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. Author manuscript; available in PMC: 2007 Sep 22.
Published in final edited form as: Heart Rhythm. 2006 Mar 27;3(7):842–850. doi: 10.1016/j.hrthm.2006.03.021

Functional expression of “cardiac-type” Nav1.5 sodium channel in canine intracardiac ganglia

Fabiana S Scornik *, Mayurika Desai *, Ramón Brugada †, Alejandra Guerchicoff *, Guido D Pollevick *, Charles Antzelevitch *, Guillermo J Pérez *
PMCID: PMC1989775  NIHMSID: NIHMS25171  PMID: 16818219

Abstract

BACKGROUND

The autonomic nervous system has been implicated in several arrhythmogenic diseases, including long QT syndrome type 3 (LQT3) and Brugada syndrome. Scarce information on the cellular components of the intrinsic cardiac ganglia from higher mammals has limited our understanding of the role of the autonomic nervous system in such diseases.

OBJECTIVES

The purpose of this study was to isolate and characterize the electrophysiologic properties of canine intracardiac neurons.

METHODS

Action potentials (APs) and ionic currents were studied in enzymatically dissociated canine intracardiac neurons under current and voltage clamp conditions. Immunohistochemical and reverse transcription-polymerase chain reaction analysis was performed using freshly isolated intracardiac ganglia.

RESULTS

APs recorded from intracardiac neurons displayed a tetrodotoxin-resistant (TTX-R) component. TTX-R APs were abolished in the absence of sodium but persisted in the absence of external calcium. Immunohistochemical studies showed the presence of TTX-R sodium channels in these ganglia. Sodium currents were characterized by two components with different affinities for TTX: a tetrodotoxin-sensitive (TTX-S) component and a TTX-R component. TTX-S current inactivation was characteristic of neuronal sodium currents, whereas TTX-R current inactivation time constants were similar to those previously reported for Nav1.5 channels. TTX sensitivity (IC50 = 1.17 μM) of the TTX-R component was in the range reported for Nav1.5 channels. Expression of Nav1.5 channels in intracardiac ganglia was confirmed by PCR analysis and sequencing.

CONCLUSION

Our results suggest that canine intracardiac neurons functionally express Nav1.5 channels. These findings open an exciting new door to our understanding of autonomically modulated arrhythmogenic diseases linked to mutations in Nav1.5 channels, including Brugada syndrome and LQT3.

Keywords: SCN5A, Tetrodotoxin, Autonomic dysfunction, Cardiac arrhythmia, Sudden cardiac death, long QT syndrome, Brugada syndrome

Introduction

Voltage-dependent sodium (Nav) channels play a critical role in membrane electrogenesis and repetitive firing of excitable cells. In the heart, the sodium current is carried largely by the Nav1.5 (or SCN5A) isoform, although the presence of “neuronal-type” sodium currents has been reported.1,2 Nav1.5 gene mutations are linked to a wide diversity of arrhythmic cardiac syndromes, including long QT syndrome type 3 (LQT3), Brugada syndrome, and progressive cardiac conduction disease. These syndromes are often associated with the development of life-threatening cardiac arrhythmias.3,4 LQT3 is often associated with sinus bradycardia, sinus pauses, and atrial standstill, which are not readily explained by the gain of function of the sodium channel responsible for QT interval prolongation.3 Although autonomic dysfunction has been invoked to explain various aspects of arrhythmogenesis attending LQT3 and Brugada syndrome,5,6 a direct link to defects in Nav1.5 affecting autonomic regulation has not been previously considered. Nav1.5 channels have been found in other noncardiac tissues, including neonatal dorsal root ganglion neurons (DRG),7 human intestinal smooth muscle and Cajal cells,8,9 and neurons from distinct regions of the brain.10,11 Direct evidence for the presence of this channel isoform in intracardiac neurons has not been provided. The present study provides evidence for the functional expression of Nav1.5 channels in intracardiac neurons of the canine heart. This evidence is supported by pharmacologic and biophysical characterization of the sodium current in isolated canine intracardiac neurons, by reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing of the channel, and by immunohistochemical studies conducted in freshly dissected ganglia.

Methods

Neuron dissociation

Principal neurons from the atrial ganglionated plexuses of the dog were obtained by standard enzymatic dissociation procedures. Dogs weighing 20 to 25 kg were anticoagulated with heparin and anesthetized with pentobarbital (30–35 mg/kg IV). The chest was opened via left thoracotomy. The heart was excised, placed in a cardioplegic solution consisting of cold (4°C) Tyrode solution containing 8.5 mM [K+]o, and transported to a dissection tray. The fat pads on the ventral, lateral, and dorsal aspects of the atrium were quickly removed and placed in a normal Krebs solution in ice. Individual ganglia were removed from the fat pads and cleaned under a dissection scope for subsequent incubation at 37°C in a mixture of 0.1% collagenase and 0.1% elastase for 50 minutes. Enzymes were washed out, and ganglia were incubated for 20 minutes in 0.2% trypsin in nominally calcium-free solution. The trypsin reaction was stopped by addition of 1 mg/mL pancreatic or lima bean trypsin inhibitor. Tissue was washed again by transferring it to a clean (no enzymes), nominal Ca2+ Krebs solution. Individual cells were obtained by triturating the remaining tissue with a Pasteur pipette. Cells were resuspended in Dulbecco modified eagle media (DMEM), supplemented with 1% fetal bovine serum (FBS), 0.1% bovine serum albumin, 100 μg/mL penicillin-streptomycin, 2 mM GlutaMAX, 10 μg/mL murine nerve growth factor (NGF) 7S, and 0.11 g/mL pyruvic acid, and plated on collagen-coated bottom glass Petri dishes (MatTek Corporation, Ashland, MA, USA). Cells were placed in a CO2 incubator at 37°C for 16 to 72 hours.

Electrophysiologic recordings of isolated cells

Current and voltage measurements were obtained using the standard whole-cell, patch clamp technique12 in voltage and current clamp modes. Electrophysiologic recordings were obtained at room temperature. Solutions were applied with a gravity flow system (speed 1–3 mL/min.) to a 500-μL bath chamber.

Data acquisition and analysis

Voltage clamp experiments were controlled with an Axopatch 200A amplifier and pClamp 9.0/Labmaster DMA TL-1 acquisition system or Digidata 1322A (Axon Instruments, Union City, CA, USA). The amplitude and kinetic characteristics of the currents were measured and analyzed with Clampfit (Axon Instruments).

Sodium conductance gNa was calculated according to the equation gNa = INa/(V − ENa), where INa = peak amplitude of the sodium current at each applied voltage pulse (V), and ENa = Nernst equilibrium potential for Na+. Activation curve data were fitted to a Boltzmann equation of the form: gNa = gNa(max)/(1 + exp[V1/2 − V]/k), where V1/2 = half-maximum activation voltage, k = slope factor of the Boltzmann equation, V = test potential, and gNa(max) = maximum value of the sodium conductance. The inactivation curve h∞ was measured using 500-ms prepulses to the indicated potentials followed by a test pulse to −20 mV. Data were fitted to a Boltzmann equation of the form: INa = INa(max)/(1 + exp[V − V1/2]/k), where V1/2 = half-maximum voltage of inactivation, INa(max) = maximum value of the current, k = slope factor, and V = voltage value of the preconditioning pulse. Activation and inactivation kinetics were evaluated using PClamp software. τfast and τslow for the tetrodotoxin-resistant (TTX-R) component were obtained from fitting the time course of the inactivation of the current, from the peak, with a second-order exponential function. The tetrodotoxin-sensitive (TTX-S) component was obtained by subtracting the current measured in 100 to 300 nM TTX from the control current. Only one exponential was used to fit this current inactivation from the peak. Total current TTX inhibition curve was obtained assuming a 1:1 binding relation. Data were best fitted with two Langmuir absorption isotherms of the form: Inhibition = [Imax1 * [TTX]/(IC501 + [TTX])] + [Imax2 * [TTX]/(IC502 + [TTX])], where Inhibition = 1 − (ITTX/I), Imax = maximum value of the inhibition, and IC50 = half-inhibition concentration for TTX. For the TTX-R TTX-dependent inhibition curve, sodium current values in the presence of increasing concentrations of TTX (ITTX) were normalized to the peak value obtained at 300 nM TTX (ITTX300). Data were best fit with a Hill function of the form: Inhibition = Inhibitionmax * [TTX]n/(IC50n + [TTX]n), where Inhibition = 1 − (ITTX/ITTX300), Inhibitionmax = maximum value of the inhibition, n = Hill coefficient, and IC50 = half-inhibition concentration for TTX.

Solutions

The following solutions (in mM) were used. Normal sodium bath solution: 140 NaCl, 3 KCl, 10 sodium N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 2.5 CaCl2, 1.2 MgCl2, pH 7.4. Low-sodium, nominal-calcium bath solution: 40–60 NaCl, 80–100 tetraethylammonium (TEA)-Cl, 3 KCl, 10 HEPES, 3.7 MgCl2, pH 7.4. No-sodium solution: 140 N-d-methylglucamine, 3 KCl, 10 HEPES, 2.5 CaCl2, 1.2 MgCl2, pH 7.4. Pipette solution: 130 KCl, CsCl, Cs-Aspartate or CsF, 10 NaCl, 10 HEPES, 0.5–1 ethylene glycol-bis(2-amino-ethylether)-N,N,N′,N′-tetra-acetic acid (EGTA), 2 Mg-ATP, pH 7.15. Glucose was added to adjust osmolarity to 320 and 310 in the bath and pipette solutions, respectively. Drugs: Tetrodotoxin (TTX; Calbiochem La Jolla, CA, USA); collagenase type I, trypsin, and trypsin inhibitor (Worthington, Lakewood, NJ, USA); DMEM, NGF 7S, FBS, and GlutaMAX (Invitrogen Corporation, Carlsbad, CA, USA). All other drugs were obtained from Sigma (St. Louis, MO, USA).

RT-PCR1:

  • Sense: 5′-GAAGATGGCAGACTTCCTGTTAC-3′

  • Antisense: 5′-CCCAGCAGTCCTGTGTCATTAG-3′

RT-PCR2:

  • Sense: 5′-GTTCTGATGCCGGGACATGTC

  • Antisense: 5′-GAACTTCACTCTCTGCTTGATG-3′

RT-PCR3:

  • Sense: 5′-GAAGAGTTGGAAGAGTCTCAC-3′

  • Antisense: 5′-GCCCAGGCTATTCTCTTCATC

RT-PCR4:

  • Sense: 5′-GATTTGAGGAAGACAAGCAGC-3′

  • Antisense: 5′-CCTGCACAAGAGCTAGGTAC-3′

RT-PCR5:

  • Sense: 5′-GACCTGCCTTTGAACTACACC-3′

  • Antisense: 5′-CTGGAAGTTGAACATGTCATC-3′

RT-PCR6:

  • Sense: 5′-CACACTGCTTCTTTGCCCTCATG-3′

  • Antisense: 5′-GTTTTAGTTCTCCTCG-3′

β-Myosin heavy-chain primers:

  • Sense: 5′-GTGAGCGGGTGCAGCTGCTGC-3′

  • Antisense: 5′-CGCTGGTGTCCTGCTCCTTCTTC-3′

Immunohistochemistry

Partially dissociated ganglia were obtained by reducing the duration of the final dispersion step. The preparations were plated and fixed immediately after dissociation. Ganglia were mounted on glass slides and stained by standard immunohistochemical technique. Briefly, after fixation with 4% paraformaldehyde, 0.2% picric acid solution and permeabilization with 0.2% Triton X-100, tissue was incubated overnight at 4°C with 1:100/200 dilutions of primary rabbit polyclonal antibodies against Nav1.5 (anti human Nav1.5), Nav1.8, and Nav1.9 channel proteins (Alomone Labs, Jerusalem, Israel). Tissue was then incubated with a 1:1,000 dilution of anti-rabbit alexa-488 conjugated secondary antibody (Molecular Probes, Eugene, OR, USA) for 2 hours at room temperature. Cells were mounted using Pro-Long antifade mounting media (Molecular Probes) and visualized under confocal microscopy using a Fluoview Olympus laser scanning confocal microscope (40× oil immersion lens) provided with an argon and a He/Ne laser.

Reverse transcription-polymerase chain reaction

Total RNA was extracted from isolated canine intracardiac neurons using RNeasy extraction Micro Kit (Qiagen Inc, USA, Valencia, CA, USA). Six sets of primers were designed to amplify six overlapping RT-PCR products covering the entire coding region of canine SCN5A.

One-step RT-PCR was performed with Superscript III Reverse Transcriptase (Invitrogen) per manufacturer’s directions. Briefly, the amplification was performed as follows: 30 minutes at 55°C and 40 cycles of 30 seconds at 94°C, 30 seconds at 58°C, and 90 seconds at 68°C; the final extension was done for 5 minutes at 68°C. The PCR product was analyzed by electrophoresis on 1% agarose gel. Double-band PCR products were extracted from the agarose gel and purified using a commercial kit (QIAEX II, Qiagen). Single-band and purified PCR products were analyzed by direct sequencing with ABIPRISM Big Dye Terminator Cycle Sequencing Reaction and the ABI 3100 Automatic DNA Sequencer.

Results

TTX-R sodium channels underlie TTX-R action potentials in isolated canine intracardiac neurons

Isolated intracardiac neurons display morphologic characteristics of parasympathetic cardiac neurons similar to those previously described in fat pad slices (Figure 1A).13-15 Electrophysiologic recordings were obtained 16 to 24 hours after dissociation from neurons lacking cellular processes. Figure 1B illustrates current clamp recordings showing action potential (AP) activity in control conditions (2.5 mM CaCl2, 140 mM NaCl) and in the presence of 300 nM TTX. Block of neuronal TTX-S channels by 300 nM TTX did not completely abolish AP activity in canine intracardiac neurons (6/7 cells). When sodium in the bath solution was replaced by N-d-methylglucamine, AP activity was abolished (n = 6; Figure 1B). Suppression of transmembrane activity under these experimental conditions (2.5 mM calcium and 0 sodium) suggests that, in canine intracardiac neurons, depolarization of TTX-R APs is mediated by sodium channel current. TTX-R–dependent APs exposed to nominal-calcium and high-magnesium (3.7–5 mM) solution were not abolished in 6 of 7 experiments (not shown). These experiments strongly suggest that TTX-R APs in canine intracardiac neurons are mediated by activation of sodium channels that are resistant to 300 nM TTX.

Figure 1.

Figure 1

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Tetrodotoxin (TTX)-resistant action potential (AP) in canine intracardiac neurons. A: Isolated neurons from adult canine cardiac ganglia at 24 hours (left) and 72 hours (right) after dissociation. Calibration bar is 10 and 30 μm (left and right, respectively). B: Representative trace showing Aps recorded from an isolated neuron in a bath solution with 140 mM NaCl and 2.5 mM CaCl2 and after sodium replacement with N-methylglucamine. APs were evoked by a 1-nA, 1-ms, current injection step in control and in the presence of 300 nM TTX.

Histologic characterization of TTX-R channels in canine intracardiac neurons

Among the TTX-R type channels, three genetically distinct isoforms (Nav1.5, Nav1.8, and Nav1.9) have been characterized. As a further test of the hypothesis that TTX-R–dependent APs in canine intracardiac neurons involve the activation of TTX-R sodium channels, immunohistochemical staining of partially dissociated ganglia was performed using polyclonal antibodies against the Nav1.5, Nav1.8, or Nav1.9 channels. Figure 2 shows representative confocal immunofluorescence images of partially dissociated ganglia stained with antibodies against Nav1.5 (top), Nav1.8 (middle), and Nav1.9 (bottom) channel proteins. Immunostaining with the cardiac-type Nav1.5 channel antibody showed specific labeling of most of the principal neurons in the ganglia. No signal was detected when the primary antibody was incubated with a blocking peptide (not shown). Similar positive staining was observed in five different experiments performed under the same experimental conditions. Positive immunostaining with antibodies against Nav1.8 and Nav1.9 was observed mainly in small cells and ganglia components other than principal neurons (Figure 2, middle and bottom). Immunohistochemical evidence of the presence of TTX-R sodium channels in canine intracardiac neurons supports the hypothesis that TTX-R channels, and more likely Nav1.5 channels, are involved in the TTX-R APs observed in canine intracardiac neurons.

Figure 2.

Figure 2

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Confocal stacked images of a partially dissociated intracardiac ganglion, taken after immunostaining for the Nav1.5 (left), Nav1.8 (middle), and Nav1.9 (right) channels. Image stacks were constructed from ten 0.5-μm optical sections for Nav1.5 and Nav1.9 channel antibodies and from twenty-nine 0.35-μm, optical sections for the Nav1.8 antibody. Photographs show the specific immunostaining (shown in green) superimposed to a transmitted light image showing nonstained components of the ganglia. Calibration bars are 20 μm.

Sodium currents from canine intracardiac neurons are characterized by a TTX-S component and a TTX-R component

In another set of experiments, we recorded sodium channel current from the isolated intracardiac neurons using whole-cell, patch clamp techniques. These experiments were conducted under conditions designed to minimize the contribution of K+ and Ca2+ currents to the total current (see Methods). Increasing depolarizing voltage steps from a holding potential of −80 mV elicited a large inward current (Figure 3). At first glance, this fast inward current was not conspicuously different from that typically identified as the “neuronal-type” TTX-S current. However, addition of 300 nM to the bath failed to completely abolish the inward current. The middle panel in Figure 3 shows the current component that remains following exposure to 300 nM TTX. These traces show that the time course of inactivation of the TTX-R current is slower than the TTX-S component (obtained by subtracting the TTX-R component from the total current). TTX-R currents were observed in 48 of 51 cells isolated from 25 different preparations.

Figure 3.

Figure 3

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Tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents from canine intracardiac neurons. Sodium current recordings from dissociated canine cardiac neurons in the absence (A) and in the presence (B) of 300 nM TTX. C: TTX-S current resulting from a point-to-point subtraction of B from A. Currents were evoked by 50-ms voltage steps from −40 mV to +10 mV in 5-mV increments from a holding potential of −80 mV. Currents were recorded in low-sodium, nominal-calcium solution.

To estimate the current density of the total sodium current and the TTX-R component, peak sodium current density was measured in the absence and in the presence of 300 nM TTX at − 20 mV in a normal (140 mM NaCl) sodium bath solution. The average membrane capacitance measured from these experiments was 72.91 ± 7.0 pF (n = 21). Current density values obtained were 181.02 ± 26 pA/pF (n = 15) and 32.17 ± 5.7 (n = 14) pA/pF for total and TTX-R currents, respectively. These findings clearly point to the presence of two different types of sodium channels (TTX-R and TTX-S) in canine intracardiac neurons. A distinguishing feature between TTX-R and TTX-S channels is that TTX-R channels are known to be blocked by micromolar concentrations of cadmium.16,17 In canine intracardiac neurons, we found that cadmium blocked 74% (200 μM Cd2+, n = 3) of the sodium current recorded in the presence of 300 nM TTX, thus providing further support for the functional expression of TTX-R channels in these neurons.

Pharmacologic and electrophysiologic characteristics of the TTX-R current component

TTX-R sodium channels are known to possess different TTX sensitivities and electrophysiologic profiles.7,18 To identify the Nav channel isoform responsible for the TTX-R current in canine intracardiac neurons, we studied the electrophysiologic and pharmacologic characteristics of this component.

Electrophysiologic distinction between TTX-R and TTX-S current components in canine intracardiac neurons

To evaluate the current/voltage relationship for the TTX-R current component, peak current values were measured in the presence of 300 nM TTX at different step potentials from a holding potential of −80 mV. Averaged normalized values shown in Figure 4A show that current was first detectable at −54.6 mV and peaked at −19.6 mV (n = 5). Voltage dependence of activation and inactivation curves for the TTX-R sodium current are shown in Figure 4B. G/Gmax values were plotted at increasing step potentials and fitted with a Boltzmann equation. The half-voltage (V1/2) activation value obtained from this fit was −33.24 ± 0.43 mV (n = 4). To evaluate the voltage dependence of the steady-state inactivation, the fractional current (I/Imax) was measured with a two-pulse protocol. Sodium current was elicited by a 50-ms voltage step to −20 mV following a series of 500-ms preconditioning pulses (from −90 to +20 mV) in the presence of 300 nM TTX. The Boltzmann fit of I/Imax values gave a V1/2 inactivation value of −59.95 ± 0.04 mV (n = 4).

Figure 4.

Figure 4

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Electrophysiologic properties of sodium currents in isolated canine intracardiac neurons: A: Normalized current/voltage relationship for peak tetrodotoxin-resistant (TTX-R) currents. Currents were evoked by 50-ms voltage steps to +5 mV in 5-mV increments from a holding potential of −80 mV, in the presence of 300 nM TTX. Peak current values were normalized to their maximal amplitude (circles). B: Voltage-dependent activation and inactivation of TTX-R currents. Open circles represent mean values of voltage-dependent activation (gNa/gNamax, n = 4). Solid circles represent mean values of voltage-dependent inactivation (INa/INamax, n = 4). Open lines are Boltzmann fits to the data. C: Half-maximal activation time determined for the voltage range between −40 and + 40 mV. Triangles represent mean values for tetrodotoxin-sensitive (TTX-S) current. Circles represent mean values for the TTX-R current. D: Inactivation time constants determined for the voltage range between −30 and 0 mV. Triangles represent inactivation time constant values for the TTX-S current. Open circles represent τfast. Solid circles represent τslow for the TTX-R current. Voltage values were corrected for junction potential error. Values are expressed as mean ± SE. Values were obtained from currents recorded in normal-sodium, nominal-calcium bath (A, B) and 40 mM NaCl, nominal-calcium, and 3.7 mM MgCl2 (C, D).

The activation and inactivation kinetics of the TTX-R current component were analyzed in a separate series of experiments. No significant differences were observed in the half-time to the peak of the two (TTX-R and TTX-S) current components, except between −40 and −20 mV, where the TTX-S component was faster (Figure 4C). In contrast, inactivation properties of the two components were markedly different. Figure 4D depicts time constant values obtained from fitting the time course of the inactivation of the TTX-R and TTX-S currents with a second- and first-order exponential function, respectively. The τslow and τfast values for the TTX-R current component were in agreement with previously reported values for the cardiac sodium channel.19-21 Moreover, the time constant values obtained for the TTX-S component were in the range reported for neuronal sodium currents.21

Pharmacologic distinction between TTX-R and TTX-S current components in canine intracardiac neurons

Although all the members of the TTX-R sodium channel family have an affinity for TTX that is significantly lower relative to that of TTX-S sodium channels, sensitivity to the toxin differs among the three subtypes of TTX-R channels.7,18 Thus, a detailed analysis of TTX-dependent inhibition of the total current was necessary to distinguish the particular type of channel underlying TTX-R currents in canine intracardiac neurons. In order to include total current values for this study, recordings were measured in a low-sodium bath solution. Peak current values were obtained as described in Figure 1 in the absence and in the presence of increasing TTX concentrations. The average normalized peak values from 5 to 15 cells are shown in the plot in Figure 5A. Inhibition values were best fitted with the sum of two Langmuir isotherms confirming the presence of two populations of channels with distinct TTX sensitivity. The TTX-S component represents 90.75% of the total current and showed an IC50 of 6.77 nM, whereas the TTX-R component represents 9.2% of the current with an IC50 of 695 nM. Because TTX affinity is known to be affected by external sodium concentration,22,23 TTX sensitivity of TTX-R current was also evaluated under normal extracellular sodium conditions. Peak current values obtained with increasing concentrations of TTX were normalized to the value measured in the presence of 300 nM TTX (Figure 3B). Data were best fit with a single Langmuir isotherm with an IC50 of 1.17 μM (n = 4), which is in the range previously reported for the cardiac Nav1.5 sodium channel (Table 1).

Figure 5.

Figure 5

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Dose-response tetrodotoxin (TTX) inhibition of sodium currents in canine intracardiac neurons. Plot shows the dose response for TTX of the total sodium current from isolated canine intracardiac neurons. Currents were elicited by a 50- to 100-ms voltage step from a holding potential of −80 mV to a final potential of −20 mV. Circles represent the portion of current inhibited by TTX at different concentrations of the toxin. Inhibition was determined from the peak current ratio in the presence and in the absence of TTX. Solid line represents the best fit of the data with two Langmuir absorption isotherms. Currents were measured in a low Na+ (40 mM) external solution. Numbers in parentheses indicate number of experiments for each concentration of TTX. B: Dose response for TTX inhibition of the tetrodotoxin-resistant component of the current in normal external Na+ (140 mM). Inset shows representative traces at increasing concentrations of TTX. Solid line represents the best fit of the data with one Langmuir absorption isotherm. Values are expressed as mean ± SE.

Table 1.

Current properties of Nav1.8, Nav1.9, and Nav1.5 channels

Current type Half-maximal activation time at −15 mV (ms) τinactivation (ms) at −15 mV V1/2 activation (mV) V1/2 inactivation (mV) IC50 TTX (μM)
Nav1.8 0.757 9.367 −15.77 −33.77 46−>1007,33,34
Nav1.9 17 12.417 −57.57 −55.27 1, 477,35
Nav1.5 0.436 τfast: 0.7-2.120,21,36-38
τslow: 4.3-720,21 		−29 to −4920,21,36-39 −66 to −9220,21,36-39 1−621,36-40
Nav1.5 in DRG neurons 0.17 0.27 −427,37 −837,37 2.17
TTX-R in canine 0.27 ± 0.04 τfast: 1.2 ± 0.16 −33.24 ± 0.43 −59.95 ± 0.04 1.17 ± 0.84
Intracardiac neurons τslow: 7.9 ± 1.1
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These findings suggest that canine intracardiac neurons express two sodium current components, which are distinguishable by their electrophysiologic and pharmacologic properties. These results also suggest that the TTX-R current component in canine intracardiac neurons is similar to the cardiac Nav1.5 sodium channel.

Molecular evidence of expression of the cardiac-type sodium channel in canine intracardiac neurons

We further investigated the molecular identity of Nav1.5 channels in the intracardiac ganglia using RT-PCR analysis. Total RNA was purified from 30 to 40 freshly isolated canine cardiac ganglia, which were individually dissected from the fat pads and freed of other tissues. To obtain the entire coding region of SCN5A expressed in ganglia, six different sets of primers were designed, overlapping with canine SCN5A cDNA sequence (accession number AJ555547). Separation of the PCR products on 1% agarose gel yielded fragments of the expected size for canine SCN5A cDNA products (Figure 6). Homology of the sequences obtained from the PCR products with canine SCN5A cDNA confirmed the expression of this gene in canine intracardiac ganglia. The fourth set of primers, which composes the region from 3,082 to 4,239 bp of the SCN5A mRNA (accession number AJ555547), showed one additional lower band (Figure 6, lane 4). Characterization of both upper and lower bands in lane 4, by gel purification and sequencing, showed a deletion of 156 bp in the lower band. This region corresponds to an alternative splicing or skipping of exon 18 of the SCN5A gene, which encodes the intracellular loop connecting domains II and III of Nav1.5, previously reported in neuroblastoma cells.24 These results suggest that canine intracardiac ganglia express two different splice variants of the SCN5A gene.

Figure 6.

Figure 6

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Molecular evidence of the expression of the “cardiac-type” Nav1.5 channel in freshly obtained canine intracardiac ganglia. Reverse transcription-polymerase chain reaction (RT-PCR) products from total canine cardiac ganglia RNA separated in agarose gel. Lanes 1–6: RT-PCR product from a pool of 30 to 40 ganglia, individually dissected from the canine “fat pads,” on the atrial surface. Lane numbers (1–6) correspond to primer sets numbers (1–6) as detailed in the Methods section. Overlapping primer sets were designed to cover the entire canine SCN5A cDNA sequence (accession number AJ555547). Single bands were obtained in lines 1–3 and 5–6. The double band observed in lane 4 corresponds to alternative splicing products (see Results section). Lane 7: 1-kb molecular ladder.

To exclude any possible contamination of our preparation with cardiac myocytes, the presence of β-myosin heavy-chain mRNA was investigated by RT-PCR. β-Myosin heavy chain was not expressed in the cardiac ganglia preparations but was highly expressed in cardiac myocytes used as a positive control (not shown).

Discussion

Intracardiac ganglia function as signal integrating centers within the heart. The final output to the heart results from the AP activity of the postganglionic neurons, determined by a combination of the intrinsic electrical properties of the neurons and neurotransmitter-induced modulation of membrane ion channel conductances.25 Sodium currents are critical determinants of the electrical activity of excitable cells. Previous voltage clamp studies involving rat and guinea pig intracardiac neurons have shown the presence of voltage-dependent TTX-sensitive sodium currents.26 However, AP studies conducted in guinea pig and intact canine intracardiac ganglia have reported that evoked APs are not totally suppressed by as much as 300 nM TTX.27,28

Our data show that in isolated canine intracardiac neurons, TTX-R APs are abolished by replacement of external sodium by the nonpermeant cation N-d-methylglucamine (Figure 1B). This, together with the observation that TTX-R APs persist in the absence of external calcium and in the presence of high magnesium concentrations, strongly suggests that TTX-R APs in canine intracardiac neurons are mediated by TTX-R sodium channel activity.

Positive immunostaining of the three types of TTX-R sodium channels (Nav1.5, Nav1.8, and Nav1.9) provides support for the participation of a TTX-R sodium channel in canine intracardiac neurons. Immunostaining of Nav1.8 and Nav1.9 occurs mainly in cellular components of the ganglia other than principal neurons, whereas immunostaining of Nav1.5 is most conspicuous in principal neurons, suggesting that Nav1.5 is largely responsible for the TTX-R component of the sodium channel in the principal neurons of the canine intracardiac ganglia.

A comparison of our data with previously reported electrophysiologic and pharmacologic properties of TTX-R channels is given in Table 1. In addition to the absence of conspicuous immunostaining of principal neurons for Nav1.9 channel protein, the inactivation kinetics of the TTX-R current in intracardiac neurons was faster than what has been reported for this type of channel (Table 1). Inactivation kinetics for both Nav1.5 and Nav1.8 channels are close to the values that we found for the TTX-R current in intracardiac neurons (Table 1). Thus, other parameters were used to distinguish between these two isoforms. Voltage-dependent activation of the TTX-R current in intracardiac neurons (V1/2 = −33 mV) was similar to that reported for the Nav1.5 channel and differs from the considerably more positive value reported for the high-voltage activated Nav1.8 channel (V1/2 = −15.7 mV; Table 1). Although the V1/2 of inactivation that we obtained (around −60mV, Figure 3A) is shifted to the right with respect to previously reported values for the cardiac-type sodium channel, it still is more negative than the V1/2 of inactivation of −33.7 reported for the Nav1.8 channel (Table 1). This observed shift could be attributed to different experimental conditions and/or to expression of different regulatory β subunits in intracardiac neurons and other tissues. More conclusive was the large difference between the IC50 value for TTX inhibition reported for the Nav1.8 channel (60–100 μM, Table 1) and the value reported for the TTX-R current in this study. The TTX inhibition curve for the total current was best fitted by the sum of two Langmuir isotherms with IC50 values separated by two orders of magnitude (~7 vs ~600 nM, Figure 5A). These data once again indicate the presence of two components of the sodium current in these cells. Moreover, the IC50 value (1.17 μM) obtained when the TTX-R current component was studied alone in normal sodium conditions falls in the TTX affinity range that had been reported for the cardiac-type sodium channel in cardiac myocytes and dorsal root ganglia neurons (Table 1). The fact that 5 μM TTX inhibited 91% of the TTX-R current (Figure 5B) makes participation of the Nav1.8 channel very unlikely as an important component of the TTX-R current in these neurons.

RT-PCR analysis and sequencing confirmed the expression of the cardiac-type Nav1.5 channel in canine intracardiac ganglia. Immunohistochemical and RT-PCR analysis were performed in freshly isolated intracardiac ganglia. These studies identified the presence of the Nav1.5 channel at both protein and mRNA levels. The acute nature of these experimental procedures ruled out the possibility that Nav1.5 channels were expressed as a consequence of the cell culture as observed in skeletal muscle following denervation.29 In addition, β-myosin heavy chain mRNA was not detected by RT-PCR analysis in the ganglia preparation. This rules out a possible contamination of the ganglia mRNA with mRNA from cardiac myocytes.

Mutations in the Nav1.5 channel are associated with several life-threatening cardiac diseases in humans. Gain or loss of function of the Nav1.5 channel leads to LQT3 or Brugada syndrome, respectively.3, 4 Autonomic activity has been shown to modulate these syndromes and to be associated with the development of malignant arrhythmias.5,30,31 Of note, LQT3 is commonly associated with sinus bradycardia, sinus pauses, and atrial standstill, phenotypes not readily explained by a gain of function of Nav1.5 in cardiac myocytes.3 These phenotypes could be explained by an increased acetylcholine release caused by enhanced AP activity of intracardiac neurons. We demonstrated that the TTX-R current in canine intracardiac neurons is sufficient to generate AP activity. Based on this observation, it may be reasonable to speculate that a gain of function of the Nav1.5 channel results in increased AP activity of these neurons. In contradistinction, loss of function mutations in Nav1.5 associated with Brugada syndrome may result in reduced ganglionic AP activity and diminished vagal output, which could have a protective effect in some cases of Brugada syndrome. In other cases in which Brugada syndrome is caused by accelerated inactivation of early INa,32 late INa might actually be augmented, leading to increased vagal tone, which could exacerbate the Brugada syndrome phenotype. The great complexity of neuronal interactions within the cardiac ganglia make it difficult to predict with certainty the effect of Nav1.5 channel mutations on overall autonomic output to the heart.

Sequencing analysis of the RT-PCR products obtained from canine intracardiac neurons showed homology with the SCN5A gene. This implies that genetic defects in the cardiac Nav1.5 channel will be equally present in intracardiac neurons. Although we are not able to provide direct evidence of a role of Nav1.5 channel in the firing properties of the intracardiac neurons at this time, we anticipate that mutations of this channel, present in these neurons, would generate an imbalance in the intracardiac ganglia activity. This autonomic imbalance operating on an already dysfunctional substrate may prove to be an important factor in the generation of arrhythmogenic activity associated with LQT3 and Brugada syndrome. Our novel observation that intracardiac neurons and cardiac myocytes share functional expression of the Nav1.5 sodium channel opens an exciting new door that implicates autonomic activity not only as a modulating influence but as part of several congenital syndromes. This knowledge may guide us to a better understanding of the basis for the development of malignant cardiac arrhythmias as well as new approaches to diagnosis and therapy.

Acknowledgments

We thank Dr. Hali Hartmann for critical review of the data; Ryan Pfeiffer for assistance in sequencing; Robert Goodrow and Andrew Pitoniak for technical support; and Judy Hefferon for assistance with the illustrations.

This work was supported in part by American Heart Association–Northeast Affiliate Grant 0335446T to Dr. Scornik, and National Institutes of Health Grant HL073161 to Dr. Pérez, Grant HL47678 to Dr. Antzelevitch, and Grant HL61669 to Dr. Brugada.

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