Skip Navigation

Cardiovascular Research 2004 63(1):77-86; doi:10.1016/j.cardiores.2004.03.007
© 2004 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
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
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Miquerol, L.
Right arrow Articles by Gros, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miquerol, L.
Right arrow Articles by Gros, D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Architectural and functional asymmetry of the His–Purkinje system of the murine heart

Lucile Miquerol*,a, Sonia Meysena, Matteo Mangonib, Patrick Boisc, Harold V.M van Rijend, Patrice Abrana, Habo Jongsmad, Joël Nargeotb and Daniel Grosa

aLaboratoire de Génétique et Physiologie du Développement, UMR 6545, Institut de Biologie du Développement de Marseille, Université de la Méditerranée, Marseille, France
bLaboratoire de Génomique Fonctionnelle, UPR 2580 Montpellier, France
cLaboratoire de Physiologie Générale, UMR 6558, Université de Poitiers, Poitiers, France
dDepartment of Medical Physiology, University Medical Center, Utrecht, The Netherlands

*Corresponding author. Tel.: +33-4-9126-9734; fax: +33-4-9126-9726. Email address: miquerol{at}ibdm.univ-mrs.fr

Received 6 January 2004; revised 19 February 2004; accepted 5 March 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: The aim of this work was to target a vital reporter gene in the mouse cardiac conduction system (CS) to distinguish this tissue from the surrounding myocardium in the adult heart. Methods: A transgenic mouse line has been created in which EGFP is expressed under the control of the Cx40 gene. Correlative investigations associating EGFP imaging and electrophysiological techniques were carried out on the adult heart and isolated cardiomyocytes. Results: In the heart of the Cx40EGFP/+ mice, EGFP signal was seen in the coronary arteries, the atria, the atrioventricular (AV) node and the His–Purkinje system. The latter was found to be structurally and functionally asymmetrical. The anatomical asymmetry was apparent in both the number of strands or fasciculi making up the His bundle branches (BBs) (1 strand on the right, 20 or so on the left), and the density (low on the right, high on the left) of the network of Purkinje fibers (PFs) that extends over the ventricular wall surfaces. The profiles of the electrical activation patterns recorded on the right and left flanks of the septum were also asymmetrical, mirroring the architecture of the branches. EGFP made it easy to identify the Purkinje cells in populations of dissociated cardiomyocytes and they were investigated using the patch-clamp technique. The hyperpolarization-activated current (If) was recorded in all spontaneously active Purkinje cells. Conclusions: This investigation provides positive evidence of the asymmetry of the His–Purkinje system of the adult mouse, and the first patch-clamp recording data on murine cardiac Purkinje cells. This mouse model opens up new perspectives for investigating the contribution of specific genes to the morphology and function of the His–Purkinje system.

KEYWORDS Conduction system; Connexins; Purkinje fiber; Transgenic animal models; Mouse


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
The rhythmic activity of the heart is set and coordinated by cardiac specialized tissues comprised of the sinoatrial (SA) node and the conduction system (CS), including the atrioventricular (AV) node, the His bundle and its two branches, and the peripheral Purkinje fibers (PFs). The architecture of the CS has been extensively described in the literature [1] and the asymmetry of the bundle branches (BBs) in the human heart and that of large mammals has been reported since the early 20th century [2]. Many cardiovascular diseases leading to fatal arrhythmias are due to anomalies of the architecture and/or function of the CS [3,4]. These anomalies result from genetic and/or environmental factors. The identification of these factors by their impact on the cardiac CS requires detailed morphological and functional investigations of this compartment. This in turn means that it is necessary to be able to clearly identify and possibly even isolate the cardiac CS from the surrounding working myocardium of animal models. The model of choice is the mouse that can be genetically modified. Surprisingly, the anatomy of the CS of the adult mouse is poorly known.

While from a histological point of view, the developing cardiac CS of the mouse has been intensively investigated [5], from the anatomical point of view, the CS of the adult mouse heart has only been very sketchily described and the most recent descriptions have been mainly limited to the proximal parts (AV node and His bundle) of this tissue [6]. On heart in toto, the use of histochemical techniques based on the detection of esterase activity has made it possible more recently to roughly visualize the thin right His BB [7,8]. However, for technical reasons, the visualization of the left BB is more difficult and requires more sophisticated techniques. Recently, genetically engineered mice have been created in which the myocytes of the cardiac CS express a reporter gene [9]. The cGATA6-lacZ transgene, driven by a heart-specific enhancer construct, is expressed in the proximal parts of the cardiac CS, and in the AV cushions [10]. Its expression in the adult heart has however not been described. The expression of the MC4/engrailed-lacZ transgene, which is integration site-dependent, resulted in one stable line in which lacZ appears to delineate the entire cardiac CS [11]. The MC4/engrailed-lacZ expression pattern is reminiscent of that of GLN2 observed in the human heart at comparable stages of development [12] but its expression area is broader. Expression of this transgene has not been described beyond the neonate stage. HF-1b-lacZ mice were created by introducing a lacZ gene into one of the loci of HF-1b, a gene essential for normal mouse growth [13]. In the adult heart, lacZ is expressed in the CS but also in the AV ring and the ventricular myocardium. The minK-lacZ mice were created by deletion of the minK gene [14]. MinK co-assembles with the pore-forming KvLQT1 protein to make a K+ channel. In the adult heart, minK-lacZ expression was found in the AV junction and in both the proximal and distal (BBs and PF network) parts of the CS [9,15]. All these investigations, with the exception of those concerning the minK-lacZ and HF-1b-lacZ mice, have paid little or no attention to the adult heart CS, either because they were focused on the development of CS during cardiogenesis, or because the transgene was not expressed at the adult stage, or both. In the HF-1b-lacZ mice, the expression of the reporter gene extends well beyond the CS which makes it impossible to clearly distinguish this compartment from the surrounding myocardium. Finally, at the adult stage, the cardiac CS would seem to be clearly visualizable and identifiable in the minK-lacZ mice only. However, the requirement of revealing the enzymatic activity of lacZ to identify the conductive myocytes kills them, and precludes any morphological and functional correlative investigations on living conductive cells. The identification and investigation of the cardiac CS on living material therefore necessitates the use of other murine models.

Among the molecular markers of the cardiac CS of the adult mouse figure connexin40 (Cx40) and connexin45 (Cx45). The characteristics of the expression pattern of Cx40 makes this Cx the best marker of the His–Purkinje system of the adult mouse [16]. Cx40 is strongly expressed in the working atrial myocytes, the central part of the AV node, the His bundle, and the distal elements of the CS [17]. It has never been detected in the ventricular myocytes. In addition, nul mutation of the Cx40 gene results in anomalies of impulse propagation at all levels of the CS, including the AV node, indicating that Cx40 plays a determinant functional role in electrical impulse transmission from the atria to the ventricles [18].

We have created transgenic mice in which the vital enhanced-green fluorescent protein (EGFP) was expressed under the control of the Cx40 gene. Investigation of the EGFP expression pattern, associated with electrophysiological techniques, made it possible to identify the His–Purkinje system of the adult mouse heart, and reveal its structural and functional asymmetry.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
The investigation conforms 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).

2.1. Generation of Cx40KIEGFP mice
The mouse Cx40 gene contains a single coding exon (exon 2) separated from the non-coding exon 1 by a large intron [19]. The targeting vector contained 2.7 and 5.7 kb of Cx40 genomic sequences flanking exon 2, as 5'- and 3'-homology arms (Fig. 1A). The EGFP coding sequence, followed by a pgk-neo cassette, was inserted in frame at the Cx40 start codon. Recombinant R1 embryonic stem (ES) cells were introduced into mouse blastocytes as described [20]. Homologous recombination at the Cx40 locus was identified by Southern blot analyses of Bgl II or Xba I digested DNA using 5' or 3' probes (Fig. 1B). Cx40EGFP/+ mice were maintained under a mixed genetic background (CD1/129Sv). Heterozygous offspring were intercrossed to obtain homozygously mutated progeny. PCR was employed to routinely genotype the mice using the following primers: 5'-CTCCAATTA ACTCCTTGTGAGCC-3' (Cx40, sense), 5'-AGGCTGAATGGTATCGCACC-3' (Cx40, antisense), 5'-CTTGCCGAA TATCATGGTGG-3' (neo, antisense).


Figure 1
View larger version (43K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Generation and characterization of Cx40EGFP/+ mice. (A) Diagrams illustrating the structure of the wild-type Cx40 locus (Cx40 WT), the targeting vector, and the knock-in allele (Cx40KIEGFP). Cx40 and EGFP coding regions and the pgk-neo cassette are represented by black, striped and dotted boxes, respectively. Black arrowheads and short thick lines indicate the position of LoxP sites and probes (5', 3' and neo), respectively. X and B indicate restriction sites for XbaI and BglII, respectively. (B) Southern blot experiments carried out with DNA extracted from recombinant ES cell clones (ES), or wild type (+/+) or heterozygous Cx40EGFP/+ (KI/+) mousetails. DNA was digested with BglII or XbaI. DNA fragments of 5.7 and 6.6 kb identify the wild-type allele; those of 7.8 and 8.7 kb, the recombined allele. A single DNA fragment was detected with the neo probe. (C) RT-PCR experiments carried out with RNA extracted from atria of Cx40EGFP/+ mice, and the Ex1–Ex2 or Ex1–EGFP primer pairs. The transcription of wild-type and recombined alleles is indicated by the amplification of 380 and 450 bp DNA fragments, respectively.

 
2.2. Transcriptional analysis of the transgene
Two micrograms of RNA extracted from atria of Cx40EGFP/+ adult mice were reverse transcribed as described [21]. The reactions of amplification, and the control experiments were also carried out as described [21]. The primers used, 5'-AGAGCAAATAACAGT-GGGCAGTTGA-3' (Ex1), 5'-ACCAGGCTGAATGGTATCG-3' (Ex2), 5'-AGAAGTCGT-GCTGCTTCATG-3' (EGFP), hybridized with Cx40 exon 1, Cx40 exon 2, and EGFP coding sequences, respectively.

2.3. Fluorescence imaging
Adult transgenic mice (8 weeks) were anesthetised by intraperitoneal injection of urethane (2g/kg body weight), then perfused through the dorsal aorta with PBS (37 °C). The heart and various other organs were examined, either in situ or after dissection, with an MZ10 stereomicroscope equipped for GFP detection. Hearts were also fixed in 4% (wt/vol) paraformaldehyde solution (1 h, 4 °C), frozen, and cryosectioned. Immunodetection of Cx40 was carried out as described [21]. Sections were examined with a fluorescence microscope (Zeiss Axiophot II).

2.4. ECG recording and mapping of impulse propagation
The techniques used for ECG recording, mapping in sinus rhythm the impulse propagation in the His BBs, and the calculation of the conduction velocities have been described previously [21,22].

2.5. In situ recording of action potentials
Hearts isolated from adult Cx40EGFP/+ mice were immersed in a standard solution containing (mmol/l): NaCl, 130; NaHCO3, 24; NaHPO4, 1.2; KCl, 4; CaCl2, 1.8; MgCl2, 1; glucose, 11; saturated with 95%O2 and 5%CO2 (pH 7.4, 35 °C). The atria were removed, the ventricular chambers opened, and the fluorescent cells were visualized with a stereomicroscope equipped for GFP detection. Action potentials (APs) were recorded with glass microelectrodes filled with 3 mol/l KCl (resistance: 15–30 M?) from EGFP-positive and -negative cardiomyocytes. The preparations were driven at pulse duration of 2 ms at 3 Hz. Acquisition and analysis of data were performed with the pClamp (Axon Instruments) and Origin (Microcal) software, respectively.

2.6. Patch-clamp recording of dissociated myocytes
Fluorescent cell networks of the ventricular chambers of Cx40EGFP/+ mice were dissected, pooled, and the cells were dissociated as described [23]. Cells were also dissociated from EGFP-negative subepicardial ventricular tissue. Dissociated cells were examined by phase-contrast and fluorescence microscopy. APs and If currents were recorded at 26 °C from isolated cardiomyocytes using the whole-cell configuration of the patch-clamp technique as described [23]. Membrane capacitance was also determined [23]. Recording electrodes, filled with a solution containing (mmol/l): KCl, 130; NaCl, 10; ATP-Na+ salt, 2; creatine phosphate, 6.6; GTP-Mg2+, 0.1; CaCl2, 0.04 (pCa 7); Hepes-KOH, 10; and adjusted to pH 7.2 with KOH, had a resistance of about 2.5 M{Omega}. Acquisition and analysis of data were performed as above.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Generation of Cx40EGFP/+ mice
Insertion of the EGFP coding sequence into the Cx40 gene locus was achieved by homologous recombination (Fig. 1A). The frequency of recombination events was 1:450. Genotyping of recombined ES cell clones, and heterozygous mutant mice indicated that the transgene was correctly inserted at a single site (Fig. 1B). Fragments of 380 and 450 bp were amplified from RNA extracted from the atria of mutant heterozygotes using the primer pairs Ex1–Ex2 and Ex1–EGFP, respectively (Fig. 1C). The size of these cDNA fragments indicated that both wild-type Cx40 and Cx40KIEGFP transcripts were present in the atrial tissue. No Cx40 gene product was detected in the homozygous mutant mice (not shown), confirming that the genotype of the heterozygous mice was indeed Cx40EGFP/+.

The Cx40EGFP/+ mice were viable, fertile and transmitted the KI allele to offspring at mendelian frequency. Their ECGs were similar to those of wild-type animals (not shown), as expected for mice expressing one wild-type allele of the Cx40 gene [22]. No gross anatomical defect has been observed in the heart of the mice investigated. Possible minor morphological alterations, such as those that occur at a low frequency in Cx40+/– mice and that vary with the genetic background [24], were not investigated.

3.2. EGFP reproduced in the adult heart the Cx40 expression pattern
Upon macroscopic examination of the organs of Cx40EGFP/+ mice, EGFP fluorescence was seen in the heart, the lungs, and the large vessels of the vascular system (Fig. 3C), i.e. in organs and tissues known to express Cx40 [25–27]. In the heart, fluorescence was observed in both atria and the coronary arteries (Fig. 2A). After excision of the atria, a strong signal was seen at the top of the IVS at the presumed site of the His bundle (Fig. 2B). EGFP expression sites in heart were further investigated on sections. In the SA node, where the cardiomyocytes do not express Cx40 [28], the only EGFP-positive structure was the nodal artery (Fig. 2C). In the AV node, fluorescence was seen in the cardiomyocytes of the central zone (Fig. 2D), and also more distally where numerous cells were EGFP-positive. These observations were in agreement with the distribution of Cx40 in the AV node [29]. Examination of sagittal sections revealed that the His bundle and its branches were strongly fluorescent (Fig. 2E). This was also the case with the PFs localized on the endocardial surfaces of the ventricular free walls (Fig. 2F). No fluorescent cardiomyocyte was ever seen within the walls or the IVS. In addition, immunofluorescence experiments have shown that both EGFP and Cx40 were co-expressed in the same cardiomyocytes (Fig. 3A,B), and the same coronary vessel endothelial cells (Fig. 3D). These results indicated that expression of EGFP faithfully reproduced in the heart the expression pattern of Cx40 [17].


Figure 3
View larger version (136K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Co-expression of Cx40 and EGFP proteins in cardiomyocytes and endothelial cells of Cx40EGFP/+ mice. (A, B) Panel A is a phase-contrast micrograph of a section in the atria. B is a merged picture of EGFP (green) and Cx40 (red) signals. The endocardial cells (arrowheads), and other cells (asterisks) that do not express Cx40, are EGFP-negative. (C) EGFP expression in the mesenteric vessels surrounding the intestine. (D) Merged picture of EGFP (green) and Cx40 (red) signals in a grazing section of a coronary artery. (m) Ventricular myocardium. Bars: 25 µm.

 

Figure 2
View larger version (97K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 EGFP imaging in the Cx40EGFP/+ mouse heart. (A) External view of an isolated heart. EGFP fluorescence is observed in both atria (a) and the coronary arteries (arrows). (B) Overhead view of the anterior part of the previous heart after excision of the atria. Asterisks indicate the entrance of the ventricular chambers. EGFP expression is observed at the top of the IVS (arrow). (C–F) Panel C illustrates a section in the right atrium at the level of the SA node (SAN). EGFP fluorescence is observed in the crista terminalis (CT), and the nodal artery (arrowhead). The nodal cells are free of signal. Panel D illustrates a section in the AV node (AVN). EGFP signal is seen in the cardiomyocytes of the central zone. IAS, interatrial septum. Expression of EGFP in the His bundle (HB) and the left (LBB) and right (RBB) branches is illustrated in panel E. EGFP signal is also seen in the Purkinje fibers (PF) localized on the endocardial surface of the ventricular free walls as shown in (F). LVW: left ventricular free wall; LVC: left ventricular chamber. Bars: 100 µm in (C, D, F); 200 µm in (E).

 
3.3. EGFP revealed extensive networks of Purkinje fibers in the ventricular cavities
Macroscopic examination of the endocardial surfaces of the ventricles of Cx40EGFP/+ mice revealed extensive and dense networks of EGFP-positive cells in both chambers. On the left septal flank (Fig. 4A), the network was composed of numerous thin strands (about 20) emerging from the His bundle, and running parallel toward the apex of the heart. The number of strands differed slightly from one mouse to another but they always covered the whole width of the septal surface. From the median part of the septum, the strands branched into a dense network of large and intermingled fascicles, forming a web overlying the apical part of the septum, as well as a large area of the left free wall. On the right septal flank (Fig. 3C), only one thin cylindrical fiber (sometimes two: 4 out of about 20 hearts examined) emerged from the His bundle, descended along the surface of the septum and intersected with the septal artery before reaching the base of the anterior papillary muscle. This branch then ramified into a web of interlaced fibers covering a small part of the right septal flank. From this web, a very few strands extended freely through the chamber to connect with a large network of peripheral fibers localized on the endocardial surface of the right free wall. Similar networks, no more extensive or dense, but with much more intense fluorescent signals, were also observed in Cx40EGFP/EGFP mice. These observations clearly indicated that the EGFP-positive cell networks seen in the left and right ventricular chambers were highly asymmetrical. This asymmetry is apparent both in the number of fasciculi making up the BBs and in the density of the Purkinje fibers network which extends over the apical parts of the IVS and the ventricular free walls. Comparison of observations carried out on cross sections of ventricles (not shown) with those described above suggested that, topographically, the fluorescent cell networks were constituted of PFs.


Figure 4
View larger version (112K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Structural and functional asymmetry of the His–Purkinje system. (A, C) EGFP-positive cell networks observed in the left (A) and right (C) ventricular chambers. In (A), the left ventricular free wall (LVW) was incised in the center from base to apex, then the two parts of the wall were pulled back on both sides to expose the left flank of the IVS (LF). The dotted line indicates the limits between the LF and the LVW. In (C), the whole right ventricular wall (RVW) was pulled back on the right. The dotted line indicates the limits between the right flank of the IVS (RF) and the RVW. Arrowhead indicates a fiber connecting the RF web to the RVW network. Small white circles indicate connecting fibers which have been cut. Insert shows details of the RBB (arrow) which emerged from the His bundle and intersected with the septal artery (star) and its ramifications. APM: anterior papillary muscle. Other legends are identical to those of Fig. 2. (B,D) Representative activation maps of the left (B) and right (D) BBs of Cx40EGFP/+ mice. The position of the electrode array on the septum flanks is indicated in the pictograms. Local electrograms recorded from the indicated electrodes (green and red dots) are shown, and stars indicate the specific BB signal. The large deflection represents activation of the septal myocardium. Color codes indicate local activation times (ms) relative to the atrial activation. Conduction velocities: 40 and 32 cm/s in (B) and (D), respectively.

 
3.4. Electrical activation patterns of the right and left His bundle branches
Fig. 4B and D are representative activation maps of the left and right BBs of Cx40EGFP/+ mice, respectively. They show clearly that the pattern of activation of the right branch and that of the left branch differ. Right BB activation was always recorded in a short and one- or two-electrode wide strand; left BB activation was always longer, wider and recorded in several electrode strands parallel to each other. Similar maps were obtained with wild-type mice [21,22]. These results are compatible with the structure of the BBs as revealed by EGFP expression. Conduction velocities were 36.5±2.1 (mean±S.E.M.; n=4), and 33.8±1.4 cm/s (n=4) for the left and right BBs, respectively. Similar values were previously calculated for wild-type mice [22].

3.5. Electrophysiological investigation of EGFP-positive ventricular cardiomyocytes
The in situ recorded APs from EGFP-positive myocytes of the right or left BBs were characterized by a rapid phase 1 repolarization and a very distinct plateau in phase 2 (Fig. 5A). In contrast, the APs of the EGFP-negative working myocytes of the ventricular free walls (VW) were triangulated and had no plateau (Fig. 5A). AP duration (APD) at 70% and 90% of repolarization of the myocytes of both BBs was significantly longer (35–50%) than that measured in the VW myocytes (Table 1). The electrophysiological characteristics of the working and EGFP-positive myocytes were further investigated after dissociation using the patch-clamp technique. The morphology of these cells is illustrated in Fig. 5B. EGFP-positive myocytes were elongated cells, easily distinguishable by their shape and aspect from working myocytes, resembling pacemaking cardiomyocytes isolated from mouse and rabbit SA node [23,30]. Their APs (n=25; N=4), elicited by suprathreshold current injections, always had longer duration than that recorded in isolated working myocytes (n=6; N=4) (Fig. 5C). Their membrane capacitance (27±3 pF, n=13; N=7) was lower than that of the working myocytes (65±20 pF, n=5; N=3). EGFP-positive myocytes frequently demonstrated slow spontaneous activity which was associated with "spike-and-dome" AP configurations and early-after depolarizations (EADs) (Fig. 5C). The hyperpolarization-activated current (If) was detected in all investigated conductive myocytes with spontaneous activity (n=9; N=3); it was never detected in working myocytes (n=5; N=3) (Fig. 5D).


Figure 5
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Electrophysiological characterization of ventricular myocytes. (A) Representative APs recorded in situ from EGFP-negative working myocytes of the ventricular free walls (VW), and EGFP-positive myocytes of the right (RBB) and left (LBB) BBs. (B) Representative phase-contrast (a, c) and corresponding fluorescence micrographs (b, d) of cardiomyocytes isolated from the VWs (a, b) and EGFP-positive cell networks (c, d). Bars: 10 µm. (C) Examples of whole-cell recordings of elicited APs from myocytes isolated from the VWs (a) and EGFP-positive cell networks (b). APs were elicited by a 3-ms-long 4 pA current injection. APs recorded from EGFP-positive cells with spontaneous activity are shown in (c) and (d). In (c), the AP displays a "spike-and-dome" configuration; the one in (d) demonstrates an EAD. (D) Whole-cell current recordings from isolated cardiomyocytes. The voltage protocol used to elicit If is shown in (a). Note the presence of If current in EGFP-positive cells with spontaneous activity (b). This current was never detected in the working myocytes (c).

 

View this table:
[in this window]
[in a new window]

  		Table 1 Parameters of APs recorded in EGFP-negative VW myocytes, and EGFP-positive myocytes of the RBB and the LBB

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
A transgenic mouse line expressing under the control of the Cx40 gene a vital fluorescent reporter gene, the EGFP gene, has been created. Expression of one allele of the reporter gene was sufficient to visualize the cell types known to express Cx40. In heart, EGFP signal was detected in the coronary arteries, the atria, and the CS, as expected.

4.1. Ventricular EGFP-positive cardiomyocytes are cardiac Purkinje cells
Examination of the endocardial surfaces of the ventricular cavities revealed dense networks of fluorescent cardiomyocytes. The distribution of these cells suggested that they were PFs. Their identity was confirmed by electrophysiological investigation. APs recorded in situ from EGFP-positive cardiomyocytes of either ventricle were similar to those recorded in the right His BB of the mouse heart [8] or the peripheral cardiac Purkinje cells of larger mammals [31]. In addition, when isolated, the ventricular EGFP-positive cardiomyocytes were spontaneously beating, and displayed EADs. Spontaneous activity was concomitant with the presence of the If current, a time-dependent inward current associated with the generation of pacemaker activity [32,33]. Spontaneous activity and If current have been previously recorded in the cardiac Purkinje cells of several other mammalian species [33]. Finally, electrical capacitance of these cells was low, and its value was similar to that determined in the spontaneous active cardiomyocytes of the mouse SA node [23] or the pacemaking spindle-shaped cells of the rabbit SA node [30]. All electrophysiological parameters thus indicate that the fluorescent cells that have been recorded are PFs, and this investigation provides the first patch-clamp recording data on this type of murine cardiomyocytes.

4.2. The His bundle branches of the murine heart are structurally and functionally asymmetrical
The asymmetry of the networks observed in the ventricular chambers of the mouse heart is reminiscent of the asymmetry of the His–Purkinje system described in detail in the human heart [1,2]. Asymmetry of the murine His–Purkinje system has been suggested [6] but has never been positively demonstrated. The structure of the right His BB, and its association with the septal artery, as they are described here, are similar to those recently reported after in toto acetylthiocholine staining [7,8]. No fluorescent myocyte penetrating into the ventricular walls was observed, suggesting that the mouse heart does not have intramural PFs in contrast to the chicken and ungulate heart [34–36].

At this point, the question arises as to whether all the myocytes of the His–Purkinje system can be detected by means of the EGFP signal. (i) Certain PFs could express only low amounts of Cx40, and EGFP, and consequently would not be visible in the heterozygous Cx40EGFP/– mouse heart. Comparative investigation of hetero- and homozygous mutant mouse hearts has revealed no significant difference in the structure of the His–Purkinje systems between the two genotypes. This indicates that a possible dose effect may play only a minor role in the visualization of the His–Purkinje system, and if it does, it might concern only a very small population of PFs. (ii) All PFs, as defined by their histological and electrophysiological characteristics, might not express Cx40, and EGFP. This is not unlikely but extremely difficult to check or prove. However, the electrical activation patterns of the BBs clearly indicated functional asymmetry of these structures that mirrored their structural asymmetry. At least for these proximal regions of the His–Purkinje system, the structure–function relationships argue against the idea that a large population of PFs would be undetectable. In addition, the similarity of the networks seen in the mouse with the His–Purkinje system as described in human suggests that at least the main structural components of the murine His–Purkinje system were visualized by EGFP expression.

4.3. EGFP does not alter the properties of the His–Purkinje system
EGFP represents a unique alternative to the β-galactosidase reporter gene because its visualization is noninvasive and can be monitored in real time in vitro and in vivo. However, depending upon its expression level, EGFP gene may or may not alter the properties of cells and tissues in which it is translated. Thus, the replacement of Cx47, a connexin expressed in oligodendrocytes, by EGFP results in no obvious morphological or behavioral abnormalities [37]. In contrast, high over-expression of EGFP in the heart of transgenic mice causes dilated cardiomyopathy [38]. This abnormality has not been seen in the heart of Cx40EGFP/+ mice where EGFP is not over-expressed. In contrast, several clues indicate that EGFP does not significantly interfere with the cardiac physiology of the Cx40EGFP/+ mice. The ECGs, the conduction velocities in the BBs, the profile of APs recorded in PFs of Cx40EGFP/+ mice were similar to those of both wild-type and Cx40–/+ mice [7,22]. These mice therefore provide a basis for specifically investigating the His–Purkinje system.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
The plasmids containing fragments of the mouse Cx40 gene were provided by Magali Théveniau-Ruissy and Sébastien Alcoléa. This work was funded by the CNRS, the Université de la Méditerranée, the European Community (contract QLG1-1999-00516) (DG, HJ), the Ministry of Education (ACI Biologie du Développement et Physiologie Intégrative) (DG, HJ), the Fondation de France (JN, DG), and the Netherlands Heart Foundation (Grant 99.200, HJ). LM and MM were recipients of postdoctoral fellowships of the Fondation pour la Recherche Médicale.


   Notes
 
Time for primary review 22 days


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

  1. Davies M.J, Anderson R.H, Becker A.E. The conduction system of the heart. (1983) London: Butterworth.
  2. Tawara S. Das Reizleitungssystem des Herzens. (1906) Jena: Gustav Fischer.
  3. Ho S.Y. Development of the cardiac conduction system. Chadwick D.J, Goode J, eds. (2003) vol. 250. Chichester: Wiley. 210–226. Novartis Foundation Symposium.[Medline]
  4. Benson W.D. Development of the cardiac conduction system. Chadwick D.J, Goode J, eds. (2003) vol. 250. Chichester: Wiley. 242–259. Novartis Foundation Symposium.[Medline]
  5. Virágh S, Challice C.E. The development of the conduction system in the mouse embryo heart: IV. Differentiation of the atrioventricular conduction system. Dev. Biol. (1982) 89:25–40.[CrossRef][Web of Science][Medline]
  6. Lev M, Thaemert J.C. The conduction system of the mouse heart. Acta Anat. (1973) 85:342–352.[Web of Science][Medline]
  7. Tamaddon H.S, Vaidya D, Simon A.M, Paul D.L, Jalife J, Morley G.E. High-resolution optical mapping of the right bundle branch in connexin40 knockout mice reveals slow conduction in the specialized conduction system. Circ. Res. (2000) 87:929–936.[Abstract/Free Full Text]
  8. Anumonwo J.M, Tallini Y.N, Vetter F.J, Jalife J. Action potential characteristics and arrhythmogenic properties of the cardiac conduction system of the murine heart. Circ. Res. (2001) 89:329–335.[Abstract/Free Full Text]
  9. Wessels A, Phelps A, Trusk T.C, et al. Development of the cardiac conduction system. Chadwick D.J, Goode J, eds. (2003) vol. 250. Chichester: Wiley. 44–67. Novartis Foundation symposium.[Medline]
  10. Davis D.L, Edwards A.V, Juraszek A.L, Phelps A, Wessels A, Burch J.B. A GATA-6 gene heart-region-specific enhancer provides a novel means to mark and probe a discrete component of the mouse cardiac conduction system. Mech. Dev. (2001) 108:105–119.[CrossRef][Web of Science][Medline]
  11. Rentschler S, Vaidya D.M, Tamaddon H, et al. Visualization and functional characterization of the developing murine cardiac conduction system. Development (2001) 128:1785–1792.[Abstract]
  12. Wessels A, Vermeulen J.L.M, Verbeek F.J, et al. Spatial distribution of "tissue-specific" antigenes in the developing human heart and skeletal muscle: III. An immunohistochemical analysis of the distribution of the neural tissue antigen GLN2 in the embryonic heart. Anat. Rec. (1992) 232:97–111.[CrossRef][Medline]
  13. Nguyêñ-Trân V.T.B, Kubalak S.W, Minamisawa S, et al. A novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages. Cell (2000) 102:671–682.[CrossRef][Web of Science][Medline]
  14. Kuperschmidt S, Yang T, Anderson M.E, et al. Replacement by homologous recombination of the minK gene with lacZ reveals restriction of minK expression to the mouse cardiac conduction system. Circ. Res. (1999) 84:146–152.[Abstract/Free Full Text]
  15. Kondo R.P, Anderson R.H, Kupershmidt S, Roden D.M, Evans S.M. Development of the cardiac conduction system as delineated by minK-lacZ. J. Cardiovasc. Electrophysiol. (2003) 14:383–391.[CrossRef][Web of Science][Medline]
  16. Myers D.C, Fishman G.I. Molecular and functional maturation of the murine cardiac conduction system. Trends Cardiovasc. Med. (2003) 13:289–295.[CrossRef][Web of Science][Medline]
  17. Miquerol L, Dupays L, Théveniau-Ruissy M, et al. Development of the cardiac conduction system. Chadwick D.J, Goode J, eds. (2003) vol. 250. Chichester: Wiley. 80–109. Novartis Foundation symposium.[Medline]
  18. Gros D, Dupays L, Alcoléa S, et al. Genetically-modified mice: tools to decode the functions of connexins in the heart, new models for cardiovascular research. Cardiovasc. Res. (2004) 62:299–308.[Abstract/Free Full Text]
  19. Seul K.H, Tadros P.N, Beyer E.C. Mouse connexin 40: gene structure and promoter analysis. Genomics (1997) 46:120–126.[CrossRef][Web of Science][Medline]
  20. Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder J.C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. (1993) 90:8424–8848.[Abstract/Free Full Text]
  21. Alcoléa S, Jarry-Guichard T, De Bakker J, et al. Replacement of connexin40 by connexin45 in the mouse. Impact on the cardiac electrical conduction. Circ. Res. (2004) 94:100–109.[Abstract/Free Full Text]
  22. Van Rijen H.V.M, van Veen T.A, van Kempen M.J, et al. Impaired conduction in the bundle branches of mouse hearts lacking the gap junction protein connexin40. Circulation (2001) 103:1591–1598.[Abstract/Free Full Text]
  23. Mangoni M.E, Nargeot J. Properties of the hyperpolarization-activated current (If) in isolated mouse sinoatrial cells. Cardiovasc. Res. (2001) 52:51–64.[Abstract/Free Full Text]
  24. Gu H, Smith F.C, Taffet S.M, Delmar M. High incidence of cardiac malformations in connexin40-deficient mice. Circ. Res. (2003) 93:201–206.[Abstract/Free Full Text]
  25. Gros D, Jarry-Guichard T, Ten Velde I, et al. Restricted distribution of connexin40, a gap junctional protein, in mammalian heart. Circ. Res. (1994) 74:839–851.[Abstract/Free Full Text]
  26. Yeh H.I, Dupont E, Coppen S, Rothery S, Severs N.J. Gap junction localization and connexin expression in cytochemically identified endothelial cells of arterial tissue. J. Histochem. Cytochem. (1997) 45:539–550.[Abstract/Free Full Text]
  27. Gustafsson F, Mikkelsen H.B, Arensbak B, et al. Expression of connexin 37, 40 and 43 in rat mesenteric arterioles and resistance arteries. Histochem. Cell Biol. (2003) 119:139–418.[Web of Science][Medline]
  28. Verheijck E.E, Van Kempen M.J.A, Veereschild M, Lurvink J, Jongsma H.J, Bouman L.N. Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution. Cardiovasc. Res. (2001) 52:40–50.[Abstract/Free Full Text]
  29. Coppen S.R, Severs N.J, Gourdie R.G. Connexin45 ({alpha}6) expression delineates an extended conduction system in the embryonic and mature rodent heart. Dev. Genet. (1999) 24:82–90.[CrossRef][Web of Science][Medline]
  30. Verheijck E.E, Wessels A, van Ginneken A.C.G, et al. Distribution of atrial and nodal Cells within the rabbit sinoatrial node: models of sinoatrial transition. Circulation (1998) 97:1623–1631.[Abstract/Free Full Text]
  31. Schram G, Pourrier M, Melnyk P, Nattel S. Differential distribution of cardiac ion channel expression as a basis for regional specialization in electrical function. Circ. Res. (2002) 90:939–950.[Abstract/Free Full Text]
  32. DiFrancesco D. Pacemaker mechanisms in cardiac tissue. Annu. Rev. Physiol. (1993) 55:455–472.[CrossRef][Web of Science][Medline]
  33. Anumonwo J.M, Jalife J. Cardiac electrophysiology. From cell to bedside. Zipes D.P, Jalife J, eds. (1995) St. Louis, Mo: Saunders. 151–164.
  34. Robb J.S. Comparative basic cardiology. (1965) New York: Grune and Stratton. 349–406.
  35. Truex R.C, Smythe M.Q. Comparative morphology of the cardiac conduction tissue in animals. Ann. N.Y. Acad. Sci. (1965) 127:19–33.[CrossRef][Medline]
  36. Oosthoek P.W, Virágh S, Lamers W.H, Moorman A.F.M. Immunohistochemical delineation of the conduction system: II. The atrioventricular node and Purkinje fibers. Circ. Res. (1993) 73:482–491.[Abstract/Free Full Text]
  37. Odermatt B, Wellershaus K, Wallraff A. Connexin-47 (Cx47)-deficient mice with enhanced green fluorescent protein reporter gene reveal predominant oligodendrocytic expression of Cx47 and display vacuolized myelin in the CNS. J. Neurosci. (2003) 213:4549–4559.
  38. Huang W.-Y, Aramburu J, Douglas P.S, Izumo S, et al. Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy. Nat. Med. (2000) 6:482–483.[CrossRef][Web of Science][Medline]

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


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
D. Gros, M. Theveniau-Ruissy, M. Bernard, T. Calmels, F. Kober, G. Sohl, K. Willecke, J. Nargeot, H. J. Jongsma, and M. E. Mangoni
Connexin 30 is expressed in the mouse sino-atrial node and modulates heart rate
Cardiovasc Res, September 4, 2009; (2009) cvp280v2.
[Abstract] [Full Text] [PDF]

Home page
 Circ Arrhythm ElectrophysiolHome page
V. M. Christoffels and A. F.M. Moorman
Development of the Cardiac Conduction System: Why Are Some Regions of the Heart More Arrhythmogenic Than Others?
Circ Arrhythm Electrophysiol, April 1, 2009; 2(2): 195 - 207.
[Full Text] [PDF]

Home page
 Circ. Res.Home page
B. J.D. Boukens, V. M. Christoffels, R. Coronel, and A. F.M. Moorman
Developmental Basis for Electrophysiological Heterogeneity in the Ventricular and Outflow Tract Myocardium As a Substrate for Life-Threatening Ventricular Arrhythmias
Circ. Res., January 2, 2009; 104(1): 19 - 31.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
N. J. Severs, A. F. Bruce, E. Dupont, and S. Rothery
Remodelling of gap junctions and connexin expression in diseased myocardium
Cardiovasc Res, October 1, 2008; 80(1): 9 - 19.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
K. Mesbah, Z. Harrelson, M. Theveniau-Ruissy, V. E. Papaioannou, and R. G. Kelly
Tbx3 Is Required for Outflow Tract Development
Circ. Res., September 26, 2008; 103(7): 743 - 750.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. Theveniau-Ruissy, M. Dandonneau, K. Mesbah, O. Ghez, M.-G. Mattei, L. Miquerol, and R. G. Kelly
The del22q11.2 Candidate Gene Tbx1 Controls Regional Outflow Tract Identity and Coronary Artery Patterning
Circ. Res., July 18, 2008; 103(2): 142 - 148.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
M. E. Mangoni and J. Nargeot
Genesis and Regulation of the Heart Automaticity
Physiol Rev, July 1, 2008; 88(3): 919 - 982.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
T. A.B. van Veen, H. V.M. van Rijen, M. J.A. van Kempen, L. Miquerol, T. Opthof, D. Gros, M. A. Vos, H. J. Jongsma, and J. M.T. de Bakker
Discontinuous Conduction in Mouse Bundle Branches Is Caused by Bundle-Branch Architecture
Circulation, October 11, 2005; 112(15): 2235 - 2244.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
K. W. Hewett, L. W. Norman, D. Sedmera, R. J. Barker, C. Justus, J. Zhang, S. W. Kubalak, and R. G. Gourdie
Knockout of the neural and heart expressed gene HF-1b results in apical deficits of ventricular structure and activation
Cardiovasc Res, August 15, 2005; 67(3): 548 - 560.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
A. Royer, T. A.B. van Veen, S. Le Bouter, C. Marionneau, V. Griol-Charhbili, A.-L. Leoni, M. Steenman, H. V.M. van Rijen, S. Demolombe, C. A. Goddard, et al.
Mouse Model of SCN5A-Linked Hereditary Lenegre's Disease: Age-Related Conduction Slowing and Myocardial Fibrosis
Circulation, April 12, 2005; 111(14): 1738 - 1746.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
G. E. Morley, S. B. Danik, S. Bernstein, Y. Sun, G. Rosner, D. E. Gutstein, and G. I. Fishman
Reduced intercellular coupling leads to paradoxical propagation across the Purkinje-ventricular junction and aberrant myocardial activation
PNAS, March 15, 2005; 102(11): 4126 - 4129.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
C. Marionneau, B. Couette, J. Liu, H. Li, M. E. Mangoni, J. Nargeot, M. Lei, D. Escande, and S. Demolombe
Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart
J. Physiol., January 1, 2005; 562(1): 223 - 234.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
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
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Miquerol, L.
Right arrow Articles by Gros, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miquerol, L.
Right arrow Articles by Gros, D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?