Deciphering the Endothelial Cell-Cardiomyocyte Crosstalk in LMNA Cardiomyopathy

项目摘要/摘要 扩张型心肌病(DCM)是心力衰竭的主要原因，也是心脏的主要原因 移植。在我们对扩张型心肌病的病理生理学和基因突变的理解上存在重大差距。 编码核膜蛋白LMNA(LMNA)的基因被认为是最多的 DCM的常见原因。然而，“心氨酸病”背后的分子机制仍然存在。 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不成比例的 对心脏的影响。使用诱导多能干细胞(IPSCs)来源的内皮细胞(IPSC-ECs)，我们 最近研究了一个受扩张型心肌病影响的家庭，其原因是LMNA中的一个移码变异，显示内皮细胞 功能障碍(Sayed等人《科学转化医学》，2020)。这种EC功能障碍可以通过以下方式逆转 用他汀类药物亚群处理IPSC-EC上调Krüppel样因子2(KLF2) 洛伐他汀。重要的是，EC功能障碍的这种改善对共同培养的IPSC- 心肌炎患者的心肌细胞(IPSC-CMS)，表明ECs之间存在复杂的串扰 LMNA心肌病的CMS。 尽管取得了令人印象深刻的进展，但几乎没有人注意到细胞间的潜在重要性。 ECs和CMS之间的信号传递，尽管ECs具有旁分泌功能，以增强 CMS，特别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，我们处于实现项目目标的有利地位 在五年内。如果成功，我们的研究将提供一个新的范式来理解 家族性扩张性心肌病的发病机制及治疗。

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.