抗心律不整藥物Amiodarone抑制斑馬魚胚胎心臟瓣膜發育之分子機制

The Molecular Mechanism of Amiodarone, an Anti-arrhythmia Drug, to Inhibit the Cardiac Valves Formation during the Embryogenesis of Zebrafish

羅道明(Dao-Ming Lo)

指導教授 ： 蔡懷楨

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10530

摘要

斑馬魚胚胎透明，易於觀察胚胎發育時組織或器官形成，胚胎也易於浸泡藥物，本實驗以斑馬魚研究Amiodarone（第三型抗心律不整藥物）對心臟發育影響。以15 μM Amiodarone從10 hours postfertilization (hpf)浸泡斑馬魚胚胎至72 hpf觀察，可見心臟血液逆流，心臟瓣膜無法發育，並以whole mount in situ hybridization發現在wild-type中，只於atrioventricular canal (AV canal) myocardium表現之similar to versican b (s-vcanb)，經浸泡Amiodarone後會異位性地在myocardium過量表現。針對瓣膜發育時期，分段進行浸泡Amiodarone，發現Amiodarone主要影響55至72 hpf之瓣膜發育，此時期為endocardial cells進行endothelial-to-mesenchymal transition，或稱invagination。偵測invagination時，endocardium需下降VE-cadherin (cdh5)基因的表現，但是浸泡Amiodarone後cdh5於AV canal endocardium卻會過量表現。進一步實驗地發現，浸泡Amiodarone的胚胎，可由注射cdh5 morpholino oligonucleotide (MO)挽救瓣膜缺失的缺陷，顯示Amiodarone藉由過量表現cdh5抑制invagination。另一方面，Snail family 的snai1b為cdh5可能之repressor，我們也發現浸泡Amiodarone後，snai1b於AV canal endocardium會消退，而且浸泡Amiodarone的胚胎，也可由過量表現snai1b致使cdh5表現下降，而挽救瓣膜的缺失。同樣地，若注射s-vcanb-MO，並浸泡Amiodarone之胚胎，cdh5表現量也會下降，瓣膜因而生成；相反地，過量表現s-vcanb的胚胎，其cdh5會over-expression，造成瓣膜缺失，顯示cardiac jelly有訊息傳遞至endocardium。進一步地，若注射snai1b-MO，則發現s-vcanb與cdh5異位過量表現，且同時注射snai1b-MO與s-vcanb-MO後，cdh5會過量異位表現，證明了s-vcanb在snai1b上游，並顯示snai1b除了扮演cdh5 repressor外，亦可使s-vcanb被回饋抑制。綜合以上得知，我們發現了一條斑馬魚心臟瓣膜發育pathway，即AV canal myocardium表現s-vcanb，造成分泌至AV canal之cardiac jelly處的s-vcanb增多，而s-vcanb進而限制AV canal endocardium表現snai1b，snai1b再調控AV canal endocardium的cdh5基因，使Cdh5蛋白質減少，並負回饋限制s-vcanb表現，最後使瓣膜順利進行invagination；而Amiodarone則藉由myocardium異位性地表現s-vcanb，使snai1b變少，進而阻斷invagination，導致瓣膜缺失。

關鍵字

Amiodarone ； 心臟瓣膜 ； 斑馬魚 ； s-vcanb ； snai1b ； cdh5

並列摘要

Due to the transparent zebrafish embryo makes the dynamic observation possible, and also makes the facilitation to study the influence of drug by soaking. We study the effect of Amiodarone, a type III anti-arrhythmia drug, on heart development. After treating embryos with 15 μM Amiodarone from 10 to 72 hours post-fertilization (hpf), a blood regurgitation between ventricle and atrium was observation owing to the defect of valves. Using whole-mount in situ hybridization, we found similar to versican b (s-vcanb), a specific gene at the myocardium of atrioventricular canal (AV canal), where the valves develop at, was ectopically over-expressed in myocardium at the Amiodarone-treated embryos. Furthermore, we treated Amiodarone on zebrafish embryos at different developmental stage, and we found Amiodarone affected the endocardial cells progressing during invagination from 55 to 72 hpf. The transcript of VE-cadherin (cdh5), which is down-regulated at endocardium through invagination, was also over-expressed at the AV canal endothelium in Amiodarone-treated embryos. In addition, knockdown of cdh5 by injecting cdh5-specific morpholino oligonucleotide (MO) can rescue the defective valves induced by Amiodarone, indicating Amiodarone may inhibit invagination by over-expressing cdh5. Besides, the transcript of snai1b, one of Snail family, was decreased at the AV canal endocardium of Amiodarone-treated embryos. But the down-regulation of cdh5 and the rescue of valves defects were observed in Amiodarone-treated embryos by over-expression of snai1b, so were by injection of s-vcanb-MO in Amiodarone-treated embryos. On contrast, the up-regulation of cdh5 and the lack of valves in wild-type embryos by over-expression of s-vcanb. Moreover, we observed that both s-vcanb and cdh5 were over-expressed after microinjection with snai1b-MO; whereas cdh5 was over-expressed after microinjection with snai1b-MO accompanied by s-vcanb-MO, suggesting snai1b functions as a repressor of cdh5 and a feedback inhibition of s-vcanb, which is an upstream modulator of snai1b. Taken together, we found a novel pathway involved in zebrafish cardiac valves formation, in which the AV canal myocardium expresses s-vcanb and secrets s-vcanb gene in cardiac jelly to regulate snai1b expressing at AV canal endocardium. After cdh5 is down-regulated by snai1b, the invagination to generate valves is processed. Therefore, we concluded that Amiodarone may inhibit the development of valves by ectopically expressing s-vcanb at the whole myocardium, promoting the down-regulating of snai1b, and causing over-expressing of s-vcanb by failing to feedback inhibition of snai1b, which in turn, the invagination is block to form cardiac valves.

並列關鍵字

Amiodarone ； cardiac valves ； zebrafish ； s-vcanb ； snai1b ； cdh5

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