Calcium Dynamics and the Mechanisms of Ventricular Tachycardia to Ventricular Fibrillation Transition in Cryoablated Rabbit Ventricles

Hwang, Gyo-Seung

Original Article

International Journal of Arrhythmia 2015;16(1): 11-18.

Introduction

   Ventricular fibrillation (VF) initiates as a well-organized waves consisting of one or two functionally re-entrant rotating waves (rotors). This stage, classically defined as Wiggers stage 1 VF, degenerates within seconds, into a more complex, less organized activation pattern classically defined as Wiggers stage 2 VF.¹ This process is thought to involve wavebreaks, wherein wavefronts are split into two or more pieces by a conduction block.
   Wavebreaks can occur when wavefronts collide with an obstacle, which can be either anatomic or functional, but the occurrence of such events depends on tissue excitability, obstacle size, and wavefront curvature.² Previous studies showed that heart wavebreaks seem to occur when wavefronts encounter zones of refractoriness created by the passage of the previous activation.^2,3
   The restitution hypothesis predicts that a small perturbation in action potential duration (APD) can result in larger APD changes on a beat-to-beat basis (i.e., alternans), which would increase the vulnerability to unidirectional conduction block and wavebreaks.^4,5 Furthermore, alternans can be spatially discordant, when the phase of alternation is out of phase with different regions of the heart, and tend to increase the dispersion of refractoriness and the propensity to conduction blocks.⁶
   While intracellular calcium (Ca_i) cycling is also increasingly recognized as an important regulator of dynamic wave instability, Ca_i dynamics may also contribute independently to initiation and break of reentry;^7,8 however, its role in wavebreak occurrence is still unclear.
   In this study, we aimed to classify the patterns of wavebreak using dual optical mapping techniques to study membrane potential (V_m) and Ca_i during the application of T-wave shocks, as well as to examine the role of Ca_i dynamics.
   To exclude the possibility that the wavebreak occurred somewhere beneath the tissue surface, we studied 10 hearts in which endocardial cryoablation left only a thin (0.5-mm) layer of surviving epicardial tissue.

Materials & Methods

Surgical Preparation and Cryoablation

   New Zealand white rabbits (N=10) weighing 3-5 kg were used in this study. After general anesthesia, the rabbit hearts were rapidly excised through midline sternotomy and immersed in cold Tyrode’s solution (NaCl 125, KCl 4.5, NaH₂PO₄ 1.8, NaHCO₃ 24, CaCl₂ 1.8, MgCl₂ 0.5, dextrose 5.5, and albumin 100 mg/L in deionized water). The ascending aorta was immediately cannulated and perfused with warm oxygenated Tyrode’s solution (36.5°C ± 0.5°C, pH 7.4 ± 0.5) at a rate of 30-40 mL/min to maintain a perfusion pressure between 80 and 95 mmHg. Two widely spaced bipolar electrodes were used for continuous pseudo-electrocardiography monitoring. Endocardial cryoablation was performed by placing a 7-cm SurgiFrost^® probe (CryoCath Technologies Inc., Montreal) into the left ventricle (LV). The probe temperature was decreased to -135°C for 5-10 minutes, during which, the epicardium was protected by the addition of warm (37°C) oxygenated Tyrode’s solution, while the entire heart was continuously Langendorff-perfused. Bipolar electrodes for S1 pacing were attached to the LV apex. Right ventricular (RV) endocardial (cathode) and LV patch electrode (anode) were placed for direct current (DC) shocks. After the optical mapping study, we perfused the coronary arteries with 1% triphenyl tetrazolium chloride (TTC) and sectioned the heart horizontally into five equally spaced sections to document the effects of cryoablation.

Optical Mapping

   We used 0.5 mg of Rhod-2 AM (Molecular Probes) dissolved in 1 mL of dimethylsulfoxide containing Pluronic F-127 (20% wt/vol) to stain Ca_i. This solution was diluted in 300 mL of Tyrode’s solution to achieve a final Rhod-2 AM concentration of 1.48 μmol/L, and was infused into the heart over a 10-min period. After perfusion with dye-free Tyrode's solution for 15-30 min to achieve Rhod-2 AM de-esterification, the heart was then stained again by direct injection of a voltage sensitive dye (RH237, Molecular Probes) into the perfusion system. The double-stained heart was excited with a laser light at 532 nm. Fluorescence was collected using two charge-coupled device (CCD) cameras (Dalsa) covering the same mapped field. The CCD cameras for V_m and Ca_i, were fitted with 715-nm long-pass and 580 ± 20 nm band-pass filters, respectively. The digital images (128 × 128 pixels) were gathered from the epicardium of the LV (25 x 25 mm² area), resulting in a spatial resolution of 0.2 x 0.2 mm² per pixel. We acquired 1,000 frames continuously with a 12-bit resolution (260-400 frames/second, or roughly 2.5-4 ms per frame). The voltage sensitive dye RH237 was used because its emission bandpass differs from that of Rhod-2, thereby preventing crosstalk between the two signals. The signal-to-noise ratio of our mapping system, as estimated from the peak-to-peak time variation in fluorescence intensity, is 40 to 1 during pacing and about 5 to 1 during VF. Cytochalasin D (cyto-D, 5-10 μmol/L) was added to the perfusate to minimize motion artifacts.
   As two CCD cameras were used, the same anatomical location may appear at different coordinates on the V_m and Ca_i maps. Therefore, we implanted four cactus needles on the epicardium as registration markers. A software program then used these markers to match the pixels on the V_m and Ca_i maps to the same locations. Data analyses were performed only with aligned maps.

Dual Optical Mapping of VT to VF Transition

   After 8 S₁ paced beats, biphasic truncated exponential waveform shocks (117 ± 62 V) of fixed pulse duration (6 ms) were delivered with a S1-shock coupling interval of 142 ± 25 ms from a Ventritex HVS-02 defibrillator on the T wave with dual optical mapping, and data recorded during the induction of VF.

Data Analysis

   The activation maps were used to examine V_m and Ca_i patterns during the induction of VF. The average fluorescence level (F) of the entire data window was calculated first, and the fluorescent level of each pixel was then compared with this average. We assigned shades of red to represent above-average fluorescence and shades of blue to represent below-average fluorescence to generate the ratio maps. All data are presented as means ± SD.

Results

Patterns of Wavebreak

   A total of 145 episodes of new wavebreaks occurred from within the mapped region 1122 ± 647 ms, resulting in VT to VF transition, with large and spatially heterogeneous variations of V_m and Ca_i occurring shortly after the shock-induced VT. In 135 of 145 episodes (93%), the wavebreaks occurred when a wavefront visited an area with persistent Ca_i elevation. The V_m map at that site showed partial or full repolarization. Figure 1 shows a typical example of a wavebreak in a high Ca_i area. Figure 1A shows V_m and Ca_i maps at the time of the shock (0 ms) and three additional snapshots 4 ms apart. In this and other color panels, the right and left lower corners were outside of the heart and were cropped. The bright dot observed at 0 ms is a light artifact used to indicate the time of the shock, after which, the Ca_i map showed high Ca_i and low Ca_i areas marked with a white and yellow asterisk, respectively, in the 236-ms frame. In the V_m map, the wavefronts propagated only from the corresponding low Ca_i area (yellow asterisk in the 240-ms frame) and formed a wavebreak (white arrow in the 244-ms frame). The black arrow shown in the phase map (Figure 1B) indicates a phase singularity formed at the boundary between the high and low Ca_i area, 244 ms after the shock. Phase singularities are points surrounded by all phases of activation that occur at the intersection of wavefronts and wavebacks, formed by wavebreak events.⁷ Figure 1C shows a ventricular myocardium after successful endocardial cryoablation. While the surviving epicardium shows a brick red color after staining with a 1% TTC solution, TTC-negative tissues show cell necrosis and contraction bands, compatible with effective cryoablation.

Figure 1D shows optical signals of V_m and Ca_i. Red and black line segments indicate the time of shock, and VT to VF transition, respectively. In 103 of the 135 episodes, the region of persistent Ca_i elevation appeared to surround part of a Ca_i sinkhole (a low Ca_i area surrounded by a high Ca_i area) which hindered wavefront propagation, and led to the occurrence of the wavebreak in the Ca_i sinkhole.

Figure 2 shows a typical pattern of a wavebreak in the Ca_i sinkhole that occurred 196 ms after the T-wave shock, shown by the white arrow on panel A. An electrical activation, marked by the yellow asterisk on the V_m Map in the 208-ms frame, arose from the corresponding Ca_i sinkhole site. The wavefront propagated (224 ms frame in panel A) and the wavebreak occurred (black arrows in panel B). However, in 4 episodes (3%), a wavebreak occurred when a wavefront encountered an area already depolarized by another wavefront, without concomitant Ca_i elevation. This is illustrated in Figure 3A by the lack of Ca_i activation observed on the Ca_i map at the corresponding site of the Y wavefront (marked by asterisk in the 832-ms frame) when the X and Y wavefronts met. Nonetheless, the wavebreak then developed at the edge of the X wavefront (white and black arrows in the 852-ms frame shown in Figure 3A and 3B). In addition, 6 (4%) wavebreak episodes developed spontaneously regardless of the presence of another wavefront and Ca_i dynamics. Figure 4 shows an example of a spontaneous wavebreak, in which the wavefront (frame 510 ms) spontaneously split to both sides (525 ms frame), resulting in a wavebreak (528-ms frame, Panel B).

Discussion

   Wiggers first proposed four distinct phases of VF based on epicardial motion visualized by high-speed cinematography in canine hearts. The 4 stages are as follows: (1) initial tachysystolic stage (0-1 s of VF), (2) stage of convulsive incoordination (1-40 s of VF), (3) stage of tremulous incoordination (40 s-3 min of VF), and (4) stage of progressive atonic incoordination (>3 min of VF).¹ The wavebreak (VT to VF transition) may occur during stage 2. The present study showed that the formation of wavebreaks at high Ca_i areas is the most common phenomenon associated with the VT to VF transition and the continuation of VF after a T-wave shock. These findings indicate that Ca_i dynamics play an important role in the VT to VF transition.

Mechanisms of Wavebreak Generation

   Tissue heterogeneity, resulting from electrical and structural remodeling, has been considered a key factor promoting wavebreaks. Recent evidence indicates that dynamic factors such as cellular membrane voltage and Ca_i cycling operate synergistically with tissue heterogeneity to promote wavebreaks. Normally, when a wave propagates through tissue, the wavefront and waveback never touch, but when they do, their point of intersection defines a wavebreak. This point is sometimes called a phase singularity,⁷ around which all phases of activation-recovery (action potential) converge.⁹
   Moreover, wavebreaks may also occur at anatomic source-sink mismatches,⁷ such as pectinate¹⁰ and papillary muscle insertions,¹¹ or at the anterior right ventricular insertion site;¹² they may also occur when electrical excitability is depressed regionally due to ischemia or drugs. However, a rotor induced by rapid pacing or a premature stimulus, may spontaneously break up into a fibrillation-like state despite homogeneous initial conditions.
   Furthermore, dynamic factors may also induce fibrillation in the normal heart.^13,14 For instance, as the wavelength is the product of APD and conduction velocity (CV), a steep APD restitution slope ( >1)^13,15 or a broad CV restitution can drive a wavebreak.¹⁶
   An alternative possible explanation for the VT to VF transition is also provided by Keldermann et al., who reported that a mechanoelectrical feedback via stretch-activated channels could induce the deterioration of an otherwise stable spiral wave into turbulent wave patterns similar to that of VF.¹⁷

Calcium Dynamics and Wavebreak

   The role of Ca_i cycling as a key factor in dynamic wave instability is well recognized, and increasing evidence suggests that, under normal physiological conditions, regulation of the contractile force of the beating heart by Ca_i is tightly controlled by V_m, such that the Ca_i transient is effectively slaved to the AP. However, calcium-induced calcium release can exhibit independent dynamics.⁷ For instance, Ca_i transients induce alternans and highly complex periodicities, despite the fact that the V_m waveform is fixed,¹⁸ and Ca_i alternans is also the predominant cause of APD alternans¹⁹ and promoter of wavebreaks.
   Moreover, Ca_i dynamics plays an important role in the mechanism of ventricular vulnerability and defibrillation. In fact, we previously showed that the first postshock activation always occurs from a Ca_i sinkhole after unsuccessful and type B successful defibrillation shocks.⁸ In this study, most wavebreaks originated around the high Ca_i area of the Ca_i sinkhole, as this region of persistent Ca_i elevation hindered wavefront propagation, generating the typical wavebreak pattern seen in Figure 2. Therefore, the results from this study add to the accumulating evidence suggesting that Ca_i dynamics plays an important role in VT to VF transition and defibrillation.

Study Limitations

   This study has a number of limitations. As with all current optical mapping studies, deeper layers of myocardium could not be examined for other possible preferential wavebreaks locations. Although, this is the reason why we performed experiments using cryoablated ventricles, the myocardial ischemia caused by subendocardial cryoablation could have affected the results of this study. It is possible that 10 wavebreak episodes were the result of subepicardial wavebreaks, regardless of Ca_i dynamics, because wavebreaks that occurred in the subepicardium could not be detected by the epicardial mapping techniques used. In addition, all experiments were performed with rabbit hearts (New Zealand white rabbits), and mechanisms underlying wavebreaks may differ in different species, as well as in ischemic and failing hearts.

Conclusion

   Wavebreaks occurring in high Ca_i areas appears to be the most common phenomenon associated with VT to VF transition and continuing VF after a T-wave shock. These findings indicate that Ca_i dynamics play an important role in the mechanisms of VT to VF transition.

References

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