開發動態三維成像技術及影像分析法於活體斑馬魚心臟之鈣離子傳導及收縮功能定量

Three-dimensional dynamic imaging for simultaneous quantification of calcium dynamics and cardiac function of a beating zebrafish heart in vivo

莊育丞(Yu-Cheng Chuang)

指導教授 ： 廖奕翰

國立交通大學/理學院/分子科學研究所/碩士(2020年)

摘要

心臟正常運作主要由電生理和心肌細胞的協調收縮決定。過去研究已知鈣離子調控失衡會引起心臟電生理及心功能異常，例如化癌藥物阿黴素 (doxorubicin) 可透過干擾調控鈣離子的相關蛋白而產生嚴重心臟毒性 (cardiotoxicity)。在活體模式生物同時測量心臟鈣離子動態及心臟功能有助於瞭解心臟生理及相關疾病。過去已有報導以「擬動態三維光學成像技術 (pseudodynamic 3D imaging)」 決定斑馬魚幼魚 (larval zebrafish) 跳動心臟腔室體積的動態變化，進而獲得各種心功能參數。然由於跳動心臟之心肌細胞會快速移動且收縮，使得定量鈣離子瞬態 (calcium transient) 及心房心室傳導 (calcium atrioventricular conduction) 有技術困難。因此迄今仍須以藥物或基因技術停止心搏才能測量心臟鈣離子動態，而同時測量鈣離子傳導及心功能更從未被報導。在此研究，我們以雙螢光通道擬動態三維光學成像技術針對於心臟同時表現 GCaMP 及 DsRed 螢光蛋白的斑馬魚幼魚心臟成像，並以影像分析演算去除影像誤差 (image artifact)，同時精確定量活體斑馬魚幼魚跳動中心臟之鈣離子傳導及心功能。應用此技術，我們探討阿黴素引起的心臟毒性，結果顯示阿黴素 (35 ppm, 48 h) 處理後，斑馬魚幼魚心室體積變小，心房到心室鈣離子傳導自 120 ms 延遲至 174 ms，心輸出量 (cardiac output) 自 0.118 nL/min 減少至 0.0394 nL/min，心室射出分率 (ventricular ejection fraction) 自 38.4 % 減少到 19.5 %。綜言之，此研究之結果未來預期可應用於藥物心臟毒性評估及基礎心臟生理及心臟病之基礎研究。

關鍵字

斑馬魚 ； 擬動態三維影像 ； 心功能 ； 鈣離子動態 ； 阿黴素

並列摘要

As heart rhythm is tightly regulated by cardiac electrophysiology and the excitation-contraction coupling, disturbance of calcium homeostasis may impair cardiac conduction and cardiac function. Simultaneous determination of cardiac calcium dynamics and cardiac function of model animals (such as zebrafish) in vivo is important to help elucidate cardiac pathophysiology. While determination of the cardiac function of larval zebrafish has been realized with pseudodynamic 3D imaging, determination of local calcium transients and calcium atrioventricular conduction has been reported only on a heart whose heartbeat are stopped. The difficulty is mainly associated with the challenge to eliminate image artifacts originated from the contraction and movement of cardiomyocytes of a rapidly beating heart. This study reports the application of dual-channel pseudodynamic 3D cardiac imaging and algorithm for elimination of motion-induced artifacts, and realize simultaneous determination of the cardiac function and calcium dynamics of larval zebrafish in vivo. Doxorubicin is a potent chemotherapeutic agent for cancer treatments, but has been reported to have cardiotoxicity through interference with regulatory proteins responsible to calcium homeostasis. We show that the cardiac chamber of larval zebrafish (4 days post fertilization) became significantly smaller, the calcium atrioventricular conduction prolonged from 120 ms to 174 ms, the cardiac output reduced from 0.118 nL/min to 0.0394 nL/min and the ejection fraction decreased from 38.4% to 19.5% after a treatment of doxorubicin (35 ppm, 48 h). Our approach, which enables simultaneous determination of cardiac function and calcium dynamics on a popular model animal, should open up a new route to investigate cardiac pathophysiology and cardiotoxicity.

並列關鍵字

Zebrafish ； Pseudodynamic 3D imaging ； Cardiac function ； Calcium dynamics ； Doxorubicin

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