Exogenous H2S Induces Hrd1 S-sulfhydration and Prevents CD36 Translocation via VAMP3 Ubiquitylation in Diabetic Hearts

Hydrogen sulfide (H2S) plays physiological roles in vascular tone regulation, cytoprotection, and ATP synthesis. HMG-CoA reductase degradation protein (Hrd1), an E3 ubiquitin ligase, is involved in protein trafficking. H2S may play a role in controlling fatty acid uptake in diabetic cardiomyopathy (DCM) in a manner correlated with modulation of Hrd1 S-sulfhydration; however, this role remains to be elucidated. The aim of the present study was to examine whether H2S can attenuate lipid accumulation and to explain the possible mechanisms involved in the regulation of the H2S-Hrd1/VAMP3 pathway. Db/db mice and neonatal rat cardiomyocytes treated with high glucose, palmitate and oleate were used as animal and cellular models of type 2 diabetes, respectively. The expression of cystathionine-γ-lyase (CSE), Hrd1, CD36 and VAMP3 was detected by Western blot analysis. In addition, Hrd1 was mutated at Cys115, and Hrd1 S-sulfhydration was examined using an S-sulfhydration assay. VAMP3 ubiquitylation was investigated by immunoprecipitation. Lipid droplet formation was tested by TEM, BODIPY 493/503 staining and oil red O staining. The expression of CSE and Hrd1 was decreased in db/db mice compared to control mice, whereas CD36 and VAMP3 expression was increased. NaHS administration reduced droplet formation, and exogenous H2S restored Hrd1 expression, modified S-sulfhydration, and decreased VAMP3 expression in the plasma membrane. Using LC-MS/MS analysis, we identified 85 proteins with decreased ubiquitylation, including 3 vesicle-associated membrane proteins, in the cardiac tissues of model db/db mice compared with NaHS-treated db/db mice. Overexpression of Hrd1 mutated at Cys115 diminished VAMP3 ubiquitylation, whereas it increased CD36 and VAMP3 expression and droplet formation. siRNA-mediated Hrd1 deletion increased the expression of CD36 in the cell membrane. These findings suggested that H2S regulates VAMP3 ubiquitylation via Hrd1 S-sulfhydration at Cys115 to prevent CD36 translocation in diabetes.


Measurement of 2-NBDG uptake
Glucose uptake was determined by a nonradioactive method using a new fluorescent analog of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) (Sigma). Cells were seeded and treated for 48 hours in 96well plates and washed three times with PBS. Then, the culture medium was removed from each well and replaced with 500 μL of glucose-free DMEM, and the cells were incubated in the presence of 10 μmol/L 2-NBDG at the indicated concentrations for 40 min. The fluorescence intensity was recorded at excitation and emission wavelengths of 485 nm and 535 nm, respectively.

Measurement of fatty acid uptake
Briefly, cells were grown on coverslips in 12-well plates, and after treatment, the cells were serum-starved for 5 hours. The medium was aspirated, and the cells were washed twice with PBS containing fatty acid-free albumin. The cells were then incubated with BODIPY 558/568C12 (1 μmol/L) for 2 min at 37 °C, and the coverslips were washed 3 times with PBS and mounted on clean glass slides using Dako antifade solution (Dako Corp, Carpinteria, CA). For confocal microscopy analysis, BODIPY-conjugated fatty acids were excited at 488 nm with a fluorescence microscope (Olympus XSZ-D2, Japan). Fluorescent images were obtained using FluoView software, and the fluorescence intensity was quantitated with ImageJ software.

Glucose tolerance test analysis
Mice were intraperitoneally injected with D-glucose (2 g/kg mass). Tail blood was collected, and blood glucose was determined using a glucometer.

Echocardiography analysis
Mouse cardiac function was assessed using an echocardiography system (GE VIVID 7 10S, St. CT., Fairfield, USA) after 6 weeks, 12 weeks and 20 weeks of treatment with NaHS. The mice were lightly anesthetized with Avertin at a dose of 240 mg· kg-1, and the mouse body temperature was maintained as close to 37 °C as possible during the entire process. Left ventricular parameters were measured, including EF % and FS %.

HPLC-MS/MS analysis
Based on our previous study, we identified lysine-ubiquitylated proteins in cardiac tissues of db/db mice and in cells treated or not treated with NaHS [1]. The peptides were dissolved in 0.1% FA and directly loaded onto a reversedphase precolumn (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversedphase analytical column (Acclaim PepMap RSLC, Thermo Scientific). The gradient was composed of an increase in solvent B (0.1% FA in 98% ACN) from 6% to 22% for 22 min, an increase from 22% to 36% for 8 min, an increase to 80% over 5 min, and a hold at 80% for the last 3 min. All steps were conducted at a constant flow rate of 300 nL/min on an EASY-nLC 1000 UPLC system. The resulting peptides were analyzed with a Q Exactive TM Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). The peptides were subjected to an NSI source followed by MS/MS in a Q Exactive TM Plus (Thermo) coupled online to UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using a normalized collision energy (NCE) setting of 28, and ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan and 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 2E4 in the MS survey scan with 10.0 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800.

Protein sequence alignments and bioinformatics analysis
To characterize the identified lysine-ubiquitylated proteins, a series of bioinformatics tools were used for protein annotation and functional analysis. Specifically, Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, domain annotation and subcellular localization prediction were performed. GO annotation was based on three categories: biological process, cellular component and molecular function. The KEGG online service tools of the KEGG map were used to annotate the proteins with KEGG pathway descriptions. Domain annotation was performed using InterProScan on the InterPro domain database via Web-based interfaces and services. For each category of proteins, the InterPro database (a resource that provides functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites) was searched using the functional annotation tool of the Database for Annotation, Visualization and Integrated Discovery (DAVID) against the background of Homo sapiens. A two-tailed Fisher's exact test was employed to test the enrichment of the protein-containing IPI entries against all IPI proteins. Correction for multiple hypothesis testing was conducted using standard false discovery rate control methods, and domains with a corrected p-value < 0.05 were considered significant.