抗心律不整藥物amiodarone影響斑馬魚胚胎心臟瓣膜的發育

Abstract

Amiodarone是一種強而有效的抗心律不整藥物，被廣泛的應用在臨床治療上，但被美國食品藥物管理局歸類為D級的藥物，孕婦服用可能造成胎兒甲狀腺機能衰退、出生體重過低與心跳速率緩慢等的副作用(Joglar and Page, 2001)。然而，當其他的抗心律不整藥物無效，而且施用amiodarone在孕婦的利益高於胎兒承受的風險時，amiodarone還是會被用在孕婦的治療上。為了能夠建立一個用來研究藥物影響胚胎心臟發育的動物模式，我們選擇斑馬魚作為實驗動物，因為斑馬魚有胚胎透明的優點以及有心臟螢光的轉殖品系可用。當斑馬魚胚胎自受精後12小時(12 hpf)開始持續浸泡15 μM amiodarone，在72 hpf的累積死亡率達到36.5%，且在某些斑馬魚胚胎中會觀察到血液逆流的情形，這種性狀在自48 hpf浸泡amiodarone至60 hpf的斑馬魚胚胎中也看得到。我們使用倍頻光學顯微術搭配雙光子螢光顯微術來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)，可以在活體直接觀察沒用amiodarone處理的斑馬魚胚胎於87 hpf有endocushion形成；但是在持續浸泡amiodarone的斑馬魚胚胎心臟，一直到87 hpf都還沒有看到endocushion的生長，呈現瓣膜發育不完全的現象。TUNEL assay證實在endocushion形成之處並無細胞凋亡的現象發生。全胚胎原位雜交染色法(whole mount in situ hybridization)顯示無論長時間(12-72 hpf)持續浸泡amiodarone或特定時間(36-72 hpf之間的任意6小時)浸泡amiodarone都會造成瓣膜發育基因versican在心臟的異位性過度表現(ectopic over-expression)，這個結果和自12 hpf持續浸泡已知的瓣膜抑制藥物cyclosporine A至72 hpf的斑馬魚胚胎是一樣的。然而在心肌細胞協助心臟內皮細胞分化的bmp4基因與協助內皮至間質轉型作用(endothelial to mesenchymal transition)的notch1b基因則沒有異位性過度表現的情形發生。總結以上所述，我們建立了一個在活體可以觀察amiodarone對心臟發育、瓣膜生成影響的動物模式系統，而且瞭解證明了抗心律不整藥物amiodarone會影響斑馬魚心臟瓣膜的正常發育，可能是因為versican在心肌細胞(myocardial cells)異位性過度表現所造成的。

Amiodarone is a potent anti-arrhythmic drug and widely used in clinic, but it is classified as FDA category D. Some adverse effects to the fetus have been reported with amiodarone, including fetal hypothyroidism, low birth weight and bradycardia. However, amiodarone is still used in case other agents are failure treatment and the benefit from using this drug in pregnant women outweighs the fetal risk. To develop an animal model for studying the drug effects on embryonic heart development, we used zebrafish due to a transgenic line is available, which has heart-specific fluorescent signal, and its transparent embryo, which makes direct observation of the spatiotemporal expression become possible. When we continuously treated embryos with 15 μM amiodarone from 12 hours post-fertilization (hpf), the accumulated mortality rate reached 36.5% at 72 hpf. A regurgitation of blood in the heart of some embryos was observed, so was observed in the embryos that were stated treatment with amiodarone from 48 hpf to 60 hpf. Based on the combination of harmonic optical microscopy and two-photon fluorescence microscopy with heart-specific HcRFP expression transgenic zebrafish Tg(cmlc2:HcRFP), we directly observed in vivo that valve failed to form completely in some amiodarone-treated embryos. The endocushion was not observed at 87 hpf. TUNEL assay confirmed that apoptosis did not occur in endocushion forming region. Whole mount in situ hybridization showed that treatment with amiodarone resulted in ectopic over-expression of versican, a valve marker, in embryonic heart; but the expression of bmp4, a marker for the differentiation of the myocardial cells, and notch1b, a marker for epithelial to mesenchymal transformation, were not affected. In addition, the embryos treated with cyclosporine A, a chemical that inhibits valve development, also produced ectopic over-expression of versican. In conclusion, we develop an animal model that enables us to demonstrate in vivo the effect of amiodarone on the valve formation during heart development. Furthermore, we suggest that amiodarone affects cardiac valve development in zebrafish heart through versican ectopic over-expression in all myocardial cells.