Function of Ca(2+) release channels in Purkinje cells that survive in the infarcted canine heart: a mechanism for triggered Purkinje ectopy

Abstract

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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) in either the subsarcolemmal or core regions of IZPCs did not differ from normal Purkinje cells, suggesting that Ca(2+) overload was not related to the frequency change. In permeabilized Purkinje cells from both groups, the event rate was related to free [Ca(2+)] in both subsarcolemmal and core, but in IZPCs, this event rate was significantly increased at each free Ca(2+), suggesting an enhanced sensitivity to Ca(2+) release. Furthermore, decays of wide long lasting Ca(2+) release events in IZPC's core were significantly accelerated compared with those in normal Purkinje cells. JTV519 (K201) suppressed IZPC cell wide Ca(2+) waves as well as normalized the enhanced event rate and its response to free Ca(2+).

Conclusions: Increased spontaneous Ca(2+) release events in IZPCs are due to uniform regionally increased Ca(2+) release channel sensitivity to Ca(2+) without a change in sarcoplasmic reticulum content. In addition, Ca(2+) reuptake in IZPCs is accelerated. These properties would lower the threshold of Ca(2+) release channels, setting the stage for the highly frequent arrhythmogenic cell wide Ca(2+) waves observed in IZPCs.

Keywords: Ca2+ waves; Purkinje cells; arrhythmias; calcium; myocardial infarction.

Figure 1

Typical 2D image of an isolated canine Purkinje cell. Amplitude of the event was calculated from ratio images. Spatial extent of the event and the event rate were calculated from binary images using IDL program (IDL6.0, Research Systems). In ratio images, arrows indicate wide long lasting event (WLE) and arrow heads indicate typical events (TEs) (Panels A, B). The amplitude and spatial extent of WLEs were markedly larger than those of typical events. A region of interest (ROI) was drawn on a confocal image and then superimposed on binary images to determine the subcellular location (SSL or Core) of events using ImageJ. N indicates nucleus.

Figure 2

Panel A shows event rate of TEs in NZPCs (white bars) and IZPCs (black bars) by region (Core and subsarcolemmal, SSL). The data are described as quartile deviation (Median, 25 percentile, 75 percentile). Panel B shows histograms of spatial extent and amplitude of TEs in NZPCs (white bars) and IZPCs (black bars) by region (Core, SSL). n= number of cells. Mann-Whitney test was used for event rate and spatial extent comparisons. Unpaired student-t test for statistics of the amplitude. NS indicates not significant.

Figure 3

Panel A Typical caffeine induced Ca²⁺ release by region in an NZPC and IZPC. Right tracings are examples from two SSL regions and one Core region in each cell represented. Panel B. Average data are plotted. NS indicates not significant.

Figure 4

Panel A Relationship between average TE rate and free Ca²⁺ concentration in the two cell groups by Region (SSL, Core). Numbers below/above each plotted average indicate number of cells used for average. Mann Whitney test was used at each free Ca²⁺ concentration. Asterisks indicate statistical difference. Panel B. Amplitude of caffeine induced Ca²⁺ transient with and without tetracaine (1mM) in saponin treated cells at 3 different free Ca²⁺. Asterisks indicate statistical difference. Unpaired student-t test was used if appropriate after two-way ANOVA. There was no significant difference in cell type (NZPC, IZPC) and location (SSL, Core) in Two-Way ANOVA test. An interaction was not observed between drug and location, drug and cell type. NS indicates not significant.

Figure 5

Panel A shows event rate, spatial extent and amplitude (mean +/-SEM) of wide long lasting events (WLEs) in intact NZPCs (white bars) and IZPCs (black bars) by region (Core and SSL). Numbers in parentheses indicate actual number of events in each group. Mann-Whitney test was carried out for statistics of the WLE event rate. Unpaired t test was carried out for statistics of the amplitude. Panel B. Time constants of decay of WLE events in both intact NZPCs and IZPCs were determined in each event/cell using Clampfit. Average Tau1 and Tau2 values are shown. Note while Tau1 values did not differ between cell groups, Tau2 was significantly reduced in IZPCs. The One-Way ANOVA and Tukey's Post Hoc Test were carried out for the statistics. Panel C. Linear profiles to the right show typical NZPC and IZPC events from both the SSL and Core. NS indicates not significant.

Figure 6

Panel A. Graph shows the incidence of cell wide Ca²⁺ waves (CWW) in NZPCs and in IZPCs in the absence and presence of JTV519 (K201) 1μM (grey bar). The One-Way ANOVA and Tukey's Post Hoc Test were carried out for the statistics. Panel B shows TE event rate, spatial extent and amplitude in IZPCs in the absence and presence of JTV519 (K201) (grey bars). Total number of events used is shown in parentheses. Kruskal-Wallis test and Steel-Dwass test were carried out for the statistics of the event rate and spatial extent. The One-Way ANOVA and Tukey's post hoc test were carried out for the statistics of the amplitude. Panel C shows typical caffeine induced Ca²⁺ release transients by region in an intact NZPC and an IZPC in the absence and presence of JTV519 (K201). The One-Way ANOVA was used. Panel D shows the effects of JTV519 (K201) on event rate in saponin treated permeabilized IZPCs. Note in the presence of JTV519 (K201) IZPC event rate was normalized to that of NZPCs (white bars) (free Ca²⁺ =100nM). Kruskal-Wallis and Steel-Dwass tests were carried out for the statistics. NS indicates not significant.

Figure 6

Panel A. Graph shows the incidence of cell wide Ca²⁺ waves (CWW) in NZPCs and in IZPCs in the absence and presence of JTV519 (K201) 1μM (grey bar). The One-Way ANOVA and Tukey's Post Hoc Test were carried out for the statistics. Panel B shows TE event rate, spatial extent and amplitude in IZPCs in the absence and presence of JTV519 (K201) (grey bars). Total number of events used is shown in parentheses. Kruskal-Wallis test and Steel-Dwass test were carried out for the statistics of the event rate and spatial extent. The One-Way ANOVA and Tukey's post hoc test were carried out for the statistics of the amplitude. Panel C shows typical caffeine induced Ca²⁺ release transients by region in an intact NZPC and an IZPC in the absence and presence of JTV519 (K201). The One-Way ANOVA was used. Panel D shows the effects of JTV519 (K201) on event rate in saponin treated permeabilized IZPCs. Note in the presence of JTV519 (K201) IZPC event rate was normalized to that of NZPCs (white bars) (free Ca²⁺ =100nM). Kruskal-Wallis and Steel-Dwass tests were carried out for the statistics. NS indicates not significant.