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Alcohol and Alcoholism Advance Access originally published online on November 25, 2008
Alcohol and Alcoholism 2009 44(1):20-24; doi:10.1093/alcalc/agn098
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© The Author 2008. Published by Oxford University Press on behalf of the Medical Council on Alcohol. All rights reserved

Ethanol Prevents Oxidant-Induced Mitochondrial Permeability Transition Pore Opening in Cardiac Cells

Kunyan Zhou1,{dagger}, Lei Zhang1,{dagger}, Jinkun Xi1,2{dagger}, Wei Tian1 and Zhelong Xu2,*

1 Heart Institute, North China Coal Medical University, Tangshan, Hebei 063000, China and
2 Department of Anesthesiology, University of North Carolina, Chapel Hill, NC 27599, USA

* Corresponding author: Department of Anesthesiology, University of North Carolina, Chapel Hill, NC 27599, USA. Tel: +1-919-843-4174; Fax: +1-919-843-3805; E-mail: zxu{at}aims.unc.edu

Received 5 August 2008; first review notified 9 September 2008; in revised form 23 September 2008; accepted 16 October 2008; advance access publication 25 November 2008


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Material and Methods
 Results
 Discussion
 References
 
Aims: The purpose of this study was to determine if ethanol prevents the mitochondrial permeability transition pore (mPTP) opening via glycogen synthase kinase 3β (GSK-3β). Methods: Cardiac H9c2 cells were exposed to ethanol (10–1000 µM) for 20 min. GSK-3β activity was determined by measuring its phosphorylation at Ser9. Mitochondrial membrane potential ({Delta}{Psi}m) was assessed by imaging (confocal microscopy) H9c2 cells loaded with tetramethylrhodamine ethyl ester (TMRE). To activate GSK-3β, cells were transfected with constitutively active GSK-3β (GSK-3β-S9A-HA) mutant plasmid. Results: Treatment of cardiac cells with low doses of ethanol (10–500 µM) significantly enhanced GSK-3β phosphorylation, indicating that ethanol can inactivate GSK-3β in H9c2 cells. The effect of ethanol on GSK-3β activity was reversed by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and ethanol could enhance Akt phosphorylation, implying that the PI3K/Akt pathway accounts for the action of ethanol. Ethanol prevented oxidant (H2O2)-induced loss {Delta}{Psi}m, an effect that was reversed by LY294002, indicating that ethanol can modulate the mPTP opening caused by oxidant stress through the PI3K/Akt pathway. Ethanol failed to preserve {Delta}{Psi}m in cells transfected with the constitutively active GSK-3β (GSK-3β-S9A-HA) mutant, suggesting that ethanol prevents the mPTP opening by inactivating GSK-3β. Conclusions: These data suggest that ethanol prevents the mPTP opening through inactivation of GSK-3β. The PI3K/Akt signaling pathway is responsible for inactivation of GSK-3β by ethanol.


   Introduction
TOP
 ABSTRACT
 Introduction
Material and Methods
 Results
 Discussion
 References
 
Epidemiological studies suggest that moderate alcohol consumption reduces morbidity and mortality from ischemic heart disease (Klatsky et al., 1990Go; Rimm et al., 1991Go; de Labry et al., 1992Go; Berger et al., 1999Go). The beneficial effect of ethanol on ischemic heart disease was initially proposed to be attributable to its effects on HDL cholesterol level (Fumeron et al., 1995Go), platelet (Renaud and de Lorgeril, 1992Go) and fibrinolytic activity (Ridker et al., 1994Go). However, recent studies have shown that ethanol has a direct cardioprotective effect on myocardium in various experimental models (Miyamae et al., 1998aGo, 1998bGo; Chen and Mochly-Rosen, 2001Go). Since KATP channels (Pagel et al., 2000Go; Zhu et al., 2000Go) and protein kinase C (PKC) (Miyamae et al., 1998bGo; Chen et al., 1999Go; Chen and Mochly-Rosen, 2001Go) have been proposed to be essential for ethanol-induced cardioprotection and these factors are also critical for ischemic preconditioning (Cohen et al., 2000Go), ethanol may induce cardioprotection by sharing many of the same signaling pathways and effector molecules that have been implicated in ischemic and pharmacological preconditioning (Lucas et al., 2005Go).

The mitochondrial permeability transition pore (mPTP) opening has been proposed as a critical determinant of myocardial ischemia/reperfusion injury (Suleiman et al., 2001Go; Weiss et al., 2003Go). Inhibition of the mPTP opening at reperfusion was demonstrated to be involved in the protective mechanism of ischemic preconditioning (Hausenloy et al., 2002Go, 2004Go; Javadov et al., 2003Go). The signaling mechanism linking preconditioning to inhibition of the mPTP opening may include PKC, PKG, Akt and AMP-activated protein kinase (AMPK) (Halestrap et al., 2007Go). Recently, Juhaszova et al. have proposed that the convergence of all these protective signals via inactivation of glycogen synthase kinase 3β (GSK-3β) on the mPTP may serve as a general mechanism for preconditioning-induced cardioprotection (Juhaszova et al., 2004Go). A recent study further reported that GSK-3β inactivation by postconditioning is required to prevent the mPTP opening (Gomez et al., 2008Go), further supporting the important role of GSK-3β in cardioprotection.

In this study, we hypothesized that alcohol prevents the mPTP opening by inactivating GSK-3β. We first examined if ethanol inactivates GSK-3β by detecting phosphorylation of GSK-3β at Ser9. We then sought to define the signaling mechanism by which ethanol inactivates GSK-3β. Lastly, we determined if ethanol prevents oxidant-induced mPTP opening through inactivation of GSK-3β.


   Material and Methods
TOP
 ABSTRACT
 Introduction
 Material and Methods
Results
 Discussion
 References
 
Cell culture
Rat heart tissue-derived H9c2 cardiac myoblast cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U penicillin/streptomycin at 37°C in a humidified 5% CO2–95% air atmosphere.

GSK-3β plasmid DNA
Two GSK-3β mutants, catalytically inactive GSK-3β (GSK-3β-KM-HA) and constitutively active GSK-3β (GSK-3β-S9A-HA) mutant plasmids containing HA-tag were kindly provided by Dr Morris Birnbaun, University of Pennsylvania School of Medicine. After purifying the plasmid DNA using an Endofree Maxi kit (Qiagen), transient transfections were performed on 12-well plates using Fugene6 with 2 µg DNA according to the manufacturer's instructions (Roche). Briefly, H9c2 cells were seeded in a 12-well plate at a 50% confluence. Two hours after cell seeding, cells were transfected with β-gal (pCDNA-His-LacZ) or the GSK-3β mutants using Fugene6 transfection reagents (DNA:reagent ratio = 1:3). Cells were replaced with fresh medium 24 h after the transfection. All experiments were done 48 h after transfection. The transfection efficiency test using a β-galactosidase assay kit (Invitrogen) revealed that over 80% of cells expressed β-galactosidase (>80% transfection; data not shown).

Confocal imaging of mitochondrial membrane potential ({Delta}{Psi}m)
{Delta}{Psi}m was measured by loading cardiomyocytes with tetramethylrhodamine ethyl ester (TMRE). TMRE is a cell permeable, cationic, nontoxic, fluorescent dye that specifically stains live mitochondria. TMRE is accumulated specifically by the mitochondria in proportion to membrane potential (Scaduto and Grotyohann, 1999Go). A number of studies have measured {Delta}{Psi}m by imaging cardiac cells loaded with TMRE (Akao et al., 2003Go; Jones et al., 2003Go; Förster et al., 2006Go). Briefly, cardiac cells cultured in a specific temperature-controlled culture dish were incubated with TMRE (100 nM) in a standard Tyrode solution containing (in mM) NaCl 140, KCl 6, MgCl2 1, CaCl2 1, HEPES 5 and glucose 5.8 (pH 7.4) for 10 min. Cells were then mounted on the stage of an Olympus FV 1000 laser scanning confocal microscope. The red fluorescence was excited with a 543-nm line of argon–krypton laser line and imaged through a 560-nm-long path filter. Temperature was maintained at 37°C. The images recorded on a computer were quantified using Image J.

Western blotting analysis
Equal amount of protein lysates were loaded and electrophoresed on SDS–polyacrylamide gel and transfected to a PVDF membrane. Membranes were probed with primary antibodies that recognize phosphorylation of GSK-3β and Akt. Each primary antibody binding was detected with a secondary antibody and visualized by the enhanced chemiluminescence (ECL) method. Equal loading of samples were confirmed by reprobing membranes with antibodies that recognize total proteins or tubulin.

Experimental protocols
Cultured cells were washed twice with PBS, and then incubated in Tyrode solution for 2 h prior to experiments. To examine the effect of ethanol on GSK-3β (or Akt) phosphorylation at Ser9, cells were exposed to ethanol for 20 min. LY294002 was given 20 min before exposure to ethanol. In the study evaluating the effect of ethanol on {Delta}{Psi}m, cells were exposed to 600 µM H2O2 for 20 min to cause the mPTP opening. Ethanol (10 µM) was given 20 min before exposure to H2O2.

Statistical analysis
Data are expressed as mean ± SD and obtained from at least six experiments. Statistical significance was determined using one-way ANOVA followed by Tukey's test. A value of P < 0.05 was considered as statistically significant.


   Results
TOP
 ABSTRACT
 Introduction
 Material and Methods
 Results
Discussion
 References
 
To determine the potential role of GSK-3β in the cardioprotective effect of ethanol, we first tested if ethanol could enhance GSK-3β phosphorylation at Ser9 in H9c2 cardiac cells. As shown in Fig. 1, treatments of cells with 10, 100 and 500 µM ethanol significantly increased GSK-3β phosphorylation at Ser9, suggesting that low doses of ethanol can inactivate GSK-3β in cardiac cells. In contrast, 1000 µM (1 mM) ethanol was not able to significantly enhance GSK-3β phosphorylation. To define the mechanism by which ethanol inactivates GSK-3β, we tested if LY294002, an inhibitor of PI3K, can alter the action of ethanol. As shown in Fig. 2, the effect of ethanol (10 µM) on GSK-3β phosphorylation was reversed by LY294002 (10 µM), indicating that the PI3K/Akt pathway may play a role in the action of ethanol. To corroborate this observation, we further examined whether ethanol could activate Akt by measuring Akt phosphorylation at Ser473. As shown in Fig. 3, ethanol (10 µM) significantly increased Akt phosphorylation, an effect that was nullified by LY294002, confirming that the PI3K/Akt signaling pathway is essential for the inhibitory action of ethanol on GSK-3β.


Figure 1
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  		Fig. 1 Western blot analysis of GSK-3β phosphorylation at Ser9 in cardiac H9c2 cells. Cells were treated with ethanol for 20 min. Ethanol (10–500 µM) significantly enhanced GSK-3β phosphorylation in H9c2 cells. Each bar is the mean ± SD of at least six experimental observations. *P < 0.05 versus control.

 


Figure 2
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  		Fig. 2 Western blot analysis of GSK-3β phosphorylation at Ser9 in cardiac H9c2 cells. Cells were treated with ethanol (10 µM) for 20 min. The PI3K inhibitor LY294002 (10 µM) was applied 20 min before exposure to ethanol. The effect of ethanol onGSK-3β phosphorylation was reversed by LY294002. Each bar is the mean ± SD of at least six experimental observations. *P < 0.05 versus control; #P < 0.05 versus ethanol.

 


Figure 3
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  		Fig. 3 Western blot analysis of Akt phosphorylation at Ser473 in cardiac H9c2 cells. Cells were treated with ethanol (10 µM) for 20 min. The PI3K inhibitor LY294002 (10 µM) was applied 20 min before exposure to ethanol. Ethanol significantly increased Akt phosphorylation, an effect that was reversed by the LY294002. Each bar is the mean ± SD of at least six experimental observations. *P < 0.05 versus control; #P < 0.05 versus ethanol.

 
To determine if ethanol can prevent the mPTP opening, we examined the effect of ethanol on oxidant-induced loss of {Delta}{Psi}m by monitoring changes in TMRE fluorescence with confocal microscopy. As shown in Fig. 4, treatment of cells with 600 µM H2O2 induced a marked decrease in TMRE fluorescence (37.6 ± 2.2% of baseline in the control group), implying that oxidant stress causes loss of {Delta}{Psi}m. Since the loss of {Delta}{Psi}m is caused by the mPTP opening, this result indicates the opening of mPTP by oxidant stress. In contrast, cells treated with 10, 100 and 500 µM ethanol showed much less decrease in TMRE fluorescence, indicating that ethanol at low doses modulates the mPTP opening. However, 1000 µM ethanol again did not significantly reduce the loss of TMRE fluorescence, suggesting that ethanol at high doses may not prevent the mPTP opening. Further experiments showed that ethanol (10 µM) was not able to prevent TMRE fluorescence loss in the presence of LY294002, implying that the PI3K/Akt pathway contributes to the protective effect of ethanol (Fig. 5). Importantly, transfection of cells with the constitutively active GSK-3β (GSK-3β-S9A-HA) mutant plasmid also nullified the protective effect of ethanol (Fig. 5), strongly suggesting that ethanol modulates oxidant-induced mPTP opening through inactivation of GSK-3β.


Figure 4
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  		Fig. 4 Summarized data for TMRE fluorescence intensity measured with confocal microscopy 20 min after exposure to H2O2 (600 µM). Confocal fluorescence images of TMRE were collected at baseline and 20 min after exposure to H2O2 in H9c2 cells. Compared to control, ethanol at low-to-moderate doses (10–500 µM) but not at the high dose (1 mM) prevented oxidant-induced TMRE fluorescence reduction, indicating that low doses of ethanol can prevent the mPTP opening. Each bar is the mean ± SD of at least six experimental observations. *P < 0.05 versus control.

 


Figure 5
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  		Fig. 5 Summarized data for TMRE fluorescence intensity measured with confocal microscopy 20 min after exposure to H2O2 (600 µM). Confocal fluorescence images of TMRE were collected at baseline and 20 min after exposure to H2O2 in H9c2 cells. The effect of ethanol (10 µM) on TMRE fluorescence was reversed by the PI3K inhibitor LY294002. The effect of ethanol on TMRE fluorescence was also lost in cells transfected with the constitutively active GSK-3β mutant (S9A), suggesting that ethanol may prevent the mPTP opening by inactivating GSK-3β. Each bar is the mean ± SD of at least six experimental observations. *P < 0.05 versus control; #P < 0.05 versus ethanol.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Material and Methods
 Results
 Discussion
References
 
This is the first study to demonstrate that ethanol at low doses modulates oxidant-induced mPTP opening by inactivating GSK-3β via the PI3k/Akt pathway in cardiac H9c2 cells. Since inhibition of the mPTP opening has been demonstrated to be a critical event in acute cardioprotection induced by preconditioning and postconditioning, the current finding may suggest that ethanol confers acute cardioprotection against acute myocardial infarction by preventing the mPTP opening via PI3K/Akt and GSK-3β.

Ethanol has been shown to induce cardioprotection against ischemia/reperfusion injury by mimicking preconditioning in various experimental models. While KATP channels (Pagel et al., 2000Go; Zhu et al., 2000Go) and PKC (Miyamae et al., 1998bGo; Chen et al., 1999Go, Chen and Mochly-Rosen, 2001Go) have been reported to be involved in ethanol-induced cardioprotection, the precise cellular and molecular events that mediate the action of ethanol remain to be elucidated. The mPTP opening has been demonstrated to be a critical determinant of myocardial ischemia/reperfusion injury (Weiss et al., 2003Go) and suppression of the mPTP opening is an important target of cardioprotection (Halestrap et al., 2004Go). The critical role of the mPTP in cardioprotection has also been demonstrated by recent reports addressing that both preconditioning and postconditioning confer cardioprotection against ischemia/reperfusion injury by inhibiting the mPTP opening (Hausenloy et al., 2002Go, 2004Go; Juhaszova et al., 2004Go; Argaud et al., 2005Go). In the present study, we demonstrated that pretreatment with cardiac cells with low doses of ethanol prevents oxidant-induced mPTP opening. Since a burst of reactive oxygen species upon myocardial reperfusion has been well described and is associated with cardiac injury (Becker, 2004Go), our finding may suggest that ethanol at low doses protects the heart by modulating the mPTP opening upon reperfusion.

In the present study, 10–500 µM ethanol was able to prevent oxidant-induced mPTP opening, whereas 1 mM ethanol failed to induce protection, suggesting that light but not heavy consumption of alcohol may be of benefit to the heart. Our finding supports the well-known view that the risk of coronary heart disease is lower when alcohol consumption is light-to-moderate and is high when alcohol consumption is higher (Lucas et al., 2005Go). We assume that high levels of ethanol may debilitate the intracellular signaling events that lead to the protection against the pore opening. The observation that 1 mM ethanol could not significantly enhance GSK-3β phosphorylation (Fig. 1) supports this assumption. In addition, it should also be noted that in the present study ethanol could induce protection against the pore opening at much lower doses (10–500 µM) than that (10 and 50 mM) used in other studies (Chen et al., 1999Go; Chen and Mochly-Rosen, 2001Go; Krenz et al., 2001Go) to protect isolated cardiomyocytes or isolated hearts. Although the reason for the discrepancy remains to be resolved, differences in cell type and experimental protocol may account for it. Since the blood alcohol concentration associated with intoxication in humans is ~8 mM or 0.04% (Zhou et al., 2002Go), the concentrations of ethanol used in this study could be obtained by light drinking.

Although the reperfusion injury salvage kinases (RISK) such as Akt and ERK have been proposed to be linked to the inhibition of the mPTP opening (Hausenloy and Yellon, 2004Go), the exact signaling mechanism by which cardioprotective interventions prevent the mPTP opening remains unclear. Recently, Juhaszova et al. proposed that GSK-3β mediates the convergence of cardioprotective signaling pathways to inhibit the mPTP opening (Juhaszova et al., 2004Go). Further studies demonstrated that inactivation of GSK-3β is crucial for prevention of the mPTP opening by preconditioning (Nishihara et al., 2007Go), postconditioning (Gomez et al., 2008Go) and bradykinin (Park et al., 2006Go). In the present study, ethanol at low doses could markedly enhance GSK-3β phosphorylation at Ser9, suggesting that ethanol can inactivate GSK-3β in cardiac cells. Accordingly, it is possible that inactivated GSK-3β may mediate the inhibitory effect of ethanol on the mPTP opening. In support, our confocal imaging study showed that transfection of cells with the constitutively active GSK-3β (GSK-3β-S9A-HA) mutant plasmid nullified the preventive effect of ethanol on oxidant-induced loss of {Delta}{Psi}m, strongly suggesting that ethanol modulates the mPTP opening by inactivating GSK-3β. In addition, the observation that the high dose (1 mM) of ethanol which failed to enhance GSK-3β phosphorylation was not able to prevent the pore opening further supports the importance of GSK-3β in the action of ethanol.

The PI3K/Akt signaling pathway plays an important role in cardioprotection (Hausenloy and Yellon, 2006Go) and is well known to inhibit GSK-3β activity (Cohen and Frame, 2001Go). We found that the effect of ethanol on GSK-3β phosphorylation was reversed by the PI3K inhibitor LY294002 and that ethanol increased Akt phosphorylation at Ser473. Moreover, the preventive effect of ethanol on the mPTP opening was abrogated by LY294002. These data clearly indicate that the PI3K/Akt signaling pathway contributes to the action of ethanol on the mPTP opening by inactivating GSK-3β. Alternatively, the PI3K/Akt pathway plays a critical role in the protective action of ethanol by serving as a negative upstream regulator of GSK-3β.

In summary (Fig. 6), our data demonstrate that ethanol at low doses can prevent oxidant-induced mPTP opening by inactivating GSK-3β via the PI3K/Akt signaling pathway. It is, therefore, reasonable to propose that light-to-moderate consumption of alcohol might be of great benefit to patients with coronary diseases. However, it should be mentioned that in this study the protective effect of ethanol was observed in cultured cardiac H9c2 cells subjected to oxidant stress, rather than in perfused heart models or conscious animal models. Obviously, further studies using perfused or open chest animal hearts are required to corroborate the current findings in the ‘bona fide’ setting of myocardial ischemia/reperfusion.


Figure 6
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  		Fig. 6 The signaling mechanism responsible for the effect of ethanol on the mPTP opening.

 

   ACKNOWLEDGEMENTS
 
This work was supported in part by grant 2007136 from Bureau of Education, Hebei Province, China and grant 20072010 from Bureau of Human Resource, Hebei Province, China.


   FOOTNOTES
 
{dagger} Contributed equally to this work. Back


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 ABSTRACT
 Introduction
 Material and Methods
 Results
 Discussion
 References
 
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