Luminal Ca2+ Controls Termination and Refractory Behavior of Ca2+-Induced Ca2+ Release in Cardiac Myocytes
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 ICa and deranged the smoothly graded ICa-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.
- sarcoplasmic reticulum
- excitation-contraction coupling
- calcium-induced calcium release
- ryanodine receptor
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 (KD ≈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
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 MΩ 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 60×1.4 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.
Macroscopic Ca2+ Transients and ICa 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 (KD 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 ICa-induced Ca2+ transients elicited by depolarization. In addition, the cells lost their ability to respond to ICa 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 ICa 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 (KD≈7 mmol/L)17 produced no significant changes in caffeine- and depolarization-induced Ca2+ transients, although it affected ICa 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.
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 (KD ≈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.
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.
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
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.
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.
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 (P≈0.025 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.
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 adaptation1,10,11,23,24⇓⇓⇓⇓; 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 ICa 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 Po, 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.
This work was supported by grants from the American Heart Association and the National Institutes of Health (HL 52620 and HL 03739).
Original received April 30, 2002; revision received July 25, 2002; accepted July 26, 2002.
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