Cardiovascular Research (2014) 102, 480–486 doi:10.1093/cvr/cvu005
Nav1.8 channels in ganglionated plexi modulate atrial fibrillation inducibility Baozhen Qi1†, Yong Wei1†, Songwen Chen1†, Genqing Zhou1, Hongli Li1, Juan Xu1, Yu Ding1, Xiaofeng Lu1, Liqun Zhao1, Feng Zhang1, Gang Chen1, Jing Zhao2‡, and Shaowen Liu1* 1
Department of Cardiology, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiaotong University, NO 100, Haining Road, Hongkou District, Shanghai 200080, China; and 2Wolfson Institute for Biomedical Research, University College London, Wing3.1, Cruciform Building, Gower Street, London WC1E 6BT, UK
Received 26 July 2013; revised 9 December 2013; accepted 27 December 2013; online publish-ahead-of-print 12 January 2014 Time for primary review: 31 days
Aims
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Vagus nerve † Sodium channel † Atrial fibrillation † Ganglionated plexus † SCN10A
1. Introduction Atrial fibrillation (AF) is the most common sustained arrhythmia and is independently associated with an increased risk of stroke, heart failure, and death.1 Autonomic innervations of the heart involve both the extrinsic and the intrinsic cardiac autonomic nervous system (ICANS). The ICANS forms a complex neural network composed of ganglionated plexi (GP) concentrated within epicardial fat pads and the interconnecting ganglia and axons, which has been implicated in cardiac conduction and AF.2,3 Studies have demonstrated that GP plays a critical role in the initiation and maintenance of AF.4 – 7 Therefore, GP ablation could be selected as a supplement to conventional pulmonary vein isolation for AF ablation, which has been verified to achieve better outcomes than pulmonary vein isolation alone in clinical trials.8,9 †
B.Q., Y.W., and S.C. contributed equally to this work.
‡
J.Z. Co-author.
SCN10A encodes NaV1.8, an alpha subunit of voltage-gated sodium channels (NaV). NaV1.8 is a tetrodotoxin-resistant sodium channel and is highly expressed in small-diameter sensory neurons of dorsal root ganglia. It is known to play a key role in generating and maintaining of action potentials in nociceptive nerve fibres.10,11 Recently, NaV1.8 has been identified in the intrinsic cardiac ganglia of mouse.12 Several genome-wide association studies showed that SCN10A was associated with cardiac conduction as increased PR interval and QRS duration on the surface ECG.13 – 16 Furthermore, the SCN10A locus was also found to be associated with AF.16,17 These observations strongly suggest a role for NaV1.8 in cardiac electrophysiology, but the detailed mechanism remains unclear. A-803467 is a selective blocker for NaV1.8 channel, which is 300- to 1000-fold more potent at blocking NaV1.8 channel than other voltagegated sodium channels, without altering other ion channels, receptors,
* Corresponding author. Tel: +86 2163240090; fax: +86 2163240090. Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2014. For permissions please email: [email protected].
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Emerging evidences indicate that SCN10A/NaV1.8 is associated with cardiac conduction and atrial fibrillation, but the exact role of NaV1.8 in cardiac electrophysiology remains poorly understood. The present study was designed to investigate the effects of blocking NaV1.8 channels in cardiac ganglionated plexi (GP) on modulating cardiac conduction and atrial fibrillation inducibility in the canine model. ..................................................................................................................................................................................... Methods Thirteen mongrel dogs were randomly enrolled. Right cervical vagus nerve stimulation (VNS) was applied to determine its effects on the sinus rate, ventricular rate during atrial fibrillation, PR interval, atrial effective refractory period, and the and results cumulative window of vulnerability. The NaV1.8 blocker A-803467 (1 mmol/0.5 mL per GP, n ¼ 7) or 5% DMSO/95% polyethylene glycol (0.5 mL per GP, n ¼ 6, control) was injected into the anterior right GP and the inferior right GP. The effects of VNS on the sinus rate, ventricular rate, PR interval, atrial effective refractory period, and the cumulative window of vulnerability were significantly eliminated at 10, 35, and 90 min after A-803467 injection. In separate experiments (n ¼ 8), A-803467 blunted the slowing of sinus rate with increasing stimulation voltage of the anterior right GP at 10 min after local injection. ..................................................................................................................................................................................... Conclusions Blockade of NaV1.8 channels suppresses the effects of VNS on cardiac conduction and atrial fibrillation inducibility, most likely by inhibiting the neural activity of the cardiac GP.
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or enzymes in a broad screening panel.18 – 21 Although A-803467 has been reported to function in isolated intrinsic cardiac neurons in vitro,12,22 there is no focus on the role of A-803467 in the cardiac GP. The exact role of NaV1.8 in cardiac electrophysiology remains poorly understood, and the effect of blocking NaV1.8 in cardiac GP has not been studied in animal models or in humans either. To fully understand NaV1.8-expressing neurons in GP, we perform the present study to elucidate the effects of local blocking NaV1.8 channels in GP by A-803467 on cardiac conduction and AF inducibility in the canine model.
2. Methods 2.1 Experimental animals and surgical preparation
2.2 Vagus nerve stimulation The right cervical vagus nerve was dissected from the carotid sheath and transected at the C3– C4 level. Right cervical vagus nerve stimulation (VNS) was applied to the distal end of the nerve (see Supplementary material online, Figure S1) by using stainless steel electrodes (Model 6491, Unipolar Pediatric Temporary Pacing Lead, Medtronic, Minneapolis, MN, USA). VNS was performed by applying high-frequency electrical stimulation (HFS, 20 Hz, 0.1 ms duration, square waves) with a Grass stimulator (S-88, Astro-Med, Warwick, MA, USA). The stimulation current amplitude has been fixed at the voltage level enough to reduce sinus rate (SR) at least by 50% (1.9 + 0.1 V). Data were obtained 10 s after the onset of VNS, and the typical duration of continuous VNS was ≤45 s.23 The VNS intensities applied before and after drug injection were the same for all dogs.
2.3 A-803467 administration A bilateral thoracotomy was performed at the fourth intercostal space, which provided access to the right posterior side of the heart through a pericardiotomy. Thirteen dogs were randomly assigned to the A-803467 group (15 – 20 kg, n ¼ 7) or the control group (15 – 20 kg, n ¼ 6). A-803467 (1 mmol/0.5 mL at each GP) or 5% DMSO/95% polyethylene glycol (PEG) (0.5 mL at each GP) was injected into the anterior right ganglionated plexi (ARGP) and the inferior right ganglionated plexi (IRGP). The GPs were identified by applying HFS using a bipolar electrode probe (AtriCure, West Chester, OH, USA). The response was a progressive slowing of the SR. The needle tip was positioned manually at several points on the epicardial surface of the GP under direct visual control ensure optimal injection. A-803467 (Sigma Aldrich, A3109), 5-(4-chlorophenyl)-N-(3,5-dimethoxyphenyl)furan-2-carboxamide, was dissolved in 5% DMSO/95% PEG 400.
The study protocol was applied at baseline and 10, 35, 90 min after drug injection during VNS. ECG and atrial electrograms were amplified and filtered from 0.05 to 500 Hz and were displayed and recorded on a Bard Computerized Electrophysiology system (CR Bard, Inc., Bard, Billerica, MA, USA). Epicardial multielectrode catheters (Capsure Epi, Medtronic, Minneapolis, MN, USA) were sutured to the right and left atrial free walls (Figure 1). Atrial pacing was then performed with twice the diastolic pacing threshold. The SR was calculated using the average R – R interval of 20 beats. Atrioventricular nodal function was evaluated by measuring ventricular rate (VR) during induced AF. The AF was induced by rapid atrial pacing (400 impulses, 20 Hz) at right and left atrial free walls during VNS.23 AF was defined as irregular atrial rhythm (.500 b.p.m.) lasting ≥5 s after the end of the burst stimulation. VR was determined from the ventricular cycle lengths over the last 20 beats during induced AF. The PR interval was defined as the interval from the onset of the P-wave to the end of the PR segment. The pacing protocol for the atrial effective refractory period (AERP) measurement was as follows. Programmed electrical stimulation was performed at a cycle length (S1– S1) of 400 ms. The S1 – S2 interval was started at 200 ms and decreased by 10 ms steps until the atrial capture failed (S1:S2 ¼ 8:1). The longest coupling interval that did not capture the atria was defined as AERP. The right atrial effective refractory period (RAERP) and left atrial effective refractory period (LAERP) were measured at right and left atrial free walls during VNS. The window of vulnerability (WOV) was defined as the longest S1– S2 minus the shortest S1– S2 that induced AF. The cumulative window of vulnerability (SWOV), defined as the sum of the individual WOVs from right and left atria free walls, was used to measure AF inducibility. To examine whether the effect of A-803467 was mediated through effects on the ICANS, we examined GP function at 10 min after A-803467 (1 mmol/ 0.5 mL) or 5% DMSO/95% PEG (0.5 mL, control) injection into the ARGP in separate experiments (n ¼ 8). Voltage – SR response curves were constructed by applying HFS (20 Hz, 0.1 ms duration, square waves) to the ARGP with incremental voltages up to the voltage that induced AF. The slowing of the SR at each voltage level was determined. The change in the SR induced by HFS of the ARGP at each voltage level was used as a surrogate marker for GP function.
2.5 Statistical analysis All data were presented as means + SEM. The repeated-measures analysis of variance (ANOVA) was used for comparisons of SR, VR, PR interval, AERP, and SWOV between groups. The slowing of the SR with increasing stimulation voltage was compared between groups using repeatedmeasures ANOVA. Significant interactions between the treatment group and time were investigated by performing an overall hypothesis test of the treatment effect after stratifying by time point for all ANOVA models, followed by pair-wise comparisons, stratified by time point. Tukey’s method was used to adjust for multiple comparisons for all pair-wise testing. The voltage required to induce AF with ARGP stimulation was compared between groups using the unpaired t-test. The Mann–Whitney U test was used for comparison of the maximal per cent change in SR. SPSS 17.0 for Windows (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Probability values of ,0.05 were considered statistically significant.
3. Results 3.1 Effects of A-803467 on SR and VR At baseline, VNS suppressed the spontaneous SR significantly in both the control and A-803467 groups (Table 1). The sinus nodal function was suppressed during VNS. The data indicated that effects of VNS on the SR are suppressed at 10, 35, and 90 min after A-803467 injection, but remained unchanged in the control group (Figure 2).
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All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996), and the study protocol was reviewed and approved by Shanghai First People’s Hospital Institutional Animal Care and Use Committee. Thirteen adult male mongrel dogs, weighing 15– 20 kg, were anaesthetized with sodium pentobarbital (initial bolus 30 mg/kg iv, 50 – 100 mg as needed for maintenance). Depth of anaesthesia was assessed by monitoring corneal reflexes, jaw tone, and alterations in cardiovascular indices. After endotracheal intubation and positive-pressure ventilation, the femoral vein was cannulated for continuous saline administration (100 mL/h). Standard surface ECG leads (II, III, aVR) and blood pressure were monitored continuously throughout the entire experiment. Intermittent arterial blood gas measurements were taken hourly (Irma TruePoint blood gas analyser, International Technidyne Corp., Edison, NJ, USA); tidal volume was adjusted and bicarbonate infused as necessary to maintain blood gas homoeostasis. An electric heating pad was used to maintain the body temperature at 36 + 18C. The dogs were euthanized at the end of the experiments by a lethal dose of pentobarbital (100 mg/kg, iv).
2.4 Electrophysiological study protocol
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Figure 1 Schematic representation of the cardiac ganglionated plexis and positions of multielectrode catheters. The labels RA and LA indicate multielectrode catheters positioned on the epicardial surface of the right atrium and left atrium. SVC, superior vena cava; RSPV, right superior pulmonary vein; RIPV, right inferior pulmonary vein; IVC, inferior vena cava; ARGP, anterior right ganglionated plexi; IRGP, inferior right ganglionated plexi; RA, right atrium; LSPV, left superior pulmonary vein; LIPV, left inferior pulmonary vein; LPA, left pulmonary artery; CS, coronary sinus; LA, left atrium.
Table 1 Effects of vagal nerve stimulation (VNS) on cardiac electrophysiological properties in the baseline state n
VNS
SR (b.p.m.)
VR (b.p.m.)
PR interval (ms)
RAERP (ms)
LAERP (ms)
SWOV (ms)
Control
6
No During
158 + 12* 68 + 5
205 + 21* 93 + 17
105 + 3* 130 + 4
85 + 7* 42 + 5
85 + 11* 47 + 6
43 + 20* 105 + 17
Treatment (A-803467)
7
No During
157 + 9* 69 + 4
211 + 15* 103 + 8
107 + 2* 128 + 3
89 + 11* 49 + 7
90 + 14* 50 + 7
39 + 17* 97 + 14
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SR, sinus rate; VR, ventricular rate during induced AF; RAERP, right atrial effective refractory period; LAERP, left atrial effective refractory period; SWOV, cumulative window of vulnerability. *P , 0.05 compared with VNS.
VNS also reduced VR before GP injection. The effect on VR reduction was decreased gradually at 10, 35, and 90 min after A-803467 injection. However, these changes were not remarkable in the control group (Figure 3).
unchanged in the control group (Figure 5C). A-803467 significantly inhibited AF inducibility and decreased the SWOV at 10, 35, and 90 min compared with control. Effects of VNS on the SWOV were as follows: 64 + 6 vs. 117 + 21, 51 + 9 vs. 108 + 15, and 53 + 13 vs. 117 + 27 at 10, 35, and 90 min, respectively (A-803467 vs. control, ms, P , 0.05).
3.2 Effects on PR interval PR interval was prolonged in the A-803467 and control groups during VNS (Table 1). A-803467 injection significantly suppressed PR interval prolongation at 10, 35, and 90 min compared with the control (Figure 4). Effects of VNS on PR interval were as follows: 115 + 2 vs. 128 + 5, 112 + 2 vs. 134 + 2, and 116 + 3 vs. 134 + 4 at 10, 35, and 90 min, respectively (A-803467 vs. control, ms, P , 0.05).
3.3 Effects on AERP and AF inducibility VNS shortened the AERPs recorded from the right and left atria (Table 1). RAERP and LAERP in the A-803467 group increased, whereas they remained relatively stable in the control group (Figure 5). AERPs were significantly higher in the A-803467 group compared with control. Effects of VNS on the RAERP were as follows: 81 + 10 vs. 47 + 4, 80 + 10 vs. 43 + 12, and 84 + 8 vs. 45 + 8 at 10, 35, and 90 min, respectively (A-803467 vs. control, ms, P , 0.05). Effects of VNS on the LAERP were as follows: 74 + 5 vs. 53 + 6, 83 + 7 vs. 52 + 8, and 83 + 10 vs. 50 + 9 at 10, 35, and 90 min, respectively (A-803467 vs. control, ms, P , 0.05). VNS widened the SWOV at baseline in both groups (Table 1). The SWOV significantly decreased in the A-803467 group, but remained
3.4 Effects on GP function Stimulation of the ARGP with incremental voltage levels induced a progressive slowing of the SR. Whereas in the control group (n ¼ 4) the SR decreased linearly with increasing stimulation voltage, the SR remained relatively flat after treatment with A-803467 (n ¼ 4, Figure 6). The treatment group by voltage interaction was significant (P ¼ 0.047), indicating that the change in SR with increasing voltage was different in the two groups. A-803467 markedly increased the voltage required for ARGP stimulation to induce AF compared with control (5.0 + 0.4 vs. 3.0 + 0.7 V, P ¼ 0.047). A-803467 decreased the maximal per cent change in SR compared with baseline by 24.3 + 3.5%, which was significantly different compared with control (50.5 + 4.1%, P ¼ 0.021).
4. Discussion In the present study, we demonstrate for the first time that blockade of NaV1.8 inhibits the decreases in SR and VR, a prolongation of PR interval, and an increase in the SWOV response to VNS. A temporary suppression of AF inducibility was achieved with A-803467, and this might be associated with preventing the decrease of AERP induced by VNS.
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Nav1.8 modulates AF inducibility
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Figure 2 Effects of VNS on the SR in the A-803467 and control groups. Tracings were lead II of surface ECG and left atrium electrogram. Compare the significant slowing of the sinus rate in the control group (A) with the blunted response in the A-803467 group (B) at 10 min after GP injection. Effects of VNS on the SR are suppressed at 10, 35, and 90 min in the A-803467 group (C). Abscissa indicates the time after GP injection (A-803467 or control). Error bars indicate 1 SEM. *P , 0.05 for A-803467 vs. control. These effects are likely mediated through inhibition of the parasympathetic ganglionic neurotransmission in the ICANS, as evidenced by the attenuation of the maximal change in SR with ARGP stimulation. This study provides the demonstration of a possible mechanism by which SCN10A/NaV1.8 regulates cardiac electrophysiology via modulating the GP function. Direct VNS has been used for decades to study the induction and maintenance of AF in experimental protocols.24,25 The vagus nerve is a mixed nerve, with two-thirds of the fibres carrying afferent sensory information from the periphery to the central nervous system and one-third carrying efferent motor information from the central nervous system to the periphery.26 To discount the possible inhibitory effects via a reflex that involves the brain, the right vagus nerve was transected, and VNS was applied at the distal end of the nerve, indicating that the antiarrhythmic effect of A-803467 is not dependent on the activation of the afferent vagus nerve fibres that project to the brain. We intentionally injected A-803467 into the GP in order to minimize systemic action on the myocardium and localize the effects on the ICANS. GPs contain entities representing both sympathetic nerves and the parasympathetic nerves.27 – 29 Nevertheless, a majority of
intrinsic cardiac neurons in GPs were found to be cholinergic.30 The functional role of the parasympathetic components is clearly dominant. Electrical stimulation of GP always evokes negative chronotropic and dromotropic effects. In this study, HFS was delivered at the ARGP and the ability of ARGP stimulation to slow the SR was used to assess the ARGP function under the influence of A-803467. A-803467 reversed the negative chronotropic responses to HFS of the ARGP parasympathetic neural elements, suggesting that A-803467 inhibits the activation of the neural elements within the ARGP. We have shown, for the first time, that A-803467 could suppress the GP activity, evidenced by counteracting the cholinergic effects of ARGP stimulation. These effects strongly support the notion that A-803467 acts as a modulator and stabilizer of the cardiac autonomic tone, protecting the heart against cholinergic overstimulation. PR interval is an amalgamated measure of atrial and atrioventricular nodal conduction, disturbances of which increase AF risk and all-cause mortality.31 Longitudinal data from the Atherosclerosis Risk in Communities Study and the Framingham Heart Study (FHS) demonstrate that PR interval prolongation is a predictor of increased AF risk.32,33 In addition, PR interval prolongation has been shown in FHS to be an independent
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Figure 3 Effects of VNS on the atrioventricular node in the A-803467 and control groups at each time point. Compare the significant slowing of the ventricular rate in the control group with the blunted response in the A-803467 group. Effects of VNS on the VR are suppressed at 10, 35, and 90 min in the A-803467 group. *P , 0.05 for A-803467 vs. control.
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Figure 4 Effects of VNS on the PR interval. Compare the marked prolongation of PR interval in the control group with the blunted response in the A-803467 group. A-803467 injection significantly suppressed PR interval prolongation compared with control. *P , 0.05 for A-803467 vs. control.
predictor in a multifactorial risk score for AF pre-disposition.33 Magnani et al. 34 identified significant relationships of PR interval to heart failure and AF in older adults. A number of clinical GWA studies have recently identified associations between genetic variants in the SCN10A and cardiac conduction (PR interval and QRS duration).13 – 16 Chambers et al. 13 found that the PR interval was shorter in Scn10a2/2 mice than in wild-type littermates. Our study showed that blockade of NaV1.8 channels in GP could blunt both vagally dependent PR interval prolongation and the inducibility of vagally mediated AF. Recently, more studies suggest that the effect of NaV1.8 on cardiac electrophysiological properties is mediated by its action on intrinsic cardiac ganglia neurons. Facer et al. 35 demonstrated the presence of
Figure 5 Effects of VNS on the atrial effective refractory period (AERP) and cumulative window of vulnerability (SWOV) in the A-803467 and control groups. (A) Effects on the right atrial effective refractory period (RAERP). (B) Effects on the left atrial effective refractory period (LAERP). AERPs in the A-803467 group increased, whereas they remained relatively stable in the control group. A-803467 significantly inhibited AF inducibility and decreased the SWOV compared with control (C). *P , 0.05 for A-803467 vs. control.
NaV1.8-immunoreactive sensory nerve fibres in human atrial myocardium. Verkerk et al. 12 also demonstrated the presence of SCN10A/ NaV1.8 in intrinsic cardiac neurons. Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labelling in intrinsic cardiac ganglia but only modest NaV1.8 expression in the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated intrinsic cardiac ganglia neurons, but no NaV1.8 expression in isolated
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underlying mechanism of NaV1.8-mediated regulation of GP function will require future additional detailed investigations. In summary, blockade of NaV1.8 channels suppresses the effects of VNS on cardiac conduction and AF inducibility, most likely by inhibiting GP function. Our findings suggest a possible mechanism for SCN10A/ NaV1.8 modulating cardiac electrophysiological properties which may be associated with regulation of the neural activity of the GP. These results not only reveal a functional role for SCN10A expression in cardiac GP, but also identify NaV1.8 as a potential target for antiarrhythmic intervention aiming at modulating the neural control of the heart.
Supplementary material Supplementary material is available at Cardiovascular Research online. Figure 6 Effects on the ganglionated plexi function at 10 min after A-803467 or 5% DMSO/95% PEG injection into the anterior right ganglionated plexi (ARGP). Compare the marked slowing of the sinus rate in the control group with the blunted response in the A-803467 group. There was a significantly different trend in the sinus rate change with increasing stimulation voltage between the two groups. *P , 0.05 for A-803467 vs. control.
Funding This work was supported by the Science and Technology Commission of Shanghai Municipality (No. 10411954800).
References 1. Benjamin EJ, Chen PS, Bild DE, Mascette AM, Albert CM, Alonso A et al. Prevention of atrial fibrillation: report from a national heart, lung, and blood institute workshop. Circulation 2009;119:606 –618. 2. Chen YJ, Chen SA, Tai CT, Wen ZC, Feng AN, Ding YA et al. Role of atrial electrophysiology and autonomic nervous system in patients with supraventricular tachycardia and paroxysmal atrial fibrillation. J Am Coll Cardiol 1998;32:732 –738. 3. Hou Y, Scherlag BJ, Lin J, Zhang Y, Lu Z, Truong K et al. Ganglionated plexi modulate extrinsic cardiac autonomic nerve input: effects on sinus rate, atrioventricular conduction, refractoriness, and inducibility of atrial fibrillation. J Am Coll Cardiol 2007;50:61 –68. 4. Chiou CW, Eble JN, Zipes DP. Efferent vagal innervation of the canine atria and sinus and atrioventricular nodes. The third fat pad. Circulation 1997;95:2573 –2584. 5. Schauerte P, Scherlag BJ, Pitha J, Scherlag MA, Reynolds D, Lazzara R et al. Catheter ablation of cardiac autonomic nerves for prevention of vagal atrial fibrillation. Circulation 2000;102:2774 –2780. 6. Scherlag BJ, Yamanashi W, Patel U, Lazzara R, Jackman WM. Autonomically induced conversion of pulmonary vein focal firing into atrial fibrillation. J Am Coll Cardiol 2005;45: 1878– 1886. 7. Po SS, Scherlag BJ, Yamanashi WS, Edwards J, Zhou J, Wu R et al. Experimental model for paroxysmal atrial fibrillation arising at the pulmonary vein-atrial junctions. Heart Rhythm 2006;3:201 –208. 8. Pappone C, Santinelli V, Manguso F, Vicedomini G, Gugliotta F, Augello G et al. Pulmonary vein denervation enhances long-term benefit after circumferential ablation for paroxysmal atrial fibrillation. Circulation 2004;109:327 –334. 9. Scherlag BJ, Nakagawa H, Jackman WM, Yamanashi WS, Patterson E, Po S et al. Electrical stimulation to identify neural elements on the heart: their role in atrial fibrillation. J Interv Card Electrophysiol 2005;13:37–42. 10. Akopian AN, Sivilotti L, Wood JN. A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature 1996;379:257 –262. 11. Akopian AN, Souslova V, England S, Okuse K, Ogata N, Ure J et al. The tetrodotoxinresistant sodium channel SNS has a specialized function in pain pathways. Nat Neurosci 1999;2:541 –548. 12. Verkerk AO, Remme CA, Schumacher CA, Scicluna BP, Wolswinkel R, de Jonge B et al. Functional NaV1.8 channels in intracardiac neurons: the link between SCN10A and cardiac electrophysiology. Circ Res 2012;111:333 –343. 13. Chambers JC, Zhao J, Terracciano CM, Bezzina CR, Zhang W, Kaba R et al. Genetic variation in SCN10A influences cardiac conduction. Nat Genet 2010;42:149 – 152. 14. Holm H, Gudbjartsson DF, Arnar DO, Thorleifsson G, Thorgeirsson G, Stefansdottir H et al. Several common variants modulate heart rate, PR interval and QRS duration. Nat Genet 2010;42:117–122. 15. Sotoodehnia N, Isaacs A, de Bakker PI, Do¨rr M, Newton-Cheh C, Nolte IM et al. Common variants in 22 loci are associated with QRS duration and cardiac ventricular conduction. Nat Genet 2010;42:1068 –1076. 16. Ritchie MD, Denny JC, Zuvich RL, Crawford DC, Schildcrout JS, Bastarache L et al. Genome- and phenome-wide analyses of cardiac conduction identifies markers of arrhythmia risk. Circulation 2013;127:1377 –1385. 17. Pfeufer A, van Noord C, Marciante KD, Arking DE, Larson MG, Smith AV et al. Genomewide association study of PR interval. Nat Genet 2010;42:153 – 159.
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ventricular myocytes. Patch-clamp studies demonstrated that A-803467 had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes, but significantly reduced INa density in intracardiac neurons.12 The results from our study now provide the evidence for a functional role for NaV1.8 in regulation of GP activity in the canine heart. The dose of A-803467 used in this study was determined based on the available literature.18,19 The plasma elimination half-life of A-803467 is 4.9 h and behavioural effects of A-803467 in the SNL model lasted at least 90 min after systemic injection.18 Injection of A-803467 into the neuronal receptive field reduced evoked discharges of WDR neurons at each time point examined (5–35 min) with the largest effect observed 35 min after injection.19 Therefore, we have reported only short-time (90 min) efficacy of A-803467 in the canine heart. The ICANS neurons synthesize many different neurotransmitters.36 Besides acetylcholine, population of ‘small intensely fluorescent’ cells synthesize most probably dopamine and serotonin, some magnocellular neurons synthesize norepinephrine and epinephrine. The presence of enzymes for histamine synthesis was also confirmed. Furthermore, various neuropeptides (e.g. cocatine and amphetamine-regulated transcript, calcitonin gene-related peptide, neuropeptide Y, tachykinins, vasoactive intestinal polypeptide) were identified in heart neurons. Nitric oxide (NO) synthase-immunoreactive neurons were also identified in the heart. Because the main purpose of this study was to provide the evidence for NaV1.8 channels in regulation of GP function, we did not pursue the exact courses of the signalling pathways within GP. It is possible that NaV1.8 channels could regulate the release of neurotransmitters and/or neuropeptides in GP. Acetylcholine and NO may represent potential candidates. Blasius et al. 37 found that mice carrying the Possum mutation in SCN10A, which enhanced NaV1.8 sodium currents and neuronal excitability, respond to ‘scruffing’ with marked sinus bradycardia and R–R variability which could be abrogated by infusion of atropine. Herring et al. 38 found that NO increased the evoked release of acetylcholine and the heart rate response to VNS via a pre-synaptic pathway involving phosphodiesterase 3 and protein kinase A. The
Conflict of interest: none declared.
486 18. Jarvis MF, Honore P, Shieh CC, Chapman M, Joshi S, Zhang XF et al. A-803467, a potent and selective NaV1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat. Proc Natl Acad Sci USA 2007;104:8520 –8525. 19. McGaraughty S, Chu KL, Scanio MJ, Kort ME, Faltynek CR, Jarvis MF. A selective NaV1.8 sodium channel blocker, A-803467 [5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide], attenuates spinal neuronal activity in neuropathic rats. J Pharmacol Exp Ther 2008;324:1204 –1211. 20. Kort ME, Drizin I, Gregg RJ, Scanio MJ, Shi L, Gross MF et al. Discovery and biological evaluation of 5-aryl-2-furfuramides, potent and selective blockers of the NaV1.8 sodium channel with efficacy in models of neuropathic and inflammatory pain. J Med Chem 2008;51:407 –416. 21. Joshi SK, Honore P, Hernandez G, Schmidt R, Gomtsyan A, Scanio M et al. Additive antinociceptive effects of the selective NaV1.8 blocker A-803467 and selective TRPV1 antagonists in rat inflammatory and neuropathic pain models. J Pain 2009;10:306 –315. 22. Yang T, Atack TC, Stroud DM, Zhang W, Hall L, Roden DM. Blocking Scn10a channels in heart reduces late sodium current and is antiarrhythmic. Circ Res 2012;111:322 –332. 23. Oh S, Choi EK, Zhang Y, Mazgalev TN. Botulinum toxin injection in epicardial autonomic ganglia temporarily suppresses vagally mediated atrial fibrillation. Circ Arrhythm Electrophysiol 2011;4:560 –565. 24. Wang Z, Page P, Nattel S. Mechanism of flecainide’s antiarrhythmic action in experimental atrial fibrillation. Circ Res 1992;71:271–287. 25. Liu L, Nattel S. Differing sympathetic and vagal effects on atrial fibrillation in dogs: role of refractoriness reterogeneity. Am J Physiol 1997;273:H805 –H816. 26. Prechtl JC, Powley TL. The fiber composition of the abdominal vagus of the rat. Anat Embryol 1990;181:101 –115. 27. Pauza DH, Skripka V, Pauziene N, Stropus R. Morphology, distribution, and variability of the epicardiac neural ganglionated subplexuses in the human heart. Anat Rec 2000;259: 353 –382. 28. Quan KJ, Lee JH, Van Hare GF, Biblo LA, Mackall JA, Carlson MD. Identification and characterization of atrioventricular parasympathetic innervation in humans. J Cardiovasc Electrophysiol 2002;13:735–739.
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29. Po SS, Nakagawa H, Jackman WM. Localization of left atrial ganglionated plexi in patients with atrial fibrillation. J Cardiovasc Electrophysiol 2009;20:1186 – 1189. 30. Hoover DB, Isaacs ER, Jacques F, Hoard JL, Page´ P, Armour JA. Localization of multiple neurotransmitters in surgically derived specimens of human atrial ganglia. Neuroscience 2009;164:1170 –1179. 31. Cheng S, Keyes MJ, Larson MG, McCabe EL, Newton-Cheh C, Levy D et al. Long-term outcomes in individuals with prolonged PR interval or first-degree atrioventricular block. JAMA 2009;301:2571 –2577. 32. Soliman EZ, Prineas RJ, Case LD, Zhang ZM, Goff DCJr. Ethnic distribution of ECG predictors of atrial fibrillation and its impact on understanding the ethnic distribution of ischemic stroke in the Atherosclerosis Risk in Communities (ARIC) study. Stroke 2009;40: 1204– 1211. 33. Schnabel RB, Sullivan LM, Levy D, Pencina MJ, Massaro JM, D’Agostino RBSr et al. Development of a Risk Score for Atrial Fibrillation (The Framingham Heart Study): a community-based cohort study. The Lancet 2009;373:739–745. 34. Magnani JW, Wang N, Nelson KP, Connelly S, Deo R, Rodondi N et al. Electrocardiographic PR interval and adverse outcomes in older adults: the Health, Aging, and Body Composition study. Circ Arrhythm Electrophysiol 2013;6:84–90. 35. Facer P, Punjabi PP, Abrari A, Kaba RA, Severs NJ, Chambers J et al. Localisation of SCN10A gene product Na(v)1.8 and novel pain-related ion channels in human heart. Int Heart J 2011;52:146 –152. 36. Kukanova B, Mravec B. Complex intracardiac nervous system. Bratisl Lek Listy 2006;107: 45 –51. 37. Blasiusa AL, Dubinb AE, Petrusc MJ, Lim BK, Narezkina A, Criado JR et al. Hypermorphic mutation of the voltage-gated sodium channel encoding gene Scn10a causes a dramatic stimulus-dependent neurobehavioral phenotype. Proc Natl Acad Sci USA 2011;108: 19413–19418. 38. Herring N, Paterson DJ. Nitric oxide-cGMP pathway facilitates acetylcholine release and bradycardia during vagal nerve stimulation in the guinea-pig in vitro. J Physiol 2001;535: 507 –518. Downloaded from by guest on November 17, 2015
Nav1.8 channels in ganglionated plexi modulate atrial fibrillation inducibility Baozhen Qi1†, Yong Wei1†, Songwen Chen1†, Genqing Zhou1, Hongli Li1, Juan Xu1, Yu Ding1, Xiaofeng Lu1, Liqun Zhao1, Feng Zhang1, Gang Chen1, Jing Zhao2‡, and Shaowen Liu1* 1
Department of Cardiology, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiaotong University, NO 100, Haining Road, Hongkou District, Shanghai 200080, China; and 2Wolfson Institute for Biomedical Research, University College London, Wing3.1, Cruciform Building, Gower Street, London WC1E 6BT, UK
Received 26 July 2013; revised 9 December 2013; accepted 27 December 2013; online publish-ahead-of-print 12 January 2014 Time for primary review: 31 days
Aims
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Vagus nerve † Sodium channel † Atrial fibrillation † Ganglionated plexus † SCN10A
1. Introduction Atrial fibrillation (AF) is the most common sustained arrhythmia and is independently associated with an increased risk of stroke, heart failure, and death.1 Autonomic innervations of the heart involve both the extrinsic and the intrinsic cardiac autonomic nervous system (ICANS). The ICANS forms a complex neural network composed of ganglionated plexi (GP) concentrated within epicardial fat pads and the interconnecting ganglia and axons, which has been implicated in cardiac conduction and AF.2,3 Studies have demonstrated that GP plays a critical role in the initiation and maintenance of AF.4 – 7 Therefore, GP ablation could be selected as a supplement to conventional pulmonary vein isolation for AF ablation, which has been verified to achieve better outcomes than pulmonary vein isolation alone in clinical trials.8,9 †
B.Q., Y.W., and S.C. contributed equally to this work.
‡
J.Z. Co-author.
SCN10A encodes NaV1.8, an alpha subunit of voltage-gated sodium channels (NaV). NaV1.8 is a tetrodotoxin-resistant sodium channel and is highly expressed in small-diameter sensory neurons of dorsal root ganglia. It is known to play a key role in generating and maintaining of action potentials in nociceptive nerve fibres.10,11 Recently, NaV1.8 has been identified in the intrinsic cardiac ganglia of mouse.12 Several genome-wide association studies showed that SCN10A was associated with cardiac conduction as increased PR interval and QRS duration on the surface ECG.13 – 16 Furthermore, the SCN10A locus was also found to be associated with AF.16,17 These observations strongly suggest a role for NaV1.8 in cardiac electrophysiology, but the detailed mechanism remains unclear. A-803467 is a selective blocker for NaV1.8 channel, which is 300- to 1000-fold more potent at blocking NaV1.8 channel than other voltagegated sodium channels, without altering other ion channels, receptors,
* Corresponding author. Tel: +86 2163240090; fax: +86 2163240090. Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2014. For permissions please email: [email protected].
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Emerging evidences indicate that SCN10A/NaV1.8 is associated with cardiac conduction and atrial fibrillation, but the exact role of NaV1.8 in cardiac electrophysiology remains poorly understood. The present study was designed to investigate the effects of blocking NaV1.8 channels in cardiac ganglionated plexi (GP) on modulating cardiac conduction and atrial fibrillation inducibility in the canine model. ..................................................................................................................................................................................... Methods Thirteen mongrel dogs were randomly enrolled. Right cervical vagus nerve stimulation (VNS) was applied to determine its effects on the sinus rate, ventricular rate during atrial fibrillation, PR interval, atrial effective refractory period, and the and results cumulative window of vulnerability. The NaV1.8 blocker A-803467 (1 mmol/0.5 mL per GP, n ¼ 7) or 5% DMSO/95% polyethylene glycol (0.5 mL per GP, n ¼ 6, control) was injected into the anterior right GP and the inferior right GP. The effects of VNS on the sinus rate, ventricular rate, PR interval, atrial effective refractory period, and the cumulative window of vulnerability were significantly eliminated at 10, 35, and 90 min after A-803467 injection. In separate experiments (n ¼ 8), A-803467 blunted the slowing of sinus rate with increasing stimulation voltage of the anterior right GP at 10 min after local injection. ..................................................................................................................................................................................... Conclusions Blockade of NaV1.8 channels suppresses the effects of VNS on cardiac conduction and atrial fibrillation inducibility, most likely by inhibiting the neural activity of the cardiac GP.
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or enzymes in a broad screening panel.18 – 21 Although A-803467 has been reported to function in isolated intrinsic cardiac neurons in vitro,12,22 there is no focus on the role of A-803467 in the cardiac GP. The exact role of NaV1.8 in cardiac electrophysiology remains poorly understood, and the effect of blocking NaV1.8 in cardiac GP has not been studied in animal models or in humans either. To fully understand NaV1.8-expressing neurons in GP, we perform the present study to elucidate the effects of local blocking NaV1.8 channels in GP by A-803467 on cardiac conduction and AF inducibility in the canine model.
2. Methods 2.1 Experimental animals and surgical preparation
2.2 Vagus nerve stimulation The right cervical vagus nerve was dissected from the carotid sheath and transected at the C3– C4 level. Right cervical vagus nerve stimulation (VNS) was applied to the distal end of the nerve (see Supplementary material online, Figure S1) by using stainless steel electrodes (Model 6491, Unipolar Pediatric Temporary Pacing Lead, Medtronic, Minneapolis, MN, USA). VNS was performed by applying high-frequency electrical stimulation (HFS, 20 Hz, 0.1 ms duration, square waves) with a Grass stimulator (S-88, Astro-Med, Warwick, MA, USA). The stimulation current amplitude has been fixed at the voltage level enough to reduce sinus rate (SR) at least by 50% (1.9 + 0.1 V). Data were obtained 10 s after the onset of VNS, and the typical duration of continuous VNS was ≤45 s.23 The VNS intensities applied before and after drug injection were the same for all dogs.
2.3 A-803467 administration A bilateral thoracotomy was performed at the fourth intercostal space, which provided access to the right posterior side of the heart through a pericardiotomy. Thirteen dogs were randomly assigned to the A-803467 group (15 – 20 kg, n ¼ 7) or the control group (15 – 20 kg, n ¼ 6). A-803467 (1 mmol/0.5 mL at each GP) or 5% DMSO/95% polyethylene glycol (PEG) (0.5 mL at each GP) was injected into the anterior right ganglionated plexi (ARGP) and the inferior right ganglionated plexi (IRGP). The GPs were identified by applying HFS using a bipolar electrode probe (AtriCure, West Chester, OH, USA). The response was a progressive slowing of the SR. The needle tip was positioned manually at several points on the epicardial surface of the GP under direct visual control ensure optimal injection. A-803467 (Sigma Aldrich, A3109), 5-(4-chlorophenyl)-N-(3,5-dimethoxyphenyl)furan-2-carboxamide, was dissolved in 5% DMSO/95% PEG 400.
The study protocol was applied at baseline and 10, 35, 90 min after drug injection during VNS. ECG and atrial electrograms were amplified and filtered from 0.05 to 500 Hz and were displayed and recorded on a Bard Computerized Electrophysiology system (CR Bard, Inc., Bard, Billerica, MA, USA). Epicardial multielectrode catheters (Capsure Epi, Medtronic, Minneapolis, MN, USA) were sutured to the right and left atrial free walls (Figure 1). Atrial pacing was then performed with twice the diastolic pacing threshold. The SR was calculated using the average R – R interval of 20 beats. Atrioventricular nodal function was evaluated by measuring ventricular rate (VR) during induced AF. The AF was induced by rapid atrial pacing (400 impulses, 20 Hz) at right and left atrial free walls during VNS.23 AF was defined as irregular atrial rhythm (.500 b.p.m.) lasting ≥5 s after the end of the burst stimulation. VR was determined from the ventricular cycle lengths over the last 20 beats during induced AF. The PR interval was defined as the interval from the onset of the P-wave to the end of the PR segment. The pacing protocol for the atrial effective refractory period (AERP) measurement was as follows. Programmed electrical stimulation was performed at a cycle length (S1– S1) of 400 ms. The S1 – S2 interval was started at 200 ms and decreased by 10 ms steps until the atrial capture failed (S1:S2 ¼ 8:1). The longest coupling interval that did not capture the atria was defined as AERP. The right atrial effective refractory period (RAERP) and left atrial effective refractory period (LAERP) were measured at right and left atrial free walls during VNS. The window of vulnerability (WOV) was defined as the longest S1– S2 minus the shortest S1– S2 that induced AF. The cumulative window of vulnerability (SWOV), defined as the sum of the individual WOVs from right and left atria free walls, was used to measure AF inducibility. To examine whether the effect of A-803467 was mediated through effects on the ICANS, we examined GP function at 10 min after A-803467 (1 mmol/ 0.5 mL) or 5% DMSO/95% PEG (0.5 mL, control) injection into the ARGP in separate experiments (n ¼ 8). Voltage – SR response curves were constructed by applying HFS (20 Hz, 0.1 ms duration, square waves) to the ARGP with incremental voltages up to the voltage that induced AF. The slowing of the SR at each voltage level was determined. The change in the SR induced by HFS of the ARGP at each voltage level was used as a surrogate marker for GP function.
2.5 Statistical analysis All data were presented as means + SEM. The repeated-measures analysis of variance (ANOVA) was used for comparisons of SR, VR, PR interval, AERP, and SWOV between groups. The slowing of the SR with increasing stimulation voltage was compared between groups using repeatedmeasures ANOVA. Significant interactions between the treatment group and time were investigated by performing an overall hypothesis test of the treatment effect after stratifying by time point for all ANOVA models, followed by pair-wise comparisons, stratified by time point. Tukey’s method was used to adjust for multiple comparisons for all pair-wise testing. The voltage required to induce AF with ARGP stimulation was compared between groups using the unpaired t-test. The Mann–Whitney U test was used for comparison of the maximal per cent change in SR. SPSS 17.0 for Windows (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Probability values of ,0.05 were considered statistically significant.
3. Results 3.1 Effects of A-803467 on SR and VR At baseline, VNS suppressed the spontaneous SR significantly in both the control and A-803467 groups (Table 1). The sinus nodal function was suppressed during VNS. The data indicated that effects of VNS on the SR are suppressed at 10, 35, and 90 min after A-803467 injection, but remained unchanged in the control group (Figure 2).
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All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996), and the study protocol was reviewed and approved by Shanghai First People’s Hospital Institutional Animal Care and Use Committee. Thirteen adult male mongrel dogs, weighing 15– 20 kg, were anaesthetized with sodium pentobarbital (initial bolus 30 mg/kg iv, 50 – 100 mg as needed for maintenance). Depth of anaesthesia was assessed by monitoring corneal reflexes, jaw tone, and alterations in cardiovascular indices. After endotracheal intubation and positive-pressure ventilation, the femoral vein was cannulated for continuous saline administration (100 mL/h). Standard surface ECG leads (II, III, aVR) and blood pressure were monitored continuously throughout the entire experiment. Intermittent arterial blood gas measurements were taken hourly (Irma TruePoint blood gas analyser, International Technidyne Corp., Edison, NJ, USA); tidal volume was adjusted and bicarbonate infused as necessary to maintain blood gas homoeostasis. An electric heating pad was used to maintain the body temperature at 36 + 18C. The dogs were euthanized at the end of the experiments by a lethal dose of pentobarbital (100 mg/kg, iv).
2.4 Electrophysiological study protocol
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Figure 1 Schematic representation of the cardiac ganglionated plexis and positions of multielectrode catheters. The labels RA and LA indicate multielectrode catheters positioned on the epicardial surface of the right atrium and left atrium. SVC, superior vena cava; RSPV, right superior pulmonary vein; RIPV, right inferior pulmonary vein; IVC, inferior vena cava; ARGP, anterior right ganglionated plexi; IRGP, inferior right ganglionated plexi; RA, right atrium; LSPV, left superior pulmonary vein; LIPV, left inferior pulmonary vein; LPA, left pulmonary artery; CS, coronary sinus; LA, left atrium.
Table 1 Effects of vagal nerve stimulation (VNS) on cardiac electrophysiological properties in the baseline state n
VNS
SR (b.p.m.)
VR (b.p.m.)
PR interval (ms)
RAERP (ms)
LAERP (ms)
SWOV (ms)
Control
6
No During
158 + 12* 68 + 5
205 + 21* 93 + 17
105 + 3* 130 + 4
85 + 7* 42 + 5
85 + 11* 47 + 6
43 + 20* 105 + 17
Treatment (A-803467)
7
No During
157 + 9* 69 + 4
211 + 15* 103 + 8
107 + 2* 128 + 3
89 + 11* 49 + 7
90 + 14* 50 + 7
39 + 17* 97 + 14
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SR, sinus rate; VR, ventricular rate during induced AF; RAERP, right atrial effective refractory period; LAERP, left atrial effective refractory period; SWOV, cumulative window of vulnerability. *P , 0.05 compared with VNS.
VNS also reduced VR before GP injection. The effect on VR reduction was decreased gradually at 10, 35, and 90 min after A-803467 injection. However, these changes were not remarkable in the control group (Figure 3).
unchanged in the control group (Figure 5C). A-803467 significantly inhibited AF inducibility and decreased the SWOV at 10, 35, and 90 min compared with control. Effects of VNS on the SWOV were as follows: 64 + 6 vs. 117 + 21, 51 + 9 vs. 108 + 15, and 53 + 13 vs. 117 + 27 at 10, 35, and 90 min, respectively (A-803467 vs. control, ms, P , 0.05).
3.2 Effects on PR interval PR interval was prolonged in the A-803467 and control groups during VNS (Table 1). A-803467 injection significantly suppressed PR interval prolongation at 10, 35, and 90 min compared with the control (Figure 4). Effects of VNS on PR interval were as follows: 115 + 2 vs. 128 + 5, 112 + 2 vs. 134 + 2, and 116 + 3 vs. 134 + 4 at 10, 35, and 90 min, respectively (A-803467 vs. control, ms, P , 0.05).
3.3 Effects on AERP and AF inducibility VNS shortened the AERPs recorded from the right and left atria (Table 1). RAERP and LAERP in the A-803467 group increased, whereas they remained relatively stable in the control group (Figure 5). AERPs were significantly higher in the A-803467 group compared with control. Effects of VNS on the RAERP were as follows: 81 + 10 vs. 47 + 4, 80 + 10 vs. 43 + 12, and 84 + 8 vs. 45 + 8 at 10, 35, and 90 min, respectively (A-803467 vs. control, ms, P , 0.05). Effects of VNS on the LAERP were as follows: 74 + 5 vs. 53 + 6, 83 + 7 vs. 52 + 8, and 83 + 10 vs. 50 + 9 at 10, 35, and 90 min, respectively (A-803467 vs. control, ms, P , 0.05). VNS widened the SWOV at baseline in both groups (Table 1). The SWOV significantly decreased in the A-803467 group, but remained
3.4 Effects on GP function Stimulation of the ARGP with incremental voltage levels induced a progressive slowing of the SR. Whereas in the control group (n ¼ 4) the SR decreased linearly with increasing stimulation voltage, the SR remained relatively flat after treatment with A-803467 (n ¼ 4, Figure 6). The treatment group by voltage interaction was significant (P ¼ 0.047), indicating that the change in SR with increasing voltage was different in the two groups. A-803467 markedly increased the voltage required for ARGP stimulation to induce AF compared with control (5.0 + 0.4 vs. 3.0 + 0.7 V, P ¼ 0.047). A-803467 decreased the maximal per cent change in SR compared with baseline by 24.3 + 3.5%, which was significantly different compared with control (50.5 + 4.1%, P ¼ 0.021).
4. Discussion In the present study, we demonstrate for the first time that blockade of NaV1.8 inhibits the decreases in SR and VR, a prolongation of PR interval, and an increase in the SWOV response to VNS. A temporary suppression of AF inducibility was achieved with A-803467, and this might be associated with preventing the decrease of AERP induced by VNS.
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Nav1.8 modulates AF inducibility
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Figure 2 Effects of VNS on the SR in the A-803467 and control groups. Tracings were lead II of surface ECG and left atrium electrogram. Compare the significant slowing of the sinus rate in the control group (A) with the blunted response in the A-803467 group (B) at 10 min after GP injection. Effects of VNS on the SR are suppressed at 10, 35, and 90 min in the A-803467 group (C). Abscissa indicates the time after GP injection (A-803467 or control). Error bars indicate 1 SEM. *P , 0.05 for A-803467 vs. control. These effects are likely mediated through inhibition of the parasympathetic ganglionic neurotransmission in the ICANS, as evidenced by the attenuation of the maximal change in SR with ARGP stimulation. This study provides the demonstration of a possible mechanism by which SCN10A/NaV1.8 regulates cardiac electrophysiology via modulating the GP function. Direct VNS has been used for decades to study the induction and maintenance of AF in experimental protocols.24,25 The vagus nerve is a mixed nerve, with two-thirds of the fibres carrying afferent sensory information from the periphery to the central nervous system and one-third carrying efferent motor information from the central nervous system to the periphery.26 To discount the possible inhibitory effects via a reflex that involves the brain, the right vagus nerve was transected, and VNS was applied at the distal end of the nerve, indicating that the antiarrhythmic effect of A-803467 is not dependent on the activation of the afferent vagus nerve fibres that project to the brain. We intentionally injected A-803467 into the GP in order to minimize systemic action on the myocardium and localize the effects on the ICANS. GPs contain entities representing both sympathetic nerves and the parasympathetic nerves.27 – 29 Nevertheless, a majority of
intrinsic cardiac neurons in GPs were found to be cholinergic.30 The functional role of the parasympathetic components is clearly dominant. Electrical stimulation of GP always evokes negative chronotropic and dromotropic effects. In this study, HFS was delivered at the ARGP and the ability of ARGP stimulation to slow the SR was used to assess the ARGP function under the influence of A-803467. A-803467 reversed the negative chronotropic responses to HFS of the ARGP parasympathetic neural elements, suggesting that A-803467 inhibits the activation of the neural elements within the ARGP. We have shown, for the first time, that A-803467 could suppress the GP activity, evidenced by counteracting the cholinergic effects of ARGP stimulation. These effects strongly support the notion that A-803467 acts as a modulator and stabilizer of the cardiac autonomic tone, protecting the heart against cholinergic overstimulation. PR interval is an amalgamated measure of atrial and atrioventricular nodal conduction, disturbances of which increase AF risk and all-cause mortality.31 Longitudinal data from the Atherosclerosis Risk in Communities Study and the Framingham Heart Study (FHS) demonstrate that PR interval prolongation is a predictor of increased AF risk.32,33 In addition, PR interval prolongation has been shown in FHS to be an independent
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Figure 3 Effects of VNS on the atrioventricular node in the A-803467 and control groups at each time point. Compare the significant slowing of the ventricular rate in the control group with the blunted response in the A-803467 group. Effects of VNS on the VR are suppressed at 10, 35, and 90 min in the A-803467 group. *P , 0.05 for A-803467 vs. control.
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Figure 4 Effects of VNS on the PR interval. Compare the marked prolongation of PR interval in the control group with the blunted response in the A-803467 group. A-803467 injection significantly suppressed PR interval prolongation compared with control. *P , 0.05 for A-803467 vs. control.
predictor in a multifactorial risk score for AF pre-disposition.33 Magnani et al. 34 identified significant relationships of PR interval to heart failure and AF in older adults. A number of clinical GWA studies have recently identified associations between genetic variants in the SCN10A and cardiac conduction (PR interval and QRS duration).13 – 16 Chambers et al. 13 found that the PR interval was shorter in Scn10a2/2 mice than in wild-type littermates. Our study showed that blockade of NaV1.8 channels in GP could blunt both vagally dependent PR interval prolongation and the inducibility of vagally mediated AF. Recently, more studies suggest that the effect of NaV1.8 on cardiac electrophysiological properties is mediated by its action on intrinsic cardiac ganglia neurons. Facer et al. 35 demonstrated the presence of
Figure 5 Effects of VNS on the atrial effective refractory period (AERP) and cumulative window of vulnerability (SWOV) in the A-803467 and control groups. (A) Effects on the right atrial effective refractory period (RAERP). (B) Effects on the left atrial effective refractory period (LAERP). AERPs in the A-803467 group increased, whereas they remained relatively stable in the control group. A-803467 significantly inhibited AF inducibility and decreased the SWOV compared with control (C). *P , 0.05 for A-803467 vs. control.
NaV1.8-immunoreactive sensory nerve fibres in human atrial myocardium. Verkerk et al. 12 also demonstrated the presence of SCN10A/ NaV1.8 in intrinsic cardiac neurons. Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labelling in intrinsic cardiac ganglia but only modest NaV1.8 expression in the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated intrinsic cardiac ganglia neurons, but no NaV1.8 expression in isolated
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underlying mechanism of NaV1.8-mediated regulation of GP function will require future additional detailed investigations. In summary, blockade of NaV1.8 channels suppresses the effects of VNS on cardiac conduction and AF inducibility, most likely by inhibiting GP function. Our findings suggest a possible mechanism for SCN10A/ NaV1.8 modulating cardiac electrophysiological properties which may be associated with regulation of the neural activity of the GP. These results not only reveal a functional role for SCN10A expression in cardiac GP, but also identify NaV1.8 as a potential target for antiarrhythmic intervention aiming at modulating the neural control of the heart.
Supplementary material Supplementary material is available at Cardiovascular Research online. Figure 6 Effects on the ganglionated plexi function at 10 min after A-803467 or 5% DMSO/95% PEG injection into the anterior right ganglionated plexi (ARGP). Compare the marked slowing of the sinus rate in the control group with the blunted response in the A-803467 group. There was a significantly different trend in the sinus rate change with increasing stimulation voltage between the two groups. *P , 0.05 for A-803467 vs. control.
Funding This work was supported by the Science and Technology Commission of Shanghai Municipality (No. 10411954800).
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ventricular myocytes. Patch-clamp studies demonstrated that A-803467 had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes, but significantly reduced INa density in intracardiac neurons.12 The results from our study now provide the evidence for a functional role for NaV1.8 in regulation of GP activity in the canine heart. The dose of A-803467 used in this study was determined based on the available literature.18,19 The plasma elimination half-life of A-803467 is 4.9 h and behavioural effects of A-803467 in the SNL model lasted at least 90 min after systemic injection.18 Injection of A-803467 into the neuronal receptive field reduced evoked discharges of WDR neurons at each time point examined (5–35 min) with the largest effect observed 35 min after injection.19 Therefore, we have reported only short-time (90 min) efficacy of A-803467 in the canine heart. The ICANS neurons synthesize many different neurotransmitters.36 Besides acetylcholine, population of ‘small intensely fluorescent’ cells synthesize most probably dopamine and serotonin, some magnocellular neurons synthesize norepinephrine and epinephrine. The presence of enzymes for histamine synthesis was also confirmed. Furthermore, various neuropeptides (e.g. cocatine and amphetamine-regulated transcript, calcitonin gene-related peptide, neuropeptide Y, tachykinins, vasoactive intestinal polypeptide) were identified in heart neurons. Nitric oxide (NO) synthase-immunoreactive neurons were also identified in the heart. Because the main purpose of this study was to provide the evidence for NaV1.8 channels in regulation of GP function, we did not pursue the exact courses of the signalling pathways within GP. It is possible that NaV1.8 channels could regulate the release of neurotransmitters and/or neuropeptides in GP. Acetylcholine and NO may represent potential candidates. Blasius et al. 37 found that mice carrying the Possum mutation in SCN10A, which enhanced NaV1.8 sodium currents and neuronal excitability, respond to ‘scruffing’ with marked sinus bradycardia and R–R variability which could be abrogated by infusion of atropine. Herring et al. 38 found that NO increased the evoked release of acetylcholine and the heart rate response to VNS via a pre-synaptic pathway involving phosphodiesterase 3 and protein kinase A. The
Conflict of interest: none declared.
486 18. Jarvis MF, Honore P, Shieh CC, Chapman M, Joshi S, Zhang XF et al. A-803467, a potent and selective NaV1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat. Proc Natl Acad Sci USA 2007;104:8520 –8525. 19. McGaraughty S, Chu KL, Scanio MJ, Kort ME, Faltynek CR, Jarvis MF. A selective NaV1.8 sodium channel blocker, A-803467 [5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide], attenuates spinal neuronal activity in neuropathic rats. J Pharmacol Exp Ther 2008;324:1204 –1211. 20. Kort ME, Drizin I, Gregg RJ, Scanio MJ, Shi L, Gross MF et al. Discovery and biological evaluation of 5-aryl-2-furfuramides, potent and selective blockers of the NaV1.8 sodium channel with efficacy in models of neuropathic and inflammatory pain. J Med Chem 2008;51:407 –416. 21. Joshi SK, Honore P, Hernandez G, Schmidt R, Gomtsyan A, Scanio M et al. Additive antinociceptive effects of the selective NaV1.8 blocker A-803467 and selective TRPV1 antagonists in rat inflammatory and neuropathic pain models. J Pain 2009;10:306 –315. 22. Yang T, Atack TC, Stroud DM, Zhang W, Hall L, Roden DM. Blocking Scn10a channels in heart reduces late sodium current and is antiarrhythmic. Circ Res 2012;111:322 –332. 23. Oh S, Choi EK, Zhang Y, Mazgalev TN. Botulinum toxin injection in epicardial autonomic ganglia temporarily suppresses vagally mediated atrial fibrillation. Circ Arrhythm Electrophysiol 2011;4:560 –565. 24. Wang Z, Page P, Nattel S. Mechanism of flecainide’s antiarrhythmic action in experimental atrial fibrillation. Circ Res 1992;71:271–287. 25. Liu L, Nattel S. Differing sympathetic and vagal effects on atrial fibrillation in dogs: role of refractoriness reterogeneity. Am J Physiol 1997;273:H805 –H816. 26. Prechtl JC, Powley TL. The fiber composition of the abdominal vagus of the rat. Anat Embryol 1990;181:101 –115. 27. Pauza DH, Skripka V, Pauziene N, Stropus R. Morphology, distribution, and variability of the epicardiac neural ganglionated subplexuses in the human heart. Anat Rec 2000;259: 353 –382. 28. Quan KJ, Lee JH, Van Hare GF, Biblo LA, Mackall JA, Carlson MD. Identification and characterization of atrioventricular parasympathetic innervation in humans. J Cardiovasc Electrophysiol 2002;13:735–739.
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29. Po SS, Nakagawa H, Jackman WM. Localization of left atrial ganglionated plexi in patients with atrial fibrillation. J Cardiovasc Electrophysiol 2009;20:1186 – 1189. 30. Hoover DB, Isaacs ER, Jacques F, Hoard JL, Page´ P, Armour JA. Localization of multiple neurotransmitters in surgically derived specimens of human atrial ganglia. Neuroscience 2009;164:1170 –1179. 31. Cheng S, Keyes MJ, Larson MG, McCabe EL, Newton-Cheh C, Levy D et al. Long-term outcomes in individuals with prolonged PR interval or first-degree atrioventricular block. JAMA 2009;301:2571 –2577. 32. Soliman EZ, Prineas RJ, Case LD, Zhang ZM, Goff DCJr. Ethnic distribution of ECG predictors of atrial fibrillation and its impact on understanding the ethnic distribution of ischemic stroke in the Atherosclerosis Risk in Communities (ARIC) study. Stroke 2009;40: 1204– 1211. 33. Schnabel RB, Sullivan LM, Levy D, Pencina MJ, Massaro JM, D’Agostino RBSr et al. Development of a Risk Score for Atrial Fibrillation (The Framingham Heart Study): a community-based cohort study. The Lancet 2009;373:739–745. 34. Magnani JW, Wang N, Nelson KP, Connelly S, Deo R, Rodondi N et al. Electrocardiographic PR interval and adverse outcomes in older adults: the Health, Aging, and Body Composition study. Circ Arrhythm Electrophysiol 2013;6:84–90. 35. Facer P, Punjabi PP, Abrari A, Kaba RA, Severs NJ, Chambers J et al. Localisation of SCN10A gene product Na(v)1.8 and novel pain-related ion channels in human heart. Int Heart J 2011;52:146 –152. 36. Kukanova B, Mravec B. Complex intracardiac nervous system. Bratisl Lek Listy 2006;107: 45 –51. 37. Blasiusa AL, Dubinb AE, Petrusc MJ, Lim BK, Narezkina A, Criado JR et al. Hypermorphic mutation of the voltage-gated sodium channel encoding gene Scn10a causes a dramatic stimulus-dependent neurobehavioral phenotype. Proc Natl Acad Sci USA 2011;108: 19413–19418. 38. Herring N, Paterson DJ. Nitric oxide-cGMP pathway facilitates acetylcholine release and bradycardia during vagal nerve stimulation in the guinea-pig in vitro. J Physiol 2001;535: 507 –518. Downloaded from by guest on November 17, 2015
