Investigating Lmod2 cardiomyopathy using human iPSC-derived cardiomyocytes

使用人 iPSC 衍生的心肌细胞研究 Lmod2 心肌病

• 批准号: 10268159
• 负责人: Jessika Iwanski
• 金额: $ 5.01万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10268159
• 关键词: Access to Information; Actins; Address; Age-Months; Alleles; Architecture; Arrhythmia; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Models; Birth; CRISPR/Cas technology; Calcium; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell physiology; Cells; Cessation of life; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; DNA Sequence Alteration; Development; Diagnosis; Diagnostic; Dilated Cardiomyopathy; Disease; Echocardiography; Family; Fiber; Filament; Fluorescent Probes; Functional disorder; Funding; Genes; Genetic; Genotype; Goals; Heart; Heart Abnormalities; Heart Transplantation; Heart failure; Human; Human Resources; Image; Immunohistochemistry; In Vitro; Kinetics; Knock-in Mouse; Knockout Mice; Knowledge; Laboratories; Lead; Length; Life; Link; Measures; Mechanics; Mediating; Mentorship; Microfilaments; Morbidity - disease rate; Mus; Muscle Contraction; Muscle Development; Mutant Strains Mice; Mutation; Myocardial dysfunction; Myocardium; Myofibrils; Myopathy; Myosin ATPase; Newborn Infant; Nonsense Mutation; Pathogenicity; Pathology; Patients; Play; Process; Property; Protein Isoforms; Proteins; Pump; Regulation; Research; Research Proposals; Review Literature; Risk; Role; Sarcomeres; Scientist; Striated Muscles; Structure; Subcellular structure; System; Therapeutic; Thick Filament; Tissues; Translating; Tropomyosin; Ventricular; base; cell type; design; disease phenotype; early onset; exome sequencing; experience; experimental study; gene correction; heart function; immunocytochemistry; in vivo; induced pluripotent stem cell; insight; interdisciplinary approach; interest; mortality; mouse model; multidisciplinary; mutant; novel; pediatric patients; polymerization; premature; release of sequestered calcium ion into cytoplasm; transcriptome; transcriptome sequencing; tropomodulin; voltage

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.

项目总结/摘要 横纹肌细胞的收缩依赖于肌球蛋白（粗）丝和肌动蛋白（细）丝的适当重叠 细丝。Leiomodin（Lmod）和tropomodulin（Tmod）是与薄的膜的尖端结合的蛋白质。 以微调它们的长度。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 它们如何促进这种疾病过程的机制在很大程度上是未知的。最近，第一个致病的 在人类中鉴定了Lmod 2中的突变。发现一个新生儿患者有一个纯合子 LMOD 2中的无义突变（c.1193G>A，p.Trp398*），其被预测导致LMOD 2中的基本上截短的 蛋白在临床上，患者在出生时出现心脏异常，并在12岁时接受了心脏移植。 10个月大。取出的心脏组织证实了扩张型心肌病的诊断。 这项研究计划的主要目的是了解这种突变对人类的影响。 心脏的结构和功能，长期目标是阐明潜在的治疗选择， Lmod 2介导的心功能不全。为此，将在体外利用各种实验方法， Lmod 2突变导致心功能不全和心功能改变， 肌节结构，由于肌动蛋白细丝的失调。为了正确解读心脏 为了确定这种人无义突变的影响，将使用两种完善的模型系统：（1）人诱导的 来自患者的多能干细胞衍生的心肌细胞（iPSC-CM）和（2）设计的新型CRISPR 携带与患者相同突变的基因敲入小鼠模型。从这两个系统中， Lmod 2的表达、结构和功能特性将通过以下生化分析来推断。 分析和功能测定：首先，将分析肌节的亚细胞结构， 使用免疫细胞化学从患者的iPSC-CM和CRISPR/Cas9测量的细丝长度 基因编辑的同基因对照。其次，钙和电压敏感的荧光探针将提供 细胞内钙动员和单细胞电记录变化的信息。在 此外，RNA测序将深入了解Lmod 2 p.Trp398* 突变对肌节的影响， 转录组网络。第三，从突变小鼠身上切下的心脏组织将用于研究肌节 通过免疫组织化学和力/Ca 2+的关系，通过孤立的单纤维力学的结构。 了解肌动蛋白丝组装是如何调节的是广泛的兴趣，因为肌动蛋白是最丰富的 蛋白质在许多细胞类型中，并参与许多重要的细胞过程。获得的结果 这个多学科的项目可能会破译Lmod 2中的单个突变如何导致人类 心肌病这也将拓宽我们对肌动蛋白丝结构和组装动力学的了解， 这被预测会影响到心肌以外的组织。