Modulation of Spontaneous Action Potential Rate by Inositol Trisphosphate in Myocytes from the Rabbit Atrioventricular Node

The atrioventricular node (AVN) is a key component of the cardiac conduction system and takes over pacemaking of the ventricles if the sinoatrial node fails. IP3 (inositol 1,4,5 trisphosphate) can modulate excitability of myocytes from other regions of the heart, but it is not known whether IP3 receptor (IP3-R) activation modulates AVN cell pacemaking. Consequently, this study investigated effects of IP3 on spontaneous action potentials (APs) from AVN cells isolated from rabbit hearts. Immunohistochemistry and confocal imaging demonstrated the presence of IP3-R2 in isolated AVN cells, with partial overlap with RyR2 ryanodine receptors seen in co-labelling experiments. In whole-cell recordings at physiological temperature, application of 10 µM membrane-permeant Bt3-(1,4,5)IP3-AM accelerated spontaneous AP rate and increased diastolic depolarization rate, without direct effects on ICa,L, IKr, If or INCX. By contrast, application via the patch pipette of 5 µM of the IP3-R inhibitor xestospongin C led to a slowing in spontaneous AP rate and prevented 10 µM Bt3-(1,4,5)IP3-AM application from increasing the AP rate. UV excitation of AVN cells loaded with caged-IP3 led to an acceleration in AP rate, the magnitude of which increased with the extent of UV excitation. 2-APB slowed spontaneous AP rate, consistent with a role for constitutive IP3-R activity; however, it was also found to inhibit ICa,L and IKr, confounding its use for studying IP3-R. Under AP voltage clamp, UV excitation of AVN cells loaded with caged IP3 activated an inward current during diastolic depolarization. Collectively, these results demonstrate that IP3 can modulate AVN cell pacemaking rate.


Introduction
The atrioventricular node (AVN) is the only pathway in structurally normal hearts for electrical activity to pass from the atria to ventricles [1,2].Relatively slow conduction through the AVN ensures that atrial contraction is complete before ventricular contraction occurs [3].During some abnormal cardiac rhythms such as atrial fibrillation (AF), slow AVN conduction limits the number of impulses transmitted to the ventricles [2,4,5].The AVN also acts as a secondary pacemaker that can take over pacemaking if the primary pacemaker (the sino-atrial node-SAN) fails [2,3,6].
From experiments on myocytes isolated from the AVN of several model species, a number of different ionic conductances have been implicated in the genesis of AVN cell pacemaker activity [6][7][8][9][10].Among these are the funny current, I f [8,11,12], rapid delayed rectifier, I Kr [6,13,14], L-type calcium current, I Ca,L [7,8,15,16], T-type calcium current, I Ca,T , [7,8], background sodium current, I B,Na , [17], and in mice the tetrodotoxin-sensitive sodium current, I Na [18,19].In the SAN, it has been established that a Ca 2+ 'clock' also contributes to generation of spontaneous activity (for reviews, see [20,21]).In respect of the AVN, inhibition of sarcoplasmic reticulum (SR) Ca 2+ release by ryanodine/thapsigargin has been shown to prolong the cycle length of isolated AVN preparations and AVN-paced hearts [18,22,23].Further, a functional role for sodium-calcium exchange (NCX) in AVN electrogenicity is supported by experiments wherein NCX activity was reduced/inhibited [24][25][26][27].Thus, it is likely that spontaneous activity in the AVN is influenced by intracellular Ca 2+ release from the SR coupled to Ca 2+ modulation of electrogenesis at the cell surface membrane.
Inositol 1,4,5 trisphosphate (IP 3 ) is a ubiquitous signalling molecule produced from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by phospholipase C (PLC) that translocates from the membrane to the cytoplasm.It is well established that IP 3 releases Ca 2+ from intracellular stores via IP 3 receptors (IP 3 -Rs) [28,29].Although cardiac myocytes generally express a much higher proportion of RyRs than IP 3 -Rs (~100:1), there is evidence that IP 3 -Rs mediate subsarcolemmal, cytoplasmic, and nuclear Ca 2+ signalling in the heart (for reviews, see [28,29]).The lower cardiac expression of IP 3 -Rs compared to RyRs does not preclude IP 3 -Rs from playing a role in setting SR Ca 2+ levels, since RyRs are open for only ~20 ms during the cardiac cycle, while an SR leak via IP 3 -R could be continuous [28].Moreover, the leak via IP 3 -Rs may be amplified by adjacent RyRs to produce Ca 2+ sparks [28].In ventricular myocytes, IP 3 -Rs are both expressed in the perinuclear region, where they are suggested to modulate gene transcription [30][31][32][33], and co-localised with RyRs in the sarcoplasmic reticulum [34,35].The close apposition of L-type Ca 2+ channels (LTCCs) and RyRs in ventricular myocyte dyads results in Ca 2+ release from the SR when LTCCs open: IP 3 -R activation increases dyadic Ca 2+ fluxes during Ca 2+ transients and increases the Ca 2+ spark rate [35].Further, cross-talk between RyRs and IP 3 -Rs increases in heart failure, which may facilitate arrhythmogenesis [34].In atrial myocytes, IP 3 -Rs are largely localised with peripheral, junctional SR [30,36], where their activity can impinge on electrical and RyR Ca 2+ signalling.Furthermore, IP 3 -Rs are central to rhythm control in a variety of non-cardiac tissues and in the spontaneous activity of embryonic cardiomyocytes (for review, see [28]).IP 3 -R stimulation has been associated with abnormal automaticity and spontaneous activity in atrial and pulmonary vein cardiomyocytes [36][37][38][39] and with Ca 2+ waves in subendocardial Purkinje cells following coronary occlusion [40].IP 3 -R inhibition has also been reported to inhibit adrenaline-mediated changes in amphibian cardiac pacemaker (sinus venosus) [41].Notably, a modulatory role for IP 3 -R2 in the SAN has been proposed in mice [28,42] and guinea-pigs [43], where it has been linked to actions of G q -coupled receptor agonists (endothelin 1 (ET-1; 43) and phenylephrine [43]).
Quantitative PCR, Western blot, and immunolabelling have shown that mouse SAN and AVN express all three IP 3 -R isoforms [42], with the predominant isoform being IP 3 -R2 [42]-this isoform has the highest IP 3 affinity [44].Application of a membrane-permeant form of IP 3 to mouse SAN increased spontaneous Ca 2+ transient rate and Ca 2+ spark frequency, whilst the IP 3 -inhibitor 2-aminoethoxydiphenyl borate (2-APB) decreased Ca 2+ transient amplitude and rate [42].Similar effects were not observed in preparations from IP 3 -R2 knock-out mice [42].Evaluation of tritiated IP 3 binding to the guinea pig heart found higher binding to the atrioventricular conducting system than to the myocardium [45].While such findings collectively raise the possibility that IP 3 may modulate AVN cellular electrophysiology, as yet there are no published data that address the question as to whether IP 3 influences AVN electrogenesis.Therefore, this study was undertaken to determine whether or not interventions targeting cellular IP 3 influence AVN spontaneous action potential (AP) rate using a well-established rabbit AVN cell preparation [6,17,46,47].

AVN Cell Isolation
Animal procedures were approved by the Animal Welfare and Ethics Review Board (AWERB) of the University of Bristol (AWERB 21/09/10,5.2.3 and 08/12/15,8.2) and conducted in accordance with UK legislation under consecutive project licences issued by the UK Home Office on 17 May 2011 and 21 April 2016.Adult male New Zealand White rabbits (2-3 kg) were killed humanely and their hearts rapidly excised.The AVN cell isolation method employed here has been described previously [15,46,47].Briefly, following excision, hearts were cannulated and secured to a Langendorff perfusion apparatus through which a series of isolation solutions were perfused, culminating in a collagenase-and protease-containing perfusate (for details, see [15,46,47]).Following enzyme perfusion, hearts were removed from the cannula and the entire AVN region within the Triangle of Koch was identified using anatomical landmarks and excised for enzymatic and mechanical dispersion of cells [15,46,47].Isolated cells were stored in Kraftbrühe (KB) solution until experimental use [15,46,48].

Electrophysiological Recording
For electrophysiological experiments, cells suspended in KB solution were placed in a recording chamber mounted on an inverted microscope (Nikon Eclipse TE2000-U, Tokyo, Japan).KB solution was replaced by gradual superfusion of Tyrode's solution containing (in mM): 140 NaCl, 4 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 glucose, and 5 HEPES (pH 7.4 using NaOH).Patch pipettes were pulled from borosilicate glass (AM Systems Inc., Sequim, WA, USA) and heat-polished to resistances of 2-3 MΩ.Pipettes were filled with a solution containing (in mM): 110 KCl, 10 NaCl, 10 HEPES, 0.4 MgCl 2 , 5 glucose, 5 K 2 ATP, and 0.5 Tris-GTP (pH 7.1 with KOH) [49,50].This solution was used for all action potential (AP) recordings.Maximum diastolic potential (MDP) was taken to be the most negative membrane potential following AP repolarization, and the slope of the diastolic depolarization was determined to be the average slope between the MDP and inflection point that marked the commencement of the AP upstroke.For recording I Ca, I Kr , and I f , 5 mM K 4 BAPTA was also included in this pipette solution [49,50].For selective recording of sodium-calcium exchange (NCX) current (I NCX ), the pipette solution contained (in mM) 110 CsCl, 10 NaCl, 0.4 MgCl 2 , 1 CaCl 2 , 5 EGTA, 10 HEPES, 5 glucose, and 20 TEACl (pH 7.2 with CsOH) [51].The external solution for I NCX recording was potassium-free Tyrode's solution containing 10 µM nitrendipine (to inhibit L-type calcium current) and 10 µM strophanthidin (to inhibit the Na + /K + pump) [26,51].Recordings were initiated with bath superfusion of cells.Once the whole-cell configuration had been obtained, control and test solutions were applied at 35-37 • C to the cell under investigation using a home-built, rapid solution exchange device [52].All electrophysiology recordings were made using an Axopatch 1D amplifier (Molecular Devices, Sunnyvale, CA, USA).Protocols were devised and applied using pClamp 10.3 software (Molecular Devices, Sunnyvale, CA, USA) via a Digidata 1322 (Molecular Devices, Sunnyvale, CA, USA) analogue-to-digital converter.Membrane currents were digitised at 10 kHz, with an appropriate bandwidth on the recording amplifier, whilst for APs, a digitization frequency of 2 kHz was used (cf.[50]).

Calcium Imaging
Most experiments in this study did not employ a Ca 2+ fluorophore in order to focus on AP recording in the absence of a potential [Ca 2+ ] i buffer.However, in one series of experiments (to investigate effects of Bt 3 -(1,4,5)IP 3 -AM), spontaneous Ca 2+ i transients were measured.For these, AVN myocytes were incubated in 2 µM Fluo-4 AM (Invitrogen, Paisley, UK; Cat No. F23917) at room temperature for 20-25 min, followed by replacement with normal Tyrode's solution for 30 min for de-esterification.Cells were then placed in the recording chamber on the stage of an LSM Pascal confocal microscope (Carl Zeiss, Jena, Germany).Fluo-4 was excited at 488 nm, with emitted fluorescence >505 nm [27].Line-scan images across the width of spontaneously beating AVN cells were obtained first in normal Tyrode's solution and then following application of Bt 3 -(1,4,5)IP 3 -AM.All confocal recordings were made at 37 • C. ImageJ (National Institutes of Health, Bethesda, MD, USA) was used for analysis.

Flash Photolysis
Caged-IP 3 (Sichem, Bremen, Germany.Cat No. cag-6-145) was included in the pipette solution.IP 3 was released from caged-IP 3 by UV flash photolysis using an OptoFlash (LED 365 nm; Cairn, Faversham, Kent, UK), which was fitted onto the microscope of patchclamping recordings.The UV flash duration was 100 ms, and intensity was 1.1 A. Single or multiple short series of flashes (up to 5) were triggered manually (about 1 s for each flash), whilst continuous repeated stimulation was automatically applied with a frequency of 1.3 Hz.

Experimental Compounds
Bt 3 -(1,4,5)IP 3 -AM (Sichem, Bremen, Germany, Cat No. 3-1-145) was dissolved in DMSO to produce a stock solution of 10 mM.In order to avoid potential confounding effects of switching artefacts and to allow some time for intracellular cleavage of the ester bond, effects of Bt 3 -(1,4,5)IP 3 -AM were evaluated after at least 110 s of its application.2-Aminoethyl diphenylborinate (2-APB, Sigma-Aldrich, Gillingham, UK, Cat No. D9754) was dissolved in DMSO to produce stock solutions of 10 and 1 mM.Xestospongin C (Xe-C, Tocris, Bristol, UK, Cat No. 1280) was dissolved in DMSO to produce a stock solution of 0.5 mM (100× stock).

Data Analysis and Statistics
Action potential and current analysis was performed using Clampfit 10.3 software (Molecular Devices, San Jose, CA, USA).Statistical analysis was performed using Microsoft Office 2016 (Professional edition) and Microsoft 365 (Version 2406) Excel (Microsoft Corporation, Redmond, WA, USA) and GraphPad Prism 7.0 and 10.2.0 (GraphPad Software, Boston, MA, USA).All data are expressed as means ± SEM.Statistical comparisons were made using two-sample paired, unpaired t-tests, one-sample t-tests, or ANOVA (or non-parametric equivalent) as appropriate.p < 0.05 was taken as statistically significant.

Immunohistochemistry
Immunocytochemistry was carried out using IP 3 -R2 and RyR2 antibodies and cell imaging using confocal microscopy.Figure 1 shows representative co-labelling with anti-IP 3 -R and RyR antibodies and also negative controls.IP 3 -R labelling is evident in transverse bands and at the cell periphery.Consistent with prior data [27], RyR labelling was also observed in transverse bands.The images in Figure 1A,B show partial overlap of IP 3 -R and RyR staining, where a magnified view in Figure 1B shows IP 3 -R labelling near the sarcolemma, RyRs, and nuclear envelope regions.Regularity of RyR and IP 3 -R labelling was quantified using Fourier analysis, for which spectral power at the first harmonic (~1.8 µm) is shown plotted in Figure 1D.For RyR2, this was 0.68 ± 0.03 (control = 0.21 ± 0.02), and for IP 3 -R2, this was 0.47 ± 0.03 (control = 0.22 ± 0.02).Co-localisation analysis yielded a Pearson's correlation coefficient of 0.31 ± 0.03 (for control images 0.20 ± 0.02; p < 0.05, Wilcoxon's rank sum).The Manders coefficient for RyR labelling overlap with IP 3 -R was 0.31 ± 0.03, and for IP 3 -R overlap with RyRs, it was 0.22 ± 0.03.In summary, rabbit AVN cells contain IP 3 -Rs, exhibiting a partial labelling overlap with RyRs.

Immunohistochemistry
Immunocytochemistry was carried out using IP3-R2 and RyR2 antibodies and cell imaging using confocal microscopy.Figure 1 shows representative co-labelling with anti-IP3-R and RyR antibodies and also negative controls.IP3-R labelling is evident in transverse bands and at the cell periphery.Consistent with prior data [27], RyR labelling was also observed in transverse bands.The images in Figure 1A and B show partial overlap of IP3-R and RyR staining, where a magnified view in Figure 1B shows IP3-R labelling near the sarcolemma, RyRs, and nuclear envelope regions.Regularity of RyR and IP3-R labelling was quantified using Fourier analysis, for which spectral power at the first harmonic (~1.8 µm) is shown plotted in Figure 1D.For RyR2, this was 0.68 ± 0.03 (control = 0.21 ± 0.02), and for IP3-R2, this was 0.47 ± 0.03 (control = 0.22 ± 0.02).Co-localisation analysis yielded a Pearson's correlation coefficient of 0.31 ± 0.03 (for control images 0.20 ± 0.02; p < 0.05, Wilcoxon's rank sum).The Manders coefficient for RyR labelling overlap with IP3-R was 0.31 ± 0.03, and for IP3-R overlap with RyRs, it was 0.22 ± 0.03.In summary, rabbit AVN cells contain IP3-Rs, exhibiting a partial labelling overlap with RyRs.

Effects of Cell Permeant IP 3 : Bt 3 -(1,4,5)IP 3 -AM
Membrane permeant esters of IP 3 have been devised [53] and are known to increase ventricular cardiomyocyte [Ca 2+ ] i (e.g., [54]) and to accelerate spontaneous AP rate in murine SAN myocytes [42].We therefore utilised this approach to determine the response of AVN cells to exogenously applied IP 3 .Figure 2 shows spontaneous APs from an AVN cell before and during exposure to 10 µM Bt 3 -(1,4,5)IP 3 -AM.In the example shown, following a brief switching artefact, spontaneous AP rate increased progressively over Cells 2024, 13, 1455 6 of 20 ~2 min in the presence of Bt 3 -(1,4,5)IP 3 -AM.This is particularly visible in the expanded time-base records shown in the lower panels of Figure 2 and mean data summarised in Table 1.Spontaneous AP rate was increased by 40.5 ± 5.7% (n = 10, p < 0.01) and the slope of diastolic depolarization was significantly increased (from 64.6 ± 8.4 mV s −1 to 102.3 ± 10.6 mV s −1 ; n = 10, p < 0.05).No significant differences were seen in maximal upstroke velocity, maximal repolarization velocity, or APD 50 (see Table 1).Despite a tendency for AP overshoot to be reduced following Bt 3 -(1,4,5)IP 3 -AM exposure, the overall reduction in AP amplitude did not attain statistical significance (Table 1).

Effects of Cell Permeant IP3: Bt3-(1,4,5)IP3-AM
Membrane permeant esters of IP3 have been devised [53] and are known to incre ventricular cardiomyocyte [Ca 2+ ]i (e.g., [54]) and to accelerate spontaneous AP rate in m rine SAN myocytes [42].We therefore utilised this approach to determine the response AVN cells to exogenously applied IP3. Figure 2 shows spontaneous APs from an AVN c before and during exposure to 10 µM Bt3-(1,4,5)IP3-AM.In the example shown, follow a brief switching artefact, spontaneous AP rate increased progressively over ~2 minu in the presence of Bt3-(1,4,5)IP3-AM.This is particularly visible in the expanded time-b records shown in the lower panels of Figure 2 and mean data summarised in Table Spontaneous AP rate was increased by 40.5 ± 5.7% (n = 10, p < 0.01) and the slope of di tolic depolarization was significantly increased (from 64.6 ± 8.4 mV s −1 to 102.3 ± 10.6 m s −1 ; n = 10, p < 0.05).No significant differences were seen in maximal upstroke veloc maximal repolarization velocity, or APD50 (see Table 1).Despite a tendency for AP ov shoot to be reduced following Bt3-(1,4,5)IP3-AM exposure, the overall reduction in AP a plitude did not attain statistical significance (Table 1).  1.   1. 71.5 ± 4.0 61.0 ± 4.4 APs were recorded using whole-cell patch clamp in control conditions and following ~2 min of exposure to Bt 3 -(1,4,5)IP 3 -AM.Comparisons were made using a paired t-test: * p < 0.05, ** p < 0.01 vs. control; (means ± SEM; n = 10).To complement the AP experiments, measurements of spontaneous Ca 2+ i transients were made from Fluo-4 loaded undialysed AVN cells (Figure 4; panel A shows control records, while panel B shows records following application of Bt3-(1,4,5)IP3-AM).The control spontaneous Ca 2+ i transient rate (1.34 ± 0.19 Hz; n = 7) was slower than those for spontaneous AP recordings shown in Table 1 and Figure 2, possibly as a result of Ca 2+ i buffering 2+

IP3-R Inhibition with Xestospongin C
We proceeded to determine effects on spontaneous AP generation of the IP3-R inhibitor xestospongin C (XeC; [55][56][57]).AVN cells were incubated in 5 µM XeC at room temperature for 1 h and 5 µM XeC was also included in the patch pipette solution.Figure 5 shows exemplar results.AP rate was evaluated immediately on commencing recording (within 30-60 s of attaining the whole-cell patch-clamp configuration) and monitored with time.After 1 min of recording, the spontaneous rate was reduced compared to that at commencement of recording.We then applied 10 µM Bt3-(1,4,5)IP3-AM, which in the absence of XeC increased spontaneous AP rate (Figure 2, Table 1).As shown in Figure 5, Bt3-(1,4,5)IP3-AM failed to increase AP rate following prior XeC treatment.Table 2 shows summary data from a total of 11 similar experiments, also including AP parameters for untreated cells (same as control data in Table 1).AP rate and slope of diastolic depolarization

IP 3 -R Inhibition with Xestospongin C
We proceeded to determine effects on spontaneous AP generation of the IP 3 -R inhibitor xestospongin C (XeC; [55][56][57]).AVN cells were incubated in 5 µM XeC at room temperature for 1 h and 5 µM XeC was also included in the patch pipette solution.Figure 5 shows exemplar results.AP rate was evaluated immediately on commencing recording (within 30-60 s of attaining the whole-cell patch-clamp configuration) and monitored with time.After 1 min of recording, the spontaneous rate was reduced compared to that at commencement of recording.We then applied 10 µM Bt 3 -(1,4,5)IP 3 -AM, which in the absence of XeC increased spontaneous AP rate (Figure 2, Table 1).As shown in Figure 5, Bt 3 -(1,4,5)IP 3 -AM failed to increase AP rate following prior XeC treatment.Table 2 shows summary data from a total of 11 similar experiments, also including AP parameters for untreated cells (same as control data in Table 1).AP rate and slope of diastolic depolarization after 1 min of recording were significantly less than measured at the start of recording.Comparison with AP parameters from untreated cells (Table 2) suggests that it was inclusion of XeC in the pipette solution rather than pre-incubation in external solution that was important for the inhibitory action of XeC on AP rate to be observed.While there was a trend for diastolic depolarization and AP upstroke velocity to be faster at the start of recording in XeC than in untreated cells, this did not attain statistical significance (p > 0.05 for both).In eight experiments in which Bt 3 -(1,4,5)IP 3 -AM was applied in the presence of XeC, there was no significant acceleration in AP rate (2.27 ± 0.25 Hz following Bt 3 -(1,4,5)IP 3 -AM compared to 1.94 ± 0.17 Hz in XeC; p = 0.27).

Effect of Photoreleased IP 3 on AP Rate
In a separate series of experiments, 100 µM caged IP 3 was introduced into cells via the pipette solution and was uncaged (to release IP 3 ) by UV flash photolysis.In the caged state, IP 3 is biologically inactive until photoreleased by exposure to UV light.We applied single, three, five, and repeated flashes of UV light in these experiments.Figure 6A shows that three successive flashes over ~3 s resulted in a transient, reversible acceleration in spontaneous AP rate.Figure 6B shows the cumulative effect of repeated UV flashes applied over approximately 20 s: with increasing numbers of flashes, the increase in spontaneous AP rate became larger.Figure 6C summarises mean results using differing extents of photostimulation: the extent of acceleration of AVN cell spontaneous AP rate increased as the extent of photostimulation was increased.This clearly demonstrates that exposure to one, three, and five flashes only partially and progressively photolysed caged IP 3 .Importantly, in cells that were not loaded with caged IP 3 , repeated application of UV flashes did not increase spontaneous AP rate (Figure 6C inset), indicating that the presence of caged IP 3 was required for UV stimulation to result in increased AVN cell AP rate.Table 3 summarises the effects of repeated UV flash stimulation of caged IP 3 .Of the AP parameters shown, only spontaneous rate and slope of diastolic depolarization were significantly increased by photoreleased IP 3 .

Effects of 2-APB
2-Aminoethoxydiphenyl borate (2-APB) exerts an inhibitory effect on constitutively active IP 3 -Rs [58][59][60] and has been utilised in the study of IP 3 -Rs in the SAN [42,43].In order to investigate whether constitutively active IP 3 -Rs may influence AVN cell rate, we applied 2-APB to spontaneously active AVN cells at two concentrations (10 µM and 1 µM).Representative results are shown in Figure 7A,B.At 10 µM, application of 2-APB rapidly led to a reduction in spontaneous AP rate, accompanied by marked depolarization of the maximal diastolic potential (MDP) and reduction in AP amplitude.Mean AP parameter data from these experiments are shown in Table 4, showing statistically significant decreases in rate (by 28.0 ± 4.3%; n = 7, p < 0.01), MDP, diastolic depolarization rate, maximal upstroke and repolarization velocities, and AP overshoot and amplitude and an increase in AP duration at 50% repolarization (APD 50 ).Application (in separate experiments) of a lower 2-APB concentration of 1 µM also led to marked reduction in AP spontaneous rate (by 25.2 ± 6.2%; n = 6, p < 0.01), with slowing of diastolic depolarization, a more modest reduction in MDP and AP upstroke velocity, and overshoot (see Table 4).
The pronounced effects of 2-APB on AP amplitude and time course, particularly at 10 µM, raised a question as to whether the compound might exert direct effects on AVN ion channels beyond effects on IP 3 signalling and hence reflect, for the purposes of this study, quite non-selective effects [29].As both I Ca,L and I Kr are critical for AVN cell activity and the observed AP effects were consistent with their partial inhibition, we tested effects of 2-APB on these two currents.Recordings of I Ca,L and I Kr were made as described in Methods.Representative currents are shown in Figure 8(Ai,Aii,Bi,Bii): marked current reductions in both I Ca,L and I Kr were evident in the presence of both 1 and 10 µM 2-APB.Mean current-voltage relationships for the two currents are included in Figure 8(Ci,Cii) for I Ca,L and Figure 8(Di,Dii) for I Kr , demonstrating reductions in each current over a range of test potentials.The higher 2-APB concentration reduced I Kr more strongly than the lower one, corresponding to the greater depolarization of MDP in AP recordings in 10 than 1 µM 2-APB.The inhibition of these two currents in the presence of a calcium chelator in the patch pipette solution is suggestive of direct inhibitory effects of 2-APB at the concentrations employed, which confounds explanation of the compound's effects on APs as being due to just IP 3 -R inhibition.4. The pronounced effects of 2-APB on AP amplitude and time course, particularly at 10 µM, raised a question as to whether the compound might exert direct effects on AVN

Effect of Photoreleased IP 3 under AP Voltage Clamp
In a final series of experiments, we investigated the effect of repeated photostimulation under AP voltage clamp (Figure 9).A standardised template sequence of 4 AVN APs (lower panel of Figure 9B) was used as the voltage command, with the recording solutions as used for AP recording.The AP command series was applied first in control conditions and then following repeated application of UV flashes.Figure 9A shows superimposed currents in control and following photorelease of IP 3 , while Figure 9B shows the IP 3 -activated current (obtained by subtraction of control from +IP 3 currents), aligned with the AP command series.As highlighted by the vertical dashed lines, an inward IP 3 -activated current was observed during the diastolic depolarization phase of the AP series.In six experiments, the mean maximal amplitude of this inward current was −0.56 ± 0.05 pA/pF.The reversal potential of this current was −35.4 ± 1.9 mV (n = 6).We also calculated the integral of the IP 3 -activated current during the diastolic depolarization phase (the time period from the MDP to the initiation of the AP upstroke phase) and found this to be −36.6 ± 3.3 fC/pF (n = 6).When comparable measurements were made without caged-IP 3 in the patch pipette, the repeated flash-sensitive current integral during diastolic depolarization was −0.45 ± 3.7 fC/pF (n = 7; p < 0.01 vs. result with caged IP 3 ).Thus, the inward current during diastolic depolarization was attributable to IP 3 release and not UV excitation per se.
of the IP3-activated current during the diastolic depolarization phase (the time period from the MDP to the initiation of the AP upstroke phase) and found this to be −36.6 ± 3.3 fC/pF (n = 6).When comparable measurements were made without caged-IP3 in the patch pipette, the repeated flash-sensitive current integral during diastolic depolarization was −0.45 ± 3.7 fC/pF (n = 7; p < 0.01 vs. result with caged IP3).Thus, the inward current during diastolic depolarization was attributable to IP3 release and not UV excitation per se.It has been established that both atrial and ventricular myocytes express IP 3 -Rs, with IP 3 -R2 predominating over other isoforms and with higher atrial than ventricular IP 3 -R levels [30].Co-staining of atrial myocytes for RyR2 and IP 3 -R2 showed subsarcolemmal IP 3 -Rs co-localised with RyRs [30].In ventricular myocytes, IP 3 -R labelling exhibits z-line regularity and co-localisation with RyRs [34,35].Atrial myocytes exhibit less regularity in IP 3 -R staining patterns [30,43].Investigation of the mouse heart has shown the presence of mRNA for all three IP 3 -R types in pacemaker regions [42], with Western blot confirming expression of both IP 3 -R1 and IP 3 -R2 at the protein level in both SAN and AVN (with higher expression of IP 3 -R2) [42].In murine SAN cells, IP 3 -R2 labelling showed some overlap with that for SERCA2a, which was used as an SR marker and exhibited a sarcomeric labelling pattern [42].Subsarcolemmal IP 3 -R2 labelling that showed some co-localisation with RyR2 also detected in SAN cells [42].Our data are broadly compatible with earlier findings and demonstrate partial overlap between RyR2 and IP 3 -R2 staining in AVN cells.

I Ca,L and I Kr Inhibition by 2-APB
2-APB was the first candidate for a membrane penetrant IP 3 -R inhibitor, with a reported IC 50 for inhibition of IP 3 -induced Ca 2+ -release from cerebellar microsomal prepa-of 42 µM [59].2-APB has been used to investigate atrial IP 3 -R signalling [36][37][38] and at low (2.5-5) µM concentrations to probe the role of IP 3 -R in the SAN [42,43].It is known that 2-APB can exert effects on calcium-release activated current (I CRAC ) [61] and members of the transient receptor potential (TRP) channel family [62].However, under the conditions of the present study, 2-APB was found to inhibit I Ca,L and I Kr in experiments in which [Ca 2+ ] i was controlled by incorporation of BAPTA in the patch pipette.These effects occurred at concentrations that overlap those used to study IP 3 -R and are reminiscent of similar of inhibition of I Ca,L and I Kr from AVN cells by the cation channel inhibitor SKF-96365 [50].2-APB has been reported to activate members of the two-pore K + channel family (TREK-1, TREK-2 and TRAAK) [63] and recently the Kv1.x family [64].However, we are unaware of any previous report of inhibition of cardiac I Kr or its molecular counterpart, hERG, by 2-APB.Due to the effects on I Ca,L and I Kr seen here, we are unable reliably to attribute slowing of spontaneous AP rate in AVN cells by 2-APB to inhibition of IP 3 -R alone.Significantly, our results with 2-APB extend the information available on non-IP 3 -R mediated effects of the compound and urge caution in its use to study the role(s) of IP 3 -R in modulating cardiac pacemaker cell excitability.

Evidence for Constitutive and IP 3 -R Activity in Modulating AVN Cell Rate
In contrast to our results with 2-APB, we observed no evidence for direct effects of Bt 3 -(1,4,5)IP 3 -AM on I Kr and I Ca,L under recording conditions comparable to those in which 2-APB was examined.Neither I f nor I NCX were directly affected by this intervention.It is therefore notable that Bt 3 -(1,4,5)IP 3 -AM application led to ~40% increase in spontaneous AP rate in our experiments, and in separate experiments also increased the rate of spontaneous Ca 2+ i transients (by >30%).When applied to murine SAN cells, IP 3 -BM increased spontaneous Ca 2+ i transient rate by 13% [42].Direct, quantitative comparison of that finding with our study is difficult because of the different species employed.However, qualitative comparison leads to our conclusion that introducing cell-permeant IP 3 accelerates the spontaneous rate of both SAN and AVN myocytes.
We are unaware of prior studies that have used XeC to study cardiac pacemaker tissue activity.Although XeC is a costly reagent, we applied it both externally (through pre-incubation) and via the patch pipette to ensure adequate exposure.This approach was serendipitous, as we observed little difference between the spontaneous rate of untreated myocytes and that of pre-treated AVN myocytes at commencement of recording.By contrast, once the whole-cell recording mode had been obtained, XeC entry into cells via the patch pipette was associated with a rapid and marked reduction in AP rate and slope of diastolic depolarization.That this effect is attributable to inhibition of IP 3 -Rs is supported by the observation that subsequent Bt 3 -(1,4,5)IP 3 -AM application to cells dialysed with XeC-containing pipette solution failed to accelerate the rate.Thus, our results with XeC support roles for constitutively generated IP 3 and raised levels of IP 3 in modulating AVN cell spontaneous AP rate.
A study of IP 3 inhibitor effects on non-cardiac cells has claimed that heparin is more effective at inhibiting IP 3 -Rs than other inhibitors [65].In additional experiments (not shown), we tested the effects of inclusion of heparin (5 mg/mL) in the pipette solution.This had a profound effect on AP morphology and produced a profound hyperpolarization of MDP, leading to quiescence in 8 out of 10 cells within 2 min.While this might be considered to be consistent with a major role for IP 3 -Rs in AVN cell pacemaking, the marked effects on multiple AP parameters and complete quiescence led us to consider that the use of heparin was problematic in this application.
Recent work on effects of IP 3 release on atrial Ca 2+ transients has highlighted the utility of the use of caged-IP 3 [43].Perhaps some of the most compelling evidence for IP 3 modulation of spontaneous AP rate in AVN cells in the present study comes from the use of this approach: the extent of observed AP rate acceleration depended on the extent of UV stimulation supplied (and was independent of UV excitation alone).UV photolysis of caged-IP 3 increased spontaneous AP rate by up to 30% after multiple flashes, which similar to the effects of Bt 3 -(1,4,5)IP 3 -AM application.Under AP voltage clamp, the current activated by caged-IP 3 was inwardly directed during the diastolic depolarization, explaining the slowing of AP rate in other experiments.While detailed investigation of the underlying basis for this current was beyond the scope of this investigation, in preliminary AP clamp experiments in which 20 µM nifedipine was applied, inward current was still observed during diastolic depolarization following release of IP 3 from caged IP 3 .

Limitations, Future Work and Conclusions
Through adopting multiple approaches to the manipulation IP 3 in AVN cells, this study demonstrates for the first time that IP 3 modulates spontaneous AVN AP generation.Our data suggest roles for both constitutive IP 3 -R activation and IP 3 changes in modulating spontaneous AP rate in pacemaking cells from this cardiac region.While this study constitutes important proof-of-concept evidence in this regard, our results leave open questions as to how AVN cell IP 3 is changed and how consequent alterations in AP rate arise physiologically.For example, ET A receptor activation is known to increase IP 3 [66] and in murine SAN cells, application of 100 nM ET-1 increased spontaneous Ca 2+ i transient rate and diastolic [Ca 2+ ] i [42].
As highlighted by others, responsiveness to intervention(s) that raise IP 3 without G q -coupled receptor activation demonstrates effects of IP 3 signalling that are independent of the activation of DAG (diacylglycerol) [43].At 10 nM, ET-1 rapidly abolishes AVN cell spontaneous activity via activation of a tertiapin-Q sensitive K + current [49].Intriguingly, AVN cells rendered quiescent by ET-1 exhibit small-amplitude spontaneous membrane potential oscillations and it is conceivable that such events involve IP 3 -R mediated Ca 2+ i mobilisation [49] and a balance between such an action and inhibitory effects of ET-1 might vary with ET-1 concentration.Future work is certainly warranted, both to reveal the mechanism(s) by which IP 3 mobilisation accelerates spontaneous AVN rate and to investigate the role of G-protein-coupled receptor activation in AVN cells in mobilizing IP 3 to modulate AVN excitability.
In this study, we focused on spontaneous AP measurement with only one series of experiments involving measurement of [Ca 2+ ] i .The rationale for this was twofold: (i) AP measurements monitor directly an electrophysiological end point and (ii) omission of a Ca 2+ fluorophore allows interrogation of IP 3 effects without potential Ca 2+ i -buffering effects due to introduction of a Ca 2+ indicator: the slower spontaneous rate of Ca 2+ i transients (Figure 4) than of spontaneous APs in whole-cell recording seems to vindicate this decision.Nevertheless, having established that IP 3 modulates spontaneous AP activity in AVN cells, it would be desirable for future studies to incorporate extensive [Ca 2+ ] i measurements, particularly as these would enable scrutiny of subcellular mechanisms of IP 3 action in the AVN (cf.[28,42]).Our AP clamp experiments demonstrate that increasing intracellular IP 3 activates an inward current during diastolic depolarization.The results of our experiments on Bt 3 -(1,4,5)IP 3 -AM suggest a lack of direct effect of IP 3 on AVN cell I NCX , but they do not preclude an indirect effect in which increased [Ca 2+ ] i activates inward I NCX .While the most likely candidate for IP 3 activated inward current may be I NCX [24,26,27], this cannot be confirmed without direct experimental evidence, and future work is required to determine the identity of this current.In studying effects of 2-APB on AVN I Ca,L and I Kr , we identified off-target effects of the compound that confound its use for studying IP 3 -R in this cardiac cell type.We did not study effects of 2-APB on other ionic currents, however, and cannot preclude the possibility that 2-APB may exert additional non-selective effects on AVN cells.

Conclusions
This is the first experimental investigation to report functional evidence for a role of IP 3 in AVN.Our key conclusion is that IP 3 can modulate AVN cell excitability: interventions that increase intracellular IP 3 were found to increase AVN cell spontaneous AP rate, while interventions expected to inhibit IP 3 -R were found to decrease AVN cell spontaneous AP rate.This information advances our knowledge of the electrophysiology of this region of heart and lays a foundation for future work.This study also provides new evidence for cardiac I Ca,L and I Kr inhibition by 2-APB, which adds to accumulating evidence that 2-APB has severe limitations as a tool with which to study cardiac IP 3 -Rs.

Bt 3 -
(1,4,5)IP 3 -AM was also applied in voltage clamp experiments in which I Ca,L , I Kr and the 'funny' current I f were measured (see Methods).Figure3Ashows closely superimposed records of I Ca,L in control and Bt 3 -(1,4,5)IP 3 -AM.Figure3Bshows overlaid current records at the end of the +20 mV command and on repolarization to −40 mV.The deactivating tail current at −40 mV shown in Figure3Bhas been demonstrated previously to represent AVN I Kr , as it is abolished by the selective I Kr inhibitor E-4031, with little evidence for the slow delayed rectifier current, I Ks , in rabbit AVN cells[13,46,47].Bt 3 -(1,4,5)IP 3 -AM did not alter I Kr tail amplitude.Figure3Cshows currents elicited on hyperpolarization to more negative voltages from the holding potential of −40 mV, showing little effect of Bt 3 -(1,4,5)IP 3 -AM on the time-dependent inward current, I f .Mean data for I CaL , I Kr and I f are shown in Figure 3E, demonstrating no significant effect of Bt 3 -(1,4,5)IP 3 -AM on the three currents.Figure 3D shows currents elicited by a descending voltage ramp protocol that was applied under selective recording conditions for I NCX .Again, currents in control solution and in Bt 3 -(1,4,5)IP 3 -AM were found to be closely superimposed.5 mM Ni 2+ was applied at the end of the recording period to confirm the identity of the measured current as I NCX , with only a small residual Ni 2+ -insensitive current visible.No change in this I NCX was observed following Bt 3 -(1,4,5)IP 3 -AM (Figure 3D,E).Consequently, the dominant effect of Bt 3 -(1,4,5)IP 3 -AM exposure was an acceleration in spontaneous AP rate, without confounding direct effects on I Kr , I Ca,L , I f or I NCX .Cells 2024, 13, x FOR PEER REVIEW 8 of 23

Figure 5 .
Figure 5.Effect of xestospongin C (Xe-C) on AVN spontaneous AP rate.(A) Trace shows a slow time-base recording of spontaneous APs.Horizontal bars indicate periods of 5 µM Xe-C in pipette solution (continuously) and subsequent external application of 10 µM Bt 3 -(1,4,5)IP 3 -AM.(B) Faster time-base extracts from the experiment showing 3 time points: at start of recording, after ~1 min recording, and after 2 min of subsequent Bt 3 -(1,4,5)IP 3 -AM application.

Figure 7 .
Figure 7. Effects of 2-APB on AVN spontaneous APs.(A) Upper panel shows a slow time-base recording (over a period of 100 s) showing the effect of rapid application of 10 µM 2-APB to a spontaneously active AVN cell.Lower panel shows faster time-base extract of APs in control (left) and in the presence (right) of 10 µM 2-APB.(B) Upper panel shows a slow time-base recording (over a period of 100 s) showing the effect of rapid application of 1 µM 2-APB to a spontaneously active AVN cell.Lower panel shows faster time-base extract of APs in control (left) and in the presence (right) of 1 µM 2-APB.Mean AP parameters are given in Table4.

Figure 7 .Figure 8 Figure 8 .
Figure 7. Effects of 2-APB on AVN spontaneous APs.(A) Upper panel shows a slow time-base recording (over a period of 100 s) showing the effect of rapid application of 10 µM 2-APB to a spontaneously active AVN cell.Lower panel shows faster time-base extract of APs in control (left) and in the presence (right) of 10 µM 2-APB.(B) Upper panel shows a slow time-base recording (over a period of 100 s) showing the effect of rapid application of 1 µM 2-APB to a spontaneously active AVN cell.Lower panel shows faster time-base extract of APs in control (left) and in the presence (right) of 1 µM 2-APB.Mean AP parameters are given in Table 4. Cells 2024, 13, x FOR PEER REVIEW 16 of

Figure 9 .
Figure 9.Effect of photoreleased IP3 on current during AP voltage clamp.(A) Superimposed net currents recorded from an AVN cell in control solution (black) and following repetitive UV excitation to photorelease caged IP3.Experimental protocol is shown vertically aligned with these traces in lower panel of (B).(B) IP3-sensitive current obtained by subtraction of control from UV-flash

Figure 9 .
Figure 9.Effect of photoreleased IP 3 on current during AP voltage clamp.(A) Superimposed net currents recorded from an AVN cell in control solution (black) and following repetitive UV excitation to photorelease caged IP 3 .Experimental protocol is shown vertically aligned with these traces in lower panel of (B).(B) IP 3 -sensitive current obtained by subtraction of control from UV-flash release current in 'A'.Vertical dashed lines highlight the diastolic depolarization phase during one cycle of spontaneous activity.Note the inward IP 3 -sensitive current during this phase.
. IP 3 -R2s in the Rabbit AVN

Table 2 .
Effects of 5 µM xestospongin C on spontaneous action potentials in rabbit AVN cells.

Table 2 .
Effects of 5 µM xestospongin C on spontaneous action potentials in rabbit AVN cells.

Table 3 .
Effects of UV flash photolysis liberation of caged IP 3 on spontaneous action potential rate in rabbit AVN cells.

Table 4 .
Effect of 2-APB on spontaneous action potentials from rabbit AVN cells.APs were recorded using whole-cell patch clamp in control conditions and in the presence of the two concentrations of 2-APB shown (10 µM, n = 7; 1 µM, n = 6).Note each concentration has its own paired control.Comparisons were made using a paired t-test: Paired t-test: * p < 0.05, ** p < 0.01 vs. control.

Table 4 .
Effect of 2-APB on spontaneous action potentials from rabbit AVN cells.