Hydrogen sulfide improves endothelial barrier function by modulating the ubiquitination degradation of KLF4 through TRAF7 S-sulfhydration in diabetic aorta.

Anomalous vascular endothelium significantly contributes to various cardiovascular diseases. VE-cadherin plays a vital role in governing the endothelial barrier. Krüppel-like factor 4(KLF4), as a transcription factor, which binds the VE-cadherin promoter and enhances its transcription. Tumor necrosis factor receptor-associated factor 7 (TRAF7) is an E3 ubiquitin ligase that has been shown to modulate the degradation of KLF4. H2S can covalently modify cysteine residues on proteins through S-sulfhydration, thereby influencing the structure and functionality of the target protein. However, the role of S-sulfhydration on endothelial barrier integrity remains to be comprehensively elucidated. This study aims to investigate whether protein S-sulfhydration in the endothelium regulates endothelial integrity and its underlying mechanism. In this study, we observed that protein S-sulfhydration was reduced in the endothelium during diabetes and TRAF7 was the main target. Overexpression of TRAF7-Cys327 mutant could mitigate the endothelial barrier damage by weakening TRAF7 interaction with KLF4 and reducing ubiquitination degradation of KLF4. In conclusion, our research demonstrates that H2S plays a pivotal role in regulating S-sulfhydration of TRAF7 at Cys327. This regulation effectively inhibits the ubiquitin-mediated degradation of KLF4, resulting in an upregulation of VE-cadherin levels. This molecular mechanism contributes to the prevention of endothelial barrier damage.

Exogenous H2S initiating Nrf2/GPx4/GSH pathway through promoting Syvn1-Keap1 interaction in diabetic hearts.

Excessive ROS accumulation contributes to cardiac injury in type 2 diabetes mellitus. Hydrogen sulfide (H2S) is a vital endogenous gasotransmitter to alleviate cardiac damage in diabetic cardiomyopathy (DCM). However, the underlying mechanisms remain unclear. In this study, we investigated the effects of NaHS administration in db/db mice via intraperitoneal injection for 20 weeks and the treatment of high glucose (HG), palmitate (PA) and NaHS in HL-1 cardiomyocytes for 48 h, respectively. H2S levels were decreased in hearts of db/db mice and HL-1 cardiomyocytes exposed to HG and PA, which were restored by NaHS. Exogenous H2S activated the nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPx4)/glutathione (GSH) pathway, suppressed ferroptosis and mitigated mitochondrial apoptosis in db/db mice. However, these effects were abrogated after Nrf2 knockdown. NaHS treatment elevated the ubiquitination level of Kelch-like ECH-associated protein (Keap1) by preserving its E3 ligase synoviolin (Syvn1), resulting in Nrf2 nuclear translocation. H2S facilitated the sulfhydration of Syvn1-cys115 site, a post-translational modification. Transfecting Syvn1 C115A in cardiomyocytes exposed to HG and PA partially attenuated the effects of NaHS on Nrf2 and cell death. Our findings suggest that exogenous H2S regulates Nrf2/GPx4/GSH pathway by promoting the Syvn1-Keap1 interaction to reduce ferroptosis and mitochondrial apoptosis in DCM.

Exogenous H2 S promotes ubiquitin-mediated degradation of SREBP1 to alleviate diabetic cardiomyopathy via SYVN1 S-sulfhydration.

BACKGROUND: Diabetic cardiomyopathy, a distinctive complication of diabetes mellitus, has been correlated with the presence of intracellular lipid deposits. However, the intricate molecular mechanisms governing the aberrant accumulation of lipid droplets within cardiomyocytes remain to be comprehensively elucidated. METHODS: Both obese diabetic (db/db) mice and HL-1 cells treated with 200 µmol/L palmitate and 200 µmol/L oleate were used to simulate type 2 diabetes conditions. Transmission electron microscopy is employed to assess the size and quantity of lipid droplets in the mouse hearts. Transcriptomics analysis was utilized to interrogate mRNA levels. Lipidomics and ubiquitinomics were employed to explore the lipid composition alterations and proteins participating in ubiquitin-mediated degradation in mice. Clinical data were collected from patients with diabetes-associated cardiomyopathy and healthy controls. Western blot analysis was conducted to assess the levels of proteins linked to lipid metabolism, and the biotin-switch assay was employed to quantify protein cysteine S-sulfhydration levels. RESULTS: The administration of H2 S donor, NaHS, effectively restored hydrogen sulfide levels in both the cardiac tissue and plasma of db/db mice (+7%, P < 0.001; +5%, P < 0.001). Both db/db mice (+210%, P < 0.001) and diabetic patients (+83%, P = 0.22, n = 5) exhibit elevated plasma triglyceride levels. Treatment with GYY4137 effectively lowers triglyceride levels in db/db mice (-43%, P = 0.007). The expression of cystathionine gamma-lyase and HMG-CoA reductase degradation protein 1 (SYVN1) was decreased in db/db mice compared with the wild-type mice (cystathionine gamma-lyase: -31%, P = 0.0240; SYVN1: -35%, P = 0.01), and NaHS-treated mice (SYVN1: -31%, P = 0.03). Conversely, the expression of sterol regulatory element-binding protein 1 (SREBP1) was elevated (+91%, P = 0.007; +51%, P = 0.03 compared with control and NaHS-treated mice, respectively), along with diacylglycerol O-acyltransferase 1 (DGAT1) (+95%, P = 0.001; +35%, P = 0.02) and 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3) (+88%, P = 0.01; +22%, P = 0.32). Exogenous H2 S led to a reduction in lipid droplet formation (-48%, P < 0.001), restoration of SYVN1 expression, modification of SYVN1's S-sulfhydration status and enhancement of SREBP1 ubiquitination. Overexpression of SYVN1 mutated at Cys115 decreased SREBP1 ubiquitination and increased the number of lipid droplets. CONCLUSIONS: Exogenous H2 S enhances ubiquitin-proteasome degradation of SREBP1 and reduces its nuclear translocation by modulating SYVN1's cysteine S-sulfhydration. This pathway limits lipid droplet buildup in cardiac myocytes, ameliorating diabetic cardiomyopathy.

Hydrogen sulfide regulates SERCA2a SUMOylation by S-Sulfhydration of SENP1 to ameliorate cardiac systole-diastole function in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is a serious complication of diabetes mellitus that eventually progresses to heart failure. The sarco(endo)plasmic reticulum calcium ATPase 2a (SERCA2a), an important calcium pump in cardiomyocytes, is closely related to myocardial systolic-diastolic function. In mammalian cells, hydrogen sulfide (H2S), as a second messenger, antioxidant, and sulfurizing agent, is involved in diverse biological processes. Despite the importance of H2S for protection against DCM, the mechanisms remain poorly understood. The aim of the present study was to determine whether H2S regulates intracellular calcium homeostasis by acting on SERCA2a to reduce cardiomyocyte apoptosis during DCM. Db/db mice were injected with NaHS for 18 weeks. Neonatal rat cardiomyocytes (NRCMs) were treated with high glucose, palmitate, oleate, and NaHS for 48 h. Compared to the NaHS-treated groups, in vivo and in vitro type 2 diabetic models both showed reduced intracellular H2S content, reduced cystathionine γ-lyase (CSE) expression, impaired cardiac function, decreased SERCA2a expression and decreased SERCA2a activity, reduced SUMOylation of SERCA2a, increased sentrin-specific protease 1 (SENP1) expression, and disruption of calcium homeostasis leading to activation of the mitochondrial apoptosis pathway. Compared to the NaHS-treated type 2 diabetes cellular model, overexpression of SENP1 C683A reduced the S-sulfhydration of SENP1, reduced the SUMOylation of SERCA2a, reduced the increased expression and activity of SERCA2a, and induced mitochondrial apoptosis in cardiomyocytes. These results suggested that exogenous H2S elevates SENP1 S-sulfhydration to increase SERCA2a SUMOylation, improve myocardial systolic-diastolic function, and decrease cardiomyocyte apoptosis in DCM.

Hydrogen Sulfide Regulates SERCA2a Ubiquitylation via Muscle RING Finger-1 S-Sulfhydration to Affect Cardiac Contractility in db/db Mice.

Hydrogen sulfide (H2S), as a gasotransmitter, is involved in various pathophysiological processes. Diabetic cardiomyopathy (DCM) is a major complication of diabetes mellitus (DM), which leads to structural and functional abnormalities of the myocardium and eventually causes heart failure (HF). Systolic and diastolic dysfunction are fundamental features of heart failure. SERCA2a, as a key enzyme for calcium transport in the endoplasmic reticulum (ER), affects the process of myocardial relaxation and contraction. H2S can protect the cardiac function against diabetic hearts, however, its mechanisms are unclear. This study found that exogenous H2S affects cellular calcium transport by regulating the H2S/MuRF1/SERCA2a/cardiac contractile pathway. Our results showed that, compared with the db/db mice, exogenous H2S restored the protein expression levels of CSE and SERCA2a, and the activity of SERCA2a, while reducing cytosolic calcium concentrations and MuRF1 expression. We demonstrated that MuRF1 could interact with SERCA2a via co-immunoprecipitation. Using LC-MS/MS protein ubiquitylation analysis, we identified 147 proteins with increased ubiquitination levels, including SERCA2a, in the cardiac tissues of the db/db mice compared with NaHS-treated db/db mice. Our studies further revealed that NaHS administration modified MuRF1 S-sulfhydration and enhanced the activity and expression of SERCA2a. Under hyperglycemia and hyperlipidemia, overexpression of the MuRF1-Cys44 mutant plasmid reduced the S-sulfhydration level of MuRF1 and decreased the ubiquitination level of SERCA2a and the intracellular Ca2+ concentration. These findings suggested that H2S modulates SERCA2a ubiquitination through MuRF1 S-sulfhydration of Cys44 to prevent decreased myocardial contractility due to increased cytosolic calcium.