2.1.

Optical Mapping of the Isolated Rabbit Heart

Six New Zealand White rabbits (weight of $3\text{to}5\mathrm{kg}$ ) were intravenously injected with 1000 units of heparin and anesthetized with ketamine $(20\mathrm{mg}∕\mathrm{kg})$ and xylazine $(5\mathrm{mg}∕\mathrm{kg})$ . After a midline sternotomy, the heart was harvested, and the ascending aorta was cannulated and perfused with $37°\mathrm{C}$ Tyrode solution (composition in mM: 125 NaCl, 4.5 KCl, 0.25 $\mathrm{Mg}{\mathrm{Cl}}_2$ , 24 $\mathrm{Na}\mathrm{H}\mathrm{C}{\mathrm{O}}_3$ , 1.8 $\mathrm{Na}{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_4$ , 1.8 $\mathrm{Ca}{\mathrm{Cl}}_2$ , and 5.5 glucose) gassed with 95% ${\mathrm{O}}_2∕5\%$ $\mathrm{C}{\mathrm{O}}_2$ . Coronary perfusion pressure was regulated between 80 and $95\mathrm{mmHg}$ , and the hearts were exposed to air. A quadripolar catheter was placed into the endocardium of the right ventricular apex through the pulmonary artery trunk for continuous bipolar recording and the induction of VF. The tissues were stained with $0.5\text{-}\mu \mathrm{M}$ di-4-ANEPPS (Molecular Probes, Eugene, Oregon). Laser light of $532\text{-}\mathrm{nm}$ wavelength (Verdi, Coherent Incorporated, Santa Clara, California) illuminated the tissues, and epifluorescence was collected through a long-pass filter with a cutoff wavelength of $600\mathrm{nm}$ with a high-speed charge-coupled device camera ( $430\text{frames}∕\mathrm{s}$ , model CA D1-0128T, Dalsa, Waterloo, Ontario, Canada). Image acquisition was controlled by a custom-designed program based on LabView and the IMAQ Vision toolset (National Instruments, Austin, Texas). We mapped the left ventricular anterior wall $(20\times 20{\mathrm{mm}}^2)$ , acquiring $128\times 128$ sites simultaneously. The electromechanical uncoupler cytochalasin D (Sigma Aldrich, Saint Louis, Missouri) was added to the perfusate at a concentration of $5\mu \mathrm{M}$ to inhibit tissue contraction.

2.2.

Multielectrode Pacing Protocols

Four pacing electrodes ( $5\mathrm{mm}$ apart, Teflon-coated stainless steel with $0.3\mathrm{mm}$ diam) were placed in the camera’s field of view. The electrodes were arranged in a line configuration in along- and across-fiber directions [Figs. 1a and 1b ]. The fiber direction was determined by visual inspection. In the synchronized pacing technique (SyncP) pacing protocol, an additional “reference site” is required in addition to the four physical pacing sites. Two different reference sites were selected at positions in line with the electrodes. The reference site was either above the top electrode (e1) or below the bottom (e4). The reference site was used to determine the timing of activation. The action potentials were monitored optically at sites directly adjacent to $(<2\mathrm{mm})$ each pacing electrode and the reference site.

Fig. 1

Pacing protocols. Four unipolar pacing electrodes (e1, e2, e3, and e4) were positioned on the left ventricular anterior wall of the isolated rabbit heart in (a) the fiber direction and (b) cross-fiber direction. A reference site positioned either above e1 or below e4 was used to detect tissue activation. (LAD:left anterior descending coronary artery.) (c) Independent SyncP mode. The first two traces correspond to optical signals registered at a reference site and near a pacing electrode. The third line indicates the time at which an electrical stimulus (S) was delivered through the pacing electrode (d) Simultaneous SyncP mode. All four electrodes fired when activation was detected at the reference site.

Figure 1c shows the SyncP algorithm. The main task of the algorithm was to detect the threshold crossing from the optical recordings at the reference site as well as the four pacing sites. Threshold levels were independently set in real time for the reference site and the pacing sites to 40% of the maximum optical potential amplitude. Two types of pacing algorithms were tested. In the independent mode [Fig. 1c], each pacing electrode was independently controlled. The pacing current was delivered when the optical potential of the pacing site was below the threshold, and the reference site was depolarized by a VF wavefront to a potential above the threshold level [Fig. 1c arrow]. In the simultaneous mode [Fig. 1d], all four electrodes fired when activation was detected at the reference site. With this latter mode, we found that at least one of the pacing sites was in the excitable gap in most cases.

VF was induced by endocardial burst pacing in the right ventricle with a pacing cycle length of $50\text{to}80\mathrm{ms}$ . The pacing protocol started within one minute of VF induction. We recorded $2.3\mathrm{s}$ (1000 frames: 500 prepacing, 500 pacing) of each pacing protocol by optical mapping. We waited at least $1\min$ before testing a new electrode configuration or reference site to minimize the effects of the previous pacing protocol. The number of pacing episodes was as follows: independent SyncP, 48 episodes (12 fiber, top reference; 12 fiber, bottom reference; 12 cross-fiber, top reference; 12 cross-fiber, bottom reference); and simultaneous SyncP, 48 episodes (same breakdown as independent SyncP).

2.3.

Data Analysis

The variance of optical potential amplitude along the line of pacing electrodes was used to quantify the degree of synchronization. The variance of the action potentials along the electrode line (line interconnecting the reference site and four electrode sites) was calculated for each frame of the prepacing and pacing periods. To calculate the variance, we first normalized the amplitude of the optical signal to values between 0 and 1 for each pixel for each frame. The variance along the electrode line was then obtained from these normalized values. Such an analysis was also performed along lines parallel, but shifted outward from the pacing line to quantify how the effect of pacing drops off with increasing distance from the pacing site.

The VF cycle length was computed at each site along the electrode line as the reciprocal of the frequency peak in the power spectrum on transforming (fast Fourier transform) the action potential to the frequency domain.

3.1.

Wavefront Synchronization along the Electrode Line

As expected, SyncP caused synchronized activation along the line of pacing electrodes. Figure 2 shows examples of independent and simultaneous SyncP in the fiber direction [Figs. 2a and 2b] and cross-fiber direction [Figs. 2c and 2d]. In Fig. 2a, when the reference site was activated (frame 581), only sites e3 and e4 were in the excitable gap. Therefore, stimulation current was delivered to e3 and e4, but not to e1 or e2. The stimulation resulted in synchronized activation (frame 587) and repolarization (frame 599). In Fig. 2b, when the reference site was activated, all electrodes fired at the same time, causing synchronized activation (frame 681) and repolarization (frame 694). The same electrode firing protocols were used in the cross-fiber configuration as shown in Figs. 2c and 2d.

Fig. 2

Voltage maps indicating linear functional block created by SyncP. The red color represents depolarization, and the blue color represents repolarization. Small circles in the maps show locations of the pacing electrodes (e1 through e4). A plus sign in the circle represents the delivery of the pacing current. The frame numbers are indicated in the corners of each map. (a) Independent SyncP, fiber direction; (b) simultaneous SyncP, fiber direction; (c) independent SyncP, cross-fiber direction; and (d) simultaneous SyncP, cross-fiber direction.

To quantify the change of synchronicity that occurred with pacing, first the variance of the optical signals for every point along the electrode line was calculated for each frame of the prepacing period. A graphical summary of the variances during the prepacing period is provided by the bell curve in Fig. 3a (mean of prepacing variances, ${\mu }_{\text{PREPACING}}=281.93$ ). The same was done for the pacing period [Fig. 3b] $({\mu }_{\text{PACING}}=281.93)$ . The shift of the distribution to the left is indicative of the decrease in variance that occurs with pacing. The drop in variance, representative of increased wavefront synchronization, is visually emphasized by the box plot of Fig. 3c. The difference in the mean of the variances for the 500 prepacing frames and the mean of the variances for the 500 pacing frames $(\Delta \text{variance}={\mu }_{\text{PREPACING}}-{\mu }_{\text{PACING}}=92.39)$ was the absolute measure of the effect of pacing on synchronization, and was normalized by dividing by the mean of the variances for the 500 prepacing frames to give the percent decrease in variance ( $\Delta \text{variance}∕{\mu }_{\text{PREPACING}}=32.8\%$ for Fig. 3).

Fig. 3

(a) The variance along the electrode line was calculated for each frame (500 in total) of the prepacing period. A graphical summary of the variances is provided by the bell curve (b) Bell curve showing distribution of variances during pacing. The shift of the distribution to the left is indicative of the decrease in variance that occurs with pacing. (c) The box plot quantifies the drop in variance that occurs with the onset of pacing. In this particular trial, the difference in the mean of the variances for the 500 prepacing frames and the mean of the variances for the 500 pacing frames is 92.39 with a 95% confidence interval of (77.66, 107.13).

Figure 4 shows the correlation between wavefront synchronization and variance. The optical traces at sites adjacent to the electrodes (p1 to p4) are shown in the panels in Figs. 4a and 4d and reveal an increased synchronicity after the pacing protocol began. The increased synchronicity was not restricted solely to the pacing sites, but also occurred along the line connecting the electrodes. This phenomenon is best illustrated by the space-time plots shown in Figs. 4b and 4e. These plots show the voltage signals at every point along the electrode line (vertical deflection) in time (horizontal deflection). Each horizontal line of varying color is an optical action potential of a single pixel along the electrode line, except the varying voltage amplitudes are instead replaced with varying colors. A homogeneous stripe of color spanning the vertical direction represents the same level of membrane potential depolarization at all points along the electrode line. This observation is quantified by calculating the variance as shown in the panels in Figs. 4c and 4f. The variance of the transmembrane potential for points along the electrode line was calculated for each frame. The synchronization effect is revealed as a relatively smaller variance during pacing compared to the prepacing period. In Fig. 4c, the mean of the variances of the prepacing period was 193.12 and the pacing period was 125.54. The decrease in variance by 67.58 $(\mathrm{P}<0.001)$ is indicative of the synchronization effect that occurred with pacing. Similarly, in Fig. 4f, the mean of the prepacing variances was 200.80, pacing variances was 143.43, and difference was 57.37 $(\mathrm{P}<0.001)$ .

Fig. 4

Effects of SyncP on wavefront synchronization. (a) Optical potentials for four points (p1, p2, p3, p4) situated along an electrode line in the fiber direction. The line marked “S” indicates the time at which an electrical stimulus was delivered through the pacing electrode (simultaneous SyncP). (b) Space-time plot of the optical potential at every point along the electrode line in time. A homogenous stripe of color spanning the vertical direction represents the same depolarization level of optical potential at all points along the electrode line. (c) A plot of the variance in time. Similar action potentials in p1 through p4 tend to correspond to a strip of uniform color in the space-time plot and a small variance. (d), (e), and (f) provide similar plots for a crossfiber, simultaneous SyncP configuration. (c) Variance decrease of 67.58 $(\mathrm{P}<0.001)$ , from 193.12 prepacing to 125.54 with pacing. (f) Variance decrease of 57.37 $(\mathrm{P}<0.001)$ , from 200.80 prepacing to 143.43 with pacing.

3.2.

Optimum Electrode Configuration for Pacing

The above-mentioned analysis was extended to all configurations to determine optimum pacing conditions. Figure 5a contrasts the amount of synchronization induced by pacing in the fiber and cross-fiber configurations. In both the independent SyncP and simultaneous SyncP modes, the fiber direction configurations showed a decrease in variance larger than the cross-fiber configurations [independent SyncP: 19.33% decrease in variance $(\Delta \text{variance}=37.89)$ versus 13.71% $(\Delta \text{variance}=30.72)$ ; simultaneous SyncP: 24.28% $(\Delta \text{variance}=47.10)$ versus 10.72% $(\Delta \text{variance}=24.98)$ , respectively]. (All $\mathrm{P}<0.001$ .) In this study, the reference site for the SyncP protocol was placed either above or below the pacing electrodes. The effect of synchronization was compared for these different electrode configurations. Figure 5b provides a summary of the synchronization abilities of all eight configurations. Among the configurations in the fiber direction, the simultaneous SyncP protocol proved more effective in the reduction of action potential variance than independent SyncP [top reference: 26.37% decrease in variance $(\Delta \text{variance}=53.26)$ versus 20.65% $(\Delta \text{variance}=42.33)$ ; bottom reference: 21.83% $(\Delta \text{variance}=40.38)$ versus 17.79% $(\Delta \text{variance}=33.09)$ , respectively]. (All $\mathrm{P}<0.001$ .)

Fig. 5

Decrease in variance due to SyncP pacing with different electrode configurations. Percent decrease in $\text{variance}=\Delta$ variance/prepacing variance. $\Delta$ variance is the difference between the prepacing variance and pacing variance. Large values for percent decrease in variance indicate that pacing has had a strong effect on synchronization. ( $\mathrm{P}<0.001$ for all configurations.)

3.3.

Effect of Pacing Drops Off Linearly with Distance from the Electrode Line

To quantify the variable degree of synchronization (the effect of pacing) away from the electrode line, the drop in variance between prepacing and pacing periods was calculated along lines parallel to, but shifted outward from, the original electrode line. Values were normalized by dividing variance drops at increasing distances from the electrode line by the variance change (between prepacing and pacing) at the electrode line. A value near 100 indicates a site at which pacing has a strong effect on encouraging wave synchronization, while a value of 0 indicates a site at which pacing has no effect.

Figures 6a and 6b show the linear dropoff in synchronization that occurred with pacing along an electrode line for the independent and simultaneous SyncP, respectively. In the independent SyncP mode, pacing was 70% as effective at $1.2\mathrm{mm}$ and 50% as effective at $2.2\mathrm{mm}$ . In the simultaneous SyncP mode, the amount of synchronization dropped to 70% at $1.5\mathrm{mm}$ , and to 50% at $2.4\mathrm{mm}$ .

Fig. 6

Effect of pacing drops off with distance from the electrode line. The effect of pacing is measured as the drop in variance between prepacing and pacing periods. A value near 100 indicates a site at which pacing has a strong effect on encouraging wave synchronization, while a value of 0 indicates a site at which pacing has no effect. Values are normalized by dividing variance drops at increasing distances from the electrode line by the variance change (between prepacing and pacing) at the electrode line. (a) Independent SyncP. (b) Simultaneous SyncP. The distances at which the pacing effect drops to 70% and 50% are provided.