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Long-term oral resveratrol intake provides nutritional preconditioning against myocardial ischemia/reperfusion injury: involvement of VDAC1 down-regulation Zhangping Liao 1, 2, Dan Liu 2, Lei Tang2, Dong Yin3, Shuhua Yin2, Songqing Lai2, Jianguo Yao2, *, Ming He1, 2, * 1
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang
330047, PR China 2
Department of Pharmacology & Molecular Therapeutics, Nanchang University School of
Pharmaceutical Science, Nanchang 330006, PR China 3
Jiangxi Provincial Key Laboratory of Molecular Medicine at the Second Affiliated Hospital,
Nanchang University, Nanchang 330006, PR China
*These authors contributed equally to this work. Corresponding author: Ming He, PhD Department of Pharmacology& Molecular Therapeutics Nanchang University School of Pharmaceutical Science Nanchang 330006, PR China Tel.: +86-791-86362231
Fax: +86-791-86361839
E-mail: [email protected] Abbreviations: CF, coronary flow; I/R, ischemia/reperfusion; LVP, left ventricular pressure; mPTP, mitochondrial permeability transition pore; TTC, triphenyl tetrazolium chloride; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labelling; VDAC, Received: 11-Oct-2014; Revised: 29-Nov-2014; Accepted: 01-Dec-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/mnfr.201400730. This article is protected by copyright. All rights reserved.
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voltage-dependent anion channel. Keywords: Ischemia-reperfusion/ Mitochondrial permeability transition pore/ Nutritional preconditioning / Resveratrol / Voltage-dependent anion channel
Abstract Scope: This study elucidates effects of long term nutritional preconditioning by resveratrol on I/R injury and its underlying mechanisms. Methods and results: Mice were treated with resveratrol at 2.0 mg/kg.day by gastric gavages for 6 weeks. Then hearts were isolated and subjected to I/R injury in a Langendorff apparatus. Resveratrol significantly improved LVP, ±dp/dtmax and CF, decreased the LDH and CPK activity, and reduced the infarction size. Additionally, long-term oral resveratrol intake prevented mPTP opening and subsequently inhibited mitochondria-mediated apoptosis, as demonstrated by decrease of cytochrome c release, inactivation of caspase-3 and reduction of TUNEL-positive cells. Furthermore, resveratrol inhibited the up-regulation of VDAC1 expression induced by I/R injury. Local left ventricle overexpression of VDAC1 by adenovirus diminished the protective effect of resveratrol against I/R injury, indicating that VDAC1 plays an important role in resveratrol-mediated cardioprotection. Conclusion: Our data revealed that long-term oral intake of resveratrol sets nutritional preconditioning in coping with myocardial I/R injury. Strikingly, we found that resveratrol down-regulates VDAC1, leading to prevention of mPTP opening and cardiomyocyte apoptosis.
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1. Introduction Despite the high-fat diet, Southern French have a low incidence of coronary heart disease and low mortality rate. This so-called “French paradox” is partly attributed to wine consumption, particularly red wine [1]. Resveratrol (trans-3,4’,5-trihydroxystilbene), a natural polyphenolic compound, is abundant in grape skins and wine, which is a well-known constituent with cardiovascular benefits [2]. Previous evidence indicated that resveratrol has multi beneficial effects, including antioxidant properties, modulation of lipid metabolism, and inhibition of platelet aggregation. Recently, it was demonstrated that resveratrol can protect the heart against ischemia/reperfusion (I/R) injury [3]. However, this study was performed in an acute setting. The long term effects of resveratrol on I/R injury still need to be evaluated. Restoration of blood flow to the ischemic myocardium may generate more tissue damage than ischemia alone, which is known as I/R injury [4]. Repeated and short sub-lethal ischemic preconditioning exerts protection of the myocardium against subsequent lethal I/R [5]. However, with a poor predictive outcome of ischemic episodes, ischemic preconditioning is not a feasible approach. Recently, Abdukeyum et.al proposed nutritional preconditioning, that nutrient intake elicits preconditioning, limits ischemic cardiac injury and myocardial infarction, thus lends cardioprotection which has similar efficacy to ischemic preconditioning [6]. Moreover, nutritional preconditioning is a safe and effective dietary approach with nutritional components. It avoids the risk of ischemic episodes and is more feasible in preventing I/R injury [6]. The mitochondrion, one of the most important organelles in cardiomyocytes, not only functions in energy production, but also activates apoptotic cascades in response to stress. Hence, mitochondria may be involved in the cardioprotection of resveratrol. Indeed, it has been reported that cardioprotection of resveratrol is associated with regulation of the mitochondrial permeability transition pore (mPTP) [7]. mPTP is a multi-protein complex This article is protected by copyright. All rights reserved.
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formed between the inner and outer mitochondrial membranes [8]. mPTP opening is critical to mitochondria-mediated apoptosis during I/R injury [9-11]. The voltage-dependent anion channel 1 (VDAC1), in the mitochondrial outer membrane, acts as a gate to control the inflow and outflow of mitochondrial metabolites and ions [12,13]. Thus, due to its key role in regulating
mPTP
opening,
we
hypothesize
VDAC1
is
a
potential
target
of
resveratrol-mediated nutritional preconditioning. In the current study, we investigated the protective effects of long-term oral administration of resveratrol on I/R injury and the mechanism underlying resveratrol-mediated cardioprotection. We found that long-term oral administration of resveratrol protects the myocardium against I/R injury at an equivalent dose to human diet intake. Furthermore, resveratrol prevented mPTP opening and inhibited I/R-induced upregulation of VDAC1. Additionally, overexpression of VDAC1 diminished the protective effect of resveratrol. These data for the first time indicate that VDAC1 is the target of resveratrol, and demonstrate that resveratrol exhibits protective effects against I/R injury and can be used as long-term nutritional preconditioning agent for I/R. This will help to advocate its use as a healthy diet supplement.
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2. Materials and methods 2.1. Animals Adult Kun-Ming mice were provided by the Animal Center of Nanchang University. Experiments was performed according to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996), and approved by the Ethics Committee of Nanchang University (No. 2012-0116). All mice were housed, two per cage, under a controlled temperature of 22°C with a humidity of 50%, in a 12 h light/dark cycle (08:00-20:00 light, 20:00-08:00 dark). 2.2 Experimental design 2.2.1 Determination of resveratrol dosage To conform to nutritional preconditioning, the dosage of resveratrol applied in our study was equivalent to that used by humans as a diet intake, according to the conversion coefficient of body surface area between human and mouse. Resveratrol was dissolved in 25% ethanol. The mice were randomly assigned to six groups (n=6 for each group, each experiment was repeated 3 times): (1) control group; (2) vehicle control group; (3) resveratrol control group; (4) I/R group; (5) resveratrol-0.5 group; (6) resveratrol-1.0 group; and (7) resveratrol-2.0 group. Three resveratrol groups were treated with resveratrol at 0.5 mg/kg.d, 1.0 mg/kg.d, or 2.0 mg/kg.d respectively by gastric gavages, for 6 weeks before I/R treatment. The control and I/R group were treated with saline (10 ml/kg.d). Mice in vehicle control or resveratrol control were administrated with 25% ethanol (2.0 mg/kg.d ) or resveratrol (2.0 mg/kg.d ) alone respectively for 6 weeks without I/R treatment. 2.2.2 Role of VDAC in resveratrol-mediated cardioprotection Six groups (n=6 for each group, the experiment was replicated 3 times) were set to investigate the role of VDAC1. (1) The sham group underwent surgical procedure but without gene transfer and ischemic injury. (2) The I/R group was subjected to 15 min of This article is protected by copyright. All rights reserved.
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ischemia followed by 30 min of reperfusion. (3) The pAd-VDAC1+I/R group. The mice were injected with adenovirus-expressing VDAC1. Five days later, the mice were sacrificed. The hearts were isolated and Langendorff-perfused to duplicate I/R model. (4) Res+I/R group. The mice were administrated with resveratrol (2.0 mg/kg.d) for 6 weeks. Then the hearts were isolated and Langendorff-perfused for the I/R model. (5) pAd-VDAC+Res+I/R group. The mice were orally administrated with resveratrol (2.0 mg/kg.d) for 6 weeks. The following treatment was same as pAd-VDAC1+I/R group. (6) pAd-Shuttle+Res+I/R group. The mice were injected with empty pAd-Shuttle vector without VDAC1 gene. Other procedures were same as pAd-VDAC+Res+I/R group. 2.3 Construction of recombinant adenovirus vectors. VDAC1 cDNA was amplified by PCR and inserted into pShuttle-CMV vector at Kpn I and Xho I sites. The recombinant pShuttle-CMV-VDAC1 was electroporated with pAdEasy1 into Escherichia coli BJ5183 cells (Stratagene). The recombinant adenoviral DNA was transfected into human embryonic kidney 293 cells with Lipofectamine (Invitrogen). Next, all of the viruses were plaque-purified and amplified. Empty adenoviral vector was used as a control. 2.4 Intramyocardial gene delivery [14] Mice were anaesthetised with intraperitoneal ketamine (100 mg/kg) and xylazine (8 mg/kg). Animals were intubated and ventilated with room air. The heart was exposed via a left anterolateral thoracotomy incision at the fourth intercostal space. Next, adenovirus with or without VDAC1 gene (2 × 1011 plaque forming units/ml) was directly injected into the left ventricular free wall (five sites, 10 μl/site)
with a 29-gauge needle. After injection, a plastic
cannula was inserted through the chest wall to evacuate residual air prior to chest closure. Sham-operated mice underwent the same procedure without gene transfer. 2.5 Ischemia-reperfusion model preparation and measurement of contractile function This article is protected by copyright. All rights reserved.
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All of the mice were anaesthetised with an intraperitoneal injection of 30 mg/kg sodium pentobarbital. After midline sternotomy, the hearts were rapidly excised and placed in cold Krebs-Henseleit (K-H) solution (4°C). Then hearts were mounted onto a modified Langendorff apparatus and perfused at a constant pressure of 80 cm H2O with K-H solution (mM: NaCl, 119.0; NaHCO3, 25.0; KCl, 4.7; KH2PO4, 1.2; MgSO4·7H2O, 1.2; CaCl2, 2.5; and glucose, 11.0). The K-H solution was saturated with 95% O2 and 5% CO2 (37°C, pH 7.4). A water-filled latex balloon was inserted into the left ventricle through the left artiotomy, and the volume was adjusted to achieve a stable left ventricular end-diastolic pressure of 5 to 6 mmHg during initial equilibration, which was maintained throughout the experiment. The left ventricular pressure (LVP, kPa) and maximal positive and negative change in the left ventricular pressure (±dp/dtmax, kPa/s) were continuously monitored and recorded by the PowerLab system (ADInstruments, Sydney, NSW, Australia). Coronary flow (CF, ml/min) was measured by the timed collection of coronary effluent. The LDH and CPK activity in CF effluent after 30 min of reperfusion was measured using a Beckman automatic biochemical analyser. I/R model was established as described previously [15]. Briefly, hearts were equilibrated in K-H solution for 30 min in the Langendorff apparatus. Subsequently, hearts were subjected to 15-min global ischemia with modified K-H solution without glucose and saturated with 95% N2 and 5% CO2 (37°C, pH 6.8), followed by 30-min reperfusion with K-H solution. Control hearts were constantly subjected to the K-H solution during ischemia and reperfusion. 2.6 Measurement of infarct size At the end of each experiment, the heart was immediately removed from the Langendorff apparatus, weighed, and frozen at -20°C. The frozen heart was manually cut into seven to eight transverse slices of approximately equal thickness (0.8 mm) and stained by incubating in 10% triphenyl tetrazolium chloride (TTC) for 30 min at room temperature (22°C). TTC This article is protected by copyright. All rights reserved.
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buffer was then replaced with 10% formaldehyde, and the slices were fixed for 4 to 6 h. The risk area was calculated as the total ventricular area minus the area of the cavities. The infarct size was calculated as a percentage of the risk area. 2.7 Isolation of mitochondria and cytosolic fractions At the end of the ischemia/reperfusion protocol, the left ventricular tissues were homogenised in an isolation medium (250 mM sucrose, 10 mM Tris-HCl, 1 mM EGTA, pH 7.4). The homogenate was centrifuged at 1000 × g for 10 min at 4°C to discard nuclei and myofibrils. The resulting supernatant was centrifuged at 10,000 × g for 15 min at 4°C, which produced a pellet that was rich in mitochondria. The pellet was washed twice with isolation medium and then resuspended in the appropriate volume of the final buffer (250 mM sucrose, 10 mM Tris-HCl, pH 7.4) to produce the final mitochondrial fraction. The supernatant was centrifuged at 100,000 × g for 60 min. The resulting supernatant was the final cytosolic fraction. All isolated mitochondria and cytosolic fractions were kept on ice and used within 3 h. 2.8 Caspase-3 activity assay Caspase-3 activity was measured in the cytosolic fraction isolated from heart homogenates as described previously. Briefly, caspase-3 activity was determined by measuring the cleavage of a caspase-3-specific substrate (acetyl-Asp-Glu-Val-Asp(DEVD)-p-nitroanilide (pNA) (DEVD-pNA)) using a caspase-3 activity assay kit (R&D Systems) according to the manufacturer's instructions. 2.9 Western blotting analysis Myocardial samples were homogenised in ice-cold lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 1 mM NaF, 1 mM Na3VO4, 0.4 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. After centrifugation at 10,000 × g for 15 min at 4°C, supernatants containing proteins were treated This article is protected by copyright. All rights reserved.
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with sample buffer, boiled for 5 min, separated using 10% SDS–PAGE, and transferred to membranes. Then the membranes were blotted with an anti-VDAC1 antibody (1:200, Abcam) in Tris-buffered saline (pH 7.6) containing 0.1% Tween-20 (TBST). To ensure equivalent protein loading, the membranes were incubated with anti-β-actin antibody (1:200, Santa Cruz) and subsequently with a corresponding horseradish peroxidase-conjugated second antibody IgG (Santa Cruz) and developed using Chemiluminescence Reagent Plus. Scan densitometric analysis was performed using the GDS-8000 UVP photo scanner and LAB WOEK45 Image software (Bio-Rad). The cytosolic cytochrome c content was evaluated using western blotting analyses with a primary antibody against cytochrome c (1:1000). 2.10 Measurement of mPTP opening mPTP opening was determined by Ca2+-induced swelling of isolated mitochondria, as described previously [16]. Opening of the pore causes mitochondrial swelling, which was measured as a reduction in absorbance at 520 nm. Isolated cardiac mitochondria were resuspended in swelling buffer (120 mmol/L KCl, 10 mmol/L Tris-HCl [pH 7.4], 20 mmol/L MOPS, and 5 mmol/L KH2PO4) to a final concentration of 0.25 mg/ml. The absorbance was measured by spectrophotometer at 520 nm, and the pore opening was induced by 200 μmol/L CaCl2 every 15-20 s. 2.11 TUNEL staining Cardiomyocyte
apoptosis
was
analysed
using
the
terminal
deoxynucleotidyl
transferase-mediated nick end labelling (TUNEL) staining method with a kit purchased from Promega. Briefly, left ventricle tissues from the area at risk were fixed in formalin for 24 h, embedded in paraffin, and 5-μm sections were obtained. The sections were deparaffinised and rehydrated with a xylene and graded alcohol series. Next, the sections were subjected to a TUNEL assay according to the manufacturer’s protocol. The number of TUNEL-positive cardiomyocytes represented the amount of apoptosis. This article is protected by copyright. All rights reserved.
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2.12 Statistical analysis The data are presented as the mean ± SEM. The difference between groups was analysed using one-way analysis of variance followed by Student–Newman–Keuls post-hoc test. P < 0.05 was considered statistically significant.
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3. Results 3.1 Effects of long-term oral resveratrol intake on I/R injury in isolated hearts LDH and CPK leakage from cells and the infarct size were used as indicia of myocardial injury. The LDH and CPK activity were measured from CF effluent after 30 min of reperfusion. TTC staining was performed to evaluate myocardial infarct size. As shown in Fig. 1, the hearts subjected to I/R injury displayed an increase in LDH and CPK activity (65.4±4.1, 100.6±6.5) compared with the control group (23.8±1.2, 44.7±2.3), which was significantly suppressed by a 6-week resveratrol pretreatment in a dose-dependent manner up to 2.0 mg/kg.d. The same protection of resveratrol was also observed in infarct size. With doses of 0.5, 1.0, and 2.0 mg/kg.d of resveratrol, a significant dose-dependent reduction in infarct size was also detected (Fig. 2).
In vehicle group, 25% ethanol did not have any
influence on LDH and CPK activity, CF effluent and infarct size compared with control group. We also found resveratrol did not have a significant effect in mice without I/R compared to control and vehicle group. 3.2 Effects of long-term oral resveratrol intake on VDAC1 expression. VDAC1 expression was detected using western blotting in mouse myocardium subjected to I/R injury with or without resveratrol pretreatment. We found that VDAC1 expression was up-regulated in I/R injury. And this up-regulation was inhibited in the resveratrol group (Fig. 3A). Furthermore, to examine the role of VDAC1 in the protective effects of resveratrol, we established an in vivo gene transfer protocol to locally increase VDAC1 levels in the mouse left ventricle. Following adenoviral injection in the left ventricular free wall, overexpression of VDAC1 was observed in mice injected with pAd-VDAC1 but not the pAd-shuttle (an empty adenoviral vector) (Fig. 3B). 3.3 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on cardiac function in response to I/R injury This article is protected by copyright. All rights reserved.
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In order to investigate the effect of various treatments on cardiac function, LVP, ±dp/dtmax and CF of isolated mouse hearts were tested in Langendorff apparatus (Fig. 4). No significant difference was observed in the pre-ischemic baseline parameters of hearts. After 15 min ischemia, LVP, ±dp/dtmax and CF decreased significantly in the I/R-group during reperfusion, indicating the damage of I/R on cardiac function. Interestingly, LVP, ±dp/dtmax and CF increased in resveratrol group compared with I/R group, indicating that resveratrol improved the recovery of heart during reperfusion. Moreover, long-term oral resveratrol treatment significantly prevented the increase of LDH and CPK activity in the CF effluent induced by I/R injury. However, overexpression of VDAC1 exacerbated I/R injury in pAd-VDAC1+I/R group, and abolished the cardioprotective effect of resveratrol after I/R injury in pAd-VDAC+Res+I/R group. The empty adenoviral vector had no effect on cardioprotection with resveratrol treatment. 3.4 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on infarct size in response to I/R injury The results and representative images of the infarct size are shown in Fig. 6. Typical heart cross sections from each treatment after TTC staining are shown in Fig. 6A. The red area represents the viable region, and the white area represents the infarct size. There was a 7.2% infarct size in the sham group after 30 min of initial perfusion. The infarct size in I/R group was significantly increased to 34.1% at the end of the 30-min reperfusion. Furthermore, when VDAC1 was overexpressed, the infarct size expanded to 45.6 % after I/R in pAd-VDAC1+I/R group. However, intake of resveratrol reduced the infarct size to 24.2% in Res+I/R group, but failed to exert protection due to VDAC1 overexpression in pAd-VDAC+Res+I/R group. 3.5 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on mPTP opening and cytochrome c release in mice subjected to I/R This article is protected by copyright. All rights reserved.
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injury Mouse cardiac mitochondria were isolated, and Ca2+-induced pore opening was detected by measuring mitochondrial swelling. The opening of the pore caused mitochondrial swelling, which was observed as a decrease in absorbance at 520 nm. In I/R-treated group, Ca2+ evoked a large decrease in A520 (Fig. 7). This effect was attenuated by resveratrol, confirming that resveratrol intake prevented mPTP opening during I/R injury. In pAd-VDAC1+I/R group, absorbance of mitochondria at 520 nm dropped even more steeply after I/R injury compared with I/R alone treated group. And due to VDAC1 overexpression, the protective effect of resveratrol on mPTP opening was abolished, indicating that VDAC1 is a key effector of resveratrol. It is well established that mPTP opening causes mitochondrial swelling and rupture of the outer mitochondrial membrane. This process results in the release of proapoptotic factors cytochrome c. As an additional index of mPTP opening, the release of cytochrome c from mitochondria was analysed by measuring the cytosolic cytochrome c content. Resveratrol reduced I/R-induced cytochrome c release from mitochondria by inhibiting mPTP opening, which is consistent with the results of mitochondrial swelling (Fig. 8). However, the effect of resveratrol was abrogated by VDAC1 overexpression. 3.6 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on caspase-3 activity in mice subjected to I/R injury To investigate the contribution of the caspase-dependent pathway on apoptosis, caspase-3 activity in the cytosol was examined. Long-term oral resveratrol intake inhibited the I/R-induced increase of caspase-3 activity. However, with overexpression of VDAC1, resveratrol failed to prevent the increase of caspase-3 activity (Fig. 9). 3.7 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on apoptosis in mice subjected to I/R injury This article is protected by copyright. All rights reserved.
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To measure
apoptosis,
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the
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TUNEL-positive cardiomyocytes
in
the
immunohistological sections. As shown in Fig.10, in the hearts of sham-group, TUNEL-positive cells were seldom identified. In hearts subjected to I/R, numerous TUNEL-positive cells were observed, and further increased after VDAC1 overexpression. Mice with resveratrol intake showed a significant reduction in the number of TUNEL-positive cells compared with I/R group. However, the protective effect of resveratrol was abolished in mice injected with adenovirus vectors expressing VDAC1.
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4. Discussion Currently, lifestyle and nutritional condition is gaining more attention to prevent and treat heart diseases.
As such, “nutritional preconditioning” has been proposed by Abdukeyum
et.al. For such preconditioning, the nutrients must exist naturally, can be obtained through daily diet and exhibit cardioprotective effects [6]. Thus, “nutritional preconditioning” could represent a low-risk solution for ischemic cardioprotection, which can limit infarct size without prediction of ischemic events [17]. Resveratrol, a polyphenolic compound in red wine, has been well-documented to provide cardiovascular protective effect. Previous epidemiological studies suggested that drinking red wine in moderation (100- 400ml per day) may provide cardioprotective effects [18-21]. The general levels of resveratrol found in red wine usually vary from 0.287 to 7.188 mg/L, depending on the grape variety and winemaking techniques [22].
Wines produced from
muscadine grapes may contain more than 40 mg/L of resveratrol [23]. We converted the daily diet intake dosage of resveratrol in human to that in mice applied in our study, according to the conversion coefficient of body surface area. It ensured the dosages of resveratrol can be obtained through daily diet, rather than acute therapeutic administration. The 6-week feeding represents a long-term habitual dietary intake and ensures the concentration of resveratrol to reach an effective steady state. In current study, mouse hearts were isolated and subjected to I/R injury in Langendorff apparatus after 6-week resveratrol intake. We found that, compared with the group without resveratrol intake, resveratrol improved LVP, ±dp/dtmax and CF, decreased LDH and CPK levels, and reduced the infarct size caused by I/R. Thus, resveratrol as a nutritional preconditioning agent can lender cardioprotection to the heart against I/R insult. The mechanisms underlying resveratrol-induced cardioprotection have been extensively studied [24]. Several signal pathways are involved including sirtuins [25], GSK-3β [7], and This article is protected by copyright. All rights reserved.
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ATP-sensitive potassium channels [26]. However, the end-effectors of resveratrol preconditioning remain unclear. Recent studies have suggested that mPTP is a convergence point of numerous pathways, including sirtuins [27], GSK-3β [28], and ATP-sensitive potassium channels [29], in ischemic preconditioning. Pathological opening of mPTP is well known to cause mitochondrial permeability transition, and consequently plays a critical role in the progression of both apoptotic and necrotic cell death. mPTP opening is associated with mitochondrial swelling. Because the surface area of the inner membrane exceeds that of the outer membrane, extensive matrix swelling with long-lasting mPTP opening can lead to the unfolding of cristae, outer membrane to rupture and irreversibly mitochondria damage [30]. In addition, outer membrane rupture releases proapoptotic molecules residing in the intermembrane space, such as cytochrome c [31]. Once released, cytochrome c activates caspase 9, which in turn activates caspase 3. This protease mediates the proteolytic cleavage of a range of proteins responsible for the rearrangement of the cytoskeleton, plasma membrane, and nucleus that are characteristic of apoptosis. Thus, inhibition of mPTP opening appears to be an effective means for cardioprotection. We found that long-term oral resveratrol intake significantly blocked mPTP opening and attenuated mitochondrial swelling, subsequently prevented cytochrome c release, caspase-3 activation and TUNEL staining effectively. Strikingly, our results revealed that resveratrol regulates mPTP opening through VDAC1. VDAC1 was remarkably up-regulated in I/R injury. Pretreatment with resveratrol offsets a rising of VDAC1 during I/R. Overexpression of VDAC1 with pAd-VDAC1 was found to exacerbate I/R damages to the heart. When VDAC1 was overexpressed, the protective effect of resveratrol was compromised, suggesting that VDAC1 is the ultimate target of resveratrol, which might be regulated at the transcriptional level. As exogenous VDAC1 in adenovirus is under the control of the CMV promoter, resveratrol can not inhibits its expression. It is This article is protected by copyright. All rights reserved.
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possible that the overexpressed level of VDAC1 surpassed the inhibitory capacity of resveratrol. In any event, our results suggest an important role of VDAC1 in I/R injury.
The
mechanism underlying apoptosis induced by overexpression of VDAC1 in I/R can be explained as follows: (1) upregulation of VDAC1 might increase outer mitochondrial membrane permeability and thereby decrease cell viability [32]; (2) VDAC1 oligomerization was found to be strongly correlated with apoptosis induction [33, 34].
Thus, overexpression
of VDAC1 results in shifting of the VDAC1 equilibrium from monomeric to oligomeric species. This VDAC1 oligomeric state allows forming a large pore causing release of pro-apoptotic proteins, such as cytochrome c [33,34]； (3) increased VDAC1 interacts with the inner membrane protein adenine nucleotide translocator at contact sites, thus forming and activating the mPTP complex [35]; (4) VDAC1 has been known as one of the mediators of oxidative stress-induced apoptosis [36, 37]. Thus, it is possible that overexpression of VDAC1 enhances ROS production and subsequently triggers apoptosis. In conclusion, we demonstrate that long-term administration of resveratrol exerts a protective effect on cardiomyocytes against I/R insult. Our data support the use of resveratrol as a nutritional preconditioning agent in preventing cardiac damage caused by I/R, which advocates its use in food/diet supplement. According to our knowledge, this is the first set of data showing that VDAC1 plays an important role in cardiac damage imposed by I/R and VDAC1 is a target of resveratrol.
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This study was supported by the Research Program of the Sate Key Laboratory of Food Science and Technology, Nanchang University (Project No. SKLF-TS-201106), and the National Natural Scientific Foundation of China (No. 30960449, 81460495).
All the authors have declared no conflicts of interest.
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5. References [1] Renaud, S.C., Gueguen, R., Schenker. J., d’Houtaud, A., Alcohol and mortality in middle-aged men from Eastern France. Epidemiology. 1998, 9, 184–188. [2] Kumerz, M., Heiss, E.H., Schachner, D., Atanasov, A.G., Dirsch, V.M., Resveratrol inhibits migration and Rac1 activation in EGF- but not PDGF-activated vascular smooth muscle cells. Mol Nutr Food Res. 2011, 55,1230-1236. [3] Yang, M., Camara, A.K., Wakim, B.T., Zhou, Y. et al., Tyrosine nitration of voltage-dependent anion channels in cardiac ischemia-reperfusion: reduction by peroxynitrite scavenging. Biochim Biophys Acta. 2012, 1817, 2049-2059. [4] Guo, J., Wang, S.B., Yuan, T.Y., Wu, Y.J. et al., Coptisine protects rat heart against myocardial ischemia/reperfusion injury by suppressing myocardial apoptosis and inflammation. Atherosclerosis. 2013, 231, 384-391. [5] Della-Morte, D., Guadagni, F., Palmirotta, R., Ferroni, P. et al., Genetics and genomics of ischemic tolerance: focus on cardiac and cerebral ischemic preconditioning. Pharmacogenomics. 2012, 13, 1741-1757. [6] Abdukeyum, G.G., Owen, A.J., McLennan, P.L., Dietary (n-3) long-chain polyunsaturated fatty acids inhibit ischemia and reperfusion arrhythmias and infarction in rat heart not enhanced by ischemic preconditioning. J Nutr. 2008,138, 1902-1909. [7] Xi, J., Wang, H., Mueller, R.A., Norfleet, E.A., Xu, Z., Mechanism for resveratrol-induced cardioprotection against reperfusion injury involves glycogen synthase kinase 3 beta and mitochondrial permeability transition pore. Eur J Pharmacol. 2009, 604, 111-116. [8] Heller, A., Brockhoff, G., Goepferich, A., Targeting drugs to mitochondria. Eur J Pharm Biopharm. 2012, 82, 1-18. [9] Kinnally, K.W., Peixoto, P.M., Ryu, S.Y., Dejean, L.M., Is mPTP the gatekeeper for This article is protected by copyright. All rights reserved.
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mitochondrial membrane by superoxide induces rapid and massive cytochrome c release. J. Cell. Biol. 2001,155, 1003-1015. [37] Simamura, E., Hirai, K., Shimada, H., Koyama, J. et al., Furanonaphthoquinones cause apoptosis of cancer cells by inducing the production of reactive oxygen species by the mitochondrial voltage-dependent anion channel. Cancer Biol. Ther. 2006, 5, 1523–1529.
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Fig. 1 Effect of long-term oral intake of resveratrol on LDH and CPK activity in isolated mouse hearts subjected to I/R. LDH and CPK activity in CF effluent was measured by a Beckman automatic biochemical analyser after 30 min of reperfusion. Long-term oral resveratrol intake suppressed the increase in LDH and CPK activity in a dose-dependent manner up to 2.0 mg/kg.d. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p> 0.05, vs. control group; b: p 0.05, vs. I/R group; d: p< 0.05, vs. I/R group.
80
LDH Activity(U/L)
b
c d
60
d 40
a
a
20
0 Cont
Vehicle
-
+
+
+
+
I/R
2.0
-
0.5
1.0
2.0
Res(mg/kg.d)
120
b
CPK Activity(U/L)
c d
90
d a
60
a
30
0 Cont
Vehicle
-
+
+
+
+
I/R
2.0
-
0.5
1.0
2.0
Res(mg/kg.d)
Fig 1
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Fig. 2 Effect of long-term oral intake of resveratrol on infarct size in isolated mouse hearts subjected to I/R. The myocardial infarct size was evaluated by TTC staining and calculated as a percentage of the risk area. Long-term administration of resveratrol reduced the infarct size in a dose-dependent manner. The most remarkable effect of resveratrol was detected at a dose of 2.0 mg/kg.day. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p> 0.05, vs. control group; b: p 0.05, vs. I/R group; d: p< 0.05, vs. I/R group.
40
b
c d
Infarct Size(%)
30
d
20
a
10
a
0 Cont
Vehicle
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+
+
+
+
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2.0
-
0.5
1.0
2.0
Res(mg/kg.d)
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Fig. 3 VDAC1 expression was detected by western blotting. (A) The effect of long-term oral resveratrol (2.0 mg/kg.d) intake on VDAC1 expression. Control mice hearts (Cont) were constantly subjected to the perfusion solution. Mice hearts in the I/R group were subjected to 15 min of global ischemia and 30 min of reperfusion. The mice in resveratrol group (Res) were orally administrated by gastric gavages with resveratrol (2.0 mg/kg.day) for 6 weeks before I/R treatment. (B) VDAC1 expression following injection of pAd-VDAC1 in the left ventricle free wall. Sham-operated mice underwent a surgical procedure but without gene transfer. pAd-VDAC1 mice were injected with adenovirus containing VDAC1 cDNA. pAd-Shuttle mice were injected with empty adenovirus vector. β-actin was used as a loading control. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. Res+I/R group.
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Fig. 5 VDAC1 overexpression abolished the cardioprotection of long-term oral resveratrol intake on LDH and CPK activity in isolated mouse hearts subjected to I/R. Long-term oral resveratrol intake decreased LDH and CPK activity in mice subjected to I/R injury. However, VDAC1 overexpression diminished the effect of resveratrol. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. I/R group; e: p> 0.05, vs. Res+I/R group.
100
b a
LDH Activity(U/L)
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c, d
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b a
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60 30 0 Sham
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Res pAd-Shuttle
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Fig. 6 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on infarct size in mice subjected to I/R injury. (A) Heart cross sections from each treatment after TTC staining. The red area represents the viable region, and the white area represents the infarct size. (B) Histogram of infarct size. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. I/R group; e: p> 0.05, vs. Res+I/R group.
A
Sham
B
I/R
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Res +I/R
pAd-VDAC1 +Res+I/R
pAd-Shuttle +Res+I/R
b a
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pAd-VDAC1 +I/R
c, d e
b
30 20 10 0 Sham
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Res pAd-Shuttle
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Fig. 7 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on mPTP opening in mice subjected to I/R injury. mPTP opening was determined by Ca2+-induced swelling of isolated mitochondria. Opening of the pore causes mitochondrial swelling, which was measured as a reduction in absorbance at 520 nm. (A) Original traces for mitochondrial swelling within 2 min. (B) The rate of mitochondrial swelling induced by 200 μmol/L CaCl2. Data are shown as a change in A520 and presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. I/R group; e: p> 0.05, vs. Res+I/R group.
A 2+
Ca 200μ mol/L
Sham
Res+I/R
A520 nm
pAd-Shuttle+Res+I/R
pAd-VDAC1+Res+I/R I/R pAd-VDAC1+I/R 2 min
B 0.6
b ΔAbsorbance(520nm)
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Fig 7 This article is protected by copyright. All rights reserved.
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Fig. 8 Cytochrome c protein expression in cytoplasmic fraction and whole lysate was analyzed by Western blotting. mPTP opening causes mitochondrial swelling and rupture of the outer mitochondrial membrane, which results in the release of cytochrome c. As an additional index of mPTP opening, the release of cytochrome c from mitochondria was analysed. (A) Representative western blots. (B) Levels of cytochrome c expression of the cytoplasmic fraction versus whole cell were quantified. Data are expressed as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. I/R group; e: p> 0.05, vs. Res+I/R group.
A
Sham
I/R
pAd-VDAC1 +I/R
Res +I/R
pAd-VDAC1 pAd-Shuttle +Res+I/R +Res+I/R
Cytoplasmic fraction Whole cell lysate β -actin
B C-Cyt c/W-Cyt c
1.2 1
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Fig. 9 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on caspase-3 activity in mice subjected to I/R injury. Caspase-3 activity was measured in the cytosolic fraction isolated from heart homogenates. Due to injection of adenovirus vectors pAd-VDAC1, long-term oral resveratrol intake failed to prevent the increase in caspase-3 activity in mice subjected to I/R injury. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. I/R group; e: p> 0.05, vs. Res+I/R group.
Caspase-3 Activity(U/mg pro)
4
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Fig. 10 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on apoptosis in mice subjected to I/R injury. (A) Hearts were sectioned and analysed for apoptosis using the TUNEL staining method. The panels display representative histological images. (B) The number of apoptotic cells evaluated by TUNEL is expressed as a percentage. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. I/R group; e: p> 0.05, vs. Res+I/R group.
Sham
I/R
pAd-VDAC1+I/R
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Long-term oral resveratrol intake provides nutritional preconditioning against myocardial ischemia/reperfusion injury: involvement of VDAC1 down-regulation Zhangping Liao 1, 2, Dan Liu 2, Lei Tang2, Dong Yin3, Shuhua Yin2, Songqing Lai2, Jianguo Yao2, *, Ming He1, 2, * 1
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang
330047, PR China 2
Department of Pharmacology & Molecular Therapeutics, Nanchang University School of
Pharmaceutical Science, Nanchang 330006, PR China 3
Jiangxi Provincial Key Laboratory of Molecular Medicine at the Second Affiliated Hospital,
Nanchang University, Nanchang 330006, PR China
*These authors contributed equally to this work. Corresponding author: Ming He, PhD Department of Pharmacology& Molecular Therapeutics Nanchang University School of Pharmaceutical Science Nanchang 330006, PR China Tel.: +86-791-86362231
Fax: +86-791-86361839
E-mail: [email protected] Abbreviations: CF, coronary flow; I/R, ischemia/reperfusion; LVP, left ventricular pressure; mPTP, mitochondrial permeability transition pore; TTC, triphenyl tetrazolium chloride; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labelling; VDAC, Received: 11-Oct-2014; Revised: 29-Nov-2014; Accepted: 01-Dec-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/mnfr.201400730. This article is protected by copyright. All rights reserved.
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voltage-dependent anion channel. Keywords: Ischemia-reperfusion/ Mitochondrial permeability transition pore/ Nutritional preconditioning / Resveratrol / Voltage-dependent anion channel
Abstract Scope: This study elucidates effects of long term nutritional preconditioning by resveratrol on I/R injury and its underlying mechanisms. Methods and results: Mice were treated with resveratrol at 2.0 mg/kg.day by gastric gavages for 6 weeks. Then hearts were isolated and subjected to I/R injury in a Langendorff apparatus. Resveratrol significantly improved LVP, ±dp/dtmax and CF, decreased the LDH and CPK activity, and reduced the infarction size. Additionally, long-term oral resveratrol intake prevented mPTP opening and subsequently inhibited mitochondria-mediated apoptosis, as demonstrated by decrease of cytochrome c release, inactivation of caspase-3 and reduction of TUNEL-positive cells. Furthermore, resveratrol inhibited the up-regulation of VDAC1 expression induced by I/R injury. Local left ventricle overexpression of VDAC1 by adenovirus diminished the protective effect of resveratrol against I/R injury, indicating that VDAC1 plays an important role in resveratrol-mediated cardioprotection. Conclusion: Our data revealed that long-term oral intake of resveratrol sets nutritional preconditioning in coping with myocardial I/R injury. Strikingly, we found that resveratrol down-regulates VDAC1, leading to prevention of mPTP opening and cardiomyocyte apoptosis.
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1. Introduction Despite the high-fat diet, Southern French have a low incidence of coronary heart disease and low mortality rate. This so-called “French paradox” is partly attributed to wine consumption, particularly red wine [1]. Resveratrol (trans-3,4’,5-trihydroxystilbene), a natural polyphenolic compound, is abundant in grape skins and wine, which is a well-known constituent with cardiovascular benefits [2]. Previous evidence indicated that resveratrol has multi beneficial effects, including antioxidant properties, modulation of lipid metabolism, and inhibition of platelet aggregation. Recently, it was demonstrated that resveratrol can protect the heart against ischemia/reperfusion (I/R) injury [3]. However, this study was performed in an acute setting. The long term effects of resveratrol on I/R injury still need to be evaluated. Restoration of blood flow to the ischemic myocardium may generate more tissue damage than ischemia alone, which is known as I/R injury [4]. Repeated and short sub-lethal ischemic preconditioning exerts protection of the myocardium against subsequent lethal I/R [5]. However, with a poor predictive outcome of ischemic episodes, ischemic preconditioning is not a feasible approach. Recently, Abdukeyum et.al proposed nutritional preconditioning, that nutrient intake elicits preconditioning, limits ischemic cardiac injury and myocardial infarction, thus lends cardioprotection which has similar efficacy to ischemic preconditioning [6]. Moreover, nutritional preconditioning is a safe and effective dietary approach with nutritional components. It avoids the risk of ischemic episodes and is more feasible in preventing I/R injury [6]. The mitochondrion, one of the most important organelles in cardiomyocytes, not only functions in energy production, but also activates apoptotic cascades in response to stress. Hence, mitochondria may be involved in the cardioprotection of resveratrol. Indeed, it has been reported that cardioprotection of resveratrol is associated with regulation of the mitochondrial permeability transition pore (mPTP) [7]. mPTP is a multi-protein complex This article is protected by copyright. All rights reserved.
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formed between the inner and outer mitochondrial membranes [8]. mPTP opening is critical to mitochondria-mediated apoptosis during I/R injury [9-11]. The voltage-dependent anion channel 1 (VDAC1), in the mitochondrial outer membrane, acts as a gate to control the inflow and outflow of mitochondrial metabolites and ions [12,13]. Thus, due to its key role in regulating
mPTP
opening,
we
hypothesize
VDAC1
is
a
potential
target
of
resveratrol-mediated nutritional preconditioning. In the current study, we investigated the protective effects of long-term oral administration of resveratrol on I/R injury and the mechanism underlying resveratrol-mediated cardioprotection. We found that long-term oral administration of resveratrol protects the myocardium against I/R injury at an equivalent dose to human diet intake. Furthermore, resveratrol prevented mPTP opening and inhibited I/R-induced upregulation of VDAC1. Additionally, overexpression of VDAC1 diminished the protective effect of resveratrol. These data for the first time indicate that VDAC1 is the target of resveratrol, and demonstrate that resveratrol exhibits protective effects against I/R injury and can be used as long-term nutritional preconditioning agent for I/R. This will help to advocate its use as a healthy diet supplement.
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2. Materials and methods 2.1. Animals Adult Kun-Ming mice were provided by the Animal Center of Nanchang University. Experiments was performed according to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996), and approved by the Ethics Committee of Nanchang University (No. 2012-0116). All mice were housed, two per cage, under a controlled temperature of 22°C with a humidity of 50%, in a 12 h light/dark cycle (08:00-20:00 light, 20:00-08:00 dark). 2.2 Experimental design 2.2.1 Determination of resveratrol dosage To conform to nutritional preconditioning, the dosage of resveratrol applied in our study was equivalent to that used by humans as a diet intake, according to the conversion coefficient of body surface area between human and mouse. Resveratrol was dissolved in 25% ethanol. The mice were randomly assigned to six groups (n=6 for each group, each experiment was repeated 3 times): (1) control group; (2) vehicle control group; (3) resveratrol control group; (4) I/R group; (5) resveratrol-0.5 group; (6) resveratrol-1.0 group; and (7) resveratrol-2.0 group. Three resveratrol groups were treated with resveratrol at 0.5 mg/kg.d, 1.0 mg/kg.d, or 2.0 mg/kg.d respectively by gastric gavages, for 6 weeks before I/R treatment. The control and I/R group were treated with saline (10 ml/kg.d). Mice in vehicle control or resveratrol control were administrated with 25% ethanol (2.0 mg/kg.d ) or resveratrol (2.0 mg/kg.d ) alone respectively for 6 weeks without I/R treatment. 2.2.2 Role of VDAC in resveratrol-mediated cardioprotection Six groups (n=6 for each group, the experiment was replicated 3 times) were set to investigate the role of VDAC1. (1) The sham group underwent surgical procedure but without gene transfer and ischemic injury. (2) The I/R group was subjected to 15 min of This article is protected by copyright. All rights reserved.
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ischemia followed by 30 min of reperfusion. (3) The pAd-VDAC1+I/R group. The mice were injected with adenovirus-expressing VDAC1. Five days later, the mice were sacrificed. The hearts were isolated and Langendorff-perfused to duplicate I/R model. (4) Res+I/R group. The mice were administrated with resveratrol (2.0 mg/kg.d) for 6 weeks. Then the hearts were isolated and Langendorff-perfused for the I/R model. (5) pAd-VDAC+Res+I/R group. The mice were orally administrated with resveratrol (2.0 mg/kg.d) for 6 weeks. The following treatment was same as pAd-VDAC1+I/R group. (6) pAd-Shuttle+Res+I/R group. The mice were injected with empty pAd-Shuttle vector without VDAC1 gene. Other procedures were same as pAd-VDAC+Res+I/R group. 2.3 Construction of recombinant adenovirus vectors. VDAC1 cDNA was amplified by PCR and inserted into pShuttle-CMV vector at Kpn I and Xho I sites. The recombinant pShuttle-CMV-VDAC1 was electroporated with pAdEasy1 into Escherichia coli BJ5183 cells (Stratagene). The recombinant adenoviral DNA was transfected into human embryonic kidney 293 cells with Lipofectamine (Invitrogen). Next, all of the viruses were plaque-purified and amplified. Empty adenoviral vector was used as a control. 2.4 Intramyocardial gene delivery [14] Mice were anaesthetised with intraperitoneal ketamine (100 mg/kg) and xylazine (8 mg/kg). Animals were intubated and ventilated with room air. The heart was exposed via a left anterolateral thoracotomy incision at the fourth intercostal space. Next, adenovirus with or without VDAC1 gene (2 × 1011 plaque forming units/ml) was directly injected into the left ventricular free wall (five sites, 10 μl/site)
with a 29-gauge needle. After injection, a plastic
cannula was inserted through the chest wall to evacuate residual air prior to chest closure. Sham-operated mice underwent the same procedure without gene transfer. 2.5 Ischemia-reperfusion model preparation and measurement of contractile function This article is protected by copyright. All rights reserved.
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All of the mice were anaesthetised with an intraperitoneal injection of 30 mg/kg sodium pentobarbital. After midline sternotomy, the hearts were rapidly excised and placed in cold Krebs-Henseleit (K-H) solution (4°C). Then hearts were mounted onto a modified Langendorff apparatus and perfused at a constant pressure of 80 cm H2O with K-H solution (mM: NaCl, 119.0; NaHCO3, 25.0; KCl, 4.7; KH2PO4, 1.2; MgSO4·7H2O, 1.2; CaCl2, 2.5; and glucose, 11.0). The K-H solution was saturated with 95% O2 and 5% CO2 (37°C, pH 7.4). A water-filled latex balloon was inserted into the left ventricle through the left artiotomy, and the volume was adjusted to achieve a stable left ventricular end-diastolic pressure of 5 to 6 mmHg during initial equilibration, which was maintained throughout the experiment. The left ventricular pressure (LVP, kPa) and maximal positive and negative change in the left ventricular pressure (±dp/dtmax, kPa/s) were continuously monitored and recorded by the PowerLab system (ADInstruments, Sydney, NSW, Australia). Coronary flow (CF, ml/min) was measured by the timed collection of coronary effluent. The LDH and CPK activity in CF effluent after 30 min of reperfusion was measured using a Beckman automatic biochemical analyser. I/R model was established as described previously [15]. Briefly, hearts were equilibrated in K-H solution for 30 min in the Langendorff apparatus. Subsequently, hearts were subjected to 15-min global ischemia with modified K-H solution without glucose and saturated with 95% N2 and 5% CO2 (37°C, pH 6.8), followed by 30-min reperfusion with K-H solution. Control hearts were constantly subjected to the K-H solution during ischemia and reperfusion. 2.6 Measurement of infarct size At the end of each experiment, the heart was immediately removed from the Langendorff apparatus, weighed, and frozen at -20°C. The frozen heart was manually cut into seven to eight transverse slices of approximately equal thickness (0.8 mm) and stained by incubating in 10% triphenyl tetrazolium chloride (TTC) for 30 min at room temperature (22°C). TTC This article is protected by copyright. All rights reserved.
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buffer was then replaced with 10% formaldehyde, and the slices were fixed for 4 to 6 h. The risk area was calculated as the total ventricular area minus the area of the cavities. The infarct size was calculated as a percentage of the risk area. 2.7 Isolation of mitochondria and cytosolic fractions At the end of the ischemia/reperfusion protocol, the left ventricular tissues were homogenised in an isolation medium (250 mM sucrose, 10 mM Tris-HCl, 1 mM EGTA, pH 7.4). The homogenate was centrifuged at 1000 × g for 10 min at 4°C to discard nuclei and myofibrils. The resulting supernatant was centrifuged at 10,000 × g for 15 min at 4°C, which produced a pellet that was rich in mitochondria. The pellet was washed twice with isolation medium and then resuspended in the appropriate volume of the final buffer (250 mM sucrose, 10 mM Tris-HCl, pH 7.4) to produce the final mitochondrial fraction. The supernatant was centrifuged at 100,000 × g for 60 min. The resulting supernatant was the final cytosolic fraction. All isolated mitochondria and cytosolic fractions were kept on ice and used within 3 h. 2.8 Caspase-3 activity assay Caspase-3 activity was measured in the cytosolic fraction isolated from heart homogenates as described previously. Briefly, caspase-3 activity was determined by measuring the cleavage of a caspase-3-specific substrate (acetyl-Asp-Glu-Val-Asp(DEVD)-p-nitroanilide (pNA) (DEVD-pNA)) using a caspase-3 activity assay kit (R&D Systems) according to the manufacturer's instructions. 2.9 Western blotting analysis Myocardial samples were homogenised in ice-cold lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 1 mM NaF, 1 mM Na3VO4, 0.4 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. After centrifugation at 10,000 × g for 15 min at 4°C, supernatants containing proteins were treated This article is protected by copyright. All rights reserved.
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with sample buffer, boiled for 5 min, separated using 10% SDS–PAGE, and transferred to membranes. Then the membranes were blotted with an anti-VDAC1 antibody (1:200, Abcam) in Tris-buffered saline (pH 7.6) containing 0.1% Tween-20 (TBST). To ensure equivalent protein loading, the membranes were incubated with anti-β-actin antibody (1:200, Santa Cruz) and subsequently with a corresponding horseradish peroxidase-conjugated second antibody IgG (Santa Cruz) and developed using Chemiluminescence Reagent Plus. Scan densitometric analysis was performed using the GDS-8000 UVP photo scanner and LAB WOEK45 Image software (Bio-Rad). The cytosolic cytochrome c content was evaluated using western blotting analyses with a primary antibody against cytochrome c (1:1000). 2.10 Measurement of mPTP opening mPTP opening was determined by Ca2+-induced swelling of isolated mitochondria, as described previously [16]. Opening of the pore causes mitochondrial swelling, which was measured as a reduction in absorbance at 520 nm. Isolated cardiac mitochondria were resuspended in swelling buffer (120 mmol/L KCl, 10 mmol/L Tris-HCl [pH 7.4], 20 mmol/L MOPS, and 5 mmol/L KH2PO4) to a final concentration of 0.25 mg/ml. The absorbance was measured by spectrophotometer at 520 nm, and the pore opening was induced by 200 μmol/L CaCl2 every 15-20 s. 2.11 TUNEL staining Cardiomyocyte
apoptosis
was
analysed
using
the
terminal
deoxynucleotidyl
transferase-mediated nick end labelling (TUNEL) staining method with a kit purchased from Promega. Briefly, left ventricle tissues from the area at risk were fixed in formalin for 24 h, embedded in paraffin, and 5-μm sections were obtained. The sections were deparaffinised and rehydrated with a xylene and graded alcohol series. Next, the sections were subjected to a TUNEL assay according to the manufacturer’s protocol. The number of TUNEL-positive cardiomyocytes represented the amount of apoptosis. This article is protected by copyright. All rights reserved.
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2.12 Statistical analysis The data are presented as the mean ± SEM. The difference between groups was analysed using one-way analysis of variance followed by Student–Newman–Keuls post-hoc test. P < 0.05 was considered statistically significant.
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3. Results 3.1 Effects of long-term oral resveratrol intake on I/R injury in isolated hearts LDH and CPK leakage from cells and the infarct size were used as indicia of myocardial injury. The LDH and CPK activity were measured from CF effluent after 30 min of reperfusion. TTC staining was performed to evaluate myocardial infarct size. As shown in Fig. 1, the hearts subjected to I/R injury displayed an increase in LDH and CPK activity (65.4±4.1, 100.6±6.5) compared with the control group (23.8±1.2, 44.7±2.3), which was significantly suppressed by a 6-week resveratrol pretreatment in a dose-dependent manner up to 2.0 mg/kg.d. The same protection of resveratrol was also observed in infarct size. With doses of 0.5, 1.0, and 2.0 mg/kg.d of resveratrol, a significant dose-dependent reduction in infarct size was also detected (Fig. 2).
In vehicle group, 25% ethanol did not have any
influence on LDH and CPK activity, CF effluent and infarct size compared with control group. We also found resveratrol did not have a significant effect in mice without I/R compared to control and vehicle group. 3.2 Effects of long-term oral resveratrol intake on VDAC1 expression. VDAC1 expression was detected using western blotting in mouse myocardium subjected to I/R injury with or without resveratrol pretreatment. We found that VDAC1 expression was up-regulated in I/R injury. And this up-regulation was inhibited in the resveratrol group (Fig. 3A). Furthermore, to examine the role of VDAC1 in the protective effects of resveratrol, we established an in vivo gene transfer protocol to locally increase VDAC1 levels in the mouse left ventricle. Following adenoviral injection in the left ventricular free wall, overexpression of VDAC1 was observed in mice injected with pAd-VDAC1 but not the pAd-shuttle (an empty adenoviral vector) (Fig. 3B). 3.3 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on cardiac function in response to I/R injury This article is protected by copyright. All rights reserved.
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In order to investigate the effect of various treatments on cardiac function, LVP, ±dp/dtmax and CF of isolated mouse hearts were tested in Langendorff apparatus (Fig. 4). No significant difference was observed in the pre-ischemic baseline parameters of hearts. After 15 min ischemia, LVP, ±dp/dtmax and CF decreased significantly in the I/R-group during reperfusion, indicating the damage of I/R on cardiac function. Interestingly, LVP, ±dp/dtmax and CF increased in resveratrol group compared with I/R group, indicating that resveratrol improved the recovery of heart during reperfusion. Moreover, long-term oral resveratrol treatment significantly prevented the increase of LDH and CPK activity in the CF effluent induced by I/R injury. However, overexpression of VDAC1 exacerbated I/R injury in pAd-VDAC1+I/R group, and abolished the cardioprotective effect of resveratrol after I/R injury in pAd-VDAC+Res+I/R group. The empty adenoviral vector had no effect on cardioprotection with resveratrol treatment. 3.4 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on infarct size in response to I/R injury The results and representative images of the infarct size are shown in Fig. 6. Typical heart cross sections from each treatment after TTC staining are shown in Fig. 6A. The red area represents the viable region, and the white area represents the infarct size. There was a 7.2% infarct size in the sham group after 30 min of initial perfusion. The infarct size in I/R group was significantly increased to 34.1% at the end of the 30-min reperfusion. Furthermore, when VDAC1 was overexpressed, the infarct size expanded to 45.6 % after I/R in pAd-VDAC1+I/R group. However, intake of resveratrol reduced the infarct size to 24.2% in Res+I/R group, but failed to exert protection due to VDAC1 overexpression in pAd-VDAC+Res+I/R group. 3.5 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on mPTP opening and cytochrome c release in mice subjected to I/R This article is protected by copyright. All rights reserved.
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injury Mouse cardiac mitochondria were isolated, and Ca2+-induced pore opening was detected by measuring mitochondrial swelling. The opening of the pore caused mitochondrial swelling, which was observed as a decrease in absorbance at 520 nm. In I/R-treated group, Ca2+ evoked a large decrease in A520 (Fig. 7). This effect was attenuated by resveratrol, confirming that resveratrol intake prevented mPTP opening during I/R injury. In pAd-VDAC1+I/R group, absorbance of mitochondria at 520 nm dropped even more steeply after I/R injury compared with I/R alone treated group. And due to VDAC1 overexpression, the protective effect of resveratrol on mPTP opening was abolished, indicating that VDAC1 is a key effector of resveratrol. It is well established that mPTP opening causes mitochondrial swelling and rupture of the outer mitochondrial membrane. This process results in the release of proapoptotic factors cytochrome c. As an additional index of mPTP opening, the release of cytochrome c from mitochondria was analysed by measuring the cytosolic cytochrome c content. Resveratrol reduced I/R-induced cytochrome c release from mitochondria by inhibiting mPTP opening, which is consistent with the results of mitochondrial swelling (Fig. 8). However, the effect of resveratrol was abrogated by VDAC1 overexpression. 3.6 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on caspase-3 activity in mice subjected to I/R injury To investigate the contribution of the caspase-dependent pathway on apoptosis, caspase-3 activity in the cytosol was examined. Long-term oral resveratrol intake inhibited the I/R-induced increase of caspase-3 activity. However, with overexpression of VDAC1, resveratrol failed to prevent the increase of caspase-3 activity (Fig. 9). 3.7 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on apoptosis in mice subjected to I/R injury This article is protected by copyright. All rights reserved.
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To measure
apoptosis,
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we scored
the
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TUNEL-positive cardiomyocytes
in
the
immunohistological sections. As shown in Fig.10, in the hearts of sham-group, TUNEL-positive cells were seldom identified. In hearts subjected to I/R, numerous TUNEL-positive cells were observed, and further increased after VDAC1 overexpression. Mice with resveratrol intake showed a significant reduction in the number of TUNEL-positive cells compared with I/R group. However, the protective effect of resveratrol was abolished in mice injected with adenovirus vectors expressing VDAC1.
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4. Discussion Currently, lifestyle and nutritional condition is gaining more attention to prevent and treat heart diseases.
As such, “nutritional preconditioning” has been proposed by Abdukeyum
et.al. For such preconditioning, the nutrients must exist naturally, can be obtained through daily diet and exhibit cardioprotective effects [6]. Thus, “nutritional preconditioning” could represent a low-risk solution for ischemic cardioprotection, which can limit infarct size without prediction of ischemic events [17]. Resveratrol, a polyphenolic compound in red wine, has been well-documented to provide cardiovascular protective effect. Previous epidemiological studies suggested that drinking red wine in moderation (100- 400ml per day) may provide cardioprotective effects [18-21]. The general levels of resveratrol found in red wine usually vary from 0.287 to 7.188 mg/L, depending on the grape variety and winemaking techniques [22].
Wines produced from
muscadine grapes may contain more than 40 mg/L of resveratrol [23]. We converted the daily diet intake dosage of resveratrol in human to that in mice applied in our study, according to the conversion coefficient of body surface area. It ensured the dosages of resveratrol can be obtained through daily diet, rather than acute therapeutic administration. The 6-week feeding represents a long-term habitual dietary intake and ensures the concentration of resveratrol to reach an effective steady state. In current study, mouse hearts were isolated and subjected to I/R injury in Langendorff apparatus after 6-week resveratrol intake. We found that, compared with the group without resveratrol intake, resveratrol improved LVP, ±dp/dtmax and CF, decreased LDH and CPK levels, and reduced the infarct size caused by I/R. Thus, resveratrol as a nutritional preconditioning agent can lender cardioprotection to the heart against I/R insult. The mechanisms underlying resveratrol-induced cardioprotection have been extensively studied [24]. Several signal pathways are involved including sirtuins [25], GSK-3β [7], and This article is protected by copyright. All rights reserved.
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ATP-sensitive potassium channels [26]. However, the end-effectors of resveratrol preconditioning remain unclear. Recent studies have suggested that mPTP is a convergence point of numerous pathways, including sirtuins [27], GSK-3β [28], and ATP-sensitive potassium channels [29], in ischemic preconditioning. Pathological opening of mPTP is well known to cause mitochondrial permeability transition, and consequently plays a critical role in the progression of both apoptotic and necrotic cell death. mPTP opening is associated with mitochondrial swelling. Because the surface area of the inner membrane exceeds that of the outer membrane, extensive matrix swelling with long-lasting mPTP opening can lead to the unfolding of cristae, outer membrane to rupture and irreversibly mitochondria damage [30]. In addition, outer membrane rupture releases proapoptotic molecules residing in the intermembrane space, such as cytochrome c [31]. Once released, cytochrome c activates caspase 9, which in turn activates caspase 3. This protease mediates the proteolytic cleavage of a range of proteins responsible for the rearrangement of the cytoskeleton, plasma membrane, and nucleus that are characteristic of apoptosis. Thus, inhibition of mPTP opening appears to be an effective means for cardioprotection. We found that long-term oral resveratrol intake significantly blocked mPTP opening and attenuated mitochondrial swelling, subsequently prevented cytochrome c release, caspase-3 activation and TUNEL staining effectively. Strikingly, our results revealed that resveratrol regulates mPTP opening through VDAC1. VDAC1 was remarkably up-regulated in I/R injury. Pretreatment with resveratrol offsets a rising of VDAC1 during I/R. Overexpression of VDAC1 with pAd-VDAC1 was found to exacerbate I/R damages to the heart. When VDAC1 was overexpressed, the protective effect of resveratrol was compromised, suggesting that VDAC1 is the ultimate target of resveratrol, which might be regulated at the transcriptional level. As exogenous VDAC1 in adenovirus is under the control of the CMV promoter, resveratrol can not inhibits its expression. It is This article is protected by copyright. All rights reserved.
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possible that the overexpressed level of VDAC1 surpassed the inhibitory capacity of resveratrol. In any event, our results suggest an important role of VDAC1 in I/R injury.
The
mechanism underlying apoptosis induced by overexpression of VDAC1 in I/R can be explained as follows: (1) upregulation of VDAC1 might increase outer mitochondrial membrane permeability and thereby decrease cell viability [32]; (2) VDAC1 oligomerization was found to be strongly correlated with apoptosis induction [33, 34].
Thus, overexpression
of VDAC1 results in shifting of the VDAC1 equilibrium from monomeric to oligomeric species. This VDAC1 oligomeric state allows forming a large pore causing release of pro-apoptotic proteins, such as cytochrome c [33,34]； (3) increased VDAC1 interacts with the inner membrane protein adenine nucleotide translocator at contact sites, thus forming and activating the mPTP complex [35]; (4) VDAC1 has been known as one of the mediators of oxidative stress-induced apoptosis [36, 37]. Thus, it is possible that overexpression of VDAC1 enhances ROS production and subsequently triggers apoptosis. In conclusion, we demonstrate that long-term administration of resveratrol exerts a protective effect on cardiomyocytes against I/R insult. Our data support the use of resveratrol as a nutritional preconditioning agent in preventing cardiac damage caused by I/R, which advocates its use in food/diet supplement. According to our knowledge, this is the first set of data showing that VDAC1 plays an important role in cardiac damage imposed by I/R and VDAC1 is a target of resveratrol.
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This study was supported by the Research Program of the Sate Key Laboratory of Food Science and Technology, Nanchang University (Project No. SKLF-TS-201106), and the National Natural Scientific Foundation of China (No. 30960449, 81460495).
All the authors have declared no conflicts of interest.
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[28] Onishi, A., Miyamae, M., Inoue, H., Kaneda, K., et al., Sevoflurane confers additive cardioprotection to ethanol preconditioning associated with enhanced phosphorylation of glycogen synthase kinase-3β and inhibition of mitochondrial permeability transition pore opening. J Cardiothorac Vasc Anesth. 2013, 27, 916-924. [29] Marina Prendes, M.G., Hermann, R., Torresin, M.E., Vélez, D., et al., Role of mitochondrial permeability transition pore and mitochondrial ATP-sensitive potassium channels in the protective effects of ischemic preconditioning in isolated hearts from fed and fasted rats. J Physiol Biochem. 2014, 70, 791-800. [30] Penna, C., Perrelli, M.G., Pagliaro, P., Mitochondrial pathways, permeability transition pore, and redox signaling in cardioprotection: therapeutic implications. Antioxid Redox Signal. 2013, 18, 556-599. [31] Griffiths, E.J., Mitochondria and heart disease. Adv Exp Med Biol. 2012, 942, 249-267. [32] Zaid, H., Abu-Hamad, S., Israelson, A., Nathan, I., Shoshan-Barmatz, V., The voltage-dependent anion channel-1 modulates apoptotic cell death. Cell Death Differ. 2005, 12, 751–760. [33] Zalk, R., Israelson, A., Garty, E., Azoulay-Zohar, H., Shoshan-Barmatz, V., Oligomeric states of the voltage-dependent anion channel and cytochrome c release from mitochondria. Biochem. J. 2005, 386, 73-83. [34] Keinan, N., Toymkin, D., Shoshan-Barmatz, V., Oligomerization of the mitochondrial protein VDAC is coupled to the induction of apoptosis. Mol Cell Biol. 2010, 30, 5698-5709. [35] De Pinto, V., Tomasello, F., Messina, A., Guarino, F. et al., Determination of the conformation of the human VDAC1 N-terminal peptide, a protein moiety essential for the functional properties of the pore. Chembiochem 2007, 8, 744-756. [36] Madesh, M., Hajnoczky, G., VDAC-dependent permeabilization of the outer This article is protected by copyright. All rights reserved.
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mitochondrial membrane by superoxide induces rapid and massive cytochrome c release. J. Cell. Biol. 2001,155, 1003-1015. [37] Simamura, E., Hirai, K., Shimada, H., Koyama, J. et al., Furanonaphthoquinones cause apoptosis of cancer cells by inducing the production of reactive oxygen species by the mitochondrial voltage-dependent anion channel. Cancer Biol. Ther. 2006, 5, 1523–1529.
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Fig. 1 Effect of long-term oral intake of resveratrol on LDH and CPK activity in isolated mouse hearts subjected to I/R. LDH and CPK activity in CF effluent was measured by a Beckman automatic biochemical analyser after 30 min of reperfusion. Long-term oral resveratrol intake suppressed the increase in LDH and CPK activity in a dose-dependent manner up to 2.0 mg/kg.d. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p> 0.05, vs. control group; b: p 0.05, vs. I/R group; d: p< 0.05, vs. I/R group.
80
LDH Activity(U/L)
b
c d
60
d 40
a
a
20
0 Cont
Vehicle
-
+
+
+
+
I/R
2.0
-
0.5
1.0
2.0
Res(mg/kg.d)
120
b
CPK Activity(U/L)
c d
90
d a
60
a
30
0 Cont
Vehicle
-
+
+
+
+
I/R
2.0
-
0.5
1.0
2.0
Res(mg/kg.d)
Fig 1
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Fig. 2 Effect of long-term oral intake of resveratrol on infarct size in isolated mouse hearts subjected to I/R. The myocardial infarct size was evaluated by TTC staining and calculated as a percentage of the risk area. Long-term administration of resveratrol reduced the infarct size in a dose-dependent manner. The most remarkable effect of resveratrol was detected at a dose of 2.0 mg/kg.day. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p> 0.05, vs. control group; b: p 0.05, vs. I/R group; d: p< 0.05, vs. I/R group.
40
b
c d
Infarct Size(%)
30
d
20
a
10
a
0 Cont
Vehicle
-
+
+
+
+
I/R
2.0
-
0.5
1.0
2.0
Res(mg/kg.d)
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Fig. 3 VDAC1 expression was detected by western blotting. (A) The effect of long-term oral resveratrol (2.0 mg/kg.d) intake on VDAC1 expression. Control mice hearts (Cont) were constantly subjected to the perfusion solution. Mice hearts in the I/R group were subjected to 15 min of global ischemia and 30 min of reperfusion. The mice in resveratrol group (Res) were orally administrated by gastric gavages with resveratrol (2.0 mg/kg.day) for 6 weeks before I/R treatment. (B) VDAC1 expression following injection of pAd-VDAC1 in the left ventricle free wall. Sham-operated mice underwent a surgical procedure but without gene transfer. pAd-VDAC1 mice were injected with adenovirus containing VDAC1 cDNA. pAd-Shuttle mice were injected with empty adenovirus vector. β-actin was used as a loading control. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. Res+I/R group.
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Fig. 5 VDAC1 overexpression abolished the cardioprotection of long-term oral resveratrol intake on LDH and CPK activity in isolated mouse hearts subjected to I/R. Long-term oral resveratrol intake decreased LDH and CPK activity in mice subjected to I/R injury. However, VDAC1 overexpression diminished the effect of resveratrol. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. I/R group; e: p> 0.05, vs. Res+I/R group.
100
b a
LDH Activity(U/L)
80
c, d
60
e
b 40 20 0 Sham
+ -
+ -
+ +
+ +
+ +
I/R
-
+ -
-
+ -
+
pAd-VDAC1
Res pAd-Shuttle
150
b a
CPK Activity(U/L)
120
c, d
90
e
b
60 30 0 Sham
+ -
+ -
+ +
+ +
+ +
I/R
-
+ -
-
+ -
+
pAd-VDAC1
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Res pAd-Shuttle
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Fig. 6 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on infarct size in mice subjected to I/R injury. (A) Heart cross sections from each treatment after TTC staining. The red area represents the viable region, and the white area represents the infarct size. (B) Histogram of infarct size. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. I/R group; e: p> 0.05, vs. Res+I/R group.
A
Sham
B
I/R
50
Res +I/R
pAd-VDAC1 +Res+I/R
pAd-Shuttle +Res+I/R
b a
40
Infarct Size(%)
pAd-VDAC1 +I/R
c, d e
b
30 20 10 0 Sham
+ -
+ -
+ +
+ +
+ +
I/R
-
+ -
-
+ -
+
pAd-VDAC1
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Res pAd-Shuttle
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Fig. 7 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on mPTP opening in mice subjected to I/R injury. mPTP opening was determined by Ca2+-induced swelling of isolated mitochondria. Opening of the pore causes mitochondrial swelling, which was measured as a reduction in absorbance at 520 nm. (A) Original traces for mitochondrial swelling within 2 min. (B) The rate of mitochondrial swelling induced by 200 μmol/L CaCl2. Data are shown as a change in A520 and presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. I/R group; e: p> 0.05, vs. Res+I/R group.
A 2+
Ca 200μ mol/L
Sham
Res+I/R
A520 nm
pAd-Shuttle+Res+I/R
pAd-VDAC1+Res+I/R I/R pAd-VDAC1+I/R 2 min
B 0.6
b ΔAbsorbance(520nm)
0.5
a
c, d
0.4 0.3
e
b 0.2 0.1 0 Sham
+ -
+ -
+ +
+ +
+ +
I/R
-
+ -
-
+ -
+
pAd-VDAC1
Res pAd-Shuttle
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Fig. 8 Cytochrome c protein expression in cytoplasmic fraction and whole lysate was analyzed by Western blotting. mPTP opening causes mitochondrial swelling and rupture of the outer mitochondrial membrane, which results in the release of cytochrome c. As an additional index of mPTP opening, the release of cytochrome c from mitochondria was analysed. (A) Representative western blots. (B) Levels of cytochrome c expression of the cytoplasmic fraction versus whole cell were quantified. Data are expressed as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. I/R group; e: p> 0.05, vs. Res+I/R group.
A
Sham
I/R
pAd-VDAC1 +I/R
Res +I/R
pAd-VDAC1 pAd-Shuttle +Res+I/R +Res+I/R
Cytoplasmic fraction Whole cell lysate β -actin
B C-Cyt c/W-Cyt c
1.2 1
b
a
c, d
0.8 0.6 0.4
e
b
0.2 0 Sham
+ -
+ -
+ +
+ +
+ +
I/R
-
+ -
-
+ -
+
pAd-VDAC1
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Fig. 9 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on caspase-3 activity in mice subjected to I/R injury. Caspase-3 activity was measured in the cytosolic fraction isolated from heart homogenates. Due to injection of adenovirus vectors pAd-VDAC1, long-term oral resveratrol intake failed to prevent the increase in caspase-3 activity in mice subjected to I/R injury. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. I/R group; e: p> 0.05, vs. Res+I/R group.
Caspase-3 Activity(U/mg pro)
4
b
3.5
a
3
c, d
2.5
b
2
e
1.5 1 0.5 0
Sham
+ -
+ -
+ +
+ +
+ +
I/R
-
+ -
-
+ -
+
pAd-VDAC1
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Fig. 10 VDAC1 overexpression abolished the cardioprotective effect of long-term oral resveratrol intake on apoptosis in mice subjected to I/R injury. (A) Hearts were sectioned and analysed for apoptosis using the TUNEL staining method. The panels display representative histological images. (B) The number of apoptotic cells evaluated by TUNEL is expressed as a percentage. Data are presented as mean ± SEM (n=6, the experiment was replicated 3 times). a: p 0.05, vs. I/R group; e: p> 0.05, vs. Res+I/R group.
Sham
I/R
pAd-VDAC1+I/R
pAd-VDAC1+Res+I/R
Res+I/R
40
pAd-Shuttle+Res+I/R
b
Apoptosis Index(%)
35 30
a
25
c, d b
20
e
15 10 5 0 Sham
+ -
+ -
+ +
+ +
+ +
I/R
-
+ -
-
+ -
+
pAd-VDAC1
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Res pAd-Shuttle
