NEDD4 in right ventricular development

项目摘要 右心室发育异常与先天性心脏病有关. 虽然RV的发育起源和初始规格一直是深入研究的主题，但关键的是， 参与随后RV生长和扩张的分子途径 形成有待探索。我们分析了组成型心肌细胞（CM）特异性基因敲除小鼠 (cKO)Nedd 4的表达揭示了一种独特的RV特异性表型。在早期胚胎阶段Nedd 4的缺失， Xmlc 2-Cre在E7.5早期发育的CM中特异性表达，在E7.5时导致显著的RV扩张， 出生后阶段，在E14.5时观察到缺陷。相反，左心室和肺血管， 看起来很正常NEDD 4是在CM中高度表达的HECT E3泛素连接酶。严重的RV特异性 Nedd 4 cKO的异常突出了NEDD 4在RV发展中的重要性。探索分子 关于CM中NEDD 4缺失导致RV发育缺陷的机制，我们进行了蛋白质组学研究， E14.5时cKO和对照心室的分析。差异表达的蛋白质富集在途径中， 参与细胞膜组织、液泡运输和囊泡介导的运输。以确定 无论Nedd 4的丢失是否破坏细胞膜组织，我们对cKO进行了TEM分析， 控制E14.5的心脏。有趣的是，内质网和核周空间的大量扩张 cKO组右室组织中可见大细胞空泡，而左室组织正常 超微结构，表明Nedd 4是必不可少的细胞膜组织在RV CM。以确定 在RV开发的不同阶段，针对NEDD 4的持续CM特定要求，我们生成了 他莫昔芬诱导的CM特异性Nedd 4敲除小鼠模型在胚胎期、围产期或 成人阶段我们的初步数据显示，在出生后或成年CM中，Nedd 4的消融并不 导致在Nedd 4 cKO中观察到的扩张的RV表型，这表明在Nedd 4 cKO中对Nedd 4的特异性需求。 胚胎CM，虽然Nedd 4控制RV发育的关键时间窗口， 其分子机制仍然未知。Nedd 4 cKO小鼠的独特RV表型 将使我们能够揭示心脏发育所需的RV特异性通路，并模拟RV的影响。 功能障碍对心脏性能的影响，与RV功能障碍相关的CHD相关。我们将测试 假设Nedd 4在CM亚细胞膜组织和RV发育中起重要作用。 我们的具体目标是：1）阐明Nedd 4在RV发育中的作用，以及 先天性RV缺陷对心脏性能的影响，通过组织学、生理学、生化和分子生物学 组成型Nedd 4 cKO小鼠的分析;和2）为了确定Nedd 4在其期间的临界窗口， 通过分析Tnnt 2 MerCreMer：Nedd 4诱导型CM特异性敲除（ticKO）小鼠，确定RV发育所需的时间。

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.