Thapsigargin, but not ryanodine, suppresses high [K⁺]_e-induced cytosolic Ca²⁺ transients in the absence, but not in the presence, of extracellular Ca²⁺.

<p><b>A</b>: Time course of cytosolic ΔF/F₀ during 30-s exposures to 80 mM [K⁺]_e in 2 mM [Ca²⁺]_e (left panel) and in the absence of external Ca²⁺. To deplete internal Ca²⁺ stores, cells were continuously incubated with thapsigargin (1 μM) starting 20 min before the first exposure to elevated [K⁺]_e. <b>B</b>: Percentage of cells exhibiting [Ca²⁺]_i transients both in 2 mM and 0 mM [Ca²⁺]_e; * <i>P</i> < 0.001 versus control by Fisher Exact test (60 cells for control and 19 cells for thapsigargin). <b>C</b>: Time course of changes in cytosolic ΔF/F₀ elicited by consecutive 30-s exposures to 80 mM [K⁺]_e in normal [Ca²⁺]_e (upper panel) and in Ca²⁺-free bath solution (with 200 μM EGTA added). The cell was continuously incubated with ryanodine (20 μM) starting 20 min before the first [K⁺]_e challenge. <b>D</b>: Percentage of cells exhibiting [Ca²⁺]_i transients both in 2 mM and 0 mM [Ca²⁺]_e; <i>P</i> = non-significant versus control by Fisher’s Exact test (60 cells for control and 14 cells for ryanodine). <b>E</b>: Ryanodine depletes caffeine-sensitive internal Ca²⁺ stores in postganglionic sympathetic neurons. Time course of changes in cytosolic ΔF/F₀ in response to a 30-s exposure to 80 mM [K⁺]_e followed by a 30-s exposure to caffeine (upper panel). Preincubation with 20 μM ryanodine abrogated the caffeine-, but not the high [K⁺]_e-, induced cytosolic Ca²⁺ transient (lower panel). Caffeine responses in the presence and absence of ryanodine were monitored in two different cells.</p

Voltage-gated Ca²⁺ channels do not conduct Na⁺ after removal of extracellular Ca²⁺.

<p><b>A</b>: Exemplar whole-cell current traces sequentially recorded in a voltage-clamped neuron during 300-ms step depolarizations to +10 mV from a holding potential of -90 mV in 2 and 0 mM [Ca²⁺]_e. The bath solution also contained 1 μM tetrodotoxin to block voltage-gated Na⁺ channels. Voltages were not corrected for liquid junction potential. Numbers in mM denote extracellular Ca²⁺ concentration. Voltage-clamp protocol is shown in the upper panel. <b>B</b>: Bar graph summarizing means of peak inward currents evoked by depolarizations to +10 mV in the presence and absence of external Ca²⁺ in the same cells. Error bars represent SEM (n = 4 cells). <i>P</i> = 0.02 versus 2 mM [Ca²⁺]_e by paired <i>t</i>-test.</p

External Cd²⁺ does not suppress high [K⁺]_e-induced ΔF/F_o transients in 0 mM [Ca²⁺]_e.

<p><b>A</b>: Irreversible increase of ΔF/F₀ in depolarized neurons in the presence of extracellular Cd²⁺. Shown are time courses of changes in cytosolic ΔF/F₀ elicited by 80 mM [K⁺]_e in the presence of 2 mM Ca²⁺ (left panel) and by 80 mM [K⁺]_e and 300 μM Cd²⁺ in a Ca²⁺-free bath solution (right panel). The increase of ΔF/F₀ does not recover after removal of high-[K⁺]_e-induced depolarization. <b>B:</b> External cadmium does not block depolarization-induced increase in cytosolic ΔF/F₀ in the absence of extracellular Ca²⁺. (<i>a</i>) reversible increase in ΔF/F₀ in response to a 30-s exposure to 80 mM [K⁺]_e in 2-mM Ca²⁺ bath solution. (<i>b</i>) and (<i>c</i>): a second exposure to 80 mM [K⁺]_e in Ca²⁺-free solution supplemented with 300 μM CdCl₂ causes a sustained increase in fluo-4 fluorescence (<i>b</i>) which is not reversed until treatment of the cell with the membrane permeable metal chelator TPEN at 100 μM (<i>c</i>). A second K⁺ challenge of the TPEN-loaded neuron in a 2-mM Ca²⁺ bath solution (<i>d</i>) shows restoration of fluo-4 Ca²⁺ responsivity. <b>C</b>: Bar graphs summarizing mean ± SEM of peak cytosolic ΔF/F₀ in sympathetic neurons before and after loading with TPEN (100 μM). <i>P</i> = 0.14 by paired <i>t</i>-test (n = 5 cells). <b>D</b>: Extracellular cadmium does not block depolarization-induced increase in ΔF/F₀ in the absence of external Ca²⁺. Left panel: reversible increase in ΔF/F₀ in response to a 30-s exposure to 80 mM [K⁺]_e in 2 mM [Ca²⁺]_e. Following TPEN loading (middle panel), a second 30-s [K⁺]_e challenge in a Ca²⁺-free bath solution containing 300 μM Cd²⁺ gives rise to a small-amplitude ΔF/F₀ transient (right panel).</p

Pharmacological inhibitors of IP₃ receptors abrogate high [K⁺]_e-induced Ca²⁺ transients in Ca²⁺-free bath solution.

<p><b>A</b>: Representative time courses of changes in cytosolic ΔF/F₀ in response to two consecutive 30-s exposures to 80 mM [K⁺]_e in 2 mM [Ca²⁺]_e (left panels) and in Ca²⁺-free bath solution (with 200 μM EGTA added; right panels) following 20-min incubation with 20 μM 2-APB or 10 μM xestospongin C. <b>B</b>: Percentage of neurons exhibiting [Ca²⁺]_i transients both in 2 mM and 0 mM [Ca²⁺]_e; * <i>P</i> < 0.001 versus control by Fisher’s Exact test (60, 6 and 16 cells for control, 2-APB and xestospongin C, respectively).</p

Sympathetic neurons possess the ability to release Ca²⁺ from intracellular stores in response to prolonged membrane depolarization in the absence of extracellular Ca²⁺.

<p><b>A</b>: Time course of cytosolic fluo-4 ΔF/F₀ in a sympathetic neuron in response to a 30-s exposure to 80 mM [K⁺]_e in 2 mM [Ca²⁺]_e and a 30-s exposure to 80 mM [K⁺]_e in Ca²⁺-free solution, followed by a second 30-s application of 80 mM [K⁺]_e in 2 mM [Ca²⁺]_e applied 70 seconds after the Ca²⁺ transient was elicited in Ca²⁺- free solution. <b>B</b>: Bar graphs of peak magnitude of the cytosolic ΔF/F₀ transients (left panel) and their rise and decay times during exposure to 80 mM [K⁺]_e in the presence and absence of extracellular Ca²⁺ (with 200 μM EGTA added). Rise times and decay times were measured as the intervals from 10% to 90% and from 90% to 10%, respectively, of peak ΔF/F₀. Values are mean ± SEM from 48 to 87 cells. *<i>P</i> < 0.0001 versus 2 mM [Ca²⁺]_e, paired <i>t</i>-test. <b>C</b>: Plots of sympathetic neuron membrane potential as a function of [K⁺]_e. Values are mean ± SEM (n = 5–8 experiments for each [K⁺]_e studied). Lines are linear fits with slopes of 67.5 mV (2 mM [Ca²⁺]_e) and 62.2 mV (0 mM [Ca²⁺]_e) per 10-fold change in [K⁺]_e. <b>D</b> and <b>E</b>: Peak magnitude of cytosolic ΔF/F₀ transients as a function of membrane potential. Shown are the relationships between membrane potential and peak ΔF/F₀ amplitude in response to 30-s exposures to 40, 60, 80 or 100 mM [K⁺]_e in 2 mM [Ca²⁺]_e (<b>D</b>) or in the absence of extracellular Ca²⁺ (<b>E</b>). Values are mean ± SEM (n = 18–87 cells for each [K⁺]_e). Solid lines represent best fits of the data to a linear function (D) and a polynomial function (E). Dashed lines denote membrane potentials generated by each [K⁺]_e. <b>B</b>: * <i>P</i> < 0.001 versus 40 mM [K⁺]_e; ** <i>P</i> < 0.03 versus 60 mM [K⁺]_e. <b>C</b>: * <i>P</i> < 0.05 versus 40 mM [K⁺]_e; repeated measures ANOVA and Dunn’s method for multiple comparisons.</p

The 1,4-dihydropyridine antagonist of L-type Ca²⁺ channels, nifedipine, does not affect Ca²⁺ transients elicited by exposure to elevated [K⁺]_e.

<p><b>A</b> and <b>B</b>: The response of peak <i>I</i>_<i>Ba</i> in a postganglionic sympathetic neuron to application of 10 and 50 μM nifedipine. <i>I</i>_<i>Ba</i> was activated by depolarizations from -80 mV to 10 mV every 10 seconds. Representative current traces are shown in A. <b>C</b>: Bar graphs represent the average peak <i>I</i>_<i>Ba</i> in control and in 10 and 50 μM nifedipine. Values are from 10 cells. *<i>P</i> < 0.05, RM ANOVA on ranks followed by Student-Newman-Keuls Method for multiple comparisons. <b>D</b>: Exemplar time courses of changes in cytosolic ΔF/F₀ elicited by 80 mM [K⁺]_e in the presence (upper panel) and absence of external Ca²⁺. The cell was continuously bathed in 50 μM nifedipine starting 20 min before the first [K⁺]_e test. <b>E</b>: Percentage of cells exhibiting [Ca²⁺]_i transients both in 2 mM and 0 mM [Ca²⁺]_e; <i>P</i> = non-significant versus control by Fisher Exact test (60 cells for control and 10 cells for nifedipine).</p

Peak magnitude of cytosolic ΔF/F₀ transients as a function of calcium channel activity.

<p><b>A</b>: Representative traces of whole-cell <i>I</i>_Ca recordings with 2 mM [Ca²⁺]_e. Currents were elicited using 300-ms depolarizations ranging between -50 and +10 mV in 5-mV steps. Pulse interval was 10 s. <b>B</b>: Peak <i>I</i>_Ca−voltage relationship. Current amplitudes were normalized to cell capacitance and plotted as mean values. Error bars represent SEM (n = 11 cells). Dashed lines indicate voltages generated by each [K⁺]_e in 2-mM [Ca²⁺]_e bath solutions. <b>C</b>: Voltage-dependence of steady-state <i>I</i>_Ca activation, <i>P</i>_act. Values are mean ± SEM for 11 cells. The solid line represents the mean of the best fit to each cell by a Boltzmann distribution, with <i>V</i>_<i>1/2</i> and <i>k</i> values of -31.2 mV and 3.4 mV, respectively. <b>D</b>: Voltage-dependence of steady-state <i>I</i>_Ca inactivation, (1 –<i>P</i>_inact). For measuring voltage-dependence of inactivation, a paired-pulse voltage protocol was used consisting of a 300-ms conditioning prepulse to voltages from -90 to 40 mV followed, after a 20-ms gap at -90 mV, by a 300-ms test pulse to 0 mV. Holding potential was -90 mV and the interval between conditioning prepulses as 10 s. For generating inactivation curve, the peak amplitudes of currents evoked by the test pulse were normalized to the current evoked during each prepulse and plotted as a function of prepulse potential. Solid line is the mean of the best fit of the descending portion of the inactivation-voltage relationship (i.e., between -111.9 and 18.1 mV) to a Boltzmann function, with <i>V</i>_<i>1/2</i> and <i>k</i> values of 55.3 mV and 16.3 mV, respectively. Values are mean ± SEM (n = 8 cells). <b>E</b>: Calculated voltage-dependence of steady-state <i>P</i>_act of high voltage-gated Ca²⁺ channels in sympathetic ganglion neurons. The curve shows the theoretical steady-state <i>P</i>_act at any potential, using the Boltzmann values for the amount of available current and the amount of current inactivation. The maximum available current was set to 1. Circles denote values for steady-state <i>P</i>_act at voltages generated by each [K⁺]_e in Ca²⁺-free bath solutions.</p

Dynamic substrate map and isopotential maps of noncontact mapping.

<p>(A) Normalized peak negative voltage (PNV) distribution of the RV in a posterior caudal view. The orange border zone rerepresents areas with voltages around 30% of the peak negative potential. (B) Isopotential map shows the activation sequence (frames 1–4). Color scale has been set so that white indicates the most negative potential and purple indicates the least negative potential. Virtual electrodes (V1-1 to V1-4) are placed along the propagation of activation wavefront from EA site (Frame 1) to BO site (Frame 4). The green arrows indicate the activation wavefron propagating from EA to BO site, then spreading out at BO site. The virtual unipolar electrograms reveal a QS pattern at the origin.</p

Noncontact mapping findings of triggers.

<p>BO = breakout; EA = earliest activation; Eg = electrogram; PNV = peak negative value; Other abbreviations are the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140167#pone.0140167.t001" target="_blank">Table 1</a>.</p><p>Noncontact mapping findings of triggers.</p

Baseline Clinical Characteristics of 35 Patients.

<p>* Measured by ventriculogram</p><p>^† Data are presented as median (range).</p><p>ARVC = arrhythmogenic right ventricular cardiomyopathy; ICD = implantable cardioverter-defibrillator; LVEF = left ventricular ejection fraction; NS = nonsignificant; PVC = premature ventricular contraction; RVEF = right ventricular ejection fraction; RVOT = right ventricular outflow tract; VT = ventricular tachycardia.</p><p>Baseline Clinical Characteristics of 35 Patients.</p