螢光蛋白基因轉殖魚在心臟疾病之研究

Application of transgenic zebrafish expressing four-and-a-half LIM(FHL) domains proteins to the study of heart disease

施繼庭(Chih-Ting Shih)

指導教授 ： 黃銓珍

國立臺灣大學/生命科學院/生化科學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10236

摘要

Four-and-a-half LIM domains(FHL) 蛋白家族在真核生物中具有很高的保守性，其結構是由四又二分之一個LIM domain所組成。LIM domain為一蛋白質交互作用motif，因此FHL蛋白能夠和許多蛋白質進行作用，包括structural proteins、kinases以及許多transcription factors，因此參與、調控了許多細胞內的活動，包括細胞存活、轉錄調控及訊息傳遞。其組織特異性的表現模式主要在骨骼肌及心肌中，因此FHL蛋白家族在體內扮演的角色主要是調控肌肉及心血管的發育及功能。
本實驗中，我們首先比較了人類、老鼠及斑馬魚的FHL蛋白胺基酸序列，顯示了高度的保守性。接著，由斑馬魚的whole mount in situ hybridization實驗所得到的結果顯示，FHL蛋白在心臟及部分肌肉皆有表現，意味著其在心臟的重要性。為了探討FHL蛋白家族對於班馬魚胚胎時期的心臟發育及功能之影響，利用實驗室所選殖出的三種FHL基因，包括FHL1、FHL2、FHL5，藉由心臟專一性表現之啟動子cmlc2過量表現於斑馬魚胚胎心臟中，並同時表達綠色螢光蛋白以利於觀察。結果發現，FHL蛋白過量表現之基因轉殖魚部分螢光斑馬魚子代產生心律不整、心跳減緩的情形，顯示FHL蛋白參與了心肌節律，調控心跳。為了嘗試治療心律不整之病症並進一步釐清FHL蛋白家族是否透過和β-adrenergic receptor signaling pathway產生作用而調控心跳，將表現不同組合FHL 蛋白之斑馬魚品系以isoprenaline此一強心劑進行處理，將5~6dpf的魚苗浸泡、飼養在不同濃度(0.01μg/μl、0.0075μg/μl、0.005μg/μl、0.001μg/μl)的isoprenaline中，並持續觀察。我們的研究結果發現，經由isoprenaline處理將可以有效地改善過表現FHL 蛋白所造成的心律不整。然而，在長時間處理之下，過表現FHL 蛋白的斑馬魚品系心律不整的情形將會復發，並且有顯著的死亡率上升。我們仍需更進一步實驗來探討此現象。
藉由本實驗，我們更清楚地瞭解FHL蛋白在心臟中之作用，以及其在心臟疾病中的角色，而我們的成果也成功地以斑馬魚建立了心律不整的生物模式，有利於將來相關藥物的篩選及開發。

關鍵字

Four-and-a-half LIM domains(FHL) 蛋白家族 ； LIM domain ； cmlc2 ； 心律不整 ； isoprenaline

並列摘要

In eukaryotes, Four-and-a-half LIM domains (FHL) protein family is highly conserved. Structurally, the FHL proteins are composed of four closely connected LIM domains arranged in tandem, which are cysteine-rich double zinc finger motifs; and an N-terminal half LIM domain, which has one zinc finger motif. The LIM domain is known for mediating protein-protein interaction. Based on its protein-protein interaction properties, FHL protein family can interact with many proteins. Such like structural proteins, kinases and many transcription factors. The presence of multiple LIM domains, suggests that FHL protein family may involve in many cellular mechanisms. Including cellular survival, transcription regulation and signal transduction. The tissue-specific expression pattern of FHL protein family is mainly at cardiac muscular and skeletal muscle. Therefore the FHL protein family plays an important role in muscle and cardiovascular differentiation and the function maintenance.
In our study, by alignment of the FHL protein sequences in human, mouse and zebrafish and the results of whole mount in situ hybridization assay. Our results shows that FHL protein family are highly conserved and mainly expressed in heart and some part of skeletal muscle, suggest the importance of FHL protein family in the cardiomyocyte. We further analysed the role of FHL protein in the heart development by overexpressed several FHL proteins fused GFP including FHL1, FHL2 and FHL5, in zebrafish heart by a cardiac-specific promoter; cmlc2. The results turned out that a certain number of the FHL-GFP-positive offspring of transgenic line had arrhythmia. This indicates that FHL protein plays a role in regulation of heart rhythm. Next, we intend to rescue the phenotype of arrhythmia. Transgenic zebrafish which overexpressed different combination of FHL proteins, were treated with isoprenaline, a cardiac stimulant. Different FHL transgenic zebrafish embryos of 5-6dpf were immersed with different concentration of isoprenaline and heart beat were recorded daily. Our results demonstrated that isoprenaline could significantly rescue arrhythmia phenotype. However, long-term treatment had resulted in dramatic drop of survival rate and the recurring of irregular heart beat of FHL overexpressed transgenic line. Therefore further studies may be required to decipher the mechanism in the future. Taken together, our studies provide more understandings about the function of FHL protein in heart, the role in the cardiac disease, and established a zebrafish arrhythmia model for cardiovascular drug selection.

並列關鍵字

Four-and-a-half LIM domains (FHL) protein ； LIM domain ； zinc finger motif ； cmlc2 ； arrhythmia ； isoprenaline

參考文獻

Arber, S., Halder, G., and Caroni, P. (1994). Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation. Cell 79, 221-231.

Asai, K., Yang, G.P., Geng, Y.J., Takagi, G., Bishop, S., Ishikawa, Y., Shannon, R.P., Wagner, T.E., Vatner, D.E., Homcy, C.J., et al. (1999). Beta-adrenergic receptor blockade arrests myocyte damage and preserves cardiac function in the transgenic G(salpha) mouse. The Journal of clinical investigation 104, 551-558.

Bach, I. (2000). The LIM domain: regulation by association. Mechanisms of development 91, 5-17.

Beckerle, M.C. (1997). Zyxin: zinc fingers at sites of cell adhesion. BioEssays : news and reviews in molecular, cellular and developmental biology 19, 949-957.

Behr, R., and Weinbauer, G.F. (2001). cAMP response element modulator (CREM): an essential factor for spermatogenesis in primates? International journal of andrology 24, 126-135.