Figure 5.

Exogenous H₂S regulated the recruitment of parkin into mitochondria by the S-sulfhydration of USP8 in cardiomyocytes under hyperglycemia and hyperlipidemia. (A) The expression of USP8 in cardiac tissues. (B) The S-sulfhydration of USP8 in cardiac tissues was examined with the biotin switch (S-sulfhydration) method. (C) Immunoprecipitation assay was used to examine the interaction between USP8 and parkin in cardiac tissues. (D) Intracellular levels of polysulfide in neonatal rat cardiomyocytes were examined by a fluorescent probe, SSP4. (E) Neonatal rat cardiomyocytes were treated with dithiothreitol (DTT, 1mM, 10 min) or high glucose (40 mM), oleate (200 μM) and palmitate (200 μM) in the presence or absence of NaHS (100 μM) for 48 h. S-sulfhydration on USP8 were examined with the Biotin switch(S- sulfhydration) method. (F) Immunoprecipitation assay was used to examine interaction between USP8 and parkin in neonatal rat cardiomyocytes treated with DTT. (G) The ubiquitination level of cytosolic parkin in neonatal rat cardiomyocytes was measured by immunoprecipitation. Values are presented as the mean ± S.D. from n = 4 replicates. *P<0.05.