Abstract

The severe cardiotoxicity of doxorubicin (Dox) significantly restricts its clinical application. Deubiquitinating enzymes (DUBs) have emerged as a research hotspot in cardiac pathophysiology due to their precise regulation of protein function, localization, and degradation. However, the specific role of ubiquitin-specific peptidase 20 (USP20) in doxorubicin-induced cardiomyopathy (DIC) remains poorly understood. In this study, we employed single-cell RNA sequencing (scRNA-seq) to delineate the cell-type-specific expression pattern of USP20 in the heart and combined it with LC-MS/MS-coupled co-immunoprecipitation (co-IP) to identify its direct, endogenous substrate proteins in cardiomyocytes. Employing cardiomyocyte-specific Usp20-knockout (Usp20-CKO) mice, we demonstrated that USP20 deficiency profoundly exacerbates DIC by driving ferroptotic cell death. Mechanistically, the ubiquitin-specific protease domain of USP20 directly interacted with human antigen R (HuR). USP20 deubiquitinated HuR at lysine 154 by specifically cleaving K48-linked polyubiquitin chains, thereby preventing its proteasomal degradation and maintaining protein stability. Subsequently, the stabilized HuR bound to GPX4 mRNA, suppressing its degradation to mitigate ferroptosis and alleviate DIC. Furthermore, AAV9-mediated targeted overexpression of USP20 in cardiomyocytes significantly attenuated DIC severity. Crucially, this cardioprotective effect was completely abolished in cardiomyocyte-specific HuR-knockout (HuR-CKO) mice, establishing HuR as an indispensable downstream effector of USP20 in DIC. In summary, our findings demonstrate that USP20 inhibits ferroptosis and mitigates DIC by stabilizing HuR through targeted deubiquitination.