Exogenous H₂S reduces the acetylation levels of mitochondrial respiratory enzymes via regulating the NAD⁺-SIRT3 pathway in cardiac tissues of db/db mice

Abstract

Hydrogen sulfide (H₂S), a gaseous molecule, is involved in modulating multiple physiological functions, such as antioxidant, antihypertension, and the production of polysulfide cysteine. H₂S may inhibit reactive oxygen species generation and ATP production through modulating respiratory chain enzyme activities; however, the mechanism of this effect remains unclear. In this study, db/db mice, neonatal rat cardiomyocytes, and H9c2 cells treated with high glucose, oleate, and palmitate were used as animal and cellular models of type 2 diabetes. The mitochondrial respiratory rate, respiratory chain complex activities, and ATP production were decreased in db/db mice compared with those in db/db mice treated with exogenous H₂S. Liquid chromatography with tandem mass spectrometry analysis showed that the acetylation level of proteins involved in the mitochondrial respiratory chain were increased in the db/db mice hearts compared with those with sodium hydrosulfide (NaHS) treatment. Exogenous H₂S restored the ratio of NAD⁺/NADH, enhanced the expression and activity of sirtuin 3 (SIRT3) and decreased mitochondrial acetylation level in cardiomyocytes under hyperglycemia and hyperlipidemia. As a result of SIRT3 activation, acetylation of the respiratory complexe enzymes NADH dehydrogenase 1 (ND1), ubiquinol cytochrome c reductase core protein 1, and ATP synthase mitochondrial F1 complex assembly factor 1 was reduced, which enhanced the activities of the mitochondrial respiratory chain activity and ATP production. We conclude that exogenous H₂S plays a critical role in improving cardiac mitochondrial function in diabetes by upregulating SIRT3.

INTRODUCTION

The worldwide incidence of type 2 diabetes mellitus is growing because of the adoption of Western-style diets and sedentary lifestyles. Diabetic cardiomyopathy (DCM) is one of the major complications of type 2 diabetes mellitus patients (22, 28). DCM is associated with changes in contractility without vascular diseases (8). An increase in free radical production and bioenergetic deficiency in mitochondria are pathological hallmarks of diabetic hearts (25, 40). Mitochondrial dysfunction generates the metabolic syndrome. The major pathophysiological changes during type 2 diabetes are reduced adenosine triphosphate (ATP) synthesis and mitochondrial mass, altered mitochondrial morphology, and increased reactive oxygen species (3). The mitochondrion plays a critical role as a platform for energy transduction, signaling, and cell death pathways related to common cardiovascular diseases such as heart failure (15). Recent evidence suggests that cardiac dysfunction in diabetic patients is linked to metabolic abnormalities and more often associated with mitochondrial dysfunction. However, the mechanisms underlying mitochondrial dysfunction and how it promotes the development of DCM are unknown.

Posttranslational modifications (PTMs), such as protein lysine acetylation, regulate a variety of physiological processes including enzyme activity, protein-protein interaction, gene expression, and subcellular localization (20, 26, 35, 41). Large-scale proteomic investigations have confirmed lysine acetylation as an important metabolic regulatory mechanism in mitochondria. The enzymes of the electron transport chain, Krebs cycle, and fatty acid oxidation are all targets of this modification (6, 13–14, 17, 35). Protein acetylation is regulated by sirtuins (SIRTs) that belong to a family of nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases. SIRTs are considered as “metabolic sensors” (11). SIRT3 is the major mitochondrial deacetylase of lysine residues. SIRT3 is highly expressed in tissues with high metabolic turnover, including those in the heart. SIRT3 has been shown to protect cardiomyocytes from stress-mediated cell death and preserve contractile function in response to a chronic increase in workload (38). However, whether SIRT3 is involved in recovering metabolic energy is still unclear.

Hydrogen sulfide (H₂S) is an endogenous gas with regulatory roles in cardiovascular function, neurotransmission, oxidative balance, and cell metabolism (10, 23, 33, 49). In mammalian tissues, the biosynthesis of H₂S is catalyzed by pyridoxal-5-phosphate-dependent enzymes, including cystathionine-β-synthetase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). The biosynthesis of H₂S in the cardiovascular system is mainly catalyzed by CSE (39). Some studies have demonstrated that H₂S modulates histone acetylation through its direct reduction of histone deacetylase (HDAC) activity in Tamm-Horsfall protein 1 (THP-1) cells (31). Xin et al. also discovered that H₂S stabilized the SIRT1-STAT3 interaction and ameliorated STAT3 acetylation, which in turn suppressed STAT3 activation in human Huh7 hepatoma cells (43). These data suggested that H₂S could regulate cellular metabolic processes through altering protein acetylation. The present study was performed to investigate mitochondrial functions in db/db mice, an established model of type 2 diabetes. Our goal was to identify the underlying molecular changes that contribute to DCM and how H₂S ameliorates energetic metabolism and its mechanisms.

MATERIALS AND METHODS

Materials

Diabetes model and treatment protocols.

Homozygous male and female 10-wk-old db/db mice on a C57BL/6 background (n = 50) and their corresponding wild-type (n = 30) littermates were used in this study. All mice were provided by the Animal Laboratory Center of Nanjing University. Animals were housed in a climate- and temperature-controlled room on a 12:12-h light-dark cycle. The db/db mice have a homozygous point mutation in the db/db gene encoding for the leptin receptor, forming a type 2 diabetic mouse model. The mice were maintained on a standard diet and water ad libitum. Half of the db/db mice and wild-type mice were placed in the sodium hydrosulfide (NaHS) treatment group and treated with NaHS (80 μmol/kg) by intraperitoneal injection every 2 days for 12 weeks. All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the China National Institutes of Health and approved by the Animal Care Committees of Harbin Medical University (Harbin, China).

Neonatal rat cardiomyocyte culture and treatment.

Neonatal rat cardiomyocytes were prepared from 2- to 3-day-old neonatal Wistar rats (Animal Research Institute of Harbin Medical University). The rats were anesthetized by immersion into 75% (vol/vol) alcohol. The hearts were removed and washed three times in D-Hanks balanced salt solution (in mM: 0.4 KCl, 0.06 KH₂PO₄, 8.0 NaCl, 0.35 NaHCO₃, and 0.06 Na₂HPO₄ 7H₂O, pH 7.2) at 4°C. Then, the hearts were cut into pieces <1 mm³ and incubated with 0.25% trypsin for 8 min at 37°C. To terminate the digestion, an equal volume of cold DMEM containing 10% (vol/vol) fetal bovine serum was added. The first digestive fluid was discarded. The latter digestion step was repeated four times. The supernatant cells were collected and then isolated by centrifugation for 10 min at 2,000 g at room temperature. The cells were resuspended in DMEM containing 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin and cultured in a humidified atmosphere containing 5% CO₂ at 37°C. After incubation for 1 h at 37°C, the attached cells were discarded and the unattached cells were cultured in new medium. The medium was replaced every 2 days.

Cellular Experimental Protocol

The cultured neonatal rat cardiomyocytes were randomly divided into groups as follows 1): control (low glucose, 5.5 mM); 2) control + trichostatin A (TSA) + nicotinamide (NAM); 3) oleate + palmitate (high glucose, 40 mM oleate, 200 μM, palmitate, 200 μM); 4) oleate + palmitate + NaHS (100 μM NaHS); and 5) oleate + palmitate + TSA + NAM. All the groups were treated for 48 h. Cells were treated with TSA (1 μM) and NAM (5 mM) for 16 and 8 h, respectively, before harvest.

Echocardiography Analysis

Transthoracic echocardiography was performed in the experimental mice using an ultrasound machine (Vivid 7GE Medical) with a 10-MHz phased array transducer to measure left ventricular (LV) function. Echocardiography was performed on self-breathing mice under anesthesia (intraperitoneal injection of 0.06 mL of 1% pentobarbital sodium/10 g body weight). The following LV parameters were measured: LV mass, LV end-diastolic volume, and ejection fraction (EF). Each parameter was calculated using M-mode view.

Measurement of Serum and Cardiac H₂S Levels

The measurement of H₂S production in serum and isolated cardiac tissues followed an established protocol (36, 42, 46). Briefly, serum was mixed with 10% trichloroacetic acid. The reaction was stopped by the addition of 1% zinc acetate, followed by incubation with N,N-dimethyl-p-phenylenediamine sulfate for 15 min. The absorbance at 670 nm was measured with a spectrophotometer (Beckman).

Detection of H₂S by 7-Azido-4-Methylcoumarin

The fluorescence response of H₂S in cardiomyocytes was detected using 7-azido-4-methylcoumarin (C-7Az; Sigma), which selectively responds to H₂S (5). Cardiomyocytes were incubated with 50 μmol/l C-7Az PBS for 30 min, and then the cells were washed with PBS. The fluorescence activation response of C-7Az to H₂S in cardiomyocytes was visualized using Zeiss LSM 510 inverted confocal microscopy and the excitation of a 720-nm laser.

Glucose Tolerance Tests

Mice were intraperitoneally injected with d-glucose (2 g/kg mass). Tail blood was collected, and blood glucose was determined using a glucometer.

ATP Production Analysis

ATP production was measured using an ATP Bioluminescence Assay Kit (Sigma) based on the luciferase-catalyzed oxidation of d-luciferin.

Mitochondria Isolation

Heart tissues (n = 6, per group) and cardiomyocytes were washed twice with ice-cold PBS, resuspended in lysis buffer (in mM: 20 HEPES/KOH pH 7.5, 10 KCl, 1.5 MgCl₂, 1.0 sodium EDTA, and 1.0 sodium EGTA, 1.0 dithiothreitol, 0.1 PMSF, and 250 sucrose), and then homogenized by a homogenizer in ice/water. After removal of the nuclei and cellular debris by centrifugation at 1,000 g for 10 min at 4°C, the supernatants were further centrifuged at 10,000 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.

Measurement of Mitochondrial Oxygen Consumption and the ADP/O Ratio

Mitochondrial oxygen consumption was measured using a Clark-type oxygen electrode (Hansatech Instruments, Norfolk, UK) at 30°C in mitochondrial respiration buffer (125 mM KCl, 5 mM K₂HPO₄, 20 mM MOPS, 2.5 mM EGTA, 1 μM Na₄P₂O₇, and 0.1% bovine serum albumin, pH 7.4) (30). Pyruvate (5 mM) and malate (5 mM) were used as substrates for complex I-containing mitochondria at a final concentration of 500 μg protein/ml. ADP-stimulated oxygen consumption (state 3 respiration) was measured in the presence of 200 μM ADP and ADP-independent oxygen consumption (state 4 respiration) was also monitored. The respiratory control ratio (RCR; state 3 respiration divided by state 4 respiration) reflects oxygen consumption by phosphorylation (coupling). The ADP/O ratio (number of ADP molecules produced for each oxygen atom consumed) is an index of oxidative phosphorylation efficiency.

Mitochondrial Complex Enzyme Activities

Complex I, complex III, and complex V enzyme activities were measured by using a spectrophotometer (GENMED, Shanghai, China). All assays were conducted according to the kit instructions.

SIRT3 Activity Analysis

The activity of SIRT3 was assessed with a Deacetylase Fluorometric Assay kits (cat. no. CY-1153V2; CycLex). Briefly, cardiac mitochondrial tissue samples (50 mg) were homogenized in 500 μl of immunoprecipitation buffer. After the immunoprecipitation of SIRT3, the final reaction mixtures (50 μl) contained 50 mM Tris-HCl (pH 8.8), 4 mM MgCl₂, 0.5 mM dithiothreitol, 0.25 M lysyl endopeptidase, 1 mM TSA, 200 μM NAD⁺, and 5 μl of extraction buffer. Fluorescence intensity was measured at 350/450 nm. Activity was presented as a relative value compared with the activity of the control group.

Acetyl-coenzyme A Analysis

Acetyl-coenzyme A was measured according to the manufacturer’s protocol (acetyl-coenzyme A assay kit; Sigma). The acetyl-coenzyme A concentration was determined by a coupled enzyme assay that produced a fluorometric product at 535-nm excitation and 587-nm emission wavelengths.

Analysis of the Level of NADs

NAD⁺ and NADH were measured in whole heart homogenates or isolated mitochondria according to the manufacturer’s protocol (NAD⁺/NADH quantification kit; Sigma) by measuring absorbance at 450 nm and comparison to a NADH standard curve. The samples were divided to measure NAD⁺_total and NADH_only separately. To detect NADH_only, NAD⁺ was decomposed by incubating the sample for 30 min at 60°C. The levels of NAD⁺_only were calculated as difference of NAD⁺_total − NADH_only.

Kac Peptide Enrichment and High-Performance Liquid Chromatography with Tandem Mass Spectrometry Analysis

To identify the lysine acetylome of three cardiac tissues each from db/db mice and those treated with NaHS, their extracted proteins were digested with proteases and then subjected to Kac peptide enrichment and high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) analysis using a previously described method (37).

Western Blotting

All mitochondria and cytoplasm were analyzed for total protein content (BCA protein assay). Samples were separated by electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose filter membranes. The antibodies used for Western blot analysis included anti-acetylated lysine (ICP0380; ImmuneChem), anti-VDAC1 (55259-1-AP; Proteintech), anti-SIRT3 (10099-1-AP; Proteintech), anti- ubiquinol cytochrome c reductase core protein 1 (UQCRQ; 14975-1-AP; Proteintech), anti-NADH dehydrogenase 1 (ND1; 19703-1-AP; Proteintech), anti-ATP synthase mitochondrial F1 complex assembly factor 1 (ATPAF1; 18016-1-AP; Proteintech), anti-CSE (12217-1-AP; Proteintech), anti-CBS (14787-1-AP; Proteintech), anti-Bax (50599-2-Ig; Proteintech), anti-Bcl2 (12789-1-AP; Proteintech), and anti-GAPDH (10494-1-AP; Proteintech). Protein complexes were incubated for 1 h at room temperature with anti-mouse/anti-rabbit IgG antibody and then detected by enhanced chemiluminescence reagent (Beyotime, China). Densitometry was conducted with the image processing and analysis program AlphaView.SA, and the data are expressed as relative units.

Immunoprecipitation Analysis

Briefly, isolated mitochondria were resuspended in PBS and diluted to a concentration of 1 mg/ml. After three freeze-thaw cycles, a total of 500 μg of protein was used per sample for immunoprecipitation. Sepharose beads were conjugated with the anti-acetylated lysine antibody (10 μg antibody/500 μg protein; ImmuneChem Pharmaceuticals) and incubated with the samples overnight at 4°C with gentle rotation. Following collection of the beads using a centrifuge and three washing steps, the precipitates were subjected to Western blotting analyses for the detection of potential interacting proteins. The acetylated protein levels were normalized to the corresponding mitochondrial protein levels, which were measured by conventional immunoblotting of the protein from isolated mitochondria.

siRNA Transfection

The H9c2 cells (80% confluent) were treated with SIRT3 short interfering RNAs (siRNAs; sc-61556; Santa Cruz Biotechnology) according to the manufacturer’s instructions for 48 h to inhibit SIRT3 expression. H9c2 cells were transfected by siRNA using Lipofectamine 3000 (Invitrogen). Briefly, SIRT3-siRNA and the transfection reagent were incubated for 20 min to form complexes that were then added to plates containing cells and medium. The cells were incubated at 37°C in a CO₂ incubator for further analysis.

Statistical Analysis

Statistics were determined using a two-tailed unpaired Student’s t-test or one-way ANOVA using GraphPad Prism software (GraphPad, La Jolla, CA). The final results were calculated as the means ± SE, unless otherwise indicated. Statistical significance is indicated in the figures by P < 0.05, P < 0.01, and P < 0.001.

RESULTS

Exogenous H₂S Ameliorated Cardiac Function in db/db Mice

Our previous study showed that exogenous H₂S can improve cardiac functions in streptozotocin-induced type I diabetes (48). Ten-week-old db/db mice and wild-type mice were intraperitoneally injected with 80 μmol/kg NaHS every 2 days for 12 weeks. We examined plasma glucose levels, glucose intolerance, plasma insulin levels, plasma triglyceride levels, and free fatty acid levels in db/db mice, recapitulating the hallmark features of type 2 diabetes (Fig. 1, A and B, and Table 1).

Fig. 1.

General indexes were detected. A: glucose was measured in 10- to 22-wk-old db/db mice, control mice, control-sodium hydroxide (NaHS) mice, and db/db-NaHS mice (n = 6). B: intraperitoneal glucose tolerance test on four groups of mice injected with 2 g glucose/kg (n = 6). C: body weight. D: plasma insulin content. E: the myocardial ultrastructure in 22-wk-old control mice, control-NaHS mice, db/db mice, and db/db-NaHS mice was observed using a transmission electron microscope. Values are means ± SE; n = 5. NS, not significant. ***P < 0.001 vs. control; ##P < 0.01 vs. db/db.

Table 1.

Plasma insulin, free fatty acid, and triglyceride

	Control	Control + NaHS	db/db	db/db + NaHS
Plasma free fatty acid, μg/l	760.46 ± 38.28	750.93 ± 39.40	883.13 ± 64.01*	697.8 ± 67.02#
Plasma triglyceride, mEq/l	0.31 ± 0.082	0.32 ± 0.12	1.17 ± 0.11***	1.11 ± 0.11
Plasma insulin, ng/ml	0.47 ± 0.36	0.35 ± 0.14	5.54 ± 1.13***	3.53 ± 0.55##
Ejection fraction, %	81.26 ± 7.15	80.57 ± 7.26	65.23 ± 4.05***	77.54 ± 4.98##
Left ventricular end-diastolic volume, μl	43.62 ± 7.61	44.45 ± 7.82	29.83 ± 4.89**	41.51 ± 6.32##
Left ventricular mass, mg	74.34 ± 10.47	76.33 ± 5.91	128.43 ± 19.50***	104.42 ± 19.3#

Values are means ± SE. Plasma insulin, free fatty acid and triglyceride were measured in three groups. Analysis of ejection fraction, left ventricular mass, and left ventricular end-diastolic volume by echocardiography (n = 6). NaHS, sodium hydroxide. Data were compared by two-way ANOVA.

^*P < 0.05 vs. control;

^**P < 0.01 vs. control;

^***P < 0.001 vs. control;

^#P < 0.05 vs. db/db;

^##P < 0.01 vs. db/db.

We found that there were no differences in the animal body weight and plasma insulin levels between male and female in db/db mice (Fig. 1, C and D). To determine whether genders affects the protection of the heart by exogenous H₂S in type 2 diabetes, we examined cardiac morphology by transmission electron microscopy; the protective effect of exogenous H₂S on cardiac morphology was no different between male and female (Fig. 1E). NaHS treatment ameliorated cardiac systolic and diastolic dysfunction in the db/db mice. We observed that EF and LV end-diastolic volume were increased and LV mass was decreased in the NaHS group compared with those in 22-wk-old db/db mice (Table 1). We also found that cardiac functions in wild-type mice were not influenced by treatment with NaHS.

H₂S level and CSE expression in cardiac tissues of db/db mice

We assessed the effects of hyperglycemia and hyperlipidemia on H₂S production in cardiac tissues of db/db mice; the expression of CSE, the H₂S-producing enzyme, was significantly reduced in db/db mice compared with the control group (Fig. 2A). With NaHS treatment, the expression of CSE was increased in db/db cardiac tissues. There was also no significant difference in CBS expression between the db/db and control groups (Fig. 2B). Endogenous H₂S levels in serum and hearts of db/db mice were both decreased compared with those in control mice and db/db mice treated with NaHS (Fig. 2, C and D). However, there was no difference in endogenous H₂S levels between genders (Fig. 2D).

Fig. 2.

Hydrogen sulfide (H₂S) content in the db/db mice and wild-type mice was intraperitoneally injected with 80 μmol/kg sodium hydroxide (NaHS) every 2 days for 12 wk was detected. A and B: cystathionine-γ-lyase (CSE) expression (A) and cystathionine-β-synthetase (CBS) expression (B) in cardiac tissues were tested by Western blotting. C: the serum H₂S level in 22-wk-old control mice, control-NaHS mice db/db mice, and db/db-NaHS mice was measured. D: cardiac H₂S levels in male and female groups in 22-wk-old control mice, control-NaHS mice, db/db mice, and db/db-NaHS mice were measured. Values are means ±SE; n = 5. NS, not significant. **P < 0.01 vs. control; ***P < 0.001 vs. control; #P < 0.05 vs. db/db; ##P < 0.01 vs. db/db; ###P < 0.001 vs. db/db.

Effect of NaHS on Mitochondrial Respiration in db/db Mice Hearts

DCM is correlated with reduced ATP synthesis and mitochondrial dysfunction (29). Therefore, we measured mitochondrial respiratory functions, including state 3 and 4 respiration, the RCR, and the ADP/O ratio. We observed that state 3 respiration, the RCR, and the ADP/O ratio in mitochondria were significantly decreased in db/db mice compared with those in mice treated with the NaHS and control groups (Fig. 3, A–D). To further demonstrate that exogenous H₂S could impact on mitochondrial respiratory chain activities, we tested the activity of complex I, complex III, and complex V. The activities of these complexes were increased in the db/db-NaHS group compared with those in the db/db mice (Fig. 3, E–G). The ATP level was increased in cardiac tissues treated with NaHS in the db/db mice (Fig. 3H). Cardiac mitochondrial respiratory functions in wild-type mice were not influenced by treatment of NaHS or by gender (Fig. 3, E–H). These results show that exogenous H₂S can improve mitochondrial respiratory functions in db/db mice.

Fig. 3.

Mitochondrial oxidative phosphorylation efficiency was evaluated in the isolated cardiac mitochondria of db/db mice. A–D: they were based on mitochondrial state 3 (A) and 4 (B) oxygen consumption, the respiratory control rate (RCR; C), and the ADP/O ratio (D). E–H: activities of mitochondrial respiration chain complexes I (E), III (F), and V (G) and cardiac ATP content (H) were measured in 22-wk-old male and female mice including control mice, control-sodium hydroxide (NaHS) mice, db/db mice, and db/db-NaHS mice. Values are means ± SE of 5 experiments; n = 5. NS, not significant. *P < 0.05 vs. control; **P < 0.01 vs. control; #P < 0.05 vs. db/db; ##P < 0.01 vs. db/db.

Cluster Analysis of Quantiles-Based Clustering for Protein Groups Involved in the Mitochondrial Respiratory Chain in db/db Cardiac Tissues

To determine the effect of exogenous H₂S on the regulation of acetylation in cardiac tissues of db/db mice, we used LC-MS/MS proteomic analysis to detect protein lysine acetylation. Our previous studies found that the acetylation level was obviously increased in db/db cardiac tissues; however, treatment with NaHS could decrease the cardiac acetylation level (37). First, proteins quantified in LC-MS/MS proteomic analysis were divided into four quantiles according to the quantification ratio to generated four quantiles: Q1 (0~25%), Q2 (25~50%), Q3 (50~75%), and Q4 (75~100%). Then, quantile-based clustering was performed (Fig. 4A).

Fig. 4.

Liquid chromatography with tandem mass spectrometry analyzed the alteration in the acetylation level in db/db mice cardiac tissues. A: distribution of quantified results. Functional enrichment-based clustering analysis of the quantified acetylome data sets from db/db mice and db/db mice treated with sodium hydroxide (NaHS). Q1–Q4, quantile 1 (0~25%), quatile 2 (25~50%), quantile 3 (50~75%), and quantile 4 (75~100%). B: cellular component-based clustering analysis. C: molecular function-based clustering analysis. D: biological process-based clustering analysis. E: protein domain-based clustering analysis was identified by bioinformatic analysis.

In the cellular component analysis, mitochondrial respiratory chain complexes and ATP synthase complex proteins were among the proteins upregulated in db/db cardiac tissues compared with the cardiac tissues of mice in the NaHS treatment group (Fig. 4B). Molecular function-based clustering results were shown in Fig. 4C. Proteins involved in ATPase activity, oxidoreductase activity, electron carrier activity, and cytochrome c oxidase activity were among those upregulated and enriched in the cardiac tissue of db/db mice compared with db/db mice treated with NaHS. To identify the cellular pathways and the protein complex related the treatment of db/db mice with NaHS, clustering analysis based on biological process and protein domain was performed. Pathways related to the ATP metabolic process, aerobic respiration, electron transport chain, and the regulation of ATPase activity were enriched in the upregulated proteins in the hearts of db/db mice and those in the hearts of mice treated with NaHS (Fig. 4, D and E). In conclusion, gene ontology enrichment-based cluster analysis indicated that more targets of the increased acetylation were involved in mitochondrial ATP production and that mitochondrial acetylation may contribute to the impairment of cardiac energy metabolism in db/db mice.

Protein Lysine Acetylation Was Elevated in Cardiac Tissues of db/db Mice

Type 2 diabetes is characterized by hyperglycemia and the production of glycated proteins (16). In addition to glycation, the role of other types of PTMs in type 2 diabetes remains to be revealed. In fact, elevated acetyl-CoA levels have been found in type 2 diabetic patients and rats (25). To verify the LC-MS/MS results, we detected the lysine acetylation level of cardiac mitochondria and observed a marked elevation in the lysine acetylation level in db/db mice relative to that in db/db mice treated with NaHS (Fig. 5A). We also tested the level of acetyl-CoA in the cardiac mitochondria of db/db mice, which was significantly increased compared with that in the control and the NaHS treatment groups (Fig. 5B). Treatment with NaHS did not affect the acetylation level of cardiac mitochondrial proteins in wild-type mice. We also found that the acetyl-CoA content in cardiac mitochondria was independent of gender (Fig. 5B). The lysine acetylation levels of the ND1, UQCRQ, and ATPAF1 proteins were elevated in db/db mice hearts compared with those in the hearts of control and db/db mice treated with NaHS (Fig. 5C). These changes were consistent with the LC-MS/MS results.

Fig. 5.

Detection of lysine acetylation level in cardiac tissues of db/db mice and db/db mice treated with sodium hydroxide (NaHS). A: cardiac mitochondrial acetylation levels were detected by Western blot analysis. B: cardiac mitochondrial acetyl-CoA content was measured in male and female group in 22-wk-old control mice, control-NaHS mice, db/db mice, and db/db-NaHS mice. C: cardiac mitochondrial lysates were immunoprecipitated (IP) with anti-acetylated lysine antibody and then immunoblotted (IB) with antibodies specific for NADH dehydrogenase 1 (ND1), ubiquinol cytochrome c reductase core protein 1 (UQCRQ), and ATP synthase mitochondrial F1 complex assembly factor 1 (ATPAF1). All values are presented as the means ± SE; n = 4. NS, not significant. **P < 0.01 vs. control; #P < 0.05 vs. db/db; ##P < 0.01 vs. db/db.

Hyperglycemia and Hyperlipidemia Reduced SIRT3 Expression and the NAD Pool in the Cardiac Mitochondria of db/db Mice

SIRT3 is in a family of NAD⁺-dependent protein deacetylases that regulates important biological pathways in eubacteria, archaea, and eukaryotes (9). SIRT3 catalyzes a deacetylation reaction that uses NAD⁺ as a cofactor and exhibits deacetylase activity (32). The expression and activity of SIRT3 in cardiac tissues were significantly reduced in the isolated mitochondria of db/db mice compared with those in the control group. The administration of exogenous H₂S for 12 wk restored SIRT3 expression and activity in the isolated cardiac mitochondria of db/db mice (Fig. 6, A and B). The expression and activity of SIRT3 were not influenced in wild-type mice by treatment with NaHS or by gender (Fig. 6B). Exogenous H₂S restored the NAD pool and elevated the NAD⁺/NADH ratio in cardiac tissues, while the NAD⁺ content and the NAD⁺/NADH ratio in the cardiac tissues of db/db mice were decreased compared with those in the control group (Fig. 6, C and D). Treatment with NaHS did not alter the content of NAD⁺ or the NAD⁺/NADH ratio in the cardiac tissues of wild-type mice. To demonstrate whether SIRT3 regulated the acetylation level of ND1, UQCRQ, and ATPAF1, immunoprecipitation was performed and the interactions of ND1, UQCRQ, and ATPAF1 with SIRT3 were all decreased in db/db mice, whereas the administration of exogenous NaHS increased the interactions between SIRT3 and ND1, UQCRQ, and ATPAF1, respectively (Fig. 6E). Exogenous H₂S reduced the acetylation level of the mitochondrial respiratory chain by activating SIRT3 in db/db mice.

Fig. 6.

Sirtuin 3 (SIRT3) expression and the NAD⁺/NADH ratio in the isolated mitochondria from the heart of db/db mice and db/db mice treated with sodium hydroxide (NaHS). A: the expression of SIRT3 in the isolated mitochondria of mice hearts. B: cardiac SIRT3 activity in male and female group in 22-wk-old control mice, control-NaHS mice db/db mice, and db/db-NaHS mice. C and D: level of NAD⁺ (C) and NAD⁺/NADH ratio (D) in isolated cardiac mitochondria. E: cardiac mitochondrial proteins were immunoprecipitated (IP) with SIRT3 and then incubated with antibodies specific for NADH dehydrogenase 1 (ND1), ubiquinol cytochrome c reductase core protein 1 (UQCRQ), and ATP synthase mitochondrial F1 complex assembly factor 1 (ATPAF1). IB, immunoblot. Values are means ± SE; n = 5. NS, not significant. *P < 0.05 vs. control; **P < 0.01 vs. control; #P < 0.05 vs. db/db; ##P < 0.01 vs. db/db.

Glucose Induced Injury in Neonatal Rat Cardiomyocytes

Neonatal rat cardiomyocytes treated with high glucose, oleate, and palmitate were used to imitate the conditions of hyperglycemia and hyperlipidemia in vivo. Previous studies have been reported that a dose of 40 mM glucose mimics hyperglycemia (7, 47). High glucose can generate oxidative stress in the body to induce cell injury. We examined the protein levels of Bax and Bcl-2 in neonatal rat cardiomyocytes. Western blotting indicated that high glucose (40 mM) significantly decreased basal levels of Bcl-2 but increased Bax protein expression (Fig. 7A). The expression levels of Bax and Bcl-2 were no different between the 5.5 mM glucose group and the 25 mM glucose group. Meanwhile, cardiomyocytes treated with 25 mM high osmotic conditions. Neonatal rat cardiomyocytes in 40 mM glucose were more sensitive to injury than those in 25 mM glucose.

Fig. 7.

Effect of exogenous hydrogen sulfide (H₂S) on regulating the mitochondrial respiratory chain in neonatal rat cardiomyocytes under hyperglycemia and hyperlipidemia. A: expression levels of Bax and Bcl2 in neonatal rat cardiomyocytes were detected by Western blotting; n = 4. **P < 0.01. B: mitochondrial acetylation level in neonatal rat cardiomyocytes treated with trichostatin A (TSA) and nicotinamide (NAM). C: level of acetyl-CoA level in mitochondria of neonatal rat cardiomyocytes. D: mitochondrial lysates were immunoprecipitated (IP) with anti-acetylated lysine antibody and then immunoblotted (IB) with antibodies specific for NADH dehydrogenase 1 (ND1), ubiquinol cytochrome c reductase core protein 1 (UQCRQ), and ATP synthase mitochondrial F1 complex assembly factor 1 (ATPAF1) in neonatal rat cardiomyocytes. E–G: activities of the mitochondrial respiration chain complexes I (E), III (F), and V (G). H: level of ATP in neonatal rat cardiomyocytes. Values are the means ± SE; n = 5. *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.001 vs. control, #P < 0.05 vs. high glucose (HG) + oleate (Ole) + palmitate (Pal); ###P < 0.001 vs. HG + Ole + Pal; &P < 0.05 vs. HG + Ole + Pal+ sodium hydroxide (NaHS); &&P < 0.01 vs. HG + Ole + Pal + NaHS; &&&P < 0.001 vs. HG + Ole + Pal + NaHS.

Impact of Lysine Acetylation on Enzymatic Activities of the Electron Transport Chain in Neonatal Rat Cardiomyocytes under Hyperglycemia and Hyperlipidemia

To identify acetylation targets that may contribute to the impairment of cardiac ATP production, cells were treated with TSA (an inhibitor of the histone deacetylases HDAC I and HDAC II) and NAM (an inhibitor of the SIRT family deacetylases). Treatment with TSA and NAM increased mitochondrial acetylation level and the acetyl-CoA level in neonatal rat cardiomyocytes under hyperglycemia and hyperlipidemia (Fig. 7, B and C). The levels of acetylated ND1, UQCRQ, and ATPAF1 were obviously increased in the high-glucose + oleate + palmitate group and after treatment with TSA and NAM (Fig. 7D). To validate the biological role of lysine acetylation, we found that acetylation can occur on the key residues of several enzymes. We detected the activities of mitochondrial complexes I, III, and V, which were significantly reduced in neonatal rat cardiomyocytes under hyperglycemia and hyperlipidemia and in those treated with treatment of TSA and NAM compared with those in cardiomyocytes treated with NaHS and the control group (Fig. 7, E–G). We also found that treatment with TSA and NAM decreased ATP production in neonatal rat cardiomyocytes under hyperglycemia and hyperlipidemia (Fig. 7H).

To further investigate whether SIRT3 regulates deacetylase activity in mitochondria, we found that neonatal rat cardiomyocytes treated with high glucose, oleate, and palmitate showed increased mitochondrial acetylation level, while the expression of SIRT3 decreased (Fig. 8A). The ratio of NAD⁺/NADH determines SIRT3 activity (44). We found that treatment with high glucose, oleate, and palmitate could decrease the ratio of NAD⁺/NADH and the activity of SIRT3 (Fig. 8, B and C) and that the effect of high glucose, oleate, and palmitate on mitochondrial acetylation was the same as that of individual TSA and NAM treatment. In addition, treatment with exogenous H₂S could elevate the NAD⁺/NADH ratio and the activity of SIRT3. In accordance with the in vivo data, the interactions between SIRT3 and ND1, UQCRQ, and ATPAF1 were decreased in the high-glucose + oleate + palmitate group and the TSA and NAM treatment group, whereas the administration of exogenous NaHS increased the interaction between SIRT3 and ND1, UQCRQ, and ATPAF1 (Fig. 8D).

Fig. 8.

Effect of exogenous hydrogen sulfide (H₂S) on regulating sirtuin 3 (SIRT3) expression and NAD⁺/NADH ratio in neonatal rat cardiomyocytes under hyperglycemia and hyperlipidemia. A: expression of SIRT3 in neonatal rat cardiomyocytes treated with trichostatin A (TSA) and nicotinamide (NAM). B: NAD⁺/NADH ratio in the isolated mitochondria of neonatal rat cardiomyocytes. C: activity of SIRT3 in isolated mitochondria of neonatal rat cardiomyocytes. D: mitochondrial proteins were immunoprecipitated (IP) with SIRT3 and then incubated with antibodies recognizing NADH dehydrogenase 1 (ND1), ubiquinol cytochrome c reductase core protein 1 (UQCRQ), and ATP synthase mitochondrial F1 complex assembly factor 1 (ATPAF1). The IgG sample of UQCRQ came from a different blot as negative control. IB, immunoblot. Values are the means ± SE; n = 5. **P < 0.01 vs. control; ***P < 0.001 vs. control; ##P < 0.01 vs. high glucose (HG) + oleate (Ole) + palmitate (Pal); &&&P < 0.001 vs. HG + Ole + Pal + sodium hydroxide (NaHS).

siRNA-Mediated Silencing of SIRT3 Increased the Acetylation Level of the Mitochondrial Electron Transport Chain

To further investigate the putative role of SIRT3 in regulating the acetylation of enzymes in the mitochondrial electron transport chain, we knocked down SIRT3 expression in H9c2 cells with siRNA. To determine whether SIRT3 knockdown had any effect on H₂S-producing enzymes and H₂S production, we measured the H₂S production and the CSE expression following SIRT3 knockdown. The H₂S probe C-7Az was used to test the H₂S content in H9c2 cells under SIRT3 knockdown conditions. The H₂S content was significantly decreased in the high-glucose + oleate + palmitate group, and recovered by treatment with NaHS (Fig. 9A). We found that H₂S content was not different in the control siRNA group and the SIRT3 siRNA group (Fig. 9B). To further observe the H₂S production following transfection with SIRT3 siRNA, the expression of CSE, the H₂S-producing enzyme, was tested. The CSE expression was not different in the SIRT3 siRNA group and the control siRNA group (Fig. 9C). SIRT3 knockdown had no effect on H₂S production and H₂S-producing enzyme. Transfection with SIRT3 siRNA for 72 h decreased SIRT3 protein expression after transfection (Fig. 10A). Reduced SIRT3 protein level also caused an increase in the levels of acetylated ND1, UQCRQ, and ATPAF1 (Fig. 10B), accompanied by a significant decrease in the activities of the mitochondrial complexes I, III, and V (Fig. 10, C–E).

Fig. 9.

Hydrogen sulfide (H₂S) content following treatment with 40 mM glucose, 200 μM oleate, and 200 μM palmitate was detected. A: fluorescence of H₂S was observed by fluorescence microscopy. B: H9c2 cells were treated with sirtuin 3 (SIRT3) siRNA, and the H₂S level was detected by fluorescence microscope. C: cystathionine-γ-lyase (CSE) level in H9c2 cells under SIRT3 knockdown conditions were determined by Western blotting (n = 5). HG, high glucose; Ole, oleate; Pal, palmitate.

Fig. 10.

Exogenous hydrogen sulfide (H₂S) regulated the mitochondrial respiratory chain by increasing sirtuin 3 (SIRT3). A: expression of SIRT3 after treatment with SIRT3 siRNA. B: mitochondrial lysate from H9c2 treated with SIRT3 siRNA was immunoprecipitated (IP) with anti-acetylated lysine antibody and then immunoblotted (IB) with antibodies specific for NADH dehydrogenase 1 (ND1), ubiquinol cytochrome c reductase core protein 1 (UQCRQ), and ATP synthase mitochondrial F1 complex assembly factor 1 (ATPAF1). C: mitochondrial respiration chain complex I activity. D: mitochondrial respiration chain complex III activity. E: mitochondrial respiration chain complex V activity. Values are means ± SE of 5 experiments. *P < 0.05 vs. control siRNA; **P < 0.01 vs. control siRNA; ***P < 0.001 vs. control siRNA.

DISCUSSION

This study identified that H₂S, a gaseous molecule, plays an important role in lysine acetylation by regulating the mitochondrial respiratory chain and ATP production, which promoted the expression of SIRT3 in the cardiac tissue of db/db mice. Our results indicated that 1) cardiac mitochondrial respiratory capacities were compromised and accompanied by reduced ATP synthesis in db/db cardiac tissues, while cardiac mitochondrial dysfunction was ameliorated by the treatment with NaHS; 2) bioinformatics analysis of the acetylated proteins indicated a role for acetylated proteins in the mitochondrial respiratory chain in db/db cardiac tissues; 3) exogenous H2S restored the NAD⁺ level, increased the expression and activity of SIRT3, and decreased the acetylation levels of the mitochondrial respiratory complex enzymes, including ND1, UQCRQ, and ATPAF1 in cardiac tissues of db/db mice; and 4) SIRT3 deletion induced the hyperacetylation of mitochondrial respiratory complexes, whereas exogenous H₂S partially restored the acetylation level of these enzymes. Collectively, these data demonstrated that H₂S plays a crucial role in regulating the lysine acetylation of mitochondrial respiratory complexes involved in regulating cardiac ATP production by activating the NAD⁺-SIRT3 pathway in type 2 diabetes.

The mitochondrion is an important organelle that maintains energy metabolism and cellular homeostasis. Deficits in mitochondrial function associated with diabetes have been linked to metabolic defects (4, 12). The cardiac demand for energy comes primarily from mitochondrial oxidative phosphorylation, which accounts for 95% of produced ATP (34). However, under a chronic diabetic state, the ability of the heart to switch between available oxidizable substrates is impaired, and under this condition, the heart depends almost exclusively on fatty acid metabolism, which increases mitochondrial damage (33). Some studies have confirmed that hyperacetylation might be an important contributor to the increased oxidative stress associated with the progression in DCM. The acetylation of metabolic enzymes is known to influence their catalytic functions (18, 45). We found that the acetylation of precursor metabolite proteins and proteins involved in energy processes, oxidation-reduction process, and cellular respiration process was enriched in db/db mice.

The constant use of fatty acids is expected to increase acetyl-CoA content and the ratio of NADH/ NAD⁺. Increased acetyl-CoA, the most likely carbon donor for protein lysine acetylation, would promote acetylation (2). In vertebrates, protein acetylation is mainly regulated by NAD⁺-dependent deacetylases of the SIRT family, of which seven members have been identified in mammals (24). An increase in the NADH/NAD⁺ ratio would impair the activity of SIRT3, which uses NAD⁺ as a cofactor. SIRT3–5 are localized in mitochondria and regulate mitochondrial metabolic pathways, such as fatty acid oxidation, the tricarboxylic acid cycle, and the electron transport chain. Our results showed that hyperglycemia and hyperlipidemia led to decrease in the expression and activity of SIRT3 and increased the acetylation of the mitochondrial enzymes ND1, UQCRQ, and ATPAF1. Therefore, a decrease in SIRT3 activity may also contribute to increased acetylation.

H₂S, an endogenous gas, is involved in multiple pathophysiological processes. Our previous study showed that Ca²⁺ stimulation causes increased CSE expression, increases H₂S and ATP production, and improves mitochondrial ATP production following hypoxia (10). Some other groups demonstrated that H₂S stimulates cellular bioenergetics, contributes to the increased reliance of cancer cells on the glycolytic pathway for ATP production, and promotes angiogenesis and cytoprotection (19, 27). Endogenous H₂S production driven by 3-MST complements and balances cellular bioenergetics and maintains electron flow in mitochondria (21). Akakura et al. (1) revealed that in cancer cells a high level of H₂S, aerobic glycolysis and the level of ATP and NAD⁺ were coordinately increased. In this study, we used db/db mice mimic type 2 diabetes mice model. After NaHS administration for 12 weeks, cardiac functions were significantly improved, as demonstrated by increased EF and LV end-diastolic volume and decreased LV mass. The administration of NaHS alone did not cause any damage either cardiac function or mitochondrial respiratory function. We also demonstrated that H₂S plays a crucial role in regulating cardiac functions independent of gender. Our previous findings suggested that the activation of SIRT3 was regulated by S-sulfhydrylation (24). Our results revealed that exogenous H₂S could regulate the expression and activity of SIRT3 by restoring the ratio of NAD⁺/NADH in the cardiac mitochondria of db/db mice; decreasing the levels of acetylated ND1, UQCRQ, and ATPAF1; and recovering their activities. SIRT3 is required to maintain mitochondrial function in the heart and may serve as a redox-sensitive rheostat that regulates ATP-generating metabolic pathways by coordinating changes in the lysine acetylation of various energy metabolic enzymes. Our results showed that exogenous H₂S affected mitochondrial dynamics through the differential modulation of mitochondrial OXPHOS acetylation levels via the NAD⁺-SIRT3 pathway.

Conclusions

Our study confirmed that changes in protein lysine acetylation had significant effects on mitochondrial functions. These acetylated proteins may offer insights into the mechanisms by which mitochondrial respiratory dysfunctions lead to the progression of DCM.

GRANTS

This study was supported by the National Natural Science Foundation of China Grants 81670344 and 81570340, Graduate Innovation Foundation of Harbin Medical University Grant YJSCX2017-7HYD), and Education Department of Heilongjiang Province Grant12531382.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Fanghao Lu and W.Z. conceived and designed research; Y.S., Z.T., X.S., Fanghao Lu, and W.Z. performed experiments; Y.S., Z.T., X.S., L.Z., J.C., B.W., Fangping Lu, N.L., M.Y., S.P., Y.W., D.Z., Y.Z., H.R., Z.C., S.D., Fanghao Lu, and W.Z. approved final version of manuscript; L.Z., J.C., B.W., Fangping Lu, and Z.C. analyzed data; N.L., M.Y., Y.W., D.Z., Y.Z., H.R., and Z.C. interpreted results of experiments; S.P. and D.Z. prepared figures; Y.Z., H.R., and SD edited and revised manuscript.