Intracellular Ca transients in rat cardiac myocytes: role of Na-Ca exchange in excitation-contraction coupling DONALD M. BERS, W. J. LEDERER, AND JOSHUA R. BERLIN Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland and Division of Biomedical Sciences, University of California, Riverside, California 92521
BERS,DONALD M.,W.J. LEDERER,ANDJOSHUA R. BERLIN. Intracellular Ca transients in rat cardiac myocytes: role of NaCa exchange in excitation-contraction coupling. Am. J. Physiol. 258 (Cell Physiol. 27): C944-C954, 1990.-Membrane current and intracellular Ca concentration ([Cal;) transients were recorded from isolated rat ventricular myocytes under voltageclamp control. The cells were dialyzed by the patch pipette solution, which contained the fluorescent Ca indicator indo-l and 0.5 mM Na. Under these experimental conditions, Ca entry via Na-Ca exchange did not appear to be appreciable even in the absence of extracellular Na. Increasing the duration of voltage-clamp pulses from 5 to 80 ms produced [Cali transients of increasing amplitude, while the peak Ca current was not changed. This duration dependence of the [Cal; transient was most demonstrable at more negative test potentials (e.g., -20 to -30 mV) and was not qualitatively modified by Na-free solutions. This latter result indicates that Ca extrusion by NaCa exchange is not responsible for the smaller [Cali transients observed when the membrane is repolarized after very brief depolarizations. Although the peak Ca current was not changed by increasing pulse duration, the integrated Ca current was increased. These observations are consistent with a Ca-release mechanism in cardiac excitation-contraction coupling in which 1) the Ca-release process can be modulated by membrane potential or 2) the Ca entering the cell via Ca channels has a preferential access [compared with Ca from the sarcoplasmic reticulum (SR)] to the site(s) that control SR Ca release. The role of Na-Ca exchange in the decline of [Cal; during relaxation was also explored. Removal of extracellular Na (Na,) resulted in 20% slowing of the decline in [Cali during relaxation. From this, we conclude that the Na-Ca exchange competes with SR to remove Ca from the cytoplasm and that under our control conditions the exchanger may account for 20% of this decline. The Na, dependence of relaxation was reduced at more positive membrane potentials and increased by SR Ca loading.
21201;
tions and intracellular Ca transients change along with changes in Ca current in a voltage-dependent manner (10, 13, 33, 38).
Further tests of the Ca-induced Ca-release theory come from experiments in which voltage-clamp pulse duration is varied. In both guinea pig (10) and cat myocytes (16), the duration of a voltage-clamp pulse had no immediate effect on the peak level of intracellular Ca concentration ([Cali) reached if the voltage pulse was terminated after the peak of the Ca current. Thus SR Ca release would seem to depend on the peak Ca current in a manner consistent with a Ca-induced Ca-release process. In contrast to those results, the peak value of the [Cali transient in rat ventricular cells was increased by increasing the duration of the voltage-clamp pulse (from 5 to 40 ms) despite a constant peak of the Ca current (at ~5 ms, Ref. 13). Cannel1 et al. (13) interpreted their results to support a possible modulatory role of membrane potential ( E,) on the process of SR Ca release in cardiac muscle. That is, they suggested that the rapid repolarization at 5-20 ms might prematurely terminate SR Ca release and hence lead to the smaller intracellular Ca transient. This is consistent with their observations and similar to the interpretation of results obtained in skeletal muscle (35). The authors could not, however, rule out the possibility that a Ca-induced Ca-release mechanism might also be modulated by E, in some indirect manner (13). In this regard, Ca extrusion via the sarcolemmal NaCa exchange is voltage dependent, such that net Ca extrusion is increasingly favored at more negative potentials (30, 31, 37, 39, 40). Thus it is possible that repolarization after a very brief depolarization described above might accelerate Ca extrusion via Na-Ca exchange and cardiac muscle; indo- 1; calcium current thereby reduce the peak level of [Cal; reached during contraction. Indeed, the rapid repolarization of the rat ventricular action potential is probably responsible for NORMAL DEPOLARIZATION of mammalian cardiac muscle the rapid extrusion of Ca observed during contraction leads to Ca influx via Ca channels (and perhaps also via (41). In the present study, we have examined whether Na-Ca exchange, e.g., Refs. 6, 37). Fabiato (20) has the duration dependence of the [Cali transient might be estimated that the amount of Ca entry via Ca current is due to a Na-Ca exchange-dependent modulation of the not sufficient to activate contraction directly (but see [Cal; transient. Refs. 59). In an elegant series of studies in mechanically The rate of relaxation and Ca extrusion in cardiac skinned cardiac myocytes, Fabiato (21-23) has charac- muscle is also accelerated at more negative potentials terized a Ca-induced Ca-release mechanism, whereby a under conditions in which Na-Ca exchange is the prismall amount of Ca influx may “trigger” release of a mary means of relaxation or Ca extrusion (1, 7, 12, 30). larger amount of Ca from the sarcoplasmic reticulum Furthermore, this Ca extrusion can make a significant (SR). Evidence from intact myocytes that supports this contribution to the amount of Ca removed from the theory of excitation-contraction coupling is that contraccytoplasmic space during relaxation (7,8,29). We, therec944
0363-6143/90
$1.50 Copyright
0 1990 the American
Physiological
Society
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CARDIAC
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TRANSIENTS
fore, attempted to determine the importance of the NaCa exchange mechanism in the time-dependent changes of [Cal; in rat ventricular myocytes during both activation and relaxation by manipulation of extracellular ion concentrations and controlling intracellular ion contents with a patch electrode in whole cell voltage-clamp mode.
METHODS
Myocyte isolation. Myocytes were isolated from adult rat hearts using a modified version of the procedure described by Mitra and Morad (36). After pretreatment with heparin sodium ( 1,000 IU/kg ip), rats were injected with pentobarbitone (50 mg/kg ip), and hearts were excised and perfused on a Langendorff apparatus for 5 min with a solution containing (in mM) 135 NaCl, 5.4 KCl, 1 MgClz, 0.33 NaHzP04, 11 glucose, and 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) at pH 7.4 and 37OC. Then collagenase (0.5 mg/ ml, Boehringer-Mannheim) and protease (0.07 mg /ml, Sigma type XIV) were added, and the heart was perfused for an additional 4-5 min. The heart was then minced and washed, and the cells were stored in the above solution at 37°C (with 0.2 mM CaClz and lacking enzymes). Fluorescence recording apparatus. The optical system was similar to that described for fura- by Cannel1 et al. (13). The cell was illuminated using a 75-W xenon arc lamp (Photon Technologies International, South Brunswick, NJ) via a 350-nm band-pass filter (10 nm full width at half-maximum transmission), through a 400-nm dichroic mirror and a x63 oil-immersion Neofluor objective (Zeiss). Optical signals from a cell were directed to two photomultiplier tubes (EM1 9789B, Thorn EMI). An adjustable window was used to restrict the area of illumination to a square area of 200 pm2 to minimize background fluorescence from the remainder of the field. The fluorescent signals to the two photomultipliers were filtered with band-pass filters at 400 and 500 nm, respectively (40 nm full width at half-maximum transmission). The patch pipette contained the fluorescent Ca indicator indo-1. After formation of a high-resistance seal between the cell membrane and the voltage-clamp electrode, background fluorescence was measured. This value (at 400 and 500 nm) was automatically subtracted from all subsequent fluorescence measurements from that cell using a digital sample and hold circuit. The membrane patch was then ruptured to establish whole cell voltage clamp and to allow diffusion of the pipette contents into the cell. The ratio of emitted fluorescence at 400/500 nm was used to calculate [Cal, based on in vitro calibrations (see below). Experimental procedure. The cells were allowed to settle onto a glass cover slip, which formed the bottom of the experimental chamber mounted on the stage of an inverted microscope (15). The cells were superfused with a modified normal Tyrode that contained (in mM) 145 NaCl, 4 KCl, 1 MgCl,, 2 CaCIZ, 10 glucose, and 10 HEPES, with pH adjusted to 7.40 with NaOH. The bath was maintained at 31 t 1°C by water jacketing up to the bath inlet. In all Na-containing solutions, 30 PM tetro-
AND
NA-CA
EXCHANGE
c945
dotoxin was included to block Na current. For Na-free solutions, Na was replaced with 145 mM N-methyl-Dglucamine (and pH was adjusted with HCl). For solutions that were also Ca free, Ca was replaced with 5 mM MgCIZ. The whole cell variant of the patch-clamp technique was used to voltage clamp the myocytes using an Axopatch 1C (Axon Instruments, Burlingame, CA). Lowresistance patch pipettes were filled with a solution containing (in mM) 100 cesium glutamate, 20 CsCl, 1 MgC1,, 2.5 KZATP, 30 piperazine-NJ/‘-bis(2-ethanesulfonic acid) (PIPES), 0.05-0.07 indo-l (acid form), and 0.5 NaOH, with pH adjusted to 7.2. To minimize the rundown of Ca current, one or more of the following were sometimes included: 9 mM creatine phosphate, 30 U/ml creatine kinase, 0.1 mM dithiothreitol, and/or 10 PM leupeptin. With this solution a junction potential of -5 mV was observed in the extracellular solutions and was not corrected for in the results. The resistances of the patch pipettes used in this study were 0.4-1.5 MQ, which allowed adequate passive loading of the cells with the indicator contained in the pipette in 55 min after rupturing the membrane patch and gaining access to the cell interior. Between voltage-clamp pulses the membrane potential was usually held at -70 mV to minimize rundown of Ca current. The holding potential was changed to -50 mV, 300 ms before test pulses were given, to inactivate Na channels. The test pulses were terminated by returning to -50 mV for 1 s. Then the cells were again repolarized to -70 mV. Data [E,, current, fluorescence, and fluorescence ratios (FdOO, F500, and F400/F500)] were recorded on videotape at 11 kHz via a Neurocorder (DR886, Neuro Data Instruments) and subsequently digitized on a computer using Vacuum software (MBC Systems, Baltimore, MD) for analysis. Calibration of indo-l fluorescence. Calibration solutions were prepared according to the method described by Bers (4). These solutions contained (in mM) 140 KCl, 5 ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 20 HEPES, and O-6 CaC12, with pH adjusted to 7.20 at 31°C with KOH and ionic equivalents (0.5 z]zilCi, where 2; and Ci are the valence and concentration of ion i, respectively) adjusted to 0.16 M with KCl. The predicted apparent association constant for Ca-EGTA (&-Eo& under these conditions was 7.03 X lo6 M-l (26). Using Scatchard analysis and an Orion Ca electrode (4), K ca-EoTA measured for these solutions was 7.73 x lo6 M-l, and the purity of EGTA was measured to be 97.0% (Sigma, lot 48F-5618). Figure 1, A and B, shows the Scatchard plot and Ca electrode calibration for these solutions. After addition of 20 PM indo-l (5OO-fold dilution), these solutions were used for calibration of indo-l fluorescence in the experimental chamber on the microscope at 31°C. The free [Cal for the calibration solutions was recalculated to include Ca binding to 20 PM indo-l [assuming dissociation constant (&) = 300 nM]. This minor correction is only important where total [Cal is close to total [EGTA] (the 2 points just below saturation). The resulting calibration is in Fig. lC, and a Hill curve is fit to the data points with the Hill coefficient = l
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C946
CARDIAC
CA,
TRANSIENTS
AND
NA-CA
400 0
Ii+ s ‘;
-40
2
Bomd
k 8 u-I
3
Ca
4
100
-160
5
-10
-8
htvl)
-6
-4
40 msec
80
msec
log [Cal 2 -c E -l.OaI 2
z=0.8 2
msec
1 set
-2
1.2
.: 0.0
20
-120
C
1.6
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+v N u c-l
-80
8 01
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9c
n
a, B z 8 iii
EXCHANGE
ii
5
50 msec
-2.o-
1
1
1
1
I
- 10 -9
-8
-7
-6
-5
1
-4
1
-3
log [Cal FIG. 1. Calibration of indo-1. A: Scatchard plot. Degree of Ca binding to EGTA was calculated via a Scatchard analysis at free [Cal measured by a Ca-sensitive electrode. Kca-EoTA was calculated to be 7.7 x lo6 M-l under the present conditions. 23: electrode measurement of free [Cal. Free Ca concentrations in the calibration solutions (see METHODS) were calculated after determination of Kca-EoTA and total EGTA concentration for these solutions (A). Slope of electrode response is 28.3 mV/decade. C: indo-l calibration curve. In vitro calibration curves were constructed with solutions used during experiments. Ratio of fluorescence emission (400/500 nm) is displayed as a function of pCa [-log (free [Cal)].
1, ratio = Rmin+ (Rmin- R,J/(l + K* /[Cal). Rminand R maxare the minimum and maximum ratio of the fluorescence at 4OO/5OOnm (0.098 and 1.48, respectively), and K* is a lumped constant (753 nM) that is proportional to the effective dissociation constant (i.e., & multiplied by the ratio of fluorescence at 500 nm of the free dye to the saturated dye, Ref. 25). It should be noted that we are using in vitro calibrations which, although carried out with great care, may not be truly representative of the dye characteristics inside the cells (e.g., Ref. 32). Konishi et al. (32) demonstrated that the related Ca indicator fura- appeared to bind extensively to intracellular proteins in skeletal muscle and that this lowered the Ca affinity and increased the fluorescence of the free dye. These effects imply that in vitro calibration curves for fura- will lead to the underestimation of [Cal;. It is possible that our indo-l calibrations could also be subject to this type of problem. RESULTS
Clamp pulse duration dependence of Cai transient with Na,. Figure 2 illustrates the influence of duration of
FIG. 2. Duration dependence of intracellular Ca concentration ([Cali) transient from a holding potential of -50 mV in normal Tyrode solution. Depolarizing pulses to -15 mV were produced every 5 s. 300 ms before a depolarization of indicated duration, holding potential was decreased from -70 to -50 mV. Membrane potential was returned to -50 mV for 1 s after the depolarization. Top: resulting [Cal; transients, which are average of 3 depolarizing pulses at indicated duration. Bottom: superimposed Ca currents recorded during [Cal; transients shown in top. Currents are extracellular Ca (Ca,)-sensitive difference currents determined by subtracting currents recorded in Na-free Ca-free solution from those recorded in the Na- and Ca-containing solution.
voltage-clamp pulses (from -50 to -20 mV) on the amplitude of Ca; transients. The cell is in a modified normal Tyrode solution with 30 PM tetrodotoxin to block residual Na current. In this experiment, single voltageclamp pulses are given at a frequency of 0.2 Hz. The duration of the pulses increases sequentially from 5 to 80 ms as illustrated in Fig. 2 and then starts again at 5 ms. Each trace in Fig. 2 is the average signal from three records at the indicated duration. Membrane currents recorded simultaneously are shown in the bottom panel of Fig. 2, and it can be seen that the inward Ca current had reached the same peak value during each pulse (3 ms after the voltage step). Despite the constant peak Ca current, the amplitude of the Cai transient increased almost twofold as the pulse duration was increased from 5 to 80 ms. This increase is not attributable to a progressive increase in SR Ca loading during the pulse protocol, since the same results were observed when 0.2Hz depolarizing pulses were given continuously (each 5ms pulse follows an 80-ms pulse) or when there was a long pause before the 5-ms pulse. Increasing pulse duration from 5 to 80 ms did not alter the rate of rise of the Cai transient but increased the time to the peak of the Ca; transient, in agreement with Cannel1 et al. (13). The kinetics of the rise in the fluorescence ratio may be partly limited by the indicator and our recording bandwidth. However, the rate of rise of [Cali can be further increased by more positive test potentials (when tested, e.g., in the cell in Fig. 6), indicating that the rate of rise of the Cai signal is not “saturated” under these conditions.
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CARDIAC
CA;
TRANSIENTS
After the results in Fig. 2 were obtained, the superfusate was changed to Na-free Ca-free medium (145 mM Nmethyl-D-glucamine, 6 mM MgCl,) for 1 min (and in other experiments for up to 5 min) to remove extracellular Na while preventing the cellular Ca load associated with Na removal. Pulses given in this Ca-free solution did not elicit any contraction, Cai transient, or Ca current. This indicates that Ca, is required for excitationcontraction coupling. The current records obtained could also be used as Ca-independent currents for leak subtraction. Clamp pulse duration dependence of Cai transient in the absence of Na,. When Ca was reintroduced (still in the absence of Na,), Cai transients and Ca currents were again observed (Fig. 3). In this situation Na,-dependent Ca extrusion is prevented, and the role of Na-Ca exchange in the duration dependence can be tested. Figure 3A shows that the Cai transient progressively increases with increasing pulse duration. However, this conclusion is complicated by a progressive rise in diastolic [Cal; (and presumably SR Ca) when Ca, is reintroduced in the absence of Na. This also precludes signal averaging, since diastolic [Cal; is changing. Despite this problem, it is clear that the duration-dependent increase in Ca; transient is occurring in the cell in Fig. 3. The traces in Fig. 3A from the 80-ms pulse and the subsequent 5-ms pulse illustrate that despite the higher Ca load at the time of the second 5-ms pulse, the amplitude of the Cai transient is smaller. Figure 3B shows superimposed Ca current traces from the first five pulses in Fig. 3A. The amplitude of the Ca current during the first 5- and lo-ms pulses is less than that for the 20-, 40-, and 80-ms pulses because the bath [Cal is still increasing to 2 mM. This could contribute to the lower Cai transients at the first 5- and 5 msec
10
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AND
c947
EXCHANGE
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lo-ms pulses. The peak Ca current at the second 5ms pulse is the same as the 80-ms pulse (Fig. 3C). Thus, with the same peak Ca current, the 5ms pulse produces a smaller Cai transient than the 80-ms pulse. In Na-free solution, all cells eventually showed progressive increases in [Cal; (even at -50 mV, e.g., Fig. 8). The rise was particularly rapid in the cell in Fig.3, so the bracketing 5-ms pulses are especially important. In other cells, the rise in [Cal; during Na-free superfusion was slow enough that diastolic [Cal; did not change from one pulse to the next (e.g., Figs. 9 and 10) or even for several cycles of the voltage-clamp protocol (Fig. 6, and see DISCUSSION).
Figure 4 illustrates the results from Figs. 2 and 3 in which the amplitude of the Cai transients are normalized to the value at the 80-ms pulse. It is clear that removal of Na, does not abolish the increase in the size of the Cai transient observed with longer depolarizations. In the absence of the complications discussed above, the Cai transient produced by the 5-ms pulse in Na-free solution would be expected to lie between the 5-ms values shown in Fig. 4 (open circles), i.e., similar to the 5-ms value in the presence of Na, (filled circle). It is therefore reasonable to expect that the duration dependence of the peak Cai transient is similar in the presence and absence of Na, (and not due to Na-Ca exchange). The duration dependence of the Cai transient (at -20 to -10 mV) was observed in each of 15 cells studied with the foregoing protocol (6 in Na-free and 15 in 145 mM Na + tetrodotoxin). In four experiments in which the identical protocols were performed in the presence and absence of extracellular Na, the peak [Cali with a 5-ms depolarization, as percent of the peak [Ca] -at the preceding 80-ms depolarization, was 42 t 8% (with Na) and 47 t 8% (Na free). In the process of obtaining the records in Figs. 2 and 3, there was significant rundown of both the Cai transient and the Ca current. In this cell, particularly in Fig. 2, the Cai transient is very small, but the Ca current is still relatively large. This raises the possibility that the clamp duration dependence of the twitch might be directly attributable to the integrated Ca current changes (rather -
1 set
FIG. 3. Duration d.ependence in Na-free soluof 1Cal; transients tions. Cell is same a in Fig. 2, with identical voltage-clamp protocol except that superfusion solution was Na free. A: [Cal; transients resulting from 6 consecutive depolarizations, each of duration indicated above the transient. [Cali at the holding potential progressively increases. Nevertheless, [Cali transient produced by the final 5-ms pulse is smaller than that produced by the preceding 80-ms depolarization. B and C: Ca,-sensitive difference currents determined by subtracting current measu red in Na-free Ca-free solution from that meas ured in 0 Na and 2mM Ca solution. B: superimposed currents for first 5 pulses. C: currents recorded from the 80-ms depolarization superimposed on current recorded from the final 5-ms depolarization.
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.I
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4. Extracellular Na (Na,)-dependent effects on duration dependence. Results from Figs. 2 (solid- circles) and 3 (open circles) are replotted as a fraction of the [Cali transient produced by 80-ms depolarization {A[ca] (% max)!. The [ Cal; trans ient produced by fi nal 5ms depolarization in Fig. 3 is also d&layed with a dashed line directly to [Cali transient produced by the 80-ms depolarization. FIG.
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C948
CARDIAC
CA;
TRANSIENTS
than changes in SR Ca release). Consideration of the quantity of Ca entry via Ca current vs. the Cai transient can be made with the following assumptions: 1) cell volume equals 30 pl (-36% as mitochondrial space, Ref. ZO), 2) intracellular Ca buffering as estimated by Fabiato (20), 3) 50 ,uM intracellular indo-l (Kd = 300 nM), and 4) [Cali is in rapid equilibrium with these buffers. The integrated Ca currents for the 5- and 80-ms pulses in Fig. 2 are 2.8 and 20.3 ,umol Ca/l cell water, respectively. In the presence of the above cellular buffering, this Ca influx is calculated to raise [ Ca]i from a diastolic value of 160 to 180 and 320 nM, respectively (compared with estimated peak [Cal; values from indo-l fluorescence of 260 and 350 nM). Thus, in this extreme case, Ca entry via Ca current cannot quite account for the Ca transients observed. However, with the assumptions involved and reservations about the precision of indo-l calibrations in the cell, we cannot rule out the possibility that the duration dependence of the Cai transient in Fig. 2 might be due to the duration dependence of integrated Ca current. A similar analysis was carried out for the results in Fig. 3 (Na free). The integrated Ca current for the second 5-ms pulse could raise [Cali from the estimated diastolic value of 284 nM to only 292 nM (rather than the estimated peak value of 410 nM) and for the 80-ms pulses from 225 to 308 nM (rather than 450 nM). Thus Ca current does not seem sufficient to explain the rise in free [Cali. Nevertheless, for the data in Fig. 3, the increase in the change of total Cai with increasing duration (calculated from the change in free [Cal;, as above) and the increase of integrated Ca current are relatively parallel (Fig. 5). The change in Ca; that may be attributed to the SR is relatively constant. Thus the change in Ca entry via Ca current may explain the change in peak [Cal; with the SR Ca release remaining relatively con-
AND
EXCHANGE
NA-CA
stant. However, changes in diastolic Ca loading may complicate this investigation. Therefore, we present an additional experiment, in which similar relative changes in the Cai transient amplitude were observed, but in which there was no appreciable increase in diastolic [Cal; during the protocol (Fig. 6). Figure 6, A and B, shows Cai transients elicited in Nafree solution from a holding potential of -50 mV to test potentials of -10 and -20 mV, respectively. In this cell the Ca current was small, and the Ca; transients were large when compared with the cell in Figs. 2 and 3 (peak Ca current = 0.4 nA in Fig. 6A). The observed cell-tocell variability did not appear to depend simply on cell size, but no rigorous analysis was done to test this possibility. Thus the integrated Ca current from this cell (Fig. 7) cannot directly explain the increase in peak [Cal; with increasing pulse duration (i.e., the integrated Ca current for the 80-ms pulse would only be expected I oooiA
2 c
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10 msec
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5. Estimation of Ca movements underlying [Cal; transients in Fig. 3. Results from Fig. 3 were used to calculate relative contribution of Ca from Ca current (Ica) and other sources to [Cal; transient recorded in Na-free solution. Total Ca influx via Ca channels was calculated by integrating the Ca,-sensitive difference current (open circles). Amount of Ca involved in each [Cal; transient (solid circles) was calculated by assuming that [Cali is in rapid equilibrium with cellular calcium buffers as described by Fabiato (20) with an additional 50 PM indo-l (&ca = 300 nM) for a 30 pl cell. Amount of Ca from other sources [triangles, from sarcoplasmic reticulum (SR)?] are calculated as difference between total Ca based on [Cal; transient and Ca influx attributable to the Ca current.
I 20
I 40
-.O
L 60
mV 0 mV
L 80
1 100
DURATION (ms)
FIG.
6. Effect of membrane potential on duration dependence of [Cali transient. Cell was superfused with Na-free solution, held at -50 mV, and depolarized for indicated duration every 5 s. A: test potential = -10 mV. iCa]i transients are signal averaged (II = 2 or 3) and filtered measured during at 80 Hz with a n &pole Bessel filter. Inset: current transients. 23: test potential = -20 mV. The same displayed [Cali protocol was used as in A except that cell was depolarized from -50 to -20 mV. C: voltage dependence. Peak [Cali during each transient is plotted as a percentage of peak [Cal; for the 80-ms depolarization in A and B. Results for test potential = 0 mV are taken from a representative cell. FIG.
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CARDIAC
From
A [Cal A&
l
______ From
Duration
- ------SR
CA; TRANSIENTS
A
--------A
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(msec)
FIG. 7. Estimation of Ca movements underlying the in Fig. 6A, at-10 mV. Contributions of Ca current sources [e.g., sarcoplasmic reticulum (SR)] to [Cal; determined for results shown in Fig. 6A. Calculations as in Fig. 5.
[Cali transients (I& and other transient were were carried out
to raise Cai by 3.4 ,umol/l cell water or [Cal; from 160 to 185 nM rather than the observed peak [Cal; of 900 nM). In this case, we estimate that the SR Ca release may account for the vast majority of the Cai transient (95%). Furthermore, changes in the amount of SR Ca released seem necessary to explain the increase of the Cai transient with increasing pulse duration (Fig. 7). Nevertheless, there is a gradation of the integrated Ca current, and this cannot be ruled out as contributing to the gradation of SR Ca release (see DISCUSSION). The cell in Fig. 6 (in contrast to that in Fig. 3) did not show a cumulative increase in diastolic [Cali (perhaps due to the exposure to Ca-free Na-free solution for 5 min before reintroduction of Ca). The amplitude of the Ca current was also the same in each pulse of the train (see Fig. 6A, inset). Thus signal averaging could be performed and the amplitude of the Ca; transients can be directly compared. The peak Cai transients are shown normalized in Fig. 6C, and it can be seen that the duration dependence of the peak [Cal; over the range 5-80 ms is more pronounced at more negative test potentials over the range -20 to 0 mV. This dependence on test potential is also apparent in the presence of Na, and could still be observed at -35 mV (not shown). When test potentials are 20 mV there is often little or no apparent duration dependence for durations 25 ms (Fig. 6C). The experiment in Fig. 6, in Na-free solution, demonstrates unequivocally that the reduction of the Cai transient by short depolarizing pulses described by Cannell et al. (13) cannot be attributed to Ca extrusion by Na-Ca exchange. It is possible that Na-Ca exchange cannot extrude Ca rapidly enough to greatly modify the peak [Cal; determined by the combination of Ca current influx and SR Ca release (or at least not rapidly enough to explain the duration dependence of the Cai transient). There is, however, good evidence that Na-Ca exchange can compete with the reaccumulation of Ca by the SR during relaxation (7, and see below). Effect of alterations of Na, on slow changes in [Cal;. We have strongly biased the experimental conditions (i.e., low intracellular Na) to minimize Ca entry via the Na-Ca exchange. However, with Na-free extracellular
AND
NA-CA
c949
EXCHANGE
solution, even a small amount of intracellular Na would thermodynamically favor Ca entry via Na-Ca exchange. On the other hand, the low [Na]i (especially with Nafree solution) would kinetically limit the quantity of Ca entry via Na-Ca exchange. To clarify this issue, we examined the [Cali changes associated with long voltage steps in the presence or absence of Na, (Fig. 8). When Na, is abruptly removed at E, = -50 mV without simultaneous Ca removal (Fig. 8A), [Cali gradually increased to a new steady level in -30-45 s (tl,, = 15 s). When Na, is reintroduced, [Cal; declines rapidly along a simple exponential (tli, = 0.97 s). The rapid Ca extrusion seemslikely to be due to Na-Ca exchange. The gradual rise in [Cal; might be due to a slow influx of Ca down its electrochemical gradient which can no longer be extruded by Na-Ca exchange. It could also be attributed to Ca entry via Na-Ca exchange, but this may be limited by the low [Na];. In the presence of Na,, depolarization leads to sustained increases in [Ca] i while hyperpolarization for several seconds leads to a decrease in [Cali (Fig. 8B, left). In this case, the increase in [Cal; could be due to either
A Y wc + cv 0 0
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5s Vc + nl c5:
20 set 0
FIG. 8. Effect of Na, on Na-Ca exchange. A: cell was held at -50 mV, and superfusing solution was changed from a 145 mM Na and 2 mM Ca Tyrode solution to a Na-free solution for period indicated by double line. B: solution change protocol in A was repeated in the same cell, but long steps (5-15 s) of membrane potential (Vm) to -100, 0, and +50 were imposed at various times.
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c950
CARDIAC
CA;
TRANSIENTS
increased Ca entry via Ca channels or Na-Ca exchange (or decreased extrusion of Ca by Na-Ca exchange). The Ca loss with hyperpolarization could be due to Na-Ca exchange. The depolarization to +50 mV produces a larger increase in [Cal; than that at 0 mV. This would be expected of the Na-Ca exchange but not of Ca flux through Ca channels. Thus Na-Ca exchange is probably involved in the [Cali changes in the presence of Na,. On the other hand, in the absence of Na, (Fig. 8B), depolarization to +50 mV decreases [Cali, while hyperpolarization to -100 mV increases [Cal;. Similar effects of E, on [Cali and tension have been reported in Purkinje fibers exposed to Na-free solutions (14). These changes in [Cal; are not expected from the Na-Ca exchange system but are consistent with uncoupled Ca movements (e.g., via Ca channels) due primarily to changes in Ca driving force. Thus the Na-Ca exchange does not seem to participate in transsarcolemmal Ca flux in the absence of Na, (with low [Na];). The experiment in Fig. 8 was done without the intermediate superfusion with Na-free Ca-free solution that was used in all the preceding experiments. This Na-free Ca-free solution would be expected to reduce [Na]i even lower, making it extremely unlikely that Na-Ca exchange mediates significant Ca flux in the Na-free solutions used in Figs. 3-7 and 9 and 10.
Effect of Na, on the decline of the Cai transient. To assess the effect of Na, on the decline of [Cali, we measured [Cal; associated with depolarizing pulses from -50 to +15 mV for either 50 or 1,000 ms (Fig. 9). The ls pulse in both control and Na-free solutions exhibits I 0.7
..:::& 44
145 Na
AND
3
0.5
i.. ..
7=
124
0 Na
T = 217
maec
0.3
= 182
maec
0.1
400 msetc
msec FIG. 9. Effect of Na, on decay of [Cal; transient. Cell was held at -50 mV and depolarized to +I5 mV for either 50 or 1,000 ms (shown by double line below each transient). Superfusion solutions contained 145 mM Na and 30 PM tetrodotoxin (left) or were Na free (right). Decay of [Cal; transients at +15 mV (ls depolarization only) and at -50 mV were fit with monoexponential functions (solid curve) with rate constants T. Bottom panels show superimposed Ca,-sensitive currents (determined as in Fig. 2) for the 50- and l,OOO-ms depolarizations.
+ N s
EXCHANGE
both a phasic rise in [Cal; (as in the 5O-ms pulse) and also a tonic component of elevated [Cal;. The Cai transients shown in Fig. 9 were selected because the diastolic [Cal; was the same in the presence and absence of Na,. When this was the case, the [Cal; at the peak of the transient and at the point of repolarization were also often similar in the presence and absence of Na,, and these cases were particularly useful for analyzing the decline of [Cali. As expected, increases in diastolic [Cali (and presumably SR Ca content) in Na-free solutions were associated with increases in the size of the Cai transient unless there were signs of cellular Ca overload. Each declining phase of [Cal; was fit with a single exponential function, and the time constants are shown in Fig. 9. In the absence of Na,, the rate of [Cali decline upon return to the holding potential is slowed in each case (by 23% after the 50-ms pulse and by 19% after the l-s pulse). Our results are consistent with Na-Ca exchange being responsible for a substantial fraction (e*g*, 20%) of the [Cali decline during relaxation. The SR is likely to be responsible for the remaining 80% of the decrease in [Cal;. Thus the Na-Ca exchange may compete with the SR to reduce [Cali. This result agrees with other recent results using relaxations after rapid-cooling contractures to assessthe relative contributions of the SR Ca pump and the Na-Ca exchange to relaxation in rabbit and guinea pig ventricular muscle (7, 8, 29). The decline in [Cali during the l-s pulse to +15 mV was not appreciably slowed in the absence of Na,. In this case, Na-Ca exchange does not seem to be involved in relaxation at +15 mV, and the SR may be primarily
0.7
n
NA-CA
400 msec
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CARDIAC
CA;
TRANSIENTS
AND
EXCHANGE
c951
be responsible for the elevation of [Cal; during the depolarization (See DISCUSSION).
responsible for reducing [Cali. This is consistent with a voltage-dependent depression of Ca extrusion via Na-Ca exchange (1, 7, 12, 30, 31) and the lack of voltage dependence of the SR Ca pump during relaxation (7). Indeed, in every cell the Na, dependence of relaxation was smaller at more positive membrane potentials. The size of the initial phasic decline of [Cal; and the tonic level of [Cal; during the l-s depolarization was variable among the 12 cells studied with this protocol. In some cells the tonic elevation of [Cal; was a small fraction of the peak [Cal; (e.g.,
BERS,DONALD M.,W.J. LEDERER,ANDJOSHUA R. BERLIN. Intracellular Ca transients in rat cardiac myocytes: role of NaCa exchange in excitation-contraction coupling. Am. J. Physiol. 258 (Cell Physiol. 27): C944-C954, 1990.-Membrane current and intracellular Ca concentration ([Cal;) transients were recorded from isolated rat ventricular myocytes under voltageclamp control. The cells were dialyzed by the patch pipette solution, which contained the fluorescent Ca indicator indo-l and 0.5 mM Na. Under these experimental conditions, Ca entry via Na-Ca exchange did not appear to be appreciable even in the absence of extracellular Na. Increasing the duration of voltage-clamp pulses from 5 to 80 ms produced [Cali transients of increasing amplitude, while the peak Ca current was not changed. This duration dependence of the [Cal; transient was most demonstrable at more negative test potentials (e.g., -20 to -30 mV) and was not qualitatively modified by Na-free solutions. This latter result indicates that Ca extrusion by NaCa exchange is not responsible for the smaller [Cali transients observed when the membrane is repolarized after very brief depolarizations. Although the peak Ca current was not changed by increasing pulse duration, the integrated Ca current was increased. These observations are consistent with a Ca-release mechanism in cardiac excitation-contraction coupling in which 1) the Ca-release process can be modulated by membrane potential or 2) the Ca entering the cell via Ca channels has a preferential access [compared with Ca from the sarcoplasmic reticulum (SR)] to the site(s) that control SR Ca release. The role of Na-Ca exchange in the decline of [Cal; during relaxation was also explored. Removal of extracellular Na (Na,) resulted in 20% slowing of the decline in [Cali during relaxation. From this, we conclude that the Na-Ca exchange competes with SR to remove Ca from the cytoplasm and that under our control conditions the exchanger may account for 20% of this decline. The Na, dependence of relaxation was reduced at more positive membrane potentials and increased by SR Ca loading.
21201;
tions and intracellular Ca transients change along with changes in Ca current in a voltage-dependent manner (10, 13, 33, 38).
Further tests of the Ca-induced Ca-release theory come from experiments in which voltage-clamp pulse duration is varied. In both guinea pig (10) and cat myocytes (16), the duration of a voltage-clamp pulse had no immediate effect on the peak level of intracellular Ca concentration ([Cali) reached if the voltage pulse was terminated after the peak of the Ca current. Thus SR Ca release would seem to depend on the peak Ca current in a manner consistent with a Ca-induced Ca-release process. In contrast to those results, the peak value of the [Cali transient in rat ventricular cells was increased by increasing the duration of the voltage-clamp pulse (from 5 to 40 ms) despite a constant peak of the Ca current (at ~5 ms, Ref. 13). Cannel1 et al. (13) interpreted their results to support a possible modulatory role of membrane potential ( E,) on the process of SR Ca release in cardiac muscle. That is, they suggested that the rapid repolarization at 5-20 ms might prematurely terminate SR Ca release and hence lead to the smaller intracellular Ca transient. This is consistent with their observations and similar to the interpretation of results obtained in skeletal muscle (35). The authors could not, however, rule out the possibility that a Ca-induced Ca-release mechanism might also be modulated by E, in some indirect manner (13). In this regard, Ca extrusion via the sarcolemmal NaCa exchange is voltage dependent, such that net Ca extrusion is increasingly favored at more negative potentials (30, 31, 37, 39, 40). Thus it is possible that repolarization after a very brief depolarization described above might accelerate Ca extrusion via Na-Ca exchange and cardiac muscle; indo- 1; calcium current thereby reduce the peak level of [Cal; reached during contraction. Indeed, the rapid repolarization of the rat ventricular action potential is probably responsible for NORMAL DEPOLARIZATION of mammalian cardiac muscle the rapid extrusion of Ca observed during contraction leads to Ca influx via Ca channels (and perhaps also via (41). In the present study, we have examined whether Na-Ca exchange, e.g., Refs. 6, 37). Fabiato (20) has the duration dependence of the [Cali transient might be estimated that the amount of Ca entry via Ca current is due to a Na-Ca exchange-dependent modulation of the not sufficient to activate contraction directly (but see [Cal; transient. Refs. 59). In an elegant series of studies in mechanically The rate of relaxation and Ca extrusion in cardiac skinned cardiac myocytes, Fabiato (21-23) has charac- muscle is also accelerated at more negative potentials terized a Ca-induced Ca-release mechanism, whereby a under conditions in which Na-Ca exchange is the prismall amount of Ca influx may “trigger” release of a mary means of relaxation or Ca extrusion (1, 7, 12, 30). larger amount of Ca from the sarcoplasmic reticulum Furthermore, this Ca extrusion can make a significant (SR). Evidence from intact myocytes that supports this contribution to the amount of Ca removed from the theory of excitation-contraction coupling is that contraccytoplasmic space during relaxation (7,8,29). We, therec944
0363-6143/90
$1.50 Copyright
0 1990 the American
Physiological
Society
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CARDIAC
CA;
TRANSIENTS
fore, attempted to determine the importance of the NaCa exchange mechanism in the time-dependent changes of [Cal; in rat ventricular myocytes during both activation and relaxation by manipulation of extracellular ion concentrations and controlling intracellular ion contents with a patch electrode in whole cell voltage-clamp mode.
METHODS
Myocyte isolation. Myocytes were isolated from adult rat hearts using a modified version of the procedure described by Mitra and Morad (36). After pretreatment with heparin sodium ( 1,000 IU/kg ip), rats were injected with pentobarbitone (50 mg/kg ip), and hearts were excised and perfused on a Langendorff apparatus for 5 min with a solution containing (in mM) 135 NaCl, 5.4 KCl, 1 MgClz, 0.33 NaHzP04, 11 glucose, and 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) at pH 7.4 and 37OC. Then collagenase (0.5 mg/ ml, Boehringer-Mannheim) and protease (0.07 mg /ml, Sigma type XIV) were added, and the heart was perfused for an additional 4-5 min. The heart was then minced and washed, and the cells were stored in the above solution at 37°C (with 0.2 mM CaClz and lacking enzymes). Fluorescence recording apparatus. The optical system was similar to that described for fura- by Cannel1 et al. (13). The cell was illuminated using a 75-W xenon arc lamp (Photon Technologies International, South Brunswick, NJ) via a 350-nm band-pass filter (10 nm full width at half-maximum transmission), through a 400-nm dichroic mirror and a x63 oil-immersion Neofluor objective (Zeiss). Optical signals from a cell were directed to two photomultiplier tubes (EM1 9789B, Thorn EMI). An adjustable window was used to restrict the area of illumination to a square area of 200 pm2 to minimize background fluorescence from the remainder of the field. The fluorescent signals to the two photomultipliers were filtered with band-pass filters at 400 and 500 nm, respectively (40 nm full width at half-maximum transmission). The patch pipette contained the fluorescent Ca indicator indo-1. After formation of a high-resistance seal between the cell membrane and the voltage-clamp electrode, background fluorescence was measured. This value (at 400 and 500 nm) was automatically subtracted from all subsequent fluorescence measurements from that cell using a digital sample and hold circuit. The membrane patch was then ruptured to establish whole cell voltage clamp and to allow diffusion of the pipette contents into the cell. The ratio of emitted fluorescence at 400/500 nm was used to calculate [Cal, based on in vitro calibrations (see below). Experimental procedure. The cells were allowed to settle onto a glass cover slip, which formed the bottom of the experimental chamber mounted on the stage of an inverted microscope (15). The cells were superfused with a modified normal Tyrode that contained (in mM) 145 NaCl, 4 KCl, 1 MgCl,, 2 CaCIZ, 10 glucose, and 10 HEPES, with pH adjusted to 7.40 with NaOH. The bath was maintained at 31 t 1°C by water jacketing up to the bath inlet. In all Na-containing solutions, 30 PM tetro-
AND
NA-CA
EXCHANGE
c945
dotoxin was included to block Na current. For Na-free solutions, Na was replaced with 145 mM N-methyl-Dglucamine (and pH was adjusted with HCl). For solutions that were also Ca free, Ca was replaced with 5 mM MgCIZ. The whole cell variant of the patch-clamp technique was used to voltage clamp the myocytes using an Axopatch 1C (Axon Instruments, Burlingame, CA). Lowresistance patch pipettes were filled with a solution containing (in mM) 100 cesium glutamate, 20 CsCl, 1 MgC1,, 2.5 KZATP, 30 piperazine-NJ/‘-bis(2-ethanesulfonic acid) (PIPES), 0.05-0.07 indo-l (acid form), and 0.5 NaOH, with pH adjusted to 7.2. To minimize the rundown of Ca current, one or more of the following were sometimes included: 9 mM creatine phosphate, 30 U/ml creatine kinase, 0.1 mM dithiothreitol, and/or 10 PM leupeptin. With this solution a junction potential of -5 mV was observed in the extracellular solutions and was not corrected for in the results. The resistances of the patch pipettes used in this study were 0.4-1.5 MQ, which allowed adequate passive loading of the cells with the indicator contained in the pipette in 55 min after rupturing the membrane patch and gaining access to the cell interior. Between voltage-clamp pulses the membrane potential was usually held at -70 mV to minimize rundown of Ca current. The holding potential was changed to -50 mV, 300 ms before test pulses were given, to inactivate Na channels. The test pulses were terminated by returning to -50 mV for 1 s. Then the cells were again repolarized to -70 mV. Data [E,, current, fluorescence, and fluorescence ratios (FdOO, F500, and F400/F500)] were recorded on videotape at 11 kHz via a Neurocorder (DR886, Neuro Data Instruments) and subsequently digitized on a computer using Vacuum software (MBC Systems, Baltimore, MD) for analysis. Calibration of indo-l fluorescence. Calibration solutions were prepared according to the method described by Bers (4). These solutions contained (in mM) 140 KCl, 5 ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 20 HEPES, and O-6 CaC12, with pH adjusted to 7.20 at 31°C with KOH and ionic equivalents (0.5 z]zilCi, where 2; and Ci are the valence and concentration of ion i, respectively) adjusted to 0.16 M with KCl. The predicted apparent association constant for Ca-EGTA (&-Eo& under these conditions was 7.03 X lo6 M-l (26). Using Scatchard analysis and an Orion Ca electrode (4), K ca-EoTA measured for these solutions was 7.73 x lo6 M-l, and the purity of EGTA was measured to be 97.0% (Sigma, lot 48F-5618). Figure 1, A and B, shows the Scatchard plot and Ca electrode calibration for these solutions. After addition of 20 PM indo-l (5OO-fold dilution), these solutions were used for calibration of indo-l fluorescence in the experimental chamber on the microscope at 31°C. The free [Cal for the calibration solutions was recalculated to include Ca binding to 20 PM indo-l [assuming dissociation constant (&) = 300 nM]. This minor correction is only important where total [Cal is close to total [EGTA] (the 2 points just below saturation). The resulting calibration is in Fig. lC, and a Hill curve is fit to the data points with the Hill coefficient = l
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C946
CARDIAC
CA,
TRANSIENTS
AND
NA-CA
400 0
Ii+ s ‘;
-40
2
Bomd
k 8 u-I
3
Ca
4
100
-160
5
-10
-8
htvl)
-6
-4
40 msec
80
msec
log [Cal 2 -c E -l.OaI 2
z=0.8 2
msec
1 set
-2
1.2
.: 0.0
20
-120
C
1.6
10 msec
+v N u c-l
-80
8 01
5 msec
9c
n
a, B z 8 iii
EXCHANGE
ii
5
50 msec
-2.o-
1
1
1
1
I
- 10 -9
-8
-7
-6
-5
1
-4
1
-3
log [Cal FIG. 1. Calibration of indo-1. A: Scatchard plot. Degree of Ca binding to EGTA was calculated via a Scatchard analysis at free [Cal measured by a Ca-sensitive electrode. Kca-EoTA was calculated to be 7.7 x lo6 M-l under the present conditions. 23: electrode measurement of free [Cal. Free Ca concentrations in the calibration solutions (see METHODS) were calculated after determination of Kca-EoTA and total EGTA concentration for these solutions (A). Slope of electrode response is 28.3 mV/decade. C: indo-l calibration curve. In vitro calibration curves were constructed with solutions used during experiments. Ratio of fluorescence emission (400/500 nm) is displayed as a function of pCa [-log (free [Cal)].
1, ratio = Rmin+ (Rmin- R,J/(l + K* /[Cal). Rminand R maxare the minimum and maximum ratio of the fluorescence at 4OO/5OOnm (0.098 and 1.48, respectively), and K* is a lumped constant (753 nM) that is proportional to the effective dissociation constant (i.e., & multiplied by the ratio of fluorescence at 500 nm of the free dye to the saturated dye, Ref. 25). It should be noted that we are using in vitro calibrations which, although carried out with great care, may not be truly representative of the dye characteristics inside the cells (e.g., Ref. 32). Konishi et al. (32) demonstrated that the related Ca indicator fura- appeared to bind extensively to intracellular proteins in skeletal muscle and that this lowered the Ca affinity and increased the fluorescence of the free dye. These effects imply that in vitro calibration curves for fura- will lead to the underestimation of [Cal;. It is possible that our indo-l calibrations could also be subject to this type of problem. RESULTS
Clamp pulse duration dependence of Cai transient with Na,. Figure 2 illustrates the influence of duration of
FIG. 2. Duration dependence of intracellular Ca concentration ([Cali) transient from a holding potential of -50 mV in normal Tyrode solution. Depolarizing pulses to -15 mV were produced every 5 s. 300 ms before a depolarization of indicated duration, holding potential was decreased from -70 to -50 mV. Membrane potential was returned to -50 mV for 1 s after the depolarization. Top: resulting [Cal; transients, which are average of 3 depolarizing pulses at indicated duration. Bottom: superimposed Ca currents recorded during [Cal; transients shown in top. Currents are extracellular Ca (Ca,)-sensitive difference currents determined by subtracting currents recorded in Na-free Ca-free solution from those recorded in the Na- and Ca-containing solution.
voltage-clamp pulses (from -50 to -20 mV) on the amplitude of Ca; transients. The cell is in a modified normal Tyrode solution with 30 PM tetrodotoxin to block residual Na current. In this experiment, single voltageclamp pulses are given at a frequency of 0.2 Hz. The duration of the pulses increases sequentially from 5 to 80 ms as illustrated in Fig. 2 and then starts again at 5 ms. Each trace in Fig. 2 is the average signal from three records at the indicated duration. Membrane currents recorded simultaneously are shown in the bottom panel of Fig. 2, and it can be seen that the inward Ca current had reached the same peak value during each pulse (3 ms after the voltage step). Despite the constant peak Ca current, the amplitude of the Cai transient increased almost twofold as the pulse duration was increased from 5 to 80 ms. This increase is not attributable to a progressive increase in SR Ca loading during the pulse protocol, since the same results were observed when 0.2Hz depolarizing pulses were given continuously (each 5ms pulse follows an 80-ms pulse) or when there was a long pause before the 5-ms pulse. Increasing pulse duration from 5 to 80 ms did not alter the rate of rise of the Cai transient but increased the time to the peak of the Ca; transient, in agreement with Cannel1 et al. (13). The kinetics of the rise in the fluorescence ratio may be partly limited by the indicator and our recording bandwidth. However, the rate of rise of [Cali can be further increased by more positive test potentials (when tested, e.g., in the cell in Fig. 6), indicating that the rate of rise of the Cai signal is not “saturated” under these conditions.
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CARDIAC
CA;
TRANSIENTS
After the results in Fig. 2 were obtained, the superfusate was changed to Na-free Ca-free medium (145 mM Nmethyl-D-glucamine, 6 mM MgCl,) for 1 min (and in other experiments for up to 5 min) to remove extracellular Na while preventing the cellular Ca load associated with Na removal. Pulses given in this Ca-free solution did not elicit any contraction, Cai transient, or Ca current. This indicates that Ca, is required for excitationcontraction coupling. The current records obtained could also be used as Ca-independent currents for leak subtraction. Clamp pulse duration dependence of Cai transient in the absence of Na,. When Ca was reintroduced (still in the absence of Na,), Cai transients and Ca currents were again observed (Fig. 3). In this situation Na,-dependent Ca extrusion is prevented, and the role of Na-Ca exchange in the duration dependence can be tested. Figure 3A shows that the Cai transient progressively increases with increasing pulse duration. However, this conclusion is complicated by a progressive rise in diastolic [Cal; (and presumably SR Ca) when Ca, is reintroduced in the absence of Na. This also precludes signal averaging, since diastolic [Cal; is changing. Despite this problem, it is clear that the duration-dependent increase in Ca; transient is occurring in the cell in Fig. 3. The traces in Fig. 3A from the 80-ms pulse and the subsequent 5-ms pulse illustrate that despite the higher Ca load at the time of the second 5-ms pulse, the amplitude of the Cai transient is smaller. Figure 3B shows superimposed Ca current traces from the first five pulses in Fig. 3A. The amplitude of the Ca current during the first 5- and lo-ms pulses is less than that for the 20-, 40-, and 80-ms pulses because the bath [Cal is still increasing to 2 mM. This could contribute to the lower Cai transients at the first 5- and 5 msec
10
msec
20
msec
40
msec
80
msec
5 msec
AND
c947
EXCHANGE
NA-CA
lo-ms pulses. The peak Ca current at the second 5ms pulse is the same as the 80-ms pulse (Fig. 3C). Thus, with the same peak Ca current, the 5ms pulse produces a smaller Cai transient than the 80-ms pulse. In Na-free solution, all cells eventually showed progressive increases in [Cal; (even at -50 mV, e.g., Fig. 8). The rise was particularly rapid in the cell in Fig.3, so the bracketing 5-ms pulses are especially important. In other cells, the rise in [Cal; during Na-free superfusion was slow enough that diastolic [Cal; did not change from one pulse to the next (e.g., Figs. 9 and 10) or even for several cycles of the voltage-clamp protocol (Fig. 6, and see DISCUSSION).
Figure 4 illustrates the results from Figs. 2 and 3 in which the amplitude of the Cai transients are normalized to the value at the 80-ms pulse. It is clear that removal of Na, does not abolish the increase in the size of the Cai transient observed with longer depolarizations. In the absence of the complications discussed above, the Cai transient produced by the 5-ms pulse in Na-free solution would be expected to lie between the 5-ms values shown in Fig. 4 (open circles), i.e., similar to the 5-ms value in the presence of Na, (filled circle). It is therefore reasonable to expect that the duration dependence of the peak Cai transient is similar in the presence and absence of Na, (and not due to Na-Ca exchange). The duration dependence of the Cai transient (at -20 to -10 mV) was observed in each of 15 cells studied with the foregoing protocol (6 in Na-free and 15 in 145 mM Na + tetrodotoxin). In four experiments in which the identical protocols were performed in the presence and absence of extracellular Na, the peak [Cali with a 5-ms depolarization, as percent of the peak [Ca] -at the preceding 80-ms depolarization, was 42 t 8% (with Na) and 47 t 8% (Na free). In the process of obtaining the records in Figs. 2 and 3, there was significant rundown of both the Cai transient and the Ca current. In this cell, particularly in Fig. 2, the Cai transient is very small, but the Ca current is still relatively large. This raises the possibility that the clamp duration dependence of the twitch might be directly attributable to the integrated Ca current changes (rather -
1 set
FIG. 3. Duration d.ependence in Na-free soluof 1Cal; transients tions. Cell is same a in Fig. 2, with identical voltage-clamp protocol except that superfusion solution was Na free. A: [Cal; transients resulting from 6 consecutive depolarizations, each of duration indicated above the transient. [Cali at the holding potential progressively increases. Nevertheless, [Cali transient produced by the final 5-ms pulse is smaller than that produced by the preceding 80-ms depolarization. B and C: Ca,-sensitive difference currents determined by subtracting current measu red in Na-free Ca-free solution from that meas ured in 0 Na and 2mM Ca solution. B: superimposed currents for first 5 pulses. C: currents recorded from the 80-ms depolarization superimposed on current recorded from the final 5-ms depolarization.
20
’
1
I
1
0
20
40
60
Durat
Ion
.I
I
80
(msed
4. Extracellular Na (Na,)-dependent effects on duration dependence. Results from Figs. 2 (solid- circles) and 3 (open circles) are replotted as a fraction of the [Cali transient produced by 80-ms depolarization {A[ca] (% max)!. The [ Cal; trans ient produced by fi nal 5ms depolarization in Fig. 3 is also d&layed with a dashed line directly to [Cali transient produced by the 80-ms depolarization. FIG.
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C948
CARDIAC
CA;
TRANSIENTS
than changes in SR Ca release). Consideration of the quantity of Ca entry via Ca current vs. the Cai transient can be made with the following assumptions: 1) cell volume equals 30 pl (-36% as mitochondrial space, Ref. ZO), 2) intracellular Ca buffering as estimated by Fabiato (20), 3) 50 ,uM intracellular indo-l (Kd = 300 nM), and 4) [Cali is in rapid equilibrium with these buffers. The integrated Ca currents for the 5- and 80-ms pulses in Fig. 2 are 2.8 and 20.3 ,umol Ca/l cell water, respectively. In the presence of the above cellular buffering, this Ca influx is calculated to raise [ Ca]i from a diastolic value of 160 to 180 and 320 nM, respectively (compared with estimated peak [Cal; values from indo-l fluorescence of 260 and 350 nM). Thus, in this extreme case, Ca entry via Ca current cannot quite account for the Ca transients observed. However, with the assumptions involved and reservations about the precision of indo-l calibrations in the cell, we cannot rule out the possibility that the duration dependence of the Cai transient in Fig. 2 might be due to the duration dependence of integrated Ca current. A similar analysis was carried out for the results in Fig. 3 (Na free). The integrated Ca current for the second 5-ms pulse could raise [Cali from the estimated diastolic value of 284 nM to only 292 nM (rather than the estimated peak value of 410 nM) and for the 80-ms pulses from 225 to 308 nM (rather than 450 nM). Thus Ca current does not seem sufficient to explain the rise in free [Cali. Nevertheless, for the data in Fig. 3, the increase in the change of total Cai with increasing duration (calculated from the change in free [Cal;, as above) and the increase of integrated Ca current are relatively parallel (Fig. 5). The change in Ca; that may be attributed to the SR is relatively constant. Thus the change in Ca entry via Ca current may explain the change in peak [Cal; with the SR Ca release remaining relatively con-
AND
EXCHANGE
NA-CA
stant. However, changes in diastolic Ca loading may complicate this investigation. Therefore, we present an additional experiment, in which similar relative changes in the Cai transient amplitude were observed, but in which there was no appreciable increase in diastolic [Cal; during the protocol (Fig. 6). Figure 6, A and B, shows Cai transients elicited in Nafree solution from a holding potential of -50 mV to test potentials of -10 and -20 mV, respectively. In this cell the Ca current was small, and the Ca; transients were large when compared with the cell in Figs. 2 and 3 (peak Ca current = 0.4 nA in Fig. 6A). The observed cell-tocell variability did not appear to depend simply on cell size, but no rigorous analysis was done to test this possibility. Thus the integrated Ca current from this cell (Fig. 7) cannot directly explain the increase in peak [Cal; with increasing pulse duration (i.e., the integrated Ca current for the 80-ms pulse would only be expected I oooiA
2 c
I
500
+ cu-
1
u c-l
1 set
-
5 msec
10 msec
20 msec
40 msec
80 msec
r
120
u I
-
loo-
, cl-
::
- - -
q ’ cl’ ___ v ____..-‘;-;I
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80
-
60
-
v*’ .’
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-
I"'e --.r"
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-20
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V ...-- V -10 Cl-
20
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60
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I l 0’ 0
(msed
5. Estimation of Ca movements underlying [Cal; transients in Fig. 3. Results from Fig. 3 were used to calculate relative contribution of Ca from Ca current (Ica) and other sources to [Cal; transient recorded in Na-free solution. Total Ca influx via Ca channels was calculated by integrating the Ca,-sensitive difference current (open circles). Amount of Ca involved in each [Cal; transient (solid circles) was calculated by assuming that [Cali is in rapid equilibrium with cellular calcium buffers as described by Fabiato (20) with an additional 50 PM indo-l (&ca = 300 nM) for a 30 pl cell. Amount of Ca from other sources [triangles, from sarcoplasmic reticulum (SR)?] are calculated as difference between total Ca based on [Cal; transient and Ca influx attributable to the Ca current.
I 20
I 40
-.O
L 60
mV 0 mV
L 80
1 100
DURATION (ms)
FIG.
6. Effect of membrane potential on duration dependence of [Cali transient. Cell was superfused with Na-free solution, held at -50 mV, and depolarized for indicated duration every 5 s. A: test potential = -10 mV. iCa]i transients are signal averaged (II = 2 or 3) and filtered measured during at 80 Hz with a n &pole Bessel filter. Inset: current transients. 23: test potential = -20 mV. The same displayed [Cali protocol was used as in A except that cell was depolarized from -50 to -20 mV. C: voltage dependence. Peak [Cali during each transient is plotted as a percentage of peak [Cal; for the 80-ms depolarization in A and B. Results for test potential = 0 mV are taken from a representative cell. FIG.
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CARDIAC
From
A [Cal A&
l
______ From
Duration
- ------SR
CA; TRANSIENTS
A
--------A
?
(msec)
FIG. 7. Estimation of Ca movements underlying the in Fig. 6A, at-10 mV. Contributions of Ca current sources [e.g., sarcoplasmic reticulum (SR)] to [Cal; determined for results shown in Fig. 6A. Calculations as in Fig. 5.
[Cali transients (I& and other transient were were carried out
to raise Cai by 3.4 ,umol/l cell water or [Cal; from 160 to 185 nM rather than the observed peak [Cal; of 900 nM). In this case, we estimate that the SR Ca release may account for the vast majority of the Cai transient (95%). Furthermore, changes in the amount of SR Ca released seem necessary to explain the increase of the Cai transient with increasing pulse duration (Fig. 7). Nevertheless, there is a gradation of the integrated Ca current, and this cannot be ruled out as contributing to the gradation of SR Ca release (see DISCUSSION). The cell in Fig. 6 (in contrast to that in Fig. 3) did not show a cumulative increase in diastolic [Cali (perhaps due to the exposure to Ca-free Na-free solution for 5 min before reintroduction of Ca). The amplitude of the Ca current was also the same in each pulse of the train (see Fig. 6A, inset). Thus signal averaging could be performed and the amplitude of the Ca; transients can be directly compared. The peak Cai transients are shown normalized in Fig. 6C, and it can be seen that the duration dependence of the peak [Cal; over the range 5-80 ms is more pronounced at more negative test potentials over the range -20 to 0 mV. This dependence on test potential is also apparent in the presence of Na, and could still be observed at -35 mV (not shown). When test potentials are 20 mV there is often little or no apparent duration dependence for durations 25 ms (Fig. 6C). The experiment in Fig. 6, in Na-free solution, demonstrates unequivocally that the reduction of the Cai transient by short depolarizing pulses described by Cannell et al. (13) cannot be attributed to Ca extrusion by Na-Ca exchange. It is possible that Na-Ca exchange cannot extrude Ca rapidly enough to greatly modify the peak [Cal; determined by the combination of Ca current influx and SR Ca release (or at least not rapidly enough to explain the duration dependence of the Cai transient). There is, however, good evidence that Na-Ca exchange can compete with the reaccumulation of Ca by the SR during relaxation (7, and see below). Effect of alterations of Na, on slow changes in [Cal;. We have strongly biased the experimental conditions (i.e., low intracellular Na) to minimize Ca entry via the Na-Ca exchange. However, with Na-free extracellular
AND
NA-CA
c949
EXCHANGE
solution, even a small amount of intracellular Na would thermodynamically favor Ca entry via Na-Ca exchange. On the other hand, the low [Na]i (especially with Nafree solution) would kinetically limit the quantity of Ca entry via Na-Ca exchange. To clarify this issue, we examined the [Cali changes associated with long voltage steps in the presence or absence of Na, (Fig. 8). When Na, is abruptly removed at E, = -50 mV without simultaneous Ca removal (Fig. 8A), [Cali gradually increased to a new steady level in -30-45 s (tl,, = 15 s). When Na, is reintroduced, [Cal; declines rapidly along a simple exponential (tli, = 0.97 s). The rapid Ca extrusion seemslikely to be due to Na-Ca exchange. The gradual rise in [Cal; might be due to a slow influx of Ca down its electrochemical gradient which can no longer be extruded by Na-Ca exchange. It could also be attributed to Ca entry via Na-Ca exchange, but this may be limited by the low [Na];. In the presence of Na,, depolarization leads to sustained increases in [Ca] i while hyperpolarization for several seconds leads to a decrease in [Cali (Fig. 8B, left). In this case, the increase in [Cal; could be due to either
A Y wc + cv 0 0
0 Na, 2 Ca 750
500
250
15
set
0
B 0 Na, 2 Ca
500
5s Vc + nl c5:
20 set 0
FIG. 8. Effect of Na, on Na-Ca exchange. A: cell was held at -50 mV, and superfusing solution was changed from a 145 mM Na and 2 mM Ca Tyrode solution to a Na-free solution for period indicated by double line. B: solution change protocol in A was repeated in the same cell, but long steps (5-15 s) of membrane potential (Vm) to -100, 0, and +50 were imposed at various times.
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c950
CARDIAC
CA;
TRANSIENTS
increased Ca entry via Ca channels or Na-Ca exchange (or decreased extrusion of Ca by Na-Ca exchange). The Ca loss with hyperpolarization could be due to Na-Ca exchange. The depolarization to +50 mV produces a larger increase in [Cal; than that at 0 mV. This would be expected of the Na-Ca exchange but not of Ca flux through Ca channels. Thus Na-Ca exchange is probably involved in the [Cali changes in the presence of Na,. On the other hand, in the absence of Na, (Fig. 8B), depolarization to +50 mV decreases [Cali, while hyperpolarization to -100 mV increases [Cal;. Similar effects of E, on [Cali and tension have been reported in Purkinje fibers exposed to Na-free solutions (14). These changes in [Cal; are not expected from the Na-Ca exchange system but are consistent with uncoupled Ca movements (e.g., via Ca channels) due primarily to changes in Ca driving force. Thus the Na-Ca exchange does not seem to participate in transsarcolemmal Ca flux in the absence of Na, (with low [Na];). The experiment in Fig. 8 was done without the intermediate superfusion with Na-free Ca-free solution that was used in all the preceding experiments. This Na-free Ca-free solution would be expected to reduce [Na]i even lower, making it extremely unlikely that Na-Ca exchange mediates significant Ca flux in the Na-free solutions used in Figs. 3-7 and 9 and 10.
Effect of Na, on the decline of the Cai transient. To assess the effect of Na, on the decline of [Cali, we measured [Cal; associated with depolarizing pulses from -50 to +15 mV for either 50 or 1,000 ms (Fig. 9). The ls pulse in both control and Na-free solutions exhibits I 0.7
..:::& 44
145 Na
AND
3
0.5
i.. ..
7=
124
0 Na
T = 217
maec
0.3
= 182
maec
0.1
400 msetc
msec FIG. 9. Effect of Na, on decay of [Cal; transient. Cell was held at -50 mV and depolarized to +I5 mV for either 50 or 1,000 ms (shown by double line below each transient). Superfusion solutions contained 145 mM Na and 30 PM tetrodotoxin (left) or were Na free (right). Decay of [Cal; transients at +15 mV (ls depolarization only) and at -50 mV were fit with monoexponential functions (solid curve) with rate constants T. Bottom panels show superimposed Ca,-sensitive currents (determined as in Fig. 2) for the 50- and l,OOO-ms depolarizations.
+ N s
EXCHANGE
both a phasic rise in [Cal; (as in the 5O-ms pulse) and also a tonic component of elevated [Cal;. The Cai transients shown in Fig. 9 were selected because the diastolic [Cal; was the same in the presence and absence of Na,. When this was the case, the [Cal; at the peak of the transient and at the point of repolarization were also often similar in the presence and absence of Na,, and these cases were particularly useful for analyzing the decline of [Cali. As expected, increases in diastolic [Cali (and presumably SR Ca content) in Na-free solutions were associated with increases in the size of the Cai transient unless there were signs of cellular Ca overload. Each declining phase of [Cal; was fit with a single exponential function, and the time constants are shown in Fig. 9. In the absence of Na,, the rate of [Cali decline upon return to the holding potential is slowed in each case (by 23% after the 50-ms pulse and by 19% after the l-s pulse). Our results are consistent with Na-Ca exchange being responsible for a substantial fraction (e*g*, 20%) of the [Cali decline during relaxation. The SR is likely to be responsible for the remaining 80% of the decrease in [Cal;. Thus the Na-Ca exchange may compete with the SR to reduce [Cali. This result agrees with other recent results using relaxations after rapid-cooling contractures to assessthe relative contributions of the SR Ca pump and the Na-Ca exchange to relaxation in rabbit and guinea pig ventricular muscle (7, 8, 29). The decline in [Cali during the l-s pulse to +15 mV was not appreciably slowed in the absence of Na,. In this case, Na-Ca exchange does not seem to be involved in relaxation at +15 mV, and the SR may be primarily
0.7
n
NA-CA
400 msec
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CARDIAC
CA;
TRANSIENTS
AND
EXCHANGE
c951
be responsible for the elevation of [Cal; during the depolarization (See DISCUSSION).
responsible for reducing [Cali. This is consistent with a voltage-dependent depression of Ca extrusion via Na-Ca exchange (1, 7, 12, 30, 31) and the lack of voltage dependence of the SR Ca pump during relaxation (7). Indeed, in every cell the Na, dependence of relaxation was smaller at more positive membrane potentials. The size of the initial phasic decline of [Cal; and the tonic level of [Cal; during the l-s depolarization was variable among the 12 cells studied with this protocol. In some cells the tonic elevation of [Cal; was a small fraction of the peak [Cal; (e.g.,
