Abstract
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.
Highlights
Type 2 diabetes is a strong risk factor for the development of cardiovascular disease and atherosclerosis, which is related to dyslipidemia
The adenoviruses were added to neonatal rat cardiomyocytes (NRCMs) directly, and after 4 hours of transfection, the medium was replaced with fresh medium
A large number of lipid droplets were produced in the db/db mice; the number of droplets was reduced by treatment with NaHS, and we postulated that H2S regulated endocytosis by S-sulfhydration of target proteins
Summary
Type 2 diabetes is a strong risk factor for the development of cardiovascular disease and atherosclerosis, which is related to dyslipidemia. H2S prevents CD36 translocation have verified that in an insulin-resistant state, TAG accumulates in the heart [3, 4]. This cardiac accumulation of TAG could be due to increased myocardial long-chain fatty acid (LCFA) uptake. Additional evidence has demonstrated that intracellular membrane trafficking and exocytosis are controlled by several superfamily proteins, including Nethylmaleimide sensitive factor (NSF); its cofactor, soluble NSF attachment protein (αSNAP); and SNAP receptors (SNAREs) [7]. TSNAREs and vesicle-associated membrane proteins (VAMPs) represent all members of the SNARE superfamily [9]. Our previous study suggested that H2S inhibits the ubiquitylation of superoxide dismutase (SOD) to promote the initiation of autophagy in diabetic cardiomyopathy [14]. Our findings raise the possibility that H2S participates in VAMP3 degradation through Hrd Ssulfhydration in the cardiac tissues of db/db mice
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