Pathological cardiac hypertrophy can lead to heart failure. The molecular mechanisms underlying pathological cardiac hypertrophy remain undefined. This study aimed to examine the role and molecular mechanisms of a deubiquitinating enzyme (DUB), ubiquitin-specific protease 20 (USP20) in cardiac hypertrophy. Hypertrophic hearts were conducted for RNA-sequencing to screen the expression profiles of DUBs. Cardiomyocyte-specific USP20 knockout and overexpression mouse models were generated to explore its function. Co-immunoprecipitation coupled with liquid chromatography-mass spectrometry/mass spectrometry were performed to screen potential USP20 substrates. Cleavage under targets and tagmentation assay with high-throughput sequencing was utilized to identify the potential downstream targets of STAT3. We identified cardiomyocyte-enriched USP20 is downregulated in cardiac hypertrophy. Cardiomyocyte-specific USP20 deficiency exacerbated cardiac hypertrophy induced by Angiotensin II and transverse aortic constriction, whereas cardiomyocyte-specific USP20 overexpression ameliorated the phenotype. We further identified STAT3 is a substrate of USP20 during cardiac hypertrophy through direct binding with DUSP2 domain. Mechanistically, USP20 removes K63 ubiquitin chains from STAT3 at the K177 site via its H645 active site, reducing STAT3 phosphorylation and nuclear translocation. This prevents STAT3 from binding to the coactivator-associated arginine methyltransferase 1 (CARM1) promoter, thereby promoting CARM1 transcription and improving cardiac hypertrophy. Importantly, we discover with STAT3 inhibitor stattic that STAT3 is a key substrate through which USP20 exerts its therapeutic effect on cardiac hypertrophy. These results elucidate a critical role for a novel USP20/STAT3/CARM1 axis in cardiomyocytes and an exciting new avenue study for therapies to treat cardiac hypertrophy.
