Mechanism of LV Hypoplasia in Hypoplastic Left Heart Syndrome Supplement

左心发育不全综合征补充剂中左室发育不全的机制

基本信息

批准号：
10091850
负责人：
CECILIA W. LO
金额：
$ 5.77万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2021-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10091850
关键词：
Affect Alleles Animal Model Aorta Apoptosis Automobile Driving Balloon Angioplasty Biological Models Blood flow Candidate Disease Gene Cardiac Cardiac Myocytes Cell Cycle Cell Cycle Arrest Cell Lineage Cell Proliferation Cell-Cell Adhesion Cells ChIP-seq Chromatin Chromatin Remodeling Factor Clinical Research Clustered Regularly Interspaced Short Palindromic Repeats Complex Conceptus Congenital Abnormality Congenital Heart Defects Cre driver Defect Development Diagnosis Echocardiography Embryo Epigenetic Process Ethylnitrosourea Gene Targeting Genes Genetic Genetic Models Genetic Predisposition to Disease Growth Growth and Development function Heart Histone Deacetylase Homeobox Human Hypoplastic Left Heart Syndrome Infant Mortality Intervention Knock-out Left Left ventricular structure Lesion Light Live Birth LoxP-flanked allele MAP Kinase Gene MAPK Signaling Pathway Pathway Mediating Mitral Valve Modeling Molecular Molecular Genetics Morbidity - disease rate Mus Mutagenesis Mutant Strains Mice Mutation Operative Surgical Procedures Pathogenesis Pathway interactions Patients Phenotype Play Prenatal Diagnosis Production Proteins Pump Recovery Recurrence Reporter Right ventricular structure Risk Role Signal Transduction Structure Survival Rate Testing Therapeutic Tissues Ventricular Zebrafish aortic valve congenital heart disorder developmental genetics druggable target experimental study fetal hemodynamics insight knock-down mortality mouse model mutant new therapeutic target palliative postnatal progenitor recruit restoration transcription factor transcriptome sequencing

项目摘要

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect (CHD) characterized by a small left ventricle (LV) and hypoplastic aorta and aortic/mitral valves. A genetic etiology for HLHS is strongly indicated by high recurrence risk, but the genetic underpinning for HLHS is poorly understood. Clinical studies suggest HLHS is multigenic and genetically heterogeneous. Insights into the genetics of HLHS has come from our recent recovery of the first mouse models of HLHS from a large-scale mouse mutagenesis screen. From 8 independent HLHS mouse lines recovered, 330 mutations were identified, with no genes shared in common between the 8 lines. These findings indicate HLHS is profoundly genetically heterogeneous, consistent with the human studies. Detailed analysis of one mutant mouse line, Ohia, showed HLSH is elicited by mutations in two genes: Sap130, a Sin3a associated protein in the chromatin modifying histone deacetylase complex (HDAC), and Pcdha9, a protocadherin mediating cell-cell adhesion. The LV hypoplasia was shown to be elicited by the Sap130 mutation, a finding confirmed with replication of a small ventricle phenotype in a CRISPR generated sap130a zebrafish mutant. The LV hypoplasia was associated with a cardiomyocyte cell proliferation defect and cardiomyocyte cell cycle arrest. In this study, we will investigate the cellular and molecular mechanisms and genetic interactions driving the LV hypoplasia in HLHS, leveraging the unique strengths of the zebrafish and mouse models. In Aim 1, we will employ lineage tracing studies in zebrafish and experiments with Cre deletion of Sap130 in mice to test the hypothesis that Sap130 functions in a cell autonomous manner to regulate ventricular/LV growth. These studies will delineate the cellular context in which Sap130 regulates LV growth. In Aim 2, we will investigate the hypothesis that the hypomorphic Sap130Ohia mutation causes LV hypoplasia via target genes that regulate cardiomyocyte cell cycle and cell proliferation. These studies will focus on Meis1, a Sap130 target gene, also known to regulate cardiomyocyte cell cycle and postnatal cell cycle arrest. In parallel, additional candidate genes identified via Sap130 ChIP-seq and RNA-seq analysis will be assessed for their role in LV hypoplasia with production and analysis of CRISPR targeted embryos and mice. In Aim 3, we will probe the interaction of chromatin modifiers with the Ras/MAPK signaling pathway in the pathogenesis of HLHS using antisense morpholino gene knockdown in zebrafish with a sensitized genetic background. Positive genetic interactions will be validated using mutant or CRISPR targeted zebrafish or mice. This study is motivated by the unexpected recovery of mutations in chromatin modifiers and Ras/MAPK pathway components in all 8 HLHS mouse lines, suggesting chromatin modifiers in combination with dysregulated Ras/MAPK signaling may contribute to the LV hypoplasia and complex genetics of HLHS. Together these studies will help to elucidate the cellular and molecular mechanisms driving the ventricular hypoplasia in HLHS, findings that may yield new therapeutic targets for fetal intervention to recover LV growth. !

左心发育不良综合征 (HLHS) 是一种先天性心脏缺陷 (CHD)，其特征是左心室小心室 (LV) 和发育不全的主动脉和主动脉/二尖瓣。强烈表明 HLHS 的遗传病因高复发风险是导致 HLHS 的主要原因，但 HLHS 的遗传基础却知之甚少。临床研究表明 HLHS 具有多基因性和遗传异质性。对 HLHS 遗传学的深入了解来自我们最近从大规模小鼠诱变筛选中恢复了第一个 HLHS 小鼠模型。 8点起独立的 HLHS 小鼠系恢复，鉴定出 330 个突变，没有共同基因 8 条线之间。这些发现表明 HLHS 具有深刻的遗传异质性，与人类研究。对一种突变小鼠系 Ohia 的详细分析表明，HLSH 是由两种小鼠中的突变引起的基因：Sap130，染色质修饰组蛋白脱乙酰酶复合物 (HDAC) 中的 Sin3a 相关蛋白，和 Pcdha9，一种介导细胞间粘附的原钙粘蛋白。左心室发育不全被证明是由 Sap130 突变，这一发现通过 CRISPR 生成的小心室表型复制得到证实 sap130a 斑马鱼突变体。左心室发育不全与心肌细胞增殖缺陷有关和心肌细胞细胞周期停滞。在这项研究中，我们将研究细胞和分子机制和遗传相互作用驱动 HLHS 中的左室发育不全，利用斑马鱼的独特优势和鼠标模型。在目标 1 中，我们将在斑马鱼中进行谱系追踪研究并使用 Cre 进行实验在小鼠中删除 Sap130，以检验 Sap130 以细胞自主方式发挥作用的假设调节心室/左室生长。这些研究将描绘 Sap130 调节 LV 的细胞环境生长。在目标 2 中，我们将研究亚等态 Sap130Ohia 突变导致 LV 的假设通过调节心肌细胞周期和细胞增殖的靶基因导致发育不全。这些研究将重点关注Meis1，Sap130的靶基因，也已知调节心肌细胞周期和产后细胞循环逮捕。与此同时，通过 Sap130 ChIP-seq 和 RNA-seq 分析鉴定出的其他候选基因将通过 CRISPR 靶向胚胎的生产和分析来评估它们在左室发育不全中的作用老鼠。在目标 3 中，我们将探讨染色质修饰剂与 Ras/MAPK 信号通路的相互作用使用具有致敏基因的斑马鱼中的反义吗啉代基因敲低研究 HLHS 的发病机制背景。将使用突变体或 CRISPR 靶向斑马鱼或小鼠来验证积极的遗传相互作用。这项研究的动机是染色质修饰剂和 Ras/MAPK 突变的意外恢复所有 8 个 HLHS 小鼠系中的通路成分，表明染色质修饰剂与 Ras/MAPK 信号传导失调可能导致 HLHS 的左室发育不全和复杂的遗传学。这些研究共同将有助于阐明驱动心室的细胞和分子机制 HLHS 发育不全，这一发现可能为胎儿干预以恢复左心室生长提供新的治疗靶点。！