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Cardiovascular Research 1999 43(2):332-343; doi:10.1016/S0008-6363(99)00118-2
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Repolarizing currents in ventricular myocytes from young patients with tetralogy of Fallot

Peter Schaffera,*, Brigitte Pelzmanna, Eva Bernharta, Petra Langa, Heinrich Mächlerb, Bruno Riglerb and Bernd Koidla

aInstitut für Medizinische Physik und Biophysik, Universität Graz, Harrachgasse 21, A-8010 Graz, Austria
bUniversitätsklinik für Chirurgie, Universität Graz, Auenbruggerplatz 5, A-8036 Graz, Austria

* Corresponding author. Tel.: +43-316-3807741; fax: +43-316-3809660 Schaffer{at}balu.kfunigraz.ac.at

Received 15 October 1998; accepted 12 February 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
Objective: It was the aim of our study to describe repolarizing currents in ventricular myocytes isolated from children with tetralogy of Fallot. This is the first report on outward currents in ventricular myocytes from children. Methods: Ventricular myocytes were isolated from tissue samples of the outflow tract of the right ventricle which were obtained during corrective surgery of tetralogy of Fallot. Action potentials and whole-cell currents were recorded with the patch clamp technique at a temperature of 36–37°C. Results: The mean resting potential was –71.7±1.92 mV, action potential amplitude was 110±2.96 mV and action potential duration at 90% repolarization was 794±99.5 ms (n=12). In four out of 12 myocytes early afterdepolarizations (EADs) were observed. Upon hyperpolarization Ba2+-sensitive inward currents similar to the inward rectifier current (IK1) could be observed. The current density at –120 mV was –22.8±2.47 pA/pF (n=14). A transient outward current (Ito1) could be recorded in all myocytes studied, the current density varied from 0.3 to 8.6 pA/pF with a mean of 3.77±0.47 pA/pF at +40 mV (n=38). Recovery of Ito1 from inactivation was fast (70% recovery within 100 ms), rate-dependent reduction amounted to 38.2% at 4 Hz. A delayed rectifier current was seen in only two out of 38 myocytes (rapid component IKr). Conclusions: The electrophysiological characteristics of right ventricular myocytes isolated from children with tetralogy of Fallot resemble in most cases subendocardial myocytes from adults. The most prominent difference is a fast recovery from inactivation as well as a small rate dependent reduction of Ito1. The observed EADs may have clinical implications.

KEYWORDS Arrhythmia (mechanisms); Congenital defects; Ion channels; Myocytes


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
In general, ventricular repolarization is the result of the combination of various different inward and outward directed currents. The contribution of individual currents to the repolarization process depends on the origin of the myocyte [1,2] and age of the patient [3–5] resulting in different action potential shapes [2] and different responses to pharmacological treatment. Outward currents in human ventricular myocytes from adults have been studied in detail by various investigators. These studies have demonstrated the presence of Ito [1,2,6–11], IK [7], the slow component of IK (IKs) [12], the rapid component of IK (IKr) [12–14] and IK1 [7,8,15]. On the other hand, ionic currents underlying ventricular repolarization in infants have not been studied. It was the aim of this study to characterize the outward currents responsible for repolarization in isolated ventricular myocytes from infants with tetralogy of Fallot (TOF) at a physiological temperature. TOF-patients are often susceptible to life threatening arrhythmias and late sudden death [16–19] whereby the underlying mechanisms are unknown at present. Studies on cellular electrophysiology of TOF myocytes are of particular interest because corrective surgery of TOF is a source of ventricular tissue from infants.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusions
 References
 
2.1 Preparation of cardiomyocytes
Pieces of tissue from the right ventricular outflow tract were obtained from nine children (age: 30.6±9.2 months; range 16–91 months) during corrective cardiac surgery. This procedure was approved by the ethical committee of the Karl-Franzens-University Graz and conformed with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–3). The cell isolation procedure, based on a method developed by Jacobson et al. [20], was described previously [21]. The isolated myocytes were stored in cell-culture medium M199 containing penicillin (50 IU/ml) and streptomycin (50 µg/ml) and were placed in an incubator at 37°C. Experiments were performed 3–24 h after cell isolation. Only quiescent rod-shaped myocytes with clear cross striation were used for experiments.

2.2 Recording technique
When placed in the experimental chamber the myocytes were continuously superfused with extracellular solution at 36–37°C. Action potential and current recordings were performed using a List L/M EPC-7 amplifier (List Electronics, Darmstadt, Germany) in the whole cell patch clamp technique [22]. When filled with internal solution patch electrodes had resistances from 2 to 4 M{Omega}. Electrode potentials were zeroed before sealing the pipette. For action potential recordings the myocytes were stimulated with minimal superthreshold current pulses of 5-ms duration at a frequency of 0.5 Hz and the voltage signals were stored on video tapes using a PCM audioprocessor (Sony, 501). For action potential data evaluation the signals were transferred to a personal computer using Axotape software (Axon Instruments, Foster City, USA) and analyzed offline.

For generation of voltage pulses, current recording and current-data evaluation pCLAMP 5.5.7 (Axon) and a digidata 1200 interface (Axon) were used. Whole cell currents were low-pass filtered at 3 kHz.

Cell capacitance was calculated by integration of the current flowing in response to a 10-mV hyperpolarizing step from –50 mV (10 ms). To correct for variations in cell size current amplitudes were divided by the cell membrane capacitance (Cm) and expressed as current density (pA/pF). Cell-capacitance (up to 100 pF) and series resistance (usually>50%) were compensated. Access resistance (Rs) was calculated (prior compensation) by dividing the time constant of the capacitive transient by the cell membrane capacitance. Rs prior compensation was 5.73±0.27 M{Omega} (n=62 cells).

In order to measure selectively outward currents, sodium current (INa) was inactivated by a 50-ms prepulse to –40 mV or by using a depolarized holding potential at which INa is almost completely inactivated. Interference with calcium currents [21] was omitted by the use of 0.1 mmol/l CdCl2 and a low CaCl2 concentration of 0.1 mmol/l in the extracellular solution [23].

2.3 Solutions
For action potential recordings the myocytes were superfused with the following external solution (in mmol/l): NaCl 137, KCl 5.4, CaCl2 1.8, MgCl2 1.1, NaHCO3 0.4, HEPES/Na+ 10, D(+)-glucose 5.6, adjusted to a pH of 7.4 with NaOH. The pipettes were filled with a solution of the following composition (in mmol/l): KCl 110, ATP/K+ 4.3, MgCl2 2, CaCl2 1, EGTA 11, HEPES/K+ 10, adjusted with KOH to a pH of 7.4 (estimated free [Ca2+]<10–8 mol/l).

The composition of the extracellular solution used for potassium current recording was (in mmol/l): NaCl 137, KCl 5.4, CaCl2 0.1, MgCl2 1.1, CdCl2 0.1, NaHCO3 0.4, HEPES/Na+ 10, D(+)-glucose 5.6, adjusted to a pH of 7.4 with NaOH. In some experiments BaCl2 (1 mmol/l) was added to the extracellular solution in order to inhibit IK1 (indicated in the text).

2.4 Statistics
All data are expressed as mean±S.E.M. Pearson correlation coefficient and two-tailed t-test were calculated by using SPSS software (SPSS Inc. Chicago, USA). A value of P<0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusions
 References
 
Right ventricular myocytes isolated from children with tetralogy of Fallot were rod-shaped with average dimensions of 78.6±1.41 µm for length (l) and 17.8±0.48 µm for width (w) yielding a mean l/w ratio of 5.0±0.13 (n=274 cells). The myocytes showed clear cross striation and were quiescent in extracellular solution containing 1.8 mmol/l external Ca2+ (without Cd2+). The cell membrane capacitance determined in 62 myocytes was 80.4±5.92 pF.

3.1 Action potential recordings
Action potentials were studied within 8 h after cell isolation. After breaking the patch and using the current clamp mode of the patch clamp technique a stable resting potential could be recorded in all cells studied. Application of short (5 ms) depolarizing current pulses at a frequency of 0.5 Hz elicited action potentials with following characteristics: Resting potential (RP) –71.7±1.92 mV, action potential amplitude 110±2.96 mV, action potential duration 612.1±84.9 ms at 50% of repolarization (APD50) and 794±99.5 ms at 90% of repolarization (APD90; n=12).

In Fig. 1 action potentials from three myocytes with differences in APD are shown. EADs as shown in Fig. 1C were found in four out of 12 myocytes (compare Ref. [21]). These differences in the action potential shape might be the consequence of differences in the repolarizing currents.


Figure 1
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  		Fig. 1 Action potentials recorded in TOF myocytes showing differences in APD (A, B) and occurrence of an EAD (C). Stimulation frequency was 0.5 Hz.

 
3.2 Steady-state current
To determine the steady state I–V relationship the myocytes were clamped for 5 s (frequency 0.1 Hz) to potentials between –90 and +60 mV in steps of 10 mV using a holding potential of –80 mV. Steady state current was measured at the end of the 5-s clamp pulses. Fig. 2 shows the I–V relationship (pA/pF) of the steady state current obtained from 14 cells. The steady state current reversed at –72.5 mV being inward directed at more negative and outward directed at more positive potentials. The slope conductance at the reversal potential was 6.45 nS. The steady state I–V relationship displayed a region of negative slope at potentials between –60 and –40 mV. The current density was –2.53±0.60 pA/pF at –90 mV, 0.35±0.07 pA/pF at –60 mV (maximum outward current at negative potentials) and 1.19±0.23 pA/pF at+40 mV (n=14).


Figure 2
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  		Fig. 2 Steady state current (pA/pF) to voltage relationship (from 14 myocytes). Current amplitude was measured at the end of 5000-ms voltage clamp steps (holding potential –80 mV).

 
When the myocytes were clamped to potentials between –50 and –140 mV (3000 ms, holding potential –40 mV) a current with a time course typical for the inward rectifier potassium current (IK1) could be recorded (Fig. 3A). The mean current density of the peak inward current was –22.8±2.47 pA/pF at –120 mV (n=14). At potentials more negative than –120 mV the inward current showed time dependent inactivation. The I–V relationship of the peak inward current at 36–37°C (open symbols, n=14) and room temperature (20–22°C, closed symbols; n=6) are shown in Fig. 3C. Superfusion with Ba2+ (1 mmol/l) inhibited a large fraction of the inward current (IK1) as shown in Fig. 3B. In all myocytes tested (n=3) a slowly activating inward current which may represent the pacemaker current (If) was observed in the presence of Ba2.


Figure 3
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  		Fig. 3 Inward currents at negative membrane potentials. (A) Current traces in response to voltage clamp steps between –50 and –140 mV (holding potential –40 mV; duration 3000 ms) under control conditions (A) and in the presence of 1 mmol/l Ba2+ (B). Ba2+ inhibited a large fraction of the inward current (IK1) and revealed the presence of a slowly activating inward current at hyperpolarized potentials similar to If (B). (C) Current (pA/pF) to voltage relationship (maximum current under control conditions) at 20–22°C (RT, filled circles, n=6) and 36–37°C (37°C, open circles, n=14).

 
3.3 Transient outward current Ito1
The calcium independent transient outward current (Ito1) was studied with 300 ms depolarizing pulses between –40 and +60 mV using a holding potential of –80 mV and a 50-ms prepulse to –40 mV in order to inactivate the sodium current INa. At potentials positive to –20 mV a fast activating outward current could be observed that decayed to a sustained level at the end of the pulse. Ito1 could be inhibited by micromolar concentrations of 4-aminopyridine (not shown). The amplitude of Ito1 was determined as the difference between the peak outward current and the current at the end of the clamp pulse. The sustained current at the end of the pulse is referred as Ilate to distinguish it from the (sustained) ultrarapid delayed rectifier current (Isus, Iso, IKur) which is present in human atrial cells [11,23–25]. A contribution of a calcium-dependent type of transient outward current (Ito2) can be ruled out because (i) the extracellular Ca2+-concentration was reduced (0.1 mmol/l), (ii) the calcium channels were blocked with external Cd2+ (0.1 mmol/l), and (iii) EGTA (11 mmol/l) was added to the pipette solution for buffering intracellular Ca2+.

Ito1 could be recorded in all myocytes (n=38) from all patients but showed marked differences in current density (even in myocytes from the same patient) ranging from 0.3 to 8.6 pA/pF with a mean of 3.77±0.42 pA/pF at +40 mV. Fig. 4 illustrates representative current traces (given as current density, panel A,B) and the current voltage relationship of Ito1 and Ilate (panel D). When the current density of Ito1 was plotted versus Ilate (Fig. 4C) no individual myocyte populations could be distinguished (subendocardial versus deeper layer). However, Ito1 current density was found to weakly correlate with Ilate current density (r=0.39; P<0.05).


Figure 4
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  		Fig. 4 Transient outward current Ito1. (A,B) Original current traces with prominent differences in current density. (C) The current density (pA/pF) of Ito1 of each individual myocyte (at +40 mV) is plotted against the amplitude of Ilate (pA/pF) (r: correlation coefficient). (D) Current (pA/pF) to voltage relationship of Ito1 and Ilate (n=38).

 
The inactivation time course of Ito1 could be fitted with a monoexponential function yielding a mean inactivation time constant ({tau}inact) of 10.9±0.87 ms at +40 mV (n=15). The inactivation time course of Ito1 was extremely temperature sensitive. Fig. 5 shows current recordings in response to a depolarization to +40 mV at 20 and 37°C (traces from different myocytes). Current amplitudes were normalized to allow a better comparison of the inactivation time course. Mean inactivation time constant at room temperature was 72.4±2.63 ms (n=3). The inactivation time course was thus 6.6 times faster at a physiological temperature than at room temperature.


Figure 5
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  		Fig. 5 Temperature dependence of Ito1. Current traces in response to a depolarization to +40 mV (holding potential –80 mV, prepulse to –40 mV) at 36–37 and 20–22°C are shown. For better comparison each current trace was normalized to its maximal value.

 
To determine the voltage dependence of activation of Ito1 conductance values were obtained by dividing the amplitudes of Ito1 by the driving force assuming a reversal potential of –58 mV [1,2]. A reliable determination of Ito1 reversal potential could not be obtained due to the small tail current amplitudes. The normalized conductances were plotted (Fig. 6, circles) against voltage and could be fitted with a Boltzmann function (Fig. 6, solid line):

Formula
where I is the normalized current, V is the membrane potential, V1/2 is the voltage of half-maximal activation and k is the slope value. The mean parameters obtained by fitting were 17.0±1.61 mV (V1/2) and 14.9±1.71 mV (k; n=38).


Figure 6
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  		Fig. 6 Voltage dependence of activation (circles) and steady state inactivation (triangles) of Ito1. The solid lines are the results of the fit with a Boltzmann function (see Results) to activation (n=38) and inactivation data (n=11).

 
3.4 Steady state inactivation of Ito1
The steady state inactivation of Ito1 was studied using a double-pulse protocol. From a holding potential of –80 mV the cells were clamped for 2 s to conditioning potentials between –100 and +20 mV followed by a 150-ms test pulse to +50 mV. These steps were separated by a 10-ms test pulse to –40 mV. The amplitudes of Ito1 in response to the test step were measured, normalized and plotted against the voltage of the conditioning pulses (Fig. 6, triangles). The data of each cell were fitted with a Boltzmann function (Fig. 6, solid line) yielding a mean half-voltage of inactivation of –29.6±0.68 mV and a slope factor of 6.22±0.60 mV (n=11).

3.5 Recovery from inactivation of Ito1
Recovery from inactivation was studied with a double pulse protocol. A conditioning pulse from a holding potential of –60 to +40 mV (duration 400 ms) was followed by a 100-ms test pulse to +40 mV, the pulse-pair being separated by intervals ranging from 10 to 20 000 ms (at –60 mV). Representative recordings are shown in Fig. 7A. The amplitude of Ito1 elicited by the test pulse was determined and normalized to Ito1 obtained at 0.05 Hz and was plotted versus the interpulse interval (Fig. 7B, n=9). The recovery of Ito1 could be described with a biexponential equation with a fast recovery time constant ({tau}r,fast) of 20.4 ms (amplitude: 0.67) and a slow recovery time constant ({tau}r,slow) of 1092 ms (amplitude: 0.32).


Figure 7
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  		Fig. 7 Recovery from inactivation of Ito1. (A) Original current traces in response to a 150-ms clamp step to +40 mV preceded by a 400-ms conditioning pulse to +40 mV. Conditioning pulse and test pulse were separated by increasing time intervals (10–20 000 ms) at a holding potential of –60 mV. (B) Mean recovery data from nine myocytes. The solid line represents a double exponential fit to the data.

 
3.6 Use dependent reduction of Ito1
To study the use dependent properties of Ito1 depolarizing steps from –60 to +40 mV with a duration of 150 ms were generated at a frequency of 4 Hz. Fig. 8A shows current traces in response to the first, second and 25th pulse at 4 Hz. The reduction of Ito1 amplitude was small and was maximal in response to the second pulse. The mean data of six experiments are shown in Fig. 8B. Reduction of Ito1 amplitude at 4 Hz amounted to 38.2±8.8% (n=6) and showed marked variations between individual myocytes ranging from 11 to 63%. These data as well as the recovery of Ito1, suggest that this current contributes to repolarization even at fast heart rates.


Figure 8
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  		Fig. 8 Rate-dependent reduction of Ito1. (A) Current traces were elicited by 150 ms depolarizations from –60 to +40 mV at a frequency of 4 Hz. Pulse 1, 2 and 25 are shown. (B) Mean normalized amplitudes of Ito1 at each pulse (n=6).

 
3.7 Delayed rectifier current IK
To test for the presence of IK the myocytes were clamped immediately after establishing an intracellular recording (to avoid any current run down) for 1 s to +40 mV followed by a 500-ms step to –30 mV using a holding potential of –40 mV. A slowly activating current at +40 mV and a tail current upon repolarization to –40 mV were taken as evidence for the presence of IK. From 38 cells tested with this protocol only two cells showed this type of current. Current traces, from a myocyte where IK was identified, in response to depolarizations between –30 and +60 mV (5000 ms) followed by repolarization to –40 mV (5000 ms) using a holding potential of –40 mV are shown in Fig. 9A. The voltage dependence of IK activation was obtained by analyzing the tail currents upon repolarization to –40 mV. The tail current amplitudes determined as the difference between the maximum tail current and the current at the end of the pulse were normalized and plotted versus the activation voltage in Fig. 9B. The mean data were fitted with a Boltzmann function yielding a V1/2 of –6.0 mV and a slope factor (k) of 5.9 mV (Fig. 9B). It has to be mentioned that Ito1 was not blocked during these experiments (Ito1 inhibitors like 4-aminopyridine may also affect IK) and that Ito1 inactivation may have partially overlaid IK activation. Therefore the activation time-course of IK was only estimated from tail current recordings (see below). The deactivation of IK could be described by a monoexponential function with deactivation time constants in the range of 300–500 ms.


Figure 9
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  		Fig. 9 Delayed rectifier potassium current IK. (A) Current traces in response to clamp potentials between –30 and +60 mV using a holding potential of –40 mV. (B) Voltage dependence of IK activation determined from tail currents. The solid line is the result of the fit with a Boltzmann function to IK activation data (see Results for details).

 
3.8 Envelope of IK tails
Fig. 10A shows current traces in response to voltage clamp steps to +40 mV of increasing duration using a holding potential of –40 mV. The activation of total IK was extremely fast, tail currents could be recorded upon repolarization. Tail current amplitudes were dependent on the duration of the depolarizing step. In Fig. 10B the normalized amplitudes (circles) of the tail currents are plotted versus the duration of the activating pulse. The solid line represents the result of a monoexponential fit of these data which yielded an activation time constant of 102.7 ms. It can be seen, that IK activation determined by tail current analysis was complete within 250 ms suggesting that the activated current was due to the rapid component of the delayed rectifier (IKr).


Figure 10
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  		Fig. 10 Envelope of tails test. (A) Current recordings in response to depolarizations from –40 to +40 mV with increasing duration. (B) Time dependence of IK activation shown as normalized tail current amplitude versus duration of depolarization (circles). The solid line represents the best fit with a single exponential function and yielded an activation time constant of 102.7 ms.

 
3.9 Influence of cell culture conditions
To allow recovery from cell isolation the myocytes were kept under primary culture conditions. Cell culture conditions may affect myocyte properties as described for guinea pig and rabbit ventricular myocytes [26–28]. A decrease of Cm which was associated with the loss of t-tubules during 24 h of cell culture were described. Moreover, a decrease of transmembrane currents was reported [27]. To evaluate possible influences of culture conditions on TOF myocytes we have compared Cm, as well as current density of Ito1 and IK1 from myocytes which were kept in culture for 3–8 or 18–24 h (Table 1). This analysis was performed on myocytes from three patients who yielded data to both groups. Statistical comparison revealed no significant effects of culture on Cm and Ito1. Due to the small number of IK1 data we did not apply statistical testing to this data set. However, our results suggest that IK1 may be affected in cell culture.


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  		Table 1 Influence of culture conditions on membrane capacitance (Cm) and current density of Ito1 (at +40 mV) and IK1 (at –120 mV)a

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusions
 References
 
Various studies reported repolarization characteristics in ventricular myocytes from adult hearts [1,2,6–15,29–31] whereas repolarizing currents of ventricular myocytes from infants have not been investigated. This report provides the first analysis of repolarizing currents in ventricular myocytes isolated from infants. Because voltage dependent ion currents are extremely temperature sensitive the experiments were performed at a physiological temperature.

4.1 Action potentials
The recorded resting potential of –72 mV is in the range of values described in adults (–71 mV [13]; –79 mV [2], –85 mV [30]; –71 mV [11]; –67 to –74 mV [15]). The APD90 (794 ms) is similar to reported APD-values ranging from 300–500 ms [1,12,15,30] to 800 ms and more [7,11,13]. Recently Näbauer et al. [2] described regional differences in action potential shape and found that subepicardial myocytes show a notch in the early repolarization phase and are shorter than subendocardial action potentials. Since a clear notch was absent in our recordings they resemble action potentials from adult subendocardial myocytes [2].

The occurrence of EADs (four of 12 myocytes) deserves particular interest. EADs are generally caused by an imbalance of inward and outward directed currents and may give rise to triggered activity and premature beats. A prolongation of the APD (probably resulting from reduced outward currents) and an alteration of inward currents (most likely L-type calcium current, ICa-L) are assumed to generate EADs [32]. As described previously [21] we found a large ICa-L window current in TOF myocytes which may be involved in the generation of EADs. It has to be mentioned, that various types of arrhythmias and late sudden death due to arrhythmias were reported for patients with tetralogy of Fallot [16–19,33]. Whether or not EADs play a role in the genesis of arrhythmias in TOF requires further investigation. We did not observe EADs in ventricular myocytes from diseased adult hearts (end stage heart failure) which often have an APD>1000 ms under the same recording conditions (our unpublished observations) which indicates that EADs are not an artifact due to the experimental conditions (conventional whole cell recording) but are specific for ventricular myocytes from children with TOF. Further studies with use of the perforated patch technique may give insight into the role of EADs in TOF myocytes.

4.2 Repolarizing currents
The steady-state current voltage relationship of ventricular myocytes from children with TOF resembles that in adult ventricular myocytes. In adult myocytes the reversal potential of the steady state current ranges from –57 mV [11] to approximately –70 mV ([8] Fig. 1), values similar to –72.5 mV as found in the present study.

Outward current at potentials between –60 and –40 mV (0.35 pA/pF at –60 mV), which is believed to be carried by IK1, was comparable to published values. In adult myocytes Amos et al. [11] described a maximum outward current density of 0.2 pA/pF at –44 mV; Konarzewska et al. [10] an IK1 current density of 0.09 and 0.07 pA/pF at –60 mV; Beuckelmann et al. [7] an IK1 current density of 0.36 µA/cm2 (0.36 pA/pF assuming a membrane capacitance of 1 µA/cm2) at –60 mV and Hoppe et al. [34] an IK1 density of 0.29 pA/pF at –60 mV. Inward currents activated during hyperpolarizing pulses were similar to IK1 (e.g. Ba2+ sensitivity) as described by various authors [8,15,34]. A direct comparison of IK1 current density between TOF (–22.8 pA/pF at –120 mV) and adult myocytes (approximately –45 pA/pF at –120 mV, at room temperature [15, Fig. 3]) is difficult because the amplitude of IK1 in our study may be underestimated since our data suggest a decrease of IK1 in cell culture.

Various cloned potassium channels give rise to currents similar to the cardiac IK1 and recently the m-RNA for four of these inward rectifier potassium channel subunits were found to be present in the ventricle of adults [37]. Which of these subunits underlie IK1 in TOF requires further investigation.

Inhibition of IK1 with external Ba2+ revealed the presence of a slowly activating inward current similar to the hyperpolarization activated pacemaker current (If) described in adult atrial [35] and ventricular myocytes [34,36]. This data suggest, that If is present in ventricular myocytes from children (although we can not exclude Ba2+ unblock of IK1 at very negative potentials which may give rise to similar current traces). However, the presence of If in non-pacemaking cardiac tissue is also discussed as a potential arrhythmogenic source [34].

4.3 Delayed rectifier potassium current IK
The presence of IK in human ventricular myocytes is a matter of debate. Some authors report its presence [7,12–14] whereas others did not find evidence for IK [8,10,11] or did not suppress IK when measuring other outward currents supposing its absence [1,2,9]. We found IK in two out of 34 myocytes. The time course of activation of IK was fast and activation was complete within 250 ms (at +40 mV) as described in adults [7]. The activation time constant derived from tail current analysis was 102.7 ms, a value reported for activation of IKr (140 ms [13]. Activation time course of IKr strongly depends on the experimental conditions as described by Iost et al. [14] who found slowing of IKr activation (657 vs. 31 ms) in the presence of Cd2+. The V1/2 of IK activation in TOF myocytes (–6 mV) is similar to the value described for IKr in undiseased adult ventricular myocytes (–5.7 mV) [14] suggesting that IKr underlies the observed IK in TOF myocytes, but it was less negative than previous published values (–14 mV [12], –29.9 mV [13]). It has to be taken into account that Cd2+, as used in the present study, also causes a positive shift of IKr activation voltage [14,38]. In only one study IKs was found to be present and a V1/2 of activation of 9.4 mV was described [12]. In undiseased human ventricular myocytes Iost et al. [14] did not find evidence for the presence of IKs which raises the question about presence and role of IKs in the human ventricle [39]. Due to the rare occurrence we did not attempt to elucidate the subtype of IK pharmacologically. The predominant lack of IK in the present study can not be attributed to the experimental conditions since using the same recording techniques a large and stable IK can be recorded in guinea pig myocytes (our unpublished observations). At present it is not clear whether the limited presence of IK is a property of ventricular myocytes from TOF-children or if IK is affected by the isolation procedure as described for canine cardiac myocytes [40].

4.4 Transient outward current Ito1
Ito1 was found in all myocytes studied but showed large variations in current density (0.3–8.6 pA/pF). In adult ventricular myocytes Ito1 density was described to depend on the origin of the myocyte with a large Ito1 in myocytes of subepicardial (and midmyocardial [6]) and a small Ito1 in myocytes of subendocardial origin [1,2]. The tissue used for myocyte isolation originates from the endocardium (outflow tract of the right ventricle), but depending on the thickness of the excised tissue more or less tissue from deeper layers was also taken for cell isolation. Since the excised tissue was rather small any further selection of sampled tissue (e.g. exclusively subendocardial sheet) was not possible. Thus variations in Ito1 current density most likely reflect differences in the origin of the myocytes. Mean Ito1 current density in TOF ventricular myocytes was in the range reported for adult subendocardial myocytes [1,2]. Voltage dependence of activation parameters and steady state inactivation parameters were also found to be similar as described for adults [1,2,6,9–11]. However, a direct comparison is difficult since most of these studies were obtained at room temperature and were performed on myocytes which did not originate from outflow tract of the right ventricle. Furthermore we do not know if human ventricular Ito1 is altered during development as described for human atrial Ito1 [3,4].

However, rate dependency and reactivation kinetics of Ito1 in TOF myocytes were strikingly different, since an extremely slow reactivation of subendocardial Ito1 has been described [1,2]. In the present study recovery of Ito1 was almost complete within 200 ms similar to epicardial Ito1 [1,2,11]. Whether this is characteristic for TOF or due to age dependent differences remains to be elucidated. In general, long term changes in repolarization may be due to alterations in the expression of specific genes coding for potassium channels [41] as shown for developmental changes in the expression of Ito1 channel isoforms [42,43]. Moreover, differences in the biophysical properties of Ito1 may reflect alterations of β-subunits [44,45]. Whether or not the observed differences in ventricular Ito1 between infants and adults can be attributed to differences in channel expression requires further investigation.

4.5 Physiological implications
The described electrophysiological properties of TOF myocytes are difficult to interpret from a clinical point of view since control data from healthy children are not available. Right ventricular hypertrophy is one of the clinical features of TOF, thus these myocytes may represent an interesting model of human ventricular hypertrophy. It is generally believed that hypertrophy (and heart failure) is accompanied by a ‘re-expression’ of fetal or neonatal genetic programs [46] which may also affect the pattern of sarcolemmal ion channels. Action potential prolongation and reduction of outward currents are well documented effects of hypertrophy [41,47,48]. A significant reduction in Ito1 density seems to play an important role in these effects [47,49]. Also in the human heart a decrease of Ito1 was also reported to occur during heart failure [7,31,41,50] and hypertrophy [29]. Since control data from healthy children are not available we do not know whether or not outward currents in TOF myocytes were affected by hypertrophy. However, the occurrence of EADs in TOF myocytes deserve interest, since a higher incidence of EADs, induced by almokalant, was found in hypertrophied myocytes from dogs with chronic complete atrioventricular block, compared to normal myocytes [48].

Various types of arrhythmias and late sudden death due to arrhythmias were reported for patients with TOF [16–19,33]. Besides right ventriculotomy scar, slow conduction and mechano-electrical alterations due to dilation [51–53] repolarization abnormalities are thought to play a role in arrhythmogenesis [54,55]. Our study provides the first cellular characterization of repolarization in infants with TOF. Although we have found several putative arrhythmogenic sources at the level of the isolated cell (e.g. EADs, heterogeneity in Ito1 current density) we do not know whether or not they have any relevance for arrhythmias in TOF patients. Thus, further studies designed to investigate potential sources for arrhythmias at the cellular and molecular level are highly requested.

The observed heterogeneity in Ito1 current density is likely to be more pronounced across the ventricular wall or between different regions of the heart [1,2,29], leading to a marked dispersion of repolarization which is generally believed to be involved in the genesis of reentrant arrhythmias [56].

Basic electrophysiological studies on TOF myocytes may also provide a rationale for ‘effective’ pharmacological treatment in TOF patients, since pharmacological suppression of arrhythmias in TOF is often unsuccessful [57].

4.6 Limitations
Since voltage activated ion currents are known to be extremely temperature sensitive we performed the experiments at a physiological temperature. This accuracy limits the comparability of our data since most published data were obtained at room temperature. A direct comparison with data obtained in myocytes from healthy children was not possible since such data are not available. Thus, all comparisons had to be made with data from adult (and most often diseased) myocytes. Differences in the origin of the myocytes between our study and studies on adult myocytes also have to be kept in mind when data are compared. Our results are a snapshot at the time of surgical repair of TOF and we do not know whether or not the electrophysiological properties of TOF myocytes change during development or in response to the disease (compare Ref. [58]), resulting in different electrophysiological characteristics late after repair where sudden death takes place. For the interpretation of our results it should be kept in mind, that potassium currents (e.g. activation and inactivation parameters) are influenced by divalent cations like Cd2+ [14,38,59] which was used to suppress ICa. We used conventional whole-cell recording which, although used as a standard technique, may have caused run-down of transmembrane currents resulting in a change of action potential parameters. As discussed above the isolation procedure could have influenced the properties of the isolated myocytes (e.g. induced the apparent absence of IK [40]).

4.7 Influence of culture conditions
The isolated myocytes were kept under primary culture conditions to allow recovery from cell isolation under fairly physiologic conditions. However, myocyte properties may change during culture as described for guinea pig and rabbit ventricular myocytes [26,27]. A decrease of Cm which was associated with the loss of t-tubules during 24 h of cell culture were described. In addition, a decrease of transmembrane currents was reported [27]. Especially IK1 seems to be affected during cell culture resulting in a decrease of whole cell IK1 [27] which is thought to be due to a reduction in the number of active channels [28]. The data shown in Table 1 indicate that culture conditions did not cause a decrease in Cm nor a reduction in the current density of Ito1. However, IK1 was of smaller amplitude in the 18–24-h group, suggesting a culture induced decrease. Further studies are requested to elucidate the influence of cell culture on human ventricular myocytes.

We used antibiotics (penicillin, streptomycin) in the culture medium, because myocyte isolation could not be performed under complete sterile conditions. Thus an influence of antibiotics on transmembrane currents, as described for neuroblastoma cells [60], can not be completely excluded.


   5 Conclusions
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
References
 
This study is the first description of repolarization currents in ventricular myocytes from children and provides the basis for future studies in this field. We found evidence for the transient outward current Ito1, a non-inactivating current Ilate, the inward rectifier current IK1, and the delayed rectifier current IK (only in two out of 38 myocytes). In addition, the hyperpolarization activated current If seems to be present in these myocytes. Most of the current characteristics resembled that of adult sub-endocardial myocytes. Further studies will have to show whether or not the observed putative arrhythmogenic sources (e.g. EADs) play a role in TOF patients.

Time for primary review 22 days.


   Acknowledgements
 
This work was supported by the Austrian Science Research Fund, P13111 [GenBank] -Med, SFB F 707 and a grant of the Federal Ministry of Science and Transport. We are grateful to the team of Cardiovascular Surgery, Universitätsklinik Graz for collaboration. The authors wish to thank Dr. M.W. Veldkamp and Dr. N. Jost for leaving us preprints of their recent manuscripts.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 

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