Development of a High-throughput Integrated In Vivo Heart Failure Assay

描述(申请人提供)：在细胞培养或无脊椎动物中，开发机械上可靠的充血性心力衰竭(CHF)生物学模型并不容易，因为这样的系统无法捕捉多细胞或多器官过程的复杂性。我们之前已经在体内开发了斑马鱼(一种可筛选的脊椎动物)的复杂多系统表型分析，并利用这些方法对遗传和化学修饰物进行了“无偏”筛选。对CHF采用这种综合方法将使我们能够识别控制CHF信号层次的基因或通过新的机制逆转CHF的小分子。这些见解可以迅速转化为CHF生物学解剖的其他模型，也可能导致新的诊断或治疗靶点。我们建议在斑马鱼中开发一套CHF的核心表型和报告器，并将这些不同的元素结合在一起，以建立针对以下特定目标的可靠的体内CHF综合检测方法：目的1.利用机械上忠实的人类CHF斑马鱼模型，建立一系列独立的斑马鱼幼体CHF的表型和报告器，我们将开发可扩展的表型工具和体内报告器，用于综合征的多个组成部分，特别是收缩功能、心输出量、血管舒缩张力以及与CHF有关的钠尿肽、肾素血管紧张素和其他心肌或全身通路。目的2.为了确定高通量筛选的最佳整合斑马鱼CHF方法，我们在斑马鱼幼体中总结了许多人类心血管疾病的通路，在体内筛选是可行的，并且心脏和血管功能的高分辨率表征是可能的。我们将验证CHF模型中的多种表型和报告线，并测试在保持特异性的同时扩展到高通量的可行性。我们将调整我们的优化分析以实现96孔格式的自动化，以生成针对HTS的具有强大z因子的集成分析。目的3.为了进行心力衰竭调节剂的中试化学筛选，将在CHF的斑马鱼模型中对7500个结构不同的小分子进行中试筛选，以验证该检测方法的大规模筛查。HITS将在高分辨率二次检测中以及现有的斑马鱼变种或扰乱心力衰竭途径的突变体中得到进一步评估。这项工作将促进对CHF遗传学和表观遗传学的研究，并将产生一系列用于操纵CHF途径的小分子。这些基因或途径探针可以快速转换到其他实验系统中，小分子靶标可能是CHF先兆的潜在药物先导。重要的是，这种体内筛选模式生理学与化学生物学或遗传学的结合可以推广到许多其他生物学问题。

DESCRIPTION (provided by applicant): The development of mechanistically faithful models of congestive heart failure (CHF) biology is not readily feasible in cell culture or invertebrates as such systems fail to capture the complexity of multi- cellular or multi-organ processes. We have previously developed in vivo assays for complex multi- system phenotypes in the zebrafish, a screenable vertebrate, and exploited these in 'unbiased' screens for genetic and chemical modifiers. Adapting this integrated approach for CHF will allow us to identify the genes controlling CHF signaling hierarchies or small molecules that reverse CHF through novel mechanisms. These insights can be rapidly translated to other models for the dissection of CHF biology, and might also lead to novel diagnostic or therapeutic targets. We propose to develop a suite of core phenotypes and reporters for CHF in the zebrafish, and to combine these different elements in order to build a robust integrated in vivo assay for CHF in the following Specific Aims; Aim 1. To develop a range of independent phenotypes and reporters for CHF in the larval zebrafish Using mechanistically faithful zebrafish models of human CHF, we will develop scalable phenotyping tools and in vivo reporters for multiple components of the syndrome, specifically; contractility, cardiac output, vasomotor tone, as well as natriuretic peptide, renin-angiotensin and other myocardial or systemic pathways implicated in CHF. Aim 2. To define the optimal integrated zebrafish CHF assay for high-throughput screening We have recapitulated many human cardiovascular disease pathways in the larval zebrafish, at a stage when in vivo screening is feasible and high-resolution characterization of cardiac and vascular function is possible. We will validate multiple phenotypes and reporter lines in CHF models and test the feasibility of scaling to high-throughput while retaining specificity. We will adapt our optimized assays for automation in 96-well format, to generate an integrated assay with a robust z-factor for HTS. Aim 3. To perform a pilot chemical screen for heart failure modifiers A pilot screen of 7,500 of structurally-diverse small molecules will be performed in a zebrafish model of CHF to validate the assay for large-scale screening. 'Hits' will be further evaluated in high-resolution secondary assays, as well as in existing zebrafish morphants or mutants that perturb myocardial heart failure pathways. This work will facilitate 'omic scale approaches to the genetics and epigenetics of CHF, and will generate an array of small molecules for manipulating CHF pathways. These genes or pathway probes can be rapidly translated into other experimental systems, and small molecule hits may represent potential drug leads for the antecedents of CHF. Importantly, this combination of screen-mode in vivo physiology with chemical biology or genetics is generalizable to many other biologic problems.