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

基本信息

批准号：
9178315
负责人：
ADAM S HELMS
金额：
$ 16.34万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
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/9178315
关键词：
Address Affect Arrhythmia Backcrossings Biological Models CRISPR/Cas technology Calcineurin Calcium Calcium Signaling Cardiac Cardiac Myocytes Cardiac Myosins Cell Culture Techniques 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 Diseases Heart failure Human Hypertrophic Cardiomyopathy Hypertrophy 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 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

项目摘要

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.

抽象的肥厚型心肌病 (HCM) 是最常见的孟德尔遗传性心脏病，可通过并发心力衰竭、心律失常、猝死。超过 60% 的遗传性 HCM 是由于 MYBPC3 突变。大多数 MYBPC3 突变会导致蛋白质过早截短，但具体的这些突变导致肥厚和心律失常的机制尚不清楚。这些突变可能导致功能丧失（单倍体不足），但也可能因截短而产生显着的负面影响 MYBPC3 蛋白。我之前的工作表明 MYBPC3 在转录本水平上有所增加，但没有蛋白质丰度的差异，反驳了功能丧失的假说。我在初步中进一步表明截短的 MYBPC3 蛋白的数据显示出显性负面影响的能力，因为它们能够融入心脏肌节，但定位错误并对收缩力产生负面影响。我假设 MYBPC3 的截短突变会产生基因型特异性显性负面效应，损害肌节组织，易发生心律失常，并激活肥厚的信号传导。假设将被探索具有三个具体目标，利用人类诱导多能干细胞衍生的心肌细胞（hiPSC-CM）研究 MYBPC3 突变的早期后果，小鼠模型研究晚期 MYBPC3 突变的后果。第一个目标是利用经过基因组工程改造的 hiPSC 系使用 CRISPR-Cas9 系统创建除等位基因谱外其他基因均相同的品系三个特定的 MYBPC3 突变。使用改良的细胞培养基质解决 HiPSC-CM 不成熟问题和微图案技术。将比较这些 hiPSC-CM 的肌节组织、收缩性、钙处理和心律失常易感性。第二个目标比较杂合 MYBPC3 敲除具有杂合 MYBPC3 敲入（截短突变）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，并将在 6 个月后与体内类似模型进行比较反映慢性不良重塑的表型。第三个目标探讨了钙调神经磷酸酶的假设 CaMKII 信号通路在肥厚和心律失常易感性的发病机制中至关重要截断 MYBPC3 中的突变，这一点得到了我在人类 HCM 中这一通路的初步数据的支持。这该提案将为截断的 MYBPC3 显性负面效应作为 HCM 中肌节功能障碍、心律失常和钙处理不当的上游机械原因。此外，由于截短 MYBPC3 突变是最常见的，因此这些发现将产生迫在眉睫的临床影响。 HCM 的常见遗传原因，因此这项研究的结果具有很大的影响未来的潜力基因型特异性治疗的开发。拟议的项目和职业发展计划也将是优秀的培训工具，可实现我成为一名独立调查员的长期职业目标将领导一个多学科团队更好地了解和治疗遗传性心脏病。