USP25 Ameliorates Pathological Cardiac Hypertrophy by Stabilizing SERCA2a in Cardiomyocytes.

BACKGROUND: Pathological cardiac hypertrophy can lead to heart failure and is one of the leading causes of death globally. Understanding the molecular mechanism of pathological cardiac hypertrophy will contribute to the treatment of heart failure. DUBs (deubiquitinating enzymes) are essential to cardiac pathophysiology by precisely controlling protein function, localization, and degradation. This study set out to investigate the role and molecular mechanism of a DUB, USP25 (ubiquitin-specific peptidase 25), in pathological cardiac hypertrophy. METHODS: The role of USP25 in myocardial hypertrophy was evaluated in murine cardiomyocytes in response to Ang II (angiotensin II) and transverse aortic constriction stimulation and in hypertrophic myocardium tissues of heart failure patients. Liquid chromotography with mass spectrometry/mass spectrometry analysis combined with Co-IP was used to identify SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2A), an antihypertrophy protein, as an interacting protein of USP25. To clarify the molecular mechanism of USP25 in the regulation of SERCA2a, we constructed a series of mutant plasmids of USP25. In addition, we overexpressed USP25 and SERCA2a in the heart with adenoassociated virus serotype 9 vectors to validate the biological function of USP25 and SERCA2a interaction. RESULTS: We revealed increased protein level of USP25 in murine cardiomyocytes subject to Ang II and transverse aortic constriction stimulation and in hypertrophic myocardium tissues of patients with heart failure. USP25 deficiency aggravated cardiac hypertrophy and cardiac dysfunction under Ang II and transverse aortic constriction treatment. Mechanistically, USP25 bound to SERCA2a directly via its USP (ubiquitin-specific protease) domain and cysteine at position 178 of USP25 exerts deubiquitination to maintain the stability of the SERCA2a protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby maintaining calcium handling in cardiomyocytes. Moreover, restoration of USP25 expression via adenoassociated virus serotype 9 vectors in USP25-/- mice attenuated Ang II-induced cardiac hypertrophy and cardiac dysfunction, whereas myocardial overexpression of SERCA2a could mimic the effect of USP25. CONCLUSIONS: We confirmed that USP25 inhibited cardiac hypertrophy by deubiquitinating and stabilizing SERCA2a.

Endothelial deubiquinatase YOD1 mediates Ang II-induced vascular endothelial-mesenchymal transition and remodeling by regulating ß-catenin.

Hypertension is a prominent contributor to vascular injury. Deubiquinatase has been implicated in the regulation of hypertension-induced vascular injury. In the present study we investigated the specific role of deubiquinatase YOD1 in hypertension-induced vascular injury. Vascular endothelial endothelial-mesenchymal transition (EndMT) was induced in male WT and YOD1-/- mice by administration of Ang II (1 µg/kg per minute) via osmotic pump for four weeks. We showed a significantly increased expression of YOD1 in mouse vascular endothelial cells upon Ang II stimulation. Knockout of YOD1 resulted in a notable reduction in EndMT in vascular endothelial cells of Ang II-treated mouse; a similar result was observed in Ang II-treated human umbilical vein endothelial cells (HUVECs). We then conducted LC-MS/MS and co-immunoprecipitation (Co-IP) analyses to verify the binding between YOD1 and EndMT-related proteins, and found that YOD1 directly bound to ß-catenin in HUVECs via its ovarian tumor-associated protease (OTU) domain, and histidine at 262 performing deubiquitination to maintain ß-catenin protein stability by removing the K48 ubiquitin chain from ß-catenin and preventing its proteasome degradation, thereby promoting EndMT of vascular endothelial cells. Oral administration of ß-catenin inhibitor MSAB (20 mg/kg, every other day for four weeks) eliminated the protective effect of YOD1 deletion on vascular endothelial injury. In conclusion, we demonstrate a new YOD1-ß-catenin axis in regulating Ang II-induced vascular endothelial injury and reveal YOD1 as a deubiquitinating enzyme for ß-catenin, suggesting that targeting YOD1 holds promise as a potential therapeutic strategy for treating ß-catenin-mediated vascular diseases.

Bicyclol Attenuates Obesity-Induced Cardiomyopathy via Inhibiting NF-κB and MAPK Signaling Pathways.

PURPOSE: Schisandra is a well-known traditional Chinese medicine in East Asia. As a traditional Chinese medicine derivative with Schisandra chinensis as raw material, bicyclol is well known for its significant anti-inflammatory effect. Chronic inflammation plays a significant part in obesity-induced cardiomyopathy. Our purpose was to explore the effect and mechanism of bicyclol on obesity-induced cardiomyopathy. METHODS: Mice fed with a high-fat diet (HFD) and cardiomyocytes stimulated by palmitic acid (PA) were used as models of obesity-related cardiomyopathy in vivo and in vitro, respectively. The therapeutic effect of bicyclol on pathological changes such as myocardial hypertrophy and fibrosis was evaluated by staining cardiac tissue sections. PCR was used to detect inflammatory factors in H9c2 cells and animal heart tissue after bicyclol treatment. Then, we used western blotting to detect the expression levels of the myocardial hypertrophy related protein, myocardial fibrosis related protein, NF-κB and MAPK pathways. RESULTS: Our results indicated that bicyclol treatment significantly alleviates HFD-induced myocardial inflammation, fibrosis, and hypertrophy by inhibiting the MAPK and NF-κB pathways. Similar to animal level results, bicyclol could significantly inhibit PA-induced inflammation and prevent NF-κB and MAPK pathways from being activated. CONCLUSION: Our results showed that bicyclol has potential as a drug to treat obesity-induced cardiomyopathy.

Tabersonine attenuates obesity-induced renal injury via inhibiting NF-κB-mediated inflammation.

Obesity-induced metabolic disorders can cause chronic inflammation in the whole body, activating the nuclear factor kappa B (NF-κB) pathway and inducing apoptosis. Therefore, anti-inflammatory strategies may be effective in preventing obesity-related renal injury. Tabersonine (Tab) has been used pharmacologically to alleviate inflammation-related symptoms. This study evaluated the therapeutic effect of Tab on obesity-related renal injury and explored the pharmacological mechanism. Tab (20 mg/kg) relieved HFD-induced renal structural disorder and alleviated renal functional decline in mice, including improvement of renal tissue fibrosis, reducing renal cell apoptosis and inflammation in renal tissues. Mechanistically, we demonstrated that Tab inhibited the activation of NF-κB signaling pathway both in vivo and in vitro, thereby improving the renal tissue lesions in the mice with obesity-related renal injury. In both the obese mouse model and the mouse glomerular mesangial cell model, the natural compound Tab ameliorated HFD- and saturated fatty acid-induced renal cell injury by inhibiting the activation of NF-κB signaling pathway. Our data suggest that Tab may become a potential candidate for the prevention and treatment of obesity-related renal injury.

Tabersonine alleviates obesity-induced cardiomyopathy by binding to Transforming growth factor activated kinase 1 (TAK1) and inhibiting TAK1-mediated inflammation.

Obesity-induced cardiomyopathy (OIC) is an increasingly serious global disease caused by obesity. Chronic inflammation greatly contributes to the pathogenesis of OIC. This study aimed to explore the role and mechanism of tabersonine (Tab), a natural alkaloid with antiinflammatory activity, in the treatment of OIC. High fat diet (HFD)-induced obese mice were administered with Tab. The results showed that Tab significantly inhibit inflammation, myocardial fibrosis, and hypertrophy to prevent heart dysfunction, without the alteration of body weight and hyperlipidemia, in HFD-induced obese mice. H9c2 cells and primary cardiomyocytes stimulated by palmitic acid (PA) were used to explore the molecular mechanism and target of Tab. We examined the effect of Tab on key proteins involved in HFD/PA-induced inflammatory signaling pathway and found that Tab significantly inhibits TAK1 phosphorylation in cardiomyocytes. We further detected the direct interaction between Tab and TAK1 at the cellular, animal, and molecular levels. We found that Tab directly binds to TAK1 to inhibit TAK1 phosphorylation, which then blocks TAK1-TAB2 interaction and then NF-κB pro-inflammatory pathway in cultured cardiomyocytes. Our results indicate that Tab is a potential agent for the treatment of OIC, and TAK1 is an effective therapeutic target for this disease.

Podocyte OTUD5 alleviates diabetic kidney disease through deubiquitinating TAK1 and reducing podocyte inflammation and injury.

Recent studies have shown the crucial role of podocyte injury in the development of diabetic kidney disease (DKD). Deubiquitinating modification of proteins is widely involved in the occurrence and development of diseases. Here, we explore the role and regulating mechanism of a deubiquitinating enzyme, OTUD5, in podocyte injury and DKD. RNA-seq analysis indicates a significantly decreased expression of OTUD5 in HG/PA-stimulated podocytes. Podocyte-specific Otud5 knockout exacerbates podocyte injury and DKD in both type 1 and type 2 diabetic mice. Furthermore, AVV9-mediated OTUD5 overexpression in podocytes shows a therapeutic effect against DKD. Mass spectrometry and co-immunoprecipitation experiments reveal an inflammation-regulating protein, TAK1, as the substrate of OTUD5 in podocytes. Mechanistically, OTUD5 deubiquitinates K63-linked TAK1 at the K158 site through its active site C224, which subsequently prevents the phosphorylation of TAK1 and reduces downstream inflammatory responses in podocytes. Our findings show an OTUD5-TAK1 axis in podocyte inflammation and injury and highlight the potential of OTUD5 as a promising therapeutic target for DKD.

Deubiquitinase OTUD6a drives cardiac inflammation and hypertrophy by deubiquitination of STING.

BACKGROUND: Cardiac hypertrophy is a crucial pathological characteristic of hypertensive heart disease and subsequent heart failure. Deubiquitinating enzymes (DUBs) have been found to be involved in the regulation of myocardial hypertrophy. OTU Domain-Containing Protein 6a (OTUD6a) is a recently identified DUB. To date, the potential role of OTUD6a in myocardial hypertrophy has not yet been revealed. METHODS AND RESULTS: We examined the up-regulated level of OTUD6a in mouse or human hypertrophic heart tissues. Then, transverse aortic constriction (TAC)- or angiotensin II (Ang II)- induced ventricular hypertrophy and dysfunction were significantly attenuated in OTUD6a gene knockout mice (OTUD6a-/-). In mechanism, we identified that the Stimulator of Interferon Genes (STING) is a direct substrate protein of OTUD6a via immunoprecipitation assay and mass spectrometry. OTUD6a maintains STING stability via clearing the K48-linked ubiquitin in cardiomyocytes. Subsequently, OTUD6a regulates the STING-downstream NF-κB signaling activation and inflammatory gene expression both in vivo and in vitro. Inhibition of STING blocked OTUD6a overexpression-induced inflammatory and hypertrophic responses in cardiomyocytes. CONCLUSION: This finding extends our understanding of the detrimental role of OTUD6a in myocardial hypertrophy and identifies STING as a deubiquinating substrate of OTUD6a, indicating that targeting OTUD6a could be a potential strategy for the treatment of cardiac hypertrophy.

Inhibition of TAK1/TAB2 complex formation by costunolide attenuates obesity cardiomyopathy via the NF-κB signaling pathway.

BACKGROUND: Chronic and persistent obesity can lead to various complications, including obesity cardiomyopathy. Inhibition of the inflammatory response is an effective measure for the intervention of obesity cardiomyopathy. Numerous studies indicate that costunolide (Cos) can reduce inflammation. However, the role of Cos in obesity cardiomyopathy and its molecular targets remains unknown. HYPOTHESIS/PURPOSE: We aimed to clarify potential cardioprotective effects and mechanism of Cos against obesity cardiomyopathy. METHODS: The model of obesity cardiomyopathy was established by feeding mice with a high-fat diet for 24 weeks. Cos at 10 and 20 mg/kg or vehicle (1% CMCNa solution) was administered once every two days via oral gavage from the 17th to 24th week. Body weight, heart weight/tibia length, cardiac function, myocardial injury markers, pathological morphology of the heart, hypertrophic and fibrotic markers, inflammatory factors were assessed. The targets of Cos were predicted through molecular docking. Pull-down assay and biolayer interferometry were used to confirm the target of Cos. RESULTS: Cos effectively reduces obesity-induced cardiomyocyte inflammation, cardiac hypertrophy and fibrosis, thereby improving cardiac function. We confirmed that Cos can interact with TAK1 and inhibit downstream NF-κB pathway activation by blocking the formation of the TAK1/TAB2 complex, thus inhibiting inflammatory cytokine release in cardiomyocytes. CONCLUSION: Our results demonstrated that Cos significantly improved myocardial remodeling and cardiac dysfunction against obesity cardiomyopathy by reducing myocardial inflammation. Therefore, Cos may serve as a promising therapeutic agent in obesity cardiomyopathy.

Deubiquitinase JOSD2 improves calcium handling and attenuates cardiac hypertrophy and dysfunction by stabilizing SERCA2a in cardiomyocytes.

Cardiac hypertrophy leads to myocardial dysfunction and represents a serious threat to global public health security. Deubiquitinating enzymes (DUBs) mainly maintain the stability of substrate proteins and are essential to cardiac pathophysiology. Here, we explored the role and regulating mechanism of a DUB, Josephin domain-containing protein 2 (JOSD2), in cardiac hypertrophy. We found that JOSD2 expression was significantly upregulated in hypertrophic myocardium. Josd2 gene knockout aggravated cardiac dysfunction and hypertrophy in mice, whereas cardiac overexpression of JOSD2 mediated by the AAV9 vector prevented angiotensin II-induced cardiac hypertrophy. A comprehensive proteome-wide quantitative analysis identified sarco/endoplasmic reticulum calcium ATPase 2a (SERCA2a) as a key substrate of JOSD2. Mechanistically, JOSD2 mediates SERCA2a deubiquitination, enhancing the stability of SERCA2a. By regulating SERCA2a, JOSD2 deficiency impairs calcium handling and promotes hypertrophy in primary cardiomyocytes. Our findings highlight the promise of JOSD2 as a beneficial therapeutic target for hypertrophic cardiomyopathy and provide an additional strategy for SERCA2a-targeted therapy.

DL-3-n-butylphthalide prevents oxidative stress and atherosclerosis by targeting Keap-1 and inhibiting Keap-1/Nrf-2 interaction.

Atherosclerosis is the common pathophysiological foundation of ischaemic stroke and myocardial ischaemia. Oxidative stress is intricately related to the progress of atherosclerosis. DL-3-n-butylphthalide (NBP) is a synthesized raceme of L-3-n-butylphthalide that is first isolated from celery. As a neuroprotective agent, NBP also exhibits potent antioxidative activity. Our research aimed to evaluate the effect of NBP on atherosclerosis and to explore the underlying antioxidative mechanisms and targets. Firstly, we detected the protective effect of NBP on ApoE-/- model of atherosclerosis. NBP showed high efficiency as a therapeutic agent against the formation of atherosclerotic plaques and oxidative events in HFD-treated ApoE-/- mice. We have also evaluated the effect of NBP on oxidized-LDL (oxLDL)-induced oxidative damage and Keap-1/ Nrf-2 interaction by utilizing rat aortic endothelial cells (ECs) and mouse primary peritoneal macrophages (MPMs). Furthermore, we investigated the possibility that NBP improves oxLDL-stimulated oxidative stress in a Keap-1- dependent way in ECs by siRNA technique. Using molecular dynamics (MD) simulation, we detected that Keap-1, a negative adaptor of Nrf-2, may be one of the target protein of NBP. Our studies show that amelioration of oxidative stress by NBP may provide a potential therapeutic strategy for atherosclerosis or cardio-cerebrovascular events from atherosclerosis.

GSDMD (Gasdermin D) Mediates Pathological Cardiac Hypertrophy and Generates a Feed-Forward Amplification Cascade via Mitochondria-STING (Stimulator of Interferon Genes) Axis.

BACKGROUND: Cardiac hypertrophy is initially an adaptive response of cardiomyocytes to neurohumoral or hemodynamic stimuli. Evidence indicates that Ang II (angiotensin II) or pressure overload causes GSDMD (gasdermin D) activation in cardiomyocytes and myocardial tissues. However, the direct impact of GSDMD on cardiac hypertrophy and its underlying mechanisms are not fully understood. METHODS AND RESULTS: In this study, we examined the aberrant activation of GSDMD in mouse and human hypertrophic myocardia, and the results showed that GSDMD deficiency reduced Ang II or pressure overload-induced cardiac hypertrophy, dysfunction, and associated cardiomyocyte pyroptosis in mice. Mechanistically, Ang II-mediated GSDMD cleavage caused mitochondrial dysfunction upstream of STING (stimulator of interferon genes) activation in vivo and in vitro. Activation of STING, in turn, potentiated GSDMD-mediated cardiac hypertrophy. Moreover, deficiency of both GSDMD and STING suppressed cardiac hypertrophy in cardiac-specific GSDMD-overexpressing mice. CONCLUSIONS: Based on these findings, we propose a mechanism by which GSDMD generates a self-amplifying, positive feed-forward loop with the mitochondria-STING axis. This finding points to the prospects of GSDMD as a key therapeutic target for hypertrophy-associated heart diseases.