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Ann Thorac Surg 2005;79:1970-1975
© 2005 The Society of Thoracic Surgeons
a Department of Cardiovascular Surgery, Istanbul, Turkey
b Department of Pathology, Marmara University School of Medicine, Istanbul, Turkey
Accepted for publication December 20, 2004.
* Address reprint requests to Dr Ak, Marmara Universitesi Hastanesi, Kalp Damar Cerrahisi AD, Tophanelioglu cad. No: 13-15, 81190 Altunizade/Istanbul, Turkey (E-mail: akkoray{at}hotmail.com).
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Abstract |
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METHODS: One hundred elective coronary artery bypass grafting patients were enrolled into the study. Right atrial tissue from all patients was sampled before cardiopulmonary bypass. Patients were monitored for the occurrence of the postoperative atrial fibrillation. Right atrial tissue samples from the atrial fibrillation group were compared with samples belonging to the patients who remained in sinus rhythm postoperatively. Evaluation for atrial histopathology and myocyte apoptosis included light microscopic and immunohistochemical studies.
RESULTS: Fourteen of 100 patients (14%) developed postoperative atrial fibrillation. On univariate analysis, the only predictor for the development of postoperative atrial fibrillation was chronic obstructive pulmonary disease (p = 0.014). Histologically, larger sized myolytic vacuoles were more common in patients who developed postoperative atrial fibrillation (p = 0.001). The percentage of apoptotic myocytes in each specimen was significantly higher in patients with atrial fibrillation (p = 0.000). Most of the specimens with positive apoptotic staining were also severely myolytic in patients with postoperative atrial fibrillation.
CONCLUSIONS: Our results suggest that degree of myolysis and increased apoptotic pattern in right atrial myocardium are significant predictors for the development of postoperative atrial fibrillation. The improvement of preoperative metabolic status of the myocardial cells may reduce the incidence of this common complication.
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Introduction |
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Until now, several risk factors have been reported for the development of postoperative AF. The most important of which are chronic obstructive pulmonary disease (COPD), advanced age, postoperative withdrawal of beta-blockers, prolonged preoperative P-wave duration, cardiopulmonary bypass (CPB), postoperative catecholamine discharge, and inadequate protection of right atrial tissue during aortic cross clamping [3–8]. However, it is difficult to explain why some patients develop AF while others having the same risk factors do not.
The exact mechanism for the postoperative AF is still unknown. There is an accumulating data that the electrophysiological mechanism behind the development of postoperative AF is in dispersion of atrial refractoriness between adjacent atrial areas that results in re-entry [3]. Current dogma suggests that re-entrant arrhythmias are predisposed by slow conduction, rapid refractoriness, and anatomic heterogeneity (either micro or macro) in the atrial tissue. It has been shown that certain pathologies like atrial ischemia, fibrosis, myolysis, myocarditis, myocyte vacuolation, and atrophy cause microscopic heterogeneity within the atrial myocardium and eventually result in atrial arrhythmias [8–12].
The purpose of this study was to relate the magnitude of right atrial appendage histopathologic alterations (such as fibrosis, vacuolation, atrophy, inflammation, and the like) and right atrial appendage myocyte apoptosis to post-coronary artery bypass grafting (CABG) AF.
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Material and Methods |
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The age of the patients ranged from 39 to 69 years old (mean, 62.1 ± 12.3 years). Seventy-two percent of the patients (72 patients) were male. In all patients, preoperative cardiac function was evaluated by transthoracic echocardiography (TTE) and left ventricular catheterization. Both right and left atrial dimensions, volumes, atrioventricular valve functions and pulmonary artery pressures were calculated routinely in all patients. Exclusion criteria for the study were as follows: any known or documented history of atrial or ventricular arrhythmias; acute myocardial infarction requiring surgical intervention; any surgical procedures concomitant to CABG except carotid artery endarterectomy. All patients were monitored continuously for the first 72 hours after CABG and then were followed up with 2 times daily electrocardiogram (ECG) strips until discharge. Preoperative medication of all patients is shown in Table 1. Perioperative prophylaxis of atrial arrhythmias is not used routinely in our institution. Postoperative beta-blocker therapy was only recommenced in patients with preoperative beta-blocker usage to prevent sudden beta-blocker withdrawal phenomenon.
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Histopathologic Evaluation
A total of 100 right atrial tissue samples were collected. Right atrial tissue samples of the patients were grouped into two. Group 1 (n = 86 patients) was the control group and consisted of tissue samples of the patients with NSR postoperatively. Group 2 (n = 14) was the study group and consisted of tissue samples of all patients with postoperative AF. Right atrial tissues were embedded in paraffin and 4-µm cut sections were obtained from each specimen. Sections were stained with hematoxylin and eosin (H&E), Masson Goldner trichrom, periodic acid shiff (PAS), and Congo red staining and were assessed under light microscopy.
Myocyte apoptosis was studied in all samples (n = 14) from group 2 and selected samples (n = 20) with varying degrees of myolysis and fibrosis from group 1. Apoptosis was examined immunohistochemically by Tdt-mediated dUTP digoxigenin nick end labeling (TUNEL) assay using a commercial kit (ApopTag Peroxidase in situ Apoptosis Detection Kit, Oncor-intergen, S7100) according to the manufacturer’s instructions. Counter staining was done using mayer hematoxylin. For positive control, lymph node tissue was used. Both negative and positive staining was done concomitantly.
For histopathologic examination, we used the modified version of the chart protocol created by Ad and colleagues [11]. A pathologist who was blinded as to which patients developed postoperative AF did a thorough examination of myocytes and connective tissue and filled out the chart. The pathologist evaluated the degree of myolysis (average size of vacuoles and frequency of vacuolation), the existence of hypertrophy, atrophy, lipofuscin pigment, and presence of apoptotic figures or necrosis in atrial myocytes. Myolysis was defined as at least 10% loss of muscle striation and replacement of contractile material by cytoplasmic vacuoles in atrial myocytes. Apoptotic nuclei were quantitatively evaluated by immunohistochemical examination. For quantification of the number of apoptotic figures, both apoptotic and total cells were counted in 10 different fields at a magnification of 400 times and the ratio of the cells stained positively with ApopTag to the total number of the cells was expressed as a percentage.
In the connective tissue; interstitial edema, mononuclear exudates, fibrosis, presence of amyloid material, fibroelastosis, and arteriolar hypertrophy were examined and graded from mild to severe semiquantitatively (grade 0 = absent; grade 1 = mild; grade 2 = moderate; and grade 3 = severe) as described by Ad and colleagues [11]. In contrast to Ad and colleagues, we did not examine the pericardial tissue.
Statistical Analysis
Statistical analysis was performed using SPSS 10.0 for Windows (SPSS, Chicago, IL). Although continuous independent variables were expressed as mean ± standard deviation (SD), categorical variables were expressed as frequency or percentage. The association between multiple clinical variables and the occurrence of postoperative AF was assessed by logistic regression test. Statistically significant univariate clinical predictors were entered into a multivariate analysis and the correlation was assessed using two-tailed Pearson correlation test. The comparison of histopathologic variables between group 1 and group 2 was assessed using chi-square and unpaired t tests. A p value less than 0.05 was considered significant.
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Results |
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In group 2, average percentage of the apoptotic nuclei in each specimen was significantly higher than group 1 (76.2% ± 22.4% versus 36.4% ± 27.3%, p = 0.000; Fig 1C). Most of the specimens with high frequency of apoptotic nuclei in group 2 were also severely myolytic, as illustrated in Figure 1D (7 of 10 patients with high frequency of apoptotic nuclei were severely myolytic).
Mild to moderate fibrosis was detected in 29 patients (33.7 %) in group 1 and 6 patients (42.8%) in group 2 (p = 0.417). Severe fibrosis was detected in only 1 patient (1.16%) in group 1.
In the connective tissue and interstitium, several histopathologic abnormalities were encountered in both groups 1 and 2, but none of these created a statistically significant difference between the groups. Edema was seen in 32 patients of group 1 (21 were mild and 11 were moderate) and 6 patients of group 2 (5 were mild and 1 was moderate). Mononuclear exudate was detected in 10 patients of group 1 and 2 patients of group 2, but none of these cases were graded as severe. Amyloid was not detected in any of the cases. Arteriolar hypertrophy was detected in 16 patients of group 1 and 5 patients of group 2.
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Comment |
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Until recently, several risk factors have been documented for the occurrence of postoperative AF in cardiac surgery. Age is the most commonly identified risk factor [7]. In the atrial myocardium, atrophy, fibrosis, decreased conduction tissue, and intracellular lipofuscin pigment storage occur as a normal consequence of aging process [14, 15]. It has been known that these age-related myocardial pathologic alterations, particularly fibrosis, constitute a substrate for the development of postoperative AF [16]. In the present study, we found important fibrosis in a significant portion of the patients in both groups (34.8% in group 1 and 42.8% in group 2), but fibrosis was not associated with the development of postoperative AF. In a study by Boldt and colleagues [17], the expression of extracellular matrix proteins (ECM) such as collagen I, collagen III, and fibronectin were studied in the left atrial tissues of patients in NSR, lone AF, and AF with underlying mitral valve disease. In this study, despite the fact that fibrosis was significantly increased in all patients with AF, collagen III expression was found to be different in each form of AF. Even though the fibrosis was not significantly increased in patients with postoperative AF in our study, it would be better to study different ECM protein expressions in the atrial tissue of these patients. Also, there was no correlation between the chronological age of the patients and each of fibrosis, atrophy or lipofuscin pigment.
In our study, the most probable explanation for low incidence of postoperative AF (14%) would be related to the relatively younger age of our patients (61.2 ± 12.3 years). Also, some patients with transient postoperative AF attacks might be missed due to the discontinuous ECG monitoring after postoperative day 3.
One of the most striking results of our study was the increased vacuolation size and frequency in patients with postoperative AF. It has been shown that myocardial vacuolation occurs either as a consequence of the normal aging process or in response to exposure to hypoxic or toxic stimuli in cardiac cells [14, 18, 19]. In animal models it has been demonstrated that atrial myolysis induced by chronic AF is an important structural alteration for the maintenance of AF [20]. In two previous studies Ad and colleagues [10, 11] found myolysis as the most important predictor for postoperative AF. They reported that the degree of myolysis and associated vacuolation in atrial myocytes reflect chronic metabolic derangement of the atrial cells and are major preoperative histopathologic determinants for the development of postoperative AF. Although all the patients in our study were in sinus rhythm and had no detected P-wave abnormality preoperatively, some of the atria revealed a possible arrhythmogenic substrate (increased vacuolation size and frequency) for the development of postoperative AF. This supports the result that the presence of such an arrhythmogenic substrate does not affect the basal rhythm, but when exposed to a triggering event such as CPB, these patients are at higher risk for postoperative atrial tachyarrhythmia.
Results obtained from both experimental and clinical studies suggest that abnormal electrical coupling between cardiomyocytes through gap junction proteins (especially connexins 40 and 43) is considered an important factor in the pathophysiology of chronic atrial fibrillation [21, 22]. Nao and colleagues [22] reported that both the expression and distribution of the gap junction protein "connexin 40" are significantly altered in the atrial appendage of patients with chronic AF. In the same study, they claimed that abnormal cell-to-cell communication caused by altered connexin 40 expression in the right atrial tissue would seem to play a role in the initiation and/or perpetuation of AF.
Apoptosis is defined as an energy-dependent form of cell death and characterized by distinct phases of ultrastructural morphologic features such as nuclear chromatin condensation, cellular shrinking, breakup of the nucleus, cellular budding, and formation of apoptotic bodies [23]. It has been demonstrated that cardiac myocytes undergo apoptosis in response to hypoxia, ischemia-reperfusion, serum and nutrient deprivation, and metabolic inhibition [23, 24]. In a canine heart failure model, Heinke and colleagues [25] reported that myocardial stress induced by rapid pacing results in a significant increase in atrial and ventricular myocytes undergoing apoptosis. Aime-Sempe and colleagues [26] also reported an association between myolytic vacuoles resulting from AF and atrial apoptotic figures; the mechanism of the death in myocytes with myolysis was shown to be through apoptotic cell death. In our study, increased right atrial myocardial apoptosis was found to be a potential marker of susceptibility to postoperative AF. Also, most of the samples with high percentage of apoptotic nuclei were also severely myolytic. Although myolysis was detected in most of the specimens in both groups, positive apoptotic staining was strictly restricted to the cases with severe myolysis and vacuolation. This suggests that a number of myocytes with severe myolysis may activate apoptotic cell death. Furthermore, Aime-Sempe and colleagues [26] also found that the cytosolic areas depleted of myofibrillar structure (myolytic vacuoles) in their patients were filled with glycogen. In our study, these spaces were not stained with PAS and did not seem to be filled with glycogen. This could be explained in two ways. The vacuoles in our study were filled with a cellular material other than glycogen or the glycogen material was dissolved in formalin solution during the preparation phase of the specimens and became invisible.
In the literature, studies questioning postoperative AF and atrial myocyte apoptosis are quite limited. In contrast to our results, Ad and colleagues [11] did not indicate any correlation between perinuclear vacuolation and apoptosis in the atrial specimens taken before and after CPB. In spite of similar patient characteristics, it is difficult to explain this great discrepancy between these two studies. It is crucial that right atrial specimens should be fixed in formalin solution immediately after excision. Also, the time interval between the fixation and paraffin embedding should be as short as possible to save pathologic alterations, especially apoptosis. In the study by Ad and colleagues [11], the unexpectedly lower number of the TUNEL-positive nuclei (1 positive nucleus in 9 of 120 slides, and 2 positive nuclei in only 1 slide) may be related to their methodology regarding tissue sampling and preparation. In the study by Aime-Sempe and colleagues [26], marked caspase-3 staining, a well-known marker of apoptosis, was observed in 6 of 36 patients with NSR.
In the present study, we examined the preoperative status of the atria morphologically. Our results suggest that preoperative morphologic alterations like atrial myocardial vacuolation and increased myocardial apoptosis may constitute a pathologic substrate for postoperative AF. These morphologic alterations may be related to the preoperative metabolic status of the atria. We think that severe myolysis (or severe vacuolation) and apoptosis are inter-related and have a "cause and effect" partnership, especially in atrial cells with chronic metabolic derangement.
Our findings correlated with the beneficial results of recent studies about the use of glucose-insulin-potassium (GIK) solution in metabolically depleted hearts and diabetic CABG patients. Lazar and colleagues [27, 28] concluded that perioperative substrate enhancement with GIK solutions decreases the incidence of AF and enhances myocardial performance postoperatively in diabetic CABG patients. Our study has some evident limitations. First, the number of the patients in the study is limited and the TUNEL staining for apoptosis was performed only in selected NSR patients (20 of 86 patients). Increasing the number of the patients studied would lead to the creation of a more accurate data analysis. Second, we sampled only the right atrial appendage and the left atrial tissue was not studied. It has been known that pulmonary veins and left atrial tissue are the most critical regions in initiating and maintaining AF [3]. However, it has been recognized that most of the pathologic alterations seen in chronic AF and human dilated cardiomyopathy predominate in the right atrial appendage tissue [19, 24, 25]. Also, in the study reported by Ad and colleagues [11], only the right atrial sampling was done and most of the right atrial appendage samples (52 of 60 patients) taken before CPB revealed varying degrees of histologic abnormalities similar to those observed in our study. And lastly, although myolysis was found to be a significant risk factor for postoperative AF in our study, the underlying mechanism of myolysis remains unknown. Studies focusing on the molecular and genetic basis of myolysis and postoperative AF are warranted to clarify the issue in detail.
In conclusion, pathophysiologic mechanisms for the occurrence of postoperative AF remain unclear. The identification of the nature of these morphologic alterations will contribute to improved treatment or prevention of this arrhythmia. Rapid perioperative sampling of right atrial appendage tissue as a frozen section may help to identify the patients at risk for postoperative AF and may influence our decision to perform concomitant preventive procedures such as radiofrequency ablation or Cox/Maze procedure. Also, further research focusing on improving the preoperative metabolic status of atrial cells may reduce the incidence of AF following CABG.
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Acknowledgments |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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References |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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