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Am J Physiol Heart Circ Physiol 293: H2168-H2177, 2007. First published August 3, 2007; doi:10.1152/ajpheart.00521.2007
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Postnatal development has a marked effect on ventricular repolarization in mice

Scott A. Grandy, Véronique Trépanier-Boulay, and Céline Fiset

Research Center, Montreal Heart Institute, and Faculty of Pharmacy, University of Montreal, Montreal, Quebec, Canada

Submitted 1 May 2007 ; accepted in final form 2 August 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To better understand the mechanisms that underlie cardiac repolarization abnormalities in the immature heart, this study characterized and compared K+ currents in mouse ventricular myocytes from day 1, day 7, day 20, and adult CD1 mice to determine the effects of postnatal development on ventricular repolarization. Current- and patch-clamp techniques were used to examine action potentials and the K+ currents underlying repolarization in isolated myocytes. RT-PCR was used to quantify mRNA expression for the K+ channels of interest. This study found that action potential duration (APD) decreased as age increased, with the shortest APDs observed in adult myocytes. This study also showed that K+ currents and the mRNA relative abundance for the various K+ channels were significantly greater in adult myocytes compared with day 1 myocytes. Examination of the individual components of total K+ current revealed that the inward rectifier K+ current (IK1) developed by day 7, both the Ca2+-independent transient outward current (Ito) and the steady-state outward K+ current (Iss) developed by day 20, and the ultrarapid delayed rectifier K+ current (IKur) did not fully develop until the mouse reached maturity. Interestingly, the increase in IKur was not associated with a decrease in APD. Comparison of atrial and ventricular K+ currents showed that Ito and IKur density were significantly greater in day 7, day 20, and adult myocytes compared with age-matched atrial cells. Overall, it appears that, in mouse ventricle, developmental changes in APD are likely attributable to increases in Ito, Iss, and IK1, whereas the role of IKur during postnatal development appears to be less critical to APD.

mouse; ventricle; K+ currents; K+ channels; action potential

AN EXTENSIVE AMOUNT OF EFFORT has been spent defining the functional properties of K+ currents that underlie repolarization in the adult mouse heart (2–5, 8, 13, 19, 22, 25, 39, 43, 44, 46–48). Studies have shown that the inward rectifier K+ current (IK1), the Ca2+-independent transient outward current (Ito), the steady-state outward K+ current (Iss), and the ultrarapid delayed rectifier K+ (IKur) determine action potential duration (APD) and the rate of repolarization (4, 6, 8, 25, 47). Studies in other species have shown that ventricular repolarization and APD change significantly between neonates and adults (7, 12, 15, 16, 30, 41, 45). However, very little is known about the effects of development on the individual K+ currents that underlie repolarization in mouse ventricular myocytes.

In mice, one study has shown that ventricular APD decreases as maturation occurs (40). Interestingly, the effects of postnatal development on K+ current in mouse ventricular myocytes has only been reported for Ito (39). Wang and Duff (39) showed a significant increase in an outward K+ current that they attributed to Ito between postnatal day 1 and postnatal day 14 and again between postnatal day 14 and adulthood (39). However, the comprehensive characterization of the development of the K+ currents that underlie ventricular repolarization (IK1, Ito, Iss, IKur) has not been performed. Therefore, it is necessary to evaluate the K+ currents that determine APD and waveform to better understand the developmental changes that occur in ventricular repolarization. Improving our understanding of how the action potential is modulated by developing K+ currents could provide insight into the differences that exist between adult and immature hearts in the mechanisms responsible for arrhythmia and other repolarization disorders. Accordingly, the main objective of this study was to characterize and compare action potential configurations and repolarizing K+ currents in ventricular myocytes isolated from day 1, day 7, day 20, and adult mice to determine the effects of postnatal development on ventricular repolarization.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Experimental animals. CD1 mice (day 1, day 7, day 20, and adult) were obtained from Charles River (St. Constant, QC, Canada). All experiments were approved by the Animal Protection Committee of the Montreal Heart Institute (protocol no. 2006-80-1) and were performed in accordance with the guidelines of the Canadian Council for Animal Care and 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).

Cell isolation. Ventricular myocytes were isolated from 7-day, 20-day, and adult mice, as previously described (9, 38). In brief, mice were heparinized (100 units ip) before being anesthetized with isoflurane. Mice were then killed by cervical dislocation (day 20 and adult) or by decapitation (day 1 and day 7). Hearts were quickly removed from the chest cavity and hung on a modified Langendorff apparatus. Hearts were perfused retrogradely through the aorta for 5 min with a Tyrode solution containing the following (in mM): 130 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 0.3 Na2HPO4, 10 HEPES, and 5.5 glucose (pH 7.4 with NaOH). Next, the heart was perfused for 10 min with a Ca2+-free Tyrode solution. The hearts were then perfused with a modified Tyrode solution containing the following: type II collagenase (73.7 U/ml; Worthington, Freehold, NJ), 0.1% bovine serum albumin (BSA; Fraction V, Sigma, St. Louis, MO), 20 mM taurine, and 0.03 mM CaCl2. Adult hearts were perfused with the modified Tyrode solution for 25 min at a flow rate of 2 ml/min, whereas hearts from 7- and 20-day-old mice were perfused at a flow rate of 1 ml/min for 30 and 45 min, respectively. Once perfusion was complete, the free wall of the right ventricle was removed, minced, and stored in a Kraft-Bruhe (KB) solution containing (in mM) the following: 100 potassium-glutamate, 2 MgSO4, 20 taurine, 5 creatine base, 0.5 EGTA, 5 HEPES, 0.1% BSA, and 20 glucose (pH 7.2 with KOH). The tissue was then gently agitated to free individual myocytes. Ventricular myocytes were stored at 4°C until needed.

Ventricular myocytes were isolated from 1-day-old neonatal mice by enzymatic dispersion using a "chunk" method and were cultured as previously described (9, 35, 38). In brief, mice were killed by decapitation, and hearts were removed under sterile conditions and placed in a Ca2+-free solution (Joklik's minimal essential medium, S-MEM; Gibco BRL, Grand Island, NY), supplemented with 24 mM NaHCO3, 0.6 mM MgSO4, and 1 mM DL-carnitine; pH 7.4. The ventricles were excised and placed in a buffer solution containing collagenase (0.23 mg/ml; Yakult, Tokoyo, Japan), 1% BSA, and 20 mM taurine, washed, and minced. The tissue was incubated in 2 ml of enzyme solution at 37°C and was continuously agitated. For the first 15 min, the supernatant was removed every 5 min and replaced with fresh enzyme solution. Subsequently, the supernatant was collected and diluted 1:1 in culture medium (M-199; Sigma) containing 10 mM HEPES, 26 mM NaHCO3, 10% fetal bovine serum, 1% penicillin-streptomycin, and 1.25 U/ml insulin (pH 7.4 with NaOH). Each preparation utilized the hearts from 30 mice.

Electrophysiological procedures and data analysis. Ventricular myocytes were placed in a recording chamber mounted on the stage of an inverted microscope. Cells were given 5–10 min to settle and adhere to the bottom of the chamber. Cells were continuously superfused with Tyrode solution. In some experiments, the Tyrode solution was supplemented with 100 µM 4-aminopyridine (4-AP). All experiments were conducted at room temperature (20–22°C).

Action potentials and K+ currents were recorded with a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Foster City, CA) in whole cell current- and voltage-clamp modes. Patch pipettes (2–4 M{Omega}) were filled with a solution containing (in mM) the following: 20 KCl, 1 MgCl2, 1 CaCl2, 10 BAPTA, 4 K2ATP, and 10 HEPES (pH 7.2 with KOH). pCLAMP 8.0 software (Axon Instruments) was used to low-pass filter (1 kHz; 4-pole Bessel analog filter), digitize (4 kHz), and store voltage-clamp currents. Capacitive transients were initiated by a 10-mV depolarizing step from a holding potential of –80 mV. The capacitive transient was integrated to provide an estimate of cell membrane area. A –10-mV correction was applied to all recorded membrane potentials to compensate for the patch pipette-bath liquid junction potential (K+-aspartate).

Action potentials were initiated with a brief stimulus current (1–3 ms; 0.4–0.7 nA) at a rate of 1–4 Hz. The current-voltage (I-V) relationship for total K+ current (Ipeak) was constructed by making consecutive 10-mV voltage steps from –110 to +50 mV. The separation of the underlying K+ currents is described in detail in Brouillette et al. (4). In brief, an inactivating prepulse at –40 mV (100 ms) was added before the voltage steps to visualize the slow component of the outward K+ current (IKslow). Ito was calculated as the difference between the current obtained with the voltage-clamp protocol with the inactivating prepulse and the protocol minus the inactivating prepulse. IKslow is composed of the 4-AP-sensitive K+ current, IKur, and the 4-AP-insensitive K+ current, Iss (4). To measure Iss and IKur, currents were initiated with the aforementioned voltage protocol with the inactivating prepulse in the presence or absence of 4-AP. Iss was the K+ current that remained in the presence of 4-AP, whereas IKur was the difference between the currents recorded in the presence and absence of 4-AP. Outward K+ currents were measured at the peak produced by the voltage step, whereas the inward current, IK1, was measured at the end of the 500-ms voltage step. All current recordings were collected at a rate of 0.1 Hz.

Real-time RT-PCR. Ventricular total RNA was isolated with a RNeasy fibrous tissue kit (Qiagen) and treated with DNase I to prevent contamination by genomic DNA. Next, cDNA was synthesized with the cloned avian myeloblastosis virus reverse transcriptase (Invitrogen) and primers specific for each K+ channel of interest Kv1.5, Kv2.1, Kv4.2, Kv4.3, Kir2.1 and the auxiliary K+ channel subunit, K+ channel interacting protein (KChIP). Melting curves for each set of primers were examined to ensure that the primers amplified a unique cDNA product. In addition, gel electrophoresis was used to ensure that the PCR fragments represented one unique product and that its molecular weight corresponded to the expected weight of the gene fragment for the K+ channel or subunit of interest. The real-time PCR reaction was performed with Platinum SYBRgreen qPCR Supermix (Invitrogen) using a real-time PCR system (MX3005P QPCR system, Stratagene). The PCR reactions were cycled 40 times using a three-step cycle procedure (denaturation at 95°C for 30 s, annealing at 50°C for 45 s, elongation at 72°C for 45 s) after the initial stage (10 min at 95°C). mRNA expression was quantified relative to the murine cyclophyllin. To ensure the validity of the results, the linearity and the efficiency criteria of the standard curves were thoroughly respected.

Statistical analysis. The sample size (n) refers to the total number of cells used for each experiment. Student t-tests or a one-way ANOVA was used when appropriate. A Tukey post hoc test was used to determine specific differences between age groups. ANOVA was performed with Origin 5.0 (OriginLab, Northhampton, MA). The data are presented as means ± SE. P values <0.05 were considered statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
APD. Initial experiments characterized and compared action potentials from ventricular myocytes isolated from the hearts of 1-, 7-, and 20-day-old mice, as well as adult mice. Representative action potentials from day 1, day 7, day 20, and adult ventricular myocytes are shown in Fig. 1, A–D. The traces show that the shape and duration of the action potential gradually changed as development progressed. Mean data for APD at 20, 50, and 90% repolarization are shown in Fig. 1E. APD was significantly longer in day 1 myocytes compared with all other ages. In addition, APD also was greater in day 7 cells compared with day 20 and adult cells. Interestingly, APD at 20, 50, and 90% repolarization were similar in day 20 and adults cells. This suggests that the action potential waveform/duration was fully developed by day 20.


Figure 1
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  		Fig. 1. Action potential duration (ADP) decreases during postnatal development. Action potentials were initiated by injecting a stimulus current (1–3 ms; 0.4–0.7 nA). Myocytes were stimulated at a rate of 4 Hz. A–D: representative action potentials for ventricular myocytes isolated from day 1, day 7, day 20, and adult mice, respectively. E: bar graph comparing mean APD at 20%, 50%, and 90% repolarization. *P < 0.001 vs. all; **P < 0.05 vs. day 20; {dagger}P < 0.01 vs. adult. Day 1, n = 8; day 7, n = 16; day 20, n = 18; adult, n = 13.

 
Ipeak. The next series of experiments examined the effects of postnatal development on Ipeak in ventricular myocytes. Ipeak was activated by 500-ms voltage-clamp steps made from a holding potential of –80 mV. Steps were made in 10-mV increments from –110 to +50 mV (Fig. 2, inset). All currents were normalized to the cell capacitance and expressed as current densities (pA/pF). Mean cell capacitances were (in pF) 13.8 ± 1.2 (day 1, n = 42), 51.9 ± 2.3 (day 7, n = 22), 110.3 ± 7.9 (day 20, n = 7.9), and 120.2 ± 12.1 (adult, n = 21). Representative traces for Ipeak from day 1, day 7, day 20, and adults cells are illustrated in Fig. 2, A–D. The mean I-V relationships are shown in Fig. 2E. The mean data show that Ipeak was significantly larger in adult cells compared with any other age. Ipeak also was significantly larger in 20-day-old myocytes compared with day 1 and day 7 myocytes. In contrast, Ipeak was similar in day 1 and day 7 myocytes. Of note, IK1 was significantly smaller in day 1 myocytes at –110 mV compared with all other groups. In line with these results, resting membrane potential was significantly more positive in day 1 cells compared with older cells (day 1 vs. all, P = 0.000). The values for resting membrane potential (mV) were as follows: day 1, –34.5 ± 3.0 (n = 19); day 7, –70.1 ± 4.0 (n = 13); day 20, –72.3 ± 3.6 (n = 11); and adult, –76.1 ± 1.4 (n = 18). Overall, it appears that IK1 develops rapidly (within 7 days of birth) compared with other components of Ipeak, which exhibit slower rates of development.


Figure 2
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  		Fig. 2. Total K+ currents (Ipeak) are markedly increased in adult ventricular myocytes compared with neonatal cells. Protocol is shown in inset. Membrane currents were initiated by 500-ms voltage-clamp steps elicited from a holding potential of –80 mV. Steps were generated in 10-mV increments from –110 to +50 mV at a rate of 0.1 Hz. A–D: representative Ipeak currents from day 1, day 7, day 20, and adult myocytes, respectively. E: mean current-voltage (I-V) curves show that Ipeak was significantly larger in adult cells compared with all other groups. Ipeak also was significantly greater in day 20 cells compared with day 1 and day 7 cells. Ipeak was similar in day 1 and day 7 cells. *P < 0.001, adult vs. all; **P < 0.001, day 20 vs. day 1 and day 7; {dagger}P < 0.01, all vs. day 1. Day 1, n = 24; day 7, n = 10; day 20, n = 15; adult, n = 17.

 
Next, we examined the effects of development on the individual outward K+ currents (Ito, IKur, and Iss). A 100-ms prepulse to –40 mV was applied before the main activation steps to inactivate Ito (see GoFig. 4, inset). Ito was then measured by subtracting pairs of currents recorded with and without the inactivating prepulse (4). Representative Ito traces for all four groups are illustrated in Fig. 3, A–D. The mean I-V relationships show that the current density for Ito was significantly less in day 1 myocytes compared with all other groups. Ito density also was significantly smaller in day 7 myocytes compared with ventricular myocytes from adult and 20-day-old mice. In contrast, the mean I-V curves for Ito were similar in day 20 and adult myocytes. These findings suggest that Ito is fully developed by day 20 in the mouse ventricle.


Figure 3
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  		Fig. 3. Transient outward current (Ito) density markedly increases between day 1 and day 20 of development. A–D: representative Ito traces from day 1, day 7, day 20, and adult ventricular myocytes, respectively. E: mean I-V curves show that Ito was similar in cells from day 20 and adult mice. However, a significant increase in Ito was observed between day 1 and day 7 and then again between day 7 and day 20. *P < 0.001, day 1 vs. day 20 and adult; **P < 0.05, day 1 vs. day 7; {dagger}P < 0.01, day 7 vs. day 20 and adult. Day 1, n = 24; day 7, n = 10; day 20, n = 15; adult, n = 16.

 

Figure 4
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  		Fig. 4. Slow component of outward K+ current (IKslow) is significantly larger in adult myocytes compared with all other developmental stages examined. IKslow was activated with the protocol shown in the inset. A–D: superimposed current traces from ventricular cells isolated from day 1, day 7, day 20, and adult mice, respectively. E: mean I-V curves recorded from the 4 age groups. IKslow was significantly larger in adult myocytes than in all other age groups. In addition, inward rectifier K+ current (IK1) was significantly smaller in day 1 myocytes compared with all other groups. *P < 0.001, adult vs. all; **P < 0.001, day 20 vs. day 7 and day 1; {dagger}P < 0.01, day 1 vs. all. Day 1, n = 30; day 7, n = 16; day 20, n = 20; adult, n = 17.

 
The current remaining after the 100-ms prepulse is denoted as IKslow (4). Figure 4, A–D, shows representative examples of IKslow in day 1, day 7, day 20, and adult myocytes. Figure 4E shows that IKslow was significantly greater in adult myocytes (–30 to +50 mV) compared with ventricular myocytes from 1-, 7- and 20-day-old mice. In addition, IKslow was greater in day 20 myocytes compared with day 7 myocytes. In contrast, IKslow was similar in ventricular myocytes from day 1 and day 7 mice.

IKslow is composed of a 4-AP-sensitive component (IKur) and a 4-AP-insensitive component (Iss) (4). We isolated Iss with a combination of 100 µM 4-AP and the Ito-inactivating prepulse (4). Figure 5, A–D, shows representative Iss traces from day 1, 7, and 20, and adult ventricular myocytes. Similar to Ito, the mean I-V curves show that Iss was similar in day 20 and adult myocytes (Fig. 5E). However, Iss was significantly smaller in day 1 and day 7 myocytes compared with myocytes isolated from day 20 and adult mice. Figure 5E also shows that the mean I-V relationships for Iss were similar in day 1 and day 7 myocytes. Thus it appears that the major development of Iss occurs between day 7 and day 20 in mouse ventricular myocytes.


Figure 5
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  		Fig. 5. Steady-state outward K+ current (Iss) in mouse ventricular myocytes is fully developed by day 20. An inactivating prepulse to –40 mV (inset) and the application of 100 µM 4-aminopyridine (4-AP) were used to obtain Iss. A–D: representative Iss traces from myocytes isolated from day 1, day 7, day 20, and adult mice, respectively. E: mean I-V relationships show that Iss density increases between day 1 and day 7 and then again between day 7 and day 20. Interestingly, Iss showed no further development between day 20 and adulthood. The mean data also shows that IK1 was significantly smaller in day 1 myocytes compared with all other groups. *P < 0.001, day 1 and day 7 vs. day 20 and adult; **P < 0.001, day 1 vs. all. Day 1, n = 15; day 7, n = 8; day 20, n = 13; adult, n = 14.

 
Next, we examined the 4-AP-sensitive K+ current (IKur), by subtracting recordings obtained in the absence and presence of 4-AP. Representative IKur traces from day 1, day 7, day 20, and adult myocytes are shown in Fig. 6, A–D. Figure 6E shows the mean I-V curves for IKur were similar in myocytes isolated from 1-, 7-, and 20-day-old mice. A significant increase in IKur density was only observed in ventricular myocytes from adult mice. Interestingly, major changes in IKur do not occur until after APD has stabilized.


Figure 6
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  		Fig. 6. Ultrapid delayed rectifier K+ current (IKur) develops between day 20 and adulthood in mouse ventricular myocytes. IKur was obtained by subtracting currents recorded in the presence of 4-AP from currents recorded in the absence of 4-AP. A–D: representative IKur recordings from day 1, day 7, day 20, and adult ventricular myocytes, respectively. E: mean data show that IKur was significantly increased in adult myocytes compared with day 1, day 7, and day 20 myocytes. Interestingly, IKur was similar in myocytes isolated from 1-day, 7-day, and 20-day-old mice. *P < 0.001, adult vs. all. Day 1, n = 15; day 7, n = 8; day 20, n = 13; adult, n = 14.

 
Expression of K+ channels in mouse ventricles during postnatal development. To determine whether the observed increases in K+ currents were attributable to changes in postnatal channel expression, the next series of experiments characterized mRNA levels for the channels corresponding to Ito (Kv4.2, Kv4.3), Iss (Kv2.1), IKur (Kv1.5), and IK1 (Kir2.1) and the accessory subunit KChIP, which also contributes to Ito, in ventricular tissue from day 1, day 7, day 20, and adult mice. Figure 7, A–F, shows the mean mRNA relative abundance for Kv4.2, Kv4.3, KChIP, Kv2.1, Kv1.5, and Kir2.1, respectively. For both Kv4.2 (Fig. 7A) and Kv4.3 (Fig. 7B), mRNA levels were significantly greater in adult and day 20 tissue compared with day 1 and day 7 tissue. Interestingly, Kv4.2 mRNA expression was similar in day 20 and adult tissue, whereas Kv4.3 expression was significantly higher in adult tissue compared with ventricular tissue from day 20 mice. Channel-interacting proteins (KChIP), which associate with Kv4.2 and Kv4.3 and can modify Ito density (18, 31, 34), also were present in the developing mouse ventricle. The results showed that KChIP mRNA expression was significantly greater in day 20 and adult tissue compared with day 1 and day 7 tissue (Fig. 7C). In addition, KChIP mRNA levels were similar in day 1 and day 7 tissue and in day 20 and adult tissue. Also, the pattern of KChIP mRNA expression was similar to that observed for Kv4.2. Thus the increases in Kv4.2, Kv4.3, and KChIP mRNA between day 1 and adulthood may be responsible in part for the increased Ito density observed during this time period.


Figure 7
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  		Fig. 7. K+ channel and K+ channel interacting protein (KChIP) mRNA expression increased markedly between day 1 and adulthood in the mouse ventricle. Quantitative real-time RT-PCR was used to determine mRNA expression levels of the K+ channel isoforms. For each channel, the cDNA samples for each age were amplified simultaneously. Values were normalized to the cyclophillin signal. Each sample was analyzed in duplicate. A–F: the comparison of the relative abundance of voltage-gated K+ (Kv) 4.2, Kv4.3, KChIP, Kv2.1, Kv1.5, and inwardly rectifying K+ (Kir) 2.1, respectively. *Significantly different from day 20, day 7, and day 1; #significantly different from day 1 and day 7; +significantly different from day 7 and day 1; **significantly different from day 1; ++significantly different from day 20: P < 0.05. Sample size: day 1, n = 3, 18 hearts/sample; day 7, n = 3, 3 hearts/sample; day 20, n = 3, 2 hearts/sample; adult, n = 3, 1 heart/sample.

 
Next, we examined mRNA expression for Kv2.1, Kv1.5, and Kir2.1. Figure 7, D–F, shows that mRNA expression of Kv2.1, Kv1.5, and Kir2.1 was significantly higher in adult and day 20 tissue compared with day 1 tissue. Interestingly, there was no increase in any of the mRNA transcripts of interest between day 1 and day 7. This suggests the significant increases in Kv2.1, Kv1.5, and Kir2.1 do not occur until after day 7.

Comparison of K+ currents in developing atrial and ventricular myocytes. Previously, our laboratory reported that K+ current density increases in mouse atrial myocytes as development occurs (38). However, it is known that K+ current density varies in different regions of the heart (1, 4, 10). Therefore, we compared IK1, Ito, Iss, and IKur density in atrial (38) and ventricular myocytes to determine whether current density and developmental patterns were similar in both tissues. Figure 8 shows the comparison of mean IK1, Ito, Iss, and IKur density in atrial [previously reported in Trépanier-Boulay et al. (38)] and ventricular myocytes isolated from 1-, 7-, and 20-day-old, and adult mice. Figure 8A shows that IK1 density was similar in atrial and ventricular myocytes for all time points (day 1, 7, and 20, and adult). In contrast, Ito density was only comparable in atrial and ventricular myocytes isolated from 1-day-old mice. In day 7, day 20, and adult myocytes, Ito was significantly greater in ventricular cells compared with age-matched atrial cells (Fig. 8B). Figure 8C shows that, in day 1 myocytes, mean Iss density was significantly smaller in ventricular myocytes compared with atrial cells, whereas Iss densities were similar in day 7, day 20, and adult atrial and ventricular myocytes. Figure 8D shows that mean IKur density did not differ between day 1 atrial and ventricular myocytes. However, IKur was significantly larger in day 7, day 20, and adult ventricular cells compared with age-matched atrial cells. These data show that both Ito and IKur are more prominent in ventricular myocytes from 7 days postbirth and that the difference in Ito and IKur density between atrial and ventricular cells becomes more accentuated at older ages, specifically in adult myocytes.


Figure 8
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  		Fig. 8. Comparison of K+ current in developing atrial and ventricular tissue. The atrial data presented in this figure represent a subset of the atrial data presented in Trépanier-Boulay et al. (38). A: for each developmental stage studied, IK1 density was similar between atrial and ventricular myocytes. Atria: day 1, n = 24; day 7, n = 13; day 20, n = 12; adult, n = 27; ventricle: day 1, n = 24; day 7, n = 15; day 20, n = 15; adult, n = 18. B: Ito density was similar in day 1 atrial and ventricular myocytes. In contrast, Ito was significantly increased in day 7, day 20, and adult ventricular cells compared with age-matched atrial cells (*P < 0.05). Atria: day 1, n = 22; day 7, n = 9; day 20, n = 11; adult, n = 19; ventricle: day 1, n = 24; day 7, n = 10; day 20, n = 15; adult, n = 16. C: in day 1 ventricular myocytes, Iss density was significantly less than in age-matched atrial cells. However, no differences in Iss density were observed between atrial and ventricular myocytes from 7-day, 20-day, and adult mice (*P < 0.05). Atria: day 1, n = 13; day 7, n = 6; day 20, n = 8; adult, n = 11; ventricle: day 1, n = 15; day 7, n = 8; day 20, n = 13; adult, n = 13. D: mean data show that IKur was similar in day 1 atrial and ventricular myocytes, but that IKur was significantly greater in day 7, day 20, and adult ventricular myocytes compared with age-matched atrial cells (*P < 0.05). Atria: day 1, n = 12; day 7, n = 6; day 20, n = 8; adult, n = 11; ventricle: day 1, n = 15; day 7, n = 8; day 20, n = 13; adult, n = 13.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study characterized and compared ventricular repolarization in myocytes from postnatal day 1, day 7, day 20, and adult mice. A significant reduction in APD was observed between day 1 and mature myocytes. The decrease in APD observed in mature myocytes corresponded to a significant increase in K+ currents and the mRNA expression of the K+ channels responsible for these currents. Examination of the K+ currents that compose Ipeak revealed that IK1, Ito, Iss, and IKur were significantly increased in mature ventricular myocytes compared with myocytes isolated from younger mice. Our data suggest that early changes in APD are attributable to increases in Ito and IK1, whereas changes in APD that occur after day 7 are primarily attributable to increases in Ito and Iss. Interestingly, significant increases in IKur did not have a significant effect on APD. This suggests that IKur is not critical for the postnatal development of APD in the mouse ventricle. Overall, it appears that the various components of Ipeak develop at different postnatal stages in the mouse ventricle and that these developmental changes are mostly reflected in APD.

Postnatal development of ventricular repolarization. Previous work has shown that ventricular APD is significantly shorter in adult mouse myocytes compared with neonatal day 1 and day 3 myocytes (29, 40). Similarly, the present study observed a decrease in APD between day 1 and day 7 myocytes. However, previous studies did not characterize APD at time points between early development and adulthood (40). This study showed APD was decreased in adult and day 20 myocytes compared with day 1 cells, but did not decrease between postnatal day 20 and adulthood. This suggests that ventricular APD stabilizes earlier (day 20) than previously thought (adult, 2–3 mo).

The repolarization phase of the cardiac action potential is dependent on K+ currents, Ito, IK1, Iss, and IKur (4). However, developmental studies have not examined effects of development on all of the individual K+ currents. This study showed that Ito and IK1 increased significantly between day 1 and day 7. This resulted in a significant decrease in APD during this time period. In contrast, neither Iss nor IKur increased between day 1 and day 7. Thus it appears that the initial reduction in APD was not dependent on either of these K+ currents. APD also decreased between day 7 and day 20. This decrease corresponded to an increase in Ito and Iss density. IKur also appeared to slightly increase during this time period, but the increase was not statistically significant. Thus the decrease in APD between day 7 and day 20 was likely attributable to increases in Ito and Iss, with IKur having little or no effect. Overall, it appears that APD develops in the first 20 days after birth and that IK1, Ito, and Iss are the primary currents underlying the changes in APD.

Interestingly, IKur was the only K+ current to increase significantly between day 20 and adulthood. This increase in IKur did not result in a reduction in APD. Thus it appears that IKur has little to no effect on ventricular repolarization in the developing heart. However, studies have shown that the blockade of IKur results in a significant prolongation of the action potential (4, 8). Furthermore, APD also is increased in transgenic animals where the K+ channel responsible for IKur (Kv1.5) has been knocked out (21). Thus IKur does play a vital role in ventricular repolarization, at least in the adult mouse. In developing ventricular myocytes, it is possible that other ionic currents and transporters that modulate APD develop in conjunction with IKur and mask the effects of IKur on APD. However, at this time, it remains unclear why increases in IKur did not have any effect on APD in developing ventricular myocytes.

Regulation of K+ channels in the developing heart. There are several possible explanations for the increase in K+ current density observed in this study. First, developmental changes in hormone levels can affect repolarization in the developing heart (27, 35). In rats, plasma levels of thyroid hormone increase between postnatal day 5 and day 20 (35). A rise in thyroid hormone is associated with an increase in mRNA for the proteins that compose Ito (Kv4.2 and Kv4.3) (35, 42). Furthermore, increases in Kv4.2 and Kv4.3 mRNA expression have been associated with an increase in Ito density (35), which implies that thyroid hormone can increase Ito in rat ventricular myocytes via the upregulation of Kv4.2 and Kv4.3. Thyroid hormone also has been shown to affect cardiac repolarization in mice (11, 14). In the present study, a significant increase in Kv4.2 and Kv4.3 mRNA was observed during postnatal development. Thus it is possible that the postnatal increase in Ito observed in this study could be attributed to developmental increases in thyroid hormone and the subsequent increase in Kv4.2 and Kv4.3 expression. Thyroid hormone also upregulates Kv1.5 expression in the ventricle (24, 28). Thus a developmental increase in thyroid hormone levels could, in part, be responsible for the increased Kv1.5 mRNA expression observed in this study. This increase in Kv1.5 expression could then result in a larger number of functional channels, which could explain the increase in IKur.

Studies also have shown that KChIP interacts with Kv4.2 and Kv4.3 and can modulate Ito (18, 31, 34). Kuo et al. (18) showed that KChIP expression is increased in adult mice compared with embryonic mice. Similarily, this study found that KChIP mRNA expression, which was low in ventricular tissue from day 1 mice, was significantly increased in day 20 tissue. This increase in KChIP mRNA also corresponded to an increase in Ito. Thus it is possible that increased modulation of Kv4.2 and Kv4.3 by KChIP also could contribute to postnatal increases in Ito.

Changes in the autonomic nervous system also could result in the developmental alterations in repolarization observed in this study. For example, significant changes in the sympathetic and parasympathetic innervation of the heart occur during development (32). It has been suggested that these changes most likely alter intracellular cAMP concentrations, which could affect the expression of genes influenced by cAMP, such as Kv1.5 (26, 27). In fact, one study has shown that cAMP regulates the transcription of Kv1.5 in primary cardiac cells (26). In addition, basal levels of cAMP are significantly increased in ventricular myocytes isolated from newborn rabbits (17). Thus it is possible that basal cAMP levels also are elevated in the ventricles of postnatal mice. If so, increased basal cAMP levels could partially explain the observed increase in Kv1.5 mRNA. This could increase Kv1.5 protein expression, which would most likely result in an increase in IKur density.

This study also found that Kv2.1 and Kir2.1 mRNA expression increased between day 1 and adulthood. The increase in Kv2.1 mRNA corresponds to an increase in Iss density. Thus the developmental increase in Iss is likely the result of an increase in Kv2.1 channel expression. In contrast, IK1 only increased between day 1 and day 7. However, there was no increase in Kir2.1 during this time period. This suggests that an increase in channel expression was not responsible for the increase in IK1. However, intracellular polyamines, such as spermine and spermidine, have been shown to result in the inward rectification of inward rectifying K+ channels (23, 36). In rats, spermine levels increase between day 3 and day 17, whereas spermidine levels are elevated for the first 10 days after birth (37). Thus it is possible that other factors, such as spermine and spermidine, also may contribute to the developmental regulation of IK1.

Overall, increases in the mRNA expression of the various K+ channels likely result in a greater number of functional K+ channels. Thus increased K+ currents and the subsequent reduction in APD are likely attributable to increased K+ channel density.

Relation to postnatal development of atrial repolarization. Studies have shown that the density of several K+ currents differ between adult atrial and ventricular myocytes (1, 4, 10). Therefore, we compared IK1, Ito, Iss, and IKur in the developing mouse ventricle with data that we previously reported on these K+ currents in developing mouse atrial tissue (38). Interestingly, the development of the action potential waveform was comparable in atrial and ventricular myocytes. Results showed that APD decreases between neonatal day 1 and day 7 in atrial (38) and ventricular myocytes. In the ventricle, this decrease was attributed to increases in Ito and IK1, whereas in the atria, only IK1 increased during the first 7 days postbirth (38). This suggests that Ito is not critical to the development of APD in early neonatal atrial cells, but is important for APD development in ventricular myocytes during the same time period. In atrial and ventricular myocytes, APD decreased further between day 7 and day 20 (38). The decrease in APD appears entirely attributable to an increase in Iss in atrial cells (38), whereas the decrease in APD in ventricular myocytes was attributed to increases in both Ito and Iss for the same period. Finally, both Ito and IKur increase significantly between day 20 and adulthood in atria, but do not have any effect on atrial APD (38). As previously mentioned, IKur also develops between day 20 and adulthood in the ventricle and has no effect on APD. However, in ventricular tissue, Ito is fully developed by day 20 and has a significant effect on APD. Overall, there are several similarities in the postnatal age at which IK1, Ito, Iss, and IKur develop in the atria and the ventricles. This suggests that the mechanisms that regulate the development of these currents may be similar in both heart chambers.

Comparison of atrial and ventricle currents showed that IK1 and Iss densities were similar in atrial and ventricular myocytes. This agrees with our previous work that shows Iss density is similar in adult atrial and ventricular cells (4). Further comparison of atrial and ventricle currents revealed that both Ito and IKur density were significantly greater in day 7, day 20, and adult ventricular myocytes compared with age-matched atrial myocytes. These data are in agreement with our laboratory's previous work, which reported that Ito and IKur density is greater in adult ventricular myocytes than in adult atrial myocytes (4). Thus, despite the similarities in the time dependence of development of Ito and IKur in ventricular and atrial tissue, there is a clear difference in Ito and IKur density between the two heart chambers.

Limitations. Cardiac structure is similar in humans and mice. For example, ion channels are highly conserved between the two species (20, 33). In fact, mice share several, but not all, cardiac K+ currents with humans. The mouse action potential is shorter and heart rate is significantly faster compared with humans (20), which results in differences in repolarization between mice and humans. Therefore, caution must be exercised when interpreting data and extrapolating the results to other species.

Summary. In summary, this study shows that APD decreases during development as a result of an increase in Ipeak density. When Ipeak was separated into its individual components,the data revealed that IK1, Ito, Iss, and IKur develop in a time-dependent manner that is not similar to one another. These changes in current density were most likely attributable to changes in channel expression, with the exception of IK1, where current increased without a corresponding increase in Kir2.1. The data also suggest that Ito plays a prominent role in the determination of APD in the developing mouse ventricle, whereas the role of IKur may be less significant. Furthermore, this study showed that the time dependence for the development of the K+ currents was similar in the atria and ventricle, but both Ito and IKur density were significantly greater in the ventricles. Overall, this work provides a foundation for understanding how K+ channel function changes as a result of postnatal development and provides a model that may be used to investigate pathological conditions in the immature heart.


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This study was supported by an operating grant from the Canadian Institute of Health Research. S. Grandy is a recipient of a Fellowship from the Fonds de la Recherche en Santé du Québec (FRSQ). C. Fiset is a Research Scholar of the FRSQ.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Fiset, Research Center, Montreal Heart Institute, 5000 Belanger St., Montreal, Quebec, Canada H1T 1C8 (e-mail: celine.fiset{at}icm-mhi.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
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