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(Circulation Research. 1999;85:623-633.)
© 1999 American Heart Association, Inc.

Integrative Physiology

Attenuation of the Slow Component of Delayed Rectification, Action Potential Prolongation, and Triggered Activity in Mice Expressing a Dominant-Negative Kv2 Subunit

Haodong Xu, Dianne M. Barry, Huilin Li, Sylvain Brunet, Weinong Guo, Jeanne M. Nerbonne

From the Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, Mo.

Correspondence to Jeanne M. Nerbonne, Department of Molecular Biology and Pharmacology, Washington University Medical School, 660 S Euclid Ave, St. Louis, MO 63110. E-mail jnerbonn{at}pharmdec.wustl.edu

	Abstract

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Abstract—An in vivo experimental strategy, involving cardiac-specific expression of a mutant Kv 2.1 subunit that functions as a dominant negative, was exploited in studies focused on exploring the role of members of the Kv2 subfamily of pore-forming () subunits in the generation of functional voltage-gated K⁺ channels in the mammalian heart. A mutant Kv2.1 subunit (Kv2.1N216) was designed to produce a truncated protein containing the intracellular N terminus, the S1 membrane–spanning domain, and a portion of the S1/S2 loop. The truncated Kv2.1N216 was epitope tagged at the C terminus with the 8–amino acid FLAG peptide to generate Kv2.1N216FLAG. No ionic currents are detected on expression of Kv2.1N216FLAG in HEK-293 cells, although coexpression of this construct with wild-type Kv2.1 markedly reduced the amplitudes of Kv2.1-induced currents. Using the -myosin heavy chain promoter to direct cardiac specific expression of the transgene, 2 lines of Kv2.1N216FLAG-expressing transgenic mice were generated. Electrophysiological recordings from ventricular myocytes isolated from these animals revealed that I_{K, slow} is selectively reduced. The attenuation of I_{K, slow} is accompanied by marked action potential prolongation, and, occasionally, spontaneous triggered activity (apparently induced by early afterdepolarizations) is observed. The time constant of inactivation of I_{K, slow} in Kv2.1N216FLAG-expressing cells (mean±SEM=830±103 ms; n=17) is accelerated compared with the time constant of I_{K, slow} inactivation (mean±SEM=1147±57 ms; n=25) in nontransgenic cells. In addition, unlike I_{K, slow} in wild-type cells, the component of I_{K, slow} remaining in the Kv2.1N216FLAG-expressing cells is insensitive to 25 mmol/L tetraethylammonium. Taken together, these observations suggest that there are 2 distinct components of I_{K, slow} in mouse ventricular myocytes and that Kv2 subunits underlie the more slowly inactivating, tetraethylammonium-sensitive component of I_{K, slow}. In vivo telemetric recordings also reveal marked QT prolongation, consistent with a defect in ventricular repolarization, in Kv2.1N216FLAG-expressing transgenic mice.

Key Words: transgenic mice • ventricle • action potential • triggered activity • arrhythmia

	Introduction

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Depolarization-activated K⁺ channels play important roles in shaping the waveforms of action potentials in myocardial cells, and in most cardiac cell types, the following 2 types of voltage-gated K⁺ currents have been distinguished: transient outward K⁺ currents, referred to as I_to, and delayed, more slowly inactivating K⁺ currents, referred to as I_K.^{1 2 3 4} These are broad classifications, however, and the detailed properties of I_to and I_K currents/channels vary in different species, as well as in different cardiac cell types isolated from the same species.^{1 2 3 4} Differences in the expression and/or properties of I_to and I_K contribute to regional variations in action potential waveforms in the mammalian heart.^{5 6} It is also now well documented that the densities, as well as the properties, of voltage-gated K⁺ currents change during normal cardiac development^{7 8} and in a variety of cardiovascular disease states.^{9 10 11 12 13} As a result, there is considerable interest in defining the molecular correlates of I_to and I_K^{3 4 5 14} and in understanding the mechanisms involved in the regulation, modulation, and functional expression of these channels.^{5 8}

Considerable experimental evidence has now been provided documenting a role for Kv subunits of the Kv4 subfamily in the generation of the rapidly inactivating and rapidly recovering I_to, now referred to as I_{to, fast} or I_{to, f},^{5 15} in rat and mouse heart.^{16 17 18 19 20} Exposure of adult rat ventricular myocytes to adenoviral constructs encoding a truncated Kv4.2 subunit, for example, selectively attenuates I_{to, f}.¹⁶ The density of rat ventricular I_{to, f} is also reduced after exposure to antisense oligodeoxynucleotides (AsODNs) targeted against Kv4.2 or Kv4.3,¹⁷ whereas only AsODNs targeted against Kv4.2 attenuate I_{to, f} in rat atrial myocytes.¹⁸ In transgenic mice expressing a pore mutant of Kv4.2 (Kv4.2W362F) that functions as a dominant negative, I_{to, f} is eliminated in both ventricular and atrial cells.^{19 20} Given that the properties of I_{to, f} in other species are similar to those of mouse and rat I_{to, f}, it seems reasonable to speculate that members of the Kv 4 subfamily underlie I_{to, f} in all cardiac cells. In support of this hypothesis, it has been reported that exposure to an AsODN targeted against Kv4.3 selectively attenuates I_{to, f} in human atrial myocytes.²¹ In some cardiac cells, more slowly inactivating and recovering (than I_{to, f}) transient outward K⁺ currents are expressed^{2 3 4 5 15 21 22 23} and are now referred to as I_{to, slow} or I_{to, s}.^{5 15} Recently, it was demonstrated that AsODNs targeted against Kv1.4 attenuate the slow transient outward K⁺ current (ie, I_{to, s}) in rabbit atrial myocytes.²¹ In addition, in mice with a targeted deletion in the Kv1.4 gene,²⁴ I_{to, s} is absent.²³ It has also been hypothesized that Kv1.4 underlies the slow transient outward K⁺ currents in ferret left ventricular endocardial myocytes.²²

Considerable progress has also been made in defining the molecular correlates of several cardiac I_K channels/currents. Two K⁺ channel genes, KvLQT1²⁵ and human ether-a-go-go or HERG,²⁶ for example, have been identified as loci of mutations in familial long-QT syndromes. HERG underlies I_Kr (the rapid I_K),^{27 28} whereas KvLQT1 contributes to I_Ks (the slow I_K).^{29 30} Other approaches have been used to explore the molecular identities of other cardiac I_K channels. Antisense strategies, for example, have been used to establish a role for Kv1.5 in the generation of the ultrarapid component of cardiac delayed rectification, I_Kur, in both human³¹ and rat¹⁸ atrial myocytes. A role for Kv1 subunits in the generation of the slowly inactivating K⁺ current, termed I_{K, slow}, in mouse ventricle has also recently been suggested.^{32 33 34} Electrophysiological recordings from ventricular myocytes isolated from transgenic mice expressing a truncated Kv1.1 subunit (Kv1.1N206Tag), which functions as a dominant negative,³⁵ revealed that I_{K, slow} is selectively attenuated.³² Detailed analysis of mouse ventricular I_{K, slow}, however, suggests the presence of 2 current components.^{15 34} The experiments described here were undertaken to test directly the hypothesis that members of the Kv 2 subfamily of subunits contribute to the generation of functional voltage-gated K⁺ channels in the mammalian heart. To achieve this, a truncation mutant of Kv2.1 was generated to produce a subunit (Kv2.1N216) that functions as a dominant negative. Transgenic mice, expressing the truncated Kv2.1 driven by the -myosin heavy chain (MHC) promoter,^{36 37 38 39} were then generated and characterized. Electrophysiological studies reveal that a component of I_{K, slow} is selectively attenuated in ventricular myocytes isolated from Kv2.1N216FLAG-expressing mice, demonstrating that the Kv2 subfamily contributes to mouse ventricular I_{K, slow}. In addition, the attenuation of I_{K, slow} leads to marked increases in action potential durations in ventricular myocytes and to prolongation of the QT interval.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of the Truncated, Epitope-Tagged Kv2.1 Subunit, Kv2.1N216FLAG Subunit
The DNA sequence (residues 1 to 648) encoding the intracellular N terminus, the S1 membrane spanning region, and a portion of the S1/S2 extracellular loop of Kv2.1 was amplified by polymerase chain reaction (PCR). The PCR primers used (in this reaction) introduced 5' SalI and 3' EcoRI sites; the 648-bp PCR product was then digested with SalI and EcoRI and subcloned into pBK-CMV (Stratagene). To epitope (FLAG) tag the truncated Kv2.1 sequence at the 3' end, 2 oligonucleotides corresponding to the sense and antisense strands of the 8–amino acid FLAG epitope were designed to contain EcoRI and XhoI overhangs at the 5' and 3' ends, respectively; these were generated by the Protein and Nucleic Acid Facility at Washington University Medical School. The annealed oligonucleotides were subcloned into pBK-CMV-Kv2.1(1 to 648) at the EcoRI and XhoI sites. The entire Kv2.1N216FLAG construct was sequenced, and no errors were detected.

Cell Lines and Transient Transfections
HEK-293 cells, from a human embryonic kidney cell line, which were obtained from the Washington University Medical School Tissue Culture Support Center, were maintained in MEM supplemented with 5% horse serum, 5% FCS, 100 U/mL penicillin, 100 U/mL stroptomycin, and 0.15% mycostatin. HEK-293 cells were transferred to serum-free MEM with 24 hours of passage and transiently transfected using calcium-phosphate with plasmids encoding Kv2.1, Kv1.4, or Kv2.1N216FLAG, together with the green fluorescent protein (GFP) (pGREENLANTERN; GIBCO-BRL); inclusion of the plasmid encoding GFP allowed transfected cells to be identified before electrophysiological recordings. Approximately 15 hours after transfection, the cells were washed and allowed to recover for 20 to 24 hours before electrophysiological recordings.

Generation of Transgenic Mice Expressing -MHC-Kv2.1N216FLAG
The Kv2.1N216FLAG coding sequence was digested from pBK-CMV-Kv2.1N216FLAG with SalI and XhoI and subcloned into the -MHC vector^{36 37 38 39} at the SalI site; restriction enzyme digests were performed to select for the proper orientation. The -MHC-Kv2.1N216FLAG plasmid was digested with NotI to isolate a 5.7-kb fragment that included the 3' exon from the ß-MHC gene, intergenic sequences, the -MHC promoter, the first 3 noncoding exons of the -MHC gene, the Kv2.1N216FLAG coding sequence, and the human growth hormone (HGH) polyadenylation signal sequences.^{36 37 38 39} After electrophoresis through a 1% agarose gel, this (5.7-kB) fragment was isolated and an equal volume of 0.3 mol/L NaCl/Tris-EDTA (TE) was added. After melting at 65°C for 30 minutes, the mixture was extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and the DNA was ethanol precipitated. The DNA was then suspended in 1 mL 0.2 mol/L NaCl/TE, purified over a Prepac column and ethanol precipitated. The recovered DNA was resuspended in 100 µL of injection buffer (10 mmol/L Tris buffer containing 10 mmol/L NaCl and 0.1 mmol/L EDTA at pH 7.4), dialyzed against a 0.1 µm Millipore filter, and diluted to a final concentration of 1 ng/µL for injection into fertilized C57BL/6 mouse blastocysts. After the (100) injections, the oocytes were transplanted into pseudopregnant ICR adult mice. A total of 37 offspring were obtained, and all were screened for expression of the transgene by PCR analysis of (tail) genomic DNA using probes directed against the HGH polyadenylation sequence (in the -MHC vector).^{36 37 38 39} Two animals positive for the transgene were identified (see Results). At 8 weeks, these animals were bred to wild-type C57BL/6 adult mice, and 2 lines of Kv2.1N216FLAG transgenic mice were established.

Expression of Kv2.1 and Kv2.1N216FLAG in Mouse Ventricles
For reverse transcription (RT)–PCR (RT-PCR) analysis, mRNA was prepared from the ventricles and brains of adult Kv2.1N216FLAG-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 mmol/L) Tris-HCl (pH 8.3) 50, MgCl₂ 5, KCl 75, sodium pyrophosphate 4, dNTPs 5, and DTT 10, 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 reverse transcriptase (Invitrogen). After a 1-hour incubation at 42°C, the reaction was terminated by heating at 95°C for 2 minutes. Approximately 2 µL of the resulting reaction mixture was used for PCR amplification. PCR was carried out in a 25-µL reaction mixture containing (in mmol/L) Tris-HCl (pH 9.0) 10, KCl 50, MgCl₂ 2, DTT 1, and dNTPs 0.2, as well as 0.5 µmol/L 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 seconds and 58°C for 45 seconds, followed by 68°C for 90 seconds. The forward and reverse primers used for RT-PCR to probe for actin, wild-type Kv2.1, and Kv2.1N216FLAG were 5'-GTGTTACGTCGCCCTTGATT-3' and 5'-GCTGGAGGTGGACAGAGAG-3' (actin), 5'-CCGAGACCAGCTCCAGTAAG-3' and 5'-CTCCACGAAGAAACCAAGC-3' (wild-type Kv2.1), and 5'-TTCAGCCAGGAGCTGGACTA-3' and 5'-CTTGTCATCTGCGTCCTTGTAGTC-3' (Kv2.1N216FLAG). The amplified PCR products were analyzed in a 1% agarose gel and stained with ethidium bromide.

Mouse ventricular membrane proteins were prepared using a protocol previously developed for the isolation of rat cardiac membrane proteins.^{40 41} After fractionation by SDS-PAGE and transfer to polyvinylidene difluoride membranes (Amersham), immunoblots were completed with a monoclonal anti-FLAG M2 antibody (Eastman Kodak) diluted 1:500 or with a polyclonal anti-Kv2.1 antibody (Upstate Biotechnology Inc, Lake Placid, NY), diluted 1:300, followed by an alkaline phosphatase-conjugated goat anti-mouse (to detect the FLAG antibody) or goat anti-rabbit secondary antibody (to detect the Kv2.1 antibody). Bound antibodies were detected using the CPSD (Tropix) chemiluminescent alkaline phosphatase substrate.^{40 41}

Electrophysiological Recordings
Whole-cell voltage-clamp recordings from GFP-positive HEK-293 cells were obtained at room temperature 48 hours after transfections. The recording pipettes contained (in mmol/L) KCl 115, KOH 15, EGTA 10, HEPES 10, and glucose 5 (pH 7.2; 300 to 310 mOsm). The bath solution contained (in mmol/L) NaCl 140, KCl 4, MgCl₂ 2, CaCl₂ 1, HEPES 10, and glucose 5 (pH 7.4; 300 to 310 mOsm). Experiments were performed using an Axopatch 1B patch-clamp amplifier (Axon Instruments) interfaced to a Gateway 350-MHz Pentium computer interfaced to the recording equipment with a Digidata 1200 analog/digital interface and the pClamp 7 software package (Axon Instruments). Data were filtered at 5 kHz before storage. 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 to 4 M and were compensated electronically by 80% to 90%; voltage errors resulting from the uncompensated series resistance were 8 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.

Ventricular myocytes were isolated from adult transgenic and wild-type animals as described previously.^{15 19 20 23} Electrophysiological experiments were conducted as described above for HEK-293 cells at room temperature on the day of cell isolation. For voltage-clamp experiments, the bath solution contained (in mmol/L) NaCl 136, KCl 4, CaCl₂ 1, MgCl₂ 2, CoCl₂ 5, HEPES 10, glucose 10, and 0.02 tetrodotoxin (TTX) (pH 7.4; 295 to 300 mOsm); TTX and Co²⁺ were eliminated when action potentials were recorded. For both current- and voltage-clamp experiments, the recording pipette solution contained (in mmol/L) KCl 135, EGTA 10, HEPES 10, and glucose 5 (pH 7.2; 295 to 310 mOsm). Outward K⁺ currents were evoked during 500-ms or 4.5-second 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 TTX-insensitive voltage-gated Na⁺ current). Inwardly rectifying K⁺ currents (I_K1) were evoked during hyperpolarizing voltage steps to test potentials between -90 and -120 mV.

Electrocardiograms
Electrocardiographic recordings were obtained from adult Kv2.1N216FLAG-expressing transgenic mice and from nontransgenic littermates using Data Sciences International implantable Physiotel TA10ETA-F20 radio frequency transmitters and receivers (placed under the cage). To implant the transmitters, animals were first anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (16 mg/kg). After opening the chest, transmitters were placed in the abdominal cavity, and the electrocardiographic leads were tunneled under the skin, sutured, and glued (veterinary tissue adhesive) 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 of the heart. After implantation of the transmitters, the animals were allowed to recover for 24 hours before recording electrocardiographic data. Analog electrocardiographic signals were collected, digitized at 1 kHz using a 16-bit Dataquest A.R.T. Gold Acquisition analog-to-digital converter (Data Sciences), and stored on CD-ROM disks. Three-minute electrocardiographic recordings every hour for 10 consecutive hours were obtained from each transgenic (n=7) and nontransgenic (n=5) animal; all animals were monitored for 5 consecutive days. Unfiltered data were analyzed, and QT, QRS, and RR intervals were measured; average values for individual animals were determined; mean±SEM values for experiments completed on transgenic (n=7) and nontransgenic (n=5) animals are reported here.

Data Analysis
Voltage-clamp 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 analyzing the decays of the capacitative transients evoked during 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 were determined by integration of the capacitative transients evoked during brief (25-ms) subthreshold (±10 mV) voltage steps from a HP of -70 mV. Mean±SEM whole-cell membrane capacitances of cells isolated from wild-type and transgenic animals were 140±7 pF (n=25) and 175±13 pF (n=17) in wild-type and Kv2.1N216FLAG-expressing cells, respectively. The mean±SEM input resistances of adult mouse wild-type (n=25) and Kv2.1N216FLAG-expressing (n=17) ventricular myocytes were 0.90±0.23 G and 0.65±0.20 G, respectively. Leak currents were always <100 pA and were not corrected. The plateau outward K⁺ current was defined as the current remaining 3 seconds after the onset of the depolarizing voltage steps, and the peak outward current was defined as the maximum value of the outward K⁺ current during 200-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.

The time constants of inactivation of the depolarization-activated outward K⁺ currents in wild-type and Kv2.1N216FLAG-expressing ventricular myocytes were determined from double exponential fits to the decay phases of the current, using the following equation: A_t=A₁xexp(-t/₁)+A₂xexp(-t/₂)+A_ss, where A_t is the amplitude of the current at time t, A₁ and ₁ and A₂ and ₂ represent the amplitudes (A) and the time constants () of the fast and slow components of current decay, and A_ss is the amplitude of the noninactivating (steady-state) component of the total outward K⁺ current. Correlation coefficients were determined to assess the quality of the fits; in all cases, correlation coefficients were 0.980. 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 2 leads. Measured QT intervals were corrected for differences in heart rate using the formula QT_c=QT_o/(RR/100)^1/2, as described by London et al.³² All data are presented as mean±SEM unless otherwise noted. Differences between wild-type and Kv2.1N216FLAG-expressing myocytes were analyzed using ANOVA and the Student's t test; probability value are presented in the text.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation and Characterization of the Dominant-Negative Kv2.1 Construct, Kv2.1N216
To generate the Kv2 dominant negative construct, mutations were introduced to the cDNA sequence to place a stop codon at amino acid residue 216; this approach is similar to that used by Folco et al³⁵ in the development of Kv1.1N206Tag. The dominant-negative Kv2 construct is referred to as Kv2.1N216 (Figure 1A). The truncated Kv2 subunit was tagged at the C terminus with the 8–amino acid FLAG epitope to allow direct detection of expression of the transgene. When transfected into HEK-293 cells, expression of Kv2.1N216FLAG was readily detected immunohistochemically using an anti-FLAG M2 antibody (data not shown). As expected, no outward K⁺ currents were evident in whole-cell voltage-clamp recordings from Kv2.1N216FLAG-expressing HEK-293 cells (Figure 1B). When the plasmid encoding Kv2.1N16FLAG is cotransfected with wild-type (full-length) Kv2.1, however, the densities of the Kv2.1-induced currents are markedly reduced (Figure 1D) compared with cells expressing wild-type Kv2.1 alone (Figure 1C). The mean±SEM peak outward K⁺ current densities at +60 mV in HEK-293 cells expressing wild-type Kv2.1 alone and wild-type Kv2.1 plus Kv2.1N216FLAG were 403±74 pA/pF (n=11) and 117±28 pA/pF (n=9), respectively; these values are significantly (P<0.001) different. The voltage dependences and the rates of activation and inactivation of the Kv2.1-induced currents in the absence (n=11) and in the presence (n=9) of Kv2.1N216FLAG, however, are indistinguishable. These observations suggest that the assembly of wild-type subunits with the truncated Kv2.1N216FLAG leads to the generation of nonfunctional channels rather than to channels with altered time- and/or voltage-dependent properties.

View larger version (27K): [in this window] [in a new window]   Figure 1. Kv2.1N216 functions as a dominant-negative subunit. A, Predicted membrane topology of the truncated Kv2.1 subunit with the intracellular N terminus, the S1 transmembrane domain, and the portion of the extracellular S1-S2 loop. B through F, Whole-cell outward K+ currents recorded from transiently transfected HEK-293 cells. Currents were evoked during 150-ms depolarizing voltage steps to potentials between -50 and +60 mV from a HP of -70 mV. B, No outward K+ currents are recorded from HEK-293 cells expressing Kv2.1N216FLAG. Coexpression of Kv2.1N216FLAG with the wild-type Kv2.1, however, reveals outward K+ currents that are markedly reduced (D), compared with currents produced on expression of Kv2.1 (C) alone. The currents recorded in HEK-293 cells cotransfected with Kv1.4 and Kv2.1N216 (F) are indistinguishable from those recorded from cells expressing Kv1.4 alone (E). Scale bars are 2 nA and 20 ms.

In contrast to the effects on Kv2.1-induced currents, coexpression of Kv2.1N216FLAG with Kv1.4, a member of the Shaker subfamily that is not expected to assemble with subunits of the Kv2 subfamily,⁴² however, has no measurable effect on the Kv1.4-induced currents (Figure 1E and 1F). The mean±SEM peak outward K⁺ current densities at +60 mV in HEK-293 cells expressing Kv1.4 alone and Kv1.4 plus Kv2.1N216 were 204±52 pA/pF (n=8) and 194±36 pA/pF (n=8), respectively; these values are not significantly different.

Generation and Characterization of Transgenic Mice Expressing Kv2.1N216FLAG
For the generation of transgenic mice expressing Kv2.1N216FLAG, the FLAG-tagged Kv2.1N216 DNA sequence was subcloned downstream of the cardiac-specific -MHC promoter in the -MHC expression vector (generously provided to us by Dr Jeffery Robbins, University of Cincinnati, Ohio). Previous work 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.^{36 37 38 39} A 5.7-kb fragment containing the -MHC promoter, the Kv2.1N216FLAG 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. Two founders (Nos. 5 and 6) were identified and were bred to wild-type C57BL/6 mice to generate 2 lines of Kv2.1N216FLAG-expressing transgenic mice.

Initial analysis of the functional consequences of Kv2.1N216FLAG expression were completed on the F₁ progeny of founder No. 5. As illustrated in Figure 2B, PCR analysis revealed that Kv2.1N216FLAG, as well as wild-type Kv2.1, expression is readily detected in the ventricles of line No. 5 transgenic animals, whereas only wild-type Kv2.1 is detected in the ventricles of nontransgenic littermates. In addition, in brains isolated from Kv2.1N216FLAG-expressing transgenic mice, only wild-type Kv2.1 is evident (Figure 2B), consistent with the cardiac-specific expression of the transgene.^{36 37 38 39} Expression of the Kv2.1N216FLAG protein was also readily detected in Western blots of fractionated ventricular membrane proteins^40,41 from line 5 transgenic mice (Figure 2C). Using an antibody targeted against the C terminus of Kv2.1, the full-length, endogenous Kv2.1 protein is evident in Western blots of fractionated mouse ventricular membrane proteins from both wild-type and transgenic animals (Figure 2C). In addition, and in contrast to the findings of London et al³² with the truncated Kv1.1N206Tag, expression of the Kv2.1N216FLAG does not to result in a detectable decrease in the amount of full-length Kv2.1 protein in the ventricles of the transgenic animals relative to wild-type hearts (Figure 2Cb) (see Discussion).

View larger version (44K): [in this window] [in a new window]   Figure 2. Expression of Kv2.1 and Kv2.1N216FLAG in mouse ventricles. Using subunit-specific probes (A) (see Materials and Methods), RT-PCR analysis revealed that Kv2.1 is readily detected in Kv2.1N216FLAG-expressing adult mouse ventricles, as well as in the ventricles of nontransgenic littermates (B); Kv2.1 is also readily detected in the brains of these animals (B). Expression of the Kv2.1N216FLAG transgene, in contrast, is only detected in the hearts of the Kv2.1N216FLAG-expressing animals (B); Kv2.1N216FLAG is not detected in the brains of these transgenic animals or in tissues from nontransgenic littermates. B, Western blot analysis (see Materials and Methods) using anti-Kv2.1 (b) and anti-FLAG (a) antibodies revealed that the Kv2.1 protein is also readily detected in wild-type and Kv2.1N216FLAG-expressing adult mouse ventricles (b), whereas Kv2.1N216FLAG protein expression is only detected in the hearts of the Kv2.1N216FLAG transgenic mice (a). wt indicates wild type, full length 2.1.

On gross examination, no obvious differences between the Kv2.1N216FLAG-expressing transgenic animals and their nontransgenic littermates were noted. Mean±SD body weights, for example, were 25.9±3.0 g (n=7) and 25.9±3.7 g (n=7) for adult (20 week) wild-type and Kv2.1N216FLAG-expressing animals; these values are not significantly different. Heart weights were also not significantly different, with mean±SD values of 102±12 mg (n=7) and 107±18 mg (n=7) for wild-type and Kv2.1N216FLAG-expressing animals, respectively. Heart-to-body weight ratios in nontransgenic and transgenic animals, therefore, were also very similar, and histological examination of hearts from Kv2.1N216FLAG-expressing animals revealed no significant differences from controls. Interestingly, however, there is a small but statistically significant (P<0.05) difference in the mean±SEM input resistances and whole-cell membrane capacitances of ventricular myocytes isolated from Kv2.1N216FLAG-expressing and wild-type animals. Mean±SEM whole-cell membrane capacitances of cells isolated from wild-type and transgenic animals were 140±7 pF (n=25) and 175±13 pF (n=17) in wild-type and Kv2.1N216FLAG-expressing cells, respectively, and mean±SEM input resistances of adult mouse wild-type (n=25) and Kv2.1N216FLAG-expressing (n=17) ventricular myocytes were 0.90±0.23 G and 0.65±0.20 G, respectively. These findings suggest that, despite the absence of gross changes in heart size and/or appearance, Kv2.1N216FLAG expression does have a small, but measurable, effect on the properties of individual mouse ventricular cells (see Discussion).

Outward K⁺ Currents are Attenuated in Kv2.1N216FLAG-Expressing Ventricular Myocytes
Whole-cell voltage-clamp recordings revealed that the waveforms of the depolarization-activated outward K⁺ currents in ventricular myocytes isolated from wild-type and transgenic littermates are distinct (Figure 3). Peak outward K⁺ current amplitudes at all test potentials, for example, are lower in cells isolated from Kv2.1N216FLAG-expressing transgenic animals (Figure 3C and 3D) compared with the currents typically recorded in myocytes isolated from nontransgenic (wild-type) littermates (Figure 3A and 3B). Similar results were obtained in 17 myocytes from 2 Kv2.1N216FLAG transgenic animals derived from line 5, and the mean (±SEM) peak outward densities at +40 mV were 50.4±4.4 pA/pF (n=25) and 36.8±3.8 pA/pF (n=17) in wild-type and Kv2.1N216FLAG-expressing myocytes, respectively (Table 1); these values are significantly (P0.05) different. In contrast to the observed reductions in peak outward K⁺ current densities in ventricular myocytes isolated from the Kv2.1N216FLAG-expressing transgenic animals, no measurable effects on the densities of either the steady-state outward K⁺ currents, I_ss, determined as the currents remaining at the end of 4.5 s voltage steps,¹⁵ or of the hyperpolarization- activated, inwardly rectifying K⁺ current, I_K1, were observed. Mean±SEM I_ss densities at +40 mV, for example, were 6.3±0.5 pA/pF (n=25) and 5.0±0.5 pA/pF (n=17) in wild-type and Kv2.1N216FLAG-expressing myocytes, respectively (Table 1). Mean±SEM peak I_K1 densities evoked at -120 mV from a HP of -70 mV in wild-type and Kv2.1N216FLAG-expressing myocytes were 11.5±0.5 pA/pF (n=25) and 10.9±0.9 pA/pF (n=17), respectively.

View larger version (23K): [in this window] [in a new window]   Figure 3. Outward K+ current waveforms are altered in Kv2.1N216FLAG-expressing transgenic mice. Whole-cell outward K+ currents, recorded from ventricular myocytes isolated from adult nontransgenic (A and B) and Kv2.1N216FLAG transgenic (C and D) littermates, were evoked during 450-ms (A and C) or 4.5-second (B and D) depolarizing voltage steps to potentials between -50 and +60 mV from a HP of -70 mV. The waveforms of the Ca2+-independent, depolarization-activated outward K+ currents in wild-type (A and B) and transgenic (C and D) myocytes are distinct; peak outward K+ current amplitudes are reduced and the slowly decaying current component, IK, slow, is much less prominent in the Kv2.1N216FLAG-expressing cell (C and D) compared with the currents recorded from the nontransgenic, wild-type, cell (A and B). The decay phases of the outward K+ currents in records such as those in panels B and D were analyzed to provide the amplitudes of Ito, f, IK, slow and Iss in individual cells (see Materials and Methods) and normalized to the whole-cell membrane capacitance (determined in the same cell). E, Mean±SEM Ito, f (, •), IK, slow (, ) and Iss (, ) densities in wild-type (filled symbols; n=25) and Kv2.1N216-expressing (open symbols; n=17) cells are plotted as a function of test potential. Mean±SEM IK, slow density is significantly different (**P<0.01) in the Kv2.1N216FLAG-expressing () than in wild-type () cells, whereas the densities of Ito, f and Iss in wild-type and transgenic cells are not significantly different (see text).

View this table: [in this window] [in a new window]   Table 1. Comparison of Outward K+ Currents in Wild-Type and Kv2.1N216FLAG Adult Mouse Ventricular Myocytes

I_{K, slow} Is Altered Selectively in Kv2.1N216FLAG-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 2 exponentials, with decay time constants (_decay) that differ by an order of magnitude, and a noninactivating, ie, steady-state, current, I_ss.^{15 23} Mean±SEM _decay for the fast and the slow components of inactivation in wild-type cells (n=25) are 74±3 ms and 1147±57 ms (Table 1), corresponding to inactivation of I_{to, f} and I_{K, slow},^{15 23 32 34} and, as reported previously,^{15 23 34} neither time constant displays any appreciable voltage dependence. Mean±SEM 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); the mean±SEM I_ss density in these cells (n=25) was 6.3±0.5 pA/pF (Table 1). The variations in the densities of the individual current components in wild-type cells are plotted as a function of test potential in Figure 3E.

Analysis of the waveforms of the depolarization-activated K⁺ currents in ventricular myocytes isolated from Kv2.1N216FLAG-expressing littermates revealed that the decay phases of the currents were also well described by the sum of 2 exponentials. In this case, however, the mean±SEM (n=17) decay time constants were 66±4 ms and 830±103 ms (Table 1) and, similar to the findings in wild-type cells,^{15 23 34} neither time constant varies with voltage. Importantly, the time constant (of 66±4 ms) for the fast component of current decay in Kv2.1N216FLAG-expressing transgenic mouse ventricular myocytes, which reflects I_{to, f},^{15 23} is not significantly different from that determined in wild-type cells (mean±SEM _decay=74±3 ms). The mean±SEM time constant of inactivation of the slower component of current decay (_decay=830±103 ms) in the Kv2.1N216FLAG-expressing cells, in contrast, is significantly (P<0.05) faster than the mean±SEM _decay of 1147±57 ms (Table 1) for I_{K, slow}^{15 23 34} in wild-type cells. This finding suggests either that I_{K, slow} inactivation is accelerated in Kv2.1N216FLAG-expressing ventricular myocytes or, alternatively, that there are 2 components of I_{K, slow} in wild-type cells (that inactivate at different rates) and that one of these is attenuated, or eliminated, in the Kv2.1N216FLAG-expressing transgenic mice (see Discussion). Analysis of the decay phases of the outward currents also revealed that the density of the slow component of current decay in Kv2.1N216FLAG-expressing ventricular cells is significantly (P<0.01) lower than in wild-type cells; mean±SEM I_{K, slow} densities (at +40 mV) were 8.7±1.3 pA/pF and 15.6±1.6 pA/pF in ventricular cells isolated from transgenic (n=17) and nontransgenic (n=25) littermates, respectively (Table 1). The voltage-dependent properties of the currents, however, are unaffected, and I_{K, slow} density is significantly lower at all test potentials in the Kv2.1N216FLAG-expressing, than in wild-type, cells (Figure 3E). No significant differences in the densities or in the properties of I_{to, f} (or I_ss) densities were observed (Table 1; Figure 3E), suggesting that I_{K, slow} is selectively attenuated in ventricular myocytes isolated from adult Kv2.1N216FLAG-expressing transgenic animals (see Discussion).

Pharmacological Properties of I_{K, slow} in Kv2.1N216FLAG-Expressing Ventricular Myocytes
In wild-type mouse ventricular myocytes, I_{K, slow} is blocked by µmol/L concentrations of 4-aminopyridine (4-AP)^{15 32 33} and by mmol/L concentrations of tetraethylammonium (TEA)¹⁵ ; I_ss is also partially blocked by millimolar concentrations of TEA but is unaffected by 4-AP at concentrations 1 mmol/L.¹⁵ Exposure of adult mouse ventricular myocytes to concentrations of 4-AP >100 µmol/L leads to further block of I_{K, slow} and also blocks I_{to, f}. In the presence of 0.5 mmol/L 4-AP, for example, I_{to, f} is attenuated by 50% and I_{K, slow} is blocked completely.¹⁵ Experiments were completed, therefore, to determine the pharmacological properties of the slowly inactivating outward current (I_{K, slow}) remaining in Kv2.1N216FLAG-expressing ventricular myocytes (Figure 4). After recording control currents (Figure 4A), cells were exposed sequentially to 25 mmol/L TEA followed by 50 µmol/L 4-AP, and the currents in the presence of 25 mmol/L TEA (Figure 4B) and 50 µmol/L 4-AP (Figure 4C) were recorded. The waveforms of the 25 mmol/L TEA-sensitive (Figure 4D) or the 50 µmol/L 4-AP-sensitive (Figure 4E) components of the currents were then determined by subtracting the currents in the presence of 25 mmol/L TEA (Figure 4B) or 50 µmol/L 4-AP (Figure 4C) from the controls (Figure 4A).

View larger version (19K): [in this window] [in a new window]   Figure 4. The TEA-sensitive component of IK, slow is eliminated in Kv2.1N216-expressing ventricular cells. Outward currents were recorded as described in legend to Figure 3 in the presence and absence of the K+ channel blockers TEA and 4-AP at 25 mmol/L and 30 µmol/L, respectively. After recording control currents (A), cells were exposed sequentially to 25 mmol/L TEA followed by 30 µmol/L 4-AP. Currents in the presence of 25 mmol/L TEA and 30 µmol/L 4-AP are presented in panels B and C. The waveforms of the 25 mmol/L TEA-sensitive (D) and the 30 µmol/L 4-AP–sensitive (E) currents were obtained by offline digital subtraction of the currents recorded in the presence of TEA (B) or 4-AP (C) from the currents of controls (A).

Analysis of the 25 mmol/L TEA-sensitive currents (Figure 4D) reveals that activation is slow and that the currents undergo little or no inactivation during the 4.5-second voltage steps. The waveforms of the 25 mmol/L TEA-sensitive currents are consistent with block of I_ss.¹⁵ In wild-type cells, the decay phases of the 25 mmol/L TEA-sensitive currents are well described by single exponentials with a mean±SEM _decay of 1234±197 ms (n=4), an observation interpreted as reflecting TEA block of I_{K, slow} in these cells.¹⁵ In wild-type cells, both I_ss and I_{K, slow} are reduced by 60% in 25 mmol/L TEA (and I_{to, f} is unaffected).¹⁵ The inactivating component of the TEA-sensitive currents seen in wild-type cells, however, is not evident in the Kv2.1N216FLAG-expressing cells (Figure 4D), suggesting that Kv2 subunits underlie this (TEA-sensitive) component of I_{K, slow} (see Discussion). Similar to findings in wild-type cells,^{15 23 32 33} the waveforms of the 50 µmol/L 4-AP-sensitive currents in Kv2.1N216FLAG-expressing cells (Figure 4E) are consistent with the selective attenuation of I_{K, slow}. Taken together, these results suggest that the component of I_{K, slow} sensitive to µmol/L concentrations of 4-AP remains in the Kv2.1N216-expressing ventricular myocytes, whereas the TEA-sensitive component of I_{K, slow} is eliminated (see Discussion).

Action Potentials and QT Intervals Are Prolonged in the Kv2.1N216FLAG-Expressing Animals
Current-clamp experiments revealed that action potentials recorded from Kv2.1N216FLAG-expressing ventricular myocytes are substantially broader than action potentials recorded from cells isolated from nontransgenic littermates (Figure 5A), with the latter phase of repolarization being particularly affected (Figure 5A). Analysis of action potential durations at 25% (APD₂₅), 50% (APD₅₀), 75% (APD₇₅), and 90% (APD₉₀) repolarization indeed revealed that only mean±SEM APD₉₀ values were increased significantly (P<0.01) in Kv2.1N216FLAG-expressing ventricular cells (Table 2). Mean±SEM APD₉₀ values were 34±5 ms (n=16) and 54±6 ms (n=22) in wild-type and Kv2.1N216FLAG-expressing cells, respectively (Table 2). In contrast, APD₂₅, APD₅₀, and APD₇₅ values in transgenic and nontransgenic cells are not significantly different (Table 2). In addition, action potential amplitudes and resting membrane potentials of the Kv2.1N216FLAG-expressing myocytes are not significantly different from those measured in wild-type cells (Table 2). The action potential prolongation seen in the Kv2.1N216FLAG-expressing ventricular cells, however, is substantially less than that observed in Kv4.2W362F-expressing mouse ventricular myocytes, which lack I_{to, f},¹⁹ and in Kv1.1N206Tag-expressing cells, in which I_{K, slow} is also attenuated³² (see Discussion). Unexpectedly, triggered activity, presumably resulting from early afterdepolarizations, was observed in 4 of the 22 transgenic Kv2.1N216FLAG-expressing ventricular cells studied (Figure 5B).

View larger version (9K): [in this window] [in a new window]   Figure 5. Whole-cell current-clamp recordings reveal marked action potential prolongation in Kv2.1N216FLAG-expressing ventricular myocytes compared with ventricular myocytes isolated from nontransgenic littermates (A). In some (4 of 22) of the Kv2.1N216FLAG-expressing ventricular cells, spontaneously triggered activity (B) was observed.

View this table: [in this window] [in a new window]   Table 2. Action Potential Prolongation in Kv2.1N216FLAG-Expressing Adult Mouse Ventricular Myocytes

Although mean±SEM APD₉₀ values are increased significantly in Kv2.1N216FLAG-expressing transgenic mice, considerable variability was observed among cells. This point is clearly evident in the histogram in Figure 6, in which APD₉₀ values determined in individual cells are displayed. In the majority (13 of 16, or 80%) of the wild-type cells, APD₉₀ values are <50 ms, and in only 1 cell was the APD₉₀ >60 ms (Figure 6). The measured APD₉₀ values were not correlated with differences in resting membrane potentials and are assumed to reflect variability among cells in the absolute densities of the depolarization-activated K⁺ currents, I_{to, f}, I_{K, slow}, and I_ss, that underlie repolarization (see Discussion). In the Kv2.1N216FLAG-expressing cells, in contrast, APD₉₀ values >50 ms were measured in half (11 of 22) of the cells (Figure 6), and, in 4 of these cells, the APD₉₀ exceeded 90 ms, ie, was significantly longer than that observed in any of the wild-type cells (Figure 6). These observations are consistent with the variable attenuation in I_{K, slow} in the Kv2.1N216FLAG-expressing ventricular myocytes (Table 1).

View larger version (15K): [in this window] [in a new window]   Figure 6. Distribution of APD90 repolarization in Kv2.1N216FLAG-expressing and wild-type adult mouse ventricular myocytes. APD90 values were measured in individual cells from records such as those presented in Figure 3 and binned in 19- to 20-ms increments. APD90 values (<50 ms) similar to those measured in the majority of wild-type cells (13 of 16) were also seen in some Kv2.1N216FLAG-expressing cells; the distribution of APD90, however, is much broader in the transgenic mice; see text).

To determine the functional consequences of the prolongation of ventricular action potentials, telemetric electrocardiographic recordings were obtained from Kv2.1N216FLAG-expressing transgenic and nontransgenic (wild-type) littermates (see Materials and Methods). Typical electrocardiographic recordings from wild-type and Kv2.1N216FLAG transgenic mice are presented in Figure 7. 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. Mean±SEM QT intervals in wild-type and transgenic animals were found to equal 27±4 ms (n=5) and 42±5 ms (n=7), respectively; these values are significantly (P<0.001) different. Neither heart rates (RR intervals) nor QRS durations, however, were affected by Kv2.1N216FLAG expression. Mean±SEM RR intervals were 100±6 ms (n=5) and 102±9 ms (n=7) in wild-type and Kv2.1N216FLAG-expressing animals, respectively; mean±SEM QRS durations were 8±1 ms (n=5) and 8±2 ms (n=7) in control and transgenic animals, respectively. When QT intervals were corrected for heart rate,³² the differences between the transgenic and the nontransgenic animals remained highly significant (at the P<0.001 level); mean±SEM QT_c intervals (see Materials and Methods) were 27±5 ms (n=5) and 42±4 ms (n=7) in control and Kv2.1N216FLAG-expressing animals, respectively. Interestingly, the QT prolongation seen in the Kv2.1N216FLAG-expressing cells is substantially less than that observed in Kv4.2W362F-expressing mouse ventricular myocytes, which lack I_{to, f},^{19 23} and in Kv1.1N206Tag-expressing cells, in which I_{K, slow} is also attenuated³² (see Discussion). As might be expected from the finding of triggered activity in single ventricular myocytes (Figure 5B), premature ventricular beats were observed in the electrocardiographic recordings from 2 of the 7 Kv2.1N216FLAG-expressing animals (see Discussion).

View larger version (11K): [in this window] [in a new window]   Figure 7. Prolongation of the QT interval is evident in electrocardiographic recordings from Kv2.1N216FLAG-expressing mice. ECGs were recorded as described in Materials and Methods with electrode position optimized to permit reliable resolution of the T waves and accurate measurements of QT intervals. P, QRS, and T waves and the QT intervals are clearly labeled in the records obtained from the nontransgenic and Kv2.1N216FLAG-expressing mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Functional Consequences of Cardiac-Specific Expression of Kv2.1N216FLAG
The results presented here reveal that the density of the slow component of current decay, referred to as I_{K, slow} in wild-type mouse ventricular cells,^{15 23 32 33 34} is reduced significantly in ventricular myocytes isolated from Kv2.1N216FLAG-expressing animals. The effect on I_{K, slow} is specific in that neither I_{to, f} nor I_ss is affected, suggesting that members of the Kv2 subunit subfamily contribute to the slow component of delayed rectification in the mouse ventricle. This finding was unexpected, because it has previously been reported that mouse ventricular I_{K, slow} is eliminated or markedly reduced^{32 34} in cells isolated from transgenic mice expressing a truncated Kv1.1 subunit, Kv1.1N206Tag, an observation that was interpreted as suggesting that members of the Kv1 subfamily of subunits underlie I_{K, slow}. These combined, and seemingly conflicting, observations could reflect strain differences in that the experiments described here were completed on C57BL/6 mice, whereas those of London et al³² were completed on FVB mice. This hypothesis, however, seems unlikely given that the properties of the various currents in ventricular myocytes isolated from these 2 strains appear to be indistinguishable.^{15 19 23 32 34}

Rather, it seems more likely that there are 2 components of I_{K, slow} in wild-type adult mouse ventricular myocytes, with similar, but not identical, properties. In support of this hypothesis, the fraction of I_{K, slow} remaining in the Kv2.1N216FLAG-expressing ventricular cells is blocked by 50 µmol/L 4-AP but is insensitive to 25 mmol/L TEA, whereas wild-type I_{K, slow} is attenuated to 50% of control by 25 mmol/L TEA, as well as by 50 µmol/L 4-AP. Taken together, these results suggest that the component of I_{K, slow} sensitive to µmol/L concentrations of 4-AP remains in the Kv2.1N216-expressing ventricular myocytes, whereas the TEA-sensitive component of I_{K, slow} is eliminated. If this interpretation is correct and the currents in wild-type C57BL/6 and FVB mice are indeed indistinguishable, it follows that a component of I_{K, slow} remains in the Kv1.1N206Tag-expressing ventricular cells and that this component reflects the expression of (TEA-sensitive) Kv2 subunits. Further experiments focused on testing this hypothesis directly and on examining the pharmacological properties of the depolarization-activated outward K⁺ currents in the Kv1.1N206Tag-expressing transgenic mice would clearly be of interest.

The results presented here also demonstrate that the rate of inactivation of the slow component of current decay in ventricular cells isolated from the Kv2.1N216FLAG-expressing animals is faster than observed (for I_{K, slow}) in wild-type ventricular cells. Again, the simplest interpretation of this finding is that there are 2 components of I_{K, slow} and that the faster component of I_{K, slow} remains in Kv2.1N216FLAG-expressing cells. If this is correct, it also follows that there is a component of I_{K, slow} remaining in Kv1.1N206Tag-expressing ventricular cells and that the time constant of inactivation of this component of I_{K, slow} (attributed to Kv2 subunit expression) must be 1100 ms, ie, similar to the mean _decay for I_{K, slow} in wild-type cells (Table 1). Further experiments aimed at testing these hypotheses directly are clearly warranted. In addition, it would be of interest to cross the Kv1.1N206Tag- and the Kv2.1N216FLAG-expressing transgenic mice to test the prediction that both components of I_{K, slow} will be eliminated in ventricular myocytes isolated from animals expressing both transgenes.

Current-clamp experiments revealed that APD₉₀ values are increased significantly in Kv2.1N216FLAG-expressing ventricular myocytes, consistent with the attenuation of I_{K, slow}. In addition, triggered activity, presumably resulting from early afterdepolarizations, was observed in 4 of the 22 Kv2.1N216FLAG-expressing ventricular cells studied. The attenuation of I_{K, slow} also results in prolongation of the QT interval, and in 2 of the 7 Kv2.1N216FLAG-expressing animals, premature ventricular beats were recorded. Expression of Kv2.1N216FLAG, however, does not appear to have any other profound physiological or pathophysiological consequences. In fact, the Kv2.1N216FLAG-expressing animals appear normal in every respect and, in experiments completed to date, we find no evidence that these animals are prone to develop arrhythmias. Interestingly, the increases in action potential durations seen in the Kv2.1N216FLAG-expressing ventricular cells and the QT prolongation in Kv2.1N216FLAG-expressing animals are substantially less than those seen in either the Kv4.2W362F-expressing transgenic mouse ventricular myocytes, which lack I_{to, f},¹⁹ or the Kv1.1N206Tag-expressing cells, in which I_{K, slow} is attenuated.³² Presumably, these difference reflect the kinetic properties and the densities of the various K⁺ currents (ie, I_{to, f} and the fast and slow components of I_{K, slow}) that are affected in Kv4.2W362F-,¹⁹ Kv1.1N206Tag-,³² and Kv2.1N216FLAG-expressing transgenic animals, respectively. It is certainly also possible that there are additional changes (such as in the properties and/or the functional expression of other ion channels, pumps, or carriers) that occur in the ventricles of these transgenic animals, and that these, in turn, contribute to prolonged action potential waveforms and QT intervals. Overexpression of the Na⁺-Ca²⁺ exchanger, for example, leads to profound changes in the waveforms of mouse ventricular action potentials.⁴³ Further experiments will be necessary to explore this possibility directly.

Relationship to Previous Studies
As noted above, London et al³² reported the generation and characterization of long-QT mice³² expressing a truncated Kv1.1 subunit, Kv1.1N206Tag, that functions as a dominant negative.³⁵ When expressed in GH3 cells, Kv1.1N206Tag coassembles with endogenous Kv1.4 and Kv1.5, and the resulting heteromeric channel complexes appear to be retained in the endoplasmic reticulum, rather than being transported to the plasma membrane or targeted for degradation.³⁵ The finding that Kv1.1N206Tag expression attenuates mouse ventricular I_{K, slow} was interpreted as suggesting that Kv1 subunits underlie I_{K, slow}.^{32 35} The sensitivity of I_{K, slow} to µmol/L concentrations of 4-AP,^{32 33} and the finding that Kv1.5 protein levels are reduced in Western blots of frac-tionated (crude) ventricular membrane proteins from Kv1.1N206Tag-expressing transgenic mice led to the specific hypothesis that Kv1.5 is the Kv1 subfamily member that underlies I_{K, slow}.³² The loss of Kv1.5 protein in these animals, however, is unexpected given that steady-state Kv1.4 and Kv1.5 protein levels in GH3 cells were unaffected by Kv1.1N206Tag expression.³⁵ These results also contrast with the results described here demonstrating that the steady-state level of Kv2.1 protein expression is not measurably altered in the ventricles of Kv2.1N216FLAG-expressing transgenic mice. Further experiments will be necessary to explore the molecular basis of these differences.

Cardiac-specific expression of Kv1.1N206Tag also results in prolonged ventricular action potentials and QT intervals, increased frequency of premature ventricular beats, ventricular arrhythmias, and spontaneous ventricular tachyarrhythmias.³² Triggered activity was seen in 4 of 22 Kv2.1N216FLAG-expressing ventricular myocytes, and premature ventricular beats were recorded in 2 of the 7 animals monitored; no evidence for ventricular arrhythmia, however, was obtained. Interestingly, the increases in action potential durations and QT intervals observed in the Kv1.1N206Tag-expressing animals are greater than observed here in the Kv2.1N216FLAG transgenic mice, and this may account for the more dramatic phenotype seen in the Kv1.1N206Tag-expressing animals. In this context, it is of interest to note that the prolongation of ventricular action potentials and QT intervals observed in Kv4.2W362F-expressing transgenic mice, which lack I_{to, f},¹⁹ are larger than in the Kv1.1N206Tag transgenic mice,³² and there is no evidence for triggered activity, premature beats, or spontaneous arrhythmias in these (KV4.2W362F) animals.¹⁹ Taken together, these results suggest that factors, in addition to prolonged ventricular repolarization, play an important role in determining the propensity to develop and to sustain arrhythmias. Studies focused on exploring the molecular mechanisms underlying the markedly different phenotypes of mice expressing Kv4.2W362F,¹⁹ Kv2.1N216FLAG, and Kv1.1N206Tag³² are clearly warranted.

	Acknowledgments

 
The financial support provided by the National Heart, Lung, and Blood Institute and the Washington University/Monsanto/Searle Biomedical Research Agreement is gratefully acknowledged. We thank Andrew Benedict for technical assistance in the generation, screening, and maintenance of the transgenic mice and Bridget Scheve for assistance with the Western blot analysis of Kv2.1 and Kv2.1N216FLAG expression. We also thank Drs Carla Weinheimer and Kathryn Yamada (Department of Medicine, Washington University Medical School) for assistance with the telemetric electrocardiographic recordings and for many helpful comments and discussions throughout the course of this work and Dr Jeffery Robbins (University of Cincinnati, Ohio) for the -MHC transgenic construct.

Received March 19, 1999; accepted July 13, 1999.

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