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.