Investigating Lmod2 cardiomyopathy using human iPSC-derived cardiomyocytes

项目摘要/摘要 横纹肌细胞收缩依赖于肌球蛋白(粗)丝和肌动蛋白(细)的适当重叠。 细丝。Leiomodin(Lmod)和Tmod(Tmod)是与薄的尖端结合的蛋白质 细丝，以便微调其长度。这些蛋白质的突变已被证明会导致 细丝长度失调、肌节解体和心肌病的发展。然而， 它们如何促进这一疾病过程的机制在很大程度上尚不清楚。最近，第一个致病的 在一名人类中发现了Lmod2的突变。发现一名新生儿患者有一种纯合子 LMOD2(c.1193G和gt；A，p.Trp398*)的无义突变，预计会导致基本上截断的 蛋白。临床上，患者在出生时出现心脏异常，并在 10个月大。移植的心脏组织证实了扩张型心肌病的诊断。 这项研究建议的主要目的是了解这种突变对 心脏的结构和功能，长期目标是阐明潜在的治疗选择 Lmod2介导的心功能不全。为了做到这一点，将利用各种实验方法在体外和 在体内解决Lmod2突变导致心脏功能障碍和心脏功能改变的假说 肌节结构，由于肌动蛋白细丝的失调。为了正确地破译心脏 对于这种人类无义突变的影响，将使用两个成熟的模型系统：(1)人类诱导 多能干细胞来源的心肌细胞(IPSC-CMS)和(2)设计的一种新的CRISPR 带有与患者相同突变的敲入小鼠模型。来自这两个系统的变化 Lmod2的表达、结构和功能特性将通过以下生化方法推导出来 分析和功能分析：首先，将分析肌节的亚细胞结构和肌动蛋白- 用免疫细胞化学方法测量患者IPSC-CMS和CRISPR/Cas9的细丝长度 基因编辑的等基因对照。第二，钙和电压敏感的荧光探针将提供 细胞内钙动员的信息和单细胞电记录的变化。在……里面 附加RNA测序将深入了解Lmod2 p.Trp398*突变对肉瘤的影响 转录组网络。第三，从突变小鼠身上摘除的心脏组织将被用于研究肌节。 结构通过免疫组织化学和力/钙的关系通过孤立的单纤维力学。 了解肌动蛋白细丝组装是如何调控的具有广泛的兴趣，因为肌动蛋白是最丰富的 蛋白质存在于多种细胞类型中，并参与许多重要的细胞过程。从以下方面获得的结果： 这个多学科的项目可能会破译Lmod2的一个单一突变是如何导致人类 心肌病。它还将拓宽我们对肌动蛋白细丝结构和组装动力学的了解， 据预测，它们的影响超出了心肌。

PROJECT SUMMARY/ABSTRACT Striated muscle cell contraction is dependent on the proper overlap of myosin (thick) filaments and actin (thin) filaments. Leiomodin (Lmod) and tropomodulin (Tmod) are proteins that bind to the pointed end of thin filaments in order to fine-tune their lengths. Mutations in these proteins have been shown to result in dysregulated thin filament lengths, sarcomere disassembly and the development of cardiomyopathies. Yet, the mechanism for how they contribute to this disease process is largely unknown. Recently, the first pathogenic mutation in Lmod2 was identified in a human. It was discovered that a newborn patient had a homozygous nonsense mutation in LMOD2 (c.1193G>A, p.Trp398*), which is predicted to result in a substantially truncated protein. Clinically, the patient presented with cardiac abnormalities at birth and received a heart transplant at 10 months of age. Explanted heart tissue confirmed the diagnosis of dilated cardiomyopathy. The main objective of this research proposal is to understand the consequences of this mutation on the structure and function of the heart, with the long-term goal of elucidating potential therapeutic options for Lmod2-mediated cardiac dysfunction. To do this, various experimental approaches will be utilized in vitro and in vivo to address the hypothesis that mutations in Lmod2 result in cardiac dysfunction and alterations in sarcomere structure, due to dysregulation of actin-thin filaments. In order to properly decipher the cardiac effects of this human nonsense mutation, two well-established model systems will be used: (1) human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from the patient and (2) a novel CRISPR designed knock-in mouse model harbouring the same mutation as the patient. From these two systems, alterations in the expression, structure and functional properties of Lmod2 will be deduced through the following biochemical analyses and functional assays: First, the subcellular structure of the sarcomere will be analyzed and actin- thin filament lengths measured using immunocytochemistry from the patient's iPSC-CMs and CRISPR/Cas9 gene edited isogenic controls. Second, calcium and voltage sensitive fluorescent probes will provide information on intracellular calcium mobilization and changes in single cell electrical recordings, respectively. In addition RNA sequencing will give insight into the effects of the Lmod2 p.Trp398* mutation on sarcomeric transcriptome networks. Third, excised heart tissue from mutant mice will be used to study sarcomere architecture via immunohistochemistry and force/Ca2+ relationships via isolated single-fiber mechanics. Understanding how actin filament assembly is regulated is of broad interest since actin is the most abundant protein in many cell types and is involved in numerous essential cellular processes. The results obtained from this multidisciplinary project will likely decipher how a single mutation in Lmod2 can lead to human cardiomyopathy. It will also broaden our knowledge about actin filament structure and assembly dynamics, which are predicted to have implications beyond cardiac muscle.