The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium

Abstract

Cardiac sodium channels are responsible for conduction in the normal and diseased heart. We aimed to investigate regional and transmural distribution of sodium channel expression and function in the myocardium. Sodium channel Scn5a mRNA and Na_v1.5 protein distribution was investigated in adult and embryonic mouse heart through immunohistochemistry and in situ hybridization. Functional sodium channel availability in subepicardial and subendocardial myocytes was assessed using patch-clamp technique. Adult and embryonic (ED14.5) mouse heart sections showed low expression of Na_v1.5 in the HCN4-positive sinoatrial and atrioventricular nodes. In contrast, high expression levels of Na_v1.5 were observed in the HCN4-positive and Cx43-negative AV or His bundle, bundle branches and Purkinje fibers. In both ventricles, a transmural gradient was observed, with a low Na_v1.5 labeling intensity in the subepicardium as compared to the subendocardium. Similar Scn5a mRNA expression patterns were observed on in situ hybridization of embryonic and adult tissue. Maximal action potential upstroke velocity was significantly lower in subepicardial myocytes (mean ± SEM 309 ± 32 V/s; n = 14) compared to subendocardial myocytes (394 ± 32 V/s; n = 11; P < 0.05), indicating decreased sodium channel availability in subepicardium compared to subendocardium. Scn5a and Na_v1.5 show heterogeneous distribution patterns within the cardiac conduction system and across the ventricular wall. This differential distribution of the cardiac sodium channel may have profound consequences for conduction disease phenotypes and arrhythmogenesis in the setting of sodium channel disease.

Electronic supplementary material

The online version of this article (doi:10.1007/s00395-009-0012-8) contains supplementary material, which is available to authorized users.

Introduction

The cardiac voltage-gated sodium channel is responsible for the fast upstroke of the action potential and is essential for proper conduction of the cardiac electrical impulse. Knowledge of its differential cardiac distribution is essential when investigating its role in the normal and diseased heart. During common pathological conditions such as myocardial ischemia and heart failure, decreased sodium current function may cause conduction disturbances and potentially life-threatening arrhythmias [9, 23, 41, 45]. In addition, mutations in the SCN5A gene encoding the pore-forming α-subunit of the cardiac sodium channel (Na_v1.5) have been shown to underlie multiple inherited arrhythmia syndromes, including long QT syndrome (type 3, LQT3), Brugada syndrome, conduction disease, sinus node dysfunction and atrial standstill [24]. Multiple biophysical effects of SCN5A mutations are considered to underlie the broad spectrum of disease symptoms observed in inherited sodium channelopathies [35].

To fully appreciate the biophysical effects of the cardiac sodium channel and the observed diversity in phenotypic expression observed in sodium channel disease, knowledge of the regional and transmural distribution of Na_v1.5 is essential. While transmural differences in action potential repolarization have previously been studied in detail, information regarding heterogeneous depolarization across the ventricular wall is rather limited [1, 12]. Inhomogeneous sodium channel expression may have profound functional consequences, including transmural heterogeneity in conduction velocity, potentially contributing to arrhythmogenesis [12, 44, 46]. In addition, sodium channel distribution in different regions of the conduction system may influence heart rate and atrioventricular and intraventricular conduction [5, 7, 30, 43].

To date, a number of studies have investigated Na_v1.5 distribution in (parts of) the conduction system, but results have been contradictory [17, 33, 47]. Other studies have been performed in enzymatically isolated cardiomyocytes, allowing for only limited interpretation of the relevance of these channels for overall cardiac conduction and function [25, 29, 31]. In the present study, we performed a comprehensive analysis of regional and transmural distribution of Scn5a mRNA and Na_v1.5 protein expression in the murine heart, combining immunohistochemistry and in situ hybridization. Our results indicate that Scn5a and Na_v1.5 show heterogeneous distribution patterns within the cardiac conduction system and across the ventricular wall. This data thus provides insight into the role of the cardiac sodium channel in normal cardiac electrophysiology and enables increased understanding of the consequences of sodium channel dysfunction for conduction disease phenotypes and arrhythmogenesis.

Methods

Animal experiments were conducted in compliance with the “Guiding Principles in the Use and Care of Animals” published by the National Institutes of Health (NIH publication No. 85–23, revised 1996) and performed in accordance with institutional guidelines for animal use in research.

Antibodies

The following primary antibodies were used: rabbit polyclonal anti-Na_v1.5 (1:200, Alomone Laboratories, ASC-005), mouse monoclonal anti-alpha-actinin (1:1000, Sigma), mouse monoclonal anti-desmin (1:200, Monosan, MON-3001), mouse monoclonal anti-Cx43 (1:200, BD Biosciences, 610061), rabbit polyclonal anti-Cx40 (1:250, Chemicon), rabbit polyclonal anti-HCN4 (1:200, Chemicon, AB5808), and rabbit polyclonal anti-cardiac troponin I (anti-cTnI, 1:1000, HytestLtd). Alexa conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were used (1:250, Molecular Probes, Invitrogen). Nuclei were stained using Sytox Green (1:30,000, Molecular Probes).

Immunocytochemistry in transfected HEK293 cells

The human sodium channel α-subunit SCN5A with GFP fused at the carboxyl end was transfected in HEK293 cells using Lipofectamine Reagent (Gibco BRL), according to manufacturer’s instructions. Transfected HEK293 cells were cultured, fixed, and stained with anti-Na_v1.5 antibody as described in the “Data supplement”.

Western blot analysis

Total membrane protein fractions were isolated from HEK293 cells transfected with the β1-subunit in the presence or absence of SCN5A. Solubilized membrane protein (50 μg) was blotted to a Hybond transfer membrane (GE Healthcare, Amersham) and incubated with anti-Na_v1.5 primary antibody (see “Data supplement”). Protein was visualized using the ECL Plus Western blotting detection kit (GE Healthcare, Amersham).

Immunohistochemistry on mouse heart sections

Adult male FVB/N mice (age 2–3 months; n = 5 hearts) were anesthetized by an intraperitoneal injection of pentobarbital, after which the heart was excised, snap-frozen in liquid nitrogen and stored at −80°C. Four-chamber view cryosections (7 μm) were mounted on 3-aminopropyltriethoxysilane (AAS) coated glass slides. Mouse embryos at embryonic day 14.5 (ED14.5; n = 4 embryos) were snap-frozen in liquid nitrogen and stored at −80°C, and cryosectioned (10 μm). Sections were permeabilized in 0.2% Triton X-100 in PBS for 1 h, whereas PFA sections were boiled for 10 min in Antigen Unmasking Solution. All sections were blocked in 2% BSA for 30 min and incubated with primary (overnight) and secondary antibody (90 min) in 10% NGS (all at RT). Confocal imaging was performed using a confocal laser scanning microscope (BioRad MRC1024) equipped with a 15 mV Krypton/Argon laser, using the 568 and 488 excitation lines and 605DF32 and 522DF35 emission filters.

In situ hybridization

Embryos (ED14.5; n = 3 embryos) and hearts from adult mice (age 2–3 months; n = 2 hearts) were fixed for 4 h to overnight in freshly prepared 4% paraformaldehyde (PFA) in PBS, dehydrated in a graded ethanol series, paraffin-embedded, sectioned, and mounted on AAS-coated slides. RNA probes complementary to the mRNAs for Scn5a, Cx43, cardiac troponin I (cTnI) and the homeodomain transcription factor Irx5 were labeled with digoxigenin-UTP, hybridized to the sections overnight and immunologically detected (for details, see “Data supplement”).

Action potential measurements in isolated myocytes

Thin slices (approximately 0.5 mm) were removed from the endocardial and epicardial surface of the left ventricular free wall, and single ventricular myocytes from subendocardial and subepicardial origin were enzymatically dissociated as previously described [34]. Action potentials were recorded at 37°C at a stimulation frequency of 2 Hz using the perforated patch-clamp technique. Tyrode’s solution (37°C) contained (in mmol/L): 140 NaCl, 5.4 KCl, 1.8 CaCl₂, 1 MgCl₂, 5.5 glucose, 5 HEPES; pH 7.4 (NaOH). Patch pipettes (~1.5 MΩ) contained (in mmol/L): 125 K-gluc, 20 KCl, 5 NaCl, 0.22 amphotericin-B, 10 HEPES; pH 7.2 (KOH). Details on action potential generation, acquisition and filtering are described elsewhere [34]. To characterize action potentials, we analyzed resting membrane potential (RMP), action potential amplitude (APA), action potential plateau phase 20 ms after the upstroke, action potential duration at 90% repolarization (APD₉₀), and maximal action potential upstroke velocity (dV/dt_max). dV/dt_max is considered a suitable index for functional sodium current availability. Although it slightly overestimates sodium channel availability at low temperatures (7–10°C), the discrepancies between sodium current and dV/dt_max are reduced at higher temperatures (26–27°C) [37] and are practically absent at physiological temperature [6]. Action potentials were measured from both subepicardial and subendocardial cells of four hearts, and parameters from ten consecutive action potentials were averaged.

Statistical analysis

Data are presented as mean ± SEM. Differences between groups were analyzed by unpaired Student’s t test. The level of statistical significance was set to P < 0.05.

Results

Anti-Na_v1.5 antibody selectivity in transfected HEK293 cells

Details on the polyclonal anti-Na_v1.5 antibody are shown in Supplemental Fig. 1. When compared to other murine sodium channel α-subunits, no relevant homology with the epitope recognized by this antibody (maximal 9/19 residues identical) was observed (Supplemental Fig. 1a). On Western blot analysis, HEK cells transfected with SCN5A showed a specific band at the approximate expected size (Supplemental Fig. 1b). Immunocytochemistry of HEK293 cells transfected with cDNA for SCN5A with GFP fused at the carboxyl end showed a high degree of overlap between the signal detected by the anti-Na_v1.5 antibody and the GFP signal (Supplemental Fig. 1c). Thus, we considered this anti-Na_v1.5 antibody selective for the cardiac sodium channel and appropriate for further immunohistochemical analysis.

Fig. 1.

a Immunohistochemical analysis of cryosections from adult murine heart showing a detailed view of the sinoatrial node (SAN; marked region). The SAN is clearly demarcated by the presence of HCN4 staining and the absence of Cx43 labeling (middle panel) and displays low levels of Na_v1.5 staining. In contrast, abundant Na_v1.5 staining is visible in the surrounding right atrial (ra) tissue. b In situ hybridization of embryonic heart (ED14.5) showing mRNA expression of Scn5a, CtnI and Cx43 (in blue). The CtnI-positive and Cx43-negative SAN (denoted by arrow) shows low Scn5a mRNA expression (pv pulmonary vein, rscv right superior caval vein). c Immunohistochemical analysis of cryosections from adult murine heart showing a detailed view of the atrioventricular node (AVN; marked region). The AVN shows intense staining for HCN4, but low expression of both Cx40 and Na_v1.5, whereas clear Na_v1.5 staining is visible in the atria (a) and working myocardium (w)

Anti-Na_v1.5 antibody selectivity in cardiac tissue

To further validate the anti-Na_v1.5 antibody, we next studied its expression in the sinoatrial node (SAN), which is generally considered to contain a limited number of Na_v1.5 sodium channels [22, 30]. Since double staining of both polyclonal antibodies anti-HCN4 and anti-Na_v1.5 was not possible, the distribution of these proteins within the SAN was examined on adjacent serial tissue sections. Na_v1.5 staining was found to be very low in the HCN4-positive SAN region, whereas clear anti-Na_v1.5 staining was observed in the surrounding atrial myocardium (Fig. 1a). This HCN4-positive region was devoid of the gap junction protein connexin43 (Cx43), as expected for the SAN. These immunohistochemical results were confirmed by in situ hybridization experiments on embryonic mouse tissue. Fig. 1b shows the region of the SAN staining positive for cardiac troponin I (cTnI) mRNA, which displayed limited Cx43 and Scn5a mRNA expression. Furthermore, Na_v1.5 staining was also low in the HCN4-positive and Cx40-negative atrioventricular node (AVN; Fig. 1c). These findings further underscore the selectivity and suitability of the anti-Na_v1.5 antibody.

Sodium channel distribution in the AV bundle and His-Purkinje system

Figure 2 displays the expression and distribution of Na_v1.5 in the conduction system in adult mouse ventricular myocardium. The atrioventricular bundle (AVB or His bundle) that displayed the expected presence of HCN4 staining and absence of Cx43 expression showed a high level of Na_v1.5 labeling, which was clearly more intense compared to that of ventricular tissue of the septum (Fig. 2a). Myocytes of both tissue types clearly showed staining of the cell contour, indicating membrane labeling of Na_v1.5 on the cell surface. The high level of Na_v1.5 expression observed in the AVB continued into a thin layer of myocytes running below the subendocardial lining of the septum (Fig. 2b). These subendocardial cells, which constitute the bundle branches and Purkinje fibers of the conduction system, showed intense desmin labeling, were Cx43-negative, Cx40-positive, and HCN4-positive (Fig. 2b, c). These findings were confirmed on Scn5a mRNA level by in situ hybridization experiments in adult hearts (Fig. 2d). A similar distribution pattern in the intraventricular conduction system was observed in embryonic mouse hearts, with high expression levels of both Na_v1.5 and Scn5a in the AVB and bundle branches (Fig. 3a, b). Overall, distribution of Na_v1.5 and Scn5a labeling intensity within the embryonic and adult heart was as follows: AVB/bundle branches > atrial/ventricular myocardium ⋙ SAN/AVN. The described distribution patterns were consistently observed in all hearts studied.

Fig. 2.

Distribution of Na_v1.5 in the atrioventricular bundle (AVB) and His-Purkinje system in adult murine myocardium. a The AVB lacks Cx43 expression, is HCN4-positive, and shows a high Na_v1.5 labeling intensity, continuing into (b) the left and right bundle braches, which show intense labeling of Na_v1.5 and desmin, but absence of Cx43 (LBB left bundle branch, RBB right bundle branch, rv right ventricle, ivs intraventricular septum). c Serial sections of Purkinje fibers (PF) show high Na_v1.5, HCN4, and Cx40 labeling intensity, but no Cx43. d In situ hybridization of adult heart shows similar high expression levels of Hcn4 and Scn5a mRNA in AVB (blue signals) in comparison with the myocardium of the intraventricular septum (ivs)

Fig. 3.

Distribution of Na_v1.5 and Scn5a in the conduction system in embryonic hearts (ED14.5). a High expression levels of Na_v1.5 protein in the AV bundle (AVB) and bundle branches (BB) in comparison with the ventricular myocardium of the intraventricular septum (ivs); the right panel represents an enlarged section of the left panel. b In situ hybridization shows prominent Scn5a mRNA expression (in blue) but absence of Cx43 mRNA in the AVB (dashed line indicates region of AVB)

Heterogeneous transmural distribution of Na_v1.5 and Scn5a in ventricular myocardium

Figure 4a and b shows double labeling of left ventricular myocardium by anti-Na_v1.5 with anti-desmin or anti-α-actinin. Although both desmin and α-actinin expression intensity appeared homogenously distributed throughout the ventricular wall, Na_v1.5 labeling showed a clear transmural gradient. Na_v1.5 expression was greatly reduced in subepicardial (denoted by arrowheads) compared to midmyocardial myocytes. Furthermore, more abundant Na_v1.5 expression was observed in subendocardial myocytes (denoted by arrows) as compared to either midmural or subepicardial cells. A similar transmural gradient (subendocardial myocardium > midmural myocardium > subepicardial myocardium) for Na_v1.5 was observed in the right ventricle (Fig. 4c). The subepicardial layer with low Na_v1.5 expression comprised a larger fraction of the total ventricular wall thickness in the right compared to the left ventricle. Furthermore, a distinct transmural distribution for Na_v1.5 was also observed in the right and left ventricle of mouse embryonic tissue (ED14.5; Fig. 5a). Again, the results from immunohistochemical analyses were confirmed by in situ hybridization experiments. Heterogeneous Scn5a mRNA expression with lower expression in the subepicardium compared to subendocardium was observed in both embryonic and adult murine ventricular tissue (Figs. 4d, 5b), in a pattern similar to Irx5, a homeodomain transcription factor with an established transmural gradient (Fig. 5b, right panel) [10, 14]. A similar substantial decrease in Scn5a/Na_v1.5 expression levels in the subepicardium compared to subendocardium was consistently observed in each independent heart studied.

Fig. 4.

Transmural distribution of Na_v1.5 in the ventricular myocardium. Low Na_v1.5 labeling intensity in ventricular epicardium (arrowheads) compared to the midmyocardial layer (m) in (a) the apex and (b) left ventricular free wall of the adult mouse heart. For comparison, α-actinin and desmin show a homogeneous transmural labeling. cLeft panel shows low Na_v1.5 expression in subepicardium (arrowheads) and high Na_v1.5 expression in subendocardium (arrows) of the right ventricle. For comparison, Cx43 shows a homogeneous transmural distribution. Also shown are details of subendocardial surface (middle panel) and subepicardial region (right panel) of the right ventricle. d In situ hybridization of adult ventricular myocardium shows clear Scn5a staining (in blue) in the subendocardium (arrows) and low Scn5a expression in the subepicardium (asterisks). The right panel depicts a magnified section of the left panel indicating higher Scn5a expression in the subendocardium (arrows) compared to midmural (m)

Fig. 5.

a Overview (left panel) and detail (right panel) of embryonic mouse heart (ED14.5) indicating low Na_v1.5 labeling intensity in ventricular subepicardium (asterisks) and abundant Na_v1.5 in the trabeculated subendocardium. b In situ hybridization of ventricular myocardium in embryonic heart (ED14.5; overview and magnified boxed section in the right panel) displaying a transmural gradient in Scn5a mRNA expression with lower expression in the subepicardium (asterisks) compared to the trabeculated subendocardium (arrows). For comparison, the homeodomain transcription factor Irx5 shows a similar transmural distribution pattern (lv left ventricle, rv right ventricle, ivs intraventricular septum)

Transmural gradient in functional sodium channel availability

To investigate the functional electrophysiological consequences of the observed differences in Scn5a mRNA and Na_v1.5 protein expression levels, we studied the action potential characteristics of subepicardial and subendocardial ventricular myocytes of four hearts. In particular, we focused on the dV/dt_max, which is a suitable index of sodium channel availability. Figure 6a shows representative action potentials recorded from a subepicardial and subendocardial cell of one heart. Subepicardial myocytes displayed considerable shorter action potentials compared to subendocardial cells, while action potential amplitude, plateau phase and resting membrane potential (important for voltage dependence of sodium channel inactivation) were not different (Fig. 6b). On average, the action potential duration at 90% repolarization (APD₉₀) was 109 ± 11 ms in subepicardial myocytes (n = 14) and 153 ± 16 in subendocardial myocytes (n = 11; P < 0.05). Figure 6c shows the first time-derivative of the action potential (dV/dt_max), reflecting the maximal action potential upstroke velocity of the cells depicted in Fig. 6a. In each heart, action potential upstroke velocity was consistently lower in subepicardial myocytes compared to subendocardial cells (Fig. 6d). Consequently, average action potential velocity was significant lower in subepicardial cells (309 ± 32 V/s, n = 14) compared to subendocardial cells (394 ± 32 V/s, n = 11; Fig. 6e), indicating decreased functional sodium channel availability in the subepicardium compared to subendocardium.

Fig. 6.

a Representative examples of action potentials of a subepicardial and subendocardial myocyte from one heart, measured at a stimulation frequency of 2 Hz. b Averaged action potential characteristics (RMP resting membrane potential, APA action potential amplitude, AP plat. action potential plateau phase 20 ms after the upstroke, APD₉₀ action potential duration at 90% repolarization, #P < 0.05 versus subendocardial). c Representative examples of the first time-derivative of the action potential (dV/dt_max), reflecting the maximal action potential upstroke velocity, measured from a subepicardial and subendocardial myocyte from one heart at a stimulation frequency of 2 Hz. d Averaged subepicardial and subendocardial action potential upstroke velocities of each heart measured (n = 4 hearts); numbers of mycoytes measured from each heart are indicated. e Average values for action potential upstroke velocities from all subepicardial and subendocardial myocytes (#P < 0.05 versus subendocardial)

Discussion

In this study, we provide a comprehensive investigation of sodium channel distribution throughout the murine heart. Our results demonstrate a unique differential distribution pattern of Scn5a mRNA and sodium channel protein Na_v1.5 expression in various parts of the conduction system. Furthermore, we observed a transmural gradient in Scn5a mRNA expression, Na_v1.5 protein expression and sodium channel availability across the ventricular wall.

Low Scn5a mRNA and Na_v1.5 protein expression in sinoatrial and atrioventricular nodes

Analysis of the murine conduction system revealed very low Scn5a mRNA and Na_v1.5 protein expression in the sinoatrial node (SAN) and atrioventricular node (AVN). Previously, low Na_v1.5 expression levels in the central atrioventricular node versus high levels of Na_v1.5 in the peripherally located transitional cells have been observed in rat and rabbit, and thus may be considered species independent [33, 47]. The low abundance of Na_v1.5 in the SAN is in accordance with the relatively low expression of Scn5a mRNA observed in the adult mouse SAN region measured by quantitative RT-PCR [30], and the previously reported low degree of Na_v1.5 staining in the SAN of the embryonic and adult mouse heart [22, 28]. We have now confirmed the presence of low Scn5a mRNA expression in the SAN using in situ hybridization, further validating these observations. Functionally, the brain-type sodium channel, and not the cardiac-type, appears essential for heart rate regulation, at least in the mouse [28]. In addition, cardiac-type sodium channels have not been found in the center of the SAN of the human heart, but only in the periphery of the SAN, where they may be essential for proper conduction to the surrounding atrial tissue [16]. Based on these observations, the clinical phenotype of sick sinus syndrome or sinus dysfunction described in some SCN5A mutations may be explained by peripheral SAN dysfunction and impaired impulse propagation to the atria [5, 7, 42]. On the other hand, the persistent inward sodium current and altered channel inactivation kinetics resulting from the SCN5A mutation 1795insD have been proposed to underlie the observed bradycardia and sinoatrial pauses [42]. Furthermore, the SCN5A mutation E161K, associated with sick sinus syndrome, was predicted to cause a reduction in sinus rate by slowing of the diastolic depolarization rate and upstroke velocity of the sinus node action potential [39]. Thus, although the expression level of sodium channels in the SAN appears limited, they may still play a role in sinus node function and heart rate regulation, perhaps even more so in the setting of sodium channel dysfunction.

Abundant Scn5a and Na_v1.5 expression in the intraventricular conduction system

We observed abundant expression of Scn5a and Na_v1.5 in the atrioventricular bundle (AVB or His bundle), bundle branches and Purkinje fibers, predicting a substantial role for Na_v1.5 in fast propagation through the intraventricular conduction system. In previous studies by others, both high and low intensities of Na_v1.5 staining have been reported in these areas [17, 47]. This discrepancy in observations may be attributed to differences in tissue handling protocols used, which may have influenced tissue binding properties of the antibody. To address this controversy, we have used in situ hybridization of Scn5a mRNA as an independent tool for assessing the distribution pattern of the cardiac sodium channel in the intraventricular conduction system. The observed pattern of Scn5a mRNA distribution matches that of Na_v1.5 protein distribution, thereby ultimately proving the presence of abundant cardiac sodium channel expression in the AVB (or His bundle), bundle branches and Purkinje fibers. Since the cardiac sodium channel is hardly expressed in the atrioventricular node (AVN), the abundance of Na_v1.5 in the AVB (or His bundle), bundle branches and Purkinje fibers is likely to be responsible for the atrioventricular and intraventricular dysfunction observed in patients with sodium channel dysfunction due to SCN5A mutations [7, 43].

Functional transmural gradient in cardiac sodium channel distribution

Another finding in the current study is the observation of a transmural gradient in Scn5a, Na_v1.5, and functional sodium channel availability. The presence and ionic mechanism of transmural differences in action potential repolarization have been investigated in detail, but studies of transmural differences in depolarization are limited. Transmural action potential heterogeneity of the left ventricle is well known, with subepicardial and midmyocardial action potentials displaying prominent phase-1 repolarization, while subendocardial action potentials do not [1]. In addition, midmyocardial cells have longer action potentials compared to subepicardial and subendocardial cells, while subepicardial action potentials are the shortest [1]. Various membrane channels contribute to these transmural differences in the time course of action potential repolarization, including the transient outward K⁺ current (I_to1) [27], the slow delayed rectifier K⁺ current (I_Ks) [26], and the sustained component of the sodium current (I_Na(sus)) [48]. Some investigators have reported reduced sodium channel density in isolated myocytes from the subepicardium as compared to the mid- and subendocardium [3, 40], whereas other studies showed either no difference [12] or rather an increased sodium current density in subepicardium versus subendocardium [21]. Similarly, inconsistent results on sodium gating properties of subendocardial versus subepicardial myocytes have been described. In the dog, subepicardial myocytes have a more negative half-inactivation voltage [12], which consequently will lead to a reduced sodium current availability at resting membrane potentials. In contrast, studies in rats revealed no differences in inactivation [3, 21], but one report showed a more negative half-activation potential in subendocardial compared to subepicardial myocytes [21]. These discrepancies may be attributed to differences in experimental protocol, myocyte isolation process and species studied. Our current immunohistochemistry results from whole mouse heart sections show the existence of a transmural gradient in Na_v1.5 distribution in both the left and right ventricle in situ. Importantly, Scn5a mRNA expression displayed a similar inhomogeneous distribution across the ventricular wall on in situ hybridization. Reduced Scn5a mRNA expression has also been demonstrated in epicardial compared to endocardial tissue in non-diseased human hearts, clearly suggesting that a transmural gradient in sodium channel availability is also present in human myocardium [18]. The functional relevance of the heterogeneous transmural Scn5a/Na_v1.5 distribution was subsequently confirmed by our demonstration of reduced functional sodium channel availability in the subepicardial compared to subendocardial cells from patch-clamp analysis in isolated myocytes. Although we did not measure sodium current characteristics in the current study, dV/dt_max is considered a suitable index for functional sodium current availability [6, 37], and these functional data are in complete accordance with our immunohistochemistry and in situ hybridization results.

Implications of transmural heterogeneity in sodium channel distribution and function

The functional implications of transmural heterogeneity in Na_v1.5 expression may be substantial. In the subepicardium, decreased inward sodium current will on the one hand slow conduction, and on the other hand allow for a greater impact of the repolarizing potassium currents, resulting in action potential shortening and subsequent transmural dispersion in repolarization. In the setting of myocardial ischemia, a gradient of Na_v1.5 expression predisposes to inhomogeneous conduction slowing, greatly increasing the risk of life-threatening arrhythmias [9]. In patients with Brugada syndrome, the typical ECG pattern of ST-segment elevation in the right precordial leads has been attributed to a transmural heterogeneity of repolarization in the right ventricle [2], where the transient outward K⁺ current (I_to1) responsible for phase-1 action potential repolarization is more prominent compared to the left ventricle [15]. In loss-of-function SCN5A mutations, present in approximately 15–30% of Brugada syndrome patients [36, 38], sodium current reduction combined with the large transient outward current (I_to) in the right ventricular subepicardium is thought to give rise to a prominent early repolarization phase and an even larger transmural repolarization gradient [2]. Brugada syndrome is also thought to arise from right ventricular conduction delay due to localized conduction slowing [4, 13]. The inhomogeneous Na_v1.5 distribution across the ventricular wall observed in our immunohistochemistry experiments may contribute to both mechanisms. Since the subepicardial layer comprises a relatively large fraction of the total right ventricular wall (as compared to the left ventricle), the effects of further sodium channel reduction is expected to be most notable in the right ventricle. Thus, in the current study, we have identified transmural heterogeneity in cardiac sodium channel expression as a novel additional pathophysiological player in the Brugada syndrome.

Cardiac distribution of Scn5a mRNA and Na_v1.5 protein expression in the mouse embryo

Cardiac expression and function of certain ion channels may be differentially regulated during murine heart development [17, 20, 32]. In our study, the observed distribution patterns of Scn5a mRNA and Na_v1.5 protein expression were similar in adult and embryonic murine hearts (ED14.5). Interestingly, the spatio-temporal expression pattern of Scn5a is similar to that of Cx40, and together they are likely responsible for fast conduction through the AVB, bundle branches and Purkinje fibers [11, 19]. Cx40, at least in embryonic hearts, displays a transmural gradient across the ventricular wall in a pattern similar to Scn5a, and its expression is known to be regulated by the T-box transcriptional activator Tbx5 [8, 19]. Sodium channel expression in the SAN is controlled by the T-box transcriptional repressor Tbx3, since Na_v1.5 is ectopically expressed in the SAN in Tbx3-deficient mice [22]. Therefore, Tbx3 and Tbx5 may act in concert to determine the unique distribution pattern of Scn5a and Na_v1.5 within the heart, with Tbx3 repressing expression in the SAN and AVN, and Tbx5 promoting expression in the atria, ventricle, AVB and bundle branches. The homeodomain transcription factor Irx5 also displays a transmural gradient similar to Scn5a and has been shown to determine the gradient of potassium channel expression and repolarization across the ventricular wall [10, 14]. Thus, it is tempting to speculate that Tbx5 and/or Irx5 are also responsible for the heterogeneous transmural distribution of the cardiac sodium channel.

Conclusion

We have presented an integrative assessment of the unique distribution pattern of the cardiac sodium channel within the murine heart. In particular, Na_v1.5 was observed to be heterogeneously expressed in the cardiac conduction system, with low abundance in the nodal tissues and high expression in the intraventricular conduction system. In addition, a transmural gradient across the ventricular wall exists, with low Na_v1.5 labeling intensity and functional sodium channel availability in the subepicardium compared to subendocardium. Since the sodium channel ensures proper electrical conduction within various regions of the myocardium, knowledge of its differential cardiac distribution is essential when investigating its role in the normal and diseased heart. The current results thus underline the importance of studying the multiple electrophysiological consequences of an SCN5A mutation in its native environment, i.e., in various regions and compartments of the heart and cardiomyocyte.