Virtual electrodes and the induction of fibrillation in Langendorff-perfused rabbit ventricles: the role of intracellular calcium

Abstract

A strong premature electrical stimulus (S₂) induces both virtual anodes and virtual cathodes. The effects of virtual electrodes on intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients and ventricular fibrillation thresholds (VFTs) are unclear. We studied 16 isolated, Langendorff-perfused rabbit hearts with simultaneous voltage and [Ca²⁺]_i optical mapping and for vulnerable window determination. After baseline pacing (S₁), a monophasic (10 ms anodal or cathodal) or biphasic (5 ms-5 ms) S₂ was applied to the left ventricular epicardium. Virtual electrode polarizations and [Ca²⁺]_i varied depending on the S₂ polarity. Relative to the level of [Ca²⁺]_i during the S₁ beat, the [Ca²⁺]_i level 40 ms after the onset of monophasic S₂ increased by 36 ± 8% at virtual anodes and 20 ± 5% at virtual cathodes (P < 0.01), compared with 25 ± 5% at both virtual cathode-anode and anode-cathode sites for biphasic S₂. The VFT was significantly higher and the vulnerable window significantly narrower for biphasic S₂ than for either anodal or cathodal S₂ (n = 7, P < 0.01). Treatment with thapsigargin and ryanodine (n = 6) significantly prolonged the action potential duration compared with control (255 ± 22 vs. 189 ± 6 ms, P < 0.05) and eliminated the difference in VFT between monophasic and biphasic S₂, although VFT was lower for both cases. We conclude that virtual anodes caused a greater increase in [Ca²⁺]_i than virtual cathodes. Monophasic S₂ is associated with lower VFT than biphasic S₂, but this difference was eliminated by the inhibition of the sarcoplasmic reticulum function and the prolongation of the action potential duration. However, the inhibition of the sarcoplasmic reticulum function also reduced VFT, indicating that the [Ca²⁺]_i dynamics modulate, but are not essential, to ventricular vulnerability.

the demonstration of virtual electrode formation after a premature stimulus (S₂) given through small epicardial electrodes has provided novel insights into the mechanisms of ventricular vulnerability (7, 15). A virtual cathode is a region with a transient depolarization of membrane potential (V_m), whereas a virtual anode is a region with a transient hyperpolarization of V_m during S₂. While virtual electrode formation was initially demonstrated by applying near-field stimulation to small epicardial electrodes, the same phenomenon was also shown in bidomain computer simulation studies (13) and after defibrillation shocks to rabbit ventricles (2). In addition to the effects on V_m, a strong electrical stimulus can also significantly change the intracellular Ca²⁺ concentration ([Ca²⁺]_i) (4). The magnitude and the spatial distribution of these changes are in part determined by the timing of the shock (10). Because heterogeneous postshock [Ca²⁺]_i changes are important in the mechanisms of ventricular defibrillation (6), it is important to study the differential effects of virtual anodes and cathodes on [Ca²⁺]_i release to better understand the mechanisms of reinduction of VF following defibrillation. As illustrated in Fig. 1, transient hyperpolarization at virtual anodes is expected to increase the driving force for Ca²⁺ entry through L-type Ca²⁺ channels (Fig. 1A), potentiating sarcoplasmic reticulum (SR) Ca²⁺ release. In contrast, transient depolarization at virtual cathodes is not expected to exert this effect (Fig. 1B), resulting in lesser SR Ca²⁺ release. The magnitude of change is increased with the increasing coupling interval (a, b, and c). These opposite effects are expected to increase the heterogeneity of [Ca²⁺]_i distribution. In contrast, after a biphasic S₂, the sites serving as virtual anodes during the first phase of the pulse became virtual cathodes during the second phase, and vice versa. Thus a biphasic shock would be expected to have more uniform effects on SR [Ca²⁺]_i release. If [Ca²⁺]_i transient heterogeneity is important in the mechanisms of ventricular vulnerability, then the induction of ventricular fibrillation (VF) should be easier with monophasic than with biphasic S₂. We hypothesize that 1) after a strong S₂ stimulus, virtual anodes cause a greater increase in [Ca²⁺]_i than virtual cathodes, and 2) the monophasic S₂ is associated with lower VF threshold (VFT) than biphasic S₂, but this difference is eliminated by the inhibition of SR function. The purpose of the present study was to test these hypotheses by simultaneously mapping V_m and [Ca²⁺]_i transients at virtual cathodes and virtual anodes during monophasic and biphasic epicardial point (near field) stimulation in Langendorff-perfused rabbit ventricles.

Fig. 1.

Schematics illustrating the proposed hypothesis. A: action potential negatively polarized at virtual anode. B: depolarization at virtual cathode. Since Ca²⁺ equilibrium potential (E_Ca) is >60 mV, the driving force for Ca²⁺ entry through L-type Ca²⁺ channels is increased at virtual anodes but decreased at virtual cathodes. The increased Ca²⁺ entry triggers additional sarcoplasmic reticulum (SR) Ca²⁺ release, potentiating the intracellular Ca²⁺ concentration ([Ca²⁺]_i) transient selectively at virtual anodes but not at virtual cathodes. The potentiation of SR Ca²⁺ release at virtual anodes is also greater at longer stimulus (S₂) coupling intervals (a, b, and c in A and B) when SR Ca²⁺ release channels have had more time to recover from refractoriness. C: methods for measuring Δ[Ca²⁺]_i. The difference of the [Ca²⁺]_i amplitude (Δ) was measured 40 ms after the S₂.

METHODS

The research protocol was approved by the Institutional Animal Care and Use Committee of Cedars-Sinai Medical Center and followed the guidelines of American Heart Association.

Tissue preparation.

New Zealand White rabbits (n = 18) weighing 3–5 kg were used in this study. The rabbits were anesthetized. The hearts were quickly removed. The ascending aorta was cannulated and secured for retrograde perfusion with warm (36.5 ± 0.5°C) and oxygenated Tyrode solution at a rate of 30–40 ml/min. The composition of the Tyrode solution (in mmol/l) at a pH of 7.4 ± 0.05 was 125 NaCl, 4.5 KCl, 1.8 NaH₂PO₄, 24 NaHCO₃, 1.8 CaCl₂, 0.5 MgCl₂, and 5.5 glucose and 50 mg/l albumin in deionized water. The coronary perfusion pressure was regulated and maintained at 80 cmH₂O, and the hearts were exposed to the air.

Stimulation protocol.

Two stimulating electrodes were placed on the left ventricle for baseline pacing (cathodal S₁) at a cycle length of 350 ms and for monophasic or biphasic S₂ stimulation 10–12 mm away using a World Precision Instrument (Sarasota, FL) model A385R constant current isolator. The line connecting S₁ and S₂ sites was parallel to the epicardial fiber orientation. The stimulating electrodes were made of stainless steel wires that were Teflon-coated except at their tips and had a diameter of 0.20 mm. We performed an in vitro testing and confirmed that a maximum of 60 mA of current can be delivered to the rabbit hearts with this stimulation system. After eight S₁ stimulations, a S₂ was applied with a stimulus strength ranging from 1 to 60 mA. The S₂ polarity was either monophasic or biphasic. The total pulse duration for either monophasic or biphasic S₂ was 10 ms. A 4-cm²-size mesh of stainless wire situated on the left ventricular posterior wall was used as the reference electrode. The biphasic S₂ was anode-cathode or cathode-anode. Each phase of the biphasic S₂ was 5 ms. The strength of the S₂ current was changed in 10-mA increments. The effective refractory period at the S₂ site was defined as the longest S₁-S₂ during which a cathodal stimulus of 10-ms duration and twice the diastolic threshold current did not initiate a propagated action potential.

The vulnerable window and the VFT.

The vulnerable window and the VFT were determined in 10 rabbits by giving S₂ with strengths of 1 to 60 mA. The shortest coupling interval that induced VF was determined by scanning the diastole starting with 60-ms S₁-S₂ coupling interval with 10-ms increments at a fixed S₂ strength. The longest coupling interval that induced VF was determined by scanning the diastole starting with 240-ms S₁-S₂ coupling interval with 10-ms decrements at a fixed S₂ strength. A defibrillation shock was used to terminate VF. The vulnerable window for that S₂ strength was defined as the duration between the shortest and the longest coupling interval that induced VF. After the shortest and the longest S₁-S₂ coupling intervals were determined, we did not apply S₂ at the in-between S₁-S₂ coupling intervals to minimize the fibrillation/defibrillation episodes that might cause tissue damage. We defined the area on the strength-interval curve associated with the induction of VF as the area of vulnerability. In 6 out of 10 hearts, we administered 1 μM ryanodine and 100 nM thapsigargin to inhibit SR Ca²⁺ cycling. The VFT was determined again by scanning the S₁-S₂ coupling interval starting from 80 ms with 10-ms increment and starting from 340 ms with 10-ms decrement at a fixed S₂ strength for the shortest and the longest coupling interval that induced VF, respectively. The S₂ strength varied from 1 to 60 mA.

Optical mapping.

Eight hearts were labeled for simultaneous dual optical mapping of V_m and [Ca²⁺]_i according to methods described in detail elsewhere (9). A Ca²⁺-sensitive dye (0.5 mg Rhod-2 AM, Molecular Probes) was infused into the heart over a 10-min period. This was followed in 15 min by a direct injection of a voltage-sensitive dye (RH 237, Molecular Probes) into the perfusion system with 10–20 μl of the 1 mg/ml solution dissolved in dimethyl sulfoxide (1). The hearts were illuminated with a solid-state, frequency-doubled laser (Verdi, Coherent). The fluorescence was acquired simultaneously with two charge-coupled device cameras (CA-D1-0128T, Dalsa) at 4 ms/frame. The digital images (128 × 128 pixels) were gathered from the epicardium of the left ventricle (20 × 20 mm² area) at 1,000 frames continuously with a 12-bit resolution. Optical signals were processed with both spatial (3 × 3 pixels) and temporal (3 frames) filtering. We used a grid to calibrate the locations of the field of view of these two cameras. Using this calibration, we can compare the recordings of V_m and [Ca²⁺]_i from the same locations. Cytochalasin D (5 μmol/l, Sigma) was added to the perfusate to inhibit muscle contraction.

Construction and interpretation of two-dimensional maps.

The ratio maps were used to examine V_m activation and changes of [Ca²⁺]_i levels. The average fluorescence level (F̄) of the entire data window was first calculated. The fluorescent level of each pixel was then compared with this average. We assigned shades of red to represent above-average fluorescence and shades of blue to represent below-average fluorescence to generate the ratio maps.

Data analysis.

We assumed that the S₁-induced action potential has a resting V_m of −75 mV and a maximum phase 0 upstroke of +15 mV. The fluorescent intensity of [Ca²⁺]_i transient was normalized from 0 (end diastolic) to 1 (peak systolic). The Δ[Ca²⁺]_i amplitude, representing the net change of [Ca²⁺]_i after S₂ stimulus compared with 7th S₁-paced beat, was measured 40 ms after S₂ (Fig. 1C). We chose to measure 40 ms after S₂ because 40 ms was sufficiently long to allow Ca²⁺-induced SR Ca²⁺ release to occur, but sufficiently short to prevent measuring the secondary [Ca²⁺]_i rise triggered by a propagated action potential in case the S₂ captured the ventricle.

Statistical analysis.

Statistical differences in Δ[Ca²⁺]_i amplitudes were tested with a two-way ANOVA. Differences among individual means were verified subsequently by Newman-Keuls post hoc tests. VFT and the S₁-S₂ coupling interval of the vulnerable window were compared by one-way ANOVA with Newman-Keuls multiple comparisons for post hoc analysis. P ≤ 0.05 was considered significant. All data are presented as means ± SD.

RESULTS

Virtual electrodes and [Ca²⁺]_i after a monophasic and biphasic S₂.

Consistent with previous studies (8, 11, 15), our optical recordings showed coexistence of virtual anode and virtual cathode after monophasic S₂ in all hearts studied. Fig. 2A shows virtual electrode formation after a monophasic anodal S₂. The V_m map shows a dog bone-shaped virtual anode perpendicular to the fiber orientation and two virtual cathodes located parallel to the fiber orientation. The simultaneous [Ca²⁺]_i map shows [Ca²⁺]_i elevation around the S₂, corresponding to the dog bone-shaped virtual anode, but no significant [Ca²⁺]_i elevation at the surrounding virtual cathodes. Figure 2B shows V_m and [Ca²⁺]_i tracings from virtual anode and virtual cathode sites, respectively. At 40 ms after the S₂ (red arrowheads in the [Ca²⁺]_i trace), before the secondary [Ca²⁺]_i transient elicited by propagated action potentials from the S₂ (blue arrowheads in the V_m trace), [Ca²⁺]_i was elevated at the virtual anode site, but not at the virtual cathode site. An activation isochronal map of the propagated response induced by S₂ is shown on the right.

Fig. 2.

Differential [Ca²⁺]_i dynamics at virtual electrodes induced by a monophasic S₂. A: S₂ polarity (anodal) and the location of stimulation electrodes in the mapped region. A double-headed arrow indicates the direction of epicardial fiber orientation. The membrane potential (V_m) map shows virtual electrode polarizations after a 60-mA, 10-ms monophasic anodal S₂ given 200 ms after the last S₁. The [Ca²⁺]_i map was taken 40 ms after the S₂. B: optical recordings at virtual anode and virtual cathode, respectively. Red arrows indicate 40 ms after S₂. Blue arrows indicate the peak of a regenerative action potential. C: S₂ polarity (cathodal) and the location of stimulation electrodes in the mapping region. The V_m map shows virtual electrode polarizations after a 60-mA, 10-ms monophasic cathodal S₂ given 200 ms after the last S₁. The [Ca²⁺]_i map shows the [Ca²⁺]_i distribution, which was taken 40 ms after the S₂. D: optical recordings at virtual anode and virtual cathode, respectively. *Location of the tip of the electrode. F, fluorescence.

In comparison, a monophasic cathodal S₂ induced a pair of virtual anodes parallel to the fiber orientation and a dog bone-shaped virtual cathode transverse to the fiber orientation (Fig. 2C). At 40 ms after S₂, there was larger [Ca²⁺]_i elevation at virtual anode than at virtual cathode sites. The region of [Ca²⁺]_i elevation corresponded directly to the shape of the virtual anode region. Figure 2D shows V_m and [Ca²⁺]_i tracings from virtual anode and virtual cathode, respectively. There was [Ca²⁺]_i elevation 40 ms after S₂ (red arrowheads) at the virtual anode site but not at the virtual cathode site. The [Ca²⁺]_i elevation at virtual anode occurred before the peak of propagated V_m (blue arrowheads). An activation isochronal map of the propagated response induced by S₂ is shown on the right.

Figure 3 shows the effects of an anodal-cathodal biphasic S₂ (anodal pulse in the first phase and cathodal pulse in the second phase). Figure 3A shows that the virtual electrode polarizations induced by the first phase of S₂ reversed polarity immediately following the second phase of S₂. The [Ca²⁺]_i map 40 ms after S₂ showed [Ca²⁺]_i elevation around the site of the S₂, but the shape did not resemble either the virtual anode or virtual cathode regions on the V_m map, and the size of [Ca²⁺]_i elevation by anodal-cathodal S₂ was smaller than that by anodal S₂ shown in Fig. 2A. Figure 3B shows V_m and [Ca²⁺]_i tracings from a virtual anode-cathode site and a virtual cathode-anode site, respectively. There was similar [Ca²⁺]_i elevation 40 ms after S₂ (red arrow) at these two sites. The [Ca²⁺]_i elevation at either site (red arrowheads) occurred before the peak of propagated V_m (blue arrowheads). An activation isochronal map of the propagated response induced by S₂ is shown on the right.

Fig. 3.

[Ca²⁺]_i dynamics after a biphasic S₂. The V_m maps in A show virtual electrode polarizations induced by anodal and cathodal phases, respectively. The S₂ was a 60-mA, 10-ms biphasic S₂ given 200 ms after the last S₁. The [Ca²⁺]_i map shows the [Ca²⁺]_i distribution, 40 ms after the S₂. B: optical recordings at virtual anode-cathode and virtual cathode-anode, respectively. Red arrows indicate 40 ms after S₂. Blue arrows indicate the onset of regenerative action potential. C: S₂ polarity and the location of stimulation electrodes in the mapped region. The V_m maps show virtual electrode polarizations induced by cathodal and anodal phases, respectively. The S₂ was a 60-mA, 10-ms biphasic S₂ given 200 ms after the last S₁. The [Ca²⁺]_i map was taken 40 ms after the S₂. D: optical recordings at virtual anode-cathode and virtual cathode-anode, respectively. *Location of the tip of the electrode.

Figure 3, C and D, shows the patterns of [Ca²⁺]_i and V_m when the biphasic waveform of the S₂ was switched to the first-phase cathodal and second-phase anodal (cathodal-anodal). An activation isochronal map of the propagated response induced by S₂ is shown on the right of Fig. 3D. When compared with the effects of the anodal-cathodal S₂ (Fig. 3A), the virtual electrodes reversed their polarity during the two phases (Fig. 3A). Figures 4 and 5 summarize the average changes in the level of [Ca²⁺]_i for both monophasic and biphasic S₂ delivered over a range of S₁-S₂ coupling intervals. Monophasic cathodal and anodal S₂ elevated [Ca²⁺]_i to a substantially greater extent at virtual anode sites compared with virtual cathode sites (Fig. 4), and the differences were larger with longer S₁-S₂ coupling intervals. In contrast, these differences were greatly attenuated with anodal-cathodal or cathodal-anodal biphasic S₂ shocks (Fig. 5).

Fig. 4.

Δ[Ca²⁺]_i amplitudes between virtual anodes and virtual cathodes at different S₁-S₂ coupling intervals. A: Δ[Ca²⁺]_i amplitude (net increase in [Ca²⁺]_i by S₂ stimulation compared with 7th S₁-paced beat) at virtual anode and virtual cathode when S₂ was monophasic anodal. The Δ[Ca²⁺]_i amplitudes progressively and significantly increased with increasing coupling interval. B: Δ[Ca²⁺]_i amplitude at virtual anode (white bars) and virtual cathode (black bars) induced by monophasic cathodal S₂. *P < 0.01 when compared with Δ[Ca²⁺]_i amplitude at virtual cathode. #P < 0.01 and †P < 0.01 when compared with Δ[Ca²⁺]_i amplitude of virtual anode and virtual cathode, respectively, at the S₁-S₂ coupling interval of 150 ms.

Fig. 5.

Comparison of Δ[Ca²⁺]_i amplitudes between virtual anode-cathode and virtual cathode-anode after biphasic S₂ stimulation. A: Δ[Ca²⁺]_i amplitudes after anodal-cathodal biphasic S₂. There was no significant difference of Δ[Ca²⁺]_i amplitudes between the 2 virtual electrode sites. However, there was progressive increase in Δ[Ca²⁺]_i amplitude with increasing S₁-S₂ coupling intervals. B: similar results of Δ[Ca²⁺]_i amplitude after cathodal-anodal biphasic S₂. The Δ[Ca²⁺]_i amplitude in both A and B progressively increased with increasing S₁-S₂ coupling intervals but did not show a significant difference between virtual anode-cathode (white bars) and virtual cathode-anode (black bars). #P < 0.01 and †P < 0.01 when compared with Δ[Ca²⁺]_i amplitude of virtual anode-cathode and virtual cathode-anode, respectively, at the S₁-S₂ coupling interval of 150 ms.

Different areas of vulnerability of monophasic S₂ and biphasic S₂.

Figure 6 shows the typical areas of vulnerability of monophasic and biphasic S₂ from one heart. The black squares show S₂ stimulus that induced VF, whereas the white squares show S₂ stimulus that failed to induce VF. No S₂ was given at the location marked by cross marks. These graphs show that the areas of vulnerability were much larger for monophasic than biphasic S₂ in this heart. Both monophasic anodal and cathodal S₂ induced VF in all 10 rabbits tested. However, VF was not inducible by either biphasic anodal-cathodal and cathodal-anodal S₂ at any S₂ strength in 3 of 10 hearts. In the remaining seven hearts, VF was inducible by both monophasic and biphasic S₂. The VFTs in these seven rabbits for monophasic S₂ were 23 ± 5 (anodal) and 19 ± 7 mA (cathodal) [P = not significant (NS)]. For biphasic S₂, the VFTs were 44 ± 10 (anodal-cathodal) and 40 ± 15 mA (cathodal-anodal) (P = NS). However, the differences between monophasic VFTs and biphasic VFTs were statistically significant (P < 0.01) for all comparisons.

Fig. 6.

Strength-interval plots of ventricular fibrillation (VF) induction at baseline. Shown are S₂ trials that induced VF (▪); S₂ trials that failed to induce VF (□), and S₂ between the shortest and the longest coupling intervals that induced VF (×). To minimize the number of fibrillation/defibrillation episodes, we did not actually deliver these S₂ to the ventricles. Ordinate (in mA) is S₂ current strengths; abscissa is coupling intervals (in ms).

In contrast to VFT, the upper fibrillation threshold (UFT) did not show a significant difference between monophasic and biphasic S₂. The UFT is defined by the highest stimulus strength that induced VF during the vulnerable period. VF was induced by 60-mA monophasic anodal or cathodal S₂ shocks in all rabbits tested, indicating that UFT was >60 mA. Similarly, the UFT for anodal-cathodal S₂ was >60 mA in all seven hearts, and the UFT for cathodal-anodal S₂ was >60 mA in all seven hearts except for one of which UFT was 50 mA. Therefore, we were not able to compare the mean UFT between monophasic and biphasic S₂.

The shortest coupling interval that induced VF averaged 130 ± 21, 120 ± 24, 151 ± 18, and 149 ± 21 ms for anodal, cathodal, anodal-cathodal, and cathodal-anodal S₂ shocks, respectively, and the longest coupling interval that induced VF averaged 177 ± 17, 183 ± 18, 180 ± 8, and 181 ± 22 ms, respectively. In seven hearts, VF was inducible by both monophasic and biphasic S₂. In these hearts, the maximum width of the vulnerable window [the difference of the longest and the shortest S₁-S₂ coupling interval that induced VF was 46 ± 18 for monophasic anodal S₂ and 74 ± 25 ms for monophasic cathodal S₂ (P < 0.01)]. The maximum width of the vulnerable window for biphasic S₂ were 29 ± 12 (anodal-cathodal) and 29 ± 29 ms (cathodal-anodal, P = NS). The differences between vulnerable windows of the monophasic S₂ and biphasic S₂ were statistically significant (P < 0.01) for all comparisons. These findings indicate that the area of vulnerability is smaller for biphasic S₂ than for monophasic S₂.

Effects of thapsigargin and ryanodine on area of vulnerability.

Pretreatment with thapsigargin and ryanodine largely eliminated the [Ca²⁺]_i transient, so that [Ca²⁺]_i mapping was not feasible. Thapsigargin and ryanodine treatment significantly prolonged the action potential duration (APD) compared with control (255 ± 22 vs. 189 ± 6 ms, P < 0.05). Thapsigargin and ryanodine eliminated the differences in VFT between monophasic and biphasic S₂ shocks. Figure 7 shows area of vulnerability of monophasic and biphasic S₂ shocks after thapsigargin and ryanodine from the same tissue shown in Fig. 6. The area of vulnerability widened after the treatment. Of note, there was a significant reduction of VFT (P < 0.01) after the treatment compared with baseline (monophasic anodal S₂, 5.8 ± 2.0; monophasic cathodal S₂, 6.7 ± 2.6; biphasic anodal-cathodal S₂, 6.7 ± 2.6; and biphasic cathodal-anodal S₂, 6.7 ± 2.6 mA), but no significant differences of VFT were detected among these four types of S₂. The UFT was >60 mA for both monophasic and biphasic S₂ in all rabbits tested. The shortest coupling interval that induced VF after thapsigargin and ryanodine was similar (127 ± 19, 127 ± 21, 135 ± 26, and 128 ± 18 ms for anodal, cathodal, anodal-cathodal, and cathodal-anodal S₂, respectively) to baseline; however, the longest coupling interval increased significantly (P < 0.01, respectively) after treatment (232 ± 64, 237 ± 34, 223 ± 36, and 228 ± 42 ms for anodal, cathodal, anodal-cathodal, and cathodal-anodal S₂, respectively).

Fig. 7.

Strength-interval plots of VF induction after the treatment of thapsigargin and ryanodine. Symbols and layout are as in Fig. 6.

DISCUSSION

The primary findings of this study are as follows: 1) a monophasic S₂ elevates [Ca²⁺]_i to a greater extent at virtual anodes than at virtual cathodes; 2) by switching the polarity in the middle of the pulse, a biphasic S₂ attenuated the differences of [Ca²⁺]_i and increased VFT; and 3) the depletion of SR Ca²⁺ stores with ryanodine and thapsigargin eliminated the VFT differences between monophasic and biphasic S₂. However, these drugs also increased APD and decreased VFT for both monophasic and biphasic S₂.

Mechanism of increased S₂-induced [Ca²⁺]_i transient heterogeneity by monophasic versus biphasic stimulus.

Figure 1 shows that at virtual anodes, the negative deflection of the membrane potential during the S₂ increases the driving force (Ca²⁺ equilibrium potential −V_m) for Ca²⁺ entry through L-type Ca²⁺ channels, thereby potentiating SR Ca²⁺ release. At virtual cathodes, however, further membrane depolarization toward the Ca²⁺ equilibrium potential decreases the net driving force, resulting in failure to potentiate SR Ca²⁺ release to the same degree. For monophasic S₂, this leads to heterogeneous [Ca²⁺]_i elevation between the virtual anode and cathode regions. For biphasic S₂, on the other hand, virtual anodes during the first phase become virtual cathodes during the second phase, and vice versa, so that the effect on SR Ca²⁺ release is more balanced. Another theoretical possibility, especially for a relatively late S₂, is that membrane depolarization at the virtual cathode could reactivate recovered L-type Ca²⁺ channels, potentiating further SR Ca²⁺ release. However, we did not observe potentiation of the [Ca²⁺]_i transient at virtual cathodes in this study.

SR Ca²⁺ cycling and ventricular vulnerability.

Coinciding with the greater [Ca²⁺]_i heterogeneity induced by monophasic versus biphasic S₂, we found that the VFT was significantly lower for monophasic S₂ than for biphasic S₂. Other investigators have demonstrated that enhancing L-type Ca²⁺ channel activity or α₁-stimulation can reduce VFT (12, 16). These findings also suggest that SR function plays an important role in modifying the ventricular vulnerability. By inhibiting SR Ca²⁺ transient with thapsigargin and ryanodine, the APD increased significantly and the differences in VFT between monophasic and biphasic S₂ disappeared. Our findings are consistent with the hypothesis that the [Ca²⁺]_i transient heterogeneity is an important determinant of VFT. The relationship between [Ca²⁺]_i and ventricular vulnerability can be partially explained by the bidirectional coupling between APD and [Ca²⁺]_i (14). It is possible that the [Ca²⁺]_i heterogeneity modulated the vulnerability to S₂ through its influence on APD heterogeneity. A second possible explanation to link between [Ca²⁺]_i and vulnerability is through afterdepolarization and triggered activity. Our laboratory (5) recently demonstrated the presence of [Ca²⁺]_i prefluorescence at the entrance of the central common pathway of S₂-induced figure-eight reentry. These findings suggest early afterdepolarization as a possible mechanism of impulse initiation at that site. Consistent with these hypotheses, the inhibition of the SR function by thapsigargin and ryanodine eliminated the differences between monophasic and biphasic S₂ on VFT. However, because SR inhibition did not eliminate ventricular vulnerability, these data also indicate that although the Ca²⁺ dynamics modulate vulnerability it is not the sole determinant of vulnerability to VF by a strong S₂ stimulus.

Shock-induced [Ca²⁺]_i changes in cultured neonatal rat myocytes.

Fast et al. (3) reported that in cultured neonatal rat myocytes, short-coupled shocks may transiently reduce [Ca²⁺]_i at sites of both positive and negative V_m changes. Raman et al. (10) extended the observations by evaluating time-dependent changes of shock on V_m and [Ca²⁺]_i in cultured neonatal rat cells. These results appear to be opposite to those obtained by us. However, those conclusions were based on the analyses of [Ca²⁺]_i changes during the shock, whereas we analyzed the [Ca²⁺]_i changes 40 ms after S₂ stimulation. The example shown in Fig. 2 of the study by Raman et al. (10) in fact showed [Ca²⁺]_i elevation in the postshock period. Furthermore, the postshock [Ca²⁺]_i elevation was larger after an anodal shock than after a cathodal shock, and there was more [Ca²⁺]_i elevation after a longer coupling interval than after a shorter coupling interval. Those postshock changes of [Ca²⁺]_i transient were consistent with the results of the present study.

Limitations.

This study was performed using small epicardial wires to give S₂. Because the stimulation electrodes are small, the current flow through these electrodes is insufficient in achieving defibrillation. Therefore, the results of these studies might not be applicable to defibrillation shocks. [Ca²⁺]_i and fluorescence do not have a linear relationship. Therefore, the absolute values shown in Figs. 4 and 5 may not reflect the correct Δ[Ca²⁺]_i levels at all coupling intervals. However, the relative values of [Ca²⁺]_i induced by anodal and cathodal stimuli and by different S₁-S₂ coupling intervals remain valid. This limitation therefore does not invalidate the conclusion of the study.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grants P01-HL-78931, R01-HL-R01-HL-78932, R01-HL-58533, R01-HL-66389, and R01-HL-71140; by Kawata, Laubisch, Price, Krannert, and Medtronic-Zipes endowments; and by American Heart Association Postdoctoral Fellowships (Western State Affiliate) 0225027Y and 0555057Y and National Scientist Development Grant 0335308N.