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Physiol. Genomics 33: 230-239, 2008. First published February 26, 2008; doi:10.1152/physiolgenomics.00143.2007
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Received 6 July 2007; accepted in final form 20 February 2008.
Physiological Genomics 33:230-239 (2008)
1094-8341/08 $8.00 © 2008 American Physiological Society

Altered heart rate control in transgenic mice carrying the KCNJ6 gene of the human chromosome 21

Jacques M. Lignon 1, Zoë Bichler 2, Bruno Hivert 1, François E. Gannier 1, Pierre Cosnay 1, José A. del Rio 2, Danièle Migliore-Samour 3 and Claire O. Malécot 1

1 Centre National de la Recherche Scientifique Unité Mixte de Recherche 6542, Physiologie des Cellules Cardiaques et Vasculaires, Université François-Rabelais, Parc Grandmont, Tours, France
2 Department of Cell Biology, Institute for Research in Biomedicine, University of Barcelona, Barcelona, and CIBERNED, Spain
3 Centre National de la Recherche Scientifique Institut de Trangénose, Orléans-La Source, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Congenital heart defects (CHD) are common in Down syndrome (DS, trisomy 21). Recently, cardiac sympathetic-parasympathetic imbalance has also been documented in DS adults free of any CHD. The KCNJ6 gene located on human chromosome 21 encodes for the Kir3.2/GIRK2 protein subunits of G protein-regulated K+ (KG) channels and could contribute to this altered cardiac regulation. To elucidate the role of its overexpression, we used homozygous transgenic (Tg+/+) mice carrying copies of human KCNJ6. These mice showed human Kir3.2 mRNA expression in the heart and a 2.5-fold increased translation in the atria. Phenotypic alterations were assessed by recording electrocardiogram of urethane anesthetized mice. Chronotropic responses to direct (carbachol) and indirect (methoxamine) muscarinic stimulation were enhanced in Tg+/+ mice with respect to wild-type (WT) mice. Alternating periods of slow and fast rhythm induced by CCPA (2-chloro-N-cyclopentyl-adenosine) were amplified in Tg+/+ mice, resulting in a reduced negative chronotropic effect. These drugs reduced the atrial P wave amplitude and area. P wave variations induced by methoxamine and CCPA were respectively increased and reduced in the Tg+/+ mice, while PR interval and ventricular wave showed no difference between Tg+/+ and WT. These results indicate that Tg+/+ mice incorporating the human KCNJ6 exhibit altered Kir3.2 expression and responses to drugs that would activate KG channels. Moreover, these altered expression and responses are limited to sino-atrial node and atria that normally express large amounts of KG channels. These data suggest that KCNJ6 could play an important role in altered cardiac regulation in DS patients.

Down syndrome; electrocardiogram; autonomic nervous system; GIRK2; cardiac


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
DOWN SYNDROME (DS, trisomy 21) is the most common genetic cause of mental retardation and occurs in approximately one out of 750 births. It is characterized by facial dysmorphology, immunodeficiency, and hypotonia, and ~40% of the children develop congenital heart defects (CHD; Refs. 1, 10, 32). Survival has greatly improved with cardiac surgical intervention and medical care (44), but altered cardiac function is a common problem in patients with DS. Asymptomatic progressive nonsurgical atrio-ventricular block and bradycardia have been reported (4, 5). Recently, impairment of cardiac regulation has been documented in adult DS patients without any cardiac malformation. This includes chronotropic incompetence and altered heart rate variability (HRV), suggesting a sympathetic-parasympathetic imbalance (13, 15, 20).

DS is caused by partial or total triplication of the human chromosome 21 (HSA21). Dosage-sensitive genes, allelic variations, and modifier genes are potential but not exclusive mechanisms accounting for DS phenotypes and phenotype variability (1, 32). Because of the life-threatening effect of CHD, cardiac morphology is often taken as a target of gene dosage imbalance. CHD have been related to an overexpression of DSCAM, COL6A1, and COL6A2 genes (32) and of DYRK1A and DSCR1, two HSA21 regulatory genes lying in the so-called Down syndrome critical region (DSCR, Ref. 2). However, impaired cardiac function and regulation resulting from potential gene dosage imbalance have not yet been explored. One gene located in the DSCR (8) and expressed in the heart (21, 35) that could contribute to altered sympathetic-parasympathetic cardiac regulation is KCNJ6, which encodes for the Kir3.2 (GIRK2) protein subunit of G protein-regulated K+ (KG) channels.

Mouse model of DS are feasible since ~80% of HSA21 is syntenic to mouse MMU16, the remaining being syntenic to MMU10 and MMU17 (19). Full mouse trisomy 16 is lethal (40) and shows alteration in the fetal heart action potential (31). DS mouse models with partial translocations of MMU16 (Ts65Dn, TsCje, and Ms1Ts65) show DS-like phenotypes including CHD (1, 10, 32, 40). Transgenic mouse models have also been constructed that carry full length or sequences of the HSA21 (10, 35). Among them, those of Smith et al. (36) cover the DSCR using four contiguous yeast artificial chromosomes (YAC). Mouse lines bearing the YAC 152F7, 230E8, and 141G6 have affected brain weight, neuron size and density, and/or behavioral responses (7, 10, 33, 37). The fourth, YAC 285E6, includes the large KCNJ6 gene (7, 33).

KG channels are activated by the stimulation of seven-transmembrane G protein-coupled receptors. They consist of four Kir3.x subunits, and most of them form hetero-tetrameric inward K+ rectifier channels that control cellular excitability (9, 43). Heterotetramers of Kir3.1 and Kir3.4 are specific to the heart and to the pancreas. Other KG channels are mostly heterotetramers of Kir3.1 and Kir3.2 and are largely expressed in the nervous system. However, Kir3.2 is ubiquitously expressed including in the heart (21, 35). In the heart, KG [K(Ach, Ado)] channels are activated by m2-muscarinic receptors and by A1-adenosine receptors (A1-AdoR). They form part of the signal transduction system leading to negative chronotropic effects in the sino-atrial node (6, 42), reduced atrial contraction and reduced atrio-ventricular (AV) conduction (3, 11) upon vagal stimulation. Their density is lower and species dependent in ventricular myocytes (11, 24, 43).

We used mouse lines carrying the YAC 285E6 as an experimental model of gene expression imbalance in the heart. The transgene expression was assessed by RT-PCR and Western blot and functional responses by electrocardiogram (ECG) recordings in urethane anaesthetized mice. Responses to cholinergic and A1-AdoR stimulation and to indirect vagal activation by methoxamine were compared in wild-type (WT) and Tg+/+ mice. Our results show expression of human Kir3.2 subunit m-RNA in transgenic mice heart, protein overexpression in the atria and modification of heart rate responses to drugs between WT and Tg+/+ mice without alteration of conduction and ventricular ECG wave-form morphology.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.
Mice were handled with the agreement of the local ethical committee and in accordance with the European Council Guidelines for the Care and Use of Laboratory animals (certificate 7320). They were housed under a 12 h/12 h light-dark cycle in François Rabelais University animal care facility (certificate C37-261-4) and fed a standard rodent chow.

We used Tg67 and Tg84 mice lines created by Smith et al. (36) by microinjection of the same human DNA fragment (inserted in the YAC 285E6) into FVB zygotes. Heterozygous lines were maintained and the WT littermates were used as control group. Homozygous animals were selected with a conventional test cross based on three generations. Experiments were performed on 1-yr-old homozygous female Tg84+/+ and Tg67+/+ that show less variability than heterozygous lines.

Genotyping and reverse transcriptase-polymerase chain reaction analysis.
PCR were performed using D21S337 primers (Table 1) as described by Smith et al. (36) on DNA extracted from tail biopsies in 10% Chelex 100 (Bio-Rad) and digested by proteinase K (0.4 mg/ml final, Promega).


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  		Table 1. Sequences of primers used for specific and unspecific amplification of Kir3.2

 
RNA extraction from heart and brain was performed with the QuickPrep total RNA extraction kit (Pharmacia Biotech) and stored in an RNAlater (Ambion) solution. Retro-transcription was performed with Titan one tube reverse transcriptase-polymerase chain reaction (RT-PCR) kit (Boehringer Mannheim). Residual DNA was checked by amplification and no retro-transcription. In a first experimental series, primers recognizing both murine and human Kir3.2 DNA were used (Ref. 19, Table 1). However, murine (m) Kir3.2 differs from human (h) Kir3.2 by only a few amino acids (14). Specific primers of the hKir3.2 DNA were thereafter designed in choosing a human-specific sequence in the untranslated part of the mRNA (Table 1). Human brain cDNA (CH-1001, CliniSciences) was used as positive control and water as negative control. A control amplification was done with specific primers of β-actin.

Western blot.
Mice were killed by cervical dislocation. Atria, ventricles, and brains were extracted and rinsed in ice-cold phosphate-buffered saline (PBS). Samples were homogenized in lysis buffer [100 mM NaCl, 10 mM HEPES (pH 7.5), 2 mM EDTA (pH 8.0), 0.1% Triton X-100, 1 mM DTT, and a mixture of proteases inhibitor (PIC set I) from Calbiochem], incubated for 30 min in ice-cold buffer, centrifuged at 2,200 g for 10 min, and stored at –20°C.

After protein quantification (BCA Protein Assay Kit, Pierce Biotechnology), samples were boiled in Laemmli buffer (25). Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membrane (Protran, Whatman). After incubation for 1 h at room temperature with blocking solution (5% nonfat milk in Tris-buffered saline 0.1% Tween 20), membranes were processed for immuno-blotting using anti-Kir3.2 primary antibodies from Alomone (1/400; Jerusalem, Israel) or from Abcam (1/500; Cambridge, UK) at 4°C overnight. Membranes were then incubated with swine anti-rabbit-HRP (1/2,500; Dako) for 1 h at room temperature and processed with chemo-luminescent reagent (ECL SuperSignal West Dura kit, Pierce Biotechnology). Membranes were then stripped, blotted with a monoclonal anti-β-tubulin antibody (1/100,000; Sigma-Aldrich), and processed with normal ECL kit (Amersham). Quantifications were performed by densitometry (Quantity one Image Software Analysis, Bio-Rad) and normalized.

ECG recordings.
ECG was recorded under urethane anesthesia (1.33 g/kg ip). Mice were placed in a supine position in a Faraday cage. Ambient air was maintained at 25–26°C, close to the neutral temperature. Three leads consisting of 50 µm thin Ag-AgCl wires were placed subcutaneously in the forelegs and the left hind leg. DI and DII derivation recordings were amplified with a 1–500 Hz bandwidth (IsoDAM8 amplifiers; WPI, Aston, UK) and continuously monitored on a Gould oscilloscope (20 MHz, type 1421). Acquisition was performed at 2 kHz (IOX software v. 1.582; EMKA Technologies, Paris, France). R, S, and RS wave amplitudes and heart rate (HR) were averaged and monitored on line every 10 s. After 1 h of control recording, drugs were injected intraperitoneally cumulatively every 20 min, a time long enough to obtain a steady-state effect.

Data analysis.
HR mode was obtained from HR averaging over 10 s. Off-line analysis of ECG was performed with ECG-auto software (v. 1.5.11 EMKA Technologies). RR/HR/SDRR analysis was performed over successive 2 min intervals by measuring average RR (RRav) and HR (HRav) with their standard error (SDRR). The ECG wave analysis used two ECG libraries made up from DI and DII original tracings. Measurements were performed every minute, and reported values are the average of five measurements made on individual wave complexes taken over the first seconds of each interval. As the mouse ECG displayed no real electrical zero, this was taken as the average of five consecutive points taken between the end of P wave and the beginning of Q (or R) wave (from time –15 ms before Q/R wave beginning) as in infants (Fig. 1). Figure 1 illustrates measurements performed on ECG recorded from mice. We followed Liu et al. (26) to label the second wave adjoining the QRS complex as a J wave. Corrected QT for RR interval changes (QTc) was calculated according to Mitchell et al. (30): QTc = QT/(RR/100)1/2.


Figure 1
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  		Fig. 1. Example of electrocardiogram (ECG) [DII derivation; wild-type (WT) mouse] showing measurements performed on wave amplitudes and durations. The isoelectric line (zero) was taken as the mean potential just preceding the R wave. J wave was labeled following Liu et al. (26). The T wave is initially negative (Tneg) and then positive (Tpos). {Delta}T and {Delta}J refer to the differences (Tpos – Tneg) and (J – Tneg ), respectively.

 
Individual dose-response curves to carbachol (CCh) were fitted with the logistic equation as provided in Origin 7.0 (Microcal Origin software): y = (A1 – A2)/[1 + (x/x0)p] + A2. In this equation, x0 represents the EC50, p is the Hill slope coefficient (nH), and A1 and A2 represent respectively the initial and final values of y.

Results are given as mean values ± SE. Statistical tests were performed with Sigma Stat software (Systat Software). We used the Student's t-test and two-way repeated-measures ANOVA followed by the Student-Newman-Keuls (SNK) multiple-comparison tests. Densitometric analysis was compared with the Mann-Whitney test, and proportions were analyzed with the Fisher exact test. Significance was set at P < 0.05.

Drugs.
Atropine sulfate, carbamoylcholine chloride (CCh), methoxamine hydrochloride, 2-chloro-N-cyclopentyl-adenosine (CCPA), atropine sulfate, urethane, Triton X-100, ethylene-diamine-tetra-acetic acid (EDTA), dithiothreitol (DTT), 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES), and PBS salts were obtained from Sigma (France). Urethane was dissolved at 20% in distilled water. All drugs, dissolved in NaCl 0.9%, were injected intraperitoneally.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Human mRNA transcription in mouse heart.
Figure 2 shows mRNA transcripts of Kir3.2 amplified with primers specific to human (Table 1). The presence of one specific band in the human brain sample (lane 3) and the absence of any amplification in WT mouse brain (lane 4) and heart (lane 5) testify to the specificity of these primers. In contrast, high-intensity bands in the heart of both Tg84 (lane 7) and Tg67 (lane 8) show specific expression of hKCNJ6 in these mice lines. On the other hand, primers recognizing both human and murine Kir3.2 generate cDNA from mouse heart and brain not only in Tg67 and Tg84 mice lines but also in WT mice though with lower intensity (not shown).


Figure 2
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  		Fig. 2. Detection of Kir3.2 transcripts amplified with human specific primers in WT, Tg84, and Tg67 mice. Lanes labeled 1–8 are respectively: β-actin amplification controls for mouse heart (1) and human brain (2), and KCNJ6 mRNA expression in human brain (3), in control (water) (4), in WT mouse brain (5), in WT mouse heart (6), in Tg84 mouse heart (7), and in Tg67 mouse heart (8). Tg67 and Tg84 heart gave strong specific bands, while no detection of Kir3.2 could be observed in WT mice with these human-specific primers. M, molecular marker.

 
Kir3.2 translation in the heart.
Figure 3 illustrates Western blots of Kir3.2 in the brain and the heart of WT and Tg84+/+ mice. Translation of Kir3.2 occurs not only in the brains but also in the atria and ventricles of both WT and Tg84+/+ mice. Quantification of the Kir3.2 bands with respect to those of tubulin shows a 2.5-fold overexpression in the brains and the atria of Tg84+/+ mice with no effect in the ventricles (Fig. 3B). Bands were obtained with anti-Kir3.2 antibody from Alomone laboratories directed against residues 374-414 (C-terminal tail) of the protein with a 45 kDa molecular weight expected from the Kir3.2 A or C sequences (14, 43). Since Kir3.2B lacks these residues, we also used antibodies directed against the N-terminal tail of the protein (Abcam Laboratories, not shown). The translation of Kir3.2 A and C at 45 kDa was confirmed, but no band was observed at the expected Kir3.2B molecular weight of 36 kDa (21, 43).


Figure 3
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  		Fig. 3. Detection of Kir3.2 protein expression in the brains, atria, and ventricles of Tg84+/+ and WT mice. Top: Western blot performed on brains, atria, and ventricles of WT (left in each panel) and Tg84+/+ (right in each panel) mice with anti-Kir3.2 antibody from Alomone laboratories (residues 374-414) and anti-tubulin. Bottom: Kir3.2 normalized ratios expression of Kir3.2 bands (45 kDa) with respect to tubulin (50 kDa). Tg84+/+ brains and atria (hatched columns) overexpress Kir3.2 with respect to WT (open columns). Data are means ± SE (n = 5 for brains and atria; n = 8 for ventricles). The Mann Whitney test P values are given by pairs.

 
WT and Tg+/+ mice ECG under control conditions.
Figure 4A illustrates typical ECG recordings of DI and DII derivations. Urethane anesthesia maintained a regular HR mode of 614 ± 11 beats/min (n = 13) in WT. Transgenic mice had a greater HR mode of 671 ± 5 beats/min (n = 12, P = 0.0001) for Tg84+/+ mice and of 664 ± 10 beats/min (n = 14, P = 0.0041) for Tg67+/+ mice, and these did not differ significantly from each other (P = 0.58). Both WT and Tg+/+ mice showed episodes of bradycardia, lasting for some seconds, that account for most of the RR interval variability (SDRR, Table 2) and an RRav larger than the modal value. Nevertheless, RRav was significantly smaller in Tg84+/+ than in WT mice. Other arrhythmias were compensated atrial extrasystoles. Irrespective of the genotype, 19% of the mice showed extrasystoles (50 ± 11/min in Tg84+/+, 7 ± 6 /min in WT; n = 3), 15% showed occasional extrasystoles (2/h).


Figure 4
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  		Fig. 4. DI and DII recordings of the ECG of a WT mouse under control conditions and the effect of carbamoylcholine chloride (CCh). A: control recording with a clear P wave. B: negative chronotropic effect of 0.22 mg/kg CCh 8 min after the injection. Note the RR prolongation and S and P wave amplitude decrease in DII. Inset: bifid morphology of the P wave in DII associated with the longer RR intervals (double arrows), while a monomorphic P wave is associated with shorter RR intervals (single arrow). The P wave is less affected in DI.

 

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  		Table 2. ECG data of WT and Tg84+/+ mice (DI and DII derivations) in control period

 
Both WT and Tg+/+ mice showed ECG wave morphology (DI and DII derivations, Fig. 4A) with sequential P, QRS, and J waves, similar to ECG of unsedated mice (26). The DII derivation showed two- to fivefold larger wave amplitudes than DI (Table 2). The mouse ECG has a large S wave, a specific J wave adjoining the QRS complex and a small negative T wave (Figs. 1 and 4A, Table 2). A Q wave was observed only in DI in 50% of the mice. The T wave was often tiny. It was monophasic negative (Tneg) in DI and biphasic (Tneg, Tpos) in DII. The QT interval was best measured in DI because of its larger Tneg wave.

It should be mentioned that during the spontaneous episodes of bradycardia, the P wave was reduced, particularly in DII. In the Tg84+/+ mice, only J wave amplitude and P wave duration and area were different from WT ones. In the Tg67+/+ mice these changes were of borderline significance.

Chronotropic responses to CCh.
CCh was used to evoke a sustained cholinergic stimulation resulting in a negative chronotropic effect and changes in ECG morphology as illustrated in Figs. 4 and 5. Figure 5 shows that whereas its negative chronotropic effect was not different between Tg84+/+ and WT at low CCh doses, the HR mode values of Tg84+/+ mice were significantly less than those of WT mice at higher submaximal doses. The EC50 of the chronotropic effects of CCh were not different between Tg67+/+, Tg84+/+, and WT mice, but the Hill slope coefficients (nH) were significantly larger (about twofold) in both Tg84+/+ and Tg67+/+ than in WT (Table 3). Atropine only reduced control HR mode values by 3.4 ± 0.8% (n = 8) in Tg84+/+ mice, but it shifted the dose-response curve for CCh to the right by one log unit (EC50 = 1.34 ± 0.34 mg/kg, n = 8; P = 0.0035) and yet maintained nH at a value of 4.14 ± 0.60 close to that of untreated Tg84+/+ (n = 8, P = 0.52).


Figure 5
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  		Fig. 5. Comparative negative chronotropic effect of cumulative doses of CCh on WT ({circ}) and Tg84+/+ mice (•). Heart rate (HR) mode at steady state is given as % of control value. Data are means ± SE (n = 8) for each curve. *Significant difference between WT and Tg84+/+ mice [Student-Newman-Keuls (SNK) test; P < 0.05].

 

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  		Table 3. Chronotropic effects of CCh in WT, Tg84+/+ and Tg67+/+ mice

 
The time course of the development of the negative chronotropic effect of CCh was irregular with brief episodes of bradycardia that resulted in prolonged RR intervals. Total duration of these episodes was dose dependent and represented 37 ± 9% of the time at 0.070 mg/kg, 55 ± 12% at 0.220 mg/kg, and 59 ± 3% at 0.470 mg/kg in WT mice (n = 8). It was significantly less in Tg84+/+ and Tg67+/+ mouse lines (Table 3) at low doses of CCh but could not be measured at higher doses because of the large negative chronotropic effect of CCh.

Chronotropic responses to methoxamine.
Methoxamine is an {alpha}1-adrenergic receptor agonist that causes vasoconstriction and increases blood pressure and induces baroreflex activation and a bradycardic vagal response. A typical response to the injection of methoxamine consisted of an initial drop of HR that decayed away to a higher steady-state value (Fig. 6). Brief episodes of bradycardia that lasted for 3–5 s were observed during this negative chronotropic effect. They consisted of a sudden increase in RR interval that decayed with an exponential-like time course over 8–12 heartbeats (Fig. 6B). The threshold of the effect was obtained at 3 mg/kg methoxamine in WT (n = 7) and Tg84+/+ (n = 9) mice. RR increased from 102.4 ± 2.1 ms to 111.1 ± 3.3 ms at peak and 107.8 ± 2.8 ms at steady-state effect in WT (n = 7). A threefold higher dose (9 mg/kg) further prolonged RR values to 160.5 ± 34.4 ms at peak and 129.4 ± 10.8 ms at the steady state in WT. Similar values were recorded in Tg84+/+ mice. Only at higher methoxamine doses (30 mg/kg) were RR intervals and SDRR peak values significantly larger in Tg84+/+ than in WT mice (Fig. 6, C and D), showing an increased chronotropic effect and an increased HRV. This is similar to the effect of CCh for which there were no difference between WT and Tg+/+ mice at threshold doses whereas large submaximal doses showed an amplified response in Tg+/+ mice.


Figure 6
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  		Fig. 6. Negative chronotropic effect of methoxamine (30 mg/kg) and its effect on the ECG in a Tg84+/+ mouse (A and B) and its quantification in WT and Tg84+/+ mice (C and D). A: DII ECG recording under control conditions (a) and after injection of methoxamine at the peak (b) and at the steady state (c) of the effects. RR interval is prolonged and P wave amplitude reduced. B: time course of the change in RR interval after methoxamine injection (arrow). The initial large increase in RR was followed by a partial recovery with short bradycardic episodes shown on an expanded time scale at bottom. C: RR intervals D: variability of the RR intervals (SDRR) measured in control (open columns), at peak methoxamine effect (hatched columns), and at steady-state effect (filled columns). Values were obtained over a 2 min period at peak effect and over three consecutive 2 min periods for control and steady state. Data are mean ± SE. (WT, n = 7; Tg84+/+, n = 9). P values are given by pairs. There is no difference between WT and Tg84+/+ mice in control or steady state, but a significant difference appears at the peak of the methoxamine effect.

 
Chronotropic responses to CCPA.
CCPA is a highly selective A1-AdoR agonist that acts mainly upon peripheral receptors and which activates the same signaling cascade as muscarinic stimulation in the heart. CCPA induced a negative chronotropic effect in both WT and Tg84+/+ mice (Fig. 7A). At low doses of CCPA, HRav are similar in WT and Tg84+/+ mice/ but for higher doses, HRav values are significantly greater in Tg84+/+ than in WT. However, in contrast to CCh, CCPA never stopped the heart and lowest HRav values were 106.8 ± 16.4 (n = 9) and 115.1 ± 9.6 (n = 10) beats/min for WT and Tg84+/+, respectively. CCh injection could then further lower the HR, whatever the mouse line (not shown).


Figure 7
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  		Fig. 7. Effect of 2-chloro-N-cyclopentyl-adenosine (CCPA) on ECG of WT and Tg84+/+ mice. A: dose-response curve of the negative chronotropic effect of CCPA as % of control value. HRav, average heart rate. B: alternating rhythm induced by 1 mg/kg CCPA. a: DII record in a Tg84+/+ mouse showing switches of high and low HR. b: Alternating long/short RR intervals in a Tg84+/+ mouse. Short RR are stable but long RR are more variable. c: Amplitude of the change in heart rate rhythm (fast rhythm – slow rhythm) given as % of the initial variation. (HRmax, highest heartbeat rate; HRmin, lowest heartbeat rate). Data are means ± SE of 7 WT and 9 Tg84+/+ mice. *Significant difference at P < 0.05 (SNK test).

 
The negative chronotropic effect of CCPA did not develop with a smooth time course. At low CCPA doses (0.01–0.1 mg/kg), brief episodes of bradycardia similar to those described above for methoxamine (sudden increase in RR interval that decayed away) were observed during this negative chronotropic effect. At higher doses, CCPA induced HR variation with alternating long RR intervals (Fig. 7B; e.g., 452 ± 19 ms; mean ± SDRR) and shorter ones (e.g., 170 ± 4 ms), resulting in low HR (HRmin) and high HR (HRmax) values, respectively. In the short RR interval periods, HRV was small. It was larger in the long RR intervals. Changes from one period to the other were rapid. The duration of these periods could be either regular or irregular, lasting for 10–30 s (Fig. 7B, a and b).

Both HRmax and HRmin decreased dose-dependently with CCPA (not shown). HRmin was not significantly different between WT and Tg84+/+ mice. However, HRmax was larger in Tg84+/+ mice than in WT at high doses of CCPA (e.g., 363 ± 47; n = 10 vs. 248 ± 28 beats/min, n = 9; P = 0.01, at 0.3 mg/kg CCPA). Figure 7Bc shows that at low CCPA doses, the difference between HRmax and HRmin increased with CCPA doses in a similar manner for Tg84+/+ and WT mice. However, at doses above 0.3 mg/kg, the amplitude of the alternating rhythm (HRmax-HRmin) significantly increased with CCPA doses to a plateau value in Tg84+/+ mice (Fig. 7Bc), whereas it decreased in WT to values not different from the control. In addition, the duration of the alternating rhythm decreased in WT mice and stopped in 50% of them at high CCPA doses while it persisted in 92% of the Tg84+/+ mice (P = 0.052).

Drugs-induced changes in ECG wave morphology and characteristics.
Figures 4, 6A, and 8A show clear-cut changes in the ECG morphology with each of the three drugs. Atrial P wave, PR interval (index of AV conduction) and ventricular complex were all modified.


Figure 8
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  		Fig. 8. Effect of CCPA on the DII derivation ECG of WT and Tg84+/+ mice. A: RR and wave morphology in control (a), and following the injection of 0.1 mg/kg (b), and 3 mg/kg (c) CCPA in a WT mouse. Notice the change in RR, S wave amplitude, and T wave deepening. B: P wave area. Data are means ± SE of 7 WT and 9 Tg84+/+ mice. *Significant difference between WT and Tg84+/+ (SNK test, P < 0.05).

 
All three drugs dose-dependently prolonged atrial P wave from 13 to 25 ms (Tables 4 and 5) in WT and Tg+/+ mice. P amplitude and area were reduced by methoxamine (Table 4) and CCPA (Table 5, Fig. 8B). Compared with WT mice, these decreases were larger in Tg84++ mice injected with methoxamine (Table 4) but smaller in CCPA-injected ones (Table 5, Fig. 8B). In addition, noticeable changes of P wave morphology were associated with the sudden changes in RR duration produced by these three drugs, irrespective of the mouse line. Associated with long RR intervals of alternating rhythm or bradycardia, a low P amplitude and/or bifid morphology was observed in DII with only a small prolongation of the P wave in DI. This is illustrated for CCh and methoxamine induced bradycardia in Figs. 4 and 6A. Similar results were obtained with CCPA (Fig. 8A).


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  		Table 4. Effects of methoxamine on ECG characteristics in WT and Tg84+/+ mice (DII derivation)

 

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  		Table 5. Effects of CCPA on ECG characteristics in WT and Tg84+/+ mice (DII derivation)

 
PR interval was dose-dependently prolonged by methoxamine and CCPA (Tables 4 and 5). CCh (0.070 and 0.220 mg/kg) increased PR from 31.9 ± 0.5 ms to 43.2 ± 6.0 and 59.6 ± 9.2 ms, respectively (n = 8). No significant difference could be observed between WT and Tg+/+ mice.

Ventricular wave parameters are reported in Tables 4 and 5. QRS was moderately prolonged by methoxamine. CCh (0.070 and 0.220 mg/kg) prolonged it from 11.0 ± 0.2 ms to 13.4 ± 1.7 ms and 15.5 ± 2.5 ms, respectively (n = 8). Nevertheless, QRS was always short and preceded by a P wave, even during the episodes of bradycardia or alternating rhythm. Changes in R and J waves were non significant for all three drugs. S was dose-dependently reduced by methoxamine and CCPA (Tables 4 and 5). CCh (0.070, 0.220 and 0.470 mg/kg) also changed S amplitude from –0.66 ± 0.14 mV to –0.50 ± 0.09 mV; –0.46 ± 0.13 mV and -0.09 ± 0.04 mV, respectively (n = 8). CCh and CCPA noticeably deepened Tneg (Fig. 8A, Table 5). All drugs prolonged the QT interval, but this prolongation was less in Tg84+/+ mice treated with 0.3 mg/kg CCPA than in WT. However, a large part of this prolongation is related to the changes in RR interval as shown by the QTc that also resolved the apparent difference between Tg84+/+ and WT mice injected with 0.3 mg/kg CCPA (Table 5). There was no significant difference in ventricular wave parameters between Tg84+/+ and WT mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major results of this study are: 1) specific human transcript of the KCNJ6 gene is detected in the heart of both transgenic mouse lines; 2) this translates into increased Kir3.2 protein expression in atria but not in the ventricle; 3) upon direct (CCh) or indirect (vagal activation by methoxamine) muscarinic cholinergic stimulation, the negative chronotropic effect is enhanced in Tg+/+ mice; 4) upon A1-purinergic stimulation (CCPA) the alternating periods of fast and slow heart rhythm is enhanced in Tg84+/+ mice, and, as a result, the mean negative chronotropic effect is reduced; 5) P wave amplitude and area variations induced by methoxamine and CCPA are respectively increased and reduced in the Tg84+/+ mice; and 6) PR intervals and ventricular wave changes induced by these drugs are not different in Tg+/+ mice with respect to WT. These results indicate that transgenic mice incorporating the human KCNJ6 exhibit altered Kir3.2 expression and responses to drugs that would activate KG channels. Moreover, these altered responses are limited to the sino-atrial node and the atria that normally express large amount of KG channels.

Expression of Kir3.2 subunit.
Our data show both expression and functionality of KCNJ6 in transgenic mouse lines Tg67+/+ and Tg84+/+ carrying YAC 285E6. Formerly restricted to CBR and PCP4 boundaries, this YAC has since been more precisely mapped to a single gene, namely KCNJ6 (7, 33). According to Smith et al. (36), the Tg67+/– line has integrated one copy of the YAC. The copy number in Tg84+/– is not known. However, our results show very similar increases in control HR and in responses to CCh in both Tg84+/+ and Tg67+/+ mice, suggesting an equivalent expression. Moreover, the similar results obtained in both lines argue for the absence of any insertional effect.

The observation of Kir3.2 m-RNA in WT mice is in agreement with their reported ubiquitous distribution in various tissues and particularly in the atrium and the ventricle (21, 35). Heart overexpression of human specific Kir3.2 m-RNA in Tg84 and Tg67 mice is in line with similar overexpression observed by Shinohara et al. (35) in mice containing a full-length HSA21. Therefore, it is likely that the observed Kir3.2 proteins overexpression in the atria leads to an increased expression of KG channels. This is supported by our data showing increased responses to submaximal doses of drugs. The KG channels could then be a mixture of conventional heterotetrameric Kir3.1/Kir3.4 channels and of KG channels including Kir3.2 subunits. Such a pluripotent assembly of Kir3.2 subunits has been described (43).

Sinus node, atrium, and ventricle modulations.
Our results show the classical negative chronotropic effects of the para-sympathomimetic drug CCh and of CCPA, a peripherally acting A1 receptor agonist (34), as well as baroreceptor activation by the {alpha}1-adrenergic agonist methoxamine. These three drugs also reduced the P wave. The noticeable differences between WT and Tg+/+ mice in the chronotropic responses and P wave suggest that Kir3.2 overexpression in Tg+/+ mice lines is functional in the sino-atrial node and in the atria. These results fit in with the known effects of Ach, CCh, and adenosine that slow HR and reduce atrial contractility by mobilizing the inhibitory Gβ{gamma} proteins and activating KG channels (3, 11, 27, 42). The observation of a greater HR drop at higher doses of CCh (increased steepness of the dose-response curve) and of methoxamine in Tg+/+ mice could be explained by the common view that moderate muscarinic stimulation mainly activates the If signaling branch of parasympathetic action, while larger stimulation activates KG channels (11, 12). Thus, at low doses, Tg+/+ and WT mice would have identical responses and differ at high doses (Figs. 5 and 6). Though as Boyett et al. (6) suggested that KG channels are the major target of muscarinic stimulation, it may be necessary to use a larger stimulation to reveal the effects of Kir3.2 subunit overexpression.

The absence of any difference between WT and Tg+/+ mice in the responses of the ventricle (reduced S wave, Tneg deepening, but constant R and J waves) is in agreement with the lower KG density in the ventricle where muscarinic and purinergic ventricular responses primarily depend upon adenylate cyclase inhibition (11, 24).

Although CCPA, CCh, and vagal stimulation with methoxamine prolonged PR interval, indicating reduced AV conduction, there were no differences between WT and Tg+/+ mice. This does not support the view of increased expression of KG channels in the AV node. The prolongation of PR interval by CCh and adenosine is usually accounted for by KG channel activation (3, 11). However, several studies have concluded that Gβ{gamma} proteins and KG channels are not critical for the action of A1-AdoR and muscarinic stimulation on AV conduction in mouse (17, 23, 42). Furthermore, changes in AV nodal conduction by A1-AdoR activation has been recently attributed to a time and voltage-dependent K+ current (Ado-IK+) different from IK+Ach, Ado (29).

It thus appears that, in agreement with chamber-specific overexpression of Kir3.2 protein, alteration in functional responses in Tg+/+ mice only occurs in those parts of the mouse heart that normally express large amounts of heterotetrameric Kir3.1-Kir3.4 channels.

Bradycardic episodes and alternating slow and fast heart rhythm under CCPA.
We found that CCPA and muscarinic stimulation reduced HR and atrial P wave amplitude and area in both WT and Tg84+/+ mice. The HR drop with muscarinic stimulation in Tg+/+ was larger than in WT and associated with episodes of bradycardia. The HR drop with CCPA was less in Tg84+/+ than in WT and associated with alternating periods of fast and slow heart rhythm. To our knowledge such clear-cut switches between high and low HR induced by CCPA have not been described in the mouse. Though, a related description of changes in minimal and maximal HR has been given by Kirchhof et al. (23) in mice overexpressing A1-AdoR. Moreover, Fu et al. (16) described a differential dependence of CCPA and CCh chronotropic effects on G{alpha}0/G{alpha}i2 in mouse and suggested that A1-AdoR activation also uses a non-Kir3.x pathway (ICaL).

During these episodes of bradycardia and periods of alternate HR, the basic P-QRS sequence of the ECG was not modified even though P wave morphology changed. P wave morphology changes may be related to pacemaker shift induced by sympathetic/parasympathetic stimulation or agonist injection (6) and to the extension of pacemaker area and pacemaker cell characteristics and coupling (28, 39). Differences between the purinergic and muscarinic types of arrhythmia could result from lower A1-AdoR density (41), the kinetics of the responses and KG gradient density (27) and/or variation in Go/i-KG signaling cascade and/or KG/adenylate cyclase balance (16). Alterations in KG channels by KCNJ6 insertion in Tg+/+ mice could then modify the pace-making pattern with respect to WT mice.

ECG of urethane anaesthetized mice.
Most anesthetics markedly depress the ECG in mouse and particularly HR (22, 30). This can even lead to unresponsiveness to cardiovascular test (23). However, urethane anesthesia is largely used to maintain cardio-respiratory control (18). In the present work, it maintained HR to values close to that recorded by telemetry in FVB mice (17). Both WT and Tg+/+ mice show episodes of bradycardia lasting for some seconds, similar to those noticed by Wickman et al. (42) in awake mice. Chronotropic responses to CCh, CCPA, methoxamine, and atropine were quantitatively similar to those reported in unsedated mice (16, 17, 42).

In control conditions, ECG wave morphology is very close to that of unsedated mice. The T wave is often illusive (26). Here, the S wave is reduced, and the late negative Tneg wave is amplified by CCPA and CCh. This contrasts with other mammals that only show changes in wave duration with sympathetic and parasympathetic drug injections.

Down syndrome.
Our data indicate that transgenic mice incorporating the human KCNJ6 gene express the corresponding h-mRNA, overexpress Kir3.2 proteins and exhibit altered cardiac responses to drugs which activate KG channels in the sino-atrial node and the atria. Our results are in agreement with other reports showing altered phenotypes in mouse models of DS carrying a limited number of genes (2, 7, 10, 33, 37). To date, cardiac effects of trisomy 21 have largely focused on CHD. Our results point out to less obvious but possibly more general effects of trisomy 21, namely altered cardiac regulation and arrhythmogenicity. Our electrocardiographic data indicate that responses to moderate vagal and purinergic stimulations are normal but responses to larger stimulations are potentiated. Our data shed some light on reports of HRV in trisomic patients free of cardiac malformations (13, 15, 20). However, sustained parasympathetic tone is absent in mouse (atropine is without effect) but is present in human (22, 38). Taking into account this specificity of the mouse, the overexpression of KCNJ6 in human might have a more sustained cardiac effect.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by the CNRS, the "Conseil Régional du Centre," and the Fondation Lejeune, France. Z. Bichler was supported by the "Région Centre" (France) and Z. Bichler and J. A. del Rio were supported by the Spanish Ministry of Science and Technology (MCYT BFU2006-13651).


   ACKNOWLEDGMENTS
 
The authors thank S. Rose and M. C. Gonzalez for expert technical assistance. They are much indebted to Dr. Ian Findlay and Prof. Pierre Roubertoux for help and criticisms.


   FOOTNOTES
 
Address for reprint requests and other correspondence: J. M. Lignon, CNRS UMR 6542, Physiologie des Cellules Cardiaques et Vasculaires, Université François-Rabelais, Parc Grandmont, 37200 Tours, France (e-mail: jacques.lignon{at}univ-tours.fr).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
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 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
 REFERENCES
 

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