THE ANATOMICAL RECORD PART A 280A:952–965 (2004) Structure-Function Relationship in the AV Junction IGOR R. EFIMOV,1* VLADIMIR P. NIKOLSKI,1 FLORENCE ROTHENBERG,1 IAN D. GREENER,2 JUE LI,2 HALINA DOBRZYNSKI,2 AND MARK BOYETT2 1 Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 2 School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom ABSTRACT In the normal heart, the atrioventricular node (AVN) is part of the sole pathway between the atria and ventricles. Under normal physiological conditions, the AVN controls appropriate frequency-dependent delay of contractions. The AVN also plays an important role in pathology: it protects ventricles during atrial tachyarrhythmia, and during sinoatrial node failure an AV junctional pacemaker can drive the heart. Finally, the AV junction provides an anatomical substrate for reentry. Using ﬂuorescent imaging with voltagesensitive dyes and immunohistochemistry, we have investigated the structurefunction relationship of the AV junction during normal conduction, reentry, and junctional rhythm. We identiﬁed molecular and structural heterogeneity that provides a substrate for the dual-pathway AVN conduction. We observed heterogeneity of expression of three isoforms of connexins: Cx43, Cx45, and Cx40. We identiﬁed the site of origin of junctional rhythm at the posterior extension of the AV node in 79% (n ⫽ 14) of the studied hearts. This structure was similar to the compact AV node as determined by morphologic and molecular investigations. In particular, both the posterior extension and the compact node express the pacemaking channel HCN4 (responsible for the IF current) and neuroﬁlament 160. In the rabbit heart, AV junction conduction, reentrant arrhythmia, and spontaneous rhythm are governed by heterogeneity of expression of several isoforms of gap junctions and ion channels. Uniform neuroﬁlament expression suggests that AV nodal posterior extensions are an integral part of the cardiac pacemaking and conduction system. On the other hand, differential expression of Cx isoforms in this region provides an explanation of longitudinal dissociation, dual-pathway electrophysiology, and AV nodal reentrant arrhythmogenesis. © 2004 Wiley-Liss, Inc. Key words: atrio-ventricular junction; atrio-ventricular arrythmia; junctional rythm; optical mapping “The AV node is the ‘soul’ of the heart, and whoever understands its anatomy and electrophysiology will unlock the key to understanding the anatomic and electrical workings of the heart itself.” So Douglas Zipes characterized the role of the AV node in his introduction to a recent book, which provided the most comprehensive “view from the millennium” on the atrial-AV nodal electrophysiology (Mazgalev and Tchou, 2000). He also concluded, paraphrasing Winston Churchill’s famous characterization of closed Stalinist Soviet society, that despite a century of scientiﬁc inquiry by a constellation of the brightest minds working in cardiac electrophysiology, the AV node remains “a riddle wrapped in a mystery inside an enigma” © 2004 WILEY-LISS, INC. (Mazgalev and Tchou, 2000). Despite publication of this impressive volume, which summarized the results of a century of AV nodal research, we hope to convince the readers that recent progress in novel imaging modalities *Correspondence to: Igor R. Eﬁmov, Washington University, St. Louis, MO. Fax: 314-935-8377. E-mail: eﬁmov@wustl.edu Received 22 April 2004; Accepted 1 July 2004 DOI 10.1002/ar.a.20108 Published online 14 September 2004 in Wiley InterScience (www.interscience.wiley.com). STRUCTURE-FUNCTION RELATIONSHIP in just 2 years yielded results that shed a new light on many longstanding theories of AV conduction and offer a plethora of novel insights worthy of further exploration. These imaging modalities include fast ﬂuorescent imaging of electrical activity with voltage-sensitive dyes, structural optical coherence tomography imaging, and highresolution immunohistochemical imaging of protein expression. The aim of this review will be to place these new insights in context with the vast body of literature that already exists. Many of these ﬁndings have been made in the rabbit model. While important differences in anatomy do exist between the rabbit and human AV node, Mazgalev et al. (2001) described how ﬁndings in the rabbit should be correlated to human AV node from a clinical perspective. PARADOXES OF STRUCTURE-FUNCTION RELATIONSHIP OF AV JUNCTION Since the discovery of the AV node by Tawara (1906), this structure still mesmerizes new generations of investigators and has been at the center of highly emotional debates between anatomists and electrophysiologists. These debates focus on several major themes reﬂecting duality of the electrophysiological nature of the AV node, which is known to be both a conductor of impulses and an oscillator: When does the AV node conduct impulses (Lewis and Master, 1925) and when does it behave as an oscillator perhaps under electronic (Meijler and Janse, 1988) or mechanical (Scherf and Cohen, 1964) modulation from the atrial inputs? What are the anatomic (Inoue and Becker, 1998) and functional substrates of the AV nodal reentry, dual-pathway physiology (Moe et al., 1956), and concealed (Langendorf, 1948) AV conduction? A profound complexity of the AV junctional area has presented a signiﬁcant challenge to anatomists throughout the entire 20th century. Scientists have disagreed on several major deﬁnitions of the components of the AV junction. Janse et al. (2000) recently acknowledged the “lack of a common terminology for the tissues of AV junction” as one of the ﬁve points highlighted to explain the fact that there were arguments as to whether the atrium was involved in nodal reentry. Attempts have been made recently to clarify the terminology (Cosio et al., 1999). As is often the case, however, it will likely take many years before clinicians, anatomists, and electrophysiologists can agree on application of this new system. Among the debated issues have been posterior-anterior (Inoue and Becker, 1998) versus inferior-superior (Anderson and Ho, 2000) anatomical orientation of the AV conduction axis; anatomic location and morphologic deﬁnition of the compact AV node itself (Anderson and Ho, 2000); existence (Racker, 1989; Inoue and Becker, 1998; Medkour et al., 1998) or nonexistence (Anderson and Ho, 2000; Bharati, 2000) of specialized conductive bundles, ﬁbers, or approaches to the AV node; existence (Anderson and Ho, 2000) or nonexistence (Bharati, 2000) of the anatomically deﬁned triangle of Koch; and existence or nonexistence (Bharati, 2000) of an isthmus, which is usually targeted during ablation (Shah et al., 2002) of AV nodal reentry and does exist from an electrophysiological point of view. Due to the lack of consensus on such fundamental issues of anatomy of the AV junction, electrophysiologists have to choose between two distinctly different anatomic and potential approaches (Shah et al., 2002) during clinical evaluation of AV conduction and therapy of AV nodal reen- 953 trant tachycardia. The anatomic approach is based on targeting the anatomical landmarks of the slow pathway, such as the isthmus between the coronary sinus oriﬁce and the tricuspid valve. The potential approach is based on targeting a characteristic slow-frequency electric potential signature of the slow pathway (Haissaguerre et al., 1992; Jackman et al., 1992). Anatomic and electrophysiological (potential) therapeutic approaches reﬂect the structure and function of the AV junction. Yet, at the present time, due to an impossibility of simultaneous application of anatomic and electrophysiological approaches that have adequate resolution, the anatomic and electrophysiological approaches remain somewhat disconnected in their interpretation and conclusions. The most striking reﬂection of this disconnect was expressed by Meijler and Janse (1988), who concluded that “we are faced with a paradoxical situation that the most typical nodal action potential has not yet been linked to the most typical nodal cell.” Thus, despite a century of anatomic, electrophysiological, and clinical research, the AV node remains, according to Zipes, “a riddle wrapped in a mystery inside an enigma”. MORPHOLOGIC AND ELECTROPHYSIOLOGICAL HETEROGENEITY OF AV JUNCTION An unprecedented technological innovation of the 20th century allowed a signiﬁcant advancement of our understanding of the electrophysiological nature of AV conduction. Similar to the clinical arena, two major approaches of research inquiry coexisted in the basic cardiac physiology laboratories: anatomic (Tawara, 1906) and electrophysiological (Watanabe and Dreifus, 1965; Shah et al., 2002). Attempts to combine the two approaches have yielded limited results (Anderson et al., 1974). Both approaches provided a signiﬁcant body of evidence testifying to the profound morphologic and electrophysiological heterogeneity of the AV junction. The ﬁrst description of the AV node was given by Tawara (1906). He identiﬁed the knoten as the atrial portion of the bundle connecting the atria and ventricles. In this work, Tawara noted that the bundle become insulated as it passed from the atrial tissues into the central ﬁbrous body and suggested that the point of insulation be taken as the boundary between the knoten and the atrioventricular bundle of His, since it was difﬁcult in the human to make this distinction using histological criteria. When Anderson (1972a, 1972b) ﬁrst illustrated the histological heterogeneity in the rabbit node, he was unaware of the criterion proposed by Tawara (1906) and suggested that the node in the rabbit could be divided into anterior enclosed and posterior open regions, a concept subsequently endorsed by Petrecca and Shrier (2000). More recently, there have been proposals that anatomists should return to the original criterion suggested by Tawara (1906) when making correlations with electrophysiology (Anderson and Ho, 2000; Mazgalev et al., 2001). Other investigators, however, have proposed that the AV junction should be deﬁned to include all structures contributing to atrial-His interval and associated ratedependent and dual-pathway properties (Billette, 2002). In this review, we will use this physiological deﬁnition of Billette’s (2002) that deﬁnes the AV junction as a heterogeneous structure, consisting of the nodal extensions and 954 EFIMOV ET AL. Fig. 1. Anatomical location of the SA node, the AV junction, and the His bundle structures in the AVN as determined by expression of neuroﬁlament 160. SVC, superior vena cava; IVC, inferior vena cava; CS, coronary sinus; PNE, posterior nodal extension; AVN, atrioventricular node; His, bundle of His; TV, tricuspid valve; SAN, sinoatrial node. Areas expressing neuroﬁlament 160 are shown in green. approaches within the triangle of Koch, along with the heterogeneous component of the conduction axis in the rabbit that is enclosed within the ﬁbrous tissues of the central ﬁbrous body (Fig. 1). We will follow the zoological orientation terminology, anterior/posterior, as opposed to superior/inferior used by clinicians. It should be noted that the rabbit is the most common animal model to study AV conduction; despite the small size of the species, its coronary sinus is large due to the existence of left precava joining the coronary sinus (similar to the left superior vena cava sometimes encountered in humans). It is also worth noting that previous investigators showed that nodal extension goes all round the tricuspid valve oriﬁce, crossing over the bundle behind the aorta (Anderson, 1972a). An important work of Mazgalev et al. (2001) showed how studies in the rabbit could be correlated with clinical ﬁndings in the human. According to Bharati (2000), histologically the AV node consists of three layers: superﬁcial or subendocardial, intermediate or mid part, and the deep or innermost layer. This three-layered structure has been associated with three different electrophysiological responses observed in the AV junction. Based on the morphology of action poten- tials recorded with microelectrodes, the following three distinct regions of the AV node have been identiﬁed (Hoffman et al., 1958; Paes de Carvalho and de Almeida, 1960): the atrionodal (AN), true nodal or compact nodal (N), and nodo-His (NH). The N response was characterized by a relatively slow rate of rise during upstroke and a small amplitude of the action potential. AN and NH action potentials showed an intermediate morphology between the N region and the atrial muscle (AN) and the N region and His bundle (NH), respectively. It is generally believed that there is an association between the morphologically typical nodal cells and the electrophysiologically typical nodal cells (N cells). Yet convincing proof of this association is still lacking. Billette (1987) presented the most comprehensive microelectrode mapping of the AV node and identiﬁed six different cell types based on action potential morphology, excitability, and refractoriness. Most importantly, Billette’s (1987) data provide convincing evidence of the presence of various electrophysiological cell types throughout almost the entire triangle of Koch, which again emphasizes inseparable structure-function integrity of the entire AV junctional area, well beyond the compact AV node. Recently presented morphological (Inoue and Becker, 1998) and electrophysiological (Medkour et al., 1998; Nikolski and Eﬁmov, 2001) evidence of the posterior nodal extensions, which in some cases can reach coronary sinus and perhaps connect directly to the Crista terminalis, could conceivably explain the presence of the nodal and transitional cells at a signiﬁcant anatomical distance from the compact AV node. Yet purely morphologic analysis of the AV junction clearly has failed to explain the complex electrophysiological behavior of the AV node. Recent progress in molecular and cellular biology research provides highly promising insights, which could one day explain normal and pathological AV conduction. MERGING OF ANATOMIC AND ELECTROPHYSIOLOGICAL APPROACHES THROUGH IMAGING So far, the major obstacle in the investigation of the structure-function relationship of the AV node has been an inability to assess simultaneously a profoundly heterogeneous ion channel expression, morphological structure, and electrophysiological responses in 3D. The results of histology and immunohistochemistry are difﬁcult to correlate with the results of electrophysiological studies, usually conducted with a limited number of electrodes. Recent application of ﬂuorescent imaging with voltage-sensitive dyes to the research of AV conduction (Eﬁmov et al., 1997) has provided a missing link between anatomy and electrophysiology (Choi and Salama, 1998; Eﬁmov and Mazgalev, 1998; Nikolski and Eﬁmov, 2001; Wu et al., 2001). Fluorescent imaging with voltage-sensitive dyes has revolutionized cardiac electrophysiology research during the last decade (Rosenbaum and Jalife, 2002). This technology allows high-resolution mapping of transmembrane voltage at various spatial scales ranging from subsubcellular to the whole heart (Rosenbaum and Jalife, 2002). In the ﬁrst application of ﬂuorescent imaging to AV conduction (Eﬁmov et al., 1997), we discovered that optical signals collected during normal sinus rhythm at the distal AV node area contain two distinct components, which could hypothetically correspond to activation of two separate layers of the rabbit AV node, similar to human tril- STRUCTURE-FUNCTION RELATIONSHIP Fig. 2. Three-dimensional (multilayer) imaging of the conduction through the AV junction. A: The AV junctional preparation was paced at atrial septum with a cycle length S1S1 ⫽ 280 ms (top) and 274 ms (bottom), which caused Wenkebach AV block. Bipolar electrograms were recorded from Crista terminalis (CrT) and bundle of His. Optical traces (OAP) represent the superimposition of signals from the septum and the distal AV nodal areas. B: Masson trichrome-stained section through the distal AV node. Places of microelectrode recordings (MAPs) are marked with black and white circles. Modiﬁed with permissions from Eﬁmov and Mazgalev (1998) and Dobrzynski et al. (2003). aminar arrangement as described by the anatomists (Tawara, 1906; Inoue and Becker, 1998; Anderson and Ho, 2000; Bharati, 2000). Our subsequent study provided proof of this hypothesis (Eﬁmov and Mazgalev, 1998). Using simultaneous microelectrode and optical recordings, we demonstrated that the two components observed in ﬂuorescent optical recordings indeed correspond to transmembrane voltage action potentials recorded with microelectrodes from different layers: superﬁcial and middle layers (Bharati, 2000). The two layers have distinctly different morphological and electrophysiological properties. The superﬁcial layers are composed of transitional AN cells, which express sodium channels and Cx43, while the middle compact node layer does not express sodium channels and relies on low-conductance Cx45 for cell-tocell coupling. As a result, the two layers have markedly dissimilar action potential morphologies, which correspond to AN and N regions, respectively. There is also a large difference in the conduction velocity, ranging from 30 to 70 and 2 to 10 cm/sec, respectively. FLUORESCENT IMAGING OF MULTILAYERED CONDUCTION Precise three-dimensional anatomic reconstruction of electrical activity from ﬂuorescent signals remains to be developed. Rapidly evolving diffusive optical tomography (Arridge, 1999) and optical coherence tomography (Huang et al., 1991) could provide such methodologies in the near future. Meanwhile, our experiments with the AV node 955 Fig. 3. Typical (slow-fast) AV node reentry induced by premature stimulation of the Crista terminalis. A: Color map of conduction during reentry produced by a single premature impulse. Only ﬁrst 120-msec interval of reentry is shown, illustrating activation of the posterior nodal extension, the compact AV node, and the fast pathway. Map is superimposed with photograph of the preparation (see B for landmarks). Diamonds illustrate position of recording sites, optical signal from which are shown in C. B: Continuation of reentrant conduction: activation of the transitional AN layer in 20 msec. The same ﬁeld of view is shown. Notice signiﬁcant difference in activation times between the two panels. C: Optical action potentials recorded along the reentry pathway. Solid-line arrow illustrates conduction in the slow pathway and fast pathway. Dashed-line arrow illustrates rapid conduction in the superﬁcial transitional layer. Time scale bars below show time intervals illustrated in A and B as conduction maps. D: Conventional bipolar electrograms recorded during last basic stimulus (S1), premature stimulus (S2), and reentrant beat. Electrograms were recorded from high Crista terminalis (hiCrT), low Crista terminalis (loCrT), interatrial septum (IAS), and the bundle of His (His). Reproduced with permission from Nikolski and Eﬁmov (2001). provided a new estimate of the depth of penetration of ﬂuorescent techniques into the tissue. Previous assessments conducted by various groups suggested that optical signals carry information from 100 to 500 m of tissue from the surface (Rosenbaum and Jalife, 2002). Experiments in the multilayered AV node provided evidence that the depth could be as large as 1–2 mm (Eﬁmov et al., 1997; Choi and Salama, 1998; Eﬁmov and Mazgalev, 1998; Wu et al., 2001). Thus, multiphasic optical signal morphology is easily explained by a longitudinal dissociation of electrical activity in the proximal but highly heterogeneous layers of the AV junction, which was ﬁrst described by Mines (1913). Figure 2 illustrates an example of characteristic signature of longitudinal dissociation: biphasic signal morphology recorded optically during conduction in the rabbit AV junction (Nikolski and Eﬁmov, 2001). It shows an example of optical action potential (OAP) and microelectrode action potential (MAP) recordings and histology from the distal AV junctional area of the rabbit heart (Eﬁmov and Mazgalev, 1998; Dobrzynski et al., 2003). These recordings were conducted during Wenkebach periodicity: the ﬁrst and third beats documented in each panel of Figure 2A conducted from Crista terminalis (CrT) to the His bundle, while the second beat failed, as evident from the His 956 EFIMOV ET AL. bundle electrograms. Optical potentials were recorded from the distal AV junction area. Figure 2B shows the histological view of this region in another preparation from the same apical area of triangle of Koch (Dobrzynski et al., 2003), which clearly shows multilayered morphology of the AV junction. One can see the thin layer of AN transitional tissue (closed circle), the compact AV node (open circle), and loosely coupled deeper layer of NH transitional cells. OAP recordings during conducted beats contained two components, representing electrical activity in the two layers. OAP recorded during AV block contained only one component. Microelectrode recordings conducted simultaneously provide evidence that the ﬁrst component of optical recording corresponds to the superﬁcial transitional AN layer (closed circle in Fig. 2) of the AV junction, while the second component represents electrical activity of the deeper nodal layer (open circle in Fig. 2). Such multiphasic optical signal morphology was recorded from all studied rabbit preparations during both antegrade and retrograde AV conduction. In our studies, we did not inject dye to mark the cells from which we obtained the electrophysiological recordings. We are currently working on microelectrode recordings in parallel with optical coherence tomography and ﬂuorescent imaging, which will allow precise 3D positioning of the microelectrode tip with accuracy of 1–3 micrometers within the studied tissue structure. We estimated the depth of origin of the two components using slow diffusion of the voltage-sensitive dye during superfusion (Nikolski and Eﬁmov, 2001). In this case, we used 1 mol/L solution of di-4-ANEPPS, which is 10 –20 times lower than the previously used concentration (Eﬁmov et al., 1997). In the ﬁrst 5–10 min, only the superﬁcial layer of tissue was stained because of the slow diffusion of the dye into deeper layers. As a result, in the beginning of staining, only the second (late) component of the optical response was observed. During retrograde conduction, this component represents activation of superﬁcial AN layer of the AV node. Activation of the deeper AV nodal structure (ﬁrst or early component) became distinguishable only after 20 min. The delivery of the dye was stopped at 40 min and washout was started. The amplitude of the ﬁrst component continued to grow due to the maintained diffusion of the dye from the superﬁcial to deeper layers. Meanwhile, the difference between the second and the ﬁrst peak started to decrease, reﬂecting dye washout from the superﬁcial layer (Nikolski and Eﬁmov, 2001). Thus, the direction of diffusion of the dye during superfusion is consistent with the anatomic location of the two layers contributing to the dual-component optical signal. FLUORESCENT IMAGING OF AV NODAL REENTRY The ﬁrst clear and elegant description of AV junctional reentry belongs to Mines (1913), who described longitudinal dissociation preceding the onset of reentry and leading to its initiation. More than half a century later, reentry was induced by premature stimulation and documented by Coumel et al. (1967) in humans and by Janse et al. (1971) in the rabbit preparation. The study by Janse et al. (1971) showed the results of the application of a brush of 10 microelectrodes during AV nodal reentry in the rabbit preparation. Now, 31 years later, despite numerous attempts, nobody has been able to repeat this enormously difﬁcult experiment (Janse et al., 1971) and reproduce his results. Microelectrode recordings, while remaining the gold standard of cellular electrophysiology, present significant challenges to those in search of spatially resolved electrical mapping of the heart. Now we can present a detailed map of AV nodal reentry using ﬂuorescent mapping of AV nodal action potentials. Figure 3 shows a map of a so-called typical AV nodal reentry also known as slow/fast reentry, reﬂecting the fact that reentrant beat propagates antegradely via the slow pathway and then retrogradely via the fast pathway. Unlike the painstaking art of several-site microelectrode recordings, which was mastered only by a few (Janse et al., 1971; Billette, 1987), optical techniques give us hundreds of recordings, providing a thorough documentation of AV nodal reentry (Nikolski and Eﬁmov, 2001; Nikolski et al., 2003). Figure 3A shows an activation color map during conduction via the slow and fast pathways, which takes 120 msec. It is followed by rapid activation by the superﬁcial AN layer (Fig. 3B), which covers the same distance in only 20 msec, bringing activation back from the fast to the slow pathway and allowing impulse to reenter the slow pathway, completing the circuit. Thus, this map, to the best of our knowledge, represents the ﬁrst complete map of activation during typical (slow/fast) AV nodal reentry. Figure 3C further illustrates these data by presenting 24 optical action potentials (out of 256 available recordings), which were selected along the reentry circuit. Similar documentation of reentry was produced in our experiments with retrograde stimulation. Figure 4 shows the preparation schematics with the trajectories of impulse conduction and 3D stack plots of dF/dt corresponding to the three types of reentry: slowfast, fast-slow, and intranodal. Stackplots are shown in projection along the main axis of the triangle of Koch and time. From this 3D projection, the rapid atrial excitation that follows S1 stimuli is seen as almost a horizontal slab while conduction through nodal structures to His bundle is presented as a sloped ﬁlament. During slow-fast pathway reentry initiation after an S2 stimulus, there was conduction block in the A/AN layer, so the horizontal slab representing atrial excitation is missing (see the left 3D stack plot in Fig. 4). Only the 3D 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/sec following S1 stimuli and 30.7 ⫾ 14.0 cm/sec following S2 stimuli (n ⫽ 6; P ⬎ 0.5). We were unable to measure conduction velocity in the AV node and the posterior nodal extension during basic beat propagation accurately 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/sec. The reentrant beat propagated at 6.9 ⫾ 2.4 cm/sec (n ⫽ 6; P ⬎ 0.5). FLUORESCENT IMAGING OF AV JUNCTIONAL AUTOMATICITY Since the ﬁrst discovery of AV nodal automaticity, the site of initial pacemaker was sought by many investigators. They found that the sites of impulse formation are located in the compact AV node (Paes de Carvalho et al., STRUCTURE-FUNCTION RELATIONSHIP 957 Fig. 4. 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 two-dimensional plots of dF/dt (Nikolski and Eﬁmov, 2001). An isosurface was built using a density threshold, which was adjusted with time in order to preserve the continuity of conduction along the pathways. Three-dimensional 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. 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. 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 3D stack plots indicate quick transmittance of the activation between dual-pathway inputs through A/AN layer. SP and FP, slow and fast pathways. Reproduced with permission from Nikolski and Eﬁmov (2001). 1961) or in the NH region (Watanabe and Dreifus, 1968). Fluorescent imaging provides a unique opportunity for locating the site of origin of the junctional heartbeat. Our data show that such sites could be found not only in the compact N region or NH region, but also in the posterior nodal extensions (PNE). Figure 5 illustrates the optical mapping of the AV nodal area during spontaneous activity when the sinus node was surgically removed. Figure 5A and B show a preparation in which the AV node drives the atrium and His bundle. As seen from Figure 5A, the impulse originates in the isthmus below the coronary sinus. Then it slowly spreads in both directions along the posterior nodal extension until it reaches a breakthrough point in the middle of the triangle of Koch. Rapid excitation of the atrium follows (see right map in Fig. 5B). Optical traces and their derivatives in Figure 5B show examples of optical action potentials recorded from the site of origin (circle and solid trace) and 958 EFIMOV ET AL. Fig. 5. Automaticity of the AV junction. A: Photograph of the preparation. White rectangle shows ﬁeld of view of the optical mapping system. The white curve circles the location of the site of the earliest excitation during junctional AV rhythm. B: Maps of activation in the deeper cell layers of AV node and posterior nodal extension (left map) and the superﬁcial AV layer (right map), following the breakthrough from the deeper layer. The traces below show optical signals and their ﬁrst derivative at the location of pacemaker (white circle) and the break- through to the surface layer (black triangle) as well as bipolar electrical recordings at the bundle of His (His) and interatrial septum (Septum). Corresponding electrodes are indicated in A with arrows. C: Map of activation during spontaneous AV junctional rhythm in the preparation with a retrograde conduction block. Only excitation of the deeper cell layers is present. The trace below shows the unﬁltered optical signal at the location of the pacemaker (white triangle). Reproduced with permission (Dobrzynski et al., 2003). the site of the breakthrough (triangle and dashed trace). One can see that the location of the earliest excitation anatomically corresponds to the posterior AV node bundle extension. In several preparations, we observed complete dissociation of the AV node from the atrium following 3– 4 hr of the experiment. Figure 5C shows an example of such activity. Despite a complete cessation of communication between the atrium and AV node, the posterior nodal extension continued robust pacemaking, which safely propagated throughout the AV nodal extension, the compact AV node, and reached His bundle. Yet no conduction was observed toward the atrium. The optical trace shown below demonstrates clear diastolic depolarization and nodal-like action potentials. We were able to record these clean nodal potentials only in such dissociated preparations, because otherwise optical action potential from the superﬁcial AN layer would mask most of the nodal action potential. needed 3D resolution. Figure 6 shows the ﬁrst attempt of the morphological 3D reconstruction of the AV nodal area by OCT. As clearly seen in Figure 6B, the OCT images are capable of visualizing the different anatomical structures in the AV junction to a depth of 1 mm or more. An advantage OCT is that this methodology can be applied in clinical electrophysiology laboratory using catheter-based OCT imagers. For example, a radio frequency ablation catheter, which will carry the OCT imager, can be developed. This will allow precise anatomic localization of the slow pathway and safe ablation of the site. At the present time, OCT is available only for structural 3D imaging without any dyes or contrast agents. Consequently, a mapping of the transmembrane voltage or other electrophysiological parameters using this technology is not presently feasible. Yet the development of such absorbance dyes is underway and we hope that 3D functionally modulated optical coherence tomography will be available within 5 years for intravascular mapping of the AV node and the slow pathway. Now OCT technology provides a bridge between the optically recorded functional electrophysiological data discussed above and new molecular approaches to characterize the anatomical data, establishing precise correspondence between the electrical activity and immunohistological images collected after cryosectioning. OPTICAL COHERENCE TOMOGRAPHY OF AV NODE Despite the impressive success of ﬂuorescent imaging with voltage-sensitive dyes, this technology remains limited in its ability to gain insights into the depth of cardiac tissue. Our data clearly indicate that optical signals carry information about the deeper layers of tissue, but precise anatomic location of the layers contributing to speciﬁc optical action potentials and perhaps reconstruction of electrical activity in 3D remains to be achieved. We have recently introduced (Gupta et al., 2002) a novel optical imaging modality, optical coherence tomography (OCT) (Huang et al., 1991), which might be able to provide MOLECULAR BASIS OF ELECTROPHYSIOLOGICAL HETEROGENEITY OF AV JUNCTION The AV node is the Cinderella of mathematical cardiology, being the only major cell type or major structure of STRUCTURE-FUNCTION RELATIONSHIP 959 Fig. 6. Optical coherence tomography of the AV junction. Optical coherence tomography was applied to isolated AV junction of the rabbit heart in vitro. A shows 3D structure of the AV junction was reconstructed from stack of 2D scans, one of which is shown in B. the heart that has not yet been characterized within a framework of an accurate mathematical model. This is in contrast to its luckier counterpart, the sinoatrial (SA) node, which inspired numerous mathematical modeling studies (Demir et al., 1999). Several attempts to explain AV conduction mathematically were based on the empirical modiﬁcations of existing models of the SA node. Molecular data suggest that electrophysiological heterogeneity of the AV node is due to heterogeneity of ion channel and gap junction expression (Petrecca and Shrier, 2000). Sodium Channels Petrecca et al. (1997) provided convincing evidence of lower levels of expression of sodium ion channels in the compact AV node region and in ovoid-type isolated cells, which exhibit an N-type electrophysiological response, including the lack of prominent INa current. Lack of INa explains characteristic slow-rising tetrodotoxin-resistant (Zipes and Mendez, 1973) upstroke of nodal cells. In addition, they presented evidence that cells lacking INa also lack Ito, but have prominent IF, which is consistent with the documented pacemaking ability of N cells (Paes de Carvalho et al., 1961). Transitional cells of AN and NH types, on the other hand, have more abundant expression of INa (Petrecca et al., 1997; Petrecca and Shrier, 2000), which explains their fast upstroke velocity and speed of conduction. Calcium Channels Calcium channels play an important role in the conduction through the AV node (Zipes and Mendez, 1973; Noma et al., 1980), thought to be responsible for impulse transmission in the absence of sodium channels. Both L-type (Munk et al., 1996) and T-type calcium channels, ICa,L and ICa,T, have been found in the AV node (Liu et al., 1993). Pharmacology of these channels has been well characterized. However, no spatial mapping of ion channel expression has been presented so far. Such data are highly desirable, because possible heterogeneity of calcium channel expression can clarify structure-function relationship of the AV node, properties of which are known to be Ca-dependent. Potassium Channels Potassium channels also appear to be nonuniformly expressed in the AV node. Transient outward current Ito, similar to INa, appears to reside primarily in the transitional layers of the AV node (Munk et al., 1996), while being more scarcely expressed in the N region (Munk et al., 1996). The classic study of Noma et al. (1980) demonstrated that the delayed rectiﬁer current IK is the major repolarizing current in the AV node. More recent studies suggested that rapid component of IKr is the predominant if not the only component (Habuchi et al., 1995; Howarth 960 EFIMOV ET AL. Fig. 7. Coexpression of Cx43 and NF160 in the AV junction: posterior nodal extension, compact AV node, and the bundle of His. Histological staining and immunolabeling was applied to 16 m neighboring cryosections from the entire AV junction. Three areas are represented: posterior nodal extension at the site of junctional pacemaker, the compact AV node, and the bundle of His. AS, atrial septum; VS, ventricular septum. et al., 1996) of the delayed rectiﬁer potassium current. In contrast to the SA node (Habuchi et al., 1995), there is little evidence of the presence of the slow component, IKs, in the AV node (Habuchi et al., 1995). The role of the inward rectifying potassium channels remains unclear. IK1 channel plays a major role in maintenance of resting potential in the atrial and ventricular cells. However, it is almost absent from the AV node (Petrecca and Shrier, 2000). On the other hand, a well-documented vagal control (Clemo and Belardinelli, 1986; Mazgalev et al., 1986) of STRUCTURE-FUNCTION RELATIONSHIP 961 Fig. 8. AV junctional rhythm originates in a neuroﬁlament 160-positive area. Top: Cryosection of the AV node dual-labeled with NF160 (green) and Cx43 (red) antibodies. NF160 labeling was used for 3D reconstruction of NF160-positive area of the AV nodal extension. AS, atrial septum; VS, ventricular septum. Bottom: Photograph of the prep- aration overlapped with the image NF160-positive area (dark gray) and an activation map of the AV junctional rhythm (colored). TT, tendon of Todaro; IAS, intra-atrial septum; TVA, tricuspid valve; CS, coronary sinus; VM, ventricular muscle; PB, penetrating bundle; BB, bundle branches; PNE, posterior nodal extension; CN, compact node. the AV node suggests a role for IK,Ach (Clemo and Belardinelli, 1986). The presence and the important role of a pacemaker “funny” current, IF, was ﬁrmly established by Noma et al. (1980) and other investigators (Habuchi et al., 1995; Petrecca et al., 1997). We have recently demonstrated that predominant isoform of genes encoding IF current (HCN4) is expressed in the AV node and posterior nodal extension of the rabbit heart (Dobrzynski et al., 2003). Gap Junctional Channels Gap junctional channels appear to play an especially prominent role in AV conduction. Three major isoforms of gap junctional channel proteins, connexins Cx43, Cx40, and Cx45, have been mapped to speciﬁc regions of the mammalian heart. Presently available data suggest that all three isoforms are expressed in the triangle of Koch and play a role in the AV conduction. The compact node (N 962 EFIMOV ET AL. most of the myocardium, speciﬁcally in the atrial and ventricular myocardium. On the other hand, transitional layers of the AV node, including both AN and NH, exhibit greater levels of expression. Our data suggest that spatial heterogeneity of Cx43 expression might be related to the dual-pathway electrophysiology of the AV node, forming cell-to-cell communication pathways, which correspond to the slow and fast pathways (Nikolski et al., 2003). Two other connexin isoforms have been reported to play a role in the conduction through the compact node (Cx45) and NH region (both Cx40 and Cx45) (Gourdie et al., 1993). CONNEXIN HYPOTHESIS OF DUALPATHWAY ELECTROPHYSIOLOGY OF AV JUNCTION Fig. 9. Summary of Cx and HCN isoforms expression in the AV junction. HCN4 and NF160 are expressed throughout the conduction system of the rabbit heart. Densities of connexins vary from area to area of the CPCS. region) has been shown to have low level of expression of the major cardiac isoform, Cx43 (Gourdie et al., 1993; Petrecca et al., 1997; Coppen and Severs, 2002), which is responsible for cell-to-cell communication throughout Fig. 10. NF160 (green) and antisarcomeric actin (red) labeling in a day 13 rabbit embryo heart. Frontal sections, left section is most posterior in the heart. Arrows indicate NF160 labeling. High-magniﬁcation images conﬁrm colocalization of both markers (not shown). Left image shows labeling in the AV junction. Sections between the left and middle sections reveal additional labeling at the leading edge of the atrial primum septum (not shown). This fuses with the positively labeled region between the atria in the middle panel. Middle image shows extensive The results of the immunohistochemical studies have provided a vast amount of information that has shaped our understanding of AV conduction and junctional rhythm, based on signiﬁcant heterogeneity of expression of various proteins, including ion channels, receptors, gap junctions, signaling molecules, etc. In particular, we have recently demonstrated that our previous ﬁndings of bifurcation in the functional reentry pathway have a molecular basis (Nikolski and Eﬁmov, 2001; Nikolski et al., 2003). Using immunolabeling of Cx43, we found Cx43-positive bundles approaching the AV node from the coronary sinus oriﬁce, surrounded by Cx43-negative tissue. Correlation with ﬂuorescent data suggested that these Cx43-positive bundles are the substrate for dual pathway and for reentry. In some preparations, we observed bifurcation of the slow pathway in the middle of the triangle of Koch (Figs. 2 and 3). Immunolabeling showed that this functionally labeling between the atria in the AV junction, at the crest of the developing interventricular septum, and on the endocardial surface of the ventricular trabeculae. Additionally, there is labeling of cardiomyocytes between the atrium and ventricle on the right atrioventricular junction. Right image shows what has been termed the retroaortic limb of the cardiac pacemaking and conduction system, which regresses in the human but is maintained in the chick. 963 STRUCTURE-FUNCTION RELATIONSHIP deﬁned bifurcating conduction pathways correlate with bifurcating Cx43-positive bundles. Recently, our observation of Cx43-positive bundles at the AVN area was conﬁrmed by Ko et al. (2004). These AV nodal data agree with the previously observed Cx43-postive bundles in the SA node (Joyner and van Capelle, 1986; Kwong et al., 1998; Coppen et al., 1999) that had previously been suggested as a mechanism of enhanced safety of the conduction from the small SA nodal area that has to drive the large atrium against an unfavorable source-sink relationship. It is worth mentioning that the pathways delineated by the connexins and NF160 immunostaining satisfy two out of the three criteria of Aschoff (1910) and Monckeberg (Monckeberg, 1910) for conducting tracts. The criteria are as follows: the cells are (immuno)histologically discrete; it is possible to follow them from section to section; and they should be insulated by ﬁbrous sheaths from nonspecialized adjacent working myocardium. The pathways we describe do not fulﬁll the third criterion because their cells intermingle with the working myocardium. Rabbits in comparison to the other animals have a unique property: cells of their cardiac pacemaking and conduction system (CPCS) express neuroﬁlament 160 (NF160). Figure 7 shows double labeling of distal AV node with immunoprobes raised against Cx43 and NF160. It shows that NF160 is expressed at high density throughout the entire AV junction, including the AV nodal extension, compact AV node, and the bundle of His. We used NF160 staining in order to determine the tissue of the cardiac pacemaking and conduction system within the AV junctional area (Fig. 8, top) and His-Purkinje network. The top panels of Figure 8 show that NF160 is expressed throughout the entire cytosol of characteristically rounded compact nodal cells. Figure 8 shows the distribution of neuroﬁlament-positive conduction tissue (dark gray) in the triangle of Koch. The posterior nodal extension corresponds to the position of the slow pathway. Cx43 immunolabeling was abundant in the working myocardium, but sparse or absent in the conduction tissue. Our data show two different tissue types in the AV node region: neuroﬁlament-negative/Cx43-positive tissue (atrial and ventricular muscle) and neuroﬁlamentpositive/(mostly Cx43-negative) tissue (conduction tissue). The color-coded activation map for the AV junctional rhythm was optically recorded for this preparation and it shows that the NF160-positive tissue provides the channel for slow conduction due to the signiﬁcant reduction of Cx43 expression in myocytes of the PNE and compact node structures. More distantly located NF160-positive myocytes show a much higher degree of Cx43 expression and it correlates with the conduction speed increase as excitation approaches the central ﬁbrous body where NF160-positive myocytes form a penetrating (His) bundle. Further investigation of the expression of other connexin isoforms in the rabbit AV junction (Dobrzynski et al., 2003; Ko et al., 2004) revealed that available Cx40 antibodies, being traditionally accepted as marker for AV nodal structures in mouse and rat hearts, cannot be successfully applied to rabbit tissue. Dobrzynski et al. (2003) reported the weak Cx40 staining and Ko et al. (2004) concluded that Cx40 cannot be detected in the rabbit AV nodal myocytes at all, while the same antibodies gave abundant signal in the vascular endothelium (Ko et al., 2004). Cx45 expression in the rabbit AV node follows the pattern reported for rat and mouse hearts (Dobrzynski et al., 2003). The relative role of different connexin isoforms in heterogeneous conduction in the AV node is yet to be determined. Figure 9 summarizes our current knowledge about connexins distribution in the AV junctional area. Figure 9 also shows that the same area expresses HCN4 —predominant isoform of IF—pacemaker “funny” current. These data correlate well with the known cellular heterogeneity of the atrioventricular node in the rabbit as described by Anderson (1972b). CONCLUSION An impressive success of ﬂuorescent imaging in visualization of the entire circuit of AV nodal reentry, the location of pacemaker during junctional rhythm, and the activation during antegrade and retrograde dualpathway conduction has already provided evidence of the strength of new imaging technology. Progress in AV nodal research achieved in just a few years (Eﬁmov et al., 1997; Choi and Salama, 1998; Eﬁmov and Mazgalev, 1998; Wu et al., 2001) will be further developed with rich new methodologies, including optical coherence tomography and diffusive optical tomography. We believe that these new extensions of voltage-sensitive imaging methodologies will allow truly three-dimensional realtime imaging of electrical activity in the AV junction, as well as in other structures of the heart. Such novel methodology will allow reconstruction of entire 3D wave fronts during various modes of conduction in the AV junction. At the same time, a rapid development of highthroughput immunohistochemistry sustains impressive stream of discoveries of different aspects of molecular mechanisms of AV conduction in normal and pathological states. An increased level of understanding of a profound complexity of heterogeneous three-dimensional structure of the AV node [initially described by Anderson (1972a), with Janse et al. (1974) later providing functional correlations] is likely to produce a theoretical basis for a comprehensive mathematical model of the AV junction. Such a model will allow an exploration of the structure-function relationship of the AV node in the near future. We believe that this is the last major piece of a puzzle that is being built by cardiac electrophysiologists within an international project known as the virtual heart. Our present ﬁndings have cast further light on the controversies described in the introduction. However, we are still far from resolving all controversies. Recently, we also applied NF160 and ﬂuorescence imaging with voltage-sensitive dye to developing rabbit hearts. Our data conﬁrm earlier ﬁndings that NF160 is expressed as early as day 9.5 (Gorza et al., 1988). Interestingly, Figure 10 shows that NF160 is a marker of the developing cardiac pacemaking and conduction system, which is already functional prior to completion of septation as early as day 13 (data not shown). At this stage of development, SA and AV junction are connected with NF160-positive conduction pathway, which disappears at later stages of development and is undetectable in the adult rabbit heart. Perhaps the structure that is known as posterior AV nodal extension is a remnant of this atrial conduction system. 964 EFIMOV ET AL. ACKNOWLEDGMENTS Supported by National Institutes of Health grant HL58808 (to I.R.E.). LITERATURE CITED Anderson RH. 1972a. The disposition and innervation of atrioventricular ring specialized tissue in rats and rabbits. J Anat 113:197–211. Anderson RH. 1972b. Histologic and histochemical evidence concerning the presence of morphologically distinct cellular zones within the rabbit atrioventricular node. Anat Rec 173:7–23. Anderson RH, Janse MJ, van Capelle FJ, Billette J, Becker AE, Durrer D. 1974. A combined morphological and electrophysiological study of the atrioventricular node of the rabbit heart. Circ Res 35:909 –922. Anderson RH, Ho SY. 2000. The atrial connection of the specialazed axis responsible for AV conduction. In: Mazgalev TN, Tchou PJ, editors. Atrial-AV nodal electrophysiology: a view from the millenium. Armonk: Futura. p 3–24. Arridge SR. 1999. Optical tomography in medical imaging. Inverse Problems 15:R41–R93. Aschoff L. 1910. Referat über die Herzstorungen in ihren Bezeihungen zu den speziﬁschen Muskelsystem des Herzens. Verh Dtsch Ges Pathol 14:3–35. Bharati S. 2000. Anatomic-morphologic relations between AV nodal structure and function in the normal and diseased heart. In: Mazgalev TN, Tchou PJ, editors. Atrial-AV nodal electrophysiology: a view from the millenium. Armonk: Futura. p 25– 48. Billette J. 1987. Atrioventricular nodal activation during periodic premature stimulation of the atrium. Am J Physiol 252:H163– H177. Billette J. 2002. What is the atrioventricular node? some clues in sorting out its structure-function relationship. J Cardiovasc Electrophysiol 13:515–518. Choi BR, Salama G. 1998. Optical mapping of atrioventricular node reveals a conduction barrier between atrial and nodal cells. Am J Physiol 274:H829 –H845. Clemo HF, Belardinelli L. 1986. Effect of adenosine on atrioventricular conduction: I, site and characterization of adenosine action in the guinea pig atrioventricular node. Circ Res 59 427– 436 Coppen SR, Kodama I, Boyett MR, Dobrzynski H, Takagishi Y, Honjo H, Yeh HI, Severs NJ. 1999. 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 47:907–918. Coppen SR, Severs NJ. 2002. Diversity of connexin expression patterns in the atrioventricular node: vestigial consequence or functional specialization? J Cardiovasc Electrophysiol 13:625– 626. Cosio FG, Anderson RH, Kuck KH, Becker A, Borggrefe M, Campbell RW, Gaita F, Guiraudon GM, Haissaguerre M, Ruﬁlanchas JJ, Thiene G, Wellens HJ, Langberg J, Benditt DG, Bharati S, Klein G, Marchlinski F, Saksena S. 1999. Living anatomy of the atrioventricular junctions: a guide to electrophysiologic mapping. Circulation 100:E31–E37. Coumel P, Carbol C, Fabiato A, et al. 1967. Tachycardie permanente par rythme reciproque. Arch Maladies Coeur Vaisseaux 60:1830 – 1864. Demir SS, Clark JW, Giles WR. 1999. Parasympathetic modulation of sinoatrial node pacemaker activity in rabbit heart: a unifying model. Am J Physiol 276:H2221–H2244. Dobrzynski H, Nikolski VP, Sambelashvili AT, Greener ID, Yamamoto M, Boyett MR, Eﬁmov IR. 2003. Site of origin and molecular substrate of atrioventricular junctional rhythm in the rabbit heart. Circ Res 93:1102–1110. Eﬁmov IR, Fahy GJ, Cheng YN, Van Wagoner DR, Tchou PJ, Mazgalev TN. 1997. High resolution ﬂuorescent imaging of rabbit heart does not reveal a distinct atrioventricular nodal anterior input channel (fast pathway) during sinus rhythm. J Cardiovasc Electrophysiol 8:295–306. Eﬁmov IR, Mazgalev TN. 1998. High-resolution three-dimensional ﬂuorescent imaging reveals multilayer conduction pattern in the atrioventricular node. Circulation 98:54 –57. Gorza L, Schiafﬁno S, Vitadello M. 1988. Heart conduction system: a neural crest derivative? Brain Res 457:360 –366. Gourdie RG, Severs NJ, Green CR, Rothery S, Germroth P, Thompson RP. 1993. The spatial distribution and relative abundance of gapjunctional connexin40 and connexin43 correlate to functional properties of components of the cardiac atrioventricular conduction system. J Cell Sci 105(Pt 4):985–991. Gupta M, Rollins AM, Izatt JA, Eﬁmov IR. 2002. Imaging of the atrioventricular node using optical coherence tomography. J Cardiovasc Electrophysiol 13:95. Habuchi Y, Han X, Giles WR. 1995. Comparison of the hyperpolarization-activated and delayed rewctiﬁer currents in rabbit atrioventricular node and sinoatrial node. Heart Vessels 9(Suppl):203–206. Haissaguerre M, Gaita F, Fischer B, Commenges D, Montserrat P, d’Ivernois C, Lemetayer P, Warin JF. 1992. Elimination of atrioventricular nodal reentrant tachycardia using discrete slow potentials to guide application of radiofrequency energy. Circulation 85: 2162–2175. Hoffman BF, Paes de Carvalho A, de Mello WC. 1958. Transmembrane potentials of single ﬁbers of the atrio-ventricular node. Nature 181:66 – 67. Howarth EC, Levi AJ, Hancox JC. 1996. Characteristics of the delayed rectiﬁer K current compared in myocytes isolated from the atrioventricular node and ventricle of the rabbit heart. Pﬂugers Arch 431:713–722. Huang D, Swanson EA, Lin CP, Schuman JS, Stinson WG, Chang W, Hee MR, Flotte T, Gregory K, Puliaﬁto CA. 1991. Optical coherence tomography. Science 254:1178 –1181. Inoue S, Becker AE. 1998. Posterior extensions of the human compact atrioventricular node: a neglected anatomic feature of potential clinical signiﬁcance. Circulation 97:188 –193. Jackman WM, Beckman KJ, McClelland JH, Wang X, Friday KJ, Roman CA, Moulton KP, Twidale N, Nazlitt HA, Proior MI. 1992. Treatment of supraventricular tachycardia due to atrioventricular nodal reentry, by radiofrequency catheter ablation of slow-pathway conduction. N Engl J Med 327:313–318. Janse MJ, Capelle Fv, Freud GE, Durrer D. 1971. Circus movement within the AV node as a basis for supraventricular tachycardia as shown by multiple microelectrode recording in the isolated rabbit heart. Circ Res 28:403– 414. Janse MJ, Capelle FJ, van, Anderson RH, Billette J, Becker AE, Durrer, D. 1974. Correlation between electrophysiologic recordings and morphologic ﬁndings in the rabbit atrioventricular node. Arch Int Physiol Biochim 82:331–332. Janse MJ, Loh P, de Bakker JM. 2000. Is the atrium involved in AV nodal reentry. In: Mazgalev TN, Tchou PJ, editors. Atrial-AV nodal electrophysiology: a view from the millenium. Armonk: Futura. p 175–182. Joyner RW, van Capelle FJ. 1986. Propagation through electrically coupled cells: how a small SA node drives a large atrium. Biophys J 50:1157–1164. Ko YS, Yeh HI, Ko YL, Hsu YC, Chen CF, Wu S, Lee YS, Severs NJ. 2004. Three-dimensional reconstruction of the rabbit atrioventricular conduction axis by combining histological, desmin, and connexin mapping data. Circulation 109:1172–1179. Kwong KF, Schuessler RB, Green KG, Laing JG, Beyer EC, Boineau JP, Safﬁtz JE. 1998. Differential expression of gap junction proteins in the canine sinus node. Circ Res 82:604 – 612. Langendorf R. 1948. Concealed A-V conduction: the effect of blocked impulses on the formation and conduction of subsequent impulse. Am Heart J 35:542–552. Lewis T, Master AM. 1925. Observations upon conduction in the mammalian heart: AV conduction. Heart 12:209 –269. Liu Y, Zeng W, Delmar M, Jalife J. 1993. Ionic mechanisms of electronic inhibition and concealed conduction in rabbit atrioventricular nodal myocytes. Circulation 88:1634 –1646. Mazgalev T, Dreifus LS, Michelson EL, Pelleg A. 1986. Vagally induced hyperpolarization in atrioventricular node. Am J Physiol 251:H631–H643. Mazgalev TN, Tchou PJ. 2000. Atrial-AV nodal electrophysiology: a view from the millenium. Armonk, NY: Futura Publishing. STRUCTURE-FUNCTION RELATIONSHIP Mazgalev TN, Ho SY, Anderson RH. 2001. Anatomic-electrophysiological correlations concerning the pathways for atrioventricular conduction. Circulation 103:2660 –2667. Medkour D, Becker AE, Khalife K, Billette J. 1998. Anatomic and functional characteristics of a slow posterior AV nodal pathway: role in dual-pathway physiology and reentry. Circulation 98:164 – 174. Meijler FL, Janse MJ. 1988. Morphology and electrophysiology of the mammalian atrioventricular node. Physiol Rev 68:608 – 647. Mines GR. 1913. On dynamic equilibrium in the heart. J Physiol 46:349 –383. Moe GK, Preston JB, Burlington H. 1956. Physiologic evidence for a dual A-V transmission system. Circ Res 4:357–375. Monckeberg JG. 1910. Beitrage zur normalen und pathologischen Anatomie des Herzens. Verh Dtsch Ges Pathol 14:64 –71. Munk AA, Adjemian RA, Zhao J, Ogbaghebriel A, Shrier A. 1996. Electrophysiological properties of morphologically distinct cells isolated from the rabbit atrioventricular node. J Physiol 493:801– 818. Nikolski V, Eﬁmov I. 2001. Fluorescent imaging of a dual-pathway atrioventricular-nodal conduction system. Circ Res 88:E23–E30. Nikolski VP, Jones SA, Lancaster MK, Boyett MR, Eﬁmov IR. 2003. Cx43 and the dual-pathway electrophysiology of the AV node and av nodal reentry. Circ Res 92:469 – 475. Noma A, Irisawa H, Kokobun S, Kotake H, Nishimura M, Watanabe Y. 1980. Slow current systems in the a-v node of the rabbit heart. Nature 285:228 –229. Paes de Carvalho A, de Almeida DF. 1960. Spread of activity through the atrioventricular node. Circ Res 8:801– 809. Paes de Carvalho A, de Mello WC, Hoffman BF. 1961. The specialized tissues of the heart. Amsterdam: Elsevier. 965 Petrecca K, Amellal F, Laird DW, Cohen SA, Shrier A. 1997. Sodium channel distribution within the rabbit atrioventricular node as analysed by confocal microscopy. J Physiol (Lond) 501:263–274. Petrecca K, Shrier A. 2000. Spatial distribution of ion channels, receptors, and innervation in the AV node. In: Mazgalev TN, Tchou PJ, editors. Atrial-AV nodal electrophysiology: a view from the millenium. Armonk: Futura. p 89 –105. Racker DK. 1989. Atrioventricular node and input pathways: a correlated gross anatomical and histological study of the canine atrioventricular junctional region. Anat Rec 224:336 –354. Rosenbaum DS, Jalife J. 2002. Optical mapping of cardiac excitation and arrhythmias. Armonk, NY: Futura Publishing. Scherf D, Cohen J. 1964. The atrioventricular node and selected cardiac arrhythmias. New York: Grune and Stratton. Shah D, Haissaguerre M, Gaita F. 2002. Slow pathway ablation for atrioventricular nodal reentry. J Cardiovasc Electrophysiol 13: 1054 –1055. Tawara S. 1906. Das Reizleitungssystem des Saugetierherzens: eine anatomische-histologische Studie über das Atrioventrikularbundel und die purkinjeschen Faden. Jena: Verlag von Gustav Fischer. Watanabe Y, Dreifus LS. 1965. Inhomogenous conduction in the A-V node: a model for reentry. Am Heart J 70:505–514. Watanabe Y, Dreifus LS. 1968. Sites of impulse formation within the atrioventricular junction of the rabbit. Circ Res 22:717–727. Wu J, Wu J, Olgin J, Miller JM, Zipes DP. 2001. Mechanisms underlying the reentrant circuit of atrioventricular nodal reentrant tachycardia in isolated canine atrioventricular nodal preparation using optical mapping. Circ Res 88:1189 –1195. Zipes DP, Mendez C. 1973. Action of manganese ions and tetrodotoxin on atrioventricular nodal transmembrane potentials in isolated rabbit hearts. Circ Res 32:447– 454.