Ca2+ Sparks and Waves in Canine Purkinje Cells
A Triple Layered System of Ca2+ Activation
We have investigated the subcellular spontaneous Ca2+ events in canine Purkinje cells using laser scanning confocal microscopy. Three types of Ca2+ transient were found: (1) nonpropagating Ca2+ transients that originate directly under the sarcolemma and lead to (2) small Ca2+ wavelets in a region limited to ≈6-μm depth under the sarcolemma causing (3) large Ca2+ waves that travel throughout the cell (CWWs). Immunocytochemical studies revealed 3 layers of Ca2+ channels: (1) channels associated with type 1 IP3 receptors (IP3R1) and type 3 ryanodine receptors (RyR3) are prominent directly under the sarcolemma; (2) type 2 ryanodine receptors (RyR2s) are present throughout the cell but virtually absent in a layer between 2 and 4 μm below the sarcolemma (Sub-SL); (3) type 3 ryanodine receptors (RyR3) is the dominant Ca2+ release channel in the Sub-SL. Simulations of both nonpropagating and propagating transients show that the generators of Ca2+ wavelets differ from those of the CWWs with the threshold of the former being less than that of the latter. Thus, Purkinje cells contain a functional and structural Ca2+ system responsible for the mechanism that translates Ca2+ release occurring directly under the sarcolemma into rapid Ca2+ release in the Sub-SL, which then initiates large-amplitude long lasting Ca2+ releases underlying CWWs. The sequence of spontaneous diastolic Ca2+ transients that starts directly under the sarcolemma and leads to Ca2+ wavelets and CWWs is important because CWWs have been shown to cause nondriven electrical activity.
In cells devoid of t tubules such as atrial and Purkinje cells (Pcells), excitation–contraction coupling (ECC) involves Ca2+ release from stores located near the sarcolemma and subsequent Ca2+-induced Ca2+ release (CICR) along a lattice of sarcoplasmic reticulum (SR) enveloping the sarcomeres, which then activate myofibrils throughout the cell.1–5 Drugs which affect SR function, such as thapsigargin and ryanodine, inhibit Ca2+ activation of Pcells.2,6 Conversely, spontaneous nonpropagating Ca2+ release and Ca2+ waves cause sarcolemma depolarization in both pacemaker cells and Pcells, which can lead to nondriven electrical activity even at normal [Ca2+]o.2,7–9 Abnormal Ca2+ release in the network of Pcells may also be involved in lethal arrhythmias after myocardial infarction.7,10 Previous observations suggested that micro Ca2+ transients, spanning only a few micrometers and traveling over short distances, initiate cell-wide Ca2+ waves (CWWs) which in turn induce nondriven electrical activity in a Pcell aggregate.7 Here, we determined the mechanistic relationships between the different subcellular spontaneous Ca2+ events in canine Pcells using confocal microscopy.
Materials and Methods
Eighteen aggregates of 2 to 6 cells were enzymatically dispersed from the Purkinje network of canine left ventricle (n=9)7 and placed in a chamber on the stage of an inverted laser scanning confocal microscope (LSCM). Fluorescence was measured only in rod-shaped Pcells with typical junctional ends, clear striations, and membranes free of blebs.2
Measurement and Analysis of Ca2+ Transients
Confocal line-scans were first positioned parallel to the long axis in the cell-center (longitudinal scans) and then moved to the lateral edge or to the top/bottom edge. The transition of fluorescence between the cytosol and extracellular fluid was used to localize the sarcolemma in transverse scans and scans through cell borders.
Local variations of [Ca2+]i along scan-lines were estimated from the pixel-to-pixel ratio F/Fo (F: instantaneous fluorescence; Fo: reference fluorescence) and analyzed using custom programs in IDL (IDL 5.4, Research Systems).
To mechanistically understand the processes that contribute to both propagating and nonpropagating Ca2+ transients, we constructed a mathematical model of release, diffusion, binding, and reuptake of Ca2+ in an array of (50) nodes. Ca2+ changes in scan line images were simulated by numerical integration of the differential equations11 for all Ca2+ fluxes. For details see the online supplement available at http://circres.ahajournals.org.
The immunocytochemical protocol used was similar to that previously described.12 Primary antibodies used were anti-IP3R1 (1:1000), an antibody that recognizes all 3 RyR isoforms, anti-RyR2 (IgG1; clone C3–33 and clone 34C, respectively; Affinity Bioreagents Inc; 1:500), and anti-RyR3 and anti-RyR1 produced and verified as described previously.13,14 For each aggregate, serial slices (2-μm intervals) through the z-axis of the entire aggregate were imaged. Antibody label density across cells was obtained from a pixel-to-pixel average of fluorescence of 30 line arrays across the cells after correction for nonspecific fluorescence (see online supplement).
Results are expressed as mean±SEM. Comparisons were performed on groups of data by ANOVA. The difference was significant when P<0.05 after Bonferroni adjustment.
Nonpropagating Ca2+ Transients
Nonpropagating Ca2+ transients were ubiquitous throughout Pcells. Most (82%) of these events (amplitude=1.85±0.02 F/Fo; duration at half maximal amplitude [T0.5]=41±1 ms; full width at half maximal amplitude [FWHM]=3.2±0.1 μm; rate=0.6±0.2 events per s per 100 μm; n=524) were similar to Ca2+ sparks reported for rabbit Pcells.6,15 The remaining nonpropagating Ca2+ transients had amplitude above 3 F/Fo (amplitude=4.9±0.2 F/Fo; T0.5=43±2 ms; FWHM: 2.4±0.2 μm; rate=0.13±0.04 events per s per 100 μm scan; n=115; Figure 1A). Clusters of consecutive and/or simultaneous Ca2+ sparks (Figure 1B) were also detected predominantly in regions below the sarcolemma and were similar to compound sparks described previously.16 Early sparks in clusters were often followed by a progressive increase in amplitude of later sparks (Figure 1B), suggesting an avalanche of multiple Ca2+ releases from a single or several adjacent sites with summation of Ca2+. However, amplitude of <5% of this compound sparks exceeded 3 F/Fo.
Transversal scans revealed the presence of spontaneous nonpropagating Ca2+ transients with various amplitudes directly under the sarcolemma (Figure 1C and supplemental Figure Is). The asymmetrical spread of these events (Figure 1D) confirmed that releases occurred directly under and against the membrane. Multiple consecutive releases could occur from the same site under the sarcolemma and produce large rises of Ca2+. The majority of the large (amplitude >3 F/Fo) nonpropagating events detected in our study actually occurred in this region (supplemental Figure Is).
Propagating Ca2+ Transients
Ca2+ transients also propagated as waves with linear fronts that extended from several micrometers to the full length of the scan-line (Figure 2). Two types of Ca2+ waves were identified: small waves or wavelets (amplitude <3 F/Fo) and large waves (amplitude ≥3 F/Fo) or CWWs.1
Wavelets had a short rise time, decayed exponentially after the peak, and lasted ≈150 ms (Figures 2 and 6⇓B and Table). Frequently wavelets started under the sarcolemma after nonpropagating Ca2+ elevations (Figure 2B and 2D) and in areas with prominent spark activity (Figure 2A). They propagated over a limited depth from the sarcolemma into the cell: 6.2±0.2 μm; n=224; 17 cells. We denote this 6-μm layer under the sarcolemma as the Sub-SL region. The wavelets traveled in the Sub-SL region over <10 μm (Table) and never triggered large nonpropagating Ca2+ transients. Interestingly, we found a significant reduction of the frequency of wavelets with 2APB (3 μmol/L), a modulator of IP3-sensitive Ca2+ release channels17,18: 0.86±0.26 (control, n=31) versus 0.39±0.18 event/μm/100 μm (2APB, n=41); P<0.001. Same 2APB effect was observed previously on nonpropagating Ca2+ transients near the sarcolemma.19
CWWs typically extended from sarcolemma to sarcolemma (Figures 2C and 3⇓), occurred at ≈5-fold lower frequency, and propagated along the aggregate at 2-fold lower velocity than wavelets (Table). CWWs lasted considerably longer than wavelets. They consistently exhibited “pseudo-plateaus” (Figures 2C, 3, and 6⇓⇓A) at a wide range of peak F/Fo (from 3 to 11 F/Fo; 21 waves) thus ruling out an artifact attributable to saturation of fluorescence at elevated [Ca2+]i.
Transverse scans showed that small [Ca2+]i increases triggered wavelets (threshold: Thr <10 nmol/L; n=6) and wavelets triggered CWWs (Figures 3 and 4⇓) at an ≈10-fold higher threshold (Thr≈70 to 120 nmol/L; n=5; Figure 3C).
Scans through an end-to-end boundary between cells confirmed the initiation sequence of Ca2+ waves and that the phenomenon perpetuates from cell to cell in the aggregate (Figure 3E).
The suggestion that Ca2+ transients start near the sarcolemma, and from these sites, propagate through the Sub-SL and/or into the cell-center, was further corroborated by x/y scanning (supplemental Figures IVA, IVB, and IVC). The 2-dimensional confocal images showed that spontaneous nonpropagating Ca2+ transients (Figure 4C; see online video clips) were common in the Sub-SL and revealed the presence of small Ca2+ transients propagating parallel to the sarcolemma within a layer of ≈6-μm thickness (Figure 4A, 2 through 4). Amplitude and propagation of these transients were similar to those of wavelets revealed by single line-scan technique (Figure 4D). Two-dimensional confocal images showed also that wavelets were frequently accompanied by diffuse increases of [Ca2+]i (“wave flag”) below the Sub-SL (see also online supplement). These “diffusional flags” were detected by single line-scans positioned further in the cell center and appeared as small events moving with same velocity than SubSL wavelets but with decreasing amplitude (see Figure 2C). On occasion, 2D animations clearly showed a wavelet initiating a CWW while traveling longitudinally in the SubSL (Figure 4B, 4 through 6⇓⇓; Figure 4D).
Our linescan observations were corroborated by fast confocal video imaging (Figure 4E), which indicates that Ca2+ wave propagation occurred on at least 2 functional subcellular levels, suggesting that elements required for propagation differ in 2 different regions.
Immunolabeling of Ryanodine and IP3 Receptors
We determined the nature and distribution of SR Ca2+ release channels in these Pcells. First, we found no labeling for IP3R2 and RyR1 (results not shown). However, colabeling with IP3R1 and RyR2 antibodies was positive and revealed 2 distinct regions: a layer of ≈2 μm thick with IP3R1 label (Figure 5A.b and 5B; green) existed directly under the sarcolemma while intense RyR2 labeling dominated most of the cell including the lateral IP3R1-positive layer (Figure 5A.b and 5C; red). Analysis of IP3R1- and RyR2-positive regions showed that only 5% of the corresponding pixels overlapped. Furthermore, Figure 5A.b and 5C illustrate a novel and typical feature of antibody staining of Pcells, in that label for both RyR2 and IP3R1 was extremely sparse in a continuous layer of 2 μm thick below the IP3R1-positive layer (see arrows). This apparent void of Ca2+ channels was observed with RyR2 antibody but not with a RyR antibody that recognized all isoforms of the channel (Figure 5A). We found that this void was filled by a specific RyR3 antibody which actually labeled a layer of 7-μm thickness below the sarcolemma (Figure 5C) and was absent in the cell-center (Figure 5A.c, 5B.c, and 5C).
Properties of the Generators of Ca2+ Transients
Using model simulations, we studied whether the large Ca2+ waves are indeed generated by nonpropagating Ca2+ releases near the sarcolemma, such as has been shown in rabbit Pcells, or are in fact caused by propagating Ca2+ releases. We also used the simulations to identify the factors that determine the properties of wavelets and CWWs.
Sparks near the sarcolemma, in the Sub-SL and cell-center could be simulated accurately using similar parameters of the release (release time ≈40 ms) and uptake functions (see online supplement for details). The correlations between simulated and experimental data were robust for all events (R2>0.97; Figure 1D and supplemental Figure IIs). The Ca2+ diffusion coefficient (Deff) required to fit such sparks was 12.1±0.3 μm2/s in both the Sub-SL and cell-center; we used this value to simulate Ca2+ wavelets and CWWs (Figure 6). Large Ca2+ sparks were reproduced accurately by assuming a larger Ca2+-release flux and a longer Ca2+-release time (up to 100 ms), without a change in other parameters.
Large Ca2+ waves are expected to travel faster than small waves; actually, we found the opposite when we compared CWWs with wavelets: wavelets propagated on average twice as fast as CWWs (Table). The simulations ruled out that this observation was caused by differences in Deff (see above) or extrusion kinetics (online supplement). The spacing20,21 of Ca2+ channels (Figure 5A.a) appeared to be similar in the Sub-SL and cell-center and cannot, therefore, explain our observation either. Finally, we tested whether the observed difference in velocity between wavelets and CWWs was caused by different Ca2+ thresholds of the generators. The velocity appeared to be inversely proportional to Thr (see supplemental Figure IIIs), and CWW propagation required a Ca2+ threshold up to 20-fold higher than the one for wavelet propagation consistent with the experimentally determined values (Figure 3). In addition, the simulations suggested 2 other aspects of the unique and distinct nature of CWWs as compared with wavelets (Figure 6): a 10-fold longer lasting Ca2+ release occurred during a CWW (200 to 500 ms) compared with that of either wavelets or sparks (20 to 45 ms), whereas the calculated total Ca2+ release in CWWs was 40-fold larger (Cf. insets Figure 6).
We show here that, at normal [Ca2+]o, Ca2+ sparks occur ubiquitously throughout canine Pcells with characteristics similar to those of ventricular myocytes,22,23 including the presence of a subpopulation of large sparks. Large sparks in this study appear, however, wider and larger than those observed in rat cardiac myocytes.24–26 The presence of compound sparks16 with, on average, amplitude <3 and large width in the population of nonpropagating Ca2+ transients may explain why, in Pcells, large nonpropagating events were slightly narrower than small events (see FWHM above).
Accuracy of the correlation between amplitude and localization of Ca2+ sparks is limited because local Ca2+ releases can occur outside the confocal plane. Nevertheless, although large single sparks were seen occasionally in the cell-center, the majority was found directly under the sarcolemma and in the SubSL region. Our observations differ from those in rabbit Pcells, where sparks are exclusively found directly under the sarcolemma.6,15 The large sparks of canine Pcells are similar to the large 2APB-sensitive Ca2+ events reported in rabbit portal vein myocytes.18 The compound sparks in the Sub-SL and repetitive Ca2+ spark generation from single sites directly under the SL (see online supplement) suggest that both near-synchronous activation of multiple Ca2+ release units20 and rapidly repetitive activation in one site may occur in these regions.
This is consistent with the hypothesis that activation of IP3R may recruit adjacent Ca2+ channels including IP3Rs and RyRs (or vice versa);18,27 our findings that these 2 types of SR-Ca2+ channels coexist (Figure 5) and that Ca2+ events are sensitive to 2APB17,19 make it probable that this IP3R/RyR interaction indeed occurs in canine Pcells.
Ca2+ Transients in the Cell-Center: Diffusion or Propagation?
Cordeiro et al observed no spontaneous Ca2+ release centrally in rabbit Pcells although RyRs were present,6,15 and thus concluded that central RyRs were “silent.” Our observations do not support the same conclusion, as we found that spontaneous Ca2+ sparks were common in the cell-center (eg, Figure 2), thus showing that RyRs are active in canine Pcells. This species difference in spontaneous Ca2+ release may also explain why canine Pcell aggregates and fibers exhibit nondriven electrical activity2,7 whereas rabbit Purkinje fibers do not.28
Cordeiro et al proposed that the large central Ca2+ elevation evoked by the action potential (AP) in rabbit Pcells resulted only from diffusion of Ca2+ released from peripheral sarcolemmal sites.6,15 Our simulations of Ca2+ release directly under the sarcolemma (Figure 1C) are consistent with this conclusion with respect to large sparks.6,15 In contrast, the spatiotemporal Ca2+ distribution of both wavelets and CWWs in canine Pcells (Figure 6) could be reproduced only by incorporating the propagation of Ca2+ release from node to node in the model, confirming that propagated CICR is responsible for these Ca2+ waves. Furthermore, our simulations show that the lower velocity of CWWs requires that Thr of Ca2+ release elements involved in the propagation be an order of magnitude higher than that of wavelets; this was consistent with our experimentally determined values (Figure 3). Finally, the typical plateau of CWW as well as the simulated Ca2+ release function are novel findings for a cardiac cell and suggest a distinct Ca2+ release mechanism. During CWWs, one or both of the following may occur: the release channels open completely, but the flux declines because of a decreased gradient across the channel.29 Alternatively, irreversible channel opening could be induced by an interaction between the permeant ion and the channel, similar to the mechanism that has been proposed for skeletal muscle.30 The determination of such mechanism is beyond the scope of this study. However, once we understand the mechanism of this persistent Ca2+ release, we should be able to reduce it in the intact Pcell. Such a reduction in the amplitude of CWWs and thus the depolarization that accompany them would be antiarrhythmic.
Mechanism of Initiation of Wavelets and CWWs
Spontaneously occurring Wavelets start commonly after sparks directly under the sarcolemma or in the Sub-SL (Figure 2), and, in turn, initiate CWWs (Figures 3 and 4⇑). The effect of 2APB observed in this study, and in the previous study of Boyden et al,19 suggests that IP3R-mediated Ca2+ release is instrumental in initiating/modulating Ca2+ wavelets. We never observed wavelets, which arrived at the sarcolemma and subsequently induced Ca2+ release directly under the sarcolemma. This directional asymmetry suggests that elements propagating wavelets and those causing Ca2+ sparks directly under the sarcolemma are functionally distinct. Furthermore, for adequate simulation of sparks near the sarcolemma, a 2-fold longer Ca2+ release pulse (supplemental Figure IIs) was required compared with that needed for wavelets (Figure 6), suggesting that Ca2+ transients directly under the sarcolemma and in the Sub-SL result from different Ca2+ release mechanisms.
Large CWWs span the entire cell suggesting that their underlying SR-Ca2+ release elements are ubiquitous. The amplitude duration and speed of the CWWs requires that the SR-Ca2+ release elements that mediate CWWs have a high CICR threshold and release a large Ca2+ flux for hundreds of ms. Faster and smaller low-threshold wavelets were found in the Sub-SL, suggesting that the SR-Ca2+ release elements that reside in this region have a low CICR threshold and release a small Ca2+ flux for tens of ms. These 2 functionally distinct Ca2+ release elements must overlap in the Sub-SL, where both CWWs and wavelets can exist. This arrangement predicts that the probability for wavelets to trigger CWWs is small, as reported previously2 (Table). On the other hand, when the high CICR threshold of the CWW generators has been surpassed, Ca2+ release elements in the SR network will propagate the wave as far as this network reaches.
In summary, we show here that canine Pcells contain 3 functionally distinct Ca2+ release systems: system (1) is restricted to a thin layer (2 μm) directly under the sarcolemma and apposed to system (2) in the Sub-SL; system (2) partially overlaps with system (3) that drives Ca2+ release from sarcolemma to sarcolemma.
Ca2+ Release Channel Elements
We demonstrate here that a sophisticated triple-layered system of SR-Ca2+ release channel architecture underlies the above-proposed hierarchy of Ca2+ wave generation. Like in atrial cells,3,4 we observed IP3R, RyR2 under the sarcolemma. However, different from atrial cells, the IP3R1 isoform was detected in Pcells. RyR2 formed a clear striated pattern in the cell, similar to the pattern shown in rabbit Pcells,6,15 as well as directly under the membrane near IP3R1s, but were virtually absent in a 2-μm layer (“void”) below the sarcolemma, in the subSL region. This void was specific for RyR2 because an antibody that recognized all three RyR isoforms showed no void. In fact, RyR3 labeling was found in high density filling the void between RyR2 and IP3R1 (Figure 5).
Although a detailed pharmacological analysis of Ca2+ transient generators in canine Pcells is beyond the scope of this study, our findings reveal that Ca2+ activation in spontaneous Pcells differs substantially from that of ventricular myocytes. It can be explained in the following way (Figure 7):
First, a layer (2 μm) directly under the sarcolemma contains both IP3Rs and RyR2,3, which are either separately or in combination responsible18,27 for the spontaneous large sparks directly under the sarcolemma. Second, the Sub-SL (6 μm thick from the sarcolemma) is a layer of RyR3s, which partially overlaps the previous IP3R/RyRs layer; RyR3s will generate sparks in the unstimulated Pcell. Because of their low threshold, RyR3s will readily respond to Ca2+ release directly under the sarcolemma by the generation of Sub-SL wavelets. RyR3s indeed show spontaneous Ca2+ activity at normal diastolic [Ca2+]i when expressed in HEK cells,31 and pure RyR3 release is more sensitive to caffeine than other RyR isoforms.31 Their distribution would make them a source of sparks and wavelets in the Sub-SL if they also exhibit a low threshold for CICR in canine Pcells.
Third, RyR3s overlap with widely distributed RyR2s, thus allowing RyR2s to respond to Ca2+ release from the RyR3 Sub-SL network and generate CWWs. RyR2 channels release a large amount of Ca2+ thereby recruiting all available Ca2+ release channels in the generation of CWWs. This model could serve as a safe unidirectional system that ensures organized activation of Pcells in response to an action potential.
In conclusion this study provides novel functional and structural evidence for a triple layered system of Ca2+ activation in canine Pcells involving IP3Rs, RyR3s, and RyR2s explaining a well recognized feature of the Pcell aggregate: the nondriven electrical activity.7
This work was supported by grants from the National Institutes of Health (HL-58860) and the Alberta Heritage Foundation for Medical Research (AHFMR); H.E.D.J.t.K. is an AHFMR Medical Scientist. We thank Dr W.G. Wier for his help during the construction of the laser scanning confocal microscope and G. Groves for his logistic support.
Original received August 10, 2004; revision received May 9, 2005; accepted June 1, 2005.
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