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Cardiovascular Research 2005 68(3):441-453; doi:10.1016/j.cardiores.2005.06.027
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Copyright © 2005, European Society of Cardiology

Role of sequence variations in the human ether-a-go-go-related gene (HERG, KCNH2) in the Brugada syndrome

Arie O. Verkerka,b,*, Ronald Wildersb, Eric Schulze-Bahrc,d, Leander Beekmana, Zahurul A. Bhuiyana,e, Jessica Bertrandc, Lars Eckardtd, Dongxin Linf, Martin Borggrefeg, Günter Breithardtc,d, Marcel M.A.M. Mannense, Hanno L. Tana, Arthur A.M. Wildea and Connie R. Bezzinaa,e

aDepartment of Experimental Cardiology, Academic Medical Center, University of Amsterdam, The Netherlands
bDepartment of Physiology, Academic Medical Center, University of Amsterdam, The Netherlands
cMolecular Cardiology, Institute for Arteriosclerosis Research, University of Münster, Germany
dDepartment of Cardiology and Angiology, University Hospital of Münster, Germany
eDepartment of Clinical Genetics, Academic Medical Center, University of Amsterdam, The Netherlands
fDepartment of Etiology and Carcinogenesis, Cancer Institute, Chinese Academy of Medical Sciences, Beijing, PR China
gDepartment of Cardiology, University Hospital Mannheim, Germany

* Corresponding author. Department of Physiology, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Tel.: +31 20 5664670; fax: +31 20 6919319. Email address: A.O.Verkerk{at}amc.uva.nl

Received 12 August 2004; revised 17 June 2005; accepted 27 June 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Background: Brugada syndrome (BrS) is an inherited electrical disorder associated with a high incidence of sudden death. In a minority of patients, it has been linked to mutations in SCN5A, the gene encoding the pore-forming {alpha}-subunit of the cardiac Na+ channel. Other causally related genes still await identification. We evaluated the role of HERG (KCNH2), which encodes the {alpha}-subunit of the rapid delayed rectifier K+ channel (IKr), in BrS.

Methods and results: In two unrelated SCN5A mutation-negative patients, different amino acid changes in the C-terminal domain of the HERG channel (G873S and N985S) were identified. Voltage-clamp experiments on transfected HEK-293 cells show that these changes increase IKr density and cause a negative shift of voltage-dependent inactivation, resulting in increased rectification. Action potential (AP) clamp experiments reveal increased transient HERG peak currents (Ipeak) during phase-0 and phase-1 of the ventricular AP, particularly at short cycle length. Computer simulations demonstrate that the increased Ipeak enhances the susceptibility to loss of the AP-dome typically in right ventricular subepicardial myocytes, thereby contributing to the BrS phenotype.

Conclusion: Our study reveals a modulatory role of IKr in BrS. These findings may provide better understanding of BrS and have implications for diagnosis and therapy.

KEYWORDS Arrhythmia; Sudden death; Ion channels; K-channel; Genetics


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
The Brugada syndrome (BrS) is an inherited primary electrical cardiac disorder associated with a high risk of sudden arrhythmic death, characterized by right precordial ST-segment elevation in the absence of ischemia, structural heart disease, or electrolyte abnormalities [1]. The only gene with a proven involvement is SCN5A (for reviews, see [2,3]), the gene encoding the pore-forming {alpha}-subunit of the human cardiac Na+ channel (INa). SCN5A mutations in BrS result in a functional reduction in INa [3]. While the pathophysiology of BrS may be multifactorial [2,3], INa reduction is believed to favour early repolarization caused by K+ currents, thereby leading to heterogeneity of repolarization in the right ventricle (RV) [4], where the transient outward K+ current (Ito) is more prominent [5]. SCN5A mutations, however, are found in only 15–30% of BrS patients [6,7], suggesting the involvement of other genes. A locus at chromosome 3p25–p22 [8], adjacent to, but not overlapping that of SCN5A, has also been linked to the disorder. However, the culprit gene within this locus remains as yet unidentified.

In this study, BrS patients in whom mutations in SCN5A had been excluded, were screened for mutations within the human ether-a-go-go-related gene (HERG, KCNH2), encoding the pore-forming {alpha}-subunit of the rapid component of the delayed rectifier K+ channel (IKr) [9]. IKr plays an important role in defining ventricular repolarization, as a wide variety of IKr inhibiting drugs prolong action potential (AP) duration and QT interval. Moreover, HERG mutations underlie long-QT syndrome type 2 (for review, see [10]). The contribution of IKr to cardiomyocyte repolarization is complex [11,12], due to its unusual kinetics characterized by slow activation and deactivation but rapid inactivation and recovery from inactivation [10]. Following the AP upstroke, IKr activates slowly, but inactivates rapidly during the plateau phase. As the membrane potential repolarizes, IKr partially recovers from inactivation before it slowly deactivates. Thus, IKr increases to a maximum during phase-3 of the AP and then decreases, as the electrical K+ driving force decreases and deactivation of the channel progresses. Inherent to its biophysical properties, IKr may also play a role in the early phase of cardiac APs [13]. IKr deactivation during diastole is relatively slow; if diastolic deactivation is incomplete, residual active IKr channels during an upstroke will be exposed to an abrupt increase in K+ driving force, resulting in a large transient peak IKr [13–15]. Experimental work on guinea-pig [16] and rabbit [17] ventricular myocytes and related theoretical studies [18] have demonstrated that such residual active IKr channels play a significant role in cardiac excitability. The modulatory role of IKr in the early phase of APs was the rationale for KCNH2 screening in BrS patients. We identified two different KCNH2 nucleotide changes in two unrelated patients with BrS, investigated their electrophysiological consequences, and present a mechanism for their potential contribution to the BrS phenotype.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgment
 References
 
2.1 Patients
The investigation conformed to the ‘Declaration of Helsinki’. The study population consisted of unrelated BrS probands recruited in Germany (n=38) and the Netherlands (n=40) in whom mutations in the SCN5A coding region and the proximal promoter [19] were excluded.

2.2 Mutational analysis
The entire coding region of KCNH2 was analyzed for mutations by PCR–DNA sequencing. DNA sequence variation was validated by sequencing two independent PCR products and, secondarily, by primer-induced restriction enzyme digestion [20]. The latter method was also used to investigate the presence of the identified nucleotide substitutions in a large sample of unrelated control individuals of the appropriate ethnicity (300 Caucasians, 500 Han Chinese).

2.3 Site-directed mutagenesis and cell culture
Nucleotide changes identified in BrS probands were introduced into HERG cDNA cloned in pGFPires as described previously [20].

HEK-293 cells were transiently transfected with 0.5-µg WT-SCN5A/pGFPIRS, or with 1-µg WT-, G873S-, or N985S-HERG/pGFPires using lipofectamine and cultured at 37 °C. In coexpression experiments, HEK cells were transfected with 0.5-µg WT- and 0.5-µg G873S- or N985S-HERG/pGFPires. Cells exhibiting green fluorescence 12–20 h post-transfection were selected for electrophysiological experiments.

2.4 Cellular electrophysiology
Cells were superfused with solution containing either (mmol/L): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5, HEPES 5.0 or NaCl 140, CsCl 10, CaCl2 2.0, MgCl2 1.0, glucose 5, sucrose 10, HEPES 10 (pH 7.4; NaOH) for HERG and SCN5A measurements, respectively. Currents were recorded at 36 ± 0.2 °C using the whole-cell patch-clamp technique (Axopatch 200B amplifier). Patch pipettes contained either (mmol/L): K-gluconate 125, KCl 20, MgCl2 1.0, EGTA 5, MgATP 5, HEPES 10 (pH 7.2; KOH) or CsCl 10, CsF 110, NaF 10, EGTA 11, CaCl2 1, MgCl2 1, Na2ATP 2, HEPES 10 (pH 7.3; CsOH), for HERG and SCN5A measurements, respectively. Voltage control, data acquisition, and analysis were accomplished using custom software. Potentials were corrected for liquid junction potential. Current density was calculated by dividing current amplitude by cell capacitance (Cm). Cm was estimated by dividing the decay time constant of the capacitive transient in response to 5-mV hyperpolarizing voltage-clamp steps from –40 mV by the series resistance (Rs). Adequate voltage control was achieved by using low-resistance pipettes (1.0–2.0 M{Omega}), Rs and Cm compensation (>80%), and small cells, especially for INa measurements (4.9 ± 1.0 pF, n=6). Without compensation, Cm and Rs were 11.2 ± 0.4 pF and 6.5 ± 0.1 M{Omega} (n=165), respectively. Signals were low-pass filtered (cut-off frequency of 5 kHz) and digitized at 5–10 kHz, except for AP-clamp measurements where it was 10 kHz and 40 kHz, respectively.

The kinetic properties of WT-, G873S-, and N985S-HERG were determined by voltage-clamp protocols as diagrammed in Fig. 3 and described previously in detail [20]. Characteristics of HERG and SCN5A currents during APs were tested using the AP-clamp technique. An AP waveform of a human ventricular cell was simulated (see below) at 400 ms cycle length (CL), digitized at 10 kHz, and stored. The AP waveform was used as command signal in voltage-clamp conditions. At least 10 consecutive waveforms were applied to reach stable electrical activity.


Figure 3
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  		Fig. 3 Biophysical properties of WT-, G873S-, and N985S-HERG currents. (A) Representative current traces. (B) Average amplitudes of steady-state (Iss) and tail currents (Itail). (C) Average activation and inactivation curves. (D) Average fully activated I–V relationship. (E) Average fully activated I–V relationship normalized to Itail at –30 mV. (F) Average fraction of slow vs. fast component of deactivation (As and Af, respectively). (G) Average time constants of activation and deactivation. (H) Average amplitudes of Ipeak. (I) Time constants of inactivation and recovery from inactivation. Voltage-clamp protocols shown as insets.

 
2.5 Computer simulations
Functional effects of the HERG variants were tested by computer simulations using the mathematical model by ten Tusscher et al. [21] (‘TNNP model’), which describes the electrophysiology of a single human ventricular myocyte of left subepicardial origin, based on recent experimental data on individual ionic currents, including INa, Ito, IKr, L-type calcium current (ICa, L), slow delayed rectifier current (IKs), and inward rectifier potassium current (IK1). The model includes pump and exchanger currents as well as "a basic calcium dynamics" [21] that we replaced with the more sophisticated description by Faber and Rudy [22]. Models for M cells and subendocardial cells were obtained by modifying IKs and Ito [21].

We modified the TNNP model equations for IKr based on our WT-HERG current data. Thus, we replaced the original steady-state activation and inactivation curves with those of Fig. 3C. Furthermore, to match our HERG current (de)activation data, we replaced the model equations for {alpha}xr1 and βxr1 [21] with {alpha}xr1=5.0+200.0/(1.0+exp(–(Vm+50.0)/10.0)) and βxr1=20.0/(1.0+exp((Vm+40.0)/10.0)). The most important of these changes is the replacement of the –88 mV half-inactivation voltage by our value of –40.6 mV. As a result, we had to scale down the maximal IKr conductance (GKr) by a factor of 4 to retain the model AP shape. Consequent to this approach, AP clamp characteristics of simulated IKr match closely those measured experimentally (data not shown).

The experimentally observed increase in IKr tail current density was implemented by a 44% increase in GKr. The experimentally observed shift in voltage dependence of IKr current inactivation was incorporated by a –12 mV shift in the IKr steady-state inactivation curve. APs were elicited by repetitive stimulation with a 1-ms two-times threshold stimulus current [21].

It has been shown that RV myocytes exhibit larger repolarizing forces than left ventricular (LV) myocytes because IKs density is twofold [23] and Ito density 2.5–4 times larger in RV [23,24], whereas IKr and IK1 densities do not differ between left and right ventricles [23]. Accordingly, we created a model of an RV subepicardial myocyte by increasing the IKs and Ito densities of the TNNP model by a factor of 2 and 4, respectively. The ATP-sensitive potassium current (IK,ATP) was incorporated as an additional ohmic potassium conductance.

2.6 Statistics
All data are presented as mean ± S.E.M. and compared using one-way repeated measures ANOVA followed by pair-wise comparison using the Student–Newman–Keuls test with a significance level of P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgment
 References
 
3.1 Mutation analysis of HERG
DNA sequencing of KCNH2 in 78 unrelated BrS probands identified two heterozygous nucleotide changes in two different individuals recruited in Germany. An A>G transition at nt 2954 (numbering from the initiation codon of cDNA NM_000238 [GenBank] ), resulting in replacement of the asparagine at position 985 by serine (AAC>AGC, N985S), was identified in a 56-year-old male patient of Caucasian origin (BRU-006). A G>A transition at nt 2617, leading to replacement of the glycine at position 873 by serine (GGC>AGC, G873S), was identified in a 57-year-old female patient of Han Chinese origin (BRU-044). The nucleotide substitution underlying the N985S change was not detected in 300 unrelated Caucasian control individuals. The nucleotide substitution underlying the G873S change was also not found in the Caucasian control population but was found in the heterozygous state in 2 of 500 unrelated Han Chinese control individuals.

3.2 Clinical details
Patient BRU-006's ECG exhibited right bundle-branch block (RBBB) morphology at rest (QRS 120 ms, normal PQ and QTc). Exercise elicited ECG changes compatible with BrS (Fig. 1A). First, a saddleback ST-segment in V1–3 appeared. At higher heart rates more RV conduction delay occurred with ST-elevation in the right precordial leads (QRS in V5 150 ms). Ajmaline testing (50 mg intravenously, not shown) caused nonspecific QRS broadening. Holter ECG revealed spontaneous, self-limiting, polymorphic ventricular tachycardia (VT) as QRS widened at fast heart rates (Fig. 1B). Programmed ventricular stimulation with three extrastimuli in the RV apex and RV outflow tract induced nonsustained polymorphic VT (20 beats). Structural heart disease was not found using transthoracic echocardiography, angiography and magnetic resonance imaging. The family history revealed sudden death in one brother. Family members refused detailed clinical and genetic investigation.


Figure 1
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  		Fig. 1 ECGs of two Brugada syndrome patients with HERG sequence variations. (A) Precordial ECG lead recordings; patient BRU-006. (B) Holter recording; patient BRU-006. (C) Precordial ECG lead recordings; patient BRU-044.

 
Patient BRU-044 had a BrS Type II ECG at rest (Fig. 1C; saddleback pattern in V2). There was no evidence for derangements in atrioventricular conduction or repolarization (QTc 390 ms). An S-wave in V4–6 indicated RBBB (QRS in V5 110 ms). Intravenous ajmaline (50 mg) induced a Type I ECG in V1–3 and QRS widening (150 ms). Programmed ventricular stimulation in the RV apex and outflow tract using three extrastimuli failed to induce ventricular tachyarrhythmias. Structural heart disease was not found using transthoracic echocardiography and angiography. The family history was negative for BrS or sudden death.

3.3 Role of HERG currents during the early action potential phases
Given the proposed importance in BrS of ion current derangements during the early AP phases [2–4], we first studied the transient peak WT-HERG current (Ipeak), and INa during AP waveforms applied at 400 and 800 ms CLs. Fig. 2A shows a typical example (left) and the average current–voltage (I–V) relationship (right) of the instantaneous Ipeak measured with a three-step protocol (inset). During the first pulse (P1), HERG channels are activated and subsequently rapidly inactivated. The second pulse (P2) allows rapid recovery from inactivation. The subsequent depolarizing step (P3) produces instantaneous currents, which are transient due to fast (re-)inactivation of open channels [13]. The Ipeak–V relationship was linear with a reversal potential near the calculated EK (dashed line). Fig. 2B shows a typical example (left) and the average Ipeak amplitudes evoked by P2 (inset) in relation to the interpulse interval (right). Longer interpulse intervals allowed more deactivation, resulting in smaller Ipeak. The decline was well fitted with a biexponential equation ({tau}s=73 and {tau}f=261 ms), predominantly reflecting fast and slow deactivation [14]. Between pulses, HERG channels were open but passed little current because the K+ driving force is minimal at –85 mV (Fig. 2A). Fig. 2C shows the applied AP waveform and resulting HERG and INa currents. Note that HERG and INa currents are measured in different cells and that both currents are independently scaled to their maximal amplitude. INa activated around –60 mV, peaked around 0 mV and subsequently decreased due to fast inactivation and reduced Na+ driving force (Fig. 2D). HERG current, however, was instantaneous and its amplitude increased upon depolarization due to larger K+ driving force (Fig. 2D). The I–V relationship between –85 and +30 mV, however, was not exactly linear as expected from changes in driving K+ force only (dashed line), suggesting substantial inactivation during the upstroke. Subsequently, the amplitude decreased around +30 mV due to further inactivation. Note that HERG current lasts longer than INa. In contrast to Ipeak, INa decreased at shorter CLs (Fig. 2D), most likely due to incomplete recovery from inactivation.


Figure 2
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  		Fig. 2 Characteristics of instantaneous HERG currents (Ipeak). Ipeak voltage-dependency (A) and interpulse interval-dependency (B): typical example (left) and average relationships (right). The typical Ipeak labeled ‘1’ and ‘2’ (B, top) are depicted on expanded time scale (B, bottom). Insets show voltage-clamp protocols. (C) Characteristics of HERG Ipeak and SCN5A (INa) currents (bottom) during an AP waveform (top). Inset (top right): AP waveform and current on condensed time scale. (D) I–V relationships of HERG Ipeak and INa currents during AP depolarization. Maximal voltage error due to uncompensated series resistance was 1.0 ± 0.2 and 1.3 ± 0.3 mV for HERG and SCN5A currents, respectively.

 
These results suggest a substantial HERG current during the early AP phases, especially at faster heart rates. We hypothesized that HERG variants, which increase Ipeak by altering density and/or kinetics, may play a role in BrS.

3.4 Biophysical properties of G873S and N985S HERG channels
Fig. 3 shows biophysical properties of WT-, G873S-, and N985S-HERG currents. Fig. 3A shows typical current traces. All currents activated upon depolarization, reached a maximum steady-state current (Iss) around –10 or –20 mV, and decreased because of the rapid onset of inactivation (rectification) at more positive potentials. During P2, all recordings displayed the typical large tail currents (Itail) due to recovery from inactivation, whereas the slow current decline is due to deactivation [9]. Iss and Itail amplitudes of G873S and N985S were both larger than WT (Fig. 3B). Fig. 3C shows average voltage-dependence of activation and inactivation. The voltage-dependence of activation of WT, G873S, and N985S was similar. The voltage for half-maximal inactivation, however, was shifted significantly from –40.6 ± 2.4 mV in WT to –51.2 ± 2.3 and –52.3 ± 3.8 mV in G873S and N985S, respectively, without changes in slope factor. Fig. 3D shows the average voltage-dependence of the fully activated HERG currents. Note the larger current amplitudes in G873S and N985S. Normalized to Itail at –30 mV (Fig. 3E), the voltage-dependence was similar at potentials negative to –30 mV, but differed at –30 mV and more positive as shown by the increased rectification in G873S and N985S. Fig. 3F and G show that the average contribution of the slow component to the deactivation process and the average time constants of activation and deactivation, did not differ between the three HERG currents. Fig. 3H shows average current densities analyzed from Ipeak after a repolarizing pulse to –100 mV where steady-state inactivation is negligible (Fig. 3C). In the variant channels, the current density in the –20 to +60 mV potential range was increased on average by 75 ± 0.5% (G873S) and 62 ± 0.5% (N985S). Fig. 3I shows that average time constants of inactivation and recovery from inactivation did not differ between WT, G873S and N985S.

3.5 HERG current in coexpression experiments
Next, we studied HERG currents in HEK-293 cells transfected with equal amounts of WT and G873S or N985S. The biophysical properties of the coexpressed HERG currents did not differ, except for voltage-dependence of inactivation and current densities (Table 1). The voltage for half-maximal inactivation was shifted significantly from –40.6 ± 2.4 mV in WT/WT to –52.9 ± 3.9 and –53.5 ± 2.8 mV in WT/G873S and WT/N985S, respectively. The current densities were significantly increased by 45% and 43%, respectively.


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  		Table 1 Biophysical properties of WT/WT and WT/variant HERG currents

 
In a final series of WT/variant coexpression experiments, we analyzed HERG current during AP waveforms at 400 and 800 ms CL. During the AP upstroke, HERG currents show large Ipeak, particularly at short CL (Fig. 4A). Additionally, HERG currents increased progressively through the AP plateau, reached a maximum towards the end of the repolarization phase and then decreased. The maximum of WT/G873S-, and WT/N985S-HERG current during phase-3 repolarization occurred at more negative potentials than WT/WT (Fig. 4B), most likely due to the negative shift in steady-state inactivation (Table 1). During the AP upstroke and phase-3 repolarization, WT/G873S- and WT/N985S-HERG current densities were larger than WT/WT (Fig. 4A and B), but were similar during the plateau phase due to the increased rectification in WT/G873S and WT/N985S.


Figure 4
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  		Fig. 4 (A) WT/WT-, WT/G873S-, and WT/N985S-HERG currents during AP-clamp protocols at 400 and 800 ms CL. (B) I–V relationships of HERG currents during AP-clamp at 400 (left) and 800 ms (right) CL.

 
3.6 Computer simulations
To assess the physiological implications of the above differences in HERG current density and kinetics, we conducted computer simulations using mathematical models of human left and right subepicardial myocytes.

Fig. 5 shows the effects of the ‘heterozygous’ increase in IKr density and negative shift in inactivation on the AP of the LV subepicardial myocyte, both separately and in combination. At 800 ms CL (75 beats/min), the AP shortened by 16 ms upon the increase in IKr density (Fig. 5A). Conversely, the negative shift in inactivation resulted in a decreased IKr availability during the AP and thus prolonged the AP by 16 ms (Fig. 5B). When combined, the effects were limited to a 4-ms prolongation (Fig. 5C). Similar results were obtained at 400 ms CLs (150 beats/min, Fig. 5D) and with subendocardial and M-cell versions of the model (not shown). Like the experiments of Fig. 4, the early transient peak IKr was larger at shorter CL and more prominent for variant IKr.


Figure 5
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  		Fig. 5 Simulated effect of HERG channel variants on the AP of human LV subepicardial myocytes. (A–D) APs and associated IKr at CL of 800 (A–C) or 400 ms (D) in case of (A) a 44% increase in HERG current density, (B) a –12 mV shift in voltage dependence of HERG channel inactivation, and (C, D) both changes. Insets show initial peak IKr on an expanded time scale.

 
The basis of BrS resides in the right ventricle. While the pathophysiology may be multifactorial [25], it has been proposed that one key cellular mechanism is loss of the AP-dome in RV subepicardial myocytes with concomitant preservation of the AP-dome in the subendocardium (all-or-none repolarization) [4]. Therefore, we assessed the susceptibility to all-or-none repolarization of RV myocytes. We did so by incorporation of a small additional potassium conductance, reflecting the experimental approach of administration of an IK,ATP agonist to unmask susceptibility to all-or-none repolarization [24]. Fig. 6 shows simulated APs of RV subepicardial myocytes with WT/WT (top) or WT/variant (‘heterozygous’) IKr (bottom) elicited at 800 ms CL (left). To reproduce the clinical observations at faster heart rate observed in BRU-006, we conducted simulations also at 400 ms CL (right). As for the LV subepicardial myocyte (Fig. 5), the incorporation of variant IKr did not induce very prominent changes in AP configuration. However, the addition of the 0.6-nS potassium conductance (dashed lines) revealed a higher susceptibility to all-or-none repolarization in case of variant IKr, particularly at 400 ms CL, where – despite the smaller Ito (insets) – the AP notch was more pronounced than at 800 ms CL due to smaller inward calcium current (not shown). The loss-of-dome was preferentially found in the RV subepicardial myocyte (not shown).


Figure 6
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  		Fig. 6 Simulated effect of HERG channel variants on susceptibility to all-or-none repolarization of human RV subepicardial myocytes, as assessed by incorporating a small additional potassium conductance of 0 (control) or 0.6 nS (solid and dashed lines, respectively). (A–D) APs and associated IKr at CL of 800 (A, C) or 400 ms (B, D) in case of (A, B) WT/WT or (C, D) WT/variant HERG channels. Insets show initial peak IKr and normalized INa and Ito.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgment
 References
 
4.1 G873S and N985S biophysical properties correlate with clinical details
We identified two HERG sequence variations, G873S and N985S, in two SCN5A mutation-negative patients with ECG features typical of BrS (Fig. 1). Patch-clamp experiments revealed that both variant proteins produced an increased IKr current density and a negative shift of inactivation leading to increased rectification (Fig. 3 and Table 1). In line with the absence of changes in QT-interval in the patients (Fig. 1), computer simulations demonstrated that the combined actions of these changes in channel properties resulted in minimal changes in AP duration (Fig. 5). Both variant proteins, however, increased Ipeak during the AP upstroke (Figs. 4 and 5Go), which led to loss of the RV epicardial AP-dome when higher stimulation rates were combined with an increase in potassium conductance (to unmask susceptibility to all-or-none repolarization) (Fig. 6). The AP-dome, however, was maintained in RV subendocardial and midmyocardial myocytes (not shown). Subsequent RV transmural current flow from endocardium to epicardium may result in ST-segment elevation, which is essentially identical to the proposed mechanisms of ST-segment elevation by SCN5A mutations [3,26,27]. This loss of the epicardial AP-dome was not found in the LV cell variant of the model. The apparent involvement of the RV is in agreement with the clinical observations that that the electrophysiological changes in BrS are mapped to the RV [25]. Furthermore, loss of the AP-dome at higher stimulation rates agrees with the occurrence of ST-segment elevation and spontaneous VT during exercise in patient BRU-006 (Fig. 1A). Moreover, patient BRU-006 had QRS broadening which worsened with exercise, suggesting that the HERG variant impacts on conduction velocity. The increase in peak IKr, which is enhanced at fast heart rates (Figs. 2 and 4Go), will oppose depolarization of the membrane potential (Fig. 2) caused by INa [14], thereby increasing the propensity to conduction slowing.

4.2 G873S and N985S are modifiers
The glycine at position 873 of the HERG channel is not conserved between human, mouse and rat HERG proteins and the nucleotide substitution leading to the G873S change was detected at a heterozygous frequency of 0.4% in Chinese control individuals (and in blacks at 0.3% [28]). G873S is therefore unlikely to constitute the primary genetic defect in patient BRU-044, but could nevertheless represent a potential modifier. However, the nucleotide change associated with the N985S substitution was not detected in 600 control chromosomes (of the appropriate ethnic origin) tested. Along with the fact that the asparagine at position 985 is conserved between human, mouse and rat HERG proteins, this could suggest that it represents the primary genetic defect underlying the disorder in patient BRU-006. However, since the electrophysiological defects associated with this change are qualitatively and quantitatively similar to the G873S variant, we hypothesize that this change also represents a sub-clinical defect, possibly a very rare variant that acts in conjunction with another genetic defect to cause the disorder. In further support of the notion that these gene variants exert modifying effects which act in concert with other genetic or environmental factors, rather than in isolation, our simulations indicate that the consequences of the G873S and N985S variants become only manifest in the presence of reduced net inward current (Fig. 6).

4.3 Limitations of the study
For the reasons discussed above, the HERG variants identified in this study are more likely to represent genetic modifiers rather than the "causal mutation" which is yet unknown in these patients. In the present study, biophysical properties of HERG variants were correlated with clinical data, using computer simulations. Computer simulation studies have improved our understanding of the ways in which various aspects of ion channel function (gating and expression) may alter cardiac excitability [29], but the properties of computer model currents are usually based on a mixture of membrane properties measured in various species and at various conditions (temperature, ion gradients). We used a recent ventricular cell model which is largely based on membrane current properties of human myocytes or expressed human genes [21]. We modified the model equations for the membrane current of interest, i.e., IKr, to ensure that its kinetics closely matched our HERG current data. Additionally, we simulated RV APs by scaling Ito and IKs densities. The scaling factors were obtained from canine left and right ventricular myocytes [23,24], because human data are lacking. Finally, we replaced the "basic calcium dynamics" of the model [21] by the more sophisticated description of the calcium dynamics of mammalian ventricular cells by Faber and Rudy [22], which was necessary to avoid numerical instabilities when simulating the RV epicardial myocyte.

4.4 Conclusions
G873S and N985S changes in HERG may contribute to BrS by increased peak IKr during phase-1 of the AP, resulting in heterogeneous loss of the epicardial AP-dome. Despite the increase in peak IKr, HERG-modulated BrS is probably best treated with conventional therapies, i.e., implantable cardioverter-defibrillators [30]. The effects of IKr blocking drugs, which have been reported in BrS management [31], are unpredictable in HERG-modulated BrS, because they have multiple actions. For instance, sotalol's IKr blocking actions would increase AP duration and, consequently, shorten the diastolic phase during which HERG currents deactivate, thereby increasing remaining peak IKr current. However, these effects may be counteracted by sotalol's heart rate slowing following its beta-blocking actions. Clearly, the identification of this genetic defect in additional BrS cases and families is required to further elucidate the significance of KCNH2 variants in this disorder.


   Acknowledgment
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
References
 
This study was supported by the Netherlands Heart Foundation grants 2000.059 (C.R.B. and A.A.M.W.) and 2002B191 (H.L.T.), the Netherlands Organization for Scientific Research grant 902-16-193 (A.A.M.W.), the Interuniversity Cardiology Institute of the Netherlands (project 27 (A.A.M.W.)), the Bekales Foundation (C.R.B. and H.L.T.), a Royal Netherlands Academy of Arts and Sciences fellowship (H.L.T.), the Deutsche Forschungsgemeinschaft (Schu1082/3-1 (E.S-B.)), the Ernst-and-Berta-Grimmke-Stiftung, Düsseldorf (E.S-B.), and the Leducq Foundation, Paris (E.S-B.).


   Notes
 
Time for primary review 24 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 

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