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1 Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425; 2 Klinik und Poliklinik für Nuklearmedizin, University of Münster, Münster, Germany 48161; 3 Krannert Institute of Cardiology, Indianapolis, Indiana 46202; and 4 Institute of Physiology, University of Lausanne, CH-1005 Lausanne, Switzerland
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 His-Purkinje system.
fish; frog; amphibian; electrophysiology; optical mapping; Rana; PSA-NCAM
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE AFRICAN TOAD (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 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.
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MATERIAL AND METHODS |
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 µmol 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 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 µmol 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 µm/pixel for Xenopus and 19 µm/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 superimposed 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 µm thickness were cut in series in frontal, transverse, and sagittal planes and stained with hematoxylin-eosin. 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 goat-anti-mouse secondary antibody (1:200, Jackson Immuno), and the nuclei were counterstained with propidium iodide (1:10,000). Polysaliac-acid neural adhesion molecule (PSA-NCAM) 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.
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RESULTS |
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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
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).
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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 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).
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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).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 His-Purkinje 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 myocardium, 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 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 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. 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 cell-cell 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.
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 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 perpendicular 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 anti-smooth 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 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.
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ACKNOWLEDGEMENTS |
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We thank Ambuja Bale and Dr. John Woodward for providing us with the Xenopus for these experiments and Dr. Jose Maria Perez-Pomares for helpful comments on the paper. D. Sedmera is particularly thankful to Dr. Lukas Kappenberger for introduction to the idea of ectopic heart pacing.
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FOOTNOTES |
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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 HD-39946 (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 16434-01. M. Biermann was supported by NIH, Indiana University, Deutsche Forschungsgemenischaft, and Westfälische Wilhems-Universität, Münster.
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: sedmerad{at}musc.edu).
1 See http://ajpheart.physiology.org/cgi/content/full/284/4/H1152/DC1.
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.
First published November 27, 2002;10.1152/ajpheart.00870.2002
Received 15 October 2002; accepted in final form 22 November 2002.
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TOP
ABSTRACT
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MATERIAL AND METHODS
RESULTS
DISCUSSION
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