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(Circulation Research. 2000;87:774.)
© 2000 American Heart Association, Inc.

Cellular Biology

Sarcoplasmic Reticulum Ca²⁺ Release Causes Myocyte Depolarization

Underlying Mechanism and Threshold for Triggered Action Potentials

Klaus Schlotthauer, Donald M. Bers

From the Department of Physiology, Loyola University Medical Center, Maywood, Ill.

Correspondence to Donald M. Bers, PhD, Department of Physiology, Loyola University Medical Center, 2160 South First Ave, Maywood, IL 60153. E-mail dbers{at}luc.edu\\ © 2000 American Heart Association, Inc.

	Abstract

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Abstract—Spontaneous sarcoplasmic reticulum (SR) Ca²⁺ release causes delayed afterdepolarizations (DADs) via Ca²⁺-induced transient inward currents (I_ti). However, no quantitative data exists regarding (1) Ca²⁺ dependence of DADs, (2) Ca²⁺ required to depolarize the cell to threshold and trigger an action potential (AP), or (3) relative contributions of Ca²⁺-activated currents to DADs. To address these points, we evoked SR Ca²⁺ release by rapid application of caffeine in indo 1-AM–loaded rabbit ventricular myocytes and measured caffeine-induced DADs (cDADs) with whole-cell current clamp. The SR Ca²⁺ load of the myocyte was varied by different AP frequencies. The cDAD amplitude doubled for every 88±8 nmol/L of [Ca²⁺]_i (simple exponential), and the [Ca²⁺]_i threshold of 424±58 nmol/L was sufficient to trigger an AP. Blocking Na⁺-Ca²⁺ exchange current (I_Na/Ca) by removal of [Na]_o and [Ca²⁺]_o (or with 5 mmol/L Ni²⁺) reduced cDADs by >90%, for the same [Ca²⁺]_i. In contrast, blockade of Ca²⁺-activated Cl^– current (I_Cl(Ca)) with 50 µmol/L niflumate did not significantly alter cDADs. We conclude that DADs are almost entirely due to I_Na/Ca, not I_Cl(Ca) or Ca²⁺-activated nonselective cation current. To trigger an AP requires 30 to 40 µmol/L cytosolic Ca²⁺ or a [Ca²⁺]_i transient of 424 nmol/L. Current injection, simulating I_tis with different time courses, revealed that faster I_tis require less charge for AP triggering. Given that spontaneous SR Ca²⁺ release occurs in waves, which are slower than cDADs or fast I_tis, the true [Ca²⁺]_i threshold for AP activation may be 3-fold higher in normal myocytes. This provides a safety margin against arrhythmia in normal ventricular myocytes.

Key Words: delayed afterdepolarization • sarcoplasmic reticulum • transient inward current • Na⁺-Ca²⁺ exchanger • arrhythmia

	Introduction

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Arrhythmias are a major cause of sudden cardiac death in heart failure (HF). Three-dimensional mapping indicates that nearly all ventricular tachycardias in nonischemic HF and 50% of those in ischemic HF are initiated by nonreentrant mechanisms.¹ These can arise from abnormal ventricular automaticity² or triggered activity. The latter consists of either early afterdepolarizations (EADs) occurring in the plateau phase of the action potential (AP) or delayed afterdepolarizations (DADs), occurring at repolarized membrane potentials (E_m). Some EADs may be attributable to reactivation of Ca²⁺ channels, which can partially recover during long APs, especially as [Ca²⁺]_i declines.^{3 4 5 6}

DADs, the focus in the present study, are generally thought to be initiated by spontaneous Ca²⁺ release from the sarcoplasmic reticulum (SR) and a Ca²⁺-activated transient, depolarizing inward current (I_ti).⁷ Three candidates for I_ti are Na⁺-Ca²⁺ exchange current (I_Na/Ca), Ca²⁺-activated Cl^– current (I_Cl(Ca)), and Ca²⁺-activated nonselective cation current (I_NS(Ca)).^{8 9 10} Although there was early evidence implicating I_NS(Ca) as underlying I_ti,⁸ more recent work has not supported a major role for I_NS(Ca) in I_ti or DADs of ventricular myocytes, favoring instead key roles for I_Cl(Ca) and I_Na/Ca.^{6 9 11 12 13 14} In canine ventricular myocytes, Zygmunt et al⁹ attributed 60% of I_ti to I_Na/Ca and 40% to I_Cl(Ca). The contributions of aforementioned currents to DAD generation may differ from those during an I_ti (where E_m is constant), because E_m changes dynamically to alter the electrochemical driving force (most notably for I_Cl(Ca)) during DADs. One goal of the present study was to measure the relative contribution of I_NS(Ca), I_Cl(Ca), and I_Na/Ca to Ca²⁺-activated depolarizations, leading to triggered APs.

Increasing SR Ca²⁺ load increases spontaneous SR Ca²⁺ release¹⁵ and DAD amplitude toward threshold to trigger an AP, the precursor of triggered arrhythmias.¹⁶ Although DADs are generally accepted to be Ca²⁺-dependent,^{12 17} the relationship between SR Ca²⁺ release and DAD amplitude has not been measured, partly because the underlying Ca²⁺ transients are hard to control.

In the present study, we used caffeine-induced SR Ca²⁺ release to simulate DADs with different [Ca²⁺]_i in a much more controlled manner and measured the resulting depolarization. These caffeine-induced DADs (cDADs) can be initiated at various SR Ca²⁺ loads (eg, by changing frequency), allowing us to measure the [Ca²⁺]_i dependence of DADs and triggered APs over a broad range of [Ca²⁺]_is. Spontaneous SR Ca²⁺ release typically occurs in waves,¹⁷ less synchronized than during application of caffeine or excitation-contraction coupling. Therefore, we injected I_ti-like current and measured membrane depolarization (E_m). These pseudo-I_tis mimic real I_tis but are Ca²⁺-independent. The resulting E_m depends on other membrane properties such as I_K1, the major background current that stabilizes resting E_m. I_K1 is reduced in HF,^{18 19} where arrhythmias are common.

The goals of the present study are to measure, for the first time, (1) the quantitative relationship between the amount of SR Ca²⁺ release and the amplitude of cDADs, (2) the amount of SR Ca²⁺ release (and E_m) required to reach threshold for an AP, (3) the specific contributions of different Ca²⁺-activated currents that cause cDADs, and (4) the effect of different I_ti kinetics on E_m and AP threshold in the absence of [Ca²⁺]_i changes. The results provide the first quantitative data on the basis of DADs and triggered APs in rabbit ventricular myocytes at 37°C.

	Materials and Methods

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Single ventricular myocytes from New Zealand White rabbits were isolated as previously described²⁰ after injection of pentobarbital (50 mg/kg) and heparin. Rabbits were obtained from Myrtle’s Rabbitry (Thompson Station, Tenn) and cared for according to AAALAC guidelines. Myocytes were stored at 22°C and plated later for experiments on laminin pretreated glass-bottomed chambers on the stage of an inverted microscope equipped for epifluorescence.²¹

Cells were loaded at 22°C with indo 1 (acetoxymethylester, 30-minute exposure, 30-minute washout). Indo 1 was excited at 365±25 nm and emitted fluorescence was measured at 405±10 and 485±10 nm. The fluorescence ratio (R=F₄₀₅/F₄₈₅) was translated as [Ca²⁺]_i=K_dß(R-R_min)/(R_max-R),²² where ß is the ratio of maximum to minimum F₄₈₅ (4.4) and K_d was 844 nmol/L.²³ R_min is R at [Ca²⁺]_i<<K_d and R_max is R at saturating [Ca²⁺]_i (0.768 and 8.45 respectively; in vivo).

Whole-cell, ruptured-patch current clamp was used to measure E_m in response to caffeine and current injection. Electrodes (borosilicate) had resistance of 2 to 20 M when filled with (mmol/L) potassium aspartate 120, KCl 8, NaCl 7, HEPES 10, MgCl₂ 1, Mg-ATP 5, Li-GTP 0.3, K₅-indo 1 0.05 (included to prevent indo 1 washout), pH 7.2 adjusted with KOH. Measurements were performed at 36±1°C. Signals were recorded with an Axopatch-1B amplifier (Axon Instruments) and pClamp (6.03) software. A Grass S44 stimulator gated the amplifier for current injection to activate APs. After membrane rupture, capacitance was measured in voltage-clamp mode.²¹ For E_m measurements, current-clamp mode was used, and APs were triggered by a 1- to 5-ms 2-nA current injection.

Normal Tyrode’s (NT) superfusate contained (mmol/L) NaCl 140, KCl 4, glucose 10, HEPES 5, MgCl₂ 2, CaCl₂ 2, pH 7.4 adjusted with NaOH at 37°C. Caffeine (10 mmol/L in NT) was applied by fast solution switching to release SR Ca²⁺. The SR Ca²⁺ load was varied either by AP frequency, rest interval, or partial reloading after a preceding caffeine application. Similar SR Ca²⁺ loads were attained for comparisons in the presence and absence of 50 µmol/L niflumate (to block I_Cl(Ca)), 5 mmol/L Ni²⁺, or 0Na/0Ca solution (replacing Na⁺ with Li⁺ and Ca²⁺ with Mg²⁺ in addition to 10 mmol/L EGTA to block I_Na/Ca). These agents were applied rapidly for 2 seconds before (and during) caffeine application.

For testing I_ti effects on E_m (independent of [Ca²⁺]_i), we applied synthetic current waveforms (dual exponential), with one chosen to match measured I_ti in rabbit myocytes (I_ti,slow). The injected artificial I_ti was varied in amplitude (and kinetics) to define a cell-dependent threshold for triggering an AP. Data are shown as mean±SE, and statistical significance was considered for values of P<0.05 (Student’s t test or ANOVA).

	Results

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Dependence of Depolarization on SR Ca²⁺ Release
SR Ca²⁺ load available for release was varied by changing AP frequency. Figure 1 shows the last steady-state (SS) AP and Ca²⁺ transient at frequencies of 1 to 4 Hz (left). As expected, there is a progressive increase in twitch [Ca²⁺]_i and a shortening of AP duration with increasing frequency. After the last stimulated AP, triggering current was switched off and 10 mmol/L caffeine was applied rapidly to induce Ca²⁺ transients and cDADs (right). With increasing frequency, the amplitude of the caffeine-induced Ca²⁺ transient increased, consistent with the expected increase in SR Ca²⁺ load. For 1- to 3-Hz stimulation, the cDADs remained subthreshold, but E_m changed as [Ca²⁺]_i rose (Figure 1, inset). At 4 Hz, depolarization was sufficient to trigger a regenerative AP with a long, low plateau. The [Ca²⁺]_i decline during this long AP was greatly slowed, presumably attributable to the decrease in driving force for inward I_Na/Ca, the major sarcolemmal Ca²⁺-removal process.²⁴

View larger version (34K): [in this window] [in a new window]   Figure 1. Figure 1. Em and [Ca2+]i in rabbit ventricular myocytes at 37°C. Graph on the left shows the last SS AP and [Ca2+]i transient. After 2 seconds, caffeine-induced [Ca2+]i transients generate subthreshold cDADs (1 to 3 Hz) and a suprathreshold cDAD/AP at 4 Hz. Inset shows that depolarization rises in phase with increasing [Ca2+]i. SS indicates steady state.

Figure 2A shows the mean effect of frequency on cDAD and [Ca²⁺]_i (subthreshold events only). Ca²⁺ transients increased progressively with frequency, and cDAD amplitude also rose with frequency from 6.6±0.8 mV at 0.5 Hz to 14.4±1.2 mV at 3 Hz. In contrast, there was no frequency-dependent change in diastolic [Ca²⁺]_i (range: 154±16 nmol/L at 0.5 Hz to 169±16 nmol/L at 2 Hz; P=0.873, ANOVA) or resting E_m (range: -76.4±0.6 mV at 1 Hz to -77.1±1.1 mV at 3 Hz; P=0.934, ANOVA).

View larger version (28K): [in this window] [in a new window]   Figure 2. Figure 2. A, Subthreshold cDAD amplitude (Em) and [Ca2+]i depend on SS stimulation frequency. Em rises with frequency from 6.6±0.8 to 14.4±1.2 mV and [Ca2+]i from 316±28 to 479±73 nmol/L (*P<0.05 vs 0.5 Hz). B, Exponential [Ca2+]i dependence of subthreshold cDADs [Em=0.4 mV·exp(K · Ca2+)]. Fits for 7 of 20 cells are shown. Mean fit (bold curve) doubles Em [ln(2)/K] for every 88±8 nmol/L [Ca2+]i (range: 21 to 156 nmol/L [Ca2+]i). Threshold [Ca2+]i to trigger an AP was 424±58 nmol/L with Em of 12.5±1.1 mV (n=12).

A wider range of subthreshold Ca²⁺ transients and cDADs were obtained by varying SR Ca²⁺ load by frequency and/or number of conditioning APs. Cells with four or more subthreshold [Ca²⁺]_i-cDAD value pairs were fit to a simple-exponential equation (Figure 2B). In 20 cells, the average [Ca²⁺]_i causing a doubling of E_m was 88±8 nmol/L [Ca²⁺]_i for subthreshold values.

In 12 cells, APs were triggered at higher [Ca²⁺]_i values. Threshold was defined as the highest [Ca²⁺]_i that failed to trigger an AP (424±58 nmol/L [Ca²⁺]_i at E_m=12.5±1.1 mV, Figure 2B; peak [Ca²⁺]_i=608±72 nmol/L and E_m=-64.7±1.2 mV). The first [Ca²⁺]_i to trigger an AP exceeded the threshold by 34±10 nmol/L [Ca²⁺]_i. Thus, the true threshold [Ca²⁺]_i may be slightly above our threshold, but within 34 nmol/L.

Separation of Inward Currents Contributing to the Generation of DAD
Three different Ca²⁺-activated currents (I_Na/Ca, I_Cl(Ca), I_NS(Ca)) could contribute to DADs. Using AP trains that produced comparable caffeine-induced [Ca²⁺]_i, we evaluated the effect of abrupt block of these different currents on the amplitude of subthreshold cDADs. Figure 3A shows that elimination of I_Na/Ca by removal of both extracellular substrates (2 seconds in 0Na/0Ca solution) nearly abolished cDADs. Because [Cl^–] was unchanged, I_Cl(Ca) should be unaffected, and the replacement of Na⁺ with Li⁺ also ensures that I_NS(Ca) is fully functional.²⁵ The half-time of [Ca²⁺]_i decline during caffeine exposure is dramatically slowed in 0Na/0Ca solution (from 120 ms to 1.7 seconds in Figure 3A). This is consistent with I_Na/Ca being responsible for >90% of [Ca²⁺]_i decline in caffeine.²⁰ Moreover, the slower [Ca²⁺]_i decline ought to enhance other Ca²⁺-activated currents (including I_Cl(Ca)), but none was evident. Thus, cDADs seem to depend on I_Na/Ca and not I_Cl(Ca) or I_NS(Ca).

View larger version (23K): [in this window] [in a new window]   Figure 3. Figure 3. cDAD before and after INa/Ca blockade by 0Na/0Ca (A) or 5 mmol/L Ni2+ (B) for 2 seconds. Traces were selected to match [Ca2+]i in a given cell before and after block of INa/Ca (0.5 to 1 Hz SS). Blockade of INa/Ca inhibited cDADs and [Ca2+]i decline in the presence of caffeine.

Ni²⁺ (5 mmol/L) is also commonly used to inhibit I_Na/Ca, although it affects Ca²⁺ and some other currents (but not I_NS(Ca)²⁶ ). Ni²⁺ superfusion for 2 seconds before and during caffeine application also blocked cDADs almost completely (Figure 3B) for a similar [Ca²⁺]_i. With 5 mmol/L Ni²⁺, the [Ca²⁺]_i decline is slowed, but to a lesser extent (3-fold versus >10-fold for 0Na/0Ca solution). This indicates less complete block of I_Na/Ca by 5 mmol/L Ni²⁺ than by 0Na/0Ca solution.

Figure 4A shows that blockade of I_Cl(Ca) by inclusion of 50 µmol/L niflumate before and during caffeine application had only a very small depressant effect on subthreshold cDADs (whereas [Ca²⁺]_i induced by caffeine was virtually identical in both cases). The Cl^– reversal potential (E_Cl) under our experimental conditions (and physiologically) is about -58 mV. Therefore, increased Cl^– conductance at resting E_m would lead to an inward current. However, as depolarization proceeds toward E_Cl (as in a DAD), the driving force for Cl^– will decrease considerably. This and the outward rectification of I_Cl(Ca) may explain why I_Cl(Ca) appears to contribute so little to the cDAD, despite the presence of this current in rabbit ventricular myocytes at 37°C.^{27 28}

View larger version (31K): [in this window] [in a new window]   Figure 4. Figure 4. A, Em and [Ca2+]i during cDAD before and after ICl(Ca) blockade with 50 µmol/L niflumate. Inset, APs recorded ±50 µmol/L niflumate. B, cDAD amplitude from experiments as in Figures 3 and 4A. 0Na/0Ca, Ni2+, and niflumate diminished cDAD amplitude by 93%, 91%, and 3%, respectively (top), for comparable [Ca2+]i (bottom; n=12, 7, and 5; paired).

I_Cl(Ca) is thought to contribute to early repolarization of the ventricular AP. Figure 4A (inset) shows that 50 µmol/L niflumate (as used in the present study) inhibits early repolarization seen as diminished notch in the AP. Niflumate also increased AP duration. These data provide an internal positive control for niflumate effects on I_Cl(Ca).

Figure 4B summarizes the effect of 0Na/0Ca, Ni²⁺, and niflumate on subthreshold cDADs. 0Na/0Ca solution and Ni²⁺ almost completely inhibited cDADs (by 93% and 91%, respectively) for comparable [Ca²⁺]_i (bottom). On the other hand, blockade of I_Cl(Ca) with niflumate did not decrease cDAD amplitude significantly (cDAD remained 97% of control) for matching [Ca²⁺]_i. We infer that I_Na/Ca is almost entirely responsible for cDADs, and by extension DADs, in rabbit ventricular myocytes.

Figure 5 shows that when a cDAD was sufficient to trigger an AP, the AP could be completely blocked by 0Na/0Ca solution (where Li⁺ can carry Na⁺ channel current). However, niflumate did not prevent the triggered AP (although it decreased early repolarization). This confirms that the key current for SR Ca²⁺ release–triggered APs is I_Na/Ca, not I_Cl(Ca) or I_NS(Ca).

View larger version (19K): [in this window] [in a new window]   Figure 5. Figure 5. Suprathreshold cDAD/AP traces. 0Na/0Ca solution prevented cDAD and triggered APs, whereas blockade of ICl(Ca) (niflumate) had little effect.

Membrane Response to Current Injection at Resting Membrane Potential
To test the Ca²⁺-independent effects of I_ti kinetics on membrane depolarization in a controlled manner, we injected inward currents mimicking Ca²⁺-activated I_ti of different amplitudes and kinetics. We used three scalable current injection templates (see Figure 6A). The slowest (I_ti,slow) resembles a measured I_ti, which is produced by a spontaneous [Ca²⁺]_i wave traveling through the cell. The faster time courses (I_ti,mid; I_ti,fast) resemble the kinetics of I_tis with greater synchronization of SR Ca²⁺ release (as induced by rapid caffeine application). These pseudo-I_tis allowed us to simulate, in a controlled manner, the depolarizing impact expected during Ca²⁺ waves where I_Na/Ca (or I_ti) is more spread out in time as [Ca²⁺]_i rises sequentially in different cellular regions.

View larger version (27K): [in this window] [in a new window]   Figure 6. Figure 6. DADs induced by pseudo-Itis. Three different current waveforms were used to simulate Itis (Iti,fast, Iti,mid, and Iti,slow). Synthetic current waveforms in panel A were defined by Y{[1-exp(-t/up)]m}[exp(-t/dn)] (Table), where Y was varied to change current integrals. A, Em responses to pseudo-Itis chosen to have equal integrals. B, Mean exponential fits (see Figure 2B) for the 3 waveforms. Symbols show typical data (from n5) for Iti,fast (filled symbols), Iti,slow (open symbols), and Iti,mid (half-filled symbols). Results are in the Table.

Figure 6A (top) shows the E_m response to these three inward current waveforms (for equal current integrals). Current amplitude was varied over a broad range until an AP was triggered. Figure 6B shows the relationship between integrated injected current, normalized to cell capacitance, and E_m. Exponential fits yielded a doubling of E_m every 0.128 C/F for I_ti,fast (n=7), whereas the I_ti,slow required 0.416 C/F to double E_m (n=17, see Table). In addition, I_ti,slow required 3 times as much charge as I_ti,fast for a given E_m. The average charge necessary to trigger an AP also increased progressively from 0.47 C/F for I_ti,fast to 1.69 C/F for I_ti,slow (Table). Threshold E_m was slightly, but not significantly, more positive in the case of I_ti,slow versus I_ti,fast (-59±1 versus -63±3 mV).

View this table: [in this window] [in a new window]   Table 1. Parameters for DADs Induced by Pseudo-Itis

The integrated I_ti,mid can be converted to an equivalent Ca²⁺ flux via I_Na/Ca. That is, 0.89 C/F corresponds to I_Na/Ca Ca²⁺ extrusion of 59.6 µmol/L cytosol (assuming a surface-to-volume ratio of 6.44 pF/pL cytosol),²⁹ requiring SR Ca²⁺ release of 64 µmol/L cytosol (assuming 93% of released Ca²⁺ is extruded by I_Na/Ca²⁰ ). Taking cytosolic Ca²⁺ buffering into account,³⁰ [Ca²⁺]_i would be raised by 428 nmol/L. This is in remarkable agreement with the [Ca²⁺]_i threshold for triggering an AP via cDAD, on the basis of data in Figure 2B (424 nmol/L).

	Discussion

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In the present study, we characterized quantitatively for the first time the relationship between [Ca²⁺]_i transients and membrane depolarization in a setting that mimics DADs, an important precursor of triggered arrhythmias. We show that DADs are almost entirely due to I_Na/Ca, whereas I_Cl(Ca) and I_NS(Ca) play little or no role in the generation of Ca²⁺-induced E_m at resting E_m.

Mechanism Underlying DADs: Ca²⁺-Activated Currents
Several Ca²⁺-activated currents have been proposed to participate in I_ti and DADs, namely I_Na/Ca, I_Cl(Ca) and I_NS(Ca).^{7 10 27 31} Our data clearly indicate that in rabbit ventricular myocytes at 37°C, DADs are almost entirely attributable to I_Na/Ca (>90%) with <10% attributable to I_Cl(Ca) and no evidence for contribution from I_NS(Ca).

Many I_ti studies use voltage clamp, where E_m is held constant. This has an inherent mechanistic bias and may not properly estimate relative current contributions to DADs (as measured in the present study). For example, with E_Cl=-58 mV under physiological conditions, an E_m change from -78 to -68 mV during a DAD would reduce the driving force for I_Cl(Ca) by 50%, leading to overestimation of the I_Cl(Ca) contribution in a voltage-clamp experiment. In contrast, E_Na/Ca is -30 mV at rest in rabbit, but as [Ca²⁺]_i rises, E_Na/Ca becomes more positive (eg, +10 mV) greatly enhancing the driving force for inward I_Na/Ca, more than offsetting the E_m-induced reduction of I_Na/Ca driving force.²⁴ Allosteric activation of I_Na/Ca by [Ca²⁺]_i³² could further stimulate inward I_Na/Ca.

In voltage-clamped rabbit ventricular myocytes, Zygmunt and Gibbons²⁷ showed the existence of a strongly outward rectifying I_Cl(Ca) in the absence of I_Na/Ca at 22°C. They used step depolarizations and excitation-contraction coupling to evoke [Ca²⁺]_i transients. Because of the highly synchronized local SR Ca²⁺ release, this would be especially effective in activating Ca²⁺-dependent currents. Laflamme and Becker¹³ confirmed a strongly outward rectifying I_Cl(Ca) even during spontaneous SR Ca²⁺ release in rabbit ventricular myocytes with no evidence of I_NS(Ca) (again with I_Na/Ca blocked and at 22°C). A marked increase of I_Cl(Ca) can be seen at 35°C versus 25°C.²⁸ These characteristics of I_Cl(Ca) do support its apparent contribution to I_to in APs (Figure 4A), despite its minor role (<10%) in the generation of DADs in our experiments (Figure 4B). These studies^{13 27 28} did not find any indication of I_NS(Ca) contributing to I_ti. In contrast, Wu and Anderson³³ observed a residual oscillatory current after blockade of both I_Na/Ca and I_Cl(Ca), which was sensitive to the removal of extracellular cations. Although I_NS(Ca) may exist in rabbit myocytes, we find no evidence for its participation in DADs. Our findings are in contrast to a study by Szigeti et al,¹⁴ who inferred that I_Cl(Ca) was the dominant Ca²⁺-dependent inward current in rabbit ventricular, atrial, and Purkinje cells. In dog ventricular myocytes, I_ti appears to be carried almost equally by I_Na/Ca (60%) and I_Cl(Ca) (40%).⁹ This could be a species difference or due to their rapid SR Ca²⁺ release. The highly synchronized SR Ca²⁺ release would produce higher local [Ca²⁺]_i, which could better activate I_Cl(Ca) (apparent K_d=150 µmol/L [Ca²⁺]_i).³⁴ On the basis of hysteresis loops of [Ca²⁺]_i versus I_Na/Ca or I_Cl(Ca), Trafford et al³⁵ inferred a closer physical location of I_Cl(Ca) to the ryanodine receptor than for I_Na/Ca in ferret myocytes.

Relationship of [Ca²⁺]_i Transient and DAD
DADs are triggered depolarizations, seen in SR Ca²⁺ overload, with spontaneous SR Ca²⁺ release being the underlying event. In the present study, we used more controlled caffeine-induced SR Ca²⁺ release (cDADs) and sacrificed the normal spontaneous nature of DADs. However, this control allowed systematic quantitative analysis of the Ca²⁺ dependence of depolarization over a broad [Ca²⁺]_i range, which cannot be achieved by spontaneous SR Ca²⁺ release. This also provides SR Ca²⁺ load data that would not be available during propagating Ca²⁺ waves associated with the spontaneous Ca²⁺ release of Ca²⁺ overload. The trade-off we make for these big advantages is that we must separately consider the impact on E_m of spreading out the Ca²⁺-induced current in time (as in slower Ca²⁺ waves). The current injection experiments in Figure 6 address this. That is, we cannot spread the Ca²⁺ transient in time (in a controlled and measured way), but we can do this with the resulting current (as pseudo I_tis) to simulate a Ca²⁺ wave–induced DADs.

I_Na/Ca is approximately linear as a function of [Ca²⁺]_i,³⁶ but the amount of depolarization produced is nonlinear because of interactions with other currents (eg, I_K1, I_Na) and membrane properties. However, we focus on the integrated E_m response because depolarization is the immediate cause of triggered APs due to SR Ca²⁺ release. An exponential equation describes well this Ca²⁺ dependence of E_m (with a doubling of DAD for every 88 nmol/L rise in [Ca²⁺]_i).

The threshold of SR Ca²⁺ release that raises [Ca²⁺]_i by 424 nmol/L, equivalent to an integrated I_Na/Ca of 0.89 C/F, is sufficient to trigger an AP with a threshold E_m of -65±1 mV. This [Ca²⁺]_i requires a total SR Ca²⁺ release of 50 to 60 µmol/L cytosol, 50% to 70% of the SR Ca²⁺ load. It is likely that at least this amount of Ca²⁺ is released during a spontaneous Ca²⁺ release under Ca²⁺-overload conditions.¹² However, several factors may limit this from triggering an AP in a normal cell. First, spontaneous SR Ca²⁺ release normally occurs as a wave,^{15 37} with [Ca²⁺]_i not rising synchronously and consequently leading to a slower rise in Ca²⁺-activated currents. Taking this into account 3 times more [Ca²⁺]_i may be required to trigger an AP under these conditions (on the basis of Figure 6). Second, even if all the SR Ca²⁺ is released during a wave, only 15% to 20% of the SR Ca²⁺ load is extruded from the cell via I_Na/Ca,^{12 38} because the SR can reaccumulate Ca²⁺ after the release channels close (unlike in our cDADs, where caffeine is present). This may limit the integrated I_Na/Ca more than peak I_Na/Ca but may nonetheless reduce the efficacy of a DAD leading to a triggered AP. Thus, the normal ventricular myocyte may have a reasonable safety margin of 3- to 4-fold against any SR Ca²⁺ release being able to trigger an AP.

An additional safety factor in the whole heart is that neighboring cells will act as current sinks, blunting the E_m effect for a given local I_Na/Ca. However, cellular changes that cause either more SR Ca²⁺ release or greater local depolarization for a given [Ca²⁺]_i increase propensity for triggered arrhythmias. That is, there would be greater chance for a cell cluster that is local enough, synchronous enough, and large enough to overcome the 3-dimensional current sink problem and trigger a propagating arrhythmia.

Possible Arrhythmogenic Role in HF
In HF, Na-Ca²⁺ exchange protein and I_Na/Ca can be doubled and I_K1 is reduced by up to 50%,^{2 18 19 39 40 41} and may overcome the safety factor above. That is, doubling I_Na/Ca will double I_ti amplitude for any given SR Ca²⁺ release and reduction of I_K1 by 50% will allow a given I_ti to be more effective in depolarizing the cell toward threshold for a triggered AP. Although SR Ca²⁺ load may be low in HF, adrenergic activity may increase Ca²⁺ load, allowing spontaneous Ca²⁺ release. Consequently, HF may greatly increase the propensity for DADs to trigger APs, with 4-fold less [Ca²⁺]_i being sufficient to trigger an AP (as seen in computer models).⁴¹ Because triggered arrhythmias cause the majority of sudden cardiac death in nonischemic HF,¹ it would be of vital interest to measure the [Ca²⁺]_i dependence of E_m and the threshold in HF to test the above-mentioned working hypothesis.

	Acknowledgments

 

This work was supported by grants from the US Public Health Service (HL-30077, HL-64724) and the Deutsche Forschungsgemeinschaft (Schl 485/2-1). The authors thank Drs S.M. Pogwizd and K.S. Ginsburg for stimulating discussions during this work.

Received June 13, 2000; revision received August 18, 2000; accepted September 5, 2000.

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