Electroporation assessment via shock induced transmembrane
- Electroporation of the heart
Double-barrelled microelectrode recordings and optical mapping techniques showed that weak stimuli, as predicted by the cable theory and generalized activating function theory, produce monotonic transmembrane potential changes (DVm) in a single cell [18,19], cell culture strands , and heart tissue [21,22]. However, reports on strong shocks of defibrillation strength sharply disagree on the morphology and amplitude of shock-induced response DVm. When a stimulus is applied to a single cell during the early plateau phase of the action potential, the optical recordings show depolarization of the cathodal end and hyperpolarization of the anodal end of the cell [23,24].
When the stimulus intensity increases, the induced hyperpolarization (or more accurately negative polarization) DVm first gradually increases in amplitude but soon starts to decay, causing elevation of cell average potential, Fig. 2 . Similar effects were observed in narrow strands of cultured rat myocytes . It was suggested that electroporation was a likely mechanism of non-monotonic hyperpolarization transients.
More direct evidence was needed. One such verification might be dye uptake through pores induced by electroporation. Uptake depends on dye concentration differences inside and outside the cells and of the net electrical charge of the dye. Because Lucifer Yellow dye uptake was not observed at 50 V/cm shock strength, which was above the 30 V/cm threshold of non-monotonic negative DVm, the conclusion was not definitive and an activation of an unknown hyperpolarization-activated channel(s) was proposed as an alternative explanation . More recently this same group has detected such uptake using another indicator: propidium iodide . However, Gillis et al.  did observe an accumulation of Lucifer Yellow dye in areas close to the anode and cathode after shocks with a field strength ³22 V/cm in a similar experimental design. Currently, the appearance of the second phase of hyperpolarizing transients (see labels 1,2 in Fig. 2A) is considered a signature of membrane electroporation.
The lack of detectable dye uptake in some of the previous studies could be related to the small exposure time and to the lower sensitivity of the Lucifer Yellow technique. Propidium iodide has 20–30 fold increases in fluorescence after binding to nucleic acids. In contrast to negative response DVm, positive polarization in a single cell and in cell culture was found to increase gradually with shock strength, saturating below 100% of action potential amplitude (APA) .
Whole heart studies revealed different types of asymmetry for the positive and negative polarizations during strong shocks (see Fig. 2). In a recent study , we also found that these effects are accompanied by epicardial post-shock elevation of diastolic potential (DP) (see Fig. 2, right panels). In our study, we determined epicardial DVm responses during high-density electric current stimuli of both polarities applied at the 6 mm diameter area of the left ventricle. We detected saturation and subsequent decay of epicardial polarizations during strong cathodal and anodal shocks applied at the area with a size of several space constants (0.8–1.5 mm at the epicardium ). Our optical recordings of negative DVm responses to high-intensity stimuli were in agreement with the results reported by others in strands of cultured myocytes [20,30], single cell , and frog heart  for hyperpolarizing stimuli. We did not observe a plateau or an increase in the depolarization transients during cathodal stimuli for the same stimulus strengths that caused a decayed hyperpolarization response. Neunlist and Tung  presented measurements of epicardial cellular responses recorded from 150 µm diameter area of stimulus application, showing hyperpolarization overshoot during anodal stimulation , similar to that found in cell strands [20,25].
Recent data  from Fast et al. showed that the initial positive polarization in virtual cathode areas in a wedge preparation changes to hyperpolarizing responses as the stimulus strength increases to 30 V/cm and above, similar to the behaviour of the middle of a single myocyte in studies by Sharma and Tung . Such observations were reported previously by Cheng et al.  and Zhou et al. , who detected hyperpolarization transients near the cathodal shock electrode. We observed the same phenomena in optical recordings from the epicardial surface . This can explain why depolarization saturation is observed at lower shock current densities than hyperpolarization saturation. Neunlist and Tung  stimulated a small area near the electrode that could affect their measurements due to a virtual electrode effect , leading to development of positive and negative polarizations at nearby locations. In contrast, our experiments were designed to overcome this limitation by stimulating a large area relative to the field of view. Similarly, we did not use transmural sections of tissue, which will interrupt fibres and thus could affect the results in a slab preparation . However, a strong cathodal stimulus resulted in hyperpolarizing responses and this response was partially reversible. We considered electroporation as the most plausible explanation of these effects. However, it would require voltage-dependent resealing of the pores during the shock application to explain the restoration of the membrane resistance.
The correlation between anodal (negative) DVm and diastolic Vm elevation was recently reported by Fast and Cheek  in myocyte cultures. A similar result was shown earlier by Neunlist and Tung  and Cheng et al. . Interestingly, Fast and Cheek  did not observe non-monotonic DVm at the cathodal end of the cell strand even at the highest shock strengths. A possible explanation for the absence of positive DVm decay in the Fast and Cheek  study was recently suggested by these authors [34,35]. They demonstrated that the absence of positive polarizations in optical recording of transmembrane potential at the edge of the preparation facing the cathodal electrode during strong shocks results from the spatial averaging of polarization in the neighbouring areas.
In our study, both stimuli polarities had the same injury threshold, judging by diastolic potential elevation . Our findings agree with earlier reports by Knisley and Grant, who showed that cell injury is independent of the intrinsic transmembrane potential ; and by Moroz et al., who reported the same electroporation threshold for monophasic and biphasic shock waveforms . These reports contradict results obtained in the single cell and in cell cultures, in which anodal sides of single cells or cell strands were affected more significantly then cathodal sides [25,36].
Application of a Ca-channel blocker resulted in an increase in the saturation level of depolarizing responses and did not affect hyperpolarizing responses in cell culture . Despite the differences in the depolarization transients behaviour mentioned above, we observed that nifedipine increased the saturation levels for positive but not negative DVm also during epicardial stimulation. Ca-channel blockers did not affect the electroporation threshold in whole cell patch studies  and only increased positive DVm in cell culture studies . Yet, there is a clear effect of nifedipine on the saturation level for the depolarization signal in our study, which means that other factors (i.e. space averaging in optical recordings) could also be responsible for saturation and reversal of the depolarizing responses with stimulus strength increase.
In agreement with earlier reports [15,16], we observed that a rather small optical hyperpolarization response could be sufficient for electroporation. Among possible reasons for this were (a) a ‘dog–bone’ virtual polarization near the pacing electrode  that could attenuate the response due to optical averaging over areas of opposite polarizations, or (b) insufficient temporal resolution of our optical mapping system  that could underestimate the true instantaneous transmembrane voltage produced by a square pulse. Our results for polarization transients recorded during 10 ms ramp waveform stimulation (no temporal resolution limitations) over a 6 mm diameter area of epicardium (opposite polarization is located 3 mm away from the central recording point) reject such explanations.
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