RBX1-CRLs Promote Perinatal Heart Development by Regulating Hippo-YAP Signaling

Abstract

Background: During development, the heart is the first organ to form, ensuring the pumping of blood through the body. Defects in cardiac morphogenesis can lead to congenital heart diseases (CHD) or predispose the heart to cardiomyopathies in adult life. The molecular mechanisms underlying this process remain incompletely understood. Cullin-RING E3 ubiquitin ligases (CRLs) are multi-subunit complexes critical to maintaining protein homeostasis by targeting diverse protein substrates for ubiquitination and subsequent degradation. In CRLs, RBX1 serves as the catalytic subunit that promotes the transfer of ubiquitin to the target protein. Germline ablation of RBX1 in worms, flies, and mice caused embryonic lethality. Deletion of RBX1 in zebrafish resulted in cardiac malformation and dysfunction, which was rescued by endothelial, but not myocardial, RBX1 overexpression. However, the cardiomyocyte-autonomous role of RBX1 in the heart in vertebrates is unknown. Therefore, we hypothesize that cardiomyocyte RBX1 is indispensable for cardiac development. Methods and Results: We generated a cardiomyocyte-specific RBX1 knockout (RBX1CKO) mouse model via αMHCCre. RBX1-deficient mouse embryos were initially morphologically indistinguishable from the littermate controls at E14.5, but began to show peripheral edema and hemorrhage at E16.5, and eventually died around E18.5. Histological analysis of RBX1CKO hearts revealed severe myocardial hypoplasia and bi-ventricular non-compaction by E14.5. Bulk RNA sequencing and proteomic analyses of RBX1CKO hearts identified perturbations in glycolytic to oxidative transition and dysregulation of sarcomeric proteins. EdU incorporation assay showed a significant reduction in cardiomyocyte proliferation in E14.5 RBX1-deficient hearts, accompanied by the impaired nuclear translocation of YAP, a co-transcriptional factor essential for cardiomyocyte proliferation and maturation. Mechanistically, RBX1 deficiency destabilized multiple CRLs and resulted in the accumulation of Hippo kinases and adaptor proteins such as SAV1, LATS1/2, and MOB1, leading to the elevated phosphorylation and inactivation of YAP. We further demonstrated that loss of RBX1 inhibited the degradation of the Hippo adaptor MOB1, likely due to suppressed ubiquitination. Conclusion: Our study identifies RBX1 as an essential regulator of ventricular maturation and found that RBX1-mediated proteolysis of Hippo kinases serves as a critical checkpoint to ensure YAP activation and, hence, cardiomyocyte proliferation and maturation during cardiac development. AHA Predoctoral Fellowship, American Heart Association, 23PRE1012641. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.