Elsevier

Heart Rhythm

Volume 3, Issue 7, July 2006, Pages 842-850
Heart Rhythm

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

https://doi.org/10.1016/j.hrthm.2006.03.021Get rights and content

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.

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.

Section snippets

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

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, 14, 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

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

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.

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