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(Chest. 2003;123:1647-1654.)
© 2003 American College of Chest Physicians

Continuous Tepid Blood Cardioplegia Can Preserve Coronary Endothelium and Ameliorate the Occurrence of Cardiomyocyte Apoptosis*

Chi-Hsiao Yeh, MD; Yao-Chang Wang, MD; Yi-Cheng Wu, MD; Jaw-Ji Chu, MD, FCCP and Pyng Jing Lin, MD, FCCP

* From the Division of Thoracic and Cardiovascular Surgery, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan.

Correspondence to: Pyng Jing Lin, MD, FCCP, Division of Thoracic and Cardiovascular Surgery, Chang Gung Memorial Hospital, 5 Fu-Hsing St, Kweishan, Taoyuan, Taiwan 333; e-mail: L0688{at}cgmh.org.tw


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objective: In modern cardiac surgery, crystalloid or blood cardioplegic solutions have been used widely for myocardial protection; however, ischemia does occur during protection with intermittent infusion of cold crystalloid or blood cardioplegic solutions. The present study was designed to evaluate the effect of different cardioplegic methods on myocardial apoptosis and coronary endothelial injury after global ischemia, cardiopulmonary bypass (CPB), and reperfusion in anesthetized open-chest dogs.

Methods: The dogs were classified into five groups to identify the injury of myocardium and coronary endothelium: group 1, normothermic CPB without cardiac arrest; group 2, hypothermic CPB with continuous tepid blood cardioplegia, and with cardiac arrest; group 3, hypothermic CPB with intermittent cold blood cardioplegia, and with cardiac arrest; group 4, hypothermic CPB with intermittent cold crystalloid cardioplegia, and with cardiac arrest; and group 5, sham-operated control group. During CPB, cardiac arrest was achieved with different cardioplegia solutions for 60 min, followed by reperfusion for 4 h before the myocardium and coronary arteries were harvested. Coronary arteries were harvested immediately and analyzed by scanning electron microscopy. Cardiomyocytic apoptosis was detected using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling, Western blot, and DNA ladder methods

Results: Regardless of the detection method used, significantly higher percentages of apoptotic cardiomyocytes were found in group 3 and group 4 than in other groups. Expression of caspase-3 correlated with increased apoptosis. Scanning electron microscopy revealed severe endothelial injury of coronary arteries in group 3 and group 4.

Conclusion: These results point to an important explanation for the difference in cardiac recovery after hypothermic ischemia and arrest with various cardioplegic solutions.

Key Words: apoptosis • cardioplegia • cardiopulmonary bypass


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ischemia and reperfusion may induce apoptosis in myocardial,1 2 cerebral,3 4 renal,5 and adrenal tissue.6 Apoptosis appears to be an active, tightly regulated, energy-requiring process under genetic control and tends to involve single cells that undergo cytoplasmic and nuclear condensation. Apoptosis of cardiomyocytes had been identified in the noninfarcted region as early as 3 h after coronary artery occlusion.7 Besides, apoptotic cells were found to be significantly more numerous in the border zones of infarcted tissue, where percentages as high as 5.1% per microscopic field were occasionally observed, which allowed considerable loss of cardiomyocytes in the vulnerable myocardial areas during the postinfarction recovery period.8 During cardiac surgery, cardiac global ischemia and reperfusion of the heart, potentially leading to apoptosis, may play a role in tissue damage and ventricular dysfunction. This process may be a precursor of heart failure.

Hyperkalemic crystalloid cardioplegic solutions have been widely used for myocardial protection in cardiac surgery, and while effective in inducing electromechanical arrest, they were only partially cardioprotective, and ventricular dysfunction has been observed.9 In the late 1970s and early 1980s, studies suggested that blood provided the best vehicle for delivery of cardioplegia in myocardium potentially injured by antecedent ischemia.10 The use of blood was based on its superior oxygen-carrying capacity, better osmotic properties and buffers, endogenous nutrients, and antioxidants compared with its crystalloid counterpart.11

The use of intermittent tepid blood cardioplegia for myocardial protection has become more popular.12 The heart is aerobically perfused and rested during tepid blood cardioplegia infusion, providing intervals during which myocardial resuscitation is believed to occur12 ; however, continuous blood cardioplegia infusion has become more and more popular,13 14 especially in those cases with prolonged myocardial ischemic time, because it provides better preservation of cardiac function than other methods when properly performed in selected cases.

The modified cardioplegic solutions and infusion methods have decreased the mortality and morbidity associated with cardiac surgery; however, the underlying pathophysiology of cardioplegia-associated ventricular dysfunction is complex and not fully understood, but it could be related, in part, to postoperative cardiomyocyte apoptosis. Accordingly, the present study was designed to compare the effect of continuous tepid blood cardioplegia, cold blood cardioplegia, and cold crystalloid cardioplegia on cardiomyocyte apoptosis.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Model of Global Cardiac Ischemia Using Cardiopulmonary Bypass
Healthy mongrel dogs (25 to 30 kg) of either sex were premedicated with ketamine (10 mg/kg IM), anesthetized with sodium pentobarbital (30 mg/kg IV injection), and then administered intermittent boluses of pentobarbital (5 mg/kg) and diazepam (5 mg) as needed during the experiment. Each dog was intubated with a cuffed endotracheal tube, and received ventilation with oxygen-enriched room air using a respirator. The left femoral artery and vein were catheterized for BP monitoring and fluid administration, respectively. The rectal temperature was monitored. After right thoracotomy, the pericardium was opened and tented to cradle the heart. The azygos vein was ligated, and umbilical tape snares were placed around the upper and lower caval veins. After heparinization (250 U/kg), the aorta was cannulated for aortic perfusion. The upper and lower caval veins were transatrially cannulated for venous return. The left ventricle was vented directly by a catheter inserted though the left atrial appendage. Cardiopulmonary bypass (CPB) was instituted by using a membranous oxygenator (Maxima Plus; Medtronic, Cardiopulmonary Division; Anaheim, CA) with a flow rate at 50 mL/kg/min. Dogs were placed on total CPB by occlusively snaring the vena cava. After stabilization, the ascending aorta was cross-clamped. A double-lumen aortic root cannula (DPL; Grand Rapids, MI) was inserted for antegrade delivery of cardioplegic solution and simultaneous measurement of infusion pressure. All infusions of cardioplegic solution were administered at approximately 40 to 60 mm Hg of pressure.

The ascending aorta was cross-clamped for 60 min. All animals received humane care in compliance with the 1985 revised guidelines of the National Institutes of Health for the care and use of laboratory animals.

Experimental Protocol
Dogs were randomly classified into five groups with six dogs in each group as follows: group 1, the body temperature during CPB was kept at approximately 37°C (cardioplegic solution was not used); group 2, the body temperature during CPB was kept at approximately 28°C (continuous tepid [28°C] blood cardioplegic solution was infused into the aortic root); group 3, the body temperature during CPB was kept at approximately 28°C (cold [4°C] blood cardioplegic solution was infused into the aortic root every 20 min); group 4, the body temperature during CPB was kept at approximately 28°C (cold [4°C] crystalloid cardioplegic solution was infused into the aortic root every 20 min); and group C, control group (sham operation). The heart was removed directly without extracorporeal circulation.

In groups 2, 3, and 4, cardioplegic solutions (10 mL/kg) were delivered for induction of cardiac arrest. The cardioplegic solution was reinfused (4 mL/kg) at 20-min intervals during global ischemia in group 3 and group 4. In group 2 and group 3, blood cardioplegia (Table 1 ) was delivered as a mixture of four parts oxygenated blood to one part crystalloid solution using a Sarns MP-4 cardioplegia delivery system (Sarns 3M Health Care Group; Ann Arbor, MI). In group 4, cold crystalloid cardioplegia (Plegisol; Abbott Laboratories; North Chicago, IL), with an electrolyte composition of calcium, 2.4 mEq/L; magnesium, 32 mEq/L; potassium, 16 mEq/L; sodium, 120 mEq/L; and chloride, 160 mEq/L was infused with systemic hypothermia (28°C). Initial induction of cardiac arrest was accomplished by using 20 mEq/L potassium blood cardioplegic solution and followed continuous infusion of 8 mEq/L potassium blood cardioplegic solution during the aortic cross-clamping period.


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  		Table 1.. Composition of Blood Cardioplegic Solutions*

 
After 60 min of aortic cross-clamping, the infusion of cardioplegia was stopped, the cross-clamp was removed, and systemic rewarming to 37°C was achieved. Then CPB was discontinued, and the mean arterial pressure was approximately 50 mm Hg. When ventricular fibrillation occurred, direct-current countershock of 10 W/s was applied. The hearts were reperfused for another 4 h in the working state before excision.

In Vitro Apoptosis Studies
Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling was performed following the method of Gavrieli et al.15 Paraffin sections were affixed to slides. Deparaffinization was performed by transferring the section to a xylene bath for 3 min three times, and then to a hydrogen peroxide bath for 3 min three times. The sections were incubated in 250 µL of proteinase K for 30 min at 37°C and immersed in terminal deoxynucleotidyl transferase (TdT) buffer (30 mmol/L Tris-HCl buffer, pH 7.2, 140 mmol/L sodium cacodylate, 1 mmol/L cobalt chloride). TdT (2.5 µL), biotin-deoxyuridine triphosphate (2.5 µL), and TdT buffer (45 µL) were added to cover the sections, which were incubated in a moist chamber at 37°C for 60 min. The reaction was terminated by transferring the slides to a buffer containing sodium chloride (300 mmol/L) and sodium citrate (30 mmol/L) for 15 min at room temperature; the sections were then covered with Avidin-FITC solution (Intergen; Burlington, MA) and stained with propidium iodide for 15 to 20 min at 4°C. After mounting with 90% glycerin and 10% phosphate-buffered saline solution, the specimens were examined by light microscopy. Labeled nuclei were easily identified from the unstained background.

Analysis of DNA Fragmentation
DNA isolation and gel electrophoresis were performed following the method reported by Facchinetti et al16 with some modification. In brief, the specimens were homogenized and lysed. After transferring 200 µL of the sample lysate to a microcentrifugal tube, extraction matrix and extraction buffer were added to the samples, which were centrifuged at 12,000g for 5 min. The volume of the aqueous phase sample transferred was estimated. After adding 0.1 volume of sodium acetate and an equal volume of isopropanol to the aqueous phase, the sample was centrifuged at 12,000g for 10 min. All samples were prepared to a volume of 20 µL that contained 20 µL of DNA and subsequently incubated with 0.1 mg/mL deoxyribonuclease-free ribonuclease (Sigma Chemical; St. Louis, MO) at 37°C for 30 min. Nuclear DNA was then solubilized in Tris-ethylenediaminetetraacetic acid (EDTA) buffer, and DNA content was quantified by spectrophotometry at 260 nm. A 4 µL volume of 0.25% bromphenol blue and 0.25% xylene cyano in 40% sucrose were added to samples at a 1:5 (volume/volume) ratio. Electrophoresis was performed at 50 V in 2% agarose gels. DNA was visualized with ethidium bromide.

Western Blot Studies
Caspase 3 has been shown to be activated in apoptotic cells and cleaves several cellular proteins, including poly-(adenosine diphosphate-ribose) polymerase (PARP) protein, the cleavage of which is a hallmark of apoptosis. Tissue levels of caspase-3 protein and PARP were determined by Western blots. Ten percent (weight/volume) tissue homogenate was prepared in the sucrose-Tris-EDTA buffer (0.32 mol/L sucrose, 5 mmol/L Tris, 2 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 5 mmol/L benzamidine and 20 µg/mL leupeptin), then centrifuged at 4°C and 14,000g for 10 min. Eighty micrograms of protein from the supernatant was separated on a 12.5% polyacrylamide denaturing gel and then electroblotted onto Hybond-C nitrocellulose membranes (Amersham Life Science; Little Chalfont, UK). Membranes were blocked by the addition of 3% bovine serum albumin in 0.1% Tween 20 Tris buffered saline solution (TTBS) at 4°C overnight before being probed with a polyclonal anti–caspase-3 and anti-PARP antibodies (PharMingen; San Diego, CA) at 1:1,000 dilution in TTBS buffer at room temperature for 2 h. Primary antibody binding was revealed using an anti-rabbit peroxidase conjugate (Dako; Carpinteria, CA) diluted at 1:2,000 in TTBS buffer for 1 h and the ECL Chemiluminescent Detection System (Amersham Life Science). Recombinant caspase-3 and PARP (PharMingen) was used to verify antibody efficacy under experimental conditions. The membranes were incubated for 1 min with ECL Detection Solution, and the levels were determined using an ECL Detection Kit (Amersham Pharmacia Biotech; Piscataway, NJ). The immunoreactive bands were quantified by digital densitometric imaging (Gel Doc 1000 with Model GS-700 Densitometer and Molecular Analyst Software; BioRad; Hercules, CA).

Scanning Electron Microscopic Studies
Specimens were obtained from myocardium and coronary arteries and used for scanning electron microscopic studies.17 18 Two dogs in each group were used for electron microscopic studies. The coronary endothelium was fixed in situ at physiologic pressure with buffered physiologic solution (pH 7.4) for 5 min of the following composition: KCl, 2.7 mmol/L; NaCl, 137.9 mmol/L; Na2HPO4, 8.1 mmol/L; and KH2PO4, 1.1 mmol/L. Glutaraldehyde (1%) in buffered physiologic solution was then infused for 10 min. Segments (2 cm in length) of left anterior descending and/or left circumflex coronary arteries were carefully harvested, kept in this iced perfusion-fixation solution, and sent for scanning electron microscopic processing. The specimens were fixed with iced 3% glutaraldehyde in 0.1 mol/L cacodylate buffer (pH 7.2 to 7.4) for 2 h. Subsequently, the specimens were rinsed with cold perfusion-fixation solution several times, and postfixed with 1% phosphate buffered osmium tetroxide (pH 7.2 to 7.4) for an additional 2 h. For electroconduction and stabilization of the surface structure, the tissues were immersed in 1% tannic acid in distilled water for 30 min at 4°C, and then transferred into 1% osmium tetroxide in distilled water for 30 min at 4°C, followed by a rinse with distilled water. The tissues were then dehydrated in graded concentrations of chilled ethanol. Then the specimens were subjected to critical point drying. After drying, samples were mounted on specimen stubs and coated with a 4-nm thickness of platinum and palladium alloy. The specimens were examined with a Hitachi S-5000 scanning electron microscope (Hitachi; Tokyo, Japan) operated at 3 kilovolts.

Scanning electron microscopy involved analysis of the luminal surface of each vessel segment. Endothelial injury was defined as presence of endothelial cell distortion or detachment, the presence of fibrin or platelets, and the attachment of leukocytes to the vascular surface.17

Data Analysis
The data were expressed as means ± SEM. Data were entered into Excel (Microsoft; Redmond, WA) and analyzed by the Statistical Package for the Social Science, version 8.0 (SPSS; Chicago, IL). A p value < 0.05 was considered statistically significant. One-way analysis of variance followed by the Tukey multiple comparison procedure was employed to compare the differences among various groups.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of Apoptosis in Myocardium by the Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate-Biotin Nick End Labeling Method
Cardiomyocyte nuclei with nicked DNA were present in all groups. Four sections (5 µm in thickness) of the left ventricle in each animal were examined to determine the percentage of apoptotic nuclei. To quantify apoptosis, 500 nuclei were identified in 10 randomly selected x 400 high-power fields (HPFs) per section. Apoptotic cell counts were expressed as a percentage of the total number of nuclei counted. In the sham operation group, the cardiomyocyte nuclei were rarely apoptotic (0.99 ± 0.26% per HPF), which was significantly lower than this percentage in the other groups (p < 0.01 for all four groups). The percentages of positive cardiomyocyte nuclei in groups 2, 3, and 4 were 3.07 ± 0.91 per HPF, 4.12 ± 0.73 per HPF, and 9.74 ± 0.26 per HPF, respectively. The percentages of nicked nuclei in group 3 and group 4 were significantly higher than that in group 1 (2.39 ± 0.60 per HPF, p < 0.001 and p < 0.001, respectively) and group 2 (p < 0.001 and p = 0.01, respectively). In group 2, the higher percentage of degraded nuclei compared with that of group 1 was insignificant (p = 0.06) [Fig 1 ].



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  		Figure 1.. Percentage of apoptotic cardiomyocytes in different groups. *p < 0.01 for group 3 vs group 1 and group 2; #p < 0.01 for group 4 vs group 1 and group 2; *p < 0.01 for control group vs the other four groups.

 
Detection of DNA Fragmentation
DNA cleavage into nucleosome-sized fragments is a hallmark of apoptosis and results from the caspase-induced activation of a specific deoxyribonuclease.19 Cardiomyocytes of groups 1, 2, and 5 exhibited a low level of DNA fragmentation. However, DNA from cardiomyocytes in group 3 and group 4 exhibited a clear nucleosomal ladder (Fig 2 ).



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  		Figure 2.. DNA laddering in various experimental groups. Lane 1, group 1; lane 2, group 2; lane 3, group 3; lane 4, group 4; lane m, marker; and lane c, control group. Obvious DNA ladders (arrows) were seen in the cardiomyocytes of group 3 and group 4. bp = base-pair.

 
Detection of Caspase-3 Activation by Western Blot
Caspase-3 plays a role as a key protease of the apoptotic machinery. In addition, we examined whether global cardiac ischemia/reperfusion actually induced caspase-3 cleavage as a prerequirement for caspase-3 enzymatic activity. As PARP protein is known to be a major substrate for active caspase-3 and PARP degradation (a hallmark of apoptosis), Western blot analysis for PARP protein was carried out. The intact 113-kd PARP was degraded, as shown by the increasing quantity of 24-kd cleavage product detected using N-terminal binding antibody (Fig 3 ). Using densitometry analyses, we found that although the activated caspase-3 in four experimental groups was significantly higher than that in group C (445 ± 136, p < 0.05), the activated caspase-3 levels in group 3 (934 ± 172) and group 4 (1048 ± 185) were even higher than in group 1 (682 ± 164, p < 0.05 and p < 0.05, respectively) and group 2 (679 ± 121, p < 0.05 and p < 0.01, respectively) [Fig 3 , center, B].



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  		Figure 3.. Western blot analysis of caspase-3 and PARP protein. Representative blot showing the levels of caspase-3 and PARP protein in different groups (Gr). The proform and major subunit of caspase-3 (top, A) and PARP proteins (bottom, C) are indicated by arrows (center, B). The protein bands were quantified by densitometry, the data were normalized to the mean control values, and the results (mean differences) were analyzed by analysis of variance. *Statistically significant increase (p < 0.05) in activated caspase-3 in group 3 and group 4 from other groups. GrC = control group.

 
Scanning Electron Microscopic Findings
Scanning electron microscopic studies were carried out with coronary arteries from each group except the sham operation group. Representative micrographs are presented in Figure 4 . As illustrated in Figure 4 , top left, A, the endothelial cells of group 1 were uniform in size, oriented with the long axis parallel to the direction of blood flow, and had prominent marginal folds at the intercellular junctions. The basic pattern remained essentially unaltered in group 2 (Fig 4 , top right, B). However, after 1 h of cold ischemia with intermittent cardioplegia infusion, with or without blood, the pattern of endothelial cell damage (denudation of the endothelial cells, exposure of the subendothelium, and platelet adhesion to the rough surface) could be found (Fig 4 , bottom left, C, and bottom right, D).



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  		Figure 4.. Scanning electron micrographs representative of coronary endothelium under various experimental conditions. Top left, A: group 1, the cells of the coronary endothelium are uniformly oriented in the direction of blood flow and prominent marginal folds are apparent (x 500 before 25% reduction). Top right, B: group 2, the coronary endothelium is well preserved without obvious damage. Bottom left, C: group 3, coronary endothelial denudation was noted occasionally and the intercellular folds faded out. Bottom right, D: group 4, coronary endothelium denudation is often found. Platelet adhesion (arrow) to the exposed subendothelium is also noted.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The use of cold, hyperkalemic cardioplegia for myocardial protection during cardiac surgery has proved to be a critical component in the development of modern cardiovascular surgery.20 During cardiac operations, myocardial preservation has remained the major concern of cardiac surgeons. However, coronary endothelial and myocardial dysfunction still occur after CPB and ischemic arrest. Our previous studies have found that endothelial responses are altered after exposure of cardiac and coronary tissue to cold crystalloid cardioplegia,18 21 which can be attenuated by warm blood cardioplegia.17 Other investigators have also provided evidence that coronary vascular22 and microvascular23 function is altered after the application of cardioplegic solutions. Few studies have ever examined the effect of cardiac arrest and cardioplegia on the cardiomyocytes, which are the fundamental units of cardiac performance.

In the present study, we have extended our understanding of the pathogenesis of endothelial cell damage and cardiomyocyte injury by examining ischemic coronary endothelium and myocardium protected with various kinds of cardioplegia. The major findings of this study are as follows: (1) continuous tepid blood cardioplegia per se causes little damage to the coronary endothelium and cardiomyocytes; (2) during hypothermic ischemia, both cold blood and crystalloid cardioplegic arrest injure coronary endothelium; (3) cardiomyocyte apoptosis did not increase during continuous tepid blood cardioplegic arrest; and (4) continued tepid blood cardioplegia attenuated the occurrence of endothelial injury during hypothermic cardiac arrest.

The composition and delivery methods of cardioplegic solutions have undergone extensive investigation to minimize myocardial dysfunction.24 In the evolution of cardioplegic myocardial protection, intermittent cold crystalloid cardioplegia had been replaced by intermittent cold blood cardioplegia in routine cardiac surgery.25 At infusion temperatures of >= 20°C or with reperfusion of acutely ischemic myocardium, blood cardioplegia appears to provide superior results.26 Progressive hypothermia shifts the oxygen-hemoglobin dissociation curve to the left, which reduces release of oxygen from blood cardioplegic solutions. Theoretically, blood cardioplegia had several advantages over the crystalloid cardioplegia: (1) a reduction in systemic hemodilution, (2) improved osmosity and buffering because of the presence of blood proteins, (3) rheologic benefits on the microvasculature, (4) oxygen-derived free radical scavenging resulting from superoxide dismutase and catalase, and (5) improved oxygen-carrying capacity.24 However, the cardiac protection of continuous tepid blood cardioplegia was better than that of intermittent cold blood cardioplegia during complex cardiac surgery with prolonged myocardial ischemic duration.27 28 29 30 Increasing experimental and clinical evidence reveals that the more prolonged cross-clamping duration is, the more myocardial protection is afforded by continuous tepid blood cardioplegia.27 Most investigators focused on the effects of various kinds of cardioplegia on the coronary endothelial function and myocardial performance. The impact of various kinds of cardioplegia on the cardiomyocytes had never been studied. In the current study, we have examined the effect of various kinds of cardioplegia solutions on the cardiomyocyte apoptosis. The percentage of apoptotic cardiomyocytes and the level of activated caspase-3 were significantly lower in the sham-operated group than in the other four groups, which implies that CPB per se with/without cardiac arrest had some negative impact on cardiomyocytes and induced myocardial apoptosis. However, the number of apoptotic cardiomyocytes and level of activated caspase-3 in the tepid blood cardioplegia and the CPB-only groups were similar, suggesting that continuous tepid blood cardioplegia could minimize injury due to cardiac arrest and preserve cardiomyocytes. Furthermore, the number of apoptotic cardiomyocytes was higher in the intermittent cold crystalloid and cold blood cardioplegia groups than in the continuous tepid blood cardioplegia group, which could explain why the latter group had better recovery than the former groups.

Another important finding of this study is that various cardioplegic solutions injured coronary endothelial cells. The scanning electron microscopic examination showed that the integrity of coronary endothelium was better preserved in the recipients of continuous tepid blood cardioplegia and without cardioplegic cardiac arrest than in those receiving either intermittent cold crystalloid or blood cardioplegia. Previous studies had revealed that intermittent infusion of the cold crystalloid or blood cardioplegia solutions induced endothelium-dependent coronary response impairment,18 21 22 25 26 coronary microvascular relaxation injuries,23 31 and histologic damage.22 32 Most studies used transmission electron microscopy to examine the integrity of the coronary endothelium. In this study, scanning electron microscopy was used to provide another view of the injury of the coronary endothelium under various cardioplegic cardiac ischemia and reperfusion conditions. The morphologic integrity of the coronary endothelium was well preserved in the groups without cardioplegia or cardiac ischemia and continuous tepid blood cardioplegia infusion. However, significant endothelial denudation and platelet adhesion were observed in the groups with intermittent cold crystalloid and blood cardioplegia infusion. Several causes of endothelial damage during cardioplegic cardiac arrest, such as high shear stress and pressure,33 neutrophil-mediated pathologic events, and oxygen-derived free radicals34 have been proposed. Nakanishi and associates22 suggested that endothelial dysfunction observed after reperfusion might be a specific event of reperfusion injury. Several potential mechanisms by which continuous tepid blood cardioplegia infusion during cardiac arrest preserves coronary endothelium integrity have been suggested. There is a possibility that continuous antegrade tepid blood cardioplegia provided adequate nutrients and oxygen, washed out all the metabolic waste, and impeded the occurrence of ischemia and reperfusion injury. Although deleterious effects of extracorporeal circulation on the coronary endothelium could not be ruled out in the present study, nearly complete preservation of the morphologic integrity of the coronary endothelium was obtained in the CPB-only and continuous tepid blood cardioplegia groups. These data suggest that in cases of CPB without cardioplegic cardiac arrest and with continuous antegrade tepid cardioplegia infusion, the morphologic integrity of the coronary endothelium is preserved, but when intermittent antegrade cold crystalloid and blood is infused, morphologic damage to the coronary endothelium is induced.

In summary, the present study demonstrated that antegrade cold crystalloid and blood cardioplegia infusion during hypothermic CPB impaired the morphologic integrity of the coronary endothelium and induced cardiomyocyte apoptosis. However, continuous antegrade tepid blood cardioplegia infusion could prevent cardiomyocyte apoptosis and preserve the integrity of the coronary endothelium well.


   Footnotes
 
Abbreviations: CPB = cardiopulmonary bypass; EDTA = ethylenediaminetetraacetic acid; HPF = high-power field; PARP = poly-(adenosine diphosphate-ribose) polymerase protein; TdT = terminal deoxynucleotidyl transferase; TTBS = Tween 20 Tris buffered saline solution

This research was supported by grant NSC87–2314 from the National Science Council of the Republic of China.

Received for publication June 17, 2002. Accepted for publication October 16, 2002.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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