Epac is required for GLP-1R-mediated inhibition of oxidative stress and apoptosis in cardiomyocytes.

ORIGINAL

RESEARCH

Epac is Required for GLP-1R-Mediated Inhibition of Oxidative Stress and Apoptosis in Cardiomyocytes Supachoke Mangmool, Piriya Hemplueksa, Warisara Parichatikanond, and Nipon Chattipakorn Department of Pharmacology (S.M., P.H., W.P.), Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand; and Cardiac Electrophysiology Research and Training Center (N.C.), Faculty of Medicine, and Excellence Center in Cardiac Electrophysiology (N.C.), Department of Physiology, Chiang Mai University, Chiang Mai 50200, Thailand

Although the cardioprotective effects of glucagon-like peptide-1 and its analogs have been reported, the exact mechanisms of the glucagon-like peptide-1 receptor (GLP-1R) signaling pathway in the heart are still unclear. Activation of the GLP-1R has been shown to increase cAMP levels, thus eliciting protein kinase A- and exchange protein activated by cAMP (Epac)-dependent signaling pathways in pancreatic ␤-cells. However, which pathway plays an important role in the antioxidant and antiapoptotic effects of GLP-1R activation in the heart is not known. In this study, we demonstrated that stimulation of GLP-1Rs with exendin-4 attenuated H2O2-induced reactive oxygen species production and increased the synthesis of antioxidant enzymes, catalase, glutathione peroxidase-1, and manganese superoxide dismutase that is dependent on Epac. Additionally, exendin-4 has an antiapoptotic effect by decreasing a number of apoptotic cells, inhibiting caspase-3 activity, and enhancing the expression of antiapoptotic protein B-cell lymphoma 2, which is mediated through both protein kinase A- and Epac-dependent pathways. These data indicate a critical role for Epac in GLP-1R-mediated cardioprotection. (Molecular Endocrinology 29: 583–596, 2015)

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lucagon-like peptide-1 receptor (GLP-1R) is the G protein-coupled receptor that stimulates insulin secretion and proinsulin gene transcription when activated (1). The stimulation of GLP-1R also exerts diverse biological effects on many target organs, such as the heart, brain, liver, muscle, and adipose tissue (2). GLP-1Rs are expressed in the heart, and several studies indicated that glucagon-like peptide-1 (GLP-1) and its analogs exert cardioprotective actions and play an important role in the regulation of heart functions. For example, administration of exendin-4 reduced the infarct size and improved the mechanical performance in isolated rat

hearts with ischemia-reperfusion injury (3). Infusion of exenatide also reduced the infarct size in a porcine model of ischemia and reperfusion injury (4). In addition, in patients admitted due to acute myocardial infarction (MI) for primary coronary angioplasty, ejection fraction, and other functional indices improved after a 72-hour infusion of GLP-1 (5). Moreover, treatment with GLP-1 inhibited apoptosis and cellular injury induced by hypoxiareoxygenation in cardiomyocytes (6). Oxidative stress is defined as an imbalance between the antioxidant defenses and the production of reactive oxygen species (ROS), which at high levels can cause cell injury, apoptosis, and

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2015 by the Endocrine Society Received November 2, 2014. Accepted February 23, 2015. First Published Online February 26, 2015

Abbreviations: AC, adenylyl cyclase; Bcl-2, B-cell leukemia-2; 6-Bnz-cAMP, N6-benzoyladenosine-3⬘-5⬘-cyclic monophosphate; CaMKII, Ca2⫹/calmodulin-dependent protein kinase II; CREB, cAMP response element-binding protein; CT, cycle threshold; DAPI, 4,6-diamidino-2-phenylindole; DCF, dichlorodihydrofluorescein; ddA, 2⬘, 5⬘-dideoxyadenosine; Epac, exchange protein directly activated by cAMP; ESCA-AM, Epac-selective cAMP analog acetoxymethyl ester; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLP-1, glucagon-like peptide-1; GLP-1R, GLP-1 receptor; GPx-1, glutathione peroxidase-1; HO-1, hemeoxygenase-1; MI, myocardial infarction; Mn-SOD, manganese superoxide dismutase; PKA, protein kinase A; PKC, protein kinase C; PKI, PKA inhibitor 14 –22 amide; PLC, phospholipase C; ROS, reactive oxygen species; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine 5-triphosphate nick end labeling.

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Mol Endocrinol, April 2015, 29(4):583–596

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Epac-Dependent GLP-1R Signaling in Cardiomyocytes

damage. ROS levels were increased in heart failure patients (7) and in animal models after MI (8). We hypothesize that cardioprotective effects of GLP-1 are due to its antioxidant and antiapoptotic properties. However, the exact mechanisms underlying cardioprotective effects following GLP-1R stimulation have not fully elucidated in the heart. After agonist binding, GLP-1R can couple with the ␣-subunit of heterotrimeric G␣s protein, which results in activation of adenylyl cyclase (AC), followed by elevation of cAMP levels. There are at least two pathways induced by cAMP, the protein kinase A (PKA)-dependent pathway and the exchange protein activated by cAMP (Epac; a cAMP regulated guanine nucleotide exchange factor)dependent pathway (9). In pancreatic ␤-cells, the binding of GLP-1 to its receptor stimulated the formation of cAMP, the activation of PKA-dependent and -independent pathways, and the rising of intracellular Ca2⫹ concentration (10, 11). Activation of GLP-1Rs decreased endogenous ROS production and increased ATP production in diabetic rat islets through the suppression of Src activation, dependently on Epac (12). Interestingly, GLP-1 and exenatide provided the protection of apoptosis in both a PKA- and Epac-dependent manner in ␤-cells (13). Moreover, the cAMP response element-binding (CREB) transcription factor was activated by GLP-1, the stimulation of which depends on the activity of cAMP/ PKA, and inhibition of the cAMP/PKA pathway diminished the protective effects of GLP-1 in endothelial cells (14). These findings suggested that both PKA- and Epacdependent pathways play a significant role in the inhibition of oxidative stress and apoptosis in several cells types. However, the specific downstream signaling mediators of PKA- and Epac-dependent pathways, which are responsible for cardioprotective effects of GLP-1 and its analogs, have not been fully elucidated. In this study, we demonstrated that the stimulation of GLP-1Rs with exendin-4 provides the cardioprotective effects via its antioxidative activity through the Epac-dependent pathway by increasing the production of antioxidant enzymes such as glutathione peroxidase-1 (GPx-1), catalase, and manganese superoxide dismutase (Mn-SOD). Moreover, the stimulation of GLP-1Rs also elicits the antiapoptosis through both PKA- and Epac-dependent manners by enhancing the production of an antiapoptotic protein, B-cell leukemia-2 (Bcl-2), and inhibiting caspase-3 activity in cardiomyocytes. We suggest that Epac participates in antioxidant and antiapoptotic effects of exendin-4, a GLP-1R agonist.


Materials and Methods Materials Exendin-4, exendin fragment 9 –39, and hydrogen peroxide were purchased from Sigma-Aldrich. Forskolin, PKA inhibitor 14 –22 amide (PKI), 2⬘,5⬘-dideoxyadenosine (ddA), N6-benzoyladenosine-3⬘-5⬘-cyclic monophosphate (6-Bnz-cAMP), and KN-93 (a selective CaMKII inhibitor) were purchased from Calbiochem. 8-pCPT-2-O-Me-cAMP-AM (ESCA-AM) was purchased from Tocris. DMEM, fetal bovine serum, 0.25% trypsin-EDTA solution, and penicillin/streptomycin solution were purchased from Gibco.

Neonatal rat cardiomyocytes isolation and culture Animal studies were approved by the Committee for Animal Care and Use of the Faculty of Pharmacy, Mahidol University, and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (eighth edition, 2011). Neonatal Sprague Dawley rats (1–2 d old; National Laboratory Animal Center, Mahidol University) were euthanized by decapitation and hearts were removed. Cardiomyocytes were dispersed from the ventricles by enzymatic digestion using collagenase type A from Roche Diagnostics. Cardiomyocytes were plated on gelatin-coated plates and cultured in DMEM containing 10% fetal bovine serum, 5 mM taurine, 1 ␮g/mL insulin, 1 ␮g/mL transferrin, 10 ng/mL selenium and 1% (vol/vol) penicillin-streptomycin solution (Gibco).

Measurement of intracellular ROS level Intracellular ROS level was quantified using a fluorescent probe dichlorodihydrofluorescein (DCF) diacetate (Sigma-Aldrich). Cardiomyocytes were seeded in a 12-well plate (1 ⫻ 105 cells/well) or 35-mm glass bottomed dishes (1 ⫻ 105 cells/dish) and treated with 100 ␮M H2O2 with or without prior incubation with 20 nM exendin-4 (Sigma-Aldrich). The cells were washed once with PBS. Thereafter 10 ␮M DCF diacetate was added and incubated with the cells at 37ºC in the dark for 30 minutes. The fluorescence intensity of DCF was quantified by a multidetection microplate reader (BioTek) with ␭485 nm excitation wavelength and ␭530 nm emission wavelengths. To monitor the ROS production in living cells, cardiomyocytes were visualized using single-line excitation (488 nm) of an IX-81 fluorescence microscope (Olympus) with a ⫻40 (NA 1.4) objective lens (Olympus).

mRNA expression analysis The extraction of RNA from cardiomyocytes was performed by using the RNeasy kits (QIAGEN). Quantitative mRNA analysis was performed on an Mx 3005p real-time PCR system (Stratagene) using the KAPA SYBR FAST one-step quantitative RT-PCR kits (KAPA Biosystems) following the manufacturer’s instructions. Primer sequences are shown in Supplemental Table 1. Relative mRNA expression levels were evaluated by the comparative cycle threshold (CT) method and normalized to an endogenous reference, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The fold increase in mRNA expression of target gene was calculated from 2⫺⌬⌬CT as recommended by the manufacturer.


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Terminal deoxynucleotidyl transferase deoxyuridine 5-triphosphate nick end labeling (TUNEL) assay TUNEL assay was performed using the in situ cell death detection kit (Roche Diagnostics) according to the manufacturer’s instructions. Briefly, cardiomyocytes plated in a 35-mm dish were treated with various agents, fixed in freshly prepared 4% paraformaldehyde, and then permeabilized in 0.1% Triton X-100. Cells were then washed twice with PBS and subjected to the TUNEL reaction at 37ºC in the dark for 60 minutes. At the end of the incubation, cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Invitrogen). The fluorescent signal, emitted by fluorescein-labeled deoxyuridine 5-triphosphate incorporated into fragmented DNA, was visualized using single-line excitation (488 nm) of a IX-81 fluorescence microscope (Olympus) with a ⫻20 (NA 1.4) objective lens (Olympus).

Caspase-3 activity assay A caspase-3 assay was performed by using caspase-3 colorimetric assay kit (Abcam) according to the manufacturer’s instructions. Briefly, cardiomyocytes were seeded in a six-well plate (1 ⫻ 106 cells/well) and treated with 200 ␮M H2O2 for 3 hours with or without prior incubation with 20 nM exendin-4 (Sigma-Aldrich). The cells were washed with PBS, resuspended in cell lysis buffer, and incubated on ice for 10 minutes. After incubation, the fractions were centrifuged for 1 minute at 10 000 ⫻ g, and the supernatants were incubated in reaction buffer containing Asp-Glu-Val-Asp p-nitroaniline (DEVD-pNA) substrate at 37ºC for 1 hour. The p-nitroaniline (p-NA) light emission can be quantified using an Infinite M200 Tecan microplate reader (Tecan) at 405 nm.

Small interfering RNA (siRNA) experiments targeting Epac Three stealth siRNA duplex oligoribonucleotides against rat Epac1 (National Center for Biotechnology Information reference sequence NM_021690.1) were synthesized by Invitrogen. The siRNA sequences are shown in Supplemental Table 2. Cardiomyocytes were transfected with 100 nM Epac1 siRNA or control siRNA using the Lipofactamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. All assays were performed at least 2 days after siRNA transfection.

Western blotting After stimulation, cells were washed once with PBS and solubilized in lysis buffer containing 20 mM Tris-HCl (pH 7.4), 0.8% Triton X-100, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 100 ␮M phenylmethylsulfonyl fluoride, 5 ␮g/mL aprotinin, and 5 ␮g/mL leupeptin. Protein concentration of cell lysates was determined using a Bio-Rad protein assay kit with BSA as standard. Samples were mixed with loading buffer and denatured by heating at 95ºC for 5 minutes prior to separation by SDS-PAGE. Separated proteins were transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories) and subjected to immunoblotting with primary antibodies to catalase (Cell Signaling Technology), Mn-SOD (Cell Signaling), Bcl-2 (Cell Signaling), GAPDH (Cell Signaling), CREB (Cell Signaling), phospho-CREB (Cell Signaling), and glutathione peroxidase-1 (Abcam). Immunoblots were visualized with an horse-


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radish peroxidase-conjugated secondary antibodies and a chemiluminescence detection system (GE Healthcare).

Statistical analysis Data are presented as mean ⫾ SEM. The statistical analysis was determined using a one-way ANOVA and a Student’s t test, and values of P ⬍ .05 were considered to be significant.

Results Exendin-4 inhibits H2O2-induced oxidative stress and apoptosis in cardiomyocytes We investigated whether stimulation of GLP-1Rs with exendin-4 inhibits H2O2-induced ROS production in cardiomyocytes. After incubation with H2O2, the levels of intracellular ROS markedly increased compared with that of the control (vehicle) group (Figure 1A). Exposure of exendin-4 significantly inhibited H2O2-induced ROS production, which had effects similar to those of vitamin C (a potent antioxidant). In contrast, exendin-4 did not decrease ROS production in the presence of exendin-(9 – 39), a GLP-1R antagonist (Figure 1A). We further quantified ROS production in intact cardiomyocytes using the fluorescent microscope. Incubation of cardiomyocytes with H2O2 induced a significant increase in the fluorescence intensity of dichlorodihydrofluorescein (DCF) as shown in green color in intact cells of the control group (Figure 1B). Treatment with exendin-4 decreased the fluorescence intensity of DCF, suggesting that exendin-4 suppressed H2O2-induced ROS production in intact cells, whereas exendin-4 had no effect in cells pretreated with exendin-(9 –39) (Figure 1B). Taken together, these data demonstrated that exendin-4 has the antioxidant effect by preventing the production of ROS induced by H2O2 in cardiomyocytes. This antioxidant effect appears to be predominantly mediated by the GLP-1R. We next investigated whether stimulation of GLP-1Rs attenuates H2O2-induced apoptosis. Exposure to H2O2 for 24 hours leads to an increase in a number of apoptotic cells as shown in the green color assayed by TUNEL (Figure 1C). Treatment with exendin-4 protected cardiomyocytes from H2O2-induced apoptosis. In contrast, exendin-4 did not exert the antiapoptotic effects in the presence of exendin-(9 –39) (Figure 1C). Moreover, stimulation of GlP-1Rs with exendin-4 also suppressed the activation of caspase-3, whereas blockade of GLP-1Rs by exendin-(9 –39) completely antagonized the action of exendin-4 (Figure 1D). These results demonstrated that stimulation of GLP-1Rs exhibits the antiapoptotic effects in a receptor-dependent fashion.


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Figure 1. Exendin-4 inhibits H2O2-induced oxidative stress and apoptosis. A and B, Cells were pretreated without or with 100 nM exendin-(9 –39) (9 –39) for 1 hour before treatment with vehicle (control), 100 ␮M vitamin C, or 20 nM exendin-4 (Ex-4) for 3 hours. Cells were then incubated with 200 ␮M H2O2 for 30 minutes. A, The intracellular ROS production was quantified, expressed as a percentage of the control, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs H2O2. B, DCF fluorescence is shown in green color. Scale bar, 10 ␮m (n ⫽ 4). C and D, Cells were pretreated without or with 100 nM exendin-(9 –39) for 1 hour before treatment with vehicle or 20 nM Ex-4 for 3 hours. C, Cells were then incubated with 250 nM H2O2 for 24 hours (C) or 200 ␮M H2O2 for 3 hours (D). C, Apoptotic cells were assessed by TUNEL stain (green) and cells counterstained with DAPI (blue) to show cell nuclei. The values are expressed as the percentage of apoptotic cells over nonstimulated cells and shown as the mean ⫾ SEM. Scale bar, 10 ␮m (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs H2O2. D, The caspase-3 activity was quantified, expressed as the value of absorbance, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs H2O2.

Exendin-4 exhibits antioxidant and antiapoptotic effects in a cAMP-dependent manner Stimulation of GLP-1Rs, which couples with G␣s protein, leads to the activation of AC, followed by the elevation of cAMP levels. Because GLP-1 was also shown to increase cAMP levels in adult rat cardiomyocytes (15), we

investigated whether the reduction of ROS production and inhibition of apoptosis by exendin-4 is dependent on cAMP. As shown in Figure 2A, the antioxidant effect of exendin-4 was inhibited by ddA (an AC inhibitor). Treatment with forskolin (an AC activator) at 0.1 ␮M tended to inhibit H2O2-induced ROS production, whereas fors-


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Figure 2. Antioxidant and antiapoptotic effects of exendin-4 is cAMP dependent. A and B, Cells were pretreated without or with 1 ␮M ddA (an AC inhibitor) for 1 hour before treatment with vehicle (control), 0.1 ␮M or 10 ␮M forskolin (Forsk; AC activator), 100 ␮M vitamin C, or 20 nM exendin-4 (Ex-4) for 3 hours. Cells were then incubated with 200 ␮M H2O2 for 30 minutes. A, The intracellular ROS production was quantified, expressed as a percentage of the control, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs H2O2. B, DCF fluorescence is shown in green color. Scale bar, 10 ␮m (n ⫽ 4). C and D, Cells were pretreated without or with 1 ␮M ddA before treatment with vehicle (control), 0.1 ␮M or 10 ␮M forskolin (Forsk; an AC activator), or 20 nM Ex-4 for 3 hours. Cells were then incubated with 250 nM H2O2 for 24 hours (C) or 200 ␮M H2O2 for 3 hours (D). C, Apoptotic cells were assessed by TUNEL stain (green) and cells counterstained with DAPI (blue) to show cell nuclei. The values are expressed as the percentage of apoptotic cells over nonstimulated cells and shown as the mean ⫾ SEM. Scale bar, 10 ␮m (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs H2O2. D, The caspase-3 activity was quantified, expressed as the value of absorbance, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 v. H2O2.

kolin at 10 ␮M significantly inhibited this effect, suggesting that the higher cAMP amount generated by AC is required for antioxidant effects. Similar results were obtained from intact cardiomyocytes by the measurement of DCF fluorescence intensity as shown in green color (Figure 2B). Collectively these results demonstrate that stimulation of GLP-1Rs by exendin-4 exhibits antioxidant effects through a cAMP-dependent pathway. Because we have shown that stimulation of GLP-1Rs elicits the antiapoptotic effects in cardiomyocytes, we therefore determined whether cAMP is involved in the

regulation of this antiapoptotic effect. The antiapoptotic effects of exendin-4 can be inhibited by blocking cAMP synthesis with ddA (Figure 2C). Moreover, pretreatment with ddA completely antagonized the inhibitory effects of exendin-4 on H2O2-induced caspase-3 activity (Figure 2D). In addition, forskolin at 10 ␮M is able to suppress H2O2-induced apoptosis and caspase-3 activity as shown by the similar results to those of exendin-4 (Figure 2, C and D). Thus, GLP-1R stimulation delivers potent antiapoptic effects through cAMP signaling by decreasing the amount of apoptotic cells and inhibiting caspase-3 activation.


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PKA is necessary for antiapoptotic effects of exendin-4 We next determined whether the PKA-dependent pathway plays a role in exendin-4-mediated inhibition of oxidative stress and apoptosis. As shown in Figure 3, A and B,


treatment with PKI (a specific PKA inhibitor) did not block the antioxidant effects of exendin-4. Moreover, treatment with 6-Bnz-cAMP (a selective PKA activator) had no effect on H2O2-induced ROS production, indicating that the antioxidant effect of exendin-4 is independent of PKA.

Figure 3. Exendin-4 prevents H2O2-mediated apoptosis in a PKA-dependent pathway. A and B, Cells were pretreated without or with 10 ␮M PKI (a PKA inhibitor) for 1 hour before treatment with vehicle (control), 1 ␮M 6-Benz-cAMP (a PKA activator), 100 ␮M vitamin C, or 20 nM exendin-4 (Ex-4) for 3 hours. Cells were then incubated with 200 ␮M H2O2 for 30 minutes. A, The intracellular ROS production was quantified, expressed as a percentage of the control, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs H2O2. B, DCF fluorescence is shown in green color. Scale bar, 10 ␮m (n ⫽ 4). C and D, Cells were pretreated without or with 10 ␮M PKI for 1 hour before treatment with vehicle, 1 ␮M 6-Benz-cAMP, 100 ␮M vitamin C, or 20 nM Ex-4 for 3 hours. Cells were then incubated with 250 nM H2O2 for 24 hours (C) or 200 ␮M H2O2 for 3 hours (D). C, Apoptotic cells were assessed by TUNEL stain (green) and cells counterstained with DAPI (blue) to show cell nuclei. The values are expressed as the percentage of apoptotic cells over nonstimulated cells and shown as the mean ⫾ SEM. Scale bar, 10 ␮m (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs H2O2. D, The caspase-3 activity was quantified, expressed as the value of absorbance, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs H2O2. E, Cell lysates were immunoblotted with antiphospho-CREB and anti-CREB antibodies. The CREB activation was quantified, expressed as fold increase over control, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control.


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Figure 4. Exendin-4 prevents H2O2-mediated oxidative stress and apoptosis in an Epac-dependent pathway. A, Cells were transfected with 100 nM Epac siRNA or control siRNA. The fold increase in mRNA expression of Epac gene are calculated from 2⫺⌬⌬CT. The mRNA levels were quantified and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control siRNA. B and C, Cells were transfected with either Epac siRNA or control siRNA. After 2 days, cells were treated with vehicle (control), 10 ␮M ESCA-AM (Epac selective cAMP analog acetoxymethyl ester), 100 ␮M vitamin C, or 20 nM exendin-4 (Ex-4) for 3 hours. Cells were then incubated with 200 ␮M H2O2 for 30 minutes. B, The intracellular ROS production was quantified, expressed as a percentage of the control, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs H2O2. C, DCF fluorescence is shown in green color. Scale bar, 10 ␮m (n ⫽ 4). D and E, Cells were transfected with either Epac siRNA or control siRNA. After 2 days, cells were pretreated without or with 10 ␮M PKI for 1 hour before treatment with vehicle, 5 ␮M ESCA-AM, or 20 nM Ex-4 for 3 hours. Cells were then incubated with 250 nM H2O2 for 24 hours (C) or 200 ␮M H2O2 for 3 hours (D). D, Apoptotic cells were assessed by TUNEL stain (green) and cells counterstained with DAPI (blue) to show cell nuclei. The values are expressed as the percentage of apoptotic cells over nonstimulated cells and shown as the mean ⫾ SEM. Scale bar, 10 ␮m (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs the H2O2. E, The caspase-3 activity was quantified, expressed as the value of absorbance, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs H2O2.

We further investigated the potential role of PKA on the antiapoptotic effects of exendin-4. We found that exendin-4-mediated antiapoptosis can be prevented by PKI, whereas treatment with 6-Bnz-cAMP alone showed the antiapoptotic actions by attenuating the amount of apoptotic cells and suppressing caspase-3 activity compared with the H2O2-treated group (Figure 3, C and D), confirming that stimulation of GLP-1Rs inhibits H2O2-induced apoptosis in a PKA-dependent manner. The downstream effector of PKA is the CREB, which is phosphorylated and activated by PKA. CREB regulates expression of several genes in conjunction with other transcription factor. As shown in Figure 3E, treatment with either exendin-4 (a GLP-1R agonist) or 6-Bnz-cAMP (a PKA activator) resulted in a significant increase in the level of phosphorylated CREB compared with the control (vehicle). Moreover, pretreatment with PKI (a specific PKA inhibitor) completely blocked exendin-4induced CREB activation, confirming that stimulation of GLP-1Rs activates the transcription factor CREB in cardiomyocytes. Epac is necessary for antioxidant and antiapoptotic effects of exendin-4 Epac serves as an important downstream effector of cAMP. We used siRNA to specifically target Epac and

examined the contribution of Epac on the exendin-4-mediated inhibition of oxidative stress and apoptosis. Two unique siRNAs suppressed the expression of endogenous Epac1 compared with nonsilencing, control siRNAtransfected cells (Figure 4A). After GLP-1R stimulation with exendin-4, the ROS level was significantly decreased in control-siRNA-transfected cells compared with that of the H2O2-treated group (Figure 4B). In contrast, exendin-4 did not decrease H2O2-induced ROS production in Epac siRNA-transfected cells (Figure 4B). Similar results were obtained from intact cardiomyocytes by the measurement of DCF fluorescence intensity, which showed as green color (Figure 4C). Moreover, activation of Epac by 8-pCPT-2-O-Me-cAMP-AM (Epac selective cAMP analog ESCA-AM) significantly attenuated H2O2-induced ROS production which had effects similar to those of exendin-4 (Figure 4, B and C). Collectively these results demonstrated that Epac is required for exendin-4-mediated inhibition of oxidative stress. GLP-1 and exenatide prevent palmitate-mediated apoptosis by the Epac-dependent pathway in ␤-cells (13). Consistent with this study, we show that treatment with either exendin-4 or ESCA-AM markedly decreased a number of apoptotic cells and caspase-3 activity induced by H2O2 (Figure 4, D and E) in cardiomyocytes. In con-


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nificantly increased the protein levels of GPx-1 and Mn-SOD and tended to increase the catalase protein level (Figure 5B), indicating that the stimulation of GLP-1Rs plays an important role in the prevention of oxidative stress by enhancing the production of these antioxidant enzymes in cardiomyocytes. Because we showed that exendin4-mediated inhibition of ROS production followed an Epac-dependent, PKA-independent manner, we further investigated whether the increase in mRNA and protein expressions of these antioxidant enzymes by exendin-4 is also dependent on Epac but not PKA. The increased mRNA expressions of GPx-1, catalase, and Mn-SOD by exendin-4 were not affected by pretreatment with PKI (Figure 6A). Exendin-4mediated induction of GPx-1, cataFigure 5. Effects of exendin-4 on the mRNA and protein expressions of antioxidant enzymes. A, lase, and Mn-SOD mRNA expresCells were stimulated with 20 nM exendin-4 (Ex-4) for 6 hours. The relative mRNA levels were sion was substantially enhanced in quantified and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs vehicle. B, Cells were stimulated control siRNA-transfected cells but with 20 nM Ex-4 for 24 hours. Cell lysates were immunoblotted with anti-GPx-1, anti-Mn-SOD, anticatalase, and anti-GAPDH antibodies. The relative protein levels were quantified, expressed as was unable to increase mRNA levels fold increase over control, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control. in the presence of Epac siRNA (Figure 5B). Moreover, exendin-4-mediated gene expression of antioxidant trast, stimulation of GLP-1Rs with exendin-4 was unable to prevent the H2O2-induced apoptotic cells and caspase-3 enzymes can be inhibited by ddA (an AC inhibitor). In activity in Epac siRNA-transfected cells (Figure 4, D and addition, depletion of Epac by Epac siRNA also supE). Moreover, exendin-4 had no inhibitory effect on pressed the protein expressions of GPx-1 and Mn-SOD, H2O2-induced apoptosis and activation of caspase-3 whereas the blockade of PKA by PKI had no effect on when blocking both Epac and PKA activities. Collectively these exendin-4-mediated induction of GPx-1 and Mn-SOD results showed that Epac is necessary for downstream GLP-1R protein expressions (Figure 6B). These results confirm that stimulation of GLP-1Rs with exendin-4 increased the signaling on inhibition of apoptosis in cardiomyocytes. production of these antioxidant enzymes in a GLP-1R/ Exendin-4-induced expression of antioxidant cAMP/Epac pathway. Ca2⫹/calmodulin-dependent proenzymes is Epac dependent tein kinase II (CaMKII) serves as an important downstream Antioxidant enzymes [eg, GPx-1, catalase, Mn-SOD, effector of Epac in cardiomyocytes (17). Overstimulation of copper-zinc superoxide dismutase (CuZn-SOD), and heme- ␤1-adrenergic receptor activates CaMKII in an Epac-depenoxygenase-1 (HO-1)] are the major components of antiox- dent manner (18, 19). A previous study also showed that idant signaling cascade in many cell types. Treatment with 8-pCPT-2⬘-O-Me-cAMP (an Epac activator) activated Epac exendin-4 suppressed hepatic oxidative stress by activating to mediate CaMKII activation (18). We next investigated catalase and GPx in the liver of diabetic mice (16). Consis- whether CaMKII plays a role on GLP-1R-mediated inductent with this previous study, we showed that stimulation of tion of antioxidant enzymes. We found that the inhibition of GLP-1Rs with exendin-4 significantly elevated the mRNA CaMKII by using KN-93 was unable to block exendin-4levels of GPx-1, catalase, and Mn-SOD (Figure 5A, upper induced mRNA and protein expressions of antioxidant enpanel). In addition, the mRNA levels of HO-1 and CuZn- zymes (Figure 6, A and B). Thus, CaMKII did not involve in SOD tended to increase after exendin-4 treatment (Figure the Epac-dependent GLP-1R signaling on the prevention of 5A, lower panel). Moreover, treatment with exendin-4 sig- oxidative stress in cardiomyocytes.


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Figure 6. Stimulation of GLP-1R enhances the mRNA and protein expressions of antioxidant enzymes in an Epac-dependent manner. A and B, Cells were transfected with either Epac siRNA or control siRNA. After 2 days, cells were pretreated without or with 1 ␮M ddA (an AC inhibitor), 10 ␮M PKI (a PKA inhibitor), or 5 ␮M KN-93 (a CaMKII inhibitor) for 1 hour before treatment with vehicle (control) or 20 nM exendin-4 (Ex-4) for 6 hours (A) or 24 hours (B). A, The relative mRNA levels were quantified and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs Ex-4-treated control siRNA. B, The relative protein levels were quantified, expressed as fold increase over control, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control.

Exendin-4-induced expression of antiapoptotic protein is Epac and PKA dependent To examine the effects of exendin-4 on the production of antiapoptotic and proapoptotic proteins, we measured the mRNA and protein expression levels of them after treatment with exendin-4. Stimulation of GLP-1Rs with exendin-4 significantly elevated the mRNA level of Bcl-2, whereas the Bcl-xL mRNA level tended to increase compared with vehicle (control) (Figure 7A). These data are in concordance with a recent study reporting the up-regulation of Bcl-2 expression after exendin-4 treatment in ␤-cells (20). However, exendin-4 did not affect the induc-

tion of proapoptotic genes, Bax and Bad (Figure 7B). In addition, exendin-4 also enhanced the protein expression of Bcl-2 in cardiomyocytes (Figure 7C). These results indicated that GLP-1R stimulation plays an important role in the prevention of apoptosis by enhancing the production of antiapoptotic protein Bcl-2. GLP-1 and exenatide prevented palmitate-mediated apoptosis in both PKA- and Epac-dependent pathways in ␤-cells (13). However, the mechanism by which signaling pathway mediates exendin-4-induced mRNA and protein expressions of Bcl-2 in cardiomyocytes is unknown. We found that blockade of PKA activity by PKI significantly


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Figure 7. Stimulation of GLP-1R induces the mRNA and protein expressions of antiapoptotic protein in PKA- and Epac-dependent manners. A and B, Cells were stimulated with 20 nM exendin-4 (Ex-4) for 6 hours. The relative mRNA levels were quantified and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs vehicle. C, Cells were stimulated with 20 nM Ex-4 for 24 hours. Cell lysates were immunoblotted with anti-Bcl-2 and anti-GAPDH antibodies. The relative protein levels were quantified, expressed as fold increase over control, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control. D and E, Cells were transfected with either Epac siRNA or control siRNA. After 2 days, cells were pretreated without or with 1 ␮M ddA (an AC inhibitor), 10 ␮M PKI (a PKA inhibitor) or 5 ␮M KN-93 (a CaMKII inhibitor) for 1 hour before treatment with vehicle (control) or 20 nM exendin-4 (Ex-4) for 6 hours (D) or 24 hours (E). D, The relative Bcl-2 mRNA level was quantified and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs Ex-4-treated control siRNA. E, The relative Bcl-2 protein level was quantified, expressed as fold increase over control, and shown as the mean ⫾ SEM (n ⫽ 4). *, P ⬍ .05 vs control; #, P ⬍ .05 vs Ex-4treated control siRNA.

inhibited the exendin-4-induced Bcl-2 synthesis (Figure 7, D and E). Similarly, depletion of endogenous Epac by Epac siRNA also attenuated this exendin-4 action. Interestingly, blockade of both PKA and Epac completely blocked exendin-4-induced Bcl-2 mRNA and protein expressions (Figure 7, D and E), suggesting that exendin-4 induced Bcl-2 synthesis via both PKA- and Epac-dependent pathways in cardiomyocytes. CaMKII can transduce signals leading to apoptosis in cardiomyocytes (19). We next determined whether CaMKII is not necessary for Epac-dependent inhibition of apoptosis after GLP-1R stimulation. Pretreatment with KN-93 (a CaMKII inhibitor) had no effect on exendin-4mediated induction of Bcl-2. These results suggested that CaMKII did not participate in the Epac-dependent

GLP-1R signaling on the inhibition of apoptosis in cardiomyocytes.

Discussion In this study, we provide a new molecular mechanism of GLP-1R signaling on inhibition of oxidative stress and apoptosis in the heart. Stimulation of GLP-1Rs with exendin-4 exhibits the antioxidant and antiapoptotic effects by showing an essential role for the Epac-signaling pathway in cardiomyocytes. GLP-1 had a direct protective effect on oxidative stress-induced senescence and was able to attenuate oxidative stress-induced DNA damage and cellular senes-


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cence in human umbilical vein endothelial cells (14). Similar to GLP-1 treatment, our study showed that exendin-4 strongly attenuated ROS production in cardiomyocytes. The previous studies indicated that GLP-1 or liraglutide enhanced cAMP level, which was diminished by the GLP-1R antagonist, exendin-(9 –39) in rodent cardiomyocytes (15, 21). In this study, we show that pretreatment with exendin-(9 –39) completely abolished the antioxidant effects of exendin-4. Data from our study and others provide evidence that antioxidant effects of GLP-1 and its analogs are GLP-1 receptor dependent. A few observations demonstrated that the activities of GLP-1R agonists may also exhibit via PKA-independent manners. For instance, GLP-1-stimulated Ca2⫹ elevation was shown to be induced without dependence of PKA (22). Furthermore, GLP-1 induced proinsulin I gene transcription was also shown to be activated independently of PKA (23). In diabetic rat islets, exendin-4 also suppressed ROS production in an Epac-dependent manner (12). Consistent with previous studies, we showed that exendin-4 inhibited ROS production through an Epac-dependent, PKA-independent pathway in cardiomyocytes. Epac has been identified as a new target of cAMP signal. Epac directly binds cAMP and exhibits guanine nucleotide exchange factor activity toward Rap (24, 25). There are two subtypes of Epac, Epac1 and Epac2 (24). Epac2 is found in the pancreas and the activation of the Epac2 pathway can involve insulin secretion (9). Epac1 is mainly expressed in the heart (26) and regulates the contraction of heart muscle cells by controlling gap junction in cardiomyocytes (27). The Epac2 to Epac1 mRNA ratio increases in adulthood compared with neonates (28), indicating it is plausible that Epac2 may have increasing biological functions with age. Thus, these data simplify that activation of Epac1 signaling seems to play several roles in the heart. We also show in this study that Epac1 is necessary for exendin-4-mediated antioxidation in neonatal rat cardiomyocytes. Interestingly, several studies demonstrated the role for Epac2 in the heart. For example, Epac2 mediates ␤1-adrenergic receptor-induced cardiac arrhythmias (29). Moreover, stimulation of GLP-1Rs with liraglutide promotes the secretion of atrial natriuretic peptide and a reduction of blood pressure through Epac2 (30). Although it is possible that Epac2 is involved in the regulation of biological processes in the heart, it will require further study to determine whether Epac2 is necessary for GLP-1R-mediated antioxidant and antiapoptotic actions in cardiomyocytes. Interestingly, the antioxidant effects of GLP-1 might be due to the induction of antioxidant genes, HO-1, and NADPH quinone oxidoreductase-1 in endothelial cells (14). Liraglutide also induced mRNA and protein expres-


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sion of HO-1 in the mice heart (21). In addition, our study showed that treatment with exendin-4 tends to induce mRNA expression of HO-1 in cardiomyocytes. We also demonstrated that exendin-4 is able to induce the mRNA expression of other antioxidant enzymes such as MnSOD. However, our study is not unlike the study by others in which Mn-SOD activity was not increased after exendin-4 treatment in islet ␤-cells (12). Further evaluation of the exendin-4-induced Mn-SOD activity in cardiomyocytes is required. Treatment with exendin-4 suppressed hepatic oxidative stress by activating catalase, SOD, and GPx in the liver of diabetic mice (16). Our data are consistent with this previous study showing that myocardial catalase and GPx-1 mRNA levels were increased after treatment with exendin-4. Interestingly, exendin-4mediated induction of mRNA and protein expressions of GPx-1 and Mn-SOD was not inhibited by either a PKA inhibitor or CaMKII inhibitor, whereas blockade of Epac strongly abolished these effects, indicating that the induction of these antioxidant enzymes by exendin-4 is mediated through the GLP-1R/cAMP/Epac pathway in cardiomyocytes. Thus, stimulation of GLP-1Rs might exert the cardioprotective effects through stimulating oxidative defense system in the heart. CaMKII is one of the important downstream effectors of Epac that are found in the heart. CaMKII expression and activity are increased in failing human heart and appear to play an important pathogenic role in the development of heart failure (31). Inhibition of CaMKII can prevent cardiac dysfunction (32). In the present study, we reported that despite CaMKII is the important effector of Epac, CaMKII does not play a role on Epac-dependent GLP-1R signaling on the inhibition of oxidative stress. This perhaps reflects distinct compartmentation of Epac signaling under different circumstances. Interestingly, protein kinase C (PKC) is capable of increasing catalase activity for controlling mitogenic cell growth in human erythrocytes, whereas CaMKII is unable to stimulate catalase activity (33). In addition, PKC has been proposed as a regulator of Mn-SOD induction because blockade of PKC inhibited the up-regulation of Mn-SOD in human adenocarcinoma cells (34). We also show in this study that blockade of CaMKII activity does not affect exendin-4-mediated induction of GPx-1 and Mn-SOD. These studies raise the possibility that stimulation of GLP-1R enhances the induction of antioxidant enzymes through the PKC-dependent Epac signaling pathway. We also proposed that Epac-mediated antioxidant effect of exendin-4 is not passing through CaMKII. Although it is likely that CaMKII is not involved in Epac-mediated GLP-1R signaling, further study will be required to investigate the precise molecular


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nitric oxide-induced apoptosis (38). Moreover, cAMP elevation has been shown to promote myocyte survival in animal model of ischemia-reperfusion injuries via activation of PKA (38, 39). The cellular response to cAMP might be associated with the cAMP effectors such as PKA and Epac. However, the divergent cellular response to cAMP and its compartmentalization is not fully elucidated. In this study, we show that the apoptotic protection of exendin-4 was abolished by ddA (an AC inhibitor), suggesting that cAMP is an important mediator in the prevention of apoptosis in cardiomyocytes. The antiapoptotic effect of GLP-1 is PKA dependent because the improvement of cell viability of GLP-1 after H2O2 exposure was blocked by H89, a PKA inhibitor (40). GLP-1 had an antiapoptotic activity mediated by a cAMP/PKA-dependent signaling pathway in ␤-cells (36) and endothelial cells (14). GLP-1 and exenatide prevent palmitate-mediated caspase-3 activation in both a PKAand Epac-dependent manner in ␤-cells (13). In this study, we show that blockade of either the PKA or Epac pathway attenuated antiapoptotic effects of exendin-4 by decreasing the numbers of apoptotic cells and suppressing caspase-3 activity in cardiomyocytes. Interestingly, dual inhibition of these two pathways completely abolished antiapoptotic effects of exendin-4. Our data support a concept whereby GLP-1R stimulation promotes antiapoptosis through PKA- and Epac-signaling pathways. Bcl-2 family is a group of apoptosis-regulating proteins that can be divided into two subgroups; antiapoptotic proteins (eg, Bcl-2, Bcl-xL, and Bcl-W) and proapoptotic proteins (eg, Bax, Bak, Bad, and Bid) (41). Bcl-2 and Bcl-xL have an important role in cell survival and can protect cardiomyocytes against deleterious consequences from various stressors. For example, treatment with exenatide increased myocardial phosphorylated Akt and Bcl-2 expression level and inhibited expression of active caspase-3 (4). The expression of Bcl-xL inhibited doxorubicin- and hypoxia-mediated apoptosis by preserving mitochondrial integrity in H9c2 cardiac cells (42). Moreover, Bcl-2 inhibited p53-mediated apoptosis in Figure 8. Schematic diagram representing the cardioprotective effects of exendin-4. Exendin-4 (Ex-4) binding to GLP-1Rs leads to Gs protein coupling, subsequently stimulates AC activity, and cardiomyocytes. In addition, overexgenerates cAMP, the second messenger. cAMP can bind to and activate both PKA and Epac pression of Bcl-2 in the heart of transsignaling. Stimulation of GLP-1Rs results in the antioxidation by inducing the synthesis of genic mice reduced ischemia/reperfucatalase, GPx-1, and Mn-SOD through Epac signaling and elicits the antiapoptosis by enhancing Bcl-2 production and inhibiting caspase-3 activity via PKA and Epac signalings. sion injury, demonstrating that Bcl-2

mechanistic of this pathway and whether any other proteins are assembled within Epac compartmentalization. Several studies also showed the GLP-1R-mediated antiapoptotic effect in the heart. For example, treatment with exendin-4 resulted in improved cell viability in response to ischemia-reperfusion injury via GLP-1R-dependent manner (35). We provide evidence that antiapoptotic effect of exendin-4 was inhibited by exendin-(9 –39). Data from our work and others suggest that the antiapoptotic effect of exendin-4 can be mediated through the GLP-1 receptor-dependent fashion. Apoptotic process is controlled by an increase in cellular cAMP levels in several cell types and can be prevented by agents that increase cAMP levels (13). The antiapoptotic effect of GLP-1 was diminished by Rp-cAMP (a PKA inhibitor) in H2O2-treated MIN6 cells (36). Liraglutide increased cAMP levels in mouse cardiomyocytes in a GLP-1R-dependent manner (21). These results strongly suggested that the antiapoptotic effects of GLP-1R agonists involve the GLP-1R/cAMP pathway in many cell types. An increase of cAMP levels inhibits apoptosis in cardiomyocytes and reduces mortalilty in acute MI (37). In addition, elevation of cAMP levels by roflumilast (a phosphodiesterase type 4 inhibitor) induced both PKAdependent CREB phosphorylation and Epac-dependent Akt phosphorylation to protect cardiomyocytes against


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exhibited the cardioprotective effect by inhibition of the mitochondrial death pathway. As we show in this study, exendin-4 increased the expression of Bcl-2 and tended to increase Bcl-xL expression, whereas the expression of Bax and Bad were not changed. We also reported here that stimulation of GLP-1Rs induces the phosphorylation and activation of CREB in the PKA-dependent way in cardiomyocytes. Previous studies have demonstrated that CREB activation is related with cellular survival and antiapoptosis (36, 43). Because Bcl-2 contains the CREB binding site and Bcl-2 expression is regulated by the transcription factor CREB (43, 44), these findings raise the possibility that CREB may be involved in the regulation of Bcl-2 expression after stimulation of GLP-1R in cardiomyocytes. It has been reported that activation of ␤2-ARs can stimulate the cAMP-Epac-Rapphospholipase C (PLC)-⑀ pathway (45). Moreover, PLC⑀ and PKC⑀ are the downstream effectors of Epac, which play a role on sarcoplasmic reticulum Ca2⫹ release in cardiomyocytes (46). Nonetheless, it remains an unsolved question whether PLC⑀ and PKC⑀ are the downstream mediators of Epac signaling after GLP-1R stimulation. GLP-1 and its analogs appear to play an important role for cardioprotection. GLP-1 analog limited the myocardial loss in the rabbit model of myocardial ischemia/reperfusion (47). Administration of liraglutide in mice before coronary artery occlusion resulted in the reduction of infarct size and the improvement of cardiac output (21). In this study, our data support a concept whereby stimulation of GLP-1R exhibits the cardioprotective effects in several in vivo studies. In conclusion, we have identified a new signaling mechanism for GLP1-R mediated the inhibitions of oxidative stress and apoptosis in cardiomyocytes (Figure 8). Stimulation of GLP-1Rs provides an antioxidation via Epac-dependent signaling pathway, which enhances the mRNA expressions of antioxidant enzymes and exerts an antiapoptosis through both PKA- and Epac-dependent signaling pathways by inducing Bcl-2 expression.

Acknowledgments Author contributions included the following: S.M. carried out the experiments and data analysis, participated in the study planning, and wrote the manuscript; P.H. performed the experiments and data analysis; W.P. carried out the data and statistical analysis and discussion; and N.C. contributed to the discussion and reviewed/edited the manuscript. Address all correspondence and requests for reprints to: Supachoke Mangmool, PhD, Department of Pharmacology, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayuthaya Road, Rajathevi, Bangkok 10400, Thailand. E-mail: supachoke. [email protected].


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This work was supported by Thailand Research Fund Grant MRG5580037 (to S.M.) and a National Science and Technology Development Agency Research Chair grant from the National Science and Technology Development Agency (to N.C.). Disclosure Summary: The authors have nothing to disclose.

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SIRT5: a safeguard against oxidative stress-induced apoptosis in cardiomyocytes.

Epac-Rap signaling reduces oxidative stress in the tubular epithelium.

mTORC2 is required for rit-mediated oxidative stress resistance.

Is BOK required for apoptosis induced by endoplasmic reticulum stress?

Humid heat exposure induced oxidative stress and apoptosis in cardiomyocytes through the angiotensin II signaling pathway.

Haptoglobin is required to prevent oxidative stress and muscle atrophy.

Hydrogen sulfide protects against apoptosis under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes.

Progranulin is required for proper ER stress response and inhibits ER stress-mediated apoptosis through TNFR2.

Redox regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress.

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reoxygenation-treated human umbilical vein endothelial cells promote apoptosis and oxidative stress in H9c2 cardiomyocytes.

Correction: Inhibition of NAPDH Oxidase 2 (NOX2) Prevents Oxidative Stress and Mitochondrial Abnormalities Caused by Saturated Fat in Cardiomyocytes.

Inhibition of NAPDH Oxidase 2 (NOX2) Prevents Oxidative Stress and Mitochondrial Abnormalities Caused by Saturated Fat in Cardiomyocytes.

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