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Cardiovascular Research 2004 62(3):500-508; doi:10.1016/j.cardiores.2004.02.004
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Copyright © 2004, European Society of Cardiology

Gene dose-dependent atrial arrhythmias, heart block, and brady-cardiomyopathy in mice overexpressing A3 adenosine receptors

Larissa Fabritz*,a,1, Paulus Kirchhof*,a,1, Lisa Fortmüllera, John A Auchampachb, Hideo A Babac, Günter Breithardta, Joachim Neumannd, Peter Boknikd and Wilhelm Schmitzd

aDepartment of Cardiology and Angiology and Institute for Arteriosclerosis Research, University Hospital Münster, Münster, Germany
bDepartment of Pharmacology and Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI, USA
cInstitute of Pathology, University of Essen, Essen, Germany
dInstitute of Pharmacology and Toxicology, University Hospital Münster, Münster, Germany

* Corresponding authors. Medizinische Klinik und Poliklinik C, Kardiologie und Angiologie, Universitätsklinikum Münster, Albert-Schweitzer-Straße 33, Münster D-48129, Germany. Tel.: +49-251-8347638; fax: +49-251-8347864. Email address: fabritzl{at}uni-muenster.de kirchhp{at}uni-muenster.de

Received 6 December 2003; revised 31 January 2004; accepted 4 February 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: An increased expression of adenosine receptors is a promising target for gene therapy aimed at protecting the myocardium against ischemic damage, but may alter cardiac electrophysiology. We therefore studied the effects of heart-directed overexpression of A3 adenosine receptors (A3ARs) at different gene doses on sinus and atrio-ventricular (AV) nodal function in mice. Methods and results: Mice with heart-specific overexpression of A3AR at high (A3high) or low (A3low) levels and their wild-type littermates were studied. Telemetric electrocardiogram (ECG) recordings in adult freely moving A3high mice showed profound sinus bradycardia resulting in either ventricular escape rhythms or an incessant bradycardia–tachycardia syndrome (minimal heart rate A3high 217±25*; WT 406±21 beats/min, all values as mean±S.E.M., n=7 per genotype, *p<0.05). Exercise attenuated bradycardia in A3high mice (maximal heart rate A3high 650±13*; WT 796±13 beats/min) and first-degree AV nodal block was present (PQ interval A3high 36±4*; WT 23±5 ms). Isolated hearts showed complete heart block (10/17 A3high* vs. 1/17 WT). Atrial bradycardia and AV block were already present 3 weeks after birth. Doppler echocardiography revealed atrial dysfunction and progressive atrial enlargement that was moderate at 3 and 8 weeks, and progressed at 12 and 21 weeks of age (all p<0.05 vs. WT). Atrial contractility and sarcoendoplasmic Ca2+ ATPase (SERCA) 2a protein expression were reduced in isolated left A3high atria at the age of 14 weeks. Fibrosis was present in left A3high atria at 14 weeks, but not at 5 weeks of age. A3low mice had first-degree AV block without arrhythmias or structural changes. Conclusions: Heart-directed overexpression of A3AR resulted in gene dose-dependent AV block and pronounced sinus nodal dysfunction in vivo. Profound bradycardia heralded atrial and ventricular dilatation, dysfunction, and fibrosis. In contrast to A1 adenosine receptors (A1AR), the effects of A3AR overexpression were attenuated during exercise. This may have implications for the physiology of sinus nodal regulation and for therapeutic attempts to increase the expression of adenosine receptors.

KEYWORDS Integrative physiology; Heart rate regulation; Autonomous nervous system; Sinus node dysfunction; AV block; Atrial cardiomyopathy; A3 adenosine receptor; Transgenic mice

Abbreviations: A3AR, A3 adenosine receptor • A1AR, A1 adenosine receptor • A3high, mice with heart-directed high-level overexpression of A3AR • A3low, mice with heart-directed low-level overexpression of A3AR • AV, atrio-ventricular • ECG, electrocardiogram • SERCA, sarcoendoplasmic Ca2+ ATPase


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Heart rate is determined by the depolarization rate of pacemaker cells in the sinus node [1,2]. The ionic currents that depolarize pacemaker cells can be modulated, for example, by β-adrenoreceptors, muscarinic receptors, and adenosine receptors. We have recently reported that heart-directed overexpression of A1 adenosine receptors (A1ARs) causes a decreased chronotropic response to exercise and first-degree atrio-ventricular (AV) nodal block [3]. In addition to A1AR, A3 adenosine receptors (A3ARs) are expressed in the human heart [4] and their pharmacological stimulation modulates cardiac function [5]. Heart-directed overexpression of A3AR in mice protects the heart against ischemia, but may also cause ventricular dysfunction and bradycardia at high levels of overexpression [6]. As pharmacological treatment with A3AR agonists [7] and gene therapy with targeted A3AR expression [6,8] have been proposed to protect the myocardium against ischemic events, electrophysiological effects of A3AR overexpression may have physiological and clinical relevance.

Here, we report that heart-directed high-level overexpression of A3AR causes severe sinus node dysfunction at rest, bradycardia–tachycardia syndrome, and complete AV block in vivo in mice. Exercise attenuates bradycardia and AV block. Electrophysiological changes accompany atrial and ventricular dysfunction and fibrosis. Low-level overexpression of A3AR, in contrast, only results in first-degree AV block at rest. A1AR and A3AR differentially affect sinus nodal and AV nodal function. High-level overexpression of A3AR may cause brady-cardiomyopathy.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
Transgenic mice were bred from transgenic mouse lines with heart-directed ({alpha}-MHC promoter) overexpression of A3AR, as described in Ref. [6]. The two mouse lines used expressed about 13 fmol (A3low) or about 67 fmol/mg (A3high) high-affinity Gi-coupled A3AR per milligram of protein [6]. All experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the local animal care committee.

2.1. Electrocardiogram (ECG) measurements in sedated mice
Mice were sedated by intraperitoneal application of ketamine/xylazine (50 mg/20 mg mixture) at 16 weeks and urethane (2 g/kg body weight) at 18 weeks of age. Gel-covered silver wire loops were attached around the four limbs of the animals to record a six-lead surface ECG. Signals were preamplified and displayed on paper (amplitude 20 mV/mm, paper speed 100 mm/s; Siemens Megacart, Erlangen, Germany) [3].

2.2. ECG recordings in awake mice
Sixteen-week-old mice were instrumented with a telemetric ECG transmitter (Data Science, Minneapolis, USA) and lead II of the ECG was continuously recorded via two subcutaneous electrodes [3,9]. In addition, we recorded short strips of surface ECGs in nonsedated 3-week-old pairs of A3high and WT mice using flexible silver wire loops that were gently placed around the limbs of the animals. After a postoperative recovery period of 10 days, telemetric ECG recordings were obtained in freely moving mice during normal activity and during a standardized exercise protocol that has been described in detail before [3]. In brief, the telemetric ECG was first continuously recorded for 1 h during daytime. Mice were then placed in a water-filled tank for 6 min (20 x 30 cm width, 15 cm depth, water temperature 34 °C). After this submaximal exercise [3,10], mice were put back into their cages. The ECG was continuously recorded during swimming and for 55 min after swimming. In addition, 24-h periods of telemetric ECG recordings were analyzed for arrhythmias. Littermate pairs of WT and transgenic mice were simultaneously subjected to the protocol.

2.3. Isolated heart measurements
Isolated Langendorff-perfused mouse hearts of mice aged 40±3 weeks were studied using published techniques [3,11]. In brief, hearts were rapidly excised and perfused on a vertical Langendorff apparatus (Hugo Sachs Harvard Apparatus, March-Hugstetten, Germany) at a constant perfusion pressure of 90–100 mm Hg (flow rate 4±1 ml/min) [12]. An octapolar murine electrophysiology catheter (CI'BER mouse; NuMED, Hopkinton, NY, USA) was inserted into the right atrium and right ventricle. The perfused and instrumented heart was allowed to stabilize for 10 min. Thereafter, spontaneous rhythm was observed for 5 min. The right atrium was paced at constant heart rates (150–750 beats/min, 1.5 min of pacing per heart rate) via the octapolar catheter. Incremental atrial pacing was performed to test the antegrade conduction properties of the AV node. The pacing protocol was repeated during infusion of orciprenaline (1.4 µmol/l continuous infusion) [3].

2.4. Isolated atria
Atria were isolated from 14-week-old animals of either genotype and superfused in a tissue bath using repeatedly published methods (e.g., Ref. [13]). Left atrial preparations were electrically stimulated to assess atrial contractility. Right atrial preparations were superfused to assess spontaneous rate of contraction. The A3AR agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB MECA; Tocris Cookson, Ellisville, MO, USA) or the {alpha},β-adrenoreceptor agonist isoproterenol were applied for 10 min at different concentrations.

2.5. ECG analysis
All analyses were performed blinded to genotype. Surface ECG recordings were manually analyzed for heart rate, PQ, QRS, and QT intervals. Telemetric ECG recordings were digitally analyzed for heart rate and PQ intervals during 5-min periods of normal activity, during the swimming period, and during 5 min, 1 h after the beginning of swimming. The program automatically determined RR intervals; all computed intervals were verified manually. PQ intervals were compared during periods of equal heart rate. All recordings were manually reviewed for arrhythmias. Isolated heart recordings were analyzed for atrial and ventricular rate, presence of higher-degree AV block, and AV nodal conduction times measured as the time from the latest atrial activation to the earliest ventricular activation [3].

2.6. Histology and protein biochemistry
Hearts were rapidly excised, immersed in formalin, and stained using Sirius red stains. Atrial cell diameter was measured in 100 cells at the height of the nucleus. Additional right and left atrial tissue samples were shock-frozen and prepared for protein biochemistry [14]. We analyzed the protein level of phospholamban, calsequestrin, and sarcoendoplasmic Ca2+ ATPase (SERCA; SERCA and calsequestrin antibodies were kind gifts of Dr. L.R. Jones, Indianapolis, IN, USA) in atrial and ventricular preparations.

2.7. Doppler echocardiography
Sedated (intraperitoneal application of ketamine S 32 mg/kg body weight and xylazine 13 mg/kg body weight) pairs of mice were studied using a Sonos 5500 echocardiography system equipped with a 15-MHz linear transducer and a 12-MHz Doppler transducer (Philips Medical Systems) using published techniques [3]. The left atrium was visualized in the parasternal long axis in the plane of the aortic root, and the left atrial diameter was measured during ventricular systole at its maximal dimension in five 2D and M-mode images. Atrial function was assessed using transmitral Doppler flow measurements. Operators were blinded to genotype.

2.8. Statistics
Variables were compared between genotypes by post-hoc Student's t test and ANOVA analyses using statistical software (SPSS and Excel). Incidences of AV block and arrhythmias were compared using Fisher's exact test. Two-sided p values <0.05 were considered significant and are marked by an asterisk (*). Continuous variables are reported as mean±S.E.M.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Atrial arrhythmias in A3high mice in vivo
Freely moving and sedated 16- and 18-week-old A3high mice (n=7 per genotype) showed severe sinus bradycardia and repetitive episodes of atrial arrhythmias during normal activity, which were not present in WT animals (Fig. 1A and C, p<0.05). We hardly found periods of normal sinus rhythm during telemetric ECG recording in sedentary A3high mice. Nonsedated A3high mice already showed marked bradycardia alternating with atrial arrhythmias during short strips of six-lead ECG recordings at the age of 3 weeks (n=4 A3high and 4 WT, p<0.05; Fig. 1B). There were no arrhythmias 5 days after birth (n=5 A3high and 5 WT).


Figure 1
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  		Fig. 1 Sinus node dysfunction, AV block, and atrial arrhythmias in A3high mice. (A) Six-lead surface ECG recording from a wild-type (left) and A3high mouse (right) at 18 weeks of age. Marked right atrial bradyarrhythmia results in ventricular escape beats in the A3high mouse. Asterisks (*) indicate P waves. (B) Lead II of a representative surface ECG recording from a nonsedated wild-type (left panel) and A3high mouse (right panel) at 3 weeks of age. (C) Representative telemetric ECG recording from a freely moving WT and A3high mouse during normal activity at 18 weeks of age. Marked sinus bradycardia is followed by a run of atrial tachycardia. The ventricular rate is determined by ventricular escape beats. (D) Representative telemetric ECG recording from a freely moving WT and A3high mouse during swimming exercise. The A3high mouse shows sinus bradycardia and first-degree AV block.

 
3.2. Bradycardia in vivo
Adult (18 weeks old) sedated A3high animals had lower heart rates, prolonged PQ intervals, and longer QT intervals than littermate WT. Heart rate was already lower 3 weeks after birth (A3high 434±25 beats/min, WT 655±9 beats/min, n=4 per genotype, p<0.01) and 2 weeks after birth (A3high 520±40 beats/min, n=5; WT 681±31 beats/min, n=5, p=0.02).

Minimal heart rate was markedly depressed. Maximal heart rate was not reduced in A3high mice during normal activity (Fig. 2). During exercise and recovery from exercise, a regular supraventricular rhythm was present in A3high mice, albeit with persistent sinus bradycardia (Figs. 1D and 2)Go.


Figure 2
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  		Fig. 2 Marked resting bradycardia is attenuated during exercise in conscious A3high mice. Minimal, mean, and maximal heart rates are given for WT (open bars, n=7) and TG mice (filled bars, n=7) during normal activity (A), swimming exercise (B), and 1 h after swimming (C). Asterisks (*) indicate significant differences between A3high and WT mice.

 
3.3. AV nodal function in vivo
In A3high mice, AV nodal conduction could often not be determined during normal activity due to lower heart rates in the atrium than in the ventricle (Fig. 1). Intermittently, complete AV block was observed in freely moving A3high mice during normal activity. During exercise, all A3high mice developed supraventricular rhythms with 1:1 conduction, albeit with marked PQ prolongation (Figs. 1D and 3)Go.


Figure 3
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  		Fig. 3 Prolonged PQ interval in conscious A3high mice. Mean PQ interval during exercise in wild-type (open bars, n=7) and A3high mice (filled bars, n=7). Asterisks (*) indicate significant differences between TG and wild type. During normal activity, PQ intervals could not be determined in vivo due to marked sinus bradycardia.

 
A3low mice showed a slight prolongation of the PQ interval (sedated mice: A3low 42±2 ms, WT 37±1 ms, n=6 per group, p<0.05; freely moving mice: A3low 37.9±1.0 ms at heart rates of 467±12 beats/min; WT 34.9±0.7 ms at heart rates of 460±7 beats/min, p<0.05). The other ECG parameters were not different between A3low and WT mice (data not shown).

3.4. Electrophysiology in isolated hearts and atria
To assess AV nodal conduction at comparable atrial heart rates and to measure intrinsic heart rate [15] and intrinsic AV conduction in the intact heart deprived of autonomic influences, we studied isolated Langendorff-perfused hearts of adult A3high (n=17), A3low (n=7), and WT (n=17) mice during spontaneous rhythm and atrial pacing. This setup allows reproducible measurement of spontaneous, intrinsic heart rate (mean heart rates between 423 and 462 beats/min in wild-type hearts in previous studies) [3,11,12]. Spontaneous atrial rate was lower in A3high than in WT hearts (A3high 245±19 beats/min; WT 423±28 beats/min, p<0.05). Ten of 17 A3high hearts showed complete AV block (Fig. 4A). The β-adrenoreceptor agonist orciprenaline (1.4 µmol/l) accelerated atrial rate in WT hearts but not in A3high hearts (A3high 170±25 beats/min; WT 618±34 beats/min, p<0.05 between genotypes). As ventricular rates were higher than atrial rates in A3high hearts during orciprenaline infusion (mean ventricular rate 325±28 beats/min), recovery from AV block could not be assessed reliably. Three A3high hearts developed spontaneous episodes of atrial fibrillation. In A3low hearts, heart rate was not different from WT during baseline and, like WT, was accelerated by orciprenaline (A3low from 423±14 to 561±20, p<0.05 for drug vs. baseline).


Figure 4
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  		Fig. 4 Bradycardia, AV block, and atrial tachyarrhythmias in isolated hearts of A3high mice. (A) Incidence of AV nodal block in isolated WT and A3high mouse hearts. Shown is the presence of complete heart block (AV block III, black bars), second-degree AV block (AV block II, striped bars), and hearts without higher-degree AV block (open bars). Asterisks indicate significant differences between WT and A3high hearts. (B) Mean AV intervals in isolated WT and TG hearts during atrial pacing measured in hearts without higher-degree AV nodal block. AV interval (ordinate) is plotted vs. pacing cycle length (abscissa) for wild-type (open boxes) and A3high mouse hearts (filled boxes). Asterisks (*) indicate significant differences between A3high and WT hearts.

 
In those seven A3high hearts without complete AV block, AV conduction times were prolonged (Fig. 4B) and antegrade AV nodal conduction was slower during incremental atrial pacing (Wenckebach point: A3high 160±18 ms, WT 79±3 ms, p<0.05). Antegrade AV nodal conduction was not altered in A3low hearts (Wenckebach point A3low 76±2 ms, n=7).

Spontaneously beating right atria (14 weeks of age) showed bradyarrhythmias in all A3high preparations studied. Spontaneous beating rates were not decreased in A3low (WT 354±7 beats/min, A3low 351±26 beats/min). The A3AR agonist IB MECA (10–7 M) reduced spontaneous rates in A3low (286±36 beats/min), but not in WT atria (352±13 beats/min, n=7, p<0.05).

3.5. Atrial morphology and contractile function
Atrial size was measured in WT and A3high mice at the age of 2 weeks (7 pairs), 8 weeks (14 pairs), 12 weeks (13 pairs), and 21 weeks (10 pairs) by serial echocardiography: Atrial size was increased by 10% in A3high mice at the age of 2 and 8 weeks and further increased at the age of 12 and 21 weeks (Fig. 5), concordant with gravimetric results (Table 1). The peak of the atrial wave of transmitral Doppler flow, generated by the contraction of the left atrium, was diminished at 8 weeks of age in A3high mice (Table 2). Histologically, we found atrial fibrosis in A3high atria at the age of 14 weeks (Fig. 5), but not at the age of 5 weeks (n=3), except for the right atrium of one mouse at 5 weeks with extreme bradyarrhythmia. Diameter of atrial cells was unaltered in A3high at the age of 5 weeks (data not shown). Ventricular contractile function as assessed by M-mode echocardiography was normal in A3high mice at 8 weeks, but reduced at 21 weeks of age (Table 2). Atrial and ventricular morphology and function as assessed by echocardiography (n=7 per group) were normal in A3low hearts (12 weeks old).


Figure 5
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  		Fig. 5 Atrial dilatation and fibrosis in A3high mice. (A) Representative echocardiographic parasternal long axis views of a WT mouse heart and A3high mouse hearts at 8, 12, and 21 weeks of age. White bars indicate the left atrial diameter. (B) Mean left atrial diameter in WT (open bars) and A3high mice (filled bars). Asterisks (*) indicate significant differences between A3high and WT. For other echocardiographic parameters, see Table 2. (C) Sirius red staining of left atria from littermates aged 14 weeks indicating fibrosis in A3high atria.

 

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  		Table 1 Gravimetric and biochemical protein measurements in left atrial and ventricular samples of wild-type (WT) and A3high mice aged 14 weeks

 

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  		Table 2 Atrial and ventricular biplane, M-mode, and Doppler echocardiographic measurements in WT and A3high mice

 
Basal force of contraction was reduced in electrically stimulated left atria from A3high mice (14 weeks old, A3high 1.03±0.13 mN, n=10 vs. WT 2.08±0.31 mN, n=8), but not in A3low (14 weeks old, 1.78±0.26 mN, n=5). The A3AR agonist IB MECA reduced force of contraction in both A3low (1.22±0.4 mN, p<0.05 vs. WT and vs. A3high) and A3high (0.52±0.09 mN, p<0.05 vs. WT and vs. A3low) compared to WT atria (2.20±0.24 mN) at concentrations of 10–7 M and higher, values given for 10–7 M. The maximum contractile response to isoproterenol was attenuated in isolated atria from both A3low and A3high mice (WT 5.2±0.4 mN or 184±9% of control, A3low 3.1±0.3 mN or 147±6% of control, A3high 2.4±3.5 mN or 135±17% of control, n=8 per group, values given for isoproterenol concentrations of 10–5 M, p<0.05 vs. WT). Similar to findings in models of heart failure, expression of SERCA was decreased in A3high atria and ventricles compared to age-matched WT at the age of 14 weeks (Table 1), but not altered in atria from A3low hearts compared to WT.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
4.1. Main findings
Heart-directed high-level overexpression of A3AR in mice caused profound resting bradycardia, AV block, and incessant bradycardia–tachycardia syndrome. These electrophysiological effects were attenuated during exercise. Low-level A3 adenosine receptor overexpression only caused mild AV nodal conduction delay during periods of low autonomic tone. Atrial arrhythmias heralded atrial and ventricular cardiomyopathy, suggestive of a brady-cardiomyopathy. These findings have implications for the physiology of sinus nodal regulation and for gene therapy aimed at increasing the function of adenosine receptors.

4.2. Bradycardia–tachycardia syndrome
The sinus node hardly contributed to heart rate in A3high mice during normal activity in vivo. Rather, profound atrial bradycardia alternated with atrial arrhythmias, and heart rate was determined by ventricular escape rhythms. These findings extend the observation of bradycardia in tailcuff blood pressure measurements in A3high mice [6]. Our in vivo ECG recordings resemble those during severe forms of bradycardia–tachycardia syndrome. We found similar changes in isolated hearts of A3high mice, a setup that allows for assessment of the intrinsic heart rate [15]. This indicates that sinus nodal and AV nodal dysfunction of A3high hearts persists in the absence of autonomic regulation.

Atrial arrhythmias in A3high mice could either be due to intermittent activity of the sinus node, or afterdepolarizations and triggered activity [16–18] that may be provoked in A3high atria. Activation of IKACh (called also IKAdo) by A3AR receptor overexpression may also decrease the threshold of atrial tachyarrhythmias [19]. Further studies are needed to elucidate the mechanism of atrial tachycardias in A3high mice.

4.3. Attenuation of bradycardia and AV block during exercise
A3high overexpression caused bradycardia and AV block predominantly at rest (Figs. 1 and 2)Go, with resumption of normal supraventricular rhythms during exercise. Of note is the fact that systemic infusion of β-adrenoreceptor agonists (orciprenaline) did not reverse atrial bradycardia in the isolated heart, suggestive of other heart rate-increasing factors in vivo (e.g., direct innervation of the sinus node or blockade of cholinergic receptors). In the present mouse model of chronic A3high overexpression, desensitization of A3AR is possible. This may preferentially suppress Gi protein-dependent effects [22,23]. Receptor–ligand interaction studies have suggested that A3AR may also act via Gq proteins [22,23] or through Rho [23,24], in addition to Gi protein-mediated effects caused by the "empty" receptor [25].

4.4 Differential regulation of heart rate and AV nodal function by A1AR and A3AR
A1AR and A3AR have different postreceptor signaling pathways: A1ARs couple to Gi proteins that attenuate the effects of adrenergic stimulation [20,21]. Among other signaling pathways, A3AR can interact with Rho and phospholipase D [24]. An important role for Rho in the postreceptor signaling of A3high mice is suggested by the similarities between the atrial phenotype of mice overexpressing a constitutively active form of Rho [26] and A3high mice.

Consistent with an attenuation of the response to catecholamines, overexpression of A1 adenosine receptors causes a decreased chronotropic response to exercise, with little attenuation of resting heart rate [3]. Overexpression of A3AR, in contrast, depresses heart rate preferentially at rest. AV nodal regulation appears to be similarly regulated: A3 adenosine receptor overexpression, albeit at high levels, caused profound AV block predominantly at rest (Figs. 3 and 4)Go, consistent with tonic depression of heart rate. A1 adenosine receptor overexpression causes first-degree AV block that is not altered during exercise, but can be partially reversed by blockade of Gi proteins (Figs. 3 and 4 in Ref. [3]). Taken together, our findings suggest that A3AR may be involved in the tonic regulation of heart rate and AV conduction, as has been suggested for the anti-ischemic action of A3AR [27]. A1AR may rather be involved in attenuation of adrenergic regulation of heart rate and AV conduction.

4.5. Atrial arrhythmias pave the way for atrial and ventricular cardiomyopathy
Incessant atrial arrhythmias were present in juvenile A3high mice prior to atrial or ventricular dysfunction or structural remodeling. Furthermore, atrial size was only slightly increased in young mice and continued to increase with age, concomitant with atrial histological and biochemical changes suggestive of heart failure [28]. Fig. 6 summarizes the time course of electrophysiological, histological, biochemical, and contractile findings in this model. In the present study and in the previous report [6], ventricular myocardial disarray, dilatation, and altered function are present in A3high mice from the age of 14 weeks, with ventricular fibrosis not being evident up to the age of 28 weeks. Reduced SERCA expression at the age of 14 weeks in A3high mice is consistent with a previous report of decreased SERCA mRNA levels in the ventricles of A3high mice [6]. This may contribute to reduced force of contraction in isolated atria of A3 high mice, in addition to a potential regulation of calcium release by A3 adenosine receptors [29]. Our findings indicate that atrial and left ventricular dilatation and dysfunction in A3high mice develop over time and may be the morphological and functional reflections of a brady-cardiomyopathy. These findings are reminiscent of the ventricular hypertrophy and cardiomyopathy observed in dogs with chronic AV nodal block [30] and suggestive of an atrial form of "brady-cardiomyopathy" that over time also extends to the ventricular level.


Figure 6
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  		Fig. 6 Time course of the electrophysiological, structural, and contractile changes in A3high mice. Plotted are the relative differences in the measured parameters (ordinate, given in percent change of A3high and wild-type mice) vs. time (abscissa, in weeks). Bradycardia and atrial arrhythmias precede atrial and ventricular dysfunction and fibrosis, and atrial size markedly increases over time (see text for details).

 
Atrial dilatation may occur in mice without concurrent arrhythmias [31]. Atrial hypertrophy may be accompanied by heart block [32], and atrial dilatation may precede atrial fibrillation in mice [33]. In other transgenic mouse models, both atrial dilatation and atrial fibrillation, or atrial dilatation and diverse forms of sinus and AV nodal dysfunction have been reported [26,32–34]. An association of atrial arrhythmias, atrial enlargement, and atrial fibrosis has been reported in mice with high-level heart-directed overexpression of RhoA [26] or junctin [34], but the time course of these alterations has not been studied. Atrial brady-cardiomyopathy in A3high mice (i.e., atrial arrhythmias preceding atrial and ventricular mechanical dysfunction) is therefore, to the best of our knowledge, a novel finding.

4.6. Implications
Cardiac expression of A3AR alters the electrophysiological function of both the sinus node and AV nodal function in vivo. In contrast to our findings in A1AR-overexpressing mice, A3AR overexpression predominantly suppresses heart rate during periods of low autonomic tone in vivo.

Some of the ventricular hemodynamic and structural changes reported in the initial characterization of A3high mice [6] may have been a consequence of cardiac arrhythmias.

Enhanced expression or function of adenosine receptors has been suggested as a target for gene therapy to prevent postischemic myocardial damage [6,35]. Although differences in rate, ionic currents, and size between murine and human hearts implicate caution when transferring results to the clinical setting, our data suggest that high-level overexpression of A3AR may adversely affect atrial electrophysiology. The potential benefits notwithstanding, careful targeting and dosing accompanied by a complete cardiovascular evaluation is necessary to obtain a maximal level of safety for gene therapy aimed at enhancing expression or function of adenosine receptors.


   Acknowledgements
 
The authors would like to thank Daniela Holtmannspötter and Marcel Tekook for excellent technical assistance. L.F. is a fellow of the Emmy Noether-Programm of the Deutsche Forschungsgemeinschaft. This work was supported by the Deutsche Forschungsgemeinschaft as part of the Sonderforschungsbereich 556 "Heart failure and arrhythmias" (to P.K., L.F., and J.N.) and by the IZKF Münster (project ZPG4, to P.K.).

We—obviously excluding G.B.—would like to dedicate this work to the 60th birthday of Günter Breithardt MD., in February 2004.


   Notes
 
1 Contributed equally. Back

Time for primary review 30 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. DiFrancesco D. The onset and autonomic regulation of cardiac pacemaker activity: relevance of the f current. Cardiovasc. Res. (1995) 29:449–456.[CrossRef][ISI][Medline]
  2. Wainger B.J, DeGennaro M, Santoro B, Siegelbaum S.A, Tibbs G.R. Molecular mechanism of cAMP modulation of HCN pacemaker channels. Nature (2001) 411:805–810.[CrossRef][Medline]
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