Pacemaker activity and ionic currents in mouse

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It is well established that pacemaker activity of the sino-atrial node (saN) initiates the heartbeat. .... along the crista terminalis and then extending in the posterior part of the lumen of the ...... ventricular tachycardia after targeted disruption of the.

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Research paper

Channels 5:3, 1-10; May/June 2011; © 2011 Landes Bioscience

Pacemaker activity and ionic currents in mouse atrioventricular node cells This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.

Laurine Marger,1-3 Pietro Mesirca,1-3 Jacqueline Alig,4 Angelo Torrente,1-3 Stefan Dubel,1-3 Birgit Engeland,4 Sandra Kanani,1-3 Pierre Fontanaud,1b-3 Jörg Striessnig,5 Hee-Sup Shin,6 Dirk Isbrandt,4 Heimo Ehmke,7 Joël Nargeot1-3 and Matteo E. Mangoni1-3,* 1 CNRS; UMR-5203; Institut de Génomique Fonctionnelle; Département de Physiologie; 1bDépartement d’Endocrinologie; Montpellier, France; 2INSERM; U661; Montpellier, France; 3Universités de Montpellier 1and 2; UMR-5203; Montpellier, France; 4Center for Obstetrics and Pediatrics; and Center for Molecular Neurobiology; RG Experimental Neuropediatrics; 7Department of Vegetative Physiology and Pathophysiology; Center for Experimental Medicine; University Medical Center Hamburg-Eppendorf; Hamburg, Germany; 5Department of Pharmacology and Toxicology; Institute of Pharmacy; Center for Molecular Biosciences; University of Innsbruck; Innsbruck, Austria; 6 Center for Neural Science; Korea Institute of Science and Technology; Cheongryang, Seoul, Republic of Korea

Key words: atrioventricular node, sino-atrial node, pacemaker activity, ion channels, electrophysiology, conduction, heart rate, Ca 2+ channels, Na+ channels, f-channels, K+ channels Abbreviations: AVN, atrioventricular node; SAN, sino-atrial node; TTX, tetrodotoxin; INa, voltage-dependent Na+ current; ICa,L , L-type Ca 2+ current; ICa,T, T-type Ca 2+ current; If, hyperpolarization-activated f-current; IKr, delayed rectifier K+ current; Ito, transient outward K+ current

It is well established that pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of INa by tetrodotoxin (TTX) or of ICa,L by isradipine abolished AVNCs pacemaker activity. TTX-resistant (INar) and TTX-sensitive (INas) Na+ currents were recorded in mouse AVNCs, as well as T-(ICa,T) and L-type (ICa,L) Ca2+ currents. ICa,L density was lower than in SANCs (51%). The density of the hyperpolarizationactivated current, (If) and that of the fast component of the delayed rectifier current (IKr) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of If by 3 μM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for If in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K+ current (Ito). In contrast, no detectable slow component of the delayed rectifier current (IKs) could be recorded in AVNCs. The lower densities of If and ICa,L, as well as higher expression of IKr in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs.

©201 1L andesBi os c i enc e. Donotdi s t r i but e. Introduction The sino-atrial node (SAN) controls the heart rhythm and rate under physiological conditions.1 The AVN is also endowed with automaticity. It is generally accepted that the AVN drives the heartbeat as a “secondary” supraventricular pacemaker in case of SAN failure.2,3 Due to its pivotal role in cardiac conduction and as a secondary pacemaker, the structure and the function of the AVN have been extensively studied (recently reviewed in ref. 3). However, little is known about mechanisms underlying AVN automaticity. Some ion channels known to be involved in SAN pacemaker activity have been found in the AVN,4,5 but their importance in the generation of automaticity in AVNC has not been investigated in depth at the cellular level. Recently, the phenotypes of genetically-modified mouse strains in which genes coding for ion channels involved in cardiac automaticity have been described (reviewed in ref. 6). Genetically-modified

mice constitute a promising approach to understand the bases of AVN automaticity, but the electrophysiological properties of spontaneously active mouse AVN cells (AVNCs) have not been investigated directly and we have limited knowledge on the functional role of ionic currents in mouse AVNCs. The aim of the present study was to isolate spontaneously active mouse AVNCs to characterize the properties of AVNCs pacemaker activity, as well as to record the major ionic currents potentially involved in AVNCs automaticity. Mouse AVNCs displayed an intrinsically slower pacemaker activity than that of SANCs. In comparison to adult rabbit7 and mouse8 SANCs, automaticity of mouse AVNCs depended on INa. A higher density than in SANCs of the fast component of the delayed rectifier current (IKr ) also characterized mouse AVNCs. Densities of the L-type Ca 2+ currents (ICa,L) were lower in AVNCs than in SANCs. Mouse AVNCs expressed lower densities of the hyperpolarization-activated current (If ) than SANCs. If showed

*Correspondence to: Matteo E. Mangoni; Email: [email protected] Submitted: 02/18/11; Revised: 02/22/11; Accepted: 02/22/11 DOI: www.landesbioscience.com Channels

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Figure 1. (A) Whole SAN-AVN preparation stained with an anti-rat HCN4 antibody effectively showed the organization of HCN4 positive SANCs and AVNCs. HCN4 positive cells are visible as stretches of cell groups along the crista terminalis and then extending in the posterior part of the lumen of the inferior vena cava. The coronary sinus was cut open for clarity. Distribution of HCN4 immunoreactivity corresponds to the extension of the HCN4-positive conduction system recently described by Dobrzynski et al. in the rabbit AVN.10 (B) HCN4-positive spindle AVNCs can be seen in a close-up view of the AVN region shown in (A). Abbreviations: right atrium (RA), left atrium (LA), aorta (Ao), crista terminalis (CT); superior vena cava (SVC); inferior vena cava (IVC); coronary sinus (CS); Fossa ovalis (FO); atrioventricular node (AVN); sino-atrial node (SAN). (C) Morphology of myocytes isolated from the mouse AVN. Rod-shaped cells (middle part) and atrial cells (right part) displayed no pacemaker activity. Spindle shaped cells (left part) were beating and were named AVNCs in this study. Note the morphological similarity between the morphology of isolated AVNCs and HCN4 positive cells in (B).

also a more negative half-activation voltage than in SANCs. However, If inhibition eventually decreased the slope of the diastolic depolarization in AVNCs, indicating that f-channels contribute to automaticity of these cells. The lower expression of key inward ionic currents in AVN (namely If and ICa,L), together with an increased expression of IKr may explain, at least in part, the slower intrinsic pacing rate of AVNCs. We expect that our work will provide a useful methodological, electrophysiological and functional framework for improving the study of the mechanisms underlying automaticity of AVNCs in genetically engineered mice. Results Functional signature of mouse AVNCs. We first attempted to develop a standard experimental approach to isolate the mouse AVN by using several anatomical landmarks (see the Material and Methods section for details). Beside these landmarks, we wished to verify independently that our AVN preparations did contain cells with a potentially high degree of automaticity, by using expression of hcn4 as a marker.9 We thus stained whole

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SAN-AVN tissue preparations with an anti-rat HCN4 ­antibody (Fig. 1A and B). HCN4 positive (presumably pacemaker) AVNCs were present in our AVNC preparations (Fig. 1A and B). In particular, anti-HCN4 immunoreactivity was identified in the SAN as stretches of HCN4-positive cells extending into the AVN region below the inferior vena cava (Fig. 1A). This is consistent with what was reported by Dobrzynski et al.10 who delineated expression of HCN4 protein in rabbit AVN. Beside the posterior nodal extension of the conduction system identified by Dobrzynski et al.10 our preparations included also the enclosed node, and a AVN sub-region defined by Boyett et al.11 that was composed by “loosely-packed” atrial cells. Our preparations did not include cells of the mouse His bundle, because the caudal cutting edge of our tissue samples was located on the tricuspid valve. We also checked in pilot experiments using Cx40EGFP/+ mice12 that the His bundle was left intact after cutting out the AVN region (see Fig. 2B in the paper by Miquerol et al.12). We have thus used all these landmarks to perform our dissections so to include the full mouse AVN region. Our next objective was to identify the functional signature of “pacemaker” AVNCs in mice. Three cellular morphologies

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Levi,4 respectively. In our mouse AVN ­preparations, rod-shaped and atrial-like cells did not appear to be spontaneously active. However, we cannot exclude the possibility that rod-shaped cells can become automatic under particular physiological conditions or in situ in the intact AVN. Only spontaneously active spindleshaped cells were used for this study (Fig. 1C). The mean input capacitance of n = 147 spindle shaped cells was 20 ± 1 pA/pF, a value similar to that reported for primary SANCs (20 pF).13 Spindle‑shaped cells from AVN of the mouse (this study), the rabbit5 and guinea pig14 have comparable cellular input capacitance (22 pF in mouse, 29 pF in rabbit and 25 pF in guinea pig). The presence of rod-shaped and atriallike cells in preparations is consistent with the cellular heterogeneity described Figure 2. Automaticity of WT AVNCs is slower than that of SANCs. (A) Examples of spontaneous in the rabbit AVN. action potentials in ANVCs and SANCs; consecutive action potentials are superimposed for clarity. Pacemaking of mouse AVNCs is The dotted line sets the zero-voltage level. (B) Histograms of pacing rate of WT mouse SANCs (filled boxes) and AVNCs (empty boxes). Perfusion of a maximal dose of ISO (0.1 μM) increased slower that that of SANCs and depends pacemaking of SANCs (C) and AVNCs (D), but AVNCs pacemaking was still slower that of SANCs on both INa and ICa,L . Comparison under the same condition. between pacemaker activity of isolated AVNCs and SANCs showed that the beating rate of mouse AVNCs was significantly lower than that Table 1. Pacemaker activity and action potential parameters of mouse of SANCs (Table 1). The maximum diastolic potential and AVNCs and SANCs the action potential threshold were significantly more negaSANCs n AVNCs n p tive in AVNCs than in SANCs (Table 1) indicating that the Rate (bpm) 260 ± 21 8 173 ± 27 8 0.0248 diastolic depolarization occurs at a more negative voltage range MDP (mV) -53 ± 1 8 -57 ± 1 8 0.0201 in AVNCs than in SANCs. To test if ISO application could Eth (mV) -35 ±1 8 -41 ± 2 8 0.0119 compensate for the slower basal rate of AVNCs, we applied ISO SDD (mV/ms) 0.09 ± 0.1 8 0.05 ± 0.01 8 0.0322 at 0.1 μM, a concentration that largely saturated the positive APA (mV) 88 ± 7 8 91 ±7 8 0.8051 chronotropic response to this agonist of mouse SANCs pacemaking (data not shown). The cellular firing rate measured dV/dt (mV/ms) 14 ± 5 8 13 ± 3 8 0.8988 after application of ISO was still higher in SANCs (C) than APD (ms) 134 ± 12 8 152 ± 18 8 0.4004 in AVNCs (D). In ISO, the beating rate of SANCs was 348 ± AVNCs displayed slower beating rate (bpm), a more negative maximum 7 bpm, while that of AVNCs measured in the same conditions diastolic potential (MDP) and action potential threshold (Eth). The slope of the diastolic depolarization (SDD), was lower in AVNCs. No significant was 222 ± 24 bpm (p < 0.05). differences were observed in the action potential amplitude (APA), Because genetic mutations affecting the cardiac INa channel upstroke velocity (dV/dt) and action potential duration (APD). isoform Nav1.5 and the L-type Cav1.3 channel isoform cause AV conduction dysfunction,15,16 we investigated the impact of inhicould be identified after enzymatic digestion of the AVN region: bition of INa and ICa,L on AVNCs pacemaking. Application of a ­spindle, rod and atrial cell morphology (Fig. 1C). In a typical 20-μM tetrodotoxin (TTX) blocked action potential discharge AVN cell preparation, 49% of the total cell population was com- (Fig. 3A). The membrane potential of AVNCs exposed to 20 prised of rod shaped cells. These cells have a length of approxi- μM TTX was stable at -59 ± 2 mV (n = 8). Inhibition of ICa,L by mately 150 μM and were quiescent. Atrial cells that were similar 0.3 μM of the L-type channel blocker isradipine stopped paceto myocytes isolated independently from the right atrium con- maker activity of AVNCs and the cell membrane potential depostituted 41% of the cellular preparation. Spindle shaped cells larized to -35 ± 3 mV (n = 6). Only low amplitude oscillations of accounted for 10% of the total cell number. Spindle cells mea- the membrane potential could be observed in isradipine treated sured about 50 μM and displayed automaticity. AVNCs (Fig. 3B). These results indicated that pacemaking of Spindle- and rod-shaped cells have been reported in the rab- mouse AVNCs required both INa and ICa,L for action potential bit and guinea-pig AVN by Munk et al. and by Hancox and discharge.

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was significantly lower in AVNCs than that recorded in SANCs (Fig. 6A). No significant If density was recorded in myocytes from the right atrium (Fig. 6A). In comparison, density of the Ba 2+ sensitive IK1 component did not significantly vary between AVNCs and SANCs (Fig. 6B). Beside specific differences in current densities, If in AVNCs was recorded at more negative potentials than in SANCs (Fig. 6C). Proper measurement of the If half-activation voltage (V1/2 ),21 showed that AVNCs If activated about 10 mV more negatively than SANCs If (-111 ± 5 n = 5 and -101 ± 1 mV,13 respectively, n = 7 p < 0.05, Fig. 7A). The slope factor (v) was not significantly affected (13 ± 1 n = 5 in AVNCs and 12 ± 1, n = 6 in SANCs, p > 0.05). We then tested if, in spite of its more negative activation curve, If contributed to pacemaking of AVNCs. We thus used 3 μM of the If inhibitor ZD-7228,22 on pacing AVNCs. Under voltageFigure 3. Automaticity of WT AVNCs is dependent on both INa and ICa,L. (A) Representative recordings of consecutive action potentials of WT mouse AVNCs in control conditions and after inhibition of INa by clamp conditions, a 32% reduction 20 μM TTX (B). of If was observed (Fig. 7B). At this concentration ZD-7228 slowed Voltage-dependent Na+ currents in mouse AVNCs. Both TTX- AVNC pacemaker activity by about 16% (Fig. 7C). We did sensitive (INa,s) and TTX-resistant (INa,r ) Na+ currents have been not test higher concentrations of ZD-7228 because of possible recorded in mouse SANCs.8,17 To test functional expression of INa,s interference with IKr inhibition. IKr, Ito and IKs in AVNCs. In mouse SANCs, IKr plays a funwe applied 0.1 μM TTX to inhibit this current.8 At this concentration, total peak INa was reduced by about 20% (Fig. 4). The net damental role in controlling the action potential duration and is peak density of INa,s blocked at 0.1 μM TTX at a test potential of -25 present during the diastolic depolarization as an outward curmV was (56 ± 12 pA/pF, n = 8), while that of residual INa was 287 rent.23 IKr was found in all AVNCs tested (n = 20). The peak IKr ± 57 pA/pF, n = 8. Residual INa after application of 0.1 μM TTX density was higher in AVNCs than that reported in a previous was not completely blocked at 20 μM TTX (Fig. 3B), demonstrat- study on mouse SANCs by Clark et al. (3.4 ± 0.4 in AVNCs ing that this current is attributable to INa,r. INa,r and INa,s displayed and 1.5 ± 0.1 pA/pF in SANCs at a test potential of +10 mV) distinct V1/2. Activation of INa,r was 11 mV negative to that of INa,s. (Fig. 8). The expression of Ito was investigated in AVNCs. In n This is consistent with a previous study in mouse SANCs.7 The V1/2 = 5 AVNCs, a net 4-AP-sensitive Ito current could be recorded of INa,s was significantly more positive than that of INa,r (-32 ± 1 mV (Fig. 8C) (2.1 ± 0.3 pA/pF at a test potential of +10 mV). These cells showed also a relatively large E-4031 and 4-AP insensitive for INa,s and -43 ± 3 mV for INa,r, p < 0.05, Fig. 4C). ICa,L and ICa,T in mouse AVNCs. Beside If, another typical outward K+ current component. This latter current is similar to hallmark of pacemaker SANCs is co-expression of “low-voltage the Ca 2+ activated K+ current mediated by SK2 channels previactivated” T- and L-type Ca 2+ channel isoforms.18,19 We thus ously described in mouse AVNCs.24 In other cells (n = 9), 4-AP measured ICa,T and ICa,L in AVNCs (Fig. 5). The peak current had only a limited effect on outward currents and no statistically density of ICa,T measured at the test potential of -30 mV was 7.9 significant 4-AP sensitive current could be recorded (not shown). ± 0.7 pA/pF, a value which is significantly higher that that of the Finally, we studied if mouse AVNCs expressed also the slow peak ICa,L (1.7 ± 0.1 pA/pF, n = 4, Vt -5 mV). The V1/2 of ICa,T was component of the delayed rectifier K+ current (IKs ). We used the -45 ± 1 mV, while that of ICa,L was -22 ± 2 mV. Current densi- IKs blocker 293B to detect a 293B-sensitive current component. ties did not significantly differ between WT C57B/6J mice (this In 11 cells tested, 293B was without significant effect on deacstudy) and that recorded in AVNCs of 129Sv mice.20 tivating tail currents at -45 mV (not shown). This observation Properties of If in mouse AVNCs. If was present in all indicates that IKs is poorly expressed or even absent in automatic spontaneously-beating AVNCs recorded. The density of If mouse AVNCs investigated in this study.

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Figure 4. TTX-sensitive (INa,s) INa, in mouse AVNCs. Examples of the effect of 0.1 (A, filled circles) and 20 μM TTX (B, empty circles) on INa traces. (C) Examples of the I-V curve of total INa (filled circles), INa, in presence of 0.1 TTX (hollow circles), INa,s (filled squares) in AVNCs. INa,s is calculated by subtracting traces recorded in 0.1 μM TTX to total INa, traces.

Discussion

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The AVN structure and function have been extensively studied (reviewed in ref. 3) however, little is known about the mechanisms underlying AVN automaticity. In this study we present, for the first time, an overview of the electrophysiological properties of spontaneously active mouse AVNCs. We expect that our work will improve the accessibility of this preparation for future studies on AVN pacemaker activity and conduction using genetically engineered mice. Furthermore, our results provide a functional signature of automatic mouse AVNCs and can be useful for studies aiming to characterize AVNCs precursors from the developing mouse heart9 or from differentiating stem cells. Pacemaking of mouse AVNCs. The AVN is a highly heterogeneous structure containing different cell types that probably serve both SAN impulse conduction and secondary pacemaker activity. Since little is known about the specific cellular mechanisms underlying AVNCs automaticity, we focused on AVNCs that displayed pacemaker activity once isolated from the intact AVN. In addition to widely accepted anatomical landmarks established in rabbit hearts (see the Materials and Methods section), we used also anti-HCN4 staining of intact AVN to identify regions of the AV junction containing automatic cells. This approach maximizes the probability of including all automatic AVNCs in our preparations, even if the fine structure of mouse AVN has not been fully explored. Even if we did not perform a pilot structure-to-function study of mouse AVNCs, we found consistency between the anatomical pattern of anti-HCN4 immunoreactivity in our preparations and previous studies on rabbit conduction system.25 Dobrzynski et al.10 have reported that the site of origin of automaticity in the rabbit AVN is predominantly located in the posterior nodal extension, even if automaticity can also originate in the enclosed node region. In our preparations, we included the mouse posterior nodal extension,

which is HCN4 positive and is thus homologous to the rabbit AVN rhythmogenic centre (Fig. 1A). Also, the gross morphology of HCN4 positive cells in situ is similar to the spindle-like shaped AVNCs used for recordings (Fig. 1B and C). In conclusion, we are confident that spontaneously beating AVNCs described in this study come from rhythmogenic centers of AVN and underlie its automaticity in vivo. The intrinsic pacemaker activity of mouse AVNCs was significantly slower than that of SANCs (Fig. 2). This difference was maintained after perfusion with ISO. This observation indicates that the slower rate of pacing of mouse AVNCs cannot be explained with a lower basal cAMP concentration, but is due to intrinsic properties of the AVNCs pacemaker mechanism. INa in mouse AVNCs. Automaticity of mouse AVNCs was highly sensitive to INa block by TTX. INa thus appears as the predominant mechanisms underlying the action potential upstroke phase. In mouse SANCs high doses of TTX do not stop pacemaker activity, but slow the action potential upstroke velocity as well as the diastolic depolarization phase.8 This suggests that the functional role of INa in mouse AVNCs can be quantitatively different from that in SANCs. Similarly to SANCs, AVNCs expressed both INa,r and INa,s. Activation of INa,r was 11 mV negative to that of INa,s. This is consistent with a previous study in mouse SANCs.8 Similarly to SANCs, preliminary data suggest that INa,s contributes to the diastolic depolarization of AVNCs, because block of TTX-sensitive channels by 0.1 μM TTX affected the slope of this phase (data not shown). At present, it is difficult to compare the densities of INa,r and INa,s between AVNCs and SANCs, because in the previous study by Lei et al.8 Na+ currents were recorded at room temperature and under reduced extracellular Na+ concentrations (30 mM). However, the high density of INa,r that we recorded in AVNCs and the prominent role of INa, in AVNCs action potential discharge are consistent with the severe dysfunction in AVN conduction observed in haplo-insufficient

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Figure 5. Recordings of ICa,T and ICa,L in isolated AVNCs of WT mice. (A) Sample ICa (ICa,T + ICa,L) traces obtained using a holding potential (Vh) of -90 mV and -55 mV (B) at different test potentials (Vt) as indicated by arrows. The voltage protocol is shown at the top of (A). (C) I–V curves of ICa,L and ICa,T obtained on WT AVNCs. ICa,T + ICa,L was recorded using a HP at -90 mV (filled symbols) and ICa,L from a HP of -55 mV (open symbols). We calculated the I–V curve of ICa,T by subtracting records at HP of -55 mV from that at HP of -90.

©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 6. If (A) and IK1 (B) in SANCs, AVNCs and right atrial cells (RACs). Currents were evoked by applying hyperpolarizing voltage steps from a HP of -30 mV, as indicated in the voltage protocol inset. (C) Isochronal I-V curves for If in AVNCs (open circles), SANCs (filled circles) and RACs (filled boxes). The peak If density measured at -130 mV was lower in AVNCs than in SANCs (16.8 ± 3 n = 5 and 42 ± 4 pA/pF, n = 6 p < 0.01). IK1 was similarly expressed in AVNCs and SANCs.

Nav1.5 mice.15 Expression of INa in AVNCs is also consistent with the fast impulse conduction in mouse AVNCs. ICa,L and ICa,T in mouse AVNCs. Automaticity of AVNCs also depended from ICa,L (Fig. 3B). Perfusion of isradipine blocked action potential discharge and depolarized the membrane potential. AVNCs thus responded to ICa,L block in a similar way as central SAN tissue strips from the rabbit heart.26 We propose that depolarization of membrane voltage upon ICa,L block can be explained, at least in part, by the loss of coupling between L-type channels and SK2 K+ channels. Such a functional coupling has been reported in mouse AVNCs,24 as well as in atrial myocytes.27 The density of ICa,L recorded in AVNCs was significantly lower than that of SANCs

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(Fig. 4). However, in spite of the strong expression of INa, ICa,L is functionally important in AVNCs, because isradipine completely stopped action potential discharge. The absolute density of ICa,T in mouse AVNCs was significantly higher than that of ICa,L (Fig. 4). This contrasts to what previously observed in rabbit SANCs, where ICa,L density is higher than that of ICa,T.18,28 High expression of ICa,T in AVNCs, can be an adaptative mechanism to sustain both high basal pacemaking and fast AV conduction in mouse AVN. Use of genetically-modified mice will be useful to understand the functional role of ICa,L and ICa,T in AVNCs automaticity. If in mouse AVNCs. In AVNCs, the density of If was significantly lower than that recorded in SANCs (Fig. 5). Activation of If

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may be a mechanism of adaptation for regulating action potential duration at high heart rates typical of this species. Some AVNCs also expressed moderate quantities of 4-AP sensitive Ito current. The exact location of Ito -expressing cells in the mouse AVN is not known. However, this current can be important in regulating the action potential duration in AVNCs involved in impulse conduction. This hypothesis would be consistent with a previous study showing that mice lacking both the fast and the slow component of Ito display second degree AV blocks.31 Materials and Methods The investigation conforms to the Guide for the care and Use of Laboratory Animals published by the US national Institute of Health (NIH Publication No. 85–23, revised 1996) and European directives (86/609/CEE). Morphological identification and definition of mouse AVN. The mouse AVN was identified as the region between the inferior vena cava (IVC), the coronary sinus (CS) and the tricuspid valve2 (Sup. Fig. 1A and B). The anatomical location of tissue samples used for cell isolation is from the mouse atrioventricular junction corresponding to the Koch’s triangle as defined in other mammals2,3,11 (Sup. Fig. 1A and B). This region corresponded to the “AVN region” identified by Rentschler et al. at the inferior part of the interatrial septum and above the mitral annulus. Indeed, in the Rentschler et al. study, hearts from transgenic mice expressing an Engrailed-LacZ reporter construct delineated the mouse cardiac conduction system from the SAN to the distal Purkinje fibers. Finally, we considered as the AVN, the entire region that has been shown to express a pattern of mRNA coding for ion channels similar to that of SAN30 and containing HCN4positive cells (see Results section). Indeed, Marionneau et al. have reported that ion channel mRNA expression pattern in the mouse AVN region is more similar to that of SAN, a finding that is consistent with the functional expression of ionic currents identified in the present study (see above). Staining of AVN tissue. Isolated mouse heart node tissue was placed immediately into 4% paraformaldehyde for 20 minutes at room temperature (RT), followed by soaking in 1x PBS for several hours. The tissue was suspended in 250 ml of 2% Bovine Serum Albumin (Sigma), 500 mg/ml digitonin (Sigma) containing rat HCN4 monoclonal antibody (SHG 1E5, Santa Cruz, 1:100) and sodium azide (0.002%, to prevent bacterial growth) for 66 hrs at RT with very gentle shaking in a LAB-TEK II Chamber Slide. The tissue was rinsed 5 x 500 ml (1x PBS) over a 5 hour period at RT. The procedure was repeated again but this time with donkey anti-rat Alexa 488 secondary (Molecular Probes, 1:500). The tissue was placed in prolong gold (Invitrogen) and a glass coverslip gently placed over the sample. Images were taken with a Leica confocal macroscope (Leica TCS LSI) at the Montpellier RIO Imaging facility of the Arnaud de Villeneuve Campus (Montpellier, France). The final images represent a snapshot of a 3D reconstructed tiled image using IMARIS software package (PC, version 7). Mouse AVNCs were isolated using the same protocol as used for patch clamping. AVNCs were placed into chambers and

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Figure 7. Properties of If in mouse AVNCs. (A) If activation curve in mouse AVNCs, measured according to DiFrancesco and Mangoni. 21 Red circles indicate curve fit simulation. (B) 3 μM of ZD-7288 inhibited If by 34.5 ± 7.3% (at -115 mV, n = 5). If was activated by hyperpolarizing steps as in Figure 6. (C) ZD-7288 reduced AVNCs beating rate by 16 ± 2% (from 286 ± 22 to 242 ± 23 bpm, n = 5, p < 0.05). ZD-7288 reduced the slope of the diastolic depolarization from 2.56 ± 0.5 to 2.17 ± 0.5, (p < 0.05). The action potential duration was not significantly affected by ZD7288 (from 301 ± 81 to 336 ± 77 ms at 3 μM ZD-7288, p > 0.05).

was shifted negatively in AVNCs by about 10 mV compared to If in SANCs. In contrast, If activation kinetics did not differ from those recorded in mouse SANCs.13,29 In a previous study, we showed that the relative abundance of HCN channel isoforms does not significantly differ between the SAN and AVN.30 It is thus possible that subtler differences exist in the regulation of HCN channels in SANCs and AVNCs. In spite of the weaker density and the more negative activation, If still contributes to automaticity of AVNCs, because ZD-7228 reduced the slope of the diastolic depolarization. The precise contribution of If is difficult to evaluate pharmacologically, because higher doses of ZD-7228 would also affect IKr. The use of genetically modified mice is thus important to evaluate the importance of If in mouse AVNCs. In mouse SANCs, IKr plays a fundamental role in controlling the AP duration and is present during the diastolic depolarization as an outward current that influences the time course of this phase.23 We found IKr in all AVNCs tested, an observation that suggests that this current can be important also for pacemaking of AVNCs. The predominance of IKr upon IKs in mouse AVNCs

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©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 8. IKr, and Ito in AVNCs. (A) IKr in AVNCs. Sample traces in control conditions (top part) and in presence of E-4031 (middle part). IKr is evaluated by subtracting traces in E-4031 from control ones (bottom part). The (*) symbol indicates the voltage protocol for records in (A). (B) Averaged IV-curves of IKr (open circles) in AVNCs, compared with total current (filled boxes) and current after E-4031 (open boxes). The E-4031-sensitive current started to activate between -40 and -30 mV and peaked at 0 mV. (C and D) Sample traces recorded in control conditions (C, top part) and in presence of 4-AP (C, middle part). Ito is evaluated by subtracting traces in 4-AP from control ones (C, bottom part). (**) symbol indicates the voltage protocol for records in (C). (D) Averaged IV-curve of total current (filled boxes) and current after 4-AP (open boxes). No significant difference was observed between currents in these conditions.

allowed to attach to Cell-Tak coated wells (3.5 μg/cm2) for 1 hr (LAB-TEK II Chamber Slide, NUNC). Cells were then fixed with 4% paraformaldehyde for 20 minutes at room temperature (RT), followed by soaking in 1x PBS for several hours. 250 μl of 2% Bovine Serum Albumin (Sigma) containing rat HCN4 monoclonal antibody (SHG 1E5, Santa Cruz, 1:100), anti mouse HA monoclonal (clone HA7, 1:250) were added and allowed to incubate overnight at 4 degrees in a humid chamber. The tissue was rinsed for 4 x 15 minutes (1x PBS). Goat anti-rat FITC secondary (Molecular Probes, 1:500), donkey anti mouse Alexa647 (Molecular Probes, 1:500), DAPI was incubated for 90 minutes at RT followed by 4 x 15 minutes (1x PBS). Prolong gold (Invitrogen) was overlaid and a glass coverslip gently placed over the sample. Images were taken with a Leica confocal microscope (Leica SPE) at the Montpellier RIO Imaging facility of the Arnaud de Villeneuve Campus (Montpellier, France). Image analysis was carried out on Metamorph Image Analysis software.

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The specificity of Rat HCN4 antibody was confirmed using a mouse monoclonal HCN4 antibody (Neuromab), which had the identical overlapping staining pattern. Furthermore, SANCs expressing HCN4-HA tagged subunits showed overlapping staining using rat HCN4/mouse HA antibodies or mouse HCN4/rat HA antibodies (data not shown). Isolation of mouse SANCs and AVNCs. Beating hearts were removed under general anesthesia, consisting of 0.01 mg/g of Xylazine (Rompun 2%, Bayer AG, Leverkusen Germany), 0.1 mg/g of Ketamine (Imalgène, Merial, Bourgelat France) and 0.2 mg/g of Na-Pentobarbital (CEVA, France). The SAN and the AVN were exposed by using the landmarks shown in Supplemental Figure S1A and B and then excised in pre-warmed (35°C) Tyrode solution containing (mM/L): NaCl, 140; KCl, 5.4; CaCl2, 1.8; MgCl2, 1; Hepes-NaOH, 5; and D-glucose, 5.5; (adjusted to pH = 7.4 with NaOH). SAN tissue strips were then transferred into a “low-Ca 2+ -low-Mg2+” solution containing (in

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mM/L): NaCl, 140; KCl, 5.4; MgCl2, 0.5; CaCl2, 0.2; KH2PO4, 1.2; taurine, 50; D-glucose, 5.5; bovine serum albumin (BSA), 1 mg/ml; Hepes-NaOH, 5; (adjusted to pH = 6.9 with NaOH). SAN and AVN tissues were digested by adding collagenase type II or IV (229 U/ml, Worthington Biochemical Corporation, Lakewood, NJ USA), elastase (1.9 U/ml, Worthington Biochemical Corporation, Lakewood, NJ, USA), protease (0.9 U/ml, Sigma, St. Quentin Fallavier, France), BSA 1 mg/ml and 200 μM CaCl2. Digestion was carried out for a variable time of 20–25 minutes at 35°C. Tissue strips were then washed and transferred into a modified “Kraftbrühe” (KB) medium33 containing (in mM/L): L-glutamic acid, 70; KCl, 20; KOH, 80; (±)D-b-OH-butyric acid, 10; KH2PO4, 10; taurine, 10; BSA, 1 mg/ml; and Hepes-KOH, 10; (adjusted to pH = 7.4 with KOH). Single SAN and AVN cells were isolated by manual agitation in KB solution at 35°C for 2–5 minutes. Cellular automaticity was recovered by re-adapting the cells to a physiological extracellular Ca 2+ concentration by addition of a solution containing (in mM/L): NaCl, 10, CaCl2, 1.8 and normal Tyrode solution containing BSA (1 mg/ml). The final storage solution contained (mM/L): NaCl, 100; KCl, 35; CaCl2, 1.3; MgCl2, 0.7; L-glutamic acid, 14; (±)D-b-OH-butyric acid, 2; KH2PO4 ; 2; taurine, 2; BSA 1 mg/ml; (pH = 7.4) and gentamycin (50 μg/ml). All chemicals were from Sigma (St. Quentin, Fallavier), except for the (±)D-β-OH-butyric acid that was from Fluka Chemika (Buchs, CH), TTX and ZD 7288 that were from Tocris Bioscience (Ellisville, USA), 293B was a generous gift by Dr. Lang and E-4031 that was a generous gift by Dr. Flavien Charpentier (L’ Institut du Thorax, Nantes). Electrophysiological recordings. For electrophysiological recordings, aliquots of the cell suspension were harvested in custom made recording chambers (working volume 500 μL) allowing unidirectional flow of solutions and mounted on the stage of an inverted microscope (Olympus, X71), and continuously perfused with normal Tyrode solution. The recording temperature was set to 36°C. The whole-cell variation of the patch-clamp technique34 was used to record cellular ionic currents, by employing an Axopatch 200A (Axon Instruments Inc., Foster USA) patchclamp amplifier, connected to the ground by an agar bridge filled with 150 mM KCl. Cellular automaticity was recorded by the perforated patch technique with escin (50 μM). Recording electrodes were fabricated from borosilicate glass, by employing a FlamingBrown microelectrode puller (Sutter, Novato, CA USA). For recording cell automaticity, as well as If and K+ currents, we used an intracellular solution containing (mM/L): K+ -aspartate, 130; NaCl, 10; ATP-Na+ salt, 2; creatine phosphate, 6.6; GTP-Mg2+, 0.1; CaCl2, 0.04 (pCa = 7); Hepes-KOH, 10; (adjusted to pH = 7.2 with KOH). Electrodes had a resistance of about 5 MΩ. Seal resistances were in the range of 2–5 GΩ. For recording of calcium currents (ICa,T, ICa,L), we replaced K-Aspartate and KCl in the intracellular solution, with an equal amount of CsCl. The extracellular solution contained (in mM/L): tetraetylammonium-chloride (TEA-Cl), 130; CaCl2, 2; MgCl2, 1; 4-amino-pyridine, 10; Hepes, 25; (adjusted to pH = 7.4 with TEAOH). IKr was recorded in Tyrode, after addition of 10 μM TTX and 0.3 μM isradipine. If was routinely recorded

in Tyrode solution containing 5 mM BaCl2 to block IK1.35 We performed data acquisition by using the pClamp software (ver. 9, Axon Instruments Inc.). Data analysis. The AP parameters were calculated as reported previously in reference 8, 36 and 37. The following action potential (AP) parameters were calculated: the AP duration (APD), the slopes of the diastolic depolarisation (SDD), the maximum diastolic potential (MDP), the AP threshold (Eth), the maximum upstroke velocity (the peak of the first derivative of the AP waveform dv/dt), and the AP amplitude (APA). The cell membrane capacitance has been monitored by applying brief (10 ms) voltage steps of ±10 mV amplitude from a holding potential (HP) of -35 mV. The If activation curve was measured according to a protocol described by DiFrancesco and Mangoni21 and fitted according to the equation: P(V) = 1/1 +exp[(V - V1/2)/v] where P(V) is the degree of activation of macroscopic If and is equivalent to the voltage dependency of the probability of opening of single f-channels,21 V1/2 is the voltage for current half activation, and v is the slope factor. Cav-mediated Ca 2+ currents and INa were analyzed as previously reported in references 20 and 37. We performed analysis by employing the Origin Lab software (ver. 7.5, Microcal Inc., Northampton, MA USA). Results are presented as means ± the standard error of the mean (SEM, number of cells). For calculating the level of significance, Student’s t-tests, the one- or two-way ANOVA tests followed by Tukey’s post-hoc tests and non-parametric Kruskal-Wallis tests were employed. When testing statistical differences results were considered significant with p < 0.05. In all figures *p < 0.05, **p < 0.01 and ***p < 0.001, respectively.

©201 1L andesBi os c i enc e. Donotdi s t r i but e. Acknowledgements

We thank Elodie Kupfer and the personnel of the IFR3 animal facility of Montpellier for breeding and managing mouse lines. We thank Isabelle Bidaud (CNRS UMR 5203, Montpellier France) for excellent technical assistance. We also thank Stéphanie Barrère-Lemaire (CNRS UMR 5203) and Emilio Carbone (University of Turin, Italy) for helpful discussion. Financial Support

This work has been supported by CavNet a Research Training Network (RTN) funded through the European Union Research Programme (6FP) MRTN-CT-2006-035367, the INSERM National Program for Cardiovascular Diseases (PNRC), the Fondation de France (Cardiovasc 2008002730), the Agence Nationale pour la Recherche (ANR-06-PHISIO-004-01), the NIH R01HL087120-A2 grant, the Deutsche Forschungsgemeinschaft (DFG, IS63/1-1/2) and the Austrian Science Fund (P-20670). Pietro Mesirca and Angelo Torrente are CavNet fellows. Note

Supplementary materials can be found at: www.landesbioscience.com/journals/channels/article/15264

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References 1.

2.

3.

4.

5.

6. 7.

8.

9.

10.

11.

12.

13.

14.

10

Boyett MR, Honjo H, Kodama I. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc Res 2000; 47:658-87. Meijler FL, Janse MJ. Morphology and electrophysiology of the mammalian atrioventricular node. Physiol Rev 1988; 68:608-47. Efimov IR, Nikolski VP, Rothenberg F, Greener ID, Li J, Dobrzynski H, et al. Structure-function relationship in the AV junction. Anat Rec A Discov Mol Cell Evol Biol 2004; 280:952-65. Hancox JC, Levi AJ. L-type calcium current in rodand spindle-shaped myocytes isolated from rabbit atrioventricular node. Am J Physiol 1994; 267:1670-80. Munk AA, Adjemian RA, Zhao J, Ogbaghebriel A, Shrier A. Electrophysiological properties of morphologically distinct cells isolated from the rabbit atrioventricular node. J Physiol 1996; 493:801-18. Mangoni ME, Nargeot J. Genesis and regulation of the heart automaticity. Physiol Rev 2008; 88:919-82. Honjo H, Boyett MR, Kodama I, Toyama J. Correlation between electrical activity and the size of rabbit sinoatrial node cells. J Physiol 1996; 496:795-808. Lei M, Jones SA, Liu J, Lancaster MK, Fung SS, Dobrzynski H, et al. Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking. J Physiol 2004; 559:835-48. Horsthuis T, Buermans HP, Brons JF, Verkerk AO, Bakker ML, Wakker V, et al. Gene expression profiling of the forming atrioventricular node using a novel tbx3based node-specific transgenic reporter. Circ Res 2009; 105:61-9. Dobrzynski H, Nikolski VP, Sambelashvili AT, Greener ID, Yamamoto M, Boyett MR, et al. Site of origin and molecular substrate of atrioventricular junctional rhythm in the rabbit heart. Circ Res 2003; 93:1102-10. Boyett MR, Inada S, Yoo S, Li J, Liu J, Tellez J, et al. Connexins in the sinoatrial and atrioventricular nodes. Adv Cardiol 2006; 42:175-97. Miquerol L, Meysen S, Mangoni M, Bois P, van Rijen HV, Abran P, et al. Architectural and functional asymmetry of the His-Purkinje system of the murine heart. Cardiovasc Res 2004; 63:77-86. Mangoni ME, Nargeot J. Properties of the hyperpolarization-activated current (If) in isolated mouse sinoatrial cells. Cardiovasc Res 2001; 52:51-64. Yuill KH, Hancox JC. Characteristics of single cells isolated from the atrioventricular node of the adult guinea-pig heart. Pflugers Arch 2002; 445:311-20.

15. Papadatos GA, Wallerstein PM, Head CE, Ratcliff R, Brady PA, Benndorf K, et al. Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a. Proc Natl Acad Sci USA 2002; 99:6210-5. 16. Platzer J, Engel J, Schrott-Fischer A, Stephan K, Bova S, Chen H, et al. Congenital deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca2+ channels. Cell 2000; 102:89-97. 17. Lei M, Goddard C, Liu J, Leoni AL, Royer A, Fung SS, et al. Sinus node dysfunction following targeted disruption of the murine cardiac sodium channel gene Scn5a. J Physiol 2005; 567:387-400. 18. Hagiwara N, Irisawa H, Kameyama M. Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J Physiol 1988; 395:233-53. 19. Irisawa H, Brown HF, Giles W. Cardiac pacemaking in the sinoatrial node. Physiol Rev 1993; 73:197-227. 20. Mangoni ME, Traboulsie A, Leoni AL, Couette B, Marger L, Le Quang K, et al. Bradycardia and slowing of the atrioventricular conduction in mice lacking CaV3.1/alpha1G T-type calcium channels. Circ Res 2006; 98:1422-30. 21. DiFrancesco D, Mangoni M. Modulation of single hyperpolarization-activated channels (If) by cAMP in the rabbit sino-atrial node. J Physiol 1994; 474:473-82. 22. BoSmith RE, Briggs I, Sturgess NC. Inhibitory actions of ZENECA ZD7288 on whole-cell hyperpolarization activated inward current (If) in guinea-pig dissociated sinoatrial node cells. Br J Pharmacol 1993; 110:343-9. 23. Clark RB, Mangoni ME, Lueger A, Couette B, Nargeot J, Giles WR. A rapidly activating delayed rectifier K+ current regulates pacemaker activity in adult mouse sinoatrial node cells. Am J Physiol Heart Circ Physiol 2004; 286:1757-66. 24. Zhang Q, Timofeyev V, Lu L, Li N, Singapuri A, Long MK, et al. Functional roles of a Ca2+-activated K+ channel in atrioventricular nodes. Circ Res 2008; 102:465-71. 25. Yamamoto M, Dobrzynski H, Tellez J, Niwa R, Billeter R, Honjo H, et al. Extended atrial conduction system characterised by the expression of the HCN4 channel and connexin45. Cardiovasc Res 2006; 72:271-81. 26. Kodama I, Nikmaram MR, Boyett MR, Suzuki R, Honjo H, Owen JM. Regional differences in the role of the Ca2+ and Na+ currents in pacemaker activity in the sinoatrial node. Am J Physiol 1997; 272:2793-806.

27. Lu L, Zhang Q, Timofeyev V, Zhang Z, Young JN, Shin HS, et al. Molecular coupling of a Ca2+-activated K+ channel to L-type Ca2+ channels via alpha-actinin2. Circ Res 2007; 100:112-20. 28. Mangoni ME, Fontanaud P, Noble PJ, Noble D, Benkemoun H, Nargeot J, et al. Facilitation of the L-type calcium current in rabbit sino-atrial cells: effect on cardiac automaticity. Cardiovasc Res 2000; 48:375-92. 29. Cho HS, Takano M, Noma A. The electrophysiological properties of spontaneously beating pacemaker cells isolated from mouse sinoatrial node. J Physiol 2003; 550:169-80. 30. Marionneau C, Couette B, Liu J, Li H, Mangoni ME, Nargeot J, et al. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J Physiol 2005; 562:223-34. 31. Guo W, Li H, London B, Nerbonne JM. Functional consequences of elimination of ito,f and ito,s: early afterdepolarizations, atrioventricular block and ventricular arrhythmias in mice lacking Kv1.4 and expressing a dominant-negative Kv4 alpha subunit. Circ Res 2000; 87:73-9. 32. Rentschler S, Vaidya DM, Tamaddon H, Degenhardt K, Sassoon D, Morley GE, et al. Visualization and functional characterization of the developing murine cardiac conduction system. Development 2001; 128:1785-92. 33. Isenberg G, Klockner U. Calcium tolerant ventricular myocytes prepared by preincubation in a “KB medium”. Pflugers Arch 1982; 395:6-18. 34. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Archiv 1981; 391:85-100. 35. DiFrancesco D, Ferroni A, Mazzanti M, Tromba C. Properties of the hyperpolarizing-activated current (If) in cells isolated from the rabbit sino-atrial node. J Physiol 1986; 377:61-88. 36. Rocchetti M, Malfatto G, Lombardi F, Zaza A. Role of the input/output relation of sinoatrial myocytes in cholinergic modulation of heart rate variability. J Cardiovasc Electrophysiol 2000; 11:522-30. 37. Mangoni ME, Couette B, Bourinet E, Platzer J, Reimer D, Striessnig J, et al. Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity. Proc Natl Acad Sci USA 2003; 100:5543-8.

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research paper

Research paper

Channels 5:3, 1-1; June/May 2011; © 2011 Landes Bioscience

Pacemaker activity and ionic currents in mouse atrioventricular node cells This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.

Laurine Marger,1-3 Pietro Mesirca,1-3 Jacqueline Alig,4 Angelo Torrente,1-3 Stefan Dubel,1-3 Birgit Engeland,4 Sandra Kanani,1-3 Pierre Fontanaud,1b-3 Jörg Striessnig,5 Hee-Sup Shin,6 Dirk Isbrandt,4 Heimo Emke,7 Joël Nargeot,1-3 Matteo E. Mangoni1-3,* 1 CNRS; UMR-5203; Institut de Génomique Fonctionnelle; Département de Physiologie; 1bDépartement d’Endocrinologie; Montpellier, France; 2INSERM; U661; Montpellier, France; 3Universités de Montpellier 1and 2; UMR-5203; Montpellier, France; 4Center for Obstetrics and Pediatrics; and Center for Molecular Neurobiology; RG Experimental Neuropediatrics; 7Department of Vegetative Physiology and Pathophysiology; Center for Experimental Medicine; University Medical Center Hamburg-Eppendorf; Hamburg, Germany; 5Department of Pharmacology and Toxicology; Institute of Pharmacy; Center for Molecular Biosciences; University of Innsbruck; Innsbruck, Austria; 6 Center for Neural Science; Korea Institute of Science and Technology; Cheongryang, Seoul, Republic of Korea

Key words: atrioventricular node, sino-atrial node, pacemaker activity, ion channels, electrophysiology, conduction, heart rate, Ca 2+ channels, Na+ channels, f-channels, K+ channels Abbreviations: AVN, atrioventricular node; SAN, sino-atrial node; TTX, tetrodotoxin; INa, voltage-dependent Na+ current; ICa,L , L-type Ca 2+ current; ICa,T, T-type Ca 2+ current; If, hyperpolarization-activated f-current; IKr, delayed rectifier K+ current; Ito, transient outward K+ current

It is well established that pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of INa by tetrodotoxin (TTX) or of ICa,L by isradipine abolished AVNCs pacemaker activity. TTX-resistant (INar) and TTX-sensitive (INas) Na+ currents were recorded in mouse AVNCs, as well as T-(ICa,T) and L-type (ICa,L) Ca2+ currents. ICa,L density was lower than in SANCs (51%). The density of the hyperpolarizationactivated current, (If) and that of the fast component of the delayed rectifier current (IKr) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of If by 3 μM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for If in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K+ current (Ito). In contrast, no detectable slow component of the delayed rectifier current (IKs) could be recorded in AVNCs. The lower densities of If and ICa,L, as well as higher expression of IKr in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs.

Supplementary Material

*Correspondence to: Matteo E. Mangoni; Email: [email protected] Submitted: 02/18/11; Revised: 02/22/11; Accepted: 02/22/11 DOI: www.landesbioscience.com Channels

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Supplemental Online Section

Fig. S1. (A). The AVN region was exposed as described by Mangoni and Nargeot {Mangoni, 2001 #106}. Dotted red lines identify the cutting edges for isolation of SAN and AVN tissue. Abbreviations: right atrium (RA), left atrium (LA), aorta (Ao), crista terminalis (CT); superior vena cava, (SVC); inferior vena cava (IVC); coronary sinus (CS); Fossa ovalis (FO); atrioventricular node (AVN); sino-atrial node (SAN). (B). Enlargement of the AVN region used for dissections.

 

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Marger et al., Fig. S1

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