Intra-sarcoplasmic reticulum Ca²⁺ oscillations are driven by dynamic regulation of ryanodine receptor function by luminal Ca²⁺ in cardiomyocytes

Abstract

During the cardiac cycle, the release of Ca²⁺ from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR2) channel complex is controlled by the levels of cytosolic and luminal Ca²⁺ and alterations in these regulatory processes have been implicated in cardiac disease including arrhythmia. To better understand the mechanisms of regulation of SR Ca²⁺ release by Ca²⁺ on both sides of the SR membrane, we investigated SR Ca²⁺ release in a wide range of cytosolic Ca²⁺ concentrations ([Ca²⁺]_cyt; 1–100 μm) in permeabilized canine ventricular myocytes by monitoring [Ca²⁺] inside the SR ([Ca²⁺]_SR). Exposing myocytes to activating [Ca²⁺]_cyt resulted in spontaneous oscillations of [Ca²⁺]_SR due to periodic opening and closing of the RyR2s. Elevating [Ca²⁺]_cyt (up to 10 μm) increased the frequency of [Ca²⁺]_SR oscillations; however at higher [Ca²⁺]_cyt (>50 μm) the oscillations diminished due to RyR2s staying perpetually open, resulting in depleted SR. Ablation of cardiac calsequestrin (CASQ2) altered the [Ca²⁺]_cyt dependence of Ca²⁺ release oscillations such that oscillations were highly frequent at low [Ca²⁺]_cyt (100 nm) but became diminished at moderate [Ca²⁺]_cyt (10 μm), as determined in myocytes from calsequestrin-null versus wild-type mice. Our results suggest that under conditions of continuous activation by cytosolic Ca²⁺, RyR2s can periodically cycle between open and deactivated states due to effects of luminal Ca²⁺. Deactivation at reduced [Ca²⁺]SR appears to involve reduction of sensitivity to cytosolic Ca²⁺ and might be mediated by CASQ2. Inactivation by cytosolic Ca²⁺ plays no detectable role in controlling SR Ca²⁺ release.

Introduction

Cardiac contractility is governed through the calibrated release of calcium (Ca²⁺) from the cisternae of the sarcoplasmic reticulum (SR) to the contractile elements in the cytosol (Bers, 2001). The molecular machinery for SR Ca²⁺ release is a multi-molecular complex that extends from the cytosol to the lumen of the SR. Among other proteins, it is composed of the cardiac ryanodine receptor (RyR2), which serves as a release channel, and the luminal-Ca²⁺ binding protein calsequestrin (CASQ2) (Terentyev et al. 2008; Zhang et al. 1997). Mutations in genes encoding these proteins have been linked to exercise-induced sudden cardiac death due to catecholaminergic polymorphic ventricular tachycardia (CPVT) (Lahat et al. 2001; Priori et al. 2001). The RyR2 channel complex is subject to intricate regulation by Ca²⁺ changes on both the cytosolic and luminal side of the SR membrane. Elevation in cytosolic Ca²⁺ ([Ca²⁺]_cyt) activates the RyR2s through a mechanism known as calcium-induced calcium release (CICR) (Bers, 2001; Fabiato, 1985). Elevations of [Ca²⁺]_cyt can also inactivate the RyR2 channel (i.e. Ca²⁺-dependent inactivation) when sufficiently high (Xu et al. 1998). Lowering luminal Ca²⁺ ([Ca²⁺]_SR) from normal, resting level (∼1 mm) reduces channel activity, whereas increasing [Ca²⁺]_SR above the normal level increases channel activity (Ching et al. 2000; Gyorke & Gyorke, 1998). The former process, termed luminal Ca²⁺- or store-dependent deactivation (Terentyev et al. 2002), is thought to contribute to termination of SR Ca²⁺ release and Ca²⁺ signalling refractoriness required for recharging of the store and contractile relaxation (Terentyev et al. 2008; Zima et al. 2008); the latter is thought to have pathological significance by accounting for arrhythmogenic spontaneous Ca²⁺ release when the SR Ca²⁺ content becomes abnormally high (Ca²⁺ overload) (Venetucci et al. 2008; Venetucci et al. 2008). Despite the clear importance of combined regulation of the RyR2 by cytosolic and luminal Ca²⁺ to both normal and pathological Ca²⁺ signalling in the heart, the mode and molecular basis of integration of these two input signals on the RyR2 remain poorly understood. This lack of knowledge is partially due to the technical difficulty of controlling [Ca²⁺]_cyt and measuring SR Ca²⁺ release while monitoring [Ca²⁺]_cyt at the same time. In the present study, we took advantage of the permeabilized myocyte preparation to measure SR Ca²⁺ release by monitoring [Ca²⁺]_SR during sustained elevation of [Ca²⁺]_cyt in the range from 100 nm to 100 μm. In addition, we investigated the role of CASQ2 in controlling SR Ca²⁺ release by using myocytes from genetically modified mice lacking this protein.

Methods

Ethical approval

The procedures for canine and murine myocyte isolations were approved by the Institutional Animal Care and Use Committee of Ohio State University and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996).

Canine myocyte preparation and imaging

Thirteen male adult dogs were anaesthetized by intravenous administration of pentobarbital sodium (150 mg kg⁻¹) followed by rapid removal of the heart once a deep plane of anaesthesia was reached. Canine ventricular myocytes were obtained via enzymatic isolation as previously described (Kubalova et al. 2005). Ca²⁺ imaging was performed with an Olympus Fluoview 1000 laser scanning confocal microscope equipped with an Olympus 60× 1.4 N.A. oil objective. In order to monitor intra-SR Ca²⁺ oscillations, cells were loaded with 10 μm Fluo-5N-AM at 37°C for 4 h in culture medium, which contained medium 199M, 5 mm creatine, 5 mm taurine, 25 mm NaHCO₃, 5 mm Hepes, 1 μg l⁻¹ gentamycin, 10 u ml⁻¹ penicillin, and 10 μg ml⁻¹ streptomycin (pH 7.3). Myocytes were permeabilized using 0.01% saponin in the control intracellular solution, which contained (mm): 120 potassium aspartate, 20 KCl, 3 MgATP, 10 phosphocreatine, 5 u ml⁻¹ creatine phosphokinase, 0.5 EGTA, 1 free [Mg²⁺], and 20 Hepes (pH 7.2), as previously described (Terentyev et al. 2008). Ten micromolar cytochalasin D was added to the experimental solutions to prevent cell contraction (Howarth et al. 1998). Ca²⁺ release oscillations were induced by decreasing EGTA from 0.5 mm to 0.05 mm. Free [Ca²⁺]_cyt was measured with a Ca²⁺ electrode. Cytosolic and intra-SR global and local Ca²⁺ release events were monitored by exciting SR-entrapped Fluo-5N and cytosolic Rhod-2 K-salt (50 μm) sequentially by 488 and 543 nm laser lines and measuring fluorescence at wavelengths of 500–530 and >560 nm, respectively. The images were acquired in the line scan mode at the rate of 5 ms per line (pixel size 0.4 μm). The images obtained by positioning the line scan along the longitudinal axis of the myocytes were cropped to a uniform size to allow the Ca²⁺ waves to be compared more accurately. In quantitative studies, the dynamics in Fluo-5N fluorescence was expressed as F_SR/F_SRmax= (F−F_caff)/(F_max−F_caff), where F represents the fluorescence at time t, F_caff represents the fluorescence level of the myocytes after application of 10 mm caffeine, and F_max represents the fluorescence level of the myocytes in the presence of caffeine and 10 mm cytosolic Ca²⁺. Ten percent quenching of Fluo-5N fluorescence by 10 mm caffeine was taken into account.

Murine myocyte isolation and imaging

To examine the role of CASQ2 in controlling CICR, ventricular myocytes from eight CASQ2-null mice (CASQ2^−/−) and seven wild-type (WT) mice were obtained by enzymatic isolation. Mice were anaesthetized with pentobarbital sodium (70 mg kg⁻¹) and once a deep plane of anaesthesia was reached (as determined by toe pinch reflex) the heart was rapidly removed and perfused via a Langendorff perfusion apparatus as previously described (Zhao et al. 2003). The isolated myocytes were permeabilized as above and Ca²⁺ waves were monitored using the control intracellular solution, which contained 30 μm Fluo-4FF, a low affinity Ca²⁺ indicator. Ca²⁺ waves were evoked by lowering the concentration of EGTA from 0.5 to 0.05 μm and were monitored by exciting Fluo-4FF with a 488 nm line of argon laser and measuring fluorescence at a rate of 5 ms per line at wavelengths of 500–600 nm. In quantitative studies, the Ca²⁺ wave signals obtained with Fluo-4FF at each pixel along the line scans were aligned at the peak and averaged to assess the effects of ablation of CASQ2 on duration of SR Ca²⁺ release during Ca²⁺ waves.

Statistics

Data are means ±s.e.m. Data were analysed by one-way ANOVA with Bonferroni comparisons or Student's t test as appropriate. P < 0.05 was considered to be significant.

Results

Intra-SR [Ca²⁺] oscillations at activating cytosolic Ca²⁺

First, we investigated the effects of different levels of activating cytosolic Ca²⁺ on SR Ca²⁺ release measured by SR-entrapped Fluo-5N in permeabilized canine ventricular myocytes (Fig. 1). [Ca²⁺]_cyt was elevated from 100 nm free [Ca²⁺] to 1–100 μm in the bathing solution. Under baseline conditions ([Ca²⁺]_cyt∼100 nm), the myocytes exhibited occasional spontaneous Ca²⁺ waves seen as decreases in intra-SR Fluo-5N fluorescence. Elevating [Ca²⁺]_cyt up to 50 μm caused oscillations of [Ca²⁺]_SR manifested as periodic unloading of the SR followed by periods of refilling due to intermittent openings and closing of RyR2s. The frequency of [Ca²⁺]_SR oscillations increased gradually with [Ca²⁺]_cyt up to 10 μm free cytosolic Ca²⁺ and declined at higher [Ca²⁺]_cyt. At the same time, the baseline [Ca²⁺]_SR progressively declined at increasingly elevated [Ca²⁺]_cyt with the SR becoming continuously unloaded at [Ca²⁺]_cyt >50 μm due to at least a fraction of RyR2s staying open (Fig. 1A and D). The degree of [Ca²⁺]_SR depletion during the unloading phases increased gradually in the range from 100 nm to 50 μm[Ca²⁺]_cyt. However, apparently [Ca²⁺]_SR depletion was incomplete even at 50 μm[Ca²⁺]_cyt, since exposure of the permeabilized myocytes to 100 μm of cytosolic Ca²⁺ did not cause an increase in the intra SR Fluo-5N fluorescence expected if Ca²⁺ equilibration across the SR membrane was complete at both of these cytosolic Ca²⁺ concentrations.

Figure 1. Changes in intra-SR [Ca²⁺] oscillations during increasing cytosolic Ca²⁺.

Intra-SR Ca²⁺ waves in permeabilized canine ventricular myocytes at different levels of cytosolic Ca²⁺. A, representative line-scan images (top) along with time-dependent profiles (bottom) of [Ca²⁺]_SR oscillations at 0.1–100 μm[Ca²⁺]_cyt. B, average basal intra-SR Ca²⁺ signal; C, average intra-SR Ca²⁺ signal at the nadir; and D, average frequency of [Ca²⁺]_SR oscillations at increasing [Ca²⁺]_cyt. n= 10, *P < 0.05.

Reversibility of the effects of high cytosolic Ca²⁺ and the potential role of calpains and CaMKII

In principle, elevated cytosolic Ca²⁺ could affect RyR2 behaviour and SR Ca²⁺ release through mechanisms other than direct action on the RyR2s. These include potential proteolytic damage to the RyR2 by the Ca²⁺-dependent proteases calpains 1 and 2 (Rardon et al. 1990; Gilchrist et al. 1992; Singh et al. 2004) and RyR2 phosphorylation by CaMKII (Li et al. 1997). However, the effects of elevated Ca²⁺ were fully reversible on reverting from 50 μm free [Ca²⁺]_cyt back to 100 nm free [Ca²⁺]_cyt (Fig. 2). To further test the potential involvement of calpains in Ca²⁺-dependent changes in [Ca²⁺]_SR oscillations, we used 20 μm of the calpain 1 and calpain 2 inhibitors ALLM and ALLN, respectively. Blocking calpain 1 and calpain 2 by these inhibitors did not produce significant change in release characteristics at high [Ca²⁺]_cyt in treated myocytes versus non-treated cells (Fig. 3). Similarly, cells pretreated with the CaMKII inhibitor KN93 (1 μm for 30 min) did not exhibit significant differences in characteristics of [Ca²⁺]_SR oscillations when exposed to high [Ca²⁺]_cyt in comparison with controls (Fig. 4). These data suggest that under our experimental conditions Ca²⁺-dependent proteases and CaMKII are not involved in Ca²⁺-dependent changes in [Ca²⁺]_SR oscillations.

Figure 2. Reversibility of changes in Ca²⁺ handling in permeabilized canine ventricular myocytes induced by increasing cytosolic Ca²⁺.

A, representative line-scan images (top) with time-dependent profiles (bottom) of [Ca²⁺]_SR oscillations at 0.1, 50 and return to 0.1 μm Ca²⁺, respectively. B, average basal intra-SR Ca²⁺ signal. n= 4, *P < 0.05.

Figure 3. The effect of calpain inhibitors ALLM and ALLN on [Ca²⁺]_SR oscillations in permeabilized canine ventricular myocytes at 100 nm and 50 μm free cytosolic Ca²⁺.

A, representative line-scan images (top) with time-dependent profiles (bottom) of [Ca²⁺]_SR oscillations at 50 μm Ca²⁺ in control and ALLM/ALLN treated myocytes. B, average basal intra-SR Ca²⁺ signal; C, average intra-SR Ca²⁺ signal at the nadir; and D, frequency of [Ca²⁺]_SR oscillations at increasing cytosolic Ca²⁺ in control (filled bars) and ALLN/ALLM treated (hatched bars) myocytes. n= 4, *P < 0.05.

Figure 4. The effect of the CaMKII inhibitor KN-93 on [Ca²⁺]_SR oscillations in permeabilized canine ventricular myocytes at 100 nm and 50 μm free cytosolic Ca²⁺.

A, representative line-scan images (top) with time-dependent profiles (bottom) of [Ca²⁺]_SR oscillations at 50 μm Ca²⁺ in control and KN-93 treated myocytes. B, average basal intra-SR Ca²⁺ signal; C, average intra-SR Ca²⁺ signal at the nadir; and D, frequency of [Ca²⁺]_SR oscillations at increasing cytosolic Ca²⁺ in control (filled bars) and KN-93 treated (hatched bars) myocytes. n= 4, *P < 0.05.

Effects of RyR2 sensitization by caffeine on Ca²⁺ sparks and blinks

Next, we explored whether the above uncovered effects of luminal Ca²⁺ on cytosolic Ca²⁺ activation takes part in shaping Ca²⁺ signalling events under normal baseline [Ca²⁺]_cyt. Normally, Ca²⁺ release during Ca²⁺ sparks (elementary Ca²⁺ release events) terminates at a constant threshold of [Ca²⁺]_SR independent of the amount of the Ca²⁺ flux or buffering capacity of the SR, leaving a substantial Ca²⁺ reserve in the SR (Terentyev et al. 2008; Zima et al. 2008). If the changes in RyR2 cytosolic Ca²⁺ sensitivity caused by a decline in [Ca²⁺]_SR are involved in termination of Ca²⁺ release during a spark, sensitizing RyR2s would be expected to affect the degree of depletion required for release termination. Therefore, we examined the degree of depletion of the SR during Ca²⁺ sparks under control conditions and in the presence of the RyR2 agonist caffeine, which is known to act by sensitizing the RyR2 to cytosolic Ca²⁺ (Rousseau & Meissner, 1989). As shown in Fig. 5, treatment of myocytes with low doses of caffeine increased the frequency of sparks and resulted in reduced baseline [Ca²⁺]_SR. Most importantly, it significantly reduced the nadir of the local SR depletion signals (Ca²⁺ blinks) during Ca²⁺ sparks. This experiment suggests that allosteric interactions between luminal and cytosolic binding sites on the RyR2 channel complex indeed occur and contribute to control of SR Ca²⁺ release during Ca²⁺ sparks.

Figure 5. The effect of caffeine on localized Ca²⁺ depletions in permeabilized canine ventricular myocytes.

A, representative line-scan images of Rhod-2 signal (top) and Fluo-5N signal (bottom) and corresponding time-dependent profiles of spontaneous Ca²⁺ sparks and corresponding local SR Ca²⁺ depletions in control and caffeine treated myocytes. B, spark amplitude in control and caffeine treated myocytes; and C, average basal and nadir intra-SR Ca²⁺ signal in control and caffeine treated myocytes. n= 49 control, 30 caffeine treated, *P < 0.05.

Effects of CASQ2 ablation on SR Ca² release oscillations

To examine the role of CASQ2 in mediating the effects of luminal Ca²⁺ on SR Ca²⁺ release, we performed experiments using cardiac myocytes isolated from genetically modified mice lacking this protein (CASQ2^−/−). If the observed store-dependent oscillations in SR Ca²⁺ release involve inhibition of RyR2s to cytosolic Ca²⁺ by CASQ2, it would be expected that CASQ2^−/− myocytes exhibit more spontaneous Ca²⁺ waves at low [Ca²⁺]_cyt (<10 μm) as a result of RyR2 lacking such an inhibitory influence and becoming sensitized to activation by cytosolic Ca²⁺. At the same time, the ability of the CASQ2^−/− myocytes to produce oscillations in Ca²⁺ release at higher [Ca²⁺]_cyt (≥10 μm) might be diminished due to an increased predisposition of RyR2s lacking inhibition by CASQ2 to remain continuously activated at elevated cytosolic Ca²⁺ levels. This is what we essentially observed in our experiments (Fig. 6A and B). Since we were unable to introduce Ca²⁺ dyes into the SR of murine myocytes for reliable measurements of SR Ca²⁺ release from inside the SR, we performed these experiments using the low-affinity Ca²⁺ indicator Fluo-4FF in the cytosolic compartment in a [Ca²⁺]_cyt range limited to 10 μm. Similar to canine myocytes, the frequency of spontaneous releases increased markedly from 100 nm to 10 μm in WT mouse myocytes. However, in CASQ2^−/− myocytes, the Ca²⁺ dependence of release frequency was manifestly altered. For example, in CASQ2^−/− myocytes, Ca²⁺ wave frequency was significantly increased at 100 nm cytosolic Ca²⁺ but decreased at 10 μm[Ca²⁺]_cyt as compared to WT cells. Additionally, the duration of SR Ca²⁺ release at 10 μm[Ca²⁺]_cyt was significantly longer in CASQ2^−/− than in WT myocytes (Fig. 6B and D). These changes in SR Ca²⁺ release were accompanied by a significant decrease in the SR Ca²⁺ content as determined by application of caffeine (10 mm) (Fig. 6E and F). Collectively, these results suggest that CASQ2 is indeed involved in the effects of luminal Ca²⁺ on the RyR2s, apparently, by reducing the channel's sensitivity to cytosolic Ca²⁺ at low [Ca²⁺]_SR.

Figure 6. Changes in Ca²⁺ release oscillations during increasing cytosolic Ca²⁺ in permeabilized isolated myocytes in wild-type and CASQ2^−/− mice.

A, representative line-scan images (top) with time-dependent profiles (bottom) of Ca²⁺ release oscillations as measured by Fluo-4FF during increasing [Ca²⁺]_cyt in wild-type and CASQ2^−/− myocytes, respectively. B, average frequency of Ca²⁺ release oscillations at increasing [Ca²⁺]_cyt in WT and CASQ2^−/− mice. C and D, average amplitude (C) and duration at half-maximal amplitude (D) of Ca²⁺ release oscillations at increasing [Ca²⁺]_cyt in WT and CASQ2^−/− mice. E, time-dependent profile of Ca²⁺ release triggered by 10 mm caffeine in 100 nm Ca²⁺; F, average amplitude of caffeine induced release in WT and CASQ2^−/− myocytes at 100 nm Ca²⁺. n= 21 WT, 9 CASQ2^−/−, *P < 0.05.

Discussion

In the present study, we investigated the effects of a wide range of cytosolic Ca²⁺ concentrations on SR Ca²⁺ release in permeabilized cardiac myocytes by monitoring [Ca²⁺] inside the SR with a low-affinity Ca²⁺ indicator. Our results show that exposing myocytes to activating Ca²⁺ in the range of 1–50 μm results in oscillations of [Ca²⁺]_SR due to periodic opening and closing of the RyR2s (Fig. 1). To our knowledge, this is the first report of Ca²⁺ release oscillations at high [Ca²⁺]_cyt made possible by using an intra-SR Ca²⁺ indicator to monitor [Ca²⁺]_SR and SR Ca²⁺ release. These [Ca²⁺]_SR oscillations disappeared at [Ca²⁺]_cyt above 50–100 μm where the RyR2s stayed perpetually open leaving the SR nearly empty of Ca²⁺. These results show explicitly that SR Ca²⁺ release does not undergo Ca²⁺-dependent inactivation, even in the presence of high cytosolic Ca²⁺. These Ca²⁺ release oscillations, suggestive of an intricate interplay between cytosolic and luminal Ca²⁺ as regulators of RyR2 channel function, were significantly influenced by the presence of CASQ2 consistent with a role of CASQ2 as an important modulator of SR Ca²⁺ release.

Store-dependent [Ca²⁺]_SR oscillations

It is well-known that cardiac myocytes can exhibit waves of spontaneous Ca²⁺ release at baseline [Ca²⁺]_cyt close to nominal physiological concentration (100 nm) (Wier et al. 1987; Lipp & Niggli, 1993; Cheng et al. 1996; Diaz et al. 1997; MacQuaide et al. 2007). These Ca²⁺ waves usually occur at elevated SR Ca²⁺ loads (Cheng et al. 1996; Diaz et al. 1997) and are ascribed to diffusion-coupled CICR through the RyR2 channels (Keizer & Smith, 1998; Song et al. 1998) sensitized by the increased luminal Ca²⁺ (Lukyanenko et al. 1996; Keller et al. 2007). The [Ca²⁺]_SR oscillations observed at high cytosolic Ca²⁺ (10–50 μm) (Fig. 1) exhibit properties substantially different from classical Ca²⁺ waves caused at low cytosolic Ca²⁺ by Ca²⁺ overload. They show a much higher frequency (dependent on [Ca²⁺]_cyt) and occur on the backdrop of reduced rather than elevated [Ca²⁺]_SR. A striking feature of these repetitive Ca²⁺ releases is that they take place even at cytosolic Ca²⁺ concentrations that are expected to continuously activate all or most of the RyR2 channels (Xu et al. 1998). These results have several important implications for better understanding of the regulation of SR Ca²⁺ release in normal and diseased hearts, which are discussed below.

Ca²⁺-dependent inactivation

First, our results show unequivocally that high levels of Ca²⁺ on the cytosolic side do not lead to inactivation of the RyR2 channel in cardiac myocytes. Ca²⁺-dependent inactivation was first proposed based on experiments in skinned cardiac cells, which showed that CICR becomes substantially inactivated at steady-state Ca²⁺ concentrations as low as 60 nm (Fabiato, 1985). However, single RyR2 channels in planar lipid bilayers reveal no evidence of Ca²⁺-dependent inactivation at physiologically relevant [Ca²⁺]_cyt (<1 mm) (Tripathy & Meissner, 1996; Xu et al. 1998). Additionally, subsequent studies on myocytes yielded controversial results about the role of this mechanism in controlling SR Ca²⁺ release (Fill et al. 2000). Nevertheless, Ca²⁺-dependent inactivation is commonly invoked as a mechanism for termination of Ca²⁺ release in both modelling and experimental studies (Stern & Cheng, 2004). Our results clearly indicate that elevated Ca²⁺ does not lead to inactivation of release during Ca²⁺ elevations as high as 100 μm, which in fact render the RyR2s perpetually open. It should be pointed out, however, that since our study was performed at steady-state cytosolic [Ca²⁺], our results do not rule out the possibility that a dynamic control mechanism such as RyR2 adaptation (Gyorke & Fill, 1993; Valdivia et al. 1995) could contribute to Ca²⁺ release deactivation during normal EC coupling when increases in [Ca²⁺] occur rapidly.

The mode of action of luminal Ca²⁺ on RyR2s

Our results also provide new insights into combined control of RyR2 function by cytosolic and luminal Ca²⁺. Based on the results in the literature and our present findings, we propose the following mechanism of RyR2 modulation by luminal Ca²⁺ and CASQ2, which is illustrated in Fig. 7 (the role of CASQ2 is discussed further below). During each release–uptake cycle, depletion of [Ca²⁺]_SR secondary to release results in deactivation of the RyR2 channels allowing the RyR2s to close temporarily so the SR can be at least partially refilled. As [Ca²⁺]_SR rises, the Ca²⁺ release channels come out of their deactivated or refractory state and become reactivated again by elevated cytosolic Ca²⁺, resulting in a new release–uptake cycle. Deactivation of the RyR2 channels on [Ca²⁺]_SR depletion appears not to be due to RyR2s entering a dominant, absorbing closed state but rather involves a shift (reduction) in RyR2 sensitivity to activation by cytosolic Ca²⁺. This notion is based on our finding that the refilling of the SR that follows Ca²⁺ release ceases to occur at [Ca²⁺]_cyt above 50–100 μm at which the RyR2 stay continuously open. In other words, high cytosolic Ca²⁺ concentrations are capable of overcoming the negative effects of reduced [Ca²⁺]_SR on the channel activity. Thus the observed [Ca²⁺]_SR oscillations appear to involve cycling of the individual RyR2s between low and high Ca²⁺-sensitivity states as determined by the filling state of the SR (low and high, respectively). At [Ca²⁺]_cyt above the dynamic response range of the desensitized, or deactivated, state, Ca²⁺ release ceases oscillating because of continual activation of the RyR2s at these high [Ca²⁺]. The shift in RyR2s is consistent with results of previous lipid bilayer experiments (Gyorke & Gyorke, 1998) as well as with the sensitizing effects of increased SR Ca²⁺ load on RyR2 channels in myocytes at physiological cytosolic Ca²⁺ levels (Lukyanenko et al. 1996; Diaz et al. 1997; Keller et al. 2007). This concept of combined dynamic regulation of RyR2 functional activity by cytosolic and luminal Ca²⁺ has been also recently elegantly exposed by MacQuaide et al. (2009) in their analysis of the effects of tetracaine on spontaneous Ca²⁺ release in permeabilized rabbit cardiomyocytes.

Figure 7. Hypothetical scheme of RyR2 cycling between activated and deactivated states mediated by store-dependent allosteric effects of CASQ2 during Ca²⁺ release oscillations in the presence of activating cytosolic Ca²⁺.

During the activation phase, the SR is filled with Ca²⁺ and CASQ2 is functionally detached from the RyR2 complex rendering the RyR2 channel sensitized to activation by cytosolic Ca²⁺ (1). Opening of the RyR2 channels results in SR Ca²⁺ release and partial unloading of the SR Ca²⁺ store (2). During the deactivation phase, reduced [Ca²⁺]_SR causes CASQ2 to functionally interact with the RyR2 complex (via triadin, TRD, and/or junctin) resulting in allosteric inhibition of the cytosolic activation site of the RyR2 channel (2). During the functional recovery (restitution) phase, refilling of the SR with Ca²⁺ by SERCA2 (1) results in dissociation of CASQ2 (2) rendering the RyR2 again functionally primed or sensitized to activation by cytosolic Ca²⁺.

Of note, in the present study, the depletion phase was significantly prolonged and followed by an incomplete albeit fast re-loading at high [Ca²⁺]_cyt (50 μm) (Fig. 1A). In general, the prolongation of the unloading phase at elevated cytosolic Ca²⁺ is consistent with the notion that luminal Ca²⁺ acts by altering the sensitivity of RyR2s to cytosolic Ca²⁺ such that at increasingly high [Ca²⁺]_cyt, reduced [Ca²⁺]_SR becomes increasingly incapable of inhibiting or deactivating the RyR2s. As to the complex kinetics of the signal, it could be due to a combination of several non-linear process affecting [Ca²⁺]_SR, including potential polymerization–depolymerization of CASQ2 (Park et al. 2004); complex relationship of [Ca²⁺]_SR with the number of open RyR2s versus sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2) activity, and dissipation of local [Ca²⁺] gradients at the cytosolic face of the RyR2s during Ca²⁺ release oscillations (Soeller & Cannell, 1997). Further studies are required to explain this interesting phenomenon.

Regulation of SR Ca²⁺ release during Ca²⁺ sparks

Although our study does not provide a quantitative measurement of the shift in RyR2 Ca²⁺ sensitivity as a function of the filling state of the SR, it shows that this shift is large enough to influence SR Ca²⁺ release during excitation–contraction coupling. In particular, we show that sensitizing the RyR2s with caffeine increases significantly the degree of local depletion during Ca²⁺ sparks with respect to that in the absence of the drug (Fig. 4). This result indicates that when the RyR2s are pharmacologically sensitized to cytosolic Ca²⁺ a larger drop in [Ca²⁺]_SR is required at a given [Ca²⁺]_cyt to bring the sensitivity of the RyR2s to the level at which RyR2 deactivation could occur to stop local SR Ca²⁺ release. These results are consistent with the notion that the regulatory influences of cytosolic and luminal Ca²⁺ on the RyR2 are allosterically linked and that luminal-dependent changes in RyR2 Ca²⁺ sensitivity play a role in termination of Ca²⁺ release during elemental Ca²⁺ signalling events, Ca²⁺ sparks.

The role of CASQ2

Finally, our study provides new insights into the molecular basis of RyR2 regulation by luminal Ca²⁺ and CASQ2. Myocytes from CASQ2^−/− mice showed marked changes in Ca²⁺ release as a function of [Ca²⁺]_cyt. Compared to WT cells which exhibited only a few Ca²⁺ waves at low [Ca²⁺]_cyt (100 nm) and a high frequency of Ca²⁺ release oscillations at elevated Ca²⁺ (10 μm), in CASQ2-deficient cells, the rate of occurrence of Ca²⁺ waves was increased at low [Ca²⁺]_cyt and decreased at elevated [Ca²⁺]_cyt (Fig. 6A and B). These results suggest that in the absence of CASQ2, the RyR2s are sensitized to activation by cytosolic Ca²⁺. Indeed, the increased frequency of Ca²⁺ waves at low Ca²⁺ on the backdrop of reduced SR Ca²⁺ content (Fig. 6E and F) may be an indication of increased sensitivity of RyR2s to CICR in CASQ2^−/− myocytes. The diminished frequency of Ca²⁺ release oscillations combined with the prolonged release phase at elevated Ca²⁺ (Fig. 6B and D) is consistent with an increased fraction of sensitized RyR2s remaining continuously open in CASQ2^−/−vs. WT myocytes. Based on these results, we propose that CASQ2 regulates SR Ca²⁺ release by stabilizing the low- sensitivity (i.e. luminal Ca²⁺ deactivated) form of the RyR2 channel (Fig. 7). Our proposed mode of regulation of the RyR2 by CASQ2 is consistent with recent results obtained on the RyR2 in lipid bilayers showing that CASQ2 affects the sensitivity of the channel to cytosolic Ca²⁺ (Qin et al. 2008). Additionally, the role of CASQ2 in luminal Ca²⁺-dependent control of SR Ca²⁺ release is consistent with our previous studies, which showed that expression of a dominant mutant interfering with the ability of CASQ2 to control RyR2 activity resulted in increased depletion during Ca²⁺ sparks (Terentyev et al. 2008). It is to be pointed out, however, that although our results provide a strong support for the role of CASQ2 in mediating the effects of luminal Ca²⁺ on RyR2s they do not rule out the possibility that other mechanisms, including potential direct effects on RyR2s, are involved in control of SR Ca²⁺ release by luminal Ca²⁺.

Of note, the altered frequency of Ca²⁺ waves could also arise from possible changes of SR Ca²⁺ buffering in myocytes deficient in CASQ2 a major Ca²⁺ binding protein in the SR. However, Knollmann et al. (2006) showed that changes in SR Ca²⁺ storage capacity are minimal due to compensatory expansion of the SR volume in the CASQ2^−/− myocytes. Additionally, this mechanism would be expected to increase the frequency of Ca²⁺ waves at all cytosolic [Ca²⁺]; however in our study, Ca²⁺ wave frequency was in fact reduced at 10 μm[Ca²⁺]_cyt in CASQ2^−/− myocytes. Based on these considerations, we do not believe a potential change in SR Ca²⁺ buffering is a significant factor in our results.

Summary

In conclusion, our study revealed a new phenomenon of Ca²⁺ release oscillations that occur at elevated, activating cytosolic Ca²⁺ concentrations and are attributable to cycling of the RyR2s between low and high cytosolic Ca²⁺ sensitivity modes assumed at low and high [Ca²⁺]_SR, respectively. Luminal Ca²⁺ acts upon the activity of the RyR2 channel by influencing the Ca²⁺ sensitivity of the cytosolic activation site, which thus serves as a point of integration of the cytosolic and luminal Ca²⁺ signals on the RyR2 channel complex. The effects of luminal Ca²⁺ on the RyR2 channel appear to be mediated by CASQ2.

Glossary

Abbreviations

Ca²⁺

calcium

[Ca²⁺]

calcium concentration

[Ca²⁺]_cyt

cytosolic calcium concentration

[Ca²⁺]_SR

luminal calcium concentration

CASQ2

cardiac calsequestrin

CASQ2^−/−

calsequestrin-null mouse

CICR

calcium-induced calcium release

CPVT

catecholaminergic polymorphic ventricular tachycardia

RyR2

cardiac ryanodine receptor

SR

sarcoplasmic reticulum

SERCA2

sarcoplasmic reticulum calcium ATPase

WT

wild-type

Author contributions

S.C.W.S., D.T., A.K., M.P. and S.G. conceived and designed the experiments, assisted significantly in revising the paper and will gave final approval of the version to be published. S.C.W.S., D.T. and S.G. interpreted the data. S.C.W.S. and D.T. analysed the data and S.C.W.S. wrote the original draft. All experiments were conducted at the Davis Heart and Lung Institute at Ohio State University.