Ryanodine Receptor Current Amplitude Controls Ca²⁺ Sparks in Cardiac Muscle

Abstract

Rationale

In cardiac muscle, Ca²⁺ induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) is mediated by ryanodine receptor (RyR) Ca²⁺ release channels. The inherent positive feedback of CICR is normally well controlled. Understanding this control mechanism is a priority because its malfunction has life-threatening consequences.

Objective

Show that CICR local control is governed by SR Ca²⁺ load largely because load determines the single RyR current amplitude that drives inter-RyR CICR.

Methods and Results

We differentially manipulated single RyR Ca²⁺ flux amplitude and SR Ca²⁺ load in permeabilized ventricular myocytes as an endogenous cell biology model of the heart. Large RyR-permeable organic cations were used to interfere with Ca²⁺ conductance through the open RyR pore. Single-channel studies show this attenuates current amplitude without altering other aspects of RyR function. In cells, the same experimental maneuver increased resting SR Ca²⁺ load. Despite the increased load, Ca²⁺ spark (inter-RyR CICR events) frequency decreased and sparks terminated earlier.

Conclusion

Spark local control follows single RyR current amplitude, not SR Ca²⁺ load per se. Spark frequency increases with load because spontaneous RyR openings at high loads produce larger currents (i.e. a larger CICR trigger signal). Sparks terminate when load falls to the point where single RyR current amplitude is no longer sufficient to sustain inter-RyR CICR. Thus, RyRs that spontaneously close no longer re-open and local Ca²⁺ release ends.

INTRODUCTION

In cardiac muscle, Ca²⁺ influx through surface membrane Ca²⁺ channels activates underlying RyR channels. This form of CICR is the basis of cardiac excitation-contraction coupling (ECC). In resting cells, single RyR channels spontaneously open with a very low frequency. The Ca²⁺ released by one of these openings may activate neighboring RyR channels. This inter-RyR CICR occurs within a cluster of RyR at discrete SR Ca²⁺ release sites and generates spontaneous non-propagating Ca²⁺ sparks. The Ca²⁺ released at one release site (if large enough) may activate RyRs at a neighboring release site. This inter-site CICR generates propagating Ca²⁺ waves that can lead to arrhythmia and even sudden death. This study defines the control of localized inter-RyR CICR (sparks) in permeabilized cardiac ventricular myocytes. Permeabilized cells are used as an endogenous cell biology model of the heart, to allow precise experimental manipulation of the cytosol and to eliminate Ca²⁺ influx through surface membrane Ca²⁺ channels (i.e. ECC-based CICR).

A long-standing unknown in spark local control is the mechanism that terminates inter-RyR CICR. Intuitively, CICR should operate with “explosive” positive feedback (released Ca²⁺ triggering further release until the SR Ca²⁺ store is empty). This does not happen in cells. Instead, local inter-RyR CICR events (sparks) terminate when SR Ca²⁺ load falls to a critical termination threshold¹, not when the SR is empty. Elevating SR Ca²⁺ load above normal levels dramatically increases spark frequency and the likelihood of arrhythmogenic Ca²⁺ waves². This modulation of sparks by SR Ca²⁺ load is commonly attributed to intra-SR (luminal) Ca²⁺ acting on luminal RyR Ca²⁺ regulatory sites^2–4. Single RyR Ca²⁺ current amplitude, however, varies proportionally to SR Ca²⁺ load and this current is what drives inter-RyR CICR^5–8. The regulatory contribution of RyR current amplitude is largely unexplored because there has been no means to differentially manipulate SR Ca²⁺ load and single RyR current in cells. Here, we use large RyR-permeable organic cations to decrease single RyR current without reducing resting SR Ca²⁺ load. Our results indicate that local SR Ca²⁺ load control of inter-RyR CICR is predominantly governed by the current, not by intra-SR Ca²⁺ acting on intra-SR sites.

METHODS

Single RyR channel function was recorded in planar lipid bilayers⁹ and Ca²⁺ sparks monitored in acutely dissociated myocytes (additional details in supplemental material).

Heavy SR microsomes were prepared from rat ventricular muscle using established methods¹⁰. These microsomes were fused into planar lipid bilayers composed of a 5:4:1 mixture of phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine, respectively. Once RyR channel activity was observed, solutions in both compartments were changed as specified in the text or figure legends. Many single RyR recordings were made in cell-like salt solutions. The cytosolic cell-like salt solution contained 120 mM K⁺, 0.1–10 μM free Ca²⁺, 1 mM free Mg²⁺ and 5 mM total ATP. The luminal cell-like salt solution contained 120 mM K⁺, 1 mM free Mg²⁺ and 0.1–1 mM free Ca²⁺. All recordings were done at room temperature with current sampled at 50 ms/pt, filtered at 1 kHz and analyzed using pCLAMP9 software (Molecular Devices, Sunnyvale, CA). No correction for missing events was made.

Sparks were recorded in single saponin-permeabilized rabbit ventricular myocytes using a fluorescent cytosolic Ca²⁺ indicator (Fluo-4). Permeabilized cells were placed in a standard recording solution containing 120 mM K-aspartate, 5 mM MgATP, 0.4 mM EGTA, 10 mM phosphocreatine, 10 mM HEPES, 5 U/ml creatine phosphokinase and pH 7.25. Cytosolic free Ca²⁺ and Mg²⁺ were 150 nM and 1mM, respectively. Spark studies were done at room temperature with Fluo-4 excited by 488 nm light and its emission monitored at >515 nm. Sparks were detected and analyzed using the SparkMaster program¹¹ with a detection threshold of 3.8. SR Ca²⁺ load was assessed by peak caffeine-evoked Ca²⁺ release or intra-SR Fluo-5N fluorescence following standard methods^12,13. Fluo-5N was excited at 488 nm and its emission measured at 500–530 nm.

Summary results are presented as mean±SEM of several individual determinations. Statistical comparisons were performed using the Student’s t-test (unpaired, p < 0.05).

RESULTS

The RyR is regulated by cytosolic Ca²⁺, Mg²⁺ and ATP as well as luminal Ca²⁺ in cells⁹. Single RyR channel function is defined here with all these important agents present at cell relevant concentrations. In these cell-like salt solutions, the RyRs cytosolic Ca²⁺ EC₅₀ is ~10 μM and sample RyR recordings with 10 μM cytosolic free Ca²⁺ (1 mM luminal Ca²⁺) are shown in Figure 1A. Single RyR channels repeatedly open (upward) and close. The open probability (Po) and mean open time (MOT) were not membrane potential dependent (Online Figure Ia) and thus this likely reflects RyR function at 0 mV (the resting SR potential in cells^14,15). Sample RyR recordings after application of cytosolic 60 mM tris(hydroxymethyl)aminomethane (Tris⁺) are also shown in Figure 1A. With Tris⁺ present, event frequency and MOT were the same (Online Figure Ia,b) but current amplitude was clearly smaller. Figure 1B shows all-points histograms showing that Tris⁺ clearly shifts the open current peak (labeled) but does not substantially change its area. Figure 1C shows RyR Po before and after Tris⁺ application when 10 μM cytosolic free Ca²⁺ is present. The Po was not significantly different with 0, 30, 60 or 120 mM cytosolic Tris⁺ present. Figure 1C also shows Po after luminal Ca²⁺ was reduced from 1 to 0.1 mM (open square) and following caffeine application (open circle). Reduced luminal Ca²⁺ did not significantly change RyR Po while caffeine potentiated it significantly. Online Table II (supplementary material) compares RyR Po and mean open time before and after Tris⁺ application with 1 or 0.1 μM cytosolic free Ca²⁺ present. All these data show that Tris⁺ reduces current amplitude without altering RyR gating in these cell-like salt solutions.

FIGURE 1.

Single cardiac RyR channel function. A. Single RyR recordings (30 mV) in our cell-like salt solutions without (control) and then with 60 mM cytosolic Tris⁺ present. Filtered at 750 Hz for presentation here. Open events upward. B. Representative all-points histograms from 4 minute recordings in cell-like salt solutions during control (filled) and after Tris⁺ (60 mM; open bars). Labeled current peaks are each ~50% of the total area. C. Single RyR open probability plotted vs. Tris⁺ concentration. Filled circles (n=5) were collected in our cell-like salt solutions. Open circle (n=5) is after adding 10 mM caffeine. Open square (n=8) is with 0.1 mM luminal Ca (instead of 1 mM). Values were statistically compared to control (n.s. is not significant; ** p<0.005). D. Single RyR conductance (filled squares; n=5) vs. Tris⁺ concentration. Values statistically compared to control (*** p<0.0001). Inset shows single RyR current-voltage (I–V) plots in our cell-like salt solutions in control (open squares; n=9) and with 30 (triangles) or 120 mM (open circles) Tris⁺ present. Current values determined from all-points histograms. Conductances determined from I–V plot (using wider voltage range than shown). Control conductance is 188 pS (reversal −2.4 mV). Conductances in 30 and 120 mM cytosolic Tris⁺ are 145 pS and 100 pS, respectively. Small filled circles (110 pS) represent data values collected in 120 mM luminal Tris⁺ (none in cytosol). E. Average dwell time histograms generated from long recordings (4–6 minutes) on 5 different RyR channels in our cell-like salt solutions with and without 60 mM Tris⁺ present. Open circles are open times without Tris⁺ and were well fit (R²>0.99) by 2 exponential components with time constants of 1.2±0.2 ms (88% of events) and 9.1±2.8 ms (12%). Filled circles are closed times without Tris⁺ (fit time constants; 1.5±0.4 ms & 8.4±1.7 ms). Black solid line is the fit of open times (data points not shown) with Tris⁺ present (fit time constant; 1.3±0.2 ms & 10.1±2.0 ms). White line is the fit of closed times (data points not shown) with Tris⁺ present (fit time constants; 1.6±0.3 ms & 11.2±1.9 ms). Arithmetic mean open time (MOT) without Tris⁺ was 8.5 ms (black arrow) and 8.2 ms with Tris⁺. Open arrow indicates average spark time-to-peak (TTP). Dashed line represents open times without Tris⁺ at a 10 fold lower cytosolic free Ca²⁺ level (i.e. 1 μM).

The attenuation of single RyR conductance by cytosolic Tris⁺ concentration is shown in Figure 1D. Conductance is significantly reduced when cytosolic Tris⁺ was >30 mM. Figure 1D (inset) plots average single RyR current amplitude (n=9) as a function of voltage in the cell-like salt solutions. In the absence of Tris⁺ (open squares), the slope conductance is 188 pS and the reversal potential is −2.4 mV. In cells, the SR membrane potential (Vm) is thought to be held near 0 mV by the SR’s high resting K⁺ permeability¹⁴. Calcium is the only RyR-permeable ion present with a trans-SR electrochemical driving force at 0 mV (the resting SR potential in cells^14,15). Thus, the 0.35 pA at 0 mV is entirely Ca²⁺ current and very similar to the RyR unit Ca²⁺ current predicted in similar cell-like condition elsewhere^16,17. Conductance is decreased to 145 and 100 pS after 30 or 120 mM cytosolic Tris⁺ is added (Fig 1D, inset). Since the SR potential rarely will stray far from 0 mV in cells¹³, only currents between +20 mV and -20 mV are shown and these vary linearly with voltage within this narrow range. Cytosolic or luminal Tris⁺ had similar action (Fig. 1D; filled & open circles) indicating this Tris⁺ action on current is not sided. The conductance of these large monovalent cations¹⁸ is typically very small (<20 pS) compared to K⁺ (>450 pS). If multiple RyR-permeable cations are present, they will compete for occupancy of the RyR pore and influence each other’s permeation^15,19. In cell-like salt solutions, Ca²⁺ primarily competes with Mg²⁺ and K⁺ for occupancy of the pore^15–17. A robust model of multi-ion RyR permeation^15,19 was used to predict single RyR Ca²⁺ flux (no Tris⁺) in cell-like salt solutions as a function of SR Ca²⁺ load (Online Figure Ic). The lumen-to-cytosol Ca²⁺ flux decreases from about 0.35 pA to 0.19 pA when load drops from 1 to 0.5 mM (as it likely does during a spark^1,13). The predicted 46% decrease in Ca²⁺ flux during a spark is about the magnitude of the RyR conductance decrease caused by 120 mM Tris⁺ (Fig. 1D). Although the precise mechanism is unclear at this point, the significant point here is that Tris⁺ attenuates RyR ion permeation without affecting RyR Po or MOT.

Single RyR MOT in cell-like salt solutions is indicated on the plot of open and closed dwell times shown in Figure 1E (arrow). The dwell times shown were measured from 5 different channels without (symbols) and with Tris⁺ (60 mM; solid lines) present. The dashed line represents open times in our cell-like salt solution with 1 instead of 10μM cytosolic Ca²⁺. Average spark time-to-peak (TTP) in our permeabilized cells is also indicated by the white arrow. With 10μM cytosolic Ca²⁺ present (like present around RyRs during a spark), greater than 96% of measured single RyR openings are shorter than spark TTP. This implies that an individual RyR likely opens and closes repeatedly during the rise time of a spark. If so, then spark termination could be due to a mechanism that limits RyR re-opening, not necessarily only one that drives RyR to close.

Spontaneous sparks were measured in saponin-permeabilized myocytes. The resting cytosolic salt composition was similar to that used in our single RyR studies. The exception is that it contained 150 nM free Ca²⁺ which supported a resting spark frequency of 12.6 ± 1.1 sparks (100 μm)⁻¹s⁻¹. Figure 2A shows representative line scan images of sparks before (control) and after the application of 60 mM cytosolic Tris⁺. The Tris⁺ dramatically reduced spark brightness (amplitude) and frequency. This action was readily reversible. It was present within 1 minute of Tris⁺ application and gone within 1 minute of its removal (Online Figure IIIa). The addition of an alternative K⁺ permeability pathway (valinomycin) to the SR had no effect on spark frequency with or without Tris⁺ present (Online Figure Ie) indicating the action of Tris⁺ on sparks is not due to a shift in SR membrane potential. Figure 2B shows that other large organic cations (L-lysine or triethanolamine) had a similar action as Tris⁺ on spark frequency and amplitude. Sparks were not altered when large uncharged organic molecules (e.g. glutamine) applied. Sample line scan images with these large organic cations present are shown in the supplementary material (Online Figure II). Thus, the spark alteration was not Tris⁺ specific but instead occurred when any large poorly RyR-permeable cation was added.

FIGURE 2.

Spontaneous sparks in permeabilized acutely dissociated ventricular myocytes. A. Line scan images before (control) and after 60 mM cytosolic Tris⁺ application. Cytosolic solution contained 150 nM free Ca²⁺. B. The large RyR-permeable organic cations (120 mM) Tris⁺, L-lysine and triethanolamine significantly reduce spark frequency (n=3 to 11) and amplitude (n=57 to 3580). Values were statistically compared to control (** p<0.005 & *** p<0.0001). A similar molecular weight neutral amino acid, Glutamine (120 mM), had not affect. C. Average spark waveforms before (black) and after (red) 60 mM Tris⁺ application. These waveforms were generated by temporally aligning (to peak) 176 control and 154 Tris⁺ (60 mM) sparks collected from 10 different cells. Only sparks within the top 25% brightest events were analyzed here. Bar graphs (mean±SEM) show the average spark decay time constant and spark time-to-peak (TTP) with 0 (control), 60 or 96 mM cytosolic Tris⁺ present. Values represent the means (±SEM) of 3574, 154 and 46 individual sparks (compared to control; n.s. means not significant, * p<0.05). D. Cumulative histograms (%) of spark TTP with 0 (white) or 96 mM (red) Tris⁺. Histograms were fit by Hill equation (50% values = 16.1 and 11.6 ms; significantly different by F-test, p<0.0001). Inset shows raw TTP distributions (normalized to peak). E. Intra-SR Ca²⁺ load (determined by Fluo-5N fluorescence) plotted as a function of time before (black circles; n=8) and after 120 mM cytosolic Tris⁺ application (red circles; n=7). Data were normalized to load at 0 minutes. Load immediately before and after Tris⁺ application are 0.95±0.14 and 1.06±0.19 (not significantly different, p=0.22). Lines were fit to the load values before and after Tris⁺ (not shown; slopes were not significantly different but initial load in Tris⁺ was significantly higher by 6%,p<0.043).

Numerous (>150) in-focus control or Tris-modified sparks were collected and temporally aligned (to their peak) to generate the average spark waveforms shown in Figure 2C (left). Average spark amplitude is about half its normal value with 60 mM Tris⁺ present. The action of Tris⁺ on spark time-to-peak (TTP) and decay time constant is presented in Figure 2C (right). Average spark decay rate was not Tris⁺ sensitive. This is expected since spark decay (local Ca²⁺ removal) is largely due to diffusion. Average spark TTP was significantly shorter at high Tris⁺ levels. The Tris^+-evoked change in spark TTP is also evident in the probability histograms presented in Figure 2D. The spark peak is the moment local Ca²⁺ release no longer out-paces local Ca²⁺ removal (diffusion). Thus, the shorter TTP implies local Ca²⁺ release ends (or terminates) sooner when Tris⁺ is present. Spark termination occurs when local SR Ca²⁺ load falls to a critical level^1,20 and this is thought to be due to intra-SR Ca²⁺ acting at intra-SR RyR Ca²⁺ regulatory sites. With Tris⁺ present, however, local intra-SR Ca²⁺ load should fall less because Tris⁺ limits single RyR current. This is evident by the decline in spark mass at high cytosolic Tris⁺ levels (Online Figure IIId). Spark mass is roughly proportional to the decrease in local load during a spark. If termination were driven by Ca²⁺ acting on intra-SR RyR regulatory sites, a smaller decline in local load would drive termination less efficiently (i.e. lengthen, not shorten, TTP). Thus, the shorter TTP in Tris⁺ suggests that a different mechanism explains the luminal Ca²⁺ sensitivity of spark termination.

The cell-wide resting SR Ca²⁺ load is established by the balance between SR Ca²⁺ uptake and resting SR Ca²⁺ leak. Any form of RyR block should reduce resting leak and thus elevate load. We evaluated the action of Tris⁺ on cell-wide SR Ca²⁺ load 3 different ways. First, SR Ca²⁺ load was directly measured using intra-SR Fluo-5N. Figure 2E shows that this acute cytosolic Tris⁺ application significantly increased Fluo-5N fluorescence (load) by 6%. Second, peak caffeine-evoked Ca²⁺ release peak was used to assess resting SR load (Online Figure IIIb). Five minutes after Tris⁺ application peak caffeine-evoked release was also significantly increased, indicating an elevation in resting load. Third, SR Ca²⁺ release waves can also be used as an indicator of SR Ca²⁺ overload^2,20. Cytosolic Ca²⁺ levels >250 nM normally result in substantial SR Ca²⁺ overload and frequent Ca²⁺ waves. Figure 3A shows that no waves are observed at increasing cytosolic free Ca²⁺ levels (images 1–4) up to 500 nM if 120 mM Tris⁺ is present. Figure 3B illustrates how load (i.e. Fluo-5N fluorescence) varies with cytosolic free Ca²⁺ concentration. Note the Fluo-5N signal is nearly saturated 150 nM cytosolic Ca²⁺ and thus the small 6% rise in load observed in Fig. 2E could be limited by indicator saturation. In any event, no waves were observed at cytosolic Ca²⁺ levels >250 nM if Tris⁺ is present (Fig. 3A). However, waves immediately occurred when the Tris⁺ was removed (Fig 3A, image 5) indicating that resting load was indeed high when the Tris⁺ was present. This Tris⁺ block/unblock process was reversible. These data indicate that wave initiation required more than just SR Ca²⁺ overload. In other words, waves are not solely governed by intra-SR Ca²⁺ acting at intra-SR RyR Ca²⁺ regulatory sites as commonly thought. We propose that a different mechanism, one associated with RyR current amplitude, is likely involved.

FIGURE 3.

High SR Ca²⁺ load does not trigger Ca²⁺ waves when single RyR current amplitude is attenuated. A. Four sequential X-Y images (1–4) of intra-SR Fluo-5N fluorescence with 120 mM Tris⁺ present. Cytosolic free Ca²⁺ was incremented (10 to 500 nM), increasing the resting SR Ca²⁺ load. No Ca²⁺ waves occurred at high loads with Tris⁺ present. However, waves occurred immediately after Tris⁺ removal and simultaneous return to 150 nM cytosolic Ca²⁺ (image 5). Waves were detected by Rhod-2 fluorescence (excited 543 nm; emitted light measured at >600 nm). After a few minutes (image 6), SR load normalized and waves were no longer observed. B. Fluo-5N fluorescence (mean±S.D.; n=5 experiments) plotted as a function of cytosolic free Ca²⁺. Solid line is Hill equation fit (EC₅₀=42.6 nM). Dashed lines are 95% confidence levels. Fluo-5N is 93% saturated at 150 nM cytosolic Ca²⁺ suggesting small 6% rise in Fluo-5N signal in Fig. 2E was limited by saturation. C. Decline of SR load (Fluo-5N fluorescence) due to SR Ca²⁺ leak following 10 μM TG application. Black circles (n=6) is load decline without Tris⁺ present (fit by single exponential, time constant = 11.1 minutes). Grey circles (n=6) is load decline with 120 mM Tris⁺ (time consant = 17.1 minutes). Loads at the 10 and 20 minutes with and without Tris⁺ were statistically different (* p<0.05, ** p<0.005).

Cell-wide resting SR Ca²⁺ load can also be altered by blocking the SR Ca²⁺ pump with thapsigargin (TG). Figure 3C shows that after TG application (at 0 minutes) the resting load declines gradually as Ca²⁺ leaks out of the SR. The time constant of this decline was 11.1 minutes when no Tris⁺ was present and slowed to 17.1 minutes when 120 mM Tris⁺ was present. Resting loads were significantly higher at the 10 and 20 minutes in Tris⁺. The slowing of SR Ca²⁺ leak by Tris⁺ is consistent with the lower spark frequency in Tris⁺ (i.e. less spark-mediated leak). There is also a RyR-mediated non-spark component of SR Ca²⁺ leak^21,22. Since Tris⁺ will limit current carried by any open RyR, it likely reduced non-spark leak as well.

The effects of reducing resting SR load and limiting single RyR current amplitude on sparks are compared in Figure 4. Average spark amplitude, spatial width (FWHM), duration (FDHM) and frequency are plotted as a function of cytosolic Tris⁺ concentration in Figure 4A (filled circles). How Tris⁺ affects the probability distributions of spark amplitude, FWHM, FDHM and decay time constant are shown in Online Figure V (these are represented by open circles in Figure 4A). The simultaneous decrease in spark amplitude and width suggests sparks are generated by fewer RyRs as cytosolic Tris⁺ levels increase. Smaller sparks are more difficult to detect. A detection problem might explain the observed reduction in spark frequency. This is not the case, however, because substantial spark frequency changes occur well above our minimal spark detection level and our control spark frequency studies nicely reproduce published values (see Fig. 4B). In Figure 4A, higher cytosolic Tris⁺ levels reduced spark frequency. A single exponential was fit to these spark frequency data (solid line; decay constant = 38.4 mM). This nonlinear reduction in spark frequency suggests that spark initiation is less likely at higher Tris⁺ levels.

FIGURE 4.

Single RyR current dependence of Ca²⁺ sparks. A. Spark amplitude (F/Fo), full-width at half maximum (FWHM), full-duration at half maximum (FDHM) and Ca²⁺ spark frequency (CaSpF) are plotted as a function of cytosolic Tris⁺ concentration. Points (black circles) are means of numerous sparks at each Tris⁺ level. For example, control values (no Tris⁺) represent 3574 sparks from 94 different cells. Values at 60 and 96 mM Tris⁺ represent 154 sparks from 54 cells and 46 sparks from 18 cells, respectively. Cumulative histograms of spark amplitude, FWHM and FDHM were generated (see supplemental material) at 0, 60 and 96 mM cytosolic Tris⁺. The 50% values obtained from Hill fits of thsse histograms are indicated by the open circles. Statistical comparison of linear and single exponential fits to the CaSpF data (F-test) indicated these data are best fit by a single exponential (p<0.0001; thick solid line, decay constant = 38.4 mM). B. Spark frequency with 0 (triangles) or 60 mM (squares) Tris⁺ is plotted as function of resting SR Ca²⁺ load. Load was experimentally manipulated using low thapsigargin doses and monitored by Fluo-5N. Stars are the 60 mM Tris⁺ points scaled by a factor of 5.6. Dashed line represents the spark results previously published spark results of Zima²⁰.

The effect of reducing resting SR Ca²⁺ load on sparks (while the RyR current is allowed to vary accordingly) was recently demonstrated by another group²¹. This group blocked the SR Ca²⁺ pump with TG and measured spark properties as the resting SR Ca²⁺ load declined. Interestingly, the effects of limiting single RyR current amplitude with Tris⁺ (Fig. 4A; filled circles) and the published action of reducing resting SR load²¹ are very similar. Specifically, both manipulations reduce spark amplitude and FWHM almost linearly. They also have little effect on spark FDHM and dramatically decrease CaSpF in non-linear fashion. This is interesting because resting load in cells normally determines single RyR current amplitude (at the start of a spark). Cytosolic Tris⁺ elevates resting load but reduces single RyR current amplitude. Thus, spark properties appear to follow changes in current not resting load. This implies sparks may be load dependent because single RyR current amplitude varies with load.

Spark frequency as a function of resting SR load without and with 60 mM cytosolic Tris⁺ is shown in Figure 4B. Spark frequency was measured at various load levels (determined using Fluo-5N) following TG application. In the absence of Tris⁺ (triangles), spark frequency increased with load exactly as reported by others²¹ (dashed line). This is commonly explained by Ca²⁺ acting on intra-SR RyR regulatory sites. In the presence of cytosolic Tris⁺, luminal Ca²⁺ was far less effective at increasing spark frequency (squares). These data sets (triangles and squares) overlap if the Tris⁺ data are scaled upward (by a factor of 5.6, not shown). This is consistent with our results that show Tris⁺ does not alter single RyR Po or dwell times (Fig. 1 & Online Figure I) but instead just reduces current amplitude. It is consistent because the reduced RyR current with Tris⁺ present would logically be less effective at triggering the inter-RyR CICR that initiates spontaneous sparks in these permeabilized cells.

DISCUSSION

There are numerous RyR inhibitors, blockers and modulators that influence RyR opening/closing⁹ (i.e. RyR gating). The few that alter RyR current amplitude also affect its gating⁹. Here, we apply a novel form of current-targeted RyR block to examine CICR local control. Single RyR current amplitude is attenuated using a large RyR-permeable organic cation (Tris⁺). We used Tris⁺ primarily but other large cations had the same effect. This cation reduces net current amplitude because it interferes/competes for occupancy of the RyR pore with other permeable cations present¹⁹ (i.e. Ca²⁺, Mg²⁺ & K⁺ in cells and in our cell-like salt solutions). This interference with permeation occurs without a change in RyR gating. We used this current-targeted block to differentially manipulate single RyR current amplitude and resting SR Ca load in cells to better define their roles in CICR local control.

It is well-established that the amplitude, width and frequency of sparks (local bouts of inter-RyR CICR) vary with resting load²¹. Also, sparks terminate after resting load falls to some critical value¹ and SR Ca²⁺ overload promotes spark activity to the point that propagating Ca²⁺ waves begin. These phenomena are often explained by intra-SR Ca²⁺ acting on intra-SR RyR Ca²⁺ regulatory sites^2–4. Changes in load will clearly drive such mechanisms. Changes in load, however, will also alter single RyR current amplitude, the current that drives local inter-RyR CICR. The role of single RyR current amplitude in CICR local control is poorly understood. One reason is that it has been difficult to independently change resting load and current in cells. Further, it is impossible to directly measure single RyR current in cells. Note that the spark represents the opening of several RyRs within an RyR cluster at a release site. Here, we applied current-targeted RyR block (proven at single channel level) to address the role of single RyR current amplitude during CICR local control. Our results indicate CICR local control is governed by SR Ca²⁺ load largely because load determines single RyR current amplitude.

A CICR local control scheme is presented in Figure 5. Although its applicability to CICR local control in intact cells remains to be established, this scheme provides a context for explaining the potential significance of our results. In this scheme, we assume spontaneous single RyR openings in resting cells result in a spark only when the opening generates a large enough local Ca²⁺ signal to trigger inter-RyR CICR (i.e. large enough to activate an adjacent RyR at the same release site). Logically, the magnitude of the local Ca²⁺ trigger signal will be determined by the single RyR current amplitude as well as the duration of the RyR opening. Spark frequency will vary with SR load because load determines single RyR current amplitude and thus local Ca²⁺ trigger signal magnitude. Spark frequency varies nonlinearly with load because the local Ca²⁺ trigger produced by 1 RyR can act on 3–4 adjacent RyRs within the RyR cluster. Also, single RyR open times are exponentially distributed (many more short than long). This means that the number of openings that have a duration that is sufficient to trigger inter-RyR (a spark) will increase exponentially with load. This is because load and current vary in parallel so when current is larger then shorter openings (exponentially more) become sufficient to trigger a spark.

FIGURE 5.

Cartoon illustrating a possible spark current control scheme. A. One RyR opens > 2 ms (spontaneous event). B. Inter-RyR CICR (activates neighboring RyRs). C. Local SR Ca²⁺ load falls as Ca²⁺ is released. D. Single RyR Ca²⁺ flux amplitude decreases and inter-RyR CICR fails (RyR recruitment ends). E. Open RyRs begin to spontaneously close. F. SR Ca²⁺ reloading and system restitution. Inset shows the published local spark-load relationship¹. Possible Spark Termination Mechanism: Local load falls to a critical level where single RyR Ca²⁺ flux becomes too small to sustain cytosolic inter-RyR CICR. Already open RyRs will spontaneously close. Ca²⁺ flux by any still open RyR is too small to re-open others.

We showed that RyR mean open time in cell-like salt solutions is substantially shorter than spark time-to-peak (see Fig. 1 and Online Table II). Indeed, very few single RyR open events (<4% at 10 μM cytosolic Ca²⁺) have durations that even approach spark time-to-peak. This suggests that individual RyRs may open and close many times during the rise time of a spark. This possibility is supported by our in vitro measurements of inter-RyR CICR after a cluster of RyRs is fused into a bilayer (Online Figure IV). During these in vitro bouts of inter-RyR CICR, the RyRs present repeatedly open and close. Note that this inter-RyR CICR is not FK506 binding protein-based coupled gating²³ because: 1) it occurs only when Ca²⁺ is the charge carrier and 2) the RyRs present do not all open and close in a “lock-step” like fashion (Online Figure IV).

Assuming individual RyRs open and close many times during a spark, our scheme (Fig. 5) suggests open RyRs that spontaneously close will re-open as long as the current carried by any of their open neighbors is sufficient to reactivate them (i.e. sufficient to drive inter-RyR CICR). As local load falls, the single RyR current amplitude (at some point) will become too small to drive this RyR reopening. At this point, any RyR that spontaneously closes will remain closed, local inter-RyR CICR will cease and the spark will terminate. This process implies that any RyR manipulation (regulatory, mutagenic, pharmacological, etc.) that alters the channel’s cytosolic CICR sensitivity or open time will alter the current level (i.e. load level) where inter-RyR CICR begins and ends. For example, the SR load where inter-RyR CICR ends would be smaller if a cardiac RyR mutation (or regulatory event) enhances the RyR CICR sensitivity because the current would need to be smaller for local CICR to cease. Once local inter-RyR CICR ends, the probability of it re-occurring at the same release site will increase over time as the SR Ca²⁺ pump restores local load (i.e. restores single RyR current amplitude).

Like early theory of CICR local control²⁴, our working local control scheme assumes the Ca²⁺ current carried by an open RyR in cells acts only on neighboring channels, not on itself⁵. Single RyRs are activated by cytosolic Ca²⁺ and thus it might be reasonable to expect that the current may feedback and prolong the opening of the RyR carrying it. Accordingly, smaller currents would reduce mean open time because there would be less self-RyR cytosolic Ca²⁺ activation. This could contribute to the reduction in spark amplitude, width, time-to-peak we observed when single RyR current was decreased. However, we have shown elsewhere that single RyR open times do not change significantly in the presence or absence of a lumen-to-cytosol Ca²⁺ current^5,25. This is consistent with the voltage independent RyR mean open time we show here (Online Figure Ia,d). Indeed, single RyRs in cells appear to be largely “immune” to their own Ca²⁺ current even though that current is more than sufficient to activate a nearby neighboring RyR⁵. This appears to be contradictory to some previous reports of RyR Ca²⁺ feedback regulation^26,27,8. However, these previous studies were carried using super-physiological Ca²⁺ fluxes, highly potentiated cytosolic Ca²⁺ sensitivity and/or pharmacologically altered channels in order to promote feedback. For example, Sitsapesan and Williams²⁶ observed RyR Ca²⁺ feedback activation only in sulmazole-activated channels. Xu and Meissner²⁷ reported that significant RyR Ca²⁺ feedback activation at physiological current levels (<0.5 pA) only when caffeine was present. Laver⁸ defined Ca²⁺ feedback of ATP-activated RyRs (no Mg²⁺ present) which have greatly potentiated cytosolic Ca²⁺ sensitivity²⁵.

Our spark local control scheme (Fig. 5) implies that some spontaneous openings will be too brief to trigger sparks (CICR). This is consistent with recent reports of non-spark RyR-mediated SR Ca²⁺ leak^21,22. It is also consistent with our previous low-dose caffeine (≤0.15 mM) study that also concluded that only relatively long RyR opening trigger sparks²⁸. Caffeine is well-known to potentiate RyR cytosolic Ca²⁺ sensitivity⁹. Caffeine (0.25 mM) has also been shown to reduce the luminal Ca²⁺ level where sparks terminate¹². This is also consistent with our scheme. Since caffeine-sensitized RyRs would be more responsive to a Ca²⁺ trigger, local load would need to fall further before the RyR current becomes insufficient to sustain inter-RyR CICR. Another rather elegant spark study has shown that cardiac sparks peak before the underlying local SR Ca²⁺ release stops¹. In other words, the active RyRs generating the spark close asynchronously over a period of time that extends past the spark peak (i.e. not all at once). Our scheme suggests open RyRs spontaneously close (asynchronously) and local CICR ends because those channels fail to reopen. Thus, some quite disparate spark phenomena are consistent with our spark current control scheme. This spark local control, however, needs to be tested in intact cells. This will likely entail perfusion of patch clamped myocytes with large organic cations and/or expressing of RyRs that have modified permeation properties.

Some form of negative control must exist to counter the inherent positive feedback of CICR²⁹. Many possible mechanisms have been proposed but none (by themselves) are sufficient to explain the termination of local SR Ca²⁺ release⁹. Consequently, there is a growing consensus that the negative control may arise from a composite of factors/processes. One of these factors/processes is luminal Ca²⁺ acting on intra-SR RyR Ca²⁺ regulatory mechanisms^4,20,30. Indeed, there is a large body of data (including our own) indicating that RyR gating is modulated by intra-SR Ca²⁺ sensing sites^4,20,30. Our new results show spark termination also occurs when luminal Ca²⁺ falls because at some point single RyR Ca²⁺ current becomes insufficient to sustain inter-RyR CICR. These factors/processes are likely not mutually exclusive because the magnitude of the local cytosolic Ca²⁺ trigger signal that governs inter-RyR CICR will depend on both unit Ca²⁺ current amplitude and single RyR open duration (Po or MOT). Any mutation, genetic manipulation, drug or disease that alters RyR gating would thus influence the efficacy of a set unit Ca²⁺ current level to drive inter-RyR CICR. For example, the higher likelihood of inter-RyR CICR during Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) might be reduced by manipulations that either reduce unit current or shorten RyR open time. Indeed, the RyR-targeted actions of flecainide and carvedilol reduce RyR open time and limit Ca²⁺ wave-evoked arrhythmias ^31,32. In resting cells, Mg²⁺ and Ca²⁺ compete for RyR activation sites (Laver and Honen, 2008). Some RyR mutations alter RyR Mg²⁺ sensitivity and lengthen RyR open time perhaps causing CPVT (Lehnart et al.,2004). These observations are quite consistent with our spark local control scheme (Fig. 5).

NOVELTY AND SIGNIFICANCE.

What Is Known?

• Sparks are normal sarcoplasmic reticulum (SR) Ca²⁺ release events that are mediated by the opening of a cluster of ryanodine receptors (RyR) channels.

• Intra-SR Ca²⁺ overload increases spark frequency and sparks terminate when intra-SR Ca²⁺ falls to a critical threshold level.

• The intra-SR Ca²⁺ dependence of sparks is often explained by intra-SR RyR regulatory sites sensing changes in intra-SR Ca²⁺ concentration.

What New Information Does This Article Contribute?

• The amplitude of the Ca²⁺ current carried by individual open RyR channels determines the likelihood of the inter-RyR Ca²⁺ induced Ca²⁺ release (CICR) that generates the spark, not the direct sensing intra-SR Ca²⁺ concentration.

• Arrhythmogenic sparks and Ca²⁺ waves occurronly when the individual RyR Ca²⁺ current was large, not when intra-SR Ca²⁺ concentration was high and the Ca²⁺ current small.

• Sparks terminate when intra-SR Ca²⁺ falls to a point where the single RyR Ca²⁺ current becomes insufficient to support ongoing inter-RyR CICR (i.e. it becomes insufficient to trigger RyR re-opening after local RyRs have spontaneously closed).

Abnormal local control of CICR can be arrhythmogenic and/or contribute to heart failure. How CICR is controlled has been debated for decades. However, it is clear that local control of CICR is governed by intra-SR Ca²⁺ concentration. This intra-SR Ca²⁺ control this is generally believed to be due to intra-SR Ca²⁺ acting at intra-SR sites. However, changes in intra-SR Ca²⁺ also vary the size of the Ca²⁺ current carried by the individual RyR channel, the current that drives the inter-RyR CICR (sparks). We devised a means to differentially manipulate current amplitude and intra-SR Ca²⁺ concentration. We report that spark local control largely follows changes in current amplitude, not intra-SR Ca²⁺ concentration. We propose that spark control by single RyR Ca²⁺ current generates the intra-SR Ca²⁺ dependency, while intra-SR Ca²⁺ acting at intra-SR sites likely modulates it. Both of these mechanisms control spontaneous sparks and waves making them clinically relevant as potential sites for pathological failure and/or therapeutic intervention.

NON-STANDARD ABREVIATIONS AND ACRONYMS

CICR

Ca²⁺ induced Ca²⁺ release

SR

Sarcoplasmic reticulum

RyR

Ryanodine receptor

ECC

Excitation contraction coupling

Po

Open probability

MOT

Mean open time

Tris⁺

Tris(hydroxymethyl)aminomethane

TTP

Time to peak

FWHM

Full-width at half maximum

FDHM

Full-duration at half maximum

CaSpF

Ca²⁺ spark frequency

CPVT

Catecholaminergic Polymorphic Ventricular Tachycardia