Excitation and injury of adult ventricular cardiomyocytes by nano- to millisecond electric shocks

Abstract

Intense electric shocks of nanosecond (ns) duration can become a new modality for more efficient but safer defibrillation. We extended strength-duration curves for excitation of cardiomyocytes down to 200 ns, and compared electroporative damage by proportionally more intense shocks of different duration. Enzymatically isolated murine, rabbit, and swine adult ventricular cardiomyocytes (VCM) were loaded with a Ca2+ indicator Fluo-4 or Fluo-5N and subjected to shocks of increasing amplitude until a Ca2+ transient was optically detected. Then, the voltage was increased 5-fold, and the electric cell injury was quantified by the uptake of a membrane permeability marker dye, propidium iodide. We established that: (1) Stimuli down to 200-ns duration can elicit Ca2+ transients, although repeated ns shocks often evoke abnormal responses, (2) Stimulation thresholds expectedly increase as the shock duration decreases, similarly for VCMs from different species, (3) Stimulation threshold energy is minimal for the shortest shocks, (4) VCM orientation with respect to the electric field does not affect the threshold for ns shocks, and (5) The shortest shocks cause the least electroporation injury. These findings support further exploration of ns defibrillation, although abnormal response patterns to repetitive ns stimuli are of a concern and require mechanistic analysis.

Excitation of murine cardiac myocytes by nanosecond pulsed electric field

“…) evidence in favor of electroporation. The longtime course of nsPEF-induced SSD can account for the progressive distortion of calcium transients under repetitive stimulation with 200 nanosecond stimuli observed in the recent study 42. Both the time course and drug sensitivity profile of SSD point to cell membrane nanoelectroporation by nsPEF as the most likely underlying mechanism.…”

“…16,17 At the same time, repeated 200-or 800-nanosecond shocks just above the stimulation threshold triggered abnormal Ca 2+ responses manifested as prolonged elevations of the resting Ca 2+ level, Ca 2+ waves, or distorted Ca 2+ transients. 16 These abnormalities were thought to be of potential concern for nsPEF use in defibrillation. The underlying mechanisms have not been explored yet and may involve permeabilization of the sarcoplasmic reticulum by nsPEF, [18][19][20] damage to voltage-gated Ca 2+ channels, 15 alteration of phosphoinositide signaling, [21][22][23] and nanoelectroporation of the cell membrane (which can be detected by the loss of membrane potential and transport of water and small ions, but not by propidium uptake).…”

“…Recent studies confirmed nsPEF efficiency for both the defibrillation in Langendorff-perfused rabbit hearts 6 and the reduction of propidium uptake (a standard marker for electroporative damage) in individual cardiomyocytes exposed to electric shocks proportionally higher than the stimulation threshold. 16,17 At the same time, repeated 200-or 800-nanosecond shocks just above the stimulation threshold triggered abnormal Ca 2+ responses manifested as prolonged elevations of the resting Ca 2+ level, Ca 2+ waves, or distorted Ca 2+ transients. 16 These abnormalities were thought to be of potential concern for nsPEF use in defibrillation.…”

Using Nanosecond Shocks for Cardiac Defibrillation

“…For isolation of mouse, rabbit, and pig cardiomyocytes, see Ref. 54 In brief, hearts were harvested and immediately arrested, and cardiomyocytes were isolated by enzymatic digestion during Langendorff perfusion (using type II collagenase or LiberaseÔ). Cells were then seeded onto glass coverslips.…”

“…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.…”