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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：
10851040
负责人：
Nazish Sayed
金额：
$ 9.19万
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 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10851040
关键词：
3-Dimensional Affect Affinity African American African American population Animal Model Attention Biomedical Engineering CRISPR/Cas technology Cardiac Cardiac Myocytes Cardiomyopathies Caucasians Cells Clinical Coculture Techniques Communities Complex Data Diagnosis Dilated Cardiomyopathy Disparity Endothelial Cells Ethnic Population Family Functional disorder Genes Genotype Goals Grant Heart Heart Diseases Heart Transplantation Heart failure Human Hypertension Impairment Incidence Individual Knowledge Lamin Type A Lovastatin Molecular Mutation Nuclear Envelope Organ Outcome Parents Pathogenesis Patients Phenotype Population Positioning Attribute Prevalence Proteomics Research Proposals Risk Signal Transduction Socioeconomic Status Tissues Translational Research United States Variant Zebrafish cardiac tissue engineering cell type cohort endothelial dysfunction env Gene Products ethnic minority population experimental study familial dilated cardiomyopathy genome editing heart function high risk improved induced pluripotent stem cell insight interdisciplinary approach lamin C mortality risk next generation sequencing novel paracrine pharmacologic racial minority population racial population single-cell RNA sequencing sound tool transcriptomics translational medicine

项目摘要

PROJECT SUMMARY Summary of Parent R01: 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. Proposed Supplement: The African American community, which represents 12.1% of the US population, is the second largest racial/ethnic minority group in the United States. When compared to other race/ethnic groups, African Americans have the highest incidence and prevalence of heart failure (HF) as well as the worst clinical outcomes. Moreover, when compared to Caucasians, they have a ~3-fold increased risk for developing dilated cardiomyopathy (DCM), and ~2-fold increased risk of death after diagnosis that is not explained by socioeconomic status and hypertension. In the proposed diversity supplement, we will extend the scope of our parent R01 to exclusively include additional patients from the African American cohort to understand this disparity at the molecular level. Specifically, we will investigate 10 additional individuals that belong to the African American community. The goal of this supplement grant would be to investigate the impact of variants, specifically in the LMNA gene, on the cardiac tissue and determine the cell-type specific signature responsible for this impaired function. For this, we will generate 3D engineered heart tissue (EHTs) from iPSC-ECs and iPSC-CMs from African American patients to characterize the effect of LMNA mutation on CM and EC function. These generated EHTs will be investigated at the single cell level to decipher the transcriptomic landscape and the impact of EC dysfunction on CMs. Furthermore, high-throughput affinity- based proteomics will be conducted to identify any secreted factors potentially involved in EC-CM crosstalk in LMNA DCM. The experiments will be carried out by Ms. Naima Turbes.

项目摘要父研究R 01总结：扩张型心肌病（DCM）是心力衰竭的主要原因，心脏移植的原因我们对扩张型心肌病的病理生理学的理解存在重大差距，编码核被膜蛋白核纤层蛋白A和核纤层蛋白C（LMNA）的基因突变被认为是DCM最常见的原因。然而，“心肌病”背后的分子机制仍然难以捉摸，而且还不知道为什么这种普遍表达的基因中的突变会有这样的作用。对心脏有不成比例的影响。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由于LMNA中的移码变体而受DCM影响的家族，内皮功能障碍（Sayed等人，Science Translational Medicine，2020）。这种EC功能障碍可能是通过用他汀类药物亚组处理iPSC-EC上调Krüppel样因子2（KLF 2）来逆转，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来自心肌病患者的iPSC-心肌细胞（iPSC-CM），表明 LMNA心肌病中的EC和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信号传导的潜在重要性在EC和CM之间，尽管事实上EC起到旁分泌功能以增强CM中的信号传导，特别是在药理学刺激的情况下。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。 3D工程心脏组织（EHTs）。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。我们已经提供了令人信服的初步数据，以支持我们的健全性，假设驱动的研究提案，我们有能力在五年内实现项目目标。如果成功，我们的研究将为理解糖尿病的发病机制和治疗提供新的范例。家族性DCM 建议补充：非洲裔美国人社区，占美国人口的12.1%，是美国第二大种族/少数民族群体。与其他种族/民族相比非裔美国人心力衰竭（HF）的发病率和患病率最高，最差的临床结果此外，与高加索人相比，他们的风险增加了3倍。发生扩张型心肌病（DCM），诊断后死亡风险增加约2倍，这可以用社会经济地位和高血压来解释在拟议的多样性补充中，我们将扩大我们的母研究R 01的范围仅包括来自非洲裔美国人队列的其他患者，在分子水平上理解这种差异。具体来说，我们将调查另外10名个人，属于非裔美国人社区。这笔补充补助金的目标是调查变异体，特别是LMNA基因，对心脏组织的影响，并确定细胞类型特异性负责此受损功能的签名。为此，我们将生成3D工程心脏组织（EHTs）来自非裔美国人患者的iPSC-EC和iPSC-CM，以表征LMNA突变的影响 CM和EC功能。这些生成的EHT将在单细胞水平上进行研究，以破译转录组景观和EC功能障碍对CM的影响。此外，高通量亲和- 将进行基于蛋白质组学的研究，以鉴定可能参与EC-CM串扰的任何分泌因子在LMNA DCM中。实验将由Naima Turbes女士进行。