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(Circulation Research. 1996;78:857-862.)
© 1996 American Heart Association, Inc.

Articles

Variability of Spontaneous Ca²⁺ Release Between Different Rat Ventricular Myocytes Is Correlated With Na⁺-Ca²⁺ Exchange and [Na⁺]_i

M.E. Díaz, S.J. Cook, J.P. Chamunorwa, A.W. Trafford, M.K. Lancaster, S.C. O'Neill, D.A. Eisner

From the Department of Veterinary Preclinical Sciences, University of Liverpool (UK).

Correspondence to Dr D.A. Eisner, Department of Veterinary Preclinical Sciences, University of Liverpool, Liverpool L69 3BX, UK. E-mail eisner@liverpool.ac.uk.

	Abstract

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Abstract We have studied the factors responsible for the variation of the frequency of "waves" caused by spontaneous Ca²⁺ release in rat ventricular myocytes. The experiments were performed in isolated myocytes using the fluorescent indicators Indo-1 (to measure [Ca²⁺]_i) and SBFI (to measure [Na⁺]_i). After electrical stimulation (either with action potentials or voltage-clamp pulses), some cells showed spontaneous Ca²⁺ release. The frequency of this release, where present, was variable. The Ca²⁺ content of the sarcoplasmic reticulum (SR) was measured by applying caffeine (10 mmol/L). The resulting increase of [Ca²⁺]_i activated the electrogenic Na⁺-Ca²⁺ exchange, and the integral of this current was used to estimate the Ca²⁺ content of the SR. The SR Ca²⁺ content was significantly higher in cells that oscillated at high rates (>10·min^-1) than in those that were quiescent. The rate of removal of Ca²⁺ from the cytoplasm by non-SR mechanisms was measured by adding caffeine (10 mmol/L) and measuring the rate constant of decay of the resulting increase of [Ca²⁺]_i. Cells that had a high rate constant of decay of [Ca²⁺]_i had a low frequency of oscillations. Measurements of [Na⁺]_i showed a positive correlation between the frequency of spontaneous SR Ca²⁺ release and [Na⁺]_i. After cessation of stimulation, there was a gradual decrease of [Na⁺]_i, which was correlated with a parallel decrease of the frequency of oscillation rate. We conclude that the variability of frequency of spontaneous SR Ca²⁺ release is due to variations of the rate of Ca²⁺ removal from the cell, which are probably due to Na⁺-Ca²⁺ exchange. The variability of Na⁺-Ca²⁺ exchange rate, in turn, is likely to result from variations of [Na⁺]_i.

Key Words: Ca²⁺ • sarcoplasmic reticulum • Na⁺ • Na⁺-Ca²⁺ exchange

	Introduction

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It is now generally accepted that during normal excitation-contraction coupling in cardiac muscle, Ca²⁺ release from the sarcoplasmic reticulum (SR) occurs via the process of Ca²⁺-induced Ca²⁺ release. In this process, the sarcolemmal Ca²⁺ current produces a "trigger" rise of [Ca²⁺]_i, which then leads to the opening of SR Ca²⁺ channels. Under some conditions, Ca²⁺ release from the SR can occur spontaneously, ie, in the absence of a trigger from the sarcolemma.^{1 2 3} This spontaneous release, which can propagate as a wave along the cell, is most commonly observed when the cell is overloaded with Ca²⁺.^{3 4} In rat ventricular myocytes, however, it is frequently seen in cells under normal conditions.⁵ The released Ca²⁺ is, in part, pumped out of the cell on the electrogenic Na⁺-Ca²⁺ exchange.^{6 7} This produces a transient depolarization, which has been implicated in abnormal pacemaker activity.^{8 9}

Spontaneous release is particularly prominent in rat myocytes. In agreement with previous observations,¹⁰ we noted that there was considerable cell-to-cell variability in the appearance of spontaneous release and, in particular, in its frequency. In principle, this could reflect variations in either the Ca²⁺ content of the cell or the properties of the SR. In the former case, any differences in cell Ca²⁺ content could reflect differences in either Ca²⁺ entering the cell or, alternatively, in the activity of the processes such as Na⁺-Ca²⁺ exchange, which remove Ca²⁺ from the cytoplasm.

The results of the present study show that the variability of appearance of oscillations depends, at least in part, on variations in the SR Ca²⁺ content and can also be related to the activity of Na⁺-Ca²⁺ exchange and to variability of [Na⁺]_i. We also found that even in a given cell, there can be considerable changes in the rate of occurrence of spontaneous release. This is particularly prominent after cessation of stimulation and, again, is correlated with a decay of [Na⁺]_i.

	Materials and Methods

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Ventricular myocytes were isolated from rats that had been killed by stunning and cervical dislocation. The methods used for cell isolation have been described previously.¹¹ Cell length was measured using a video edge detector.

Measurement of [Ca²⁺]_i
Measurement of [Ca²⁺]_i was accomplished by loading the cells for 5 minutes in a solution containing 2.5 µmol/L of the acetoxymethyl ester of the Ca²⁺ indicator Indo-1 (Molecular Probes). The cells were excited with 340-nm light, and emission was measured at 400 and 500 nm. Details of the apparatus have been published previously.¹² In the experimental records, the ratio of fluorescence emitted at 400 and 500 nm (F₄₀₀:F₅₀₀) is used as a measure of [Ca²⁺]_i.

Measurement of [Na⁺]_i
The Na⁺ indicator SBFI (Molecular Probes) was used and was loaded as the acetoxymethyl ester (for 1 hour at 10 µmol/L). As described previously,¹³ the cell was alternately excited with light at 340 and 380 nm, and emission was measured at 510 nm. The ratio F₃₄₀:F₃₈₀ was measured and converted to absolute [Na⁺] using an in situ calibration. This was performed at the end of the experiment by exposing the cell to a series of divalent-free solutions (zero Ca²⁺ and Mg²⁺ with 1 mmol/L EGTA) containing gramicidin D (1 µg·mL^-1), ouabain (1 mmol/L), and various Na⁺ concentrations (at constant [Na⁺]+[K⁺] of 140 mmol/L), titrated to pH 7.4 (10 mmol/L HEPES). The records shown are displayed on a linear [Na⁺]_i scale.

Voltage Clamp
Voltage clamp was performed by the perforated-patch method,¹⁴ as described previously,¹⁵ using a switch clamp (Axoclamp 2A). Membrane current was stored digitally on a VCR-based system (Medical Systems).

Measurement of SR Ca²⁺ Content
SR Ca²⁺ content was estimated by releasing SR Ca²⁺ with caffeine (10 mmol/L). Caffeine was applied by switching between solutions in two fine tubes positioned close to the cell. The speed of application of the drug could be monitored from the quench of Indo-1 as the caffeine entered the cell.¹² The amount of Ca²⁺ pumped out of the cell by the Na⁺-Ca²⁺ exchange can be estimated by integrating the resulting inward electrogenic Na⁺-Ca²⁺ exchange current.¹⁶ This was related to cell volume. As described previously,¹⁷ the cell surface area was estimated from capacitance (assuming a specific capacitance of 1 µF·cm^-2). Volume was calculated from area by taking a value of 0.5 µm^-1 for the surface-to-volume ratio.¹⁸ In addition, the results were corrected by multiplying by a factor of 1.5 to allow for factors other than Na⁺-Ca²⁺ exchange (such as the sarcolemmal Ca²⁺-ATPase and the mitochondria) that contribute to the decay of [Ca²⁺]_i after caffeine application.¹⁷ The values of SR Ca²⁺ content are expressed as micromoles per liter of total cell volume.

Solutions
The experimental solution contained (mmol/L) NaCl 134, KCl 4, MgCl₂ 1.2, HEPES 10, glucose 11, and CaCl₂ 1, titrated to pH 7.4 with NaOH and equilibrated with air. All experiments were carried out at 27°C. Perforated patch-clamp experiments¹⁴ were performed using amphotericin B. Microelectrodes were made from borosilicate glass and had resistances of 2.5 to 3.0 M when filled with (mmol/L) KCH₃O₂S 125, KCl 20, NaCl 12, HEPES 10, MgCl₂ 5, and K₂EGTA 0.1, titrated to pH 7.2 with NaOH. Amphotericin B was dissolved in dimethyl sulfoxide and added to the pipette filling solution to a final concentration of 240 µg·mL^-1.

	Results

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In the experiments carried out in the present study, there was a marked variability in the frequency of occurrence of spontaneous Ca²⁺ waves (as observed either by direct observation of the cell or from [Ca²⁺]_i or current records). Some cells were quiescent, even when observed for periods of up to 10 minutes, whereas others had oscillation rates of up to 25·min^-1. Examples of this variability are shown in Fig 1A. The records of cell length show three cells: one is not oscillating, one is oscillating at 6·min^-1, and one is oscillating at 14·min^-1. This variability is emphasized in the histogram of Fig 1B.

View larger version (21K): [in this window] [in a new window]   Figure 1. Variability of oscillation frequency between cells. A, Original data. Records show measurements of cell shortening from three different cells. B, Frequency distribution of spontaneous sarcoplasmic reticulum release between different cells. The bars show the fraction of cells that had oscillation rates in the ranges shown on the x axis. The total number of cells was 63.

One fact to be determined was whether the variability in oscillation rate reflects variations in SR Ca²⁺ content or, alternatively, differences in the ease with which the SR releases Ca²⁺. The experiment illustrated in Fig 2 shows records from two cells, of which one is quiescent (Fig 2A) and the other (Fig 2B) is undergoing spontaneous SR Ca²⁺ release at a rate of 4·min^-1. In Fig 2, the oscillations are demonstrated by the inward Na⁺-Ca²⁺ exchange currents in panel B, although oscillations of [Ca²⁺]_i were also evident (not shown for this cell, but see Fig 4). Fig 2 also shows the effects of applying caffeine on membrane current. The cumulative integrals (bottom traces) show that the SR Ca²⁺ content of the oscillating cell (cell B) is greater than that of the cell that is not oscillating (cell A). Fig 3 shows similar data from several experiments comparing the SR content of the following populations of cells: cells that are not oscillating, cells that are oscillating at <10·min^-1, and cells that are oscillating at >10·min^-1. The SR Ca²⁺ content of the nonoscillating cells is significantly less (P<.05 by ANOVA) than that of the fastest oscillating cells. The other comparisons are not statistically significant.

View larger version (11K): [in this window] [in a new window]   Figure 2. Comparison of sarcoplasmic reticulum (SR) Ca2+ content and oscillation rate. Panels A and B show records from two different cells. In both panels, the top trace shows membrane current. The record in panel B shows spontaneous inward currents due to SR Ca2+ release. The lower panels show the effects of adding caffeine (10 mmol/L) on membrane current (top) and the calculated integrated net efflux of Ca2+ from the cell (bottom). The membrane potential was held at -80 mV throughout.

View larger version (13K): [in this window] [in a new window]   Figure 4. The relationship between the decay rate of the caffeine response and the frequency of spontaneous sarcoplasmic reticulum Ca2+ release. A and B, Records of [Ca2+]i obtained from two cells. To permit comparison between the two cells, the fluorescence ratio records have been normalized to the resting level (defined as 1.0). The top traces show [Ca2+]i recorded in the absence of stimulation. The cell in panel B is oscillating spontaneously, whereas that in panel A is not. The lower traces show the effects of adding caffeine (10 mmol/L) for the time indicated by the solid bar. C, Caffeine responses (taken from panels A and B) normalized in amplitude and displayed on a faster time scale. Membrane potential was -80 mV throughout.

View larger version (34K): [in this window] [in a new window]   Figure 3. Correlation of sarcoplasmic reticulum (SR) Ca2+ content and oscillation rate. The cells have been binned according to their oscillation rates (from left to right): not oscillating (11 cells), oscillating at <10·min-1 (20 cells), and oscillating at >10·min-1 (8 cells). The SR Ca2+ content of the fastest oscillating cells (125±11.3 µmol/L cell volume) is significantly greater than that of the nonoscillating ones (91±9 µmol/L) (P<.05, ANOVA). The membrane potential was held at -80 mV in all cells.

The conclusion from the experiments described above is that cells that oscillate do so because they have an elevated SR Ca²⁺ content. This could come about in two ways: (1) variations in SR properties, such as enhanced SR Ca²⁺-ATPase activity or decreased release channel opening, and (2) differences in the sarcolemma, such as decreased Ca²⁺ removal from the cell. The next series of experiments was designed to investigate whether the variability in oscillation rate could be attributed to differences in Ca²⁺ handling by the sarcolemma. In principle, one should investigate both the mechanisms allowing Ca²⁺ to enter the cell and those responsible for its removal. The mechanisms allowing Ca²⁺ entry into the resting cell are not well characterized and presumably include a combination of "leak" channels,¹⁹ sporadic opening of voltage-gated channels, and a contribution from Na⁺-Ca²⁺ exchange. Ca²⁺-removal mechanisms are more amenable to experimental study. This can be done by elevating [Ca²⁺]_i by releasing Ca²⁺ from the SR with caffeine and then measuring the rate of decay of [Ca²⁺]_i.^{20 21} This was carried out for the cells illustrated in Fig 4. For both cells (A and B, in corresponding panels), the application of caffeine resulted in an increase of [Ca²⁺]_i, which then decayed spontaneously (Fig 4, middle traces). There is a clear difference in the rate of decay of [Ca²⁺]_i, with that in Fig 4A decaying more quickly than that illustrated in Fig 4B. The difference in time course is emphasized by the normalized traces in Fig 4C. This suggests that Ca²⁺-removal processes are operating more quickly in cell A than in cell B. The top traces in Fig 4 were recorded from the same cells in the absence of stimulation. No spontaneous release is seen in cell A, whereas cell B oscillated at a frequency of 8·min^-1. Therefore, the quiescent cell has a faster rate of Ca²⁺ removal than the cell that is oscillating. This can also be seen in Fig 2, where the cell that is not oscillating shows a faster decay of the caffeine-evoked current (and therefore presumably of [Ca²⁺]_i). This apparent association between the rate of Ca²⁺ removal and the frequency of oscillations is tested more rigorously in Fig 5. Fig 5A shows oscillation frequency plotted as a function of the rate constant of decay of the caffeine response. It is clear that the greater the rate constant of decay of the caffeine response, the lower the frequency of oscillations. The graph shown in Fig 5B shows the data normalized to the mean values of both parameters on that experimental day. This normalization removes some scatter but, otherwise, shows the same general trend: those cells with the lowest rate of recovery of the caffeine response have the highest frequency of spontaneous oscillations.

View larger version (10K): [in this window] [in a new window]   Figure 5. The relationship between the rate constant of decay of the caffeine response and the frequency of oscillations. A, Unnormalized data. The ordinate shows the frequency of oscillations plotted as a function of the rate constant of decay of the caffeine response. The data have been binned by the rate constant of decay of the caffeine response. B, Normalized data. The data from all the cells on one day have been normalized to the mean value of oscillation frequency and rate constant of decay on that day. In both graphs, the data are taken from 34 cells in total drawn from 8 days. The different numbers of points in the two graphs reflect the different binning.

The correlation of Fig 5 shows that those cells that have the greatest ability to remove Ca²⁺ have the lowest oscillation frequency. In subsequent work, we investigated the origin of the decreased Ca²⁺ removal. Previous work²¹ has shown that the major component of Ca²⁺ removal is provided by Na⁺-Ca²⁺ exchange. In addition, we have shown that inhibiting the Na⁺-K⁺ pump with ouabain slows Ca²⁺ removal.²² Therefore, it seemed appropriate to investigate whether the variability in oscillation rate could be related to variations in [Na⁺]_i. In the experiment illustrated in Fig 6A, the cell was initially stimulated. When stimulation was discontinued, the resting [Na⁺]_i was 9 mmol/L, and the oscillation rate was 4·min^-1. In the cell illustrated in Fig 6B, the level of [Na⁺]_i immediately after stopping stimulation was 18 mmol/L, and the oscillation rate was also higher (10·min^-1) than that in Fig 6A. This correlation between [Na⁺]_i and oscillation rate is shown for all 16 cells in the graph shown in Fig 7. The solid symbols show that oscillation frequency is highly correlated with [Na⁺]_i. The straight line through the data was obtained by linear regression on the raw data and has a slope that is significantly different from zero (P<.02).

View larger version (21K): [in this window] [in a new window]   Figure 6. The effects of discontinuing stimulation on oscillation rate and [Na+]i. The data in panels A and B are taken from two different cells. In each part, the panels show the following (from top to bottom): [Na+]i, cell length, and oscillation frequency calculated over 1-minute periods. The cell was initially stimulated externally at 0.5 Hz. With the slow time base of the record, the contractions fuse into a continuous envelope. On cessation of stimulation, spontaneous oscillations can be seen.

View larger version (13K): [in this window] [in a new window]   Figure 7. The relationship between the frequency of oscillations and the level of [Na+]i. The data show frequency of oscillation plotted as a function of [Na+]i. The data have been binned according to [Na+]i. The number of cells in each bin is four. The solid symbols were obtained in the first minute after stopping stimulation. The straight line is a linear regression to this original data and has a slope that is significantly (P<.02) different from zero. The open symbols were obtained at the end of the rest (10-minute duration). The symbol shapes correspond; eg, the open triangles indicate the same collection of cells at the end of a rest from which the data at the start of the rest are depicted by the closed triangles.

The influence of [Na⁺]_i on oscillation rate can also be seen within a single cell. In both cells illustrated in Fig 6, [Na⁺]_i decays with time after ceasing stimulation. This is accompanied by a decay of oscillation rate. The influence of rest on [Na⁺]_i and oscillation rate is also shown in Fig 7. The open symbols show data at the end of a rest for the same cells illustrated (at the beginning of a rest) with the closed symbols. For all but the point corresponding to the lowest [Na⁺]_i (circles), the rest decreases both [Na⁺]_i and oscillation rate. The effects of changes of [Na⁺]_i within a cell can be estimated from the gradient between the pairs of open and closed symbols. These are at least as steep as the regression line (apart from the circles) and are consistent with the idea that much of the intercell variability in oscillation rate can be attributed to [Na⁺]_i.

	Discussion

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The results of the present study provide an explanation for the variability in the frequency of spontaneous SR Ca²⁺ release that is observed between ventricular cells. We show that this is correlated with differences between cells in the rate at which Ca²⁺ ions are removed from the cytoplasm and that this, in turn, appears to be related to differences in [Na⁺]_i.

Previous work has shown that there is a considerable degree of variability in the frequency of spontaneous SR Ca²⁺ release. Thus, Capogrossi et al¹⁰ found (also in rat ventricular myocytes) that 15% of cells did not display spontaneous Ca²⁺ release, 50% had rates of between 1·min^-1 and 3·min^-1, and the remainder had higher rates, including some at frequencies of >20·min^-1. The factors underlying this variability have, to date, remained unexplained.

Correlation of Oscillation Frequency and SR Ca²⁺ Content
The frequency of spontaneous release is known to increase with factors that increase the degree of cellular "Ca²⁺ overload."^{3 4} The measurements in the present study show that those cells that had the highest oscillation rate had larger SR Ca²⁺ contents than those that did not oscillate. However, there was no statistically significant difference between cells oscillating at lower frequencies and those either not oscillating or oscillating at high rates. There are at least two possible reasons for the limited correlation: (1) It may simply be produced by experimental scatter. (2) Alternatively, it may arise because the SR only releases Ca²⁺ spontaneously when its Ca²⁺ content reaches a certain level. In this case, all cells that have an SR Ca²⁺ content less than this level will be quiescent, and one would not therefore expect any correlation over this range.

Relationship Between the Rate of Ca²⁺ Removal and Oscillation Frequency
The experiments (Fig 5) show that the frequency of spontaneous oscillations is greater in those cells that have the lowest rate of Ca²⁺ removal after caffeine application. One might argue that the Indo-1 ratio that is being used as a measure of [Ca²⁺]_i is a nonlinear Ca²⁺ indicator and, therefore, that any differences between cells in the concentration range over which [Ca²⁺]_i changes could affect the rate constant of decay of the Indo-1 ratio. However, it is worth noting that the Na⁺-Ca²⁺ exchange current, which is linearly related to [Ca²⁺]_i²³ and therefore provides a measure of the time course of decay of [Ca²⁺]_i, also decays more slowly in cells that are oscillating at higher frequencies (Fig 2). The present experiments do not identify the component of Ca²⁺ removal, which varies between different cells. However, given the observation in the present study that [Na⁺]_i is positively correlated with the frequency of spontaneous oscillations and the fact that elevating [Na⁺]_i decreases the rate of Ca²⁺ removal,²² it seems most probable that the rate of Na⁺-Ca²⁺ exchange is varying between different cells. Presumably, those with the greatest Na⁺-Ca²⁺ exchange activity have the greatest Ca²⁺ removal and thus the lowest rate of spontaneous oscillations. To balance a constant Ca²⁺ entry, a cell with a lower rate constant of decay of [Ca²⁺]_i during caffeine application (and therefore a lower Na⁺-Ca²⁺ exchange rate) will require a higher time-averaged [Ca²⁺]_i. One way of achieving this will be for the cell to have spontaneous SR Ca²⁺ release at a higher frequency. This will arise because the decreased rate of Ca²⁺ extrusion will lead to a greater cell and therefore SR Ca²⁺ content, and the SR will therefore be more likely to release Ca²⁺ spontaneously. Furthermore, although we cannot exclude the possibility that the degree of expression of the Na⁺-Ca²⁺ exchange varies between different cells or that other modulators are involved, it is tempting to assume that the variations of [Na⁺]_i account for the variation of the Na⁺-Ca²⁺ exchange rate.

Relationship Between [Na⁺]_i and Oscillation Frequency
The experiments show that there is a marked variation in [Na⁺]_i between different cells that, by the mechanisms discussed above, is correlated with the variations of oscillation frequency. This relationship could arise because the variations in [Na⁺]_i are the initiating factor, which then leads to differences of Ca²⁺ removal and thus oscillation frequency. Alternatively, variations of Ca²⁺ entry between cells could be the primary event, which then affects Na⁺ entry on Na⁺-Ca²⁺ exchange and thus [Na⁺]_i. Our experiments do not allow a distinction between these alternatives, but in either case, oscillation frequency is related to [Na⁺]_i. If cells are rested for long periods, then there is both a fall in [Na⁺]_i and a fall in the frequency of spontaneous oscillations. Although the scatter of the data (Fig 7) preclude an exact analysis of the relationship between [Na⁺]_i and oscillation frequency, we find that in a given cell, during the decay of [Na⁺]_i in a rest, the slope of the relationship is steep enough to account for the slope found between cells. It should be noted that this comparison assumes that during the decay of [Na⁺]_i on stopping stimulation, cell and therefore SR Ca²⁺ content reaches equilibrium with the prevailing level of [Na⁺]_i. If this is not the case, then the spontaneous release itself will decrease SR Ca²⁺ content and thus the frequency.

Our data provide no information about the origin of the variations in [Na⁺]_i. Variations could come about because of differences in either (1) Na⁺-K⁺ pump density or activity or (2) processes that allow Na⁺ to leak into the cell. If the tendency for cells to have different frequencies of spontaneous SR Ca²⁺ release can be attributed to differences between these cells in their Na⁺ content, it is worth considering whether these differences of Na⁺ content are also present in different cells in the intact ventricle or whether they are simply a product of cell isolation. Although we cannot exclude the latter possibility, it is worth noting that there are significant electrophysiological differences between cells in, for example, epicardium and endocardium,²⁴ and there also appear to be differences in Na⁺ regulation.²⁵

	Acknowledgments

 
This study was supported by grants from The Wellcome Trust. Dr Díaz was supported by Consejo Nacional de Investigaciones Científicas y Technológicas (CONICIT; Venezuela) and an Overseas Research Student Award (ORS). Dr Chamunorwa is a Fellow of the Beit Trust.

Received December 18, 1995; accepted February 1, 1996.

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