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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix
  9. Supporting Information

Spontaneous Ca2+ waves in cardiac muscle cells are thought to arise from the sequential firing of local Ca2+ sparks via a fire–diffuse–fire mechanism. This study compares the ability of the ryanodine receptor (RyR) blocker ruthenium red (RuR) to inhibit these two types of Ca2+ release in permeabilised rabbit ventricular cardiomyocytes. Perfusing with 600 nm Ca2+ (50 μm EGTA) caused regular spontaneous Ca2+ waves that were imaged with the fluorescence from Fluo-5F using a laser-scanning confocal microscope. Addition of 4 μm RuR caused complete inhibition of Ca2+ waves in 50% of cardiomyocytes by 2 min and in 100% by 4 min. Separate experiments used 350 μm EGTA (600 nm Ca2+) to limit Ca2+ diffusion but allow the underlying Ca2+ sparks to be imaged. The time course of RuR-induced inhibition did not match that of waves. After 2 min of RuR, none of the characteristics of the Ca2+ sparks were altered, and after 4 min Ca2+ spark frequency was reduced ∼40%; no sparks could be detected after 10 min. Measurements of Ca2+ within the SR lumen using Fluo-5N showed an increase in intra-SR Ca2+ during the initial 2–4 min of perfusion with RuR in both wave and spark conditions. Computational modelling suggests that the sensitivity of Ca2+ waves to RuR block depends on the number of RyRs per cluster. Therefore inhibition of Ca2+ waves without affecting Ca2+ sparks may be explained by block of small, non-spark producing clusters of RyRs that are important to the process of Ca2+ wave propagation.

Abbreviations 
RuR

ruthenium red

SERCA

sarcoplasmic/endoplasmic reticulum Ca2+-ATPase

SR

sarcoplasmic reticulum

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix
  9. Supporting Information

In ventricular cardiomyocytes, a small influx of Ca2+ via the L-type Ca2+ channel triggers a larger global release of Ca2+ from the sarcoplasmic reticulum (SR) inducing contraction. Release from the SR is mediated by Ca2+ release channels (ryanodine receptor type 2, RyR) that exist in clusters of channels ∼2 μm apart along the longitudinal axis of the cell and ∼1 μm apart radially (Chen-Izu et al. 2006). The number of RyRs in each cluster is uncertain, with estimates ranging from 10 to 270 (Bridge et al. 1999; Franzini-Armstrong et al. 1998, 1999; Soeller et al. 2007). The RyRs in a cluster are thought to be functionally coupled such that the status of one channel (e.g. open) enhances the rate constant for opening of neighbouring RyR channels in the cluster (Marx et al. 2001; Yin et al. 2005). This cooperative activity is thought to be important in determining the duration of the release event (Sobie et al. 2002) and may be altered by phosphorylation of RyR either by A-kinase or CaM-kinase (Marx et al. 2001; Yin et al. 2005).

Ca2+ release from individual clusters can be observed experimentally as Ca2+ sparks (Cheng et al. 1993). These localised spontaneous Ca2+ release events are thought not only to underlie the basic Ca2+ release unit of excitation–contraction (E–C) coupling, but also to mediate a significant fraction of diastolic SR Ca2+ release (Santiago et al. 2010). Under conditions of cellular Ca2+ overload, larger spontaneous Ca2+ release events known as Ca2+ waves are observed. The Ca2+ wave is thought to be initiated when a spontaneous Ca2+ spark triggers the regenerative propagation of Ca2+ release from one RyR cluster to another. Release eventually propagates through the entire heart cell via a saltatory, ‘fire–diffuse–fire’ mechanism (Keizer & Smith, 1998). This regenerative form of SR Ca2+ release during diastole can depolarise the sarcolemma via Ca2+-sensitive currents, and has been implicated in initiation of ventricular arrhythmias (Weir & Hess, 1984).

Recent work suggests that an alternative mode of Ca2+ leak may occur via smaller clusters of RyRs distributed between the Z-lines outside the dyadic junction (Lukyanenko et al. 2007). It is hypothesized that spontaneous openings of these non-junctional or ‘rogue’ RyRs do not produce detectable Ca2+ sparks but contribute to RyRs Ca2+ leak and may influence Ca2+ wave propagation. Computational modelling of the activity of smaller clusters of RyRs that may be appropriate for rogue RyRs suggests that the reduced cooperative activity in these clusters would result in a higher sensitivity to manipulations that affect RyR activity, such as phosphorylation (Sobie et al. 2006).

In this study, we investigated the effects of an RyR inhibitor, ruthenium red (RuR), on Ca2+ sparks and waves. We present data which demonstrate that Ca2+ wave activity is more sensitive than Ca2+ spark activity to inhibition by RuR. The more rapid inhibition by RuR is not due to a more rapid depletion of the SR when Ca2+ waves occur. Modelling the effect of blocking individual channels within RyR clusters of variable size predicts that those containing high numbers (e.g. 30) of RyRs resist the effects of accumulating block better than those with fewer RyRs (e.g. 5). These data suggest that Ca2+ waves require the participation of small (sub-spark) clusters of RyR for robust initiation and propagation.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix
  9. Supporting Information

Cell isolation and permeabilisation

All procedures and were approved by the local (University of Glasgow) ethical committee complied with both Home Office regulations and the policies of The Journal of Physiology as set out by (Drummond, 2009). New Zealand White rabbits (2–2.5 kg) were given an intravenous injection of 500 U heparin together with an overdose of sodium pentobarbitone (100 mg kg−1). The hearts were rapidly excised, weighed and cannulated onto a Langendorff perfusion column via the aorta. Ventricular myocytes were isolated from Langendorff perfused rabbit hearts by enzymatic digestion as previously described (McIntosh et al. 2000) and kept in a modified Krebs solution buffered with no Ca2+ added at a concentration of 104 cells ml−1 until use. The cells were allowed to settle onto the coverslip at the base of a small bath. β-Escin (Sigma, St Louis, MO, USA) was added from a freshly prepared stock solution to the cell suspension to give a final concentration of 0.1 mg ml−1 for 0.5–1 min and the β-escin subsequently removed by perfusion with a mock intracellular solution with the following composition (mm): 100 KCl, 5 K2ATP, 5 Na2CrP, 5.5 MgCl2, 25 Hepes, 0.05 K2EGTA, pH 7.0 (20–21°C).

[Ca2+] measurements

A mock intracellular solution containing 10 μm Fluo-5F, 600 nm free Ca2+ and either 50 μm EGTA (to allow Ca2+-waves) or 350 μm EGTA (to suppress waves but allow Ca2+ sparks). Intracellular fluorescence was monitored using a Bio-Rad 2000 laser scanning confocal microscope (LSCM). The fluorophore was excited at 488 nm (Ar laser) and measured at >515 nm using linescan mode at a rate of 500 Hz. The cells were imaged using the epifluorescence optics of a Nikon Eclipse inverted microscope with a 60× water objective lens (NA 1.2). The iris diameter was set at 1.9, providing an axial (z) resolution of about 0.9 μm and x–y resolution of about 0.5 μm based on full-width, half-maximal amplitude measurements of images of 0.1 μm fluorescent beads (Molecular Probes/Invitrogen). Data were acquired in line-scan mode at 2 ms per line scan; the pixel dimension was 0.3 μm (512 pixels per scan; zoom 1.4). The scanning laser line was oriented parallel to the long axis of the cell and placed approximately equidistant between the outer edge of the cell and the nucleus/nuclei, to ensure the nuclear area was not included in the scan line.

To enable this trace to be converted to free Ca2+ concentration ([Ca2+]) a series of calibration solutions were used at the end of each experiment incorporating 10 mm EGTA as previously described (Currie et al. 2004). Ca2+ sparks were measured in periods of 15 s every minute. Ca2+ spark parameters were measured using MacSpark: (http://www.gla.ac.uk/departments/nbs/researchinterests/academicstaff/godfreysmith/macspark/), a program that used a previously established algorithm for spark detection (Cheng et al. 1999) and the detection criterion (CRI value) was set at 3.5 after an initial 5 median filter. Spark frequency was calculated per 100 μm s−1. The influence of false event detection was minimised by subtracting the number of events detected in an identical perfusing solution after the cell had been exposed to a 1.5 min application of caffeine (10 mm) and thapsigargin (25 μm). Normally, Ca2+ sparks are measured at a cytoplasmic [Ca2+] of 100–150 nm Ca2+, and under these conditions sparks can easily be detected because of the low background fluorescence (using Fluo-3/4). But for this study it was important to measure Ca2+ sparks under similar conditions to those used to examine Ca2+ waves. Therefore we increased the Ca2+ buffer (EGTA) sufficiently to prevent Ca2+ waves but maintained the perfusing [Ca2+] at 600 nm. Spark detection under these conditions is technically difficult due to high background fluorescence. Using Fluo-3 as an indicator the background fluorescence at 600 nm would be ∼3× higher than that at 120 nm and the noise on the fluorescence signal would increase by ∼2. In this study Fluo-5F (Kd= 1.1 μm) (Loughrey et al. 2002) was used rather than Fluo-3 (Kd= 0.55 μm) to reduce the background fluorescence signal and the associated noise at 600 nm thereby facilitating spark detection. The use of Fluo-5F as opposed to Fluo-3 reduced the background fluorescence and associated noise at 600 nm by ∼30%. Despite the lower background signal the events were difficult to detect by eye, and therefore detected events including the perimeter in linescan are highlighted using a white fill as indicated in Supplementary Fig. 1. This format is used to display linescan images containing detected spark events (Fig. 3).

image

Figure 3. Inhibition of Ca2+ sparks by RuR Spark-mediated Ca2+ release persists subsequent to RuR inhibition of Ca2+ waves. A, linescan image showing cessation of Ca2+ waves (a), with persistent spark release shown in b (see zoomed region outlined within the black rectangle). The detected events are shown in white. B, example linescan images taken at specified periods after application of RuR. Ca2+ sparks measured under 600 nm free Ca2+ and high (350 μm) EGTA. Images shown have been filtered using a 15 point Savitzky–Golay filter (spatial) and 7 boxcar (temporal); the detected events are shown in white. C, Average temporal (a) and spatial (b) profiles of the largest 10% spark at different times after addition of RuR Da, summary plot of spark frequency vs. time normalised to each cell's control and by subtracting the spark frequency of each cell after thapsigargin (25 μm) and caffeine (10 mm) application for 2 min. Db, SR content changes (expressed as F/Fo,cs). RuR added 10 s after time zero. Dc, mean spark amplitude (upper), width (middle) and duration (lower), all plots normalised to control.

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In experiments to monitor SR Ca2+, cells were incubated with Fluo-5N-AM (10 μm) for 3 h at 37°C. Changes in SR Ca2+ are expressed as relative change in caffeine-sensitive fluorescence (F/Fo,cs), where the data are normalised to the inter-wave fluorescence level after subtraction of the level measured during caffeine application (Fo,cs). Fluorescence signals were monitored using linescan parameters used to measure a cytoplasmic signals to ensure that the Fluo-5N signal tracked the Ca2+ wave. Long term measurements of luminal Ca2+ signal were made using the confocal microscope in frame-scan mode every 30 s with the pin-hole aperture opened to the maximum. This procedure gave good signal/noise with minimal bleaching over a 10–15 min of continuous data capture.

Ca2+ wave characteristics were obtained using semi-automated analysis software written by one of the authors (N.M.).

Modelling

Mathematical modelling was used to gain insight into how inhibition of RyRs by RuR affected Ca2+ spark properties and Ca2+ wave propagation. The primary goal was to determine what characteristics of RyR clusters were required to reproduce the unexpected observation that RuR abolished Ca2+ waves while simultaneously causing only minor changes in Ca2+ spark frequency and amplitude (see below). To avoid conclusions that depended on specific RyR gating mechanisms, we purposely employed a simplified model with a limited number of free parameters.

The basic assumption of the modelling was that the drug renders a certain percentage of channels within the RyR cluster inactive. Thus, the behaviour of a RyR cluster with (for example) 20% of the channels inhibited by RuR is equivalent to that of a cluster with 20% fewer channels physically present. Given this assumption, we systematically varied the number of channels in the RyR cluster to generate predictions of how RuR affected spark amplitude and Ca2+ wave propagation.

The first step was to predict the local Ca2+ release fluxes resulting from RyR clusters of different sizes. The basic structure of the model used for these simulations was similar to that described previously (Sobie et al. 2002); however, to avoid complications caused by stochastic RyR gating, we assumed that all RyRs opened for a fixed duration during Ca2+ sparks (as in Sobie et al. 2005). Besides the number of RyRs in the cluster, the most important free parameter in these simulations was the rate of refilling from network to junctional SR, which determined the degree of local SR depletion during the spark. Next, the calculated local Ca2+ release fluxes were used as the input to an established model of Ca2+ diffusion in the cytosol, binding to intracellular buffers including the fluorescent indicator Fluo-5F, and blurring by the optical apparatus (Smith et al. 1998; Sobie et al. 2002). The resulting simulated Ca2+ sparks were analysed to derive the predicted relationship between the fraction of inactive RyRs and Ca2+ spark amplitude.

The final step was to predict how RuR-induced changes in the local SR Ca2+ release flux affected the propagation of Ca2+ waves. We assumed that 50 clusters were arranged linearly, and that waves failed to propagate if a single cluster out of 50 was not triggered by its immediate predecessor. We further assumed that each cluster would be activated if the total quantity of Ca2+ released by the preceding cluster was above a certain threshold. Although this threshold was somewhat arbitrary, its absolute level could be varied over a sizeable range without affecting the main conclusions (see supplementary Fig. S1). To calculate the probability of propagation failure, we used the binomial distribution to compute the probability that at least one cluster out of 50 released less Ca2+ than the threshold amount. For instance, if N channels are present in each cluster, and p per cent of channels are inhibited by a drug, then the probability P that exactly k channels are inhibited is:

  • image

After calculating P for all values of k, we computed the probability that the inhibition of one or more clusters out of 50 was sufficient to decrease the quantity of Ca2+ released below the threshold level.

Statistics

All data are presented as means ± standard error of the mean (s.e.m.) with the number of myocytes examined as n. All the results presented were statistically significant at P < 0.05 using Student's t test or one-way ANOVA.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix
  9. Supporting Information

The effects of RuR on wave propagation

Rabbit ventricular myocytes were permeabilised with β-escin and perfused with a mock intracellular solution containing a free [Ca2+] of approximately 600 nm. This level of Ca2+ induced spontaneous Ca2+ waves with a frequency of ∼0.4 Hz for periods in excess of 15 min (data not shown). This level was chosen to produce regular Ca2+ waves every 2–3 s to allow accurate resolution of the time taken for RuR to inhibit Ca2+ waves. Lower perfusing [Ca2+] would reduce the wave frequency and thereby reduce time resolution. Higher perfusing [Ca2+] would produce higher frequency waves and better time resolution but a higher background fluorescence. As shown in Fig. 1, addition of 4 μm RuR to the perfusate caused inhibition of Ca2+ waves within 4 min. Figure 1A demonstrates the mode of wave inhibition routinely observed on application of 4 μm RuR. Initially, regular Ca2+ waves were observed (Fig. 1A), with no apparent change in frequency or amplitude of the events. After ∼4 min, propagation was interrupted, after which no further globally propagating waves were observed. The increase in minimum Ca2+ that occurs after Ca2+ wave inhibition is due to the absence of an uptake phase that precedes the Ca2+-release phase of the wave. This transient period of rapid sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)-mediated Ca2+ uptake is sufficient to briefly reduce the [Ca2+] within the permeabilised preparation below the ambient [Ca2+].

image

Figure 1. Inhibition of Ca2+ waves by RuR The time course of RuR effects on wave propagation in permeabilised myocyte. Aa, linescan showing propagating Ca2+ waves during RuR application (upper). Red and blue F/Fo signals derived from sections indicated by the red and blue bars at either side of the image above (lower). RuR blockade of RyR causes propagation to fail, followed by total failure of initiation event. Ac and d, linescans and associate F/Fo from grey bars indicated in regions i and ii of Aa. B, time course of wave inhibition by RuR.

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When a higher concentration of RuR (15 μm) was used, propagating Ca2+ waves were abolished in all cells within 1 min. Figure 1B summarises this relationship, showing that by 2 min of continuous perfusion with 4 μm RuR, Ca2+ waves were blocked in 50% of cells, by 3 min waves were blocked in ∼85% of cells, and after 4 min propagation was blocked in 100% of cells. Prior to the moment of RuR-induced inhibition of the Ca2+ waves, the velocity and amplitude of these events were similar to control values. Comparing the velocity and amplitude of the last three Ca2+ waves prior to RuR addition with the last three waves prior to RuR-induced wave block, no significant differences were evident (velocity: 102.2 ± 4.19 vs. 94.6 ± 12.55 μms−1 and amplitude (ΔF/Fo): 3.28 ± 0.19 vs. 3.37 ± 0.31). Therefore there was no evidence of progressive effects before RuR-induced block.

By applying caffeine (10 mm) rapidly via a glass pipette at the end of the cell, distal to the perfusion inflow, wave propagation was successfully reinstated temporarily (Fig. 2A). The local SR Ca2+ release caused by caffeine triggered a propagating Ca2+ wave in the remainder of the cell. The first wave initiated in this way propagated with a higher velocity (148.0 ± 16.3% of control, n= 5, P < 0.05) but was of similar amplitude (115 ± 26%). This observation, seen in all cells studied, indicated that RuR rapidly blocked the initiation of Ca2+ waves, prior to preventing longitudinal propagation.

image

Figure 2. Caffeine responses after RuR inhibition Local caffeine application allows reinitiation of propagating Ca2+ waves. A, linescan and F/Fo showing propagation block, then local caffeine application (black arrows) reinitiates the propagated release. Ba, a more detailed view of region i from A, showing the first 2 propagating waves initiated by caffeine. Bb, region ii from A showing failure of caffeine-initiated propagation events, but still allowing SR Ca2+ release. C, Ca2+ measurement with SR loaded Fluo-5N-AM during Ca2+ waves. a, SR [Ca2+] rise observed expressed as F/Fo,cs followed by caffeine induced Ca2+ depletion. RuR applied 10s after beginning of trace. b, mean F/Fo,cs from 8 cells.

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To determine whether RuR abolished Ca2+ waves by reducing SR [Ca2+], cells were loaded with the low affinity indicator Fluo-5N and perfused after permeabilisation with the same solutions as used in the measurements shown in Fig. 2A and B. On addition of RuR, the averaged Fluo-5N signal increased over the initial 2–4 min (Fig. 2C). At the end of 4 min, when Ca2+ waves had been inhibited in 100% of cells (Fig. 1B) SR [Ca2+] was 116 ± 2% of control (n= 8, P < 0.05).

The effects of RuR on Ca2+ sparks

Figure 3A shows that for a considerable time after cessation of Ca2+ waves, Ca2+ sparks still occurred at a frequency comparable to that seen in control cells (∼7.2 sparks (100 μm−1 s−1). In separate experiments, permeabilised cells were exposed to the same free [Ca2+] (∼600 nm), but with propagated release prevented by increasing cytoplasmic Ca2+ buffering (350 μm EGTA). Figure 3B shows representative traces before and at various times following RuR application, illustrating that the number of events progressively decreased during perfusion with RuR. Figure 3Ca and b shows average profiles of sparks recorded at different time points indicating no major changes in amplitude and width despite a decrease in frequency. Figure 3Da shows mean data from all cells studied (n= 18 cells from 4 hearts). Under control conditions in the absence of RuR Ca2+ spark frequency was 9.1 ± 1.2 sparks (100 μm)−1 s−1, and this value was obtained after subtracting the ‘false events’ detected after caffeine/thapsigargin treatment (1.2 ± 0.33 sparks (100 μm)−1 s−1). After 2 min, Ca2+ sparks occurred with a similar frequency to control (87.1 ± 7.0%). By 4 min of RuR perfusion, spark frequency had fallen significantly (to 39.8 ± 9.5%, P < 0.05) and finally at 10 min, spark activity was not significantly different from zero (1.4 ± 6.8% of the control rate). Figure 3Dc shows that no significant changes in spark amplitude or duration, and only a small change in spark width, were observed as the number of detectable sparks decreased. This is in contrast to earlier work measuring Ca2+ sparks at ∼100 nm where spark amplitude decreased substantially in 5 μm RuR (Lukyanenko et al. 2000). As with the Ca2+ wave protocol, cells were loaded with Fluo-5N to assess SR [Ca2+] during RuR exposure in a parallel set of measurements using identical solutions. The mean (n= 8 cells) is shown in Fig. 3Db. Fluorescence increased gradually to 114.2 ± 4.0% (P < 0.05 n= 8) over a 5 min period, indicating an increase in SR content similar to that observed during the Ca2+ wave protocol. To check that the SR load was comparable under these two conditions (600 nm Ca2+ in the presence of 50 or 350 μm EGTA), caffeine induce Ca2+ release was measured in the absence of RuR. Using the cytoplasmic Fluo-5F signal and allowing for the extra cytoplasmic buffering due to EGTA (MacQuaide et al. 2009), SR content was 192 ± 17 μm (l cell volume)−1 (n= 7) in the presence of 50 μm EGTA and 205 ± 32 μm (l cell volume)−1 (n= 5) in the presence of 350 μm EGTA. This indicates that there were no major differences in SR Ca2+ content in the two experimental groups.

Comparison of Ca2+ wave and Ca2+ spark inhibition

To illustrate the differences in time course of the inhibitory action of RuR on Ca2+ waves and Ca2+ sparks, the relative changes in wave incidence and Ca2+ spark frequency for each 1 min interval are plotted in Fig. 4. The data indicated that, prior to changes in Ca2+ spark frequency, RuR was able to significantly reduce the incidence of Ca2+ waves. This suggests that the mode of action of the RuR-induced block of Ca2+ waves was not simply via inhibition of Ca2+ sparks. In parallel studies, the relationship between Ca2+ sparks and Ca2+ waves was determined using other methods of blocking Ca2+ waves: (i) reduced RyR sensitivity using tetracaine and (ii) SERCA inhibition via tetrabutylquinone (TBQ). Each of these interventions produced roughly parallel changes in Ca2+ waves and Ca2+ sparks, in marked contrast to the highly non-linear relationship observed with RuR.

image

Figure 4. Relationship between wave occurrence and spark frequency measured after inhibition of RyR or SERCA Parallel experiments with 600 nm free Ca2+, for spark and wave experiments, with respective low and high EGTA concentrations (see Methods). Experiments with inclusion of TBQ (open symbols), tetracaine (filled grey symbols) and RuR (filled black symbols) are plotted as spark frequency vs. no. of cells exhibiting waves. In both cases the measurements represent steady state effects of both TBQ and tetracaine.

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Computational modelling of Ca2+ wave propagation

Computational modelling was used to explore potential mechanisms underlying the differential effects of RuR on Ca2+ sparks and waves. The simulations generated predictions of how altering the number of RyRs in each cluster affected Ca2+ spark amplitude and the probability of wave propagation. Given the assumption that RuR inhibition makes channels essentially inactive, altering the cluster size is equivalent to simulating different degrees of RuR inhibition. Figure 5B shows the predicted spark amplitude, a quantity closely related to the total Ca2+ released during the spark (see supplementary Fig. S3), versus the percentage of available RyRs, assuming that either 30 RyRs or five RyRs were present in the control cluster. This illustrates the relative insensitivity to RyR availability in the former case (30 RyRs) compared to the latter (5 RyRs), which occurs because the local SR Ca2+ depletion during sparks is faster and more complete when greater numbers of RyRs are present in the cluster.

image

Figure 5. Probabalistic model of propagating Ca2+ release A, illustration of the computational model of the propagation of a Ca2+ wave along arrays of 50 RyR clusters (R1–50), containing 30 (upper) or 5 (lower) RyRs per cluster. The model examined the effects of progressive block of RyRs; when the magnitude of release from one site fell below the threshold level, propagation failed. B, data from previously published model of Ca2+ release during a Ca2+ spark (Sobie et al. 2002); this formulation was used to describe the Ca2+ release at each site in the cluster. The graph shows model output of Ca2+ spark amplitude from clusters with 5 (blue) and 30 (red) RyRs. Arbitrary threshold set for proportion of cluster RyRs’ available Ca2+ activation required for propagation (indicated by the dotted line). C, model output showing percentage of cells with propagating waves, given a cluster size of 5 (blue) and 30 (red). Experimental data from Fig. 4 is superimposed for comparison.

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Additional calculations were performed to predict the effects of RuR block on the propagation of Ca2+ waves. Assuming that a 35% decrease in SR Ca2+ release in one RyR cluster prevented wave propagation (see Methods), Fig. 5C shows that when 30 RyRs per cluster is assumed, Ca2+ wave propagation remains close to control values until ∼60% of RyRs are blocked by RuR. In contrast, Ca2+ wave probability drops to zero with only moderate levels of RyR inhibition when each RyR cluster is assumed to contain only five channels. This demonstrates how cluster size can dramatically influence the predicted response to RyR blockade.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix
  9. Supporting Information

In this study, the RyR blocker RuR was used to compare and contrast the time course of inhibition of spontaneous Ca2+ waves with that of spontaneous Ca2+ sparks. The primary mode of block of RyR by RuR is a reduction in the overall availability of channels by inducing long closures (Rousseau & Meissner, 1989; Ashley & Williams, 1990; Lindsay & Williams, 1991; Xu et al. 1998; Lukyanenko et al. 2000). Thus on addition of RuR there are fewer RyRs available for activation within each cluster at any time. Perfusion with RuR leads to the rapid inhibition of Ca2+ waves despite the continued presence of Ca2+ sparks. When the incidence of Ca2+ waves was reduced to 50% of control values, Ca2+ spark characteristics were not significantly altered. When wave incidence was only ∼5% of control, spark frequency was decreased by only 40% (Ca2+ spark amplitude, duration and width were unchanged). These differences occurred despite similar levels of intra-SR [Ca2+] under the two conditions. This highlights a previously unreported discrepancy in the response of unitary and propagating events to this mode of blockade.

The Ca2+ wave is a consequence of a series of ‘fire–diffuse–fire’ events between clusters of RyRs after an initiating Ca2+ spark. Therefore the data indicate that the inhibition of Ca2+ waves by RuR is not caused by the absence of an initiating Ca2+ spark but rather interference with the ‘diffuse’ or subsequent triggering of a ‘fire’ event.

A possible explanation for the disparity between wave and spark activity comes from computational modelling of the behaviour of variable sizes of RyR clusters. RuR block of Ca2+ waves cannot be explained by the action of RuR on large clusters of RyRs (>30) that generate detectable Ca2+ sparks. Instead the sensitivity suggests the involvement of much smaller clusters of RyRs (<5 RyRs) than those thought to generate the archetypical Ca2+ sparks (Bridge et al. 1999; Franzini-Armstrong et al. 1998, 1999; Soeller et al. 2007). Thus non-spark RyR activity may be an essential component in the formation and propagation of Ca2+ waves.

Inhibition of Ca2+ sparks by RuR

The experimental protocols used in this study made use of the slow diffusion of RuR into myocytes, and consequently RyR activity became attenuated gradually over 5–10 min, consistent with a previous study (Lukyanenko et al. 2000). This gradual time course of block allowed fine resolution of its effects on Ca2+ sparks and Ca2+ waves. Spark frequency was reduced to ∼50% within 5 min of RuR exposure, falling to ∼1% of control after 10 min in the presence of 600 nm Ca2+ (350 μm EGTA). Ca2+ spark duration and width decreased with an even slower time course, while amplitude remained relatively constant. The changes are consistent with those observed in a previous study in the presence of ∼150 nm Ca2+ (100 μm EGTA) (Lukyanenko et al. 2000), with the exception of amplitude which showed a reduction to approximately one-third of control in this earlier study. Differences in bathing [Ca2+] and indicator (Fluo-3 vs. Fluo-5F) may be responsible for the disparity between the two studies.

In the present study, the consequence of the slow onset of effects of 4 μm RuR is that after 2 min of exposure to RuR, no significant changes in spark frequency, amplitude, width or duration were observed (i.e. changes were < 3%). Parallel measurements of SR Ca2+ indicated that during this time SR Ca2+ content increased. The SR Ca2+ level is a balance between uptake via SERCA and release via Ca2+ leak pathways, and thus a rise of SR Ca2+ (in the absence of SERCA stimulation) suggests a reduction in the flux through the Ca2+ leak pathways, yet no changes in Ca2+ sparks were evident. Two possible options to explain the lack of detectable changes in Ca2+ spark characteristics are:

  • 1
    Autoregulation. Inhibition of Ca2+ sparks would quickly lead to an increase in SR Ca2+ which would in turn lead to an increase in RyR activity and spark frequency back to normal via SR luminal regulation of RyR activity. In the steady state, SR Ca2+ efflux would return to normal in the presence of a higher SR Ca2+. This type of autoregulation has been previously used to explain the biphasic action of the drug tetracaine (Gyorke et al. 1997). On addition of tetracaine, spark frequency decreased for 1–2 min before returning to the control rate over the subsequent 5 min in parallel with a rise in SR Ca2+ levels. Given this time course, it seems unlikely that autoregulation of Ca2+ sparks occurs in the current study since even over a period of 1 min, no detectable changes in Ca2+ spark activity were observed, and therefore this was not the mechanism underlying increased SR Ca2+ levels.
  • 2
    Sub-sparks. The sparks detected may represent the largest of a range of spark size. RuR might inhibit a subpopulation of smaller sparks/release events that could not be detected above the background fluorescence signal but contribute to SR leak. If these undetectable sub-sparks are crucial to Ca2+ wave formation, their inhibition by RuR may explain the poor correlation between Ca2+ wave events and detectable Ca2+ sparks. In a recent study (Zima & Blatter, 2009), SR Ca2+ efflux was proposed to occur via three distinct pathways: (i) Ca2+ sparks via RyR clusters, (ii) a non-spark pathway mediated via RyR, and (iii) a non-spark pathway not mediated via RyR. The exact molecular identities for (ii) and (iii) are not certain, but the increase in SR Ca2+ content in the absence of changes in Ca2+ spark characteristics observed in the current study could be explained in terms of inhibition of a non-spark, RyR-mediated pathway.

Interruption of Ca2+ wave propagation by RuR

Superfusion with RuR caused a rapid inhibition of Ca2+ waves without obvious changes in wave velocity, amplitude or frequency. This contrasts with other manipulations known to inhibit Ca2+ waves (Gyorke et al. 1997; Trafford et al. 2000; Smith & O’Neill, 2001; MacQuaide et al. 2007). In these cases, tetracaine caused an initial slowing of wave propagation before complete block. In the continued presence of tetracaine, Ca2+ waves returned, in parallel with a rise in SR Ca2+ content (Gyorke et al. 1997). In contrast, the immediate and sustained inhibitory effect of RuR without inhibition of Ca2+ sparks suggests interference with the events initiating a Ca2+ wave, i.e. the ‘diffuse’ or ‘fire’ events associated with propagation of a Ca2+ wave. Rapid local application of caffeine after complete inhibition of Ca2+ waves nonetheless caused a large release of Ca2+ from the SR. This provides further evidence that the mechanism of Ca2+ wave inhibition by RuR is more complicated than the drug simply making all RyRs inactive. Initially, the local release was able to propagate along the length of the cell at wave velocities comparable to the Ca2+ waves prior to RuR addition, but within 5–10 s this propagated release was inhibited. Why RuR should block wave initiation before propagation is unclear; one possible explanation is provided by a modelling study that suggests that wave initiation may involve regions of the cell with marginally shorter sarcomere lengths, where initial longitudinal propagation can occur with a higher probability (Izu et al. 2006). RuR may be able to reduce the probability of propagation in these regions before propagation between clusters in regions of longer sarcomere length. Further work is required to investigate this issue.

Slowly propagating waves were not observed either just prior to block or during caffeine triggered wave production. One possible explanation is the concomitant increase of intra-SR Ca2+ during RuR perfusion. This will result in larger amplitude Ca2+ waves that would tend to propagate faster if no other changes were taking place. Thus the absence of significant changes in velocity may result from two opposing effects, RuR-induced inhibition of RyR activity that slows the last one to two wave events and increased SR load that tends to produce larger Ca2+ release events. Unfortunately, the poor sensitivity of Fluo-5F signals to the [Ca2+] reached at the peak of the wave prevents significant changes in Ca2+ wave amplitude being easily detected.

RuR is also an effective inhibitor of the mitochondrial uniporter, and therefore parallel inhibition of mitochondrial Ca2+ uptake must occur on addition of RuR. This action does not underlie the inhibition of Ca2+ waves in the present study because: (i) similar effects of RuR were observed when Ca2+ waves were initiated in the presence of mitochondrial inhibitors (1 μm FCCP, 1 μm oligomycin, data not shown) and (ii) rapid application of 10 μm Ru360, a drug with similar effects on the mitochondrial uniporter but no effects on RyR, did not block Ca2+ waves (data not shown).

Differential sensitivity of sparks and waves

When the frequency of Ca2+ sparks at particular times after addition of RuR was plotted against the incidence of Ca2+ waves at the same time points, a highly non-linear relationship was observed (Fig. 4). This was in marked contrast to the relationships observed in the steady state (8–10 min) with increasing concentrations of the RyR inhibitor tetracaine or the SERCA inhibitor TBQ. In either the former case, with progressive reduction in RyR sensitivity, or the latter, with progressive SR Ca2+ pump activity, the inhibition of Ca2+ waves paralleled a decrease in Ca2+ spark frequency. In particular, in the presence of tetracaine, Ca2+ waves remained despite a considerable decrease in Ca2+ spark frequency. This seems in direct contrast to the effects of RuR, but the action of the two inhibitors on RyR are quite different. Previous work has shown that tetracaine reduces the open probability of RyR, but this effect can be reversed by an increase in SR Ca2+ (Gyorke et al. 1997). Therefore in the steady state, RyR activity was restored, albeit at a higher SR [Ca2+]. No such competitive inhibition has been shown for RuR. The reduced incidence of Ca2+ waves and Ca2+ sparks in tetracaine reflects the longer period of Ca2+ uptake required after both events before sufficiently high SR Ca2+ is reached to activate release. The raised SR Ca2+ and restoration of the activity of RyR will apply to Ca2+ release both at the major clusters involved in spark activity and any sub-spark units and this may explain why Ca2+ waves persist on perfusion of tetracaine.

Previous studies using SERCA inhibitors reported a lowered frequency and eventual inhibition of Ca2+ waves (O’Neill et al. 2004) and sparks (Lukyanenko et al. 2000); the present study reproduced these data and showed that under identical conditions, spark frequency and the proportion of cells exhibiting Ca2+ waves decreased in parallel. This is consistent with the concept that SR Ca2+ levels are a major factor in the triggering of both events; reduced SERCA activity will increase the time necessary for SR [Ca2+] to reach a threshold level. Lower SERCA activity has also been reported to reduce the threshold for Ca2+ release (O’Neill et al. 2004), and therefore the net effect of SERCA inhibition will depend on the balance of both these factors.

Rogue RyRs

One mechanism which could explain the disparity between Ca2+ wave and Ca2+ spark inhibition by RuR is the involvement of ‘extra-dyadic’ or ‘rogue’ RyRs as an SR Ca2+ leak pathway as proposed by several groups (Sobie et al. 2006; Lukyanenko et al. 2007; Hayashi et al. 2009). The existence of smaller clusters which do not cause detectable spark events yet may substantially contribute to diastolic leak has been suggested (Sobie et al. 2006; Zima & Blatter, 2009). While not influencing the Ca2+ spark activity, these smaller clusters may act to facilitate the propagation of the Ca2+ signal between the large clusters of RyR at the dyad. A simple computational model of Ca2+ wave propagation, based on a previously published description of the cardiac RyR cluster (Sobie et al. 2002), was constructed to examine the relationship between cluster size and the extent of inhibition of SR Ca2+ release. The simulations showed that inhibition of large (30 RyR) clusters hardly affected spark size, whereas the smaller (5 RyR) clusters were much more sensitive to inhibition. Because depletion of local SR [Ca2+] is faster and more complete with large RyR clusters, inhibition of a few RyRs in this case has little effect on either the total quantity of Ca2+ released or the Ca2+ spark amplitude. This agrees with the relatively small changes in spark size detected in our experiments. This suggests that cardiac cells contain both large and small RyR clusters. Most of the release events identifiable as Ca2+ sparks result from the coordinated opening of relatively large RyR clusters, and RuR inhibition of these ryanodine receptors can reduce Ca2+ spark frequency while barely affecting spark amplitude (e.g. at 3 min in Fig. 3C). However, Ca2+ wave propagation may also require the participation of small clusters of only a few RyRs. Ca2+ release events produced by these small clusters may be too small to be detected and therefore contribute an ‘invisible’ SR Ca2+ leak (Sobie et al. 2006). Their small size, however, makes them more susceptible to moderate degrees of inhibition by RuR, for two reasons. (i) The relationship between total Ca2+ released and RyR availability (Fig. 5B) is non-linear for large clusters, which implies that with 30 RyRs per cluster, a larger percentage of channels need to be inhibited to reduce released Ca2+ below a given threshold level. (ii) In addition, simple probabilistic arguments make small RyR clusters more likely to be blocked even when the overall percentage of channels inhibited by RuR is small. This mechanism does not require that rogue RyRs be more sensitive to RuR block than channels in larger clusters. An alternative explanation is that differences in phosphorylation state or regulation by the calsequestrin/triadin/junctin complex may make the rogue RyRs more susceptible to inhibition by RuR. Further work is required to distinguish these possiblities.

In summary, this study is the first to demonstrate a dramatically different time course of inhibition of Ca2+ wave and Ca2+ spark events in ventricular myocytes. RuR increased SR Ca2+ levels consistent with a block of SR Ca2+ leak, but no significant change in Ca2+ spark size or frequency was observed. Over a similar time period the incidence of Ca2+ waves was dramatically decreased. These data suggest that another non-spark Ca2+ leak pathway may be blocked by RuR, and this leak pathway is important in the mechanisms that allow Ca2+ sparks to initiate and propagate a Ca2+ wave. The occurrence of Ca2+ waves during diastole has been linked to the generation of delayed after-depolarisation and subsequent arrhythmias. Our results suggest that there may be a pharmacological strategy that can inhibit the initiation and propagation of these events without dramatically affecting the activity of Ca2+ release from the larger clusters of RyRs in the dyad that is essential for E–C coupling.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix
  9. Supporting Information

Appendix

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix
  9. Supporting Information

Author contributions

N.M., experimental work, data analysis and writing. H.E.R. computational modelling. E.S. computational modelling and writing. G.L.S. experimental design and writing.

Acknowledgements

The authors acknowledge the financial support of the British Heart Foundation (RG/04/07) to GLS and the National Institutes of Health (HL076230) to EAS.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix
  9. Supporting Information

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

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