Deciphering the Endothelial Cell-Cardiomyocyte Crosstalk in LMNA Cardiomyopathy

项目总结 父母R01摘要：扩张型心肌病(DCM)是心力衰竭的主要原因，而 心脏移植的原因。我们对扩张型心肌病的病理生理学和 编码核膜蛋白LMNA(LMNA)的基因突变被认为是 是DCM最常见的原因。然而，“心氨酸病”背后的分子机制 仍然难以捉摸，也不知道为什么这种无处不在的表达基因的突变会有如此 对心脏有不成比例的影响。使用诱导多能干细胞(IPSCs)来源的内皮细胞 (IPSC-ECs)，我们最近研究了一个由于LMNA移码变体而受到DCM影响的家庭，结果显示 内皮功能障碍(Sayed等人《科学转化医学》，2020)。这种EC功能障碍可能是 通过用他汀类药物的子集处理IPSC-ECs上调Krüppel样因子2(KLF2)而逆转， 包括洛伐他汀。重要的是，这种EC功能障碍的改善对共培养有积极的影响 IPSC-心肌炎患者的心肌细胞(IPSC-CMS)，表明 LMNA心肌病的ECs和CMS 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信号转导的潜在重要性。 在ECs和CMS之间，尽管ECs在CMS中发挥旁分泌功能以增强信号传递， 尤其是在药物刺激的背景下。这一知识差距阻碍了我们全面的 从多细胞水平理解器官功能障碍。我们提案的首要目标是使用 集成人类IPSCs、生物工程工具、基因组编辑和NGS的多学科方法 对扩张型心肌病的发病机制有了新的认识。使用人类的ipscs，我们建议破译受损的人 LMNA心肌病患者ECs和CMS之间的相互作用及他汀类药物的有益等级效应 在改善EC-CM信号转导作为调节心脏功能的关键因素。我们将追求三个具体的目标 目标。在目标1：我们将建立一个实验平台来研究基因和表型之间的关联 ECs和CMS上的LMNA突变。为此，我们将总结一下LMNA iPSC派生的EC-CM串扰 细胞与3D工程心脏组织(EHTS)。目标2：我们将破译EC-CM串扰的机制 在LMNA中，IPSC使用单细胞方法(scRNA-seq和scATAC-seq)衍生EHTS。在目标3中：我们将 应用CRISPR技术验证LMNA心肌病EC-CM串扰的关键调控因子 和斑马鱼动物模型。我们已经提供了令人信服的初步数据来支持我们的 假设驱动的研究提案，我们处于有利地位，可以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式。 家族性扩张性心肌病。 拟议补充：占美国人口12.1%的非洲裔美国人社区 美国第二大种族/少数民族群体。与其他种族/民族相比 在这些群体中，非裔美国人心力衰竭(HF)的发病率和流行率最高， 最糟糕的临床结果。此外，与高加索人相比，他们患癌症的风险增加了约3倍 罹患扩张型心肌病(DCM)，确诊后死亡风险增加~2倍 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文件中，我们将延长 我们的母公司R01的范围仅包括来自非洲裔美国人队列的额外患者 在分子水平上理解这种差异。具体地说，我们将调查另外10名 属于非裔美国人社区。这项补充拨款的目标将是调查 变异，特别是在LMNA基因中，对心脏组织的影响，并确定细胞类型特异性 对此功能受损负责的签名。为此，我们将产生3D工程化心脏组织(EHTS) 从非洲裔美国人的IPSC-ECs和IPSC-CMS中鉴定LMNA突变的作用 关于CM和EC的功能。这些产生的超高温超导将在单细胞水平上进行调查，以破译 转录剪除格局和EC功能障碍对CMS的影响。此外，高吞吐量亲和力- 将进行基于蛋白质组学的研究，以确定任何可能涉及EC-CM串扰的秘密因素 在LMNA DCM中。实验将由纳伊玛·图布斯女士进行。

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.