Published online before print October 16, 2003, doi: 10.1161/01.RES.0000101913.95604.B9
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© 2003 American Heart Association, Inc.
Integrative Physiology |
Site of Origin and Molecular Substrate of Atrioventricular Junctional Rhythm in the Rabbit Heart
From the Department of Biomedical Engineering (H.D., V.P.N., A.T.S., I.R.E.), Case Western Reserve University, Cleveland, Ohio; School of Biomedical Sciences (H.D., I.D.G., M.Y., M.R.B.), University of Leeds, Leeds, UK.
Correspondence to Igor R. Efimov, PhD, Cardiac Bioelectricity Research and Training Center, Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio. E-mail ire{at}case.edu
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Abstract |
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During failure of the sinoatrial node, the heart can be driven by an atrioventricular (AV) junctional pacemaker. The position of the leading pacemaker site during AV junctional rhythm is debated. In this study, we present evidence from high-resolution fluorescent imaging of electrical activity in rabbit isolated atrioventricular node (AVN) preparations that, in the majority of cases (11 out of 14), the AV junctional rhythm originates in the region extending from the AVN toward the coronary sinus along the tricuspid valve (posterior nodal extension, PNE). Histological and immunohistochemical investigation showed that the PNE has the same morphology and unique pattern of expression of neurofilament160 (NF160) and connexins (Cx40, Cx43, and Cx45) as the AVN itself. Block of the pacemaker current, If, by 2 mmol/L Cs+ increased the AV junctional rhythm cycle length from 611±84 to 949±120 ms (mean±SD, n=6, P<0.001). Immunohistochemical investigation showed that the principal If channel protein, HCN4, is abundant in the PNE. As well as the AV junctional rhythm, the PNE described in this study may also be involved in the slow pathway of conduction into the AVN as well as AVN reentry, and the predominant lack of expression of Cx43 as well as the presence of Cx45 in the PNE shown could help explain its slow conduction.
Key Words: ablation • electrophysiology • surgery • arrhythmia • imaging
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Introduction |
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Since Tawara’s discovery of the atrioventricular node (AVN) nearly a century ago,1 anatomists and electrophysiologists have established that the AVN is the only conduction pathway between the atria and ventricles in the normal heart.2 The AVN has unique slow and frequency-dependent conduction properties.2 Under normal physiological conditions, the AVN determines the appropriate frequency-dependent delay of conduction between the atria and ventricles and, during atrial fibrillation, the AVN filters high-frequency excitation, thus protecting the ventricular myocardium.3 The AVN has dual inputs (fast and slow pathways) from the atrial myocardium and this may be the substrate for AVN reentry.4,5 The AVN also has pacemaking ability: during failure of the sinoatrial node, the heart can be driven by an atrioventricular (AV) junctional pacemaker, although the position of the leading pacemaker site is debated.6,7
Recent application of fluorescent imaging with voltage-sensitive dyes5,8–12 has provided new insights into the electrophysiology of the AV junction. With fluorescent imaging, we have recently shown how the fast and slow pathways of conduction support normal conduction, 9 AVN echo,5 and AVN reentry.12 Application of immunohistochemical imaging has shown that the expression of ion channels13 and gap junction channel isoforms14,15 can explain the electrophysiology of the AVN. In particular, a lack of or a low density of Na+ channels in the compact node (CN) can explain the slow upstroke and low amplitude of the action potential in CN.13 Similarly, in CN, a lack of or a low density of low impedance isoforms of gap junction channels (Cx43 and Cx40) and the presence of a high-impedance isoform (Cx45)14,15 can explain the extremely slow conduction through the AVN.16 Our recent finding of Cx43-positive bundles in juxtaposition to Cx43-negative tissue may help to explain the dual inputs into the AVN17 and/or longitudinal dissociation.18 For a more detailed review of ion channel expression in the AVN, see Schram et al.19
In this study, we have applied both imaging techniques—fluorescent imaging of transmembrane potential with the voltage-sensitive dye, di-4-ANNEPS, and the immunohistochemical imaging of neurofilament160 (NF160; a marker of the conductive system20–22), the principle connexins of the heart (Cx40, Cx43, and Cx45), and the If channel (HCN) responsible for pacemaking23—in order to establish the site of origin and molecular substrate of AV junctional rhythm. We excised the sinoatrial node (SAN) and focused on "escape" rather than "accelerated" AV junctional rhythm.
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Materials and Methods |
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Fluorescent Imaging
We studied AV junctional rhythm in isolated right atrial preparations from the rabbit heart. The preparations were prepared as described previously.9 We used the excitation-contraction uncoupler 2,3-butanedione monoxime (15 mmol/L), which does not depress AVN conduction.24 To investigate the role of If in the junctional pacemaker activity, we added 2 mmol/L Cs+ to the perfusate. Electrical activity was monitored using bipolar electrodes at the crista terminalis (CrT), atrial septum (AS), and the His bundle (HB). We stained preparations with di-4-ANEPPS and recorded voltage-sensitive fluorescent signals at a rate of 1500 frames/sec using a 16x16 photodiode array as previously described.12 For details, see the expanded Materials and Methods section in the online data supplement available at http://www.circresaha.org. All quantitative data are expressed as mean±SD.
Antibodies
Various primary antibodies were used: (1) monoclonal anti-NF160 raised in mouse (Chemicon, USA); (2) monoclonal anti-Cx43 raised in mouse (Chemicon); (3) polyclonal anti-Cx43 raised in rabbit (Sigma, USA); (4) polyclonal anti-Cx45 raised in guinea-pig Q14 (GP42)25; (5) polyclonal anti-Cx40 raised in guinea-pig V15K (GP318)25; and (6) polyclonal anti-HCN4 raised in rabbit (Alomone Labs, Jerusalem, Israel). Anti-Cx45 and anti-Cx40 were obtained from N.J. Severs and S.R. Coppen (Imperial College London, UK).
Histology and Immunohistochemistry
After fluorescent imaging, AVN preparations from four rabbits were cryosectioned. Five adjacent serial sections (20 µm thick) were cut at 1-mm intervals as indicated in Figure 1C by the vertical cyan lines. One of the five sections at each 1-mm interval was stained with a modified Masson’s trichrome technique. Sections through the leading pacemaker site, CN and HB (marked by thick vertical cyan lines in Figure 1C) were used for immunohistochemistry as previously described.25 See expanded Materials and Methods for details.
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Specificity of the Immunolabeling
With all primary antibodies, it was checked that single labeling produced the same pattern of labeling as double labeling. Negative control experiments included omission of either primary or secondary antibodies. In all negative control experiments, there was no signal produced above background fluorescence. The specificity of the Chemicon anti-Cx43, the anti-Cx45 antiserum Q14 (GP42), and the anti-Cx40 antiserum V15K (GP318) has been proven previously.26,27 In the rabbit heart, NF160 is only expressed in the cells of the pacemaker and conduction system20–22; the same pattern of expression was observed in the present study with the anti-NF160 primary antibody used. Western blot of HCN4 in rat brain (Alomone data sheet) and HEK 293 cells heterologously expressing HCN428 shows a single band of the correct molecular weight. As a further test of the anti-HCN4 antibody, we labeled for HCN4 sections through the rat AVN.
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Pacemaker Activity of the AVN Originates Posterior to CN and Is Sensitive to Block of If by Cs+
After excision of the SAN, preparations showed AV junctional rhythm. In three preparations, the cycle length changed from 334±32 to 627±111 ms after excision of the SAN. Figure 1A shows a photograph of a typical preparation. The magenta box and the black grid shows the 16x16 photodiode array mapping area. The blue oval marks the location of the junctional pacemaker—note that it was immediately above the tricuspid valve and was posterior to CN (recording site No. 3). Figure 1B presents optical recordings from seven different points of the mapped area (black boxes in Figure 1A) showing the initiation and conduction of the junctional beat. Electrograms recorded at HB, AS, and CrT (shown in Figure 1B) follow the characteristic sequence of activation corresponding to junctional rhythm. HB signal appeared first, despite the fact that the pacemaker was located closer to CrT electrode near the coronary sinus orifice (see Figure 1A). This is because the excitation initially traveled anteriorly along a "channel" toward CN. In terms of its position, the channel corresponds to the posterior nodal extension (PNE)29–31 or slow pathway into CN. At CN, one wavelet went to HB while another broke through to the atrium via the fast pathway. This activation sequence is better shown by the activation maps before (left) and after (right) the excitation breakthrough to the atrium in panels C and D of Figure 1. The online data supplement provides an animation of excitation in this preparation (online Movie 1, available at http://www.circresaha.org) as well as optical recordings and a movie of anterograde conduction (online Figure 1 and online Movie 2). In the majority of the cases (11 of 14 preparations), the junctional rhythm originated in the PNE, posterior to CN as shown in Figure 1. In 4 out of 14 preparations, the junctional rhythm originated in the N/NH region rather than the PNE (see online Movie 3). Figure 2A shows an activation map and an optical signal recorded at the PNE pacemaker site, when there was a block of retrograde conduction through both slow and fast pathway exits (due to the long duration of the experiment and overstaining with the dye). It shows typical pacemaker action potentials without overlapping atrial excitation. If is known to play an important role in cardiac pacemaking,32,33 and has been observed before in cells isolated from the AVN.34,35 In six preparations, 2 mmol/L Cs+ was used to block If (1 to 2 mmol/L Cs+ is considered to be a selective blocker of If).36 Cs+ greatly slowed pacemaker activity originating from the PNE (Figure 2B shows one example): the cycle length was increased on average from 611±84 to 949±120 ms (n=6, P<0.001).
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Pacemaker Activity Originates in a Group of Small Nodal-Like Cells, Which Predominantly Do Not Express Cx43, but Do Express NF160 and Cx45 and a Lower Amount of Cx40
In four preparations, after fluorescent imaging and identification of the junctional pacemaker site (in the PNE in each case), preparations were cryosectioned. Data from one of the four preparations are shown here, but similar data were obtained from the other preparations. Sections through the junctional pacemaker site (recording site No. 1 in Figure 1A) and, for comparison, through CN (recording site No. 3 in Figure 1A) and HB (recording sites No. 5 to 6 in Figure 1A) were stained by a modified Masson’s trichrome technique to view the anatomy of the different regions. Figures 3A, 4A, and 5
A show examples of the three regions. At HB, a crescent of HB cells (lightly stained purple-red) was situated above the ventricular muscle (stained red) and below fibrous tissue (stained blue) (Figure 3A). At CN, an oval of compact node cells was situated next to the central fibrous body (Figure 4A). At the junctional pacemaker site, there was a cluster of loosely packed morphologically nodal-like cells (separated by connective/fatty tissue; Figure 5A); the cluster of cells was situated next to the ventricular septum, but separated from it by a substantial layer of connective/fatty tissue (Figure 5A). To identify the cell type at the pacemaker site, the sections were immunolabeled for NF160, Cx43, Cx45, and Cx40; it is well known that NF160 is a marker of the pacemaker and conduction system in the rabbit, 20–22 and the SAN and ventricular conduction system expresses a different complement of connexins to the working myocardium.25,37–41 Figures 3 through 5 show the expression of NF160 and Cx43. NF160 was not expressed in the working myocardium (Figures 3B, 4B, 4E, and 5B), but it was expressed in HB and compact node (see Figures 3B and 4 B); the labeling was present in the cytosol (see yellow arrows in Figures 3D and 4D). Cx43 was expressed in the working myocardium at intercalated discs (see white arrow in Figure 3E). Cx43 was also expressed in HB; labeling was characteristically spotty (Figure 3D). In contrast, Cx43 was largely absent from CN (Figure 4C and 4F), although it could be present on the margins of CN (Figure 4C and 4G). Figure 6 shows expression of Cx45 (the tissues were double labeled for Cx43 and Cx45), and it shows that whereas Cx45 was absent from the ventricular muscle (Figure 6A, VS), a patchy and low level of expression of Cx45 was found in some regions of AS (Figure 6D). Cx45 was also present in HB (Figure 6C). Cx45 was much more abundant in CN, in which Cx43 was predominantly absent (Figure 6B). Online Figures 2 and 3 show expression of Cx40 (the tissues were double labeled for Cx43 and Cx40). Cx40 was absent in the ventricular muscle (online Figure 2C, VS), possibly present (above background staining) in the atrial muscle (online Figure 3A), abundant in HB (online Figure 2C, HBB), and present at a low density in CN (online Figure 2B).
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At the junctional pacemaker site, the cells showed the same pattern of expression as in CN: NF160 was expressed at high density (Figure 5B) in the cytosol (Figure 5D), Cx43 was predominantly not expressed (Figures 5C and 5E), Cx45 was highly abundant (Figure 6A) and Cx40 was present in a lower amount (online Figure 2A). It is concluded that the cells at the leading pacemaker site are nodal-like, and this is consistent with the cells comprising the PNE.
Cells at the Junctional Pacemaker Site Express the If Channel Protein, HCN4
The suppression of pacemaker activity by 2 mmol/L Cs+ (Figure 2B) suggests that If plays an important role in the pacemaker activity in the AV junctional area. In murine as well as in rabbit SAN, HCN4 has been reported to be the main HCN channel protein responsible for If.42–44 Figure 7 shows examples of labeling of HCN4 and Cx43 in one preparation (similar results were obtained from another three preparations). No or little labeling (above background fluorescence) of HCN4 was detected in atrial (Figure 7D) and ventricular muscle (Figure 7A, VS), whereas Cx43 was expressed in the atrial and ventricular muscle (eg, Figure 7D). HCN4 labeling above background fluorescence was detected in HB (Figure 7C). High HCN4 expression was observed in CN (Figure 7B). Once again, the pattern of expression at the junctional pacemaker site was the same as in CN, and HCN4 was highly abundant (Figure 7A); this is consistent with the results from the fluorescent imaging. In CN and at the junctional pacemaker site, HCN4 labeling was located around the cell membrane and in the cytosol (Figures 7A through 7C). This is not surprising because another If channel protein, HCN1, has been observed before to be expressed on the cell membrane and in the cytosol in single rabbit SAN cells.45 To check the HCN4 labeling observed in rabbit, sections through rat AVN were immunolabeled for HCN4: online Figure 4 shows that, as expected, there was bright labeling of AVN tissue, but there was no signal above background detected in the working myocardium.
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Figure 8 shows a schematic diagram of the AV junction and summarizes the labeling of NF160, Cx43, Cx45, Cx40, and HCN4 at the junctional pacemaker site (PNE) and in CN and HB.
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Position of the Leading Pacemaker Site
We found that the junctional pacemaker site in the majority of cases was located not in CN or nodal-His area as suggested earlier,6,7 but in the area posterior to CN between the coronary sinus and the tricuspid valve. It is well known that the leaflets of the tricuspid valve show pacemaker activity.46,47 However, in the present study, the pacemaker site was always located near the coronary sinus, whereas the valve leaflets extended along the whole width of the preparation (see Figure 1). Earlier work showed that the coronary sinus can show rhythmic activity.48 However, in the present study, the pacemaker site corresponded to AVN-like cells. During premature stimulation and reentry, conduction occurs along the slow pathway12 (PNE), and in the present study, the junctional pacemaker site corresponded to this pathway in most preparations (Figure 1C; also compare online Movies 1 and 2). This suggests that the junctional pacemaker site is a part of the PNE. This conclusion is supported by the histology: Masson’s trichrome showed that the junctional pacemaker site corresponded to a cluster of small nodal-like cells extending from CN toward the coronary sinus along the tricuspid valve. These are the anatomical characteristics of the PNE as described by others.29–31,49 In the rabbit, NF160 is known to be expressed in other parts of the conduction system.20–22 In the present study, we observed immunolabeling of NF160 at the junctional pacemaker site (as well as in CN and HB) and this is additional evidence that the junctional pacemaker site is a part of the PNE. At the junctional pacemaker site, Cx43 was largely not expressed, whereas Cx45 was expressed and this is the same pattern of expression as in CN (Figure 6); this is further evidence. As found in this study, Coppen and Severs37 reported that all three connexins (Cx43, Cx45, and Cx40) are expressed in variable amounts in the rabbit compact node. In other species,39,41 all three connexins (Cx43, Cx45, and Cx40) are also expressed in variable amounts in the AVN and HB branches. The slowing of pacemaker activity by Cs+ (Figure 2B) showed that If is important in pacemaking, and at the junctional pacemaker site, only the nodal-like cells expressed the If channel protein, HCN4. In expressing HCN4, the cells of the junctional pacemaker site are like the cells of CN, and this is confirmation that the junctional pacemaker site is an integral part of the PNE and the conduction system of the heart.
We observed a different pacemaker location in 4 of 14 preparations. In two cases, the rhythm was initiated in the area of HB near the edge of the preparation. One of these two preparations exhibited initially PNE rhythm only, then PNE and HB pacemakers fired concurrently, and later HB pacemaker prevailed. We cannot reject the possibility that HB pacemaker activity was related to injury during dissection. In the two other cases, the first detectable excitation was in the area of CN, although we cannot reject the possibility that a high noise level did not allow us to detect a signal from the PNE, which always was significantly lower in amplitude (see Figure 1).
If in the AVN
If has been recorded from ovoid and rod-shaped cells isolated from the rabbit AVN.34,35,50,51 Hancox and Levi35 reported that 80% to 90% of isolated rabbit AVN cells do not express If. However, Habuchi et al34 and Munk et al51 stated that 90% of isolated rabbit AVN cells do express If, and this suggests that If does play an important role in AV junctional pacemaking. It is possible that there are regional differences in If in the AV junctional region: Munk et al51 found If to be approximately 25 times greater in ovoid cells (possibly from CN) than in rod-shaped cells (possibly AN or NH cells). Habuchi et al34 showed that in rabbit the density of If is greater in SAN cells than in AVN cells, and this suggests that If is more important in the SAN. However, the present study suggests that the role of If may be more important in the PNE than in the SAN: in the rabbit SAN, 2 mmol/L Cs+ slows pacemaker activity by 
12%36 (whereas it caused a 36% slowing in the present study). In two preparations with a non-PNE pacemaker, Cs+ increased the pacemaker cycle length from 545 to 774 ms (42% increase) and from 592 to 798 ms (35% increase). The response developed more slowly than for PNE pacemakers: the half-response time was 20 to 30 minutes. Block of If did not stop pacemaker activity in any preparation and this demonstrates that, as in the SAN, currents other than If are also involved in pacemaker activity. In murine and rabbit SAN, HCN4 has been reported to be the main HCN protein responsible for the If channel.42–44 Altomare et al28 reported that HCN1 may also contribute to the If channel in the rabbit SAN. However, the abundance of mRNA for HCN channel isoforms (including HCN1) other than HCN4 is low compared with that for HCN4.43 In the present study, HCN4 (the main HCN channel protein) was abundantly expressed at the junctional pacemaker site. In the present study, anti-HCN1 (Alomone Labs) did not produce a signal above background fluorescence (unpublished data).
Nature of the Slow Pathway
The PNE is not only responsible for junctional rhythm (this study), it is also the slow pathway for conduction into CN, and as such, it plays an important role in AV node reentry.12 This study has shown for the first time that the slow pathway expresses NF160 (confirming that the tissue is similar to that of CN). This study has also shown that the slow pathway expresses Cx45, and a low amount of Cx40, but predominately it does not express Cx43 (Figure 8), which is present at high densities in adjacent bundles and tissue layers of the triangle of Koch.12 The expression of Cx45, but not Cx43, could help explain the low conduction velocity of the pathway. The lack of Na+ channels in the AVN may also contribute to the slow conduction. However, it is unlikely to be as important as connexin expression, because a reduction of Na+ conductance can produce only a three-fold reduction in conduction velocity, whereas a decrease in intercellular coupling can result in 100-fold reduction.16 In addition, a decrease in intercellular coupling leads to a paradoxical improvement of the safety of conduction.52 In a previous study, we described the presence of horizontally orientated bundles of Cx43-expressing cells.12 Such bundles were also observed in this study. However, these bundles express Cx43 and not NF160 (online Figure 5), and they are distinct from the Cx43-negative tract described in this study: they run parallel to the Cx43-negative tract but superior to it (eg, see Figure 5A and online Figure 5A). Based on location, the Cx43-negative tract, rather than bundles of atrial-like Cx43-expressing cells, probably comprises the slow pathway and, therefore, constitutes the PNE. However, it is still possible that the bundles of Cx43-expressing cells may play a role in AV node reentry,12 because a significant difference in the conduction velocity of the two adjacent bundles could be responsible for longitudinal dissociation and reentry.
Limitations
It is possible that the prominence of the PNE pacemaker in our preparation was related to dissection trauma or exposure to BDM and di-4-ANNEPS. Although this is unlikely due to the normal electrophysiological parameters of the preparation only future in vivo studies can finally resolve the issue. The AV junction of the rabbit differs from that of the human in the location of CN53 and the extent of the PNE.54 The PNE is more pronounced in the rabbit than in the human,54 although the length of the PNE in the human increases with age.30 It remains to be determined whether the PNE in the human is the origin of pacemaker activity as in the rabbit.
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Acknowledgments |
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Acknowledgments
This work was supported by the NIH (HL58808 to I.R.E.) and the British Heart Foundation (RG/2001/009 to M.R.B.). We would like to thank Minh Lam for his help with confocal imaging.
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Footnotes |
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*Both authors contributed equally to this study.
Original received August 5, 2003; resubmission received September 30, 2003; accepted October 7, 2003.
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References |
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