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

基本信息

批准号：
10462968
负责人：
Sonette Steczina
金额：
$ 4.47万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
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/10462968
关键词：
3-Dimensional 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 Head 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 Regulation Respiration Sampling Sarcomeres Structure Surface Symptoms Testing Therapeutic Thick Filament Thrombin Tissue constructs Tissues University Hospitals Variant Ventricular base 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 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

项目摘要

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 25% 的 HCM 患者会出现左心室壁增厚和心源性猝死的可能性。突变发生在肌节蛋白心肌肌球蛋白结合蛋白-C (cMyBP-C)，目前尚无突变。治愈 HCM，仅治疗症状和疾病进展，左心室梗阻手术，或因此，非常需要更好地了解心脏移植的病理机制。为了更好地为该项目的靶向治疗的开发提供信息，我将研究特定的 HCM 突变。研究 cMyBP-C 中的高度渗透突变，c.772G>A (p.E258K)，该突变在为了更好地探索突变的直接影响，我在意大利东北部的托斯卡纳地区培养了一名患者。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已进行了初步研究。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，但利用我们的患者 iPSC 系，我可以提供更多信息。我建议从几乎无限的组织特异性细胞中彻底探究 HCM 的机制。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 突变的机制以及研究性别和年龄等其他因素如何影响在 Careggi 大学医院的 E258K 患者队列中，发病率可能会影响上述机制。肌切除术样本显示全长 cMyBP-C 蛋白的表达始终较低，表明在肌节水平上，肌切除样本表明潜在的单倍体不足疾病机制。加速过桥骑行，伴随着产生张力的更大的能量消耗。总的来说，我承认 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如果成功，这项研究将有助于揭示使用工程心脏组织（EHT）构建的张力。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。