Deciphering the Endothelial Cell-Cardiomyocyte Crosstalk in LMNA Cardiomyopathy

破译 LMNA 心肌病中的内皮细胞-心肌细胞串扰

• 批准号: 10276748
• 负责人: Nazish Sayed
• 金额: $ 39.35万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10276748
• 关键词: 3-Dimensional; ATAC-seq; Affect; Animal Model; Attention; Biomedical Engineering; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Physiology; Cell Communication; Cell Line; Cells; Chromatin; Coculture Techniques; Collaborations; Communication; Complex; Computational Biology; Data; Dilated Cardiomyopathy; Disease; Endothelial Cells; Environment; Family; Functional disorder; Gene Expression; Genes; Genomic DNA; Genotype; Goals; Heart; Heart Diseases; Heart Transplantation; Heart failure; Human; Impairment; In Vitro; Individual; Kinetics; Knowledge; Lamin Type A; Lead; Ligands; Lovastatin; Machine Learning; Mediating; Modeling; Molecular; Morbidity - disease rate; Mutation; Nuclear Envelope; Oligonucleotides; Organ; Paracrine Communication; Pathogenesis; Pathogenicity; Patients; Pharmacology; Phenotype; Positioning Attribute; Regulator Genes; Research Proposals; Signal Transduction; Site; Technology; Tissue Engineering; Translational Research; Variant; Wing; Work; Zebrafish; base; cardiac tissue engineering; cell type; endothelial dysfunction; env Gene Products; experimental study; familial dilated cardiomyopathy; genome editing; heart function; improved; in vivo; induced pluripotent stem cell; induced pluripotent stem cell technology; insight; interdisciplinary approach; knock-down; lamin C; loss of function; mechanical properties; mortality; next generation sequencing; novel; paracrine; predictive modeling; promoter; receptor; single cell technology; single-cell RNA sequencing; sound; stem cell biology; tool; transcription factor; translational medicine

Project Summary/Abstract Dilated cardiomyopathy (DCM) is a leading cause of heart failure and the leading reason for heart transplantation. Major gaps exist in our understanding of the pathophysiology of DCM and mutations in the gene that encodes the nuclear envelope proteins lamin A and C (LMNA) are considered to be the most common cause of DCM. However, the molecular mechanisms that underlie “cardiolaminopathy” remain elusive, and it is unknown why mutations in this ubiquitously expressed gene have such a disproportionate effect on the heart. Using induced pluripotent stem cell (iPSCs)-derived endothelial cells (iPSC-ECs), we recently studied a family affected by DCM due to a frameshift variant in LMNA, which showed endothelial dysfunction (Sayed et al. Science Translational Medicine, 2020). This EC dysfunction could be reversed by upregulating Krüppel-like Factor 2 (KLF2) by treatment of iPSC-ECs with a subset of statins, including lovastatin. Importantly, this improvement in EC dysfunction had a positive effect on co-cultured iPSC- cardiomyocytes (iPSC-CMs) from cardiolaminopathy patients, indicating an intricate crosstalk between the ECs and CMs in LMNA cardiomyopathy. Despite impressive progress, little attention has been given to the potential importance of cell-to-cell signaling between ECs and CMs, despite the fact that ECs serve a paracrine function to enhance signaling in CMs, especially in context to pharmacological stimulation. This knowledge gap impedes our comprehensive understanding of organ dysfunction at a multi-cellular level. The overarching goal of our proposal is to use a multidisciplinary approach that integrates human iPSCs, bioengineering tools, genome editing, and NGS to gain novel insights into the pathogenesis of DCM. Using human iPSCs, we propose to decipher the impaired cross-talk between ECs and CMs in LMNA cardiomyopathy and elucidate the beneficial class effects of statins in improving the EC-CM signaling as a key factor in regulating cardiac function. We will pursue three specific aims. In Aim 1: we will establish an experimental platform to study the genotype-phenotype association of LMNA mutations on ECs and CMs. For this, we will recapitulate the EC-CM crosstalk in LMNA iPSC-derived cells with 3D engineered heart tissues (EHTs). In Aim 2: we will decipher the mechanism of EC-CM crosstalk in LMNA iPSC-derived EHTs using single-cell approaches (scRNA-seq and scATAC-seq). In Aim 3: we will validate the key regulatory players of EC-CM crosstalk in LMNA cardiomyopathy by using CRISPR technology and zebrafish animal model. We have provided compelling preliminary data to support the soundness of our hypothesis-driven research proposal, and we are well positioned to achieve the project goals within five years. If successful, our studies will provide a new paradigm for understanding the pathogenesis and treatment of familial DCM.

项目总结/摘要 扩张型心肌病（DCM）是心力衰竭的主要原因，也是心脏病的主要原因。 移植我们对扩张型心肌病的病理生理学和心肌细胞中的突变的理解存在重大差距。 编码核被膜蛋白层蛋白A和C（LMNA）的基因被认为是最重要的基因 DCM的常见原因然而，“心肌病”的分子机制仍然存在， 难以捉摸，目前还不清楚为什么这种普遍表达的基因中的突变具有如此不成比例的表达。 对心脏的影响使用诱导多能干细胞（iPSC）衍生的内皮细胞（iPSC-ECs），我们 最近研究了一个家族，该家族由于LMNA中的移码变异而受到DCM的影响， 功能障碍（Sayed et al. Science Translational Medicine，2020）。这种EC功能障碍可以通过以下方式逆转： 通过用他汀类药物的子集处理iPSC-EC来上调Krüppel样因子2（KLF 2），包括 洛伐他汀。重要的是，EC功能障碍的这种改善对共培养的iPSC-100有积极的影响。 心肌细胞（iPSC-CM），表明EC之间存在复杂的串扰 和CMs在LMNA心肌病中的作用 尽管取得了令人印象深刻的进展，但很少有人注意到细胞间相互作用的潜在重要性。 尽管EC具有旁分泌功能以增强EC和CM之间的信号传导， CM，特别是在药理学刺激的背景下。这种知识差距阻碍了我们的全面 在多细胞水平上理解器官功能障碍。我们建议的首要目标是使用一个 多学科方法，整合人类iPSC，生物工程工具，基因组编辑和NGS， 获得对DCM发病机制的新见解。使用人类iPSC，我们建议破译受损的 LMNA心肌病中EC和CM之间的串扰，并阐明他汀类药物的有益类效应 改善EC-CM信号传导作为调节心脏功能的关键因素。我们将追踪三个具体的 目标。目的一：建立一个研究基因型-表型关联的实验平台， EC和CM上的LMNA突变。为此，我们将概括LMNA iPSC衍生物中的EC-CM串扰。 细胞与3D工程心脏组织（EHT）。在目标2中：我们将破译EC-CM串扰的机制 在LMNA iPSC衍生的EHT中使用单细胞方法（scRNA-seq和scATAC-seq）。目标3：我们将 通过使用CRISPR技术验证LMNA心肌病中EC-CM串扰的关键调控因素 和斑马鱼动物模型。我们提供了令人信服的初步数据，以支持 我们的假设驱动的研究建议，我们有能力实现项目目标 五年内如果成功的话，我们的研究将为理解 家族性扩张型心肌病的发病机制和治疗。