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.

项目总结/摘要 先天性心脏病（CHD）是最常见的先天畸形。5%的冠心病包括 房室间隔缺损（AVSD）。然而，大多数AVSD的分子病因并非如此 明白此外，CHD可在以后的生活中引起心血管疾病，导致心律失常， 中风和过早死亡为了开发能够预防CHD和治愈特定CHD的新疗法， 心血管组织，这是至关重要的，以获得指导正常的基本机制的理解 心腔发育和再生。因此，我们实验室的长期目标是了解 保守的机制，指导发展的个别心腔和腔室特异性 脊椎动物的再生机制。已知需要很少的信号来专门指导心房肌 心房再生的特定调节因子尚不清楚。具体目标是 我们的建议是阐明同线长非编码RNA（lncRNA）as-oca限制 斑马鱼心房发育和再生通过抑制Nr 2f 1a翻译。这方面的研究 这些建议与人类健康有关，因为最近的基因组分析表明， 核受体Nr 2f 2与人类的AVSD相关。虽然Nr 2f 2基因敲除小鼠和体外研究 与人类干细胞的研究揭示了心房发育中对Nr 2f 1和Nr 2f 2的需求， Nr 2f蛋白指导正常心房发育的机制还不完全清楚。 重要的是，目前还没有了解调控Nr 2f的表观遗传lncRNA依赖性机制。 proteins.我们对新的斑马鱼突变橡子虫（aco）的初步分析表明， as-oca的表达特异性地限制了晚期分化的第二心野（SHF）衍生的 心房细胞在目标1中，我们将使用囊胚细胞移植，体内心肌细胞分化测定， 基因组编辑，以确定心房缺陷的细胞需求和as增加的原因， ACO突变体中的OCA表达。我们不知道as-oca如何抑制aco突变体中的Nr 2f 1a翻译。在 目的2，我们将使用RNA和核糖体结合技术和功能丧失方法来确定是否作为- oca通过与nr 2f 1a 3'非翻译区相互作用抑制nr 2f 1a翻译。除了它们 在发育过程中的需求，我们发现as-oca和nr 2f 1a特异性地表达在心房， 成年斑马鱼在目的3中，我们将测试as-oca对胚胎和成人心脏模型的影响， 再生由于Nr 2f转录因子在所有脊椎动物的心房发育中起保守作用， 这些研究将极大地提高我们对转录后机制的理解， 脊椎动物心房发育和人类AVSD和ASD的分子病因学。最终，这些 这些研究将为改进能够预防儿童CHD的治疗方法奠定基础， 修复成年人受伤的心脏