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
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.
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.
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.
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: Rmax and Rmin (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. Kd equaled 844 nmol/L,19 and Sf/Sb, 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, 1.25×threshold) 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.
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%.
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.
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.
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⇓).
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.
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).
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 5A⇓c). In all 4 of these cells, 5 μmol/L KBR abolished the spontaneous activity (Figure 5B⇓d) but left the strophanthidin-induced inotropy largely intact (compare Figure 5B⇓, b and d). KBR also partially restored diastolic cell length.
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.
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.
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 (Em) 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 NCX9 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 Em 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 ms1 33 34 and (2) the AP differences are at an Em 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 Em (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).
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 Em and favoring net Ca2+ influx at rest and Ca2+ overload. Spontaneous Ca2+ release at the resting Em 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.
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.
- Copyright © 2000 by American Heart Association
Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. Dordrecht, the Netherlands: Kluwer Academic Press; 1991.
Satoh H, Hayashi H, Noda N, Terada H, Kobayashi A, Hirano M, Yamashita Y, Yamazaki N. Regulation of [Na+]i and [Ca2+]i in guinea pig myocytes: dual loading of fluorescent indicators SBFI and fluo 3. Am J Physiol. 1994;266:H568–H576.
Kohmoto O, Levi AJ, Bridge JHB. The relationship between reverse Na-Ca exchange and SR Ca2+ release in guinea-pig ventricular myocytes. Circ Res. 1994;74:550–554.
Levi AJ, Spitzer KW, Kohmoto O, Bridge JHB. Depolarization-induced Ca entry via Na-Ca exchange triggers SR release in guinea pig cardiac myocytes. Am J Physiol. 1994;266:H1422–H1433.
Hancox JC, Evans SJ, Levi AJ. The Fura-2 transient can show two types of voltage dependence at 36°C in ventricular myocytes isolated from the rat heart. Pflugers Arch. 196;432:215–224.
Leblanc N, Hume JR. Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum. Science. 1990;248:372–376.
Sham JK, Cleeman L, Morad M. Gating of the cardiac Ca2+ release channel: the role of Na+ current and Na+/Ca2+ exchange. Science. 1992;255:850–853.
Sipido KR, Maes MM, Van de Werf F. Low efficiency of Ca2+ entry through the Na/Ca exchanger as trigger for Ca2+ release from the sarcoplasmic reticulum. Circ Res. 1997;81:1034–1044.
Terada H, Hayashi H, Satoh H, Katoh H, Yamazaki N. Simultaneous measurement of [Na+]i and Ca2+ transients in an isolated myocyte: effects of strophanthidin. Biochem Biophys Res Commun. 1994;203: 1050–1056.
Iwamoto T, Watano T, Shigekawa M. A novel isothiourea derivative selectively inhibits the reverse mode of Na+/Ca2+ exchange in cells expressing NCX1. J Biol Chem. 1996;271:22391–22397.
Hayashi H, Miyata H, Noda N, Kobayashi A, Hirano M, Kawai T, Yamazaki N. Intracellular Ca2+ concentration and pHi during metabolic inhibition. Am J Physiol. 1992;262:C628–C634.
Iwamoto T, Shigekawa M. Differential inhibition of Na+/Ca2+ exchanger isoforms by divalent cations and isothiourea derivative. Am J Physiol. 1998;275:C423–430.
Shattock MJ, Bers DM. Rat vs rabbit ventricle: Ca flux and intracellular Na assessed by ion-selective microelectrodes. Am J Physiol. 1989;256:C813–C822.
Bouchard RA, Clark RB, Giles WR. Role of sodium-calcium exchange in activation of contraction in rat ventricle. J Physiol. 1993;472:391–413.
Vornanen M, Shephed N, Isenburg G. Tension-voltage relations of single myocytes reflect Ca release triggered by Na/Ca exchange at 35°C but not 23°C. Am J Physiol. 1994;267:C623–C632.
Yao A, Nonaka A, Zubair I, Lu L, Philipson KD, Bridge JHB, Barry H. Effects of overexpression of the Na+-Ca2+ exchanger on [Ca2+]i transients in murine ventricular myocytes. Circ Res. 1998;82:657–665.
Adachi-Akahane S, Lu L, Li Z, Frank JS, Philipson KD. Calcium signaling in transgenic mice overexpressing cardiac Na+-Ca2+ exchanger. J Gen Physiol. 1997;109:717–729.
Györke S, Fill M. Ryanodine receptor adaptation: control mechanism of Ca2+-induced Ca2+ release in heart. Science. 1993;260:807–809.
Satoh H, Blatter LA, Bers DM. Effects of [Ca2+]i, SR Ca2+ load and rest on Ca2+ spark frequency in ventricular myocytes. Am J Physiol. 1997;272:H657–H668.
Bouchard RA, Clark RB, Giles WR. Effects of action potential duration on excitation-contraction coupling in rat ventricular myocytes: action potential voltage-clamp measurements. Circ Res. 1995;76:790–801.
Cannell MB, Berlin JR, Lederer WJ. Effect of membrane potential changes on the calcium transient in single rat cardiac muscle cells. Science. 1987;238:1419–1423.
Santana LF, Gomez AM, Lederer WJ. Ca2+ flux through promiscuous cardiac Na+ channels: slip-mode conductance. Science. 1998;279:1027–1033.
Nuss NB, Marban E. Whether “slip-mode conductance” occurs. Science. 1999;284:711.
Altamirano J, DeSantiago J, Bers DM. The inotropic effect of acetyl- strophantidin and digoxin in ferret ventricular myocytes requires Na/Ca exchanger function. Biophys J. 1999;76:A300. Abstract.