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Nitric Oxide j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i o x

Hydrogen sulfide protects against apoptosis under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes Dan Wu a,1, Qingxun Hu a,1, Xinhua Liu a, Lilong Pan a, Qinghui Xiong a, Yi Zhun Zhu a,b,* a b

Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai, China Department of Pharmacology, Loo Yong Lin School of Medicine, National University of Singapore, Singapore

A R T I C L E

I N F O

Article history: Received 5 September 2014 Revised 31 October 2014 Accepted 10 November 2014 Available online Keywords: Hydrogen sulfide Oxidative stress Sirtuin-1 Mitochondrial function

A B S T R A C T

Oxidative stress plays a great role in the pathogenesis of heart failure (HF). Oxidative stress results in apoptosis, which can cause the damage of cardiomyocytes. Hydrogen sulfide (H2S), the third gasotransmitter, is a good reactive oxygen species (ROS) scavenger, which has protective effect against HF. Sirtuin-1 (SIRT1) is a highly conserved nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase that plays a critical role in promoting cell survival under oxidative stress. The purpose of this article is to investigate the interaction between H2S and SIRT1 under oxidative stress in H9c2 cardiomyocytes. Oxidative stress was induced by hydrogen peroxide (H 2 O 2 ). Treatment with NaSH (25–100 μmol/L) dosedependently increased the cell viability and improved the cell apoptosis induced by H2O2 in H9c2 cardiomyocytes. The protective effect of NaSH against the apoptosis could be attenuated by SIRT1 inhibitor Ex 527 (10 μmol/L). Treatment with NaSH (100 μmol/L) could increase the expression of SIRT1 in time dependent manner, which decreased by different concentration of H2O2. NaSH (100 μmol/L) increased the cellular ATP level and the expression of ATPase. These effects were attenuated by Ex 527 (10 μmol/L). After NaSH (100 μmol/L) treatment, the decrease in ROS production and the enhancement in SOD, GPx and GST expression were observed. Ex 527 (10 μmol/L) reversed these effects. In conclusion, for the first time, this article can identify antioxidative effects of H2S under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Accumulating evidences suggest that apoptosis is one of the important cases for progressive cardiomyocytes death, which can lead to various cardiac diseases [1,2]. Apoptosis can be induced by oxidative stress and results in a further decrease in cardiac function [3–5]. Despite amassing clinical and experimental evidences, the mechanism and signaling pathway in cardiac diseases induced by oxidative stress are largely unexplored. Thus, identification of novel regulatory mediators is crucial for new therapies to protect against myocardial dysfunction after injury. Hydrogen sulfide (H2S) is commonly considered as an environmental contaminant. However, studies show that H2S plays a pivotal role as an important endogenous modulator [6,7]. H2S is endogenously generated by cystathionine-β-synthase, cystathionine-γlyase and cysteine aminotransferase along with 3-mercaptopyrucate

Abbreviations: H2S, hydrogen sulfide; H2O2, hydrogen peroxide; NaSH, sodium hydrogen sulfide; NO, nitric oxide; ROS, reactive oxygen species; SIRT1, Sirturn-1; HF, heart failure. * Corresponding author. 826 Zhangheng Road, Shanghai 201203, China. Fax: +86-21-51980018. E-mail address: [email protected]; [email protected] (Y.Z. Zhu). 1 These authors contributed equally to this work.

sulfurtransferase. H2S is identified as the third gasotransmitter like nitric oxide (NO) and carbon monoxide (CO) in the cardiovascular system of mammals [8]. There are evidences indicating that H2S has the antioxidant properties [9,10]. H2S can attenuate oxidative stress in hypoxic pulmonary hypertension and remove oxygen free radicals to protect the heart against ischemic injury [11,12]. Sirtuin-1 (SIRT1) is a highly conserved nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase that plays a critical role in aging, inflammation and cell cycle progression [13]. SIRT1 mediates cellular homeostasis through deacetylating many target proteins, such as eNOS, PGC-1α, and FOXOs [14–16]. Studies suggest that SIRT1 is sensitive to intracellular redox radicals and may be a key regulator of cell survival in response to oxidative stress [17]. SIRT1 prevents p53-dependent apoptosis resistance to oxidative stress [18,19]. SIRT1 promotes cell survival through increasing the ability of FOXO3 in response to oxidative stress [20,21]. Although both of H2S and SIRT1 are shown to play a significant role in apoptosis, there is no information about the interaction between them. In order to test this puzzle, the H9c2 cardiomyocytes were exposed to hydrogen peroxide (H2O2) and the role of SIRT1 was determined in the presence of H2S or not. In this study, we firstly found that H2S protected H9c2 cardiomyocytes against H2O2 induced apoptosis through SIRT1 pathway.

http://dx.doi.org/10.1016/j.niox.2014.11.006 1089-8603/© 2014 Elsevier Inc. All rights reserved.

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2. Methods and materials 2.1. Cell culture and treatments H9c2 cardiomyocytes (American Type Culture Collection) were grown in completed Dulbecco’s minimum essential medium supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin and 100 U/ml penicillin at 37 °C in 5% CO2. H2O2 150 μmol/L was used to induce apoptosis. Sodium hydrogen sulfide (NaSH) was added 4 h before H2O2 treatment. 2.2. Chemicals and antibodies The following antibodies were used: anti-SIRT1 antibody (CST); anti-caspase 9 antibody (CST); anti-ATPase antibody (Santa Cruz); anti-SOD antibody, anti-GPx antibody and anti-GST antibody (Santa Cruz); anti-PGC 1α antibody, anti-p-eNOS antibody and antieNOS antibody (CST); anti-GAPDH antibody (Santa Cruz). All drugs were purchased from Sigma unless otherwise stated.

antibody and detected with chemiluminescence (ECL, Millipore). Detection of GAPDH was used as a loading control. Quantitative analysis of immunoblotted bands was performed using Alpha Ease software. 2.6. ATP levels The ATP levels of H9c2 cardiomyocytes were measured by luciferin-luciferase method with ATP Assay Kit (Beyotime Institute of Biotechnology) according to the manufacturer’s protocol. Briefly, after H2O2 treated, H9c2 cardiomyocytes pretreated with or without NaSH (25–100 μmol/L) or Ex 527 (10 μmol/L) were schizolysised and centrifuged at 12,000 g for 5 min. In 96-well plates, 100 μl of each supernatant was mixed with 100 μl ATP detection working dilution. Luminance was measured by the Microplate reader (Infinite 1000, TECAN). Standard curves were also generated and the protein concentration was determined using BCA Protein Assay Kit (Pierce). Total ATP levels were expressed as μmol/mg protein. 2.7. Detection of intracellular oxidants generation

2.3. Cell viability (MTT assay) H9c2 cardiomyocytes were seeded at a density of 1 × 104 cells/ well in 96-wells micro-plates and cultured with NaSH (25, 50 and 100 μmol/L) and Ex 527 (10 μmol/L). After 4 h, H2O2 was added and maintained for 4 h. At the end of experiment point, the samples were incubated with 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-Htetrazolium bromide (MTT) at a final concentration of 0.5 mg/ml. After 4 h incubation at 37 °C, 5% CO2, 150 μl DMSO was added in each well to dissolve the formazan product. Absorbance at 570 nm was measured by the Microplate reader (Infinite 1000, TECAN). Data were then normalized to the control group and were presented as the percentage of control. 2.4. Analysis of apoptosis The activation of caspase-9 in H9c2 cardiomyocytes was measured with caspase-9 fluorometric assay kit (BioVison) according to the manufacturer’s protocol. Briefly, after H2O2 treated, H9c2 cardiomyocytes pretreated with or without NaSH (100 μmol/L) or Ex 527 (10 μmol/L) were resuspended in 50 μl chilled Cell Lysis Buffer and incubated in ice for 10 min. In 96-well plates, 50 μl of each sample was mixed with 50 μl 2 × Reaction Buffer (containing 10 mmol/L DTT and 50 μmol/L LEHD-AFC). Fluorescence intensity was measured by the Microplate reader (Infinite 1000, TECAN) with a 400-nm excitation filter and 505-nm emission filter. Standard curves were also generated and the protein concentration was determined using BCA Protein Assay Kit (Pierce). Fold-increase in caspase-9 activity was determined by comparing these results with the level of the control group. Apoptotic cells were also identified by staining with Hoechst 33258 (Beyotime Institute of Biotechnology). Samples were viewed in a darkness room with fluorescence microscope (Zeiss). Images were taken from three randomly selected fields. 2.5. Western blot H9c2 cardiomyocytes were lysed in the lysis buffer (Pierce) and centrifuged at 12,000 g for 30 min at 4 °C. The protein concentration was determined using BCA Protein Assay Kit (Pierce). Proteins samples (30 μg) were separated with 10% SDS-PAGE and transferred to nitrocellulose membranes (Millipore). The membranes were blocked with 5% non-fat milk (0.1% Tween-20) for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight. After washing with TBST (0.1% Tween-20), the membranes were incubated with a horseradish peroxidase conjugated secondary

Intracellular oxidants levels were detected by measuring the oxidative conversion of cell permeable 2′,7′-dichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF). After drug treatment, cells were washed twice by phosphate-buffered saline and then incubated with 10 μmol/L DCFA-DH (Beyotime Institute of Biotechnology) at 37 °C for 20 min according to the manufacturer’s instructions. DCF fluorescence was detected by the Microplate reader (Infinite 1000, TECAN) at the excitation wavelength of 488 nm and the emission wavelength of 535 nm. At the meantime, images were taken from three randomly selected fields. 2.8. Lactate dehydrogenase (LDH) leakage rate H9c2 cardiomyocytes were seeded at a density of 1 × 104 cells/ well in 96-wells micro-plates and cultured with NaSH (25, 50 and 100 μmol/L) and Ex 527 (10 μmol/L). After 4 h, H2O2 was added and maintained for 4 h. After drug treatment, the platelet was centrifuged at 400 g for 5 min. The supernatants were used to detect the LDH leakage rate according to the manufacturer’s protocol (Beyotime Institute of Biotechnology). 2.9. Statistical analysis Statistical analysis was performed using one-way ANOVA analysis or Student’s t test. Post hoc pairwise comparisons were performed using GraphPad software. All the data were presented as means ± SEM. A P value < 0.05 was considered significant. 3. Results 3.1. EX 527 prevented the anti-apoptotic effect of NaSH The first step of our experiments is to confirm that whether the anti-apoptosis effect of NaSH has the relationship with SIRT1. H9c2 cardiomyocytes were exposed to 150 μmol/L H2O2 for 4 h to induce apoptosis. It played different protective effects of H 2 S for cardiomyocytes at different times (Fig. 1A). When pretreatment of 4 h, H2S achieved the optimal cytoprotection effect. And 25, 50 and 100 μmol/L NaSH (an exogenous H2S donor) were added for 4 h before H2O2 treatment. Then the cell viability was measured by MTT assay. NaSH could protect H9c2 cardiomyocytes against H2O2induced oxidative stress in a dose-dependent manner, but this effect was attenuated by 10 μmol/L SIRT1 inhibitor, Ex 527 (Fig. 1C and 1D). At the meantime, NaSH could reduce the LDH leakage rate in H2O2-induced oxidative stress in a dose-dependent manner, but this

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Fig. 1. NaSH decreased the apoptosis through the SIRT1 pathway in H9c2 cardiomyocytes. (A) Cell viability after NaSH treatment in different pre-treated time point. H9c2 cardiomyocytes were pre-treated with NaSH (100 μmol/L) for 1–8 h. (B) LDH leakage rate after NaSH treatment. H9c2 cardiomyocytes were pre-treated with or without NaHS (25, 50 and 100 μmol/L) and Ex 527 (10 μmol/L) for 4 h, and then treated with 150 μmol/L H2O2 for 4 h. (C and D) Cell viability after NaSH treatment. H9c2 cardiomyocytes were pre-treated with or without NaSH (25, 50 and 100 μmol/L) and Ex 527 (10 μmol/L) for 4 h, and then treated with 150 μmol/L H2O2 for 4 h. (E) Measurement of the apoptotic cell in H9c2 cardiomyocytes with indicated treatments. H9c2 cardiomyocytes were treated with or without NaSH (25–100 μmol/L) and Ex 527 (10 μmol/L) for 4 h, then incubated with 150 μmol/L H2O2 for 4 h. Hochest 33258 (blue) was used to detect the karyon morphology of H9c2 cardiomyocytes. The arrows indicated the apoptotic cells. Data represent mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01 compared with H2O2 treated group. ##P < 0.01.

effect was blocked by 10 μmol/L SIRT1 inhibitor, Ex 527 (Fig. 1B). In addition, we used Hoechst 33258 to determine apoptotic cell. As shown in Fig. 1E, treatment with different concentrations of NaSH (25, 50 and 100 μmol/L) for 4 h dose-dependently decreased nuclear shrinkage and improved cells apoptosis induced by H2O2. In contrast, pretreatment with Ex 527 (10 μmol/L) for 1 h promoted the apoptosis compared with the 100 μmol/L NaSH treated group. Caspase 9 is a member of the caspase family, which functions as the initiator and effector of apoptosis. Pro-caspase 9 is cleaved by apoptosis stimulation and it is the major initiator of caspase in programmed cell apoptosis [22]. To further determine the relationship between H2S and SIRT1, the activity and the expression of caspase 9 were also be measured. It showed that treatment of NaSH

caused a large increase in the activity of caspase 9. Treatment with NaSH also resulted in an increase in the protein level of procaspase 9 and a decrease in the expression of cleaved-caspase 9. However, these effects were attenuated by the addition of Ex 527 (Fig. 2). The data indicated that H2S reduced apoptosis through SIRT1 pathway. 3.2. NaSH increased SIRT1 level in H2O2 induced cells In order to study the mechanism for anti-apoptotic effect of H2S with SIRT1, we determined the effects of NaSH on SIRT1 protein levels. As shown in Fig. 3A, treatment of H9c2 cardiomyocytes with different H2O2 (100, 150 and 200 μmol/L) for 4 h dose-dependently

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Fig. 2. NaSH decreased the activity of caspase 9 via the SIRT1 pathway in H9c2 cardiomyocytes. (A) The protein expression of pro-caspase 9 and cleaved-caspase 9 after NaSH or Ex 527 treatment. Lysates of H9c2 cardiomyocytes were subjected to Western Blot with anti-caspase 9 antibodies. GAPDH was used as a loading control. The protein levels of caspase 9 were quantified and analyzed by Alpha Ease software. (B) The activity of caspase 9 after NaSH treatment. H9c2 cardiomyocytes were treated with or without NaSH (25–100 μmol/L) and Ex 527 (10 μmol/L) for 4 h, followed by 150 μmol/L H2O2 stimulation for 4 h. Data represent mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01 compared with H2O2 treated group. ##P < 0.01.

decreased the SIRT1 level. The time course study showed that treatment with H2O2 for different time (1, 2 and 4 h) decreased the SIRT1 level in a time dependent manner. In contrast, pretreatment with NaSH (25, 50, 100 μmol/L) for 4 h significantly inhibited H2O2induced SIRT1 decreased (Fig. 3). In addition, this effect was attenuated by pretreatment with 10 μmol/L Ex 527 for 1 h (Fig. 4A). In the other hand, NaSH could increase SIRT1 levels dosedependently without the existing of H2O2 (Fig. 4B). Endogenous H2S was generated by CSE in cardiomyocytes. In order to determine the SIRT1 response in the absence of endogenous H 2 S, H9c2 cardiomyocytes was treated with the CSE inhibitor PAG under oxidative stress. Cell viability was reduced in the treatment of PAG compared with the treatment of H2O2 (Fig. 4C). In the meantime, PAG could reduce SIRT1 level compared with H2O2 group (Fig. 4D). The data showed that the absence of endogenous H2S could increase the damage of H2O2 and decrease the expression level of SIRT1. These results indicated that H2S might directly regulate the protein level of SIRT1 under H2O2-induced oxidative stress. 3.3. NaSH inhibited ROS generation to attenuate apoptosis through SIRT1 pathway Reactive oxygen species (ROS) accumulation is an important factor of apoptosis induced by H2O2 [23]. To determine whether the protective effect of H2S in apoptosis might be related with SIRT1, we measured the effect of NaSH on H2O2-induced intracellular oxidants generation and relative proteins expression without or with the SIRT1 inhibitor. Intracellular oxidants generation was detected by DCFH-DA probe. As shown in Fig. 5A and B, treatment with 150 μmol/L H2O2 for 4 h caused a large increase in intracellular

oxidants level. In addition, the intracellular oxidants level in the different concentration of NaSH (100, 150 and 200 μmol/L) pretreatment group was dose-dependently decreased. However, Ex 527 abolished the inhibitory effect of NaSH on ROS accumulation under oxidative stress. In order to explore the mechanism of the interaction between H2S and SIRT1 on ROS depletion, we continued to test the effect of NaSH on the expressions of superoxide dismutase (SOD), glutathione S-transferase (GST) and glutathione peroxidase (GPx) without or with the SIRT1 inhibitor. They act as antioxidants and protect cellular components from being oxidized by ROS [24]. Treatment with 150 μmol/L H2O2 for 4 h significantly decreased the protein level of SOD. On the contrary, NaSH treatment markedly increased SOD level. However, this effect was reversed by Ex 527. The similar effect was also observed in the expression of GST and GPx (Fig. 5C). 3.4. NaSH promoted the expression of ATPase, PGC-1α and eNOS via SIRT1 The regulating of mitochondrial biogenesis and function is important for preventing apoptosis induced by H2O2 [23,25]. In order to study whether the mechanism of anti-apoptotic effect of H2S might be through SIRT1 pathway on mitochondrial homeostasis, we next investigated the effects of NaSH without or with the SIRT1 inhibitor on the ATP level and the expression of ATPase, PGC-1α and eNOS under oxidative stress. ATP is the product of mitochondrial biosynthesis and transports chemical energy within cells for metabolism [26]. As shown in Fig. 6B, treatment with different concentrations of NaSH (25, 50 and 100 μmol/L) for 4 h concentration dependently increased the

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Fig. 3. NaSH elevated the expression of SIRT1 in H9c2 cardiomyocytes under oxidative stress. The levels of SIRT1 were increased by NaSH in a time dependent manner. Cell lysates of H9c2 cardiomyocytes treated with or without 100 μmol/L NaSH plus 100–200 μmol/L H2O2 for the indicated time were subjected to Western Blot using antiSIRT1 antibody. The quantitative results of SIRT1 protein levels were analyzed by Alpha Ease software. Data represent mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01 compared with H2O2 treated group. ##P < 0.01.

ATP level under oxidative stress in H9c2 cardiomyocytes. However, the promotional effect of ATP level was not observed in Ex 527 treated group (Fig. 6C). The similar effect was also shown in the expression of ATPase (Fig. 6A). PGC-1α and eNOS play a critical role in mitochondrial biogenesis and function [27,28]. In addition, studies show that PGC-1α and eNOS are the target proteins of SIRT1. Treatment with 100 μmol/L NaSH significantly increased the level of PGC-1α under oxidative stress. However, this effect was reversed by Ex 527 (Fig. 7A). As shown in Fig. 7B, the cytotoxicity of 150 μM H2O2 caused the decrease of eNOS expression; however, the expression of p-eNOS was decreased much more. After addition of NaSH, the ratio of p-eNOS/ eNOS was raised. These data showed that SIRT1 pathway contributed to the protective effect of H2S on mitochondrial function (Fig. 8). 4. Discussion Studies show that oxidative stress plays a key role in various cardiovascular diseases [3,29]. However, the mechanism and signaling pathway are poorly understood. SIRT1, a NAD + -dependent deacetylase, is an important regulator in many cellular processes, such as aging, proliferation and inflammation. It has been reported that SIRT1 suppresses cell apoptosis to protect cardiovascular function [17]. H2S, a novel third gasotransmitter, can regulate physiological functions of cardiovascular and neuronal systems. H2S is reported to be a potent actor of SIRT1 in anti-aging, but the mechanism is poorly known [30,31]. In this study, we for the first time

investigated that whether H2S had a direct anti-apoptosis effect on H9c2 cardiomyocytes through SIRT1 pathway. To identify this hypothesis, H9c2 cardiomyocytes were exposed to H2O2 to induce apoptosis. NaSH could promote cell survival and reduce LDH leakage rate in a dose-dependent manner. But the treatment with Ex 527, an effective inhibitor of SIRT1, caused an increase of cell apoptosis, suggesting that SIRT1 played a great role in the protective effect of NaSH in H9c2 cardiomyocytes. In order to identify this result, we next detected the activity and the expression of caspase 9, which are the markers of apoptosis. Although NaSH could decrease caspase 9 activity and increase the expression of procaspase 9 under oxidative stress, decrease of activity of caspase 9 and increase of pro-caspase 9 level were observed in Ex 527 treatment group. Thus, SIRT1 was associated with protective effects of NaSH in H9c2 cardiomyocytes under H2O2-induced apoptosis. Studies indicate that the expression of SIRT1 and its activity are mediated by H2S [30,32]. Suo et al. found that H2S protected against oxidative stress-mediated senescence through modulation of SIRT1 activity [33]. In our study, the expression of SIRT1 was decreased under oxidative stress, and NaSH could elevate its level in dose dependent and time dependent manner. However, Ex 527 largely reversed this effect. NaSH could increase SIRT1 level in the absence of H2O2, and the absence of endogenous H2S could reduce SIRT1 level and cell viability under oxidative stress. Results of our study indicated that NaSH directly mediated the expression of SIRT1 under oxidative stress. The sulfhydration has been proposed to emerge as a major functional modification of proteins [34]. It is believed that

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Fig. 4. NaSH increased SIRT1 expression directly. (A) The levels of SIRT1 increased by NaSH could be reversed by Ex 527. Cell lysates of H9c2 cardiomyocytes treated with or without NaSH (100 μmol/L) and Ex 527 (10 μmol/L) for 4 h, and then treated with 150 μmol/L H2O2 for 4h were subjected to Western Blot using anti-SIRT1 antibody. (B) The levels of SIRT1 could be increased by NaSH without H2O2 treatment. Cell lysates of H9c2 cardiomyocytes treated with or without NaSH (25, 50 and 100 μmol/L) for 4 h were subjected to Western Blot using anti-SIRT1 antibody. (C) Cell viability after removing endogenous H2S. H9c2 cardiomyocytes were treated with or without CSE inhibitor PAG (5 mmol/l) for 1 h and then incubated with H2O2 for 4 h. (D) Removing endogenous H2S could decrease SIRT1 expression. Cell lysates of H9c2 cardiomyocytes treated with or without PAG (5 mmol/l) for 1 h and then incubated with H2O2 for 4 h were subjected to Western Blot using anti-SIRT1 antibody. The quantitative results of SIRT1 protein levels were analyzed by Alpha Ease software. Data represent mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01 compared with H2O2 treated group. ##P < 0.01.

oxidation-sensitive cysteines are easily sulfhydrated. Zee et al. demonstrated that SIRT1 was regulated by S-glutathiolation of specific Cys residues [35]. We are currently exploring the target Cys residues of H2S on SIRT1 protein. It will open an exciting new avenue of H2S regulation and better understand the physiological role of SIRT1. Oxidative stress induced by H2O2 causes ROS overproduction. H2S, a potent reducing agent, plays an antioxidant role to quench the ROS in a wide array of cells and tissues. In addition, studies show that ROS is also regulated by SIRT1. SOD are enzymes which catalyze the dismutation of superoxide (O2−) into oxygen and hydrogen peroxide. Tanno et al. demonstrated that resveratrol increased Mn-SOD levels via nuclear SIRT1 [36]. It is best known for GST’s ability to catalyze the conjugation of the reduced form of glutathione to xenobiotic substrates for the purpose of detoxification. The main biological role of GPx is to protect the organism from oxidative damage [24]. All of superoxide dismutase (SOD), GST and GPx have a pivotal role in the detoxification of ROS. Our results showed that NaSH significantly decreased intracellular oxidants levels induced

by H2O2. Consistently, NaSH increased the expression of SOD, GST and GPx under oxidative stress. All these effects were largely reversed by Ex 527 treatment. These finding indicated that the activation of SIRT1-mediated redox signaling pathway contributed to the antioxidant function of H2S. The mitochondrial electron transport chain is the major source of ROS and mitochondrial ROS disorder can increase markedly cellular apoptosis [37]. In order to stay mitochondrial homeostasis, H2S is used as an energy substrate. A recent study showed that mammalian cells utilized H2S to generate ATP under hypoxic stress [38]. And apoptosis is mediated by cellular ATP level. ATP synthesis depends on the content of NAD+. Nicotinamide phosphoribosyltransferase (NAMPT) is an enzyme required for NAD+ synthetic salvage pathway [39]. SIRT1 plays a great role in the gene regulatory effect of NAMPT, and the inhibition of SIRT1 results in the down-regulatory of NAMPT and the decrease of ATP generation [40]. The present study showed that NaSH significantly increased cellular ATP levels and the expression of ATPase under oxidative stress, but this effect was attenuated by Ex 527. These results in H9c2

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Fig. 5. NaSH decreased the reactive oxygen species (ROS) accumulation through SIRT1 in H9c2 cardiomyocytes. (A) Intracellular oxidants level changes caused by NaSH. H9c2 cell were treated with or without NaSH (100 μmol/L) and Ex 527 (10 μmol/L) for 4 h, and then treated with 150 μmol/L H2O2 for 4 h. DCFH-DA was incubated with cells for 20 min at 37 °C and ROS level was represented by DCF intensity (B). (C) Expression levels of SOD, GST and GPx after NaSH treatment. Cell lysates of H9c2 cardiomyocytes treated with or without NaSH (100 μmol/L) and Ex 527 (10 μmol/L) for 4 h, and then treated with 150 μmol/L H2O2 for 4 h were subjected to Western Blot using anti-SOD, anti-GST and anti-GPx antibodies. The quantitative results of SOD, GST and GPx protein levels were analyzed by Alpha Ease software. Data represent mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01 compared with H2O2 treated group. ##P < 0.01.

cardiomyocytes were similar to that in skeletal muscle cells [41], which suggest that inhibition of SIRT1 activity decreased intracellular ATP levels. PGC-1α is a transcriptional coactivator that regulates the genes involved in energy metabolism, which is a regulator of mitochondrial biogenesis and function [16,42,43]. eNOS catalyzes the production of nitric oxide (NO), which is a key regulator of many physiological functions [44]. eNOS and PGC-1α play a critical role in mediating mitochondrial biogenesis, which are important for antiapoptosis. Our results suggested that NaSH increased the expression of eNOS and PGC-1α after H2O2 treatment, but Ex 527 could decrease the eNOS activity and PGC-1α expression. Consistently, eNOS and PGC-1α are the target proteins of SIRT1. These findings indicated that NaSH associated with the protective effect of SIRT1 in H9c2 cardiomyocytes under oxidative stress.

In summary, we demonstrated for the first time that H2S inhibited apoptosis induced by H2O2 via SIRT1 pathway. These novel results may help to understand the important physiological role of H2S and light a new strategy for oxidative stress-related diseases.

Acknowledgments This work was supported by the National Basic Research Program of China (973 Program, No. 2010CB912603); The key laboratory program of the Education Commission of Shanghai Municipality (ZDSYS14005);The Key Program of Shanghai Committee of Science and Technology in China (No. 10431900100); The National Science and Technology Major Project (No. 2012Z X 09501001-001-003).

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Fig. 6. NaSH increased ATP level in H9c2 cardiomyocytes. (A) The level of ATPase protein increased by NaSH. Cell lysates of H9c2 cardiomyocytes treated with or without NaSH (100 μmol/L) and Ex 527 (10 μmol/L) for 4 h, and then treated with 150 μmol/L H2O2 for 4 h were subjected to Western Blot using anti-ATPase antibodies. The quantitative results of ATPase protein levels were analyzed by Alpha Ease software. (B) ATP levels of H9c2 were measured after being treated with or without NaSH (25–100 μmol/ L) and Ex 527 (10 μmol/L) for 4 h, followed by 150 μmol/L H2O2 incubation for 4 h. Data represent mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01 compared with H2O2 treated group. ##P < 0.01.

Fig. 7. NaSH promoted the expression of PGC-1α and activity of eNOS through SIRT1 pathway in H9c2 cardiomyocytes. Expression levels of PGC-1α (A) and p-eNOS/eNOS (B) after NaSH treatment. Cell lysates of H9c2 cardiomyocytes treated with or without NaSH (100 μmol/L) and Ex 527 (10 μmol/L) for 4 h, and then treated with 150 μmol/L H2O2 for 4 h were subjected to Western Blot using anti-PGC-1α and anti-eNOS antibodies. The quantitative results of PGC-1α, p-eNOS and eNOS protein levels were analyzed by Alpha Ease software. Data represent mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01 compared with H2O2 treated group. ##P < 0.01.

Please cite this article in press as: Dan Wu, et al., Hydrogen sulfide protects against apoptosis under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes, Nitric Oxide (2014), doi: 10.1016/j.niox.2014.11.006

ARTICLE IN PRESS D. Wu et al./Nitric Oxide ■■ (2014) ■■–■■

Fig. 8. Schematic diagram of H2S protected against apoptosis through SIRT1 pathway under oxidative stress. Under oxidative stress, the decline of SIRT1 expression resulted in apoptosis. H2S increased the expression of SIRT1, which could lead to the activation of the downstream pathway. H2S could enhance the expression of PGC1α and eNOS, inducing the ATP generation and ROS scavenging. SIRT1 inhibition attenuated the protective effect of H2S against apoptosis.

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Please cite this article in press as: Dan Wu, et al., Hydrogen sulfide protects against apoptosis under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes, Nitric Oxide (2014), doi: 10.1016/j.niox.2014.11.006