Mechanisms of cardiomyocyte dysfunction due to the E258K-MYBPC3 mutation modeled in patient-derived cardiomyocytes

肥厚型心肌病(HCM)是最常见的遗传性心脏病，其特点是进行性 左室壁增厚和心源性猝死的可能性。25%的HCM 肌节蛋白心肌肌球蛋白结合蛋白-C(cMyBP-C)发生突变。目前，没有 治疗肥厚性心肌梗死，仅对症状和疾病进展进行管理，左心室梗阻手术，或 心脏移植。因此，很有必要更好地了解不充分的病理机制 特定的HCM突变，以便更好地为靶向治疗的发展提供信息。对于这个项目，我将 研究cMyBP-C，c.772G&gt；A(p.E258K)的一个高穿透性突变，它在 意大利托斯卡纳东北部地区。为了更好地探索突变的直接影响，我产生了患者 6例携带E258K突变的HCM患者和1例代表性患者的诱导多能干细胞(IPSCs) 使用CRISPR/Cas9通过纠正突变获得等基因细胞系。已经对以下方面进行了初步研究 然而，上述6名携带E258K突变的肥厚型心肌病患者中有3名患者的肌切取样本 组织是有限的，并提供来自疾病晚期的结果。利用我们的耐心IPSC线路，我可以做更多 从几乎无限的组织特异性细胞中彻底探索HCM的潜在机制。我建议 为了研究多个患者来源的具有相同E258K突变的IPSC系，允许我探索 E258K突变的机制以及性别和年龄等其他因素如何 发病时间的长短可能影响上述机制。在卡雷吉大学医院的E258K患者队列中， 肌切除术样本显示全长cMyBP-C蛋白的表达一直较低，表明 潜在的单倍体功能不全疾病机制。在肌节水平上，肌肉切除样本表明 加速跨桥骑行，伴随着更大的紧张产生的能量成本。已被占用 总之，我假设E258K突变破坏了cMyBP-C招募和调节cMyBP-C的能力 肌球蛋白，导致肌节cMyBP-C表达和/或掺入减少 (单倍体功能不全)和2)在收缩过程中使肌节进入过度利用ATP的状态 (精力充沛的低效)为了验证这一假设，我将使用我们的患者IPSCs分化为心肌细胞， 和它们的等基因控制线，培养在直线排列的底物表面上，以促进成熟 心肌细胞的结构和功能。我的假设将通过多种方式得到验证：肌原纤维跨桥 动力学，通过停止血流(无序松弛状态与超松弛状态)对肌球蛋白确认的评估， 使用基于质谱仪(MS)的蛋白质组学在肌节中的cMyBP-C表达和化学计量， 细胞代谢通过海马试验，底物利用通过基于MS的代谢组学和能量成本 使用工程化心脏组织(EHT)构造产生张力。如果成功，这项研究将有助于揭示 这种高渗透性HCM突变的机制，并为临床前筛选潜在的治疗方法提供信息。

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease, characterized by progressive thickening of the left ventricular walls and potential for sudden cardiac death. Twenty-five percent of HCM mutations occur in the sarcomere protein cardiac myosin binding protein-C (cMyBP-C). Currently, there is no cure for HCM, only management of symptoms and disease progression, left ventricular obstruction surgery, or heart transplantation. As such, there is great need to better understand the pathological mechanisms that underly specific HCM mutations in order to better inform development of targeted therapeutics. For this project, I will study a highly penetrant mutation in cMyBP-C, c.772G>A (p.E258K), that has an identified founder effect in the north-east Tuscany region of Italy. To better explore the direct impacts of the mutation, I have generated patient induced pluripotent stem cells (iPSCs) from six HCM patients carrying the E258K mutation and a representative isogenic cell line using CRISPR/Cas9 by correcting the mutation. Initial studies have been performed on myectomy samples from three of the above six HCM patients with the E258K mutation, however, such patient tissue is limited and provides results from late stage of disease. Utilizing our patient iPSC lines, I can more thoroughly probe mechanisms underlying HCM from an almost unlimited supply of tissue specific cells. I propose to study multiple patient-derived iPSC lines all harboring the same E258K mutation, allowing me to probe the mechanism of the E258K mutation as well as investigate how other factors such as gender and age of onset may affect said mechanisms. Within the E258K patient cohort at the Careggi University Hospital, myectomy samples demonstrate consistently lower expression of full-length cMyBP-C protein, suggesting a potential haploinsufficiency disease mechanism. At the level of the sarcomere, myectomy samples indicate accelerated cross-bridge cycling, accompanied by a greater energetic cost of tension generation. Taken together, I hypothesize that the E258K mutation 1) destabilizes cMyBP-C’s ability to recruit and regulate myosin, leading to reduced expression and/or incorporation of cMyBP-C into the sarcomere (haploinsufficiency) and 2) shifts the sarcomere to a state of excessive ATP utilization during contraction (energetic inefficiency). To test this hypothesis, I will use our patient iPSCs differentiated to cardiomyocytes, and their isogenic control lines, cultured on linear, aligned substrate surfaces to enhance maturation of cardiomyocyte structure and function. My hypothesis will be tested with multiple modalities: myofibril cross-bridge kinetics, evaluation of myosin confirmations by stopped flow (disordered relaxed state vs. super-relaxed state), cMyBP-C expression and stoichiometry in the sarcomere using mass spectrometry (MS) based proteomics, cellular metabolism via Seahorse assay, substrate utilization via MS based metabolomics and energetic cost of tension generation using engineered heart tissue (EHT) constructs. If successful, this study will help uncover the mechanism of this highly penetrant HCM mutation and inform preclinical screening of potential therapeutics.