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Cardiovascular Research 2005 65(4):842-850; doi:10.1016/j.cardiores.2004.11.028
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Copyright © 2004, European Society of Cardiology

Temporal and spatial expression pattern of β1 sodium channel subunit during heart development

Jorge N. Domíngueza, Francisco Navarroa, Diego Francoa, Robert P. Thompsonb and Amelia E. Aránegaa,*

aDepartment of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Paraje de las Lagunillas, s/n, 23071 Jaén, Spain
bDepartment of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC, USA

* Corresponding author. Tel.: +34 953 212604; fax: +34 953 212141. Email address: aaranega{at}ujaen.es

Received 27 April 2004; revised 26 October 2004; accepted 24 November 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 
Objectives: The aim of this study is to analyze Scn1b mRNA expression levels and protein distribution of Scn1b, a putative modulator of the pore-forming Na+ channel subunit in the heart, during mouse cardiac development.

Methods: Scn1b mRNA levels were determined by real-time RT-PCR using embryonic hearts ranging from E9.5 to E18.5 as well as in postnatal and adult heart. Scn1b protein distribution and subcellular localization during cardiogenesis were analyzed by immunohistochemistry and confocal microscopy.

Results: Scn1b mRNA showed a dynamic expression pattern, peaking at stage E12.5 and decreasing at E15.5. Scn1b mRNA increased at later embryonic and neonatal stages, being maximal in the adult heart. Immunohistochemistry experiments revealed comparable distribution of Scn1b protein between the different cardiac chambers at early embryonic stages. With further development, Scn1b protein showed an enhanced expression in the trabeculated myocardium and the bundle branches. At the subcellular level in later embryonic and postnatal mouse cardiomyocytes, Scn1b was present in T-tubules as identified by immunostaining of {alpha}-actinin, and in the intercalated disks as identified by immunostaining of connexin 43.

Conclusion: These results demonstrate that Scn1b is expressed during mouse heart development, suggesting it can play an important role in the action potential configuration of the cardiomyocytes during heart morphogenesis.

KEYWORDS Gene expression; Developmental biology; Ion channel; Purkinje fibers; Sarcolemma


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 References
 
Cardiac development is a complex process leading to the formation of a four-chambered heart with synchronous contraction from a single peristaltoid tubular heart [1]. After cardiac tube formation, the heart loops towards the right side and five different areas can be distinguished: two fast-contracting regions (atria and ventricles) and three slow-contracting regions (the outflow tract, the atrioventricular canal and the inflow tract) [1,2]. With further development, the septation of the heart is initiated, and the atrial and ventricular chambers acquire a coordinate contraction through a specialized tissue network, the cardiac conduction system (CCS). The components of CCS include: the sinoatrial (SA) node that generates a pacemaker impulse, the atrioventricular (AV) node that delays the electrical impulse separating therein the contraction of the atrial and ventricular chambers, and the His bundle that continues propagating the electrical impulse through left and right bundle branches to the Purkinje fibers, to rapidly activated both ventricles from apex to base.

Voltage-gated sodium channels are responsible for the rapid increase in membrane sodium permeability that occurs during the initial phase of the action potential in cardiac myocytes [3]. The sodium channel is a multi-subunit protein complex composed of a single large pore forming {alpha}-subunit along with the smaller additional β ancillary subunits [4]. Nav1.5 (voltage-gated sodium channel type V) has been described as the main cardiac {alpha}-subunit sodium channel, encoded by Scn5a gene [5–7].

The auxiliary β-subunits do not form ion-conducting pores, but they have been shown to be important modulators of Na+ channels in brain and skeletal muscle. These auxiliary subunits, named β1 to β4, encoded by the genes Scn1b, Scn2b, Scn3b and Scn4b, respectively, have been demonstrated to modulate channel gating, interact with extracellular matrix and play a role as cell adhesion molecules [8–13]. β1 sodium channel subunit (Scn1b) expression was demonstrated in adult rat and human heart, as well as in fetal and adult sheep heart [8,9,14,15]. Northern blot experiments in adult sheep heart showed that Scn1b mRNA is expressed in the myocardium of all four chambers, including Purkinje fibers [14]. Curiously no expression of Scn1b has been detected in mouse heart [16]. However, recently immunohistochemistry experiments have described the expression of Scn1b in SA node in adult mouse heart and isolated mouse adult ventricular myocytes [17,18].

Although the β-subunits are expressed in the heart, the functional role of these auxiliary subunits in this tissue is still uncertain [6]. Scn1b and Scn5a co-expression in Xenopus oocytes cause a small but significant acceleration in the recovery from inactivation [19,20], and several mutations of Scn5a associated with long QT and Brugada syndrome appear to be modulated by co-expression of Scn1b [21]. However, purified preparations of cardiac sodium channels from chicken and rat do not show detectable associated β-subunits after immunoprecipitation with {alpha}-subunits antibodies [22,23].

Similar to the adult heart, during cardiac development the distinct heart regions display differences in the action potential configuration, which are correlated with distinct molecular phenotypes [2,24]. In the last years some studies have demonstrated that ion channel expression in the developing mouse heart can be age- and chamber-specific [25,26]. For example, the potassium channel β-subunits Kcne1, Kcne2 and Kcne3 have been described as showing a dynamic expression pattern during development, while potassium channel {alpha}-subunits display a homogeneous expression, suggesting a major role of β-subunit in establishing potassium current heterogeneity during cardiogenesis [26].

However, there is scant information concerning the expression pattern of the β1-subunit sodium channel, Scn1b, during cardiogenesis, when many changes in the electrophysiology and patterns of gene expression are observed [27].

The aim of this study is to investigate the mRNA expression and protein distribution of Scn1b, a putative modulator of the {alpha}-subunit in the heart, during mouse heart development. Using quantitative RT-PCR we describe the dynamic expression levels of Scn1b from the beginning of cardiogenesis to adult heart stage. Immunohistochemistry experiments revealed similar distribution of Scn1b protein in different cardiac chambers during early mouse heart development, with an enhanced expression in the right and left bundle branches and the trabeculated ventricular myocardium (including Purkinje fibers) at later stages of cardiogenesis. At subcellular level, Scn1b labelling was detected in T-tubules and intercalated disks in late embryonic and postnatal mouse heart. We demonstrate that Scn1b is expressed in the developing myocardium and thus it can have an important role in the establishment of a mature pattern of ventricular activation and in the coupling of cell surface depolarization to contraction during early cardiomyocyte maturation.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 References
 
2.1. Embryos and adults mice
Balb/c female mice were sacrificed and whole hearts ranging from embryonic day (E) 9.5 to E18.5 were isolated. The day of vaginal plug was taken as E0.5. Adult and neonatal hearts (1 and 15 days) were also obtained. Hearts for RNA isolation were dissected including myocardial components of outflow and inflow tracts and stored in liquid nitrogen. For immunohistochemistry experiments, four embryos for each stage, as well as four neonatal and adult hearts, were fixed in fresh DENT'S solution (80% methanol, 20% DMSO) overnight, dehydrated in absolute ethanol, and embedded in paraplast. Thereafter, the samples were sectioned at 8 µm and mounted in 3-aminopropyltriethoxy-silane coated glasses. 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).

2.2. mRNA extraction and reverse transcription
RNA extraction was performed from five pooled hearts at each stage, ranging from E9.5 to E17.5 embryos, and from three hearts of neonate and adult mice using the eukaryotic Perfect RNA mini-kit (Eppendorf) according to the manufacturer's guidelines. Contaminating genomic DNA was removed by treatment with RNase-free DNase (Roche) for 1 h at 30 °C. First-strand cDNA was synthesized at 37 °C for 1 h using 3 µg of RNA, oligo-dT primers and Superscript RNase H– reverse transcriptase (Invitrogen), according to manufacturer's instructions. As a negative control for genomic DNA contamination each sample was subjected to the same reaction without reverse transcriptase.

2.3. Quantitative real time PCR (Q-PCR)
The primers used to detect mouse Scn1b, Nkx2.5 and β-actin were obtained from Genotek (Bonsai Technologies Group, Spain).


Primers Sequence Size cDNA amplification (bp)
Scn1b (+78F) 5'-CTG CGT GGA GGT GGA TTC CG-3' 623
Scn1b (–711R) 5'-GGC TGG CTC TTC CAT GAG GC-3'
βACTIN (+374F) 5'-TGA GGA GCA CCC TGT GCT-3' 143
βACTIN (–517R) 5'-CCA GAG GCA TAC AGG GAC-3'
Nkx2.5 502 (F) 5'-GCG CAG GTC TAC GAG CTG GAG-3' 176
Nkx2.5 303 (R) 5'-CCC AGA AGC TCC AGA GTC TGG-3'

Real-time PCR was performed within an iCycler PCR thermocycler (Bio-Rad, Spain) and SYBR Green detection system. Reactions were performed in 96-well plates with optical sealing tape (Bio-Rad) in 20 µl total volume containing SYBR Green Mix (Bio-Rad) and cDNA corresponding to 150 ng of total RNA. Mouse β-actin was used in parallel for each run as internal control [28]. Amplification conditions were: 95 °C for 5 min; 40 cycles of 95 °C for 30 s, 58.8 °C for 30 s, 72 °C for 30 s; and 72 °C for 10 min. In the case of Nkx2.5, 64 °C was used as annealing temperature. Each PCR reaction was performed at least five times to obtain a representative average. The relative level of expression of the Scn1b gene was calculated as the ratio of the extrapolated levels of expression of Scn1b and β-actin mRNAs. For hearts from E12.5, E15.5 and E17.5 stages, the relative level of expression of Scn1b was also calculated as the ratio of the extrapolated levels of expression of Scn1b and the cardiac specific gene Nkx2.5. The amplification PCR products were analyzed in agarose gel electrophoresis and each band was subcloned and confirmed by sequencing from both directions.

2.4. Immunohistochemistry
Sections were deparaffinised, hydrated through graded ethanol steps, briefly rinsed in water and blocked at room temperature using TBSA-BSAT (10 mM Tris, 0.9% NaCl, 0.02% sodium azide, 2% bovine serum albumin and 0.1% Triton-x100 detergent). The Scn1b rabbit polyclonal antibody (a kind gift from Lori Isom, University of Michigan) was raised against the peptide sequence, GGCVEVDSETEAVYGMTF, spanning from amino acid 19 to amino acid 36 of the rat Scn1b protein sequence [8]. Protein sequence comparison between rat Scn1b peptide and mouse protein displays about 94.4% homology, showing only a conservative Glu (rat) to Asp (mouse) amino acid replacement. The rabbit anti-mouse connexin40 (Cx40) IgG was raised against the cytoplasmic C-terminal domain (19 amino acids) (Gentaur Molecular Products, Alpha Diagnostic, Belgium) [29]. Polyclonal antibody against desmin (Sigma, Spain) was used to delimit the myocardium within distinct cardiac chambers. Polyclonal antibody against {alpha}-actinin was purchased from Sigma. The polyclonal antibody recognizing connexin 43 (Cx43) was kindly gifted by Lucile Miquerol (IGBD, Marseille, France).

Rehydrated sections were incubated overnight at room temperature with primary antibodies at 1:100 (Scn1b, desmin, Cx43 and {alpha}-actinin) and 1:40 dilution (Cx40). Following rinsing, sections were incubated for 5 h with anti-rabbit Cy3 secondary antibody (Jackson Labs, USA) diluted in TBSA-BSAT (1:100). Control sections included omission of primary antibody or preincubation with Cx40 antigenic peptide. Nuclear staining was performed using DRAQ-5TM (Red Fluorescen Cell-Permeable DNA probe, Biostatus Limited, United Kingdom). Immunofluorescence analysis was performed using a Leica TCS SL confocal microscope (Leica LCS Version 2.0).


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 References
 
3.1. Scn1b mRNA expression during heart development
Quantitative RT-PCR was used to measure Scn1b and β-actin mRNA expression in whole embryonic, neonatal and adult mouse hearts. This enabled a comparison of the quantitative expression of Scn1b mRNA at different developmental stages to be made. These data are expressed as a ratio of Scn1b to β-actin expression levels, taking as 100% the ratio at E12.5 (Fig. 1). Our results show a weak expression of the β1 subunit sodium channel as early as stage E9.5. With further development, there is an increase in cardiac expression levels of Scn1b transcripts, thus, during cardiac embryonic development Scn1b mRNA peaks around E12.5, decreasing at E13.5–E15.5 and raising again after E15.5. The amount of Scn1b mRNA increases gradually in postnatal hearts being maximal in adult heart stage (Fig. 1).


Figure 1
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  		Fig. 1 Scn1b mRNA expression levels in the heart normalized to β-actin during mouse cardiogenesis as measured by quantitative RT-PCR. Differences in Scn1b mRNA levels are expressed as percentages relative to E12.5 values. Results are averaged from five independent experiments in all stages. Standard deviations included in the figure were less than 10% in all stages. During cardiac embryonic development Scn1b mRNA levels peak at stage E12.5. A decrease of Scn1b transcripts is observed at E13.5 and E15.5, increasing at later embryonic, neonatal and adult stages. Embryonic day (E) 9.5 to 17.5; N1: 1 day neonate mouse heart; N15: 15 day neonate heart; A: adult mouse heart.

 
To discard that the decrease of Scn1b mRNA levels at E13.5 and E15.5 stages is due to an increase in non-myocardial cells observed during cardiac development at these stages [30,31], we used a specific myocardial gene (Nkx2.5) to normalize Scn1b values. As shown in Fig. 2, Scn1b expression pattern from E12.5 to E17.5 stages is maintained using Nkx2.5 as internal control. These results demonstrate that decrease of Scn1b mRNA levels at embryonic stages (E13.5–E15.5) is unlikely to be due to differences in the ratio between myocardial and non-myocardial cells.


Figure 2
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  		Fig. 2 Scn1b mRNA level normalized to Nkx2.5 (black) and to β-actin (white) mRNA at embryonic day (E) 12.5, E15.5 and E17.5. Differences in Scn1b mRNA levels are expressed as percentages relative to E12.5. Results are averaged from five independent experiments in all stages. Standard deviations included in the figure were less than 10% in all stages. At these stages, changes in Scn1b mRNA levels normalized to Nkx2.5 mRNA and normalized to β-actin mRNA display similar pattern.

 
3.2. Scn1b protein distribution during cardiogenesis
In order to examine the protein distribution of Scn1b during mouse heart development, immunohistochemistry experiments were performed using antibodies raised against β1 sodium channel subunit during heart development, ranging from E9.5 to the adult stage.

In the early embryonic stages (E9.5) Scn1b expression was similar in all cardiac compartments, i.e. atrial and ventricular myocardium (Fig. 3A–D). The signal observed in slow conducting cardiac regions (outflow tract and atrio-ventricular canal) was also similar to that seen in other areas of the heart such as atrial and ventricular myocardium (Fig. 3B and D). At E10.5–E11.5 stages, although Scn1b staining remains in all cardiac compartments, Scn1b expression within the ventricular compartment is higher in trabeculated myocardium and the prospective areas that will give rise right and left bundle branches on either side of the ventricular septum as compared with the nascent interventricular septum (Fig. 3E–G).


Figure 3
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  		Fig. 3 Distribution of Scn1b protein in embryonic mouse hearts. (B, D) At E9.5 Scn1b protein expression is similar in all cardiac compartments, including the atrioventricular canal (AVC) and the outflow tract (OFT). (F, G) At E10.5–E11.5 Scn1b expression remains in all cardiac chambers and a lower expression in nascent interventricular septum can be observed. Scn1b expression in the prospective areas that will give to the right and left bundle branches (arrows in F). (H) High expression of Scn1b is observed in ventricular trabeculations and in prospective right and left bundle branches (arrows) at E12.5. (I–M) At E13.5 expression of Scn1b is higher in the right and left bundle branches and the trabeculated ventricular component as compared to the ventricular compact myocardium and the interventricular septum. J–M are close-ups of the framed areas depicted in I. (O) Negative control for Scn1b antibody. A, C, E and N illustrate desmin expression as a marker of myocardial boundaries. Nuclei in blue. A: common atria; V: common ventricle; RV: right ventricle; RA: right atria; LV: left ventricle; LA: left atria; IVS: interventricular septum; OFT: outflow tract; AVC: atrioventricular canal.

 
Such differences become more pronounced with further development. Thus, by E12.5 stage, when the budding interventricular septum could more clearly be seen between the primitive right and left ventricles and the appearance of discrete bundle branches could be visualized [32], a higher Scn1b protein expression can be observed in the developing right and left bundle branches as well as in the ventricular trabeculations (Fig. 3H) from which the peripheral Purkinje fibers are derived. At stage 13.5 (Fig. 3I–M), when the communication between the cavities of the right and left ventricles is almost fully closed [33], the highest Scn1b expression is observed in the right and left bundle branches and in the ventricular trabeculated myocardium (Fig. 3L–M) as compared to the compact ventricular myocardium and the interventricular septum (Fig. 3J–K).

The expression profile of Scn1b protein remains similar at later embryonic stages (E17.5) (Fig. 4A). In addition, we show Scn1b labelling in other components of conduction system (SA and His bundle) at E17.5 (Fig. 4E–I), when distinction between working myocardium and conduction system myocardium can be easily established [34]. To identify the SA, sections were labeled with an antibody recognizing Cx43, which stain the atrial and the ventricular muscle cells but not SA [17]. AV node and the His bundle were identified by a less intense staining with desmin antibody [35,36]. At this stage, Scn1b shows a strongly staining in the periphery of the SA node but not in the central SA node (Fig. 4E,F). Scn1b displays also a low levels of expression in AV node (data not show) and in the His bundle (Fig. 4H,I) as compared with others components of ventricular conduction system, such as the right and left bundle branches.


Figure 4
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  		Fig. 4 Scn1b protein expression at later embryonic stages (E17.5) (A–I) and Scn1b protein expression in the neonatal heart (J–N). (A) At embryonic day 17.5 high expression of Scn1b is observed in the trabecular ventricular components. (B and C) Similar labelling in trabecular ventricular myocardium for Scn1b and Cx40 antibodies (brackets) is observed. (D) negative control for Cx40 using peptide preabsorbed antibody. (E and F) Scn1b expression in the SA node; F is close-ups of the framed area depicted in E. (G) Section labeled with anti-connexin 43 to illustrate the lack of expression in the SA node (asterisk). (H) Scn1b expression is weaker in the His bundle (arrow) compared with Scn1b staining in the left bundle branches (arrowhead) (E17.5). (I) Sister sections of H stained with anti-desmin polyclonal antibody, delineating the His bundle (arrow). (J–N) Distribution of Scn1b protein in a 1 day neonate heart (N1): Scn1b staining in right and left atrial appendages (J, N), compact wall of right and left ventricle (K, M) and interventricular septum (L). Arrowhead in 1 day neonate (N1) shows the signal in the right bundle branch (L). Nuclei in blue. RV: right ventricle; RA: right atria; LV: left ventricle; LA: left atria; IVS: interventricular septum.

 
To demonstrate specific expression of Scn1b protein in the Purkinje system, a comparative immunohistochemistry study using β1 sodium channel and anti-Cx40 antibody was performed in E17.5 embryos. Cx40 is a member of connexin family that forms gap junction channels in mammalian cardiomyocytes [36–38] and is localized mainly in the Purkinje fibers [40,41]. The results obtained showed a similar labelling in the trabeculated ventricular myocardium for Scn1b and Cx40 (Fig. 4B–D). Thus, Scn1b staining in trabeculated myocardium is consistent with the presence of this β-subunit in Purkinje fibers network.

With further development, i.e. the postnatal and adult heart stages, no changes in the expression pattern of protein for Scn1b was detected (Fig. 4J–N), including SA, the His bundle and AV nodes (data not shown).

3.3. Subcellular localization of Scn1b during cardiogenesis
To investigate Scn1b subcellular localization in developing myocardium, higher magnification analysis was performed. As shown in Fig. 5, changes in the immunolocalization pattern of Scn1b occur during heart development. In longitudinal sections from hearts at embryonic stages (E12.5), Scn1b shows a punctuate labelling in the cytoplasm and the cell surface (Fig. 5A and B). During subsequent stages, as maturation of the myocardial cells take place, changes in the Scn1b subcellular localization can be observed. Thus, in a postseptation stage (E17.5), Scn1b is distributed in a striated pattern within the cardiomyocytes (Fig. 5C) and remains unchanged in neonatal and the adult heart stages (Fig. 5E and G). This striated pattern is very similar to that of {alpha}-actinin, a marker for cardiac muscle Z-lines, in ventricular tissue [42] (Fig. 5F). In adult hearts, Scn1b displays an intermittent long stripes staining delineating the surface of cardiac muscle fibers.


Figure 5
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  		Fig. 5 Subcellular immunolocalization of Scn1b during mouse heart development. (A and B) Cardiac sections at E12.5 using sodium channel anti-β1 antibody. (C) Scn1b is localized in the cardiac fibers at E17.5 in the T-tubular system, and a signal was detected in the intercalated disks (arrow). (D) Negative control at E17.5. (E and G) Similar Scn1b immunolocalization was observed in the muscle fibers in 1 day neonate (N1) (E) and adult heart (G). Fibers from adult heart displayed a linear label delineating the surface of cardiac cells and in the intercalated disks. (F and H) {alpha}-actinin and Cx43 staining, respectively, in adult heart. Arrows in C, G and H illustrate intercalated disks. Nuclei in blue.

 
In addition to the distribution of Scn1b in a striated pattern, Scn1b signal began to be observed in zones of cell–cell contacts at E17.5 (Fig. 5C) displaying a similar pattern to that of connexin 43 (Fig. 5H), a marker of intercalated disk in cardiac muscle [18]. This additional Scn1b expression is maintained in neonatal and the adult heart (Fig. 5E and G).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
References
 
The heart is the first functional organ during embryogenesis and develops from a peristaltoid-contracting tube into a synchronously contracting four-chambered heart. Initially, the rudimentary heart tube exhibits a rhythmic propagation of action potential with posterior–anterior polarity [43]. After looping begins, three regions display electromechanical properties similar to those of the early tubular heart (AV canal, sinoatrial region and outflow tract) and two faster conducting segments becomes distinguishable (the atrium and ventricle) [2,44]. At this stage we have detected the initial weak expression of the sodium channel β-subunit Scn1b transcripts. An enhanced expression of Scn1b mRNA was observed at embryonic stage (E12.5). Immunohistochemical analysis revealed a similar Scn1b protein distribution within the different myocardial chambers during early embryonic stages (E9.5). Similar expression patterns for different ion channels subunits at these stages of cardiac development have been reported [26]. Thus, these results demonstrate that Scn1b is present in early embryonic heart development and its expression pattern suggests that this Na+ β-subunit may not have a distinctly relevant role in the regional electromechanical differences found at these stages as suggested for the potassium currents [26].

Although with further development Scn1b protein expression remains in all cardiac compartments, at E12.5 stage we observed an enhanced expression in right and left bundle branches, coinciding with the increase of Scn1b mRNA levels reported in this study and the switching from an immature base-to-apex pattern of epicardial activation to a mature apex-to-base pattern [32,45]. Thus, Scn1b could have a role at the beginning of the apical activation of the ventricles during cardiac development. In our immunohistochemical analysis at embryonic stages (E13.5) we observed that Scn1b expression is higher in the trabeculated myocardium while expression in the compact myocardium showed a less intensive labelling at these stages. Thus, the development of a compact myocardium in ventricles during cardiogenesis might explain the decrease of cardiac Scn1b mRNA expression observed at E13.5 and E15.5 stages.

In postseptation embryonic stages, we observed a strong labelling for Scn1b in the periphery of the SA node. This finding is in line with a recent study [46] which shows that the main cardiac {alpha}-subunit sodium channel Nav1.5 is absent from the centre of SA node but present in the periphery of the SA node and that cardiac Nav1.5 isoform is involved in the propagation of the action potential from the SA node to the surrounding atrial muscle. We found additionally in this paper that Scn1b protein expression is clearly reduced in AV node and His bundle in comparison with others areas of the ventricular conduction system. These results are in line with studies which demonstrate that sodium channel {alpha}-subunit Nav1.5 is present in Purkinje fibers but absent in AV node [47]. In addition, enhanced expression of Scn1b protein in trabeculated myocardium is maintained during later cardiac development and we show that Scn1b expression coincides with expression profile of gap-junction Cx40, which is expressed mainly in His–Purkinje system [39–41,48] in agreement with previous Northern blot analysis in the adult sheep heart [14]. This expression pattern suggests that Scn1b might be involved in the faster conduction through Purkinje fibers network as compared with the working ventricular myocardium. Indeed, co-expression of Scn1b with Scn5a has been correlated with a small but significant acceleration in recovery from inactivation [19,20]. Furthermore, co-expression of Scn1b in Xenopus oocytes leads to a higher current amplitude, suggesting that it may increase the efficiency with which the mature channel is targeted to the plasma membrane [14]. Our results suggest that Scn1b subunit would have an important role in establishment of the mature conduction system of the ventricles during development.

In our study, immunolocalization analysis revealed that Scn1b is present in the cytoplasm and cell surface of the nascent cardiomyocytes at embryonic stages. However, at postseptation and neonatal stages, when cardiomyocytes start to acquire a mature appearance we observed Scn1b labelling in T-tubules system as identified by in {alpha}-actinin staining, remaining as such in neonatal and adult stages. The presence of Scn1b in the T-tubules system is consistent with enhanced Scn1b mRNA levels at later embryonic and neonatal stages as well as in adult heart found in this study. In addition, we demonstrate Scn1b labelling in the intercalated disks in later embryonic, neonatal and adult cardiomyocytes as well as an additional Scn1b staining along the long axis of the cardiac fibers in adult heart consistent with the delineated surface of the cells. This subcellular distribution of Scn1b in adult heart is in agreement with recent studies in isolated adult mouse ventricular myocytes [18].

The Scn1b subcellular expression pattern reported herein is consistent with previous studies which show that transverse tubules develop in cardiomyocytes during later fetal and early postnatal mammalian cardiogenesis and that, at these stages, intercalated disk becomes increasingly complex [49,50]. Recent immunological studies have shown that isolated cardiac adult rat and mouse ventricular myocytes express several different Na+ channel isoforms, with the "cardiac" pore-forming {alpha}-subunit Nav1.5 located in T-tubules system and at the intercalated disks but with the "brain" isoforms Nav1.1, Nav1.3 and Nav1.6 expressed in T-tubules, suggesting that the T-tubules may represent a region of the cell where the regulation of Na+ is different from that of the surface sarcolemma [18,42]. Our results show that the presence of Scn1b in T-tubular system and intercalated disks would indicate an important modulation of this Na+ β-subunit in the coupling between excitation–contraction in cardiac cells and in action potential propagation from cell to cell.

In summary, we demonstrate that Scn1b is expressed in developing cardiac myocardium by E9.5 and that Scn1b expression is increased during cardiogenesis. Additionally, we show an enhanced expression of this Na+ ion channel β-subunit in the right and left bundle branches and trabeculated ventricular components. At the subcellular level Scn1b is present in the T-tubular system and intercalated disks, additionally, Scn1b staining is consistent with the presence of the β1 sodium channel subunit in the sarcolemma. Thus, Scn1b could have a role in the establishment of the global pattern of conduction and contraction of the developing four-chambered heart.


   Acknowledgements
 
We thank Lori Isom (University of Michigan, USA) and Lucile Miquerol (IGBM, Marseille, France) for supplying the anti β1 sodium channel subunit and anti-connexin 43 antibody, respectively, and Nieves de la Casa for excellent technical assistance in the Leica Confocal Microscope (Confocal Microscopy Core Facility, University of Jaén, Spain). This work was supported by a grant of Ministry of Science and Technology (SAF2001-3634-C02-01, Spain) and a grant of Junta de Andalucía (CTS446, Spain).


   Notes
 
Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 

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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.
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L.S. Meadows and L.L. Isom
Sodium channels as macromolecular complexes: Implications for inherited arrhythmia syndromes
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