NEDD4 in right ventricular development

NEDD4 在右心室发育中的作用

• 批准号: 10472746
• 负责人: Xi Fang
• 金额: $ 50.53万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10472746
• 关键词: Ablation; Adult; Agreement; Animal Model; Anterior; Binding; Biochemical; C2 Domain; Cardiac; Cardiac Myocytes; Cellular Membrane; Critical Pathways; Data; Defect; Development; Embryo; Endoplasmic Reticulum; Family; Growth; Heart; Heart Atrium; Histologic; Human; Knock-out; Knockout Mice; Left ventricular structure; Mediating; Membrane; Mesoderm; Modeling; Molecular; Molecular Analysis; Morphology; Mus; N-terminal; Pathway interactions; Performance; Perinatal; Phenotype; Phospholipids; Physiological; Play; Proline; Proteins; Proteomics; Pulmonary vessels; Right Ventricular Dysfunction; Right ventricular structure; Role; Tamoxifen; Testing; Time; Tissues; Transmission Electron Microscopy; Tube; UBE3A gene; Vacuole; Ventricular; Vesicle; cardiogenesis; congenital heart disorder; differential expression; early embryonic stage; heart function; malformation; member; mouse model; mutant; nerve stem cell; novel; postnatal; ubiquitin-protein ligase

PROJECT SUMMARY Abnormalities in right ventricle (RV) development are associated with congenital heart disease (CHD). Although developmental origins and initial specification of the RV has been a subject of intense study, critical molecular pathways involved in subsequent RV growth and expansion at later stages following chamber formation remain to be explored. Our analysis of mice with constitutive cardiomyocyte (CM)-specific knockout (cKO) of Nedd4 revealed a unique RV-specific phenotype. Deletion of Nedd4 during early embryonic stages by Xmlc2-Cre, which specifically expresses in early developing CMs from E7.5, resulted in dramatic RV dilation at postnatal stages, with defects observed at E14.5. Conversely, the LV, as well as the pulmonary vessels, appeared normal. NEDD4 is a HECT E3 ubiquitin ligase highly expressed in CMs. The severe RV-specific abnormalities in Nedd4 cKO highlight the importance of NEDD4 in RV development. To explore molecular mechanisms by which loss of NEDD4 in CMs resulted in RV development defects, we performed proteomics analysis of cKO and control ventricles at E14.5. Differentially expressed proteins were enriched in pathways involved in cellular membrane organization, vacuolar transport and vesicle-mediated transport. To determine whether loss of Nedd4 disrupts cellular membrane organization, we performed TEM analysis on cKO and control hearts at E14.5. Intriguingly, a massive expansion of endoplasmic reticulum and perinuclear space accompanied by large cellular vacuoles was observed in RV tissues of cKOs, while the LV displayed normal ultrastructure, suggesting that Nedd4 is essential for cellular membrane organization in RV CMs. To determine the ongoing CM-specific requirement for NEDD4 in RV development at different stages, we generated tamoxifen inducible CM-specific Nedd4 knockout mouse models to ablate Nedd4 at embryonic, perinatal or adult stages. Our preliminary data revealed that ablation of Nedd4 in either postnatal or adult CMs did not result in the dilated RV phenotype observed in Nedd4 cKOs, suggesting a specific requirement for Nedd4 in embryonic CMs, although the critical time window during which Nedd4 controls RV development, and detailed molecular mechanisms by which it does so remain unknown. The unique RV phenotype of Nedd4 cKO mice will allow us to uncover RV-specific pathways required for heart development, and to model the impact of RV dysfunction on cardiac performance, of relevance to CHDs associated with RV dysfunction. We will test the hypothesis that Nedd4 plays essential roles in CM subcellular membrane organization and RV development. Our Specific Aims are: 1) To elucidate the role of Nedd4 in RV development, as well as the impact of congenital RV defects on cardiac performance, by histological, physiological, biochemical, and molecular analyses of constitutive Nedd4 cKO mice; and 2) To determine the critical window during which Nedd4 is required for RV development by analysis of Tnnt2MerCreMer:Nedd4 inducible CM-specific knockout (ticKO) mice.

项目摘要 右心室（RV）发育异常与先天性心脏病（CHD）有关。 尽管RV的发育起源和初始规范是一项激烈研究的主题，但关键 腔室后后期参与随后的RV生长和扩张涉及的分子途径 编队仍有待探索。我们用组成型心肌细胞（CM）特异性敲除的小鼠分析 NEDD4的（CKO）揭示了独特的RV特异性表型。在早期胚胎阶段删除NEDD4 XMLC2-CRE在E7.5的早期开发CM中专门表达，导致RV急剧扩张 产后阶段，在E14.5处观察到缺陷。相反，LV以及肺部血管 看起来正常。 NEDD4是在CMS中高表达的Hect E3泛素连接酶。严重的RV特异性 NEDD4 CKO的异常强调了NEDD4在RV开发中的重要性。探索分子 CMS中NEDD4丢失导致RV发育缺陷的机制，我们进行了蛋白质组学 E14.5的CKO和控制心室的分析。差异表达的蛋白在途径中富含 参与细胞膜组织，液泡转运和囊泡介导的转运。确定 NEDD4的损失是否破坏细胞膜组织，我们对CKO和 在E14.5控制心。有趣的是，内质网和核周空间的大量扩张 在CKO的RV组织中观察到伴随着大细胞液泡，而LV显示正常 超微结构，表明NEDD4对于RV CMS中的细胞膜组织至关重要。确定 在不同阶段的RV开发中，NEDD4的持续CM特定要求，我们生成了 他莫昔芬可诱导的CM特异性NEDD4基因敲除小鼠模型在胚胎，围产期或 成人阶段。我们的初步数据表明，nedd4在产后或成人CMS中的消融均未 导致在NEDD4 CKO中观察到的扩张的RV表型，这表明对NEDD4的特定要求 胚胎CMS，尽管NEDD4控制RV开发的关键时间窗口，并详细介绍 这样做的分子机制仍然未知。 NEDD4 CKO小鼠的独特RV表型 将使我们能够发现心脏发育所需的RV特异性途径，并建模RV的影响 与RV功能障碍相关的心脏性能功能障碍，与CHD相关。我们将测试 NEDD4在CM亚细胞膜组织和RV开发中起着重要作用的假设。 我们的具体目的是：1）阐明NEDD4在RV开发中的作用，以及 先天性RV缺陷心脏性能，组织学，生理，生化和分子 组成型NEDD4 CKO小鼠的分析； 2）确定NEDD4为的关键窗口 通过分析TNNT2Mercremer：NEDD4诱导CM特异性敲除（Ticko）小鼠所需的RV开发所需。