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(Circulation Research. 2004;95:807.)
© 2004 American Heart Association, Inc.

Cellular Biology

Sarcoplasmic Reticulum Ca²⁺ Refilling Controls Recovery From Ca²⁺-Induced Ca²⁺ Release Refractoriness in Heart Muscle

Peter Szentesi, Christophe Pignier, Marcel Egger, Evangelia G. Kranias, Ernst Niggli

From the Department of Physiology (P.S., C.P., M.E., E.N.), University of Bern, Bern, Switzerland; the Department of Physiology (P.S.), Medical and Health Science Center, University of Debrecen, Debrecen, Hungary; and the Department of Pharmacology and Cell Biophysics (E.G.K.), University of Cincinnati, Cincinnati, Ohio.

Correspondence to E. Niggli, Dept of Physiology, University of Bern, Buehlplatz 5, CH-3012 Bern, Switzerland. E-mail niggli{at}pyl.unibe.ch

	Abstract

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In cardiac muscle Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca²⁺ influx via L-type Ca²⁺ channels. At present, the mechanisms underlying termination of SR Ca²⁺ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca²⁺ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca²⁺ release, we analyzed Na⁺-Ca²⁺ exchange currents reflecting cytosolic Ca²⁺ signals induced by UV-laser flash-photolysis of caged Ca²⁺. Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, ß-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by 2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 µmol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca²⁺ refilling mediated by the SR Ca²⁺-pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca²⁺ sensitivity of CICR appears to be regulated by SR Ca²⁺ content, possibly resulting from a change in the steady-state Ca²⁺ sensitivity and in the gating kinetics of the SR Ca²⁺ release channels (ryanodine receptors). During Ca²⁺ release, the concomitant reduction in Ca²⁺ sensitivity of the ryanodine receptors might also underlie Ca²⁺ spark termination by deactivation.

Key Words: calcium-induced calcium release • excitation-contraction coupling • cardiac myocyte • heart • sarcoplasmic reticulum • calcium content • photolysis of caged calcium

	Introduction

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The contraction of ventricular myocytes is the final step of excitation-contraction (EC) coupling in heart muscle. During this process, Ca²⁺ entering via L-type Ca²⁺ channels induces a massive liberation of sarcoplasmic Ca²⁺ (Ca²⁺-induced Ca²⁺ release [CICR]) through Ca²⁺ release channels (ryanodine receptors [RyRs]).^1,2 CICR occurs by a concerted activation of numerous Ca²⁺-release events (Ca²⁺ sparks).³ Relaxation requires both the termination of Ca²⁺ sparks and the removal of Ca²⁺ from the cytosol.^4,5 Mechanisms terminating Ca²⁺ release from the sarcoplasmic reticulum (SR) are presently not well characterized, but several possibilities have been discussed. These include Ca²⁺-induced inactivation of RyRs, adaptation of RyRs, stochastic attrition, and functional depletion of the SR Ca²⁺ stores.^2,4,6,7 A substantial body of indirect experimental evidence indicates that one or more mechanisms leading to refractoriness of the CICR must exist. However, studies examining the time course of recovery of cardiac force after a twitch contraction have generally failed to detect a refractoriness of CICR.⁸ But a genuine and slowly recovering refractoriness of CICR may be difficult to detect with a technique that relies on the Ca²⁺ current, because this trigger itself exhibits a recovery from inactivation. Using a different approach, we have recently observed that activation of SR Ca²⁺ release by UV-laser flash photolysis of caged Ca²⁺ indeed unmasks a genuine refractoriness of CICR.⁹ With photochemically generated Ca²⁺-concentration jumps, CICR can be triggered independently of L-type Ca²⁺ currents.¹⁰ We then suggested that Ca²⁺ store depletion may underlie, at least in part, the observed refractoriness, and that SR Ca²⁺ content may determine the Ca²⁺ sensitivity of the RyRs on the cytosolic side of the channels.

The goal of the present study was to examine whether SR Ca²⁺ content may indeed determine RyR Ca²⁺ sensitivity and whether SR Ca²⁺ refilling is the rate-limiting step determining the recovery from refractoriness. For this purpose, we applied pharmacological interventions to correlate SR Ca²⁺ pump (SERCA) activity with the time course of recovery from refractoriness. To this end, we elicited Ca²⁺ release from the SR by using UV-laser flash photolysis of caged Ca²⁺ as the trigger. The resulting Na⁺-Ca²⁺ exchange currents (I_NCX) were analyzed to evaluate SR Ca²⁺ release events and to characterize the refractoriness of CICR in terms of its temporal features.⁹ Analogous experiments were also performed in myocytes from phospholamban (PLB)-deficient mice. PLB is a protein, which regulates SR Ca²⁺ cycling in cardiomyocytes by reversibly inhibiting the SERCA.¹¹ We found that SR Ca²⁺ refilling is strongly influencing recovery from CICR refractoriness. Thus, the Ca²⁺ sensitivity of the RyRs appears to be regulated by SR Ca²⁺ content. Preliminary reports describing this work have been presented in abstract form.^12,13

	Materials and Methods

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Cell Isolation
All experiments were performed according to the guidelines of the Swiss Animal Protection Law and with the permission of The State Veterinary Office, Bern, Switzerland. Cardiac ventricular myocytes were isolated from adult guinea pigs and mice (Schneider, Rubigen, Switzerland) using established enzymatic procedures.¹⁴ The superfusion solution contained (in mmol/L): 140 NaCl, 5 KCl, 0.5 BaCl₂, 1.8 CaCl₂, 1 CsCl, 10 HEPES, 10 glucose, pH 7.4 (NaOH). The pipette filling solution contained (in mmol/L): 120 Cs-aspartate, 20 tetraethylammonium chloride, 2 Na₄-DM-nitrophen (1-(2-nitro-4,5-dimethoxyphenyl)-1,2-diaminoethane-N,N,N',N'-tetraacetic acid, 4Na⁺), 2 reduced glutathione, 0.05 Fluo-3, 0.5 CaCl₂, 5 K₂-ATP, 20 HEPES, pH 7.2 (CsOH). In experiments with mouse cells, the Na₄-DM-nitrophen concentration was 4 mmol/L. All experiments were performed at room temperature (20°C to 22°C). Where required, the superfusion solution contained isoproterenol (1 mmol/L; Sigma) or cyclopiazonic acid (1, 5 or 10 µmol/L; Sigma).

Electrophysiological Recordings
Cells were voltage-clamped in the whole-cell configuration of the patch-clamp technique and held at –80 mV. The SR was loaded with Ca²⁺ by six depolarizing steps to 10 mV lasting 200 ms, at 1 Hz, to elicit L-type Ca²⁺ currents. I_NCX were recorded at –40 mV to estimate changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i). Within the [Ca²⁺]_i jumps reached under our conditions, I_NCX is known to depend linearly on [Ca²⁺]_i.¹⁵ The SR Ca²⁺ content was estimated by integrating the I_NCX induced by rapid application of 20 mmol/L caffeine. All values are expressed as means±SEM. Student unpaired t tests were used to test for statistical significance (P<0.01, as indicated).

Flash Photolysis
Pairs of UV-light flashes were used to photolyse intracellular DM-nitrophen in an epi-illumination arrangement and were generated with a frequency-tripled neodymium: yttrium-aluminum garnet laser (Nd:YAG laser) (wavelength 355 nm, maximal energy 20.7 mJ, repetition rate up to 10 Hz; Shurelite II; Continuum). Photoconsumption of DM-nitrophen in our setup was determined as 4% at maximum tolerable laser power.

	Results

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UV-laser flash photolysis of caged Ca²⁺ was used to activate CICR with a paired-pulse protocol. This approach allowed us to examine the refractoriness of SR Ca²⁺ release and to determine the time course of recovery from refractoriness. As we have shown before, this technique has the advantage that two trigger signals of identical amplitude can be applied to unmask and analyze the CICR refractoriness.⁹ For the experiments shown below, the photolytic triggers have the additional advantage of remaining constant during ß-adrenergic stimulation of the myocytes, unlike the L-type Ca²⁺ currents, which would increase in amplitude.

ß-Adrenergic Stimulation Accelerates Recovery From Refractoriness
In the following experiments, we used guinea pig ventricular myocytes to record I_NCX generated by the electrogenic Na⁺-Ca²⁺ exchanger, as estimates of changes in the intracellular Ca²⁺ concentration, [Ca²⁺]_i. I_NCX has been shown to be linearly dependent on [Ca²⁺]_i over the range of concentrations covered by UV-flash photolysis of caged Ca²⁺.^9,15 Laser-flash photolysis of DM-nitrophen caused a rapid and homogeneous Ca²⁺ concentration jump (<30 µs) that resulted in an almost immediate activation of inward I_NCX.¹⁶ The fast photochemical Ca²⁺ release from DM-nitrophen was reflected in a rapidly rising I_NCX component, followed by a slower increase in I_NCX (I_NCX-slow), continuing for 50 ms before I_NCX started to decline, in parallel with the decrease in [Ca²⁺]_i. The slow component of I_NCX activation arises from CICR triggered by the photolytic Ca²⁺ release, and the amplitude of I_NCX-slow can be used to estimate the magnitude of CICR (see also inset in Figure 1 Ba).⁹ Figure 1A shows the loading protocol, which was applied to ensure comparable SR Ca²⁺ load conditions. In this series of experiments, we applied isoproterenol as an agonist for the ß-adrenergic receptors to accelerate the SERCA. Subsequent to receptor activation, Protein kinase A–dependent phosphorylation of PLB is known to relieve the suppression of the SERCA, thereby, accelerating the pump and SR Ca²⁺ refilling. Thus, we used the SERCA stimulation to test the hypothesis that recovery from CICR refractoriness is governed by SR Ca²⁺ refilling.

View larger version (20K): [in this window] [in a new window]   Figure 1. Effects of ß-adrenergic stimulation on CICR refractoriness. A, Loading protocol used to study SR Ca2+ release refractoriness in control and after application of 1 µmol/L isoproterenol. Isoproterenol superfusion was started before reloading of the SR (6 prepulses). B, Representative INCX obtained at –40 mV induced by applying a pair of UV-flashes to photolyse caged Ca2+ (DM-nitrophen). The inset (right) shows the superimposed and expanded INCX given at the left. 1, Photolytic component for both signals; 2, CICR component of first signal; 3, CICR component of second signal. B (a), INCX obtained under conditions where CICR is activated by the photolytic Ca2+ trigger. The SR-Ca2+ release component has a slower time course compared with the fast photolytic Ca2+–release component. B (b), Control experiment in the presence of 10 µmol/L ryanodine and 0.1 µmol/L thapsigargin showing blocked CICR. The photolytic Ca2+ trigger did not exhibit refractoriness, and only the fast INCX component attributed to the photolytic Ca2+ release was activated. C, Representative INCX obtained at –40 mV at different intervals (200 ms and 500 ms) in control (left) and after isoproterenol treatment (right). D, Averaged data showing the recovery of CICR refractoriness in control conditions () and in the presence of isoproterenol (). Normalized Ca2+ release represents the ratio of the slow component (CICR) of the first and the second INCX. Solid lines represent a least-square fit of a monoexponential function to the averages (control, =717.9±60.9 ms; isoproterenol, =342.0±15.8 ms; mean±SEM; n=4 to 34). The INCX exhibited a for the current decay of 162.4±13.8 ms (n=18).

Figure 1C compares the results from experiments with pairs of UV-laser flashes in control conditions (left panels) and in the presence of 1 µmol/L isoproterenol (right panels). The concentration of isoproterenol used was 100-fold the EC₅₀ and was chosen to rapidly activate a large fraction of the ß-adrenergic receptors.¹⁷ When the interval between successive flashes was longer than 500 ms, I_NCX exhibited approximately the same peak amplitude, particularly in the presence of ß-adrenergic stimulation (Figure 1C). However, when the interval between photolytic trigger signals was gradually reduced, the second I_NCX in a pair became progressively smaller in both cases (Figure 1C and 1D). Previous experiments in the presence of ryanodine and thapsigargin have confirmed that the photolytic component and, therefore I_NCX per se, was not smaller after the second flash.⁹ (An example is also shown in Figure 1Bb.) The reduction of the slow component of I_NCX at short intervals thus indicates that the CICR was curtailed and refractory after the second pulse of a pair (see inset, Figure 1B). In control conditions, SR Ca²⁺ release exhibited refractoriness recovering with a of 717.9±60.9 ms (Figure 1D). Interestingly, the I_NCX decayed faster with a of 162.4±13.8 ms. Even though this time course reflects the decay of [Ca²⁺]_I near the sarcolemma, it allows an, albeit very indirect, estimate of SR refilling. The recovery of CICR was significantly accelerated by isoproterenol treatment (=342.0±15.8 ms). Although this result is entirely consistent with our hypothesis, unfortunately, ß-adrenoceptor stimulation is thought to alter many cellular processes involved in cardiac Ca²⁺ signaling, including RyR phosphorylation and changes of steady-state SR Ca²⁺ content via the intended SERCA stimulation.^18–21 Both mechanisms could, in principle, lead to modifications of the Ca²⁺ sensitivity of the RyRs and thus of CICR, which might change the time course of recovery from CICR refractoriness independently of the rate of SR refilling.

Inhibition of the SERCA Slows Recovery From CICR Refractoriness
Because the accelerated recovery from refractoriness after ß-adrenoceptor stimulation could result from several modifications of the Ca²⁺-signaling machinery, we intended to apply a more specific pharmacological approach to test the hypothesis that SERCA activity governs CICR recovery. To slow down the SERCA several inhibitors are available. Cyclopiazonic acid (CPA), an inhibitor of the SERCA, could be a pharmacological tool to tune its function in a graded fashion.²² In SR vesicles derived from cardiac muscle, CPA inhibits Ca²⁺ uptake at nanomolar concentrations. In contrast to thapsigargin, the inhibitory effect of CPA is reversible. We applied CPA at low concentrations to slow down, but not completely inhibit, the SERCA. This would be expected to subsequently slow down the recovery from CICR refractoriness, at least when SR Ca²⁺ refilling via the SERCA was the rate-limiting step. Before carrying out experiments on the recovery from CICR refractoriness, we assessed the steady-state SR Ca²⁺ content during application of CPA. The voltage-clamp protocol outlined in Figure 1A was applied, and the SR was again loaded Ca²⁺ by applying a train of six I_Ca,L. But instead of using UV flashes after the loading protocol, the cells were briefly exposed to 20 mmol/L caffeine to empty the SR. The amount of Ca²⁺ released by the application of caffeine was estimated by integrating the caffeine-induced I_NCX recorded at –40 mV. Figure 2A shows the relative SR Ca²⁺ content after 1 minute application of 1 µmol/L, 5 µmol/L, and 10 µmol/L CPA. The lowest concentration of CPA used is smaller than the IC₅₀ in intact cells and was selected to inhibit just a fraction of the SERCAs. It has been shown previously that this concentration causes 25% decrease in the twitch tension of guinea pig atria.²³ In the presence of CPA the SR Ca²⁺ content decreased in a concentration-dependent manner. The data revealed that these CPA concentrations were sufficient to gradually slow down the SERCA function and subsequently lower the SR Ca²⁺ content. To verify that we could still release Ca²⁺ from the SR and thus activate CICR with flash photolytic Ca²⁺ concentration jumps, we determined the ratio of CICR (I_NCX-slow) before drug application and in the presence of CPA. Figure 2B shows that SR Ca²⁺ release could still be elicited photolytically despite a 1 minute exposure to a low concentration of CPA

View larger version (44K): [in this window] [in a new window]   Figure 2. Effects of CPA on SR Ca2+ content and SR Ca2+ release. A, SR Ca2+ content determined by integrating (over time, dt) INCX induced by rapid caffeine application (20 mmol/L) in control and in the presence of 1, 5, and 10 µmol/L CPA (64.1±5.7%, n=14; 47.1±7.5%, n=10; and 38.7±4.6%, n=18, respectively). B, SR Ca2+ release obtained from the CICR component of INCX (INCX-slow) in control and in the presence of 1, 5, and 10 µmol/L CPA triggered by a single UV-flash (83.5±4.8%, n=21; 76.4±5.2%, n=11; and 63.5±5.4%, n=15, respectively). *P<0.01. Data are mean±SEM.

Recovery From CICR Refractoriness in the Presence of CPA
To assess the effect of slowing down (but not completely inhibiting) Ca²⁺ uptake via the SERCA, we performed refractoriness recovery experiments in the presence of various concentrations of CPA (Figure 3). For this purpose, experiments analogous to those described in Figure 1 were performed, now in the presence of 1 µmol/L and 5 µmol/L CPA. In this series of experiments, CICR recovered with a of 720.0±31.8 ms in control conditions, and the recovery was slowed down in the presence of 1 µmol/L CPA (=792.2±23.6 ms). This effect of CPA was concentration dependent, and by increasing CPA to 5 µmol/L, the time constant for recovery was further prolonged to 943.9±34 ms. A CPA concentration of 10 µmol/L almost completely inhibited the SERCA (Figure 3, inset), similar to our previous observations with thapsigargin (not shown). This high CPA concentration thus prevented sufficient refilling of the SR within the time window of our experiments.

View larger version (24K): [in this window] [in a new window]   Figure 3. Slowing SR Ca2+ refilling with CPA. Averaged data showing the recovery from CICR refractoriness under control conditions () and in the presence of 1 µmol/L () and 5 µmol/L () CPA. Solid lines are the least-square fit of a monoexponential function to the averages (control, =720.0±31.8 ms; 1 µmol/L CPA, =792.2±23.6 ms; 5 µmol/L CPA, =943.9±34 ms; mean±SEM, n=5 to 27). The inset shows the effects of 10 µmol/L CPA () on recovery from CICR refractoriness.

Effect of Phospholamban on Recovery from CICR Refractoriness in Genetically Modified Mice
Both the acceleration of CICR recovery after ß-adrenergic stimulation and the retardation of recovery during SERCA inhibition are consistent with the notion that SR refilling plays an important role for the time course of recovery from refractoriness. To validate this conclusion we wanted to make use of an approach not entirely relying on pharmacological tools. To this end, we performed refractoriness experiments on myocytes isolated from PLB knockout (PLB-KO) mice and their respective wild-type (WT) littermates. The experiments were analogous to those described in Figures 1 and 3. The left column of Figure 4 (A and B) shows examples of exchange currents elicited by pairs of UV-laser flashes with a shorter (200 ms) and a longer (900 ms) interval recorded in myocytes from WT (Figure 4A) and PLB-KO (Figure 4B) mice. Compared with I_NCX from WT, the traces recorded from PLB-KO mice showed a more rapid decay, as expected because of the abbreviated Ca²⁺ transient present in these animals. The application of isoproterenol and CPA in WT mice resulted in changes of the time course of recovery similar to those observed in guinea pigs (Figure 4A: control, 239.5±8.2 ms; isoproterenol, 175.5±7.1 ms; CPA, 531.3±9.4 ms). Compared with WT myocytes, the recovery was accelerated almost 5-fold in PLB-KO cells (Figure 4B). As expected, ß-adrenergic stimulation could not further accelerate the recovery in PLB-KO mice, whereas CPA slowed it down, as in guinea pigs and WT mice (Figure 4B: control, 54.9±3.7 ms; isoproterenol, 54.5±1.9 ms; CPA, 231.5±13.1 ms).

View larger version (30K): [in this window] [in a new window]   Figure 4. Effects of PLB on CICR refractoriness. Representative INCX obtained at –40 mV induced by applying a pair of UV-flashes to photolyse caged Ca2+ with 200 and 900 ms intervals in myocytes from WT mice (A) and PLB-KO mice (B) under control conditions (left). Averaged data showing the recovery of CICR refractoriness in control conditions () and in the presence of 1 µmol/L isoproterenol () and 1 µmol/L () CPA (right). Solid lines represent the least-square fit of a monoexponential function to the averages (WT [A]: control, =239.5±8.2 ms; isoproterenol, =175.5±7.1 ms; CPA, =531.3±9.4 ms; n=6 to 36; PLB-KO [B]: control, =54.9±3.7 ms; isoproterenol, =54.5±1.9 ms; CPA, =231.5±13.1 ms; n=5 to 23; mean±SEM). The INCX exhibited a for the current decay of 113.3±29.6 ms (n=14) in WT and 84.4±15.1 ms (n=17) in PLB-KO mice, respectively.

	Discussion

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EC-coupling in cardiac muscle is inherently unstable because of its positive feedback arising from CICR. However, to ensure stable EC-coupling behavior, one or more mechanisms terminating Ca²⁺ release from the SR are required.⁴ To date, it is not clear to what extent Ca²⁺-induced inactivation or adaptation of RyRs, stochastic attrition, or functional depletion of the SR Ca²⁺ stores contribute and interact to terminate SR Ca²⁺ release.^{2,4,6,9,24–27} However, Ca²⁺-induced inactivation of RyRs has been difficult to demonstrate in intact cells, presumably because it may only occur at the very high Ca²⁺ concentrations prevailing in the microenvironment of the dyadic cleft.^28,29 Also the recovery from CICR refractoriness has been difficult to examine because of the temporal overlap of this process with the recovery of I_Ca,L from inactivation.^8,29 However, recently we were able to unmask a genuine and slowly recovering refractoriness of CICR when activating SR Ca²⁺ release by UV-laser flash photolysis of caged Ca²⁺.⁹ This technique produces a coherent and homogeneous trigger for CICR and is an ideal approach to examine refractoriness of CICR because it bypasses I_Ca,L, thus avoiding inactivation of the trigger signal and effects of isoproterenol on I_Ca,L.

Based on the observed discrepancies between slow global and fast local recovery from CICR refractoriness, we proposed that the SR Ca²⁺ content may play a regulatory role for the Ca²⁺ sensitivity of the RyRs. In other words, if Ca²⁺-induced inactivation (or adaptation) of RyRs would be the dominant mechanism for CICR refractoriness, then it should be seen not only in global but also in localized CICR events. Interestingly, slowly recovering refractoriness was not observed on the level of local Ca²⁺-release signals. Because both inactivation and adaptation are even functioning on the level of a single RyR channel, they do not require activation of the entire SR network. This conclusion is also supported by our observation of flash-photolytically induced alternans of Ca²⁺ release.⁹ This type of alternans implies that the rise of cytosolic Ca²⁺ concentration alone is insufficient to inactivate the RyRs. Instead, release of Ca²⁺ from the SR is required to terminate CICR and cause refractoriness. Therefore, we proposed that recovery from CICR refractoriness is determined by the time course of SR Ca²⁺ refilling subsequent to global Ca²⁺ release.⁹ In principle, two scenarios can be imagined how SR Ca²⁺ refilling could relieve refractoriness: (1) the SR Ca²⁺ concentration could directly affect the RyR Ca²⁺ sensitivity and thus the recovery from refractoriness by binding to a specific site within the SR; and (2) additional rate-limiting steps could be required after SR Ca²⁺ refilling, such as conformational changes of one or more proteins.

In the present study, we successfully distinguished between these two possibilities by examining how luminal SR Ca²⁺ content can control recovery from CICR refractoriness. When Ca²⁺ inside the SR exerts its effect on the RyR Ca²⁺ sensitivity without subsequent rate-limiting steps, modifications of the SERCA activity should result in immediate changes in the rate of recovery from CICR refractoriness, which was observed. Nevertheless, some additional, but not rate-limiting, steps may be required after SR refilling (see below). As expected for an immediate effect, acceleration of the SERCA by isoproterenol hastened the recovery from CICR refractoriness. Unfortunately, ß-adrenergic stimulation is not specific for the SERCA, and this observation could arise from protein kinase A–mediated phosphorylation of various proteins other than PLB.^18–21 To clarify this question, we initially tried to target the SERCA function more specifically with thapsigargin. Unfortunately, SERCA inhibition by thapsigargin led to almost no recovery from refractoriness, probably as a result of an excessive inhibition of the SERCA or a reduction of the SR Ca²⁺ content below a level critical for CICR.³⁰ However, the use of low and intermediate concentrations of CPA permitted better control over SERCA inhibition. At high concentrations of CPA, the recovery from CICR refractoriness was again minimal and similar to that observed in the presence of thapsigargin. Even the first flash did not trigger a substantial SR Ca²⁺ release; thus CICR already exhibited a state similar to refractoriness, probably attributable to reduced SR Ca²⁺ content. This was confirmed by the application of caffeine puffs, which revealed that the SR contained significantly less Ca²⁺ in presence of high concentrations of CPA. However, at low concentrations of CPA, recovery from CICR refractoriness was slowed down in a dose-dependent manner. In the presence of CPA the recovery from CICR refractoriness remained incomplete, as expected. This may have several reasons: (1) significant photoconsumption of the caged compound which is not replenished from the patch-pipette between two flashes leads to a reduced second I_NCX; (2) in the presence of CPA, the SR may not refill to the same extent after a flash as during the vigorous SR loading protocol with several Ca²⁺ currents; or (3) there may be also a slower phase of recovery of some RyRs, which could take several seconds.³¹

In experiments performed in myocytes obtained from PLB-KO mice, we found an almost 5-fold acceleration of recovery from refractoriness, when compared with WT myocytes. PLB is known to suppress the SERCA, and the ablation of PLB has been described to accelerate SR refilling.^11,32 The lack of any further acceleration of recovery from refractoriness by exposure to isoproterenol in PLB-KO mice suggests that SERCA activation and subsequent changes of SR Ca²⁺ refilling are the dominant effects of ß-adrenergic stimulation under our experimental conditions. However, this observation does not allow for the inference that phosphorylation of proteins other than PLB (eg, the RyRs) has no effect on CICR features other than the amplitude of the transient (eg, on the gating cooperativity or open probability of the RyRs). Based on our results, we conclude that Ca²⁺ refilling of the SR via the SERCA is a rate-limiting step governing recovery from CICR refractoriness. However, I_NCX signals reflecting subsarcolemmal [Ca²⁺]_I and, indirectly, SR refilling, decay more rapidly than recovery of CICR occurs. This may reflect subsarcolemmal Ca²⁺ depletion resulting from the Na⁺-Ca²⁺ exchange activity,³³ but could also be explained by one or more additional steps required for the recovery of CICR subsequent to SR refilling. Taken together, the most likely explanation for our observations is that CICR refractoriness results from SR Ca²⁺ depletion during Ca²⁺ release and that store emptying leads to deactivation and termination of the Ca²⁺ release by reducing the Ca²⁺ sensitivity of the RyRs on the cytosolic side of the channels.³⁴ This notion is also supported by recent findings of prolonged Ca²⁺ spark durations after increasing the buffer capacity and Ca²⁺ content of the SR, either by adding chemical buffers or by overexpression of calsequestrin.^27,35

An important unsolved question is how the EC-coupling machinery can sense the releasable SR Ca²⁺ content or the free Ca²⁺ concentration in the SR. In any case, SR Ca²⁺ content–dependent regulation and termination of CICR requires the presence of Ca²⁺ sensors inside the SR or on the luminal side of the RyRs. In bilayer experiments, the presence of Ca²⁺-binding sites on the luminal side of the RyR has been reported³⁶ (but this notion has not remained unchallenged³⁷). Alternatively, this signaling pathway may involve Ca²⁺ binding to proteins inside the SR, like calsequestrin or other Ca²⁺-binding proteins. This in turn may induce conformational changes, which are relayed to the RyRs via allosteric interactions and/or via other proteins, such as triadin and junctin.^38,39 This finally could lead to a change in the sensitivity of the RyRs for Ca²⁺.²⁷ In fact, very recently it was shown that by adding calsequestrin, triadin, and junctin to purified RyRs incorporated in lipid bilayers, the dependence of the channels on luminal Ca²⁺ can be restored.⁴⁰ In the future, additional studies in intact cells will be required to identify and characterize the signaling pathway that connects local and global SR Ca²⁺ content with the Ca²⁺ sensitivity of the RyRs for incoming trigger signals. A detailed understanding of the mechanisms regulating RyR Ca²⁺ sensitivity is not only a prerequisite for comprehending EC-coupling, but also for appreciating the importance of pathological alterations in EC-coupling during diseases.⁴¹ In addition, disruption of the normal function of SR Ca²⁺ buffering has recently been reported to be causally related to potentially lethal arrhythmias.⁴²

	Acknowledgments

 
This work was supported by the Swiss National Science Foundation (grant SNF 31-61'344.00 to E.N.), the Swiss Cardiovascular Research and Training Network, the Swiss Federal Office for Science and Education, and the National Institutes of Health (grants HL2657 and HL64018 to E.G.K.). We thank D. Lüthi for technical assistance.

	Footnotes

 
Original received March 25, 2004; first resubmission received May 27, 2004; second resubmission received August 24, 2004; revised resubmission received September 13, 2004; accepted September 13, 2004.

	References

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