Paradoxical loss of excitation with high intensity pulses during electric field stimulation of single cardiac cells

Abstract

Transmembrane potential responses of single cardiac cells stimulated at rest were studied with uniform rectangular field pulses having durations of 0.5-10 ms. Cells were enzymatically isolated from guinea pig ventricles, stained with voltage sensitive dye di-8-ANEPPS, and stimulated along their long axes. Fluorescence signals were recorded with spatial resolution of 17 microm for up to 11 sites along the cell. With 5 and 10 ms pulses, all cells (n = 10) fired an action potential over a broad range of field amplitudes (approximately 3-65 V/cm). With 0.5 and 1 ms pulses, all cells (n = 7) fired an action potential for field amplitudes ranging from the threshold value (approximately 4-8 V/cm) to 50-60 V/cm. However, when the field amplitude was further increased, five of seven cells failed to fire an action potential. We postulated that this paradoxical loss of excitation for higher amplitude field pulses is the result of nonuniform polarization of the cell membrane under conditions of electric field stimulation, and a counterbalancing interplay between sodium current and inwardly rectifying potassium current with increasing field strength. This hypothesis was verified using computer simulations of a field-stimulated guinea pig ventricular cell. In conclusion, we show that for stimulation with short-duration pulses, cells can be excited for fields ranging between a low amplitude excitation threshold and a high amplitude threshold above which the excitation is suppressed. These results can have implications for the mechanistic understanding of defibrillation outcome, especially in the setting of diseased myocardium.

FIGURE 1

Cell excitation with 10 ms duration pulses. The cell shown was stimulated in the indicated direction with S1 pulse of 10 ms and variable amplitude, and V_m responses were optically recorded from seven sites spaced equally along the cell length. The four sets of recordings show V_m responses for the various S1 pulses. The numbers beneath and above the S1 portion of the recordings indicate the site number to which that trace corresponds. The amplitude of the S1 pulses is shown alongside the recordings. The circled numbers on the right of the traces indicate the sequence in which the recordings were obtained. The time bar for all recordings is shown alongside the 56 V/cm traces.

FIGURE 2

Loss of excitation for 0.5 ms pulses. The cell shown was stimulated with a series of 0.5 ms pulses of variable amplitude. The cell could be excited with 44 and 60 V/cm pulses but not with the 64 V/cm pulse. Excitation was restored when field strength was decreased back to 60 and 43 V/cm. The circled numbers on the right of the traces indicate the sequence in which the S1 pulses were applied. Also indicated are the recordings corresponding to sites 1 and 6. The time bar shown for the 43 V/cm recordings is common to all five sets of traces.

FIGURE 3

Delay and loss of excitation for 1 ms pulses. The cell shown was stimulated with a series of S1 pulses of increasing amplitude. The cell fired normally for 36 and 48 V/cm pulses. For the 53 V/cm pulse, a delay of ∼6 ms (measured from the S1 break) was observed before cell excitation occurred. For the 60 V/cm pulse, the cell failed to fire an action potential. The circled numbers on the right of the traces indicate the sequence in which the recordings were obtained. Also numbered in each set of traces are responses corresponding to sites 1 and 8. The time bar for the 60 V/cm recordings is common to all sets of recordings.

FIGURE 4

Return of excitation with increase in pulse duration. (Upper box) The cell shown was stimulated successively with three S1 pulses of 50, 55, and 50 V/cm. The cell was excited with the 50 V/cm pulses, but not with the 55 V/cm pulse. The time bar on the right applies to all three sets of recordings. (Lower box) With S1 amplitude fixed at 55 V/cm, the S1 amplitude was increased from 0.5 ms to 1 ms, 5 ms, and 10 ms. The cell did not fire an action potential for the 0.5 ms and 1 ms pulses but fired for the 5 ms and 10 ms pulses. Note the delay in excitation for the 5 ms pulse. The circled numbers indicate sequence of stimulation. The recordings corresponding to sites 1 and 8 have also been numbered in each set of traces.

FIGURE 5

Summary of data for seven cells stimulated with short (0.5 and 1 ms) pulses. The zone of excitation and lack thereof for each of the seven cells is shown individually. Each circle represents an applied field stimulus. Open circles represent the field stimuli for which excitation occurred, and solid circles represent the fields for which a cell failed to excite. For cell 2, the data point with an asterisk represents the stimulus corresponding to trace 3 of Fig. 3 for which excitation occurred after a considerable delay from the stimulus pulse. Note that the ordinate is the scaled electric field and represents an equivalent field experienced by a 120-μm long cell, which is the nominal length of a guinea pig cell. Cell lengths for various cells are shown at the top of the plot.

FIGURE 6

Responses of a model cell for 1 ms pulses near the LLE and near the ULE. Panel A shows the schematic of the model cell divided into 201 equal sized patches, but for simplicity only 11 (every one in 20 patches) are shown. Panel B shows the response of the central patch to three low amplitude pulses near LLE. Panel C shows the superimposed responses from the same patch for three pulses near ULE. The cell was excited with 58 V/cm but not with 59 and 60 V/cm pulses. The takeoff potential for 6.28 and 58 V/cm pulses is the same (∼−55 mV) and is equal to the Na⁺ channel activation threshold. However, the overshoot potentials are different (+37 and +25 mV, respectively).

FIGURE 7

V_m response and ionic currents (I_Na and I_K1) of a model cell stimulated with a field pulse at LLE. The model cell of Fig. 6 A was stimulated with a 5 ms field pulse of ∼6.3 V/cm in the indicated direction. The V_m responses from the 11 representative patches are shown in the topmost set of traces, and I_Na and I_K1 from the various patches are shown in the traces directly below. The numbers adjacent to the three sets of traces indicate the patch number to which that recording corresponds. For clarity, only traces from the end patches have been numbered. At the bottom is a schematic cell that shows the flow of I_Na and I_K1 in the various patches. The various arrows signify the relative amplitudes of the corresponding currents.

FIGURE 8

Paradoxical loss of excitation in a model cell for fields near ULE. The left column shows V_m, I_Na, and I_K1 for the 11 patches of the model cell for a 58 V/cm, 1 ms field pulse. The right column shows V_m, I_Na, and I_K1 for a pulse also of 1 ms duration but with amplitude increased to 59 V/cm. The numbers indicate the patch numbers to which various traces correspond. For clarity, only traces from the end patches are numbered for V_m and I_K1. For I_Na, intermediate patches with complex temporal behavior are also indicated. Time bar is applicable to all sets of traces. I_Na and I_K1 amplitude bars are applicable to both 58 and 59 V/cm traces. At the bottom is a schematic cell that shows the flow of I_Na and I_K1 in the various patches. The various arrows signify the relative amplitudes of the corresponding currents. The simulations in this figure are the same as in Fig. 6 C.

FIGURE 9

Return of excitation with increasing pulse duration. Left, middle, and right columns show V_m, I_Na, and I_K1 for three pulses of equal amplitude (59 V/cm) but increasing duration (1, 2, and 4 ms, respectively). The cell was unexcited for a pulse duration of 1 ms but excited with 2 and 4 ms pulses. For clarity, only traces from the end patches are numbered for V_m and I_K1. For I_Na, intermediate patches with complex temporal behavior are also numbered. Time bar in each column is applicable to all sets of traces at that duration. I_Na and I_K1 amplitude bars are applicable to all three durations.

FIGURE 10

Change in ULE (ΔULE) of a model cell with varying I_Na and I_K1 (ΔI). The I_Na and I_K1 in the model cell of Fig. 6 A were varied and ULE determined by gradually increasing the amplitude of a 1 ms field pulse. The ordinate shows ΔULE compared to that of a cell having nominal I_Na and I_K1.