Publication details

Irreversible electroporation effect on cardiac cells investigated through phenotypic response


CALUORI Guido TEDESCO Mariateresa PEŠL Martin RAITERI Roberto STÁREK Zdeněk

Year of publication 2019
Type Article in Proceedings
Conference Poster session 2 - 43rd EWGCCE Meeting, EP Europace, Volume 21, Issue Supplement_2, March 2019,
MU Faculty or unit

Faculty of Medicine

Keywords Irreversible electroporation cardiomyocytes atomic force microscopy microelectrode arry
Description Radiofrequency (RF) ablation is the most common thermal-based approach for interventional resolution of cardiac arrhythmias. Albeit effective, RF ablation is limited in the cardiac chambers, the procedure is relatively long and can unselectively damage adjacent structures. Irreversible electroporation (IRE) has been proposed as a suitable, muscle-selective alternative to RF ablation. Despite the encouraging data from in vivo investigation, no systematic study for its effect and cell death mechanism on cardiac models has yet been performed. Purpose: To apply IRE in a combined in vitro electroporation system and model of cardiac tissue. To observe the IRE effect on cells. To observe and describe the eventual peri-ablation altered transition zone. Methods: Primary rat cardiomyocytes were isolated from newborn pups via enzymatic digestion and differential adhesion. Cardiac muscle cells were plated on the surface of micro-electrode array (MEA, 30µm electrode radius, 200µm inter-electrode spacing). After 3 days, we selectively applied IRE symmetrical biphasic stimuli (500V/cm for 120µs, 1 Hz, up to 120 pulses) to spontaneously activating cultures. Calcium fluorescent dye was loaded to observe signal conduction with a CMOS camera (frame rate 50Hz). After IRE stimulation, cultures were stained with annexin V and propidium iodide (PI) kit to selectively visualize apoptosis and necrosis. Results were processed offline and results are shown as mean?±?standard deviation. Results: Cellular electroporation was confirmed by EFP shape change. Signals recorded from active electrodes gradually decreased in amplitude during sequential stimuli application. After 10-20 stimuli, the cultures rhythm was no longer captured. Transient electroporation was observed up to 400 µm in distance from electrodes, due to the presence of mixed intracellular and extracellular field potentials. Calcium imaging showed a progressive state of intracellular calcium overload during IRE application, which plateaued in rapid monomorphic action potential firing. After stimulation stop, calcium ions moved out from the cytoplasm of affected cells to lower levels and tachyarrhythmia terminated. AnnexinV/PI staining showed localized exposure of apoptotic marker phosphatidylserine around the application electrode areas (108.2?±?14.91µm, n?=?14) with significant difference from electrode free from applications (p?<?0.0001, n?=?12). Co-staining with PI in the nucleus of healthy-shaped cells showed that also the nuclear membrane is affected by IRE. Conclusions: We showed how IRE produces fast and effective impairing of cardiac muscle, with localized apoptosis in the surrounding of the application sites. Furthermore, we have observed how, at least in short-range, IRE causes electrical or calcium conduction disturbances, which can in principle constitute a transient substrate for arrhythmia and stunning during ablation therapies, if not addressed, probably by pharmacological prevention.
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