NEDD4 in right ventricular development

项目总结 右室发育异常与先天性心脏病(CHD)有关。 尽管房车的发展起源和最初的规格一直是一个密集研究的主题，但关键是 参与房室后后期RV生长和扩张的分子途径 形成机制仍有待探索。我们对结构性心肌细胞(CM)特异性基因敲除小鼠的分析 Nedd4的CKO具有独特的RV特异性表型。Nedd4基因在早期胚胎阶段的缺失 Xmlc2-Cre在e7.5开始的早期CMS中特异表达，导致RV在 出生后阶段，在E14.5观察到缺陷。相反，左心室和肺血管， 看起来很正常。NEDD4是一种在CMS中高表达的Hect E3泛素连接酶。严重的轮状病毒特异性 Nedd4 CKO的异常突出了NEDD4在RV发育中的重要性。探索分子 在CMS中丢失NEDD4导致RV发育缺陷的机制，我们进行了蛋白质组学研究 E14.5的CKO和对照脑室的分析。差异表达的蛋白质在通路中得到丰富 参与细胞膜的组织、空泡转运和囊泡介导的转运。要确定 Nedd4缺失是否破坏细胞膜组织，我们对CKO和CKO进行了透射电子显微镜分析 在E14.5控制心跳。耐人寻味的是，内质网和核周空间的大规模扩张 CKO的右室组织中可见大的细胞空泡，而左室未见异常。 提示Nedd4在RV CMS细胞膜组织中起重要作用。要确定 在RV开发的不同阶段，正在进行的CM特定于NEDD4的需求，我们生成了 他莫昔芬诱导CM特异性Nedd4基因敲除小鼠模型在胚胎、围产期或 成人期。我们的初步数据显示，无论是出生后还是成年CMS，Nedd4的消融都没有 结果在Nedd4 cKO中观察到扩大的RV表型，提示对Nedd4在 胚胎CMS，尽管Nedd4控制RV发育的关键时间窗口，并详细 它这样做的分子机制仍然不清楚。Nedd4 CKO小鼠独特的轮状病毒表型 将使我们能够发现RV特定的心脏发育所需的途径，并对RV的影响进行建模 功能障碍对心脏功能的影响，与RV功能障碍相关的冠心病有关。我们将测试 假设Nedd4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Nedd4在RV发展中的作用以及对RV的影响 先天性RV缺陷对心脏功能影响的组织学、生理学、生化和分子生物学研究 Nedd4基因CKO小鼠的分析；2)确定Nedd4基因的临界窗口 通过对Tnnt2MerCreMer：Nedd4诱导的CM特异性基因敲除(TicKO)小鼠的分析，发现RV发育所需的基因。

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.