YTHDF3 as a critical regulator of cardiac function

YTHDF3 作为心脏功能的关键调节因子

• 批准号: 10676427
• 负责人: Charles P. Rabolli
• 金额: $ 4.29万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676427
• 关键词: Address; Adenosine; Adopted; Adult; Affect; Binding; Binding Proteins; Biology; Cardiac; Cardiac Myocytes; Cause of Death; Cell Nucleus; Chemicals; Co-Immunoprecipitations; Data; Development; Dilated Cardiomyopathy; Disease; Drug Design; Echocardiography; Enhancers; Enzymes; Eukaryota; Event; Family; Functional disorder; Future; Gene Expression; Gene Expression Regulation; Genetic Transcription; Growth; Health; Heart; Heart Diseases; Heart Hypertrophy; Heart Injuries; Heart failure; Histologic; Homeostasis; Hypertrophy; Immunoprecipitation; Injury; Knock-out; Knockout Mice; Knowledge; Mass Spectrum Analysis; Mediating; Messenger RNA; Methylation; Modeling; Modification; Molecular; Morbidity - disease rate; Mus; Muscle Cells; Myocardial; Names; Nuclear; Pathologic; Pathology; Patients; Phenotype; Physiological; Positioning Attribute; Property; Proteins; Proteome; RNA; RNA Binding; RNA methylation; RNA-Binding Proteins; Reader; Regulation; Research; Research Personnel; Role; Stress; Testing; Therapeutic; Time; Transcript; Translations; Work; Workload; aorta constriction; biological adaptation to stress; coping mechanism; crosslink; epitranscriptome; experimental study; heart function; improved; improved outcome; innovation; member; mortality; mouse model; novel; novel therapeutics; posttranscriptional; pressure; protein protein interaction; response; transcription factor; transcriptome sequencing

PROJECT SUMMARY As the global leading cause of death, heart failure is a major challenge for researchers in their quest to discover therapeutics that can save countless lives. After cardiac injury, the heart begins to remodel itself in a way that is initially adaptive, but this innate coping mechanism may over time expedite heart failure onset. Elucidating the mechanisms which underly the progression from adaptive cardiac hypertrophic remodeling to heart failure will dramatically impact the discovery of novel therapeutics for this deadly disease. While regulation of gene expression through transcription of messenger RNA (mRNA) has been extensively studied, only recently an appreciation for the importance of chemical modifications that can occur on mRNA has emerged. This proposal focuses on the methylation of the N6-Adenosine of mRNA (m6A), which is the most abundant internal mRNA modification in eukaryotes. Previous research from our lab has shown that modulation of m6A content in the heart is sufficient to drive cardiac remodeling and to affect the ability of the heart to respond to stress. Despite this, the exact mechanisms through which this occurs is not well understood. The fate of m6A-modified mRNAs is regulated by members of the YTH Domain Family (YTHDF). We found that YTHDF3 is specifically important in cardiomyocytes, where it localizes to the nucleus and binds to Myocyte Enhancer Factor 2D (MEF2D), which is an important transcription factor regulating hypertrophic cardiac growth. Further, we have found that knockout of YTHDF3 mitigates pathological remodeling following pressure overload injury. Given these preliminary data, we hypothesize that YTHDF3 regulates cardiomyocyte size and stress-induced remodeling by modulating the processing of m6A-modified mRNAs transcribed by MEF2D. To test this hypothesis, we already generated and validated a new mouse line in which YTHDF3 can be selectively deleted in cardiomyocytes (YTHDF3-cKO). In Aim 1, we will investigate the role of YTHDF3 at baseline and in the stressed murine heart using longitudinal echocardiography analysis, and assessing histological and molecular signs of pathology at the terminal time point. In Aim 2, we will determine the mechanism through which YTHDF3 regulates the fate of specific subsets of MEF2D-transcribed m6A-mRNAs in cardiomyocytes. First, we will further characterize the binding between YTHDF3 and MEF2D by defining the respective domains involved. Then, we will dissect the binding of YTHDF3 to MEF2D mRNA targets and determine consequent stability, export, and translation of these transcripts. Finally, in Aim 3, we will undertake an unbiased approach to more globally investigate the role of YTHDF3 in regulating mRNA biology in healthy and stressed adult cardiomyocytes by cross-linking immunoprecipitations of YTHDF3-bound mRNAs followed by sequencing (CLIP-seq). Our approach is innovative and significant, as it will be the first project to define the role of YTHDF3 in the heart, which may lead to a new field of therapeutics based on the biology of mRNA methylation.

项目概要 作为全球主要死亡原因，心力衰竭是研究人员寻求发现的重大挑战 可以挽救无数生命的疗法。心脏损伤后，心脏开始以以下方式进行自我重塑： 最初是适应性的，但随着时间的推移，这种先天的应对机制可能会加速心力衰竭的发生。阐明 从适应性心脏肥厚重塑到心力衰竭的进展机制将 极大地影响了这种致命疾病的新疗法的发现。在基因调控的同时 通过信使 RNA (mRNA) 转录进行的表达已被广泛研究，直到最近 人们已经认识到 mRNA 上发生的化学修饰的重要性。这个提议 专注于 mRNA N6-腺苷 (m6A) 的甲基化，这是最丰富的内部 mRNA 真核生物中的修饰。我们实验室之前的研究表明，m6A 含量的调节 心脏足以驱动心脏重塑并影响心脏对压力的反应能力。尽管 然而，发生这种情况的确切机制尚不清楚。 m6A 修饰 mRNA 的命运 受 YTH 域家族 (YTHDF) 成员监管。我们发现YTHDF3特别重要 在心肌细胞中，它定位于细胞核并与肌细胞增强因子 2D (MEF2D) 结合， 是调节肥大心脏生长的重要转录因子。进一步，我们发现淘汰赛 YTHDF3 可以减轻压力超负荷损伤后的病理重塑。鉴于这些初步数据， 我们假设 YTHDF3 通过调节心肌细胞大小和应激诱导的重塑 MEF2D 转录的 m6A 修饰 mRNA 的加工。为了检验这个假设，我们已经 生成并验证了一个新的小鼠品系，其中 YTHDF3 可以在心肌细胞中选择性删除 (YTHDF3-cKO)。在目标 1 中，我们将研究 YTHDF3 在基线和应激小鼠心脏中的作用 使用纵向超声心动图分析，并评估病理学的组织学和分子迹象 终点时间点。在目标2中，我们将确定YTHDF3调节命运的机制 心肌细胞中 MEF2D 转录的 m6A-mRNA 的特定子集。首先，我们将进一步表征 通过定义所涉及的各自域来绑定 YTHDF3 和 MEF2D。然后，我们将剖析 YTHDF3 与 MEF2D mRNA 靶标的结合，并确定这些靶标随后的稳定性、输出和翻译 成绩单。最后，在目标 3 中，我们将采取公正的方法在全球范围内调查 YTHDF3 通过交联调节健康和应激成年心肌细胞的 mRNA 生物学 对 YTHDF3 结合的 mRNA 进行免疫沉淀，然后进行测序 (CLIP-seq)。我们的方法是创新的 而且意义重大，因为这将是第一个定义 YTHDF3 在心脏中的作用的项目，这可能会导致一个新的研究 基于 mRNA 甲基化生物学的治疗学领域。