L Carr1,2, C Palego2, D Arnaud-Cormos1, P Leveque1 1) Univ. Limoges, CNRS, XLIM, UMR 7252, F-87000 Limoges, France 2) School of Electronic Engineering, Bangor University, UK Influence of pulse duration on intracellular calcium concentration for sub-10 ns pulses Introduction This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 737164 SUMCASTEC. Calcium ions (Ca2+) play a key role in a wide range of cellular processes. Intracellular calcium concentrations [Ca2+]i are known to increase following nanosecond pulsed electric field (nsPEF) exposure [1]. This increase is caused by Ca2+ entering the cell from the extracellular media and/or being released from intracellular calcium stores, such as the E.R. [2]. In both cases membrane poration is believed to be critical for the movement of Ca2+. In this study we characterise changes in [Ca2+]i following the application of single sub-10 ns pulses. We also attempt to identify the source of calcium involved in the post-pulse increase. 1. Batista Napotnik 2016, Bioelectrochemistry 110: 1– 12; 2. Semenov I 2013 Biochim Biophys Acta 1828:981–9; 3. Charuk 1990, Analytical Biochemistry, 188(1), 123–131. Results Amplitude of [Ca2+]i increase is dependent on pulse duration Figure 3. A: Change in fluorescence over time plot, from live cell imaging, for U87 cells loaded with 0.5 µM FLUO-4 exposed to a single 250 kV/cm pulse of durations varying between 2-10 ns. Each line shows average fluorescence change from at least 20 cells from a minimum of 5 separate experiments. Error bars show SE. B: Representative live cell images of U87 cells loaded with 0.5 µM FLUO-4 before and after exposure to a single 2 or 10 ns, 250 kV/cm pulse. Cells have been pseudo coloured for clarity and scale shows fluorescence in arbitrary units. -5000 0 5000 10000 15000 20000 25000 0 20 40 60 80 100 120 140 Fluorescence change (arbitrary units) Time (seconds) Pulse 10 ns 4 ns 6 ns 8 ns 2 ns 0 ns A. Time post pulse Pre pulse 3 s 32 s 100 s 10 ns 2 ns B. • Immediate increase in [Ca2+]i following pulse • The amplitude of this increase is dependent on pulse length • Return towards baseline within 60 seconds of pulse Both extracellular and intracellular calcium sources contribute to [Ca2+]i increase -500 0 500 1000 1500 2000 2500 0 20 40 60 80 100 120 140 Fluorescence change (arbitrary units) Time (seconds) Pulse 10 ns 4 ns 6 ns 8 ns 2 ns 0 ns -5000 0 5000 10000 15000 20000 25000 0 20 40 60 80 100 120 140 Fluorescence change (arbitrary units) Time (seconds) Buffer with Ca2+ Buffer without Ca2+ 10 ns pulse Figure 4. Change in fluorescence over time plot for U87 cells loaded with 0.5 µM FLUO-4 and pulsed in a Ca2+ free buffer. Cells were exposed to a single 250 kV/cm pulse of durations varying between 2- 10 ns. Each line shows average fluorescence change from at least 20 cells from a minimum of 5 separate experiments. Error bars show SE. • Pulses of 4 ns and longer result in a release of intracellular calcium stores • Similar maximum fluorescence for 4, 6 and 8 ns pulses, but different kinetics • Extracellular calcium is the dominant source of the [Ca2+]i increase To see if intracellular calcium stores contribute to the [Ca2+]i increase, cells were pulsed in a calcium free extracellular buffer. Figure 5. Comparison of the FLUO-4 response for cells exposed to a single 10 ns, 250 kV/cm pulse in the presence or absence of extracellular calcium. Each line shows average fluorescence change from at least 20 cells from a minimum of 5 separate experiments. Error bars show SE. Nanopores may not be the only way Ca2+ passes through the plasma membrane -500 0 500 1000 1500 2000 2500 3000 3500 4000 0 20 40 60 80 100 120 140 160 180 Fluorescence change (arbitrary units) Time (seconds) 10 ns 4 ns 6 ns 8 ns 2 ns 0 ns Pulse 2 ns + 50 µM RR 2 ns Pulse -5000 0 5000 10000 15000 20000 25000 0 20 40 60 80 100 120 140 Fluorescence change (arbitrary units) Time (seconds) 10 ns 10