Novel Approach to Enhance Myocardial Performance and Improve Heart Failure Outcome

心力衰竭（HF）是发病率和死亡率的主要原因，在美国，每9例死亡中就有1例是由HF引起的。 因此，对新的HF疗法存在巨大的需求，其只能通过发现新的治疗方法来出现。 治疗目标在过去，开发了急性增强心肌性能的正性肌力药物 但是现在大多数都是禁忌的，因为它们会使HF的长期结局恶化。最近， 我们开发了一种新的培养方法，称为基质胶床垫，它允许同时评估 单个人诱导多能干细胞衍生的收缩性能和钙动力学 心肌细胞（hiPSC-CM）。使用Matrigel床垫方法作为化学筛选的基础 在这个平台上，我们发现小分子EGM显著增强了小鼠的肌力和Lustropy。 hiPSC-CM和改善的体内心脏功能。与传统的正性肌力药不同，EGM不影响钙 (Ca)循环，细胞cAMP浓度或增加节拍率，这表明它的作用从根本上说， 新机制为了解开EGM药理学的机制基础，我们进行了一项 生物化学下拉测定和鉴定法呢基二磷酸合酶（FDPS），所需的蛋白质 异戊烯化，作为EGM的候选靶标。与先前的研究一致，表明FDPS有助于 在动物模型中，我们发现FDPS基因中自然发生的变异是高度相关的。 在范德比尔特大学医学中心的电子健康记录关联DNA数据库中，的 基于真实的世界临床数据的后一结果提出了调节FDPS水平的令人兴奋的可能性， 一个人一生中的活动可以显著改变HF的自然史;并且像EGM这样的化合物 抑制FDPS可能改善长期HF结局。基于这些发现，我们假设EGM 通过抑制FDPS增强心肌性能，并且FDPS抑制改善急性 心功能和长期HF结局。在这里，我们提出了创新的化学和功能基因组 方法阐明FDPS在EGM功能中的作用。在目标1中，我们将进行结构活动 EGM类似物的相关性（SAR）研究，以确定FDPS抑制是否是EGM功能所必需的。 在目标2中，我们将采用CRISPR/Cas9介导的基因组编辑来确定是否消融FDPS。 基因概括了EGM在hiPSC-CM中的独特药理学。在目标3中，我们将利用CRISPR/Cas9- 定向同源定向修复（HDR）以引入对应于CHF-1的核苷酸变化。 相关FDPS变体，并评估其对hiPSC-CM性能和FDPS功能的影响。的 拟议的研究将描述FDPS调制对心肌性能的影响，并可能确定 EGM的其他目标。这项研究利用EGM的独特药理学为以下方面奠定了基础： 对心肌调节的新理解和“双重目的”药物的新治疗范例， 急性缓解HF症状并改善长期HF结局。

Heart failure (HF) is a leading cause of morbidity and mortality, contributing to 1 in 9 deaths in the US. Consequently, there is an enormous need for new HF therapies, which can only emerge from discovery of new therapeutic targets. In the past, inotropic drugs that enhance myocardial performance acutely were developed to treat HF, but most of them are now contraindicated because they worsen HF outcomes long-term. Recently, we developed a novel culturing method, termed Matrigel Mattress, which allowed the simultaneous assessment of contractile performance and calcium dynamics in individual human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using the Matrigel Mattress method as a basis for a chemical screening platform, we discovered that the small molecule EGM significantly enhanced both inotropy and lusitropy in hiPSC-CMs and improved cardiac function in vivo. Unlike the traditional inotropes, EGM did not affect calcium (Ca) cycling, cellular cAMP concentrations or increase the beat rate, suggesting it acts by a fundamentally novel mechanism. To unlock the mechanistic underpinnings of EGM's pharmacology, we carried out a biochemical pull-down assay and identified farnesyl diphosphate synthase (FDPS), required for protein prenylation, as a candidate target of EGM. Consistent with prior studies demonstrating that FDPS contributes to hypertrophy and HF in animal models, we found that naturally occurring variants in the FDPS gene were highly associated with HF in Vanderbilt University Medical Center's electronic health record-linked DNA database. The latter result, based on real world clinical data, raises the exciting possibility that modulating the level of FDPS activity over a course of a person's life can significantly alter HF natural history; and that compounds like EGM that inhibit FDPS may improve long-term HF outcomes. Based on these findings, we hypothesize that EGM enhances myocardial performance by inhibiting FDPS, and that FDPS inhibition improves both acute cardiac function and long-term HF outcome. Here, we propose innovative chemical and functional genomic approaches to elucidate the role of FDPS in EGM function. In Aim 1, we will carry out a structure activity relationship (SAR) study of EGM analogs to determine whether FDPS inhibition is essential for EGM function. In Aim 2, we will employ the CRISPR/Cas9-mediated genome editing to determine whether ablating the FDPS gene recapitulates EGM's unique pharmacology in hiPSC-CMs. In Aim 3, we will utilize the CRISPR/Cas9- directed homology directed repair (HDR) to introduce the nucleotide changes corresponding to the CHF- associated FDPS variants, and evaluate their impact on hiPSC-CM performance and FDPS function. The proposed study will delineate the effects of FDPS modulation on myocardial performance, and possibly identify additional targets of EGM. This study leverages the unique pharmacology of EGM to lay the foundation for a new understanding of myocardial regulation and the new therapeutic paradigm of “dual purpose” drugs that acutely relieve HF symptoms as well as improve long-term HF outcomes.