Avalable online at www.sciencedirect.com ）D科能eT年 Joumal of Molecular and Cellular Cardiology ELSEVIER Joeml of Mokcular and Celklr Cardiology 36(004)561-566 Original Article Cardioprotective effect of chronic low dose ethanol drinking: Insights into the concept of ethanol preconditioning Annabelle Guiraud,Michel de Lorgeril Frangois Boucher.Corinne Berthonneche. Andry Rakotovao.Joel de Leiris Laboair Nutrition Wetlene et Mlher Candmarviaims (NVMCY)Lmeriu fourph Faurier Ginceie Freer Booeivd 17 Nvemer 2003:vd invd fom S Febrary 200cetd Febrry 2004 Ahstract The reaon why low-to-moderale alcobol driaking is associated with reduced cardiowascalar mortality is sot elacidaled.While dat sgerted tha ethanol driiing my have prurectire fecon glol cunfiac inchemin.the effect chonie ow done cthunol drinking C2hDa用mocanlal ifant ner hr n时样n eaated in a model af mrgianl irhemia.Using in isolated rat heart m3ed the effect of various in io condounders we hine sudied the effeet of CLEhD on infnet sine (I5)and lef ventricular function after 30 min of regonal ischemiaand 120 min of reperfusion.The effoct of CLEthD was compared with ischemi preoonditoning(PC)and protein kinae C (PKC)soforms were analysed in the myocardium betore the 30-min ischemia.Ethanol-fed rats received 9%(w)ethanol in their drinking water for 7 weeks.Four gromps of rats were studiod:(1)conrolL (2)cthanol.(comol IPC.(4)ethanol IPC.Compared with controls 59±10队5(percent of risk 2one)was smaller in the cthan(39±6)and IP℃(3引±8)oups (both pc005)Combino0 fethanol and IPC in the same rats further decreased IS (-46%ys.ethanol.p<005)PKC analyses did aot show sustained c isoform translocarion in that model.These data indcale that chroric low dose cthanol drinking actually induces in the rat heart a cheonie prolective stle that is independert from anefecton the Irditical (lipid and cgultion)risk factors.Fther shudiesa required toclucide the mechismf protecton 2004 Ebevier Lad.All nights reserved. Krywondr Ethnde Akuhel:Coronary heart dicae:Myocardial isfarction.Lek ventriculr functia:Ischemic prccundioning:Prolcin kinae C 1.Introduction dium.In fact,chronie ethanol consumption mary mimic the classical ischemic preconditioning (IPC)and proect against A number of buman studies have reported decreased car. ischemia and reperfusion injury [16]Imestigaors however. diowascular disease among moderate acohol drinkersas compared with abstainers.[1-8]Even very low consumpion failed [17.]to reproduce the ethanol cardioprocection re of alcohol (one drink per week in certain studies)appears to ported by others in various experimental settings [19-271. These conflicting data might be related to differences in be protective.which is quite difficult to explain through the animal species,experimental model (glohal versus regional "alcohol-lipid-haemostasis"theory.Indeed,the protective effect of aleohol has beem principally explained by an effect ischemia.acute versus chronic ethanol exposure)and ethanol doage.Also,in mo studies reporting proeection,only car- on blood lipids 9]and plaelets [10,111.with increases in high density lipoproleins (HDL)and decreased platelet ag- diac function recovery or cardiac enzyme release was studied 19-25]and a reduction in the size of the infarcted tissue was gregation,resulting in a reduced rate of coronary artery ohstruction.Other mechanisms are probably involved.For not measured raising doubt on the reality of a direct cffect of instance.recent clinical studies have shown that moderate ethanol drinking (especially at low dosage)on the ischemc drinking may improve early outcome after acune myocardial myocardium (17.18]. infarction [12-14]and peevent sudden cardiae death 15]. Tnep国mary aim of触s study was therefore to investigate suggesting a direct effect of cthanol on the ischemic myocar the effect of chronic low dose echanol drinking(CLEthD)on both infaret sice and left ventricular function recovery in a ral ·Cemespou4 agathor:De.M出Larpel NVMCV.UFR de Madecine model of regional ischemia and reperfunsion.The rat species et Parmacie.Demraine de l Mercl.38706 La Tronche (Cmnohel Prance was selected because it is omnnorous and known to metabo E.wlad:michel dclorgerl ajf-prencbde(M.de Loe) lise ethanol in a way similar to that of bumans 21.We used GOa包evier LA.All righes reserved 4001同30020国3
Original Article Cardioprotective effect of chronic low dose ethanol drinking: Insights into the concept of ethanol preconditioning Annabelle Guiraud, Michel de Lorgeril *, François Boucher, Corinne Berthonneche, Andry Rakotovao, Joël de Leiris Laboratoire Nutrition, Vieillissement et Maladies Cardiovasculaires (NVMCV), Université Joseph Fourier, Grenoble, France Received 17 November 2003; received in revised form 5 February 2004; accepted 9 February 2004 Abstract The reason why low-to-moderate alcohol drinking is associated with reduced cardiovascular mortality is not elucidated. While data suggested that ethanol drinking may have a protective effect on global cardiac ischemia, the effect of chronic low dose ethanol drinking (CLEthD) on myocardial infarct size has not been evaluated in a model of regional ischemia. Using an isolated rat heart model to exclude the effect of various in vivo confounders, we have studied the effect of CLEthD on infarct size (IS) and left ventricular function after 30 min of regional ischemia and 120 min of reperfusion. The effect of CLEthD was compared with ischemic preconditioning (IPC) and protein kinase C (PKC) isoforms were analysed in the myocardium before the 30-min ischemia. Ethanol-fed rats received 9% (v/v) ethanol in their drinking water for 7 weeks. Four groups of rats were studied: (1) control, (2) ethanol, (3) control + IPC, (4) ethanol + IPC. Compared with controls (59 ± 10), IS (as percent of risk zone) was smaller in the ethanol (39 ± 6) and IPC (31 ± 8) groups (both p < 0.05). Combination of ethanol and IPC in the same rats further decreased IS (–46% vs. ethanol, p < 0.05). PKC analyses did not show sustained e isoform translocation in that model. These data indicate that chronic low dose ethanol drinking actually induces in the rat heart a chronic protective state that is independent from an effect on the traditional (lipid and coagulation) risk factors. Further studies are required to elucidate the mechanisms of that protection. © 2004 Elsevier Ltd. All rights reserved. Keywords: Ethanol; Alcohol; Coronary heart disease; Myocardial infarction; Left ventricular function; Ischemic preconditioning; Protein kinase C 1. Introduction A number of human studies have reported decreased cardiovascular disease among moderate alcohol drinkers as compared with abstainers.[1-8] Even very low consumption of alcohol (one drink per week in certain studies) appears to be protective, which is quite difficult to explain through the “alcohol-lipid-haemostasis” theory. Indeed, the protective effect of alcohol has been principally explained by an effect on blood lipids [9] and platelets [10,11], with increases in high density lipoproteins (HDL) and decreased platelet aggregation, resulting in a reduced rate of coronary artery obstruction. Other mechanisms are probably involved. For instance, recent clinical studies have shown that moderate drinking may improve early outcome after acute myocardial infarction [12–14] and prevent sudden cardiac death [15], suggesting a direct effect of ethanol on the ischemic myocardium. In fact, chronic ethanol consumption may mimic the classical ischemic preconditioning (IPC) and protect against ischemia and reperfusion injury [16]. Investigators, however, failed [17,18] to reproduce the ethanol cardioprotection reported by others in various experimental settings [19–27]. These conflicting data might be related to differences in animal species, experimental model (global versus regional ischemia, acute versus chronic ethanol exposure) and ethanol dosage. Also, in most studies reporting protection, only cardiac function recovery or cardiac enzyme release was studied [19–25] and a reduction in the size of the infarcted tissue was not measured raising doubt on the reality of a direct effect of ethanol drinking (especially at low dosage) on the ischemic myocardium [17,18]. The primary aim of this study was therefore to investigate the effect of chronic low dose ethanol drinking (CLEthD) on both infarct size and left ventricular function recovery in a rat model of regional ischemia and reperfusion. The rat species was selected because it is omnivorous and known to metabolise ethanol in a way similar to that of humans [28]. We used * Corresponding author: Dr. M de Lorgeril, NVMCV, UFR de Médecine et Pharmacie, Domaine de la Merci, 38706 La Tronche (Grenoble), France. E-mail address: michel.delorgeril@ujf-grenoble.fr (M. de Lorgeril). Journal of Molecular and Cellular Cardiology 36 (2004) 561–566 www.elsevier.com/locate/yjmcc © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2004.02.003
62 A.Gainsf er at.Jaumal eyf Maleewiwe and Celllar Conlidogy M (2000)561-566 an isolated heart model to study the specific effect of ethanol 2.I.Experimental prodocol on the myocardium itself,independent from the confounding effects of ethanol on other organs (liver)or blood compo- Two series of experiments were performed:one tor hemo- nemts (lipids.leakocytes.platelets).Our second aim was to dymamic and infarrdt sine measurements (n 50)and one to compare the effect of CLEthD and IPC and to examine analyse PKC (u 16).The hemodynamic-infarct study was whether their combinatioe would result in synergy or antago performed in the following groups:controls (n 8).cthanol nism.In the same line of reasoning.we hanve imestigaled the (n =17),IPC (n 8)and ethanol+IPC (n 17)The four changes oocurring in the sub-cellular locallisation of the pro roups were similar for all aspec电of the protocol except fe可 tein kinase C (PKC)ioforms because activation and trans- ethanol in the drinking waker.A 5-0 silk snare was passed locatiom of PKC are thought to be imolved both in IPC nder the left-coronary arery close to its origin.Afer 15-min [29-31]and in response to ethanol (21-25]although there is equilbeation period and normoie perfusion.the left- no data so far reganding CLErhD. coronary artery was occhaded by tightening the snare for 30 minutes and then reperfused for 120 mimtes.In the IPC group.price to the 30-mimne regional ichemia.the hearts 2.Methods were submitted tuice to 5-minute global ischemia and 10- minute reperfusion.All hearts were kept at 37C in a thermo- All procedures were in accordance with recommendations statically controlled glass chamber throughout the experi published in the Guide for the Care ond U're of Laboratory mental protocol.For each heart,myocardial function was Anials,National Academic Press,Washington,DC.1996. recocded after 15-min stabilization and then every 10 min- Adalt male Wistar rats (IFFA Credo.France)were divided es Regarding the PKC study.myocardial samples were into ethamol drinking group (=42)and age-mtched water obeained just before the 30-minute regional ischemia in each drinking control 24).Ethanol-fed rats received 9(wv) group.quickly frozen in liquid nitrogen and stored at-80C until use. ethanol in their drinking water for 7 weeks.Pilot studies in our laboratory have indeed suggested that 7 weeks of drink- ing and 9%ethanol are the manimal duration and dosage to 2.2.Risk zone ond infarct size see a proteetive effeet without inducing metabolie abno- malities.Because we suspected (after the pilot shadies)a After 120 minutes of reperfusion and retighening of the smaller effect of chromic low dose cthanol drinking (com- coronary snare,a solution of Evans Blue was injected through the aorta to delineate the non-stained risk zone.The pared with ethanol imfusion)on infaret size.the groups drink- ing ethanol were larger (n17)than those drinking water hearts were then brety frozen in lquid nitrogen and stored 8).All animals received standard solid food (UAR. at-20℃They were then cut im6n7 ravere slices o可 France)ad libitum.The average consumption of ethanol (and 1 mm thickness.Slices were incubuted in 1%triphemyhetra- other foods)was checked every 4 days and expressed as colium chloride in sodium phosphate buffer at 37C daring 20 minutes to stain viable cells in the risk zoee.Volume of peroent of total energy intake.Rats were weighed once a infarct and risk zoees年都calculated using image software week and boused under conditions of constant lemperabure. (NIH AutoEutractor 1.51).Risk zone was expressed as per humidity and standard light-dark cycle(12h/12h) cent of total ventricular volume and infaret size as pereent of Heart preparation and perfusion were carried out accord- risk zone. ing to methods described(32-34]withall animals being sacrificed between 8 and 10 am Briefly.rats were anaesthe. 2.3.PKC isoform assay ticed with pentobarbital sodium (Sanofi:40 mg/kg.i.p.).and heparinined (Sigma:100 Ul/rat,i.v.).Hearts were exeised. PKC activation is associated with translocatice from the washed in cold (+4C)Krebs-Henseleit butfer and canulated soluble to the particulate fraction [29-31).So.to test whether via the aorta.After removing sinus node.heart was poced at cardioprotectioe induced by CLEthD is mediated by PKC Ssu30 0bpm)via a monopo山r electrode p山ced on the left activatioe in our model,we deterined the sub-cellular dis atrial wall and connected to a stimulator(6021 SRI,UK)and tribution of u.6 and PKC in the soluble (S)and particulate perfused at a constant pressure of 9.81 kPa (Im H,0)wing (P)fractions of hearts from the 4 groups of rats.Subcellular the Langendorfr mode with Krehs-Henseleit crystalloid fractionation study was made at 4'C.Frozen bearts (about buffer (contaiming in mM:NaCl 118:KCl 4.75:NaHCO,25. 200 mg)were minced and homogenized in TEE buffer Mgs07H01.14KHP0,1.18Ca,2H0136md (50 mM Tris-HCI 5 mM EDTA.10 mM EGTA.0,3% glucose 11.1)and equilihrated with a mixtare of O CO. B-mercaploethanol.10 mM benzamidine and 50 up/ml 95泽/5%)端37℃pH7.4.LV pressure was measured with a PMSF.pH 7.5)with a homogeniser at its maximal speed transducer (Statham P231D.Goald,France)comnected to a three times.Homogenates were centrifaged at 100000 g for non-compliant water-fil led ultra thin balloon introduced into 60 min and the supernatant was frozen at-80C.The pellet the LV cavity with volume adjusted to preset a baseline was resuspended in TEET buffer (TEE buffer with 0.2% end-diastolic pressure of 4 mmHg Triton X-100).incubated for 60 min and ceatnfuged at
an isolated heart model to study the specific effect of ethanol on the myocardium itself, independent from the confounding effects of ethanol on other organs (liver) or blood components (lipids, leukocytes, platelets). Our second aim was to compare the effect of CLEthD and IPC and to examine whether their combination would result in synergy or antagonism. In the same line of reasoning, we have investigated the changes occurring in the sub-cellular localisation of the protein kinase C (PKC) isoforms because activation and translocation of PKC are thought to be involved both in IPC [29–31] and in response to ethanol [21–25] although there is no data so far regarding CLEthD. 2. Methods All procedures were in accordance with recommendations published in the Guide for the Care and Use of Laboratory Animals, National Academic Press, Washington, DC, 1996. Adult male Wistar rats (IFFA Credo, France) were divided into ethanol drinking group (n = 42) and age-matched water drinking controls (n = 24). Ethanol-fed rats received 9% (v/v) ethanol in their drinking water for 7 weeks. Pilot studies in our laboratory have indeed suggested that 7 weeks of drinking and 9% ethanol are the minimal duration and dosage to see a protective effect without inducing metabolic abnormalities. Because we suspected (after the pilot studies) a smaller effect of chronic low dose ethanol drinking (compared with ethanol infusion) on infarct size, the groups drinking ethanol were larger (n = 17) than those drinking water (n = 8). All animals received standard solid food (UAR, France) ad libitum. The average consumption of ethanol (and other foods) was checked every 4 days and expressed as percent of total energy intake. Rats were weighed once a week and housed under conditions of constant temperature, humidity and standard light-dark cycle (12h/12h). Heart preparation and perfusion were carried out according to methods described[32–34] with all animals being sacrificed between 8 and 10 am. Briefly, rats were anaesthetized with pentobarbital sodium (Sanofi; 40 mg/kg, i.p.), and heparinized (Sigma; 100 UI/rat, i.v.). Hearts were excised, washed in cold (+4°C) Krebs-Henseleit buffer and canulated via the aorta. After removing sinus node, heart was paced at 5 Hz (300 bpm) via a monopolar electrode placed on the left atrial wall and connected to a stimulator (6021 SRI, UK) and perfused at a constant pressure of 9.81 kPa (1m H20) using the Langendorff mode with Krebs-Henseleit crystalloid buffer (containing in mM: NaCl 118; KCl 4.75; NaHCO3 25; MgSO4.7 H20 1.19; K H2PO4 1.18; CaCL2.2H20 1.36 and glucose 11.1) and equilibrated with a mixture of O2/CO2 (95%/5%) at 37°C, pH 7.4. LV pressure was measured with a transducer (Statham P23ID, Gould, France) connected to a non-compliant water-filled ultra thin balloon introduced into the LV cavity with volume adjusted to preset a baseline end-diastolic pressure of 4 mmHg. 2.1. Experimental protocol Two series of experiments were performed: one for hemodynamic and infarct size measurements (n = 50), and one to analyse PKC (n = 16). The hemodynamic-infarct study was performed in the following groups: controls (n = 8), ethanol (n = 17), IPC (n = 8) and ethanol+IPC (n = 17). The four groups were similar for all aspects of the protocol except for ethanol in the drinking water. A 5-0 silk snare was passed under the left-coronary artery close to its origin.After 15-min equilibration period and normoxic perfusion, the leftcoronary artery was occluded by tightening the snare for 30 minutes and then reperfused for 120 minutes. In the IPC group, prior to the 30-minute regional ischemia, the hearts were submitted twice to 5-minute global ischemia and 10- minute reperfusion. All hearts were kept at 37°C in a thermostatically controlled glass chamber throughout the experimental protocol. For each heart, myocardial function was recorded after 15-min stabilization and then every 10 minutes. Regarding the PKC study, myocardial samples were obtained just before the 30-minute regional ischemia in each group, quickly frozen in liquid nitrogen and stored at –80°C until use. 2.2. Risk zone and infarct size After 120 minutes of reperfusion and retightening of the coronary snare, a solution of Evans Blue was injected through the aorta to delineate the non-stained risk zone. The hearts were then briefly frozen in liquid nitrogen and stored at –20°C. They were then cut into 6 to 7 transverse slices of 1 mm thickness. Slices were incubated in 1% triphenyltetrazolium chloride in sodium phosphate buffer at 37°C during 20 minutes to stain viable cells in the risk zone. Volume of infarct and risk zones was calculated using image software (NIH AutoExtractor 1.51). Risk zone was expressed as percent of total ventricular volume and infarct size as percent of risk zone. 2.3. PKC isoform assay PKC activation is associated with translocation from the soluble to the particulate fraction [29–31]. So, to test whether cardioprotection induced by CLEthD is mediated by PKC activation in our model, we determined the sub-cellular distribution of a, d and e PKC in the soluble (S) and particulate (P) fractions of hearts from the 4 groups of rats. Subcellular fractionation study was made at 4°C. Frozen hearts (about 200 mg) were minced and homogenized in TEE buffer (50 mM Tris-HCl, 5 mM EDTA, 10 mM EGTA, 0,3% b-mercaptoethanol, 10 mM benzamidine and 50 µg/ml PMSF, pH 7,5) with a homogeniser at its maximal speed three times. Homogenates were centrifuged at 100,000 g for 60 min and the supernatant was frozen at –80°C. The pellet was resuspended in TEET buffer (TEE buffer with 0.2% Triton X-100), incubated for 60 min and centrifuged at 562 A. Guiraud et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 561–566
A.Giraud er at.Jaumal of Malecuier and Celalar Candiology 36 (200)561-566 563 50 detected with a chimiluminesent delection kt (Super Signal Wesf5co.P巧icrce)..The amounts of PKC isoforms o通h 40间 immunoblots were quantitated using image software (NIH AutoExtractor 1.51) 2.4.Sranistics Data are expressed as mean SEM.End points included cardiac function parameters and infarct size in the 4 groups. Measuremenes were analysed by ANOVA with between- group differences tested by post-hoc application of Tukey's est.For all tests,p<005 was considered significant. 234 56 Weeks Fg 1.Efect of emhanel drinking on body weighe in chanol-fod and comtol 3.Results groups Open triangles.comol:clased squares,ethanol.fed ns. As shown in Figure 1.the increase in body weidhe was not 100,000 g for 60 min.The superatant was stored in ice and different in the echanol-fed and control groups:39983 g the pellet was resuspended in TEET buffer.incubaled for and 42140 g respectively after 7 weks.Also.there was no 60 min and centrifuged at 100.000g for 20 min.The TEE and difference between groups in blood lipids(toal choleserol: TEET supermatants were designated as solble (S)and par- Q64±009 pL in controls and051±00明in the cthanol ticulate (P)fractions.The total protein concentration in each oup：平triglycerides:147±0.35gLx163±0.97 and HDL fraction was deterined with a kit (Pieree,Rockford UK) cholesterol:01.32 0.05 p/L vs.0 29+0.07 in the ethanol and To quantitate PKC isoforms.the S and P fractions were control groups respectively)and blood ethanol levels aver boiled with 50%glycerol,5%SDS.250mM Tris base. aged 1.70.I mgll in ethanol-fed rats at the time of 50mM Tris HCI B-mercapooethanol and 5%hromophe- sacnfice. nol blae,pH 6.8.Each fraction (15 pg)was kaded on 105 LV function at baseline during nommoxie perfusion and SDS-polyacrylamide gel electrophoresis and transferred before ischemia was not different in the ethanol and control electrophoeetically to a nitrocellulose membrane.The mem- groups (Table 1).After 120 min of reperfusioe (Table 2) brane was incuhated with primary antibodics (Transduction there was no significant difference between groups in coro- Laboratories.Prance)for one night at 4C.After washing in nary flow,LVDevP and diastolic pressure. IBS-T buffer (20 mM Tris base,137mM NaCl and 0.05% The risk zone was not different in the four groups (Figure Tween 20,pH 7.4)with 1%non fat malk,the memhranes 2A).As shon in Figure 2B,infaret size (as percent of risk were incubated with peroxidase-linked secoedary antibodies zone)was significantly (p <0.05)smaller in the IPC (318) (Interchim.Prance)for one hour.After being washed in and ethanol (39 6)groups compared with the controls TBS-T (4 times for 7 minules each).PKC isoforms were (5910).The combination of cthanol and IPC further Table 1 eh vertricular function道ae家in the feer group of 3 perimental grasp四 Cartrdd ie Ethanod (n 17) IPC ia -8I EthanalelPC ie 17)ANOVA P LVDeP (mmHg 1302±60 12w0±20 1178±20 127%±12 034 Dianiolic prroure (mmHg) 444026 429主0.14 40主00 41%主00 049 4d山de (mmHgh 6375主683 5915主25 5444主33 5766主231 03 出mmle 45110 9916d 莲球经 0期64 067 Carosary New imlAl 165车06 1524m055 14.75a067 15aa044 064 LVDev:leh vertricubr developed presuure. Tahde 2 lemodysamics after10na0e中efa Experimental groaps Cemdrd (n w8i EaM▣小的 IPC (8 Enenel+lPC (n 17) ANOVA P LYDevP (mmHg 663主4.3 673t14 566主58 634主21 0I6 Daniolic prrour (mmHg) 线63主之第 94行7主2燃 5线00主73 2377支71 031 +dwa具e间 219±305 363生24w 213金246 530±167 037 -doldr immHg) 2519主164 2175±17m 300%±211 2306±142 043 Canr与ewe (mL/min 1000±0s7 962±057 84士02 92%±045 073
100,000 g for 60 min. The supernatant was stored in ice and the pellet was resuspended in TEET buffer, incubated for 60 min and centrifuged at 100,000 g for 20 min. The TEE and TEET supernatants were designated as soluble (S) and particulate (P) fractions. The total protein concentration in each fraction was determined with a kit (Pierce, Rockford, UK). To quantitate PKC isoforms, the S and P fractions were boiled with 50% glycerol, 5% SDS, 250mM Tris base, 50mM Tris.HCl, 5% b-mercaptoethanol and 5% bromophenol blue, pH 6,8. Each fraction (15 µg) was loaded on 10% SDS-polyacrylamide gel electrophoresis and transferred electrophoretically to a nitrocellulose membrane. The membrane was incubated with primary antibodies (Transduction Laboratories, France) for one night at 4°C. After washing in TBS-T buffer (20 mM Tris base, 137mM NaCl and 0,05% Tween 20, pH 7,4) with 1% non fat milk, the membranes were incubated with peroxidase-linked secondary antibodies (Interchim, France) for one hour. After being washed in TBS-T (4 times for 7 minutes each), PKC isoforms were detected with a chimiluminesent detection kit (Super Signal West Pico, Pierce). The amounts of PKC isoforms on the immunoblots were quantitated using image software (NIH AutoExtractor 1.51). 2.4. Statistics Data are expressed as mean ± SEM. End points included cardiac function parameters and infarct size in the 4 groups. Measurements were analysed by ANOVA with betweengroup differences tested by post-hoc application of Tukey’s test. For all tests, p < 0.05 was considered significant. 3. Results As shown in Figure 1, the increase in body weight was not different in the ethanol-fed and control groups: 399 ± 83 g and 421 ± 40 g respectively after 7 weks. Also, there was no difference between groups in blood lipids (total cholesterol: 0.64 ± 0.09 g/L in controls and 0.51 ± 0.09 in the ethanol group; triglycerides: 1.47 ± 0.35 g/L vs. 1.63 ± 0.97 and HDL cholesterol: 0.32 ± 0.05 g/L vs. 0.29 ± 0.07 in the ethanol and control groups respectively) and blood ethanol levels averaged 1.7 ± 0.1 mg/dL in ethanol-fed rats at the time of sacrifice. LV function at baseline during normoxic perfusion and before ischemia was not different in the ethanol and control groups (Table 1). After 120 min of reperfusion (Table 2), there was no significant difference between groups in coronary flow, LVDevP and diastolic pressure. The risk zone was not different in the four groups (Figure 2A). As shown in Figure 2B, infarct size (as percent of risk zone) was significantly (p < 0.05) smaller in the IPC (31 ± 8) and ethanol (39 ± 6) groups compared with the controls (59 ± 10). The combination of ethanol and IPC further Body Weight (g) 0 100 200 300 400 500 Weeks 0 7 1 2 4 6 3 5 Fig. 1. Effect of ethanol drinking on body weight in ethanol-fed and control groups. Open triangles, control; closed squares, ethanol-fed rats. Table 1 Left ventricular function at baseline in the four groups of rats Experimental groups Control (n = 8) Ethanol (n = 17) IPC (n = 8) Ethanol+IPC (n = 17) ANOVA P LVDevP (mmHg) 130.2 ± 6.0 128.0 ± 2.0 117.8 ± 2.0 127.8 ± 3.2 0.34 Diastolic pressure (mmHg) 4.44 ± 0.26 4.29 ± 0.14 4.00 ± 0.00 4.18 ± 0.10 0.49 +dp/dt (mmHg/s) 6375 ± 683 5915 ± 235 5444 ± 383 5766 ± 237 0.93 –dp/dt (mmHg/s) 4531 ± 310 3929 ± 168 3838 ± 92 3909 ± 164 0.67 Coronary Flow (mL/s) 16.85 ± 0.36 15.24 ± 0.55 14.75 ± 0.67 15.82 ± 0.44 0.64 LVDevP: left ventricular developed pressure. Table 2 Hemodynamics after 120 min of reperfusion in the four groups of rats Experimental groups Control (n = 8)) Ethanol (n = 17)) IPC (n = 8) Ethanol+IPC (n = 17) ANOVA P LVDevP (mmHg) 66.3 ± 4.3 67.3 ± 2.4 56.6 ± 5.8 63.4 ± 2.1 0.16 Diastolic pressure (mmHg) 33.63 ± 2.88 29.47 ± 2.08 33.00 ± 7.93 23.77 ± 3.71 0.31 +dp/dt (mmHg/s) 3219 ± 305 3463 ± 248 2813 ± 246 3300 ± 167 0.37 –dp/dt (mmHg) 2519 ± 164 2175 ± 178 2006 ± 211 2206 ± 142 0.43 Coronary flow (mL/min) 10.00 ± 0.57 9.62 ± 0.57 8.94 ± 0.82 9.26 ± 0.45 0.73 LVDevP: left ventricular developed pressure. A. Guiraud et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 561–566 563
564 A.Gainad er at Joamal of Moleevlwr and Cellalar Canliology 36 (2004)561-566 A 01 807 601 0 20 efunal drinking on isfart省k p<0s:转Pc00gct山dp←n的Ldod decreased infaret size (21 3.p <005 vs.ethanol)In fact. fect of ethaol were in fact conducted in models of global the effect of ethamol on infarct size was the same in the ischemia and infarct size was not measured.In studies evalu abence (-34%vs.controls)or presence (a further decrease ating the effect of ethanol on infaret size in models of re. of 32%)of IPC and the effect of IPC on infarct size was the gional ischemia.results were negative [17.18]o,when they same when rats were drinkers (a further decrease of 46)or were positie,imvestigators used acute (not chronic)ethanol not (-47%vs.controls). exposure of the myocardium 34]or high (not low)dose of To test whether chreeie candiopeotectiomn induced by ethanol [26271.Regarding ethanol dosage (a major point in CLEthD is associated with PKC activation.we determined terms of elinical implication),the dosage lested in the peesent the sub-cellalar distribution of a.and c PKC ia the soluble study corresponds to less than 15%of total daily energy (S):md particulane (P)fractions of hearts from the 4 groups of intake,when expressing ethanol drinking in terms of human rats.Tahle 3 shows each PKC isoform in cach fraction. matrition.Thus.it is the effect of low-o-moderale chromic Compared with coetrols,the drinking groupes (as well as the drinking that is stodied here whereas imvestigators who re. IPC group)did not show signifiean difference foe the s poeted a significant effeet on infaret size had used very high isoform although there was a small (borderline significant) dosage.higher than 0%of total energy intake [26.27].Such ditference in the ethanol+IPC group regarding the a isocorm. high dosages correspond to heay drinking in humans.a conditioe ofen associated with major clinical and biological side effects and a poor overall prognosis [35].This is remi. 4.Diseussion niscent of studies testing the effect of acue ethanol exposure on infarct size |18.34]where ethanol was iatravenously in- The present data show that chronic and low dose ethanol fused prior to ischemia and failed to affect infarct size.In drinking actuallyts in a significant reduction of infarct contrast,ethanol exposure of perfused hearts followed by size in the ex vivo heart rat model,a procective effect similar washout or suficien time to metabolise the aleobol price to to that ohserved with IPC. ischemia induced a significant reduction of infarct size[34. Thus,if present in sufficient coecentration theroughout is- 4.1 Erhanol avd Ieeforer size chemia ethanol was not procective aad even abolished the protection induced by IPC [34].This also probably esplains Published data about ethanol and the ischemic myocar the failure to show any peotective effect of ethanol using in dium are confusing.Most studies reporting a prodective ef- vivo model of myocardial ischemia where average blood Tahle 3 Disirhution of PKC aofoem in the four groups cl rats PKCe K降 PKCe Salahde Partiulate Seluhle Particulate Soluble Particslate Cantrdl 4纵1主87 503±发7 819士50 181主50 9明2士1海 6B生6 Ethanol 463生132 537±132 643±150 3线7±50 641±128 359±128 c 线97.7 561.71 6的4t14.1 06a141 75373 247473 LPC+Ethonel 132a109 568金109 场%主95 4线1主95 51.7主4.3 463玉43钟 ANONA p fer cn0 005 tread iPe且0 Swrun coetrl
decreased infarct size (21 ± 3, p < 0.05 vs. ethanol). In fact, the effect of ethanol on infarct size was the same in the absence (–34% vs. controls) or presence (a further decrease of 32%) of IPC and the effect of IPC on infarct size was the same when rats were drinkers (a further decrease of 46%) or not (–47% vs. controls). To test whether chronic cardioprotection induced by CLEthD is associated with PKC activation, we determined the sub-cellular distribution of a, d and e PKC in the soluble (S) and particulate (P) fractions of hearts from the 4 groups of rats. Table 3 shows each PKC isoform in each fraction. Compared with controls, the drinking groups (as well as the IPC group) did not show significant difference for the e isoform although there was a small (borderline significant) difference in the ethanol+IPC group regarding the a isoform. 4. Discussion The present data show that chronic and low dose ethanol drinking actually results in a significant reduction of infarct size in the ex vivo heart rat model, a protective effect similar to that observed with IPC. 4.1. Ethanol and Infarct size Published data about ethanol and the ischemic myocardium are confusing. Most studies reporting a protective effect of ethanol were in fact conducted in models of global ischemia and infarct size was not measured. In studies evaluating the effect of ethanol on infarct size in models of regional ischemia, results were negative [17,18] or, when they were positive, investigators used acute (not chronic) ethanol exposure of the myocardium [34] or high (not low) dose of ethanol [26,27]. Regarding ethanol dosage (a major point in terms of clinical implication), the dosage tested in the present study corresponds to less than 15% of total daily energy intake, when expressing ethanol drinking in terms of human nutrition. Thus, it is the effect of low-to-moderate chronic drinking that is studied here whereas investigators who reported a significant effect on infarct size had used very high dosage, higher than 30% of total energy intake [26,27]. Such high dosages correspond to heavy drinking in humans, a condition often associated with major clinical and biological side effects and a poor overall prognosis [35]. This is reminiscent of studies testing the effect of acute ethanol exposure on infarct size [18,34] where ethanol was intravenously infused prior to ischemia and failed to affect infarct size. In contrast, ethanol exposure of perfused hearts followed by washout or sufficient time to metabolise the alcohol prior to ischemia induced a significant reduction of infarct size [34]. Thus, if present in sufficient concentration throughout ischemia, ethanol was not protective and even abolished the protection induced by IPC [34]. This also probably explains the failure to show any protective effect of ethanol using in vivo model of myocardial ischemia where average blood Fig. 2. Comparison of risk zone in the 4 experimental groups, from left to right Control, Ethanol, IPC, and Ethanol + IPC, respectively (A). Effect of IPC and ethanol drinking on infarct size (B). *:p < 0.05; **: p < 0.001 vs. controls and †: p < 0.05 vs. ethanol. Table 3 Distribution of PKC isoforms in the four groups of rats PKCe PKCd PKCa Soluble Particulate Soluble Particulate Soluble Particulate Control 49.7 ± 8.7 50.3 ± 8.7 81.9 ± 5.0 18.1 ± 5.0 93.2 ± 1.6 6.8 ± 1.6 Ethanol 46.3 ± 13.2 53.7 ± 13.2 64.3 ± 15.0 35.7 ± 15.0 64.1 ± 12.8 35.9 ± 12.8 IPC 43.9 ± 7.7 56.1 ± 7.7 69.4 ± 14.1 30.6 ± 14.1 75.3 ± 7.3 24.7 ± 7.3 IPC+Ethanol 13.2 ± 10.9 86.8 ± 10.9 56.8 ± 9.5 43.2 ± 9.5 53.7 ± 4.3* 46.3 ± 4.3* ANOVA p for trend ns ns ns ns <0,05 <0,05 Each PKC fraction is expressed as percentage of total (particulate + soluble) of this isoform. * : p < 0.05 versus control. 564 A. Guiraud et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 561–566
A.Gainsf er at.Jaumal eyf Maleewiwe and Celllar Conlidogy M (2000)561-566 568 akohol level during ischemia was very high [17].Taken tion of the c isoform.In that view.the t isodorm may serve as together,these data (inclading ours)are in mutual agreemeat a trigger (sustained activation-translocation is not required to and underline the importance of the way of drinking alcobol indoce cardioprotection)rather than a mediator (sustained (regular versus hinging.moderale versus heavy)to induce activation is required)of the IPC.or ethanol-induced cardio. cardiac protection.The point should be constantly in mind protection.Alo,we have no explanatiomn for the change in when "celebeating"the health benetits of alcohol drinking. the a isoform obsenved in one group and not in the other other point is that,切h3fa东here wels no signif groups with reduction of infaret size.Funther studies are camt difference hetwee切ops in blood lipids.This isa圆 required to re-cxamine this (and other)possible mecha- indireet comfratlo that ethamel ltake was actsally low nism(s) and did mo resalr in obrious merabolle afreration in tese 4.3.Ethanol and leff venmcular fwecnion animals.Tis is in lime wish wos hwon date since significant chowges in blood市or的ermw5 mh的shudy,there wos no difference切LVDerP and asaily are ouly observed beyond a certain level of etheo diastolic presmre between te IPC and ethonol groups in cewSmprio. owe side and the conrrod growp in the other side.This may be quite surprising.However in mowry other snidies ffor 4.2.Mechanism nf cthanol preconditioning iustancee in /34/).limitation of infaret size (following either ethanolor ischemic preconditioning)was not associated with In our isolated heart model.we used a crystallid buffer. improvement in cardac function.The point has been comm. therefoce elimnating the problem o the presence of ethanol mented in some review articles such as.for instance.by during ischemia [341.Additionally,the cffect of ethanol in Kleer and Jennings [38)Actually.brief episodes of is. the present study was independent from an effeet om lipopr- chemia (preconditioning ischemia)can have both a negative teins.haemostasis and other circulating blood factors (that effect on the heart in terms of function (the stunning phenom are often peesented is the mediators of the protective effect enon)and a protective etfect against cell necrosis (the so- induced by moderte drinking)since they were not present in called ischemic preconditioning).Thus.in our study.despite the perfusion buffer Thus,CLEhD actually induced a direct a smaller infarct size (compared with the control group),the procective effect on the ischemic myocardium that resembles preconditoeed hearts (following brief ischemia or chroie [PC.However.IPC is a response to an acute stress whereas ethanol drinking)might have developed a certain degree of the protective effect of CLEthD is the coesequence of a myocardial stunning that could have masked the procective negular exposure.This raises the question of whether IPC and effect resulting from tissue salvage.As a result no difference cthanol preconditioning imolve the same biological mecha- in cardiac function was detected between the groups in our nism(s)or,in other words,the same signalling pathway. study Although the present sudy was not designed to fully investigate the mechanism of ethnol preconditioning.t is 4.4.Climical iplicatio and comcherion noteworthy that several investigators have proposed that both During the paot 25 years,mamy drugs have been claimed echanol and IPC activate PKC and that PKC activation is to protect the ischemic myocardium.but few of these results responsible for the protection through its effect on ATP. have been reproducible and none has been tramslated into dependent potassium channels [19-31].As a matter of fact. clinical therapies.Furthermore.any potentially effective the degree of protection (of redaction of infanet size)ob- drug needs to he given prioe to the acute coronary attack tained with kow dose cthanol drinking in our model is in the (prior to ischemia)in order to protect the myocardium.This same range as the protection observed with IPC.In addition. form of drug treatment is usually not poesable in most pa- both IPC and CLEthD were associated with similar sub tients with acute myocardial infarction.These difficulties cellular fractionation peofiles of PKC isoforms.For instance. have prompted many invesligalors to try developing non- in our model.both types of preconditioning were not associ- pharmacologic approoches aimed at maintaining the heart in aed with obvsos change of the c isoform although previous a chronically proeected state,such that ischemic damage investigators claimed that this specific isoform might ply a would be attenualed whenever ischemia occurs.To this cnd. primary role in boch IPC and ethanol preconditoning 21232529-311. some groups are using pene therapy to emulate the genetic changes that underlie the late phase of IPC.The present One porsaihility,as cardiae PKC activity has heen shown study.in association with a large body of evidence provided [36.37]to translocate very rapidly (as earty as I minute after by clinical and epidemiological studies [1-151.sugpests that the onset of ischemia),but transiently (with a rapid relocarion chronic low dose ethanol drinking may represent a simple ocytosol after every cycl of reperfusion).is th满a中ind way to induce a chronic cardoprotectrve slale. transkcation of the c isoform is not necessry to induce cardioprotection in that model [37].This suggests that the Acknowledgements wo short episodes of ischemia that were used in our IPC proocol (as well as the low ethanol dosape that s tested in We thank the Institut de Recherches Scientifiques sur ks this study)were no sufficient to induce sustained transloca. Boissons (IREB)for its help to complete that work
alcohol level during ischemia was very high [17]. Taken together, these data (including ours) are in mutual agreement and underline the importance of the way of drinking alcohol (regular versus binging, moderate versus heavy) to induce cardiac protection. The point should be constantly in mind when “celebrating” the health benefits of alcohol drinking. Another point is that, in this study, there was no signifi- cant difference between groups in blood lipids. This is an indirect confirmation that ethanol intake was actually low and did not result in obvious metabolic alteration in these animals. This is in line with most human data since significant changes in blood lipids (or liver enzymes) usually are only observed beyond a certain level of ethanol consumption. 4.2. Mechanism of ethanol preconditioning In our isolated heart model, we used a crystalloid buffer, therefore eliminating the problem of the presence of ethanol during ischemia [34]. Additionally, the effect of ethanol in the present study was independent from an effect on lipoproteins, haemostasis and other circulating blood factors (that are often presented as the mediators of the protective effect induced by moderate drinking) since they were not present in the perfusion buffer. Thus, CLEthD actually induced a direct protective effect on the ischemic myocardium that resembles IPC. However, IPC is a response to an acute stress whereas the protective effect of CLEthD is the consequence of a regular exposure. This raises the question of whether IPC and ethanol preconditioning involve the same biological mechanism(s) or, in other words, the same signalling pathway. Although the present study was not designed to fully investigate the mechanism of ethanol preconditioning, it is noteworthy that several investigators have proposed that both ethanol and IPC activate PKC and that PKC activation is responsible for the protection through its effect on ATPdependent potassium channels [19–31]. As a matter of fact, the degree of protection (of reduction of infarct size) obtained with low dose ethanol drinking in our model is in the same range as the protection observed with IPC. In addition, both IPC and CLEthD were associated with similar subcellular fractionation profiles of PKC isoforms. For instance, in our model, both types of preconditioning were not associated with obvious change of the e isoform although previous investigators claimed that this specific isoform might play a primary role in both IPC and ethanol preconditioning [21,23–25,29–31]. One possibility, as cardiac PKC activity has been shown [36,37] to translocate very rapidly (as early as 1 minute after the onset of ischemia), but transiently (with a rapid relocation to cytosol after every cycle of reperfusion), is that a sustained translocation of the e isoform is not necessary to induce cardioprotection in that model [36,37]. This suggests that the two short episodes of ischemia that were used in our IPC protocol (as well as the low ethanol dosage that was tested in this study) were not sufficient to induce sustained translocation of the e isoform. In that view, the e isoform may serve as a trigger (sustained activation-translocation is not required to induce cardioprotection) rather than a mediator (sustained activation is required) of the IPC- or ethanol-induced cardioprotection. Also, we have no explanation for the change in the a isoform observed in one group and not in the other groups with reduction of infarct size. Further studies are required to re-examine this (and other) possible mechanism(s). 4.3. Ethanol and left ventricular function In this study, there was no difference in LVDevP and diastolic pressure between the IPC and ethanol groups in one side and the control group in the other side. This may be quite surprising. However, in many other studies (for instance in [34]), limitation of infarct size (following either ethanol or ischemic preconditioning) was not associated with improvement in cardiac function. The point has been commented in some review articles such as, for instance, by Kloner and Jennings [38]. Actually, brief episodes of ischemia (preconditioning ischemia) can have both a negative effect on the heart in terms of function (the stunning phenomenon) and a protective effect against cell necrosis (the socalled ischemic preconditioning). Thus, in our study, despite a smaller infarct size (compared with the control group), the preconditioned hearts (following brief ischemia or chronic ethanol drinking) might have developed a certain degree of myocardial stunning that could have masked the protective effect resulting from tissue salvage. As a result, no difference in cardiac function was detected between the groups in our study. 4.4. Clinical implication and conclusion During the past 25 years, many drugs have been claimed to protect the ischemic myocardium, but few of these results have been reproducible and none has been translated into clinical therapies. Furthermore, any potentially effective drug needs to be given prior to the acute coronary attack (prior to ischemia) in order to protect the myocardium. This form of drug treatment is usually not possible in most patients with acute myocardial infarction. These difficulties have prompted many investigators to try developing nonpharmacologic approaches aimed at maintaining the heart in a chronically protected state, such that ischemic damage would be attenuated whenever ischemia occurs. To this end, some groups are using gene therapy to emulate the genetic changes that underlie the late phase of IPC. The present study, in association with a large body of evidence provided by clinical and epidemiological studies [1–15], suggests that chronic low dose ethanol drinking may represent a simple way to induce a chronic cardioprotective state. Acknowledgements We thank the Institut de Recherches Scientifiques sur les Boissons (IREB) for its help to complete that work. A. Guiraud et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 561–566 565
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