High throughput platform for simultaneous multiparametric assessment of cardiac physiology for heart failure drug development

心力衰竭影响2-3%的美国人口，并且仍然是死亡的单一最大原因。 尽管存在大量未满足的需求，但心力衰竭药物开发是出了名的困难， 在过去的十年里，类药物已经被批准。人诱导多能干细胞（hiPSC）- 基于心脏病的模型被广泛认为具有巨大的发展潜力， 因为它们忠实地模拟疾病表型并反映个体患者 遗传学然而，它们用于药物筛选的效用是有限的，因为用于评估的技术 疾病改善效果对于大规模筛选来说太麻烦且通量低。 可视化心力衰竭的基因和药物的疾病修饰活性需要动力学读出 心肌细胞功能与收缩力和静息张力相关钙（Ca 2+）循环 来揭示心脏收缩和舒张功能障碍缺乏现成的同时解决方案 这些参数的测量是一个关键的差距，阻碍了基础研究的步伐， 疾病机制和药物开发。尽管现代高容量成像系统可以 获得必要的快速动力学数据集，主要的障碍是现有的数据分析工具缺乏 关键功能，吞吐量太低，和/或需要大量的编码专业知识来实现， 大规模的基因研究和药物发现。 通过将强大的工具交给科学家，解决这一障碍将是变革性的 没有编码专业知识，使他们能够使用iPSC和成人开发基因和药物筛选， 心力衰竭的心肌细胞模型。我们将提供一个软件、试剂和 同时测量细胞内和亚细胞内Ca 2+的标准化方案 具有收缩力和静息张力的动力学，可与亚细胞特征重叠 检测-所有与384孔板格式兼容-以模拟收缩和舒张心脏功能。 该软件将具有用户友好的图形用户界面，可完全自动化测量Ca 2+。 收缩力曲线（以绝对µM和nN表示）。一个关键特征 将是单独的，逐个细胞的分析，这将增加动态范围，并允许识别 制备物中的细胞异质性使得具有多个细胞的现实培养模型成为可能 类型我们还将开发一个病毒载体工具包，以提供基因编码的绝对传感器。 细胞内Ca 2+浓度，而不产生稳定的细胞系。的 整合平台将根据试点药物和基因的当前软件进行验证和基准测试 显示能够量化收缩期和舒张期心脏疾病改善活动的屏幕 疾病定量性能指标将评价检测试剂盒的就绪性。

Heart failure affects 2-3% of the US population and remains the single largest cause of mortality. Despite the large unmet need, heart failure drug development is notoriously difficult, and few first-in- class drugs have been approved in the past decade. Human induced pluripotent stem cell (hiPSC)- based models of heart disease are widely considered to hold tremendous potential for the development of heart failure drugs because they faithfully model disease phenotypes and reflect individual patient genetics. However, their utility for drug screening is limited because the technology for assessing disease modifying effects are too cumbersome and low throughput for large-scale screens. Visualizing disease-modifying activity of genes and drugs for heart failure requires kinetic read outs of cardiomyocyte function that correlate calcium (Ca2+) cycling with contractile force and resting tension to reveal systolic and diastolic heart dysfunction. The lack of off-the-shelf solutions for simultaneous measurement of these parameters is a critical gap that has hindered the pace of basic research into disease mechanisms and drug development. Although modern high content imaging systems can acquire the requisite fast kinetic datasets, the major roadblock is that available data analysis tools lack key capabilities, are too low throughput, and/or require substantial coding expertise to implement in large-scale genetic studies and drug discovery. Resolving this roadblock will be transformative by placing powerful tools in the hands of scientists without coding expertise, enabling them to develop gene and drug screens using iPSC and adult cardiomyocyte models of heart failure. We will deliver an integrated toolbox of software, reagents, and standardized protocols for contemporaneous measurement of intracellular and subcellular Ca2+ dynamics with contractile force and resting tension that can be overlaid with subcellular feature detection – all compatible with 384-well plate format – to model systolic and diastolic heart function. The software will have a user-friendly graphical user interface to fully automate measurements of Ca2+- contractility force curves (in absolute µM and nN terms) from beating cardiomyocytes. A key feature will be individual, cell-by-cell analysis that will increase dynamic range and allow the recognition of cellular heterogeneity in the preparations making possible realistic culture models with multiple cell types. We will also develop a toolkit of viral vectors to deliver genetically encoded sensors of absolute intracellular Ca2+ concentrations in cardiomyocyte populations without generating stable cell lines. The integrated platform will be validated and benchmarked against current software in pilot drug and gene screens that demonstrate ability to quantify disease-modifying activities for systolic and diastolic heart disease. Quantitative performance measures will evaluate assay readiness.