The endothermic state of mammals and birds requires high heart rates to accommodate the high rates of oxygen consumption. These high heart rates are driven by very similar conduction systems consisting of an atrioventricular node that slows the electrical impulse and a His-Purkinje system that efficiently activates the ventricular chambers. While ectothermic vertebrates have similar contraction patterns, they do not possess anatomical evidence for a conduction system. This lack amongst extant ectotherms is surprising because mammals and birds evolved independently from reptile-like ancestors. Using conserved genetic markers, we found that the conduction system design of lizard (Anolis carolinensis and A. sagrei), frog (Xenopus laevis) and zebrafish (Danio rerio) adults is strikingly similar to that of embryos of mammals (mouse Mus musculus, and man) and chicken (Gallus gallus). Thus, in ectothermic adults, the slow conducting atrioventricular canal muscle is present, no fibrous insulating plane is formed, and the spongy ventricle serves the dual purpose of conduction and contraction. Optical mapping showed base-to-apex activation of the ventricles of the ectothermic animals, similar to the activation pattern of mammalian and avian embryonic ventricles and to the His-Purkinje systems of the formed hearts. Mammalian and avian ventricles uniquely develop thick compact walls and septum and, hence, form a discrete ventricular conduction system from the embryonic spongy ventricle. Our study uncovers the evolutionary building plan of heart and indicates that the building blocks of the conduction system of adult ectothermic vertebrates and embryos of endotherms are similar.
Citation: Jensen B, Boukens BJD, Postma AV, Gunst QD, van den Hoff MJB, et al. (2012) Identifying the Evolutionary Building Blocks of the Cardiac Conduction System. PLoS ONE 7(9): e44231. doi:10.1371/journal.pone.0044231
Editor: Marie Jose Goumans, Leiden University Medical Center, The Netherlands
Received: May 25, 2012; Accepted: July 30, 2012; Published: September 11, 2012
Copyright: © Jensen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the European Community’s Seventh Framework Programme contract (‘CardioGeNet’ 223463 to VMC) and the Netherlands Heart Foundation (NHS 2008B062 to VMC). Tobias Wang and Bjarke Jensen were supported by The Danish Council for Independent Research | Natural Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The hearts of mammals and birds maintain high rates of contraction  that in concert with high systemic blood pressures accommodate their high rates of oxygen consumption due to their endothermic state . The high heart rates, the timing of sequential atrial and ventricular contractions and the rapid spread of the activating impulse over the avian and mammalian ventricles are possible because of a specialized cardiac conduction system . However, while the sequential activation of the cardiac chambers and appropriate matching of the atrial and ventricular contractions are similar across all vertebrate groups, there is no anatomical evidence for a specialized conduction system in hearts of reptiles or other ectothermic vertebrates , . Because mammals and birds evolved independently from reptilian ancestors, the evolutionary origin of their specialized conduction systems has remained unclear; either their conduction systems evolved independently or primordial components of the system were already present in the ancestral reptiles (Fig. 1).
Figure 1. Background and hypothesis.
(A) Anatomical works concluded that the specialized cardiac conduction system evolved independently in mammals and birds because similar structures could not be found in ectothermic vertebrates. (B) We are testing the hypothesis that a primordial version of the specialized cardiac conduction system can be found in ectothermic vertebrates.doi:10.1371/journal.pone.0044231.g001
Like the hearts of ectothermic vertebrates, embryonic mammalian and avian hearts also exhibit regulated sequential activation patterns in the absence of a morphological conduction system , , . This suggests that the functional components for conduction system are established early in development and in evolution, but are not represented by anatomically distinguishable components as in the mature hearts of endothermic vertebrates. Instead, the components may reflect an intrinsic part of the building plan of the heart.
Figure 2. The atrioventricular junction in ectotherms is not interrupted by an insulating plane.
Picro-sirius red stain for collagen (red) on 10 µm sections of hearts of adult ectotherms showing the atrioventricular canal to be in full communication (+) with the ventricle. l(r)a, left(right) atrium.doi:10.1371/journal.pone.0044231.g002
Figure 3. The phenotype of the slow propagating atrioventricular canal is evolutionary conserved.
Numbers in the phylogenetic tree indicate time in millions of years since major splits in tetrapod evolution. (A–E) The hearts of mature ectotherms (blue) and embryonic endotherms (red) maintain complete muscular connection in the atrioventricular canal (avc). (F–J) Markers of fast propagating chamber myocardium (Nppa and Gja5) are absent from the atrioventricular canal (arrowheads). Note that the specimen in H is contracted, obscuring the spongy design otherwise visible. Scale bars in (A–E), 300 µm; (F–J), 100 µm.doi:10.1371/journal.pone.0044231.g003
Tbx2 and Tbx3 belong to an ancient family of transcription factors  expressed in the embryonic atrioventricular canal from human to primitive fish , . In the embryo, the atrioventricular canal delays the impulse from atrium to ventricle. Tbx2/3 suppresses chamber genes including Nppa and Gja5, encodes for connexin40 that is required for fast conduction , and hence inhibits differentiation of the atrioventricular canal to fast-conducting chamber myocardium , . Tbx3 remains expressed in the mature conduction system components of mammals, including the atrioventricular node that derives from the atrioventricular canal . Bmp2/4 are expressed in the atrioventricular canal of early embryonic mammals, birds and fish, and are crucial for activation of Tbx2/3 (Fig. S1) –. Hence, Tbx2/3, Bmp2/4 and Gja5/Nppa represent evolutionary conserved positive and negative markers, respectively, that discriminate the embryonic slow-conducting atrioventricular myocardium and fast-conducting chamber myocardium. The ventricular His-Purkinje system of mammals and birds is specifically marked by expression of Gja5 (and Nppa in mammals) , .
Here, we carried out in situ hybridization analysis using evolutionary conserved genetic markers and provide a three-dimensional reconstruction of the key components of the conduction system. The ventricular conduction pattern was visualized using optical imaging of activation. The cardiac expression and conduction patterns of a reptile were then compared to those of mammals, chicken, and other ectothermic vertebrates, frog and fish. We find an anatomic, genetic and physiologic conserved building plan where hearts of adult ectothermic vertebrates are similar to embryos of the endothermic mammals and birds. This indicates that primordial components of the cardiac conduction system were present in the ancestral reptiles.
Materials and Methods
All experimental procedures on adult material complied with national and institutional guidelines and were approved by Institutional Animal Care and Use Committee of the University of Amsterdam. The approval is registered as “DAE101617” for optical mapping of the ectotherms and “DAE101532” for optical mapping of developing mice. In The Netherlands experiments with non-mammalian embryos (that are not autonomously viable) do not require approval from the Institutional Animal Care and Use Committee.
Figure 4. The developmental gene programme of amniotes is maintained in the mature heart of Anolis.
(A–D) Stage 17/19 Anolis hearts show complementary expressions of Bmp2 and Tbx3 to Gja5 in the developing myocardial atrioventricular canal (arrowheads). (E–H) The developmental expression of Bmp2, Tbx3 and Gja5 is maintained in the mature myocardial atrioventricular canal (left side shown). la, left atrium; ra, right atrium; ven, ventricle. Scale bars are 100 µm.doi:10.1371/journal.pone.0044231.g004
Adult zebrafish were provided by the Hubrecht Laboratory, Utrecht, the Netherlands, and adult Xenopus laevis from Leiden University, the Netherlands. Mice and Xenopus laevis embryos were raised in the AMC. Green and brown anole (Anolis carolinensis and A. sagrei) eggs and adults and fertilized chicken eggs, were bought commercially in the Netherlands. Xenopus embryos were staged according to Nieuwkoop , Anolis embryos according to Sanger et al. , chicken according to Hamburger and Hamilton  and mice from days post coitus.
Figure 5. Three-dimensional reconstructions of Tbx3 (yellow) expression in amniotes reveal a shared design.
Reconstructions are based on in-situ hybridizations of serial sections, except in human (based on immunohistochemistry, modified from , ). The Tbx3 domains are strikingly similar in the early phases of chamber formation (upper panel). The Tbx3 expression of the Anolis ventricle is very similar to that associated with ventricular septation (black arrows) in the other amniotes (lower panel).doi:10.1371/journal.pone.0044231.g005
Optical mapping was performed at 25°C in the ectothermic vertebrates and we used specific ringer solutions for zebrafish and Xenopus (in mmol/l: NaCl 115, Tris 5, NaH2PO4 1, KCl 2.5, MgSO4 1, CaCl2 1.5, Glucose 5, pH adjusted to 7.2 with HCl) and Anolis (adopted from ; in mmol/l: NaCl 95, Tris 5, NaH2PO4 1, KCl 2.5, MgSO4 1, CaCl2 1.5, Glucose 5, pH adjusted to 7.5 with HCl). For embryonic mouse hearts we used Tyrode’s solution at 37°C (in mmol/l: NaCl 128, KCl 4.7, CaCl2 1.45, MgCl2 0.6, NaHCO3 27, NaH2PO4 0.4, Glucose 11[pH maintained at 7.4 by equilibration with a mixture of 95% O2 and 5% CO2]). Excised hearts from sedated animals were incubated in the specific solution containing 15 mmol/l di-4-ANEPPS (voltage sensitive). Excitation light was provided by a 5-watt power LED (filtered 510±20 nm). Fluorescence (filtered >610 nm) was transmitted through a tandem lens system on CMOS sensor (100×100 elements; MICAM Ultima). Activation patterns were measured during sinus rhythm. Optical action potentials were analyzed with custom software.
Figure 6. Tbx2 expression in the atrioventricular canal of the formed heart of the zebrafish (in-situ hybridization).
Nppa is expressed in a complementary pattern. a, atrium; ven, ventricle.doi:10.1371/journal.pone.0044231.g006
Figure 7. Tbx2 expression (in-situ hybridization) in developing Xenopus (st 48).
Tbx2 is expressed in the atrioventricular canal and base of the myocardial outflow tract and complementary to Nppa, marker of chamber myocardium. In ectotherms primitive myocardium (p), remnants of the embryonic heart tube, can be recognized by its smooth surface as opposed to the trabeculated myocardium (t) formed during chamber formation. The primitive myocardium of the ventricular base of st 48 Xenopus hearts already has the adult configuration. The trabecular component is far from fully developed. a, atrium; avc, atrioventricular canal; c, conus arteriosus (myocardial outflow tract); p, primitive (atrabecular) myocardium; t, ventricular trabeculated myocardium; ven, ventricle.doi:10.1371/journal.pone.0044231.g007
In situ Hybridization
All embryos and hearts were fixed in 4% paraformaldehyde for one day and then kept in 70% ethanol until imbedding in paraffin and then sectioned at 7–12 µm for in situ hybridization. Methodology of the non-radioactive in situ hybridization analysis has been described previously ,  and so has probes for Zebrafish , Xenopus , , chicken – and mouse . Probes for Anolis were made in house based on the following coordinates using UCSC Genome Browser on Lizard May 2010 (Broad AnoCar2.0/anoCar2) Assembly; Tnnt2 (chr4:131,362,830–131,375,015), Bmp2 (chr1:136,434,179–136,445,741), Gja5 (chr3:162,064,640–162,065,729), Tbx3 (chrUn_GL343338:1,255,354–1,255,812), Tbx2 (regarded as Tbx3, chrLGb:3,261,791–3,267,757), Tbx5 (chrUn_GL343338:1,127,724–1,145,671). Briefly, we generated a cDNA library with standard TRIzol RNA extractions  from freeze-fixed specimens of developmental stages 5–9 and GOI cDNA were obtained by PCR amplification and cloned into pBluescript SK_ (Stratagene, La Jolla, CA). Digoxigenine-labeled antisense mRNA were then produced by in vitro transcription according to the manufacturer’s instructions (Roche, Mannheim, Germany).
Figure 8. Development of compact walls.
The development of the compact walls (Nppa and Gja5 negative) of mammals and birds leaves the trabeculated myocardium (Nppa and Gja5 positive) as a thin inner lining of the ventricular lumina in the fully formed hearts. Nppa is not expressed birds. Scalebars, 100 µm. ivs, interventricular septum; lv, left ventricle.doi:10.1371/journal.pone.0044231.g008
Figure 9. Trabeculated ventricles are activated from base to apex.
(A, D, G, J) Markers of fast propagating myocardium (Nppa and Gja5) are homogenously expressed in the ventricular trabeculated myocardium from base to apex (ap). (B, E, H) Ventricular activation occurs from base to apex. Early activation is red, late activation is blue. Note that the time-colour coding in panel E is different from that in panels B and H. (K) In species with thick compact myocardium, surface breakthrough of the activation front is earlier in the apical region than in the base. (C, F, I, L) Graphs show the average activation time of the apex and base and the total ventricular activation time. Note that in zebrafish, Xenopus and Anolis, the ventricular base is activated earlier than the apex whereas in mice the ventricular base is activated later than the apex (* Significantly different (one-way ANOVA P<0.05)). n is 3, 6, 9 and 2, respectively. Scale bars in (B, E, H, K) indicate respectively 0.2, 1, 0.5 and 0.1 millimetre. avc, atrioventricular canal; ven, ventricle.doi:10.1371/journal.pone.0044231.g009
Results and Discussion
In adult lizards, the sequential chamber contractions and an atrioventricular delay are well-established , but we found no insulating plane or insulated atrioventricular node in Anolis (Fig. 2). Instead, the atrioventricular canal was entirely myocardial (Fig. 3). This differs from the adult hearts of mammals and birds, where the atrioventricular myocardium has largely disappeared and an insulating plane of fibrous-fatty tissue has ingressed between the atria and ventricles except at the atrioventricular node and His bundle, which provide the sole electrical communication between the atria and the ventricles , , . To explain the atrioventricular delay in reptiles, we hypothesized, therefore, that the atrioventricular canal of Anolis has a molecular phenotype that differs from that of the chambers.
Table 1. Ventricular activation pattern in ectothermic vertebrates investigated by optical mapping(OP), ECG(ECG), electrodes(E) or markers (M).doi:10.1371/journal.pone.0044231.t001
In embryos of mammals, birds, frog and fish, Nppa and/or Gja5 mark the rapid propagating atrial and ventricular chamber myocardium, whereas the atrioventricular canal is negative for these markers ,  (Nppa is lost in birds and all reptiles except turtles , ). Focusing on the atrioventricular gene programme during Anolis development, we found Tbx3 and Bmp2 to be expressed in the developing atrioventricular canal myocardium, exactly complementary to Gja5 in the adjacent chambers (Fig. 4A–D). Tbx5, known to promote differentiation into Nppa- and Gja5-expressing chamber myocardium , was also present in the atrioventricular canal and the chambers (Fig. S2) .
Figure 10. The trabecular myocardium is activated from base to apex in all vertebrates.
(A–C) The trabecular myocardium gives rise to the His-Purkinje system in mammals and birds and remains activated from base to apex. (C) On the epicardial surface of septated and thick-walled ventricles, as in the formed hearts of mammals and birds, activation is seen to occur from apex to base and the luminal base-to-apex activation is obscured.doi:10.1371/journal.pone.0044231.g010
We then examined the adult atrioventricular region in Anolis, and observed that Bmp2, which in mammals and birds is expressed in the atrioventricular canal only at embryonic stages, remained expressed throughout ontogeny (Fig. 4B,F). Tbx3, which marks the cardiac conduction system in mature mammals , was found within the same Bmp2-positive atrioventricular domain (Fig. 4C,G). Three-dimensional reconstructions of the expression patterns of Tbx3 in early developing hearts of Anolis, chicken and mammals revealed a striking resemblance (Fig. 5, Fig. S3). In all species, a similarly-shaped atrioventricular ring was observed. The Tbx3 expression domain extended into the sinus venosus, marking the sinus node primordium. This expression pattern did not substantially change in lizards just prior to hatching, whereas in mammals and birds, it became more complex with further development as the morphology of the heart, and particularly the sinu-atrial region, changed (Fig. 5).
We then pursued this building plan of the atrioventricular canal to older vertebrate classes, represented by Xenopus, an amphibian and thus a non-amniotic member of tetrapods, as well as zebrafish. In both species, the atrioventricular canal is composed of myocardium in continuity with the atrium and ventricle (Fig. 3). An insulating plane and an insulated atrioventricular node were not found (Fig. 2). In adult zebrafish and stage 48 Xenopus, we found Tbx2 in the atrioventricular canal and Nppa (whose spatio-temporal pattern strongly resembles that of Gja5 in mammals) in the atria and ventricles, the patterns resembling those of Tbx3 and Gja5, respectively, in the amniotic vertebrates (Figs. 6, 7). No cardiac Tbx3 expression was found (Fig. S4).
Next, we examined the His-Purkinje system in Anolis. Gja5 was used as marker for the mature His-Purkinje system conserved in mammals and chicken . The developing His bundle does not express Gja5 until late stages in mammals  and birds , but can nonetheless be identified very early by the expression of Tbx3 . In mouse  and chicken, the Tbx3-positive and Gja5-negative myocardium of the developing His bundle extends from the atrioventricular canal ventrally and dorsally into the ventricular myocardium and unto the crest of the ventricular septum (Fig. 5). The region formed by the dorsal and ventral extension and the crest is referred to as the primary ring . The Anolis ventricle is not septated, but shows Tbx3 expression into the ventricle ventrally and dorsally, indicating the presence of a primordial, but incomplete, primary ring that lacks the septal crest component (Fig. 5).
The ventricular wall in mammalian and avian embryos is composed of a trabecular inner layer and a thin compact outer layer. Initially, both of these layers express natriuretic peptides (Nppa and Nppb) and Gja5 , , but halfway through development, Nppa and Gja5 expression ceases in the strongly expanding compact layer. After birth (or hatching in birds) the expression of Nppa and Gja5 is limited to the His-Purkinje network that eventually constitutes only a small fraction of the ventricular mass (Fig. 8) , . The ventricular wall of fish, amphibian and reptilian hearts does not display such an overt distinction in expression pattern between an inner trabecular wall and a compact outer layer. Their ventricular wall typically is composed of a spongy or trabecular type of myocardium (Figs. 2, 3). In adult Anolis hearts, Gja5 was homogenously expressed throughout the trabecular ventricular wall. This suggests an absence of tracts of preferential conduction leading to the ventricular apex and such condition resembles that of early mammalian and avian embryos (Fig. 9). Further back in evolution, as represented by Xenopus and zebrafish, we observed homogenous Nppa expression in their trabecular ventricular wall (Fig. 9A,D), while Tbx3 did not identify a primordial atrioventricular bundle (Fig. S4).
Mammalian and avian hearts have an elaborate Gja5/Nppa-expressing ventricular conduction system that activates the ventricles from apex to base , , . The homogenous Gja5/Nppa expression patterns in Anolis, Xenopus and zebrafish trabecular ventricles suggest that the electrical activation front may spread from the vicinity of the atrioventricular canal, i.e. the ventricular base, and reach the apex later. Such an activation pattern would be reminiscent of early embryonic mammalian and avian ventricles . We used optical mapping to measure epicardial activation patterns in Anolis. The first point of activation always occurred in the cranial third of the ventricle, i.e. the base, and later at the apex (Fig. 9H). This activation pattern is consistent with most previous ECG and electrode investigations on reptiles and very similar to the activation patterns of chamber-forming hearts of mouse (E8–10) and chicken (E2–5) (Table 1) –. At these stages in mammals and birds, a morphologically distinct conduction system has yet to form and ventricular septation is only starting to take place , . Assuming that the dorsal and ventral activation patterns share the same time point of activation at the apex, we could synchronize the activation patterns and infer that the dorsal base is activated prior to the ventral base in the ectothermic vertebrates (Fig. 9B,E). Dorsal activation of the ventricular base has been reported in chicken hearts prior to septation and seemingly occurs in embryonic mouse as well , –. In Xenopus and zebrafish, the activation front travels from the dorsal base to the apex. In Anolis, Xenopus and zebrafish the location of the first point of activation varied within the dorso-basal region (Fig. S5). The activation maps and expression data indicate that the trabeculated ventricular wall of the ectothermic vertebrates function essentially as an isotropic conduction network. Consistently, conduction on the luminal surface of the adult mammalian and avian ventricles through the Gja5-positive His-Purkinje system also proceeds from base-to-apex , . On the epicardial side, however, the activation front reaches the apex first and then the base (Fig. 9J–L) . In mammals and birds, the developmental change in activation pattern to apex-to-base observed at the epicardial side coincides with the development of the Gja5-negative compact ventricular wall, which therefore may contribute to this developmental change in activation pattern.
Hearts of ectothermic species and of embryos of endothermic species do not have anatomically marked conduction system components. In this study we used expression patterns of conserved genetic markers and identified molecular conduction system components in developing and adult lizards. We found them to be similar to the components in embryonic mammals and birds, indicating they constitute an integral part of the building plan of the heart. Therefore, the conduction systems found in mature mammals and birds most likely evolved from the components of this shared building plan, and did not evolved independently.
In mature birds and mammals, left-over traces of the atrioventricular canal muscle in addition to the atrioventricular node can be found. Birds have a well-developed right-sided atrioventricular ring bundle that communicates with the ventricle anteriorly through the so-called recurrent branch , . The mammalian heart also maintains a molecularly distinct atrioventricular ring bundle above the insulating plane . Interestingly, in congenital corrected transposition of the human heart, the insulating plane disrupts the normal posterior atrioventricular communication, whereas the anterior communication is abnormally maintained , . The anterior part, then, resembles the recurrent branch of the bird heart.
The adult lizards and ectothermic vertebrates in general maintain important aspects of the embryonic vertebrate building plan. The Bmp2/4-Tbx2/3-positive, Gja5/Nppa-negative atrioventricular canal myocardium is maintained in adult ectothermic vertebrates. This provides an electrical insulation between atrium and ventricle in these hearts that lack an insulating plane of connective tissue. The developing hearts of mammals and birds have great tolerance to ischemia and regenerative potential, which is lost around birth , . Interestingly, many ectothermic vertebrates (e.g. newts and zebrafish) retain the regenerative capacity and ischemia tolerance throughout life . It is therefore tempting to speculate that the maintenance of important aspects of the embryonic programme in adult ectothermic vertebrates may be involved in the retention of these capacities.
Our study provides a plausible scenario of the evolution of the hearts of mammals and birds. The spongy myocardium of ectothermic adult vertebrates, as well embryonic mammals and birds allows for high ejection fractions and also serves to conduct the ventricular depolarization (Fig. 10). However, a transition to compact myocardium was necessary when pressure and heart rate increased. This rendered the early trabecules secondary on force generation, but available to differentiate into fibres of poor contractility and high propagation speeds. Furthermore, mammals and birds develop a compact ventricular septum whereby the early trabecules come to drape the septal surfaces and thus form the characteristic bundle branches of the His bundle (Fig. 10). Our study, therefore, suggests that the parallel evolution of virtually identical conduction systems and cardiac designs in birds and mammals can be traced back to the existence of a primordial conduction system of the ancestral reptile heart.
Gene program of the developing atrioventricular canal in chicken. Tbx5, known to induce Gja5, is present in the atrioventricular canal but Gja5 is absent where Bmp2 and Tbx3 are expressed. la, left atrium; lv, left ventricle; ra, right atrium; rv, right ventricle.
Gene program of the developing atrioventricular canal in Anolis. Despite expression of Tbx5 the atrioventricular canal does not initiate chamber program and expresses the transcription repressor Tbx3 along with Bmp2. avv, atrioventricular valves; la, left atrium; ra, right atrium; t, trachea (positive for Tbx3).
Tbx3 expression shown in interactive 3D pdfs. Tbx3 expression (yellow) and lumen cast (red) of all models used in Figure 5.
Tbx3 expression (in-situ hybridization) in developing and adult Xenopus. Tbx3 was only found outside the heart (e.g. developing trachea, t) of stage 40 embryos (top row). No Tbx3 was only found in the heart of adults (lower row). a, atrium; avc, atrioventricular canal; c, conus; ven, ventricle; t, developing trachea (positive for Tbx3).
Summary of individual ventricular activation maps. Red marks the earliest epicardial breakthrough of the activation front which was consistently at the ventricular base. Earliest and latest activation (red and white dots respectively) from each specimen is projected onto one specimen.
We thank Corrie de Gier-de Vries and Marc Sylva for technical assistance, Gert van den Berg for stimulating discussions, Jeroen Bakkers for providing zebrafish, and Tony Durston for providing adult Xenopus.
Conceived and designed the experiments: BJ AVP AFMM VMC. Performed the experiments: BJ BJDB QDG. Analyzed the data: BJ BJDB AFMM VMC. Contributed reagents/materials/analysis tools: MJBvdH AFMM VMC. Wrote the paper: BJ MJBvdH AFMM TW VMC.
- 1. Lillywhite HB, Zippel KC, Farrell AP (1999) Resting and maximal heart rates in ectothermic vertebrates. Comp Biochem Physiol A Mol Integr Physiol 124: 369–382. doi: 10.1016/s1095-6433(99)00129-4
- 2. Burggren W, Farrell A, Lillywhite H (2010) Vertebrate Cardiovascular Systems. Comprehensive Physiology. John Wiley & sons, Inc.
- 3. Christoffels VM, Smits GJ, Kispert A, Moorman AF (2010) Development of the pacemaker tissues of the heart. Circ Res 106: 240–254. doi: 10.1161/circresaha.109.205419
- 4. Davies F, Francis ET (1946) The conducting system of the vertebrate heart. Biol Rev Camb Philos Soc 21: 173–188. doi: 10.1111/j.1469-185x.1946.tb00323.x
- 5. Martinez-Palomo A, Alanis J (1980) The amphibian and reptilian hearts: impulse propagation and ultrastructure. In: Bourne GH, editor. Hearts and heart-like organs; Comparative anatomy and development. 1 ed. New York: Academic Press. 171–197.
- 6. Poss KD (2010) Advances in understanding tissue regenerative capacity and mechanisms in animals. Nat Rev Genet 11: 710–722. doi: 10.1038/nrg2879
- 7. Moorman AFM, Christoffels VM (2003) Cardiac chamber formation: development, genes and evolution. Physiol Rev 83: 1223–1267.
- 8. Naiche LA, Harrelson Z, Kelly RG, Papaioannou VE (2005) T-Box Genes in Vertebrate Development. Annu Rev Genet 39: 219–239. doi: 10.1146/annurev.genet.39.073003.105925
- 9. Kokubo N, Matsuura M, Onimaru K, Tiecke E, Kuraku S, et al. (2010) Mechanisms of heart development in the Japanese lamprey, Lethenteron japonicum. Evol Dev 12: 34–44. doi: 10.1111/j.1525-142x.2009.00389.x
- 10. Miquerol L, Beyer S, Kelly RG (2011) Establishment of the mouse ventricular conduction system. Cardiovasc Res 91: 232–242. doi: 10.1093/cvr/cvr069
- 11. Chi NC, Shaw RM, De VS, Kang G, Jan LY, et al. (2008) Foxn4 directly regulates tbx2b expression and atrioventricular canal formation. Genes Dev 22: 734–739. doi: 10.1101/gad.1629408
- 12. Aanhaanen WT, Mommersteeg MT, Norden J, Wakker V, de Gier-de Vries C, et al. (2010) Developmental origin, growth, and three-dimensional architecture of the atrioventricular conduction axis of the mouse heart. Circ Res 107: 728–736. doi: 10.1161/circresaha.110.222992
- 13. Yamada M, Revelli JP, Eichele G, Barron M, Schwartz RJ (2000) Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: evidence for BMP2 induction of Tbx2. Dev Biol 228: 95–105. doi: 10.1006/dbio.2000.9927
- 14. Ma L, Lu MF, Schwartz RJ, Martin JF (2005) Bmp2 is essential for cardiac cushion epithelial-mesenchymal transition and myocardial patterning. Dev 132: 5601–5611. doi: 10.1242/dev.02156
- 15. Auman HJ, Coleman H, Riley HE, Olale F, Tsai HJ, et al. (2007) Functional modulation of cardiac form through regionally confined cell shape changes. PLoS Biol 5: e53. doi: 10.1371/journal.pbio.0050053
- 16. Verhoeven MC, Haase C, Christoffels VM, Weidinger G, Bakkers J (2011) Wnt signaling regulates atrioventricular canal formation upstream of BMP and Tbx2. Birth Defects Res A Clin Mol Teratol 91: 435–440. doi: 10.1002/bdra.20804
- 17. Singh R, Hoogaars WM, Barnett P, Grieskamp T, Rana MS, et al.. (2011) Tbx2 and Tbx3 induce atrioventricular myocardial development and endocardial cushion formation. Cell Mol Life Sci.
- 18. Nieuwkoop PD, Faber J (1956) Normal table of Xenopus laevis (Daudin). 1 ed. Amsterdam: North-Holland Publishing Company.
- 19. Sanger TJ, Losos JB, Gibson-Brown JJ (2008) A developmental staging series for the lizard genus Anolis: a new system for the integration of evolution, development, and ecology. J Morphol 269: 129–137. doi: 10.1002/jmor.10563
- 20. Hamburger V, Hamilton JL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88: 49–92. doi: 10.1002/jmor.1050880104
- 21. Skovgaard N, Moller K, Gesser H, Wang T (2009) Histamine induces postprandial tachycardia through a direct effect on cardiac H2-receptors in pythons. Am J Physiol Regul Integr Comp Physiol 296: R774–R785. doi: 10.1152/ajpregu.90466.2008
- 22. Moorman AFM, Houweling AC, de Boer PAJ, Christoffels VM (2001) Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol. J Histochem Cytochem 49: 1–8. doi: 10.1177/002215540104900101
- 23. Aanhaanen WT, Brons JF, Dominguez JN, Rana MS, Norden J, et al. (2009) The Tbx2+ primary myocardium of the atrioventricular canal forms the atrioventricular node and the base of the left ventricle. Circ Res 104: 1267. doi: 10.1161/circresaha.108.192450
- 24. Moorman AFM, de Boer PAJ, Small EM, Krieg PA, Christoffels VM (2005) Evolutionary conservation of atrial natriuretic factor (Anf) expression, cardiac chamber formation and the heart-forming region. In: Artman M, Woodrow Benson D, Srivastava D, Nakazawa M, editors. Cardiovascular development and congenital malformations. Molecular & genetic mechanisms. Massachusetts: Blackwell Publishing, Inc. 84–87.
- 25. Takabatake Y, Takabatake T, Takeshima K (2000) Conserved and divergent expression of T-box genes Tbx2-Tbx5 in Xenopus. Mech Dev 91: 433–437. doi: 10.1016/s0925-4773(99)00329-9
- 26. Hoogaars WMH, Tessari A, Moorman AFM, de Boer PAJ, Hagoort J, et al. (2004) The transcriptional repressor Tbx3 delineates the developing central conduction system of the heart. Cardiovasc Res 62: 489–499. doi: 10.1016/j.cardiores.2004.01.030
- 27. Houweling AC, Somi S, van den Hoff MJ, Moorman AFM, Christoffels VM (2002) Developmental pattern of ANF gene expression reveals a strict localization of cardiac chamber formation in chicken. Anat Rec 266: 93–102. doi: 10.1002/ar.10042
- 28. Gibson-Brown JJ, Agulnik I, Silver LM, Papaioannou VE (1998) Expression of T-box genes Tbx2-Tbx5 during chick organogenesis. Mech Dev 74: 165–169. doi: 10.1016/s0925-4773(98)00056-2
- 29. Somi S, Buffing AA, Moorman AFM, van den Hoff MJB (2004) Dynamic patterns of expression of BMP isoforms 2,4,5,6, and 7 during chicken heart development. Anat Rec A Discov Mol Cell Evol Biol 279: 636–651. doi: 10.1002/ar.a.20031
- 30. Beyer EC (1990) Molecular cloning and developmental expression of two chick embyro gap junction proteins. J Biol Chem 265: 14439–14443.
- 31. Chomszynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159. doi: 10.1016/0003-2697(87)90021-2
- 32. Soufan AT, van den Berg G, Moerland PD, Massink MMG, van den Hoff MJB, et al. (2007) Three-dimensional measurement and visualization of morphogenesis applied to cardiac embryology. J Microsc 225: 269–274. doi: 10.1111/j.1365-2818.2007.01742.x
- 33. Mullen RK (1967) Comperative Electrocardiography of the Squamata. Physiological Zoo 40: 114–126.
- 34. Wessels A, Markman MWM, Vermeulen JLM, Anderson RH, Moorman AFM, et al. (1996) The development of the atrioventricular junction in the human heart. Circ Res 78: 110–117. doi: 10.1161/01.res.78.1.110
- 35. Kolditz DP, Wijffels MC, Blom NA, van der LA, Hahurij ND, et al. (2008) Epicardium-derived cells in development of annulus fibrosis and persistence of accessory pathways. Circulation 117: 1508–1517. doi: 10.1161/circulationaha.107.726315
- 36. Houweling AC, Somi S, Massink MPG, Groenen MA, Moorman AFM, et al. (2005) Comparative analysis of the natriuretic peptide precursor gene cluster in vertebrates reveals loss of ANF and retention of CNP-3 in chicken. Dev Dyn 233: 1076–1082. doi: 10.1002/dvdy.20423
- 37. Takei Y, Inoue K, Trajanovska S, Donald JA (2011) B-type natriuretic peptide (BNP), not ANP, is the principal cardiac natriuretic peptide in vertebrates as revealed by comparative studies. Gen Comp Endocrinol 171: 258–266. doi: 10.1016/j.ygcen.2011.02.021
- 38. Bruneau BG, Nemer G, Schmitt JP, Charron F, Robitaille L, et al. (2001) A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106: 709–721. doi: 10.1016/s0092-8674(01)00493-7
- 39. Koshiba-Takeuchi K, Mori AD, Kaynak BL, Cebra-Thomas J, Sukonnik T, et al. (2009) Reptilian heart development and the molecular basis of cardiac chamber evolution. Nature 461: 95–98. doi: 10.1038/nature08324
- 40. Bakker ML, Boukens BJ, Mommersteeg MTM, Brons JF, Wakker V, et al. (2008) Transcription factor Tbx3 is required for the specification of the atrioventricular conduction system. Circ Res 102: 1340–1349. doi: 10.1161/circresaha.107.169565
- 41. Gourdie RG, Green CR, Severs NJ, Anderson RH, Thompson RP (1993) Evidence for a distinct gap-junctional phenotype in ventricular conduction tissues of the developing and mature avian heart. Circ Res 72: 278–289. doi: 10.1161/01.res.72.2.278
- 42. Hall CE, Hurtado R, Hewett KW, Shulimovich M, Poma CP, et al. (2004) Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart. Dev 131: 581–592. doi: 10.1242/dev.00947
- 43. Christoffels VM, Moorman AF (2009) Development of the cardiac conduction system: why are some regions of the heart more arrhythmogenic than others? Circ Arrhythm Electrophysiol 2: 195–207. doi: 10.1161/circep.108.829341
- 44. Sedmera D, Reckova M, Rosengarten C, Torres MI, Gourdie RG, et al. (2005) Optical mapping of electrical activation in the developing heart. Microsc Microanal 11: 209–215. doi: 10.1017/s1431927605050452
- 45. Sedmera D (2011) Function and form in the developing cardiovascular system. Cardiovasc Res 91: 252–259. doi: 10.1093/cvr/cvr062
- 46. Rentschler S, Zander J, Meyers K, France D, Levine R, et al. (2002) Neuregulin-1 promotes formation of the murine cardiac conduction system. Proc Natl Acad Sci U S A 99: 10464–10469. doi: 10.1073/pnas.162301699
- 47. Valderrabano M, Chen F, Dave AS, Lamp ST, Klitzner TS, et al. (2006) Atrioventricular ring reentry in embryonic mouse hearts. Circulation 114: 543–549. doi: 10.1161/circulationaha.106.633727
- 48. Reckova M, Rosengarten C, DeAlmeida A, Stanley CP, Wessels A, et al. (2003) Hemodynamics is a key epigenetic factor in development of the cardiac conduction system. Circ Res 93: 77–85. doi: 10.1161/01.res.0000079488.91342.b7
- 49. Chuck ET, Meyers K, France D, Creazzo TL, Morley GE (2004) Transitions in ventricular activation revealed by two-dimensional optical mapping. Anat Rec A Discov Mol Cell Evol Biol 280: 990–1000. doi: 10.1002/ar.a.20083
- 50. Miquerol L, Meysen S, Mangoni M, Bois P, Van Rijen HV, et al. (2004) Architectural and functional asymmetry of the His-Purkinje system of the murine heart. Cardiovasc Res 63: 77–86. doi: 10.1016/j.cardiores.2004.03.007
- 51. Durrer D, van Dam RT, Freud GE, Janse MJ, Meijler FL, et al. (1970) Total excitation of the isolated human heart. Circulation 41: 899–912. doi: 10.1161/01.cir.41.6.899
- 52. Murakami T, Saito I, Mochizuki K (1985) The atrioventricular conducting system of the avian heart. Nihon Juigaku Zasshi 47: 73–79. doi: 10.1292/jvms1939.47.73
- 53. Anderson RH, Becker AE, Arnold R, Wilkinson JL (1974) The conducting tissues in congenitally corrected transposition. Circulation 50: 911–923. doi: 10.1161/01.cir.50.5.911
- 54. Moorman AFM, Soufan AT, Hagoort J, de Boer PAJ, Christoffels VM (2004) Development of the building plan of the heart. Ann N Y Acad Sci 1015: 171–181. doi: 10.1196/annals.1302.014
- 55. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabe-Heider F, et al. (2009) Evidence for cardiomyocyte renewal in humans. Science 324: 98–102. doi: 10.1126/science.1164680
- 56. Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, et al. (2011) Transient regenerative potential of the neonatal mouse heart. Science 331: 1078–1080. doi: 10.1126/science.1200708
- 57. Sizarov A, Ya J, de Boer BA, Lamers WH, Christoffels VM, et al. (2011) Formation of the building plan of the human heart: morphogenesis, growth, and differentiation. Circulation 123: 1125–1135. doi: 10.1161/circulationaha.110.980607
- 58. Sizarov A, Devalla HD, Anderson RH, Passier R, Christoffels VM, et al. (2011) Molecular Analysis of the Patterning of the Conduction Tissues in the Developing Human Heart. Circ Arrhythm Electrophysiol 4: 532–542. doi: 10.1161/circep.111.963421
- 59. Arbel ER, Liberthson R, Langendorf R, Pick A, Lev M, et al. (1977) Electrophysiological and anatomical observations on the heart of the African lungfish. Am J Physiol 232: H24–H34.
- 60. Azarov JE, Shmakov DN, Vityazev VA, Roshchevskaya IM, Roshchevsky MP (2007) Activation and repolarization patterns in the ventricular epicardium under sinus rhythm in frog and rabbit hearts. Comp Biochem Physiol A Mol Integr Physiol 146: 310–316. doi: 10.1016/j.cbpa.2006.10.036
- 61. Burggren WW (1978) Influence of intermittent breathing on ventricular depolarization patterns in chelonian reptiles. J Physiol 278: 349–364.
- 62. Chi NC, Shaw RM, Jungblut B, Huisken J, Ferrer T, et al. (2008) Genetic and physiologic dissection of the vertebrate cardiac conduction system. PLoS Biol 6: e109. doi: 10.1371/journal.pbio.0060109
- 63. Christian E, Grigg GC (1999) Electrical activation of the ventricular myocardium of the crocodile Crocodylus johnstoni: a combined microscopic and electrophysiological study. Comp Biochem Physiol A Mol Integr Physiol 123: 17–23. doi: 10.1016/s1095-6433(99)00024-0
- 64. Dillon S, Morad M (1981) A new laser scanning system for measuring action potential propagation in the heart. Science 214: 453–456. doi: 10.1126/science.6974891
- 65. Furman KI (1960) The electrocardiogram of the South African clawed toad (Xenopus laevis) with special reference to temperature effects. S Afr J Med Sci 25: 109–118.
- 66. Gray SW (1950) Pattern of contraction and relaxation in tortoise ventricle. Am J Physiol 162: 249–258.
- 67. Harris AS (1941) The Spread of Excitation in Turtle, Dog, Cat and Monkey Ventricles1. Am J Physiol 134: 319–332.
- 68. Heaton-Jones TG, King RR (1994) Characterization of the Electrocardiogram of the American Alligator (Alligator mississippiensis). Journal of Zoo and Wildlife Medicine 25: 40–47.
- 69. Lewis T (1916) The spread of the Excitatory Process in the Vertebrate Heart. Parts 1–5. Philosophical Transactions of the Royal Society of London 207: 221–310. doi: 10.1098/rstb.1916.0006
- 70. McDonald HS, Heath JE (1971) Electrocardiographic observations on the tuatara, Sphenodon punctatus. Comp Biochem Physiol A Comp Physiol 40: 881–892. doi: 10.1016/0300-9629(71)90277-5
- 71. Meek WJ, Eyster AE (1912) The Course of the Wave of Negativity which passes over the Tortoise’s Heart during the normal Beat. Am J Physiol 31: 31–46.
- 72. Mullen RK (1974) Electrocardiographic characteristics of four anuran amphibia. Comp Biochem Physiol A Comp Physiol 49: 647–654. doi: 10.1016/0300-9629(74)90892-5
- 73. Noseda V, Chiesa F, Marchetti R (1962) Intracardiac electrocardiography in fishes. Exp 18: 380–381. doi: 10.1007/bf02172263
- 74. Noseda V, Chiesa F, Marchetti R (1963) Vectocardigram of Anguilla anguilla L. and Pleurodeles waltlii Mich. Nature 197: 816–818. doi: 10.1038/197816a0
- 75. Peters JA, Mullen RK (1966) Electrocardiography in Caecilia guentheri (Peters). Physiological Zoo 39: 193–201.
- 76. Sedmera D, Reckova M, DeAlmeida A, Sedmerova M, Biermann M, et al. (2003) Functional and morphological evidence for a ventricular conduction system in zebrafish and Xenopus hearts. Am J Physiol Heart Circ Physiol 284: H1152–H1160.
- 77. Syme DA, Gamperl K, Jones DR (2002) Delayed depolarization of the cog-wheel valve and pulmonary-to-systemic shunting in alligators. J Exp Biol 205: 1843–1851.
- 78. Valentinuzzi ME, Hoff HE, Geddes LA (1969) Electrocardiogram of the snake: effect of the location of the electrodes and cardiac vectors. J Electrocardiol 2: 245–252. doi: 10.1016/s0022-0736(69)80084-1
- 79. Vaykshnorayte MA, Azarov JE, Tsvetkova AS, Vityazev VA, Ovechkin AO, et al. (2011) The contribution of ventricular apicobasal and transmural repolarization patterns to the development of the T wave body surface potentials in frogs (Rana temporaria) and pike (Esox lucius). Comp Biochem Physiol A Mol Integr Physiol 159: 39–45. doi: 10.1016/j.cbpa.2011.01.016
- 80. Vaikshnoraite MA, Belogolova AS, Vitiazev VA, Azarov I, Shmakov DN (2008) [Cardiac electric field at the period of depolarization and repolarization of the frog heart ventricle]. Zh Evol Biokhim Fiziol 44: 173–179. doi: 10.1134/s0022093008020084
- 81. Zhao-Xian W, Ning-Zhen S, Wei-Ping M, Jie-Ping C, Gong-Qing H (1991) An Analysis of Electrocardiogram of Alligator Sinensis. Comp Biochem Physiol 98A: 89–95. doi: 10.1016/0300-9629(91)90582-w