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

基本信息

批准号：
10688257
负责人：
Nazish Sayed
金额：
$ 39.42万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
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/10688257
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 Chromosome Mapping Coculture Techniques Collaborations Communication Complex Computational Biology Data Dilated Cardiomyopathy Disease Endothelial Cells Environment Family Functional disorder Gene Deletion Gene Expression Genes Genomic DNA Genotype Goals Heart Heart Diseases Heart Transplantation Heart failure Human Impairment In Vitro Individual Kinetics Knowledge Lamin Type A Ligands Lovastatin Machine Learning Mediating Modeling Molecular Morbidity - disease rate Mutation Nuclear Envelope Oligonucleotides Organ Paracrine Communication Pathogenesis Pathogenicity Patients Phenotype Positioning Attribute Research Proposals Signal Transduction Site Technology Tissue Engineering Translational Research Variant Wing Work Zebrafish cardiac tissue engineering cell type endothelial dysfunction env Gene Products experimental study familial dilated cardiomyopathy gene regulatory network genome editing heart function improved in vivo induced pluripotent stem cell induced pluripotent stem cell derived cardiomyocytes induced pluripotent stem cell technology insight interdisciplinary approach knock-down lamin C loss of function mechanical properties mortality next generation sequencing novel paracrine pharmacologic promoter receptor single cell technology single-cell RNA sequencing sound stem cell biology tool transcription factor translational medicine

项目摘要

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.

项目概要/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我们对 DCM 的病理生理学和突变的理解存在重大差距。编码核膜蛋白核纤层蛋白 A 和 C (LMNA) 的基因被认为是最重要的。然而，“心肌胺病”的分子机制仍然存在。难以捉摸，并且尚不清楚为什么这个普遍表达的基因中的突变具有如此不成比例的使用诱导多能干细胞 (iPSC) 衍生的内皮细胞 (iPSC-EC)，我们研究了对心脏的影响。最近研究了一个因 LMNA 移码变异而受到 DCM 影响的家庭，该变异显示内皮细胞功能障碍（Sayed 等人，Science Translational Medicine，2020）。通过用他汀类药物的子集治疗 iPSC-EC 来上调 Krüppel 样因子 2 (KLF2)，包括重要的是，EC 功能障碍的改善对共培养的 iPSC 具有积极作用。来自心肌病患者的心肌细胞 (iPSC-CM)，表明 EC 之间存在复杂的串扰和 LMNA 心肌病中的 CM。尽管取得了令人印象深刻的进展，但很少有人关注细胞间的潜在重要性尽管 EC 具有旁分泌功能以增强 EC 和 CM 之间的信号传导 CM，特别是在药理刺激方面，这种知识差距阻碍了我们的全面了解。在多细胞水平上理解器官功能障碍我们建议的总体目标是使用整合人类 iPSC、生物工程工具、基因组编辑和 NGS 的多学科方法我们建议使用人类 iPSC 来破译受损的 DCM 发病机制。 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别效应在改善 EC-CM 信号传导作为调节心脏功能的关键因素方面，我们将追求三个具体目标。目标1：我们将建立一个实验平台来研究基因型-表型关联。 EC 和 CM 上的 LMNA 突变为此，我们将概括 LMNA iPSC 衍生的 EC-CM 串扰。目标 2：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）在目标 3 中：我们将利用 CRISPR 技术验证 LMNA 心肌病中 EC-CM 串扰的关键调控因子我们提供了令人信服的初步数据来支持其合理性。我们的假设驱动的研究提案，我们有能力实现项目目标如果成功的话，我们的研究将在五年内提供一个新的范式来理解这一点。家族性 DCM 的发病机制和治疗。