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Cardiovascular Research 2002 53(1):89-96; doi:10.1016/S0008-6363(01)00421-7
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Copyright © 2001, European Society of Cardiology

Heterogeneous expression of connexins in rabbit sinoatrial node cells: correlation between connexin isotype and cell size

Haruo Honjoa,*, Mark R Boyettb, Steven R Coppenc, Yoshiko Takagishia, Tobias Opthofd, Nicholas J Seversc and Itsuo Kodamaa

aResearch Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
bSchool of Biomedical Sciences, University of Leeds, Leeds, UK
cNational Heart and Lung Institute, Imperial College School of Medicine, London, UK
dDepartment of Medical Physiology, University Medical Center, Utrecht, The Netherlands

* Corresponding author. Tel.: +81-52-789-5007; fax: +81-52-789-5003 honjo{at}riem.nagoya-u.ac.jp

Received 14 February 2001; accepted 30 July 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Intercellular coupling through gap junctions allows the morphologically and functionally heterogeneous sinoatrial node to synchronize and drive the atrial muscle. The purpose of this study was to identify the connexin isotypes expressed by sinoatrial node cells and to analyse the density of connexins in relation to cell size. Methods: Labeling for the different connexins using isotype-specific antibodies was assessed in cells isolated from the rabbit sinoatrial node by immunoconfocal microscopy. Results: Sinoatrial node cells with a cell projection area smaller than 800 µm2 were devoid of immunolabeling for connexin43. Such small cells showed high levels of connexin45 labeling (compared to that in large cells) and low levels of connexin40 labeling. Sinoatrial node cells with a projection area between 800 and 1200 µm2 had a lower amount of connexin45 label and again a small amount of connexin40 but an increased amount of connexin43 label. In the larger sinoatrial node cells, some colocalization of connexin45 and connexin43 immunolabeled spots was observed. Conclusions: Rabbit sinoatrial node cells are heterogeneous in terms of connexin expression, and there is a clear cell size-dependence in pattern of connexin expression. Small (putative central) cells express connexin45 but not connexin43, whereas larger (putative peripheral) cells express both connexin45 and connexin43. The co-localization of connexin43 and connexin45 in larger cells raises the possibility that heterotypic or heteromeric connexin43/connexin45 channels could be present in gap junctions at the periphery of the sinoatrial node.

KEYWORDS Conduction (block); Gap junctions; Histo(patho)logy; Impulse formation; Sinus node


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The sinoatrial (SA) node requires a delicate balance of electrical coupling in order to maintain normal function [1]. Too much electrical coupling will turn the SA node silent, because its membrane will be ‘clamped’ to a more negative potential than the normal maximal diastolic potential despite its intrinsic pacemaker potency by the surrounding atrial muscle. Too little coupling may prevent successful activation of the surrounding atrium. The predominant constituent of gap junctions in the heart, connexin43 (Cx43), is lacking in the very center of the guinea-pig and rabbit SA node [2,3]. Other types of connexins (connexin40 [Cx40], connexin45 [Cx45] and connexin46 [Cx46]) have been reported in the rabbit SA node by immunohistochemical methods [3–5]. Our previous study on rabbit SA node tissue sections has revealed that Cx45 is a major connexin in the central area (leading pacemaker site) and that a distinct region of merger exists between the Cx45-expressing myocytes of the node and the Cx43-expressing myocytes of the surrounding atrium [3]. Because of the complex cell-packing architecture of the SA node, it was impossible to elucidate the relationship between nodal cell morphology and expression of connexin isotypes.

Based on extensive experimental studies and computer simulations, we recently proposed a ‘gradient model’ to explain regional differences in electrical activity between the periphery and center of the SA node [6,7]. The model predicts a gradual transition in intrinsic electrical properties of individual cells from the periphery to the center of the SA node. Because cells in the center of the SA node are thought to be smaller than those in the periphery [8,9], a correlation between cellular dimensions and gap junction connexin expression would provide information to shed light on the cellular organization of the SA node.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell isolation
Single SA node pacemaker cells were isolated from adult New Zealand White rabbits (1.2–1.5 kg) by methods similar to those described previously [10,11]. In brief, the SA node tissue (~4x4 mm), located in the intercaval region and rising up the endocardial face of the crista terminalis [3,6,12], was cut into several strips perpendicular to the crista terminalis, and atrial muscle and fat tissue on the epicardial surface were carefully removed. The tissue strips were then treated with collagenase (350–400 U/ml, Yakult, Tokyo, Japan) and elastase (12–15 U/ml, type IIA, Sigma, USA) for 30 min. The digested tissues were gently triturated in Kraftbrühe (K-B) solution to produce the cell suspension. Atrial cells were isolated by enzymatic digestion of small tissue pieces (~5x5 mm) dissected from the left and right atrium. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Antibodies
For immunolabeling of Cx45 and Cx40, Cx45 guinea-pig antiserum Q14E/GP42 (raised against residues 354–367 of human Cx45) and Cx40 guinea-pig antiserum V15K/GP318 (raised against residues 256–270 of rat Cx40) were used. The specificity of these antibodies has been demonstrated previously [13,14]. Detection of connexin43 (Cx43) was performed with a commercially available mouse monoclonal Cx43 antibody of well-established specificity (raised against residues 252–270 of rat Cx43; Chemicon, Harrow, UK). In some experiments, mouse monoclonal anti-desmin antibody (Sigma, USA) was applied to detect intermediate filaments of muscle cells (muscle cell marker).

2.3 Immunoconfocal microscopy
Isolated cells were placed on carbon-coated glass cover slips and fixed with methanol at –20°C for 5–10 min. After blocking with 10% normal donkey serum in PBS for 1 h, cells were incubated with primary antibodies (anti-Cx45, 1:100 dilution; anti-Cx40, 1:500 dilution; anti-Cx43, 1:1000 dilution; anti-desmin 1:1000 dilution; all dilutions in 1% BSA in PBS) for 1 h at room temperature. After washing with PBS, cells were incubated with FITC-conjugated secondary antibodies (anti-mouse or anti-guinea pig IgG, depending on the primary antibody; 1:200 dilution in 1% BSA in PBS) for 1 h. For double labeling of Cx43 and Cx45, cells were incubated sequentially with the primary antibodies, the anti-Cx43 being applied second; the secondary antibodies, a mixture of FITC-conjugated anti-mouse IgG and rhodamine-conjugated anti-guinea pig IgG, were applied simultaneously. Cells were washed several times with PBS and mounted on slides. Samples processed with either no primary antibody or the inappropriate secondary antibody for each primary antibody served as negative controls. There was no significant labeling in the control experiments.

Immunolabeled cells were examined using a confocal laser scanning microscope (Leica TCS SP) equipped with Ar and Kr lasers, which allowed excitation at 488 and 568 nm wavelength for the detection of FITC and rhodamine fluorescence, respectively. In the case of double labeling, the images were recorded sequentially with excitation at 488 and 568 nm. The images presented in the Results section are projections of 5–15 consecutive single optical sections taken at 1-µm intervals through the full thickness of cells. The density of connexin was evaluated semi-quantitatively from projection images by calculating an index of the density of immunolabeling (the area labeled with anti-connexin antibody normalized by the cell projection area) using Scion Imaging Software (Scion Corporation, USA) [15,16].

2.4 Statistics
All results are expressed as mean±S.E.M. (number of cells). Statistical significance was determined by Student's t-test and a probability <0.05 was considered to indicate a significant difference.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Isolated SA node pacemaker cells
Dissociated cells from the SA node region showed a wide variation in morphology: there were spindle- and spider-shaped cells, rod-shaped atrial cells with clear striations and clusters of small round cells identified as endothelial cells. We have previously demonstrated that among these various types of cells, spindle and spider cells show the typical electrophysiological characteristics of pacemaker cells, including the presence of the hyperpolarization-activated current (If) and absence of the inwardly-rectifying K+ current (IK,1) as well as spontaneous beating under physiological conditions [6,10,11,17]. In the present study, we used only spindle and spider cells for observations on immunolabeling of connexins.

3.2 Immunolabeling of Cx43
Representative examples of projection images of cells immunolabeled with Cx43 antibody are shown in Fig. 1A,B,D,E. Immunofluorescent spots for Cx43 were virtually absent in relatively small spindle and spider SA node cells (Fig. 1A,B). These spindle and spider SA node cells were intensely labeled with anti-desmin antibody, indicating that they were myocytes (Fig. 1C). In contrast to the lack of immunolabeling of Cx43 in small SA node cells, larger spindle SA node cells showed clear albeit small immunofluorescent spots for Cx43 scattered over the cell surface (Fig. 1D). The immunofluorescent spots for Cx43 in medium-sized SA node cells (Fig. 1D) were small and sparse compared with those in atrial cells. For comparison, Fig. 1E shows immunolabeling of Cx43 in a left working atrial cell (similar immunolabeling was obtained in right atrial cells).


Figure 1
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  		Fig. 1 (A, B) Immunolabeling of Cx43 in a small spindle-shaped SA node cell (A) and a spider-shaped (B) SA node cell. (C) Immunolabeling of desmin in a small spindle-shaped SA node cell. (D) Immunolabeling of Cx43 in a medium-sized spindle SA node cell. (E) Immunolabeling of Cx43 in a left atrial cell. (F, G) Immunolabeling of Cx45 in spider SA node cells. (H) Immunolabeling of Cx45 (top) and Cx43 (bottom) in a left atrial cell (cell double-labeled). Projection images shown in all panels. Scale bar, 20 µm.

 
Fig. 2A summarises these data: the density of Cx43 immunolabeling is plotted against the size of cells isolated either from the SA node region (open circles) or from the left atrium (filled squares). Cx43 was undetectable in the majority of SA node cells (33/39, 85%). Cx43 was not detected in any small pacemaker cells that had a cell projection area of less than ~800 µm2, whereas Cx43 was detected in some larger cells (6/39, 15%). The density of Cx43 immunolabeling tended to be higher in larger SA node cells, but the average Cx43 density (0.0088±0.0028, n=6) was significantly less in Cx43-positive SA node cells than in working atrial cells isolated from the left atrium (0.0325±0.0036, n=7; P<0.05) (Fig. 2A).


Figure 2
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  		Fig. 2 Correlation between density of Cx43 (A) and Cx45 (B) immunolabeling and cell size. Area of cell labeled with Cx43 or Cx45 antibody (calculated from projection image), normalised for cell projection area, plotted against cell projection area. Open circles, data for SA node cells. Filled squares, data for left atrial cells.

 
3.3 Immunolabeling of Cx45
Fig. 1F–H show representative examples of projection images of cells immunolabeled with Cx45 antibody. Small immunofluorescent spots were detected over the cell surface in all spindle and spider cells isolated from the SA node region (Fig. 1F,G). In contrast, left atrial cells with abundant Cx43 immunolabeling showed no detectable immunofluorescent spots for Cx45 (Fig. 1H). Fig. 2B summarises these data: the density of Cx45 immunolabeling is plotted against the size of cells isolated either from the SA node region (open circles) or from the left atrium (filled squares). We examined a total of 36 SA node cells and all cells showed Cx45 immunolabeling of varying density; the Cx45 immunolabeling density tended to be higher in smaller cells. In contrast, all left atrial cells were devoid of immunolabeling for Cx45 (n=5).

3.4 Double immunolabeling of Cx43 and Cx45
Immunolabeling data for Cx43 and Cx45 described so far suggest a possibility of co-expression of Cx43 and Cx45 in some large spindle SA node cells and this was tested by double labeling. Fig. 3A shows an example. Projection images labeled for Cx43 (first image) and for Cx45 (second) are superimposed (third). Yellow spots of the superimposed image suggest some colocalization of the Cx43 and Cx45 signals, but the distribution of the two connexins was not identical, with examples of isolated spots showing label for Cx43 only (red) and a few for Cx45 only (green). Similar co-localization of a proportion of the Cx43 and Cx45 spots was also demonstrated in a single optical plane (Fig. 3A, fourth image). Essentially the same results for co-expression of Cx43 and Cx45 were obtained in three other large spindle SA node cells.


Figure 3
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  		Fig. 3 (A) Immunolabeling of both Cx43 and Cx45 in a large spindle SA node cell (cell double-labeled). Projection image of Cx43 (first image), projection image of Cx45 (second), superimposed projection image of Cx43 and Cx45 (third) and superimposed image of Cx43 and Cx45 at a single optical plane sectioned at the level close to the top surface (fourth) shown. (B,C) Immunolabeling of Cx40 in spider SA node cells. (D) Immunolabeling of Cx40 in a left atrial cell. Projection images shown in B–D. Scale bar, 20 µm.

 
3.5 Immunolabeling of Cx40
Representative examples of projection images of SA node cells immunolabeled with Cx40 antibody are shown in Fig. 3B–D. Tiny immunofluorescent spots for Cx40 were always sparsely detected in spindle and spider SA node cells (n=25) (Fig. 3B,C) and atrial cells (n=6) (Fig. 3D). Quantitative image analysis was not feasible because of the small size of the immunofluorescent spots for Cx40.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
4.1 Major findings
In the present study, we have shown a clear correlation between connexin expression and the size of SA node cells — there was dispersed punctate immunolabeling of Cx45 in small cells and the labeling decreased with increase in cell size. In contrast, there was no immunolabeling of Cx43 in small cells, but there was labeling (dispersed and punctate) in larger cells. These larger cells showed immunolabeling for Cx45 as well as Cx43, with co-localization of signal for both connexins in some spots. Although a relationship between cell morphology and differential expression of connexin isotypes has previously been noted in the rabbit SA node [5], Cx45 was not examined in this context, and no quantitative data correlating SA node cell size with connexin expression has previously been presented in any species.

4.2 Heterogeneity of the SA node
The correlations between connexin expression and cell size in the present study extend results from earlier studies on the rabbit SA node, which provide the basis for the ‘gradient model’ [6,7]. There are correlations between the densities of the Na+ current (INa), L-type Ca2+ current (ICa,L), transient outward K+ current (Ito), a sustained outward current (Isus), rapid delayed rectifier K+ current (IK,r), slow delayed rectifier K+ current (IK,s) and If and cell size (in all cases the current density was less in smaller cells) [6,10,11,17] and the differences in current density are sufficient to explain the differences in action potential characteristics [7,18]. In addition to our own data, Wu et al. [19] have recently shown that small spider (putative central) cells have a less negative diastolic potential and a smaller amplitude of the action potential than larger spindle (putative peripheral) cells. In contrast to our data, however, they [19] have shown that the If density is higher and the spontaneous rate is faster in small spider cells. Wilders et al. [20] have reported that there is no clear correlation between cell size and the densities of ICa,L, If and IK. Lei et al. [21] have recently shown correlations between the expression of various Ca2+-handling proteins (Na+/Ca2+ exchanger, RYR2 and SERCA2) and cell size (the density of immunolabeling of all these Ca2+-handling proteins was less in smaller cells). The differences in connexins observed in the present study constitute a new element in this perspective of differential protein expression (and properties) in relation to cell size in the SA node.

The intact SA node is a heterogeneous tissue in terms of structure and function [6]. Previous morphological studies have suggested that there is a gradient in cell size in the intact rabbit SA node; the cell size is reported to increase from the center toward the periphery adjacent to the atrial muscle [8,9]. Lei et al. [21] demonstrated more direct evidence by measuring dimensions of rabbit SA node cells isolated from different regions; cells from the center were ~51 µm in length, whereas those from the periphery (close to the crista terminalis) were ~88 µm. Verheijck et al. [22] established a roughly equal proportion of (small) spider cells, (larger) spindle and elongated spindle cells as well as atrial cells in the dominant pacemaker area and in latent pacemaker areas (Table 1 in Ref. [22]). However, since they did not measure absolute cell dimensions, this does not exclude that cells in the peripheral areas are larger than cells in the dominant pacemaker area.

4.3 Connexin phenotypes in the SA node
The majority of previous studies have reported that Cx43 immunolabeling is absent from the rat [23,24], cow [24], dog [4], guinea-pig [2] as well as human [24,25] SA node (see reviews [6,26]). In the dog and human SA node, one group has reported Cx40 to be present [4,25]; Cx45 was also reported to be present, but the interpretation of these findings is hampered by the authors’ use of an antibody raised against the same sequence as that used for an anti-Cx45 antibody shown to cross-react with Cx43 [13]. Indeed, another study using this debated antibody reports apparent Cx45 immunolabeling only in cells in which Cx43 immunolabeling was present, and apparent Cx45 and Cx43 labeling were exactly coincident [27]. In all these studies on the SA node, no attempt was made to distinguish between the center and periphery of the SA node and it was assumed that the center of the SA node was studied. Although two early studies claimed that Cx43 is present in the hamster and rabbit SA node [28,29], the results from the majority of studies agree that Cx43 immunolabeling is absent from the SA node (or at least the center of the SA node).

Our finding that the gap junctions (as revealed by immunolabeling of Cx40, Cx43 and Cx45) in the rabbit are small, punctate and dispersed over the entire cell surface accords with previous observations on SA nodal cells of other species [2,23–25,27–29] (see review [26]) and is in marked contrast to the gap junctions seen in ventricular and atrial cells, which are abundant and largely confined to intercalated discs. The smallness of gap junctions in the SA node as revealed by immunolabeling is consistent with the size of gap junctions in the SA node as shown by electron microscopy [30]. According to the morphological estimation by Masson-Pévet et al. [30], each gap junction plaque found at the center of the rabbit SA node contains ~90 channels on average. This value is close to the lower limit of immunolabeling detection (40–75 channels per gap junction [2] or a gap junction plaque size of ~80 nm [31]).

It might be suggested that cell isolation procedures could alter the expression and distribution of connexins, thereby compromising extrapolation of our data to the intact tissue. When adult cardiomyocytes are dissociated, the gap junctions are not split into their component membranes but retained as intact bimembranous structures with one or other of the cells originally sharing them [32], leaving a hole in the apposing plasma membrane which is rapidly sealed over [33]. The gap-junctional membrane either remains surface-located (but sealed over) or is endocytosed as a vesicle of gap-junctional membrane. In rabbit myocytes, there is no endocytosis of the surface-located junctions, no migration of the gap-junctional vesicles and no formation of new junctions over a period as long as 22 h [32]. Thus, once dissociation has taken place the junctions remain stable and, with fixation carried out at just 30 min after isolation in the present study, they will be preserved close to their original location. At the resolution of immunoconfocal microscopy, the localization of fluorescent spots will thus faithfully reflect the original distribution of junctions and their component connexins, though individual isolated cells will show fewer junctions than the number that each cell shared in the intact tissue.

4.4 Heterogeneous distribution of connexin phenotypes in the SA node
The present results accord with previous studies on intact rabbit SA node tissue sections [4,34]. Coppen et al. [4] showed that Cx45 and Cx40, but not Cx43, were expressed in the center of the SA node, and that there was a restricted zone expressing both Cx43 and Cx45 at the border between the SA node and atrial muscle in the crista terminalis. Relatively large spindle cells expressing both Cx43 and Cx45 are probably derived from this transitional zone. Verheule et al. [5] investigated the correlation of different cell morphologies and immunolabeled connexin isoforms in the rabbit SA node, and showed that Cx43 was absent in typical nodal-type spindle and spider cells, but present in transitional-type elongated spindle cells.

In the study of Kwong et al. [27] ~35% of spindle or spider cells (assumed to be SA node cells) isolated from the dog SA node showed Cx43 immunolabeling, whereas the remainder did not. Most cells (atrial and SA node) showed Cx40 immunolabeling. In tissue sections, tracts of Cx43-immunolabeled SA node cells were seen to course through SA node not expressing Cx43. It is possible that the Cx43-expressing SA node cells in dogs are the exit route for the action potential from the SA node as proposed for the Cx43-expressing transitional SA node cells of the rabbit [5].

4.5 Colocalization of Cx43 and Cx45
In large spindle SA node cells, we observed that a proportion of immunolabeled spots for Cx43 and Cx45 overlapped even when viewed in single confocal planes, indicating instances of colocalization of both connexins in the same gap junction. The presence of two connexins within the same gap-junctional plaque is compatible with a variety of structural arrangements of connexins at the level of the individual gap-junctional channel, including heterotypic gap junction channels, in which one connexon is constructed from Cx43 and the partner connexon from Cx45, or heteromeric channels comprising various mixtures of Cx43 and Cx45. Whether such putative heterotypic or heteromeric channels, which are known to have different properties from homomeric channels in vitro [35,36], might contribute to modulation of electrical coupling at the periphery of the SA node to help maintain normal pacemaker function is at present unclear. Cx43/Cx45 heterotypic channels are reported to exhibit rectification properties [35] and so it might be speculated that such channels, if formed in the right orientation, could in theory facilitate normal conduction from the SA node in a manner that prevents reentrant arrhythmias [37]. However, there is as yet no evidence for the existence of heterotypic Cx43/Cx45 channels at the periphery of the SA node, and this aspect will require further investigation.

Time for primary review 22 days.


   Acknowledgements
 
The present study was partly supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and a grant from the Vehicle Racing Commemorative Foundation in Japan. NJS is supported by the European Commission (QLRT-1999-00516).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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