Published online before print September 18, 2003, doi: 10.1161/01.RES.0000096362.60730.AE
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© 2003 American Heart Association, Inc.
Cellular Biology |
Kv1.5 Is an Important Component of Repolarizing K+ Current in Canine Atrial Myocytes
From the Department of Physiology (D.F., J.E., D.F.S.), University of British Columbia, Vancouver, British Columbia, Canada; Cardiome Pharma Corp (D.F., J.C.H.), Vancouver, British Columbia, Canada; and Department of Cardiology (M.L., L.C., D.R.V.W.), Cleveland Clinic Foundation, Cleveland, Ohio.
Correspondence to Dr David Fedida, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3. E-mail fedida{at}interchange.ubc.ca
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Abstract |
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Although the canine atrium has proven useful in several experimental models of atrial fibrillation and for studying the effects of rapid atrial pacing on atrial electrical remodeling, it may not fully represent the human condition because of reported differences in functional ionic currents and ion channel subunit expression. In this study, we reassessed the molecular components underlying one current, the ultrarapid delayed rectifier current in canine atrium [IKur(d)], by evaluating the mRNA, protein, immunofluorescence, and currents of the candidate channels. Using reverse transcriptase-polymerase chain reaction, we found that Kv1.5 mRNA was expressed in canine atrium whereas message for Kv3.1 was not detected. Western analysis on cytosolic and membrane fractions of canine tissues, using selective antibodies, showed that Kv3.1 was only detectable in the brain preparations, whereas Kv1.5 was expressed at high levels in both atrial and ventricular membrane fractions. Confocal imaging performed on isolated canine atrial myocytes clearly demonstrated the presence of Kv1.5 immunostaining, whereas that of Kv3.1 was equivocal. Voltage- and current-clamp studies showed that 0.5 mmol/L tetraethylammonium had variable effects on sustained K+ currents, whereas a compound with demonstrated selectivity for hKv1.5 versus Kv3.1, hERG or the sodium channel, fully suppressed canine atrial IKur tail currents and depressed sustained outward K+ current. This agent also increased action potential plateau potentials and action potential duration at 20% and 50% repolarization. These results suggest that in canine atria, as in other species including human, Kv1.5 protein is highly expressed and contributes to IKur.
Key Words: potassium channels • hKv1.5 • Kv3.1 • canine heart • atrium
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Introduction |
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In the heart, opening of voltage-activated K+ channels after the initial Na+ and Ca2+ current-driven depolarizations governs the amplitude and duration of the cardiac action potential. In human atria, as well as in other species, the atrial action potential is abbreviated with respect to the ventricular action potential and the plateau is at a lower potential because of a greater density of repolarizing K+ currents compared with inward currents. Progress has been made in understanding the molecular correlates of the rapid early repolarization current, Ito, which is observed in both atrial and ventricular myocytes1 and is now thought to be attributable to differential expression across species of Kv4.2/4.3 and Kv1.4 subfamily
subunits.2,3 An additional rapidly activating K+ current, IKur, is important in speeding the repolarization of atrial myocytes in many species.4,5 Although the Kv1.5 channel that underlies IKur is expressed at both the mRNA6,7 and protein levels8 in human ventricular tissue, albeit at low levels,9 its functional activity seems to be specific to the atrium.10 Thus, block of IKur may produce an atrial-selective increase in action potential prolongation and refractoriness.11 Drugs that modulate IKur may have useful selectivity and safety advantages over compounds that block ionic currents like IKr present in both atrial and ventricular myocytes. IKur blockade may thus represent a promising new approach to treating atrial fibrillation (AF).12 Like the transient outward current, there seems to be species variation in the molecular correlates of IKur. Kv1.5 is thought to underlie this current in human and rat atrium,2,10,13–15 whereas Kv1.2 has an important role in rat IK,slow.16 Studies in canine atrium have suggested that Kv1.5 is absent in that species17 and that Kv3.1 is the primary potassium channel subunit contributing to the so-called IKur(d) in dog.17,18
Because the canine model has been widely used in AF studies, it is important to be certain whether Kv1.5, Kv3.1, or both underlie this current in canine atria. In this study we have tested the hypothesis that Kv1.5 is present in canine atrium and have found Kv1.5 to be expressed at high levels in both canine atrium and ventricle. Surprisingly, we were unable to find biochemical evidence of Kv3.1 expression in these tissues, although current sensitive to low concentrations of tetraethylammonium (TEA) was present in some canine atrial myocytes. Based on immunofluorescence and pharmacological data, Kv1.5 would seem to contribute significantly also to canine IKur, at least in the dogs tested here. These results have important implications for the use of the dog model in the study of the atrial action potential and for the treatment of AF. The differences between our results and those reported by Yue et al17 raise the possibility that there is significant variation in channel expression from animal to animal.
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Materials and Methods |
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hKv1.5 channels were studied in a human embryonic kidney cell line (HEK293) as reported previously.14 Rat Kv4.2, rat Kv2.1, rat Kv3.1, and human H1 Na+ channels were also stably expressed in HEK293 cells. Atrial myocytes were dissociated from canine atrial specimens using a chunk dissociation technique.19 In paired blots shown in Figures 1B and 1D, for comparison of membrane and cytosol fractions, the protein concentration in the membrane and cytosol was measured using the bicinchoninic acid method (Pierce) and then equal amounts were loaded on the gels (30 µg for Kv3.1 and Kv1.5 blots). Other Western blots were performed as described in the online data supplement (available at http://www.circresaha.org).20 Blots were probed with a polyclonal anti-Kv1.5 antibody (Upstate Biotechnologies, Lake Placid, NY; 1:200 dilution) or with a Kv1.5 antibody generated in our laboratory, a rabbit antibody against the C-terminus of hKv1.5 (aa 537-553 EQGTQSQGPGLDRGVQR; 1:10 000). This antibody was chosen for its unique sequence region at the C-terminus that is not shared with other Kv channels. It shares 12 of 17 residues with canine Kv1.5 and detected canine Kv1.5 under several experimental conditions (see Figures 1 and 4
). Blots for Kv3.1 were probed with anti-Kv3.1 (Alomone Labs, Jerusalem, Israel; 1:5000). Detection was by HRP-labeled goat anti-rabbit antibody (Jackson Immunoresearch, West Grove, Pa) and Renaissance Western Blot Chemiluminescence Reagent Plus (NEN).
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RNA was isolated from heart or brain samples using the RNeasy Midiprep kit (Qiagen) according to the manufacturer’s instructions. Invitrogen’s SuperScript One-Step reverse transcriptase-polymerase chain reaction (RT-PCR) with Platinum Taq kit was used for most RT-PCR experiments. For additional experimental details, see the online data supplement.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
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Results |
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Detection of Kv1.5 and Kv3.1 in Dog Atrium
Figure 1A shows representative Western blots of untransfected HEK293 cells and HEK cells stably expressing Kv1.5, Kv3.1, Kv1.2, and Kv1.4 as well as canine atrial, ventricular, and brain tissue preparations, all probed with our C-terminal Kv1.5 antibody. A band migrating at almost the same position as Kv1.5 in transfected HEK293 cells was abundantly expressed in all canine crude membrane fractions (20 000-g pellet). Identical results were obtained using cardiac tissue from each of four canine atrial preparations tested. These bands from canine atrium and also from rat ventricle were prevented after incubation with the blocking peptide used to generate the antibody (online Figure 1, available in the online data supplement). Using the Upstate polyclonal antibody, channel protein was found in the crude membrane fraction of paired samples of canine atrium and ventricle (Figure 1B). Although a strong band was detected in each sample used, it was conceivable that some of the Kv1.5 originated from noncardiac muscle tissue. Therefore, we used protein extracts from isolated atrial myocytes in additional Western blots to confirm Kv1.5-specific expression in myocytes (Figure 1C). These blots showed weaker bands because of the relatively low yield of atrial myocytes, but an identically sized band is clearly detected from the hKv1.5 HEK293 stable line and from the canine atrial myocytes.
Given that Yue et al17 have attributed canine rapid delayed rectifier, IKur(d), to the presence of Kv3.1 in that tissue, we sought to confirm the biochemical presence of Kv3.1 in canine heart and expected a 97-kDa Kv3.1 band, as detected by Yue et al.17 However, paired Western blots from the same protein preparations used for the Kv1.5 experiments consistently failed to uncover a band of the expected size for Kv3.1 in either atrial or ventricular extracts (Figure 1D), although an 85-kDa band was evident in rat brain (RB) membrane preparations. Bands of 
70, 60, and 50 kDa were present in cytosolic and membrane fractions, respectively, of the canine heart preparations. It seems unlikely that these smaller bands were immature or less glycosylated Kv3.1 protein, because they were still present (marked by arrows) after incubation with the blocking peptide (Figure 1E, right). In addition, although Kv3.1 could be immunoprecipitated from dog brain preparations, no band was pulled down in several attempts to immunoprecipitate the protein from cardiac preparations (data not shown).
To additionally characterize the expression of Kv1.5 and Kv3.1 in canine atria, RT-PCR experiments were conducted (Figure 2). For Kv1.5, an oligonucleotide pair designed to amplify a 754-bp portion of that gene’s transcript was used. For Kv3.1, oligonucleotide pairs identical to those used to clone the channel were used,17 as were other oligonucleotides (see the online data supplement). Amplified Kv1.5 transcript was readily detectable from DNAaseI-treated dog atrial RNA preparations, as shown by the bright band adjacent to the ladder in Figure 2. No band was detectable if the RNA preparations were treated with RNAase A before RT-PCR (data not shown). Kv3.1 from brain was readily detected using primers 1 and 7 or 4 and 10 (online data supplement), but products could not be amplified from atrium in >10 attempts using any of the primer pairs tested. In Figure 2, for Kv3.1, primer pairs 4 and 10 were used and a 725-bp transcript was expected. As a control for the quality of the heart RNA, we amplified a 588-bp segment of dystroglycan message across a splice junction (Figure 2, far right lane). Although this is expected for amplification from mRNA, the primers are more than 17 kbp apart in the dog genome.21
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Kv1.5 Expression on the Surface of Canine Atrial Myocytes
Immunocytochemistry and confocal imaging using an hKv1.5 C-terminal antibody confirmed Kv1.5 expression at the cell surface of canine atrial myocytes. This antibody was raised to a unique sequence in Kv1.5 not shared by other Kv channels. We also tested commercial Kv1.5 antibodies from Alomone Labs and the polyclonal antibody from Upstate Biotechnology. Control experiments (Figure 3A) showed that the C-terminal antibody did not react with untransfected HEK293 cells but localized to the surface of dual-labeled cells expressing a T7-tagged Kv1.5 protein, as did the T7 antibody, revealing extensive colocalization (Figure 3A, far right). The Alomone antibody showed diffuse cytosolic staining and some faint membrane localization in these highly expressing cells (Figure 3C, far right). The Upstate monoclonal antibody failed to detect T7-tagged hKv1.5 at the cell surface (Figure 3C, right). The Kv3.1 antibody did not stain untransfected HEK cells but detected cells transfected with Kv3.1 (Figure 3B, far left and left, respectively). Neither Kv3.1 nor Kv1.5 antibodies stained HEK cells transfected with the other channel (Figure 3B, right), and staining by both antibodies in their concordant transfected cell lines was blocked by preincubation with the immunogenic peptides (Figure 3C, left). In some cases in Figure 3, DAPI was used to identify cell nuclei and render cells visible.
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Strong membrane staining of canine atrial myocytes with the C-terminal Kv1.5 antibody is apparent from single slices of two different myocytes (Figures 4A and 4C), and projection images demonstrate increased staining at cell ends near intercalated discs (Figures 4B and 4D). A myocyte cross section confirms little detection of intracellular protein (Figure 4B, inset). Kv1.5 labeling was specifically inhibited by preincubation with the antigenic peptide (Figure 4E). In contrast to these bright images, the Upstate polyclonal Kv1.5 antibody showed only patchy nuclear staining (Figure 4F). The Alomone Kv3.1 antibody showed faint membrane and intracellular staining of myocytes (Figure 4G), but this was still present after preincubation with the Kv3.1 peptide (Figure 4H). Control experiments with only secondary antibodies did not reveal any nonspecific staining of myocytes (data not shown).
Pharmacological Studies Suggest That Kv1.5 Contributes to IKUR(d)
Although biochemical and imaging experiments demonstrate that Kv1.5 is widely expressed at the surface of isolated canine atrial myocytes, they give little insight into the role of Kv1.5 in IKUR(d) current and during canine atrial action potentials. A pharmacological approach was therefore taken to understand this role. Kv1.5 and Kv3.1 differ markedly in sensitivity to extracellularly applied TEA. Kv3.1, expressed in mammalian cells, has reported IC50s of 130 µmol/L17 and 200 µmol/L22, whereas blockade of Kv1.5 requires 500-fold higher concentrations of TEA.22 Thus, if Kv3.1 is the only component of IKUR(d), the current should be blocked by 0.5 mmol/L TEA. We also tested the effects of a new drug that blocks Kv1.5 selectively. C9356 was synthesized at Cardiome Pharma Corp in Vancouver. It is a potent Kv1.5 blocker (IC50 4.4 µmol/L) and very selective for Kv1.5 over other channels like Kv2.1, which is quite TEA-sensitive, Kv4.2, and Kv3.1 (Figure 5). Concentration-response relationships predict that 10 µmol/L C9356 will block 75% to 80% of Kv1.5 with no effect on Kv3.1 and minor effects on Kv2.1 and Kv4.2.
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Data in Figure 6 show the effects of C9356 on canine atrial K+ currents. Both 0.5 mmol/L TEA and C9356 reduced the sustained outward currents (Figure 6A), as shown by difference currents in the bottom panels, with only minor inhibition of the transient outward current. However, C9356 had a more prominent action on tail currents than TEA (Figure 7A). These effects were readily reversible on washout. Importantly, with respect to TEA-sensitive current, two populations of cells were present, as shown in Figure 6B. Most cells had little TEA-sensitive current, whereas the amount of C9356-sensitive current was more normally distributed. Overall, mean drug-sensitive difference currents were similar for the two compounds, although mean TEA-sensitive currents were subject to greater variation (Figure 6C). Higher concentrations of TEA had essentially similar effects (Figure 7). Control currents during clamp steps are shown in Figure 7A along with a rather modest action of 5 mmol/L TEA. Addition of 10 µmol/L C9356 in the presence of TEA resulted in substantial inhibition of the sustained current in this example, as shown by drug-sensitive currents in the bottom panel. Overall, difference currents sensitive to the two drugs are similar (Figure 7B) but again seem not to be fully additive in individual myocytes. This finding was confirmed when the drugs were added in a different order (Figures 7C and 7D). Addition of a second compound has a lesser effect on the sustained outward current than that produced by the first compound. One obvious difference between the two drugs is that the TEA-sensitive currents are activated at 20 mV more positive potentials than the C9356-sensitive current (Figure 7B).
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C9356 has substantial effects on the canine atrial action potential (Figure 8). Control APs are shown at 30, 60, and 120 bpm (Figure 8A). C9356 effects alone and in combination with 0.5 mmol/L TEA are shown at these stimulation rates in Figures 8B through 8D. In every case, C9356 markedly prolonged the plateau (rather than late repolarization) of the canine AP, whereas further addition of 0.5 mmol/L TEA was without effect. All drug effects were reversible on washout, as shown. Rate-dependent effects of C9356 (in the absence of TEA) on the canine AP are summarized in Figure 8E. Action potential duration was measured at 20%, 50%, and 90% repolarization, as shown in the three rows of the graph. APD50 is the parameter most affected by the compound, consistent with a block of sustained delayed rectifier K+ current in these cells.
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Detection of Kv1.5 in Canine Cardiac Myocytes
There is little doubt from the data presented in the Figures 1 through 4
that Kv1.5 was amply expressed in our samples of canine heart. Kv1.5 message was readily detectable in canine atrium (Figure 2), and protein was found in membrane fractions of homogenized whole atrial or ventricular heart tissue from a variety of different sources and in isolated canine myocytes (Figure 1) using two different antibodies. Immunofluorescence experiments using our own C-terminal antibody confirmed the presence of Kv1.5 on the cell membrane and concentrated at the intercalated discs (Figure 4). This antibody was independently validated against T7-tagged Kv1.5 expressed in HEK293 cells (Figure 3) and also detected Kv1.5 in Western blots (Figure 1A). Others have shown Kv1.5 present at the intercalated discs in human atrial myocytes and also expressed in a punctuate fashion at the cell surface in newborn tissue.8 This increases our confidence that the C-terminal antibody correctly labels Kv1.5 in canine atrium. Several commercial antibodies were variably successful in detecting Kv1.5 at the surface of HEK cells and canine cardiac myocytes. The Upstate Biotechnologies’ polyclonal antibody was successful in detecting Kv1.5 in Western blots but stained primarily nuclei in canine myocytes (Figure 4F). The Alomone antibody was unsuccessful in Western blots as well but showed faint staining in transfected HEK cells that could be attributed to Kv1.5 in cells expressing Kv1.5 highly (Figure 3C). Because of its poor activity against Kv1.5, no staining was attempted in canine myocytes. This low activity may provide an explanation for the failure of Yue et al17 to detect Kv1.5 in canine myocytes, because they used the Alomone antibody for their Western blot experiments. It is also possible that this antibody, although weakly detecting human Kv1.5, does not detect canine Kv1.5.
Failure to Detect Kv3.1 Using Molecular Methods
Previous reports on the presence of Kv3 channels in canine heart have been conflicting. Dixon et al23 reported that Kv3.1, Kv3.2, and Kv3.3 gene transcripts were all expressed at negligibly low levels in canine hearts, with the exception of Kv3.4, which was weakly detectable in canine ventricle. On the other hand, the channel was clearly present in the cardiac tissues tested by Yue et al,17 and a range of pharmacological experiments as well as actual molecular cloning of the channel supported its presence of canine atrium.17,18 However, in agreement with Dixon et al,23 we could find no definitive trace of it in any of the dog hearts we tested. Although the Kv3 subfamily consists of only four genes (Kv3.1, Kv3.2, Kv3.3, and Kv3.4), each gene may encode multiple products by alternative splicing of 3' ends.24–26 Several alternative Kv3 transcripts are known to be expressed in neuronal populations within the central nervous system, all of which can have different sizes.27,28 For Kv3.1, these seem to be between 85 and 97 kDa. In heart, the band detected by Yue et al17 was 97 kDa, but the short cytosolic/microsomal bands detected in our experiments (Figures 1D and 1E) in canine atrium were <75 kDa and remained after incubation with the antigenic peptide (Figure 1E) and so are unlikely to be related to the presence of Kv3.1. That we were unable to immunoprecipitate either band from myocytes strengthens the case that these are not Kv3.1 isoforms. Finally, in atrial myocytes, immunofluorescent labeling of Kv3.1 was equivocal at best (Figures 4G and 4H).
Pharmacological Properties of IKUR(d) and Prolongation of the Canine Atrial Action Potential
Our interpretation of the molecular nature of the sustained atrial delayed rectifier current that we have recorded from canine atrial myocytes is more complicated. The current and also the APD50 were sensitive to C9356, which selectively blocks Kv1.5 (Figures 5 through 8). We conclude that Kv1.5 is not only present (protein, messenger RNA) in our canine atrial myocytes but that it contributes to membrane currents and action potential repolarization. A recent study has also shown that a large component of IKur(d) in adult canine atrium is resistant to 5 mmol/L TEA,29 and our data suggest that this component may well be Kv1.5.
The IKur(d) in some atrial myocytes was also partially sensitive to 0.5 mmol/L TEA, a concentration that will block most Kv3.1 channels, in agreement with reports of such a current in canine atrium.18 Higher concentrations of TEA (5 mmol/L, Figure 7) had little additional effect on outward current, which supports a role for Kv3.1 in these myocytes. Another candidate channel that is relatively sensitive to TEA is Kv2.1. Although Kv2.1 has not been reported to be expressed in canine heart, only ventricle has been tested.23 It is difficult to definitively identify the TEA-sensitive current that we have observed as Kv3.1, because we were unable to detect the channel using molecular methods in several different canine heart preparations in two separate laboratories. However, there was considerable variation in the presence of TEA-sensitive current,30 so the tissues that we used for biochemistry may have lacked Kv3.1. Cells used for patch-clamp experiments were used for up to 6 to 8 hours after isolation, whereas the tissue for biochemistry was immediately flash frozen. It is possible that the expression of the two channels could change over time or be modulated by factors such as the age of the animals.29 Both our subject animals and those of Yue et al17 were mongrels of indefinite age, and breed differences in ion channel expression may exist, or presently unknown differences in channel distribution in various regions of the atrium might explain the differences between results from the two groups.
Implications
Kv1.5 is a prominent repolarizing current in human atria. In atrial myocytes from patients with valvular disease and persistent AF, we observed that the sustained K+ current was reduced by 50%, and there was a parallel reduction in the expression of Kv1.5 protein.15 A similar conclusion was reached by Brandt et al.31 Although the Kv1.5 protein and functional K+ currents are reduced in AF, this does not imply that drugs that suppress this current would not be effective in prolonging atrial refractoriness, because there is typically an even greater reduction (60% to 70%) in the density of the L-type Ca2+ current.19 Thus, it is possible that drugs that suppress Kv1.5 currents might be more, rather than less, effective in AF patients than in controls. Studies designed to evaluate this hypothesis need to be performed in an appropriate model system. This study suggests that the canine model, already well characterized from an electrophysiological perspective, may be useful in evaluating the efficacy and utility of this new therapeutic approach. It should be noted that the electrophysiological role of the Kv1.5 expressed in canine ventricle (Figure 1A) remains to be clarified.
Conclusions
Kv1.5 protein and messenger RNA can be clearly detected in the atria of dogs using Western blotting, RT-PCR, and specific Kv1.5 immunofluorescence. In addition, a sustained delayed rectifier current [IKUR(d)] is detectable in canine atria that can be pharmacologically attributed, at least in part, to Kv1.5. This current seems to have a functional role in canine atrial repolarization, because blockade leads to significant action potential prolongation. Our results suggest that canine models of atrial disease may prove valuable in the study of the corresponding human pathologies.
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Acknowledgments |
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The authors acknowledge project support to D.F. from the Canadian Institutes of Health Research (CIHR) and the Heart and Stroke Foundation of British Columbia and Yukon and to D.V.W. from the NIH (National Heart, Lung, and Blood Institute, 1RO1-HL-65412). They thank Paul Murray for access to canine tissues and Dan Minor and Bruce Tempel for the Kv1.2 clones. Jie Liu provided molecular biology support, Shunping Lin provided HEK293 electrophysiology, and Anu Khurana provided cell preparation.
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Footnotes |
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This manuscript was sent to Michael R. Rosen, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received February 28, 2003; revision received August 27, 2003; accepted September 9, 2003.
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