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(Circulation. 2000;101:1441.)
© 2000 American Heart Association, Inc.

Basic Science Reports

KB-R7943 Block of Ca²⁺ Influx Via Na⁺/Ca²⁺ Exchange Does Not Alter Twitches or Glycoside Inotropy but Prevents Ca²⁺ Overload in Rat Ventricular Myocytes

Hiroshi Satoh, MD, PhD; Kenneth S. Ginsburg, PhD; Ke Qing, PhD; Hajime Terada, MD, PhD; Hideharu Hayashi, MD, PhD; Donald M. Bers, PhD

From the Third Department of Internal Medicine (H.S., K.Q., H.T.) and Photon Medical Research Center (H.H.), Hamamatsu University School of Medicine, Hamamatsu, Japan, Department of Physiology (K.S.G., D.M.B.), Loyola University Chicago, Maywood, Ill.

Correspondence to Hiroshi Satoh, MD, PhD, Third Department of Internal Medicine, Hamamatsu University School of Medicine, 3600 Handa-Cho, Hamamatsu 431-3192, Japan. E-mail satoh36{at}hama-med.ac.jp

	Abstract

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Background—The Na⁺/Ca²⁺ exchange (NCX) extrudes Ca²⁺ from cardiac myocytes, but it can also mediate Ca²⁺ influx, load the sarcoplasmic reticulum with Ca²⁺, and trigger Ca²⁺ release from the sarcoplasmic reticulum. In ischemia/reperfusion or digitalis toxicity, increased levels of intracellular [Na⁺] ([Na⁺]_i) may raise levels of intracellular [Ca²⁺] ([Ca²⁺]_i) via NCX, leading to cell injury and arrhythmia.

Methods and Results—We used KB-R7943 (KBR) to selectively block Ca²⁺ influx via NCX to study the role of NCX-mediated Ca²⁺ influx in intact rat ventricular myocytes. Removing extracellular Na⁺ caused [Ca²⁺]_i to rise, due to Ca²⁺ influx via NCX, and this was blocked by 90% with 5 µmol/L KBR. However, KBR did not alter [Ca²⁺]_i decline due to NCX. Thus, we used 5 µmol/L KBR to selectively block Ca²⁺ entry but not efflux via NCX. Under control conditions, 5 µmol/L KBR did not alter steady-state twitches, Ca²⁺ transients, Ca²⁺ load in the sarcoplasmic reticulum, or rest potentiation, but it did prolong the late low plateau of the rat action potential. When Na⁺/K⁺ ATPase was inhibited by strophanthidin, KBR reduced diastolic [Ca²⁺]_i and abolished the spontaneous Ca²⁺ oscillations, but it did not prevent inotropy.

Conclusions—In rat ventricular myocytes, Ca²⁺ influx via NCX is not important for normal excitation-contraction coupling. Furthermore, the inhibition of Ca²⁺ efflux alone (as [Na⁺]_i rises) may be sufficient to cause glycoside inotropy. In contrast, Ca²⁺ overload and spontaneous activity at high [Na⁺]_i was blocked by KBR, suggesting that net Ca²⁺ influx (not merely reduced efflux) via NCX is involved in potentially arrhythmogenic Ca²⁺ overload.

Key Words: myocytes • ion exchange • sarcoplasmic reticulum • arrhythmia

	Introduction

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Na⁺/Ca²⁺ exchange (NCX) is the main mechanism of Ca²⁺ extrusion from cardiac myocytes, and 7% to 28% of cardiac relaxation is due to NCX.^{1 2 3} Less quantitative information is available concerning Ca²⁺ influx via the NCX. On the basis of thermodynamic considerations, Ca²⁺ influx via NCX is most likely to occur during the very early phase of the action potential (AP).¹ Normally, little Ca²⁺ enters via the NCX, but Ca²⁺ entry can increase greatly when intracellular [Na⁺] ([Na⁺]_i) increases; this can occur as a result of Na⁺/K⁺ ATPase inhibition or during ischemia and reperfusion.^{4 5}

Ca²⁺ entry via NCX during the AP, although normally small, could be sufficient to trigger Ca²⁺ release from the sarcoplasmic reticulum (SR), because depolarization increases the driving force for Ca²⁺ entry via NCX.^{6 7 8 9 10} Ca²⁺ entry can also occur when the Na⁺ current causes local subsarcolemmal [Na⁺]_i to rise.^{11 12} However, the physiological relevance of SR Ca²⁺ release triggered by NCX is controversial.^{13 14}

Inhibition of Na⁺/K⁺ ATPase by cardiac glycosides causes [Na⁺]_i to increase, resulting in increased cell Ca²⁺ load via NCX. Positive inotropy or even Ca²⁺ overload and arrhythmias can result.¹ Strophanthidin increases intracellular [Ca²⁺] ([Ca²⁺]_i) and Ca²⁺ transients as [Na⁺]_i rises; when arrhythmias occur, Ca²⁺ transient amplitude decreases while [Na⁺]_i and basal [Ca²⁺]_i continue to increase.¹⁵ An increase of [Ca²⁺]_i in response to increased [Na⁺]_i can occur because either Ca²⁺ efflux via the NCX is reduced (failing to match Ca²⁺ influx) or [Na⁺]_i levels are high enough (NCX mediates net Ca²⁺ influx).

KB-R7943 (KBR) is a novel agent that reportedly preferentially blocks the Ca²⁺ influx mode of the cardiac NCX rather than the Ca²⁺ extrusion mode.^{16 17} We used this property of KBR to examine the likely role of Ca²⁺ influx via NCX in cellular Ca²⁺ handling during excitation-contraction (E-C) coupling and during the genesis of strophanthidin-induced inotropy and Ca²⁺ overload. KBR had no effect on normal E-C coupling, but it blocked Ca²⁺ entry via NCX. KBR also blocked the spontaneous activity caused by strophanthidin-induced increased [Na⁺]_i, without preventing the inotropy. Thus, inotropy can be due to a reduction of Ca²⁺ efflux via NCX, whereas Ca²⁺ overload may require net Ca²⁺ influx.

	Methods

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Preparation of Ventricular Myocytes
Ventricular myocytes were isolated from male Sprague-Dawley rats (weighing 200 to 240 g) and loaded with indo 1-acetoxymethylester at 23°C.¹⁸ Then, they were placed in a chamber on the stage of an inverted microscope (TMD, Nikon) with a perfusate containing (in mmol/L): NaCl 137, KCl 4, MgSO₄ 1.2, glucose 10, HEPES 10, and CaCl₂ 1.5 (pH adjusted to 7.4 with NaOH). Myocytes were field-stimulated through Ag-AgCl electrodes at 0.5 Hz.

Apparatus
The indo-1 fluorescence ratio (405/485 nm emission with 340 nm excitation)² was used after background subtraction to measure [Ca²⁺]_i using the following formula:

R_max and R_min (maximum and minimum indo-1 fluorescence ratios) were determined in cells exposed to ionomycin (10 µmol/L) and either CaCl₂ (5 mmol/L) or EGTA (10 mmol/L), respectively. K_d equaled 844 nmol/L,¹⁹ and S_f/S_b, the ratio of Ca-free to Ca-bound 485-nm fluorescence, was 1.096. Contraction was measured using an edge detection system (Hamamatsu Photonics) with transilluminated red light (>600 nm).

AP Recording and Analysis
An Axopatch 200A amplifier (Axon Instruments) was used in current-clamp mode to record APs in a perforated patch configuration (extracellular Ca²⁺ concentration was 1 mmol/L). Pipettes (1 to 1.5 megohms) were filled with the following (in mmol/L): KCl 40, K-glutamate 80, NaCl 0 or 10, and HEPES 10 (pH 7.2); amphotericin B (120 µg/mL) was added only in the backfill. APs were evoked by injecting a depolarizing current (0.5 ms, 1.25xthreshold) via the recording electrodes (access resistance, 4 to 7 megohms). AP durations at 50% and 90% repolarization (APD₅₀ and APD₉₀) were measured from synchronized averages of 10 to 20 steady-state APs.

Test Reagent
The KBR was a gift from the New Drug Research Laboratories, Kanebo Co, Ltd (Osaka, Japan), and stock solution was dissolved in dimethyl sulfoxide at 10 mmol/L. The final concentration of dimethyl sulfoxide in 5 or 10 µmol/L KBR was 0.1%.

Statistical Analyses
Results were expressed as mean±SEM for the number of isolated myocytes. Student’s t test or 1-way ANOVA were used for analyses; P<0.05 was considered significant.

	Results

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KBR Blocks Ca²⁺ Influx but Not Ca²⁺ Efflux
For the control experiments (shown in Figure 1) only, SR function was completely blocked by pretreatment with 10 µmol/L ryanodine and 1 µmol/L thapsigargin.

View larger version (31K): [in this window] [in a new window]   Figure 1. KBR reduces Ca2+ influx but not Ca2+ efflux via NCX in intact rat ventricular myocytes. A, Ca2+ influx via NCX, induced by rapid switch to Na+-free solution for 15 s, was blocked by KBR. B, KBR did not alter [Ca2+]i decline during twitch Ca2+ transients due to NCX (in which SR function was abolished by 1 µmol/L thapsigargin and 10 µmol/L ryanodine). Ca2+ influx was increased by administration of 100 nmol/L BayK 8644. indicates time constant of [Ca2+]i decline. C, Block of Ca2+ influx was quantified by % inhibition of 0Na-induced rise in [Ca2+]i and block of Ca2+ efflux was measured as % change of rate constant of [Ca2+]i decline (1/). Results are mean±SEM from 5 to 8 experiments.

To assess Ca²⁺ influx via NCX, extracellular Na⁺ was abruptly removed (replaced by tetramethylammonium) for 15 s, which changed the thermodynamic driving force on NCX to favor Ca²⁺ influx. Figure 1A shows that this caused [Ca²⁺]_i to rise continuously, as was expected for Ca²⁺ entry via NCX. When extracellular Na⁺ was returned, [Ca²⁺]_i recovered with a similar time course. KBR dose-dependently inhibited the Na-free–induced increase in [Ca²⁺]_i (measured at 15 s). Washout of KBR effects was slow (>30 minutes) and often incomplete.

To test KBR effects on Ca²⁺ efflux via NCX, twitches were activated so that [Ca²⁺]_i decline was almost entirely due to NCX.² For this experiment (without SR function), Ca²⁺ influx via sarcolemmal Ca²⁺ channels was increased with 100 nmol/L BayK 8644 (Figure 1B). Twitch [Ca²⁺]_i declined with a time constant of 1.6 s, as previously reported in rat ventricular myocytes.² KBR (even 10 µmol/L) did not change the time constant of the Ca²⁺ decline.

Figure 1C shows that with 5 µmol/L KBR, Ca²⁺ influx via NCX was strongly inhibited (to 9.9±6.6% of control [Ca²⁺]_i rise), whereas Ca²⁺ efflux via NCX was unaffected (111±13% of control rate constant of [Ca²⁺]_i decline). Similar control data were obtained at 36°C, where 5 µmol/L KBR depressed peak [Ca²⁺]_i on Na⁺ removal (to 19.9±6.4% of control, n=3) but left the NCX-mediated rate of [Ca²⁺]_i decline unchanged (94±16% of control, n=4). We concluded that 5 µmol/L KBR could be used to selectively inhibit Ca²⁺ influx via NCX in subsequent experiments.

KBR Does Not Affect Twitch Contractions or Ca²⁺ Transients at 23°C
To test whether Ca²⁺ influx via NCX is physiologically important, we measured the effects of KBR on 0.5-Hz twitch contractions, both steady-state (SS) and after 30 s of rest (PR), and Ca²⁺ transients. Figure 2 shows that 5 µmol/L KBR (given over 8 minutes) did not alter twitch Ca²⁺ transients or contractions (whether SS or PR, n=12; see the Table).

View larger version (21K): [in this window] [in a new window]   Figure 2. KBR does not affect SS or PR twitches or [Ca2+]i transients. A, Simultaneous records of shortening and [Ca2+]i during SS and PR twitches±KBR at room temperature (23°C). B, Time courses of PR potentiation of twitch contractions in control () and with 5 µmol/L KBR (•). Results are mean±SEM from 4 paired experiments (P=NS by ANOVA).

View this table: [in this window] [in a new window]   Table 1. Effects of KBR on SS, PR Twitches and SR Ca2+ Load at 23°C and 36°C

Figure 3A shows that although peak twitch [Ca²⁺]_i was not altered by KBR, [Ca²⁺]_i decline was slightly slowed (P<0.01, paired t test, n=12 cells; see the Table). This slowing of twitch [Ca²⁺]_i decline with KBR was not seen when cells were depleted of [Na⁺]_i and studied with Li⁺ replacing extracellular Na⁺ (thereby blocking NCX; data not shown). This indicates that NCX is responsible for the slower twitch [Ca²⁺]_i decline with KBR. Without external Na⁺, [Ca²⁺]_i decline is almost entirely due to the SR Ca-ATPase,² so this result also demonstrates that SR Ca²⁺ transport is not altered by KBR.

View larger version (18K): [in this window] [in a new window]   Figure 3. SS twitch and caffeine-induced Ca2+ transient amplitudes are unaltered by KBR. A, SS twitch Ca2+ transients; B, caffeine-induced Ca2+ transients±KBR at 23°C. TTP indicates time to peak [Ca2+]i; and , time constant of [Ca2+]i decline. In B, perfusate [Ca2+] was reduced to 0.8 mmol/L to prevent Ca2+ overload.

Caffeine-induced contractures and Ca²⁺ transients were also used to assess SR Ca²⁺ content (Figure 3B). Caffeine (10 mmol/L) was rapidly applied in Na⁺-free (tetramethylammonium-substituted), Ca²⁺-free, 1 mmol/L EGTA solution (to block NCX). KBR (5 µmol/L) did not affect the amplitude (Table) or other properties (data not shown) of caffeine-induced Ca²⁺ transients (n=8 cells).

KBR Prolongs the Low Plateau of the AP
Figures 4A and 4B show that APs, recorded in SS at 0.5 Hz, were reversibly prolonged by KBR in the late low plateau phase (when Ca²⁺ influx via NCX is not expected). Moreover, the same prolongation of the low plateau was still seen whether pipette [Na⁺] was 0 or 10 mmol/L (Figure 4, A versus B). This suggests that the AP prolongation was not due to altered NCX current. The APD₅₀ was not significantly changed by KBR (n=8), whereas the APD₉₀ was increased by KBR from 74.5±10.0 ms to 123.8±17.5 ms (P<0.05). These effects began in several seconds, maximized within 3 to 4 minutes, and were reversible with a similar time course. The resting potential (-79.6±2.6 mV) depolarized by 0.96 mV with KBR in 8 cells (not significant).

View larger version (16K): [in this window] [in a new window]   Figure 4. KBR lengthens only low plateau of rat ventricular AP. APs were evoked by injecting inward current at 0.5 Hz and recorded in whole cell perforated patch at 23°C. A and B, APs with 10 mmol/L and 0 mmol/L pipette [Na+], respectively (latter blocks NCX-mediated Ca2+ influx), ±5 µmol/L KBR. C, Mean APD50 and APD90. Results are mean±SEM from 8 experiments. *P<0.05 by paired t test.

KBR Does Not Affect Twitch Contractions or Ca²⁺ Transients at 36°C
NCX current is temperature-dependent,²⁰ and the role of Ca²⁺ entry via NCX in triggering SR Ca²⁺ release may be greater at 37°C than at 23°C to 25°C.^{8 9} Therefore, we repeated the above experiments at 36°C, but KBR still failed to change the amplitudes of Ca²⁺ transients or twitches, either SS or PR (Table).

These data suggest that under physiological conditions in rat ventricular myocytes, Ca²⁺ influx via NCX does not modulate SS contractions. Our data further argue against the hypothesis that the PR potentiation observed in the rat (and some other species) is due to Ca²⁺ influx via NCX. The data do not address Ca²⁺ efflux via the NCX, because 5 µmol/L KBR did not affect NCX-mediated Ca²⁺ extrusion.

KBR Reduces Spontaneous Activity During Na⁺ Loading
Figure 5 shows the effect of 50 µmol/L strophanthidin on cell contraction and [Ca²⁺]_i during SS 0.5 Hz stimulation. Blocking Na⁺/K⁺ ATPase with strophanthidin should cause [Na⁺]_i to rise gradually, shifting the thermodynamic balance on NCX toward Ca²⁺ entry. In all 16 cells studied, strophanthidin increased both twitch contraction and Ca²⁺ transient amplitudes. In 4 of these cells (Figure 5), spontaneous contractions and [Ca²⁺]_i oscillations occurred within 10 minutes, indicating Ca²⁺ overload; also, in these cells, twitch contractions decreased (Figure 5Ac). In all 4 of these cells, 5 µmol/L KBR abolished the spontaneous activity (Figure 5Bd) but left the strophanthidin-induced inotropy largely intact (compare Figure 5B, b and d). KBR also partially restored diastolic cell length.

View larger version (19K): [in this window] [in a new window]   Figure 5. KBR blocks strophanthidin-induced arrhythmia but not inotropy. A, Continuous recording of twitch cell shortening from typical experiment with 50 µmol/L strophanthidin and 5 µmol/L KBR added as indicated by bars. B, Ca2+ transients recorded during control perfusion (a), 5 minutes after starting strophanthidin perfusion (b), when arrhythmia appeared (11 minutes, indicated by bar; c), and 3 minutes after addition of KBR (d).

In the 12 cells that did not show spontaneity, KBR reduced Ca²⁺ transients toward predrug control amplitude in only 3 cells; the same inotropic state was sustained in the other 9 cells. Furthermore, as shown in Figure 6, the application of 5 µmol/L KBR 5 minutes before strophanthidin administration did not prevent the increase in amplitude of twitch contractions and Ca²⁺ transients, and Ca²⁺ overload was not observed.

View larger version (33K): [in this window] [in a new window]   Figure 6. Pretreatment with KBR does not prevent strophanthdin-induced inotropy. Simultaneous records of SS contractions and Ca2+ transients (23°C) for cell preincubated with KBR for 5 minutes, then exposed to strophanthidin in continuous presence of KBR.

The failure of KBR to block strophanthidin inotropy suggests that Ca²⁺ influx via NCX is not essential for the positive inotropic effect of strophanthidin. That is, simply reducing Ca²⁺ efflux (due to increased [Na⁺]_i) is sufficient. However, insofar as spontaneous activity is a marker of a transition to Ca²⁺ overload in response to elevated [Na⁺]_i, the block of all spontaneous activity by KBR suggests that Ca²⁺ influx via NCX may cause the overload.

	Discussion

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KBR as a Tool to Study NCX
KBR can selectively inhibit Ca²⁺ influx (versus efflux) via NCX under appropriate conditions. In guinea pig ventricular myocytes, KBR inhibited 50% of outward and inward NCX current at 0.3 and 17 µmol/L, respectively.¹⁶ Directional selectivity was less in sarcolemmal vesicles,¹⁷ and there may be extracellular Ca²⁺ sensitivity to the block.^{17 21 22} Although the mechanism of directional selectivity is not clear, Figure 1 shows the selective block of Ca²⁺ influx via NCX by 5 µmol/L KBR under our conditions.

Ca²⁺ Influx Via NCX and E-C Coupling
The lack of effect of KBR on normal E-C coupling (Table and Figures 2 through 4) is not consistent with a role for Ca²⁺ entry via NCX. Compared with the L-type Ca²⁺ channel, NCX mediates Ca²⁺ entry, which is smaller and slower, making it a less efficient trigger for SR Ca²⁺ release, even for a given Ca²⁺ influx.¹⁴ This may be because (1) NCX does not couple tightly with the SR Ca²⁺ release channel, as does the L-type Ca²⁺ channel, and (2) 1000 NCX molecules are required to produce the flux of one L-type Ca²⁺ channel. We conclude that the role of Ca²⁺ entry via NCX is minimal in rat ventricular E-C coupling.

Ca²⁺ entry via NCX can occur during the cardiac AP upstroke, when the membrane potential (E_m) transiently exceeds the NCX reversal potential.²³ Indeed Ca²⁺ can enter via NCX during depolarizations and can trigger SR Ca²⁺ release,^{1 4 6 7 9 10} although evidence is most compelling when the Ca²⁺ current is blocked. If an L-type Ca²⁺ channel opens, local [Ca²⁺]_i around the NCX will rapidly become high enough to prevent net Ca²⁺ entry by NCX. This occurs very early in the AP because of the rapid activation of Ca²⁺ channels. Tetrodotoxin-sensitive Na⁺ influx could raise subsarcolemmal [Na⁺]_i sufficiently to promote Ca²⁺ entry via NCX and trigger Ca²⁺ release,^{11 12} but this remains controversial.^{13 24 25}

Lower temperature reportedly limits the activation of SR Ca²⁺ release by NCX^{9 26} ; however, we found no E-C coupling depression at 23°C or 36°C. Species differences could alter the effectiveness of Ca²⁺ entry via NCX. This remains possible: we found KBR (5 µmol/L) slightly reduced contractions and Ca²⁺ transients in guinea pig myocytes (unpublished data). In transgenic mice overexpressing NCX current 3-fold, evidence supporting NCX-induced SR Ca²⁺ release is conflicting.^{27 28}

A limitation in previous work was a difficulty in inhibiting Ca²⁺ entry via NCX without altering Ca²⁺ current. KBR may be an imperfect agent, but our results in rat ventricular myocytes clearly indicate that no E-C coupling change occurred when blocking Ca²⁺ entry via NCX.

Potentiation of PR contractions and Ca²⁺ transients in the rat²⁹ could result from Ca²⁺ entry via NCX loading SR, secondary to [Na⁺]_i elevation, after a train of stimuli.^{1 23} Resuming stimulation could then give a negative staircase as cell and SR Ca²⁺ content decline.^{1 3 23} Because PR potentiation was unaltered by KBR (Figure 2A and the Table), Ca²⁺ influx via NCX does not explain PR potentiation. PR potentiation also occurs without increased SR Ca²⁺ load or Ca²⁺ current.^{29 30} This is likely due to a slow recovery of SR Ca²⁺ release channels after an activated release.^{29 31 32}

Changes in AP
KBR lengthened the low AP plateau (Figure 4), reflecting either less outward current and/or greater inward current. Although the inhibition of outward NCX current by KBR is in the correct direction, it is unlikely that NCX current is outward during the low plateau of the AP (or at rest).¹ Moreover, because these same KBR effects were seen with Na⁺-free pipette solution, NCX is probably not involved.

Although KBR blocks outward NCX current, it blocks other currents at higher concentrations.¹⁶ Preliminary perforated patch voltage clamp recordings made during our AP recordings suggest some decrease in composite outward K⁺ and inward Ca²⁺ currents (data not shown). More work is needed to clarify which currents cause the AP changes, but modest inhibition of K⁺ current could explain both a depolarized resting E_m and the longer AP.

Prolongation of the late phase of the AP with KBR is unlikely to alter SR Ca²⁺ release because (1) Ca²⁺ release is independent of duration after 20 ms^{1 33 34} and (2) the AP differences are at an E_m that does not activate SR Ca²⁺ release. However, the slower repolarization with KBR could contribute to the slower twitch [Ca²⁺]_i decline, because Ca²⁺ extrusion by NCX is slower at a more positive E_m (and the NCX reversal potential is rather negative in the rat).¹ This could slow Ca²⁺ extrusion, even if the total amount extruded was unchanged (such that SR Ca²⁺ content is unchanged, as observed).

In some cells, KBR reduced the peak of the AP (Figure 4A). This could be explained by a small reduction in Na⁺ current,¹⁶ which could also explain the higher AP threshold with KBR (data not shown).

Ca²⁺ Influx Via NCX Under Na⁺ loading: Inotropy and Ca²⁺ Overload
Na⁺/K⁺ pump blockade by cardiac glycosides increases [Na⁺]_i, which enhances contractility by increasing cellular Ca²⁺ due to NCX.^{1 15} This could simply be caused by reduced Ca²⁺ efflux by the NCX as [Na⁺]_i rises. That is, less Ca²⁺ extrusion for a given Ca²⁺ influx would increase cell Ca²⁺. The increased [Na⁺]_i could also increase Ca²⁺ influx via NCX. The apparent preferential block of Ca²⁺ influx via NCX by KBR allows unique insights into this functional distinction.

Our results indicate that Ca²⁺ influx via NCX is not important under normal conditions in the rat ventricle, although resting [Na⁺]_i in the rat is high compared with the rabbit or guinea pig ventricle.^{23 35} However, as [Na⁺]_i increases, Ca²⁺ influx is more favored and Ca²⁺ efflux is less favored thermodynamically. The ability of KBR to block oscillations attributable to Ca²⁺ overload, without preventing the inotropic effect of strophanthidin, leads us to propose that inhibiting Ca²⁺ efflux via NCX is sufficient to produce the inotropic effect of cardiac glycosides. In contrast, the arrhythmogenic effects of glycosides may depend on [Na⁺]_i rising high enough to cause the net Ca²⁺ entry via NCX to be favored.

For a resting [Na⁺]_i level of 12 to 14 mmol/L and a [Ca²⁺]_i level of 100 nmol/L, the predicted NCX reversal potential is -67 to -79 mV; thus, Ca²⁺ efflux at rest is slightly favored. If [Na⁺]_i rose by just 3 mmol/L, the reversal potential would become -85 to -95 mV, negative to resting E_m and favoring net Ca²⁺ influx at rest and Ca²⁺ overload. Spontaneous Ca²⁺ release at the resting E_m would drive Ca²⁺ efflux (lessening Ca²⁺ overload),³⁶ but also produce transient inward NCX current delayed afterdepolarization, and triggered arrhythmias.¹

Santana et al³⁷ recently suggested that glycosides cause Ca²⁺ influx through Na⁺ channels. However, this possibility remains controversial,³⁸ and we found that glycoside inotropy does not occur without a functioning NCX.³⁹ Thus, this possibility is unlikely to complicate our interpretations.

We conclude that in rat ventricular myocytes, Ca²⁺ influx via NCX is not important in SS twitches, rest potentiation, or glycoside inotropy. However, KBR can prevent the arrhythmogenic effects associated with glycoside toxicity (which may rely on net Ca²⁺ influx via NCX). This latter effect of KBR makes it a potentially useful adjunct to digitalis treatment and justifies further investigation.

	Acknowledgments

 
Supported by the Japan Heart Foundation and Pfizer Pharmaceuticals Grant for Research on Coronary Artery Disease, and the National Institutes of Health (HL-30077). The authors thank the Pharmaceuticals R & D Center of Kanebo, Ltd, Osaka, Japan, for supplying the KBR. They also thank Dr Hideki Katoh for general support and the critical reading of this manuscript.

Received June 29, 1999; revision received October 14, 1999; accepted October 14, 1999.

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