extracellular sodium dependence of the conduction velocity ...nov 05, 2015 · p and increases cv....

CALL FOR PAPERS Arrhythmias, Electrophysiology, and Optical Mapping

Extracellular sodium dependence of the conduction velocity-calciumrelationship: evidence of ephaptic self-attenuation

Sharon A. George,1 Mohammad Bonakdar,1,2 Michael Zeitz,3 Rafael V. Davalos,1,2 James W. Smyth,3

and Steven Poelzing1,3

1Department of Biomedical Engineering and Mechanics, Virginia Polytechnic Institute and State University, Blacksburg,Virginia; 2Department of Mechanical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia;and 3Virginia Tech Carilion Research Institute and Center for Heart and Regenerative Medicine, Roanoke, Virginia

Submitted 5 November 2015; accepted in final form 2 March 2016

George SA, Bonakdar M, Zeitz M, Davalos RV, Smyth JW,Poelzing S. Extracellular sodium dependence of the conduction ve-locity-calcium relationship: evidence of ephaptic self-attenuation. AmJ Physiol Heart Circ Physiol 310: H1129–H1139, 2016. First pub-lished March 4, 2016; doi:10.1152/ajpheart.00857.2015.—Our labo-ratory previously demonstrated that perfusate sodium and potassiumconcentrations can modulate cardiac conduction velocity (CV) con-sistent with theoretical predictions of ephaptic coupling (EpC). EpCdepends on the ionic currents and intercellular separation in sodiumchannel rich intercalated disk microdomains like the perinexus. Wesuggested that perinexal width (WP) correlates with changes in extra-cellular calcium ([Ca2�]o). Here, we test the hypothesis that increas-ing [Ca2�]o reduces WP and increases CV. Mathematical models ofEpC also predict that reducing WP can reduce sodium driving forceand CV by self-attenuation. Therefore, we further hypothesized thatreducing WP and extracellular sodium ([Na�]o) will reduce CVconsistent with ephaptic self-attenuation. Transmission electron mi-croscopy revealed that increasing [Ca2�]o (1 to 3.4 mM) significantlydecreased WP. Optically mapping wild-type (WT) (100% Cx43)mouse hearts demonstrated that increasing [Ca2�]o increases trans-verse CV during normonatremia (147.3 mM), but slows transverseCV during hyponatremia (120 mM). Additionally, CV in heterozy-gous (�50% Cx43) hearts was more sensitive to changes in [Ca2�]o

relative to WT during normonatremia. During hyponatremia, CVslowed in both WT and heterozygous hearts to the same extent.Importantly, neither [Ca2�]o nor [Na�]o altered Cx43 expression orphosphorylation determined by Western blotting, or gap junctionalresistance determined by electrical impedance spectroscopy. Narrow-ing WP, by increasing [Ca2�]o, increases CV consistent with en-hanced EpC between myocytes. Interestingly, during hyponatremia,reducing WP slowed CV, consistent with theoretical predictions ofephaptic self-attenuation. This study suggests that serum ion concen-trations may be an important determinant of cardiac disease expres-sion.

ion concentration; sodium; calcium; conduction; ephaptic coupling

NEW & NOTEWORTHY

This study is the first demonstration of perinexal width andconduction velocity modulation in hearts by varying extracel-lular calcium without measurable changes in gap junctional(GJ) coupling. Also, provided here is the first experimentalevidence for ephaptic self-attenuation, as predicted by mathe-

matical models that incorporate the effects of ephapticcoupling.

THE SYNCHRONIZED SPREAD OF electrical activity is important forcoordinating efficient cardiac contraction, and slowed conduc-tion is a well-established substrate for lethal cardiac arrhyth-mias. GJ coupling, tissue excitability, intercalated disk micro-architecture, and extracellular ion concentrations have beenidentified as important determinants of cardiac conduction (1,11, 19, 29, 46). Many studies have now demonstrated thatsimultaneously altering more than one determinant of cardiacconduction can synergistically modulate conduction velocity(CV) significantly more than even pathological alterations of asingle parameter (12, 42, 47, 48).

More interestingly, our laboratory recently determined thataltering perfusate composition within concentration rangesconsistent with murine plasma ionic concentrations producescomplex effects on CV that are interdependent on sodium,potassium, and functional expression of the principal ventric-ular gap junction protein connexin 43 (Cx43) (11, 12). Inprevious papers, our laboratory suggested that the effects areconsistent with the theory of ephaptic coupling (EpC), as wellas GJ coupling. EpC, which can facilitate electrical couplingbetween myocytes through electric fields that develop in re-stricted extracellular spaces like the perinexus (12, 47), isdependent on the volume of intercalated disk microdomains, aswell as rate of charge depletion from these regions (12, 26).Specifically, our laboratory previously demonstrated that widerperinexi, increased intercellular separation, with low extracel-lular sodium concentration ([Na�]o) and high extracellularpotassium concentration, both of which can reduce rate ofcharge depletion from the perinexus, was associated withslower impulse propagation through myocardium (12).

The perinexus has been suggested to be a candidate structureof a cardiac ephapse and an important modulator of CV (12,47). Specifically, previous studies demonstrated that changingtissue hydration in guinea pig with mannitol or albumin canalter bulk interstitial edema (48), and, in the case of mannitol,also increase perinexal width (WP) (47) to modulate cardiacconduction in a manner most consistent with the theoreticalpredictions of EpC. More recently, our laboratory found thatperfusate ion composition can also modulate WP and CV inmouse (12), and we speculated that increasing extracellularcalcium concentration ([Ca2�]o) might decrease WP, since

Address for reprint requests and other correspondence: S. Poelzing, VirginiaTech Carilion Research Institute, 2 Riverside Circle, Roanoke, VA 24016(e-mail: [email protected]).

Am J Physiol Heart Circ Physiol 310: H1129–H1139, 2016.First published March 4, 2016; doi:10.1152/ajpheart.00857.2015.

0363-6135/16 Copyright © 2016 the American Physiological Societyhttp://www.ajpheart.org H1129

by 10.220.32.247 on Septem

ber 26, 2017http://ajpheart.physiology.org/

Dow

nloaded from

mailto:[email protected]

http://ajpheart.physiology.org/

intercellular adhesion is calcium sensitive. Therefore, in thisstudy, we hypothesize that [Ca2�]o modulates WP.

Mathematical models of EpC also predict a phenomenontermed self-attenuation, which is similarly dependent on inter-cellular separation within the intercalated disk and extracellularion composition (22, 26, 28). More specifically, the mechanismof self-attenuation has been described as the process by whichsodium current (INa) attenuates itself by reducing driving forcewhen the cleft between myocytes is sufficiently narrow (22).This delays myocyte activation and manifests as macroscopicconduction slowing. The driving force for INa is the differencebetween the transmembrane potential (Vm) and the Nernstpotential for sodium (ENa). Either increasing Vm or decreasingENa can reduce the driving force and thereby INa. Therefore,two important components that are required to support self-attenuation are 1) reduced sodium driving force, and 2) re-duced width of intercalated disk spaces like the perinexus.Based on these predictions, we also hypothesize that decreas-ing WP and reducing sodium driving force slows cardiacconduction by a mechanism consistent with ephaptic self-attenuation.

The results of this study demonstrate that increasing [Ca2�]o

decreases WP. Furthermore, decreasing WP during normona-tremia increased CV, consistent with our laboratory’s previousstudies (12, 47), and decreasing WP during hyponatremiadecreased CV, consistent with computational predictions ofephaptic self-attenuation.

METHODS

All protocols were approved by the Institutional Animal Care andUse Committee at Virginia Polytechnic Institute and State Universityand conform to the guidelines of the National Institutes of HealthGuide for the Care and Usage of Laboratory Animals.

Langendorff Preparations

Mice were anesthetized by inhalation of isoflurane vapors from anisoflurane-soaked cotton gauze in a custom-designed closed chamber.Cervical dislocation was performed upon cessation of respiration andwas immediately followed by thoracotomy and excision of the heart.Wild-type (WT; 100% Cx43) and heterozygous (HZ; � 50% Cx43)mice, 10–30 wk old, on the C57BL/6 background were cannulatedand Langendorff perfused, as previously described (12), with solu-tions containing (in mM) 118.3 NaCl, 29 NaHCO3, 4.7 KCl, 1.4KH2PO4, 1 MgSO4, 10 glucose at pH 7.4. CaCl2 concentration wasvaried from 1 to 3.4 mM. The perfusate and the bath solution were thesame at any given time. The perfusion pressure was maintained at�70 mmHg, and the heart was suspended in a bath maintained at�37°C along with the perfusates. The process was repeated duringhyponatremia (NaCl 91 mM). The osmolarities of the various perfu-sates were measured using a Wescor VAPRO5520 Vapor PressureOsmometer and are reported in Table 1.

Optical Mapping

Hearts (8 solutions � 2 mouse types � 5/7 replicates, N � 96)were perfused with the voltage-sensitive dye, di-4-ANEPPS at aconcentration of 4 �M, and excess dye was washed out. Each heartwas serially perfused with four solutions of either increasing ordecreasing calcium concentration. Hearts were perfused with eachsolution for �8 min before being optically mapped. Motion wasreduced by 2,3-butanedionemonoxime (10 mM), and the heart wasstabilized against the front glass of the bath by applying slightpressure to the posterior surface. Hearts were paced using a unipolarsilver wire placed on the anterior surface and a reference wire at the

back of the bath. Stimuli of 1-V amplitude and 1-ms duration at abasic cycle length of 150 ms were applied. CV longitudinal (CVL) andtransverse (CVT), anisotropic ratio (AR), and action potential duration(APD) were quantified as previously described (12). Briefly, the heartwas excited by 510-nm light, and the excited light passed through a610-nm filter and then was recorded using the MiCam Ultima CMOSL-camera at a sampling rate of 1,000 frames/s. The maximum rate ofrise of the optical action potentials was assigned as activation timesand CV vectors were determined by fitting the activation times atevery pixel to a parabolic surface. Vectors up to 5 pixels away froma user-defined line indicating the direction of propagation, which fellwithin �3 or 5 mm (CVT and CVL, respectively) from the pacing site,not including the first 2 rows, and whose direction was not more than7.5° from the direction of propagation, were included in the analysis.This region is roughly illustrated by the orange and green boxes in theisochrones maps (see Figs. 2 and 4). APD was calculated as the timeinterval between activation and 90% repolarization.

Transmission Electron Microscopy

Hearts (7 solutions � 2 mouse types � 3 replicates � 15 images,N � 630) were perfused with the respective solution for 30 min, and1-mm3 cubes were then fixed overnight in 2.5% glutaraldehyde at4°C. Samples were washed in PBS and processed for transmissionelectron micrographs, as previously described (12). The samples werethen sectioned onto copper grids, and the sections were imaged at�150,000 magnification using a JEOL JEM 1400 transmission elec-tron microscope. Perinexal images were then analyzed in a blindedmanner by ImageJ to determine WP. It has to be noted here that theWP measurements in this study refer to the intermembrane separationadjacent to the GJ plaque as our laboratory previously reported inmice (12) and guinea pigs (47) and not the spatial extent (distancefrom the gap junction edge) of the perinexus, as reported by Rhett etal. (35). Additionally, based on our previous studies, WP changes inthe plateau portion of this nanodomain (30–105 nm away from theedge of the gap junction) best correlated with CV, and, therefore,values reported here are averages of six measurements at 15-nmintervals from within this region.

Western Immunoblotting

Hearts (4 solutions � 2 mouse types � triplicates run twice, N �48), perfused with the respective solutions for 30 min, were snapfrozen. Tissue was homogenized in a lysis buffer containing (in mM)50 HEPES, 150 KCl, 1 EDTA, 1 EGTA, 1 DTT, 1 NaF, 0.1 Na3VO4,and 0.5% Triton X-100. Protein concentration was normalized fol-lowing quantification with the Bio-Rad DC protein assay. SDS-PAGEelectrophoresis was performed as previously described (40) using4–20% NuPage Bis-Tris gels, which were then transferred to a PVDFmembrane and blocked with 5% BSA in TNT buffer (0.1% Tween 20,150 mM NaCl, 50 mM Tris, pH 8.0) at room temperature for 1 h.Membranes were then incubated with rabbit anti-phospho-Cx43Ser368

(1:1,000 in 5% BSA TNT, no. 3511 Cell Signaling Technology)primary antibody overnight at 4°C. Membranes were washed and

Table 1. Perfusate osmolarity

[Na�]o, mM [Ca2�]o, mM Osmolarity, mosM

147.3 1.0 304.3147.3 1.8 303.0147.3 2.6 301.3147.3 3.4 301.3120 1.0 250.3120 1.8 253.0120 2.6 250.3120 3.4 246.3

The osmolarity of the perfusates with varying [Na�]o and [Ca2�]o are listed.

H1130 CALCIUM AND SODIUM IN CARDIAC CONDUCTION

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00857.2015 • www.ajpheart.org

by 10.220.32.247 on Septem


Dow

nloaded from


incubated with goat anti-rabbit horseradish peroxidase secondaryantibody (1:5,000, Abcam) for 1 h at room temperature, washed, andbound antibody detected using Clarity Western ECL Substrate (Bio-Rad) and imaged using the Bio-Rad Chemidoc MP system. To detecttotal Cx43, membranes were first stripped with Re-Blot Plus Strong(Millipore), according to the manufacturer’s instructions. Followingblocking for 1 h at room temperature in 5% milk in TNT buffer,membranes were incubated with primary antibodies against Cx43(1:4,000, C6219 rabbit; Sigma Aldrich), and mouse anti-GAPDH(1:4,000 RDI-TRK5G4-6C5, mouse; Research Diagnostics) overnightat 4°C diluted in 5% milk TNT. Membranes were then washed andincubated with the secondary antibodies goat anti-mouse Alexa Fluor555 and goat anti-rabbit Alexa Fluor 647 (both 1:1,000 in milk TNT,Life Technologies) for 1 h at RT. Following several washes in TNT,membranes were imaged using the Bio-Rad Chemidoc MP System,and protein expression was quantified by densitometry using Image-Lab software (Bio-Rad). For quantification, all samples were runtogether on 26-well gels. Cx43 expression levels were normalized toGAPDH and pCx43 to total Cx43 to compare between samples.

Impedance Spectroscopy

GJ resistance (RGJ) was estimated using the four-point electrodetechnique as previously described in cardiac tissue (5, 16, 31, 38, 39, 43).The electrical impedance spectra in the mice hearts (4 solutions � 2mouse types � 5/3 replicates for WT/HZ, N � 64) were measured duringperfusion with different solutions. The four-point measurement techniquewas implemented to reduce the effect of electrode polarization andfacilitate measurement at low frequencies. Due to the small size of theheart, a custom-made electrode array with interelectrode distance of�200 �m was used. For each measurement, the electrodes penetratedto a depth of 500 �m from the heart surface in an orientationapproximately parallel to the epicardial fiber orientation. The rest ofthe electrode length was insulated to minimize the parasitic conduc-tivity path induced by the perfusion fluid on the heart exterior. TheGamry Instruments Interface 1000 was used in the galvanostatic modeto introduce a 500-�A alternating current between the two outerelectrodes, and the two inner electrodes measured the change involtage, over a range of 1 Hz to 1 MHz.

The resistance-reactance curves between 1 and 100 kHz, obtainedfrom these recordings, were fitted with a circle to determine an index of

RGJ, as previously described (2, 4–6). The x-intercepts of the circle weredetermined, as they correspond to the intracellular resistance (rightintercept) and the cytoplasmic resistance (left intercept). The differencebetween the two is reported as RGJ below. All resistance values arereported as percent change from control (solution containing, 147.3 mM[Na�]o and 1 mM [Ca2�]o), except for the comparison between WT andHZ RGJ, where absolute values were used.

Due to potential inconsistencies associated with anisotropic natureof the tissue and different fiber orientations between animals, data arecompared in a paired fashion within hearts to determine whetherimpedance spectroscopy can detect changes in RGJ due to perfusatecomposition. Unpaired comparisons between WT and HZ heart wereused to determine whether the measurement could detect a 50%reduction in Cx43 protein. Finally, positive control impedance esti-mates were made in hearts treated with 50 �M carbenoxolone (CBX)or subjected to 30 min of no-flow ischemia (ISC).

Statistical Analysis

All data are reported as means � SD, unless stated otherwise.Single-factor ANOVA was used to detect differences between WTand HZ WP. Two-tailed, equal sample size and variance, paired/unpaired Student’s t-test was performed to determine significantdifference in WP, CV, protein expression, as well as RGJ data.Bonferroni correction was applied for multiple comparisons.

RESULTS

Normonatremia-Perinexal Width

To investigate whether [Ca2�]o alters intercellular separa-tions within the intercalated disk, perfusate [Ca2�]o was in-creased from 1 to 3.4 mM, and WP was quantified fromtransmission electron micrographs (Fig. 1A) in hearts perfusedwith normonatremic solutions. WP was not significantly dif-ferent between WT and Cx43 HZ hearts at any [Ca2�]o, asrevealed by a single-factor ANOVA (P � 0.45), and allreported data and comparisons include both WT and HZ WP

values.Summary data demonstrate that WP was inversely correlated

to [Ca2�]o in the range of 1 to 3.4 mM (Fig. 1B). A single-

200 nm

1 3.42.61.8[Ca2+]o (mM)[Na+]o (mM) 147.3

WP (

nm)

*B

[Ca2+]o (mM)

A

WTHZ

1.0 1.8 2.6 3.40

10

20

30

40

50

Fig. 1. Extracellular calcium-perinexal width relation-ship during normonatremia. A: representative electronmicrographs of hearts perfused with varying [Ca2�]o.Perinexi are highlighted in yellow. B: summary data ofWP as a function of [Ca2�]o from WT (black) and HZhearts (gray). Means and SD are indicated by red lines.n � 3 � 15 images. *P � 0.05 between [Ca2�]o � 1 and3.4 mM by unpaired Student’s t-test.

H1131CALCIUM AND SODIUM IN CARDIAC CONDUCTION


by 10.220.32.247 on Septem


Dow

nloaded from


factor ANOVA reveals a significant relationship between[Ca2�]o and WP (P � 0.001). Post hoc comparison between 1and 3.4 mM [Ca2�]o reveals that WP is significantly narrowerat the higher calcium concentration (17.8 � 0.8 and 13.8 � 0.9nm, means � SE, respectively). Additionally, increasing[Ca2�]o from 1 to 1.8 mM visually appears to increase in WP;however, this was not significant (P � 0.252). Importantly, theWP values reported here are consistent with those observed inour laboratory’s previous study in mouse hearts exposed to asimilar range of [Ca2�]o (12).

Normonatremia-Conduction Velocity

Our laboratory previously demonstrated under conditions ofphysiological [Na�]o in mice as well as guinea pig hearts thatWP and CV are inversely correlated (12, 47). Specifically,decreasing WP was associated with faster CV. Representativeconduction isochrones in Fig. 2A demonstrate the response ofCV to increasing [Ca2�]o, decreasing WP. CVL and CVT

vectors were averaged from two regions each that are indicatedby green and orange boxes, respectively.

The relationship between CV, [Ca2�]o, and Cx43 expressionis complex, as illustrated in Fig. 2B. Increasing [Ca2�]o overthe range of 1–3.4 mM did not significantly alter CVL in eitherWT or HZ hearts. On the other hand, increasing [Ca2�]o overthe same range appears to sigmoidally modify CVT in WT

hearts. Specifically, increasing [Ca2�]o from 1 to 1.8 mM didnot significantly alter CVT. However, increasing [Ca2�]o to 2.6or 3.4 mM significantly increased CVT (0.22 � 0.03 to 0.28 �0.04 or 0.31 � 0.03 m/s, respectively) relative to 1 mM[Ca2�]o.

In HZ hearts, increasing [Ca2�]o over the range of 1–3.4mM only increased CVT for very low [Ca2�]o. Specifically,CVT significantly increased for [Ca2�]o raised from 1 to 1.8mM (0.21 � 0.03 and 0.27 � 0.03, respectively), but CVT wasnot different between [Ca2�]o of 1.8 and 3.4 mM (0.27 � 0.03,0.28 � 0.02, and 0.27 � 0.03 m/s). While summary data of ARvisually suggest that AR may decrease in both WT and HZhearts with increasing [Ca2�]o, this relationship was not sig-nificant.

Our laboratory’s previous studies have suggested that loss offunctional gap junctions increases CV sensitivity to cardiachydration. To explore the unique responses of CVT to [Ca2�]o,the relative change in CVT for WT and HZ hearts is comparedfor [Ca2�]o between 1 and 1.8, and 1 and 3.4 mM (Fig. 2C).The data reveal that CVT increases more in HZ hearts for[Ca2�]o between 1 and 1.8 mM. Interestingly, the difference isattenuated when comparing CVT for [Ca2�]o between 1 and3.4 mM, suggesting that CV is more sensitive to WP changesduring reduced Cx43 GJ coupling (12, 47). The sensitivity of

65

70

75

80

85

90

65

70

75

80

85

90

65

70

75

80

85

70

75

80

85

90

WT

70

75

80

85

90

1 mm

70

75

80

85

90

70

75

80

85

90

70

75

80

85

90

95

HZ0 ms

25

0

0.1

0.2

0.3

0.4

1 1.8 2.6 3.4

CV

T (m

/s)

0

0.2

0.4

0.6

1 1.8 2.6 3.4

CV L

(m/s

)

0

1

2

3

1 1.8 2.6 3.4

AR

[Ca2+]o (mM)

WTHZ4

1 3.42.61.8[Ca2+]o (mM)[Na+]o (mM) 147.3

WT HZ

A

B

C

*# #*#

ΔC

VT (%

)

WT HZ

*

0

20

40

60

1 - 1.8 mM [Ca2+]o

1 - 3.4 mM [Ca2+]o

0

20

40

60

0.8

Fig. 2. Extracellular calcium modulates conduction velocityduring normonatremia. A: representative activation maps ob-tained by optically mapping a single heart during the serialperfusion of solutions with increasing [Ca2�]o. Orange boxesto the top and bottom of the pacing site indicate the regionsfrom which CVT was averaged, and green boxes to the left andright indicate regions from which CVL was averaged. B:average CVT, CVL, and AR (anisotropic ratio) from WT (blackline) and HZ (gray line) hearts. C: percent change in CVT

between [Ca2�]o � 1–1.8 mM and 1–3.4 mM in WT and HZhearts demonstrates increased sensitivity of HZ hearts to WP inthe former and not the latter range. Values are means � SD;n � 5. P � 0.05 relative to [Ca2�]o � 1 mM in *WT and #HZby paired Student’s t-test.



by 10.220.32.247 on Septem


Dow

nloaded from


Cx43 HZ hearts to [Ca2�]o appears to be limited by a mech-anism preventing further CV increases even as WP decreases.

Hyponatremia-Perinexal Width

Interestingly, computational models of EpC predict that therelationship between CV and intercalated disk separation maybe biphasic, particularly when GJ coupling is reduced (22, 28).In short, for very narrow intercellular separations, conductioncan also slow by a process termed “self-attenuation.” To probewhether ephaptic self-attenuation can occur, WP was reducedin the setting of reduced sodium reversal potential and drivingforce (hyponatremia).

First, the structural relationship between WP-[Ca2�]o wasquantified in hearts perfused with a hyponatremic solution([Na�]o � 120 mM), and representative micrographs arepresented in Fig. 3A. Once again, a single-factor ANOVAreported significant differences in WP between solutions (P �0.05) and [Ca2�]o (1–3.4 mM, Fig. 3B). Further post hocanalysis revealed significantly wider WP at the lowest relativeto highest [Ca2�]o (19.4 � 0.6 vs. 17.2 � 0.6 nm, means �SE). Additionally, WP was not different between WT and HZhearts (P � 0.83) during hyponatremia as well.

Hyponatremia-Conduction Velocity

First, it is important to note that hyponatremia was associ-ated with significantly slower CVT relative to normonatremiain both WT and HZ hearts at all [Ca2�]o. Furthermore, repre-sentative isochrones maps in Fig. 4A suggest that increasing[Ca2�]o during hyponatremia now decreases CV, and thisrelationship is different from the normonatremic cases pre-sented in Fig. 2. Lastly, the isochrones suggest increasedlikelihood of conduction block into the left ventricle in bothWT and HZ hearts, particularly at the highest [Ca2�]o. Underthese conditions, CV was measured from the right ventricle.The number of hearts in which conduction block was observedis indicated above the isochrones in Fig. 4A, and, in these

hearts, CVL and CVT were averaged only in one direction each,as indicated by the green and orange box in Fig. 4A.

In WT hearts, increasing [Ca2�]o from 1 to 1.8 or 2.6 mMdid not significantly alter CVL, CVT, or AR. However, increas-ing [Ca2�]o from 1 to 3.4 mM slowed CVT (0.17 � 0.03 to0.12 � 0.03 m/s) and increased AR (1.6 � 0.2 vs 2.0 � 0.5)without measurably changing CVL. The finding that increasing[Ca2�]o during hyponatremia has the opposite effect on CVrelative to normonatremia is consistent with theoretical predic-tions of ephaptic self-attenuation. Specifically, CV increased inhearts with narrow perinexi and high ENa, but decreased inhearts with narrow perinexi and low ENa.

CV slowing was also observed in HZ hearts in response toincreasing [Ca2�]o during hyponatremia. Increasing [Ca2�]o

slowed CVT (0.17 � 0.02 to 0.11 � 0.03, 0.12 � 0.03 or0.12 � 0.03 m/s, respectively) without measurably alteringCVL or AR. Interestingly, hyponatremia did not produce asignificantly greater CVT response in HZ relative to WThearts at the lowest [Ca2�]o of 1 and 1.8 mM (Fig. 4C). Thisfinding is consistent with the predictions of mathematicalmodels that, in the self-attenuation range, the effect of GJcoupling on CV is reduced (22, 26, 28).

Extracellular Calcium and GJ Coupling

Cx43 expression and phosphorylation. Calcium ions are animportant component of several cell signaling pathways andcan modulate the functional states of proteins like Cx43 (17,27, 36). The effect of varying [Ca2�]o and [Na�]o on totalCx43 and phosphorylated Cx43 at Ser368 (pCx43) were quan-tified in this study to determine whether [Ca2�]o might altergap junction expression levels after 30 min of perfusion.Representative and summary data are illustrated in Fig. 5.

Perfusion with the extremes of the [Ca2�]o and [Na�]o usedin this study (Fig. 5A) did not alter the expression of total Cx43or pCx43 (Fig. 5, B and C). However, it must be noted that, inthe HZ hearts, increasing [Ca2�]o to 3.4 mM trended to

200 nm

[Ca2+]o (mM)[Na+]o (mM)A

B

WP (

nm)

WTHZ

[Ca2+]o (mM)

*

1.0 1.8 2.6 3.4

1 3.42.61.8120

0

10

20

30

40

50

Fig. 3. Extracellular calcium-perinexal width relationship dur-ing hyponatremia. A: representative electron micrographs ofhearts perfused with varying [Ca2�]o during hyponatremia.Perinexi are highlighted in yellow. B: summary data of WP asa function of [Ca2�]o from WT (black) and HZ hearts (gray).Means and SD are indicated by red lines. n � 3 � 15 images.*P � 0.05 between [Ca2�]o � 1 and 3.4 mM by unpairedStudent’s t-test.



by 10.220.32.247 on Septem


Dow

nloaded from


decrease total Cx43 expression at both high and low [Na�]o

(P � 0.080 and 0.094, respectively), but these changes did notreach statistical significance after correction for multiple com-parisons. As expected, HZ hearts expressed significantly lesstotal Cx43 expression (Fig. 5D, data normalized to GAPDH),but the ratio of pCx43 to total Cx43 was similar to that of WThearts (Fig. 5E, data normalized to total Cx43).

GJ resistance. Since protein levels do not necessarily cor-relate to its function, GJ coupling was estimated by tissueimpedance spectroscopy. Figure 6A shows a representativeresistance-reactance curve with the best circle fit. In Fig. 6B,the percent change in RGJ relative to control, during perfusionof 50 �M CBX and after 30 min of no-flow ISC, are reportedas positive controls. As expected, both CBX and ISC signifi-cantly increased RGJ relative to control. Furthermore, theincrease in RGJ during inhibition of Cx43 by CBX is consistentwith a previous study with guinea pig that reported a similarincrease in RGJ in the presence of CBX (5).

The percent change in RGJ relative to control, during perfu-sion of different solutions in WT and HZ hearts, are presentedin Fig. 6C. Varying [Na�]o and [Ca2�]o in the range used inthis study did not significantly change RGJ. As an additionalpositive control for demonstrating that impedance spectros-copy can detect loss of functional Cx43, as previously demon-strated (6), RGJ was compared between WT and HZ hearts.

Summary data in Fig. 6D demonstrate that RGJ in WT heartswas significantly lower relative to HZ, as expected.

The impedance data coupled with the immunoblotting sug-gest that the perfusates used in this study either did not alterCx43 functional expression, or altered it below the resolutionfor detection. Taken together, the data suggest that varying[Ca2�]o and [Na�]o in the specified range may not change GJcoupling, while these interventions did produce measureablechanges in cardiac conduction.

Action Potential Duration

Altering calcium concentration has been demonstrated tomodify the functioning of several ion channels like Nav1.5 (21,44) and the small-conductance calcium-activated potassiumchannels (52). Modulation of these ion channels can thenmanifest as changes in APD and morphology, and also alterCV. However, alterations in these proteins have been reportedfor much larger [Ca2�]o, and the effect of small variations, asused in this study, has not been explored. Therefore, wequantified APD in WT and HZ hearts perfused with 1 and 3.4mM [Ca2�]o solutions.

Representative action potentials from the same heart per-fused with 1 and 3.4 mM [Ca2�]o and 120 mM [Na�]o arepresented in Fig. 7A and the summary of APD in Fig. 7B.Importantly, APD did not significantly change in response to

1 1.8 2.6 3.40

1

2

3

4

55

60

65

70

75

80

85

90

95

100

105

110

55

60

65

70

75

80

85

90

95

100

105

110

55

60

65

70

75

80

85

90

95

100

105

110

55

60

65

70

75

80

85

90

95

100

105

110

60

65

70

75

80

85

90

95

100

105

110

60

65

70

75

80

85

90

95

100

105

110

60

65

70

75

80

85

90

95

100

105

110

60

65

70

75

80

85

90

95

100

105

110

1mm

0 ms

55

1 3.42.61.8[Ca2+]o (mM)

WT

HZ

CV

T (m

/s)

CV

L (m

/s)

AR

[Ca2+]o (mM)

WTHZ

[Na+]o (mM) 120

C

B

1 1.8 2.6 3.40

0.1

0.2

0.3

0.4# *#

1 1.8 2.6 3.40

0.2

0.4

0.6

5/4 4/4 4/2 5/4

*# *

A

Conduction Block (WT/HZ)

WT HZ

ΔC

VT (%

)

-60

1 - 1.8 mM [Ca2+]o

-80

-40

-20

0

0.8

Fig. 4. Extracellular calcium modulates conduction velocityduring hyponatremia. A: representative activation maps ob-tained by optically mapping a single heart during the serialperfusion of solutions with increasing [Ca2�]o during hypona-tremia. The orange box to the top of the pacing site indicatesthe region from which CVT was averaged, and the green boxto the left indicates the region from which CVL was averagedin hearts with CV block over the left ventricle. B: averageCVT, CVL, and AR (anisotropic ratio) from WT (black line)and HZ hearts (gray line). C: percent change in CVT between[Ca2�]o � 1 and 1.8 mM in WT and HZ hearts. Values aremeans � SD; n � 5. P � 0.05 relative to [Ca2�]o � 1 mM in*WT and #HZ by paired Student’s t-test.



by 10.220.32.247 on Septem


Dow

nloaded from


[Ca2�]o or [Na�]o, as determined from a single-factorANOVA (P � 0.9 and 0.2 for WT and HZ, respectively).Additionally, Fig. 7B also demonstrates that Cx43 expressionlevels do not significantly alter APD for any concentration of[Ca2�]o or [Na�]o.

DISCUSSION

The results of this study demonstrate that [Ca2�]o canmodulate WP and CV as hypothesized. More specifically,increasing [Ca2�]o from 1 to 3.4 mM reduces WP during bothnormonatremia as well as hyponatremia. However, the rela-tionship between [Ca2�]o and CV is more complex and depen-dent on [Na�]o. Specifically, increasing [Ca2�]o can increaseCV during normonatremia and decrease CV during hyponatre-mia. Taken together, these findings suggest that [Ca2�]o mod-ulates WP, which modulates CV differently during normona-tremia and hyponatremia by a mechanism that is not gapjunction dependent.

Extracellular Calcium and Perinexal Width

At several points along the intercalated disk, transmembraneproteins that form gap junctions, desmosomes, or adherensjunctions bind to their counterparts on the apposing cell mem-brane, structurally holding the two membranes together (24).Several proteins that compose these junctions like N-cadherins,desmocollin, and desmoglein, among others, have calcium-dependent adhesion properties (3, 49, 51). In the absence ofcalcium or during hypocalcemia, the binding affinity of these

proteins could be reduced, making these junctions looser pointsof contact, which in turn results in greater separation betweenthe membranes at the intercalated disk (10, 14).

Varying [Ca2�]o in this study resulted in WP modulation.Specifically, increasing calcium ion concentration was associ-ated with narrower perinexi or, in other words, WP and [Ca2�]o

were inversely correlated. However, this correlation wasweaker during hyponatremia.

Ephaptic Coupling

Cellular excitation of cardiac myocytes occurs when trans-membrane potential (Vm), which is the difference between theintracellular and extracellular potentials (Vm � i o),depolarizes the cell sufficiently to activate voltage-gated so-dium channels. The postjunctional cell Vm can rise by eitherdecreasing o or increasing i. The most commonly acceptedmethod for raising postjunctional Vm is by electrotonicallyincreasing i via gap junctions. On the other hand, o can bealtered by the depletion or accumulation of charge in theextracellular space caused by, for example, withdrawal ofsodium or increased potassium in intercellular clefts during theactivation of the prejunctional cell. Such a non-GJ, nonsynapticform of electrical coupling between myocytes is what is termedas EpC, and several more mechanisms for EpC have beenmathematically proposed (41).

One important requirement for EpC is the presence ofmicrodomains with closely abutting cell membranes. Severalsuch intercalated disk structures like the perinexus (47) and

pCx4

3/C

x43

(AU

)

Cx4

3/G

APD

H (A

U)

[Ca2+]o (mM)[Na+]o (mM)Solution 1

120432

147.3 147.3 1203.4 3.41 1

1 432 1 432

WT HZ

Cx4

3/G

APD

H (A

U)

WT HZ

GAPDH

Cx43

Cx43-pSer368

A

B

D

*

WT HZ

WT HZ

E

pCx4

3/C

x43

(AU

)

WT HZ

70

0

140

70

0

140

80

40

120

0

140

80

40

120

0

140

C

Fig. 5. Cx43 expression during variation in [Na�]o and[Ca2�]o. A: upper and lower limits of the range over which[Na�]o and [Ca2�]o were varied in this study and the colorcode for the panels below. B: representative Western immuno-blots of total and pSer368 Cx43. C: total Cx43 normalized toGAPDH (left) and pCx43 normalized to total Cx43 (right)illustrate that varying [Na�]o and [Ca2�]o do not significantlyalter Cx43 expression. D: total Cx43 is significantly reducedin HZ hearts relative to WT. E: the ratio of pCx43 to totalCx43 is not different between WT and HZ hearts. Values aremeans � SD; n � 3. *P � 0.05 by unpaired Student’s t-test.AU, arbitrary units.



by 10.220.32.247 on Septem


Dow

nloaded from


connexome (23) have been proposed as possible cardiac ep-hapses with volumes that can be modulated. The study byVeeraraghavan et al. (47) also identified the consequences thatWP modulation has on CV by pharmacological as well asmathematical methods. Specifically, narrower perinexi pro-mote faster transmission of electrical impulses from the pre- tothe postjunctional cell due to closer spacing of the two mem-branes. Thus increasing [Ca2�]o, as we did in this study, couldincrease CV in normonatremic hearts by narrowing perinexiand improving EpC between cells consistent with our laborat-ory’s previous works (12, 47).

In this study, transverse CV was more sensitive to experi-mental interventions that altered WP and sodium driving force.To understand this finding, it is important to consider that gapjunctional uncoupling preferentially alters transverse CV, be-cause the propagating wavefront in the transverse directionencounters more GJs per unit length relative to the longitudinaldirection. Parallel to this concept, perinexi, which are theoret-ical EpC junctions, are also localized to the intercalated disk.Therefore, a wavefront propagating transverse to fibers willencounter more gap and ephaptic junctions relative to thelongitudinal direction of propagation. In short, inhibiting EpCis expected to likewise preferentially affect transverse CV.

Self-attenuation

Self-attenuation was previously suggested in mathematicalmodels of cardiac conduction incorporating extracellular fieldeffects as well as a polarized voltage-gated sodium ion channeldistribution to the intercalated disk, both of which are essentialto support EpC (22, 28). Self-attenuation was described as theprocess by which peak INa attenuates itself due to reduced cleftwidth and INa driving force. The INa driving force, as describedabove, can be reduced by either increasing Vm or decreasingENa. While two models (22, 26) predict self-attenuation solelybased on changes in Vm, the Peskin (28) model additionally

tracks ion concentrations and Nernst potentials. Therefore, themodels that incorporate both Vm overshoot and changes in ENa

within the intercalated disk may be more sensitive to ephapticself-attenuation (28).

In the present study, cleft width was reduced by increasing[Ca2�]o, and sodium driving force was reduced by reducing[Na�]o. In the case of the high [Na�]o solutions, where sodiumdriving force was relatively larger, altering WP alone increasedCV, consistent with our previous work in guinea pigs and mice,possibly due to modulation of EpC outside of the self-attenu-ation range reported by mathematical models. Importantly, this

0

20

40

60

80

100

100 200 300 400 500 600 700Resistance (Rs, Ω)

-Rea

ctan

ce (-

Xs, Ω

)

Rc Ri

100 Hz1 MHz

ΔR

GJ (

%)

0

20

40

60

80

CBX ISC

ΔR

GJ (

% o

f Sol

n. 1

)

-100

-50

0

50

100

0

200

400

600

800

RG

J (Ω

)

WT HZ

A B

C D

*

*

*WT HZ

[Ca2+]o (mM)[Na+]o (mM)Solution

120432

147.3 1203.4 3.41

1147.3

1120

432147.3 120

3.4 3.41

100

Fig. 6. Tissue impedance during variation in [Na�]o and[Ca2�]o. A: representative resistance (Rs)-reactance (Xs)curve with circle fit. B: percent change (�) in gap junctionalresistance (RGJ) increases after treatment with 50 �M car-benoxolone (CBX) and 30 min of ischemia (ISC). C: aver-age RGJ values determined during perfusion of solutionswith different [Na�]o and [Ca2�]o. D: RGJ resistance issignificantly increased in HZ hearts relative to WT. Valuesare means � SD. *P � 0.05 by paired Student’s t-test in Band C, and unpaired Student’s t-test in D.

[Ca2+]o=1mM[Ca2+]o=3.4mM

25 ms

147.3 [Na+]o (mM)120 147.3 120

WT HZ

0

20

40

60

80

APD

(ms)

A

B

Fig. 7. Action potential duration (APD) not affected by [Na�]o, [Ca2�]o, orCx43 expression. A: representative action potentials from the same heartperfused with [Ca2�]o � 1 mM (black) and 3.4 mM (gray). B: neither [Na�]o,[Ca2�]o, nor Cx43 expression alter mean epicardial APD.



by 10.220.32.247 on Septem


Dow

nloaded from


study for the first time identifies that, in the setting of reducedsodium driving force (low [Na�]o solutions), the relationshipbetween [Ca2�]o, WP, and CV is consistent with self-attenua-tion. Specifically, our data are consistent with the hypothesisthat reducing WP during reduced [Na�]o further reduces avail-able [Na�]o in the perinexus during sodium-mediated depolar-ization. Under these conditions, depolarization of the prejunc-tional cell withdraws a large number of available sodium ionsfrom the intercalated disk microdomain. This charge with-drawal greatly reduces the INa driving force for the postjunc-tional cell. The reduced driving force then delays propagationof the electrical impulse from the pre- to the postjunctionalmyocyte, resulting in CV slowing.

It is difficult to compare the results of this work withprevious studies investigating the relationship between CV and[Ca2�]o due to significant experimental differences. For exam-ple, elevating [Ca2�]o from 1 to 2 mM did not alter CV, whileraising [Ca2�]o from 2 to 8 mM significantly slowed CV infalse tendons isolated from dog ventricles (33). Another studyreported nonsignificant changes in CVL and CVT in dogpapillary muscle when [Ca2�]o was varied similar to concen-trations used in our study (45). Therefore, while previousstudies in different animal models, tissue preparations, andwith different perfusate compositions demonstrated that rais-ing [Ca2�]o within some narrow window can decrease or notchange CV, to our knowledge this is the first study todemonstrate that CV and [Ca2�]o can positively or nega-tively correlate, depending on [Na�]o, in a manner consis-tent with the mathematical predictions of ephaptic self-attenuation (22, 26, 28).

GJ Coupling

Calcium ions are involved in the regulation of severalphysiological functions and play a key role in various signalingpathways (55). Intracellular calcium homeostasis is tightlyregulated by a highly developed system that involves severalintracellular calcium stores (9, 20). However, during certainpathophysiological states, intracellular calcium increases,which then results in modulation of several factors that canthen alter CV (7). For example, intracellular calcium concen-tration has been shown to modulate GJ coupling (17, 27, 36).Elevated [Ca2�]i alters Cx43 gating by a Ca2�/calmodulin-dependent pathway and reduces intercellular coupling (54).Cx43 uncoupling has been associated with slow CV when theextracellular ionic composition is altered to weaken EpC (8,12, 15, 19). However, this study demonstrates that modulating[Ca2�]o (1–3.4 mM) does not change either total or phosphor-ylated Cx43 expression during normonatremia as well as hy-ponatremia. Additionally, varying [Ca2�]o did not alter RGJ,and increasing [Ca2�]o was also associated with faster CVduring normonatremia. Furthermore, the changes in electro-physiology were measured at a much earlier time point (8 min)compared with Cx43 expression measurements (30 min).These findings suggest that GJ coupling may not be themechanism by which [Ca2�]o modulates CV in this study.

Limitations

Modulating [Ca2�]o could have several physiological impli-cations in the heart, which includes altered contraction, cal-cium handling, cell signaling pathways, and apoptosis (55).

[Ca2�]o was varied from 1 to 3.4 mM in this study, which is arange wider than the normal physiological [Ca2�]o, whichtypically falls between 1.7 and 2.5 mM in mice (34). Previousstudies investigating the effects of hypercalcemia have re-ported significant changes in GJ coupling at much higher[Ca2�]o (�5 mM) (27, 53). Such [Ca2�]o increases could alsoalter ionic currents, which could in turn modulate CV (21, 44,52), even though changes in APD were not significantlydifferent between interventions.

The perinexus has been identified as the site of denselocalization of several ion channels, and it is possible thatdownregulation of Cx43 in HZ mice could affect the structureof the perinexus. First, this study demonstrates that intercellu-lar separation within the perinexus (WP) is not different be-tween WT and HZ hearts for any intervention. Furthermore, ithas been previously demonstrated in the same mouse modelthat the structure of GJ plaques in HZ mice was not differentcompared with WT, although the number of GJ plaques wasreduced (37). This suggests that at least the GJ plaque andintercellular separation are conserved, even when GJs aregenetically reduced. However, this does not exclude the pos-sibility that the spatial extent of the perinexus, distance fromthe GJ edge, is altered in HZ animals, and this requires furtherinvestigation, specifically of the three-dimensional structure ofthe perinexus.

Finally, application of the four-electrode tissue impedancespectroscopy technique in whole heart preparations to estimateGJ coupling has many limitations based on anisotropic cellsize, space constant, and extracellular conductance. Thesefactors can significantly confound estimates of RGJ (18).Therefore, RGJ in this paper should be interpreted as an indexof GJ coupling, and not an absolute estimate of gap junctionconductance. Importantly, recent studies by Drs. Pollard andBarr demonstrated that impedance spectroscopy can well esti-mate RGJ in cardiac tissue when electrode spacing and diam-eters fall below the space constant for cardiac tissue (32, 50).Specifically, we used electrodes made with 180-�m-diameterKingli acupuncture needles with 200 �M spacing betweenelectrodes and an exposed needle depth of 500 �M. Further-more, estimates of RGJ that are compared in this study aremade between recordings obtained from the same electrodeposition in the same tissue (except WT to HZ) to minimize theaforementioned confounding variables.

Conclusions

Extracellular ion concentration is a crucial component inmodulating cardiac conduction. This study identifies that ex-tracellular sodium and calcium composition could differentlymodulate CV during health and conditions like hyponatremiacommonly associated with several diseases that include con-gestive heart failure (30), kidney (25), and liver (13) disorders.Specifically, while reducing WP by increasing [Ca2�]o wasbeneficial and increased CV during normonatremia, it slowedCV during hyponatremia. Furthermore, the changes in CVreported in this study were independent of Cx43 protein ex-pression, phosphorylation, or RGJ, suggesting the role of anon-GJ-mediated mechanism for CV modulation like EpC.

These data reinforce the importance of regulating serum ionconcentration during health and, more importantly, duringdisease. Furthermore, there is now mounting evidence that



by 10.220.32.247 on Septem


Dow

nloaded from


modulating parameters predicted to alter EpC can be used as atool in the treatment of conduction-based disorders in the heart.

ACKNOWLEDGMENTS

We thank Dr. Robert G. Gourdie for assistance in determining tissuemorphology from electron microscopy images.

GRANTS

This work was supported by an R01-HL-102298 awarded to S. Poelzing,and a Virginia Tech Carilion Research Institute Medical Research ScholarAward, an American Heart Association Predoctoral fellowship, and the DavidW. Francis and Lillian Francis Scholarship Fund awarded to S. A. George. R.Davalos acknowledges the support from the Institute for Critical Technologiesand Applied Sciences of Virginia Tech.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: S.A.G., M.B., M.Z., R.D., J.W.S., and S.P. concep-tion and design of research; S.A.G., M.B., M.Z., and J.W.S. performedexperiments; S.A.G., M.B., M.Z., R.D., and J.W.S. analyzed data; S.A.G.,M.B., M.Z., R.D., J.W.S., and S.P. interpreted results of experiments; S.A.G.,M.B., M.Z., J.W.S., and S.P. prepared figures; S.A.G., M.B., M.Z., J.W.S., andS.P. drafted manuscript; S.A.G., M.B., M.Z., R.D., J.W.S., and S.P. edited andrevised manuscript; S.A.G., M.B., M.Z., R.D., J.W.S., and S.P. approved finalversion of manuscript.

REFERENCES

1. Ballantyne F 3rd, Davis LD, Reynolds EW Jr. Cellular basis forreversal of hyperkalemic electrocardiographic changes by sodium. Am JPhysiol 229: 935–940, 1975.

2. Chapman RA, Fry CH. An analysis of the cable properties of frogventricular myocardium. J Physiol 283: 263–282, 1978.

3. Chitaev NA, Troyanovsky SM. Direct Ca2�-dependent heterophilicinteraction between desmosomal cadherins, desmoglein, and desmocollin,contributes to cell-cell adhesion. J Cell Biol 138: 193–201, 1997.

4. Cooklin M, Wallis WR, Sheridan DJ, Fry CH. Changes in cell-to-cellelectrical coupling associated with left ventricular hypertrophy. Circ Res80: 765–771, 1997.

5. Dhillon PS, Chowdhury RA, Patel PM, Jabr R, Momin AU, Vecht J,Gray R, Shipolini A, Fry CH, Peters NS. Relationship between connexinexpression, and gap-junction resistivity in human atrial myocardium. CircArrhythm Electrophysiol 7: 321–329, 2014.

6. Dhillon PS, Gray R, Kojodjojo P, Jabr R, Chowdhury R, Fry CH,Peters NS. Relationship between gap-junctional conductance, and con-duction velocity in mammalian myocardium. Circ Arrhythm Electro-physiol 6: 1208–1214, 2013.

7. Di Diego JM, Antzelevitch C. High [Ca2�]o-induced electrical heteroge-neity, and extrasystolic activity in isolated canine ventricular epicardium.Phase 2 reentry. Circulation 89: 1839–1850, 1994.

8. Eloff BC, Lerner DL, Yamada KA, Schuessler RB, Saffitz JE, Rosen-baum DS. High resolution optical mapping reveals conduction slowing inconnexin43 deficient mice. Cardiovasc Res 51: 681–690, 2001.

9. Fearnley CJ, Roderick HL, Bootman MD. Calcium signaling in cardiacmyocytes. Cold Spring Harb Perspect Biol 3: a004242, 2011.

10. Ganote CE, Grinwald PM, Nayler WG. 2,4-Dinitrophenol (DNP)-induced injury in calcium-free hearts. J Mol Cell Cardiol 16: 547–557,1984.

11. George SA, Poelzing S. Cardiac conduction in isolated hearts of geneti-cally modified mice–connexin43 and salts. Prog Biophys Mol Biol 120:189–198, 2016.

12. George SA, Sciuto KJ, Lin J, Salama ME, Keener JP, Gourdie RG,Poelzing S. Extracellular sodium, and potassium levels modulate cardiacconduction in mice heterozygous null for the Connexin43 gene. PflugersArch 467: 2287–2297, 2015.

13. Gines P, Guevara M. Hyponatremia in cirrhosis: pathogenesis, clinicalsignificance, and management. Hepatology 48: 1002–1010, 2008.

14. Greve G, Rotevatn S, Saetersdal T, Oksendal AN, Jynge P. Ultrastruc-tural studies of intercalated disc separations in the rat heart during thecalcium paradox. Res Exp Med (Berl) 185: 195–206, 1985.

15. Guerrero PA, Schuessler RB, Davis LM, Beyer EC, Johnson CM,Yamada KA, Saffitz JE. Slow ventricular conduction in mice heterozy-gous for a connexin43 null mutation. J Clin Invest 99: 1991–1998, 1997.

16. Jain SK, Schuessler RB, Saffitz JE. Mechanisms of delayed electricaluncoupling induced by ischemic preconditioning. Circ Res 92: 1138–1144, 2003.

17. Kagiyama Y, Hill JL, Gettes LS. Interaction of acidosis, and increasedextracellular potassium on action potential characteristics and conductionin guinea pig ventricular muscle. Circ Res 51: 614–623, 1982.

18. Kleber AG, Riegger CB. Electrical constants of arterially perfused rabbitpapillary muscle. J Physiol 385: 307–324, 1987.

19. Kleber AG, Rudy Y. Basic mechanisms of cardiac impulse propagation,and associated arrhythmias. Physiol Rev 84: 431–488, 2004.

20. Kohlhaas M, Maack C. Calcium release microdomains and mitochon-dria. Cardiovasc Res 98: 259–268, 2013.

21. Koval OM, Snyder JS, Wolf RM, Pavlovicz RE, Glynn P, Curran J,Leymaster ND, Dun W, Wright PJ, Cardona N, Qian L, Mitchell CC,Boyden PA, Binkley PF, Li C, Anderson ME, Mohler PJ, Hund TJ.Ca2�/calmodulin-dependent protein kinase II-based regulation of voltage-gated Na� channel in cardiac disease. Circulation 126: 2084–2094, 2012.

22. Kucera JP, Rohr S, Rudy Y. Localization of sodium channels inintercalated disks modulates cardiac conduction. Circ Res 91: 1176–1182,2002.

23. Leo-Macias A, Liang FX, Delmar M. Ultrastructure of the intercellularspace in adult murine ventricle revealed by quantitative tomographicelectron microscopy. Cardiovasc Res 107: 442–452, 2015.

24. Li J, Radice GL. A new perspective on intercalated disc organization:implications for heart disease. Dermatol Res Pract 2010: 207835, 2010.

25. Lin CS, Cheng CJ, Shih KC, Lin SH. Recurrent hyponatremia in apatient with chronic kidney disease. J Nephrol 19: 394–398, 2006.

26. Lin J, Keener JP. Microdomain effects on transverse cardiac propaga-tion. Biophys J 106: 925–931, 2014.

27. Maurer P, Weingart R. Cell pairs isolated from adult guinea pig and rathearts: effects of [Ca2�]i on nexal membrane resistance. Pflügers Arch409: 394–402, 1987.

28. Mori Y, Fishman GI, Peskin CS. Ephaptic conduction in a cardiac strandmodel with 3D electrodiffusion. Proc Natl Acad Sci U S A 105: 6463–6468, 2008.

29. Nygren A, Giles WR. Mathematical simulation of slowing of cardiacconduction velocity by elevated extracellular. Ann Biomed Eng 28: 951–957, 2000.

30. Oren RM. Hyponatremia in congestive heart failure. Am J Cardiol 95:2B–7B, 2005.

31. Padilla F, Garcia-Dorado D, Rodriguez-Sinovas A, Ruiz-Meana M,Inserte J, Soler-Soler J. Protection afforded by ischemic preconditioningis not mediated by effects on cell-to-cell electrical coupling duringmyocardial ischemia-reperfusion. Am J Physiol Heart Circ Physiol 285:H1909–H1916, 2003.

32. Pollard AE, Barr RC. A structural framework for interpretation offour-electrode microimpedance spectra in cardiac tissue. Conf Proc IEEEEng Med Biol Soc 2014: 6467–6470, 2014.

33. Pressler ML, Elharrar V, Bailey JC. Effects of extracellular calciumions, verapamil, and lanthanum on active and passive properties of caninecardiac purkinje fibers. Circ Res 51: 637–651, 1982.

34. Research Animal Resources University of Minnesota. Reference Valuesfor Laboratory Animals. Normal Hematology Values (Online). http://www.ahc.umn.edu/rar/refvalues.html [2013].

35. Rhett JM, Ongstad EL, Jourdan J, Gourdie RG. Cx43 associates withNa(v)1.5 in the cardiomyocyte perinexus. J Membr Biol 245: 411–422,2012.

36. Rudisuli A, Weingart R. Electrical properties of gap junction channels inguinea-pig ventricular cell pairs revealed by exposure to heptanol.Pflügers Arch 415: 12–21, 1989.

37. Saffitz JE, Green KG, Kraft WJ, Schechtman KB, Yamada KA.Effects of diminished expression of connexin43 on gap junction number,and size in ventricular myocardium. Am J Physiol Heart Circ Physiol 278:H1662–H1670, 2000.

38. Sanchez JA, Rodriguez-Sinovas A, Fernandez-Sanz C, Ruiz-MeanaM, Garcia-Dorado D. Effects of a reduction in the number of gapjunction channels or in their conductance on ischemia-reperfusion arrhyth-mias in isolated mouse hearts. Am J Physiol Heart Circ Physiol 301:H2442–H2453, 2011.

39. Smith WT, Fleet WF, Johnson TA, Engle CL, Cascio WE. The Ibphase of ventricular arrhythmias in ischemic in situ porcine heart is related



by 10.220.32.247 on Septem


Dow

nloaded from

http://www.ahc.umn.edu/rar/refvalues.html

http://www.ahc.umn.edu/rar/refvalues.html


to changes in cell-to-cell electrical coupling. Experimental CardiologyGroup, University of North Carolina. Circulation 92: 3051–3060, 1995.

40. Smyth JW, Hong TT, Gao D, Vogan JM, Jensen BC, Fong TS,Simpson PC, Stainier DY, Chi NC, Shaw RM. Limited forward traf-ficking of connexin 43 reduces cell-cell coupling in stressed human, andmouse myocardium. J Clin Invest 120: 266–279, 2010.

41. Sperelakis N. An electric field mechanism for transmission of excitationbetween myocardial cells. Circ Res 91: 985–987, 2002.

42. Stein M, van Veen TA, Remme CA, Boulaksil M, Noorman M, vanStuijvenberg L, van der Nagel R, Bezzina CR, Hauer RN, de BakkerJM, van Rijen HV. Combined reduction of intercellular coupling andmembrane excitability differentially affects transverse and longitudinalcardiac conduction. Cardiovasc Res 83: 52–60, 2009.

43. Stiles DK, Oakley BA. Four-point electrode measurement of impedancein the vicinity of bovine aorta for quasi-static frequencies. Bioelectromag-netics 26: 54–58, 2005.

44. Tan HL, Kupershmidt S, Zhang R, Stepanovic S, Roden DM, WildeAA, Anderson ME, Balser JR. A calcium sensor in the sodium channelmodulates cardiac excitability. Nature 415: 442–447, 2002.

45. Veenstra RD, Joyner RW, Rawling DA. Purkinje and ventricular acti-vation sequences of canine papillary muscle. Effects of quinidine andcalcium on the Purkinje-ventricular conduction delay. Circ Res 54: 500–515, 1984.

46. Veeraraghavan R, Gourdie RG, Poelzing S. Mechanisms of cardiacconduction: a history of revisions. Am J Physiol Heart Circ Physiol 306:H619–H627, 2014.

47. Veeraraghavan R, Lin J, Hoeker G, Keener J, Gourdie R, Poelzing S.Sodium channels in the Cx43 gap junction perinexus may constitute a

cardiac ephapse: an experimental and modeling study. Pflugers Arch 467:2093–2105, 2015.

48. Veeraraghavan R, Salama ME, Poelzing S. Interstitial volume modu-lates the conduction velocity-gap junction relationship. Am J PhysiolHeart Circ Physiol 302: H278–H286, 2012.

49. Vleminckx K, Kemler R. Cadherins, and tissue formation: integratingadhesion and signaling. Bioessays 21: 211–220, 1999.

50. Waits CM, Barr RC, Pollard AE. Sensor spacing affects the tissueimpedance spectra of rabbit ventricular epicardium. Am J Physiol HeartCirc Physiol 306: H1660–H1668, 2014.

51. Wallis S, Lloyd S, Wise I, Ireland G, Fleming TP, Garrod D. The alphaisoform of protein kinase C is involved in signaling the response ofdesmosomes to wounding in cultured epithelial cells. Mol Biol Cell 11:1077–1092, 2000.

52. Wang H, Li T, Zhang L, Yang Y, Zeng XR. [Effects of intracellularcalcium alteration on SK currents in atrial cardiomyocytes from patientswith atrial fibrillation]. Zhongguo Ying Yong Sheng Li Xue Za Zhi 30:296–300, 305, 2014.

53. Weingart R. The actions of ouabain on intercellular coupling, andconduction velocity in mammalian ventricular muscle. J Physiol 264:341–365, 1977.

54. Xu Q, Kopp RF, Chen Y, Yang JJ, Roe MW, Veenstra RD. Gating ofconnexin 43 gap junctions by a cytoplasmic loop calmodulin bindingdomain. Am J Physiol Cell Physiol 302: C1548–C1556, 2012.

55. Zarain-Herzberg A, Fragoso-Medina J, Estrada-Aviles R. Calcium-regulated transcriptional pathways in the normal and pathologic heart.IUBMB Life 63: 847–855, 2011.



by 10.220.32.247 on Septem


Dow

nloaded from


extracellular sodium dependence of the conduction velocity ...nov 05, 2015 · p and increases cv....

Documents