Association of Anti–Heat Shock Protein 65 Antibodies With Development of Postoperative Atrial Fibrillation
Background— Atrial fibrillation (AF) is a frequently encountered arrhythmia after cardiac surgery, but its underlying mechanisms are still unclear. We hypothesize that autoimmune and inflammatory responses against heat shock protein 65 (HSP65) may be involved and hence examined the relationship between HSP65 autoantibodies and occurrence of postoperative AF.
Methods and Results— A prospective study of 329 patients undergoing elective primary CABG was undertaken. Cardiovascular risk factors, ECG characteristics, medications, and intraoperative and postoperative features were documented. Anti-HSP65 antibodies and C-reactive protein levels were measured in all preoperative blood samples with ELISA. Postoperative AF was defined as the characteristic arrhythmia, lasting for at least 15 minutes and confirmed on 12-lead ECG and occurring within the first postoperative week. This occurred in 62 patients (19%). In univariate analysis, HSP65 antibodies were significantly higher in patients with postoperative AF (P=0.02). History of previous myocardial infarction, duration of bypass, number of distal anastomosis, and duration of ventilation were also associated with AF (P<0.05), but C-reactive protein levels were not (P=0.13). Multivariate analysis confirmed the positive association of HSP65 antibodies with postoperative AF (OR, 1.41; P=0.04) independent of age, sex, other cardiovascular risk factors, severity of coronary artery disease, duration of ventilation, duration of bypass, and left ventricular function.
Conclusions— We report a novel association between anti-HSP65 antibodies and occurrence of postoperative AF, indicating a possible role for antibody-mediated immune response in its pathogenesis.
Received February 23, 2004; de novo received April 13, 2004; revision received June 4, 2004; accepted June 4, 2004.
Atrial fibrillation (AF) is a common complication after cardiac surgery. Its incidence after CABG ranges from 15% to 37% and is associated with significant morbidity.1 Histological examination of atrial myocardium of chronic AF patients has confirmed the presence of myocarditis accompanied by an inflammatory infiltrate.2 Aviles and colleague3 have recently shown that C-reactive protein (CRP), an inflammatory marker, is associated with paroxysmal AF, and Gaudino et al4 have demonstrated that −174G/C interleukin-6 gene polymorphism influences the occurrence of AF after CABG. These findings support the involvement of “inflammation” in development of AF.
Heat shock proteins (HSPs) are a family of chaperone proteins that assist in the preservation of cellular integrity by maintaining proteins in their correctly folded state5 under stressful conditions. HSPs in most cell types, including myocytes, are expressed on the cell membrane only after exposure to stressful stimuli,6 eg, surgery. In individuals with high preoperative levels of HSP65 antibodies, a harmful autoimmune reaction might occur when these antibodies encounter stressed cells, expressing HSPs on their surface.7 This could lead to myocyte injury, formation of electroanatomical substrate, and finally AF. To test this hypothesis, we examined the relationship between preoperative anti-HSP65 antibodies and incidence of postoperative AF.
We prospectively studied 329 patients admitted for elective primary CABG. The ECG characteristics and cardiovascular risk profile were documented. Exclusion criteria were preceding history of AF, other associated operations, acute coronary event within 6 weeks before surgery, active infection, history of autoimmune diseases, and use of immunosuppressive drugs. The ethics committee at St George’s Hospital approved the study, and informed consent was obtained from all patients. Preoperative blood samples were collected, and plasma was stored at −80°C for subsequent analysis.
HSP65 IgG Assay
The ELISA technique used was based on previously described protocols.8,9 Duplicate blanked absorbance values for each sample were averaged and reported in absorbance units (AU). The interassay and intra-assay coefficient of variation was <10%.
CRP concentrations were measured by immunoturbidometry with a Beckman Synchro LX-20 Pro Automatic Analyser. The interassay and intra-assay coefficient of variation was <4%.
Similar surgical and anesthetic protocols were followed. CABG was performed with either an on-pump or an off-pump technique. Cold, blood-based antegrade cardioplegia at a systemic hypothermia of 32°C was used in all cases requiring cardiopulmonary bypass. Off-pump surgery was performed at near normothermia (35°C). Intraoperative features and postoperative complications were noted.
Postoperatively, heart rate and rhythm were continuously monitored for the first 48 hours and at 4-hour clinical observations thereafter until discharge. Patients were subjected to daily morning 12-lead ECGs. Additional recordings were done if there was a suspicion of arrhythmia. For this study, postoperative AF for this study was defined as the characteristic arrhythmia lasting for ≥15 minutes, occurring within first week after surgery, and requiring treatment for cardioversion. The date of onset and duration of AF suffered were documented. All hemodynamically stable patients were started on atenolol 25 mg on the first postoperative day. Patients with AF were treated with amiodarone after correction of electrolyte and acid-base imbalances.
Differences between the 2 groups were tested with an unpaired t test for continuous variables and a χ2 test for categorical variables. HSP65 antibodies and CRP were not normally distributed and were log-transformed for analysis. Strength of association between HSP65 antibodies and postoperative AF was assessed with multifactorial logistic regression analysis. Criteria for entry and exclusion from the multivariate analysis were P<0.05 and P>0.1, respectively. In addition, we included variables that have been associated with postoperative AF in previous studies.1,10,11 The goodness of fit for the multivariate models was tested by Pearson’s χ2 and likelihood ratio tests.
Postoperative atrial tachyarrhythmia developed in 62 patients (19%). Of these, 58 patients (18%) developed AF only, and 4 (1%) had episodes of atrial flutter interposed with AF. Median onset of AF was on the second postoperative day, and its median duration was 12 hours (range, 5.5 to 33.5 hours). Amiodarone was sufficient for cardioverting 57 patients. Three patients required electrical cardioversion. AF was refractory to both treatments in 2 patients, who were discharged on warfarin.
Univariate analysis (Table 1) showed significant associations between postoperative AF and preoperative levels of HSP65 antibodies, history of previous myocardial infarction, duration of cardiopulmonary bypass, number of distal anastomosis, and duration of ventilation. Preoperative CRP levels were similar in the 2 groups (P=0.13) and did not correlate with HSP65 antibodies (P=0.20). No significant differences were found with respect to age, sex, various cardiovascular risk factors, severity of coronary artery disease, left ventricular function, and P-wave duration
Multivariate analysis (Table 2) confirmed the significant positive association of anti-HSP65 antibodies with AF (OR, 1.41; 95% CI, 1.00 to 1.99; P=0.04) independent of other risk factors. Grafting the left anterior descending artery seemed to decrease the occurrence of postoperative AF in the overall analysis (OR, 0.23; 95% CI, 0.06 to 0.85; P=0.03).
Subgroup analysis (on-pump CABG, n=215) reaffirmed the significant association of HSP65 antibodies with postoperative AF (Table 2). In the off-pump CABG patients (n=114), the same trends were seen but did not reach statistical significance.
Postoperative AF is a common but vexing problem. Recent reports from several groups demonstrate the influence of inflammation on the development of AF. In this study, we provide the first evidence demonstrating the association of circulating anti-HSP65 antibodies with postoperative AF. These results suggest that serum HSP65 antibody levels may be a marker for subsequent development of AF. Alternatively, our findings may highlight their role in the pathogenesis of postoperative AF.
HSP60 has been shown to be expressed only on the surface of stressed cells, eg, cytokine-stimulated endothelial cells7 or hypoxic cardiomyocytes.6 The surface-expressed HSP60 can be attacked by circulating HSP65 antibodies, which are cross-reactive12 and are known to mediate complement-dependent cytotoxicity,13 causing cell damage and apoptosis. Although it is unknown whether circulating anti-HSP65 antibodies are cytotoxic to atrial myocytes, we speculate that such antibody-mediated autoimmune responses can damage the cell membrane of perioperatively stressed myocytes. This could alter their transmembranous conductance, cause increased anisotropy, and generate the critical electroanatomical substrate required for AF.
Preoperative CRP levels were similar in both groups, which is a finding consistent with the results of Bruins et al.14 Aviles and colleagues3 have demonstrated the association between CRP and AF only in patients with persistent or chronic AF, not in patients with a single episode of short-term AF after surgery.
We included patients undergoing both on-pump and off-pump cardiac surgery in our study because the role of extracorporeal circulation-related inflammation in the causation of postoperative AF is not definitive.10,15 Moreover, the consistency of association between HSP65 antibodies and postoperative AF in both the overall and subgroup analyses validates our conclusion regardless of the operative technique used.
The results of our study must be viewed in light of its limitations. Continuous ECG monitoring was used only for the first 48 hours. This could have resulted in missing some episodes of transient asymptomatic AF. However, such episodes should occur with similar frequency in both groups, and their confounding effects are likely to be minimal.
In conclusion, we report for the first time a novel association between HSP65 antibodies and the occurrence of postoperative AF. This finding lends support to the possible role of inflammation and cross-reactive autoimmunity in the development of AF.
This work was supported by the British Heart Foundation and the Oak Foundation.
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