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

摘要 肥厚型心肌病是孟德尔人最常见的遗传性心脏病，可 并发心力衰竭、心律失常和猝死。超过60%的基因定义的HCM是由于 MYBPC3基因突变。大多数MYBPC3突变会导致蛋白质过早截断，但特定的 这些突变导致肥大和心律失常的机制尚不清楚。这些突变可能 导致功能丧失(单倍体功能不全)，但也可能因截断而产生显性负面影响 MYBPC3蛋白。我之前的工作表明，MYBPC3在记录水平上有所增加，而不是 蛋白质丰度的差异，反驳了功能丧失假说。我已经在预赛中进一步展示了 截短MYBPC3蛋白的数据显示出具有显性负效应的能力，因为它们能够 合并到心肌肌节，但错误定位并对收缩产生负面影响。我假设 MYBPC3基因截短突变对肌节产生基因特异性显性负效应 组织，易发生心律失常，并激活肥大信号。我们将探索这一假说 有三个特定的目的，这两个目的利用人类诱导的多能干细胞来源的心肌细胞 (hiPSC-CMS)研究MYBPC3突变的早期后果，并研究晚期小鼠模型 MYBPC3突变的后果。第一个目标是利用已经进行基因组工程的HiPSC系 使用CRISPR-Cas9系统创建除了等位基因谱之外在遗传上相同的品系 三个特定的MYBPC3突变。使用改良的细胞培养底物解决HiPSC-CM的不成熟问题 和微图案化技术。这些HiPSC-CMS将在肌节组织、收缩能力、 钙的处理和心律失常的易感性。第二个目的是比较杂合子MYBPC3基因敲除 小鼠模型与杂合子MYBPC3敲入(截断突变)小鼠模型 第一个目标的HiPSC-CM模型的推论，并将在6个月后进行体内类似的比较 反映慢性不良重塑的表型。第三个目标是探索钙调神经磷酸酶-- CaMKII信号通路在高血压肥厚和心律失常易感性的发病机制中起关键作用 截断MYBPC3的突变，这得到了我在人类HCM中这一途径的初步数据的支持。这个 该提案将提供令人信服的证据，证明截断的MYBPC3显性负面效应作为 肥厚性心肌病肌节功能障碍、心律失常和钙离子处理不当的上游机械性原因。 此外，这些发现将产生迫在眉睫的临床影响，因为截断MYBPC3突变是最 肥厚型心肌病的共同遗传原因，因此这项研究的结果对未来有很大的影响潜力 特定于基因型别的治疗发展。拟议的项目和职业发展计划也将是 优秀的培训载体，实现了我长期的职业目标，成为一名独立的调查员 将领导一个多学科团队，更好地了解和治疗遗传性心脏病。

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.