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.

项目概要家长 R01 总结：扩张型心肌病 (DCM) 是心力衰竭的主要原因，也是导致心力衰竭的主要原因我们对 DCM 的病理生理学的理解存在重大差距。编码核膜蛋白核纤层蛋白 A 和 C (LMNA) 的基因突变被认为是 DCM 的最常见原因，然而，“心肌胺病”的分子机制。仍然难以捉摸，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大的影响使用诱导多能干细胞 (iPSC) 衍生的内皮细胞对心脏产生不成比例的影响。 (iPSC-ECs)，我们最近研究了一个因 LMNA 中的移码变异而受到 DCM 影响的家族，结果表明内皮功能障碍（Sayed et al. Science Translational Medicine, 2020）。通过用他汀类药物的子集治疗 iPSC-EC，上调 Krüppel 样因子 2 (KLF2) 来逆转，重要的是，EC 功能障碍的改善对共培养产生了积极影响。来自心肌病患者的 iPSC 心肌细胞 (iPSC-CM)，表明细胞之间存在复杂的串扰 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 EC 具有旁分泌功能以增强 CM 中的信号传导，尤其是在药理学刺激方面，这种知识差距阻碍了我们的全面发展。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我们的合理性。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疾病的发病机制和治疗提供新的范例家族性 DCM。拟议补充：占美国人口 12.1% 的非裔美国人社区与其他种族/族裔相比，美国第二大种族/族裔群体。在所有群体中，非裔美国人的心力衰竭 (HF) 发病率和患病率最高，并且此外，与白人相比，他们的风险增加了约 3 倍。发展为扩张型心肌病 (DCM)，诊断后死亡风险增加约 2 倍通过社会经济地位和高血压来解释。在拟议的多样性补充中，我们将扩展。我们的母体 R01 的范围专门包括非裔美国人队列中的其他患者具体来说，我们将调查另外 10 名个人，这笔补助金的目标是调查非裔美国人社区的情况。变异（特别是 LMNA 基因）对心脏组织的影响，并确定细胞类型特异性为此，我们将生成 3D 工程心脏组织 (EHT)。来自非裔美国患者的 iPSC-EC 和 iPSC-CM 来表征 LMNA 突变的影响这些生成的 EHT 将在单细胞水平上进行研究，以破译其功能。转录组景观和 EC 功能障碍对 CM 的影响此外，高通量亲和力 - 将进行基于蛋白质组学的研究，以确定任何可能涉及 EC-CM 串扰的分泌因子 LMNA DCM 实验将由 Naima Turbes 女士进行。