U D K 5 9 9 . 3 2 3 . 4 : 6 3 7 . S 0/ 8' 831 Originalresearchpaper EVALUATION OF THE POTENTIAL ANTTARRHYTHMIG EFFECT OF FISH MEAT DURING MYOGARDIAL ISGHEMIA.REPERFUSION !P ft1ATS NikolettMorvayl,JuditPeter-Sz[icsn62, LjiljanaM. Kostadinovic3, OliveraM. Duragic3, BojanaM. Kokict,lstvanCsengerio,lstvanLepranl. lDepartment Facultyof Medicine,University of Pharmacology, of Szeged,Hungary 2lnstitute Animal Facultyof Agriculture, of SciencesandWildlifeManagement, University of Szeged,Hungary 3University of Novi Sad, Instituteof FoodTechnology,NoviSad, Serbia oResearch (HAKI)Szarvas,Hungary Aquaculture Institute for Fisheries, and lrrigation r "Corresponding author: E-maiI address:lepran.istvan@med. u-szeged.hu ABSTRACT:The potentialantiarrhyihmiceffectof dietaryfish meat feedingwas investigatedin rats. Fish meat,harvestedfrom carpsfed experimental foods enrichedwith essentialfatty acids,was mixed rat food and fed to ratsfor 4 weeks.At the end of the feedingperiodin anesthetized with a comrnercial inducedarrhythmiaswere inducedby occlusionof the coronary rats myocardialischemia-reperfusion fatty arteryfor 6 min, followedby repertusion.Supplyingfish meat, rich in n-3 type polyunsaturated inducedarrhythmias acids,significantly decreasedthe incidenceof myocardialischemia-reperfusion that feeding in the rat, and increasedthe survivalrate. These resultsconfirmpreviousinvestigations fish oil or fish meat may offer cardioprotective effect,and consumptionof the farmed fish, like carp, may resultin similarprotectiveeffectsto the marinefood. Key words: rafs, myocardialischemia-reperfusion, fishmeat diet, cardioprotection INTRODUGTION diseasesare the leading Cardiovascular cause of death both in developed and d e v e l o p i n gc o u n t r i e s( W H O , 2 0 0 8 ) . l n most cases ischemicheart disease,acute myocardialinfarctionand associatedventricular arrhythmiasstand in the background. The restorationof blood flow is commonlyused therapyin case of acute myocardial infarction. Early repertusion after a short lastingmyocardialischemia, however,can result in arrhythmias,tissue injury and cell death, called reperfusion injuryffellon and Hausenloy,2007). emphasizethe imSeveralinvestigations portanceof n-3 polyunsaturated fatty acid (PUFA) consumptionand its cardioprotectiveeffects(Psotaet al., 2006), both in primaryand secondarypreventionof cardiovascularcomplications(Saravananet aI.,2010).Thesehumanstudiesaboutthe beneficial effects of PUFA feeding are supportedby in viva investigationsin rats (Lepr6rnet al., 1981; Mclennanet al., 1990;Leprdnand Szekeres,1992;Zhu et al., 1994;Fdldeset al,, 2006),marmosets (Charnock,1994) and dogs (Oskarsson, 1993).Long chainn-3 PUFA has also triglyceride lowering effect, and decrease the synthesisof inflammatoryeicosanoids and cytokines(reviewedby Calder,2AA4). The human body cannotsynthesizethese essentialfatty acids,thus we need to take with consumingfood rich in PUFA.Marine and dofoodstuff,rich in eicosapentaenoic source main acids, is the cosahexaenoic 71 NikolettMorvayef ai., Evaluationof the potentialantiarrhythmic effectof fish meat duringmyocardialischemia-reperfusion in rats,Foodand Feed Research40 (2),71-76,2013 of n-3 fatty acids for human beings. However there are many continentalcountries without the opportunityto consume fresh marine food, thereby people suggested to eat farmed fish more, e.g. the carp.The aim of the presentinvestigations was to study the effects of feeding carp meat, harvested from fishes after using different fish food composition,on myocardialischaemia-reperfusion inducedarrhythmiasin rats. EXPERIMENTAL Animals and diet The experimentswere performedon male Sprague-Dawley rats weighing between 250-300 g. The animalswere housed5 to a cage, and fed carp meat enrichedchow as follows.All carp-meat enrichedrat food (RF2,RF3, RF4, RFs) was preparedusing frozen fish filletsmixed with corn flake in a ratio of 3:1 of fish meat and corn flake. This fillet mixture was then grinded and pelleted after mixing with commercialrat chaw ingredients.Rat food RF1 was prepared by using commercialfish meal with added lard to equalize the energy contents.The final experimentalrat diets containedapproximately 10%of fish flesh. Fish flesh used for preparing rat food (RF2-RFS)was taken from carps, fed on "experimentalextruded expanded foods (carp food, CF2-CF5), enrichedwith different oils (linseedoil; fish oil and Mortierella alpineoil, containing40% arachidonicacid (ARA))or eicosapentaenoic (EPA)and docosahexaenoic(DHA) ethyl esters. The amountsof the addedoils were as follows: CF2:6.4% Linseedoil (LSO);CF3:3.5% i Ij LSO + 1.5%Arachidonic acid(ARA)+ 1% D H A ; C F 4 .6 , 0 % F i s ho i l ( F O ) ;C F 5 :4 . 5 % FO + 1.5%ARA. Detaileddata of fish food analysisand other observationsobtained duringthe fish feedingexperiments will be presentedelsewhere. The weeklyportionsof the rat foods were sealedundervacuumin plasticbags and stored in frozenform until use. The control rats (CF) were fed a standard rat chow (CRLT/Nrodentfood, Bonafarm,Babolna, Hungary;containing20.0%crudeprotein, 4.4% crude fat and 4.3% crude fiber), supplemented with 10 weight%of porkfat, rich in saturatedfatty acids. Rats were fed for 4 weeks with the experimentaldiets. Duringthistimethe animalsallowedto eat the experimentalfood and to drink tap water ad libitum.The rat experimentswere pedormed accordingto the protocolreviewed by the EthicalCommitteefor the Protectionof Animals in Researchof the Universityof Szeged,Hungary. Composition of the rat foods Proximatecompositionand metabolizable energy contents of the experimentalrat foods,RF1 to RFs, as determinedby standard laboratorymethods (AOAC, 2005), are presentedin Tabie 1. Biological assessment of modified fish meat in rats Blood samples were taken for the measurementof plasmatriglyceride(TG), total cholesterol(TC), high density lipoprotein(HDL) and low densitylipoprotein (LDL) cholesterolusing commerciallaboratory tests (Roche Diagnostics,Switzerland). T a b l e1 . Proximatecomposilion(gikg)and metabolizable energycontentof the rat foods Rat foods Connposition RF1 RF2 RF3 RF4 Dry rnatter 876 B6B 870 874 Crudeprotein 234 244 241 233 Crude fat 55 62 54 52 Crudefiber 31 34 36 35 Crudeash 73 68 65 65 N-free extract 483 460 474 489 Starch 264 291 286 310 ME (MJ/kg) 16.0 16.2 16.1 16.0 I ,l ;l 72 RF5 877 238 60 32 64 483 321 16.3 NikolettMoruayef a/., Evaluationof the potentialantiarrhythmiceffect of fish meat during myocardialischemia-reperfusion in rats, Food and Feed Research40 tZ\,71-76, 2O1Z Myocardial ischemia-reperfusion induced arrhythmias in anesthetizedrats arrhythmiascore was used, includingthe incidenceand durationof differentarrhythmias by giving a grade to each animal (Lepranand Szekeres,1992). Animals were anaesthetizedwith pentobarbitoneand the left carotid artery was cannulated for measuring the blood pressure.The tracheawas cannulatedfor artificial ventilation (Harvar:d Ventilator, Model 603) and the chest was opened in the fourth intercostalspace.The heartwas exposed and a loose loop of atraumatic silk was placed around the left main coronaryartery,approximately2 mm from its origin. Both ends of the ligaturewere led out of the thoracic cavity through flexible tubing(Lepranand Szekeres,1992). The standard electrocardiogram (lead ll, ECG) and the changesin blood pressure were recordedcontinuouslyand displayed after A/D conversion (Chart5, ADlnstruments, United Kingdom).After finishing the preparationthe animalswere allowed to stabilizef or 10 min, then the loose loop of the coronaryartery ligaturewas tightened and fixed by clampingon the silk and thus regional myocardial ischemia was produdedfor 6 min and then followedby reperfusion. Statistical analysis The survivalrate was coinparedby using the Xz-methodwith Yates-correction.All other parameters were expressed as mean + standarderror of the mean (SE) and after analysisof variancewere compared by means of the modified 't'-statisticalmethod. RESULTS AND DISCUSSION The consumptionof the amountof expe-' rimental rat food was not diffe;ent from that of the controlrat food. The final body weight of the animalsafter 4-week dietary period was not different among the different experimentalgroups. The plasma triglyceride,total cholesterol,high density lipoproteincholesteroland the low density lipoproteincholesterolconcentrations were also not significantlydifferentafterfeeding different experimentaldiets in rats. The baseline heart rate and blood pressure also did not differamongthe experimental groupsstudied(datanot shown). Coronaryarteryocclusionin rats pr:oduced a slowly developingregional myocardial ischemiathat inducedvariousarrhythmias within 4-5 min (Table 2). There were no significantdifferencesin the survivalrate, incidenceof arrhythmiasduring myocardial ischemiain differentdiet fed groups, as comparedto the control. The survival rate and the incidence of arrhythmiaswere analysedin accordance with the Lambeth Conventions(Walker, ,. 1.988),i.e. ventricularfibrillation(VF), ventriculartachycai'dia(VT) and other types of arrhythmias,includingsingleextrasystoles (VEB), bigeminia,and salvos.The duration of VF, VT and other types of arrhythmia attacks were also measured. An T able 2. Survival rate and the incidence of arrhythmias durinq 6 min myocardial ischemia in anesthetized rats Group cF R F1 R F2 RF3 RF*4_ " _ RFs Survived 16 15 15 '13 15 14 In c i d ence of i schemi a i nduced arrhythmi as None VF VT Other 15 19 94 3 1173533427853853 1280853427640426 15 12 92 2 1510053300746960 tbo 5 36 14 1 6 8 50 1275 1 B I 69 6 46 1 T T 50 9 64 Score 1.7510.36 2.1310.65 1.6710.64 1,62+0.42 1.20x0.26 1.0010.30 N = total numberof animalsin a given grq_up; n = numberof animalsshowingthe given response.None = no arrhythmiaoccurred;VF = ventricularfibrillation; VT = ventriculartachycardia;Other= othertypesof arrhythmias, includingventricularextrasystoles, bigeminia. 73 in effectof fish meat duringmyocardialischemia-reperfusion NikolettMorvayet al., Evaluationof the potentialantiarrhythmic rats,Foodand FeedResearch40 (2),71-76,2013 Table 3. Survivalrate and the incidenceof arrhythmiasduringreperfusionafter6 min myocardialischemiain rats anesthetized Incidenceof reperfusioninduced arrhythmias Score Group Survived None VF VT Other o/ to % CF RF1 RF2 RF3 RF4 RF5 15 5 33 11982*2 12975 127581 15 5 33 148570 0 1 1 0 18 B 8 7 0 o/ to 13 4 7 B 13 9 87 36* 58 67 87 64 % 15 7 B 11 13 14 100 64* 67* 92 87 100 % 11 6 10 7 B 12 73 55 83 58 53 86 5,40t0.27 3.09+0.62. 3.92t0.57* 4.17t0,61 5.1310.46 4.43l.0.47 For abbreviations see Table2. Asteriskdenotesstatistically significant differencecomparedto the controlrat food ( CF )f ed an i m a l s(P< 0 .0 5 ) Reperfusionafter 6 min myocardialischemia inducedrapidlydeveloping,severearrhythmias(Table3). RF1 and RF2 diets significantly decreasedthe incidenceof reperfusioninducedarrhythmiasafter myocardialischemia The other diets offered somewhat less protection.Althoughthe survivalrate after feeding RF4 diet was significantlylower and the incidenceof ventricularfibrillation was higher after.feeding RF4 diet as comparedto the otherRF diets,the overall survivalduring myocardialischemia and reperfusiondid not change significantly (e.g. RF1=60%,RF2=60%, RF3=54%, RF5=57vs. RF4=33o/o, no significantdiffe. rence),due to the higher survivalduring coronary artery occlusion in the RF4 group. Preliminaryfindingsfrom parallelinvestigations on the compositionof fish meat after differentfish food suggest that fish meat used for preparingthe rat food of the RF4 groupwas originatedfrom carp gioup fed on fish oil fortified feed showed the best growth, however, producing the leanestfish fillet. This is represented by the smallest amountof crudefat in RF4 (52 g/kg,Table 1) in the present investtigations.As a result,this group of rats were fed smaller amount of fish fat, in spite of adding the same weight percenttageof fish meat to the rat food. The detailedanalysisof fish meat and the changesin the composition of rat tissueas a resultof dietarytreatmentneeds further investigations. 74 GONCLU$ION We may concludethat as a resultof the 4-week long feeding period in rats there were no significantdifferencesamong the five dietarygroups,i.e. feedingfish meat after different fish food composition,as concerningthe body weight,the baseline electrocardiogram and bloodpressure,the .orthe responseto parameters, bloodlipid 6 min myocardialischemiaand 5 min reperfusionin rats. These data suggestthat each of the present experimental diets (RF1-RF3,RFs) contained enough polyunsaturatedfatty acids to evoke protectionagainstthe developmentof myocardialischemia-reperfusion induced arrhythmiasin anaesthetized rats, as compared to the control, saturatedfattyacid richdiet. Supplyingfish meat, rich in n-3 type polyunsaturatedfatty acids, significantlydecreasedthe incidenceof myocardialischemia-reperfusioninduced arrhythmiasin the rat, and increasedthe survivalrate. These results confirm previous investtigationsthat feedingfish oil or fish meat may offer cardioprotectiveeffect. The present results support that consumptionof farmed fish, like carps, may result in similar protectiveeffects to the marinefood, therebyofferinga good alternative to human dietary suggestionsfor continentalcountries, not having fresh marine food. However,the observed differences in the effects of fish meat containingdiets in the presentinvestigations NikolettMoruayef a/., Evaluationof the potentialantiarrhythmiceffect of'fish meat during myocardialischemia-reperfusionin rats, Food and Feed Research40 (2),71-76, ZO13 are warning,that when using oil enriched foods care should be taken both aboutthe quantityand the balanced availabilityof the long chain n-G and n-3 fatty acids in the diet. AKNOWLEDGEMENT This work has been co-financed by the EuropeanUnion through projectHUSRB1042-214-12A ("Researchcooperationon developing innovative fish feed for promotion of healthy food ih the region") within the Hungary-Serbia IPA CrossborderCo-operationProgramme. REFERENGES 1. AOAC (2005). OfficialMethodsof Analysisof AOAC International, 18'ned. Associationof OfficialAnalyticalChemists,WashingtonDC, USA. 2. Calder,P.C. 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