High content assay for zebrafish cardiomyocyte development

描述（申请人提供）：心脏细胞治疗和再生是心力衰竭心脏修复的新领域。研究集中在开发影响干细胞心脏分化的药物，以及增强内源性心脏再生能力。很少有心脏小分子被描述，没有一个处于临床前开发阶段。斑马鱼已成为药物发现过程中多个步骤的重要动物模型。我们设计并使用荧光心脏的生长和大小来筛选斑马鱼胚胎的表型，并进行了稳健且可重复的中试筛选。我们的最终目标是进行高通量筛选，以识别新的和有效的化合物，这些化合物可以在临床前环境中通过药物化学进一步开发。因此，我们建议建立一个高通量和含量的分析设计，以满足所有严格的要求。特别地，本提案首先旨在开发一种测量心脏大小的自动解决方案，并将该分析配置为高通量筛选设置。接下来，将建立一系列二级和反筛选试验，并用于评估从初级筛选中鉴定出的小分子的选择性、效力和细胞毒性。我们提出的检测和筛选开发的结果将有助于识别诱导心脏干细胞产生或心肌细胞增殖的小分子。我们希望这些药物线索的发现可以为衰竭心脏的心脏细胞补充开辟新的途径。

DESCRIPTION (provided by applicant): Cardiac cell therapy and regeneration are promising new frontiers for cardiac repair in heart failure. Studies have intensively focused on developing drug leads to influence cardiac differentiation from stem cells, as well as enhance the endogenous cardiac regenerative capacities. Very few cardiogenic small molecules have been described, and none are in preclinical development. Zebrafish has emerged as an important animal model in multiple steps of the drug discovery process. We have designed and performed a robust and reproducible pilot screen using the growth and size of fluorescent hearts as screening phenotypes in zebrafish embryos. Our ultimate goal is to perform a high throughput screen to identify novel and potent compounds that can be further developed with medicinal chemistry in a preclinical environment. We thus propose to establish a high throughput and content assay designed to meet all rigorous requirements. Specially, this proposal first aims to develop an automatic solution for measuring heart size and configure the assay into a high throughput screening setting. Next, series of secondary and counter-screen assays will be established and used to evaluate selectivity, potency, and cytotoxicity of small molecules identified from the primary screen. The results of our proposed assay and screen development will facilitate to identify small molecules in inducing production of cardiac stem cells or proliferation of cardiomyocytes. It is our hope that discovery of these drug leads may open new avenues for replenishing cardiac cells in failing hearts. PUBLIC HEALTH RELEVANCE: Heart disease remains the number one leading cause of death in USA. The proposed studies may provide an efficient approach for identifying small- molecules useful for potential therapeutic treatments of heart failure.