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: 10548738
• 负责人: Sangita Choudhury
• 金额: $ 85.56万
• 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/10548738
• 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; Cytoprotection; 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; Link; Malignant Neoplasms; Measures; Methods; Modeling; Mutagens; Mutation; Myocardial dysfunction; Myocardium; Neurons; Newborn Infant; Organ; Oxidative Stress; Oxidative Stress Induction; 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; postmitotic; 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.

项目总结/摘要 最近的证据表明，在一生中引起体细胞突变的环境因素具有 更关键的作用，不仅在癌症，而且在其他常见疾病，包括心力衰竭。最近的研究 还表明，体细胞单核苷酸变异体（sSNV）甚至在非分裂细胞中积累，如 在人类大脑皮层的神经元中，导致老年时每个神经元基因组中有数千个sSNV。然而，在这方面， 衰老心肌细胞（CM）中的基因组DNA变化仍然知之甚少。体细胞的积累 DNA随时间的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情况和作用 通过采用一种新技术，可以对从单个CM中分离的DNA进行深度全基因组测序， 冷冻的死后心脏上找到的本研究的第一个目的是评估体细胞突变负荷 （sSNV）在衰老CM基因组中的表达。我们还将比较CM突变负荷与来自另一种细胞的有丝分裂后细胞， 器官（神经元），以确定在老化过程中的积累率的差异。在第二个目标中，我们将问什么 是衰老人类心脏中的突变特征和突变形成机制， 心脏突变特征不同于大脑突变特征。进一步概括突变 我们将在体外培养模型中直接诱导氧化应激 主要CM最后的目标将侧重于评估儿童期后CM的辐射遗传毒性效应 放射治疗和辐射在过早衰老中的作用。该研究对我国的 全面、基于结果地制定战略，以了解自然衰老和疾病 在人类心中的进步。连同突变特征的计划表征， 预期的结果可能为开发预防心脏病的新策略提供知识 与衰老有关。这项拟议中的研究之所以成为可能，是因为一系列的创新， 时间，唯一提供给我们的研究小组，1）一种新的方法，以分离单个CM核从冷冻 基于CM倍体和核心肌肌钙蛋白T表达的心肌。2)一项重大突破， 开发“里拉”和“PhaseDel”算法，以确信地从四倍体细胞中识别sSNV和sSV， 考虑scWGS数据中DNA测序的细胞特异性深度分布和等位基因丢失率。为 第一次，我们的研究将揭示体细胞突变的景观，衰老过程中和之后的基因组变化 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 提供了一些见解，可能允许阻止一些突变过程， 心肌功能障碍