This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 91/5/414    most recent 01.RES.0000032490.04207.BDv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Terentyev, D. Articles by Györke, S. Search for Related Content PubMed PubMed Citation Articles by Terentyev, D. Articles by Györke, S. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL MALEIC ACID Related Collections Calcium cycling/excitation-contraction coupling Cell signalling/signal transduction Ion channels/membrane transport

(Circulation Research. 2002;91:414.)
© 2002 American Heart Association, Inc.

Cellular Biology

Luminal Ca²⁺ Controls Termination and Refractory Behavior of Ca²⁺-Induced Ca²⁺ Release in Cardiac Myocytes

Dmitry Terentyev, Serge Viatchenko-Karpinski, Héctor H. Valdivia, Ariel L. Escobar, Sandor Györke

From Texas Tech University HSC (D.T., S.V.-K., A.L.E., S.G.), Lubbock, Tex; and the University of Wisconsin (H.H.V.), Madison, Wis.

Correspondence to Sandor Györke, Dept of Physiology, Texas Tech University HSC, 3601 4th St, Lubbock, TX 79430. E-mail sandor.gyorke{at}ttuhsc.edu

	Abstract

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Despite extensive research, the mechanisms responsible for the graded nature and early termination of Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac muscle remain poorly understood. Suggested mechanisms include cytosolic Ca²⁺-dependent inactivation/adaptation and luminal Ca²⁺-dependent deactivtion of the SR Ca²⁺ release channels/ryanodine receptors (RyRs). To explore the importance of cytosolic versus luminal Ca²⁺ regulatory mechanisms in controlling CICR, we assessed the impact of intra-SR Ca²⁺ buffering on global and local Ca²⁺ release properties of patch-clamped or permeabilized rat ventricular myocytes. Exogenous, low-affinity Ca²⁺ buffers (5 to 20 mmol/L ADA, citrate or maleate) were introduced into the SR by exposing the cells to "internal" solutions containing the buffers. Enhanced Ca²⁺ buffering in the SR was confirmed by an increase in the total SR Ca²⁺ content, as revealed by application of caffeine. At the whole-cell level, intra-SR [Ca²⁺] buffering dramatically increased the magnitude of Ca²⁺ transients induced by I_Ca and deranged the smoothly graded I_Ca-SR Ca²⁺ release relationship. The amplitude and time-to-peak of local Ca²⁺ release events, Ca²⁺ sparks, as well as the duration of local Ca²⁺ release fluxes underlying sparks were increased up to 2- to 3-fold. The exogenous Ca²⁺ buffers in the SR also reduced the frequency of repetitive activity observed at individual release sites in the presence of the RyR activator Imperatoxin A. We conclude that regulation of RyR openings by local intra-SR [Ca²⁺] is responsible for termination of CICR and for the subsequent restitution behavior of Ca²⁺ release sites in cardiac muscle.

Key Words: sarcoplasmic reticulum • excitation-contraction coupling • calcium-induced calcium release • ryanodine receptor

	Introduction

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In cardiac muscle, the process of excitation-contraction (EC) coupling is mediated by Ca²⁺ influx through voltage-dependent Ca²⁺ channels in the plasmalemma triggering Ca²⁺-induced Ca²⁺ release (CICR) from the SR.^1,2 This Ca²⁺ release is the result of spatial and temporal summation of brief and localized Ca²⁺ release events, ie, "Ca²⁺ sparks" that can be visualized by fluorescence imaging.^3–5 Each spark involves 6 to 30 individual Ca²⁺ release channels/ryanodine receptors (RyRs).^6–8 The concerted openings of multiple RyRs appears to be due to cross-activation of channels by released Ca²⁺ and may also involve more direct interactions between neighboring channels, ie, "coupled gating."⁹ A fundamental question about CICR is how Ca²⁺ release is terminated. Once initiated, Ca²⁺ release from the regenerative clusters should continue until the Ca²⁺ stores are emptied. However, Ca²⁺ sparks feature abrupt termination. Such a robust termination of Ca²⁺ release is necessary for the relaxation of the cardiac muscle.

Previous studies have indicated that termination of Ca²⁺ release cannot be simply accounted for by such passive extinguishing mechanisms as depletion of SR Ca²⁺ or stochastic closure of the release channels.^10,11 Instead, it appears that release termination is more likely to involve changes in open probability of RyRs, either caused by Ca²⁺-dependent inactivation/adaptation of RyRs or after changes in SR luminal [Ca²⁺] ([Ca²⁺]_SR). The resting free [Ca²⁺] in the SR has been estimated to be approximately 1 mmol/L.^2,12 To some extent, [Ca²⁺]_SR appears to be buffered by low-affinity endogenous Ca²⁺ binding proteins, such as calsequestrin (K_D 0.6 mmol/L), that are expressed in the SR lumen.² If depletion of luminal Ca²⁺ was an important factor in termination of Ca²⁺ release, increasing luminal Ca²⁺ buffering would be expected to prolong Ca²⁺ release duration leading to enlarged and prolonged cytosolic Ca²⁺ transients. In disagreement with this scenario, overexpressing calsequestrin in mouse heart has been shown to result in marked reduction in cellular Ca²⁺ transients and contractions, although the total SR Ca²⁺ content (estimated by caffeine application) increased dramatically.^13,14 Furthermore, the mice overexpressing calsequestrin developed cardiac hypertrophy and failure. Although overexpressed calsequestrin should clearly increase intra-SR [Ca²⁺] buffering, the interpretation of the results of these studies is complicated by potential compensatory changes and direct effects of calsequestrin on RyR activity¹⁵ in transgenic myocytes.

In the present study, we investigated the effects of low-affinity exogenous Ca²⁺ chelators loaded into the lumen of the SR on properties of global and local Ca²⁺ release in dialyzed patch-clamped and permeabilized rat ventricular myocytes. Inclusion of exogenous Ca²⁺ buffers into the SR should stabilize intra-SR [Ca²⁺], thus enabling us to determine the relative importance of changes of [Ca²⁺]_SR in Ca²⁺ release termination. In addition, this strategy should allow us to probe the role of luminal Ca²⁺ buffers in the absence of potential nonspecific changes induced by protein overexpression in transgenic animals. We show that at variance with the results obtained in calsequestrin-overexpressing myocytes, enhanced luminal Ca²⁺ buffering increases the magnitude of Ca²⁺ release and at the same time slows the dynamics of functional recharging of SR Ca²⁺ stores. Based on our results, we conclude that termination of CICR and the subsequent restitution behavior of Ca²⁺ release sites in cardiac muscle are determined by changes in intra-SR [Ca²⁺] regulating RyR openings.

	Materials and Methods

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Single ventricular myocytes were obtained from adult male Sprague-Dawley rat (Charles River Labs, Raleigh, NC) hearts by enzymatic dissociation.¹⁶ Rats were handled in accordance with the guidelines of the Texas Tech University HSC Animal Care and Use Committee. Experiments were performed either in patch-clamped or in permeabilized myocytes at room temperature (21 to 23°C). Whole-cell patch-clamp recordings of transmembrane currents were performed using an Axopatch 200B amplifier (Axon Instruments, USA). External solution contained (in mmol/L) 140 NaCl, 5.4 KCl, 1 CaCl₂, 0.5 MgCl₂, 10 HEPES, 5.6 glucose, and 0.02 TTX (pH 7.3). Micropipettes made from borosilicate glass (Sutter Instrument Co, USA; 1 to 3 M resistance) were filled with a solution that contained (in mmol/L) 120 Cs-aspartate, 20 CsCl, 3 Na₂ATP, 3.5 MgCl₂, 5 HEPES, 0.4 EGTA, 0.05 Fluo-3, and pCa 7 (pH 7.3). Pipette solutions containing various Ca²⁺ chelators (20 mmol/L of citrate, maleate or 5,5'-dinitro-BAPTA) were prepared by substituting Cs-aspartate with osmotically equivalent amounts of Cs-salts of the chelators. Free [Mg²⁺] in the pipette solutions was measured fluorometrically (with Mag-Fluo-4 [5 µmol/L], Molecular Probes) and adjusted to 1 mmol/L. Voltage pulses were applied from a holding potential of -50 mV at 1-minute intervals, unless specified otherwise. Cardiac myocyte permeabilization was performed using saponin as described previously.¹⁶ The basic internal solution contained (in mmol/L) 100 K-aspartate, 20 KCl, 3 MgATP, 0.5 EGTA, 0.81 MgCl₂, 10 phosphocreatine, 20 HEPES, 0.03 Fluo-3 K-salt (TefLabs), and 5 U/mL creatine phosphokinase, pCa 7 (pH 7.2). K-salts of citrate, maleate, acetamido iminodiacetic acid (ADA), and 5,5'-dinitro-BAPTA (dn-BAPTA) were added into the internal solution at specified concentrations by replacing osmotically equivalent amounts of K-aspartate. Free [Mg²⁺] was adjusted to 1 mmol/L in the solutions. Cells were imaged using a Bio-Rad Laser Scanning Confocal system (Bio-Rad MRC-1024ES interfaced to an Olympus IX-70 inverted microscope) with an Olympus 60x1.4 NA oil objective.¹⁶ Fluo-3 was excited by light at 488 nm, and the fluorescence was acquired at wavelengths of >515 nm in the line-scan mode of the confocal system at rate of 2.0 ms per scan. Ca²⁺ spark parameters were quantified using a detection/analysis computer algorithm.⁷ All chemicals unless specified otherwise were from Sigma. The local changes in cytosolic and luminal [Ca²⁺] were simulated using a multicompartmental diffusion-reaction model of Ca²⁺ dynamics in the cytosolic and SR compartments. A detailed description of the model can be found in the online data supplement available at http://www.circresaha.org.

	Results

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Macroscopic Ca²⁺ Transients and I_Ca Measured Under Voltage Clamp
First, we explored the effects of dialysis of myocytes with internal solution containing the low-affinity Ca²⁺ chelators citrate and maleate (K_D values=0.47 and 11 mmol/L, respectively)¹⁷ on EC coupling in patch-clamped ventricular myocytes. These anions may be effectively transported into the SR by an anion transporter, thus increasing the Ca²⁺ buffering capacity of the SR. Indeed, dialysis of the cells with solutions containing 20 mmol/L citrate or maleate for 20 minutes dramatically increased the amount of Ca²⁺ stored in the SR, as indicated by increased caffeine-induced Ca²⁺ transients (Figure 1A; see also online Table 2, which can be found in the online data supplement available at http://www.circresaha.org). This increase in the total SR Ca²⁺ content was associated with a marked enhancement of the amplitude of I_Ca-induced Ca²⁺ transients elicited by depolarization. In addition, the cells lost their ability to respond to I_Ca in a graded manner, because even small Ca²⁺ currents triggered maximal Ca²⁺ transients (Figure 1B). Similar effects of intracellular citrate on Ca²⁺ transients have been observed previously in atrial myocytes.¹⁸ The increases in Ca²⁺ transients were more pronounced with citrate, which has a higher affinity for Ca²⁺, than with maleate. This is consistent with the notion that the effects of these Ca²⁺ chelators result from an increased Ca²⁺ storage capacity of the SR. Citrate should bind more Ca²⁺ ions in the SR lumen at free [Ca²⁺] of 1 mmol/L, which is believed to correspond to the level of free [Ca²⁺] in the SR at steady-state.^2,12 These effects on Ca²⁺ transients were not accompanied by any significant changes in the amplitude of the Ca²⁺ currents, although I_Ca decay rate was slowed 1.4- to 2.7-fold, apparently due to interference of the buffers with Ca²⁺-dependent inactivation (Figures 1A and 1B; online Table 2). Exposure of the myocytes to the high–molecular weight Ca²⁺ buffer dn-BAPTA (K_D7 mmol/L)¹⁷ produced no significant changes in caffeine- and depolarization-induced Ca²⁺ transients, although it affected I_Ca inactivation in a manner similar to that observed with maleate and citrate (Figure 1B; online Table 2). These results may be due to inability of this compound to cross the SR membrane, a feature that would restrict its actions to the cytosolic compartment. These findings support the possibility that the bulk of the effects of maleate and citrate on Ca²⁺ transients was through the action of these compounds in the luminal rather than in the cytosolic compartment. When citrate-dialyzed myocytes were stimulated periodically (at 0.5 Hz), they responded by alternating large and small Ca²⁺ transients. As illustrated by line-scan images (Figure 1C), the large-size Ca²⁺ transients were produced either by a massive and prolonged release of Ca²⁺, activated synchronously throughout the cell, or by a smaller, spatially inhomogeneous response eventually turning into full-scale regenerative Ca²⁺ release. Qualitatively, similar effects were observed in cells dialyzed with maleate but not dn-BAPTA, which had no significant impact on the periodic Ca²⁺ transients (not shown). In a separate series of 2-pulse experiments, intracellular dialysis with maleate or citrate also slowed the restitution of steady-state Ca²⁺ transients after Ca²⁺ release (online Figure 1, which can be found in the online data supplement available at http://www.circresaha.org). Taken together, these results suggest that exogenous Ca²⁺ buffers in the SR affect the processes of release and recharging of the SR Ca²⁺ stores by increasing the functional size of the stores. One significant limitation of the whole-cell dialysis experiments is that potential cytoplasmic effects of the chelators, such as effects on cytosolic Ca²⁺-dependent inactivation of release, complicate interpretation of the results.

View larger version (42K): [in this window] [in a new window]   Figure 1. Ca2+ release in myocytes dialyzed with low-affinity Ca2+ chelators. A, Representative traces of ICa and Ca2+ transients, induced by depolarizing steps from -50 to 0 mV, along with traces of Ca2+ transients, induced by caffeine (10 mmol/L), in 3 different cells dialyzed with control solution or with solutions containing 20 mmol/L maleate or citrate. B, Voltage-dependence of Ca2+ transients and ICa in myocytes dialyzed with control pipette solution and with solutions containing 20 mmol/L maleate, citrate, or dn-BAPTA. C, Line-scan images along with averaged temporal profiles of fluorescence changes acquired in 2 different periodically stimulated myocytes dialyzed either with control or citrate-containing pipette solutions. Myocytes were stimulated at 0.5 Hz by voltage-clamp steps from -50 to 0 mV.

Ca²⁺ Sparks in Permeabilized Myocytes
Therefore, to gain further mechanistic insights into the action of luminal Ca²⁺ buffers, we explored the effects of the same Ca²⁺ chelators as well as ADA (K_D 0.2 mmol/L)¹⁷ on the Ca²⁺ storage capacity of the SR and Ca²⁺ release in permeabilized cardiac myocytes. Figure 2A shows traces of caffeine-induced Ca²⁺ transients in saponin-permeabilized myocytes that were incubated for various times with 20 mmol/L citrate or 5 mmol/L ADA before application of caffeine. Caffeine (10 mmol/L) was applied within 1 minute after reverting to the control, Ca²⁺ chelator-free experimental solution. Exposure of the permeabilized myocytes to these Ca²⁺ chelators led to a gradual increase in the magnitude of the caffeine-induced Ca²⁺ transients, signaling a corresponding increase in the amount of Ca²⁺ sequestered in the SR. Again, the effects were most pronounced with the chelator that has the highest affinity for Ca²⁺ (ADA) and no significant changes were observed with the presumably SR impermeable dn-BAPTA (Figure 2B). After washout of the chelators from the cytosolic compartment, they appeared to remain transiently entrapped inside the SR, as indicated by the slow return of the amplitude of the caffeine-induced Ca²⁺ transients back the control levels. These results confirm that indeed certain low–molecular weight Ca²⁺ chelators including maleate, citrate, and ADA can be introduced into the SR to buffer intra-SR [Ca²⁺]. The slow rate of loss of the chelators from the SR provides a time window of several minutes during which the effects of luminal buffers on properties of Ca²⁺ release can be studied at relatively steady conditions and in the absence of buffers on the cytosolic side.

View larger version (28K): [in this window] [in a new window]   Figure 2. Effects of exposure to low-affinity Ca2+ chelators on the total SR Ca2+ content in permeabilized myocytes. A, Representative traces of Ca2+ transients induced by caffeine (10 mmol/L) before and at different times after addition and removal to/from the internal solution of 20 mmol/L citrate or 5 mmol/L ADA. B, Averaged amplitude of caffeine-induced Ca2+ transients as a function of time on addition and removal of citrate (20 mmol/L), maleate (20 mmol/L), dn-BAPTA (20 mmol/L), or ADA (5 mmol/L). Each data point represents average from 2 to 7 determinations in different cells.

Figure 3 illustrates the effects of addition and subsequent removal from the internal solution of some of the aforementioned Ca²⁺ chelators on properties of spontaneous Ca²⁺ sparks. The brightness of the fluorescence events was reduced on addition of the chelators, apparently due to the action of buffers on the cytosolic side of the SR. However, removal of citrate and ADA from the internal solution after 30 minutes incubation of the myocytes with the chelators resulted in dramatic increases in the magnitude of Ca²⁺ sparks. The magnitude of events returned to control levels within 45 minutes in parallel with restoration of the total SR Ca²⁺ content (Figure 2B). We attribute this potentiation of sparks to enhanced buffering of luminal [Ca²⁺] by the chelators entrapped in the SR.

View larger version (91K): [in this window] [in a new window]   Figure 3. Effects of exposure to low-affinity Ca2+ chelators on Ca2+ sparks in permeabilized myocytes. Representative images of Ca2+ sparks acquired before and after exposure of the permeabilized myocytes to 20 mmol/L citrate or 5 mmol/L ADA, and at 2 different times after washout. Traces below the images plot F/F0 at the center of sparks.

The effects of intraluminal buffers on spatiotemporal properties of sparks are summarized in Figure 4 (see also online Table 3). ADA (5 mmol/L) and citrate (20 mmol/L) were loaded into the SR by a 30-minute incubation; sparks were then measured within 3 minutes after washout of the chelators from the experimental chamber, as shown in Figure 3. The images of sparks in Figure 4A were obtained by averaging the brightest events (5% of all events in each group, with most likely localization in the center of the line-scan⁷) measured in control as well as in the presence of luminal ADA and citrate. Similar to the effects on caffeine-induced Ca²⁺ transients, the effects on Ca²⁺ sparks were most pronounced with ADA, which has the highest capacity to buffer luminal Ca²⁺. The effects of the exogenous luminal buffers on the magnitude and time course of the sparks are further illustrated in Figure 4B. In the inset, the signals were scaled to the same peak amplitude to better illustrate the effects on rise times. In addition to increasing the amplitude and the overall duration of the signal, the luminal buffers dramatically increased the rise time of sparks (about 1.74- and 2.35-fold for citrate and ADA, respectively). Figure 4D shows the effects of luminal buffers on the distribution of spark rise times for all the events. Luminal citrate and ADA produced significant shifts in the distribution of spark rise times to the right. The duration of the rising phase of the spark has been shown to provide a good estimate of the duration of underlying Ca²⁺ release.¹⁹ Therefore, the observed increase in duration of the rising phase of the events is a clear indication that the release was prolonged in the presence of the luminal buffers. The changes in Ca²⁺ release fluxes are further emphasized by the prolonged duration of the first derivatives of the fluorescent signals in the presence of the exogenous chelators in the SR (Figure 4C). The first derivative of the fluorescent signal can be considered to be an approximation of the rate of local Ca²⁺ release underlying the spark.²⁰

View larger version (39K): [in this window] [in a new window]   Figure 4. Effects of buffering [Ca2+]SR with luminal citrate or ADA on properties of Ca2+ sparks in permeabilized myocytes. A, Images of Ca2+ sparks obtained before (top) and after loading the SR with 20 mmol/L citrate (middle) or 5 mmol/L ADA (bottom). Signals were obtained by averaging 22, 16, and 19 individual sparks for reference and luminal citrate or ADA, respectively. B, Temporal profiles of Ca2+ sparks presented in A at peak. Inset, Traces were scaled to the same peak amplitude. C, First derivatives of the F/F0 traces shown in B. D, Histograms of the rise times of Ca2+ sparks in reference (green), luminal citrate (red), or ADA (blue) for 450, 350, and 390 events, respectively.

Simulated Ca²⁺ Release
To further evaluate the effects of luminal Ca²⁺ chelators on Ca²⁺ release, we used a theoretical model of Ca²⁺ release that simulates changes in local [Ca²⁺] in both the cytosolic and luminal compartments. Because this approach relies on assumptions regarding binding and diffusion parameters for different Ca²⁺ binding molecules (online Table 1), it is intended only as a qualitative analysis tool. As shown in Figure 5, activation of the release unit for a fixed period of time (5 ms) produces a luminal to cytosolic Ca²⁺ flux and results in an increase in the local cytosolic [Ca²⁺] and a decline in the local SR luminal [Ca²⁺]. Increasing luminal Ca²⁺ buffering has only minor effects on the magnitude and time course of the local cytosolic Ca²⁺ transients and no effect on their rise time; however, the extent of depletion of local [Ca²⁺]_SR is reduced substantially (Figures 5C and 5D, left traces). To approximately reproduce the experimentally observed increases in the magnitude and the time-to-peak of Ca²⁺ sparks in the presence of ADA and citrate, the release unit open time had to be increased about 3- and 4-fold, respectively (Figures 5C and 5D, right traces). Thus, our experimental and modeling results suggest that increased buffering profoundly influences the duration of local Ca²⁺ release fluxes, apparently by influencing RyR open probability. Another important point suggested by our simulations is that in addition to affecting release flux duration the exogenous buffers should also slow dramatically the recovery of luminal [Ca²⁺] after release. Intuitively, this is expected because of the increased filling capacity of the SR. This would imply that if termination of local Ca²⁺ release is indeed controlled by luminal [Ca²⁺], the presence of exogenous buffers in the SR should influence the apparent refractoriness of release sites by affecting the dynamics of recovery of local [Ca²⁺]_SR after sparks.

View larger version (26K): [in this window] [in a new window]   Figure 5. Simulations of luminal Ca2+ buffer effects on Ca2+ sparks. Composite conductance of the release unit (A), local Ca2+ release flux from the SR (B), profiles of Ca2+ simulated sparks (C), and luminal [Ca2+] ([Ca2+]SR) versus time (D) under control conditions (black lines) and in the presence of 20 mmol/L luminal citrate (red lines) or 5 mmol/L luminal ADA (blue lines). In simulations presented on the left, the duration of composite RyR openings was set at fixed value; in simulations presented on the right, the duration of composite RyR openings was increased from 5 ms to 16 and 22 ms for luminal citrate and ADA, respectively, to reproduce the experimentally observed effects of these chelators on Ca2+ sparks. E, Simulated effects of luminal citrate and ADA on spatiotemporal properties of Ca2+ sparks.

Repetitive Activity Induced by IpTxA
To explore the effect of luminal buffers on the ability of release sites to fire repeatedly, we preformed experiments with the high-affinity RyR activator Imperatoxin A (IpTxA). In bilayer experiments, IpTxA has been shown to activate skeletal and cardiac RyRs by inducing long-lived openings with 1/3 conductance, and by increasing the frequency of transitions from closed to fully open states²¹ (H.H. Valdivia and S. Györke, unpublished observations, 2002). In accordance with the single channel effects, the toxin induces Ca²⁺ sparks with long-lasting tails and generates repetitive sparks at individual release sites in skeletal muscle.²² We found that IpTxA has qualitatively similar effects on Ca²⁺ sparks in cardiac myocytes. Thus, IpTxA increased the overall frequency of events 3.5-fold, mainly by increasing the number of repetitive events at the same individual sites (Figure 6C); 14 of a total of 2381 events in the presence of IpTxA featured tails similar to those described in skeletal muscle (not shown). In the present study, we focused on the repetitive activity induced by the toxin.

View larger version (70K): [in this window] [in a new window]   Figure 6. Combined effects of IpTxA and luminal maleate or citrate on properties of Ca2+ sparks in permeabilized myocytes. A, Images of repetitive Ca2+ sparks measured in the presence of 10 nmol/L IpTxA under reference conditions and after loading the SR with 20 mmol/L maleate or citrate. Traces present temporal profiles of F/F0 at arrows. B, Histograms of interspark intervals in sequences of events at individual sites under reference conditions and in the presence of luminal maleate or citrate, for 266, 247, and 250 counts, respectively. C, Average frequency of events recorded in the presence and absence of IpTxA (10 nmol/L) for reference conditions as well as for luminal maleate or citrate. Maleate and citrate were loaded into the SR by 30 minutes incubation and sparks measured within 5 minutes after washout of the chelators from the cytosolic compartment.

An example of repetitive activity of a release site in the presence of 10 nmol/L IpTxA is shown in Figure 6A. Repeated events are apparently ignited by a single toxin-modified RyR that acts as a pacemaker for the whole release unit. Interestingly, the amplitude of the recurrent events exhibited high variability, presumably due to a variable number of channels involved in the events and variations in the local [Ca²⁺]_SR. Loading the SR with 20 mmol/L maleate or citrate led to a dramatic prolongation of the time period between repetitive sparks induced by IpTxA (average interspark intervals increased 2.3- and 5-fold, respectively; Figure 6B). Accordingly, the overall frequency of events was decreased by 38% and 54%, respectively (Figure 6C). At the same time, luminal chelators had only a small effect on the frequency of Ca²⁺ sparks measured in the absence of the toxin (Figure 6C). We attribute the differential effects of the luminal Ca²⁺ buffers on repeated IpTxA-induced sparks and spontaneous sparks to differences in the dynamics of local luminal [Ca²⁺]. The capacity of IpTxA to activate repeatedly the same release sites depends on the rate of refilling of the Ca²⁺ storage sites, which is in turn influenced by binding of Ca²⁺ to luminal buffers. On the other hand, in the absence of IpTxA the frequency of sparks arising spontaneously at the same site is very low (P0.025 s^-1 100 µm^-1, assuming a spark frequency of 5 s^-1 100 µm^-1 in the whole line scan and 200 release sites contained in the volume scanned by the confocal microscope⁷), and the Ca²⁺ storage sites have sufficient time to fully recharge even when the loading capacity of the stores is increased by the exogenous Ca²⁺ chelators. These results indicate that [Ca²⁺]_SR strongly influences not only the termination of local release but also the restitution behavior of release sites after Ca²⁺ sparks in cardiac myocytes.

	Discussion

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How CICR is terminated is an outstanding question in the field of cardiac EC coupling. During the Ca²⁺ release process, [Ca²⁺] on the cytosolic side of the SR increases, whereas [Ca²⁺] in the SR lumen decreases. Accordingly, two types of mechanisms have been discussed in the literature to explain Ca²⁺ release termination: (1) binding of Ca²⁺ to inhibition sites on the RyR reduces channel activity through processes referred to as Ca²⁺-dependent inactivation or adaptation^{1,10,11,23,24}; and (2) dissociation of Ca²⁺ from luminal activation sites decreases RyR open probability by a process that can be termed luminal Ca²⁺-dependent deactivation.^25–27 In this study, we have loaded low-affinity Ca²⁺ buffers into the SR luminal compartment to probe the mechanisms responsible for termination of local Ca²⁺ release events, ie, Ca²⁺ sparks, in permeabilized rat ventricular myocytes. Such buffers should stabilize or "clamp" [Ca²⁺]_SR, thus enabling us to address the importance of changes in luminal versus cytosolic Ca²⁺ in termination of Ca²⁺ sparks. We found that buffering luminal [Ca²⁺] dramatically increases the amplitude and rise time of sparks as well as the duration of local Ca²⁺ release fluxes underlying sparks, thus implying that duration of Ca²⁺ release depends on changes in [Ca²⁺]_SR. The buffers also reduced the frequency of repetitive activity induced at individual release sites by the high-affinity RyR activating peptide IpTxA, although they did not considerably affect the frequency of events occurring randomly throughout the cells. These results indicate that the restitution behavior, ie, "refractoriness," of release sites after discharge is related to local depletion and refilling of SR Ca²⁺ storage sites by Ca²⁺. In whole-cell patch-clamped myocytes, the SR lost its ability to respond to I_Ca in a graded manner in the presence of SR-permeable Ca²⁺ buffers. The buffers also slowed the recovery of cell-averaged Ca²⁺ transients in experiments with two pulses. Taken together our results suggest that termination of Ca²⁺ release and the subsequent restitution behavior of release sites in cardiac muscle is controlled by local intra-SR [Ca²⁺] regulating RyR openings.

Previous studies from our and other laboratories have shown that luminal Ca²⁺ enhances RyR activity by 2acting at sites localized at the luminal side of the RyR complex.^25,28,29 The effects of luminal Ca²⁺ have been also observed in intact and permeabilized myocytes as increases in frequency of Ca²⁺ sparks on elevating the SR Ca²⁺ load.^3,16,30 The luminal Ca²⁺ sensor have been shown to continuously regulate the functional activity of the SR Ca²⁺ stores by linking SR Ca²⁺ content to the activity of the RyRs.^16,29 This control mechanism, although possessing a certain time lag (due to the dynamics of changing SR load), stabilizes Ca²⁺ cycling when either Ca²⁺ release or uptake is altered. For example, when the RyRs are inhibited by tetracaine, reduced leak leads to elevation of SR Ca²⁺ content.^16,31 Increased luminal [Ca²⁺] then enhances RyR P_o, countering the inhibitory action of tetracaine. In the present study, we have shown that a luminal Ca²⁺ sensor also regulates RyR activity in a more immediate fashion, directly accounting for or contributing to termination of CICR at both local and global levels.

Our finding that Ca²⁺ spark termination is governed by luminal Ca²⁺ reconciles a body of apparently contradicting results. Several studies have found that SR Ca²⁺ release is turned off during sustained Ca²⁺ trigger stimuli before the SR would exhaust its supply of Ca²⁺ (as assessed by caffeine).^11,23 Consequently, it has been proposed that release termination is caused by mechanisms such as Ca²⁺-dependent inactivation or adaptation of RyRs, which depend on elevation of cytosolic Ca²⁺, or channel activation per se, rather than by a loss of Ca²⁺ from the SR.^1,11,24 The present study shows that even a partial Ca²⁺ depletion can terminate Ca²⁺ sparks, and that the apparent refractoriness after release can be ascribed to the time required for partially depleted SR to be refilled with Ca²⁺. It is important to note that the Ca²⁺ release channels on partial depletion of stores do not become absolutely refractory, ie, desensitized to the cytosolic Ca²⁺ trigger. Instead, changes in luminal [Ca²⁺] reduce the sensitivity of the release channels to cytosolic [Ca²⁺],^16,25,32 leaving the channels potentially responsive to larger Ca²⁺ concentrations. Therefore, our finding could account for the ability of Ca²⁺ stores to respond transiently to multiple incremental increases in the trigger signal, a phenomenon termed "quantal" or "adaptive" Ca²⁺ release. The mechanisms underlying this phenomenon, demonstrated in both ryanodine- and IP₃-sensitive stores, have not been defined thus far, although several schemes have been discussed (eg, Fill et al²⁴ and Koizumi et al³²), including Ca²⁺ release from functionally heterogeneous Ca²⁺ stores, inactivation/adaptation of RyRs, and control of RyR openings by Ca²⁺ within the lumen of the SR. Local [Ca²⁺]_SR changes and the subsequent changes in RyR open probability could also account for our previous observations that the decay of back-calculated Ca²⁺ release fluxes underlying sparks increase with increasing SR Ca²⁺ load.¹⁰ Indeed, faster releases from more fully loaded SR Ca²⁺ stores could lead to a more rapid and synchronous activation of RyRs composing the release unit, resulting in a faster decline of luminal [Ca²⁺]. Effects of luminal Ca²⁺ on RyR functional activity is also the most likely explanation for the differences in restitution behavior of the SR Ca²⁺ stores after their global versus local activation by photolysis of caged Ca²⁺, as suggested by DelPrincipe et al.²⁶

Wang et al¹³ demonstrated that 10- to 20-fold overexpression of calsequestrin leads to impaired Ca²⁺ release and a reduction of spontaneous Ca²⁺ sparks in transgenic mouse cardiomyocytes. The reasons for the discrepancies between our results and those with overexpressed calsequestrin are not known. We can speculate that in calsequestrin-overexpressing myocytes, the free [Ca²⁺]_SR may be chronically lowered due to exceptionally heavy Ca²⁺ buffering, leading to depressed RyR activity. Considering a concentration of intra-SR Ca²⁺ sites of 10 mmol/L that is almost entirely due to the presence calsequestrin, ^2,12 a 10- to 20-fold overexpression of this protein would result in an increase of concentration of intra-SR Ca²⁺ sites to 100 to 200 mmol/L. With such a massive Ca²⁺ buffering, it might be difficult to attain the steady-state free [Ca²⁺]_SR characteristic of normal myocytes. However, it is also possible that the alterations of release in transgenic myocytes are caused by direct effects of calsequestrin on RyR¹⁵ and/or some nonspecific changes induced by protein overexpression.

In conclusion, in the present study, we have shown that termination of SR Ca²⁺ release and the subsequent restitution behavior of Ca²⁺ release sites in cardiac muscle is governed by local intra-SR [Ca²⁺] regulating RyR openings. Thus, our study may provide a solution to the longstanding problem in the field of cardiac EC coupling: how is CICR terminated? It is also relevant to understanding intracellular Ca²⁺ signaling in other cell types, including neurons, skeletal muscle fibers, and smooth muscle cells, where Ca²⁺ sparks and CICR have been described.

	Acknowledgments

 
This work was supported by grants from the American Heart Association and the National Institutes of Health (HL 52620 and HL 03739).

Received April 30, 2002; revision received July 25, 2002; accepted July 26, 2002.

	References

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