Published online before print February 4, 2008, doi: 10.1161/CIRCULATIONAHA.107.752220
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(Circulation. 2008;117:866-875.)
© 2008 American Heart Association, Inc.
Arrhythmia/Electrophysiology |
Deficient Zebrafish Ether-à-Go-Go–Related Gene Channel Gating Causes Short-QT Syndrome in Zebrafish Reggae Mutants
From the Department of Internal Medicine III (D.H., E.P.S., N.T., S.J., B.M., E.Z., S.M., B.V., C.A.K., H.A.K., W.R.), University Hospital Heidelberg, Heidelberg, Germany; Department of Physiology and Pathophysiology (O.F.), University of Heidelberg, Heidelberg, Germany; Institute of Biomedical Engineering (D.L.W., G.S.), University of Karlsruhe, Karlsruhe, Germany; and Cardiovascular Research Center (M.C.F.), Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Mass.
Correspondence to Wolfgang Rottbauer, Department of Internal Medicine III, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. E-mail wolfgang.rottbauer{at}med.uni-heidelberg.de
Received May 23, 2007; accepted December 7, 2007.
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Abstract |
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Background— Genetic predisposition is believed to be responsible for most clinically significant arrhythmias; however, suitable genetic animal models to study disease mechanisms and evaluate new treatment strategies are largely lacking.
Methods and Results— In search of suitable arrhythmia models, we isolated the zebrafish mutation reggae (reg), which displays clinical features of the malignant human short-QT syndrome such as accelerated cardiac repolarization accompanied by cardiac fibrillation. By positional cloning, we identified the reg mutation that resides within the voltage sensor of the zebrafish ether-à-go-go–related gene (zERG) potassium channel. The mutation causes premature zERG channel activation and defective inactivation, which results in shortened action potential duration and accelerated cardiac repolarization. Genetic and pharmacological inhibition of zERG rescues recessive reg mutant embryos, which confirms the gain-of-function effect of the reg mutation on zERG channel function in vivo. Accordingly, QT intervals in ECGs from heterozygous and homozygous reg mutant adult zebrafish are considerably shorter than in wild-type zebrafish.
Conclusions— With its molecular and pathophysiological concordance to the human arrhythmia syndrome, zebrafish reg represents the first animal model for human short-QT syndrome.
Key Words: arrhythmia • fibrillation • genetics • ion channels
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Introduction |
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Over the last decade, it has been found that the propensity to human cardiac arrhythmias is often inherited and caused by gene mutations in various ion channels that control cardiac depolarization and repolarization; however, detailed characterization of the underlying pathophysiological mechanisms is often hindered by the lack of genetic animal models that exhibit action potential characteristics comparable to human cardiomyocytes and an arrhythmia phenotype that corresponds to those in humans with specific mutations. For instance, murine hearts beat 7 to 10 times faster than the human heart. In mice, cardiac repolarization, therefore, is driven primarily by the rapidly activating Ito current and not, as in humans, by delayed currents such as IKr.1
Clinical Perspective p 875
Human short-QT syndrome (SQTS) is a recently described hereditary ion channelopathy that is a cause of sudden cardiac death.2,3 SQTS is associated with a high risk of arrhythmic events, particularly atrial and ventricular fibrillation, due to a pathological shortening of action potential duration and refractoriness.4 To date, distinct gain-of-function mutations in 3 major repolarizing potassium channels, namely, hERG (KCNH2), KvLQT1 (KCNQ1), and Kir2.1 (KCNJ2), have been linked to SQTS.4 Pharmacological treatment options are still a matter of debate because a valid in vivo animal model has been lacking.4,5
Here, we present zebrafish mutant reggae (reg) as the first animal model of congenital SQTS. Zebrafish reg displays a distinct phenotype of intermittent atrial fibrillation and accelerated cardiomyocyte repolarization (QT shortening) reminiscent of human SQTS. By positional cloning, we find that reg mutants carry a missense mutation (L499P) in the voltage sensor of the zebrafish ether-à-go-go–related gene (zERG) potassium channel. Genetic and pharmacological inhibition of zERG rescues recessive reg mutant embryos, which implies a gain-of-function effect of the reg mutation on zERG channel function. Electrophysiologically, the reg mutation mainly impedes channel inactivation, thereby functionally increasing repolarizing currents, shortening action potential duration and refractoriness. Accordingly, QT intervals are found to be notably shortened both in heterozygous and in homozygous reg mutant adult zebrafish. With its molecular and pathophysiological concordance to human SQTS type 1 (SQT1), zebrafish reg may prove to be a valuable model to investigate disease mechanisms genetically and to test new pharmacological treatment options in high-throughput screens.
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Histology, Immunostaining, Calcium Imaging, and Electrical Stimulation
For histology, embryos were fixed in 4% paraformaldehyde and embedded in JB-4 (Polysciences, Inc, Warrington, Pa). Five-micrometer sections were cut, dried, and stained with hematoxylin/eosin. For whole-mount immunostaining, zebrafish embryos (72 hours after fertilization [hpf]) were fixed in Dent’s fixative. Monoclonal antibodies MF20 and S46 were used.6 Calcium imaging was performed as described previously.7 Electrical stimulation was performed essentially as described previously8 with a pulsar stimulator (HSE Stimulator P, Hugo Sachs Elektronik, March-Hugstetten, Germany).
Genetic Mapping, Positional Cloning, Mutation Detection, and Generation of Expression Clones
DNA from 24 reg mutant and 24 wild-type embryos was pooled, and bulked segregation analysis was performed as described.9 The genotyping of 1403 mutant embryos for polymorphic markers in the area defined the critical genomic interval for reg. RNA from reg mutant and wild-type embryos, which was isolated with TRIZOL reagent (Life Technologies/Invitrogen, Carlsbad, Calif) and reverse-transcribed. Polymerase chain reaction pools from 3 independent reverse-transcribed polymerase chain reactions from mutant and wild-type reg complementary DNA were sequenced. Genomic DNA from reg mutant and wild-type embryos was sequenced around the point mutation. Full-length wild-type and reg mutant zkcnh2 sequence was cloned into pDESTCS2+ for sequencing and subsequent RNA synthesis with the mMessage mMachine Kit (Ambion, Austin, Tex).
Injections and Terfenadine Treatment
Two different morpholino-modified oligonucleotides were directed against either the translational start site (5'-GCGGCGCACGGGC-ATTTTTCACGCG-3') or the intron 7 splice donor site (5'-GGAAAGCCTCACCTCATCT GAACCG-3') of zERG. A standard control morpholino antisense oligonucleotide (MO control; Gene Tools, LLC, Philomath, Ore) was injected at the same concentrations.
A 1-mmol/L terfenadine (Sigma-Aldrich, St Louis, Mo) stock solution in DMSO was diluted to the desired final concentrations in embryo medium. Final DMSO concentration did not exceed 0.1%. Embryos (72-hpf reg–/– embryos) were chosen phenotypically, bathed for 
1 hour in terfenadine, and subsequently phenotypically scored after a 1- to 2-minute observation period.
Voltage-Clamp Experiments
Double-electrode voltage-clamp experiments in Xenopus laevis oocytes were performed in a K+ solution (in mmol/L: 5 KCl, 100 NaCl, 1.5 CaCl2, 2 MgCl2, and 10 HEPES; pH adjusted to 7.4 with NaOH) at room temperature. Microelectrodes had tip resistances that ranged from 1 to 5 M
. Data digitalization and acquisition were performed as described previously.10 The human ether-à-go-go–related gene (hERG) clone (GenBank accession No. u04270) was a gift from M.T. Keating (Boston, Mass). Preparation and injection of hERG complementary RNA, as well as site-directed mutagenesis, were performed as described previously.10 The investigation conformed to the "Guide for the Care and Use of Laboratory Animals" published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). hERG activation curves were fitted to a Boltzmann function: y={1+exp[(V1/2–V)/k]}–1, with V1/2 representing the half-maximal activation potential, y the degree of activation, and k the slope factor. Statistical data are expressed as mean±SEM, with n representing the number of experiments performed.
Electrophysiological Modeling
An electrophysiological model of a human left ventricular midmyocardial myocyte11 was used to reconstruct cellular electrophysiological properties by mathematical equations, as described previously.12 Simulations with the cell models were performed with the Euler method for numeric integration with a time step of 10 µs. Results after the 100th stimulation were analyzed for each simulation. For the adaptation of the hERG model to the measured data, an iterative Powell method was implemented that used the root-mean-square error to find the optimal fit for measured and simulated data. First, the "physiological" equations were fitted to the wild-type measurements with consideration of temperature and expression density changes. Then, the mutant data were fitted, and the previous result was used to reconstruct the mutant behavior by readjusting the temperature and channel density. Eelectrophysiological modeling was performed for a myocyte being homozygous for the reg mutation.
Compound Action Potential Recordings
In vivo compound action potential recordings (cAPs) were performed with the potentiometric mode of a GeneClamp 500 2-microelectrode voltage-clamp amplifier (Axon Instruments, Foster City, Calif) in Ringer solution containing (in mmol/L) 5 KCl, 140 NaCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4), with temperature adjusted to 31°C to guarantee a sufficient heart rate in mutant zebrafish larvae. Microelectrode preparation was performed as described previously.13 After the zebrafish were paralyzed by titration of tricaine (Sigma), the sharp microelectrode was impaled in the pericardium and driven toward the heart until potential changes synchronous with the ventricular contraction were observed. Potential traces were recorded as described previously.13 Because the extracellular location of the microelectrode reflects potential changes that originate from the superposed activity of many excitable cells, they are referred to as cAPs. Repolarization was quantified as follows: From each recording, 3 consecutive cAPs were chosen at random, analyzed manually, and averaged. Time intervals from onset of depolarization until 30% (cAPD30), 70% (cAPD70), and 90% (cAPD90) of repolarization were measured and corrected for heart rate with the Bazett formula. Whereas wild-type zebrafish displayed a fast heart rate (235.3±15.5 bpm at 31°C), mean heart rate was slow in reg mutants (82.8±28.5 bpm; n=6).
ECG Recordings
Wild-type and mutant (m230+/– and m230–/–) 9-month–old adult zebrafish were anesthetized by titration of tricaine solution (Sigma) for 2 minutes. Cisatracurium (0.01 mL, 0.2 mg/mL; GlaxoSmithKline, London, United Kingdom) was injected into the intraperitoneal cavity of the anesthetized zebrafish to paralyze both the skeletal and the gill musculature, and the zebrafish were placed sidelong into a wettish recording chamber. Noninvasive ECG recordings were performed with small-plane metal electrodes (4-mm diameter) that were placed laterally on both sides of the zebrafish gills. All experiments were performed at room temperature. The ECG was amplified with a custom-built differential amplifier and digitized in 40-second sweeps at a sampling rate of 500 Hz with a Digidata 1200 interface and pCLAMP6 software (Axon Instruments, Union City, Calif). Original ECG traces were notch-filtered (center frequency 55 Hz), baseline-corrected, and analyzed with Clampfit9.0 software (Axon Instruments). QT measurements were performed according to Goldenberg et al.14 Briefly, the QT interval was defined as the time between the initial deflection of the QRS complex and the end of the T wave. Heart rate correction was performed with the Bazett formula with the R-R cycle length of the preceding heart beat. All ECG recordings were blinded and were analyzed by an expert in the field. Genotypes of the analyzed fish were reconfirmed by tail clipping and subsequent genotyping. The study protocol was approved by the responsible advisory board. All experiments conformed to the "Guide for the Care and Use of Laboratory Animals" (NIH publication No. 85-23, revised 1996).
Statistical Analysis
To evaluate the association of terfenadine concentration and rescue capability, we applied a logistic regression model using a dichotomized data set (reg phenotype/no reg phenotype). An estimated probability value of <0.05 was considered to be statistically significant. Analyses were performed with the SAS Statistics & Data Analysis Software package (SAS, Cary, NC). For the comparison of cAPs between wild-type and m230–/– larvae, statistical significance was evaluated with a 1-sided unpaired Student t test. Differences were considered significant if the probability value was <0.05 and highly significant if the probability value was <0.01. For ECG recordings, statistical comparison among groups was performed by ANOVA and a post hoc analysis with Bonferroni correction. Both the Student t test and ANOVA were performed with Origin software (OriginLabs, Northampton, Mass).
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Zebrafish Reg Mutants Display Sinuatrial Block and Atrial Fibrillation
We isolated the zebrafish mutation reg in a large-scale ethylnitrosourea (ENU)-mutagenesis screen for recessive lethal mutations that affect cardiac function.15 Reg mutant embryos display intermittent cardiac arrest accompanied by cessation of blood circulation (Movie I in the online-only Data Supplement). Besides their cardiac phenotype, reg mutant embryos are not noticeably affected, and, similar to other zebrafish mutants with impaired circulation, they usually die 6 to 7 days after fertilization.16 Rarely, homozygous reg mutant embryos survive to adulthood, become fertile, and, when intercrossed, produce 100% reg mutant offspring.
Key steps of early zebrafish heart development, such as heart jogging, heart looping, chamber demarcation, and maturation of chamber myocardium, proceed normally in homozygous reg mutant embryos (Figure 1A through 1F). By 72 hpf, reg atrial and ventricular chambers are well defined, separated by the atrioventricular ring, and display proper endocardial and myocardial layers. By that time, the ventricle has looped to the right and displays multilayered myocardium, which indicates proper growth by addition of myocardial cells (Figure 1C and 1D). Atrial and ventricular cardiomyocytes appear rather terminally differentiated, expressing myosin heavy chains in the usual heart-chamber–specific fashion (Figure 1E and 1F).
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During normal zebrafish development, cardiac contractions begin at 24 hpf as a peristaltic wave initiated at the sinus venosus and propagated toward the future bulbus arteriosus. Sequential atrial and ventricular contractions become evident by 36 hpf. By contrast, reg heart tubes appear rather silent. Only very rarely a contraction wave is initiated and then properly transmitted through the reg heart tube. As revealed by monitoring of intracellular calcium levels (calcium transients), cardiac electrical impulse generation normally arises from sinus venosus tissue. In reg mutant hearts, periodic rhythmic contractions of a small patch of cardiomyocytes can be seen in the sinus venosus tissue, beating at a rate of 128±5 bpm. Most of the time, this impulse is not propagated to surrounding atrial cells, and neither the atrium nor the ventricle contracts. This type of arrhythmia resembles human sinus node exit block. No escape rhythm is evident (online-only Data Supplement Movie II).
Phases of sinus exit block are frequently interrupted by sporadic, uncoordinated electrical and contractile activity of atrial cells that resembles atrial fibrillation. This phenotype is similar to that observed in other zebrafish mutants such as island beat8 and tremblor7,17 (online-only Data Supplement Movie II). This form of atrial electrical activity is not sufficient to excite the ventricle. Rarely, periods occur of regular sequential atrial and ventricular contractions of reg mutant hearts. In these phases, no atrioventricular block can be observed (online-only Data Supplement Movies I and II). An overview of the various types of arrhythmias, their relative frequency, and their duration is summarized in Figure 1H for 10 different 72-hour–old reg mutant embryos, which were observed by video light microscopy for 10 minutes (Figure 1H and 1I). Patterns of occurrence and duration of the various arrhythmias were different for each reg mutant embryo.
Reg Mutant Atrial and Ventricular Cardiomyocytes Are Not Electromechanically Uncoupled
To further define the electrophysiological alterations of reg myocardium, we performed a set of electrophysiological experiments in reg mutants in vivo. To visualize the cardiac electrical wave front in stages of regular cardiac activity, as well as in stages of constrained cardiac activity, we injected the calcium indicator Calcium Green dextran into 1-cell–stage reg mutant zebrafish embryos. Usually, in wild-type embryos, in synchronization with the heart beat, a cytosolic calcium signal, which starts at the inflow tract of the atrium, is propagated throughout the atrium and ventricle, accompanied by immediate sequential contraction of first the atrium and then the ventricle (Figure 1G). By contrast, during phases of cardiac mechanical arrest, calcium waves are absent in reg mutant hearts, which indicates that atrial and ventricular cardiomyocytes are not properly electrically excited and therefore do not contract (Figure 1G). During phases of atrial fibrillation, similar to zebrafish tremblor7,17 mutants, subtle changes in intracellular calcium can be observed in the fibrillating atrial cardiomyocytes, whereas again, no coordinate calcium wave traversing the ventricle can be observed. However, whenever a coordinate contraction of first the atrium and then the ventricle occurs in reg mutant hearts, it is accompanied by physiological calcium transients that propagate first throughout the atrial chamber and then throughout the ventricle (Figure 1G). These findings indicate that electromechanical uncoupling does not account for loss of atrial/ventricular contractility of reg hearts.
To further evaluate whether reg also has an effect on cardiac excitability in general, we electrically stimulated wild-type and reg mutant hearts. In wild-type hearts, when the atrial chamber was touched with the electrode and paced at a rate of 180 bpm, all external electrical stimuli were followed by atrial and consecutive ventricular contractions. Similarly, when the atrial chamber of reg mutants was stimulated during phases of sinus exit block, all externally applied electrical stimuli were followed immediately by atrial and ventricular contractions. Interestingly, even during atrial fibrillation, electrical stimulation led to regular atrial and ventricular contraction, which suggests preliminary electrical cardioversion of atrial fibrillation by the first test pulse. These findings demonstrate that the underlying reg cardiac tissue is capable of normal conduction and contraction but is predisposed to an arrhythmia.
The Reggae (regm230) Locus Encodes a Potassium Channel Highly Homologous to the Cardiac hERG Channel
We identified the ENU-induced mutation that caused the recessive reg mutant phenotype by a positional walk (Figure 2A). By bulked segregant analysis, we mapped reg to zebrafish linkage group 3. Recombination analysis of 1403 reg mutant embryos restricted reg to a genomic interval flanked by the 2 microsatellite markers z54701 and z68142. According to genomic sequence alignment, the physical contig that covers the reg locus contains 4 open reading frames that encode a class III intermediate filament precursor protein (GenBank accession No. AY397679), a WD-repeat domain 68 protein (GenBank accession No. NP_956363), an unknown RIKEN GK001 protein (GenBank accession No. NP_001004551), and a potassium channel protein (zERG; GenBank accession No. NM_212837.1), which is highly homologous to the hERG protein in humans (Figure 2A).
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-helical transmembrane domains (S1–S6). The reg mutation resides within the mainly positively charged S4 domain and is highlighted in red. E, Amino acid sequence alignment of the human (hERG), zebra fish (zERG), and Drosophila melanogaster (dERG) ERG S4 domain demonstrates high amino acid homology across species. Black boxes indicate amino acid identity; gray boxes indicate amino acids with similar chemical properties. The red bar underneath the sequence indicates amino acids building the S4 domain. The reg mutation zERGL499P is highlighted by a red box and resides within the evolutionary highly conserved S4 domain.
To identify the site of the ENU-induced mutation in reg, we sequenced the entire zebrafish coding sequence from wild-type and reg mutant complementary DNA and genomic DNA of all open reading frames in the interval and identified the regm230 mutation to be a nucleotide transversion in exon 8 (CTG
CCG) of zerg predicted to change the highly conserved amino acid leucine to a proline at amino acid position 499 (L499P; Figure 2B and 2C). zERG is encoded by 16 exons. The protein consists of 1186 amino acids and shows overall 60% amino acid identity to hERG, whereas in specific domains such as the transmembrane regions S4, S5, and S6 of the protein, a significantly higher homology (up to 100%) can be observed.18 As in other species, zERG consists of 6 transmembrane domains (S1 through S6). Whereas domains S1 through S4 are thought to be mainly involved in sensing of the plasma membrane potential, domains S5 and S6 contribute to the potassium-selective pore of the channel. The reg mutation resides within the S4 domain, which is thought to be involved in voltage sensing (Figure 2D and 2E). To test whether the reg mutation interferes with zerg RNA stability, thereby leading to loss of zERG function in reg mutants, we assayed RNA levels of zerg in wild-type and reg mutant hearts at different developmental stages and found that similar to the situation in wild-type hearts, robust zerg RNA expression in reg mutant hearts occurred at different developmental time points (24, 48, and 72 hpf), which indicates that the reg mutation does not interfere with zerg RNA stability (data not shown).
zERGL499P Mutation Leads to Increased zERG Function in Reg Mutants
To further evaluate how zERGL499P interferes with zERG channel function, we first abolished zERG function in wild-type zebrafish by morpholino-modified antisense oligonucleotide injection. However, after injection of 4.6 ng of MO-zerg directed against the translational start site of zERG, 81% of the injected embryos displayed atrioventricular blockage at various degrees (from 2:1 to complete atrioventricular block), but none of the injected embryos developed either sinus exit block or atrial fibrillation, the main characteristics of the cardiac arrhythmia in reg mutants. Consistent with the present findings, others have recently observed a similar cardiac phenotype after antisense-oligonucleotide–mediated knockdown of zERG.18
Langheinrich et al18 recently reported a zERG mutation (I59S) to be responsible for the zebrafish breakdance (bre) phenotype. Bre mutants display intermittent atrioventricular block similar to the zERG morphants, which indicates that the bre mutation abolishes zERG function. Accordingly, the intercrossing of heterozygous reg mutant and bre mutant zebrafish did not result in a cardiac arrhythmia phenotype in the offspring, which indicates that the reg and bre alleles can complement each other and which implies opposing effects of the reg and bre mutations on zERG channel function.
Therefore, to further evaluate whether channel activity might be increased by the reg mutation, we injected MO-zerg into reg mutant embryos. After 4 ng of MO-zerg was injected, 47.8% (n=11) of reg mutant embryos no longer revealed any obvious cardiac phenotype. Interestingly, some of the genotypically homozygous reg mutants even displayed atrioventricular block, which indicates marked loss of zERG function (Figure 3B). After injection of higher amounts of MO-zerg (6 ng), the percentage of genotypically homozygous reg mutants that displayed the characteristic reg phenotype decreased even further (36.4%, n=4), whereas the percentage of reg mutant embryos that displayed atrioventricular block increased from 21% (n=5) at 4 ng to 45% (n=5; Figure 3B).
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Furthermore, to confirm the gain-of-function effect of the reg mutation on zERG, we incubated reg homozygous mutant zebrafish with terfenadine, an antihistamine known to potently reduce both hERG and zERG potassium currents.18,19 Similar to our antisense experiments, when reg mutant embryos were incubated at 72 hpf for 1 hour in various concentrations of terfenadine (0.5, 1, 5, 10, and 25 µmol/L), rescue rates varied from 18% at 0.5 µmol/L to 54% at 10 µmol/L (P<0.05), whereas at a terfenadine concentration of 25 µmol/L, rescue rates of reg homozygous decreased slightly to 50%, and an additional 14% of the incubated reg mutant embryos displayed overcompensation and a loss-of-zERG-function phenotype by displaying 2:1 atrioventricular block (Figure 3C). Taken together, the present knockdown experiments and chemical inhibition of zERG function in reg mutant embryos clearly implicate a gain-of-function effect of the reg mutation on zERG channel function.
In Vitro Characterization Confirms Gain-of- Function Effects of the Reggae Mutation on Zebrafish and Human Ether-à-Go-Go Currents
As outlined above, the present in vivo findings strongly suggest a gain-of-function effect of the reg mutation on zERG channel function. Hence, to further dissect the molecular basis of the observed gain-of-function effect, we heterologously expressed wild-type and reg mutant ether-à-go-go (ERG) channels from zebrafish (L499P) and humans (L532P) in Xenopus oocytes and analyzed their basic biophysical characteristics, such as channel activation and inactivation kinetics. As shown in Figure 4A through 4D, similar to human ERG channels with the human SQT1 mutation N588K,20 reg mutant zebrafish and human ERG channels conducted larger activation currents followed by smaller and faster deactivating tail currents than wild-type channels, which indicates impaired channel inactivation (Figure 4B and 4D).
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) display a low activation threshold. They open at membrane potentials (–70 mV) just above the resting potential (–80 mV), whereas wild-type channels (
) show a typical activation threshold of –40 mV in oocytes. The physiological range of a cardiomyocyte membrane potential is indicated (shaded area). F, Current-voltage relationship of wild-type and reg mutant tail currents of hERG channels (activation curves). Compared with wild-type (Usually, hERG channels open at a membrane potential of –40 mV. By contrast, reg mutant channels activate at just above the resting membrane potential (approximately –70 mV; Figure 4C through 4F). Thus, in reg mutants, repolarizing electrical forces via hERG are already active at a stage at which depolarization of the cell is still in progress.
hERG channels then undergo a rapid, strong inactivation process during the early plateau phase of the action potential, which leads to well-restricted potassium currents that merely balance the influx of calcium. Hence, by 10 mV, 97±0.2% of the hERG channels are inactivated (n=6; Figure 4G). By contrast, reg mutant hERG channels are inactivated to a much lesser extent (at 10 mV, 61±4% inactivation, n=6; Figure 4G), thereby mediating excessive potassium efflux. Voltage dependence of reg channel inactivation is shifted by 50 mV toward more positive potentials. Thus, compared with wild-type hERG, fewer mutant hERG channels inactivate within the range of physiological membrane potentials, which presumably mediates the gain-of-function effect observed in reg mutant hearts.
Furthermore, to investigate the effects of altered channel gating on hERG current during defined phases of the cardiac action potential, a voltage protocol simulating the action potential of a ventricular cardiomyocyte was applied to oocytes expressing wild-type or mutant hERG channels. As expected, wild-type hERG channels conducted a characteristic potassium current with rapid onset, a constant flow of potassium during plateau phase, and a prominent increase in outward current during final repolarization (Figure 4H). By contrast, hERGL532P channels instantaneously conducted prominent potassium currents, which implies stronger repolarizing electrical forces.
The in vitro electrophysiological characterization of mutant hERG channels strongly supports the present in vivo findings. In contrast to hERG wild type, mutant channels exhibit facilitated activation and deficient inactivation kinetics, thereby conducting early and increased repolarizing potassium currents, which presumably contributes to a gain of function of the reg mutation on ERG-mediated ion currents.
regL499P Induces SQTS in Zebrafish by Reducing Cardiac Action Potential Duration
Pronounced prolongation or shortening of the cardiac action potential is the substrate for malignant cardiac arrhythmias in both long-QT syndrome and SQTS. Accordingly, to study the effects of the reg mutation (hERGL532P) on action potential duration, current traces of both wild-type and mutant hERG channels (Figure 4C and 4D) were first integrated into a modified Priebe-Beuckelmann model to calculate their impact on the cardiac action potential.12 To validate the model, we simulated time courses of wild-type and reg mutant hERG currents during a given ventricular action potential. As shown in Figure 5A, calculated hERG current traces were highly similar to current traces obtained from our previous voltage-clamp experiments (Figure 4H). Subsequent in silico modeling of an action potential of a homozygous reg mutant midventricular cardiomyocyte revealed significant action potential shortening (Figure 5B).21
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To verify the predicted effects of the reg mutation on cardiac action potential duration, we next recorded ventricular cAPs in vivo from wild-type and reg mutant zebrafish larvae. Again, in contrast to wild-type zebrafish, cardiac action potential duration was found to be significantly reduced in reg mutants in vivo (Figure 6A and 6B). When corrected for heart rate with the Bazett formula, cAPD30, cAPD70, and cAPD90 were reduced in reg mutants by 29%, 25%, and 19%, respectively (n=6). Repolarization was significantly accelerated, from 289.1±17.6 to 186.3±10 ms for cAPD30 (P<0.01, n=6), from 352.8±27.3 to 265.5±13.6 ms for cAPD70 (P<0.05, n=6), and from 406±67.0 ms to 328±53.9 ms for cAPD90 (P<0.05, n=6).
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To determine whether shortening of the cardiac action potential duration in reg mutants is also reflected by a shortening of the QT interval in ECGs of adult reg mutant zebrafish, we next recorded ECGs from 9-month-old adult wild-type, heterozygous, or homozygous reg mutant zebrafish and measured the corresponding corrected QT (QTc) intervals. In wild-type adult zebrafish, mean QTc interval length was found to be 405±30.7 ms (n=6), whereas ECG recordings from heterozygous reg mutant adult zebrafish (m230+/–) revealed considerably shorter QTc intervals (344±28.0 ms; n=6; Figure 6C and 6D). The effect of the reg mutation on QTc interval length was even more pronounced in homozygous reg mutant zebrafish (m230–/–), in which QTc interval lengths were found to be significantly shortened to 279±35.9 ms (n=6; P<0.05).
In summary, both in silico modeling and in vivo measurements revealed a significant shortening of the time course of cardiac repolarization due to the reg mutation in ERG channels, thus establishing the zebrafish reg line as the first animal model for human SQTS.
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Discussion |
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We isolated here the zebrafish mutation reg, which displays clinical features of human SQTS. The reg mutation resides within the voltage sensor of zERG and causes defective hERG channel inactivation, which results in accelerated cardiac repolarization, reflected by a shortening of cAPs and QTc intervals in reg mutant embryos and adult zebrafish. Genetic and pharmacological inhibition of zERG rescues recessive reg mutant embryos, which confirms the gain-of-function effect of the reg mutation on zERG channel function in vivo. Hence, with its molecular and pathophysiological concordance to the human arrhythmia syndrome, zebrafish reg represents the first animal model for human SQTS.
Ion channel inactivation is a molecular process that transfers a channel from an open state to a stable, nonconducting state. Conformational changes of the channel protein that lead to a closure of the outer mouth have been suggested as the mechanism for the rapid inactivation of cardiac hERG potassium channels during the early phase of the cardiac action potential.22 Interestingly, whereas mutations located between the S5-P linker and the outer half of the S6 domain have been associated with altered inactivation gating, the precise role of the voltage-sensing S4 domain for hERG inactivation has remained unclear.23 We have identified here for the first time a mutation (zERGL499P) that resides within the highly conserved voltage-sensing S4 domain of ERG channels and leads to altered activation gating and marked changes in inactivation of the ERG channel. Altered inactivation gating of hERG channels has recently been linked to the human hereditary channelopathy SQT1 caused by the hERGN588K mutation.20 Similar to the reg mutation hERGL532P, electrophysiological studies revealed marked biophysical changes of hERGN588K mutant channels, including a marked loss of inactivation within the range of physiological relevance. Interestingly, inactivation kinetics, as well as hERG currents during a ventricular action potential (Figure 4H), are very similar for hERGL532P and hERGN588K mutant channels.12
Atrial fibrillation is the most prevalent arrhythmia phenotype in patients with SQTS.4 The zebrafish mutant reg also exhibits long-lasting episodes of atrial arrhythmia that resemble atrial fibrillation. Similar to other zebrafish mutants with atrial fibrillation, such as tremblor7,17or island beat8, when Calcium Green dextran is used as an indicator, subtle, uncoordinated changes in cytosolic calcium concentrations can be observed in atrial cardiomyocytes of reg mutants that indicate uncoordinated excitation of atrial cardiomyocytes. For the induction of atrial fibrillation, a pathological substrate is usually required. Shortening of action potential duration and hence effective refractory period increases the vulnerability of the atrium and forms a potential substrate for reentry mechanisms.24,25 Thus, atrial fibrillation can result not only from changes in ion channel conduction due to pharmacological influences or nerval stimulation but also from channel mutations. In human SQTS, a gain-of-function mutation in the hERG-conducted IKr current has been identified that leads to a marked shortening of the action potential duration and the effective refractory period.12 As a result, atrial fibrillation is a common clinical finding in patients with SQTS.4 Similarly, the reg mutation zERGL499P leads to a shortening of action potential duration, thereby presumably generating a pathological substrate for the development of reentry circuits that maintain atrial fibrillation. Whereas atrial fibrillation is a common clinical finding, episodes of ventricular tachycardia and ventricular fibrillation occur to a lesser extent in patients who have SQTS.4 Invasive electrophysiological testing in SQTS patients revealed a high probability for the induction of ventricular fibrillation.4 In zebrafish mutant reg, no spontaneous episodes of ventricular tachyarrhythmias were observed. Assays to evaluate the inducibility of ventricular tachyarrhythmias have not yet been established in zebrafish embryos.
Zebrafish reg mutants exhibit no ventricular excitation during episodes of atrial fibrillation. Electrical stimulation experiments in reg mutants revealed normal atrioventricular sequential excitation on external stimulation. Given the fact that hERGL532P mutant channels lead to a hyperpolarizing load, it seems reasonable to propose that atrioventricular conduction might be impaired by hyperpolarization, especially during stages of irregular and weak depolarizing wave fronts during atrial fibrillation.
Taken together, the present results demonstrate that the cardiac phenotype of zebrafish mutant reg is caused by a missense mutation in the voltage sensor of the zERG potassium channel, which leads to altered activation and inactivation kinetics. Impaired inactivation gating results in a significant decrease of cardiac action potential duration, thereby leading to atrial fibrillation. An additional shift of the activation threshold might contribute to the promotion of episodes of intermittent sinus exit block and atrioventricular conduction disturbances. With its molecular and pathophysiological concordance to human SQTS type 1, reg may prove to be the first valuable animal model to investigate disease mechanisms genetically and to test new pharmacological treatment options.
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Acknowledgments |
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We thank Stephanie Kolb and Ramona Bloehs for excellent technical support.
Sources of Funding
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ro2173/1-1 and Ro2173/2-1) and Bundesministerium für Bildung und Forschung (01GS0420) to Dr Rottbauer, as well as the Klaus-Georg and Sigrid Hengstberger Stipendium (to Dr Rottbauer), and a postdoctoral fellowship of the medical faculty of the University of Heidelberg (to Drs Scholz and Zitron).
Disclosures
None.
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Footnotes |
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*The first 3 authors contributed equally to this article.
Guest Editor for this article was Harvey D. White, DSc.
The online-only Data Supplement, consisting of movies, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA. 107.752220/DC1.
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