1Department of Pathophysiology, Harbin Medical University, Harbin, China. 2Department of Forensic Medicine, Harbin Medical University, Harbin, China. 3Department of Urologic Surgery, First affiliated hospital of Harbin Medical University, Harbin, China. 4Department of Cardiology, First affiliated hospital of Harbin Medical University, Harbin, China. 5Key Laboratory of Cardiovascular Medicine Research (Harbin Medical University), Ministry of Education, Harbin, China
Hydrogen sulfide (H2S), an important gasotransmitter, regulates cardiovascular functions. Mitochondrial damage induced by the overproduction of reactive oxygen species (ROS) results in myocardial injury with a diabetic state. The purpose of this study was to investigate the effects of exogenous H2S on mitophagy formation in diabetic cardiomyopathy. In this study, we found that exogenous H2S could improve cardiac functions, reduce mitochondrial fragments and ROS levels, enhance mitochondrial respiration chain activities and inhibit mitochondrial apoptosis in the hearts of db/db mice. Our results showed that exogenous H2S facilitated parkin translocation into mitochondria and promoted mitophagy formation in the hearts of db/db mice. Our studies further revealed that the ubiquitination level of cytosolic parkin was increased and the expression of USP8, a deubiquitinating enzyme, was decreased in db/db cardiac tissues. S-sulfhydration is a novel posttranslational modification of specific cysteine residues on target proteins by H2S. Our results showed that the S-sulfhydration level of USP8 was obviously decreased in vivo and in vitro under hyperglycemia and hyperlipidemia, however, exogenous H2S could reverse this effect and promote USP8/parkin interaction. Dithiothreitol, a reducing agent that reverses sulfhydration-mediated covalent modification, increased the ubiquitylation level of parkin, abolished the effects of exogenous H2S on USP8 deubiquitylation and suppressed the interaction of USP8 with parkin in neonatal rat cardiomyocytes treated with high glucose, oleate and palmitate. Our findings suggested that H2S promoted mitophagy formation by increasing S-sulfhydration of USP8, which enhanced deubiquitination of parkin through the recruitment of parkin in mitochondria.
Figure 1. Exogenous H2S promoted autophagy in the hearts of db/db mice and in neonatal rat cardiomyocytes. (A) The ultrastructure of cardiac tissues was observed using a transmission electron microscope. The red arrow indicates mitophagosomes. (B) Data are presented as the number of autophagosomes in cardiac tissue in the control, db/db and db/db+NaHS groups (n=5). (C) The expression of Beclin1, Atg7 P62 and LC3II/I were examined in db/db cardiac tissues by western blotting. (D) The expression of Beclin1, ATG7, P62 and LC3II/I was examined by Western blotting following the treatment of Bafilomycin A1 in neonatal rat cardiomyocytes. (E) Autophagosomes were detected by the MDC test in neonatal rat cardiomyocytes (green). Values are presented as the mean ± S.D. from n = 5 replicates. *P<0.05, **P<0.01, ***P<0.001.
Figure 2. Exogenous H2S promoted mitophagy in the hearts of db/db mice and in neonatal rat cardiomyocytes. (A) Mitophagosomes were detected in neonatal rat cardiomyocytes by mitophagy detection kit. Red fluorescence represents the mitophagosomes and green fluorescence represents the fusion of mitophagosomes and lysosomes. (B) The expression of LC3B was examined in the mitochondria of db/db cardiac by Western blotting. (C) The expression of LC3B in mitochondria was examined by western blotting following the treatment of neonatal rat cardiomyocytes with Bafilomycin A1. Values are presented as the mean ± S.D. from n = 4 replicates. *P<0.05, **P<0.01, ***P<0.001.
Figure 3. Exogenous H2S protected mitochondria by maintaining of mitochondrial dynamics. (A) The expression levels of the mitochondrial dynamics-related proteins, P-Drp1/Drp1, Fis1 and Mfn2, were measured in cardiac mitochondria by Western blotting. (B) The expression levels of the mitochondrial dynamics-related proteins, P-Drp1/Drp1, Fis1 and Mfn2, were examined in neonatal rat cardiomyocytes by Western blotting. (C) The mitochondrial morphology of neonatal rat cardiomyocytes was measured by MitoTracker green assay. (D) JC-1 assay was used to examine the mitochondrial membrane potential of neonatal rat cardiomyocytes. (E) The expression of Parkin, PINK1, Beclin1, Atg7, P62 and LC3II/I was detected in neonatal rat cardiomyocytes with Mito-Tempo and Midivi-1 treatment in by Western blotting. Values are presented as the mean ± S.D. from n = 5 replicates. *P<0.05, **P<0.01, ***P<0.001.
Figure 4. Exogenous H2S promoted mitophagy under hyperglycemia and hyperlipidemia. (A) Western blotting analysis and quantification of mitochondrial and cytoplasmic parkin protein in cardiac tissues. (B) Western blotting analysis and quantification of mitochondrial and cytoplasmic parkin protein in neonatal rat cardiomyocytes under hyperglycemia and hyperlipidemia. (C) The ubiquitination level of cytosolic parkin in cardiac tissues was examined by immunoprecipitation. (D) Immunoprecipitation assay was used to examine the interaction between PINK1 and parkin in cardiac tissues. (E) Western blot analysis detected the expression of parkin and PINK1 in the mitochondria of neonatal rat cardiomyocytes. Values are presented as the mean ± S.D. from n = 5 replicates. *P<0.05, **P<0.01, ***P<0.001.
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.
Figure 6. Exogenous H2S upregulated mitophagy through the activation of the USP8 signaling pathway. (A) The ubiquitination level of cytosolic parkin was examined following USP8 siRNA treatment by immunoprecipitation. (B) The ubiquitination level of mitochondrial Mfn2 was examined following USP8 siRNA treatment by immunoprecipitation. (C) Western blotting analysis and quantification of mitochondrial and cytoplasmic parkin protein under USP8 siRNA treatment. Values are presented as the mean ± S.D. from n = 4 replicates. **P<0.01vs Control, ***P<0.001 vs Control. (D) The expression of parkin in cytoplasma under USP8 siRNA and MG132 treatment. (E) Western blotting analysis of mitochondrial LC3B protein under USP8 siRNA treatment. Values are presented as the mean ± S.D. from n = 4 replicates. *P<0.05, **P<0.01.
Figure 7. The role of H2S in the regulation of cardiac mitophagy by the S-sulfhydration of USP8 in a type 2 diabetes model.
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