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

描述（由申请人提供）：在细胞培养或无脊椎动物中，开发机制可靠的充血性心力衰竭（CHF）生物学模型是不容易实现的，因为这样的系统无法捕捉多细胞或多器官过程的复杂性。我们之前已经在斑马鱼（一种可筛选的脊椎动物）中开发了复杂的多系统表型的体内分析，并利用这些“无偏”筛选遗传和化学修饰剂。将这种综合方法用于CHF将使我们能够识别控制CHF信号层次的基因或通过新机制逆转CHF的小分子。这些见解可以迅速转化为其他模型来解剖CHF生物学，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的核心表型和报告基因，并将这些不同的元素结合起来，以便在以下特定目标中建立一个强大的CHF体内综合检测方法；目的1。利用人类CHF的斑马鱼模型，我们将开发可扩展的表型工具和体内报告器，用于该综合征的多个组成部分，特别是；收缩力、心输出量、血管舒缩张力，以及利钠肽、肾素-血管紧张素和其他与CHF有关的心肌或全身通路。目标2。为了确定用于高通量筛选的最佳综合斑马鱼CHF测定方法，我们在斑马鱼幼虫中重现了许多人类心血管疾病途径，此时体内筛选是可行的，心脏和血管功能的高分辨率表征是可能的。我们将在CHF模型中验证多种表型和报告系，并测试在保持特异性的同时扩大到高通量的可行性。我们将调整我们优化的分析方法，以实现96孔格式的自动化，以生成具有强大z因子的HTS集成分析。目标3。将在CHF斑马鱼模型中对7500种结构多样的小分子进行中试筛选，以验证该试验是否可以进行大规模筛选。“hit”将在高分辨率二级分析中进一步评估，以及在现有的斑马鱼突变体或扰乱心肌心力衰竭途径的突变体中进行评估。这项工作将促进对CHF的遗传学和表观遗传学的“组学尺度”方法，并将产生一系列操纵CHF途径的小分子。这些基因或途径探针可以快速翻译到其他实验系统中，小分子命中可能代表潜在的药物线索，用于CHF的前体细胞。重要的是，这种筛选模式体内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.