Published online before print October 11, 2007, doi: 10.1161/CIRCRESAHA.107.162735
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© 2007 American Heart Association, Inc.
UltraRapid Communication |
Spatiotemporal Relationship Between Intracellular Ca2+ Dynamics and Wave Fragmentation During Ventricular Fibrillation in Isolated Blood-Perfused Pig Hearts
From the Nora Eccles Harrison Cardiovascular Research and Training Institute (M.W., A.G.S., A.V.Z.), University of Utah, Salt Lake City; and the Institute for Cardiovascular Research (J.F.H.), SUNY Upstate Medical University, Syracuse, NY.
Correspondence to Alexey V. Zaitsev, PhD, Nora Eccles Harrison CVRTI, University of Utah, 95 South 2000 East, Salt Lake City, UT 84112-5000. E-mail zaitsev{at}cvrti.utah.edu
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Abstract |
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Normal "master–slave" relationship between the action potential (AP) and intracellular Ca2+ transient (CaiT) is sometimes altered during ventricular fibrillation (VF). The nature of AP/CaiT dissociation during VF and its role in inducing wavebreaks (WBs) remain unclear. We simultaneously mapped AP (RH237) and CaiT (Rhod-2) during VF in blood-perfused pig hearts. We computed AP and CaiT dominant frequency (DF) and CaiT delay in each AP cycle. We identified WBs as singularity points in AP phase movies and sites of conduction block (CB) as sites where an AP wavefront failed to propagate. We analyzed spatiotemporal relationship between abnormal AP/CaiT sequences and CB sites. We used a calcium chelator (BAPTA-AM) to abolish CaiT and test its involvement in WB formation. During VF, the DF difference between AP and CaiT was <10% of the respective values in 95% of pixels, and 80% of all CaiT upstrokes occurred during the initial 25% of the excitation cycle. Aberrant sequences of AP and CaiT occurred almost exclusively near CB sites but could be traced to normal wavefront sequences away from CB sites. Thus, apparent AP/CaiT dissociation was largely attributable to spatial uncertainty of the absolute position of block of each wave. BAPTA-AM reduced CaiT amplitude to 30.5±12.9% of control and the DF of AP from 12.2±1.6 to 10.4±1.3Hz (P<0.01), but did not significantly alter WB incidence (0.76±0.19 versus 0.72±0.19SP/mm2). These results do not support presence of spontaneous, non–voltage-gated CaiTs during VF and suggest that AP/CaiT dissociation is a consequence rather than a cause of wave fragmentation.
Key Words: ventricular fibrillation • pig heart • action potential • calcium transient • wavebreak
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Introduction |
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Recurrent wave fragmentation, or wavebreak (WB), is a hallmark of ventricular fibrillation (VF).1,2 Wavebreak occurs when a propagating wavefront encounters an obstacle which may result from electrophysiological heterogeneities,3–6 intrinsic repolarization instabilities,7,8 anatomic structures,9 or regional ischemia.10 Repolarization instabilities in the form of action potential duration (APD) alternans are at least in part mediated by beat-to-beat changes in L-type Ca2+ current (ICa,L) and Na+-Ca2+ exchanger (NCX).2,11 These currents are bidirectionally coupled to intracellular Ca2+ (Cai) cycling, so that Ca2+ influx via ICa,L triggers Ca2+ release from the sarcoplasmic reticulum (SR), whereas an increase in Cai modulates inactivation of ICa,L and the transsarcolemmal current generated by NCX.12 SR Ca2+ cycling can exhibit intrinsic dynamics in the form of Cai transient (CaiT) alternans (even when the AP waveform is fixed under voltage clamp)13 and in the form of spontaneous, non–voltage-gated Ca2+ releases.12 It was hypothesized that such intrinsic dynamics of CaiT can promote APD fluctuations at high excitation rates and thus contribute to mechanisms of WB during VF.2,13 To test this hypothesis, Omichi et al14 simultaneously recorded optical AP and CaiT (AP/CaiT) during VF in isolated porcine right ventricle. They reported that in the areas of wave fragmentation, Cai waves bore no apparent relationship to voltage waves, so that CaiT could follow as well as precede the AP upstroke. The authors speculated that the observed AP/CaiT dissociation could be in part attributable to "non–voltage-gated Ca2+ release",14 a mechanism commonly associated with delayed after-depolarizations (DADs)15 underlying ectopic arrhythmias. In support of this hypothesis they presented 2 numerical paradigms of AP/CaiT coupling in the setting of VF. In 1 model, CaiT was solely triggered by AP, and in the other CaiT was not exclusively triggered by AP but could sustain independent oscillations in a given region of the parameter space. Although both models were capable of recreating WB and complex wavelet dynamics, the latter case generated a significantly higher number of WBs. The authors concluded that "localized non–voltage-gated Ca2+ release promoted wavebreak" in the latter computer model.14
Despite the important contributions of the aforementioned studies and recent theoretical work related to the dynamics of Cai,16 the mechanism of abnormal CaiT events as well as their cause-effect relationship with WBs during VF remain unknown. In this study we sought to address the following questions: (1) What is the spatiotemporal organization of abnormal CaiT waves during VF and, in particular, the sites where the AP and CaiT waves diverge or new CaiT waves appear? and (2) how do the dynamics and spatiotemporal organization of AP waves during VF change when CaiT is abolished by chelating Cai?
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Isolated Blood-Perfused Heart
Animals were used according to NIH guidelines. Hearts were obtained from pigs (15 to 25 kg) of either sex premedicated with ketamine 350 mg, azaperone 80 mg, and atropine 0.5 mg (i.m.), and anesthetized with pentobarbital 20 mg/kg (i.v.). After isolation via midline sternotomy, the heart was Langenddorff-perfused with blood as described previously.10 All the outflow of perfused blood was collected for recirculation by means of tubes inserted into the left and right ventricles via the atrial appendages.10 After sealing the blood circulation system, the heart was placed in a chamber with a heated water-jacketed transparent glass wall and superfused with warm (37±0.5°C) oxygenated Tyrode solution.
Optical Recordings
We simultaneously recorded fluorescence from voltage-sensitive dye RH-237 and Ca2+ sensitive dye Rhod-2 similar to method described previously.17 Briefly, the red (Rhod-2) and infrared (RH-237) fluorescence signals were separated using a dichroic mirror (634DCLP, Omega Optical), and were further filtered with a 585±20-nm band-pass and a 720-nm long-pass filter, respectively. Two synchronized digital CCD cameras (DALSA, 64x64 pixels, 300 frames/s) were aligned using a grid pattern with an accuracy of 1 pixel. Additionally, to exclude the Ca2+ buffering effect of Rhod-2 on VF dynamics, in a different group of hearts we recorded fluorescence of voltage-sensitive dye Di-4-ANEPPS as previously described.10 All dyes were excited with green light (532 nm) emitted by a 5-watt DPSS laser (Coherent) and delivered to the mapped area via fiber optics light guide (Edmund Optics). The field of view was approximately 35 mm in diameter and covered the epicardial area of the anterior LV with the upper left corner just viewing the left anterior descending coronary artery. To minimize motion artifacts, the heart was gently pressed against the glass chamber wall during acquisition of a 5-second movie.10
Experimental Procedures and Protocols
We used a total of 12 hearts. In all experiments, episodes of VF were induced by a short (
1 sec) application of 9V DC current. Measurements were performed at least 5 minutes after VF induction to allow steady state conditions to be attained.10,18 Other experimental procedures and measurements were different among experiments and are summarized in the Table. We recorded dual AP and CaiT movies during baseline VF in 7 hearts. After defibrillation, BAPTA-AM was delivered to 4 of these hearts, and in 2 additional hearts where we imaged Di-4-ANEPPS fluorescence. In these experiments, VF was reinduced 10 to 15 minutes after BAPTA-AM loading, and data obtained during VF before and after BAPTA-AM loading were compared. A sham procedure was performed in 2 hearts, 1 loaded with RH-237 and Rhod-2 and the other 1 was loaded with Di-4-ANEPPS. In the latter heart, the sham procedure was performed before infusion with BAPTA-AM. Contractility changes were monitored in 3 hearts perfused with BAPTA-AM and in the 2 sham experiments. To determine the Rhod-2 fluorescence changes caused by the dye internalization and leak out of the cells, in 2 experiments we recorded CaiT fluorescence movies during 2 hours of VF after injection of Rhod-2. The time course of the Rhod-2 signal evolution was essentially the same in the 2 experiments. The average of 2 such curves, normalized to the maximal CaiT amplitude, provided a calibration curve that was used to correct the difference in CaiT amplitude observed before and after BAPTA-AM perfusion.
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Dye and Drug Delivery to the Blood-Perfused Heart
Di-4-ANEPPS, Rhod-2-AM, RH237, and BAPTA-AM were obtained from Molecular Probes. Stock solution of Di-4-ANEPPS (5 mg/mL in DMSO) was diluted in Tyrode solution in a volume ration 1:1000, and 30 to 60 mL of the final solution was directly infused into the aortic cannula. Direct injection of Rhod-2 into blood failed to produce a detectable time-varying signal, presumably attributable to absorption of Rhod-2 by blood cells. However, previously Qian et al19 were able to detect Ca-sensitive Rhod2-AM signal in blood-perfused rabbit hearts when the dye was delivered during perfusion with Tyrode solution before switching to blood perfusion. Therefore, for optimal delivery of the dyes into the cardiac tissue, the following procedure was developed. Aliquots of RH237 (50 to 100 µL of a 5 mg/mL solution in DMSO) and Rhod2-AM (1 mL of 2 mg/mL solution in DMSO) were dissolved in warm oxygenated Tyrode solution (120 mL). Before dye delivery, perfusion was switched to a warm oxygenated Tyrode solution (120 mL) to clear the blood perfusate. Subsequently, we administered 120 mL of the dye cocktail followed by an infusion of additional 120 mL of Tyrode solution, before returning back to blood circulation. A continuous cardiac perfusion was maintained throughout the entire procedure (2 to 4 minutes) during which the heart outflow was discarded. BAPTA-AM was delivered in a similar manner. Specifically, we dissolved 1600 µL of the stock solution (25 mg BAPTA-AM in 2 mL DMSO) into 1.5 L of Tyrode solution. The entire volume of solution was circulated through the heart (10 minutes) and was discarded before switching back to blood circulation. In 2 sham experiments, we repeated exactly the same procedure but without adding BAPTA-AM to the Tyrode solution.
Myocardial Contractility
Given the strong Ca2+ buffering properties of BAPTA-AM, we confirmed the drug delivery to the myocardium by assessing the LV contractility. In each experiment we had a collector tubing inserted in the LV to collect a small amount of outflow (3 to 4 mL/min) from Thebesian veins. This outflow kept the LV filled with blood at a constant end-diastolic pressure determined by the height of the maximal elevation of the outflow tubing with respect to the mid-LV cavity (5 cm water). A pressure gauge was inserted in series with the LV outflow tubing. Measurements were done during sinus rhythm before and after dye infusion, and after 10-minute perfusion with BAPTA-AM or Tyrode solution (sham experiments). The outflow tubing was blocked for several seconds, just enough to record several beats. The difference between peak systolic pressure and the end-diastolic pressure was averaged in 5 consecutive beats and was normalized to the baseline value.
Data Analysis
Both RH237 and Rhod-2 fluorescent signals were filtered in an identical manner as follows. Spatial filtering by weighted averaging of neighboring pixels was performed by convolution with pyramid-shaped kernel (cone width, 3 pixels). In addition, a temporal median filter (length, 3 frames) was applied. To remove drift (if present), we first applied a running average filter with a large kernel size (100 frames) to the signal. The output of this filter which represented slow frequency components (< 1Hz) was subsequently subtracted from the original signal.
Analysis of the recorded fluorescent signals in the frequency domain has been previously described in detail.20 The dominant frequency (DF) was defined as the frequency corresponding to the highest peak in the power spectrum. Phase movies21 were generated by applying the Hilbert transform5,22 to the AP fluorescent signal. Singularity point (SP) was defined as a point where all phases converge.21 SP trajectories were determined in phase movies using SCROLL software (Sergey Mironov, Institute for Cardiovascular Research, SUNY at Syracuse). A starting point of a SP trajectory was considered as the site of a new WB. The total number of WBs per 1024 frames (3.41 s) was determined for each movie of VF.
Phase Delay Between AP and CaiT
Custom software first detected all robust local minima and maxima in each individual pixel recording of both AP and CaiT movies.18 In each pulse, the amplitude was defined as the difference between the respective local maximum and minimum. The pulses with amplitudes less than 10% of the difference between the global maximum and global minimum in the same pixel were discarded from this analysis. We determined the time of the onset of both the AP and the CaiT at the 50% of the upstroke of the respective pulses. The time delay between the AP and CaiT upstrokes in each excitation cycle (see Figure 2, inset) was then plotted as a time delay distribution histogram. We also calculated the time delay normalized to the time interval between the onset of the preceding AP and the onset of the following AP and expressed as a percentage.
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Identification of Abnormal AP/CaiT Wavefront Sequences
We generated wavefront movies of AP and CaiT signals by initially determining the wavefront of each wave as the data points belonging to the range between 10% and 90% of the ascending phase of the respective pulses (Figure 3F). The pulses with amplitudes less than 10% of the difference between the global maximum and global minimum in the same pixel were discarded. We then superimposed the AP and CaiT wavefronts and coded AP wavefronts with green color, CaiT wavefronts with blue, and their overlap with white (see Figure 3F). For each dual wavefront movie, we plotted a series of vertical and horizontal time-space plots (TSPs)18,23 at 4-pixel intervals. During normal propagation, the AP and CaiT wavefronts visit all pixels in each excitation cycle, the CaiT wavefronts lagging behind the AP wavefronts. In this case each TSP should show continuous bands of both AP and CaiT wavefronts spanning the entire row or column of pixels. Conversely, an AP band which does not span the entire range of pixels indicates conduction block (CB) at the pixel where it stops. (Topological relationship between CB sites, WBs and reentry are explained diagrammatically in supplemental Figure I, available online at http://circres.ahajournals.org). All vertical and horizontal TSPs were inspected to determine the spatiotemporal relationship between the AP and CaiT wavefronts, and the association between the CB sites and abnormal AP/CaiT wavefront sequences. Abnormal wavefront sequences at the periphery (within 4 pixels of edge) were discarded from further analysis.
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The 10% rejection threshold used for identification of the AP and CaiT impulses could potentially affect the results. Supplemental Figures IV and V indicate that the distribution of CaiT delays and the relationship between the AP and CaiT wavefronts did not change appreciably when the amplitude rejection threshold for the AP and CaiT was varied between 5 and 20%.
Statistical Analysis
Data are presented as mean±SD. Differences between parameters measured before and after BAPTA-AM perfusion were assessed with the paired Student t test. A value of P<0.05 was considered statistically significant.
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Dominant Frequency of the AP and CaiT During VF
DF maps of AP and CaiT were very similar (Figure 1A), and the mean DF across all pixels in AP and CaiT maps was not statistically different (12.57±1.66 versus 12.56±1.57, Figure 1B). Pixel-to-pixel comparison showed that the DF difference between AP and CaiT was less than 10% (less than 1 Hz) of the respective values in 95% of pixels. However, the average pixel-to-pixel absolute difference of AP and CaiT DF values reached statistical significance despite the fact that it was very small (0.12±0.09Hz). Thus, the DF distribution of the AP and CaiT was not fully identical but the discrepancy was at least an order of magnitude smaller than previously reported (5.02±1.22 Hz).14 Accordingly, the pixel-to-pixel cross-correlation coefficient between AP and CaiT DF values was larger than 0.99 in all hearts. Overall, these data indicated the same or a similar number of AP and CaiT cycles per unit of time in each pixel. The next question we addressed was whether any consistent phase relationship existed between the onset of the AP and CaiT.
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Time Delay Between AP and CaiT
Figure 2 shows histograms of time delay between the AP and CaiT upstrokes expressed in the number of frames. It is clear that in all experiments (n=7) the majority of CaiT were tightly coupled to the upstroke of the preceding AP. The peak of the distribution occurred either at 2 or 3 frames (6.7 to 10 ms), and in 80% of excitation cycles the CaiT delay was at most 8 frames (26.7 ms). We also calculated the CaiT delay as the percentage of the excitation cycle where it occurred. The normalized CaiT delay had similar distribution (not shown). In particular, in 80% of all excitation cycles, CaiT upstroke occurred within the initial 25% of the corresponding cycle. However, in each experiment there was also a thin "floor" in the histogram reflecting a small fraction of CaiTs which had an apparently random phase in the AP cycle. Thus, we sought to explain those seemingly "non–voltage-gated" CaiT events.
Spatiotemporal Organization of Abnormal AP/CaiT Wavefront Sequences Near CB Sites
Omichi et al14 used previously mutual information (MI) as the main tool to quantify the degree of temporal correlation between the AP and CaiT. We also performed a limited analysis of MI which is presented in supplemental Figure VII. However, MI does not take into account spatial factors. To investigate the relationship between the AP and CaiT wavefronts during VF in both space and time, we constructed dual wavefront movies (see Methods and Figure 3F). Figure 3A shows a snapshot of one such movie with three short-living reentrant circuits (a, b, and c) in the field of view. It is seen that CaiT followed AP (green-white-blue sequence) along the paths of the reentrant circuits. Figure 3D shows that the AP and CaiT recordings taken from pixels distant from the core of the reentrant circuits (pixels 2 and 4) show a consistent phase relationship between the AP and CaiT. Specifically, every AP is followed, after a delay, by one and only one CaiT, even though the shape of both the AP and CaiT impulses varies from cycle to cycle. In contrast, the recordings taken in the vicinity of the centers of reentrant circuits b and c (pixels 1 and 3) show bursts of a highly discordant and apparently random relationship between the AP and CaiT.
For a more systematic analysis of the spatiotemporal relationship between the AP and CaiT wavefronts, we carefully inspected series of vertical and horizontal TSPs such as those shown in Figure 3B and 3C. We first identified all the sites of CB in the TSPs. Examples of CBs are marked with red arrowheads in Panels E-I, E-II, and E-III of Figure 3, which show expanded portions of the TSPs presented in Figure 3B and 3C. The topological relationship between sites of CB in TSPs and various reentrant and nonreentrant patterns during VF is explained in supplemental Figure I. In particular, the core area of a spiral wave reentry will manifest itself as an instance of CB once for each half of a rotation. For example, the reentrant circuit b indicated in Figure 3A makes approximately two and a half rotations (not shown). Accordingly, the vertical TSP (see Figure 3B and 3E-I) constructed for a line crossing the reentrant circuit b (vertical dashed line in Figure 3A) reveals 5 instances of CB (see red arrowheads in Figure 3E-I). In some cases, however, CB sites could not be identified with a reentrant circuit, including incomplete reentry and a pattern in which waves coming from 2 or more different directions converged to a CB area, forming a "propagation sinkhole". For example, the 3 instances of CB marked with red arrowheads in Figure 3C are associated with a "propagation sinkhole" pattern (not shown).
Regardless of whether or not they corresponded to an identifiable reentry, the CB sites were the predominant locations of dissociation between the AP and CaiT. The TSPs shown in Figure 3 clearly demonstrate that the normal AP/CaiT wavefront sequence (CaiT follows AP) is present everywhere with the exception of the vicinity of the CB sites (red arrowheads in Figure 3E). Close examination reveals that in the center of the core/CB areas the dual AP/CaiT wavefronts coming from different directions converge and overlap in an apparently random manner, such that the exact stop positions of the AP and CaiT wavefronts do not coincide in the same wavefront pair and vary between consecutive wavefront pairs. This results in a poor association between the AP and CaiT signals in those pixels which are close (and when they are close) to the CB sites. For example, the transient AP/CaiT dissociation observed in pixel 1 (dashed rectangle "I" in Figure 3D) corresponds to the time interval when this pixel is close to the core/CB area of the transient reentrant circuit b, which is manifested in the vertical TSP by 5 instances of CB (red arrowheads in Figure 3E-I). Similarly, the longer streak of AP/CaiT dissociation observed in pixel 3 (dashed rectangle "II & III" in Figure 3D) is associated with abundant occurrences of CB in the vicinity of this pixel (red arrowheads in Figure 3E-II and 3E-III). Note that a seemingly non–voltage-triggered CaiT wave observed in pixel 3 and in the vertical TSP near pixel 3 (red cross in Figure 3B, 3D, and 3E-II), can be traced to a master AP wave in the horizontal TSP (red cross in Figure 3C and 3E-III). Supplemental Figure III presents detailed pixel-to-pixel analysis of the AP and CaiT waves in the vicinity of pixel 3. It shows that despite the apparently random sequence of impulses in the AP and CaiT recordings from pixel 3, every deflection in these recordings could be traced in space and back in time to a normal AP/CaiT wave. These observations underscore importance of spatial information for understanding the sources of the AP/CaiT dissociation during VF.
Based on the analysis of dual wavefront representation shown in Figure 3, we found that all abnormal AP/CaiT wavefronts patterns could be reduced to 4 distinct types: "solitary CaiT", "solitary AP", "AP/CaiT crossover", and "CaiT breakthrough". Specifically, a "solitary CaiT" is the pattern where a CaiT wave triggered by AP propagates beyond the site where AP wave stops (ie, CB site). "Solitary AP" is the pattern where AP wavefront is not followed by a CaiT. "AP/CaiT crossover" is the pattern where CaiT wavefront initially triggered by an AP eventually breaks ahead of the AP wavefront. "CaiT breakthrough" is the pattern where a CaiT wavefront appears "de novo" and cannot be traced to any preceding AP wave. Obviously, normal wavefront pattern is the one whereby CaiT wavefront follows the AP wavefront along its entire span at all times. We found that in the vicinity of CB sites the abnormal AP/CaiT patterns were either of solitary CaiT, or solitary AP, or AP/CaiT crossover type. The first 2 types were related to an unequal depth of penetration of the respective AP and CaiT wavefronts into a CB area. Examples of a solitary AP and solitary CaiT can be seen in the second and the third lower waves in Figure 3E-I. AP/CaiT crossover occurred when an AP-triggered CaiT wave apparently short-circuited the CB area, while its master AP wave took a longer path around that CB area. As a result, the CaiT wave transiently appeared ahead of the respective AP wave. Two examples of AP/CaiT crossover can be seen in the first and in the last waves shown in Figure 3E-III. Note that in the respective dual complexes recorded from pixel 3 (the first and the last complexes inside the dashed rectangle "II & III" in Figure 3D), the CaiT upstroke appears to be ahead of the respective AP upstroke (phase inversion), which could lead us to an erroneous assumption of a non–voltage-gated CaiT in the absence of spatial information. Association of such phase inversion with the CB sites was confirmed using an alternative definition of the AP-CaiT phase difference based on Hilbert transform (see supplemental Figure VI for details).
Abnormal AP/CaiT Wavefront Sequences Outside the CB Sites
In rare occasions we observed abnormal AP-CaiT wavefront sequences which could not be associated with CB sites. Figure 4A presents an example of "solitary AP" event (ie, CaiT block) not associated with a CB site (arrows). Figure 4B shows examples of an "AP/CaiT crossover" (left oblique arrow) and a "CaiT breakthrough" (right oblique arrow). "AP/CaiT crossover" patterns not associated with CB sites occurred in areas of slow propagation, so that in a few pixels the starting point of the CaiT upstroke "broke ahead" of the starting point of the AP upstroke for 1 or 2 frames. However, the geometry of the dual wavefronts before and after this temporary event maintained the normal master–slave configuration (not shown). We counted total of 6 apparent "CaiT breakthrough" patterns in all 7 experiments. None of those caused a detectable disturbance (eg, conduction delay or block) in the following AP wavefront. The case shown in Figure 4B and 4C is the only one where CaiT breakthrough immediately precedes an AP wave (small deflection in Cai signal preceding the AP upstroke denoted with right oblique arrow in Figure 4B). Figure 4C shows selected snapshots from the respective AP/CaiT wavefront movie. Initially (0ms), a normal AP/CaiT wave invades the field of view from the upper-right corner. At 10 ms, the AP wave is blocked immediately beyond the site of the pixel recording. The AP wave rotates around the area of block and propagates further out of the field of view. CaiT wave does not invade the area around the pixel of interest (20 ms). After a silent period (43 ms), there is a small CaiT breakthrough (50 ms), which is immediately followed by an AP breakthrough (53ms). An instant later (60 ms), the AP wave is breaking ahead of CaiT wave toward the left side of the field. Finally, a large AP/CaiT wave incoming from the upper right corner invades the region (66 ms) and fuses with the apparently focal AP wave which was preceded by the CaiT breakthrough. A possibility that this unique occurrence of focal CaiT preceding an AP represents a DAD-like ectopic depolarization is analyzed in the Discussion.
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Abnormal AP/CaiT Events Summary
Figure 5A shows the distribution of different types of abnormal AP/CaiT wavefront sequences near and far from the CB sites. It can be seen that 90.2% of all abnormal events are associated with the CB sites ("solitary AP", 43.2%; "solitary CaiT", 39.5%; AP/CaiT crossover, 7.5%). In contrast, only 9.8% of the abnormal events could not be associated with CB sites. Among those the "AP/CaiT crossover" type was the most abundant (6.6%), followed by cases of "solitary AP" (2.8%). Finally, "CaiT breakthrough" patterns were extremely rare (0.4%) and, as previously mentioned, they did not have any detectable impact on the subsequent AP wavefronts.
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Because the abnormal AP/CaiT events are predominantly associated with the CB sites, an increase in wave fragmentation should lead to an increase in the abnormal AP/CaiT events. In general, CB lines are associated with WBs which can be identified as SPs in phase movies (this is explained diagrammatically in supplemental Figure I). Thus, it is not unexpected that there was a significant correlation between the total number of abnormal AP/CaiT events and the total number of SPs identified in each heart (R2=0.90, P=0.0013, Figure 5B). Note however, that this correlation does not indicate whether abnormal AP/CaiT wavefront sequences are a cause or a consequence of wave fragmentation during VF.
VF Dynamics in the Presence of BAPTA-AM
To investigate mechanistically a possibility that the dynamics of Cai cycling contributes to wave fragmentation during VF as previously proposed,14 we buffered Cai with BAPTA-AM. The pixel recording in Figure 6A shows that BAPTA-AM markedly reduced the CaiT signal amplitude but did not change the amplitude of the AP complexes. In the presence of BAPTA-AM, the residual level of CaiT signal in general was approaching noise level and did not form any propagating waves (supplemental Movie I). Despite virtual abolishment of the CaiT signal, the 2 snapshots of AP phase movies during VF before an after BAPTA-AM (Figure 6B) show multiple wavelets flanked by SPs under both experimental conditions (supplemental Movies II and III). Identification of SP in all hearts perfused with BAPTA-AM demonstrated that WB formation was not reduced after chelating Cai. Figure 7A shows a total SP number normalized to the total area before and after BAPTA-AM in all experiments (0.76±0.19 versus 0.72±0.19 SP/mm2, P>0.05). Interestingly, BAPTA-AM caused a small but significant reduction in the DF of excitation (12.2±1.6 to 10.4±1.3 Hz, P<0.01, Figure 7B). As a result, SP incidence normalized to DF showed a small increase after BAPTA-AM which however did not reach statistical significance (0.018±0.005 SP/(Hz x mm2) versus 0.021±0.007 SP/(Hz x mm2), P>0.05). Figure 7C shows that BAPTA-AM significantly reduced the maximal amplitude of CaiT averaged over the mapped area (to 30.5±12.9% of control, P<0.002). We used the developed pressure in the LV as an index of BAPTA-AM effectiveness independent of fluorescence imaging. Figure 7D shows that BAPTA-AM reduced the LV pressure to 12.2% of control (P<0.001) in hearts loaded with RH237 and Rhod-2, whereas the contractility was practically unaltered in the sham experiments.
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This study sheds light on the nature of dissociation between the AP and CaiT during VF14,24 and its possible role in VF maintenance. Specifically, it shows that abnormal AP/CaiT wavefront sequences are restricted to CB sites, giving rise to apparently non–voltage-gated CaiT events, which can however be traced to a region outside of the CB area where the AP leads CaiT. Further, it shows that buffering CaiT with BAPTA-AM does not reduce incidence of WBs. Taken together, these findings speak against the presence of DAD-like depolarizations during VF and diminish the possible role of intrinsic Cai dynamics2 in WB formation.
AP/CaiT Coupling During VF
Our data clearly show that during VF CaiT closely tracks the AP in the vast majority of VF cycles despite the high excitation frequencies (11 to 15 Hz). The predominant time lag between the AP and CaiT upstrokes measured during VF in our study is in the range of previously reported values (11 to 26 ms) in isolated porcine myocytes steadily paced at frequencies 0.5 to 1 Hz.25 Values outside this range during VF were most likely attributable to the spatial effects observed at the CB sites (see Figure 3 in the main text and supplemental Figure III). Nevertheless, we cannot exclude that the delay of CaiT with respect to the AP upstroke is altered during activation at high frequencies. In any case, the normal triggering mechanism of CaiT seem to be largely preserved in our model of VF.
Omichi et al14 used MI as a measure of coupling between the AP and CaiT. In particular, these authors reported that during VF the average MI measured as the function of time shift between the AP and CaiT signals in selected locations was not different from a random relationship. Although the main conclusions of our study are not based on the MI measurements, we have performed a limited analysis of MI presented in supplemental Figure VII. We measured MImax which represents the best possible match between the shape of the AP and the CaiT, minimizing the influence of the time delay inherently present between the 2 waveforms. Indeed, the time lag at which MImax was achieved agreed well with the CaiT delay determined by the analysis of the AP and CaiT upstrokes (not shown). On the contrary, averaging MI over a range of time lags different from the optimal one enhances the influence of non-periodic modulations of both AP and CaiT signals, which clearly occur during VF. Although in our experiments MImax was heterogeneous in different pixels (supplemental Figure VII), in all AP/CaiT recordings obtained from each single pixel in 7 experiments, MImax values was higher than the respective values after random shuffling of the AP signal in respective pairs (supplemental Figure VII). To our opinion, these data cast serious doubts with regard to the notion of predominantly random relationship between the AP and CaiT during VF.14
Omichi et al14 also reported large differences in the DF distribution measured in the AP and CaiT movies, whereas in our experiments the respective DF distributions were very similar. The average pixel-to-pixel absolute difference of AP and CaiT DF values in our study was 0.12±0.09Hz versus 5.02±1.22 Hz reported by Omichi et al.14 These discrepancies may be explained by differences in the experimental model, although the species is the same (pig). In particular, Omichi et al14 used a low Ca2+ concentration in the perfusate (0.54 mmol/L) to reduce the motion artifact. For that purpose, we used a short-duration mechanical restraint, but we perfused the heart with blood containing normal Ca2+ concentration (1.8 mmol/L). In addition, we mapped the LV in a whole blood-perfused heart, instead of an isolated RV preparations perfused with crystalline solution. For these or other reasons, it is possible that in the Omichi et al14 study the intensity of wave fragmentation (and hence the incidence of WB and CB) was much larger than in our model, so that the irregular AP/CaiT sequences were the prevailing pattern everywhere in the preparation. Lack of WB incidence statistics in the report by Omichi et al14 prevents us from testing this assumption. However, the diversity of the dynamics of electrical waves during VF under different experimental conditions is clearly recognized nowadays18,26,27 leading to a possibility that Cai dynamics during VF may also depend on even subtle differences in the experimental conditions. Thus we should emphasize that the results presented here are specific to VF electrically induced in the intact, normoxemic, blood-perfused porcine heart. This pattern may not be universal in different species and under different experimental conditions. In particular, the degree of coupling may be different in the presence of ischemia or cardiac disease, a common context of VF in the clinical setting.
Dissociation Between the AP and CaiT Wavefronts at CB Sites
A tight coupling between the AP and CaiT during cell depolarization was markedly reduced in the vicinity of CB sites. Importantly, the spatial extent of the AP/CaiT dissociation was restricted to the immediate vicinity (1 to 3 pixels or 0.5 to 1.5 mm) of the CB sites. In contrast, Omichi et al14 reported examples of complete dissociation between the AP and CaiT waves in much larger area (30x30 mm, see their Figure 6A). As we mentioned above, we cannot exclude that in Omichi et al14 study the incidence of WB and CB was much larger than in our study, so that the disorganized patterns associated with CB/WB sites were prevalent.
Our systematic analysis of the AP/CaiT phase in space and time revealed that AP/CaiT dissociation at the CB sites was attributable to an apparently random penetration of the AP and CaiT waves into the blocked area. A very detailed pixel-to-pixel analysis of one example showed that the uncorrelated waves inside the region of CB could be traced back in time to normal sequences of AP/CaiT waves outside of this region (see supplemental Figure III). These observations suggest that, in our model of VF, the AP/CaiT dissociation is a consequence rather than a cause of wave fragmentation.
The relevance of the observed variable penetration of the AP and CaiT waves into CB sites (see Figure 3 in the main text and supplemental Figure III) remains uncertain. It is important to emphasize that the difference in penetration of AP and CaiT waves into CB sites were never more than 1 to 3 pixels in size (0.5 to 1.5 mm). This may be at the limit of the effective spatial resolution of conventional wide field, epi-fluorescence optical mapping, which is determined by photon diffusion in the depth of the myocardium.28,29 The integration of both RH237 and Rhod-2 fluorescence signals through subepicardial layers could potentially distort our results. Because RH237 emits at a longer wavelength than Rhod-2, the fluorescence representing the AP signal could have been collected from a larger depth than the fluorescence representing CaiT. Larger contribution of deeper layers of tissue to the AP signal as compared with the CaiT signal could have affected the surface manifestation of AP/CaiT dissociation. This seems unlikely, however, given that we observed a similar prevalence of "solitary AP waves" and "solitary CaiT waves" (see Figure 5A). Nonetheless, we cannot exclude that the presence of low-amplitude CaiT waves at the CB sites was underestimated as compared with AP waves. In addition, spatial integration of the optical signals in our system could have lead to "averaging out" abnormal Ca2+ events at the spatial scale less than approximately 0.5 mm. However, if microscopic Ca2+ responses during VF were predominantly abnormal, it would lead to a global macroscopic dissociation between the AP and CaiT waves, which was not the case in our study.
At a more conceptual level, we should note that the nature and fine structure of CB sites during VF is not fully understood. It is customary in the field to use interchangeably terms "conduction block", "wavebreak", and "the core of a spiral wave". However, these are not necessarily the same. Whereas some of CB sites could be identified with a reentrant circuit (and therefore formally corresponded to the "spiral wave core"), other CB sites corresponded to incomplete reentry, and still other CB sites corresponded to "propagation sinkholes" where no reentrant circuit could be identified. The nature of the core of a scroll wave is also far from understood. According to one concept, the spiral wave core is a 2-dimensional area which is at rest, ie, it is "not excited but excitable".30 However, another computational study demonstrated that the core of a spiral wave can have a circular or linear shape depending on the parameters of the excitable medium.31 Experimentally, an excitable core was demonstrated only under very specific conditions of atrial tachycardia, with a single fixed reentrant circuit in the mapped region.32 However, our own experience (eg, supplemental Figure III) as well as data by others23 do not provide evidence for core areas which are at rest. Rather, a common observation is the presence of an elongated central area of block33 which is continuously depolarized because of variable penetration of the reentrant wave toward the center of the blocked area. Regardless of the true nature of the spiral wave core, our data indicate that the penetration of Cai waves inside the spiral core region may be different from penetration of the AP waves. Regenerative Cai waves can propagate through multicellular preparations because of Ca2+-induced Ca2+ release and Ca2+ diffusion between cells.34 Thus, it is possible that during VF Ca2+ waves might propagate beyond the site where the AP wave is blocked thus contributing to the observed AP/CaiT dissociation. Further progress in understanding the nature of the AP/CaiT dissociation during VF will most likely require spatial resolution beyond the limit of the conventional wide-field illumination optical mapping technique, such as the resolution achievable with confocal imaging systems.35
Does Spontaneous Ca2+ Release Play a Role in VF Dynamics?
Intracellular calcium overload-induced spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) causes afterdepolarizations which can trigger action potentials and ectopic arrhythmias.15 In ferret hearts perfused with crystalline solution, pacing induced VF lead to a rapid and marked increase in Cai.36 Others demonstrated that manipulations leading to Cai overload readily induced VF.37,38 Those studies, however, did not provide definitive data as to the role of Cai oscillations in the maintenance of VF (this topic is discussed in Ref. 14). A number of computational studies suggested a possibility for a significant contribution of spontaneous SR Ca2+ release secondary to Ca2+ overload into the destabilization of spiral waves either during the initiation or maintenance of VF, depending on certain parameters of Cai formulations used in those studies.13,14,39,40 On the experimental side, Omichi et al14 speculated that "the failure of Cai to faithfully track Vm during VF might be explained by spontaneous Ca-induced Ca2+ release becoming dominant over Ca2+ release triggered by the L-type Ca2+ current during the AP".
Whereas direct quantitative comparison of our data with numerical and experimental results discussed above is hardly possible, we can discuss the extent at which our data are consistent with the proposed role of spontaneous Ca2+ release during VF. We assert that the following experimental observations would support an active role of spontaneous Cai oscillations in the mechanism of VF: (1) identification of non–voltage-gated Cai events during VF; (2) effects of non–voltage-gated Cai events with respect to the following excitation wavefront; (3) a decrease or elimination of wave fragmentation after abolishment of CaiT. Our data does not support any of these notions. We found only 6 "CaiT breakthrough" patterns in all 7 experiments (0.4% of all abnormal CaiT events). Neither of those had any detectable influence on the propagation of the following AP wave. In only one case we observed a "CaiT breakthrough" which preceded an AP wave (Figure 4), which is topologically compatible with a DAD-like ectopic activation. However, in that case the CaiT breakthrough appeared in the area not invaded by either the AP or CaiT wave in the previous cycle (Figure 4C). This raises a possibility that the apparent CaiT breakthrough in that case is associated with a 3-dimensional reentry, which would make it similar to "AP/CaiT crossover" events we observed in clear cases of reentry on the surface (see Figure 3 in the main text and supplemental Figure III). Even if the case in discussion is a true DAD-like event, the rarity of this event speaks against its importance in the maintenance of VF in our model. Finally, we found that abolishing CaiT with BAPTA-AM did not alter significantly the number of WBs during VF. BAPTA-AM was previously shown to eliminate Ca2+ overload-induced DADs in paced ferret papillary muscles,41 and completely inhibited the onset of APD alternans in rapidly paced rabbit myocytes.11 Thus, failure of BAPTA-AM to suppress wavebreak speaks against the idea that the dynamics of Cai fluctuations and the related changes in the APD are major factors of VF maintenance.2,13,14
It is important to emphasize that the results obtained with BAPTA-AM allow us to dissect the relative role of specific components of the Cai handling system in the VF dynamics. CaiT can influence the AP during VF mainly through Ca-induced inactivation of ICa,L and activation of the NCX.2 Buffering of the global Cai concentration with BAPTA-AM should effectively eliminate the feedback mechanism involving the NCX. BAPTA may also eliminate a small component of Ca-dependent inactivation of ICa,L by the bulk cytosolic Cai42 (which might explain a slight decrease in VF excitation frequency we observed in the presence of BAPTA-AM, see Figure 7B). However, buffering of the global Cai does not affect the main component of Ca2+-dependent inactivation of ICa,L caused by Ca2+ influx through ICa,L and by Ca2+ release from the SR.43 In contrast to minor effects on VF caused by buffering CaiT with BAPTA-AM in our study, blockade of ICa,L in previous studies led to very significant stabilization of rotors during VF, culminating in complete elimination of wave break and conversion of VF into monomorphic VT.27,45 This suggests that, among possible feedback mechanisms between CaiT and the AP during VF,2,27,44,45 the mechanism involving inactivation of ICa,L predominates over the mechanism involving the NCX.
Limitations
In addition to limitations mentioned above in relation to specific topics of the discussion, we should mention the following. Our conclusions are based on dual AP and CaiT recordings from the subepicardial layers, and thus we cannot exclude the possibility that in the depth of the tissue or on the endocardium, the relationship between AP and CaiT is different from that recorded in the present study. We did not specifically address the role of Cai cycling during VF initiation, because we focused on the established steady-state phase of VF. Although suppressing of CaiT by BAPTA-AM did not prevent immediate induction of VF by a brief application of direct current, we cannot exclude that in different modes of VF initiation, intact Cai dynamics is more critical for the onset of VF. Lastly, our study was performed in normal hearts. Alteration of Cai dynamics related to ischemia or heart failure could change the relationship between the AP and CaiT during VF. Further studies are needed to resolve these issues.
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Acknowledgments |
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Sources of Funding
This study was supported by American Heart Association Scientist Development Grant 0230281N and Nora Eccles Treadwell Foundation Research Grant (to A.V.Z.).
Disclosures
None.
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Footnotes |
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Original received January 24, 2007; resubmission received August 28, 2007; revised resubmission received September 27, 2007; accepted October 1, 2007.
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