Termination of Cardiac Ca²⁺ Sparks

Abstract

Ca²⁺ release from cardiac sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) is regulated by dyadic cleft [Ca²⁺] and intra-SR free [Ca²⁺] ([Ca²⁺]_SR). Robust SR Ca²⁺ release termination is important for stable excitation–contraction coupling, and partial [Ca²⁺]_SR depletion may contribute to release termination. Here, we investigated the regulation of SR Ca²⁺ release termination of spontaneous local SR Ca²⁺ release events (Ca²⁺ sparks) by [Ca²⁺]_SR, release flux, and intra-SR Ca²⁺ diffusion. We simultaneously measured Ca²⁺ sparks and Ca²⁺ blinks (localized elementary [Ca²⁺]_SR depletions) in permeabilized ventricular cardiomyocytes over a wide range of SR Ca²⁺ loads and release fluxes. Sparks terminated via a [Ca²⁺]_SR-dependent mechanism at a fixed [Ca²⁺]_SR depletion threshold independent of the initial [Ca²⁺]_SR and release flux. Ca²⁺ blink recovery depended mainly on intra-SR Ca²⁺ diffusion rather than SR Ca²⁺ uptake. Therefore, the large variation in Ca²⁺ blink recovery rates at different release sites occurred because of differences in the degree of release site interconnection within the SR network. When SR release flux was greatly reduced, long-lasting release events occurred from well-connected junctions. These junctions could sustain release because local SR Ca²⁺ release and [Ca²⁺]_SR refilling reached a balance, preventing [Ca²⁺]_SR from depleting to the termination threshold. Prolonged release events eventually terminated at a steady [Ca²⁺]_SR, indicative of a slower, [Ca²⁺]_SR-independent termination mechanism. These results demonstrate that there is high variability in local SR connectivity but that SR Ca²⁺ release terminates at a fixed [Ca²⁺]_SR termination threshold. Thus, reliable SR Ca²⁺ release termination depends on tight RyR regulation by [Ca²⁺]_SR.

In cardiac muscle, excitation–contraction coupling is initiated by membrane depolarization and Ca²⁺ entry via voltage-gated Ca²⁺ channels that activates sarcoplasmic reticulum (SR) Ca²⁺ release channels (ryanodine receptors [RyRs]). This process is known as Ca²⁺-induced Ca²⁺ release (CICR).^1,2 Elementary SR Ca²⁺ release events, which arise from the simultaneous opening of clustered RyRs in a single SR Ca²⁺ release site, can be measured as Ca²⁺ sparks,^3–5 a locally restricted increase in cytosolic Ca²⁺; or Ca²⁺ blinks,⁶ the corresponding local intra-SR Ca²⁺ ([Ca²⁺]_SR) depletion that accompanies a spark. Following the synchronized initiation of elementary Ca²⁺ release during excitation–contraction coupling, sparks summate in space and time and give rise to the global Ca²⁺ transient required for contraction. Ca²⁺ sparks also occur spontaneously because RyRs have a stochastic open probability in a resting myocyte. Despite the inherent positive feedback of CICR that may lead to the complete depletion of the SR Ca²⁺ store, compelling data have shown that during global Ca²⁺ transients and Ca²⁺ sparks, intra-SR [Ca²⁺] only partially depletes.^6–8 This raises the critical question of what terminates SR Ca²⁺ release.

RyRs underlie a complex regulation by both [Ca²⁺] in the dyadic cleft ([Ca²⁺]_cleft), as well as intra-SR free [Ca²⁺].^9,10 SR Ca²⁺ release can occur in response to an increase in [Ca²⁺]_cleft (eg, during excitation–contraction coupling) but can also occur spontaneously when [Ca²⁺]_SR is high (eg, during SR Ca²⁺ overload), which can lead to triggered arrhythmias. Partial depletion of [Ca²⁺]_SR is considered a key factor involved in SR release termination.^7,11–13 In fact, Terentyev et al¹⁴ have recently shown that release termination of Ca²⁺ blinks occurs at a relatively constant [Ca²⁺]_SR level even after calsequestrin overexpression. However, dynamic changes in [Ca²⁺]_cleft, which occur during Ca²⁺ release, may also modulate the mechanism of release termination.^15–18 The aim of this study was: (1) to investigate the functional importance of [Ca²⁺]_SR depletion for the termination of elementary Ca²⁺ release; (2) to investigate whether the SR Ca²⁺ release flux (and therefore [Ca²⁺] in the dyadic cleft) regulates the termination mechanism; and (3) to test whether Ca²⁺-independent SR Ca²⁺ release termination can occur when local [Ca²⁺]_SR remains constant during release over a prolonged period of time.

We simultaneously measured Ca²⁺ sparks and corresponding [Ca²⁺]_SR depletions (Ca²⁺ blinks⁶) in permeabilized rabbit ventricular myocytes over a wide range of SR Ca²⁺ loads and release fluxes. We found that Ca²⁺ sparks terminated at a particular absolute [Ca²⁺]_SR depletion threshold (at ≈60% of resting [Ca²⁺]_SR under control conditions), independent of either SR Ca²⁺ load or release flux. This suggests that Ca²⁺ spark termination is mainly determined by a [Ca²⁺]_SR-dependent mechanism and not by release flux or [Ca²⁺]_cleft. Local [Ca²⁺]_SR refilling after a blink was mainly attributable to intra-SR Ca²⁺ diffusion from nearby regions rather than SR Ca²⁺ reuptake. Despite a consistent [Ca²⁺]_SR nadir and highly reproducible blink kinetics at any given site, there was remarkably wide variation in blink recovery kinetics among sites. This is consistent with high variation in how well a given junction is connected within the SR network. When SR Ca²⁺ release flux was substantially reduced by partial RyR inhibition, well-connected SR release sites exhibited prolonged SR Ca²⁺ release events. At these sites, local [Ca²⁺]_SR was maintained above the termination threshold as local SR Ca²⁺ release and replenishment reached a steady state. These long events eventually terminated by a slower, [Ca²⁺]_SR-independent termination mechanism. These results provide direct experimental evidence for dynamic regulation of release termination by [Ca²⁺]_SR, release flux, and intra-SR Ca²⁺ diffusion in a cellular environment.

Materials and Methods

Ventricular myocytes were isolated from New Zealand White rabbits as previously described.⁷ All procedures were approved by the Institutional Animal Care and Use Committee. For simultaneous recording of [Ca²⁺]_SR and cytosolic [Ca²⁺], we used the low-affinity Ca²⁺ indicator Fluo-5N and the high-affinity Ca²⁺ indicator Rhod-2, respectively. Following the incubation of cardiomyocytes with Fluo-5N/acetoxymethylester (AM) under conditions that promote dye accumulation within the SR,^7,19 the sarcolemma of Fluo-5N/AM-loaded myocytes was permeabilized with saponin as described before.²⁰ The saponin-free internal solution was composed of (in mmol/L unless otherwise indicated): K aspartate 100; KCl 15; KH₂PO₄ 5; MgATP 5; EGTA 0.35; CaCl₂ 0.12; MgCl₂ 0.75; phosphocreatine 10; HEPES 10; Rhod-2 pentapotassium salt 0.04; creatine phosphokinase 5 U/mL; dextran (M_r 40 000) 8% and pH 7.2 (KOH). Free [Ca²⁺] and [Mg²⁺] of this solution were 150 nmol/L and 1 mmol/L, respectively. For the experiments shown in Figure 5A, the free [Ca²⁺] was adjusted to 200 nmol/L by adding appropriate amounts of CaCl₂. Membrane permeabilization has been used extensively in the study of cardiac SR Ca²⁺ release^20–22 and provides several advantages in the context of our experiments: the cytosolic milieu is tightly controlled (eg, ion concentrations and energy supply) and can easily be manipulated during the experiments. Furthermore, any cytosolic Fluo-5N is washed out after permeabilization providing a purely SR-derived Fluo-5N fluorescence signal. All experiments were performed at room temperature (20 to 23°C).

Figure 5.

Ca²⁺ sparks and blinks at increased [Ca²⁺]_SR and at inhibited SR release flux. A, Line scan images and corresponding profiles of Ca²⁺ sparks and blinks under control conditions and during increased [Ca²⁺]_SR. In these experiments, [Ca²⁺]_SR was increased by elevation of cytosolic [Ca²⁺] from 150 (Control) to 200 nmol/L (High Ca²⁺). B, Effect of increased initial [Ca²⁺]_SR on blink nadir. C, Ca²⁺ sparks and blinks under control conditions and during application of 0.7 mmol/L tetracaine. The region marked in orange shows [Ca²⁺]_SR depletion in a neighboring, quiescent region of the SR. Recordings of different release sites were made from the same cell. D, Effect of release flux inhibition with tetracaine (Tetrac) on blink nadir. Maximum SR Ca²⁺ load (Max) was obtained during stimulation of SR Ca²⁺ uptake by the catalytic subunit of PKA (5 U/mL) and complete inhibition of RyR with ruthenium red (20 μmol/L). SR Ca²⁺ load shown in gray with blink nadir shown in black (B and C). *P<0.05.

Changes in cytosolic [Ca²⁺] and [Ca²⁺]_SR were measured simultaneously with a laser-scanning confocal microscope (Radiance 2000 MP, Bio-Rad) equipped with a ×40 oil immersion objective lens (NA=1.3). Fluo-5N was excited with the 488-nm laser line of an argon ion laser, and fluorescence was measured at 515±15 nm. Rhod-2 was excited with the 543-nm laser line of a HeNe laser and fluorescence was measured at wavelengths >600 nm. Images were acquired in line scan mode (3 ms per scan; pixel size, 0.12 μm). Ca²⁺ sparks were detected and analyzed using SparkMaster.²³ For each detected Ca²⁺ spark, corresponding changes of the Fluo-5N signal were analyzed. The profiles of local [Ca²⁺]_SR depletions (Ca²⁺ blinks) were fitted with the product of 2 exponential functions to the decay and recovery phase, respectively, as described previously for spark²⁴ and blink analysis.⁶ Blink amplitudes and kinetic parameters were obtained from the fit of the experimental data. Amplitudes of sparks and blinks are expressed as F/F₀, where F₀ is the initial fluorescence before release. All Fluo-5N signals (blinks) were corrected for the Ca²⁺-insensitive component of the Fluo-5N fluorescence after complete SR Ca²⁺ depletion by 10 mmol/L caffeine. This concentration of caffeine fully activates RyRs²⁵ and leads to the synchronized release of the total Ca²⁺ stored in the SR. Thapsigargin was used to block the sarcoplasmic Ca²⁺ ATPase (SERCA). The complete inhibition of SERCA was confirmed by repeated SR Ca²⁺ load measurements using rapid application of 10 mmol/L caffeine. Thapsigargin (10 μmol/L) completely inhibited SERCA in permeabilized cells within 2 minutes (data not shown).

The maximum SR Ca²⁺ release flux was calculated from the peak of the first derivative of the cytosolic fluorescence intensity and expressed as d(ΔF/F₀)/ms^13,20,26,27 where ΔF=F–F₀. Although this approach lacks the accuracy of methods previously used to calculate release flux,^17,28 the waveform of directly measured SR Ca²⁺ release using the “Ca²⁺ spike” method is very similar to the d(ΔF/F₀)/dt waveform.²⁷ This method therefore provides a valid measure of the SR Ca²⁺ release flux.

To assess the degree of interconnection between individual SR Ca²⁺ release sites, the rate of Fluo-5N photobleaching was measured. [Ca²⁺]_SR and cytosolic [Ca²⁺] were simultaneously measured as described above but the excitation intensity of the 488-nm laser line was 10 times higher than for normal scans to induce Fluo-5N photobleaching. No obvious cellular damage was induced using this protocol. We only compared photobleaching in SR release sites that showed approximately the same Fluo-5N fluorescence intensity before bleaching. The resulting rate of Fluo-5N signal decay is determined by Fluo-5N photobleaching and Fluo-5N diffusion from neighboring SR regions that replenish bleached Fluo-5N. Because the rate of Fluo-5N photobleaching is considered identical for all bleached release sites in an image, differences in the decay rate of the fluorescence intensity result from differences in Fluo-5N diffusion that occurs during the photobleaching. The decay rate can therefore be considered an index of SR connectivity for individual SR release sites (fast decay rates denoting little interconnection and slow decay rates denoting extensive interconnection, respectively). The time course of the Fluo-5N fluorescence intensity decline during photo-bleaching could be well-fitted using a monoexponential function.

Global cytosolic Ca²⁺ transients and [Ca²⁺]_SR depletions were measured in intact, field-stimulated rabbit ventricular myocytes after loading with Fluo-5N/AM or Rhod-2/AM, respectively.¹⁹ To avoid motion artifacts, the scan line was positioned along the short axis in the central region of the cell where cell motion is minimal during contraction. Stimulation frequency was 0.5 Hz.

All chemicals were purchased from Sigma-Aldrich (St Louis, Mo). Fluo-5N/AM and Rhod-2 were purchased from Molecular Probes/Invitrogen (Carlsbad, Calif).

Data are presented as means±SEM of n measurements. Statistical comparisons between groups were performed with the Student t test. Differences were considered statistically significant at P<0.05. Coefficient of variation was calculated as SD/mean.

Results

Properties of Ca²⁺ Sparks and Blinks Under Control Conditions

To investigate the role of [Ca²⁺]_SR depletion in terminating local SR Ca²⁺ release (Ca²⁺ sparks), we simultaneously measured cytosolic [Ca²⁺] and concurrent intra-SR [Ca²⁺] depletions (Ca²⁺ blinks⁶) in isolated ventricular myocytes that were permeabilized using standard protocols (Figure 1A).^20–22

Figure 1.

Properties of Ca²⁺ sparks and corresponding Ca²⁺ blinks. A, Two examples (left and right) of simultaneously measured Ca²⁺ sparks and Ca²⁺ blinks recorded from different release sites. Top, Normalized (F/F₀) line scan images of Rhod-2 fluorescence and corresponding profiles of Ca²⁺ sparks obtained by averaging fluorescence from the 1-μm-wide regions marked by red boxes. Bottom, Normalized line scan images of Fluo-5N fluorescence and corresponding profiles of Ca²⁺ blinks obtained by averaging fluorescence from the regions marked by green boxes. Blink profiles were fitted by exponential functions to the rising and decay phase, respectively (dashed blue line). B, a, Average time-to-peak (TTP) of sparks and time-to-nadir (TTN) of blinks. b, Average time constants of spark decay and blink recovery. c, Relationship between the time constants of spark decay and blink recovery. d, Average FDHM of sparks and blinks. C, Left, Fluo-5N signal during Ca²⁺ blink and complete depletion of the SR after the application of 10 mmol/L caffeine. Right, Distribution and mean±SD of [Ca²⁺]_SR at the nadir of blinks ([Ca2+]_{SR, nadir}). Histogram was fitted with a single Gaussian function (red line).

On average, the time-to-nadir of Ca²⁺ blinks was approximately twice as long as the time-to-peak of the associated Ca²⁺ sparks (68±4 versus 31±1 ms; 86 events; P<0.05; Figure 1B, a). This demonstrates that SR Ca²⁺ release terminates well after the peak of a Ca²⁺ spark (also see elsewhere^6,12). The average time constant of local SR Ca²⁺ refilling after release was ≈3.5 times slower than the cytosolic [Ca²⁺] decline of the corresponding Ca²⁺ spark (161±13 versus 46±2 ms; 86 events; P<0.05; Figure 1B, b) and did not correlate with spark decay time (R²=0.14; Figure 1B, c). This is also evident in Figure 1A where the 2 Ca²⁺ sparks had essentially the same decay time constants (48 and 50 ms), whereas the recovery time constants of the blinks were 101 and 351 ms, respectively. The longer time-to-nadir and slower [Ca²⁺]_SR refilling resulted in a ≈3.5 times longer Ca²⁺ blink duration (full duration at half-maximum [FDHM]) compared to Ca²⁺ sparks (175±15 versus 50±1 ms; 86 events; P<0.05; Figure 1B, d). The blink duration had a much higher variability than the more stereotypical Ca²⁺ spark (ie, the coefficient of variation [SD/mean] was 4 times higher for Ca²⁺ blink duration than for Ca²⁺ spark duration).

Despite the high variability in Ca²⁺ blink kinetics, [Ca²⁺]_SR at the blink nadir showed remarkably little variation (59.9±0.9% of caffeine releasable Ca²⁺; 86 events; Figure 1C). Indeed, there was no correlation between blink nadir [Ca²⁺]_SR and either SR Ca²⁺ release flux [d(ΔF/F₀)/dt]or spark width (μm), but larger release flux correlated significantly with faster blink recovery rates (data not shown). Here, and elsewhere blink nadir is reported as percentage [Ca²⁺]_SR with respect to diastolic [Ca²⁺]_SR under control conditions (taken as 100%).

Global [Ca²⁺]_SR depletions in intact cardiomyocytes during excitation–contraction coupling had a duration that was only slightly longer than Ca²⁺ blinks (FDHM, 323±14 ms) and also exhibit a relatively similar constant nadir at 63.4±2.8% (Figure 2, n=6 cells; also see elsewhere^7,19). In contrast, Ca²⁺ sparks are much briefer and smaller in amplitude compared to global cytosolic Ca²⁺ transients. The finding that a substantial amount of Ca²⁺ remains in the SR at the end of the release rules out total SR Ca²⁺ depletion as the mechanism of Ca²⁺ spark termination. Rather, the consistent [Ca²⁺]_SR at the blink nadir and the known luminal Ca²⁺-sensitivity of RyR gating^29,30 suggests that release terminates when [Ca²⁺]_SR decreases to a critical threshold.

Figure 2.

Global and local SR Ca²⁺ release events. A, Global cytosolic Ca²⁺ transients and [Ca²⁺]_SR depletions measured in field-stimulated, intact rabbit ventricular cardiomyocytes at a stimulation frequency of 0.5 Hz. Measurements were performed in different cells and time-aligned to illustrate the relationship between Ca²⁺ transients and global SR Ca²⁺ depletions. Line scan images and profile plots (obtained by averaging the entire width of the line scan) are shown. B, Comparison between cytosolic [Ca²⁺] and [Ca²⁺]_SR depletion on a global (cytosolic Ca²⁺ transients and global intra-SR Ca²⁺ depletion, black traces) and local level (Ca²⁺ spark and Ca²⁺ blink, red traces). Original traces representative for average experimental values are shown.

To test whether the residual variability of [Ca²⁺]_SR depletion during Ca²⁺ sparks was characteristic of an individual release site or whether variability occurred randomly, we recorded repeated release events from the same site. At a given release site termination occurred at a very consistent [Ca²⁺]_SR (Figure 3A and 3B) even when release started from a lower initial [Ca²⁺]_SR early after a preceding event, eg, second event in Figure 3B. From release site to release site, however, the [Ca²⁺]_SR at the nadir of a blink varied over a modest range (between 43% and 73% in 48 different sites, average depletion was to 59.2±1.0%; Figure 3C). Notably, despite some variation in [Ca²⁺]_SR at the nadir, the blink nadir of repeated release events at a given site was highly correlated (R²=0.85; n=48). This is consistent with a critical [Ca²⁺]_SR threshold being the main factor responsible for SR Ca²⁺ release termination.

Figure 3.

Ca²⁺ blinks originating from the same release site. A, Line scan images and corresponding profiles of 2 consecutive Ca²⁺ sparks and blinks originating from the same release site. Profiles of sparks and blinks were obtained by averaging fluorescence from the regions marked by the 1-μm-wide red and green boxes, respectively. B, Similar recordings of multiple release events and corresponding blinks from a different SR Ca²⁺ release site. C, Relationship between [Ca²⁺]_SR at the nadir of consecutive blinks ([Ca²⁺]_{SR, nadir}), originating from the same release site. D, Relationship between blink recovery time constants (black data points) and spark decay time constants (red data points) of consecutive release events from the same release site. Time constants were measured from concurrent sparks and blinks.

In contrast, the Ca²⁺ refilling time constants of Ca²⁺ blinks varied at different release sites over more than a 10-fold range (values varied from 30 to 358 ms in 48 successively measured blinks; average time constant, 139±14 ms, SD/mean=0.668) but were remarkably consistent at each individual release site (R²=0.98; n=48; Figure 3D, black data points). In comparison, the decay time constants of the simultaneously measured Ca²⁺ sparks varied over a much narrower range (average time constant, 44±2 ms; SD/mean=0.318; Figure 3D, red data points).

These results show that the rate of local [Ca²⁺]_SR replenishment varies throughout the SR but is constant for any given release site. The variability of blink recovery among release sites (despite a similar extent of depletion) and the consistency of blink recovery at a given site, could either result from differences of Ca²⁺ uptake into the SR via the SERCA or could result from differences in intra-SR diffusion between release sites. These 2 possibilities were tested in the following experiments.

Ca²⁺ Sparks and Blinks at Decreased SR Ca²⁺ Load

To test whether the [Ca²⁺]_SR threshold for SR Ca²⁺ release termination depends on the initial [Ca²⁺]_SR before release occurs, we performed experiments in which we varied SR Ca²⁺ load over a wide range. To decrease SR Ca²⁺ load, we applied the selective SERCA inhibitor thapsigargin (10 μmol/L), which completely inhibits SR Ca²⁺ uptake within 2 minutes (see Materials and Methods). On thapsigargin application, [Ca²⁺]_SR and Ca²⁺ spark frequency progressively decreased until Ca²⁺ sparks ceased when [Ca²⁺]_SR had declined to ≈50% (data not shown). Interestingly, this [Ca²⁺]_SR was close to the termination threshold of Ca²⁺ blinks and may be an additional manifestation of the same RyR-gating regulation by [Ca²⁺]_SR.

As resting [Ca²⁺]_SR declined after thapsigargin application, the amplitudes of sparks and blinks, as well as the peak SR Ca²⁺ release flux, progressively decreased. Average values of release flux under different experimental conditions are presented in the Table. However, complete SERCA inhibition by thapsigargin did not prevent local [Ca²⁺]_SR recovery during blinks. [Ca²⁺]_SR at the blink nadir remained constant at 62.3±0.9% of the initial SR Ca²⁺ load under control conditions, consistent with a critical absolute [Ca²⁺]_SR threshold for release termination that is independent of the initial [Ca²⁺]_SR and release flux (Figure 4A). At release sites where repeated sparks and blinks could be measured at different initial [Ca²⁺]_SR (Figure 4B, abscissa), [Ca²⁺]_SR at the nadir did not change (Figure 4B, ordinate) as the initial SR Ca²⁺ load decreased. When data were grouped according to the initial SR Ca²⁺ load (Figure 4C), the [Ca²⁺]_SR at the nadir during blinks was constant (black bars in Figure 4C). These results are in line with the results from the previous experiments showing that termination occurred at the same [Ca²⁺]_SR threshold when [Ca²⁺]_SR had not fully recovered after a preceding release (Figure 3B; second event). Thus, even when the initial SR Ca²⁺ load is low and release flux is low, release terminates at the same absolute [Ca²⁺]_SR as under control conditions.

Table.

SR Ca²⁺ Release Flux and Corresponding [Ca²⁺]_SR at the Blink Nadir Measured Under Different Experimental Conditions

	SR Ca2+ Release Flux [d (ΔF/F0)/ms]	[Ca2+]SR at the Blink Nadir (% of Initial [Ca2+]SR at Control)	No. of Events
Control	0.049±0.002	59.9±0.9	89
Thapsigargin	0.036±0.03*	62.3±0.9	17
High Ca2+	0.070±0.05*	55.8±2.4	14
Tetracaine	0.029±0.02*†	90.5±2.0*†	24

Thapsigargin: 10 μmol/L; high Ca²⁺: 200 nmol/L; tetracaine: 0.7 mmol/L.

^*P<0.05 vs control

^†P<0.05 vs Thapsigargin

Figure 4.

Ca²⁺ sparks and blinks at decreased [Ca²⁺]_SR. A, Line scan images and corresponding profiles of Ca²⁺ sparks and blinks originating from the same release site under control conditions and at different times following thapsigargin (TG) (10 μmol/L) application. Spark and blink profiles were fitted with a double exponential function (black, blue, orange, and purple lines). B, Relationship between the initial [Ca²⁺]_SR and the [Ca²⁺]_SR at the blink nadir ([Ca²⁺]_{SR, nadir}). Data were obtained from different Ca²⁺ release sites (indicated by different colors) during thapsigargin application and were normalized to the initial SR Ca²⁺ load before thapsigargin application (100%). C, Summary of results shown in B with data grouped into control (100%), intermediate (85% to 95%), and low (70% to 80%) initial [Ca²⁺]_SR level. Initial [Ca²⁺]_SR (SR Ca²⁺ load before release) represented in gray with [Ca²⁺]_SR at the blink nadir shown in black. D, Normalized decay of Ca²⁺ sparks and corresponding recovery of blinks under control conditions (black) and in the presence of 10 μmol/L thapsigargin (orange). The profiles were obtained from exponential fits of experimental traces in A (control and thapsigargin, 5 minutes). *P<0.05.

Contribution of Ca²⁺ Uptake and Intra-SR Diffusion to Ca²⁺ Blink Recovery

With SR Ca²⁺ uptake blocked by thapsigargin, local [Ca²⁺]_SR recovery must be attributable to diffusion of Ca²⁺ from neighboring SR regions. Figure 4D shows the normalized Ca²⁺ spark decay and the corresponding blink recovery under control conditions and in the presence of 10 μmol/L thapsigargin (exponential fits taken from Figure 4A; control and thapsigargin, 5 minutes). SERCA blockade increased the time constant of spark decline by 36.2±7.2% (n=5), in agreement with previous data indicating that the cytosolic [Ca²⁺] decline of sparks is dominated by cytosolic Ca²⁺ diffusion rather than Ca²⁺ reuptake into the SR.³¹ Furthermore, thapsigargin increased the time constant of local [Ca²⁺]_SR refilling by a similar 33.4±7.6% (n=5; Figure 4D). These data indicate that diffusion of Ca²⁺ within the SR network rather than SR Ca²⁺ uptake dominates recovery of local [Ca²⁺]_SR. Therefore, the large variation in [Ca²⁺]_SR recovery kinetics between individual release sites results mainly from differences in local intra-SR Ca²⁺ diffusion. Better interconnected SR release sites within the SR (ie, where diffusion is faster between that junction and the rest of the SR) would therefore exhibit faster blink recovery as intra-SR Ca²⁺ diffusion is facilitated. Conversely, more isolated SR release sites would show slower local [Ca²⁺]_SR refilling.

Ca²⁺ Sparks and Blinks at Increased SR Ca²⁺ Load

To complement the experiments with decreased [Ca²⁺]_SR, we next measured Ca²⁺ blinks at increased SR Ca²⁺ load. For this purpose, cytosolic [Ca²⁺] was elevated from 150 to 200 nmol/L (thereby enhancing SERCA activity³²). This increased [Ca²⁺]_SR to 110.4±2.8% (n=12; P<0.05; Figure 5A and 5B), increased Ca²⁺ spark frequency by 46% [from 9.7±0.9 to 14.2±1.4 sparks×(100 μm)⁻¹×sec⁻¹; n=12; P<0.05], and increased maximal SR Ca²⁺ release flux by 43% [from 0.049±0.002 (n=89) to 0.070±0.05 d(ΔF/F₀)/ms; n=14; P<0.05; Table]. Despite the increased [Ca²⁺]_SR and maximal SR release flux, release still terminated at a similar [Ca²⁺]_SR (56±2%; n=14) as control (60±1%; n=89; Figure 5B and Table). These results argue for a stable absolute [Ca²⁺]_SR termination threshold that is not affected by the initial SR Ca²⁺ load and not regulated by the SR release flux which was altered in these experiments over a wide range (Table).

Ca²⁺ Sparks and Blinks at Partially Inhibited RyRs

We have shown previously that partial inhibition of SR Ca²⁺ release flux in combination with increased SR Ca²⁺ load induces long-lasting release events in some SR Ca²⁺ release sites.²⁰ These prolonged release events can sustain a constant SR Ca²⁺ release flux for several hundred milliseconds before termination occurs. We simultaneously measured cytosolic [Ca²⁺] and intra-SR [Ca²⁺] at partially inhibited RyRs to: (1) test the hypothesis that additional releasable Ca²⁺ can become available from neighboring SR regions via intra-SR Ca²⁺ diffusion, when the release site is well connected within the SR network; and (2) test whether the termination of prolonged release events is [Ca²⁺]_SR-dependent or -independent.

We partially inhibited RyRs using tetracaine, Mg²⁺, or ruthenium red at concentrations that decrease the open probability (P_o) of RyRs reconstituted in lipid bilayers by 80% to 90%.²⁰ We and others have shown previously that these RyR inhibitors decrease RyR P_o without changing mean open time, single-channel amplitude or inducing subconductance states.^20,33,34 SR Ca²⁺ release flux during local release events in the presence of tetracaine was significantly reduced compared to control conditions or to experiments when SR Ca²⁺ load was depleted with thapsigargin (Table).

Partial RyR inhibition initially abolished SR Ca²⁺ release, but as [Ca²⁺]_SR increased, spark frequency gradually recovered to a new steady state within 4 to 6 minutes because of RyR activation via higher [Ca²⁺]_SR [spark frequency decreased from 9.3±1.2 sparks×sec⁻¹×(100 μm)⁻¹ under control conditions to 4.0±1.2 sparks×sec⁻¹×(100 μm)⁻¹ 6 minutes after tetracaine application; n=10; P<0.05]. Under these conditions, elementary release events with durations similar to sparks under control conditions occurred, but additional long-lasting Ca²⁺ release events originating from some of the SR Ca²⁺ release sites were observed.²⁰ Figure 5C shows Ca²⁺ sparks from different release sites and corresponding local [Ca²⁺]_SR depletions before and after tetracaine application, including long-lasting release events. Tetracaine (0.7 mmol/L, 6 minutes) increased SR Ca²⁺ load to 119±2% (n=8; Figure 5D). Intra-SR Fluo-5N was not saturated under these conditions because fluorescence could be further increased to 136±5% (n=10) by SERCA stimulation using the PKA catalytic subunit (5 U/mL) and complete RyR inhibition with 20 μmol/L ruthenium red (Figure 5D).

When the release flux was decreased by partial RyR inhibition, the decrease in [Ca²⁺]_SR during release failed to reach the nadir seen for control blinks (Figure 5C and 5D). Short and prolonged release events in the presence of tetracaine terminated at 85±4% (n=10) and 94±3% (n=14) of the initial [Ca²⁺]_SR under control conditions, respectively, much higher than for blinks observed in the absence of RyR inhibition (59.9±0.9%; n=86; P<0.05). We infer that the threshold [Ca²⁺]_SR for release termination may be higher in the presence of tetracaine (which is known to alter RyR Ca²⁺ sensitivity³⁵). During long-lasting release events (second and fourth image in Figure 5C), [Ca²⁺]_SR decreased to a relatively stable level from which termination eventually occurred, suggesting a [Ca²⁺]_SR- and [Ca²⁺]_i-independent mechanism of release termination (because neither [Ca²⁺] is changing). The sites that showed long-lasting SR Ca²⁺ release events in the presence of tetracaine could have maintained local [Ca²⁺]_SR during release because at a reduced SR Ca²⁺ release flux, there may be sufficient luminal Ca²⁺ diffusion from neighboring SR regions to prevent [Ca²⁺]_SR from decreasing to the critical [Ca²⁺]_SR threshold that terminates release. This could occur if these release sites are especially well connected within the SR. Direct evidence for intra-SR diffusion and Ca²⁺ supply from neighboring release sites is presented in the right image of Figure 5C, which shows [Ca²⁺]_SR depletion in a quiescent SR region ≈1.8 μm lateral to a release site where prolonged Ca²⁺ release occurred (marked in orange). The distance at which the depletion in the neighboring SR region was observed was close to the sarcomere length and indicates that the depletion was indeed spreading to a neighboring release site. Similar [Ca²⁺]_SR depletion in quiescent neighboring junctions was observed during 5 long-lasting release events. Additionally, we found that the time constant of [Ca]_SR recovery was much faster after long release events (τ=73.4±11.8 ms; n=9) than after short events (τ=113.4±29.2 ms; n=10; P<0.05) in the presence of tetracaine. This provides evidence that faster diffusional SR refilling occurs at sites where long-lasting events occur. Next, we tested directly whether SR release sites that show long-lasting release events with partial RyR inhibition are especially well connected within the SR network.

Heterogeneity of SR Ca²⁺ Release Site Interconnection Within the SR

If the long-lasting events evoked by partial RyR inhibition correlate with more extensive Ca²⁺ release site interconnection within the SR (ie, limiting local depletion), then Fluo-5N should also diffuse more rapidly into these sites during local Fluo-5N photobleaching. To test this hypothesis, we locally photobleached intra-SR Fluo-5N and compared the fluorescence decline kinetics of release sites which showed prolonged release events with sites that exhibited short release events during partial RyR inhibition.

The 2 traces at the top of Figure 6A show the cytosolic fluorescence at 2 different release sites along the same scan line in a permeabilized myocyte. Release flux was partially inhibited with 0.7 mmol/L tetracaine (Table). The long-lasting release event originating from site 1 (s1, black trace) had a duration of ≈500 ms, whereas the 2 short release events occurring at site 2 (s2, gray trace) were comparable to typical sparks (FDHM≈80 ms). The bottom of Figure 6A shows the simultaneously measured Fluo-5N fluorescence intensity, which was similar at both sites at the beginning of the measurement. When the laser power in these experiments was increased to induce photobleaching, the Fluo-5N fluorescence decreased with an exponential time course at both sites. Both the time course and the extent of bleaching were different at the 2 sites. Despite the photobleaching, the [Ca²⁺]_SR depletions, which were associated with the release events shown in the top trace are easily identifiable. The SR Ca²⁺ release site exhibiting the long-lasting release event (s1, black) had slower and less extensive decay of fluorescence than the site from which the short release events originated (s2, gray). The relationship between the time constant of Fluo-5N fluorescence decline and the duration of cytosolic release events measured in 35 different release sites is presented in Figure 6B. Both parameters correlated remarkably well (R²=0.78), and a similar correlation was seen for the extent of photo-bleaching during a 5 second period (data not shown). A potential explanation for the Fluo-5N photobleaching variability is presented schematically in Figure 6A (right). The SR is organized as a network of release junctions connected by free SR. It has been shown that the typical SR junction connects to the free SR at 4 or 5 locations along its periphery and that some junctions may have fewer connections,⁶ thus indicating variability in connectivity of individual SR junctions. A release site that is well connected within the SR network (s1, top) shows a slower and less extensive decrease in fluorescence because diffusion from neighboring sites replenish the dye into the release site that is being bleached. In contrast, if a release site is relatively isolated within the SR (s2, bottom), diffusion of Fluo-5N into this site is restricted, resulting in a faster and more extensive decline in fluorescence during photobleaching. These results suggest that the wide variation in Ca²⁺ spark/blink duration at partial RyR inhibition (Figure 6B) and that the variation in the Ca²⁺ blink recovery time constant (Figures 1 and 3) reflects variance in the extent of individual SR Ca²⁺ release site interconnection within the SR network.

Figure 6.

Local Fluo-5N photobleaching in permeabilized myocytes. A, Left, Ca²⁺ sparks and corresponding blinks during the Fluo-5N bleaching protocol. Recordings were made from 2 different SR Ca²⁺ release sites (s1, black trace; s2, gray trace) along the same scan line during 0.7 mmol/L tetracaine application. Experimental data were fitted by a monoexponential function (blue lines). Right, Potential mechanism of Fluo-5N bleaching variability. Fluorescence in a well-connected SR release site (s1) decays slowly because of fast diffusion of dye (blue arrows) from adjacent regions of the SR. An isolated SR release site (s2) bleaches fast because of restricted diffusion of the dye. The degree of intra-SR connectivity was symbolized by the number of free SR connections and diameter of free SR. B, The relationship between Ca²⁺ spark duration and the rate of Fluo-5N bleaching after tetracaine application.

Discussion

Robust termination of SR Ca²⁺ release is required to ensure stable physiological excitation–contraction coupling in the heart. Failure of SR Ca²⁺ release termination has been linked to arrhythmogenesis in both acquired (eg, hypertrophy and heart failure)^36,37 and inherited pathological conditions (eg, catecholaminergic polymorphic ventricular tachycardia).³⁸ However, despite intensive study, the mechanism of release termination in cardiac muscle is still unclear.

In the present study, we investigated the importance of local [Ca²⁺]_SR depletion and release flux in the termination of elementary SR Ca²⁺ release in permeabilized rabbit ventricular cardiomyocytes. The main findings were: (1) local [Ca²⁺]_SR depletions during sparks were similar in amplitude and duration to global SR Ca²⁺ depletions; (2) under control conditions, SR Ca²⁺ release terminated when the local [Ca²⁺]_SR declined to a fixed absolute termination threshold; (3) this termination threshold was independent of the initial [Ca²⁺]_SR and release flux; and (4) decreasing the release flux by inhibiting RyR led to the occurrence of long-lasting SR Ca²⁺ release events that originated from especially well-connected SR release sites and terminated independently of [Ca²⁺]_SR depletion.

Intra-SR Ca²⁺ Diffusion Shapes the Kinetics of Ca²⁺ Blinks

A remarkable feature of Ca²⁺ blinks is that on average they are similar in amplitude and duration to global SR Ca²⁺ depletions, which occur when release is triggered simultaneously at all release sites during excitation–contraction coupling (Figure 2 and elsewhere^6,7,19). This contrasts sharply with Ca²⁺ sparks, which are much smaller in amplitude and duration than Ca²⁺ transients. The explanation may be that Ca²⁺ sparks summate extensively in space and time during the synchronized activation of SR Ca²⁺ release during excitation–contraction coupling, forming the high-amplitude cell-wide Ca²⁺ transient. In contrast, most of the Ca²⁺ released during excitation–contraction coupling or during a spark is available locally from individual SR release sites. Because Ca²⁺ diffusion in the SR is slower than in the cytosol,^39–41 and [Ca²⁺]_SR is buffered by calsequestrin only minor spatiotemporal summation or overlap of intra-SR release events occurs, resulting in the relative similarity of local and global SR Ca²⁺ depletions. Also, Ca²⁺ spark decline and Ca²⁺ blink recovery kinetics are substantially influenced by cytosolic and intra-SR Ca²⁺ buffering, respectively. Increased intra-SR Ca²⁺ buffering (eg, by overexpressing calsequestrin) leads to a slower [Ca²⁺]_SR recovery after the nadir of a blink.¹⁴ Ca²⁺ blink recovery was only modestly prolonged when SERCA activity was completely inhibited (Figure 4), clearly showing that intra-SR Ca²⁺ diffusion rather than Ca²⁺ uptake into the SR is the main factor responsible for local [Ca²⁺]_SR recovery. Although the entire SR network and nuclear envelope are part of one large Ca²⁺ compartment throughout which diffusion occurs,^39,41 the large variability in blink recovery kinetics (Figures 1A, 1B, and 3D) indicates that there is substantial variation in how isolated a particular SR release site is and how well diffusion can occur between neighboring release sites. A junction that is well connected within the SR and allows for faster intra-SR Ca²⁺ diffusion would then replenish faster than a more isolated junction. The fact that we found consistent recovery kinetics for any given release site (Figure 3D) suggests that the variation in release site recovery is based on relatively fixed structural inhomogeneities rather than on alternating functional or stochastic differences. The importance of release site interconnection for release termination is discussed below.

The properties of the Ca²⁺ blinks described here differ substantially from the initial description of Ca²⁺ blinks by Brochet et al,⁶ who described blinks with much faster kinetics similar to the kinetics of cytosolic Ca²⁺ sparks. Several factors may be responsible for these differences: the present study was performed in permeabilized myocytes, whereas the study by Brochet et al was performed in intact myocytes. Although certain cytosolic proteins may be washed out during permeabilization, it is unlikely that the fundamental differences in blink properties can be attributed to the cell permeabilization because Ca²⁺ spark kinetics are virtually identical in intact and permeabilized ventricular cardiomyocytes.²¹ It is also possible that Brochet et al preferentially detected smaller and shorter depletions, especially in experiments in which Ca²⁺ blinks were not measured simultaneously with Ca²⁺ sparks. All Ca²⁺ blinks in our study were measured simultaneously with cytosolic [Ca²⁺], which enabled us to unequivocally prevent false-positive Ca²⁺ blink detections (which may be of short duration). Finally, the Ca²⁺ blink characteristics here agree closely with other published data.^14,36

[Ca²⁺]_SR Depletion to a Fixed Threshold Terminates SR Ca²⁺ Release

A major finding of the present study is that SR Ca²⁺ release robustly terminated at a seemingly fixed [Ca²⁺]_SR threshold of ≈60% of the diastolic [Ca²⁺]_SR value under control conditions. The termination threshold was stable for individual SR Ca²⁺ release sites but varied to some extent from site to site. These results confirm previous experimental findings^6,8,14 and predictions from mathematical modeling^12,42 that the termination involves changes in RyR gating rather than exhaustion of releasable Ca²⁺. Our findings also make random release termination (such as stochastic attrition⁴³) unlikely, because this would result in varying termination levels.

Both cytosolic [Ca²⁺] and [Ca²⁺]_SR are known to regulate RyR gating^9,10 through modulation of activation, as well as termination of SR Ca²⁺ release.^18,29,44,45 Therefore, it is likely that termination is the consequence of a dynamic balance between luminal and cytosolic [Ca²⁺] regulation of RyRs during Ca²⁺ release. The [Ca²⁺] in the dyadic cleft is mainly determined by the SR Ca²⁺ release flux,¹⁸ which, in turn, is determined by SR Ca²⁺ loading and the number of active channels in a release site. Our results, however, argue against an important dynamic regulation of spark termination by [Ca²⁺]_cleft because the threshold [Ca²⁺]_SR for SR release termination remained constant when the SR Ca²⁺ load and therefore the release flux was varied over a wide range (Figures 4 and 5A and Table). These findings suggest that partial depletion of intra-SR [Ca²⁺] to a fixed threshold represents the main mechanisms of cardiac Ca²⁺ spark termination via a [Ca²⁺]_SR-dependent mechanism.

Release Termination at Partial RyR Inhibition

Partial RyR inhibition has 2 important consequences that influence the termination process. Firstly, the number of active RyRs (or total RyR P_o) in a SR release site is substantially reduced, leading to a decreased SR Ca²⁺ release flux and [Ca²⁺]_cleft for any given SR Ca²⁺ load in comparison to control conditions. Secondly, because SERCA activity remains unaltered by selective RyR inhibition, the SR Ca²⁺ content increases.

As in the experiments with elevated cytosolic Ca²⁺ (Figure 5A), release started from a similarly elevated SR Ca²⁺ load. However, release did not terminate at the [Ca²⁺]_SR threshold that we consistently observed in the absence of RyRs inhibition (Figures 1, 3, 4, and 5A). Rather, termination occurred at a much higher [Ca²⁺]_SR in both short and prolonged release events. Tetracaine acts primarily by inhibiting RyR activation rather than by promoting channel closing in reconstituted RyRs in lipid bilayers (prolonging closings without changing mean open time).^33,35 Thus, it is likely that tetracaine directly alters the threshold at which SR Ca²⁺ release terminates. This may be why brief Ca²⁺ blinks under this condition terminated at higher [Ca²⁺]_SR in tetracaine versus control conditions. We cannot exclude the possibility that the reduced SR Ca²⁺ release flux with tetracaine causes less RyR stimulation by [Ca²⁺]_cleft (which could contribute to release termination at a smaller [Ca²⁺]_SR depletion). However, this would require a very steep relationship between release flux and RyR stimulation by [Ca²⁺]_cleft, because the reduction in SR Ca²⁺ release flux that occurred at low SR Ca²⁺ loads after SERCA inhibition did not influence the termination threshold.

Intra-SR Ca²⁺ Diffusion Provides [Ca²⁺]_SR for Prolonged Release Events

A remarkable feature of prolonged release events is that [Ca²⁺]_SR decreases to a stable level from which termination occurs (Figure 5C, fourth image). The first question that arises is how a single release site can supply sufficient Ca²⁺ to maintain ongoing SR Ca²⁺ release over several hundred milliseconds without getting depleted. The answer likely involves the extent of interconnection that individual release sites have within the SR. As discussed above, differences in intra-SR interconnection determine the kinetics of [Ca²⁺]_SR refilling after the nadir of a blink. In addition, a well connected release site may also draw Ca²⁺ from a neighboring SR region and limit local [Ca²⁺]_SR decline, allowing continued release because the [Ca²⁺]_SR termination threshold is not reached when the release flux is low. Supporting data for this hypothesis is shown in the fourth image of Figure 5C: the long-lasting release event does not only deplete its own junction but also partially depletes a neighboring junction. Although this local intra-SR Ca²⁺ diffusion would also be expected to occur in well-connected release sites under control conditions, it may be difficult to detect experimentally because of the brevity of corresponding blinks and the smaller depletion expected at that time and distance. Under control conditions there was a significant correlation between release flux and spark width, such that some sites release more total Ca²⁺ (despite unaltered [Ca²⁺]_SR nadir). These sites of higher release flux exhibited faster blink recovery rates, consistent with the idea that larger amounts of Ca²⁺ release are in fact enabled by better connected SR (ie, greater diffusive flux through the region before the release threshold is reached). Otherwise greater Ca²⁺ release might be expected to result in slower refilling.

The photobleach experiments further test whether the sites that exhibit long-lasting release events (with tetracaine) are especially well connected within the SR network. Similar to intra-SR Ca²⁺, Fluo-5N can diffuse freely within the sarcoplasmic reticulum.^39,46 The rate of local Fluo-5N photo-bleaching provides direct insight into the functional interconnection of individual release sites within the SR: isolated release sites would exhibit faster and more extensive fluorescence decrease during photobleaching, whereas SR release sites that are well connected with neighboring SR would show slower and less extensive decrease in fluorescence because intra-SR Fluo-5N diffuses into the release sites that are being bleached (see schematic diagram in Figure 6A, right). The junctions where partial RyR inhibition led to long-lasting release events had photobleaching characteristics expected for well-connected release sites. On the other hand, the bleaching kinetics of release sites that showed short release events in the presence of partial RyR inhibition had the hallmarks of isolated domains. These data support our previous conclusion that there are substantial differences in the degree of release site interconnection within the SR which was based on the large variation of Ca²⁺ blink recovery kinetics and the relatively small contribution of Ca²⁺ uptake to local [Ca²⁺]_SR refilling (Figures 1, 3, and 4). Well-connected release sites therefore have a functionally higher [Ca²⁺]_SR reserve because Ca²⁺ can be supplied from neighboring release sites. In this sense, the prolongation of SR Ca²⁺ release with partially inhibited RyRs and increased [Ca²⁺]_SR resembles the prolongation of release at enhanced intra-SR Ca²⁺ buffering.^13,47 At enhanced intra-SR buffering, additional Ca²⁺ is available for release from the buffers in the SR release site and this delays approach to the termination [Ca²⁺]_SR termination threshold.

Termination of Prolonged SR Release Events: [Ca²⁺]_SR-Independent Termination of Release

The remaining question is how long-lasting release events terminate. The long duration of a relatively sustained [Ca²⁺]_SR depletion from which termination occurs (Figure 5C, fourth image) would suggest a slower, [Ca²⁺]_SR- and [Ca]_i-independent mechanism of release termination. One such mechanism is stochastic attrition, where the simultaneous closure of all RyRs in a release cluster leads to release termination by breaking the inherent positive feedback of Ca²⁺-induced Ca²⁺ release. Because stochastic attrition is very sensitive to the number of RyRs available for activation,⁴³ it is probably not relevant for the termination of SR Ca²⁺ release under control conditions. However, this mechanism may become relevant for Ca²⁺ spark termination during RyR inhibition when a smaller than normal number of RyRs are active. Additionally, RyR adaptation¹⁵ may be involved in the termination of prolonged SR Ca²⁺ release events because the RyRs in a junction that exhibits a long-lasting release event are exposed to a relatively high cleft [Ca²⁺] for a long period of time. Adaptation was initially described to occur with a very slow time constant (>1 second) but can also occur on a timescale relevant for the termination of prolonged release events.⁴⁸

In conclusion, this study demonstrates that SR Ca²⁺ release terminates at a critical [Ca²⁺]_SR threshold that is not dynamically regulated by SR Ca²⁺ release flux or cleft [Ca²⁺]. Intra-SR Ca²⁺ diffusion can supply Ca²⁺ from neighboring SR regions and thereby maintain SR Ca²⁺ release when the release flux is low. Multiple release termination mechanisms exist in the heart contributing to the stability of cardiac excitation–contraction coupling.