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Am J Physiol Heart Circ Physiol 294: H1896-H1905, 2008. First published February 29, 2008; doi:10.1152/ajpheart.00986.2007
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Moderate hypothermia increases the chance of spiral wave collision in favor of self-termination of ventricular tachycardia/fibrillation

Masahide Harada,1,* Haruo Honjo,1,* Masatoshi Yamazaki,1 Harumichi Nakagawa,1 Yuko S. Ishiguro,1 Yusuke Okuno,1 Takashi Ashihara,2 Ichiro Sakuma,3 Kaichiro Kamiya,1 and Itsuo Kodama1

1Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya; 2Department of Cardiovascular Medicine, Shiga University of Medical Science, Otsu; and 3Graduate School of Engineering, The University of Tokyo, Tokyo, Japan

Submitted 27 August 2007 ; accepted in final form 23 February 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In cardiac arrest due to ventricular fibrillation (VF), moderate hypothermia (MH, 33°C) has been shown to improve defibrillation success compared with normothermia (NR, 37°C) and severe hypothermia (SH, 30°C). The underlying mechanisms remain unclear. We hypothesized that MH might prevent reentrant excitations rotating around functional obstacles (rotors) that are responsible for the genesis of VF. In two-dimensional Langendorff-perfused rabbit hearts prepared by cryoablation (n = 13), action potential signals were recorded by a high-resolution optical mapping system. During basic stimulation (2.5–5.0 Hz), MH and SH caused significant prolongation of action potential duration and significant reduction of conduction velocity. Wavelength was unchanged at MH, whereas it was shortened significantly at SH at higher stimulation frequencies (4.0–5.0 Hz). The duration of direct current stimulation-induced ventricular tachycardia (VT)/VF was reduced dramatically at MH compared with NR and SH. The spiral wave (SW) excitations documented during VT at NR were by and large organized, whereas those during VT/VF at MH and SH were characterized by disorganization with frequent breakup. Phase maps during VT/VF at MH showed a higher incidence of SW collision (mutual annihilation or exit from the anatomical boundaries), which caused a temporal disappearance of phase singularity points (PS-0), compared with that at NR and SH. There was an inverse relation between PS-0 period in the observation area and VT/VF duration. MH data points were located in a longer PS-0 period and a shorter VT/VF duration zone compared with SH. MH causes a modification of SW dynamics, leading to an increase in the chance of SW collision in favor of self-termination of VT/VF.

optical mapping; ventricular fibrillation

SUCCESSFUL USE OF THERAPEUTIC hypothermia after cardiac arrest in humans was described in the late 1950s as a procedure to improve the clinical outcomes but then almost abandoned because of uncertain benefits and difficulties with its use (17). It was demonstrated in 2002 by two prospective randomized trials that the induction of therapeutic hypothermia in patients, who had been resuscitated after cardiac arrest due to ventricular fibrillation (VF), increased neurological recovery and reduced mortality (2, 12a). This has led to recent guideline recommendations that all unconscious adult patients with spontaneous circulation after out-of-hospital cardiac arrest should be cooled to 32–34°C for 12–24 h when the initial rhythm was VF (1). In experiments using swines with induced VF, Boddicker et al. (3) demonstrated that defibrillation success of electrical shocks and resuscitation outcomes were significantly improved under moderate hypothermia (MH, 33°C) compared with normothermia (NR, 37°C) and that the benefit of MH was larger than severe hypothermia (SH, 30°C) (3). The underlying mechanisms of this amelioration are not well understood. Temperature-dependent amelioration of ischemic myocardial injury may contribute to the beneficial effect of hypothermia for resuscitation, but this does not explain an advantage of MH over SH (10). Defibrillation success or failure depends on the balance between shock-induced extinction and generation of such rotors (9). We hypothesized that MH may have substantial effects on the rotor dynamics in favor of its termination. To test this hypothesis, we investigated temperature-dependent modification of spiral wave (SW) dynamics in a perfused two-dimensional (2-D) layer of rabbit ventricle by using our custom-made, high-resolution optical action potential mapping system.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Experimental model. The protocol was approved by the Institutional Animal Care and Use Committee at Nagoya University. Experiments were performed in hearts from Japanese white rabbits of both sexes weighing 1.7–2.0 kg (n = 13). The experimental model is essentially the same as reported previously (26). In brief, rabbits were anesthetized with pentobarbital sodium (10–15 mg/kg), and the hearts were rapidly removed. The isolated hearts were continuously perfused on a Langendorff apparatus with modified Krebs-Ringer solution equilibrated with 95% O2-5% CO2 to maintain pH at 7.3–7.4. Complete atrioventricular (AV) block was produced by ligation of His bundle. Two-dimensional epicardial layer of ventricular myocardium (1.0 ± 0.2 mm thick) was created by cryoprocedure. At the end of experiment, the thickness of surviving epicardial myocardium was confirmed by staining the heart with 2,3,5-triphenyltetrazolium chloride. This model has an advantage over intact three-dimensional (3-D) heart to visualize the SW reentry on the epicardial surface.

High-resolution optical mapping. The optical mapping system used in this study is similar to that described previously, except for inducing the upgraded digital video camera (26). After endocardial freezing, the hearts were stained with a voltage-sensitive dye, 4-{β-[2-(di-n-butylamino)-6-naphthyl]vinyl}pyridinium (di-4-ANEPPS). To minimize motion artifacts, 15 mM 2,3-butanedione monoxime (BDM) was added unless otherwise specified. Bipolar electrograms were recorded through widely spaced electrodes to monitor the whole ventricular excitations. The signals were filtered from 1.5 to 1,000 Hz and digitized at 1,000 Hz.

The hearts were illuminated with bluish-green light-emitting diodes (LEDs); the emitted fluorescence was recorded with a solid-state image-sensing digital video camera (Fastcam-Max, Photron, Japan) to acquire 10-bit gray scale images from 256 x 256 sites simultaneously at a speed of 1,000 frames/s. The images acquired (30 x 30 mm) covered the anterolateral surface of the left ventricle (LV). Each acquisition lasted for 2–10 s.

To reveal the action potential signal, the background fluorescence was subtracted from each frame and low-pass spatial filtering was applied. Action potential configuration was analyzed after the application of a five-point median filter to the spatially averaged data, and the data were then normalized to the range of the maximum and the minimum values in the respective 800-frame sample. A time point at 10% depolarization in the upstroke phase and a time point at 90% repolarization in its repolarization phase were identified for each action potential signal, and the interval was measured as action potential duration at 90% repolarization (APD90). Wave-front tail dynamics during ventricular tachycardia (VT)/VF were visualized by connecting the 10% depolarization points in the action potential upstrokes as the wave front (red) in the recording area and by connecting the 90% repolarization points at the wave tail (green). The wave propagation pattern during VT/VF was quantified with the aid of the phase-mapping methods described by Gray et al. (12). Phase singularity (PS) was defined as the point at which all phases converged.

Myocardial cooling of heart. The heart was sequentially cooled from 37°C (NR) to 33°C (MH), and then to 30°C (SH) by controlling temperature of the perfusate and thermoregulated water chamber. The target temperature was decided in accordance with the in vivo experimental study by Boddicker et al. (3). We confirmed in pilot experiments of five rabbit hearts that the surface temperature of LV free wall monitored by thermography (TVS-200 Nippon Avionics, Tokyo) was kept homogeneous at the respective target temperature (Fig. 1), and the effects of cooling on the basal electrophysiological properties reached a steady state at ~10 min. The cooling-induced changes were reversible upon rewarming to NR.


Figure 1
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  		Fig. 1. Thermography images of a heart at normothermia (NR, 37°C), moderate hypothermia (MH, 33°C) and severe hypothermia (SH, 30°C). The temperature images recorded by thermography are shown as color gradients, ranging from red (40°C) to light blue (25°C).

 
Experimental protocols. Steady-state APD and its distribution were measured under constant stimulation (S1) from the LV apex at basic cycle length (BCL) of 200–400 ms. Effective refractory period (ERP) was estimated at the LV apex by a standard extrastimulus (S1-S2) protocol at BCL of 400 ms. Steady-state conduction velocity (CV) was measured under S1 stimulation at the center of LV free wall at BCL of 200–400 ms. A monopolar electrode made of platinum wire (diameter, 0.5 mm) was used for stimulation. The pulses applied were 2 ms in duration and 1.2 times the diastolic threshold for S1 and twice the diastolic threshold for S2.

CV was measured in a square (24 x 16 mm) around the stimulation site at the center of the LV free wall. The longitudinal and transverse directions (LD and TD) of propagation were determined from the activation maps elicited by S1 stimulation. The long and short axes of the CV measurement square were set parallel to LD and TD, respectively. CVs in LD (CVL) and TD (CVT) were calculated from the slope of a linear least-square fit of the activation time plotted against the distance.

We used the dynamic pacing method to characterize the restitution properties of APD and CV (15). With regard to APD, the LV apex was initially paced at a BCL of 500 ms, and the BCL was progressively shortened in steps of 10–100 ms. A minimum of 60 stimuli were delivered at a given BCL. APD restitution at a site of 3 mm distant from the stimulation site on the anterior LV free wall was analyzed. The APD90 of the last two paced action potentials was measured, and the differences >5 ms was defined as APD alternans. The BCL was shortened until either a 2:1 block or higher-order periodicity occurred. The APD restitution curve was constructed by plotting APD90 against the preceding diastolic interval, and the curve was fitted to a single exponential function (ORIGIN 7.0, Microcal Software, Northampton).

To evaluate the CV restitution, the center of the LV free wall was initially paced at a BCL of 700 ms, and the BCL was progressively shortened in steps of 10–200 ms. A minimum of 60 stimuli were delivered at a given BCL. CVL and CVT in the 24 x 16 mm2 around the stimulation site were measured for the last 10 excitations from the time-distance plots and then averaged. The restitution curves of CVL and CVT plotted as a function of BCLs were fitted to a single exponential function, and they were characterized by measuring "breadth" and "depth" (5). The breadth was defined as the BCL range corresponding to CV recovery of 90% or less. The depth was defined as the CV range between the minimum value and 90% recovery.

VT/VF resulting from SW excitation was induced by modified cross-field stimulation. Eighteen S1 stimuli at a BCL of 400 ms were applied to the LV apex through a pair of contiguous bipolar electrodes. A 10-ms monophasic rectangular pulse of constant voltage (10–20 V) was generated by a direct current (DC) power unit and delivered from the unit through a 6,700-pF capacitor connected to a power MOS-FET switch. A pair of Ag-AgCl paddle electrodes (7 mm in diameter) was placed on the lateral surface of both ventricles for the DC stimulation (DS2) to induce an electrical filed in a direction roughly perpendicular to the S1 excitation from the apex (26). The S1-DS2 coupling interval was shortened progressively in 10-ms steps to cover the whole vulnerable window of the final S1 excitation at the respective temperature (from 100 ms longer than ERP until reaching the loss of ventricular capture). The sequence was repeated in each heart at three stages of DS2 intensity (10, 15, and 20 V). The incidence of VT/VFs was presented as percentage of the episodes over the number of DS2 applied.

Statistical analysis. Group data were expressed as means ± SD. Statistical comparisons were performed by ANOVA with multiple comparison, t-test, or {chi}2 test when appropriate. Differences were considered significant when the probability value was <0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Basal electrophysiological properties. Representative changes in APD under constant stimulation (BCL, 400 ms) are shown in Fig. 2A; APD90 values in the entire mapping area are displayed as color gradients (left), and action potential signals obtained from 16 sites covering 18 x 18 mm square are superimposed (right). Figure 2B summarizes the temperature-dependent changes in APD90 values. When APD alternans was induced at shorter BCLs, the shorter and longer APD90 values were averaged. Cooling to 33°C (MH) and 30°C (SH) resulted in an almost homogeneous prolongation of APD. APD90 values at a BCL of 400, 250, and 200 ms were increased significantly by 11–30% at MH and by 13–43% at SH. The dispersion of APD90 among the 16 recording sites at BCL of 400 ms was slightly increased from 18 ± 3 ms at NR to 22 ± 4 ms at MH (not significant) and 25 ± 6 ms at SH (P < 0.05 vs. NR). ERP at BCL of 400 ms was increased in parallel with APD90, from 149 ± 18 ms at NR to 182 ± 17 ms at MH (P < 0.05 vs. NR) and to 203 ± 23 ms at SH (P < 0.05 vs. NR).


Figure 2
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  		Fig. 2. Basal electrophysiological properties. A: action potential duration at the 90% repolarization (APD90) in the recording area (anterolateral surface of left ventricular free wall) under constant stimulation from apex [basic cycle length (BCL) of 400 ms]. A, left: APD90 color gradient maps, ranging from red (shortest) to blue (longest). A, right: superimposed action potential signals (BCL, 400 ms) recorded from the 16 sites covering 18 x 18 mm area (white dots in the left-end map). B: APD90 at BCL of 200, 250, and 400 ms (means ± SD, n = 6). C: conduction velocity in longitudinal (CVL; top) and transverse (CVT; bottom) directions at BCL of 200, 250, and 400 ms (means ± SD, n = 6). D: wave length during longitudinal (WLL; top) and transverse (WLT; bottom) propagation at BCL of 200, 250, and 400 ms (means ± SD, n = 6). *P < 0.05 vs. 37°C; {dagger}P < 0.05 vs. 33°C.

 
In experiments to examine conduction properties, the isochrones of activation front exhibited a smooth, symmetric, elliptical pattern; the long axis corresponded to the fiber orientation of subepicardial cardiac muscle. MH and SH caused significant reduction of both CVL and CVT in steps (Fig. 2C). The reduction of CVs at SH was enhanced at shorter BCLs, and the enhancement was more prominent in CVT than in CVL (Fig. 2C), giving rise to an increase of the anisotropic ratio (CVL/CVT) at a BCL of 200 ms from 1.9 ± 0.3 for NR to 2.1 ± 0.6 for MH (not significant) and to 3.5 ± 1.6 for SH (P < 0.05 vs. NR).

We estimated wavelength (WL = APD90 x CV) during constant pacing (Fig. 2D). At a BCL of 400 ms, both WLL and WLT at MH and SH were significantly longer than those at NR. In contrast, at BCL of 250 and 200 ms, WLL and WLT at SH were significantly shorter than those at NR and MH.

We used the dynamic pacing method to characterize the restitution properties. The maximal APD restitution slopes at MH and SH were larger than that at NR (see supplemental Fig. 1A and supplemental Table 1; note: all supplemental material may be found with the online version of his article). With regard to CV restitution, the breadth and depth (5) were unchanged or increased slightly at MH, whereas they were increased largely by two- to threefold at SH (supplemental Fig. 1B and supplemental Table 1).

VT/VFs induced by DC stimulation. The VT/VFs induced in 13 hearts by DS2 were categorized into two types in terms of their duration: 1) nonsustained, lasting ≥5 beats and <30 s and 2) sustained, lasting ≥30 s (Fig. 3A). In terms of ECG morphology (distant bipolar electrogram), VT was categorized into monomorphic type and polymorphic type. In the present experiments, polymorphic VT lasting ≥30 s was defined as VF. At NR, 71 VT/VFs were induced by 241 DS2s (total incidence, 29%): 59 (83%) were nonsustained (46 monomorphic and 13 polymorphic), and 12 (17%) were sustained (all monomorphic). At MH, 108 VT/VFs were induced by 289 DS2s (total incidence 37%): 107 (99%) were nonsustained (all polymorphic VTs), and 1 (1%) was sustained (VF). At SH, 89 VT/VFs were induced by 203 DS2s (total incidence 44%): 78 (88%) were nonsustained (all polymorphic VTs), and 11 (12%) were sustained (all VFs). There were no significant differences among the three temperature conditions in the average intensity of DS2 to induce VT/VFs (16.0 ± 3.7 at NR, 15.2 ± 3.9 at MH, and 14.8 ± 3.8 V at SH, P = 0.14). Thus, although the total incidence of VT/VFs was increased in steps by cooling (Fig. 3A), the relative incidence of sustained form over the total VT/VF episodes was decreased dramatically at MH (P < 0.001) (Fig. 3B).


Figure 3
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  		Fig. 3. Incidence and duration of shock-induced ventricular tachycardia (VT)/ventricular fibrillation (VF). A: total incidence of VT/VFs induced by direct current (DC) stimulation (DS2) in 13 hearts at 37°, 33°, and 30°C. The VT/VFs were categorized into 2 types; nonsustained (≥5 beats, <30 s), and sustained (≥30 s). B: percentage of sustained VT/VFs over total VT/VFs.

 
Dynamics of SW excitation. Optical images of excitation during VT/VF were analyzed in 10 hearts exhibiting visible SWs at NR as well as at MH and SH. At NR, some forms of SW excitation (single-loop or figure-eight reentry) were documented in 17 episodes (59%) of 29 VTs available for image analysis. The remaining 12 VTs showed one-way propagation of wave fronts traversing the observation area. At MH and SH, multiple SW excitations were always (100%) documented in 32 and 46 VT/VF episodes, respectively.

Figure 4 shows representative isochrone maps. In the record at NR (Fig. 4A), a clockwise rotation of wave front around a line of functional block (FBL, ~3.0 mm) can be seen at a cycle length (CL) of 146–148 ms. The circuit was stable for >30 s and exhibited a small meandering. Distant bipolar electrogram (ECG) showed monomorphic ventricular excitations. Action potential signals (sites a–d) showed regular configurations with no isoelectric segments, and there were clear double potentials on the FBL (site e) as reported previously (26).


Figure 4
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  		Fig. 4. Spiral wave (SW) excitations during DC stimulation-induced VT/VF. A: isochrone maps of 3 consecutive activations during sustained monomorphic VT at 37°C (4-ms intervals, blue lines for earlier wave fronts, green lines for later wave fronts). Clockwise rotation around a line of functional block (FBL, yellow) in the anterior left ventricular free wall was repeated at a cycle length (CL) of 146 to 148 ms. A–C, top: distant bipolar electrogram (ECG) during the VT. A–C, right: optical action potential signals from 5 sites (sites a–e in the isochrone map of beat iii). B: isochrone maps of 3 activations during nonsustained polymorphic VT at 33°C. Single or multiple SW excitations circulated around FBLs with marked beat-to-beat variations (CLs varied from 153 to 224 ms). C: isochrone maps of 3 activations during sustained VF at 30°C. Multiple SWs circulated around more complex FBLs with tremendous beat-to-beat variations (CLs varied from 124 to 216 ms). All the records of A–C were obtained from the same heart. Numbers on the white dotted arrows in isochrone maps (B and C) indicate the connection of respective activation waves.

 
Figure 4B shows activation patterns during a short polymorphic VT (lasting for 5 s) at MH. SWs changed their circuits in a beat-to-beat manner with CLs varying from 153 to 224 ms. In beat i, there were two reentry circuits. In the upper region, a wave front coming down from the base faced a long FBL, giving rise to its extension toward the center to maintain the clockwise rotation. In the lower region, a wave toward the right margin was divided into two pathways of counterrotation around a long tortuous FBL. The upper limb of this lower circuit shared a common pathway with the lower limb of the upper circuit. The lower circuit ended by collision at the opposite side of the FBL. In beat ii, a single clockwise rotation remained around a Y-shaped FBL. In beat iii, double circuits appeared again around two long L-shaped FBLs. The wave fronts finally terminated by collision (sites c and e). The action potentials recorded from the circuits showed substantial beat-to-beat variation, and some of them had very long durations.

Figure 4C shows more complex activation patterns during VF (lasting ≥30 s, CL varying from 124 to 216 ms) at SH. In beat i, a wave front of counterclockwise rotation passed through between two curved FBLs near the apex and then merged with another wave front coming down from the left upper region, resulting in two wave fronts of opposite direction. In beat ii, the opposite wave fronts created an additional very-long FBL, and several wave fronts coexisted around three FBLs. In beat iii, the multiple wave fronts merged with each other around the center, giving rise to a labyrinth associated with collision (site d). The action potential signals recorded from the circuits showed tremendous beat-to-beat changes.

The modification of SW dynamics by cooling was investigated more extensively by phase mapping analysis. At NR, SW excitations terminated spontaneously when the two PSs of opposite chirality collided with each other (mutual annihilation) or when the PS collided against an anatomical boundary (AV groove), but the chance was rare as reported previously (26). At MH, the chance of collision was increased because of large meandering and further generation of new PSs. Figure 5A shows SW extinction patterns in the final beat of a VT episode (the same experiment as in Fig. 4B). A PS of clockwise rotation (PS1) moved back and forth in the middle upper region of LV (4,570–4,592 ms). A pair of new PSs of opposite chirality (PS2 and PS3) was then generated (4,592 ms) due to front-tail interactions (supplemental Fig. 2A). The distance between the two PSs initially increased and then decreased, culminating in mutual annihilation. After considerable meandering, PS1 was finally pushed out of the AV groove (supplemental movie 1). The trajectories of three PSs were plotted on 2-D (x-y) axes (Fig. 5B) as well as on 3-D (x-y-time) axes (Fig. 5C).


Figure 5
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  		Fig. 5. Self-termination of SW reentry at 33°C. A: 4 snapshots of phase maps of the final beat of a short polymorphic VT episode (the same experiment as in Fig. 4B). Phase singularities (PSs) are indicated by circles (white for clockwise rotation; black for counterclockwise rotation). A pair of new PSs (PS2 and PS3) were generated from a SW excitation circulating around PS1 via front-tail interactions causing breakup (#). PS2 and PS3 terminated soon by mutual annihilation, whereas PS1 terminated by collision with atrioventricular (AV) groove after meandering. B: trajectories of the 3 PSs on two-dimensional (2-D) (x, y) axes (red for clockwise rotation; blue for counterclockwise rotation). C: trajectories of the 3 PSs plotted on space (x, y) and time axes. The half-tone wall on the left indicates AV groove.

 
Figure 6A shows four sequential phase maps of a VF episode (the same experiment as in Fig. 4C). There were six PSs at 2,232 ms. A pair of counterrotating PSs in the central region (PS1, PS2) approached each other to a very close distance (2,250 ms) and then separated without mutual annihilation, and the sequence was repeated (supplemental Fig. 2B and movie 2). Figure 6B illustrates trajectories of the 11 PSs in 2-D (x-y) axes. PS4, PS5, and PS6 were pushed out of the observation area after meandering. Two pairs of counterrotating PSs (PS3–PS11 and PS9–PS10) dissipated at last by mutual annihilation. PS1, PS2, PS7, and PS8 survived the period. Figure 6C shows 3-D time-space (x, y) plots of the trajectories of PS1 and PS2. The distance between PS1 and PS2 was plotted against time in Fig. 6D. The two PSs approached each other twice to a distance of ~0.5 mm but survived without annihilation. This behavior contrasts with the SW dynamics at MH, causing mutual annihilation easily.


Figure 6
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  		Fig. 6. Coexistence of multiple SW excitations at 30°C. A: 4 snapshots of phase maps during an episode of sustained VF (the same experiment as in Fig. 4C). Eleven PSs were recognized in the observation area (white for clockwise rotation; black for counterclockwise rotation). B: trajectories of the 11 PSs on 2-D (x, y) axes. Some of them were pushed out of the observation area after meandering (PS4, PS5, and PS6) or dissipated by mutual annihilation (PS3–PS11, and PS9–PS10), but others (PS1, PS2, PS7, and PS8) survived the period. C: trajectories of the 2 PSs (PS1 and PS2) in the central dotted square were plotted on space (x, y) and time axes (red for clockwise rotation; blue for counterclockwise rotation). D: temporal change in the distance between PS1 and PS2. *Closest distance.

 
We compared the closest distance (Dc) between a pair of counterrotating PSs constructing figure-eight reentry without mutual annihilation at MH and SH. Average values of Dc at SH were significantly less than those at MH (0.5 ± 0.2 vs. 1.3 ± 0.6 mm, n = 6, P < 0.05).

PS dynamics and VT/VF termination. We analyzed the PS dynamics more quantitatively to elucidate their roles in perpetuation and termination of rotors. Figure 7A, left, shows representative changes in PS number during 2,000 frames (2 s) of VT/VF episodes. At NR, the number of PSs was normally 1 and, rarely, increased to 2 for short periods. At MH and SH, the number of PSs increased frequently to 2–8, resulting in an increase of average PSs/2 s. Pooled data are summarized in Fig. 7A, right.


Figure 7
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  		Fig. 7. PS dynamics and VT/VF duration. A: representative changes in PS number during 2,000 frames (2 s) of VT/VF episodes and averaged number of PSs/2 s at 37°, 33°, and 30°C (means ± SD, n = 6, *P < 0.05 vs. 37°C, {dagger}P < 0.05 vs. 33°C). B: relationship between temporal PS dissipation (total PS-0 period/2 s) and VT/VF duration. Seventy-eight VT/VF episodes (32 at 33°C, and 46 at 30°C) were analyzed. The data points were fitted by an exponential curve.

 
In Fig. 7A, left, it was also evident that temporal PS dissipation from the observation area (PS-0 period) occurred more frequently at MH than at NR and SH. Such temporal PS dissipation would favor spontaneous termination of VT/VF depending on rotors. We, therefore, plotted the duration of 78 VT/VF episodes at MH and SH against the respective total PS-0 period/2 s (Fig. 7B). There was an inverse relation between the two parameters. MH data points were located in a longer PS-0 period and shorter VT/VF duration zone compared with SH.

Spatial excitable gap during SW excitation. The basal electrophysiological data at short BCLs (200–250 ms) showed that WLs at SH were significantly shorter than those at MH and NR (Fig. 2D). This seems to suggest that WLs during rotation of SW at SH are short, allowing a relatively wide excitable gap. During SW reentry, however, both APD and CV are influenced by wave-front curvature, which increases progressively toward the rotation center (19). We, therefore, estimated the spatial excitable gap (SEG) of SW by measuring an area circumscribed by wave front, wave tail, and an arc of a certain radius (distance) from the SW tip. Representative results are shown in Fig. 8A. At NR, there was a dynamic variation of SEG during a single rotation; the value at 0.5 mm from the rotation center ranged from 0.11 to 0.43 mm2. SEG of SW at MH showed a similar variation, although the measurements did not cover a full rotation due to meandering of the circuit. SEG of SW at SH was characterized by a less variation with a larger minimal value (0.27 mm2 at 0.5 mm). Figure 8B compares the minimal SEG (SEGmin) of six hearts (for a distance at 0.5 mm). SEGmin at SH was significantly larger than those at NR and MH for a distance of 0.5–1.0 mm, whereas there were no significant differences among the three temperatures for a distance ≥ 2.0 mm (1.8 ± 0.9 at NR, 1.3 ± 0.4 at MH, and 2.0 ± 0.6 mm2 at SH, for a distance of 2.0 mm, P = 0.12). This behavior at SH would help SWs form small circuits and coexist without mutual annihilation.


Figure 8
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  		Fig. 8. Spatial excitable gap (SEG) of SW close to the rotation center. A: representative changes of SEG during rotation of SWs. SEG was estimated by measuring an area circumscribed by wave front (red), wave tail (green), and an arc with a certain distance (radius) from the spiral tip at 10-ms intervals. A, top: front-tail maps of SWs at NR (37°C), MH (33°C), and SH (30°C). SW selected for the analysis showed rotation >180° and were distant (>5 mm) away from any other SWs in the observation area. A, bottom: sequential changes of SEG at a distance of 0.5 mm (red asterisks indicate minimal values). B: average values of minimal SEG at a distance of 0.5 mm from the spiral tip. Data were obtained from 5–9 SWs in each heart at the respective temperature (means ± SD, n = 6, *P < 0.05 vs. 37°C, {dagger}P < 0.05 vs. 33°C).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings in the present study are as follows. First, MH and SH caused a significant prolongation of APD and a reduction of CV; WL was unchanged at MH, whereas it was shortened at SH at higher stimulation frequencies. Second, the duration of VT/VFs was reduced dramatically at MH compared with that at NR and SH. Third, SWs were destabilized at MH and SH with different behaviors; those at MH were characterized by more frequent collisions, leading to longer temporal disappearance of PSs (PS-0) in favor of self termination, compared with those of SH.

Cooling-induced changes in the basal electrophysiological properties. The temperature-dependent prolongation of APD and the reduction of CV in response to cooling are the well-known properties of cardiac muscle. More than several mechanisms are involved in the APD prolongation. Voltage-clamp studies have indicated that the major factor is a decrease in the delayed rectifier K+ current (IK) with a temperature coefficient (Q10) of 4.4 (14). The decrease of IK is the result in part of its delayed activation (14). Delayed inactivation of L-type Ca2+ current (Q10 of 2.3) and a decrease of inward rectifier K+ current (Q10 of 1.5) contribute as well to the APD prolongation (6, 14). A decrease of time-independent outward current has also been described (14). The reduction of CV is most likely explained by a decrease of Na+ current availability via its temperature-dependent slowing of activation/inactivation kinetics (Q10 of ~3) (13). A reduction of gap junction conductance (Q10 of 1.4) by cooling may also contribute to the CV reduction (4).

Destabilization of rotor dynamics: undesirable or beneficial? Hypothermia caused temperature-dependent destabilization of SW dynamics. This may be attributed in part to a steeper slope of APD restitution engaged with increased depth and breadth of CV restitution, which is known to cause an enhancement of wave-front tail interactions (25). Nevertheless, we cannot rule out other factors affecting SW stability; those include short-term cardiac memory, electrotonic current, and intracellular Ca2+ cycling (24). The effect of cooling on these latter factors remains to be quantified. In any case, an increased dynamic instability would favor the generation of functional reentry. Our results are consistent with this prediction since the total incidence of VT/VF elicited by DC stimulation was increased in steps from NR to MH and SH.

Another consequence of enhanced dynamic instability is the destruction of existing waves, promoting self-termination of VT/VF. The mechanisms of SWs termination by an interaction with other waves were first demonstrated by Davidenko et al. (8). In a theoretical study on myocardial tissue model of finite size, Qu (20) has demonstrated that an increase of dynamic instability created by steep APD restitution slope promotes wave breaks, maintaining fibrillation, but it also causes the waves to extinguish, facilitating spontaneous termination of fibrillation. They showed three ways by which a wave self-terminates: 1) two waves can annihilate each other if their tips collide, 2) a wave can run into a region of refractoriness from a previous wave, or 3) a wave can move off a tissue boundary (20).

In the present study, the relative incidence of sustained VT/VF over the total VT/VF episodes at MH was much less than those at NR and SH, indicating considerable promotion of self-termination at MH. In the phase maps, the average number of PSs/2 s was increased in steps with cooling, reflecting a more frequent generation of new SWs. However, the PS-0 period resulting from temporal dissipation of SWs was also increased especially at MH. Therefore, the promotion of self-termination of VT/VF at MH is explained most likely by an increased dissipation of existing SWs, which offsets the increased generation of new SWs.

The dissipation of existing rotors at MH was the result of either mutual annihilation of 2 PSs or a collision of PSs with the AV groove after considerable meandering. Such dissipation of existing SWs was less frequent at SH. This may be the consequence of substantial shortening of WL at SH, reflected in the lower value of the critical distance (Dc) between a pair of counterrotating PSs without mutual annihilation. Under constant stimulation at a long BCL (400 ms), WLs at MH and SH were both increased compared with NR, reflecting hypothermia-induced APD prolongation. At shorter BCLs (250 and 200 ms), however, WLs at MH were similar to NR because of the concomitant decrease of CV; WLs at SH were shortened significantly because of more pronounced reduction of CV. In the SW excitations during VT/VF, the SEGmin at MH close to the rotation center (≤1 mm) was comparable with NR, whereas the value at SH was significantly larger than NR and MH, suggesting a substantial shortening of WLs. However, the values at a distance ≥ 2.0 mm did not differ among the three temperature conditions. Smaller SEGmin at a distance ≤ 1 mm at NR and MH would facilitate wave-front tail interaction just around the rotation center, whereas larger SEGmin at SH would prevent it. However, these values would not affect the whole dynamics of SW excitations at a distance ≥ 2.0 mm. Wave-front tail interactions at a distance away from the core, causing SW destabilization at MH and SH, may be attributable to other factors, including restitution kinetics, cardiac memory, or Ca2+ dynamics (24).

In short, the hypothermia-induced enhancement of dynamic instability may have dual effects: an increase of ventricular vulnerability on one side and a destruction of existing SWs by collision on the other side. MH causing moderate dynamic instability without shortening of WLs would favor the latter effect, whereas SH causing more extensive dynamic instability in association with significant shortening of WLs would compromise the latter effect. Nevertheless, further experimental and simulation model studies will be required to substantiate this interpretation.

Most previous studies in humans and animals have shown that hypothermia is associated with increased susceptibility for ventricular arrhythmias and VF (18, 23), and this effect has been related to the slowing of conduction, a heterogeneous increase in ventricular repolarization and the dispersion of refractoriness (21). However, there is experimental evidence suggesting the antiarrhythmic therapeutic potential of hypothermia (7, 10). In pigs subjected to defibrillation shocks and cardiopulmonary resuscitation following unsupported VF, Boddicker et al. (3) showed that the defibrillation success and subsequent resuscitation outcome were improved by preexisting MH and SH, and the effect was most remarkable at MH. Some unidentified mechanical, metabolic, or electrophysiological properties were suggested to be involved in the beneficial effect of hypothermia in terms of prevention of refibrillation (3). In the present study, a certain modification of SW dynamics at MH could contribute to the in vivo prevention of refibrillation following DC shocks.

Limitations. First, we investigated the temperature-dependent alterations of DC stimulation-induced SW dynamics, and the results have revealed a destabilization and an early termination of SW excitations at MH. This observation may not directly translate to the mechanisms of defibrillation, where many other factors are involved (11). Second, the experiments were carried out using a 2-D subepicardial layer of rabbit ventricular myocardium. Extending these results to 3-D hearts, especially in larger animals including humans, is not straightforward. If there is sufficient tissue mass, the chance of spontaneous termination of rotors by mutual annihilation or collision with anatomical boundaries would be reduced, whereas the enhancement of wave breakup would increase the generation of new rotors in favor of VT/VF perpetuation. The greater structural discontinuities and functional heterogeneities in diseased hearts would also alter the spatial requirements of spontaneous rotor termination. Finally, we used BDM as an excitation-contraction uncoupler that is known to alter ionic currents, to reduce APD restitution slope and to affect intracellular Ca2+ dynamics (16). However, this does not seem to invalidate the present results, because the characteristic temperature-dependent modification of SW dynamics was preserved in the absence of BDM (see supplemental Fig. 3). Despite these limitations, the present study provides a new perspective for therapeutic potential of hypothermia in the treatment of serious recurrent VT/VF (electrical storm).


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This study was supported by the Health and Labor Science Research Grant for Research on Medical Devices for Analyzing, Supporting and Substituting the Function of Human Body from the Ministry of Health, Labor and Welfare of Japan (H15-19 Physi-001) and by the Suzuken Memorial Foundation.


   ACKNOWLEDGMENTS
 
We thank Prof. Jose Jalife (SUNY, Syracuse, NY) for critical reading of our manuscript. We also thank Dr. Kazuo Nakazawa (National Cardiovascular Center, Suita, Japan) for theoretical advice in completing the study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. Kodama, Dept. of Cardiovascular Research, Research Inst. of Environmental Medicine, Nagoya Univ., Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan (e-mail: ikodama{at}riem.nagoya-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* M. Harada and H. Honjo contributed equally to this work. Back


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