Molecular mechanisms of atrial development and regeneration

心房发育和再生的分子机制

• 批准号: 9363356
• 负责人: Joshua Waxman
• 金额: $ 39.75万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-21 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9363356
• 关键词: 3' Untranslated Regions; ATAC-seq; Ablation; Adult; Affect; Affinity Chromatography; Antisense RNA; Arrhythmia; Biological Assay; Birth; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cardiovascular system; Cell Lineage; Cell Transplantation; Cells; Cessation of life; Child; Chromatin; Complex; Congenital Abnormality; Congenital Heart Defects; Data; Defect; Development; Diagnosis; Disease; Embryo; Engineering; Epigenetic Process; Ethylnitrosourea; Etiology; Foundations; Gene Expression Regulation; Generations; Genetic Complementation Test; Genomics; Glean; Goals; Health; Heart; Heart Atrium; Heart Hypertrophy; Heart Injuries; Human; Impairment; In Vitro; Individual; Knockout Mice; Knowledge; Life; Methods; MicroRNAs; Modeling; Molecular; Morphology; Mus; Mutagenesis; Mutation; Natural regeneration; Neonatal Mortality; Newborn Infant; Nuclear Hormone Receptors; Nuclear Orphan Receptor; Orphan; Pathway interactions; Play; Positioning Attribute; Prevalence; Production; Proteins; RNA; Ribosomes; Role; Signal Transduction; Site; Stroke; Surface; Techniques; Testing; Tissues; Transcript; Transcriptional Activation; Translations; Transposase; Untranslated RNA; Ventricular; Vertebrates; Zebrafish; apoAI regulatory protein-1; atrioventricular septal defect; blastocyst; cardiac regeneration; cardiogenesis; genome editing; healing; human stem cells; improved; in utero diagnosis; in vivo; inhibitor/antagonist; injured; loss of function; mutant; next generation sequencing; novel; novel therapeutics; premature; prevent; progenitor; promoter; regenerative; repaired; transcription factor; transcriptome sequencing

Project Summary/Abstract Congenital heart defects (CHDs) are the most common congenital malformations. 5% of CHDs comprise atrioventricular septal defects (AVSDs). However, the molecular etiology underlying most AVSDs are not understood. Furthermore, CHDs can cause cardiovascular diseases later in life, resulting in arrhythmias, stroke, and premature death. In order to develop novel therapies able to prevent CHDs and heal specific cardiovascular tissues, it is critical to garner understanding of fundamental mechanisms directing normal cardiac chamber development and regeneration. Therefore, long-term goals of our lab are to understand conserved mechanisms that direct the development of the individual cardiac chambers and chamber-specific mechanisms of regeneration in vertebrates. Few signals are known to be required that specifically direct atrial development, with specific regulators of atrial regeneration not being understood. The specific aims of this proposal are to elucidate the mechanisms by which a syntenic long non-coding RNA (lncRNA) as-oca restricts atrial development and regeneration through inhibition of Nr2f1a translation in zebrafish. The studies in this proposal are relevant to human health as recent genomic analysis indicates that mutations in the orphan nuclear receptor Nr2f2 are associated with AVSDs in humans. While Nr2f2 knockout mice and in vitro studies with human stem cells have revealed requirements for both Nr2f1 and Nr2f2 in atrial development, the mechanisms by which Nr2f proteins direct proper atrial development are not completely understood. Importantly, there is currently no understanding of epigenetic lncRNA-dependent mechanisms regulating Nr2f proteins. Our preliminary analysis of the novel zebrafish mutant acorn worm (aco) indicate that excess expression of as-oca specifically restricts the addition of later differentiating second heart field (SHF)-derived atrial cells. In Aim 1, we will use blastula cell transplantation, in vivo cardiomyocyte differentiation assays, and genome editing to determine the cellular requirements underlying the atrial defects and cause of increased as- oca expression in aco mutants. We do not understand how as-oca inhibits Nr2f1a translation in aco mutants. In Aim 2, we will use RNA and ribosomal association techniques and loss of function methods to determine if as- oca inhibits Nr2f1a translation through interactions with the nr2f1a 3' untranslated region. In addition to their requirements during development, we find that as-oca and nr2f1a are specifically expressed in the atria of adult zebrafish. In Aim 3, we will test the effects of as-oca on embryonic and adult models of cardiac regeneration. Because Nr2f transcription factors play conserved roles in atrial development of all vertebrates, these studies will dramatically improve our understanding of posttranscriptional mechanisms regulating normal vertebrate atrial development and the molecular etiology of AVSDs and ASDs in humans. Ultimately, these studies will generate a foundation for improved therapies capable of preventing CHDs in children and efficiently repairing injured hearts in adults.

项目摘要/摘要 先天性心脏缺陷（CHD）是最常见的先天性畸形。 CHD的5％包括 心室间隔缺陷（AVSD）。但是，大多数AVSD的基础分子病因不是 理解。此外，CHD会在以后的生活中引起心血管疾病，导致心律不齐， 中风和过早死亡。为了开发能够预防CHD并治愈特定疗法的新型疗法 心血管组织，至关重要的是要获得对指导正常的基本机制的理解 心脏腔室的发展和再生。因此，我们实验室的长期目标是了解 指导单个心脏室和室特异性发展的保守机制 脊椎动物再生的机制。已知很少需要专门直接房屋的信号 开发，不了解房屋再生的特定调节剂。这个特定的目的 提案是为了阐明同步长的非编码RNA（LNCRNA）AS-OCA限制的机制 通过抑制斑马鱼中的NR2F1A翻译，心房发育和再生。关于这一点的研究 提案与人类健康有关，因为最近的基因组分析表明孤儿的突变 核受体NR2F2与人类的AVSD有关。而NR2F2敲除小鼠和体外研究 随着人类干细胞的需求，对心房发育中NR2F1和NR2F2的需求， NR2F蛋白直接适当心房发育的机制尚未完全理解。 重要的是，目前尚不了解调节NR2F的表观遗传学依赖性机制 蛋白质。我们对新型斑马鱼突变橡胶蠕虫（ACO）的初步分析表明过多 AS-OCA的表达明确限制了后来区分第二心脏场（SHF）衍生的添加 心房细胞。在AIM 1中，我们将使用囊泡细胞移植，体内心肌细胞分化测定和 基因组编辑以确定心房缺陷的基础细胞需求，并导致AS-增加的原因 ACO突变体中的OCA表达。我们不了解AS-OCA如何抑制ACO突变体中的NR2F1A翻译。在 AIM 2，我们将使用RNA和核糖体关联技术以及功能方法的丧失来确定是否as- OCA通过与NR2F1A 3'未翻译区域的相互作用来抑制NR2F1A的翻译。除了他们 在开发过程中的要求，我们发现AS-OCA和NR2F1A在心房中特别表达 成人斑马鱼。在AIM 3中，我们将测试AS-OCA对心脏胚胎和成人模型的影响 再生。由于NR2F转录因子在所有脊椎动物的心房发育中起着保守的作用，所以 这些研究将极大地提高我们对调节正常情况的转录后机制的理解 脊椎动物心房发育以及人类AVSD和ASD的分子病因。最终，这些 研究将为改进能够预防儿童CHD并有效的治疗方法奠定基础 修复成人受伤的心脏。