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: 10421080
• 负责人: Jessika Iwanski
• 金额: $ 5.08万
• 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/10421080
• 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; 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; induced pluripotent stem cell derived cardiomyocytes; 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.

项目摘要/摘要 条纹肌肉细胞的收缩取决于肌球蛋白（厚）丝的适当重叠和肌动蛋白（薄） 细丝。 Leiomodin（LMOD）和Tropomodulin（TMOD）是与薄尖端结合的蛋白质 细丝以微调其长度。这些蛋白质中的突变已显示导致 失调的细丝长度，肌膜拆卸和心肌病的发展。但是， 它们如何促进这种疾病过程的机制在很大程度上尚不清楚。最近，第一个病原体 在人类中发现了LMOD2中的突变。发现新生儿患有纯合子 LMOD2（C.1193G> a，p.trp398*）中的废话突变，预测会导致基本截断 蛋白质。临床上，患者出生时出现心脏异常，并接受了心脏移植 10个月大。外植的心脏组织证实了扩张心肌病的诊断。 该研究建议的主要目的是了解该突变对 心脏的结构和功能，其长期目标是阐明潜在的治疗选择 LMOD2介导的心脏功能障碍。为此，将在体外使用各种实验方法 体内解决了以下假设：LMOD2中的突变导致心脏功能障碍和改变 肌节结构，由于肌动蛋白细丝的失调。为了正确破译心脏 这种无意义的突变的影响，将使用两个建立的模型系统：（1）人类诱导的 来自患者的多能干细胞衍生的心肌细胞（IPSC-CM）和（2）新型CRISPR设计 敲入与患者相同突变的小鼠模型。从这两个系统中，改变 LMOD2的表达，结构和功能特性将通过以下生化推导 分析和功能分析：首先，将分析肌动蛋白的亚细胞结构 使用患者IPSC-CMS和CRISPR/CAS9的免疫细胞化学测量的细丝长度 基因编辑的同源控制。其次，钙和电压敏感荧光探针将提供 有关细胞内钙动员和单细胞电记录的变化的信息。在 添加的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，突变小鼠切除的心脏组织将用于研究肌膜 通过免疫组织化学和力/Ca2+关系通过孤立的单纤维力学进行体系结构。 了解肌动蛋白细丝组件的调节是广泛的，因为肌动蛋白是最丰富的 许多细胞类型中的蛋白质，参与了许多必需的细胞过程。从 这个多学科项目可能会破译LMOD2中的单个突变如何导致人类 心肌病。它还将扩大我们对肌动蛋白丝结构和组装动态的了解， 预计这具有超出心脏肌肉的影响。