Comprehensive Analysis of Allelic, Cellular and Molecular Heterogeneity in Human 3-Dimensional Cardiac Microtissues with MYH7 Mutations

具有 MYH7 突变的人三维心脏微组织等位基因、细胞和分子异质性的综合分析

• 批准号: 9983170
• 负责人: John Travis Hinson
• 金额: $ 53.99万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9983170
• 关键词: 3-Dimensional; ATAC-seq; Actins; Alleles; Architecture; Arginine; Binding; Biological Assay; Biological Markers; Biomimetics; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cell Line; Cells; Chromatin; Computing Methodologies; Consumption; Data; Disease; Disease model; Engineering; Epigenetic Process; Future; Gene Expression; Gene Expression Profile; Gene Mutation; Gene Targeting; Genes; Genetic; Genetic Transcription; Genotype-Tissue Expression Project; Glutamine; Goals; Heart failure; Heterogeneity; High-Throughput Nucleotide Sequencing; Human; Human Engineering; Hypertrophic Cardiomyopathy; Image; Immunofluorescence Immunologic; Individual; Inherited; Kinetics; Knock-out; Knowledge; Laboratories; Lead; Link; Location; Mechanics; Metabolic; Methionine; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Molecular Motors; Mutation; Myocardium; Myofibrils; Myosin Heavy Chains; Nuclear; Pathogenesis; Pathologic; Pathology; Pathway Analysis; Patients; Pattern; Phenotype; Prevalence; Proteins; RNA analysis; Regulatory Element; Research; Resolution; Risk; Role; Sarcomeres; Secondary to; Stress; Structure; Symptoms; Therapeutic; Three-dimensional analysis; Tissues; Transcript; Transposase; Treatment Efficacy; Valine; Variant; beta-Myosin; cell type; cohort; disease heterogeneity; genetic variant; genomic tools; heart dimension/size; heart function; in vivo; induced pluripotent stem cell; insight; new therapeutic target; novel; programs; response; single-cell RNA sequencing; stem cell differentiation; stem cell model; sudden cardiac death; therapeutic development; therapeutic target; transcriptome; transcriptome sequencing; treatment response

PROJECT SUMMARY/ABSTRACT Hypertrophic cardiomyopathy (HCM) patients are at risk for sudden cardiac death and progressive heart failure (HF), and there are no effective therapeutics, due in part to our limited genetic understanding of HCM pathogenesis. There are critical gaps in our current knowledge of the molecular mechanisms that link specific mutations in the beta myosin heavy chain gene (MYH7) to pathological thickening of the heart muscle that is associated with HCM. Our long-term goals are to utilize genomic tools combined with 3-dimensional cardiac microtissues derived from human induced pluripotent stem cells to interrogate mechanisms of HCM secondary to specific MYH7 variants, and to utilize these insights to identify new disease biomarkers and therapeutic targets for specific HCM patients. Our previous studies identified that two HCM-associated MYH7 variants, arginine 403 to glutamine and valine 606 to methionine that are located in the actin-binding domain of beta myosin heavy chain protein (MHC- β), generate increased microtissue contraction force with associated abnormalities in contraction kinetics. Studies by others have indicated that MYH7 variants located in distinct structural domains of MHC-β cause distinct phenotypes. These results lead to our central hypothesis that HCM is a heterogeneous disorder, in which patient symptoms and therapeutic responses are dependent on the location of the causative MYH7 variant(s) within the gene and on cell-type specific transcriptional and epigenetic programs, which initiate from abnormalities in contractile function. Guided by our comprehensive preliminary data, we propose to pursue three Specific Aims to determine multi-scale insights into HCM pathogenesis: (1) to characterize functional consequences of MYH7 variants localized to the actin-binding, ATP-binding and converter domains of MHC- β, (2) to identify cell type-specific transcriptional and epigenetic mechanisms of HCM in microtissues using paired single-cell RNA-seq and ATAC-seq and (3) to interrogate the function of C1ORF105, a nuclear-encoded mitochondrial protein that is associated with HCM and is specifically expressed in human cardiomyocytes. In summary, the execution of these aims will provide a more precise understanding of the functional role of MYH7 variant localization, generate novel cell-type specific and molecular mechanisms of HCM and identify critical molecular linkages between sarcomere and mitochondrial function that will broadly impact the field of HCM and heart failure.

项目摘要/摘要 肥厚的心肌病（HCM）患者有猝死和进行性心脏的风险 失败（HF），没有有效的治疗，部分原因是我们对HCM的遗传学有限的理解 发病。我们目前对分子机制的知识存在关键的差距 β肌球蛋白重链基因（MYH7）的突变对心肌的病理增厚 与HCM相关。我们的长期目标是利用基因组工具与三维心脏 从人类诱导的多能干细胞衍生的微作用，以询问HCM次级机制 特定的MYH7变体，并利用这些见解来识别新的疾病生物标志物和治疗靶标 针对特定的HCM患者。 我们以前的研究表明，两个与HCM相关的MYH7变体，精氨酸403至谷氨酰胺和 valine 606至甲硫氨酸，位于β肌球蛋白重链蛋白的肌动蛋白结合结构域（MHC- β），在收缩动力学中与相关异常产生增加的微动收缩力。 其他人的研究表明，位于MHC-β不同结构结构域的MyH7变体引起 独特的表型。这些结果导致了我们的中心假设，即HCM是一种异质性疾病，其中 患者症状和治疗反应取决于病因MYH7变体的位置 在基因和细胞类型的特定转录和表观遗传程序中，从 收缩功能异常。在我们的全面初步数据的指导下，我们建议追求三个 确定对HCM发病机理的多尺度见解的具体目的：（1）表征功能 MYH7变体的后果位于MHC-的肌动蛋白结合，ATP结合和转换器域 β，（2）使用配对的细胞类型特异性转录和表观遗传机制 单细胞RNA-SEQ和ATAC-SEQ以及（3）询问C1orf105的功能，核编码 与HCM相关的线粒体蛋白，并在人类心肌细胞中特别表达。 总而言之，这些目标的执行将为功能角色提供更精确的理解 MYH7变体定位，生成新型的细胞类型特异性和分子机制，并识别 肌膜和线粒体功能之间的关键分子联系，将广泛影响 HCM和心力衰竭。