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Selective elimination of I_K,slow1 in mouse ventricular myocytes expressing a dominant negative Kv1.5 subunit

Departments of¹Molecular Biology and Pharmacology and ²Internal Medicine, Washington University Medical School, St. Louis, Missouri 63110

Submitted 15 July 2003 ; accepted in final form 23 September 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Although previous studies have revealed a role for the voltage-gated K⁺ channel -subunit Kv1.5 (KCNA5) in the generation of the 4-aminopyridine (4-AP)-sensitive component of delayed rectification in mouse ventricles (I_K,slow1), the phenotypic consequences of manipulating I_K,slow1 expression in vivo in different (mouse) models are distinct. In these experiments, point mutations were introduced in the pore region of Kv1.5 to change the tryptophan (W) at position 461 to phenylalanine (F) to produce a nonconducting subunit, Kv1.5W461F, that is shown to function as a Kv1 subfamily-specific dominant negative (Kv1.5DN). With the use of the -myosin heavy chain promoter to direct cardiac-specific expression, three lines of Kv1.5DN-expressing (C57BL6) transgenic mice were generated and characterized. Electrophysiological recordings from Kv1.5-DN-expressing left ventricular myocytes revealed that the micromolar 4-AP sensitive I_K,slow1 is selectively eliminated. The attenuation of I_K,slow1 is accompanied by increased ventricular action potential durations and marked QT prolongation. In contrast to previous findings in mice expressing a truncated (DN) Kv1.1 transgene; however, no electrical remodeling is evident in Kv1.5DN-expressing ventricular myocytes, and the (Kv1.5DN-induced) elimination of I_K,slow1 does not result in spontaneous ventricular arrhythmias.

repolarization; K⁺ currents; long QT

All available evidence suggests that Kv channel pore-forming () subunits of the Kv4 subfamily underlie the rapidly inactivating and rapidly recovering (i.e., fast) transient outward K⁺ current, I_to,f (3, 7, 10, 13, 28, 31) and that Kv1.4 encodes the slowly recovering transient outward K⁺ current, I_to,s (10, 11, 27). Considerable progress has also been made in defining the molecular correlates of several types of cardiac I_K channels (23). In mouse ventricles, two components of delayed rectification, I_K,slow, were distinguished based on differential sensitivities to micromolar concentrations of 4-aminopyridine (4-AP) and millimolar tetraethylammonium (TEA) (30). Subsequent studies that exploited dominant negative and targeted gene deletion strategies, have revealed roles for Kv2- and Kv1-subunits in the generation of the micromolar 4-AP- and the millimolar TEA-sensitive I_K,slow components (17, 18, 29), now referred to as I_K,slow1 and I_K,slow2 (33).

Interestingly, the phenotypic consequences of manipulating I_K,slow1 expression in vivo are distinct. Voltage-clamp recordings from ventricular myocytes isolated from transgenic mice expressing a truncated Kv1.1-subunit, Kv1.1N206Tag, which functions as a dominant negative (Kv1.1DN) (18), for example, revealed that I_K,slow1 is selectively attenuated and that I_K,slow2 is upregulated (33). In addition, Kv1.1DN mice display spontaneous arrhythmias and inducible reentrant ventricular tachycardia (2, 4, 12, 18). Targeted deletion of Kv1.5 (in Kv1.1/1.5 SWAP mice) also results in the elimination of I_K,slow1 and upregulation of I_K,slow2, although, the elimination of I_K,slow1 in this mouse model is not proarrhythmic (17).

Taken together, these findings suggest the interesting possibility that the increased arrhythmia susceptibility in Kv1.1DN mice reflects effects of transgene expression other than (or in addition to) the loss of I_K,slow1. To explore this hypothesis, a pore mutant of Kv1.5, Kv1.5W461F, was developed and shown to function as a Kv1 subfamily-specific DN (Kv1.5DN). Transgenic mice expressing Kv1.5DN driven by the -myosin heavy chain (-MHC) promoter were generated, and electrophysiological studies on ventricular myocytes from these animals revealed that the 4-AP-sensitive component of I_K,slow (I_K,slow1) is selectively eliminated. In contrast with previous models (17, 18, 33), however, no electrical remodeling is evident in Kv1.5DN-expressing ventricular cells. In addition, the functional knockout of I_K,slow1 in Kv1.5DN animals leads to marked prolongation of ventricular action potentials and QT intervals although, in contrast with Kv1.1DN-expressing ("long QT") mice (2, 4, 12, 14, 18, 33), spontaneous arrhythmias are not evident in Kv1.5DN transgenic animals, revealing that the loss of I_K,slow1 is not arrhythmogenic per se.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Construction of Kv1.5W461F-Myc. PCR-based site-directed mutagenesis using Accu-Taq DNA polymerase (Sigma) was employed to generate mouse Kv1.5W461F. Mutagenesis primers were designed to introduce two nucleotide base changes at residues 1381 and 1382 in the region encoding the pore of (wild-type mouse) Kv1.5 [in the pBk-cmv vector (Invitrogen)]. These two mutations change the codon for amino acid in position 461 from the wild-type TGG (tryptophan, W) to TTT (phenylalanine, F); the mutations were confirmed by sequencing. In addition, the stop codon was removed, and the mutant construct (Kv1.5W461F) was subcloned into pcDNA3.1 (Invitrogen) in frame with the downstream sequence in the vector encoding the Myc epitope. After heterologous expression in human embryonic kidney-293 (HEK-293) cells, the properties of the resulting construct, Kv1.5W461F-Myc, were characterized in electrophysiological experiments and in immunohistochemical studies using anti-Kv1.5 and anti-Myc antibodies (see below). All animal care procedures followed the guidelines in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Functional characterization of Kv1.5W461F. HEK-293 cells, obtained from the Washington University Medical School Tissue Culture Support Center and maintained as described previously (9), were transiently transfected using the calcium-phosphate method with plasmids encoding Kv1.4, Kv1.5, Kv4.2, or Kv1.5W461F-Myc, together with a plasmid-encoding enhanced green fluorescent protein (pEGFP-N1; Clontech) to allow transfected cells to be identified before electrophysiological recordings.

Generation of transgenic mice expressing -MHC-Kv1.5W461F-Myc. The Kv1.5W461F-Myc coding sequence was subcloned into the -MHC vector (24, 25) at the SalI site. The resulting -MHC-Kv1.5W461F plasmid was digested to isolate a 7.5-kb fragment containing the -MHC promoter (25), the Kv1.5W461F-myc coding sequence, and the human growth hormone (hGH) polyadenylation signal sequences. After purification, this fragment was resuspended in 100 µl of injection buffer (10 mM Tris buffer containing 10 mM NaCl and 0.1 mM EDTA at pH 7.4) and dialyzed against a 0.1 µm Millipore filter. The fragment was then diluted to a concentration of 1 ng/µl and injected into (100) fertilized C57BL6 mouse blastocysts. After the injections, the oocytes were transplanted into pseudopregnant FVB adult mice. Thirty-six offspring were obtained, and all were screened for integration of the transgene by PCR analysis of (tail) genomic DNA using probes directed against the hGH polyadenylation sequence. Eight founders were identified and crossed with wild-type C57BL6 animals to establish lines for subsequent analysis.

Expression of Kv1.5 and Kv1.5W461F-Myc. For RT-PCR analysis, mRNA was prepared from the ventricles and brains of adult Kv1.5W461F-Myc-expressing transgenic and nontransgenic (control) littermates using the Micro-FasTrack mRNA isolation kit (Invitrogen). cDNA was synthesized in a 20-µl reaction mixture containing (in mM) 50 Tris·HCl (pH 8.3), 5 MgCl₂, 75 KCl, 4 sodium pyrophosphate, 5 dNTPs, and 10 DTT, as well as 0.5 µg of oligo(dT)_12–18, 0.2 µg of mRNA, 10 units of RNase inhibitor, and 5 units of avian myeloblastosis virus-RT (Sigma). After a 1-h incubation period at 42°C, the reaction was terminated by heating at 95°C for 2 min. Approximately 2 µl of the resulting reaction mixture was used for PCR amplification. PCR was carried out in a 20-µl reaction mixture containing (in mM) 10 Tris·HCl (pH 9.0), 50 KCl, 2 MgCl₂, 1 DTT, and 0.2 2-deoxynucleotide 5'-triphosphate (dNTPs), as well as 0.5 µM of each primer (see below), and 1 unit of Taq DNA polymerase (Sigma). The reaction proceeded for 30 cycles as follows: 94°C for 30 s and 58°C for 45 s, followed by 68°C for 90 s. The forward and reverse primers used for actin, Kv1.5 and Kv1.5W461F were 5'-GTGTTACGTCGCCCTTGATT and 5'-GCTGGAGGTGGACAGAGAG (actin), 5'-GACCACTGTAGGCTATTT and 5'-AGCAGATAAGCAAGCTCAAG (wild-type Kv1.5), and 5'-GACCACTGTAGGCTATGG and 5'-AGATCCTCTTCTGAGATGAG (Kv1.5W461F-Myc). The amplified PCR products were analyzed in a 1% agarose gel and stained with ethidium bromide.

To assay Kv1.5W461F-Myc expression, HEK-293 cells transfected with pcDNA3.1 Kv1.5W461F-Myc were fixed in 4% paraformaldehyde in PBS at pH 7.4 for 1 h. After a 1-h incubation in blocking buffer (PBS containing 5% normal goat serum, 0.2% Triton X-100, and 0.1% NaN₃), cells were incubated overnight at 4°C with the anti-Myc (Calbiochem) or anti-Kv1.5 (Transduction Labs) antibody diluted 1:250 in blocking buffer. After being washed with PBS, cells were then exposed for 1 h at room temperature to a Cy3-conjugated goat anti-mouse IgG secondary antibody (Chemicon) diluted 1:1,000 in blocking buffer; labeling was visualized under epifluorescence illumination.

To examine Kv1.5 and Kv1.5W461F-Myc protein expression, membrane proteins were prepared from mouse ventricles and brains using previously described protocols (9–11, 15). After fractionation by SDS-PAGE and transfer to polyvinylidene difluoride membranes (Bio-Rad), immunoblots were completed with an anti-Myc, anti-Kv1.5 (Transduction) or anti-Kv2.1 (UBI) antibody diluted 1:250–500, followed by an alkaline phosphatase-conjugated goat anti-mouse (for anti-Myc and anti-Kv1.5) or anti-rabbit (for anti-Kv2.1) secondary antibody. Bound antibodies were detected using CPSD (Tropix), a chemiluminescent alkaline phosphatase substrate.

Electrophysiological recordings. Whole cell voltage-clamp recordings from GFP-positive HEK-293 cells were obtained at room temperature within 48 h of transfection. The recording pipettes contained (in mM) 115 KCl, 15 KOH, 10 EGTA, 10 HEPES, and 5 glucose (pH 7.2; 300 to 310 mosM). The bath solution contained (in mM) 140 NaCl, 4 KCl, 2 MgCl₂, 1 CaCl₂, 10 HEPES, and 5 glucose at pH 7.4; 300 to 310 mosM. Experiments were performed using an Axopatch 1B patch-clamp amplifier (Axon Instruments) interfaced to a Gateway 400-MHz Pentium computer interfaced to the recording equipment with a Digidata 1200 analog-to-digital interface and the pCLAMP version 7 software package (Axon Instruments). The recording electrodes were fabricated from soda lime glass (Kimble) coated with Sylgard (Dow Corning) and fire polished; tip resistances were 1.5 to 2.5 M. Series resistances were in the range of 3–4 M and were compensated electronically by 80–90%; voltage errors resulting from the uncompensated series resistance were always 6 mV and were not corrected. Outward K⁺ currents in transiently transfected HEK-293 cells were evoked during 140-ms depolarizing voltage steps to test potentials between –40 and +60 mV from a holding potential (HP) of –70 mV; data were filtered at 5 kHz and stored off-line for subsequent analysis.

Ventricular myocytes were isolated from wild-type and Kv1.5DN-expressing adult (6–8 wk) C57BL6 mice, as described previously (10, 11, 29, 30). Briefly, the animals were anesthetized with 3% halothane (97% O₂), and, once deep anesthesia was confirmed, the hearts were rapidly removed, cannulated, and perfused with collagenase-containing buffer solutions. After perfusion, each heart was microdissected to separate the left ventricular free wall, and, in some experiments, the ventricular septum was isolated (10, 11, 30). The isolated tissue pieces were incubated briefly in the collagenase-containing buffer solution, and individual left ventricular myocytes were obtained by gentle trituration. The resulting cells were collected by low-speed centrifugation and were resuspended in serum-free medium 199 (Irvine), plated on laminin-coated coverslips and maintained in a 95% O₂-5% CO₂ incubator.

Whole cell voltage- and current-clamp recordings were obtained from isolated left ventricular myocytes at room temperature within 24 h of cell isolation. For voltage-clamp experiments, the bath solution contained (in mM) 136 NaCl, 4 KCl, 1 CaCl₂, 2 MgCl₂, 5 CoCl₂, 10 HEPES, 10 glucose, and 0.02 tetrodotoxin (TTX) (pH 7.4; 295 to 300 mosM); the TTX and Co²⁺ were eliminated when action potentials were recorded. For current- and voltage-clamp experiments, the pipette solution contained (in mM) 135 KCl, 10 EGTA, 10 HEPES, and 5 glucose (pH 7.2; 295 to 300 mosM). Outward K⁺ currents were evoked during 500-ms or 4.5-s depolarizing voltage steps to test potentials between –40 and +60 mV from a HP of –70 mV after a 20-ms prepulse to –20 mV to eliminate the residual (20 µM TTX insensitive) voltage-gated Na⁺ current. Inwardly rectifying K⁺ currents (I_K1) were recorded in response to hyperpolarizing voltage steps to test potentials between –90 and –120 mV from a HP of –70 mV. Action potentials were recorded under current clamp in response to brief depolarizing current injections.

Electrocardiograms. ECG recordings were obtained from adult (10–12 wk) wild-type (n = 8) and Kv1.5DN-expressing (n = 8) C57BL6 mice using Data Sciences International implantable Physiotel TA10ETA-F20 or TA10EA-F20 radio frequency transmitters and receivers, as previously described (10). Briefly, after an animal was anesthetized [intraperitoneal injection of ketamine (80 mg/kg) and xylazine (16 mg/kg)], the chest was opened and transmitters were placed in the abdominal cavity. Leads were tunneled under the skin, sutured, and glued to muscles in the thorax. Cathodal leads were routinely placed on the upper right portion of the thorax, and anodal leads were placed on the chest wall near the apex. After implantation of the transmitters, the animals were allowed to recover for at least 72 h. Four-minute recordings every hour for 72 consecutive hours were obtained from each animal; data were acquired at 1 kHz. Unfiltered data were analyzed off-line, and QT, QRS, PR, and RR intervals were measured. Average values for individual animals were determined, and mean ± SE values for experiments completed on age-matched wild-type (n = 8) and Kv1.5-DN (n = 8) animals are reported.

Data analysis. Voltage- and current-clamp data were compiled and analyzed using Clampfit (Axon Instruments) and Excel (Microsoft). For each cell, the spatial control of the membrane voltage was assessed by analysis of the decays of the capacitative transients evoked during brief (10 ms) subthreshold (±10 mV) voltage steps from the HP (–70 mV); only cells with capacitative transients well described by single exponentials were analyzed further. Whole cell ventricular myocyte membrane capacitances, determined by integration of the capacitative transients, were not significantly different in cells isolated from wild-type and Kv1.5DN-expressing animals: mean ± SE whole cell capacitances of wild-type and Kv1.5DN-expressing ventricular cells were 140 ± 7 pF (n = 25) and 145 ± 6 pF (n = 25), respectively. The mean ± SE input resistances of Kv1.5DN-expressing (1.11 ± 0.07 G; n = 25) and wild-type (1.07 ± 0.09 G; n = 25) left ventricular myocytes were also not significantly different.

The plateau outward K⁺ current at each test potential in each cell was defined as the current remaining 4.5 s after the onset of the depolarizing voltage steps; peak outward current was defined as the maximum amplitude of the outward K⁺ current during the 500-ms voltage steps. Current amplitudes, measured in individual cells, were normalized to cell size (whole cell membrane capacitance), and current densities (in pA/pF) are reported. Activation time constants were determined from single exponential fits to the rising phases of the outward K⁺ currents evoked during depolarizing voltage steps to test potentials between +10 and +60 mV from a HP of –70 mV. Inactivation time constants were determined from (single or double) exponential fits to the decay phases of the outward K⁺ currents recorded during 4.5-s depolarizing voltage steps.

For analysis of ECGs, the onsets and offsets of the P, Q, R, S, and T waves were determined by measuring the earliest (onset) and the latest (offset) times from the two leads. QT intervals were measured as previously described (16, 17) and were corrected for differences in heart rate using the formula QTc = QT/(RR/100)^1/2 (20). All data are presented as means ± SE unless otherwise noted. Differences between wild-type and Kv1.5-GFP-expressing myocytes/animals were analyzed with the use of ANOVA and the Student's t-test; P values are presented in the text.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Generation and characterization of the dominant negative Kv1.5DN. To generate the putative dominant negative Kv1.5 construct, Kv1.5W461F, mutations were introduced into (mouse) Kv1.5 to change the tryptophan (W) residue at position 461 in the pore region of Kv1.5 to phenylalanine (F). This mutation is analogous to the one made in Kv4.2 to generate Kv4.2W362F, which was shown to function as a Kv4-subfamily-specific dominant negative (3). The Kv1.5W461F construct (Fig. 1A) was tagged at the COOH-terminus with the 10 amino acid Myc epitope tag to allow direct detection of transgene expression. When transiently transfected into HEK-293 cells, expression of Kv1.5W461F-Myc is readily detected immunohistochemically using either an anti-Kv1.5 antibody or an anti-Myc antibody (Fig. 1E).

View larger version (32K): [in this window] [in a new window]   Fig. 1. Kv1.5W461F functions as a Kv1 subfamily-specific dominant negative subunit transgene (Kv1.5DN). A, left: schematic of Kv1.5DN is illustrated; the point mutation [tryptophan (W) to phenylalanine (F)] at residue 461 is indicated by the arrow. A–D: whole cell voltage-gated outward K+ currents evoked during 150-ms depolarizing voltage steps to potentials between –50 and +60 mV from a holding potential (HP) of –70 mV were recorded from transiently transfected human embryonic kidney (HEK)-293 cells. No outward K+ currents are recorded from HEK-293 cells expressing Kv1.5DN alone (A, right). Coexpression of Kv1.5DN with wild-type Kv1.5 (B, right) or Kv1.4 (C, right), however, markedly attenuates outward K+ current amplitudes compared with the currents produced on expression of Kv1.5 (B, left) or Kv1.4 (C, left) alone. D: in contrast, the currents recorded in HEK-293 cells expressing Kv4.2 alone (left) or Kv4.2 and Kv1.5DN (right) are indistinguishable. E: expression of Kv1.5DN in transiently transfected HEK-293 cells is readily detected using an anti-Myc antibody.

Whole cell voltage-clamp recordings from Kv1.5W461F-Myc-expressing HEK-293 cells revealed no outward K⁺ currents (Fig. 1B). When equal amounts of the plasmids encoding wild-type Kv1.5 and Kv1.5W461F-Myc were cotransfected into HEK-293 cells; however, the densities of the Kv1.5-encoded K⁺ currents are reduced significantly (P < 0.001) compared with cells expressing wild-type Kv1.5 alone (Fig. 1B). The mean ± SE peak outward K⁺ current densities at +40 mV in HEK-293-expressing wild-type Kv1.5 alone or wild-type Kv1.5 plus Kv1.5W461F-Myc (1:1) were 596 ± 55 (n = 10) and 46 ± 5 (n = 10), respectively. The voltage dependence and the kinetic properties of the Kv1.5-encoded K⁺ currents in the absence (n = 10) and in the presence (n = 10) of Kv1.5W461F-Myc, however, are indistinguishable. These observations suggest that the assembly of wild-type subunits with Kv1.5W461F-Myc leads to the generation of nonfunctional channels rather than to channels with altered time- and/or voltage-dependent properties (i.e., Kv1.5W461F functions as a dominant negative, Kv1.5DN).

Coexpression of Kv1.5DN with another member of the Kv1 subfamily Kv1.4, also results in marked attenuation of the K⁺ currents produced on expression of Kv1.4 alone (Fig. 1C). The mean ± SE peak outward K⁺ current densities at +40 mV in HEK-293-expressing wild-type Kv1.4 and in cells coexpressing wild-type Kv1.4 and Kv1.5DN were 483 ± 31 pA/pF (n = 8) and 129 ± 17 (n = 8), respectively. In contrast, coexpression of Kv1.5DN with Kv4.2 has no measurable effect on Kv4.2-encoded K⁺ current densities or properties (Fig. 1D). The mean ± SE peak outward K⁺ current densities at +40 mV in HEK-293 cells expressing Kv4.2 alone (768 ± 103 pA/pF; n = 8) or Kv4.2 plus Kv1.5DN (755 ± 107 pA/pF; n = 8) were indistinguishable. Similar results were obtained on coexpression of Kv1.5DN with Kv2.1, revealing that Kv1.5DN functions as a Kv1 subfamily-specific dominant negative.

Generation and characterization of transgenic mice expressing Kv1.5DN. For the generation of transgenic mice expressing Kv1.5DN, the Myc-tagged Kv1.5W461F DNA sequence was subcloned downstream of the cardiac-specific -MHC promoter in the -MHC expression vector (24, 25). Previous work (24) has documented that this promoter is cardiac specific and that constitutive expression of -MHC, as well as transgenes driven by this promoter, are detected in mouse ventricles and atria from the time of birth. A 7.5-kb fragment containing the -MHC promoter, the Kv1.5W461F-Myc sequence, and the hGH polyadenylation signal sequence was isolated and injected into C57BL/6 mouse blastocysts. For screening, genomic tail DNA was prepared, and transgene incorporation was assayed by PCR using probes directed against the hGH polyadenylation signal sequence (3, 29). Eight founders were identified, and all were bred to wild-type C57BL6 mice. Three lines (1, 3, and 5) of Kv1.5W461F-Myc (Kv1.5DN)-expressing transgenic mice were established. Initial experiments were completed on animals/cells from line 5.

PCR analysis revealed that Kv1.5DN, as well as wild-type Kv1.5, is readily detected in the ventricles of (line 5) Kv1.5DN-expressing transgenic animals, whereas only wild-type Kv1.5 is detected in the ventricles of nontransgenic littermates (Fig. 2A). In brains isolated from Kv1.5DN-expressing transgenic mice, however, only wild-type Kv1.5 is evident (Fig. 2A), consistent with the cardiac-specific expression of the transgene. With the use of an anti-Myc antibody, Kv1.5DN protein expression was also readily detected in Western blots of fractionated ventricular membrane proteins from Kv1.5DN-expressing transgenic animals (Fig. 2B). On gross examination, there were no apparent differences between Kv1.5DN-expressing transgenic and nontransgenic littermates. The mean ± SD body weights of 8-wk (adult) wild-type and Kv1.5DN-expressing animals, for example, were 24.6 ± 3.2 g (n = 6) and 25.1 ± 3.5 g (n = 6), respectively. Heart weights were also not significantly different, with mean ± SD values of 104 ± 13 mg (n = 6) and 108 ± 19 mg (n = 6) for wild-type and Kv1.5DN-expressing adult animals, respectively. Heart-to-body weight ratios in nontransgenic and transgenic animals, therefore, were also very similar, and histological examination of hearts from Kv1.5DN-expressing animals revealed no detectable morphological differences from controls (not shown). In addition, the input resistances and whole cell membrane capacitances of left ventricular myocytes isolated from Kv1.5DN-expressing and wild-type animals were indistinguishable (see MATERIALS AND METHODS).

View larger version (69K): [in this window] [in a new window]   Fig. 2. Cardiac-specific expression of Kv1.5DN. A: RT-PCR analysis revealed that wild-type Kv1.5 (wt Kv1.5) is readily detected in the ventricles and the brains of wild-type and Kv1.5DN-expressing transgenic animals, whereas Kv1.5DN is only detected in the ventricles of the Kv1.5DN-expressing animals. B: Western blots of fractionated ventricular membrane proteins prepared from wild-type and Kv1.5DN-expressing animals were probed with an anti-Myc, anti-Kv1.5, or anti-Kv2.1 antibody. As expected, the Myc-tagged Kv1.5DN is detected only in ventricular membrane proteins from the Kv1.5DN-expressing animals. Wild-type Kv1.5 and Kv2.1, in contrast, are detected in the blots of fractionated wild-type and transgenic ventricular membrane proteins. IB, immunoblot.

Outward K⁺ currents are attenuated in Kv1.5DN-expressing ventricular myocytes. As illustrated in Fig. 3, the waveforms of the depolarization-activated outward K⁺ currents in left ventricular myocytes isolated from (line 5) wild-type (Fig. 3A) and Kv1.5DN-expressing transgenic (Fig. 3B) littermates are distinct. Peak outward K⁺ current amplitudes at all test potentials, for example, are significantly (P < 0.005) lower in cells isolated from Kv1.5DN-expressing transgenic animals compared with the currents recorded in myocytes isolated from nontransgenic (wild-type) littermates (Fig. 3C). The mean ± SE peak outward densities at +40 mV, for example, were 50.4 ± 4.4 (n = 25) and 36.5 ± 3.2 pA/pF (n = 25) in wild-type and Kv1.5DN-expressing left ventricular myocytes, respectively (Table 1). In contrast to the observed reductions in peak outward K⁺ current densities in ventricular myocytes isolated from the Kv1.5DN-expressing animals, no measurable effects on the densities of either the steady-state outward K⁺ currents (I_ss), which was determined as the currents remaining at the end of 4.5-s voltage steps (Fig. 3, arrows), or of the hyperpolarization-activated I_K1 (not shown) were observed. Mean ± SE I_ss densities at +40 mV, for example, were 6.0 ± 0.5 pA/pF (n = 25) and 4.8 ± 0.3 pA/pF (n = 25) in wild-type and Kv1.5DN-expressing myocytes, respectively (Table 1). Mean ± SE peak I_K1 densities evoked at –120 mV from a HP of –70 mV were 11 ± 0.5 pA/pF; n = 10 in wild-type and 12 ± 0.5 pA/pF; n = 10 in Kv1.5DN-expressing myocytes, respectively. Similar results were obtained in experiments completed on left ventricular cells isolated from lines 1 and 3 Kv1.5DN-expressing transgenics.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Outward K+ current waveforms are altered in Kv1.5DN-expressing ventricular myocytes. Whole cell outward K+ currents, recorded from ventricular myocytes isolated from nontransgenic (A) and Kv1.5DN-expressing transgenic (B) littermates were evoked during 450-ms (top) or 4.5-s (bottom) depolarizing voltage steps to potentials between –50 and +60 mV from an HP of 70 mV (see MATERIALS AND METHODS). The waveforms of the Ca2+-independent, depolarization-activated outward K+ currents in wild-type (A) and Kv1.5DN-expressing transgenic (B) myocytes are distinct: peak outward K+ current (Ipeak) amplitudes are reduced and the slowly decaying current component (IK,slow) is much less prominent in Kv1.5DN-expressing (B) than in wild-type (A) cells. C and D: mean ± SE peak outward K+ current (C), fast transient outward current (Ito,f), IK,slow and steady-state outward current (ISS) (D) densities are plotted as a function of test potential. As is evident, means ± SE peak outward K+ current (C) and IK,slow (D) densities are significantly (P < 0.001) lower in Kv1.5DN-expressing (open symbols), than in wild-type (solid symbols), mouse ventricular cells. Ito,f and ISS densities (D) in wild-type (solid symbols) and Kv1.5DN-expressing (open symbols) ventricular cells, in contrast, are similar.

I_K,slow is attenuated in Kv1.5DN-expressing ventricular myocytes. In wild-type adult mouse ventricular myocytes, analysis of the decay phases of the outward K⁺ currents evoked during long (4.5 s) depolarizations reveals that current decay is well described by the sum of two exponentials, with decay time constants (_decay) that differ by more than an order of magnitude, and a noninactivating, i.e., I_ss (30). Mean ± SE _decay determined here for the fast and the slow components of inactivation in wild-type cells (n = 25) were 74 ± 3 ms and 1,147 ± 57 ms (Table 1), corresponding to inactivation of I_to,f and I_K,slow, and, as reported previously, neither time constant displays any appreciable voltage dependence (10, 11, 30). Mean ± SE I_to,f and I_K,slow densities (at +40 mV) in wild-type ventricular cells (n = 25) were 28.2 ± 2.7 pA/pF and 15.5 ± 1.6 pA/pF, respectively (Table 1). Analysis of the decay phases of the outward currents in Kv1.5DN-expressing ventricular cells revealed that the density of the slow component of current decay is significantly (P < 0.005) lower than in wild-type cells (Fig. 3D); the mean ± SE I_K,slow density (at +40 mV) in Kv1.5DN-expressing ventricular cells was 6.6 ± 0.8 pA/pF (Table 1). The voltage-dependent properties of the currents, however, are indistinguishable, and I_K,slow densities are significantly lower at all test potentials in Kv1.5DN-expressing than in wild-type left ventricular cells (Fig. 3D). In contrast to the attenuation of I_K,slow, no significant differences in the densities or the voltage-dependent properties of I_to,f (or I_ss) were observed (Fig. 3D), suggesting that I_K,slow is selectively attenuated in ventricular myocytes isolated from adult Kv1.5DN-expressing transgenic animals.

The decay phases of the depolarization-activated K⁺ currents in Kv1.5DN-expressing ventricular myocytes were also well described by the sum of two exponentials, with mean ± SE (n = 25) decay time constants were 86 ± 5 ms and 1,818 ± 136 ms (Table 1). Similar to the findings in wild-type cells, neither time constant displays any measurable voltage dependence. The time constant for the fast component of current decay (86 ± 5 ms) is similar to the _decay for wild-type I_to,f (Table 1). The mean ± SE time constant of inactivation of the slower component of current decay (_decay = 1,818 ± 136 ms) in the Kv1.5DN-expressing cells, however, is significantly (P < 0.005) larger than the mean ± SE _decay of 1,147 ± 57 ms for I_K,slow in wild-type cells (Table 1). These findings are similar to those obtained previously in experiments on ventricular myocytes isolated from SWAP mice lacking Kv1.5 (17), suggesting the selective elimination of one component of I_K,slow, the µM 4-AP-sensitive I_K,slow1, with Kv1.5DN expression.

Selective elimination of I_K,slow1 in Kv1.5DN-expressing ventricular myocytes. Previous studies demonstrated the presence of two distinct components of I_K,slow in mouse ventricular myocytes (17, 29, 30), now referred to as I_K,slow1 and I_K,slow2 (33). I_K,slow2 is blocked selectively by millimolar concentration of TEA, whereas I_K,slow1 is selectively blocked by micromolar concentrations of 4-AP (17, 29, 30). Experiments were completed, therefore, to test the hypothesis that the micromolar 4AP-sensitive I_K,slow1 is selectively attenuated in Kv1.5DN-expressing ventricular myocytes (Fig. 4). After recording control currents (Fig. 4, A and D), cells were exposed to 50 µM 4-AP or 30 mM TEA, and the currents in the presence of 50 µM 4-AP (Fig. 4B) or 30 mM TEA (Fig. 4E) were recorded. The waveforms of the 50 µM 4-AP-sensitive (Fig. 4C) or the 30 mM TEA-sensitive (Fig. 4F) components of the currents were then determined by subtracting the currents in the presence of 50 µM 4-AP (Fig. 4B) or 30 mM TEA (Fig. 4E) from the controls (Fig. 4, A and D).

View larger version (29K): [in this window] [in a new window]   Fig. 4. The 50 µM 4-aminopyridine (4-AP)-sensitive component of IK,slow, IK,slow1, is selectively eliminated in Kv1.5DN-expressing ventricular myocytes. Outward currents were recorded as described in Fig. 3 under control conditions and after exposure to one of the K+ channel blockers 4-AP or tetraethylammonium (TEA) at 50 µM or 30 mM, respectively. After recording control currents (A and D), cells were exposed to either 50 µM 4-AP (B) or 30 mM TEA (E). The waveforms of the 50 µM 4-AP-sensitive (C) and the 30 mM TEA-sensitive (F) currents were obtained by off-line digital subtraction of the currents recorded in the presence of 4-AP (B) or TEA (E) from the corresponding control currents (A and D) recorded in the same cell(s).

As illustrated in Fig. 4, A–C, a small fraction of the rapidly activating and inactivating outward current is blocked by 50 µM 4-AP. Analysis of the decay phases of the 50-µM 4-AP-sensitive currents in Kv1.5DN-expressing cells (Fig. 4C) reveals that the decay phase of the currents are well described by a single exponential with a mean ± SE (n = 5) inactivation time constant (_decay) of 75 ± 5 ms, suggesting partial block of I_to,f by 50 µM 4-AP in these cells. The slowly inactivating component of the 50 µM 4-AP-sensitive current evident in wild-type cells, however, is not evident in the Kv1.5DN-expressing cells (Fig. 4C), an observation interpreted as revealing that I_K,slow1 is selectively eliminated in Kv1.5DN-expressing ventricular myocytes. The decay phases of the 30 mM TEA-sensitive currents (Fig. 4F) are well described by single exponentials with a mean ± SE _decay of 1,906 ± 243 ms (n = 4). In wild-type cells, both I_ss and I_K,slow are reduced by 50% in 30 mM TEA, whereas I_to,f is unaffected (29, 30). The waveforms of the 30 mM TEA-sensitive currents in Kv1.5DN-expressing cells (Fig. 4F) are consistent with the selective attenuation of I_K,slow2, and, interestingly, the density of I_K,slow2 (6.6 ± 0.8 pA/pF; n = 25) in the Kv1.5DN cells is similar to the density of the 30 mM TEA-sensitive component of I_K,slow (7.3 ± 1.0 pA/pF; n = 8), i.e., I_K,slow2, in wild-type cells, suggesting that electrical remodeling is not evident in ventricular cells expressing Kv1.5DN (see DISCUSSION).

Elimination of I_to,s in Kv1.5DN-expressing ventricular septum cells. As illustrated in Fig. 1C, expression of Kv1.5 DN also attenuates Kv1.4-encoded K⁺ currents heterologously expressed in HEK-293 cells. In previous studies (10, 11), we have shown that Kv1.4 encodes the slow transient outward K⁺ current, I_to,s, that is expressed in mouse ventricular septum cells (30). Although these results and the finding that Kv1.5 encodes mouse ventricular I_K,slow1 (17) reveal that Kv1.4 and Kv1.5 do not coassemble to form heteromeric K⁺ channels in adult mouse ventricle in situ, it seemed important to determine whether I_to,s was also affected by the (over) expression of Kv1.5DN. Whole cell voltage-clamp recordings obtained from septum cells isolated from Kv1.5DN-expressing adult transgenic mice (n = 2) revealed that I_to,s is eliminated (n = 16). These findings suggest that although Kv1.4 and Kv1.5 do not normally coassemble in adult mouse ventricles, the (over) expression of the Kv1.5DN transgene appears to be sufficient to override the normal physiological regulation of homomeric assembly of Kv1.4-subunits (see DISCUSSION).

Ventricular action potentials and QT intervals are prolonged with Kv1.5DN expression. Current-clamp experiments revealed that action potentials recorded from Kv1.5DN-expressing ventricular myocytes are substantially broader than action potentials recorded from cells isolated from nontransgenic (wild-type) littermates, with the latter phase of repolarization being particularly affected (Fig. 5A). In contrast, action potential amplitudes and resting membrane potentials measured in Kv1.5DN-expressing myocytes are not significantly different from those seen in wild-type cells (Table 2). Analysis of action potential durations at 50% (APD₅₀), 75% (APD₇₅), and 90% (APD₉₀) repolarization indeed revealed that mean ± SE APD₇₅ and APD₉₀ are increased significantly (P < 0.005) in Kv1.5DN-expressing ventricular cells compared with (APD₇₅ and APD₉₀ in) wild-type cells (Table 2). Mean ± SE APD₇₅ and APD₉₀ repolarization, for example, were 10.1 ± 1.1 ms and 34.4 ± 4.2 ms (n = 16) in wild-type cells and 15.9 ± 2.1 ms and 54.1 ± 5.6 ms (n = 16) in Kv1.5DN-expressing cells (Table 2). In contrast, APD₅₀ values in Kv1.5DN-expressing and wild-type cells are not significantly different (Table 2).

View larger version (21K): [in this window] [in a new window]   Fig. 5. Action potential durations at 75 and 95% repolarization (APD75 and APD95) are increased in Kv1.5DN-expressing cells and QT intervals are prolonged in Kv1.5DN-expressing animals. A: action potentials were recorded under current-clamp conditions from individual wild-type and Kv1.5DN-expressing ventricular myocytes in response to brief depolarizing current injections. Representative recordings from wild-type and Kv1.5DN-expressing cells are displayed. Analysis of evoked action potentials revealed that mean ± SE APD75 and APD90 values are significantly (P < 0.005) longer in Kv1.5DN-expressing, compared with wild-type, ventricular cells (Table 2). B: telemetric ECG recordings, obtained as described in MATERIALS AND METHODS, from representative wild-type and Kv1.5DN-expressing animals are displayed; QT intervals are indicated below the records. Analysis of ECG recordings revealed that the mean ± SE corrected QT interval is significantly (P < 0.001) longer in Kv1.5DN-expressing, than in wild-type, animals (Table 3).

To determine the functional consequences of the action potential prolongation evident in Kv1.5DN-expressing ventricular cells, in vivo telemetric ECG recordings were obtained from Kv1.5DN-expressing transgenic animals and compared with recordings from nontransgenic (wild-type) littermates. Representative ECG recordings from wild-type and Kv1.5DN-expressing animals are presented in Fig. 5B. As is immediately evident on visual inspection of the records, there is a marked prolongation of the QT interval in the transgenic animals, consistent with a defect in ventricular repolarization. The mean ± SE QT interval (62 ± 2 ms; n = 8) is significantly (P < 0.001) longer in Kv1.5-expressing animals compared with the mean ± SE value (49 ± 1 ms; n = 8) in nontransgenic littermates. Neither heart rates (RR intervals) nor QRS durations, however, were measurably affected by Kv1.5DN expression (Table 3). When QT intervals were corrected for heart rate (see MATERIALS AND METHODS), the differences between the transgenic and the nontransgenic animals remained highly significant (at the P < 0.001 level); mean ± SE QT_c intervals were 48 ± 1 ms (n = 8) and 61 ± 2 ms (n = 8) in control and Kv1.5DN-expressing animals, respectively (Table 3).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Functional consequences of cardiac-specific expression of Kv1.5DN. The results presented here demonstrate that the slowly decaying outward K⁺ current I_K,slow is attenuated in Kv1.5DN-expressing left ventricular myocytes. The effect on I_K,slow is specific in that neither I_to,f nor I_ss is measurably affected by the expression of Kv1.5DN. In addition, the time constant of decay of the I_K,slow remaining in Kv1.5DN-expressing left ventricular myocytes is larger than the decay time constant for wild-type I_K,slow (Table 1). Because previous studies have demonstrated that I_K,slow reflects the presence of two distinct components, I_K,slow1 and I_K,slow2, characterized by differences in inactivation kinetics and pharmacological sensitivities to µM 4-AP and mM TEA (30, 33), the simplest interpretation of the kinetic analyses appears to be that I_K,slow1 is eliminated and only I_K,slow2 remains in cells expressing Kv1.5DN. Consistent with this interpretation, subsequent pharmacological experiments revealed that the fraction of I_K,slow remaining in Kv1.5DN-expressing ventricular cells is blocked by 30 mM TEA, but is insensitive to 50 µM 4-AP. Taken together, therefore, the results presented here demonstrate the selective elimination of I_K,slow1 in Kv1.5DN-expressing left ventricular myocytes.

In addition to the loss of I_K,slow1, further voltage-clamp experiments revealed that I_to,s is also eliminated in Kv1.5DN-expressing adult mouse ventricular septum cells (n = 16). Although previous studies have clearly demonstrated that (wild-type) Kv1.4 and Kv1.5 do not coassemble to form heteromeric channels in adult mouse ventricles (10, 11, 17), experiments in heterologous systems reveal that Kv1.4 and Kv1.5 can coassemble and that coexpression of Kv1.5DN attenuates Kv1.4-encoded K⁺ currents (see, for example, Fig. 1). The simplest interpretation of the observed elimination of I_to,s in Kv1.5DN-expressing adult mouse ventricular septum cells, therefore, is that the robust (over) expression of the Kv1.5DN transgene overwhelms the normal physiological mechanisms, presumably involving requirements for subunit cotranslation, regulating the homomeric assembly of Kv1.4-subunits and preventing the heteromeric assembly of Kv1.5 and Kv1.4-subunits in wild-type adult mouse ventricles.

Consistent with the elimination of I_K,slow1, current-clamp experiments revealed that APD₇₅ and APD₉₀ values are increased significantly (P < 0.005) in Kv1.5DN-expressing left ventricular myocytes. The elimination of I_K,slow1 also results in marked QT prolongation. Expression of Kv1.5DN, however, does not appear to have any other measurable physiological or pathophysiological consequences. In fact, the Kv1.5DN-expressing transgenic animals appear normal in every respect, and, in experiments completed to date (involving 72-h telemetric ECG recordings from freely moving animals), we find no evidence for the presence of spontaneous ventricular arrhythmias. Further experiments will be necessary to determine whether the Kv1.5DN animals display increased sensitivity to arrhythmogenic stimuli.

Relationship to previous studies. The suggestion that Kv1.5 underlies I_K,slow in mouse ventricular myocytes was originally advanced by Fiset and colleagues (6). Experimental support for a role for Kv1-subunits was provided in studies demonstrating attenuation of I_K,slow in ventricular myocytes isolated from (Kv1.1DN) transgenic mice expressing a dominant negative Kv1.1 construct, Kv1.1N206Tag (18). Subsequent studies (4, 33) revealed that the micromolar 4-AP-sensitive I_K,slow1 is selectively eliminated and, in addition, that I_K,slow2 density is increased in Kv1.1DN-expressing ventricular cells. Because heterologous coexpression (in GH3 cells) of Kv1.1N206Tag had previously been shown to result in the accumulation of wild-type Kv1.5 in the endoplasmic reticulum (8), the finding that Kv1.5 protein expression is reduced in Kv1.1DN-expressing ventricles was interpreted as suggesting a specific role for Kv1.5 in the generation of mouse ventricular I_K,slow1 (4, 18). To test this hypothesis directly, London and colleagues (17) exploited gene targeting in vivo to eliminate Kv1.5, and replace it with (rat) Kv1.1 (SWAP mice). Electrophysiological studies on ventricular cells isolated from SWAP mice revealed that I_K,slow1 was eliminated (17), thereby providing direct evidence for the functional importance of Kv1.5 in the generation of I_K,slow1. In addition, and similar to the findings in Kv1.1DN-expressing ventricular cells (33), I_K,slow2 density is increased in SWAP ventricular myocytes lacking I_K,slow1 (17).

Although similar changes in I_K,slow1 and I_K,slow2 densities are observed, there are rather marked differences in the properties of Kv1.1DN-expressing and SWAP ventricular cells and in the phenotypes of the Kv1.1DN-expressing and SWAP mice (4, 12, 14, 17, 18, 33). Action potential durations, for example, are prolonged significantly in Kv1.1DN-expressing ventricular myocytes (4, 18) but are not measurably different from controls in SWAP myocytes (17). Similarly, QT intervals are increased in Kv1.1DN, but not in SWAP, animals (4, 12, 14, 17, 18). The normalization of action potential durations and QT intervals in SWAP animals was attributed to the upregulation of I_K,slow2 (17).

The findings that neither I_K,slow2 nor the Kv2.1 protein (which encodes I_K,slow2) (29) is upregulated in Kv1.5DN mice may explain the phenotypic differences between the SWAP and Kv1.5DN-expressing animals. It is not clear, however, why similar normalizing effects are not seen in Kv1.1DN mice, given that I_K,slow2 is also upregulated in Kv1.1DN-expressing ventricular myocytes (33). Similarly, it is unclear why spontaneous ventricular arrhythmias are observed in Kv1.1DN transgenics (4, 12, 18). The results presented here, however, clearly suggest that this (pathophysiological) effect cannot be attributed to the loss of I_K,slow1, as has been previously suggested (4, 12, 18). Taken together, however, the findings here do suggest the interesting possibility that the increased incidence of and susceptibility to ventricular arrhythmias in Kv1.1DN mice reflects effects of transgene expression other than (or in addition to) the loss of I_K,slow1. The results presented here clearly also demonstrate that the loss of I_K,slow1 is not arrhythmogenic per se.

It may be important that the present study involved a different strategy for generating a Kv1 subfamily-specific dominant negative than that employed by London and colleagues (18). In the experiments here, point mutations were introduced into the pore region of Kv1.5 to produce Kv1.5W461F. The rationale for this approach was the hypothesis that little or no change in the structure of the individual Kv-subunit or the resulting Kv-subunit-encoded K⁺ channels would be desirable to avoid unwanted effects that might be due to abnormal folding or assembly of subunits and/or to perturbations in the normal functional interactions between the Kv-subunits/channels and other accessory (membrane and/or cytoplasmic) proteins. These concerns seem particularly relevant to consider for in situ studies that typically involve multiple copies of transgenes and robust overexpression of transgenic proteins. In this context, it is of interest to note that it has been demonstrated that outward K⁺ waveforms in ventricular myocytes isolated from transgenic mice overexpressing Kv1.5 (Kv1.5-GFP) are distinct from those recorded from wild-type cells (15). Specifically, although pharmacological experiments revealed a selective increase in the density of the µM 4-AP-sensitive current in Kv1.5-GFP-expressing cells, the rates of inactivation of the overexpressed Kv1.5-encoded K⁺ currents are significantly slower than wild-type I_K,slow1 (15). Importantly, experiments in heterologous expression systems revealed that the properties of Kv1.5- and Kv1.5-GFP-encoded K⁺ currents are indistinguishable (15). The simplest interpretation of the in situ results, therefore, is that the overexpressed Kv1.5 subunits fail to assemble properly or in the correct stoichiometry with accessory or modulatory subunits that contribute to the formation of the wild-type Kv1.5-encoded mouse ventricular I_K,slow1.

Although Kv accessory and other modulatory proteins that interact with Kv1-subunits have been described (23), it is unclear whether any of these subunits plays a role in the generation of functional Kv1.5-encoded mouse ventricular I_Kslow1 channels. It is also unclear whether the expression levels or the distributions of other proteins involved in the generation of I_Kslow1 are affected in the hearts of Kv1.1DN-expressing transgenic mice, primarily because these issues have not been explored directly. Importantly, however, the fact that the expression of the wild-type Kv1.5 protein is affected in Kv1.1DN ventricles (18) raises the concern that the functional consequences of expression of the Kv1.1N206Tag truncation mutant may arise at least in part from effects on the expression and/or distribution of wild-type proteins other than the targeted Kv1.5-subunit. Further experiments will be needed to test this specific hypothesis and to explore the hypothesis that targeting the pore region for site-directed mutagenesis is an effective strategy for creating dominant negative Kv-subunits that do not affect the expression of wild-type Kv-subunits or Kv channel accessory or modulatory subunits.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Franck Aimond for many helpful discussions and Rick Wilson for expert technical assistance.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-034161 and HL-066388.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. M. Nerbonne, Dept. of Molecular Biology and Pharmacology, Washington Univ. Medical School, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail: jnerbonne{at}msnotes.wustl.edu).

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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