Skip Navigation

Cardiovascular Research 2006 72(2):271-281; doi:10.1016/j.cardiores.2006.07.026
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Yamamoto, M.
Right arrow Articles by Boyett, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamamoto, M.
Right arrow Articles by Boyett, M. R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2006, European Society of Cardiology

Extended atrial conduction system characterised by the expression of the HCN4 channel and connexin45

Mitsuru Yamamotoa, Halina Dobrzynskib, James Tellezb, Ryoko Niwaa, Rudi Billeterc, Haruo Honjoa, Itsuo Kodamaa and Mark R. Boyettb,*

aResearch Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan
bCardiovascular Research Group, School of Medicine, University of Manchester, Core Technology Facility, 46 Grafton Street, Manchester M13 9NT, UK
cInstitute of Membrane and Systems Biology, University of Leeds, Leeds, LS2 9JT, UK

* Corresponding author. Tel.: +44 161 275 1192; fax: +44 161 275 1233. Email address: mark.boyett{at}manchester.ac.uk

Received 8 June 2006; revised 20 July 2006; accepted 25 July 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Objective: In the heart, there are multiple supraventricular pacemakers involved in normal pacemaking as well as arrhythmias and the objective was to determine the distribution of HCN4 (major isoform underlying the pacemaker current, If) in the atria.

Methods: In the atria of the rat, the localisation of HCN4 and connexins was determined using immunohistochemistry, and electrical activity was recorded using extracellular electrodes.

Results: As expected, HCN4 and Cx45 (but not Cx43) were expressed in the sinoatrial node extending from the superior vena cava down the crista terminalis. The same pattern of expression of HCN4 and connexins was observed in a novel tract of nodal-like cells extending from the superior vena cava down the interatrial groove. Although the sinoatrial node was usually the leading pacemaker site, the novel tract of HCN4-expressing cells was capable of pacemaking and could act as the leading pacemaker site; there was evidence of a hierarchy of pacemakers. The same pattern of expression of HCN4 and connexins was also observed in the atrioventricular ring bundle (including the atrioventricular node) encircling the tricuspid valve, but not in the atrioventricular ring bundle encircling the mitral valve. HCN4 was not expressed in the pulmonary veins.

Conclusions: The widespread distribution of HCN4 can explain the widespread location of the leading pacemaker site during sinus rhythm, the extensive region of tissue that has to be ablated to stop sinus rhythm, and the widespread distribution of ectopic foci responsible for atrial tachycardia.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
In the heart, there are multiple supraventricular tissues capable of showing pacemaker activity including the sinoatrial node (SAN), atrioventricular node (AVN), superior vena cava [1–4], AV ring bundles [5–12] and pulmonary veins [13]. These tissues constitute the normal pacemaker (SAN), back-up pacemakers (e.g. AVN) and ectopic foci (e.g. pulmonary veins) responsible for atrial tachycardia and fibrillation. The hyperpolarization-activated current, If, plays a major role in pacemaker activity in the heart [14] and If is known to play a significant role in the pacemaker activity of many of the atrial pacemakers [7,15,16]. Four cDNAs encoding If-type currents have been cloned from vertebrates (HCN1-4); HCN4 has been reported to be a major isoform in the cardiac conduction system (SAN, AVN, Purkinje fibres) [15,17,18]. The aim of the present study was to determine the distribution of HCN4 in the atria. A characteristic feature of the SAN and AVN is that there is little or no expression of connexin43 (Cx43; medium conductance gap junction protein), which is abundantly expressed in the remainder of the heart, and instead there is expression of connexin45 (Cx45; low conductance gap junction protein). This results in poor electrical coupling in the nodal tissues and this is considered essential to protect the pacemaking tissue from the inhibitory electrotonic influence of the surrounding non-pacemaking atrial muscle [19]. The distribution of Cx43 and Cx45 in the atria was also determined in the present study. As well as in the SAN and AVN, HCN4 and Cx45 (but not Cx43) were expressed in tracts of nodal-like cells in the interatrial groove and around the tricuspid valve, but surprisingly not in the pulmonary veins.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Wistar rats (male; 250–300 g) and New Zealand white rabbits (male; 1.9–2 kg; used for histology and quantitative PCR) were sacrificed humanely according to the United Kingdom Animals (Scientific Procedures) Act, 1986; in addition, the investigation conformed 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). For the recording of membrane potential from the pulmonary veins, Japanese white rabbits (of either sex; 1.5–2.0 kg) were anesthetized with pentobarbital (30–40 mg/kg, iv); animal procedures were approved by the Animal Care and Use Committee, Research Institute of Environmental Medicine, Nagoya University. Following death of the animals, the heart was quickly removed. From the rat hearts, the atria (or AV junction) were isolated. Some of the rat preparations were frozen and cryosectioned; the sections were subsequently stained with Masson's trichrome or immunolabelled for HCN4, Cx43 and Cx45 using standard techniques [20]. Alternatively, extracellular electrodes were used to measure the activation sequence and spontaneous beating rate of the rat atria [21]. One rabbit heart was frozen and cryosectioned and the sections stained with Masson's trichrome. From seven rabbit hearts, total RNA was isolated from the proximal and distal pulmonary veins and the right atrium; cDNA was generated from the total RNA. The relative abundance of HCN1 and HCN4 cDNA (a measure of the abundance of HCN1 and HCN4 mRNA in the original samples) was then measured with quantitative PCR. From three rabbit hearts, the right superior pulmonary vein was isolated and the membrane potential recorded with glass microelectrodes. Data are presented as means±SEM. A one- or two-way ANOVA completed by a Bonferroni t-test was used to test differences; a difference was considered significant if P<0.05. Further details of the techniques are available in the Electronic Supplementary Material.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 References
 
3.1. Superior vena cava and rear wall of right atrium
Isolated atrial preparations were serially cryosectioned in a plane perpendicular to the axis from the superior to the inferior vena cava. Representative results from one heart are shown in Figs. 1–3GoGo; similar results were obtained from four hearts. Fig. 1 shows adjacent sections at the level of the junction of the superior vena cava with the right atrium (see inset in Fig. 1A). The dorsal region of the junction showed strong red staining with Masson's trichrome, but in the ventral region, there were cells with paler staining (Fig. 1A). The adjacent section was labelled for HCN4 and the cells showing strong Masson's trichrome staining showed no labelling of HCN4, whereas the cells showing pale Masson's trichrome-staining showed strong HCN4 labelling (see tissue demarcated by dashed lines in Fig. 1A,B). At high magnification, labelling of HCN4 was observed at the cell membrane and, in addition, there was substantial intracellular labelling (perhaps protein just translated or being trafficked) (Fig. 1C). Another adjacent section was double labelled for Cx43 and Cx45 – the cells showing strong Masson's trichrome staining showed labelling of Cx43, but not of Cx45 (data not shown), whereas the cells showing pale Masson's trichrome staining showed labelling (punctate) of Cx45, but not of Cx43 (Fig. 1D). The strong Masson's trichrome-stained/HCN4-negative/Cx43-positive/Cx45-negative cells are atrial cells and the pale Masson's trichrome-stained/HCN4-positive/Cx43-negative/Cx45-positive cells are SAN cells.


Figure 1
View larger version (88K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Expression of HCN4 and connexins at base of superior vena cava. A, Masson's trichrome stained section of junction of superior vena cava with right atrium showing nodal cells (lightly stained; demarcated by dashed lines) surrounded by atrial cells (darkly stained). Dashed lines in inset show location of sections through right atrium in Figs. 1, 2 and 3Go; red bar highlights section in this figure. B, HCN4 labelling in region identified by box in A. Dashed lines highlight area strongly expressing HCN4 (dull green labelling outside this region may be background labelling only). C, high-magnification image of HCN4 labelling in box in B. D, high-magnification image of Cx45 (red) and Cx43 (green) labelling in box in B. Scale bars, 200 µm (A, B) and 50 µm (C, D). In this and all figures: AS, atrial septum; AVN, atrioventricular node; CS, coronary sinus; CT, crista terminalis; D, dorsal; IVC, inferior vena cava; L, left; LA, left atrium; LAA, left atrial appendage; LSARB, left sinoatrial ring bundle; LV, left ventricle; MV, mitral valve; PV-P and PV-D, proximal and distal parts of pulmonary vein; R, right; RA, right atrium; RAA, right atrial appendage; RSARB, right sinoatrial ring bundle; RS-PV, right superior pulmonary vein; RV, right ventricle; SAN, sinoatrial node; SEP, interatrial septum; SVC, superior vena cava; TV, tricuspid valve; V, ventral; VS, ventricular septum.

 

Figure 2
View larger version (83K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Expression of HCN4 and connexins below base of superior vena cava. A, Masson's trichrome stained section. Dashed circles highlight two pale Masson's-trichrome stained nodal/nodal-like regions. Inset shows location of section in right atrium (red bar). B, C, high-magnification images of labelling of HCN4 (B) and Cx45 (red) and Cx43 (green) (C) from tract of nodal-like cells in interatrial groove (upper box in A). Note that myocytes highlighted by arrows expressed both HCN4 (B) and Cx45 (C). D, E, high-magnification images of labelling of HCN4 from tract of nodal-like cells in interatrial groove (D) and SAN (E) (from boxed regions in F). F, low-magnification image of HCN4 labelling in another preparation. Level of section was similar to that of A. Dashed circles highlight two regions strongly expressing HCN4. White line marks cavity of superior vena cava. Scale bars, 200 µm (A, F) and 50 µm (B–E).

 

Figure 3
View larger version (74K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Expression of HCN4 and connexins at level of right superior pulmonary vein. A, Masson's trichrome stained section including right superior pulmonary vein. Inset shows location of section in right atrium (red bar). B, C, high-magnification images of labelling of HCN4 (B) and Cx45 (red) and Cx43 (green) (C) from tract of nodal-like cells in interatrial groove (right box in A). Note that group of myocytes highlighted by arrows expressed both HCN4 (B) and Cx45 (C). D, E, high-magnification images of labelling of HCN4 (D) and Cx45 (red) and Cx43 (green) (E) from proximal right superior pulmonary vein (left box in A). Scale bars, 200 µm (A) and 50 µm (B–E).

 
Fig. 2 shows sections at a more inferior level (Fig. 2A, inset). At this level, there were two groups of nodal (or nodal-like) cells: a large group of nodal cells was located alongside the crista terminalis (this group constitutes the SAN [20]) and a smaller group of nodal-like cells was located on the epicardial surface next to the interatrial groove. Fig. 2A shows that both groups (tissue within dashed lines in the main panel of Fig. 2A) appeared pale when stained with Masson's trichrome (unlike surrounding atrial muscle). Fig. 2F shows that both groups (tissue within dashed lines in Fig. 2F) showed strong expression of HCN4. Details of the HCN4 labelling are shown in Fig. 2D,E. Both groups also showed expression of Cx45 and no expression of Cx43. As an example, Fig. 2C shows labelling of Cx43 and Cx45 in the nodal-like cells next to the interatrial groove – in these cells, note the close correspondence between the labelling of HCN4 and Cx45 (Fig. 2B,C; see cells highlighted by arrows).

Fig. 3 shows sections at a more inferior level – at the level of the right superior pulmonary vein (Fig. 3A, inset). Fig. 3A shows part of the rear wall of the right atrium and the neighbouring right superior pulmonary vein (RS-PV) – the crista terminalis is not included. The small group of HCN4-positive/Cx43-negative/Cx45-positive nodal-like cells next to the interatrial groove was still present at this level (Fig. 3B,C). At this level, a group of SAN cells was also still present next to the crista terminalis (data not shown). The results presented in Figs. 1–3GoGo show that nodal/nodal-like tissue forms an inverted U shape centred on the superior vena cava: one arm of the U forms the SAN and the other forms a tract of nodal-like cells extending down the interatrial groove.

3.2. Functional importance of tract of HCN4-expressing nodal-like cells in interatrial groove
Under control conditions, the location of the leading pacemaker site was investigated in 18 preparations. Fig. 4 shows representative activation maps (epicardial views shown). In 11 of 18 preparations, the leading pacemaker site was located in the SAN next to the crista terminalis and Fig. 4A,B shows two such examples (see left hand activation maps obtained under control conditions) – the leading pacemaker site (asterisk) was located on or next to the crista terminalis (grey shaded bundle) and the right sinoatrial ring bundle (RSARB). However, in the remaining seven preparations, the leading pacemaker site was located in the interatrial groove and Fig. 4C (left) shows one example – the leading pacemaker site (asterisk) is next to the left sinoatrial ring bundle (LSARB). Next, we investigated the effect of 0.05 µM isoproterenol (ISO) on the leading pacemaker site. In four of five preparations, the leading pacemaker site shifted from the SAN adjacent to the crista terminalis to the junction between the superior vena cava and the interatrial groove (ISO site) – Fig. 4A,B shows examples. Of these four preparations, in the presence of ISO, two exhibited a permanent pacemaker shift to the ISO site and in the other two the leading pacemaker site shifted back and forth between the SAN and the ISO site. In one of five preparations, the leading pacemaker site shifted from the interatrial groove to the ISO site (Fig. 4C). This distribution of leading pacemaker sites (under control conditions and in presence of ISO) corresponds to the distribution of HCN4 described above.


Figure 4
View larger version (50K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Activation sequence in absence and presence of isoprenaline. A–C, activation sequence from three representative cases under control conditions (left) and in presence of 0.05 µM isoprenaline (ISO; right). Asterisks indicate position of leading pacemaker site and isochrones (dashed lines; at 2.5 ms intervals) show spread of activation.

 
We compared the pacemaker activity of the SAN and the tract of nodal-like cells in the interatrial groove. Preparations were divided into two as shown in Fig. 5 (inset). One part included the SAN and the other part included the interatrial groove (Fig. 5, inset; endocardial view of tissue shown). Under control conditions, after the division of the tissue into two, the preparation containing the interatrial groove showed spontaneous beating for a brief period (0.5–5 min), whereas the preparation containing the SAN showed continuous beating. The cycle length of the preparation including the SAN was 362±26 ms and it was not changed by the separation of the tissue into two. In the presence of 0.05 µM ISO (Fig. 5A, left), the preparation including the interatrial groove started beating spontaneously and the cycle length was similar to that of the preparation including the SAN in the presence of ISO (166±8 and 174±8 ms, respectively; not significantly different). In the presence of 0.05 µM ISO, 2 mM Cs+ was applied to block If, for which HCN4 is responsible (Fig. 5A, right). Cs+ caused both preparations to slow down: in the preparation including the SAN, the cycle length was prolonged by 31±5% to 228±16 ms (P<0.05 versus control) and, in the preparation including the interatrial groove, the cycle length was prolonged by 23±7% to 204±18 ms (P<0.05 versus control) (Fig. 5A,B). This suggests that HCN4 plays an important role in pacemaking in both regions.


Figure 5
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Comparison of intrinsic pacemaker activity of SAN and tract of nodal-like cells in interatrial groove. Preparation was divided into two parts as shown in inset. One part included SAN and second part included interatrial groove. A, cycle length of two preparations in presence of 0.05 µM isoprenaline (ISO; left) and 0.05 µM isoprenaline with 2 mM Cs+ (right). Individual data points as well as means±SEM (n=6) shown. B, mean (+SEM; n=6) percentage increase in cycle length of two preparations on addition of Cs+. *P<0.05; NS, not significant.

 
3.3. AV ring bundles and AVN
In the atria, there are rings of nodal-like cells around the atrioventricular valves (AV ring bundles) [5–12]. Fig. 6A shows a Masson's trichrome stained sagittal section through the right and left atria and ventricles. Small nodal-like cells were present alongside both the tricuspid and mitral valves – in the boxed areas B and C. Fig. 6B (from boxed area B in Fig. 6A) shows that in the AV ring bundle around the tricuspid valve, the expression pattern of HCN4, Cx45 and Cx43 was similar to that of the SAN, although the amount of HCN4 and Cx45 was lower than in the SAN. The expression pattern of HCN4, Cx45 and Cx43 in the AVN (seen in Fig. 6A) was similar to that of the AV ring bundle around the tricuspid valve (data not shown). This is not surprising, because the AVN can be thought of as part of the AV ring bundle [22]. Fig. 6C (from boxed area C in Fig. 6A) shows that in the AV ring bundle around the mitral valve, the expression of HCN4 was low or absent, whereas the expression of Cx45 and absence of Cx43 were similar to those in the AV ring bundle around the tricuspid valve. Similar results were obtained from four hearts.


Figure 6
View larger version (76K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Expression of HCN4 and connexins at AV junction. A, Masson's trichrome stained section (cut along sagittal plane) showing AV junction. B, C, high-magnification images of labelling of HCN4 (left), Cx45 (red; right) and Cx43 (green; right) at tricuspid (B) and mitral (C) valves. Location of images shown by boxes in A. Scale bars, 1 mm (A) and 50 µm (B, C).

 
3.4. Pulmonary veins
In four rat hearts, expression of HCN4 and Cx45 was not detected in the myocardial sleeves of the pulmonary veins, whereas Cx43 was expressed throughout the myocardial sleeves (Fig. 3D,E). This expression pattern was same as that of the right and left atrial free walls. The absence of the HCN4 in the pulmonary veins is surprising, because in the embryonic mouse (ED 12) HCN4 mRNA has been observed in tissue that will give rise to the common pulmonary vein [23]. The presence of HCN4 in the pulmonary veins was, therefore, checked in another species. The abundance of HCN4 (and also HCN1) mRNA was measured in the proximal and distal parts of the pulmonary veins of the rabbit (Fig. 7A) using quantitative PCR (it was not possible to immunolabel HCN4 protein as in previous experiments, because the antibody used was raised in the rabbit). In the proximal and distal parts of the rabbit pulmonary veins, the abundance of both HCN1 and HCN4 mRNA was comparable with that in right atrial muscle, i.e. the abundance of HCN4 mRNA in the pulmonary veins was low (Fig. 7B). We have previously shown that, in the presence of ryanodine, rapid stimulation routinely induces pacemaker activity in the pulmonary veins from the rabbit [13]; similar behaviour has been observed in the dog [24]. Fig. 7C shows action potentials recorded from the rabbit right superior pulmonary vein after exposure to 0.5 µM ryanodine. Following a rest period of 1 min, the preparation was stimulated at 3.3 Hz for 20 pulses; this resulted in a substantial depolarization of the membrane (Fig. 7C). When 3.3 Hz stimulation was stopped, a train of spontaneous action potentials was recorded; a pacemaker potential was observed between action potentials (Fig. 7C). Block of If by 2 mM Cs+ had no effect on this behaviour: in the absence and presence of Cs+, the train of spontaneous action potentials continued for 28.9±5.7 s (n=3) and 26.6±3.6 s (n=3), respectively (Fig. 7C). This demonstrates that If plays no role in pacemaker activity in the pulmonary veins.


Figure 7
View larger version (54K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 HCN4 and pulmonary veins of rabbit. A, Masson's trichrome stained section through right superior pulmonary vein. Proximal and distal parts of pulmonary vein are shown. Scale bar, 1 mm. B, abundance of HCN1 and HCN4 mRNAs in right atrial muscle and proximal and distal parts of pulmonary veins as detected by quantitative PCR. Means±SEM (n=7) shown. C, effect of rapid pacing (20 pulses at 3.3 Hz) on membrane potential of right superior pulmonary vein treated with 0.5 µM ryanodine. Left, stimuli and slow time base recording of membrane potential before (top) and after (bottom) the application of 2 mM Cs+. Preparation was stimulated at 2 Hz, rested for 1 min, and then briefly stimulated at 3.3 Hz (20 pulses). Burst of spontaneous action potentials was induced by rapid pacing. Right, superimposed recordings of spontaneous action potentials before and after application of Cs+ (from times shown on left).

 
3.5. Summary
Fig. 8A,B shows ventral and dorsal views of the atria and summarises the distribution of HCN4 and Cx45 (and Cx43) observed in the present study. Nodal or nodal-like cells (as defined by the expression of HCN4 and Cx45 and lack of Cx43) looped around the ventral surface at the junction of the superior vena cava with the right atrium and extended from here down the intercaval region, both next to the crista terminalis (to form the SAN) and the interatrial groove. This roughly corresponds to the distribution of leading pacemaker sites observed in 18 preparations under control conditions (asterisks in Fig. 8C). Fig. 8A,B also shows the expression of HCN4 and Cx45 in the AV ring bundles as well as the AVN; as indicated by the grey scale, expression of HCN4 in the SAN>expression in the AV ring bundle encircling the tricuspid valve>expression in the AV ring bundle encircling the mitral valve, whereas the expression of Cx45 was the same in the three tissues.


Figure 8
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 Summary of HCN4 and Cx45 distribution in atria and position of leading pacemaker site. A, B, expression patterns of HCN4 (A) and Cx45 (B). Ventral view of atria is shown on left and dorsal view is shown on right. Dark area has abundant expression and dotted area has less abundant expression. Tissues expressing Cx45 did not express Cx43. C, dorsal view of atria showing location of leading pacemaker site (asterisks) under control conditions in 18 preparations.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 References
 
In the present study, in the atrial muscle of the rat, Cx43 was expressed, whereas HCN4 and Cx45 were not expressed. However, HCN4 and Cx45 were expressed, whereas Cx43 was not expressed, in tracts of nodal-like cells extending down the interatrial groove and surrounding the tricuspid valve, as well as in the SAN and AVN (Fig. 8). Evidence showed that the tract of HCN4-expressing cells in the interatrial groove has probable If-dependent pacemaker activity and in some circumstances can be the leading pacemaker site (taking over from SAN) ((Figs. 4, 5 and 8C)GoGo).

4.1. Tract of nodal-like cells in interatrial groove
This study showed the presence of a tract of HCN4-positive/Cx43-negative/Cx45-positive small nodal-like cells extending downwards from the superior vena cava in the interatrial groove (Figs. 2 and 3Go). The tract has not been described previously and is distinct from the SAN, which consists of a tract of HCN4-positive/Cx43-negative/Cx45-positive small nodal cells extending downwards from the superior vena cava next to the crista terminalis (Fig. 2). By separating the interatrial groove from the SAN (Fig. 5), the nodal-like cells in the interatrial groove were demonstrated to be capable of independent pacemaker activity. However, the pacemaker activity of the nodal-like cells in the interatrial groove was less robust than that of the SAN, because, in isolated preparations, they did not show pacemaker activity under control conditions (ISO was required), whereas the SAN did (Fig. 5). This explains why in intact preparations under control conditions, the SAN was normally the leading pacemaker, although in some preparations the tract of nodal-like cells in the interatrial groove was the leading pacemaker site (Figs. 4C and 8CGo). HCN4 is responsible for the Cs+-sensitive pacemaker current, If, and it was demonstrated that the pacemaker activity of both the SAN and the nodal-like cells in the interatrial groove is Cs+-sensitive (Fig. 5). Although under control conditions the pacemaker activity of the nodal-like cells in the interatrial groove was less robust than that of the SAN, in the presence of ISO the pacemaker activity of the nodal-like cells in the interatrial groove was as fast as that of the SAN (Fig. 5) and this perhaps explains why in intact preparations the leading pacemaker shifted from the SAN to the interatrial groove (Fig. 4). In the dog, Boineau et al. [25] (see also [26]) reported a consistent relationship between the leading pacemaker site and the heart rate: vagal stimulation decreased the heart rate and resulted in a caudal shift of the leading pacemaker site, whereas β-adrenergic stimulation increased the heart rate and resulted in a cranial shift. They suggested that different regions of the SAN are specialised for different heart rates. Our data are consistent with these observations.

The tract of HCN4-expressing nodal-like cells in the interatrial groove may be important pathologically: recently, Yamada et al. [27] showed that atrial tachycardia in a group of patients could be attributed to a focus in the interatrial groove on the rear wall of the right atrium. Perhaps the focus was a tract of HCN4-expressing nodal-like cells in the interatrial groove (as observed in the present study – Fig. 8). In another study, Kistler et al. [28] showed that tachycardia, in one group of patients, could be attributed to a focus in the crista terminalis and, in a second group, to a focus in the interatrial septum. Perhaps in the first group of patients the focus was the SAN itself, whereas in the second group the focus was once again a tract of HCN4-expressing nodal-like cells in the interatrial groove.

4.2. AV ring bundles
It has been reported that in several mammalian species, including the human, myocytes in the atrioventricular annuli have automatic or triggered activities [5–12]. The automaticity in rabbit tricuspid valve myocytes is sensitive to Cs+ block of If (Cs+ results in a slowing of the beating rate [6,7]) and, furthermore, If has been recorded from the tricuspid valve myocytes [7]. In the rabbit, pacemaker activity is faster in the tricuspid annulus than in the mitral annulus [5]. Consistent with these observations, in the present study, we demonstrated expression of HCN4 in the AV ring bundle of the tricuspid valve (Fig. 6). In the AV ring bundle of the mitral valve there was little or no expression of HCN4 (Fig. 6). This finding may account for the difference in pacemaker activity between the two annuli. In the AV ring bundles of both the tricuspid and mitral valves, we observed expression of Cx45 (and no expression of Cx43; Fig. 6) (cf. [8,22]). The tract of HCN4-expressing nodal-like cells around the tricuspid valve may again be important pathologically: Kistler et al. [28] showed that atrial tachycardia in another group of patients (see above) could be attributed to a focus in the right atrium around the tricuspid valve – this could correspond to a tract of HCN4-expressing nodal-like cells surrounding the tricuspid valve (as observed in the present study – Fig. 8).

4.3. Pulmonary veins
The pulmonary veins are capable of pacemaking and pulmonary vein cells can have a pacemaker potential [13]. In the present study, no significant expression of HCN4 protein or mRNA was observed in the pulmonary veins (Figs. 3D and 7BGo) and 2mM Cs+ had no effect on spontaneous activity of the pulmonary veins (Fig. 7C). Consistent with this, Ehrlich et al. [29] reported that If is not present in dog pulmonary vein cells. These observations suggest that If plays no role in the pacemaker activity of the pulmonary veins and in this case pacemaking must be the result of other ionic currents.

4.4. SAN and superior vena cava
In the present study, HCN4 and Cx45, but not Cx43, were expressed in the SAN as has been observed previously [17,30]. In the present study, the SAN extended downwards from the superior vena cava next to the crista terminalis. In many experimental animals (rabbit, guinea-pig, monkey, mouse, cat, pig), the SAN has been reported to be located in this position [16,31–35]; for example, see our three-dimensional anatomical model of the SAN of the rabbit [20]. In contrast, in the human, early studies by James [36] and Truex et al. [37] described the SAN as a discrete tissue at the junction of the superior vena cava with the right atrium (see well known diagrams of Netter). However, subsequently, in the human, it has been shown that the SAN can have a tail projecting down the crista terminalis [38]. Furthermore, the leading pacemaker site in the human (as well as dog and pig) can be located at many sites: as well as at the junction between the superior vena cava and the right atrium as expected from the work of James [36] and Truex et al. [37], it can be located at any point between the superior and inferior vena cava along the line of the sulcus terminalis (a groove marking the border of the crista terminalis) in the rear wall of the right atrium [25,26,39–42]. This widespread distribution of leading pacemaker sites is consistent with the distribution of SAN tissue observed in the present study (Fig. 8C). The dog SAN is considered to be similar to the human SAN and, in the dog, total ablation of SAN pacemaker function requires the destruction of a block of tissue ~4 cm long and 1.5 cm wide extending from the lateral junction of the superior vena cava with the right atrial appendage, along the crista terminalis to the inferior vena cava [26,43]. Once again, this corresponds to the distribution of SAN tissue as described in the present study (Fig. 8). The superior vena cava has been reported to have ectopic pacemaker activity [1–4]; it is possible that this is the result of abnormal pacemaker activity in the nodal cells that are wrapped around the ventral surface of the superior vena cava (Fig. 8). The tract of nodal-like cells in the interatrial groove is a continuation of the tract of nodal cells wrapped around the ventral surface of the superior vena cava (Fig. 8).

Specialised internodal pathways (between the SAN and AVN) have been proposed. Could nodal or nodal-like cells form the pathways? There was not a continuous tract of HCN4-expressing cells between the SAN and AVN (Fig. 8) and this is consistent with the finding that an increase in extracellular K+ (which abolishes the action potential in atrial muscle but not in nodal tissue) blocks action potential transmission from the SAN to the AVN [44].

4.5. AVN
In the present study, HCN4 and Cx45, but not Cx43, was expressed in the AVN (c.f. [15]). The AVN can be considered to be part of the AV ring bundle of the tricuspid valve (Fig. 8A,B). The posterior (or inferior) nodal extension is the slow pathway into the AVN, and the posterior nodal extension is the part of the AV ring bundle proximal to the AVN. Both the AVN and the posterior nodal extension had a similar expression of HCN4 and connexins as the rest of the AV ring bundle of the tricuspid valve (data not shown). Recently, we have shown that, after the destruction of the SAN, the junctional rhythm, which is Cs+-sensitive (i.e. If-dependent), originates in the posterior nodal extension [15].

4.6. Conclusion
In the atria, HCN4 is expressed in four structures: (i) a tract of nodal-like cells in the interatrial groove, (ii) a tract of nodal-like cells encircling the tricuspid valve, (iii) the SAN and (iv) the AVN. This will be of importance for normal and abnormal pacemaking in the atria.


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2006.07.026.


   Notes
 
Time for primary review 18 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 

  1. Tsai C.F., Tai C.T., Hsieh M.H., Lin W.S., Yu W.C., Ueng K.C., et al. Initiation of atrial fibrillation by ectopic beats originating from the superior vena cava: electrophysiological characteristics and results of radiofrequency ablation. Circulation (2000) 102:67–74.[Abstract/Free Full Text]
  2. Liu T.Y., Tai C.T., Lee P.C., Hsieh M.H., Higa S., Ding Y.A., et al. Novel concept of atrial tachyarrhythmias originating from the superior vena cava: insight from noncontact mapping. J Cardiovasc Electrophysiol (2003) 14:533–539.[CrossRef][ISI][Medline]
  3. Shah D.C., Haissaguerre M., Jais P., Clementy J. High-resolution mapping of tachycardia originating from the superior vena cava: evidence of electrical heterogeneity, slow conduction, and possible circus movement reentry. J Cardiovasc Electrophysiol (2002) 13:388–392.[CrossRef][ISI][Medline]
  4. Goya M., Ouyang F., Ernst S., Volkmer M., Antz M., Kuck K.H. Electroanatomic mapping and catheter ablation of breakthroughs from the right atrium to the superior vena cava in patients with atrial fibrillation. Circulation (2002) 106:1317–1320.[Abstract/Free Full Text]
  5. Rozanski G.J., Jalife J. Automaticity in atrioventricular valve leaflets of rabbit heart. Am J Physiol (1986) 250:H397–H406.[ISI][Medline]
  6. Rozanski G.J. Electrophysiological properties of automatic fibers in rabbit atrioventricular valves. Am J Physiol (1987) 253:H720–H727.[ISI][Medline]
  7. Anumonwo J.M., Delmar M., Jalife J. Electrophysiology of single heart cells from the rabbit tricuspid valve. J Physiol (1990) 425:145–167.[Abstract/Free Full Text]
  8. McGuire M.A., de Bakker J.M., Vermeulen J.T., Moorman A.F., Loh P., Thibault B., et al. Atrioventricular junctional tissue. Discrepancy between histological and electrophysiological characteristics. Circulation (1996) 94:571–577.[Abstract/Free Full Text]
  9. Bassett A.L., Fenoglio J.J. Jr., Wit A.L., Myerburg R.J., Gelband H. Eelctrophysiologic and ultrastructural characteristics of the canine tricuspid valve. Am J Physiol (1976) 230:1366–1373.[Abstract/Free Full Text]
  10. Wit A.L., Fenoglio J.J. Jr., Hordof A.J., Reemtsma K. Ultrastructure and transmembrane potentials of cardiac muscle in the human anterior mitral valve leaflet. Circulation (1979) 59:1284–1292.[Free Full Text]
  11. Wit A.L., Fenoglio J.J. Jr., Wagner B.M., Bassett A.L. Electrophysiological properties of cardiac muscle in the anterior mitral valve leaflet and the adjacent atrium in the dog. Possible implications for the genesis of atrial dysrhythmias. Circ Res (1973) 32:731–745.[Abstract/Free Full Text]
  12. Makarychev V.A., Kosharskaia I.L., Ul'ianinskii L.S. Automatic activity of pacemaker cells in the atrioventricular valves of the rabbit heart. Biulleten Eksperimentalnoi Biologii I Meditsiny. (1976) vol. 81:517–520.
  13. Honjo H., Boyett M.R., Niwa R., Inada S., Yamamoto M., Mitsui K., et al. Pacing-induced spontaneous activity in myocardial sleeves of pulmonary veins after treatment with ryanodine. Circulation (2003) 107:1937–1943.[Abstract/Free Full Text]
  14. DiFrancesco D. Pacemaker mechanisms in cardiac tissue. Annu Rev Physiol (1993) 55:455–472.[CrossRef][ISI][Medline]
  15. Dobrzynski H., Nikolski V.P., Sambelashvili A.T., Greener I.D., Boyett M.R., Efimov I.R. Site of origin and molecular substrate of atrioventricular junctional rhythm in the rabbit heart. Circ Res (2003) 93:1102–1110.[Abstract/Free Full Text]
  16. Boyett M.R., Honjo H., Kodama I. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc Res (2000) 47:658–687.[Abstract/Free Full Text]
  17. Biel M., Schneider A., Wahl C. Cardiac HCN channels: structure, function, and modulation. Trends Cardiovasc Med (2002) 12:206–212.[CrossRef][ISI][Medline]
  18. Han W., Bao W., Wang Z., Nattel S. Comparison of ion-channel subunit expression in canine cardiac Purkinje fibers and ventricular muscle. Circ Res (2002) 91:790–797.[Abstract/Free Full Text]
  19. Joyner R.W., van Capelle F.J.L. Propagation through electrically coupled cells: how a small SA node drives a large atrium. Biophys J (1986) 50:1157–1164.[Abstract/Free Full Text]
  20. Dobrzynski H., Li J., Tellez J., Greener I.D., Nikolski V.P., Wright S.E., et al. Computer three-dimensional reconstruction of the sinoatrial node. Circulation (2005) 111:846–854.[Abstract/Free Full Text]
  21. Yamamoto M., Honjo H., Niwa R., Kodama I. Low frequency extracellular potentials recorded from the sinoatrial node. Cardiovasc Res (1998) 39:360–372.[Abstract/Free Full Text]
  22. Coppen S.R., Severs N.J., Gourdie R.G. Connexin45 ({alpha}6) expression delineates an extended conduction system in the embryonic and mature rodent heart. Dev Genet (1999) 24:82–90.[CrossRef][ISI][Medline]
  23. Garcia-Frigola C., Shi Y., Evans S.M. Expression of the hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 during mouse heart development. Gene Expr Patterns (2003) 3:777–783.[CrossRef][Medline]
  24. Chou C.C., Nihei M., Zhou S., Tan A., Kawase A., Macias E.S., et al. Intracellular calcium dynamics and anisotropic reentry in isolated canine pulmonary veins and left atrium. Circulation (2005) 111:2889–2897.[Abstract/Free Full Text]
  25. Boineau J.P., Schuessler R.B., Roeske W.R., Autry L.J., Miller C.B., Wylds A.C. Quantitative relation between sites of atrial muscle origin and cycle length. Am J Physiol (1983) 245:H781–H789.[ISI][Medline]
  26. Kalman J.M., Lee R.J., Fisher W.G., Chin M.C., Ursell P., Stillson C.A., et al. Radiofrequency catheter modification of sinus pacemaker function guided by intracardiac echocardiography. Circulation (1995) 92:3070–3081.[Abstract/Free Full Text]
  27. Yamada T., Murakami Y., Muto M., Okada T., Okamoto M., Toyama J., et al. Electrophysiologic characteristics of atrial tachycardia originating from the right pulmonary veins or posterior right atrium: double potentials obtained from the posterior wall of the right atrium can be useful to predict foci of atrial tachycardia in right pulmonary veins or posterior right atrium. J Cardiovasc Electrophysiol (2004) 15:745–751.[CrossRef][ISI][Medline]
  28. Kistler P., Haqqani H., Fynn S., Stevenson I., Vohra J., Sparks P., et al. RF ablation of focal atrial tachycardia – anatomic distribution and long term follow-up in a large consecutive single center series. Heart Rhythm. (2004) vol. 1:S236.
  29. Ehrlich J.R., Cha T.J., Zhang L., Chartier D., Melnyk P., Hohnloser S.H., et al. Cellular electrophysiology of canine pulmonary vein cardiomyocytes: action potential and ionic current properties. J Physiol (2003) 551:801–813.[Abstract/Free Full Text]
  30. Coppen S.R., Kodama I., Boyett M.R., Dobrzynski H., Takagishi Y., Honjo H., et al. Connexin45, a major connexin of the rabbit sinoatrial node, is co-expressed with connexin43 in a restricted zone at the nodal-crista terminalis border. J Histochem Cytochem (1999) 47:907–918.[Abstract/Free Full Text]
  31. Opthof T., de Jonge B., Mackaay A.J.C., Bleeker W.K., Masson-Pevet M., Jongsma H.J., et al. Functional and morphological organization of the guinea-pig sinoatrial node compared with the rabbit sinoatrial node. J Mol Cell Cardiol (1985) 17:549–564.[CrossRef][ISI][Medline]
  32. Alings A.M.W., Abbas R.F., de Jonge B., Bouman L.N. Structure and function of the simian sinoatrial node (Macaca fascicularis). J Mol Cell Cardiol (1990) 22:1453–1466.[CrossRef][ISI][Medline]
  33. Opthof T., de Jonge B., Masson-Pevet M., Jongsma H.J., Bouman L.N. Functional and morphological organization of the cat sinoatrial node. J Mol Cell Cardiol (1986) 18:1015–1031.[CrossRef][ISI][Medline]
  34. Opthof T., de Jonge B., Jongsma H.J., Bouman L.N. Functional morphology of the pig sinoatrial node. J Mol Cell Cardiol (1987) 19:1221–1236.[CrossRef][ISI][Medline]
  35. Lei M., Jones S.A., Liu J., Lancaster M.K., Fung S.S., Dobrzynski H., et al. Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking. J Physiol (2004) 559:835–848.[Abstract/Free Full Text]
  36. James T.N. Anatomy of the human sinus node. Anat Rec (1961) 141:109–139.[CrossRef][Medline]
  37. Truex R.C., Smythe M.Q., Taylor M.J. Reconstruction of the human sinoatrial node. Anat Rec (1967) 159:371–378.[CrossRef][Medline]
  38. Sanchez-Quintana D., Anderson R.H., Cabrera J.A., Climent V., Martin R., Farre J., et al. The terminal crest: morphological features relevant to electrophysiology. Heart (2002) 88:406–411.[Abstract/Free Full Text]
  39. Boineau J.P., Canavan T.E., Schuessler R.B., Cain M.E., Corr P.B., Cox J.L. Demonstration of a widely distributed atrial pacemaker complex in the human heart. Circulation (1988) 77:1221–1237.[Abstract/Free Full Text]
  40. Boineau J.P., Schuessler R.B., Hackel D.B., Miller C.B., Brockus C.W., Wylds A.C. Widespread distribution and rate differentiation of the atrial pacemaker complex. Am J Physiol (1980) 239:H406–H415.[ISI][Medline]
  41. Betts T.R., Ho S.Y., Sanchez-Quintana D., Roberts P.R., Anderson R.H., Morgan J.M. Three-dimensional mapping of right atrial activation during sinus rhythm and its relationship to endocardial architecture. J Cardiovasc Electrophysiol (2002) 13:1152–1159.[CrossRef][ISI][Medline]
  42. Betts T.R., Roberts P.R., Ho S.Y., Morgan J.M. High density endocardial mapping of shifts in the site of earliest depolarization during sinus rhythm and sinus tachycardia. Pacing Clin Electrophysiol (2003) 26:874–882.[CrossRef][Medline]
  43. Euler D.E., Jones S.B., Gunnar W.P., Loeb J.M., Murdock D.K., Randall W.C. Cardiac arrhythmias in the conscious dog after excision of the sinoatrial node and crista terminalis. Circulation (1979) 59:468–475.[Free Full Text]
  44. Sanchis J., Opthof T., Wilde A.A.M., Janse M.J. Mutual electrotonic interaction between the rabbit sinoatrial and atrioventricular node. J Cardiovasc Electrophysiol (1990) 1:318–326.

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Physiol. Rev.Home page
M. E. Mangoni and J. Nargeot
Genesis and Regulation of the Heart Automaticity
Physiol Rev, July 1, 2008; 88(3): 919 - 982.
[Abstract] [Full Text] [PDF]

Home page
 Circ Arrhythmia ElectrophysiolHome page
S. A. Jones, M. Yamamoto, J. O. Tellez, R. Billeter, M. R. Boyett, H. Honjo, and M. K. Lancaster
Distinguishing Properties of Cells From the Myocardial Sleeves of the Pulmonary Veins: A Comparison of Normal and Abnormal Pacemakers
Circ Arrhythmia Electrophysiol, April 1, 2008; 1(1): 39 - 48.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Yamamoto, M.
Right arrow Articles by Boyett, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamamoto, M.
Right arrow Articles by Boyett, M. R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?