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(Circulation Research. 2002;90:182.)
© 2002 American Heart Association, Inc.

Cellular Biology

Na⁺-Ca²⁺ Exchange Current and Submembrane [Ca²⁺] During the Cardiac Action Potential

Christopher R. Weber, Valentino Piacentino, III, Kenneth S. Ginsburg, Steven R. Houser, Donald M. Bers

From the Department of Physiology (C.R.W., K.S.G., D.M.B.), Loyola University Chicago, Stritch School of Medicine, Maywood, Ill; Department of Physiology (V.P., S.R.H.), Temple University School of Medicine, Philadelphia, Pa.

Correspondence to Donald M. Bers, PhD, Department of Physiology, Loyola University Chicago, 2160 S First Ave, Maywood, IL 60153.E-mail dbers{at}lumc.edu

	Abstract

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Na⁺-Ca²⁺ exchange (NCX) is crucial in the regulation of [Ca²⁺]_i and cardiac contractility, but key details of its dynamic function during the heartbeat are not known. In the present study, we assess how NCX current (I_NCX) varies during a rabbit ventricular action potential (AP). First, we measured the steady-state voltage and [Ca²⁺]_i dependence of I_NCX under conditions when [Ca²⁺]_i was heavily buffered. We then used this relationship to infer the submembrane [Ca²⁺]_i ([Ca²⁺]_sm) sensed by NCX during a normal AP and [Ca²⁺]_i transient (when the AP was interrupted to produce an I_NCX tail current). The [Ca²⁺]_i dependence of I_NCX at -90 mV allowed us to convert the peak inward I_NCX tail currents to [Ca²⁺]_sm. Peak [Ca²⁺]_sm measured via this technique was >3.2 µmol/L within <32 ms of the AP upstroke (versus peak [Ca²⁺]_i of 1.1 µmol/L at 81 ms measured with the global Ca²⁺ indicator indo-1). The voltage and [Ca²⁺]_sm dependence of I_NCX allowed us to infer I_NCX during the normal AP and Ca²⁺ transient. The early rise in [Ca²⁺]_sm causes I_NCX to be inward for the majority of the AP. Thus, little Ca²⁺ influx via NCX is expected under physiological conditions, but this can differ among species and in pathophysiological conditions.

Key Words: Na⁺-Ca²⁺ exchanger • action potential • rabbit • calcium

	Introduction

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The Na⁺-Ca²⁺ exchanger (NCX) of cardiac sarcolemma, transports 3 Na⁺ for 1 Ca^2+,1 is the main mechanism of Ca²⁺ extrusion from ventricular myocytes, and contributes to relaxation.^2,3 NCX exhibits a thermodynamically defined reversal potential (E_NCX) analogous to those of ion channels (eg, E_Na and E_Ca). Based on the transport stoichiometry, E_NCX=3E_Na-2E_Ca. Thus, when the membrane potential (E_m) exceeds E_NCX, Ca²⁺ entry (outward I_NCX) is favored. The exact role of Ca²⁺ influx via outward I_NCX during the cardiac action potential (AP) is controversial, but it can contribute to sarcoplasmic reticulum (SR) Ca²⁺ loading and Ca²⁺-induced Ca²⁺ release from the SR.^2,4,5 More detailed information about how NCX varies during the AP is essential for understanding its physiological role in health and disease.

Because there is no selective I_NCX blocker, I_NCX is often recorded after other currents are blocked, sometimes accompanied by controls using nonselective I_NCX blockers (eg, Ni²⁺), to isolate I_NCX.⁶ Unfortunately, this prevents the direct measurement of I_NCX during an AP because blockade of contaminating currents will necessarily alter the electrochemical gradients for Ca²⁺ and Na⁺, which drive I_NCX.^7,8 In particular, blockade of I_Ca or I_Na, or altering SR release, will alter the electrochemical gradients for Ca²⁺ and Na⁺, making it difficult to interpret the significance of I_NCX as measured. Also, nonspecific blockers may impair assessment of NCX in intact cells.

The alternative to directly measuring I_NCX is to model I_NCX based on its steady-state dependence on [Ca²⁺]_i and E_m.⁹ A limitation with this approach is that the submembrane [Ca²⁺]_i, as sensed by NCX, ([Ca²⁺]_sm), can differ from global cytosolic [Ca²⁺]_i sensed by fluorescent indicators.¹⁰ For example, when caffeine is rapidly applied to a cell, SR Ca²⁺ is released, and inward I_NCX increases quickly, even before fluorescent [Ca²⁺] indicators sense a change in bulk [Ca²⁺]_i. During spontaneous SR Ca²⁺ release at -80 mV, Trafford et al¹⁰ observed a 133-ms lag between the submembrane compartment containing I_NCX and bulk [Ca²⁺]_i.

Our goals were to measure the [Ca²⁺]_sm sensed by NCX during a normal AP and Ca²⁺ transient and to determine the time course and amplitude of inward and outward I_NCX during the AP. We hypothesize that local elevations in [Ca²⁺]_sm, due to I_Ca or SR Ca²⁺ release occurring near NCX proteins, will limit Ca²⁺ influx via outward I_NCX during the majority of the AP, such that I_NCX is primarily a Ca²⁺ extrusion mechanism in the cardiac myocyte.

Our method combined both experimental data and modeling. First, we measured the steady-state dependence of I_NCX on [Ca²⁺]_i and E_m under conditions when [Ca²⁺]_i was heavily buffered (ie, [Ca²⁺]_sm=[Ca²⁺]_i). We then used this relationship and the fact that I_NCX can be measured at a fixed E_m as a bioassay for [Ca²⁺]_sm during a normal Ca²⁺ transient and AP at 37°C. Cells were voltage-clamped with an AP waveform, which was interrupted at different times by hyperpolarization to reveal I_NCX tail currents.¹¹ The time course of [Ca²⁺]_sm during an AP, combined with our knowledge of the E_m and [Ca²⁺]_i dependence of I_NCX, allowed us to infer I_NCX during an AP.

Our results suggest that [Ca²⁺]_sm reaches >3.2 µmol/L, peaking <32 ms after the AP upstroke. This limits outward I_NCX to the first 19 ms of the AP, and it is largely driven by depolarization before [Ca²⁺]_sm has reached its peak. These data indicate that during almost the entire rabbit cardiac cycle I_NCX extrudes Ca²⁺.

	Materials and Methods

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Cell Isolation, Voltage Clamping, and [Ca²⁺]_i Measurement
Rabbit myocytes were isolated, currents (whole-cell patch clamp), and [Ca²⁺]_i (using indo-1 K⁺-salt) were measured as previously described.⁶ New Zealand White rabbits (Myrtle’s Rabbitry, Thompson Station, Tenn) were cared for and used according to AAALAC guidelines.

Steady-State I_NCX Dependence on [Ca²⁺]_i and E_m
Cells were pretreated for 12 minutes with Tyrode’s solution containing (in mmol/L) thapsigargin 0.001, NaCl 140, CaCl₂ 2, KCl 4, MgCl₂ 1, glucose 10, and HEPES 5, pH 7.4 with NaOH. Patch electrodes (1 to 3 M) were tip-dipped and back-filled with solution containing (mmol/L) CsCl 40, cesium glutamate 80, MgCl₂ 0.92, CaCl₂ 1.13, HEPES 10, NaCl 10, MgATP 5, LiGTP 0.3, BAPTA 5, Br₂BAPTA 1, and 1 K⁺ indo-1 ([Ca²⁺]43 nmol/L), with pH set to 7.2 using CsOH at 37°C. In some experiments, K⁺ indo-1 was replaced with 500 µmol/L K⁺ SBFI to monitor [Na⁺]_i. Pipettes were sealed to myocytes in Tyrode’s solution containing (in mmol/L) NaCl 140, KCl 4, glucose 10, HEPES 5, and MgCl₂ 1, and CaCl₂ 2, with pH set to 7.4 at 37°C using NaOH. After access to the cytosol was attained, external solution was switched to Tyrode’s solution containing 20 µmol/L nifedipine (to block Ca²⁺ current), 30 µmol/L niflumic acid (to block Ca²⁺-activated Cl^- current), 4 µmol/L N-acetylstrophanthidin (to block Na⁺/K⁺ ATPase), and CsCl replacing KCl (to block K⁺ currents). After 20 minutes of dialysis, 800-ms voltage ramps from +100 to -100 mV were repeated every 5 seconds. Holding E_m between the ramps was either +40 or -90 mV driving Ca²⁺ entry or exit via I_NCX. [Ca²⁺]_i did not change during a single ramp but gradually changed between successive ramps (by 50 to 100 nmol/L) because E_m differed from E_NCX. Identical ramps were also applied after the addition of 10 mmol/L Ni²⁺, giving Ni²⁺-sensitive I_NCX.

Time Course of [Ca²⁺]_sm During an AP
Patch electrodes and pipette solutions were identical to above, but without BAPTA and Br₂BAPTA, and with only 100 µmol/L K⁺ indo-1. Bath Tyrode’s solution (after patch rupture) contained 30 µmol/L niflumic acid, 4 µmol/L N-acetylstrophanthidin, and CsCl (replacing KCl). Unlike the experiments above, SR function was not blocked in these experiments.

A typical rabbit AP, recorded in current clamp with physiological solutions at 1 Hz and 37°C,¹² was used as a template for AP clamp. The AP clamp was interrupted at 10, 25, 50, 100, 200, 300, and 400 ms by hyperpolarization to -90 mV, allowing uncontaminated I_NCX tail current recordings. The interrupted AP templates were also inverted for P/8 capacitance subtraction. The AP clamp interrupted at 10 ms was always recorded before loading the SR with Ca²⁺ by conditioning pulses (below). This gave the tail current component that was independent of SR Ca²⁺ load. This current, attributable to I_Ca deactivation at -90 mV, decayed monoexponentially with 1 ms, and was not apparent at interruptions 25 ms. Subsequently, cells were conditioned with ten 200-ms depolarizations to +40 mV at 1 Hz and then either a full or interrupted AP was applied, followed by the respective P/8 protocol.

Data Acquisition and Analysis
Currents and fluorescence signals were recorded using PClamp6.4 and PClamp8 software (Axon Instruments). All experiments were performed at 37°C.

I_NCX Steady-State Relationship
I_NCX was represented as described previously.⁶

K_m values are the Na⁺ and Ca²⁺ dissociation constants for intracellular (i) and extracellular (o) Na⁺ and Ca²⁺. is the position of the energy barrier of NCX in the membrane electric field, k=F/RT, and k_sat is a factor that controls the saturation of I_NCX at negative E_m. Fixed parameters were as follows: K_mCaAct=125 nmol/L, K_mNao=87.5 mmol/L, K_mNai=12.3 mmol/L, K_mCai=3.6 µmol/L, K_mCao=1.30 mmol/L, k_sat=0.32, =0.27, and T=37°C. V_max (in A/F) varied and was determined for each cell. When Ni²⁺ block was not recorded, a linear leak component was also included in I_NCX fits. Equation 1 was used in steady-state conditions ([Ca²⁺]_sm=[Ca²⁺]_i) to solve for V_max and [Na⁺]_i for measured I_NCX, E_m, and [Ca²⁺]_i. In the interrupted AP clamp experiments, it was used to predict [Ca²⁺]_sm at each AP interruption, from the measured I_NCX. It was also used to calculate I_NCX during the entire AP, using E_m and the predicted [Ca²⁺]_sm (in place of [Ca²⁺]_i). [Ca²⁺]_i and [Ca²⁺]_sm were smoothed to purely empirical functions for use with Equation 1 (eg, [Ca²⁺]_i=a/{1+exp[-(t-b+d/2)/d]}{1-1/[1+exp(-(t-b-c/2)/e)]}+f; a-f constant, t=time).

	Results

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I_NCX Steady-State Relationship
Figure 1 shows the steady-state I_NCX dependence on [Ca²⁺]_i and E_m using voltage ramps where [Ca²⁺]_i was heavily buffered. [Ca²⁺]_i was gradually driven up by outward I_NCX at E_m=+40 mV between ramps (Figure 1A) but did not change during a single ramp (Figure 1D). Figures 1B through 1D show the first, fifth, and tenth ramps recorded from a single myocyte using this protocol. Figures 1E and 1F show that the Ni²⁺-sensitive I_NCX data are well fit by Equation 1, either as the E_m dependence of I_NCX at different [Ca²⁺]_i or as the [Ca²⁺]_i dependence of I_NCX at different E_m. We repeated this protocol on cells loaded with SBFI and found that [Na⁺]_i also decreased by several mmol/L over a similar time course as [Ca²⁺]_i rose to >1 µmol/L (data not shown). In Figure 1, integration of I_NCX over the entire 46 seconds indicates net influx of 645 µmol/L total Ca²⁺ into the cell (and extrusion of 2 mmol/L Na⁺), as [Ca²⁺]_i rises by 852 nmol/L. Although this strong Ca²⁺ buffering is somewhat less than expected for the pipette solution, it allows characterization of I_NCX under relatively static conditions. It is also in reasonable agreement with the measured [Na⁺]_i decline (above) and the inferred [Na⁺]_i changes based on E_NCX and [Ca²⁺]_i measurements at successive ramps. There was large cell-to-cell variation in V_max (V_max=17.8 A/F±4.8 SEM; range=3.6 to 33.7; n=14), but other parameters of Equation 1 did not need to be varied. Thus, Equation 1 can be used with confidence in subsequent experiments to describe the [Ca²⁺]_i and E_m dependence of I_NCX.

View larger version (16K): [in this window] [in a new window]   Figure 1. Steady-state INCX dependence on [Ca2+]i and Em. [Ca2+]i was heavily buffered with BAPTA, Br2BAPTA, and indo-1. A, Voltage ramps from +100 to -100 mV (held at +40 mV for 4 seconds between ramps to promote Ca2+ entry via outward INCX). B through D, Em, INCX, and [Ca2+]i for first (a), fifth (b), and tenth (c) voltage ramps. Time scale as in C. E, INCX versus Em for first, fifth, and tenth voltage ramps with fits (dashed line) to Equation 1. F, INCX versus [Ca2+]i for all 10 voltage ramps with fits (dashed line) to Equation 1 (with Vmax=3.7 A/F and [Na+]i for ramps 1 through 10 (in mmol/L): 13.5, 11.8, 10.9, 10.6, 10.6, 10.4, 10.4, 10.4, 10.1, 10.2.

Determination of [Ca²⁺]_sm During an AP
Equation 1 describes I_NCX as a function of static [Ca²⁺]_i but also allows us to infer the [Ca²⁺]_sm that drives I_NCX during a normal Ca²⁺ transient. Note that fluorescent indicators cannot detect [Ca²⁺]_i elevations local to the NCX molecules¹⁰ and that [Ca²⁺]_sm does not refer to a physical volume that we can describe. Rather, it is the average [Ca²⁺]_i, sensed by all NCX molecules in the cell (see Discussion). Figure 2 shows the voltage protocol, I_NCX tail currents, and [Ca²⁺]_i for determining [Ca²⁺]_sm during an AP. The AP clamp interruptions produce different inward I_NCX tails. For clarity, and to illustrate how hyperpolarization accelerates [Ca²⁺]_i decline, only three [Ca²⁺]_i transients are shown for AP interruptions (Figure 2C).

View larger version (26K): [in this window] [in a new window]   Figure 2. Protocol to determine [Ca2+]sm during an AP. A, AP was interrupted and clamped to -90 mV at the indicated times in ms. B, INCX tails at -90 for the protocol in panel A. C, [Ca2+]i for 10-, 100-, and 400-ms AP interruptions. Data were smoothed using empirical fits.

Figure 3A shows I_NCX as a function of [Ca²⁺]_i for the record in Figure 2. At early time points, I_NCX far exceeds the value that would be expected from the global [Ca²⁺]_i (which is shown as the steady-state [SS] curve for this cell). This reflects the fact that [Ca²⁺]_sm sensed by NCX is much higher than bulk [Ca²⁺]_i. We used the peak value of I_NCX immediately upon hyperpolarization to indicate [Ca²⁺]_sm at the moment of hyperpolarization. Inward I_NCX usually rises during the initial few milliseconds, probably reflecting the noninstantaneous transition to -90 mV. For earlier interruptions, however, it may also represent an actual rise in [Ca²⁺]_sm during continued SR Ca²⁺ release. Because we cannot differentiate between these two possibilities, we recorded both the time and the value of I_NCX at its peak (see Discussion: Sources of Error). The steady-state relationship for I_NCX versus [Ca²⁺]_i for this cell (SS curve) is defined by the region where all traces of I_NCX versus [Ca²⁺]_i converge (at 300 ms) and is fit by Equation 1 with a leak offset.

View larger version (20K): [in this window] [in a new window]   Figure 3. INCX is driven by [Ca2+]sm. A, INCX versus [Ca2+]i from Figure 2. Multiple traces intersect the steady-state (SS) INCX versus [Ca2+]i relationship for this cell (Equation 1: Vmax=17.3 A/F, [Na+]i=10 mmol/L, Em=-90 mV, including a leak offset of 0.59 A/F). [Ca2+]i was smoothed. Arrows illustrate extrapolation from peak inward INCX to [Ca2+]sm (superimposed with [Ca2+]i in panel B).

V_max values obtained in this manner (V_max=10.6 A/F±2.5 SEM; range=3.0 to 23.1; n=8) were not significantly different from values obtained under buffered conditions. For each AP interruption, the [Ca²⁺]_sm is inferred by extending the peak I_NCX value to the SS curve (arrows in Figure 3A). Figure 3B shows [Ca²⁺]_sm together with [Ca²⁺]_i. Variations in [Ca²⁺]_sm were not correlated with variations in V_max.

Determination of I_NCX During an AP
Figure 4B shows pooled [Ca²⁺]_sm values (n=8 cells) calculated as in Figure 3 and an average [Ca²⁺]_i transient, during the representative full-length AP (Figure 4A). Peak [Ca²⁺]_sm is 3.2 µmol/L at 32 ms. Figure 4C shows I_NCX based on [Ca²⁺] and E_m, as predicted by Equation 1, using the average V_max value of these same cells and 10 mmol/L [Na⁺]_i. If we use [Ca²⁺]_i to define I_NCX, we predict outward I_NCX for 142 ms of the AP. If we use [Ca²⁺]_sm, I_NCX is inward after only 19 ms. The dotted lines show I_NCX calculated using a linear extrapolation of [Ca²⁺]_sm to [Ca²⁺]_rest (at 2 ms). Figure 4D shows currents from Figure 4C integrated and converted to cumulative [Ca²⁺]_T (using 6.4 pF/pL cytosol).¹³ When [Ca²⁺]_i is used to calculate I_NCX, 285 ms are needed to extrude the 4.4 µmol/L Ca²⁺ that entered via outward I_NCX. When [Ca²⁺]_sm is used to calculate I_NCX, only 0.6 µmol/L Ca²⁺ enters the cell, and all of this Ca²⁺ has been extruded by 90 ms. Therefore, at 1 Hz, >98% of the cardiac cycle is spent with I_NCX functioning to extrude Ca²⁺, which enters predominantly through I_Ca.

View larger version (22K): [in this window] [in a new window]   Figure 4. Pooled data. A, Em during an AP. B, Mean [Ca2+]sm calculated as in Figure 3 (n=8); [Ca2+]i represents an average of 5 cells. Broken lines in panels B and C represent linear extrapolations of [Ca2+]sm to [Ca2+]rest (at 2 ms). Data empirically smoothed (see Materials and Methods). C, INCX for [Ca2+]i (thin trace) or [Ca2+]sm (thick trace) and Em, using Equation 1 (Vmax=10.6 A/F, [Na+]i=10.0 mmol/L). D, Integrated INCX in panel C converted to [Ca2+]tot.13

Simple Method to Transform [Ca²⁺]_i to [Ca²⁺]_sm
It is not always possible to measure [Ca²⁺]_sm as we have done here. Therefore it may be useful to predict how [Ca²⁺]_sm may change during cellular Ca²⁺ transients. Trafford et al,¹⁰ using spontaneous [Ca²⁺]_i transients, transformed [Ca²⁺]_i to [Ca²⁺]_sm using a single time constant for diffusion between cytosol and submembrane space:

where was 133±47 ms. When applied to our data, Equation 2 described the rising phase of [Ca²⁺]_sm adequately (in our case with =110 ms), but the [Ca²⁺]_sm declined too rapidly (Figure 5A). More comprehensive mathematical compartment models¹⁴ are surely needed to describe [Ca²⁺]_sm ideally, but we have found a simple extension of Equation 2, which allows a reasonable description of the [Ca²⁺]_sm based solely on the [Ca²⁺]_i transient. As shown in Figure 5, we use Equation 2 up to the point where [Ca²⁺]_sm is maximum (ie, the maximum rate of [Ca²⁺]_i rise or peak d[Ca²⁺]_i/dt) and then, for the declining phase, switch to a simple exponential decay for the [Ca²⁺]_sm versus [Ca²⁺]_i difference:

View larger version (17K): [in this window] [in a new window]   Figure 5. Transfer function expressing [Ca2+]sm in terms of [Ca2+]i. A, Thick trace: [Ca2+]i+d[Ca2+]i/dt10; thin trace: decay of [Ca2+]sm after peak d[Ca2+]i/dt (t=24.5 ms) represented empirically as [Ca2+]sm=[Ca2+]i+Aexp{-(t-to)/}. B, Thick trace: d[Ca2+]i/dt (=110 ms); thin trace: Aexp{-(t-to)/}, where A=2770 nmol/L, =92 ms, and t0=24.5 ms.

where A=[Ca²⁺]_sm-[Ca²⁺]_i at the transition time (t_o, dotted line) and =92 ms. Figure 5B shows that Equations 2 and 3 coincide at t_o. This simple description matched the average data in Figure 4, in which [Ca²⁺]_sm was measured experimentally.

	Discussion

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Our results assess rather directly the actual I_NCX that flows during a normal rabbit ventricular myocyte AP. Because NCX can produce both Ca²⁺ influx and Ca²⁺ efflux, this type of information is essential in the understanding of Ca²⁺ and force regulation in cardiac muscle. This importance is reinforced by recent work suggesting that increased NCX expression and altered NCX function contribute to contractile dysfunction and arrhythmias in pathophysiological conditions.^15–18

I_NCX is inward during the majority of the normal rabbit ventricular AP. In demonstrating this, we also showed how I_NCX can be used as a bioassay for local [Ca²⁺]_sm (after careful characterization of the [Ca²⁺]_i and E_m dependence of I_NCX). During the AP, [Ca²⁺]_sm peaks earlier and higher than [Ca²⁺]_i. This high [Ca²⁺]_sm drives I_NCX inward by 19 ms into the AP. Before this time, outward I_NCX may be driven by membrane depolarization as [Ca²⁺]_sm rises.

Steady-State Relationship for I_NCX
We used the I_NCX equation published by Weber et al⁶ to describe E_m, [Na⁺], and [Ca²⁺] dependence of I_NCX under conditions when [Ca²⁺]_i is not changing appreciably. The E_m dependence of I_NCX in Equation 1 is the same as in the Luo and Rudy⁹ equation for I_NCX, but the electrochemical component of our equation considers both intracellular and extracellular ion dependencies at the transport sites as well as an allosteric Ca²⁺ activation factor. In our previous study,⁶ we characterized allosteric Ca²⁺ activation in ferret myocytes (K_mCaAct=125 nmol/L), and we see evidence of similar Ca²⁺ activation in rabbit myocytes using similar protocols (not shown). The present study illustrates that Equation 1 (including both the allosteric K_mCaAct=125 nmol/L and Ca²⁺ transport K_mCai=3.6 µmol/L) describes very well the E_m and [Ca²⁺]_i dependence of I_NCX (Figure 1).

Comparison to Earlier Studies
Our peak inward I_NCX was 1.2 A/F, occurring roughly at the end of AP phase 3 repolarization (Figure 4). This peak value is close to that predicted by Luo and Rudy⁹ in guinea pig at a similar degree of AP repolarization. However, their published model does not consider [Ca²⁺]_sm, as sensed by the NCX or binding of Ca²⁺ to internal transport or allosteric sites. The Luo and Rudy model has outward I_NCX lasting 100 ms. We believe I_NCX should be driven inward much earlier (19 ms), as a result of elevated [Ca²⁺]_sm. If we use [Ca²⁺]_i instead of [Ca²⁺]_sm, we predict I_NCX to turn inward at 143 ms, comparable with Luo and Rudy.⁹

Several studies have used pharmacological techniques to characterize I_NCX during the AP. Grantham and Cannell⁷ measured I_NCX during AP clamp by applying nifedipine rapidly after conditioning the cell with a train of APs with I_Ca unblocked. They recorded outward I_NCX through most of the AP, but the rate of rise and peak of the [Ca²⁺]_i transient were markedly decreased in the presence of nifedipine. It follows that [Ca²⁺]_sm would also be much less than normal, and this would greatly favor more outward I_NCX during most of the AP. Thus, this type of experiment does not provide data on I_NCX during a normal AP and [Ca²⁺]_i transient. In ferret ventricular APs, Janvier et al⁸ showed that AP duration was reduced when I_NCX was blocked by rapid substitution of external Na⁺ with Li⁺ or buffering [Ca²⁺]_i with BAPTA-AM. While these data might reflect mainly inward I_NCX during an AP (as we find), it is not easy to quantify I_NCX in absolute terms using their techniques.

Egan et al¹¹ interrupted APs by repolarizing back to resting E_m and usually found that inward I_NCX tail currents were largest at interruptions between 50 and 100 ms after the start of the AP. This is later than the average time (32 ms) at which we observed maximal inward I_NCX tails. They did not measure [Ca²⁺]_i but used an indirect conversion factor (1 µmol · L^-1 · nA^-1) based on data of Kimura et al³ to predict [Ca²⁺]_sm. Their reported [Ca²⁺]_sm peaked at 100 ms and 1.6 µmol/L, similar to [Ca²⁺]_i as we measured with fluorescent indicators (Figure 4). Our data indicate that [Ca²⁺]_sm peaks earlier (32 ms) and is much higher.

Sources of Error
Up to this point, we have assumed that [Na⁺]_i is constant and equal to pipette [Na⁺] (10 mmol/L). In reality, submembrane [Na⁺]_i ([Na⁺]_sm) may be elevated transiently by I_Na and I_NCX. For example, a 10-ms triangular Na⁺ current, peaking at 50 nA, flowing into a submembrane volume equivalent to 2% of cytosolic volume (0.6 pL)¹³ could increase local [Na⁺]_i by a maximum of 4 mmol/L (ignoring diffusion from the space). To assess the possible effect of elevated [Na⁺]_sm on our calculated [Ca²⁺]_sm, we assumed that [Na⁺]_sm peaks at >14 mmol/L, 2 ms into the AP, and declines to 10 mmol/L with a tau of 8 ms (Figure 6B). This is an upper limit for [Na⁺]_sm because the NCX is not well colocalized with Na⁺ channels.¹⁹ Transient elevations in [Na⁺]_sm could reduce inward I_NCX, which would lead us to underestimate [Ca²⁺]_sm based on the steady-state relationship for I_NCX, which was always measured after submembrane elevations would have dissipated. Using Equation 1, our first value for [Ca²⁺]_sm in Figure 3 (at t=17.9 ms) may be underestimated by 8.1% ([Ca²⁺]_sm=2021 at [Na⁺]_sm=10.7 mmol/L versus 1858 nmol/L using [Na⁺]_sm=10.0 mmol/L), but subsequent points will not be greatly affected (Figure 6C). Thus, our technique used to determine [Ca²⁺]_sm should be relatively insensitive to these [Na⁺]_sm changes. This is partly because the electrochemical determinants for inward I_NCX at -90 mV are driven largely by [Ca²⁺]_sm.⁶

View larger version (16K): [in this window] [in a new window]   Figure 6. Elevated [Na+]sm raises both predicted [Ca2+]sm and INCX during an AP. A, Em during an AP. B, Hypothetical [Na+]sm and [Na+]i. C, [Ca2+]sm using [Na+]sm or [Na+]i. D, INCX for [Na+]sm or [Na+]i, and Em, using Equation 1 (Vmax=10.6 A/F). Broken lines in panels C and D represent linear extrapolations of [Ca2+]sm to [Ca2+]rest (at 2 ms).

On the other hand, elevated [Na⁺]_sm can substantially impact predicted outward I_NCX during an AP. It has been suggested that I_Na may augment the ability for I_NCX to trigger SR Ca²⁺ release.⁵ Using [Ca²⁺]_sm, E_m, and [Na⁺]_sm, Figure 6A ±shows increased outward I_NCX by 86% for our first data point (Figure 6D). Extrapolating [Ca²⁺]_sm and I_NCX to earlier times (dotted traces) would predict even greater outward I_NCX. This, however, does not confirm the ability of I_NCX to trigger SR Ca²⁺ release (see Physiological Implications).

Our protocol for measuring [Ca²⁺]_sm involves large voltage steps to -90 mV from E_m during an AP that cannot be instantaneous. We usually observe about a 7-ms delay between our desired hyperpolarization and the measured peak inward I_NCX. During hyperpolarization, the driving force (E_m-E_NCX) for inward I_NCX will increase as the true E_m reaches the commanded value (-90 mV). Because inward I_NCX flows during this transition, [Ca²⁺]_sm is necessarily different from that at the instant hyperpolarization started, and therefore, our values of [Ca²⁺]_sm are likely to be underestimated. To estimate this possible error, we linearly extrapolated I_NCX from its measured inward peak back to the exact time of hyperpolarization. For the record in Figure 2B, [Ca²⁺]_sm based on extrapolated I_NCX (using 40 ms of data after the peak), was on average 14% higher for interruptions up to 200 ms, but not much different for later interruptions, where the voltage steps were smaller and [Ca²⁺]_sm was lower. Thus, a 7-ms delay and an underestimation of [Ca²⁺]_sm imply that the real I_NCX during an AP is less outward than indicated in Figures 4C and 6D.

Allosteric Ca²⁺ activation is taken as an instantaneous process in Equation 1.⁶ However, delayed activation could contribute to the slow rise in I_NCX tail currents. This will be particularly true for the earlier values of I_NCX recorded immediately after SR release, when I_NCX is changing from about half activation to full activation. This could have caused us to underestimate the rate of rise and peak [Ca²⁺]_sm and could also explain why the curves in Figure 3A diverge from one another. However, the steepness (supralinear) of the early interruption curves (versus SS) cannot be attributed to allosteric regulation.

Taking elevated [Na⁺]_sm, noninstantaneous hyperpolarization, and noninstantaneous kinetics of allosteric activation together, all could cause us to underestimate the peak [Ca²⁺]_sm reported in Figure 4 and overestimate the time at which it is achieved. Thus [Ca²⁺]_sm probably reaches >3.5 µmol/L in <25 ms during an AP in rabbit ventricular myocytes. This would shift I_NCX inward even earlier than indicated in Figure 4.

Physiological Implications
Our results suggest that submembrane elevations in [Ca²⁺] drive I_NCX to function predominantly as a Ca²⁺ efflux mechanism throughout the cardiac cycle in rabbit (Figure 4). If [Ca²⁺]_i, rather than [Ca²⁺]_sm, is used to predict I_NCX, outward I_NCX flows for 142 ms, bringing 4.4 µmol Ca²⁺/L cytosol into the cell. An additional 152 ms is required just to extrude the portion of Ca²⁺ that entered on I_NCX. At a physiological frequency of 2 Hz, this would leave only 200 ms for I_NCX to extrude the 10 µmol/L Ca²⁺ entry that occurs on I_Ca. This seems implausible, even with the help of the sarcolemmal Ca²⁺ pump. When [Ca²⁺]_sm is used in Equation 1 to predict I_NCX, all of the Ca²⁺ that enters via outward I_NCX is extruded by 90 ms, leaving plenty of time for inward I_NCX to extrude the additional Ca²⁺ that entered via I_Ca.

With AP depolarization, outward I_NCX may bring Ca²⁺ into the cell for up to 15 ms. It is important to realize, however, that these data are based on average [Ca²⁺]_sm, as sensed by all of the NCX molecules in the sarcolemma. These data do not directly determine [Ca²⁺] within a physical compartment, nor does our data directly address the role of I_NCX in triggering SR release. However, the NCX molecules most likely to trigger SR Ca²⁺ release (those closest to the dyadic cleft) would sense even higher [Ca²⁺]_sm earlier during the AP, causing I_NCX to become inward even earlier at that location. For example, as soon as an L-type Ca²⁺ channel opens, cleft [Ca²⁺] may rise to >10 µmol/L in <1 ms.²⁰ Furthermore, SR Ca²⁺ release may be activated rapidly (<1 ms) by local I_Ca,²¹ and [Ca²⁺]_i in the junctional cleft may rise to >42 µmol/L in 5 to 10 ms based on computer simulations.²² Thus, cleft [Ca²⁺] may greatly exceed [Ca²⁺]_sm and limit the functional ability for outward I_NCX to trigger SR Ca²⁺ release. There are two important counterpoints. First, outward I_NCX carried by exchanger molecules outside the cleft may raise [Ca²⁺]_sm enough to slow diffusion of high cleft [Ca²⁺], which would enhance the efficacy of an I_Ca trigger for SR Ca²⁺ release. Second, in the latent period before opening of any L-type Ca²⁺ channel at a junction (or if I_Ca fails locally or is blocked), outward I_NCX may become an alternative, albeit less efficient, means to trigger SR release.²³ However, L-type Ca²⁺ channel latency is very brief under normal conditions at positive E_m. Moreover, L-type channels are believed to be located close to ryanodine receptors, while NCX molecules may not be.¹⁹ Thus, although Ca²⁺ influx via NCX might help to raise local cleft [Ca²⁺] in triggering SR Ca²⁺ release and even synergize with I_Ca, its contribution is likely to be limited to the time before I_Ca activation, a time where it is probably insufficient by itself to trigger release.²³

Pathophysiological Implications
Alterations in [Na⁺]_i can profoundly affect I_NCX, and species-dependent differences in [Na⁺]_i exist. For example, [Na⁺]_i may be as high as 16 mmol/L in rat²⁴ and mouse²⁵ versus 5 to 10 mmol/L in rabbit^24,25 or guinea pig ventricle.²⁶ [Na⁺]_i is elevated during hypertrophy,²⁷ heart failure (HF),^27a and glycoside therapy. In HF, Ca²⁺ transient amplitude is also decreased and AP duration is prolonged.^17,18,28 All three of these changes in HF (higher [Na⁺]_i, lower peak [Ca²⁺]_i, and longer AP duration) would increase Ca²⁺ influx via I_NCX during the AP. Thus, in HF, Ca²⁺ entry via NCX may be more important than under normal physiological conditions.¹⁶

Greater Ca²⁺ influx during the AP via I_NCX in HF would leave less time for net Ca²⁺ extrusion via I_NCX, which is required to balance the Ca²⁺ influx via I_Ca (and NCX). However, increased NCX expression in HF^17,29 may act compensatorily to enhance Ca²⁺ extrusion during diastole. In fact, basal SR Ca²⁺ content is reduced in HF³⁰ (as a result of increased NCX and reduced SR Ca²⁺-ATPase function).^15,18,29 Although elevated NCX expression may be compensatory (in limiting Ca²⁺ overload), when Ca²⁺ overload and spontaneous SR release do occur (eg, during ß-adrenergic activation), NCX can also produce more transient inward current, thereby promoting triggered arrhythmias.¹²

	Conclusions

Top Abstract Introduction Materials and Methods Results Discussion Conclusions References

 
We used inward I_NCX to predict the time course of [Ca²⁺]_sm, sensed by the NCX, during a normal rabbit ventricular AP under physiological conditions. Elevated [Ca²⁺]_sm (peaking at >3.2 µmol/L) should prevent outward I_NCX within <19 ms of the AP upstroke. Before that time, some outward I_NCX might flow, but much less outward I_NCX will flow on NCX molecules located closer to the dyadic cleft (where local [Ca²⁺]_i would be higher). Although these results suggest limited outward I_NCX during the majority of the cardiac cycle, it is not yet possible to define the role of NCX in excitation-contraction coupling before SR release. These results emphasize the importance of spatial [Ca²⁺]_i heterogeneities in myocyte Ca²⁺ fluxes. Furthermore, the balance of Ca²⁺ fluxes on NCX may differ among species and under pathological conditions.

	Acknowledgments

 
This work was supported by NIH grants HL30077 and HL64098 (D.M.B.) and American Heart Association predoctoral fellowship 0010180Z (C.R.W.). We thank Dr Sanda Despa for her help with measurement of [Na⁺]_i and Jorge Acevedo for his rabbit myocyte isolations.

Received August 20, 2001; revision received December 6, 2001; accepted December 7, 2001.

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