Role of RAF1 in human cardiogenesis and congenital heart defects

RAF1 在人类心脏发生和先天性心脏缺陷中的作用

• 批准号: 10930198
• 负责人: Fabrice Jaffré
• 金额: $ 46.77万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-23 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10930198
• 关键词: 3-Dimensional; Ablation; Apoptosis; Biochemical; Biological Models; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Differentiation process; Cell Nucleus; Cell Proliferation; Cell model; Cell physiology; Cells; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Congenital Abnormality; Congenital Heart Defects; Data; Defect; Development; Epigenetic Process; Follow-Up Studies; Generations; Genetic; Genetic Transcription; Genomics; Goals; Grant; Heart Abnormalities; Human; Hypertrophic Cardiomyopathy; Impairment; Infant; Intervention; Investigation; Knowledge; Lead; Mediator; Mesoderm; Modeling; Molecular; Morphogenesis; Mutagenesis; Mutation; Newborn Infant; Noonan Syndrome; Nuclear; Oncogenic; Pathway interactions; Patients; Phenotype; Play; Process; Proliferating; Protein Isoforms; Protein Kinase; Proteins; Proteomics; RAF1 gene; Resolution; Role; Running; Serine; Signal Pathway; Signal Transduction; Solid; Specific qualifier value; Testing; Therapeutic; Threonine; candidate identification; cardiogenesis; design; directed differentiation; epigenome; experimental study; gain of function; gene network; genome editing; loss of function; novel; novel therapeutics; pharmacologic; phosphoproteomics; prevent; progenitor; programs; raf-1 Protein; single-cell RNA sequencing; therapeutic target; transcriptome sequencing

Project Summary The goal of this project is to discover fundamental RAF1-dependent mechanisms that regulate early steps of cardiogenesis to further delineate the molecular basis of congenital heart defects (CHDs) and cardiomyopathies. RAF1 (or CRAF) is a serine/threonine-specific protein kinase that is ubiquitously expressed and controls cell proliferation, apoptosis, cell differentiation, and oncogenic transformation. Newborns with RAF1 germline loss- of-function have been recently identified and present with severe CHDs. Moreover, infants with Noonan syndrome (NS) associated RAF1 mutations present with obstructive hypertrophic cardiomyopathy and a variety of CHDs. Currently, no specific treatment exists for NS children with CHDs or cardiomyopathy, therefore there is an urgent need to understand the molecular mechanisms underlying cardiac developmental defects to identify specific therapeutic strategies. Using hiPSCs and cardiac-directed differentiation as a developmental model, we discovered that RAF1 was required for human cardiogenesis by regulating cardiac mesoderm specification and showed that NS RAF1 mutations impacted this process. How RAF1 regulates early human cardiogenesis programs and how NS mutations, such as those found in the RAF1 gene, impair cardiac development remains elusive. Hence, the overall goal of this proposal is to discover signaling, genetic and epigenetic networks modulated by RAF1 and impacted by NS RAF1 mutations during early cardiogenesis. We seek to perform an unprecedented in-depth investigation into the signaling and epigenetic networks altered by loss of RAF1 or NS RAF1 mutations at early stages of human cardiac differentiation. Toward that goal, we have compiled a “toolbox” of hiPSC lines generated by genome editing (CRISPR-Cas9) to enable a comprehensive analysis of gain-and loss-of-function phenotypes, with a solid track record for such analyses. Our proposal will break ground beyond current knowledge by achieving the following aims: Aim 1: Discover the function of RAF1 in early human cardiogenesis. Aim 2: To interrogate the impact of Noonan syndrome RAF1 mutations on human cardiac development. Aim 3: Delineate the role of nuclear RAF1 in early human cardiogenesis. Successful completion of our proposal will illuminate the fundamental role of RAF1 in human cardiogenesis and the molecular mechanisms underlying CHDs and cardiomyopathy in NS RAF1 at an unprecedented resolution. It will also provide fundamental new knowledge regarding the transcriptional, epigenetic and protein networks controlling human cardiac development and will pave the way for follow up studies to design new therapies for NS children with heart defects. Finally, we anticipate that our study will open new avenues of investigation of other developmental defects observed in NS and will highlight hiPSCs as a powerful model system to decipher the molecular mechanisms underlying CHDs.

项目摘要 该项目的目的是发现基本的RAF1依赖机制，该机制调节了早期步骤 心脏病发生，以进一步描述先天性心脏缺陷（CHD）和心肌病的分子基础。 RAF1（或CRAF）是一种丝氨酸/苏氨酸特异性蛋白激酶，它是普遍表达的，并控制细胞 增殖，凋亡，细胞分化和致癌转化。具有RAF1系种损失的新生儿 - 最近已经确定了功能，并存在严重的CHD。此外，有Noonan的婴儿 综合征（NS）相关的RAF1突变，具有阻塞性肥厚性心肌病和一种多样性 CHD。目前，对于NS CHD或心肌病儿童尚无特定治疗，因此 迫切需要了解心脏发育缺陷的分子机制以识别 具体的治疗策略。使用HIPSC和心脏指导的分化作为发展模型，我们 发现通过调节心脏中胚层规范和 表明NS RAF1突变影响了这一过程。 RAF1如何调节早期人类心脏病发生 程序以及NS突变（例如RAF1基因中发现的突变）如何损害心脏发展 难以捉摸。因此，该提案的总体目标是发现信号，遗传和表观遗传网络 由RAF1调节，并受到早期心脏病期间NS RAF1突变的影响。我们试图执行 对RAF1或NS的损失改变了对信号传导和表观遗传网络的前所未有的深入研究 RAF1突变在人类心脏分化的早期阶段。为了实现这一目标，我们编制了一个“工具箱” 基因组编辑（CRISPR-CAS9）生成的HIPSC线的全面分析 功能丧失的表型，具有此类分析的稳固记录。我们的提议将跨越地面 通过实现以下目标来实现当前知识：目标1：发现RAF1在早期人类中的功能 心脏病发生。目标2：询问Noonan综合征RAF1突变对人类心脏的影响 发展。目标3：描述核RAF1在早期人类心脏病中的作用。成功完成 我们的提议将阐明RAF1在人类心脏发生和分子中的基本作用 NS RAF1中的CHD和心肌病的机制以前所未有的分辨率。它也会 提供有关控制的转录，表观遗传和蛋白质网络的基本新知识 人类心脏发展，并将为后续研究铺平道路，为NS儿童设计新疗法 有心脏缺陷。最后，我们预计我们的研究将开放其他对其他调查的途径 在NS中观察到的发展缺陷，并将强调HIPSC作为强大的模型系统，以破译 CHD的分子机制。