Exogenous H2S prevents the nuclear translocation of PDC‐E1 and inhibits vascular smooth muscle cell proliferation in the diabetic state

Abstract Hydrogen sulphide (H2S) inhibits vascular smooth muscle cell (VSMC) proliferation induced by hyperglycaemia and hyperlipidaemia; however, the mechanisms are unclear. Here, we observed lower H2S levels and higher expression of the proliferation‐related proteins PCNA and cyclin D1 in db/db mouse aortae and vascular smooth muscle cells treated with 40 mmol/L glucose and 500 μmol/L palmitate, whereas exogenous H2S decreased PCNA and cyclin D1 expression. The nuclear translocation of mitochondrial pyruvate dehydrogenase complex‐E1 (PDC‐E1) was significantly increased in VSMCs treated with high glucose and palmitate, and it increased the level of acetyl‐CoA and histone acetylation (H3K9Ac). Exogenous H2S inhibited PDC‐E1 translocation from the mitochondria to the nucleus because PDC‐E1 was modified by S‐sulfhydration. In addition, PDC‐E1 was mutated at Cys101. Overexpression of PDC‐E1 mutated at Cys101 increased histone acetylation (H3K9Ac) and VSMC proliferation. Based on these findings, H2S regulated PDC‐E1 S‐sulfhydration at Cys101 to prevent its translocation from the mitochondria to the nucleus and to inhibit VSMC proliferation under diabetic conditions.


| INTRODUC TI ON
Vascular complications, such as atherosclerosis, in individuals with diabetes mellitus (DM) increase patient morbidity and mortality. 1 As a main component of the artery wall, the proliferation of vascular smooth muscle cells (VSMCs) plays a pivotal role in the initiation and development of diabetic vascular complications. 2 Recent studies have confirmed that chronic hyperglycaemia/high glucose (HG)-enhanced reactive oxygen species (ROS) production accelerates the progression of VSMC proliferation. 3 However, the mechanisms of VSMC proliferation have not been completely explained. Due to the complexity of pathogenic mechanisms, new insights are required to explore hyperglycaemia/high glucose (HG)-induced VSMC proliferation. Based on accumulating evidence, epigenetic regulation of gene expression is essential for cell proliferation and differentiation through the modifications of core histones by methylation, phosphorylation and acetylation. 4 In metazoan cells, the biosynthesis of acetyl-coenzyme A (CoA) in different subcellular compartments, such as in mitochondria and the nucleus, has been affirmed. ATP citrate lyase (ACL) has been reported to be present in the nucleus. ACL, the main enzyme for nuclear acetyl-CoA generation in mammalian cells, utilizes mitochondrial citrate as its substrate. 5 According to recent studies, the pyruvate dehydrogenase complex (PDC) might be translocated from the mitochondria to the nucleus in response to mitochondrial stress or growth factor stimulation in tumour cells. PDC catalyses pyruvate conversion to acetyl-CoA in mitochondria and the nucleus. 4 However, researchers have not determined whether PDC regulates histone acetylation.
Hydrogen sulphide (H 2 S), an important gasotransmitter in the cardiovascular system, is involved in vascular relaxation and antioxidant defences. Our previous study revealed that H 2 S sustained mitochondrial ATP production by regulating key enzymes in the mitochondrial tricarboxylic acid (TCA) cycle. 6 Novel studies have shown that H 2 S covalently modifies cysteine residues on target proteins, through a process known as S-sulfhydration. This modification modulates protein structure and activity. 7,8 As shown in our previous study, exogenous H 2 S inhibits VSMC proliferation under diabetic conditions. 9 However, its mechanism remains unclear. The aim of the present study was to explore whether H 2 S regulates PDC translocation from the mitochondria to the nucleus to alter histone acetylation and subsequently inhibit VSMC proliferation.

| Animals
Leptin receptor-deficient (db/db) mice (8-10 weeks old, n = 60) and wild-type C57BL/6 mice (n = 40) were purchased from the Animal Model Institute of Nanjing (Nanjing, China). The animals were housed under diurnal lighting conditions and provided standard mouse chow and water throughout the study period. Half of the db/db mice were allocated to the NaHS treatment group and intraperitoneally in-

| Cell count
Treated cells were prepared as a suspension that was slowly dripped onto the edge of the counting plate to fill the gap between the counting plate and the cover slip. After cells had spread on the counting plate, cells were observed and counted under a low power lens (10 × 10 times).

| Functional nuclear isolation
Nuclei were isolated with the nuclei isolation kit: nuclei PURE prep from Sigma-Aldrich. Briefly, VSMCs were washed with PBS and scraped from the plate in the presence of lysis buffer. VSMCs were carefully placed on top of a 1.8 mol/L sucrose gradient, and the resulting suspension was centrifuged at 30 000 g for 45 minutes in a precooled swinging bucket ultracentrifuge. Nuclei were collected as a white pellet at the bottom of the centrifuge tube and washed with nuclei storage buffer (provided with the kit). The purity of nuclei was assessed using immunoblotting. Isolated nuclei were used immediately in functional experiments.

| Mitochondria isolation
Vascular smooth muscle cells were washed twice with ice-cold PBS, resuspended in lysis buffer (mmol/L: 20 Hepes/KOH, pH 7.5, 10 KCl, 1.5 MgCl 2 , 1.0 sodium EDTA, 1.0 sodium EGTA, 1.0 dithiothreitol, 0.1 PMSF and 250 sucrose) and then homogenized with a homogenizer in an ice/water bath. After removing the nuclei and cell debris by centrifugation at 1000 g for 10 minutes at 4℃, the supernatants were subsequently centrifuged at 10 000 g for 10 minutes at 4℃.
The resulting mitochondrial pellets were resuspended in lysis buffer.
The supernatants and mitochondrial fractions were stored at −80℃.

| Detection of H 2 S in VSMCs using the H 2 S probe 7-azido-4-methylcoumarin
The fluorescence reaction of the sulphate diester in VSMCs was tested using 7-azido-4 methylcoumarin (C-7Az, Sigma), which has been shown to selectively respond to H 2 S. 10 VSMCs were incubated with 50 μmol/L C-7Az PBS for 30 minutes and then washed with PBS. The fluorescence response of C-7Az in VSMCs was detected using a fluorescence microscope (Olympus, XSZ-D2) after excitation with a 720 nm laser. The results indicated that excitation fluorescence imaging was useful for detecting H 2 S by triggering the fluorescence reaction of C-7Az.

| Flow cytometry analysis of the cell cycle
Cells in the logarithmic growth phase were inoculated in 24well plates with 1 mL of media or in 6-well plates at a density of 1 × 10 6 cells/mL with 2 mL of media. The required treatment was performed (such as adding HG+Pal or NaHS), the culture was stopped at a specific time-point, and the next experiment was performed. Cells were centrifuged at 68 g for 5 minutes, the supernatant was discarded, and the cell pellet was collected, washed twice with pre-chilled PBS and fixed with pre-chilled 70% ethanol at 4℃ for more than 4 hours. The sample was centrifuged at 239 g for 5 minutes, and the supernatant was discarded. The cell pellet was washed once with 3 mL of PBS and then incubated with 400 μL of CCAA solution (PI stain solution, green) and 100 μL of RNase A (100 μg/mL) at 4℃ in the dark for 30 minutes. Samples were detected using a flow cytometer with standard procedures, and generally 20 000 to 30 000 cells were counted. The results were analysed using the cell cycle fitting software ModFit.

| Immunofluorescence staining
Vascular smooth muscle cells were fixed with 4% paraformaldehyde for 30 minutes and then permeabilized with 0.5% Triton X-100 for 30 minutes. Cells on the coverslips were blocked with 5% BSA for 1 hour at 37℃, incubated with anti-PDC-E1 antibodies overnight at 4℃ and incubated with anti-rabbit IgG for 1 hour.
Analyses and photomicrography were performed using a fluorescence microscope.

| Immunoprecipitation
Briefly, isolated mitochondria and nuclei were resuspended in PBS and diluted to a concentration of 1 mg/mL. After three freeze-thaw cycles, 500 μg of protein from each sample was used for immunoprecipitation. Sepharose beads were conjugated with the anti-HSP70 antibody (10 μg of antibody/500 μg of protein) and incubated with samples overnight at 4℃ with gentle rotation. Following the collection of beads using a centrifuge and three washing steps, precipitates were subjected to Western blot analyses to detect potential interacting proteins.

| Analysis of mitochondrial and cellular ROS levels
Mitochondrial ROS and cellular ROS levels were analysed.
Mitochondrial ROS production was measured using the MitoSOX Red mitochondrial superoxide indicator (Invitrogen). VSMCs were treated with control, HG, NaHS and MitoTempo for 24 hours. Cells were loaded with 5 μmol/L MitoSOX Red at 37℃ for 30 minutes.

| Point mutation of PDC-E1
Adenoviruses expressing GFP and PDC-E1-GFP were purchased The cells received different treatments 24 hours after transfection, and the related proteins were detected using Western blotting.

| Pyruvate dehydrogenase activity assay
The PDH activity in the nuclear lysates was measured with a colorimetric PDH activity assay kit (GENMED) according to the manufacturer's instructions, and the absorbance at 450 nm was measured kinetically for approximately 30 minutes at 37℃ after the addition of PDH developer and PDH substrate. Values for the blank sample (without PDH substrate) were subtracted from the sample readings, and PDH activity (nmol NADH/min) was normalized to mg of protein.

| Acetyl-coenzyme A analysis
Acetyl-coenzyme A was measured according to the manufacturer's protocol (acetyl-coenzyme A assay kit, Sigma). All samples and standards were analysed in duplicate. Ten microlitres of each sample was added to duplicate wells of a 96-well plate. Samples were brought to a final volume of 50 μL with acetyl-CoA assay buffer. A blank sample for each sample was prepared by omitting the Conversion Enzyme in the Reaction Mix. Ten microlitres of acetyl-CoA quencher was added to each sample, standard and sample blank well to correct for the background signal created by free coenzyme A and succinyl-CoA. Plates were incubated at room temperature for 5 minutes. Two microlitres of Quench Remover was added to each well, mixed well and incubated for an additional 5 minutes. The plate was mixed well using a horizontal shaker or by pipetting, and the reaction proceeded for 10 minutes at 37℃ in the dark. The fluorescence intensity was subsequently measured (λex = 535/λem = 587 nm).

| S-sulfhydration assay
S-sulfhydration was performed using a previously described method. 7  After an incubation for 3 hours at 25℃, biotinylated proteins were precipitated with streptavidin-agarose beads, which were subsequently washed with HEN buffer. The biotinylated proteins were eluted by SDS-PAGE and subjected to Western blot analysis using antibodies against PDC-E1.

| Measurement of intracellular polysulphide levels
Intracellular production of polysulphide was monitored using a newly developed fluorescent probe, SSP4, with slight modifications. 11 Briefly, VSMCs were loaded with 50 μmol/L SSP4 in serumfree DMEM containing 0.003% Cremophor EL for 15 minutes at 37℃ in the dark. After washing, SSP4 was detected using a fluorescence microscope (Olympus, XSZ-D2).

| Statistical analysis
Statistical analyses of the data were performed using GraphPad Prism software. Two groups were compared using unpaired Student's t tests, and multiple groups were compared using one-way ANOVA followed by multiple comparison tests. Statistical significance was detected at the P < .05 level. The results are presented as the means ± SEM of multiple experiments.

| CSE expression and H 2 S levels in vascular smooth muscle cells under diabetic conditions
In mammalian cells, the generation of H 2 S mainly depends on cystathionineγ-lyase (CSE) and cystathionineβ-synthase (CBS).
A tissue-specific distribution of the two enzymes required for H 2 S production has been observed. CBS is the main enzyme required for H 2 S generation in the nervous system. CSE is the predominant enzyme for H 2 S production in the cardiovascular system. However, a recent study found that CBS also generates H 2 S in the cardiovascular system. 12 As shown in our previous study, the expression of CSE, an enzyme that generates H 2 S, and H 2 S levels was decreased in the mesenteric artery of animals with STZ-induced type 1 diabetes. 13 In this study, we chose db/db mice as the animal model of type 2 diabetes. The expression of CSE in the thoracic artery was detected.
A lower level of the CSE protein was detected in db/db mice than in control mice. CSE protein levels were restored by the NaHS treatment in mice (db/db+NaHS) ( Figure 1A). The expression of CBS was similar among the different groups ( Figure S1).

| Effects of H 2 S on the proliferation and migration of VSMCs stimulated with high glucose and palmitate
We examined whether H 2 S levels regulated VSMC proliferation.
Proliferation assays showed that high glucose and palmitate induced cell growth, whereas exogenous H 2 S blocked cell growth (Figure 2A, B). H 2 S significantly retarded cell progression at G1 phase, and the number of cells in S phase decreased ( Figure 2C and Table 1). Next, the protein levels of cyclin D1, a G1/S checkpoint protein, and PCNA, which is responsible for governing the G1/S transition, were assessed. Levels of the cyclin D1 and PCNA proteins were significantly increased in the thoracic arteries of db/db mice and VSMCs treated with high glucose and palmitate compared F I G U R E 1 Exogenous H 2 S alters CSE expression and the H 2 S content. A, CSE expression in tissues was quantified by performing Western blotting (n = 4). B, vascular smooth muscle cells (VSMCs) were treated with HG (40 mmol/L) + palmitate (Pal, 500 μmol/L), HG+Pal+NaHS (100 μmol/L) or HG+Pal+PPG (10 nmol/L, an irreversible competitive CSE inhibitor) for 24 h. The CSE protein expression level was determined using Western blotting (n = 5). C, Staining with the 7-azido-4-methylcoumarin probe was used to measure H 2 S levels in VSMCs (n = 4). Values are presented as the means ± SD *P < .05, **P < .01 and ***P < .001 to those treated with exogenous H 2 S ( Figure 2D, E). Additionally, HG and palmitate significantly enhanced VSMC migration rates two-and threefold at 6, 12 and 24 hours, respectively, compared to the control groups using the scratch assay. PPG also increased VSMC migration rates. However, exogenous H 2 S inhibited VSMC migration ( Figure S3).
Hyperproliferative SMCs induce a phenotypic switch of VSMCs.
The biomarkers α-SMA, which represents the contractile type of SMCs, OPN and MMP2/9, which represent the synthetic phenotype, were examined. The expression of α-SMA was significantly attenuated in db/db mice and HG-and palmitate-stimulated VSMCs compared to cells treated with exogenous H 2 S. In contrast, the expression of OPN and MMP2/9 was significantly increased in the thoracic arteries of db/db mice and VSMCs treated with high glucose and palmitate. Pretreatment with PPG increased OPN, MMP2 and MMP9 expression ( Figure 2F, G). Taken together, these results validated that the decrease in H 2 S levels promoted VSMC proliferation in the diabetic state.

| H 2 S inhibits PDC-E1 translocation from the mitochondria to the nucleus under high glucose and palmitate conditions
Pyruvate dehydrogenase complex is composed of three catalytic enzymes: pyruvate dihydrolipoamide (E1), dihydrolipoamide transacetylase (E2), and dihydrolipoamide dehydrogenase (E3). PDC-E1 is responsible for catalysing the conversion of pyruvate to acetyl-CoA.
According to some studies, PDC-E1 translocates from the mitochondria to the nucleus in response to mitochondrial stress. 14 We first examined the nuclear level of PDC-E1 in the thoracic aorta and VSMCs exposed to hyperglycaemia and hyperlipidaemia. We extracted nuclei from VSMCs, and the Western blot analysis showed a higher level of the PDC-E1 protein in db/db mice and VSMCs stimulated with high glucose and palmitate than that in the control and exogenous H 2 S treatment groups ( Figure 3A, B). We also observed that PDC-E1 was evidently localized within the nucleus (marked with DAPI) upon the administration of high glucose and palmitate using immunofluorescence microscopy ( Figure 3C). We extracted the mitochondria and then measured the level of the PDC-E1 protein to further confirm that PDC-E1 detected in the nucleus was derived from mitochondria. We observed a lower level of the PDC-E1 protein in mitochondria from the HG+Pal group than in the control and NaHS groups ( Figure 3D). These results suggested that high glucose and palmitate promoted the translocation of PDC-E1 from the mitochondria to the nucleus; however, exogenous H 2 S reduced PDC-E1 translocation.
We separated nuclei from VSMCs through high-sucrose gradient centrifugation, avoiding mitochondrial contamination, to evaluate whether nuclear PDC-E1 was functional. The activities of PDC-E1 in nuclei were tested. The activity of PDC-E1 in the HG+Pal group was significantly increased compared to the control and NaHS treatment groups ( Figure 3E). We then detected the acetyl-CoA concentration in the nuclei and detected obviously higher levels in the HG+Pal group than in the control and exogenous H 2 S groups ( Figure 3F).
Based on these results, PDC-E1 translocation from the mitochondria to the nucleus after treatment with high glucose and palmitate was involved in acetyl-CoA production.

| Nuclear PDC-E1 promotes histone acetylation and cell proliferation
We showed that nuclear PDC-E1 generated acetyl-CoA and then confirmed whether acetyl-CoA modified histones. We first extracted the nuclei from the thoracic aorta and VSMCs and examined the acetylation level. The acetylation level in db/db mice and VSMCs treated with high glucose and palmitate was higher than that in the control and cells treated with NaHS ( Figure 4A, B). Next, we detected the specific acetylation site of H3K9, which is involved F I G U R E 2 Effects of exogenous H 2 S on proliferation and phenotypic changes. A, vascular smooth muscle cells (VSMCs) were treated with HG (40 mmol/L) + palmitate (Pal, 500 μmol/L), HG+Pal+NaHS (100 μmol/L) or HG+Pal+PPG (10 nmol/L, an irreversible competitive CSE inhibitor). Cell counting was performed to detect the number of cells after 24 h of treatment (n = 4). B, CCK-8 assays were used to detect the effects of different drug treatments on cell viability (n = 6). C, Flow cytometry assay of the cell cycle of VSMCs (n = 3). D, PCNA and CyclinD1 expression levels in aortas from control mice, db/db mice and db/db mice treated with NaHS (n = 4). E, PCNA and CyclinD1 expression levels in VSMCs treated with HG (40 mmol/L) + palmitate (Pal, 500 μmol/L), HG+Pal+NaHS (100 μmol/L), or HG+Pal+PPG (10 nmol/L, an irreversible competitive CSE inhibitor) (n = 5). (F and G) Expression levels of the α-SMA, MMP2, MMP9 and OPN proteins in tissues (F) and VSMCs (G) were determined using western blot analyses (n = 4). Values are presented as the means ± SD. *P < .05, **P < .01 and # P < .05 compared with the HG+Pal group in regulating gene expression in proliferating VSMCs. After exposure to high glucose and palmitate, the level of H3K9 acetylation was increased compared to that in the control and NaHS-treated groups ( Figure 4C, D). ATP citrate lyase (ACL) is an acetyl-CoAgenerating enzyme localized in the cytosol and nuclei that provides an acetyl group for histone acetylation. We used ACLI (ACL inhibitor, SB204990, 50 μmol/L) and a PDC-E1 inhibitor (PDHI, CPI-613, 100 μmol/L) to study the relative importance of these two enzymes in nuclear acetylation. PDHI ( Figure 4E) and ACLI ( Figure S4)

| mtHSP70 assists with the nuclear translocation of PDC-E1 from mitochondria
Mitochondria adapt to stress induced by increased ROS levels, which leads to increased expression of heat shock proteins and mitochondrial transporters, promoting communication with the nucleus. 15 We examined whether high glucose and palmitate induced oxidative stress. We measured intracellular and mitochondrial reactive HG+Pal group, was upregulated by the NaHS treatment ( Figure 5C).
Our data reinforced the concept that exogenous H 2 S attenuated oxidative stress in VSMCs.
Next, we investigated whether mitochondrial chaperones are involved in PDC-E1 translocation. We chose mtHSP70 (mitochondrial heat shock protein 70) based on previous studies showing that HSP70 participates in the nuclear transport of several mitochondrial proteins. 16,17 We extracted mitochondria, and Western blot assays revealed a significantly higher level of the mtHSP70 protein in the HG+pal group than in the control, exogenous H 2 S and MitoTempo groups ( Figure 5D). We also confirmed a noticeable increase in the level of nuclear mtHSP70 in the HG+pal group ( Figure 5E). We performed immunoprecipitation to detect the interaction between mtHSP70 and PDC-E1. Our results suggested that mtHSP70 may bind to PDC-E1 in the nucleus and mitochondria ( Figure 5F, G). Based on these results, mtHSP70 assisted with PDC-E1 translocation induced by mitochondrial oxidative stress.

| Exogenous H 2 S regulates PDC-E1 Ssulfhydration to inhibit VSMC proliferation
As shown in Figure 3E, exogenous H 2 S decreased the activity of PDC-E1. We detected PDC-E1 S-sulfhydration to further study how exogenous H 2 S inhibits PDC-E1 activity. The H 2 S modification of specific cysteine residues of proteins, which is referred to as Ssulfhydration, has been extensively studied. Protein S-sulfhydration, a post-translational modification, is involved in alterations in protein structure and activity. SSP4, a fluorescent probe, was used to detect S-sulfhydration. S-sulfhydration was obviously observed after treatment with exogenous H 2 S. Dithiothreitol (DTT, 1 mmol/L), an inhibitor of S-sulfhydration, reduced the effect of NaHS on Ssulfhydration ( Figure 6A). Furthermore, a biotin-switch assay was also used to measure S-sulfhydration in proteins. NaHS evidently increased the S-sulfhydration of PDC-E1, whereas DTT abolished the S-sulfhydration of PDC-E1 ( Figure 6B). We used bioinformatics methods to analyse and predict the structure of the active centre of PDC-E1. PDC-E1 consists of 390 amino acids and contains 11 cysteine residues. Based on the bioinformatics analysis, Cys101 of PDC-E1 in the active centre was mutated to alanine ( Figure S5) and a mutant PDC-E1-Cys101 (PDC-E1m) overexpression plasmid was constructed. After PDC-E1m overexpression, H 2 S did not reduce the expression of cyclin D1 and PCNA in the high glucose and palmitate groups or the exogenous H 2 S treatment group ( Figure 6C).
Moreover, after PDC-E1m overexpression, the level of H3K9 acetylation was increased in the HG+Pal and exogenous H 2 S groups ( Figure 6D). Taken together, H 2 S inhibited VSMC proliferation by increasing PDC-E1 S-sulfhydration and reducing PDC-E1 translocation ( Figure 7).

| D ISCUSS I ON
In our study, PDC-E1 was present and functional in the nucleus in VSMCs stimulated with high glucose and palmitate. Nuclear PDC-E1 produced acetyl-CoA that was used for histone acetylation to promote VSMC proliferation. Exogenous H 2 S inhibited PDC-E1 translocation from the mitochondria to the nucleus via PDC-E1 Ssulfhydration to decrease VSMC proliferation.
F I G U R E 3 H 2 S inhibits pyruvate dehydrogenase complex-E1 (PDC-E1) translocation from the mitochondria to the nucleus of cells stimulated with high glucose and palmitate. A and B, Nuclei were extracted from tissues (A) and cells (B) separately to detect the level of the PDC-E1 protein in the nucleus (n = 4). C, Immunofluorescence staining was used to detect the nuclear localization of PDC-E1. D, The levels of PDC-E1 in the mitochondria of VSMCs (n = 5). E, The activity of PDC-E1 in the nuclei of VSMCs (n = 4). F, Detection of the acetyl-CoA content in VSMCs (n = 4). Values are presented as the means ± SD *P < .05 and **P < .01 Relocalization of PDC-E1 has been observed in cancer cells that F I G U R E 4 Nuclear pyruvate dehydrogenase complex-E1 (PDC-E1) promotes histone acetylation and cell proliferation. A and B, Relative acetylation levels in the nuclei of the thoracic aorta (A) and VSMCs (B). C, The levels of H3 and H3K9ac in nuclear extracts of the thoracic aorta (n = 4). D, The levels of H3, H3K9ac and H3K18ac in nuclear extracts of VSMCs (n = 4). E, PDC inhibitor (PDHI, 100 μmol/L) inhibited the proliferation of VSMCs (n = 3). Values are presented as the means ± SD *P < .05 F I G U R E 5 Exogenous H 2 S inhibits oxidative stress in vascular smooth muscle cells (VSMCs). A, The total ROS level was measured using DCFH (green fluorescence), and B, mitochondrial ROS level was measured using MitoSOX (red fluorescence). The mean fluorescence intensity was measured. C, Mitochondrial Mito-CAT and Mn-SOD levels were examined using western blotting (n = 4). D, The mitochondria were extracted from VSMCs, and the expression of the mtHSP70 protein in each group was detected using western blotting (n = 4). E, Nuclear proteins were extracted from VSMCs, and the expression of the mtHSP70 protein in each group was detected using western blotting (n = 4). F, The mitochondrial extracts from VSMCs were immunoprecipitated with an anti-mtHSP70 antibody and then immunoblotted with antibodies specific for PDC-E1. G, The nuclear extracts from VSMCs were immunoprecipitated with an anti-mtHSP70 antibody and then immunoblotted with antibodies specific for PDC-E1. Values are presented as the means ± SD. *P < .05 were serum-starved or stimulated with epidermal growth factor or a mitochondrial respiration inhibitor (rotenone). 22 PDC-E1 converts mitochondrial pyruvate into acetyl-CoA. Thus, the concentration of acetyl-CoA in mitochondria may be 20-to 30-fold greater than that in the cytoplasm and nucleus because acetyl-CoA is membrane-impermeable. 23 Therefore, PDC-E1 must be translocated to the nucleus from mitochondria to generate acetyl-CoA. We confirmed that mitochondrial oxidative stress promoted The expression levels of PDC-E1, CyclinD1 and PCNA were detected using western blotting (n = 5). D, The levels of H3 and H3K9ac were detected using western blotting after the transfection of the mutant (n = 5). Values are presented as the means ± SD. *P < .05, **P < .01 and ***P < .001 Hydrogen sulphide plays a crucial role in the physiology and pathophysiology of the cardiovascular system. In our study, we noticed that the expression of CSE, a key enzyme to produce H 2 S in cardiovascular system, was decreased in db/db mice ( Figure 1A, B).
Immunoprecipitating to detect the interaction between CSE and ubiquitin, We found CSE was modified by ubiquitination and degraded by ubiquitin-proteasome system, thus reduced the expression level of CSE. Exogenous H 2 S alleviated the ubiquitination and degradation of CSE, thereby increasing the expression of CSE ( Figure S2).
The persulphidation or S-sulfhydration of reactive cysteines (ie Cys-SSH) has recently been shown to contribute to cellular redox homeostasis. Persulphidation or S-sulfhydration is generated by the transsulphuration pathway, which catabolizes cysteine and cystathionine to generate hydrogen sulphide (H 2 S) and H 2 S-related sulphane sulphur compounds (referred to as H 2 Sn). 24,25 This pathway is of particular importance for the vascular system because it expresses CSE. 26 Based on emerging data, the hydropersulphide moiety (-SSH) in the active cysteine residues of target proteins mediates cellular functions. In our study, exogenous H 2 S modified PDC-E1 via Ssulfhydration, which inhibited its activity and nuclear translocation.
The administration of DTT, a blocker of S-sulfhydration, decreased the S-sulfhydration level of PDC-E1. We overexpressed PDC-E1 in which Cys101 was mutated to Ala and showed that treatment with exogenous H 2 S decreased the PDC-E1 S-sulfhydration level and increased PDC-E1 translocation to the nucleus. Thus, S-sulfhydration of PDC-E1 at Cys101 might be one of the specific mechanisms by which H 2 S reduced PDC-E1 translocation to the nucleus and histone acetylation and inhibited VSMC proliferation (Figure 7).
In this study, PDC-E1, a key mitochondrial TCA cycle-related enzyme, translocated to the nucleus, and the relationship between this change in metabolic enzyme localization and waves of transcriptional activity was confirmed. This study provided evidence that H 2 S increased PDC-E1 S-sulfhydration at Cys101 to regulate PDC-E1 translocation from the mitochondria to the nucleus, thereby preventing VSMC proliferation under hyperglycaemic and hyperlipidaemic conditions. H 2 S may be a useful therapeutic strategy for cardiovascular diseases in the future.

ACK N OWLED G EM ENTS
This work was supported by National Science Foundation of China (81970317, 81970411) and Graduate Innovation Foundation of Harbin Medical University (YJSKYCX2019-08HYD).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated during this study are included in this published article.