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Introduction

Hydrogen sulfide (H2S), a well-known toxic gas with a characteristic smell of rotten eggs, has been previously described as an endogenously produced labile diffusible gasotransmitter which plays multiple roles in the cardiovascular system in general health and also in diseases (1–6). For example, in murine models of ischemia-induced heart failure, endogenous and exogenous H2S clearly were shown to exert protective effects against left ventricular structural and functional impairment caused by ischemia-induced heart failure (7). Wang et al reported that H2S attenuated ventricular dysfunction and arrested the progression of heart failure following myocardial infarction (MI) in a rat model (8). In addition, in relation to plasma H2S levels in patients with coronary heart disease (CHD), a significant inverse correlation with the severity of CHD and changes in the coronary artery has been noted (9). Previously, we demonstrated that exogenous H2S protects H9c2 cardiomyocytes against chemical hypoxia- (10,11) or doxorubicin-induced (12–14) injury. The roles of H2S in diabetes-related cardiovascular complications have attracted considerable attention, due to the following findings. First, lower circulating H2S concentrations have been noted in animal models of diabetes (5,15,16) and patients with type 2 diabetes mellitus (DM) (5,6). Second, low blood H2S levels may be associated with the vascular inflammation observed in diabetes since the supplementation of H2S prevents the secretion of inflammatory factors by monocytes cultured in high-glucose (HG) medium (5). Third, exogenous H2S protects against the development of HG-induced endothelial dysfunction (15). Fourth, exogenous H2S alleviates myocardial ischemia/reperfusion (IR) injury in db/db mice (17). Fifth, H2S has been shown to exert protective effects against myocardial I/R-induced damage in diabetic rats (18). Furthermore, our recent studies demonstrated that exogenous H2S protects H9c2 cardiac cells against HG-induced injury and inflammation (19–21). Although we have reported that several factors, including antioxidant, anti-apoptotic and anti-inflammatory effects, mitochondrial protection, and the inhibition of certain intracellular signaling pathways, such as mitogen-activated protein kinase (MAPK) (19), leptin (20) and nuclear factor-κB (NF-κB) (21), contribute to the protective effects of H2S against HG-induced cardiomyocyte injury, the mechanisms responsible for these cardioprotective effects of H2S remain unclear. Since previous studies have indicated that H2S activates ATP-sensitive K+ (KATP) channels in both the heart (22,23) and vascular tissues (24) and that KATP channels are cardioprotective (23,25–29), we thus hypothesized that KATP channels are involved in the protective effects which H2S exerts against HG-induced injury in H9c2 cardiomyocytes.

KATP channels are abundant in cardiac tissue (30). Cardiomyocytes contain KATP channels in both the sarcolemma [surface membrane (31)] and mitochondria (32,33). The opening of sarcolemmal KATP channels is associated with the shortening of cardiac action potential, and a decrease in intracellular Ca2+ loading and cardioprotection during ischemia (34–36). The opening of mitochondrial KATP channels contributes to the regulation of cardiac mitochondrial function (37) and cardio-protection induced by ischemic preconditioning (23,37,39). In addition, mitochondrial KATP channels ameliorate the apoptosis induced by oxidative stress in cardiac cells (26). Notably, previous research has revealed that DM is associated with the dysfunction of the cardiovascular KATP channels (40). Hyperglycemia, as well as DM, is harmful to the vasodilation mediated by KATP channels in human vascular smooth muscle cells (41–43). However, the roles of both sarcolemmal KATP channels and mitochondrial KATP channels in HG-induced cardiomyocyte injury, in particular, in relation to the cardio-protective effects of exogenous H2S, remain unclear.

Based on our recent studies (19–21) and other previous studies (5,6,15,17,18,22–29), we hypothesized that H2S exerts cardioprotective effects by modulating the activation of KATP channels in HG-treated cardiomyocytes. Therefore, the present study was designed to examine the following points: i) the effect of HG on the expression of cardiac KATP channels; ii) the roles of both sarcolemmal KATP channels and mitochondrial KATP channels in HG-induced cardiomyocyte injury; iii) whether exogenous H2S protects cardiomyocytes against HG-induced injury by modulating KATP channel activity; and iv) the role of reactive oxygen species (ROS) in the inhibitory effect of HG on the expression of cardiac KATP channels.

Materials and methods

Materials and reagents

Anti-kir6.1 (R-14) antibody (sc-11224) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); anti-cleaved caspase-3 antibody (#9662) was purchased from Cell Signaling Technology, Inc. (Boston, MA, USA); anti-GAPDH antibody (10494-1-AP) was purchased from Proteintech Group, Inc. (Wuhan, China); horseradish peroxidase (HRP) conjugated secondary antibody and the BCA Protein assay kit were obtained from KangChen Biotech (Shanghai, China). Diazoxide (DZ), pinacidil (Pin), 5-hydroxydecanoic acid (5-HD) and glibenclamide (Gli) were all purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Sodium hydrosulfide (NaHS; a donor of H2S) was obtained from Sigma-Aldrich (St. Louis, MO, USA), and was protected from sunlight and stored at 2–4°C. The cell counting kit-8 (CCK-8) was supplied by Dojindo Laboratories (Kumamoto, Japan). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraeth-ylbenzimidazolylcarbocyanine iodide (JC-1), Hoechst 33258 and N-acetyl-L-cysteine (NAC) were all purchased from Sigma-Aldrich. Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) medium were obtained from Gibco-BRL (Grand Island, NY, USA). The enhanced chemiluminescence (ECL) solution was purchased from KeyGen Biotech (Nanjing, China). The H9c2 cardiac cells were supplied by the Sun Yat-sen University Experimental Animal Center (Guangzhou, China).

Cell culture and treatment

The H9c2 cardiac cells, a rat cardiac myoblast cell line, were cultured in DMEM, supplemented with 10% FBS in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The culture medium was replaced with fresh medium every 2–3 days and expanded to new culture plates when the cells reached approximately 80% confluence.

To investigate the role of KATP channels in HG-induced cardiomyocyte injury, the H9c2 cardiac cells were treated with 100 µM DZ (a mitochondrial KATP channel opener) or 50 µM Pin (a non-selective KATP channel opener) for 30 min prior to exposure to 35 mM glucose for 24 h. To explore the protective effects of H2S on HG-induced injury, the H9c2 cells were conditioned with 400 µM NaHS for 30 min prior to exposure to HG for 24 h. To further determine whether the protective effects of NaHS were associated with the activation of KATP channels, the cells were conditioned with 100 µM 5-HD (a mitochondrial KATP channel blocker) or 1 mM Gli (a non-selective KATP channel blocker) for 30 min prior to treatment with NaHS and 35 mM glucose for 24 h. To confirm whether there is an antagonistic interaction between ROS and KATP channels, the H9c2 cells were treated with 500 µM NAC (a scavenger of ROS) for 60 min prior to exposure to HG.

Cell viability assay

The H9c2 cells were seeded in 96-well plates at a concentration of 1×104 cells/ml and incubated at 37°C. CCK-8 assay was employed to assess the viability of the cells. After being subjected to the above-mentioned treatments, the cells were washed with phosphate-buffered saline (PBS), and 10 µl CCK-8 solution at 10% dilution was added to each well, and the plate was then incubated for approximately 2 h in an incubator. The absorbance at 450 nm was assayed using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The means of the optical density (OD) of 3 wells in the indicated groups were used to calculate the percentage of cell viability according to the following formula: cell viability (%) = (ODtreatment group/ODcontrol group) ×100. The experiment was repeated 5 times.

Hoechst 33258 nuclear staining for the assessment of apoptosis

Apoptotic cell death was assessed using the Hoechst 33258 staining method followed by photofluorography. The H9c2 cells were plated in 35-mm dishes at a density of 1×106 cells/well. After being subjected to the indicated treatments, the cells were harvested and fixed with paraformaldehyde in 0.1 mol/l PBS (pH 7.4) for 10 min. The slides were then washed 5 times with PBS. After rinsing with PBS, the nuclear DNA was stained with 5 mg/ml Hoechst 33258 for 10 min before being rinsed briefly with PBS and then visualized under a fluorescence microscope (BX50-FLA; Olympus, Tokyo, Japan). The viable H9c2 cells exhibited a uniform blue fluorescence throughout the nucleus, whereas the apoptotic cells had fragmented and condensed nuclei. The experiment was carried out 3 times.

Measurement of intracellular ROS levels

The determination of intracellular ROS levels was performed by measuring the fluorescence product formed by the oxidation of DCFH-DA, as prevoiusly described (19). Briefly, the culture medium was removed and the cells were then washed 3 times with PBS. Following the addition of fresh culture medium, the cells were incubated with DCFH-DA at a final concentration of 10 µM, for 30 min at 37°C. The cells were then washed 5 times with PBS, and the relative amount of fluorescence product was assessed using a fluorescence microscope connected to an imaging system (BX50-FLA; Olympus). The mean fluorescence intensity (MFI) from 5 random fields was measured using ImageJ 1.47i software, and the MFI was used as an index for the amount of ROS. The experiment was carried out 5 times.

Measurement of mitochondrial membrane potential (MMP)

As previousy described (19), MMP was assessed using a fluorescent dye, JC-1, which is a cell-permeable cationic dye that enters the mitochondria based on a highly negative MMP. The depolarization of MMP results in the loss of JC-1 from the mitochondria and a decrease in intracellular green fluorescence. The H9c2 cardiac cells were cultured on a slide with Eagle's minimal essential medium (EMEM). After being subjected to the indicated treatments, the slides were washed 3 times with PBS. The H9c2 cells were incubated with 1 mg/l JC-1 at 37°C for 30 min in an incubator and washed 3 times with PBS. JC-1 fluorescence was then measured over the entire field of vision using a fluorescence microscope connected to an imaging system (BX50-FLA; Olympus). The MFI of JC-1 from 5 random fields was analyzed using ImageJ 1.47i software and was taken as an index of the levels of MMP. The experiment was carried out 5 times.

Western blot analysis

After being subjected to the indicated treatments, the H9c2 cardiac cells were harvested and lysed with cell lysis solution at 4°C for 30 min. Total proteins in the cell lysates were quantified using a BCA protein assay kit. Loading buffer was added to the cytosolic extracts and, after boiling for approximately 5 min, equal amounts of supernatant from each sample were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Total proteins in the gel were transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for approximately 90 min at room temperature in fresh blocking buffer [0.1% Tween-20 in Tris-buffered saline (TBS-T) containing 5% fat-free milk] and then incubated with either anti-KATP (1:1,000 dilution), or anti-cleaved caspase-3 antibody (1:1,000 dilution) in freshly prepared TBS-T with 3% fat-free milk overnight with slow agitation at 4°C. Following 3 washes with TBS-T, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:2,500 dilution; KangChen Bio-tech) in TBS-T with 3% fat-free milk for 90 min at room temperature. GAPDH was used as an internal control. The membranes were then washed 3 times with TBS-T solution for 15 min. The immunoreative signals were visualized by ECL detection. In order to quantify protein expression, the X-ray films were scanned and analyzed using ImageJ 1.47i software. Each experiment was repeated 3 times.

Statistical analysis

All data are presented as the means ± SEM. Differences between groups were analyzed by one-way analysis of variance (ANOVA) using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) followed by the least significant difference (LSD) post hoc comparison test. A P-value <0.05 was considered to indicate a statistically significant difference.

Results

NaHS attenuates the HG-induced decrease in protein expression of KATP channels in H9c2 cardiac cells

In order to investigate the influence of HG (35 mM glucose) on the protein expression of KATP channels in H9c2 cardiac cells, a time-response experiment to determine the protein expression levels of KATP channels was performed. As shown in Fig. 1A and B, the cells were exposed to HG for 1, 3, 6, 9, 12 and 24 h, respectively. Following exposure to HG for 6 h, the protein expression level of KATP channels began to decrease, and the maximum decrease in expression levels was observed after the cells were exposed to HG for 12 and 24 h. Based on these results, the expression levels of KATP channels were detected at 12 h following exposure to HG in the subsequent experiments.

Figure 1 Sodium hydrogen sulfide (NaHS) reduces the high-glucose (HG)-induced decrease in the protein expression levels of ATP-sensitive potassium (KATP) channels in H9c2 cardiac cells. (A and C) KATP channel protein expression levels were semi-quantified by western blot analysis. (A and B) Time course of changes observed in KATP channel expression levels induced by HG (35 mM glucose) over a 24-h time period. (C and D) H9c2 cardiac cells were exposed to HG for 12 h with or without treatment with 400 µM NaHS for 30 min prior to exposure to HG. (B and D) Densitometric analysis of the KATP channel expression levels in (A and C). Data are represented as the means ± SEM (n=3). **P<0.01 vs. control (Con) group; ++P<0.01 vs. the HG-treated group.

It is important to note that the decrease in the KATP channel levels was ameliorated by treatment with 400 µM NaHS (a donor of H2S) for 30 min prior to exposure to HG for 12 h (Fig. 1C and D). However, the basal expression level of KATP channels was not markedly altered by treatment with 400 µM NaHS alone for 30 min. These data indicate that exogenous H2S alleviates the decrease in the protein expression levels of KATP channels induced by HG in H9c2 cardiac cells.

KATP channels are involved in the protective effects which H2S exerts against HG-induced cytotoxicity to H9c2 cardiac cells

Consistent with our recent studies (19–21), treatment of the cells with 400 µM NaHS for 30 min prior to exposure to HG for 24 h significantly inhibited HG-induced cytotoxicity, leading to an increase in cell viability (Fig. 2). To explore the role of KATP channels in HG-induced cytotoxicity, the cells were treated with 100 µM DZ (a mitochondrial KATP channel opener) or 50 µM Pin (a non-selective KATP channel opener) for 30 min prior to exposure to HG. As shown in Fig. 2, pre-treatment of the H9c2 cardiac cells with DZ or Pin considerably reduced HG-induced cytotoxicity, as evidenced by an increase in cell viability. To further investigate the role which KATP channels play in the protective effects exerted by H2S against HG-induced cytotoxicity, the cells were treated with 100 µM 5-HD (a mitochondrial KATP channel blocker) or 1 mM Gli (a non-selective KATP channel blocker) for 30 min prior to treatment with NaHS and exposure to HG. It was demonstrated that the blockade of KATP channels with 5-HD or Gli markedly reduced the protective effects of NaHS against HG-induced cytotoxicity, resulting in a decrease in cell viability (Fig. 2). Alone, 5-HD and Gli did not significantly alter cell viability. These data suggest that KATP channels mediate the protective effects of H2S against cytotoxicity to H9c2 cardiac cells induced by HG.

Figure 2 Effects of different treatments on the high-glucose (HG)-induced cytotoxicity in H9c2 cardiac cells. The cells were treated with 35 mM glucose for 24 h with or without pre-treatment with 400 µM sodium hydrogen sulfide (NaHS) or 100 µM diazoxide (DZ) or 50 µM pinacidil (Pin) or 100 µM 5-hydroxydecanoic acid (5-HD) or 1 mM glibenclamide (Gli) for 30 min. Cell viability was detected using the CCK-8 assay. Data are presented as the means ± SEM (n=6). **P<0.01 vs. control (Con) group; ++P<0.01 vs. the HG-treated group; △△P<0.01 vs. the NaHS + HG-treated group.

KATP channels are involved in the protective effects which H2S exerts against HG-induced apoptosis in H9c2 cardiac cells

In agreement with our recent studies (19–21), exposure of the cells to HG for 24 h markedly increased the number of apoptotic cells (Fig. 3A, panel b), leading to an increase in the percentage of apoptotic cells (Fig. 3A, panel m). The increased number of apoptotic cells was decreased by pre-treatment with NaHS, Pin or DZ (Fig. 3A, panels c, d and e). Similarly, exposure of the cells to 35 mM glucose for 24 h markedly enhanced the expression level of cleaved caspase-3 (Fig. 3B). However, the increased expression level of cleaved caspase-3 was attenuated by treatment with 400 µM NaHS or 50 µM Pin or 100 µM DZ for 30 min prior to exposure to HG for 24 h (Fig. 3B, panels a to d). Furthermore, treatment of the H9c2 cardiac cells with 100 µM 5-HD (Fig. 3A, panel f and B, panels c and d) or 1 mM Gli (Fig. 3A, panel g and B, panels c and d) for 30 min prior to treatment with NaHS and exposure to HG blocked the above-mentioned anti-apoptotic effects of NaHS, as evidenced by the increase in the percentage of apoptotic cells (Fig. 3A, panels f and g) and the increase in cleaved caspase-3 expression (Fig. 3B, panels c and d). Alone, NaHS, Pin, DZ, 5-HD and Gli did not significantly affect the percentage of apoptotic cells or the basal expression level of cleaved caspase-3.

Figure 3 Role of ATP-sensitive K+ (KATP) channels in the protective effects of H2S against the high-glucose (HG)-induced apoptosis in H9c2 cardiac cells. (A) Hoechst 33258 nuclear stating followed by fluorescence imaging was performed to examine cell apoptosis. (B) The expression level of cleaved caspase-3 was semi-quantified by western blot analysis. (B, panels b and d) Densitometric analysis of the cleaved caspase-3 expression level in (B, panels a and c). (A, panel a) Control group. H9c2 cells were (panel b) treated with 35 mM glucose (HG) for 24 h; (panel c) treated with 400 µM sodium hydrogen sulfide (NaHS) or (panel d) 50 µM pinacidil (Pin) or (panel e) 100 µM diazoxide (DZ) for 30 min prior to exposure to HG; (panel f) treated with 100 µM 5-hydroxydecanoic acid (5-HD) or (panel g) 1 mM glibenclamide (Gli) for 30 min prior to treatment with NaHS and exposure to HG; (panel h) treated with 400 µM NaHS or (panel i) 50 µM Pin or (panel j) 100 µM DZ for 30 min followed by 24 h of culture; (panel k) treated with 100 µM 5-HD or (panel l) 1 mM Gli for 30 min followed by 24 h of culture. (m) The apoptosis rate was analyzed using a cell counter and ImageJ 1.47i software. Data are presented as the means ± SEM (n=3). **P<0.01 vs. control (Con) group; ++P<0.01 vs. the HG-treated group; △△P<0.01 vs. the NaHS + HG-treated group.

KATP channels are implicated in the protective effects exerted by H2S against HG-induced oxidative stress in H9c2 cardiac cells

In agreement with our recent findings (19–21), treatment of the cells with 35 mM glucose for 24 h significantly increased the intracellular production of ROS (Fig. 4B and M). The increased ROS production was ameliorated by treatment with 400 µM NaHS for 30 min prior to exposure to HG for 24 h (Fig. 4C and M). Similarly, treatment with 50 µM Pin (Fig. 4D and M) or 100 µM DZ (Fig. 4E and M) for 30 min prior to exposure to HG for 24 h also attenuated the generation of ROS. To confirm the role played by KATP channels in the protective effects of NaHS against HG-induced oxidative stress, the cells were treated with 100 µM 5-HD or 1 mM Gli for 30 min prior to exposure to NaHS and HG. Our data indicated that pre-treatment with 5-HD (Fig. 4F and M) or Gli (Fig. 4G and M) blocked the inhibitory effects which NaHS exerted on the generation of ROS induced by HG, suggesting that the KATP channels contribute to the protective effects which H2S exerts against the HG-induced overproduction of ROS.

Figure 4 ATP-sensitive K+ (KATP) channels contribute to the inhibitory effecta of H2S on the high-glucose (HG)-induced reactive oxygen species (ROS) generation in H9c2 cardiac cells. (A–L) After the cells were subjected to the indicated treatments, intracellular ROS generation was measured by 2′7′-dichlorodi-hydrofluoresein diacetate (DCFH-DA) staining followed by photofluorography. (A) Control group. H9c2 cardiac cells were (B) treated with 35 mM glucose (HG) for 24 h; (C) treated with 400 µM sodium hydrogen sulfide (NaHS) or (D) 50 µM pinacidil (Pin) or (E) 100 µM diazoxide (DZ) for 30 min prior to exposure to HG; (F) treated with 100 µM 5-hydroxydecanoic acid (5-HD) or (G) 1 mM glibenclamide (Gli) for 30 min prior to treatment with NaHS and exposure to HG; (H) treated with 400 µM NaHS or (I) 50 µM Pin or (J) 100 µM DZ for 30 min followed by 24 h of culture; (K) treated with 100 µM 5-HD or (L) 1 mM Gli for 30 min followed by 24 h of culture. (M) Quantitive analysis mean fluorescence intensity (MFI) in (A-L) using ImageJ 1.47i software. Data are presented as the means ± SEM (n=5). **P<0.01 vs. control (Con) group; ++P<0.01 vs. the HG-treated group; △△P<0.01 vs. the NaHS + HG-treated group.

KATP channels are linked to the protective effects of H2S against HG-induced mitochondrial insults in H9c2 cardiac cells

Consistent with our recent studies (19–21), treatment of the cells with 35 mM glucose for 24 h markedly induced mitochondrial damage, as evidenced by the loss of MMP (Fig. 5A, panel b and 5M). In addition, we noted that the HG-induced decrease in MMP was reversed by treatment of the cells with 400 µM NaHS for 30 min prior to exposure to HG (Fig. 5A, panel c and 5M). Notably, treatment of the cells with 50 µM Pin (Fig. 5A, panel d and 5M) or 100 µM DZ (Fig. 5A, panel e and 5M) for 30 min prior to exposure to HG also blocked the HG-induced loss of MMP. Subsequently, we further explored the role of KATP channels in the protective effects of NaHS against the dissipation of MMP induced by HG. Our findings revealed that treatment with 100 µM 5-HD or 1 mM Gli for 30 min prior to treatment with NaHS and exposure to HG considerably reduced the inhibitory effects of NaHS on the HG-induced dissipation of MMP (Fig. 5A, panels f and g and 5M). These results demonstrate that KATP channels play a critical role in the protective effects of H2S against the HG-induced mitochondrial damage.

Figure 5 ATP-sensitive K+ (KATP) channels mediate the protective effects of H2S against the high-glucose (HG)-induced dissipation of mitochondrial membrane potential (MMP) in H9c2 cardiac cells. (A) After the cells were subjected to the indicated treatments, MMP was examined using the lipophilic cationic probe, JC-1, followed by photofluorography. (Panel a) Control group. H9c2 cardiac cells were (panel b) treated with 35 mM glucose (HG) for 24 h; (panel c) treated with 400 µM sodium hydrogen sulfide (NaHS) or (panel d) 50 µM pinacidil (Pin) or (panel e) 100 µM diazoxide (DZ) for 30 min prior to exposure to HG; (panel f) treated with 100 µM 5-hydroxydecanoic acid (5-HD) or (panel g) 1 mM glibenclamide (Gli) for 30 min prior to treatment with NaHS and exposure to HG; (panel h) treated with 400 µM NaHS or (panel i) 50 µM Pin or (panel j) 100 µM DZ for 30 min followed by 24 h of culture; (panel k) treated with 100 µM 5-HD or (panel l) 1 mM Gli for 30 min followed by 24 h of culture. (B) Quantitive analysis of the ratio of red/green fluorescence intensity, in (A) using ImageJ 1.47i software. Data are presented as the means ± SEM (n=5). **P<0.01 vs. control (Con) group; ++P<0.01 vs. the HG-treated group; △△P<0.01 vs. the NaHS + HG-treated group.

ROS scavenger blocks the HG-induced downregulation of the expression of KATP channels in H9c2 cardiac cells

Since hyperglycemia, as well as DM, has been reported to impair KATP channels in human vascular smooth muscle cells via ROS (41–43), we investigated the role of ROS in the HG-induced decrease in KATP channel expression in the H9c2 cardiac cells. As shown in Fig. 6, treatment of the cells with 500 µM NAC (a scavenger of ROS and NAC) for 60 min prior to exposure to 35 mM glucose for 12 h blocked the inhibitory effects of HG on the expression levels of KATP channels. Alone, NAC did not significantly alter the basal expression level of KATP channels. These results suggest that ROS participates in the HG-induced downregulation of the expression of KATP channels.

Figure 6 Reactive oxygen species (ROS) is implicated in the high glucose (HG)-induced decrease in the expression level of ATP-sensitive K+ (KATP) channels in H9c2 cardiac cells. The cells were exposed to 35 mM glucose (HG) for 12 h with or without treatment with 500 µM NAC (a scavenger of ROS) for 60 min prior to exposure to HG. (A) KATP channel expression was semi-quantified by western blot analysis. (B) Densitometric analysis of the expression levels of KATP channel in (A). Data are represented as the means ± SEM (n=3). **P<0.01 vs. control (Con) group; ++P<0.01 vs. the HG-treated group.

Discussion

Cardiac KATP channels are key sensors and effectors of the metabolic status of cardiomyocytes, and their roles in HG-induced cardiomyocyte injury and in the cardioprotective effects of H2S are noteworthy. The major findings of this study can be summarized as follows: i) HG markedly downregulated the expression levels of cardiac KATP channels; ii) exogenous H2S attenuated the inhibitory effects of HG on the expression of cardiac KATP channels; iii) the KATP channel openers, DZ (a mitochondrial KATP channel opener) and Pin (a non-selective KATP channel openner), ameliorated the HG-induced cardiomyocyte injury, including cytotoxicity, apoptosis, ROS generation and the dissipation of MMP; iv) the KATP channel antagonists, namely 5-HD (a mitochondrial KATP channel antagonist) and Gli (a non-selective KATP channel antagonist), blocked the cardio-protective effects of exogenous H2S against the HG-induced cardiomyocyte injury; v) the ROS scavenger, NAC, reduced the inhibition of cardiac KATP channel expression induced by HG. These results strongly suggest that the impairment of KATP channels is implicated in HG-induced cardiomyocyte injury and that the activation of KATP channels is linked to the protective effects which exogenous H2S exerts against HG-induced cardiomyocyte injury.

KATP channels have the unique ability to regulate membrane excitability in response to changes in the energetic status of cells (31,44,45). This ability serves to decrease myocardial energy consumption and vulnerability to stress (44,45). Previous research has indicated that the ability of cardiac KATP channels to affect cellular excitability and function depends on their abundance at the membrane surface (46,47). For example, an increase in cardiac sarcolemmal KATP channels enhances the speed and degree of shortening of action potentials and reduces cardiac energy consumption in response to escalating workloads (46). Several studies have indicated a correlation between dysfunction in KATP channel gating and insulin secretory disorders (40,48), such as neonatal diabetes (48). Furthermore, in human vascular smooth muscle cells, HG impairs vasorelaxation by inhibiting the activity of KATP channels (41–43). However, our knowledge of the roles of KATP channels in HG-induced cardiac cells remains incomplete.

In order to explore this issue, in the present study, we first investigated the effects of HG on the expression levels of KATP channels in H9c2 cardiac cells. Our data demonstrated that the exposure of the cells to HG markedly reduced the expression levels of KATP channels. This reduced expression level of KATP channels indirectively suggests a decrease in their presence in the HG-treated cardiac cells. Since a previous study indicated that a reduction in the number of sarcolemmal KATP channels slows cardiac action potential during shortening under hypoxia (47),. Thus, we hypothesized that the inhibition of cardiac KATP channels is a critical mechanism which underlies HG-induced cardiomyocyte injury. To confirm this hypothesis, we observed the influence of KATP channel activation on HG-induced injury. In agreement with our recent studies (19–21), the findings of this study demonstrated that treatment of the H9c2 cardiac cells with HG induced considerable injuries, including a decrease in cell viability, an increase in apoptotic cells, cleaved caspase-3 expression and ROS generation, as well as the loss of MMP. However, treatment of the H9c2 cardiac cells with DZ (a mitochondrial KATP channel opener) or Pin (a non-selective KATP channel opener) markedly attenuated the above-mentioned HG-induced injuries, as evidenced by an increase in cell viability, and a decrease in the number of apoptotic cells, decreased cleaved caspase-3 expression and ROS generation, as well as the loss of MMP. The above-mentioned results suggest that HG impairs the function of cardiac KATP channels, which contributes to HG-induced injuries. In support of our results are experimental studies which demonstrate that hyperglycemia damages the functionality of the human ether-ago-go-related gene (HERG) K+ channels, reduces the transient outward K+ current, enhances the intracellular concentration of Ca2+, and impairs the excitation-contraction coupling in the heart (49,50). It has also been noted that the opening of the mitochondrial KATP channels plays an important role in the regulation of cardiac mitochondrial function (37) and attenuates the oxidative stress-induced apoptosis of cardiac cells.

Of note, sulfonylurea drugs have been shown to stimulate insulin secretion by closing KATP channels (51,52). Despite the fact that their target is not completely clear, these drugs have been used to treat type 2 DM since the 1950s and they are still used today. Thus, the findings of this study drive us to explore the effects of sulfonylurea drugs on HG-induced cardiomyocyte injury in future experiments.

Another important result of this study relates to the role of the activation of KATP channels in the cardioprotective effects of exogenous H2S against HG-induced cardiac injuries. Previously, the protective effects of H2S on DM-related cardiovascular insults have received much attention. Exogenous H2S attenuates I/R-induced injury in db/db mice (17) and diabetic rats (18). Our recent studies have also examined the protective effects of exogenous H2S against HG-induced injury and inflammation in H9c2 cardiac cells (19–21). The mechanisms responsible for these cardioprotective effects of H2S are associated with the inhibition of the MAPK (19), leptin (20) and NF-κB (21) pathways. However, the protective mechanisms of H2S are complex, and other factors are likely involved in these cardioprotective effects of H2S. Since the KATP channels in the heart (22,23) have been reported to be activated by H2S, this study further investigated that roles KATP channels play in the protective effects which H2S exerts against HG-induced cardiomyocyte injury. In agreement with our recent studies, the findings of this study demonstrated that exogenous H2S exerted protective effects against HG-induced cardiomyocyte injuries, as indicated by an increase in cell viability, and a decrease in the number of apoptotic cells, decreased cleaved caspase-3 expression and ROS generation, as well as the loss of MMP. Notably, our results demonstrated that exogenous H2S markedly reduced the downregulation of KATP channel expression by HG and that both 5-HD (a mitochondrial KATP channel blocker) and Gli (a non-selective KATP channel blocker) blocked the cardioprotective effects of H2S mentioned above. These results suggest that KATP channels, in particular mitochondrial KATP channels, play a critical role in the cardioprotective effects of H2S. Similarly, Zhao et al demonstrated the protective effects which H2S exerts against arrhythmia by opening mitochondrial KATP channels (24). However, Bian et al (23) revealed that sarcolemmal KATP channels, but not mitochondrial KATP channels, mediate the cardioprotective effects of H2S in isolated cardiac myocytes exposed to simulated ischemia solution. Therefore, the roles of subtypes of KATP channels involved in the cardioprotective effects of H2S in the present study differ from the ones reported by Bian et al (23), although we did not use a sarcolemmal KATP channels blocker or MCC-134 that opens sarcolemmal KATP channels and blocks minochondrial KATP channels. One possible explanation for this difference in the results may lie in the use of different experimental models.

In the present study, we also investigated the interaction between ROS and cardiac KATP channel activation. As aforementioned, both DZ and Pin ameliorated HG-induced ROS generation, suggesting that KATP channel activity had an inhibitory effect on ROS generation. A previous study reporting that KATP channel opener (DZ) reduces mitochondrial ROS production at reoxygenation (53) supports our results. On the other hand, we noted that NAC, a ROS scavenger, blocked the decrease in the expression of KATP channel protein induced by HG. Collectively, these results suggest that there is an interaction between ROS and KATP channels in HG-treated cardiac cells. The elucidation of the molecular mechanism underlying this interaction may be significant for the treatment and prevention of DM-related cardiovascular complications.

In conclusion, the present study provides novel evidence that the impairment of KATP channels is associated with HG-induced multiple cardiac injuries, including cytotoxicity, apoptosis, oxidative stress and mitochondrial damage. Therefore, the inhibition of cardiac KATP channels by insulin secretagogues should be considered to increase cardiac risk. Importantly, the present study has demonstrated that exogenous H2S protects cardiomyocytes against HG-induced injuries by activating KATP channels. Based on the results of the present study and previous studies (5,6,15,16), we suggest that low levels of endogenous H2S and the impairment of KATP channels are an important pathophysiological mechanism underlying huperglycemia-induced cardiovascular complications. Therefore, supplementation with H2S and modulation of the H2S-KATP channel pathway should be considered a potential approach to attenuating hyperglycemia-induced cardiomyocyte injury.

Acknowledgments

The present study was supported by grants from the National Natural Science Foundation of China (no. 81270296) and the Guangdong Province Finance Science and Technical Fund (no. 2014Sc107).

References