Genome-Engineered Stem Cell Models to Determine Disease Mechanisms in MYBPC3 Hypertrophic Cardiomyopathy

基因组工程干细胞模型确定 MYBPC3 肥厚性心肌病的疾病机制

• 批准号: 9321380
• 负责人: ADAM S HELMS
• 金额: $ 16.33万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9321380
• 关键词: Acute; Address; Affect; Alleles; Arrhythmia; Backcrossings; Biological Models; CRISPR/Cas technology; Calcineurin; Calcium; Calcium Signaling; Cardiac; Cardiac Myocytes; Cardiac Myosins; Cell Culture Techniques; Cell Line; Cell model; Cells; Chronic; Clinical; Data; Development; Development Plans; Disease; Dominant-Negative Mutation; Electrophysiology (science); Etiology; Functional disorder; Future; Genes; Genetic; Genome; Genome engineering; Genotype; Goals; Heart; Heart Abnormalities; Heart Diseases; Heart failure; Human; Hypertrophic Cardiomyopathy; Hypertrophy; Impairment; Induced Mutation; Inherited; Knock-in; Knock-in Mouse; Knock-out; Knockout Mice; Knowledge; Lead; Link; Measures; Modeling; Mutation; Myocardium; Myopathy; Organ; Pathogenesis; Pathway interactions; Patients; Phenotype; Phosphotransferases; Population; Predisposition; Production; Protein Truncation; Proteins; Research Personnel; Role; Sarcomeres; Signal Pathway; Signal Transduction; Stem cells; Stimulus; Structure; Sudden Death; System; Techniques; Testing; Therapeutic; Tissue Sample; Training; Transcript; Up-Regulation; Work; calmodulin-dependent protein kinase II; career; career development; citrate carrier; disorder control; heart rhythm; human disease; human stem cells; in vivo; induced pluripotent stem cell; inhibitor/antagonist; loss of function; mouse model; multidisciplinary; mutant; myosin-binding protein C; premature; skills; therapeutic development; transcriptome sequencing

ABSTRACT Hypertrophic cardiomyopathy (HCM) is the most common Mendelian inherited cardiac disease and can be complicated by heart failure, arrhythmias, and sudden death. Over 60% of genetically-defined HCM is due to mutations in MYBPC3. Most MYBPC3 mutations cause premature protein truncations, but the specific mechanisms by which these mutations lead to hypertrophy and arrhythmias is elusive. These mutations may lead to loss of function (haploinsufficiency) but may also exert dominant negative effects from truncated MYBPC3 protein. My previous work has demonstrated an increase in MYBPC3 at the transcript level and no difference in protein abundance, countering the loss of function hypothesis. I have further shown in preliminary data that truncated MYBPC3 proteins demonstrate a capacity for dominant negative effects since they are able to incorporate in the cardiac sarcomere but mislocalize and negatively influence contractility. I hypothesize that truncating mutations in MYBPC3 exert genotype-specific dominant negative effects that impair sarcomere organization, predispose to arrhythmia, and activate hypertrophic signalling. The hypothesis will be explored with three specific aims, which leverage both human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) to investigate early consequences of MYBPC3 mutations, and mouse models to investigate late consequences of MYBPC3 mutations. The first aim utilizes hiPSC lines that have been genome-engineered using the CRISPR-Cas9 system to create lines that are genetically identical except for an allelic spectrum of three specific MYBPC3 mutations. HiPSC-CM immaturity is addressed using modified cell culture substrates and micropatterning techniques. These hiPSC-CMs will be compared for sarcomere organization, contractility, calcium handling, and arrhythmia susceptibility. The second aim compares a heterozygous MYBPC3 knock-out mouse model with a heterozygous MYBPC3 knock-in (truncating mutation) mouse model, which are direct corollaries for the hiPSC-CM models in the first aim, and will be compared at 6 months for analogous in vivo phenotypes that reflect chronic adverse remodeling. The third aim explores the hypothesis that the calcineurin- CaMKII signaling pathway is critical in the pathogenesis of hypertrophy and arrhythmia susceptibility due to truncating mutations in MYBPC3, as supported by my preliminary data for this pathway in human HCM. The proposal will provide convincing evidence for the role of truncated MYBPC3 dominant negative effects as a mechanistic upstream cause of sarcomere dysfunction, arrhyhthmias, and calcium mishandling in HCM. Furthermore, the findings will have imminent clinical impact since truncating MYBPC3 mutations are the most common genetic cause of HCM, and therefore results of this study have high potential for influencing future genotype-specific therapeutic development. The proposed project and career development plan will also be an excellent training vehicle to achieve my long-term career goal of becoming an independent investigator who will lead a multidisciplinary team to better understand and treat inherited heart disease.

抽象的 肥厚性心肌病（HCM）是最常见的孟德尔遗传性心脏病，可以是 因心力衰竭，心律不齐和猝死而复杂。超过60％的遗传定义的HCM是由于 MYBPC3中的突变。大多数MyBPC3突变会导致过早的蛋白质截断，但特定于 这些突变导致肥大和心律不齐的机制难以捉摸。这些突变可能 导致功能丧失（单倍弥补），但也可能造成截短的显着负面影响 mybpc3蛋白。我以前的工作表明在笔录级别上有MYBPC3的增加，没有 蛋白质丰度的差异，反对功能假设的丧失。我进一步显示了 截断MyBPC3蛋白的数据显示出具有主要负面影响的能力，因为它们能够 将其纳入心脏肌膜中，但不定位并对收缩性产生负面影响。我假设这一点 MYBPC3中的截断突变发挥基因型特异性的显性负面影响，损害了肌膜 组织，心律不齐，并激活肥厚的信号传导。将探讨该假设 以三个特定的目的，它利用了两个人类诱导的多能干细胞衍生的心肌细胞 （HIPSC-CMS）研究MYBPC3突变的早期后果和小鼠模型以研究迟 MYBPC3突变的后果。第一个目标利用了经过基因组工程的HIPSC线 使用CRISPR-CAS9系统来创建遗传上相同的线，除了等位基因谱 三个特定的MyBPC3突变。 HIPSC-CM不成熟是使用修饰的细胞培养基材解决的 和微观图案。这些HIPSC-CMS将在肌节组织，收缩力， 钙处理和心律不齐的敏感性。第二个目标比较了杂合MYBPC3淘汰赛 具有杂合MYBPC3敲进（截断突变）小鼠模型的小鼠模型，该模型是直接的 第一个目标中HIPSC-CM模型的推论，将在6个月的体内进行比较 反映慢性不良重塑的表型。第三个目的探讨了钙调神经蛋白酶的假设 CAMKII信号通路对于由于 MybPC3中的截断突变，在人类HCM中该途径的初步数据支持。这 提案将提供令人信服的证据证明MybPC3截断的作用主要负面影响 HCM中的肌节功能障碍，心血症和钙不当的机械上游原因。 此外，这些发现将产生临床影响，因为截断MYBPC3突变是最大的 HCM的常见遗传原因，因此这项研究的结果具有影响未来的很高潜力 基因型特异性治疗发育。拟议的项目和职业发展计划也将是 出色的训练工具，以实现成为成为独立调查员的长期职业目标 将带领一个多学科团队更好地理解和治疗遗传的心脏病。