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(Circulation Research. 2003;92:469.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Cx43 and Dual-Pathway Electrophysiology of the Atrioventricular Node and Atrioventricular Nodal Reentry

Vladimir P. Nikolski, Sandra A. Jones, Matthew K. Lancaster, Mark R. Boyett, Igor R. Efimov

From the Department of Biomedical Engineering (V.P.N., I.R.E.), Case Western Reserve University, Cleveland, Ohio; and the School of Biomedical Sciences (S.A.J., M.K.L., M.R.B.), University of Leeds, Leeds, UK.

Correspondence to Igor R. Efimov, PhD, Cardiac Bioelectricity Research and Training Center, Dept of Biomedical Engineering, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106-7207. E-mail ire{at}po.cwru.edu

	Abstract

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Fluorescent imaging has revealed that posterior nodal extensions provide the anatomical substrate for the dual-pathway electrophysiology of the atrioventricular (AV) node during normal conduction and reentry. The reentry can be intranodal, or as well as the posterior nodal extensions, it can involve an endocardial layer of atrial/atrial-nodal (A/AN) cells as part of the AV nodal reentry (AVNR) circuit. Using fluorescent imaging with a voltage-sensitive dye and immunolabeling of Cx43, we mapped the electrical activity and structural substrate in 3 types of AVNR induced by premature atrial stimulation in 8 rabbit hearts. In 6 cases, the AVNR pathway involved (1) a fast pathway (FP), (2) the A/AN layer, and (3) a slow pathway (SP). In 4 cases, reentry took the path (1) SP, (2) A/AN layer, and (3) FP. In 2 cases, reentry was intranodal, propagating between the 2 posterior nodal extensions. Immunolabeling revealed that the FP and SP are formed by Cx43-expressing bundles surrounded by tissue without Cx43. Cx43-expressing posterior nodal extensions are the substrate of AVNR during both intranodal and extranodal reentry.

Key Words: ablation • electrophysiology • arrhythmia • imaging

	Introduction

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Despite a long history of research^1,2 and a wealth of electrophysiological observations³ on atrioventricular (AV) nodal reentrant tachycardia (AVNRT) from both clinical and basic investigations, the anatomical correlates of AVNRT reentry circuit have yet to be agreed on. Two main controversies remain: (1) the anatomical substrate of the dual-pathway electrophysiology underlying AVNRT, and (2) whether AVNRT is exclusively an intranodal phenomenon or whether it involves part(s) of the atrium. Recent advances in understanding the molecular basis of cardiac arrhythmias have added a new dimension to these controversies: is there a molecular substrate, such as variations of the protein expression pattern within the triangle of Koch, which might contribute to AVNRT?

Advances in the radiofrequency therapy of this arrhythmia with preservation of AV conduction^4,5 support the concept of reentry involving not only the compact AV node but also atrial tissue⁶ and nodal extensions⁷ or bundles,⁸ which could provide the anatomical substrate for the fast and slow pathway—FP and SP, respectively.^9,10 The anatomical site of the SP, that is clinically localized using anatomical criteria, shows characteristic signature of slow potentials^4,5 identified during programmed stimulation or sinus rhythm. Inoue and Becker⁷ proposed an anatomical structure that could be the substrate for the SP. Inoue et al¹¹ presented evidence that successful clinical radio frequency ablation of AVNRT interrupted an inferior extension of the compact AV node. Similar anatomical structures were identified in canine⁸ and rabbit⁹ hearts. Several groups suggested that more than two AV nodal inputs/pathways could exist. Scherlag et al¹² concluded "that the persistence of AV conduction, albeit modified, after FP and SP ablation, suggests the existence of multiple AV nodal inputs, whereas retrograde conduction relies mainly on dual exits from the AV node to the atria" (page 753). The controversy remains whether involvement of atrial tissue well outside the specialized nodal area is essential for the reentry. Loh et al¹³ found that the reentrant pathway during ventricular echoes in dog is confined to the AV node, and the tissue that connects the node to the endocardial exit sites is excluded from the reentrant circuit responsible for single echoes. Also disagreement exists whether the fast pathway arises as a separate branch of the posterior nodal extension,⁸ or originates from the compact AV node.^7,14

The objective of this study was to extend our previous study¹⁰ that visualized reentry pathways during retrograde excitation (ventricular echo) to reentry produced by more physiological anterograde programmed stimulation. We aimed to identify the role and anatomical localization of the FP in AV nodal reentry. We also hypothesized that specialized conduction tracks of the two pathways identified with fluorescent imaging may have a molecular basis. Specifically, heterogeneity of expression of connexins¹⁵ could spatially correlate with reentrant pathways identified using fluorescent imaging. We applied immunohistochemistry techniques to determine the possible role of the major cardiac connexin isoform, Cx43, in providing the substrate for AVNRT.

	Materials and Methods

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This study conformed to the guidelines of the American Heart Association. We used isolated AV node preparations from 12 New Zealand White rabbits (age 1 to 3 months, weight 1.3 to 1.8 kg, Harlan, Indianapolis, Ind). Preparations were superfused with 37°C Tyrode’s solution containing 15 mmol/L 2,3-butanedione monoxime.^10,16,17 Preparations were paced with two leads of a quadruple electrode at the crista terminalis or the interatrial septum. A test premature stimulus (S2) was applied after 20 basic 300 ms (S1) beats at progressively shorter coupling intervals (S1-S2=300 to 50 ms). Coupling intervals below 110 ms were varied with a step of 5 ms. Another two leads of the electrodes at the crista terminalis and interatrial septum and the bipolar electrode at the bundle of His (His) were used to monitor excitation at the triangle of Koch boundaries and to construct conduction curves. A floating bipolar electrode was used to map electrical activity in the area of the compact AV node.

To stain preparations, we added to the perfusate di-4-ANEPPS at a final concentration of 1 µmol/L for 40 minutes as previously described.¹⁰ Optical fluorescent signals (F) were recorded from a 5x5- to 8x8-mm area at the triangle of Koch at a rate of 1894 frames/second using a 16x16 photodiode array. The signals were low-pass filtered at 50 Hz, differentiated (dF/dt), normalized by the basic beat recordings, plotted as 2-dimensional intensity graphs, and overlapped as frames with the image of the preparation to produce animations. Wavefronts of activation were visualized in these animations in order to identify the anatomical location of the reentry circuit.¹⁰ The centers of wavefronts propagating along the SP and FP were identified for reconstruction of trajectories of impulse conduction in 3-dimensional (3-D) stack plots.¹⁰ IDL software (Research Systems) was used to visualize these 3-D trajectories. Stack plots allowed visualization of the direction and speed of excitation in the triangle of Koch as spatiotemporal patterns. Each preparation was digitally photographed and fields of view were identified with an accuracy of 0.2 mm. All quantitative data are expressed as mean±SD.

As shown in our previous article,¹⁸ optical action potential contains signal from both the surface of the preparation and from deeper layers of the triangle of Koch such as the compact node. More recently,¹⁰ we extended this finding to the entire triangle of Koch, demonstrating multiple components of optical signals during AV nodal conduction and during AV nodal reentry. Results from various groups of investigators^19–21 characterized the depth of the penetration during optical mapping technique. The depth estimate ranges from 300 µm to 2 mm from the surface.

Immunolabeling of connexin43 (Cx43, a gap junction protein) was performed as previously described.²² A commercially available monoclonal antibody (raised against residues 252 to 270 of rat Cx43; MAB3068; Chemicon) was used at 1:500 dilution, and FITC-conjugated rabbit anti-mouse IgG secondary antibody (DACO A/S, Denmark) was used at 1:500 dilution. Frozen sections (20 µm thick, 100 µm between sections) were cut from frozen AV node preparations embedded in Tissue-Tek OCT compound. Sections were taken perpendicular to the endocardium and to the main axis of the triangle of Koch, which is usually parallel to the slow pathway. Thus, the transmural sections were taken perpendicular to the slow pathway. The sections were mounted on poly-L-lysine-coated glass slides, which were then stored at -65°C until use. The sections were fixed by immersing the slides in 4% formaldehyde at room temperature for 20 minutes and then washed 3 times for 10 minutes with PBS. Permeabilization was done by incubation of the slides with 0.1% Triton X-100 in PBS for 20 minutes. Blocking was carried out for 1 hour with 25% horse serum in PBS before incubation with primary antibody (diluted in 1% bovine serum albumin (BSA) with 10% horse serum in PBS) for 12 hours at +4°C. After washing, four times with PBS for 15 minutes, the sections were incubated with secondary antibody (diluted in 1% BSA with 10% horse serum in PBS) for 1 hour. Finally, the slides were washed 4 times with PBS for 15 minutes, mounted with Vectashield mounting medium (Vector Laboratories, Inc), and the coverslips were sealed with nail polish. Immunolabeling of Cx43 was examined by confocal laser scanning microscopy using a Zeiss LSM 510 instrument and by epi-fluorescent microscopy using a Nikon E600FN fluorescence microscope equipped with a B2-A filter cube (excitation 450 to 490 nm; dichroic mirror 505 nm; emission >520 nm). Specificity of the primary antibody binding was documented in a rabbit atrium previously.²² The control for the level of background fluorescence signal due to nonspecific secondary antibody binding was tested by excluding primary antibody from staining procedure, and it was less then the Cx43 signal from Cx43-negative structures (valves, fat, central fibrous body) after full staining protocol.

An expanded Materials and Methods section can be found in the online data supplement (online Figures 1 through 3 and 7 and online Movie 1) available at http://www.circresaha.org that provides further details regarding the preparations, protocols, electrode positions, optical mapping, dF/dt animation techniques, and 3-D stack plot visualization.¹⁰

	Results

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Using fluorescent imaging with voltage-sensitive dye, we observed 3 types of excitation pathways during AVN reentry induced by premature atrial stimulation. Twelve rabbit hearts were studied. However, in 4 hearts, reentry was not inducible at any premature interval. In 6 hearts, we observed slow-fast reentry: during the premature beat the excitation propagated anterogradely along the SP toward the AV node, exited retrogradely through the FP to a surface layer of atrial/atrial-nodal (A/AN) cells, and then reentered the SP. In 4 hearts, we observed fast-slow reentry: excitation engaged the AV node anterogradely via the FP, then went retrogradely via the SP, excited the A/AN layer, and finally reentered the FP. In 2 hearts among these 2 groups, both types of reentry were observed: slow-fast and fast-slow. In addition, in 2 hearts from the reentrant group, we observed intranodal reentry. In these cases, reentrant excitation excited the His bundle without exciting the A/AN layer and atria.

Figure 1 shows an example of the electrical recording at high and low crista terminalis sites (hiCrT, loCrT), interatrial septum (IAS), His bundle (His), and AV nodal area (AVN) for the 3 types of reentry observed: slow-fast, fast-slow, and intranodal. Application of stimuli is clearly seen as prominent artifact spikes on a sensing pair of leads in quadruple electrodes when stimuli were delivered through the other pair of leads. "S1" and "S2" mark the last basic and premature stimuli and show which electrode was used for pacing. For types 1 and 2 reentry, all reentry responses in His bundle electrograms (marked as H3) were preceded by an atrial excitation as one can see from IAS, LoCri, and HiCri recordings. For type 3 reentry, the last reentrant excitation of the His bundle (marked as H4) has no "parent" atrial activation. Conduction curves for all preparations studied are shown in Figure 1C. Premature stimuli resulting in reentry are marked with circles. There was no obvious relationship between the shape of the conduction curve and occurrence of reentry. Online Figures 4 through 6 and online Movies 2 through 4 in the online data supplement provide dF/dt animations, which dynamically show the conduction pathways in these examples.

View larger version (27K): [in this window] [in a new window]   Figure 1. Bipolar electrograms recorded during the 3 types of AV nodal reentry and anterograde conduction curves. A, Bipolar recordings with electrodes, which positions are marked on the preparation schematic in B with black dots. loCrT and hiCrT indicate low and high crista terminalis areas; IAS, interatrial septum; His, bundle of His; and AVN, compact AV node area. Anatomical landmarks: CS indicates coronary sinus; tT, tendon of Todaro; TrV, tricuspid valve; FO, fossa ovalis; IVC, inferior vena cava. S1 and S2 indicates last basic and premature stimuli; H1 and H2, His signals caused by basic and premature stimuli; H3 and H4, His signals caused by reentry. Recordings marked with S1 and S2 correspond to the pacing artifact recorded from 2 sensing leads of a quadruple electrode when the 2 other leads are used for pacing. Preparation with slow/fast type reentry was paced from an electrode at the high crista terminalis area with basic/premature stimulation intervals of 300/110 ms. Fast/slow and intranodal reentry were induced during pacing from the septum electrode with basic/premature stimulation intervals of 300/110 ms and 300/100 ms. C, Conduction curves measured during programmed atrial pacing in the 12 preparations studied. Reentry occurrences are marked with circles. Solid black lines correspond to reentry-inducible preparations; dashed conduction curves, the 4 noninducible preparations.

Figure 2 illustrates the our technique of 3-D stack plots of dF/dt used for the visualization of conduction pathways during the slow-fast pathway reentry from Figure 1 in comparison with the traditional representation by isochronal activation maps and overlapping signal traces from the whole reentrant circuit.

View larger version (56K): [in this window] [in a new window]   Figure 2. Comparison of the AV nodal reentry visualization by 3-D stack plot of optical signal derivatives vs traditional methods of representation of conduction. Isochronal activation maps (top) show the activation sequence for the conduction during slow-fast pathway reentry. Preparation schematic and electrode recording abbreviations are described in Figure 1. Middle panels present the reconstruction of conduction by 3-D stack plot of optical signal derivatives and graph of overlapping signal traces from the whole reentrant circuit. Signals were low path filtered at 50 Hz.

Figure 3 shows the preparation schematics with the trajectories of impulse conduction and 3-D stack plots of dF/dt corresponding to the 3 types of reentry shown at the Figure 1: slow-fast, fast-slow, and intranodal. Stack plots are shown in projection along the main-axis of the triangle of Koch and time. From this 3-D projection, the rapid atrial excitation that follows S1 stimuli is seen as almost a horizontal slab, whereas conduction through nodal structures to His bundle is presented as a sloped filament. During slow-fast pathway reentry initiation after S2 stimulus, there was conduction block in the A/AN layer, so the horizontal slab representing atrial excitation is missing (see the left 3-D stack plot). Only the 3-D filament corresponding to the slow-pathway conduction is present. The slope of the filament reflects the conduction velocity: the steeper the slope, the slower the propagation. The conduction velocity in the A/AN layer within the triangle of Koch was 35.1±17.2 cm/s after S1 stimuli and 30.7±14.0 cm/s after S2 stimuli (n=6, P>0.5). We were unable to accurately measure conduction velocity in the AV node and the posterior nodal extension during basic beat propagation due to superimposition of optical signals from the AV node and the A/AN layer. During premature stimulation and reentry, the conduction velocity in the AV node and the posterior nodal extension (the SP) was slower and permitted measurements. Premature beat H2 resulted from an impulse propagating at 7.9±3.4 cm/s. The reentrant beat propagated at 6.9±2.4 cm/s (n=6, P>0.5).

View larger version (61K): [in this window] [in a new window]   Figure 3. Visualization of AV nodal reentry. Reentry trajectories obtained for the cases presented in Figure 2 together with space-time stack plots of the dual-pathway AVN conduction are shown. Data were collected from a square field of view containing the triangle of Koch as shown on the schematics. Three-dimensional volumes were built by stacking the sequentially recorded 2-dimensional plots of dF/dt.10 An isosurface was built using a density threshold, which was adjusted with time in order to preserve the continuity of conduction along the pathways. 3-D stack plot is visualized in a projection perpendicular to the preparation plane so that horizontal axis of the stack plot corresponds to the horizontal axis of the preparation schematic and vertical axis corresponds to time that increases from top to bottom. Details of the technique are presented in online Figure 7. Arrows S1 and S2 show the times of the last basic and premature stimuli. H1, H2, H3, and H4 show the times of His signal appearances (see Figure 2). Quick A/AN layer activation corresponds to almost horizontal areas on the plot. Solid lines represent conduction trajectory for basic and premature pacing. Dashed lines show reentry pathways. Bold arrows on schematics and 3-D stack plots indicate quick transmittance of the activation between dual pathway inputs through A/AN layer. SP and FP indicate slow and fast pathways.

Figure 4 shows reentrant circuits during slow-fast reentry in 2 different preparations superimposed with endocardial projections of the stack plots. These 2 representative examples illustrate apparent anatomical variations of the FP during slow-fast reentry in different preparations. The right panels show that the conduction via FP splits off the SP clearly before the latter enters the AV node. This type of reentry circuit was observed in 3 preparations. The observed reentry circuit suggests that the posterior nodal extension bifurcates forming the anatomical substrate for the reentry circuit, which does not include the compact AV node itself. This observation is consistent with the morphological characterization of proximal and superior AV bundles in the canine AV node, presented by Racker and Kadish.⁸ The left panels illustrate findings from 5 remaining preparations, in which we were unable to separate the FP from the AV node. In 3 of them, the reentry circuit ran through the apex of the triangle of Koch apparently including the compact AV node. It remains to be determined whether this difference is due to the limited spatial resolution of our imaging techniques or to anatomical variation.

View larger version (75K): [in this window] [in a new window]   Figure 4. Activation maps of conduction during slow/fast AV nodal reentry in 2 preparations showing 2 different exit locations. Activation maps are superimposed with the preparation photographs. Top panels, Conduction before and during breakthrough (red color). Bottom panels, Subsequent quick engagement of the A/AN layer after breakthrough. SP and FP indicate slow and fast pathway.

In order to determine a possible link between expression of Cx43 and the 2 bundles forming the SP and the FP, we immunolabeled Cx43 and analyzed its distribution throughout the triangle of Koch. Figure 5 shows the results of Cx43 labeling of a preparation exhibiting fast-slow reentry. The photograph of the preparation is superimposed on the activation map during reentry. Only a part of the reentry circuit at the posterior nodal extensions is shown. Activation of the A/AN layer is not shown for the sake of simplicity. Green vertical lines indicate the position of the histological sections through the bifurcation of slow pathway into slow and fast pathway bundles (section 4), nodal area (section 6), and penetrating bundle (section 10). The positions for all 14 sections are shown. Color bars on a photograph indicate location of different structures circled with different colors on all 14 microphotographs at the left. As shown earlier,^15,23,24 the compact AV node is Cx43 negative, whereas the His bundle is Cx43 positive. We originally expected to see the posterior nodal extensions corresponding to the SP and the FP to be Cx43 negative. Paradoxically, the opposite was observed: a Cx43-positive bundle like structure was revealed in the fast-slow reentry circuit. Online Figures 8 and 9 show additional images of the immunolabeling of the preparation, and online Figure 10 shows additional activation maps.

View larger version (81K): [in this window] [in a new window]   Figure 5. Activation maps for conduction during fast/slow AV nodal reentry and immunolabeling of Cx43 in different areas of the triangle of Koch. Cx43 immunolabeling of sections at 400-µm intervals (for the positions marked on the preparation photograph; bottom left). Color bars on a photograph indicate location of different structures circled with different colors on all 14 sections at the left. Two Cx43-positive bundles (circled with magenta lines) can be seen in 3 sections 1.2 mm posterior to the compact AV node (circled with white lines) (Cx43-negative area) before they merged together (circled with cyan and magenta) approaching the crista terminalis. His bundle (circled with black lines) shows moderate Cx43 labeling. Photograph of the preparation is also superimposed with the activation map during reentry. Only a part of the reentry circuit at the posterior nodal extensions is shown. Activation of the A/AN layer is not shown for the sake of simplicity. Green vertical lines indicate the position of the histological sections through the bifurcation of slow pathway into slow and fast pathway bundles (section 4), nodal area (section 6), and penetrating bundle (section 10). RA and LA indicate right and left atria; RV and LV, right and left ventricle; VS, ventricular septum.

The immunohistochemical study demonstrates that our previous findings of bifurcation in functional reentry pathway has a molecular basis. We observed the bifurcation of Cx43-positive bundles at the point corresponding to the bifurcation of reentry pathway as determined by fluorescent imaging of AV nodal reentry. Figure 6, right, shows a stack plot visualizing the reentry circuit in the AV node area during the premature stimulation beat and the reentry beat. Cx43 labeling Figure 6, left, shows that the Cx43-positive bundles correspond to the optically detected activation pathways, corresponding to the FP (top) and the SP (bottom) bundles.

View larger version (39K): [in this window] [in a new window]   Figure 6. Correspondence between the stack plot of the optical potential derivatives at the triangle of Koch during premature stimulation and reentry and Cx43 labeling. Right, 3-D spatiotemporal visualization of conduction in the AV node area during premature stimulation and reentry (stack plot of first derivative of the optical potential signal is shown). Histological section on the left shows that Cx43-labeled bundles correspond to the optically detected activation pathways. SP and FP indicate slow and fast pathway bundles.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our study presents the first functional visualization, obtained with voltage-sensitive dye and optical imaging, of the entire reentry circuit induced by premature atrial stimulation in the rabbit heart. We have earlier presented the reentry pathway for the retrograde-induced ventricular echo phenomenon¹⁰; now we have extended our findings to the more clinically relevant anterogradely-induced AV nodal reentry. Our data are consistent with the electrode mapping data of AV nodal reentry from Billette’s group^9,25,26 and the optical imaging data of canine AV nodal reentry from Zipes group.⁶ Our results provide support for the dual-pathway electrophysiology theory by presenting images of the two AV nodal conduction pathways. However, unlike our previous data from retrograde stimulation,¹⁰ we did not observe jumps in the conduction curves associated with the dual-pathway electrophysiology.

AV nodal reentry was induced and mapped in 8 out of 12 rabbit preparations (67%). Four preparations (33%) were not inducible. We observed and characterized in rabbit preparations 3 types of reentry²⁷: slow-fast, fast-slow, and a posterior type of reentry that can be termed intranodal due to a lack of atrial participation in the reentry circuit. Fluorescent imaging revealed the entire trajectory of reentrant impulse propagation during these 3 types of AV nodal reentry in the rabbit heart, which appear to correlate with most commonly occurring clinical forms of AVNRT²⁷: (1) slow-fast reentry, clinically known as "common type,"²⁷ was observed in 6 preparations (50%). In this case, the reentry circuit included: slow anterograde conduction via the SP, retrograde conduction via the FP, and fast retrograde conduction via the endocardial A/AN layer. (2) Fast-slow reentry, clinically known as the "uncommon type,"²⁷ was observed in 4 preparations (33%), two of which were also exhibiting the slow-fast reentry. The fast-slow reentry circuit encompassed: antegrade conduction via the FP, retrograde conduction via the SP, and anterograde conduction via the endocardial A/AN layer. (3) Intranodal reentry, which may correspond to clinical "slow-slow" type or "posterior type,"²⁷ was observed in 2 preparations (17%), one of which also supported slow-fast reentry, and the other supported fast-slow reentry. During intranodal reentry, the reentry circuit was located within the posterior nodal extension(s). Activation of the His bundle was not accompanied by A/AN layer excitation.

Our data appear to agree with morphological data from the canine heart.⁸ The 2 bundles termed by Racker and Kadish⁸ as the proximal and the superior AV bundles correspond to the SP and FP determined functionally in our study. Our data are also consistent with the morphological characterization of the posterior nodal extension by Becker’s group,^7,28 which provides the substrate for the SP.⁹ Our data suggest that this structure is commonly present in the rabbit heart. Interestingly, the FP exhibits anatomical variability. We observed 2 types of reentry circuit during AV nodal reentry, which might reflect morphological differences in the triangle of Koch. Figure 4 shows 2 representative examples. In the type shown in the left panels of Figure 4, no specific FP was detected. Fast pathway exit was observed at the apex of the triangle of Koch. This type of reentry might be explained by the 2 nodal extensions originating in the AV node or only one posterior nodal extension, documented by Inoue and Becker.⁷ In the other type of AV nodal reentry, the conduction pathway bifurcated clearly before reaching the apex of the triangle of Koch (right panels in Figure 4). The latter case appears to agree with bifurcation of the proximal AV bundle into the superior AV bundle and the compact AV node as shown by Racker and Kadish.⁸ Interestingly, intranodal reentry was present only in preparations with the Racker-Kadish posterior bifurcation of the proximal AV bundle. The impulse was entrapped between the two bundles, located posterior to the compact AV node.

Morphological evidence^8,9,28 supports our functional characterization of the reentry circuit. However, morphology alone cannot explain experimentally observed complex multiphasic waveforms of electrograms^4,5,29 and optical signals^6,16,18,30 from the SP area and the AV node. Our data provide one of the possible molecular factors that could play a role in conduction heterogeneity. Immunolabeling of Cx43 in a preparation with bifurcated conduction pathways revealed in the triangle of Koch the presence of several Cx43-positive structures surrounded by Cx43-negative tissue: an endocardial A/AN layer and one or two deeper bundles. The Cx43-positive A/AN layer may provide the substrate for the rapidly propagating wide wavefront observed as the fast component in electrograms^4,5 or the first hump in optical signals^6,16,18,30 during anterograde conduction. The deeper Cx43-positive bundle(s) may provide the substrate for the SP and the FP. Furthermore, the structure of the Cx43-positive bundles appears to agree with the Racker-Kadish bifurcating AV bundle morphology.⁸

Mapping of connexins in the sinoatrial node has revealed a complex arrangement of different connexin isoforms.^22,31 In the atrial muscle surrounding the sinoatrial node, Cx43 alone is expressed; whereas in the center of the sinoatrial node, Cx43 is absent and instead Cx45 is expressed. However, in the periphery of the sinoatrial node at the interface between the two tissues, both Cx43 and Cx45 are expressed. Our data indicate that a similar degree of complexity in connexin expression may be present in the AV node and surrounding tissues. It is possible that the Cx43-positive posterior extensions of the AV node correspond to the Cx43-positive periphery of the sinoatrial node. In the sinoatrial node, the presence of Cx43 in the periphery may provide a gradient in electrical coupling from the atrial muscle (with good coupling) to the center (with poor coupling), a feature that is considered vital for the proper functioning of the sinoatrial node.³² A complex pattern of connexin expression may also play an important role in normal and abnormal AV conduction and may provide the substrate for AVNRT. Further studies are required to map other connexins isoforms and also ion channels and to elucidate their role in AV node conduction.

Limitations
In the present study, we evaluated the expression of Cx43 only. However, studies of Cx40 knockout mice have directly implicated this gap junction protein in AV nodal conduction.^33,34 It was also shown that Cx40 and Cx45 are the major connexins in the rabbit SA node,²² which suggests that both isoforms could play a role in AV conduction. Further studies are required to elucidate the role that these two low and high conductance gap junctions play in the critical reentrant pathways of the AV node.

To eliminate the movement artifact during optical recording, we had to use 2,3-butanedione monoxime, which could potentially affect AV node conduction and reentry inducibility. However, previous studies of AV nodal conduction in rabbit¹⁷ and canine heart,^13,35 provided no evidence of such effects on AV conduction.

	Acknowledgments

 
This project was supported by Grant HL58808 (I.R.E.) from the National Institute of Health, National Heart, Lung, and Blood Institute.

Received September 5, 2002; revision received January 22, 2003; accepted January 22, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
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