………………………………………………………………………33考文獻………………………………………………………………………46表……………………………………………………………………………55錄……………………………………………………………………………6