Skip Navigation

Correction for Franco and Icardo, Cardiovasc Res 50 (3) 613.
Cardiovascular Research 2001 49(2):417-429; doi:10.1016/S0008-6363(00)00252-2
© 2001 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Erratum (v50,p613)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Franco, D.
Right arrow Articles by Icardo, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Franco, D.
Right arrow Articles by Icardo, J. M.
Related Collections
Right arrowRelated Article
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2000, European Society of Cardiology

Molecular characterization of the ventricular conduction system in the developing mouse heart: topographical correlation in normal and congenitally malformed hearts

Diego Francoa,* and Jose Manuel Icardob

aDepartment of Experimental Biology, Faculty of Experimental and Health Sciences, University of Jaén, Paraje Las Lagunillas s/n, 23071 Jaén, Spain
bDepartment of Anatomy and Cell Biology, University of Cantabria, Santander, Spain

* Corresponding author. Tel.: +31-20-566-4926; fax: +31-20-697-6177 d.franco{at}ujaen.es

Received 16 June 2000; accepted 21 September 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Within the adult heart, it is convention to distinguish the conduction system and working (atrial and ventricular) myocardium. The adult conduction system (CS) comprises the sinoatrial (SAN), and atrioventricular (AVN) nodes, the atrioventricular bundle (AVB), the bundle branches and the peripheral Purkinje fibers, each of which display distinct functional properties and distinct profile of gene expression. Characterization of the mouse cardiac conduction system during development is rudimentary at present, even though genetically-modified mice are an increasing source of information regarding cardiac function and embryonic heart development. Methods: We have performed a detailed study of the pattern of expression of myosin heavy chain (MHC), myosin light chain (MLC), troponin I (TnI) isoforms, connexin 43 (Cx43), desmin and alpha-smooth muscle actin ({alpha}-SMA), in the ventricular conduction system of normal and congenitally malformed mouse hearts (iv background) from embryonic day 14.5 to 19.5. Results: The AVN is characterized by co-expression of MHC and MLC isoforms and no detectable expression of Cx43, desmin or {alpha}-SMA. The AVB expresses βMHC and MLC2v, but no {alpha}MHC, MLC2a, Cx43, desmin or {alpha}-SMA. The right and left bundle branches display enhanced expression of desmin and {alpha}-SMA but no Cx43. The normal expression profile is maintained in congenitally malformed hearts such as double-outlet right ventricle and common atrioventricular canal. Three-dimensional reconstruction of the conduction system shows normal arrangement of the bundle branches in congenitally malformed hearts, but abnormal location and/or extension of the AVN. Conclusions: Molecular characterization allows to follow the development of the CS in both, normal and malformed mouse hearts. Normal phenotypic expression of the CS is independent of heart situs but shows minor modifications in the presence of heart malformations. It is concluded that the AVN derives from the atrioventricular canal myocardium, the bundle of His from the ventricular myocardium, and the bundle branches from the ventricular trabeculations. Our results do not provide evidence to support an extra-cardiac origin of the ventricular CS.

KEYWORDS Conduction system; Congenital defects; Gene expression


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Within the cardiac muscle two types of myocardial components are conventionally distinguished: the conduction system (CS) and the working (atrial and ventricular) myocardium. The adult CS comprises separate morphological components which perform distinct functions. The sinoatrial (SAN) and atrioventricular (AVN) nodes are pacemaking and slow-conducting myocardial regions, whereas the atrioventricular bundle (bundle of His; AVB), the right and left bundle branches and the peripheral Purkinje fiber network (PPN), are fast-conducting pathways (for a review see Ref. [1]).

In the early tubular heart (‘primary myocardium’), no morphological components of the CS can be traced although there is already a leading pacemaker activity at the venous pole of the heart [2,3]. With further development, the heart acquires five different functional compartments [4] and a sequential activation of atrial and ventricular segments can be recorded as an ECG [3]. The origin of the cardiac CS is still debated. A myocardial origin of the CS has been advocated by us, a principal argument being that an ECG can already be recorded before neural crest cells have arrived in the heart [1,4]. Others have suggested that the CS has a neural crest origin [5–7].

The mouse has classically been considered an excellent model to study different aspects of cardiac function and heart development. Furthermore, genetically-modified mice are an increasing source of information for these purposes. Several studies have dealt with the morphological characterization of the murine CS [8,9], however, molecular characterization of the mouse CS is rudimentary at present [10–12]. It is widely documented that different sarcomeric genes, as well as some other muscle-enriched genes, are differentially expressed in the different parts of the CS myocardium of different species, including man [13,14]. For instance, co-expression of MHC isoforms is a characteristic of the nodal tissue (SAN and AVN) phenotype in several species including man [13,14]. Another characteristic of the nodal structures is the low of expression of Cx43 [15], which correlates with the slow conduction velocity [16–18] of this myocardial tissue.

We have therefore taken advantage of the fact that several genes are differentially expressed in other species and we have studied here the expression pattern of several molecular markers that could be useful in identifying the emergence of the different components of the CS in developing mouse embryos. Furthermore, these molecular markers would also be useful to identify the cellular origin of the CS. Lineage-tracing studies based on viral constructs have been used in the past to investigate the cellular origin of the components of the chick CS [19,20]. While identification of the cellular origin of the CS based on phenotypic characteristics is not unequivocal, it is not unreasonable to assume that, if the CS is of myocardial origin, differential expression of sarcomeric and muscle-enriched genes will delineate the different components of the ventricular CS as they differentiate from the rest of the myocardium.

We used in this study control mice, and mice of the iv/iv mutant strain, which display randomization of cardiac looping [21] and a large number of cardiac malformations [22,23]. Our results localize the developing ventricular CS and indicate that it derives from the myocardium. The present data also show that the expression profile of the different molecular markers is similar in normal and in congenitally malformed hearts (double-outlet right ventricle, DORV, and common atrioventricular canal, CAVC), indicating that neither cardiac situs nor the presence of malformations modifies substantially the differentiation program of the CS. We have also observed that the extension and location of the AVN are modified in most specimens with impaired atrioventricular development, but not in those with defective outflow tract septation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Embryos
Ten control C57BL6/J (Charles Rivers, Belgium) and 56 homozygous SI (iv mutant strain) [21] embryos ranging from embryonic day (E) 14.5 to E19.5 were analyzed. A total of 45 embryos were processed for immunohistochemistry and 21 embryos were processed for in situ hybridization. The day of vaginal plug was taken as E0.5. Embryos were excised from the uterus and the thoracic wall was partly removed to allow maximal penetration of fixatives. These experiments were performed with the approval of the ethical committee of the University of Amsterdam and conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Specimens were fixed overnight (4°C) in 4% freshly-prepared formaldehyde (in situ hybridization) or methanol/acetone/water (40:40:20 v/v; immunohistochemistry), rinsed twice in phosphate-buffered saline (PBS), dehydrated in increasing graded ethanol steps and embedded in paraffin. Serial sections of 7 µm were cut, mounted onto RNAse-free aminopropyltriethosixylane-coated slides (in situ hybridization) or poly-lysine-coated slides (immunohistochemistry) and stored at room temperature.

2.2 In situ hybridization
Complementary RNA probes against rat {alpha}-MHC [24,25], rat β-MHC [25], mouse MLC2a [26], mouse MLC2v [27], mouse slow skeletal troponin I [28], mouse cardiac troponin I [28] and rat connexin43 [15] mRNAs were radiolabelled with 35S-UTP by in vitro transcription according to standard protocols [29]. Hybridization conditions were as detailed elsewhere [29]. The specificity of the hybridization signal using these probes has been previously documented [15,28,32,34].

2.3 Immunohistochemistry
Serial sections were incubated with specific monoclonal antibodies against {alpha}- and β-MHC isoforms [14], MLC2a [26], MLC2v [30] (kindly provided by W. Franz, Lübeck, Germany), desmin (Monosan), {alpha}-smooth muscle actin ({alpha}-SMA, Sigma), and fibronectin (Brunschwig Chemie, Germany). In essence, the detection method used is as described by Wessels et al. [14]. Alternatively, detection of the primary antibody was performed using a goat anti-mouse β-galactosidase-coupled secondary antibody as described by Franco et al. [29]. The specificity of the antibodies has been previously described [14,33,35]. Moreover, the specificity of the immunohistochemical reaction can be monitored using the valve mesenchyme as internal negative control.

2.4 Three-dimensional reconstruction
The spatial distribution of the developing ventricular conduction system was studied in three-dimensional reconstructions. The contours of MHC and MLC distribution in serially incubated sections of the embryos were traced onto acetate sheets using a projection microscope and a camera lucida. Computer reconstructions were made using according to the method described by Verbeek et al. [31].


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
This study analyzes differential gene expression in the cardiac CS of the developing mouse. Since, at present, no molecular markers have been described in mice that can distinguish the SAN, the internodal tracts or the PPN from the working myocardium, the study has been restricted to the AVN, bundle of His and left/right bundle branches. We report findings in normal mice and in mice of the iv/iv mutant strain. First evidence of a morphologically definable ventricular CS is not observed until the stage E14.5, when the formation of the heart septa is yet incomplete. However, the separation between the working myocardium and conduction system myocardium cannot be fully established until the stage E17.5, when septation of the heart is finished. Consequently, data are presented considering two cardiac developmental periods: an early septation stage (E14.5–E16.5) and a late septation, fetal stage (E17.5–E19.5).

The expression patterns of the gene products analyzed in this study, as well as the topographical location of the ventricular CS, was similar in both control mice and in normal iv/iv mice (irrespective of heart situs). We report first the expression pattern in normal mouse embryos (findings summarized in Fig. 1) and, then, in congenitally malformed hearts. Most malformed hearts included in this study displayed double outlet right ventricle (DORV) and/or common atrioventricular canal (CAVC). These are the most common malformations observed in iv/iv mice and they often occur in association [22].


Figure 1
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Schematic representation of the patterns of expression in the fetal conduction system of the mouse heart as compared to the atrial and ventricular myocardium, respectively. Black filling indicates high expression level, gray filling indicates low expression level and no filling indicates no detectable expression. AVN, atrioventricular node; AVB, bundle of His; R/L BB, right and left bundle branches.

 
3.1 Pattern of gene expression during early cardiac septation (E14.5–E16.5)
First morphological evidence of the ventricular CS is observed at E14.5. At this stage, the precise final localization of the AVN cannot be assessed as the insulation of the atrial and ventricular chambers is not complete yet and, therefore, the atrioventricular canal remains partly as a distinct anatomical entity. The cardiac ventricles consist of two distinct myocardial components that show different morphological and molecular characteristics: a trabeculated layer and a compact layer. The trabeculations adjacent to the ventricular septum acquire a characteristic spatial conformation, running from the top of the ventricular septum to the ventricular apex (Fig. 2A,B). Desmin expression is high in the ventricular trabeculations, well above levels in the compact myocardium. Desmin allows identification of the developing left and right bundle branches along the left and right aspects of the ventricular septum (Fig. 2C,D). In contrast, the expression of MLC2v and slow skeletal troponin I is lower in the trabeculated component than in the compact myocardium (data not shown). Also at this stage, expression of βMHC is lower in the bundle of His and in the bundle branches than in the rest of the ventricular myocardium (Fig. 2A). In addition, {alpha}-SMA expression is negative in the bundle of His, but positive in the bifurcation of the two bundle branches (Fig. 2B). Interestingly, {alpha}-SMA expression is more intense in the compact than in the trabeculated myocardium.


Figure 2
View larger version (130K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Molecular delineation of the left/right bundle branches at E14.5 (A–D) and E16.5 (E) normal mouse hearts. Immunohistochemical detection of βMHC (A), {alpha}-SMA (B) and desmin (C–D). In situ hybridization in four-chambered sections against slow skeletal troponin I mRNA (E). Expression of βMHC is mostly confined to the ventricular myocardium at this stage. The expression in the right and left bundle branches is lower (arrows) than in the rest of the ventricular myocardium (A). Scattered expression of {alpha}-SMA is observed in the atrial and ventricular myocardium. Note that expression of {alpha}-SMA is enhanced in the bundle branches (arrows, panel B). Desmin expression is high in the bundle branches (arrow, panels, C,D) and in the early trabeculae (arrowheads, panels C,D). Expression of slow skeletal troponin I mRNA (ssTnI) is high in the working ventricular myocardium and low in the working atrial myocardium. Also, the expression of slow skeletal troponin I is lower in the bundle branches than in the working ventricular myocardium (arrowheads, panel E). RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; IVS, ventricular septum.

 
The differences in gene expression between the CS and the working myocardium become progressively highlighted. Thus, at the end of this developmental period (E16.5), desmin expression is very high in the bundle of His and in the left and right bundle branches, {alpha}-SMA delineates the left and right bundle branches and βMHC is expressed in all ventricular CS components. On the contrary, the expression of other genes, such as slow skeletal troponin I (Fig. 2E) and MLC2v mRNAs, displays a clear steep gradient of expression between the left/right bundle branches and the working myocardium.

3.2 Gene expression pattern in the fetal heart (E17.5–E19.5)
From E17.5 onwards, the differentiation pattern of the three components (AVN, AVB, left/right bundle branches) of the ventricular CS diverges from that of the working myocardium, and a clear separation can be established.

3.2.1 The AVN and the bundle of His
The AVN is located at the posterior (dorsal) aspect of the ventricular septum, just above the boundary between the atrial and the ventricular working myocardium. {alpha}MHC and MLC2a are mostly confined to the atrial myocardium, whereas βMHC and MLC2v are mostly confined to the ventricular myocardium (for a review see Ref. [32]). The myocardium forming the AVN is characterized by co-expression of {alpha}MHC and βMHC (data not shown) and of MLC2a and MLC2v transcripts (Fig. 3). The expression of MHC (data not shown) and MLC isoforms in the AVN is markedly lower than in the working myocardium (Fig. 3A–D). Such a pattern is reminiscent of that observed in the primary myocardium in the early stages of cardiac development. Other sarcomeric genes such as cardiac troponin I (Fig. 3C) and slow skeletal troponin I (data not shown) display similar expression levels in the AVN and in the working myocardium. The transition of the AVN myocardium into the bundle of His is marked by the lack of expression of MLC2a (Fig. 3A,D), although it shows low levels of MLC2v transcripts (Fig. 3B,E). The AVN and the bundle of His lack Cx43 mRNA expression (Fig. 3F). At the protein level, the AVN myocardium is characterized by co-expression of MHC (Fig. 4) and MLC (data not shown) isoforms. Expression of {alpha}MHC is higher in the AVN than in the surrounding ventricular working myocardium (Fig. 4A), whereas expression of βMHC is lower (Fig. 4B). The AVN shows faint levels of {alpha}-SMA whereas the adjacent blood vessels are highly positive (Fig. 4C). Expression of desmin (Fig. 4D) is similar in the AVN and in the working ventricular myocardium, being of no help in delineation of the AVN tissue. Desmin expression at these stages is higher in the atrial than in the ventricular myocardium.


Figure 3
View larger version (166K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Delineation of the AVN and bundle of His in the early fetal stage (E17.5) corresponding to normal mouse hearts. In situ hybridization in four-chambered sections against MLC2a (A,D), MLC2v (B,E), cardiac troponin I (C) and Cx43 (F) mRNAs. MLC2a expression is high in the atrial working myocardium, lower in the AVN (arrow, panel A,D), and undetectable in the bundle of His (dotted arrow, panel D). High expression of MLC2v is observed in the working ventricular myocardium and in the bundle of His (arrow, panel E), being somewhat lower in the AVN (arrow, panel B; dotted arrow, panel E). Expression of cardiac troponin I (cTnI) mRNA is homogeneous in the ventricular working and CS myocardium (panel C). The arrow in panel C indicates the AVN. In panel F, the absence of Cx43 mRNA in the AVN (dotted arrow) and bundle of His (arrow) allows definition of the boundary between the ventricular CS and working myocardium. RA, right atrium; RV, right ventricle; LV, left ventricle; IVS, interventricular septum.

 

Figure 4
View larger version (125K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Protein expression profile in the ventricular CS at late fetal stages (E18.5) corresponding to normal mouse hearts. Immunohistochemistry detection of {alpha}MHC (A,E), βMHC (B,F), {alpha}-SMA (C,G) and desmin (D,H). Panels A–D and E–H correspond to serial sections of the same heart. Expression of {alpha}MHC is clearly observed in the atrial myocardium and in the AVN (arrow, panel A), whereas just detectable levels of expression are observed in the bundle of His (arrow), bundle branches and the ventricular working myocardium (panel E). Expression of βMHC is observed in the ventricular myocardium, with barely detectable levels in the atrial myocardium (panel B). βMHC expression in the AVN (arrow, panel B), the bundle of His (arrow, panel F) and the bundle branches is distinctly weaker as compared to the working ventricular myocardium (panel F). Expression of {alpha}-SMA is observed mainly in the ventricular myocardium and the developing cardiac vessels (panel C). The AVN lacks expression of {alpha}-SMA (arrow, panel C) whereas only some peripheral cells of the bundle of His (arrow, panel G) are {alpha}-SMA positive. Enhanced expression is observed in the bundle branches (arrowheads, panel G). G' is a close-up of the boxed area in G. Desmin expression is higher in the atrial than in the ventricular myocardium (panel D). Similar expression levels are observed in the AVN (arrow, panel D) the bundle of His (arrow, panel H) and the ventricular working myocardium. Enhanced expression of desmin is observed in the bundle branches (arrowheads, panel H). H' is a close-up of the boxed area in H. RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; IVS, interventricular septum.

 
The bundle of His originates in the most posterior (dorsal) region of the ventricular septum and runs along the crest of the ventricular septum. The bundle of His can be identified because it shows a looser cell arrangement than the surrounding working myocardium and appears insulated by a fibronectin-rich matrix (data not shown). Within the bundle of His, {alpha}MHC (Fig. 4E) (protein and transcripts) is no longer expressed, whereas βMHC (protein and transcripts) displays a weaker expression than the surrounding myocardium (Fig. 4F). The MLC isoforms (protein and transcripts) show similar expression patterns to those observed for the MHC isoforms. MLC2a is no longer expressed in the bundle of His whereas MLC2v shows lower expression than the surrounding working ventricular myocardium. The bundle of His lacks expression of {alpha}-SMA except for some peripheral cells (Fig. 4G), shows similar desmin (Fig. 4H) and cardiac troponin I mRNA expression as the working myocardium, has lower levels of slow skeletal troponin I transcripts, and lacks Cx43 (data not shown).

3.2.2 Left and right bundle branches
The left and right bundle branches are located on either side of the ventricular septum, coursing from the top of the septum to the ventricular apex (Fig. 4). The expression of sarcomeric genes in the bundle branches is limited to those isoforms which are either expressed ubiquitously in myocardium (cardiac troponin I and slow skeletal troponin I) or those which are confined to the ventricular myocardium (βMHC and MLC2v). Thus, no expression of {alpha}MHC (Fig. 4E) or MLC2a is observed in the left/right bundle branches. Interestingly, βMHC (Fig. 4F), MLC2v, cTnI and ssTnI display a lower level of expression in the bundle branches than in the working myocardium. At the protein level, the differences observed for MHC and MLC isoforms are similar to those described at the mRNA level. In contrast, {alpha}-SMA (Fig. 4G) and desmin (Fig. 4H) levels are higher in the bundle branches than in the ventricular working myocardium.

3.3 Congenital heart disease in the iv background
We have analyzed 56 embryos derived from the iv/iv background ranging from E14.5 to E19.5. Twenty three specimens displayed normal cardiac morphology (situs solitus and situs inversus). The remaining 33 showed different types of congenital heart malformations, in line with previous reports [22,23]. We have centered our attention on the study of the molecular delineation of the ventricular CS in two types of cardiac malformations, double-outlet right ventricle (DORV) and common atrioventricular canal (CAVC): five specimens with isolated DORV, four specimens with isolated CAVC, and 11 specimens with CAVC associated with malformations in the arterial pole of the heart (DORV in nine cases and transposition of the great arteries, TGA, in two cases). A single specimen displayed anteroposterior disposition of the ventricular chambers, and the findings observed in this particular embryo are discussed separately. We have chosen specimens corresponding to the fetal stage (E17.5–E19.5) to clearly demarcate the boundaries of each component of the ventricular CS.

3.3.1 Ventricular conduction system in isolated DORV
The expression pattern of the different molecular markers in the distinct components of the ventricular CS in embryos with isolated DORV is similar to that observed in normal control embryos. Thus, the AVN is characterized by co-expression of MHC and MLC isoforms, the bundle of His is characterized by low expression levels of βMHC and MLC2v as compared to the surrounding ventricular working myocardium, and the left/right bundle branches are rich in desmin and {alpha}-SMA protein (data not shown). The topographical location of the bundle of His and of the left/right bundle branches is similar to that observed in normal hearts, although the extension (but not the location) of the AVN is reduced in some specimens with DORV. Also, the hearts with DORV display a more extensive network of desmin-positive and {alpha}-SMA-positive left/right bundle branches, probably due to anomalies in ventricular compactation.

3.3.2 Ventricular conduction system in CAVC
The molecular phenotype of the ventricular CS components in hearts with CAVC (isolated form or associated with other cardiac malformations), is similar to that observed in control embryos. The topographical location of the bundle of His and of the left/right bundle branches is tightly linked to the position of the ventricular septum (Fig. 5). The bundle of His remains located along the crest of the ventricular septum while the two bundle branches run along the surface of the ventricular septum (Fig. 5). Interestingly, the location and extension of the AVN is variable, depending on whether there is a symmetrical or an asymmetrical connection with the right and left ventricular orifices. In cases with an asymmetrical AV connection (e.g. double inlet left ventricle) (Fig. 5D–F), the AVN myocardium, characterized by the co-expression of MLC (data not shown) and MHC (Fig. 6A,B) isoforms, is larger than normal and can be seen extending longitudinally along the posterior wall of the atrioventricular junction. Most of the elongated AVN is located below the unfused dorsal endocardial cushion and, in some cases, part of the AVN myocardium is immersed into the cushion mesenchyme (Fig. 6C–F). The AVN shows a more heterogeneous composition, with cells loosely arranged. The AVN displays an overall normal expression of {alpha}-SMA (Fig. 6C,E) and desmin (Fig. 6D,F). However, some cells within the ANV and also in its periphery show {alpha}-SMA expression while the innermost cells do not show substantial staining (Fig. 6C–E).


Figure 5
View larger version (40K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Three-dimensional reconstruction of normal (A–C) and malformed (CAVC, D–F) E18.5 mouse embryonic hearts. Panels B and E correspond to dorsal views of the ventricular CS in normal and CAVC, respectively. Panels C and F correspond to ventral views of the same specimens. Green lining corresponds to the atrial myocardium; turquoise lining corresponds to the ventricular myocardium; red lining correspond to the vascular profiles. RA, right atrium; LA, left atrium; LV, left ventricle; AVN, atrioventricular node; LSCV, left superior cava vein; L, left side; R, right side.

 

Figure 6
View larger version (137K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Protein expression in E18.5 hearts with CAVC. Immunohistochemical localization of βMHC (A), {alpha}MHC (B), {alpha}-SMA (C,E) and desmin (D,F). The AVN is elongated and co-expresses {alpha}MHC (arrow, panel B) and βMHC (arrow, panel A). Desmin expression (panels D,F) is similar to that observed in the ventricular myocardium of normal hearts (see Fig. 2, panel D). The myocardial cells forming the AVN are more loosely arranged that the ventricular working myocardial cells. Peripheral cells of the AVN are {alpha}-SMA positive cells whereas only some scattered cells within the AVN are {alpha}-SMA positive (arrow, panel E). LA, left atrium; LV, left ventricle.

 
3.3.3 Ventricular conduction system in a heart with antero-posterior arrangement of the ventricular chambers
In this particular specimen, the right ventricle was located anterior to the left ventricle, the atrial septation was impaired, and the two atrial orifices drained into the left ventricle (Fig. 7). We were unable to delineate the AVN within the atrioventricular junction in this case. However, a loose arrangement of the myocardial cells, plus some molecular characteristics, such as the detection of low expression levels of βMHC (Fig. 7A,D), were found in a restricted area associated with the developing papillary muscles of the tricuspid valve (Fig. 7). This may represent an ectopic AVN/bundle of His complex, although not all molecular markers were consistent with the molecular delineation of the AVN. For instance, this area showed high expression levels of {alpha}-SMA (Fig. 7B,E) and the absence of {alpha}MHC expression (data not shown). Clearly, the location of this AVN is completely aberrant as compared to the rest of the congenitally malformed hearts. Molecular delineation of the left/right bundle branches was possible in this specimen by enhanced expression of desmin (Fig. 7F).


Figure 7
View larger version (141K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Transversal sections at the ventricular level corresponding to a E17.5 heart with anteroposterior arrangement of the ventricular chambers. Sections are immunostained for βMHC (A,D), {alpha}-SMA (B,E) and desmin (C,F). Panels D–F correspond to the areas marked in A–C. Arrows in A–C indicate an area of the developing tricuspid papillary muscle apparatus that shows looser cell arrangement and lower expression of βMHC than the surrounding myocardium. Expression of {alpha}-SMA (B) and desmin (C) is higher in this subset of cells than in the rest of ventricular myocardium. Arrows in D, E and F indicate the course of the left/right bundle branches; arrowheads indicate the ectopic position of the AVN-like myocardium. RV, right ventricle; LV, left ventricle; IVS, interventricular septum.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Differential gene expression within the adult working myocardium (for a review see Ref. [32]) and within the adult CS has been extensively documented (for a review see Ref. [1]). In the adult heart, sarcomeric genes such as MLC and MHC isoforms display restricted expression in the atrial and ventricular chambers, a pattern that becomes established in the prototypical embryonic stage when the heart is formed by five distinct functional domains; inflow tract, atrium, atrioventricular canal, ventricle and outflow tract [32–34]. However, at present little information is available regarding the molecular characteristics of the developing central CS in the mouse [10–12,36]. We have studied here the differential gene expression of the central ventricular CS during normal and abnormal mouse cardiogenesis. Using morphological and molecular characteristics, we have been able to distinguish the AVN, the bundle of His and the left/right bundle branches. Furthermore, within the limits of the techniques used (see Introduction), we have been able to establish a correlation between the fetal expression pattern profile of the components of the CS and their putative developmental origin.

Morphological studies in the developing mouse embryo have described the origin of the AVN from the embryonic atrioventricular canal (AVC) [9]. The AVN is characterized by co-expression of MLC and MHC isoforms (this study) and no detectable expression of Cx43 mRNA, in line with previous reports in the embryonic rat heart [15]. This expression profile is similar to that observed in the derivatives of the primary myocardium, i.e. inflow tract, AVC and outflow tract [15,17,33]. Unlike the inflow and outflow tracts, however, the AVN shares with the AVC myocardium the absence of {alpha}-SMA expression. Thus, our observations are in line with the notion that the AVN is derived from the embryonic AVC myocardium. In the canine heart, three distinct components have been described based on their distinct morphological and electrophysiological characteristics: a proximal AV bundle, an AV node and a distal AV bundle (also named His or AV bundle) [37]. The presence of a distinct proximal AV bundle has not been unequivocally demonstrated in other species, including humans [38,39], and we could not demonstrate it in the murine heart either.

The developmental origin of the bundle of His is still controversial. Previous morphological studies have suggested that the bundle of His originates from the AVC myocardium, as a caudal extension of the AVN [9,40]. However, other authors have proposed that the bundle of His differentiates in situ from the left sinus horn [41,42], or as an extension of the ventricular myocardium towards the AVN [9,43,44]. The present results indicate that the molecular characteristics of the bundle of His are different from those of the AVN. The molecular phenotype of the bundle of His shows most of the characteristics of the working ventricular myocardium, such as the absence of expression of sarcomeric atrial specific genes, although it does not fully reproduce the expression pattern of the ventricular myocardium (i.e. low expression of ventricular specific sarcomeric genes and the absence of Cx43). Our results suggest that the bundle of His originates from the ventricular myocardium, most probably from the apical portion of the ventricular septum.

There is also some controversy regarding the origin of the left and right bundle branches. Early morphological work indicated that the entire wall of the primitive heart acted as conducting tissue, conducting capabilities being later restricted to the trabecular layer [44]. It was suggested that, from these primitive trabeculae, the bundle of His and bundle branches, and the intramural and subendocardial Purkinje fibers, were retained as the definitive central conduction tissue [45]. Recent experiments in chicken indicate that trabecular muscle cells adjacent to the ventricular septum are transformed into specialized cells of the CS as the trabeculae coalesce to form the developing ventricular septum and the muscular trabecular cells withdraw from the cell cycle [45–47]. These studies agree with the present observations. While most early myocardial trabeculae are desmin-positive, desmin expression becomes progressively restricted to the trabeculations adjacent to the ventricular septum. These trabeculae are incorporated into the developing ventricular septum to form the right and left bundle branches, which remain desmin-positive at later stages. As pointed out by Moorman et al. [1], the coalescence of trabeculations may result in the formation of the left and right bundle branches. This notion is also underscored by the expression profile of Cx40 [12], Cx43 [15] and Cx45 [10,11] mRNAs during the ventricular cardiac development, although some controversy remains as to the extent of expression of Cx45 in the central conduction system in fetal stages [48].

In summary, these findings are in line with a developmental model in which the components of the central ventricular CS are derived from myocardial components of the embryonic heart [1], without the need of extra-cardiac non-myocardial components [7,49]. Gourdie et al. [20] have elegantly demonstrated that the peripheral Purkinje fibers become differentiated from a pre-existing pool of myocardial cells in the embryonic heart. Recently, a contribution of ventral neural crest cells entering through the venous pole of the heart to the vicinity of the developing AVN in chickens has suggested a possible role for these neural-derived cells in the morphological differentiation of the central CS [50]. This hypothesis is difficult to reconcile, in mice, given the fact that a normal morphological and molecular central CS is observed in splotch mutant mice (D. Franco, unpublished data), which are deficient in Pax3 gene expression and present alterations in the neural crest migratory pathway [51]. We suggest that neural crest cells are not required for normal differentiation of the central CS in mice, in line with recent cell lineage tracing studies in chicken embryos [19,20].

4.1 Conduction system and congenital heart disease (CHD)
Descriptive studies of the course of the adult CS have been reported in human CHD such as common atrioventricular canal (CAVC) [52–54], atrial septal defects (ASD) [55] ventricular septal defects (VSD) [56] and double inlet left ventricle (DILV) [56–58]. For example, a small AVN with a bundle of His of variable length appears to be common in CAVC in humans [58]. The AVN has been found displaced anteriorly and laterally to the VSD in cases with DILV [56], whereas it is displaced posteriorly in cases with ASD [55]. Based on morphological findings in pathological specimens some developmental considerations have been inferred [59,60]. The fact that many human CHD are accompanied by functional abnormalities of the CS [61] suggests the presence of morphological anomalies. However, scarce information is available about the course of the CS in CHD in mice. It has been recently documented that isomerism of the right atrial appendages is accompanied, both in humans and mice, by bilateral sinus nodes and by anomalies in the histological characteristics of the nodal tissue [8].

We have explored the molecular delineation of the ventricular CS in malformed mouse hearts. The molecular characteristics of the AVN, the bundle of His and the bundle branches are mostly similar in normal and malformed hearts, indicating that the basic facts of the developmental program of the CS are not modified by randomization of heart situs or by the occurrence of CHD. The fact that, in the presence of malformations of the AVC, {alpha}-SMA shows a non-uniform distribution within the AVN, suggests that myocardial cell recruitment and/or some final tuning in the differentiation process of the AVN may be altered. On the other hand, we have found modifications in the topography of the AVN. In our series, a large proportion of specimens with CAVC presented enlargement of the AVN. This contrasts with earlier observations carried out in humans with similar malformations [58]. We have also found that severe cases of CAVC, coursing with a common atrium, showed an AVN displaced to the posterior atrial wall. This agrees with previous observations in humans [53]. Curiously, in our series of hearts with isolated DORV, the molecular characteristics and the topography of the AVN were identical to those observed in control mice. This also agrees with the cases described in humans [62]. All these data reinforce the concept that the AVN develops from the AVC. The observation that the extension and location of the bundle of His are not affected in cases of isolated DORV (or of single VSD), reinforces the notion that the bundle of His represents a structure derived not from the embryonic AVC but from the ventricular myocardium. Minor contribution of the ventricular myocardium to the AVN cannot be discarded at the present time. However, it is unlikely since abnormal septation of the AVC leads to abnormal location and/or extension of the AVN, but not of the bundle of His and of the two bundle branches.

The special characteristics of the AVN in the heart with antero-posterior arrangement of the ventricular chambers deserve special attention. It is possible that, in extremely malformed hearts, where looping and the establishment of the atrioventricular connections are severely modified, not only the location but also some of the differentiation parameters of the AVN are modified. Whether this may be due to the loss of spatial information, to the presence (or absence) of specific differentiation signals, or to defects in the process of atrio-ventricular insulation, remains to be established. It is possible that establishment of an adequate differentiation program and of proper connections between the AVN and the bundle of His needs reciprocal interactions between the two components of the CS. However, the occurrence of a single extreme case does not allow definitive conclusions to be drawn.

Time for primary review 31 days.


   Acknowledgements
 
The authors wish to thank Dr Robert P. Thompson for access to unpublished observations and to Dr Antoon F.M. Moorman for critical reading of the manuscript. D. Franco is supported by NWO (902-16-219) and Dutch Heart Foundation (97206) grants. J.M. Icardo is supported by Ministry of Education and Culture (PB98-1418-C02-02) and ‘Marqués de Valdecilla’ Foundation (15/99) grants.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Moorman A.F.M., de Jong F., Denyn M.M.F.J., Lamers W.H. Development of the conduction system. Circ Res (1998) 82:629–644.[Free Full Text]
  2. Kamino K., Hirota A., Fujii S. Localization of pacemaking activity in early embryonic heart monitored using voltage-sensitive dye. Nature (1981) 290:595–597.[CrossRef][Medline]
  3. Van Mierop L.H.S. Localization of pacemaker in chick embryo heart at the time of initiation of heartbeat. Am J Physiol (1967) 212:407–415.[Free Full Text]
  4. Moorman A.F.M., Lamers W.H. Molecular anatomy of the developing heart. Trends Cardiovasc Med (1994) 4:257–264.[CrossRef][ISI]
  5. Gorza L., Saggin L., Sartore S., Ausoni S. An embryonic-like myosin heavy chain is transiently expressed in nodal conduction tissue of the rat heart. J Mol Cell Cardiol (1988) 20:931–941.[CrossRef][ISI][Medline]
  6. Gorza L., Vitadello M. Distribution of conduction system fibers in the developing and adult rabbit heart revealed by an antineurofilament antibody. Circ Res (1989) 65:360–369.[Abstract/Free Full Text]
  7. Gorza L., Vettore S., Vitadello M. Molecular and cellular diversity of heart system myocytes. Trends Cardiovasc Res (1994) 4:153–159.[CrossRef]
  8. Ho S.Y., Seo J.-W., Brown N.A., Cook A.C., Fagg N.L.K., Anderson R.H. Morphology of the sinus node in human and mouse hearts with isomerism of the atrial appendages. Br Heart J (1995) 74:437–442.[Abstract/Free Full Text]
  9. Viragh S., Challice C.E. The development of the conduction system in the mouse heart. Dev Biol (1982) 89:25–40.[CrossRef][ISI][Medline]
  10. Coppen S.R., Dupont E., Rothery S., Severs N.J. Connexin45 expression is preferentially associated with the ventricular conduction system in mouse and rat heart. Circ Res (1998) 82:232–243.[Abstract/Free Full Text]
  11. Coppen S.R., Severs N.J., Gourdie R.G. Connexin45 ({alpha}6) expression delineates and extended conduction system in the embryonic and mature rodent heart. Dev Genet (1999) 24:82–90.[CrossRef][ISI][Medline]
  12. Delorme B., Dahl E., Jarry-Guichard T., et al. Expression pattern of connexin gene products at the early developmental stages of mouse cardiovascular system. Circ Res (1997) 81:423–437.[Abstract/Free Full Text]
  13. De Groot I.J.M., Hardy G.P.M.A., Sanders E., Los J.A., Moorman A.F.M. The conducting tissue in the adult chicken atria: a histological and immunohistochemical analysis. Anat Embryol (1985) 172:239–245.[CrossRef][Medline]
  14. Wessels A., Vermeulen J.L.M., Virágh S., Kálmán F., Lamers W.H., Moorman A.F.M. Spatial distribution of ‘tissue specific’ antigens in the developing human heart and skeletal muscle. II. An immunohistochemical analysis of myosin heavy chain isoform expression patterns in the embryonic heart. Anat Rec (1991) 229:355–368.[CrossRef][Medline]
  15. van Kempen M.J.A., Vermeulen J.L.M., Moorman A.F.M., Gros D.B., Paul D.L., Lamers W.H. Developmental changes of connexin40 and connexin43 mRNA distribution patterns in the rat heart. Cardiovasc Res (1996) 32:886–900.[Abstract/Free Full Text]
  16. Argüello C., Alanis J., Pantoja O., Valenzuela B. Electrophysiological and ultrastructural study of the atrioventricular canal during the development of the chick embryo. J Mol Cell Cardiol (1986) 18:499–510.[CrossRef][ISI][Medline]
  17. De Jong F., Opthof T., Wilde A.A.M., et al. Persisting zones of slow impulse conduction in developing chicken hearts. Circ Res (1992) 71:240–250.[Abstract/Free Full Text]
  18. McGuire M.A., de Bakker J.M.T., Vermeulen J.T., et al. Atrioventricular junctional tissue. Discrepancy between histological and electrophysiological characteristics. Circulation (1996) 94:571–577.[Abstract/Free Full Text]
  19. Cheng C., Litchenberg W.H., Cole G.J., Mikawa T., Thompson R.P., Gourdie R.G. Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage. Development (1999) 126:5041–5049.[Abstract]
  20. Gourdie R.G., Mima T., Thompson R.P., Mikawa T. Terminal diversification of the myocyte lineage generates Purkinje fibers of the cardiac conduction system. Development (1995) 121:1423–1431.[Abstract]
  21. Hummel K.P., Chapman D.B. Visceral inversion and associated anomalies in the mouse. J Hered (1959) 50:9–13.[Free Full Text]
  22. Icardo J.M., Sanchez de Vega M.J. Spectrum of heart malformations in mice with situs solitus, situs inversus and associated visceral heterotaxy. Circulation (1991) 84:2547–2558.[Abstract/Free Full Text]
  23. Seo J.W., Brown N.A., Ho S.Y., Anderson R.H. Abnormal laterality and congenital cardiac anomalies. Relations of visceral and cardiac morphologies in the iv/iv mouse. Circulation (1992) 86:642–650.[Abstract/Free Full Text]
  24. Schiaffino S., Samuel J.L., Sassoon D. Non-synchronous accumulation of {alpha}-skeletal actin and β-myosin heavy chain mRNAs during early stages of pressure overload-induced cardiac hypertrophy demonstrated by in situ hybridization. Circ Res (1989) 64:937–948.[Abstract/Free Full Text]
  25. Boheler K.R., Chassagne C., Martin X., Wisnewsky C., Schwartz K. Cardiac expressions of {alpha}- and β-myosin heavy chains and sarcomeric {alpha}-actins are regulated through transcriptional mechanisms. J Biol Chem (1992) 267:12979–12985.[Abstract/Free Full Text]
  26. Kubalak S.W., Miller-Hance W.C., O'Briens T.X., Dyson E., Chien K.R. Chamber specification of atrial myosin light chain-2 expression precedes septation during murine cardiogenesis. J Biol Chem (1994) 269:16961–16970.[Abstract/Free Full Text]
  27. O'Brien T.X., Lee K.J., Chien K.R. Positional specification of ventricular myosin light chain 2 expression in the primitive murine heart tube. Proc Natl Acad Sci USA (1993) 90:5157–5161.[Abstract/Free Full Text]
  28. Ausoni S., de Nardi C., Moretti P., Gorza L., Schiaffino S. Developmental expression of rat cardiac troponin I mRNA. Development (1991) 112:1041–1051.[Abstract]
  29. Franco D, de Boer PAJ, de Gier-de Vries C, Lamers WH, Moorman AFM. Methods on in situ hybridization, immunohistochemistry and β-galactosidase reporter gene detection. Eur J Morphol 2000 (in press).
  30. Katus H.A., Hurrell J.G., Matsueda G.R., et al. Increased specificity in human cardiac-myosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay. Mol Immunol (1982) 19:451–455.[CrossRef][ISI][Medline]
  31. Verbeek F.J., Huysmans D.P., Baeten R.J.A.M., Schoutsen N.J.C., Lamers W.H. Design and implementation of a database and program for 3D-reconstruction from serial sections: a data-driven approach. Microsc Res (1995) 30:496–512.[CrossRef]
  32. Franco D., Lamers W.H., Moorman A.F.M. Patterns of gene expression in the developing myocardium: towards a morphologically integrated transcriptional model. Cardiovasc Res (1998) 38:25–53.[Free Full Text]
  33. Franco D., Markman M.W.M., Wagenaar G.T.M., Ya J., Lamers W.H., Moorman A.F.M. Myosin light chain 2a and 2v identifies the embryonic outflow tract myocardium in the developing rodent heart. Anat Rec (1999) 254:135–146.[CrossRef][Medline]
  34. Lyons G.E. In situ analysis of the cardiac muscle gene programme during embryogenesis. Trends Cardiovasc Med (1994) 4:70–77.[CrossRef][ISI]
  35. Ya J., Markman M.W., Wagenaar G.T., Blommaart P.J., Moorman A.F., Lamers W.H. Expression of the smooth-muscle proteins alpha-smooth-muscle actin and calponin, and of the intermediate filament protein desmin are parameters of cardiomyocyte maturation in the prenatal rat heart. Anat Rec (1997) 249:495–505.[CrossRef][Medline]
  36. Gourdie R.G., Kubalak S., Mikawa T. Conducting the embryonic heart: orchestrating development of specialized cardiac tissues. Trends Cardiovasc Med (1999) 9:18–26.[CrossRef][ISI][Medline]
  37. Racker D.K., Kadish A.H. Proximal atrioventicular bundle, atrioventricular node, and distal atrioventricular bundle are distinct anatomic structures with unique histological characteristics and innervation. Circulation (2000) 101:1049–1059.[Abstract/Free Full Text]
  38. Becker A.E., Anderson R.H. Conduction system of the heart. Wellens H.J., Lee K.I., Janse M.J., eds. (1976) Philadelphia: Lea and Febiger.
  39. Anderson R.H., Becker A.E., Brechenmacher C., Davies M.J., Rossi L. The human atrioventricular junctional area. A morphological study of the A-V node and bundle. Eur J Cardiol (1975) 3:11–25.[Medline]
  40. Mall F.P. On the development of the human heart. Am J Anat (1912) 13:249–298.[CrossRef][ISI]
  41. Doménech-Mateu J.M., Arnó-Palau A., Martínez-Pozo A. Desarrollo del nodo atrioventricular y del haz de His en el período embrionario humano. Res Esp Cardiol (1993) 46:421–430.
  42. Patten B.M. The development of the sinoventricular conduction system. Univ Mich Med Bull (1956) 22:1–21.[Medline]
  43. James T.N. Cardiac conduction system: Fetal and postnatal development. Am J Cardiol (1970) 25:213–226.[CrossRef][Medline]
  44. Viragh S., Challice C.E. The development of the conduction system in the mouse embryo heart. II. Histogenesis of the atrioventricular node and bundle. Dev Biol (1977) 56:397–411.[CrossRef][ISI][Medline]
  45. Robb J.S. The development of the sinoventricular conducting system. Univ Mich Med Bull (1956) 22:1–21.[Medline]
  46. Thompson R.P., Kanai T., Germroth P.G., et al. Developmental mechanisms of congenital heart disease. Clark E.B., Markwald R.R., Takao A., eds. (1995) Armonk, NY: Futura. 269–279.
  47. Thompson R.P., Soles-Rosenthal P., Cheng G. Proceedings 5th international symposium on etiology and morphogenesis of congenital heart disease. Clark E.B., Takao A., eds. (2000) Armonk, NY: Futura. in press.
  48. Alcoléa S., Théveniau-Ruissy M., Jarry-Guichard T., et al. Downregulation of connexin 45 gene products during mouse heart development. Circ Res (1999) 84:1365–1379.[Abstract/Free Full Text]
  49. Vitadello M., Vettore S., Lamar E., Chien K.R., Gorza L. Neurofilament M mRNA is expressed in conduction system myocytes of the developing and adult rabbit heart. J Mol Cell Cardiol (1997) 28:1833–1844.[CrossRef][ISI]
  50. Poelmann R.E., Gittenberger-de Groot A.C. A subpopulation of apoptosis-prone cardiac neural crest cells targets to the venous pole: multiple functions in heart development? Dev Biol (1998) 207:271–286.[CrossRef][ISI]
  51. Conway S.J., Henderson D.J., Copp A.J. Pax3 is required for cardiac neural crest migration in the mouse: Evidence from the splotch (Sp2H) mutant. Development (1997) 124:505–514.[Abstract]
  52. Ho S.Y., Fagg N., Anderson R.H., Cook A., Allan L. Disposition of the atrioventricular conduction tissues in hearts with isomerism of the atrial appendages — its relationship to congenitally complete heart block. J Am Coll Cardiol (1992) 20:904–910.[Abstract]
  53. Thiene G., Wenink A.C.G., Frescura C., et al. The surgical anatomy of the conduction tissues in atrioventricular defects. J Thorac Cardiovasc Surg (1981) 82:928–937.[ISI][Medline]
  54. Anderson R.H., Ho S.Y., Becker A.E. The surgical anatomy of the conduction tissues. Thorax (1983) 38:408–420.[Abstract]
  55. Lev M. Conduction system in congenital heart disease. Am J Cardiol (1968) 21:619–627.[CrossRef][ISI][Medline]
  56. Anderson R.H., Arnold R., Thaper M.K., Jones R.S., Hamilton D.I. Cardiac specialized tissues in hearts with an apparent single ventricular chamber (double inlet left ventricle). Am J Cardiol (1974) 33:95–106.[CrossRef][ISI][Medline]
  57. Wenink A.C.G. The conduction tissues in primitive ventricle with outlet chamber: two different possibilities. J Thorac Cardiovasc Surg (1978) 75:747–753.[Abstract]
  58. Essed C.E., Ho S.Y., Hunter S., Anderson R.H. Atrioventricular conduction system in univentricular heart of right ventricular type with right-sided rudimentary chamber. Thorax (1980) 35:123–127.[Abstract]
  59. Doménech-Mateu J.M., Arnó-Palau A., Martínez-Pozo A. Study of the development of the atrioventricular conduction system as a consequence of observing an extra atrioventricular node in the normal heart of a human fetus. Anat Rec (1991) 230:73–85.[CrossRef][Medline]
  60. Wenink A.C.G. Proceedings of the IX Congreso della Societa Italiana de Cardiologia Pediatrica. (1979).
  61. Blink-Boelkens M.T.E., Begstra A., Landsman M.L.J. Functional abnormalities of the conduction system in children with an atrial septal defect. Int J Cardiol (1988) 20:263–272.[CrossRef][ISI][Medline]
  62. Bharati S., Lev M. The conduction system in double outlet right ventricle with pulmonary ventricular septal defect and related hearts (the Taussing–Bing Group). Circulation (1976) 54:459–467.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?

Related Article

Erratum to: Molecular characterization of the ventricular conduction system in the developing mouse heart: topographical correlation in normal and congenitally malformed hearts: [Cardiovascular Research 2001;49:417–429]
Diego Franco and Jose Manuel Icardo
Cardiovasc Res 2001 50: 613. [Extract] [Full Text] [PDF]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
J. N. Dominguez, A. de la Rosa, F. Navarro, D. Franco, and A. E. Aranega
Tissue distribution and subcellular localization of the cardiac sodium channel during mouse heart development
Cardiovasc Res, April 1, 2008; 78(1): 45 - 52.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Erratum (v50,p613)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Franco, D.