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，其特征是进行性进展 左心室壁增厚和心源性猝死的可能性。 25% 的 HCM 突变发生在肌节蛋白心肌肌球蛋白结合蛋白-C (cMyBP-C) 中。目前，没有 治愈 HCM，仅治疗症状和疾病进展，左心室梗阻手术，或 心脏移植。因此，非常需要更好地了解其背后的病理机制。 特定的 HCM 突变，以便更好地为靶向治疗的开发提供信息。对于这个项目，我将 研究 cMyBP-C 中的高度渗透突变，c.772G>A (p.E258K)，该突变在 意大利东北托斯卡纳地区。为了更好地探索突变的直接影响，我生成了患者 来自六名携带 E258K 突变的 HCM 患者和一名代表性患者的诱导多能干细胞 (iPSC) 使用 CRISPR/Cas9 纠正突变的同基因细胞系。初步研究已进行 上述 6 名 HCM 患者中的 3 名携带 E258K 突变的心肌切除样本，然而，此类患者 组织有限，只能提供疾病晚期的结果。利用我们的患者 iPSC 细胞系，我可以更多 从几乎无限供应的组织特异性细胞中彻底探索 HCM 的机制。我建议 研究多个源自患者的 iPSC 系，它们都含有相同的 E258K 突变，使我能够探索 E258K 突变的机制，并研究性别和年龄等其他因素如何影响 发病的时间可能会影响所述机制。在 Careggi 大学医院的 E258K 患者队列中， 子宫肌瘤切除样本显示全长 cMyBP-C 蛋白的表达始终较低，这表明 潜在的单倍体不足疾病机制。在肌节水平，肌切除样本表明 加速过桥骑行，伴随着产生张力的更大的能量消耗。采取 总之，我假设 E258K 突变 1) 破坏了 cMyBP-C 招募和调节能力的稳定性 肌球蛋白，导致 cMyBP-C 表达减少和/或掺入肌节 （单倍体不足）和 2) 将肌节转变为收缩期间过度利用 ATP 的状态 （能量效率低下）。为了检验这个假设，我将使用我们的患者 iPSC 分化为心肌细胞， 及其同基因对照系，在线性、对齐的基质表面上培养，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进行检验：肌原纤维跨桥 动力学，通过停止流动评估肌球蛋白确认（无序松弛状态与超松弛状态）， 使用基于质谱 (MS) 的蛋白质组学分析肌节中的 cMyBP-C 表达和化学计量， 通过海马测定进行细胞代谢，通过基于 MS 的代谢组学进行底物利用以及能量消耗 使用工程心脏组织（EHT）结构产生张力。如果成功的话，这项研究将有助于揭示 这种高渗透性 HCM 突变的机制，并为潜在治疗方法的临床前筛选提供信息。