Exogenous H2S Preventing PDC-E1 Translocation to Nucleus and Inhibiting Vascular Smooth Muscle Cell Proliferation in Diabetic State

The poliferation of vascular smooth muscle cells (VSMCs) is the main cause of diabetic vascular complications. Hydrogen sulde (H 2 S), a gaseous molecule, is involved in modulating multiple physiological functions. H 2 S could inhibit VSMCs proliferation induced by hyperglycemia and hyperlipidemia, however, the mechanisms are unclear. Our results showed that H 2 S level was lower and expression of proliferative protein for PCNA and CyclinD1 was higher in db/db mice aorta and VSMC treated by glucose and palmitate, whereas, exogenous H 2 S decreased PCNA and CyclinD1 expression. We found that mitochondrial pyruvate dehydrogenase complex-E1 (PDC-E1) was signicantly translocated to the nucleus of VSMCs with the treatment of high glucose and palmitate, and it increased the level of acetyl-CoA and histone acetylation (H3K9Ac). Exogenous H 2 S inhibited PDC-E1 translocation from mitochondria to nucleus, due to PDC-E1 being modied via S-sulfhydration. In addition, PDC-E1 was mutated at Cys101. Overexpression of PDC-E1 mutated at Cys101 enhanced histone acetylation (H3K9Ac) and VSMCs proliferation. These ndings suggested that H 2 S regulated PDC-E1 S-sulfhydration at Cys101 to prevent its translocation from mitochondria to nucleus and inhibit VSMCs proliferation in diabetic conditions. The indicate that excitation imaging can be used to detect H 2 S by the triggering uorescence reaction of C-7Az.


Introduction
Vascular complications, such as atherosclerosis, in 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 con rmed that chronic hyperglycemia / high glucose (HG) enhancing reactive oxygen species (ROS) accelerates the progress of VSMCs proliferation [3]. However, the mechanisms of VSMCs proliferation could not be fully explained. Due to the complexity of pathogenic mechanisms, a new insight is required to explore hyperglycemia/high glucose (HG)-induced VSMCs proliferation.
Increasing evidence demonstrated that epigenetic regulation of gene expression is essential for cell proliferation and differentiation to explore modi cations 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 nucleus, has been a rmed. ATP-citrate lyase (ACL) have been reported to be present in the nucleus. ACL, as the main enzyme for nuclear acetyl-CoA generation in mammalian cells, utilizes mitochondrial citrate as its substrate [5]. Recent studies have demonstrated that the pyruvate dehydrogenase complex (PDC) might be translocated from mitochondria to the nucleus in response to mitochondrial stress or the stimulus of growth factor in tumor cells. PDC catalyzes pyruvate to produce acetyl-CoA in mitochondria and nucleus [4]. However, whether PDC regulates histone acetylation is still unknown.
Hydrogen sul de (H 2 S), as an important gasotransmitter in the cardiovascular system, is involved in vascular relaxation and anti-oxidation. Our previous study revealed that H 2 S sustained mitochondrial ATP production through regulating the key enzymes of the mitochondrial tricarboxylic acid (TCA) cycle [6].
The novel studies have demonstrated that H 2 S covalently modi es cysteine residues on target proteins, which is named as S-sulfhydration. This modi cation could modulate protein structure and activity [7,8].
Our group has also found that exogenous H 2 S inhibited VSMCs proliferation in diabetic conditions [9].
However, its mechanism is not still clear. The aim of the present study was to explore whether H 2 S could regulate PDC translocation from mitochondria to nucleus to effect on histone acetylation to inhibit VSMCs proliferation.

Materials And Methods
Animals.
Leptin receptor-de cient (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 fed standard mouse chow and water throughout the study period. Half of the db/db mice were put in the NaHS treatment group and treated with NaHS (80 μmol/kg) by intraperitoneal injection every 2 days for 12 weeks. All animal experiments were performed by the Guide for the Care and Use of Laboratory Animals published by the China National Institute of Health and approved by the Animal Care Committees of Harbin Medical University, China.

Cell count
Make cell suspension from the treated cells. Slowly drip from the edge of the counting plate to ll the gap between the counting plate and the cover sheet. Wait a while, the counting plate under a low power lens (10×10 times) to observe and count.

Functional nuclear Isolation
Isolating nucleus was performed with the nuclei isolation kit: nuclei PURE prep from Sigma Aldrich. Brie y, 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 M sucrose gradient and the resulting suspension was centrifuged at 30,000 g for 45 min in a precooled swinging bucket ultracentrifuge. Nuclei were collected as a white pellet at the bottom of the centrifuge and washed with nuclei storage buffer (provided with the kit). Purity of nuclei was assessed by immunoblot. For functional experiments, isolated nuclei were used immediately.

Mitochondria isolation
VSMCs 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 by a homogenizer in ice/water. After removing the nuclei and cell debris by centrifugation at 1000 g for 10 min at 4 °C, the supernatants were further centrifuged at 10000 g for 10 min at 4°C. The resulting mitochondrial pellets were resuspended in lysis buffer. The supernatants and mitochondrial fractions were stored at -80 °C.

Detection of H 2 S in VSMCs using H 2 S probe 7-Azido-4-Methylcoumarin
The uorescence reaction of the sulfate diester in VSMCs was tested using 7-azido-4 methyl coumarin (C-7Az, Sigma), which has been shown to selectively respond to H 2 S [10]. VSMCs were incubated with 50 μmol/LC-7Az PBS for 30 minutes and then the cells were washed with PBS. The uorescence response of C-7Az in VSMCs was detected using a uorescence microscope (Olympus, XSZ-D2, Tokyo, Japan) excited by a 720 nm laser. The results indicate that excitation uorescence imaging can be used to detect H 2 S by the triggering uorescence reaction of C-7Az.
Flow cytometric analysis of cell cycle Take cells in the logarithmic growth phase, inoculate 24 well plates or 2 mL in 6 well plates at 1×10 6 cells/mL with 1mL, perform the required treatment (such as adding HG+Pal, NaHS), stop the culture after a speci c time, and proceed to the next experiment. Centrifuge at 800 rpm for 5 min, collect the cell pellet, discard the supernatant, wash twice with pre-chilled PBS, add pre-chilled 70% ethanol, and x at 4 °C for more than 4 h. Centrifuge at 1500 rpm for 5 min, discard the supernatant, wash once with 3 mL of PBS, add 400 μL of CCAA solution (PI stain solution, green), 100 μL RNase A (100 ugs/mL), and incubate at 4°C in the dark for 30 min. Flow analysis: It is detected by ow cytometry with standard procedures and generally counts 20,000 to 30,000 cells, and the results are analyzed by cell cycle tting software ModFit.
Immuno uorescence staining VSMCs were xed in 4% paraformaldehyde for 30 minutes and then permeabilized with 0.5% Triton X-100 for 30 min. The coverslips were blocked with 5% BSA for 1 h at 37 °C. Cells were incubated with anti-PDC-E1 antibodies overnight at 4 °C and incubated with anti-rabbit IgG for 1 h. Analysis and photomicrography were performed using a uorescence microscope.

Immunoprecipitation
Brie y, isolated mitochondria and nuclues were resuspended in PBS and diluted to a concentration of 1 mg/mL. After three freeze-thaw cycles, a total amount of 500 μg of protein was used per sample for immunoprecipitation. Sepharose beads were conjugated with the anti-HSP70 antibody (10 μg antibody/500 μg protein) and incubated with samples overnight at 4 °C with gentle rotation. Following collecting of beads using centrifuge and three washing steps, precipitates were subjected to Western blotting analyses for detection of potential interacting proteins.
Mitochondrial and cellular ROS level analysis Mitochondrial ROS and cellular ROS level analysis. Mitochondrial ROS production was measured using MitoSOX Red mitochondrial superoxide indicator (Invitrogen). VSMCs were treated with control, HG, NaHS and MitoTempo for 24 h. Cells were loaded with 5 μM MitoSOX Red at 37 °C for 30 min. Red uorescence was measured at 583 nm following excitation at 488 nm using a uorescence microscope. Intracellular ROS levels were examined using the DCFH-DA staining method based on the conversion of nonuorescent DCFH-DA to the highly uorescent DCF upon intracellular oxidation by ROS. VSMCs were seeded on coverslips and incubated (45 min, 37 °C, in the dark) in serum-free media containing DCFH-DA incubation, respectively leave at 4 °C overnight. All antibodies were from Proteintech Group, Inc, USA. Protein complexes were incubated for 1 hour at room temperature with an anti-mouse / anti-rabbit antibody. Densitometry was conducted with image processing and analysis program AlphaView SA and the data were expressed as relative units.

S-sulfhydration Assay
S-sulfhydration was performed as described previously [7]. VSMCs were homogenized in HEN buffer solution containing 250 mmol / L Hepes-NaOH, 1 mmol / L EDTA, 0.1 mmol / L neocuproine and 100 mmol / L deferoxamine to adjust the pH to 7.7. Cell lysate contained HEN buffer, 0.5% CHAP, 0.1% SDS, 20 mmol / L methyl methanethiosulfonate, 10 μg / mL leupetin, 5 μg / mL aprotinin and 1 mM protease inhibitor PMSF. Quantitative analysis was after the lysis of cells. VSMCs were added to the blocking buffer (HEN buffer with 2.5% SDS and 20 mM methyl methanethiosulfonate) at 50 °C for 60 min with frequent vortexing. Added 4 volumes of cold acetone to each 15 mL centrifuge tube and incubated at -20°C for 1 hour. Then, centrifuged at 2000 g at 4 °C for 10 minutes. Removed the cold acetone. The cells were resuspended in 90 μL of HEN buffer (containing 1% SDS) and transferred to a new 1.5 mL EP tube. 4 mM N-[6-(biotinamido) hexyl]-3-(2-pyridyldithio) propionamide (biotin-HPDP stop solution) was added and incubated at 25 °C for 1 hour. After incubation for 3 h at 25 °C, biotinylated proteins were precipitated by streptavidin-agarose beads, which were later washed with HEN buffer. The biotinylated proteins were eluted by SDS-PAGE and subjected to western blotting analysis using antibodies against PDC-E1.

Measurement of intracellular levels of polysul de
Intracellular production of polysul de was monitored using a newly developed uorescent probe, SSP4, with slight modi cations [11]. Brie y, VSMCs were loaded with 50 μmol·L -1 SSP4 in a serum-free DMEM medium containing 0.003% Cremophor EL for 15 min at 37 °C in the dark. After being washed, SSP4 was detected using the uorescence microscope (Olympus, XSZ-D2, Tokyo, Japan).

Statistical analysis
Statistical analysis of data was performed using Graphpad Prism. Two groups were compared using unpaired Student's t-tests and multiple groups using One-way ANOVA followed by multiple comparison tests. Statistical signi cance was detected at P < 0.05 level. Results were expressed as Mean ± SEM of multiple experiments.

CSE expression and H 2 S level in vascular smooth muscle cells in diabetic condition
In mammalian cells, the generation of H 2 S is mainly dependent on cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS). The distribution of two enzymes for H 2 S production has tissue speci city. CBS is the main enzyme for H 2 S generation in nervous system. CSE is predominant enzyme for H 2 S production in cardiovascular system. However, recent study has found that CBS also generates H 2 S in cardiovascular system [12]. Our previous study has shown that the expression of CSE, an enzyme to generate H 2 S, and H 2 S level were decreased in the mesenteric artery in STZ-induced type 1 diabetes [13]. In this study, we chose db/db mice as the type 2 diabetes animal model. The expression of CSE in the thoracic artery was detected. The protein level of CSE was lower in db/db mice, compared to control mice. CSE protein level was recovered with the treatment of NaHS (db/db+NaHS) mice (Fig. 1A). The expression of CBS was similar among different groups (SFig. 1).
Vascular smooth muscle cells of mice aorta were cultured with high glucose (HG, 40 mM) and Palmitate (Pal, 500 µM) for 24 h to mimic hyperglycemia and hyperlipidemia of type 2 diabetes. Similarly, CSE expression was signi cantly decreased with the treatment of HG and Pal while exogenous H 2 S recovered its expression. DL-propargylglycine (PPG, 10 nM) is an inhibitor of CSE. PPG treatment decreased the protein level of CSE (Fig. 1B). To explain the decrease of CSE protein level in HG+Pal group, we tested ubiquitylation level of CSE with immunoprecipitation. MG132 is an inhibitor of 26S proteasome. Our data showed that the ubiquitylation level of CSE was signi cantly increased in HG+Pal group, compared to control and NaHS-treated and MG132 groups (SFig. 2). H 2 S uorescence probe, 7-azido-4-methyl coumarin (C-7Az) was used to test H 2 S content in VSMCs. The H 2 S level in HG+Pal and PPG groups was signi cantly lowered than that of control and exogenous H 2 S groups (Fig. 1C). These results indicated that endogenous H 2 S production in VSMCs decreased, due to CSE degradation, in hyperglycemia and hyperlipidemia conditions.

Effects of H 2 S on proliferation and migration in VSMCs induced by high glucose and palmitate
We examined whether H 2 S level regulated VSMCs proliferation. Proliferation assay showed that high glucose and palmitate could induce cell growth, whereas, exogenous H 2 S blocked cell growth ( Fig. 2A,B). H 2 S signi cantly retarded cell progression at the G1 phase and the number of cells in the S phase decreased (Fig. 2C, Table1). Next, the protein level of CyclinD1, G1/S-checkpoint protein, and PCNA, which is responsible for governing the G1/S transition, was assessed. Our results showed that the protein level of CyclinD1 and PCNA was signi cantly increased in the thoracic arteries of db/db mice and VSMCs treated by high glucose and palmitate, compared to that with the treatment of exogenous H 2 S (Fig. 2D,E).
Additionally, we found that HG and Palmitate signi cantly enhanced VSMCs migration rates of two and three folds for 6, 12, and 24 h, respectively, compared to control groups, using the scratch assay. PPG also increased VSMCs migration rates. However, exogenous H 2 S inhibited VSMCs migration rates (SFig.

3).
Hyper-proliferated SMCs induced 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. Our results showed that α-SMA expression in db/db mice and HG and Palmitatestimulated VSMCs was signi cantly attenuated compared to that the treatment of exogenous H 2 S. By contrast, the expression of OPN and MMP2/9 signi cantly increased in the thoracic arteries of db/db mice and VSMCs treated by high glucose and palmitate. Pre-treatment of PPG increased OPN, MMP2 and MMP9 expression (Fig. 2F,G). Taken together, these results validated that the decrease of H 2 S promoted VSMCs proliferation in the diabetic state.

H 2 S inhibiting PDC-E1 translocation from mitochordria to nucleus under high glucose and palmitate
state.
PDC is composed of three catalytic enzymes: pyruvate dihydrolipoamide (E1), dihydrolipoamide transacetylase (E2), and dihydrolipoamide dehydrogenase (E3). PDC-E1 is responsible for catalyzing pyruvate to acetyl-CoA. Some study has demonstrated that PDC-E1 translocated from mitochondria to the nucleus under mitochondrial stress [14]. We rst examined the nuclear presence of PDC-E1 in thoracic aorta and VSMCs under hyperglycemia and hyperlipidemia. We extracted the nucleus from VSMCs, Western blot analyzed that the protein level of PDC-E1 in db/db mice and VAMCs under high glucose and palmitate condition was higher than that in control and with the treatment of exogenous H 2 S group (Fig.   3A,B). We also observed that PDC-E1 evidently localized within the nucleus (marked by DAPI) with the administration of high glucose and palmitate by immuno uorescence microscopy (Fig. 3C). To further con rm that PDC-E1 within the nucleus derived from mitochondria, we extracted the mitochondria and then measured its protein level. Our results found that the protein level of PDC-E1 in mitochondria of the HG+Pal group was lower than that in control and with the treatment of NaHS groups (Fig. 3D). These results suggested that high glucose and palmitate promoted the translocation of PDC-E1 from mitochondria to the nucleus, however, exogenous H 2 S could reduce PDC-E1 translocation.
To evaluate whether nuclear PDC-E1 was functional, we separated nuclei from VSMCs by high sucrose gradient centrifuged methods, avoiding of mitochondrial contamination. The activities of PDC-E1 in nuclei were tested, respectively. Our results found that the activity of PDC-E1 in HG+Pal group signi cantly increased, compared to control and with the treatment of NaHS (Fig. 3E). We then detected the content of acetyl-CoA in nuclei and found it in HG+Pal group was obviously higher than that in control and exogenous H 2 S group (Fig. 3F). These results suggested that PDC-E1 translocation from mitochondria to nucleus with the treatment of high glucose and palmitate could be involved in acetyl-CoA production.

Nuclear PDC-E1 promoting Histone acetylation and cell proliferation
We have demonstrated that nuclear PDC-E1 generated acetyl-CoA, and then con rmed whether acetyl-CoA could modify histone. We rst extracted the nuclei from thoracic aorta and VSMCs and examined the acetylation level. Our results showed that the acetylation level in db/db mice and administration of high glucose and palmitate was higher than that in control and treated as NaHS (Fig. 4A,B). Next, we detected the speci c acetylation site of H3K9, which is involved in regulating VSMCs proliferative gene expression. Our results showed that after exposure to high glucose and palmitate, the acetylation level of H3K9 had increased compared to control and treated with NaHS groups (Fig. 4C,D). ATP-citrate lyase (ACL) is acetyl-CoA generating enzyme localized in cytosol and nuclei, which provides acetyl group for histone acetylation. We used ACLI (ACL inhibitor, SB204990, 50 μM ) and PDC-E1 inhibitor (PDHI, CPI-613, 100 μM ) to study the relative importance of these two enzymes in nuclear acetylation. Our data showed that PDHI (Fig. 4E) and ACLI (SFig. 4) obviously reduced the expression of Cyclin D1 and PCNA in VSMCs nucleus under diabetic condition.These results demonstrated that exogenous H 2 S decreased the acetylation level of H3K9 via inhibiting PDC-E1 translocation from mitochondria to the nucleus.

mtHSP70 assisting PDC-E1 nuclear translocation from mitochondria
Evidence shows that mitochondria adapt to stress inducing by increased ROS level, which leads to the enhanced expression of heat shock proteins and mitochondrial transporters, promoting the communication with the nucleus [15]. We examined whether high glucose and palmitate can induce oxidative stress. We measured intracellular and mitochondrial reactive oxygen species (ROS) with uorescent probe DCFH and MitoSOX. We found that quanti cation of DCFH and MitoSOX uorescent intensity showed similar elevation with adminstration of high glucose and palmitate, while exogenous NaHS, NAC and MitoTempo signi cantly attenuated ROS levels in intracellular and mitochondria (Fig.  5A,B). MitoTempo is a suppressor of ROS production in mitochondria. The expression of mitochondrial catalase (Mito-CAT) and manganese-dependent superoxide dismutase (Mn-SOD) were tested to further examine the role of exogenous H 2 S on ROS production in VSCMs treated with HG and palmitate. The results showed that the expression of Mito-CAT and Mn-SOD, which was downregulated in HG+pal group, was upregulated by NaHS treatment (Fig. 5C). Our data reinforced the concept that exogenous H 2 S attenuated oxidative stress in VSMCs.
Next, to investigate whether mitochondrial chaperone is involved in PDC-E1 translocation. We chose mtHSP70 (mitochondrial heat shock protein 70) based on previous studies that HSP70 took part in the nuclear transportation of several mitochondrial proteins [16,17]. We extracted mitochondria and Western blot assay found that the protein level of mtHSP70 was signi cantly higher in HG+pal group, compared to control, exogenous H 2 S and MitoTempo groups (Fig. 5D). We also con rmed that expression of mtHSP70 in nuclear also obviously increased in HG+pal group (Fig. 5E). We performed immunoprecipatation to detect interaction between mtHSP70 and PDC-E1. Our results suggested that mtHSP70 may bind to PDC-E1 in nucleus and mitochondria (Fig. 5F,G). These results demonstrated that mtHSP70 assisted PDC-E1 translocation induced by mitochondria oxidative stress. 6. Exogenous H 2 S regulating PDC-E1 S-sulfhydration to inhibit VSMCs proliferation PDC-E1.
In Fig. 3E, our results demonstrated that exogenous H 2 S decreased the activity of PDC-E1. To further study how exogenous H 2 S inhibiting PDC-E1 activity, we detected PDC-E1 S-sulfhydration. H 2 S modifying speci c cysteine residues of proteins, which is referred to S-sulfhydration, has been extensively studied. Protein S-sulfhydration as a kind of post-translational modi cation is involved in the alterations of protein structure and activity. SSP4, as a uorescent probe, was used to detect the production of Ssulfhydration. The S-sulfhydration group, was obviously observed in the treatment of exogenous H 2 S. Dithiothreitol (DTT, 1 mM), as an inhibitor of S-sulfhydration formation, reduced the effect of NaHS on Ssulfhydration formation (Fig. 6A). Furthermore, a biotin-switch assay was also used to measure protein modi cation of S-sulfhydration. Our results demonstrated that NaHS evidently enhanced PDC-E1 Ssulfhydration formation, whereas, DTT abolished the S-sulfhydration level of PDC-E1 (Fig. 6B). We used bioinformatics methods to analyze and predict the structure of the active center of PDC-E1. PDC-E1 consists of 390 amino acids and contains 11 cysteine residues. Based on bioinformatics analysis, the cysteine Cys101 of PDC-E1 in the active center was mutated to alanine (SFig. 5). Mutant of PDC-E1-Cys101 (PDC-E1m) overexpression plasmid was constructed. After PDC-E1m overexpression, H 2 S could not reduce the expression of CyclinD1 and PCNA between high glucose and palmitate group and exogenous H 2 S treatment group (Fig. 6C). Moreover, after PDC-E1m overexpression, the acetylation level of H3K9 had increased in HG+Pal and exogenous H 2 S groups (Fig. 6D). Taken together, these results indicated that H 2 S could inhibit VSMCs proliferation via elevating PDC-E1 S-sulfhydration modi cation and reducing PDC-E1translocation .

Discussion
In our study, we demonstrated that PDC-E1 was present and functional in the nucleus in VSMCs under high glucose and palmitate condition. Nucleus PDC-E1 produced acetyl-CoA used for histone acetylation to promote VSMCs proliferation. Exogenous H 2 S inhibited PDC-E1 translocation from mitochondria to the nucleus via PDC-E1 S-sulfhydration to decrease VSMCs proliferation.
Growing evidence has con rmed that VSMCs proliferation is characteristics in type 2 diabetes patients. When VSMCs proliferation occurs, VSMCs switch from contractile to synthetic phenotype. The cell cycle associated genes are overexpressed. In our study, the expression of PCNA and cyclin D1 was signi cantly increased under high glucose and palmitate condition. Some studies have revealed that when stress reaction happens, such as oxygen de ciency or high metabolic state, they induce the alteration of the post-translational modi cations (PTMs) of histone [18,19]. PTMs have been considered as key contributors to control targeted gene expression in both physiological and pathophysiological conditions. For example, histone acetylation via histone acetyltransferase contributes to active transcription via rendering gene promoters more accessible to the transcription machinery [20]. Evidence demonstrates the emergence of metabolic enzymes as crucial modulators, such as ATP-citrate lyase and PDC-E1, are involved in cell proliferation. PDC-E1 is mitochondrial complex, as a gatekeeper to regulate pyruvate ux from the cytosol to mitochondria, which couples glycolysis to OXPHOS [21]. Relocalization of PDC-E1 has been observed in cancer cells that were serum-starved or stimulated by 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 can be 20-30 fold greater than that in the cytoplasm and nucleus because acetyl-CoA is membrane impermeable [23]. Therefore, PDC-E1 is necessary to translocate to the nuclei to generate acetyl-CoA from mitochondria. We con rmed that mitochondrial oxidative stress promoted PDC-E1 translocation to nuclei. In our study, we demonstrated that the activity of PDC-E1 and the acetyl-CoA content in the nucleus increased in the HG+Pal group. Exogenous H 2 S reduced PDC-E1 activity and the acetyl-CoA content in the nucleus. H3K9 acetylation level was signi cantly increased were upregulated under hyperglycemia and hyperlipidemia condition, due to PDC-E1 translocation; however, exogenous H 2 S reduced H3K9 acetylation level. These ndings indicated that acetyl-CoA, as an important substrate for histone acetylation can be generated in the nucleus from pyruvate, which is dependant on PDC-E1 translocation in the nucleus.
Hydrogen Sul de plays a crucial role in physiology and pathophysiology in cardiovascular systems. Recent studies showed that the persul dation or S-sulfhydration of reactive cysteines (i.e., Cys-SSH) contributes to cellular redox homeostasis. Persul dation or S-sulfhydration are generated by the transsulfuration pathway which catabolizes cysteine and cystathionine to generate hydrogen sul de (H2S) and H2S-related sulfane sulfur compounds (referred to as H2Sn) [24,25]. This pathway is of particular importance for the vascular system as CSE [26]. Emerging data suggest that hydropersul de moiety (-SSH) in the active cysteine residues of target proteins can mediate cellular functions. In our study, we demonstrated that exogenous H 2 S can modify PDC-E1 via S-sulfhydration, which inhibited its activity and its translocation. Administration of DTT, a blocker of S-sulfhydration, decreased the Ssulfhydration level of PDC-E1. We overexpressed PDC-E1 in which Cys101 was mutated to Ala and demonstrated that treatment with exogenous H 2 S decreased PDC-E1 S-sulfhydration level and increased PDC-E1 translocation to the nuclei. This nding suggested that S-sulfhydration of PDC-E1 at Cys101 Page 12/17 might be one of the detailed mechanisms by which H 2 S reduced PDC-E1 translocation to the nuclei and histone acetylation and inhibits VSMCs proliferation (Fig. 7).
In this study, it is noteworthy that PDC-E1, a key mitochondrial TCA cycle-related enzyme, translocated to the nuclei and that the relationship between this change in metabolic enzyme localization and waves of transcriptional activity has been con rmed. This study has provided evidence that H 2 S increased PDC-E1 S-sulfhydration at Cys101 to regulate PDC-E1 translocation from mitochordria to the nucleus, thereby preventing VSMCs proliferation under hyperglycemia and hyperlipidemia condition. H 2 S may be a useful therapeutic strategy in cardiovascular diseases in the future.

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