RBX1-Cullin RING Ubiquitin Ligases Are Indispensable For Perinatal Cardiac Development Through Regulating The Hippo-YAP Signaling Pathway

Defective cardiac development can lead to congenital heart diseases, the primary cause of congenital mortality. Recent insights into transcriptional regulatory mechanisms have greatly expanded our understanding of the signaling networks that shape cardiac development for postnatal life. However, the role of posttranslational mechanisms in this process remains largely unexplored. Cullin-RING ubiquitin E3 ligases (CRLs) are multi-subunit complexes composed of a catalytic subunit (RBX1/RBX2), a scaffold cullin protein, and a substrate receptor. By facilitating the ubiquitination and degradation of various intracellular proteins, CRLs regulate numerous cellular and pathophysiological processes. Yet, the specific role of CRLs, particularly their catalytic subunit RBX1, in mammalian heart development remains largely unknown. Here, we demonstrate that RBX1 is a dosage-sensitive regulator of cardiac morphogenesis. RBX1 is robustly expressed in fetal mouse hearts but downregulated in adult hearts. Surprisingly, mice with germline deletion of one allele of the RBX1 gene presented severe cardiac morphological abnormalities and died at perinatal stage. To evaluate the cell autonomous role of RBX1 in cardiogenesis, we deleted one allele of RBX1 via a cardiac-specific Cre line Xmlc2-Cre that is highly active beginning at E8.5. The RBX1 haploinsufficient mice presented hypoplastic ventricular myocardium that emerged as early as E13.5 and discernable ventricular septum defects at E18.5. These cardiac morphological abnormalities result in heart failure, evidenced by significantly compromised cardiac contractility revealed by echocardiography and eventually lethality around P5. Interestingly, deletion of one allele of RBX1 via αMHC-Cre, which turns on later than Xmlc2-Cre in cardiomyocytes, were viable, fertile, and showed no gross abnormalities at adulthood, indicating a crucial dosage effect of RBX1 on early cardiac morphogenesis. In contrast, homozygous deletion of RBX1 via αMHC-Cre, resulted in myocardial hypoplasia and biventricular non-compaction by E16.5, culminating in embryonic heart failure and lethality at E18.5. The severe cardiac phenotypes in both models were associated with diminished cardiomyocyte proliferation and cardiomyocyte immaturity, the latter being characterized by disrupted sarcomere assembly, deficits in metabolic transition from glycolysis to oxidative metabolism and impaired ion channel switch (Kir2.1 to HCN4). Mechanistically, loss of RBX1 destabilized cullin proteins and impaired the activities of CRLs, leading to accumulation of Hippo kinases (LATS1/2, MST1, and MST2) and their adaptor proteins (SAV and MOB1). Consequently, YAP failed to enter the nucleus and initiate the expression of genes essential for cardiac morphogenesis. We further reveal that RBX1 targets MOB1 for ubiquitination and degradation. In conclusion, our findings demonstrate the indispensable role of RBX1 in embryonic cardiac development. Sustained and high levels of RBX1 are necessary to regulate the Hippo-YAP pathway thus promoting cardiomyocyte proliferation and maturation, and ventricular chamber development. Our findings also highlight that multiple aspects of embryonic cardiac morphogenesis and function are exquisitely sensitive to small changes in RBX1 expression levels. This work was funded by the American Heart Association Predoctoral Fellowship to J.Z.C (23PRE1012641). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.