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The Journal of Physiology logoLink to The Journal of Physiology
. 2015 May 26;593(Pt 12):2649–2663. doi: 10.1113/JP270082

Regulation of calcium clock-mediated pacemaking by inositol-1,4,5-trisphosphate receptors in mouse sinoatrial nodal cells

Nidhi Kapoor 1, Andrew Tran 1, Jeanney Kang 1, Rui Zhang 1, Kenneth D Philipson 2, Joshua I Goldhaber 1,✉
PMCID: PMC4500350  PMID: 25903031

Abstract

Cardiac pacemaking initiated by the sinus node is attributable to the interplay of several membrane currents. These include the depolarizing ‘funny current’ (If) and the sodium-calcium exchanger current (INCX). The latter is activated by ryanodine receptor (RyR)-mediated calcium (Ca2+) release from the sarcoplasmic reticulum (SR). Another SR Ca2+ release channel, the inositol-1,4,5-triphosphate receptor (IP3R), has been implicated in the generation of spontaneous Ca2+ release in atrial and ventricular cardiomyocytes. Whether IP3R-mediated Ca2+ release also influences SAN automaticity is controversial, in part due to the confounding influence of periodic Ca2+ flux through the sarcolemma accompanying each beat. We took advantage of atrial-specific sodium–calcium exchanger (NCX) knockout (KO) SAN cells to study the influence of IP3 signalling on cardiac pacemaking in a system where periodic intracellular Ca2+ cycling persists despite the absence of depolarization or Ca2+ flux across the sarcolemma. We recorded confocal line scans of spontaneous Ca2+ release in WT and NCX KO SAN cells in the presence or absence of an IP3R blocker (2-aminoethoxydiphenyl borate, 2-APB), or during block of IP3 production by the phospholipase C inhibitor U73122. 2-APB and U73122 decreased the frequency of spontaneous Ca2+ transients and waves in WT and NCX KO cells, respectively. Alternatively, increased IP3 production induced by phenylephrine increased Ca2+ transient and wave frequency. We conclude that IP3R-mediated SR Ca2+ flux is crucial for initiating and modulating the RyR-mediated Ca2+ cycling that regulates SAN pacemaking. Our results in NCX KO SAN cells also demonstrate that RyRs, but not NCX, are required for IP3 to modulate Ca2+ clock frequency.

Key points

  • Inositol-1,4,5-trisphosphate receptors (IP3Rs) modulate pacemaking in embryonic heart, but their role in adult sinoatrial node (SAN) pacemaking is uncertain.

  • We found that stimulation of IP3Rs accelerates spontaneous pacing rate in isolated mouse SAN cells, whereas inhibition of IP3Rs slows pacing.

  • In atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells, where the Ca2+ clock is uncoupled from the membrane clock, IP3R agonists and antagonists modulate the rate of spontaneous Ca2+ waves, suggesting that IP3R-mediated Ca2+ release modulates the Ca2+ clock.

  • IP3R modulation also regulates Ca2+ spark parameters, a reflection of ryanodine receptor open probability, consistent with the effect of IP3 signalling on Ca2+ clock frequency.

  • Modulation of Ca2+ clock frequency by IP3 signalling in NCX KO SAN cells demonstrates that the effect is independent of NCX.

  • These findings support development of IP3 signalling modulators for regulation of heart rate, particularly in heart failure where IP3Rs are upregulated.

Introduction

The primary cardiac pacemaker resides in the sinoatrial node (SAN). Specialized SAN pacemaker cells generate ionic currents that contribute to diastolic depolarization until the membrane potential reaches the threshold for L-type Ca2+ channels (LCCs) to open and produce an action potential (AP). Two ‘clocks’ drive pacemaker activity in the SAN: the ‘membrane clock’ and the ‘Ca2+ clock’. The ‘membrane clock’ uses ion channels in the membrane, most notably the funny current (If) through hyperpolarization activated cyclic nucleotide-gated cation channel 4 (HCN4), to drive diastolic depolarization (DiFrancesco, 1995). The ‘Ca2+ clock’ uses periodic ryanodine receptor (RyR)-mediated Ca2+ release from the sarcoplasmic reticulum (SR) to drive SAN automaticity (Lipsius et al. 2001; Lakatta et al. 2003, 2006; Lipsius & Bers, 2003; Vinogradova et al. 2004). This local Ca2+ release (LCR) by RyRs leads to a depolarizing current carried by the electrogenic sodium–calcium exchanger (NCX) as it removes cytoplasmic Ca2+ (Vinogradova et al. 2006). In addition to RyRs, several lines of evidence suggest that Ca2+ flux through inositol 1,4,5-trisphosphate receptors (IP3Rs) on the SR may play a modulatory role in cardiac pacemaking (Bramich et al. 2001; Ju et al. 2011; Ju et al. 2012). While both the membrane and the Ca2+ clock mechanisms have been extensively investigated, the role of IP3Rs in pacemaking remains poorly understood. This may be of particular importance in the setting of heart failure where IP3R expression in the SAN is increased and HCN4 expression is decreased (Verkerk et al. 2003; Zicha et al. 2005; Yanni et al. 2011; Ju et al. 2012).

Studying the effects of IP3 signalling on intracellular Ca2+ and pacemaking can be challenging because of the confounding influence of periodic Ca2+ flux through the sarcolemma with every beat. In addition, pharmacological blockers of ion channels and transporters are often non-specific (Bootman et al. 2002; Reuter et al. 2002; Brustovetsky et al. 2011; Abramochkin & Vornanen, 2014; Wiczer et al. 2014). To avoid these confounders, we took advantage of the atrial-specific NCX KO mouse where the entire atrium and SAN lack NCX (Groenke et al. 2013). NCX KO SAN cells are healthy with intact If and preserved SR Ca2+ stores (Groenke et al. 2013). Although these cells exhibit the periodic intracellular Ca2+ release events (Ca2+ waves and Ca2+ sparks) indicative of a functioning Ca2+ clock, they lack spontaneous action potentials (Groenke et al. 2013). This is because the Ca2+ clock is ‘uncoupled’ from the membrane due to the absence of NCX. Ca2+ flux across the sarcolemmal membrane is therefore practically eliminated (i.e. no NCX, and insignificant Ca2+ flux through LCCs due to lack of depolarization). Nevertheless, intracellular Ca2+ release events occur at a similar frequency to the spontaneous Ca2+ transients observed in WT SAN cells (Groenke et al. 2013). Using this system, we found that IP3 signalling modulates RyRs and thus pacemaker rate by influencing the ‘Ca2+ clock’ mechanism of SAN pacemaking.

Methods

Ethical approval

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mouse experiments were approved by the Institutional Animal Care and Use Committee at Cedars-Sinai Medical Center (IACUC #: 003574). We anaesthetized the mice with isoflurane prior to heart removal.

Isolation of SAN myocytes from adult mouse hearts

We enzymatically isolated murine SAN cells from 8- to 12-week-old, male and female NCXfx/fx mice (referred to throughout as wild type or WT) and atrial-specific NCX KO mice using an established protocol (Groenke et al. 2013). Following isolation, we plated the cells on laminin-coated glass bottom Petri dishes, and used the cells within 6 h of isolation, the time frame during which the cells were healthy and viable based on trypan blue staining and stable rhythmic beating.

Ca2+ measurements and confocal microscopy

We measured [Ca2+]i using the Ca2+-sensitive indicator Cal-520/AM (AAT Bioquest, Sunnyvale, CA, USA). Cal-520 is a BAPTA-based Ca2+ indicator that has a higher signal to noise ratio than Fluo-4 (Tada et al. 2014). Consequently we were able to use lower concentrations of dye to avoid buffering. We loaded cells previously plated on laminin-coated glass bottom dishes with Cal-520/AM (0.5 μm) dissolved in a modified tyrodes solution for 45 min at 20–22°C, followed by a 15 min wash in dye-free tyrodes. The tyrodes solution, which we also used to perfuse cells throughout experiments, contained (in mm): NaCl 140; KCl 5.4; CaCl2 1.5; MgCl2 1.5; glucose 10; Hepes 5 (pH adjusted to 7.4 with NaOH). We carried out these experiments at room temperature (20–22°C). We used the line scan mode of a Leica TCS-SP5-II confocal microscope (Leica Microsystems, Wetzlar, Germany) to perform spatiotemporal recordings of [Ca2+]i (Chantawansri et al. 2008; Groenke et al. 2013). The dye was excited at 488 nm and fluorescence emission light was collected at >515 nm. We used a 63× water objective lens (Leica: HCX OL APO 63×/1.20W CORR CS), and a line scan frequency of 400 Hz. Fluorescence (F) was ratioed to baseline (F0) after background subtraction. We used ImageJ 1.34 (Schneider et al. 2012) and GraphPad Prism 4 software (La Jolla, CA, USA) to analyse the image data. We also used SparkMaster (Picht et al. 2007) to detect and analyse Ca2+ sparks in our images. The spontaneous Ca2+ transient frequency in WT SAN cells was stable for 6 min, the maximum time course for experiments included in this study.

Chemicals

All chemicals including phenylephrine (PE), caffeine, tetracaine (TET) and 2-aminoethoxydiphenyl borate (2-APB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). We purchased ryanodine (Ry), U73122 and U73343 from Tocris Biosciences (Bristol, UK).

Immunocytochemistry

We fixed SAN cells with 4% paraformaldehyde and permeabilized them with 0.1% Triton X-100. We then incubated the cells with the appropriate primary antibody as follows: rabbit anti-HCN4 (abcam (ab69054); 1:500), mouse anti-α-sarcomeric actin (Sigma (A2172); 1:400) rabbit anti-IP3R2 (Abcam Inc., Cambridge, MA, USA (ab5805); 1:100). Secondary antibodies used were goat anti-rabbit Alexa 488 or goat anti-mouse Alexa 568 (Invitrogen, Carlsbad, CA, USA; A-11034 and A-11031; 1:1000).

Statistics

Results are presented as mean ± SEM and n is the number of experiments. Statistical differences were determined by Student’s t-test or one-way ANOVA with Holm–Sidak’s multiple comparisons test and considered significant at P < 0.05.

Results

Periodic [Ca2+]i cycling in NCX KO SAN cells

Enzymatically isolated NCX KO SAN cells have no spontaneous APs or Ca2+ transients, but they do display periodic Ca2+ sparks suggestive of Ca2+ clock activity (Groenke et al. 2013). We confirmed this result using a high efficiency Ca2+ dye (0.5 μm of the Ca2+ indicator Cal-520/AM (Tada et al. 2014)) to examine Ca2+ cycling. WT SAN cells displayed rapid upstroke Ca2+ transients, indicative of depolarization, at the typical published rate (Groenke et al. 2013; Herrmann et al. 2013) (Fig.1A and C). NCX KO SAN cells had no Ca2+ transients, but instead exhibited periodic Ca2+ sparks and Ca2+ waves at frequencies similar to WT (Fig.1B and C). Previously we had only observed Ca2+ sparks when using Fluo 4, suggesting that the low concentration of Cal-520 avoids Ca2+ buffering. The similarity in frequency between WT Ca2+ transients and KO Ca2+ sparks and waves is consistent with NCX KO cells possessing a functioning Ca2+ clock that is ‘uncoupled’ from the membrane, as we reported previously (Groenke et al. 2013). To exclude the possibility that overload of SR Ca2+ stores was responsible for the Ca2+ waves in KO cells, we recorded Ca2+ release in response to application of caffeine (20 mm). Caffeine releases all Ca2+ from the SR, and the amplitude of the caffeine-induced transient is an indicator of SR Ca2+ content. We found no difference in the amplitude of the caffeine-induced transients between WT and NCX KO SAN cells (Fig.2A–C), indicating similar SR Ca2+ stores in both WT and KO.

Figure 1.

Figure 1

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Ca2+ oscillations in WT and NCX KO SAN cells

A and B, confocal linescan images and corresponding fluorescence intensity plots of a representative WT SAN cell (A), and a representative NCX KO SAN cell (B). C, summary plots of Ca2+ oscillation frequency in WT SAN cells (transients; n =24 cells) and NCX KO SAN cells (waves; n =32 cells). Lines in each plot indicate the mean frequency ± SEM. P = n.s., unpaired Student’s t-test.

Figure 2.

Figure 2

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Caffeine releasable SR Ca2+ stores

A and C, confocal linescan images and corresponding fluorescence intensity plots of a representative WT (A) and an NCX KO SAN cell (C) during application of 20 mm caffeine. B, pooled data showing no significant difference in caffeine-induced Ca2+ transient amplitude in WT (n =8) and NCX KO SAN cells (n =5). D, Ca2+ transient decay (τ) was markedly slowed in NCX KO SAN cells, consistent with the absence of NCX. E, representative confocal linescan images and corresponding fluorescence intensity plots showing caffeine-induced Ca2+ transients in a WT SAN cell under control conditions (left) and another WT SAN cell during superfusion with 2-APB (right). F, summary plots showing no significant difference in the caffeine-induced Ca2+ transient amplitude in control conditions (n =8) and in 2-APB (n =10). *P < 0.05, unpaired Student’s t-test.

Blocking IP3Rs inhibits intracellular Ca2+ Cycling

Ju et al. (2011) previously identified IP3Rs in the murine SAN and also reported that IP3 signalling could alter pacemaker frequency. However, the expression and distribution of IP3Rs in SAN cells isolated from NCX KO SAN is not known. Using immunocytochemistry, we confirmed that IP3Rs were present in NCX KO SAN cells, with a similar cellular distribution to IP3Rs in WT SAN cells (Fig.3).

Figure 3.

Figure 3

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Immunostaining of SAN cells with antibodies against HCN4, α-sarcomeric actin and IP3R2

A, HCN4 and α-sarcomeric actin staining of WT and NCX KO SAN cells confirm the cardiac pacemaker phenotype of these cells. B, α-sarcomeric actin (α-SA) and IP3R2 staining of WT and NCX KO SAN cells show similar IP3R2 distribution in NCX KO SAN cells and WT SAN cells. C, co-immunostaining of a WT SAN cell with only the secondary antibodies. The signal for the secondary antibodies could not be detected, confirming the specificity of the primary antibodies used.

We then tested the effects of the widely used IP3R blocker, 2-APB, on spontaneous Ca2+ transients in SAN cells. We used a low concentration of 2-APB (2 μm) that is known to block IP3R-mediated Ca2+ release in ventricular myocytes without affecting evoked Ca2+ transients (Peppiatt et al. 2003; Kapur & Banach, 2007). This is also a much lower concentration than what has been used in previous studies examining the role of IP3Rs on SAN pacing (Bramich et al. 2001; Ju et al. 2011). We found that 2-APB led to an 82.7% decrease (from 1.92 ± 0.32 to 0.33 ± 0.22 Hz; n = 9, P < 0.05) in the frequency of Ca2+ transients (Fig.4A and C), without depleting RyR-mediated intracellular Ca2+ stores (Fig.2E and F). In 7 of the 9 cells tested, Ca2+ transients were completely abolished by 2-APB. These results suggest that IP3R-mediated Ca2+ release is required for SAN pacemaking.

Figure 4.

Figure 4

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Blocking IP3Rs inhibits pacing and the Ca2+ clock

A and B, confocal linescan images and corresponding fluorescence intensity plots of a representative WT (A) and an NCX KO SAN cell (B) before (CONTROL) and after superfusion with 2-APB (2 μm). C and D, summary plots showing the effect of 2-APB in nine WT SAN cells (C) and in nine NCX KO SAN cells (D). ∗P < 0.05, paired Student’s t-test.

Although 2-APB (2 μm) is reportedly specific for IP3Rs (Peppiatt et al. 2003; Kapur & Banach, 2007), we cannot exclude the possibility that 2-APB has non-specific effects on the interplay between LCCs, NCX, RyRs or even If that might also retard pacemaking. Therefore, we repeated this experiment using NCX KO SAN cells where the Ca2+ clock is still functioning but is ‘uncoupled’ from the plasma membrane. In NCX KO SAN cells, blocking IP3Rs with 2-APB led to a 64.3 ± 7.5% decrease (n =9) in the frequency of spontaneous Ca2+ waves (Fig.4B and D; P < 0.05), similar to the reduction in the frequency of depolarization-associated Ca2+ transients in WT cells. There was no change in caffeine-releasable SR Ca2+ content after 2-APB in the SAN cells that could explain the reduction in Ca2+ oscillation frequency. These results are consistent with direct effects of IP3Rs on the Ca2+ clock. We also found that 2-APB had no effect on the velocity of Ca2+ wave propagation in NCX KO SAN cells, suggesting that IP3R-mediated Ca2+ release is crucial for the initiation of the Ca2+ waves but has no significant effect on their propagation velocity.

Modulation of pacing rate by phospholipase C

Phospholipase C (PLC) activation generates IP3 that ultimately binds to IP3Rs to release Ca2+ from intracellular stores. Conversely, inhibition of PLC decreases IP3 levels. We hypothesized that inhibition of PLC would reduce IP3 and thus slow pacing and Ca2+ oscillation frequency similar to the IP3R blocker 2-APB. To test this hypothesis, we superfused WT and KO cells with the PLC antagonist U73122. Similar to IP3R blockade with 2-APB, inhibition of PLC by U73122 (1 μm) suppressed spontaneous Ca2+ transient frequency in WT SAN cells by 80.8 ± 11.8% (Fig.5A and B, n =5, P < 0.05) and suppressed spontaneous Ca2+ wave frequency in the NCX KO SAN cells by 66.2 ± 13.8% (Fig.5C and D; n =6, P < 0.05). This included 3 of the 5 WT SAN cells in which Ca2+ transients were completely abolished by U73122. In contrast, U73343 (1 μm), the inactive analogue of U73122, had no effect on [Ca2+]i oscillations, confirming that the effect of U73122 was specific (Fig.5E and F, n =5).

Figure 5.

Figure 5

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Blocking PLC inhibits pacing and the Ca2+ clock

A and C, confocal linescan images and corresponding fluorescence intensity plots of a representative WT (A) and an NCX KO SAN cell (C) before (CONTROL) and after superfusion with the PLC blocker U73122 (1 μm). B and D, summary plots showing the effects of U73122 in WT SAN cells (B; n =5) and NCX KO SAN cells (D; n =6). E and F, representative confocal linescans showing that U73343, the inactive analogue of U73122, has no effect on spontaneous Ca2+ transients in WT SAN cells (n =5). ∗P < 0.05, paired Student’s t-test.

To confirm that PLC could influence pacing by changing IP3 levels, we stimulated IP3 production using the α-1 adrenergic receptor agonist PE (10 μm). PE activates the Gq-PLC pathway (Scholz et al. 1992), which results in downstream generation of IP3. In WT SAN cells, receptor-mediated stimulation of IP3 production by PE for 3 min had a positive chronotropic effect on spontaneous Ca2+ transients (3.2-fold increase in frequency, Fig.6A and C; n =6, P < 0.05). The positive chronotropic effect was then blocked by 2-APB (2 μm; Fig.6A and C; n =7, P < 0.05), underlining the relevance of IP3 and IP3R-mediated Ca2+ release to this signalling pathway. In NCX KO SAN cells, PE had a similar effect and led to a significant increase (2.2-fold) in the frequency of Ca2+ waves that could also be blocked by 2-APB (2 μm; Fig.6B and D, n =5, P < 0.05). We did not detect any significant changes in Ca2+ wave velocities during application of these agents (Fig.6E; control, 65.15 ± 2.4; PE, 72.74 ± 2.97; PE + 2-APB, 64.97 ± 2.37 μm s−1; P = n.s.). Thus, while stimulating or inhibiting IP3R-mediated Ca2+ release can alter Ca2+ transient or wave frequency, it does not alter the velocity of Ca2+ wave propagation through the cell.

Figure 6.

Figure 6

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Stimulating IP3 production accelerates pacing and the Ca2+ clock

A and B, confocal linescan images and corresponding fluorescence intensity plots of a representative WT (A) and an NCX KO SAN cell (B) before (CONTROL), during subsequent superfusion with phenylephrine (PE, 10 μm), and during superfusion with PE (10 μm) + 2-APB (2 μm). C and D, summary plots showing the effects of PE and subsequent application of PE +2-APB for WT (C; n =7) and NCX KO SAN cells (D; n =5). E, mean Ca2+ wave velocity in NCX KO SAN cells was unchanged by the pharmacological interventions (PE, PE + 2-APB) in comparison to the controls. ∗P < 0.05, one-way ANOVA with Holm–Sidak’s multiple comparisons test.

IP3 signalling in the presence of RyR blockers

IP3Rs could influence pacemaker activity either (1) directly by releasing Ca2+ in close proximity to NCX, thereby generating inward NCX current (INCX) to depolarize the cell, or (2) indirectly by triggering neighbouring RyRs to release Ca2+ to activate inward INCX. Our results in NCX KO cells, which do not depolarize spontaneously, clearly demonstrate that NCX-induced depolarization by IP3R-mediated Ca2+ release is not necessary for IP3 to modulate Ca2+ wave (e.g. Ca2+ clock) frequency (Figs4D, 5D and 6D). To further study the role of RyRs in the mechanism of IP3-mediated modulation of the Ca2+ clock, we recorded Ca2+ transients during application of PE (10 μm) while blocking RyRs using Ry (at a blocking concentration of 100 μm to avoid depleting SR Ca2+ stores). In WT SAN cells Ry blocked spontaneous Ca2+ transients in all five cells tested, and prevented PE from restoring them (Fig.7). Caffeine-induced Ca2+ transients recorded at the end of the protocol indicated that SR Ca2+ was not depleted (Fig.7). Similar results were obtained in NCX KO SAN cells where blocking RyRs with TET (1 mm) caused cessation of Ca2+ waves, and subsequent addition of PE failed to restore them (data not shown). Taken together, these results are consistent with the hypothesis that IP3-mediated Ca2+ release from IP3Rs increases the likelihood of SR Ca2+ release by RyRs to modulate pacemaker rate.

Figure 7.

Figure 7

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IP3Rs require RyRs to induce pacemaking

Representative confocal linescan images (left) and corresponding fluorescence intensity plots (right) in a WT SAN cell under control conditions (CONTROL), during subsequent superfusion with ryanodine (Ry; 100 μm) alone, and then Ry + phenylephrine (PE; 10 μm). Caffeine (20 mm) was applied at the end of the experiment where indicated by the black line to assess releasable Ca2+ in the SR.

Modulation of Ca2+ sparks by IP3R-mediated Ca2+ release

Spontaneous LCRs by RyRs in SAN cells are thought to trigger depolarization by stimulating forward mode NCX. A single LCR involves several Ca2+ sparks fired by neighbouring Ca2+ release units via fire–diffuse–fire propagation (Maltsev et al. 2011). To further test the hypothesis that IP3R-mediated Ca2+ release induces Ca2+ release from RyR channels, we examined how Ca2+ sparks in WT and NCX KO SAN cells are influenced by IP3 signalling. We observed localized Ca2+ release events with characteristics of Ca2+ sparks (Cheng & Lederer, 2008) in 47% (86 out of 184 cells) of WT and 56% (65 out of 116 cells) of NCX KO SAN cells. We then tested the effects of IP3R block on Ca2+ sparks in WT (n =6) and NCX KO (n =3) SAN cells. Superfusion of both WT and NCX KO SAN cells with 2-APB resulted in a significant decrease in the frequency and amplitude, but not width or duration, of the Ca2+ sparks (Fig.8).

Figure 8.

Figure 8

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IP3R-mediated Ca2+ release modulates Ca2+ Sparks

A, representative confocal linescan images showing Ca2+ sparks in an NCX KO SAN cell under control conditions (upper panel) and during superfusion with 2-APB (2 μm; lower panel). B, higher magnification images of Ca2+ sparks occurring at a single location on the linescan in A (indicated by the dotted box) under control conditions (upper) and during superfusion with 2-APB (lower). C, fluorescence intensity plots (black, control; grey, 2-APB) for the sparks shown in B. D–K, summary plots showing the effect of 2-APB on spark amplitude (D, H), frequency (E, I), width (F, J) and duration (G, K), in WT and KO SAN cells, respectively. ∗P < 0.05, unpaired Student’s t-test. FDHM, full duration at half-maximum; FWHM, full width at half-maximum.

The reduction in spark amplitude upon superfusion with 2-APB could not be attributed to a change in SR Ca2+ content, as caffeine-releasable Ca2+ stores were similar before and after application of the drug (Fig.2E and F). This suggests instead that blocking IP3Rs decreases RyR Po (open probability) and thus recruitment of functional RyR Ca2+ release units in a couplon (Lukyanenko et al. 2000; Cheng & Lederer, 2008; MacQuaide et al. 2010; Lee et al. 2013).

In contrast to 2-APB, PE increased Ca2+ spark frequency after 3 min in WT and NCX KO SAN cells (Fig.9). Again, this effect was reversed by 2-APB (Fig.9). These results support the hypothesis that IP3R-mediated Ca2+ release participates in the triggering of Ca2+ release from RyRs and thus potentiates the ‘Ca2+ clock’ pathway of pacemaking.

Figure 9.

Figure 9

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Effect of phenylephrine on Ca2+ sparks

A and B, summary plots showing the effect of phenylephrine (PE; 10 μm) and the subsequent application of PE (10 μm) + 2-APB (2 μm) on the frequency of Ca2+ sparks in WT (A) and NCX KO SAN cells (B). WT (n =3) and KO cells (n =3) both show an increase in spark frequency upon superfusion with PE that was reduced by subsequent addition of 2-APB. ∗P < 0.05, one-way ANOVA with Holm–Sidak’s multiple comparisons test.

Discussion

SAN pacemaker activity is thought to be driven by the coupled activity of two cellular ‘clocks’: a membrane clock driven by the inward funny current (If) through HCN4 channels, and a calcium clock driven by the inward current through NCX (INCX) in response to local SR calcium release by RyRs (Santoro & Tibbs, 1999; Lakatta et al. 2003, 2006, 2010; Vinogradova et al. 2005). In the embryonic heart, an alternative signalling pathway involving IP3 has been shown to play a crucial role in pacemaker activity (Mery et al. 2005; Kapur & Banach, 2007; Kapoor et al. 2014). In the adult heart, IP3Rs release Ca2+ from intracellular Ca2+ stores and participate in the pathogenesis of both ventricular hypertrophy (Barac et al. 2005; Wang et al. 2005; Luo et al. 2006; Roderick & Bootman, 2007; Harzheim et al. 2009; Nakayama et al. 2010; Arantes et al. 2012) and heart failure (Gutstein & Marks, 1997; Woodcock et al. 1998; Guatimosim et al. 2002; Fauconnier et al. 2005; Harzheim et al. 2009; Hohendanner et al. 2015). In atrial myocardium, where IP3R expression levels are 6–10 fold higher than ventricle (Lipp et al. 2000; Mackenzie et al. 2002), IP3 is thought to contribute to EC coupling by ‘facilitating’ RyR Ca2+ release (Zima & Blatter, 2004), although it may also trigger atrial arrhythmias characterized by spontaneous Ca2+ waves. The potential of IP3 as a regulator of cardiac pacemaker activity in the adult SAN has been controversial (Ju et al. 2011; Vinogradova, 2011). In the current study we clearly show that IP3 signalling can alter the frequency of pacemaking in murine SAN cells. In WT cells, blockers of IP3 production or IP3Rs reduced pacing rate (Figs4 and 5), whereas stimulating IP3 signalling accelerated it (Fig.6). Similar effects of these agents on Ca2+ wave frequency in NCX KO mice (Figs6) indicate that the mechanism involves changes in Ca2+ clock cycle length mediated by IP3R-mediated Ca2+ release near RyRs. Finally, our results in NCX KO mice indicate unequivocally that the effect of IP3 signalling on the ‘Ca2+ clock’ does not depend upon NCX-mediated depolarization in direct response to Ca released by IP3Rs.

Unlike ventricular myocytes, where spontaneous RyR-mediated Ca2+ release and Ca2+ waves are often the consequence of elevated SR Ca2+ load (Jiang et al. 2004), RyRs in SAN myocytes release SR Ca2+ in response to normal SR refilling and content in accordance with the Ca2+ clock mechanism (Vinogradova et al. 2004, 2005). In WT cells this locally released Ca2+ is removed by NCX, which generates a depolarizing inward current contributing to an AP and a corresponding Ca2+ transient. Because NCX KO mice lack any mechanism to remove Ca2+ rapidly across the sarcolemma, local Ca2+ release from the SR can either (1) facilitate activation of adjacent RyRs and propagate as a Ca2+ wave, or (2) fail to propagate and instead generate Ca2+ sparks (Groenke et al. 2013). Notably the Ca2+ waves that occur in NCX KO SAN cells do so even though SR Ca2+ content is not significantly increased (Fig.2A–C). The lack of SR Ca2+ overload despite the absence of NCX is consistent with decreased Ca2+ entry through LCCs and increased Ca efflux through the plasma membrane Ca2+ pump, which we have shown previously in NCX KO SAN cells (Groenke et al. 2013) and NCX KO ventricular myocytes (Pott et al. 2005). SAN pacemaker rate can slow down when SR Ca2+ load is reduced (Vinogradova et al. 2002; Maltsev et al. 2011). We found no evidence of SR Ca2+ depletion during IP3R block with 2-APB (Fig.2E and F) that could explain the reduction in rate we observed in WT and NCX KO cells. Similar to the reduction in spontaneous pacing rate in WT cells, we observed a decrease in the frequency and amplitude of Ca2+ sparks upon blocking IP3Rs with 2-APB (Fig.8). Reductions in Ca2+ spark frequency are typically attributed to decreases in RyR P0 (Zima et al. 2008). While this can be caused by reductions in SR Ca2+ content, drugs that reduce RyR Po, such as ruthenium red, ryanodine and tetracaine, can also reduce spark frequency without lowering SR Ca2+ content (Lukyanenko et al. 2000; MacQuaide et al. 2010). As SR Ca2+ content remained unaffected by 2-APB (Fig.2E and F), we conclude that blocking IP3R-mediated Ca2+ release decreased the local concentration of Ca2+ sensed by RyRs, thereby decreasing RyR Po and consequently spark amplitude and frequency. Conversely, our data show that Ca2+ spark frequency increased after activating the IP3 signalling pathway with PE (Fig.9). The effect of PE could be blocked with 2-APB, suggesting that the effect was specific for IP3Rs. Taken together, these results are consistent with IP3Rs regulating pacemaker rate through modulation of local Ca2+ in the vicinity of RyRs and thus RyR Po. A similar mechanism has been described to explain how IP3 facilitates EC coupling in atrial myocytes (Zima & Blatter, 2004). The effect is likely to be enhanced in SAN for two reasons: first, SAN cells have higher levels of cAMP in comparison to the rest of the myocardium (Vinogradova et al. 2008), and high concentrations of cAMP sensitize IP3Rs to IP3 via a direct effect (Tovey et al. 2010; Tovey & Taylor, 2013). Second, IP3Rs are more abundant in atria and the SAN compared to the ventricle (Lipp et al. 2000; Mackenzie et al. 2002; Ju et al. 2011). Thus, it seems likely that IP3Rs of SAN cells are poised to provide a relatively large source of Ca2+ to nearby RyRs.

In the present study we were able to slow Ca2+ cycling and pacing rate in WT and NCX KO SAN cells using two different blockers of IP3Rs or IP3 production. Blocking or activating PLC could potentially have complicating effects as PLC activation leads to hydrolysis of membrane-associated phosphatidylinositol (4,5)-bisphosphate (PIP2) into diacylglycerol (DAG) as well as IP3. However, our data show that the effect of PLC activation by PE is blocked by 2-APB (Fig.6), suggesting that the effect is mediated via IP3 and IP3Rs, as DAG is not known to activate IP3Rs. Our data on Ca2+ spark modulation with 2-APB also suggest that there is a basal level of IP3R activity sufficient to provide nearby RyRs with enough Ca2+ to trigger their activation. On the other hand, stimulation of IP3 signalling with PE increased the Ca2+ cycling rate in WT and KO SAN cells (Fig.6). PE leads to an increase in intracellular IP3 (Remus et al. 2006), which results in Ca2+ release by the IP3Rs. We assume that additional Ca2+ released by IP3Rs in close proximity to the RyRs could increase their P0 and thereby increase the ‘Ca2+ clock’ frequency.

Although we found that IP3 signalling could modulate the frequency of Ca2+ transients in WT, and Ca2+ sparks and waves in KO, we saw no change in the velocity of Ca2+ wave propagation in KO upon stimulation or inhibition of IP3Rs (Fig.6E). The reason for this apparent contradiction is not clear, but prior studies in ventricular and atrial cells have shown similar disparities. For example, MacQuaide et al. (2010) have shown in permeabilized ventricular myocytes that the RyR blocker ruthenium red can reduce Ca2+ wave frequency without retarding wave velocity. A similar phenomenon has been reported in atrial myocytes where block of IP3Rs reduces Ca2+ spark frequency but not Ca2+ transient propagation velocity from cell periphery to centre (Li et al. 2005).

Our results in WT and KO mouse SAN cells are consistent with a report by Ju et al. (2011) describing decreased sinus rate in IP3R2 KO mice, although the reduction in rate that we observed with IP3R blockade was much higher. There are several potential explanations for this. First, we conducted our experiments at 20–22°C whereas Ju et al. (2011) conducted their experiments at 37°C. The lower temperature we used resulted in a slower and more variable Ca2+ transient and wave rate at baseline, and may have increased the sensitivity of our isolated SAN cells to the IP3R blocker. Second, despite the finding that HCN4 expression is unchanged in the IP3R2 KO mice, it is possible that these mice have compensatory adaptations in other membrane channels (e.g. ICa, IK, INa) that might influence and support pacemaker rate in the absence of IP3R2. Finally, Ju et al. (2011) tested the effects of 2-APB on intact SAN tissue preparations whereas we used single SAN cells. It has been our experience that the intact SAN requires higher drug concentrations to achieve the same effect as a lower concentration in single cells. Moreover, it is well known that the SAN is composed of a heterogeneous population of cells with respect to shape, size and electrophysiology and Ca2+ handling proteins (Boyett et al. 2000; Musa et al. 2002; Lancaster et al. 2004). In the absence of IP3R2, it is possible that latent pacemaker cells with less dependence on IP3 signalling take over to maintain automaticity. We have observed this heterogeneity in our cells with regard to sensitivity to the IP3R blocker, 2-APB. In 7 of 9 cells, spontaneous Ca2+ transients were completely abolished by 2-APB while in the 2 others 2-APB only slowed the rate (see Fig.4C).

Limitations

At high concentrations, 2-APB is known to have non-specific effects in addition to blocking IP3Rs (Wilcox et al. 1998; Bootman et al. 2002). However, we used a low concentration (2 μm) that does not have any detectable effect on the amplitude or duration of electrically evoked Ca2+ transients in isolated ventricular myocytes (Peppiatt et al. 2003; Kapur & Banach, 2007). Thus, the concentration that we used should have minimal off-target effects. Furthermore, we used alternative methods of accessing the IP3 signalling system, including PE and U73122, which yielded consistent effects. We did not use IP3R blockers such as xestospongin C (Oka et al. 2002), as they clearly inhibit voltage-dependent Ca2+ and K+ currents (Ozaki et al. 2002) as well as SR calcium transport ATPase (SERCA) (Castonguay & Robitaille, 2002) at concentrations used to inhibit IP3Rs.

Conclusion

Our findings support the hypothesis (Bramich et al. 2001; Ju et al. 2011, 2012) that functional cross-talk between IP3Rs and RyRs provides a secondary mechanism of SAN pacemaker regulation. Based on our results in NCX KO cells, we also conclude that NCX is not required for IP3Rs to modulate Ca2+ cycling, and thus ‘Ca2+ clock’ frequency. Thus, we suggest that IP3R-mediated SAN pacemaker regulation is controlled primarily by the Ca2+ clock rather than the membrane clock. As has been suggested previously (Ju et al. 2011, 2012), IP3R-mediated pacemaker regulation could have particular importance in the setting of heart failure where IP3R expression in the SAN is increased (Yanni et al. 2011) and HCN4 expression is decreased (Verkerk et al. 2003; Zicha et al. 2005). Therefore, our findings may support the development of innovative strategies for using modulators of IP3 signalling to regulate heart rate in failing hearts where IP3Rs are increased. Given that IP3Rs are broadly expressed in the heart, further study is needed to address this possibility.

Glossary

AP

action potential

2-APB

2-aminoethoxydiphenyl borate

[Ca]i

cytosolic free Ca2+ concentration

DAG

diacylglycerol

HCN4

hyperpolarization activated cyclic nucleotide-gated cation channel 4

If

funny current

INCX

sodium–calcium exchanger current

IP3

inositol-1,4,5-trisphosphate

IP3R

IP3 receptor

LCC

L-type Ca2+ channel

LCR

local Ca2+ release

NCX

sodium–calcium exchanger

PE

phenylephrine

PIP2

phosphatidylinositol (4,5)-bisphosphate

Po

open probability

Ry

ryanodine

RyR

ryanodine receptor

SAN

sinoatrial node

SERCA

sarcoplasmic/endoplasmic reticulum Ca2+ ATPase

SR

sarcoplasmic reticulum

TET

tetracaine

Additional information

Competing interests

None declared.

Author contributions

Conception and design of the experiments: N.K., K.D.P., J.I.G. Collection, analysis and interpretation of data: N.K., A.T., J.K., R.Z., K.D.P., J.I.G. Drafting the article: N.K., K.D.P., J.I.G. All authors have approved the final version of the manuscript.

Funding

This work was supported by National Institutes of Health grants R01 HL048509 to J.I.G. and K.D.P., R01 HL070828 to J.I.G. and the Dorothy and E. Phillip Lyon Chair for Laser Research to J.I.G.

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