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Circulation Research. 2000;87:797-804

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(Circulation Research. 2000;87:797.)
© 2000 American Heart Association, Inc.

Integrative Physiology

The Role of Electroporation in Defibrillation

Ayman Al-Khadra, Vladimir Nikolski, Igor R. Efimov

From the Department of Cardiology (A.A.-K.), Cleveland Clinic Foundation, and Department of Biomedical Engineering (V.N., I.R.E.), Case Western Reserve University, Cleveland, Ohio.

Correspondence to Igor R. Efimov, PhD, Department of Biomedical Engineering, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106-7207. E-mail ire{at}cwru.edu\\ © 2000 American Heart Association, Inc.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Abstract—Electric shock is the only effective therapy against ventricular fibrillation. However, shocks are also known to cause electroporation of cell membranes. We sought to determine the impact of electroporation on ventricular conduction and defibrillation. We optically mapped electrical activity in coronary-perfused rabbit hearts during electric shocks (50 to 500 V). Electroporation was evident from transient depolarization, reduction of action potential amplitude, and upstroke dV/dt. Electroporation was voltage dependent and significantly more pronounced at the endocardium versus the epicardium, with thresholds of 229±81 versus 318±84 V, respectively (P=0.01, n=10), both being above the defibrillation threshold of 181.3±45.8 V. Epicardial electroporation was localized to a small area near the electrode, whereas endocardial electroporation was observed at the bundles and trabeculas throughout the entire endocardium. Higher-resolution imaging revealed that papillary muscles (n=10) were most affected. Electroporation and conduction block thresholds in papillary muscles were 281±64 V and 380±79 V, respectively. We observed no arrhythmia in association with electroporation. Further, preconditioning with high-energy shocks prevented reinduction of fibrillation by 50-V shocks, which were otherwise proarrhythmic. Endocardial bundles are the most susceptible to electroporation and the resulting conduction impairment. Electroporation is not associated with proarrhythmic effects and is associated with a reduction of vulnerability.


Key Words: electric stimulation • fibrillation • arrhythmia • ventricle • imaging


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Electric shock, the only effective treatment for ventricular fibrillation, was originally thought to be effective by temporarily suppressing cardiac electrical function, resulting in cessation of any activity including fibrillation for seconds until excitability is recovered.1 An alternative theory originally proposed by Gurvich and Yuniev2 3 suggested that "momentary electrical stimulation of the fibrillating heart" by a shock abolishes fibrillation by "synchronization of separate heart elements."4 However, Gurvich and Yuniev2 3 acknowledged that defibrillation had a suppressive effect on cardiac function, which may be injurious and thus necessitated limiting the magnitude of shock current.

Clinical evidence suggests the existence of both excitatory and suppressive effects of shocks on the myocardium. Stimulating effects range from the induction of transient ectopy or tachycardia to the induction of ventricular fibrillation.5 6 7 Such stimulating effects also take place during defibrillation in intact heart models.8 9 10 Virtual electrode polarization11 12 13 may be one of the underlying mechanisms of these effects.9

There is also evidence for depression of cardiac electrical and mechanical function after the delivery of shocks.14 15 16 Bradycardia, complete heart block, and increased pacing thresholds are known to occur, particularly at high energies.6 17 18 In addition, mechanical dysfunction (stunning) has been demonstrated in both the atria and ventricles and appeared to be directly related to the strength of shocks.19 20 21 22 23

Thus, both stimulating and inhibiting effects of electric shocks are believed to participate in defibrillation. However, their relative contribution to both proarrhythmic and antiarrhythmic effects remains controversial.24 25

Electroporation is the disruption of the lipid matrix and the creation of aqueous pathways (electropores) in the cell membrane, resulting from the delivery of high-voltage electrical pulses. That the depressive effect of a strong electric shock via electroporation lasts for seconds has been demonstrated in both isolated cells26 and cardiac tissue preparations.14 15 16 However, the localization and spatial extent of electroporation in intact hearts remains unknown.

Electroporation is more likely to develop in sites with maximal shock-induced transmembrane polarization. The occurrence and amplitude of electroporation have been related to the external field gradient.16 Heterogeneity of myocardial structure has also been implicated in development of strong shock-induced transmembrane polarization.27 28 29 30 Thus, electroporation may develop in sites with maximal external field gradient as well as in sites with maximal structural heterogeneity.

We hypothesized that in normal heart the trabeculated endocardium might be most susceptible to electroporation as a result of its heterogeneous structure. In this study we used optical mapping techniques to assess and compare the endocardial and epicardial cellular responses to high-energy shocks so that we might delineate the relationship between myocardial structure and electroporation and evaluate its effect on electrical activity.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Experimental Preparation
Experiments were performed in vitro on rabbit coronary-perfused cardiac preparations (n=27). The techniques have been described previously10 31 and will be restated briefly. Rabbits were anesthetized by sodium pentobarbital (50 mg/kg). The heart was removed and Langendorff-perfused. Average heart weight was 7.5±0.7 g. The temperature and pH in the chamber were maintained at 37±0.5°C and 7.30±0.05, respectively. The chamber was filled with Tyrode’s solution to cover the heart and the active areas of the defibrillation electrodes. Two types of electrodes were used to deliver shocks. Coil defibrillation electrodes 10 mm long and 2 mm in diameter were used to deliver electroporation shocks. A pair of mesh 35x95-mm electrodes positioned at both sides of the heart were used to investigate the vulnerability.

The experimental preparation was stained by bolus injection of 350 µL of a 2 mmol/L solution of di-4-ANEPPS (Molecular Probes) in DMSO (Fisher Scientific). Movement artifacts were suppressed by 15 mmol/L 2,3-butanedione monoxime (BDM, Sigma) added to perfusate. In 6 experiments, measurements were conducted before and after administration of BDM.

Experimental Protocol
In the first group (n=10), measurements were performed first in intact hearts at the epicardium and then after dissection at the endocardium. The field of view ranged from 4x4 to 12x12 mm. In the second group (n=9), the measurements were focused on the conduction in papillary muscles and septum and thus were performed in dissected preparations only (n=10 papillary muscles). In the third group (n=8), vulnerability was evaluated in intact hearts.

The dissection procedure in the second group was performed after staining and perfusion with BDM. The right ventricular free wall was dissected along its septal border, sparing its basal septal attachments to protect the right coronary blood flow. The free portion was then lifted up to expose the right side of the interventricular septum. At least 1 septal papillary muscle 1.1±0.3 mm in diameter was selected for mapping. An area of 4x4 or 5x5 mm was mapped so that the base and most of the papillary muscles constituted at least a third of the mapped surface, along with an area of the septum on 1 or either side of it. An active fixation bipolar pacing electrode (Medtronic, Inc) was fixed to the basal septum. The heart was paced at a cycle length of 300 ms.

One of the shocking leads was placed in the right or left ventricular cavity. The other lead was placed in the bath on either side of the heart with an approximate interelectrode distance of 2 cm. Monophasic shocks were applied 50 and 100 ms after the onset of the last basic beat stimulus by a defibrillator (VHS-02; Ventritex). Truncated exponential monophasic shocks were 8 ms in duration. A total of 6 to 10 monophasic shocks starting with 50 V and incrementing by 50 V (up to a maximum of 500 V), both anodal and cathodal and at both coupling intervals, were applied. A period of 3 minutes or more was allowed between successive shocks. We measured impedance for different distances between electrodes and different locations of electrodes. An increase of distance between the 2 electrodes placed in the bath outside of the heart from 0.5 to 4 cm resulted in an increase of impedance from 45 to 70 {Omega}. Similar measurements conducted with 1 electrode inside the right and left ventricles and with the reference electrode outside of the heart resulted in a range of impedances of 60 to 85 and 70 to 85 {Omega}, respectively, for distances 0.5 to 4 cm.

Optical Mapping Techniques
A previously described32 imaging system was used in our experiments. The magnification was adjusted to focus on an area from 250x250 to 312x312 µm per diode. The entire field of view ranged from 4x4 to 5x5 mm depending on the size of the papillary muscle. Mapping of intact hearts was performed from a field of view up to 12x12 mm.

Each scan contained 1 to 3 seconds of data sampled at 1 or 2 kHz. When electroporation was observed, longer scans that allowed observation of recovery were performed at a lower frame rate.

Endpoint Definition
Electroporation was considered to have occurred if at least 10% reduction of one of the following parameters was observed in the first postshock beat compared with the beat preceding the shock, in any of the recorded 256 voltage signals: (1) resting membrane potential, (2) action potential amplitude, and (3) rate of rise (dV/dt) of the upstroke of the action potential.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Evidence of Electroporation
Figure 1Down shows a representative trace recorded during the delivery of a 500-V shock. As in microelectrode data of Moore and Spear,33 one can see a reduction of postshock optical action potential (OAP) amplitude and an upward shift in optical resting membrane potential. The lower trace shows postshock reduction of (dV/dt)max. All of these 3 effects were fully or partially reversible, with recovery time ranging from 5 seconds to minutes. These effects were voltage dependent (see Figure 2Down). Such observations are consistent with previously reported effects observed in cardiac cells during strong electric shocks and associated with electroporation.14 16 33 34 Thus, we identified these observations as electroporation and will refer to them as such throughout the article.



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  		Figure 1. Figure 1Up. Evidence of shock-induced electroporation. Optical recording of transmembrane potential V (upper trace) shows dramatic reduction of resting membrane potential and action potential amplitude, which both fully or partially recover with time after shock. Maximal upstroke rate of rise (dV/dt) is also dramatically reduced and slowly recovers after shock (lower trace).



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  		Figure 2. Figure 2Up. Comparison of maximal observed electroporation at the endo- and epicardium in the same heart. Interelectrode distance (2 cm) and position of electrodes with respect to corresponding fields of view were similar in both cases. No polarity dependence of electroporation was detected (compare 2 left columns).

Difference Between the Epicardium and Endocardium
A comparison of epicardial and endocardial electroporation revealed significant difference. Electroporation was detected in both the epicardium and the endocardium and was shock voltage dependent. However, endocardial electroporation was detectable at significantly lower voltages. Figure 2Up shows typical examples of responses with maximal electroporation recorded from the endocardium and epicardium of the same heart. This observation was consistent in all 10 hearts in which we conducted measurements at both the epicardium and the endocardium. Electroporation thresholds were 229±81 and 318±84 V (P=0.01) for the endocardium and the epicardium, respectively. It is interesting to note the lack of effect of shock polarity. The same figure shows that both anodal and cathodal shocks produced similar effects on postshock electrical activity.

In addition, the epicardial and endocardial surfaces had different spatial distributions of areas of electroporation. When the lead was in the right ventricle, epicardial electroporation was first evident near the shocking electrode. In contrast, endocardial electroporation was observed throughout the entire endocardium, with most of the effect at the bundles and papillary muscles, independent of the position of the electrode within the left ventricle or outside of the heart (see online Figure 1Up; data supplement available at http://www.circresaha.org).

This finding led us to hypothesize that endocardial trabeculae; small papillary muscles; and other bundles, including the bundle brunches and the Purkinje network, might be most vulnerable to electroporation during defibrillation shocks. Thus, electrical activity of these structures might be selectively suppressed for seconds to minutes after the shock. To test this hypothesis, we conducted the next phase of the study with higher spatial resolution, concentrating on single papillary muscles with a field of view of 4x4 or 5x5 mm.

Impulse Conduction in Papillary Muscles
Similar to our previous observations in the atrioventricular node35 and at the ventricular epicardium during ventricular arrhythmias,36 we have observed OAPs with a peculiar, dual-humped morphology (Figure 3BDown, upper trace). As is evident from Figure 3Down, such dual-humped signals were detected only in the area of papillary muscle, whereas flat areas of endocardium produced OAPs with a normal morphology (Figure 3BDown, lower trace). Such observations were reproduced in all 10 papillary muscles from 9 hearts. The diameter of papillary muscles was 1.1±0.3 mm.



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  		Figure 3. Figure 3Up. OAP morphology recorded in proximity to a papillary muscle. A, Photograph of the 4x4-mm field of view (large box) and surrounding area. B, Examples of 2 types of optical recordings observed in this field of view: "dual-humped" response (upper trace) was recorded at the papillary muscle ({square}, see location in panel A), and normal action potential (lower trace) was recorded at the septum ({blacksquare}). C, Raw optical recordings from the entire field of view. Notice 2 components in upstrokes recorded at the bundle, whereas only 1 component recorded elsewhere.

We hypothesized that the 2 components of OAP upstrokes recorded from the papillary muscle belong to the muscle itself and the underlying septal myocardium. Figure 4Down shows evidence supporting such a hypothesis. The left panel shows optical recordings conducted from the septum (dotted line) and a papillary muscle before (solid line) and after (dashed line) insertion of a piece of opaque plastic film, which shielded the septum from the muscle, eliminating the optical component that carried the electrophysiological signature of the septum. This test was conducted in 2 preparations.



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  		Figure 4. Figure 4Up. Optical signal from the papillary muscle (P.M.) with and without shielding of the underlying septum. Photographs of 8x8-mm field of view are on the right. Upper panel, Control. Lower panel, Optical signal with plastic shielding film inserted between the papillary muscle and the septum. Left, Photodiode current traces in pA from areas on papillary muscle and septum.

Using a previously developed method,35 36 we reconstructed 2-layered activation patterns based on the 2 maximums of the temporal derivative of the upstrokes. Figure 5CDown illustrates an example of such a reconstruction conducted on the basis of optical recordings from a 4x4-mm area of septum with a single papillary muscle within the field of view. Online Figures 2Up and 3Up provide additional support of the 2-layered-conduction hypothesis (data supplement available at http://www.circresaha.org). Similar patterns of conduction were consistently observed in all 10 studied papillary muscles during ventricular pacing.



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  		Figure 5. Figure 5Up. Shock-induced block of conduction in the papillary muscle. A, Photograph of 4x4-mm field of view and surrounding structures of the right ventricular septum. Circle indicates position of recording site illustrated in panel B. B, Optical recording at the papillary muscle during application of a 300-V 8-ms monophasic shock. Notice 2 components in the upstrokes of all responses except the first postshock action potential. C, Activation isochronal maps (1 ms) reconstructed from the 2 components of optical recordings (see text for details).

Evidence of Shock-Induced Block in Papillary Muscles
Using the techniques described above, we investigated the effect of electroporation on postshock electrical activity.

Figure 5Up shows representative data recorded during a 300-V shock. All upstrokes recorded at the papillary muscle except the first postshock response had 2 components (Figures 3Up and 5BUp). The left pair of maps in Figure 5CUp illustrates spread of activation in the right ventricular septum (upper map) followed by the activation of the papillary muscle (lower map). Application of the shock resulted in electroporation, as is evident from the depolarization of the optical resting membrane potential. In addition, the first postshock response had dramatically reduced amplitude as a result of the loss of its second component, which then recovered by the next beat (compare with Figure 5Up). The slow recovery of the amplitude of the first component of the upstroke illustrates recovery from electroporation at the septum. The lack of a second component and the all-or-none behavior can be explained by transient conduction block in the papillary muscle caused by selective electroporation of the papillary muscle. The second pair of maps in Figure 5CUp shows the activation pattern during the first postshock beat, which was present only at the septum. Conduction was restored in the papillary muscle during the second postshock beat (Figure 5CUp).

The online data supplement illustrates shock-induced block in the papillary muscle in greater details (see online movie 2 and online Figures 4Up and 5Up, available at http://www.circresaha.org). Such a block was observed in all 10 papillary muscles. The duration of conduction block was voltage dependent. Table 1Down summarizes the data.


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  		Table 1. Thresholds of Electroporation and Block in Papillary Muscles

An important finding of this study is the lack of any evidence of spontaneous arrhythmias (reentrant or focal) associated with electroporation of the endocardium or the papillary muscles.

Antiarrhythmic Preconditioning With High-Energy Shocks
The importance of the involvement of different bundles, including the Purkinje system, in the maintenance of arrhythmias is generally accepted.37 38 Our results suggest that the temporary incapacitation of such bundles after shock may suppress the genesis and maintenance of postshock arrhythmias. We conducted a separate series of 8 experiments in intact hearts to test this hypothesis. In these experiments, we evaluated the hypothesis that strong electric shocks that are known to cause electroporation and temporary incapacitation of bundles would reduce vulnerability to arrhythmias provoked by a shock applied during the period of electroporation.

The protocol included uninterrupted pacing at a coupling interval of 300 ms. Initially, the lower limit of vulnerability, upper limit of vulnerability, and the defibrillation threshold (DFT) were determined using an up-down protocol.39 Determination of the lower limit of vulnerability was limited by the minimum output of the defibrillator, which was 50 V. Two shocks were applied 100 ms after the action potential upstroke. The first "preconditioning" strong shock was applied from the pair of coil electrodes. These anodal shocks were 1x, 2x, and in one experiment 3x DFT measured in each heart separately (DFT=181.3±45.8 V [n=8]). Test shocks were applied from a separate pair of mesh electrodes using the average of the low and upper limits of vulnerability. Average voltage of test shocks was 72.9±20.0 V (n=105). Coupling interval between the 2 shocks was 1200 and 1500 ms.

Figure 6Down shows that test shock without preconditioning resulted in arrhythmia in 94.8%. Most of these arrhythmias were sustained. Preconditioning with 1x DFT and 2x DFT shock reduced arrhythmia inducibility to 70% and 42.9%, respectively. Preconditioning with 3x DFT completely abolished the ability of the test shock to induce arrhythmia. Online Figure 6Down (data supplement available at http://www.circresaha.org) provides representative optical recordings obtained during this study. Table 2Down summarizes these findings. As is evident from these data, increment in preconditioning shock intensity resulted in a decrease in vulnerability to arrhythmias. It is interesting that such a decrease in vulnerability associated with 1x DFT and 2x DFT preconditioning is accompanied by an increase in nonsustained arrhythmias compared with the control. Such an increase may indicate that electroporation reduces the probability of shock-induced scroll wave to degenerate into sustained fibrillation, perhaps by reducing the mass of tissue participating in the conduction.



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  		Figure 6. Figure 6Up. Fibrillation inducibility vs intensity of preconditioning shock applied 1200 or 1500 ms before profibrillatory test shock.


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  		Table 2. Preconditioning With High-Intensity Shocks Reduced Vulnerability to Ventricular Fibrillation

Effect of BDM
Use of the excitation-contraction decoupler BDM made it possible to optically image the effects of shocks on myocardium. Yet BDM is known to block ionic currents. To investigate the impact of BDM on this effect, we conducted 6 experiments in which the shocks were applied before and after the administration of BDM, and the results were compared. Both the block of conduction in the papillary muscle and the preconditioning effects of strong electric shocks were observed both with and without perfusion with BDM. Data shown in Figure 6Up and Tables 1Up and 2Up included both buffer measurements containing BDM and those containing no BDM.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In this report, we have presented experimental evidence suggesting that electroporation is an important component of defibrillation, as follows: (1) electroporation has been detected preferentially in trabeculated endocardial structures; (2) selective transient impairment of electrical activity in endocardial bundles is caused by electroporation; and (3) electroporation might transiently reduce myocardial vulnerability to arrhythmias, as is evident from the preconditioning effect of supra-DFT shocks on postshock vulnerability.

Factors Contributing to Shock-Induced Transmembrane Polarization
The external electric field is capable of stimulating the myocardium through its ability to induce transmembrane polarization. Until recently, the electric field gradient has been considered the principal factor contributing to this effect.40 Recent studies have suggested that the heterogeneity or asymmetry of myocardial structure is also an important factor contributing to shock-induced polarization.28 41 42 43 The theoretical concept of a generalized activating function provides conceptual unification of both myocardial structure and the field gradient.29 Myocardial structure actively interacts with the field, producing local sources of current, known as the secondary sources,28 and resulting in virtual electrode polarization during point stimulation11 44 and during defibrillation shocks.9

The seminal work of Weidmann45 demonstrated that a discontinuity in current flow arising at the boundary between myocardium and the bath results in strong transmembrane polarization, which exponentially decays with spatial scale known as the space constant. This effect is important during defibrillation, resulting in significant transmembrane polarization produced by shocks at the surface of the myocardium.42 46 47 Yet this effect does not affect the bulk of the myocardium, where fiber curvature or other syncytial heterogeneities are the likely causes of transmembrane polarization. Nevertheless, in the far field, Weidmann’s45 effect appears to produce transmembrane polarization of significantly stronger amplitude compared with that resulting from alternative mechanisms.48 Furthermore, the curvature of tissue surface may be an important factor modulating the effect demonstrated by Wiedmann.45 Theory predicts that smaller bundles with higher surface curvature are subject to stronger polarization by far-field stimulation when compared with their less-curved counterparts.49 Curvature and branching of such bundles is likely to further contribute to their polarization50 and electroporation in the far field.

Implications of the Postshock Block of Electrical Activity in Bundles for Defibrillation: Is Electroporation a Proarrhythmic or Antiarrhythmic Phenomenon?
The relative volume affected by the effect of Weidmann45 is not significant in large hearts, because it is confined proximal to the surfaces of myocardium. Yet because of the important role of some surface structures such as the Purkinje network in the genesis and maintenance of arrhythmias,37 38 this effect might still play an important role in defibrillation. Selective disruption and depolarization of these structures might have both proarrhythmic and antiarrhythmic effects. Sustained depolarization of a bundle electrically coupled with the normal excitable myocardium can hypothetically result in abnormal repolarization and arrhythmogenesis via a focal mechanism.51 Alternatively, impairment of electrical activity in the Purkinje system and other conductive bundles might exclude them from participation in reentrant circuits. Berenfeld and Jalife38 recently suggested that the Purkinje system is an important factor in the maintenance of ventricular fibrillation. We demonstrated earlier that subthreshold stimulation applied at the Purkinje system terminates arrhythmias in the guinea pig heart.37 Thus, exclusion of such bundles may contribute to the antiarrhythmic effects of shocks. Which of the 2 effects plays the major role in clinical defibrillation remains to be determined. Our data support the latter rather than the former.

Role of Electroporation in Defibrillation
The success or failure of defibrillation therapy has usually been attributed to 1 or both of the following mechanisms: (1) success in extinguishing ongoing fibrillatory activity52 and (2) failure to reinitiate a new arrhythmia.53 Our data suggest that electroporation may be actively involved in both of these processes. Transient impairment of the conduction system may facilitate termination of ongoing fibrillation and reduce probability of virtual electrode-induced reentry10 to degenerate into sustained fibrillation. Demonstrated preconditioning with supra-DFT shocks indicates that such impairment may indeed be acutely antifibrillatory.

On the other hand, high-energy shocks are known to produce a permanent damage, perhaps associated with electroporation. This effect of electroporation may provide the substrate for arrhythmogenesis.54

Limitations
Our model of defibrillation cannot be directly scaled to the human heart. Block of conduction was evident in all of the papillary muscles in the rabbit heart, yet the larger size of human papillary muscles might prevent this effect from occurring to the same extent. It appears that the space constant is a good estimate of the size of the affected papillary muscles and bundles, which could exhibit transient block after shock. If the thickness of the fiber is comparable to the spatial depth of electroporation, then the failure of conduction is more likely to occur in thinner, compared with thicker, bundles. The latter might be electroporated less and only at the surface while the large functional core is preserved where electroporation cannot reach. Thus, we suggest that the right and left bundles, as well as the entire Purkinje system in humans, are likely to be affected by the selective blockade associated with electroporation. Clinical evidence of postshock bradycardia, asystole, and widening of QRS is consistent with such a hypothesis.6 17 Thus, despite the scale limitation, our model predicts that a similar mechanism may play an important antifibrillatory role in clinical defibrillation.

As a result of limited resolution of experimental techniques, we are unable to provide direct evidence that pores were actually formed in cellular membranes during the shock, which would prove electroporation. It appears that such evidence remains unachievable in vitro and in vivo at the present level of experimental methodology. Yet we base our conclusion on previous reports, which provide similar evidence of electroporation from optical16 34 and microelectrode33 recordings. Neunlist and Tung16 have presented a thorough analysis of several alternative hypotheses of observed changes in optical recordings and conclude that electroporation is the most grounded explanation. Specifically, they addressed the hypothetical effects of the field on fluorescent dye and movement artifacts and rejected these as possible explanations.

The optical techniques at this resolution provide only a limited assessment of the absolute mV amplitude of the observed elevation of resting potential as in Figure 1Up and polarizations during shocks as in Figure 2Up. Optical signal presents an average fluorescence collected from many layers of cells. Surface layers of cells are more likely to be electroplated compared with deeper layers as a result of the tissue-bath interface effect, governed by the space constant.42 Thus, traces as in Figures 1Up and 2Up could represent layers of cells, which were completely electroporated at the surface of myocardium (epi- or endocardium), as well as deeper layers, which underwent partial electroporation or no electroporation at all. Yet their contribution to the signal will be of a lower weight because of the absorption of fluorescence light on its way to the detector. Because of this limitation, we did not attempt to quantify absolute levels of transmembrane voltage at which electroporation does occur.

Similarly, recordings of electrical activity from trabeculated structures at the endocardium are likely to pick signals from opposite sides of papillary muscles or fibers, which would undergo opposite polarization. The total signal will represent an average of opposite signals. Yet this average is unlikely to be 0, because shock-induced polarization is known to be highly polarity asymmetric with negative polarization {approx}1.5 to 2 times stronger than positive polarization for the same field amplitudes.55 Thus, an average of the 2 opposite polarizations is likely to be biased toward the negative polarization for both shock polarities as seen in Figure 2Up.

Accurate assessment of slow recovery of the resting potential after the shock was also unattainable as a result of low-frequency noise and AC coupling ({tau}=30 seconds) of our instrumentation. In addition, electroporation might be irreversible in a part of the myocardium, which is also likely to contribute to the irreversible shift in the optical "resting potential."


*    Acknowledgments
 

Financial support from NIH (Grants R01HL58808 and R01HL59464) and the American Heart Association (Ohio Valley affiliate Grant-in-Aid 9806201) is gratefully acknowledged.

Received August 7, 2000; revision received August 31, 2000; accepted September 1, 2000.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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