ARTICLE |
Correspondence to: Mark R. Boyett, School of Biomedical Sciences, Univ. of Leeds, Leeds LS2 9JT, UK. E-mail: m.r.boyett@leeds.ac.uk
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Summary |
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The functionally important effects on the heart of ACh released from vagal nerves are principally mediated by the muscarinic K+ channel. The aim of this study was to determine the abundance and cellular location of the muscarinic K+ channel subunits Kir3.1 and Kir3.4 in different regions of heart. Western blotting showed a very low abundance of Kir3.1 in rat ventricle, although Kir3.1 was undetectable in guinea pig and ferret ventricle. Although immunofluorescence on tissue sections showed no labeling of Kir3.1 in rat, guinea pig, and ferret ventricle and Kir3.4 in rat ventricle, immunofluorescence on single ventricular cells from rat showed labeling in t-tubules of both Kir3.1 and Kir3.4. Kir3.1 was abundant in the atrium of the three species, as shown by Western blotting and immunofluorescence, and Kir3.4 was abundant in the atrium of rat, as shown by immunofluorescence. Immunofluorescence showed Kir3.1 expression in SA node from the three species and Kir3.4 expression in the SA node from rat. The muscarinic K+ channel is activated by ACh via the m2 muscarinic receptor and, in atrium and SA node from ferret, Kir3.1 labeling was co-localized with m2 muscarinic receptor labeling throughout the outer cell membrane.
(J Histochem Cytochem 49:12211234, 2001)
Key Words: ion channels, acetylcholine, sinus node, pacemaker, heart
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Introduction |
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Acetylcholine (ACh) released from vagal nerves has many effects on the heart. In the ventricle and atrium, ACh exerts a negative inotropic effect, i.e., it causes a decrease in the strength of contraction (e.g.,
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Materials and Methods |
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Preparations
Tissue from different regions of the heart from rats, ferrets, and guinea pigs, isolated heart cells from rats, and Chinese hamster ovary (CHO) cells were used. Procedures were carried out under license and in accordance with the regulations of the UK Animals (Scientific Procedures) Act 1986. Rats and guinea pigs of either sex weighing 0.20.3 kg were sacrificed by stunning and cervical dislocation, and ferrets weighing 1 kg were anesthetized with IP sodium pentobarbital (90 mg/kg). Left ventricular free wall tissue was taken for Western blotting and immunofluorescence labeling and right atrium (including the crista terminalis and right atrial appendage) was taken for Western blotting. Preparations of SA node with some surrounding atrial muscle (Fig 1A) were dissected as previously described (
20 µm were cut and mounted on poly-L-lysine-coated glass slides (BDH; Poole, UK) and stored at -80C until use for immunofluorescence labeling. Single ventricular and atrial cells were isolated from rats as previously described (
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Antibodies
Primary antibodies used included the following: (a) polyclonal antibody against the C-terminus of mouse Kir3.1 (amino acids 436501) raised in rabbit (Alomone Labs; Jerusalem, Israel); (b) polyclonal antibody against the N-terminus of Kir3.4 (CIR-N2, amino acids 1932) raised in rabbit (gift from Dr. Grigory B. Krapivinsky; Harvard Medical School, Boston, MA); (c) monoclonal antibody against the i3 loop of the m2 muscarinic receptor (amino acids 225359) raised in rat (Chemicon; Harrow, UK); (d) monoclonal antibody against rat cardiac connexin43 (amino acids 252270; Cx43) raised in mouse (Chemicon). For immunofluorescence the anti-Kir3.1 primary antibody was used at 1:100, anti-Kir3.4 primary antibody was used at 1:100, anti-m2 muscarinic receptor primary antibody was used at 1:1000, and anti-Cx-43 primary antibody was used at 1:1000. Secondary antibodies used included the following: anti-rabbit or anti-rat conjugated to TRITC or FITC (used at 1:100), and anti-mouse conjugated to Cy5 (used at 1:500). All secondary antibodies were obtained from either Sigma or Chemicon. For Western blotting the anti-Kir3.1 primary antibody was used at 1:1000 and goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Dako; High Wycombe, UK) was used at 1:3000.
Western Blotting, Immunofluorescence Labeling, and Confocal Microscopy
Western blotting was carried out as previously described (
Immunolabeled tissue sections and single heart cells were examined by a confocal laser scanning microscope (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, and Cy5, respectively. The images recorded were single optical sections and in the case of double or triple labeling the images were recorded either simultaneously or sequentially. The images were processed using Corel Photo-Paint and Corel Draw software (Corel; Ottawa, ONT, Canada). Immunolabeled CHO cells were viewed using a Nikon epifluorescence microscope under 488-nm light for GFP and 514-nm light for TRITC.
Control Experiments
Various control experiments were conducted to check the validity of the data from the present study. The specificity of the anti-Kir3.1 antibody was tested by transfecting CHO cells with plasmid vectors for Kir3.1 as well as GFP (see above for full details). In CHO cells showing GFP fluorescence (presence indicates successful transfection), Kir3.1 labeling was detected, whereas in CHO cells not showing GFP fluorescence no Kir3.1 labeling was detected (not shown). This experiment shows that the anti-Kir3.1 antibody detects Kir3.1 protein.
In Western blotting experiments, when anti-Kir3.1 antibody was pre-incubated with the antigenic peptide (before application to tissue) no bands were detected in ventricle, atrium, and intercaval region from three rats and in ventricle and atrium from one ferret (not shown). Furthermore, in our laboratory, in Western blotting experiments, when the secondary antibody only is applied (i.e., primary antibody not applied) to various heart tissues (including SA node) from different species, no bands are detected.
In immunofluorescence experiments, when the anti-Kir3.1 antibody was pre-incubated with the antigenic peptide (before application to tissue) no labeling in tissue sections (from ventricle, atrium, and SA node from rat, ferret, and guinea pig) and single rat atrial and ventricular cells was detected (not shown).
The anti-Kir3.4 antibody was obtained from Dr. Grigory Krapivinsky and was previously characterized by Western blotting on Sf9 cells (
When a secondary antibody only was applied (i.e., primary antibody not applied), no labeling of tissue sections (from ventricle, atrium, and SA node from rat, ferret, and guinea pig), single rat atrial and ventricular cells, and CHO cells was again detected (not shown). This control experiment was carried out for all primary and secondary antibodies used.
No labeling of tissue sections (from ferret ventricle, atrium and SA node) was detected when the wrong secondary antibodies to detect the primary antibodies were applied (not shown). In most immunofluorescence experiments, tissue was double or triple labeled. With all primary antibodies, it was checked that the single labeling produced the same pattern of labeling. The labeling patterns in single and multiple labeling experiments were always identical (not shown).
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Results |
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Detection of Kir3.1 Protein by Western Blotting in Different Regions of Heart
Western blotting was carried out on gel samples prepared from ventricle (left ventricular free wall used), atrium (crista terminalis and right atrial appendage used), and intercaval region of rat, ferret (with the exception of the intercaval region), and guinea pig heart. Fig 1A shows a preparation from rat (preparations from ferret and guinea pig similar) including the intercaval region (tissue between the entrances of the superior and inferior venae cavae into the right atrium), the crista terminalis (a thick band of atrial muscle that runs principally along the edge of the intercaval region), and part of the right atrial appendage. In Fig 1A the dashed line outlines the intercaval region, the tissue used for Western blotting. The SA node in the rat and guinea pig is located in the intercaval region alongside the crista terminalis. The tissue of the intercaval region from these species used for Western blotting therefore includes SA node tissue (however, it also includes atrium-like tissue that lies in the intercaval region towards the interatrial septum; SEP in Fig 1A). The SA node (as judged by no Cx43 immunolabeling; see below) in the ferret is located on the crista terminalis, and therefore the intercaval region from ferret was not used for Western blotting.
Fig 1B (Lanes 18) shows typical Western blots in which equal amounts of protein (determined by densitometry) from different tissues were loaded and the level of exposure of the enhanced chemiluminescence (ECL) film was the same for each. The anti-Kir3.1 antibody detected two major bands at the expected molecular weights of 50 and 55 kD (corresponding to non-glycosylated and glycosylated forms of Kir3.1;
50 and
55 kD were detected; an example is shown in Fig 1B (Lane 9). This shows that there is a very low expression of Kir3.1 in ventricle from rat at least.
Detection of Kir3.1 Protein by Immunofluorescence and Confocal Microscopy in Ventricle
The cellular distribution of Kir3.1 was determined by immunofluorescence and confocal microscopy. In some cases, tissues were labeled for the m2 muscarinic receptor as well as Kir3.1 (to investigate whether Kir3.1 and the m2 muscarinic receptor are co-localized) and connexin43 (a gap junction protein ubiquitously expressed in the heart apart from in nodal tissue). Fig 2A and Fig 2B show a section of ferret ventricle triple labeled for Kir3.1, m2 muscarinic receptor, and Cx43. In ventricle from rat (not shown), ferret (Fig 2A), and guinea pig (not shown), Kir3.1 labeling was not detected. Fig 2B shows that m2 muscarinic receptor labeling was present in ventricle from ferret (rat and guinea pig ventricle not tested). The m2 muscarinic receptor labeling was present in the outer cell membrane. Ventricular cells have well-developed t-tubules but little or no labeling of the m2 muscarinic receptor was apparent in the t-tubules. Cx43 labeling was also detected in ventricle from rat (not shown), ferret (inset in Fig 2A), and guinea pig (not shown) and, as expected, was located at the intercalated disks. Similar results were obtained from two rats, two ferrets, and two guinea pigs. Fig 2C and Fig 2D show ventricular cells isolated from rat hearts labeled for either Kir3.1 or the m2 muscarinic receptor. Kir3.1 labeling was either absent or very weak. In cells in which weak Kir3.1 labeling was present, Kir3.1 labeling was absent from the outer cell membrane but was present in the t-tubules (Fig 2C). On the other hand, m2 muscarinic receptor labeling was present in the outer cell membrane and, in general, there was weaker labeling of the m2 muscarinic receptor in the t-tubules (Fig 2D). Similar results were obtained from ten (Kir3.1) and five (m2 receptor) rat ventricular cells. In summary, the immunofluorescence data show low expression of Kir3.1 in ventricle.
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Detection of Kir3.1 Protein by Immunofluorescence and Confocal Microscopy in Atrium
Sections of atrium from rat, ferret, and guinea pig either double labeled for Kir3.1 and Cx43 (rat and guinea pig) or triple labeled for Kir3.1, m2 muscarinic receptor, and Cx43 (ferret) are shown in Fig 3A3D. In atrium from all species, Kir3.1 labeling was present in the outer cell membrane (Fig 3A3C). Fig 3D shows that m2 muscarinic receptor labeling was present in atrium from ferret (rat and guinea pig atrium not tested) in the outer cell membrane. Cx43 labeling was also present in atrium from all species and, as expected, was located at the intercalated disks (Fig 3A3C, insets). Similar results were obtained from three rats, two ferrets, and two guinea pigs. Fig 3E and Fig 3F show an atrial cell double labeled for Kir3.1 and the m2 muscarinic receptor. Both Kir3.1 (Fig 3E) and m2 muscarinic receptor (Fig 3F) labeling was present in the outer cell membrane, and it is clear that the labeling was co-localized. Similar results were obtained from eight rat atrial cells. In summary, the immunofluorescence data are in agreement with the Western blotting data in showing a high abundance of Kir3.1 in atrium.
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Detection of Kir3.1 Protein by Immunofluorescence and Confocal Microscopy in SA Node
Fig 4 shows sections of SA node double labeled for Kir3.1 and Cx43 (rat and guinea pig), and Fig 5 shows sections of SA node triple labeled for Kir3.1, m2 muscarinic receptor, and Cx43 (ferret). Cx43 is not expressed in the center of the SA node (e.g.,
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Detection of Kir3.4 Protein by Immunofluorescence and Confocal Microscopy in Rat Ventricle, Atrium, and SA Node
The cellular distribution of Kir3.4 in rat ventricle, atrium, and SA node was determined by immunofluorescence and confocal microscopy. Fig 7A shows a section of rat ventricle double labeled for Kir3.4 and Cx43 (Cx43 labeling not shown). In ventricle from rat, Kir3.4 labeling was not detected (Fig 7A). Similar results were obtained from three rats. Fig 7C shows a ventricular cell isolated from a rat heart single labeled for Kir3.4. Although Kir3.4 labeling was absent from the outer cell membrane, it was present in t-tubules. Similar results were obtained from 10 rat ventricular cells. Why Kir3.4 labeling should be present in single cells but absent in ventricular tissue sections is considered in the Discussion. In summary, the immunofluorescence data show a low abundance of Kir3.4 in ventricle. Fig 7B shows a section of rat atrium double labeled for Kir3.4 and Cx43 (Cx43 labeling not shown). In atrium from rat, Kir3.4 labeling was present in the outer cell membrane (Fig 7B). Similar results were obtained from three rats. Fig 7D shows an atrial cell single labeled for Kir3.4; labeling was present in the outer cell membrane (Fig 7D). Similar results were obtained from eight rat atrial cells. In summary, the immunofluorescence data show a high abundance of Kir3.4 in atrium. Fig 8 shows sections of rat SA node double labeled for Kir3.4 and Cx43. In the tissue shown in Fig 8A, Cx43 labeling was absent (no Cx43 label in inset in Fig 8A), showing the tissue to be SA node. In the tissue section shown in Fig 8B, Cx43 labeling was absent in the middle, but was present in the periphery of the section (inset in Fig 8B). In cells in which there was no Cx43 labeling (SA node cells), Kir3.4 labeling, although weak, was detected in the outer membrane of cells (Fig 8). Similar results were obtained from three rats.
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Discussion |
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This study has shown the following: (a) little or no Kir3.1 protein expression in ventricle from rat, ferret, and guinea pig, as revealed by Western blotting, and little Kir3.1 and Kir3.4 protein expression in ventricle from rat, as revealed by immunofluorescence; (b) a high abundance of Kir3.1 protein in atrium from rat, ferret, and guinea pig, as revealed by Western blotting and immunofluorescence, and presence of Kir3.4 protein in atrium from rat, as revealed by immunofluorescence; (c) a high abundance of Kir3.1 protein in ferret SA node, but an apparently lower abundance in rat and guinea pig SA node, and presence of Kir3.4 protein in rat SA node as revealed by immunofluorescence; and (d) co-localization of Kir3.1 protein with the m2 muscarinic receptor protein in the outer cell membrane in ferret atrium and SA node, as revealed by immunofluorescence. The significance of the distribution of Kir3.1 and Kir3.4 for the vagal control of the heart via the muscarinic K+ channel is discussed below.
Abundance of Kir3.1 and Kir3.4 Proteins in Ventricle
Our previous functional studies have shown that ACh has a negative inotropic effect on rat, ferret, and dog ventricle, and this was shown to be the result of the activation of the muscarinic K+ channel (
In summary, this study shows that, whereas the m2 muscarinic receptor is abundant in the ventricle, the muscarinic K+ channel is much less abundant. This is not unreasonable because it is known that the muscarinic receptor couples to other effectors, e.g., adenylate cyclase, that are known to be present in the ventricle.
Abundance of Kir3.1 and Kir3.4 Proteins in Atrium
Functional studies have shown that ACh has a negative inotropic effect in atrium, and this may be the result of the activation of the muscarinic K+ current (
Abundance of Kir3.1 and Kir3.4 Proteins in SA Node
ACh slows the spontaneous activity of the SA node primarily by activating the muscarinic K+ channel (
If the reasons above are correct, it is unclear why the density of the muscarinic K+ channel should be higher in the SA node in ferret. This species difference may be the result of the species difference of the location of the SA node, in the crista terminalis in the case of the ferret and in the intercaval region in the case of the rat and guinea pig.
There is another possible reason for the apparent low abundance of Kir3.1 and Kir3.4 in rat and guinea pig SA node. It is not known whether the epitope recognized by the anti-Kir3.1 antibody is identical between the species. It is possible, therefore, that the antibodyantigen interaction may be the limiting factor rather than a lower abundance of the Kir.3.1 protein in rat and guinea pig SA node compared to that in ferret SA node. However, the similar labeling of Kir3.1 in atrial muscle of the three species (Fig 3) is not in accord with a species difference in the epitope.
It is interesting that the distribution of the Kir3.1Kir3.4 K+ channel in atrium and SA node (present study) is different from the distribution of the Kv1.5 K+ channel in the two tissues (
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Acknowledgments |
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Supported by the British Heart Foundation, the Ministry of Education, Science and Culture of Japan, and the Japan Society for the Promotion of Science.
We wish to thank Dr Z. Shui, Dr C.F. Howarth, and Dr L. Davis for isolation of heart cells, and Dr I. Khan for transfection of CHO cells.
Received for publication December 19, 2000; accepted June 16, 2001.
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