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(Circulation Research. 2004;94:1589.)
© 2004 American Heart Association, Inc.

Cellular Biology

Effects of Electrical Shocks on Ca_i²⁺ and V_m in Myocyte Cultures

Vladimir G. Fast, Eric R. Cheek, Andrew E. Pollard, Raymond E. Ideker

From the Department of Biomedical Engineering (V.G.F., E.R.C., A.E.P., R.E.I.) and Departments of Medicine and Physiology (R.E.I.), University of Alabama at Birmingham.

Correspondence to Vladimir G. Fast, PhD, University of Alabama at Birmingham, 1670 University Blvd, VH B149, Birmingham, AL 35294. E-mail fast{at}crml.uab.edu

	Abstract

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Changes in intracellular calcium concentration (Ca_i²⁺) induced by electrical shocks may play an important role in defibrillation, but high-resolution Ca_i²⁺ measurements in a multicellular cardiac tissue and their relationship to corresponding V_m changes (V_m) are lacking. Here, we measured shock-induced Ca_i²⁺ and V_m in geometrically defined myocyte cultures. Cell strands (width=0.8 mm) were double-stained with V_m-sensitive dye RH-237 and a low-affinity Ca_i²⁺-sensitive dye Fluo-4FF. Shocks (E5 to 40 V/cm) were applied during the action potential plateau. Shocks caused transient Ca_i²⁺ decrease at sites of both negative and positive V_m. Similar Ca_i²⁺ changes were observed in an ionic model of adult rat myocytes. Simulations showed that the Ca_i²⁺ decrease at sites of V⁺_m was caused by the outward flow of I_CaL and troponin binding; at sites of V^–_m it was caused by inactivation of I_CaL combined with extrusion by Na–Ca exchanger and troponin binding. The important role of I_CaL was supported by experiments in which application of nifedipine eliminated Ca_i²⁺ decrease at V⁺_m sites. Largest Ca_i²⁺ were observed during shocks of 10 V/cm causing simple monophasic V_m. Shocks stronger than 20 V/cm caused smaller Ca_i²⁺ and postshock elevation of diastolic Ca_i²⁺. This was paralleled with occurrence of biphasic negative V_m that indicated membrane electroporation. Thus, these data indicate that shocks transiently decrease Ca_i²⁺ at sites of both V^–_m and V⁺_m. Outward flow of I_CaL plays an important role in Ca_i²⁺ decrease in the V⁺_m areas. Very strong shocks caused smaller negative Ca_i²⁺ and postshock elevation of diastolic Ca_i²⁺, likely caused by membrane electroporation.

Key Words: defibrillation • fluorescent imaging • membrane potential • intracellular calcium

	Introduction

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Calcium ions play crucial roles in regulation of cardiac excitation and contractility, and they may be an important determinant of the tissue response to defibrillation shocks. The interaction between electrical field and Ca_i²⁺ may affect the outcome of a defibrillation attempt in several ways. First, it was suggested that very strong shocks cause calcium overload, which can lead to abnormal impulse generation, re-induction of rapid arrhythmias,¹ and defibrillation failure.² Second, it was reported that relatively weak shocks with an energy below the defibrillation threshold applied during fibrillation can prevent the loss of cardiac contractility often observed after successful defibrillation, so-called pulseless electrical activity syndrome.^3,4 In a related study, it was reported that cardiac contractility is enhanced when shocks are applied during the absolute refractory period.⁵ Two alternative mechanisms were proposed to explain these effects: stimulation of intracardiac sympathetic nerves by the shock⁴ or an increase of peak Ca_i²⁺ concentration caused by a direct effect of the shock on myocytes.⁶

The direct assessment of the mechanisms of shock-Ca_i²⁺ interaction and its role in defibrillation requires measurements of shock-induced Ca_i²⁺ and V_m changes with high spatial and temporal resolution. Previously, shock-induced Ca_i²⁺ changes were measured in single myocytes^7,8 and at a single point in whole hearts.⁶ No spatially resolved data on shock-induced Ca_i²⁺ changes and colocalized V_m changes in multicellular cardiac tissue are currently available. Such data are especially important because of the known complexity of shock-induced V_m in the heart. It is well established that shocks produce highly nonuniform patterns of V_m with areas of positive, negative, or negligible polarizations present in different parts of cardiac tissue,^9,10 suggesting that Ca_i²⁺ changes may also be nonuniform. In addition, shocks produce different types of V_m responses that depend on the shock strength and the tissue geometry. With the exception of very weak shocks, shocks applied during the action potential (AP) plateau produce nonlinear V_m of 2 main types.¹¹ Shocks of moderate strength induce asymmetric V_m, with negative V_m being much larger than positive V_m.^10,12,13 Stronger shocks can induce V_m of another type, which is characterized by a nonmonotonic behavior of negative V_m in which strong hyperpolarization is followed by a return of V_m to more positive levels.^11,14 In cell cultures, the transition from one nonlinear V_m type to another with increasing shock strength was paralleled by the occurrence of postshock arrhythmias originating from the area of shock-induced negative polarization¹ as well as with membrane electroporation in the same areas.¹⁵ It can be hypothesized that these 2 types of nonlinear V_m are associated with different Ca_i²⁺ changes as well.

The purpose of the present study was to use high-resolution optical mapping of V_m and Ca_i²⁺ to determine spatio-temporal Ca_i²⁺changes during and after shocks, as well as the relationship between these Ca_i²⁺ and different types of V_m in multicellular cardiac tissue. Because shock-induced V_m strongly depend on the tissue geometry,^11,16 experiments were performed in geometrically defined cultured cell monolayers. To elucidate the ionic mechanisms of Ca_i²⁺ changes, shock effects were also investigated in a computer model of rat ventricular myocardium.

	Methods

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Cell Cultures
Growth substrates containing linear strands (width=0.8 mm) were prepared as described previously.¹⁷ Neonatal rat myocytes were obtained from 1- to 2-day-old Wistar rats and cultured according to the previously published procedures.¹¹ Cell cultures were incubated in UltraCulture medium (BioWhittaker) supplemented with 20 µg/mL vitamin B₁₂, antibiotics, 0.1 mmol/L of bromodeoxyuridine, and 0.5 µmol/L of epinephrine at 37°C in a humidified atmosphere containing 4% CO₂. Culture medium was exchanged on the next day after preparation and every second day thereafter. Experiments were performed after 4 to 6 days in culture.

Optical Mapping of V_m and Ca_i²⁺
V_m and Ca_i²⁺ changes were measured using a previously described optical mapping technique¹⁸ with some modifications. Cells were first stained with 5 µmol/L of a Ca_i²⁺-sensitive dye (see later) by incubating with a dye solution for 30 to 45 minutes. After that, cultures were transferred into experimental chamber and superfused with Hanks solution (Life Technologies) having a pH of 7.4 and a temperature of 36°C. Cells were stained for 5 minutes with 2.5 µmol/L of V_m-sensitive RH-237 (Molecular Probes). To reduce leakage of the Ca_i²⁺ dye, 1 mmol/L of probenecid was added to the staining and perfusion solutions.¹⁸

Previous Ca_i²⁺measurements using dye Fluo-3 demonstrated very long durations of Ca_i²⁺ transients (CaD).¹⁸ We hypothesized that such long CaD were caused by the high affinity of Fluo-3 to Ca²⁺ ions. Because this can affect measurements of shock-induced Ca_i²⁺, we examined dyes with different affinities to Ca²⁺ ions from 2 major groups. The first group included Fluo-3 analogs: Fluo-4, Fluo-5F, Fluo-4FF, and Fluo-5N (Molecular Probes) that have nominal dissociation constants (K_d) of 0.345, 2.3, 40, and 90 µmol/L, respectively. The second group included Rhod-2 and Rhod-FF, with K_d of 0.57 and 17 µmol/L, respectively. Based on these measurements (see Results), the dye Fluo-4FF was selected for measurement of shock-induced Ca_i²⁺ and V_m in double-stained preparations.

Ca_i²⁺ and V_m were measured sequentially using different sets of optical filters. For V_m measurements, fluorescence was excited at 560/55 nm and emitted light was measured at >650 nm. For Ca_i²⁺ measurements using Fluo dyes, the respective wavelength ranges were 480/40 nm and 530/50 nm. With Rhod dyes, they were 530/40 nm and 580/40 nm. Tests described previously¹⁸ showed that there was no optical cross-talk between RH-237 and Fluo dyes. Fluorescence was measured using a 16x16-photodiode array (Hamamatsu) and a microscopic mapping system¹¹ at a sampling rate of 2 to 5 kHz/channel and spatial resolution of 110 or 55 µm/diode.

Cells were paced at a cycle length of 500 ms. Rectangular uniform-field shocks with a duration of 10 ms were delivered via 2 platinum plate electrodes. The field strength (E) was measured using a bipolar electrode. Delivery of shocks was synchronized with stimulation pulses. The delay between AP upstrokes and shocks was 10 to 20 ms. At each mapping location, 3 to 4 shocks of different strengths selected from the values of 5, 10, 20, 30, and 40 V/cm were applied. A shock of the same strength was applied twice to measure V_m and Ca_i²⁺. For shocks of 20 V/cm or stronger, a 3-minute interval was allowed after shocks for tissue recovery.¹ Before shock application, control (no shocks) V_m and Ca_i²⁺ recordings were made. To check for data reproducibility, at the end of each series, measurements with the initial shock strength were repeated. In some cases, shocks of the same strength were applied repeatedly. Both V_m and Ca_i²⁺ measurements were highly reproducible. Changing the shock polarity resulted in symmetric reversal of V_m and Ca_i²⁺ responses.

A shock-induced V_m was measured as the difference between a linear fit of the plateau phase and the magnitude of the shock response 9 ms after the shock onset and normalized by the action potential amplitude.¹⁶ To measure a shock-induced change in Ca_i²⁺, 2 recordings of Ca_i²⁺ transients taken with and without a shock were normalized according to their levels at the moment preceding the shock. This normalization compensated for changes in optical signals caused by dye washout and photobleaching. The Ca_i²⁺ was calculated at the shock end (t=9 ms) as the difference between the 2 signals and expressed as a percentage of the amplitude of the control Ca_i²⁺ transient (% ACa).

An activation time was measured at the level of 50% change of the AP upstroke amplitude. The onset of Ca_i²⁺ transients was measured at the level of 30% change. The difference between the 2 times was taken as the V_m–Ca_i²⁺ delay. The durations of AP and Ca_i²⁺ transients were measured as time intervals between their respective onsets and as 50% or 80% levels of signal recovery.

Data were expressed as mean±SD. Differences were compared using the 2-tailed t test or the 1-way ANOVA test. Results were considered statistically significant if P<0.05.

Computer Modeling of Shock Effects
Computer simulations were performed in a 1-dimensional cable with ionic model of adult endocardial rat ventricular myocytes.¹⁹ The Ca-handling system included I_CaL, Na–Ca exchange (NCX), sarcoplasmic reticulum (SR), sarcolemmal pump current (I_CaP), and buffering by troponin (Tpn). SR and Tpn buffering were slightly modified according to a previous publication.²⁰ Full description of ionic model and cable parameters is presented in the online data supplement at http://circres.ahajournals.org. Cable equations were solved numerically using previously described procedures.²¹ The cable was stimulated at all nodes simultaneously. After 15 ms of the stimulus, a current was injected at one cable end and the same current was withdrawn from the other end. Such current injection is equivalent to application of extracellular uniform-field shocks.

	Results

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Selection of Ca_i²⁺-Sensitive Dye
Figure 1 illustrates the effects of dye affinity on optical measurements of Ca_i²⁺ transients. Calcium transients (Figure 1A) measured with high-affinity dyes Fluo-4 and Fluo-5F were much longer than those measured with low-affinity Fluo-4FF. The average CaD₈₀ (Figure 1B) measured with Fluo-4 and Fluo-5F (243±29 ms, n=6, and 220±15, n=7, number of strands) was 80% longer than with Fluo-4FF (132±10 ms, n=6; P<0.001). A further reduction of dye affinity by using Fluo-5N did not reduce CaD₈₀ (141±11 ms, n=7) in comparison to Fluo-4FF. In all series of measurements, AP durations were not significantly different (Figure 1B, right panel). Decreasing the dye affinity caused reduction of the signal-to-noise ratio (Figure 1A).

View larger version (39K): [in this window] [in a new window]   Figure 1. Effects of dye affinity on optical measurements of Cai2+ transients. A, Optical recordings of Cai2+ and Vm in double-stained cell monolayers using analogs of Fluo-4. B, Dependence of Cai2+ transient duration (CaD80) on dye affinity (left panel). Action potential duration (APD80) was similar in these series of measurements (right panel). C, Dependence of Cai2+ rise time (left) and Vm–Cai2+ delay (right) on dye affinity. D and E, Cai2+ transients (D) and CaD80 (E) measured with dyes Rhod-2 and Rhod-FF. *P<0.05.

Contrary to the recovery phase of Ca_i²⁺ transients, the parameters of the rising phase were not dependent on the dye choice. As shown in Figure 1C, the rise-times of Ca_i²⁺ transients measured using high-affinity and low-affinity dyes Fluo-4 and Fluo-4FF dyes were 16.3±1.0 ms and 17.5±1.3 ms (n=6, NS), respectively. The respective delays between AP and Ca_i²⁺ upstrokes were 3.1±0.1 and 3.1±0.3 (n=6, NS).

Similar data were obtained using Rhod dyes. The low-affinity Rhod-FF resulted in markedly shorter Ca_i²⁺ transients than the high-affinity Rhod-2 (Figure 1D). The CaD₈₀ values measured by these 2 dyes (Figure 1E) were similar to those measured by Fluo-4FF and Fluo-4, respectively.

These data indicate that the dye affinity plays an important role in optical measurements of Ca_i²⁺. Long Ca_i²⁺ transients measured with high-affinity dyes can be explained by dye saturation (see Discussion). Therefore, in conditions in which in situ dye calibration compensating for this saturation is not available, low-affinity dyes are more appropriate for the faithful reproduction of Ca_i²⁺ time course. Because Fluo-4FF exhibited a much higher signal-to-noise ratio than Fluo-5N, it was selected for measurements of shock-induced Ca_i²⁺ and V_m in double-stained preparations described later.

Shock-Induced Ca_i²⁺ Changes and Monophasic Asymmetric V_m
It was previously shown that depending on the field strength, shocks applied during AP plateau produce nonlinear V_m of 2 main types, monophasic asymmetric V_m and biphasic V_m.^10–13 Figure 2 illustrates V_m of the first type and accompanying Ca_i²⁺ changes induced by a 10-V/cm shock. The shock caused negative and positive V_m on the opposite strand edges with a gradual transition of V_m amplitude in between (Figure 2A). The V_m distribution was strongly asymmetric with maximal V^–_m (trace 1) being 2.5-times larger than maximal V⁺_m (trace 7). At the strand edges and at most other locations, polarizations had simple monophasic shapes. A more complex V_m shape was observed at the boundary between areas of positive and negative polarizations (trace 5), reflecting electrotonic interactions with areas of V^–_m and V⁺_m. After the shock, V_m returned to levels similar to those in the control.

View larger version (34K): [in this window] [in a new window]   Figure 2. Shock-induced Cai2+ with monophasic asymmetric Vm. A, Vm induced by 10-V/cm shock. Locations of recordings are shown in the inset in C. B, Changes in Cai2+ during the shock in comparison to control recordings. C, Longer recordings of Cai2+ transients.

As shown in Figure 2B, a shock of the same strength reduced Ca_i²⁺ at sites of both negative (traces 1 to 3) and positive polarizations (traces 6, 7). At the intermediate location where V_m was small (trace 5), Ca_i²⁺ was negligible. Ca_i²⁺ reduction was larger at sites of negative, rather than at positive, V_m, with Ca_i²⁺ magnitude being –25% and –11% ACa at sites of maximal V^–_m (trace 1) and V⁺_m (trace 7), respectively.

After the shock, Ca_i²⁺ continued to decrease for 4 ms at sites of negative V_m, where Ca_i²⁺ could reach –40%ACa (trace 1). After that, Ca_i²⁺ began to rise and, at most locations, Ca_i²⁺ returned to the control level soon after the shock (Figure 2C). A slight postshock increase of peak Ca_i²⁺ amplitude was observed at some locations (trace 6). Similarly, the duration of Ca_i²⁺ transients could be slightly prolonged (traces 6, 7). The diastolic level of Ca_i²⁺ was not changed.

Shock-Induced Ca_i²⁺ and Biphasic V^–_m
Figure 3 illustrates Ca_i²⁺ changes during a shock (E=29 V/cm) that produced another nonlinear V_m type. In this case, the negative V_m waveforms (Figure 3A, traces 1 to 3) were biphasic, exhibiting a strong positive shift after the initial large hyperpolarization. At the same time, the positive V_m at the cathodal strand edge (trace 7) was not different from the weaker shock.

View larger version (34K): [in this window] [in a new window]   Figure 3. Shock-induced Cai2+ with biphasic Vm. A, Biphasic Vm induced by 29-V/cm shock. B and C, Cai2+ recordings during the same shock in comparison to control recordings. The arrow indicates elevation of the diastolic Cai2+ level.

Similar to the effect of the weaker shock, Ca_i²⁺ was transiently reduced at sites of both negative and positive V_m (Figure 3B), but these changes were smaller than Ca_i²⁺ during the weaker shock. At sites of maximal V^–_m and V⁺_m, the decreases of Ca_i²⁺ were 16% and 5% ACa, respectively. Another difference was that the duration of Ca_i²⁺ transients was somewhat prolonged in the areas of both positive and negative V_m. A small elevation of the diastolic Ca_i²⁺ level was observed, more prominent at the anodal edge of the strand where it was 12% ACa 300 ms after the rising phase (Figure 3C, trace 1). In addition, the shock induced an extra beat, with a coupling interval of 360 ms.

Shock-induced V_m and Ca_i²⁺ changes were measured in a total of 19 cell strands from 15 cell monolayers. Each shock strength was examined in 7 to 10 strands. In all strands, V_m and Ca_i²⁺ responses of the 2 types described were observed. Thus, all shocks caused Ca_i²⁺ decreases at sites of both V⁺_m and V^–_m (Figure 4B). The Ca_i²⁺magnitude was always larger at V^–_m sites than at V⁺_m sites. The largest –Ca_i²⁺ (16% ACA) were observed at a shock strength of 10 V/cm, and they decreased with increasing shock strength (P<0.05, 40-V/cm group versus 10-V/cm group).

View larger version (28K): [in this window] [in a new window]   Figure 4. Dependence of Cai2+ on shock strength. A, Definitions of end-shock (Cai2+end), diastolic (Cai2+dia) Cai2+ changes as well as changes of Cai2+ transient duration (CaD50). Cai2+end was measured 9 ms after the shock onset; Cai2+ dia was measured 300 ms after the Cai2+ transient rising phase; CaD50 was measured at the 50% level of Cai2+ transient amplitude. B, C, and D, Plots of mean Cai2+end, Cai2+dia and CaD50 as functions of shock strength, respectively. The number of measurements (number of cell strands) for each shock strength was 7 to 10. *P<0.05 from zero.

The average duration of Ca_i²⁺ transients was increased in a strength-dependent fashion with mean CaD₅₀ at the strand edges reaching 25% to 30% at 40-V/cm shocks (Figure 4D). The diastolic Ca_i²⁺ measured 300 ms after the Ca_i²⁺ rising phase was not significantly affected by shocks with strength of 10 V/cm or less (Figure 4C). Increasing the shock strength to 20 V/cm caused elevation of diastolic Ca_i²⁺, but the magnitude of this elevation was very small (3% to 4% ACa). Measurements of diastolic Ca_i²⁺ changes by shocks stronger than 20 V/cm were precluded in the majority of cases because of occurrences of extra beats with relatively short coupling intervals. In 1 case, when a 30-V/cm shock induced an extra beat with a coupling interval longer than 300 ms, a more substantial elevation of diastolic Ca_i²⁺ level was registered, with Ca_i²⁺_dia measuring 12% and 7% ACa at the anodal and cathodal edges, respectively.

Computer Modeling
Figure 5 illustrates effects of shocks in a computer model. A shock caused V_m and Ca_i²⁺ that were qualitatively similar to those observed in experiments. Thus, shock-induced polarizations were asymmetric with V^–_m>V⁺_m and Ca_i²⁺ was decreased at sites of both V^–_m and V⁺_m (Figure 5A). I_CaL recordings showed that shocks caused rapid inactivation of this current in the V^–_m area (trace 1), which contributed to Ca_i²⁺ decrease at this location. In the V⁺_m region, I_CaL remained active, but it changed its direction from inward to outward (trace 2), causing removal of Ca²⁺ ions from the intracellular space.

View larger version (26K): [in this window] [in a new window]   Figure 5. Computer simulations of shock-induced Cai2+ and Vm. A, Recordings of Vm, Cai2+, and ICaL at cathodal (site 1) and anodal (site 2) ends of a cable during application of current with I=360 µA/cm2. B, Effects of blocking different Ca2+-handling pathways on Cai2+. ICaL indicates L-type calcium current; ICaP, sarcolemmal calcium pump current; NCX, Na-Ca exchanger; SR, sarcoplasmic reticulum; Tpn, troponin binding. C, Effects of permanent block of ICaL by 60% on Vm and Cai2+. D, Dependence of Cai2+ on current strength. E, Dependence of Cai2+ on Vm.

To examine the roles of various Ca_i²⁺ handling pathways in shock-induced Ca_i²⁺ decrease, these pathways were selectively inhibited during the shock. As shown in Figure 5B, Ca_i²⁺ decrease in V^–_m area (site 1) became smaller when NCX or Tpn buffering was disabled (green and blue traces). Inhibition of SR (both release and uptake), I_CaL, or I_CaP did not reduce Ca_i²⁺. In the area of V⁺_m (site 2), Ca_i²⁺ decrease became smaller after disabling of I_CaL or Tpn. Blocking other pathways (NCX, SR, I_CaP) produced no or minor effects.

The effect of I_CaL inhibition on Ca_i²⁺ was more pronounced when I_CaL was partially inhibited during the whole simulation. With I_CaL reduced by 60%, a shock caused practically no change in Ca_i²⁺ in the V⁺_m area (Figure 5C). In these conditions, V⁺_m and V^–_m became equal. Thus, block of I_CaL resulted in elimination of V_m asymmetry.

With increasing shock strength, Ca_i²⁺decrease became larger in the V^–_m area (Figure 5D). In the V⁺_m area, Ca_i²⁺ dependence was nonmonotonic, with Ca_i²⁺ becoming positive at stronger shocks. This Ca_i²⁺ reversal was eliminated by blocking NCX (not shown), indicating that positive Ca_i²⁺ was caused by inflow of Ca²⁺ via reversed NCX at large V⁺_m. Within the range of V⁺_m observed experimentally (<100 mV), however, shock-induced Ca_i²⁺ were negative (Figure 5E).

Effects of Nifedipine, Caffeine, and Thapsigargin on Shock-Induced Ca_i²⁺
The roles of I_CaL and SR in Ca_i²⁺ were investigated in cell cultures using drug application. Figure 6 illustrates the effects of 1 µmol/L nifedipine on Ca_i²⁺. Nifedipine completely eliminated the shock-induced Ca_i²⁺ decrease at the cathodal side (Figure 6A, traces 1). This effect was reversible (not shown). Similar results were obtained in 7 monolayers. As shown in Figure 6B, nifedipine caused radical reduction of the average cathodal Ca_i²⁺ (P<0.05 from control), which became not different from zero (NS); the average anodal Ca_i²⁺ was reduced by 37% (P<0.05). In 2 monolayers, V_m were measured together with Ca_i²⁺. In accordance with a previous publication,¹⁵ nifedipine reduced ratio of V^–_m/V⁺_m caused by an increase of V⁺_m (Figure 6C).

View larger version (29K): [in this window] [in a new window]   Figure 6. Effects of nifedipine on Cai2+. A, Cai2+ transients at anodal (site 1) and cathodal (site 2) strand edges in control and 5 minutes after perfusion with 1 µmol/L nifedipine. To improve signal-to-noise ratio, signals from 7 edge diodes were averaged. Black and gray traces depict recordings with and without shocks. B, Effect of nifedipine on end-shock Cai2+ measured in 5 monolayers during shocks with E=10.1±1.0 V/cm. *P<0.05. C, Recordings of Vm in the same monolayer.

Figure 7 demonstrates the effects of caffeine (10 mmol/L) and thapsigargin (1 µmol/L) on Ca_i²⁺. Although both drugs caused slowing of rising and recovery phases of Ca_i²⁺ transients, they did not eliminate shock-induced Ca_i²⁺ decrease (Figure 7A and 7B). The average Ca_i²⁺ was not strongly affected by both drugs (Figure 7C and 7D).

View larger version (39K): [in this window] [in a new window]   Figure 7. Effects of caffeine and thapsigargin on Cai2+. A and B, Cai2+ transients at anodal (site 1) and cathodal (site 2) edges in control, during application of 10 mmol/L caffeine (A) or 1 µmol/L thapsigargin (B), and after washout. C and D, Effects of drugs on end-shock Cai2+ measured in 6 monolayers. E=10.9±1.5 V/cm (C) and 9.9±1 V/cm (D). *P<0.05.

Measurements of Shock-Induced Ca_i²⁺ With High-Affinity Dyes
As shown in Figure 1, dye affinity affects measurements of Ca_i²⁺ transient duration. To examine whether it also affects measurements of shock-induced Ca_i²⁺, these measurements were repeated using a high-affinity dye Fluo-4. As shown in Figure 8A, use of this dye resulted in measurements of much smaller negative Ca_i²⁺ than those measured with Fluo-4FF. A 9-V/cm shock caused only 5% decrease of Ca_i²⁺ at the anodal strand edge (trace 1) and no Ca_i²⁺ change at the cathodal edge (trace 2). In addition, Fluo-4 resulted in measurements of larger postshock Ca_i²⁺ changes. Thus, the 29-V/cm shock caused elevation of the anodal diastolic Ca_i²⁺ level by 65% ACa (Figure 8B). Qualitatively similar results were observed in a total of 4 cell monolayers stained with Fluo-4.

View larger version (16K): [in this window] [in a new window]   Figure 8. Shock-induced Cai2+ measured with high-affinity dye Fluo-4. A, Cai2+ transients with shocks of different strength in comparison to control recordings (black traces) at anodal (site 1) and cathodal (site 2) strand edges. C, Corresponding longer Cai2+ recordings. The arrow indicates apparent elevation of the diastolic Cai2+ level.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study presents high-resolution measurements of Ca_i²⁺ and V_m changes caused by electrical shocks in cultured cell monolayers. The main findings are: (1) shocks induced transient decreases of Ca_i²⁺ in areas of both V^–_m and V⁺_m; computer simulations and experiments with channel blockers indicate that I_CaL played an important role in Ca_i²⁺ decrease; (2) the magnitude of Ca_i²⁺ had a nonmonotonic dependence on shock strength, decreasing at stronger shocks; this was paralleled with the occurrence of biphasic negative V_m; and (3) optical measurements of Ca_i²⁺ changes were strongly dependent on the affinity of the Ca_i²⁺-sensitive dye.

Role of Dye Affinity in Ca_i²⁺ Measurements
It is well-recognized that dye affinity plays an important role in Ca_i²⁺ measurements.²² Although there is no "gold" standard for validation of fluorescent dyes, it is generally agreed that low-affinity dyes are more appropriate for dynamic Ca_i²⁺ imaging.²² High-affinity dyes may misrepresent Ca_i²⁺ transients because of several factors, including (1) saturation at high Ca²⁺ concentrations generated by cardiac cells and (2) slow dye dissociation from Ca_i²⁺ ions. Without in situ calibration compensating for dye saturation, using high-affinity dyes may result in "clipping" of the upper portion of Ca_i²⁺ transients, which can explain long measured Ca_i²⁺ transient durations (see online supplement for detailed explanation). This mechanism may also exaggerate small Ca_i²⁺ changes near the resting level, which can explain the large elevation of diastolic Ca_i²⁺ level measured with high-affinity dyes. The fact that these dyes may underestimate Ca_i²⁺ changes near the peak of the Ca_i²⁺ transient contributes to the apparent small shock-induced Ca_i²⁺.

Shock-Induced Ca_i²⁺ and Relationship With V_m
This is the first study in which spatio-temporal Ca_i²⁺ changes were measured and directly related with colocalized V_m changes during electrical shocks in multicellular cardiac tissue. It was found that shocks of all strengths caused transient decreases of Ca_i²⁺ in the areas of both negative and positive polarizations. Qualitatively similar results were obtained in a computer model of rat myocytes in which shocks induced asymmetric V_m and Ca_i²⁺ decrease at sites of V^–_m and V⁺_m within the range of V_m observed experimentally. The model allowed evaluating roles of different Ca_i²⁺-handling pathways in shock-induced Ca_i²⁺. Simulations showed that in V^–_m areas, Ca_i²⁺ reduction was caused by inactivation of I_CaL combined with extrusion of Ca²⁺ ions by NCX and troponin binding. Unexpectedly, SR played no role in Ca_i²⁺ decrease. This could be because of the fact that SR activity is relatively low in this model; it is responsible for only 30% of Ca_i²⁺ transient amplitude.

In regions of V⁺_m, I_CaL remained active but it changed its direction from inward to outward when V_m exceeded the I_CaL reversal potential causing a reduction of Ca_i²⁺ at these locations. This mechanism accounted for a significant portion of shock-induced Ca_i²⁺ decrease with the remaining portion being caused by troponin binding. NCX did not play a role in Ca_i²⁺ decrease in this area because NCX in this model reverses at potentials above 0 mV.

Experiments in cell cultures supported the important role of I_CaL in Ca_i²⁺. Thus, inhibition of I_CaL by nifedipine caused elimination of Ca_i²⁺ decrease at the cathodal strand side indicating that the reversed flow of I_CaL contributed to Ca_i²⁺ decrease in this area in control conditions. In accordance with the model, disabling of SR by caffeine and thapsigargin did not affect Ca_i²⁺. This may suggest that, similar to the model, SR played a negligible role in Ca_i²⁺ in cell cultures, which is consistent with the relatively low level of SR development in neonatal rat myocytes.^23,24 Alternatively, the lack of drug effects may be explained by their inhibition of both Ca_i²⁺ release and uptake. This may affect amplitudes of Ca_i²⁺ transients and Ca_i²⁺ to the same extent, leaving relative Ca_i²⁺ unchanged.

The fact that Ca_i²⁺ was decreased during shocks at the majority of locations is in apparent contradiction with previous studies in whole hearts reporting an increase of peak Ca_i²⁺ in response to shocks applied during the absolute refractory period.⁶ Also, no Ca_i²⁺ decrease during shocks was reported in a study in isolated cells.⁷ These discrepancies may be caused by a combination of several factors. First, previous studies used high-affinity dyes that, because of their saturation and slow dissociation, may underestimate negative Ca_i²⁺ near peak of Ca_i²⁺ transient. Second, these studies did not assess regional differences in Ca_i²⁺ changes; according to the present work, Ca_i²⁺ are nonuniform and may remain unchanged during shocks in areas where V_m are small. Therefore, the definitive resolution of the discrepancy between this and the previous studies requires spatially and temporally resolved measurements of shock-induced Ca_i²⁺ and V_m in whole hearts or whole tissue preparations using low-affinity Ca_i²⁺ dyes.

It was previously reported that shocks with a strength below the defibrillation threshold applied during the absolute refractory period or during fibrillation can cause a positive inotropic effect, alleviating postshock "pulseless electrical activity."^3–5 Two alternative mechanisms were proposed to explain this effect. First, shocks could have a direct effect on myocytes by increasing their peak Ca_i²⁺. Second, shocks could affect myocytes indirectly via stimulation of sympathetic intracardiac nerves. The shock strength used in these studies was below the defibrillation threshold, which is estimated at 5 V/cm.²⁵ According to the present study, shocks of such strength should not increase Ca_i²⁺ directly. Instead, the main effect of 5-V/cm shocks was a decrease of Ca_i²⁺ during the shocks at sites of both positive and negative V_m (Figure 4). There was also a small increase of the Ca_i²⁺ transient duration (by 10%), but such an effect is unlikely to cause an increase in peak force. Therefore, these data do not support the hypothesis relating inotropic shock effect with the direct increase of Ca_i²⁺. More likely, it is caused by parasympathetic nerve stimulation.

Strong shocks (E 20 V/cm) that caused biphasic V_m produced smaller Ca_i²⁺decreases than the weaker shocks that caused monophasic V_m. The stronger shocks also often caused a small but statistically significant postshock elevation of Ca_i²⁺, which was more prominent in the areas of negative V_m (Figure 4). Shocks of such strength were shown to cause membrane electroporation.¹⁵ Therefore, both Ca_i²⁺ changes can be explained by shock-induced electroporation causing entry of Ca²⁺ ions via membrane pores. This interpretation is also supported by simulations in a model without membrane electroporation in which increasing shock strength caused larger negative Ca_i²⁺ at sites of V^–_m. At sites of V⁺_m, Ca_i²⁺ dependence was nonmonotonic, with negative Ca_i²⁺ first increasing and then decreasing because of Ca²⁺ entry via reverse NCX. Because this effect was associated with relatively large positive V_m, which were not observed experimentally, it is unlikely that this mechanism played a large role in Ca_i²⁺ in cell cultures.

Shock-induced Ca_i²⁺ overload has been implicated in the generation of postshock focal arrhythmias¹ via activation of calcium-dependent inward currents.²⁶ The threshold for these arrhythmias in 0.8-mm-wide cultured cell strands is 20 V/cm.¹ Although such shocks caused a statistically significant increase of diastolic Ca_i²⁺, the change was too small (4%) to make a definitive conclusion about possible contribution of such Ca_i²⁺ changes to arrhythmia generation. It is more likely that Ca_i²⁺ elevation plays a role in arrhythmias induced by stronger shocks.

Postshock Ca_i²⁺ elevation was previously described in a study on isolated myocytes.⁷ In that work, Ca_i²⁺ elevation comparable to the amplitude of Ca_i²⁺ transients (100%ACa) was observed after shocks with strengths of 30 V/cm and higher. Such Ca_i²⁺ changes are much larger than the diastolic Ca_i²⁺ changes observed here at comparable shock strengths (Figure 4C). This difference may be partially attributed to the high affinity of Fura-2 used in these experiments. When dye Fluo-4 with a similar affinity was used here, significant postshock elevation of Ca_i²⁺ was observed after 30-V/cm shocks, also (Figure 7). This and the arguments presented indicate that previous measurements might have substantially overestimated shock-induced elevation of diastolic Ca_i²⁺.

Limitations
Ca_i²⁺ changes were measured in strands of only one width. Because Ca_i²⁺ changes are mediated by V_m changes, shock-induced Ca_i²⁺ in strands of other widths can be estimated using the dependence of V_m magnitude on the strand width published previously.¹¹ There are species- and age-dependent differences in ionic currents and handling of intracellular calcium^23,24,27 that may affect shock-induced Ca_i²⁺ changes. On the qualitative level, however, calcium and other ionic currents have similar voltage dependence and kinetic properties in different cell types, indicating that the effects of shocks on Ca_i²⁺ should be qualitatively similar as well.

	Acknowledgments

 
This work was supported by NIH Grants HL67748 and HL67961 and AHA Grant 0255025B. We thank Reuben Collins for help with preparation of cell cultures.

	Footnotes

 
Original received October 21, 2003; resubmission received January 29, 2004; revised resubmission received May 5, 2004; accepted May 7, 2004.

	References

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