Targeted Replacement of Kv1.5 in the Mouse Leads to Loss of the 4-Aminopyridine–Sensitive Component of IK,slow and Resistance to Drug-Induced QT Prolongation
Abstract—The K+ channel mKv1.5 is thought to encode a 4-aminopyridine (4-AP)–sensitive component of the current IK,slow in the mouse heart. We used gene targeting to replace mKv1.5 with the 4-AP–insensitive channel rKv1.1 (SWAP mice) and directly test the role of Kv1.5 in the mouse ventricle. Kv1.5 RNA and protein were undetectable, rKv1.1 was expressed, and Kv2.1 protein was upregulated in homozygous SWAP hearts. The density of the K+ current IK,slow (depolarizations to +40 mV, pA/pF) was similar in left ventricular myocytes isolated from SWAP homozygotes (17±1, n=27) and littermate controls (16±2, n=19). The densities and properties of Ipeak, Ito,f, Ito,s, and Iss were also unchanged. In homozygous SWAP myocytes, the 50-μmol/L 4-AP–sensitive component of IK,slowwas absent (n=6), the density of the 20-mmol/L tetraethylammonium-sensitive component of IK,slow was increased (9±1 versus 5±1, P<0.05), and no 100- to 200-nmol/L α-dendrotoxin–sensitive current was found (n=8). APD90 in SWAP myocytes was similar to controls at baseline but did not prolong in response to 30 μmol/L 4-AP. Similarly, QTc (ms) was not prolonged in anesthetized SWAP mice (64±2, homozygotes, n=9; 62±2, controls, n=9), and injection with 4-AP prolonged QTc only in controls (63±1, homozygotes; 72±2, controls; P<0.05). SWAP mice had no increase in arrhythmias during ambulatory telemetry monitoring. Thus, Kv1.5 encodes the 4-AP–sensitive component of IK,slow in the mouse ventricle and confers sensitivity to 4-AP–induced prolongation of APD and QTc. Compensatory upregulation of Kv2.1 may explain the phenotypic differences between SWAP mice and the previously described transgenic mice expressing a truncated dominant-negative Kv1.1 construct.
Transgenic and gene-targeting technologies have led to marked advances in the understanding of the molecular basis of cardiac repolarization in the mouse.1 2 Dominant-negative transgenic mice overexpressing mutated Kv1.x, Kv2.x, Kv4.x, and HERG α subunits have less repolarizing K+ current, varying degrees of cardiac action potential duration (APD) and QT prolongation, and arrhythmias3 4 5 6 7 In these experiments, the transgene may interact with several related K+ channels in the heart, the phenotype may depend on the details of the transgene design,5 6 and the relationship of individual gene products to the phenotype may be unclear. Gene targeting of K+ channels using embryonic stem (ES) cells circumvents several of these difficulties by directly knocking out a single gene product.8 9 10 11 12 13 However, gene targeting usually leads to loss of the gene in multiple organs and throughout development and is still subject to compensatory upregulation of other genes. Cross-mating lines of mice with different mutations provides one mechanism to sort out these interactions.14
We previously reported dominant-negative transgenic mice that overexpress in the heart an N-terminal fragment of the rat brain K+ channel rKv1.1, have QT prolongation, and lack a rapidly activating, slowly inactivating, 4-aminopyridine (4-AP)–sensitive K+ current, IK,slow, in their ventricular myocytes.3 15 These mice have both spontaneous and inducible ventricular arrhythmias, attributable at least in part to increased dispersion of repolarization and refractoriness between the apex and the base of the heart.16 17 Although these mice have decreased protein levels of Kv1.5, the transgene may disrupt other cardiac K+ channels, including Kv1.4, which has been shown to encode Ito,s.9 14 Thus, the precise role of the loss of Kv1.5 in the pathogenesis of the phenotype is uncertain. In addition, nothing is known about the relationship of Kv1.5 to Kv2.x, the subunits responsible for the 4-AP–resistant component of IK,slow.4
Here we report gene-targeted mice in which mKv1.5 is replaced by the 4-AP–insensitive channel subunit rKv1.1 (SWAP mice). The 4-AP–sensitive component of IK,slow is absent in ventricular myocytes isolated from these animals, proving definitively that Kv1.5 underlies this current. Of note, total IK,slow is unchanged at least in part because of the upregulation of the tetraethylammonium (TEA)-sensitive component encoded by Kv2.1. As a result, SWAP mice have normal cellular APDs and QT intervals on baseline electrocardiograms (EKGs) and resistance to drug-induced prolongation of APD and QT intervals after exposure to 4-AP.
Materials and Methods
All animal experiments were approved by the Institutional Animal Care and Use Committees at the University of Pittsburgh or Washington University School of Medicine.
The mouse Kv1.5 gene (mKv1.5) was cloned (genomic SV129 library, Stratagene), restriction-mapped, and sequenced. A targeting construct was engineered using a 2-kb 5′ arm consisting of the promoter and 5′-untranslated region (UTR) of mKv1.5, the rat Kv1.1 K+ channel (rKv1.1) tagged with the 9-amino-acid hemagglutinin tag (HA) and cloned into the SmaI site located at position −6 of mKv1.5 (relative to the ATG start codon), a neomycin resistance cassette (NeoR), a 3-kb 3′ arm starting at the XbaI site in the 3′-UTR of mKv1.5, and the thymidine kinase gene (TK) for negative selection (Figure 1A⇓). Homologous recombination with this construct should yield rKv1.1 driven by the mKv1.5 promoter, although the effect of the NeoR cassette is unknown, and any 3′ regulatory elements may be lost.
Electroporation of ES cells, identification of ES cell lines heterozygous for the targeted allele, blastocyst injection to obtain chimeras, and mating with C57BL/6 mice to obtain germ-line transmission and mice heterozygous for the targeted allele (SWAP heterozygotes) were done as previously described.8 Mice were backcrossed into the C57BL/6 line two additional generations. Male and female SWAP heterozygotes were then mated to yield the 2- to 7-month-old SWAP homozygotes, SWAP heterozygotes, and wild-type littermate controls used in the subsequent experiments. All mice were genotyped using genomic Southern blots.
The coding region of mKv1.5 was cloned into the high-expression oocyte vector pGH19K after addition of a Kozak consensus sequence.18 The HA epitope was added to the 3′ end of rKv1.1 by polymerase chain reaction (PCR) (rKv1.1-HA). Channel properties were tested in Xenopus oocytes injected with in vitro transcribed cRNA (50 ng/oocyte) using the 2-microelectrode voltage-clamp technique as previously described.19
Single ventricular myocytes were isolated either from the entire left ventricle or separately from the left ventricular apex and septum, and whole-cell voltage- and current-clamp studies were performed at room temperature or 35°C using a Dagan 3900A (Dagan Corporation) or an Axopatch-1D (Axon Instruments) patch-clamp amplifier interfaced to a microcomputer equipped with a Digidata 1200 Series analog-to-digital interface and pClamp 7 software (Axon Instruments).9 20 Membrane capacitance and series resistance were compensated electronically (>85%). Voltage errors were <6 mV and not corrected. Only data from cells with input resistances >0.7 GΩ were analyzed.
EKGs and Ambulatory Telemetry
Three lead EKGs (leads I, II, and AP) were performed on mice anesthetized with avertin (0.5 mg/kg) using subcutaneous electrodes, a differential amplifier (Warner DP301), and an analog-to-digital converter (MacLab, ADInstruments). Signals were digitized at 1 kHz and stored on computer. QT interval (ms) was determined as the time to 95% return to baseline by an individual blinded to genotype and corrected for heart rate using the formula QTc=QT/(√ RR/100).3 21 At least 6 days after implantation of the telemetry device (TA10ETA-F20, Data Sciences), 24-hour ambulatory EKG recordings were performed digitized at 400 Hz, stored on disk, and analyzed by hand for arrhythmias and using custom software designed to determine heart rate and heart rate variability.
Voltage-clamp data were analyzed using Clampfit 6.0.5 (Axon Instruments), and current densities, normalized for cell capacitance (pA/pF), are reported. The amplitudes of the IK,slow, Ito,f, Ito,s, and ISS were determined by fitting the decay phase of the outward K+ currents to the sum of two or three exponentials, as described previously.9 14 20 Correlation coefficients were determined, and the χ2 test was used to assess the quality of the fits. The amplitudes of the drug-sensitive currents were obtained by offline digital subtraction of the records obtained before and after drug application. All data are presented as mean±SEM. Statistical significance was determined using one-way ANOVA followed by Student-Neuman-Keuls test for comparison between groups or Student’s t test with correction for multiple comparisons, with P<0.05 defined as significant.
Expression of mKv1.5 and rKv1.1-HA In Vitro
In vitro transcribed cRNA from mKv1.5 yielded delayed rectifier currents with minimal inactivation (Figure 1B⇑). Steady-state inactivation curves (generated in 50 mmol/L rubidium chloride from isochronal tail current measurements) gave a total gating valence (z=4.6±0.1 e−) and a voltage of 10% activation (V10%=−32±2 mV) (n=6). This gating valence is 23% lower and V10% is shifted 20 mV to the right compared with rKv1.1 (P<0.01).
Addition of the 9-amino-acid HA epitope to rKv1.1 did not affect the currents when injected into Xenopus oocytes. We subcloned rKv1.1-HA into the prokaryotic/eukaryotic vector pBK-CMV (Stratagene, California) and transfected the construct into COS cells. The HA epitope was easily detected using monoclonal antibodies both on Western blots and by immunofluorescence (data not shown).
Properties of SWAP Mice
Two lines of targeted ES cells were identified (SWAPL77 and SWAPL46; Figure 1C⇑). Additional insertion sites of the rKv1.1 transgene were excluded by genomic Southern blot using a probe within the transgene. The targeted band in the SWAPL46 ES line was reproducibly weaker than the native band, suggesting that the targeted allele was less abundant. This could result from either a mixed population of ES cells or a degree of aneuploidy. Both ES lines underwent germ-line transmission, however, and heterozygotes had one copy of each allele in equal abundance. Experimental results were confirmed on both lines of mice.
SWAP homozygotes from both lines appeared phenotypically normal, and there was no evidence of increased mortality. Hearts were not hypertrophied, and histology of the hearts was normal (data not shown). No overt neurological phenotype was evident in the mice.
K+ Channel Expression in SWAP Mice
Kv1.5 RNA and protein were absent in hearts from SWAP homozygotes (Figures 1D⇑ and 1E⇑). KV1.1 RNA was detected in SWAP homozygotes by Northern blot (Figure 1D⇑), and reverse transcriptase (RT)-PCR was used to confirm the expression of the transgene (Figure 1F⇑). Using antibodies against the HA epitope, we detected a specific band at ≈62 kDa in the hearts of SWAP mice that was identical in size to the band produced by transfecting rat neonatal cardiac myocytes with rKv1.1-HA (Figure 1E⇑). We did not detect rKv1.1-HA protein in the brain, although native mKv1.1 protein was quite abundant and runs at ≈72 kDa, as previously described (data not shown).22
Kv2.1 RNA expression was not significantly changed in homozygous SWAP compared with control ventricles (0.95±0.14, n=3 each; Figure 1D⇑), but Kv2.1 protein was increased by Western blot analysis (n=3 each; Figure 1E⇑).
K+ Current Densities Are Unchanged in SWAP Myocytes
Figure 2⇓ shows representative currents from voltage-clamped, randomly dispersed, left ventricular myocytes isolated from SWAP homozygotes and wild-type controls obtained using 4-second depolarizing steps from a holding potential of −70 mV to potentials between −20 and +40 mV. No differences are apparent in the amplitudes or inactivation rates at either 25°C or 35°C. The mean±SEM peak outward K+ current densities at +40 mV at 25°C were 46±2 pA/pF in cells isolated from SWAP homozygotes (n=27), 45±2 pA/pF in cells from SWAP heterozygotes (n=8), and 47±3 pA/pF in cells from littermate controls (n=19).
The decay phases of the outward currents recorded from these left ventricular myocytes were well-fitted by the sum of 2 exponentials, consistent with the expression of Ito,f, IK,slow, and the steady-state noninactivating current ISS.9 20 The density of IK,slow (at +40 mV) was similar for cells from SWAP homozygotes (17±1 pA/pF; n=27), SWAP heterozygotes (14±2 pA/pF; n=8), and controls (16±2 pA/pF; n=19). In addition, the decay time constants for IK,slow (at +40 mV) were indistinguishable for cells isolated from SWAP homozygotes (1259±51 ms; n=27), SWAP heterozygotes (1177±82 ms; n=8), and controls (1270±48 ms; n=19).
Similar experiments were performed on cells isolated separately from either the left ventricular apex or septum of SWAP mice. The mean±SEM peak outward K+ current densities in the left ventricular apex (n=14) and septum (n=8) were 60±6 and 29±3 pA/pF, respectively. These values, as well as the densities of Ito,f, IK,slow, and ISS, are indistinguishable from those measured in wild-type apex and septal cells.9 14 20 In addition, analysis of the currents in septal cells isolated from SWAP mice revealed that the mean±SEM Ito,s density (8.2±0.8 pA/pF) and inactivation time constant (τdecay=199±21 ms) are not significantly different from those determined for Ito,s in wild-type cells.9 14 20 These results are consistent with previous findings demonstrating that Kv1.4 underlies Ito,s and does not contribute to IK,slow. They also suggest that Kv1.5 and Kv1.4 do not coassemble to form functional heteromultimeric voltage-gated K+ channels in mouse ventricular myocytes.
4-AP–Sensitive Component of IK,slow Is Absent in SWAP Myocytes
After exposure to 50 μmol/L 4-AP, wild-type myocytes showed a marked decrease in IK,slow (Figure 3A⇓), as has been shown in previous studies.3 4 9 15 20 This low concentration of 4-AP also blocked Ito,f by ≈20%, an observation also consistent with previous studies.20 The 50-μmol/L 4-AP–sensitive component of IK,slow, however, was completely absent in myocytes isolated from SWAP homozygotes (n=6). This provides strong evidence that Kv1.5 encodes an α subunit necessary for this current. In contrast, the component of IK,slow sensitive to 20 mmol/L TEA (at +40 mV; Figure 3B⇓) was increased in myocytes from SWAP homozygotes (9±1 pA/pF, n=11) compared with controls (5±1 pA/pF, n=6; P<0.05). At this concentration, TEA partially blocks the component of IK,slow encoded by Kv2.1 channels.4 20 Together with the Western blot data (Figure 1E⇑), these findings suggest that functional Kv2.1 channels are upregulated in response to the loss of Kv1.5.
We were unable to identify any currents in myocytes from either wild-type or SWAP mice that were sensitive to 100 to 200 nmol/L α-dentrotoxin (DTX, n=8). Because Kv1.1 is highly sensitive to DTX, this suggests that functional Kv1.1 channels are not expressed at significant levels in SWAP myocytes.
Action Potentials in SWAP Myocytes Are Insensitive to 4-AP
APD90 values determined at 1-Hz stimulation were similar in current-clamped myocytes isolated from SWAP homozygotes (35±2 ms, n=8, at 25°C; 27±3 ms, n=7, at 35°C) and wild-type controls (36±3 ms, n=16, at 25°C; 25±1 ms, n=13; P=NS). As previously reported, wild-type myocytes show marked APD prolongation in response to low concentrations of 4-AP (Figure 4⇓).15 20 As predicted from the voltage-clamp studies, myocytes from SWAP mice were insensitive to 30 μmol/L 4-AP (n=3).
QT Interval in SWAP Mice Is Insensitive to 4-AP
The effects of 4-AP on QT intervals are shown in Figure 5⇓. Baseline QT interval and QTc were not prolonged in anesthetized homozygous SWAP mice compared with age- and strain-matched controls (QTc: 64±2 ms, homozygotes, n=9; 60±2 ms, heterozygotes, n=4; 62±2 ms, controls, n=9). Injection with 4-AP (10 μmol/kg IP) prolonged QTc in controls but not in SWAP homozygotes (63±1 ms, homozygotes; 66±2 ms, heterozygotes; 72±2 ms, controls; P<0.05). The heterozygotes had an intermediate degree of QTc prolongation when injected with 4-AP. Injection with 4-AP did not induce arrhythmias in either control or SWAP mice. Heart rate was not significantly changed in anesthetized SWAP homozygotes compared with controls, and injection with saline had no effect on QTc (data not shown).
SWAP Mice Have No Increase in Arrhythmias
Mean heart rate, measured using ambulatory telemetry monitors in unanesthetized untethered mice, was similar in SWAP homozygotes compared with littermate controls (610 versus 622 bpm, n=3 each). Standard deviation of cycle length over 24 hours, a gross measure of heart rate variability, was also similar in these mice (11±1 versus 10±7 ms). In addition, SWAP homozygotes had no increase compared with littermate controls (n=4 each) in the frequency of premature atrial complexes (11±3 versus 9±6 per day), premature ventricular complexes (5±1 versus 5±2 per day), or episodes of second-degree atrioventricular block (18±10 versus 22±5 per day). During the 192 hours of telemetry screened, one SWAP homozygote had an atrial couplet and a ventricular triplet, whereas one control mouse had a ventricular couplet.
Kv1.5 Encodes the 4-AP–Sensitive Component of IK,slow
The mouse cardiac K+ channel subunit mKv1.5 encodes a rapidly activating, very slowly inactivating current when heterologously expressed in Xenopus oocytes (Figure 1B⇑). Similar Kv1.5 channel subunits have been cloned from rat and human and are highly sensitive to 4-AP and resistant to TEA.23 24 25 On the basis of these pharmacological and kinetic characteristics, Kv1.5 channels were proposed as the molecular basis for IKur in human atrium and IK,slow in mouse ventricle.26 27 Antisense experiments in cultured heart cells have provided direct experimental evidence that Kv1.5 contributes to IKur in rat and human.28 29
We previously used a dominant-negative transgenic strategy to disrupt channels of the Kv1.x family in the heart, leading to a mouse that lacks the 4-AP–sensitive component of IK,slow.3 15 These mice have decreased protein levels of Kv1.5, probably because of increased degradation. The transgene does, however, affect other channels, and these experiments do not completely prove the relationship between mKv1.5 and IK,slow. In the present study, we have used gene targeting to selectively eliminate mKv1.5 and showed the selective loss of the 4-AP–sensitive portion of IK,slow. We chose the strategy to replace Kv1.5 with the pharmacologically different channel Kv1.1 with the intention of minimizing other potential changes. The findings presented here definitively link Kv1.5 to a component of IK,slow in the mouse ventricle.
Ectopic Expression of Kv1.1 in the Hearts of SWAP Mice
Kv1.1 mRNA and protein are expressed in the hearts of transgenic SWAP mice under the control of the Kv1.5 promoter. (Figures 1D⇑ through 1F). We were not able to detect any DTX-sensitive currents, however. This suggests that very few functional Kv1.1 channels are present on the extracellular membranes of mouse ventricular myocytes and that the mouse is functionally acting as a Kv1.5 knockout, although we cannot fully exclude the possibility that heteromultimeric channels containing Kv1.1 are not DTX-sensitive. The smaller size of the rKv1.1 protein expressed in the mouse heart (62 kDa) compared with native mKv1.1 in the brain (72 kDa) points to tissue-specific differences in posttranslational processing.22 Recent studies have highlighted the importance of helper proteins and β subunits in transporting K+ channels to the surface membrane.30 31 Kv1.1 is not normally expressed at high levels in the heart (Figure 1D⇑), and the mechanism to process and successfully insert physiological levels of expressed channels into the surface membrane may be absent.
Kv2.1 Upregulation Compensates for the Loss of Kv1.5 in the Hearts of SWAP Mice
Despite the loss of the 50 μmol/L 4-AP component of IK,slow in myocytes isolated from SWAP mice, there was no decrease in the overall density of IK,slow or of the total outward current compared with controls. SWAP myocytes had an increased density of the 20 mmol/L TEA-sensitive component of IK,slow, and Western blots showed increased Kv2.1 protein in SWAP hearts. Kv2.1 α subunits produce slowly activating, slowly inactivating, or noninactivating K+ currents when expressed heterologously in tissue culture, and antibodies to Kv2.1 block currents of this type in hippocampal neurons.32 Previous studies have shown that the TEA-sensitive component of the rapidly activating, slowly inactivating cardiac current IK,slow is selectively eliminated in transgenic mice overexpressing the dominant-negative Kv2.1N216Flag construct in the heart.4 Taken together, these data suggest that upregulation of Kv2.1 is one compensatory mechanism for the loss of Kv1.5 in the ventricles of the SWAP mice. We cannot be certain that the entire compensation in IK,slow is attributable to upregulation of Kv2.1. In addition, the differences in the time- and voltage-dependent properties of Kv2.1 between tissue culture cells, hippocampal neurons, and cardiac myocytes likely reflect differences in accessory subunits or posttranslational processing.
Kv2.1 RNA levels are not changed in the SWAP mice, whereas protein levels are increased. The mechanism by which the loss of Kv1.5 leads to posttranscriptional upregulation of Kv2.1 is unknown. The feedback could be based on the action potential shape and ionic currents. Alternatively, proteins that bind to the ion channel subunits could directly mitigate subunit processing, transport to the membrane, or stability.
Difference Between Dominant-Negative Transgenic and Gene-Targeted Mice
Both Kv1.x dominant-negative transgenic and Kv1.5 homozygous SWAP mice lack the 4-AP–sensitive component of IK,slow.3 15 The Kv1.x dominant-negative transgenic mice have QT prolongation and arrhythmias.3 15 16 17 In this study, we show that targeted mice lacking mKv1.5 have no QT interval prolongation and no arrhythmias. In fact, these mice are resistant to QT prolongation on exposure to the Kv1.5-blocking agent 4-AP. Likely explanations for the differences include both the effect of the Kv1.x transgene on other cardiac K+ channels known to be important for repolarization, such as Kv1.4,8 9 14 and compensatory regulation of other K+ channels, such as Kv2.1, in the SWAP mouse. Clearly, loss of Kv1.5 alone is insufficient to lead to a highly arrhythmogenic phenotype. These findings highlight the fact that transgenic and gene-targeting techniques give different and complementary information on the role of ion channels in cardiac function.
Additional study of both models, along with mating of different strains to form double mutants, should lead to both a better understanding of the role of individual genes in repolarization and susceptibility to arrhythmias and to insights into the mechanisms by which K+ channel gene expression is regulated in vivo in the heart.
These studies were supported in part by National Heart, Lung, and Blood Institute grants R01 HL58030 (to B.L.), R01 34161 (to J.M.N.), and K08 HL03908 (to J.A.H.); a Grant-in-Aid (to B.L.), Scientist Development Grant (to V.S.), and Postdoctoral Research Fellowship (to W.G.) from the American Heart Association; and grants from the Veterans Administration, Procter & Gamble Corporation, and the Roy J. Carver Charitable Trust (to J.A.H.). The sequence of the mKv1.5 genomic clone is available in GenBank (accession No. AF302768). We would like to thank Dr Alexandre F.R. Stewart for his assistance and suggestions.
Original received August 11, 2000; revision received April 3, 2001; accepted April 3, 2001.
- © 2001 American Heart Association, Inc.
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