Cardiomyocyte Apoptosis After Antegrade and Retrograde Cardioplegia

DOI: 10.1016/j.athoracsur.2005.05.057 2005;80:2229-2234 Ann Thorac Surg

Kentala, Markku Kallajoki and Timo Savunen Tommi Vähäsilta, Antti Saraste, Ville Kytö, Markus Malmberg, Jan Kiss, Erkki

Cardiomyocyte Apoptosis After Antegrade and Retrograde Cardioplegia

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ardiomyocyte Apoptosis After Antegrade andetrograde Cardioplegia

ommi Vähäsilta, MD, Antti Saraste, MD, PhD, Ville Kytö, MD, PhD,arkus Malmberg, MB, Jan Kiss, MB, Erkki Kentala, MD, PhD,arkku Kallajoki, MD, PhD, and Timo Savunen, MD, PhD

epartments of Cardiothoracic Surgery, Anaesthesiology and Intensive Care, and Pathology, Turku University Central Hospital,nd Research Centre of Applied and Preventive Cardiovascular Medicine and Department of Anatomy, Turku University, Turku,

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Background. Retrograde cardioplegia alone is oftensed in aortic valve and aortic root surgery. Due to theifferences in venous anatomy between the right and the

eft side of the heart, retrograde cardioplegia is associatedith incomplete protection of the right side. Since some

poptotic cardiomyocyte death is inevitable during anpen heart surgery, we compared the extent of cardiomy-cyte apoptosis in the left and right ventricles afterntegrade and retrograde cardioplegia in a pig ischemia-eperfusion model.

Methods. Pigs (n � 16, mean weight 30 kg) were openlyssigned into the groups of antegrade and retrogradeardioplegia. After aortic cross-clamping, 500 mL of coldrystalloid (modified St Thomas) cardioplegia was ad-inistered into the ascending aorta or the coronary sinus.ortic cross-clamp time was 30 minutes. Cardiomyocyte

poptosis was measured using the terminal transferaseediated ddUTP nick end-labeling (TUNEL) assay and

mmunohistochemical (IHC) staining for active caspase-3

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ddress correspondence to Dr Vähäsilta, Turku University Central Hos-ital, FIN-20520, Turku, Finland; e-mail: [email protected].

2005 by The Society of Thoracic Surgeonsublished by Elsevier Inc

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n myocardial biopsies obtained before ischemia andfter 90 minutes of reperfusion.Results. Apoptotic cardiomyocytes were significantly

ncreased after ischemia-reperfusion as shown by bothhe TUNEL assay and caspase-3 activation. In the rightentricle, retrograde cardioplegia was associated with a.4-fold higher amount (TUNEL assay) of apoptotic car-iomyocytes as compared with antegrade cardioplegia

0.107% vs 0.032%, p < 0.05). A similar difference was alsoound in the left ventricle, although at a lower level0.027% vs 0.012%, p < 0.05).

Conclusions. Increased apoptotic death of cardiomyo-ytes after retrograde cardioplegia as compared with thentegrade procedure implicates that retrograde cardio-legia alone provides inferior cardioprotection against

rreversible ischemia-reperfusion injury both in the rightnd the left ventricle.

(Ann Thorac Surg 2005;80:2229–34)

yocardial protection during cardiac operations de-pends on adequate delivery of cardioplegia solu-

ion to all regions of the heart, most effectively providedy combined retrograde and antegrade cardioplegia. In

he aortic root and aortic valve surgery, however, only aetrograde administration of cardioplegia is frequentlysed [1]. In the retrograde procedure, the cardioplegicolution is administered through the coronary sinus intohe venous system of the heart. Retrograde cardioplegias associated with partial shunting of the cardioplegiaolution through the arteriosinusoidal system and thehebesian veins into ventricular cavities, particularly tohe right ventricle, without perfusing the myocardium2]. Indeed, experimental studies have shown that retro-rade cardioplegia is associated with incomplete perfu-ion [3, 4], depressed functional recovery [5, 6], andmpaired preservation of energy metabolism [7] in theight ventricle. However, the differences in the effects ofetrograde and antegrade cardioplegia on irreversibleyocardial ischemia-reperfusion injury remain largely

ccepted for publication May 17, 2005.

nknown. In the porcine heart, cardioplegia can beuccessfully delivered through the coronary sinus, andonsequently this has been a widely used experimentalodel to study the effects of retrograde cardioplegia [8].Apoptosis is a morphologically distinct type of cell

eath which involves a series of genetically controlledolecular and cellular events [9–11]. The biochemical

allmarks of apoptosis are internucleosomal DNA frag-entation and activation of caspase enzymes. Apoptosis

s also responsible, at least in part, for the loss ofardiomyocytes during acute myocardial infarction andschemia-reperfusion injury [12, 13]. Apoptosis of cardi-myocytes is also induced during cardioplegic ischemiassociated with open heart surgery in animal models14–21] and in humans [22–25]. It is not known, however,hether apoptosis is particularly associated with the usef retrograde cardioplegia.We hypothesized that retrograde cardioplegia pro-

ides inferior protection against the programmed celleath (apoptosis) of cardiomyocytes as compared with

he antegrade procedure. In this study, we compared theccurrence of apoptosis in cardiomyocytes after ante-rade and retrograde cardioplegia, using the porcine

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2230 VAHASILTA ET AL Ann Thorac SurgCARDIOPLEGIA AND APOPTOSIS 2005;80:2229–34

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aterial and Methods

n this study, 16 domestic Finnish landrace pigs (29 kgean weight) were openly assigned into the groups of

ntegrade and retrograde cardioplegia. All animals re-eived humane care in compliance with the Europeanonvention on Animal Care. In the retrograde group,ne animal was excluded from the final data analysisecause of technical difficulties in the sinus coronariusannulation and incomplete cardiac arrest. The studyrotocol was approved by the Ethical Committee fornimal Research at the University of Turku.

urgical Procedurehe animals were premedicated with intramuscular in-

ection of S-ketamine (1,000 mg Ketanest-S; Pfizer AB,aby, Sweden). The peripheral vein in the ear wasannulated and 10 mg of diazepam (Stesolid Novum, A/Sumex, Denmark) was given intravenously. The tracheaas surgically exposed and intubation performed openly.or the intubation, animals received 4 mg intravenousolus of pancuronium (Pavulon, Organon, The Nether-

ands). The animals were connected to a respirator (Res-iration Pump model 607, Harvard Apparatus, Millis,A; with the tidal volume 450 mL/minute, frequency 16

o 18/minute) that was set according to blood gas analysisABL 50, Radiometer A/S, Copenhagen, Denmark). An-sthesia was maintained with continuous intravenousnfusion of S-ketamine and pancuronium. Arterial bloodressure and central venous pressure, electrocardio-ram, and heart rate were monitored throughout thexperiment (Uniflow pressure monitoring kit 43-600F;axter, Uden, Holland, and Olli 530; Kone Co, Espoo,inland). Internal carotid artery and external jugular veinere cannulated for hemodynamic monitoring and blood

ampling.Medial sternotomy was performed and pericardium

pened and lifted. Purse string sutures were placed onhe ascending aorta, the superior vena cava, and the righttrium. After a 100 mg bolus of heparin (Heparin; Lövens,allerup, Denmark), the aorta and the caval veins wereannulated and the animal was connected to cardiopul-onary bypass (CPB). The flow in the aortic line was

djusted to 2.5–3 L/minute (85–100 mL/kg/minute) ac-ording to blood gas analysis. A pediatric membranexygenator (Midiflo Pediatric D705; Dideco, Mirandola,taly) was primed with 1,500 mL of fresh pig bloodontaining 3,800 mg of sodium citrate and 50 mg ofeparin. The left hemiazygos vein draining to the coro-ary sinus was ligated. In the antegrade group, an 18Gannula (Venlon 2; Viggo AB, Helsingborg, Sweden) waslaced in the ascending aorta. In the retrograde group,

he sinus coronarius was cannulated with a 6F retrogradeardioplegia cannula introduced through a pursestringuture at the origin of the sinus coronarius (DLP,

edtronic Inc, Minneapolis, MN). This method has beenhown to improve cardioplegia distribution in the rightentricle and in the interventricular septum [8].A single dose of 500 mL of modified St Thomasospital No II cold (10°C) crystalloid cardioplegic solu-

ion was given after the clamping of the aorta in both p

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roups. No additional topical cooling of the heart wassed, and the temperature of the heart was not assessed.ystemic normothermia was maintained at 36°C with aeat exchanger attached to CPB. The pressure in theardioplegia line was maintained between 100 and 120m Hg in the antegrade group and under 50 mm Hg in

he retrograde group. The aortic cross-clamp time was 30inutes in both groups. After declamping of the aorta,

he animals were kept under partial CPB for 90 minutes.n order to standardize the hemodynamic state duringhis follow-up and avoid the use of inotropic agentswhich may induce apoptosis), no weaning from the CPBas performed. After the experiment, all animals were

acrificed with a potassium chloride injection given di-ectly to the left atrium.

Transmyocardial samples were collected from the freeall of left and right ventricles before ischemia (preisch-

mic samples) and after the 90-minute reperfusion pe-iod at the end of the experiment (ischemic samples). A4G Tru-cut biopsy needle (Pharmaseal, Allegianceealthcare Corp, McGaw Park, IL) was used to obtainreischemic biopsies. To minimize the risk of bleeding,nly one preischemic biopsy was taken from each ani-al, but it was repeated if the first biopsy was technically

nsuccessful. One postischemic sample (1 cm3 in size)as obtained. All tissue samples were fixed in bufferedeutral formalin overnight, embedded in paraffin, andut into 4-�m sections for analysis of apoptosis.

ssessment of Apoptosispoptosis was detected using the TUNEL (terminal

ransferase mediated ddUTP nick end-labeling) assay, asreviously described [10, 12]. In brief, paraffin-embeddedyocardial sections were heated in sodium citrate solu-

ion and digested with proteinase-K to expose DNA. TheNA strand breaks were then labeled using terminal

ransferase with digoxigenin-conjugated ddUTP and vi-ualized using alkaline phosphatase immunohistochem-stry (IHC). To confirm optimal sensitivity of the assay, itas standardized with the use of serial sections treatedith DNase I to induce enzymatic DNA fragmentation

positive control of apoptosis).We also used IHC to analyze apoptosis-specific activa-

ion of caspase-3 with a polyclonal antibody specific forarge (17–20 kDa) fragments of cleaved caspase-3 (Cellignaling Technology, Beverly, MA). In brief, deparaf-ned hydrated sections were treated in a microwaveven for 10 minutes in sodium citrate buffer (pH 6.0) toxpose antigens, followed by inhibition of endogenouseroxidase activity by 1% H2O2. The primary antibody

1:100) was visualized using the Vectastain ABC Elite KitVector Laboratories, Burlingame, CA) according to the

anufacturer’s instructions, using the avidine-biotin im-unoperoxidase technique with diaminobenzine as the

hromogen. Sections of inflamed human tonsil showingositive staining in some lymphocytes served as positiveontrols. Negative control sections incubated without

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uantifying Apoptotic Cellshe numbers of TUNEL-positive cardiomyocytes andardiomyocytes containing cleaved caspase-3 were calcu-ated using light microscopy (�250) with an ocular grid.ll analyses were done in a blinded manner, the pathol-gist being unaware of the study group of the individualnimals. The proportions of TUNEL-positive cardiomyo-ytes are expressed as percentages of the total number ofardiomyocyte nuclei counted in the serial DNase-reated sections. The average number of microscopyelds analyzed in each animal was 39 (range, 8–112) in

he preischemic samples and 286 (152–524) in the isch-mic samples. The average number of cardiomyocyteuclei per field was 205 (170–285) in the preischemicamples and 206 (158–290) in the ischemic samples. Theroportions of caspase-3 positive cardiomyocytes are alsoxpressed as percentages of the total number of cardio-yocytes. The average number of fields studied in each

aspase-3 stained preischemic sample was 31 (8–72) and68 (96–496) in the ischemic sample. The average num-er of myocytes per field was 174 (135–201) in the former,nd 165 (135–190) in the latter. The cardiomyocyte originf the cells was confirmed by the presence of myofila-ents. In some cases, consecutive histologic sectionsere studied or TUNEL-stained sections were stainedsing IHC for cardiac myosin.

tatistical Analysisata are expressed as mean � SD. The differencesetween the groups were tested with the two-tailedtudent=s t test, and values p less than 0.05 were consid-red statistically significant.

esultshere were no statistical differences in hemodynamiceasurements between the two groups. In the baselineeasurements, blood pressure was 92 � 9/51 � 4 mm Hg

n the antegrade group and 87 � 9/56 � 7 mm Hg in theetrograde group. Central venous pressure was 2 � 2 mmg and 4 � 1 mm Hg, and heart rate was 134 � 4/minute

nd 133 � 7/minute, respectively. After opening of theorta, five animals in both groups spontaneouslychieved sinus rhythm. Three animals in both groupsequired defibrillation because of ventricular fibrillation,nd sinus rhythm was achieved in all these animals.In the preischemic samples, apoptotic cardiomyocytesere rarely detected, in contrast to the postischemic

amples, where scattered myocytes with intenselyUNEL–positive nuclei or cytoplasmic and nuclearaspase-3 expression were detected in both the right andeft ventricles (Fig 1). The proportion of apoptotic cardi-myocytes (measured by the TUNEL assay) in the pre-

schemic biopsies was very low in both the left ventricle0.001% � 0.004% [n � 13]) and the right ventricle (0.001%

0.005% [n � 13]). Using IHC for activated caspase-3,he proportions of apoptotic cardiomyocytes were 0.002%

0.008% (n � 13) in the left ventricle and 0.002% �.005% (n � 10) in the right ventricle.After ischemia-reperfusion, the proportion of apopto-

ic cardiomyocytes (TUNEL assay) in both groups was s

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ignificantly increased in the left ventricle (0.019% �.013, p � 0.05) and in the right ventricle (0.067% �.074%, p � 0.05), as compared with the preischemic

ig 1. (A) Positive TUNEL staining (arrow) in the nucleus of a car-iomyocyte in the right ventricle after ischemia-reperfusion. (B) Theame cell (arrow) seen in a consecutive tissue section stained withan Gieson. (C) Positive immunohistochemical staining for activatedaspase-3 in a myocyte (arrow).

amples. As detected by IHC for activated caspase-3, this

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ncrease was even more marked in both the left (0.062%0.031%, p � 0.05) and the right ventricles (0.300% �

.263%, p � 0.05).Retrograde cardioplegia was associated with signifi-

antly increased cardiomyocyte apoptosis in both theight and left ventricles, as compared with the antegradeardioplegia. As measured by the TUNEL assay, thereas a 3.4-fold higher proportion of apoptotic myocytes in

he right ventricle after retrograde cardioplegia than afterntegrade cardioplegia (0.107% � 0.093% vs 0.032% �.021%, p � 0.05) (Fig 2). This increase was 2.3-fold in theeft ventricle (0.027% � 0.012% vs 0.012% � 0.010%, p �.05). A similar pattern of increased apoptosis was de-ected when analyzed using the activated caspase-3 IHC.ompared with antegrade cardioplegia, the proportionf caspase-3 positive cells after retrograde cardioplegiaas 2.2-fold higher (0.438% � 0.353%, p � 0.05) in the

ight ventricle and 1.7-fold higher (0.080% � 0.036%, p �.05) in the left ventricle (Fig 3).

omment

his study analyzed the influence of antegrade andetrograde cardioplegia on the development of pro-rammed cell death (apoptosis) in cardiac myocytessing the experimental pig model of open heart surgery.he results unequivocally demonstrate that apoptoticyocytes increased significantly more after retrograde

han antegrade cardioplegia in this model. This providesirect experimental evidence to substantiate the concept

hat retrograde cardioplegia alone provides inferior car-ioprotection against irreversible ischemia-reperfusion

njury in both the right and the left ventricles, as com-ared with the antegrade approach. This has importantlinical implications as are discussed later.

Apoptosis is a morphologically distinct type of celleath, involving a series of genetically controlled molec-lar and cellular events [9–11]. The biochemical markersf apoptosis are internucleosomal DNA fragmentationnd activation of caspase enzymes. In order to specifi-ally detect apoptotic cardiac myocytes, we used two

ig 2. The percentages of apoptotic cardiomyocytes detected by theUNEL assay after 30 minutes of cardioplegic ischemia and 90 min-tes of reperfusion.

omplementary methods; the TUNEL assay and immu- c

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ohistochemical demonstration of activated caspase-3nzyme, which is essential for apoptosis. Consonant witheveral previous experimental studies, we found thatpoptosis was induced in cardiac myocytes during car-ioplegic ischemia in this experimental open heart sur-ery model [14–20]. Indeed, apoptotic myocytes wereignificantly increased after ischemia-reperfusion asompared with the preoperative (baseline) biopsies,hen analyzed by the TUNEL assay and using IHC

taining for caspase-3. The difference was more pro-ounced with the caspase-3 staining, suggesting thataspase activation precedes the DNA fragmentation,hich is in alignment with the known sequence of events

n the programmed cell death. For full quantification ofpoptosis after the operation, more prolonged reperfu-ion time is likely to be needed.

At present, a selection of clinical antiapoptotic inter-entions is already available for the cardiac surgeon.hese include (a) prompt coronary revascularization, (b)eduction of myocardial ischemia time, (c) avoiding CPBhen possible, (d) avoiding sustained use of cat-

cholamines, and (e) liberal and early insertion of anntraaortic balloon pulsation or a ventricular assist device26]. A variety of other potentially effective ways ofttenuating apoptosis in cardiac surgery exist, includinghe use of caspase inhibitors, antioxyradical stress agents,denosine, carbon monoxide, ischemic preconditioning,nd monoclonal antibodies against neutrophil adhesion14–20, 27]. A recent study demonstrated that using bloodardioplegia instead of crystalloid reduced caspase-3ctivation and enhanced antiapoptotic signaling in thesolated heart [28]. Whether any of these new experimen-al antiapoptotic therapies are clinically useful in hu-

ans, remains to be seen.Myocardial protection during cardiac operations de-

ends on adequate delivery of cardioplegia solution to allegions of the heart. Retrograde administration of cardio-legia is often used alone in the aortic root and aorticalve surgery [1]. In retrograde cardioplegia, the solutions administered through the coronary sinus into theardiac venous system. Most of the veins in the heartrain to the right atrium through the coronary sinus.

ig 3. The percentages of myocytes demonstrating activated

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owever, a smaller part of the cardiac venous returnrains directly to the cardiac chambers through thenterior cardiac veins and the thebesian veins (venaeordis minima), particularly in the right side [2]. Thus, inetrograde cardioplegia, the solution is shunted into theentricular cavity without myocardial perfusion. Indeed,oth experimental and human studies have shown thatetrograde cardioplegia is associated with incompleteerfusion [4], depressed functional recovery [5, 6], and

mpaired preservation of energy metabolism [7, 29] in theight ventricle. At the tissue level, only mild to moderatend usually reversible ultrastructural changes have beenetected in the myocardium [30]. However, the signifi-ance of retrograde versus antegrade cardioplegia inroducing irreversible myocardial ischemia-reperfusion

njury remains largely unknown. Our results clearlyemonstrate that more cardiac myocytes are lost bypoptosis when retrograde cardioplegia is used alone, asompared with antegrade cardioplegia. Indeed, signifi-antly more numerous apoptotic myocytes were foundot only in the right, but also in the left ventricle. This

mplicates an incomplete distribution of cardioplegia inoth ventricles and suggests that retrograde cardioplegiarovides inferior protection in both ventricles as com-ared with the antegrade procedure.The significance of increased apoptosis of myocytes

fter cardiac surgery remains to be clarified in the clinicaletting in terms of cardiac function, surgical complica-ions, and long-term survival. In chronic cardiac diseaseseg, severe heart failure and myocarditis), a correlationas been shown between disease severity and apoptosis

31–33]. On the other hand, this type of correlation hasot been confirmed in acute surgical ischemia-

eperfusion injury. However, in right atrial biopsies theelease of cytochrome-C from mitochondria (involved inhe induction of apoptosis) was shown to be associatedith the severity of immediate postischemic dysfunction

stunning) in patients who underwent cardiac surgery23]. Notably, the amount of cardiomyocyte apoptosis inur study was higher in the right than in the left ventricle,

rrespective of the cardioplegia type used. This mayndicate that right ventricular myocardium is more sen-itive to ischemia-reperfusion injury. Since moderateypothermia has been shown to reduce apoptosis, it mayell be that warming of the right ventricle during theperation (due to its anterior location) predisposes it topoptosis [15].To conclude, the present study demonstrates that ap-

ptosis is substantially increased after retrograde cardio-legia in this porcine model. Because of the common usef retrograde cardioplegia in the clinical routine, thisnding may have important clinical implications.learly, more studies are needed to evaluate the impor-

ance of apoptotic cell death and the effect of antiapop-otic interventions on the clinical outcome after cardio-ulmonary bypass surgery.

his study was supported by a grant from the Finnish Culturaloundation and by the Clinical Research (EVO) funding of

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3. Saraste A, Pulkki K, Kallajoki M, et al. Cardiomyocyteapoptosis and progression of heart failure to transplantation.

NVITED COMMENTARY

poptosis or program cell death has been previouslyeported to occur during cardioplegic arrest and cardio-ulmonary bypass (CPB), suggesting that apoptosis may,t least in part, contribute to myocardial stunning.ähäsilta and colleagues [1] confirmed these previousndings in their in-vivo pig model and further tested theypothesis that differences from retrograde and ante-rade cardioplegia might translate into differences inppearance of apoptosis. In this interesting and impor-ant study, the authors found that retrograde cardiople-ia induced higher amount of apoptosis cardiomyocyteeath than antegrade cardioplegia. We believe that this is

he first report to show that retrograde cardioplegia isnferior to antegrade cardioplegia in inhibiting myocar-ial apoptosis in both the right and left ventricles. Thisnding has very important clinical implications becauseetrograde cardioplegia has been commonly used in theortic root and aortic valve surgery. These data mayartially explain why retrograde cardioplegia is oftenssociated with incomplete perfusion, depressed func-ional recovery, and impaired preservation of energy

etabolism in the right ventricle.However, there are still several limitations in this

tudy. First, the authors used a relatively short period (30inutes) of ischemic arrest induced by cold cardioplegia

10°C). They did not measure the myocardial tempera-ure during the 30-minute ischemic arrest. Apoptosis isot only an ischemia-reperfusion event, but also timend temperature dependent. It has been demonstratedhat mild or moderate hypothermia protects myocardiumgainst myocardial dysfunction, necrosis, apoptosis, andpoptosis-related gene expression. Second, the authorssed cold crystalloid cardioplegia with normothermic

36°C) CPB. In clinical practice, cold crystalloid cardio-legia is often combined with hypothermic CPB to pro-

ect ischemic myocardium. Third, the authors used only

taining is known to be nonspecific, which has beenecently commented by several authors in this journalnd others. Measurement of activated caspase-3 withmmunohistochemistry was very helpful in this study,ut the authors used an unusual and complicatedethod to quantify activated caspase-3. Western blots for

apase-3 cleavage, a pre-requirement for caspase-3 en-ymatic activity, poly-(adenosine diphosphate-ribose)olymerase degradation, a major substrate for activatedaspase-3, and cytoplasmic cytochrome c releases arexcellent methods for detecting myocytes apoptosis. Be-ause different phases of apoptosis may present differentfaces of apoptosis,” multiple approaches will be veryelpful to identify real apoptotic cells.Irrespective of these limitations, this is an important

nd relevant study because the changes in apoptosis mayot only contribute to short-term functional deterioration,ut more important may also contribute to the long-termeneficial effects. Thus, this may affect clinical practice,ainly by the prevention of myocardial apoptosis.

un Feng, MD, PhDrank W. Sellke, MD

ivision of Cardiothoracic Surgeryeth Israel Deaconess Medical Center30 Brookline Aveoston, MA 02215-mail: [email protected];

[email protected]

eference

. Vähäsilta T, Saraste A, Kytö V, et al. Cardiomyocyte apoptosisafter antegrade and retrograde cardioplegia. Ann Thorac

0003-4975/05/$30.00doi:10.1016/j.athoracsur.2005.07.009

by on June 7, 2013 als.org

DOI: 10.1016/j.athoracsur.2005.05.057 2005;80:2229-2234 Ann Thorac Surg

Kentala, Markku Kallajoki and Timo Savunen Tommi Vähäsilta, Antti Saraste, Ville Kytö, Markus Malmberg, Jan Kiss, Erkki

Cardiomyocyte Apoptosis After Antegrade and Retrograde Cardioplegia

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