Septic cardiomyopathy is a life-threatening complication of sepsis, and an uncontrolled inflammatory response represents a key pathogenic mechanism. PARP7 negatively regulates the IFN-I signaling pathway through a mono-ADP-ribosylation-dependent interaction with TBK1. Here, through comprehensive analysis of the expression profile of the PARP family in LPS-treated myocardial tissues, we propose that PARP7 may be associated with septic cardiomyopathy. Then, we demonstrate that PARP7 deficiency exacerbates LPS-induced septic cardiomyopathy in vivo. Integrated single-nucleus and single-cell RNA sequencing analyses demonstrate that PARP7 is predominantly upregulated in macrophages in the hearts of LPS-treated mice. Using an AAV9-based delivery system, we further validated the cardioprotective role of macrophage-specific PARP7 in murine models of sepsis induced by either LPS or CLP. Mechanistically, PARP7 interacts with TBK1 to mediate its ADP-ribosylation, thereby suppressing the TBK1-driven inflammatory response in macrophages. The snRNA-seq and cytokine array data collectively support a critical role for PARP7 as a molecular "brake" that constrains excessive macrophage inflammation. In conclusion, this work identifies a macrophage-specific PARP7-TBK1 regulatory axis in septic cardiomyopathy and highlights the therapeutic potential of macrophage-specific PARP7 overexpression.

Background Septic acute kidney injury (AKI) is a common clinical complication associated with high mortality, and inflammation plays a central role in its pathophysiology. PARP7, a member of the poly ADP-ribose polymerase (PARP) family, functions as a negative feedback regulator of the IFN-I pathway by interacting with TBK1 in a mono-ADP-ribosylation-dependent manner. Methods and results In this study, we first confirmed upregulated PARP7 expression in septic AKI and found that increased PARP7 levels were predominantly localized in renal proximal tubular epithelial cells. We further demonstrated that global deletion of PARP7 exacerbated lipopolysaccharide (LPS)-induced renal inflammation and acute kidney injury, whereas specific overexpression of PARP7 in renal proximal tubular epithelial cells attenuated septic AKI in mice. Using single-cell RNA sequencing (scRNA-seq), we revealed that PARP7 suppresses the LPS-induced inflammatory response in renal tubular epithelial cells. Mechanistically, PARP7 interacts with TBK1 and mediates its ADP-ribosylation in LPS-stimulated renal tubular epithelial cells. The anti-inflammatory effects of PARP7 are primarily mediated through targeting TBK1 via its catalytic residue H532. Conclusions Collectively, our findings highlight the renoprotective role of PARP7 in AKI and provide a theoretical basis for developing therapies targeting the PARP7-TBK1 axis.

As the roles of poly-ADP-ribose polymerase 7 (PARP7) in tumor immune evasion become increasingly well defined, PARP7 inhibitors have emerged as a promising class of anticancer therapeutics. To date, phase I clinical trials of PARP7 inhibitors have reported no significant cardiovascular adverse events. However, PARP7’s involvement in doxorubicin (DOX)-induced cardiotoxicity (DIC), a major clinical limitation of widely used chemotherapeutics, remains poorly understood. Elucidating the functional role of PARP7 in this pathological context is essential for evaluating the therapeutic safety of pharmacological PARP7 inhibition. This study aimed to define the functional role and molecular mechanism of PARP7 in DIC. We demonstrate that PARP7 is robustly and selectively upregulated in cardiomyocytes following DOX exposure. Genetic ablation of PARP7 markedly attenuates DIC. Mechanistically, we identify glutathione peroxidase 4 (GPX4) as a direct ADP-ribosylation substrate of PARP7. PARP7 catalyzes mono-ADP-ribosylation of GPX4, leading to its loss of enzymatic activity. In addition, this suppression of GPX4 is both necessary and sufficient for PARP7-driven ferroptosis in cardiomyocytes. Importantly, cardiomyocyte-specific PARP7 knockdown, achieved via AAV9-cTNT-mediated delivery of short hairpin RNA, effectively reverses established DIC, confirming its therapeutic relevance in clinical translation. Collectively, these findings establish PARP7 as a druggable, cardiomyocyte- specific regulator of ferroptosis and define the PARP7-GPX4 axis as a mechanistically grounded, therapeutically targetable pathway in cardio-oncology.

INTRODUCTION:Gasdermin D (GSDMD) and the pyroptosis it mediates are importantly involved in cardiovascular diseases (CVDs). Identifying and developing new inhibitors of GSDMD could be a promising strategy for treating pyroptosis-mediated diseases, such as atherosclerosis. OBJECTIVES:We aimed to develop new inhibitor of GSDMD in atherosclerosis, as well as clarify the mechanisms underlying this inhibiting effect. METHODS:Surface plasmon resonance and pull-down assay were used to identify the amino acid sites of GSDMD inhibited by GI-Y2. A mouse model of atherosclerosis was established by feeding a high-fat diet for 12 weeks. After treating mice with GI-Y2 (10 or 20 mg/kg, i.g.), the lipid plaque area on the arterial intimal surface, lipid deposition, collagen deposition and pyroptosis levels in aortic root sections were evaluated. Additionally, further treatment of atherosclerotic mice with macrophage membrane-encapsulated GI-Y2 was conducted to enhance the targeting ability of GI-Y2 to atherosclerotic plaques. RESULTS:In this study, we confirmed GI-Y2 as a novel inhibitor of GSDMD via structure-based virtual screening and pharmacological validation. Mechanistically, GI-Y2 directly interacts with the Arg10 residue of GSDMD and reduces the membrane binding of GSDMD-N. Functionally, we revealed that GI-Y2 inhibits the formation of atherosclerotic plaques by targeting GSDMD. Similarly, GI-Y2 reduces pyroptosis and macrophage infiltration in atherosclerosis. Furthermore, we constructed macrophage membrane-coated GI-Y2 nanoparticles to enhance the targeting of GI-Y2 to macrophages in atheromatous plaques and demonstrated its vascular protective effect in vivo. CONCLUSION:This work demonstrated that GI-Y2 can potentially alleviate CVDs by targeting GSDMD and provided a new compound for the study of GSDMD-mediated pyroptosis. KEY POINTS:We preliminarily confirmed GI-Y2 as a novel inhibitor of GSDMD via structure-based virtual screening and pharmacological validation. GI-Y2 directly interacts with GSDMD and reduces the membrane binding of GSDMD-N via the Arg10 residue. GI-Y2 inhibits the formation of atherosclerotic plaques by targeting GSDMD and GI-Y2 reduces pyroptosis and macrophage infiltration in atherosclerosis. We constructed macrophage membrane-coated GI-Y2 nanoparticles to enhance the targeting of GI-Y2 to macrophages in atheromatous plaques and demonstrated its vascular protective effect in vivo.

Increased level of angiotensin II (Ang II) plays a central role in the development of hypertensive vascular remodeling. In this study, we identified the deubiquitinating enzyme Josephin domain-containing protein 2 (JOSD2) as a protective factor and investigated its molecular mechanism in Ang II-induced vascular remodeling. First, we found that JOSD2 was upregulated in aortic smooth muscle cells, but not in endothelial cells of Ang II-challenged mouse vascular tissues. Whole-body knockout of JOSD2 significantly deteriorated Ang II-induced vascular remodeling in mice. Conversely, Ang II-induced vascular remodeling was reversed by vascular smooth muscle cell (VSMC)-specific JOSD2 overexpression. In vitro, JOSD2 deficiency aggravated Ang II-induced fibrosis, proliferation, and migration VSMCs, while these changes were reversed by JOSD2 overexpression. RNA-seq analysis showed that the protective effects of JOSD2 in VSMCs were related to the TGFβ-SMAD pathway. Furthermore, the LC-MS/MS analysis identified SMAD7, a negative regulator in the TGFβ-SMAD pathway, as the substrate of JOSD2. JOSD2 specifically bound to the MH1 domain of SMAD7 to remove the K48-linked ubiquitin chains from SMAD7 at lysine 220 to sustain SMAD7 stability. Taken together, our finding reveals that the JOSD2-SMAD7 axis is critical for relieving Ang II-induced vascular remodeling and JOSD2 may be a novel and potential therapeutic target for hypertensive vascular remodeling.

BACKGROUND AND PURPOSE:Myocardial dysfunction is a significant complication associated with sepsis. However, there are currently no specific and effective treatments available. Inhibiting gasdermin D (GSDMD)-mediated pyroptosis has shown promise in mitigating sepsis-induced myocardial dysfunction. The GSDMD inhibitor Y2 (GI-Y2) has been demonstrated to directly bind to GSDMD. Nonetheless, it remains uncertain whether GI-Y2 offers a cardioprotective effect in the context of sepsis-induced myocardial dysfunction. EXPERIMENTAL APPROACH:A mouse model of sepsis was created using lipopolysaccharide (LPS), caecal ligation and puncture. Following treatment with GI-Y2 or macrophage membrane-encapsulated GI-Y2 nanoparticles (GI-Y2@MM-NPs), myocardial dysfunction and pyroptosis levels in heart tissues were assessed. Transcriptome sequencing revealed the molecular mechanism of GI-Y2 in treating septic cardiomyopathy. KEY RESULTS:We observed that GI-Y2 alleviated myocardial dysfunction and attenuated cardiac inflammation in mice induced by LPS, caecal ligation and puncture. GI-Y2 reduced macrophage pyroptosis and attenuated macrophage-mediated cardiomyocyte injury induced by LPS/nigericin. Concurrently, we confirmed the protective effect of GI-Y2 against LPS-induced cardiac dysfunction was abolished in the absence of GSDMD. Additionally, GI-Y2 attenuated the mitochondrial damage induced by LPS by inhibiting GSDMD in the mitochondria. Furthermore, we developed GI-Y2@MM-NPs to enhance the targeting capability of GI-Y2 towards macrophages in heart tissues and demonstrated its protective effect in vivo. CONCLUSION AND IMPLICATIONS:These findings indicate that GI-Y2 alleviates septic myocardial injury and dysfunction by specifically targeting GSDMD, thereby inhibiting GSDMD-mediated pyroptosis and mitochondrial damage. Both GI-Y2 and GI-Y2@MM-NPs may serve as promising therapeutic options for addressing septic myocardial dysfunction.

BACKGROUND/AIMS:Activation of hepatic stellate cells (HSCs) is key to the development of liver fibrosis. Recent studies have highlighted the role of deubiquitinating enzymes (DUBs) in regulating protein stability and function, closely related to liver fibrosis. In this study, we screened out a key DUB, ubiquitin-specific peptidase 13 (USP13), in HSCs activation and explored its role and underlying mechanism. METHODS:Gene Expression Omnibus (GEO) public database were used to demonstrate the correlation of USP13 with HSC activation. Mice with adeno-associated virus (AAV)-mediated HSC-specific USP13 deficiency are proceeded to carbon tetrachloride (CCl4) or common bile duct ligation (BDL) models. Co-immunoprecipitation combined with mass spectrometry (Co-IP/MS) was used to identify the substrate for USP13. RESULTS:We first found that USP13 expression was upregulated in activated HSCs and in both CCl4- and BDL-induced liver fibrosis mice. HSC-specific knockdown of USP13 alleviates liver fibrosis and HSC activation in mice caused by CCl4 or BDL. Mechanistically, we identified SMAD3 as a potential substrate for USP13 by Co-IP/MS. USP13 binds to the MH2 domain of SMAD3 and deubiquitinates SMAD3. USP13 cysteine at position 345 (C345) promotes the stability of SMAD3 by removing the K48 ubiquitin chain from the lysine at position 13 (K13) of SMAD3, enhancing SMAD3 protein activity and inducing transcription of downstream profibrotic genes, which finally leads to HSCs activation and liver fibrosis. CONCLUSIONS:This study illustrates an HSC-specific USP13-SMAD3 axis in regulating liver fibrosis and presents USP13 as a potential target for the treatment of liver fibrosis.

Hypertension engenders numerous complications, including hypertensive renal disease (HRD). Recent studies have underscored the significance of redox homeostasis in hypertension-associated kidney disorders. Carnosol (Car) has been identified as a potent antioxidant in other diseases. Nevertheless, its role in HRD remains uncertain. This research evaluated the therapeutic impact of Car on HRD and explored its pharmacological mechanism. Car (40 mg/kg) effectively ameliorated kidney injury and oxidative stress (OS) induced by angiotensin II (Ang II) both in vitro and in vivo. Mechanistically, Car facilitates the phosphorylation of p62 through its interaction with mTOR, leading to the degradation of KEAP1, the release of NRF2, and the subsequent upregulation of antioxidant genes. Blocking mTORC1 eliminated Car-induced p62 activation and its antioxidant functions. In summary, our research has demonstrated that Car activates the p62-KEAP1-NRF2 pathway to alleviate the kidney injury and OS caused by Ang II. Consequently, Car may emerge as a promising candidate for the prevention and treatment of Ang II-induced kidney injury.

Doxorubicin (Dox) is an anthracycline drug widely applied in various malignancies. However, the fatal cardiotoxicity induced by Dox limits its clinical application. Post-transcriptional protein modification via ubiquitination/deubiquitination in cardiomyocytes mediates the pathophysiological process in Dox-induced cardiotoxicity (DIC). In this study, we aimed to clarify the regulatory role and mechanism of a deubiquitinating enzyme, ubiquitin-specific peptidase 13 (USP13), in DIC. RNA-seq analysis and experimental examinations identified that cardiomyocyte-derived USP13 positively correlated with DIC. Mice with cardiac-specific deletion of USP13 were subjected to Dox modeling. Adeno-associated virus serotype 9 (AAV9) carrying cTNT promoter was constructed to overexpress USP13 in mouse heart tissues. Cardiomyocyte-specific knockout of USP13 exacerbated DIC, while its overexpression mitigated DIC in mice. Mechanistically, USP13 deubiquitinates the stimulator of interferon genes (STING) and promotes the autolysosome-related degradation of STING, subsequently alleviating cardiomyocyte inflammation and death. Our study suggests that USP13 serves a cardioprotective role in DIC and indicates USP13 as a potential therapeutic target for DIC treatment.

Diabetic cardiomyopathy (DCM) is a leading cause of diabetes-related mortality. Identifying new functional proteins in DCM pathology and elucidating the underlying mechanisms may provide new therapeutic targets for this disease. Here, we observed that the expression of the deubiquitinating enzyme USP13 was significantly downregulated in DCM mouse heart tissues. We discovered that the expression of USP13 was predominantly localized in cardiomyocytes. Cardiomyocyte-specific knockout of USP13 exacerbated myocardial injury in both type I and type II diabetic mice. Conversely, overexpression of USP13 in cardiomyocytes via recombinant adeno-associated virus 9 (AAV9) showed therapeutic effects against DCM in mice. Interestingly, using co-precipitation and LC-MS/MS analysis, we identified the NOD-like receptor family pyrin domain containing 3 (NLRP3) as a target protein of USP13 in cardiomyocytes. Mechanistically, we have illustrated that USP13 removes the K63-linked ubiquitin chain at K557 of NLRP3 to inhibit NLRP3-ASC interaction, thereby inhibiting ASC polymerization and the activation of NLRP3 inflammasome complex, ultimately alleviating pyroptosis in HG + PA challenged cardiomyocytes. Importantly, we showed that the cardioprotective effects of USP13 overexpression depended on NLRP3, as evidenced by the loss of protection in NLRP3-deficient diabetic mice. Taken together, this study identifies the protective impact and molecular regulation of USP13 in DCM pathology, uncovering a novel cardiomyocyte-specific USP13-NLRP3 axis in DCM.

Cardiac hypertrophy leads to ventricular dysfunction and heart failure. Deubiquitinating enzymes are responsible for preserving the substrate protein stability and are essential to myocardial hypertrophy. In this study, we aimed to explore the role and regulatory mechanism of a cardiomyocyte-derived deubiquitinating enzyme, USP13, in cardiac hypertrophy. Here we show that USP13 was increased in hypertrophic myocardium and was mainly distributed in cardiomyocytes. Cardiomyocyte-specific Usp13 knockout aggravated TAC or Ang II-induced myocardial hypertrophy and dysfunction in male mice. Correspondingly, USP13 overexpression by AAV9 in hearts exerted a therapeutic impact on cardiac hypertrophy in male mice. Mechanistically, we identified STAT1 as a substrate of USP13 through interactome analysis. USP13 deubiquitinated STAT1, thereby reducing its degradation. Subsequently, USP13 promoted the STAT1-targeted Nppb gene transcription and enhanced mitochondrial function in cardiomyocytes. This study illustrated a beneficial effect of USP13 in hypertrophic cardiomyocytes and identified a cardiomyocyte-specific USP13-STAT1 axis in regulating cardiac hypertrophy.

Rationale: Myocardial ischemia/reperfusion (I/R) injury leads to irreversible cardiomyocyte death and aggravates myocardial infarction. Deubiquitinating enzymes (DUBs) are essential for maintaining substrate protein stability and functionality, playing significant roles in cardiac pathophysiology. In this study, we aimed to clarify the regulatory role of a DUB, Myb-like, SWIRM, and MPN domains 1 protein (MYSM1), in myocardial I/R injury and explore the molecular mechanism behind. Methods and Results: Firstly, it was found that the expression of MYSM1 positively correlates with myocardial I/R injury. Genetic knockdown of MYSM1 significantly conferred protection against I/R injury in hearts. Correspondingly, AAV9-mediated cardiomyocyte-specific knockdown of MYSM1 had a therapeutic effect on myocardial I/R injury. Through a comprehensive proteome-wide quantitative analysis, we identified signal transducer and activator of transcription 1 (STAT1) as the direct substrate of MYSM1. Mechanistically, MYSM1 mediated the K63-linked deubiquitination and stabilization of STAT1 at position K379 via its MPN metalloprotease domain. Additionally, MYSM1 initiates the expression of necroptosis-related genes by promoting the transcription factor function of STAT1. Conclusion: This study illustrated a MYSM1-STAT1 axis in regulating myocardial I/R injury and identified MYSM1 as a pharmacological target for myocardial I/R injury.

BackgroundSeptic cardiomyopathy is a frequent complication in patients with sepsis and is associated with a high mortality rate. Given its clinical significance, understanding the precise underlying mechanism is of great value.Methods and resultsOur results unveiled that Z-DNA binding protein 1 (ZBP1) is upregulated in myocardial tissues of lipopolysaccharide (LPS)-treated mice. Single-cell mRNA sequencing (scRNA-seq) and single-nucleus mRNA sequencing (snRNA-seq) indicated that Zbp1 mRNA in endothelial cells, fibroblasts and macrophages appeared to be elevated by LPS, which is partially consistent with the results of immunofluorescence. Through echocardiography, we identified that global deletion of ZBP1 improves cardiac dysfunction and the survival rate of LPS-treated mice. Mechanistically, snRNA-seq showed that ZBP1 is mainly expressed in macrophages and deletion of ZBP1 promotes the macrophage polarisation towards M2-subtype, which reduces inflammatory cell infiltration. Notably, myeloid-specific deficiency of ZBP1 also promotes M2 macrophage polarisation and improves cardiac dysfunction, validating the role of macrophage-derived ZBP1 in septic myocardial dysfunction. Finally, we revealed that LPS increases the transcription and expression of ZBP1 through signal transducer and activator of transcription 1 (STAT1). Fludarabine, the inhibitor of STAT1, could also promote M2 macrophage polarisation and improve cardiac dysfunction of LPS-treated mice.ConclusionsOur study provides evidence of a novel STAT1-ZBP1 axis in macrophage promoting septic cardiomyopathy, and underscores the potential of macrophage-derived ZBP1 as a therapeutic target for septic cardiomyopathy.Key points Macrophage-derivedZBP1 exacerbates LPS-induced myocardial dysfunction and inflammatory cellinfiltration. Deletionof ZBP1 promotes macrophage polarisation from M1 to M2. STAT1-ZBP1axis promotes septic cardiomyopathy. ZBP1has emerged as a potential therapeutic target for inflammationand septic cardiomyopathy.

BACKGROUND:Cardiac hypertrophy constitutes the primary pathological basis for heart failure and exerts a considerable influence on morbidity and mortality. Deubiquitinating enzymes are crucial regulators of protein degradation and play a pivotal role in cardiac pathophysiology. This study aimed to clarify the involvement of a deubiquitinating enzyme, MYSM1 (Myb-like, SWIRM [Swi3p, Rsc8p and Moira], and MPN [Mpr1/Pad1 N-terminal] domains 1), in cardiac hypertrophy and to explore the underlying mechanism. METHODS:Cardiac hypertrophy was developed by angiotensin II or transverse aortic constriction surgery. Cardiomyocyte-specific knockdown of MYSM1 was accomplished using the adeno-associated virus serotype 9-cTNT-Mysm1-shRNA. Co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry analysis was utilized to identify potential substrates of MYSM1. RESULTS:First, we discovered that the expression of MYSM1 increases during cardiac hypertrophy. MYSM1 knockdown mitigated cardiac dysfunction and hypertrophy after angiotensin II administration. Cardiomyocyte-specific knockdown of MYSM1 with adeno-associated virus serotype 9 alleviated myocardial dysfunction and hypertrophy caused by transverse aortic constriction surgery. Through co-immunoprecipitation and LC-MS, poly (ADP-ribose) polymerase 1 (PARP1) was identified as a potential substrate protein of MYSM1. PARP1 initiates a novel form of programmed cell death termed parthanatos, which is characterized by excessive PARylation, nuclear translocation of AIF, and depletion of nicotinamide adenine dinucleotide. MYSM1 deubiquitinates and stabilizes PARP1 in an MPN domain-dependent manner. In addition, MYSM1 mediates cardiac hypertrophy through PARP1-dependent cardiomyocyte parthanatos. CONCLUSIONS:This study identified the role of the MYSM1-PARP1 axis in mediating cardiac hypertrophy and suggested that MYSM1 is a promising pharmacological target for the treatment of cardiac hypertrophy.

Macrophage is a vital factor in determining the fate of abdominal aortic aneurysm (AAA). The crosstalk between macrophage and other cells plays a crucial role in the development of aneurysm. Gasdermin D (GSDMD) is a vital executive protein of pyroptosis, which is a novel programmed cell death associated with inflammation. In this study, we identified aortic macrophage as the main expressing cell of GSDMD in AAA. Using Gsdmd-/-ApoE-/- mouse and AAV-F4/80-shGSDMD, we demonstrated the potential role of macrophage-derived GSDMD in AAA and aortic pyroptosis induced by Ang II in vivo. In vitro experiments showed that GSDMD promotes the pyroptosis of mouse primary peritoneal macrophages (MPMs), murine aortic vascular smooth muscle cells (MOVAS) and primary smooth muscle cells. Mechanistically, a mouse cytokine antibody array showed that Gsdmd-/- inhibited LPS + nigericin (LN)- induced secretion of multiple cytokines from MPMs. Furthermore, GSDMD is involved in the crosstalk between MPMs and MOVAS via cytokine secretion. This study provides a novel fundamental insight into macrophage-derived GSDMD in AAA and showed that GSDMD could be a promising therapeutic target for AAA.

Benign laryngotracheal stenosis is widely managed with minimally invasive endoscopic interventions, such as laser incision or excision scar, and dilation. However, various endoscopic treatments are significantly associated with a high recurrence rate. Local auxiliary measures, including inhalation of steroids, injection of steroids, and local topical application of mitomycin C, have been studied in order to increase the success rate. To compare the efficacy of endoscopic treatments with and without local adjuncts in patients with benign laryngotracheal stenosis, and analyze their clinical outcomes, recurrence, and complications. In the meta-analysis, databases including PubMed, EMBASE, OVID, and Web of Science were searched for papers comparing the outcomes of adjunct therapy with non-adjunct therapy in patients with laryngotracheal stenosis. The duplicate publications, reviews, comments or letters, conference abstracts, and case reports were excluded. The random effect model was used for assessing the pooled risk estimates. Eight studies (1204 cases) referring to two prospective randomized controlled studies, two prospective cohort studies, and four retrospective cohort studies were ultimately included in the meta-analysis. Three delivery modes of adjuncts were identified, including intralesion steroid injection (n = 2), inhaled steroid (n = 2), and topical application of mitomycin C (n = 4). The decreased risk estimates of recurrence rate were detected in patients receiving endoscopic treatments with steroid injection or inhaled steroid, compared with endoscopic interventions alone (P < 0.05). Additionally, patients undergoing adjunct therapies had lower risk estimates of recurrence, compared to those receiving endoscopic procedures alone (P < 0.05), based on the subgroup of prospective cohort studies, subglottis, Mayer-Cotton scale of I–II degree, and stenosis length of < 3 cm. The high heterogeneity of the pooling risk estimates perhaps was due to factors of auxiliary drug, clinical characteristics of patients, and methodology. No discernible difference in the incidence of complication was identified. Local application of steroids to minimally invasive interventions appear to reduce the recurrence rate of laryngotracheal stenosis. Various adjuncts available, including steroids and mitomycin C, appear to be safe and associated with a low risk estimate of adjuncts-specific complication rate. High quality multi-center randomized controlled studies are needed, with sufficient periods for follow-up and subjective and objective outcome indicators, to properly evaluate the efficacy, safety, and cost-effectiveness of adjuvant drugs.

Rationale: Cardiac hypertrophy is an important pathological basis for heart failure. Most physiological activities of cardiomyocytes are regulated by proteins and their post-translational modification. Deubiquitinating enzymes (DUBs) are involved in protein stability maintenance and closely related to myocardial hypertrophy. In this study, we aimed to clarify the regulatory role of a DUB, ubiquitin-specific peptidase 28 (USP28), in cardiac hypertrophy and explore the molecular mechanism behind. Methods: Transcriptome and single-cell mRNA sequencing was used to demonstrate the association of USP28 and cardiac hypertrophy. Cardiomyocyte-specific USP28 knockout mice (USP28CKO) were subjected to angiotensin II (Ang II) infusion or transverse aortic constriction (TAC) models. Coimmunoprecipitation combined mass spectrum analysis (Co-IP/MS) was applied to screen out the substrate of USP28. Results: We first showed the up-regulation of USP28 in cardiac hypertrophy, and its cellular localization of cardiomyocytes. USP28CKO protects mouse heart against Ang II- or TAC-induced cardiac dysfunction and hypertrophy. Mechanistically, we identified tripartite motif-containing protein 21 (TRIM21) as the potential substrate of USP28 by Co-IP/MS analysis. Cardiomyocyte USP28 deubiquitinates and stabilizes TRIM21 to negatively regulate nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response, increasing oxidative stress in cardiomyocytes and promoting cardiac hypertrophy and injury. Finally, using a selective USP28 inhibitor Otilonium Bromide, we confirmed the therapeutic effect of pharmacological inhibition of USP28 against TAC-induced established hypertrophic heart failure. Conclusion: Our study illustrates a cardiomyocyte-specific USP28-TRIM21 axis in regulating hypertrophic cardiomyopathy and presents USP28 as a potential target for the treatment of cardiac hypertrophy.

BACKGROUND: Macrophages play a crucial role in atherosclerotic plaque formation, and the death of macrophages is a vital factor in determining the fate of atherosclerosis. GSDMD (gasdermin D)-mediated pyroptosis is a programmed cell death, characterized by membrane pore formation and inflammatory factor release. METHODS: ApoE −/− and Gsdmd −/− ApoE −/− mice, bone marrow transplantation, and AAV (adeno-associated virus serotype 9)-F4/80-shGSDMD (shRNA-GSDMD) were used to examine the effect of macrophage-derived GSDMD on atherosclerosis. Single-cell RNA sequencing was used to investigate the changing profile of different cellular components and the cellular localization of GSDMD during atherosclerosis. RESULTS: First, we found that GSDMD is activated in human and mouse atherosclerotic plaques and Gsdmd −/− attenuates the atherosclerotic lesion area in high-fat diet–fed ApoE −/− mice. We performed single-cell RNA sequencing of ApoE −/− and Gsdmd −/− ApoE −/− mouse aortas and showed that GSDMD is principally expressed in atherosclerotic macrophages. Using bone marrow transplantation and AAV-F4/80-shGSDMD, we identified the potential role of macrophage-derived GSDMD in aortic pyroptosis and atherosclerotic injuries in vivo. Mechanistically, GSDMD contributes to mitochondrial perforation and mitochondrial DNA leakage and subsequently activates the STING (stimulator of interferon gene)-IRF3 (interferon regulatory factor 3)/NF-κB (nuclear factor kappa B) axis. Meanwhile, GSDMD regulates the STING pathway activation and macrophage migration via cytokine secretion. Inhibition of GSDMD with GSDMD-specific inhibitor GI-Y1 (GSDMD inhibitor Y1) can effectively alleviate the progression of atherosclerosis. CONCLUSIONS: Our study has provided a novel macrophage-derived GSDMD mechanism in the promotion of atherosclerosis and demonstrated that GSDMD can be a potential therapeutic target for atherosclerosis.

Aim: Hypertensive nephropathy is a common complication of hypertension. However, no effective measures are currently available to prevent the progression of renal insufficiency. Gasdermin D (GSDMD) is a crucial mediator of pyroptosis that induces an excessive inflammatory response. In the present study, we aimed to determine the effect of GSDMD on the pathogenesis of hypertensive nephropathy, which may provide new insights into the treatment of hypertensive nephropathy. Methods: C57BL/6 (wild-type, WT) and Gsdmd knockout (Gsdmd(-/-)) mice were subcutaneously infused with angiotensin II (Ang II) via osmotic mini-pumps to establish a hypertensive renal injury model. Recombinant adeno-associated virus serotype 9 (AAV9) carrying GSDMD cDNA was used to overexpress GSDMD. Renal function biomarkers, histopathological changes, and inflammation and fibrosis indices were assessed. Transcriptome sequencing (RNA-seq) and cleavage under targets and mentation (CUT & Tag) experiments were performed to identify the downstream pathogenic mechanisms of GSDMD in hypertensive nephropathy. Results: GSDMD was activated in the kidneys of mice induced by Ang II (P < 0.001). This activation was primarily observed in the renal tubular epithelial cells (P < 0.0001). GSDMD deficiency attenuated renal injury and fibrosis induced by Ang II (P < 0.0001), whereas Gsdmd overexpression promoted renal injury and fibrosis (P < 0.01). Mechanistically, GSDMD increased Ang II-induced GATA binding protein 2 (GATA2) transcription factor expression (P < 0.01). GATA2 also bound to the aquaporin 4 (Aqp4) promoter sequence and facilitated Aqp4 transcription (P < 0.001), leading to renal injury and fibrosis. Moreover, treatment with GI-Y1, an inhibitor of GSDMD, alleviated Ang II-induced renal injury and fibrosis (P < 0.01). Conclusion: GSDMD plays an important role in the development of hypertensive nephropathy. Targeting GSDMD may be a therapeutic strategy for the treatment of hypertensive nephropathy.

Anthracycline antitumor drug doxorubicin (DOX) induces severe cardiotoxicity. Deubiquitinating enzymes (DUBs) are crucial for protein stability and function and play a significant role in cardiac pathophysiology. By comparing RNA sequencing datasets and conducting functional screening, we determined that Myb-like, SWIRM, and MPN domains 1 (MYSM1) is a key regulator of DOX-induced cardiotoxicity. In this study, we aimed to explore the function and regulatory mechanisms of MYSM1 in DOX-induced cardiotoxicity. Genetic knockdown of MYSM1 significantly mitigated DOX-induced cardiomyopathy. Correspondingly, cardiomyocyte-specific knockdown of MYSM1 by AAV9 protected the heart from DOX-induced cardiotoxicity. Gain- and loss-of-function analysis verified that MYSM1 mediated DOX-induced cardiomyocyte injury in vitro. Through a Co-IP combined with LC-MS/MS analysis, we discovered that MYSM1 directly interacted with tripartite motif-containing protein 21 (TRIM21). Mechanistic investigations revealed that MYSM1 regulates the deubiquitination and the stability of TRIM21 via its MPN domain. Furthermore, MYSM1 exacerbated DOX-induced cardiotoxicity by enhancing ferroptosis. This study identified MYSM1 as a potential therapeutic target for DOX-induced cardiotoxicity and illustrated a MYSM1-TRIM21-ferroptosis axis in regulating DOX-induced cardiotoxicity.