Abstract Body: Ubiquitin (Ub)-dependent signaling regulates virtually every aspect of cellular function by maintaining protein homeostasis. Disruption of this tightly controlled system contributes to human diseases, including cardiomyopathy and heart failure. OTUD6B is an Ovarian Tumor (OTU) family deubiquitinase (DUB) that removes Ub from substrates. OTUD6B has been implicated in regulating cell-cycle progression, apoptosis, inflammation, and DNA repair, and mutations in OTUD6B are associated with intellectual disability and congenital heart defects, yet its cardiac role remains undefined.
Here we demonstrated that OTUD6B is essential for perinatal ventricular development and adult cardiac homeostasis. Germline Otud6b deletion in mice caused ventricular septal defects, cardiac hypoplaxia, and perinatal lethality, due to impaired cardiomyocyte cell-cycle progression and dysregulation of cell-cycle regulators (Cyclin E). To define its cardiomyocyte-autonomous functions beyond development, we generated αMHC-Cre-mediated, cardiomyocyte-specific OTUD6B knockout (CKO) mice. While cardiac function was preserved at 3 months, CKO mice exhibited reduced ejection fraction by 4 months, progressing to severe dilated cardiomyopathy, myocardial fibrosis, and heart failure by 7 months. Bulk-RNAseq prior to overt cardiac dysfunction revealed suppression of oxidative phosphorylation, fatty acid metabolism, and cardiac contraction, with GSEA confirming downregulation of mitochondrial gene programs. In cultured cardiomyocytes, silencing Otud6b impaired mitochondrial respiration, increased mitochondrial ROS, and markedly reduced CCCP-induced mitochondrial ubiquitination, accompanied by destabilization of PINK1 and Parkin—key regulators of mitophagy. These findings identify OTUD6B as a regulator of mitochondrial quality control. To assess the relevance of human OTUD6B mutations, we generated a cardiomyocyte-specific knock-in (CKI) model expressing the disease-associated R116* truncation (αMHC-Cre/+:Otud6b^KI/flox). CKI mice phenocopied CKO model, developing progressive cardiomyopathy and heart failure, supporting pathogenicity of the mutation.
In summary, we establish OTUD6B as a key regulator of heart development and adult cardiac function, acting in part through maintenance of mitochondrial homeostasis. These findings suggest that OTUD6B loss-of-function variants may increase cardiovascular risk, and the CKI model provides a platform for mechanistic investigation and therapeutic development.