Postnatal development of atrial repolarization in the mouse

Abstract

Objectives and methods: This study examines postnatal development of action potential duration (APD) and voltage-dependent K⁺ currents in mouse atrial myocytes and compares the expression levels of corresponding K⁺ channels between adult and neonatal mouse atrial tissues. APD and K⁺ currents were compared between atrial myocytes isolated from postnatal Day-1, Day-7, Day-20, and adult mice. Results: All K⁺ currents examined underwent significant up-regulation during postnatal life in mouse atrium, resulting in a dramatic shortening of the APD. The ultrarapid delayed rectifier (I_Kur) was absent in the developing mouse heart and only contributed to repolarization in the adult mouse atrium, whereas the density of the other K⁺ currents increased earlier during the developmental period. Indeed, the major changes in the expression of the inward rectifier current (I_K1) occurred within the first week of life, the density of the Ca²⁺-independent transient outward K⁺ current (I_to) gradually increased while the development of the steady-state outward K⁺ current (I_ss) was completed within the first 3 weeks of life. Results of RNase protection assay and Western blot analysis confirmed that the postnatal development of the mouse atrial K⁺ currents correlates with an increase in expression levels of underlying K⁺ channel isoforms. Conclusion: These findings indicate that in mouse atrium, each K⁺ current exhibits a specific postnatal development, suggesting that regulatory factors taking place during development are major determinants of the functional role of K⁺ channels in cardiac repolarization. The mouse atrium is, therefore, a very interesting model to gain information on the mechanisms regulating K⁺ channel activity.

Mouse heart, Atrial myocytes, Repolarization, Postnatal development, K⁺ currents

1. Introduction

A number of recent studies have focused on the expression of K⁺ currents in adult mouse ventricular myocytes [3,12,13,16,20,21,26,28–30,32]. However, despite the fact that it is well recognized that there is difference in the K⁺ currents expressed in cells isolated from different regions of the heart in the same species [1,2,6,11,18], at present, studies of ionic currents controlling atrial repolarization in mouse are much less detailed. Before being able to study the regulation of cardiac repolarization in mouse atrium, we have to understand the ionic and molecular properties of the main K⁺ currents governing repolarization in this tissue. To our knowledge, until now only few studies have reported the presence of outward K⁺ currents in adult mouse atrial cells [5,19,31].

Despite the widespread interest in the mouse as a model for genetically manipulated cardiovascular disease, no comprehensive study has been reported regarding the functional properties and expression levels of K⁺ channels throughout postnatal development in mouse atrium. Moreover, functional changes in the heart take place in the first few days or weeks of postnatal life. Understanding these developmental changes and the factors regulating them might give significant insight into the mechanisms controlling specific cardiac functions such as cardiac K⁺ channel physiology. Indeed, changes in K⁺ channel expression during cardiac development are likely to reflect alterations in cardiac repolarization.

So far, there are some reports related to embryonic developmental changes in K⁺ currents/channels in mouse atrial and ventricular myocytes [10,15]. However, after birth relatively little is known concerning the functional properties and expression levels of the different K⁺ channels present in mouse atrium. Although no data are available yet on atrial repolarization throughout postnatal development in mouse, Bou-Abboud et al. [5] have compared K⁺ currents in atrial myocytes isolated from adult and postnatal day 15 (P15) C57BL6 mice. They documented a small but statistically significant difference in the current density of the outward K⁺ current between the two groups. In fact, the densities of the three outward K⁺ currents were higher in P15 than in adult cells. Since the current amplitudes were not different between P15 and adult cells, they attributed these differences to smaller membrane capacitance in P15 mice.

The present study was initially directed toward electrophysiological characterization of the action potential configuration and the K⁺ currents present in mouse atrium throughout postnatal development. We then examined the expression levels of different K⁺ channel isoforms in mouse atrium, providing further evidence for the molecular basis responsible for these K⁺ currents.

2. Materials and methods

2.1. Animals

Animal handling followed the guidelines of the Canadian Council of Animal Care and conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. All experiments were performed on postnatal Day-1, Day-7, Day-20 and adult CD1 mice obtained from Charles River (St-Constant, Qc, Can). Adult mice were heparinized (100 U, I.P.) before being anaesthetized by inhalation of isoflurane and then sacrificed by cervical dislocation. Neonatal mice were sacrificed by decapitation.

2.2. Isolation of mouse atrial myocytes

2.2.1. Neonatal mouse atrial myocytes

The isolation procedure and primary cell culture technique used for the 1-day-old mouse atrial myocytes was identical to the one used for rat myocytes [14]. Briefly, the 1-day-old mouse myocytes were obtained by enzymatic dispersion using a “chunk” method. For each cell preparation, 30 animals were used. Both left and right atria were excised quickly under sterile conditions and placed in a Ca²⁺-free buffer solution (Joklik's Minimal Essential Medium (S-MEM; Gibco BRL, Grand Island, NY, USA) supplemented with 24 mM NaHCO₃, 0.6 mM MgSO₄ and 1 mM dl-carnitine, pH 7.4) at room temperature. The atria were washed and minced gently in buffer solution containing 0.23 mg/ml collagenase (Yakult, Tokyo, Japan), 1% bovine serum albumin and 20 mM taurine. The tissue was then incubated with 2 ml of the enzyme solution and continuously agitated at 37 °C. During the first 15–20 min of incubation, the supernatant was removed and replaced with fresh enzymatic solution every 5 min. Subsequently the supernatant was collected and diluted 1:1 in culture medium (M-199; Sigma) supplemented with 10 mM HEPES, 26 mM NaHCO₃, 10% fetal bovine serum, 1% penicillin/streptomycin, 1.25 U/ml insulin; pH was adjusted to 7.4 with NaOH.

2.2.2. Day-7, Day-20, and adult mouse atrium myocytes

Single atrial myocytes isolated from Day-7, Day-20 and adult mouse heart were dissociated by enzymatic dispersion as previously described [7,8,13,26]. In brief, the hearts were rapidly removed, and retrogradely perfused through the aorta on a modified Langendorff apparatus with the following solutions: (i) HEPES-buffered Tyrode solution containing (in mM): 130 NaCl, 5.4 KCl, 1 CaCl₂, 1 MgCl₂, 0.3 Na₂HPO₄, 10 HEPES, 5.5 glucose (pH adjusted to 7.4 with NaOH) for 5 min; (ii) Tyrode solution without added Ca²⁺ (“Ca²⁺-free”) for 10 min. The adult hearts were digested with the enzymatic solution Ca²⁺-free Tyrode solution containing 73.7 U ml⁻¹ collagenase type 2, Worthington, Freehold, NJ, USA; 0.1% bovine serum albumin, BSA; Fraction V, Sigma, St. Louis, MO, USA; 20 mM taurine and 30 μM CaCl₂) for 25 min at a flow rate of 2 ml/min. Day-7 and Day-20 hearts were digested for 30 min (Day-7) and for 45 min (Day-20) at a flow rate of 1 ml/min. At the end of the perfusion, the atria were removed from the heart, rod-shape single atrial myocytes were obtained by gentle agitation and stored in KB solution (in mM): 100 K-glutamate, 10 K-aspartate, 25 KCl, 10 KH₂PO₄, 2 MgSO₄, 20 taurine, 5 creatine base, 0.5 EGTA, 5 HEPES, 0.1% BSA, 20 glucose (pH adjusted to 7.2 with KOH) at 4 °C until being used 2–6 h later.

2.3. Electrophysiological recording procedures and data analysis

The myocytes were superfused with HEPES-buffered Tyrode solution (see above section on adult mouse myocytes). Pipettes were filled with the following solution (mM): 110 K⁺-aspartate, 20 KCl, 8 NaCl, 1 MgC1₂, 1 CaC1₂, 10 BAPTA, 4 K₂ATP and 10 HEPES (pH 7.2 with KOH). Whole-cell voltage and current recording methods, data acquisition, voltage-clamp protocols and analysis methods were identical to those described previously [7,26] All experiments were carried out at room temperature (20–22 °C). K⁺ currents were recorded in absence of sodium or L-type Ca²⁺ channels blockers to allow recordings of K⁺ currents and action potential on the same myocyte. Furthermore, under these recording conditions (e.g. room temperature), I_Ca is small. Also, the very fast activation and inactivation of the fast sodium current (which represents the largest part of the I_Na) [17,24] prevents interference with K⁺ currents. However, to rule out the possibility that the K⁺ currents were contaminated, in some experiments we measured K⁺ currents first in the absence of sodium and calcium channel blockade and then again in the presence of Na⁺ and Ca²⁺ blockers (tetrodotoxin and cadmium, respectively) in the same cells. These experiments were carried out using 7-day-old and adult mice. In both age groups, K⁺ currents obtained with and without the Na⁺ and Ca²⁺ channel blockers were not significantly different (data not shown).

2.4. RNase protection assay

An RNase protection assay was used to compare the mRNA levels for Kv1.5, Kv2.1, Kv4.2, Kv4.3, Kir2.1, KvLQT1, and minK K⁺ channels in mouse atrium obtained from Day-1 and adult mice. The preparation of the RNA and the RNase protection assay were performed as described previously [26]. For each total RNA samples, tissues were pooled from 5–8 pairs of atria. Transcription was carried out using the MAXIscript In Vitro transcription kit (Ambion, Austin, TX, USA). The RNase protection assays were performed using the RPAIII kit (Ambion).

2.5. Statistical analysis

Values are presented as mean±S.E.M., and n refers to the number of different cells. One-way analysis of variance (ANOVA) with a Tukey post-test was used to compare the differences in mean values of all pairs of age groups. P-values smaller than 0.05 were considered to be statistically significant.

3. Results

3.1. Action potentials in adult and developing mouse atrial myocytes

Representative action potential recordings obtained from Day-1, Day-7, Day-20 and adult mouse atrial myocytes are illustrated in Fig. 1A. The duration and configuration of the action potential dramatically changed during postnatal development. The bar graphs in Fig. 1B summarize the mean action potential duration (APD) data measured at 20%, 50%, and 90% of repolarization in each of the four age groups. The APDs were found to be significantly longer in neonatal atrial myocytes when compared with values obtained in the three older age groups (see Fig. 1). It should be noted that all of the recordings in this study were made with patch pipettes containing a high concentration of BAPTA, hence it is unlikely that any electrogenic processes that depend on changes in internal [Ca²⁺], e.g., Na⁺–Ca²⁺ exchange would contribute to the waveform of the AP.

Fig. 1

Age-related changes in APDs in mouse atrial myocytes. (A) Typical examples of action potential recorded in Day-1, Day-7, Day-20, and adult mouse atrial myocytes recorded at a rate of 4 Hz. (B) Bar graph showing mean APD₂₀, APD₅₀ and APD₉₀ (±S.E.M.) in the four different age groups. For the examples shown in panel A, resting membrane potentials were Day-1: −63.5 mV, Day-7: −73.6 mV, Day-20: −72.5 mV and adult: −79.1 mV.

3.2. Age-related changes in total K⁺ current in mouse atrial myocytes

We then examined the developmental changes of the K⁺ currents present in mouse atrium. Fig. 2A illustrates typical examples of family of total K⁺ currents obtained in all the four age groups of mouse atrial myocytes. These families of current records were elicited by series of 500-ms voltage-clamp steps from −110 to +50 mV, in 10-mV increments, from a holding potential of −80 mV. All current amplitudes were normalized to the cell capacitance and expressed as densities (pA/pF). The mean cell capacitances were (in pF) 10±0.5 (Day-1, n=25), 21±2 (Day-7, n=28), 29±3 (Day-20, n=25), and 72±2 (Adult, n=45). The current records in Fig. 2A show outward current magnitude that increased monotonically with depolarization for voltages positive to about −40 mV. On the other hand, steps to voltages between −90 and −110 mV produced inward currents that activated rapidly and maintained their amplitude throughout the duration of the steps. The currents displayed inward rectification in the voltage range around the resting potential. Fig. 2B summarizes the current–voltage (I–V) relationships of the peak currents for the four age groups. Our data show a small I_K1 in the neonatal mouse atrial cells with a less pronounced rectification relative to the data obtained in the other age groups. The current density of I_K1 measured at −110 mV was about twofold smaller in neonatal atrial cells compared to that in the older age groups. In fact, I_K1 (in pA/pF) increased from Day-1 (−8.5±1.3) to Day-7 (−17.2±1.7, p<0.001) without any further increase thereafter (Day-20: −20.1±2.6, Adult: −16.8±1.0). Consistent with these data, the resting membrane potentials (in mV) increased from −60.6±2.0 (n=25) to −73.4±1.3 (n=28) (p<0.001) during that period but was comparable between Day-7 and the two older groups (Day-20: −73.8±1.7, n=25 and Adult: −73.9±1.1, n=45). Also shown on this figure, there was a progressive postnatal development in the current density of the peak outward K⁺ current (I_peak) in mouse atrium. For instance, at +30 mV, there was more than a threefold increase in the density of the current (in pA/pF) (Day-1: 10.5±0.9; Day-7: 14.9±2; Day-20: 22.6±2; Adult: 33.7±3) between Day-1 and adult atrial myocytes.

Fig. 2

Postnatal development of I_peak in mouse atrial myocytes. (A) Family of K⁺ currents from voltage-clamped mouse atrial myocytes isolated from Day-1 to adult mice were recorded at a rate of 0.1 Hz. (B) Mean I–V relationships for the peak K⁺ current (I_peak) for the cells in the four age groups presented in A.

3.3. Postnatal development of I_Kslow in mouse atrial myocytes

We then examined the age-dependent changes of individual outward K⁺ currents. First, we eliminated the transient portion (or I_to) by applying an inactivating prepulse (100 ms, −40 mV) right before the main activation steps. The current remaining is denoted I_Kslow and is composed of a 4-aminopyridine (4-AP)-sensitive component (I_Kur) and a 4-AP-resistant component (I_ss). Indeed, as we previously reported for mouse ventricular myocytes [7,26], we use the difference in sensitivities of I_Kur and I_ss to 4-AP to isolate these two components of I_Kslow in mouse atrial myocytes (see below). Representative traces of I_Kslow recorded from Day-1, Day-7, Day-20 and adult mouse atrial myocytes are shown in Fig. 3A. The mean I–V curves for I_Kslow in all age groups are presented in Fig. 3B. I_Kslow was very small during the first postnatal week (in pA/pF) (at +30 mV: Day-1, 8.4±0.7; Day-7, 8.2±1.3, p=NS) and then there was almost a twofold increase at Day-20 (15.4±1.1, p<0.001) and after that, I_Kslow increased even further in the adult group (19.4±1.2, p<0.001).

Fig. 3

Postnatal development of I_Kslow (I_Kur+I_ss) in mouse atrial myocytes. (A) Superimposed current traces of I_Kslow in mouse atrial myocytes isolated from Day-1 to adult mice. I_Kslow was activated by the protocol shown in inset. Note that the rapid inactivation phase of the outward current (I_to) was abolished by the prepulse. (B) Mean I–V curves for I_Kslow recorded in the four age groups.

3.4. Postnatal development of I_to in mouse atrial myocytes

Fig. 4 shows the time course of postnatal development of I_to in mouse atrium which was obtained by subtracting the current traces measured with and without the inactivating prepulse (subtraction of I_Kslow from I_peak) [7,26]. Panel A illustrates the changes in I_to at postnatal days 1, 7, 20, and adult. Fig. 4B presents the corresponding I–V curves. These results show that I_to density (in pA/pF) was very small and comparable between Day-1 (at +300 mV, 2.0±0.6) and Day-7 (2.1±0.7), but thereafter there was a significant and gradual increase in its density (Day-20: 6.4±2; Adult: 13.9±2.2).

Fig. 4

Postnatal development of I_to in mouse atrial myocytes. (A) Family of current records illustrating I_to in Day-1, Day-7, Day-20, and adult mice. I_to was obtained by subtracting corresponding current records with (Fig. 3) and without (Fig. 2) the inactivating prepulse. (B) Mean I–V curves for I_to recorded from the different age groups.

3.5. Postnatal development of I_ss in mouse atrial myocytes

We applied the inactivating prepulse (which eliminates I_to) in combination with 100 μM 4-AP (which blocks I_Kur) [7,13,22,26] and recorded the 4-AP-resistant K⁺ current, or I_ss. Data presented in Fig. 5 illustrate the postnatal development of I_ss in mouse atrial cells and demonstrate that in this tissue I_ss activated with much slower time course than I_to or I_Kur (see below) at a given membrane potential. The rate of activation of the steady-state current increased with more depolarized potentials, but it inactivated very little during the 500-ms voltage steps. The properties of this current closely resembled those of the slowly activating, 4-AP-insensitive delayed rectifier K⁺ current that we observed in mouse ventricular myocytes, designated I_ss[7,26]. As we can see in Fig. 5B, which depicts the mean I–V relationships for I_ss, there was no difference between the density of I_ss (in pA/pF) recorded from Day-1 (at +30 mV, 6.2±0.6) and Day-7 mice (7.4±1.4) but in the two older groups the density of the current was significantly greater (Day-20: 14.4±1.4, Adult: 13.4±1.3).

Fig. 5

Postnatal development of I_ss in mouse atrial myocytes. (A) Representative examples steady-state K⁺ current in Day-1 to adult mouse atrial myocytes. These currents were measured after the application of 100 μM of 4-AP using the inactivation prepulse protocol. (B) Mean I–V curves for I_ss recorded from the four groups.

3.6. Postnatal development of I_Kur in mouse atrial myocytes

Similar to the effect of 4-AP on adult mouse ventricular [7,26], a low concentration of 4-AP completely blocked a rapidly activating and slowly inactivating outward K⁺ current in the adult mouse atrial myocytes providing strong evidence that I_Kur is present in these cells [19]. We then determined the developmental changes of the 4-AP-sensitive current (or I_Kur) in mouse atrial cells. Fig. 6A presents superimposed current traces of I_Kur in the four age groups. These records were obtained by subtracting the currents recorded before (Fig. 3) and after (Fig. 5) the addition of 100 μM of 4-AP. As illustrated on these recordings, I_Kur was virtually absent in neonatal mouse atrial myocytes and remained barely visible during the first 3 weeks of postnatal life (in pA/pF) (at +30 mV: Day-1, 0.4±0.3; Day-7, not detectable; Day-20: 1.2±0.7) but then there was a significant increase in I_Kur density in the adult group (Adult: 5.9±1.1, p<0.001 vs. all).

Fig. 6

Postnatal development of I_Kur in mouse atrial myocytes. (A) Typical examples of the ultrarapid delayed rectifier in Day-1 to adult mouse atrial myocytes obtained by subtracting corresponding current records obtained with (Fig. 5) and without (Fig. 3) the application of 100 μM 4-AP. (B) Mean I–V curves for I_Kur recorded from all age groups.

3.6.1. Effect of low concentration of 4-AP on APD in developing and adult mouse atrium

We examined the effect of a 100 μM 4-AP on the APD of mouse atrial myocytes. Fig. 7A,C shows representative examples of action potential recorded at 4 Hz in Day-20 and adult mouse atrial myocytes under control conditions and in the presence of 100 μM 4-AP. Addition of this concentration of 4-AP, which blocks I_Kur without affecting I_to (see inset Fig. 7), significantly prolonged APD₅₀ and APD₉₀ only in the adult myocytes as shown in Fig. 7B,D. This effect of 4-AP on APD clearly demonstrates that I_Kur contributes to the repolarization phase of the action potential only in the adult myocytes.

Fig. 7

Effect of 100 μM 4-AP on the APD in Day-20 and adult mouse atrial myocytes. (A) Examples of action potential recordings obtained in Day-20 atrial myocytes under control conditions and after the application of 100 μM 4-AP. (B) Histograms presenting the mean data for the APD₂₀, APD₅₀ and APD₉₀ measured in Day-20 atrial myocytes. (C) Examples of action potential recordings in adult atrial myocytes under control conditions and after the application of 100 μM 4-AP. (D) Histograms showing the mean data for the APD₂₀, APD₅₀ and APD₉₀ in adult atrial myocytes. The figure presented in inset compares current recordings before and after the addition of 100 μM 4-AP. The amplitude of I_to, determined by subtracting the current record obtained with the inactivating prepulse from the current record obtained without the prepulse, was unaffected by 100 μM 4-AP (I_to: −4-AP, 100% of I_to amplitude; +4-AP, 95%±2% of I_to amplitude; n=4, p=0.2).

3.6.2. Postnatal development of the expression of K⁺ channels in mouse atrium

To establish whether the postnatal development of the different K⁺ currents in mouse atrium correlates with an increase in expression levels of underlying K⁺ channel isoforms, we examined the developmental changes in mRNA expression of several K⁺ channels using RNase protection assays. Fig. 8A shows gels in which mRNA expression levels of atrial K⁺ channels were compared between Day-1 and adult mice. These results demonstrated a significant postnatal development in the mRNA expression of the different K⁺ channels studied. The bar graphs in Fig. 8B, which show ratio of adult-to-neonatal expression levels for each K⁺ channel genes, confirm the large difference in Kv1.5, Kv2.1, Kv4.2, Kv4.3, and Kir2.1 K⁺ channel mRNA expression between Day-1 and adult mouse atrium. For instance, Kv4.2 transcripts were almost sixfold more abundant in adult than in neonatal atrium. This was the largest difference in K⁺ channel expression between the two groups. Kv1.5 and Kv4.3 expression was more than threefold superior in adults while the expression of Kv2.1 and Kir2.1 was more then double in adult tissues. Our mRNA data also show that although message for KvLQT1 can be detected in atrium of adult and neonatal mice at about equal level, minK signal was absent from both tissues.

Fig. 8

Comparison of K⁺ channels mRNA expression between neonatal and adult mouse atrium. (A) Representative autoradiographs of mRNA levels for different K⁺ channel isoforms (Kv1.5, Kv2.1 Kv4.2, Kv4.3, Kir2.1, KvLQT1, and minK) measured in 2 RNA samples isolated from Day-1 and adult atria (2.5 μg of total RNA/sample; 5–8 pairs of atria/sample). Yeast represents negative controls and β-actin the internal control. (B) Graph bars illustrating the relative abundance of the different K⁺ channel mRNA transcripts in adult and neonatal atrial tissues. Adult-to-neonatal ratios for each K⁺ channel gene are shown (n=3 different samples; 5–8 pairs of atria/sample). Values were normalized to β-actin signal.

4. Discussion

4.1. Summary of main findings

The results obtained in this study describe and compare the major repolarizing K⁺ currents that are expressed in mouse atrium throughout postnatal development. In addition, this work compares the expression level of different K⁺ channels between neonatal and adult atrial tissues. Our electrophysiological results indicate that there are four K⁺ currents present in adult mouse atrial myocytes: I_to, I_Kur, I_ss, and I_K1. All of these currents are carried by K⁺ ions as confirmed by cesium experiments (data not shown) and they are also all present in adult mouse ventricular myocytes [7,26]. There was a general increase in K⁺ channel activity during development, which correlates well with the observed shortening of the APD and more negative resting membrane potential. Furthermore, the pattern of developmental changes of each of the different K⁺ currents present in mouse atrium was different; suggesting that expression of each of them is individually regulated. In line with the electrophysiological data, the expression studies revealed that the level of expression of the corresponding K⁺ channels underwent significant up-regulation during postnatal life in mouse atrium.

4.2. Postnatal development in K⁺ current expression in the mouse atrium

The major developmental change in I_K1 expression occurred within the first week of life, the density of I_to gradually increased and the development of I_ss was completed within the first 3 weeks of life. In contrast, I_Kur was absent in the developing mouse heart and was only present in the adult mouse atrium. I_Kur density was fairly small (e.g. at +30 mV: 5.9±1 pA/pF) in adult cells, however, the effect of a low concentration of 4-AP on the APD clearly demonstrates that I_Kur contributes to the repolarization phase of the action potential in the adult myocytes but does not play a significant role in the developing mouse atrium.

The Kv1.5 mRNA level was more abundant in adult mouse atrium compared to the neonatal mouse atrium, compatible with our voltage-clamp data supporting the hypothesis that the underlying K⁺ channel responsible for I_Kur in adult mouse atrium is Kv1.5. Moreover, electrophysiological characteristics of the current and complete inhibition of I_Kur by very low concentrations of 4-AP [13] also suggest that the underlying isoform of I_Kur in mouse atrium is Kv1.5. This is in accordance with data from Bou-Abboud et al. [5] showing that Kv1.5 contributes to repolarization in mouse atrium.

4.3. Relation to previous studies

Most of the available data on the ionic mechanism(s) underlying repolarization in mouse heart describe excitability and ion channel expression in ventricular myocytes [3,12,13,16,20,21,23,26,28–30,32]. These recent studies characterize K⁺ currents in adult mouse ventricular myocytes in some detail. However, previous studies of atrial repolarization in adult mouse are much less extensive. Xu et al. [31] described the presence of three Ca²⁺-independent, depolarization activated outward K⁺ currents in adult mouse atrial myocytes. These include: a fast transient K⁺ current, a slowly inactivating K⁺ current, and a steady-state current. Using adult mice expressing a dominant-negative Kv4 α-subunit, they demonstrated that members of Kv4 subfamily underlie I_to in mouse atrium [31]. The same group has also reported that both Kv1.5 and Kv2.1 would also contribute to atrial repolarization in mouse [5]. Finally, very recently Lomax et al. [19] have compared the K⁺ currents expressed in left and right atrial myocytes isolated from adult mouse. Results obtained in these studies are all in keeping with findings presented here in adult mouse atrial myocytes.

Very few reports are available concerning developmental changes in atrial repolarization in mouse. A first study reported changes in K⁺ current expression in mouse atrial and ventricular cells during embryogenesis [10]. In embryonic atrial cells, the K⁺ currents identified include I_K1, I_to; I_ss, the slow and the rapid components of the delayed rectifier K⁺ current: I_Ks and I_Kr. More recently, another group has examined the expression of α- and β-subunits associated with I_Kr and I_Ks during embryonic and fetal life in mouse heart, showing that the pattern of expression for I_Kr and I_Ks was consistent with that of their β-subunits in embryonic mouse ventricle and atrium [15]. Thus, in the present study, it was not surprising to see that although KvLQT1 was present in mouse atrium, in the absence of minK, I_Ks was not detected. These findings are also in agreement with other studies showing that the pattern of expression of I_Ks was dependable on that of minK [4,9]. However, until now, the functional properties and expression levels of the different K⁺ channels present in mouse atrium have never been evaluated throughout postnatal life, although there is one report comparing outward K⁺ currents in atrial myocytes isolated from adult and postnatal day-15 mice [5]. The present study is therefore the first report focusing on cardiac repolarization in mouse atrium throughout postnatal development.

4.4. Relation to human atrium

In addition to being expressed in adult mouse atrial and ventricular myocytes [7,13,26], I_Kur has also been previously identified in human atrial myocytes [25,27]. Thus, results obtained in this study reveal that many of the major K⁺ currents expressed in human atrium (e.g., I_Kur, I_to, I_K1) are also present in adult mouse atrium. This suggests that the mouse atrium may become a potentially very useful model for analyzing regulation of K⁺ channel expression in transgenic models of human cardiovascular disease, for studying the effects of hormonal regulation of the expression of K⁺ channels and as a model for development of cardiac drugs targeting selected K⁺ channel isoforms such as Kv1.5.

4.5. Potential significance

Our findings indicate that in mouse atrium, each K⁺ current exhibits a specific postnatal development, suggesting that regulatory factors taking place during postnatal development are major determinants of the functional role of K⁺ channels in cardiac repolarization and that different regulatory factors may affect different channels in various ways. The mouse atrium is therefore a very interesting model to gain information on the mechanisms regulating K⁺ channel activity.

In summary, our data show that in mouse atrium, the developmental increase in K⁺ currents/channels is physiologically relevant and contributes to the dramatic changes in the duration and configuration of the action potential that occur during postnatal development. This work provides an essential foundation for the study of the regulation of cardiac K⁺ channel activity under normal and pathophysiological conditions. Finally, the presence of I_Kur/Kv1.5 in adult mouse atrium implies that the mouse atrium may become very useful as a model system for human atrium, which also expresses I_Kur and Kv1.5.

Acknowledgements

This study was supported by operating grants and personnel awards from the Canadian Institute of Health Research, the Heart and Stroke Foundation of Canada, and the Fonds de la Recherche en Santé du Québec. The authors wish to thank Judith Brouillette for critical reading of the manuscript and Maya Mamarbassi for skill technical assistance.