it has been recognized that cardiac autonomic nervous system activity is of great significance in the initiation and maintenance of atrial fibrillation ( af ) .
stimulation or inhibition of selective extracardiac neural structures may be a useful therapeutic option for cardiac arrhythmias , including af , and other abnormalities .
as the major part of the intrinsic cardiac autonomic nervous system , most cardiac ganglionated plexus ( gps ) embedded in the fat pad around 4 pulmonary veins ( pvs ) , play an important role in the early stage of af.1 increased gp activity can induce the increase of both parasympathetic and sympathetic activity , which promotes rapid focal firing in the pv myocardium.2 , 3 clinical evidence also suggests that additional gp ablation to pv isolation could increase success rates in eliminating af.4 , 5 
  as a tetrodotoxinresistant periphery nerve voltagegated sodium channel , nav1.8 ( encoded by scn10a ) plays a significant part in the upstroke of action potential in neurons , and is responsible for repetitive firing.6 it is found primarily expressed in small and mediumdiameter nociceptive sensory neurons , which mediate pain perception.7 genomewide association study highlights the role of nav1.8 in cardiac conduction and arrhythmic diseases .
in addition to the canonical cardiac sodium channel nav1.5/scn5a , nav1.8 is recently considered a  new cardiac sodium channel.8 
 scn10a is adjacent to scn5a on the same chromosome , and there is 70.4% of similarity in the amino acid sequence between these 2 sodium channels .
our previous research shows nav1.8 could physically interact with nav1.5 by using coimmunoprecipitation when both are expressed in  vitro.9 others prove that nav1.8 could modulate the activity and expression of nav1.5 at the transcriptional level , which may be intermediated by tbx3/5.10 , 11 
  increasing evidence indicates that nav1.8/scn10a plays a critical role in af .
both common and rare variants of scn10a were associated with the risk of af.12 , 13 some rare scn10a variants have been identified in patients with earlyonset af ( rs141207048 , rs202143516 , rs202192818 , rs139861061 , et  al ) , and the relevant mechanism implied by functional study in  vitro might be through the modulation of peak sodium current ( ina , p ) and late sodium current ( ina , l ) of nav1.8.14 certain common variants , such as rs6795970 , have been proved to be related to af susceptibility.12 , 13 one recent study has suggested that blockade of nav1.8 suppresses vagalmediated af most likely by inhibiting the neural activity of gp.15 however , the exact electrophysiological role of nav1.8 in af , especially at the early stage of acute af , is uncertain , and the relevant mechanisms still need to be assessed . in this study
, we aim to explore the expression of nav1.8 and nav1.5 in canine cardiac ganglia , and to evaluate the role of the nav1.8 blocker a803467 in cardiac gp in an acute af canine model .
we have further investigated the changes of the channel density and kinetic characteristics in the presence of a803467 when the scn5a and scn10a coexpressed in  vitro , which might provide the potential mechanism to explain its effect in a canine af model .
experiments were approved by the animal ethics committee of wuhan university under approval number 20150072 and followed the guidelines outlined by the care and use of laboratory animals of the national institutes of health .
sixteen mongrel dogs weighing from 20 to 25  kg were included in this study .
surgeries were performed under anesthesia with sodium pentobarbital with an initial dose of 50  mg / kg and an additional dose of 2  mg / kg per hour .
bilateral thoracotomy was conducted at the fourth intercostal space , as previously described16 , 17 ( figure  1a and 1b ) . in brief ,
multielectrode catheters were sutured to obtain recordings at the surface of the atrium and pvs and to pace at the left atrial appendage ( laa ) .
all recordings were displayed on a computerized bard electrophysiology system ( cr bard inc , billerica , ma ) .
highfrequency stimulation ( 20  hz , 0.1  ms duration , square waves ) was applied at the fat pad to identify gps by a bipolar electrode stimulator ( grasss88 ; astromed , west warwick , ri ) .
anterior right ganglionated plexi ( argp ) was located at the rspvatrial junction and superior left ganglionated plexi  ( slgp ) at the left superior pv ( lspv)atrial junction .
a  successful gp stimulation was marked at the 50% sinus rate slowing or second or thirddegree atrioventricular block developing .
schematic catheter positions in the atria and pulmonary veins and representative traces . a , left thoracotomy approach .
electrode catheters were sutured to the left superior pulmonary vein ( lspv ) , left inferior pulmonary vein ( lipv ) , left atrium ( la ) , left atrial appendage ( laa ) , right superior pulmonary vein ( rspv ) , right inferior pulmonary vein ( ripv ) , right atrium ( ra ) , and right atrial appendage ( raa ) .
argp indicates anterior right ganglionated plexi ; ilgp , inferior left ganglionated plexi ; irgp , inferior right ganglionated plexi ; ivc , inferior vena cava ; lpa , left pulmonary artery ; lv , left ventricle ; slgp , superior left ganglionated plexi ; svc , superior vena cava ; rv , right ventricle .
c and d , representative traces in the electrophysiological study before and after administrating a803467 at ganglionated plexus .
two groups were set in this study based on injecting saline ( 0.5  ml per gp ) or the selective nav1.8 blocker a803467 ( 1  mol/0.5  ml per gp ) into both argp and slgp .
after drug administrating , rap was delivered at the laa ( 20  hz , 10 diastolic threshold ) for 6  hours . at the end of rap , targeted parameterseffective refractory period ( erp ) and window of vulnerability ( wov ) were evaluated as previously described.16 , 18 briefly , we measured erp by using s1s1 programmed stimulating at 330  ms interval and s1s2 interval from 150  ms with decrementing at 10 and 1  ms when approaching the refractory period ( s1:s2=8:1 , 10  th ) . to delineate af inducibility ,
erp dispersion was assessed as the coefficient of variation of erp at all 8 recording sites .
af was defined as irregular atrial rates related with irregular atrioventricular conduction at more than 500  bpm lasting at least 5  seconds .
af cycle length was measured as the averaged first 10 fibrillation waves at the onset of af .
the number of af episodes and averaged af duration were also recorded at each group .
representative traces in the electrophysiological study before and after administrating a803467 at gps are shown in figure  1c and figure  1d .
cardiac ganglia were quickly obtained from 3 canine heart fat pads located at the atria posterior wall .
after that , they were placed into normal krebs solution in ice , dissected under a stereomicroscope , and put into liquid nitrogen for flash freezing .
cryosections from embedded ganglia tissue were fixed in 4% paraformaldehyde for 10  minutes , permeabilized in 0.3% triton x100 in pbs for 15  minutes , and all blocked in 5% donkey serum for 30  minutes at room temperature .
tissue sections were incubated at 4c overnight with primary antibody specific for rabbit polyclonal antinav1.8 ( alomone labs , jerusalem , israel ) , rabbit polyclonal antinav1.5 ( alomone labs , jerusalem , israel ) , goat polyclonal antipgp9.5 ( abcam , cambridge , ma ) , goat polyclonal anticholine acetyltransferase ( chat , chemicon international , temecula , canada ) , and mouse monoclonal antityrosine hydroxylase ( th , alpha diagnostic international , san antonio , tx ) .
alexa 488conjugated secondary antibody was then added according to a different source of primary antibody and incubated for 90  minutes at room temperature .
stained samples were visualized under an olympus fluoview laser scanning confocal microscope ( olympus , orangeburg , ny ) .
tsa201 cells transfected with scn5a , scn10a , and scn3b plasmids were used for patch clamp study . a plasmid encoding egfp was used to identify transfected cells .
briefly , transient transfection using fugene 6 ( roche diagnostics , indianapolis , indiana ) was carried out with scn10a , scn5a , and scn3b with a molar ratio of 5:5:1 ( for a total of 2.25  g of dna ) .
15140 ] , life technologies ) on polylysinecoated 35  mm culture dishes ( cell+ , sarstedt , newton , nc ) .
cells were placed in a 5% co2 incubator at 37c for 24 to 48  hours prior to patch clamp study .
all recordings were obtained at room temperature ( 2022c ) using an axopatch 200b amplifier equipped with a cv201a head stage ( axon instruments inc./molecular devices , union city , ca ) .
currents were filtered with a 4pole bessel filter at 5  khz and digitized at 50  khz .
series resistance was compensated at around 80% to assure that the command potential was reached within microseconds with a voltage error < 2  mv .
cells were allowed to stabilize for 10  minutes after establishment of the wholecell configuration before current was measured .
macroscopic wholecell na+ current was recorded by using bath solution perfusion containing ( in mmol / l ) 140 nacl , 4 kcl , 1.8 cacl2 , 1 mgcl2 , 10 hepes , and 10 dextrose ( ph 7.35 with naoh ) . osmolarity was adjusted to 310  mmol / kg with sucrose .
patch pipettes were fabricated from 1.5  mm od borosilicate glass capillaries ( fisher scientific inc , hampton , nh ) .
they were pulled using a gravity puller ( model pp830 , narishige international usa , inc , east meadow , ny ) to obtain resistances between 0.8 and 2.2  m when filled with a pipette solution containing ( in mmol / l ) 10 naf , 105 csf , 20 cscl , 2 egta , and 10 hepes with a ph of 7.35 adjusted with csoh and an osmolality of 300  mmol / kg with sucrose .
specific voltageclamp protocols assessing channel activation and fast inactivation were used , as depicted in the figure insets .
cardiac sodium channel current ( ina ) was elicited by depolarizing pulses ranging from 90 to + 40  mv in 5  mv increments with a holding potential of 120  mv .
peak currents ( ina , p ) were measured and ina densities ( pa / pf ) were attained by dividing the obtained cell capacitance .
activation properties were determined from i / v relationships by normalizing ina , p to driving force and maximal ina , and plotting normalized conductance versus vm .
voltage dependence of steadystate inactivation was obtained by plotting the normalized ina , p ( 40ms test pulse to 0  mv after a 1000ms conditioning pulse from 140 to 10  mv with the holding potential of 120  mv ) versus vm .
the steadystate channel availability and inactivation curves were fitted to the boltzmann equation , i / imax=1/(1+exp((vv1/2)/ ) ) , to determine the membrane potential for halfmaximal activation / inactivation ( v1/2 ) and the slope factor (  ) .
pulses for recovery from inactivation were of 100  ms duration for p1 and 50  ms for p2 .
the peak current elicited during the second pulse was normalized to the value obtained during the initial test pulse .
it was analyzed by fitting data to a double exponential function : i(t)/imax = af(1exp(t/f))+as(1exp(t/s ) ) , where af and as are the fractions of fast and slow inactivating components , respectively , and f and s are their time constants . all data acquisition and analysis were performed using pclamp version 9.2 ( molecular devices , sunnyvale , ca ) , excel ( microsoft , redmond , wa ) , and origin 7.5 ( microcal software , ge healthcare , pittsburgh , pa ) .
experiments were approved by the animal ethics committee of wuhan university under approval number 20150072 and followed the guidelines outlined by the care and use of laboratory animals of the national institutes of health .
sixteen mongrel dogs weighing from 20 to 25  kg were included in this study .
surgeries were performed under anesthesia with sodium pentobarbital with an initial dose of 50  mg / kg and an additional dose of 2  mg / kg per hour .
bilateral thoracotomy was conducted at the fourth intercostal space , as previously described16 , 17 ( figure  1a and 1b ) . in brief ,
multielectrode catheters were sutured to obtain recordings at the surface of the atrium and pvs and to pace at the left atrial appendage ( laa ) .
all recordings were displayed on a computerized bard electrophysiology system ( cr bard inc , billerica , ma ) .
highfrequency stimulation ( 20  hz , 0.1  ms duration , square waves ) was applied at the fat pad to identify gps by a bipolar electrode stimulator ( grasss88 ; astromed , west warwick , ri ) .
anterior right ganglionated plexi ( argp ) was located at the rspvatrial junction and superior left ganglionated plexi  ( slgp ) at the left superior pv ( lspv)atrial junction .
a  successful gp stimulation was marked at the 50% sinus rate slowing or second or thirddegree atrioventricular block developing .
schematic catheter positions in the atria and pulmonary veins and representative traces . a , left thoracotomy approach .
electrode catheters were sutured to the left superior pulmonary vein ( lspv ) , left inferior pulmonary vein ( lipv ) , left atrium ( la ) , left atrial appendage ( laa ) , right superior pulmonary vein ( rspv ) , right inferior pulmonary vein ( ripv ) , right atrium ( ra ) , and right atrial appendage ( raa ) .
argp indicates anterior right ganglionated plexi ; ilgp , inferior left ganglionated plexi ; irgp , inferior right ganglionated plexi ; ivc , inferior vena cava ; lpa , left pulmonary artery ; lv , left ventricle ; slgp , superior left ganglionated plexi ; svc , superior vena cava ; rv , right ventricle .
c and d , representative traces in the electrophysiological study before and after administrating a803467 at ganglionated plexus .
two groups were set in this study based on injecting saline ( 0.5  ml per gp ) or the selective nav1.8 blocker a803467 ( 1  mol/0.5  ml per gp ) into both argp and slgp .
after drug administrating , rap was delivered at the laa ( 20  hz , 10 diastolic threshold ) for 6  hours . at the end of rap , targeted parameterseffective refractory period ( erp ) and window of vulnerability ( wov ) were evaluated as previously described.16 , 18 briefly , we measured erp by using s1s1 programmed stimulating at 330  ms interval and s1s2 interval from 150  ms with decrementing at 10 and 1  ms when approaching the refractory period ( s1:s2=8:1 , 10  th ) . to delineate af inducibility ,
erp dispersion was assessed as the coefficient of variation of erp at all 8 recording sites .
af was defined as irregular atrial rates related with irregular atrioventricular conduction at more than 500  bpm lasting at least 5  seconds .
af cycle length was measured as the averaged first 10 fibrillation waves at the onset of af .
the number of af episodes and averaged af duration were also recorded at each group .
representative traces in the electrophysiological study before and after administrating a803467 at gps are shown in figure  1c and figure  1d .
cardiac ganglia were quickly obtained from 3 canine heart fat pads located at the atria posterior wall .
after that , they were placed into normal krebs solution in ice , dissected under a stereomicroscope , and put into liquid nitrogen for flash freezing .
cryosections from embedded ganglia tissue were fixed in 4% paraformaldehyde for 10  minutes , permeabilized in 0.3% triton x100 in pbs for 15  minutes , and all blocked in 5% donkey serum for 30  minutes at room temperature .
tissue sections were incubated at 4c overnight with primary antibody specific for rabbit polyclonal antinav1.8 ( alomone labs , jerusalem , israel ) , rabbit polyclonal antinav1.5 ( alomone labs , jerusalem , israel ) , goat polyclonal antipgp9.5 ( abcam , cambridge , ma ) , goat polyclonal anticholine acetyltransferase ( chat , chemicon international , temecula , canada ) , and mouse monoclonal antityrosine hydroxylase ( th , alpha diagnostic international , san antonio , tx ) .
alexa 488conjugated secondary antibody was then added according to a different source of primary antibody and incubated for 90  minutes at room temperature .
stained samples were visualized under an olympus fluoview laser scanning confocal microscope ( olympus , orangeburg , ny ) .
tsa201 cells transfected with scn5a , scn10a , and scn3b plasmids were used for patch clamp study . a plasmid encoding egfp was used to identify transfected cells . briefly , transient transfection using fugene 6 ( roche diagnostics , indianapolis , indiana ) was carried out with scn10a , scn5a , and scn3b with a molar ratio of 5:5:1 ( for a total of 2.25  g of dna ) .
15140 ] , life technologies ) on polylysinecoated 35  mm culture dishes ( cell+ , sarstedt , newton , nc ) .
cells were placed in a 5% co2 incubator at 37c for 24 to 48  hours prior to patch clamp study .
all recordings were obtained at room temperature ( 2022c ) using an axopatch 200b amplifier equipped with a cv201a head stage ( axon instruments inc./molecular devices , union city , ca ) .
currents were filtered with a 4pole bessel filter at 5  khz and digitized at 50  khz .
series resistance was compensated at around 80% to assure that the command potential was reached within microseconds with a voltage error < 2  mv .
cells were allowed to stabilize for 10  minutes after establishment of the wholecell configuration before current was measured .
macroscopic wholecell na+ current was recorded by using bath solution perfusion containing ( in mmol / l ) 140 nacl , 4 kcl , 1.8 cacl2 , 1 mgcl2 , 10 hepes , and 10 dextrose ( ph 7.35 with naoh ) . osmolarity was adjusted to 310  mmol / kg with sucrose .
patch pipettes were fabricated from 1.5  mm od borosilicate glass capillaries ( fisher scientific inc , hampton , nh ) .
they were pulled using a gravity puller ( model pp830 , narishige international usa , inc , east meadow , ny ) to obtain resistances between 0.8 and 2.2  m when filled with a pipette solution containing ( in mmol / l ) 10 naf , 105 csf , 20 cscl , 2 egta , and 10 hepes with a ph of 7.35 adjusted with csoh and an osmolality of 300  mmol / kg with sucrose .
specific voltageclamp protocols assessing channel activation and fast inactivation were used , as depicted in the figure insets .
cardiac sodium channel current ( ina ) was elicited by depolarizing pulses ranging from 90 to + 40  mv in 5  mv increments with a holding potential of 120  mv .
peak currents ( ina , p ) were measured and ina densities ( pa / pf ) were attained by dividing the obtained cell capacitance .
activation properties were determined from i / v relationships by normalizing ina , p to driving force and maximal ina , and plotting normalized conductance versus vm .
voltage dependence of steadystate inactivation was obtained by plotting the normalized ina , p ( 40ms test pulse to 0  mv after a 1000ms conditioning pulse from 140 to 10  mv with the holding potential of 120  mv ) versus vm .
the steadystate channel availability and inactivation curves were fitted to the boltzmann equation , i / imax=1/(1+exp((vv1/2)/ ) ) , to determine the membrane potential for halfmaximal activation / inactivation ( v1/2 ) and the slope factor (  ) .
pulses for recovery from inactivation were of 100  ms duration for p1 and 50  ms for p2 .
the peak current elicited during the second pulse was normalized to the value obtained during the initial test pulse .
it was analyzed by fitting data to a double exponential function : i(t)/imax = af(1exp(t/f))+as(1exp(t/s ) ) , where af and as are the fractions of fast and slow inactivating components , respectively , and f and s are their time constants .
all data acquisition and analysis were performed using pclamp version 9.2 ( molecular devices , sunnyvale , ca ) , excel ( microsoft , redmond , wa ) , and origin 7.5 ( microcal software , ge healthcare , pittsburgh , pa ) .
as shown in figure  2 , adult canine cardiac ganglias were partially dissected and labeled by neuron marker pgp9.5 , th , and chat . both adrenergic and cholinergic
neurons can be found in those ganglias ( figure  2 g through 2j ) .
staining of nav1.8 and nav1.5 separately proved their expression on canine gp ( figure  2a through 2d ) .
the 2 sodium channels can be observed in both membrane and cytoplasm under high magnification and no significant differences were found in regard to the channel distribution in adrenergic and cholinergic neurons ( data not shown ) .
immunochemical study of nav1.8 , nav1.5 , pgp9.5 , anticholine acetyltransferase ( chat ) , and antityrosine hydroxylase ( th ) in partial dissected intracardiac ganglia .
protein expression of nav1.8 ( a and b ) , nav1.5 ( c and d ) , pgp9.5 ( e and f ) , chat ( g and h ) , and th ( i and j ) present in intracardiac ganglia .
calibration bars in ( a through d ) are 20  m and in ( e through j ) are 50  m .
representative traces in the electrophysiological study before and after administrating a803467 at gps are displayed in figure  1c and figure  1d .
mean erp at baseline between the control and a803467 groups was similar at all 8 recording sites ( 97.986.86 versus 97.957.30  ms , p>0.05 , 10x threshold ) .
after administrating saline or a803467 , respectively , to the 2 groups at argp and slgp , rapid pacing was implemented at laa .
the erps were reevaluated at the end of 6hour rap by the same protocol . compared with the control group ,
the shortening of erp at all pv and atrial sites was suppressed in the a803467 group ( figure  3 , p<0.05 ) .
mean effective refractory period ( erp ) values are shown at all 8 recording sites in 2 study groups .
at all sites ( a through h ) , mean erp decreased significantly after 6  hours of rap ( not significant [ ns ] , p>0.05 ; * p<0.05 , vs rap+saline at the same time point ) .
la indicates left atrium ; lipv , left inferior pulmonary vein ; lspv , left superior pulmonary vein ; ra , right atrium ; raa , right atrial appendage ; ripv , right inferior pulmonary vein ; rspv , right superior pulmonary vein . erp dispersion and cumulative wov ( wov )
were calculated and af inducibility was compared between the 2 groups before and after rap .
no baseline differences at erp dispersion or wov were found between the 2 groups before rap ( erp dispersion : 0.150.15 versus 0.140.13  ms ; wov : 19.303.51 versus 18.132.34  ms ; p>0.05 [ figure  4d and 4e ] ) .
after 6hour rap , both erp dispersion and wov were significantly increased in the control group . however , injecting a803467 at gps attenuated the increase in erp dispersion and wov apparently at all sites , underlying the decrease in af inducibility ( figure  4d and 4e , erp dispersion : 0.290.15 versus 0.150.13 , wov : 180.7020.47 versus 21.645.84  ms ; p<0.01 ) .
changes in programmed stimulating parameters in 2 groups before and after 6hour rapid atrial pacing ( rap ) . a through c , atrial fibrillation ( af ) duration , cycle length , and episodes after 6hour rap .
d and e , effective refractory period ( erp ) dispersion and cumulative window of vulnerability ( wov ) changes at baseline ( 0 hour ) and after 6hour rap .
not significant ( ns ) , p>0.05 ; * p<0.05 ; * * p<0.01 vs rap+saline at the same time point . in rap+saline , rap resulted in significant decreases in erp and increases in erp dispersion , wov , and af inducibility . in rap+a803467 , however , these changes were significantly attenuated .
after 6hour rap , spontaneous af occurred in 7 dogs in the control group ( n=8 ) , whereas it occurred in only 2 dogs in the a803467 group ( n=8 ) , with an incidence of 87.50% versus 25.0% ( figure  4c ) .
in addition , administrating a803467 can also shorten af duration and prolong af cycle length in contrast to that in the control group ( af duration : 47.0510.45  seconds versus 18.186.59  seconds , p<0.05 ; af cycle length : 79.211.24 versus 129.7717.41  ms , p<0.05 [ figure  4a and 4b ] ) .
previous reports have shown the abundant expression of 3 subunit ( scn3b ) in dorsal root ganglion neurons and have emphasized that the 3 subunit can promote the trafficking of nav1.8 and help its expression on membrane in comparison to other  subunits without changing gating properties.19 , 20 based on that , scn5ascn10ascn3bwt was cotransfected into tsa201 cells in the present study to depict the electrophysiological characteristics of the sodium channel composed of scn5ascn10a under 100  nmol / l a803467 .
a significant reduction was shown with 100  nmol / l a803467 on the currentvoltage relationship , whereas no difference was found at the same concentration with scn5ascn3bwt ( figure  5a ) .
other and our previous results have reported the extremely low current density when scn10a was transfected into tsa201 cells , with or without sodium  subunit.9 , 21 hence , the blocking effect of a803467 was not performed on scn10ascn3bwt transfected tsa201 cells .
electrophysiological effect of a803467 at 100  nmol / l on scn10ascn5ascn3b coexpressed tsa201 cells .
a , representative in 
 a , p traces in scn5awt 
 + scn3bwt transfected cells , and scn10awt 
 + scn5awt 
 + scn3bwt cotransfected cells with and without 100  nmol / l a803467 separately .
b and c , currentvoltage relationship and in 
 a , p current density depicting in 
 a , p recording from coexpression of scn10awt 
 + scn5awt 
 + scn3bwt before and after perfusing with 100  nmol / l a803467 .
d , representative traces and blocking effect of 100  nmol / l a803467 on in 
 a , l , as the percentage of in 
 a , p . * p<0.05 compared 2 groups .
e , comparison of normalized current of in 
 a , p and in 
 a , l between the control and a803467 100  nmol / l groups .
a803467 reduced maximum ina , p by 45% ( ina , p , from 758.77173.48 to 414.7592.44  pa / pf , n=8 ; p<0.01 ) with similar iv curve ( ina , p amplitudes at 35  mv ) between the control and a803467 groups ( figure  5b and 5c ) .
in addition , ina , l was measured under 100  nmol / l a803467 between 295 and 300  ms by the test pulse at 20  mv from hp of 120  mv at the rate of 0.2  hz ( figure  5d ) .
relative ina , l ( percentage of ina , p ) decreased from 0.52% to 0.20% .
the inhibitions of ina , p and ina , l under the same protocol were also compared .
both the ina , p and ina , l in control conditions were regarded as 100% . as presented in figure  5e , after applying a803467 , a more pronounced inhibition with ina , l than ina
, p was shown : ina , p was decreased 42.023.88% and ina , l was decreased 68.575.86% ( n=8 for each group , p=0.02 ) . figure  6a shows a hyperpolarized shift of steadystate inactivation by the influence of 100  nmol / l a803467 under a 100  ms prepulse to various potentials in 10  mv step followed by a 20  ms test pulse to 0  mv .
a803467 caused a prominent negative shift of the v1/2 of inactivation from 83.762.38 to 95.773.20  mv ( n=12 , p<0.05 ) , predicting less availability of channels open at physiological resting potential or sustained depolarization with the drug .
as shown in figure  6b and the table , there was no significant difference in steadystate activation before and after applying the drug ( v1/2 : 49.522.74 and 51.951.38  mv , n=12 [ p=0.44 ] ;  : 5.210.48 and
5.780.24 , n=12 [ p=0.30 ] ) . effect of a803467 at 100  nmol / l on the kinetics of scn10ascn5ascn3b coexpressed tsa201 cells .
a , steadystate inactivation curves for the control and a803467 groups . administrating a803467 can hyperpolarize the inactivation curve by 10  mv ( p<0.05 ) .
boltzmann analysis showed that the slope factor (  ) and the halfactivation potential did not differ significantly between groups .
c , representative recovery traces elicited from 120  mv holding potential to 100  ms inactivating pulse followed by variable duration ( 11480  ms ) to allow the recovery with and without a803467 .
d , effects of a803467 on recovery from inactivation between the 2 groups by 2 exponential fitting . both fast and slow time constants were increased after applying a803467 .
effects of a803467 on the kinetics gating parameters in scn10ascn5ascn3b coexpression tsa201 cells parameters of inactivation and activation were calculated from the boltzmann function .
v1/2 is the voltage for halfmaximal availability or activation and  is the slope factor .
recovery from inactivation was also assessed before and after drug administration by using a standard 2pulse protocol : first test 10  mv for 100  ms then recovering at 120  mv for a various duration before a second pulse at 10  mv for 50  ms . as displayed in the representative traces ( figure  6c ) ,
both the fast ( f ) and slow ( s ) time constants were much slower in the presence of 100  nmol / l a803467 ( f : 2.870.38 versus 6.460.58 , n=14 [ p<0.01 ] ; s : 20.510.61 versus 64.3715.47 , n=14 [ p=0.01 ] , figure  6d , table ) .
for further study , usedependent block ( udb ) was investigated in the absence and presence of a803467 via a series of 40 pulses to 20  mv from 120  mv holding potential at the rate of 1 , 2 , and 10  hz .
the proportion of the 40th and first current amplitudes was compared between control and drug conditions under these 3 different frequencies . for the control group , the proportions of ina ,
p were 97.38% , 95.80% , and 93.89% , and for the drug were 95.95% , 89.81% , and 83.96% , at the rate of 1 , 2 , and 10  hz , respectively ( figure  7b ) .
the inhibition effect of a803467 was more evident at faster rates ( reduction at 1/2/10  hz was 3.68%/6.06%/12.78% , n=9 [ p<0.01 ] ) .
both traces of the 2 conditions were positioned at the same scale so that the noticeable reduction under drug influence was more obvious in comparison .
together with the inhibition of recovery from inactivation , a803467 may interact with the inactivated state of these sodium channels .
figure  7d shows the relative current proportion of ina , l at 20  mv in the rate of 2  hz . in the control condition
l than that in ina , p , with 68.993.67% versus 95.801.75% remaining after the 40th pulse . when the drug was applied , the value of ina , l and ina , p decreased to 60.643.98% and 89.432.06% ( reduced rate of ina , l versus ina , p ; 1204% versus 62% , n=8 for each group [ p=0.04 ] ) .
the result indicates preferential udb of a803467 on ina , l than ina , p .
usedependent block ( udb ) in the control and a803467 100  nmol / l groups .
pulse duration was 20  ms , and the holding potential was 120  mv and given at a rate of 0.1  seconds ( 10  hz ) .
c , udb of in 
 a , p in the control and a803467 groups at 1 , 2 , and 10  hz .
d , udb of in 
 a , l in the control and a803467 groups at 2  hz .
as shown in figure  2 , adult canine cardiac ganglias were partially dissected and labeled by neuron marker pgp9.5 , th , and chat . both adrenergic and cholinergic
neurons can be found in those ganglias ( figure  2 g through 2j ) .
staining of nav1.8 and nav1.5 separately proved their expression on canine gp ( figure  2a through 2d ) .
the 2 sodium channels can be observed in both membrane and cytoplasm under high magnification and no significant differences were found in regard to the channel distribution in adrenergic and cholinergic neurons ( data not shown ) .
immunochemical study of nav1.8 , nav1.5 , pgp9.5 , anticholine acetyltransferase ( chat ) , and antityrosine hydroxylase ( th ) in partial dissected intracardiac ganglia .
protein expression of nav1.8 ( a and b ) , nav1.5 ( c and d ) , pgp9.5 ( e and f ) , chat ( g and h ) , and th ( i and j ) present in intracardiac ganglia .
calibration bars in ( a through d ) are 20  m and in ( e through j ) are 50  m .
representative traces in the electrophysiological study before and after administrating a803467 at gps are displayed in figure  1c and figure  1d
. mean erp at baseline between the control and a803467 groups was similar at all 8 recording sites ( 97.986.86 versus 97.957.30  ms , p>0.05 , 10x threshold ) .
after administrating saline or a803467 , respectively , to the 2 groups at argp and slgp , rapid pacing was implemented at laa .
the erps were reevaluated at the end of 6hour rap by the same protocol . compared with the control group ,
the shortening of erp at all pv and atrial sites was suppressed in the a803467 group ( figure  3 , p<0.05 ) .
mean effective refractory period ( erp ) values are shown at all 8 recording sites in 2 study groups .
at all sites ( a through h ) , mean erp decreased significantly after 6  hours of rap ( not significant [ ns ] , p>0.05 ; * p<0.05 , vs rap+saline at the same time point ) .
la indicates left atrium ; lipv , left inferior pulmonary vein ; lspv , left superior pulmonary vein ; ra , right atrium ; raa , right atrial appendage ; ripv , right inferior pulmonary vein ; rspv , right superior pulmonary vein .
erp dispersion and cumulative wov ( wov ) were calculated and af inducibility was compared between the 2 groups before and after rap .
no baseline differences at erp dispersion or wov were found between the 2 groups before rap ( erp dispersion : 0.150.15 versus 0.140.13  ms ; wov : 19.303.51 versus 18.132.34  ms ; p>0.05 [ figure  4d and 4e ] ) .
after 6hour rap , both erp dispersion and wov were significantly increased in the control group . however , injecting a803467 at gps attenuated the increase in erp dispersion and wov apparently at all sites , underlying the decrease in af inducibility ( figure  4d and 4e , erp dispersion : 0.290.15 versus 0.150.13 , wov : 180.7020.47 versus 21.645.84  ms ; p<0.01 ) .
changes in programmed stimulating parameters in 2 groups before and after 6hour rapid atrial pacing ( rap ) . a through c , atrial fibrillation ( af ) duration , cycle length , and episodes after 6hour rap .
d and e , effective refractory period ( erp ) dispersion and cumulative window of vulnerability ( wov ) changes at baseline ( 0 hour ) and after 6hour rap .
not significant ( ns ) , p>0.05 ; * p<0.05 ; * * p<0.01 vs rap+saline at the same time point . in rap+saline , rap resulted in significant decreases in erp and increases in erp dispersion , wov , and af inducibility . in rap+a803467 , however , these changes were significantly attenuated .
after 6hour rap , spontaneous af occurred in 7 dogs in the control group ( n=8 ) , whereas it occurred in only 2 dogs in the a803467 group ( n=8 ) , with an incidence of 87.50% versus 25.0% ( figure  4c ) .
in addition , administrating a803467 can also shorten af duration and prolong af cycle length in contrast to that in the control group ( af duration : 47.0510.45  seconds versus 18.186.59  seconds , p<0.05 ; af cycle length : 79.211.24 versus 129.7717.41  ms , p<0.05 [ figure  4a and 4b ] ) .
previous reports have shown the abundant expression of 3 subunit ( scn3b ) in dorsal root ganglion neurons and have emphasized that the 3 subunit can promote the trafficking of nav1.8 and help its expression on membrane in comparison to other  subunits without changing gating properties.19 , 20 based on that , scn5ascn10ascn3bwt was cotransfected into tsa201 cells in the present study to depict the electrophysiological characteristics of the sodium channel composed of scn5ascn10a under 100  nmol / l a803467 .
a significant reduction was shown with 100  nmol / l a803467 on the currentvoltage relationship , whereas no difference was found at the same concentration with scn5ascn3bwt ( figure  5a ) .
other and our previous results have reported the extremely low current density when scn10a was transfected into tsa201 cells , with or without sodium  subunit.9 , 21 hence , the blocking effect of a803467 was not performed on scn10ascn3bwt transfected tsa201 cells .
electrophysiological effect of a803467 at 100  nmol / l on scn10ascn5ascn3b coexpressed tsa201 cells .
a , representative in 
 a , p traces in scn5awt 
 + scn3bwt transfected cells , and scn10awt 
 + scn5awt 
 + scn3bwt cotransfected cells with and without 100  nmol / l a803467 separately .
b and c , currentvoltage relationship and in 
 a , p current density depicting in 
 a , p recording from coexpression of scn10awt 
 + scn5awt 
 + scn3bwt before and after perfusing with 100  nmol / l a803467 . * p<0.05 compared 2 groups .
d , representative traces and blocking effect of 100  nmol / l a803467 on in 
 a , l , as the percentage of in 
 a , p . * p<0.05 compared 2 groups .
e , comparison of normalized current of in 
 a , p and in 
 a , l between the control and a803467 100  nmol / l groups .
a803467 reduced maximum ina , p by 45% ( ina , p , from 758.77173.48 to 414.7592.44  pa / pf , n=8 ; p<0.01 ) with similar iv curve ( ina , p amplitudes at 35  mv ) between the control and a803467 groups ( figure  5b and 5c ) .
in addition , ina , l was measured under 100  nmol / l a803467 between 295 and 300  ms by the test pulse at 20  mv from hp of 120  mv at the rate of 0.2  hz ( figure  5d ) .
relative ina , l ( percentage of ina , p ) decreased from 0.52% to 0.20% .
the inhibitions of ina , p and ina , l under the same protocol were also compared .
both the ina , p and ina , l in control conditions were regarded as 100% . as presented in figure  5e , after applying a803467 , a more pronounced inhibition with ina , l than ina , p was shown : ina , p was decreased 42.023.88% and ina , l was decreased 68.575.86% ( n=8 for each group , p=0.02 ) . figure  6a shows a hyperpolarized shift of steadystate inactivation by the influence of 100  nmol / l a803467 under a 100  ms prepulse to various potentials in 10  mv step followed by a 20  ms test pulse to 0  mv .
a803467 caused a prominent negative shift of the v1/2 of inactivation from 83.762.38 to 95.773.20  mv ( n=12 , p<0.05 ) , predicting less availability of channels open at physiological resting potential or sustained depolarization with the drug .
as shown in figure  6b and the table , there was no significant difference in steadystate activation before and after applying the drug ( v1/2 : 49.522.74 and 51.951.38  mv , n=12 [ p=0.44 ] ;  : 5.210.48 and 5.780.24 , n=12 [ p=0.30 ] ) . effect of a803467 at 100  nmol /
boltzmann analysis showed that the slope factor (  ) and the halfactivation potential did not differ significantly between groups .
c , representative recovery traces elicited from 120  mv holding potential to 100  ms inactivating pulse followed by variable duration ( 11480  ms ) to allow the recovery with and without a803467 .
d , effects of a803467 on recovery from inactivation between the 2 groups by 2 exponential fitting .
effects of a803467 on the kinetics gating parameters in scn10ascn5ascn3b coexpression tsa201 cells parameters of inactivation and activation were calculated from the boltzmann function .
v1/2 is the voltage for halfmaximal availability or activation and  is the slope factor .
recovery from inactivation was also assessed before and after drug administration by using a standard 2pulse protocol : first test 10  mv for 100  ms then recovering at 120  mv for a various duration before a second pulse at 10  mv for 50  ms .
as displayed in the representative traces ( figure  6c ) , a803467 inhibited the process of recovery from inactivation .
a 2exponential equation  was used for fitting , and time constants were compared . both the fast ( f ) and slow ( s ) time constants were much slower in the presence of 100  nmol
/ l a803467 ( f : 2.870.38 versus 6.460.58 , n=14 [ p<0.01 ] ; s : 20.510.61 versus 64.3715.47 , n=14 [ p=0.01 ] , figure  6d , table ) . for further study , usedependent block ( udb )
was investigated in the absence and presence of a803467 via a series of 40 pulses to 20  mv from 120  mv holding potential at the rate of 1 , 2 , and 10  hz .
the proportion of the 40th and first current amplitudes was compared between control and drug conditions under these 3 different frequencies . for the control group , the proportions of ina , p were 97.38% , 95.80% , and 93.89% , and for the drug were 95.95% , 89.81% , and 83.96% , at the rate of 1 , 2 , and 10  hz , respectively ( figure  7b ) .
the inhibition effect of a803467 was more evident at faster rates ( reduction at 1/2/10  hz was 3.68%/6.06%/12.78% , n=9 [ p<0.01 ] ) .
both traces of the 2 conditions were positioned at the same scale so that the noticeable reduction under drug influence was more obvious in comparison .
together with the inhibition of recovery from inactivation , a803467 may interact with the inactivated state of these sodium channels .
figure  7d shows the relative current proportion of ina , l at 20  mv in the rate of 2  hz . in the control condition , there was more inhibition of ina ,
l than that in ina , p , with 68.993.67% versus 95.801.75% remaining after the 40th pulse . when the drug was applied , the value of ina , l and ina , p decreased to 60.643.98% and 89.432.06% ( reduced rate of ina , l versus ina , p ; 1204% versus 62% , n=8 for each group [ p=0.04 ] ) .
the result indicates preferential udb of a803467 on ina , l than ina , p .
usedependent block ( udb ) in the control and a803467 100  nmol / l groups
pulse duration was 20  ms , and the holding potential was 120  mv and given at a rate of 0.1  seconds ( 10  hz ) .
c , udb of in 
 a , p in the control and a803467 groups at 1 , 2 , and 10  hz .
d , udb of in 
 a , l in the control and a803467 groups at 2  hz .
in this study , we demonstrate that , in a canine acute af model , pharmacological inhibition of nav1.8 on gp can prevent electrical remodeling at pv and atrial myocardium and further reduce the incidence of paroxysmal af .
sodium channel properties were obviously affected by the nav1.8 blocker when scn5ascn10ascn3bwt coexpressed in  vitro , which indicates a possible underlying mechanism of our animal model results .
the transfected sodium channel complex with 100  nmol / l a803467 displayed decreased ina , and faster inactivation , slower recovery , and stronger udb on both ina , p and ina , l in contrast to the characteristics before the drug . 
 
one study by nattel and colleagues22 , 23 verified that gps played a vital role in the experimental af related to atrial tachycardia remodeling . compared with small effect on af vulnerability by pv isolation , gp ablation could reduce af vulnerability and attenuate af maintenance by prolonging erp , reducing af duration and dominant frequency of fibrillatory activity .
in addition , gp stimulation increases vagal and sympathetic tone , which induces early afterrepolarization formation , enhances ca transient facilitating trigger activity , and shortens action potential duration , especially at pv myocardium , increasing heterogeneous repolarization and prompting substrate and reentry within atria.3 , 24 , 25 rap can shorten erp and increase erp dispersion and wov , which were all reversed after gp ablation . implementing gp ablation or autonomic blockers ( atropine or propranolol ) followed by rap
can prevent induced spontaneous af and atrial electrical remodeling.26 , 27 in our study , administrating a803467 can effectively attenuate erp shortening and suppress increased erp dispersion and wov , indicating a pharmacological denervation effect by this selective nav1.8 blocker .
voltagegated sodium channels are responsible for generating and propagating action potentials and are critical in neuronal firing and excitability .
therefore , our results suggest that blocking nav1.8 can decrease gp firing , inhibit its activity , and prevent the initiation and maintenance of rapinduced af .
similar findings were observed by qi and colleagues.15 they applied a different afinduced model by vagus nerve stimulation ( vns ) to evaluate cardiac conduction and af inducibility after injecting a803467 on argp and inferior right ganglionated plexi at the same concentration .
they concluded that blockade of nav1.8 could suppress the effect of vns on sinus rate , pr interval , and ventricular rate when af occurred during vns .
the values of erp in the control group after gp injection were lower than ours probably because of vns .
previous study demonstrated that argp and slgp in the intrinsic cardiac autonomic nerve system collected the nerve from both vagosympathetic trunk as integration center , and either argp or slgp ablation could eliminated erp shortening during vagosympathetic trunk stimulation .
it identified that argp and slgp are the main connections in the neural pathway to control intrinsic autonomic activity in af.28 therefore , ablation ( drug denervation ) of argp and slgp could be sufficient to show the effect of af prevention . in the present study ,
we chosen argp and slgp for injection and applied 6hour rap after injection to assess the effect of blocking nav1.8 on acute af directly .
decreased erps were found in all atrium and pulmonary recording sites , which were suppressed when a803467 were administrated in advance .
this could suggest that blocking nav1.8 could avoid functional substrate forming in pv myocardium , probably as a result of inhibiting gp function and atrial electrical remodeling .
although we could not deny the fact that sodium pentobarbital has been reported vagolytic and may increase the erp value when applying,29 , 30 the effect of a803467 on erp in the present study is still established , since both groups were treated equally with the anesthetic to eliminate bias between groups , and the minimum dosage was used to reduce the possibility of unnecessary influence , as in our previously published studies.18 , 26 , 31 , 32 
  scherlag et  al33 injected lidocaine into gps , leading to the loss of af inducibility in 6 of 7 dogs .
our results displayed an apparent inhibition with 25.0% af incidence when applying a803467 , compared with 87.5% in the control group ( odds ratio=21.00 ) .
as a class i antiarrhythmic drug and local anesthetic , lidocaine proved to have a pronounced effect in inhibiting nav1.8 current , enhancing udb , and regulating gating properties.34 a803467 , as a selective nav1.8 blocker , is used in alleviating nav1.8involved neuropathic pain . in recombinant hek293 cells , a803467 was blocked human nav1.8 ( ic50=8  nmol / l ) , and was > 100fold selective versus human nav1.2 , nav1.3 , nav1.5 , and nav1.7 ( ic50 > 1  mol / l).35 in the present study , tsa201 cells were transfected with scn5ascn10ascn3b , a803467 at 100 
nmol / l potently inhibited ina , p by 42.02% , and it suppressed more in ina , l by 68.57% .
gating properties were also changed under a803467 with hyperpolarized steadystate inactivation and delayed recovery from inactivation .
interestingly , the leftshifted steadystate inactivation was quite consistent with the effect of a803467 in isolated mouse intracardiac neurons delineated by verkerk et  al.36 like other sodium channel blockers , our results support that a803467 might block sodium channels in an inactivated state and the number of trapped inactivated sodium channels might be increased during repeated stimulation .
obvious delay of recovery was displayed as 2 times slower in f and more than 3 times in s .
the changes in gating properties , together with udb , indicate reduced availability of the sodium channel under sustained depolarization or repetitive stimulation , especially in a rapinduced af model . in the present study
, we utilized the scn5ascn10ascn3b cotransfected model to analyze the effect of a803467 on sodium current and gating properties to provide a possible explanation for the results we found in animal experiments . compared with scn5ascn3b transfected tsa201 cells ,
the current density was significantly increased and was sensitive to a803467 when scn10a were added together with scn5ascn3b .
since others ' and our studies have reported the extremely low current density in scn10ascn3b transfected cell lines,9 , 21 it is reasonable to deduce that the increased current is not an arithmetic addition of current component produced by scn10a .
it implies that scn10a might interact with scn5a as a new sodium complex ( figure  8) , and a803467 could have an effect on scn10ascn5a complex , as indicated in our patch clamp study .
although the complicated protein network of this new complex needs to be further explored , this model cells might be help us to understand the electrophysiological role of nav1.8 in arrhythmias .
our previous results demonstrated the association between nav1.8 and nav1.5 at the protein level when expressed separately ex  vivo.9 in the present study , a803467 have apparent effects on current density and gating kinetics when coexpressed sodium complex of scn10ascn5ascn3b , which indirectly show the functional interaction between the 2 alpha sodium units .
the detailed components of the complicated sodium complex in the cytoplasm need to be specifically explored .
given their proximity to one another , scn5a and scn10a may be subject to common regulatory mechanisms , such as transcriptional control by tbx3 and tbx5 .
many association studies have discovered the intensive relationship between scn10a variants and af phenotype or incidence .
in addition to the significant effect on gp , nav1.8 might directly influence the electrophysiological characteristics of cardiac tissue with suppressed trigger activity and reduced substrate during af ( figure  8) .
the decreased current in ina , p by a803467 might prolong erp in atrium by virtue of postrepolarization refractoriness and reduce excitability at the fast rate due to udb .
the significantly reduced ina , l seen in our study might suppress the incidence of early afterrepolarization during af .
yang et  al21 also showed that 30  nmol / l a803467 can remarkably reduce ina , l in mouse and rabbit myocytes with less effect on ina , p .
in addition , the inhibition of sodium channel by a803467 can reduce na in myocytes during repolarization , which may prevent the ca loading to inhibit delay after repolarization .
the suppressed trigger activity and reduced substrate could also be potential mechanisms for inhibiting af initiation and maintenance .
several independent loci of scn10a were indicated to increase pr and/or qrs intervals , which were regarded as intermediate phenotypes , suggesting the risk of conduction disease and arrhythmia susceptibility.37 , 38 there is an increased number of studies that focus on the relationship of scn10a / nav1.8 with cardiac diseases . in spite of several disputes such as the expression of nav1.8 in the heart,36 ,
our previous study first reported 17 putative pathogenic scn10a variants in 25 of 150 brugada syndrome probands with a positive proband yield of 16.7% , approaching our historical yield of 20.1% for scn5a .
subsequent studies from others also emphasize the importance of scn10a in both rare and common variants in brugada syndrome.9 one recent study concluded that rs6795970 might be associated with cardiac conduction abnormalities in patients with hypertrophic cardiomyopathy.40 therefore , it has come a long way as the  new cardiac sodium channel ,  and the exact roles of nav1.8 in arrhythmia still have a long way to go .
it can be considered as a novel target in understanding cardiac electrophysiology , and its selective blocker or other relative drug might be promising in scn10arelated arrhythmias .
we did not record gp activity directly in our rapinduced canine af model in the absence or presence of a803467 .
however , previous studies have demonstrated that blocking gp activity can inhibit rapinduced atrial electrical remodeling and af inducibility . after we administrated a803467 in gps , the shortening of the erp in all recording sites and the increase in erp dispersion and wov were all attenuated , potently indicating the suppressed gp activity under the effect of a803467 .
since neurotransmission is not static and circumscribed , the nav1.8 blockade within the gps on af can have effects throughout the cardiac neuraxis . due to the plasticity in the nervous system , based on present data , we can not deduce that the observed effects may be limited only to the gps .
meanwhile , our previous study showed the colocalization of nav1.5 and nav1.8 when we transfected their cdna separately into tsa201 cells .
it would be more desirable if we could demonstrate this relationship in canine gp directly .
however , our patch clamp study displayed that scn5a could evidently increase scn10a current in tsa201 cells , which can be blocked by a803467 .
we did not record gp activity directly in our rapinduced canine af model in the absence or presence of a803467 .
however , previous studies have demonstrated that blocking gp activity can inhibit rapinduced atrial electrical remodeling and af inducibility . after we administrated a803467 in gps , the shortening of the erp in all recording sites and the increase in erp dispersion and wov were all attenuated , potently indicating the suppressed gp activity under the effect of a803467 . since neurotransmission is not static and circumscribed , the nav1.8 blockade within the gps on af can have effects throughout the cardiac neuraxis . due to the plasticity in the nervous system , based on present data
, we can not deduce that the observed effects may be limited only to the gps .
meanwhile , our previous study showed the colocalization of nav1.5 and nav1.8 when we transfected their cdna separately into tsa201 cells .
it would be more desirable if we could demonstrate this relationship in canine gp directly .
however , our patch clamp study displayed that scn5a could evidently increase scn10a current in tsa201 cells , which can be blocked by a803467 .
in the present study , we found that the nav1.8 selective inhibitor could decrease the incidence of acute af with suppressed electrical remodeling of atrial and pv myocardium in a rapinduced af model .
nav1.8 could be a promising target in early af , as gp activity were more important in early electrical remodeling process of  af begets af  compared with progressed stages with prominent fibrosis and structural remodeling . in addition , we displayed that nav1.8 could probably interact with nav1.5 , which may form a novel functional complex affecting cardiomyocyte and ( or ) cardiac gp properties , as shown in figure  8 . and it could be supported by our electrophysiological study with noticeable changes in ina and gating kinetics under a803467 .
this study was supported by national natural science foundation of china ( no . 81670304 ) , china ; independent research project of wuhan university ( no .
2042016kf0138 ) , china ; national natural science foundation of china ( no . 81530011 ) , china ; and the masons of new york , florida , massachusetts , connecticut , maryland , wisconsin , and rhode island , usa .
 backgroundganglionated plexus have been developed as additional ablation targets to improve the outcome of atrial fibrillation ( af ) besides pulmonary vein isolation . 
 recent studies implicated an intimate relationship between neuronal sodium channel nav1.8 ( encoded by scn10a ) and af . 
 the underlying mechanism between nav1.8 and af remains unclear . 
 this study aimed to determine the role of nav1.8 in cardiac electrophysiology in an acute af model and explore possible therapeutic targets.methods and resultsimmunohistochemical study was used on canine cardiac ganglionated plexus . 
 both nav1.5 and nav1.8 were expressed in ganglionated plexus with canonical neuronal markers . 
 sixteen canines were randomly administered either saline or the nav1.8 blocker a803467 . 
 electrophysiological study was compared between the 2 groups before and after 6hour rapid atrial pacing . 
 compared with the control group , administration of a803467 decreased the incidence of af ( 87.5% versus 25.0% , p<0.05 ) , shortened af duration , and prolonged af cycle length . 
 a803467 also significantly suppressed the decrease in the effective refractory period and the increase in effective refractory period dispersion and cumulative window of vulnerability caused by rapid atrial pacing in all recording sites . 
 patch clamp study was performed under 100  nmol / l a803467 in tsa201 cells cotransfected with scn10awt , scn5awt , and scn3bwt . in 
 a , 
 p was reduced by 45.34% at 35  mv , and in 
 a , l by 68.57% at 20  mv . 
 evident fast inactivation , slow recovery , and usedependent block were also discovered after applying the drug.conclusionsour study demonstrates that nav1.8 could exert its effect on electrophysiological characteristics through cardiac  ganglionated plexus . 
 it indicates that nav1.8 is a novel target in understanding cardiac electrophysiology and scn10arelated arrhythmias . 
Introduction
Methods
Animal Preparation
Study Protocol
Tissue Preparation and Immunofluorescence
Coexpression of NaV1.5 and NaV1.8 and Patch Clamp Study
Statistics
Results
Na
Effect of A803467 on ERP and AF Inducibility in GPs
Characterization of A803467 at 100nmol/L on 
Discussion
Study Limitations
Conclusions
Sources of Funding
Disclosures