Excitation of murine cardiac myocytes by nanosecond pulsed electric field

Abstract

Introduction Opening of voltage‐gated sodium channels takes tens to hundreds of microseconds, and mechanisms of their opening by nanosecond pulsed electric field (nsPEF) stimuli remain elusive. This study was aimed at uncovering the mechanisms of how nsPEF elicits action potentials (APs) in cardiomyocytes. Methods and Results Fluorescent imaging of optical APs (FluoVolt) and Ca2+‐transients (Fluo‐4) was performed in enzymatically isolated murine ventricular cardiomyocytes stimulated by 200‐nanosecond trapezoidal pulses. nsPEF stimulation evoked tetrodotoxin‐sensitive APs accompanied or preceded by slow sustained depolarization (SSD) and, in most cells, by transient afterdepolarization waves. SSD threshold was lower than the AP threshold (1.26 ± 0.03 vs 1.34 ± 0.03 kV/cm, respectively, P < 0.001). Inhibition of l‐type calcium and sodium‐calcium exchanger currents reduced the SSD amplitude and increased the AP threshold ( P < 0.05). The threshold for Ca 2+‐transients (1.40 ± 0.04 kV/cm) was not significantly affected by a tetrodotoxin‐verapamil cocktail, suggesting the activation of a Ca 2+ entry pathway independent from the opening of Na + or Ca 2+ voltage‐gated channels. Removal of external Ca 2+ decreased the SSD amplitude ( P = 0.004) and blocked Ca 2+‐transients but not APs. The incidence of transient afterdepolarization waves was decreased by verapamil and by removal of external Ca 2+ ( P = 0.002). Conclusions The study established that nsPEF stimulation caused calcium entry into cardiac myocytes (including routes other than voltage‐gated calcium channels) and SSD. Tetrodotoxin‐sensitive APs were mediated by SSD, whose amplitude depended on the calcium entry. Plasma membrane electroporation was the most likely primary mechanism of SSD with additional contribution from l‐type calcium and sodium‐calcium exchanger currents.

Using Nanosecond Shocks for Cardiac Defibrillation

“…To measure calcium transients, a calcium-sensitive fluorescent dye was added to the medium, and fluorescence measured under a microscope. 33,38,54,55 The electromechanical uncoupler blebbistatin was added to the medium to prevent movement artifacts and propidium iodide (PI) was added to measure membrane permeability.…”

“…A separate study found that nanosecond stimulation caused both calcium entry into cardiac myocytes, including routes other than voltage-gated calcium channels, and slow sustained depolarization (SSD). 33 Tetrodotoxin-sensitive action potentials were mediated by SSD, whose amplitude depended on the calcium entry. Plasma membrane electroporation was the most likely primary mechanism of SSD with additional contribution from L-type calcium and sodiumcalcium exchanger currents.…”

“…Stimulation is a viable proxy on theoretical grounds, because the success of defibrillation depends on whether a shock activates enough of the cardiac tissue 15,16,31 (but our data hereunder suggest that it has limitations). Stimulation of individual myocytes with nanosecond pulses is possible, 32,33 and the mechanism of stimulation likely involves electroporation of the plasma membrane. 33,34 The possible adverse effects of electric shocks can be assessed both in isolated myocytes and in cardiac tissue.…”

“…Stimulation of individual myocytes with nanosecond pulses is possible, 32,33 and the mechanism of stimulation likely involves electroporation of the plasma membrane. 33,34 The possible adverse effects of electric shocks can be assessed both in isolated myocytes and in cardiac tissue. The main mechanism of damage is electroporation, 14,[34][35][36] and since the pores formed by nanosecond shocks tend to be smaller than those formed by millisecond shocks, [37][38][39][40][41] the electroporative damage may also be reduced.…”

Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca2+ and Ba2+ Ions

“…Cytosolic Ca 2+ level is tightly controlled, hence, in quiescent cells, the dye fluorescence can be stable for a long time. However, Ca 2+ entry through nanopores should be separated from the activation of cation channels which can admit Ca 2+ [24,29,[34][35][36], as well as from receptor-and store-operated Ca 2+ entry [37]. Moreover, a modest Ca 2+ entry through the electropermeabilized membrane may trigger an overwhelmingly strong response by activating calcium-induced calcium release (CICR), followed by massive active pumping of excess Ca 2+ from the cytosol [33].…”