Single cell, whole genome analysis of the aging human cardiomyocyte

衰老人类心肌细胞的单细胞、全基因组分析

• 批准号: 10326403
• 负责人: Sangita Choudhury
• 金额: $ 87.2万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-15 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10326403
• 关键词: Adopted; Adult; Age; Aging; Algorithms; Alleles; Autopsy; Brain; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Diseases; Cardiovascular Pathology; Cell Nucleus; Cell Respiration; Cells; Copy Number Polymorphism; DNA; DNA Damage; DNA Sequence Alteration; DNA sequencing; Data; Development; Disease; Disease Progression; Dropout; Elderly; Environmental Risk Factor; Enzyme-Linked Immunosorbent Assay; Freezing; Genetic; Genome; Genomic DNA; Genomics; Goals; Heart; Heart Diseases; Heart failure; Human; In Vitro; Individual; Inherited; Interphase Cell; Knowledge; Lead; Link; Longitudinal Studies; Malignant Neoplasms; Measures; Methods; Modeling; Mutagens; Mutation; Myocardial dysfunction; Myocardium; Neurons; Newborn Infant; Organ; Oxidative Stress; Pathologic; Patients; Pattern; Phenotype; Ploidies; Point Mutation; Premature aging syndrome; Process; Radiation; Radiation therapy; Research; Resolution; Risk Factors; Role; Sampling; Series; Single Nucleotide Polymorphism; Somatic Mutation; Techniques; Testing; Time; Troponin T; Variant; age related; aged; base; cancer radiation therapy; cardiovascular risk factor; cell type; disorder risk; epidemiology study; genome analysis; genome sequencing; genome-wide; genotoxicity; human old age (65+); innovation; insight; novel; prevent; radiation during childhood; radiation effect; whole genome

Project Summary/Abstract Recent evidence suggests that environmental factors causing somatic mutations during the lifetime have a more crucial role, not only in cancer but also in other common diseases, including heart failure. Recent studies have also shown that somatic single nucleotide variants (sSNV) accumulate even in nondividing cells, such as neurons in the human cortex, resulting in thousands of sSNV per neuronal genome by old age. However, genomic DNA changes in aging cardiomyocytes (CM) remain poorly understood. The accumulation of somatic DNA mutations over time has recently been demonstrated to be a hallmark of aging in many human cell types. The current study aims to determine the landscape and role of somatic mutations in aging and cardiac disease by adopting a new technique that allow deep whole-genome sequencing of DNA isolated from single CM taken from the frozen postmortem heart. The first Aim of this study is to evaluate the somatic mutational burden (sSNVs) in aging CM genome. We will also compare CM mutational burden with postmitotic cells from another organ (neurons) to define differences in accumulation rate during aging. In the second Aim, we will ask what are the mutational signature and the mechanisms of mutation formation in the aging human heart and if the heart mutational signature is different than the brain mutational signature. Further to recapitulate the mutational signature and related phenotype in the heart we will directly induce oxidative stress in an in vitro culture model of primary CMs. The final Aim will focus on evaluating the genotoxic effect of radiation in CMs after childhood radiation therapy and the role of radiation in premature aging. The proposed research is significant for the comprehensive, results-based development of strategies for understanding natural aging and disease progression in the human heart. Together with the planned characterization of mutational signatures, the anticipated results may provide knowledge to develop new strategies for preventing the heart disease associated with aging. The proposed study is only possible because of a series of innovations that are, at this time, uniquely available to our research team, 1) a novel method to isolate single CM nuclei from frozen myocardium based on CM ploidy and nuclei cardiac troponin T expression. 2) A major breakthrough by developing “LiRA” and “PhaseDel” algorithm to call sSNV and sSV confidently from tetraploid cells that considers cell-specific depth distributions of DNA sequencing and allele-dropout rates in scWGS data. For the first time, our study will reveal the landscape of somatic mutations, genomic changes during aging and after radiation therapy in human heart muscle cells in a single-cell resolution. In the long term, this study will provide insights that might allow blocking some of the mutational processes ameliorating age-related myocardial dysfunction.

项目摘要/摘要 最近的证据表明，在人的一生中导致体细胞突变的环境因素 更关键的作用，不仅在癌症中，而且在包括心力衰竭在内的其他常见疾病中。最新研究 还表明，即使在未分裂的细胞中，体细胞单核苷酸变体(SSNV)也会积累，例如 人类大脑皮层中的神经元，导致每个神经元基因组到老年时有数千个sSNV。然而， 衰老心肌细胞(CM)基因组DNA的变化仍然知之甚少。体细胞的积累 随着时间的推移，DNA突变最近被证明是许多人类细胞类型衰老的标志。 目前的研究旨在确定体细胞突变在衰老和心脏病中的格局和作用 通过采用一种新技术，对从单个CM提取的DNA进行深度全基因组测序 来自冰冻的死后心脏。本研究的第一个目的是评估体细胞突变的负担。 (SSNvs)在老化的CM基因组。我们还将比较CM突变负担与来自另一种细胞的有丝分裂后细胞 器官(神经元)来定义衰老过程中蓄积率的差异。在第二个目标中，我们将询问 是人类老化心脏中的突变特征和突变形成机制，如果 心脏突变的特征不同于大脑的突变特征。进一步概括一下突变 我们将在体外培养模型中直接诱导心脏的氧化应激 主要不育系的数量。最终目标将集中在评估辐射对儿童后CMS的遗传毒性影响。 放射治疗和辐射在早衰中的作用。这项拟议的研究对 全面、注重结果地制定了解自然衰老和疾病的战略 人类心脏的进展。与计划中的突变特征特征一起， 预期的结果可能会为开发预防心脏病的新策略提供知识 与衰老相关的。拟议的研究之所以可能，是因为在这一点上有一系列的创新 我们的研究团队独有的时间，1)从冰冻中分离单个CM核的新方法 心肌基于CM倍体和细胞核的心肌肌钙蛋白T的表达。2)重大突破： 开发“Lira”和“PhaseDel”算法，从四倍体细胞中可靠地调用sSNV和SSV 在scWGS数据中考虑DNA测序和等位基因丢失率的细胞特定深度分布。对于 我们的研究将首次揭示衰老过程中和衰老后的体细胞突变、基因组变化的图景 放射治疗在人类心肌细胞中的单细胞分辨率。从长远来看，这项研究将 提供一些见解，可能允许阻止一些改善与年龄相关的突变过程 心肌功能不全。