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：
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
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/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

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.

项目摘要/摘要扩张的心肌病（DCM）是心力衰竭的主要原因，也是心脏的主要原因移植。我们对DCM的病理生理学和突变的理解存在主要差距编码核包膜蛋白层层粘连蛋白A和C（LMNA）的基因被认为是最大的 DCM的常见原因。但是，“心脏疾病”基础的分子机制仍然存在难以捉摸，尚不清楚为什么在这个普遍表达的基因中的突变如此不成比例对心脏的影响。使用诱导的多能干细胞（IPSC）衍生的内皮细胞（IPSC-EC），我们最近由于LMNA中的移码变体而研究了一个受DCM影响的家庭，该家庭显示了内皮功能障碍（Sayed等人，科学转化医学，2020年）。这种EC功能障碍可能会被通过以汀类药物为包括的IPSC-EC来上调Krüppel样因子2（KLF2），包括他汀类药物 lovastatin。重要的是，EC功能障碍的这种改善对共培养的IPSC-有积极影响心脏瘤患者的心肌细胞（IPSC-CMS），表明ECS之间存在复杂的串扰和LMNA心肌病中的CMS。尽管进步令人印象深刻，但很少关注细胞到细胞的潜在重要性 ECS和CMS之间的信号传导，DOS EC具有旁分泌功能以增强信号传导的事实 CM，尤其是在药物刺激的背景下。这种知识差距阻碍了我们的全面在多细胞水平上了解器官功能障碍。我们建议的总体目标是使用多学科的方法将人类IPSC，生物工程工具，基因组编辑和NGS整合到对DCM的发病机理获得新的见解。使用人IPSC，我们建议破译受损的 LMNA心肌病中的EC和CMS之间的串扰，并阐明了他汀类药物的有益类作用在改善EC-CM信号作为控制心脏功能的关键因素时。我们将追求三个特定的目标。在AIM 1中：我们将建立一个实验平台来研究 EC和CMS上的LMNA突变。为此，我们将概括以LMNA IPSC衍生的EC-CM串扰具有3D工程心组织（EHT）的细胞。在AIM 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）。在目标3：我们将通过使用CRISPR技术来验证LMNA心肌病中EC-CM串扰的关键监管参与者和斑马鱼动物模型。我们提供了引人入胜的初步数据，以支持我们的假设驱动的研究建议，我们在实现项目目标方面处于良好状态五年之内。如果成功，我们的研究将为理解家族DCM的发病机理和治疗。