Functional and morphological evidence for a ventricular conduction ...

Am J Physiol Heart Circ Physiol 284: H1152–H1160, 2003. First published November 27, 2002; 10.1152/ajpheart.00870.2002.

Functional and morphological evidence for a ventricular conduction system in zebrafish and Xenopus hearts David Sedmera,1 Maria Reckova,1,2,4 Angela deAlmeida,1,2,4 Martina Sedmerova,1 Martin Biermann,2,3 Jiri Volejnik,1 Alexandre Sarre,4 Eric Raddatz,4 Robert A. McCarthy,1 Robert G. Gourdie,1 and Robert P. Thompson1 1

Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425; 2Klinik und Poliklinik fu¨r Nuklearmedizin, University of Mu¨nster, Mu¨nster, Germany 48161; 3Krannert Institute of Cardiology, Indianapolis, Indiana 46202; and 4 Institute of Physiology, University of Lausanne, CH-1005 Lausanne, Switzerland Submitted 15 October 2002; accepted in final form 22 November 2002

Sedmera, David, Maria Reckova, Angela deAlmeida, Martina Sedmerova, Martin Biermann, Jiri Volejnik, Alexandre Sarre, Eric Raddatz, Robert A. McCarthy, Robert G. Gourdie, and Robert P. Thompson. Functional and morphological evidence for a ventricular conduction system in zebrafish and Xenopus hearts. Am J Physiol Heart Circ Physiol 284: H1152–H1160, 2003. First published November 27, 2002; 10.1152/ajpheart.00870.2002.—Zebrafish and Xenopus have become popular model organisms for studying vertebrate development of many organ systems, including the heart. However, it is not clear whether the single ventricular hearts of these species possess any equivalent of the specialized ventricular conduction system found in higher vertebrates. Isolated hearts of adult zebrafish (Danio rerio) and African toads (Xenopus laevis) were stained with voltage-sensitive dye and optically mapped in spontaneous and paced rhythms followed by histological examination focusing on myocardial continuity between the atrium and the ventricle. Spread of the excitation wave through the atria was uniform with average activation times of 20 ⫾ 2 and 50 ⫾ 2 ms for zebrafish and Xenopus toads, respectively. After a delay of 47 ⫾ 8 and 414 ⫾ 16 ms, the ventricle became activated first in the apical region. Ectopic ventricular activation was propagated significantly more slowly (total ventricular activation times: 24 ⫾ 3 vs. 14 ⫾ 2 ms in zebrafish and 74 ⫾ 14 vs. 35 ⫾ 9 ms in Xenopus). Although we did not observe any histologically defined tracts of specialized conduction cells within the ventricle, there were trabecular bands with prominent polysialic acid-neural cell adhesion molecule staining forming direct myocardial continuity between the atrioventricular canal and the apex of the ventricle; i.e., the site of the epicardial breakthrough. We thus conclude that these hearts are able to achieve the apex-to-base ventricular activation pattern observed in higher vertebrates in the apparent absence of differentiated conduction fascicles, suggesting that the ventricular trabeculae serve as a functional equivalent of the HisPurkinje system.

(Xenopus laevis) and zebrafish (Danio rerio) are becoming model organisms representing the

amphibians and teleosteans, respectively. Recent advances in molecular genetics, together with ease of experimental manipulations not feasible in higher vertebrates, make these species useful model systems for studying heart development and the role of different genes in heart (mal)formation and (mal)function. Although the structure of the heart in these species is well characterized (10, 11, 18, 32), and the frog heart has long been used as a model system for heart physiology (e.g., Refs. 9 and 25), it is not entirely clear how closely findings from such systems can be applied to understanding the human heart. One of the important questions about the lower vertebrate heart is whether it possesses pacemaking and conduction tissues comparable with those of higher vertebrates. The cardiac pacemaker of frogs and fish and its neurohumoral regulation have been found to be comparable functionally with the sinoatrial node of mammals (reviewed in Ref. 12). It is also agreed that the atrioventricular myocardium serves the same role as the atrioventricular node, i.e., to create a delay between the atrial and ventricular contraction and prevent atrial tachyarrhythmias from reaching the ventricle (1). However, controversy persists as to the existence of a fast-conducting component of the ventricular myocardium (21, 23, 27) or the functional equivalent of the His-Purkinje system. Most previous investigators (1, 23) searched conduction system equivalents histologically, searching for pale cells arranged in bundles such as those found in the hearts of birds and mammals. This search was complicated by the fact that neither anurans nor fish possess a defined interventricular septum in which those fascicles are to be found (27). So far, we are unaware of any morphological study that would unequivocally demonstrate the existence of specialized ventricular conduction tissue in lower vertebrates. However, the lack of a morphologically distinct structure does not necessarily imply that the function

Address for reprint requests and other correspondence: D. Sedmera, Dept. of Cell Biology and Anatomy, Medical Univ. of South Carolina, 173 Ashley Ave., BSB 601, Charleston, SC 29425 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

fish; frog; amphibian; electrophysiology; optical mapping; Rana; PSA-NCAM

THE AFRICAN TOAD

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VENTRICULAR CONDUCTION SYSTEM IN LOWER VERTEBRATES

is absent, as demonstrated by developmental studies performed in higher vertebrates. In the chick, morphologically distinct bundles are not present before stage 31 [7 days of incubation (33)], but specific staining can identify the precursors of these structures much earlier as a diffuse network (6), and, indeed, the function of a His-Purkinje network can be demonstrated by conversion of ventricular activation patterns at around stages 29–31 [6–7 days (5)]. In the mouse, the situation is similar, and apex-to-base activation pattern is established well before completion of ventricular septation (26), implicating ventricular trabeculae in the process of accelerated impulse propagation (34). The question of presence or absence of functionally defined specialized ventricular conduction tissue is of importance from both phylogenetic and practical points of view. Is the fast-conducting component a neomorphic feature of homeothermic vertebrates or does it have more ancient roots? Similarly, its existence would strengthen the case for using various zebrafish mutants for the study of the genetic mechanisms of cardiac arrhythmias. We have therefore undertaken a functional study of the spread of normal and artificially induced activation in the adult heart of zebrafish and Xenopus, coupled with histological and immunohistochemical examination of atrioventricular connections. We hypothesized that the existence of specialized conduction tissue would be revealed by apex-to-base activation pattern and by prolonged ventricular activation time of ectopic heartbeats. In both species, we were able to document ventricular activation rapidly spreading from apex to base, whereas ectopic activation induced by electrical pacing took significantly more time. Histological examination did not reveal any morphologically distinct tracts of specialized conduction cells. However, the ventricular trabeculae formed straight myocardial continuity between the myocardium of the atrioventricular canal and the ventricular apex, which was the earliest activated region of the ventricle. We conclude that both species possess the functional equivalent of a ventricular conduction system. MATERIAL AND METHODS

Animals. This study conforms to the principles of Declaration of Helsinki and “Guiding Principles for Research Involving Animals and Human Beings” of the American Physiological Society. The zebrafish were maintained by standard methods (36). Three-year-old adult zebrafish (n ⫽ 10) were transferred to a petri dish and euthanized by rapid cutting between the head and vertebral column, followed by brain pithing. The pericardial cavity was opened, and the torso was transferred to 0.0015% solution of 4-[␤-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS) with 40 ␮mol cytochalasin D in freshwater fish saline [composition (in g/l): 9.88 NaCl, 0.37 KCl, 0.33 CaCl2, 0.14 MgCl2, 0.17 Na2HPO4, and 0.04 NaH2PO4; pH 7.2] bubbled with 100% oxygen for 10 min. After a brief rinse in the same saline, the torso was pinned on the silicone bottom of a custom-made dish, which was positioned on the temperature-controlled fixed stage (Biostage AJP-Heart Circ Physiol • VOL

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600, 20/20 Technology) of a Leica DMLB-FS upright microscope. The Xenopus toads (females, 2–3 yr old, n ⫽ 5) were supplied by Xenopus Express (Homosassa, FL) and maintained under 12:12-h light-dark conditions. Instead of the usual euthansia by anesthetic overdose, they were rapidly decapitated and despinalized, and their hearts were excised and rinsed of excess blood in amphibian Ringer saline gassed with 100% oxygen [composition (in g/l): 6.5 NaCl, 0.14 KCl, 0.12 CaCl2, and 0.2 NaHCO3; pH 7.2] and stained for 10 min in 0.003% di-4-ANEPPS with 40 ␮mol cytochalasin D in the same saline. The isolated heart was pinned through the distal bulbus and apex to achieve the required orientation in the same custom chamber positioned on a temperature-controlled stage of Leica MZFLIII epifluorescence microscope. For observation and additional illumination, we used a Schott KLD-1500 battery-powered light source with a green interference filter. Pacing at the ventricular apex was performed after the recordings in spontaneous rhythm were obtained. A platinum electrode (bipolar for Xenopus, pseudounipolar with anode in the tissue bath for zebrafish) was positioned at the apex of the ventricle by using a Narishige micromanipulator. The heart was stimulated in overdrive mode at 125% of the intrinsic rate with 2-ms pulses twice the diastolic threshold, which was between 1.5 and 2.5 mA for both species. Experimental protocol. All experiments were performed at 26°C (standard laboratory temperature to which the animals were adapted) with continuous bubbling of the bath with 100% oxygen, which was interrupted only during recording. Under these conditions, the hearts were electrophysiologically stable (spontaneously beating in regular rhythm) for at least 1 h; however, no more than 3 min of total illumination with the green light was possible because of the development of arrhythmias and photobleaching. For these reasons, only the recordings from the first 2 min were used for analysis; therefore, not all possible positions (dorsal, ventral, left, and right lateral views for Xenopus; ventral and both lateral views for zebrafish) were obtained from all hearts. Additionally, five zebrafish hearts were cut open with fine scissors (2 in frontal, 3 in sagittal plane) following initial imaging, and the hearts were restained and imaged again to reveal endocardial activation patterns. Optical mapping was performed by illuminating the specimen with green light (546 ⫾ 10 nm). Emitted light was passed through a 590-nm LP barrier filter and detected with a 12-bit intensified Neurocam camera (EG&G-Wallac/Olympus). The decrease in intensity of emitted fluorescence corresponds to changes in membrane potential (17). The magnifications used resulted in a spatial resolution of 78 ␮m/pixel for Xenopus and 19 ␮m/pixel for zebrafish in the binned mode used for data analysis. The frame rates used were either 500 (full resolution, 80 ⫻ 80 pixels) or 1,000 (with binning) frames per second depending on camera settings. Data processing. The recordings were digitally processed by using UltraView and Universal Mapping software. A typical cardiac cycle was selected (usually the first in each recording, because it usually had the best signal-to-noise ratio, but there were no beat-to-beat differences in any individual heart), and a time course of fluorescence intensity from individual pixels was digitally filtered by using a Butterworth low-pass (100 Hz) filter. The first derivative was computed (dV/dt), and its peak corresponding to maximal dV/dt was used for detection of the action potential upstroke. To account for virtual electrode effect, the pixels adjacent to the stimulation site were excluded from analysis. Isochronal contour maps of activation were constructed and superim-

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Table 1. Electrophysiological parameters of adult isolated zebrafish and Xenopus hearts Ventricular Activation Time, ms

Species

Heart Rate, beats/min

Atrial Activation Time, ms

AV Delay, ms

Spontaneous

Paced

Zebrafish (Danio rerio) African toad (Xenopus laevis)

123 ⫾ 12 67 ⫾ 10

20 ⫾ 2 50 ⫾ 2

47 ⫾ 8 414 ⫾ 16

14 ⫾ 2 35 ⫾ 9

24 ⫾ 3* 74 ⫾ 14*

Values are means ⫾ SD; n ⫽ 10 zebrafish hearts 5 and Xenopus hearts. AV, atrioventricular. * P ⬍ 0.01 vs. spontaneous beat.

posed on images of the hearts taken at maximal spatial resolution of the camera. Electrophysiological recordings. To validate our data using standard electrophysiological techniques, five green frogs (Rana clamitans) were prepared by brain and spinal cord pithing for electrocardiographic recording. The active electrode was clamped on the ventricular apex, and a reference electrode was positioned in the mouth. The recordings were performed at room temperature (25°C), and the skin was kept moist with paper towels soaked with tap water. Ventricular extrasystole was delivered by using a silver pseudounipolar suction electrode positioned at the ventricular apex at the end of T wave during continuous ECG recordings using custom software running on a Macintosh LC II computer. ECG intervals were then measured on digital recordings of spontaneous and paced beats. Histological examination. For morphological evaluation, the hearts were fixed after the experiments in Dent’s fixative (80% methanol-20% DMSO) and processed into paraffin. Sections at 5 ␮m thickness were cut in series in frontal, transverse, and sagittal planes and stained with hematoxylineosin. Selected sister sections were stained with antibodies directed against smooth muscle actin (1:1,000, Sigma) or myosin (9:1, MF20, Developmental Studies Hybridoma Bank) diluted with 10 mM Tris buffer, 0.9% NaCl, 0.001% sodium azide, 2% bovine serum albumin, and 0.1% Triton-X. The antibody binding was detected using Cy2-coupled goatanti-mouse secondary antibody (1:200, Jackson Immuno), and the nuclei were counterstained with propidium iodide (1:10,000). Polysaliac-acid neural adhesion molecule (PSANCAM) IgM monoclonal antibody (1:200, 5A5, Developmental Studies Hybridoma Bank), a marker for developing conduction system in the embryonic chick (6) was detected by Cy5-coupled anti-mouse IgM secondary antibody (Jackson Immuno). Observations were made on a Bio-Rad MRC1024 confocal microscope using appropriate lasers and filter sets. In addition, we refer to scanning electron micrographs from our earlier studies (10, 11, 29). Because of its size, overview pictures of the Xenopus heart were obtained by scanning the slides at 2,400 dpi in transparency mode using a UMAX PowerLook II scanner. Statistical evaluation. All values are expressed as means ⫾ SD. For statistical comparisons of spontaneous versus paced activation time, we used a paired t-test with values of P ⬍ 0.05 considered as significant. RESULTS

Zebrafish. The heart rate and other parameters of isolated heart function are expressed in Table 1. The activation of the atria progressed from its junction with the sinus venosus in an isotropic manner. The average time necessary for activation of the entire atrium (measured as a temporal difference between activation of the first and the last pixel) was 20 ⫾ 2 ms (n ⫽ 10). After a delay caused by slow conduction through the AJP-Heart Circ Physiol • VOL

atrioventricular canal (Table 1), the ventricle first became activated in the apical region (Fig. 1). This directionality could be discriminated in all views. On average, 14 ⫾ 2 ms were necessary for the activation of the entire ventricular surface. The activation wave elicited by ectopic ventricular pacing propagated across the ventricular surface isotropically and took 24 ⫾ 3 ms (P ⫽ 0.0001 vs. spontaneous beat) to traverse the ventricle. The propagation of the action potential across the ventricular surface can be seen in the online movie supplement.1 Imaging of the exposed endocardial surfaces revealed the activation wave spreading rapidly through the radial trabecular network toward the outer contour of the heart (Fig. 1E). Attempts of ablation of the two connections between the atrioventricular canal myocardium and the main trabecular bundles (Figs. 1E and 2, E and F) showed rapid compensation by impulse propagating from the other connection, indicating a degree of redundancy. If both connections were severed, complete conduction block ensued (Fig. 1F). Histological examination showed myocardial continuity among the sinus venosus, atrium, and the ventricle (Fig. 2). There were two distinct trabecular bands running from the anterior and posterior side of the atrioventricular canal on each side, which then joined together (Fig. 2E). These bands showed stronger PSANCAM staining than the remaining trabeculae. The bulboventricular valve was surrounded by myocardium (Fig. 2C); however, the bulbus arteriosus was entirely nonmyocardial (Fig. 2A). The arrangement of the myocytes within the atrioventricular canal was predominantly circumferential and the canal connected directly to the ventricular trabeculae, although there was a wedge of connective tissue separating it from the outer compact myocardium (Fig. 2B). Xenopus. The functional parameters of isolated hearts are summarized in Table 1. The activation of the atrium spread isotropically from the sinoatrial junction toward the atrioventricular canal and took 50 ⫾ 2 ms (n ⫽ 5). The first activated region of the ventricle was located near the apex on the left side (Fig. 3). From there, the wave of excitation spread over and around the ventricle toward the bulbus, taking 35 ⫾ 9 ms to activate the entire ventricular surface. The action potential obtained from the bulbus was of small amplitude and difficult to interpret due to complex spatial arrangement, but the spread of activation 1

See http://ajpheart.physiology.org/cgi/content/full/284/4/H1152/DC1.

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arteries. However, there were small arterial branches within the atrioventricular and bulbar myocardium, which reacted positively for smooth muscle actin (Fig. 4, D and F). The myocardium of the atrioventricular canal showed a predominantly circular arrangement of myocytes and was connected directly with the radially arranged trabeculae, which stretched all the way to the apex (Fig. 4A). Similar to the zebrafish, the trabeculae connecting immediately to the atrioventricular canal showed stronger anti-PSA-NCAM staining (Fig. 4E). The bulbus arteriosus was myocardial in its proximal part, which contained the spiral valve. ECG recordings in R. clamitans. The rate of the spontaneously beating, blood-perfused heart in situ was 50 ⫾ 8 beats/min (n ⫽ 5). The P wave lasted 70 ⫾ 5 ms, the P-Q interval duration was 298 ⫾ 43 ms, and the QRS complex took 106 ⫾ 42 ms. The QRS complex induced by apical pacing differed in shape from the spontaneous complex and was significantly longer (160 ⫾ 30 ms) than that occurring in spontaneous rhythm (P ⫽ 0.00001). DISCUSSION

Fig. 1. Isochronal maps of ventricular activation in zebrafish. a: Frontal view (cranial end up), spontaneous rhythm. *Earliest activated region, A, atrium; BA, bulbus arteriosus; V, ventricle. b: Apical pacing (arrow). c: Right lateral view of the same heart, spontaneous rhythm. d: Apical pacing (arrow shows position of electrode). Note apex-to-base activation pattern and slower propagation of the paced impulse. e: Endocardial activation pattern revealed by sagittal bisection of the heart. Note spreading of the impulse through the two main trabecular bundles (arrowheads) from the atrioventricular (AV) canal and then joining of the two wavefronts and further spread through the trabecular network. f: Optically recorded action potentials (raw, unfiltered data) from the atrium and ventricle before (top) and after (bottom) ablation of the two main trabecular bundles connected to the AV junction (arrowheads in e). Complete AV conduction block was produced by severing of both connections. Scale bar is 100 ␮m. The isochrones are at 2-ms intervals. See online movie supplement at http://ajpheart.physiology.org/cgi/content/full/284/4/ H1152/DC1.

was much slower than that in the ventricle. The average time necessary for pacing-induced activation to traverse the entire ventricular surface was 74 ⫾ 14 ms (P ⫽ 0.0026 vs. spontaneous beat). Histological examination confirmed the extremely thin nature of the ventricular compact myocardium (Fig. 4), which was insulated from the atrial myocardium by a substantial wedge of connective tissue, and, unlike zebrafish (10, 11), was devoid of any coronary AJP-Heart Circ Physiol • VOL

Methodology. Optical mapping is a well-established approach for studying activation patterns in both developing (15, 26) and adult (20, 24, 31) hearts. When compared with traditional microelectrode recordings, optical mapping offers the advantage of a far greater array of recording points and relative noninvasiveness. The main drawbacks are the cytotoxicity of the dye and products of its photo breakdown, artifacts caused by heart movements, and inability to image areas that are not in the same focal plane or are obscured by other compartments (sinus venosus and atrioventricular canal). Also, the epicardial activation maps do not provide direct information about the exact three-dimensional movement of the excitation wavefront. These problems can be addressed by limiting the dye concentration and exposure time, use of motion inhibition drugs [excitation-contraction uncoupling (13)], and partial dissection of the heart in different planes (31). Functional evidence of ventricular conduction system. Our data indicate, by means of isochronal activation maps showing an apex-to-base epicardial activation pattern, that there exists a specialized conduction pathway used for impulse propagation in the hearts of lower vertebrates. The presence of such a pattern was used as an indicator of the functional presence of HisPurkinje system during chick embryonic development (5), although the epicardial activation pattern does not directly account for the actual excitation pathway. We tried to overcome this pattern by imaging partly open hearts, which showed ventricular activation progressing from the endocardium to the epicardium. This finding is further strengthened by longer ventricular activation time of ectopic ventricular beat, demonstrated both optically and electrophysiologically, similar to the situation in the adult mouse heart (24). The normal heartbeat propagates from the sinus venosus (or the sinoatrial node) throughout the atrial myocar-

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Fig. 2. Immunohistochemical staining for myosin (green) shows myocardial continuity between the atrium (A) the ventricle (V) in the adult zebrafish heart. a: Sagittal section with higher magnification (b) showing myocardial connection (arrow) between the AV ring (AV) and trabeculae (Tr) as well as the insulating wedge of the connective tissue (arrowhead) in the AV sulcus. SV, sinus venosus; Co, ventricular compact layer. c: Frontal section through the valve plane. d: Higher power view of the AV canal showing the circular arrangement of myocytes and the connection (arrows) to the trabeculae. The red color shows nuclear counterstaining with propidium iodide. e: Sagittal section showing the PSA-NCAM-positive (green) main trabecular bands between the AV canal and the ventricular apex. Arrowheads indicate the target of ablation. f: Histological confirmation of successful ablation of the conduction pathway (see Fig. 1, e and f). Scale bars are 100 ␮m.

dium, with acceleration along the internodal tracts. Transmission to the ventricle is through the atrioventricular node (or canal), which conducts very slowly, thus assuring delay necessary for ventricular filling. Within the ventricle of higher vertebrates, the His bundle, bundle branches, and network of Purkinje fibers assure the rapid and coordinated activation of the working myocardium in the apex-to-base direction (9). The rationale for using ectopic pacing is that should no specialized conduction system be present, the total ventricular activation time and isochronal spacing would be the same as for the spontaneous beat. If, however, a faster-conducting pathway is present and used for propagation of the normal activation, the AJP-Heart Circ Physiol • VOL

paced beat would take longer to activate the entire ventricle. This was the approach used in an elegant study in the mouse by Nygren et al. (24), where presence of fast-conducting ventricular conduction tissue is well established from early embryonic stages (25). Our data correspond well with a study performed in the African lungfish (1), where the authors found that ventricular activation proceeded from the apex to the base of the heart and increased width of the QRS complex associated with ventricular premature beats. These results were also used to infer presence of a specialized ventricular conduction system in this species. Histology of conduction tissue. Extensive histological study failed to show any organized intraventricular

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conduction pathways in the African lungfish (1) and neither did similar studies performed in different fish species (21, 23). Mohsen and colleagues (21) described the presence of pale cells in the sinus and atrioventricular canal but made no mention about possible intraventricular conduction pathways. Nair (23) studied the development of innervation of the carp heart and noted the presence of nerve fibers associated with the central trabecular column, similar to the location of the main trabecular bundles described in zebrafish by Hu and colleagues (11). Our finding of stronger PSA-NCAM staining in these bundles corresponds well with their proposed role of a preferential conduction pathway, because this staining is considered an early marker of developing conduction tissue (5, 6). Given the spongy nature of the ventricular myocardium composed mainly of slender trabecular bands, fibrous insulation is not necessary because of their physical separation.

Fig. 3. Activation maps of Xenopus ventricle. a and b: Ventral view in spontaneous and apical pacing (arrow). Note the rapid sweep of the spontaneous activation from the left side of the ventricle. c and d: Dorsal view in spontaneous rhythm and apical pacing (arrow), respectively. e and f: Left lateral view showing propagation of the spontaneous and paced beat. * Site of epicardial breakthrough, LA, left atrium; RA, right atrium. Isochrones are at 8-ms intervals, and scale bar is 1 mm. AJP-Heart Circ Physiol • VOL

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The muscular connections between the atrioventricular canal and the ventricle in different vertebrate species were also studied by Keith and Flack (16). They noted the presence of distinct fibers radiating from the canal and fusing later on with the ventricular myocardium in hearts of fish, frogs, and reptiles. Whereas their morphology was found to be different from the rest of the canal myocardium in the eel (larger, more pale, and less striated), no difference was noted in the frog. However, partial fibrous insulation from the ventricular myocardium was noted in the proximal part of their course in the frog heart, which corresponds well with our results (cf. Fig. 4, C and E). Ontogenesis of ventricular conduction system. In the embryonic hearts of birds and mammals, ventricular trabeculae were hypothesized to play a role in the spreading of the action potential (7, 34). This hypothesis was based on the conspicuously radial arrangement of the trabeculae, which is well suited for such a function, and their direct attachment to the myocardium of the atrioventricular canal. Our previous (10, 29) and present study showed that this arrangement is also present in the hearts of fish and anurans. Already, Benninghoff (3) had noted this parallel and hypothesized that the inner trabeculae of the heart tube (“Konturfasern” or contour fibers) comprise the primitive ventricular conduction system in lower vertebrates as well as in embryonic mammals. The PSA-NCAM-positive main trabecular bands spanning the entire ventricle from the atrioventricular junction to the apex are good candidates for a functional equivalent of the His bundle and its branches in the zebrafish, because their ablation resulted in complete heart block. The remaining trabeculae are shorter and run in different directions (11, 29). PSA-NCAM is, together with HNK-1, a cell surface carbohydrate known to participate in cellcell and cell-substrate interactions in the development of the nervous system. In the developing chick heart, it preferentially stains components of the early ventricular conduction system (6). Its presence in a subset of trabeculae in the hearts of lower vertebrates suggests that this trabecular subpopulation is concerned with rapid propagation of excitation within the ventricle. It should be noted that most of the myocardial mass is contained in the trabeculae in lower vertebrates and embryonic higher vertebrates, stressing the other roles of these tissues in ventricular contractility and myocardial oxygenation (29). In Xenopus, the central trabecular sheet (Fig. 4B) is in the best position to serve as a conduction shortcut and also corresponds with the future location of the interventricular septum, where the largest fascicles of the conduction system of higher vertebrates are found. It also stained more strongly with PSA-NCAM antibody than the remaining trabeculae. The arrangement of these trabecular bands could explain the epicardial breakthrough pattern near the ventricular apex, but as we demonstrated by direct imaging of cut hearts, the activation is ultimately spread by all the trabeculae, which thus form an equivalent of the Purkinje network. This explains shorter ventricular activation time in spontaneous rhythm.

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Fig. 4. Histological analysis of adult Xenopus heart. a and b: Frontal sections stained with hematoxylin-eosin showing the general disposition of the chambers. Arrowhead in b points to incomplete interatrial septum; arrows point to trabecular band spanning from the AV junction to the ventricular apex. c: Sagittal section showing connection of the AV myocardium to the trabeculae (arrow). Note insulating fibrous tissue in the AV sulcus (arrowheads). Anti-myosin staining (green) with nuclear counterstaining with propidium iodide (red) in shown. Extracellular matrix in the valves also shows red staining. d: Sister section showing higher power view of the AV myocardium containing small arterial branches (arrows) demonstrated by smooth muscle actin antibody (green). e: Frontal section through AV junction showing the polysaliac acid-neural adhesion molecule immunoreactivity (blue) in the subendocardial region adjacent to the AV canal (arrows). This staining is stronger than in the neighboring trabeculae. Green staining, myosin antibody; red staining, tissue autofluorescence in the valve. f: Smooth muscle actin staining (green) of BA shows the transition of the myocardial bulbus arteriosus to the systemic and pulmocutaneous arteries as well as numerous small arteries (arrows) supplying its myocardium. Scale bars are 1 mm in a, b, c, and f and 100 ␮m in d and e.

Role of myocardial architecture in impulse propagation. Histological specialization is not the only substrate of anisotropic ventricular conduction. Tissue geometry could perhaps account for anisotropy of impulse propagation. In the elegant study of Kucera and colleagues (19), effects of tissue geometry were examined in patterned cardiomyocyte cultures. The speed of propagation was faster in trabeculae-like “struts” and slowed down when an expansion or “sink” was encountered. The side branches also slowed down conduction AJP-Heart Circ Physiol • VOL

but made it more reliable. This could explain why the excitation travels preferentially through the inner trabecular bundles, which have few branches before reaching the apical compact myocardium, as well as slow propagation of the paced beat on the ventricular surface, to which numerous trabeculae are attached (Figs. 2 and 4). The myofiber geometry in the compact layer plays a role in anisotropy of impulse propagation (20, 22), being more rapid in a parallel direction than perpen-

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dicular to the predominant bundle orientation. The orientation of muscle fiber in large marine fish hearts was described by Sanchez-Quintana and Hurle (28), and the orderly alignment of the trabeculae in the dogfish shark supported their role in impulse propagation, but the interspecies differences were considerable. Unlike in mammals, neither species examined showed distinct spiraling in the compact layer, which correlates with the observed isotropy of propagation of the paced beat. Furthermore, the compact myocardium is considerably thinner in the species examined in our study, making the existence of spiral systems that usually arise with complex organization of the compact layer (14) even less likely. Indeed, varying the position of the stimulation electrode on the ventricular surface did not change the isochronal spacing or total ventricular activation time (unpublished data) in contrast to the situation in the mouse (22). Heart blood supply in lower vertebrates. The anuran ventricle is considered to be “coronary less” (8); however, two coronary trunks originating from the carotid arch were described to spread over the thick myocardial wall of bulbus. In this study, we report the existence of additional branches, highlighted by antismooth muscle actin immunostaining, in the similarly thick myocardium of the atrioventricular canal. This feature can explain the development of atrioventricular block after 1 h in culture and larger atrioventricular delay (over 400 ms) compared with the 300-ms P-Q interval of the blood-perfused R. clamitans heart in situ at comparable heart rates. It seems that slow conduction through these two regions is more dependent on oxygen concentration. The alternating arrangement of fast (atria and ventricle) and slow-conducting segments (atrioventricular canal and bulbus arteriosus) is remarkably similar to the embryonic chick heart (7). Considerably longer atrioventricular delay between atrial and ventricular contraction in anurans compared with zebrafish could be explained by the necessity of maintaining strictly laminar blood flow in these larger hearts to assure efficient compartmentalization of oxygenated and deoxygenated blood within the ventricle (4). On the other hand, the longer duration of the QRS complex in R. clamitans compared with Xenopus can be explained by methodological difference; whereas ECG gives a more precise estimate of total activation time of the ventricular myocardium, it is impossible to image the entire ventricle (including inside) from any given view using optical mapping. Concluding comments. The existence of the functional equivalent of a specialized network of conduction cells (albeit diffuse) in the ventricle of lower vertebrates has interesting phylogenetic and mechanistic implications. First, it suggests that the emergence of pathways for preferential spread of electrical excitation in the ventricle is not a recent innovation in higher vertebrates. In this respect, it is noteworthy that the existence of myocytes specialized for conduction have been described in the primitive tube heart of the tunicate Ciona (2), a genus whose long extinct ancestors are thought to sit at the base of the chordate family AJP-Heart Circ Physiol • VOL

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tree. Second, the demonstration of an organized network of conduction cells in the lower vertebrate heart could present new opportunities for deepening our understanding of the developmental biology of such tissues. Indeed, because proper heart function is not required for initial survival in zebrafish, this model could be exploited to study morphological and electrophysiological heart phenotypes that would cause early lethality in mammalian models (30, 35). Such innate advantages, together with powerful tools already available for probing genetics in zebrafish, could provide a basis for exciting new advances in this field in coming years. We thank Ambuja Bale and Dr. John Woodward for providing us with the Xenopus for these experiments and Dr. Jose Maria PerezPomares for helpful comments on the paper. D. Sedmera is particularly thankful to Dr. Lukas Kappenberger for introduction to the idea of ectopic heart pacing. This work was supported by National Institutes of Health (NIH) Grant RO1 HL-50582 (to R. P. Thompson), National Science Foundation Shared Instrumentation Grant and NIH Grant PO1 HD39946 (to R. G. Gourdie), and a University Research Committee grant (to D. Sedmera). D. Sedmera was also supported by South Carolina Center of Biomedical Research Excellence Grant RR 1643401. M. Biermann was supported by NIH, Indiana University, Deutsche Forschungsgemenischaft, and Westfa¨ lische Wilhems-Universita¨ t, Mu¨ nster. REFERENCES 1. Arbel ER, Liberthson R, Langendorf R, Pick A, Lev M, and Fishman AP. Electrophysiological and anatomical observations on the heart of the African lungfish. Am J Physiol Heart Circ Physiol 232: H24–H34, 1977. 2. Becker DL, Cook JE, Davies CS, Evans WH, and Gourdie RG. Expression of major gap junction connexin types in the working myocardium of eight chordates. Cell Biol Int 22: 527– 543, 1998. ¨ ber die Beziehung des Reitzleitungssystems 3. Benninghoff A. U und der Papillarmusklen zu den Konturfasern des Herzschlauches. Anat Anz 57: 185–208, 1923. 4. Burggren WW. Cardiac design in lower vertebrates: what can phylogeny reveal about ontogeny? Experientia 44: 919–930, 1988. 5. Chuck ET, Freeman DM, Watanabe M, and Rosenbaum DS. Changing activation sequence in the embryonic chick heart. Implications for the development of the His-Purkinje system. Circ Res 81: 470–476, 1997. 6. Chuck ET and Watanabe M. Differential expression of PSANCAM and HNK-1 epitopes in the developing cardiac conduction system of the chick. Dev Dyn 209: 182–195, 1997. 7. de Jong F, Opthof T, Wilde AA, Janse MJ, Charles R, Lamers WH, and Moorman AF. Persisting zones of slow impulse conduction in developing chicken hearts. Circ Res 71: 240–250, 1992. 8. de la Cruz MV, Moreno-Rodriguez R, and Angelini P. Phylogeny of the coronary arteries. In: Coronary Artery Anomalies: a Comprehensive Approach, edited by Angelini P. Philadelphia, PA: Lippincott Williams & Wilkins, 1999, p. 1–9. 9. Gattuso A, Mazza R, Pellegrino D, and Tota B. Endocardial endothelium mediates luminal ACh-NO signaling in isolated frog heart. Am J Physiol Heart Circ Physiol 276: H633–H641, 1999. 10. Hu N, Sedmera D, Yost HJ, and Clark EB. Structure and function of the developing zebrafish heart. Anat Rec 260: 148– 157, 2000. 11. Hu N, Yost HJ, and Clark EB. Cardiac morphology and blood pressure in the adult zebrafish. Anat Rec 264: 1–12, 2001. 12. Irisawa H. Comparative physiology of the cardiac pacemaker mechanism. Physiol Rev 58: 461–498, 1978.

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