Initiation and propagation of ectopic waves: insights from an in vitro model of ischemia-reperfusion injury

Abstract

The objective of the present study was to directly visualize ectopic activity associated with ischemia-reperfusion and its progression to arrhythmia. To accomplish this goal, we employed a two-dimensional network of neonatal rat cardiomyocytes and a recently developed model of localized ischemia-reperfusion. Washout of the ischemia-like solution resulted in tachyarrhythmic episodes lasting 15-200 s. These episodes were preceded by the appearance of multiple ectopic sources and propagation of ectopic activity along the border of the former ischemic zone. The ectopic sources exhibited a slow rise in diastolic calcium, which disappeared upon return to the original pacing pattern. Border zone propagation of ectopic activity was followed by its escape into the surrounding control network, generating arrhythmias. Together, these observations suggest that upon reperfusion, a distinct layer, which consists of ectopically active, poorly coupled cells, is formed transiently over an injured area. Despite being neighbored by a conductive and excitable tissue, this transient functional layer is capable of sustaining autonomous waves and serving as a special conductive medium through which ectopic activity can propagate before spreading into the surrounding healthy tissue.

Fig. 1

Generation of ectopic beats in the border zone. A: diagram on the left shows the experimental field and the injury zone (I-zone). The I-zone (shown in gray) was created by flows of control and ischemia-like solutions using a custom-designed superfusion chamber (2). Recordings of calcium transients (CaTs) were obtained from the border zone, inside the blue box shown on the left. The pseudocolor reflects increasing calcium concentrations as measured by fluo 4 fluorescence. a, Image was obtained during application of injury (I) solution. It shows a propagating wave of CaTs (CaT wave) that travels through the control area but does not penetrate into the I-zone. Black arrows point in the direction of CaT wave propagation. b, Appearance of ectopic clusters upon initiation of reperfusion. Three large clusters and a small one can be seen in this frame. c, Spreading of ectopic activity into surrounding network. Black arrows point in the direction of CaT wave propagation. B: A, b was used to plot the intensity profiles for regions where ectopic activity was observed. Two rectangular regions with their corresponding profiles are shown on the left. The position of these regions relative to the rest of the field can be seen on the right. The white dotted line marks the border of the I-zone.

Fig. 2

Progression of ectopic CaTs into arrhythmic episodes. A: diagram of the experimental field. Recordings from the border area inside the blue box are shown in B and were used to create the isochronal maps in C. B: initial stages of propagation of ectopic activity. Three consecutive images show the initial stages of ectopic cluster expansion. Elapsed time (in s) is shown in the boxes. The pseudocolor reflects increasing calcium concentrations. C: isochronal maps illustrating the growth and spread of ectopic activity. Each isochronal map is composed of several sequential frames (110 ms between frames) with the areas of increased intracellular calcium ${(\text{Ca}}_{\mathrm{i}}^{2+})$ depicted in color. The colors correspond to the times shown on the right. The isochronal map on the left shows expansion of the ectopic activity presented in B. At that time (~219 s), the activity failed to spread into the surrounding cell network, and the event presented in this isochronal map is reflected as a distinct ectopic beat by the corresponding CaT trace. The following ectopic beats, including the one illustrated on the right isochronal map, propagated into the control network, causing an arrhythmia. D: CaTs collected from the control and ischemic areas (black and red squares in C). Dotted lines indicate timing of pacing stimuli. Initial 10 s of the recording shows that, during local ischemia, cells within the control network (black trace) exhibited regular CaTs, whereas cells within the I-zone were silent (red trace). Upon reperfusion, ectopic activity from the border zone (red trace, with arrows pointing to the individual beats) spread into the control network, causing an arrhythmia. *Additional peaks on the control trace between pacing stimuli, which were caused by the spread of ectopic activity from the border zone. Shaded regions correspond with the isochronal maps shown in C. AUF, arbitrary units of fluorescence.

Fig. 3

Changes in diastolic ${\text{Ca}}_{\mathrm{i}}^{2+}$ associated with ectopic activity. A: two traces from control (black) and border (red) zones illustrating an entire tachyarrhythmic episode are shown. Reperfusion was initiated at 550 s. The black arrow points to the first ectopic beat. B: relative position of the five regions of interest that used to acquire the traces on the right. The traces (shown in corresponding colors) reveal individual CaTs during the initial and late stages of the tachyarrythmic episode. The arrows point to the respective timing of the selected intervals. The red arrows point to a positive slope in diastolic ${\text{Ca}}_{\mathrm{i}}^{2+}$ observed in ectopic regions. C: the averaged diastolic slope of CaTs from the ectopic regions (red column) was significantly greater (P = 0.003) than that from the control zone (black column). Fluo 4 fluorescence was converted to ${\text{Ca}}_{\mathrm{i}}^{2+}$, as described in materials and methods, to calculate these slopes.

Fig. 4

Repetitive injury increases the incidence of reperfusion arrhythmias. A: multiple brief superfusions with a heptanol-free ischemic solution increased the incidence of reperfusion arrhythmias in proportion to the number of injury events. Statistical data are from 35 experiments. B, top: continuous CaTs collected from the control zone (black) and I-zone (red) during six consecutive injury events, each consisting of 10 min of ischemia, followed by 10 min of reperfusion. The top traces are rather compressed and mainly illustrate changes in the relative amplitudes of CaTs. Acidic pH of the I-solution resulted in a downward shift of the fluo 4 baseline signal, due to its known pH dependence (28). The two insets below have an expanded time scale and reveal individual CaTs and changes in frequency. Specifically, the left inset shows that reperfusion after the second injury event recovers CaTs in the I-zone without changing the rhythmic control pattern. The right inset shows that reperfusion after the fifth injury event failed to restore CaTs inside the I-zone and induced an arrhythmia in the control zone. In this particular experiment, arrhythmic episodes occurred after the fourth, fifth, and sixth injury events (*).

Fig. 5

Border zone propagation. Consecutive frames illustrating the propagation of ectopic activity along the border zone during a microreperfusion protocol are shown. In this protocol, only a narrow, 300-µm width of border zone was reperfused with control Tyrode solution, whereas the remaining I-zone cells continued to be superfused with I-solution. A: microreperfusion caused the appearance of ectopic CaTs, which propagated alongside the border zone with an apparent speed of 0.1–0.4 cm/s. This sequence illustrates a wave of CaTs that receded without activating the surrounding control zone. B: spread of CaTs into the surrounding control network, where they were propagated at a faster velocity of 6–9 cm/s. A complete sequence of events can be seen in the online supplement.