Toxicology in Vitro 28 (2014) 1461–1473

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a-Lipoic acid protected cardiomyoblasts from the injury induced by sodium nitroprusside through ROS-mediated Akt/Gsk-3b activation Surong Jiang a,b, Weina Zhu a, Jun Wu a, Chuanfu Li c, Xiaojin Zhang a, Yuehua Li d, Kejiang Cao b, Li Liu a,⇑ a

Department of Geriatrics, First Affiliated Hospital with Nanjing Medical University, Nanjing 210029, China Department of Cardiology, First Affiliated Hospital with Nanjing Medical University, Nanjing 210029, China c Department of Surgery, East Tennessee State University, Johnson City, TN 37614, United States d Department of Pathophysiology, Nanjing Medical University, Nanjing 210029, China b

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Article history: Received 23 March 2014 Accepted 14 August 2014 Available online 2 September 2014 Keywords: a-Lipoic acid Sodium nitroprusside Nitric oxide Apoptosis Akt/Gsk-3b signaling Reactive oxygen species

a b s t r a c t It has been long noted that cardiac cell apoptosis provoked by excessive production of nitric oxide (NO) plays important roles in the pathogenesis of variant cardiac diseases. Attenuation of NO-induced injury would be an alternative therapeutic approach for the development of cardiac disorders. This study investigated the effects of a-lipoic acid (LA) on the injury induced by sodium nitroprusside (SNP), a widely used NO donor, in rat cardiomyoblast H9c2 cells. SNP challenge significantly decreased cell viability and increased apoptosis, as evidenced by morphological abnormalities, nuclear condensation and decline of mitochondrial potential (DWm). These changes induced by SNP were significantly attenuated by LA pretreatment. Furthermore, LA pretreatment prevented the SNP-triggered suppression of Akt and Gsk-3b activation. Blockade of Akt activation with triciribin (API) completely abolished the cytoprotection of LA against SNP challenge. In addition, LA moderately increased intracellular ROS production. Interestingly, inhibition of ROS with N-acetylcysteine abrogated Akt/Gsk-3b activation and the LA-induced cytoprotection following SNP stimulation. Taken together, the results indicate that LA protected the SNP-induced injury in cardiac H9c2 cells through, at least in part, the activation of Akt/Gsk-3b signaling in a ROS-dependent mechanism. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction It is well known that the cardiac cell apoptosis contributes to a variety of cardiac disorders, such as cardiac ischemia/reperfusion, heart failure, diabetes, and dilated cardiomyopathy (Aharinejad et al., 2008; Cheng et al., 2010; Frustaci et al., 2000; Zhang et al., 2011). Although there are multiple pathogenic molecular events leading to apoptosis in cardiac cells, one of the most important culprits is the excessive produced nitric oxide (NO) (Andreka et al., 2001; Li et al., 2007; Niu et al., 2011; Shafaroodi et al., 2010; Taimor et al., 2000). NO itself is far less toxic at physiological relevant concentrations and is essential for the regulation of a diverse range of physiological processes such as modulating blood flow, thrombosis, and neural activity. These physical roles exerted by NO are mediated through at least two distinct pathways: cGMPdependent and cGMP-independent. The production of cGMP by ⇑ Corresponding author. Address: Department of Geriatrics, First Affiliated Hospital with Nanjing Medical University, Guangzhou Road 300, Nanjing 210029, China. Tel.: +86 25 83718836x5021; fax: +86 25 83724440. E-mail address: [email protected] (L. Liu). http://dx.doi.org/10.1016/j.tiv.2014.08.006 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.

guanylate cyclase is the major signal transduction mechanism of NO, while cGMP-independent effects occur mainly via S-nitrosylation (Martinez-Ruiz et al., 2011; Ziolo, 2008). However, excessive NO is believed to be a mediator of cardiotoxicity, which will cause cell apoptosis by increasing mitochondrial permeability, changing structure and functions of important proteins, and disrupting the calcium transportation system (Lim et al., 2009; Tatsumi et al., 2000; Wang et al., 2007b). Therefore, it is needed to develop effective means for the management of NO-induced apoptosis on cardiomyocytes. The decreased levels of phosphorylated Akt were observed in variant cells (e.g. HL-60 cells and BGC-823 cells) that carrying high levels of NO (Sang et al., 2011; Wang et al., 2007a), suggesting a possible involvement of Akt suppression in the NO-induced cytotoxicity. Akt is a serine/threonine kinase, which coordinates a variety of intracellular signals, controls cell responses to extrinsic stimuli and regulates cell proliferation and survival (Li et al., 2010; Song et al., 2009). Phosphorylation of Thr308 and Ser473 on Akt is necessary for its full activation. Activated Akt has been shown to protect hearts from variant stimuli such as myocardial ischemia/reperfusion, sepsis/septic shock and doxorubicin

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challenge (Das et al., 2011; Song et al., 2009; Zhou et al., 2011). Glycogen synthase kinase 3b (GSK-3b) is one of the important downstream targets of Akt, which is phosphorylated by Akt at serine 9 and inactivated (Das et al., 2011; Song et al., 2009; Zhou et al., 2011). Alpha-lipoic acid (LA) is a naturally occurring dithiol compound synthesized enzymatically in the mitochondria and is also available as a dietary supplement. LA has been shown an effective protection in cardiac diseases such as cardiac ischemia/reperfusion and diabetic cardiomyopathy (He et al., 2012; Navarese et al., 2011). The mechanisms responsible for the cardioprotection of LA involve antioxidant property and activation of prosurvival signaling pathways including Akt/Gsk-3b signaling (Wang et al., 2011b, 2010). Interestingly, though LA has long been considered as an antioxidant (Bobermin et al., 2013; Della Croce et al., 2003), evidence suggest that LA acts as a powerful antioxidant only following long-term treatment, whereas it may function as a mild prooxidant following short-term treatment (Dicter et al., 2002). In supporting this, we and others have demonstrated that LA can moderately increase intracellular ROS content, which was positively correlated with the improvement of cell survival and proliferation (Wang et al., 2011a, 2010; Yalcin et al., 2010; Yao et al., 2012). Moreover, we reported recently that the moderate ROS induction is required for the LA-induced Akt activation (Wang et al., 2011b, 2010). Therefore, it is possible, that LA may protect cardiac cells from excessive NO-induced injury through ROS-mediated activation of Akt/Gsk-3b signaling pathway. To test this possibility, we examined the effects of LA on cytotoxicity caused by sodium nitroprusside (SNP), a widely used NO donor (Lee et al., 2001; Nakahashi et al., 1995), in rat cardiac H9c2 cells. We observed that LA pretreatment significantly attenuated SNP-induced H9c2 cell death. This action of LA was through, at least in part, activation of Akt/Gsk-3b signaling in a ROS-dependent manner. 2. Methods and materials 2.1. Reagents LA, SNP, 20 ,70 -Dichlorofluorescein diacetate (DCFH-DA), N-acetylcysteine (NAC), Triciribin (API) and primary antibody for a-Tubulin were purchased from Sigma–Aldrich (St Louis, MO). Hoechst 33342 reagent, Dulbecco’s Modified Eagle Medium (DMEM), fetal calf serum (FCS) and DHR 123 Molecular Probe were obtained from Invitrogen (Carlsbad, CA). 5,50 ,6,60 -tetrachloro-1, 10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was obtained from Biovision Inc (Mountain View, CA). Primary antibodies for nitrotyrosine, phospho-Akt (p-Akt, Ser473) and Akt, phospho-Gsk-3b (p-Gsk-3b, Ser9) and Gsk-3b were from Cell Signaling Technology (Beverly, MA). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide reagent (MTT) was from Bio Besic Inc (Markham, Ontario, Canada). Protease inhibitor cocktail was from Roche (Mannheim, Germany). BCA protein assay kit and supersignal west pico chemiluminescent substrate were obtained from Pierce (Rockford, IL). 2.2. Cell culture and treatment Rat cardiomyoblast H9c2 cells were obtained from ATCC and maintained in DMEM supplemented with 10% FCS. Cells were assigned to four groups when they reached 75–80% confluence: (1) untreated control group (Con); (2) LA group: cells were treated with LA (500 lM) for indicated times; (3) SNP group: cells were stimulated with SNP (1 mM) for indicated hours; (4) LA + SNP group: cells were pretreated with LA (500 lM) for 12 h and

followed by stimulation with SNP (1 mM) for indicated hours. For Akt inhibition or ROS inhibition experiments, cells were treated with API (1.5 lM) or NAC (6 mM), respectively, 30 min prior to LA administration. 2.3. Examination of cell viability and morphology After stimulation with SNP (1 mM) for 24 h, cell viability was determined by MTT assay as described previously (Wang et al., 2011b; Yao et al., 2012). In another set of experiments, cell morphology was examined using phase-contrast light microscope with a magnification of 200  (Zeiss Ltd., Germany). 2.4. Examination of nuclear condensation Nuclear condensation was examined by Hoechst 33342 staining. After stimulation with SNP (1 mM) for 48 h, cells were fixed with methanol/acetone (50%/50%) for 10 min and incubated with Hoechst 33342 (0.4 lg/ml) for 5 min at room temperature. The stained nuclei were examined under a fluorescence microscope with a magnification of 400  (Zeiss Ltd., Germany). Six fields in each well were randomly examined. The condensed nuclei were expressed as a percentage of total nuclei. 2.5. Evaluation of mitochondrial transmembrane potential (DWm) We used JC-1 to evaluate DWm. JC-1 dye exhibits a DWm-dependent accumulation in mitochondria, and aggregates at high DWm from a green-fluorescent monomeric form to a red-fluorescence ‘‘J-aggregates’’. The monomeric JC-1 and ‘‘J-aggregates’’ could be detected at excitation wavelength/emission wavelength of 485 nm/528 nm and 530 nm/590 nm, respectively (Ma et al., 2010; Salido et al., 2007). In our measurement system, JC-1 (10 lg/ml) was added to the culture for 15 min in the dark after the cells were treated with SNP for 24 h. After washing thoroughly with PBS, the fluorescence intensity was detected and quantified using a fluorometer (Synergy HT, BIO-TEK, USA) at excitation wavelength/emission wavelengths as described above. DWm was expressed as the ratio of OD530 nm/590 nm over OD485 nm/528 nm. After the fluorescence quantification, the images were also captured using fluorescence microscope with a magnification of 200  (Zeiss Ltd., Germany). 2.6. Western blot Western blot was performed as previously described methods (Yao et al., 2012; Zhang et al., 2010). For the examination of PI3K/Akt activation, the cytosolic extracts were prepared from H9c2 cells after treatment with SNP (1 mM) for 1 h. In a separate experiment, cells were collected 6 h after SNP administration for the analysis of the levels of tyrosine-nitrated proteins. Briefly, Equal amount of protein extracts were separated on 10% SDS–PAGE gel and transferred onto immobilon-p membrane (Milipore Corp., Bedford, MA). After blocking, the membrane was incubated with primary antibody at 4 °C overnight and followed by incubation with an appropriate secondary antibody. The same membrane was also probed with anti-a-Tubulin for loading control. The blots were detected with ECL kit and the signals were quantified by scanning densitometry. 2.7. Measurement of intracellular ROS content Intracellular ROS content was measured by DCFH-DA assay as described in our previous studies (Wang et al., 2011b; Zhang et al., 2010). After treatment with LA for the indicated times, DCFH-DA (10 lM) was introduced to culture for 30 min. The

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intensity of fluorescence were then quantified by fluorometer (BIO-TEK, USA) at an excitation/emission wavelength of 485/ 530 nm.

analysis of variance (ANOVA). Tukey’s procedure for multiple range tests was performed. P < 0.05 was considered to be significant. 3. Results

2.8. Detection of peroxynitrite and NO levels

3.1. LA improves cell survival following SNP challenge 

The formation of peroxynitrite (ONOO ) was determined by using dihydrorhodamine 123 (DHR 123) (Chun and Low, 2012). Cells were treated with SNP (1 mM) with or without LA for 12 h. DHR 123 (50 lM) was then introduced to culture for 30 min. The intensity of fluorescence was then quantified by fluorometer (BIO-TEK, USA) at an excitation/emission wavelength of 485/530 nm. The concentration of NO was measured as nitrite (the final stable state of NO) accumulation in the culture medium using a NO assay kit according to the manufacture’s instruction. Briefly, supernatant medium was collected 24 h after SNP (1 mM) stimulation. A volume of 100 ll supernatant medium was subjected to colorimetric reaction with Griess reagents. After reaction, the NO concentration was quantified by absorbance at 570 nm using a microplate reader (BIO-TEK, USA). 2.9. Statistical analysis Results are expressed as means ± standard deviation ( x  SD). Comparison of data between groups was performed using one-way

(A)

0.5

We first examined the dose–response of SNP on the viability in H9c2 cells. As shown in Fig. 1A, 0.75 mM of SNP did not significantly change cell viability when compared with the untreated controls. However, when the concentrations of SNP increased to 1, 1.5 and 3 mM, cell viability was decreased by 43.56%, 79.46% and 96.41%, respectively, in comparison with the untreated controls (P < 0.01). Based on these results, 1 mM of SNP was selected in the following experiments. We then evaluated whether LA could improve cell survival following SNP challenge. As shown in Fig. 1B, LA pretreatment increased viability by 42.66% in SNP-challenged cells compared with the cells challenged with SNP alone (P < 0.01). The protective effects of LA on cell survival were confirmed by the observations in cell morphology (Fig. 1C). SNP challenge resulted in obviously morphological changes, including applanate and shrunken shapes with loss of cellular integrity in comparison with the untreated controls. However, LA pretreatment pronouncedly attenuated the SNP-induced morphological abnormalities. Administration alone with LA did not induce morphological changes.

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Fig. 1. LA attenuated injury in SNP-challenged H9c2 cell. (A) SNP decreased cell viability in a dose-dependent manner. H9c2 cells were stimulated with SNP at indicated concentrations for 24 h. Cell viability was examined by MTT assay. *P < 0.01 vs. untreated control (0 mM). n = 12 per group. (B) LA increased viability in SNP-challenged cells. H9c2 cells were pretreated with LA (500 lM) for 12 h and then exposed to SNP (1 mM) for 24 h. Cell viability was evaluated by MTT assay. *P < 0.01, n = 6 per group. (C) LA maintained morphological integrity in SNP-challenged cells. H9c2 cells were pretreated with LA (500 lM) for 12 h and then exposed to SNP (1 mM) for 24 h. Cell morphology was examined by phase-contrast microscope at a magnification of 200. n = 6 per group.

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3.2. LA reduces SNP-induced apoptosis Nuclear condensation is a widely used marker for apoptotic cells (Yao et al., 2012). As shown in Fig. 2A, SNP increased the percentage of condensed nuclei by 34.38-fold compared with the untreated controls (P < 0.01). However, LA pretreatment significantly decreased the SNP-induced nuclear condensation by 70.27% compared with the cells stimulated with SNP alone (P < 0.01). Solely administration with LA did not induce nuclear condensation.

The loss or collapse of DWm is a critical step for the activation of mitochondria-associated apoptotic signaling (Green and Reed, 1998). As shown in Fig. 2B, SNP decreased DWm by 12.67% compared with the untreated controls (P < 0.01). However, the SNP-induced loss of DWm was significantly attenuated by LA pretreatment. LA increased DWm by 10.54% in SNP-treated cells compared with the cells treated with SNP alone (P < 0.01). The right panel of Fig. 2B shows the representative images of JC-1 fluorescence shifting.

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LA+SNP Fig. 2. The SNP-triggered apoptosis was attenuated by LA. (A) LA attenuated the SNP-triggered nuclear condensation. H9c2 cells were pretreated with LA (500 lM) for 12 h and then exposed to SNP (1 mM) for 48 h. The nuclear condensation was examined by Hoechst 33342 staining. Images of nuclei staining were taken under fluorescence microscope at a magnification of 400 . Arrows indicate condensed and coalesced nuclei with a brighter blue fluorescence. *P < 0.01, n = 6 per group. (B) LA prevented the loss of mitochondrial potential (DWm) following SNP stimulation. H9c2 cells were treated with LA 12 h prior to stimulation with SNP. DWm was determined by the shifting of JC-1 fluorescence at 24 h after SNP (1 mM) stimulation. The levels of DWm were expressed as the ratio of OD530 nm/590 nm (red fluorescence) over OD485 nm/528 nm (green fluorescence). The right panel shows the representative images of JC-1 fluorescence shifting (200 ). Red fluorescence represents aggregated JC-1 (J-aggregates) in mitochondria and green fluorescence represents monomeric JC-1 in the cytosol. *P < 0.01, #P < 0.05, n = 4 per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.3. Effects of LA on SNP-induced NO production Fig. 3 shows that the NO content was significantly increased by 2.96-fold in SNP-challenged cells when compared with the untreated controls (P < 0.01). LA pretreatment showed no significant effects on SNP-induced NO generation (P > 0.05). Solely administration with LA did not significantly change NO level in H9c2 cells. 3.4. Effects of LA on SNP-induced peroxynitrite production As shown in Fig. 4, SNP increased peroxynitrite (ONOO) generation by 0.68-fold in cells when compared with the untreated controls (P < 0.01). However, pretreatment with LA markedly decreased the level of ONOO by 31.12% in SNP-challenged cells compared with the cells challenged with SNP alone (P < 0.01). Solely administration with LA did not significantly change ONOO level in H9c2 cells.

0.22 ± 0.02 vs. 0.23 ± 0.01, P > 0.05; p-Gsk-3b/Gsk-3b: 0.22 ± 0.01 vs. 0.25 ± 0.04, P > 0.05). We then examined whether Akt inhibition with API will affect the protective effects of LA against SNP stimulation. Indeed, the results of MTT assay showed that API abolished LA-induced protection in cell viability following SNP challenge (Fig. 7A). The viability was significantly reduced by 35.48% in API + LA + SNP group compared with LA + SNP group (P < 0.01). Furthermore, no significant difference of cell viability was observed between API + LA + SNP group and SNP group (0.20 ± 0.06 vs. 0.22 ± 0.05, P > 0.05). Consistently, API abrogated the protection of LA in cell morphology following SNP stimulation (Fig. 7B). Treatment with API alone did not change cell viability and morphology. Fig. 7C shows that API removed the protection of LA against SNP-induced apoptosis. API administration increased nuclear condensation in LA + SNP group by 2.26-fold compared with the 20

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3.5. LA inhibited the SNP-induced increase in nitrotyrosine production Fig. 5 shows that SNP challenge significantly increased the level of nitrotyrosine by 4.44-fold compared with untreated controls (P < 0.01). However, pretreatment with LA markedly decreased the level of nitrotyrosine by 64.21% in SNP-challenged cells compared with the cells challenged with SNP alone (P < 0.01). There were no significant differences in the levels of nitrotyrosine between untreated controls and LA-treated cells.

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3.7. Blockade of Akt activation abrogates the cytoprotective effects of LA To determine the role of Akt activation in the cytoprotection of LA against SNP challenge, we pretreated H9c2 cells with API (a selective inhibitor of Akt) 30 min before LA administration. As shown in Fig. 6B, API significantly decreased the levels of p-Akt and p-Gsk-3b in Con, LA, SNP and LA + SNP groups, respectively, in comparison with the untreated controls (P < 0.01). Importantly, API administration prevented the LA-induced increases in p-Akt and p-Gsk-3b levels following SNP challenge. No significant difference of p-Akt and p-Gsk-3b levels was detected between LA + SNP group and SNP group in the presence of API (p-Akt/Akt:

ONOO− generation (% of Con)

Activation of Akt is well known in promoting cell survival under variant pathological circumstances (Parcellier et al., 2008). We therefore examined whether Akt activation was involved in the cytoprotection of LA against SNP challenge. Fig. 6A shows a significant decrease in p-Akt level in SNP-challenged cells compared with the untreated controls (0.51 ± 0.06 vs. 1.00 ± 0.08, P < 0.01). Interestingly, the SNP-induced decrease in Akt phosphorylation was significantly attenuated by LA. LA pretreatment increased the p-Akt level by 79.85% in SNP-challenged cells compared with the cells challenged with SNP alone (P < 0.01). Solely treatment with LA also significantly increased p-Akt level compared with the untreated controls (P < 0.01). Gsk-3b is an important downstream target of Akt. Phosphorylation of GSK3b at the inactivating residue serine-9 by Akt results in GSK3b inactivation. Similar with the observations in Akt phosphorylation, SNP also significantly decreased p-Gsk-3b level compared with untreated controls (0.49 ± 0.05 vs. 1.00 ± 0.01, P < 0.01). However, the SNP-induced decrease in Gsk-3b phosphorylation was attenuated by 86.56% by LA pretreatment (Fig. 6A).

Fig. 3. Effects of LA on SNP-induced NO production. H9c2 cells were pretreated with LA for 12 h and then exposed to SNP (1 mM) for 24 h. The NO content in culture medium was evaluated by the Griess reaction. *P < 0.01, n = 12 per group.

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Fig. 4. LA suppressed the SNP-induced peroxynitrite generation. H9c2 cells were pretreated with LA for 12 h and then exposed to SNP (1 mM) for 12 h. The peroxynitrite (ONOO) generation was evaluated by dihydrorhodamine 123 (DHR 123). *P < 0.01, n = 6 per group.

7.0 6.0

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3.6. LA rescues the SNP-provoked decreases in the levels of phosphorylated Akt and Gsk-3b

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Fig. 5. LA attenuated the SNP-induced increase in nitrotyrosine content. H9c2 cells were pretreated with LA for 12 h and then exposed to SNP (1 mM). The cell lysates were prepared at 6 h after SNP exposure and used for Western blots. *P < 0.01, n = 3 per group.

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cells treated with LA + SNP alone (P < 0.01). Moreover, no significant difference of nuclear condensation was observed between API + LA + SNP group and SNP group (57.23 ± 1.85% vs. 59.10 ± 2.41%, P > 0.05). The results of JC-1 assay showed that API also abrogated LA-induced preservation of DWm in SNP-challenged cells (Fig. 7D).

a second messenger for Akt activation (Wang et al., 2011a; Yalcin et al., 2010). Fig. 8A shows that compared with untreated controls, ROS levels were significantly increased by 17.78%, 14.94%, 11.21%, 7.50% and 7.38%, respectively, in the cells that treated with LA for 1, 3, 6, 12 and 24 h (P < 0.01 or 0.05). To determine the role of ROS in LA-induced activation of Akt, we employed NAC (a commonly used inhibitor for ROS) in the experiments. As shown in Fig. 8B, the induction of ROS by LA was completely removed by NAC pretreatment. Most importantly, inhibition of ROS with NAC abolished the LA-induced increase in

3.8. LA-induced Akt activation requires ROS production To investigate how LA activated Akt, we examined whether LA will induce ROS production because ROS has been considered as

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Fig. 6. LA prevented the SNP-induced decreases in Akt and Gsk-3b phosphorylation. (A) LA activated Akt and Gsk-3b. H9c2 cells were pretreated with LA (500 lM) for 12 h and then exposed to SNP (1 mM). The cell lysates were prepared at 1 h after SNP exposure and used for Western blots. *P < 0.01, n = 5 per group. (B) The LA-induced activation of Akt and Gsk-3b was inhibited by API. H9c2 cells were treated with API (1.5 lM) 30 min prior to LA administration. Twelve hours after LA administration, cells were stimulated with SNP (1 mM) for 1 h. The cell lysates were then prepared for Western blots. Please note that the phosphorylation levels of Akt and Gsk-3b were comparable between LA + SNP group and SNP group in the presence of API. *P < 0.01 vs. untreated control group (Con). n = 3 per group.

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Fig. 7. Akt inhibition with API removed the cytoprotection of LA against SNP challenge. H9c2 cells were treated with API (1.5 lM) 30 min prior to LA administration. Twelve hours after LA administration, cells were stimulated with SNP (1 mM) for the indicated hours and followed by the following experiments. (A) Cell viability. Cell viability was examined by MTT assay at 24 h after SNP stimulation. *P < 0.01 and #P < 0.05, n = 5 per group. (B) Morphological study. It was performed at 24 h after SNP stimulation. Cell morphology was examined by phase-contrast microscope at a magnification of 200 . n = 6 per group. (C) Nuclear condensation. It was evaluated by Hoechst 33342 staining after the cells were exposed to SNP for 48 h. Images of nuclei staining were taken under fluorescence microscope at a magnification of 400 . Arrows indicate condensed and coalesced nuclei with a brighter blue fluorescence. *P < 0.01, n = 6 per group. (D) DWm. It was examined by JC-1 staining at 24 h after SNP challenge. DWm was determined by the shifting of JC-1 fluorescence. The levels of DWm were expressed as the ratio of OD530 nm/590 nm (red fluorescence) over OD485 nm/528 nm (green fluorescence). The right panel shows the representative images of JC-1 fluorescence shifting (200 ). *P < 0.01 and #P < 0.05, n = 4 per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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the levels of phosphorylated Akt and Gsk-3b (P < 0.01) (Fig. 8C). NAC decreased the levels of p-Akt by 48.76% and p-Gsk-3b by 48.04% in LA-treated cells compared with the cells treated with LA alone. 3.9. ROS inhibition abrogates the cytoprotection of LA Fig. 9A shows that NAC removed the LA-induced protection in cell viability following SNP challenge. NAC significantly decreased cell viability by 26.03% in LA + SNP group compared with the cells treated with LA + SNP alone (P < 0.01). Moreover, no significant difference in viability was observed between SNP group and NAC + LA + SNP group (0.22 ± 0.03 vs. 0.23 ± 0.02, P > 0.05). Consistently, NAC pretreatment abolished the LA-induced protection in cell morphology following SNP challenge (Fig. 9B). NAC pretreatment also abrogated the LA-induced protection against SNP-induced apoptosis in H9c2 cells. As shown in Fig. 9C, NAC increased nuclear condensation by 2.19-fold in LA + SNP group compared with the cells treated with LA + SNP alone. The NAC + LA + SNP group showed comparable nuclear condensation with SNP group (56.06 ± 1.38% vs. 59.10 ± 2.42%, P > 0.05). Furthermore, NAC abolished the LA-induced preservation in DWm following SNP challenge. As shown in Fig. 9D, DWm was significantly lower in NAC + LA + SNP group compared with LA + SNP group (P < 0.01). Treatment solely with NAC showed no significant effects on cell viability, morphology, nuclear condensation and DWm. 4. Discussion We demonstrated in the present study that LA pretreatment resulted in a significant attenuation of SNP-induced injury in

cardiomyoblasts. We also observed that LA pretreatment activated Akt/Gsk-3b signaling following SNP stimulation. Moreover, inhibition of Akt abrogated LA-induced protection against SNP challenge. We confirmed that LA-induced activation of Akt/Gsk-3b signaling was mediated by a moderate production of ROS, which served as a signaling molecule. Our results indicate that LA attenuated SNP-induced injury in cardiac H9c2 cells through ROS-mediated activation of Akt/Gsk-3b signaling. It has long been noted that overproduction of NO is existed in the myocardial tissues during cardiac ischemia/reperfusion, cardiac allograft vasculopathy, mechanical trauma and sepsis/septic shock (Hasegawa et al., 2009; Li et al., 2007; Wang et al., 2007b). These excess NO will react with superoxide anion to form reactive nitrogen species (RNS), such as peroxynitrite (ONOO). Peroxynitrite’s nitrating and oxidizing properties produce significant cellular toxicity that will cause apoptosis in cardiac cells and subsequently result in myocardial modification and destruction (Hasegawa et al., 2009). In this study, we observed a significant higher NO content, nitrotyrosine expression and cell death in SNP-challenged cells compared with untreated controls. Pretreatment with LA significantly attenuated the SNP-induced increase in the level of peroxynitrite and nitrotyrosine content, suggesting a role of LA in the suppression of ONOO generation. Importantly, LA significantly reduced the SNP-provoked cell injury as evidenced by increase in cell survival and decrease in apoptosis. We observed that SNP significantly decreased the levels of phosphorylated Akt and Gsk-3b in H9c2 cells. Akt is a serine/ threonine protein kinase. Phosphorylation at serine or threonine residues results in the activation of Akt which in turn phosphorylates and inactivates its downstream targets including Gsk-3b.

J-aggregates

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LA 8 7 6 5 4 3 2 1 0

#

*

*

*

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LA

API

SNP

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API+ LA+ SNP

SNP

LA+SNP

API+ LA+SNP Fig. 7 (continued)

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Decreased phosphorylation of Akt and Gsk-3b has been demonstrated to mediate the endotoxin-induced myocardial injury, pleiotrophin-potentiated cardiomyocyte apoptosis and local anesthetics-induced neurotoxicity (Jiang et al., 2013; Wang et al., 2012; Zhou et al., 2011). Conversely, increased phosphorylation of Akt and Gsk-3b has been shown to promote cell survival under variant pathological stimuli (Aikawa et al., 2000; Matsui et al., 1999; Wang et al., 2012; Zhou et al., 2011). Akt has been shown to exert protective effects through multiple pathways. For example, it is an upstream regulator of apoptotic signaling and antiapoptotic molecules (Datta et al., 1999; Tsuruta et al., 2002). In addition, it has been reported that. Akt activation mediates nitrative/oxidative stress suppression via improving antioxidants capacity such as SOD (Ji et al., 2010). Indeed, we observed that LA prevented SNP-provoked decreases in the levels of phosphorylated Akt and Gsk-3b in H9c2 cells. Furthermore, inhibition of Akt with API, a widely used and highly selective inhibitor for Akt phosphorylation (Yang et al., 2004), abrogated the LA-induced cytoprotection against SNP-triggered H9c2 cell death. Our data suggest that the cytoprotection of LA against SNP stimulation was through an Akt-dependant signaling pathway. Extensive evidence indicates that the role of Akt in regulating cell survival involves the inter-connection with mitochondria, which are also recognized as important players in apoptotic events

(A)

(B)

4000 3500

*

*

*

#

#

ROS Contents

ROS Contents

(Parcellier et al., 2008). The prolonged opening of the pore responsible for mitochondrial permeability transition (MPT) will cause the dissipation of the DWm which in turn leads to the release of pro-apoptotic proteins from mitochondrial inter-membrane space (Chelli et al., 2004). The decrease or even collapse of DWm reflects the point of no return of the process of mitochondria-associated apoptosis. Interestingly, active Akt has been shown to prevent the disruption of DWm by decreasing the mitochondrial out membrane permeability (Yamaguchi and Wang, 2001). Indeed, as demonstrated in this study, pretreatment with LA activated Akt and prevented the loss of DWm in SNP-challenged H9c2 cell. Moreover, inhibition of Akt with API abrogated the LA-induced preservation of DWm following SNP stimulation, suggesting an inter-play between Akt and mitochondria in the cytoprotection of LA against SNP challenge. We then asked how LA activated Akt in H9c2 cells. Evidence indicates that transient or moderate ROS production serves as a second messenger for Akt activation in variant types of cells such as neurons, hepatocytes and hematopoietic stem cells (Wang et al., 2011a, 2010; Yalcin et al., 2010; Yao et al., 2012). Comparing with other non-classical antioxidants such as flavonoids, LA meets all the criteria for an ideal antioxidant. In addition to its direct radical scavenging properties in both aqueous and lipid portions of the cell, LA was reported to exert indirect anti-oxidant properties by

3000 2500 2000 1500 1000 500 0

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p-Akt Akt p-Gsk-3 β Gsk-3 β α- tubulin 1.4

*

1.4

*

p-Gsk-3b/Gsk-3b

p-Akt/Akt

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Fig. 8. LA-induced Akt activation required ROS production. (A) LA moderately and transiently increased ROS contents within H9c2 cells. H9c2 cells were treated with LA (500 lM) for indicated hours. The intracellular ROS contents were examined by DCFH-DA assay. *P < 0.01 and #P < 0.05 compared with untreated control (0 h group), n = 4 per group. (B) NAC inhibited the ROS induction by LA. H9c2 cells were pretreated with NAC (6 mM) for 30 min followed by administrated with LA (500 lM) for 1 h. The intracellular ROS contents were examined by DCFH-DA assay. *P < 0.01, n = 6 per group. (C) ROS inhibition with NAC abrogated the LA-induced activation of Akt and Gsk-3b. H9c2 cells were pretreated with NAC (6 mM) for 30 min and then administrated with LA. Thirteen hours after LA administration, cells were collected for Western blot analysis. *P < 0.01, n = 3 per group.

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(A)

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LA Apoptotic Cells (%)

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70 60 50

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*

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NAC+ LA+ SNP

Fig. 9. ROS inhibition with NAC abrogated the LA-induced cytoprotection against SNP-provoked death in H9c2 cells. H9c2 cells were pretreated with NAC (6 mM) for 30 min and then administrated with LA (500 lM). Twelve hours after LA administration, cells were stimulated with SNP (1 mM) for indicated hours and followed by the following measurements. (A) Cell viability. Cell viability was examined by MTT assay at 24 h after SNP stimulation. *P < 0.01, n = 5 per group. (B) Morphological study. It was performed at 24 h after SNP stimulation. Cell morphology was examined by phase-contrast microscope at a magnification of 200 . n = 6 per group. (C) Nuclear condensation. It was evaluated by Hoechst 33342 staining after the cells were exposed to SNP for 48 h. Images of nuclei staining were taken under fluorescence microscope at a magnification of 400 . Arrows indicate condensed and coalesced nuclei with a brighter blue fluorescence. *P < 0.01, n = 6 per group. (D) DWm. It was examined by JC-1 staining at 24 h after SNP challenge. The levels of DWm were expressed as the ratio of OD530 nm/590 nm (red fluorescence) over OD485 nm/528 nm (green fluorescence). *P < 0.01 and #P < 0.05, n = 4 per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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J-aggregates

Monomeric JC-1

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(D) Con

LA #

8 *

7

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*

Ψm

6 5

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4 3 2 1 0

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NAC

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SNP

NAC+ LA+ SNP

LA+SNP

NAC+ LA+SNP Fig. 9 (continued)

(1) regenerating of other antioxidants such as GSH (Bustamante et al., 1998; Khanna et al., 1999); (2)chelating redox-active metals (Ou et al., 1995;Suh et al., 2005); (3) regulating signaling pathways. As examples, we and others have reported that the antioxidant property of LA is due to its mild prooxidant activity (Dicter et al., 2002), which could in turn leads to its modulation of signal transduction and gene transcription to improve cellular antioxidant status (Fujita et al., 2008; Moini et al., 2002b; Wang et al., 2011b; Yao et al., 2012). Indeed, we observed in the present study that LA moderately increased ROS content in H9c2 cells. So far little is known about how LA increases ROS production. We have reported recently that most DCF-fluorescence spots in LA-treated cardiac cells were colocalized with MitoTrackerÒ Red CMXRos, a marker for mitochondria (Yao et al., 2012). In addition, LA has been shown to promote mitochondrial permeability transition in permeabilized hepatocytes and isolated rat liver mitochondria (Moini et al., 2002a; Saris et al., 1998), indicating that mitochondria-related mechanism is quite probably involved in the ROS induction by LA. More significantly, we found that ROS inhibition with NAC completely abrogated LA-induced activation of Akt. Also, NAC abolished the LA-induced protection against SNP-provoked cell death. The data suggest that LA protected H9c2 cells from SNP challenge through ROS-mediated activation of Akt/Gsk-3b signaling. In conclusion, our results demonstrate that pretreatment with LA protected H9c2 cells from SNP-triggered injury. This action of LA was mediated by the activation of Akt/Gsk-3b signaling through a ROS-dependent mechanism.

Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.

Acknowledgements This work was supported by National Natural Science Foundation of China (30972856, 81071752, 81071067 and 81170321), Jiangsu Province’s outstanding Medical Academic Leader program (LJ201124), post-graduate innovation projects of Jiangsu Province (CXZZ11_0700), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and a project funded by Collaborative Innovation Center for Cardiovascular Disease Translational Medicine. References Aharinejad, S., Andrukhova, O., Lucas, T., Zuckermann, A., Wieselthaler, G., Wolner, E., Grimm, M., 2008. Programmed cell death in idiopathic dilated cardiomyopathy is mediated by suppression of the apoptosis inhibitor Apollon. Ann. Thorac. Surg. 86, 109–114, discussion 114.

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