Cardiomyocyte Apoptosis After Antegrade and Retrograde Cardioplegia Tommi Vähäsilta, Antti Saraste, Ville Kytö, Markus Malmberg, Jan Kiss, Erkki Kentala, Markku Kallajoki and Timo Savunen Ann Thorac Surg 2005;80:2229-2234 DOI: 10.1016/j.athoracsur.2005.05.057
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The Annals of Thoracic Surgery is the official journal of The Society of Thoracic Surgeons and the Southern Thoracic Surgical Association. Copyright © 2005 by The Society of Thoracic Surgeons. Print ISSN: 0003-4975; eISSN: 1552-6259.
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Tommi Vähäsilta, MD, Antti Saraste, MD, PhD, Ville Kytö, MD, PhD, Markus Malmberg, MB, Jan Kiss, MB, Erkki Kentala, MD, PhD, Markku Kallajoki, MD, PhD, and Timo Savunen, MD, PhD Departments of Cardiothoracic Surgery, Anaesthesiology and Intensive Care, and Pathology, Turku University Central Hospital, and Research Centre of Applied and Preventive Cardiovascular Medicine and Department of Anatomy, Turku University, Turku, Finland
Background. Retrograde cardioplegia alone is often used in aortic valve and aortic root surgery. Due to the differences in venous anatomy between the right and the left side of the heart, retrograde cardioplegia is associated with incomplete protection of the right side. Since some apoptotic cardiomyocyte death is inevitable during an open heart surgery, we compared the extent of cardiomyocyte apoptosis in the left and right ventricles after antegrade and retrograde cardioplegia in a pig ischemiareperfusion model. Methods. Pigs (n ⴝ 16, mean weight 30 kg) were openly assigned into the groups of antegrade and retrograde cardioplegia. After aortic cross-clamping, 500 mL of cold crystalloid (modified St Thomas) cardioplegia was administered into the ascending aorta or the coronary sinus. Aortic cross-clamp time was 30 minutes. Cardiomyocyte apoptosis was measured using the terminal transferase mediated ddUTP nick end-labeling (TUNEL) assay and immunohistochemical (IHC) staining for active caspase-3
in myocardial biopsies obtained before ischemia and after 90 minutes of reperfusion. Results. Apoptotic cardiomyocytes were significantly increased after ischemia-reperfusion as shown by both the TUNEL assay and caspase-3 activation. In the right ventricle, retrograde cardioplegia was associated with a 3.4-fold higher amount (TUNEL assay) of apoptotic cardiomyocytes as compared with antegrade cardioplegia (0.107% vs 0.032%, p < 0.05). A similar difference was also found in the left ventricle, although at a lower level (0.027% vs 0.012%, p < 0.05). Conclusions. Increased apoptotic death of cardiomyocytes after retrograde cardioplegia as compared with the antegrade procedure implicates that retrograde cardioplegia alone provides inferior cardioprotection against irreversible ischemia-reperfusion injury both in the right and the left ventricle. (Ann Thorac Surg 2005;80:2229 –34) © 2005 by The Society of Thoracic Surgeons
M
unknown. In the porcine heart, cardioplegia can be successfully delivered through the coronary sinus, and consequently this has been a widely used experimental model to study the effects of retrograde cardioplegia [8]. Apoptosis is a morphologically distinct type of cell death which involves a series of genetically controlled molecular and cellular events [9 –11]. The biochemical hallmarks of apoptosis are internucleosomal DNA fragmentation and activation of caspase enzymes. Apoptosis is also responsible, at least in part, for the loss of cardiomyocytes during acute myocardial infarction and ischemia-reperfusion injury [12, 13]. Apoptosis of cardiomyocytes is also induced during cardioplegic ischemia associated with open heart surgery in animal models [14 –21] and in humans [22–25]. It is not known, however, whether apoptosis is particularly associated with the use of retrograde cardioplegia. We hypothesized that retrograde cardioplegia provides inferior protection against the programmed cell death (apoptosis) of cardiomyocytes as compared with the antegrade procedure. In this study, we compared the occurrence of apoptosis in cardiomyocytes after antegrade and retrograde cardioplegia, using the porcine open heart surgery model.
yocardial protection during cardiac operations depends on adequate delivery of cardioplegia solution to all regions of the heart, most effectively provided by combined retrograde and antegrade cardioplegia. In the aortic root and aortic valve surgery, however, only a retrograde administration of cardioplegia is frequently used [1]. In the retrograde procedure, the cardioplegic solution is administered through the coronary sinus into the venous system of the heart. Retrograde cardioplegia is associated with partial shunting of the cardioplegia solution through the arteriosinusoidal system and the thebesian veins into ventricular cavities, particularly to the right ventricle, without perfusing the myocardium [2]. Indeed, experimental studies have shown that retrograde cardioplegia is associated with incomplete perfusion [3, 4], depressed functional recovery [5, 6], and impaired preservation of energy metabolism [7] in the right ventricle. However, the differences in the effects of retrograde and antegrade cardioplegia on irreversible myocardial ischemia-reperfusion injury remain largely
Accepted for publication May 17, 2005. Address correspondence to Dr Vähäsilta, Turku University Central Hospital, FIN-20520, Turku, Finland; e-mail:
[email protected].
© 2005 by The Society of Thoracic Surgeons Published by Elsevier Inc
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Cardiomyocyte Apoptosis After Antegrade and Retrograde Cardioplegia
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Material and Methods
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In this study, 16 domestic Finnish landrace pigs (29 kg mean weight) were openly assigned into the groups of antegrade and retrograde cardioplegia. All animals received humane care in compliance with the European Convention on Animal Care. In the retrograde group, one animal was excluded from the final data analysis because of technical difficulties in the sinus coronarius cannulation and incomplete cardiac arrest. The study protocol was approved by the Ethical Committee for Animal Research at the University of Turku.
Surgical Procedure The animals were premedicated with intramuscular injection of S-ketamine (1,000 mg Ketanest-S; Pfizer AB, Taby, Sweden). The peripheral vein in the ear was cannulated and 10 mg of diazepam (Stesolid Novum, A/S Dumex, Denmark) was given intravenously. The trachea was surgically exposed and intubation performed openly. For the intubation, animals received 4 mg intravenous bolus of pancuronium (Pavulon, Organon, The Netherlands). The animals were connected to a respirator (Respiration Pump model 607, Harvard Apparatus, Millis, MA; with the tidal volume 450 mL/minute, frequency 16 to 18/minute) that was set according to blood gas analysis (ABL 50, Radiometer A/S, Copenhagen, Denmark). Anesthesia was maintained with continuous intravenous infusion of S-ketamine and pancuronium. Arterial blood pressure and central venous pressure, electrocardiogram, and heart rate were monitored throughout the experiment (Uniflow pressure monitoring kit 43-600F; Baxter, Uden, Holland, and Olli 530; Kone Co, Espoo, Finland). Internal carotid artery and external jugular vein were cannulated for hemodynamic monitoring and blood sampling. Medial sternotomy was performed and pericardium opened and lifted. Purse string sutures were placed on the ascending aorta, the superior vena cava, and the right atrium. After a 100 mg bolus of heparin (Heparin; Lövens, Ballerup, Denmark), the aorta and the caval veins were cannulated and the animal was connected to cardiopulmonary bypass (CPB). The flow in the aortic line was adjusted to 2.5–3 L/minute (85–100 mL/kg/minute) according to blood gas analysis. A pediatric membrane oxygenator (Midiflo Pediatric D705; Dideco, Mirandola, Italy) was primed with 1,500 mL of fresh pig blood containing 3,800 mg of sodium citrate and 50 mg of heparin. The left hemiazygos vein draining to the coronary sinus was ligated. In the antegrade group, an 18G cannula (Venlon 2; Viggo AB, Helsingborg, Sweden) was placed in the ascending aorta. In the retrograde group, the sinus coronarius was cannulated with a 6F retrograde cardioplegia cannula introduced through a pursestring suture at the origin of the sinus coronarius (DLP, Medtronic Inc, Minneapolis, MN). This method has been shown to improve cardioplegia distribution in the right ventricle and in the interventricular septum [8]. A single dose of 500 mL of modified St Thomas Hospital No II cold (10°C) crystalloid cardioplegic solution was given after the clamping of the aorta in both
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groups. No additional topical cooling of the heart was used, and the temperature of the heart was not assessed. Systemic normothermia was maintained at 36°C with a heat exchanger attached to CPB. The pressure in the cardioplegia line was maintained between 100 and 120 mm Hg in the antegrade group and under 50 mm Hg in the retrograde group. The aortic cross-clamp time was 30 minutes in both groups. After declamping of the aorta, the animals were kept under partial CPB for 90 minutes. In order to standardize the hemodynamic state during this follow-up and avoid the use of inotropic agents (which may induce apoptosis), no weaning from the CPB was performed. After the experiment, all animals were sacrificed with a potassium chloride injection given directly to the left atrium. Transmyocardial samples were collected from the free wall of left and right ventricles before ischemia (preischemic samples) and after the 90-minute reperfusion period at the end of the experiment (ischemic samples). A 14G Tru-cut biopsy needle (Pharmaseal, Allegiance Healthcare Corp, McGaw Park, IL) was used to obtain preischemic biopsies. To minimize the risk of bleeding, only one preischemic biopsy was taken from each animal, but it was repeated if the first biopsy was technically unsuccessful. One postischemic sample (1 cm3 in size) was obtained. All tissue samples were fixed in buffered neutral formalin overnight, embedded in paraffin, and cut into 4-m sections for analysis of apoptosis.
Assessment of Apoptosis Apoptosis was detected using the TUNEL (terminal transferase mediated ddUTP nick end-labeling) assay, as previously described [10, 12]. In brief, paraffin-embedded myocardial sections were heated in sodium citrate solution and digested with proteinase-K to expose DNA. The DNA strand breaks were then labeled using terminal transferase with digoxigenin-conjugated ddUTP and visualized using alkaline phosphatase immunohistochemistry (IHC). To confirm optimal sensitivity of the assay, it was standardized with the use of serial sections treated with DNase I to induce enzymatic DNA fragmentation (positive control of apoptosis). We also used IHC to analyze apoptosis-specific activation of caspase-3 with a polyclonal antibody specific for large (17–20 kDa) fragments of cleaved caspase-3 (Cell Signaling Technology, Beverly, MA). In brief, deparaffined hydrated sections were treated in a microwave oven for 10 minutes in sodium citrate buffer (pH 6.0) to expose antigens, followed by inhibition of endogenous peroxidase activity by 1% H2O2. The primary antibody (1:100) was visualized using the Vectastain ABC Elite Kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions, using the avidine-biotin immunoperoxidase technique with diaminobenzine as the chromogen. Sections of inflamed human tonsil showing positive staining in some lymphocytes served as positive controls. Negative control sections incubated without primary antibody showed no staining.
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Quantifying Apoptotic Cells
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The numbers of TUNEL-positive cardiomyocytes and cardiomyocytes containing cleaved caspase-3 were calculated using light microscopy (⫻250) with an ocular grid. All analyses were done in a blinded manner, the pathologist being unaware of the study group of the individual animals. The proportions of TUNEL-positive cardiomyocytes are expressed as percentages of the total number of cardiomyocyte nuclei counted in the serial DNasetreated sections. The average number of microscopy fields analyzed in each animal was 39 (range, 8 –112) in the preischemic samples and 286 (152–524) in the ischemic samples. The average number of cardiomyocyte nuclei per field was 205 (170 –285) in the preischemic samples and 206 (158 –290) in the ischemic samples. The proportions of caspase-3 positive cardiomyocytes are also expressed as percentages of the total number of cardiomyocytes. The average number of fields studied in each caspase-3 stained preischemic sample was 31 (8 –72) and 268 (96 – 496) in the ischemic sample. The average number of myocytes per field was 174 (135–201) in the former, and 165 (135–190) in the latter. The cardiomyocyte origin of the cells was confirmed by the presence of myofilaments. In some cases, consecutive histologic sections were studied or TUNEL-stained sections were stained using IHC for cardiac myosin.
Statistical Analysis Data are expressed as mean ⫾ SD. The differences between the groups were tested with the two-tailed Student=s t test, and values p less than 0.05 were considered statistically significant.
Results There were no statistical differences in hemodynamic measurements between the two groups. In the baseline measurements, blood pressure was 92 ⫾ 9/51 ⫾ 4 mm Hg in the antegrade group and 87 ⫾ 9/56 ⫾ 7 mm Hg in the retrograde group. Central venous pressure was 2 ⫾ 2 mm Hg and 4 ⫾ 1 mm Hg, and heart rate was 134 ⫾ 4/minute and 133 ⫾ 7/minute, respectively. After opening of the aorta, five animals in both groups spontaneously achieved sinus rhythm. Three animals in both groups required defibrillation because of ventricular fibrillation, and sinus rhythm was achieved in all these animals. In the preischemic samples, apoptotic cardiomyocytes were rarely detected, in contrast to the postischemic samples, where scattered myocytes with intensely TUNEL–positive nuclei or cytoplasmic and nuclear caspase-3 expression were detected in both the right and left ventricles (Fig 1). The proportion of apoptotic cardiomyocytes (measured by the TUNEL assay) in the preischemic biopsies was very low in both the left ventricle (0.001% ⫾ 0.004% [n ⫽ 13]) and the right ventricle (0.001% ⫾ 0.005% [n ⫽ 13]). Using IHC for activated caspase-3, the proportions of apoptotic cardiomyocytes were 0.002% ⫾ 0.008% (n ⫽ 13) in the left ventricle and 0.002% ⫾ 0.005% (n ⫽ 10) in the right ventricle. After ischemia-reperfusion, the proportion of apoptotic cardiomyocytes (TUNEL assay) in both groups was
Fig 1. (A) Positive TUNEL staining (arrow) in the nucleus of a cardiomyocyte in the right ventricle after ischemia-reperfusion. (B) The same cell (arrow) seen in a consecutive tissue section stained with Van Gieson. (C) Positive immunohistochemical staining for activated caspase-3 in a myocyte (arrow).
significantly increased in the left ventricle (0.019% ⫾ 0.013, p ⬍ 0.05) and in the right ventricle (0.067% ⫾ 0.074%, p ⬍ 0.05), as compared with the preischemic samples. As detected by IHC for activated caspase-3, this
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increase was even more marked in both the left (0.062% ⫾ 0.031%, p ⬍ 0.05) and the right ventricles (0.300% ⫾ 0.263%, p ⬍ 0.05). Retrograde cardioplegia was associated with significantly increased cardiomyocyte apoptosis in both the right and left ventricles, as compared with the antegrade cardioplegia. As measured by the TUNEL assay, there was a 3.4-fold higher proportion of apoptotic myocytes in the right ventricle after retrograde cardioplegia than after antegrade cardioplegia (0.107% ⫾ 0.093% vs 0.032% ⫾ 0.021%, p ⬍ 0.05) (Fig 2). This increase was 2.3-fold in the left ventricle (0.027% ⫾ 0.012% vs 0.012% ⫾ 0.010%, p ⬍ 0.05). A similar pattern of increased apoptosis was detected when analyzed using the activated caspase-3 IHC. Compared with antegrade cardioplegia, the proportion of caspase-3 positive cells after retrograde cardioplegia was 2.2-fold higher (0.438% ⫾ 0.353%, p ⫽ 0.05) in the right ventricle and 1.7-fold higher (0.080% ⫾ 0.036%, p ⬍ 0.05) in the left ventricle (Fig 3).
Comment This study analyzed the influence of antegrade and retrograde cardioplegia on the development of programmed cell death (apoptosis) in cardiac myocytes using the experimental pig model of open heart surgery. The results unequivocally demonstrate that apoptotic myocytes increased significantly more after retrograde than antegrade cardioplegia in this model. This provides direct experimental evidence to substantiate the concept that retrograde cardioplegia alone provides inferior cardioprotection against irreversible ischemia-reperfusion injury in both the right and the left ventricles, as compared with the antegrade approach. This has important clinical implications as are discussed later. Apoptosis is a morphologically distinct type of cell death, involving a series of genetically controlled molecular and cellular events [9 –11]. The biochemical markers of apoptosis are internucleosomal DNA fragmentation and activation of caspase enzymes. In order to specifically detect apoptotic cardiac myocytes, we used two complementary methods; the TUNEL assay and immu-
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nohistochemical demonstration of activated caspase-3 enzyme, which is essential for apoptosis. Consonant with several previous experimental studies, we found that apoptosis was induced in cardiac myocytes during cardioplegic ischemia in this experimental open heart surgery model [14 –20]. Indeed, apoptotic myocytes were significantly increased after ischemia-reperfusion as compared with the preoperative (baseline) biopsies, when analyzed by the TUNEL assay and using IHC staining for caspase-3. The difference was more pronounced with the caspase-3 staining, suggesting that caspase activation precedes the DNA fragmentation, which is in alignment with the known sequence of events in the programmed cell death. For full quantification of apoptosis after the operation, more prolonged reperfusion time is likely to be needed. At present, a selection of clinical antiapoptotic interventions is already available for the cardiac surgeon. These include (a) prompt coronary revascularization, (b) reduction of myocardial ischemia time, (c) avoiding CPB when possible, (d) avoiding sustained use of catecholamines, and (e) liberal and early insertion of an intraaortic balloon pulsation or a ventricular assist device [26]. A variety of other potentially effective ways of attenuating apoptosis in cardiac surgery exist, including the use of caspase inhibitors, antioxyradical stress agents, adenosine, carbon monoxide, ischemic preconditioning, and monoclonal antibodies against neutrophil adhesion [14 –20, 27]. A recent study demonstrated that using blood cardioplegia instead of crystalloid reduced caspase-3 activation and enhanced antiapoptotic signaling in the isolated heart [28]. Whether any of these new experimental antiapoptotic therapies are clinically useful in humans, remains to be seen. Myocardial protection during cardiac operations depends on adequate delivery of cardioplegia solution to all regions of the heart. Retrograde administration of cardioplegia is often used alone in the aortic root and aortic valve surgery [1]. In retrograde cardioplegia, the solution is administered through the coronary sinus into the cardiac venous system. Most of the veins in the heart drain to the right atrium through the coronary sinus.
Fig 3. The percentages of myocytes demonstrating activated caspase-3 after ischemia-reperfusion.
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This study was supported by a grant from the Finnish Cultural Foundation and by the Clinical Research (EVO) funding of Turku University Central Hospital.
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However, a smaller part of the cardiac venous return drains directly to the cardiac chambers through the anterior cardiac veins and the thebesian veins (venae cordis minima), particularly in the right side [2]. Thus, in retrograde cardioplegia, the solution is shunted into the ventricular cavity without myocardial perfusion. Indeed, both experimental and human studies have shown that retrograde cardioplegia is associated with incomplete perfusion [4], depressed functional recovery [5, 6], and impaired preservation of energy metabolism [7, 29] in the right ventricle. At the tissue level, only mild to moderate and usually reversible ultrastructural changes have been detected in the myocardium [30]. However, the significance of retrograde versus antegrade cardioplegia in producing irreversible myocardial ischemia-reperfusion injury remains largely unknown. Our results clearly demonstrate that more cardiac myocytes are lost by apoptosis when retrograde cardioplegia is used alone, as compared with antegrade cardioplegia. Indeed, significantly more numerous apoptotic myocytes were found not only in the right, but also in the left ventricle. This implicates an incomplete distribution of cardioplegia in both ventricles and suggests that retrograde cardioplegia provides inferior protection in both ventricles as compared with the antegrade procedure. The significance of increased apoptosis of myocytes after cardiac surgery remains to be clarified in the clinical setting in terms of cardiac function, surgical complications, and long-term survival. In chronic cardiac diseases (eg, severe heart failure and myocarditis), a correlation has been shown between disease severity and apoptosis [31–33]. On the other hand, this type of correlation has not been confirmed in acute surgical ischemiareperfusion injury. However, in right atrial biopsies the release of cytochrome-C from mitochondria (involved in the induction of apoptosis) was shown to be associated with the severity of immediate postischemic dysfunction (stunning) in patients who underwent cardiac surgery [23]. Notably, the amount of cardiomyocyte apoptosis in our study was higher in the right than in the left ventricle, irrespective of the cardioplegia type used. This may indicate that right ventricular myocardium is more sensitive to ischemia-reperfusion injury. Since moderate hypothermia has been shown to reduce apoptosis, it may well be that warming of the right ventricle during the operation (due to its anterior location) predisposes it to apoptosis [15]. To conclude, the present study demonstrates that apoptosis is substantially increased after retrograde cardioplegia in this porcine model. Because of the common use of retrograde cardioplegia in the clinical routine, this finding may have important clinical implications. Clearly, more studies are needed to evaluate the importance of apoptotic cell death and the effect of antiapoptotic interventions on the clinical outcome after cardiopulmonary bypass surgery.
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INVITED COMMENTARY Apoptosis or program cell death has been previously reported to occur during cardioplegic arrest and cardiopulmonary bypass (CPB), suggesting that apoptosis may, at least in part, contribute to myocardial stunning. Vähäsilta and colleagues [1] confirmed these previous findings in their in-vivo pig model and further tested the hypothesis that differences from retrograde and antegrade cardioplegia might translate into differences in appearance of apoptosis. In this interesting and important study, the authors found that retrograde cardioplegia induced higher amount of apoptosis cardiomyocyte death than antegrade cardioplegia. We believe that this is the first report to show that retrograde cardioplegia is inferior to antegrade cardioplegia in inhibiting myocardial apoptosis in both the right and left ventricles. This finding has very important clinical implications because retrograde cardioplegia has been commonly used in the aortic root and aortic valve surgery. These data may partially explain why retrograde cardioplegia is often associated with incomplete perfusion, depressed functional recovery, and impaired preservation of energy metabolism in the right ventricle. However, there are still several limitations in this study. First, the authors used a relatively short period (30 minutes) of ischemic arrest induced by cold cardioplegia (10°C). They did not measure the myocardial temperature during the 30-minute ischemic arrest. Apoptosis is not only an ischemia-reperfusion event, but also time and temperature dependent. It has been demonstrated that mild or moderate hypothermia protects myocardium against myocardial dysfunction, necrosis, apoptosis, and apoptosis-related gene expression. Second, the authors used cold crystalloid cardioplegia with normothermic (36°C) CPB. In clinical practice, cold crystalloid cardioplegia is often combined with hypothermic CPB to protect ischemic myocardium. Third, the authors used only two histochemical markers to measure apoptosis. TUNEL
staining is known to be nonspecific, which has been recently commented by several authors in this journal and others. Measurement of activated caspase-3 with immunohistochemistry was very helpful in this study, but the authors used an unusual and complicated method to quantify activated caspase-3. Western blots for capase-3 cleavage, a pre-requirement for caspase-3 enzymatic activity, poly-(adenosine diphosphate-ribose) polymerase degradation, a major substrate for activated caspase-3, and cytoplasmic cytochrome c releases are excellent methods for detecting myocytes apoptosis. Because different phases of apoptosis may present different “faces of apoptosis,” multiple approaches will be very helpful to identify real apoptotic cells. Irrespective of these limitations, this is an important and relevant study because the changes in apoptosis may not only contribute to short-term functional deterioration, but more important may also contribute to the long-term beneficial effects. Thus, this may affect clinical practice, mainly by the prevention of myocardial apoptosis. Jun Feng, MD, PhD Frank W. Sellke, MD Division of Cardiothoracic Surgery Beth Israel Deaconess Medical Center 330 Brookline Ave Boston, MA 02215 e-mail:
[email protected];
[email protected]
Reference 1. Vähäsilta T, Saraste A, Kytö V, et al. Cardiomyocyte apoptosis after antegrade and retrograde cardioplegia. Ann Thorac Surg 2005;80:2229 –34.
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Cardiomyocyte Apoptosis After Antegrade and Retrograde Cardioplegia Tommi Vähäsilta, Antti Saraste, Ville Kytö, Markus Malmberg, Jan Kiss, Erkki Kentala, Markku Kallajoki and Timo Savunen Ann Thorac Surg 2005;80:2229-2234 DOI: 10.1016/j.athoracsur.2005.05.057 Updated Information & Services
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