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(Circulation Research. 2001;89:526.)
© 2001 American Heart Association, Inc.

Cellular Biology

Effects of Cytosolic ATP on Ca²⁺ Sparks and SR Ca²⁺ Content in Permeabilized Cardiac Myocytes

Zhaokang Yang, Derek S. Steele

From the School of Biology, University of Leeds, Leeds, UK.

Correspondence to Derek S. Steele, PhD, School of Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK. E-mail D.steele{at}Leeds.ac.uk

	Abstract

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Abstract — Confocal imaging was used to study the influence of cytosolic ATP on the properties of spontaneous Ca²⁺ sparks in permeabilized ventricular myocytes. Cells were perfused with mock intracellular solutions containing fluo 3. Reducing [ATP] to <0.5 mmol/L decreased the frequency but increased the amplitude of spontaneous Ca²⁺ sparks. In the presence of 20 µmol/L ATP, the amplitude increased by 48.7±10.9%, and the frequency decreased by 77.07±3.8%, relative to control responses obtained at 5 mmol/L ATP. After exposure to a solution containing zero ATP, the frequency of Ca²⁺ sparks decreased progressively and approached zero within 90 seconds. As ATP washed out of the cell, the sarcoplasmic reticulum (SR) Ca²⁺ content increased, until reaching a maximum after 3 minutes. Subsequent introduction of adenylyl imidodiphosphate precipitated a burst of large-amplitude Ca²⁺ sparks. This was accompanied by a rapid decrease in SR Ca²⁺ content to 80% to 90% of the steady-state value obtained in the presence of 5 mmol/L ATP. Thereafter, the SR Ca²⁺ content declined much more slowly over 5 to 10 minutes. The effects of ATP withdrawal on Ca²⁺ sparks may reflect reduced occupancy of the adenine nucleotide site on the SR Ca²⁺ channel. These effects may contribute to previously reported changes in SR function during myocardial ischemia and reperfusion, in which ATP depletion and Ca²⁺ overload occur.

Key Words: ATP • sarcoplasmic reticulum • Ca²⁺ sparks

	Introduction

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In recent studies on isolated cardiac cells, microscopic sarcoplasmic reticulum (SR) Ca²⁺–release events (sparks) have been detected by using laser scanning confocal microscopy.¹ A Ca²⁺ spark occurs when an SR "release unit" is activated, resulting in a localized flux of Ca²⁺ from the SR to the cytosol. Current evidence suggests that a release unit is composed of a cluster (perhaps 10 to 20) of SR Ca²⁺ channels (ryanodine [RyR] receptors [RyRs]) activated in concert.^2–4 Sparks arise predominantly in the junctional regions of the SR, where the Ca²⁺ channels are concentrated, and the membrane is in close apposition to sarcolemmal L-type Ca²⁺ channels, particularly at the T tubules.⁵ It has been proposed that (1) recruitment of SR release units, during the Ca²⁺ transient, results from the time- and voltage-dependent opening of neighboring sarcolemmal Ca²⁺ channels, and (2) the macroscopic [Ca²⁺]_i transient reflects the temporal and spatial summation of many Ca²⁺ sparks, each activated by the opening of a surface membrane Ca²⁺ channel.⁶

Although the systolic Ca²⁺ transient reflects the concerted activation of many Ca²⁺-release units triggered by Ca²⁺ influx, sparks with similar spatial and temporal properties also occur spontaneously in quiescent cells.² Previous studies have shown that the frequency of spontaneous Ca²⁺ sparks increases as a function of SR Ca²⁺ content.⁷ In electrically stimulated cells, spontaneous Ca²⁺ sparks occur during diastole, and the frequency of these events increases as a function of the SR Ca²⁺ load.⁸ It has been suggested that spontaneous Ca²⁺ sparks constitute a significant "Ca²⁺ leak" pathway,⁹ which may influence or, in some circumstances, limit the SR Ca²⁺ content. Consistent with this suggestion, pharmacological desensitization of the SR Ca²⁺ channel with tetracaine reduces the frequency of spontaneous Ca²⁺ sparks and markedly increases the SR Ca²⁺ content.^10,11

A variety of cytosolic factors including H⁺, calmodulin, cADP-ribose, and ATP are known to influence the gating properties of isolated SR Ca²⁺ channels.^12–14 Recent work has shown that cADP-ribose and calmodulin increase but that H⁺ reduces the frequency of spontaneous Ca²⁺ sparks in isolated cardiac myocytes.^15–17 These changes in spark frequency are accompanied by corresponding changes in the SR Ca²⁺ content. The possible effect of cytosolic ATP on spontaneous Ca²⁺ sparks has not yet been characterized.

The aim of the present study was to investigate the influence of cytosolic ATP on the properties of Ca²⁺ sparks. Experiments were carried out in permeabilized myocytes, and localized changes in [Ca²⁺] were detected with the use of fluo 3. Decreasing [ATP] to <0.5 mmol/L markedly reduced the frequency of Ca²⁺ sparks and increased the SR Ca²⁺ content. Complete withdrawal of ATP resulted in a profound decrease in Ca²⁺ spark activity and a prolonged increase in SR Ca²⁺ content. This suggests that ATP-dependent spontaneous spark activity influences the SR Ca²⁺ leak and, therefore, the [Ca²⁺] gradient across the SR membrane. These effects may have relevance to ischemia and subsequent reperfusion, during which cytosolic ATP depletion and Ca²⁺ overload occur.

	Materials and Methods

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Cell Permeabilization and Composition of Mock Intracellular Solutions
Male Wistar rats (200 to 250 g) were killed by concussion, followed by cervical dislocation. Ventricular myocytes were isolated as previously described.¹⁸ Cells were exposed to saponin (10 mg/mL) in a "mock intracellular" solution (as described below) for 6 minutes, before centrifugation and resuspension. Experiments were performed at room temperature (20°C to 22°C). Unless otherwise stated, chemicals were obtained from Sigma Chemical Corp. H⁺ and Ca²⁺ were buffered with HEPES and EGTA, respectively. The basic solution contained KCl (100 mmol/L), HEPES (25 mmol/L), EGTA (0.36 mmol/L), phosphocreatine (10 mmol/L), ATP (0 to 5 mmol/L), and fluo 3 (5 µmol/L). The free [Ca²⁺] was adjusted to the desired level by the addition of CaCl₂.

Because ATP buffers Mg²⁺ strongly, it was necessary to ensure that the free [Mg²⁺] remained constant (at 1 mmol/L) when [ATP] was altered. The [Mg²⁺] was measured directly by using furaptra, as previously described.¹⁸ In the absence of ATP, it was necessary to add 1.2 mmol/L total Mg²⁺ to produce a free concentration of 1 mmol/L in the standard experimental solution (as described above). This is because 0.2 mmol/L Mg²⁺ is bound to 10 mmol/L phosphocreatine. In solutions with 5 mmol/L ATP and 10 mmol/L phosphocreatine, it was necessary to add 5.75 mmol/L total Mg²⁺ to achieve a free [Mg²⁺] of 1 mmol/L. In solutions containing 5 mmol/L adenylyl imidodiphosphate (AMP-PNP), [Mg²⁺] was reduced by 1.7 mmol/L to maintain the free [Mg²⁺] at 1 mmol/L.

Where necessary, the equilibrium concentrations of metal ions in the calibration solutions were calculated by using the affinity constants for H⁺, Ca²⁺, and Mg²⁺ for EGTA, as previously reported,^19,20 with use of the REACT program.²¹ Azide (5 mmol/L) was included in the solutions to inhibit possible mitochondrial activity. However, azide had no apparent influence on the effects of ATP reported in the present study.

Confocal Microscopy
The bath was placed on the stage of a Nikon Diaphot Eclipse inverted microscope, and the cells were viewed with a x40 oil immersion lens (Nikon Plan Fluor DLL, numerical aperture 1.3) or a x60 water immersion lens (Plan Apo, numerical aperture 1.2). A confocal laser-scanning unit (Microradiance 2000, Bio-Rad) was attached to the side port of the microscope. The x-y resolution of the system was 0.45 µm, as measured from the point-spread function of fluorescent microspheres (diameter 0.175 µm, Molecular Probes). The aperture size was set to the size of the Airy disk (1.8 mm for x60 objective, 1.07 mm for x40 objective) to optimize the z-axis resolution. Fluo 3 was excited with the 488-nm line of an argon ion laser and fluorescence measured at >515 nm. Images were acquired in line-scan mode (at intervals of 6 or 2 ms) along the longitudinal axis of the cell. To reduce possible laser damage, the position of the line was changed after two or three scans. Sparks were identified by applying an automatic spark detection program to the line-scan images as previously described.²² As in previous studies on skinned cells, events were detected with the detection threshold set at 2.6xSD.¹⁶ Image processing and analysis were performed by using IDL (Research Systems Inc) and Laserpix (Bio-Rad) software. Gaussian curves were fitted by the use of Origin (Microcal).

	Results

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Effects of Single Large Decrease in [ATP] on Spontaneous Ca²⁺ Sparks
Figure 1A shows line-scan images of spontaneous Ca²⁺ sparks in a saponin-permeabilized ventricular myocyte. Selected plots of resting fluorescence (F/F_o) are shown to the right of each image. Under control conditions, the myocyte was exposed to a solution containing 5 mmol/L ATP and 0.36 mmol/L EGTA, at a free bathing [Ca²⁺] of 200 nmol/L. This level of EGTA has been shown to block propagation of Ca²⁺ waves, without altering the time course or spatial characteristics of localized Ca²⁺ sparks.¹⁶ In the presence of 5 mmol/L ATP, regular localized Ca²⁺ sparks were clearly visible in the line-scan image. The cell was then equilibrated with a solution containing 0.02 mmol/L ATP. In the presence of 0.02 mmol/L ATP, the frequency of spontaneous Ca²⁺ sparks was markedly reduced, whereas the amplitude increased. When [ATP] was again increased to 5 mmol/L ATP, the amplitude and frequency of Ca²⁺ sparks returned to control levels.

View larger version (46K): [in this window] [in a new window]   Figure 1. Ca2+ sparks obtained in the presence of 5 or 0.02 mmol/L ATP. A, Representative longitudinal line-scan images obtained from a permeabilized myocyte in the presence of 5 mmol/L ATP (top), 0.02 mmol/L ATP (middle), and after reexposure to 5 mmol/L ATP (bottom). Horizontal and vertical calibration bars correspond to 10 µm and 200 ms, respectively. Selected plots of F/Fo are shown to the right of each image. These were obtained by averaging 3 pixels, centered on a Ca2+ release event. B, Amplitude histograms of Ca2+ sparks obtained in the presence of 5 and 0.02 mmol/L ATP.

Figure 1B shows histograms of Ca²⁺ spark amplitudes obtained in the presence of 5 and 0.02 mmol/L ATP. The abscissa indicates the spark amplitude expressed relative to F/F_o. Reducing [ATP] to 0.02 mmol/L was associated with a shift in the peak of the gaussian distribution toward higher mean amplitudes (from 0.7 to 1.1 F/F_o). This was accompanied by a broadening of the distribution, as indicated by an increase in the standard deviation from 0.21 to 0.4 U.

Characteristics of Ca²⁺ Sparks at Various Levels of [ATP]
Figure 2A shows surface plots of averaged Ca²⁺ sparks obtained at various ATP concentrations. As [ATP] decreased, the amplitude of spontaneous Ca²⁺ sparks increased. At 0.01 mmol/L ATP, the descending phase of the Ca²⁺ spark was also significantly prolonged, whereas spark width appeared largely unaffected. Accumulated data illustrating the relationship between [ATP] and the frequency and amplitude of Ca²⁺ sparks are given in Figure 2B. As [ATP] was reduced to <0.5 mmol/L, there was a progressive increase in spark amplitude and a decrease in frequency. In the presence of 0.02 mmol/L ATP, the amplitude increased by 48.7±10.88%, and the frequency decreased by 77.07±3.82% relative to control responses obtained at 5 mmol/L ATP. As shown in Figure 2C, reducing [ATP] had no significant influence on spark width. However, the descending phase of the Ca²⁺ sparks was significantly prolonged at [ATP] 0.1 mmol/L. At 0.01 mmol/L ATP, the descending phase of the spark increased from 25.5±1.2 ms (n=17) to 32.2±1.4 ms (n=8). Prolongation of the Ca²⁺ sparks at very low [ATP] is consistent with inhibition of the SR Ca²⁺ pump.^22a

View larger version (46K): [in this window] [in a new window]   Figure 2. Accumulated data showing the effects of ATP on the characteristics of Ca2+ sparks. A, Surface plot images of averaged Ca2+ sparks (18 to 22) obtained in the same preparation after equilibration with solutions containing 5, 0.5, 0.1, or 0.01 mmol/L ATP. B, Accumulated data showing the effects of ATP on the frequency and amplitude of spontaneous Ca2+ sparks. C, Accumulated data illustrating the effect of bathing [ATP] on the width and duration of spontaneous Ca2+ sparks. Responses are expressed relative to the mean control values obtained in the presence of 5 mmol/L ATP. The duration and width were measured at half-maximal amplitude. The number of myocytes at each [ATP] ranged from 8 to 25. *P<0.05, **P<0.02, and ***P<0.005. Error bars represent the mean±SEM.

It is possible that a reduction in Ca²⁺ buffering by ATP might explain or contribute to the increase in the amplitude of Ca²⁺ sparks or the Ca²⁺ transient induced by caffeine. However, in further experiments, it was found that a large increase in [ATP], from 5 to 10 mmol/L, produced only a small (5.2±2.9%, n=4) decrease in spark amplitude. Furthermore, computer modeling suggests that the reduction in Ca²⁺ buffering due to ATP withdrawal should increase spark amplitude only by 5% to 6% (M.B. Cannell, PhD, C. Soeller, PhD, written communication, July 2001).

Changes in Steady-State SR Ca²⁺ Content as Function of [ATP]
In previous studies, we have shown that the SR Ca²⁺ content progressively increases in response to stepwise decreases in [ATP].¹⁸ Figure 3 shows the protocol used to assess the relationship between [ATP] and SR Ca²⁺ content under the conditions of the present study. Permeabilized myocytes were initially equilibrated with a control solution containing 5 mmol/L ATP. Caffeine was then rapidly applied during a longitudinal line scan. Figure 3A shows line-scan images obtained in the presence of 5 and 0.1 mmol/L ATP. Figure 3B shows several responses obtained at a range of [ATP]. Typically, the transients exhibited a rapid uniform increase in [Ca²⁺] followed by a slower and variable decline. As in previous studies,¹⁸ the amplitude of the early [Ca²⁺] peak was used as an index of the SR Ca²⁺ content. Reducing [ATP] resulted in a progressive increase in the amplitude of the response.

View larger version (27K): [in this window] [in a new window]   Figure 3. Relationship between [ATP] and steady-state SR Ca2+ content. A, Longitudinal line-scan images obtained from permeabilized myocytes during a rapid exposure to 20 mmol/L caffeine in the presence of 5 mmol/L (top) or 0.1 mmol/L (bottom) ATP. B, Fluorescence transients obtained by integrating the line-scan images during a caffeine application, at various levels of [ATP]. C, Accumulated data illustrating the effect of bathing [ATP] on the amplitude of the caffeine-induced fluorescence transient, used as an index of SR Ca2+ content. Responses are expressed relative to the mean control values in the presence of 5 mmol/L ATP. The number of myocytes at each [ATP] ranged from 10 to 25. **P<0.02 and ***P<0.005.

The accumulated data (Figure 3C) show that stepwise decreases in [ATP] resulted in corresponding increases in the amplitude of the caffeine-induced fluorescence response. At 0.01 mmol/L ATP, the steady-state amplitude of the caffeine-induced transient was 59.2±8.1% higher than that of controls in the presence of 5 mmol/L ATP. Also shown is the mean amplitude of the caffeine-induced response 1 minute after complete ATP withdrawal. Unexpectedly, ATP withdrawal resulted in a further, more pronounced, increase in amplitude (see below).

Spark Characteristics After Complete Withdrawal and Introduction of AMP-PNP
It has been suggested that Ca²⁺ sparks may contribute to the resting Ca²⁺ leak from the SR.⁹ Therefore, the increase in SR Ca²⁺ content, which occurs as [ATP] is reduced, may reflect a decrease in spark frequency (eg, Figure 1). However, the relationship between spark frequency and the unidirectional Ca²⁺ leak is difficult to assess, because of the continual reaccumulation of Ca²⁺ by the SR Ca²⁺ pump. Therefore, experiments were carried out after complete withdrawal of ATP, which will ultimately abolish SR Ca²⁺ uptake.

Figure 4A shows line-scan images obtained from a permeabilized myocyte. At the top are images taken in the presence of 5 mmol/L ATP and then 30 or 60 seconds after complete withdrawal of ATP from the bathing medium (below). In this example, spark frequency decreased markedly within 30 seconds of exposure to zero ATP. After 60 seconds, sparks were rarely detected. At this point in the protocol, SR Ca²⁺ content is likely to be higher than the control level in the presence of 5 mmol/L ATP (Figure 3). Therefore, reintroduction of ATP would be expected to precipitate the reappearance of Ca²⁺ sparks.

View larger version (28K): [in this window] [in a new window]   Figure 4. Effects of ATP withdrawal and subsequent introduction of AMP-PNP on Ca2+ sparks. A, Representative longitudinal line-scan images obtained from a permeabilized myocyte under control conditions in the presence of 5 mmol/L ATP and at 30 and 60 seconds after complete ATP withdrawal. Sequential line-scan images are also shown at various time intervals after introduction of 5 mmol/L AMP-PNP, 90 seconds after ATP withdrawal. Horizontal and vertical calibration bars correspond to 20 µm and 100 ms, respectively. Selected plots of F/Fo are shown to the right of each image. B, Accumulated data showing the changes in spark frequency and amplitude as a function of time after complete ATP withdrawal and after subsequent introduction of 5 mmol/L AMP-PNP.

In this example, the nonhydrolyzable ATP analogue AMP-PNP (5 mmol/L) was introduced into the perfusing solution. AMP-PNP substitutes for ATP at the adenine nucleotide binding site with a similar efficacy²³ but does not support Ca²⁺ uptake via the SR ATPase. Ninety seconds after ATP withdrawal, introduction of AMP-PNP resulted in a rapid burst of spark activity. These events were initially more frequent and of larger amplitude than sparks obtained under control conditions, in the presence of 5 mmol/L ATP. Large propagating Ca²⁺ waves were not generally observed. However, several neighboring sites were sometimes activated, resulting in "macro sparks" or closely grouped multiple peaks. These large frequent Ca²⁺ sparks suggest that the SR Ca²⁺ content, at the point of introduction of AMP-PNP, was higher than the control level. However, spark activity rapidly decreased again, and the frequency approached zero within 30 seconds of exposure to AMP-PNP.

Cumulated data illustrating the effect ATP withdrawal and reintroduction of AMP-PNP is shown in Figure 4B. Spark frequency under control conditions and at 30 or 60 seconds after complete ATP withdrawal is shown at the top. Spark frequency approached zero at 60 seconds after ATP withdrawal. Introduction of AMP-PNP 90 seconds after ATP withdrawal resulted in a transient increase in spark activity, which was initially of higher frequency than under control conditions, in the constant presence of 5 mmol/L ATP. However, in the continued presence of AMP-PNP, spark frequency decreased rapidly and approached zero after 30 seconds. After ATP withdrawal, spark amplitude increased progressively as the frequency declined. The largest sparks were obtained on introduction of AMP-PNP, which presumably reflected a higher SR Ca²⁺ content (see below).

Substitution of ATP With AMP-PNP
Introduction of AMP-PNP 90 seconds after complete withdrawal of ATP precipitated a transient burst of large-amplitude Ca²⁺ sparks (Figure 4). However, different results were obtained when ATP was substituted with AMP-PNP and when the step involving exposure to zero ATP was omitted. Figure 5 shows line-scan images obtained from a permeabilized myocyte under control conditions in the presence of 5 mmol/L ATP and at 15, 45, or 60 seconds after the substitution of ATP with 5 mmol/L AMP-PNP. After exposure to AMP-PNP, there was a progressive decrease in the frequency of Ca²⁺ sparks, and spontaneous events were rare after 60 seconds. Approximately 90 seconds after the cell was returned to a solution with 5 mmol/L ATP, the frequency of the spontaneous Ca²⁺ sparks approached control levels. Accumulated data illustrating the decrease in spark frequency as a function of time, after substitution of ATP with AMP-PNP, are shown in Figure 5B.

View larger version (40K): [in this window] [in a new window]   Figure 5. Effects of ATP withdrawal and subsequent introduction of AMP-PNP on Ca2+ sparks. A, Longitudinal line-scan images obtained from a permeabilized myocyte under control conditions in the presence of 5 mmol/L ATP, at various intervals after substitution of 5 mmol/L ATP with 5 mmol/L AMP-PNP, and finally, 90 seconds after reintroduction of 5 mmol/L ATP. Horizontal and vertical calibration bars correspond to 200 ms and 20 µm, respectively. Selected plots of F/Fo (to the right of each image) were obtained by averaging 3 pixels, centered on a Ca2+ release event. B, Accumulated data showing the changes in spark frequency and amplitude as a function of time after substitution of 5 mmol/L ATP with 5 mmol/L AMP-PNP. **P<0.02 and ***P<0.005.

Changes in SR Ca²⁺ Content After Complete ATP Withdrawal and Substitution With AMP-PNP
Step decreases in [ATP] result in corresponding maintained increases in the steady-state SR Ca²⁺ content (Figure 3). This demonstrates that the SR Ca²⁺ pump can maintain a substantial SR [Ca²⁺] gradient in the presence of micromolar [ATP]. However, the situation is more complex after complete withdrawal of ATP or substitution with AMP-PNP because abolition of SR Ca²⁺ uptake will ultimately occur. Therefore, experiments were carried out to investigate the changes in SR Ca²⁺ content that occur as a function of time, after (1) complete withdrawal of ATP, (2) substitution of ATP with AMP-PNP, or (3) introduction of AMP-PNP after exposure to zero ATP.

Figure 6 shows accumulated data illustrating changes in SR Ca²⁺ content after withdrawal of ATP or substitution of 5 mmol/L ATP with 5 mmol/L AMP-PNP. The SR Ca²⁺ content was assessed from the amplitude of the caffeine-induced fluo 3 fluorescence transient obtained in line-scan images (Figure 3). After withdrawal of ATP (or its substitution with AMP-PNP), SR Ca²⁺ uptake was rapidly impaired. As a consequence, caffeine application resulted in a single response, which could not be repeated (not shown). Therefore, to obtain the time-dependent changes in SR Ca²⁺ content, several control responses were obtained in the presence of ATP. The solution was then changed to one lacking ATP (or one containing AMP-PNP), and caffeine was rapidly applied after a set period of time. The cell was then reexposed to the control solution, and the process was repeated with a different time interval.

View larger version (29K): [in this window] [in a new window]   Figure 6. Effects of ATP complete withdrawal on the SR Ca2+ content. A, Accumulated data showing changes in SR Ca2+ content as a function of time after (1) ATP withdrawal, (2) ATP withdrawal followed (after 3 minutes) by introduction of 5 mmol/L AMP-PNP, and (3) replacement of 5 mmol/L ATP with 5 mmol/L AMP-PNP. B, Accumulated data showing changes in SR Ca2+ content as a function of time after (1) ATP withdrawal, (2) introduction of 5 µmol/L thapsigargin in the continued presence of ATP, and (3) ATP withdrawal followed (after 3 minutes) by reintroduction of ATP with 5 µmol/L thapsigargin. SR Ca2+ content was assessed from the amplitude of the caffeine-induced fluorescence transient. Points represent mean±SEM (n=8).

As shown in Figure 6A, after complete withdrawal of ATP, the SR Ca²⁺ content increased progressively and peaked after 3 minutes. In the continued absence of ATP, the SR Ca²⁺ content declined slowly. However, even after 10 minutes of exposure to zero ATP, the SR Ca²⁺ content was higher than that under control conditions, in the constant presence of 5 mmol/L ATP. When ATP was replaced by AMP-PNP (open triangles, Figure 6A), the SR Ca²⁺ content declined only slowly. Indeed, after 10 minutes of exposure to AMP-PNP, the SR Ca²⁺ content had fallen by <50% of the steady-state control level in the presence of 5 mmol/L ATP.

Figure 6A also shows data from a series of experiments in which ATP was withdrawn completely for 3 minutes, before introduction of 5 mmol/L AMP-PNP. Introduction of 5 mmol/L AMP-PNP was associated with a rapid decline in the SR Ca²⁺ content. Approximately 60 seconds after introduction of AMP-PNP, the SR Ca²⁺ content had decreased to 88.4±15.9% of the control level. Thereafter, the SR Ca²⁺ content declined much more slowly, with a similar profile to that obtained, when ATP was immediately substituted with AMP-PNP.

Figure 6A shows that the SR Ca²⁺ content increases for 3 minutes after complete ATP withdrawal. Consequently, the SR Ca²⁺ pump must be capable of net SR Ca²⁺ accumulation during this period. Figure 3 shows that the SR can sustain a higher than normal SR Ca²⁺ content in the constant presence of 10 µmol/L ATP. This suggests that it takes 3 minutes for [ATP] in the vicinity of the SR Ca²⁺ pump to decline from 5 mmol/L to a level that cannot sustain the activity of the SR Ca²⁺ pump (<10 µmol/L). However, it seems likely that the local rephosphorylation of ADP by bound creatine kinase will prolong pump activity after a decrease in the bulk solution [ATP].²⁴

A rapid decrease in SR Ca²⁺ content was also obtained in experiments in which ATP and the SR Ca²⁺ pump inhibitor thapsigargin were simultaneously introduced 3 minutes after ATP withdrawal (Figure 6B). Addition of ATP plus 5 µmol/L thapsigargin will activate the adenine nucleotide site on the SR Ca²⁺ channel but block Ca²⁺ uptake. Furthermore, immediate exposure to 5 mmol/L ATP and thapsigargin was followed by a slow decrease in the SR Ca²⁺ content with a time course similar to that obtained when ATP was substituted with AMP-PNP.

	Discussion

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Effects of Cytosolic ATP on Frequency of Spontaneous Ca²⁺ Sparks
Previous work has shown that a number of cytosolic factors, including cADP-ribose, calmodulin, and H⁺, affect the frequency and amplitude of spontaneous Ca²⁺ sparks.^15–17 The present study shows that cytosolic ATP also influences the properties of Ca²⁺ sparks. Stepwise reductions in [ATP] to <0.5 mmol/L resulted in corresponding decreases in the frequency of spontaneous Ca²⁺ sparks (Figures 1 and 2). Complete ATP withdrawal abolished spontaneous Ca²⁺ sparks. This effect is consistent with experiments on isolated channels and SR vesicles, showing that activation of RyRs by cytosolic Ca²⁺ is impaired in the absence of ATP.^12,25,26 However, a further possibility is that ATP depletion may reduce the influence of luminal Ca²⁺ on the RyR. The open probability of isolated SR Ca²⁺ channels increases in response to raised luminal [Ca²⁺].²⁷ However, luminal Ca²⁺ has no apparent influence on channel gating, when ATP is absent from the solution bathing the cytosolic face of the RyR. Such a reduction in the sensitivity of the RyR to luminal [Ca²⁺] might also explain why ATP depletion results in a maintained reduction in spark frequency, despite a marked increase in SR Ca²⁺ content (Figure 3). This differs from the inhibitory effects of tetracaine on the RyR, where spark frequency decreases on exposure to the drug but then increases again as the SR Ca²⁺ content rises.¹⁰

Effects of ATP on SR Ca²⁺ Content
The steady-state SR Ca²⁺ content reflects a balance between uptake (via the SR Ca²⁺-ATPase) and efflux (via Ca²⁺ leak pathways).²⁸ In the present study, the steady-state SR Ca²⁺ content increased progressively after stepwise reductions in [ATP] from 0.5 mmol/L to 10 µmol/L (Figure 3). The increase in SR Ca²⁺ content occurred over the same [ATP] range as the decrease in Ca²⁺ spark frequency, suggesting a causal relationship.

Results obtained after complete ATP withdrawal provided information regarding the relative effects of ATP on the SR Ca²⁺ uptake and efflux mechanisms. As shown in Figure 4, spark frequency decreased markedly within 60 seconds of complete ATP withdrawal. At this point, the SR Ca²⁺ content was higher than under control conditions, and it continued to increase, reaching a maximum 3 minutes after ATP withdrawal (Figure 6). The continued rise in the SR Ca²⁺ content suggests that it takes 3 minutes for the cytosolic [ATP] to fall from 5 mmol/L to a level that cannot sustain the activity of the SR Ca²⁺ pump (ie, <10 µmol/L).

Taken together, these results suggest that the rise in SR Ca²⁺ content occurs because the Ca²⁺ efflux associated with the RyRs is more sensitive to ATP than the Ca²⁺ pump. After ATP withdrawal, the decrease in spark frequency alters the balance between the uptake and efflux pathways, favoring net Ca²⁺ accumulation. Although the rate of SR Ca²⁺ uptake decreases as [ATP] falls to <0.1 mmol/L,¹⁸ the Ca²⁺ content will continue to rise because of the reduction in Ca²⁺ leak.

Effects of Introduction of AMP-PNP After ATP Withdrawal
After 3 minutes of exposure to zero ATP, the SR Ca²⁺ content was substantially higher than in the presence of 5 mmol/L ATP, and net Ca²⁺ uptake had ceased. At this point, introduction of AMP-PNP caused a rapid decrease in the SR Ca²⁺ content (Figure 6A). Within 60 seconds of exposure to AMP-PNP, the Ca²⁺ content decreased to a level that was 13% below the steady-state value obtained in the presence of 5 mmol/L ATP. Thereafter, the SR Ca²⁺ content declined much more slowly and followed a profile that was similar to the profile that occurred when ATP was substituted with AMP-PNP. As shown in Figure 4, withdrawal of ATP resulted in the abolition of spontaneous Ca²⁺ sparks, and subsequent reintroduction of AMP-PNP precipitated the transient appearance of large frequent Ca²⁺ sparks. Therefore, it seems likely that the rapid decline in SR Ca²⁺ content after the introduction of AMP-PNP results from a transient burst of Ca²⁺ sparks. A similar rapid decrease in SR Ca²⁺ content was obtained when 5 mmol/L ATP and 5 µmol/L thapsigargin were simultaneously introduced 3 minutes after ATP withdrawal (Figure 6B). The subsequent and much slower decrease in SR Ca²⁺ content over 7 to 8 minutes (Figure 6) occurs under conditions in which spark frequency is very low (Figure 5). This slow loss of SR Ca²⁺ may result from undetected Ca²⁺ efflux via the RyRs or efflux via other Ca²⁺ leak pathways.²⁸

In the Absence of SR Ca²⁺ Uptake, Ca²⁺ Sparks Result in Incomplete Discharge of SR Ca²⁺
One interesting feature of these experiments is that after ATP withdrawal, reintroduction of AMP-PNP did not fully deplete the SR (Figure 6A). After the introduction of AMP-PNP, the SR Ca²⁺ content declined rapidly for 40 to 60 seconds. However, the rate of decline then slowed abruptly. As shown in Figure 4, Ca²⁺ sparks persisted for 60 seconds after introduction of AMP-PNP. Therefore, the profound decrease in spark frequency probably accounts for the slowing in the rate of decline of the SR Ca²⁺ content. Consistent with previous studies, it seems likely that spark frequency decreases because the SR Ca²⁺ content declines. However, after 60 seconds, when sparks were rarely observed, the SR Ca²⁺ content was still 87% of the steady-state value in the presence of 5 mmol/L ATP (Figure 6A). A similar value was obtained in experiments in which ATP and thapsigargin were introduced 3 minutes after ATP withdrawal (Figure 6B).

One possible explanation for this is that in intact cells, the gain of the SR Ca²⁺ channel increases dramatically as the SR Ca²⁺ content rises to >70% to 80% of the maximum SR Ca²⁺ content attainable in the presence of 5 mmol/L ATP.²⁹ Hence, when the SR Ca²⁺ content is at or above this level, the high gain of the SR Ca²⁺ channel may precipitate the appearance of Ca²⁺ sparks. Conversely, when the SR Ca²⁺ content is at or below this critical level, Ca²⁺ sparks are abolished, or they appear to occur at very low frequencies.

Possible Relevance to Myocardial Ischemia or Reperfusion
Reperfusion of ischemic tissue is commonly associated with spontaneous Ca²⁺ release from the SR, symptomatic of Ca²⁺ overload. Such spontaneous Ca²⁺ release has been shown to activate a transient inward current, leading to afterdepolarizations and "triggered" arrhythmias.³⁰ The initiation of spontaneous Ca²⁺ release is induced by a localized Ca²⁺ spark, which propagates across the cell as a wave.³¹ We have shown previously that reducing [ATP] increases the amplitude but decreases the frequency of spontaneous Ca²⁺ waves.¹⁸ The present study suggests that the decrease in the frequency of spontaneous Ca²⁺ waves at low [ATP] may reflect a decrease in spark frequency because of desensitization of the RyR. Similarly, the increase in amplitude of spontaneous Ca²⁺ waves probably reflects the increase in spark amplitude that occurs at low [ATP], secondary to a rise in SR Ca²⁺ content (Figure 3). Hence, at low [ATP], spontaneous Ca²⁺ release is less frequent, but when it occurs, the larger Ca²⁺ release may be more likely to trigger arrhythmias.

Recent work has shown that in cyanide-induced metabolic blockade, the SR Ca²⁺ content increases but that Ca²⁺ sparks are abolished.³² The results of the present study suggest that the decrease in [ATP] that occurs in metabolic blockade may explain this effect. However, other cytosolic modulators of the SR Ca²⁺ channel, such as Mg²⁺ and H⁺, may also contribute.

	Acknowledgments

 
This study was supported by the British Heart Foundation and the Wellcome Trust.

Received April 23, 2001; accepted July 18, 2001.

	References

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