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 该项目的目标是发现调节细胞凋亡早期步骤的基本RAF 1依赖性机制。 心脏发生，以进一步阐明先天性心脏缺陷（CHD）和心肌病的分子基础。 RAF 1（或CRAF）是一种丝氨酸/苏氨酸特异性蛋白激酶，广泛表达并控制细胞凋亡。 增殖、凋亡、细胞分化和致癌转化。RAF 1种系丢失的新生儿- 功能丧失最近已被确定，并与严重的CHD。此外，患有努南病的婴儿 与梗阻性肥厚型心肌病相关的RAF 1突变以及各种 的CHD。目前，对于患有CHD或心肌病的NS儿童没有特异性治疗，因此， 迫切需要了解心脏发育缺陷的分子机制， 具体的治疗策略。使用hiPSCs和心脏定向分化作为发育模型，我们 发现RAF 1通过调节心脏中胚层特化而为人类心脏发生所需， 表明NS RAF 1突变影响了这一过程。RAF 1如何调节人类早期心脏发生 NS基因突变，如RAF 1基因中发现的突变，如何损害心脏发育， 难以捉摸。因此，本提案的总体目标是发现信号传导、遗传和表观遗传网络 在早期心脏发生过程中受RAF 1调节并受NS RAF 1突变影响。我们寻求执行一个 对RAF 1或NS缺失改变的信号传导和表观遗传网络进行了前所未有的深入研究 RAF 1基因在人类心脏分化早期的突变为了实现这一目标，我们编制了一个“工具箱”， 通过基因组编辑（CRISPR-Cas9）产生的hiPSC系，以实现对获得和表达的全面分析。 功能丧失表型，具有此类分析的可靠记录。我们的提议将突破 目的1：发现RAF 1在早期人类中的功能 心脏发生目的2：探讨努南综合征RAF 1基因突变对人类心脏的影响。 发展目的3：阐明核RAF 1在人类早期心脏发生中的作用。成功完成 我们的建议将阐明RAF 1在人类心脏发生中的基本作用， 在NS RAF 1中，CHD和心肌病的潜在机制得到了前所未有的解决。它还将 提供有关转录，表观遗传和蛋白质网络控制的基本新知识 人类心脏发育，并将为后续研究铺平道路，为NS儿童设计新的治疗方法 有心脏缺陷最后，我们预计，我们的研究将开辟新的途径，调查其他 在NS中观察到的发育缺陷，并将突出hiPSC作为一个强大的模型系统，以破译 冠心病的分子机制