Cardioprotection With Alcohol

Ann. N.Y. Acad. Sci.

957: 122–135 (2002). ©2002 New York Academy of Sciences.

Cardioprotection with Alcohol

Role of Both Alcohol and Polyphenolic Antioxidants

MOTOAKI SATO, NILANJANA MAULIK, AND DIPAK K. DAS

Cardiovascular Research Center, University of Connecticut School of Medicine, Farmington, Connecticut 06030-1110, USA

A

BSTRACT

: Both epidemiological and experimental studies indicate that mild-to-moderate alcohol consumption is associated with a reduced incidence ofmortality and morbidity from coronary heart disease. The consumption ofwine, particularly red wine, imparts a greater benefit in the prevention of cor-onary heart disease than the consumption of other alcoholic beverages. Thecardioprotective effects of red wine have been attributed to several polyphe-nolic antioxidants including resveratrol and proanthocyanidins. The results ofour study documented that the polyphenolic antioxidants present in red wine,for example, resveratrol and proanthocyanidins, provide cardioprotection bytheir ability to function as

in vivo

antioxidants while its alcoholic component oralcohol by itself imparts cardioprotection by adapting the hearts to oxidativestress. Moderate alcohol consumption induced significant amount of oxidativestress to the hearts which was then translated into the induction of the expres-sion of several cardioprotective oxidative stress-inducible proteins includingheat shock protein (HSP) 70. Feeding the rats with red wine extract or itspolyphenolic antioxidants as well as alcohol resulted in the improvement ofpostischemic ventricular function. Additionally, both wine and alcohol trig-gered a signal transduction cascade by reducing proapoptotic transcriptionfactors and genes such as JNK-1 and c-Jun thereby potentiating an anti-deathsignal. This resulted in the reduction of myocardial infarct size and cardiomy-ocyte apoptosis. The results, thus, indicate that although both wine and alcoholalone reduce myocardial ischemic reperfusion injury, the mechanisms of car-dioprotection differ from each other.

K

EYWORDS

: alcohol; wine; polyphenols; flavonoids; antioxidants; free radicals;nitric oxide; heat shock proteins; adaptation; oxidative stress

INTRODUCTION

Recently, a number of studies have been devoted to understanding the cause ofthe so-called French Paradox, the anomaly which means that in several partsof France and other Mediterranean countries the morbidity and mortality fromcoronary heart diseases, both absolutely and relative to other causes of death is sig-nificantly lower than that is in other developed countries, despite of the high con-sumption of fat and saturated fatty acids.

1–10

This cardioprotective effect is believed

Address for correspondence: Dipak K. Das, Ph.D., Cardiovascular Research Center, Uni-versity of Connecticut, School of Medicine, Farmington, CT 06030-1110, USA. Voice: 860-679-3687; fax: 860-679-4606.

[email protected]

123SATO

et al.

: CARDIOPROTECTION WITH ALCOHOL

to be in part related to the regular consumption of wine. Wines, especially red wines,have about 1,800–3,000 mg/L of polyphenolic compounds.

11

Many polyphenoliccompounds are potent antioxidants capable of scavenging free radicals and inhibit-ing lipid peroxidation both

in vitro

and

in vivo

. It has been shown that flavonoids aswell as non-flavonoids present in wines inhibit the oxidation of low-density lipopro-teins, eicosanoid synthesis, and platelet aggregation as well as promote nitric oxidesynthesis.

4,12,13

Over the past several years, various epidemiological studies have strongly sug-gested that mild-to-moderate alcohol consumption is associated with a reduced inci-dence of mortality and morbidity from coronary heart disease.

14–19

Proposedmechanisms for such cardioprotective effects have included among others anincrease in high-density lipoprotein (HDL) cholesterol,

20

reduction/inhibition ofplatelet aggregation,

21

reduction in clotting factor concentrations,

4

reduction inthromboxane synthesis,

5

increase in vasodilatory prostacyclin synthesis,

6

inhibitionof low-density lipoprotein (LDL) oxidation,

3

and

free radical scavenging.

22

During the past 20 years studies in different ethnic groups starting from an Amer-ican cohort and including the recently performed analysis in the MONICA-projectprovided evidence for a decreased morbidity and mortality from coronary heart dis-ease at one to three drinks a day when compared to total abstainers.

23

Although it iscomprehensible that antioxidants like flavonoids and polyphenols found in winesmay explain the cardioprotective effects of wine consumption, the mechanism ofcardioprotection afforded by alcohol alone remains obscure. The present study com-pared the effects of wine (alcohol-free red wine extract) and alcohol on cardiopro-tection. The results demonstrated that both wine and alcohol can protect the heartagainst ischemia/reperfusion injury to similar extent, but by different mechanisms.

MATERIALS AND METHODS

Red Wine Extract

We vacuum evaporated 0.75L of red wine (under 30

o

C) to obtain 14.5g of jelly-like extract. One gram of this extract was then solubilized in deionized double-distilled water. The resulting fluid was diluted 1,000-fold with double-distilledwater. Vacuum evaporation was performed so as to facilitate the exclusive study ofthe biological effects of red wine constituents other than ethanol without alcoholinterference. The composition of red wine (Syrah region, North Italy) extract usedin our study: trans-resveratrol, 110ppm; quercitin, 2.9ppm; salycylic acid; non-detectable.

Trans-resveratrol and all other chemicals used were obtained from Sigma Chem-ical Company (St. Louis, MO), unless otherwise specified.

Animals

Male Sprague-Dawley rats (275–300g, Harlan Sprague-Dawley) were used inthis study. They were provided with food and water

ad libitum

up until the start ofthe experimental procedure. Rats were randomly assigned to one of several groups,A (control, water only), B (red wine extract), C (alcohol), D (proanthocyanidin) and

124 ANNALS NEW YORK ACADEMY OF SCIENCES

E (resveratrol). They were given orally (once a day) either water (control) or theabove-mentioned experimental compounds for three weeks. All animals receivedcare in compliance with the principles of laboratory animal care formulated by theNational Society for Medical Research and

Guide for the Care and Use of Labora-tory Animals

prepared by the National Academy of Sciences and published by theNational Institute of Health (publication no. NIH 80-23, revised 1978).

Isolated Rat Heart Model

The perfusion buffer used in this study consisted of a modified Krebs-Henseleitbicarbonate buffer (KHB) (in mM: 118 NaCl, 4.7 KCl, 1.2 MgSO

4

, 1.2 KH

2

PO

4

,25 NaHCO

3

, 10 glucose, and 1.7 CaCl

2

, gassed with 95% O

2

–5% CO

2

, pH 7.4)which was maintained at a constant temperature of 37

°

C and was gassed continuous-ly for the entire duration of the experiment. Rats were anesthetized with sodium pen-tobarbital (80 mg/kg b.w., i.p. injection, Abbott Laboratories), anticoagulated withheparin sodium (500IU/kg b.w., i.v. injection, Elkins-Sinn, Inc.). After ensuring suf-ficient depth of anesthesia, thoracotomy was performed, hearts were excised andimmersed in ice-cold perfusion buffer. Both the aorta and the pulmonary vein werecannulated as quickly as possible and the hearts were perfused in the retrograde Lan-gendorff mode at a constant perfusion pressure of 100cm H

2

O for a stabilizationperiod of 10 minutes. Then the circuit was switched to the antegrade working modeas described previously.

24

The hearts pumped against an afterload of 100cm H

2

Operfused with a constant preload of 17cm H

2

O. Aortic pressure was measured usinga Gould P23XL pressure transducer connected to a sidearm of the aortic cannula andthe signal was amplified using a Gould 6600 series signal conditioner and monitoredon a real-time data acquisition and analysis system (CORDAT II.). Heart rate, dev-eloped pressure (defined as the difference of the maximum systolic and diastolicaortic pressures) and the first derivative of developed pressure were all derived orcalculated from the continuously obtained pressure signal. Aortic flow was measuredusing a calibrated flowmeter (Gilmont Instruments Inc.) and coronary flow was mea-sured by timed collection of the coronary effluent dripping from the heart. At the endof 10 minutes, after the attainment of steady state cardiac function, baseline func-tional parameters were recorded and coronary effluent collected for biochemicalassays. The circuit was then switched back to the retrograde mode and the heartswere perfused with either KHB (Group A), red wine extract (Group B), or trans-resveratrol (Group C), the latter at a concentration of 10

µ

M, for a duration of15 minutes. At the end of this period, hearts were subjected to global ischemia for30 minutes followed by a two-hour reperfusion. The first 10 minutes of reperfusionwas in the retrograde mode to allow for post-ischemic stabilization and thereafter inthe antegrade working mode to allow for assessment of functional recovery, whichwas recorded at 15 minutes, 30 minutes, 60 minutes, and 120 minutes into reperfu-sion. Coronary effluent samples for biochemical assays were also collected at theabove timepoints and stored at

−

20

o

C.

Infarct Size Estimation

Hearts to be used for infarct size calculations (

n

=

6 per group) were taken upontermination of the experiment and immersed in 1% triphenyl tetrazolium solution

125SATO

et al.


in phosphate buffer (Na

2

HPO

4

88mM, NaH

2

PO

4

1.8mM) preheated to 37

°

C for20 minutes and then stored at

−

70

°

C for later processing. Frozen hearts (includingonly ventricular tissue) were sliced in a plane perpendicular to the apico-basal axisinto approximately 1 mm thick sections, blotted dry, placed in between microscopeslides and scanned on a single pass flat bed scanner (Hewlett-Packard Scanjet 5p).

25

Using the NIH Image 1.6.1 image processing software, each image was subjected tobackground subtraction, contrast enhancement and grayscale conversion forimproved clarity and distinctness. Total as well as infarct zones of each slice weretraced and the respective areas were calculated in terms of pixels and cm

2

,the respective volumes being obtained in cm

3

. The weight of each slice was thenrecorded to facilitate the expression of total and infarct masses of each slice ingrams. The total and infarct masses of each heart were then calculated by taking aweighted average. Infarct size was taken to be the percent infarct mass of total massfor any one heart.

Estimation of Malonaldehyde (MDA) Formation

Malonaldehyde was assayed as described previously, to monitor the developmentof oxidative stress.

26

In short, coronary effluents were collected in 2mL of a solutioncontaining 20% trichloroacetic acid, 5.3mM sodium bisulfite, kept on ice for 10 min,and centrifuged at 3,000

×

g

for 10 min; supernatants were then collected, derivatizedwith 2,4-dinitrophenylhydrazine (DNPH), and extracted with pentane. Aliquots of25

µ

l in acetonitrile were injected onto a Beckman Ultrasphere C

18

(3

µ

m) column.The products were eluted isocratically with a mobile phase containing aceto-nitrile:water:acetic acid (40:60:0.1, v/v/v) and measured at three different wave-lengths (307nm, 325nm and 356nm) using a Waters M-490 multichannel UVdetector. The peak for malonaldehyde was identified by co-chromatography withDNPH derivative of the authentic standard, peak addition, UV pattern of absorptionat the three wavelengths, and by GC-MS.

Evaluation of Apoptosis

To examine apoptosis,

cardiomyocytes were obtained by well-establishedmethods.

27

Following experiments, a group of hearts were quickly placed into achilled dissociation buffer containing (mM): NaCl, 137; KCl, 5.4; CaCl

2

, 1.8;MgCl

2

, 1.0; KH

2

PO

4

, 0.44; Na

2

HPO

4,

0.34; dextrose, 5.6; HEPES buffer (pH 7.5),20N; penicillin, 50 U/mL; and streptomycin, 50

µ

g/mL. The ventricles were cut into1–2mm cubes and dissociated by trypsinization (0.05% trypsin-EDTA at 37

°

Cfor 10 min). Unfreed cells from the first treatment were discarded, and the sequencewas repeated until all tissue was dissociated (approximately five times). Freed cellswere collected in cold Dulbecco’s modified Eagle’s medium (DMEM, GIBCO BRL,Gaithersburg, MD) supplemented with 0.5% fetal calf serum (FCS) and 0.002%DNAse, and washed in the same medium. Immunohistochemical detection of apo-ptotic cells was carried out using TUNEL

27

assay method in conjunction withan antibody against myosin heavy chain for specifically identifying apoptoticcardiomyocytes.


Western Blot Analysis

To quantify the abundance of p38MAPK, JNK1 and c-Jun protein as well heatshock proteins (HSPs), the heart tissues were homogenized and suspended(5 mg/mL) in sample buffer [10 mM HEPES, pH 7.3, 11.5% sucrose, 1mM EDTA,1mM EGTA, 1mM diisopropylfluorophosphate (DFP), 0.7mg/ml pepstatin A,10mg/mL leupeptin, 2 mg/mL aprotinin].

28

Proteins were then solubilized withthe addition of same amount of 2

×

Laemmli solution [9% (w/v) SDS; 6% (v/v)

β

-mercaptoethanol; 10% (v/v) glycerol; and a trace amount of bromophenol bluedye in 0.196M Tris/HCl (pH 6.7)]. The cellular proteins (50

µ

L samples) were elec-trophoresed through 10% SDS-PAGE and then transferred to Immobilon-P mem-branes (Millipore Corp.) using a semidry transfer system (Bio-Rad). Pre-stainedprotein standards (Bio-Rad) were run in each gel. The blots were blocked in Tris-buffered saline/Tween-20 (TBS-T containing 20mM Tris base, pH 7.6; 137mMNaCl; 0.1% Tween-20) supplemented with 5% BSA for 1 hour, incubated with1:1000 diluted primary rabbit antibodies specifically against either c-Jun or JNK 1(Santa Cruz Biotech) for two hours, and then with 1:10000 diluted secondary anti-bodies of horseradish peroxidase-conjugated anti-rabbit IgG (Boehringer MannheimCorp., Inc) for one hour at room temperature. After three washes of five minuteseach, blots were treated with Enhanced Chemi-Luminescence (ECL from Amer-sham) reagents and the JNK1 and c-Jun were detected by autoradiography for vari-able lengths of time (15sec to 3min) with Kodak X-Omat film. Each blot wasscanned with a scanning densitometer, and the results are shown as means

±

SEM ofsix different blots per group.

STATISTICAL ANALYSIS

For statistical analysis, a two-way analysis of variance (ANOVA) followed byScheffe’s test was first carried out to test for any differences between groups. If dif-ferences were established, the values were compared using Student’s

t

-test for paireddata. The values were expressed as mean

±

SEM. The results were considered sig-nificant for

p

<

0.05.

RESULTS

Postischemic Ventricular Function

The effects of alcohol and red wine extract on post-ischemic left ventricular func-tion are summarized in T

ABLE

1. All data are presented as the mean

±

the standarderror of the mean. As expected, except for heart rate and coronary flow, postischemicventricular function was depressed in all three groups. For example, after 60 min ofreperfusion AF, LVDP, and LVdp/dt were 12.7

±

0.5mL/min, 32

±

7mm Hg, and1454

±

78mm Hg/min, respectively, in the control group compared to those at thebaseline levels (47.2

±

2.7mL/min, 103

±

6mm Hg, and 3378

±

167mm Hg/sec,respectively). Although similar trends were noticed for other groups, these values

127SATO

et al.


were significantly higher for the alcohol group (28.3

±

0.7mL/min, 67

±

4mm Hg,and 2305

±

67mm Hg/min, respectively) and red wine group (30.7

±

0.9mL/min,62 ± 3mm Hg and 2008

±

55mm Hg/sec) compared to control group. This trendremained the same up to 120 min of reperfusion. Thus, both alcohol and red winedisplayed significant recovery of post-ischemic myocardial function as compared tocontrol group.

Infarct Size

In this particular study, hearts were arrested by global ischemia for 30 min; there-fore, the whole ventricle was considered as the area of risk. Control hearts subjectedto ischemia and reperfusion (I/R) had an infarct size of 34.9

±

3.3% (see F

IGURE

1)compared to almost zero when a heart was subjected to 2.5 hours perfusion withoutischemia/reperfusion (control) Percent infarct size was significantly reduced foralcohol (21.2

±

1.8%) and red wine (26

±

2.2%). The red wine polyphenolic compo-nents, proanthocyanidin (21.1

±

1.5%) and resveratrol (13.9

±

1.0%) also significant-ly reduced the myocardial infarct size.

T

ABLE

1. Effects of feeding alcohol and wine on postischemic left ventricular functiona

aRats in three groups were fed orally wince, alcohol, or water (control) for three weeks.Isolated rat hearts were subjected to 30 min ischemia followed by two hours of reperfusion atworking mode. Ventricular function was measured at baseline and at 30, 60, and 120 min of rep-erfusion. *p < 0.05 versus control.

Reperfusion at

Baseline 30 min 60 min 120 min

Heart rate(beats/min)

Control 310 ± 11 293 ± 10 293 ± 8 290 ± 9

Alcohol 312 ± 8 298 ± 8 296 ± 11 293 ± 8

Wine 309 ± 7 300 ± 10 298 ± 6 290 ± 5

Coronary flow(ml/min)

Control 26.7 ± 1.5 19.5 ± 0.7 16.9 ± 0.8 14.2 ± 1.0

Alcohol 27.0 ± 1.2 20.6 ± 1.0 18.8 ± 0.9 17.3 ± 1.1

Wine 26.5 ± 0.8 21.5 ± 1.2 17.4 ± 1.0 15.9 ± 0.8

Aortic flow(ml/min)

Control 47.2 ± 2.7 14.5 ± 0.3 12.7 ± 0.5 9.8 ± 0.2

Alcohol 50.4 ± 3.1 30.4 ± 1.1* 28.3 ± 0.7* 23.5 ± 1.0*

Wine 48.7 ± 2.8 32.2 ± 0.6* 30.7 ± 0.9* 25.1 ± 0.5*

LVDP(mm Hg)

Control 103 ± 6 35 ± 4* 32 ± 7* 25 ± 3*

Alcohol 110 ± 12 70 ± 9* 67 ± 4* 56 ± 7*

Wine 104 ± 8 65 ± 6* 62 ± 3* 51 ± 5*

LVmaxdp/dt

(mm Hg/sec)

Control 3,378 ± 167 1,634 ± 121 1,454 ± 78 1,234 ± 103

Alcohol 3,445 ± 123 2,411 ± 89* 2,305 ± 67* 2,223 ± 45*

Wine 3,389 ± 120 2,255 ± 105* 2,008 ± 55* 1,988 ± 45*


MDA Formation

The production of malonaldehyde (MDA) is an indicator for lipid peroxidationand development of oxidative stress. As shown in FIGURE 2, the MDA of the heartincreased significantly within four hours of alcohol feeding. The MDA contentincreased further after 12 hours, but came down significantly within 24 hours of alco-hol feeding. After 72 hours, the MDA content of the alcohol-fed heart remained at thebaseline level. Feeding of red wine also increased the MDA content slightly, whichreadily came down to the baseline levels. The results, thus, indicate that alcoholcauses the development of oxidative stress in the heart; however, such oxidative stresswas only transient.

Expression of Cardioprotective HSPs

Alcohol also caused the induction of the expression of HSP 70 as shown inFIGURE 3. Among the three HSPs examined, only HSP 70 protein was increasedcompared to control. There was no change in the induction of HSP 27 and HSP 90.Wine did not affect any of the HSPs.

FIGURE 1. Effects of wine, its polyphenolic components, proanthocyanidin and res-veratrol, and alcohol on myocardial infarct size. Rats were fed orally with red wine extract,proanthocyanidin, resveratrol or alcohol for three weeks. At the end of three weeks, ratswere anesthetized and isolated perfused working hearts were subjected to 30 min ofischemia followed by two hours of reperfusion. The infarct size was measured by TTCstaining as described in the METHODS section. Results are expressed as means ± SEM of atleast six hearts per group. Representative infarcts are shown (top): the light-colored areas(white to yellow stain) represent amount of infarct. The results expressed in a bar graph areshown below. * p < 0.05 versus I/R. † p < 0.05 versus control.

129SATO et al.: CARDIOPROTECTION WITH ALCOHOL

Cardiomyocyte Apoptosis

The number of apoptotic cells were significantly higher (19.5%) in theischemic/reperfused myocardium compared to the control hearts (see FIGURE 4).Wine and its polyphenolic components proanthocyanidin and resveratrol as well asalcohol all were able to reduce the number of apoptotic cardiomyocytes comparedto ischemic/reperfused myocardium.

JNK1 and c-Jun Expression

Thirty minutes of ischemia followed by two hours of reperfusion significantlyenhanced the amount of the protein levels of p38 MAPK, JNK1, and c-Jun (seeFIGURE 5). The ischemia/reperfusion–mediated enhancement of c-Jun and JNK-1protein levels, but not p38 MAPK, were significantly reduced by wine or alcoholtreatment.

DISCUSSION

In this report, we demonstrated that an alcohol-free red wine extract as wellas alcohol can protect the hearts from detrimental effects of ischemia reperfusioninjury, as evidenced by improved post-ischemic ventricular function and reducedmyocardial infarction. Red wine extract and its polyphenolic components, proantho-cyanidin and resveratrol reduced the amount of oxidative stress in the heart, as

FIGURE 2. Effects of wine, its polyphenolic components, proanthocyanidin and res-veratrol, and alcohol on the MDA content of the heart. Rats were fed orally with red wineextract, proanthocyanidin, resveratrol, or alcohol for three weeks. At the end of threeweeks, rats were anesthetized and isolated perfused working hearts were subjected to 30min ischemia followed by two hours of reperfusion. The MDA content was measured byHPLC as described in the METHODS section. Results, means ± SEM of at least six hearts pergroup, are expressed in a bar graph. * p < 0.05 versus baseline (0 hour) or wine.


indicated by decreased MDA formation. Alcohol, on the other hand, initiallyincreased the MDA in the heart. However, the MDA content decreased significantlyafter 24 hours and reduced to baseline level after 72 hours. Both wine and alcoholtriggered a signal transduction cascade resulting in the inhibition of proapoptoticfactors Jnk and c-Jun, leading to the reduction of cardiomyocyte apoptosis. Myocar-dial infarct size was also reduced in all groups of hearts. Alcohol, but not the redwine or the polyphenols, induced the expression of cardioprotective HSP 70 protein.

The results of this study suggest that the mechanism of cardioprotection affordedby wine and alcohol is quite different. While, polyphenolic antioxidants includingresveratrol and proanthocyanidin are responsible for cardioprotection found in wine,alcohol protects the heart by adapting this organ to oxidative stress. Alcoholconsumption appears to induce oxidative stress which is subsequently translatedinto oxidative stress-inducible proteins. Indeed, alcohol feeding resulted in the

FIGURE 3. Effects of wine, its polyphenolic components, proanthocyanidin and res-veratrol, and alcohol on the induction of the expression of HSP 27, HSP 70, and HSP 90protein content of the heart. Rats were fed orally with red wine extract, proanthocyanidin,resveratrol or alcohol for three weeks. At the end of three weeks, rats were anesthetizedand isolated perfused working hearts were subjected to 30 min ischemia followed by twohours of reperfusion. The HSPs was measured by Western blot analysis as described in theMETHODS section. Results are representative of at least six hearts per group.


induction of the expression of cardioprotective HSP 70 protein. A large number ofevidence exists in the literature indicating cardioprotective abilities of HSPs includ-ing HSP 70.29–32 Currie and colleagues were probably the first group to demonstrateenhanced postischemic ventricular recovery after subjecting the heart to heatstress.29 The results of many recent studies including our own now support the ear-lier observation by Currie and further indicate that the same heat shock protein, HSP70, can also be induced by other stresses such as oxidative stress as shown in thepresent study.33,34 The expression of HSP 70 was associated with improved post-ischemic ventricular function, and decreased infarct size and cardiomyocyteapoptosis.

The mechanism of cardioprotection by red wine is likely to be mediated byits polyphenolic components, resveratrol and proanthocyanidin. As shown in thisstudy wine as well as resveratrol and proanthocyanidin decreased myocardial infarctsize and reduced apoptotic cell death to the same extent. Antioxidants have longbeen known to protect against the damaging effects of free radical–mediated tissueinjury, especially ischemia reperfusion injury of the heart and other organs.35,36 Sub-stantial evidence exists to support the notion that ischemia and reperfusion generateoxygen free radicals which contribute to the pathogenesis of ischemic reperfusioninjury.37,38 The presence of reactive oxygen species were confirmed directly by esti-mating free radical formation and indirectly by assessing lipid peroxidation andDNA breakdown products.39 Among the oxygen free radicals, superoxide anion

FIGURE 4. Effects of wine, its polyphenolic components, proanthocyanidin and res-veratrol, and alcohol on cardiomyocyte apoptosis. Rats were fed orally with red wineextract, proanthocyanidin, resveratrol or alcohol up to a period of three weeks. At the endof three weeks, rats were anesthetized and isolated perfused working hearts were subjectedto 30 min ischemia followed by two hours of reperfusion. The apoptotic cardiomyocyteswere detected by Tunnel staining in conjunction with a specific antibody against α-myosinheavy chain to specifically stain the cardiomyocytes as described in the METHODS section.Representative apoptotic cells were detected by laser scanning microscopy ( top). Theresults (average of at least six/group) expressed in a bar graph are shown below. * p < 0.05versus I/R or control.


( ) is the most innocent free radical, while the hydroxyl radical (�OH) is the mostdetrimental to the cells. In spite of their relatively low oxidizing ability compared to�OH radicals, in biological systems, organic peroxyl radicals could be extremelydamaging to the tissues.40 Tissues like heart are protected from the detrimentalactions of peroxyl radicals by the presence of naturally occurring antioxidants suchas bilirubin and biliverdin as well as plasma antioxidants.41 Ascorbic acid andvitamin E comprise the other potent peroxyl radical traps for biological systems.42

Generally, lipid soluble antioxidants can scavenge chain-carrying lipid peroxylradicals thereby preventing propagation of lipid peroxidation after the initiation ofthe lipid peroxidation. Recent study from our laboratory demonstrated that not only

O2–

FIGURE 5. Effects of wine, its polyphenolic components, proanthocyanidin and res-veratrol, and alcohol on the induction of the expression of p38 MAPK, JNK-1 and c-Fosprotein content of the heart. Rats were fed orally with red wine extract, proanthocyanidin,resveratrol or alcohol for three weeks. At the end of three weeks, rats were anesthetized andisolated perfused working hearts were subjected to 30 min of ischemia followed by twohours of reperfusion. P38 MAPK, JNK-1 and c-Fos were measured by Western blot analysisas described in the METHODS section. Results are representative of at least six hearts pergroup.


are red wine extract and proanthocyanidin and resveratrol potent scavengers of per-oxyl radicals, but also they reduced the extent of lipid peroxidation in the ischemicreperfused myocardium.43–47 These findings seem to be important because theseperoxyl radicals are formed in vivo in membranes and lipoproteins as intermediateproducts of lipid peroxidation.

Wine as opposed to other sources of polyphenols and antioxidants is unique in anumber of ways. First of all, resveratrol and a few other polyphenols are virtuallyabsent from commonly consumed fruits and vegetables, and thus the consumptionof red wine would constitute their only source in the diet. The only other resveratrolsource in human consumption is peanuts. Secondly, the various procedures involvedin wine production further enrich its polyphenol content. Furthermore, the increasedalcohol content as a result of the fermentation process allows for enrichment of totalpolyphenol content and also for better solubilization of the polyphenols resulting ingreater bioavailability than in other foodstuffs.48 All in all, red wine might possiblybe the richest effective source of natural polyphenol antioxidants. Secondary to theantioxidant properties of wines are alcoholic vasodilation, decreases in plateletaggregability, changes in prostacyclin/thromboxane ratios and increased fibrinolyticactivities which should be considered as additional benefits caused by mild-to-moderate alcohol consumption.

In summary, the results of the present study indicate that the cardioprotectiveeffects of red wine and alcohol are mediated by two different mechanisms. It appearsthat wine exerts its cardioprotective abilities through its polyphenolic antioxidants,but alcohol protects the heart from cellular injury by adapting the organ to oxidativestress. Cardioprotective proteins such as HSP 70 produced during the myocardialadaptation to oxidative stress may at least be partially responsible for cardioprotec-tion. This is further supported by the ability of alcohol to trigger a signal transduc-tion cascade potentiating an antideath signal through the reduction of proapoptoticfactors JNK anf c-Jun thereby leading to the decrease in cardiomyocyte apoptosis.

ACKNOWLEDGMENTS

This study was supported by NIH HL 34360, HL 22559, HL 33889, HL 56803,and a grant from the California Table Grape Association.

REFERENCES

1. RIMM, E.B., E.L. GIOVANNUCCI, W.C. WILLETT, et al. 1991. Prospective study of alco-hol consumption and risk of coronary disease in men. Lancet 338: 464–486.

2. GAZIANO, J.M., J.E. BURING, J.L. BRESLOW, et al. 1993. Moderate alcohol intake,increased levels of high-density lipoprotein and its sub-fractions and decreased riskof myocardial infarction. N. Engl. J. Med. 329: 1829–1834.

3. FRANKEL, E.N., J. KANNER & J.B. GERMAN. 1993. Inhibition of human low-densitylipoprotein by phenolic substances in red wine. Lancet 341: 454–457.

4. RIDKER, P.M., D.E. VAUGHAN, M.J. STAMPFER, et al. 1994. Association of moderatealcohol consumption and plasma concentration of tissue-type plasminogen activator.JAMA 272: 929–933.


5. MIKHAILIDIS, D.P., J.Y. JEREMY, M.A. BARRADAS, et al. 1983. Effect of ethanol on vas-cular prostacyclin synthesis, platelet aggregation and platelet thromboxane release.Br. Med. J. 287: 1495–1498.

6. LANDOLFI, R. & M. STEINER. 1984. Ethanol raises prostacyclin in vivo and in vitro.Blood 64: 679-682.

7. SAIJA, A., M. SCALESE, M. LAIZA, et al. 1995. Flavonoids as antioxidant agents: impor-tance of their interaction with biomembranes. Free Radical Biol. Med. 19: 481–486.

8. FANCONNEAU, B., P. WAFFO-TEGNO, F. HUGNET, et al. 1997. Comparative study of rad-ical scavenger and antioxidant properties of phenolic compounds from vitis viniferacell cultures using in vitro tests. Life Sci. 61: 2103–2110.

9. RENAUD, S. & M. DE LORGERIL. 1992. Wine, alcohol, platelets and the French Paradoxfor coronary heart disease. Lancet 339: 1523–1526.

10. KLATSKY, A.L., M.A. ARMSTRONG & G.D. FRIEDMAN. 1986. Relations of alcoholicbeverage use to subsequent coronary artery disease hospitalization. Am. J. Cardiol.58: 710–714.

11. GOLDBERG, D., E. TSANG, A. KARUMANCHIRI, et al. 1996. Method to assay the concen-trations of phenolic constituents of biological interests in wines. Anal. Chem. 68:1688–1694.

12. GRYGLEWSKI, R.J., R. KORBUT, J. ROBAK & J. SWIES. 1987. On the mechanism of anti-thrombotic action of flavonoids. Biochem. Pharmacol. 36: 317–322.

13. RENAUD, S.C., A.D. BESWICK, A.M. FEHILY, et al. 1992. Alcohol and platelet aggrega-tion: the Caerphilly prospective heart disease study. Am. J. Clin. Nutr. 55: 1012–1017.

14. ST. LEGER, A.S., A.L. COCHRANE & F. MOORE. 1979. Factors associated with cardiacmortality in developed countries with particular reference to the consumption ofwine. Lancet 1: 1017–1020.

15. HERTOG, M.G.L., E.J.M. FESKENS & D. KROMHOUT. 1997. Antioxidant flavonols andcoronary heart disease risk. Lancet 349: 699.

16. KLATSKY, A.L., M.A. ARMSTRONG & G.D. FRIEDMAN. 1990. Risk of Cardiovascularmortality in alcohol drinkers, ex-drinkers and non-drinkers. Am. J. Cardiol. 66:1237–1242.

17. GOLDBERG, D.M., S.E. HAHN & J.G. PARKS. 1995. Beyond alcohol: beverage con-sumption and cardiovascular mortality. Clin. Chim. Acta 237: 155–187.

18. KANNEL, W.B. & R.C. ELLISON. 1996. Alcohol and coronary heart disease: the evi-dence for a protective effect. Clin. Chim. Acta 246: 59–76.

19. CRIQUI, M.H. 1996. Alcohol and coronary heart disease: consistent relationship andpublic health implications. Clin. Chim. Acta 246: 51–57.

20. FUHRMAN, B., A. LAVY & M. AVIRAM. 1995. Consumption of red wine with mealsreduces the susceptibility of human plasma and low-density lipoprotein to lipid per-oxidation. Am. J. Clin. Nutr. 61: 549–554.

21. GALLI, C., S. COLLI & G. GIANFRANCESHI. 1984. Acute effects of ethanol, caffeine, orboth on platelet aggregation, thromboxane formation and plasma free fatty acids innormal subjects. Drug-Nutr. Interact. 3: 6107.

22. SAIJA, A., D. MARZULLO, M. SCALESE, et al. 1995. Flavonoids as antioxidant agents:importance of their interaction with biomembranes. Free Radical Biol. Med. 19:481–486.

23. CRIQUI, M.H. & B.L. RINGEL. 1994. Does diet or alcohol explain the French Paradox?Lancet 344: 1719–1723.

24. ENGELMAN, D., M. WATANABE, R. ENGELMAN, et al. 1995. Hypoxic preconditioningpreserves antioxidant reserve in the working rat heart. Cardiovasc. Res. 29: 133–140.

25. YOSHIDA, T., N. MAULIK, R.M. ENGELMAN, et al. 1997. Glutathione peroxidase knock-out mice are susceptible to myocardial ischemia reperfusion injury. Circulation96(11): 216–220.

26. CORDIS, G.A., N. MAULIK & D.K. DAS. 1995. Detection of oxidative stress in heart byestimating the dinitrophenylhydrazine derivative of malonaldehyde. J. Mol. Cell.Cardiol. 27: 1645–1653.

27. MAULIK, N., H. SASAKI, S. ADDYA & D.K. DAS. 2000. Regulation of cardiomyocyteapoptosis by redox-sensitive transcription factors. FEBS Lett. 485: 7–12.


28. MAULIK, N., R.M. ENGELMAN, J.A. ROUSOU, et al. 1999. Ischemic preconditioningreduces apoptosis by upregulating anti-death gene Bcl-2. Circulation 100(II): 369–375.

29. CURRIE, R.W. 1987. Effects of ischemia and reperfusion on the synthesis of stress-induced (heat shock) proteins in isolated and perfused rat hearts. J. Mol. Cell. Car-diol. 19: 795–808.

30. DAS, D.K., R.M. ENGELMAN & Y. KIMURA. 1993. Molecular adaptation of cellulardefences following preconditioning of the heart by repeated ischemia. Cardiovasc.Res. 27: 578–584.

31. HUTTER, M.M., R.E. SIEVERS, V. BARBOSA & C.L. WOLFE. 1994. Heat shock proteininduction in rat hearts. A direct correlation between the amount of heat-shock pro-tein induced and the degree of myocardial protection. Circulation 89: 355–360.

32. KNOWLTON, A.A., P. BRECHER & C.S. APSTEIN. 1991. Rapid expression of heat shockprotein in the rabbit after brief cardiac ischemia. J. Clin. Invest. 87: 139–147.

33. LIU, X., R.M. ENGELMAN, I.I. MORARU, et al. 1992. Heat shock: a new approach formyocardial preservation in cardiac surgery. Circulation 86 (Suppl. 2): 358–363.

34. MAULIK, N., X. LIU, G.A. CORDIS, et al. 1994. The reduction of myocardial ischemiareperfusion injury by amphetamine is linked with its ability to induce heat shock.Mol. Cell. Biochem. 137: 17–24.

35. DAS, D.K. & N. MAULIK. 1994. Evaluation of antioxidant effectiveness in ischemiareperfusion tissue injury methods. Methods Enzymol. 233: 601–610.

36. DAS, D.K. & N. MAULIK. 1995. Protection against free radical injury in the heart andcardiac performance. In Exercise and Oxygen Toxicity. C.K. Sen, L. Packer, O. Han-ninen, Eds.: 359–388. Elsevier Science. Amsterdam.

37. ARROYO, C.M., J.H. KRAMER, B.F. DICKENS & W.B. WEGLICKI. 1987. Identification offree radicals in myocardial ischemia/reperfusion by spin trapping with nitroneDMPO. FEBS Lett. 221: 101–104.

38. TOSAKI A., D. BAGCHI, D. HELLEGOUARCH, et al. 1993. Comparisons of ESR andHPLC methods for the detection of hydroxyl radicals in ischemic/reperfused hearts.A relationship between the genesis of oxygen-free radicals and reperfusion-inducedarrhythmias. Biochem. Pharmacol. 45: 961–969.

39. CORDIS, G.A., G. MAULIK, D. BAGCHI, et al. 1998. Detection of oxidative DNA dam-age to ischemic reperfused rat hearts by 8-hydroxydeoxyguanosine formation. Mol.Cell. Cardiol. 30: 1939–1944.

40. CHANCE, B., H. SIES & A. BOVERIS. 1979. Hydroperoxide metabolism in mammalianorgans. Physiol. Res. 59: 527–540.

41. DAS, D.K., N. MAULIK & I.I. MORARU. 1995. Gene expression in acute myocardialstress. Induction by hypoxia, ischemia, reperfusion, hyperthermia and oxidativestress. J. Mol. Cell. Cardiol. 27: 181–193.

42. STOCKER, R. & E. PETERHANS. 1989. Synergistic interaction between vitamin E and thebile pigments bilirubin and biliverdin. Biochim. Biophys. Acta 1002: 238–243.

43. SATO, M., G. MAULIK, P.S. RAY, et al. 1999. Cardioprotective effects of grape seedproanthocyanidin against ischemic reperfusion injury. J. Mol. Cell. Cardiol. 31:1289–1297.

44. SATO, M., P.S. RAY, G. MAULIK, et al. 2000. Myocardial protection with red wineextract. J. Cardiovasc. Pharmacol. 35: 263–268.

45. SATO, M., D. BAGCHI, A. TOSAKI & D.K. DAS. 2001. Grape seed proanthocyanidinreduces cardiomyocyte apoptosis by inhibiting ischemia/reperfusion-induced activa-tion of JNK-1 and c-Jun. Free Radical Biol. Med. 16: 729–739.

46. RAY, P.S., G. MAULIK, G.A. CORDIS, et al. 1999. The red wine antioxidant resveratrolprotects isolated rat hearts from ischemia reperfusion injury. Free Radical Biol. Med.27: 160–169.

47. PATAKI, T., I. BAK, P. KOVACS, et al. Grape seed proanthocyanidins reducedischemia/reperfusion-induced injury in isolated rat hearts. Am. J. Clin. Nutr. Inpress.

48. VRHOVSEK, U., S. WENDELIN & R. EDER. 1997. Effects of various vinification tech-niques on the concentration of cis- and trans-resveratrol and resveratrol glucose iso-mers in wine. Am. J. Viticul. 48: 214–220.

Cardioprotection With Alcohol

Documents

Transcript of Cardioprotection With Alcohol