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

摘要 肥厚型心肌病（HCM）是最常见的孟德尔遗传性心脏病， 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 MYBPC 3突变。大多数MYBPC 3突变导致过早的蛋白质截短，但特定的MYBPC 3突变导致蛋白质截短。 这些突变导致肥大和心律失常的机制尚不清楚。这些突变可以 导致功能丧失（单倍不足），但也可能产生截短的显性负效应 MYBPC 3蛋白。我以前的工作已经证明了MYBPC 3在转录水平上的增加， 蛋白质丰度的差异，反驳功能丧失假说。我在初步研究中进一步表明， 数据表明，截短的MYBPC 3蛋白表现出显性负效应的能力，因为它们能够 并入心脏肌节中，但错误定位并对收缩性产生负面影响。我假设 MYBPC 3中的截短突变发挥基因型特异性显性负效应，损害肌节 组织，易患心律失常，并激活肥大信号。我们将探讨这一假设 有三个具体的目标，利用人类诱导多能干细胞衍生的心肌细胞， （hiPSC-CM）来研究MYBPC 3突变的早期后果，以及小鼠模型来研究MYBPC 3突变的晚期后果。 MYBPC 3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系统来创建除了以下等位基因谱之外在遗传上相同的品系： 三个特定的MYBPC 3突变使用改良的细胞培养基质解决HiPSC-CM不成熟问题 和微图案化技术。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。第二个目的是比较杂合MYBPC 3敲除 小鼠模型与杂合MYBPC 3敲入（截短突变）小鼠模型，其直接 第一个目标中hiPSC-CM模型的推论，并将在6个月时比较类似的体内 反映慢性不良重塑的表型。第三个目的是探讨钙调神经磷酸酶的假设- CaMKII信号通路在肥大和心律失常易感性的发病机制中至关重要， MYBPC 3中的截短突变，这得到了我对人类HCM中该途径的初步数据的支持。的 该提案将提供令人信服的证据，证明截短的MYBPC 3显性负效应作为一种 HCM中肌节功能障碍、心律失常和钙处理不当的机制上游原因。 此外，这些发现将产生迫在眉睫的临床影响，因为截短MYBPC 3突变是最重要的。 HCM的常见遗传原因，因此这项研究的结果具有很高的影响未来的潜力。 基因型特异性治疗开发。拟议的项目和职业发展计划也将是一个 优秀的培训工具，以实现我的长期职业目标，成为一个独立的调查员， 将领导一个多学科团队，以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.