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: 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
• 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

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.

肥厚性心肌病（HCM）是最常见的遗传性心脏病，以进行性为特征