NEDD4 in right ventricular development

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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.