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Cardiovascular Research Advance Access first published online on January 31, 2008
This version [Corrected Proof] published online on February 26, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn020
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Cardiac-specific overexpression of the human type 1 angiotensin II receptor causes delayed repolarization

Katy Rivard1,2, Pierre Paradis3,{dagger}, Mona Nemer3 and Céline Fiset1,2,*

1 Research Centre, Montreal Heart Institute, 5000 Rue Bélanger, Montréal, QC, Canada H1T 1C8
2 Université de Montréal, Faculty of Pharmacy, Montréal, QC, Canada
3 Institut de Recherche Clinique de Montréal, Montréal, QC, Canada

* Corresponding author. Tel: +1 514 376 3330 (3025); fax: +1 514 376 1355. E-mail address: celine.fiset{at}icm-mhi.org

Received 8 December 2007; revised 8 January 2008; accepted 11 January 2008

Time for primary review: 41 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Mice with cardiac-specific overexpression of human angiotensin II type 1 receptor (AT1R) undergo cardiac remodelling and die prematurely of sudden death. Since excessive QT prolongation is a major risk factor for ventricular arrhythmias and sudden death, we hypothesize that chronic stimulation of AT1R might contribute to sudden death by promoting delayed repolarization and ventricular arrhythmias.

Methods: In the present study, a detailed analysis of ventricular repolarization parameters was undertaken in AT1R mice.

Results: Measurement of K+ currents in ventricular myocytes isolated from 6–8 months AT1R male mice revealed a significant reduction of the Ca2+-independent transient outward (Ito), the ultra-rapid delayed rectifier (IKur), and the inward rectifier (IK1) K+ currents compared with littermate controls (CTL). The expression of the underlying K+ channels was also decreased in AT1R ventricles. Moreover, reactivation of Ito was slower in AT1R mice. Consistent with these findings, AT1R mice presented a longer action potential duration (APD90, CTL: 19.0 ± 1.8 ms; AT1R: 39.1 ± 4.7 ms, P = 0.0001) and QTc interval (CTL: 53.6 ± 1.5 ms, AT1R: 64.2 ± 1.4 ms, P = 0.0005). In addition, spontaneous ventricular arrhythmias were reported in the AT1R mice. Importantly, the increased incidence of arrhythmia and the repolarization defects also occurred in much younger AT1R mice that do not present signs of hypertrophy, confirming that these arrhythmogenic changes are not secondary to cardiac remodelling.

Conclusion: These results strongly suggest that chronic stimulation of AT1R directly leads to an increased incidence of cardiac arrhythmia associated with delayed repolarization.

KEYWORDS Angiotensin II; K+ currents; Ventricular repolarization; Arrhythmias; Transgenic mice


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Angiotensin II (ANG II), the active end product of the renin–angiotensin system (RAS), has numerous physiological effects on the cardiovascular system. Unfortunately, under pathological conditions, the increased activity of RAS contributes to the development of ventricular hypertrophy and remodelling.1–5 To date, most of the cardiac damaging effects of ANG II appear to be mediated by the ANG II type 1 receptor (AT1R).6 Although it is well recognized that ANG II is involved in the initiation of cardiac remodelling,7–10 relatively little is known concerning the role of ANG II in the higher propensity of lethal cardiac arrhythmias associated with heart disease.4,5,11 Activity of the RAS appears to be able to alter repolarization in myocardium.12 However, while transgenic mouse models with cardiac restricted overexpression of different components of the RAS have been instrumental in studying the role of ANG II in cardiac remodelling,10,13,14 no detailed characterization of cardiac cellular excitability has been realized using these genetically manipulated mouse models. Thus far, only a small number of studies have documented an increased incidence of cardiac arrhythmias and sudden death in these transgenic mice.15–17 Blockade of AT1R (AT1R antagonist, ARA) has been shown to decrease the arrhythmia morbidity in mice with ventricular hypertrophy.4 In addition, angiotensin-converting enzyme inhibitors (ACEI) have been reported to restore normal ventricular action potential duration (APD), refractoriness, and reduced susceptibility to ventricular fibrillation.5 Recently, using transgenic mice with cardiac-specific ANG II overproduction, Domenighetti et al.13 reported an increased incidence of ventricular arrhythmias and QT interval prolongation associated with a reduced density of inward rectifier K+ current in mouse. However, they did not examine any of the other K+ currents/channels involved in mouse ventricular repolarization.

Transgenic mice overexpressing the human AT1Rs specifically in cardiomyocytes have been shown to develop hypertrophy and heart failure in the absence of hypertension.10 Moreover, these AT1R mice die prematurely and spontaneously, suggesting that under pathological conditions, ANG II might contribute to cardiac sudden death by promoting severe cardiac arrhythmias. Since excessive QT prolongation, which reflects delayed repolarization, is a major risk factor for ventricular arrhythmias and sudden death, we conducted the present study to determine whether there was a causal relationship between chronic AT1R activation, delayed repolarization, and cardiac arrhythmias. Findings presented here reveal that overexpression of the AT1R in the myocardium leads to delayed repolarization which could contribute to the increased incidence of cardiac arrhythmia.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
A detailed description of all the following methods used in this study can be found in the online data supplement.

2.1 Animals
Experiments were performed on male C57BL/6 AT1R mice aged of either 6–8 months or 50 ± 5 days. This transgenic mouse model overexpressing the human AT1R receptor (200-fold in the ventricles of the transgenic mice of both age groups) specifically in cardiomyocytes was described previously.10 Heterozygous transgenic mice (AT1R) and age-matched wild-type littermates (CTL) were used. All experiments were conducted in accordance with the Canadian Council Animal Care guidelines and 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).

2.2 Ventricular myocyte isolation
The ventricular myocyte isolation protocol has been described previously.18

2.3 Electrophysiology
2.3.1 Cellular electrophysiology
All experiments were carried out at room temperature (20–22°C). The whole-cell voltage- and current-clamp recording methods, data acquisition, voltage-clamp protocols, and analysis methods have been described previously.19

2.3.2 Surface ECG
Mice were anaesthetized with isoflurane. Electrodes were placed subcutaneous and surface ECG were acquired in lead I configuration. The QT intervals were corrected (QTc) for the heart rate using the formula for mice (QTc=QT/(RR/100)1/2).20

2.3.3 Telemetry recording
Telemetry was used to record spontaneous ventricular arrhythmias in conscious free moving unanaesthetized CTL and AT1R mice. ECG lead placement represented lead II configuration.

2.4 Western blots analysis
Protocols used for isolation of sarcolemmal-enriched protein and Western blot analysis were identical to those reported previously.18,21

2.5 Real-time PCR
RNA levels of the different K+ channels (Kv1.5, Kv2.1, Kv4.2, KChIP2, and Kir2.1) were determined by real-time PCR as described previously.22 mRNA expression was quantified relative to cyclophyllin.

2.6 Echocardiography
Echocardiography was performed as described previously.23 This was done to determine the left ventricular internal dimension in systole and diastole (LVIDs and LVIDd, respectively) and the fractional shortening (FS).

2.7 Statistical analysis
Results are expressed as mean ± SEM. An unpaired Student's t-test was used to compare means. P-value of <0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Alteration of K+ currents in 6–8 months AT1R mouse ventricular myocytes
Figure 1A shows that the total K+ current (Ipeak) was significantly smaller in AT1R myocytes compared with CTL cells (at +30 mV, CTL = 68.1 ± 9.2 pA/pF and AT1R = 28.2 ± 4.6 pA/pF; P = 0.001). The individual K+ conductance contributing to Ipeak in mouse ventricular tissue were then compared. These K+ currents correspond to (i) the Ca2+-independent transient outward (Ito), (ii) the ultra-rapid delayed rectifier (IKur), (iii) the steady-state outward (Iss), and (iv) the inward rectifier (IK1) K+ currents.19 Mean current–voltage (I–V) relationships (Figure 1B) show a significant decrease of Ito in AT1R cells compared with CTL myocytes (at +30 mV, CTL = 31.9 ± 3.9 pA/pF and AT1R = 9.8 ± 1.4 pA/pF; P = 0.00004). As shown in Figure 1C, IKur also was significantly decreased in AT1R mice (at +30 mV, CTL = 27.3 ± 5.5 pA/pF and AT1R = 9.8 ± 1.4 pA/pF; P = 0.04). In contrast, the third component of the outward K+ current, Iss, was similar in both groups (P = NS) (Figure 1D). Figure 1A and D also presents data on IK1. The inward portion of IK1 was significantly smaller in AT1R myocytes compared with CTL cells (at –110 mV, CTL = –21.3 ± 1.2 pA/pF and AT1R = –11.9 ± 1.1 pA/pF, P = 0.00009). The outward part of IK1 (–80 to –40 mV) was also significantly decreased in AT1R myocytes (at –60 mV, CTL = 1.6 ± 0.2 pA/pF and AT1R = 0.9 ± 0.1 pA/pF, P = 0.003).


Figure 1
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  		Figure 1 K+ currents in 6–8 months AT1R ventricular mouse myocytes. (A) The total K+ current (Ipeak). Left: representative family of Ipeak recorded from CTL and AT1R ventricular myocytes using the protocol shown in inset. Right: comparison of mean I–V curves for Ipeak (*P < 0.05). (B) Ito—left. Superimposed current records corresponding to Ito calculated by subtraction of current traces recorded with and without an inactivating prepulse (100 ms, –40 mV) applied before the main activation steps (protocol shown in inset, D).19 Right: mean I–V curves for Ito (*P < 0.05). (C) IKur—left. After inactivation of Ito the remaining outward current (recorded with the protocol shown in inset, D) is composed of IKur and Iss and can be separated with 100 µM of 4-aminopyridine (4-AP). Family of membrane currents presented in the left panel represents IKur (the 4-AP-sensitive component) in CTL and AT1R cells. Right: corresponding mean I–V curves for IKur (*P < 0.05). (D) Iss and IK1—left. Typical examples of IK1 and the 4-AP resistant component of the outward current, Iss, recorded in CTL and AT1R myocytes. Right: corresponding I–V curves for IK1 and Iss showing that IK1 was significantly reduced in AT1R mice (*P < 0.05), whereas Iss was similar in CTL and AT1R myocytes. Electrophysiological protocols used to record the different K+ currents are described in the Supplementary material.

 
Figure 2A compares the voltage dependence of steady-state inactivation of Ito in CTL and AT1R ventricular myocytes. The right panel presents Boltzmann functions fitted to mean data. The steady-state inactivation of Ito was similar in both groups (V1/2, CTL = –53.2 ± 0.7 mV; AT1R = –52.4 ± 0.7 mV, P = 0.3; slope factor (k), CTL = 5.9 ± 0.5 mV; AT1R = 7.3 ± 0.8 mV, P = 0.2). Figure 2B shows the recovery from inactivation of Ito in AT1R mice. Examples of the family of currents that illustrate the time course of recovery at –80 mV for CTL and AT1R myocytes are shown in the left panel. The recovery from inactivation was significantly slower in AT1R myocytes (time constant: 79 ± 7 ms) compared with controls (34 ± 2 ms) (P = 0.000001).


Figure 2
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  		Figure 2 Steady-state inactivation and reactivation of Ito in 6–8 months AT1R ventricular mouse myocytes. (A) Voltage dependence of steady-state inactivation of Ito. Left: typical examples of family of currents. Right: plot of voltage dependence of the steady-state inactivation of Ito. The amplitude of Ito for each pre-pulse membrane potential was determined by subtracting each test pulse current with that obtained with the –40 mV pre-pulse. The Ito test pulse amplitude was normalized to the amplitude at the most negative pre-pulse potential (I/Imax). (B) Reactivation of Ito. Left: example of family of membrane currents showing the time course of recovery of Ito from inactivation. A 500 ms inactivating pulse (+50 mV) was followed at intervals of 10, 20, 30, 40, 50, 60, 80, 100, 200, 400, and 600 ms by an identical test pulse. Right: membrane potential dependence of recovery from inactivation of Ito. P2/P1 is the ratio of test pulse current/pre-pulse current amplitudes. Ito amplitude was measured as the difference between peak outward current and the current 150 ms after the peak. The smooth lines are best-fit single exponential functions.

 
We also studied the steady-state inactivation and the recovery from inactivation of IKur in AT1R mice. The voltage dependence of steady-state inactivation of IKur was comparable in CTL (n = 12) and AT1R (n = 6) cardiomyocytes (V1/2, CTL = –41.4 ± 1.1 mV and AT1R = –41.1 ± 1.1 mV, P = 0.9; k, CTL = 6.0 ± 0.4 mV and AT1R = 6.2 ± 1.0 mV, P = 0.9). The recovery from inactivation of IKur was also comparable in CTL and AT1R myocytes, with a time constant of 552 ± 59 ms in CTL (n = 12) and 490 ± 33 ms in AT1R (n = 9) (P = 0.4). These results indicate that the reduced IKur density observed in AT1R ventricular myocytes cannot be explained by an alteration in IKur inactivation kinetic properties.

3.2 Changes in K+ channels expression in AT1R mouse ventricle
Using Western blot analysis, we compared the protein expression levels of the K+ channels (Kv1.5, Kv2.1, Kv4.2/Kv4.3, and Kir2.1) corresponding to the murine ventricular K+ currents (IKur, Iss, Ito, and IK1, respectively).24–27 In line with the electrophysiological data, protein expression of Kv1.5, Kv4.2, and Kir2.1 was decreased in AT1R ventricle compared with CTL (Figure 3A). However, Kv4.3 protein expression was similar in the two groups, whereas Kv2.1 was increased in AT1R ventricle compared with controls.


Figure 3
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  		Figure 3 K+ channel expression in 6–8 months AT1R mouse ventricles. (A) Protein expression of K+ channels. Western blot analysis of Kv1.5 (1:500), Kv2.1 (1:500), Kv4.2 (1:1000), Kv4.3 (1:5000), and Kir2.1 (1:500) on sarcolemmal-enriched fraction (100 µg/lane, one ventricle/sample) isolated from CTL and AT1R ventricle. Equal protein loading was confirmed by ponceau-S stained membranes. Bar graph (right) represents relative protein expression of the K+ channels determined by densitometry. Relative abundance was calculated with the value of CTL as a reference of 100% (*P < 0.05). (B) mRNA expression of K+ channels. Bar graph comparing the abundance of Kv1.5, Kv2.1, Kv4.2, KChIP2, and Kir2.1 mRNA transcripts in CTL and AT1R mice determined by real-time PCR. Each sample was analysed in triplicate (*P < 0.05).

 
To determine whether the changes observed in the protein level could be explained by transcriptional or post-transcriptional mechanisms, real-time PCR analysis was carried out on the K+ channel isoforms that exhibit differences at the protein level. Since both density and voltage-dependent kinetics of recovery from inactivation of Ito can also be influenced by the expression of the KChIP2 ancillary subunit, we also determined whether the transcript level of KChIP2 was affected by the AT1R overexpression. In support of a transcriptional regulation for Kv4.2 and KChIP2, the real-time PCR experiments revealed a significant reduction of Kv4.2 and KChIP2 mRNA transcripts in AT1R compared with CTL ventricles. In contrast, the mRNA levels for Kv1.5, Kv2.1, and Kir2.1 were similar between the two groups (Figure 3B), suggesting a post-transcription mechanism of regulation for these subunits.

3.3 Physiological implication of the decrease of the K+ currents in AT1R mice
Consistent with the decrease of the K+ currents in AT1R ventricles, APD was significantly longer in AT1R myocytes compared with those measured in CTL myocytes (in ms) (APD20, CTL = 2.5 ± 0.2; AT1R = 4.3 ± 0.3, P = 0.00006), (APD50, CTL = 5.2 ± 0.9; AT1R = 9.7 ± 1.2, P = 0.007), (APD90, CTL = 19.0 ± 1.8; AT1R = 39.1 ± 4.7, P = 0.0005) (Figure 4A). Moreover, in keeping with the prolongation of the ventricular APDs, both QT and QTc intervals were significantly prolonged in AT1R compared with CTL mice (Figure 4B). However, heart rate was comparable between both groups. Of note, the QRS complex and the QRS corrected for the heart rate (QRSc) were significantly longer in the AT1R mice. However, even after subtracting the QRS (or QRSc) from the QT (or QTc), the prolongation was still statistically significant (respectively, P = 0.02 and 0.01) (Figure 4B), confirming that the repolarization process is markedly delayed in the AT1R mice.


Figure 4
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  		Figure 4 Ventricular repolarization and cardiac rhythm in 6–8 months AT1R mice. (A) Comparison of APDs between CTL and AT1R mice. Left: typical examples of AP recorded at 4 Hz. Bar graph (right) shows mean APD20, APD50, and APD90 (±SEM) (*P < 0.01). (B) Table comparing mean values for ECG parameters between CTL and AT1R mice obtained with surface ECG recordings. (C) Representative ECG recordings obtained with telemetry monitoring in CTL and AT1R mice. These examples illustrate polymorphic (b) and monomorphic (c) irregular rhythms observed in two AT1R mice, whereas no rhythm abnormalities were recorded in CTL (a).

 
ECG recordings have been obtained using a telemetry system on free moving CTL and AT1R mice to determine whether the overexpression of the AT1R was associated with spontaneous arrhythmias. Figure 4C illustrates typical examples of ECG traces recorded from control (a) and AT1R (b–c) mice aged 6–8 months. While none of the six CTL mice experienced rhythm abnormalities, a mixture of spontaneous ventricular arrhythmias was observed in AT1R mice. The examples presented in Figure 4C illustrate different irregular rhythms observed in AT1R mutant mice: a polymorphic ventricular arrhythmia and a normal sinus rhythm interrupted by episodes of irregular rhythm with wider QRS complexes. Similar arrhythmia episodes have been observed in five out of six AT1R mice. These arrhythmias were seen throughout the 24 h recording period.

3.4 Cardiac remodelling in 6–8 months AT1R mice
A common electrical alteration present in heart diseases is the prolongation of APD,28–30 a known risk factor for severe ventricular arrhythmias. Thus, it was important to determine whether the arrhythmogenic changes described in the AT1R mice were the result of alterations in the context of cardiac remodelling or a direct consequence of chronic AT1 stimulation. To address this question, young adult mice (50 ± 5 days) also were studied. We first examined cardiac structure and function of these AT1R mice. The results showed that compared with CTL, the 6–8 months AT1R mice exhibit an increased left ventricular mass (LVmass) to body weight (BW) ratio [LVmass/BW (mg/g), CTL: 3.39 ± 0.20; AT1R: 4.26 ± 0.25; P = 0.01] (Figure 5A). Cell capacitances (an indication of cell volume) of ventricular myocytes also were larger in AT1R mice (CTL: 162.8 ± 8.5 pF; AT1R: 216.4 ± 23.5 pF; P = 0.01) (Figure 5B). Thus, these data indicate that the hearts from AT1R mice exhibit hypertrophy. Using ventricular internal dimensions in systole and diastole (determined by echocardiography), the FS was calculated. A significant reduction of this marker of cardiac function was observed in AT1R hearts (CTL: 55.6 ± 0.9%; AT1R: 27.4 ± 1.3%, P < 0.00001) (Figure 5C and D). Taken together, these data demonstrate that there was clear evidence of both cardiac hypertrophy and failure in 6–8 months AT1R mice.


Figure 5
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  		Figure 5 Cardiac function in 6–8 months AT1R mice. (A) Bar graph compares the left ventricular mass (LV mass) corrected for body weight (BW) between CTL and AT1R (P = 0.01). (B) Comparison of the cell capacitance measured in CTL and AT1R ventricular myocytes (P = 0.01). (C) Typical example of an M-mode echocardiography used to calculate the fractional shortening. (D) Left ventricular fractional shortening = (LVIDd – LVIDs/LVIDd) x 100 (P < 0.00001).

 
3.5 Ventricular repolarization defects and cardiac arrhythmias in younger AT1R mice
To ascertain that the repolarization defects seen in AT1R mice were not secondary to cardiac hypertrophy or failure, we characterized ventricular repolarization in much younger AT1R mice (50 ± 5 days) that have similar AT1R expression as 6–8 months AT1R mice, but do not present cardiac remodelling. Capacitance of the ventricular myocytes of these young AT1R mice was comparable with that of age-matched littermate controls (CTL: 149.2 ± 6.9 pF, n = 20; AT1R: 139.5 ± 8.0 pF, n = 19, P = 0.4) further confirming the absence of hypertrophy at the cellular level. The results presented in Figure 6 indicate that the younger transgenic mice exhibit a repolarization defect similar to that observed in the 6–8 months AT1R mice. Indeed, compared with their age-matched controls, the 50 days AT1R mice had longer QTc interval (CTL: 46.0 ± 1.4 ms; AT1R: 56.9 ± 2.1 ms, P = 0.0004) and APD (APD20, CTL: 2.2 ± 0.1 ms; AT1R: 4.2 ± 0.3 ms, P < 0.00005), (APD50 CTL: 4.2 ± 0.2 ms; AT1R: 12.1 ± 1.4 ms, P < 0.00005), (APD90, CTL: 16.7 ± 0.8 ms; AT1R: 34.9 ± 3.1 ms, P < 0.00005) (Figure 6A and B). In addition, 50 days AT1R mice exhibited a decrease in density of the outward component of Ipeak (at +30 mV; CTL: 64.2 pA/pF and AT1R: 28.4 ± 3.1 pA/pF, P < 0.00001). Of note, this reduction was similar to that observed in 6–8 months AT1R mice (Figure 6C). Similar to what was seen in the older group, the 50 days AT1R mice also had a reduced density of Ito and IKur compared with their age-matched control counterparts (Figure 6D). However, Iss was decreased in 50 days AT1R (at +30 mV, CTL: 14.8 ± 0.7 pA/pF and AT1R: 11.3 ± 0.8 pA/pF, P = 0.002), but IK1 was similar (at –110 mV, CTL = –19.7 ± 1.4 pA/pF and AT1R = –18.0 ± 1.0 pA/pF, P = 0.3).


Figure 6
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  		Figure 6 Comparison of the ventricular repolarization phenotype between 6–8 months (6 Mo) and 50 days (50 Do) AT1R male mice. (A) Comparison of the QTc interval between the CTL and AT1R mice at 50 days and 6–8 months (*P < 0.05). (B) Comparison of APD between 50 days CTL and AT1R mice. Typical examples of AP recorded at 4 Hz are presented. Bar graph presents mean APD (±SEM) (*P < 0.05). (C) Comparison of Ipeak in CTL and AT1R at 6–8 months and 50 days mice. Left: typical examples of Ipeak recorded in CTL and AT1R myocytes at 50 days. Right: mean I–V curves obtained in CTL and AT1R mice in the two age groups. (square: 6–8 months mice, circle: 50 days mice) (*P < 0.05 for young AT1R group). (D) Comparison of individual K+ currents in 50 days CTL and AT1R mice. Representative family of currents (top) and comparison of mean I/V curve between CTL and AT1R myocytes (bottom) for Ito, IKur, and Iss/IK1 are presented (*P < 0.05).

 
Telemetry monitoring of young AT1R mice revealed that these electrophysiological defects were associated with a variety of spontaneous arrhythmias in three out of three young AT1R mice tested (data not shown). These arrhythmias were similar to those observed in the older AT1R animals. In contrast, no arrhythmias were observed in age-matched littermate CTL (zero out of four).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
The present work provides important new insight into the electrical remodelling that occurs as a consequence of chronic stimulation of myocardial AT1R. First, our results obtained in a mouse model of cardiac-specific overexpression of human AT1R revealed the occurrence of ventricular arrhythmias and the electrophysiological consequences of AT1 overexpression. We found prolonged APD, delayed repolarization, and alterations in several K+ currents/channels. Secondly, studies in younger mice, prior to the development of overt cellular hypertrophy, indicate that these electrical changes precede the anatomic disturbances, suggesting that the arrhythmogenic changes do not occur as a consequence of cardiac remodelling, but are directly associated with overstimulation of the AT1R. Thirdly, the electrophysiological alterations described here also occur independently of haemodynamic changes,10 ruling out the involvement of peripheral vascular changes in the phenotype observed.

In both age groups, the QTc interval and APD were significantly prolonged in the AT1R mice. The K+ currents affected in the older AT1R mice were Ito, IKur, and IK1, whereas those affected in the younger animals were Ito, IKur, and Iss. The fact that Iss was reduced in the 50 days AT1R mice implies that this current is affected by chronic stimulation of the AT1R. However, in the older group, Iss was similar between CTL and AT1R mice. It is possible that the density of Iss increases with time to compensate the reduction of the other outward K+ currents. Compensatory upregulation of Kv2.1 (Iss) has been reported in previous studies.31,32 Indeed, following targeted replacement of Kv1.5, RNA and protein of Kv1.5 were undetectable and Kv2.1 protein was increased in a transgenic model of delayed repolarization.31,32 In the present study, the increased Kv2.1 protein expression seen in the older AT1R mice also supports a time-dependent compensatory upregulation of Kv2.1. Furthermore, because Iss does not contribute as much as Ito or IKur at the peak of the total K+ current, the difference in the density of Iss in AT1R mice between 50 days and 6–8 months mice is not sufficient to result in a significant change in the amplitude of Ipeak.

The other noticeable difference between the younger and older AT1R mice relates to IK1. In the 6–8 months AT1R myocytes, IK1 was smaller compared with the controls. In contrast, in the younger AT1R mice, IK1 was not altered. The fact that younger and older AT1R mice display a similar density of AT1R implies that the difference between the two age groups is not due to the degree of overexpression. In fact, the IK1 data strongly suggest that changes in the density of this K+ current in the older AT1R mice might result from alterations in intracellular signalling associated with cardiac hypertrophy and would not be directly due to the increase stimulation of AT1R. In a separate study, we observed that overexpression of wild-type {alpha}1B-adrenergic receptor ({alpha}1B-AR) in mice did not affect the density of IK1 (unpublished data, Rivard et al., 2008). Of note, the late-onset heart failure phenotype observed in {alpha}1B-AR mice was not preceded by longstanding cardiac hypertrophy. Taken together these findings suggest that IK1 might be altered only in the presence of hypertrophy.

In a recent study, Domenighetti et al.13 studied 50–60 weeks transgenic mice with cardiac-specific ANG II overproduction. They reported that these mice develop hypertrophy associated with prolonged QT interval and APD. These observations were attributed to a decrease in IK1, which was the only current examined in their study. A reduction of IK1 in that model of hypertrophy would agree with a relationship between downregulation of IK1 and the presence of cardiac remodelling. However, although they reported that the animals studied presented ventricular hypertrophy, they failed to establish a correlation between cell capacitance and the density of IK1. Based on that observation, they concluded that the reduction of IK1 was independent of cell size and hypertrophy. However, to clearly establish the contribution of hypertrophy on IK1 density it would have been more informative to test younger animals that do not present signs of hypertrophy.

The results obtained in our expression studies indicate the chronic stimulation of the AT1R decreased Ito density and altered its kinetics of reactivation through alteration of Kv4.2 and KChIP2 transcription. However, Kv4.3 expression levels were unaffected in AT1R mice. We also observed that overexpression of {alpha}1B-AR in mouse heart decreased Ito, Kv4.2 and KChIP2 without any effect on Kv4.3 (unpublished data). Our expression studies also suggest that the decreased density of IKur and IK1 observed in the AT1R mice could be explained by post-transcriptional regulation as suggested by decreased protein expression of Kv1.5 (IKur) and Kir2.1 (IK1) without changes in the mRNA levels.

Fischer et al.33 have recently reported that hypertensive transgenic rats harbouring the human renin and angiotensinogen genes developed cardiac damage leading to sudden death. They also observed that ventricular tachycardia was common in these animals. Although they did not have any cellular electrophysiological data, they used non-invasive cardiac magnetic field mapping to report that depolarization and repolarization were prolonged and inhomogeneous. These data are in strong support of the findings reported here. However, because of the presence of hypertension in that rat model, it is hard to determine whether the observed electrical remodelling occurs as a consequence of ANG II stimulation or results from hypertension.

Our results are also consistent with previous reports showing a relationship between ANG II and ionic currents. In vascular smooth muscle cells, ANG II has been shown to inhibit several K+ channels, including KATP, BKca, and Kv channels.34–36 Similarly, different groups have reported that cardiac K+ currents are attenuated after application of ANG II.37–39 Furthermore, in some studies, the effects of ANG II on ionic currents could be reversed when the action of ANG II was blocked with ACEI or ARA.39 Findings reported here also agree with previous reports showing beneficial effects of ACEI or ARA in preventing arrhythmic mortality.4,5

The primary objective of the telemetry experiments was to detect spontaneous arrhythmias and nearly all AT1R mice studied (eight out of nine; old and young AT1R mice altogether) exhibited spontaneous cardiac arrhythmias during the telemetry monitoring. However, none of these mice died during the 24 h recording period. Clearly, that recording time was sufficient to detect spontaneous arrhythmias; however, a much longer recording period would be necessary to obtain ECG evidence for spontaneous sudden death events.

Taken together the association of ventricular repolarization defects with the increased incidence of arrhythmias in AT1R mice is in support of a ventricular origin for the observed arrhythmias. However, we cannot exclude the possibility that conduction abnormalities could also contribute to some rhythm disturbances seen in these animals. Additional experimental studies are required to address this question.

In conclusion, data presented here reveal that overexpression of the human AT1R in the myocardium leads to an increased incidence of cardiac arrhythmia associated with delayed repolarization in mice. Importantly, these changes occurred before the development of cardiac remodelling and in absence of hypertension. These studies provide new insight into the role of chronic AT1R stimulation in the pathogenesis of cardiac arrhythmia and sudden death. These results could help to develop and understand therapeutic strategies preventing arrhythmic mortality associated with heart disease.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
This study was supported by an operating grant from the Canadian Institutes of Health Research (MOP-64621; CF). K.R. has a studentship award from the Fonds de la Recherche en Santé du Québec (FRSQ). C.F. is a Research Scholar of the FRSQ.


   Acknowledgements
 
The authors are thankful to M.A. Lupien, M.A. Gillis, and M. Laprise for skilled technical assistance and to Hao Wang for the measurements of the AT1R levels.

Conflict of interest: none declared.


   Notes
 
{dagger} Present address: Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal. Back


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

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