Inducible cardiac arrhythmias caused by enhanced β₁-adrenergic autoantibody expression in the rabbit

Abstract

Previous studies demonstrated burst pacing and intravenous infusion of ACh induced sustained atrial tachycardia when rabbits were immunized to produce β₂-adrenergic receptor (β₂AR)-activating autoantibodies. The objective of this study was to examine the arrhythmogenic effect of β₁-adrenergic receptor (β₁AR)-activating autoantibodies in the rabbit. Eight New Zealand white rabbits were immunized with a β₁AR second extracellular loop peptide to raise β₁AR antibody titers. A catheter-based electrophysiological study was performed on anesthetized rabbits before and after immunization. Arrhythmia occurrence was determined in response to burst pacing before and after ACh infusion in incremental concentrations of 10 μM, 100 μM, and 1 mM. The baseline sinus heart rate before and after immunization averaged 149 ± 17 per min and 169 ± 16 per min, respectively (P < 0.05). In the preimmune studies, there were five sustained (≥10 s) arrhythmias in 32 induction attempts, which occurred in only four of eight rabbits. In the postimmune studies, there were 22 sustained arrhythmias in 32 induction attempts, which occurred in all eight rabbits (P < 0.0001 for the independent effect of immunization). Of the 22 sustained arrhythmias postimmunization, 15 were sinus tachycardia compared with only two before immunization (P < 0.01 for the independent effect of immunization). Postimmune (but not preimmune) rabbit sera demonstrated specific binding to β₁AR and induced significant β₁AR activation in transfected cells in vitro. No cross-reactivity with β₂AR was observed. In conclusion, in contrast with rabbits with β₂AR-activating autoantibodies that demonstrate predominantly atrial tachycardias, enhanced autoantibody activation of β₁AR in the rabbit leads to tachyarrhythmias mainly in the form of sustained sinus tachycardia.

increasing data support a significant pathophysiological role for autoantibodies that activate β₁- and β₂-adrenergic receptors (β₁/₂AR) in atrial and ventricular tachyarrhythmias (14, 15). Attention has been focused on the role of β₁AR-activating autoantibodies in the etiology and maintenance of dilated cardiomyopathy (8, 13). There is ample evidence these same activating autoantibodies are related to tachyarrhythmias in dilated cardiomyopathy (10), Graves′ disease (19), primary ventricular arrhythmias (7), and inappropriate sinus tachycardia (6).

The relative densities of β₁AR and β₂AR vary in different regions of the atrium. β₂AR density in human atria was found to be significantly higher in the sinoatrial node than in the working atrial myocardium (18). In rabbits, this situation is reversed (20). Consistent with these observations, we demonstrated that burst pacing in conjunction with intravenous infusion of ACh induced sustained atrial rather than sinus tachycardias in rabbits with activating autoantibodies to β₂AR (16). In the present study we instituted the same protocol to examine the arrhythmogenic effect of β₁AR-activating autoantibodies in the rabbit. This study has permitted us to test the hypothesis that the relative regional concentrations of cardiac β₁AR and β₂AR may in part mediate localization of cardiac tachyarrhythmias.

METHODS

This study protocol was approved by the Institutional Animal Care and Use Committee of the Oklahoma City Veterans Affairs Medical Center and University of Oklahoma Health Sciences Center and conforms to international standards for animal safety and comfort.

Catheter electrophysiological study.

Eight young New Zealand white rabbits (3- to 4 mo old) were anesthetized with ketamine-xylazine (35 mg/5 mg/kg) and subjected to a catheter-based electrophysiological study. Standard electrocardiograms (Leads 1-aVF) were continuously monitored. After the neck area was shaved and application of betadine antiseptic, the right jugular vein was dissected and cannulated with a 4 Fr multi-electrode catheter. Under electrographic control, the catheter was passed into the right atrium to record atrial potentials in conjunction with the standard 6-lead ECG. Atrial tachyarrhythmia susceptibility was tested by bursts of stimuli (3–5 s duration) at a high frequency (20 Hz) and voltages that were at least twice the diastolic pacing threshold before and after the infusion of ACh in three incremental concentrations (10 μM, 100 μM, and 1 mM) at a rate of 1 ml/min. These were applied to a site in the right atrium at which a discrete atrial potential with little or no far-field ventricular potentials was recorded. Nonsustained (<10 s) and sustained (≥10 s) arrhythmia occurrence was determined in response to burst pacing at 2× diastolic threshold, at baseline, and then with each of the three concentrations of ACh infusion for 2 min before initiating burst pacing. The number of burst pacing ranged from 3 to 10 (median = 6). It should be noted that the number of burst pacing in the preimmune state was most likely to be closer to 10 because it was more difficult to induce any nonsustained or sustained arrhythmia with or without ACh infusions. On the other hand, after immunization, particularly with ACh infusion two to three burst pacing events readily induced either nonsustained or sustained arrhythmias. Figure 1 shows the flow chart depicting the protocols for these studies before and after each rabbit was immunized to produce β₁AR-activating autoantibodies. When this study was completed, the wound was closed and antibiotic treatment was instituted. A second electrophysiological study was performed after the 6-wk immunization interval.

Fig. 1.

Flow chart depicting the protocol of the procedures performed in the rabbits during the preimmune and postimmune state.

Definition of arrhythmias in the rabbit heart.

Nonsustained arrhythmia is any arrhythmia lasting <10 s.

Sustained arrhythmia is any arrhythmia lasting ≥10 s. The 10-s cutoff was determined empirically since the majority of arrhythmias induced in the preimmune state did not exceed 10 s, whereas the majority of induced arrhythmias after immunization invariably exceeded 10 s, many lasting up to several minutes.

Sinus tachycardia is a regular, rapid heart rate ≥250 beats/min showing 1:1 atrioventricular (AV) conduction arising from the sinus node (upright P waves in leads II, III, and aVF) with the earliest atrial electrogram occurring at least 10 ms before the onset of the P wave (Figs. 2 and 3).

Fig. 2.

Illustration, as an inset (top left), showing the location of the multi-electrode catheter in the right atrium (RA) and the sequence of bipolar electrograms from the superior vena cava (SVC) just above the right atrial entrance, high right atrium (HRA), mid-right atrium (MID RA), and the area of the AV junction (AVJ) during sinus rhythm at a rate of 197 beats/min (bpm). ECG leads I through aVF are also shown. LA, left atrium; LV, left ventricle; RV, right ventricle; SAN, sinoatrial node; AVN, atrioventricular node; aVR, aVL, and aVF, augmented limb leads.

Fig. 3.

Atrial burst pacing-induced ventricular premature contractions followed by sinus tachycardia at a rate of 263 beats/min. Note the same sequence of activation as seen in the electrograms during sinus rhythm (Fig. 2).

Junctional tachycardia is a regular, rapid heart rate ≥200 beats/min showing 1:1 short AV interval arising from the AV junction with an altered sequence of activation and morphology of atrial electrograms compared with sinus rhythm (Fig. 4).

Fig. 4.

Atrial burst pacing-induced junctional tachycardia at an initial rate of 448 beats/min. The sequence of atrial activation starts at the AVJ area and proceeds toward the HRA (↑). Note that the atrial electrogram at the SVC is coincident with the ventricular activation until at the end of the trace, when the junctional rate increases to 462 beats/min. At this point the SVC electrogram appears before the ventricular activation (↓).

Atrial tachycardia is a regular, rapid heart rate ≥250 beats/min showing a constant A-A interval with 2:1 AV conduction with different P wave and electrogram morphologies compared with sinus rhythm or junctional tachycardia (Fig. 5).

Fig. 5.

Atrial burst pacing-induced atrial tachycardia at a rate of 509 beats/min with a constant A-A interval (118 ms) and 2:1 atrioventricular block.

Atrial fibrillation is rapid, irregular, fractionated atrial electrograms with a rapid but irregular ventricular response.

Ventricular tachycardia is three or more beats arising from the ventricles at a rate ≥250 beats/min.

Immunization.

The eight rabbits were immunized with 1 mg of the highly conserved second extracellular loop (ECL2) peptide for β₁AR (HWWRAESDEARRCYNDPKCCDFVTNR) in 0.5 ml of complete Freund's adjuvant. The animals were boosted with the same peptide plus incomplete Freund's adjuvant (1 mg/0.5 ml) at 2 and 4 wk. Pre- and postimmune sera were obtained from all animals for ELISA and activity assays of the expected antibodies generated during immunization.

ELISA.

Antibodies produced in the sera were detected by ELISA. Briefly, microtiter plates were coated with β₁AR ECL2 or β₂AR ECL2 (for cross-reactivity testing) (16) peptide at 10 μg/ml in coating buffer. To determine antibody titer, sera were diluted 1:10,000 in 1% BSA in PBS and thereafter serially diluted twofold. Goat anti-rabbit IgG conjugated with alkaline phosphatase (Sigma, St. Louis, MO) and its substrate para-nitrophenyl-phosphate 104 were used to detect antibody binding. Titers were determined as the highest dilution with an optical density (OD) value of 0.10 at 60 min.

Immunofluorescence microscopy.

Chinese hamster ovary (CHO) cells expressing human β₁AR were cultured on glass cover slips in 6-well plates for 24 h. The cells were fixed with 4% paraformaldehyde, blocked with 5% normal goat sera, and incubated with preimmune or postimmune rabbit anti-β₁AR sera (1:100) for 1 h, followed by incubation with Alexa Fluor 594-labeled goat anti-rabbit IgG (Invitrogen, Grand Island, NY). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were obtained using a fluorescence microscope (Olympus).

cAMP assay.

Rabbit sera were tested for activation of β₁AR using the cAMP Hunter eXpress GPCR Assay kit (DiscoveRx, Fremont, CA). Briefly, 30,000 CHO cells expressing human β₁AR were dispensed into each well of 96-well culture plate and incubated overnight. The medium was removed and assay buffer containing the cAMP antibody and rabbit sera (1:100) in the presence and absence of βAR blocker propranolol (1 μM) were sequentially added and incubated for 30 min. Preincubation of sera with a 10-fold excess of β₁AR ECL2 peptide was also tested for neutralization studies. cAMP standard, negative (buffer), and positive (isoproterenol 100 nM) controls were included in each assay. Samples were tested in triplicate. After sample treatment, cAMP detection reagent and solution were added, and luminescent signal was read on a TD-20/20 Luminometer (Turner BioSystems). The cAMP values are expressed as percentage of buffer baseline to normalize the individual data.

Statistical analysis.

Data are presented as means ± SD. Differences in baseline heart rates, those induced by ACh infusions, and cAMP production were assessed by a paired Student's t-test or repeated-measures ANOVA as appropriate. Repeated-measures logistic regression was used to assess differences in occurrence of a sustained arrhythmia for each rabbit at baseline and at all concentrations of ACh infusion before and after immunization. All of these analyses were performed using the generalized estimating equations procedure implemented in GENLIN in SPSS (IBM SPSS Statistics v. 20.0; IBM, Armonk, NY). A P value of <0.05 was considered statistically significant.

RESULTS

Electrophysiological studies.

Table 1 summarizes the results of the electrophysiological studies performed before and after immunization with the β₁AR ECL2 peptide to induce production of β₁AR-activating antibodies. The antibody titers ranged from 1:320,000 to 1:1.28 million in the postimmune studies and were undetectable in the preimmune studies. Using each rabbit as its own control, we found that the baseline heart rate after immunization was significantly increased (preimmune: 149 ± 17 vs. postimmune: 169 ± 16 beats/min; P < 0.05). Arrhythmias were induced by burst pacing at baseline and at each infused concentration of ACh. The rates of the induced sustained arrhythmias varied over a wide range from 250 to 385 per min. If no arrhythmia could be elicited, no response (NR) was registered. At each incremental concentration of ACh, there was a progressive increase in heart rate, each of which was significantly greater than the baseline value. The importance of these findings will be discussed below as the basis for the use of ACh as an adjunct to the provocative effect of burst pacing to induce arrhythmias in the presence of β₁AR-activating antibodies.

Table 1.

Rabbit response to acetylcholine and burst pacing: preimmune and postimmune studies

	Preimmune		Postimmune
Acetylcholine	Arrhythmias	HR	Arrhythmias	HR	Anti-β1AR Titer
Rabbit 1
0	NR	160	NR	163	1:1.28 million
10 μM	NR	205	NR	208
100 μM	NR	205	NR	215
1 mM	NR		sus VT
Rabbit 2
0	NR	158	sus ST	195	1:640,000
10 μM	NR	175	NR	237
100 μM	NR	183	sus ST	195
1 mM	NS AF	197	sus ST	240
Rabbit 3
0	NR	155	NS AT	172	1:320,000
10 μM	NR	175	NS VT	208
100 μM	NS AT	179	sus VT	205
1 mM	sus AT	179	sus VT	199
Rabbit 4
0	NR	179	NR	165	1:1.28 million
10 μM	NR	215	NR	209
100 μM	NR	230	sus ST	226
1 mM	NS AT/AF	230	sus ST	215
Rabbit 5
0	NR	137	sus JT/ST	188	1:320,000
10 μM	NS ST/AT/AF, VPC	145	NS ST	160
100 μM	sus JT	199	sus ST	159
1 mM	NS AF/JT	196	sus JT/ST	190
Rabbit 6
0	NS AFL	133	sus ST	155	1:640,000
10 μM	NS AFL	205	sus ST	213
100 μM	NS AFL	170	sus ST	183
1 mM	NS ST	219	sus ST	287
Rabbit 7
0	NR	129	NR	146	1:640,000
10 μM	NR	181	NR	204
100 μM	sus ST	184	sus ST	205
1 mM	sus ST	260	sus ST	245
Rabbit 8
0	NR	144	NR	167	1:640,000
10 μM	NS VT	190	sus AT/AF	221
100 μM	sus AF	208	NS AT	221
1 mM	NS ST/AT	208	sus ST	229

β₁AR, β₁-adrenergic receptor; AF, atrial fibrillation; AFL, atrial flutter; AT, atrial tachycardia; HR, heart rate; JT, junctional tachycardia; NR, no response; NS, nonsustained; ST, sinus tachycardia; sus, sustained; VPC, ventricular premature contraction; VT, ventricular tachycardia.

In the preimmune studies, 15 episodes of nonsustained supraventricular tachycardia (SVT) and 1 episode of nonsustained ventricular tachycardia (VT) were induced by burst pacing during the baseline state and three incremental concentrations of infused ACh. There were five episodes of sustained SVT, including two sinus tachycardia (ST), one atrial tachycardia (AT), one junctional tachycardia (JT), and one atrial fibrillation (AF). In contrast, in the postimmune studies, there were four episodes of nonsustained tachyarrhythmias, but there were 22 burst pacing inductions of sustained tachyarrhythmias, including 15 ST, 2 JT, 1 AT, 1 AF, and 3 VT. From a pathophysiological standpoint, we compared the induction of various sustained cardiac arrhythmias between the preimmune and postimmune state for each rabbit with each rabbit serving as its own control. After the effect of dose was accounted for, the proportion of burst pacing (8 rabbits, 4 dosages) showing sustained arrhythmias before immunization was 5/32 (16%), whereas after immunization the proportion of burst pacing showing sustained arrhythmias was 22/32 (69%) (P < 0.0001 for the independent effect of immunization). If the results are collapsed across dose and whether a rabbit showed sustained arrhythmias at any dosage is considered then the proportion of rabbits showing sustained arrhythmias before immunization was 4/8 (50%), whereas after immunization the proportion of rabbits showing sustained arrhythmias was 8/8 (100%) (P < 0.05 for the effect of immunization). Similarly, we found 15 sustained ST were induced in the 32 events after immunization compared with two such sustained arrhythmias induced in the preimmune state (P < 0.01 for the independent effect of immunization; P < 0.05 after collapsing across dose: 6/8 after immunization vs. 1/8 before immunization).

Figure 2 shows, as an inset, the location of the multi-electrode catheter in the right atrium and the sequence of bipolar electrograms from the superior vena cava (SVC) just above the right atrial entrance, the high right atrium, the mid-right atrium, and the area of the AV junction during sinus rhythm at a rate of 197 beats/min. ECG leads I through aVF are also shown. Figure 3 shows atrial burst pacing induced ventricular premature contractions (VPC) followed by a ST at a rate of 263 beats/min. Note the same sequence of activation as seen in the electrograms during sinus rhythm (Fig. 2). In Fig. 4, atrial burst pacing induced a JT at an initial rate of 448 beats/min. The sequence of atrial activation starts at the area of the AV junction and proceeds toward the high right atrium. Note that the atrial electrogram at the SVC is coincident with the ventricular activation until at the end of the trace, when the junctional rate increased to 462 beats/min. The SVC electrogram appears before the ventricular activation. In Fig. 5, atrial burst pacing induced an AT at a rate of 509 beats/min with a constant A-A interval (118 ms) and 2:1 AV block.

β₁AR antibody binding and activity.

All eight rabbits developed high antibody titers to β₁AR ranging from 1:320,000 to 1:1.28 million after peptide immunization. To analyze antibody binding to β₁AR, immunofluorescence was performed with CHO cells expressing human β₁AR. A representative stain is shown in Fig. 6. Rabbit antisera strongly reacted with β₁AR in CHO cells, whereas preimmune sera did not show any significant reactivity. Preincubation of rabbit antisera with an excess of the β₁AR ECL2 peptide diminished fluorescence signal in CHO cells (data not shown), confirming the specific reactivity to β₁AR.

Fig. 6.

Immunofluorescence staining of β₁-adrenergic receptor (β₁AR) in Chinese hamster ovary (CHO) cells. CHO cells expressing human β₁AR were stained with rabbit preimmune sera, rabbit anti-β₁AR sera, and Alexa 594 (red)-labeled secondary antibody. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The anti-β₁AR sera demonstrated strong reactivity, whereas the preimmune sera did not show any significant binding. All images were obtained at 40× magnification.

Rabbit antisera were able to stimulate cAMP production in β₁AR-transfected CHO cells in vitro (Fig. 7A). Sera-induced β₁AR activation was abolished by the nonselective βAR blocker propranolol and by preincubation of the sera with the β₁AR ECL2 peptide. No significant increase in cAMP production was found with the preimmune sera compared with buffer baseline.

Fig. 7.

A: rabbit sera-induced cAMP production in β₁AR-CHO cells. When compared with the preimmune sera, the anti-β₁AR sera significantly increased cAMP production (*P < 0.01; n = 3), whereas the β-blocker propranolol or preincubation with the second extracellular loop (ECL2) peptide for β₁AR both effectively blocked the sera-induced β₁AR activation of cAMP production (**P < 0.01; n = 3). B and C: binding and agonist specificity of rabbit anti-β₁AR sera. Rabbit anti-β₁AR sera were tested for reactivity with β₁AR versus β₂AR ECL2 peptide in ELISA (B) and activation of cAMP production in β₁AR-CHO versus β₂AR-CHO cells in cAMP assay (C). The anti-β₁AR sera demonstrated β₁AR-specific reactivity and activity. No cross-reactivity with β₂AR was observed. OD, optical density.

To check the specificity of rabbit antisera, rabbit anti-β₁AR sera were examined for cross-reactivity with β₂AR by ELISA and cAMP assay. As shown in Fig. 7, B and C, the anti-β₁AR sera reacted specifically with the β₁AR, and not the β₂AR ECL2 peptide in ELISA. The anti-β₁AR sera also stimulated significant cAMP production in β₁AR-transfected CHO cells, whereas no significant cAMP stimulation was observed in β₂AR-transfected CHO cells.

DISCUSSION

We have used ACh and burst atrial pacing for induction of arrhythmias in conjunction with β₁AR-activating antibodies. The rationale for this combination is based on the observation, as shown in Table 1, that infusion of incremental concentrations of ACh consistently increases heart rate. ACh is known to have a direct action on the sinus node to slow the heart rate and also cause vasodilation which would, by a baroreflex action, increase sinus rate. In fact, within the first minute of the ACh infusion, there was a consistent increase in the heart rate compared with baseline levels, and this correlated with the concentration of ACh that was infused in the preimmune state. This pattern was qualitatively similar after immunization, which induced β₁AR-activating autoantibodies. However, the baseline and ACh values for heart rate were significantly higher in response to β₁AR-activating autoantibodies using each animal as its own control. We waited for 2 min when heart rate returned toward baseline values before instituting burst pacing under both circumstances. In a recent series of experiments in which we monitored both heart rate and blood pressure (unpublished data), we did indeed find the inverse baroreflex relationship so that the increase in heart rate was directly associated with a decrease in systolic and diastolic blood pressure. However, the increase in sympathetic activity based on the baroreflex effect could not account for the qualitative and quantitative differences, i.e., preponderance of induced sustained sinus tachycardia, in response to ACh and burst pacing seen in the postimmune state when each animal in the preimmune state showed no comparable arrhythmogenic effect due to the same protocol. Also, it should be noted that ACh released at presynaptic junctions can activate sympathetic neurons resulting in sinus rate acceleration. The significant increase in heart rate at all concentrations of infused ACh resulted in a positive chronotropic effect, which could also contribute to the initial sympathetic acceleration. There was a significant difference in the increased sinus rate with each concentration of infused ACh between the preimmune and postimmune state due to the direct agonistic action of the induced β₁AR-activating autoantibodies. In regard to burst pacing, this standard provocative intervention was unable to induce a substantial number of sustained arrhythmias in the preimmune state supposedly due to the tissue levels of catecholamines not being high enough even with the increase due to ACh infusions. On the other hand, with the combination of high titers of β₁AR-activating antibodies and increased secondary sympathetic action of ACh infusions, burst pacing now induced significant numbers of sustained supraventricular arrhythmias particularly arising from the sinus node. It is interesting to note that some of these sustained STs terminated spontaneously (n = 3) or were terminated by burst pacing (n = 5). Others were not terminated by several attempts with burst pacing (n = 7). There were no noticeable changes in the ECG parameters such as QRS or QT interval. Some of the rabbits did show changes in T wave amplitudes associated with increased sympathetic activity after immunization due to ACh infusions or to β₁AR antibody induction.

In a previous study we induced sustained atrial tachycardia in rabbits expressing high titers of β₂AR-activating autoantibodies (16). In the present report, using a similar protocol but with rabbits expressing β₁AR-activating autoantibodies, we were able to induce sustained supraventricular and ventricular tachyarrhythmias. Moreover, the majority of the supraventricular arrhythmias were of sinus origin (15/32 episodes) and two episodes of junctional origin. Only one episode of atrial tachycardia was observed, whereas there were three instances of sustained ventricular tachycardia noted. In the baseline state, i.e., before ACh infusion or burst pacing, there was a significant increase in the basal heart rate between the preimmune and postimmune studies. It could be argued that the increased heart rate was due to an increase in sympathetic activity associated with the initial surgery and subsequent trauma; however, in a similar protocol previously published (16) in which β₂AR-activating autoantibodies were induced, there was no significant increase in the baseline sinus rate before and after immunization. Thus it indicates that β₁AR-activating autoantibodies in the present study were directly associated with the significant increase in the sinus rate.

These findings correspond to previous studies of the βAR densities. In the rabbit, as opposed to human (18), the highest density of β₁AR was found in the specialized tissues, i.e., sinus node and other sites of pacemaker activity (20). Although β₁AR and β₂AR coexist in the rabbit atria, β₁AR is predominant in the rabbit ventricles (2, 4). It is interesting to note that there were three episodes of sustained VT after expression of β₁AR autoantibodies, whereas no sustained VT was induced in the preimmune state. These differences should be taken into consideration when applying these observations to the human. An important issue is the marked specificity of the autoantibodies, and the dependence of the site of the arrhythmia appears to be at least in part associated with the preponderance of the β_1/2AR subtype for that region. These observations have been more difficult to demonstrate in previous studies that use only partially specific orthosteric agonists and antagonists.

Lee et al. (15) addressed the evidence that autoantibodies targeting the cardiac autonomic receptors are involved in the development of various cardiovascular disorders and specifically associated cardiac arrhythmias. These autoantibodies display agonist-like properties in vitro and primarily target the second extracellular loops of the respective receptors. The sympathomimetic anti-βAR autoantibodies have been reported to be associated with primary ventricular arrhythmias (7), inappropriate sinus tachycardia (6), and a high incidence of VT and sudden death in dilated cardiomyopathy (10), whereas the parasympathomimetic autoantibodies to M2 muscarinic receptor are associated with both bradyarrhythmias and tachyarrhythmias, such as idiopathic sinus node dysfunction (5) and AF (1).

The importance of the previous report and the present study is to provide a model with direct evidence linking specific forms of supraventricular arrhythmias to receptor-specific activating autoantibodies as opposed to the indirect or observed associations described clinically. Furthermore, these models may provide specific targets for drugs that will neutralize the arrhythmogenic effects of the activating autoantibodies.

Limitations of the study.

Animal models have played an important role in our understanding of the impact of these autoantibodies on human disease (3, 9, 11, 12, 17). These studies have primarily focused on their cardiomyopathic effects, whereas study of their impact on cardiac arrhythmias has been more complicated owing to the small heart size of smaller rodents and their difficulty in sustaining complex tachyarrhythmias compared with that observed in larger hearts in animals such as the dog or pig. However, these latter animals do not lend themselves to immunological study for a variety of reasons, and this has impaired research in this venue. We have found the immunized rabbit presents a fertile avenue for examination of a variety of tachyarrhythmias (16). However, it is important to recognize there may be species-related differences in their manifestations of the tachyarrhythmias to make appropriate conclusions from the available data in regard to the human.

The previous and present studies were intended to provide prototypical models to examine the specific role of β-adrenergic activation on triggering and perpetuation of the arrhythmias. They also provide evidence for alteration of the substrate through remodeling associated with the persistent β₁AR antibody-induced tachycardia. Future studies will focus on using combinations of β₁AR- and β₂AR-activating autoantibodies with other receptor activators to simulate more complex arrhythmias present in chronic human autoimmune diseases, application of more selective testing of alterations in substrate susceptibility, and use of specific peptide antagonists for the activating autoantibodies to block their activity without altering their target receptor function.

We recognize that anesthesia can have an effect on cardiac electrophysiology and arrhythmogenesis. It should be noted that the induction and maintenance of anesthesia was the same in the pre- and postimmune states, thus, it seems unlikely that the differences in baseline heart rates and induction of sustained arrhythmias using each animal as its own control was based on the anesthetic employed. Moreover, in a previous report in which a similar protocol was instituted and β₂AR-activating autoantibodies were induced, burst pacing significantly increased the incidence of sustained atrial tachycardia rather than sinus tachycardia.

Conclusions

In summary, we have demonstrated the presence of β₁AR-activating autoantibodies predisposes the heart to tachyarrhythmias arising mainly at the sinus node. These data are consistent with the previous demonstration of a relative predominance of β₁AR in this region of the rabbit heart. The high specificity of these receptor-activating autoantibodies makes them useful for examination of the regional pathophysiology and mechanistic basis for cardiac arrhythmias.

GRANTS

This study was supported by the Heart Rhythm Institute at the University of Oklahoma Health Sciences Center; the Helen and Will Webster Arrhythmia Research Fund of the University of Oklahoma Foundation; the Talley Research Award of the Harold Hamm Diabetes Center at the University of Oklahoma; National Heart, Lung, and Blood Institute Grant HL-56267; American Heart Association Postdoctoral Fellowship; and a VA Merit Review grant.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: H.L., B.J.S., D.C.K., and X.Y. conception and design of research; H.L., B.J.S., A.B., X.S., and X.Y. performed experiments; H.L., B.J.S., D.C.K., A.B., X.S., M.W.C., R.L., C.E.A., and X.Y. analyzed data; H.L., B.J.S., D.C.K., M.W.C., R.L., C.E.A., and X.Y. interpreted results of experiments; H.L., B.J.S., and X.Y. prepared figures; H.L., B.J.S., D.C.K., and X.Y. drafted manuscript; H.L., B.J.S., D.C.K., M.W.C., R.L., C.E.A., and X.Y. edited and revised manuscript; H.L., B.J.S., D.C.K., A.B., X.S., M.W.C., R.L., C.E.A., and X.Y. approved final version of manuscript.