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1Bioelectricity Laboratory, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston 02115; 2Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110; and 3Cardiovascular Engineering Inc., Holliston, Massachusetts 01746
Submitted 3 April 2003 ; accepted in final form 13 August 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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cardiac arrhythmia; electrophysiology; potassium channels; long QT syndrome
Kv1DN mice were created by overexpression of a truncated Kv1.1 (Kv1.1N206Tag) in the heart and are characterized by a prolonged QT interval and a spontaneous and inducible ventricular tachycardia (VT) (7, 10). Optical mapping of Kv1DN hearts ex vivo revealed substantially shorter effective refractory periods at the apex of left ventricles compared with the base, causing spatial dispersion of refractoriness and repolarization that formed the base for reentrant arrhythmias (2). Interestingly, none of the other mouse models in which the IKr (1), IKs (9), or Kv4.2 (3) channel was functionally knocked out has been reported to have proarrhythmic activity.
There is much interest in determining the roles of different channels in the pathogenesis of arrhythmias and the interrelationships between long QT, electrical remodeling, and the pathogenesis of cardiac arrhythmias. Crossbreeding of Kv1DN mice with Kv4DN mice resulted in mice (Kv1/Kv4DN) with a substantial prolongation of the action potential duration (APD) and QT interval that was associated with suppression of spontaneous and inducible arrhythmias, likely due to the homogenous prolongation of the APD across the left ventricle. Recently, we demonstrated that suppression of the rapidly activating, slowly inactivating current (IK,slow1) in Kv1DN mice led to electrical remodeling with the upregulation of Kv2.1-encoded slowly inactivating outward current (IK,slow2) in apical cardiomyocytes (16), resulting in regional heterogeneities in repolarization. We reasoned that this spatially restricted electrical remodeling might underlie the enhanced dispersion of repolarization observed in Kv1DN hearts (2) and therefore the spontaneous and inducible arrhythmias in these animals. For this study, we crossbred Kv1DN mice with Kv2DN mice to eliminate currents encoded by Kv2.1 to examine whether this modification influences spontaneous and inducible arrhythmias to an extent similar to that produced by crossbreeding with Kv4DN animals. We hypothesized that this would also help delineate the influence of APD/QT prolongation and regional heterogeneities caused by differential expression of Kv1.5, Kv2.1, and Kv4.2 channels in the mouse heart.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Solutions and chemicals. In all experiments, the following physiological solution was used (in mM): 135 NaCl, 5.4 KCl, 0.33 NaH2PO4, 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose (pH was adjusted to 7.35 with NaOH). The L-type Ca2+ and TTX-sensitive Na+ channels were blocked by the application of 2 mM CoCl2 and 20 µM TTX, respectively. The cells in the chamber were superfused with the appropriate external solution by means of a peristaltic pump (Peri-Star). The pipette solution contained (in mM) 140 KCl, 1 MgCl2, 10 HEPES, 5 EGTA, 5 Mg2ATP, 0.1 GTP (pH 7.2 with KOH). The contamination by ATP-sensitive potassium currents was avoided with the use of a high concentration of ATP in the intracellular solution. Stock solutions of TTX, 4-aminopyridine (4-AP), and tetraethylammonium (TEA) in 2 to 10 mM concentration were prepared. Collagenase type I was purchased from Worthington Biochemical (Lakewood, NJ). All other chemicals were purchased from Sigma (St. Louis, MO).
Cellular electrophysiology and data analysis. Mouse ventricular myocytes were obtained as described previously (10, 15). Calcium-tolerant, rod-shaped ventricular myocytes with clear striations were selected randomly for electrophysiological studies at 35°C. Membrane currents were recorded using the whole cell mode of the patch-clamp technique (6). The patch electrodes (P87; Sutter) were pulled from 1.5-mm-diameter borosilicate glass capillaries (WPI; Sarasota, FL). Their resistances were 0.8 to 2 M
when filled with a standard pipette solution. An Axopatch 200B amplifier in combination with pCLAMP version 8.1 software and DigiData 1322A (Axon Instruments) was used for data acquisition. Raw traces are shown without correction for leakage currents. Experiments showing a significant leak, which was recognized after depolarizing steps, were discarded. In this study, the cell capacitances were 134.5 ± 6.8 pF (n = 28) in wild-type (WT) myocytes, 151.6 ± 12.8 pF (n = 25) in Kv1DN, 208.5 ± 28.7 (n = 7) in Kv2DN, and 204.2 ± 17.1 in Kv1/Kv2DN (n = 24). Statistical ANOVA revealed that capacitance of cardiomyocytes expressing Kv2DN transgene was higher than that of myocytes derived from WT or Kv1DN (P < 0.05). The series resistances were in the range of 2–10 M and were compensated electronically by 75%. Data analysis and graphs were obtained with Clampfit 8.1 (Axon Instruments), Excel 2000 (Microsoft), and Origin version 7.0 software (Microcal).
ECG recordings in awake, free-moving mice. ECG recordings of awake, free-moving mice were obtained as previously described (7). Briefly, adult (3–10 mo old) animals were sedated with pentobarbital (50 mg/kg ip) and transmitters (model EA-F20, DataSciences) were implanted subcutaneously on the back of the animals. The positive lead was tunneled to the left anterior chest wall above the apex of the heart and the negative lead to the right shoulder. This configuration approximates lead II on the surface ECG. After 24 h of recovery, ECGs were recorded continuously for 24 h with a 500 Hz analog-to-digital converter; acquired data were stored on a standard IBM-compatible personal computer using custom-designed software. Semiautomatic offline analysis of the whole recording was performed with the use of an algorithm with automatic R wave and premature beat detection (11). Ventricular premature beats (VPBs) were identified when at least two of three criteria were met: 1) atypical QRS configuration with alteration or inversion of the T wave; 2) postextrasystolic pause; and 3) atrioventricular dissociation. For bradycardia, a pause of 
300 ms was considered significant. All ECGs were analyzed by a blinded operator.
Measurement of heart rate and QT intervals. Heart rate and QT intervals were measured from the 24-h recordings; the measurements were taken every 20 min, resulting in 72 measurements per animal. Intervals were measured on screen with calipers on an averaged and overlaid signal derived from all beats during a 4-s period, a method that has been shown to reduce artifacts without significantly affecting measurements (11). In case of noise or movement artifacts during the 4-s period, the measurement was taken 1 min later. QT intervals were corrected for heart rate using an established formula for awake, free-moving mice (11).
Right ventricular electrophysiological stimulation in sedated mice. Electrophysiological studies were performed by transvenous right ventricular stimulations in anesthetized mice (50 mg/kg ip pentobarbital) using an extended protocol as described previously (4). A full ventricular stimulation protocol was performed sequentially three times: 1) without medication (except for anesthesia), 2) after intraperitoneal atropine, and 3) after an intraperitoneal injection of isoproterenol. Right ventricular refractory periods were assessed with 2-ms decrements after a 10-beat stimulation train of 100-ms cycle length. The stimulation protocol consisted of 20-beat trains with up to three extrastimuli; each extrastimulus was initially coupled with the basic cycle lengths (BCLs), and the coupling interval was subsequently decreased in 10-ms steps until the shortest possible interval was reached. Each ventricular stimulation was performed at two different BCLs of 100 ms and either 70 or 130 ms, depending on the ventricular refractory periods. After the ventricular stimulations and the assessment of refractory times, atropine (20 µg) was injected, and a minimum of 3 min was allowed for stabilization of the heart rate; the ventricular stimulation (with two different BCLs) was then repeated. Thereafter, isoproterenol (10 µg) was injected intraperitoneally to increase the heart rate by at least 30%. After a minimum of 2 min for the stabilization of the heart rate, the ventricular stimulation (again with 2 BCLs) was repeated. As previously stated (4), an induced VT had to last >5 beats, and shorter runs of induced VPBs were counted as premature complexes but not classified as VT. All induced VTs had to be confirmed by a second stimulation to be counted. During the procedure, heart rate and respiration were continuously monitored, and the animals were placed under a heating lamp to maintain body temperature. For all stimulations, the ECG leads I, II, and III were recorded on a personal computer for offline analysis. Only mice surviving at least 24 h (after the stimulation) were included. All in vivo electrophysiological studies were performed and analyzed by a blinded operator.
Statistical analysis. Statistical analysis employed one-way and two-way ANOVA for continuous variables, with Bonferroni's test for post hoc analysis. The Kruskal-Wallis test (with Dunn's post hoc test) was used for non-Gaussian distributed values; 
2-tests were performed for categorical values. Student's paired t-tests were used for differences between two data groups. Analysis was performed with Prism version 3.03 (GraphPad; San Diego, CA) and SPSS version 10.0.1 (SPSS; Chicago, IL) software packages. The data are presented (unless otherwise noted) as means ± SE, and n corresponds to the number of experiments. A P value < 0.05 was considered significant.
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RESULTS |
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We next used a pharmacological approach to prove that the two components of IK,slow were eliminated in Kv1/Kv2DN mice. The sequential use of 50 µM 4-AP and then 50 µM 4-AP and 5 mM TEA in WT myocytes (Fig. 2, B and C) blocked outward currents encoded by Kv1.5 and Kv2.1, respectively. Subtraction of currents inhibited by these drugs revealed that the amplitudes of 4-AP- and TEA-sensitive currents were 11.3 ± 2 pA/pF (n = 17) and 8.9 ± 1.8 pA/pF (n = 15), respectively (Fig. 2, D–F, left panels). Application of 4-AP and TEA revealed that both components were eliminated in Kv1/Kv2DN mice (Fig. 2, D–F, right panels).
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Action potentials are further prolonged in Kv1/Kv2DN mice. The voltage-clamp experiments described above showed that both components of IK,slow were eliminated in left ventricular myocytes derived from Kv1/Kv2DN mice. To determine the effects of elimination of these currents on the repolarization phase of Kv1/Kv2DN myocytes, action potential waveforms in left ventricular myocytes were recorded at physiological temperature. As illustrated in Fig. 3, A–D, current-clamp experiments revealed that action potentials recorded at 1 Hz in physiological temperatures from Kv1/Kv2DN cells are broader than action potentials recorded from WT, Kv1DN, and Kv2DN myocytes. APD at 90% repolarization (APD90) was increased in a stepwise fashion, although the APD90 of Kv1DN myocytes did not differ significantly from that of Kv2DN myocytes (Fig. 3E). No differences in action potential amplitude or in resting membrane potential were observed (data not shown). Importantly, none of the WT cells (n = 28) showed early afterdepolarizations, whereas 20 of 33 Kv1/Kv2DN myocytes exhibited this phenomenon (P < 0.01).
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Corrected QT intervals are prolonged in stepwise fashion. Using implanted transmitters in awake, free-moving mice, we assessed heart rate and QT intervals during the 24-h period from 6–8 animals per group as a repeated measure by averaging the heart rate from a 4-s screen every 20 min of the 24-h recording, giving 72 measurements (levels) per animal. Overall, 2,075 heart rate/QT pairs were measured. The mean heart rates of the animals were (±SD) WT: 613.8 ± 74.3 beats/min, Kv1DN: 600.7 ± 90.3 beats/min, Kv2DN: 594.3 ± 68.9 beats/min, and Kv1/Kv2DN: 599.7 ± 78.6 beats/min. By using two-way ANOVA (unweighted means analysis), we found a significant influence of time of day on heart rate (P = 0.0012) and a highly significant influence of genotype on heart rate (P < 0.0001).
To analyze the QT intervals we used a previously established formula for correction to heart rates; the corrected QT (QTc) intervals showed a significant stepwise increase from WT to Kv1/2DN: WT: 55.85 ± 5.1 ms, Kv1DN: 58.87 ± 6.4 ms, Kv2DN: 64.15 ± 5.6 ms, Kv1/Kv2DN: 67.38 ± 6.4 ms (overall P < 0.001, P < 0.001 between all groups) (Fig. 4C). These differences were maintained throughout the day, with virtually parallel stacked QTc intervals, as shown in Fig. 4D. With the use of two-way ANOVA, both time of day (P < 0.01) and genotype (P < 0.0001) were shown to significantly influence the QTc interval; however, we found no significant difference in the influence of time of day on QT intervals between the different genotypes.
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Spontaneous and inducible arrhythmias. A blinded analysis of spontaneously occurring arrhythmias during the 24-h monitoring was performed in 7 or 8 animals per group. Differences in the mean number of single VPBs was not statistically significant between the groups (WT: n = 2.5 ± 1.9, Kv1DN: n = 3.4 ± 3.1, Kv2DN: n = 1.3 ± 0.9, Kv1/Kv2DN: n = 2.0 ± 1.2, P = not significant). Couplets, triplets, and quadruplets were seen in two of seven Kv1DN, three of eight Kv2DN, and in one of eight Kv1/Kv2DN. By contrast, none of the WT animals had complex arrhythmias (P = not significant). There were no significant differences in the incidence of bradyarrhythmias. Both pauses longer than 300 ms and Mobitz type II and III atrioventricular (AV) block were observed in all groups: second-degree AV block was seen on one or more instances in 4 WT, 2 Kv1DN, 4 Kv2DN, and 5 Kv1/Kv4DN animals (P = not significant). Complete AV block was detected in 2 WT, 2 Kv1DN, 4 Kv2DN, and 1 Kv1/Kv2DN mice, and ventricular pauses (of any kind) longer than 300 ms were seen in 5, 5, 6, and 8 animals, respectively (P = not significant).
In vivo electrophysiological studies in sedated mice were successfully completed in 10 WT, 8 Kv1DN, 10 Kv2DN, and 10 Kv1/Kv2DN animals, with an average age of 142.2 ± 5.1, 175.9 ± 17.6, 200.3 ± 29.8, and 193.1 ± 23.8 days, respectively (P = not significant). Similarly, the weight at stimulation did not differ significantly. When right ventricular refractory periods were assessed with 2-ms steps, significant prolongation was observed in the Kv2DN and the Kv1/Kv2DN groups (WT: 44.6 ± 3.8, Kv1DN: 57.4 ± 3.4; Kv2DN 63.0 ± 2.5 Kv1/Kv2DN: 58.4 ± 3.9 ms; overall ANOVA, P < 0.005; WT vs. Kv2DN and WT vs. Kv1Kv2DN: P < 0.05). Before the application of atropine or isoproterenol, VTs lasting longer than five beats were induced in one animal in the WT and one in the Kv1DN group (Fig. 4D), but in none of the animals in the other groups. The Kv1DN mouse exhibited a fast, sustained VT at VR ejection fraction testing. Thereafter, intraperitoneal atropine was administered, which increased the mean heart rate from 379.5 ± 12.3 to 399.1 ± 13.0 beats/min (P < 0.001) without significant differences between groups. One WT mouse was inducible for sustained VT (>5 beats) after atropine, compared with none in the other groups. A brief run of a VT (<5 beats) was induced in a single Kv1/Kv2DN mouse (Fig. 4E). The administration of isoproterenol increased the mean heart rate from 406.7 ± 14.6 beats/min to 531.0 ± 15.5 beats/min (P < 0.05) without significant differences between groups. During the stimulation after isoproterenol, 3 WT, 2 Kv1DN, 2 Kv2DN, and 3 Kv1/Kv2DN animals were inducible for VT lasting longer than 5 beats (P = not significant). In summary, VT was induced in 5 of 10 WT, 3 of 8 Kv1DN, 2 of 10 Kv2DN, and 3 of 10 Kv1/Kv2DN mice (P = not significant). In inducible mice, both polymorphic and rapidly alternating VTs were seen, sometimes with a change in morphology during the tachycardia in the same mouse. All induced arrhythmias except for one in a Kv1DN mouse ended spontaneously; this mouse died as a consequence of the arrhythmia.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Single-cell patch-clamp experiments showed that peak outward currents decreased in Kv1/Kv2DN mice. Analyses of the outward potassium currents revealed that overexpression of Kv1N206 and Kv2N216 in the heart resulted in the elimination of the 4-AP-sensitive component (IK,slow1) and the TEA-sensitive component (IK,slow2), respectively. In addition, expression of Kv2N206 was associated with a significant reduction in peak currents, whereas Iss remained unchanged. The reduction in peak current likely reflects a reduction in Ito,f in these mice. This reduction is associated with increased cell capacitance and may therefore reflect a hypertrophic-like response of the cardiomyocytes to the expression of Kv2DN transgene. The inhibition of IK,slow1 and IK,slow2 permits a more direct assessment of the kinetics of inactivation of Ito,f. Indeed, the inactivation phase can be fitted with one exponent that resembles that of Kv4.2 channels.
As we reported earlier, Kv2N216 abolished the upregulation of TEA-sensitive currents in Kv1DN myocytes (16), and as anticipated, both Kv1DN and Kv2DN transgenes prolonged the APD. The effect seen in Kv1DN mice was more pronounced than that seen in Kv2DN mice, although it did not reach statistical significance. The in vivo data showed that the APD prolongation was reflected in QT/QTc prolongation. Kv1/Kv2DN mice had a substantially prolonged QT interval, which correlated with prolongation of the APD. Interestingly, Kv2DN mice had a slightly prolonged QTc compared with Kv1DN mice, whereas APD was not significantly different due to the large variation in APD. Clearly, several factors influence the length of the QTc interval on the surface ECG compared with single-cell APDs; among these are regional differences in the expression of repolarizing potassium channels and the vector and subsequent downslope of the T wave (which influences the measurement of the T end).
The in vivo electrophysiological studies revealed high inducibility in WT crossbred mice as well as in all other genotypes. Thus the elimination of the TEA-sensitive component of IK,slow in Kv1DN mice did not have an antiarrhythmic effect, whereas in a previous study the elimination of Ito,f in (Kv1/Kv4DN) mice with an otherwise identical genetic background was associated with a significant antiarrhythmic effect (4). Importantly, the QT interval was prolonged to the same extent in Kv1/Kv2DN and in the previously reported Kv1/Kv4DN mice. Thus the QT prolongation per se was not sufficient to suppress spontaneous or inducible arrhythmias in Kv1/Kv2DN. We therefore conclude that although Kv2.1-encoded currents are upregulated in the apex of Kv1DN mice, this upregulation per se does not explain the dispersion of repolarization and refractoriness observed in these mice. By contrast, attenuation of Kv4-encoded currents in Kv1/Kv4DN mice was sufficient to abolish arrhythmias. Collectively, these observations suggest an important role for Kv4-encoded currents in the pathogenesis of reentrant arrhythmias in mice, contrasting with the observations of Kuo et al. (8), who reported a proarrhythmic effect in mice with knockout of KCHIP and elimination of Ito,f.
In conclusion, we have shown that the dominant negative constructs eliminated the 4-AP- and TEA-sensitive components of IK,slow in mouse ventricular myocytes, resulting in prolongation of the APD and QT intervals. The more marked prolongation of APD and QT interval was not associated with suppression of spontaneous or inducible arrhythmias.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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subunit. Circ Res 83: 560–567, 1998. subunit. Circ Res 85: 623–633, 1999.This article has been cited by other articles:
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), fast component transient outward current (Ito,f; 
), and steady-state outward current (Iss; 
).
).
