ARTICLE |
Correspondence to:
Mark R. Boyett, School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, U.K. E-mail:
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Summary |
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The aim of this study was to establish, using immunolabeling, whether the Kv1.5 K+ channel is present in the pacemaker of the heart, the sinoatrial (SA) node. In the atrial muscle surrounding the SA node and in the SA node itself (from guinea pig and ferret), Western blotting analysis showed a major band of the expected molecular weight, ~64 kD. Confocal microscopy and immunofluorescence labeling showed Kv1.5 labeling clustered in atrial muscle but punctate in the SA node. In atrial muscle, Kv1.5 labeling was closely associated with labeling of Cx43 (gap junction protein) and DPI/II (desmosomal protein), whereas in SA node Kv1.5 labeling was closely associated with labeling of DPI/II but not labeling of Cx43 (absent in the SA node) or Cx45 (another gap junction protein present in the SA node). Electron microscopy and immunogold labeling showed that the Kv1.5 labeling in atrial muscle is preferentially associated with desmosomes rather than gap junctions. (J Histochem Cytochem 48:769780, 2000)
Key Words: pacemaker, ion channels, desmosomes, heart
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Introduction |
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PACEMAKING in the sinoatrial (SA) node, the normal pacemaker of the heart, is caused by the interaction of many time- and voltage-dependent ionic currents (
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Materials and Methods |
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Although the guinea pig was the principal species investigated, other species (ferret, mouse) had to be used because of the nature of the antibodies used and the type of experiment carried out. Procedures were carried out under license and in accordance with the regulations of the UK Animals (Scientific Procedures) Act 1986.
SA Node Tissue Dissection
Guinea pigs of either sex weighing 0.20.25 kg were sacrificed by stunning and cervical dislocation. Ferrets weighing 1 kg were anesthetized with IP sodium pentobarbital (90 mg/kg). The chest was opened and the heart was excised rapidly and placed in Tyrode's solution at 3234C. The right atrium was separated from the ventricles and then the left atrium. The separated right atrium was opened by a longitudinal incision in the ventral wall (between the inferior and superior venae cavae) to expose the endocardial surface. The right atrium was trimmed to leave a preparation approximately 10 x 12 mm in the case of the guinea pig SA node and 15 x 15 mm in the case of the ferret SA node. Finally, the preparation, which included the entire SA node and some of the surrounding atrial muscle, was pinned down (endocardial surface up) on silicon rubber. Tyrode's solution contained (in mM): NaCl 93; NaHCO3 20; Na2HPO4 1; KCl 5; CaCl2 2; MgSO4 1; sodium acetate 20; glucose 10; insulin 5 U/ml; equilibrated with 95% O2 and 5% CO2 to give pH 7.4. After dissection, two guinea pig SA node preparations were electrophysiologically mapped at 32C as described previously (
Antibodies
Various primary antibodies were used: (a) a polyclonal antibody against the NH2 terminus of the human Kv1.5 K+ channel raised in rabbit (
Western Blotting
After the SA node tissue dissection described above (from eight guinea pigs and one ferret), the thin intercaval region between the superior and inferior venae cavae (where the SA node is located) was separated from the posterior wall of the right atrium (including the crista terminalis and atrial appendage). A part of the left ventricular wall was also dissected, and all three types of cardiac tissue (intercaval region, posterior wall of right atrium, left ventricle) were rapidly frozen in liquid N2. The tissues were stored at -80C until the experiment. Whole tissue homogenates were prepared by pulverizing the frozen tissues on dry ice using a mortar and pestle. The powdered frozen tissue was thawed and lysed in SB20 (20% SDS, 0.15 M Tris, pH 6.8) and centrifuged at 4000 x g for 15 min. The supernatant from each tissue sample was separated from the pellets and 4 x Laemlli sample buffer was added to each supernatant sample; 4 x Laemlli sample buffer contained Tris-HCl 10 mM; EDTA 1 mM; glycerol 40%; SDS 2.5%; DTT 1 mM; ß-mercaptoethanol 2%; bromophenol blue 0.01%, pH 6.8. Each sample was vortexed and warmed in a water bath at 80C for 15 min. Approximately 10 µl of supernatant from each tissue sample was run on a 12% SDS polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane. The membrane was blocked with 5% dried skimmed milk in PBS containing 0.1% Tween-20, pH 7.5 (PBS-T). PBS contained (in mM): NaH2PO4 20; Na2HPO4 80; NaCl 100, pH 7.5. After washing in PBS-T, the membrane was incubated with the anti-Kv1.5 primary antibody [diluted 1:5000 in PBS-T, 1% bovine serum albumin (BSA), and 2 mM NaN3] for 0.5 hr at room temperature (RT), overnight at 4C, and then again for 0.5 hr at RT. The membrane was thoroughly washed in PBS-T and incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (diluted 1:3000 in PBS-T; Dako, High Wycombe, UK) for 1 hr. The membrane was again thoroughly washed and the enzyme activity was revealed using a chemiluminescence system (Supersignal ECL; Pierce, Rockford, IL). An equivalent gel was also run on 12% SDS polyacrylamide gel and stained with Coomassie Blue for total protein estimation in each sample. Gels and ECL films were scanned and the amount of protein in each sample was estimated using a custom-written densitometry program.
Immunofluorescence Double and Triple Labeling
After dissection and, in some cases, mapping of the activation sequence, the SA node preparation, still pinned down on silicon rubber, was photographed. The preparation was embedded at RT in 10% gelatin (porcine, type A; Sigma, Poole, UK) and preheated to 37C in 0.1 M phosphate buffer, which contained (in mM): Na2HPO4 77; NaH2PO4 23. The preparation was raised to allow penetration of the gelatin beneath the epicardial surface of the tissue. The gelatin-embedded tissue was allowed to cool to 4C to solidify the gelatin. The tissue was cut out with a surround of gelatin and all the pins (except a pin that indicated the primary pacemaker site in the electrophysiologically mapped preparations) were removed. The preparation was frozen at -50C in isopentane and stored at -80C until it was ready to be cryosectioned. A part of the liver from two guinea pigs was also removed, rapidly frozen in liquid N2, and stored at -80C until ready to be cryosectioned. Frozen serial sections (~20-µm thick) were cut from the tissue. In three guinea pig SA node preparations, sections were cut at ~1-mm intervals from the superior to the inferior part of the preparation perpendicular to the crista terminalis and through the intercaval region (see Fig 2A). In preparations of two other guinea pig and one ferret SA node, sections were cut at the level of the main branch from the crista terminalis (the level at which the center of the SA node is usually located). After cryosectioning, the sections were mounted on poly-L-lysine-coated glass slides (BDH; Poole, UK) and stored at -80C until use.
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The sections were fixed by immersing the slides in methanol at -20C for 5 min and were washed three times with PBS, pH 7.4, over 0.5 hr. PBS contained (in mM): Na2HPO4 7.7; NaH2PO4 2.3; NaCl 150. The sections were blocked with 5% non-fat dried milk and 10 % normal donkey serum in PBS, either overnight at 4C or 1 hr at RT. The sections were then briefly washed in PBS. For double labeling, the sections were incubated with the primary antibodies (anti-Kv1.5, anti-Cx43, mouse anti-DPI/II, and rabbit anti-DPI/II diluted in 0.25% saponin, 10% normal donkey serum, and 6.15 mM NaN3 in PBS) for ~24 hr at 37C. In one experiment, sections were triple labeled. The sections were incubated with anti-Cx45 primary antibody (diluted in 1% BSA in PBS) for 2 hr at RT, followed by a mixture of anti-Kv1.5 and anti-Cx43 primary antibodies (diluted in 10% normal donkey serum and 0.25% saponin in PBS) for 2 hr at 37C. After incubation in the primary antibodies, the sections were washed several times in PBS over 0.51 hr and incubated with secondary antibodies conjugated to fluorescent markers (diluted either in 1.5% normal donkey serum and 1% BSA or 1% BSA in PBS) for 1 hr at RT. Secondary antibodies conjugated to FITC, TRITC, Cy3, and Cy5 were used. Sections were washed several more times in PBS over 0.51 hr and mounted with either Vectashield (H-1000; Vector Laboratories, Peterborough, UK) or Citifluor (AF1; Agar Scientific, Standsted, UK) mounting medium. Finally, coverslips were sealed with nailpolish.
Anti-Kv1.5 antibody was used at 1:250, antiCx-43 antibody at 1:1000, anti-Cx45 antibody at 1:50, rabbit anti-DPI/II antibody at 1:100, and mouse anti-DPI/II antibody was purchased in a ready-to-use form. All secondary antibodies were obtained from either Chemicon (Temecula, CA) or Dako (Carpinteria, CA). Anti-rabbit or anti-mouse secondary antibody conjugated to TRITC or FITC was used at 1:100 and anti-guinea pig secondary antibody conjugated to Cy3 or anti-mouse secondary antibody conjugated to Cy-5 was used at 1:500.
Immunolabeled sections were examined by confocal laser scanning microscopy (Leica TCS 4D or Leica TCS SP) equipped with argon, krypton, and heliumneon lasers, which allowed excitation at 488-, 568-, and 633-nm wavelengths for the detection of FITC, TRITC/Cy3, and Cy5, respectively. The images recorded were single optical sections and, in the case of double or triple labeling, the images were recorded sequentially. The images were saved and were processed using Corel Photo-Paint and Corel Draw software (Corel; Ottawa, ONT, Canada).
In most experiments, sections were double or triple labeled as described above. With all primary antibodies, it was checked that single labeling produced the same pattern of labeling.
Postembedding Immunogold Labeling for Electron Microscopy
For immunogold labeling, samples were freeze-substituted and embedded at low temperature in Lowicryl K4M (
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Results |
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Detection of Kv1.5 K+ Channel Protein by Western Blotting Analysis
Western blotting analysis was used to detect the presence of the Kv1.5 K+ channel protein and to test the specificity of the anti-Kv1.5 antibody. Fig 1 shows Western blotting analysis of tissue samples prepared from different regions of guinea pig and ferret heart. The antibody detected a single or double band at the expected molecular weight (~64 kD) in the left ventricle, right atrium, and intercaval region (where the SA node is located) from guinea pig, and in the left ventricle and right atrium from ferret (see arrow in Fig 1A). In some experiments, there were other bands labeled corresponding to proteins with molecular weights of ~30 and ~130 kD (the latter can be seen in some lanes in Fig 1A). The former is probably a proteolytic product of Kv1.5, because it was much more prominent if tissue was not thawed into a high SDS solution, whereas the latter could be a dimer of Kv1.5. No bands were detected in guinea pig left ventricle in a control experiment in which the primary antibody was omitted (not shown). Densitometry of blots (e.g., Fig 1A) and parallel Coomassie-stained gels (not shown) was performed, and Kv1.5 labeling was normalized for protein loading and expressed as a fraction of that seen in the right atrium. Mean results from eight guinea pig hearts are shown in Fig 1B. The amount of Kv1.5 K+ channel protein in the SA node was not significantly different (ANOVA) from that in the right atrium and left ventricle (Fig 1B).
Detection of Kv1.5 K+ Channel Protein by Immunofluorescence Labeling
This series of experiments was carried out on guinea pig tissue. Fig 2A shows a diagram of a guinea pig SA node preparation. The SA node is located in the intercaval region (between the entrances of the superior and inferior venae cavae into the right atrium) and is bounded by the crista terminalis (a thick bundle of atrial muscle) on one side and by the atrial septum on the other. To the right of the crista terminalis (left in Fig 2A) is the right atrial appendage. The preparation was electrophysiologically mapped, and the isochrones in Fig 2A represent the time for the action potential to spread from the leading pacemaker site (asterisk) in the SA node. The leading pacemaker site was in the intercaval region, as expected. To study the expression of the Kv1.5 K+ channel protein in detail, confocal microscopy and immunofluorescence labeling were used. Sections were cut perpendicular to the crista terminalis (at level of arrows in Fig 2A) through the right atrial appendage, the crista terminalis, and the intercaval region (where the SA node is located).
Fig 3A and Fig 3B show Kv1.5 and Cx43 labeling in the atrial muscle of the crista terminalis. Cx43 is a gap junction protein located throughout much of the heart to provide electrical coupling between cells. In the guinea pig and other species, Cx43 is absent in the SA node (
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Fig 4A shows Kv1.5 and Cx43 labeling at the leading pacemaker site (as identified electrophysiologically; asterisk in Fig 2A) in the SA node. As expected, Cx43 signal was largely absent from the tissue (a small amount of Cx43 labeling in green can be seen in Fig 4A) but, in contrast, Kv1.5 labeling was detected (red labeling in Fig 4A). The pattern of Kv1.5 labeling in the SA node was different from that in the atrial muscle. Whereas in the atrial muscle Kv1.5 labeling was in clusters (Fig 3A), in the SA node Kv1.5 labeling was punctate (Fig 4A). A similar pattern of Kv1.5 labeling was seen in SA node tissue from five guinea pigs.
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In the intercaval region, towards the interatrial septum, Cx43 labeling was again detected (not shown). In this region, Kv1.5 labeling was also detected (not shown). The pattern of Kv1.5 and Cx43 labeling was similar to that in the crista terminalis. The two labels were closely associated at the level of resolution afforded by confocal microscopy, and appeared in clusters.
Fig 2B summarizes the distribution of Kv1.5 and Cx43 labeling in different regions of the guinea pig SA node. The result shown is typical of three preparations, two of which were electrophysiologically mapped. The black zone represents the SA node, in which punctate Kv1.5 labeling was detected but Cx43 labeling was not detected. The width of the black zone varies from the superior to the inferior part of the preparation. The dark gray zone is the region of the crista terminalis and the intercaval region, in which both Kv1.5 and Cx43 labeling was detected. The extent of the black zone in Fig 2B, in which there was punctate Kv1.5 labeling but no Cx43 labeling, is outlined in Fig 2A (dotted line). The leading pacemaker site (asterisk) was located in this zone.
No immunofluorescence in the atrial muscle or the SA node was detected when the primary antibodies were omitted (not shown).
Is Kv1.5 Co-localized with Another Gap Junction Protein in SA Node?
In atrial muscle, Kv1.5 labeling was closely associated with Cx43 labeling (Fig 3A and Fig 3B), whereas in the SA node, although Cx43 labeling was mainly absent, Kv1.5 labeling was still observed (Fig 4A). Other connexins, e.g., Cx45, are known to be present in the SA node (
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Is Kv1.5 Co-localized with Desmosomal Proteins?
In the SA node, because Kv1.5 labeling was only sometimes spatially associated with Cx45 labeling, it is possible that Kv1.5 is associated with a connexin other than Cx43 and Cx45. However, it would be surprising if Kv1.5 was actually located within gap junctions. A more likely explanation is that, in atrial muscle, Kv1.5 is present in another structure alongside Cx43-containing gap junctions at the intercalated disc. Desmosomes are also present at the intercalated disc in atrial muscle. Guinea pig tissue was double labeled with anti-Kv1.5 and anti-DPI/II antibodies (DPI/II are abundant desmosomal proteins;
It could be argued that the anti-Kv1.5 antibody binds nonspecifically to desmosomes. To test this possibility, guinea pig liver (in which desmosomes are known to be abundant and the presence of voltage-dependent K+ channels is unlikely) was double labelled for Kv1.5 and DPI/II. Fig 6 shows pronounced DPI/II labeling in guinea pig liver (Fig 6B) but no Kv1.5 labeling (Fig 6A). In the liver, no labeling was detected when the anti-Kv1.5 and anti-DPI/II antibodies were omitted. These results suggest that the Kv1.5 antibody detects Kv1.5 rather than binding nonspecifically to desmosomes.
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DPI/II in guinea pig tissue was also studied using another antibody to DPI/II raised in rabbit (not shown). DPI/II labeling was once again observed throughout the atrial muscle and SA node. In the atrial muscle, DPI/II labeling again appeared in clusters, whereas in the SA node, in which Cx43 labeling was not detected, DPI/II labeling was punctate. Because both the anti-Kv1.5 and anti-DPI/II antibodies were raised in rabbit, association of Kv1.5 and DPI/II labeling could not be studied. In the atrial muscle and intercaval region, no immunofluorescence was detected when the rabbit anti-DPI/II antibody was omitted.
Taken together, these results suggest that Kv1.5 must be located at or close to desmosomes.
Subcellular Immunolocalization of Kv1.5
To detect the precise site of binding of the anti-Kv1.5 antibody at high resolution, we carried out immunogold labeling of ultrathin sections of freeze-substituted, Lowicryl-embedded mouse atria. Experiments had to be carried out on mouse tissue because attempts to process guinea pig tissue did not result in adequate tissue preservation. Electron microscopic examination of sections labeled with anti-Kv1.5 antibody demonstrated high levels of labeling at the intercalated disc membrane (Fig 7A and Fig 7B). The gold label was not uniformly distributed within the disc, however, but was preferentially associated with desmosomes (Fig 7C and Fig 7D). Lower levels of labeling occurred along the membranes of fasciae adherentes junctions and non-junctional regions of the discs, with no labeling (above background) at gap junctions. As previously reported (
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Discussion |
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In this study, at the confocal microscopic level, immunolabeling of Kv1.5 revealed Kv1.5 immunofluorescence labeling in SA node in close association with DPI/II and occasionally Cx45 labeling and in atrial muscle in close association with DPI/II and Cx43 labeling and, at the electron microscopic level, immunogold labeling in atrial muscle preferentially associated with desmosomes and other parts of the intercalated disc membrane rather than gap junctions.
Validity of Data
In Western blotting experiments, labeling of a protein of the expected molecular weight was observed (Fig 1). In Western blotting, immunofluorescence, and immunogold experiments, no labeling was observed in the absence of the anti-Kv1.5 antibody. In a previous study using the same antibody as used in the present study,
Subcellular Location of Kv1.5
In atrial muscle, the intercalated disc is an extensive and irregular structure containing gap junctions, desmosomes and fasciae adherents (
The immunogold findings at the electron microscopic level demonstrate preferential binding of the Kv1.5 antibody at desmosomes (Fig 7). Although the preferential binding of Kv1.5 at desmosomes is unexpected, the presence of extracellular space between the adjacent membranes at desmosomes is substantial (Fig 7), and therefore the channel might be able to function. Our finding that Kv1.5 labeling was preferentially associated with desmosomes does not exclude the presence of Kv1.5, at lower levels, in other regions of the intercalated disc and lateral plasma membrane. This possibility has been suggested by
A number of ion channels, Kv1.2, Kv2.1, Kv4.2, and rH1 (Na+ channel), apart from Kv1.5, as well as the Na+-K+ pump (unpublished observations) and IP3 have been reported to be concentrated at the intercalated disc (
Possible Physiological Role of Kv1.5 in SA Node
Kv1.5 is responsible for ultra-rapid delayed rectifying K+ current (iK,ur) sensitive to block by 4-AP (e.g.,
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Acknowledgments |
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Supported by the British Heart Foundation, by the Ministry of Education, Science and Culture of Japan, by the Japan Society for the Promotion of Science, and by the NIH (grant number HL49330).
We wish to thank Ms J. Higgins and Mr D. Harrison for technical assistance and Dr S. Jones for a control Western blot experiment. The rabbit anti-DPI/II antibody was a gift from Dr A.I. Magee (University College London).
Received for publication February 9, 2000; accepted February 9, 2000.
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