Potassium Channel Subunit Remodeling in Rabbits Exposed to Long-Term B
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《循环学杂志》
the Research Center and Department of Medicine, Montreal Heart Institute and University of Montreal, Montreal, Quebec, Canada (Y.T., S.Z., X.Y.Q., S.N.)
Department of Pharmacology, McGill University, Montreal, Quebec, Canada (S.Z., S.N.)
Department of Circulation, Research Institute of Environmental Medicine, Nagoya University, Nagoya City, Japan (I.K.).
Abstract
Background— Sustained heart rate abnormalities produce electrical remodeling and susceptibility to arrhythmia. Uncontrolled tachycardia produces heart failure and ventricular tachyarrhythmia susceptibility, whereas bradycardia promotes spontaneous torsade de pointes (TdP). This study compared arrhythmic phenotypes and molecular electrophysiological remodeling produced by tachycardia versus bradycardia in rabbits.
Methods and Results— We evaluated mRNA and protein expression of subunits underlying rapid (IKr) and slow (IKs) delayed-rectifier and transient-outward K+ currents in ventricular tissues from sinus rhythm control rabbits and rabbits with AV block submitted to 3-week ventricular pacing either at 60 to 90 bpm (bradypaced) or at 350 to 370 bpm (tachypaced). QT intervals at matched ventricular pacing rates were longer in bradypaced than tachypaced rabbits (eg, by 50% at 60 bpm; P<0.01). KvLQT1 and minK mRNA and protein levels were downregulated in both bradypaced and tachypaced rabbits, whereas ERG was significantly downregulated in bradypaced rabbits only. Kv4.3 and Kv1.4 were downregulated by tachypacing only. Patch-clamp experiments showed that IKs was reduced in both but IKr was decreased in bradypaced rabbits only. Continuous monitoring revealed spontaneous TdP in 75% of bradypaced but only isolated ventricular ectopy in tachypaced rabbits. Administration of dofetilide (0.02 mg/kg) to mimic IKr downregulation produced ultimately lethal TdP in all tachypaced rabbits.
Conclusions— Sustained tachycardia and bradycardia downregulate IKs subunits, but bradycardia also suppresses ERG/IKr, causing prominent repolarization delays and spontaneous TdP. Susceptibility of tachycardia/heart failure rabbits to malignant tachyarrhythmias is induced by exposure to IKr blockers. These results point to a crucial role for delayed-rectifier subunit remodeling in TdP susceptibility associated with rate-related cardiac remodeling.
Key Words: arrhythmia electrophysiology ion channels long-QT syndrome torsade de pointes
Introduction
Sustained heart rate abnormalities produce ventricular electrical remodeling and susceptibility to cardiac arrhythmia. Uncontrolled tachycardia produces heart failure (HF), along with a risk of nonsustained ventricular tachycardia (VT) and/or ventricular fibrillation (VF).1,2 Chronic AV block (AVB) produces susceptibility to torsade de pointes (TdP) in animals,3–6 and sustained bradycardia resulting from AVB is a well-known precipitator of TdP in humans.7
Clinical Perspective p 355
Repolarization delays caused by ionic remodeling are common in hypertrophy and HF.8,9 Reduced transient-outward current (Ito) is common in HF.8,9 Transcriptional downregulation of Kv4.3 subunits underlies Ito reduction in human HF.10,11 In chronic AVB-induced remodeling, Ito is unchanged, but decreased rapid (IKr) and slow (IKs) delayed-rectifier K+ currents are seen.6,12 Reduced IKs appears to be due to transcriptional downregulation of the -subunit KvLQT1 and the -subunit minK in canine AVB.13 Limited information is available on other K+ channel subunit expression changes in animal models of sustained tachycardia and bradycardia, and the spontaneous arrhythmic syndromes associated with such models have not been directly compared.
Here, we directly compare the consequences of sustained tachycardia and bradycardia on rabbit QT intervals and spontaneous arrhythmias and relate these to mRNA and membrane-protein expression changes in subunits underlying IKr, IKs, and Ito. Because of discrepancies in previous findings, we also evaluate functional changes in IKr and IKs.
Methods
Animal handling followed the Guide for the Care and Use of Laboratory Animals (NIH publication 85–23); procedures were approved by the Animal-Experimentation Ethics Committee of the Montreal Heart Institute.
Experimental Protocol
New Zealand white rabbits (2.0 to 3.0 kg) were used. We created AVB and implanted programmable right ventricular (RV) pacemakers (Medtronic) as previously described.6 AVB was created in all 44 bradycardia rabbits and in 32 tachycardia rabbits (to monitor their rhythm at heart rates similar to those of bradycardia rabbits). Fifteen additional tachypaced rabbits without AVB were studied to assess ECG interval changes. After AVB creation, ventricular pacing (180 bpm, near-physiological rabbit heart rate) was performed for 5 days. A day 0 baseline ECG was then recorded under ketamine (25 mg/kg)/xylazine (5 mg/kg) anesthesia. In tachycardia rabbits, tachypacing was then programmed (350 bpm for 1 week, then 370 bpm for 2 weeks), as previously described.14 Bradycardia rabbits were subjected to VVI pacing (90 bpm for 1 week, followed by 60 to 80 bpm for 2 weeks).6 Compatible with previous echocardiographic findings in these models,6,14 all tachycardia rabbits had signs of HF, including biventricular dilation, ascites, and pleural effusions. Bradycardia rabbits had no signs of HF but biventricular hypertrophy.
At the end of bradypacing or tachypacing periods, rabbits were euthanized (pentobarbital, 40 mg/kg IV). RV and left ventricular (LV) free walls were removed. LV samples were divided midway (because of the thinness of rabbit LVs, we could not separate distinct midmyocardial, endocardial, and epicardial samples) and frozen in liquid N2.
Competitive RT-PCR and Western blotting were applied15 to evaluate mRNA and membrane protein expression of KvLQT1, minK, ERG, KChIP2, Kv4.3, and Kv1.4 subunits in RV and LV from 10 sham-operated sinus rhythm control, 13 bradycardia, and 10 tachycardia rabbits. IKr and IKs were measured by whole-cell patch clamp in 8 control, 5 bradycardia, and 4 tachycardia rabbits.
ECG Recordings
Once weekly (days 0, 7, 14, and 21), ECGs were obtained under ketamine/xylazine anesthesia at pacing rates of 180 and 60 bpm and then recorded continuously for 30 minutes at pacing rates of 60 to 90 bpm to observe spontaneous ventricular arrhythmias. ECG parameter measurements were based on averages of 3 consecutive beats in the lead providing the clearest QT-interval definition. QT dispersion (QTd) was defined as the time between peak and end of T waves.
A total of 44 bradycardia and 32 tachycardia rabbits entered the protocol. The detection function in Medtronic Kappa was set to detect and store ventricular high-rate episodes (>400 bpm) in 24 bradycardia rabbits and in 10 tachycardia rabbits with pacemakers reprogrammed to 90 bpm at day 21. In addition, 24-hour ECG monitoring was performed by implanted telemetry in 2 bradycardia and 7 tachycardia rabbits. Ventricular tachyarrhythmias were defined as 8 consecutive beats at >400 bpm.
Competitive RT-PCR
Total RNA was isolated from 0.5- to 1.0-g samples with TRIzol reagent (Invitrogen), followed by chloroform extraction and isopropanol precipitation. Genomic DNA was eliminated by incubation in DNase I (0.1 U/μL, 37°C) for 30 minutes, followed by acid phenol-chloroform extraction. RNA was quantified by spectrophotometric absorbency (260 nm); purity was confirmed by the A260/A280 ratio; and integrity was evaluated by ethidium bromide staining on a denaturing agarose gel. RNA mimics were synthesized as previously described,15 including 460-bp rabbit -actin sequences flanked by gene-specific primers and an 8-nucleotide linker homologous to the 3'-end of T7 promoter. Primer sequences and PCR conditions are provided in Table 1. Serial mimic dilutions were mixed with 1-μg RNA samples in 20-μL reaction mixtures for RT. RT products were used as templates for subsequent PCR with gene-specific primers. PCR products were visualized under ultraviolet light with ethidium bromide, captured with a Nighthawk camera, and quantified with Quantity-One software (PDI). A DNA mass marker (100 ng) was used to determine size and quantity of DNA bands and to create standard curves for absolute quantification. Logarithmic plots {LN[(target)/(mimic)] versus LN[(mimic)]} were fit by linear regression to quantify mRNA.15
Western Blots
Membrane fractions were prepared as previously described.15 Protein samples were separated with SDS PAGE, transferred to Immobilon-P polyvinylidene fluoride membranes, and blocked in Tris-buffered saline/5% nonfat milk. Primary antibodies were incubated for 18 hours at 4°C (minK) or 1 to 2 hours at room temperature (KvLQT1, ERG, Kv4.3, and Kv1.4). Antibodies to KvLQT1 (sc-10646; goat), minK (sc-16796; goat), ERG-1a (sc-15968; goat), and Kv4.3 (sc-11686; goat) were purchased from Santa Cruz Biotechnology; and anti-Kv1.4 (APC007; rabbit) was purchased from Alomone. Monoclonal KChIP2 antibody was kindly supplied by James Trimmer. Secondary antibodies were horseradish peroxidase–conjugated donkey anti-goat IgG and anti-rabbit IgG (Santa Cruz). Band signals were detected and quantified with Western-Lightning Chemiluminescence Reagent Plus, laser scanning, and Quantity-One software.
Patch-Clamp Experiments
LV cardiomyocytes were isolated as described previously.6,14 Sham-operated sinus rhythm rabbits were used as controls. All rabbits for cell isolation were euthanized at day 21. Membrane currents were recorded by tight-seal whole-cell patch clamp with Na+-free, K+-free extracellular solutions including N-methyl-D-glucamine to eliminate inward-rectifier K+ current (IK1).6,14 Cell capacitance was determined with ramp pulses (0.5 V/s) from –50 to 70 mV. Cell capacitance and series resistance (RS) were compensated by 50% to 70%. IKr was recorded in the presence of the highly selective IKs blocker HMR-1566 (1 μmol/L)16 and IKs in the presence of the IKr blocker E-4031 (5 μmol/L).
Data Analysis
All data are expressed as mean±SEM. Each biochemical determination was performed on an individual rabbit. Western blot band intensities are expressed after background subtraction as optical density units (ODUs) normalized to GAPDH signal intensity for the same sample. There were no significant differences between LV endocardial and epicardial results, so data from each rabbit were averaged and presented as a single LV value. For the ECG parameters, 2-way repeated-measures ANOVA was performed using mixed model methodology with group and time as main effects. In case of a significant interaction between the 2 main effects, Dunnett contrasts were used to compare them over time within each group. The mixed procedure and multiple comparison with Dunnett’s test were used to evaluate differences in mRNA and protein expression among groups over time. Statistical comparisons for ionic current data were obtained by ANOVA, followed by Tukey’s test. Values of P<0.05 indicated statistical significance. All probability values in the figures are based on ANOVA, followed by Dunnett’s or Tukey’s test.
Results
ECG Changes
Figure 1 shows ECGs at matched pacing rates in a brady-paced and a tachypaced rabbit. Day 21 QT intervals were prolonged in the bradypaced rabbit but were unchanged in the tachypaced rabbit. Figure 1C summarizes ECG changes. QRS duration did not change. In bradycardia rabbits, QT intervals increased from 256±3 ms at 60 bpm (day 0) to nearly steady-state values (332±9 ms) by day 7. In tachycardia rabbits, QT intervals decreased from 256±5 ms at day 0 to 223±5 ms at day 7. During sinus rhythm recording (available only for tachypaced rabbits not subjected to AVB), corrected QT intervals increased from 145±3 ms (day 0) to 158±2 ms (day 21; P<0.05). QTd increased only in bradycardia rabbits.
Spontaneous Ventricular Arrhythmias
Of 44 rabbits entering the bradycardia group, 23 died before day 21. Sixty-one percent (14 of 23) died suddenly, with ECG recordings showing spontaneous TdP. The remaining 9 died of unknown causes. The bradypaced rabbits surviving for 21 days had 66±17 (range, 2 to 232) episodes per rabbit of TdP. Thirteen and 5 bradypaced rabbits were euthanized for molecular studies and patch-clamp experiments, respectively, at day 21. Spontaneous TdP was documented in 33 of 44 bradypaced rabbits (75%). Figure 2A shows intracardiac electrograms stored by implanted pacemakers in 2 brady-paced rabbits. Rabbit 1 developed polymorphic VT at day 7 and died the next day. Rabbit 2 had frequent TdP-like VT before death at day 16. Figure 2B shows continuous telemetry from a bradypaced rabbit. Five days after pacing was set to 60 bpm, spontaneous TdP occurred and degenerated to VF. All rabbits were subjected to a 30-minute ECG recording on day 21. Of 21 bradypaced rabbits surviving to day 21, 16 (76%) showed spontaneous TdP during this recording.
Of the 32 tachypaced rabbits, 21 survived for 21 days, and 11 died prematurely. No tachyarrhythmias were detected with the episode report function of implanted pacemakers. Continuous 24-hour telemetry ECG recording of 7 tachycardia rabbits after pacing rate reduction to 60 to 90 bpm showed a single 7-complex run in 1 (Figure 2C), isolated ventricular premature beats in 3, and no arrhythmia in 3. Of 21 tachypaced rabbits monitored by ECG for 30 minutes on day 21, none showed spontaneous VT (versus 76% of bradypaced rabbits; P<0.001).
Expression of IKs Subunits
Examples of competitive RT-PCR gels for KvLQT1 and minK are shown in Figure 3A. Lane 0 contains 100 ng of DNA mass ladder to create the standard curve. Lanes 1 through 6 were obtained with serial dilutions of the RNA mimic, along with 1 μg total RNA per lane. Top bands represent RNA mimic products. Bottom bands are target bands coamplified with mimics in the same reaction tube. As mimic concentration decreases from left to right, the target band becomes stronger, demonstrating mimic-target competition. Overall, mRNA expression was significantly reduced for both IKs subunits in bradypaced and tachypaced rabbits (Figure 3A, bottom). In bradypaced rabbits, KvLQT1 expression was reduced by averages of 76% and 53% (RV and LV, respectively) and minK by 58% and 43%. In tachypaced rabbits, KvLQT1 expression was reduced by 64% and 51% (RV and LV) and minK by 74% and 43%.
Figure 3B shows KvLQT1 and minK Western blots on 1 gel each. A prominent KvLQT1 band was observed at the expected molecular mass of 75 kDa. Antibody preincubation with antigenic peptide eliminated the signal. The minK blot showed a faint signal at the expected molecular mass of 27 kDa, consistent with previous reports of human, guinea pig, and rabbit minK signals at 27 kDa.15 This signal disappeared on preincubation with antigenic peptide, unlike the higher-molecular-mass signals. KvLQT1 protein expression was reduced by 72% and 53% in the RV and LV, respectively, among bradypaced rabbits and by 78% and 53% for tachypaced rabbits. MinK was downregulated similarly in tachypaced (by 45% and 37% in the RV and LV) and bradypaced (by 37% and 33%) rabbits.
ERG Expression
A typical ERG competitive RT-PCR gel for a control rabbit is shown in Figure 4A. ERG mRNA was significantly downregulated in bradypaced rabbits only (Figure 4B; expression in bradycardia rabbits reduced by 59% in the RV and 52% in the LV). Representative ERG Western blots are shown in Figure 4C. A clear band at the expected molecular mass of 165 kDa was suppressed in bradypaced rabbits, and lower-molecular-weight bands sometimes appeared (3 of 7). All signals were eliminated by antibody preincubation with antigenic peptide. ERG protein expression was significantly downregulated in bradypaced rabbits only (Figure 4D; by 50% in the RV and 37% in the LV).
Expression of Ito Subunits
Figure 5A shows representative competitive RT-PCR gels for Kv4.3 and Kv1.4, as well as mean data. Kv4.3 and Kv1.4 mRNA expression was downregulated in tachy-paced rabbits (Kv4.3: by 53% in RV, 26% LV; Kv1.4: by 77% in RV, 59% LV). Kv4.3 mRNA was unchanged in bradypaced rabbits, whereas Kv1.4 mRNA showed a small decrease of borderline statistical significance in the RV and no significant change in the LV. Kv4.3 antibody revealed bands at 78 and 70 kDa (Figure 5B), consistent with long and short splice variants in rabbit ventricle.17 For Kv1.4, 2 clear bands were detected close to the expected molecular mass of 97 kDa. Ito subunit protein expression was significantly reduced in tachypaced rabbits only, by 60% and 74% (Kv4.3 and Kv1.4, respectively) in the RV and 43% and 36% (Kv4.3, Kv1.4) in the LV. KChIP2 expression was comparable in control, tachypaced, and bradypaced rabbits (eg, averages in the LV were 3.4±0.8, 3.1±1.1, and 3.2±0.6 ODUs, respectively).
Changes in IKr and IKs
Previous data on delayed-rectifier current downregulation in tachypacing-induced HF have varied.14,18 To correlate directly delayed-rectifier subunit expression and current alterations in our rabbits, we performed patch-clamp experiments. Typical IKr tail currents were seen in cells from all groups but were smaller in bradypaced rabbits (Figure 6A). IKs was smaller in both bradypaced and tachypaced rabbits (Figure 6B). Figure 7 shows activation voltage dependence (top) and tail current-voltage relations (bottom). IKr (Figure 7A) activated at more negative voltages than IKs (Figure 7B), following well-recognized properties.6,18 IKr activation voltage dependence was not altered in either bradypaced (V1/2, –7.0±2.5 mV) or tachypaced (V1/2, –8.7±2.0 mV; control, –5.5±1.9 mV) rabbits. IKs activation voltage dependence was similarly unaffected (V1/2, 9.3±4.4 mV for bradypaced, 17.8±3.9 mV for tachypaced, 14.7±1.7 mV for control; P=NS). IKr tail current density was reduced significantly in bradypaced rabbits (by 40%) but was unchanged in tachypaced rabbits (Figure 7C). IKs tail current density was reduced by 70% and 55% in bradypaced and tachypaced rabbits, respectively (Figure 7D). There were no differences among groups in IKr and IKs deactivation kinetics (Table 2).
Response of Tachypaced Rabbits to IKr Inhibition
Our arrhythmia-monitoring data showed that bradypaced but not tachypaced rabbits were highly susceptible to spontaneous TdP. IKs and its subunits KvLQT1 and minK were downregulated similarly by bradypacing and tachypacing, but only bradypacing suppressed ERG/IKr. We hypothesized that IKr reduction might be crucial for differential TdP susceptibility in bradypaced rabbits. We therefore tested the effect of selective IKr blockade with a single intravenous dose of dofetilide (0.02 mg/kg), followed by telemetric monitoring for 3 hours during pacing at 90 bpm in tachypaced rabbits, to mimic IKr downregulation in bradypaced rabbits. The dose of dofetilide was based on previous studies showing that normal rabbits receiving the same dose per minute (total dose 60 times as large) developed TdP and VF with incidences of 2%5 and 17%, respectively.19 Figure 8A shows continuous monitoring after dofetilide administration to a tachypaced rabbit. The QT interval was markedly prolonged, and TdP occurred frequently. All 6 tachypaced rabbits given dofetilide displayed TdP that degenerated into VF. Spontaneous ventricular tachyarrhythmia prevalence in bradypaced rabbits, tachypaced rabbits without dofetilide exposure, and tachy-paced rabbits treated with dofetilide is summarized in Figure 8B. Dofetilide increased VT occurrence in tachypaced rabbits from 0% to 100% (P<0.0001, Fisher’s exact test) to a level comparable to bradypaced rabbits (75%). QT intervals and QTd at 600-ms cycle length were much smaller in tachypaced than bradypaced rabbits in the absence of dofetilide (Figure 8C); however, dofetilide exposure increased values in tachy-paced rabbits to levels comparable to those in bradypaced animals.
Discussion
In this study, we compared directly the K+ channel subunit remodeling and arrhythmic phenotypes resulting from persistent bradycardia versus tachycardia in rabbits. Whereas bradypaced rabbits displayed downregulation of both rapid and slow components of the delayed-rectifier current system, only slow-component subunits and current were suppressed in tachypaced rabbits. This differential ion channel regulation was associated with differential susceptibility to spontaneous ventricular tachyarrhythmias, with bradypaced rabbits showing frequent TdP and tachypaced rabbits manifesting limited spontaneous ventricular arrhythmia. The arrhythmic response of tachypaced rabbits to dofetilide, which transformed their arrhythmic phenotype toward that of bradycardia rabbits, supports the notion that the particular TdP diathesis of bradycardic rabbits is due to IKr downregulation.
Relationship to Previous In Vivo Studies of Rate-Related Remodeling
The tachypacing model of HF is well established for studies of electrophysiological remodeling.2,8–10,14,17,18,20,21 Ito downregulation is the most consistent finding. The response of delayed-rectifier currents has been more variable; although decreases in IKs have been observed consistently,14,18,21 some studies reported decreased IKr14 and others indicated no change.18,21 In bradycardia-related remodeling, IKs is consistently downregulated.6,22 IKr has been reported to be decreased in bradycardic rabbits6 and decreased in RVs but not LVs of bradycardic dogs.22 In this study, IKs was reduced in both bradycardic and tachycardic rabbits, but IKr was reduced only in bradycardia, consistent with changes in mRNA and protein expression of corresponding subunits. In a previous study,14 IKr was reportedly downregulated in tachycardic rabbits; however, 50% activation voltages for IKr and IKs were similar, suggesting possible contamination of E-4031 sensitive currents by IKs rundown.
The propensity of bradycardia-remodeled hearts to TdP is well documented.3–6 Pak et al2 observed nonsustained VT and 1 recorded lethal ventricular tachyarrhythmia in 25 dogs with tachypacing-induced HF. A propensity to early afterdepolarization-related arrhythmias can be unmasked in tachypaced HF by repolarization-prolonging interventions,2,23 suggesting decreased repolarization reserve consistent with the reductions in IKs and associated subunits observed in the present study.
There has been limited work on remodeling of ion channel subunits in animal models involving sustained heart rate abnormalities. Ramakers et al13 observed decreases in both KvLQT1 and minK expression in bradycardic dogs. We were unable to identify other studies of K+ channel subunit remodeling in bradycardic animal models. In tachycardia remodeling, downregulation of Kv4.x subunits is consistently observed.17,20,24 Akar et al24 found a tendency to reduced ERG mRNA in tachypaced dog hearts, although a band of unspecified molecular weight was increased on Western blot. No changes in IKs subunits were seen. Similarly, Rose et al20 did not find significant changes in delayed-rectifier K+ channel subunits in tachypaced rabbits despite significantly decreased delayed-rectifier currents (IKr and IKs were not separated electrophysiologically). In addition to decreased ERG mRNA, our results point to possible posttranslational changes in bradypaced rabbits, because in some cases we observed lower-molecular-weight bands possibly corresponding to immature ERG protein.
Novel Findings and Potential Significance
The present study is the first to compare directly arrhythmic phenotypes and ion channel remodeling in bradycardic versus tachycardic animal models. We have observed substantial differences in spontaneous ventricular arrhythmias, with TdP being ubiquitous in bradycardia-dependent remodeling and never occurring in tachypaced animals. This discrepancy appears to be due to differences in ERG/IKr remodeling. Evidence for the importance of bradycardia-specific ERG remodeling included reduced IKr and ERG mRNA and protein, as well as the transformation of the ECG and arrhythmic phenotype of tachypaced rabbits toward that of bradypaced rabbits with IKr inhibition.
These experimental phenotypes are likely relevant to several clinical phenomena. AVB has been recognized since the 1920s25 as a precipitant of TdP, and TdP is a recognized presentation of congenital heart block.26 Patients with AVB and spontaneous TdP require pacing to at least 70 bpm to prevent excess QT prolongation.27 It has long been thought that the promotion of TdP by bradycardia is due to the acute heart rate–APD relationship, but our studies suggest an intriguing additional possibility: downregulation of ion channel subunits (and in particular ERG) by bradycardia. This notion may also account for another potentially important phenomenon: the ability of chronic -blocker therapy to increase APD. Unexplained APD-prolonging effects of chronic (but not acute) in vivo -blockade have been observed in animal28 and human29 studies, as well as in isolated human atrial myocytes.30 Such effects are potentially relevant to mechanisms of -blocker antiarrhythmic actions and to the varying efficacy of -blockade in patients with congenital long-QT syndrome.
We did not observe spontaneous TdP in tachypaced rabbits with HF; however, TdP occurred in all rabbits on dofetilide exposure. These observations may parallel the uncommon occurrence of TdP in HF patients, despite a clearly increased risk of TdP in HF patients exposed to IKr-blocking drugs.31 These findings are consistent with the concept of "repolarization reserve."32 Downregulation of IKs subunit expression and function may not in itself be sufficient to cause spontaneous TdP, but in the setting of superimposed IKr inhibition or downregulation, repolarization may become unstable and lead to early afterdepolarizations and related tachyarrhythmias.
Potential Limitations
We cannot exclude the possibility that the abnormal activation sequence caused by ventricular pacing contributed to ion channel remodeling. However, if dyssynchrony were a prime factor, we would have expected to see differential remodeling of LV endocardium, epicardium, and RV, which was not the case. It is unlikely that AVB per se contributed to ion channel remodeling, because KvLQT1 protein expression was reduced to the same extent in tachycardia rabbits without (0.12±0.03 ODU) and with (0.10±0.02 ODU) AVB compared with controls (0.36±0.09 ODU).
We were surprised to see QT shortening in tachypaced rabbits with AVB (Figure 1C). This may have resulted because the pacing rate (180 bpm) during the 5-day recovery interval after AVB, before tachypacing, was slower than the spontaneous sinus rate (250 bpm), possibly causing APD prolongation at day 0. This notion is supported by the fact that QT intervals during sinus rhythm increased slightly but significantly over time in tachypaced rabbits without AVB (Figure 1C). The small QT interval increase in the face of significant downregulation of KvLQT/minK and Kv4.3/Kv1.4 subunits encoding IKs and Ito may be due to several factors. IKs is small in the rabbit because of low-level minK expression,15 and pure IKs suppression fails to increase APD in the rabbit.33 In addition, because of the very slow recovery kinetics of rabbit Ito, it contributes very little current at physiological heart rates.34
Our minK protein bands were very weak, consistent with known low-level expression in the rabbit,15 but limiting the reliability of the measurement and requiring caution in interpretation. Therefore, conclusions about the protein expression of this subunit need to be particularly guarded. We elected to perform patch-clamp studies only for delayed-rectifier currents because of previous discrepancies in the literature. Because Ito has been consistently found to be downregulated in HF,2,9,14,18,20,21 a result consistent with our molecular determinations, we did not prepare additional rabbits for Ito measurement and therefore lack such results for direct correlation with biochemical data.
The response of tachypaced rabbits to dofetilide supports the notion that IKr downregulation is the major factor determining arrhythmic phenotype differences between brady-paced and tachypaced animals. We studied a limited number of ion channel subunits and currents. The potential roles of other ion channels and transporters, structural remodeling, differences in autonomic or neurohumoral response, altered activation sequence, mechanical factors, etc, require further exploration. In addition, we did not explore the issue of whether tachypaced rabbits were more susceptible to dofetilide-induced TdP than control rabbits. A full exploration of the potential electrophysiological and arrhythmic consequences of reduced repolarization reserve in brady-paced and tachypaced rabbits would have required extensive additional experiments and, while interesting and important, is beyond the scope of the present paper.
We were unable to separate a distinct midmyocardial layer because of the thinness of the rabbit ventricle. We did evaluate separately ion channel expression changes in subepicardial versus subendocardial tissues and did not observe any significant differences.
The arrhythmic phenotype we noted in tachypaced HF rabbits was different from that reported previously by Pogwizd et al35 in rabbits with HF after aortic banding/regurgitation. The differences are likely due to the different methods of HF induction but emphasize the importance of not generalizing excessively from a single animal model of HF. Differences in underlying cause, duration, and severity of HF likely differentially influence the resulting electrophysiological remodeling and arrhythmic phenotype of HF in humans.
Acknowledgments
Y. Tsuji was a research fellow of Heart and Stroke Foundation of Canada and was supported by Japan Heart Foundation & Bayer Yakuhin Research Grant Abroad. We thank Medtronic Japan for providing pacemakers; Pfizer Pharmaceuticals for dofetilide; the Canadian Institutes of Health Research and Quebec Heart and Stroke Foundation for funding; Chantal Maltais, Marc-Antoine Gillis, Evelyn Landry, and Chantal St-Cyr for technical assistance; and France Theriault for secretarial support.
Disclosures
None.
References
Nerheim P, Birger-Botkin S, Piracha L, Olshansky B. Heart failure and sudden death in patients with tachycardia-induced cardiomyopathy and recurrent tachycardia. Circulation. 2004; 110: 247–252.
Pak PH, Nuss HB, Tunin RS, Kaab S, Tomaselli GF, Marban E, Kass DA. Repolarization abnormalities, arrhythmia and sudden death in canine tachycardia-induced cardiomyopathy. J Am Coll Cardiol. 1997; 30: 576–584.
Vos MA, Verduyn SC, Gorgels AP, Lipcsei GC, Wellens HJ. Reproducible induction of early afterdepolarizations and torsade de pointes arrhythmias by d-sotalol and pacing in dogs with chronic atrioventricular block. Circulation. 1995; 91: 864–872.
Vos MA, de Groot SH, Verduyn SC, van der Zande J, Leunissen HD, Cleutjens JP, van Bilsen M, Daemen MJ, Schreuder JJ, Allessie MA, Wellens HJ. Enhanced susceptibility for acquired torsade de pointes arrhythmias in the dog with chronic, complete AV block is related to cardiac hypertrophy and electrical remodeling. Circulation. 1998; 98: 1125–1135.
Volders PG, Sipido KR, Vos MA, Kulcsar A, Verduyn SC, Wellens HJ. Cellular basis of biventricular hypertrophy and arrhythmogenesis in dogs with chronic complete atrioventricular block and acquired torsade de pointes. Circulation. 1998; 98: 1136–1147.
Tsuji Y, Opthof T, Yasui K, Inden Y, Takemura H, Niwa N, Lu Z, Lee JK, Honjo H, Kamiya K, Kodama I. Ionic mechanisms of acquired QT prolongation and torsades de pointes in rabbits with chronic complete atrioventricular block. Circulation. 2002; 106: 2012–2018.
Jackman WM, Friday KJ, Anderson JL, Aliot EM, Clark M, Lazzara R. The long QT syndromes: a critical review, new clinical observations and a unifying hypothesis. Prog Cardiovasc Dis. 1988; 31: 115–172.
Nabauer M, Kaab S. Potassium channel down-regulation in heart failure. Cardiovasc Res. 1998; 37: 324–334.
Tomaselli GF, Marban E. Electrophysiological remodeling in hypertrophy and heart failure. Cardiovasc Res. 1999; 42: 270–283.
Kaab S, Dixon J, Duc J, Ashen D, Nabauer M, Beuckelmann DJ, Steinbeck G, McKinnon D, Tomaselli GF. Molecular basis of transient outward potassium current downregulation in human heart failure: a decrease in Kv4.3 mRNA correlates with a reduction in current density. Circulation. 1998; 98: 1383–1393.
Borlak J, Thum T. Hallmarks of ion channel gene expression in end-stage heart failure. FASEB J. 2003; 17: 1592–1608.
Volders PG, Sipido KR, Vos MA, Spatjens RL, Leunissen JD, Carmeliet E, Wellens HJ. Downregulation of delayed rectifier K(+) currents in dogs with chronic complete atrioventricular block and acquired torsades de pointes. Circulation. 1999; 100: 2455–2461.
Ramakers C, Vos MA, Doevendans PA, Schoenmakers M, Wu YS, Scicchitano S, Iodice A, Thomas GP, Antzelevitch C, Dumaine R. Coordinated down-regulation of KCNQ1 and KCNE1 expression contributes to reduction of I(Ks) in canine hypertrophied hearts. Cardiovasc Res. 2003; 57: 486–496.
Tsuji Y, Opthof T, Kamiya K, Yasui K, Liu W, Lu Z, Kodama I. Pacing-induced heart failure causes a reduction of delayed rectifier potassium currents along with decreases in calcium and transient outward currents in rabbit ventricle. Cardiovasc Res. 2000; 48: 300–309.
Zicha S, Moss I, Allen B, Varro A, Papp J, Dumaine R, Antzelevich C, Nattel S. Molecular basis of species-specific expression of repolarizing K+ currents in the heart. Am J Physiol Heart Circ Physiol. 2003; 285: H1641–H1649.
Thomas GP, Gerlach U, Antzelevitch C. HMR 1556, a potent and selective blocker of slowly activating delayed rectifier potassium current. J Cardiovasc Pharmacol. 2003; 41: 140–147.
Rose J, Armoundas AA, Tian Y, Disilvestre D, Burysek M, Halperin V, O’Rourke B, Kass DA, Marban E, Tomaselli GF. Molecular correlates of altered expression of potassium currents in failing rabbit myocardium. Am J Physiol Heart Circ Physiol. 2005; 288: H2077–H2087.
Li GR, Lau CP, Ducharme A, Tardif JC, Nattel S. Transmural action potential and ionic current remodeling in ventricles of failing canine hearts. Am J Physiol Heart Circ Physiol. 2002; 283: H1031–H1041.
Lu HR, Van Ammel K, Vlaminckx E, De Clerck F. QT and JT dispersion in the drug-induced long QT syndrome in anesthetized rabbits is accurately detected by a three-lead surface ECG measurement. J Pharmacol Toxicol Methods. 2004; 49: 71–79.
Zicha S, Xiao L, Stafford S, Cha TJ, Han W, Varro A, Nattel S. Transmural expression of transient outward potassium current subunits in normal and failing canine and human hearts. J Physiol Lond. 2004; 561: 735–748.
Han W, Chartier D, Li D, Nattel S. Ionic remodeling of cardiac Purkinje cells by congestive heart failure. Circulation. 2001; 104: 2095–2100.
Volders PG, Sipido KR, Vos MA, Spatjens RL, Leunissen JD, Carmeliet E, Wellens HJ. Downregulation of delayed rectifier K(+) currents in dogs with chronic complete atrioventricular block and acquired torsades de pointes. Circulation. 1999; 100: 2455–2461.
Jones DL, Petrie JP, Li HG. Spontaneous, electrically, and cesium chloride induced arrhythmia and afterdepolarizations in the rapidly paced dog heart. Pacing Clin Electrophysiol. 2001; 24: 474–485.
Akar FG, Wu RC, Juang GJ, Tian Y, Burysek M, Disilvestre D, Xiong W, Armoundas AA, Tomaselli GF. Molecular mechanisms underlying potassium current down-regulation in canine tachycardia-induced heart failure. Am J Physiol Heart Circ Physiol. 2005; 288: H2887–H2896.
Kerr WJ, Bender WL. Paroxysmal ventricular fibrillation with cardiac recovery in a case of auricular fibrillation and complete heart block while under quinidine sulphate therapy. Heart. 1922; 9: 269–281.
Nikolic G, Arnold J, Coles DM. Torsade de pointes and asystole in a child with complete heart block and prolonged QT interval. Aust Paediatr J. 1983; 19: 187–191.
Kurita T, Ohe T, Marui N, Aihara N, Takaki H, Kamakura S, Matsuhisa M, Shimomura K. Bradycardia-induced abnormal QT prolongation in patients with complete atrioventricular block with torsades de pointes. Am J Cardiol. 1992; 69: 628–633.
Raine AE, Vaughan Williams EM. Adaptation to prolonged beta-blockade of rabbit atrial, Purkinje, and ventricular potentials, and of papillary muscle contraction: time-course of development of and recovery from adaptation. Circ Res. 1981; 48: 804–812.
Edvardsson N, Olsson SB. Effects of acute and chronic beta-receptor blockade on ventricular repolarisation in man. Br Heart J. 1981; 45: 628–636.
Workman AJ, Kane KA, Russell JA, Norrie J, Rankin AC. Chronic beta-adrenoceptor blockade and human atrial cell electrophysiology: evidence of pharmacological remodelling. Cardiovasc Res. 2003; 58: 518–525.
Lehmann MH, Hardy S, Archibald D, Quart B, MacNeil DJ. Sex difference in risk of torsade de pointes with d,l-sotalol. Circulation. 1996; 94: 2535–2541.
Roden DM. Taking the "idio" out of "idiosyncratic": predicting torsades de pointes. Pacing Clin Electrophysiol. 1998; 21: 1029–1034.
Lengyel C, Iost N, Virag L, Varro A, Lathrop DA, Papp JG. Pharmacological block of the slow component of the outward delayed rectifier current (I(Ks)) fails to lengthen rabbit ventricular muscle QT(c) and action potential duration. Br J Pharmacol. 2001; 132: 101–110.
Wang Z, Fermini B, Feng J, Nattel S. Role of chloride currents in repolarizing rabbit atrial myocytes. Am J Physiol. 1995; 268: H1992–H2002.
Pogwizd SM, Schlotthauer K, Li L, Yuan W, Bers DM. Arrhythmogenesis and contractile dysfunction in heart failure: roles of sodium-calcium exchange, inward rectifier potassium current, and residual -adrenergic responsiveness. Circ Res. 2001; 88: 1159–1167.(Yukiomi Tsuji, MD; Stephen Zicha, PhD; X)
Department of Pharmacology, McGill University, Montreal, Quebec, Canada (S.Z., S.N.)
Department of Circulation, Research Institute of Environmental Medicine, Nagoya University, Nagoya City, Japan (I.K.).
Abstract
Background— Sustained heart rate abnormalities produce electrical remodeling and susceptibility to arrhythmia. Uncontrolled tachycardia produces heart failure and ventricular tachyarrhythmia susceptibility, whereas bradycardia promotes spontaneous torsade de pointes (TdP). This study compared arrhythmic phenotypes and molecular electrophysiological remodeling produced by tachycardia versus bradycardia in rabbits.
Methods and Results— We evaluated mRNA and protein expression of subunits underlying rapid (IKr) and slow (IKs) delayed-rectifier and transient-outward K+ currents in ventricular tissues from sinus rhythm control rabbits and rabbits with AV block submitted to 3-week ventricular pacing either at 60 to 90 bpm (bradypaced) or at 350 to 370 bpm (tachypaced). QT intervals at matched ventricular pacing rates were longer in bradypaced than tachypaced rabbits (eg, by 50% at 60 bpm; P<0.01). KvLQT1 and minK mRNA and protein levels were downregulated in both bradypaced and tachypaced rabbits, whereas ERG was significantly downregulated in bradypaced rabbits only. Kv4.3 and Kv1.4 were downregulated by tachypacing only. Patch-clamp experiments showed that IKs was reduced in both but IKr was decreased in bradypaced rabbits only. Continuous monitoring revealed spontaneous TdP in 75% of bradypaced but only isolated ventricular ectopy in tachypaced rabbits. Administration of dofetilide (0.02 mg/kg) to mimic IKr downregulation produced ultimately lethal TdP in all tachypaced rabbits.
Conclusions— Sustained tachycardia and bradycardia downregulate IKs subunits, but bradycardia also suppresses ERG/IKr, causing prominent repolarization delays and spontaneous TdP. Susceptibility of tachycardia/heart failure rabbits to malignant tachyarrhythmias is induced by exposure to IKr blockers. These results point to a crucial role for delayed-rectifier subunit remodeling in TdP susceptibility associated with rate-related cardiac remodeling.
Key Words: arrhythmia electrophysiology ion channels long-QT syndrome torsade de pointes
Introduction
Sustained heart rate abnormalities produce ventricular electrical remodeling and susceptibility to cardiac arrhythmia. Uncontrolled tachycardia produces heart failure (HF), along with a risk of nonsustained ventricular tachycardia (VT) and/or ventricular fibrillation (VF).1,2 Chronic AV block (AVB) produces susceptibility to torsade de pointes (TdP) in animals,3–6 and sustained bradycardia resulting from AVB is a well-known precipitator of TdP in humans.7
Clinical Perspective p 355
Repolarization delays caused by ionic remodeling are common in hypertrophy and HF.8,9 Reduced transient-outward current (Ito) is common in HF.8,9 Transcriptional downregulation of Kv4.3 subunits underlies Ito reduction in human HF.10,11 In chronic AVB-induced remodeling, Ito is unchanged, but decreased rapid (IKr) and slow (IKs) delayed-rectifier K+ currents are seen.6,12 Reduced IKs appears to be due to transcriptional downregulation of the -subunit KvLQT1 and the -subunit minK in canine AVB.13 Limited information is available on other K+ channel subunit expression changes in animal models of sustained tachycardia and bradycardia, and the spontaneous arrhythmic syndromes associated with such models have not been directly compared.
Here, we directly compare the consequences of sustained tachycardia and bradycardia on rabbit QT intervals and spontaneous arrhythmias and relate these to mRNA and membrane-protein expression changes in subunits underlying IKr, IKs, and Ito. Because of discrepancies in previous findings, we also evaluate functional changes in IKr and IKs.
Methods
Animal handling followed the Guide for the Care and Use of Laboratory Animals (NIH publication 85–23); procedures were approved by the Animal-Experimentation Ethics Committee of the Montreal Heart Institute.
Experimental Protocol
New Zealand white rabbits (2.0 to 3.0 kg) were used. We created AVB and implanted programmable right ventricular (RV) pacemakers (Medtronic) as previously described.6 AVB was created in all 44 bradycardia rabbits and in 32 tachycardia rabbits (to monitor their rhythm at heart rates similar to those of bradycardia rabbits). Fifteen additional tachypaced rabbits without AVB were studied to assess ECG interval changes. After AVB creation, ventricular pacing (180 bpm, near-physiological rabbit heart rate) was performed for 5 days. A day 0 baseline ECG was then recorded under ketamine (25 mg/kg)/xylazine (5 mg/kg) anesthesia. In tachycardia rabbits, tachypacing was then programmed (350 bpm for 1 week, then 370 bpm for 2 weeks), as previously described.14 Bradycardia rabbits were subjected to VVI pacing (90 bpm for 1 week, followed by 60 to 80 bpm for 2 weeks).6 Compatible with previous echocardiographic findings in these models,6,14 all tachycardia rabbits had signs of HF, including biventricular dilation, ascites, and pleural effusions. Bradycardia rabbits had no signs of HF but biventricular hypertrophy.
At the end of bradypacing or tachypacing periods, rabbits were euthanized (pentobarbital, 40 mg/kg IV). RV and left ventricular (LV) free walls were removed. LV samples were divided midway (because of the thinness of rabbit LVs, we could not separate distinct midmyocardial, endocardial, and epicardial samples) and frozen in liquid N2.
Competitive RT-PCR and Western blotting were applied15 to evaluate mRNA and membrane protein expression of KvLQT1, minK, ERG, KChIP2, Kv4.3, and Kv1.4 subunits in RV and LV from 10 sham-operated sinus rhythm control, 13 bradycardia, and 10 tachycardia rabbits. IKr and IKs were measured by whole-cell patch clamp in 8 control, 5 bradycardia, and 4 tachycardia rabbits.
ECG Recordings
Once weekly (days 0, 7, 14, and 21), ECGs were obtained under ketamine/xylazine anesthesia at pacing rates of 180 and 60 bpm and then recorded continuously for 30 minutes at pacing rates of 60 to 90 bpm to observe spontaneous ventricular arrhythmias. ECG parameter measurements were based on averages of 3 consecutive beats in the lead providing the clearest QT-interval definition. QT dispersion (QTd) was defined as the time between peak and end of T waves.
A total of 44 bradycardia and 32 tachycardia rabbits entered the protocol. The detection function in Medtronic Kappa was set to detect and store ventricular high-rate episodes (>400 bpm) in 24 bradycardia rabbits and in 10 tachycardia rabbits with pacemakers reprogrammed to 90 bpm at day 21. In addition, 24-hour ECG monitoring was performed by implanted telemetry in 2 bradycardia and 7 tachycardia rabbits. Ventricular tachyarrhythmias were defined as 8 consecutive beats at >400 bpm.
Competitive RT-PCR
Total RNA was isolated from 0.5- to 1.0-g samples with TRIzol reagent (Invitrogen), followed by chloroform extraction and isopropanol precipitation. Genomic DNA was eliminated by incubation in DNase I (0.1 U/μL, 37°C) for 30 minutes, followed by acid phenol-chloroform extraction. RNA was quantified by spectrophotometric absorbency (260 nm); purity was confirmed by the A260/A280 ratio; and integrity was evaluated by ethidium bromide staining on a denaturing agarose gel. RNA mimics were synthesized as previously described,15 including 460-bp rabbit -actin sequences flanked by gene-specific primers and an 8-nucleotide linker homologous to the 3'-end of T7 promoter. Primer sequences and PCR conditions are provided in Table 1. Serial mimic dilutions were mixed with 1-μg RNA samples in 20-μL reaction mixtures for RT. RT products were used as templates for subsequent PCR with gene-specific primers. PCR products were visualized under ultraviolet light with ethidium bromide, captured with a Nighthawk camera, and quantified with Quantity-One software (PDI). A DNA mass marker (100 ng) was used to determine size and quantity of DNA bands and to create standard curves for absolute quantification. Logarithmic plots {LN[(target)/(mimic)] versus LN[(mimic)]} were fit by linear regression to quantify mRNA.15
Western Blots
Membrane fractions were prepared as previously described.15 Protein samples were separated with SDS PAGE, transferred to Immobilon-P polyvinylidene fluoride membranes, and blocked in Tris-buffered saline/5% nonfat milk. Primary antibodies were incubated for 18 hours at 4°C (minK) or 1 to 2 hours at room temperature (KvLQT1, ERG, Kv4.3, and Kv1.4). Antibodies to KvLQT1 (sc-10646; goat), minK (sc-16796; goat), ERG-1a (sc-15968; goat), and Kv4.3 (sc-11686; goat) were purchased from Santa Cruz Biotechnology; and anti-Kv1.4 (APC007; rabbit) was purchased from Alomone. Monoclonal KChIP2 antibody was kindly supplied by James Trimmer. Secondary antibodies were horseradish peroxidase–conjugated donkey anti-goat IgG and anti-rabbit IgG (Santa Cruz). Band signals were detected and quantified with Western-Lightning Chemiluminescence Reagent Plus, laser scanning, and Quantity-One software.
Patch-Clamp Experiments
LV cardiomyocytes were isolated as described previously.6,14 Sham-operated sinus rhythm rabbits were used as controls. All rabbits for cell isolation were euthanized at day 21. Membrane currents were recorded by tight-seal whole-cell patch clamp with Na+-free, K+-free extracellular solutions including N-methyl-D-glucamine to eliminate inward-rectifier K+ current (IK1).6,14 Cell capacitance was determined with ramp pulses (0.5 V/s) from –50 to 70 mV. Cell capacitance and series resistance (RS) were compensated by 50% to 70%. IKr was recorded in the presence of the highly selective IKs blocker HMR-1566 (1 μmol/L)16 and IKs in the presence of the IKr blocker E-4031 (5 μmol/L).
Data Analysis
All data are expressed as mean±SEM. Each biochemical determination was performed on an individual rabbit. Western blot band intensities are expressed after background subtraction as optical density units (ODUs) normalized to GAPDH signal intensity for the same sample. There were no significant differences between LV endocardial and epicardial results, so data from each rabbit were averaged and presented as a single LV value. For the ECG parameters, 2-way repeated-measures ANOVA was performed using mixed model methodology with group and time as main effects. In case of a significant interaction between the 2 main effects, Dunnett contrasts were used to compare them over time within each group. The mixed procedure and multiple comparison with Dunnett’s test were used to evaluate differences in mRNA and protein expression among groups over time. Statistical comparisons for ionic current data were obtained by ANOVA, followed by Tukey’s test. Values of P<0.05 indicated statistical significance. All probability values in the figures are based on ANOVA, followed by Dunnett’s or Tukey’s test.
Results
ECG Changes
Figure 1 shows ECGs at matched pacing rates in a brady-paced and a tachypaced rabbit. Day 21 QT intervals were prolonged in the bradypaced rabbit but were unchanged in the tachypaced rabbit. Figure 1C summarizes ECG changes. QRS duration did not change. In bradycardia rabbits, QT intervals increased from 256±3 ms at 60 bpm (day 0) to nearly steady-state values (332±9 ms) by day 7. In tachycardia rabbits, QT intervals decreased from 256±5 ms at day 0 to 223±5 ms at day 7. During sinus rhythm recording (available only for tachypaced rabbits not subjected to AVB), corrected QT intervals increased from 145±3 ms (day 0) to 158±2 ms (day 21; P<0.05). QTd increased only in bradycardia rabbits.
Spontaneous Ventricular Arrhythmias
Of 44 rabbits entering the bradycardia group, 23 died before day 21. Sixty-one percent (14 of 23) died suddenly, with ECG recordings showing spontaneous TdP. The remaining 9 died of unknown causes. The bradypaced rabbits surviving for 21 days had 66±17 (range, 2 to 232) episodes per rabbit of TdP. Thirteen and 5 bradypaced rabbits were euthanized for molecular studies and patch-clamp experiments, respectively, at day 21. Spontaneous TdP was documented in 33 of 44 bradypaced rabbits (75%). Figure 2A shows intracardiac electrograms stored by implanted pacemakers in 2 brady-paced rabbits. Rabbit 1 developed polymorphic VT at day 7 and died the next day. Rabbit 2 had frequent TdP-like VT before death at day 16. Figure 2B shows continuous telemetry from a bradypaced rabbit. Five days after pacing was set to 60 bpm, spontaneous TdP occurred and degenerated to VF. All rabbits were subjected to a 30-minute ECG recording on day 21. Of 21 bradypaced rabbits surviving to day 21, 16 (76%) showed spontaneous TdP during this recording.
Of the 32 tachypaced rabbits, 21 survived for 21 days, and 11 died prematurely. No tachyarrhythmias were detected with the episode report function of implanted pacemakers. Continuous 24-hour telemetry ECG recording of 7 tachycardia rabbits after pacing rate reduction to 60 to 90 bpm showed a single 7-complex run in 1 (Figure 2C), isolated ventricular premature beats in 3, and no arrhythmia in 3. Of 21 tachypaced rabbits monitored by ECG for 30 minutes on day 21, none showed spontaneous VT (versus 76% of bradypaced rabbits; P<0.001).
Expression of IKs Subunits
Examples of competitive RT-PCR gels for KvLQT1 and minK are shown in Figure 3A. Lane 0 contains 100 ng of DNA mass ladder to create the standard curve. Lanes 1 through 6 were obtained with serial dilutions of the RNA mimic, along with 1 μg total RNA per lane. Top bands represent RNA mimic products. Bottom bands are target bands coamplified with mimics in the same reaction tube. As mimic concentration decreases from left to right, the target band becomes stronger, demonstrating mimic-target competition. Overall, mRNA expression was significantly reduced for both IKs subunits in bradypaced and tachypaced rabbits (Figure 3A, bottom). In bradypaced rabbits, KvLQT1 expression was reduced by averages of 76% and 53% (RV and LV, respectively) and minK by 58% and 43%. In tachypaced rabbits, KvLQT1 expression was reduced by 64% and 51% (RV and LV) and minK by 74% and 43%.
Figure 3B shows KvLQT1 and minK Western blots on 1 gel each. A prominent KvLQT1 band was observed at the expected molecular mass of 75 kDa. Antibody preincubation with antigenic peptide eliminated the signal. The minK blot showed a faint signal at the expected molecular mass of 27 kDa, consistent with previous reports of human, guinea pig, and rabbit minK signals at 27 kDa.15 This signal disappeared on preincubation with antigenic peptide, unlike the higher-molecular-mass signals. KvLQT1 protein expression was reduced by 72% and 53% in the RV and LV, respectively, among bradypaced rabbits and by 78% and 53% for tachypaced rabbits. MinK was downregulated similarly in tachypaced (by 45% and 37% in the RV and LV) and bradypaced (by 37% and 33%) rabbits.
ERG Expression
A typical ERG competitive RT-PCR gel for a control rabbit is shown in Figure 4A. ERG mRNA was significantly downregulated in bradypaced rabbits only (Figure 4B; expression in bradycardia rabbits reduced by 59% in the RV and 52% in the LV). Representative ERG Western blots are shown in Figure 4C. A clear band at the expected molecular mass of 165 kDa was suppressed in bradypaced rabbits, and lower-molecular-weight bands sometimes appeared (3 of 7). All signals were eliminated by antibody preincubation with antigenic peptide. ERG protein expression was significantly downregulated in bradypaced rabbits only (Figure 4D; by 50% in the RV and 37% in the LV).
Expression of Ito Subunits
Figure 5A shows representative competitive RT-PCR gels for Kv4.3 and Kv1.4, as well as mean data. Kv4.3 and Kv1.4 mRNA expression was downregulated in tachy-paced rabbits (Kv4.3: by 53% in RV, 26% LV; Kv1.4: by 77% in RV, 59% LV). Kv4.3 mRNA was unchanged in bradypaced rabbits, whereas Kv1.4 mRNA showed a small decrease of borderline statistical significance in the RV and no significant change in the LV. Kv4.3 antibody revealed bands at 78 and 70 kDa (Figure 5B), consistent with long and short splice variants in rabbit ventricle.17 For Kv1.4, 2 clear bands were detected close to the expected molecular mass of 97 kDa. Ito subunit protein expression was significantly reduced in tachypaced rabbits only, by 60% and 74% (Kv4.3 and Kv1.4, respectively) in the RV and 43% and 36% (Kv4.3, Kv1.4) in the LV. KChIP2 expression was comparable in control, tachypaced, and bradypaced rabbits (eg, averages in the LV were 3.4±0.8, 3.1±1.1, and 3.2±0.6 ODUs, respectively).
Changes in IKr and IKs
Previous data on delayed-rectifier current downregulation in tachypacing-induced HF have varied.14,18 To correlate directly delayed-rectifier subunit expression and current alterations in our rabbits, we performed patch-clamp experiments. Typical IKr tail currents were seen in cells from all groups but were smaller in bradypaced rabbits (Figure 6A). IKs was smaller in both bradypaced and tachypaced rabbits (Figure 6B). Figure 7 shows activation voltage dependence (top) and tail current-voltage relations (bottom). IKr (Figure 7A) activated at more negative voltages than IKs (Figure 7B), following well-recognized properties.6,18 IKr activation voltage dependence was not altered in either bradypaced (V1/2, –7.0±2.5 mV) or tachypaced (V1/2, –8.7±2.0 mV; control, –5.5±1.9 mV) rabbits. IKs activation voltage dependence was similarly unaffected (V1/2, 9.3±4.4 mV for bradypaced, 17.8±3.9 mV for tachypaced, 14.7±1.7 mV for control; P=NS). IKr tail current density was reduced significantly in bradypaced rabbits (by 40%) but was unchanged in tachypaced rabbits (Figure 7C). IKs tail current density was reduced by 70% and 55% in bradypaced and tachypaced rabbits, respectively (Figure 7D). There were no differences among groups in IKr and IKs deactivation kinetics (Table 2).
Response of Tachypaced Rabbits to IKr Inhibition
Our arrhythmia-monitoring data showed that bradypaced but not tachypaced rabbits were highly susceptible to spontaneous TdP. IKs and its subunits KvLQT1 and minK were downregulated similarly by bradypacing and tachypacing, but only bradypacing suppressed ERG/IKr. We hypothesized that IKr reduction might be crucial for differential TdP susceptibility in bradypaced rabbits. We therefore tested the effect of selective IKr blockade with a single intravenous dose of dofetilide (0.02 mg/kg), followed by telemetric monitoring for 3 hours during pacing at 90 bpm in tachypaced rabbits, to mimic IKr downregulation in bradypaced rabbits. The dose of dofetilide was based on previous studies showing that normal rabbits receiving the same dose per minute (total dose 60 times as large) developed TdP and VF with incidences of 2%5 and 17%, respectively.19 Figure 8A shows continuous monitoring after dofetilide administration to a tachypaced rabbit. The QT interval was markedly prolonged, and TdP occurred frequently. All 6 tachypaced rabbits given dofetilide displayed TdP that degenerated into VF. Spontaneous ventricular tachyarrhythmia prevalence in bradypaced rabbits, tachypaced rabbits without dofetilide exposure, and tachy-paced rabbits treated with dofetilide is summarized in Figure 8B. Dofetilide increased VT occurrence in tachypaced rabbits from 0% to 100% (P<0.0001, Fisher’s exact test) to a level comparable to bradypaced rabbits (75%). QT intervals and QTd at 600-ms cycle length were much smaller in tachypaced than bradypaced rabbits in the absence of dofetilide (Figure 8C); however, dofetilide exposure increased values in tachy-paced rabbits to levels comparable to those in bradypaced animals.
Discussion
In this study, we compared directly the K+ channel subunit remodeling and arrhythmic phenotypes resulting from persistent bradycardia versus tachycardia in rabbits. Whereas bradypaced rabbits displayed downregulation of both rapid and slow components of the delayed-rectifier current system, only slow-component subunits and current were suppressed in tachypaced rabbits. This differential ion channel regulation was associated with differential susceptibility to spontaneous ventricular tachyarrhythmias, with bradypaced rabbits showing frequent TdP and tachypaced rabbits manifesting limited spontaneous ventricular arrhythmia. The arrhythmic response of tachypaced rabbits to dofetilide, which transformed their arrhythmic phenotype toward that of bradycardia rabbits, supports the notion that the particular TdP diathesis of bradycardic rabbits is due to IKr downregulation.
Relationship to Previous In Vivo Studies of Rate-Related Remodeling
The tachypacing model of HF is well established for studies of electrophysiological remodeling.2,8–10,14,17,18,20,21 Ito downregulation is the most consistent finding. The response of delayed-rectifier currents has been more variable; although decreases in IKs have been observed consistently,14,18,21 some studies reported decreased IKr14 and others indicated no change.18,21 In bradycardia-related remodeling, IKs is consistently downregulated.6,22 IKr has been reported to be decreased in bradycardic rabbits6 and decreased in RVs but not LVs of bradycardic dogs.22 In this study, IKs was reduced in both bradycardic and tachycardic rabbits, but IKr was reduced only in bradycardia, consistent with changes in mRNA and protein expression of corresponding subunits. In a previous study,14 IKr was reportedly downregulated in tachycardic rabbits; however, 50% activation voltages for IKr and IKs were similar, suggesting possible contamination of E-4031 sensitive currents by IKs rundown.
The propensity of bradycardia-remodeled hearts to TdP is well documented.3–6 Pak et al2 observed nonsustained VT and 1 recorded lethal ventricular tachyarrhythmia in 25 dogs with tachypacing-induced HF. A propensity to early afterdepolarization-related arrhythmias can be unmasked in tachypaced HF by repolarization-prolonging interventions,2,23 suggesting decreased repolarization reserve consistent with the reductions in IKs and associated subunits observed in the present study.
There has been limited work on remodeling of ion channel subunits in animal models involving sustained heart rate abnormalities. Ramakers et al13 observed decreases in both KvLQT1 and minK expression in bradycardic dogs. We were unable to identify other studies of K+ channel subunit remodeling in bradycardic animal models. In tachycardia remodeling, downregulation of Kv4.x subunits is consistently observed.17,20,24 Akar et al24 found a tendency to reduced ERG mRNA in tachypaced dog hearts, although a band of unspecified molecular weight was increased on Western blot. No changes in IKs subunits were seen. Similarly, Rose et al20 did not find significant changes in delayed-rectifier K+ channel subunits in tachypaced rabbits despite significantly decreased delayed-rectifier currents (IKr and IKs were not separated electrophysiologically). In addition to decreased ERG mRNA, our results point to possible posttranslational changes in bradypaced rabbits, because in some cases we observed lower-molecular-weight bands possibly corresponding to immature ERG protein.
Novel Findings and Potential Significance
The present study is the first to compare directly arrhythmic phenotypes and ion channel remodeling in bradycardic versus tachycardic animal models. We have observed substantial differences in spontaneous ventricular arrhythmias, with TdP being ubiquitous in bradycardia-dependent remodeling and never occurring in tachypaced animals. This discrepancy appears to be due to differences in ERG/IKr remodeling. Evidence for the importance of bradycardia-specific ERG remodeling included reduced IKr and ERG mRNA and protein, as well as the transformation of the ECG and arrhythmic phenotype of tachypaced rabbits toward that of bradypaced rabbits with IKr inhibition.
These experimental phenotypes are likely relevant to several clinical phenomena. AVB has been recognized since the 1920s25 as a precipitant of TdP, and TdP is a recognized presentation of congenital heart block.26 Patients with AVB and spontaneous TdP require pacing to at least 70 bpm to prevent excess QT prolongation.27 It has long been thought that the promotion of TdP by bradycardia is due to the acute heart rate–APD relationship, but our studies suggest an intriguing additional possibility: downregulation of ion channel subunits (and in particular ERG) by bradycardia. This notion may also account for another potentially important phenomenon: the ability of chronic -blocker therapy to increase APD. Unexplained APD-prolonging effects of chronic (but not acute) in vivo -blockade have been observed in animal28 and human29 studies, as well as in isolated human atrial myocytes.30 Such effects are potentially relevant to mechanisms of -blocker antiarrhythmic actions and to the varying efficacy of -blockade in patients with congenital long-QT syndrome.
We did not observe spontaneous TdP in tachypaced rabbits with HF; however, TdP occurred in all rabbits on dofetilide exposure. These observations may parallel the uncommon occurrence of TdP in HF patients, despite a clearly increased risk of TdP in HF patients exposed to IKr-blocking drugs.31 These findings are consistent with the concept of "repolarization reserve."32 Downregulation of IKs subunit expression and function may not in itself be sufficient to cause spontaneous TdP, but in the setting of superimposed IKr inhibition or downregulation, repolarization may become unstable and lead to early afterdepolarizations and related tachyarrhythmias.
Potential Limitations
We cannot exclude the possibility that the abnormal activation sequence caused by ventricular pacing contributed to ion channel remodeling. However, if dyssynchrony were a prime factor, we would have expected to see differential remodeling of LV endocardium, epicardium, and RV, which was not the case. It is unlikely that AVB per se contributed to ion channel remodeling, because KvLQT1 protein expression was reduced to the same extent in tachycardia rabbits without (0.12±0.03 ODU) and with (0.10±0.02 ODU) AVB compared with controls (0.36±0.09 ODU).
We were surprised to see QT shortening in tachypaced rabbits with AVB (Figure 1C). This may have resulted because the pacing rate (180 bpm) during the 5-day recovery interval after AVB, before tachypacing, was slower than the spontaneous sinus rate (250 bpm), possibly causing APD prolongation at day 0. This notion is supported by the fact that QT intervals during sinus rhythm increased slightly but significantly over time in tachypaced rabbits without AVB (Figure 1C). The small QT interval increase in the face of significant downregulation of KvLQT/minK and Kv4.3/Kv1.4 subunits encoding IKs and Ito may be due to several factors. IKs is small in the rabbit because of low-level minK expression,15 and pure IKs suppression fails to increase APD in the rabbit.33 In addition, because of the very slow recovery kinetics of rabbit Ito, it contributes very little current at physiological heart rates.34
Our minK protein bands were very weak, consistent with known low-level expression in the rabbit,15 but limiting the reliability of the measurement and requiring caution in interpretation. Therefore, conclusions about the protein expression of this subunit need to be particularly guarded. We elected to perform patch-clamp studies only for delayed-rectifier currents because of previous discrepancies in the literature. Because Ito has been consistently found to be downregulated in HF,2,9,14,18,20,21 a result consistent with our molecular determinations, we did not prepare additional rabbits for Ito measurement and therefore lack such results for direct correlation with biochemical data.
The response of tachypaced rabbits to dofetilide supports the notion that IKr downregulation is the major factor determining arrhythmic phenotype differences between brady-paced and tachypaced animals. We studied a limited number of ion channel subunits and currents. The potential roles of other ion channels and transporters, structural remodeling, differences in autonomic or neurohumoral response, altered activation sequence, mechanical factors, etc, require further exploration. In addition, we did not explore the issue of whether tachypaced rabbits were more susceptible to dofetilide-induced TdP than control rabbits. A full exploration of the potential electrophysiological and arrhythmic consequences of reduced repolarization reserve in brady-paced and tachypaced rabbits would have required extensive additional experiments and, while interesting and important, is beyond the scope of the present paper.
We were unable to separate a distinct midmyocardial layer because of the thinness of the rabbit ventricle. We did evaluate separately ion channel expression changes in subepicardial versus subendocardial tissues and did not observe any significant differences.
The arrhythmic phenotype we noted in tachypaced HF rabbits was different from that reported previously by Pogwizd et al35 in rabbits with HF after aortic banding/regurgitation. The differences are likely due to the different methods of HF induction but emphasize the importance of not generalizing excessively from a single animal model of HF. Differences in underlying cause, duration, and severity of HF likely differentially influence the resulting electrophysiological remodeling and arrhythmic phenotype of HF in humans.
Acknowledgments
Y. Tsuji was a research fellow of Heart and Stroke Foundation of Canada and was supported by Japan Heart Foundation & Bayer Yakuhin Research Grant Abroad. We thank Medtronic Japan for providing pacemakers; Pfizer Pharmaceuticals for dofetilide; the Canadian Institutes of Health Research and Quebec Heart and Stroke Foundation for funding; Chantal Maltais, Marc-Antoine Gillis, Evelyn Landry, and Chantal St-Cyr for technical assistance; and France Theriault for secretarial support.
Disclosures
None.
References
Nerheim P, Birger-Botkin S, Piracha L, Olshansky B. Heart failure and sudden death in patients with tachycardia-induced cardiomyopathy and recurrent tachycardia. Circulation. 2004; 110: 247–252.
Pak PH, Nuss HB, Tunin RS, Kaab S, Tomaselli GF, Marban E, Kass DA. Repolarization abnormalities, arrhythmia and sudden death in canine tachycardia-induced cardiomyopathy. J Am Coll Cardiol. 1997; 30: 576–584.
Vos MA, Verduyn SC, Gorgels AP, Lipcsei GC, Wellens HJ. Reproducible induction of early afterdepolarizations and torsade de pointes arrhythmias by d-sotalol and pacing in dogs with chronic atrioventricular block. Circulation. 1995; 91: 864–872.
Vos MA, de Groot SH, Verduyn SC, van der Zande J, Leunissen HD, Cleutjens JP, van Bilsen M, Daemen MJ, Schreuder JJ, Allessie MA, Wellens HJ. Enhanced susceptibility for acquired torsade de pointes arrhythmias in the dog with chronic, complete AV block is related to cardiac hypertrophy and electrical remodeling. Circulation. 1998; 98: 1125–1135.
Volders PG, Sipido KR, Vos MA, Kulcsar A, Verduyn SC, Wellens HJ. Cellular basis of biventricular hypertrophy and arrhythmogenesis in dogs with chronic complete atrioventricular block and acquired torsade de pointes. Circulation. 1998; 98: 1136–1147.
Tsuji Y, Opthof T, Yasui K, Inden Y, Takemura H, Niwa N, Lu Z, Lee JK, Honjo H, Kamiya K, Kodama I. Ionic mechanisms of acquired QT prolongation and torsades de pointes in rabbits with chronic complete atrioventricular block. Circulation. 2002; 106: 2012–2018.
Jackman WM, Friday KJ, Anderson JL, Aliot EM, Clark M, Lazzara R. The long QT syndromes: a critical review, new clinical observations and a unifying hypothesis. Prog Cardiovasc Dis. 1988; 31: 115–172.
Nabauer M, Kaab S. Potassium channel down-regulation in heart failure. Cardiovasc Res. 1998; 37: 324–334.
Tomaselli GF, Marban E. Electrophysiological remodeling in hypertrophy and heart failure. Cardiovasc Res. 1999; 42: 270–283.
Kaab S, Dixon J, Duc J, Ashen D, Nabauer M, Beuckelmann DJ, Steinbeck G, McKinnon D, Tomaselli GF. Molecular basis of transient outward potassium current downregulation in human heart failure: a decrease in Kv4.3 mRNA correlates with a reduction in current density. Circulation. 1998; 98: 1383–1393.
Borlak J, Thum T. Hallmarks of ion channel gene expression in end-stage heart failure. FASEB J. 2003; 17: 1592–1608.
Volders PG, Sipido KR, Vos MA, Spatjens RL, Leunissen JD, Carmeliet E, Wellens HJ. Downregulation of delayed rectifier K(+) currents in dogs with chronic complete atrioventricular block and acquired torsades de pointes. Circulation. 1999; 100: 2455–2461.
Ramakers C, Vos MA, Doevendans PA, Schoenmakers M, Wu YS, Scicchitano S, Iodice A, Thomas GP, Antzelevitch C, Dumaine R. Coordinated down-regulation of KCNQ1 and KCNE1 expression contributes to reduction of I(Ks) in canine hypertrophied hearts. Cardiovasc Res. 2003; 57: 486–496.
Tsuji Y, Opthof T, Kamiya K, Yasui K, Liu W, Lu Z, Kodama I. Pacing-induced heart failure causes a reduction of delayed rectifier potassium currents along with decreases in calcium and transient outward currents in rabbit ventricle. Cardiovasc Res. 2000; 48: 300–309.
Zicha S, Moss I, Allen B, Varro A, Papp J, Dumaine R, Antzelevich C, Nattel S. Molecular basis of species-specific expression of repolarizing K+ currents in the heart. Am J Physiol Heart Circ Physiol. 2003; 285: H1641–H1649.
Thomas GP, Gerlach U, Antzelevitch C. HMR 1556, a potent and selective blocker of slowly activating delayed rectifier potassium current. J Cardiovasc Pharmacol. 2003; 41: 140–147.
Rose J, Armoundas AA, Tian Y, Disilvestre D, Burysek M, Halperin V, O’Rourke B, Kass DA, Marban E, Tomaselli GF. Molecular correlates of altered expression of potassium currents in failing rabbit myocardium. Am J Physiol Heart Circ Physiol. 2005; 288: H2077–H2087.
Li GR, Lau CP, Ducharme A, Tardif JC, Nattel S. Transmural action potential and ionic current remodeling in ventricles of failing canine hearts. Am J Physiol Heart Circ Physiol. 2002; 283: H1031–H1041.
Lu HR, Van Ammel K, Vlaminckx E, De Clerck F. QT and JT dispersion in the drug-induced long QT syndrome in anesthetized rabbits is accurately detected by a three-lead surface ECG measurement. J Pharmacol Toxicol Methods. 2004; 49: 71–79.
Zicha S, Xiao L, Stafford S, Cha TJ, Han W, Varro A, Nattel S. Transmural expression of transient outward potassium current subunits in normal and failing canine and human hearts. J Physiol Lond. 2004; 561: 735–748.
Han W, Chartier D, Li D, Nattel S. Ionic remodeling of cardiac Purkinje cells by congestive heart failure. Circulation. 2001; 104: 2095–2100.
Volders PG, Sipido KR, Vos MA, Spatjens RL, Leunissen JD, Carmeliet E, Wellens HJ. Downregulation of delayed rectifier K(+) currents in dogs with chronic complete atrioventricular block and acquired torsades de pointes. Circulation. 1999; 100: 2455–2461.
Jones DL, Petrie JP, Li HG. Spontaneous, electrically, and cesium chloride induced arrhythmia and afterdepolarizations in the rapidly paced dog heart. Pacing Clin Electrophysiol. 2001; 24: 474–485.
Akar FG, Wu RC, Juang GJ, Tian Y, Burysek M, Disilvestre D, Xiong W, Armoundas AA, Tomaselli GF. Molecular mechanisms underlying potassium current down-regulation in canine tachycardia-induced heart failure. Am J Physiol Heart Circ Physiol. 2005; 288: H2887–H2896.
Kerr WJ, Bender WL. Paroxysmal ventricular fibrillation with cardiac recovery in a case of auricular fibrillation and complete heart block while under quinidine sulphate therapy. Heart. 1922; 9: 269–281.
Nikolic G, Arnold J, Coles DM. Torsade de pointes and asystole in a child with complete heart block and prolonged QT interval. Aust Paediatr J. 1983; 19: 187–191.
Kurita T, Ohe T, Marui N, Aihara N, Takaki H, Kamakura S, Matsuhisa M, Shimomura K. Bradycardia-induced abnormal QT prolongation in patients with complete atrioventricular block with torsades de pointes. Am J Cardiol. 1992; 69: 628–633.
Raine AE, Vaughan Williams EM. Adaptation to prolonged beta-blockade of rabbit atrial, Purkinje, and ventricular potentials, and of papillary muscle contraction: time-course of development of and recovery from adaptation. Circ Res. 1981; 48: 804–812.
Edvardsson N, Olsson SB. Effects of acute and chronic beta-receptor blockade on ventricular repolarisation in man. Br Heart J. 1981; 45: 628–636.
Workman AJ, Kane KA, Russell JA, Norrie J, Rankin AC. Chronic beta-adrenoceptor blockade and human atrial cell electrophysiology: evidence of pharmacological remodelling. Cardiovasc Res. 2003; 58: 518–525.
Lehmann MH, Hardy S, Archibald D, Quart B, MacNeil DJ. Sex difference in risk of torsade de pointes with d,l-sotalol. Circulation. 1996; 94: 2535–2541.
Roden DM. Taking the "idio" out of "idiosyncratic": predicting torsades de pointes. Pacing Clin Electrophysiol. 1998; 21: 1029–1034.
Lengyel C, Iost N, Virag L, Varro A, Lathrop DA, Papp JG. Pharmacological block of the slow component of the outward delayed rectifier current (I(Ks)) fails to lengthen rabbit ventricular muscle QT(c) and action potential duration. Br J Pharmacol. 2001; 132: 101–110.
Wang Z, Fermini B, Feng J, Nattel S. Role of chloride currents in repolarizing rabbit atrial myocytes. Am J Physiol. 1995; 268: H1992–H2002.
Pogwizd SM, Schlotthauer K, Li L, Yuan W, Bers DM. Arrhythmogenesis and contractile dysfunction in heart failure: roles of sodium-calcium exchange, inward rectifier potassium current, and residual -adrenergic responsiveness. Circ Res. 2001; 88: 1159–1167.(Yukiomi Tsuji, MD; Stephen Zicha, PhD; X)