USP20 mitigates doxorubicin-induced cardiotoxicity by deubiquitinating and stabilizing HuR

USP20 mitigates doxorubicin-induced cardiotoxicity by deubiquitinating and stabilizing HuR | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article USP20 mitigates doxorubicin-induced cardiotoxicity by deubiquitinating and stabilizing HuR Zhouqing Huang, Yunxuan Chen, Shuoning Wu, Lang Deng, Yixin Zhou, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6151078/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The severe cardiotoxicity of doxorubicin (Dox) significantly restricts its clinical application. Deubiquitinating enzymes (DUBs) play pivotal roles in cardiac pathophysiology because of their precise regulation of protein function, localization and degradation. Objectives The objective of this study was to investigate the role and molecular mechanism of ubiquitin-specific peptidase 20 (USP20), a DUB, in doxorubicin-induced cardiotoxicity. Methods Cardiomyocyte-specific USP20-knockout (USP20-CKO) mice were utilized to assess the role of USP20 in doxorubicin-induced cardiomyopathy (DIC). Coimmunoprecipitation (co-IP) combined with liquid chromatography‒mass spectrometry/mass spectrometry (LC‒MS/MS) analysis was employed to screen the substrate protein of USP20. Furthermore, mutant plasmids of USP20 were constructed to elucidate the molecular mechanism underlying the regulation of human antigen R (HuR) by USP20. Finally, an AAV9 vector was used to overexpress USP20 in the hearts of cardiac-specific HuR-knockout mice to assess the interaction between USP20 and HuR. Results The results revealed a decrease in USP20 expression in Dox-stimulated mouse cardiomyocytes. Cardiomyocyte-specific USP20 knockout resulted in increased cardiomyocyte ferroptosis and led to DIC. Mechanistically, USP20 directly interacted with HuR through its ubiquitin-specific protease structural domain. Deubiquitination at position 154 was crucial for maintaining HuR protein stability by cleaving K48 ubiquitin chains and inhibiting proteasomal degradation. Additionally, HuR bound to GPX4 mRNA to suppress its degradation, thereby mitigating ferroptosis and contributing to alleviating DIC. Furthermore, targeted USP20 overexpression via AAV9 in cardiomyocytes significantly alleviated DIC. However, in mice with cardiomyocyte-specific HuR knockout, USP20 no longer had an anti-DIC effect, indicating that HuR, as a downstream target protein of USP20, plays an irreplaceable role in DIC. Conclusions Our findings indicate that USP20 enhances the stability of the HuR protein through deubiquitination, thereby inhibiting ferroptosis and mitigating DIC. Health sciences/Diseases/Cardiovascular diseases/Cardiomyopathies Biological sciences/Cell biology/Proteolysis/Deubiquitylating enzymes USP20 Doxorubicin-induced cardiomyopathy Deubiquitinating enzyme Cardiomyocytes Ferroptosis HuR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Doxorubicin (Dox) is a highly efficacious and extensively used chemotherapeutic agent that is currently used in the management of various neoplastic conditions, such as breast cancer, ovarian cancer, lymphoma, and sarcoma 1 , 2 , 3 . However, both its acute and chronic toxic effects considerably limit the clinical application of Dox 4 , 5 . The dose-dependent cardiotoxicity associated with doxorubicin leads to damage to cardiomyocytes and subsequent cardiac dysfunction, ultimately culminating in heart failure and severely impairing the quality of life of cancer patients during or following treatment with doxorubicin. Despite extensive research on the mechanisms underlying Dox-induced cardiotoxicity over the years, these processes remain poorly understood. Therefore, the identification of new therapeutic targets that may alleviate Dox-induced cardiotoxicity is urgently needed. Ferroptosis, a form of iron-dependent nonapoptotic cell death predominantly induced by lipid peroxidation triggered by excessive intracellular iron accumulation, plays a pivotal role in the pathogenesis of Dox-induced cardiomyopathy (DIC) 6 . Consequently, modulating ferroptosis in cardiomyocytes may offer a novel approach for preventing and treating DIC. Ubiquitination, a dynamic and multifaceted posttranslational modification, plays a pivotal role in various crucial physiological processes, such as transcriptional regulation, cellular signalling, and DNA damage repair 7 , 8 . Furthermore, the precise regulation of protein degradation, localization, and function is facilitated by deubiquitinating enzymes (DUBs), which reverse protein ubiquitination. Currently, approximately 100 identified DUBs within the human proteome are categorized into seven families. Among these families, the ubiquitin-specific protease (USP) family represents the largest subfamily of deubiquitinases, which are critically involved in various pathological processes, including inflammation, metabolic disorders, cancer, and cardiovascular diseases 9 , 10 , 11 . Recent studies have demonstrated that USP19 exerts a protective effect against DIC by deubiquitinating TRAF2 to prevent its degradation 12 . However, evidence has indicated that doxorubicin accelerates the progression of DIC by activating the USP36-mediated deubiquitination of PARP1 13 . The USP family clearly plays a pivotal role in elucidating the molecular mechanism underlying DIC. Elucidating the roles and mechanisms of deubiquitinases in DIC will enhance our understanding of this phenomenon and provide novel insights for clinical treatment strategies. The deubiquitinating enzyme USP20, a member of the USP family, has been recognized for its critical involvement in diverse biological processes through the modulation of deubiquitination modifications on specific substrates. Many studies have reported findings related to cancer, atherosclerosis, lipid metabolism, and other related fields 14 , 15 , 16 , 17 . Nevertheless, the role of USP20 in cardiac biology remains largely underexplored and represents a promising avenue for future investigations. Our team focused on the regulatory mechanism of DUBs in the pathogenesis of cardiovascular diseases. We compared DUB gene expression profiles in myocardial tissue from DIC patients in the GEO database and detected a significantly decreased level of the DUB ubiquitin-specific protease 20 (USP20), indicating the potential involvement of USP20 in the pathogenesis of DIC. Furthermore, our investigation of Universal Protein data revealed that USP20 is markedly expressed in the heart, thereby providing additional evidence for the potential involvement of USP20 in cardiac biology. Herein, we investigated the role of USP20 in DIC, with the aim of clarifying its regulatory molecular mechanism. Our findings identify USP20 as a potential target for future clinical treatment of DIC. Materials and methods Animal experiments All experimental protocols received approval from the Laboratory Animal Ethics Committee and the Laboratory Animal Centre of the First Affiliated Hospital of Wenzhou Medical University (WYYY-IACUC-AEC-2024-097). The experiments were conducted utilizing cardiomyocyte-specific USP20-knockout mice (USP20CKO) and cardiomyocyte-specific HuR-knockout mice (HuRCKO), both of which were on a C57BL/6J genetic background, and flox mice (USP20 fl/fl and HuR fl/fl ) derived from the same litter. Randomization was employed for animal grouping, and analyses were performed by blinded experimenters. To elucidate the role of USP20 in doxorubicin-induced cardiotoxicity, 8-week-old male mice were divided into four groups: (1) USP20 fl/fl + saline; (2) USP20CKO + saline; (3) USP20 fl/fl + Dox; and (4) USP20CKO + Dox. Mice were subjected to weekly intraperitoneal injections of either saline or Dox at a dosage of 5 mg/kg, which were administered over a duration of four weeks. The mice were housed under controlled conditions at a temperature of 25°C with a 12-h light/dark cycle and had ad libitum access to standard chow. Adeno-associated virus (AAV) infection To achieve targeted USP20 overexpression in cardiomyocytes, male mice were administered adeno-associated virus serotype 9 containing troponin-specific promoters. This vector carried either an empty vector (AAV9-cTnT-EV) or Usp20 cDNA (AAV9-cTnT-USP20 oe ). The mice were separated into three experimental groups: (1) WT + AAV9-cTnT-EV + saline, (2) WT + AAV9-cTnT-EV + Dox, and (3) WT + AAV9-cTnT-USP20 oe + Dox. AAV9 was introduced via the tail vein at a dosage of 2E + 11 v.g./mouse two weeks prior to the initiation of the modelling process. A four-week treatment regimen with either saline or Dox was subsequently delivered through the peritoneal cavity in accordance with previously established protocols. Echocardiography Echocardiography was conducted four weeks after the administration of Dox. The hair in the left anterior thoracic region of each mouse was removed using a depilatory cream, and the mice were subsequently anaesthetized with isoflurane. Cardiac function was evaluated through M-mode echocardiography utilizing the Visual Sonics Vevo 3100 Small Animal Ultrasound Imaging System, allowing for the determination of the ejection fraction (EF) and shortening fraction (FS), as well as other cardiac function parameters. Serum biochemical analysis The levels of lactate dehydrogenase (LDH) were measured using an LDH assay kit (BC0685, Solarbio, Beijing, China). The levels of creatine kinase isoenzyme (CK-MB) were assessed with a CK-MB assay kit (E006-1-1; Jiancheng Biological Engineering Institute, Nanjing, China). The concentration of mouse cardiac troponin T (cTnT/TNNT2) was evaluated utilizing a cTnT assay kit (E-EL-M1801, Elabscience, Wuhan, China). Additionally, the level of ferrous iron was measured using a ferrous iron assay kit (E-BC-K773-M, Elabscience, Wuhan, China). All measurements were conducted in accordance with the product manuals provided for each respective kit. Histological analysis Cardiac tissues were fixed in 4% paraformaldehyde for 24 h, subsequently embedded in paraffin, and sectioned into 5 µm slices. The sections were dewaxed, hydrated, and stained with haematoxylin and eosin (H&E, G1120, Solarbio, China) as well as Sirius Red (G1472, Solarbio, China). Images were captured using an Olympus Corporation microscope. Immunohistochemical (IHC) staining Following dewaxing, the tissue sections were immersed in a boiling citrate buffer solution (0.01 M, pH 6.0) for 10 min at 100°C. The sections were allowed to cool to ambient temperature before being incubated in a 3% hydrogen peroxide solution for 15 min. The sections were subsequently incubated with 5% BSA for 30 min to block nonspecific binding. After the BSA was removed, the sections were incubated with a diluted anti-4-HNE antibody (1:200, MAB3249, R&D Systems, Shanghai, China) overnight at 4°C. The following day, the sections were incubated with the corresponding secondary antibody at room temperature for one hour. Finally, the sections were stained with DAB staining solution for 8 min, followed by haematoxylin counterstaining for 1 min. The stained sections were mounted with an appropriate mounting medium and visualized under a microscope (Olympus Corporation, Tokyo, Japan). Immunofluorescence staining Frozen sections were fixed with 4% paraformaldehyde for 20 min. Subsequently, 3% hydrogen peroxide was used to inhibit endogenous peroxidase activity, and nonspecific binding was blocked with 5% BSA. After the BSA was aspirated, the sections were incubated with diluted anti-USP20 (rabbit, 1:200, A301-189A-M, Bethyl), anti-α-SMA (mouse, 1:200, 48838s, CST), anti-vimentin (mouse, 1:200, EM0401, Huabio) and anti-F4/80 (mouse, 1:200, sc-377009, Santa) antibodies overnight at 4°C. On the following day, the sections were incubated with an iFluor™ 488-coupled goat anti-rabbit IgG polyclonal antibody (1:2000, HA1122, Hua An) and an iFluor™ 594-coupled goat anti-mouse IgG polyclonal antibody (1:2000, HA1125, Hua An) for 1 h at room temperature. Finally, nuclei were stained with DAPI (36308ES11; Yeasen Biotech). Images were acquired with a microscope (Olympus Corporation, Tokyo, Japan). TUNEL assay Apoptotic cells in cardiac tissues were detected using the One-Step TUNEL Apoptosis Detection Kit (C1090, Beyotime, China) according to the manufacturer's protocol. After staining, positive cells were visualized using a confocal microscope. Five random fields were chosen for the quantification of apoptotic cells. Cell culture and transfection The NIH/3T3 and H9C2 cell lines were incubated in DMEM (Gibco, USA, 4.5 g/L) supplemented with 10% foetal bovine serum (Gibco, USA), 100 U/ml penicillin, and 100 U/ml streptomycin at 37°C in a 5% CO 2 atmosphere. Once reaching a confluence of approximately 80%, the cells were incubated with Dox (0.5 µg/ml, 25316-40-9, MCE) for 24 h to establish the model. Primary cardiomyocytes were isolated from neonatal Sprague‒Dawley rats. The hearts were excised under aseptic conditions, subsequently washed with phosphate-buffered saline (PBS), and minced into small fragments. The myocardial tissue was then digested using trypsin and collagenase at 37°C. Following digestion, the fibroblasts were eliminated via differential adhesion, and the remaining cells were cultured in Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum for a period of 48 h prior to subsequent experiments. A USP20 overexpression plasmid was introduced into cardiomyocytes via Lipofectamine 3000 (Lot No. 3039420; Thermo Fisher Scientific, Germany) transfection reagent in accordance with the manufacturer's instructions. siRNAs were transfected into cardiomyocytes using Lipofectamine 2000 (Lot No. 3035197; Thermo Fisher Scientific, Germany) transfection reagent, in accordance with the manufacturer's instructions, to suppress the expression of USP20. CCK-8 assay Cell viability was assessed using Cell Counting Kit-8 (CCK-8; C0037; Beyotime, China) following the manufacturer's protocol. After treatment, 10 µl of CCK-8 solution was added to each well, after which the cells were incubated for 1 h at 37°C. Absorbance was measured at 450 nm using a microplate reader to determine cell viability. PCR analysis Total RNA was extracted from cells via TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized using a reverse transcription kit (Vazyme R333-01), and quantitative PCR was performed using SYBR Green reagent (Takara, DRR037A). GAPDH was used as an internal control. The sequences of all the primers used are listed in Supplementary Table S1 . Western blot analysis Protein samples from cell lysates and myocardial tissues were prepared using a lysis buffer containing phosphatase inhibitors. The protein concentration was determined with a bicinchoninic acid (BCA) assay. The samples were subsequently subjected to SDS‒PAGE, and separated proteins were transferred onto nitrocellulose membranes, which were subsequently incubated overnight at 4°C with the following primary antibodies: anti-USP20 (1:1000, 17491-1-AP, Proteintech), anti-6xHis-Tag (1:1000, 66005-1-ig, Proteintech), anti-DykDDDDk-tag (1:1000, 20543-1-AP, Proteintech), anti-UB (1:1000, sc-8017, Santa Cruz Biotechnology), anti-HA-Tag (1:1000, sc-57592, Santa Cruz Biotechnology), anti-GPX4 (1:1000, ET-706-45, Huabio), anti-HuR (1:1000, ET1705-81, Huabio) and anti-GAPDH (1;1000, ET1601-4, Huabio). The following day, the membranes were incubated with horseradish peroxidase (HRP)-labelled secondary antibodies (1:2000, A0208 or A0216, Beyotime). Bands were visualized via an enhanced chemiluminescence (ECL) luminescent solution. Immunoprecipitation (Co-IP) After the complete lysis of cell samples or animal tissues using a protein lysis buffer, magnetic beads were introduced into the lysates to selectively eliminate nonspecific binding proteins. A portion of each lysate was subsequently retained as an input sample. Appropriate antibodies were added to the resulting sample, which was then incubated overnight at 4°C. On the following day, fresh magnetic beads were added, and the sample was incubated for 2 h at 4°C. The sample was then centrifuged, after which the precipitate was retained and washed multiple times with PBS to ensure thorough removal of contaminants. Finally, the pellet was resuspended in sodium dodecyl sulfate sample buffer for 10 min to obtain a sample suitable for subsequent western blot analysis. LC‒MS/MS analysis Plasmids encoding Usp20 cDNA or an empty vector were transfected into NIH/3T3 cells. Subsequently, cell lysates were prepared for immunoprecipitation as described previously 11 . The resulting samples were subjected to liquid chromatography‒tandem mass spectrometry (LC‒MS/MS) analysis, which was conducted by Shanghai Bioprofile (Shanghai, China). RNA immunoprecipitation (RIP) RNA-binding protein immunoprecipitation (RIP) assays were conducted using an RNA-binding protein immunoprecipitation kit (Millipore, USA) according to the manufacturer's instructions. Anti-HuR (ET1705-81, Huabio, Hangzhou) and anti-rabbit IgG were used as antibodies in these assays. The expression level of GPX4 mRNA was quantified by quantitative PCR (qPCR). ROS measurement To assess the levels of reactive oxygen species (ROS) in cells and tissues, we used an MDA lipid oxidation kit (S0131M, Beyotime, China), a total SOD activity assay kit (S0101S, Beyotime, China), and GSH and GSSG measurement kits (S0053, Beyotime, China). These kits were used in accordance with the manufacturer's instructions. Propidium iodide (PI) staining Cell death was evaluated via propidium iodide (PI; CA1120; Solarbio, China) staining. Following treatment, the cells were washed with PBS and subsequently incubated in a 5 µg/ml PI solution for 25 min at room temperature in the dark. The stained cells were then visualized under a fluorescence microscope, and images were captured to assess the percentage of PI-positive cells. Dihydroethidium (DHE) staining Superoxide levels in cardiomyocytes were quantified using dihydroethidium (DHE; S0063; Beyotime, China) staining. Cells were treated and subsequently incubated with 5 µM DHE at 37°C for 30 min. Following incubation, the cells were washed with PBS, and images were obtained using a fluorescence microscope to evaluate ROS levels. Construction of USP20-deficient NIH/3T3 cells (gUSP20-NIH/3T3) A lentiviral vector containing Cas9 and guided RNA targeting USP20 was generated from NIH/3T3 cells. Monoclonal cell lines were established following the transformation process and subsequent colony selection. The lentivirus packaging plasmid was transfected into 293T cells, after which the culture supernatant containing the lentivirus was collected 48 h later. NIH/3T3 cells were then infected with the viral supernatant and selectively cultured in medium supplemented with blastocide. The expression levels of USP20 in gUSP20-NIH/3T3 cells were analysed via western blotting. Determination of mRNA stability We treated cells with actinomycin D (#S8964, Selleck, Shanghai) for 0, 2, 4, 6, and 8 h and then extracted RNA to quantify GPX4 mRNA levels using qPCR. Statistical analysis The data are expressed as means ± standard deviations (SDs). Statistical analyses were performed using GraphPad Prism 8 software (GraphPad, San Diego, CA). For comparisons involving more than two groups of data, two-way analysis of variance (ANOVA) followed by Tukey's correction was employed, while the t test was used for comparisons between two groups of data. A significance level of 0.05 was considered to indicate statistical significance. Results USP20 expression is downregulated in DIC The aim of this investigation was to elucidate the protective role of DUBs in DIC. A comprehensive analysis of downregulated DUB genes was conducted using microarray datasets (GSE42177 and GSE207737) obtained from the Gene Expression Omnibus (GEO) database, with a focus on genes activated by Dox. We identified genes whose expression differed by more than 1.5-fold across both datasets and performed a cross-tabulation analysis (Fig. 1 A). The mRNA levels of USPL1, USP2, USP10, USP20, USP39, and USP42 were significantly lower in the Dox-treated group than in the control group. Neonatal rat primary cardiomyocytes (NRCMs) were subsequently isolated and subjected to Dox stimulation. The qRT‒PCR results revealed that the mRNA expression of USP20 decreased the most substantially following Dox treatment (Fig. 1 B ) . Consequently, USP20 was selected as the focal point for further exploration in this study. Additionally, we confirmed the downregulation of USP20 protein expression in cardiac tissues and primary cardiomyocytes. Notably, a pronounced reduction in USP20 protein expression was observed in heart tissue of mice post-Dox treatment compared with that in wild-type mice ( Fig. 1 C-D ) . Similarly, decreased USP20 protein expression was noted in Dox-stimulated primary cardiomyocytes (Fig. 1 E-F). In contrast, we found that USP20 protein expression was largely unchanged in primary fibroblasts (Fig. 1 G-H) and macrophages (Fig. 1 I-J). Accordingly, we hypothesized that USP20 is predominantly expressed in cardiomyocytes in murine models. To test this hypothesis, single-cell sequencing was performed using myocardial tissues from the Dox treatment group, revealing the predominant localization of USP20 in cardiomyocytes (Fig. 1 K ‒L ). We subsequently analysed the expression pattern of USP20 in cardiomyocytes (Fig. 1 M-N). The distribution of USP20 expression in cardiomyocytes decreased following Dox treatment. Moreover, via t-distributed stochastic neighbour embedding (t-SNE) analysis, we found that USP20 was predominantly localized to cardiomyocytes undergoing remodelling after Dox treatment (Fig. 1 O). Furthermore, colocalization fluorescence analysis revealed that the labelled USP20 protein was significantly distributed primarily within cardiomyocytes, with a marked decrease in fluorescence intensity following Dox treatment (Fig. 1 P), corroborating findings from previous experiments. Cardiomyocyte-specific USP20 deficiency exacerbates the progression of DIC via ferroptosis in vivo To elucidate the role of USP20 in DIC, we constructed USP20CKO mice ( Supplementary Fig. 1A ) and established a mouse model of DIC (Fig. 2 A). Subsequently, we verified myocardium-specific USP20 knockout in mice ( Supplementary Fig. 1B-C ). On the day preceding the execution of the experiment, a noninvasive transthoracic echocardiogram was performed on all experimental mice to analyse cardiac function and the extent of myocardial damage. The echocardiography results revealed further deterioration of cardiac function in USP20CKO mice under Dox stimulation, particularly in terms of a decrease in the ejection fraction (EF) and fractional shortening (FS) (Fig. 2 B-D). H&E staining revealed that Dox stimulation resulted in the disorganization of cardiac muscle fibres in wild-type mice, an effect that was further exacerbated in USP20-knockout mice following Dox treatment (Fig. 2 E). Furthermore, Sirius red staining (Fig. 2 F) and TUNEL staining (Fig. 2 G-H) revealed that intermyocardial fibrosis and the degree of cardiomyocyte death were similarly altered. A number of myocardial injury-related markers, including cTnI, CK-MB, and LDH, were subsequently assessed to provide further insights into the extent of myocardial injury. The results of the serum biochemical assays indicated that USP20CKO significantly increased cTnT, CK-MB, and LDH levels (Fig. 2 I-K) in mice following Dox treatment. These findings indicate that myocardial injury is markedly elevated in USP20 knockout mice following Dox treatment. To elucidate the precise mode of death regulated by USP20 in the context of DIC, a comprehensive literature review was conducted, revealing a substantial body of evidence that identified ferroptosis as a critical mechanism in DIC 6 , 23 . To determine whether USP20 regulates cardiac ferroptosis, we quantified the concentration of Fe²⁺ in mouse serum (Fig. 2 L). Notably, following Dox treatment, Fe²⁺ levels were significantly higher in USP20-CKO mice than in wild-type controls. Consistent with these findings regarding Fe²⁺, reactive oxygen species (ROS) levels in myocardial tissues were also markedly increased in USP20-knockout mice, as evidenced by the upregulation of malondialdehyde (MDA) expression and downregulation of superoxide dismutase (SOD) expression, suggesting enhanced myocardial lipid peroxidation under doxorubicin treatment conditions. We subsequently conducted Western blot analysis and immunohistochemical staining of cardiac tissues (Fig. 2 M-N). The results revealed the significant downregulation of the expression of GPX4, a critical protein implicated in ferroptosis, following USP20 knockout in the context of Dox-induced modelling (Fig. 2 O-P). Furthermore, the staining intensity for another ferroptosis marker, 4-hydroxy-2-nonenal (4-HNE), was markedly increased (Fig. 2 Q). Taken together, these findings indicate that USP20 deficiency obviously accelerates the progression of DIC via ferroptosis. USP20 mitigates Dox-induced ferroptosis in cardiomyocytes To verify the role of USP20 in vitro , we transfected neonatal rat primary cardiomyocytes (NRCMs) with si-USP20, followed by stimulation with Dox. The results demonstrated that, in the context of Dox stimulation, cell activity (CCK8) exhibited a more pronounced decline in the si-USP20-transfected group (Fig. 3 A), whereas cell permeability (LDH) increased (Fig. 3 B). These findings indicate that the absence of USP20 intensifies the deleterious effects of Dox on cardiomyocytes in vitro . Furthermore, the involvement of USP20 in ferroptosis was assessed in vitro . The cells in each group were analysed for Fe 2+ , SOD, MDA, and GSH levels (Fig. 3 C-F). The results demonstrated that Dox stimulation resulted in elevated Fe 2+ levels, increased SOD and GSH levels, and decreased MDA levels. However, transfection with si-USP20 led to further intensification of these alterations. These findings indicate that the degree of intracellular lipid peroxidation increased after USP20 silencing. In addition, under Dox-stimulated conditions, the transfection of si-USP20 further reduced the expression of GPX4, a pivotal protein in the process of ferroptosis (Fig. 3 G-H). To gain a more detailed understanding of the changes occurring in the cells, we performed DHE and PI staining using cardiomyocytes (Fig. 3 I-J), and the results revealed that USP20 silencing in vitro significantly increased both the cellular reactive oxygen species (ROS) level and mortality rate. In addition, we constructed a USP20-overexpression plasmid, which we transfected into NRCMs, followed by stimulation with Dox. As expected, USP20 overexpression alleviated the decrease in cellular activity (CCK-8) and increase in cell permeability (LDH) (Fig. 3 K-L), increased both the Fe²⁺ concentration and lipid peroxidation level (Fig. 3 M-P), and abrogated downregulated GPX4 expression in Dox-induced cardiomyocytes (Fig. 3 Q-R). Furthermore, the ability of USP20 to attenuate the doxorubicin-induced increase in ROS and apoptosis levels was visualized via the fluorescence staining of DHE and PI in cells (Fig. 3 S-T). Thus, we validated the role of USP20 in Dox-induced cardiomyocytes in terms of both interference and overexpression and provided necessary evidence from experiments conducted at the cellular level indicating that the absence of USP20 significantly exacerbates Dox-induced cardiotoxicity and ferroptosis. Restoration of cardiac USP20 alleviates DIC by attenuating ferroptosis To elucidate the therapeutic function of USP20 in DIC, we generated recombinant AAV9 vectors carrying Usp20 cDNA and specifically overexpressed USP20 in the cardiomyocytes of wild-type mice via the tail vein injection of AAV9-cTnT-USP20 ( Supplementary Fig. 3A ). Dox was subsequently administered intraperitoneally to establish a DIC model (Fig. 4 A). We next measured cardiac function by echocardiography. Compared with that in Dox-stimulated wild-type mice, in USP20-overexpressing mice, Dox-induced myocardial contractile dysfunction was alleviated (Fig. 4 B), which led to a reduction in the left ventricular ejection fraction (EF) and fractional shortening (FS) (Fig. 4 C-D). We subsequently observed that USP20 overexpression ameliorated myocardial fibre disorders, intermyocardial fibrosis and cardiomyocyte apoptosis, as evidenced by H&E, Sirius red and TUNEL staining of histopathological sections of the myocardium, respectively (Fig. 4 E-G). Furthermore, USP20 overexpression in heart tissues was shown to have a protective effect against Dox-induced myocardial injury. The levels of myocardial injury markers, including cTnI, CK-MB and LDH, were reduced (Fig. 4 I-K). Moreover, the serum Fe²⁺ concentration and lipid peroxidation level in the myocardial tissues of the mice were examined, and USP20 overexpression alleviated the Dox-induced increase in the Fe²⁺ concentration and lipid peroxidation level (Fig. 4 L-N), mitigated the decrease in GPX4 protein expression (Fig. 4 O-P) and reduced 4-HNE expression in the hearts of Dox-induced mice, as determined via immunohistochemical staining (Fig. 4 Q). Taken together, these results suggest that the restoration of cardiac USP20 expression ameliorates cardiac remodelling and dysfunction and impedes cardiac ferroptosis in DIC. USP20 directly interacts with HuR DUBs regulate a range of biological activities by affecting the degradation or function of substrate proteins. To identify target proteins regulated by USP20 during DIC, we employed immunoprecipitation (co-IP) coupled with mass spectrometry to screen for potential substrate proteins of USP20 (Fig. 5 A-C). Substrate proteins with scores exceeding 100 were selected for screening, and it was determined that only ELAVL1 (HuR) has been documented both in the heart and associated with Dox. Accordingly, we hypothesized that HuR is the substrate protein of USP20 in the context of DIC. To test this hypothesis, USP20 was cotransfected with an HuR plasmid into NIH/3T3 cells. The initial results indicated that HuR could bind to USP20 (Fig. 5 D). We subsequently conducted endogenous validation using neonatal rat primary cardiomyocytes and myocardial tissues that had been injected with AAV9-cTnT-USP20 (Fig. 5 E-F), the results of which confirmed the binding of USP20 to HuR. USP20 comprises three distinct structural domains: the USP structural domain and two tandemly linked DUSP structural domains (Fig. 5 G). To elucidate the specific structural domain of USP20 that interacts with HuR, we constructed USP20 plasmids containing mutations in the three structural domains. The cotransfection of HuR with each of the three USP20 mutants in NIH/3T3 cells revealed that the deletion of amino acids 145–687 resulted in the loss of HuR binding, whereas mutations in the other structural domains did not affect HuR binding (Fig. 5 H). These results provide direct evidence that USP20 acts through its USP structural domain by binding directly to HuR. The C154 active site of USP20 deubiquitinates HuR and maintains its stability We speculate that USP20 may alleviate DIC by regulating HuR. The expression of the HuR protein was also upregulated upon USP20 overexpression, and this increase was not caused by a change in the transcript level (Fig. 6 A-B). We thus postulated that USP20 inhibits HuR protein degradation by deubiquitination. To test this hypothesis, we generated USP20 knockout NIH/3T3 cells using CRISPR/Cas9 technology ( Supplementary Fig. 4A ). We subsequently observed a significant increase in the degradation rate of the HuR protein in the USP20-knockout group upon treatment with cycloheximide (Fig. 6 C-D). As a member of the DUB family, USP20 may play a regulatory role by removing ubiquitin molecules from HuR. To determine the manner in which USP20 acts, we cotransfected UB-K48, UB, HuR, and USP20 into NIH/3T3 cells and inhibited the proteasome degradation pathway with MG132. The results demonstrated that USP20 effectively removed K48-linked ubiquitin molecules, thereby inhibiting the degradation of target proteins via the proteasomal pathway (Fig. 6 E). DUBs are capable of hydrolysing amide bonds between ubiquitin molecules and substrate proteins through active sites, including cysteines and histidines. To identify the active site through which USP20 acts, we constructed USP20 plasmids with mutations at cysteine 154 and histidine 645 (Fig. 6 F). The two mutants were cotransfected with the HuR plasmid into NIH/3T3 cells, which were then treated with MG132. The capacity to stabilize the HuR protein was diminished when the cysteine at position 154 was mutated (Fig. 6 G-H). Furthermore, the plasmid mutation did not affect binding to the target protein (Fig. 6 I). Accordingly, we initially postulated that the cysteine at position 154 represents the active site of USP20, through which HuR is regulated. We subsequently cotransfected a UB plasmid and an HuR plasmid with the two mutant plasmids into NIH/3T3 cells. Our findings revealed that USP20 was unable to exert its deubiquitinating effect following the mutation of the C154 cysteine residue. However, the H645 mutant retained the deubiquitinating effect of USP20 (Fig. 6 J). Furthermore, both mutants were overexpressed in HL-1 cells stimulated with Dox. A cell activity assay (CCK-8) and a cell permeability assay (LDH) were subsequently conducted (Fig. 6 K-L). The results demonstrated that the C154 mutant was no longer capable of mitigating Dox-induced cardiotoxicity. Additionally, we conducted assays to measure the levels of Fe²⁺, SOD, MDA, and GSH. The results demonstrated that in the context of the C154 mutation, no decrease in the Dox-induced Fe²⁺ concentration or lipid peroxidation level was observed in the presence of USP20 ( Supplementary Fig. 5A-D ). The protein expression of GPX4 was also assessed ( Supplementary Fig. 5E ), and the results were consistent with those described above. In conclusion, these findings confirm that C154 of USP20 plays a pivotal role in the deubiquitination of HuR. The anti-DIC effect of USP20 is dependent on HuR To investigate the dependence of the anti-DIC function of USP20 on HuR, we created heart-specific HuR-knockout mice. AAV9-cTnT-USP20 was injected via the tail vein to specifically overexpress USP20 in the heart. Compared with the injection of AAV9-cTnT-EV (empty vector), the injection of AAV9-cTnT-USP20 did not improve cardiac function under Dox modelling conditions (Fig. 7 A). Furthermore, no significant differences were observed in EF or FS (Fig. 7 B-C). The H&E staining results indicated that the overexpression of USP20 in HuR-CKO mice did not ameliorate myocardial fibre disorganization ( Fig. 7 D). Similarly, we conducted Sirius Red and TUNEL staining of pathological cardiac sections, and the results revealed that the degree of interstitial fibrosis and the number of cardiomyocyte deaths in HuR-CKO mice following Dox stimulation remained unaltered following USP20 overexpression in cardiomyocytes (Fig. 7 E-G). Subsequently, serum myocardial injury marker assays revealed that the extent of myocardial injury (including cTnI, LDH, and CK-MB) was not significantly different between the two groups (Fig. 7 H-J). In addition, serum iron levels (Fig. 7 K), lipid peroxidation marker levels (Fig. 7 L-M), and 4-hydroxyenoaldehyde (4-HNE, a measure of ferroptosis) levels (Fig. 7 N) were assessed. The results showed that AAV9-cTnT-USP20 did not significantly attenuate ferroptosis in the Dox model group. Preliminary verification in animals indicated that targeting HuR via USP20 plays a role. To further corroborate our conclusions, we extracted NRCMs and subsequently transfected them with siRNAs targeting HuR along with a USP20 overexpression plasmid. Next, the NRCMs were treated with Dox. Compared with Dox treatment alone, si-HuR treatment led to a decrease in cellular activity (CCK8) and an increase in cellular permeability (LDH) ( Supplementary Fig. 6C-D ). However, this alteration was not alleviated by the overexpression of USP20 (Fig. 7 O-P). Similarly, the concentration of Fe²⁺ (Fig. 7 Q), the level of lipid peroxidation (Fig. 7 R-T), and the expression of GPX4 (Fig. 7 U-V) exhibited similar alterations. Moreover, USP20 overexpression in heart-specific HuR-knockout mice did not reduce apoptosis or ROS levels, as reflected by DHE and PI staining. (Fig. 7 W-X). In conclusion, the ability of USP20 to target HuR to alleviate DIC was validated both in vivo and in vitro . The question thus arises as to how HuR exerts its influence through ferroptosis. We discovered that HuR, an RNA-binding protein, can bind to GPX4 mRNA and that this binding is reduced under Dox stimulation. Backfilling USP20 reversed these alterations ( Supplementary Fig. 7A ). Furthermore, we demonstrated that the overexpression of HuR slows the degradation of GPX4 mRNA ( Supplementary Fig. 7B ). Therefore, we propose that USP20 reduces the degradation of HuR by deubiquitination, thereby promoting GPX4 mRNA stability and regulating ferroptosis. Discussion In this study, we observed that USP20 expression was significantly downregulated in both myocardial tissues and primary cardiomyocytes under Dox stimulation. Myocardial-specific USP20 deficiency results in the increased severity of cardiac dysfunction in Dox-induced mice. In contrast, restoring USP20 by the AAV approach alleviated Dox-induced cardiac remodelling and dysfunction, suggesting that USP20 plays an irreplaceable role in combating Dox-induced cardiomyopathy. In addition, via mass spectrometry and coimmunoprecipitation, we identified HuR as a key substrate protein of USP20. These results were verified in myocardia-specific HuR-deficient mice in which USP20 was overexpressed; HuR loss diminished the protective effect of USP20 against Dox-induced cytotoxicity. Mechanistically, USP20 binds to HuR through its USP domain and cysteine 154, facilitating the deubiquitination of HuR and ultimately stabilizing its expression, which leads to a reduction in ferroptosis. Hence, the findings of this study reveal novel insights into the effect of USP20 in DIC and provide a promising therapeutic strategy for DIC by demonstrating the essential role of USP20 in preventing ferroptosis and maintaining cardiac function. To date, the deubiquitinating enzyme family contains approximately 100 proteins; as crucial regulatory mediators of posttranslational modifications that deubiquitinate various substrates, they have garnered increasing attention. DUBs play crucial roles in various fields of cell biology, and their expression is highly correlated with pathologies such as neurodegenerative diseases, cancer, and cardiovascular disease 18 . Despite the growing number of reports on DUBs in cardiovascular diseases in recent years 19 , 20 , our understanding of their mechanisms of action in DOX-induced cardiotoxicity remains limited. Even different deubiquitinating enzymes exhibit diametrically opposed functions in DIC. For example, Rimpy Dhingra reported that USP19 alleviated DIC by deubiquitinating TRAF2 and preventing its degradation 12 . However, Wang demonstrated that Dox can promote the progression of DIC by activating the USP36-mediated deubiquitination of PARP1 13 [13]. Our research revealed that endogenous USP20 in the heart was located and more abundant in cardiomyocytes and served as a protective regulator involved in DIC. USP20 depletion exacerbates whereas USP20 overexpression rescues cardiac remodelling and function in DIC. Moreover, we confirmed that USP20 regulated the degradation of HuR, a deubiquitinase, in Dox-induced cardiomyocytes and verified that HuR, a key downregulated protein of USP20, is crucial for the effect of USP20 on DIC, as evidenced by turnover experiments in which HuR was lost in USP20-overexpressing mice. In addition, we revealed the linked motif between USP20 and HuR, in which the domain of USP20 is directly bound to HuR, stabilizing HuR through K48-linked deubiquitination. Overall, we demonstrated that USP20, as a favourable regulatory protein, participates in the pathogenesis of DIC by stabilizing HuR expression, thereby expanding the understanding of USP20 as a crucial signalling molecule in cardiomyocytes and offering potential therapeutic avenues for targeted gene therapy for DIC. The pathophysiology of DIC is complex and is characterized by the increased production of ROS, mitochondrial dysfunction, autophagy, and cardiomyocyte apoptosis and ferroptosis, resulting in cardiac dysfunction 21 , 22 . Among these mechanisms, cardiac ferroptosis is one of the main forms of cell death in DIC 23 . Consistent with a previous report, we found that cardiac ferroptosis and ROS levels largely increased, whereas these alterations were further exacerbated by USP20 deficiency and reversed by USP20 overexpression in vivo and in vitro , suggesting that USP20 can effectively mitigate DIC through cardiac ferroptosis. However, the precise underlying mechanism remains elusive. The RNA-binding protein HuR is widely distributed and selectively binds to AU-rich domains in various mRNA molecules, thereby influencing their stability. HuR plays a pivotal role in the pathogenesis and progression of diverse diseases 24 , 25 , 26 . For example, Zhong et al. reported that Ang II modulated HuR activity through P2X7 receptor activation, consequently influencing the stability of HO-1 and GPX4 mRNAs and subsequently regulating ferroptosis as well as ventricular remodelling 27 . Notably, HuR plays a pivotal role not only in cardiomyocytes but also in cardiac fibroblasts. Guo reported that NF-κB-repressing factor (NKRF) interacts with HuR and then regulates ventricular remodelling in cardiac fibroblasts by modulating the stability of MMP2 and MMP9 mRNA 28 . Additionally, recent studies have demonstrated the involvement of the HuR protein in the regulation of Dox-induced ferroptosis within the context of DIC 29 . In this study, we discovered that USP20 enhances the stability of the HuR protein through deubiquitination and that the overexpression of this protein via the AAV approach in cardiocyte-specific HuR-knockout mice abrogated the cardioprotective benefits of USP20 and diminished the inhibitory effect of USP20 against cardiac ferroptosis, suggesting that HuR is critical for USP20-mediated cardiac ferroptosis. Mechanistically, HuR bound to GPX4 mRNA, and this binding decreased upon Dox stimulation but was partially reversed by USP20 overexpression. Additionally, we validated the impact of HuR on GPX4 mRNA stability and observed that overexpressing HuR alleviated the Dox-induced decrease in GPX4 mRNA stability. These findings suggest that USP20 regulates ferroptosis in DIC by modulating the binding capacity of HuR to its target mRNA. In summary, our findings provide evidence that USP20 mitigates DIC by exerting inhibitory effects against cardiac ferroptosis. Moreover, we identified that there is an interaction between USP20 and HuR, which markedly decreased HuR ubiquitination and thus led to the increased binding of HuR to GPX4 and affected GPX4 mRNA stability. In addition, cysteine 154 in USP20 is key for its deubiquitination by HuR. In addition, we ascertained that HuR deletion abrogates USP20-induced cardiac ferroptosis and its cardioprotective benefits in DIC. These data strongly support a previously undescribed USP20–HuR–GPX4 axis, which plays a critical role in cardiac ferroptosis and ROS during DIC, providing a possible therapeutic option for preventing Dox-induced cardiac injury. Study limitations The results of the present study indicated that USP20 restoration with AAV therapy might be a potential approach for treating Dox-induced cardiotoxicity; however, this translational concept has not been validated in nonhuman primates. In addition, we suggest that USP20 supplementation is a potential treatment strategy for DIC; therefore, specific agonists that target USP20 warrant further investigation. Finally, although we determined that USP20 interacts with HuR, we did not explore whether HuR has other direct binding sites that influence ferroptosis. Data availability All data supporting the conclusions of this study are presented in this manuscript or the supplementary information. The materials described in this study are either commercially available or available upon reasonable request from the corresponding authors. Declarations Data availability All data supporting the conclusions of this study are presented in this manuscript or the supplementary information. The materials described in this study are either commercially available or available upon reasonable request from the corresponding authors. Author contributions Zhouqing Huang, Shanshan Dai and Yinuo Lin contributed to the literature search and study design. Yunxuan Chen, Shuoning Wu, Lang Deng, Yixin Zhou, Yucong Zhang, Jiaxuan Mei, Fang Wang, Sirui Shen and Zimin Fang performed the experiments and analysed the data. Shanshan Dai and Yinuo Lin provided technical help. Yunxuan Chen participated in the drafting of the article. All authors agree to be accountable for all aspects of the work, ensuring its integrity and accuracy. Funding This work was supported by the National Natural Science Foundation of China (Grant Nos. 82070446 and 82202380), the Natural Science Foundation of Zhejiang Province (Grant Nos. LY22H020004 and LQ23H310005), the Project of the Health Commission of Zhejiang Province (Grant No. WKJ-ZJ-2540), and the Science and Technology Project of Wenzhou (Grant No. Y20210136). Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Wu Y, Wang Z, Han L, Guo Z, Yan B, Guo L, et al. PRMT5 regulates RNA m6A demethylation for doxorubicin sensitivity in breast cancer. Mol Ther 2022, 30(7): 2603–2617. Butowska K, Han X, Gong N, El-Mayta R, Haley RM, Xue L, et al. Doxorubicin-conjugated siRNA lipid nanoparticles for combination cancer therapy. Acta Pharm Sin B 2023, 13(4): 1429–1437. Curigliano G, Cardinale D, Dent S, Criscitiello C, Aseyev O, Lenihan D, et al. Cardiotoxicity of anticancer treatments: Epidemiology, detection, and management. CA Cancer J Clin 2016, 66(4): 309–325. Carvalho FS, Burgeiro A, Garcia R, Moreno AJ, Carvalho RA, Oliveira PJ. Doxorubicin-induced cardiotoxicity: from bioenergetic failure and cell death to cardiomyopathy. Med Res Rev 2014, 34(1): 106–135. 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Additional Declarations There is no duality of interest Supplementary Files Supplementaryfiles3.23.docx Supplementary Information WB.pdf WB sup1.tif Supplementary Figure 1 sup2.tif Supplementary Figure 2 sup3.tif Supplementary Figure 3 sup4.tif Supplementary Figure 4 sup5.tif Supplementary Figure 5 sup6.tif Supplementary Figure 6 sup7.tif Supplementary Figure 7 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6151078","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":435141340,"identity":"8ee2b110-f60a-4433-ad1c-5abf1a3ccb47","order_by":0,"name":"Zhouqing 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expression is downregulated in DIC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Transcriptome sequencing was used to reveal the expression profile of DUBs in DIC. Red points indicate the DUBs whose expression was downregulated (compared with the control group).\u003c/p\u003e\n\u003cp\u003e(B) mRNA expression levels of \u003cem\u003eUsp20\u003c/em\u003ein neonatal rat cardiomyocytes (NRCMs) from mice treated with Dox (n=3).\u003c/p\u003e\n\u003cp\u003e(C–D) USP20 expression in heart tissues from mice treated with Dox was assessed by western blotting(C) and statistically analysed (D) (n=3).\u003c/p\u003e\n\u003cp\u003e(E–F) USP20 expression in NRCMs treated with Doxwas assessed by western blotting(E) and statistically analysed (F) (n=3).\u003c/p\u003e\n\u003cp\u003e(G–H) USP20 expression in primary fibroblasts treated with Dox was assessed by western blotting(G) and statistically analysed (H) (n=3).\u003c/p\u003e\n\u003cp\u003e(I–J) USP20 expression in macrophages treated with Dox was assessed by western blotting(I) and statistically analysed (J) (n=3).\u003c/p\u003e\n\u003cp\u003e(K-L) tSNE dimensionality reduction in heart cells. The main cell types were identified. The population identities were determined on the basis of marker gene expression. t-SNE dimensional reduction showing the expression pattern of USP20 among the different cell types in the heart (K). The colourscale represents expression levels in biaxial scatter plots (grey: low; red: high) (L).\u003c/p\u003e\n\u003cp\u003e(M-N) tSNE dimensionality reduction in reclustered subpopulationcells in the control group. The main cell types were identified. The populationidentities were determined on the basis of marker gene expression (M). t-SNE dimensional reduction in reclustered subpopulation cells in the Dox-stimulated group (N).\u003c/p\u003e\n\u003cp\u003e(O) Dot plot illustrating relative USP20 expression in 4 distinct cardiomyocyte populations.\u003c/p\u003e\n\u003cp\u003e(P) Representative images of immunofluorescence staining for USP20 (green), α-actin (red), vimentin (red) and F4/80 (red) in heart sections from Dox-treated mice. Yellow in the merged images denotes regions of colocalization.\u003c/p\u003e\n\u003cp\u003eSignificance is defined as *P \u0026lt; 0.05, **P\u0026lt;0.01, and ***P \u0026lt; 0.001. The abbreviation “ns” denotes no statistical significance.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/f411b78eaef2507913025730.png"},{"id":80722287,"identity":"4af5a99c-993a-424d-85b9-6d772acd0e2f","added_by":"auto","created_at":"2025-04-16 11:15:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":751353,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCardiomyocyte-specific USP20 deficiency exacerbates the progression of DIC via ferroptosis \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of the Dox-stimulated mouse model.\u003c/p\u003e\n\u003cp\u003e(B) Representative echocardiography (Echo) of Dox model mice.\u003c/p\u003e\n\u003cp\u003e(C–D) Myocardial function parameters, i.e., ejection fraction (EF) (C) and fractional shortening (FS) (D) levels, in Dox-treated mice were measured by echocardiography (n = 6).\u003c/p\u003e\n\u003cp\u003e(E) Representative images of H\u0026amp;E-stained heart tissue from mice treated with Dox (magnification × 400).\u003c/p\u003e\n\u003cp\u003e(F) Representative images of Sirius Red staining of heart tissue from mice treated with Dox (magnification × 400).\u003c/p\u003e\n\u003cp\u003e(G-H) Representative images of TUNEL-stained heart tissue from Dox-treated mice (magnification × 400) (G) and statistical results (H).\u003c/p\u003e\n\u003cp\u003e(I-K) The levels of the creatine kinase isoenzymes MB (CK-MB) (I), lactate dehydrogenase (LDH) (J) and cardiac troponin I (cTnI) (K) in the serum of Dox-treated mice (n = 6).\u003c/p\u003e\n\u003cp\u003e(L) Levels of ferrous ions (Fe\u003csup\u003e2+\u003c/sup\u003e) (L) in the serum of mice treated with Dox (n = 6).\u003c/p\u003e\n\u003cp\u003e(M-N) The levels of malondialdehyde (MDA) (M) and superoxide dismutase (SOD) (N) in the heart tissues of mice treated with Dox (n=6).\u003c/p\u003e\n\u003cp\u003e(O‒P) Expression levels of GPX4 proteins in heart tissues were assessed via Western blot analysis. GAPDH was used as a loading control (O). The results were statistically analysed (P) (n = 6).\u003c/p\u003e\n\u003cp\u003e(Q) Representative images of 4-HNE immunofluorescence staining of heart tissue from mice treated with Dox (magnification × 400).\u003c/p\u003e\n\u003cp\u003eSignificance is defined as *P \u0026lt; 0.05, **P\u0026lt;0.01, and ***P \u0026lt; 0.001. The abbreviation “ns” denotes no statistical significance.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/388eda0b2412591c545d0222.png"},{"id":80723920,"identity":"e7c83920-42c0-4c92-a7ed-436030c0b4db","added_by":"auto","created_at":"2025-04-16 11:31:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":427313,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUSP20 mitigates Dox-induced ferroptosis in cardiomyocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Cell counting kit-8 (CCK8) results (A) and lactate dehydrogenase (LDH) levels (B) in the culture supernatants from each group (n = 3).\u003c/p\u003e\n\u003cp\u003e(C-F) The levels of ferrous ions (Fe\u003csup\u003e2+\u003c/sup\u003e) (C), superoxide dismutase (SOD) (D), malondialdehyde (MDA) (E) and glutathione (GSH) (F) in NRCMs from each group (n=3).\u003c/p\u003e\n\u003cp\u003e(G-H) Representative western blot analysis of GPX4 in NRCMs; GAPDH was used as a loading control (G); the results were statistically analysed (H) (n=3).\u003c/p\u003e\n\u003cp\u003e(I) Representative images of dihydroethidium (DHE) staining from each group (magnification × 200).\u003c/p\u003e\n\u003cp\u003e(J) Representative images of propidium iodide (PI) staining from each group (magnification × 200).\u003c/p\u003e\n\u003cp\u003e(K-L) Cell counting kit-8 (CCK8) results (K) and lactate dehydrogenase (LDH) levels (L) in the culture supernatants from each group (n = 3).\u003c/p\u003e\n\u003cp\u003e(M-P) The levels of ferrous ions (Fe\u003csup\u003e2+\u003c/sup\u003e) (M), superoxide dismutase (SOD) (N), malondialdehyde (MDA) (O) and glutathione (GSH) (P) in NRCMs from each group (n=3).\u003c/p\u003e\n\u003cp\u003e(Q‒R) Representative western blot analysis of GPX4 in NRCMs; GAPDH was used as a loading control (Q); the results were statistically analysed (R) (n=3).\u003c/p\u003e\n\u003cp\u003e(S) Representative images of dihydroethidium (DHE) staining from each group (magnification × 200).\u003c/p\u003e\n\u003cp\u003e(T) Representative images of propidium iodide (PI) staining from each group (magnification × 200).\u003c/p\u003e\n\u003cp\u003eSignificance is defined as *P \u0026lt; 0.05, **P\u0026lt;0.01, and ***P \u0026lt; 0.001. The abbreviation “ns” denotes no statistical significance.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/bcd51a7349749cc0b036a0e1.png"},{"id":80723919,"identity":"b42691cf-5585-4214-809e-1adb6ab3d0a4","added_by":"auto","created_at":"2025-04-16 11:31:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":608248,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRestoration of cardiac\u003c/strong\u003e \u003cstrong\u003eUSP20 alleviates DIC by attenuating ferroptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Dox was administered intraperitoneally to establish a DIC model, and AAV9-USP20 was injected via the tail vein to specifically overexpress USP20 in the heart.\u003c/p\u003e\n\u003cp\u003e(B) Representative echocardiography (Echo) of the mice from each group.\u003c/p\u003e\n\u003cp\u003e(C–D) Myocardial function parameters, i.e., ejection fraction (EF) (C) and fractional shortening (FS) (D) levels, in Dox-treated mice were measured by echocardiography (n = 6).\u003c/p\u003e\n\u003cp\u003e(E) Representative images of H\u0026amp;E-stained heart tissue from Dox-treated mice (magnification × 400).\u003c/p\u003e\n\u003cp\u003e(F) Representative images of Sirius Red-stained heart tissue from Dox-treated mice (magnification × 400).\u003c/p\u003e\n\u003cp\u003e(G-H) Representative images of TUNEL-stained heart tissue from Dox-treated mice (magnification × 400) (G) and statistical results (H).\u003c/p\u003e\n\u003cp\u003e(I-K) The levels of lactate dehydrogenase (LDH) (I), creatine kinase isoenzyme MB (CK-MB) (J) and cardiac troponin I (cTnI) (K) in the serum from mice in each group (n = 6).\u003c/p\u003e\n\u003cp\u003e(L-N) The levels of ferrous ions (Fe\u003csup\u003e2+\u003c/sup\u003e) (L), superoxide dismutase (SOD) (M) and malondialdehyde (MDA) (N) in heart tissues from each group (n=6).\u003c/p\u003e\n\u003cp\u003e(O‒P) The protein expression levels of GPX4 in heart tissue were assessed via Western blot analysis. GAPDH was used as a loading control (O); the results were statistically analysed (P) (n = 6).\u003c/p\u003e\n\u003cp\u003e(Q) Representative images of 4-HNE immunofluorescence staining of heart tissue from mice treated with Dox (magnification × 400).\u003c/p\u003e\n\u003cp\u003eSignificance is defined as *P \u0026lt; 0.05, **P\u0026lt;0.01, and ***P \u0026lt; 0.001. The abbreviation “ns” denotes no statistical significance.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/54444a9dc9322bf9ec1c3b56.png"},{"id":80722296,"identity":"f0a1e128-3122-4687-bf09-7d05bd565e98","added_by":"auto","created_at":"2025-04-16 11:15:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":306004,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUSP20 directly interacts with HuR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of a comprehensive combined ubiquitinome, proteome and interactome analysis for USP20 substrate screening.\u003c/p\u003e\n\u003cp\u003e(B) Two-dimensional plot with log10 (signal intensity) values of the quantified proteins, revealing an enrichment in USP20 immunoprecipitation versus IgG immunoprecipitation, on the y-axis, and the molecular mass (MM) of the proteins, on the x-axis.\u003c/p\u003e\n\u003cp\u003e(C) Mass spectrometry approach to screen for potential substrate proteins of USP20.\u003c/p\u003e\n\u003cp\u003e(D) Coimmunoprecipitation of USP20 and HuR in NIH/3T3 cells cotransfected with Flag-USP20 and His-HuR plasmids. Exogenous USP20 was immunoprecipitated with an anti-Flag antibody. GAPDH was used as a loading control.\u003c/p\u003e\n\u003cp\u003e(E-F) Coimmunoprecipitation of USP20 and HuR in primary cardiomyocytes (E) and heart tissue (F). Endogenous USP20 was immunoprecipitated with an anti-USP20 antibody. IgG, immunoglobulin G. GAPDH was used as a loading control.\u003c/p\u003e\n\u003cp\u003e(G) Schematic illustration of the USP20 domain deletion construct used in H.\u003c/p\u003e\n\u003cp\u003e(H) Coimmunoprecipitation of wt-USP20, mut-USP20, and HuR in NIH/3T3 cells cotransfected with Flag-wtUSP20, Flag-mut-USP20 and His-HuR overexpression plasmids. Exogenous normal or mutated USP20 was immunoprecipitated with an anti-Flag antibody. GAPDH was used as a loading control.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/2ff8afb896087fcc2056ede2.png"},{"id":80722299,"identity":"1c2f256f-70e5-4296-aa3b-770913909aa0","added_by":"auto","created_at":"2025-04-16 11:15:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":378673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe C154 active site of USP20 deubiquitinates HuR and maintains its stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A–B) Representative western blotting (A) and real-time qPCR (B) results for USP20 and HuR expression in NIH/3T3 cells transfected with a Flag-USP20 overexpression plasmid (n=3).\u003c/p\u003e\n\u003cp\u003e(C-D) Representative western blots of HuR in control (gCtrl) or USP20-knockout (gUSP20) NIH/3T3 cells subjected to a CHX pulse-chase assay (C) and densitometric quantification of HuR (D). ns, gUSP20-3 h group vs. gCtrl-3 h group; ***, gUSP20-6 h group vs. gCtrl-6 h group; ###, gUSP20-9 h group vs. gCtrl-9 h group (n=3).\u003c/p\u003e\n\u003cp\u003e(E) Immunoprecipitation of HuR in gCtrl or gUSP20 NIH/3T3 cells that were cotransfected with His-HuR, HA-Ub or HA-K63 overexpression plasmids and then treated with MG132 (10 μM). Ubiquitinated HuR was detected by immunoblotting using an His-specific antibody to investigate the ubiquitination pattern of HuR regulated by USP20.\u003c/p\u003e\n\u003cp\u003e(F) Schematic illustration of the USP20 active site deletion construct.\u003c/p\u003e\n\u003cp\u003e(G-H) Representative western blot showing HuR protein expression in gUSP20 NIH/3T3 cells transfected with Flag-USP20, Flag-USP20C154A, or Flag-USP20H645A overexpression plasmids (G) and densitometric quantification of HuR (H). ns, USP20-C154A-6 h vs. NC-6 h; ***USP20-H645A-6 h vs. NC-6 h; ###, USP20\u003csup\u003eoe\u003c/sup\u003e-6 h vs. NC-6 h; NS, USP20-C154A-9 h vs. NC-9 h; %%, USP20-H645A-9 h vs. NC-9 h; \u0026amp;\u0026amp;\u0026amp;, USP20\u003csup\u003eoe\u003c/sup\u003e-9 h vs. NC-9 h (n=3).\u003c/p\u003e\n\u003cp\u003e(I) Coimmunoprecipitation of USP20C154A, Flag-USP20H645A and HuR in NIH/3T3 cells cotransfected with the Flag-USP20C154A, Flag-USP20H645A and His-HuR plasmids. Exogenous normal or mutated USP20 was immunoprecipitated with an anti-Flag antibody.\u003c/p\u003e\n\u003cp\u003e(J) Immunoprecipitation of HuR in gUSP20 NIH/3T3 cells cotransfected with His-HuR, HA-Ub, Flag-USP20, Flag-USP20C154A and Flag-USP20H645A overexpression plasmids. Exogenously ubiquitinated HuR was detected by immunoblotting with an anti-His-specific antibody to identify the active site of USP20 that regulates the ubiquitination of HuR.\u003c/p\u003e\n\u003cp\u003e(K–L) Cell Counting Kit-8 (CCK-8) results (K) and lactate dehydrogenase (LDH) levels (L) in the culture supernatants from each group (n = 3).\u003c/p\u003e\n\u003cp\u003eSignificance is defined as *P \u0026lt; 0.05, **P\u0026lt;0.01, and ***P \u0026lt; 0.001. The abbreviation “ns” denotes no statistical significance.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/aa66ae9c7808f82f43e8de57.png"},{"id":80723187,"identity":"4308123a-8aa5-41e7-8cc4-533fb1314228","added_by":"auto","created_at":"2025-04-16 11:23:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":571916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe anti-DIC effect of USP20 is dependent on HuR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative echocardiograph (Echo) of mice in each group.\u003c/p\u003e\n\u003cp\u003e(B-C) Myocardial function parameters, i.e., ejection fraction (EF) (B) and fractional shortening (FS) (C) levels, in Dox-treated mice were measured by echocardiography (n = 6).\u003c/p\u003e\n\u003cp\u003e(D) Representative images of H\u0026amp;E-stained heart tissue from Dox-treated mice (magnification × 400).\u003c/p\u003e\n\u003cp\u003e(E) Representative images of Sirius Red-stained heart tissue from Dox-treated mice (magnification × 400).\u003c/p\u003e\n\u003cp\u003e(F-G) Representative images of TUNEL-stained heart tissue from Dox-treated mice (magnification × 400) (F) and statistical results (G).\u003c/p\u003e\n\u003cp\u003e(H-J) The levels of the creatine kinase isoenzymes MB (CK-MB) (H), lactate dehydrogenase (LDH) (I) and cardiac troponin I (cTnI) (J) in the serum from mice in each group (n = 6).\u003c/p\u003e\n\u003cp\u003e(K–M) Ferrous ion (Fe\u003csup\u003e2+\u003c/sup\u003e) (K), superoxide dismutase (SOD) (L) and malondialdehyde (MDA) (M) levels in heart tissues of mice from each group (n=6).\u003c/p\u003e\n\u003cp\u003e(N) Representative images of 4-HNE immunofluorescence staining of heart tissue from Dox-treated mice (magnification × 400).\u003c/p\u003e\n\u003cp\u003e(O‒P) Cell counting kit-8 (CCK8) results (O) and lactate dehydrogenase (LDH) levels (P) in the serum of mice from each group (n = 3).\u003c/p\u003e\n\u003cp\u003e(Q-T) The levels of ferrous ions (Fe\u003csup\u003e2+\u003c/sup\u003e) (Q), superoxide dismutase (SOD) (R), malondialdehyde (MDA) (S) and glutathione (GSH) (T) in NRCMs from each group (n=3).\u003c/p\u003e\n\u003cp\u003e(U-V) Representative western blot results. GAPDH was used as a loading control. (U) Densitometric quantification of Flag, HuR, and GPX4 in NRCMs (V) (n=3).\u003c/p\u003e\n\u003cp\u003e(W) Representative images of dihydroethidium (DHE) staining from each group (magnification × 200).\u003c/p\u003e\n\u003cp\u003e(X) Representative images of propidium iodide (PI) staining from each group (magnification × 200).\u003c/p\u003e\n\u003cp\u003eSignificance is defined as *P \u0026lt; 0.05, **P\u0026lt;0.01, and ***P \u0026lt; 0.001. The abbreviation “ns” denotes no statistical significance.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/d55fd5fbb671ec98a06d170f.png"},{"id":81277377,"identity":"4b463a66-656c-4848-bba3-e5519e2b940b","added_by":"auto","created_at":"2025-04-24 09:33:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5352475,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/da3bbf6a-efda-4015-98d4-b815c74f276b.pdf"},{"id":80722284,"identity":"ce1d1210-3090-4fef-85dd-f8ce5ab4fbed","added_by":"auto","created_at":"2025-04-16 11:15:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":61415,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Information\u003c/p\u003e","description":"","filename":"Supplementaryfiles3.23.docx","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/1bd7bb66984da883fe41f2aa.docx"},{"id":80722290,"identity":"67187fcc-c15b-46cd-986a-4aa134ab9186","added_by":"auto","created_at":"2025-04-16 11:15:41","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1606975,"visible":true,"origin":"","legend":"\u003cp\u003eWB\u003c/p\u003e","description":"","filename":"WB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/9287d52ce1cbdd762755c762.pdf"},{"id":80722293,"identity":"d7df9ed3-bfea-4f51-9378-b1b919d5311b","added_by":"auto","created_at":"2025-04-16 11:15:41","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":199546,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 1\u003c/p\u003e","description":"","filename":"sup1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/3286e7e563ea1a73aa7b429c.tif"},{"id":80722292,"identity":"48c806bb-b10f-479f-b988-53bf433145ba","added_by":"auto","created_at":"2025-04-16 11:15:41","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":34948,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 2\u003c/p\u003e","description":"","filename":"sup2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/ecb6786ef352c0fe5f1cf0bf.tif"},{"id":80723185,"identity":"eef9f1a9-86ca-44a9-989f-90ebacba949e","added_by":"auto","created_at":"2025-04-16 11:23:41","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":9819,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 3\u003c/p\u003e","description":"","filename":"sup3.tif","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/43937235b7dc7c70674c5bbf.tif"},{"id":80722294,"identity":"58140d97-ede0-46f9-ab43-069b59013827","added_by":"auto","created_at":"2025-04-16 11:15:42","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":8036,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 4\u003c/p\u003e","description":"","filename":"sup4.tif","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/a5459c9ad96e79cd150b0c2f.tif"},{"id":80722326,"identity":"330ae123-b271-42f7-8807-f49c49ffd82b","added_by":"auto","created_at":"2025-04-16 11:15:43","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":60624,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 5\u003c/p\u003e","description":"","filename":"sup5.tif","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/11bf7edad16edacae16c4627.tif"},{"id":80722298,"identity":"6bceb241-0ab6-4120-b1c3-34a12743945d","added_by":"auto","created_at":"2025-04-16 11:15:42","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":37774,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 6\u003c/p\u003e","description":"","filename":"sup6.tif","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/981735d7446d074f75337d51.tif"},{"id":80722312,"identity":"d5bbd35a-7ee9-46b3-ac0a-a3a4831d6ce5","added_by":"auto","created_at":"2025-04-16 11:15:42","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":37030,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 7\u003c/p\u003e","description":"","filename":"sup7.tif","url":"https://assets-eu.researchsquare.com/files/rs-6151078/v1/13c83f26ebb276f0cb360273.tif"}],"financialInterests":"There is no duality of interest","formattedTitle":"USP20 mitigates doxorubicin-induced cardiotoxicity by deubiquitinating and stabilizing HuR","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDoxorubicin (Dox) is a highly efficacious and extensively used chemotherapeutic agent that is currently used in the management of various neoplastic conditions, such as breast cancer, ovarian cancer, lymphoma, and sarcoma \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, both its acute and chronic toxic effects considerably limit the clinical application of Dox \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The dose-dependent cardiotoxicity associated with doxorubicin leads to damage to cardiomyocytes and subsequent cardiac dysfunction, ultimately culminating in heart failure and severely impairing the quality of life of cancer patients during or following treatment with doxorubicin. Despite extensive research on the mechanisms underlying Dox-induced cardiotoxicity over the years, these processes remain poorly understood. Therefore, the identification of new therapeutic targets that may alleviate Dox-induced cardiotoxicity is urgently needed. Ferroptosis, a form of iron-dependent nonapoptotic cell death predominantly induced by lipid peroxidation triggered by excessive intracellular iron accumulation, plays a pivotal role in the pathogenesis of Dox-induced cardiomyopathy (DIC) \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Consequently, modulating ferroptosis in cardiomyocytes may offer a novel approach for preventing and treating DIC.\u003c/p\u003e \u003cp\u003eUbiquitination, a dynamic and multifaceted posttranslational modification, plays a pivotal role in various crucial physiological processes, such as transcriptional regulation, cellular signalling, and DNA damage repair \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Furthermore, the precise regulation of protein degradation, localization, and function is facilitated by deubiquitinating enzymes (DUBs), which reverse protein ubiquitination. Currently, approximately 100 identified DUBs within the human proteome are categorized into seven families. Among these families, the ubiquitin-specific protease (USP) family represents the largest subfamily of deubiquitinases, which are critically involved in various pathological processes, including inflammation, metabolic disorders, cancer, and cardiovascular diseases \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Recent studies have demonstrated that USP19 exerts a protective effect against DIC by deubiquitinating TRAF2 to prevent its degradation \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, evidence has indicated that doxorubicin accelerates the progression of DIC by activating the USP36-mediated deubiquitination of PARP1 \u003csup\u003e13\u003c/sup\u003e. The USP family clearly plays a pivotal role in elucidating the molecular mechanism underlying DIC. Elucidating the roles and mechanisms of deubiquitinases in DIC will enhance our understanding of this phenomenon and provide novel insights for clinical treatment strategies.\u003c/p\u003e \u003cp\u003eThe deubiquitinating enzyme USP20, a member of the USP family, has been recognized for its critical involvement in diverse biological processes through the modulation of deubiquitination modifications on specific substrates. Many studies have reported findings related to cancer, atherosclerosis, lipid metabolism, and other related fields \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the role of USP20 in cardiac biology remains largely underexplored and represents a promising avenue for future investigations.\u003c/p\u003e \u003cp\u003eOur team focused on the regulatory mechanism of DUBs in the pathogenesis of cardiovascular diseases. We compared DUB gene expression profiles in myocardial tissue from DIC patients in the GEO database and detected a significantly decreased level of the DUB ubiquitin-specific protease 20 (USP20), indicating the potential involvement of USP20 in the pathogenesis of DIC. Furthermore, our investigation of Universal Protein data revealed that USP20 is markedly expressed in the heart, thereby providing additional evidence for the potential involvement of USP20 in cardiac biology. Herein, we investigated the role of USP20 in DIC, with the aim of clarifying its regulatory molecular mechanism. Our findings identify USP20 as a potential target for future clinical treatment of DIC.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003e All experimental protocols received approval from the Laboratory Animal Ethics Committee and the Laboratory Animal Centre of the First Affiliated Hospital of Wenzhou Medical University (WYYY-IACUC-AEC-2024-097). The experiments were conducted utilizing cardiomyocyte-specific USP20-knockout mice (USP20CKO) and cardiomyocyte-specific HuR-knockout mice (HuRCKO), both of which were on a C57BL/6J genetic background, and flox mice (USP20\u003csup\u003efl/fl\u003c/sup\u003e and HuR\u003csup\u003efl/fl\u003c/sup\u003e) derived from the same litter. Randomization was employed for animal grouping, and analyses were performed by blinded experimenters.\u003c/p\u003e \u003cp\u003eTo elucidate the role of USP20 in doxorubicin-induced cardiotoxicity, 8-week-old male mice were divided into four groups: (1) USP20\u003csup\u003efl/fl\u003c/sup\u003e + saline; (2) USP20CKO\u0026thinsp;+\u0026thinsp;saline; (3) USP20\u003csup\u003efl/fl\u003c/sup\u003e + Dox; and (4) USP20CKO\u0026thinsp;+\u0026thinsp;Dox. Mice were subjected to weekly intraperitoneal injections of either saline or Dox at a dosage of 5 mg/kg, which were administered over a duration of four weeks. The mice were housed under controlled conditions at a temperature of 25\u0026deg;C with a 12-h light/dark cycle and had ad libitum access to standard chow.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAdeno-associated virus (AAV) infection\u003c/h3\u003e\n\u003cp\u003eTo achieve targeted USP20 overexpression in cardiomyocytes, male mice were administered adeno-associated virus serotype 9 containing troponin-specific promoters. This vector carried either an empty vector (AAV9-cTnT-EV) or \u003cem\u003eUsp20\u003c/em\u003e cDNA (AAV9-cTnT-USP20\u003csup\u003eoe\u003c/sup\u003e). The mice were separated into three experimental groups: (1) WT\u0026thinsp;+\u0026thinsp;AAV9-cTnT-EV\u0026thinsp;+\u0026thinsp;saline, (2) WT\u0026thinsp;+\u0026thinsp;AAV9-cTnT-EV\u0026thinsp;+\u0026thinsp;Dox, and (3) WT\u0026thinsp;+\u0026thinsp;AAV9-cTnT-USP20\u003csup\u003eoe\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;Dox. AAV9 was introduced via the tail vein at a dosage of 2E\u0026thinsp;+\u0026thinsp;11 v.g./mouse two weeks prior to the initiation of the modelling process. A four-week treatment regimen with either saline or Dox was subsequently delivered through the peritoneal cavity in accordance with previously established protocols.\u003c/p\u003e\n\u003ch3\u003eEchocardiography\u003c/h3\u003e\n\u003cp\u003eEchocardiography was conducted four weeks after the administration of Dox. The hair in the left anterior thoracic region of each mouse was removed using a depilatory cream, and the mice were subsequently anaesthetized with isoflurane. Cardiac function was evaluated through M-mode echocardiography utilizing the Visual Sonics Vevo 3100 Small Animal Ultrasound Imaging System, allowing for the determination of the ejection fraction (EF) and shortening fraction (FS), as well as other cardiac function parameters.\u003c/p\u003e\n\u003ch3\u003eSerum biochemical analysis\u003c/h3\u003e\n\u003cp\u003eThe levels of lactate dehydrogenase (LDH) were measured using an LDH assay kit (BC0685, Solarbio, Beijing, China). The levels of creatine kinase isoenzyme (CK-MB) were assessed with a CK-MB assay kit (E006-1-1; Jiancheng Biological Engineering Institute, Nanjing, China). The concentration of mouse cardiac troponin T (cTnT/TNNT2) was evaluated utilizing a cTnT assay kit (E-EL-M1801, Elabscience, Wuhan, China). Additionally, the level of ferrous iron was measured using a ferrous iron assay kit (E-BC-K773-M, Elabscience, Wuhan, China). All measurements were conducted in accordance with the product manuals provided for each respective kit.\u003c/p\u003e\n\u003ch3\u003eHistological analysis\u003c/h3\u003e\n\u003cp\u003eCardiac tissues were fixed in 4% paraformaldehyde for 24 h, subsequently embedded in paraffin, and sectioned into 5 \u0026micro;m slices. The sections were dewaxed, hydrated, and stained with haematoxylin and eosin (H\u0026amp;E, G1120, Solarbio, China) as well as Sirius Red (G1472, Solarbio, China). Images were captured using an Olympus Corporation microscope.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical (IHC) staining\u003c/h2\u003e \u003cp\u003eFollowing dewaxing, the tissue sections were immersed in a boiling citrate buffer solution (0.01 M, pH 6.0) for 10 min at 100\u0026deg;C. The sections were allowed to cool to ambient temperature before being incubated in a 3% hydrogen peroxide solution for 15 min. The sections were subsequently incubated with 5% BSA for 30 min to block nonspecific binding. After the BSA was removed, the sections were incubated with a diluted anti-4-HNE antibody (1:200, MAB3249, R\u0026amp;D Systems, Shanghai, China) overnight at 4\u0026deg;C. The following day, the sections were incubated with the corresponding secondary antibody at room temperature for one hour. Finally, the sections were stained with DAB staining solution for 8 min, followed by haematoxylin counterstaining for 1 min. The stained sections were mounted with an appropriate mounting medium and visualized under a microscope (Olympus Corporation, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eFrozen sections were fixed with 4% paraformaldehyde for 20 min. Subsequently, 3% hydrogen peroxide was used to inhibit endogenous peroxidase activity, and nonspecific binding was blocked with 5% BSA. After the BSA was aspirated, the sections were incubated with diluted anti-USP20 (rabbit, 1:200, A301-189A-M, Bethyl), anti-α-SMA (mouse, 1:200, 48838s, CST), anti-vimentin (mouse, 1:200, EM0401, Huabio) and anti-F4/80 (mouse, 1:200, sc-377009, Santa) antibodies overnight at 4\u0026deg;C. On the following day, the sections were incubated with an iFluor\u0026trade; 488-coupled goat anti-rabbit IgG polyclonal antibody (1:2000, HA1122, Hua An) and an iFluor\u0026trade; 594-coupled goat anti-mouse IgG polyclonal antibody (1:2000, HA1125, Hua An) for 1 h at room temperature. Finally, nuclei were stained with DAPI (36308ES11; Yeasen Biotech). Images were acquired with a microscope (Olympus Corporation, Tokyo, Japan).\u003c/p\u003e\n\u003ch3\u003eTUNEL assay\u003c/h3\u003e\n\u003cp\u003eApoptotic cells in cardiac tissues were detected using the One-Step TUNEL Apoptosis Detection Kit (C1090, Beyotime, China) according to the manufacturer's protocol. After staining, positive cells were visualized using a confocal microscope. Five random fields were chosen for the quantification of apoptotic cells.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfection\u003c/h2\u003e \u003cp\u003eThe NIH/3T3 and H9C2 cell lines were incubated in DMEM (Gibco, USA, 4.5 g/L) supplemented with 10% foetal bovine serum (Gibco, USA), 100 U/ml penicillin, and 100 U/ml streptomycin at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Once reaching a confluence of approximately 80%, the cells were incubated with Dox (0.5 \u0026micro;g/ml, 25316-40-9, MCE) for 24 h to establish the model.\u003c/p\u003e \u003cp\u003ePrimary cardiomyocytes were isolated from neonatal Sprague‒Dawley rats. The hearts were excised under aseptic conditions, subsequently washed with phosphate-buffered saline (PBS), and minced into small fragments. The myocardial tissue was then digested using trypsin and collagenase at 37\u0026deg;C. Following digestion, the fibroblasts were eliminated via differential adhesion, and the remaining cells were cultured in Dulbecco's modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% foetal bovine serum for a period of 48 h prior to subsequent experiments.\u003c/p\u003e \u003cp\u003eA USP20 overexpression plasmid was introduced into cardiomyocytes via Lipofectamine 3000 (Lot No. 3039420; Thermo Fisher Scientific, Germany) transfection reagent in accordance with the manufacturer's instructions. siRNAs were transfected into cardiomyocytes using Lipofectamine 2000 (Lot No. 3035197; Thermo Fisher Scientific, Germany) transfection reagent, in accordance with the manufacturer's instructions, to suppress the expression of USP20.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCCK-8 assay\u003c/h2\u003e \u003cp\u003eCell viability was assessed using Cell Counting Kit-8 (CCK-8; C0037; Beyotime, China) following the manufacturer's protocol. After treatment, 10 \u0026micro;l of CCK-8 solution was added to each well, after which the cells were incubated for 1 h at 37\u0026deg;C. Absorbance was measured at 450 nm using a microplate reader to determine cell viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePCR analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells via TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized using a reverse transcription kit (Vazyme R333-01), and quantitative PCR was performed using SYBR Green reagent (Takara, DRR037A). GAPDH was used as an internal control. The sequences of all the primers used are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eProtein samples from cell lysates and myocardial tissues were prepared using a lysis buffer containing phosphatase inhibitors. The protein concentration was determined with a bicinchoninic acid (BCA) assay. The samples were subsequently subjected to SDS‒PAGE, and separated proteins were transferred onto nitrocellulose membranes, which were subsequently incubated overnight at 4\u0026deg;C with the following primary antibodies: anti-USP20 (1:1000, 17491-1-AP, Proteintech), anti-6xHis-Tag (1:1000, 66005-1-ig, Proteintech), anti-DykDDDDk-tag (1:1000, 20543-1-AP, Proteintech), anti-UB (1:1000, sc-8017, Santa Cruz Biotechnology), anti-HA-Tag (1:1000, sc-57592, Santa Cruz Biotechnology), anti-GPX4 (1:1000, ET-706-45, Huabio), anti-HuR (1:1000, ET1705-81, Huabio) and anti-GAPDH (1;1000, ET1601-4, Huabio). The following day, the membranes were incubated with horseradish peroxidase (HRP)-labelled secondary antibodies (1:2000, A0208 or A0216, Beyotime). Bands were visualized via an enhanced chemiluminescence (ECL) luminescent solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eAfter the complete lysis of cell samples or animal tissues using a protein lysis buffer, magnetic beads were introduced into the lysates to selectively eliminate nonspecific binding proteins. A portion of each lysate was subsequently retained as an input sample. Appropriate antibodies were added to the resulting sample, which was then incubated overnight at 4\u0026deg;C. On the following day, fresh magnetic beads were added, and the sample was incubated for 2 h at 4\u0026deg;C. The sample was then centrifuged, after which the precipitate was retained and washed multiple times with PBS to ensure thorough removal of contaminants. Finally, the pellet was resuspended in sodium dodecyl sulfate sample buffer for 10 min to obtain a sample suitable for subsequent western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLC‒MS/MS analysis\u003c/h2\u003e \u003cp\u003ePlasmids encoding \u003cem\u003eUsp20\u003c/em\u003e cDNA or an empty vector were transfected into NIH/3T3 cells. Subsequently, cell lysates were prepared for immunoprecipitation as described previously \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The resulting samples were subjected to liquid chromatography‒tandem mass spectrometry (LC‒MS/MS) analysis, which was conducted by Shanghai Bioprofile (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA immunoprecipitation (RIP)\u003c/h2\u003e \u003cp\u003e RNA-binding protein immunoprecipitation (RIP) assays were conducted using an RNA-binding protein immunoprecipitation kit (Millipore, USA) according to the manufacturer's instructions. Anti-HuR (ET1705-81, Huabio, Hangzhou) and anti-rabbit IgG were used as antibodies in these assays. The expression level of GPX4 mRNA was quantified by quantitative PCR (qPCR).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eROS measurement\u003c/h2\u003e \u003cp\u003eTo assess the levels of reactive oxygen species (ROS) in cells and tissues, we used an MDA lipid oxidation kit (S0131M, Beyotime, China), a total SOD activity assay kit (S0101S, Beyotime, China), and GSH and GSSG measurement kits (S0053, Beyotime, China). These kits were used in accordance with the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePropidium iodide (PI) staining\u003c/h2\u003e \u003cp\u003eCell death was evaluated via propidium iodide (PI; CA1120; Solarbio, China) staining. Following treatment, the cells were washed with PBS and subsequently incubated in a 5 \u0026micro;g/ml PI solution for 25 min at room temperature in the dark. The stained cells were then visualized under a fluorescence microscope, and images were captured to assess the percentage of PI-positive cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDihydroethidium (DHE) staining\u003c/h2\u003e \u003cp\u003eSuperoxide levels in cardiomyocytes were quantified using dihydroethidium (DHE; S0063; Beyotime, China) staining. Cells were treated and subsequently incubated with 5 \u0026micro;M DHE at 37\u0026deg;C for 30 min. Following incubation, the cells were washed with PBS, and images were obtained using a fluorescence microscope to evaluate ROS levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of USP20-deficient NIH/3T3 cells (gUSP20-NIH/3T3)\u003c/h2\u003e \u003cp\u003eA lentiviral vector containing Cas9 and guided RNA targeting USP20 was generated from NIH/3T3 cells. Monoclonal cell lines were established following the transformation process and subsequent colony selection. The lentivirus packaging plasmid was transfected into 293T cells, after which the culture supernatant containing the lentivirus was collected 48 h later. NIH/3T3 cells were then infected with the viral supernatant and selectively cultured in medium supplemented with blastocide. The expression levels of USP20 in gUSP20-NIH/3T3 cells were analysed via western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of mRNA stability\u003c/h2\u003e \u003cp\u003eWe treated cells with actinomycin D (#S8964, Selleck, Shanghai) for 0, 2, 4, 6, and 8 h and then extracted RNA to quantify GPX4 mRNA levels using qPCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SDs). Statistical analyses were performed using GraphPad Prism 8 software (GraphPad, San Diego, CA). For comparisons involving more than two groups of data, two-way analysis of variance (ANOVA) followed by Tukey's correction was employed, while the t test was used for comparisons between two groups of data. A significance level of 0.05 was considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eUSP20 expression is downregulated in DIC\u003c/h2\u003e \u003cp\u003eThe aim of this investigation was to elucidate the protective role of DUBs in DIC. A comprehensive analysis of downregulated DUB genes was conducted using microarray datasets (GSE42177 and GSE207737) obtained from the Gene Expression Omnibus (GEO) database, with a focus on genes activated by Dox. We identified genes whose expression differed by more than 1.5-fold across both datasets and performed a cross-tabulation analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The mRNA levels of USPL1, USP2, USP10, USP20, USP39, and USP42 were significantly lower in the Dox-treated group than in the control group. Neonatal rat primary cardiomyocytes (NRCMs) were subsequently isolated and subjected to Dox stimulation. The qRT‒PCR results revealed that the mRNA expression of USP20 decreased the most substantially following Dox treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Consequently, USP20 was selected as the focal point for further exploration in this study. Additionally, we confirmed the downregulation of USP20 protein expression in cardiac tissues and primary cardiomyocytes. Notably, a pronounced reduction in USP20 protein expression was observed in heart tissue of mice post-Dox treatment compared with that in wild-type mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D\u003cb\u003e)\u003c/b\u003e. Similarly, decreased USP20 protein expression was noted in Dox-stimulated primary cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). In contrast, we found that USP20 protein expression was largely unchanged in primary fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H) and macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J). Accordingly, we hypothesized that USP20 is predominantly expressed in cardiomyocytes in murine models. To test this hypothesis, single-cell sequencing was performed using myocardial tissues from the Dox treatment group, revealing the predominant localization of USP20 in cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK\u003cb\u003e‒L\u003c/b\u003e). We subsequently analysed the expression pattern of USP20 in cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM-N). The distribution of USP20 expression in cardiomyocytes decreased following Dox treatment. Moreover, via t-distributed stochastic neighbour embedding (t-SNE) analysis, we found that USP20 was predominantly localized to cardiomyocytes undergoing remodelling after Dox treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO). Furthermore, colocalization fluorescence analysis revealed that the labelled USP20 protein was significantly distributed primarily within cardiomyocytes, with a marked decrease in fluorescence intensity following Dox treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eP), corroborating findings from previous experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCardiomyocyte-specific USP20 deficiency exacerbates the progression of DIC via ferroptosis\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the role of USP20 in DIC, we constructed USP20CKO mice (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e) and established a mouse model of DIC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Subsequently, we verified myocardium-specific USP20 knockout in mice (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B-C\u003c/b\u003e). On the day preceding the execution of the experiment, a noninvasive transthoracic echocardiogram was performed on all experimental mice to analyse cardiac function and the extent of myocardial damage. The echocardiography results revealed further deterioration of cardiac function in USP20CKO mice under Dox stimulation, particularly in terms of a decrease in the ejection fraction (EF) and fractional shortening (FS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). H\u0026amp;E staining revealed that Dox stimulation resulted in the disorganization of cardiac muscle fibres in wild-type mice, an effect that was further exacerbated in USP20-knockout mice following Dox treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Furthermore, Sirius red staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) and TUNEL staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H) revealed that intermyocardial fibrosis and the degree of cardiomyocyte death were similarly altered. A number of myocardial injury-related markers, including cTnI, CK-MB, and LDH, were subsequently assessed to provide further insights into the extent of myocardial injury. The results of the serum biochemical assays indicated that USP20CKO significantly increased cTnT, CK-MB, and LDH levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-K) in mice following Dox treatment. These findings indicate that myocardial injury is markedly elevated in USP20 knockout mice following Dox treatment. To elucidate the precise mode of death regulated by USP20 in the context of DIC, a comprehensive literature review was conducted, revealing a substantial body of evidence that identified ferroptosis as a critical mechanism in DIC\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To determine whether USP20 regulates cardiac ferroptosis, we quantified the concentration of Fe\u0026sup2;⁺ in mouse serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL). Notably, following Dox treatment, Fe\u0026sup2;⁺ levels were significantly higher in USP20-CKO mice than in wild-type controls. Consistent with these findings regarding Fe\u0026sup2;⁺, reactive oxygen species (ROS) levels in myocardial tissues were also markedly increased in USP20-knockout mice, as evidenced by the upregulation of malondialdehyde (MDA) expression and downregulation of superoxide dismutase (SOD) expression, suggesting enhanced myocardial lipid peroxidation under doxorubicin treatment conditions. We subsequently conducted Western blot analysis and immunohistochemical staining of cardiac tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM-N). The results revealed the significant downregulation of the expression of GPX4, a critical protein implicated in ferroptosis, following USP20 knockout in the context of Dox-induced modelling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO-P). Furthermore, the staining intensity for another ferroptosis marker, 4-hydroxy-2-nonenal (4-HNE), was markedly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eQ). Taken together, these findings indicate that USP20 deficiency obviously accelerates the progression of DIC via ferroptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eUSP20 mitigates Dox-induced ferroptosis in cardiomyocytes\u003c/h2\u003e \u003cp\u003eTo verify the role of USP20 \u003cem\u003ein vitro\u003c/em\u003e, we transfected neonatal rat primary cardiomyocytes (NRCMs) with si-USP20, followed by stimulation with Dox. The results demonstrated that, in the context of Dox stimulation, cell activity (CCK8) exhibited a more pronounced decline in the si-USP20-transfected group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), whereas cell permeability (LDH) increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These findings indicate that the absence of USP20 intensifies the deleterious effects of Dox on cardiomyocytes \u003cem\u003ein vitro\u003c/em\u003e. Furthermore, the involvement of USP20 in ferroptosis was assessed \u003cem\u003ein vitro\u003c/em\u003e. The cells in each group were analysed for Fe\u003csup\u003e2+\u003c/sup\u003e, SOD, MDA, and GSH levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F). The results demonstrated that Dox stimulation resulted in elevated Fe\u003csup\u003e2+\u003c/sup\u003e levels, increased SOD and GSH levels, and decreased MDA levels. However, transfection with si-USP20 led to further intensification of these alterations. These findings indicate that the degree of intracellular lipid peroxidation increased after USP20 silencing. In addition, under Dox-stimulated conditions, the transfection of si-USP20 further reduced the expression of GPX4, a pivotal protein in the process of ferroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H). To gain a more detailed understanding of the changes occurring in the cells, we performed DHE and PI staining using cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-J), and the results revealed that USP20 silencing \u003cem\u003ein vitro\u003c/em\u003e significantly increased both the cellular reactive oxygen species (ROS) level and mortality rate. In addition, we constructed a USP20-overexpression plasmid, which we transfected into NRCMs, followed by stimulation with Dox. As expected, USP20 overexpression alleviated the decrease in cellular activity (CCK-8) and increase in cell permeability (LDH) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-L), increased both the Fe\u0026sup2;⁺ concentration and lipid peroxidation level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM-P), and abrogated downregulated GPX4 expression in Dox-induced cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eQ-R). Furthermore, the ability of USP20 to attenuate the doxorubicin-induced increase in ROS and apoptosis levels was visualized via the fluorescence staining of DHE and PI in cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eS-T). Thus, we validated the role of USP20 in Dox-induced cardiomyocytes in terms of both interference and overexpression and provided necessary evidence from experiments conducted at the cellular level indicating that the absence of USP20 significantly exacerbates Dox-induced cardiotoxicity and ferroptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eRestoration of cardiac USP20 alleviates DIC by attenuating ferroptosis\u003c/h2\u003e \u003cp\u003eTo elucidate the therapeutic function of USP20 in DIC, we generated recombinant AAV9 vectors carrying \u003cem\u003eUsp20\u003c/em\u003e cDNA and specifically overexpressed USP20 in the cardiomyocytes of wild-type mice via the tail vein injection of AAV9-cTnT-USP20 (\u003cb\u003eSupplementary Fig.\u0026nbsp;3A\u003c/b\u003e). Dox was subsequently administered intraperitoneally to establish a DIC model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We next measured cardiac function by echocardiography. Compared with that in Dox-stimulated wild-type mice, in USP20-overexpressing mice, Dox-induced myocardial contractile dysfunction was alleviated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), which led to a reduction in the left ventricular ejection fraction (EF) and fractional shortening (FS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). We subsequently observed that USP20 overexpression ameliorated myocardial fibre disorders, intermyocardial fibrosis and cardiomyocyte apoptosis, as evidenced by H\u0026amp;E, Sirius red and TUNEL staining of histopathological sections of the myocardium, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G). Furthermore, USP20 overexpression in heart tissues was shown to have a protective effect against Dox-induced myocardial injury. The levels of myocardial injury markers, including cTnI, CK-MB and LDH, were reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-K). Moreover, the serum Fe\u0026sup2;⁺ concentration and lipid peroxidation level in the myocardial tissues of the mice were examined, and USP20 overexpression alleviated the Dox-induced increase in the Fe\u0026sup2;⁺ concentration and lipid peroxidation level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL-N), mitigated the decrease in GPX4 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO-P) and reduced 4-HNE expression in the hearts of Dox-induced mice, as determined via immunohistochemical staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eQ). Taken together, these results suggest that the restoration of cardiac USP20 expression ameliorates cardiac remodelling and dysfunction and impedes cardiac ferroptosis in DIC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eUSP20 directly interacts with HuR\u003c/h2\u003e \u003cp\u003eDUBs regulate a range of biological activities by affecting the degradation or function of substrate proteins. To identify target proteins regulated by USP20 during DIC, we employed immunoprecipitation (co-IP) coupled with mass spectrometry to screen for potential substrate proteins of USP20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Substrate proteins with scores exceeding 100 were selected for screening, and it was determined that only ELAVL1 (HuR) has been documented both in the heart and associated with Dox. Accordingly, we hypothesized that HuR is the substrate protein of USP20 in the context of DIC. To test this hypothesis, USP20 was cotransfected with an HuR plasmid into NIH/3T3 cells. The initial results indicated that HuR could bind to USP20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). We subsequently conducted endogenous validation using neonatal rat primary cardiomyocytes and myocardial tissues that had been injected with AAV9-cTnT-USP20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F), the results of which confirmed the binding of USP20 to HuR. USP20 comprises three distinct structural domains: the USP structural domain and two tandemly linked DUSP structural domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). To elucidate the specific structural domain of USP20 that interacts with HuR, we constructed USP20 plasmids containing mutations in the three structural domains. The cotransfection of HuR with each of the three USP20 mutants in NIH/3T3 cells revealed that the deletion of amino acids 145\u0026ndash;687 resulted in the loss of HuR binding, whereas mutations in the other structural domains did not affect HuR binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). These results provide direct evidence that USP20 acts through its USP structural domain by binding directly to HuR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eThe C154 active site of USP20 deubiquitinates HuR and maintains its stability\u003c/h2\u003e \u003cp\u003eWe speculate that USP20 may alleviate DIC by regulating HuR. The expression of the HuR protein was also upregulated upon USP20 overexpression, and this increase was not caused by a change in the transcript level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). We thus postulated that USP20 inhibits HuR protein degradation by deubiquitination. To test this hypothesis, we generated USP20 knockout NIH/3T3 cells using CRISPR/Cas9 technology (\u003cb\u003eSupplementary Fig.\u0026nbsp;4A\u003c/b\u003e). We subsequently observed a significant increase in the degradation rate of the HuR protein in the USP20-knockout group upon treatment with cycloheximide (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D). As a member of the DUB family, USP20 may play a regulatory role by removing ubiquitin molecules from HuR. To determine the manner in which USP20 acts, we cotransfected UB-K48, UB, HuR, and USP20 into NIH/3T3 cells and inhibited the proteasome degradation pathway with MG132. The results demonstrated that USP20 effectively removed K48-linked ubiquitin molecules, thereby inhibiting the degradation of target proteins via the proteasomal pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). DUBs are capable of hydrolysing amide bonds between ubiquitin molecules and substrate proteins through active sites, including cysteines and histidines. To identify the active site through which USP20 acts, we constructed USP20 plasmids with mutations at cysteine 154 and histidine 645 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). The two mutants were cotransfected with the HuR plasmid into NIH/3T3 cells, which were then treated with MG132. The capacity to stabilize the HuR protein was diminished when the cysteine at position 154 was mutated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-H). Furthermore, the plasmid mutation did not affect binding to the target protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Accordingly, we initially postulated that the cysteine at position 154 represents the active site of USP20, through which HuR is regulated. We subsequently cotransfected a UB plasmid and an HuR plasmid with the two mutant plasmids into NIH/3T3 cells. Our findings revealed that USP20 was unable to exert its deubiquitinating effect following the mutation of the C154 cysteine residue. However, the H645 mutant retained the deubiquitinating effect of USP20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Furthermore, both mutants were overexpressed in HL-1 cells stimulated with Dox. A cell activity assay (CCK-8) and a cell permeability assay (LDH) were subsequently conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK-L). The results demonstrated that the C154 mutant was no longer capable of mitigating Dox-induced cardiotoxicity. Additionally, we conducted assays to measure the levels of Fe\u0026sup2;⁺, SOD, MDA, and GSH. The results demonstrated that in the context of the C154 mutation, no decrease in the Dox-induced Fe\u0026sup2;⁺ concentration or lipid peroxidation level was observed in the presence of USP20 (\u003cb\u003eSupplementary Fig.\u0026nbsp;5A-D\u003c/b\u003e). The protein expression of GPX4 was also assessed (\u003cb\u003eSupplementary Fig.\u0026nbsp;5E\u003c/b\u003e), and the results were consistent with those described above. In conclusion, these findings confirm that C154 of USP20 plays a pivotal role in the deubiquitination of HuR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe anti-DIC effect of USP20 is dependent on HuR\u003c/h3\u003e\n\u003cp\u003eTo investigate the dependence of the anti-DIC function of USP20 on HuR, we created heart-specific HuR-knockout mice. AAV9-cTnT-USP20 was injected via the tail vein to specifically overexpress USP20 in the heart. Compared with the injection of AAV9-cTnT-EV (empty vector), the injection of AAV9-cTnT-USP20 did not improve cardiac function under Dox modelling conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Furthermore, no significant differences were observed in EF or FS (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C). The H\u0026amp;E staining results indicated that the overexpression of USP20 in HuR-CKO mice did not ameliorate myocardial fibre disorganization \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Similarly, we conducted Sirius Red and TUNEL staining of pathological cardiac sections, and the results revealed that the degree of interstitial fibrosis and the number of cardiomyocyte deaths in HuR-CKO mice following Dox stimulation remained unaltered following USP20 overexpression in cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-G). Subsequently, serum myocardial injury marker assays revealed that the extent of myocardial injury (including cTnI, LDH, and CK-MB) was not significantly different between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH-J). In addition, serum iron levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK), lipid peroxidation marker levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL-M), and 4-hydroxyenoaldehyde (4-HNE, a measure of ferroptosis) levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eN) were assessed. The results showed that AAV9-cTnT-USP20 did not significantly attenuate ferroptosis in the Dox model group. Preliminary verification in animals indicated that targeting HuR via USP20 plays a role. To further corroborate our conclusions, we extracted NRCMs and subsequently transfected them with siRNAs targeting HuR along with a USP20 overexpression plasmid. Next, the NRCMs were treated with Dox. Compared with Dox treatment alone, si-HuR treatment led to a decrease in cellular activity (CCK8) and an increase in cellular permeability (LDH) (\u003cb\u003eSupplementary Fig.\u0026nbsp;6C-D\u003c/b\u003e). However, this alteration was not alleviated by the overexpression of USP20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eO-P). Similarly, the concentration of Fe\u0026sup2;⁺ (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eQ), the level of lipid peroxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eR-T), and the expression of GPX4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eU-V) exhibited similar alterations. Moreover, USP20 overexpression in heart-specific HuR-knockout mice did not reduce apoptosis or ROS levels, as reflected by DHE and PI staining. (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eW-X). In conclusion, the ability of USP20 to target HuR to alleviate DIC was validated both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. The question thus arises as to how HuR exerts its influence through ferroptosis. We discovered that HuR, an RNA-binding protein, can bind to GPX4 mRNA and that this binding is reduced under Dox stimulation. Backfilling USP20 reversed these alterations (\u003cb\u003eSupplementary Fig.\u0026nbsp;7A\u003c/b\u003e). Furthermore, we demonstrated that the overexpression of HuR slows the degradation of GPX4 mRNA (\u003cb\u003eSupplementary Fig.\u0026nbsp;7B\u003c/b\u003e). Therefore, we propose that USP20 reduces the degradation of HuR by deubiquitination, thereby promoting GPX4 mRNA stability and regulating ferroptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we observed that USP20 expression was significantly downregulated in both myocardial tissues and primary cardiomyocytes under Dox stimulation. Myocardial-specific USP20 deficiency results in the increased severity of cardiac dysfunction in Dox-induced mice. In contrast, restoring USP20 by the AAV approach alleviated Dox-induced cardiac remodelling and dysfunction, suggesting that USP20 plays an irreplaceable role in combating Dox-induced cardiomyopathy. In addition, via mass spectrometry and coimmunoprecipitation, we identified HuR as a key substrate protein of USP20. These results were verified in myocardia-specific HuR-deficient mice in which USP20 was overexpressed; HuR loss diminished the protective effect of USP20 against Dox-induced cytotoxicity. Mechanistically, USP20 binds to HuR through its USP domain and cysteine 154, facilitating the deubiquitination of HuR and ultimately stabilizing its expression, which leads to a reduction in ferroptosis. Hence, the findings of this study reveal novel insights into the effect of USP20 in DIC and provide a promising therapeutic strategy for DIC by demonstrating the essential role of USP20 in preventing ferroptosis and maintaining cardiac function.\u003c/p\u003e \u003cp\u003eTo date, the deubiquitinating enzyme family contains approximately 100 proteins; as crucial regulatory mediators of posttranslational modifications that deubiquitinate various substrates, they have garnered increasing attention. DUBs play crucial roles in various fields of cell biology, and their expression is highly correlated with pathologies such as neurodegenerative diseases, cancer, and cardiovascular disease \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Despite the growing number of reports on DUBs in cardiovascular diseases in recent years \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, our understanding of their mechanisms of action in DOX-induced cardiotoxicity remains limited. Even different deubiquitinating enzymes exhibit diametrically opposed functions in DIC. For example, Rimpy Dhingra reported that USP19 alleviated DIC by deubiquitinating TRAF2 and preventing its degradation \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, Wang demonstrated that Dox can promote the progression of DIC by activating the USP36-mediated deubiquitination of PARP1 \u003csup\u003e13\u003c/sup\u003e [13]. Our research revealed that endogenous USP20 in the heart was located and more abundant in cardiomyocytes and served as a protective regulator involved in DIC. USP20 depletion exacerbates whereas USP20 overexpression rescues cardiac remodelling and function in DIC. Moreover, we confirmed that USP20 regulated the degradation of HuR, a deubiquitinase, in Dox-induced cardiomyocytes and verified that HuR, a key downregulated protein of USP20, is crucial for the effect of USP20 on DIC, as evidenced by turnover experiments in which HuR was lost in USP20-overexpressing mice. In addition, we revealed the linked motif between USP20 and HuR, in which the domain of USP20 is directly bound to HuR, stabilizing HuR through K48-linked deubiquitination. Overall, we demonstrated that USP20, as a favourable regulatory protein, participates in the pathogenesis of DIC by stabilizing HuR expression, thereby expanding the understanding of USP20 as a crucial signalling molecule in cardiomyocytes and offering potential therapeutic avenues for targeted gene therapy for DIC.\u003c/p\u003e \u003cp\u003eThe pathophysiology of DIC is complex and is characterized by the increased production of ROS, mitochondrial dysfunction, autophagy, and cardiomyocyte apoptosis and ferroptosis, resulting in cardiac dysfunction \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Among these mechanisms, cardiac ferroptosis is one of the main forms of cell death in DIC \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Consistent with a previous report, we found that cardiac ferroptosis and ROS levels largely increased, whereas these alterations were further exacerbated by USP20 deficiency and reversed by USP20 overexpression \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e, suggesting that USP20 can effectively mitigate DIC through cardiac ferroptosis. However, the precise underlying mechanism remains elusive. The RNA-binding protein HuR is widely distributed and selectively binds to AU-rich domains in various mRNA molecules, thereby influencing their stability. HuR plays a pivotal role in the pathogenesis and progression of diverse diseases \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. For example, Zhong et al. reported that Ang II modulated HuR activity through P2X7 receptor activation, consequently influencing the stability of HO-1 and GPX4 mRNAs and subsequently regulating ferroptosis as well as ventricular remodelling \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Notably, HuR plays a pivotal role not only in cardiomyocytes but also in cardiac fibroblasts. Guo reported that NF-κB-repressing factor (NKRF) interacts with HuR and then regulates ventricular remodelling in cardiac fibroblasts by modulating the stability of MMP2 and MMP9 mRNA \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Additionally, recent studies have demonstrated the involvement of the HuR protein in the regulation of Dox-induced ferroptosis within the context of DIC \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In this study, we discovered that USP20 enhances the stability of the HuR protein through deubiquitination and that the overexpression of this protein via the AAV approach in cardiocyte-specific HuR-knockout mice abrogated the cardioprotective benefits of USP20 and diminished the inhibitory effect of USP20 against cardiac ferroptosis, suggesting that HuR is critical for USP20-mediated cardiac ferroptosis. Mechanistically, HuR bound to GPX4 mRNA, and this binding decreased upon Dox stimulation but was partially reversed by USP20 overexpression. Additionally, we validated the impact of HuR on GPX4 mRNA stability and observed that overexpressing HuR alleviated the Dox-induced decrease in GPX4 mRNA stability. These findings suggest that USP20 regulates ferroptosis in DIC by modulating the binding capacity of HuR to its target mRNA.\u003c/p\u003e \u003cp\u003eIn summary, our findings provide evidence that USP20 mitigates DIC by exerting inhibitory effects against cardiac ferroptosis. Moreover, we identified that there is an interaction between USP20 and HuR, which markedly decreased HuR ubiquitination and thus led to the increased binding of HuR to GPX4 and affected GPX4 mRNA stability. In addition, cysteine 154 in USP20 is key for its deubiquitination by HuR. In addition, we ascertained that HuR deletion abrogates USP20-induced cardiac ferroptosis and its cardioprotective benefits in DIC. These data strongly support a previously undescribed USP20\u0026ndash;HuR\u0026ndash;GPX4 axis, which plays a critical role in cardiac ferroptosis and ROS during DIC, providing a possible therapeutic option for preventing Dox-induced cardiac injury.\u003c/p\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eStudy limitations\u003c/h2\u003e \u003cp\u003eThe results of the present study indicated that USP20 restoration with AAV therapy might be a potential approach for treating Dox-induced cardiotoxicity; however, this translational concept has not been validated in nonhuman primates. In addition, we suggest that USP20 supplementation is a potential treatment strategy for DIC; therefore, specific agonists that target USP20 warrant further investigation. Finally, although we determined that USP20 interacts with HuR, we did not explore whether HuR has other direct binding sites that influence ferroptosis.\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data supporting the conclusions of this study are presented in this manuscript or the supplementary information. The materials described in this study are either commercially available or available upon reasonable request from the corresponding authors.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the conclusions of this study are presented in this manuscript or the supplementary information. The materials described in this study are either commercially available or available upon reasonable request from the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhouqing Huang, Shanshan Dai and Yinuo Lin contributed to the literature search and study design. Yunxuan Chen, Shuoning Wu,\u0026nbsp;Lang Deng, Yixin Zhou, Yucong Zhang, Jiaxuan Mei, Fang Wang, Sirui Shen and Zimin Fang performed the experiments and analysed the data. Shanshan Dai and Yinuo Lin provided technical help. Yunxuan Chen participated in the drafting of the article. All authors agree to be accountable for all aspects of the work, ensuring its integrity and accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant Nos. 82070446 and 82202380), the Natural Science Foundation of Zhejiang Province (Grant Nos. LY22H020004 and LQ23H310005), the Project of the Health Commission of Zhejiang Province (Grant No. WKJ-ZJ-2540), and the Science and Technology Project of Wenzhou (Grant No. Y20210136).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWu Y, Wang Z, Han L, Guo Z, Yan B, Guo L, \u003cem\u003eet al.\u003c/em\u003e PRMT5 regulates RNA m6A demethylation for doxorubicin sensitivity in breast cancer. 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Arterioscler Thromb Vasc Biol 2018, 38(10): 2295\u0026ndash;2305.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu XY, Shi XJ, Hu A, Wang JQ, Ding Y, Jiang W, \u003cem\u003eet al.\u003c/em\u003e Feeding induces cholesterol biosynthesis via the mTORC1-USP20-HMGCR axis. Nature 2020, 588(7838): 479\u0026ndash;484.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrigan JA, Jacq X, Martin NM, Jackson SP. Deubiquitylating enzymes and drug discovery: emerging opportunities. Nat Rev Drug Discov 2018, 17(1): 57\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan J, Lin L, Fang Z, Ye B, Han X, Xu J, \u003cem\u003eet al.\u003c/em\u003e Cardiomyocyte-derived USP28 negatively regulates antioxidant response and promotes cardiac hypertrophy via deubiquitinating TRIM21. \u003cem\u003eTheranostics\u003c/em\u003e 2024, 14(16): 6236\u0026ndash;6248.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan J, Fang Z, Han B, Ye B, Lin W, Jiang Y, \u003cem\u003eet al.\u003c/em\u003e Deubiquitinase JOSD2 improves calcium handling and attenuates cardiac hypertrophy and dysfunction by stabilizing SERCA2a in cardiomyocytes. Nat Cardiovasc Res 2023, 2(8): 764\u0026ndash;777.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong CY, Guo Z, Song P, Zhang X, Yuan YP, Teng T, \u003cem\u003eet al.\u003c/em\u003e Underlying the Mechanisms of Doxorubicin-Induced Acute Cardiotoxicity: Oxidative Stress and Cell Death. 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Redox Biol 2024, 72: 103154.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo C, Ji W, Yang W, Deng Q, Zheng T, Wang Z, \u003cem\u003eet al.\u003c/em\u003e NKRF in Cardiac Fibroblasts Protects against Cardiac Remodeling Post-Myocardial Infarction via Human Antigen R. Adv Sci (Weinh) 2023, 10(30): e2303283.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Ren J, Zhang J, Shi H, Wang J, Yan Y. FTO ameliorates doxorubicin-induced cardiotoxicity by inhibiting ferroptosis via P53-P21/Nrf2 activation in a HuR-dependent m6A manner. Redox Biol 2024, 70: 103067.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"USP20, Doxorubicin-induced cardiomyopathy, Deubiquitinating enzyme, Cardiomyocytes, Ferroptosis, HuR","lastPublishedDoi":"10.21203/rs.3.rs-6151078/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6151078/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe severe cardiotoxicity of doxorubicin (Dox) significantly restricts its clinical application. Deubiquitinating enzymes (DUBs) play pivotal roles in cardiac pathophysiology because of their precise regulation of protein function, localization and degradation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eObjectives\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe objective of this study was to investigate the role and molecular mechanism of ubiquitin-specific peptidase 20 (USP20), a DUB, in doxorubicin-induced cardiotoxicity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCardiomyocyte-specific USP20-knockout (USP20-CKO) mice were utilized to assess the role of USP20 in doxorubicin-induced cardiomyopathy (DIC). Coimmunoprecipitation (co-IP) combined with liquid chromatography‒mass spectrometry/mass spectrometry (LC‒MS/MS) analysis was employed to screen the substrate protein of USP20. Furthermore, mutant plasmids of USP20 were constructed to elucidate the molecular mechanism underlying the regulation of human antigen R (HuR) by USP20. Finally, an AAV9 vector was used to overexpress USP20 in the hearts of cardiac-specific HuR-knockout mice to assess the interaction between USP20 and HuR.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe results revealed a decrease in USP20 expression in Dox-stimulated mouse cardiomyocytes. Cardiomyocyte-specific USP20 knockout resulted in increased cardiomyocyte ferroptosis and led to DIC. Mechanistically, USP20 directly interacted with HuR through its ubiquitin-specific protease structural domain. Deubiquitination at position 154 was crucial for maintaining HuR protein stability by cleaving K48 ubiquitin chains and inhibiting proteasomal degradation. Additionally, HuR bound to GPX4 mRNA to suppress its degradation, thereby mitigating ferroptosis and contributing to alleviating DIC. Furthermore, targeted USP20 overexpression via AAV9 in cardiomyocytes significantly alleviated DIC. However, in mice with cardiomyocyte-specific HuR knockout, USP20 no longer had an anti-DIC effect, indicating that HuR, as a downstream target protein of USP20, plays an irreplaceable role in DIC.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur findings indicate that USP20 enhances the stability of the HuR protein through deubiquitination, thereby inhibiting ferroptosis and mitigating DIC.\u003c/p\u003e","manuscriptTitle":"USP20 mitigates doxorubicin-induced cardiotoxicity by deubiquitinating and stabilizing HuR","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 11:15:37","doi":"10.21203/rs.3.rs-6151078/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"084ff715-3984-4afb-bbf6-35d36f3dfb93","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46334978,"name":"Health sciences/Diseases/Cardiovascular diseases/Cardiomyopathies"},{"id":46334979,"name":"Biological sciences/Cell biology/Proteolysis/Deubiquitylating enzymes"}],"tags":[],"updatedAt":"2025-05-09T06:00:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-16 11:15:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6151078","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6151078","identity":"rs-6151078","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}