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Am J Physiol Heart Circ Physiol 286: H310-H318, 2004. First published September 4, 2003; doi:10.1152/ajpheart.00092.2003
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Shock-induced arrhythmogenesis is enhanced by 2,3-butanedione monoxime compared with cytochalasin D

Yuanna Cheng,1,* Li Li,2,* Vladimir Nikolski,2 Don W. Wallick,1 and Igor R. Efimov2

1Department of Cardiovascular Medicine, Cleveland Clinic Foundation, Cleveland 44195; and 2Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106

Submitted 28 January 2003 ; accepted in final form 3 September 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Investigation of the mechanisms of arrhythmia genesis and maintenance has benefited from the use of optical mapping techniques that employ excitation-contraction uncouplers. We investigated the effects of the excitation-contraction uncouplers 2,3-butanedione monoxime (BDM) and cytochalasin D (Cyto D) on the induction and maintenance of arrhythmia by electric shocks. Electrical activity was optically mapped from anterior epicardium of rabbit hearts (n = 9) during shocks (–100 V, 8 ms) applied from a ventricular lead at various phases of action potential duration (APD). Restitution curves were obtained using S1-S2 protocol and measurement of APD values at 70% of repolarization. Compared with Cyto D, BDM significantly shortened APD at 90% of repolarization, although no significant difference in dispersion of repolarization was observed. Wavelength was also shortened with BDM. In general, shock-induced arrhythmias with BDM and Cyto D were ventricular tachycardic in nature. With respect to shock-induced sustained arrhythmias, the vulnerable window was wider and the incidence was higher with BDM than with Cyto D. There was also a difference in the morphology of ventricular tachycardia (VT) between the two agents. The arrhythmias with BDM usually resembled monomorphic VT, especially those that lasted >30 s. In contrast, arrhythmias with Cyto D more resembled polymorphic VT. However, the average number of phase singularities increased under Cyto D vs. BDM, whereas no significant difference in the dominant frequency of shock-induced sustained arrhythmia was observed. BDM reduced the slope of the restitution curve compared with Cyto D, but duration of arrhythmia under BDM was significantly increased compared with Cyto D. In conclusion, BDM increased arrhythmia genesis and maintenance relative to Cyto D.

excitation-contraction uncouplers; cardiac vulnerability; action potential duration restitution; optical mapping

OPTICAL IMAGING OF ELECTRICAL activity with voltage-sensitive dyes is commonly used in studies of arrhythmia induction, maintenance, and termination. The advantages of these techniques compared with conventionally used electrode-based mapping include the ability to map entire action potentials from hundreds or thousand of sites, immunity from electrical stimulus-induced artifacts, and adjustable spatial and temporal resolutions (18).

However, these advantages come at a price. The major difficulty in applying optical mapping techniques is the motion artifact, which is caused by vigorous contraction of the myocardium. One way to overcome motion artifacts is to use excitation-contraction uncouplers such as 2,3-butanedione monoxime (BDM; Refs. 5, 15, 19, 25, 28) to abolish the myocardial contraction. However, BDM is known to inhibit many ionic currents at concentrations that are effective at uncoupling contractile activity, and it causes a significant shortening of action potential duration (APD) in canine (7, 42), sheep (32), guinea pig (32), and swine (31) hearts. Cytochalasin D (Cyto D) was recently proposed as a novel excitation-contraction uncoupler for optical mapping studies (7, 42). Cyto D has been shown to block contractions without affecting action potential shape or duration in rat (39, 44), canine (7, 42), and swine (31) hearts. Cyto D is becoming an excitation-contraction uncoupling agent of choice in optical mapping studies despite its effects on mouse action potentials (26) and its significant cost and toxicity. Thus additional characterization of this agent is warranted to evaluate its wider applicability in the field.

The recently formulated restitution hypothesis suggests that BDM has an antifibrillatory effect. Riccio et al. (37) and Lee et al. (31) showed that BDM prevented the induction of ventricular fibrillation (VF), caused less dynamic complexity of fibrillation, and converted existing VF into ventricular tachycardia (VT). They also suggested that this conversion of VF to VT was due to a reduction of the slope of the APD restitution curve by BDM. On the other hand, the slope of the restitution curve with Cyto D has been shown to be steeper than that of BDM (1, 31). Therefore, investigation of the effects of the two agents on the restitution curve was used to elucidate mechanisms of arrhythmia maintenance. However, a recent report by Banville and Gray (2) illustrates that alternans and arrhythmia dynamics in rabbit hearts are affected by the spatial dispersion of APD restitution as well as conduction velocity (CV) restitution and not simply by the slope of APD restitution.

Qin et al. (36) recently reported the effects of major interventions of heart isolation, voltage-sensitive dye, and excitation-contraction uncouplers used during optical mapping on VF in pig hearts. Despite the importance of optical mapping in vulnerability/defibrillation research, the comparative effects of BDM and Cyto D on shock-induced arrhythmia genesis and maintenance have not yet been systematically evaluated. The purpose of this study was 1) to compare the effects of BDM and Cyto D on optically recorded action potentials in Langendorff-perfused rabbit hearts at the effective concentrations routinely used to immobilize the heart, 2) to examine the effects of BDM and Cyto D on control monophasic action potentials under the same conditions as the optical mapping, 3) to assess the effects of BDM and Cyto D on shock-induced vulnerability and the duration of induced arrhythmia, 4) to compare the dynamics and characteristics of shock-induced arrhythmias between BDM and Cyto D, and 5) to determine whether these changes occur in parallel with changes in the slopes of APD restitution curves and wavelengths.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Our experimental protocol was approved by the Animal Research Committee of the Cleveland Clinic Foundation. All animals used in this study received humane care in compliance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.

Experimental preparation. Initially, nine Langendorff-perfused hearts from young rabbits (60 ± 5 days old) were used in the study. Details of the experimental preparation have been described in our previous publications (13, 14, 17, 19, 20–22). Briefly, after administration of anesthesia, the heart was removed and placed on a Langendorff apparatus, where it was retrogradely perfused with oxygenated (95% O2-5% CO2) modified Tyrode solution with the following composition (in mM): 128.2 NaCl, 1.3 CaCl2, 4.7 KCl, 1.05 MgCl2, 1.19 NaH2PO4, 25 NaHCO3, and 11 glucose. Temperature and pH were continuously maintained at 36 ± 0.5°C and 7.30 ± 0.05, respectively.

A custom-made 10-mm platinum coil electrode (Guidant) was inserted into the right ventricular cavity through the pulmonary artery. A second similar electrode was positioned in the bath 1 to 2 cm above and 1 to 2 cm behind the heart. The heart was stained with 10 µM 4-[{beta}-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS) (Molecular Probes) over 5–10 min. In each experiment, the heart was perfused in the following sequence: 1) control: modified Tyrode's solution; 2) BDM: modified Tyrode solution plus 15 mM BDM (Fisher Scientific); 3) BDM washout: modified Tyrode solution without BDM; and 4) Cyto D: modified Tyrode solution plus 20 µM Cyto D (Sigma). We added BDM first in the perfusate because its effects can be fully washed out (7, 31), unlike the effects of Cyto D (7). However, in two separate experiments, the effects of Cyto D were tested without prior perfusion of BDM. We found no significant difference in the slopes of restitution curves or other characteristics of the restitution properties obtained in the presence of Cyto D after washout of BDM versus those obtained without prior perfusion of BDM. Thus the experimental protocol described (see Experimental protocol) was first executed with BDM and then with Cyto D. In each experiment, the heart was equilibrated in the chamber for ~30 min before the start of the experiment. The experimental protocol was started 10–15 min after administration of either BDM or Cyto D. The BDM washout period was usually 30 min. In general, the entire study lasted <3 h, which is well within the normal physiological survival range of Langendorff-perfused hearts.

Optical mapping. Di-4-ANEPPS fluorescence was excited by a direct current-powered light source at a wavelength ({lambda}) of 520 ± 45 nm (Oriel). The emission was collected above 610 nm by a 16 x 16-element square matrix of photodiodes (Hamamatsu) coupled to a computerized data-conditioning and -acquisition system (Microstar Laboratories). Data were filtered at 1 kHz and sampled at a rate of 1,894 frames/s to yield a temporal resolution of 528 µs. The field of view was 17.5 x 17.5 mm in all experiments. Optical action potentials were recorded before, during, and after the application of shocks.

Experimental protocol. The heart was positioned in a temperature-controlled glass chamber with the anterior wall facing the optical apparatus and was paced at a basic cycle length of 300 ms from the apex of the heart. To obtain the shock-induced vulnerability in BDM and Cyto D, truncated exponential cathodal monophasic shocks (intensity, 100 V; duration, 8 ms) were delivered at various phases of APD from a defibrillator (HVS-02, Ventritex) between the two electrodes described. We chose to use a –100-V shock, because in our previous study (43), we found that the arrhythmia incidence induced by a –100-V shock reached 100% when applied at the vulnerable phase of APD.

Standard APD restitution curves (30) for each rabbit heart in the presence of BDM and Cyto D were measured using an S1-S2 stimulation protocol, and APD values at 70% of repolarization (APD70) were averaged from all 256 optical channels. Single test pulses (S2) were delivered after every 20th basic pulse (S1) at a basic cycle length (S1-S1) of 300 ms, and the stimulus protocol was repeated with progressively shorter S1-S2 intervals. We began with an S1-S2 interval of 500 ms, and then we tested intervals of 400, 350, 300, 250, 220, and 200 ms. After that, the S1-S2 value was decreased in 10-ms steps until the premature pulse was blocked. The S1-S2 coupling interval was then increased by 5–10 ms to restore capture and was subsequently shortened in 5-ms increments until S2 was blocked. The duration of the response to S2 was measured at APD70 and plotted as a function of the preceding diastolic interval (DI). We used the following formula to calculate the DI: DI = TI2 – TI1 – ADP70, where TI2 is the time interval from the last S1 stimulus to the peak upstroke of the response induced by S2, TI1 is the time interval from the last S1-stimulus to the peak upstroke of the response induced by the last S1, and APD70 is the APD of the second-to-last S1-induced action potential. We measured DI using APD70 induced by the second-to-last S1 stimulus instead of the last S1 stimulus because this gave us more accurate and reliable DI measurements. We assumed that APD was stabilized under steady-state pacing of the heart. The parameters of APD70 and DI were automatically calculated using a custom-built data analysis program based on LabView (National Instruments).

Monophasic action potential recordings. Because control action potentials cannot be faithfully recorded in the absence of excitation-contraction uncouplers during optical mapping, we used eight additional Langendorff-perfused rabbit hearts to record control monophasic action potentials (MAP) in the absence of voltage-sensitive dye. They were compared with those under 15 mM BDM and in the presence of 20 µM Cyto D. In each heart, a pressure MAP catheter (model 1675PS, EP Technologies) was inserted into the left ventricular cavity via a cut in the left atria. The heart was kept at the same conditions as those described in Optical mapping. Because Cyto D is not completely washable and because we aimed to limit the duration of each experiment to exclude any adverse effects of prolonged MAP recordings, we divided eight rabbits into two groups of four rabbits. One group was used to compare MAP values between control solutions and in the presence of BDM, whereas the other group was used to compare MAP values between control measurements and in the presence of Cyto D.

Classification of shock-induced arrhythmia. VT/VF is not readily sustainable in normal young rabbit hearts either in vivo (34) or in vitro (6, 33, 35) and spontaneously terminates, perhaps due to the small heart size compared with the wavelength and/or due to the lack of fibrosis. In some cases, no extra beats resulted from the shock. Occurrence of one or more extra beats was defined as shock-induced arrhythmia, which can be further divided into sustained or nonsustained arrhythmias. In accordance with Fabritz et al. (23), we defined shock-induced arrhythmias as sustained if a sequence of >=6 regular or irregular fast (cycle length <160 ms) responses (extra beats) were present. Induction of 1–5 repetitive responses (extra beats) was regarded as nonsustained arrhythmia. We also measured a number of arrhythmias that lasted >5 s under BDM and Cyto D conditions.

Characterization of shock-induced arrhythmias under BDM and Cyto D. We applied fast Fourier transform (FFT) analysis (38) to study the dynamics of the shock-induced sustained arrhythmias (>=6 extra beats) with BDM and Cyto D. Depending on the length of the recorded arrhythmias, the length of the data analyzed was either 750 or 1,500 ms. For each arrhythmia instance, FFT of the optical data was calculated for all 256 channels. The FFT values at 0-Hz frequency were set to 0. The sum of the FFT curves of all the channels was called the composite FFT. In the composite FFT, the frequency value that had the highest amplitude was considered the dominant frequency. For the single-channel FFT, the frequency value that had the highest amplitude was considered the local dominant frequency. The standard deviations of the local dominant frequencies for all channels were calculated for each arrhythmia instance and were named the dominant frequency standard deviation. We also defined the monomorphic or polymorphic nature of VT using visual inspection of optical data that was performed independently by two investigators based on morphology and periodicity of recordings (cf. Fig. 5, A–C).



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  		Fig. 5. Representative examples of sustained arrhythmia under BDM and Cyto D. Shown are a sustained arrhythmia that lasted >30 s under BDM (A), a sustained arrhythmia that self-terminated after 11 s under BDM (B), and a sustained arrhythmia that lasted >30 s under Cyto D (C) conditions. Snapshots of transmembrane voltage selected at one-fourth and three-fourths duration of the data set are represented (top). Phase singularity with clockwise reentry around it ({circ}) and phase singularity with counterclockwise reentry around it ({bullet}) are indicated in corresponding phase maps. Optical traces were picked from the center of the fields of view with data length of 1.57 s. Number of phase singularity (PS) vs. time (T) is shown. Composite fast Fourier transforms for the shown data set are illustrated (bottom).

 

Electrical activity was analyzed in the phase space using Bray-Wikswo algorithms (9–11). Using phase representation, we identified phase singularities and measured the dynamics and average numbers in the field of view. For each arrhythmia data set, we averaged the number of phase singularities over time.

Data analysis and visualization. The signal analysis software programs used in this study were previously described (13, 19, 20). These programs automatically calculated maps of activation, repolarization, and APD from all 256 optical recordings. Activation time (AT) was subtracted from coupling interval (CI), which is defined as the time difference between the stimulus and the shock application to calculate fractional (percent) APD in each channel in which the shock was applied according to the following formula: %APD = (CI – AT)/APD x 100. The dispersion of repolarization was defined as the difference between the shortest and longest repolarization times across the field of view (6). The vulnerable window was defined by excluding the CI at which arrhythmia incidence was <50% (12, 43).

Statistical analysis. Group data were expressed as mean values ± SDs. Statistical comparisons were performed using the paired or unpaired t-test. Differences were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of BDM and Cyto D on optically recorded APDs. In the present study, we tested BDM and Cyto D at concentrations that effectively abolish contractions in the intact rabbit heart. In the past, we routinely used BDM at a concentration of 15 mM to immobilize the heart (13, 14, 17, 19, 20–22). To determine the effective concentration of Cyto D, we conducted a preliminary study in two hearts. The effects of 10, 20, and 30 µM Cyto D on contractility of intact hearts were assessed by measuring left ventricular pressure with a latex balloon and comparing the responses to those in the presence of 15 mM BDM. Figure 1 shows a representative result from one heart. Addition of 15 mM BDM abolished most of the contraction, making the optical action potential recording possible. Upon washout of BDM, contractility of the heart returned to the control level. Subsequent addition of 10 µM Cyto D largely eliminated contraction, whereas 20 and 30 µM Cyto D almost completely abolished contraction. Thus we chose 20 µM Cyto D in this study, because 10 µM Cyto D left significant motion artifacts at the edges of the field of view in some hearts.



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  		Fig. 1. Representative traces of optical transmembrane action potential (AP), left ventricular (LV) pressure (LVP), and electrocardiograms (EKG) in controls (A) and during perfusion with 2,3-butanedione monoxime (BDM; B), during BDM washout (C), and with administration of three concentrations of cytochalasin D (Cyto D; D–F). Three consecutive optical action potentials were taken from 1 of the 256 channels recorded during steady-state ventricular pacing at a cycle length of 300 ms. LV pressure traces were measured with a latex balloon in the LV cavity.

 

Figure 2A shows an example of superimposed optical action potentials recorded with 15 mM BDM and 20 µM Cyto D from two different optical channels in the same heart. APDs in the presence of BDM were consistently shorter than those recorded in the presence of Cyto D. In all hearts (n = 9), we found that 15 mM BDM significantly (P < 0.001) shortened the APDs at 50, 70, and 90% of repolarization (APD50, APD70, and APD90, respectively) compared with 20 µM Cyto D at the basic cycle length of 300 ms. On average, the reduction in APD50 was 23%, APD70 was 20%, and APD90 was 17%. See Table 1 for details. Accordingly, the average repolarization time across the field of view defined at APD90 was also significantly reduced by BDM compared with Cyto D (208.8 ± 9.9 vs. 235 ± 13.6 ms; P < 0.001). However, no significant difference in dispersion of repolarization was observed between the two groups (53.7 ± 23.1 in BDM vs. 48.5 ± 55.5 ms in Cyto D; P = 0.68). Furthermore, the average conduction times across the field of view (minimum AT – maximum AT) were 33.2 ± 7.3 and 28.3 ± 5.8 ms (P < 0.05; n = 9 hearts) in BDM and Cyto D, respectively. CV values across the field of view (field of view divided by conduction time) were reduced in BDM relative to Cyto D. However, the activation patterns for both agents remained similar. The wavelength ({lambda} = APD90 x CV) was significantly shorter with BDM compared with Cyto D (9.0 ± 1.9 vs. 12.5 ± 2.6 cm; P < 0.01). See Table 1 for details.



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  		Fig. 2. Representative traces of optical transmembrane action potentials and monophasic action potentials. Superimposed representative optical recordings of action potentials during the BDM and Cyto D taken from 2 out of 256 channels (A). Heart was paced at basic cycle length of 300 ms. Photograph of the preparation (middle) shows the 17.5 x 17.5-mm field of view (inset), where recording sites from which illustrated action potentials were taken are indicated ({blacksquare}). Also in this image, the bipolar electrode that was used for pacing is shown at the apex of the heart. Representative examples of monophasic action potential recordings under control, BDM, and Cyto D are shown (B).

 

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  		Table 1. Optical action potential recordings during steady-state ventricular pacing at cycle length of 300 ms

 

These optical action potential recordings lacked controls, because we were unable to faithfully record them in the absence of excitation-contraction uncouplers. Therefore, in an additional study that included eight Langendorff-perfused rabbit hearts, we recorded control MAPs and compared them with those in the presence of 15 mM BDM (n = 4) or 20 µM Cyto D (n = 4). Compared with control, 15 mM BDM significantly shortened APD50, APD70, and APD90 by 18, 15, and 14%, respectively. In contrast, 20 µM Cyto D slightly but significantly prolonged APD90 by 8%. The percentage reductions of APD in BDM compared with Cyto D are virtually identical between optical recordings and MAP recordings (compare last rows of Tables 1 and 2). Typical examples of MAP recordings are shown in Fig. 2B. Summarized data are shown in Table 2.


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  		Table 2. Monophasic action potential recordings during steady-state ventricular pacing at 300-ms cycle length

 

Shock-induced vulnerability by BDM and Cyto D. We compared the incidences of shock-induced arrhythmias under two conditions: perfusion with BDM and with Cyto D. Figure 3 shows the results. Monophasic shocks (–100 V, 8 ms) were delivered during various phases of the action potential during steady-state pacing at a cycle length of 300 ms. Figure 3A shows the incidence of shock-induced arrhythmias, which includes both sustained and nonsustained arrhythmias, and Fig. 3B shows the incidence of sustained arrhythmias only (6 or more extra beats). In both cases, perfusion with BDM resulted in more frequent shock-induced arrhythmias. All shock-induced arrhythmias (nonsustained and sustained) by BDM and Cyto D fell in the range of 20–90% of APD. Within this range, the overall incidences of shock-induced arrhythmias were 72% in the presence of BDM and 50% in the presence of Cyto D. Similarly, shock-induced sustained arrhythmia incidences were 37% with BDM vs. 16% with Cyto D. The range of the vulnerable window was increased from 40 to 80% of APD in Cyto D to 30 to 80% of APD in BDM for all arrhythmias and from 60 to 70% in Cyto D to 40 to 70% in BDM for sustained arrhythmias.



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  		Fig. 3. Comparison of incidence of shock-induced arrhythmias by BDM and Cyto D. Arrhythmias were induced by –100-V, 8-ms monophasic shocks delivered at various phases of the action potential duration (APD). Horizontal bars represent ranges of vulnerable windows. See text for more details.

 

It is important to emphasize that BDM also promoted maintenance of arrhythmia. In BDM, 18% of shock-induced arrhythmias lasted >5 s, whereas in the presence of Cyto D, only 5% of arrhythmias lasted >5 s. We also observed a significant difference in the number of arrhythmias that lasted >30 s and required defibrillation: 10 vs. 4% for BDM vs. Cyto D, respectively. Table 3 summarizes these results.


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  		Table 3. Effects of BDM and Cyto D on arrhythmia genesis and maintenance

 

Furthermore, we compared the total numbers of shock-induced arrhythmias by the two agents with the control conditions. Because we cannot faithfully record action potentials before the application of uncouplers in the control, we are only able to calculate the total numbers of shocks that induced the arrhythmias in control hearts after hearts were stained with the voltage-sensitive dye di-4-ANEPPS. Extra beats immediately following the shocks were identified by carefully inspecting and counting the number of upstrokes of recorded optical signals, which are immune to the effects of motion artifacts caused by contraction of the heart. In control conditions in 9 hearts, 62 shock-induced arrhythmias were identified. Of those, 55 arrhythmias were nonsustained, whereas 7 were sustained. In comparison, in the presence of BDM, there was a total of 73 shock-induced arrhythmias; 36 of these were nonsustained, whereas 37 were sustained. In the presence of Cyto D, a total of 63 arrhythmias were observed. Of these, 43 were nonsustained and 20 were sustained. Therefore, the number and characteristics of shock-induced arrhythmias in the presence of Cyto D were closer to control than were those in the presence of BDM.

Comparison of characteristics of shock-induced arrhythmias under BDM and Cyto D. We performed FFT analysis of all shock-induced sustained arrhythmias in BDM or Cyto D from nine hearts, which we found to be tachycardic in nature, demonstrated by the fact that most of the frequency components were <10 Hz (longer than the 100-ms cycle length) as shown in Table 4. We further characterized the type of shock-induced VT under BDM and Cyto D. As summarized in Fig. 4, the shock-induced arrhythmias in BDM usually resembled monomorphic VT; this was especially true of those >30 s. In contrast, shock-induced arrhythmias in Cyto D more resembled a polymorphic tachyarrhythmia. Among the 37 shock-induced sustained arrhythmias under BDM, 23 were monomorphic VT and 14 were polymorphic VT. In comparison, among the 20 shock-induced sustained arrhythmias under Cyto D, the numbers of monoand polymorphic VTs were 1 and 19, respectively (see Fig. 4A). This difference was especially pronounced after 30 s. Arrhythmias lasting >30 s were all monomorphic under BDM and all polymorphic under Cyto D (see Fig. 4B). This indicates that initially unstable polymorphic arrhythmia under BDM stabilizes during this 30-s period if it survives. In contrast, it always remains polymorphic under Cyto D. Typical examples of sustained arrhythmias under BDM and Cyto D conditions are shown in Fig. 5. Figure 5A represents the dynamics of a sustained arrhythmia lasting >30 s in the presence of BDM. The optical trace picked from the center of the field of view was regular and repeated exactly from beat to beat. The average number of phase singularities was 0 over the 1.57-s time interval. Dominant frequency was 5.33 Hz, which corresponds to a cycle length of 188 ms. It was a typical monomorphic VT. Figure 5B illustrates a sustained polymorphic arrhythmia that self-terminated after 11 s under BDM conditions. The average number of phase singularities was 1.88 over the 1.57-s time interval. Dominant frequency was 7.33 Hz with a larger dispersion than that in Fig. 5A. Figure 5C shows a sustained arrhythmia that lasted >30 s under Cyto D conditions. It was polymorphic with an average of 2.47 phase singularities. Its dominant frequency was 7.33 Hz, and it had the largest dispersion among all the examples. As summarized in Table 4, the average number of phase singularities under Cyto D conditions was greater than under BDM (2.2 ± 0.4 vs. 1.5 ± 0.5; P < 0.05; Cyto D vs. BDM). No statistically significant differences were found in dominant frequencies under BDM and Cyto D conditions (7.8 ± 0.9 vs. 7.0 ± 0.5 Hz; P = 0.16; BDM vs. Cyto D). However, the standard deviation of dominant frequency under Cyto D was significantly larger than that under BDM (1.12 ± 0.28 vs. 0.34 ± 0.13 Hz; P < 0.01; Cyto D vs. BDM). Both the increased average number of phase singularities and the dispersion of dominant frequencies were consistent with increased fractionation of arrhythmia wave fronts under Cyto D compared with BDM conditions.


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  		Table 4. Characteristics of shock-induced arrhythmias with BDM and Cyto D

 


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  		Fig. 4. Number of monomorphic (Mono) and polymorphic (Poly) sustained arrhythmias under BDM and Cyto D. Numbers of sustained arrhythmias (>=6 extra beats) from 9 hearts (A) are compared with numbers of sustained arrhythmias that lasted >30 s, also from 9 hearts (B).

 

Effects of BDM and Cyto D on APD restitution curves. Finally, we constructed APD restitution curves from nine hearts in the presence of BDM and Cyto D. Figure 6A shows a representative curve from one heart and an averaged curve from nine hearts (Fig. 6B). The average values for maximum slope from nine hearts were 0.91 ± 0.38 under BDM and 1.51 ± 0.18 under Cyto D (P < 0.05). From this result, it is apparent that BDM flattened the restitution slope compared with Cyto D as was reported by others (1, 2, 31).



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  		Fig. 6. Representative and average APD restitution curves for BDM and Cyto D. APD at 70% of repolarization (APD70) was plotted against diastolic interval (DI). In the representative example (A), the maximum slopes for BDM and Cyto D are 0.98 and 1.51, respectively. In the averaged curves (B), the maximum slopes are 0.90 and 1.62 (BDM vs. Cyto D, respectively). See text for more details.

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we showed that BDM (15 mM) produced a significant reduction of APD50, APD70, and APD90 when compared with the effects of 20 µM Cyto D in the whole rabbit heart, which is in agreement with findings from others (7, 31). Furthermore, APD90 in control MAP recording was slightly yet significantly shorter than that under Cyto D but was significantly longer than that for BDM, which is also in agreement with a recent report (2). We demonstrated that BDM resulted in a significant increase of vulnerability to shock-induced arrhythmias compared with Cyto D. This was evident from a widening of the vulnerable window and increased propensity to initiate arrhythmias. We did not observe any significant increase of ventricular repolarization heterogeneity between the two agents, yet we observed that BDM shortened the wavelength compared with Cyto D with a mean of 9 vs. 12.5 cm. Also CV across the field of view was slower in BDM. We found that shock-induced arrhythmias in BDM and Cyto D were tachycardic in nature. In addition to a different propensity to induce arrhythmias between these two agents, there was a difference in morphology in shock-induced VT. Namely, the arrhythmias in BDM usually resembled monomorphic VTs; this was especially true of those >30 s. In contrast, arrhythmias in Cyto D more resembled polymorphic tachyarrhythmias. In agreement with the results of others, we confirm that BDM flattened the restitution curve (1, 31, 41), which also agrees with the restitution theory that suggests reduced propensity to polymorphic tachyarrhythmia. We also confirm that in agreement with the restitution hypothesis, BDM reduced fractionation of wave fronts and average numbers of phase singularities compared with Cyto D. However, such reduction was accompanied by both increased vulnerability to shock-induced arrhythmias and increased duration of arrhythmia in BDM compared with Cyto D. This poses a question regarding usefulness of flattening the restitution curve as an antiarrhythmic drug-development strategy. Our point of view is in agreement with a recent report by Gray et al. (2).

The recently formulated restitution hypothesis states that the slope of the restitution curve is an important factor in the development of wave breaks (dynamic wave-front fractionation), which are suggested to be a primary mechanism of the transition from VT to VF as well as the mechanism responsible for the maintenance of VF (41). Furthermore, the restitution hypothesis suggests that strategies for antiarrhythmic drug development should be based on targeting the slope of the restitution curve and the range of diastolic intervals at which this slope exceeds the critical value of 45° (8, 24, 27, 29, 30, 40).

On the other hand, such flattening can be achieved by shortening the APD at long coupling intervals or by prolonging the APD at short coupling intervals. The former preserves the wavelength, whereas the latter shortens the wavelength. We suggest that the two possibilities will have different impacts on arrhythmia maintenance despite similar effects on the slope of the restitution curve. Indeed, our results show that despite the flattened restitution curve under BDM, arrhythmia duration was increased.

Shock-induced arrhythmogenesis is governed by reentrant mechanisms. We have previously demonstrated that such arrhythmias are induced by virtual electrode-induced phase-singularity mechanisms (13, 19, 20). Phase singularities are observed in shock-induced arrhythmias under both BDM and Cyto D, which suggests that the mechanism for maintenance of the arrhythmias is also reentry. Decreased dispersion of the standard deviation of the dominant frequency and decreased average numbers of phase singularities in BDM compared with Cyto D indicate that BDM caused more regular patterns of activation in optical recordings during arrhythmia and decreased fractionation of wave fronts. This is in agreement with the restitution hypothesis (slope <1), which suggests less-frequent fractionation. Yet a more-regular reentrant pattern (monomorphic VT) under BDM conditions was maintained longer. This could result from significant shortening of the wavelength and slowing of CV by BDM.

Furthermore, our study demonstrates that BDM enhanced vulnerability to arrhythmia induced by shock compared with Cyto D. We believe that this might be due to suppression of several ionic currents, including 1) the Ca2+ current, which was suggested as a target for flattening of the restitution slope (41); and 2) the Na+ current. BDM produces a significant reduction of APD50, APD70, and APD90 compared with Cyto D. Cyto D is known to have no significant effect on the APD morphology in various species (7, 31, 39, 42). However, compared with controls, Cyto D slightly but significantly prolonged APD90 in rabbit hearts as reported in this and another study (2). The ionic basis for this prolongation needs to be further investigated. Considering that there is no significant increase of ventricular repolarization heterogeneity between BDM and Cyto D, we speculate that an enhanced susceptibility to deexcitation due to shortening of APD by BDM might contribute to the difference in vulnerability between BDM and Cyto D that was observed in this study. Shock-induced arrhythmias are critically dependent on the ability of the shock to deexcite tissue. We have recently demonstrated that deexcitation during the early plateau phase is prevented by Ca2+ current (43). Therefore, suppression of Ca2+ current could potentially enhance deexcitation and vulnerability. Although additional studies are required to explore this at the cellular level, we suggest that targeting Ca2+ channels for restitution flattening has to be approached with care. In addition, suppression of Na+ channels results in slowing of conduction, which enhances arrhythmogenesis.

It is noteworthy that in our study we used young rabbit hearts (average age of rabbit, 2 mo) as in most of our previous studies (13, 14, 17, 19–22). We observed that the majority of shock-induced arrhythmias in these young rabbits with BDM and Cyto D were transient. For example, 82% of shock-induced arrhythmias in BDM and 95% of arrhythmias in Cyto D lasted <5 s. This is consistent with reports of others without BDM or Cyto D (6, 33–35). On the other hand, Manoach et al. (34) demonstrated that verapamil converted transient fibrillation into sustained fibrillation in young rabbits. Similarly, verapamil exerted a profibrillatory effect in the goat model of atrial fibrillation (16).

Finally, both BDM and Cyto D are frequently used to suppress motion artifacts in optical mapping studies. Our results indicate that although both agents have effects on control MAPs in rabbit heart, the relative prolongation of MAP duration with Cyto D is less pronounced than the shortening and triangulation of the MAP associated with BDM (see Table 2 and Fig. 2). The number and characteristics of shock-induced arrhythmias under Cyto D are also closer to those of the control hearts. Thus for studies on rabbit hearts, Cyto D more closely approximates the control conditions. In view of already-published reports (36), it is apparent that this result may not be generalizable from species to species or perhaps even from ventricular to atrial preparations within the same species.

In conclusion, BDM shortens APD and thus flattens the restitution curve. In contrast, Cyto D slightly but significantly prolongs APD. In addition, BDM results in slowing of conduction compared with Cyto D. As a result, wavelength is significantly shorter under BDM than under Cyto D. In agreement with the restitution hypothesis, flattening of the restitution curve by BDM is associated with decreased fractionation of arrhythmia wave fronts and the more monomorphic nature of arrhythmia compared with polymorphic arrhythmias under Cyto D. However, our findings indicate that BDM enhances vulnerability to shock-induced arrhythmia and increases its duration compared with Cyto D. This could be a result of the reduction of wavelength. Thus flattening of the restitution curve as an antiarrhythmic drug-development strategy did not work in this model.

Study limitations. Because the main focus of this study was to compare the shock-induced arrhythmias under BDM and Cyto D, we did not evaluate the dynamic restitution relations in the present study, which other studies have already reported (Refs. 1, 31).


   ACKNOWLEDGMENTS
 
The authors thank Dr. David R. Van Wagoner for careful review of the manuscript and helpful suggestions on improvement. The authors are grateful to Brian Wollenzier for excellent technical support.

GRANTS

This study was supported by Grant 9960384V from the American Heart Association Southern and Ohio Valley Research Consortium and Grant 0235172N from the National American Heart Association (to Y. Cheng) and National Institutes of Health Grant HL-67322 (to I. R. Efimov).


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Cheng, Dept. of Cardiovascular Medicine, Desk FF10, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195 (E-mail: chengy{at}ccf.org).

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.

* Y. Cheng and L. Li contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Banville I and Gray RA. Effect of cytochalasin D on action potential duration restitution (Abstract). Pacing Clin Electrophysiol 23: 609, 2000.
  2. Banville I and Gray RA. Effect of action potential duration and conduction velocity restitution and their spatial dispersion on alternans and the stability of arrhythmias. J Cardiovasc Electrophysiol 13: 1141–1149, 2002.[CrossRef][Web of Science][Medline]
  3. Banville I, Gray RA, Ideker RE, and Smith WM. Shock-induced figure-of-eight reentry in the isolated rabbit heart. Circ Res 85: 742–752, 1999.[Abstract/Free Full Text]
  4. Behrens S, Li C, and Franz MR. Effects of myocardial ischemia on ventricular fibrillation inducibility and defibrillation efficacy. J Am Coll Cardiol 29: 817–824, 1997.[Abstract]
  5. Biermann M, Rubart M, Wu J, Moreno A, Josiah-Durant A, and Zipes DP. Effects of cytochalasin D and 2,3-butanedione monoxime on isometric twitch force and transmembrane action potentials in isolated canine right ventricular trabecular fibers. J Cardiovasc Electrophysiol 9: 1348–1357, 1998.[Web of Science][Medline]
  6. Boyett MR and Jewell BR. Analysis of the effects of changes in rate and rhythm upon electrical activity in the heart. Prog Biophys Mol Biol 36: 1–52, 1980.[Web of Science][Medline]
  7. Bray MA, Lin SF, Aliev RR, Roth BJ, and Wikswo JP Jr. Experimental and theoretical analysis of phase singularity dynamics in cardiac tissue. J Cardiovasc Electrophysiol 12: 716–722, 2001.[CrossRef][Web of Science][Medline]
  8. Bray MA and Wikswo JP. Considerations in phase plane analysis for nonstationary reentrant cardiac behavior. Phys Rev E Stat Nonlin Soft Matter Phys 65: 051902, 2002.[Medline]
  9. Bray MA and Wikswo JP. Use of topological charge to determine filament location and dynamics in a numerical model of scroll wave activity. IEEE Trans Biomed Eng 49: 1086–1093, 2002.[CrossRef][Web of Science][Medline]
  10. Cheng Y, Mowrey KA, Nikolski V, Tchou PJ, and Efimov IR. Mechanisms of shock-induced arrhythmogenesis during acute global ischemia. Am J Physiol Heart Circ Physiol 282: H2141–H2151, 2002.[Abstract/Free Full Text]
  11. Cheng Y, Mowrey KA, Van Wagoner DR, Tchou PJ, and Efimov IR. Virtual electrode induced re-excitation: a basic mechanism of defibrillation. Circ Res 85: 1056–1066, 1999.[Abstract/Free Full Text]
  12. Cheng Y, Nikolski V, and Efimov IR. Reversal of repolarization gradient does not reverse the chirality of shock-induced reentry in the rabbit heart. J Cardiovasc Electrophysiol 11: 998–1007, 2000.[Web of Science][Medline]
  13. Davidenko JM, Pertsov AV, Salomonsz R, Baxter W, and Jalife J. Stationary and drifting spiral waves of excitation in isolated cardiac muscle. Nature 355: 349–351, 1992.[CrossRef][Medline]
  14. Duytschaever MF, Garratt CJ, and Allessie MA. Profibrillatory effects of verapamil but not of digoxin in the goat model of atrial fibrillation. J Cardiovasc Electrophysiol 11: 1375–1385, 2000.[CrossRef][Web of Science][Medline]
  15. Efimov IR, Aguel F, Cheng Y, Wollenzier B, and Trayanova N. Virtual electrode polarization in the far field: implications for external defibrillation. Am J Physiol Heart Circ Physiol 279: H1055–H1070, 2000.[Abstract/Free Full Text]
  16. Efimov IR and Cheng Y. Mechanisms of defibrillation. 3. Virtual electrode-induced wave fronts and phase singularities; mechanisms of success and failure of internal defibrillation. In: Optical Mapping of Cardiac Excitation and Arrhythmias, edited by Rosenbaum DS and Jalife J. Armonk, NY: Futura, 2001, ch. 22, p. 407–432.
  17. Efimov IR, Cheng YN, Biermann M, Van Wagoner DR, Mazgalev T, and Tchou PJ. Transmembrane voltage changes produced by real and virtual electrodes during monophasic defibrillation shock delivered by an implantable electrode. J Cardiovasc Electrophysiol 8: 1031–1045, 1997.[Web of Science][Medline]
  18. Efimov IR, Cheng Y, Van Wagoner DR, Mazgalev T, and Tchou PJ. Virtual electrode-induced phase singularity: a basic mechanism of failure to defibrillate. Circ Res 82: 918–925, 1998.[Abstract/Free Full Text]
  19. Efimov IR, Cheng Y, Yamanouchi Y, and Tchou PJ. Direct evidence of the role of virtual electrode induced phase singularity in success and failure of defibrillation. J Cardiovasc Electrophysiol 11: 861–868, 2000.[Web of Science][Medline]
  20. Efimov IR, Sidorov VY, Cheng Y, and Wollenzier B. Evidence of 3D scroll waves with ribbon-shaped filament as a mechanism of ventricular tachycardia in the isolated rabbit heart. J Cardiovasc Electrophysiol 10: 1452–1462, 1999.[Web of Science][Medline]
  21. Fabritz CL, Kirchhof PF, Behrens S, Zabel M, and Franz MR. Myocardial vulnerability to T wave shocks: relation to shock strength, shock coupling interval, and dispersion of ventricular repolarization. J Cardiovasc Electrophysiol 7: 231–242, 1996.[Web of Science][Medline]
  22. Gilmour RF Jr, Otani NF, and Watanabe MA. Memory and complex dynamics in cardiac Purkinje fibers. Am J Physiol Heart Circ Physiol 272: H1826–H1832, 1997.[Abstract/Free Full Text]
  23. Gray RA, Jalife J, Panfilov A, Baxter WT, Cabo C, Davidenko JM, and Pertsov AM. Nonstationary vortexlike reentrant activity as a mechanism of polymorphic ventricular tachycardia in the isolated rabbit heart. Circulation 91: 2454–2469, 1995.[Abstract/Free Full Text]
  24. Jalife J, Morley GE, Tallini NY, and Vaidya D. A fungal metabolite that eliminates motion artifacts. J Cardiovasc Electrophysiol 9: 1358–1362, 1998.[Web of Science][Medline]
  25. Karagueuzian HS, Khan SS, Hong K, Kobayashi Y, Denton T, Mandel WJ, and Diamond GA. Action potential alternans and irregular dynamics in quinidine-intoxicated ventricular muscle cells. Implications for ventricular proarrhythmia. Circulation 87: 1661–1672, 1993.[Abstract/Free Full Text]
  26. Knisley SB, Hill BC, and Ideker RE. Virtual electrode effects in myocardial fibers. Biophys J 66: 719–728, 1994.[Web of Science][Medline]
  27. Kobayashi Y, Peters W, Khan SS, Mandel WJ, and Karagueuzian HS. Cellular mechanisms of differential action potential duration restitution in canine ventricular muscle cells during single versus double premature stimuli. Circulation 86: 955–967, 1992.[Abstract/Free Full Text]
  28. Koller ML, Riccio ML, and Gilmour RFJ. Dynamic restitution of action potential duration during electrical alternans and ventricular fibrillation. Am J Physiol Heart Circ Physiol 275: H1635–H1642, 1998.[Abstract/Free Full Text]
  29. Lee MH, Lin SF, Ohara T, Omichi C, Okuyama Y, Chudin E, Garfinkel A, Weiss JN, Karagueuzian HS, and Chen PS. Effects of diacetyl monoxime and cytochalasin D on ventricular fibrillation in swine right ventricles. Am J Physiol Heart Circ Physiol 280: H2689–H2696, 2001.[Abstract/Free Full Text]
  30. Liu Y, Cabo C, Salomonsz R, Delmar M, Davidenko J, and Jalife J. Effects of diacetyl monoxime on the electrical properties of sheep and guinea pig ventricular muscle. Cardiovasc Res 27: 1991–1997, 1993.[Abstract/Free Full Text]
  31. MacConaill M. Ventricular fibrillation thresholds in Langendorff perfused rabbit hearts: all or none effect of low potassium concentration. Cardiovasc Res 21: 463–468, 1987.[Web of Science][Medline]
  32. Manoach M, Netz H, Erez M, and Weinstock M. Ventricular self-defibrillation in mammals: age and drug dependence. Age Ageing 9: 112–116, 1980.[Abstract/Free Full Text]
  33. Merillat JC, Lakatta EG, Hano O, and Guarnieri T. Role of calcium and the calcium channel in the initiation and maintenance of ventricular fibrillation. Circ Res 67: 1115–1123, 1990.[Abstract/Free Full Text]
  34. Qin H, Kay MW, Chattipakorn N, Redden DT, Ideker RE, and Rogers JM. Effects of heart isolation, voltage-sensitive dye, and electromechanical uncoupling agents on ventricular fibrillation. Am J Physiol Heart Circ Physiol 284: H1818–H1826, 2003.[Abstract/Free Full Text]
  35. Riccio ML, Koller ML, and Gilmour RF Jr. Electrical restitution and spatiotemporal organization during ventricular fibrillation. Circ Res 84: 955–963, 1999.[Abstract/Free Full Text]
  36. Samie FH, Mandapati R, Gray RA, Watanabe Y, Zuur C, Beaumont J, and Jalife J. A mechanism of transition from ventricular fibrillation to tachycardia: effect of calcium channel blockade on the dynamics of rotating waves. Circ Res 86: 684–691, 2000.[Abstract/Free Full Text]
  37. Undrovinas AI and Maltsev VA. Cytoskeleton disruption results in electromechanical dissociation in rat ventricular cardiomyocytes. J Am Coll Cardiol 29: 404A–405A, 1997.
  38. Varro A, Saitoh H, and Surawicz B. Effects of antiarrhythmic drugs on premature action potential duration in canine ventricular muscle fibers. J Cardiovasc Pharmacol 10: 407–414, 1987.[Web of Science][Medline]
  39. Weiss JN, Garfinkel A, Karagueuzian HS, Qu Z, and Chen PS. Chaos and the transition to ventricular fibrillation: a new approach to antiarrhythmic drug evaluation. Circulation 99: 2819–2826, 1999.[Abstract/Free Full Text]
  40. Wu J, Biermann M, Rubart M, and Zipes DP. Cytochalasin D as excitation-contraction uncoupler for optically mapping action potentials in wedges of ventricular myocardium. J Cardiovasc Electrophysiol 9: 1336–1347, 1998.[Web of Science][Medline]
  41. Yamanouchi Y, Cheng Y, Tchou PJ, and Efimov IR. The mechanisms of vulnerable window: the role of virtual electrodes and shock polarity. Can J Physiol Pharmacol 79: 25–33, 2001.[CrossRef][Web of Science][Medline]
  42. Yang X, Salas PJ, Pham TV, Wasserlauf BJ, Smets MJ, Myerburg RJ, Gelband H, Hoffman BF, and Bassett AL. Cytoskeletal actin microfilaments and the transient outward potassium current in hypertrophied rat ventriculocytes. J Physiol 541: 411–421, 2002.[Abstract/Free Full Text]



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