HTS assay for zebrafish embryonic heart function (RMI)

HTS 检测斑马鱼胚胎心脏功能 (RMI)

• 批准号: 6879877
• 负责人: C. Geoffrey Burns
• 金额: $ 8.75万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879877
• 关键词: cardiovascular pharmacology; drug discovery /isolation; drug screening /evaluation; heart contraction; heart function; heart rate; high throughput technology; molecular probes; small molecule; technology /technique development; vertebrate embryology; zebrafish

DESCRIPTION (provided by applicant): Embryonic heart function is a complex biological process regulated by a host of cardiac proteins and, directly or indirectly, by proteins and pathways in other organ systems. If technology existed for automated determination of embryonic heart function in a high-throughput molecular screening (HTS) assay, then screening for small molecules (novel compounds and FDA approved drugs) that influence embryonic cardiac physiology would become possible. The small molecules identified in such screens would have important applications in cardiovascular physiology, toxicology, and drug discovery. An automated assay for zebrafish embryonic heart rate has been developed that utilizes a 96-well format and transgenic embryos (cmlc2::GFP) expressing GFP specifically in the embryonic heart. Briefly, an automated fluorescence microscope scans a 96-well plate, identifies GFP+ hearts, and acquires short digital videos. Raw data from image analysis are used, in conjunction with a Fast Fourier Transform function, to quickly and automatically determine heart rates. Heart rates are outputted in a simple format. Experiments in specific Aim I will determine the reproducibility, responsiveness, and selectivity of assay-response by screening the NINDS custom collection (approximately 1,000 bioactive molecules) available from the Harvard Institute of Chemistry and Cell Biology. Experiments in specific aim II will investigate the viability of incorporating indices of embryonic heart contractility into the assay so that two parameters, heart rate and contractility, can be assay simultaneously thereby yielding a more-complete measure of embryonic heart function. In its current incarnation, the assay has important applications in cardiovascular physiology, toxicology, and drug discovery. The breadth of applications would increase if contractility were incorporated. The long-term goal is to perform high throughput screening to identify small-molecule-modifiers of embryonic heart function for use as biological probes and as lead compounds in the treatment of human heart disease.

描述（由申请人提供）：胚胎心脏功能是一个复杂的生物学过程，由许多心脏蛋白质调节，直接或间接地通过其他器官系统中的蛋白质和途径进行调节。 如果在高通量分子筛选（HTS）测定中自动确定胚胎心脏功能的技术存在，则将对影响胚胎心脏生理学的小分子（新颖的化合物和FDA认可的药物）进行筛查。 在此类筛选中鉴定出的小分子将在心血管生理学，毒理学和药物发现中具有重要的应用。 已经开发了用于使用96孔格式和转基因胚胎（CMLC2 :: GFP）的斑马鱼胚胎心率的自动测定，该测定法在胚胎心脏中专门表达GFP。 简而言之，自动荧光显微镜扫描了96孔板，标识了GFP+心脏，并获得了简短的数字视频。 使用图像分析的原始数据与快速傅立叶变换函数结合使用，以快速自动确定心率。 心率以简单的格式输出。 特定目标中的实验将通过筛选哈佛化学和细胞生物学研究所可提供的NINDS自定义收集（约1,000个生物活性分子）来确定测定反应的可重复性，响应性和选择性。 特定目标II中的实验将研究将胚胎心脏收缩率纳入指标的生存能力，以便可以同时分析两个参数，心率和收缩力，从而产生更完整的胚胎心脏功能量度。 在目前的化身中，该测定在心血管生理学，毒理学和药物发现中具有重要的应用。 如果合并收缩力，申请的广度将增加。 长期的目标是进行高吞吐量筛查，以鉴定胚胎心脏功能的小分子模型，以用作生物探针和治疗人心脏病的铅化合物。