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.

项目总结 作为全球主要的死亡原因，心力衰竭是研究人员探索发现的一大挑战。 可以拯救无数生命的治疗学。心脏损伤后，心脏开始以一种方式自我重塑 最初是适应性的，但随着时间的推移，这种与生俱来的应对机制可能会加速心力衰竭的发生。澄清 从适应性心脏肥厚重塑进展到心力衰竭的机制将 极大地影响了这种致命疾病的新疗法的发现。而基因的调控 信使核糖核酸(Mrna)的转录表达已被广泛研究，直到最近和 人们已经意识到可以对信使核糖核酸进行化学修饰的重要性。这项建议 重点关注最丰富的内部mRNA(M6A)的N6-腺苷的甲基化 真核生物中的修饰。我们实验室之前的研究表明，M6A含量的调制 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 这种情况发生的确切机制还没有被很好地理解。M6A修饰的mRNAs的命运 由YTH域家族(YTHDF)的成员管理。我们发现YTHDF3特别重要 在心肌细胞中，它定位于细胞核并与心肌细胞增强因子2D(MEF2D)结合，后者 是一种重要的转录因子，调节肥厚性心脏生长。此外，我们还发现了这种基因敲除 YTHDF3可减轻压力超负荷损伤后的病理重塑。根据这些初步数据， 我们假设YTHDF3通过调节心肌细胞大小和应激诱导的重构来调节心肌细胞的大小和应激诱导的重构 MEF2D转录的m6A修饰的mRNA的处理。为了检验这一假设，我们已经 建立并验证了一个新的小鼠系，在该系中YTHDF3可以在心肌细胞中选择性缺失 (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，并评估病理组织学和分子征象 终端时间点。在目标2中，我们将确定YTHDF3通过什么机制来调控 心肌细胞中MEF2D转录的m6A-mRNAs的特定亚群。首先，我们将进一步描述 通过定义各自涉及的结构域，在YTHDF3和MEF2D之间结合。然后，我们将剖析 YTHDF3与MEF2D信使核糖核酸靶标的结合及其稳定性、输出和翻译 成绩单。最后，在目标3中，我们将采取不偏不倚的方法，更全面地调查 YTHDF3交联剂对健康和应激成年心肌细胞mRNA生物学的调节作用 YTHDF3结合的mRNAs的免疫沉淀和测序(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，这可能导致一个新的 以信使核糖核酸甲基化生物学为基础的治疗领域。