應用「擬動態三維影像技術」評估斑馬魚心臟功能及藥物對心臟功能影響

Abstract

斑馬魚具有飼育容易、胚胎透明、遺傳學工具成熟等諸多優點，有潛力成為活體動物藥物測試的實驗動物。相較於傳統生化或細胞層級的藥物測試，使用斑馬魚做藥物測試更能反映藥物對人體的效果；與其他實驗動物例如老鼠相比，斑馬魚也具有低成本、給藥容易、高通量等優勢。近來斑馬魚已被應用於藥物對心臟疾病的療效或藥物心臟毒性的評估。決定心功能需要量測心臟腔室之動態體積變化。 雖然心功能量測非常重要，但由於斑馬魚心律極快，精確量測心臟腔室之動態體積變化有技術上困難，因此大多數斑馬魚的研究多利用二維顯微影像估算其心功能。本研究利用「擬動態三維影像技術」重建活體斑馬魚心臟跳動時之三維影像，並決定心室之動態體積變化。並首開先例精確量測斑馬魚心臟之射血分率 (ejection fraction)、心輸出量 (cardiac output)、心室質量 (ventricular mass） 及心室充盈曲線 (diastolic filling curve) 等臨床上重要的心功能參數。我在此研究中將我們的方法與「二維顯微影像-橢圓球近似法」所得的結果做有系統的比較，發現我們三維影像方法具有多種優勢。為展現此方法在藥物篩選的應用，本研究中也量測腎上腺素等藥物刺激所導致的心搏速率及心室輸出功能變化，展示此技術可以評估藥物對心臟功能的改變。並以抗癌藥物 - 阿黴素 (doxorubicin) 所導致的心臟損傷模型，展示應用此技術檢測具心臟毒性之藥物。我們預期此技術未來可應用於藥物之效力及心臟毒性之測試以及探索心臟病理機制等研究領域。

The zebrafish (Danio rerio) possesses various attractive features including rapid development, ease of genetic manipulation and low cost of maintenance, and has become a popular animal model for the study of cardiovascular diseases and for the screening of drugs with therapeutic or adverse effects. Herein, we demonstrate a novel application of pseudo-dynamic three-dimensional (3D) cardiac imaging for precise determination of the cardiac function of zebrafish. With this technique, we particularly determined individual important parameters of the cardiac function of zebrafish larvae (including ventricular stroke volume, ejection fraction, cardiac output, heart rate, diastolic filling function and ventricular mass). We compared critically the cardiac parameters obtained with our approach with those derived with conventional 2D approximation termed “fit-to-ellipse”, and show that our 3D approach is superior in several respects. To demonstrate potential applications of our approach to pharmaceutical development, we evaluated specifically the inotropic and chronotropic response of the heart of zebrafish subject to pharmacological interventions of epinephrine, esmolol and doxazosin. Our results show that zebrafish exhibited pharmacological responses to these cardioactive drugs in a way similar to human beings do. We revealed also an impaired cardiac function of a zebrafish model of cardiomyopathy induced by a treatment of doxorubicin. Given the growing interest in the application of zebrafish in both basic and applied biomedical research, we anticipate that our approach should have widespread applications in not only pharmaceutical development but also studies of cardiac development, pathophysiology or therapies targeting human heart diseases.