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European Journal of Heart Failure 2008 10(12):1172-1176; doi:10.1016/j.ejheart.2008.09.014
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© 2008 European Society of Cardiology

Direct pro-arrhythmogenic effects of angiotensin II can be suppressed by AT1 receptor blockade in human atrial myocardium

Dirk von Lewinskia,*,1, Jens Kockskämpera,1, Sven-Ulrich Rübertusb, Danan Zhub, Jan D. Schmittoc, Friedrich A. Schöndubec, Gerd Hasenfussb and Burkert Pieskea

a Department of Cardiology, Medical University Graz Graz, Austria
b Department of Cardiology and Pneumology, Georg-August-University Göttingen, Germany
c Department of Thoracic and Cardiovascular Surgery, Georg-August-University Göttingen, Germany

* Corresponding author. Abteilung Kardiologie, Medizinische Universität Graz, Auenbruggerplatz 15, 8036 Graz, Austria. Tel.: +43 316 385 2544; fax: +43 316 385 3733. E-mail address: dirk.von-lewinski{at}meduni-graz.at (D. von Lewinski).


   Abstract
Top
 Notes
 Abstract
1. Background
 2. Aims
 3. Methods
 4. Results
 5. Conclusions
 References
 
Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. Indirect evidence from clinical trials demonstrates that chronic inhibition of the renin–angiotensin-system (RAS) significantly reduces the incidence of AF. Since mechanisms of this protective effect of RAS-blockade are poorly understood, we directly tested proarrhythmic effects of angiotensin II (Ang II) in human atrial myocardium.

Methods: Isolated trabeculae from human atrial appendages (n = 80) were electrically stimulated. We assessed isometric force and incidence of arrhythmic extra contractions (AECs) with and without increasing concentrations of Ang II (1–1000 nmol/L) in the absence or presence of receptor-blockade by saralasin (non-specific ATR-antagonist), irbesartan (AT1R-antagonist) or PD123319 (AT2R-antagonist).

Results: Twitch force and AECs concentration-dependently increased with Ang II. Effects became significant at concentrations >1 nmol/L Ang II and were maximal at 1000 nmol/L (increase in twitch force to 157±14% and AECs from 0 to 80%) saralasin and irbesartan partially prevented the inotropic effect of 100 nmol/L Ang II (by 4±12% and 68±6%; p<0.05), and completely prevented the occurrence of AECs.

Conclusion: Ang II exerts direct pro-arrhythmic effects in human atrial myocardium. These effects are mediated by AT1-receptors and can be prevented by AT1R-blockade. This mechanism may contribute to the beneficial effects of RAS-blockade on AF in clinical trials.

Key Words: Atrial fibrillation • Heart failure • Angiotensin • Remodelling

Received February 1, 2008; Revised June 9, 2008; Accepted September 22, 2008


   1. Background
Top
 Notes
 Abstract
 1. Background
2. Aims
 3. Methods
 4. Results
 5. Conclusions
 References
 
AF is the most common cardiac arrhythmia in clinical practice with increased risk for hospitalisation or death [1]. Despite its clinical significance, the cellular mechanisms that initiate AF are poorly understood. AF is associated with increased expression of Ang II and altered expression of Ang II-receptors [2]. Inhibition of the renin-angiotensin-system is effective against AF presumably via the reduction of atrial fibrosis. Thus, current evidence suggests that Ang II may increase the likelihood of AF in the long-term via an indirect effect, i.e. induction of structural atrial remodelling. The presented data indicate that Ang II may also induce arrhythmias in human atrial myocardium via a direct mechanism.


   2. Aims
Top
 Notes
 Abstract
 1. Background
 2. Aims
3. Methods
 4. Results
 5. Conclusions
 References
 
According to our hypothesis Ang II would induce and ATR antagonists would prevent the Ang II-induced arrhythmias in isolated trabeculae. Such a mechanism would create an additional rationale for the treatment of patients at risk of AF with AT1R antagonists.


   3. Methods
Top
 Notes
 Abstract
 1. Background
 2. Aims
 3. Methods
4. Results
 5. Conclusions
 References
 
Experiments were performed in muscle strips from right atrial appendages of 28 patients undergoing cardiac surgery (valve replacement (n=5), bypass surgery (n=23); 13 male). Appendages were excised before extracorporeal circulation was started. 25 patients had sinus rhythm and 3 patients had AF before surgery and average ejection fraction was 55±3%. The mean age of the patients was 64±3 years.

Premedication consisted of ACE-inhibitors or AT1R-blockers in 23, beta-blockers in 21, cardiac glycosides in 4, and diuretics in 14 patients, respectively. This study was approved by the Hospital Ethics Committee and all patients gave written informed consent to participate in the study.

3.1. Muscle strip preparation
Small trabeculae were dissected and connected to an isometric force transducer as described previously [3]. Preparations were superfused with Tyrode's solution and electrically stimulated at 1 Hz (37 °C).

Appendages were transported to the laboratory in ice cold cardioplegic Tyrode's solution containing (in mmol/L): Na+ 152, K+ 3.6, Cl– 135, HCO3– 25, Mg2+ 0.6, H2PO4– 1.3, SO42– 0.6, Ca2+ 0.2, glucose 11.2, Insulin 10 IU/L and 2,3-butanedione-monoxime (BDM) 30, equilibrated with carbogen (95% O2, 5% CO2) to a pH of 7.4. This solution protects the myocardium during transportation and from cutting injury at the time of dissection with full reversibility of the cardioplegic effects upon washout. Small endocardial trabeculae ("muscle strips", cross-sectional area <0.5 mm2) were dissected with the help of a stereo-microscope.

Muscles strips were mounted in special chambers between miniature hooks, connected to an isometric force transducer (Scientific Instruments, Germany) and superfused with modified Tyrode's solution (37 °C) of the composition given above except that BDM was omitted and [Ca2+]o was stepwise increased to 2.5 mmol/L. Isometric twitches were evoked through electrical stimulation with a stimulation voltage 25% above threshold (1 Hz, pulse duration 5 ms) and preparations were gradually stretched to 2-2.5 mN/mm2 diastolic tension.

3.2. Drugs
Inotropic and arrhythmogenic effects of Ang II (0.1, 1, 10, 100 or 1000 nmol/L) were tested. In subsequent experiments Ang II (100 nmol/L) was applied for 60 min in the absence or presence of saralasin (non-specific ATR-antagonist 1000 nmol/L), irbesartan (AT1R-antagonist 100 nmol/L) or PD123.319 (AT2R-antagonist 100 nmol/L).

3.3. Statistical analysis
Data are expressed as mean±SEM. Differences were compared by paired or unpaired Student's-t-test as appropriate. Statistical significance was taken as p<0.05.


   4. Results
Top
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 Abstract
 1. Background
 2. Aims
 3. Methods
 4. Results
5. Conclusions
 References
 
In human atrial myocardium from patients with sinus rhythm, Ang II induced a concentration-dependent positive inotropic (PIE) and arrhythmogenic effect as shown in Fig. 1: Ang II (100 nmol/L) increased force of contraction briefly after administration and induced the occurrence of AECs about 1 min later.


Figure 01
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  		Fig. 1 Original recording demonstrating typical effects of Ang II in a human atrial muscle strip. Isometric twitch force is shown at two time scales (see 2 min and 10 s scale bars). At higher time resolution and the zoomed inlay, individual twitches are easily visible. Following application of 100 nmol/L Ang II an initial increase in twitch tension was followed by the initiation of arrhythmic extra contractions (AECs indicated by arrows, the first AEC is marked by the asterisk).

 
Fig. 2 summarizes average data of the effects of Ang II on force of contraction (left) and AECs (right). The PIE of Ang II started at 10 nmol/L and was maximal at 1000 nmol/L, the highest concentration applied. At 1000 nmol/L, the force of contraction increased to 157±14% of baseline. Assuming a maximum PIE of Ang II at 1000 nmol/L, the EC50 of Ang II was 20 nmol/L and the Hill coefficient 0.7.


Figure 02
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  		Fig. 2 Left: Concentration-dependent positive inotropic effect of Ang II (1-1000 nmol/L). Each muscle was exposed to a single concentration of Ang II. Numbers in parentheses indicate number of trabeculae studied. *p<0.05 vs. baseline. Right: Arrhythmic extra contractions (AECs) of 1-1000 nmol/L Ang II. Each muscle was exposed to a single concentration of Ang II. Numbers in parentheses indicate number of trabeculae studied. *p<0.05 vs. baseline.

 
The concentration-dependent effects of Ang II on AECs are shown in Fig. 2 (right). At 10 nmol/L, 3 out of 8 (38%) muscles showed arrhythmias, and this increased to 10/14 (72%) at 100 nmol/L and 7/9 (78%) at 1000 nmol/L Ang II. The EC50 of Ang II on AECs was 23 nmol/L with a Hill coefficient of 1.3. These values did not differ significantly from the EC50 for force of contraction. Three of the muscle strips used were taken from three patients who were not receiving AT1R antagonists or ACE-inhibitors. All three muscles showed AECs, although the Ang II induced PIE was smaller than the average of all muscles.

To elucidate the ATR subtypes involved in the inotropic and arrhythmogenic effect, Ang II (100 nmol/L) was added alone or in the presence of the nonselective ATR-antagonist saralasin, the selective AT1R-antagonist irbesartan or the selective AT2R-antagonist PD123319. As can be seen from Fig. 3A, maximal inotropic response to Ang II was significantly reduced in the presence of saralasin or irbesartan (from 140±7% to 116±7% and 101±2%), whereas PD123319 (123±8%) did not significantly attenuate the inotropic response to Ang II (p=0.11).


Figure 03
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  		Fig. 3 A: Positive inotropic effect of Ang II without ATR inhibition (Cont) and after 30 min pre-incubation with saralasin (Sar), irbesartan (Irb) or PD123319 (PD). Maximum increase is shown. Numbers in parentheses indicate number of trabeculae studied. *p<0.05 vs. baseline; #p<0.05 vs. Cont. B: Arrhythmic extra contractions (AECs) induced by Ang II (100 nmol/L) without (Cont) and after 30 min pre-incubation with saralasin (Sar), irbesartan (Irb) and PD123319 (PD). Average numbers of AECs after the first 20 min is shown on the left (early), AECs after 40-60 min is shown on the right (late). *p<0.05 vs. baseline; #p<0.05 vs. Cont.

 
AECs began to develop a few minutes after Ang II application. Several muscles displayed sustained arrhythmias, others recovered to regular rhythmic contractions. Fig. 3B therefore shows both early (first 20 min after Ang II administration) and late (from 40-60 min after Ang II) AECs. Ang II induced 11.2±4.9 AECs/min (early, left) and 5.8±2.7 (late, right) AECs/min, respectively (both p<0.05). Both saralasin and irbesartan almost completely prevented the arrhythmic effects of Ang II both during the early and late phase, while PD123319 had no effect.

An additional subset of experiments was performed in eight muscle strips of patients with AF. In these preparations PIE after administration of 1000 nmol/L Ang II was considerably lower (to 117±4% of basal value) compared to patients without AF. None of these muscles developed AECs.


   5. Conclusions
Top
 Notes
 Abstract
 1. Background
 2. Aims
 3. Methods
 4. Results
 5. Conclusions
References
 
Inhibition of the angiotensin-system significantly reduces AF in large cohorts [4,5]. In addition, a prospective study [6] has shown that the AT1R-antagonist irbesartan reduced the incidence of recurrent AF in patients after electrical cardioversion if given in addition to amiodarone. This finding was confirmed with the ACE-inhibitor enalapril in a second trial [7].

Atrial tissue exerts an elevated propensity for the development of fibrosis compared to ventricular myocardium [8-10] and atrial fibrosis is a substrate for AF. Inhibitors of the renin-angiotensin system prevent adverse remodelling and exert anti-fibrotic effects [11] and this might explain the beneficial effects of AT1R-blockade on AF in clinical trials.

Acute direct arrhythmogenic effects of Ang II, however, have not been demonstrated before in human atrium. A small in-vivo study in humans did not show any effects of Ang II on atrial electrophysiology in 12 patients treated with Ang II infusions [12]. However, plasma levels were low in this study (maximum ~0.5 nmol/L), a concentration which did not exert AECs in our study, either. It is of note that Ang II concentration in the interstitial fluid of the heart is much higher than in plasma as shown in dogs, potentially due to autocrine-paracrine secretion [13]. In addition, AF induced by rapid atrial pacing induces shortening of the atrial effective refractory period (AERP) and a loss of rate-related AERP shortening increasing the inducibility and stability of AF. In a dog model, ACE-inhibitors as well as AT1R-blockers prevent shortening of the AERP [14].

Our data support this hypothesis showing that in parallel to the known PIE [15], Ang II concentration-dependently induces arrhythmias in isolated human atrial myocardium within minutes after application. The arrhythmogenic effect is inhibited by the unselective ATR-antagonist saralasin as well as the selective AT1R-antagonist irbesartan, whereas PD123319, a selective AT2R-blocker, has no effects. This indicates that the direct arrhythmogenic effect of Ang II is mainly mediated via AT1R signalling. A possible cellular mechanism for this direct arrhythmogenic effect of Ang II may be altered sarcoplasmic reticulum (SR) Ca2+ release. In isolated human atrial myocytes, Ang II increased the frequency of spontaneous ryanodine receptor (RyR)-mediated elementary Ca2+ release events, i.e. Ca2+ sparks [16]. This effect was mediated by AT1Rs since it was blocked by candesartan. Furthermore, we showed recently that in human atrial myocardium, endothelin-1 elicits arrhythmias via ETARs coupling to PLC-IP3 signalling [17]. IP3-induced SR Ca2+ release triggers further SR Ca2+ release through RyRs and induces arrhythmias through Na+/Ca2+ exchanger-mediated membrane depolarization. Since AT1Rs, like ETARs, couple to the IP3 pathway, we hypothesize that Ang II-induced atrial arrhythmias are mediated by IP3-dependent diastolic SR Ca2+ release, activation of electrogenic Na+/Ca2+-exchange, and induction of delayed afterdepolarizations (DADs). Interestingly, in human AF AT1R density is down-regulated, whereas AT2R density is up-regulated [2]. Since both direct (acute) and indirect (long-term) arrhythmogenic effects of Ang II appear to be mediated largely by AT1Rs, this may be considered a compensatory mechanism in the face of elevated expression of ACE and Ang II in this disease. This notion is supported by the observation that only a smaller PIE and no AECs were induced in muscle strips from patients with pre-existing AF.

In conclusion, the presented data demonstrate, for the first time in intact human myocardium, that Ang II induces direct AT1R-mediated arrhythmogenic effects in human atrium. They support the notion that AT1R-antagonists not only prevent AF due to long-term attenuation of atrial fibrosis but also that they may inhibit acute arrhythmogenic effects of Ang II.


   Acknowledgement
 
This work was supported by Sanofi-Aventis Deutschland GmbH.


   Notes
Top
 Notes
Abstract
 1. Background
 2. Aims
 3. Methods
 4. Results
 5. Conclusions
 References
 
1 These authors contributed equally. Back


   References
Top
 Notes
 Abstract
 1. Background
 2. Aims
 3. Methods
 4. Results
 5. Conclusions
 References
 

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