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Cardiovascular Research 2005 67(3):467-475; doi:10.1016/j.cardiores.2005.05.017
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Copyright © 2005, European Society of Cardiology

HERG mutation predicts short QT based on channel kinetics but causes long QT by heterotetrameric trafficking deficiency

Aimé D.C. Paulussena,*,1, Adam Raesb,1, Roselie J. Jongbloeda, Ron A.H.J. Gilissenc, Arthur A.M. Wilded, Dirk J. Snydersb, Hubert J.M. Smeetsa and Jeroen Aerssensc

aDepartment of Genetics and Cell Biology (CARIM/NUTRIM), University of Maastricht, P.O. Box 616 (#16), 6200 MD Maastricht, The Netherlands
bLaboratory for Molecular Biophysics, Physiology and Pharmacology, University of Antwerp (UIA), Belgium
cJohnson and Johnson Pharmaceutical Research and Development, Beerse, Belgium
dExperimental and Molecular Cardiology Group, Academic Medical Centre, Amsterdam, The Netherlands

* Corresponding author. Tel.: +31 43 3882982; fax: +31 43 3884573. Email address: aimee.paulussen{at}gen.unimaas.nl

Received 9 March 2005; revised 2 May 2005; accepted 18 May 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Mutations in the KCNH2 (hERG, human ether-à-go-go related gene) gene may cause a reduction of the delayed rectifier current IKr, thereby leading to the long QT syndrome (LQTS). The reduced IKr delays the repolarisation of cardiac cells and renders patients vulnerable to ventricular arrhythmias and sudden death. We identified a novel mutation in a LQTS family and investigated its functional consequences using molecular and microscopic techniques.

Methods and results: Genetic screening in the LQTS family revealed a heterozygous frameshift mutation p.Pro872fs located in the C-terminus of the KCNH2 gene. The mutation leads to a premature truncation of the C-terminus of the hERG protein. p.Pro872fs channels lack 282 amino acids at the C-terminus and possess an extra 4-amino acid tail. Both the kinetic and biochemical properties of the p.Pro872fs and p.Pro872fs/WT channels were studied in HEK293 cells and resulted in a novel proof of concept for heterozygous LQTS mutations: homotetrameric p.Pro872fs channels displayed near-normal expression, trafficking, and channel kinetics. Unexpectedly, upon co-expression of p.Pro872fs and WT channels, the repolarising power (the proportion of hERG current contributing to the action potential as the percentage of the total current available) was substantially higher during action potential clamp experiments as compared to WT channels alone. This would lead to a shorter rather than a prolonged QT interval. However, at the same time, heterotetramerisation of p.Pro872fs and WT channels also caused a dominant negative effect on trafficking by an increase in ER retention of these heterotetrameric channels, which surpassed the former gain in repolarising power.

Conclusion: The LQTS phenotype in the studied family is caused by a mutation with novel properties. We demonstrate that a KCNH2 mutation that clinically leads to long QT syndrome causes at the cellular level both a "gain" and a "loss" of HERG channel function due to a kinetic increase in repolarising power and a decrease in trafficking efficiency of heteromultimeric channels.

KEYWORDS Arrhythmia; Congenital defects; Ion channels; Long QT syndrome


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
The long QT syndrome (LQTS) is an inherited cardiac arrhythmia that usually becomes apparent through symptoms like syncope, loss of consciousness and sudden death [1]. The clinical manifestation of the disease shows itself by prolongation of the QT interval on the surface ECG, which is indicative for aberrant lengthening of the cardiac action potential [2]. Mutations in seven genes, namely KCNQ1 (KvLQT1, LQT1), KCNH2 (hERG, LQT2), SCN5A (LQT3), ANKB (LQT4), KCNE1 (MinK, LQT5), KCNE2 (MiRP1, LQT6) and KCNJ2 (LQT7) have been identified to cause LQTS or related syndromes [3–9].

The KCNH2 gene encodes the {alpha}-subunit of the rapidly activating, delayed rectifier potassium ion channel IKr, an important determinant of the action potential duration [10,11]. Functional studies performed by various groups have indicated that mutations in KCNH2 may result in reduced outward potassium current due to various mechanisms. These mechanisms have recently been classified by Delisle et al. [12] in accordance with other inherited ion channel diseases. The proportion of nonsense and/or frameshift mutations detected in LQT2 families is currently around 30% (nonsense: 12%, frameshifts: 18%, http://pc4.fsm.it:81/cardmoc), yet very few of these have been functionally characterised [13,14]. To a degree this is understandable, as many mutations cause deletions of functionally important transmembrane or pore regions, precluding measurable channel function. However, also several mutations creating premature stopcodons in the C-terminus of the hERG protein have been reported and non-functionality of these proteins may not be obvious. In addition, the common view that a truncated protein, if able to coassemble with wild type (WT), will cause merely a loss of function, might not always be true, as we demonstrate in this study. We investigated the underlying mechanisms of a truncating frameshift mutation that causes long QT syndrome in a Dutch LQTS family.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. LQTS family
A pedigree of the Dutch family of Caucasian origin with diagnosed long QT syndrome is presented in Fig. 1. Based on the clinical symptoms of the proband, other family members were initially evaluated by 12 lead ECG registrations. Informed consent for participation in this study was obtained from 23 family members (14 females and 9 males). The investigation conformed with the principles outlined in the Declaration of Helsinki [15].


Figure 1
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  		Fig. 1 Long QT syndrome family pedigree of p.Pro872fs mutation carriers. The arrow indicates the proband. Circles indicate females; squares indicate males; symbols with a slash represent deceased family members. QTc-intervals (in ms) are indicated below the symbols, where available.

 
2.2. Genetic analysis
Genomic DNA was extracted from peripheral blood lymphocytes using standard techniques. All exons, encompassing the entire coding region of the KCNH2 gene, were amplified by PCR and mutation screening was performed using either direct sequencing or DHPLC analysis as described previously [16]. Aberrant DHPLC elution profiles were subsequently analysed by sequencing. After identification of the causative mutation in the proband, additional mutation carriers were identified in the LQTS families by direct sequencing (p.Pro872fs). Involvement of mutations in other LQTS genes (KCNQ1, KCNE1, KCNE2 and SCN5A) or compound heterozygosity was excluded (data not shown).

2.3. Site-directed mutagenesis and KCNH2 constructs
The wild type (WT) cDNA hERG/pcDNA3 expression construct was kindly donated by Dr. Craig January and Dr. Zhengfeng Zhou (University of Wisconsin-Madison). The p.Pro872fs mutation was generated by site-directed mutagenesis as described previously [17]. Briefly, primer 5'-CGT-ACT-GCC-GG-GGA-GCC-C-3' (p.Pro872fs) was used to create the mutant construct (mutation underlined). Wild type (GFP-WT) and p.Pro872fs (GFP-p.Pro872fs) hERG cDNAs were also subcloned downstream of green fluorescent protein (GFP) in pcDNA3.1_NT_GFP. The ATG start codon of the HERG cDNA was 24 bp downstream of the final GFP codon.

2.4. Cell culture and transfection
HEK293 cells were cultured at 37 °C as described previously [17] and transiently transfected with wild type (WT) or p.Pro872fs constructs. Heterozygous carrier status was mimicked by transient cotransfections as well as by transfections of the p.Pro872fs construct on a stable WT hERG cell line. The amount of mutant construct transfected was chosen as such that if it were transfected alone, it would yield the same amount of current as the stable WT cell line. The procedure of transfecting on a stable cell line was applied to minimize the variation caused by the cotransfections as it is often observed that not both constructs are taken up in equal amounts by the cell. The transfection on the stable cell line has the advantage that cells positive for transfection are certainly heterozygous.

2.5. Voltage clamp and data analysis
Potassium current recordings were made with an Axopatch-200B amplifier in the whole cell configuration using suction pipettes as described previously [18]. All current recordings were performed at room temperature (20–22 °C). Solutions were identical as described previously [19]. The access resistance varied between 2.5 and 8 M{Omega}. Series resistance was compensated to ensure that voltage errors were <5 mV. No leak subtraction was applied and the holding potential was –80 mV. HEK293 cells displayed a small-amplitude of endogenous current during depolarising steps, which was not present during the recordings of the tail current [18]. The voltage dependence of channel activation was determined by fitting a Boltzmann equation to the maximal tail currents. Time constants were obtained by curve fitting with an exponential function using a non-linear least-squares (Gauss–Newton) algorithm. The inactivation curve was obtained as described by Zou et al. [20] but was also obtained with the recovery pulse adapted to 3 times the recovery time constant (see pulse protocol in Fig. 4B). The action potential waveform was obtained from a recording of an action potential of guinea-pig ventricular myocyte. Data are presented as mean ± SEM, the Student's t-test was used for statistical analysis and n represents the number of cells.


Figure 4
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  		Fig. 4 Voltage dependence of inactivation. The voltage dependence of inactivation was determined as in Zou et al. [19] and by a modified protocol (inside panel B). (A) The inactivation curves were obtained with a protocol as shown in the inset. The midpoint of inactivation amounted –70.4 ± 1.6 mV with a slope factor of 16.0 ± 0.4 mV (n = 9) for WT, –57.3 ± 3.3 mV with a slope factor of 20.8 ± 2.3 mV (n = 6) for p.Pro872fs, –58.5 ± 3.6 mV with a slope factor of 19.1 ± 1.4 mV (n = 5) for WT+p.Pro872fs. (B) Inactivation curves for WT, p.Pro872fs and WT/p.Pro872fs heterotetrameric channels with adapted protocol as depicted in the inset. (C) The inactivation time constants were obtained with a voltage protocol as shown in the inset. After a prepulse to +50 mV, a short pulse to –100 mV recovered the inactivated channels. A subsequent third pulse to various voltages allowed determining the inactivation time constant by fitting an exponential function to the decaying currents during the final pulse.

 
2.6. Confocal imaging
HEK293 cells were cultivated on cover slips as described [17]. HEK293 cells were transiently transfected with GFP-WT or GFP-p.Pro872fs constructs. A red fluorescent endoplasmic reticulum (DsRed-ER) marker was cotransfected to distinguish ER retained from membrane bound proteins as described previously [19]. To investigate potential dominant negative effects on protein trafficking, equal amounts of WT and p.Pro872fs constructs were transiently cotransfected, with either one of the constructs tagged with GFP. Confocal images were obtained 48 h after transfection on a Zeiss CLSM 510, equipped with an argon laser for the visualisation of GFP and DsRed tagged proteins.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Clinical characteristics
Of the 23 available members (Fig. 1), 9 individuals presented with either symptoms and/or aberrant ECGs. Subject III:9 (proband) experienced syncopes immediately after giving birth to her first child. Initially torsades de pointes were registered in the ambulance, which later deteriorated into cardiac arrest. The patient was successfully resuscitated and an ICD was implanted. Subject III:3 experienced several syncopes during menstruations. Beta-blocker therapy was initiated in all mutation carriers after genetic diagnosis.

3.2. Genetic analysis
The proband of described LQTS family was carrier of a deletion of a single nucleotide (2616delC) in exon 11 (subject III:9). This single base pair deletion introduces a frame shift that leads to a premature stop codon at amino acid position 877 (p.Pro872fs). The mutation is located downstream of the cNBD and causes a truncation of the hERG protein of 282 amino acids and the addition of an aberrant translated tail of four amino acids. Genetic screening of 23 members identified 11 mutation carriers who were heterozygous for this mutation (Fig. 1).

3.3. Current recordings of WT and p.Pro872fs channels
Representative current recordings of homotetrameric hERG WT and p.Pro872fs channels are shown in Fig. 2. p.Pro872fs mutant channels produced currents comparable to WT hERG (Fig. 2B), although with reduced current amplitudes (Fig. 2C). Steady-state I–V plots (Fig. 2D) or steady state activation curves (Fig. 2F) indicated no significant differences between WT and p.Pro872fs homotetrameric channels. Heterozygous carrier status was mimicked by transient cotransfections and by transfections of the p.Pro872fs construct on a stable WT hERG cell line. No significant differences in the voltage dependence of activation were observed. However, by cotransfection of equal amounts of cDNA on WT cells a significant decrease in current densities of heterotetrameric channels was detected (Fig. 2E) as well as an increase in the level of the steady state current compared to the tail current amplitude (Fig. 2B, C and the inset of Fig. 3). The steady-state current level was normalized to the peak tail amplitude at –40 mV to quantify this behaviour (Fig. 3A). The steady state current level at +20 mV represented ~40% of the peak tail amplitude for WT channels as compared to ~60% for homotetrameric p.Pro872fs or heterotetrameric WT/p.Pro872fs channels. Fig. 3B shows the voltage dependence of the time constants for activation and deactivation. Heterotetrameric channels showed a small but significant speeding of the activation at moderate voltages and more pronounced speeding of deactivation was observed at negative voltages. The midpoint of inactivation for both homo-and heterotetrameric p.Pro872fs channels was shifted about 14 mV towards positive voltages compared to WT (Fig. 4A). The procedure to determine the inactivation curve used a second voltage step of fixed duration, which may affect the shape of the inactivation curve because of the different recovery time constants. To correct for this we used an alternate method in which the duration of second step was set to 3 times the recovery time constant as obtained in Fig. 5. The shift disappeared after adaptation of the protocol (Fig. 4B). Homotetrameric p.Pro872fs channels inactivated slightly faster than WT or heterotetrameric hERG channels (Fig. 4C). Homotetrameric p.Pro872fs channels also recovered faster from inactivation than WT channels but surprisingly heterotetrameric channels recovered even faster than p.Pro872fs homotetramers (Fig. 5). Action potential clamp experiments were performed to determine the importance of these subtle changes in kinetics with protocols as previously described (Fig. 6A) [21]. The time course of the current during the action potential displayed no major differences between channels (Fig. 6B). However, when the raw current traces were normalized to the total current available, obtained from the peak tail current amplitude from voltage clamp experiments, a pronounced difference was observed. The ratios of the peak current of the action potential to the peak tail current from voltage clamp experiments were 32 ± 6% for WT, 21 ± 8% for p.Pro872fs and 67 ± 10% for heterotetrameric channels. This calculation was performed for the whole action potential (Fig. 6C) resulting in a total amount of hERG current activation of about 30% for both homotetrameric channels compared to almost 70% for heterotetrameric channels.


Figure 2
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  		Fig. 2 Voltage clamp recordings of hERG WT and p.Pro872fs channels. (A) Voltage-clamp protocol, representative current recordings from transiently transfected cells with WT (B), and p.Pro872fs constructs (C). (D) Steady state amplitude I–V plots for HERG WT and p.Pro872fs channels. I–V plots were determined by plotting the current level at the end of the 5 s depolarising step versus the command voltage. (E) Current densities obtained from a triple pulse protocol as shown in the inset of Fig. 4C, by transfection of 2.5 µg WT or p.Pro872fs constructs and cotransfection of 1.25 µg WT and 1.25 µg P872fs constructs. *Difference in densities between WT and WT/p.Pro872fs co-transfected cells was significant, p<0.001. (F) Voltage dependence of steady-state activation. Relative tail current amplitudes were derived from the currents as shown in (B) and (C), and were fitted with a Boltzmann function to determine midpoint of activation (V1/2) and slope factor (k). HERG WT (n = 7): V1/2=–3.01 ± 1.5 mV, k = 8.09 ± 0.34 mV, HERG p.Pro872fs (n = 8): V1/2=–0.03 ± 2.22 mV, k = 8.13 ± 0.17 mV, HERG WT/p.Pro872fs (n = 12): V1/2=1.88 ± 0.57 mV, k = 7.65 ± 0.07 mV. n is the number of cells.

 

Figure 3
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  		Fig. 3 Inward rectification and time constants of activation/deactivation. (A) The inward rectification was determined by normalizing the steady-state current amplitude at the end of the voltage step by the peak tail amplitude at –40 mV (n>5, currents of WT/p.Pro872fs heterotetrameric channels are shown in the inset. (B) The time constants of activation were determined from current traces as shown in Fig. 3 by fitting a single exponential function to the activating currents between –20 and 60 mV. The slow and fast component of current deactivation were determined by fitting a double exponential function to deactivating tail currents (obtained with a protocol as shown in the inset).

 

Figure 5
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  		Fig. 5 Time constants of recovery from inactivation. The recovery of inactivation was obtained from the first 50 ms after the prepulse (protocol shown in the inset of Fig. 4B) from current traces as shown in the inset. Time constants of recovery from inactivation were obtained by fitting the recovery of the current with an exponential function.

 

Figure 6
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  		Fig. 6 Action potential waveform kinetics. (A) Action potential waveform pulse protocol and the raw current tracings for WT channels. (B) Normalized currents (current traces divided by the maximal amplitude obtained during the action potential, Imax, AP), n>5 for WT, p.Pro872fs and WT/p.Pro872fs channels. (C) Normalization of the current during the action potential to the maximal tail current amplitude during voltage clamp experiments (Imax tail, VC). The curves were obtained by multiplying the curves of panel B with the ratio of the maximal current amplitude recorded during the action potential and the total amplitude of the tail current recorded by voltage clamp.

 
3.4. Confocal imaging
Confocal images showed the presence of WT protein in the plasma membrane (Fig. 7A), whilst the red fluorescence was situated purely in the ER (Fig. 7B). Homotetrameric p.Pro872fs protein was present within the plasma membrane (Fig. 7E–F), although small portions accumulated in clusters (green dots) in the ER. Upon co-expression of equal amounts of WT and tagged p.Pro872fs constructs (Fig. 7G–H) or tagged WT and p.Pro872fs constructs (Fig. 7I–J) retainment of heterotetramers was prominent.


Figure 7
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  		Fig. 7 Subcellular distribution of WT and p.Pro872fs channels. Confocal imaging of GFP tagged WT and mutant channels in HEK293 cells. Cells were transfected with GFP tagged constructs in combination with a DsRed tagged, ER-targeted construct. Upper panels, GFP fluorescence images of GFP-WT (A), GFP-p.Pro872fs (C), GFP-p.Pro872fs+WT (E) and GFP-WT+p.Pro872fs (G) channels. Lower panels, super-imposed images of double stained (green GFP and red DsRed) transfected cells. Note that the WT and p.Pro872fs channels show normal membrane staining but the heterotetramers do not, irrespective of the location of the GFP tag on the WT or the mutant subunit.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
In this study we investigated the pathophysiological consequences of a novel frameshift mutation p.Pro872fs that was detected in a Dutch LQTS family. The mutation leads to a premature stop codon at amino acid 877 (p.Pro872fs) with a non-hERG tail of four amino-acids (AVRS in stead of GSTE). Nonsense mutations in LQTS genes leading to truncated proteins have been reported frequently [16], but functional characterization has been reported only for few of them [13,14,22–24]. Many of these mutations lead to deletions of important regions of the hERG protein, such as the pore region or parts of the transmembrane domains, and may therefore appear of less interest for further studies as these proteins are expected to be non-functional.

p.Pro872fs homotetrameric channels produced WT-like currents. These findings were supported by confocal microscopy results, which showed that these channels were visible at the level of the cell membrane, albeit with some clustering of the proteins in the endoplasmic reticulum (ER). Functionality of these truncated homomultimeric channels is in agreement with a study by Aydar and Palmer [25], who demonstrated that at least the first 881 amino acids were required for channel function as a deletion of 278 amino acids resulted in functional channels while deletions of 311 and 344 amino acids resulted in non-functional channels. Mutant R1014X channels with a deletion of 146 amino acids, recently reported by Gong et al., [24] also showed WT-hERG like currents also with reduced amplitudes. The p.Pro872fs protein has a deletion of 282 amino acids (total length hERG protein 1159 amino-acids) and therefore even shorter hERG proteins than reported by Aydar and Palmer can form functional channels. Aydar and Palmer further observed accelerated deactivation rates for protein truncations larger than 236 amino acids, which was also observed for the p.Pro872fs channels. Recently, another frameshift KCNH2 mutation p.Pro1122fs, detected in a Japanese family, was functionally characterized. Despite the additional 147 falsely translated amino acids, p.Pro1122fs homotetramers were also able to form functional channels, with no changes to activation and deactivation, but with accelerated inactivation kinetics as compared to WT channels. Also these channels showed reduced current densities, but no dominant negative effects were observed with co-expression of WT and p.Pro1122fs channels [26].

Because all the LQTS patients of the family with the p.Pro872fs mutation were heterozygous mutation carriers (Romano–Ward LQTS), co-expression experiments were performed to study the effects of tetramer formation. Co-expression of WT and p.Pro872fs channels produced unexpected results, as the typical tail currents were reduced as compared to the steady state level indicating that heterotetramers were kinetically different from WT and p.Pro872fs homotetramers which was indeed the case. The speed of inactivation for heterotetrameric channels was comparable to WT, but coincided with a faster activation at voltages from 0 to –30 mV and a faster recovery from inactivation compared to homotetrameric WT and p.Pro872fs channels. These small but significant differences in kinetics were evaluated by action potential clamp experiments. Interestingly, the repolarising power of hERG current during the action potential mounted much higher for heterotetrameric channels (~70%) than for homotetrameric WT or p.Pro872fs channels (~30%). This difference can be explained by the observed altered inactivation and recovery from inactivation kinetics. Overall, the repolarising power was markedly increased for heterotetrameric channels compared to WT and p.Pro872fs homotetrameric channels. As patients carry both a WT and mutated allele, the subunits composition is likely heterotetrameric and the results therefore suggest increased hERG current amplitude during the action potential. As the IKr current regulates the duration of the plateau phase and thus contributes to the duration of the action potential [11,27] the biophysical properties of WT/p.Pro872fs heterotetrameric channels would be expected to result in a shorter rather than a longer QT interval. However, upon co-expression of WT and p.Pro872fs subunits, retainment of hERG heterotetrameric channels was increased drastically, indicating a huge dominant negative effect of subunit co-assembly on trafficking efficiency. This dominant negative effect on trafficking was also observed for the R1014X/WT heterotetramers [24], with the important distinction that these heterotetramers did not show an increased repolarising power and an expected short QT interval.

The dominant negative effect on trafficking may be explained by C-terminal elements important for trafficking ability such as the RGR retention signal [12,28,29]. In the case of WT channels the signal could be masked by the tetramerisation while for homotetrameric mutant channels it is absent since it is located in the C-terminal truncated part. In the case of heterotetrameric channels, we hypothesize that the retention signal of the WT subunits is not masked by the mutant subunits. Although tetramerisation seemed to effect current repolarising power in a positive way, the effect on trafficking was dominant negative. The resulting decrease in numbers of channels inserted in the cell membrane exceeded the positive effect on channel repolarising power during the action potential.

Trafficking deficiency could be rescued by specific drug treatment as earlier studies have shown that misfolding and subsequent transport of mutated subunits could be corrected for some KCNH2 mutations [17,30–32]. As these drugs at the same time also block the HERG channel and thereby could cause drug-induced LQTS, treatment seemed not obvious, although a later study by Delisle et al. showed that the drug thapsigargin could selectively rescue trafficking of mutant G601S and F805C channels without blocking them [33]. In the case of our studied mutation p.Pro872fs one may question which treatment, if any, could be a solution. Rescuing of the WT/p.Pro872fs channels without channel blockade could lead to a final "gain in repolarising power" during the action potential, maybe leading to Short QT Syndrome as described for KCNH2 mutation N558K [34].

In conclusion, the biological pathology for the LQTS phenotype in the examined family is the result of novel counteracting properties of heterotetrameric WT/p.Pro872fs channels; a "loss" of function caused by retention of the heterotetramers superseded the "gain" in repolarising power of these heterotetramers during the action potential. To our knowledge this is the first dominant Romano–Ward KCNH2 mutation with both "gain" and "loss" of function properties and provides a novel proof of concept for heteromultimeric channels.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We thank Ann Mariën, Wim Keysers and Evy Mayeur for their excellent technical support and we thank Jean-Pierre Timmermans for the use of the confocal microscope. This work was supported by the Johnson and Johnson Pharmaceutical Research and Development, Flanders Institute for Biotechnology Grant PRJ05, National Institutes of Health/National Heart, Lung, and Blood Institute Grant HL59689 and the Netherlands Heart Foundation (NHS grant 95.014).


   Notes
 
1 Both authors contributed equally to this work. Back

Time for primary review 20 days


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

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