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

在患者来源的心肌细胞中建模 E258K-MYBPC3 突变引起的心肌细胞功能障碍的机制

• 批准号: 10794930
• 负责人: Sonette Steczina
• 金额: $ 4.57万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2025-09-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10794930
• 关键词: 3-Dimensional; Acceleration; Actins; Address; Affect; Age of Onset; Back; Binding; Biochemistry; Biological Assay; CRISPR/Cas technology; Cardiac Myocytes; Cardiac Myosins; Cell Line; Cells; Clinical; Custom; Data; Development; Disease; Disease Progression; Evaluation; Extracellular Matrix; Fibrin; Founder Effect; Functional disorder; Gender; Generations; Genetic; Genus Hippocampus; Heart Diseases; Heart Transplantation; Heart failure; Hypertrophic Cardiomyopathy; Induced Mutation; Inherited; Italy; Kinetics; Lead; Left; Length; Link; Mass Spectrum Analysis; Metabolism; Missense Mutation; Modality; Modeling; Mutation; Myocardium; Myofibrils; Myosin ATPase; Obstruction; Operative Surgical Procedures; Pathogenicity; Pathologic; Patients; Pattern; Phenotype; Phosphocreatine; Prevalence; Production; Proteins; Proteomics; Quality of life; Relaxation; Respiration; Sampling; Sarcomeres; Structure; Surface; Symptoms; Testing; Therapeutic; Thick Filament; Thrombin; Tissue constructs; Tissues; University Hospitals; Variant; Ventricular; cardiac muscle disease; cardiac tissue engineering; citrate carrier; cohort; cost; genetic variant; heart function; individualized medicine; induced pluripotent stem cell; insight; liquid chromatography mass spectrometry; metabolic profile; metabolomics; mutant; mutation correction; myosin-binding protein C; pre-clinical; protein expression; protein protein interaction; recruit; screening; stem cell differentiation; stoichiometry; sudden cardiac death; symptom management; targeted treatment; young adult

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.

肥厚型心肌病（HCM）是最常见的遗传性心脏病，其特征在于进行性心肌梗死， 左心室壁增厚和心源性猝死的可能性。HCM的25% 突变发生在肌节蛋白心脏肌球蛋白结合蛋白-C（cMyBP-C）中。目前尚无 治愈HCM，仅管理症状和疾病进展，左心室梗阻手术，或 心脏移植因此，非常需要更好地了解导致这些疾病的病理机制。 特定的HCM突变，以便更好地为靶向治疗的开发提供信息。为了这个项目，我将 研究cMyBP-C中的高度渗透突变c.772G>A（p.E258K），该突变在MyBP-C中具有确定的奠基者效应， 位于意大利东北部的托斯卡纳地区。为了更好地探索突变的直接影响，我已经产生了病人 来自六名携带E258 K突变的HCM患者的诱导多能干细胞（iPSC）和代表性的 通过校正突变，使用CRISPR/Cas9在等基因细胞系中表达。初步研究已经进行， 来自上述六名具有E258 K突变的HCM患者中的三名的肌切除术样品，然而，这样的患者 组织是有限的，并提供来自疾病晚期的结果。利用我们的患者iPSC细胞系，我可以 从几乎无限的组织特异性细胞供应中彻底探测HCM的潜在机制。我提议 研究多个患者来源的iPSC系，所有这些细胞都携带相同的E258 K突变， E258 K突变的机制，以及研究其他因素，如性别和年龄， 可能会影响上述机制。在Careggi大学医院的E258 K患者队列中， 肌切除术样本显示全长cMyBP-C蛋白的表达持续较低，提示 潜在的单倍性不足疾病机制。在肌节水平，肌切除样本表明 加速的跨桥循环，伴随着产生张力的更大能量消耗。采取 总之，我假设E258 K突变1）使cMyBP-C的募集和调节能力不稳定， 肌球蛋白，导致cMyBP-C表达减少和/或掺入肌节 （单倍不足）和2）在收缩期间将肌节转移到过度ATP利用的状态 （效率低下）。为了验证这一假设，我将使用我们的患者分化为心肌细胞的iPSC， 和它们的同基因对照系，在线性排列的基质表面上培养， 心肌细胞的结构和功能。我的假设将通过多种方式进行测试：肌原纤维交叉桥 动力学，通过停止流动评价肌球蛋白确认（无序松弛状态vs.超松弛状态）， 使用基于质谱（MS）的蛋白质组学，在肌节中cMyBP-C表达和化学计量， 通过Seahorse测定的细胞代谢、通过基于MS的代谢组学的底物利用和 使用工程化心脏组织（EHT）构建体产生张力。如果成功，这项研究将有助于揭示 这一高度渗透性HCM突变的机制，并告知潜在的治疗药物的临床前筛选。