Cardiomyocyte-enriched USP20 ameliorates pathological cardiac hypertrophy by targeting STAT3-CARM1 axis

doi:10.21203/rs.3.rs-5264946/v1

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Cardiomyocyte-enriched USP20 ameliorates pathological cardiac hypertrophy by targeting STAT3-CARM1 axis

https://doi.org/10.21203/rs.3.rs-5264946/v1

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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.

Biological sciences/Molecular biology

Health sciences/Cardiology

Pathological cardiac hypertrophy is an adaptive response to various forms of cardiovascular stress, temporarily enhancing cardiac function.^1–3 Although this compensatory response is initially advantageous, it leads to heart failure if the underlying stimulus persists.^4–6 Currently, classical medicine such as β-adrenergic receptor blockers and renin-angiotensin-aldosterone system inhibitors can improve pathological cardiac hypertrophy, but the occurrence is still high, strongly suggesting that modulator proteins or the other signaling pathways also contribute to pathological cardiac hypertrophy.^7–11 Therefore, investigating the pathogenesis of cardiac hypertrophy and identifying novel therapeutic targets from diverse perspectives remain a significant challenge in the field of cardiology.

Accumulating evidence illustrates that protein ubiquitination plays a crucial role in various cardiac pathologies, including cardiac hypertrophy.^12–15 ischemic heart disease.^16,17 and heart failure.^18,19 Protein ubiquitination is a complex and dynamic post-translational modification and is mediated by both E3 ligases and deubiquitinating enzymes (DUBs), which regulate protein function, localization, and degradation.²⁰ The ubiquitin itself can form various types of chains by linking through any of its seven lysine residues (K6, K11, K27, K29, K33, K48, and K63). The two most common types are K48-linked and K63-linked chains. K48-linked ubiquitin chains typically signal for proteasomal degradation, whereas K63-linked chains are more often involved in non-degradative processes such as DNA repair and signaling transduction.²¹ Recent studies have identified two DUBs, ubiquitin-specific peptidase 25 (USP25) and josephin domain containing 2 (JOSD2), both capable of regulating the stability of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 2a (serca2a) to protect hearts from hypertrophy.^22,23 Additionally, USP28 maintains mitochondrial homeostasis in diabetic cardiomyopathy by modulating the PPARα-Mfn2 axis.²⁴ Hence, DUBs have emerged as potential therapeutic targets for cardiac hypertrophy and heart failure.

As a member of the deubiquitinase family, USP20, is crucial for regulating protein stability.²⁵ and metabolic processes.²⁶ Snail homolog 2 (SNAI2), a transcription factor that promotes metastasis, is a highly unstable protein subject to degradation via the ubiquitin-proteasome pathway; USP20 regulates SNAI2 ubiquitination and promotes its stability, leading to breast cancer metastasis.²⁵ In addition, USP20 promotes cancer progression by inhibiting autophagy and enhancing cell proliferation.²⁷ While previous studies on USP20 have largely focused on its involvement in cancer signaling pathways, however, its role in cardiovascular diseases, particularly in cardiac hypertrophy remains undefined.

Signal transducer and activator of transcription 3 (STAT3) serves as a pivotal member of the STAT family and acts as a transcription factor, playing an essential role in both cardiac physiology and pathophysiology.²⁸ including cell survival, energetics, and metabolism.^29,30 Moreover, STAT3 protein can be stabilized and activated through post-translational ubiquitination modifications, further enhancing its function.^31–33 In the cardiovascular diseases, the mechanisms by which DUBs modulate STAT3 ubiquitination and subsequently impact its transcriptional regulatory capacity remain poorly understood.

In this study, we identified cardiomyocyte-enriched USP20 is a protective regulator in human and mouse cardiac hypertrophy. Cardiomyocyte-specific deficiency of USP20 exacerbated cardiac hypertrophy and dysfunction, whereas cardiomyocyte-specific overexpression of USP20 ameliorated cardiac function upon hypertrophy. Mechanistically, we identified STAT3 as a substrate of USP20 in cardiomyocytes. USP20 impedes the nuclear transcription activity of STAT3 by removing its K63-linked ubiquitin chains. Our findings reveal a novel cardiomyocyte-specific USP20-STAT3-CARM1 (coactivator-associated arginine methyltransferase 1) axis that regulates cardiac hypertrophy and heart failure, and suggest that USP20 may serve as a potential therapeutic target for cardiac hypertrophy and heart failure.

Identification of cardiomyocyte-enriched USP20 as a downregulated factor in mouse and human cardiac hypertrophy

Recent studies suggest that deubiquitinating enzymes (DUBs) contribute to the development of pathological cardiac hypertrophy.³⁴ We first analyzed the mRNA profiles of DUBs from seven families in the hypertrophic myocardium of mice challenged with Angiotensin (Ang II), and found significant alterations in the gene expression of Otud1, Usp28, and Usp20 (Fig. 1a). As the effects of OTUD1 and USP28 in diseased hearts have been previously documented.^24,31 therefore, we sought to examine the unknown role of USP20 in cardiac pathophysiology. We confirmed Usp20 mRNA (Fig. 1b, c) and protein (Fig. 1d, e) expression levels were both attenuated in mice hearts induced by Ang II and transverse aortic constriction (TAC), compared to the control or sham group respectively. Importantly, similar results of USP20 were obtained from human hypertrophic myocardium of patients with heart failure (Fig. 1f, g).

To determine the cellular source of downregulated USP20 in the hypertrophic myocardium, we conducted single-cell RNA sequencing (scRNA-Seq) on approximately 16928 single cells from Ang II-administered mice hearts. Based on specific marker genes expression, we categorized nine cell types: cardiomyocytes (CMs), endothelial cells (ECs), fibroblasts (FBs), neutrophil (NPs), macrophages (MPs), T/NK cells, smooth muscle cells (SMCs), Lymphatic endothelial cells (Lym Ecs) and B cells. Significantly, Usp20 mRNA expression was predominantly observed in cardiomyocytes (Fig. 1h, i). Similarly, USP20 protein expression was significantly higher in cardiomyocytes compared to non-cardiomyocytes (Fig. 1j). In addition, the protein expression of USP20 in neonatal rat cardiomyocytes (NRCMs) was downregulated upon Ang II treatment in a time-dependent manner (Supplementary Fig. 1a, b). Furthermore, immunofluorescence staining of mouse heart stimulated by Ang II and TAC showed that the downregulated USP20 was primarily localized in α-actinin⁺ cardiomyocytes, rather than in CD68⁺ macrophages or vimentin⁺ fibroblasts (Fig. 1k, l and supplementary Fig. 2c, d). Taken together, we identified USP20 as a cardiomyocyte-enriched factor, downregulated in mouse and human hypertrophic hearts.

Cardiomyocyte-specific deficiency of USP20 exacerbates cardiac hypertrophy and dysfunction induced by Ang II

We generated cardiomyocyte-specific USP20 knockout mice (USP20 CKO) by breeding USP20^fl/fl mice with Myh6-Cre mice (Supplementary Fig. 2a) and assessed the expression levels of USP20 across various organs and tissues in USP20 CKO mice (Supplementary Fig. 2b, c). USP20 CKO mice, along with littermate USP20^fl/fl mice (as control counterparts), were implanted subcutaneously with osmotic mini-pumps delivering Ang II for 4 weeks (Fig. 2a). During Ang II-induced cardiac hypertrophy, serum Ang II levels did not affect the body weight (BW) of the mice (Supplementary Fig. 3a, b). Both USP20 CKO and USP20^fl/fl mice exhibited a significant increase in systolic blood pressure (SBP), but no significant differences between the two groups (Supplementary Fig. 3c, d). Non-invasive echocardiography results showed that cardiomyocyte deficiency of USP20 caused more pronounced cardiac dysfunction in the mice administrated with Ang II (Fig. 2b). This was characterized by decreased ejection fraction and fractional shortening (Fig. 2c, d and Supplementary Table 3). USP20 CKO mice showed exacerbated Ang II-induced heart enlargement (Fig. 2e), and increased the ratio of heart weight (HW) to BW (Fig. 2f), as well as the ratio of HW to tibial length (TL) (Fig. 2g). Cardiomyocyte size, as shown by H&E staining (Fig. 2h) and wheat germ agglutinin (WGA) staining (Fig. 2i, j), exhibited similar patterns of hypertrophy. Furthermore, cardiac fibrosis was significantly increased in Ang II-administered USP20 CKO mice compared to USP20^fl/fl mice assessed with masson's trichrome staining (Fig. 2k, l and Supplementary Fig. 3e, f). Additionally, the mRNA levels of hypertrophic markers Myh7 and Anp were significantly increased in the heart tissues of Ang II-administered USP20 CKO mice compared to USP20^fl/fl mice (Fig. 2m). These findings reveal that cardiomyocyte-specific knockout of USP20 exacerbates cardiac hypertrophy and dysfunction induced by Ang II.

We then determined the effect of USP20 on cardiomyocyte hypertrophy in vitro. We showed that increasing protein level of USP20 further reduced the expression of hypertrophic markers (β-MYHC, ANP in protein; Myh7, Anp in mRNA) (Supplementary Fig. 4a, b, c). The Ang II-induced increase in cardiomyocyte surface area was also decreased (Supplementary Fig. 4d, e). In contrast, decreasing USP20 protein expression using siRNA (si-USP20) led to opposite effects, with an increase in hypertrophic marker expression and cardiomyocyte surface area (Supplementary Fig. 4f-j).

Deficiency of cardiomyocyte-specific USP20 aggravates cardiac hypertrophy and dysfunction induced by TAC

Next, we investigated whether USP20 played an important role in transverse aortic constriction (TAC)-induced cardiac hypertrophy in both USP20 CKO mice and USP20^fl/fl mice (Fig. 3a). Post-procedure, we assessed the aortic blood flow velocity at the construction site using flow doppler ultrasound, observing a significant increase in blood flow velocity following TAC compared to the sham group (Supplementary Fig. 5a, b). Echocardiography showed that TAC-induced USP20 CKO mice exhibited more severe cardiac dysfunction than TAC-induced USP20^fl/fl control mice (Fig. 3b, c, d; Supplementary Table 4). Moreover, USP20 CKO mice appeared exacerbated cardiac hypertrophic phenotype in response to TAC, such as gross heart size (Fig. 3e), HW/BW (Fig. 3f), and HW/TL (Fig. 3g). Consistently, pathological examination further confirmed that the deficiency of USP20 resulted in more pronounced myocardial hypertrophy and fibrosis (Fig. 3i-l; Supplementary Fig. 5c, d). Furthermore, the mRNA levels of Myh7 and Anp in USP20 CKO mice were significantly higher than those in USP20^fl/fl mice subjected to TAC (Fig. 3m). These findings (Fig. 2 and Fig. 3) suggest that USP20 exerts a protective role in both TAC-induced and Ang II-induced cardiac hypertrophy in cardiomyocytes, suggesting that USP20 is a potential therapeutic target for preventing pathological cardiac hypertrophy.

Identification of STAT3 as a substrate of USP20 in cardiomyocytes

DUBs influence biological activities by modulating the degradation or function of substrates.²⁰ To identify the candidate substrates modulated by USP20 in cardiac hypertrophy, we utilized myocardial tissues from Ang II-induced mice in vivo and mouse-derived HL-1 cells in vitro to perform co-immunoprecipitation (Co-IP) coupled with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) to screen potential USP20 substrates, the workflow for this interactome analysis is illustrated in Fig. 4a. Excluding peptides related to antibody light and heavy chains and selecting target proteins with a fold change greater than 2.0, we identified that 55 USP20 physically binding proteins in HL-1 cells (dataset I, Fig. 4b), and 43 ones in heart tissues (dataset II, Fig. 4c). By comparing these two interactomes, we pinpointed potential substrates of USP20: STAT3, DSP, JUP, XRRA1. Interestingly, KEGG enrichment analysis indicated that the JAK-STAT pathway is significantly involved in USP20-mediated cardiac hypertrophy (Supplementary Fig. 6a). Therefore, our results suggest that USP20 may prevent cardiac hypertrophy through STAT3 as a substrate.

We next performed validation of the mass spectrometry and found that USP20 interacts with STAT3 in cardiac tissues and neonatal rat cardiomyocytes (NRCMs) following Ang II treatment (Fig. 4d, e), suggesting that USP20 directly binds to the STAT3 protein in cardiomyocytes. Next, we verified the interaction between USP20 and STAT3 by co-transfection of Flag-USP20 and His-STAT3 plasmids into NIH3T3 cells (Fig. 4f). Additionally, immunofluorescence analysis confirmed the co-localization of both exogenous and endogenous USP20 and STAT3 in NIH3T3 cells (Fig. 4g, h) and NRCMs (Supplementary Fig. 6b, d). USP20 is composed of four distinct structural domains: the N-terminal zinc-finger ubiquitin-binding domain (ZnF-UBP), the catalytic (USP) domain, and two tandem DUSP domains (DUSP1, DUSP2).³⁵ Given that the ZnF-UBP domain of USP20 may play a physiological role unrelated to its ubiquitin-binding capacity.³⁵ we generated three USP20 mutants to delineate which domain interacts with STAT3 (Fig. 4i). By co-transfecting STAT3 and the mutated USP20 plasmids into NIH3T3 cells, we found that USP20 could not bind to STAT3 when amino acids 791 to 894 were deleted, whereas mutations in other domains did not affect binding (Fig. 4j). Taken together, we identified STAT3 is a substrate of USP20 in cardiomyocytes, in which USP20 directly binds to STAT3 through its DUSP2 domain.

USP20 attenuates the K63-linked deubiquitination of STAT3 at residue K177 through the active site H645

Previous studies have demonstrated that STAT3 can be modified by K63-linked ubiquitin chains, which in turn affects its phosphorylation and protein function.³⁶ This post-translational modification plays a crucial role in regulating the activity of STAT3 in various cellular processes, including its stability, localization, and interaction with other proteins.³⁷ Given that USP20 is a deubiquitinase that directly binds to STAT3, we determined whether USP20 could modulate the K63 ubiquitination of STAT3, thereby influencing its biological activity. Overexpression of Flag-USP20 did not increase the STAT3 protein level in NIH3T3 cells (Fig. 5a, b). The same phenomenon was observed in Ang II-induced USP20 CKO mice (Fig. 5c, d). We then generated USP20-knockout NIH3T3 cells (gUSP20) by using CRISPR/Cas9 technology. When cycloheximide (CHX) was added to USP20-knockout cells to inhibit new protein synthesis, the degradation rate of STAT3 protein remained unchanged (Fig. 5e, f). Ubiquitin has seven Lys residues (K6, K11, K27, K29, K33, K48, and K63) that can form polyubiquitin chains, with K48 and K63 linkages being the most prevalent.²¹ Thus, we transfected gUSP20 cells with mutated ubiquitin plasmids containing only active K48 (HA-K48) or K63 (HA-K63) sites and observed that the ubiquitin molecules on STAT3 were significantly increased in gUSP20 cells transfected with the HA-K63 plasmid compared to wild-type cells (Fig. 5g). Moreover, the K63 regulated ubiquitination level of STAT3 protein was much higher in myocardium tissues of Ang II-induced USP20 CKO mice than those of USP20^fl/fl mice (Fig. 5h). Together, these results demonstrate that USP20 removes K63-linked ubiquitin molecules from STAT3.

DUBs can cleave the amide bonds between ubiquitin molecules and substrate using active sites such as cysteine and histidine.³⁸ To explore the specific roles of these active sites in USP20, we generated mutants by substituting glycine for cysteine at position 154 and histidine at position 645 (Fig. 5i), both of which are highly conserved across species (Supplementary Fig. 7a). Then the mutants, USP20^C154A and USP20^H645A, were evaluated for their ability to bind and deubiquitinate STAT3. Our results showed that both mutants could still bind to STAT3 (Supplementary Fig. 7b). However, the USP20^C154A mutant retained its deubiquitination activity, while the USP20^H645A could no longer remove ubiquitn molecules from STAT3 (Fig. 5k). To further elucidate the role of the H645 site in cardiomyocytes, we assessed the impact of the H645 mutation on USP20's protective effects against Ang II-induced cardiomyocyte hypertrophy. The mutation of H645 abolished USP20's protective effect against Ang II-induced cardiomyocyte hypertrophy (Supplementary Fig. 7c-g). These findings reveal that USP20 promotes STAT3 deubiquitination and function through the active site H645, and this regulation is crucial for its protective role against cardiac hypertrophy.

STAT3 consists of six domains: N domain (ND), Coil-coil domain (CCD), DNA binding domain (DBD), Linker domain (LD), SH2 domain (SH₂D), and TAD domain (TAD).³⁹ We created six STAT3 truncation mutants, each lacking one of these domains (Supplementary Fig. 8a), to determine which domain is responsible for binding USP20. After co-transfection of these mutants with Flag-USP20 plasmid in HEK293T cells, it showed that the absence of amino acids 130–320, which constitute the CCD domain, disrupted the binding between STAT3 and USP20 (Supplementary Fig. 8b). These results suggest that STAT3 binds to USP20 through its CCD domain, which is known to regulate the phosphorylation and nuclear translocation of STAT3 via allosteric regulation of SH2.⁴⁰ As for the ubiquitination sites on STAT3 involved in pathological cardiac hypertrophy have not been reported, we utilized affinity-based ubiquitin peptide enrichment ubiquitomics to investigate the specific ubiquitination sites on STAT3 modulated by USP20 (Supplementary Fig. 9a). Through analyzed the results, we identified 15 potential ubiquitination lysine residues K87, K97, K161, K177, K244, K294, K348, K354, K548, K551, K601, K615, K626, K631 and K707 in STAT3 (Supplementary Fig. 9b). Given that USP20 binds to the CCD domain (130-320aa) of STAT3, we focused on lysine within this region (K161, K177, K244, and K294) and constructed mutant plasmids with lysine-to-arginine substitutions to simulate the deubiquitinated state of STAT3. Interestingly, the expression of p-STAT3 in nuclear is significantly decreased when K177, but not the other lysine residues, was mutated (Supplementary Fig. 9c, d). We then selected the K177 and examined its role in the USP20-mediated STAT3 ubiquitination in Ang II-incubated cardiomyocytes. We observed that the K177R mutation did not disrupt USP20-STAT3 binding but resulted in significantly lower STAT3 ubiquitination compared to the wild-type, and this reduction was not further decreased by USP20 overexpression (Fig. 5k). Taken together, USP20 eliminates the K63-linked deubiquitination of STAT3 at residue K177 through its active site H645 (Fig. 5l).

USP20 protects heart from cardiac hypertrophy by inhibiting STAT3 nuclear transcription to promote CARM1 expression

STAT3 serves multiple biological functions, primarily acting as a transcription factor that regulates the expression of numerous genes coding for various proteins.²⁸ Therefore, we further evaluated whether USP20 could enhance the nuclear translocation of STAT3. Previous study reported that STAT3 activation is controlled by phosphorylation at tyrosine 705 (pTyr705).^41,42 Therefore, we determined the level of p-STAT3 at Try705 following USP20 overexpression in Ang II-stimulated NRCMs. Overexpression of USP20 remarkably decreased the level of p-STAT3 without affecting the total STAT3 expression (Fig. 6a, b). Notably, USP20 overexpression reduced the nuclear translocation of p-STAT3 in Ang II-incubated NRCMs, whereas silencing USP20 significantly increased p-STAT3 level in nuclear (Fig. 6c, d). Interestingly, inhibition STAT3 by STAT3 inhibitor stattic attenuates STAT3 phosphorylation and nuclear translocation as well (Supplementary Fig. 10a, b). Karyopherin alpha 3 (KPNA3) has been identified as an auxiliary protein to facilitate STAT3 nuclear translocation⁴³, we then determined whether USP20 binding to STAT3 could interfere with the STAT3-KPNA3 association, thereby reducing nuclear translocation of STAT3. IP assay showed that Ang II stimulation enhanced the interaction between STAT3 and KPNA3, however, this interaction was diminished when USP20 was overexpressed in NRCMs (Supplementary Fig. 10c).

To identify the potential downstream targets of STAT3 following Ang II stimuli, we performed cleavage under targets and tagmentation (CUT&Tag) assay with high-throughput sequencing. The results showed that STAT3 peak-related genes significantly decreased within 1000 bp (Promotor 1k) of the gene transcriptional start site (TSS) (Fig. 6E, from 17.45–13.86%). Subsequently, we analyzed the up-regulated genes in EV + Ang II vs. USP20^OE + Ang II conduction and identified significant differences in the expression of cardiac function-related genes such as Stk3, Pten, Eno1, Atf4, Alkbh5, coactivator-associated arginine methyltransferase 1 (Carm1) and Pcna (Supplementary Fig. 10d). RT-qPCR analysis showed that, apart from increased expression of Carm1 and Atf4, the expression levels of other genes did not show significant alterations in USP20^OE-Ang II-incubated NRCMs (Supplementary Fig. 10e). CARM1 is a crucial factor for cardiac homeostasis and regulates multiple aspects of cardiomyocyte maturation, including cellular hypertrophic growth and myofibril expansion.⁴⁴ We hypothesized that USP20-STAT3 axis prevents from cardiac hypertrophy through transcriptional regulation of Carm1. To determine the interaction motif between STAT3 and Carm1, we predicted the binding sites of STAT3 to the Carm1 promoter regions by JASPAR (Fig. 6f). We analyzed the potential binding site which clustered in the − 1659 ~ 1650 bp of the Carm1 promoter regions. Using CUT&Tag and qPCR assays, we found that STAT3 directly bound to the promoter regions of Carm1, which was significantly increased by USP20 silencing (Fig. 6g, h). On the other hand, this binding was decreased when USP20 was overexpressed (Supplementary Fig. 10f, g). We also predicted that STAT3 peak enriched to the promoter region of Carm1 by using ENCODE database (Fig. 6i). Luciferase assays were then performed by cloning the mutated − 1659 to -1650 bp fragments of the Carm1 promoter (Mut-Carm1), which lacked the putative STAT3-binding site, and using the wild-type fragment as a control (Fig. 6j). Transfection of STAT3-overexpressing in NIH3T3 cells with either wild-type or mutant Carm1 promoter plasmids showed increased luciferase expression in vectors containing WT-Carm1, but not in those with Mut-Carm1 (Fig. 6k). Taken together, these results suggest that USP20 inhibits STAT3 transcriptional activity in nuclear and promotes Carm1 expression in cardiomyocytes (Fig. 6l).

Cardiomyocyte-specific overexpression of USP20 improves cardiac hypertrophy and dysfunction induced by Ang II

We next evaluated whether USP20 overexpression has a therapeutic effect on myocardial hypertrophy. We prepared a cardiomyocyte-targeting adeno-associated virus serovar 9 (AAV9) vector and the wide type mice were subjected to AAV9 carrying USP20 (USP20^OE) or empty vehicle (EV) respectively via tail vein injection. After 4 weeks, the mice were administered with Ang II to induce hypertrophy and cardiac dysfunction (Fig. 7a) and succeeded (Supplementary Fig. 11a). AAV9 administration did not alter both body weight (Supplementary Fig. 11b) and blood pressure in Ang II-challenged mice (Supplementary Fig. 11c, d). USP20 overexpression strikingly alleviated cardiac dysfunction induced by Ang II (Fig. 7b-g; Supplementary Table 5), and also resulted in reductions in cardiomyocyte size (Fig. 7h-j), fibrosis (Fig. 7k-l; Supplementary Fig. 11e-h), and hypertrophic markers (Fig. 7m), suggesting that overexpression of USP20 prevents Ang II-induced heart remodeling. More importantly, USP20 overexpression did not change the protein level of STAT3 (Supplementary Fig. 11g), and as expected, USP20 overexpression decreased the K63-ubiquitination of STAT3 in heart tissues (Supplementary Fig. 11h). Taken together, these results suggest that USP20 overexpression improves cardiac hypertrophy and dysfunction.

USP20 ameliorates cardiac hypertrophy and dysfunction by inhibiting STAT3

To assess whether USP20 can improve cardiac hypertrophy by regulating STAT3, we administered wild type and USP20 CKO mice with or without STAT3 inhibitor stattic (10mg/kg, i.g.). Stattic is a cell-permeable inhibitor that specifically targets the STAT3 protein, preventing its activation and dimerization.⁴⁵ By blocking the SH2 domain, stattic prevents STAT3 from translocating to the nucleus and initiating the transcription of target genes. USP20 CKO mice was injected with the AAV9-USP20 and the empty vehicle for 4 weeks to overexpressed the level of USP20 in the hearts. Then these mice were implanted with Ang II osmotic pumps to induce cardiac hypertrophy for 4 weeks, and administered with static every day (10mg/kg, i.g., Fig. 8a). Ang II levels rose in all mice administered with Ang II (Supplementary Fig. 12a), leading to increased systolic blood pressure (Supplementary Fig. 12b, c). Stattic did not alter the body weight (Supplementary Fig. 12d). Cardiac function assessment demonstrated that STAT3 inhibitor stattic protected heart from dysfunction caused by USP20 deficiency, and more importantly, the USP20 expression in the heart failed to ameliorate Ang II-induced cardiac dysfunction when the stattic was administered (Fig. 8b, c, d; Supplementary Table 6). Similar results were observed for hypertrophic morphology (Fig. 8e-g, the cardiomyocyte size (Fig. 8h-j), the fibrosis level (Fig. 8k-l; Supplementary Fig. 12e, f), and hypertrophic markers (Fig. 8m). These results reveal that USP20 exerts a therapeutic effect on cardiac hypertrophy and dysfunction by abolishing STAT3. It was also confirmed in vitro that USP20 affects cardiomyocyte hypertrophy by regulating STAT3 (Supplementary Fig. 12g-k). Therefore, these data demonstrate that USP20 ameliorates cardiac hypertrophy by inhibiting STAT3.

Pathological cardiac hypertrophy can progress to heart failure, a significant global health issue.^46,47 Although β-adrenergic receptor blockers and renin-angiotensin-aldosterone system inhibitors offer some benefit, the incidence remains high.⁴⁸ Understanding the molecular mechanisms driving cardiac hypertrophy is essential for developing better treatments. Deubiquitination, which is an important post-translational modification and regulated by deubiquitinating enzymes (DUBs), modulates signal transduction and prevents the degradation of substrates by altering ubiquitin linkages.^49,50 Elucidating the regulatory mechanisms of DUBs in cardiac hypertrophy may provide a new therapeutic strategy. Recent studies have highlighted the role of DUBs like USP25.²² JOSD2.²³ OTUD1.³¹ UCHL1.⁵¹ and OTUD6a.⁵² in cardiovascular diseases. In our study, we identified USP20 derived from cardiomyocytes as a crucial down-regulated factor in cardiac hypertrophy. USP20 has been recognized for its functions in other organs.^25,26 especially in cancer.^25,53 USP20 attenuates TNF- and IL-1β-induced atherogenic signaling in smooth muscle cells (SMCs) by deubiquitinating receptor-interacting protein kinase 1 (RIPK1) and other signaling intermediates.⁵⁴ Here, we illustrated that USP20 plays a protective role which attenuates cardiac hypertrophy and cardiac dysfunction. Moreover, our studies revealed that down-regulated USP20 is predominantly located in cardiomyocytes.

The function of DUBs is related to the roles of their substrate.⁵⁵ We identified STAT3 is a direct substrate of USP20 on myocardial hypertrophy tissues and mouse-derived HL-1 cells. STAT3 is crucial for survival, growth, sarcomere architecture, energy dynamics, and metabolism.^29,56 Since activated STAT3 promotes heart failure.^57,58 STAT3 has become a pivotal therapeutic target for heart failure, with gene therapy approaches showing promising results in patients with cardiac hypertrophy.⁵⁹ However, STAT3 regulates numerous biological processes across almost all human cells and organs, making it an unsuitable direct therapeutic target.⁶⁰ Constant activation of STAT3 induces hypertrophy and inflammation in the heart and other organs, such as the gut, and promote cellular de-differentiation, potentially leading to cancer.⁶¹ Conversely, reducing or blocking STAT3 below a certain threshold can lead to diminished cardiac vasculature, increased fibrosis, and heart failure.⁵⁶ Thus, cell-type-specific STAT3 regulation is required for beneficial effects, a task that is likely challenging to achieve through direct pharmacological interventions targeting STAT3. Here, we identified a cardiomyocyte-specific USP20-STAT3 axis with important roles in cardiac hypertrophy (Fig. 9).

The type of ubiquitin chain attaches to a substrate protein dictates the fate of the ubiquitinated substrate.⁵⁵ Consequently, different patterns of deubiquitination on substrate proteins impart distinct functional roles to DUBs. Generally, USP20 regulates the K48-linked ubiquitination on substrate proteins to modulate their stability, while K63 or other ubiquitin chains are rarely reported.^62,63 K63-linked ubiquitination serves as a molecular scaffold for protein-protein interactions, playing a crucial role in activating protein kinase signaling, receptor endocytosis, protein trafficking, and DNA damage repair.^64,65 It has been shown that K63-linked polyubiquitination of STAT3 influences its phosphorylation and nuclear translocation.^36,37 Our data revealed that K63-linkage is crucial for USP20-regulated STAT3 deubiquitination. We demonstrated K177 as key for USP20 directly binds to STAT3 deubiquitination using ubiquitylome analysis and validation. USP20 binds to the coiled-coil domain (CCD), which regulates STAT3 phosphorylation and nuclear translocation through allosteric modulation of the SH2 domain.⁴⁰ It is noteworthy that K177 is in the CCD of STAT3, and therefore we speculated that the ubiquitination level at the K177 site affect the binding of STAT3 with target gene promoters.

Although STAT3 is recognized as a transcription factor that promotes gene transcription, however, increasing evidence suggests that STAT3 also suppresses gene transcription.^66,67 One of the mechanisms by which STAT3 inhibits gene expression is the acetylation of the K685 site in the SH2 domain, which is crucial for the interaction between STAT3 and DNA methyltransferase 1, leading to the methylation and silencing of different promoters.⁶⁷ However, the transcriptional regulation of STAT3 in cardiomyocytes remains to be elucidated. Our results provide the first evidence that STAT3 inhibits the transcription of Carm1 through directly bound to its promoter regions (Fig. 9). Notably, this inhibitory effect was significantly increased by inhibition of USP20 or diminished by overexpression of USP20. Interestingly, this study is the first to demonstrate a functional link between STAT3 and CARM1 in the pathogenesis of pathological myocardial hypertrophy. Whether other transcription factors involve in the USP20-STAT3-CARM1 axis will be explored in our future studies.

There are some limitations to our study. First, our studies have confirmed that STAT3 is a substrate of USP20; however, we cannot exclude the possibility that USP20 may regulate other substrate proteins to mediate cardiomyocyte hypertrophy. Further investigation is needed to elucidate the other mechanisms by which cardiomyocyte-derived USP20 regulates cardiac hypertrophy. Second, it is general believe that protein levels are modulated by gene expression and post-translational modifications (PTMs).²⁰ Currently, the transcription factors regulating USP20 gene expression are still poorly understood. Our data show that USP20 expression diminishes as early as 3 hours following Ang II treatment, suggesting the involvement of PTMs. Ubiquitination, a key PTM regulating protein degradation, may contribute to alter USP20 protein level. Additional research is necessary to investigate the involvement of PTMs in the USP20 mediated pathogenesis of cardiac hypertrophy.

Taken together, we elucidated that cardiomyocyte-derived USP20 regulated K63-linked ubiquitination of STAT3. We demonstrated that the cardiomyocyte-specific USP20-STAT3-CARM1 axis plays a protective role in cardiac hypertrophy, suggesting that targeting USP20 with cardiac-specific gene therapy could be a promising strategy for treating cardiac hypertrophy.

Detailed experimental materials and methods are available in the Supplementary Data.

DATA AVAILABILITY

The data underlying this article will be shared on reasonable request by the corresponding author.

AUTHOR CONTRIBUTIONS

L.F.Z. and S.S.D. designed and carried out the most experiments, performed statistical analysis, and generated the figures. L.F.Z. and S.S.D. performed most animal assay supervised by J.H.C. and D.L.Y.; S.S.D. assisted L.F.Z. for primary cardiomyocyte preparation and part of cell assays; F.Y., Q.Y.G., Y.C.Z., J.-S.D. and Z.Y.L. helped with the experiments; Z.X.T. and F.Z.G. assisted S.S.D. for the single-cell sorting and helped with image data analysis; D.R.C. assisted L.F.Z. for Co-IP and CUT&Tag experiments. L.Y.H. provided technical assistance to L.F.Z. and S.S.D.; L.F.Z., S.S.D. and D.L.Y. conceived the study; L.F.Z., S.S.D. and Q.Y.G. wrote the initial manuscript; D.L.Y. and J.H.C. provided important discussions and essential revisions. G.P.S., J.H.C., J.A.W. and D.L.Y. supervised the study; J.A.W. and D.L.Y. designed and oversaw the study. All authors have read and approved the article.

ACKNOWLEDGEMENTS

The authors thank all the colleagues in our research team for technical support and constructive discussion. All authors thank Ms. Xiangmin Kong for technical support.

SUPPLEMENTARY DATA

Supplementary data is available at European Heart Journal online.

CONFLICT OF INTEREST

All authors declare no disclosure of interest for this contribution.

FUNDING

This study was supported by the National Science Foundation of China (82170242, 82030014, 82370256, 81570454, U22A20267, U21A20338), State Key Laboratory of Transvascular Implantation Devices (012024015, 012024010), National Key R&D Program of China (2023YFA1800700).

ETHICAL APPROVAL

All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Second Affiliated Hospital, Zhejiang University School of Medicine (AIRB-2024-1663) and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experimental protocols involving humans were approved by the Ethics Committee of the Second Affiliated Hospital Zhejiang University School of Medicine (IRB-2024-1270).

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Cardiomyocyte-enriched USP20 ameliorates pathological cardiac hypertrophy by targeting STAT3-CARM1 axis

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