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* Experimental and Molecular Cardiology Group and the Departments of Physiology and Clinical Genetics, Academic Medical Center, University of Amsterdam, The Netherlands
Correspondence: Address reprint requests to G. Berecki, Dept. of Clinical and Experimental Cardiology, Academic Medical Center, University of Amsterdam, Rm. M01-217, Meibergdreef 9, 1105 AZ Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands. Tel.: 31-20-566-7547; Fax: 31-20-691-9319; E-mail: g.berecki{at}amc.uva.nl.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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). Type 2 of the congenital long-QT (LQT2) syndrome is linked to mutations in the human ether-a-go-go-related gene (HERG), which encodes the pore-forming 
-subunit of the rapid delayed rectifier potassium channel (Curran et al., 1995; Sanguinetti et al., 1995; Trudeau et al., 1995). Properties of current through HERG channels (IHERG) are similar to those of the rapidly activating component of delayed rectifier K+ current (IKr) that contributes to the final repolarization of the ventricular action potential (AP) (Sanguinetti and Jurkiewicz, 1990). Investigations of various (wild-type and mutant) HERG channels in heterologous expression systems such as Xenopus laevis oocytes or mammalian tissue cells in culture have provided remarkable results in understanding the congenital forms of the LQT2 syndrome. It is apparent that the mechanisms by which HERG mutations cause the clinically observed electrical disease are various. For some HERG mutants, the observed differences in HERG channel kinetics and/or IHERG density are evident and translation into effects that these mutated channels would have on the ventricular AP are obvious. Conversely, in several cases, results of voltage-clamp experiments do not provide satisfactory explanation to how structural changes of the channel protein would affect cardiac AP repolarization and ultimately lead to the observed clinical phenotype in affected patients. In such cases, where the observed differences between the wild-type and mutant channels are less clear, one can in existing computer models of the human ventricular AP (Priebe and Beuckelmann, 1998) modify IKr according to what was found for the mutant and determine the resulting change(s) in AP characteristics. It is then, often implicitly, assumed that the mathematical description of the IKr fully covers the properties of this current. Besides, this approach is restrained by the lack of quantitative data on the complex kinetics of the IKr and IHERG at physiological temperature. The mathematical description is therefore merely an approximation, despite recent advances in modeling (Clancy and Rudy, 2001), and results from simulations in which HERG channel properties have been changed should be interpreted circumspectly. In this study, we introduce a novel electrophysiological technique to assess the functional implications of ion channel mutations. We hypothesize that rapid and unambiguous interpretation of the altered channel function is possible with an experimental setting in which mutant channels are allowed to follow a natural time course of membrane potential (Vm) change, upon being simultaneously allowed to contribute current to the AP as they would have when incorporated into a real ventricular cell. With our dynamic action potential clamp (dAPC) technique, the native IKr of a ventricular myocyte or cell model is effectively replaced with IHERG recorded from a transfected HEK-293 cell that is voltage-clamped by the free-running AP of the ventricular cell. To this end, the native IKr is pharmacologically blocked (or set to zero in case of a model cell) and IHERG is applied to the ventricular cell as an external current input. When wild-type (WT) IHERG is added to the net membrane current of this ventricular cell, the resulting AP should be considered as normal, whereas a mutant IHERG should cause distortion of the AP.
We applied our dAPC technique to the R56Q (arginine to glutamine) mutation, a defect known to increase the rate of deactivation most profoundly (Chen et al., 1999). Previously, R56Q HERG channels had only been expressed in Xenopus oocytes, and characterized at room temperature (Chen et al., 1999). We studied WT and mutant channels in HEK-293 cells also by conventional whole-cell voltage-clamp technique, at both 23°C and 36°C. At physiological temperature, the mutant channels showed both faster deactivation, which would lengthen the AP, and faster activation, which by itself would shorten the AP. However, our dAPC experiments directly and unambiguously demonstrate that the net effect of the mutation is an increase in action potential duration (APD).
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MATERIALS AND METHODS |
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HEK-293 cells were either superfused with Tyrode's solution containing (mmol/L): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 5.5 glucose, 5 HEPES (pH 7.4 with NaOH), or with a modified Tyrode's solution with 4.5 instead of 5.4 mmol/L KCl (see below). Membrane currents were recorded with an Axopatch 200B amplifier (Axon Instruments, Union City, CA) in the whole-cell configuration of the patch-clamp technique at 23 ± 0.5°C and 36 ± 0.5°C. Voltage control, data acquisition, and analysis were accomplished using custom software. Patch pipettes (1.5–3 M
) were filled with solution containing (mmol/L): 125 K-gluconate, 20 KCl, 1 MgCl2, 5 EGTA, 5 MgATP, 10 HEPES (pH 7.2 with KOH), resulting in a K+ equilibrium potential (EK) of –87.7 mV at 36°C. To obtain a better match between the EK of the experimental solutions and the model cell's maximum diastolic potential of –90.7 mV, we also used 4.5 mmol/L KCl in the Tyrode solution (resulting in an EK of –92.5 mV). All figures showing APs in the model-cell mode (see below) were obtained with this modified Tyrode solution. The pH of solutions was corrected for temperature; potentials were corrected for liquid junction potential. Membrane currents and potentials were low-pass filtered (cutoff frequency 2 kHz) and digitized at 5 kHz. The current-voltage (I-V) relationships, and IHERG kinetics were determined by voltage-clamp protocols, as diagrammed in Figs. 2 and 3, and as described previously (Sanguinetti et al., 1995; Smith et al., 1996; Snyders and Chaudhary, 1996) and in the Supplementary Material. APs from freshly isolated rabbit left-ventricular myocytes were measured at 36°C with the solutions described above (5.4 mmol/L KCl in the Tyrode solution; EGTA was omitted in the pipette solution), as described previously (Verkerk et al., 1996) and detailed in the Supplementary Material.
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), model clamp (Wilders et al., 1996), and dynamic clamp (Sharp et al., 1993) techniques. The development of these techniques is built on the concept that an isolated (cardiac) cell can be electrically coupled to either another isolated cardiac cell or to a model analog that mimics the electrical properties of the cardiac myocyte. As diagrammed in Fig. 1, a single cardiac ventricular cell and a transfected HEK-293 cell can be electrically coupled by means of an electrical circuit. The ventricular cell (with IKr blocked) is in current clamp mode on one patch-clamp setup, whereas the HEK-293 cell is in voltage-clamp mode on another setup. The command potential for the HEK-293 cell is the Vm of the ventricular cell (action potential clamp), and the current input applied to the ventricular cell is the IHERG recorded from the transfected HEK-293 cell, a connection resulting in dAPC condition (Fig. 1 A). We performed two kinds of dAPC experiments, defined as the model-cell mode and the real-cell mode.
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) of a single human ventricular myocyte that is computed in real-time. We extended the model clamp (Wilders et al., 1996) and dynamic clamp (Sharp et al., 1993) techniques, implementing dAPC with a real-time Linux operating system (Barabanov and Yodaiken, 1997) as a software platform according to Christini et al. (1999). To attain simultaneous control and recording of Vm and IHERG and to resolve the time-critical tasks of analog-to-digital conversion of IHERG, calculation of the model, and digital-to-analog conversion of Vm, we developed a user program (DynaClamp). This was used with a real-time module that operated on a 1.8-GHz Pentium-4 PC with a 16-bit National Instruments PCI-6052E data acquisition board (National Instruments, Austin, TX) under real-time Linux, and communicated through shared memory and/or first-in, first-out queues. This allows a guaranteed-timing real-time process (i.e., 40-µs periodic time steps with the PB cell model). In all dAPC experiments, IKr of the model cell is set to zero. We first determine maximal IHERG amplitude in the HEK-293 cell in voltage-clamp configuration, with 4-s depolarizing voltage steps to –10, 0, and 10 mV, from a holding potential of –80 mV, at 36 ± 0.5°C. Considering the unusual kinetics of HERG channels (Lu et al., 2001a), we measure IHERG amplitudes at the end of 4-s pulses rather than from tail current amplitudes. The largest outward current value is then used to estimate the scaling factor (Fs) for the IHERG input to the PB model cell. In our standard protocol, WT as well as R56Q IHERG amplitude are scaled to 47.6 pA (equivalent to the original IKr amplitude in the PB model). After appropriate scaling, the program establishes dAPC configuration between the model cell and the HEK-293 cell for 10 s, during which a series of 2-ms, 4-nA, 1-Hz suprathreshold stimuli are applied to the computer model cell. The recorded IHERG and computed PB model variables (Vm and ionic currents) and settings of the DynaClamp program are stored on disk for off-line analysis. The time-dependent changes in Vm of the ventricular model cell are derived from WT and/or mutant IHERG input and the model equations. The combination of the cell model and WT IHERG will then result in a normal AP. Using the same method for HEK-293 cells with mutant channels will reveal an AP, which resembles the ventricular AP of the patient from which the mutant was derived.
Real-cell mode
In real-cell mode, the model cell is replaced with a rabbit left-ventricular myocyte (Fig. 1 C). The procedure to define Fs is as follows: we measure IHERG amplitude in the HEK-cell (as described above) and, simultaneously, estimate IKr density in the rabbit cell (as E-4031 sensitive current). We elicite APs in the myocyte at 1 Hz in the presence of E-4031, and then establish coupling between the myocyte and the HEK-293 cell, implementing scaled WT IHERG. A proper Fs value would result in IHERG density comparable to that of the IKr density in the myocyte and in a typical AP duration at 90% repolarization (APD90) value of 230.8 ± 4.5 ms at 1 Hz (see Table 2 in Supplementary Material), characteristic for these cells. Ca2+ loading of the myocytes exhibiting long action potentials in the presence of E-4031 (as in Fig. 8 B) is likely. However, this process loses its grip when the scaled IHERG is implemented and APD is shortened to its initial value (to same APD as before the addition of E-4031) where Ca2+ loading will be ruled out.
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Statistics
Data are expressed as mean ± SE (n, number of cells) and considered significantly different if P < 0.05 in ANOVA and Student's t-test.
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RESULTS |
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). The deactivation time course of R56Q channels was markedly faster than for those of WT at both temperatures, as shown by the diminution of both (fast and slow) time constants (Fig. 3 A; and see Table 1 in the Supplementary Material). The finding that the mutation causes faster deactivation is in agreement with the results of Chen et al. (1999). Time constants of inactivation and recovery from inactivation (Fig. 3 B) did not differ significantly between WT and R56Q (see Table 2 in the Supplementary Material). Our results demonstrate that acceleration of the R56Q HERG activation remains obscured at 23°C and highlight the importance of investigating HERG kinetics at physiological temperature.
Replacing IKr of the model cell with WT and R56Q IHERG
In the comprehensive human subepicardial ventricular cell model by Priebe and Beuckelmann (1998), description of IKr is based on data from human ventricular cells (Li et al., 1996). With model-cell IKr set to zero, the AP prolongs (Fig. 4 A). When WT IHERG from a HEK-293 cell replaces IKr, AP characteristics are restored and the AP can be considered as normal (Fig. 4, A and C). Similar results were obtained when the KCl content of the Tyrode solution was modified to 5.4 mmol/L (see, in Supplementary Material, Fig. 1 and Table 3). The time course of the scaled IHERG compares well to that of IKr of the model cell (Fig. 4 B) except that the initial time course of IHERG differs from that of the mathematically described IKr, which is due to the model assumption that IKr inactivation is instantaneous. Many HERG channels are still in the open state at –90 mV as a result of slow deactivation (Lu et al., 2001a), and this results in an initial transient peak (asterisk), reflecting the sudden increase of the electrochemical driving force for K+ during the AP upstroke. After a fast decay of the transient peak amplitude, caused by inactivation during the overshoot of the AP and by the decreasing driving force for K+ at less depolarized Vm, current increases progressively as channels rapidly recover from inactivation, a process faster than the deactivation (Sanguinetti et al., 1995; Trudeau et al., 1995; Smith et al., 1996; Zhou et al., 1998). With repolarization progressing, HERG channels dwell in a highly stable open state before closing (Wang et al., 1998), resulting in a resurgent current. Altered HERG channel properties in long-QT syndrome generally reduce the magnitude of this resurgent current (Chen et al., 1999; Sanguinetti et al., 1996). Both IKr and IHERG reach maximum value 
–40 mV, then rapidly deactivate in a time- and voltage-dependent manner.
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Action potential heterogeneity in the PB model cell with WT and R56Q IHERG
The heterogeneity of the electrical properties of the myocytes in the different layers of the human left ventricle is now well established. As in our previous model studies (Bernus et al., 2002; Conrath et al., 2004), we generated subendocardial, midmyocardial (M), and subepicardial model cells by adjusting selected membrane ionic currents in the PB model cell (Table 2). When, in a dAPC experiment, WT IHERG replaced model-cell IKr, APs of different shape and duration could still be reproduced (Fig. 5). The major consequence of the R56Q mutation on the AP characteristics of these cell types was AP prolongation (Fig. 6). We analyzed in detail AP characteristics of the epicardial model cell (Fig. 7), comparing the frequency dependence of APD90 values generated with model-cell IKr to values obtained with WT or R56Q IHERG. These values are comparable when WT IHERG replaces IKr, whereas R56Q IHERG causes frequency-dependent APD90 prolongation (Fig. 7 A). APD90 with the cotransfected WT/R56Q channels showed intermediate values (not shown). The role of WT or R56Q IHERG in shaping the AP was evaluated by phase plane analysis (Sperelakis and Shumaker, 1968), plotting membrane currents against membrane potential (Fig. 7 B). With input IHERG scaled for identical amplitudes for both WT and R56Q, the consequence of the mutation is apparent. The most notable changes are detected during phase-3 repolarization, with a reduction of the net membrane current (Itotal).
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), thus we first demonstrate IKr presence as the E-4031 sensitive current (Clay et al., 1995). Currents during depolarization as well as tails were markedly diminished in the presence of E-4031, resulting in prolonged repolarization and early after-depolarizations in all cells tested (n = 9) (Fig. 8 B). dAPC experiments (n = 5) were performed with a single myocyte coupled first to a HEK-293 cell with WT IHERG (Fig. 8 C), and then to a HEK-293 cell with R56Q IHERG (Fig. 8 D). In both cases, AP parameters were determined at different stimulation frequencies (Fig. 9; see also Table 4 in the Supplementary Material). The measured resting Vm of the myocytes was –82.9 ± 2.7 mV. APs were effectively reconstituted in a dAPC experiment with WT IHERG. APs with R56Q IHERG exhibited significant APD prolongation at 0.2 and 1 Hz (Fig. 9 B). These experiments also revealed that WT IHERG consists of an early fast transient outward current followed by a sustained outward current (Figs. 8 C and 9). Transient IHERG may contribute importantly to AP dynamics during tachycardia (Lu et al., 2001a). Amplitude of the transient component showed positive frequency dependence (Fig. 9 C), whereas that of the sustained component peaked during the terminal AP repolarization, in a reverse frequency-dependent manner between 1 and 5 Hz (Fig. 9 D). Although frequency dependence of the sustained R56Q IHERG was similar to that of WT IHERG, frequency dependence of the R56Q IHERG transient component was absent, consistent with the impaired deactivation kinetics of these channels.
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DISCUSSION |
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; Lu et al., 2001a; Zhou et al., 1998) and, as shown in the present study, even better during dAPC condition (i.e., by letting them shape the ventricular action potential), in line with their normal function.
The NH2 terminus of the -subunit of the channel regulates deactivation gating and represents a mechanism by which functional diversity is generated in HERG and related channels (Wang et al., 1998). Our electrophysiological experiments demonstrate that the R56Q mutation impairs not only deactivation (Chen et al., 1999) but activation kinetics as well, the latter becoming apparent only at 36°C (Fig. 3 A). Intriguingly, a faster activation and a positive shift in the voltage dependence of channel availability (Fig. 2 B), would actually act to shorten AP duration. Characteristics of the heteromultimeric (WT/R56Q) channels suggest that some of the functional effects are not simply combined, but that a dominant negative interaction can also occur between the WT and R56Q HERG channels (see activation time constants at 36°C in Fig. 3 A). Along the same lines with the impaired biophysical properties, certain mutations in the Per-Arnt-Sim domain might actually cause an HERG protein trafficking defect (Paulussen et al., 2002). However, we did not find significant differences in IHERG densities of WT and/or R56Q channels, suggesting that the primary defect in mutant channel properties is attributable to altered gating.
MinK-related peptide (MiRP1)/HERG complexes have received considerable support as molecular correlates for native IKr (Abbott et al., 1999, 2001). We did not coexpress MiRP1 for reconstitution of native IKr by HERG, as properties of IHERG in mammalian systems are similar in many ways to those of native IKr, and discrepancies that remain cannot be fully abolished by coexpression with MiRP1 (Weerapura et al., 2002).
Most experimental data on cardiac ion channel (dys)function have been obtained in expression systems, away from the cellular environment where these channels function to generate the cardiac action potential. Table 3 shows a comparison of IKr in the various systems: 1), PB model; 2), human ventricle; 3), rabbit ventricle; and 4), HEK-293 cells. The relatively few studies of human ventricular IKr make it difficult to fully validate such comparison. Nevertheless, the study of Iost et al. (1998) provides data on IKr in human ventricular tissue obtained from healthy patients not receiving medication. Despite the apparent differences between some properties of IHERG and IKr in the present study and previous results in the literature, mammalian cell lines generally provide an adequate environment for HERG channels. Here, experiments should be performed at physiological temperatures, as HERG channel gating at 36°C more closely resembles endogenous IKr (Zhou et al, 1998; this study). Necessarily, the Xenopus system can be an alternative when channels do not express well in a mammalian cell line, although 36°C for oocytes is not physiological, and observed differences in the behavior of the expressed cardiac potassium channel proteins suggest that endogenous factors in oocytes dictate channel properties to some extent (Seebohm et al., 2001).
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Superimposed phase plane plots of the repolarization phases of model-cell APs indicate that the net membrane current is severely affected by the mutation during the late repolarization phase. APD90 values with R56Q IHERG were increased at lower stimulation rates and unchanged at higher frequencies. Consistent with the role of HERG in the suppression of arrhythmias initiated by premature beats (Lu et al., 2001a), the technique revealed the presence of an early fast, frequency-dependent transient WT IHERG. The frequency-dependent increase of this current component was absent with R56Q channels. APs with R56Q IHERG were generally longer (Fig. 9 B), which can be explained by the faster deactivation. However, the reason why the faster activating, thus initially larger R56Q IHERG does not have significant effect on the AP plateau is less obvious. It is likely that the faster activation of the R56Q IHERG in the myocyte causes a slightly modified membrane potential in the early plateau phase of the AP, influencing activation of other currents. Computer simulations using either the PB model or the recently published human ventricular cell model by Ten Tusscher et al. (2004) also predict little or no effect of a moderate increase in IKr during the plateau phase of the action potential (data not shown). On the other hand, even small changes of the myocyte's membrane potential can cause significant changes in activation of voltage-dependent currents, such as the transient outward current, Ito (Greenstein et al., 2000) and calcium current, ICa (Fülöp et al., 2003).
In summary, both the computed model of the human ventricular cell as well as a freshly isolated myocyte can effectively be used in dAPC experiments. Kinetic features that are difficult to investigate with standard voltage-clamp protocols become apparent with the dAPC technique. The model-cell mode offers an outstanding reproducibility of the results during experimentation, as the input WT or mutant IHERG is the only variable. However, the technically more difficult real-cell mode can reveal AP waveforms and IHERG kinetics that can be considered truly physiological. Additionally, the real-cell mode offers the advantage that stimulation rates above 2.5 Hz (maximal value in the model cell) can easily be achieved. Theoretically, any individual conductance in the model cell or in a real ventricular cell (if a specific blocker for the investigated conductance is available) can be replaced by a surrogate input current from an expression system.
In the model-cell variant of the technique, it is a straightforward operation to test the effect of interventions directed at counteracting the effects of the mutations in HERG, e.g., increasing the slow repolarizing component (IKs) of the delayed rectifier K+ current.
Data presented here on the behavior of WT and R56Q HERG channels may have implications for further studies, where differences between WT and mutant channels are subtle. With our approach, the contribution of (mutated) channels to the AP is determined without making assumptions with regard to the kinetic properties of the channels, and the altered shape of AP directly reflects the effect of the mutation. The dAPC technique allows other cardiac ion channels than HERG (e.g., SCN5A, KvLQT1) to be studied as well.
General considerations
The inherent limitations of the PB model and of simulations when creating transmural AP heterogeneity on the basis of experimental findings have been discussed before (Bernus et al., 2002; Priebe and Beuckelmann, 1998). During dAPC experiments, in both model-cell and real-cell modes, we assumed that the defect in the R56Q channel is attributed to altered gating. Accordingly, we scaled WT and mutant input IHERG to similar magnitudes.
We are aware that it is potentially conceivable that a mutation in an ion channel gene could result in compensatory changes in other ion channel genes in vivo, representing a general limitation of any heterologous expression system. Short-term alteration of mRNA levels of ion channels, caused by rapid pacing, is well documented (Yamashita et al., 2000). Libbus et al. (2004) provide direct evidence for Ito remodeling in the ventricle caused by reduced AP upstroke amplitude, on a surprisingly short timescale.
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ACKNOWLEDGEMENTS |
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Submitted on June 8, 2004; accepted for publication September 29, 2004.
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slow, triangles) and fast and slow time constant of deactivation (
