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. !

低塑性左心综合症（HLHS）是先天性心脏缺陷（CHD），其特征是小左脑室（LV）和型心理主动脉和主动脉/二尖瓣。强烈指出了HLHS的遗传病因出于高复发风险，但对HLHS的遗传基础知之甚少。临床研究表明 HLHS是多基因和遗传异质性的。对HLHS遗传学的见解来自我们最近从大规模小鼠诱变筛选中恢复了HLHS的第一个小鼠模型。从8 恢复了独立的HLHS小鼠线，鉴定了330个突变，没有共同的基因共享在8行之间。这些发现表明HLHS在遗传上是非常异构的，与人类研究。对一条突变小鼠系OHIA的详细分析显示，HLSH是通过两个突变引起的基因：SAP130，染色质中的SIN3A相关蛋白，修饰组蛋白脱乙酰基酶复合物（HDAC），和PCDHA9，一种介导细胞 - 细胞粘附的原钙粘蛋白。 LV发育不全被证明是由 SAP130突变，通过在CRISPR中复制的小心室表型证实这一发现 SAP130a斑马鱼突变体。 LV发育不全与心肌细胞增殖缺陷有关和心肌细胞周期停滞。在这项研究中，我们将研究细胞和分子机制以及驱动HLHS中LV发育不全的遗传相互作用，利用斑马鱼的独特优势和鼠标模型。在AIM 1中，我们将在斑马鱼和CRE实验中采用谱系追踪研究在小鼠中删除SAP130的删除，以测试SAP130以细胞自主方式起作用的假设调节心室/LV生长。这些研究将描述SAP130调节LV的细胞环境生长。在AIM 2中，我们将调查以下假设：肌sap130ohia突变会导致LV 通过调节心肌细胞周期和细胞增殖的靶基因下降质基因。这些研究会专注于Meis1，一种SAP130靶基因，也已知可以调节心肌细胞周期和产后细胞周期逮捕。同时，通过SAP130 CHIP-SEQ和RNA-SEQ分析确定的其他候选基因将通过生产和分析CRISPR靶向胚胎和老鼠。在AIM 3中，我们将探测染色质修饰符与RAS/MAPK信号通路的相互作用斑马鱼中使用反义的形态敲除HLHS的发病机理，具有敏感的遗传背景。阳性遗传相互作用将使用突变体或CRISPR靶向斑马鱼或小鼠进行验证。这项研究是由染色质修饰剂和RAS/MAPK突变意外恢复的动机所有8 HLHS小鼠系中的途径成分，表明染色质修饰符与 RAS/MAPK信号失调可能有助于HLHS的LV发育不全和复杂的遗传学。这些研究共同有助于阐明驱动心室的细胞和分子机制 HLHS中的低流质，可能产生新的治疗靶标的胎儿干预措施以恢复LV生长。呢