抗心律不整藥物Amiodarone抑制斑馬魚胚胎 心臟瓣膜發育之受器與其調控機制

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

羅浩展(Hao-Chan 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(2012年)

https://doi.org/10.6342/NTU.2012.10598

摘要

Amiodarone一種抗心律不整藥物會誘導斑馬魚胚胎心臟s-vcanb增加，抑制snail-1b在AV canal的表現，造成cdh-5異位性過量增加而導致endocardial cell無法進行invagination，最後造成心臟瓣膜的缺失。然而，在myocardium的s-vcanb如何經過cardiac jelly而影響endocardium的invagination其受器以及分子機制尚未清楚。首先我們注射多量失去功能的dominant-negative form的s-vcanb (dn-s-vcanb)至胚胎中，結果顯示在心臟無法抑制snail-1b的表現，而使snail-1b以及cdh-5的表現與wild-type類似；表示myocardial cell中的S-vcanb透過其蛋白上的domain而影響endocardial cell中的snail-1b以及cdh-5。當我們注射vcana MO，發現抑制vcana不影響正常snail-1b與cdh-5在AV canal的表現，也不影響Amiodarone的結果。所以，我們證明Amiodarone 增加S-vcanb表現是透過其蛋白上的domain而誘導訊息傳導路徑影響下游基因的表現，而vcana則否。另一方面，若用receptor inhibitor，發現其下游snail-1b不在斑馬魚胚胎心臟表現，使得cdh-5不受抑制而增加，其結果與浸泡Amiodarone的胚胎相同；顯示Amiodarone促使S-vcanb過量表現後，S-vcanb會與receptor結合使之呈現dimer狀態，而抑制活性。進一步，我們發現不論是浸泡Amiodarone或是receptor inhibitor皆會讓GSK3β磷酸化程度下降；若將胚胎注射s-vcanb MO並浸泡Amiodarone，發現GSK3β磷酸化就不會下降。綜合以上證據 ，我們的總結是Amiodarone使斑馬魚胚胎心臟myocardium增加S-vcanb表現，蛋白分泌至cardiac jelly後，抑制receptor signaling，使得GSK3β因磷酸化下降而增加活性，進而抑制了snail-1b的表現，使cdh-5過量增加，導致endocardial cell進行invagination受阻，前後造成斑馬魚心臟瓣膜缺失。

關鍵字

抗心律不整藥物 ； 瓣膜發育

並列摘要

Amiodarone, an anti-arrhythmia drug, stimulates s-vcanb over-expression at zebrafish embryonic heart, promotes cdh-5 over-expression by inhibited snail-1b at AV canal, and causes invagination of endicardial cell is block to form cardiac valve. However, the receptor and mechanism of s-vcanb affected endocardium invagination through cardiac jelly is still unknown. First, we inject large amount of dominant-negative form of s-vcanb (dn-s-vcanb) into embryo, over-expression of dn-s-vcanb embryos can not inhibit snail-1b expression in the heart field, whereas snail-1b and cdh-5 expression pattern are similar to wild-type; indicating S-vcanb in myocardial cell affects the expression of snail-1b and cdh-5 in endocardial cell by domain. When inject vcana MO, we found that snail-1b and cdh-5 expression in wild-type and Amiodarone-treated embryo was not affected by knockdown vcana. So, we prove that S-vcanb, but not vcana, via domain affect expression of downstream gene in endocardium. Next; Using receptor inhibitor, we found expression of snail-1b at AV canal was missing, and making cdh-5 over-expression, similar to Amiodarone-treated embryo and overexpression wild-type s-vcanb embryo. Therefore, we suggesting that Amiodarone induce over-expression of S-vcanb, then S-vcanb bind to receptor but inhibit receptor activity. Moreover, we found that phosphorylation of GSK3β was decresased in Amiodarone-treated embryo and receptor inhibitor-treated embryo; while Amiodaorone-treated embryo inject with s-vcanb MO, phosphorylation of GSK3β was not decreased; indicating Amiodarone inhibit receptor signaling by inducing s-vcanb over-expression, promoting GSK3β activity, and causing cdh-5 over-expression by suppressing snail-1b. Taken together, we concluded that Amiodarone induces s-vcanb overexpression at myocardium, Amiodarone stimulates S-vcanb overpression and inhibits receptor signaling, elevates GSK3β activity by decreasing its phosphorylation, and causes cdh-5 over-expression by inhibited snail-1b, with in turn, the invagination of endocardial cell is blocked, resulting defect of zebrafish cardiac valve.

並列關鍵字

Anti-arrhythmia Drug ； valve development

參考文獻

黃昱愷（2007）。抗心律不整藥物Amiodarone影響斑馬魚胚胎心臟瓣膜的發育。台灣大學分子與細胞生物學研究所碩士論文。

陳大淵（2008）。抗心律不整藥物Amiodarone藉由抑制表皮間質轉換而干擾斑馬魚胚胎心臟瓣膜發育。台灣大學分子與細胞生物學研究所碩士論文。

羅道明（2010）。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。台灣大學分子與細胞生物學研究所碩士論文。

Babatin, M, Lee, SS, Pollak, PT. (2008) Amiodarone hepatotoxicity:review. Current Vascular Pharmacology .6:228-36

Bachelder, RE, Yoon, SO, Franci, C, de Herreros, AG, Mercurio, AM. (2005) Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial-mesenchymal transition. The Journal of Cell Biology.168:29-33.