Published online before print September 13, 2008, doi: 10.1161/CIRCEP.107.758110
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Original Articles |
Function of Ca2+ Release Channels in Purkinje Cells That Survive in the Infarcted Canine Heart
A Mechanism for Triggered Purkinje Ectopy
From the Department of Pharmacology (M.H., W.D., P.A.B.), Center for Molecular Therapeutics, Columbia University, New York, NY; University of Calgary (H.E.D.J.K), Calgary, Alberta; and Department of Medicine/Biomedical Sciences (B.D.S.), Memorial University, St John’s, NL, Canada.
Correspondence to Dr Penelope A. Boyden, Department of Pharmacology Columbia College of Physicians and Surgeons, 630 West 168th St, New York, NY 10032. E-mail pab4{at}columbia.edu
Received December 4, 2007; accepted August 8, 2008.
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Abstract |
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Background— Triggered Purkinje ectopy can lead to the initiation of serious ventricular arrhythmias in post–myocardial infarction patients. In the canine model, Purkinje cells from the subendocardial border of the healing infarcted heart can initiate ventricular arrhythmias. Intracellular Ca2+ abnormalities underlie these arrhythmias, yet the subcellular reasons for these abnormalities remain unknown.
Methods and Results— Using 2D confocal microscopy, we directly quantify and compare typical spontaneous Ca2+ events in specific subcellular regions of normal Purkinje cells with those Purkinje cells from the subendocardium of the 48-hour infarcted canine heart (IZPCs). The Ca2+ event rate was higher in the subsarcolemmal region of IZPCs when compared with normal Purkinje cells; IZPC amplitudes were higher, yet the spatial extents of these events were similar. The amplitude of caffeine-releasable Ca2+ in either the subsarcolemmal or core regions of IZPCs did not differ from normal Purkinje cells, suggesting that Ca2+ overload was not related to the frequency change. In permeabilized Purkinje cells from both groups, the event rate was related to free [Ca2+] in both subsarcolemmal and core, but in IZPCs, this event rate was significantly increased at each free Ca2+, suggesting an enhanced sensitivity to Ca2+ release. Furthermore, decays of wide long lasting Ca2+ release events in IZPC’s core were significantly accelerated compared with those in normal Purkinje cells. JTV519 (K201) suppressed IZPC cell wide Ca2+ waves as well as normalized the enhanced event rate and its response to free Ca2+.
Conclusions— Increased spontaneous Ca2+ release events in IZPCs are due to uniform regionally increased Ca2+ release channel sensitivity to Ca2+ without a change in sarcoplasmic reticulum content. In addition, Ca2+ reuptake in IZPCs is accelerated. These properties would lower the threshold of Ca2+ release channels, setting the stage for the highly frequent arrhythmogenic cell wide Ca2+ waves observed in IZPCs.
Key Words: arrhythmias • calcium • myocardial infarction • Ca2+ waves • Purkinje cells
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Introduction |
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Recently, several reports have appeared about the occurrence and ablation attempts of ventricular premature beats in patients with and without structural heart disease.1,2 In some, mapping has implicated the Purkinje system as the cause of these abnormal ventricular beats. In particular, the Haissaguerre group has reported about Purkinje ectopy initiating ventricular tachycardia in patients with Brugada syndrome, long QT and RVOT syndromes. In these studies, ablation of the early Purkinje network activity has been successful.2a More recent reports, in which similar approaches have been taken, suggest that for some clinically mapped ventricular premature beats in patients post–myocardial infarction (MI), the earliest activation occurred in the Purkinje network at the so-called endocardial "border zone."3–5 Ablation at some of these sites successfully reduced electrical storm arrhythmias post-MI as well as those ventricular tachycardias with a focal origin in the distal His-Purkinje system.3,4,6 Thus, triggered Purkinje ectopy can lead to initiation of serious storms of ventricular arrhythmias in post-MI patients.
Clinical Perspective see p 387
Our laboratory and others7 have studied the mechanisms of these arrhythmias in the canine post-MI model. In particular, we have developed the model of the isolated Purkinje cell aggregate from both the normal and 48-hour infarcted myocardium (IZPCs).8,9 These Purkinje myocytes isolated from the subendocardial border zone are the cells that initiate these arrhythmias.10 Like ventricular myocytes, cardiac Purkinje cells respond to an action potential by an overall rise in intracellular Ca2+ ([Ca2+]i). However, in Purkinje cells, Ca2+ rises first just at the periphery of the cell below the membrane (subsarcolemmal [SSL]). This is then followed by an elevation of [Ca2+] in the cell’s core. Such centripetal activation is uniform in normal Purkinje cells (NZPCs). In IZPCs, the same initial peripheral Ca2+ increase occurs in response to an action potential but often the subsequent large central Ca2+ elevation does not happen.11,12 Importantly using global epi-fluorescent techniques, we found that such nonuniform Ca2+ activation increased the likelihood of IZPCs generating large cell wide Ca2+ waves, which underlie delayed afterdepolarizations, spontaneous action potentials, and arrhythmias.11 However, these studies did not provide precise information about the altered subcellular regional Ca2+ release events in Purkinje cells. One probable cause is a fundamental and regional change in sarcoplasmic reticulum (SR) Ca2+ release elements. Therefore, in this study, we have examined using the enhanced spatial resolution of confocal microscopy, IZPCs to quantify directly the release of Ca2+ from the SR at sites in the SSL area as well as core. Thus, we compared the local Ca2+ release events in different subcellular regions of NZPCs and IZPCs using confocal microscopy and fluorescence Ca2+ imaging techniques.
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Methods |
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Preparation of Intact Purkinje Cell Aggregates
MI was induced in the dog13 and Purkinje cells were prepared as described before.11,12,14 Aggregates of 2 to 6 cells were enzymatically dispersed from the Purkinje network of canine left ventricle and were placed in a perfusion chamber on the stage of inverted microscope equipped, respectively, with a rapid 2D confocal spinning disk system. Fluorescence was measured only in rod-shaped Purkinje cells with typical junctional ends, clear striations, and membranes free of blebs.11,15 Cells were superfused with Tyrode solution ([mM]: 137 NaCl, 24 NaHCO3, 1.8 NaH2PO4, 0.5 MgCl2, 2CaCl2, 4 KCl, 5.5 dextrose, pH 7.4, 24°C) for
15 minutes before the experiment.
Preparation of Permeabilized Purkinje Cells
Intact cells were superfused with Tyrode containing 2 mmol/L Ca2+ for 10 minutes. Tyrode solution was then replaced with a Ca2+ free "internal solution" (see below) for 10 minutes before cells were permeabilized by exposure to 0.01% saponin in a Ca2+ free mock intracellular solution (60 s). Fluo 4-K+ salt [Ca2+] containing intracellular solution was then superfused for at least 5 minutes. The composition of the internal solution was (mM): 100 potassium aspartate, 15 KCl, 5 KH2PO4, 0.5 EGTA, 0.75 MgCl2, 5 MgATP, 10 phosphocreatine, 10 HEPES, 5 U/mL creatine phosphokinase, and 8% dextran, with a pH of 7.2. Solutions with different free [Ca2+] were prepared by adding appropriate amounts of CaCl2. The free [Ca2+] and [Mg2+] were calculated using a computer program (MAXChelator 2.50), and the free [Mg2+] was adjusted to 1 mmol/L. The free [Ca2+] was verified using calcium calibration buffer kits (Molecular Probes).
Confocal Ca2+ Imaging
Spatial and temporal characteristics of local Ca2+ events were measured in Purkinje cell aggregates by using 2D spinning disk (Nipkow) confocal microscopy.15 Ca2+ concentration was monitored using the fluorescence of the Ca2+ indicator Fluo-4. The cells were loaded with 5 µmol/L Fluo-4AM for 30 minutes and washed for 15 to 20 minutes with Tyrode solution. The intensity of the Ca2+-related fluorescence was captured from the illumination field and light intensity was collected using a fast charge-coupled device camera (ORCA-ER C4742-95, Hamamatsu Photonics KK, Japan). The system was attached to the video port of an inverted microscope equipped with 60x oil objective lens. In the 2DCM system, the fluorescence images were captured at 17 to 30 fps. [Ca2+]i was estimated from the pixel-to-pixel ratio F/Fo (F, intensity of instantaneous fluorescence; Fo, intensity of reference fluorescence) and analyzed using custom IDL programs (IDL 5.4, Research Systems, Boulder, Colo). In caffeine experiments, [Ca2+]i was given by Kd(F/F0)/[Kd/[Ca2+]i,rest +1-F/F0]·[Ca2+]i rest.
Data Analysis and Processing
Apparent nonpropagating events were detected and selected for counting as follows: 20 consecutive frames of the 2D imaged intact cell or 33 frames of a permeabilized cell (F images) were divided by basal fluorescence image (F0 image) to make ratio images (F/F0)15 (Figure 1). The basal fluorescence image was made by averaging all frames. Ca2+ release events were defined as elevations of basal [Ca2+] equivalent to F/F0 3.4 SD over F0 using a computer-based detection algorithm modified from Cheng et al.16 Only frames free of Ca2+ waves were analyzed with the exception of some of the JTV519 (K201) studies. The events were counted, confirmed visually, and compared with the results of the computer detection. The sensitivities were 95.3±2.3% in intact cell (n=8) and 97.4±0.3% in permeabilized cell (n=5). Probabilities of a detecting false event were 6.0±2.3% in intact cell and 4.8±0.6% in permeabilized cell.
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Determination of Event Location and Measurements
Regions of interest (ROIs) were drawn on a cell image and then superimposed on binary cell images to determine the subcellular location (SSL or core) of events (ImageJ, National Institutes of Health) (Figure 1). Amplitudes of events were calculated from ratio images. Spatial extent of an event and the event rate were calculated from binary images using IDL functions (IDL 6.0, Research Systems). Typical events (TEs) were defined as those nonpropagating events occurring at the same site within one frame.15
Caffeine Experiments
SR-Ca2+ load was estimated in intact Purkinje cells by rapid exposure of a cell to Tyrode solution containing 20 mmol/L caffeine, 10 mmol/L BDM, and 30 µmol/L tretrodotoxin. The regional SR-Ca2+ load was measured in the SSL and core and was given by the peak of the caffeine-induced Ca2+ transient. A similar protocol was used to estimate SR-Ca2+ load in a subset of saponin-treated Purkinje cells at 3 different free [Ca2+]. In some experiments, solutions were supplemented with 1 mmol/L tetracaine to block Ca2+ loss through ryanodine receptors.17
JTV519 (K201) Protocol
The events in the absence and presence of JTV519 (K201) (1 µmol/L) were determined by 2D imaging of aliquots of cells in the absence and after 10 minutes superfusion with JTV519 (K201) from each of several preparations. JTV519 (K201) was provided by Aetas Pharmaceuticals.
Statistics
For comparisons of data of this study, SPSS (version 11.0.1J, SPSS Inc) and R (GNU, http://www.r-project.org/) were used. The Shapiro-Wilk test was performed to test normal distribution. When the distribution was normal, the results were described as mean±SE. When the distribution was not normal, the results were described as quartile deviation (median [25th percentile, 75th percentile]). The Levene test was performed to test homoscedasticity. In Figures 2A, 2B, 3B, 4A, 4B, and 5
A, a 2-way ANOVA was first completed, and if appropriate, a post hoc test (Tukey post hoc test, Kruskal-Wallis test, and Steel-Dwass test) was carried out (see figure legends). In Figures 2A, 2B, 3B, 4A, 4B, and 5A, an interaction was not observed between cell types (NZPC and IZPC) and location (SSL and core). In Figure 4B, an interaction was not observed between drug and location, drug and cell types.
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All authors had full access and take responsibility for the integrity of these data. All authors have read and agree to the manuscript as written.
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Results |
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Intact Cell Data
In intact cells at [Ca2+]o (2 mmol/L), the majority (94% NZPCs; 92% IZPCs) of spontaneous Ca2+ release events (TE) were detected within a single frame. The TE rate in all regions of IZPCs (0.00428 ev/µm2/s [0.00178, 0.00741]) was significantly higher than that in NZPC (0.00222 ev/µm2/s [0.00118, 0.00590]; P<0.05, Mann-Whitney test). The cell areas were not different between 2 cell groups (4414±170 versus 4888±268 µm2; P=0.142, unpaired t test). To investigate the subcellular regionality of the event rates, SSL and core region were independently analyzed in both cell types (Figure 2A). The TE rates in core were not different between NZPCs and IZPCs (0.00067 ev/µm2/s [0.00018, 0.00184] versus 0.00143 ev/µm2/s [0.00073, 0.00310]; P=0.270, Mann-Whitney). The TE rate in SSL region of IZPC (1198 events, n=30 cells, 0.01101 ev/µm2/s [0.00447, 0.02818]; black bars, Figure 2A) was significantly higher than that in NZPC (747 events, n=34, 0.00512 ev/µm2/s [0.00152, 0.00889]; P<0.05, Mann-Whitney). TE amplitude in IZPCs was also significantly higher (9% to 10%) than that of NZPCs in both SSL (1.32±0.004 versus 1.21±0.004 F/F0) and core (1.30±0.006 versus 1.18±0.004 F/F0) (Figure 2B), whereas spatial extent did not differ (SSL: 9.35±0.35 versus 9.04±0.40 µm2; core: 9.11±0.59 versus 8.99±0.68 µm2).
To determine whether the observed changes in the incidence of Ca2+ events were due to regional differences in the SR-Ca2+ load, we assessed Ca2+ transients evoked by rapid caffeine exposure (Figure 3). The Ca2+ level was calculated by integrating fluorescence over a selected ROI. The time course of [Ca2+] in the ROI was obtained from serial frames acquired at video rate during the caffeine response. Average [Ca2+] data for each ROI was obtained for every frame. The amplitude of the Ca2+ transient was measured in 3 different ROIs: 2 in the SSL and 1 in the core (Figure 3A). No significant difference in the amount of Ca2+ released by caffeine was detected between the SSL and the core and between NZPCs and IZPCs (Figure 3B). Thus, changes detected in IZPC TE rates were not caused by a regional variation in the (caffeine sensitive) SR Ca2+ content.
Saponin Cell Data
Cytosol and SR luminal Ca2+ levels are both critical factors that affect individual Ca2+ release sites as well as interactions between sites. Therefore, a change in the Ca2+ sensitivity of SR Ca2+ release function could explain the augmented event rate in IZPCs. The Ca2+ sensitivity of SR Ca2+ release was tested by measuring the Ca2+ event rate in permeabilized NZPCs and IZPCs at various free Ca2+ concentrations ranging from 25 to 150 nM. This technique allows for control of cytoplasmic milieu (ionic composition) while maintaining SR structurally and functionally. TEs were counted in the SSL and core of cells from both groups. Figure 4A shows that the relationship between TE rate and free Ca2+ is shifted leftward and upward in both the core and the SSL of IZPCs suggesting a greater sensitivity of Ca2+ release function to cytosolic Ca2+.
To rule out that permeabilization of cells exposed to varying free [Ca2+] affected IZPCs differently than NZPCs, SR-Ca2+ content was estimated using a rapid caffeine (20 mmol/L) application to saponin-treated cells (Figure 4B). Our analyses show that under these conditions, SR content varied depending on free [Ca2+] but did not differ between NZPCs and IZPCs (Figure 4B). However, tetracaine clearly increased caffeine-induced Ca2+ release, while reducing the Ca2+ event rate significantly (NZPCs drug free: 100 nM, TE rate 0.0198±0.01434 ev/µm2/s [n=28]); plus tetracaine: TE rate 0±0 (n=10); IZPCs drug free: 100 nM, TE rate 0.04041±0.0246 ev/µm2/s [n=21]; plus tetracaine: TE rate 0±0 [n=10]).
Other Ca2+ Release Events
Events that lasted over several frames (120 to 300 ms) and had a greater spatial extent and yet failed to propagate within the confocal plane were called "wide, long lasting events" (WLEs)15 (Figure 5). The amplitude and event rate of WLEs in SSL of IZPCs were significantly greater than those in NZPCs (Figure 5A), whereas these events had similar spatial extents. Core and SSL WLE Ca2+ transients decayed with multiple time constants (Figure 5B and 5C). However, in IZPCs, the decay of core WLEs was significantly accelerated compared with that of NZPCs.
In sum, increased spontaneous Ca2+ release TEs in IZPCs seem to result from a uniform increase of Ca2+ release channel sensitivity without a change in SR content. In addition, Ca2+ reuptake in IZPCs is accelerated consistent with other data.18 These properties could set the stage for the arrhythmogenic cell wide Ca2+ waves observed in IZPCs.11
Effects of JTV519 (K201)
We previously reported that JTV519 (K201) can suppress the incidence of cell wide Ca2+ waves in arrhythmogenic IZPCs19 (Figure 6A). Because our results suggest that there is an enhanced Ca2+ sensitivity to Ca2+ release events in IZPCs, we tested whether JTV519 (K201) (1 µmol/L) was effective in altering the regional Ca2+ event rate in both intact and saponin-treated IZPCs. JTV519 (K201) significantly reduced TE rate of intact IZPCs without changing either their spatial extent or the amplitude of caffeine-releasable pools of Ca2+ (Figure 6B and 6C). Furthermore, the TE rate in response to [Ca2+] in saponin-skinned IZPCs was normalized to NZPC values (free [Ca2+]=100 nM) by JTV519 (K201) (Figure 6D).
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Discussion |
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The major findings of this study are that typical Ca2+ events in intact Purkinje cells from the subendocardium of the 48-hour infarcted heart are larger in amplitude and more frequent in the SSL, but not in the core when compared with the same in NZPCs. This increase reflects a distribution of events to a population with a larger amplitude and occurs without a change in regional caffeine-releasable Ca2+ pools. It is unlikely to result from a larger membrane Ca2+ influx because global ICaT and ICaL are reduced by 55% in IZPCs.9 Core TE Ca2+ events in IZPCs varied but most were larger and decayed faster than their NZPC counterparts. WLEs in IZPCs were also more frequent, larger in amplitude and decayed faster (Figure 5).
Clearly, fundamental Ca2+ events change in IZPCs without an altered SR content. Hence, we went on to determine whether the threshold for Ca2+ release events was changed in IZPCs using saponin-skinned Purkinje cells under conditions where we manipulated free [Ca2+] bathing the cell ([Ca]cyto). We found that there was a leftward and upward shift in the relation between Ca2+ event frequency and free [Ca2+] in IZPCs (Figure 4A). Again, we determined that under these conditions, caffeine-releasable Ca2+ pools in IZPCs were not lower than those in NZPCs (Figure 4B). However, tetracaine significantly increased these pools, after reducing the event rate. These findings imply that a difference in SR Ca2+-content is probably not the cause of the IZPC increased event rate. On the other hand, the difference in event rate between IZPCs and NZPCs does not seem to have a large effect on the SR Ca-content. It is possible that the SR depleting effect of spontaneous SR Ca2+ release may have been offset by faster SR Ca2+ uptake as is witnessed by the faster decay of the IZPC Ca2+ transients (Figure 5B). JTV519 (K201) normalized the IZPC increased sensitivity to Ca2+ release to NZPC values as it also reduced incidence of the arrhythmogenic cell wide Ca2+ waves (Figure 6A).
An important limitation is that varying [Ca]cyto did not induce Ca2+ release directly but changing [Ca]cyto could increase luminal [Ca]SR which then could elicit Ca2+ release by store operated induced Ca2+ release.20 We did show that the SR-Ca2+ content is similar in IZPCs compared with NZPCs (Figure 3). However, it is still reasonable to conclude that IZPCs are more sensitive to [Ca]cyto and/or the Ca2+ content of the SR than NZPCs, implying that the cytosolic and/or luminal Ca2+ sensitivity of the Ca2+ release channels in the SR of IZPCs may be increased.
Comparison With Other Studies Regarding Sparks in Cells from Diseased Hearts
Most subcellular Ca2+ release studies have been completed using ventricular cells from diseased hearts,21–25 and most findings are consistent with a diseased induced reduction in global cellular Ca2+ transient. Most have suggested that a reduced SR load may be a reason for the reduced spark amplitude in CHF cells and only one study suggests that Ryanodine sensitivity (single-channel recordings) to Ca2+ is enhanced in heart failure cells.26 This latter finding can account for the increase in spark rate as well as the reported reduced SR content in heart failure cells.
To our knowledge, no one has described spontaneous Ca2+ sparks in Purkinje cells from diseased hearts. Our data suggest that typical Ca2+ events in IZPCs differ from those of NZPCs cells in that they are larger, and more frequent than NZPC sparks/events, particularly in the SSL region. In addition, IZPCs sparks occur in a substrate that shows no change in SR content but still has enhanced sensitivity to Ca2+ release. Further, wide long lasting core events in IZPCs show an acceleration in decay suggesting enhanced SR uptake. Thus, defective Ca2+ regulation in IZPCs leads to increased frequency of Ca2+ events; however, SR content is maintained due to enhanced rate of Ca2+ uptake to SR. Clearly, our findings using these diseased Purkinje cells differ from those of ventricular cells and suggest that specific therapeutic modalities could be developed for Purkinje cell Ca2+ dependent arrhythmias.
When we used a drug, JTV519 (K201), to ameliorate the disorder of defective Ca2+ regulation of the SR in intact IZPCs (similar to that shown for expression systems20), we found that this drug not only reversed the enhanced sensitivity to Ca2+ in saponin-treated IZPCs but also reduced the incidence of cell wide Ca2+ waves in the intact IZPCs (Figure 6). An action to reduce the incidence of cell wide Ca2+ waves would be antiarrhythmic and is consistent with JTV519 (K201) being protective in models of ischemia reperfusion27–29 where a disorder of Ca2+ handling has been shown. Also consistent with findings in ventricular cells, we found that JTV 519(K201) has no effect on Purkinje SR Ca2+ load30 yet suppresses excess Ca2+ release events (as inferred from "noise analysis") and cell wide Ca2+ waves in Purkinje cells.19 JTV519 (K201)’s effect in these diseased Purkinje cells is consistent with the theory that this drug binds to specific domains of the ryanodine receptor protein31 to stabilize this Ca2+ release channel’s mediated Ca2+ release.
Limitations
No polymerase chain reaction or protein analysis was completed on these small Purkinje cell samples; thus, we cannot, at this time, detail whether there is profound structural remodeling of proteins in IZPCs. However, since we found JTV519 (K201) to quickly reverse the observed IZPC functional abnormalities, it is unlikely that profound structural remodeling occurs.
We used saponin in some of our studies so we could then "equilibrate" cytosolic Ca2+(Cacyto) and SR Ca2+ and test SR dependence on Cacyto. This required exposing Purkinje cells to saponin to form 30 nm pores in the sarcolemma and expose the SR directly to the superfusion solutions. This technique has distinct advantages over the isolated lipid bilayer ryanodine receptor preparation that studies Ca2+ release channel activity in isolation. We as well as others show that Ca2+ release activity increases in all cell regions under these conditions32 presumably due to the loss of unknown protein/protein interactions. Nevertheless, we saponin-treated NZPCs and IZPCs the same way to complete Ca2+ dependent release experiments and found a difference between the 2 cell types.
Studies here were completed on isolated Purkinje cells and not intact Purkinje tissues. Nevertheless, our findings of abnormal intracellular Ca2+ regulation underlying arrhythmias in Purkinje cells that have survived in the infarcted heart are consistent with those showing the antiarrhythmic effects of agents that alter intracellular Ca2+ release on the triggerability of intact Purkinjes that survive in the infarcted subendocardium.10,19
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Acknowledgments |
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Sources of Funding
This work was supported by grant HL58860 from the National Heart, Lung, and Blood Institute; grants 81150 (B.D.S), 135573, and 104907 (H.E.D.J.t.K.) from the Canadian Institute of Health Research; and grant 20040163 from the Alberta Heritage Foundation for Medical Research, Canada (H.E.D.J.t.K.).
Disclosures
None.
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Related Article
CLINICAL PERSPECTIVE
Recently, premature ventricular contractions originating from the Purkinje system have been shown to be responsible for the initiation of ventricular fibrillation in patients with early post–myocardial infarction, and a radio frequency ablation of the triggering premature ventricular contractions for those patients can prevent ventricular fibrillation. Although the precise mechanisms of the triggering premature ventricular contraction generation had not been shown, our current work on Purkinje cells that have survived in the infarcted heart elucidate one of the cellular mechanisms for these arrhythmias. Also, we show here that a drug that stabilize Ca2+ release channels, JTV-519 (K201), is effective in correcting the abnormality and as such may also be effective for Ca2+ dependent arrhythmias in post–myocardial infarction patients.
Circ Arrhythm Electrophysiol 2008 1: 387-395.
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