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Cardiovascular Research 2003 59(2):339-350; doi:10.1016/S0008-6363(03)00360-2
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Copyright © 2003, European Society of Cardiology

Developmental changes in IKr and IKs contribute to age-related expression of dofetilide effects on repolarization and proarrhythmia

M.N. Maria N. Obreztchikovaa,1, E.A. Eugene A. Sosunova,1, Alexei Plotnikova,1, E.P. Evgeny P. Anyukhovskya,1, Ravil Z. Gainullina, Peter Danilo, Jr.a, Zi-Ho Yeomb, Richard B. Robinsona and Michael R. Rosena,*

aDepartments of Pharmacology and Pediatrics, Center for Molecular Therapeutics, College of Physicians and Surgeons of Columbia University, New York, NY, USA
bC&C Research Laboratories, 146-141 Annyung-ri, Taean-ub, Hwasung-gun, Kyunggi-do 445-970, South Korea

* Corresponding author. College of Physicians and Surgeons of Columbia University, Department of Pharmacology, 630 West 168 Street, PH 7West-321, New York, NY 10032, USA. Tel.: +1-212-305-8754; fax: +1-212-305-8351. mrr1{at}columbia.edu

Received 7 November 2002; accepted 20 March 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Clinical and experimental studies suggest that immature hearts are as or more sensitive than adult hearts to adverse effects of IKr blocking drugs. We hypothesized that age-dependent changes in IKr and IKs contribute to the different repolarization reserves and proarrhythmic effects of IKr blockers in the young and adult heart. Methods: Dogs aged 1–150 days and adults were used to study (1) proarrhythmic effects in situ of the IKr blocker dofetilide; (2) dofetilide effects on action potential duration (APD) recorded with microelectrodes from left ventricular (LV) slabs; (3) IKr and IKs in single LV myocytes using whole-cell voltage clamp. Results: In situ, dofetilide-induced proarrhythmia occurred in 40% of adults, 86% of young (20–150 day) dogs and 0% of neonatal (1–19 day) dogs (P<0.05). Isolated tissue experiments showed no transmural gradient for repolarization from neonate through 3 months of age, after which the gradient increased through adulthood. In the presence of dofetilide, the greatest APD prolongation occurred in neonates. Yet, transmural dispersion did not increase in neonates but significantly increased in young and adults. Dofetilide-induced early afterdepolarization (EAD) incidence was 23% in adults, 59% in young and 8% in neonates (P<0.05). IKr but not IKs was expressed at <30 days, whereas both currents were present in adult myocardium. Conclusions: Our data suggest that a lack of IKs results in a greater dependence on IKr for repolarization in neonates and is associated with exaggerated effects of IKr-blockade on APD. However, APD prolongation alone is insufficient for expression of proarrhythmia, which also requires transmural dispersion of repolarization and EADs. The extent to which APD prolongation, transmural dispersion and EADs are manifested at various ages in the absence and presence of IKr blocking drugs appears to be the ultimate determinant of proarrhythmia.

KEYWORDS Arrhythmia (mechanisms); Antiarrhythmic agents; Developmental biology; Membrane potential; Membrane currents; Delayed rectifier current


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
IKr blocking agents can induce torsades de pointes (TdP), the signature arrhythmia of acquired long QT syndrome [1]. Such drugs lengthen action potential duration (APD) and prolong the electrocardiographic QT interval, the consequences of which can be antiarrhythmic or proarrhythmic [1,2]. Clinical evidence suggests that immature hearts are as or more sensitive than adult hearts to the effects of IKr blockade. For example, sotalol reportedly induces excess QT prolongation and a significant risk of proarrhythmia in about 10% of children as compared to 2–5% of adults [3–7]. Moreover, the QT-prolonging effect of sotalol tends to be greatest in the smallest children [8]. Similar results have been reported for newborns, infants and children after treatment with either cardiac or non-cardiac IKr-blocking drugs [9–12].

Experimental animal studies also indicate that the immature mammalian myocardium is more sensitive to the APD-prolonging effects of IKr blockers than the adult [13–16]. In rodents, the IKr blocker almokalant had no effect on maternal cardiac rhythm or APD of adult cardiac cells, but induced arrhythmias and early afterdepolarizations in embryonic ventricular and atrial tissues [17]. Similar results were found in rabbits [18,19].

Based on these data we hypothesized that the sensitivity of the neonatal and young heart to proarrhythmic effects of IKr blockade results from age-dependent changes in the relative contributions of IKr and IKs to repolarization. In keeping with the repolarization reserve concept [20] this would imply that the net contributions of both currents to repolarization in the neonate are such that the lesser IKs in the presence of a greater dependence on IKr results in enhanced sensitivity to the APD-prolonging effects of IKr-blocking agents. We therefore studied intact dogs, as well as isolated tissues and single myocytes to: (1) examine the effect of the IKr blocker dofetilide to induce QT prolongation and arrhythmia in intact neonatal, young and adult dogs; (2) compare the effects of dofetilide on APD in left ventricular (LV) myocardium of neonatal, young and adult dogs; (3) study IKr and IKs in single myocytes isolated from the LV of young and adult dogs.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
The investigation conforms with 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). All protocols were approved by the Columbia University Institutional Animal Care and Use Committee.

2.1 In situ experiments
Mongrel dogs, of either sex were divided into three groups: neonatal (8.8±1.6 days, range 5–14 days, n = 6), young (69±10.4 days, range 30–120 days, n = 7) and adult (3–5 years, n = 5). Following anesthesia with thiopental 17 mg/kg i.v., animals were intubated, and ventilated with isoflurane 1.5–3.0% and oxygen. The femoral artery was catheterized to monitor blood pressure and both cephalic veins were catheterized to maintain drug infusion and facilitate collection of blood samples. A heating pad was used to maintain body temperature.

2.1.1 Electrocardiographic recordings
Seven electrocardiographic leads were recorded using "Dr. Vetter PC-EKG" software (Dr. Vetter, Germany). Recordings were made before and after dofetilide infusion. ECG parameters were measured from at least 10 consecutive complexes at every experimental time point and averaged values were analyzed. Rate correction of the QT interval was done using Bazett's formula. QT and QTc dispersion were calculated from seven ECG leads (I-aVF and V10) as difference between maximal (QTmax) and minimal (QTmin) intervals measured in simultaneously recorded leads. Data were analyzed as average dispersion calculated in three consecutive complexes.

2.1.2 Dofetilide administration, blood sampling and measurements of drug plasma concentrations
Dofetilide (a gift of Helopharm, Berlin, Germany) was administered as a 0.1 mg/kg i.v. bolus followed by 0.1 mg/kg/min infusion for 10 min. The infusion was terminated if ventricular arrhythmias developed.

In young and adult dogs, 1–2 ml of blood was drawn before dofetilide administration and at the end of infusion. In neonates, blood was drawn only at the end of drug infusion. When an arrhythmia occurred, a blood sample was obtained at that time and the infusion was terminated. Blood samples were heparinized and centrifuged at 10,000 rpm for 30 min and stored at –70°C.

Plasma drug levels were determined by high-performance liquid chromatography (HPLC). Dofetilide was extracted from plasma with a liquid–liquid extraction column (Extrelut® NT1, Merck, Germany) as follows: 600 µl of plasma sample was applied to the column and equilibrated for 10 min, followed by diethyl ether 1 ml for 10 min. This elution process was repeated three times. Eluted solvent was evaporated under a stream of nitrogen. The dry residue was resuspended in 200 µl of acetonitrile (J.T. Baker, USA) (gradient A) and 0.1% trifluoroacetic acid (Sigma, USA) (gradient B). Finally, a 30-µl aliquot was injected into the HPLC system.

Dofetilide concentrations were measured with a HP 1100 series system, (Hewlett-Packard, Germany) and a Hypersil BDS-C18, 125x2.0 mm I.D., 3 µm particle size column (Agilent, USA). The UV detection wavelength was 224±5 nm, and flow-rate, 0.25 ml/min. The calibration was linear from 10 to 1250 ng/ml.

2.2 Experiments with multicellular preparations
2.2.1 Left ventricular preparations
Dogs were selected and age-matched to the groups described above: neonate (8.9±1.7 days, range 1–19 days, n = 10), young (80±8.8 days, range 20–150 days, n = 23) and adult (3–5 years, n = 6) (for all ages P>0.05 compared to respective in situ group). Dogs were anesthetized with sodium pentobarbital, 30 mg/kg i.v. (young and adult) or 40 mg/kg i.p. (neonate). Their hearts were removed through a left lateral thoracotomy and immersed in cold Tyrode's solution equilibrated with 95% O2–5% CO2 and containing (mmol/l): NaCl 131, NaHCO3 18, KCl 4, CaCl2 2.7, MgCl2 0.5, NaH2PO4 1.8 and dextrose 5.5. Epicardial, endocardial, and midmyocardial strips (~10x5x0.5–1 mm) were filleted with surgical blades parallel to the surface of the left ventricular free wall. The preparations were placed in a tissue bath and superfused with control Tyrode's solution (37°C, pH 7.35±0.05). Solution was pumped at 12 ml/min, changing chamber content three times/min. The bath was connected to ground via a 3 M KCl/Ag/AgCl junction.

2.2.2 Action potential recordings
Preparations were impaled with 3 mol/l KCl-filled glass capillary microelectrodes having tip resistances equal to 10–20 M{Omega}. The electrodes were coupled by an Ag/AgCl junction to an amplifier with high input impedance and capacity neutralization. Transmembrane action potentials were digitized with an analog-to-digital converter (D-210, DATAQ Instruments) and stored on a personal computer for subsequent analysis. For stimulation of preparations, standard techniques were used to deliver square-wave pulses 1.0 ms in duration and 1.5 times threshold through bipolar Teflon-coated silver electrodes. To investigate frequency-dependence of drug effects, preparations were driven at cycle lengths (CLs) from 4000 to 250 ms in sequence. Each frequency was maintained for 3 min before data were collected.

Experiments began after 3 h of equilibration in control Tyrode's solution. The effects of dofetilide 10–8–10–6 mol/l and of the IKr and IKs blocker azimilide [21] (a gift of Procter and Gamble) (5x10–6 mol/l in the presence of dofetilide, 10–6 mol/l) were studied. Measurements of drug effects commenced after 30 min equilibration at each concentration.

2.3 Experiments with single ventricular myocytes
2.3.1 Myocyte preparation
Canine LV subepicardial myocytes were isolated from five young dogs (27–31 days) and four adults. We focused on the young rather than the neonatal age group, and in particular the early portion of this age range because the intact tissue studies (see Results) suggested this was a critical transitional period. We used a collagenase perfusion method reported previously [22]. Briefly, a wedge of left ventricular free wall was dissected, and the first or second branch of the left circumflex coronary artery was cannulated. After 10–15 min of collagenase perfusion for adults and 6 min for young, the epicardial layer (~1–2 mm) was removed, placed in a beaker, minced, incubated in fresh collagenase solution and agitated with 95% O2–5% CO2 for 5–15 min. Incubation was repeated 3–5 times, and the supernatant from each digestion was centrifuged. Isolated cells were stored at room temperature in buffer solution.

2.3.2 Current recordings
Myocytes were placed in a heated bath and superfused with a modified Tyrode's solution containing (mmol/l): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1, HEPES 5, glucose 10 (pH 7.4 adjusted with NaOH) at 35°C. Delayed rectifier potassium current (IK) was recorded via whole cell patch clamp using a personal computer equipped with pClamp 8 software, DigiData 1200 series interface and Axopatch 1D amplifier (Axon Instruments). Borosilicate glass pipettes had tip resistances of 1–2.5 M{Omega}. L-type Ca2+ current was blocked with 10–6 mol/l nisoldipine. Na+ current was inactivated by holding cells at –40 mV. The pipette solution contained (mmol/l) KOH 60, KCl 80, aspartate 40, HEPES 5, EGTA 10, MgATP 5, Na creatinine phosphate 5, CaCl2 0.65 (pH was adjusted to 7.2 with 1 M NaOH). Currents were recorded during 5 s depolarizing test pulses ranging from –20 to +55 mV in 15 mV increments and upon repolarization to holding potential –40 mV. Pulses were applied at 20-s intervals to ensure deactivation of tail currents. The protocol was run before and after exposure of a cell to dofetilide, 10–6 mol/l. Where indicated, chromanol 293B (a gift of Aventis Pharma, Germany), 10–5 mol/l, was added to dofetilide and the protocol was repeated. IKr was obtained by digital subtraction from controls of currents recorded in the presence of dofetilide. The dofetilide-resistant current was considered IKs.

In adult myocytes, in which we observed IKs, we checked for current rundown by comparing total IK tail at +25–55 mV from 2 to 8 min after rupture (measurement window). No significant rundown was seen. We did not observe IKs in most young myocytes. We tested the possibility of this being a rundown artifact by recording from three myocytes using the perforated patch method [22]. We observed IKs, defined as a chromanol-sensitive outward tail current, in one of the three cells. This incidence is comparable to that which we found using ruptured patch in two of 10 young myocytes (see Results).

The current–voltage (I–V) relationship was determined by first fitting tail currents normalized to cell capacitance, with a double-exponential fit of the form It=A0+A1e–t/{tau}1+A2e–t/{tau}2. The voltage dependence of IKr activation was then determined by fitting these amplitude values with a Boltzmann function I = Imax/{1+exp[(V1/2–Vt)/k]} to obtain Imax, V1/2 and k. Statistical comparison was done on the average of the individual fit parameters. Each cell's activation curve was then normalized by that cell's Imax value to generate an average conductance graph (see Fig. 9).


Figure 9
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  		Fig. 9 IKr (A) and IKs (B) current–voltage relationships in subepicardial ventricular myocytes from adult and young dogs. Tail currents were measured upon repolarization to –40 mV from the indicated membrane potentials. * P<0.05 versus adult at the same membrane potential. (C) Voltage dependence of IKr activation in adult and young dogs. Curves were obtained by fitting tail current data from each cell with a Boltzmann distribution (see Methods). Mean values of V1/2 were –3.8±2 mV (adult) and –11.8±2 mV (young) (P<0.05). The value of k did not differ significantly between adult and young (7.1±1.3 and 6.0±2.6 mV, respectively). The lines in panel C are the best fit to the plotted summary data. In all panels, test voltages ranged from –20 to 55 mV in 15 mV increments; n = 16 for adult and 10 for young in all panels.

 
2.4 Statistical analysis
Data are expressed as mean±S.E.M. Values for ionic currents were corrected for cell capacitance, which was 20±1 pF (n = 10) for young and 207±6 pF (n = 16) for adult myocytes. One-way or two-way analysis of variance for repeated or non-repeated measures were used for data analysis, with Bonferroni's test when the F-value permitted. Significance of incidence of early afterdepolarizations was evaluated with Fisher's exact test. Post-hoc analysis was used to estimate dofetilide effects on ECG parameters across age groups using the Bonferroni test where variances were equal and Games–Howell where variances were unequal. P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effects of dofetilide in the intact animal
Plasma dofetilide concentrations did not differ (P>0.05) among the three groups: neonate=107±27, young=74±29, and adult=89±9 ng/ml. Dofetilide effects on the ECG are shown in Table 1 and Fig. 1. Dofetilide decreased heart rate significantly at all ages, but no changes in P wave duration, PR interval, or the QRS complex were observed. Dofetilide prolonged the uncorrected and rate-corrected QT intervals at all ages. However dispersion of the QT (Table 1) and QTc (Fig. 1) intervals was increased in adults only (P<0.05) (Fig. 1). A significant incidence of proarrhythmia occurred in adults, but the greatest incidence was in young animals and there was no proarrhythmia in the neonates (Fig. 2).


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  		Table 1 Effects of dofetilide on the ECG (for QTc dispersion see Fig. 1)

 

Figure 1
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  		Fig. 1 Dofetilide-induced changes in QTc dispersion with age. * P<0.05 versus neonate; + P<0.05 cf. respective control values, which were 33±5 for neonates; 25±4 for young and 24±6 for adults.

 

Figure 2
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  		Fig. 2 Incidence of proarrhythmia during dofetilide administration. VPD, Ventricular premature depolarizations; TdP, torsade de pointes. * P<0.05 versus respective values for neonates.

 
3.2 Studies of isolated myocardium
Control values for action potential characteristics for APD90 are shown in Table 2 for cycle lengths of 500, 1000 and 4000 ms. At a CL of 500 ms, there were no differences in APD90 among age groups in any type of tissue. Prolongation of CL from 500 to 1000 ms produced APD90 lengthening in all tissues and at all ages. When CL increased from 1000 to 4000 ms, so did APD90 in young and adult dogs but not neonates. More prolongation was observed in adults, so that at CL=4000 ms, APD90 in adults was significantly longer than in younger animals. There were no age dependent differences in action potential amplitude in all tissues. At CL=1000 ms, the maximum diastolic potential for all neonatal cells studied was –81±1 mV (n = 30). This increased to –85±1 mV in young (n = 69, P<0.05) and to –85±1 mV in adults (n = 18, P<0.05).


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  		Table 2 Control APD90 (ms) in all tissues and age groups

 
Representative action potentials from epicardium, midmyocardium and endocardium for each of the three age groups are shown in Fig. 3A at CL=4000 ms. Note the age-related emergence of a transmural gradient for repolarization. Summary data at the same cycle length are presented in Fig. 3B, demonstrating both the age-associated increase in action potential duration and in transmural dispersion of action potential duration. The effects of dofetilide on repolarization and transmural dispersion also were age-dependent. As shown in Fig. 3C, prolongation of action potential duration occurred with dofetilide at all ages, but the greatest increase in transmural dispersion was seen in adults.


Figure 3
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  		Fig. 3 (A) Representative transmembrane potentials recorded from epicardial (Epi), midmyocardial (Mid), and endocardial (Endo) sites of 3-day-old, 90 day-old and adult dogs under steady-state conditions at CL=4000 ms. (B) APD90 for all sites and all ages at CL=4000 ms. Inset: TDR is transmural dispersion of repolarization (maximum difference in APD90 among Epi, Mid and Endo was calculated in each dog and averaged for each age group). (C) Percent lengthening of APD90 in the presence of dofetilide, 10–7 mol/l, for all sites and all ages at CL=4000 ms. n for each tissue=10, 23, and 6 for neonate, young and adult, respectively. * P<0.05 versus Endo and Epi in the same group. + P<0.05 versus Young and Neonate in the same tissue.

 
There was greater complexity than is shown in Fig. 3 in the relationship between age and action potential duration in the response to dofetilide at different cycle lengths. The extent of this complexity is shown in Fig. 4. In Fig. 4A (representative neonatal and adult action potentials at CL=1000 ms), note the greater degree of action potential prolongation in the neonate rather than the adult. Fig. 4B makes two points: first, at CL=1000 ms the increase in action potential duration was significant for all tissues, all ages and all dofetilide concentrations. Second, the greatest increase in duration occurred in the neonates. Fig. 4C considers the same data in light of cycle length. At CL=500 and 1000 ms (and—although not shown—at 250 ms as well) the greatest prolongation of repolarization in all tissues occurs in the neonates. However, at the longest cycle length (4000 ms) the greatest action potential prolongation is in adult midmyocardium. Hence, the magnitude of dofetilide-induced action potential prolongation varies with cycle length and with age: at the longest cycle length (4000 ms), the greatest prolongation is in adults; at shorter cycle lengths the greatest prolongation is in neonates.


Figure 4
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  		Fig. 4 (A) Representative experiments illustrating effects of 10–6 mol/l dofetilide at CL=1000 ms in epicardium of 6-day-old and adult dogs (Contr=control). (B) Concentration-dependent effects of dofetilide on APD90 at CL=1000 ms all sites and all ages. The prolongation produced by dofetilide was statistically significant at all concentrations in all tissues and age groups. * P<0.05 versus two older groups at same dofetilide concentration. (C) Age-dependent effects of dofetilide, 10–6 mol/l, in all tissues at all ages at CL=500, 1000, and 4000 ms (note a different scale for APD prolongation in the right panel). * P<0.05 versus two older groups in the same type of tissue. n = 6 to 16 per group. Control values are in Table 2.

 
Given the association between increases in cycle length and in occurrence of drug-induced early afterdepolarizations (EADs) and triggered activity, we next explored the relationships of age and cycle length to the incidence of EADs. Fig. 5B demonstrates a low EAD incidence through day 20 of life; a far higher incidence between days 20–90, and thereafter an incidence indistinguishable from adults. Fig. 5C provides data from all experiments on dofetilide-induced prolongation of action potential duration at CL=1000 ms as a frame of reference. It must be emphasized in considering these data that while neonates showed the greatest dofetilide-induced increase in action potential duration (Fig. 5C); the actual duration of the action potential was greatest in the adults. This can be appreciated by considering the data in Fig. 5C in light of the control values in Table 2.


Figure 5
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  		Fig. 5 (A) Action potential of epicardial cell of 49-day-old dog at CL=4000 ms in control (Contr) and dofetilide, 10–6 mol/l. Note dofetilide-induced EADs. (B) Incidence of EAD in all types of tissues. * P<0.05 versus predrug control. P<0.05 versus 1–20, 90–150, and adult. + P<0.05 versus 1–20. (C) Prolongation of APD90 induced by 10–6 mol/l dofetilide obtained from each impalement in epicardium (Epi), midmyocardium (Mid), and endocardium (Endo) at CL=1000 ms for all dogs studied.

 
The effect of dofetilide to prolong action potential duration in the neonate more than the adult might be expected in a setting where IKr and/or IKs were lower in the neonate, resulting in the equivalent of a reduced repolarization reserve [20]. As a first step to testing the possibility of age-dependent differences in these currents in the dog, we studied the effects of adding the IKr/IKs blocker, azimilide, to the IKr blocker, dofetilide, in LV epicardium of neonatal and adult dogs. A representative experiment is shown in Fig. 6A and summary data in Fig. 6B. Initial superfusion with dofetilide, 10–6 mol/l, prolonged action potential duration in neonates and adults, but addition of azimilide increased duration further in adults only. This implies that there is less (or no) functional IKs in the neonate as compared to the adult.


Figure 6
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  		Fig. 6 (A) Action potentials of epicardial cells of 7-day-old and adult dogs at CL=2000 ms in control (Contr), in dofetilide, 10–6 mol/l (Dof), and in dofetilide plus azimilide, 5x10–6 mol/l (Dof+Azi). (B) Prolongation of APD90 induced by 5x10–6 mol/l azimilide in the presence of 10–6 mol/l dofetilide in two age groups at various CL. * P<0.05 versus dofetilide alone at same CL (n = 6/group).

 
3.3 Voltage clamp experiments
We next tested the hypothesis that IKs would be greater in the adult, while IKr would be present across the age range represented in the study. Two age groups, young and adult were included in these experiments. Under baseline conditions, IK tail currents were seen in myocytes from young (Fig. 7A) and adult (Fig. 7C) dogs. Dofetilide completely abolished IK tail currents in the young myocyte (Fig. 7B) and reduced them in the adult (Fig. 7D). Addition of the IKs blocker chromanol 293B completely eliminated tail currents in the adult (Fig. 7E).


Figure 7
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  		Fig. 7 Representative recordings of IK in one young and one adult canine subepicardial ventricular myocyte under basal conditions (A and C, respectively), after exposure to 10–6 mol/l dofetilide (B and D, respectively) and after exposure to 10–5 mol/l chromanol in the presence of dofetilide (E, for the adult myocyte only). Currents were recorded during 5-s pulses to –20, –5, +10, +25, +40 and +55 mV applied from a holding potential (HP)=–40 mV. Tail currents were obtained on return to –40 mV from indicated test potentials.

 
The critical changes that occur in IKs in young dogs, aged 25–31 days, are shown in Fig. 8. Note the trend for IKr to decrease over this age range and the minimal magnitude for IKs throughout, except for two of the three myocytes at age 30–31 days. Pooled normalized IKr and IKs current density data from all adult and young myocytes are shown in Fig. 9. IKr tail current density was significantly greater in the young animals (Fig. 9A). In contrast, IKs tail currents were seen in all adult but only two of 10 young myocytes. The averaged IKs of all 10 young cells was significantly smaller than in adults (Fig. 9B). Fig. 9C shows voltage-dependent activation properties of IKr in both groups. Current in young myocytes activated at more negative voltages than in adult cells.


Figure 8
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  		Fig. 8 Age dependence of IKr and IKs at +40 mV. For young animals, individual values for IKr (A) and IKs (B) are shown for every cell studied (n = 10); for adults, mean values±S.E.M. are shown (n = 16). Linear regression line and correlation coefficient are presented for IKr. Note, for IKs, that two cells have achieved adult values by 1 month of age.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Our study focuses on the developmental electrophysiologic remodeling of the myocardium and the effects on this of the IKr blocker, dofetilide. The impact of developmental remodeling on cardiac rhythm can be appreciated in considering the response of the heart to proarrhythmic drugs. It has been estimated that IKr-blocking antiarrhythmic drugs are proarrhythmic in up to 30% of adult patients [23], and that the classes of drugs having IKr-blocking and proarrhythmic actions are diverse, among them antipsychotics, macrolide antibiotics, antifungals and antihistamines [1].

Although not generally appreciated, proarrhythmic drugs carry a major risk for the young as well as adults [10,12] and their deleterious effects may be greatest in young children [24], who experience not only TdP, but other ventricular arrhythmias, conduction block and bradycardia [4]. Moreover, as emphasized by our study, the mechanisms for proarrhythmia in the young are more subtle than simple IKr blockade and are not completely understood.

Considering mechanism, among the prime variables for induction of proarrhythmia is prolongation of repolarization and slowing—or pauses—in heart rate. Both of these are "moving targets" in that growth and development incorporate a changing spectrum of repolarizing and pacemaker currents. Moreover, in light of the postnatal development of Ito [25] and of M cells (see Refs. [26,27]) we find little-to-no transmural gradient for repolarization in the neonatal dog. We have shown in the rat [28] and surmise in the human that sympathetic neural growth and development are central to both the evolution of Ito and the occurrence of a transmural gradient for repolarization. Moreover, in studies of rat [17] and mouse [29] ventricle, there is a greater APD-prolonging effect of IKr-blocking drugs in the fetus than at older ages. IKr appears to be the major repolarizing current in the fetal mouse ventricle [30–33], but in contrast to the dog, IKs in mouse is seen prenatally and decreases postnatally [32–34]. All these factors provide a background for our current study, which incorporates measurements in intact animals through currents in a large animal model in an attempt to understand the postnatal changes in IKr blockade on the heart.

Our isolated tissue experiments clearly demonstrate developmental expression of transmural differences in ventricular repolarization. In earlier large animal experiments, Rodriguez-Sinovas et al. [35] reported no cells with long APD in midmyocardium of 4–8-week-old pigs. In contrast, Stankovocova et al. [36] identified longer APD in some cells isolated from left ventricular midmyocardium and subepicardium of 14–22-week-old pigs. Our data show that neonatal canine heart is homogeneous with respect to APD and that transmural differences in repolarization are not distinct until about 3 months of age (i.e., if the young age group is broken out further, dispersion is evident after about 90 days). Differences in APD between midmyocardium and epicardium or endocardium then increase through adulthood. Importantly, the effects of dofetilide on transmural differences in repolarization differ with age. In neonates, dofetilide prolonged APD to the same extent transmurally such that increased dispersion of repolarization did not occur. In contrast, in older animals having a transmural gradient of repolarization in control conditions, dofetilide prolonged APD more in midmyocardium than epicardium or endocardium, significantly increasing transmural dispersion at long cycle lengths.

Our voltage-clamp data on epicardial myocytes suggest these dofetilide actions derive from developmental regulation of IK. Only IKr is functionally expressed in the majority of cells from young (27 to 31 days old) canine ventricles whereas both IKr and IKs are present in adult myocardium. In addition, IKr density is greater and it activates at more negative voltages in young myocytes than in adult. All this suggests a greater dependence on IKr for repolarization in young than in adult dogs. The molecular basis for the developmental positive shift in IKr is unknown, but a similar shift occurs when ERG channels associate with the beta subunit MiRP1 [37] or when membrane PIP2 is depleted by autonomic activation of phospholipase C [38]. Whether developmental changes in MiRP1 or autonomic signaling (perhaps associated with development of sympathetic innervation) contribute to the developmental changes seen in delayed rectifier current remains to be tested.

Our action potential data with azimilide compliment the voltage-clamp findings. When IKr was blocked with dofetilide, the IKr/IKs blocker azimilide prolonged APD further in adult but not neonatal tissue, suggesting that IKs contributes to repolarization in older animals only. This finding has two levels of importance with regard to the interpretation of our studies: first, it suggests strongly that the finding of little to no IKs in young myocytes is both valid and characteristic of the neonate as well. Second, it provides supportive evidence that the lack of IKs in the young myocytes was not an effect of rundown. Thus, a greater dependence on IKr for repolarization can exaggerate dofetilide effects on APD in neonates.

The effect of dofetilide to induce EAD in ventricular tissue is also age-dependent: minimum incidence was detected in the neonates and maximum incidence at 20–90 days. At least three factors may explain these differences in EADs: first, EADs are usually generated at slow pacing rates and after prolongation of repolarization: the greater the prolongation, the greater the probability of EAD development [39]. Second, intracellular calcium handling is important for induction of EADs [40,41] in that block of calcium release from sarcoplasmic reticulum suppresses E-4031-induced EADs in isolated ventricular tissue [40,42] and the induction of torsades de pointes in the intact canine heart [43]. Third, ultrastructural and functional studies [44–48] have shown that sarcoplasmic reticulum is underdeveloped in the newborn mammalian heart and its expression increases with age. In the dog, the Ca-handling apparatus is effectively absent at birth and does not mature until approximately 5 months of age [47,48]. Thus, an immature sarcoplasmic reticulum may determine the low incidence of EADs in the neonates. That the incidence of EADs significantly increases at 20 through 90 days of age may reflect the initial development of sarcoplasmic reticulum in these animals, which—if EAD incidence is any hallmark—is a process that might be functionally developed by 90 days. Clearly, the rate of maturation of uptake and release processes and their roles in intracellular Ca handling need further investigation if we are to understand the changing pattern of EAD incidence with age.

Our in vivo data demonstrating a significant age dependence in dofetilide induction of ventricular arrhythmias are in good accord with in vitro results. Both induction of EAD and amplification of transmural dispersion of repolarization are believed to be critical to the development of torsades de pointes [27,49,50]. In the neonates, where a low incidence of EADs and no transmural gradient for repolarization were found in vitro in the presence of dofetilide (despite a large magnitude of action potential prolongation), no ventricular arrhythmias were seen in vivo. The increased proarrhythmia in the young dogs in vivo effectively parallels the effect of dofetilide to induce a maximum incidence of EADs as well as an increase in transmural dispersion of repolarization in vitro at this age. In adult tissues, the incidence of EADs was significantly lower than in the young, but a further, prominent increase in transmural dispersion of repolarization occurred.

These results show that developmental changes in the effects on repolarization of IKr blocking drugs have a complex etiology and complex expression in arrhythmias. They suggest that in neonates, the repolarization reserve differs significantly from that in adults, as a result of differences in both IKr and IKs shown in this study, as well as differences in Ito demonstrated earlier [26]. However, this change in repolarizing current is reflected as an increase in duration of repolarization, alone, but not in its dispersion. The young animals show a significantly higher incidence of EADs than adults, and begin to manifest dispersion—but not as much as adults. Finally, the adults have a lower incidence of EADs than the young but greater transmural dispersion. These findings emphasize that prolongation of repolarization is not enough to induce proarrhythmia: the additional factors that appear needed are dispersion and EADs, and these vary with age. The extent to which APD prolongation, dispersion and EADs are manifested across the life cycle in the absence and presence of IKr blocking drugs (and in the absence or presence of disease) would therefore appear to be the ultimate determinants of proarrhythmia expression.

Limitations of the study: (1) given that the dog at birth is in many ways less mature than the human, neonatal canines may offer a parallel to premature human neonates, and young canines may be more like the full-term human neonate.

(2) We cannot discount the possibility that rundown of IKs was greater in the small neonatal cells than in adult myocytes. However, our control experiments using the perforated patch technique (see Methods), as well as our earlier study [22] suggest that rundown of IKs is not the reason for our failure to observe this current component in young myocytes. The validity of our IKs results is also suggested by the azimilide studies in isolated neonatal and adult tissues.

(3) Quantitative differences in IKs during patch recording versus effects of the current in the intact heart also could result from the non-physiologic conditions required for current isolation, including the long time between voltage tests and buffering of intracellular calcium. Nonetheless, the complete consonance of patch clamp and isolated tissue experiments minimize this concern.

Time for primary review 28 days.


   Acknowledgements
 
The authors express their gratitude to Dr. Nimee Bhat for her assistance in performing certain of the studies and to Ms. Eileen Franey for her careful attention to the preparation of the manuscript. Studies were supported by USPHS-NHLBI grants HL-28958 and HL-67101, and by C&C Pharmaceuticals.


   Notes
 
1 Contributed equally as first author. Back


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

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