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

Abstract

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 I_Ca,L, I_Kr, I_f or I_NCX. 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 I_Ca,L and I_Kr, 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.

1. 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 Ca²⁺ ‘clock’ also contributes to generation of spontaneous activity (for reviews, see [20,21]). In respect of the AVN, inhibition of sarcoplasmic reticulum (SR) Ca²⁺ 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 Ca²⁺ release from the SR coupled to Ca²⁺ modulation of electrogenesis at the cell surface membrane.

Inositol 1,4,5 trisphosphate (IP₃) is a ubiquitous signalling molecule produced from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) by phospholipase C (PLC) that translocates from the membrane to the cytoplasm. It is well established that IP₃ releases Ca²⁺ from intracellular stores via IP₃ receptors (IP₃-Rs) [28,29]. Although cardiac myocytes generally express a much higher proportion of RyRs than IP₃-Rs (~100:1), there is evidence that IP₃-Rs mediate subsarcolemmal, cytoplasmic, and nuclear Ca²⁺ signalling in the heart (for reviews, see [28,29]). The lower cardiac expression of IP₃-Rs compared to RyRs does not preclude IP₃-Rs from playing a role in setting SR Ca²⁺ levels, since RyRs are open for only ~20 ms during the cardiac cycle, while an SR leak via IP₃-R could be continuous [28]. Moreover, the leak via IP₃-Rs may be amplified by adjacent RyRs to produce Ca²⁺ sparks [28]. In ventricular myocytes, IP₃-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 Ca²⁺ channels (LTCCs) and RyRs in ventricular myocyte dyads results in Ca²⁺ release from the SR when LTCCs open: IP₃-R activation increases dyadic Ca²⁺ fluxes during Ca²⁺ transients and increases the Ca²⁺ spark rate [35]. Further, cross-talk between RyRs and IP₃-Rs increases in heart failure, which may facilitate arrhythmogenesis [34]. In atrial myocytes, IP₃-Rs are largely localised with peripheral, junctional SR [30,36], where their activity can impinge on electrical and RyR Ca²⁺ signalling. Furthermore, IP₃-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]). IP₃-R stimulation has been associated with abnormal automaticity and spontaneous activity in atrial and pulmonary vein cardiomyocytes [36,37,38,39] and with Ca²⁺ waves in subendocardial Purkinje cells following coronary occlusion [40]. IP₃-R inhibition has also been reported to inhibit adrenaline-mediated changes in amphibian cardiac pacemaker (sinus venosus) [41]. Notably, a modulatory role for IP₃-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 IP₃-R isoforms [42], with the predominant isoform being IP₃-R2 [42]—this isoform has the highest IP₃ affinity [44]. Application of a membrane-permeant form of IP₃ to mouse SAN increased spontaneous Ca²⁺ transient rate and Ca²⁺ spark frequency, whilst the IP₃-inhibitor 2-aminoethoxydiphenyl borate (2-APB) decreased Ca²⁺ transient amplitude and rate [42]. Similar effects were not observed in preparations from IP₃-R2 knock-out mice [42]. Evaluation of tritiated IP₃ 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 IP₃ may modulate AVN cellular electrophysiology, as yet there are no published data that address the question as to whether IP₃ influences AVN electrogenesis. Therefore, this study was undertaken to determine whether or not interventions targeting cellular IP₃ influence AVN spontaneous action potential (AP) rate using a well-established rabbit AVN cell preparation [6,17,46,47].

2. Materials and Methods

2.1. 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].

2.2. 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 CaCl₂, 1 MgCl₂, 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 MgCl₂, 5 glucose, 5 K₂ATP, 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 K₄BAPTA 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 MgCl₂, 1 CaCl₂, 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]).

2.3. Calcium Imaging

Most experiments in this study did not employ a Ca²⁺ fluorophore in order to focus on AP recording in the absence of a potential [Ca²⁺]_i buffer. However, in one series of experiments (to investigate effects of Bt₃-(1,4,5)IP₃-AM), spontaneous Ca²⁺_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 Bt₃-(1,4,5)IP₃-AM. All confocal recordings were made at 37 °C. ImageJ (National Institutes of Health, Bethesda, MD, USA) was used for analysis.

2.4. Flash Photolysis

Caged-IP₃ (Sichem, Bremen, Germany. Cat No. cag-6-145) was included in the pipette solution. IP₃ was released from caged-IP₃ by UV flash photolysis using an OptoFlash (LED 365 nm; Cairn, Faversham, Kent, UK), which was fitted onto the microscope of patch-clamping 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.

2.5. Immunohistochemistry

AVN cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilised with 0.1% Triton X-100 for 10 min, then blocked with 10% goat serum for 1 h at room temperature. The cells were then incubated overnight at 4 °C with 2% goat serum containing primary antibodies of rabbit anti-IP₃-R2 (1:25; Alomone labs, Jerusalem, Israel, Cat No. ACC-116) and mouse anti-RyR2 (1:100; Invitrogen, Paisley, UK; Cat No. MA3-916) or no primary antibody as negative control. Secondary antibodies were incubated for 2 h at room temperature with goat anti-rabbit Alexa Fluor 488 (1:200; Invitrogen, Cat No. A-11008) and goat anti-mouse Alexa Fluor 594 (1:200; Invitrogen, Cat No. A-11005). The cells were mounted in Vectashield mounting medium (Vector Laboratories, Newark, CA, USA) on slides. The IP₃-R- and RyR-labelled AVN cells were imaged with an LSM880 confocal microscope (Carl Zeiss, Jena, Germany) using a 63× (NA 1.4, oil immersion) objective sampled at ~0.15 μm/pixel and z-stacks (~0.4 μm step size) obtained. Signals were detected at 600–650 nm under 594 nm laser illumination, followed by detection at 495–550 nm under 488 nm laser illumination. Confocal images were deconvolved using the Richardson–Lucy algorithm, with fast Fourier transform (FFT), Pearson, and Mander analyses performed in MATLAB (R2023a, Mathworks, Natick, MA, USA).

2.6. Experimental Compounds

Bt₃-(1,4,5)IP₃-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 Bt₃-(1,4,5)IP₃-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).

2.7. 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.

3. Results

3.1. 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,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.

3.2. Effects of Cell Permeant IP₃: Bt₃-(1,4,5)IP₃-AM

Membrane permeant esters of IP₃ have been devised [53] and are known to increase ventricular cardiomyocyte [Ca²⁺]_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 IP₃. Figure 2 shows spontaneous APs from an AVN cell before and during exposure to 10 µM Bt₃-(1,4,5)IP₃-AM. In the example shown, following a brief switching artefact, spontaneous AP rate increased progressively over ~2 min in the presence of Bt₃-(1,4,5)IP₃-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. Effects of 10 µM Bt₃-(1,4,5)IP₃-AM on spontaneous action potentials in rabbit AVN cells.

Parameter	Control	10 µM Bt3-(1,4,5)IP3-AM
Spontaneous AP rate (beats s−1) (Percentage increase %)	2.85 ± 0.17	3.96 ± 0.21 ** (40.5 ± 5.7%)
Slope of pacemaker diastolic depolarization (mV s−1)	64.6 ± 8.4	102.3 ± 10.6 *
Maximal upstroke velocity (Vmax, V s−1)	4.67 ± 0.80	3.89 ± 1.14
Maximal repolarization velocity (Vrep, V s−1)	−1.22 ± 0.08	−1.09 ± 0.08
AP duration at 50% repolarization (APD50, ms)	77.4 ± 3.5	73.3 ± 3.8
Maximal diastolic potential (MDP, mV)	−51.8 ± 2.8	−49.4 ± 1.9
Overshoot (mV)	20.6 ± 2.1	11.6 ± 2.9 *
AP amplitude (mV)	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 Bt₃-(1,4,5)IP₃-AM. Comparisons were made using a paired t-test: * p < 0.05, ** p < 0.01 vs. control; (means ± SEM; n = 10).

. 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 APD₅₀ (see Table 1). Despite a tendency for AP overshoot to be reduced following Bt₃-(1,4,5)IP₃-AM exposure, the overall reduction in AP amplitude did not attain statistical significance (Table 1).

Bt₃-(1,4,5)IP₃-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). Figure 3A shows closely superimposed records of I_Ca,L in control and Bt₃-(1,4,5)IP₃-AM. Figure 3B shows 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 Figure 3B has 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]. Bt₃-(1,4,5)IP₃-AM did not alter I_Kr tail amplitude. Figure 3C shows currents elicited on hyperpolarization to more negative voltages from the holding potential of −40 mV, showing little effect of Bt₃-(1,4,5)IP₃-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 Bt₃-(1,4,5)IP₃-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 Bt₃-(1,4,5)IP₃-AM were found to be closely superimposed. 5 mM Ni²⁺ 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 Ni²⁺-insensitive current visible. No change in this I_NCX was observed following Bt₃-(1,4,5)IP₃-AM (Figure 3D,E). Consequently, the dominant effect of Bt₃-(1,4,5)IP₃-AM exposure was an acceleration in spontaneous AP rate, without confounding direct effects on I_Kr, I_Ca,L, I_f or I_NCX.

To complement the AP experiments, measurements of spontaneous Ca²⁺_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 Ca²⁺_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 Ca²⁺_i buffering by the Ca²⁺ fluorophore used for these experiments. Nevertheless, spontaneous rate increased (to 1.78 ± 0.2 Hz; n = 7, p < 0.05) following Bt₃-(1,4,5)IP₃-AM exposure. Over the time of laser scanning in the continuous presence of Bt₃-(1,4,5)IP₃-AM, there was a small upward shift in diastolic fluorescence that overall was not statistically significant (by 16.6 ± 10.0%, p > 0.05, n = 7). Ca²⁺_i transient amplitude did not change significantly (F/F0: 2.25 ± 0.56 in control, and 2.13 ± 0.60 with Bt₃-(1,4,5)IP₃-AM; p > 0.05, n = 7), nor did the peak of the Ca²⁺_i transient (F/F0: 3.25 ± 0.56 in control, and 3.28 ± 0.70 with Bt₃-(1,4,5)IP₃-AM; p > 0.05, n = 7).

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

Parameter	Untreated Cells	Xestospongin C (Start of Recording)	Xestospongin C (~1 min of Recording)
Spontaneous AP rate (beats s−1) (Percentage decrease %)	2.85 ± 0.17	2.63 ± 0.14 (7.7%, vs. untreated cells)	1.94 ± 0.17 * ## (23.1 ± 8.7%, vs. Xe-C At Start)
Slope of pacemaker diastolic depolarization (mV s−1)	64.6 ± 8.4	97.8 ± 13.2	51.0 ± 5.2 **
Maximal upstroke velocity (Vmax, V s−1)	4.67 ± 0.80	8.66 ± 1.30	8.47 ± 1.42 #
Maximal repolarization velocity (Vrep, V s−1)	−1.22 ± 0.08	−1.61 ± 0.16	−1.49 ± 0.12
AP duration at 50% repolarization (APD50, ms)	77.4 ± 3.5	84.7 ± 3.5	83.5 ± 3.1
Maximal diastolic potential (MDP, mV)	−51.8 ± 2.8	−53.4 ± 2.1	−57.6 ± 2.3 **
Overshoot (mV)	20.6 ± 2.1	31.8 ± 2.2	30.5 ± 2.2 ##
AP amplitude (mV)	71.5 ± 4.0	85.2 ± 4.0	88.1 ± 4.3 #

APs with XeC containing pipette solution were recorded using whole-cell patch clamp immediately after gaining whole-cell access and ~1 min after gaining access (n = 11). For comparison, AP parameters for cells untreated with XeC (same data as Table 1 control, n = 10) are included. Paired t-test: * p < 0.05, ** p < 0.01, Xe-C after ~1 min vs. Xe-C at start; group t-test: ^# p < 0.05, ^## p < 0.01, Xe-C after ~1 min vs. untreated cells.

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 Bt₃-(1,4,5)IP₃-AM was applied in the presence of XeC, there was no significant acceleration in AP rate (2.27 ± 0.25 Hz following Bt₃-(1,4,5)IP₃-AM compared to 1.94 ± 0.17 Hz in XeC; p = 0.27).

3.4. Effect of Photoreleased IP₃ on AP Rate

In a separate series of experiments, 100 µM caged IP₃ was introduced into cells via the pipette solution and was uncaged (to release IP₃) by UV flash photolysis. In the caged state, IP₃ 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 IP₃. Importantly, in cells that were not loaded with caged IP₃, repeated application of UV flashes did not increase spontaneous AP rate (Figure 6C inset), indicating that the presence of caged IP₃ was required for UV stimulation to result in increased AVN cell AP rate.

Table 3. Effects of UV flash photolysis liberation of caged IP₃ on spontaneous action potential rate in rabbit AVN cells.

Parameter	Control	With UV Excitation of Caged IP3
Spontaneous AP rate (beats s−1) (Percentage increase %)	2.30 ± 0.37	2.90 ± 0.36 * (32.1 ± 9.5%)
Slope of pacemaker diastolic depolarization (mV s−1)	73.4 ± 17.6	110.5 ± 14.0 *
Maximal upstroke velocity (Vmax, V s−1)	6.55 ± 1.49	7.73 ± 1.66
Maximal repolarization velocity (Vrep, V s−1)	−1.86 ± 0.15	−1.82 ± 0.17
AP duration at 50% repolarization (APD50, ms)	69.1 ± 5.1	71.0 ± 4.5
Maximal diastolic potential (MDP, mV)	−65.8 ± 1.9	−65.3 ± 2.3
Overshoot (mV)	26.1 ± 4.5	27.9 ± 4.4
AP amplitude (mV)	91.9 ± 4.5	93.2 ± 4.3

APs were recorded using whole-cell patch clamp in control conditions and following repetitive application of UV flash excitation to release IP₃ from caged IP₃ (100 µM in pipette solution). Comparisons were made using a paired t-test: * p < 0.05, control vs. UV flash (mean ± SEM; n = 7).

summarises the effects of repeated UV flash stimulation of caged IP₃. Of the AP parameters shown, only spontaneous rate and slope of diastolic depolarization were significantly increased by photoreleased IP₃.

3.5. Effects of 2-APB

2-Aminoethoxydiphenyl borate (2-APB) exerts an inhibitory effect on constitutively active IP₃-Rs [58,59,60] and has been utilised in the study of IP₃-Rs in the SAN [42,43]. In order to investigate whether constitutively active IP₃-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. Effect of 2-APB on spontaneous action potentials from rabbit AVN cells.

Parameter	Control	10 μM 2-APB	Control	1 μM 2-APB
Spontaneous AP rate (beats s−1) (Percentage decrease %)	3.10 ± 0.36	2.28 ± 0.37 ** (28.0 ± 4.3%)	2.78 ± 0.22	2.07 ± 0.22 ** (25.2 ± 6.2%)
Slope of pacemaker diastolic depolarization (mV s−1)	79.2 ± 18.9	24.6 ± 7.7 **	82.0 ± 9.2	34.8 ± 3.6 **
Maximal upstroke velocity (Vmax, V s−1)	5.50 ± 1.05	0.78 ± 0.12 **	7.03 ± 2.14	4.55 ± 1.54 **
Maximal repolarization velocity (Vrep, V s−1)	−1.34 ± 0.11	−0.66 ± 0.05 **	−2.49 ± 0.50	−2.43 ± 0.51
AP duration at 50% repolarization (APD50, ms)	68.4 ± 3.1	117.8 ± 11.6 **	75.2 ± 5.7	90.7 ± 5.0 **
Maximal diastolic potential (MDP, mV)	−51.8 ± 2.5	−38.9 ± 3.4 **	−62.7 ± 2.8	−58.2 ± 3.5 *
Overshoot (mV)	24.5 ± 3.5	−0.19 ± 3.1 **	23.0 ± 4.3	13.6 ± 5.2 **
AP amplitude (mV)	76.3 ± 5.5	38.7 ± 3.0 **	85.5 ± 6.6	71.8 ± 8.4 **

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.

, 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 (APD₅₀). 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 IP₃ 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 IP₃-R inhibition.

3.6. Effect of Photoreleased IP₃ 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 IP₃, while Figure 9B shows the IP₃-activated current (obtained by subtraction of control from +IP₃ currents), aligned with the AP command series. As highlighted by the vertical dashed lines, an inward IP₃-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 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.

4. Discussion

4.1. IP₃-R2s in the Rabbit AVN

It has been established that both atrial and ventricular myocytes express IP₃-Rs, with IP₃-R2 predominating over other isoforms and with higher atrial than ventricular IP₃-R levels [30]. Co-staining of atrial myocytes for RyR2 and IP₃-R2 showed subsarcolemmal IP₃-Rs co-localised with RyRs [30]. In ventricular myocytes, IP₃-R labelling exhibits z-line regularity and co-localisation with RyRs [34,35]. Atrial myocytes exhibit less regularity in IP₃-R staining patterns [30,43]. Investigation of the mouse heart has shown the presence of mRNA for all three IP₃-R types in pacemaker regions [42], with Western blot confirming expression of both IP₃-R1 and IP₃-R2 at the protein level in both SAN and AVN (with higher expression of IP₃-R2) [42]. In murine SAN cells, IP₃-R2 labelling showed some overlap with that for SERCA2a, which was used as an SR marker and exhibited a sarcomeric labelling pattern [42]. Subsarcolemmal IP₃-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 IP₃-R2 staining in AVN cells.

4.2. I_Ca,L and I_Kr Inhibition by 2-APB

2-APB was the first candidate for a membrane penetrant IP₃-R inhibitor, with a reported IC₅₀ for inhibition of IP₃-induced Ca²⁺-release from cerebellar microsomal preparations of 42 µM [59]. 2-APB has been used to investigate atrial IP₃-R signalling [36,37,38] and at low (2.5–5) µM concentrations to probe the role of IP₃-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 [Ca²⁺]_i was controlled by incorporation of BAPTA in the patch pipette. These effects occurred at concentrations that overlap those used to study IP₃-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 IP₃-R alone. Significantly, our results with 2-APB extend the information available on non-IP₃-R mediated effects of the compound and urge caution in its use to study the role(s) of IP₃-R in modulating cardiac pacemaker cell excitability.

4.3. Evidence for Constitutive and IP₃-R Activity in Modulating AVN Cell Rate

In contrast to our results with 2-APB, we observed no evidence for direct effects of Bt₃-(1,4,5)IP₃-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 Bt₃-(1,4,5)IP₃-AM application led to ~40% increase in spontaneous AP rate in our experiments, and in separate experiments also increased the rate of spontaneous Ca²⁺_i transients (by >30%). When applied to murine SAN cells, IP₃-BM increased spontaneous Ca²⁺_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 IP₃ 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 IP₃-Rs is supported by the observation that subsequent Bt₃-(1,4,5)IP₃-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 IP₃ and raised levels of IP₃ in modulating AVN cell spontaneous AP rate.

A study of IP₃ inhibitor effects on non-cardiac cells has claimed that heparin is more effective at inhibiting IP₃-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 IP₃-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 IP₃ release on atrial Ca²⁺ transients has highlighted the utility of the use of caged-IP₃ [43]. Perhaps some of the most compelling evidence for IP₃ 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-IP₃ increased spontaneous AP rate by up to 30% after multiple flashes, which is similar to the effects of Bt₃-(1,4,5)IP₃-AM application. Under AP voltage clamp, the current activated by caged-IP₃ 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 IP₃ from caged IP₃.

4.4. Limitations, Future Work and Conclusions

Through adopting multiple approaches to the manipulation IP₃ in AVN cells, this study demonstrates for the first time that IP₃ modulates spontaneous AVN AP generation. Our data suggest roles for both constitutive IP₃-R activation and IP₃ 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 IP₃ is changed and how consequent alterations in AP rate arise physiologically. For example, ET_A receptor activation is known to increase IP₃ [66] and in murine SAN cells, application of 100 nM ET-1 increased spontaneous Ca²⁺_i transient rate and diastolic [Ca²⁺]_i [42].

As highlighted by others, responsiveness to intervention(s) that raise IP₃ without G_q-coupled receptor activation demonstrates effects of IP₃ 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 IP₃-R mediated Ca²⁺_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 IP₃ mobilisation accelerates spontaneous AVN rate and to investigate the role of G-protein-coupled receptor activation in AVN cells in mobilizing IP₃ to modulate AVN excitability.

In this study, we focused on spontaneous AP measurement with only one series of experiments involving measurement of [Ca²⁺]_i. The rationale for this was twofold: (i) AP measurements monitor directly an electrophysiological end point and (ii) omission of a Ca²⁺ fluorophore allows interrogation of IP₃ effects without potential Ca²⁺_i-buffering effects due to introduction of a Ca²⁺ indicator: the slower spontaneous rate of Ca²⁺_i transients (Figure 4) than of spontaneous APs in whole-cell recording seems to vindicate this decision. Nevertheless, having established that IP₃ modulates spontaneous AP activity in AVN cells, it would be desirable for future studies to incorporate extensive [Ca²⁺]_i measurements, particularly as these would enable scrutiny of subcellular mechanisms of IP₃ action in the AVN (cf. [28,42]). Our AP clamp experiments demonstrate that increasing intracellular IP₃ activates an inward current during diastolic depolarization. The results of our experiments on Bt₃-(1,4,5)IP₃-AM suggest a lack of direct effect of IP₃ on AVN cell I_NCX, but they do not preclude an indirect effect in which increased [Ca²⁺]_i activates inward I_NCX. While the most likely candidate for IP₃ 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 IP₃-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.

5. Conclusions

This is the first experimental investigation to report functional evidence for a role of IP₃ in AVN. Our key conclusion is that IP₃ can modulate AVN cell excitability: interventions that increase intracellular IP₃ were found to increase AVN cell spontaneous AP rate, while interventions expected to inhibit IP₃-R were found to decrease AVN cell spontaneous AP rate. This information advances our knowledge of the electrophysiology of this region of the 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 IP₃-Rs.