‘Eventless’ InsP₃-dependent SR-Ca²⁺ release affecting atrial Ca²⁺ sparks

Abstract

Augmented inositol 1,4,5-trisphosphate receptor (InsP₃R) function has been linked to a variety of cardiac pathologies, including cardiac arrhythmia. The contribution of inositol 1,4,5-trisphosphate-induced Ca²⁺ release (IP3ICR) in excitation-contraction coupling (ECC) under physiological conditions, as well as under cellular remodelling, remains controversial. Here we test the hypothesis that local IP3ICR directly affects ryanodine receptor (RyR) function and subsequent Ca²⁺-induced Ca²⁺ release in atrial myocytes. IP3ICR was evoked by UV-flash photolysis of caged InsP₃ under whole-cell configuration of the voltage-clamp technique in atrial myocytes isolated from C57/BL6 mice. Photolytic release of InsP₃ was accompanied by a significant increase in the Ca²⁺ release event frequency (4.14 ± 0.72 vs. 6.20 ± 0.76 events (100 μm)⁻¹ s⁻¹). These individual photolytically triggered Ca²⁺ release events were identified as Ca²⁺ sparks, which originated from RyR openings. This was verified by Ca²⁺ spark analysis and pharmacological separation between RyR and InsP₃R-dependent sarcoplasmic reticulum (SR)-Ca²⁺ release (2-aminoethoxydiphenyl borate, xestospongin C, tetracaine). Significant SR-Ca²⁺ flux but eventless SR-Ca²⁺ release through InsP₃R were characterized using SR-Ca²⁺ leak/SR-Ca²⁺ load measurements. These results strongly support the idea that IP3ICR can effectively modulate RyR openings and Ca²⁺ spark probability. We conclude that eventless and highly efficient InsP₃-dependent SR-Ca²⁺ flux is the main mechanism of functional cross-talk between InsP₃Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

Key points

• Inositol 1,4,5-trisphosphate receptors (InsP₃Rs) are functionally expressed in cardiac myocytes.

• The influence of inositol 1,4,5-trisphosphate-induced sarcoplasmic reticulum (SR)-Ca²⁺release (IP3ICR) on atrial excitation-contraction coupling (ECC) under physiological and pathophysiological conditions remains elusive.

• The present study focuses on local IP3ICR and its functional consequences for ryanodine receptor (RyR) activity and subsequent Ca²⁺-induced Ca²⁺ release in atrial myocytes.

• Here we show significant SR-Ca²⁺ flux, but eventless SR-Ca²⁺ release through InsP₃Rs.

• We suggest a new mechanism based on eventless and highly efficient InsP₃-dependent SR-Ca²⁺ flux as a crucial mechanism of functional cross-talk between InsP₃Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

Introduction

In cardiac myocytes two mechanisms for sarcoplasmic reticulum (SR)-Ca²⁺ release have been established, Ca²⁺-induced Ca²⁺ release (CICR) as the central mechanism for excitation-contraction coupling (ECC) and inositol 1,4,5-trisphosphate-induced Ca²⁺ release (IP3ICR), Ca²⁺ release via inositol 1,4,5-trisphosphate receptor (InsP₃R) activation. However, although InsP₃Rs are functionally expressed in cardiac preparations (Mackenzie et al. 2004; Zima & Blatter, 2004; Domeier et al. 2008; Harzheim et al. 2009), their role under physiological as well as under pathophysiological conditions in ECC and CICR is still a matter of an ongoing debate.

It has been suggested that SR-Ca²⁺ release via InsP₃R may effectively modulate ECC in ventricular cardiomyocytes, even though the expression of InsP₃Rs is only a fraction of ryanodine receptor (RyR) expression levels (Lipp et al. 2000; Kockskämper et al. 2008). These aspects gain even more importance in atrial myocytes, where InsP₃Rs are expressed 3–10 times more compared with ventricle myocytes (Lipp et al. 2000; Domeier et al. 2008). In addition, this hypothesis is supported by two key observations. First, under pathophysiological conditions, during cellular remodelling, InsP₃R expression is upregulated and may cause delayed after depolarizations, which lead to arrhythmogenicity (Zima & Blatter, 2004; Bootman et al. 2007). Second, InsP₃R-deficient hearts are largely protected from pro-arrhythmogenic stress (Li et al. 2005). Supporting data that encourage a significant contribution of IP3ICR to ECC and CICR are colocalization studies of RyRs and InsP₃Rs (Lipp et al. 2000; Mackenzie et al. 2002), the microarchitecture of atrial myocytes (Yamasaki et al. 1997), and functional studies that have been carried out (Mackenzie et al. 2002; Zima & Blatter, 2004; Li et al. 2005).

In atrial myocytes normal Ca²⁺ transient propagation by concentric Ca²⁺ waves, which are not pro-arrhythmogenic, may be facilitated by IP3ICR (Mackenzie et al. 2004). Local Ca²⁺ signalling between InsP₃Rs/RyRs and vice versa might mediate a functional Ca²⁺ cross-talk between the two types of SR-Ca²⁺ release channels. This was conceptualized for smooth muscle cells, where local RyR Ca²⁺ release events, ‘Ca²⁺ sparks’, were activated via CICR subsequent to InsP₃R activation (Gordienko & Bolton, 2002).

In atrial cells, RyR is the major Ca²⁺ release channel, organized in clusters and functionally coupled (Niggli & Shirokova, 2007). On a local scale, RyR openings give rise to ‘Ca²⁺ sparks’, the building blocks of global Ca²⁺ transients in cardiac cells (Cheng et al. 1993), while coordinated openings of clustered InsP₃Rs will form ‘Ca²⁺ puffs’ with distinct properties (Yao et al. 1995). Along with microscopic Ca²⁺ release events (Ca²⁺ sparks and Ca²⁺ puffs), eventless Ca²⁺ release has been discovered (‘Ca²⁺ quarks’, ‘Ca²⁺ blips’; Lipp et al. 1996; Parker et al. 1996; Brochet et al. 2011). However, these largely invisible Ca²⁺ release events and concomitant Ca²⁺ fluxes may have a significant impact on Ca²⁺ signalling in subcellular microdomains, and may be involved in the functional cross-talk between RyRs and InsP₃Rs, which is still enigmatic.

The present study focuses on local IP3ICR and its functional consequences for RyR activity in atrial myocytes. We examined local IP3ICR triggered by photoactivation of caged InsP₃ in isolated atrial myocytes from mice under whole-cell voltage-clamp conditions. This highly specific approach bypasses sarcolemmal membrane receptor activation and subsequent signalling cascades. Surprisingly, we found that although no individual IP3ICR events could be visualized, InsP₃ release significantly affected the Ca²⁺ spark probability and fosters that RyR Ca²⁺ release can be modulated by Ca²⁺ release via InsP₃Rs in intracellular microdomains. This suggested a new mechanism based on highly efficient but ‘eventless’ InsP₃-dependent SR-Ca²⁺ release. We conclude that invisible InsP₃-dependent SR-Ca²⁺ events are the main mechanism of functional cross-talk between InsP₃Rs and RyRs.

Methods

Atrial myocytes from adult male C57/BL6 mice, obtained from the Central Animal Facility, University Hospital, University of Bern, were freshly isolated by the Langendorff perfusion technique. Hearts were removed after animals were killed by cervical dislocation. The number of animals (N) and myocytes (n) are given in the figure legends. All experiments were performed at room temperature and approved by the State Veterinary Office of Bern, Switzerland, according to Swiss Federal Animal Protection Law. Whole-cell voltage-clamp was combined with confocal Ca²⁺ imaging and UV-flash photolysis. The pipette solution contained (in mmol l⁻¹): CsAsp, 120; Hepes, 10; tetraethylammonium chloride, 20; potassium-ATP, 5; MgCl₂, 1; K₅-fluo-3 (Biotium), 0.1; GSH, 2; caged InsP₃–6Na, (0.03, 0.06, 0.24; Sichem); pH 7.2 with CsOH. Cells were perfused with external solution containing (in mmol l⁻¹): NaCl, 140; Hepes, 5; MgCl₂, 1.1; KCl, 5.4; glucose, 10; CaCl₂, 1.8; BaCl₂, 0.5; CsCl, 1; pH 7.4 NaOH. Pharmacological experiments used 5 μm 2-aminoethoxydiphenyl borate (2APB; Fluka), 5 μm xestospongin C (Xesto; A.G Scientific Inc.), 1 mm tetracaine (TET; Sigma) or 10 mm caffeine (Sigma) added to the external solution. For Ca²⁺ leak/load experiments (Shannon et al. 2002), atrial myocytes were loaded with fluo-3 AM (Biotium).

Data are reported as means ± SEM. Where the data were normally distributed, paired or unpaired, where appropriate, Student's t test was applied. For not normally distributed data the Wilcoxon Matched-Pairs Signed-Ranks Test was applied. A detailed Materials and Methods section is available in the Online Data Supplement.

Results

Endothelin-1 (ET-1)-induced InsP₃R Ca²⁺ release in atrial myocytes

Figure 1 shows that rapid administration of 100 nm ET-1, an InsP₃ pathway activator, caused a significant increase in spontaneous local as well as global Ca²⁺ release events in resting mouse atrial cardiomyocytes. This increase was 2APB (InsP₃R blocker) sensitive (Fig. 1B) and was also absent in control (Fig. 1C). Observations agreed with previously performed experiments from other groups (Mackenzie et al. 2004; Zima & Blatter, 2004; Li et al. 2005), that ET-1-mediated IP3ICR could be obtained in various mammalian species including mouse atrial myocytes. ET-1-triggered IP3ICR is based on a complex signalling cascade that involves G-protein-coupled phospholipase C activation followed by diacylglycerol and InsP₃ production. To bypass this complex signal transduction pathway, InsP₃Rs were activated by rapid and transient intracellular InsP₃ elevation induced by photorelease of InsP₃ from caged InsP₃.

Figure 1. Endothelin-1 (ET-1)-induced InsP₃R Ca²⁺ release in atrial myocytes.

Time series of InsP₃-induced Ca²⁺ release events expressed as F/F₀ of fluo-3 fluorescence obtained in atrial myocytes. Cells were loaded with 5 μm fluo-3 AM (Biotium) for 20 min and left for de-esterification for 15 min before time series were collected in resting cells. A, the InsP₃ pathway was stimulated with 100 nm ET-1 causing a substantial increase in SR-Ca²⁺ release event activity. B, inhibition of ET-1-triggered Ca²⁺ events with 5 μm 2-aminoethoxydiphenyl borate (2APB). C, spontaneous Ca²⁺ event activity under control conditions. SR-Ca²⁺ load was measured at the end of each experiment and found to be not significantly different in the various conditions (Ctrl: 3.45 ± 0.28 F/F₀; ET-1: 3.89 ± 0.48 F/F₀; 2APB + ET-1: 3.84 ± 0.53 F/F₀). Bar graphs represent averaged F/F₀ of the first 20 s (Ctrl) and 160–200 s; scale bars represent 10 μm, N= 3, n= 4–9.

InsP₃-induced Ca²⁺ release triggered by UV-flash photolysis of caged InsP₃

Atrial myocytes were voltage-clamped in the whole-cell configuration of the patch-clamp technique and held at –80 mV. Caged InsP₃ in combination with the Ca²⁺ indicator fluo-3 were dialysed into the cell through the patch pipette. A SR-Ca²⁺ loading protocol based on L-type Ca²⁺ current activation elicited by depolarization steps from –80 mV to 0 mV (up to 10 times) was applied to ensure comparable SR-Ca²⁺ content, before line scan images were recorded (Fig. 2A). Under these conditions spontaneous Ca²⁺ event activity of about 3.7 ± 0.92 events (100 μm)⁻¹ s⁻¹ was observed.

Figure 2. Inositol 1,4,5-trisphosphate (InsP₃)-induced Ca²⁺ release by UV-flash photolysis of caged InsP₃ in atrial myocytes.

A, SR-Ca²⁺ loading protocol performed by I_Ca,L (10 times). B, representative line scan images of voltage-clamped atrial myocytes in whole-cell configuration before and after UV-flash photolysis in control and with 30, 60, 240 μm caged InsP₃. C, the frequency of Ca²⁺ release events counted after UV-flash photolysis normalized to the spontaneous Ca²⁺ release events. D, averaged spatio-temporal Ca²⁺ spark properties after uncaging of InsP₃ (240 μm) normalized to control. Cells were divided into two groups, white bars represent cells that showed little (<1.3) increase in Ca²⁺ release frequency (0.98 ± 0.08 events (100 μm)⁻¹ s⁻¹), black bars represent cells that showed >1.3 increase in Ca²⁺ release frequency (3.24 ± 0.55 events (100 μm)⁻¹ s⁻¹). Amplitude (ΔF/F₀), full-width at half-maximum amplitude (FWHM; μm), full-duration at half-maximum amplitude (FDHM; ms) showed no significant difference after flash photolysis in both groups (see Supplementary Table 1). Full width (μm) showed a significant increase (from 3.80 ± 0.3 μm to 4.50 ± 0.4 μm) in cells with low Ca²⁺ event frequency in response to photolysis. *P < 0.05 vs. control, N= 6–7, n= 6–17, mean ± SEM.

In order to evoke IP3ICR in a highly specific manner, global UV-flashes were applied to photorelease InsP₃ in the entire cytosol, which was followed by an increase of local Ca²⁺ event frequency triggered in a dose-dependent manner (Fig. 2C). The number of Ca²⁺ release events observed after photorelease of InsP₃ was normalized to the number of spontaneous Ca²⁺ releases counted before InsP₃ uncaging. Caged InsP₃ concentrations of 30 μm and 60 μm caused a slight but not significant increase in the frequency of Ca²⁺ release events (30 μm: 2.73 ± 0.51 to 3.06 ± 0.71 events (100 μm)⁻¹ s⁻¹; 60 μm: 4.03 ± 0.73 to 4.20 ± 0.58 events (100 μm)⁻¹ s⁻¹). However, photolysis of 240 μm caged InsP₃ triggered a significant (P= 0.02) increase in frequency of Ca²⁺ release events from 4.14 ± 0.72 to 6.20 ± 0.76 events (100 μm)⁻¹ s⁻¹ (Fig. 2B and C). It is to be mentioned that the effective InsP₃ concentration after UV-flash application is orders of magnitude lower but in proportion to the loading concentration of the caged compound in the pipette solution (see online data supplement). Figure 2C shows control experiments lacking the caged compound, indicating that UV-flash application alone had no effect on the frequency of events. We expected that at least part of the total number of photolytically triggered Ca²⁺ events was based on IP3ICR (e.g. Ca²⁺‘puffs’). Although Ca²⁺ sparks show similarities with Ca²⁺ puffs, it has been shown that Ca²⁺ puffs exhibit significantly distinct spatio-temporal properties that are different from Ca²⁺ sparks (Cheng et al. 1993; Yao et al. 1995; Tovey et al. 2001; Niggli & Shirokova, 2007). At maximal event activity (240 μm caged InsP₃), local Ca²⁺ release events were analysed in more detail for amplitude, full-duration at half-maximum amplitude (FDHM), full-width at half-maximum amplitude (FWHM), full duration, full width (fullwidth) and rise time (Picht et al. 2007). Cells were divided into two groups: one group showed little (<1.3 events (100 μm)⁻¹ s⁻¹) increase (frequency: 0.98 ± 0.08 events (100 μm)⁻¹ s⁻¹); a second group showed >1.3 events (100 μm)⁻¹ s⁻¹ increase in Ca²⁺ release events (frequency: 3.24 ± 0.55 events (100 μm)⁻¹ s⁻¹) after photolytic InsP₃R activation. Ca²⁺ release events obtained in both groups showed no significant difference in their temporal characteristics. The fullwidth was significantly larger in the group of cells that showed little increase in the frequency of Ca²⁺ release events, from 3.80 ± 0.29 to 4.51 ± 0.38 μm (Fig. 2D), an indication for contribution of IP3ICR in Ca²⁺ spark formation. Nevertheless, Ca²⁺ event analysis did not show the anticipated two classes of local Ca²⁺ release events (i.e. Ca²⁺ sparks and Ca²⁺ puffs). The obtained parameters of Ca²⁺ events were comparable to those reported for Ca²⁺ sparks (Niggli & Shirokova, 2007) and are summarized in Supplementary Table 1 in the online material.

However, control measurements revealed photolytically triggered local InsP₃ Ca²⁺ events (Ca²⁺ puffs) in neonatal rat cardiomyocytes. In other words, experimental conditions can be assumed to be appropriate for detecting microscopic subcellular Ca²⁺ events including Ca²⁺ puffs (see Supplementary Fig. 2S).

While photorelease of InsP₃ lead to an increased Ca²⁺ spark frequency (Fig. 2), the precise mechanism by which this occurred still remained unclear. Therefore, pharmacological interventions were used to distinguish between Ca²⁺ sparks and Ca²⁺ puffs by selective inhibition of either RyRs or InsP₃Rs. To inhibit IP3ICR, cells were incubated for at least 20 min in 5 μm Xesto, or 5 μm 2APB was acutely applied. TET (1 mm) was applied to inhibit RyR Ca²⁺ release (Fig. 3A). Photolytic increase of Ca²⁺ release events was inhibited by 2APB and Xesto, without affecting the spontaneous Ca²⁺ release event activity.

Figure 3. Identification of inositol 1,4,5-trisphosphate (InsP₃)-evoked Ca²⁺ sparks.

A, experimental protocol, sarcoplasmic reticulum (SR)-Ca²⁺ loading protocol performed by I_Ca,L (10 times); atrial myocytes were voltage-clamped under whole-cell conditions, the pipette solution contained 240 μm caged InsP₃; line scans are numbered 1–3: (1) control; (2) pharmacological interventions; and (3) pharmacological interventions combined with photolytically released InsP₃. Modified protocols were used for the application of 2-aminoethoxydiphenyl borate (2APB) and tetracaine (TET). B, representative data showing line scan images of fluo-3 fluorescence under whole-cell conditions taken at selected time points: (1) control; (2) in the presence of 2APB or TET; (3) pharmacology and photolytic release of InsP₃. C, frequency of Ca²⁺ release events evoked by uncaging of caged InsP₃ normalized to control (spontaneous frequency in rest). The increase in Ca²⁺ spark frequency was inhibited by the application of either 2APB or xestospongin (Xesto; InsP₃R inhibitors) and by TET (RyR inhibitor). D, SR-Ca²⁺ content measured by rapid caffeine (10 mm) application. E, change in background fluo-3 fluorescence F/F₀ after InsP₃ uncaging, in the presence of TET (dark grey) or 2APB (red). Fluorescence F/F₀ was normalized to background fluorescence before InsP₃ release (right). Background fluorescence increase after UV-flash photolysis of caged InsP₃ in the presence of TET was blocked by 2APB. *P < 0.05 vs. control, N= 2–5; n= 6–13, mean ± SEM.

Surprisingly, in the presence of TET a complete inhibition of all Ca²⁺ release events (Fig. 3B and C) was observed. This observation is in full agreement with the Ca²⁺ event analysis given in Fig. 2, showing that all provoked SR-Ca²⁺ release events can be classified as Ca²⁺ sparks. Taken together, photolytically activated IP3ICR efficiently facilitates RyR Ca²⁺ release.

SR-Ca²⁺ load and invisible InsP₃R Ca²⁺ release (SR-Ca²⁺ leak)

Ca²⁺ spark probability strongly depends on luminal Ca²⁺ ([Ca²⁺]_SR). RyRs and InsP₃Rs may share, at least in part, the same luminal Ca²⁺ pool, suggesting that [Ca²⁺]_SR may have a potential role in the functional cross-talk of both Ca²⁺ release channels, which was examined using caffeine-induced Ca²⁺ transients. InsP₃R block (Fig. 3D) did not significantly affect the SR-Ca²⁺ content (2APB: 1 ± 0.18 to 1.09 ± 0.08 a.u., P= 0.8; and Xesto: from 1.13 ± 0.07 to 0.78 ± 0.09 a.u., P= 0.07), whereas TET caused a significant increase in the SR-Ca²⁺ content from 1 ± 0.07 to 1.4 ± 0.1 a.u., P= 0.02, presumably by suppressing Ca²⁺ leak (Shannon et al. 2002). Besides the SR-Ca²⁺ content, SR-Ca²⁺ leak is an important determinant for Ca²⁺ spark frequency. Indirect evidence that InsP₃R stimulation may play a role in SR-Ca²⁺ leak came from the observation that in the presence of TET subsequent InsP₃ release caused a slow but significant increase in background fluorescence, which was not seen in the presence of 2APB (Fig. 3E). This result suggests that ‘eventless’ Ca²⁺ flux through the InsP₃R, which cannot be resolved as local Ca²⁺ release events, may be more pronounced in atrial cardiomyocytes than previously expected.

In Fig. 4 we addressed the question whether InsP₃Rs may contribute to the SR-Ca²⁺ leak and thus affect the Ca²⁺ spark appearance by performing a modified SR-Ca²⁺ leak protocol introduced by Shannon et al. (2002). Atrial myocytes were field stimulated for 45 s at 1 Hz, followed by a rapid solution switch from 1.8 mm extracellular Ca²⁺ concentration ([Ca²⁺]_o) to a nominally [Ca²⁺]_o free and [Na⁺]_o free solution. After 10 s, 10 μm cyclopiazonic acid (CPA; a potent SERCA blocker) was added and finally, 30 s later, the SR-Ca²⁺ content was measured with rapid caffeine application (Fig. 4A). Atrial myocytes were divided into two groups. The control group was recorded under conditions given above, the second group was pre-incubated with 100 nm ET-1 for at least 120 s before CPA was applied (Fig. 4A). CPA causes an inhibition of the SERCA function. Hence, in combination with the blocked Na⁺/Ca²⁺ exchanger function, any Ca²⁺ leaking from the SR is trapped in the cytoplasm and mirrored by an increase in the cytosolic Ca²⁺ concentration [Ca²⁺]_i. This increase was measured and defined as SR-Ca²⁺ leak.

Figure 4. Invisible InsP₃-dependent sarcoplasmic reticulum (SR)-Ca²⁺ release unmasked by SR-Ca²⁺ leak-load relationship.

A, representative data showing averaged F/F₀ of fluo-3 fluorescence changes (control: red; endothelin-1 (ET-1): black) and B, corresponding line scan images of fluo-3 fluorescence recorded in atrial myocytes. Cells were field stimulated for at least 45 s at 1 Hz before line scan images were recorded. After pre-pulse protocol, superfusion was rapidly switched from 1.8 mm[Ca²⁺]_o to a nominally [Ca²⁺]_o- and [Na⁺]_o-free extracellular solution for 15 s, then 10 μm cyclopiazonic acid (CPA) was added for 20 s, before 10 mm caffeine was applied. The protocol was repeated in the presence of 100 nm ET-1 after pre-incubation with 100 nm ET-1 for at least 120 s. The increase in background fluorescence when CPA was applied represents SR-Ca²⁺ leak (Δ[Ca²⁺]_i). C, averaged data, SR-Ca²⁺ leak (Δ[Ca²⁺]_i), SR-Ca²⁺ content and SR-Ca²⁺ leak/SR-Ca²⁺ load relationship (Δ[Ca²⁺]_i/SR-Ca²⁺ load) compared with control (no InsP₃ pathway stimulation). In the presence of ET-1 there was a significant increase in the SR-Ca²⁺ leak (Δ[Ca²⁺]_i) when CPA was added, unmasking a substantial contribution of the InsP₃R opening to the total SR-Ca²⁺ leak. *P < 0.02–009 vs. control, N= 3; n= 12–13, mean ± SEM.

When CPA was added in the presence of ET-1 there was a significant increase in SR-Ca²⁺ leak Δ[Ca²⁺]_i) compared with the leak in control (0.16 ± 0.03 to 0.38 ± 0.09 ΔF/F₀, P= 0.02). The SR-Ca²⁺ leak/SR-Ca²⁺ load relationship increased accordingly, although not significantly (Δ[Ca²⁺]_i/SR-Ca²⁺ load: 0.047 ± 0.007 to 0.086 ± 0.020, P= 0.09). This may be due to the additional Ca²⁺ influx induced by ET-1 (He et al. 2000), causing an increased SR-Ca²⁺ content (3.30 ± 0.24 to 4.53 ± 0.26 F/F₀, P= 0.001). However, reduced SERCA activity unmasked a substantial contribution of InsP₃R activity to the total SR-Ca²⁺ leak (Fig. 4), which was not seen before. Taken together, eventless InsP₃R-Ca²⁺ release flux is part of SR-Ca²⁺ leak and facilitates SR-Ca²⁺ spark probability.

Discussion

The physiological role of atrial myocytes is to ensure ventricular blood refilling especially under conditions of physical stress and activity. This involves stimulation by direct autonomic nervous system innervation, neuro-humoral effects and interactions with hormones released by the vasculature that boost ECC on a cellular level. Similar to ventricular myocytes, the fundamental mechanism for ECC in atrial myocytes is driven by CICR. However, the contribution of IP3ICR in initiation, propagation and amplification of local and global SR-Ca²⁺ release and thus CICR in atrial myocytes remains controversial (Blatter et al. 2003; Mackenzie et al. 2004).

Our results obtained in atrial myocytes showed significant alterations in Ca²⁺ sparks (width and frequency) during InsP₃R activation. This emphasizes that local Ca²⁺ release from InsP₃Rs may play a significant role in the modulation of Ca²⁺-dependent RyR activity in atrial myocytes. Our approach, based on the combination of different biophysical methods, revealed some discrepancies between InsP₃R activation and the appearance of local SR-Ca²⁺ release events, such as apparent invisible, ‘eventless’ Ca²⁺ release although InsP₃R activation occurred. From these discrepancies we derived a novel conclusion regarding functional cross-talk between RyRs and InsP₃Rs in atrial myocytes based on highly efficient but ‘eventless’ IP3ICR.

Identification of Ca²⁺ sparks

We analysed individual Ca²⁺ release events triggered by photorelease of InsP₃. RyRs Ca²⁺ sparks are characterized by amplitudes of up to 2 ΔF/F₀, widths ≍2 μm, FDHM ≍30 ms, whereas InsP₃ Ca²⁺ puffs are characterized by smaller amplitudes, increased widths ≍6 μm and a prolonged FDHM of about 100–600 ms (Zima & Blatter, 2004; Niggli & Shirokova, 2007; Cheng & Lederer, 2008). Our spark analysis revealed Ca²⁺ sparks but did not show a second distinct class of Ca²⁺ release events that could be ad hoc assigned to Ca²⁺ puffs, respectively. In addition, selective pharmacological inhibition of either InsP₃Rs or RyRs let us conclude that the detected InsP₃-triggered Ca²⁺ release events can be classified as Ca²⁺ sparks. However, the absence of photolytically triggered IP3ICR events was surprising.

Functional cross-talk of InsP₃R and RyR2 via invisible SR-Ca²⁺ release

Despite the evidence that IP3ICR occurs in cardiac cells (Zima & Blatter, 2004), reports of elementary Ca²⁺ signals arising from InsP₃R openings have been rare, which may be due to the Ca²⁺ spark dominance. In atrial myocytes, InsP₃Rs are colocalized with RyRs, preferentially in the subsarcolemmal space and around the nucleus (Lipp et al. 2000; Mackenzie et al. 2002). The expression of InsP₃Rs is known to be regulated in a highly dynamic manner, notably during development and cellular remodelling in pathophysiological situations (Janowski et al. 2006).

RyRs are arranged in functional arrays (10–300 RyRs) and subject to stochastic cluster assembly processes (Baddeley et al. 2009). The synchronized openings of clustered RyRs result in local Ca²⁺ sparks. Under resting conditions, in diastole, spontaneous Ca²⁺ sparks reflect the finite open probability of RyRs, which is controlled by [Ca²⁺]_i, luminal Ca²⁺ ([Ca²⁺]_SR), phosphorylation state and numerous other factors (Eisner et al. 1998). A comparable configuration of spatially segregated clusters of 10–100 InsP₃Rs is believed to be responsible for the generation of ‘Ca²⁺ puffs’, activated by InsP₃ and Ca²⁺ (Foskett et al. 2007), but direct ultrastructural evidence is yet missing (Shuai et al. 2007). In addition, there are species differences in human/rat/rabbit versus mouse in the InsP₃R cluster dynamics and microarchitecture that shape the InsP₃ Ca²⁺ event characteristics (Diambra & Marchant, 2011). It has been suggested that InsP₃Rs are mobile in the SR membrane and can form clusters upon stimulation (Pantazaka & Taylor, 2011). There is also evidence that InsP₃R Ca²⁺ release sites represent pre-established, stable clusters of InsP₃Rs (Smith et al. 2009). Under this view, brief exposure to InsP₃ should trigger Ca²⁺ puffs, which was not the case in our study. We suggest that InsP₃Rs in intact mouse atrial myocytes may not form sufficient cluster size to facilitate Ca²⁺ puffs due to a low level of InsP₃R expression and/or intercluster distance. InsP₃Rs may rather be distributed near RyRs to facilitate Ca²⁺ sparks. This view is supported by our immunohistochemistry study, showing that InsP₃Rs seem to be rather homogeneously distributed (Supplementary Fig. 3S). According to our data, expression of functional InsP₃Rs in atrial myocytes may not necessarily lead to the formation of distinguished Ca²⁺ puffs.

Thus, how can the increase in Ca²⁺ spark frequency after InsP₃ photorelease be explained? The prevalent view that Ca²⁺ sparks and Ca²⁺ puffs are elementary events becomes more comprehensive, as for both types of local Ca²⁺ release events, even smaller elementary Ca²⁺ release events have been confirmed. Compared with Ca²⁺ sparks, openings of single RyRs may result in smaller but more frequent events (‘Ca²⁺ quarks’; Lipp et al. 1996; Brochet et al. 2011). At the same time, similar to Ca²⁺ quarks, InsP₃R-dependent elementary Ca²⁺ release events arising from single InsP₃R openings, termed ‘Ca²⁺ blips’, have been discovered (Parker et al. 1996). Opposed to triggered events, which are experimentally easy to detect, ‘blips’ and ‘quarks’ are largely invisible because these Ca²⁺ events are below detection threshold (Sobie et al. 2002; Williams et al. 2011). This is due to the low signal-to-noise ratio given by fluorescent Ca²⁺ measurements. The absence of visible Ca²⁺ puffs in our measurements, together with the observed increase in spark frequency mediated by InsP₃Rs, suggests a significant contribution of ‘invisible’ or ‘eventless’ IP3ICR to the occurrence of Ca²⁺ sparks (and possible Ca²⁺ wave propagation). We propose that these eventless InsP₃R openings may be responsible for microdomain [Ca²⁺]_i increase that either sensitize RyRs for CICR or lead to direct RyR activation. Functional cross-talk between InsP₃R and RyR can operate in both directions. Hence, microdomain [Ca²⁺]_i elevations could sensitize InsP₃R for InsP₃, which would favour InsP₃R openings (Foskett et al. 2007).

Thus, the contribution of eventless SR-Ca²⁺ release via InsP₃R could be more substantial for the regulation of Ca²⁺ signalling in cellular microdomains than previously thought. This is consistent with a number of not entirely explained experimental reports regarding functional interactions of InsP₃R and RyR. It has been reported that InsP₃R activity increases [Ca²⁺]_i in the vicinity of RyRs and thus facilitates CICR during ECC in adult cat atrial myocytes (Zima & Blatter, 2004), and that InsP₃-dependent Ca²⁺ release has a positive inotropic effect on ECC by facilitating Ca²⁺ release through RyR clusters in rabbit ventricle myocytes (Domeier et al. 2008). Furthermore, there is evidence that Ca²⁺ leak through InsP₃Rs is present at sites where RyRs are located and that this Ca²⁺ leak can modulate RyR Ca²⁺ release events (Gordienko & Bolton, 2002).

This ‘invisible’ SR-Ca²⁺ leak, respectively, SR-Ca²⁺ flux via InsP₃R, was further examined in a series of SR-Ca²⁺ leak measurements. Here, InsP₃R function was stimulated with ET-1. Compared with control, ET-1 may stimulate various Ca²⁺ influx pathways (e.g. via TRPC; Treves et al. 2004), which may have led to an increased SR-Ca²⁺ load before the nominally Ca²⁺- and Na⁺-free solution was added. This would lead per se to a transient increase in SR-Ca²⁺ leak until a new steady-state with matched leak/load relationship is reached. Nevertheless, increased InsP₃R open probability will then concomitantly produce an ‘excess’ SR-Ca²⁺ leak in the Ca²⁺- and Na⁺-free solution compared with control as seen in Fig. 4A. This is largely independent of the total SR-Ca²⁺ content and results in an increased leak/load relationship.

Taken together, experiments using photorelease of InsP₃ and SR-Ca²⁺ leak measurements suggest that ‘eventless’ InsP₃-dependent SR-Ca²⁺ leak is the main mechanism of functional cross-talk between InsP₃Rs and RyRs.

Pathophysiological implications

A change in the SR-Ca²⁺ leak/load relationship can effectively change [Ca²⁺]_SR transiently or even under steady-state conditions. It is well established that [Ca²⁺]_SR is involved in various forms of cellular instability and affected during cellular remodelling in response to pathophysiological stress (Bers, 2003; Shannon et al. 2003; Wehrens et al. 2005). Under chronic atrial fibrillation, InsP₃Rs are targeted during cellular remodelling and were found to be upregulated, which may have positive inotropic effects on the global level (Zima & Blatter, 2004; Li et al. 2005; Bootman et al. 2007; Harzheim et al. 2009). Recently, subcellular mechanism(s) linking InsP₃R activity to the development of atrial fibrillation and cardiac hypertrophy has been demonstrated (Higazi et al. 2009; Nakayama et al. 2010). Cardiac-specific blockage of the InsP₃R pathway, therefore, could offer a new therapeutic strategy for treatment of atrial arrhythmogenicity.

Glossary

2APB

2-aminoethoxydiphenyl borate

CICR

Ca²⁺-induced Ca²⁺ release

CPA

cyclopiazonic acid

ECC

excitation-contraction coupling

ET-1

endothelin-1

FDHM

full-duration at half-maximum amplitude

fullwidth

full width

InsP₃R

inositol 1,4,5-trisphosphate receptor

IP3ICR

inositol 1,4,5-trisphosphate-induced Ca²⁺ release

RyR

ryanodine receptor

SR

sarcoplasmic reticulum

TET

tetracaine

Xesto

xestospongin C

Author contributions

All experiments were performed at the Department of Physiology, University of Bern, Switzerland. M.E. conceived and designed experiments. T.H. collected and analysed the experimental data. M.E., T.H. and N.D.U. drafted and revised the article. All authors approved the final version for publication.

Supplementary material

Supporting Information