Eur J Cardiothorac Surg 2004;25:801-806
© 2004 Elsevier Science NL
Significant damage of the conduction system during cardioplegic arrest is due to necrosis not apoptosis
Friedhelm Sayka,b*,
Stefan Krügerb,
J.F. Matthias Bechtela,
Alfred C. Fellerb,
Hans H. Sieversa,
Claus Bartelsa
a Clinic for Cardiovascular Surgery, University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany
b Institute of Pathology, University of Luebeck, Luebeck, Germany
Received 27 October 2003;
accepted 9 January 2004.
* Corresponding author. Tel.: +49-451-500-2331; fax: +49-451-500-2328
e-mail: friedhelm.sayk{at}innere1.uni-luebeck.de
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Abstract
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Objectives: Ventricular conduction disturbances following cardioplegic arrest remains a serious, yet unsolved problem. In the present study we examined whether myocardial conduction cells (MCC, Purkinje fibers) are more vulnerable to ischemia/reperfusion injury than working myocardial cells and whether the damage is due to necrosis or apoptosis. Methods: Mini-pigs were subjected to 60 min of crystalloid (St Thomas; n=15, group I) or blood (Buckberg; n=6, group II) cardioplegic arrest followed by 3 h of reperfusion. Animals not subjected to either procedures served as controls (n=5). Ventricular myocardial specimens were investigated by hematoxylin and eosin (HE) and periodic acid Schiff (PAS) staining and immunohistochemical labeling of apoptosis-associated proteins (Bax, Bcl-2, Caspase-3). DNA-breaks were visualized by in situ end labeling (terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling, TUNEL). Electron microscopy confirmed apoptosis or necrosis. Results: MCC of control hearts intrinsically expressed Bax, Bcl-2, and Caspase-3 without signs of either apoptotic or necrotic damage. Subendocardial Purkinje fibers of groups I and II hearts exhibited focal damage, with reduced labeling of apoptosis-associated proteins, glycogen loss, karyopycnosis and increased eosinophilia (15/21 hearts). The majority of damaged MCC displayed nuclear TUNEL-positivity (2.8±2.5% of MCC), whereas the average TUNEL-rate of the adjacent working myocardium was low (<0.1%). Electron microscopy demonstrated ischemic changes in MCC consistent with cellular necrosis. Conclusions: Ischemia/reperfusion injury due to cardioplegic arrest inflicts significant damage on subendocardial MCC, but not on working myocardium. Ultrastructural and light-microscopic findings are consistent with coagulation necrosis, rather than apoptosis.
Key Words: Purkinje fibers • Conduction system • Cardioplegia • Apoptosis • Oncosis • Necrosis
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1. Introduction
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Modern myocardial protection techniques have significantly improved clinical outcome after cardiac surgical procedures [1]. Cardioplegia, however, still poses a major problem. Current cardioplegic techniques appear to be more effective in preserving myocardial pump function rather than the electrophysiologic integrity of the heart. After both, blood and crystalloid cardioplegic arrest, transient or persistent rhythm and conduction disturbances represent the most frequent complication following cardiac surgery [2,3]. Fascicular blocks without any evidence of perioperative myocardial infarction are reported to predominate [2,3]. The mechanisms underlying postoperative conduction disturbances are poorly understood. Electrical arrest is reported to be incomplete in subendocardial conduction fibers (Purkinje fibers) during cardioplegic arrest [4]. The vulnerability of myocardial conduction cells (MCC) to ischemia/reperfusion injury is disputed [5,6].
Apoptosis and necrosis are two distinct, widely recognized modes of ischemic cardiomyocyte death [7–11]. Necrotic death with ischemic cell swelling is also termed oncosis [11]. Both mechanisms, apoptosis and oncosis, have been addressed in the context of cardioplegic arrest during open heart surgery [8,12–14] but not specifically in alterations of the conduction system. The differentiation between apoptosis and oncosis would implicate therapeutic consequences aiming to reduce the incidence of postoperative conduction disturbances, e.g. the addition of apoptosis inhibitors to the cardioplegic solution or modifications of the cardioplegic strategy.
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2. Materials and methods
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2.1. Study protocol
Conventional median sternotomy and cardiopulmonary bypass were performed on 26 mini-pigs (mean weight 29.4±12.7 kg) preanesthetized with intramuscular ketamine followed by continuous intravenous administration of propofol. The animals were intubated and volume-controlled mechanical ventilation was performed until cardiopulmonary bypass was instituted. Cardioplegic arrest was induced and maintained for 60 min under moderate systemic hypothermia (32 °C) by intermittent (20 min intervals) cold antegrade either crystalloid (St Thomas Hospital II; n=15) or blood-containing (Buckberg; n=7) hyperkalemic solutions (all solutions: Köhler Chemie, Alsbach-Hähnlein, Germany). After cross-clamp release and resumption of spontaneous myocardial activity animals were weaned from cardiopulmonary bypass followed by 180 min of reperfusion. Animals subjected to sternotomy alone served as controls (n=5). All animals received humane care in compliance with the European Convention and National Institutes of Health revised Guidelines for the Care and Use of Laboratory Animals being approved by the relevant institutional ethics committee. After termination hearts were immediately removed. Subendocardial specimens were harvested from the interventricular septum, the left ventricular free wall and the right ventricular septomarginal band, known to contain numerous MCC.
2.2. Histologic methods
For histological examination 5–10 tissue samples per animal were fixed in pH-buffered formalin and paraffin wax embedded (2–3 blocks per animal). Hematoxylin and eosin (HE), periodic acid Schiff (PAS, cytoplasmic glycogen) and alpha-naphthol-AS-D-chloracetate-esterase (ASD, presence of polymorphonuclear cells) staining was performed on serial sections according to standard protocols.
For immunohistochemical labeling of Bax, Bcl-2 and Caspase-3, serial paraffin sections were heat-pretreated and endogenous peroxidase activity was blocked with H2O2. The first antibody reaction was performed at room temperature for 60 min using rabbit polyclonal antibodies against Bax (1:200, Santa Cruz, USA) and Caspase-3 protein (1:20, Pharmingen, Germany) and a monoclonal antibody against Bcl-2 protein (1:50, Dako, Denmark), respectively. Slides were then exposed to biotinylated secondary antibodies and peroxidase-labeled streptavidin. Visualization was performed with diaminobenzidine followed by slight counterstaining with hematoxylin. For negative control, the primary antibody was replaced by a non-specific mouse or rabbit IgG. To further confirm the specificity of Bax staining, sections were incubated with an anti-Bax antibody, which had been preabsorbed by excess Bax-epitope oligopeptides (Santa Cruz). Porcine lymphoid (Bcl-2, Caspase-3) and cerebellar tissue (Purkinje-cells, Bax [15]) served as positive controls.
Capillary endothelium was visualized using biotinylated Dolichus biflorus agglutinin (Vector laboratories, USA) peroxidase-labeled streptavidin and diaminobenzidine.
The TUNEL assay (terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling; Boehringer Mannheim/Roche, Germany) was performed as previously described with some modifications [16]. In brief, paraffin sections were pretreated with proteinase K (20 µg/ml; Boehringer Mannheim/Roche, Germany), then incubated for 90 min at 37 °C with a TdT (50 U/ml)–biotinylated dUTP (0.01 µM/ml) containing cacodylate buffer. After washes in citrate buffer and phosphate-buffered saline (PBS), specimens were incubated with peroxidase-labeled streptavidin for 30 min at 37 °C and stained with diaminobenzidine and hematoxylin. All steps were performed in a humidified atmosphere. Lymphoid tissue and samples pretreated with DNAse I served as positive controls; for negative control either TdT or dUTP-16-biotin was omitted.
2.3. Quantification of cardiomyocyte damage
In each specimen the percentage of TUNEL-positive subendocardial MCC nuclei was determined by direct cell count. For quantification of working myocardial damage all TUNEL-positive working cardiomyocyte nuclei per specimen were counted. Cardiomyocyte density was calculated in at least 30 high-power microscopic fields per specimen using an ocular reticle and the result multiplied by the specimen's surface area [16]. Working myocardial TUNEL rates are expressed in percent (Table 1).
2.4. Transmission electron microscopy
For electron microscopy subendocardial tissue samples were immediately immersed in 6% phosphate-buffered glutaraldehyde for 24 h at 4 °C. After postfixation in phosphate-buffered OsO4, specimens were embedded in araldite. Areas of interest were selected from toluidine-blue-stained semithin sections. Ultrathin sections were mounted in slot grids and double stained in uranyl acetate and lead citrate. They were then examined with a Zeiss EM 109 (Carl Zeiss, Germany) transmission electron microscope at magnifications ranging from x1100 to x20,000. Cellular damage was evaluated according to the graduation scheme suggested by Axford-Gatley et al. [13].
2.5. Data analysis
Data are presented as mean±SE. Values from different cardioplegic groups were compared using the Mann–Whitney U-test. A P-value <0.05 was considered to indicate statistical significance. Multiple comparisons were accounted for using Bonferoni's method. All tests were two-sided and performed using SPSS for Windows (SPSS Inc., Chicago, Ill, USA).
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3. Results
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3.1. Surgical procedure
Application of cardioplegic solutions produced complete mechanical arrest in all pigs without resumption of activity during the infusion intervals. Postbypass myocardial function returned to preoperative values rapidly and remained adequate throughout the reperfusion period without the need for excess catecholamines except in one animal of the blood cardioplegic group which suffered from severe postpump syndrome with low systemic vascular resistance. Because the administration of high-dose catecholamines might enhance cardiomyocyte death, this animal was excluded from further analysis (blood n=6).
3.2. Histopathology (HE, PAS, ASD)
Focal Purkinje fiber damage was observed in 11/15 animals following crystalloid and 3/6 following blood cardioplegia. Foci of damaged MCC were found predominantly in subendocardial (rather than intramural) portions of both ventricles (LV: crystalloid 10/15, blood 1/6; RV: crystalloid 9/15, blood 3/6), demonstrating increased cytoplasmic eosinophilia, karyopycnosis, loss of sarcomeric cross-striation (HE staining), and depletion of glycogen (PAS)—features consistent with early coagulation necrosis (Figs. 1A and 2A)
. Accumulations of infiltrating polymorphonuclear cells in foci of damaged Purkinje fibers were disclosed in two hearts (ASD). In contrast, the adjacent working myocardium remained largely unaltered in light-microscopy. Controls showed no damage to either MCC or working myocardial cells.
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Fig. 1. Damaged myocardial conduction cells (MCC, asterisk) next to normal-appearing MCC (#) in the right-ventricular septomarginal band showing (A) features of coagulation necrosis (HE) as well as (B) nuclear TUNEL positivity (x600).
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Fig. 2. Serial sections with focal damage of subendocardial Purkinje fibers next to normal conduction myocytes showing (A) loss of glycogen (PAS), (B) reduced Bax labeling and (C) TUNEL positivity in the damaged myocytes (x200).
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3.3. Immunohistochemistry of apoptosis-associated proteins
In contrast to the working myocardium, MCC of controls displayed strong intrinsic expression of the apoptosis-promoting factor Bax, apoptosis-inhibitor Bcl-2 and the pro-enzyme Caspase-3. In damaged MCC, however, immunohistochemical labeling of these proteins was markedly reduced (Fig. 2B). The specificity of the immunohistochemical reaction was validated by adequate positive or negative staining in appropriate control tissues. No Bax signaling was detected in myocardial specimens after preabsorption of the antibody with Bax-epitope oligopeptides.
3.4. TUNEL
The mean number of subendocardial MCC nuclei investigated by the TUNEL assay was 800.9±377.5 per animal. The average TUNEL rate of subendocardial MCC nuclei was 2.8±2.5% (crystalloid 2.87±2.69%; blood 3.06±2.59%; P=0.682), comprising the vast majority of pycnotic nuclei observed in damaged Purkinje fibers in corresponding serial sections (Figs. 1B and 2C). Control hearts exhibited no TUNEL positivity in MCC (crystalloid or blood vs. control P<0.001). In contrast to MCC, the average morphometric TUNEL rate of the adjacent working myocardium was below 0.1% in the two cardioplegic groups and controls (Table 1).
3.5. Electron microscopy
Ultrastructural analysis of subendocardial tissue revealed varying degrees of ischemic changes in MCC following cardioplegic arrest ranging from normal to irreversible (Fig. 3)
[13]. Severe ischemic damage was characterized by extreme intracellular edema with irreversible mitochondrial changes, sarcoplasmic vacuolization, disintegration of myofilaments and focal disturbances of the integrity of the cytoplasmic membrane—features consistent with oncotic cell death. Nuclei were shrunken and stellate with condensation and margination of the chromatin into ill-defined clumps (Fig. 3A). In contrast, no apoptotic changes (e.g. chromatin margination into sharply delineated huge masses, budding, blebbing or apoptotic bodies) could be detected in any MCC. In the adjacent working myocardium only mild (reversible) mitochondrial changes were observed.
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Fig. 3. (A) Irreversible ischemic injury of a subendocardial conduction cell showing severe mitochondrial damage (extreme swelling with membrane rupture, fragmentation of cristae and electron-dense calcium deposits in the matrix), sarcoplasmic vacuolization, myofibrillar disorganization and stellate nucleus with chromatin clumping into ill-defined masses (x7000). (B) Normal conduction cell with well-preserved cytoplasmic and nuclear structures (x7000).
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3.6. Comment
It is crucial for the clinician to identify and prevent the events that underlie postoperative conduction disturbances. In the present study we found structural and histochemical evidence that MCC are highly vulnerable to ischemia/reperfusion injury associated with cardioplegic arrest. Irreversible subendocardial MCC damage was oncotic but not apoptotic. No differences regarding cell preservation were detected between the blood and crystalloid cardioplegic groups.
Oncotic myocyte changes after cardioplegic arrest and reperfusion have been repeatedly observed by other investigators in animal models and humans [5,13,14]. Schmitt et al. [12] recently suggested the occurrence of apoptosis in the postcardioplegic atrial myocardium due to evidence of mitochondrial cytochrome c release, which is known to be associated with early apoptosis [10]. Global ischemia and reperfusion are major injurious factors associated with cardioplegic arrest. Each produces a mixture of pro-apoptotic and necrosis-promoting signals. And because they are inducers of both pathways, there may well be some overlap between these two processes under cardioplegic conditions [8,10]. Discrimination between apoptotic and oncotic death is difficult, necessitating a combined methodological approach [7]. Currently, morphological findings are thought to provide the most reliable evidence of apoptosis [10].
In the present study, damaged MCC exhibited morphological features consistent with coagulation necrosis/oncosis rather than apoptosis, including cytoplasmatic eosinophilia, loss of sarcomeric cross-striation and karyopycnosis at the light-microscopic level. This interpretation was strengthened by the evidence of infiltrating polymorphonuclear cells in foci of damaged Purkinje fibers in two hearts, whereas in other hearts the reperfusion period of 3 h might have been too short for the distinct inflammatory demarcation of necrotic tissue [17].
In contrast to the working myocardium, MCC displayed strong intrinsic expression of the apoptosis-associated proteins Bax, Bcl-2 and Caspase-3. Following cardioplegic arrest with reperfusion, such immunohistochemical signaling was markedly reduced in damaged MCC. Denaturation and coagulation of proteinateous epitopes is an early event in the course of oncotic cell death, whereas proteins remain well preserved until the late stages of apoptosis [9,18]. The biological meaning of the intrinsic expression of Bax- and Bcl-2 documented not only in MCC but in other long-living cells with high ischemic vulnerability (e.g. cerebellar Purkinje cells) [15] remains to be clarified.
The specificity of light-microscopic findings is known to be limited [10]. In order to confirm oncosis or apoptosis, ultrastructural studies of subendocardial specimens were performed disclosing irreversible mitochondrial, sarcoplasmic, membranous and nuclear changes in damaged MCC distinctive of oncotic cell death (Fig. 3A), usually not found during apoptosis [10,18]. In addition, we observed no early or late apoptotic changes (e.g. apoptotic bodies), although a high proportion of TUNEL-positive nuclei were observed. The TUNEL assay, however, does not appear to be suited for distinguishing between apoptotic internucleosomal and non-specific (oncotic) DNA degradation [19], although it is extremely helpful for the quantification of irreversibly damaged cells [10].
MCC are characterized by their high glycogen content and high activity of those enzymes which favour anaerobic metabolism. This has led to the postulation that MCC are highly resistant to hypoxia [6]. In contrast to this assumption, we found a significantly higher DNA-fragmentation rate (TUNEL) in subendocardial conduction fibres than in working myocardium. Consistent with our findings, Schnabel et al. [5] demonstrated in the ischemic canine heart that cardioplegic solutions failed to sufficiently protect the conduction system as they did the working myocardium.
Several factors may account for the increased vulnerability and insufficient protection of subendocardial MCC: particularly in ungulates but also in humans, the size of MCC is considerably increased as compared to working cardiocytes [6]. Moreover, the capillary density is much lower in the subendocardial tissue as compared to the working myocardium (Fig. 4)
, thus hampering delivery of the protective cardioplegic agent. In addition, oxygen diffusion from the ventricular cavity, which might be crucial for subendocardial layers, subsides during cardioplegic arrest [5].
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Fig. 4. Myocardial conduction cells (MCC) in the subendocardium with low capillary density as compared to the adjacent working myocardium (WM) in a control heart (Dolichus biflorus agglutinin staining, x200).
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3.7. Limitations of the study
The morphologic findings of our study provide no evidence that apoptosis contributed to the postcardioplegic damage of the conduction system. However, biochemical assays (e.g. Northern or Western blot to confirm m-RNA or protein-levels of apoptosis associated factors, ‘DNA-laddering’, or assays for annexin V or cytochrome c release) are often considered helpful to further validate and quantify oncosis or apoptosis. In the present study damage on subendocardial Purkinje fibers occurred focally and thus, it was only microscopically detectable. Therefore, the selection of myocardial probes to perform whole tissue-lysate assays would have been unreliable and not suited to distinguishing and quantifying working myocardial or MCC damage. Counting of TUNEL-positive cardiomyocyte nuclei in large histologic samples in contrast enabled us to exactly quantify severe (irreversible) ischemic changes involving multiple DNA-breaks, leaving, however, the rate of mildly (reversibly) damaged MCC unknown.
We did not correlate our morphologic findings with the presence or absence of electrocardiographic abnormalities. Consistent with our findings, though, Cohen et al. [20], using sophisticated electrophysiological techniques in mini-pigs, reported significant slowing of ventricular activation due to derangements of electrical impulse propagation after cardioplegic arrest.
In conclusion, the present findings clearly indicate that subendocardial MCC are insufficiently protected during cardioplegic arrest, rendering Purkinje fibers much more vulnerable than working myocardium to ischemia/reperfusion injury associated with cardioplegic arrest. Furthermore, our data suggest that MCC damage is due to oncosis rather than apoptosis.
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Abstract
1. Introduction
