Ca²⁺ spark-dependent and -independent sarcoplasmic reticulum Ca²⁺ leak in normal and failing rabbit ventricular myocytes

Abstract

Sarcoplasmic reticulum (SR) Ca²⁺ leak is an important component of cardiac Ca²⁺ signalling. Together with the SR Ca²⁺-ATPase (SERCA)-mediated Ca²⁺ uptake, diastolic Ca²⁺ leak determines SR Ca²⁺ load and, therefore, the amplitude of Ca²⁺ transients that initiate contraction. Spontaneous Ca²⁺ sparks are thought to play a major role in SR Ca²⁺ leak. In this study, we determined the quantitative contribution of sparks to SR Ca²⁺ leak and tested the hypothesis that non-spark mediated Ca²⁺ release also contributes to SR Ca²⁺ leak. We simultaneously measured spark properties and intra-SR free Ca²⁺ ([Ca²⁺]_SR) after complete inhibition of SERCA with thapsigargin in permeabilized rabbit ventricular myocytes. When [Ca²⁺]_SR declined to 279 ± 10 μm, spark activity ceased completely; however SR Ca²⁺ leak continued, albeit at a slower rate. Analysis of sparks and [Ca²⁺]_SR revealed, that SR Ca²⁺ leak increased as a function of [Ca²⁺]_SR, with a particularly steep increase at higher [Ca²⁺]_SR (>600 μm) where sparks become a major pathway of SR Ca²⁺ leak. At low [Ca²⁺]_SR (<300 μm), however, Ca²⁺ leak occurred mostly as non-spark-mediated leak. Sensitization of ryanodine receptors (RyRs) with low doses of caffeine increased spark frequency and SR Ca²⁺ leak. Complete inhibition of RyR abolished sparks and significantly decreased SR Ca²⁺ leak, but did not prevent it entirely, suggesting the existence of RyR-independent Ca²⁺ leak. Finally, we found that RyR-mediated Ca²⁺ leak was enhanced in myocytes from failing rabbit hearts. These results show that RyRs are the main, but not sole contributor to SR Ca²⁺ leak. RyR-mediated leak occurs in part as Ca²⁺ sparks, but there is clearly RyR-mediated but Ca²⁺ sparks independent leak.

Introduction

During cardiac excitation–contraction coupling (ECC), simultaneous activation of sarcoplasmic reticulum (SR) ryanodine receptor (RyR; type 2) Ca²⁺ release channels generates global Ca²⁺ transients required for activation of contraction. After termination of SR Ca²⁺ release, a significant portion of cytosolic Ca²⁺ is sequestered back into the SR by the Ca²⁺-ATPase (SERCA) leading to cardiac muscle relaxation. During diastole, RyRs are not completely quiescent, thus providing a pathway for significant SR Ca²⁺ leak. Therefore, the finite balance between SR Ca²⁺ uptake and leak determines the amount of Ca²⁺ stored in the SR. Because the fractional SR Ca²⁺ release steeply depends on SR Ca²⁺ load (Bassani et al. 1995), a small shift in this balance can lead to substantial changes in SR Ca²⁺ load and, therefore, Ca²⁺ transient amplitude. While it is well established that SR Ca²⁺ uptake is entirely mediated by SERCA pump activity, the specific pathways of SR Ca²⁺ leak have not been characterized in detail.

In ventricular myocytes SR Ca²⁺ release during ECC occurs at specialized release sites where L-type Ca²⁺ channels in the T-tubules are closely associated with the RyR clusters of the junctional SR. The release cluster contains dozens of, possibly more than 100, RyRs (Franzini-Armstrong & Protasi, 1997) and their simultaneous activation produces the elementary release events termed Ca²⁺ sparks (Cheng et al. 1993). During ECC, the global Ca²⁺ transient is the result of spatio-temporal summation of Ca²⁺ release from thousands of these individual release sites. Ca²⁺ sparks can also occur spontaneously during rest or diastole providing an important pathway for SR Ca²⁺ leak. It has been proposed that Ca²⁺ release in the form of sparks can explain almost the entire diastolic SR Ca²⁺ leak (Cheng et al. 1993; Bassani & Bers, 1995). However, a growing body of evidence suggests that SR Ca²⁺ leak may also occur as undetectable openings of RyRs (non-spark RyR-mediated SR Ca²⁺ leak). In ventricular myocytes, Ca²⁺ sparks are rare events during diastole suggesting that SR Ca²⁺ release events with amplitudes that are significantly smaller than typical sparks are responsible for a major part of SR Ca²⁺ leak (Santiago et al. 2010). Potential mechanisms include spontaneous openings of single RyR in a release cluster without activation of the remaining channels in the cluster (Lipp & Niggli, 1996), activation of isolated unclustered RyRs (Sobie et al. 2006), or non-RyR leak pathways. However, it remains elusive to what degree these different types of Ca²⁺ release events contribute to the global SR Ca²⁺ leak and how these release events are dependent on SR Ca²⁺ load.

Heart failure (HF) is commonly associated with decreased contractile function due to alterations of the activity of several important Ca²⁺ transport systems, including the RyR. Increased RyR-mediated Ca²⁺ leak during HF has been implicated in reduction of SR Ca²⁺ content and triggering of Ca²⁺-dependent arrhythmias (George, 2008). Abnormal phosphorylation of RyR by either protein kinase A (Marx et al. 2000) or Ca²⁺–calmodulin-dependent kinase II (Ai et al. 2005) has been shown to contribute to enhanced SR Ca²⁺ leak in HF. Furthermore, redox modification of the RyR also plays a functional role in changes of SR Ca²⁺ leak in failing hearts (Terentyev et al. 2008). In addition, contributions from non-RyR Ca²⁺ leak need to be considered. A prime candidate in this context is the inositol-1,4,5-trisphosphate receptor (IP₃R) SR Ca²⁺ release channel. Although expressed at lower densities compared to RyRs, IP₃R-dependent Ca²⁺ release modulates ECC and contributes to arrhythmogenesis (Proven et al. 2006; Domeier et al. 2008). IP₃Rs are upregulated during HF (Go et al. 1995; Ai et al. 2005) and thus may contribute to the enhanced SR Ca²⁺ leak. Therefore, it is critically important to characterize the mechanisms of SR Ca²⁺ leak for better understanding of cardiac Ca²⁺ signalling under normal conditions and in disease states.

In this study, we investigated mechanisms of SR Ca²⁺ leak in rabbit ventricular myocytes, with a particular interest in the role of Ca²⁺ sparks. Spontaneous Ca²⁺ sparks have been used extensively as the index of SR Ca²⁺ leak in cardiomyocytes. Although previous studies provide important information about RyR regulation and Ca²⁺ signalling, it remains unclear whether or not Ca²⁺ sparks are the major pathway of SR Ca²⁺ leak. Here, we employed a newly developed approach to directly measure SR Ca²⁺ leak as changes of [Ca²⁺]_SR after complete SERCA inhibition. To measure [Ca²⁺]_SR we used the low affinity Ca²⁺ indicator Fluo-5N entrapped within the SR. After permeabilization of the sarcolemma, the high affinity Ca²⁺ indicator Rhod-2 was added to the cytosol to measure Ca²⁺ sparks simultaneously. This experimental approach allowed us to directly and continuously study the functional relationship between Ca²⁺ spark properties and SR Ca²⁺ leak. We found that in rabbit ventricular myocytes RyRs were the main pathway for SR Ca²⁺ leak which occurred in the form of Ca²⁺ sparks, but also as spark-independent Ca²⁺ leak. At low [Ca²⁺]_SR, leak occurred mostly in the absence of sparks, whereas at high [Ca²⁺]_SR, Ca²⁺ sparks became a significant contributor to SR Ca²⁺ leak. The spark-independent Ca²⁺ leak consisted of at least two components: undetectable Ca²⁺ release through RyRs and Ca²⁺ efflux through pathways other than RyRs and IP₃Rs; however stimulation with IP₃ increased RyR-independent leak. We also found that SR Ca²⁺ leak was significantly increased in ventricular myocytes from failing hearts. This effect was attributed to an increased RyR activity. Part of this work has been published in abstract form (Zima & Blatter, 2009).

Methods

Myocyte isolation

Ventricular myocytes were isolated from New Zealand White rabbits (30 animals, 2.5 kg; Myrtle's Rabbitry, Thompsons Station, TN, USA) or rabbits with non-ischaemic HF induced by combined aortic insufficiency and stenosis (5 animals, for detailed description of HF model see Pogwizd, 1995). The procedure of cell isolation was approved by the Institutional Animal Care and Use Committee, and complies with US and UK regulations on animal experimentation (Drummond, 2009). Adult rabbits were anaesthetized with sodium pentobarbital (50 mg kg⁻¹i.v.). Following thoracotomy hearts were quickly excised, mounted on a Langendorff apparatus, and retrogradely perfused with collagenase-containing solution at 37°C according to the procedure described previously (Domeier et al. 2009). All chemicals and reagents were purchased from Sigma-Aldrich (St Louis, MO, USA).

Confocal microscopy

For simultaneous recording of [Ca²⁺]_SR and [Ca²⁺]_i we used the low affinity Ca²⁺ indicator Fluo-5N and the high affinity Ca²⁺ indicator Rhod-2, respectively (indicators obtained from Molecular Probes/Invitrogen, Carlsbad, CA, USA). Myocytes were incubated with 5 μm of Fluo-5N/AM for 2.5 hours at 37°C as described before (Zima et al. 2008b; Domeier et al. 2009). Fluo-5N/AM loaded myocytes were permeabilized with saponin (Zima et al. 2008a) to remove cytosolic Fluo-5N. The saponin free internal solution was composed of (in mm): potassium aspartate 100; KCl 15; KH₂PO₄ 5; MgATP 5; EGTA 0.35; CaCl₂ 0.12; MgCl₂ 0.75; phosphocreatine 10; Hepes 10; Rhod-2 tripotassium salt 0.04; creatine phosphokinase 5 U ml⁻¹; dextran (MW: 40,000) 8%; pH 7.2 (KOH). Free [Ca²⁺] and [Mg²⁺] of this solution were 150 nm and 1 mm, respectively. All experiments were performed at room temperature (20–24°C).

Changes in [Ca²⁺]_i and [Ca²⁺]_SR were measured with laser scanning confocal microscopy (Radiance 2000 MP, Bio-Rad, UK) equipped with a ×40 oil-immersion objective lens (NA = 1.3). Fluo-5N was excited with the 488 nm line of an argon ion laser and fluorescence was measured at 515 ± 15 nm. Rhod-2 was excited with the 543 nm line of a He–Ne 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). The axial resolution was 1.1 μm at full-width half-maximum (point-spread function determined experimentally).

Measurements of Ca²⁺ sparks

Ca²⁺ sparks were detected and analysed using SparkMaster (Picht et al. 2007). To exclude false positive events, a threshold criterion for spark detection of 3.8 was chosen. At this threshold no events were detected when SR Ca²⁺ was emptied after simultaneous application of caffeine (10 mm) and thapsigargin (TG; 10 μm). Analysis of Ca²⁺ sparks included frequency (sparks (100 μm)⁻¹ s⁻¹), amplitude (ΔF/F₀), full duration at half-maximal amplitude (FDHM; ms), and full width at half-maximal amplitude (FWHM; μm). F₀ is the initial fluorescence recorded under steady-state conditions and ΔF=F−F₀. Spark frequency was corrected for missing events as described previously (Song et al. 1997; see online Supplemental Material). Ca²⁺ release flux (Sipido & Wier, 1991) and signal mass (Chandler et al. 2003) were used to estimate releasable Ca²⁺ during individual spark. The SR Ca²⁺ release flux was estimated from the peak of the first derivative of the cytosolic fluorescence intensity and expressed as d(ΔF/F₀)/dt (ms⁻¹). Signal mass of Ca²⁺ sparks was calculated according to the formula: SM_spark= 1.206 ×ΔF/F₀× FWHM³. By measuring Ca²⁺ sparks in control and Fluo-5N-loaded cells, we also found that loading of the SR with Fluo-5N did not significantly affect spark properties and SR Ca²⁺ load (see online Supplemental Material).

Measurements of SR Ca²⁺ leak

To minimize indicator photobleaching, Fluo-5N was excited with minimum laser energy. To improve the signal-to-noise ratio of the low intensity Fluo-5N signal, fluorescence was collected with an open pinhole (non-confocal settings) and averaged over the entire cellular width of line-scan image (Figs 1A and 4A). At the end of each experiment minimum (F_min) and maximum (F_max) Fluo-5N fluorescence were estimated. F_min was measured after depletion of the SR with 10 mm caffeine in the presence of 5 mm EGTA ([Ca²⁺]∼5 nm). F_max was measured following an increase of [Ca²⁺] to 10 mm. Caffeine (10 mm) keeps RyRs open allowing [Ca²⁺] equilibration across the SR membrane (Shannon et al. 2003a). To prevent irreversible cell contraction during application of high [Ca²⁺], cells were pretreated for 5 min with the muscle contraction uncouplers 2,3-butanedione monoxime (10 mm) and blebbistatin (10 μm). Caffeine (10 mm) decreased Fluo-5N fluorescence by 16% due to chemical quenching of the dye. Therefore, F_max and F_min values were corrected accordingly. After correction, the Fluo-5N signal was converted to [Ca²⁺] using the formula: [Ca²⁺]_SR=K_d× (F−F_min)/(F_max−F), where K_d (Ca²⁺ dissociation constant) was 390 ± 35 μm (n= 5 cells) based on in vivo calibrations (see Supplemental Material). SR Ca²⁺ leak was measured as the changes of total [Ca²⁺]_SR ([Ca²⁺]_SRT) over time (d[Ca²⁺]_SRT/dt) after complete SERCA inhibition with TG. [Ca²⁺]_SRT was calculated as: [Ca²⁺]_SRT=B_max/(1 +K_d/[Ca²⁺]_SR) +[Ca²⁺]_SR; where B_max and K_d were 2700 μm and 630 μm, respectively (Shannon et al. 2000b). The rate of SR Ca²⁺ leak (d[Ca²⁺]_SRT/dt) was plotted as a function of [Ca²⁺]_SR for each time point (30 s) during [Ca²⁺]_SR decline. The complete SERCA inhibition was confirmed by measuring [Ca²⁺]_SR recovery after SR Ca²⁺ depletion with caffeine and then [Ca²⁺]_i elevation to drive Ca²⁺ uptake. TG (10 μm) completely prevented [Ca²⁺]_SR recovery within 1 min (data not shown). Based on Fluo-5N sensitivity to Ca²⁺, it seems unlikely that Fluo-5N trapped in mitochondria significantly contributed to the measured signal, because under our experimental conditions mitochondrial [Ca²⁺] has been reported to be lower than 100 nm (Andrienko et al. 2009).

Figure 1. Simultaneous measurements of Ca²⁺ sparks and [Ca²⁺]_SR in permeabilized ventricular myocytes.

A, line-scan images and corresponding profiles (F/F₀) of Rhod-2 (red) and Fluo-5N (green) fluorescence in control conditions and at different times after application of thapsigargin (TG; 10 μm). Fluo-5N was recorded with open pinhole (non-confocal setting) whereas Rhod-2 was recorded confocally. The Ca²⁺ spark profiles were obtained by averaging fluorescence from the 1 μm wide region marked by the red box. Fluo-5N profiles were obtained by averaging fluorescence over the entire width of the line-scan images. At the end of the experiment, F_min and F_max were measured (see Methods). B, relationship between initial [Ca²⁺]_SR and Ca²⁺ spark frequency measured under control conditions (before TG application) from 16 different cells. Frequency correlated positively with [Ca²⁺]_SR measured under control conditions (R²= 0.78). C, effects of SERCA inhibition on spark frequency and [Ca²⁺]_SR. Measurements were made from the same cell shown in panel A.

Figure 4. Effects of RyR stimulation by low-dose caffeine on Ca²⁺ sparks, [Ca²⁺]_SR and SR Ca²⁺ leak.

A, line-scan images and corresponding profiles of Rhod-2 (red) and Fluo-5N (green) fluorescence in control conditions, in the presence of caffeine (200 μm) and after subsequent application of thapsigargin (TG; 10 μm). Fluo-5N was recorded with open pinhole (non-confocal setting) whereas Rhod-2 was recorded confocally. B, effect of caffeine (200 μm) followed by SERCA inhibition (10 μm TG) on spark frequency and [Ca²⁺]_SR. Application of 10 mm caffeine at the end of the experiment indicates complete depletion of the SR. Measurements were made from the same cell shown in panel A. C, average spark frequency and [Ca²⁺]_SR in control conditions, immediately (initial) and 2 min (late) after exposure to caffeine (200 μm). D, the relationships between SR Ca²⁺ leak rate and [Ca²⁺]_SR in control conditions (back) and in the presence of caffeine (red).

Measurements of Ca²⁺ blinks

Localized [Ca²⁺]_SR depletions or Ca²⁺ blinks (Brochet et al. 2005; Zima et al. 2008b) and the corresponding local cytosolic Ca²⁺ elevations (Ca²⁺ sparks) were recorded simultaneously with confocal microscopy (Fig. 3C and D) using Rhod-2 as the cytosolic and Fluo-5N as the SR Ca²⁺ indicator, respectively. For each detected Ca²⁺ spark with Rhod-2, the corresponding local changes of the Fluo-5N signal were analysed. The profiles of Ca²⁺ blinks were fitted with the product of two exponential functions to the declining and recovery phase, respectively, as described previously (Zima et al. 2008b). Blink amplitudes were obtained from the fit of the experimental data.

Figure 3. Contribution of Ca²⁺ sparks to total SR Ca²⁺ leak.

A, relationships between SR Ca²⁺ leak rate and [Ca²⁺]_SR (n= 16 myocytes). Black circles and black line represent experimentally measured total leak. The leak data points between [Ca²⁺]_SR of 25 and 325 μm were fitted with a single sigmoid function and represents the non-spark-mediated leak (green line). Spark-mediated leak as function of [Ca²⁺]_SR was obtained by subtracting non-spark-mediated leak (green line) from total SR Ca²⁺ leak (black circles). The calculated points (red circles) could be fitted with a single exponential function (red line). B, dependence of total spark signal mass (black circles) and total spark-mediated Ca²⁺ release flux (red circles) from [Ca²⁺]_SR. Signal mass and Ca²⁺ release flux of all detected sparks were summated and normalized to the recording time (4.5 s). Spark signal mass and spark-mediated release flux were calculated as described in Methods. C, simultaneously recorded Ca²⁺ spark and blink. Top, line-scan image of Rhod-2 fluorescence and corresponding spark profile (F/F₀). Bottom, line-scan image of Fluo-5N fluorescence and corresponding blink profile. Spark and blink profiles were obtained by averaging fluorescence from the 1 μm the wide regions marked by the red and green box, respectively. D, distribution of [Ca²⁺]_SR at the nadir of blinks (44 events).

Statistics

Data are presented as means ±s.e.m. of n measurements. Statistical comparisons between groups were performed with Student's t test. Differences were considered statistically significant at P < 0.05.

Results

The effect of [Ca²⁺]_SR on Ca²⁺ spark properties

To investigate the relationship between Ca²⁺ sparks and SR Ca²⁺ load, we simultaneously measured cytosolic [Ca²⁺] and [Ca²⁺]_SR in permeabilized ventricular myocytes after complete SERCA inhibition with thapsigargin (TG). Figure 1A depicts line-scan images of Rhod-2 and Fluo-5N fluorescence with corresponding profiles of local [Ca²⁺]_i (including Ca²⁺ sparks) and cell-averaged [Ca²⁺]_SR. The recordings were made in control conditions and at different times after application of TG (10 μm). In these experiments, local [Ca²⁺]_SR depletions, Ca²⁺ blinks (Brochet et al. 2005; Zima et al. 2008b), were not resolved because Fluo-5N fluorescence was acquired in non-confocal mode. This strategy allowed us to use very low laser intensity to avoid dye photobleaching and improved the signal-to-noise ratio of Fluo-5N (see Methods). Under control conditions, Ca²⁺ spark frequency and [Ca²⁺]_SR had average values of 10.1 ± 0.8 sparks (100 μm)⁻¹ s⁻¹ and 760 ± 22 μm (n= 16), respectively. Spark frequency correlated positively with [Ca²⁺]_SR measured under control conditions (no TG present; Fig. 1B). After SERCA inhibition, [Ca²⁺]_SR and Ca²⁺ spark frequency gradually declined until sparks ceased completely at [Ca²⁺]_SR= 279 ± 10 μm (n= 16 cells) (Fig. 1C). After the disappearance of Ca²⁺ sparks, [Ca²⁺]_SR continued to decline until full depletion (verified as lack of response to stimulation with 10 mm caffeine). These results suggest that SR Ca²⁺ leak can occur in form of sparks, but there is also spark-independent leak.

We then analysed how luminal [Ca²⁺] affects the properties of Ca²⁺ sparks. For each individual cell studied under conditions illustrated in Fig. 1A, spark frequency, amplitude, width and duration were plotted as a function of [Ca²⁺]_SR. [Ca²⁺]_SR affected spark frequency, amplitude and width in a dose-dependent manner (Fig. 2A–C); however, spark duration was not affected (Fig. 2D). Ca²⁺ spark frequency was most sensitive to [Ca²⁺]_SR. Changes of [Ca²⁺]_SR from 400 to 800 μm increased spark frequency, amplitude and width by 10, 1.8 and 1.6 times, respectively. The relationship between spark frequency and [Ca²⁺]_SR was similar to that previously observed when SERCA was not blocked (Fig. 1B), indicating that TG did not directly affect SR Ca²⁺ release. We corrected spark frequency for missing events using a previously tested approach (Song et al. 1997). The correction for missing events did not significantly change the relationship between spark frequency and [Ca²⁺]_SR (see Supplemental Material).

Figure 2. Effect of [Ca²⁺]_SR on Ca²⁺ spark properties.

The dependence of spark frequency (A), spark amplitude (B), spark width (C) (measured at half-maximal amplitude, FWHM) and spark duration (D) (measured at half-maximal amplitude, FDHM) on [Ca²⁺]_SR (bin size 50 μm; n= 16 myocytes).

These results demonstrate that Ca²⁺ spark frequency, amplitude and width are highly dependent on [Ca²⁺]_SR. These might be due to changes of Ca²⁺ release flux as the Ca²⁺ gradient across the SR membrane changes and also could be due to luminal Ca²⁺-dependent RyR regulation (Sitsapesan & Williams, 1994; Gyorke & Gyorke, 1998).

Contribution of Ca²⁺ sparks to total SR Ca²⁺ leak

Next, we examined to what extent SR Ca²⁺ release through Ca²⁺ sparks contributes to total SR Ca²⁺ leak. Free luminal [Ca²⁺] after SERCA inhibition was converted to total [Ca²⁺]_SR ([Ca²⁺]_SRT) based on the known intra-SR Ca²⁺-buffer capacity (Shannon et al. 2000b). SR Ca²⁺ leak rate, which was measured as changes of [Ca²⁺]_SRT over time (d[Ca²⁺]_SRT/dt), was plotted against the corresponding free [Ca²⁺]_SR to obtain the relationship between SR Ca²⁺ leak rate and [Ca²⁺]_SR (Fig. 3A). We found that the SR Ca²⁺ leak increased as a function of [Ca²⁺]_SR, with a particularly steep increase at higher [Ca²⁺]_SR (>600 μm). This increase in the leak rate can be attributed to higher Ca²⁺ spark frequency that occurred at higher [Ca²⁺]_SR (Fig. 2A). Higher [Ca²⁺]_SR also led to increased spark amplitude and width (Fig. 2B and C). Spark signal mass, which is proportional to amplitude and width, has been used previously as a measure of releasable Ca²⁺ during an individual spark (Chandler et al. 2003). Therefore, the overall rate of spark-mediated Ca²⁺ leak should be proportional to total spark signal mass. Figure 3B shows the relationship between [Ca²⁺]_SR and total spark signal mass. The experimental points were well fitted with a single exponential function with a growth constant of 148 μm (black line). Alternatively, SR Ca²⁺ release flux during a spark can be estimated from the maximum of the first derivative of the cytosolic fluorescence intensity (Sipido & Wier, 1991). The spark-mediated leak would then be proportional to total spark-mediated release flux. The data were binned and plotted as total spark-mediated release flux versus[Ca²⁺]_SR (Fig. 3B, red symbols). Similar to the results using the signal mass, the experimental points were well fitted with a single exponential function with a growth constant of 167 μm (red line). These two functions matched well, suggesting that both describe the same process of Ca²⁺ spark-mediated leak. When [Ca²⁺]_SR was depleted below 300 μm, the total spark signal mass and the total spark-mediated release flux became insignificant, indicating the absence of spark-mediated leak. Therefore, at low [Ca²⁺]_SR (<300 μm) SR Ca²⁺ leak occurred mostly as non-spark-mediated leak. The experimental points of SR Ca²⁺ leak at low [Ca²⁺]_SR (between 25 and 325 μm) were best fitted with a Hill function (Fig. 3A, green line) with K_0.5 of 135 μm and V_max of 3.9 μm s⁻¹. Next, the spark-mediated leak as a function of [Ca²⁺]_SR was estimated by subtracting the non-spark-mediated leak rate (green line) from the measured total SR Ca²⁺ leak rate (black points). The obtained points could be fitted with a single exponential function with a growth constant of 145 μm (red line), which agrees with the spark-mediated Ca²⁺ leak estimated from the properties of Ca²⁺ sparks (signal mass and release flux) shown in Fig. 3B.

In the following experiments, we analysed [Ca²⁺]_SR remaining in the SR at the nadir of Ca²⁺ blinks (Brochet et al. 2005; Zima et al. 2008b). Figure 3C shows a representative example of a Ca²⁺ spark and corresponding Ca²⁺ blink simultaneously recorded with confocal microscopy. We have shown previously that Ca²⁺ sparks terminate at an absolute [Ca²⁺]_SR depletion threshold that was independent of SR Ca²⁺ load (Zima et al. 2008b). Therefore, it is reasonable to predict that Ca²⁺ spark activity, and therefore spark-mediated Ca²⁺ leak, would cease completely at [Ca²⁺]_SR lower than the average spark termination threshold. Fluo-5N signals during Ca²⁺ blinks were converted to [Ca²⁺] and distribution of [Ca²⁺]_SR at the nadir of blinks was analysed (Fig. 3D). On average, Ca²⁺ sparks terminated at 305 ± 11 μm of [Ca²⁺]_SR (n= 44 events). This value matched well above estimates of the threshold for spark-mediated Ca²⁺ leak measured from the SR Ca²⁺ leak rate (Fig. 3A) and spark properties (Fig. 3B).

These results demonstrate that at least two components of SR Ca²⁺ leak can be identified in rabbit ventricular myocytes: Ca²⁺ leak in the form of sparks and non-spark-mediated leak. Depending on [Ca²⁺]_SR, these two components contribute to a different degree to the total SR Ca²⁺ leak. At low [Ca²⁺]_SR, Ca²⁺ leak occurred mostly as non-spark-mediated leak. At high [Ca²⁺]_SR, however, Ca²⁺ sparks became a significant pathway of SR Ca²⁺ leak.

Contribution of RyR-mediated Ca²⁺ leak to total SR Ca²⁺ leak

In the following experiments, we investigated whether spark-independent Ca²⁺ leak still occurred through RyRs. To this end, we studied the effects of the RyR agonist (caffeine) and RyR antagonists (ruthenium red (RuR), Mg²⁺, or tetracaine) on [Ca²⁺]_SR, spark frequency and SR Ca²⁺ leak.

Initially, we studied the effect of a low dose of caffeine, which does not evoke global SR Ca²⁺ release, but substantially sensitizes RyRs. Figure 4A shows representative confocal line-scan images of Ca²⁺ sparks and averaged [Ca²⁺]_SR under control conditions, immediately and 2 min after application of caffeine (200 μm), as well as after subsequent addition of TG (10 μm). Caffeine transiently increased Ca²⁺ spark activity and partially depleted the SR (Fig. 4B). On average (Fig. 4C), spark frequency initially increased from 8.5 ± 0.9 to 14.9 ± 1.2 (n= 6; P < 0.05), then decreased to 6.1 ± 0.9 sparks (100 μm)⁻¹ s⁻¹ (n= 6; P < 0.05). After 2 min of caffeine application, [Ca²⁺]_SR decreased from 869 ± 48 to 615 ± 66 μm (n= 6; P < 0.05; Fig. 4C). At the same time when [Ca²⁺]_SR reached a new steady-state level, spark amplitude and width were decreased by 26% (n= 6; P < 0.05) and by 21% (n= 6; P < 0.05), respectively. In the presence of caffeine, SERCA inhibition resulted in a faster decline of [Ca²⁺]_SR (by 45%) and Ca²⁺ spark frequency (by 79%) than in the absence of RyR stimulation (Fig. 4B). Sparks ceased completely when [Ca²⁺]_SR decreased below 229 ± 32 μm (n= 6 cells), which is significantly lower than under control conditions. We measured leak rate as a function of [Ca²⁺]_SR in the presence of caffeine (Fig. 4D, red symbols) and compared with the leak rate under control conditions (Fig. 4D, black symbols). These data show that sensitization of RyRs with caffeine significantly increased SR Ca²⁺ leak. Therefore, caffeine decreased [Ca²⁺]_SR by stimulation of RyR-mediated Ca²⁺ leak and consequently led to a decrease in SR Ca²⁺ release due to a luminal Ca²⁺-dependent mechanism (Trafford et al. 2000; Lukyanenko et al. 2001). Notably at [Ca²⁺]_SR below 170 μm, caffeine did not affect Ca²⁺ leak (Fig. 4D). This observation suggests that either caffeine does not activate RyRs at low [Ca²⁺]_SR or that at this [Ca²⁺]_SR Ca²⁺ leak occurs via pathways other than RyRs.

In the next set of experiments, we studied the effects of RyR inhibitors on SR Ca²⁺ leak. We used RuR, Mg²⁺, or tetracaine at concentrations which completely inhibit RyRs reconstituted in lipid bilayers (Xu et al. 1996; Lukyanenko et al. 2000; Zima et al. 2008a) as well as SR Ca²⁺ release in myocytes (Gyorke et al. 1997; Lukyanenko et al. 2000). RuR (50 μm) completely abolished Ca²⁺ sparks and almost doubled [Ca²⁺]_SR, confirming that RyRs provide an important SR Ca²⁺ leak pathway under resting conditions (Fig. 5A). Similar results were obtained when RyRs were inhibited with either 15 mm Mg²⁺ (Fig. 5B) or 1 mm tetracaine (data not shown). Average results of RyR inhibitors on [Ca²⁺]_SR under control conditions (no TG present) are shown in Fig. 5C. Application of TG (10 μm) in the presence of RyR inhibitors resulted in decline of [Ca²⁺]_SR, which reached complete depletion within approximately 40 min (Fig. 5A and B). Compared to control conditions (dashed lines in Fig. 5A and B), RyR inhibition significantly decreased SR Ca²⁺ leak rate but did not prevent leak. Increasing RuR concentration to 100 μm or combining RuR (50 μm) and Mg²⁺ (15 mm) had no additional effect on Ca²⁺ leak. Figure 5D shows effects of RuR (50 μm), Mg²⁺ (15 mm), and tetracaine (1 mm) on SR Ca²⁺ leak over a wide range of [Ca²⁺]_SR. For all RyR inhibitors tested here, RuR had the most pronounced effect on SR Ca²⁺ leak. In the presence of RuR, the leak rate was best fitted with a Hill function with K_0.5 of 148 μm and V_max of 1.5 μm s⁻¹. By subtracting this RuR-insensitive Ca²⁺ leak from the total leak we estimated RyR-mediated Ca²⁺ leak as a function of [Ca²⁺]_SR. Figure 6A shows different components of SR Ca²⁺ leak in permeabilized rabbit ventricular myocytes.

Figure 5. Effects of RyR inhibitors on Ca²⁺ sparks, [Ca²⁺]_SR and SR Ca²⁺ leak.

Effect of ruthenium red (RuR; 50 μm) (A) and Mg²⁺ (15 mm) (B) on spark frequency and [Ca²⁺]_SR before and after SERCA inhibition. For comparison, the dashed lines indicate the decline of [Ca²⁺]_SR in the absence of RyR inhibition (data from Fig. 1C). C, average effect of RuR (50 μm), Mg²⁺ (15 mm) and tetracaine (1 mm) on [Ca²⁺]_SR in the absence of TG. D, the relationships between SR Ca²⁺ leak rate and [Ca²⁺]_SR in control conditions (back), in the presence of RuR (green), tetracaine (blue) and Mg²⁺ (red). For presentation purposes only the fit to the data is shown for tetracaine and Mg²⁺.

Figure 6. Components of SR Ca²⁺ leak and role of IP₃Rs in SR Ca²⁺ leak.

A, different components of SR Ca²⁺ leak rate as a function of [Ca²⁺]_SR. Grey line represents the total RyR-mediated Ca²⁺ leak (spark and non-spark). This component was obtained by subtracting RuR-insensitive Ca²⁺ leak (green line) from the total Ca²⁺ leak (black line). Ca²⁺ spark-mediated leak (red line) was obtained as described in Fig. 3A. Non-spark RyR-mediated Ca²⁺ leak (blue line) was obtained by subtracting Ca²⁺ spark-mediated leak (red line) from the total RyR-mediated Ca²⁺ leak (grey line). B, the relationship between SR Ca²⁺ leak rate and [Ca²⁺]_SR in the presence of RuR (50 μm; black circles), in the presence of RuR plus 2-APB (20 μm; open circles) and in the presence of RuR plus IP₃ (10 μm; red circles).

We also tested whether spontaneous openings of IP₃Rs were responsible for RyR-independent Ca²⁺ leak. The IP₃R inhibitors 2-APB (20 μm; Fig. 6B) or heparin (0.5 mg ml⁻¹; not shown) had no additional inhibitory effect on SR Ca²⁺ leak when added together with RuR (50 μm) suggesting that the residual SR Ca²⁺ leak was not the result of IP₃R activity. However, application of IP₃ (20 μm) increased RyR-independent Ca²⁺ leak. At [Ca²⁺]_SR= 780 μm SR Ca²⁺ leak nearly doubled from 1.2 to 2.2 μm s⁻¹ (Fig. 6B).

These results show that RyRs are the main, but not the sole, contributor to SR Ca²⁺ leak in rabbit ventricular myocytes. Under basal conditions (in the absence of IP₃ production), IP₃R-mediated leak is minimal; however, there is SR Ca²⁺ leak that is RyR and IP₃R independent through a yet to be determined mechanism.

The properties of SR Ca²⁺ leak in ventricular myocytes from failing heart

In the following experiments, we studied properties of SR Ca²⁺ leak in permeabilized ventricular myocytes isolated from failing hearts. Similar to previous studies using the same HF model (Pogwizd et al. 2001; Guo et al. 2007; Domeier et al. 2009), we found that resting [Ca²⁺]_SR was significantly lower in HF myocytes. In normal (nonfailing) myocytes, resting [Ca²⁺]_SR was 760 ± 15 μm (n= 44), whereas under identical experimental conditions [Ca²⁺]_SR was 683 ± 27 μm in HF myocytes (n= 14; P < 0.05). Furthermore, the rate of decline of [Ca²⁺]_SR and spark frequency after SERCA inhibition was faster in HF myocytes than in normal myocytes (Fig. 7A, compare with Fig. 1C). The SR Ca²⁺ leak rate was analysed as a function of [Ca²⁺]_SR in HF myocytes (Fig. 7B, red symbols) and compared to normal myocytes (Fig. 7B, black symbols). We found that at [Ca²⁺]_SR higher than 200 μm Ca²⁺ leak was markedly increased in HF myocytes.

Figure 7. Properties of SR Ca²⁺ leak in HF myocytes.

A, changes of spark frequency and [Ca²⁺]_SR after SERCA inhibition with TG (10 μm) in myocyte from failing hearts. B, the total (circles) and RuR-insensitive (squares) SR Ca²⁺ leak as a function of [Ca²⁺]_SR in normal (black) and in HF myocytes (red). C, Ca²⁺ spark frequency in normal and HF myocytes measured at the same [Ca²⁺]_SR (680 μm).

In the presence of RuR (50 μm), SR Ca²⁺ leak was not significantly different between normal and HF myocytes (Fig. 7B) suggesting that the increased SR Ca²⁺ leak in HF myocytes was mainly due to higher RyR activity. We then studied if increased SR Ca²⁺ leak in HF myocytes was a result of higher Ca²⁺ spark activity. When Ca²⁺ sparks were analysed for these two groups at the same [Ca²⁺]_SR (680 μm), spark frequency was higher by 21% in HF myocytes (9.3 ± 0.9 sparks (100 μm)⁻¹ s⁻¹; n= 14) compared to normal myocytes (7.7 ± 0.8 sparks (100 μm)⁻¹ s⁻¹; n= 16; Fig. 7C). However, RyR-mediated SR Ca²⁺ leak (estimated as the difference between the total and RuR-insensitive Ca²⁺ leak) measured at the same [Ca²⁺]_SR (680 μm) was significantly higher (by 40%) in HF myocytes (Fig. 7B). These results suggest that the modifications of RyRs which occur during HF lead to augmentation of both spark- and non-spark-mediated Ca²⁺ leak.

Discussion

SR Ca²⁺ leak is generally defined as ‘basal’ Ca²⁺ efflux from the SR during rest or diastole. Despite its low flux rate relative to SR Ca²⁺ release during systole, diastolic SR Ca²⁺ leak plays an important role in modulating SR Ca²⁺ load. The RyR is the primary Ca²⁺ release channel of the SR and considered the key pathway of Ca²⁺ leak in ventricular myocytes. Abnormal activity of RyRs has been suggested to be involved in numerous cardiac pathologies, including HF (George, 2008) and catecholaminergic polymorphic ventricular tachycardias (Chelu & Wehrens, 2007). In spite of its importance, the mechanisms of SR Ca²⁺ leak have not been characterized in detail. Here we measure directly and continuously (in real time) SR Ca²⁺ leak properties in normal and HF ventricular myocytes, and determine the role of RyR and Ca²⁺ sparks in SR Ca²⁺ leak. The main findings of this study are that (1) RyR is the key channel of SR Ca²⁺ leak which occurs in part as Ca²⁺ sparks, but there is also spark-independent Ca²⁺ leak; (2) at low [Ca²⁺]_SR leak occurs mostly as non-spark-mediated leak, whereas at high [Ca²⁺]_SR sparks became a significant contributor to SR Ca²⁺ leak; (3) there is also a significant component of SR Ca²⁺ leak that is insensitive to RyR and IP₃R inhibitors (although IP₃R activation can increase leak significantly); and (4) RyR-mediated Ca²⁺ leak is significantly increased in ventricular myocytes from failing heart.

Novel approach to measure SR Ca²⁺ leak

In ventricular myocytes, SR Ca²⁺ leak has been previously measured either from characteristics of Ca²⁺ transients (Balke et al. 1994), from the rate of decline in SR Ca²⁺ load after complete SERCA blockade (Bassani & Bers, 1995) or as Ca²⁺ spark properties (Cheng et al. 1993). Another method that has been widely used to measure SR Ca²⁺ leak quantifies the decrease of cytosolic [Ca²⁺] and increase in SR Ca²⁺ content upon acute RyR block with tetracaine (Shannon et al. 2002). In the present study, we employed a new approach to measure SR Ca²⁺ leak. We combined direct continuous measurement of [Ca²⁺]_SR using Fluo-5N (Shannon et al. 2003a; Belevych et al. 2007; Domeier et al. 2009) with cytosolic Ca²⁺ sparks using Rhod-2 (Brochet et al. 2005; Zima et al. 2008b). This allowed simultaneous measurement of total SR Ca²⁺ leak flux (the rate of [Ca²⁺]_SR decline with SERCA fully blocked) and appearance of Ca²⁺ sparks (one cytosolic readout of SR Ca²⁺ leak).

This approach has several advantages compared to previous studies. First, SR Ca²⁺ leak is measured directly and continuously as changes of [Ca²⁺]_SR after SERCA inhibition with TG. It is essential for the determination of SR Ca²⁺ leak that the SERCA-mediated Ca²⁺ uptake was completely blocked by TG. Here we confirmed that TG (10 μm) completely and irreversibly inhibits SERCA within 1 min (Bassani et al. 1995; Zima et al. 2008b). Second, SR Ca²⁺ leak and [Ca²⁺]_SR can be measured simultaneously over the full physiological range, because [Ca²⁺]_SR gradually declines until full depletion upon SERCA blockade (Fig. 1). Third, using permeabilized myocytes allows cytosolic Fluo-5N washout (providing a more pure [Ca²⁺]_SR signal), cytosolic [Ca²⁺] can be precisely controlled (so global [Ca²⁺]_i does not change as [Ca²⁺]_SR declines) and cytosolic Ca²⁺ indicator (Rhod-2) can be introduced to measure cytosolic [Ca²⁺] and Ca²⁺ sparks. Therefore, in this study for the first time, SR Ca²⁺ load, SR Ca²⁺ leak and Ca²⁺ sparks were measured simultaneously. This is particularly important because Ca²⁺ sparks are often considered to be the main pathway of SR Ca²⁺ leak.

Ca²⁺ sparks are not the sole pathway of SR Ca²⁺ leak

The observation that after SERCA inhibition Ca²⁺ spark frequency declined significantly faster than [Ca²⁺]_SR (Fig. 1B) suggests a non-linear relationship between spark frequency and SR Ca²⁺ load. Although it is generally accepted that spark frequency depends on [Ca²⁺]_SR (Satoh et al. 1997; Lukyanenko et al. 2001), the spark–load relationship had not been rigorously studied because simultaneous measurements of these two characteristics were technically difficult until now. We found that Ca²⁺ spark amplitude and width are linear functions of [Ca²⁺]_SR (Fig. 2B and C) suggesting that these parameters are mainly determined by the SR to cytosol [Ca²⁺] gradient. In contrast, Ca²⁺ spark frequency was exponentially dependent on [Ca²⁺]_SR (Fig. 2A), suggesting a more complex regulation by [Ca²⁺]_SR. Increasing evidence indicates that luminal Ca²⁺ regulates RyR gating by at least two different mechanisms. Ca²⁺ can directly activate RyR from the luminal side of the channel (Sitsapesan & Williams, 1994; Gyorke & Gyorke, 1998), perhaps due to interaction with the Ca²⁺ binding protein calsequestrin (Gyorke et al. 2004). Luminal Ca²⁺ can also indirectly regulate RyR by acting on the cytosolic Ca²⁺ activation site of neighbouring channels by a ‘feed-through’ mechanism (Laver, 2007).

An important finding of this study was that Ca²⁺ sparks entirely disappeared at relatively constant [Ca²⁺]_SR (279 ± 10 μm; Fig. 1). Because Ca²⁺ spark detection relies on their amplitude, the absence of sparks at low [Ca²⁺]_SR might have been the result of a failure to detect Ca²⁺ sparks of smaller amplitudes (i.e. if they fall below the detection threshold). However, we think that is not why Ca²⁺ sparks disappeared at ∼300 μm[Ca²⁺]_SR, for two major reasons. First, the average spark amplitude at the point of disappearance was ∼0.5 ΔF/F₀ (Fig. 2B). By analysing the sensitivity of our detection algorithm (see Supplemental Material), we confirmed that at this amplitude the vast majority of sparks (∼70%) are readily detected. Moreover, we have corrected for missed events, and this correction at 0.5 ΔF/F₀ is very small. Second, analysis of [Ca²⁺]_SR at the nadir of Ca²⁺ blinks (Fig. 3D) showed that Ca²⁺ sparks terminate at a relatively constant [Ca²⁺]_SR (305 ± 11 μm), and this termination threshold was independent of SR Ca²⁺ load (Zima et al. 2008b). Presumably this Ca²⁺ spark termination threshold would also prevent spark initiation (i.e. it would immediately stop). Thus, this disappearance of Ca²⁺ sparks as [Ca²⁺]_SR falls below ∼300 μm would be quite consistent with abolition of spark initiation at the same [Ca²⁺]_SR at which Ca²⁺ sparks terminate. If SR Ca²⁺ leak is solely mediated by Ca²⁺ spark activity, then leak should abruptly stop at [Ca²⁺]_SR∼300 μm. However, SR Ca²⁺ leak continued below this [Ca²⁺]_SR suggesting that non-spark mediated Ca²⁺ leak also exists in ventricular myocytes.

SR Ca²⁺ leak can occur as spark-independent RyR openings

We separated SR Ca²⁺ leak into RyR-dependent and RyR-independent components (Fig. 6A). The latter is small and persists in the presence of RyR inhibition (Fig. 5); it will be discussed in the next section. The RyR-dependent leak is composed of both a Ca²⁺ spark component (which starts at [Ca²⁺]_SR≥ 300 μm and rises steeply at higher [Ca²⁺]_SR) and a spark-independent component (apparent at low [Ca²⁺]_SR reaching a plateau at the [Ca²⁺]_SR where Ca²⁺ sparks appear; Fig. 6A). The steep [Ca²⁺]_SR dependence of the spark-mediated leak can be explained by the fact that [Ca²⁺]_SR affects both the probability of Ca²⁺ release events (spark frequency) in a non-linear manner (Fig. 2A) and the amount of Ca²⁺ released during individual sparks (spark amplitude and width), which increase linearly with [Ca²⁺]_SR (Fig. 2B and C). Interestingly, the non-spark RyR mediated leak in our analysis is best fitted with a K_0.5∼135 μm[Ca²⁺]_SR (Fig. 6A), raising the possibility that the same luminal Ca²⁺ site might influence both RyR-dependent pathways (although further tests would be required). The non-spark RyR leak flux seems to be maximal at ∼2.5 μm s⁻¹, which is almost 2 times higher than the non-RyR-mediated leak. As [Ca²⁺]_SR rises the Ca²⁺ spark-mediated leak is increasingly dominant (accounting for ∼16, 43 and 77% of RyR-mediated leak at 600, 800 and 1000 μm, respectively).

The RyR-mediated leak curve resembles the tetracaine-sensitive SR Ca²⁺ leak reported by Shannon et al. (2002) except for two features. First, the spark-independent pedestal component that we see for 100–500 μm[Ca²⁺]_SR was not identified, but they had only one data point for [Ca²⁺]_SR <500 μm. So, this component may have been missed by Shannon et al. Second, they found that at [Ca²⁺]_SR= 1200 μm leak rose almost vertically and leak reached >21 μmol (litre cytosol)⁻¹ s⁻¹. In the present study it was difficult to push [Ca²⁺]_SR that high, in part because some leak occurs as TG block of SERCA is being achieved. If we would extrapolate our curves from Fig. 6A up to 1200 μm[Ca²⁺]_SR we may project a similar and very steep leak–load relationship as implied by Shannon et al. (2002). This reinforces the notion that there may be a limiting SR Ca²⁺ load due to SR Ca²⁺ leak (Diaz et al. 1997), unless leak is blocked by RuR or tetracaine (as in Fig. 5C).

So why is some RyR-mediated Ca²⁺ release not spark mediated? The spark-independent RyR leak may still arise from the same RyR clusters responsible for Ca²⁺ spark generation if at low [Ca²⁺]_SR the RyR openings are insufficient to recruit neighbouring RyRs to form a spark. At low [Ca²⁺]_SR RyR openings are briefer (∼1/6 as long), carry less current (∼1/3 as much), are less sensitive to [Ca²⁺]_i-dependent activation (∼10-fold) and have longer latency (∼3-fold) (Gyorke & Gyorke, 1998). These aspects could possibly explain the failure of RyR-mediated flux to initiate Ca²⁺ sparks at low [Ca²⁺]_SR. At high [Ca²⁺]_SR, however, the chance that a single RyR opening can trigger a spark would substantially increase because RyR activation can generate a Ca²⁺ flux large enough to activate the rest of the channels in the cluster. There is also a higher probability that two adjacent channels in the cluster can open simultaneously and increase cytosolic [Ca²⁺] to the critical level that triggers a spark.

RyR-independent pathways of SR Ca²⁺ leak

Another important finding of this study is that complete block of RyRs did not abolish SR Ca²⁺ leak (Fig. 5). Although the existence of RyR-independent Ca²⁺ leak in ventricular myocytes has been suggested previously, at ∼10% of the RyR-mediated Ca²⁺ leak (Neary et al. 2002) the mechanisms have not been identified. Our data agree with this and show that the RyR-independent Ca²⁺ leak is a larger fraction of leak at low [Ca²⁺]_SR. We tested several potential pathways of RyR-independent Ca²⁺ leak. We have shown previously that IP₃Rs are also expressed in rabbit ventricular myocytes (Wu & Bers, 2006; Domeier et al. 2008) and may contribute to this residual SR Ca²⁺ leak. However, IP₃R inhibitors (2-APB and heparin) did not prevent RyR-independent Ca²⁺ leak (Fig. 6B) suggesting that IP₃Rs are not contributing to RyR-independent Ca²⁺ leak in our experimental conditions (presumably because IP₃R activation requires IP₃). However, despite low IP₃R expression levels in ventricular myocytes, these channels can participate in diastolic SR Ca²⁺ leak during stimulation of the phospholipase C–IP₃ signalling cascade (e.g. ET-1 receptor activation). In support of this notion, we found that activation of IP₃Rs by IP₃ application nearly doubled RyR-independent SR Ca²⁺ leak (Fig. 6B). The ‘backflux’ mode of SERCA (Shannon et al. 2000b) cannot contributes to the RyR-independent Ca²⁺ leak because all our measurements were carried out with SERCA completely blocked in a dead-end complex by TG. It has been suggested that phospholamban (PLB) pentamers can function as Ca²⁺ channels in lipid bilayers (Kovacs et al. 1988). However, Ca²⁺ leak measured from SR vesicles isolated from wild-type and PLB-knockout mouse was not significantly different (Shannon et al. 2001). Additionally, it has been shown that the translocon of the rough endoplasmic reticulum is an important Ca²⁺ leak pathway in smooth muscle cells (Amer et al. 2009). On the contrary, we did not find any differences in SR Ca²⁺ leak rate when the translocon was opened with puromycin or blocked with anisomycin (data not shown). Therefore, further studies are required to determine the exact molecular mechanisms of RyR-independent SR Ca²⁺ leak and its physiological relevance in cardiomyocytes.

Physiological and pathological significance of SR Ca²⁺ leak

The steep [Ca²⁺]_SR dependence of Ca²⁺ spark dependent leak is paralleled by the efficacy of Ca²⁺-induced Ca²⁺ release during ECC (Bassani et al. 1995; Diaz et al. 1997; Shannon et al. 2000a). For [Ca²⁺]_SR below the threshold for Ca²⁺ spark termination that we report here, L-type Ca²⁺ current cannot induce appreciable SR Ca²⁺ release. Moreover, as [Ca²⁺]_SR increases on the steep part of the Ca²⁺ sparks vs.[Ca²⁺]_SR relationship described here, there is an increasingly steep increase in fractional SR Ca²⁺ release for a given Ca²⁺ current. We hypothesize that the same luminal Ca²⁺ sensor increases the probability of spontaneous Ca²⁺ sparks and fractional SR Ca²⁺ release during ECC.

A common characteristic of almost every HF models is a decrease in SR Ca²⁺ content, caused by some combination of decreased SERCA pump function, enhanced Na⁺–Ca²⁺ exchange (NCX) function and SR Ca²⁺ leak (George, 2008). Here we eliminated the sarcolemmal Ca²⁺ flux and SERCA effects to directly evaluate SR Ca²⁺ leak in HF myocytes. We found that in HF myocytes SR Ca²⁺ leak for a given [Ca²⁺]_SR was increased to a similar degree as in control cells during exposure to low concentration of caffeine (37 vs. 42%, respectively). As a result of this, steady state [Ca²⁺]_SR decreased to a similar level in both groups (683 μm in HF vs. 615 μm in the presence of caffeine). Thus, enhanced RyR-mediated leak by itself could largely explain the reduced SR Ca²⁺ load in HF, although functional changes in SERCA, NCX and [Na⁺]_i regulation can contribute to the resulting SR Ca²⁺ load in intact HF myocytes (Pogwizd et al. 1999; Shannon et al. 2003b; Despa et al. 2002). The increased SR Ca²⁺ leak in HF has been attributed to phosphorylation of the RyR by CaMKII (Ai et al. 2005) or protein kinase A (Marx et al. 2000) although work in this area is controversial. RyR gating in HF was found to have altered luminal Ca²⁺-dependent regulation (Kubalova et al. 2005), a mechanism that is responsible for Ca²⁺ spark termination (Zima et al. 2008b) and spark activation (see above). In the same HF model studied here we reported previously that SR Ca²⁺ load is reduced in HF (without altered intra-SR Ca²⁺ buffering), that SR Ca²⁺ release is sensitized to trigger in HF (Guo et al. 2007) and that Ca²⁺ sparks terminate at lower [Ca²⁺]_SR (Domeier et al. 2009). These may all be interrelated changes in RyR function in HF and this contributes to both altered diastolic and systolic cardiac function in HF.

Conclusion

The fact that RyR inhibition greatly increases SR [Ca²⁺]_SR (Fig. 5C) indicates that a significant RyR-mediated Ca²⁺ leak exists under resting conditions and that it limits SR Ca²⁺ load. This implies that at rest SERCA cannot achieve its maximal thermodynamic efficiency and that some ATP is wasted in a futile pump–leak balance. Increased SR Ca²⁺ leak has been implicated in HF and may contribute to triggered arrhythmias (Marx et al. 2000; Ai et al. 2005). Thus, inhibition of diastolic SR Ca²⁺ release (without inhibiting systolic Ca²⁺ release) would be a potentially important therapeutic strategy. It could have benefits with respect to enhancing energetic efficiency, reducing triggered arrhythmias, limiting myocyte death and limiting the progression from cardiac hypertrophy to HF.

Glossary

Abbreviations

2-APB

2-aminoethoxydiphenyl borate

[Ca²⁺]_i

cytosolic free calcium concentration

[Ca²⁺]_SR

sarcoplasmic reticulum free calcium concentration

ECC

excitation–contraction coupling

HF

heart failure

IP₃R

inositol-1,4,5-trisphosphate receptor

NCX

Na⁺–Ca²⁺ exchanger

PLB

phospholamban

RuR

ruthenium red

RyR

ryanodine receptor

SERCA

sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase

SR

sarcoplasmic reticulum

TG

thapsigargin

Author contributions

A.V.Z., E.B., L.A.B. and D.M.B. contributed to the conception and design of the study, interpretation of data and writing of the manuscript. A.V.Z. and E.B. performed the experimental work and analysis of results. All authors have approved the version to be published. All experiments were carried out at Loyola University Chicago and Rush University Medical Center, Chicago.