ARTICLE |
Correspondence to: Nicholas J. Severs, Cardiac Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK.
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Summary |
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The pacemaker of the heart, the sinoatrial (SA) node, is characterized by unique electrical coupling properties. To investigate the contribution of gap junction organization and composition to these properties, the spatial pattern of expression of three gap junctional proteins, connexin45 (Cx45), connexin40 (Cx40), and connexin43 (Cx43), was investigated by immunocytochemistry combined with confocal microscopy. The SA nodal regions of rabbits were dissected and rapidly frozen. Serial cryosections were double labeled for Cx45 and Cx43 and for Cx40 and Cx43, using pairs of antibody probes raised in different species. Dual-channel scanning confocal microscopy was applied to allow simultaneous visualization of the different connexins. Cx45 and Cx40, but not Cx43, were expressed in the central SA node. The major part of the SA nodalcrista terminalis border revealed a sharply demarcated boundary between Cx43-expressing myocytes of the crista terminalis and Cx45/Cx40-expressing myocytes of the node. On the endocardial side, however, a transitional zone between the crista terminalis and the periphery of the node was detected in which Cx43 and Cx45 expression merged. These distinct patterns of connexin compartmentation and merger identified suggest a morphological basis for minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node while maintaining a communication route for directed exit of the impulse into the crista terminalis. (J Histochem Cytochem 47:907918, 1999)
Key Words: gap junctions, connexins, heart, sinoatrial node, confocal microscopy
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Introduction |
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THE PACEMAKER OF THE HEART, the sinoatrial (SA) node, has unique electrical coupling properties which remain poorly understood. The SA node has to drive the large mass of atrial muscle that surrounds it and yet not be electrotonically suppressed by the more hyperpolarized atrial muscle. Modeling has shown this to be difficult (
The morphological structures responsible for intercellular coupling in the heart are gap junctions, clusters of transmembrane channels that span the plasma membranes of neighboring myocytes (
To advance our understanding of how the expression of different connexin types may contribute to the specialized properties of the SA node, this study set out to identify which connexin or connexins are expressed in SA nodal myocytes in the rabbit and to determine how the connexin expression patterns of the SA node and surrounding atrial myocytes are spatially connected. We were particularly interested in establishing whether, by simultaneous visualization of different connexin types combined with the high resolution afforded by confocal microscopy, evidence might be found for the existence of gradients of coupling at the SA nodecrista terminalis border. A prerequisite for this study was a set of reliable connexin-specific antibody probes, raised in different species to permit specific secondary detection in double label experiments and suitable for application in the rabbit (
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Materials and Methods |
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Animals and Tissue Preparation
Rabbits weighing 1.52.0 kg were anesthetized with IV sodium pentobarbital (3040 mg/kg). The chest was opened and the heart was rapidly excised and placed in KrebsRinger solution at 34C. To dissect SA nodal preparations for cryosectioning and connexin localization, the following procedure was used. The right atrium was separated from the remainder of the heart and opened by a longitudinal incision in the free wall to expose the endocardial surface. With the preparation pinned to a silicon base, the atrium was trimmed to leave a preparation approximately 15 x 15 mm, which included the SA node and some of the surrounding atrial muscle. The SA node is located in the intercaval region between the superior and inferior venae cavae (Figure 1). Laterally, it is bounded by the atrial septum on one side and the crista terminalis and the atrial appendage on the other. Towards the superior vena cava, a thick muscle bundle (the central pectinate muscle) projects from the crista terminalis towards the atrial appendage. The leading pacemaker site in the SA node is typically located ~1 mm caudal to the lower edge of this muscle bundle and 0.51.0 mm from the crista terminalis. All animal procedures were conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986.
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Dissected SA node preparations, still pinned to their base, were immersed in melted gelatin. The preparation was raised to allow penetration of the gelatin beneath the tissue. After brief cooling at 4C to set the gelatin, the silicon rubber and pins were removed and the preparation frozen at -50C in isopentane for subsequent serial cryosectioning.
Supporting histological observations were made on Evans blue-stained cryosections, and on semithin sections of epoxy resin-embedded tissue after Toluidine Blue staining. For the latter, the tissue was fixed in phosphate-buffered 2% paraformaldehyde/2.5% glutaraldehyde for 1 hr, rinsed in phosphate buffer, postfixed in 1% osmium tetroxide (phosphate-buffered) for 1 hr, dehydrated, and embedded in epoxy resin.
For Western blot analysis, the SA node, crista terminalis, and atrial septum were carefully dissected under a binocular microscope and rapidly frozen in liquid nitrogen. Samples of left ventricle and right ventricle were frozen in parallel as controls.
Antibodies
Polyclonal antibodies against Cx45 and Cx40 were raised in guinea pig. The anti-Cx45 antiserum Q14E(GP42) and the anti-Cx40 antiserum V15K(GP318) were prepared, purified and characterized as described previously (
For Western blotting, alkaline phosphatase-conjugated secondary antibody to mouse IgGs (used at 1:5000 dilution) was purchased from Pierce & Warriner (Chester, UK). All the fluorophore-conjugated (CY3 and CY5) secondary antibodies for immunoconfocal localization were purchased from Chemicon (used at 1:500 dilution). The fluorophore-conjugated antibodies were adsorbed against IgGs from a range of species before use to ensure specificity and avoid crossreactivity in double labeling experiments.
Sample Preparation and Western Blotting
For Western blotting, whole tissue homogenates were prepared by pulverizing the frozen specimens under liquid nitrogen using a mortar and pestle. After lysing the powdered frozen tissues in SB20 (20% SDS, 0.15 mol/liter Tris, pH 6.8), an aliquot was removed for protein estimation (Bio-Rad DC protein assay) and 2-mercaptoethanol added to the remainder to a final concentration of 2.5%.
Ten µg of total protein per lane was run on 12.5% SDS-PAGE and electrophoretically transferred overnight to PVDF membrane at constant voltage. Transfer was assessed by Ponceau S staining and the membrane blocked with TBS/0.1% Tween 20 with 5% dried milk powder. The membrane was incubated with primary antibody, diluted in TBS/0.1% Tween/1% BSA, for 1 hr at room temperature (RT), washed, and incubated with alkaline phosphatase-conjugated secondary antibody, diluted in TBS/0.1% Tween/10% normal goat serum, for 1 hr. The membrane was washed and the enzyme activity revealed using a freshly prepared NBT/BCIP substrate solution (Pierce and Warriner).
Immunofluorescent Labeling
Frozen sections (10 µm thick) were cut from four frozen SA nodal preparations. The sections were mounted on poly-L-lysine-coated glass slides, which were then stored at -65C until use. The sections were fixed by immersing the slides in methanol at -20C for 5 min and were then washed three times with PBS. Blocking was carried out for 1 hr with 1% bovine serum albumin (BSA) in PBS before incubation with primary antibody (diluted in 1% BSA in PBS) for 2 hr (GP42 and GP318) or 1 hr (monoclonal anti-Cx43). After washing five times with PBS over 30 min, the sections were incubated with CY3-conjugated secondary antibodies (diluted in 1% BSA in PBS) for 1 hr. The slides were washed a further five times with PBS over 30 min, mounted with Citifluor mounting medium (Agar; Essex, UK), and the coverslips sealed with clear nail polish.
For double labeling of Cx45 and Cx43 and of Cx40 and Cx43, the sections were incubated sequentially with the primary antibodies, the monoclonal anti-Cx43 being applied second. The secondary antibodies (CY3-conjugated anti-guinea pig IgG and FITC-conjugated anti-mouse IgG) were applied together in the presence of 10% normal rabbit serum. Double labeling for Cx40 and Cx45 was not feasible because both antibodies were raised in the same species.
For peptide inhibition experiments, the antibodies were incubated with 50 µg/ml peptide at RT for 30 min before application to the tissue sections or Western blots. Controls for the immunofluorescent labeling experiments were (a) omission of the primary antibody (one or both in the case of double labeling), (b) using the inappropriate secondary antibody for each individual primary antibody, and (c) using a single primary followed by both secondary antibodies.
Immunolabeled sections were examined by confocal laser scanning microscopy using a Leica TCS 4D instrument equipped with an argon/krypton laser and fitted with the appropriate filter blocks for detection of CY3 and FITC fluorescence. The images recorded were projections of five consecutive single optical sections taken at 0.5-µm intervals.
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Results |
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Isolated SA node preparations from four rabbits were serially cryosectioned in a plane perpendicular to the crista terminalis, as illustrated in Figure 1A. The serial sections were taken through the zone marked X to Y (Figure 1A). Toluidine Blue staining of selected semithin sections of epoxy resin-embedded tissue helped to determine the histological orientation and the extent of the SA node tissue (Figure 1B and Figure 1C). The SA node tissue appears lighter in color than the atrial muscle and occupies the thin intercaval region with an extension onto the endocardial surface of the crista terminalis (the periphery of the SA node; Figure 1C).
On imaging Cx43 labeling at low magnification by confocal microscopy, the SA nodal region was readily identifiable as a conspicuous immunonegative zone between the strongly Cx43-positive crista terminalis and the atrial myocardium on the septal side (Figure 2A and Figure 2B). The boundary between Cx43-positive and Cx43-negative zones was sharply delineated, and although some bundles of Cx43-expressing atrial myocytes penetrated the nodal region, appearing as "islands" in sectional view (Figure 2A), no extensive interdigitation between the zones was apparent. The lack of Cx43 in the SA node compared with the crista terminalis, atrial septum, and ventricular myocardium was confirmed by Western blotting (Figure 3).
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The typical extent of the Cx43-negative region, as reconstructed from serial sections, is indicated in Figure 1A. Survey montage views such as those in Figure 2 were used for mapping the features of connexin expression observed at high magnification throughout this region. The montages illustrated in Figure 2A and Figure 2B come from sections across the inferior edge and center of the SA node, respectively. Because multiple montages of this type were used for mapping connexin distribution in detail, an idealized diagram summarizing the montage features (Figure 4) is used here to illustrate the locations of all the high-magnification panels presented in Figure 5 Figure 6 Figure 7 Figure 8, which have been drawn from different representative sections through the nodal region.
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Dual-channel imaging of double labeled preparations was used to permit high-resolution simultaneous visualization of Cx45 with Cx43 and of Cx40 with Cx43. With this approach, close inspection throughout the Cx43-negative SA nodal region revealed extensive, widely distributed Cx45 labeling in the form of small but sharply defined spots (Figure 5A), and a similar pattern of labeling was observed for Cx40 (Figure 5B). The relative amounts of Cx45 and Cx40 labeling in the SA node varied from one heart to the next, but the overall level of labeling for these connexins was always substantially lower than that observed for Cx43 in the surrounding atrial myocardium (Figure 5A, inset). The labeling for Cx45 and Cx40 differed from that of Cx43 by being widely dispersed rather than aggregated into intercalated disk clusters and by comprising individual spots of much smaller size.
The double labeling approach disclosed two distinctive expression patterns at the boundary of Cx43-expressing regions of the crista terminalis. The first, involving a sharp demarcation of Cx43 from both Cx45 and Cx40, characterized the epicardial side of the crista terminalis extending inwards. Here, Cx45 and Cx43 labeling were each confined to discrete, nonoverlapping zones (Figure 6A). A corresponding distinct boundary was present between Cx40- and Cx43-labeled zones (Figure 6B). An intervening, discontinuous connective tissue layer occupied stretches of the boundary (Figure 6A and Figure 6B; see also Figure 1C), although strands of Cx45/Cx40-expressing nodal cells were in places closely approximated to groups of Cx43-expressing cells of the crista terminalis. A similar sharp delineation between Cx43 and Cx45/Cx40 was apparent at the border of the node with "islands" of penetrating myocyte bundles (Figure 6C and Figure 6D).
The second distinctive pattern, in contrast to these clearly delineated zones, involved a zone in which Cx43 and Cx45 expression merged and overlapped (two examples are shown in Figure 7 and Figure 8). This co-expressing zone was exclusively on the endocardial surface of the crista terminalis, extending beyond the right branch of the SA ring bundle (Figure 7A and Figure 8A), and was continuous with the Cx45-positive nodal tissue on one side and the Cx43-expressing atrial myocytes of the crista terminalis on the other. Deeper zones of the crista terminalis showed only Cx43 (Figure 7B and Figure 8B). Cx40 was not detectable in the co-expressing zone (Figure 7C and Figure 8C). This general pattern of expression was apparent in all the SA node preparations studied, although some variation in the width of the Cx45-positive region penetrating the crista terminalis was encountered from one heart to the next, as illustrated in the two examples in Figure 7 and Figure 8. Cx45 and Cx43 co-expression was also apparent in the right and left branches of the SA ring bundle.
In all experiments, the specificity of the localization was confirmed by peptide inhibition and by controls in which (a) the primary antibody was omitted and (b) treatment with a single primary antibody was followed by the nonmatching secondary antibody or both secondary antibodies.
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Discussion |
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The principal features of the spatial distribution of connexins 45, 43, and 40 in the SA nodal region of the rabbit, as determined from the double label confocal imaging results, are summarized diagrammatically in Figure 9. Key findings are that Cx45 and Cx40, but not Cx43, are expressed in the central SA node and that distinctive zones of differential connexin expression exist at the SA nodal/crista terminalis border. The border zones are of two types; a sharply demarcated boundary between Cx43 and Cx45/Cx40 and a transitional zone adjacent to the right branch of the SA ring bundle in which Cx43 and Cx45 expression merges. These findings provide new insights into a possible structural basis for the unique electrophysiological properties of the SA node.
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Previous studies on connexin distribution in the SA node in different species present a confusing picture. Our finding of a lack of immunodetectable Cx43 in rabbit SA node contrasts with earlier reports in both rabbit (
By using a new, rigorously characterized Cx45 antibody (
Theoretical models have proposed that successful pacing and conduction of the action potential into the surrounding atrial tissue requires a central zone of weakly coupled cells, surrounded by a ring across which a gradient of increased coupling occurs (
Our ability to image pairs of connexin types simultaneously sheds new light on the possible mechanism by which the pacemaker is protected from the hyperpolarizing influence of the surrounding tissue. Although the border between the SA node and crista terminalis presents a potentially large area of contact, our findings indicate that the two tissues at this site remain compartmentalized in terms of connexin expression and with a discontinuous connective tissue layer, apart from a single conspicuous region of contact characterized by Cx43 and Cx45 expression. Such an arrangement could achieve both minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node, and maintenance of a communication route for directed exit of the impulse into the crista terminalis. The differential patterns of connexin expression we show here, comprising predominantly Cx43 expression in the atrial muscle, Cx43 and Cx45 co-expression in the region of contact (at the periphery of the SA node), and Cx45/Cx40 co-expression in the SA node itself, may thus provide the morphological basis of the gradient of coupling proposed by
Our ability to demonstrate a transitional zone of connexin expression at the nodal periphery depended critically on the availability of a reliable, specific Cx45 antibody and the ability to undertake double labeling combined with dual-channel scanning for simultaneous visualization of Cx45 and Cx43. Previous studies concluding the absence of a morphological gradient in gap junction coupling did not examine connexins other than Cx43 (
Mounting evidence suggests that connexin compatibility is a key mechanism for regulating intercellular communication and coupling between tissue compartments of different function. Cx45 has been shown to alter channel properties when transfected into cells that endogenously express Cx43 (
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Acknowledgments |
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Supported by project grants (nos. PG/93136 and PG/97175; NJS) and a program grant (no. RG/96004; MRB) from the British Heart Foundation and by a grant from the Ministry of Education, Science and Culture of Japan.
We thank Dr Robert Gourdie (Medical University of South Carolina) for the anti-Cx40 antiserum and Stephen Rothery for invaluable assistance with animal work and immunoconfocal microscopy.
Received for publication October 27, 1998; accepted January 29, 1999.
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