Author + information
- Received February 4, 2000
- Revision received August 12, 2000
- Accepted October 2, 2000
- Published online February 1, 2001.
- Michikado Iwata, MDa,
- Tsutomu Yoshikawa, MDa,* (, )
- Akiyasu Baba, MD, PhDa,
- Toshihisa Anzai, MDa,
- Hideo Mitamura, MDa and
- Satoshi Ogawa, MDa
- ↵*Reprint requests and correspondence: Dr. Tsutomu Yoshikawa, Cardiology Division, Department of Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan
We sought to define the clinical and long-term prognostic implications of autoantibodies that act against the second extracellular loop of beta1-adrenergic receptors (ARs) in patients with idiopathic dilated cardiomyopathy (IDC).
Although autoantibodies directed against various domains of beta-ARs are found in patients with IDC, only a subgroup against the second extracellular domain of beta1-ARs exerts intrinsic sympathomimetic-like actions on human beta-ARs. It is suggested that the autoantibodies take part in the pathophysiology of IDC and may affect long-term prognosis of patients with this disorder.
Sera from 104 patients with IDC were screened for autoantibodies that act against the second extracellular loop of beta1-ARs by enzyme-linked immunosorbent assay, using a synthetic peptide corresponding to the domain. Relations of the autoantibodies to clinical variables and long-term prognosis were assessed by multivariate analysis.
Autoantibodies were detected in 40 patients (38%). Multifocal ventricular premature contractions (p < 0.01) and ventricular tachycardia (VT; p < 0.01) were more common in autoantibody-positive than in autoantibody-negative patients, although no differences in cardiac function or neurohormonal levels were demonstrated. The presence of autoantibodies (p = 0.001) and a low left ventricular ejection fraction (LVEF <30%; p = 0.02) were independent predictors of VT. Sudden death was independently predicted by the presence of autoantibodies (p = 0.03), as well as by LVEF <30% (p = 0.01), whereas total mortality was predicted only by LVEF <30% (p = 0.001).
Autoantibodies directed against the second extracellular loop of beta1-ARs were closely related to serious ventricular arrhythmias in patients with IDC, and the presence of autoantibodies independently predicted sudden death. These autoantibodies may contribute to electrical instability in patients with IDC.
Autoantibodies that act against beta1-adrenergic receptors (ARs) can be detected in ∼30% to 50% of patients with idiopathic dilated cardiomyopathy (IDC) (1–4). These autoantibodies are directed against various domains of beta-ARs as an epitope (1–4). Among them, only a subgroup of autoantibodies directed against the second extracellular domain of beta1-ARs has been shown to exert agonist-like actions on human beta-ARs, inhibiting radioligand binding and enhancing receptor-mediated signaling in vitro (4). These results suggest that autoantibodies, indeed, take part in the pathophysiology of IDC and may affect the long-term prognosis of patients with this disorder.
Immunoapheresis to remove autoantibodies against beta1-ARs from the blood has ameliorated symptoms and improved hemodynamic variables in patients with IDC (5). In contrast, a high prevalence of autoantibodies that act against beta-ARs has been found among patients with primary electrical cardiac abnormalities (6). Several studies have reported relations between clinical variables and the autoantibodies in IDC (4,7). However, the characteristics of a subset of patients with IDC who have the autoantibodies have not been subjected to multivariate analysis to assess their interactions. We used this analytic method to better characterize patients with IDC with autoantibodies directed against the second extracellular loop of beta1-ARs.
One hundred four patients with IDC who were clinically stable with conventional therapy, including digitalis glycosides, diuretics, angiotensin-converting enzyme inhibitors or beta-blockers, were treated in our clinic and were recruited to participate in the present study between 1995 and 1999. The patients’ mean age was 56 ± 14 years. The diagnosis of IDC was based on World Health Organization/International Society and Federation of Cardiology criteria established in 1995 (8). A left ventricular ejection fraction (LVEF) <55% was confirmed by radionuclide ventriculography (mean LVEF 30 ± 10%). Coronary angiography was performed in 87 patients (84%), and myocardial biopsy was performed in 78 patients (75%). Patients with >50% stenosis of the major coronary arteries, myocarditis or one of the other specific cardiomyopathies were excluded. The study protocol was approved by an institutional Review Committee, and all patients gave written, informed consent to participate in the present study.
The autoantibodies of interest were measured in patient sera with an enzyme-linked immunosorbent assay (ELISA), using a synthetic peptide corresponding to the putative sequence of the second extracellular loop of human beta1-ARs (amino acid sequence number, 197 to 222; His-Trp-Trp-Arg-Ala-Glu-Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-Val-Thr-Asn-Arg). Briefly, 50 μl of peptide (50 μg/ml in 0.1 mol/liter Na2CO3) was coated on microtiter plates. The wells were then saturated with phosphate-buffered saline (PBS), including 3% skim milk, 0.1% polysorbate-20 (Tween-20) and 0.01% thimerosal (Merthiolate) (PMT). Fifty microliters of patient serum dilutions beginning at 1:20 were then added to the coated and saturated microtiter plates. After the wells were washed three times with PBS, an affinity-purified biotinylated rabbit anti-human immunoglobulin G antibody solution diluted 1:1000 in PMT was allowed to react for 1 h. After another three washes, bound biotinylated antibodies were detected using streptavidin peroxidase (1 μg/ml), and as substrates H2O2(2.5 mmol/liter) with 2,2′-azino-diethylbenzthiazoline sulfonic acid (2 mmol/liter). After 30 min, optical densities were determined at 405 nm in an ELISA reader. Positivity was defined as 2.5 times the background density.
Clinical variables, including findings from ultrasonic echocardiography and Holter monitoring, were recorded for all patients within one month of serum sampling for measurement of autoantibodies. Echocardiography was performed to assess left ventricular end-diastolic dimensions, end-systolic dimensions and fractional shortening. The Holter electrocardiogram (ECG) was analyzed by using the Marquette system (SXP 8000 or MARS 8000, Marquette Medical Systems Inc., Milwaukee, Wisconsin) to assess the total number of supraventricular premature contractions (SVPCs) and ventricular premature contractions (VPCs). The morphology of VPC, presence of ventricular tachycardia (VT), maximal number of runs of VT, longest VT duration and fastest VT rate were examined. Data were averaged from two or more sets of recordings. Ventricular tachycardia was defined as a run of three or more consecutive VPCs with a heart rate ≥100 beats/min, which was noted in at least one of the recordings. Furthermore, VT lasting ≥3 s or VT with a maximal rate ≥165 beats/min was defined as high risk VT, according to each median value, as previously described (9). Blood samples for measurement of plasma norepinephrine and serum potassium were collected from a peripheral vein after the patient had rested in the supine position for at least 20 min.
The patients studied were followed for a mean of 31 months (range 0.2 to 52.5). Deaths were classified as follows: sudden death was defined as death within 1 h without previous worsening of symptoms of heart failure. Unexpected death occurring during sleep was considered to be sudden when patients were found dead in the morning by family members sharing the same room. We generally classified other unwitnessed deaths as unknown. When death occurred as a consequence of progressive deterioration of heart failure, acute pulmonary edema or cardiogenic shock, we classified it as due to pump failure (10).
Data are expressed as the mean value ± SD. Comparison between two groups was performed by using the nonpaired ttest or the nonparametric means test for continuous variables and by the chi-square test for discrete variables. Logistic regression analysis was used to assess predictors of VT. Twelve variables, including those with p values <0.10 by univariate analyses, were further assessed by multivariate logistic regression analysis. Kaplan-Meier survival curves for cardiac mortality, sudden death and pump failure death were calculated according to presence or absence of autoantibodies, and the differences were analyzed by the log-rank test. Long-term prognosis was assessed by using the Cox proportional hazards model. Statistical significance was set at p < 0.05. StatView 5.0 software (SAS Institute Inc., Cary, North Carolina) was used for statistical analyses.
Patient characteristics and autoantibodies
Autoantibodies against the second extracellular loop of beta1-ARs were detected in 40 patients in our study group (38%). No difference was noted for age, duration of illness, gender, New York Heart Association (NYHA) functional class, concomitant medications, cardiac function or plasma norepinephrine levels between autoantibody-positive and autoantibody-negative patients (Table 1).
Holter ECG findings are summarized in Table 2. No difference was evident in the number of SVPCs or VPCs between the autoantibody-positive and autoantibody-negative patients. The presence of VT was more common in the autoantibody-positive than in autoantibody-negative patients, with multifocal VPCs being predominant. The maximal numbers of runs of VT were similar between the two groups, although the maximal heart rate during VT was greater in autoantibody-positive than in autoantibody-negative patients.
Determinants of VT
Univariate analyses were performed for the 17 variables listed in Table 3, which showed that the presence of autoantibodies, LVEF <30% and the use of diuretics were related to the presence of VT (Table 3). Multivariate logistic regression analysis showed the presence of autoantibodies to be the strongest independent predictor of overall VT. Poor left ventricular function (LVEF <30%) also independently predicted overall VT. For high risk VT, however, the presence of autoantibodies was the sole independent predictor (Table 4).
Subgroups representing autoantibody-positive and autoantibody-negative patients were analyzed further with regard to the presence of VT. In autoantibody-positive patients, no additional factor significantly predicted overall VT, although the use of beta-blockers was a negative predictor of high risk VT (odds ratio [OR] 0.11, 95% confidence interval [CI] 0.01 to 0.89; p = 0.04). For autoantibody-negative patients, LVEF <30% was an independent predictor of overall VT (OR 7.28, 95% CI 1.26 to 42.18; p = 0.03), but no significant predictor of high risk VT was identified (data not shown).
Determinants of cardiac mortality and sudden death
After a mean follow-up period of 31 ± 15 months, 82 patients (79%) were alive. The 22 deaths included 10 sudden deaths, 11 deaths due to pump failure and 1 death due to cancer, with no deaths from unknown causes. Ten cardiac deaths occurred in autoantibody-negative patients (3 sudden deaths and 7 pump failure deaths), and 11 cardiac deaths occurred in autoantibody-positive patients (7 sudden deaths and 4 pump failure deaths). Six patients without VT had cardiac deaths (2 sudden deaths and 4 pump failure deaths), and 15 cardiac deaths occurred in patients with VT (8 sudden deaths and 7 pump failure deaths).
Figure 1compares the Kaplan-Meier survival curves for cardiac mortality, sudden death and pump failure death according to the presence or absence of autoantibodies. Sudden deaths were more common among autoantibody-positive as compared with autoantibody-negative patients, although no difference in overall cardiac mortality or pump failure death was found between the two groups. Total cardiac deaths (p = 0.007 by the log-rank test) and sudden deaths (p = 0.016) were more common in patients with VT than in those without VT, although the occurrence of pump failure death did not differ between the two groups. High risk VT was associated with a higher incidence of cardiac death in general (p = 0.023) and of sudden death (p = 0.016), as compared with low risk VT or no VT (data not shown).
A Cox proportional hazards model for cardiac mortality or sudden deaths was used to assess the significance of autoantibodies and other clinical variables for long-term outcome in patients with IDC. First, univariate analyses with the proportional hazards model were performed for 18 variables, including the presence of VT, as well as for the variables listed in Table 3, with the result that LVEF <30% and the presence of VT each were related to an increase in overall cardiac mortality, whereas LVEF <30%, the presence of VT and the presence of autoantibodies were related to an increase in sudden death. Other variables were not significantly related to either total cardiac mortality or sudden death (p > 0.10). Accordingly, multivariate Cox proportional hazards analysis was performed for the seven variables listed in Table 5. Left ventricular ejection fraction <30% was the only independent predictor of overall cardiac mortality. For sudden death, the presence of autoantibodies and LVEF <30% were independent predictors. By multivariate analysis, the presence of overall VT or high risk VT was not an independent predictor for either cardiac mortality or sudden death.
We characterized the clinical features of patients with autoantibodies directed against the second extracellular loop of beta1-ARs in a large group (n = 104), demonstrating that the presence of autoantibodies was associated with high risk ventricular arrhythmias likely to result in sudden cardiac death in patients with IDC.
Detection of autoantibodies directed against beta-adrenergic receptors
A number of studies have shown that autoantibodies directed against beta-ARs can be detected in 26% to 95% of all patients with IDC, depending on antibody detection methods (3,4). Jahns et al. (4)demonstrated that autoantibodies selective for the amino terminal or carboxy terminal domains of beta1-ARs and those selective for the domains of beta2-ARs detected by ELISA were nonspecific antibodies that were unable to affect the function of native human receptors. In addition, autoantibodies selective for the second extracellular loop of beta2-ARs detected by ELISA were also nonspecific. Only a subgroup of autoantibodies directed against the second extracellular domain of beta1-ARs impaired receptor ligand binding and enhanced receptor-mediated signaling in native human beta-ARs. These findings suggest that functional autoantibodies are detected by a peptide corresponding to the second extracellular domain of beta1-ARs. In their study, the positivity of ELISA was defined as the upper limit of the 95% CI of healthy subjects, showing that 26% of the autoantibodies directed against the second extracellular loop of beta1-ARs, as detected by ELISA, were nonspecific. In contrast, the present study defined positivity as 2.5 times the background density. In the study by Jahns et al. (4), immunoglobulin G preparations from patients with IDC that showed >2.5 times the background density in ELISA substantially enhanced cyclic adenosine monophosphate (cAMP) production in sf9 cells expressing human beta1-ARs; there was a positive correlation between immunoreactivity of ELISA and the increase in cAMP production. Moreover, Magnusson et al. (1)confirmed that the positive response of ELISA, according to the definition in the present study, was a marker for the recognition of human beta1-ARs by using Western blots and immunohistochemistry. These observations support the fact that ELISA, using a peptide corresponding to the second extracellular loop of beta1-ARs, appropriately detects functional autoantibodies by defining 2.5 times the background density as a positive criterion.
Physiologic actions of autoantibodies
Affinity-purified autoantibodies directed against the second extracellular loop of beta1-ARs from patients with IDC were observed to increase the beating frequency of cultured rat heart myocytes, without inducing desensitization for more than 6 h; in contrast, isoproterenol induced desensitization after stimulation (2). In addition, these autoantibodies have been found to inhibit radioligand binding to beta-ARs by decreasing the numbers or the affinity and to enhance receptor-mediated signaling by increasing both basal and isoproterenol-stimulated cAMP levels in cells expressing human beta-ARs (4). These phenomena induced by the autoantibodies were completely abolished by the beta1-selective antagonist, bisoprolol. These results show that the autoantibodies exert a sustained intrinsic sympathomimetic-like action through beta1receptors. Long-term stimulation of beta1-ARs by autoantibodies may induce electrophysiologic instability through Ca2+overload. Ventricular arrhythmias are induced by sympathetic stimulation through triggered activity of both early and delayed afterdepolarizations (11,12).
Clinical profile of autoantibody-positive patients with IDC
Matsui et al. (7)examined autoantibodies directed against the second extracellular loop of beta1-ARs in 28 patients with IDC; no difference in NYHA functional class or LVEF was apparent between autoantibody-positive and autoantibody-negative patients, although the issue of ventricular arrhythmias was not considered. Chiale et al. (6)reported a high prevalence of autoantibodies directed against the domain of beta1-ARs and beta2-ARs among patients with primary electrical cardiac abnormalities. These autoantibodies were detected in 52.3% of patients with ventricular arrhythmias. These authors suggested that autoantibodies found in patients with IDC were associated with ventricular arrhythmias through beta1-ARs, but not through beta2-ARs, as based on the functional test. This finding is consistent with the previous reports that the autoantibodies exert agonist-like actions through beta1-AR in vitro (2,4). In contrast, ventricular arrhythmias may be associated with cardiac dysfunction (13)or may be affected by various clinical factors, including age, neurohormonal levels and concomitant medications. A recent study reported that the presence of autoantibodies directed against beta1-ARs was associated with poorer left ventricular function in patients with IDC (4), but clinical factors likely to affect cardiac function may not be evenly distributed between patient groups. Therefore, we performed multivariate analysis to assess the interactions between clinical factors in large samples.
In our study, autoantibodies directed against the second extracellular loop of beta1-ARs were associated with multifocal VPCs and the presence of VT, but not with the total number of VPCs. The association may reflect electrophysiologic actions of the autoantibodies. Ventricular tachycardia was independently predicted by the presence of autoantibodies and poor left ventricular function, whereas high risk VT was predicted only by the presence of autoantibodies. In other studies, complex ventricular arrhythmia was a major risk factor for overall cardiac death (14,15)and sudden death (16)in patients with IDC. Moreover, Szabo et al. (9)documented that relatively persistent VT with a particularly rapid rate predicted sudden cardiac death in patients with left ventricular dysfunction, including IDC. In the present study, the presence of VT was also associated with increased overall cardiac mortality and sudden death, but VT was not an independent predictor of these events. A previous study concluded that the presence of VT in 24-h ECGs was an independent predictor of sudden death in patients with IDC (17). This discrepancy may be explained by the presence of autoantibodies as an additional variable to predict (on multivariate analysis) sudden death in the present study. Multivariate analysis showed that the presence of autoantibodies was an independent predictor of sudden death, as was left ventricular dysfunction; the latter finding was in agreement with the previous studies (14,15,18).
The beneficial effects of beta-blockers on symptoms, cardiac function and overall survival in patients with heart failure have been confirmed in large clinical trials (19–21). In our study, the use of beta-blockers was associated with a lower incidence of high risk VT, but not overall VT, in autoantibody-positive patients, suggesting that beta-blockers may attenuate electrophysiologic instability induced by the autoantibodies, as demonstrated in vitro (2). Recent clinical trials of beta-blockers have demonstrated that these drugs can help to prevent sudden cardiac death in patients with congestive heart failure (19–21). The autoantibody hypothesis is one possible explanation for the decrease in sudden death in such patients.
The present study demonstrated that autoantibodies directed against the second extracellular loop of beta1-ARs were closely related to serious ventricular arrhythmias, but not to cardiac function or neurohormone levels, in patients with IDC. The presence of these autoantibodies and poor left ventricular function were independent predictors of sudden cardiac death. Autoantibodies directed against the second extracellular loop of beta1-ARs may be at least partly responsible for electrical instability in patients with IDC.
☆ This study was supported in part by a grant (no. 09670748) from the Ministry of Education, Sciences, and Culture of Japan (Dr. Yoshikawa).
- adrenergic receptor
- cyclic adenosine monophosphate
- enzyme-linked immunosorbent assay
- idiopathic dilated cardiomyopathy
- left ventricular ejection fraction
- New York Heart Association
- phosphate-buffered saline
- supraventricular premature contractions
- ventricular premature contractions
- ventricular tachycardia
- Received February 4, 2000.
- Revision received August 12, 2000.
- Accepted October 2, 2000.
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