There is growing evidence that H2S has beneficial effects in treatment of various cardiovascular diseases. However, it remains unclear whether H2S can attenuate the development of diabetic cardiomyopathy (DCM). The present study was designed to investigate the protective effects of H2S against DCM. Diabetic rats were induced by intraperitoneal injection of streptozotocin and administered with the H2S donor sodium hydrosulfide (NaHS) for 16 weeks. Neonatal rat cardiomyocytes (NRCMs) transfected with nuclear factor erythroid 2-related factor 2 (Nrf2)-specific siRNA or pre-treated with SP600125, SB203580 or LY294002 prior to high glucose exposure were used to confirm the involvement of Nrf2/antioxidant response element (ARE), mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K)/Akt signalling pathways in the protective effects of H2S. The echocardiographical and histopathological data indicated that H2S improved left ventricular function and prevented cardiac hypertrophy and myocardial fibrosis in diabetic rats. H2S was also found to attenuate hyperglycaemia-induced inflammation, oxidative stress and apoptosis in the cardiac tissue. In addition, H2S could activate the Nrf2/ARE signalling pathway and up-regulate the expression of antioxidant proteins haem oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) in the diabetic myocardium. Moreover, H2S was found to reduce high glucose-induced apoptosis both in vitro and in vivo by inhibiting c-Jun N-terminal kinase (JNK) and p38 MAPK pathways and activating PI3K/Akt signalling. In conclusion, our study demonstrates that H2S alleviates the development of DCM via attenuation of inflammation, oxidative stress and apoptosis.

CLINICAL PERSPECTIVES

  • There is growing evidence that H2S has beneficial effects in treatment of various cardiovascular diseases. However, it remains unclear whether H2S can attenuate the development of DCM. The present study was designed to investigate the protective effects of H2S against DCM.

  • Our findings indicated that H2S improved left ventricular function and prevented cardiac hypertrophy and myocardial fibrosis in diabetic rats. H2S was also found to reduce high glucose-induced oxidative stress by activating Nrf2/ARE pathway and exert anti-apoptotic effects in diabetic myocardium by inhibiting JNK and p38 MAPK pathways and activating PI3K/Akt signalling.

  • Our study demonstrates that H2S alleviates the development of DCM via attenuation of inflammation, oxidative stress and apoptosis. These findings will provide valuable insights into the application of H2S in prevention and treatment of DCM.

INTRODUCTION

Diabetic cardiomyopathy (DCM), which has been defined as ventricular dysfunction that occurs independently of coronary artery disease and hypertension, carries a substantial risk for the subsequent development of heart failure and increased mortality. DCM is characterized by a variety of morphological changes, including myocyte hypertrophy, myofibril depletion, interstitial fibrosis and intramyocardial microangiopathy. Previous studies have demonstrated that hyperglycaemia, lipid accumulation, increased oxidative stress, impaired calcium handling, mitochondrial dysfunction, renin–angiotensin system activation, myocardial inflammation and apoptosis are probably involved in the pathogenesis of DCM [13].

H2S, the third gaseous signalling molecule identified after nitric oxide and carbon monoxide, is endogenously generated from cysteine by the pyridoxal-5′-phosphate-dependent enzymes, including cystathionine β-synthase and cystathionine γ-lyase [4]. In the recent years, accumulating evidence has demonstrated that H2S plays critical roles in the physiology and pathophysiology of cardiovascular system [5,6]. H2S attenuates myocardial ischaemia-reperfusion injury by preservation of mitochondrial function and reduces the morbidity and mortality associated with ischaemia-induced heart failure [7,8]. Decreased endogenous production of H2S predisposes to vascular remodelling and early development of atherosclerosis [9]. In the present study, we established a streptozocin-induced diabetic rat model to investigate whether H2S has protective effects against DCM.

MATERIALS AND METHODS

Animal model and grouping

All experiments and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Soochow University. Male Sprague–Dawley rats, weighing 200–250 g, were obtained from the Experimental Animal Center of Soochow University and were housed in a room at 22±2°C and 50±5% relative humidity with an alternating 12-h light/dark cycle. The diabetic rat model was induced with a single intraperitoneal injection of streptozotocin [65 mg/kg dissolved in 0.1 M citrate buffer (Sigma)]. Blood glucose levels were measured 3 and 5 days after streptozotocin injection using a hand-held glucometer (Accu-Chek, Roche Applied Science) by tail vein puncture blood sampling. Only rats with blood glucose levels ≥16.7 mM on both days were defined as diabetic and used in the present study. Diabetic rats were then randomly devided into two groups. One group was intraperitoneally administered H2S donor sodium hydrosulfide (NaHS) at a dose of 14 μmol/kg per day for 16 weeks [diabetes mellitus (DM) + NaHS group]. The other group was intraperitoneally injected with an equivalent volume of physiological saline for 16 weeks (DM group). In addition, two groups of sex- and age-matched normal rats were intraperitoneally injected with NaHS solution (NaHS group) or physiological saline (control group).

Cell culture and treatment

Primary cultures of neonatal rat cardiomyocytes (NRCMs) were prepared as previously described [10]. To investigate the anti-apoptosis effect of H2S, NRCMs were administered 100 μM NaHS for 30 min prior to exposure to 30 mM glucose for 24 h. To further determine whether H2S reduces high glucose-induced cardiomyocyte apoptosis by inhibiting mitogen-activated protein kinase (MAPK) signalling and activating PI3K (phosphoinositide 3-kinase)/Akt pathway, NRCMs were pre-conditioned with 10 μM SP600125 [c-Jun N-terminal kinase (JNK) inhibitor], 20 μM SB203580 (p38 inhibitor) or 10 μM LY294002 (PI3K inhibitor) for 60 min, followed by exposure to high glucose. SP600125, SB203580 and LY294002 were purchased from Cell Signaling.

Measurement of H2S content

After 16 weeks of streptozotocin injection, the plasma and myocardial levels of H2S were determined by the Methylene Blue method, as described previously [11]. This method is based on the reaction of sulfide with N,N-dimethyl-p-phenylenediamine, in a ferric chloride catalysed reaction with a 1:2 stoichiometric ratio to give the Methylene Blue dye, which is detected spectrophotometrically.

Echocardiographic study

Cardiac structure and function were evaluated by 2D echocardiography performed as previously described [12]. Left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD) and left ventricular posterior wall thickness (LVPWT) were measured from the parasternal long-axis view. Left ventricular mass was calculated using the Devereux-modified method [13]. Left ventricular mass index (LVMI) was calculated as the ratio of left ventricular mass to body weight. Left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) were determined to assess left ventricular systolic function. The mitral peak-flow velocities at early diastole and atrial contraction were recorded by pulsed Doppler technique and the E/A ratio was calculated to reflect left ventricular diastolic function. All measurements were averaged for three consecutive cardiac cycles by an experienced technician who was blinded to study grouping.

Histological analysis and immunohistochemical staining

Rat left ventricular tissue was fixed in 10% buffered formalin, embedded in paraffin and sliced into 5-μm-thick sections. Slides were then stained with haematoxylin and eosin (H&E) and Masson's Trichrome and observed under a light microscope. Myocyte cross-sectional area (CSA) and collagen volume fraction (CVF) were measured using an image analysis software (Image-Pro Plus, Media Cybernetics). The expression of types I and III collagen in cardiac tissue was examined by immunohistochemistry. After endogenous peroxidase quenching and antigen retrieval, the sections were incubated in 10% normal goat serum for 30 min at room temperature to block non-specific binding sites and then were incubated with rabbit anti-rat collagen I and III antibodies (Santa Cruz Biotechnology) at 4°C overnight. After washing with PBS, the slides were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (Invitrogen) at room temperature for 30 min. Finally, the sections were exposed to diaminobenzidine peroxidase substrate for 5 min and counterstained with Mayer's haematoxylin.

Detection of inflammatory cytokines

The levels of inflammatory markers in myocardial tissue, including tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8, were determined using commercial ELISA kits (R&D Systems) according to the manufacturer's instructions.

Measurement of oxidative stress

Oxidative stress was evaluated by detecting malondialdehyde (MDA) levels, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and reactive oxygen species (ROS) generation according to the instructions of detection kits (Jiancheng Biotech).

EMSA

Nuclear protein was extracted from cardiac tissue using the NE-PER nuclear extraction kit (Thermo Scientific). Equal amounts of nuclear protein were incubated with biotin-labelled oligonucleotide probes of antioxidant response element (ARE) for 30 min at room temperature. The oligonucleotide pairs of ARE were 5′-TTTATGCTGTGTCATGGTT-3′ and 5′-AACCATGACACAGCATAAA-3′. The DNA–protein complexes were separated on a non-denaturating polyacrylamide gel and then transferred on to a nylon membrane. The transferred DNA was cross-linked to the membrane, incubated with horseradish peroxidase-conjugated streptavidin and visualized by enhanced chemiluminescence. To assess the specificity of the reaction, competition assay was performed with 100-fold excess of unlabelled consensus oligonucleotide pairs of ARE. In antibody super-shift assay, nuclear factor erythroid 2-related factor 2 (Nrf2) antibody was added to the reaction mixture and incubated for 3 h at 4°C prior to the addition of probes.

siRNA transfection

NRCMs were transfected with Nrf2-specific siRNA or non-specific siRNA using Lipofectamine 2000 reagents (Invitrogen). Subsequently, the cells were exposed to high glucose (30 mM) for 24 h after incubation with or without NaHS (100 μM) for 30 min. The sense and antisense sequences of Nrf2 siRNA were as follows: 5′-GGAAACCUUACUCUCCCAGUGAGUA-3′ and 5′-UACUCACUGGGAGAGUAAGGUUUCC-3′. Non-specific siRNA was used to determine the efficiency of Nrf2-specific siRNA transfection.

TUNEL staining

Cardiomyocyte apoptosis was detected with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) In Situ Cell Death Detection Kit (Promega). The apoptotic index was calculated as the percentage of TUNEL-positive cells divided by the total number of cells. At least ten representative fields were evaluated for each group and the average value was calculated.

Annexin V–FITC/propidium iodide staining

Cellular apoptosis was determined using the Annexin V–FITC apoptosis detection kit (BD Pharmingen) according to the manufacturer's instructions. NRCMs were stained with Annexin V–FITC and propidium iodide (PI) and then subjected to flow-cytometry analysis. The apoptotic rate was calculated as the percentage of Annexin V-positive and PI-negative cells divided by the total number of cells in the gated region.

PI3K activity assay

PI3K activity was measured using a commercial ELISA kit (Echelon Biosciences) according to the manufacturer's instructions. In this method, PI3K activity was evaluated by detecting the conversion of phosphatidylinositol (4,5)-bisphosphate (PIP2) into phosphatidylinositol (3,4,5)-trisphosphate (PIP3).

Western blot analysis

Protein homogenates were prepared from cardiac tissue and protein concentrations were quantified with the BCA protein assay kit (Thermo Scientific). Equal amounts of protein (80 μg) were separated by SDS/PAGE (10% gel), transferred on to nitrocellulose membranes and blocked with 5% non-fat milk. The membranes were incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: anti-Nrf2, anti-HO-1 (haeme oxygenase-1), anti-NQO1 [NAD(P)H:quinone oxidoreductase 1], anti-Bax, anti-Bcl-2, anti-caspase-3, anti-cleaved caspase-3 (Asp175) (Santa Cruz Biotechnology) and anti-JNK, anti-phospho-JNK (Thr183/Tyr185), anti-p38, anti-phospho-p38 (Thr180/Tyr182), anti-Akt, anti-phospho-Akt (Ser473), anti-phospho-Bad (Ser136) and anti-phospho-caspase-9 (Ser196) (Cell Signaling Technology). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The immunocomplexes were visualized with an enhanced chemiluminescence detection kit (Amersham Biosciences).

Statistical analysis

All data in the present study are expressed as means ± S.D. and differences between groups were analysed using analysis of variance with SPSS version 15.0 (SPSS). Post-hoc analysis was used if the analysis of variance was significant. A value of P<0.05 was considered statistically significant.

RESULTS

As shown in Figure 1, H2S levels in plasma and myocardial tissue were significantly lower in the DM group than in the control group, whereas in the DM + NaHS group, H2S levels were remarkably higher than in the DM group. In addition, H2S content was not markedly increased in the NaHS group compared with that in the control group.

H2S content in plasma (A) and myocardial tissue (B) measured by the Methylene Blue method

Figure 1
H2S content in plasma (A) and myocardial tissue (B) measured by the Methylene Blue method

*P<0.05 compared with control; **P<0.05 compared with DM (n=10).

Figure 1
H2S content in plasma (A) and myocardial tissue (B) measured by the Methylene Blue method

*P<0.05 compared with control; **P<0.05 compared with DM (n=10).

The echocardiographic data are presented in Table 1. LVEDD, LVESD, LVPWT and LVMI were significantly increased in the DM group and decreased in the DM + NaHS group. LVFS, LVEF and E/A ratio, indicators of left ventricular systolic and diastolic function, were found to be remarkably lower in the DM group, whereas NaHS administration could improve left ventricular function in the DM + NaHS group.

Table 1
Echocardiographic study

E, peak velocity of early left ventricular filling; A, peak velocity of transmitral flow during atrial contraction. Data are expressed as means ± S.D. (n=10). *P<0.05 compared with control; **P<0.05 compared with DM.

Parameter Control DM DM + NaHS NaHS 
LVEDD (mm) 7.63±0.34 8.79±0.54* 8.31±0.42** 7.79±0.30 
LVESD (mm) 4.17±0.26 5.47±0.38* 5.01±0.31** 4.28±0.28 
LVPWT (mm) 1.32±0.08 1.63±0.15* 1.46±0.13** 1.35±0.10 
LVMI (mg/g) 1.75±0.12 2.23±0.26* 1.95±0.19** 1.68±0.14 
LVFS (%) 44.64±1.54 38.32±1.68* 41.77±1.94** 43.96±1.70 
LVEF (%) 74.55±2.34 66.23±2.52* 71.16±2.89** 73.85±2.72 
E/A ratio 1.58±0.09 1.30±0.11* 1.40±0.09** 1.54±0.06 
Parameter Control DM DM + NaHS NaHS 
LVEDD (mm) 7.63±0.34 8.79±0.54* 8.31±0.42** 7.79±0.30 
LVESD (mm) 4.17±0.26 5.47±0.38* 5.01±0.31** 4.28±0.28 
LVPWT (mm) 1.32±0.08 1.63±0.15* 1.46±0.13** 1.35±0.10 
LVMI (mg/g) 1.75±0.12 2.23±0.26* 1.95±0.19** 1.68±0.14 
LVFS (%) 44.64±1.54 38.32±1.68* 41.77±1.94** 43.96±1.70 
LVEF (%) 74.55±2.34 66.23±2.52* 71.16±2.89** 73.85±2.72 
E/A ratio 1.58±0.09 1.30±0.11* 1.40±0.09** 1.54±0.06 

Left ventricular tissue was stained with H&E and Masson's Trichrome and the representative images are shown in Figure 2. CSA and CVF were found to be significantly increased in the DM group and decreased in the DM + NaHS group, suggesting that H2S could attenuate cardiac hypertrophy and myocardial fibrosis in the diabetic rats.

H2S prevented cardiac hypertrophy and myocardial fibrosis in diabetic rats

Figure 2
H2S prevented cardiac hypertrophy and myocardial fibrosis in diabetic rats

Representative images of left ventricular tissue sections stained with H&E (A) and Masson's Trichrome (B). Quantitative analysis of myocyte CSA (C) and collagen volume fraction (D). *P<0.05 compared with control; **P<0.05 compared with DM.

Figure 2
H2S prevented cardiac hypertrophy and myocardial fibrosis in diabetic rats

Representative images of left ventricular tissue sections stained with H&E (A) and Masson's Trichrome (B). Quantitative analysis of myocyte CSA (C) and collagen volume fraction (D). *P<0.05 compared with control; **P<0.05 compared with DM.

The expression of types I and III collagen in left ventricular tissue was determined by immunohistochemistry and the representative images are shown in Figure 3. Types I and III collagen expression was significantly up-regulated in the DM group, whereas NaHS treatment could markedly down-regulate collagen expression in the DM + NaHS group.

H2S reduced collagen expression in the myocardium of diabetic rats

Figure 3
H2S reduced collagen expression in the myocardium of diabetic rats

Representative immunohistochemical staining of collagen I and collagen III in left ventricular tissue (A and B). Quantitative analysis of the positive staining of collagen I and collagen III (C and D). *P<0.05 compared with control; **P<0.05 compared with DM.

Figure 3
H2S reduced collagen expression in the myocardium of diabetic rats

Representative immunohistochemical staining of collagen I and collagen III in left ventricular tissue (A and B). Quantitative analysis of the positive staining of collagen I and collagen III (C and D). *P<0.05 compared with control; **P<0.05 compared with DM.

The levels of inflammatory markers in cardiac tissue were measured by ELISA and the results are presented in Table 2. TNF-α, IL-1β, IL-6 and IL-8 levels were significantly elevated in the myocardium of the diabetic rats, whereas NaHS administration was associated with reduced levels of these inflammatory cytokines.

Table 2
Detection of inflammatory cytokines

Data are expressed as means ± S.D. (n=10). *P<0.05 compared with control; **P< 0.05 compared with DM.

Cytokine Control DM DM + NaHS NaHS 
TNF-α (pg/mg of protein) 72.97±13.20 429.69±29.40* 234.94±20.77** 63.76±10.51 
IL-1β (pg/mg of protein) 17.81±4.77 69.86±9.47* 32.57±7.43** 14.40±4.19 
IL-6 (pg/mg of protein) 20.45±4.19 76.20±10.26* 38.49±8.75** 16.74±6.08 
IL-8 (pg/mg of protein) 16.22±3.99 67.43±7.87* 40.14±6.61** 18.09±4.63 
Cytokine Control DM DM + NaHS NaHS 
TNF-α (pg/mg of protein) 72.97±13.20 429.69±29.40* 234.94±20.77** 63.76±10.51 
IL-1β (pg/mg of protein) 17.81±4.77 69.86±9.47* 32.57±7.43** 14.40±4.19 
IL-6 (pg/mg of protein) 20.45±4.19 76.20±10.26* 38.49±8.75** 16.74±6.08 
IL-8 (pg/mg of protein) 16.22±3.99 67.43±7.87* 40.14±6.61** 18.09±4.63 

Oxidative stress was evaluated by detecting MDA levels and SOD and GSH-Px activities in myocardial tissue and the results are presented in Table 3. There were marked increases in MDA levels and decreases in SOD and GSH-Px activities in the DM group, whereas NaHS treatment was associated with decreased MDA levels and increased SOD and GSH-Px activities in the DM + NaHS group.

Table 3
Measurement of oxidative stress

Data are expressed as means ± S.D. (n=10). *P<0.05 compared with control; **P<0.05 compared with DM.

Parameter Control DM DM + NaHS NaHS 
MDA (nmol/mg of protein) 6.84±1.16 13.62±2.00* 8.25±1.10** 6.06±1.49 
SOD (U/mg of protein) 17.33±2.67 7.14±1.99* 13.53±2.34** 18.53±2.20 
GSH-Px (U/mg of protein) 24.90±4.18 11.12±3.14* 16.39±3.52** 22.10±4.77 
Parameter Control DM DM + NaHS NaHS 
MDA (nmol/mg of protein) 6.84±1.16 13.62±2.00* 8.25±1.10** 6.06±1.49 
SOD (U/mg of protein) 17.33±2.67 7.14±1.99* 13.53±2.34** 18.53±2.20 
GSH-Px (U/mg of protein) 24.90±4.18 11.12±3.14* 16.39±3.52** 22.10±4.77 

The activity of Nrf2, a crucial regulator of the antioxidative stress response was determined by Western blotting and EMSA (Figure 4). The results showed that Nrf2 protein accumulated in the nucleus of cardiomyocytes and Nrf2–ARE binding activity was remarkably elevated in the myocardium of diabetic rats following administration of NaHS. Consequently, the protein expression of two downstream targets of Nrf2, HO-1 and NQO1 was significantly increased in the DM + NaHS group.

Effect of H2S on the Nrf2/ARE pathway in diabetic myocardium

Figure 4
Effect of H2S on the Nrf2/ARE pathway in diabetic myocardium

Detection of Nrf2–ARE binding activity by electrophoretic mobility shift assay (A). Representative immunoblots and densitometric analysis of Nrf2 in the nucleus and cytosol (B) and its downstream targets HO-1 and NQO1 (C). *P<0.05 compared with Control; **P<0.05 compared with DM (n=5).

Figure 4
Effect of H2S on the Nrf2/ARE pathway in diabetic myocardium

Detection of Nrf2–ARE binding activity by electrophoretic mobility shift assay (A). Representative immunoblots and densitometric analysis of Nrf2 in the nucleus and cytosol (B) and its downstream targets HO-1 and NQO1 (C). *P<0.05 compared with Control; **P<0.05 compared with DM (n=5).

To confirm whether H2S suppresses high glucose-induced oxidative stress in an Nrf2-dependent manner, we transfected NRCMs with Nrf2-specific siRNA and then subjected them to high glucose. We found that Nrf2-specific siRNA-transfected cells that had been exposed to high glucose and NaHS exhibited reduced expression of Nrf2 and increased production of ROS than cells that had not been transfected with Nrf2-specific siRNA (Figure 5).

Effect of NaHS and Nrf2 siRNA on ROS generation in NRCMs exposed to high glucose

Figure 5
Effect of NaHS and Nrf2 siRNA on ROS generation in NRCMs exposed to high glucose

Representative immunoblots and densitometric analysis of Nrf2 in NRCMs (A). Pre-conditioning of cardiomyocytes with NaHS reduced high glucose-induced oxidative stress; Nrf2-specific siRNA-transfected cells exposed to NaHS and high glucose exhibited increased ROS production (B). *P<0.05 compared with cells treated with normal glucose; **P<0.05 compared with cells treated with high glucose; #P<0.05 compared with cells treated with high glucose and NaHS (n=5).

Figure 5
Effect of NaHS and Nrf2 siRNA on ROS generation in NRCMs exposed to high glucose

Representative immunoblots and densitometric analysis of Nrf2 in NRCMs (A). Pre-conditioning of cardiomyocytes with NaHS reduced high glucose-induced oxidative stress; Nrf2-specific siRNA-transfected cells exposed to NaHS and high glucose exhibited increased ROS production (B). *P<0.05 compared with cells treated with normal glucose; **P<0.05 compared with cells treated with high glucose; #P<0.05 compared with cells treated with high glucose and NaHS (n=5).

Cardiomyocyte apoptosis was evaluated by TUNEL staining and the apoptotic index was found to be higher in the DM group and lower in the DM + NaHS group (Figures 6A and 6B). In addition, cleaved caspase-3 expression and Bax/Bcl-2 ratio were significantly increased in the myocardium of diabetic rats and decreased following the administration of NaHS (Figures 6C and 6D).

Effect of H2S on cardiomyocyte apoptosis and the expression of apoptosis-related genes in diabetic rats

Figure 6
Effect of H2S on cardiomyocyte apoptosis and the expression of apoptosis-related genes in diabetic rats

Cardiomyocyte apoptosis was determined by TUNEL staining (A) and the apoptotic index was calculated (B). Arrows indicate apoptotic cardiomyocytes. Representative immunoblots and densitometric analysis of apoptosis regulatory proteins Bax, Bcl-2 and caspase-3 (C and D). *P<0.05 compared with control; **P<0.05 compared with DM (n=5).

Figure 6
Effect of H2S on cardiomyocyte apoptosis and the expression of apoptosis-related genes in diabetic rats

Cardiomyocyte apoptosis was determined by TUNEL staining (A) and the apoptotic index was calculated (B). Arrows indicate apoptotic cardiomyocytes. Representative immunoblots and densitometric analysis of apoptosis regulatory proteins Bax, Bcl-2 and caspase-3 (C and D). *P<0.05 compared with control; **P<0.05 compared with DM (n=5).

The phosphorylation of JNK and p38 MAPK in cardiac tissue was analysed by Western blotting (Figure 7). The results showed that protein levels of phospho-JNK and phospho-p38 MAPK were significantly elevated in the DM group, whereas NaHS treatment was found to inhibit the phosphorylation of MAPKs in the DM + NaHS group.

Representative immunoblots and densitometric analysis of JNK (A) and p38 MAPK (B) phosphorylation in cardiac tissue

Figure 7
Representative immunoblots and densitometric analysis of JNK (A) and p38 MAPK (B) phosphorylation in cardiac tissue

*P<0.05 compared with control; **P<0.05 compared with DM (n=5).

Figure 7
Representative immunoblots and densitometric analysis of JNK (A) and p38 MAPK (B) phosphorylation in cardiac tissue

*P<0.05 compared with control; **P<0.05 compared with DM (n=5).

To determine whether H2S reduces high glucose-induced apoptosis via blockade of MAPK pathways, NRCMs were pre-conditioned with NaHS, SP600125 or SB203580 prior to exposure with high glucose. As shown in Figure 8, pre-treatment of the cells with 100 μM NaHS for 30 min significantly inhibited MAPK phosphorylation and reduced high glucose-induced apoptosis. Similarly, pre-conditioning of NRCMs with either 10 μM SP600125 or 20 μM SB203580 for 60 min also inhibited apoptosis induced by high glucose.

Effect of NaHS, SP600125 and SB203580 on apoptosis in NRCMs treated with high glucose

Figure 8
Effect of NaHS, SP600125 and SB203580 on apoptosis in NRCMs treated with high glucose

Western blot analysis of JNK and p38 MAPK phosphorylation in NRCMs (A and B). Pre-treatment of cardiomyocytes with NaHS, SP600125 or SB203580 reduced high glucose-induced apoptosis (C). Representative images of cardiomyocyte apoptosis determined by flow cytometry (D). *P<0.05 compared with cells exposed to normal glucose; **P<0.05 compared with cells exposed to high glucose (n=5).

Figure 8
Effect of NaHS, SP600125 and SB203580 on apoptosis in NRCMs treated with high glucose

Western blot analysis of JNK and p38 MAPK phosphorylation in NRCMs (A and B). Pre-treatment of cardiomyocytes with NaHS, SP600125 or SB203580 reduced high glucose-induced apoptosis (C). Representative images of cardiomyocyte apoptosis determined by flow cytometry (D). *P<0.05 compared with cells exposed to normal glucose; **P<0.05 compared with cells exposed to high glucose (n=5).

PI3K activity in cardiac tissue was measured by ELISA and the expression of phospho-Akt and its downstream target proteins was detected by Western blotting (Figure 9). PI3K activity was found to be significantly decreased in the DM group and increased in the DM + NaHS group. Furthermore, the protein expression of phospho-Akt, phospho-caspase-9 and phospho-Bad was remarkably down-regulated in the diabetic rats and up-regulated after treatment with NaHS.

Effect of H2S on the PI3K/Akt pathway in diabetic myocardium

Figure 9
Effect of H2S on the PI3K/Akt pathway in diabetic myocardium

PI3K activity in myocardial tissue was measured by ELISA (A). Western blot analysis of Akt phosphorylation (B) and its downstream targets phospho-caspase-9 and phospho-Bad (C and D). *P<0.05 compared with control; **P<0.05 compared with DM (n=5).

Figure 9
Effect of H2S on the PI3K/Akt pathway in diabetic myocardium

PI3K activity in myocardial tissue was measured by ELISA (A). Western blot analysis of Akt phosphorylation (B) and its downstream targets phospho-caspase-9 and phospho-Bad (C and D). *P<0.05 compared with control; **P<0.05 compared with DM (n=5).

To confirm whether H2S inhibits high glucose-induced apoptosis via activation of PI3K/Akt pathway, NRCMs were pre-conditioned with NaHS and LY294002 prior to exposure to high glucose. As shown in Figure 10, pre-treatment of the cells with 100 μM NaHS for 30 min significantly increased PI3K activity and reduced high glucose-induced apoptosis. However, pre-conditioning of NRCMs with NaHS and 10 μM LY294002 for 60 min prior to high glucose exposure increased the apoptotic rate compared with pre-treatment with NaHS alone.

Effect of NaHS and LY294002 on apoptosis in NRCMs exposed to high glucose

Figure 10
Effect of NaHS and LY294002 on apoptosis in NRCMs exposed to high glucose

PI3K activity in NRCMs was detected by ELISA (A). Western blot analysis of Akt phosphorylation (B). Pre-treatment of cardiomyocytes with NaHS reduced high glucose-induced apoptosis; pre-conditioning with NaHS and LY294002 prior to high glucose exposure increased cardiomyocyte apoptosis compared with pre-treatment with NaHS alone (C). Representative images of cardiomyocyte apoptosis determined by flow cytometry (D). *P<0.05 compared with cells treated with normal glucose; **P<0.05 compared with cells treated with high glucose; #P<0.05 compared with cells treated with high glucose and NaHS (n=5).

Figure 10
Effect of NaHS and LY294002 on apoptosis in NRCMs exposed to high glucose

PI3K activity in NRCMs was detected by ELISA (A). Western blot analysis of Akt phosphorylation (B). Pre-treatment of cardiomyocytes with NaHS reduced high glucose-induced apoptosis; pre-conditioning with NaHS and LY294002 prior to high glucose exposure increased cardiomyocyte apoptosis compared with pre-treatment with NaHS alone (C). Representative images of cardiomyocyte apoptosis determined by flow cytometry (D). *P<0.05 compared with cells treated with normal glucose; **P<0.05 compared with cells treated with high glucose; #P<0.05 compared with cells treated with high glucose and NaHS (n=5).

DISCUSSION

In the present study, we established a streptozocin-induced diabetic rat model to investigate the protective effects of H2S against DCM. Our findings showed that endogenous generation of H2S was decreased in the diabetic rats, whereas exogenous administration of NaHS increased H2S contents in both plasma and myocardial tissue. The echocardiographic and histopathologic studies were performed to evaluate the beneficial effects of H2S in the prevention of DCM and the results indicated that H2S improved left ventricular function and attenuated cardiac hypertrophy and myocardial fibrosis in diabetic rats.

There is a growing evidence that inflammation, oxidative stress and apoptosis play critical roles in the pathogenesis of DCM [13]. Cardiac inflammation is an early and notable response to diabetes and actively involved in the development and progression of DCM. Several inflammatory cytokines such as TNF-α, IL-1β, IL-6 and IL-8 have been found to be increased in the diabetic myocardium [14,15]. Hyperglycaemia-induced ROS generation decreases the antioxidant capacity in the diabetic myocardium, contributing significantly to oxidative stress and the resultant myocardial damage, including myocyte cell death, hypertrophy, fibrosis, disordered calcium homoeostasis and endothelial dysfunction, which eventually lead to cardiac morphological and functional abnormalities [16,17]. It has been well documented that increased oxidative and nitrative stress in the diabetic heart may trigger activation of stress signalling pathways and consequently result in cardiomyocyte apoptosis, which is critically involved in the pathogenesis of DCM [18]. In the present study, our findings suggested that H2S attenuated inflammation, oxidative stress and apoptosis in the diabetic rat heart, which might be important protective mechanisms against DCM.

Nrf2, a member of the nuclear factor erythroid 2 family of nuclear basic leucine-zipper transcription factors, regulates the gene expression of several antioxidative enzymes by binding to ARE [19]. Under normal physiological conditions, Nrf2 is confined to the cytoplasm associated with the suppressor protein Keap1. Oxidative and electrophilic stress factors stimulate dissociation of the Nrf2–Keap1 complex, thereby promoting the release and translocation of Nrf2 into the nucleus to up-regulate expression of Nrf2/ARE-linked antioxidant genes [20]. In the present study, H2S was found to increase the binding activity of Nrf2–ARE and up-regulate the protein expression of HO-1 and NQO1, which consequently enhanced resistance to oxidative stress in the diabetic rat heart.

MAPK signalling pathways mediate cellular responses to a wide range of extracellular stimuli, including growth factors, hormones, cytokines and stress [21]. JNK and p38 MAPK have been proved to be key regulators of apoptosis in the cardiac myocyte [22]. In the present study, our findings suggested that JNK and p38 MAPK signalling was activated in the diabetic myocardium, which might be an important molecular mechanism responsible for cardiomyocyte apoptosis. Moreover, H2S was found to reduce high glucose-induced apoptosis both in vitro and in vivo by inhibiting phosphorylation of JNK and p38 MAPK.

The PI3K/Akt pathway plays critical roles in the regulation of cell survival and apoptosis [23]. Activation of PI3K results in the synthesis of PIP3 generated by the phosphorylation of PIP2. PIP3 activates the kinase PDK1 (phosphoinositide-dependent kinase-1), which in turn activates the kinase Akt (also known as protein kinase B). Phosphorylated Akt modulates cellular processes by phosphorylation of a number of substrates, including IκB kinase, Bad, caspase-9 and forkhead transcription factors [24]. In addition, Akt can also activate the mammalian target of rapamycin (mTOR) which acts to promote cell survival [25]. A previous study has reported that a novel H2S-releasing molecule GYY4137 probably protects H9c2 cells against high glucose-induced cytotoxicity by activation of the AMPK (5’ AMP-activated protein kinase)/mTOR signal pathway [26]. In the present study, PI3K/Akt signalling was inhibited in the diabetic myocardium, which might be another molecular mechanism involved in cardiomyocyte apoptosis. Furthermore, H2S was found to reduce high glucose-induced apoptosis both in vitro and in vivo by activating PI3K/Akt signalling.

In conclusion, our study demonstrates that H2S alleviates the development of DCM via attenuation of inflammation, oxidative stress and apoptosis. Moreover, H2S may reduce high glucose-induced oxidative stress by activating Nrf2/ARE pathway and may exert anti-apoptotic effects in diabetic myocardium by inhibiting JNK and p38 MAPK pathways and activating PI3K/Akt signalling.

Abbreviations

     
  • ARE

    antioxidant response element

  •  
  • CSA

    cross-sectional area

  •  
  • CVF

    collagen volume fraction

  •  
  • DCM

    diabetic cardiomyopathy

  •  
  • DM

    diabetes mellitus

  •  
  • GSH-Px

    glutathione peroxidase

  •  
  • H&E

    haematoxylin and eosin

  •  
  • HO-1

    haem oxygenase-1

  •  
  • IL

    interleukin

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • LVEDD

    left ventricular end-diastolic diameter

  •  
  • LVEF

    left ventricular ejection fraction

  •  
  • LVESD

    left ventricular end-systolic diameter

  •  
  • LVFS

    left ventricular fractional shortening

  •  
  • LVMI

    left ventricular mass index

  •  
  • LVPWT

    left ventricular posterior wall thickness

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MDA

    malondialdehyde

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • NaHS

    sodium hydrosulfide

  •  
  • NQO1

    NAD(P)H:quinone oxidoreductase 1

  •  
  • NRCM

    neonatal rat cardiomyocyte

  •  
  • Nrf2

    nuclear factor erythroid 2-related factor 2

  •  
  • PI

    propidium iodide

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PIP2

    phosphatidylinositol (4,5)-bisphosphate

  •  
  • PIP3

    phosphatidylinositol (3,4,5)-trisphosphate

  •  
  • ROS

    reactive oxygen species

  •  
  • SOD

    superoxide dismutase

  •  
  • TNF-α

    tumour necrosis factor-α

  •  
  • TUNEL

    terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling

AUTHOR CONTRIBUTION

Xiang Zhou performed the experiments, analysed the data and wrote the manuscript. Guoyin An performed the experiments and analysed the data. Xiang Lu conceived and designed the experiments.

FUNDING

This work was supported by the National Natural Science Foundation of China [grant numbers 81470501 and 81400292].

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