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Cardiovascular Research 1998 38(2):424-432; doi:10.1016/S0008-6363(98)00002-9
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

L-type calcium current in human ventricular myocytes at a physiological temperature from children with tetralogy of Fallot

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

aInstitut für Medizinische Physik und Biophysik, Karl-Franzens-Universität, Harrachgasse 21/IV, A-8010 Graz, Austria
bUniversitätsklinik für Chirurgie, Karl-Franzens-Universität, Graz, Austria

* Corresponding author.Tel.: +43 (316) 380-7741; Fax: +43 (316) 380-9660; E-mail: pelzmann@balu.kfunigraz.ac.at

Received 21 July 1997; accepted 9 December 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aim was to investigate the electrophysiological properties of the L-type calcium current (ICa,L) in ventricular myocytes at a physiological temperature (36–37°C) isolated from children undergoing surgical repair of tetralogy of Fallot. Methods: ICa,L was recorded with the patch-clamp technique in the single electrode whole-cell mode at a physiological calcium concentration (1.8 mmol/l) at 36–37°C. Results: Under these conditions, maximum current density averaged –5.80±0.45 pA/pF. ICa,L showed a bell-shaped current–voltage relationship: the current activated at –37.7±1.36 mV, peaked at +9.41±1.60 mV and reversed at +57.7±2.12 mV (n=17). At +10 mV, time to peak of ICa,L was 5.23±0.46 ms. Membrane potentials for half-maximal steady-state activation and inactivation of ICa,L were –6.02 and –20.4 mV, respectively, the slope factors were 7.16 mV for steady-state activation and 6.49 mV for steady-state inactivation. ICa,L did not completely inactivate and showed a big window current between –45 and +40 mV. The inactivation of ICa,L showed a biexponential time course with a fast time constant ranging from 9.11 to 12.9 ms and a slow time constant ranging from 60.9 to 220 ms between –30 and +30 mV. Only the slow time constant showed a pronounced voltage dependency. The recovery from inactivation of ICa,L was biphasic with a fast time constant of 60.7 ms and a slow time constant of 619 ms. β-Adrenergic stimulation with isoprenaline (1 µmol/l) increased the ICa,L density from –5.71±1.55 to –13.8±1.96 pA/pF (142%; P<0.05) at +10 mV. Conclusions: The present study demonstrates that most of the electrophysiological properties of ICa,L in ventricular myocytes isolated from children with tetralogy of Fallot resemble those of adult ventricular cells. The existence of a big calcium window current could be involved in the occurrence of early afterdepolarizations which could lead to the high incidence of arrhythmias after surgical repair of tetralogy of Fallot.

KEYWORDS Human; Ventricular myocyte; Calcium current; Isoprenaline; Whole cell clamp; Tetralogy of Fallot; Hypertrophy


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The properties of the L-type calcium current (ICa,L) and its role in excitation and contraction in cardiac cells have been extensively documented (for review see [1, 2]). In mature cardiomyocytes, the L-type calcium channel is the dominant Ca2+ channel. Several physiological roles of ICa,L are established in the heart. So it is an important determinant for action potential duration and refractoriness in all cardiac cells, for diastolic depolarization and cardiac rhythm in pacemaker cells, and for action potential upstroke and conduction velocity in nodal cells. Furthermore, Ca2+ ions entering through cardiac calcium channels are essential for excitation–contraction coupling and initiate contraction by triggering calcium release from the sarcoplasmic reticulum. This wide range of functions makes the calcium channel an important target for clinically important Ca2+-channel blockers or catecholamines. Under pathological conditions such as hypertension, hypertrophy and heart failure, channel density and function may be altered [3–5].

The electrophysiological properties of ICa,L in healthy and diseased human ventricular myocytes have been extensively investigated [6–13]. However little information concerning ICa,L in ventricular myocytes isolated from children is available [14]. Excised tissue from surgical correction of tetralogy of Fallot is thus an interesting source of ventricular tissue from children, which is also hypertrophied. Beyond that, it is well known that the surgical repair of the tetralogy is often followed by the occurrence of arrhythmias and late sudden death due to arrhythmias [15, 16]. The aim of the present study was to describe the electrophysiological characteristics of the L-type calcium current in ventricular myocytes derived from this tissue. Due to the marked temperature dependence of voltage- and time-dependent properties of ion channels, ICa,L was studied at a physiological temperature. It was shown that the main characteristics of ICa,L in myocytes obtained from Fallot patients are not very different from normal adult human myocytes. Only the occurrence of a significant calcium window current could be a possible link to the understanding of the high incidence of arrhythmic electrical activity in these patients.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Isolation of cardiomyocytes
Pieces of human right ventricular tissue of the outflow tract were obtained from 7 children undergoing surgical correction of tetralogy of Fallot. The mean age was 35±12 months (16–91 months). The use of human tissue was approved by the ethical committee of the University of Graz and conformed with the principles outlined in the Declaration of Helsinki. The samples were transported to the laboratory in saline containing in mmol/l: NaCl 90, KCl 30, NaHCO3 2, HEPES/Na+ 2, D(+)-glucose 5.5, sucrose 42, adjusted to pH 7.4 with NaOH (‘saline 1’). Cells were enzymatically isolated as described previously for adult human atrial myocytes [17]. The tissue pieces were cut into fragments of about 1 mm3 and washed with saline 1 to remove calcium from the tissue. The fragments were transferred to a dissociation vessel [18]containing 0.25% trypsin in saline 1 (Sigma, Deisenhofen, Germany). After 7 min of treatment, the trypsin solution was replaced by saline 1 containing 300 IU/ml collagenase (Worthington CLS 2, Worthington Biochemical Corporation, New Jersey, USA).

The supernatant was replaced by this collagenase solution every 15 min. After the third change, the collagenase concentration was reduced to 150 IU/ml. Dissociation steps were carried out at 37°C. Isolated cells were collected and washed in saline 1 in which the calcium ion concentration was raised stepwise. After transferring the isolated myocytes into cell culture medium M199 (Sigma) containing penicillin (50 IU/ml), streptomycin (50 µg/ml) and fetal calf serum (5%), they were stored in an incubator at 37°C for up to 24 h.

2.2 Electrophysiological recordings and data analysis
Membrane currents were recorded in the whole-cell single-electrode voltage-clamp configuration of the patch-clamp technique [19]using a List L/M-EPC 7 amplifier (List, Darmstadt, Germany). Action potentials were recorded in the current clamp mode. Myocytes were placed into an experimental chamber and superfused with extracellular solution at 36–37°C with a flow rate of about 1.5 ml/min. Only quiescent rod-shaped cells with clear cross striation were used for voltage-clamp experiments. When filled with standard internal solution and placed into standard external solution, patch-pipette tip resistances were 2–4 M{Omega}. After formation of a seal, the electrode capacitance was compensated. After breaking the patch membrane, the cell membrane capacitance (Cm) was determined by integration of the capacitive transient elicited by a 10-mV hyperpolarizing pulse from –50 mV. Cm was compensated up to 100 pF. Series resistance (Rs) was calculated by dividing the time constant of the capacitive transient by Cm. Rs prior to compensation was 5.81±0.36 M{Omega} (n=41). Rs was compensated by at least 50%. Thus, a current of 1 nA resulted in a voltage error of less than 3 mV. Voltage-clamp pulses were generated with an IBM compatible computer connected to a D/A and A/D converter (Digidata 1200, Axon Instruments, Foster City, USA). Data acquisition and analyses were performed using the pCLAMP software (Axon Instruments). In order to allow equilibration of the pipette solution with the cytosol, current recordings were started 5 min after rupture of the membrane patch. Current amplitudes were divided by Cm and expressed as current density (pA/pF) in order to compensate for variations in cell size. In order to study L-type Ca2+ current without contamination of Na+ current (INa), a 50-ms prepulse to –40 mV from a holding potential of –80 mV was used to inactivate INa. For determination of steady-state inactivation parameters, a holding potential of –45 mV was chosen. Potassium currents were suppressed by substituting K+ with Cs+ in both, the extracellular and the patch pipette solution. The composition of the extracellular solution was (in mmol/l): NaCl 137, CsCl 5.4, NaHCO3 2.2, MgCl2 1.1, NaH2PO4 0.4, CaCl2 1.8, HEPES/Na+ 10, D(+)-glucose 5.6, adjusted to pH 7.4 with NaOH. The patch pipette solution contained (in mmol/l): CsCl 110, EGTA 11, MgCl2 2, CaCl2 1, HEPES/Na+ 10, ATP (sodium salt) 4.3, adjusted to pH 7.4 with NaOH. During action potential recording, K+ was not substituted by Cs+ in the extracellular and the pipette solution.

Averaged data are expressed as means±s.e.m., n=number of cells. Error bars in figures represent s.e.m. Statistical significance was determined by Student's t-test for paired data. Differences were considered significant when P<0.05.

2.3 Drugs
A stock solution of the bitartrate salt of (–)-isoprenaline (Sigma) was prepared daily.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Cell dimension and cell capacitance
Ventricular myocytes isolated from children with tetralogy of Fallot were 78.7±1.65 µm long and 18.2±0.56 µm wide (n=212). The membrane capacitance (Cm) of the cells was 92.6±8.17 pF (41 cells of 7 patients in the age of 35±12 months).

3.2 Voltage dependence of ICa,L
Fig. 1A shows representative Ca2+ current traces recorded in a ventricular myocyte isolated from a patient with tetralogy of Fallot. Peak inward current was maximal upon depolarization to +10 mV, smaller currents were recorded at more negative and more positive potentials. The amplitude of ICa,L was measured as the difference between peak inward current and the current at the end of the depolarizing pulse. In order to compensate for variations in cell size, ICa,L was normalized to Cm and expressed as current density. Mean L-type calcium current density as a function of voltage is shown in Fig. 1B (voltage-clamp protocol, see inset). Maximum current density was –5.80±0.45 pA/pF when recorded in the presence of 1.8 mmol/l Ca2+ at a physiological temperature of 36–37°C. The current voltage (I/V) relationship showed a bell-shaped pattern typical for ICa,L. The current activated positive to –37.7±1.36 mV, peaked at +9.41±1.6 mV and reversed at +57.7±2.12 mV (n=17). ICa,L activated rapidly with a time to peak (measured as the time difference between the initiation of the depolarizing voltage step and the peak inward current) of 5.23±0.46 ms at +10 mV (n=17, see Fig. 2). At more negative and more positive membrane potentials, the time to peak increased.


Figure 1
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  		Fig. 1 A: L-type Ca2+ current elicited by voltage steps from –40 mV to various membrane potentials as indicated in the figure. Inactivation of INa was achieved by a prestep from –80 to –40 mV (50 ms). B: L-type Ca2+ current density as a function of voltage (n=17). Inset: voltage-clamp protocol.

 

Figure 2
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  		Fig. 2 Time to peak of ICa,L as a function of voltage. Time to peak was measured as the time difference between the initiation of the depolarizing voltage step and the peak inward current.

 
3.3 Voltage dependence of the steady-state activation and inactivation of ICa,L
Fig. 3 shows the steady-state activation and inactivation curves of ICa,L. In order to determine the activation at different potentials, peak values of ICa,L were divided by the driving force and normalized. A curve was fitted to mean normalized data (n=13) according to


Formula

where V is the membrane potential, V1/2 the membrane potential of half maximum activation and k is the slope of the activation curve. V1/2 was –6.02 mV and k 7.16 mV. Maximum activation of ICa,L was at +20 mV. A double-pulse protocol was used to determine the steady-state inactivation curve. ICa,L was activated with test pulses to +10 mV (400 ms), which were preceded by 400 ms conditioning prepulses to different potentials from a holding potential of –45 mV. The pulse pair was separated by a short (10-ms) repolarizing step to –45 mV. ICa,L during the test step was normalized and plotted as a function of prepulse potential. Increasing the potential of the conditioning prepulses led to a decrease in calcium current amplitude during the test pulses indicating that an increasing number of calcium channels were inactivated. However ICa,L did not inactivate completely within the potential range investigated (–45 to +40 mV). Inactivation reached a maximum at +20 mV. At prepulse potentials more positive than +20 mV, the extent of inactivation decreased resulting in an U-shaped inactivation curve of ICa,L. A curve was fitted to the mean normalized data (n=14) within the potential range of –45 to +20 mV according to the following equation


Formula

where V is the membrane potential, V1/2 the membrane potential of half maximum inactivation and k is the slope of the inactivation curve. A1 represents the maximal amplitude and A2 the amplitude of the non-inactivating component of ICa,L. V1/2 was –20.4 mV and k was 6.49 mV.


Figure 3
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  		Fig. 3 Steady-state activation (circles, n=13) and inactivation (squares, n=14) curves of ICa,L. In order to determine the steady-state activation curve, ICa,L peak values were divided by driving force and normalized. The steady-state inactivation curve of ICa,L was evaluated with test pulses to +10 mV (400 ms in duration), which were preceded by 400-ms conditioning prepulses from a holding potential of –45 mV. The pulses were separated by a 10-ms repolarizing step to –45 mV. ICa,L during the test step was normalized and plotted as a function of the prepulse potential. For fitting procedures, see text.

 
Due to incomplete inactivation, there existed a rather big overlap region of steady-state activation and inactivation, i.e. a big ‘window’ current.

3.4 Inactivation time course of ICa,L
The inactivation of ICa,L followed a biexponential time course. A fast and a slow inactivation time constant ({tau}f and {tau}s) could be separated at all membrane potentials investigated (Fig. 4A). {tau}f exhibited no obvious voltage dependence between –30 and +30 mV and varied from 9.11 to 12.9 ms. In contrast, {tau}s showed a pronounced voltage dependence and ranged from 60.9 to 220 ms. The voltage dependence of the slow time constant was U-shaped with a minimum near +10 mV (coinciding with maximum current density) and several-fold larger values positive and negative to +10 mV. The inactivation phase of ICa,L was fitted by a double exponential process according to the equation


Formula

where t is the time, Af and As are the amplitudes of the rapidly and the slowly decaying components, {tau}f and {tau}s the corresponding time constants. Aoff is the amplitude of the offset. The inset of Fig. 4A shows the inactivation phase of a ICa,L trace elicited by a voltage pulse to +10 mV (dots) superimposed by a biexponential fit (open circles). Fig. 4B shows the relative contribution of the fast and the slow inactivating process of ICa,L to the total charge movement as a function of membrane potential. The charge areas were analyzed by integrating the inactivation functions of the fast and the slow inactivating process (0–350 ms), determined by the amplitudes and time constants of inactivation, derived from the fitting procedure.


Figure 4
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  		Fig. 4 A: biphasic inactivation time course of ICa,L as a function of membrane potential. The fast inactivation phase ({tau}f, diamonds) exhibited no voltage dependency. In contrast, the slow inactivation phase ({tau}s, squares) was strongly dependent on voltage. Inset: inactivation phase of calcium current trace elicited by a voltage step from –40 to +10 mV (dots). Open circles represent a biexponential fit of calcium current decay. For fitting procedures, see text. B: relative contribution of the fast (circles) and the slow (squares) inactivating process to the total charge movement as a function of membrane potential (n=17).

 
3.5 Recovery from inactivation of ICa,L
The time dependence of ICa,L recovery from inactivation was studied using a double-pulse protocol (Fig. 5, inset): two depolarizing pulses to +10 mV with varying interpulse intervals (10–5000 ms) were applied from a holding potential of –40 mV every 8 s. The extent of recovery at each interpulse interval was obtained by expressing the amplitude of ICa,L in response to the test pulse as a fraction of the calcium current amplitude elicited by the conditioning pulse (Fig. 5). Averaged data from 6 myocytes were plotted as a function of interpulse interval, and the data were fitted by a biexponential function according to the equation


Formula

where t is the time, Af and As are the amplitudes of the fast and the slow recovery phase, {tau}f and {tau}s the corresponding time constants. The time constants were 60.7 ms for the fast and 619 ms for the slow phase of recovery.


Figure 5
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  		Fig. 5 Biexponential recovery of ICa,L from inactivation (n=5). Inset: from a holding potential of –60 mV two depolarizing pulses to +10 mV with a varying interpulse interval (10–5000 ms) were applied every 8 s. Ratio of current amplitudes elicited by the test pulse to current amplitudes elicited by the conditioning pulse was plotted as a function of interpulse interval. For fitting procedures, see text.

 
3.6 Effects of β-adrenergic stimulation on ICa,L
Fig. 6 shows the I/V relationships of ICa,L under control conditions and 5 min after exposure to 1 µmol/l isoprenaline (n=4). Superfusion of the myocytes with isoprenaline increased the mean peak calcium current from –5.71±1.55 to –13.8±1.96 pA/pF (an increase of 142%; P<0.05). The inset shows original current traces in response to depolarizing pulses to +10 mV from a holding potential of –40 mV and illustrates the large increase of peak ICa,L induced by the drug.


Figure 6
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  		Fig. 6 Current–voltage relationships of ICa,L under control conditions (open circles) and after exposure to 1 µmol/l isoprenaline (ISO, filled circles, n=4). Inset: original current traces in response to a depolarizing pulse to +10 mV from a holding potential of –40 mV. The voltage dependence of peak ICa,L was not changed significantly by isoprenaline.

 
3.7 Action potential recordings
Using K+-containing extra- and intracellular solutions (c.f. Section 2: Methods) a resting membrane potential of –74.3±1.4 mV could be recorded. Action potentials were elicited by electrical stimulation with short (5 ms) current pulses at a frequency of 0.5 Hz. The average action potential amplitude was 113.3±3.0 mV, the action potential duration at 90% repolarization (APD90) was 853.2±167.5 ms (n=6). In one of these myocytes, the spontaneous occurrence of early afterdepolarizations (EADs) was observed (Fig. 7). APD90 of the action potential shown was 1767 ms.


Figure 7
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  		Fig. 7 Occurrence of early afterdepolarizations in one of six investigated ventricular myocytes.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Isolated ventricular myocytes from patients with tetralogy of Fallot are for several reasons of special interest: (1) the excised tissue represents hypertrophied tissue of the right ventricle; (2) the tissue stems from children; and (3) it is known that the long-term success of surgical repair of tetralogy of Fallot is often hindered by the occurrence of arrhythmias which may lead to late sudden death [15, 16]. Basic studies in these cells could provide insight into cellular effects of hypertrophy and developmental changes of electrophysiological properties of current components. In addition, they could be a way to understand the factors underlying the high incidence of arrhythmias. Because of the important role of ICa,L in many physiological processes, the basic electrophysiological characteristics of this current were studied at a physiological temperature since it is well known that voltage-activated ionic currents are highly temperature-sensitive. Therefore, we had to take into account that a comparison of our results with published data is limited by the fact that most studies on human myocytes were performed at room temperature. To our knowledge, this is the first description of the electrophysiological properties of ICa,L in isolated human ventricular myocytes at a physiological temperature.

Ventricular myocytes isolated from children with tetralogy of Fallot had a mean length of 79 µm and a mean width of 18 µm (n=212). Data on myocyte dimensions from children are very sparse in the literature. Bustamante et al. [20]reported a myocyte length of 118 µm and a width of 16.7 µm, but they did not provide any patient characteristics (number of patients, disease, age). Human ventricular myocytes from adults give a broad range of dimensions. Hypertrophy and dilatation are thought to lead to an increase in cell size beside an increase in muscle mass and ventricular cavity volume. This would explain why many investigators studying myocytes from failing hearts reported larger cell dimensions than reported here [11, 20–23]. On the other hand longer myocytes were also reported from non-failing hearts [24, 25]and small cells similar to ours from failing hearts [10]. An explanation for the small cell size found in our study could be that this is a specific characteristic of cells isolated from children. Hypertrophy of the ventricle could also have not affected cell size at this age. It also has to be considered that different isolation procedures could have favored the survival of myocytes with certain dimensions.

According to the small dimensions of the cardiomyocytes, cell capacitance (79 pA/pF) was lower than that of adult human ventricular cells [11, 12, 26, 27]. Similar values were given by Bénitah et al. [10]and Konarzewska et al. [28], while Varró et al. [23]reported a still smaller cell capacitance. Cell capacitance, however, is not a good estimation of cell size variations because cells of a certain size could be selected for the experiments (e.g. small cells are used for voltage-clamp experiments to improve voltage control).

Using a physiological calcium concentration (1.8 mmol/l) and temperature (36–37°C), ICa,L showed a bell-shaped I/V relationship with a maximum current density of –5.8 pA/pF. The current activated at –38 mV, peaked at +9.4 mV and reversed at +58 mV (n=17). At +10 mV, time to peak was 5.2 ms. This voltage dependence of ICa,L is not different from that reported by others in adult human ventricular myocytes [8, 10, 12, 14, 29]. In our study, the current density was found to be slightly higher and the time-to-peak shorter when compared to other studies [10, 12]. The most likely explanation for these differences is the physiological temperature used in our experiments. Therefore, we cannot conclude if calcium current density is affected by hypertrophy in our patients. Current densities were reported to be not significantly different between diseased and healthy adult human ventricular cells [7, 12]or decreased in diseased myocytes [13]. In animal hypertrophy models, the calcium current density has been reported to be either unchanged [30–32]or increased [33]. Only in severe ventricular hypertrophy a significant reduction of current density has been reported [34].

Steady-state activation and inactivation parameters of ventricular myocytes from children with tetralogy of Fallot were found to be in the range of values given in the literature. ICa,L showed a half maximum activation at –6.02 mV with a slope of the activation curve of 7.16 mV. Maximum activation occurred at +20 mV. Half maximum activation was less negative in our study than that reported by others in pediatric ventricular cells (–10.06 mV) [14]and more negative than that reported in myocytes from adults by Bénitah et al. (–0.14 mV) [10]. Values similar to ours were given by Mewes and Ravens [12]who could not find a significant difference of activation parameters in adult ventricular myocytes from failing and non-failing hearts. The steady-state inactivation curve of ICa,L showed half maximum inactivation at –20.4 mV which is more negative than that reported for failing and less than that for non-failing hearts [12]. However, the shift of the inactivation curve towards positive potentials due to failure reported by Mewes and Ravens [12]was not significant. In myocytes from animals, showing mild hypertrophy, a positive shift of the inactivation curve has been found [30, 35, 36]. Cohen and Lederer [14]described a significant age-dependent shift of the steady-state inactivation curve. They showed that half maximum inactivation occurred at more negative potentials in adult human ventricular myocytes compared to pediatric cells, thus giving rise to a larger window current in pediatric myocytes. In human atrial myocytes, no difference in the voltage-dependence of steady-state inactivation between adult and pediatric cells could be found [37]. The value for half maximum inactivation reported in this study is more negative than that reported for pediatric and less than that for adult ventricular cells [14]. Values similar to ours were given by Bénitah et al. [10]. In the present study, steady-state inactivation of calcium current was found to be incomplete over a pulse duration comparable to the action potential plateau. Maximum inactivation was reached at about +20 mV. At prepulse potentials more positive than +20 mV, the extent of inactivation decreased resulting in an U-shaped inactivation curve of ICa,L comparable to that in human ventricular myocytes [8, 12]. In accordance with our results, Bénitah et al. [10]reported that steady-state inactivation never reached zero (the mean residual current in this study was around 3% of the maximal amplitude). Also in this study, after a minimum, an increase was observed although this was less pronounced. Similar findings were also shown in pediatric ventricular cells [14]. Due to the incomplete inactivation, there existed a significant region where steady-state activation and inactivation overlapped, giving rise to a distinct window current. This pronounced window current could be a source for prolonged calcium influx leading to a prolongation of the action potential plateau thus favoring the occurrence of early afterdepolarizations (EADs) [38]. Several authors have described shifts of the calcium inactivation variable to more positive potentials due to hypertrophy which would increase the calcium window current [30, 35, 36]and therefore favor EADs. Other changes in the properties, like prolonged inactivation [30, 33, 35, 39]and more rapid recovery of the current [35], could also favor EADs.

Action potentials in ventricular myocytes of children with tetralogy of Fallot lasted for 850 ms, which is much longer than the action potential duration of 300 ms reported by Cohen and Lederer [14]in pediatric ventricular cells. This discrepancy still gains significance by the fact that we recorded long-lasting action potentials at 37°C, whereas Cohen and Lederer [14]found shorter action potentials at 25°C. The pronounced calcium window current found in ventricular myocytes from children with tetralogy of Fallot could account for the long action potential duration. A possible pathophysiological implication of this window current was supported by the fact that EADs were observed in one out of six myocytes. In general, EADs are thought to be a main source for arrhythmias. However, if EADs contribute to the incidence of arrhythmias after surgical repair of tetralogy of Fallot requires further investigation. Beuckelmann et al. [40]reported a significant prolongation in myopathic human adult ventricular myocytes (1090 ms) compared with control cells (650 ms). Substantially longer action potentials (~1000 ms) are also described by others in diseased human ventricular cells [41, 42]compared with undiseased myocytes [40]. A long APD and a flat plateau phase is generally believed to promote EADs. Although extremely long action potentials have been reported in isolated human ventricular myocytes, no spontaneous occurrence of EADs has been described up to now. In action potential recordings in myocytes from explanted hearts, using the same experimental conditions as in the present study, we frequently found action potentials with an APD90 in the range of 800–1000 ms. However, in none of these myocytes could EADs be observed. It is thus tempting to speculate that a combination of a long APD (e.g. due to a reduction of repolarizing currents in heart failure [43]) and a large calcium window current could play a substantial role in the occurrence of EADs described in the present study.

In ventricular myocytes from children with tetralogy of Fallot, the time course of inactivation of ICa,L was biexponential at all investigated membrane potentials. The fast inactivation time constant ({tau}f) ranging from 9.11 to 12.9 ms showed no obvious voltage dependence, whereas the voltage dependence for the slow time constant ({tau}s) was U-shaped with {tau}s becoming progressively longer at membrane potentials positive and negative to +10 mV. {tau}s ranged from 60.91 to 219.58 ms. A biexponential decay of calcium current with {tau}f showing no voltage dependence in contrast to {tau}s was also described by others [10, 12], but these values are not comparable to ours because we performed the experiments at a physiological temperature.

Data from hypertrophied animal myocytes show considerable variation regarding calcium current inactivation. Some studies claim a lack of difference between control and hypertrophy groups [31, 32, 34, 36]. Others found the fast inactivation component unchanged and the slow component prolonged [30, 33]. In some studies, both components were described to be prolonged in hypertrophy [35, 39].

Our results showed that the time dependence of ICa,L recovery from inactivation followed a biexponential time course. The time constants were 60.71 ms for the fast phase and 618.79 ms for the slow phase of recovery. Complete recovery from inactivation was reached in 2 s. Bénitah et al. [10]found the recovery from inactivation to be very slow at room temperature. Reactivation of 100% occurred in about 4000 ms with a 50% recovery time of 318 ms. Nánási et al. [11]reported a monoexponential recovery, in contrast to Bénitah et al. [10], ICa,L showed complete recovery at about 900 ms at room temperature.

In our study, ICa,L exhibited a clear sensitivity to β-adrenergic stimulation. Addition of 1 µmol/l isoprenaline (ISO) increased ICa,L by 142% (n=4). A similar increase of ICa,L by the same concentration was shown by Cheng et al. [29]in human ventricular cells, a smaller increase by lower concentrations of ISO was reported by others [8, 10]. The signal transduction pathway of the β-adrenoceptor-mediated catecholamine response is known to be impaired at several levels in the failing heart. Receptor density and sensitivity were shown to be decreased [44, 45], the stimulatory GTP-binding protein Gs was reported to be reduced [46], and the inhibitory Gi protein to be increased [47]. Recently, Harding et al. [24, 48]reported a reduction in β-adrenergic agonist response in single atrial and ventricular cells from failing human hearts.

Kozlik-Feldmann et al. [49]reported a reduction of β-adrenoceptor density in right atrial myocardium in infants and children with severe congenital heart disease. Mewes and Ravens [12], however, found a similar increase of calcium current by forskolin in failing and non-failing hearts and conclude that the signal transduction cascade beyond the level of GTP-binding proteins is not impaired in failure. Our data suggest that the β-adrenergic signal transduction pathway seems to be qualitatively intact in these myocytes.

The main limitation of our study is the lack of a direct comparability of our data with data from healthy children due to the unavailability of undiseased pediatric myocardium. Therefore comparisons had to be made with adult and, in most cases, diseased myocardium.

The calcium current in ventricular myocytes isolated from children with tetralogy of Fallot resembles the L-type calcium current in adult human myocytes including a clear sensitivity to β-adrenergic stimulation. Our results suggest that this disease has little influence on the electrophysiological properties of ICa,L. The existence of a big window current over a wide potential range, however, could be a possible link to the understanding of the high incidence of arrhythmic electrical activity in these patients.

Time for primary review 25 days.


   Acknowledgements
 
This work was supported by the Austrian Science Fund P11131 [GenBank] -Med, SFB 007 and a grant of the Austrian Federal Ministry of Science and Transport. We are grateful to the team of Cardiovascular Surgery, Universitätsklinik Graz for their collaboration and we thank Dr. M. Kriebel for critical reading of the manuscript.


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

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