OTUD6B Regulates Ventricular Chamber Maturation and Is Required for Mouse Embryonic Development

Deubiquitinases (DUBs) regulate protein stability and various cellular processes by removing ubiquitin from substrate proteins. OTUD6B, a member of the OTU DUB family, has been linked to intellectual disabilities and congenital heart defects through compound heterozygous or homozygous variants in the OTUD6B gene. However, its molecular and pathophysiological roles in the heart remain poorly understood. Here, we demonstrate that OTUD6B is essential for ventricular chamber development and postnatal survival. OTUD6B is ubiquitously expressed in mouse embryos. Hypomorphic Otud6b neo/neo mice, with a 90% reduction in OTUD6B protein, were viable, fertile, and showed no gross abnormalities up to 24 weeks. In contrast, Otud6b -/- mice, with biallelic loss of OTUD6B, exhibited growth retardation and failed to survive beyond postnatal day 1 (P1). Histological analysis revealed significant cardiac defects in Otud6b-/- mice, including ventricular septal defects (VSD) and severe bi-ventricular wall thinning as early as embryonic day 16.5 (E16.5). Echocardiography at E20.5 showed increased cardiac stiffness, a hallmark of heart failure. The severe cardiac phenotype arises from reduced cardiomyocyte proliferation due to defective cell cycle progression, characterized by S-phase arrest and decreased binucleation in OTUD6B-deficient cardiomyocytes. Unbiased transcriptomic analysis of E14.5 hearts reveal perturbations in cell cycle regulation, fatty acid metabolism, myogenesis and cardiomyocyte maturation in Otud6b -/- mice. HIF1α plays a pivotal role in cardiac development by regulating cardiomyocytes proliferation and metabolic transition from glycolysis to oxidative metabolism. Interestingly, overlapping OTUD6b-regulated transcriptome with HIF1a-regulated transcriptome (GSE61209) or HIF1a potential targets (GSE61247) suggest persistent activation of HIF1a signaling in Otud6b -/- hearts. Moreover, homozygous Otud6b knockin mice ( Otud6b KI/KI ) bearing pathogenic variants R145* in OTUD6B gene recapitulated the myocardial hypoplasia, ventricular septal defects, and perinatal lethality observed in Otud6b -/- mice, mirroring cardiac defects seen in patients with R145* mutations. At the molecular level, loss of OTUD6B reduced cyclin D/E protein levels while increasing HIF1a proteins, which may contribute to the abnormal cell cycle progression and disrupted cardiomyocyte metabolism. Loss of OTUD6B did not affect the transcripts of cyclin D/E and HIF1a, suggesting OTUD6B’s role in regulation of their proteolysis, directly or indirectly. Taken together, our findings establish an essential role for OTUD6B in perinatal ventricular chamber development and embryogenesis, likely by fine-tuning cell cycle regulators and HIF1α during heart development. This work is supported by NHLVI 2R01HL124248-06A1 and NIH/NHLBI 1R01HL16505. 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.