Shock Induced A Vm in Cell Strands

Cell bundles and cell layers represent another common type of structural tissue organization at the microscopic level. The laminar structure might be especially important because it is present in the intramural bulk of ventricular myocardium where cardiac cells are organized into layers with thickness varying from tens to hundreds of microns.21'33 Boundaries of cell layers form resistive barriers to current flow and, therefore, may provide substrates for secondary sources during shock application. Similar to cell borders, the magnitude of laminar secondary sources and their relevance for defibrillation depend on multiple factors, including layer thickness, density of interlayer connections, electrotonic space constant, and so forth. The direct experimental observation of microscopic secondary sources in the intact tissue is not currently feasible. Therefore, to estimate their role in defibrillation, researchers mimicked the laminar type of structure in cell cultures, using linear cell strands of variable width, and measured their filed responses, using optical mapping.

Optical measurements of shock-induced AVm were performed in cell strands with width varying between 0.2 and 2 mm.34-36 As expected, shocks applied during AP plateau depolarized cells facing the cathode and hyperpolarized cells facing the anode (Fig. 4). Similar to the predictions of the linear cable model, weak shocks applied to narrow strands produced linear Vm responses with equal magnitudes of positive and negative AVm (Fig. 4a). The symmetry of Vm response was maintained for AVm below approximately 40% APA.34 Increasing the shock strength and/or the strand width resulted in an increase of AVm magnitude and a loss of AVm linearity. One such change was that polarizations became asymmetric where negative AVm significantly exceeded positive AVm (Fig. 4b, thin black traces). When the shock strength was further increased, another change in AVm shape was observed. In these cases, AVm became nonmonotonic, with negative AVm exhibiting shift to more positive levels, which reduced the degree of negative AVm asymmetry (Fig. 4b, thick black traces). Besides changes in AVm shapes, both positive and negative AVm exhibited saturation at high shock strength. The saturation level depended on the AVm polarity: positive AVm reached saturation at a relatively low level of ~ 100% APA, whereas negative AVm saturated above 200% APA.

The nonlinear features of Vm responses described above, including AVm asymmetry, nonmonotonic AVm shape, and saturation, were also observed in the intact myocardium.37-40 These effects may have important implications for defibrillation. For instance, since during fibrillation most of the myocardium is in the depolarized state, the effects of electrical shocks on Vm are predicted to be asymmetric, with a larger portion of myocardium undergoing negative rather than positive polarizations. It was shown that an interaction between areas of hyper- and depolarization might determine the success or the failure of defibrillation.41'42 Therefore, AVm asymmetry affects the outcome of a defibrillation shock. The knowledge of ionic mechanisms involved in shock-induced AVm might provide an opportunity a E b E

Figure 4: Shock-induced AVm in cell strands. (a) Linear AVm produced by weak shocks in narrow cell strands. Strand width equals 200 ^m; shock strength E = 1.9V/cm-1. Insert shows schematics of a cell strand with photodiode locations. (b) Nonlinear asymmetric (thin black tracées) and nonmonotonic (thick tracées) AVm. Gray tracées show Vm recordings without shocks (from Refs. 34,36)

Figure 4: Shock-induced AVm in cell strands. (a) Linear AVm produced by weak shocks in narrow cell strands. Strand width equals 200 ^m; shock strength E = 1.9V/cm-1. Insert shows schematics of a cell strand with photodiode locations. (b) Nonlinear asymmetric (thin black tracées) and nonmonotonic (thick tracées) AVm. Gray tracées show Vm recordings without shocks (from Refs. 34,36)

for pharmacological modulation of AVm asymmetry and, therefore, of defibrillation efficacy.

Until now ionic mechanisms of nonlinear Vm responses were investigated only in cell cultures34'35'43-45 and isolated single cells.46 The AVm asymmetry with larger AVm than AVj+ reflects an increase in the net outward current. Inhibition of potassium currents in cell cultures using barium chloride (blocker of inward rectifier current), dofetilide (delayed rectifier current), and 4-AP (transient outward current) did not change AVm significantly,34'35 indicating that none of these outward currents was responsible for the AVm asymmetry. In contrast, it was found that the asymmetric behavior of AVm was partially reversed by inhibition L-type calcium current.35 As shown in Fig. 5, application of nifedipine in cell strands increased positive AVm while leaving negative AVm unaffected, thus reducing the degree of AVm asymmetry. These findings indicate that AVm asymmetry is caused by the outward flow of Ica,L in the depolarized portions of strands. Normally, Ica,L is inward but it changes the direction when Vm exceeds the ICa L reversal potential. According to patch clamp studies, the ICa,L reversal potential in rat and rabbit myocytes is 45-50 mV.47,48 Therefore, positive AVm with magnitudes larger than ^50 mV should be reduced by the outward flow of ICa,L, which explains why blocking of ICa,L with nifedipine increases AV+.

The important role of ICa,L in AVm asymmetry was corroborated by measurements of shock-induced Ca2+ changes.44 According to this mechanism, shocks should decrease Ca2+ in the area of positive AVm due to the outward flow of Ca2+ ions through of L-type channels. To test this prediction, shock-induced ACa2+ were measured in cultured cell strands. As shown in Fig. 6, shocks applied during AP plateau transiently decreased Ca2+ in areas of both positive and negative AVm. Inhibition of ICa,L by nifedipine eliminated shock-induced Ca2+ decrease at sites of positive AVm (not shown). On the other side, inhibition of sarcoplasmic reticulum by either caffeine or thapsigargin had no effect on

Computer simulations in an ionic model of rat ventricular myocytes further supported these experimental findings.44'45 Similar to experiments, application of shocks in the model during the early AP produced (1) negatively asymmetric AVm and (2) decrease of Ca2+ in areas of both AV+ and AVm. Selective inhibition of sarcoplasmic reticulum had no effect on ACa2+. In contrast, inhibition of ICa,L increased AV+, reduced AVm asymmetry, and eliminated shock-induced Ca2+ decrease in the AVj+ area. Thus, both experiments and computer simulations support the hypothesis about the role of ICa,L in negative AVm asymmetry and shock-induced Ca2+ decrease.

The second type of nonlinear Vm response in cell cultures characterized by nonmonotonic negative AVm can be due to an inward ionic current activated at negative Vm or due to a nonspecific leakage current caused by membrane electroporation. The occurrence of such nonmonotonic AVm in cell cultures was paralleled with diastolic elevation of Vm as well as with induction of postshock arrhythmias.36 The polarization threshold for nonmonotonic AVm was approximately 200% APA, which corresponds to a Vm level of approximately —180 mV. There are two inward currents that are open at such Vm levels: "funny" current (If) and inward rectifier current (IK1). Their role in nonlinear AVm was examined using channel blockers. It was found that inhibition of IK1 by barium chloride and of If by cesium chloride caused no effect on AVm shape,43 indicating that these currents were not responsible for nonmonotonic AVm. The role of membrane electroporation was examined by measuring shock-induced uptake of a cell impermeable dye, propidium iodide, which becomes fluorescent after entering cells and binding to nucleic acids. It was found that application of a series of shocks with strength similar to the one inducing nonmonotonic AVm caused cell uptake of propidium iodide at the anodal side of cell strands where negative AVm were induced but not on the cathodal side (Fig. 7).43 Shock-induced dye uptake paralleled with nonmonotonic AVm and diastolic Vm elevation were also observed

Figure 5: Mechanism of asymmetric AVm. (a) Effect of nifedipine application on shock-induced AVm. Shock strength was ~10.7V/cm-1. (b) Isopotential maps of AVm distribution. (c) Spatial profiles of AVm across the strand. (d) Effect of nifedipine on magnitudes of AVj+, AVm, and asymmetry ratio AVm/AV+. Shock strength was 9.3 ± 0.8V/cm-1. *Statistically significant difference from control value (p < .05) (from Ref. 35)

Figure 5: Mechanism of asymmetric AVm. (a) Effect of nifedipine application on shock-induced AVm. Shock strength was ~10.7V/cm-1. (b) Isopotential maps of AVm distribution. (c) Spatial profiles of AVm across the strand. (d) Effect of nifedipine on magnitudes of AVj+, AVm, and asymmetry ratio AVm/AV+. Shock strength was 9.3 ± 0.8V/cm-1. *Statistically significant difference from control value (p < .05) (from Ref. 35)

Figure 6: Effects of shocks on Ca2+. (a) AVm induced by a 10-V/cm 1 shock in a cultured cell strand (width = 0.8 mm). Locations of recordings are shown in the inset in (c). ( b) Changes in Ca2+ during the shock in comparison to control recordings. (c) Longer recordings of Ca2+ transients (from Ref. 44)

Figure 6: Effects of shocks on Ca2+. (a) AVm induced by a 10-V/cm 1 shock in a cultured cell strand (width = 0.8 mm). Locations of recordings are shown in the inset in (c). ( b) Changes in Ca2+ during the shock in comparison to control recordings. (c) Longer recordings of Ca2+ transients (from Ref. 44)

on epicardial surface of rabbit hearts.49 These data in combination with the results of experiments with ionic channel blockers indicate that nonmonotonic negative AVm were due to membrane electroporation.

Strong shocks may induce arrhythmias,50-53 which can explain the reduced defibrillation efficacy of very strong shocks.54 The field threshold for postshock arrhythmias in cell cultures was very close to the thresholds for nonmonotonic negative AVm and electroporation.36 Optical mapping in cell strands with local expansions demonstrated that postshock arrhythmias were focal, and that the arrhythmia source was located in the hyperpolarized area of strands (Fig. 8),36 indicating that postshock arrhythmias were caused by membrane electroporation.

Figure 7: Mechanism of nonmonotonic AVm: shock-induced uptake of cell-impermeable dye propidium iodide. (a1) Phase contrast image of a cell strand (width = 0.7mm). (a2) Image of dye fluorescence after control dye application for 4 min (no shocks). (a3) Image of dye fluorescence after application of a series of shocks with a strength of 31 V/cm—1 and interval of 2 s. (a4) Difference between images in (a2) and (a3). The resulting image was filtered with a median filter. (b) Average horizontal profiles of fluorescent intensity (in arbitrary units) from images in (a2) (control), (a3) (shocks), and (a4) (difference) (from Ref. 43)

Figure 7: Mechanism of nonmonotonic AVm: shock-induced uptake of cell-impermeable dye propidium iodide. (a1) Phase contrast image of a cell strand (width = 0.7mm). (a2) Image of dye fluorescence after control dye application for 4 min (no shocks). (a3) Image of dye fluorescence after application of a series of shocks with a strength of 31 V/cm—1 and interval of 2 s. (a4) Difference between images in (a2) and (a3). The resulting image was filtered with a median filter. (b) Average horizontal profiles of fluorescent intensity (in arbitrary units) from images in (a2) (control), (a3) (shocks), and (a4) (difference) (from Ref. 43)

Figure 8: Localization of the shock-induced arrhythmias. (a) Phase contrast image of a narrow cell strand with an area of local expansion. (b) Recordings of Vm during application of 35V/cm-1 shock. (c, d) Vm recordings at selected sites during shock application and during the postshock extrabeat. The arrow in (d) depicts the direction of activation spread. (e) Isopotential map of shock-induced AVm distribution 5 ms after the shock onset. (f) Isochronal map of activation spread during the extra beat (from Ref. 36)

Figure 8: Localization of the shock-induced arrhythmias. (a) Phase contrast image of a narrow cell strand with an area of local expansion. (b) Recordings of Vm during application of 35V/cm-1 shock. (c, d) Vm recordings at selected sites during shock application and during the postshock extrabeat. The arrow in (d) depicts the direction of activation spread. (e) Isopotential map of shock-induced AVm distribution 5 ms after the shock onset. (f) Isochronal map of activation spread during the extra beat (from Ref. 36)

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