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Cardiovascular Research Advance Access originally published online on January 4, 2008
Cardiovascular Research 2008 78(1):45-52; doi:10.1093/cvr/cvm118
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Tissue distribution and subcellular localization of the cardiac sodium channel during mouse heart development

Jorge N. Domínguez, Ángel de la Rosa, Francisco Navarro, Diego Franco and Amelia E. Aránega*

Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Paraje de las Lagunillas, s/n, Jaén 23071, Spain

* Corresponding author. Tel: +34 953 212604; fax: +34 953 211875. E-mail address: aaranega{at}ujaen.es

Received 10 April 2007; revised 12 December 2007; accepted 19 December 2007

Time for primary review: 26 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: The aim of this study was to analyse the mRNA expression levels and protein distribution of the cardiac sodium channel Scn5a/Nav1.5 during mouse cardiogenesis.

Methods and results: Scn5a mRNA levels were determined by real-time RT–PCR using embryonic hearts ranging from E9.5 to E17.5 as well as postnatal and adult hearts. In addition, Scn5a protein (Nav1.5) distribution was analysed by immunohistochemistry and confocal microscopy. Scn5a mRNA levels displayed a peak at stage E11.5, decreased during the subsequent stages and then steadily increased from E17.5 onwards, and throughout the postnatal to the adult stages. Immunohistochemistry experiments revealed comparable distribution of Nav1.5 between the different cardiac chambers at early embryonic stages. During the foetal stages, Nav1.5 showed an enhanced expression in the trabeculated myocardium and in the bundle branches. At the subcellular level, Nav1.5 and Scn1b double-immunostaining analysis is consistent with the presence of both sodium channel subunits in the T-tubule system and the intercalated discs.

Conclusion: Our results demonstrate that the cardiac sodium channel, Nav1.5, shows a dynamic expression pattern during mouse heart development, indicating that it could play an important role in the acquisition of a mature pattern of conduction and contraction during cardiogenesis.

KEYWORDS Ion channels; Gene expression; Cell differentiation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
The heart is the first functional organ during embryonic development and is initially formed as a simple linear tube located at the ventral midline of the embryo. This embryonic heart initiates rhythmic contractions at about the eight to nine somite stage [mouse embryonic day 8 (E8)]1 and begins to function as a peristaltic pump despite lacking valves and conduction system.1,2 During subsequent development, the tube undergoes a complex series of movements and tissue remodelling events that lead to the formation of the mature synchronously contracting four-chambered heart.

Synchronous contraction of the heart is coordinated by conduction of electrical excitation through the specialized tissues of the cardiac conduction system (CCS).2 This rhythmic sequence of activation initiates at the sinoatrial node (SAN) and is conducted as an action potential across the atrial chambers to the atrioventricular node (AVN). With a delay in the AVN the impulse is then rapidly transmitted, via the bundle branches, to the Purkinje fibres to ensure a coordinated apex-to-base activation of the ventricular myocardium.

In the adult heart, voltage-gated Na+ channels determine the amplitude and slope of the action potential upstroke in atrial and ventricular myocytes.3 The sodium channel is a multi-subunit protein complex composed of a single large {alpha}-subunit along with additional smaller β-subunits.4 Eleven genes encoding sodium channel {alpha}-subunits are widely expressed in excitable tissues.4,5 Although other sodium channel {alpha}-subunits, such as Nav1.1, Nav1.3, and Nav1.6, have been reported in isolated ventricular myocytes from adult mammalian heart,6–8 Nav1.5 is the major {alpha}-subunit in the heart, being called the cardiac sodium channel.9 Scn5a encodes the voltage-dependent sodium channel subunit protein Nav1.53,10 and is responsible for the generation of the inward sodium current (INa) that underlies excitability and conduction in working myocardium and cardiac conduction tissue cells.11,12 Mutations in the Scn5a sodium channel {alpha}-subunit gene are associated with multiple arrhythmogenic syndromes, including long QT syndrome,10 Brugada syndrome,13 an inherited cardiac conduction defect (Lenegre disease)14,15 in addition to with dilated cardiomyopathy with atrial fibrillation.16

Differential expression and localization of sodium channel subunits are likely to be important determinants of electric excitability of cardiac myocytes. It is well-documented that relevant electrophysiological and gene expression changes occur during the embryonic development of mammalian hearts.1 Recently, we have described the expression pattern of the β1 sodium channel subunit, Scn1b, during mouse heart development.17 However, scant information is available about the expression pattern and subcellular localization of the primary sodium channel subunit isoform, Nav1.5, during cardiogenesis.

The purpose of this study is to systematically analyse mRNA expression levels and protein distribution of the cardiac sodium channel subunit, Scn5a/Nav1.5, during mouse heart development. Our results demonstrate a dynamic mRNA expression pattern of Scn5a during cardiogenesis. At the protein level Nav1.5 is distributed throughout atrial and ventricular myocardium during development, showing higher expression levels in the bundle branches and Purkinje fibres of the CCS. Moreover, Nav1.5 expression is higher in the atrial chambers than in ventricles from postnatal stages onwards. These results suggest that Nav1.5 could play an important role in the establishment of ventricular activation during development. In addition, coexpression of Nav1.5 and the β1-subunit Scn1b in T-tubules system and intercalated discs of adult cardiac fibres indicate that β1-subunit could modulate Nav1.5 function.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Tissue preparation
Balb/c embryos ranging from embryonic day (E) 9.5 to E18.5 were removed from the uteri of time-controlled pregnant females. The day of vaginal plug was taken as E0.5. Adult and neonatal (1 and 15 days) hearts were also obtained.

For RNA isolation, hearts were dissected, including myocardial components of outflow and inflow tracts, and stored in liquid nitrogen. For immunohistochemistry experiments, four embryos from each stage were processed as previously described.17 The procedures used in this investigation conform to 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).

2.2 mRNA extraction and reverse transcription
Total RNA was isolated and reverse transcribed from pooled hearts at each stage (n = 5 per stage), ranging from E9.5 to E17.5 embryos, and from neonate and adult mice (n = 3 per stage) as previously described.17

2.3 Quantitative real-time polymerase chain reaction
Quantitative real-time PCR was performed in a I-Cycler thermocycle (Bio-Rad) using a SYBR Green PCR kit (Bio-Rad). The primers used to detect mouse Scn5a, Nkx2.5, and β-actin (summarized in Table 1) were obtained from Genotek (Bonsai Technologies Group). Reactions were performed as described previously.17


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  		Table 1 Primers used for quantitative real-time polymerase chain reaction analysis

 
The annealing temperature used was 61.7ºC for Scn5a and β-actin, and 64ºC for Nkx2.5. Each PCR reaction was performed in triplicate and repeated at least five times to obtain a representative average. The reactions were assayed during their exponential phase. The expression of Scn5a was normalized to β-actin. In addition, the relative levels of expression of the cardiac sodium channel were also calculated as the ratio of the expression of Scn5a and the cardiac-specific transcription factor Nkx2.5 for hearts at E12.5, E15.5, and E17.5 stages. PCR products were verified by sequencing.

2.4 Immunohistochemistry
Serial tissue sections from littermates were cut for parallel analysis and the distribution of sodium channel {alpha}- and β-subunits and marker proteins were analysed by immunohistochemistry and confocal microscopy as previously described.17 The anti-Nav1.5 antibody (developed in rabbit, Alomone Labs) was generated against the peptide corresponding to residues 493–511 (Accession P15389 [GenBank] ) of I/II intracellular loop domain of Nav1.5.18 The Anti-Nav1.5, developed in goat, and anti-Scn1b were obtained from Santa Cruz and Cell Applications, respectively. Anti-desmin and anti-{alpha}-actinin were purchased from Sigma; anti-connexin 40 was obtained from Gentaur Molecular Products, and anti-connexin 43 was kindly gifted by Lucile Miquerol (IGBD, Marseille, France). Nuclear staining was performed using DRAQ-5TM (Red Fluorescent Cell-Permeable DNA probe, Biostatus Limited).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Scn5a mRNA expression during mouse heart development
In this study, we investigated the mRNA expression levels of the cardiac sodium channel, Scn5a, during mouse cardiogenesis by quantitative RT–PCR. Scn5a mRNA was detected as early as stage E9.5, showing low expression levels (Figure 1A and B). An enhanced expression of Scn5a mRNA is found in intermediate embryonic stages, peaking at stage E11.5 and subsequently decreasing around E12.5. However, increased levels are again observed in later foetal stages (E17.5) and throughout postnatal and adult stage (Figure 1A and B).


Figure 1
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  		Figure 1 Scn5a mRNA expression levels during mouse heart development as measured by quantitative real-time polymerase chain reaction. Data are expressed as ratio of Scn5a mRNA to β-actin mRNA (A and B) and as ratio of Scn5a mRNA to β-actin and Nkx2.5 (C). Differences in Scn5a mRNA levels are expressed as percentages relative to E17.5 values in (A and B) and E11.5 in (C). Results are averaged from five independent experiments in each stage. (C) Scn5a mRNA levels at various embryonic stages were normalized to Nkx2.5 mRNA (white bars), and showed a similar pattern compared with data normalized to β-actin mRNA (blue bar). Embryonic day (E) 9.5 to 17.5. N1, 1 day neonate mouse heart; N15, 15 day neonate heart; A, adult mouse heart.

 
In order to establish that the decrease of Scn5a mRNA expression observed from stages E12.5 to E15.5 is not due to the relative contribution of non-myocardial cells from the endocardial cushion and/or by the invasion of epicardial-derived cells,19,20 we normalized Scn5a mRNA expression levels to myocardial-enriched transcription factor expression Nkx2.5, as previously reported.17 The Scn5a expression profile from E11.5 to E15.5 stages displays a similar pattern using Nkx2.5 as internal control (Figure 1C), demonstrating that changes in mRNA expression levels at embryonic stages (E12.5–E15.5) are unlikely to be due to differences in the ratio between myocardial and non-myocardial cells.

3.2 Nav1.5 protein distribution during cardiogenesis
We have analysed the cardiac sodium channel protein expression pattern by immunohistochemistry from the early embryonic stages (E9.5) to the adult mouse stage using a specific anti-Nav1.5 antibody.

Identical but weak expression of the cardiac sodium channel, Nav1.5, is first observed at E9.5 throughout the atrial and ventricular myocardium, as well as in the slow conducting myocardial regions such as the outflow tract (Figure 2A). Similar results were obtained at stage E11.5 and E12.5 (Figure 2B and C). Desmin expression has been described to be higher in the developing left and right bundle branches and in the ventricular trabeculations.21 Therefore, although Nav1.5 expression can be observed similarly in all cardiac compartments, the prospective right and left bundle branches areas begin to show an enhanced expression of the Nav1.5 compared with the surrounding working myocardium, and thus display a similarity to desmin staining (Figure 2E–G).


Figure 2
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  		Figure 2 Sodium channel protein expression in embryonic mouse hearts. Immunohistochemical detection of Nav1.5 (A–D) staining in transverse sections of mouse embryos of E9.5 (A), E11.5 (B), E12.5 (C) and E13.5 (D). Nav1.5 staining in the embryo is observed homogeneously distributed in the atrial and ventricular myocardium from stages E9.5 to E12.5 (A–C). Nav1.5 enhanced expression begins to be observed in the bundle branches at stage E12.5 (arrows in C). At E13.5 (D), Nav1.5 enhanced expression become prominent in the bundle branches (arrows in the left bundle branch) and in the ventricular trabeculated myocardium (arrowheads), as compared to the ventricular compact myocardium (asterisk). Desmin immunostaining illustrates the myocardial boundaries (E–H). Nuclei in blue. CV, common ventricle; LCCA, left component of the common atria; RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; OFT, outflow tract; IVS, interventricular septum; E9.5, E11.5, E12.5, and E13.5, embryonic day (E) 9.5, 11.5, 12.5, and 13.5.

 
With further development, atrial and ventricular septation is almost complete (stage E13.5–E15.5), and the heart is divided into four distinct chambers. During this period, the conduction system starts also to be morphologically well established.22,23 Concomitant with these morphogenetic changes, Nav1.5 protein expression shows an important heterogeneity in the ventricular chambers at stage E13.5, as compared to the observations in previous stages (E11.5 and E12.5). Although cardiac sodium channel expression is maintained throughout the entire myocardium at this stage (E13.5), an enhanced Nav1.5 expression is observed in the ventricular trabeculation, including the future bundle branches as compared to the surrounding compact myocardium that stains far less intensely (Fig. 2D). This was confirmed by semi-quantitative assessment of Nav1.5 labelling (Supplementary material online Figures S1 and 2).

Cardiac sodium channel expression remains unaltered at foetal stages (E17.5) (Fig. 3) as compared to the previous stage (E13.5). At this stage (E17.5), when the distinction between working and conduction system myocardium can be easily established,21 we performed protein expression analysis of Nav1.5 in distinct components of the conduction system (Figure 3A, D, E and H).


Figure 3
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  		Figure 3 Expression profile of Nav1.5 in the different components of the cardiac conduction system at stage E17.5. Nav1.5 (A) staining reveals lower expression of Nav1.5 in the central region of the sinoatrial node. The absence of Cx43 protein in the sinoatrial node (B) allows definition of the pacemaker region. Desmin immunostaining illustrates the myocardial boundaries at the sinoatrial node region (C). Expression of the cardiac sodium channel, Nav1.5 in the His bundle (D) and bundle branches (E) at foetal stage (E17.5): His bundle shows lower Nav1.5 staining (D) as compared to bundle branches (arrow, E), identified with desmin immunostaining (arrow in F and G). At embryonic day 17.5, higher expression of Nav1.5 is observed in the ventricular trabeculated myocardium (H), similar that observed for Cx40 protein expression (I). (J) Negative control for Cx40 using an aliquot of antibody preabsorbed with the corresponding peptide. Nuclei in blue. RV, right ventricle; RA, right atria; LV, left ventricle; LA, left atria; IVS, interventricular septum; SAN, sinoatrial node; HB, His bundle; LBB, left bundle branch; E17.5, embryonic day (E) 17.5.

 
Connexin 43 (Cx43) is expressed in the atrial and ventricular myocardium, but is not detected at the SAN.8 Thus, Cx43 protein expression is commonly used to identify the pacemaker region8 (Figure 3B). Our results demonstrate a higher Nav1.5 immunostaining in the periphery of the SAN as compared with the central SAN region (Figure 3A) (see semi-quantitative assessment in Supplementary material online Figures S1 and 2).

On the other hand, desmin shows less intense labelling at the AVN and the His bundle, being used to identify these components of the ventricular conduction system.17 Comparative analysis with desmin immunoassaying (Figure 3F and G) reveals lower presence of the Nav1.5 cardiac sodium channel in the AVN (data not shown) and in the His bundle (Figure 3D), in contrast with a robust staining in the left and right bundle branches (Figure 3E). In addition, Connexin 40, a member of connexin family mainly expressed in the trabeculated ventricular myocardium,24 display a similar staining pattern that Nav1.5 at this stage (E17.5) (Figure 3H–J). Therefore, a higher level of Nav1.5 staining in trabeculated myocardium is maintained with respect to compact myocardium at E17.5 (Figure 3H), showing a clear transmural gradient of Nav1.5 expression in the developing ventricles (see semi-quantitative assessment in Supplementary material online Figures S1 and 2).

From neonate day 1 onwards, while Nav1.5 protein expression remains unchanged in the CCS components (data not shown), atrial and ventricular myocardium begin to display differences, revealed by higher Nav1.5 expression in the atrial as compared to ventricular chambers in postnatal and adult hearts (Figure 4A and B).


Figure 4
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  		Figure 4 Nav1.5 protein expression differences between cardiac chambers during postnatal stages (A and B) and subcellular immunolocalization of Nav1.5 during development (C, E and G–H). Immunohistochemical detection of Nav1.5 at neonate 1 day (A) and in adult heart (B) displays higher Nav1.5 expression in the walls of the atrium as compared with the ventricles. Cardiac sections at E12.5 using sodium channel anti-Nav1.5 (C). (E) At neonatal stages sodium channel {alpha}-subunits begin to show a striated pattern of expression (white arrows) corresponding with the developing T-tubule system. (D and F) {alpha}-actinin staining at E12.5 and neonate 1 day, respectively. (G and H) Cardiac sections from adult mouse heart immunostained with anti-Nav1.5 antibody. Fibres from adult heart display a striated pattern of Nav1.5 expression (G) similar to the labelling of {alpha}-actinin used as a Z-lines marker (I). Immunolocalization of Nav1.5 in zones of cell contacts (arrow in H) similar to Cx43 staining (J). Nuclei in blue. RV, right ventricle; RA, right atria; LV, left ventricle; LA, left atria; IVS, interventricular septum; E12.5: embryonic day (E) 12.5; N1, 1 day neonate mouse heart; A, adult heart.

 
3.3 Subcellular localization of the cardiac sodium channel during cardiogenesis
The distribution of Nav1.5 at the subcellular level during mouse heart development was observed by immunohistochemistry and confocal laser scanning microscopy at high magnification. Concomitant with maturation of the myocardial cells, Nav1.5 displays changes in the subcellular localization. As shown in Figure 4C, at E12.5 embryonic stage Nav1.5 is localized in the cytoplasm and at the cardiac cell surface.

This localization profile pattern remains similar during the subsequent foetal stages (data not shown). However, with further cardiogenesis (neonate 1 day), changes in Nav1.5 protein localization are observed in the myocardial cell. During perinatal life, Nav1.5 protein distribution begins to display a striated pattern within the cardiomyocytes (Figure 4E). This striated pattern, located along the Z-line as identified by anti-{alpha}-actinin labelling6,7 (Figure 4D, F and I), becomes more distinguishable in adult cardiac fibres (Figure 4G), supporting localization of this {alpha}-subunit in T-tubules. In addition, Nav1.5 is also observed in the intercalated discs (Figure 4H), as identified by immunostaining of Cx43 (Figure 4J).

The β1 sodium channel subunit has been described as one of the most important sodium channel auxiliary subunits in the nervous system and skeletal muscle and its expression has been previously described in the T-tubules system and in the intercalated discs of developing and adult mouse heart.17 However, its spatial-temporal relation to Nav1.5 remains poorly understood. Double-fluorescent immunostaining was performed in order to determine whether if the cardiac sodium channel, Nav1.5, and Scn1b, the β1 sodium channel subunit, co-localize. As illustrated in Figure 5, our results reveal that Nav1.5 co-localizes with the β1 sodium channel subunit in the T-tubule system and intercalated discs levels in adult cardiomyocytes.


Figure 5
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  		Figure 5 Subcellular immunocolocalization of Nav1.5 and β1 sodium channel subunits, Scn1b, in the T-tubule system (A, B and C) and in the intercalated discs (arrows in D, E and F) of the adult mouse heart. Double immunostaining of Nav1.5 and β1 sodium channel subunits were performed using an anti-Nav1.5 antibody developed in goat (Santa Cruz; see section Methods) and the anti-Scn1b antibody developed in rabbit. The signal was detected using two distinct fluorochromes conjugated antibodies, a Cy2 conjugated anti-goat for Nav1.5 and Cy3 conjugated anti-rabbit for Scn1b. (C and F) Merged images of (A and B) and (D and E), respectively. Nuclei in blue; Nav1.5 staining in green; β1 sodium channel subunit staining in red. A, adult heart.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
4.1 Nav1.5 during embryonic cardiogenesis
Scn5a encodes Nav1.5, also called the cardiac sodium channel, which generates the INa that underlies excitability and conduction in atrial and ventricular myocardial cells and cardiac conduction tissue.3,10 Although it has been reported that Nav1.5 is the most prominent sodium {alpha}-subunit in the cardiac tissue,25,26 there is scant information about its detailed expression pattern during cardiogenesis. We report herein the mRNA expression profile and protein distribution of the main cardiac sodium channel {alpha}-subunit, Nav1.5, during heart development by quantitative real-time PCR and immunohistochemistry.

Important electrophysiological changes occur during the embryonic development of mammalian and avian hearts such as the shift from peristaltic-like contractions of the primitive heart tube to more sequential contractions of the atria and ventricles in the foetal and adult heart.1 Our real-time RT–PCR analysis revealed a dynamic expression profile of the cardiac sodium channel, Scn5a, during cardiogenesis, starting as early as stage E9.5 peaking at embryonic stage (E11.5) and increasing gradually in postnatal and adult stages. These results are in line with increases in Scn5a transcripts previously reported in foetal and adult mouse heart9 and suggest that Nav1.5 could play a developmental role in determining electrical excitation of cardiac muscle cells. In addition, Scn5a expression profiles are similar to that previously reported for Scn1b,17 supporting the notion that both genes could have a concomitant transcriptional regulation during cardiogenesis.

Immunohistochemical experiments revealed comparable distribution of Nav1.5 between different cardiac chambers at early embryonic stages. These results are thus in agreement with previous reports which found no differences in Na+ current between different regions of developing murine heart at early stages.25,26 At later embryonic stages (E12.5–E13.5), coinciding with the initiation of ‘apex-to-base’ pattern of ventricular activation,22 Nav1.5 expression becomes heterogeneous in the ventricular trabeculations including the prospective right/left bundle branches as compared to the compact ventricular myocardium. In fact, the importance of its role in the electrophysiological properties of the embryonic heart at these stages is evident since homozygous Scn5a knock-out mice die at E11.5 due to impaired ventricular morphogenesis. Moreover, differential expression of Nav1.5 in distinct components of the CCS is in concordance with the fact that Scn5a heterozygous mice display a delay in the cardiac conduction.27

At the later developmental stage (E17.5), our immunohistochemical analysis demonstrates a lower Nav1.5 expression in the central region of the SAN as compared to the SAN periphery. This data are consistent with previous electrophysiological and immunohistochemical analysis in the mouse showing that dependent Na+ channel current (INa) density as well as Nav1.5 staining were greater in the periphery of SA node than in the centre.8,28 Furthermore, immunohistochemical analysis of the Nav1.5 expression in the AVN and the His bundle revealed a weaker expression in comparison to other components of the ventricular conduction system such as the left bundle branch in line with previous studies in adult rat heart.29 In contrast, Nav1.5 remains highly expressed in the bundle branches and the ventricular trabeculated myocardium at these stages (as shown by Cx40 immunostaining comparison), in accordance with previous northern blot30 and RT–PCR data in adult sheep and human hearts, respectively.31 Taken together, these results indicate that Nav1.5 seems to be an important sodium channel {alpha}-subunit involved in the heterogeneous propagation of the action potential along distinct cardiac compartments; i.e. in the establishment of the rapid conduction of the electrical impulse throughout the CCS to the ventricles during cardiogenesis.

Interestingly, our data revealed important differences in Nav1.5 distribution between cardiac chambers at postnatal and adult heart stages. Nav1.5 expression is higher in atrial myocardium as compared to the ventricular myocardium at postnatal stages, including neonatal and adult hearts. Curiously, it has been reported that the density of INa was 50% greater in atrial than in ventricular pig adult myocytes as well as an enhancement excitability of atrial vs. ventricular myocardial cells32 in line with our protein expression data. Thus, the enhancement of expression of the cardiac sodium channel Nav1.5 in atria would be essential for the increased excitability of the atrial myocardial cells.

4.2 Subcellular localization of Nav1.5 during heart development
The heart undergoes marked ultrastructural changes during development.33,34 Our data, obtained using high magnification confocal microscopy reveals changes in the Nav1.5 subcellular localization during mouse cardiogenesis. Nav1.5 was present in the cytoplasm and cell surface of the immature cardiomyocytes at embryonic stages. Consistent with previous studies which indicate that, after birth, transverse tubules develop and intercalated discs become increasingly complex,33,34 we found Nav1.5 expression displays a striated pattern; such expression pattern resembles the T-tubules system distribution (as identified by {alpha}-actinin staining) at neonatal stages. This striated pattern of expression is clearly established in the adult cardiac fibres in which Nav1.5 becomes clearly located in the T-tubules. Moreover, a lateralized Nav1.5 distribution to intercalated discs localization was detected from postnatal stages, similarly to that observed for Cx43.35 Taken together, these results indicate the possibility that Nav1.5 could be involved in the propagation of action potential from the surface to the centre of myocyte and in the isotropic propagation between myocytes. The progressive localization of Nav1.5 in the T-tubules system and intercalated discs is in agreement with the increase of Scn5a mRNA levels at foetal, neonatal, and adult stages as reported in this study.

Furthermore, Nav1.5 subcellular localization in transverse tubules and intercalated discs are in agreement with those reported by Cohen et al. (1996)36 who found Nav1.5 in the T-tubular system in cross-sections of rat adult cardiac muscle. Others authors have found that Nav1.5 is preferentially localized in the intercalated discs but not in transverse tubules of adult isolated mouse ventricular cells,6,7 in contrast to our results using tissue sections. The use of distinct antibodies sources of technical procedures might underline such differences, although such controversy remains to be further dissected.

4.3 Localization of Nav1.5 and β1-subunit during cardiogenesis by double immunolabelling
The pore forming {alpha}-subunit interacts with smaller accessory subunits known as β-subunits (Scn1b, Scn2b, Scn3b, and Scn4b) that might modulate their function.6 Although the functional role of sodium channel β-subunits in the heart is still uncertain, recent data revealed concomitant changes of Nav1.5 and β1-subunit expression levels in hearts from an animal model of dilated cardiomyopathy associated to slow cardiac conduction.37 Interestingly, Scn5a expression profile and protein distribution during heart development described here overlaps to that of Scn1b expression profile previously reported by us.17 Our results show that, at adult stages, Nav1.5 and Scn1b are both co-expressed in T-tubular system and intercalated discs of cardiac fibres. Our results do not determine whether others β-subunits are also associated with Nav1.5. However, in contrast to β2- and β4-subunits, previous works have revealed that coexpression of β1-subunits with Nav1.5 {alpha}-subunits has a substantial functional effect.38–40 Thus, our data suggest that β1-subunit could be a good candidate to modulate Nav1.5 function during cardiogenesis.

In conclusion, in this study we describe the expression pattern of the main cardiac {alpha}-sodium channel subunit, Nav1.5, from early embryonic (E9.5) to adult stages. Our results demonstrate a dynamic expression pattern during cardiogenesis and reveal an important enhanced Nav1.5 expression in different components of the CCS during development. Subcellular localization demonstrates that the cardiac sodium channel, Nav1.5, co-localizes with the β1 sodium channel subunit in the T-tubules system and in the intercalated discs, suggesting that both subunits may play an important role in the establishment of the global pattern of conduction and contraction of the developing four-chambered heart.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
This work was partially supported by grants BFU2005-07727 (Ministerio de Educacion y Cultura, Gobierno de España) and CTS446 (Junta de Andalucia).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

  1. Moorman AFM, Lamers WH. Molecular anatomy of the developing heart. Trends Cardiovasc Med (1994) 4:257–264.[CrossRef][Web of Science]
  2. Chien KR. Development of the pacemaking and conduction system. Braunwald's Molecular Basis of Cardiovascular Disease—Zipes DP, Libby P, Bonow RO, Braunwald E, eds. (2005) 2nd ed. Philadelphia: Elsevier Saunders. 225–238.
  3. Marban E, Yamagishi T, Tomaselli GF. Structure and function of voltage-gated sodium channels. J Physiol (1998) 508:647–657.[Abstract/Free Full Text]
  4. Catterall WA. From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron (2000) 26:13–25.[CrossRef][Web of Science][Medline]
  5. Goldin AL, Barchi RL, Caldwell JH, Hofmann F, Howe JR, Hunter JC, et al. Nomenclature of voltage-gated sodium channels. Neuron (2000) 28:365–368.[CrossRef][Web of Science][Medline]
  6. Maier SK, Westenbroek RE, McCormick KA, Curtis R, Scheuer T, Catterall WA. Distinct subcellular localization of different sodium channel alpha and beta subunits in single ventricular myocytes from mouse heart. Circulation (2004) 109:1421–1427.[Abstract/Free Full Text]
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