Effects of Cytosolic ATP on Ca2+ Sparks and SR Ca2+ Content in Permeabilized Cardiac Myocytes
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.
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
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.
The bath was placed on the stage of a Nikon Diaphot Eclipse inverted microscope, and the cells were viewed with a ×40 oil immersion lens (Nikon Plan Fluor DLL, numerical aperture 1.3) or a ×60 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 ×60 objective, 1.07 mm for ×40 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 2.6×SD.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).
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/Fo) 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.
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/Fo. 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 ΔF/Fo). 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
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.
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.
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.
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.
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.
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.
This study was supported by the British Heart Foundation and the Wellcome Trust.
Original received April 23, 2001; revision received June 26, 2001; accepted July 18, 2001.
Cheng H, Cannell MB, Lederer WJ. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science. 1993; 262: 740–744.
Cannell MB, Cheng H, Lederer WJ. Spatial nonuniformities in [Ca2+]i during excitation-contraction coupling in cardiac myocytes. Biophys J. 1994; 67: 1942–1956.
Bridge JH, Ershler PR, Cannell MB. Properties of Ca2+ sparks evoked by action potentials in mouse ventricular myocytes. J Physiol. 1999; 518: 469–478.
Lukyanenko V, Gyorke I, Subramanian S, Smirnov A, Wiesner TF, Gyorke S. Inhibition of Ca2+ sparks by ruthenium red in permeabilized rat ventricular myocytes. Biophys J. 2000; 79: 1273–1284.
Shacklock PS, Wier WG, Balke CW. Local Ca2+ transients originate at transverse tubules in rat-heart cells. J Physiol. 1995; 487: 601–608.
Lopez-Lopez JR, Shacklock PS, Balke CW, Wier WG. local calcium transients triggered by single L-type calcium-channel currents in cardiac-cells. Science. 1995; 268: 1042–1045.
Lukyanenko V, Gyorke I, Gyorke S. Regulation of Ca2+ release by calcium inside the sarcoplasmic reticulum in ventricular myocytes. Pflugers Arch. 1996; 432: 1047–1054.
Satoh H, Blatter LA, Bers DM. Effects of [Ca2+]i, SR Ca2+ load, and rest on Ca2+ spark frequency in ventricular myocytes. Am J Physiol. 1997; 272: H657–H668.
Bassani RA, Bers DM. Rate of diastolic Ca release from the sarcoplasmic reticulum of intact rabbit and rat ventricular myocytes. Biophys J. 1995; 68: 2015–2022.
Gyorke S, Lukyanenko V, Gyorke I. Dual effects of tetracaine on spontaneous calcium release in rat ventricular myocytes. J Physiol. 1997; 500: 297–309.
Overend CL, O’Neill SC, Eisner DA. The effect of tetracaine on stimulated contractions, sarcoplasmic reticulum Ca2+ content and membrane current in isolated rat ventricular myocytes. J Physiol. 1998; 507: 759–769.
Rousseau E, Smith JS, Henderson JS, Meissner G. Single channel and Ca-45 flux measurements of the cardiac sarcoplasmic-reticulum calcium-channel. Biophys J. 1986; 50: 1009–1014.
Sitsapesan R, McGarry SJ, Williams AJ. Cyclic ADP -ribose competes with ATP for the adenine-nucleotide binding-site on the cardiac ryanodine receptor Ca2+ release channel. Circ Res. 1994; 75: 596–600.
Xu L, Mann G, Meissner G. Regulation of cardiac Ca2+ release channel (ryanodine receptor) by Ca2+, H+, Mg2+, and adenine-nucleotides under normal and simulated ischemic conditions. Circ Res. 1996; 79: 1100–1109.
Cui Y, Galione A, Terrar DA. Effects of photoreleased cADP-ribose on calcium transients and calcium sparks in myocytes isolated from guinea-pig and rat ventricle. Biochem J. 1999; 342: 269–273.
Lukyanenko V, Gyorke S. Ca2+ sparks and Ca2+ waves in saponin-permeabilized rat ventricular myocytes. J Physiol. 1999; 521 (pt 3): 575–585.
Balnave CD, Vaughan-Jones RD. Effect of intracellular pH on spontaneous Ca2+ sparks in rat ventricular myocytes. J Physiol. 2000; 528 (pt 1): 25–37.
Yang Z, Steele DS. Effects of cytosolic ATP on spontaneous and triggered Ca2+-induced Ca2+ release in permeabilised rat ventricular myocytes. J Physiol. 2000; 523 (pt 1): 29–44.
Fabiato A, Fabiato F. Calculator programs for computing the composition of solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris). 1979; 75: 463–505.
Smith GL, Miller DJ. Potentiometric measurements of stoichiometric and apparent affinity constants of EGTA for protons and divalent ions including calcium. Biochem Biophys Acta. 1985; 839: 287–299.
Duncan L, Burton FL, Smith GL. REACT: calculation of free metal and ligand concentrations using a Windows-based computer program. J Physiol. 1999; 517P. Abstract.
Cheng H, Song LS, Shirokova N, Gonzalez A, Lakatta EG, Rios E, Stern MD. Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method. Biophys J. 1999; 76: 606–617.22a. Gomez AM, Cheng H, Lederer WJ, Bers DM. Ca2+ diffusion and sarcoplasmic reticulum transport both contribute to [Ca2+]i decline during Ca2+ sparks in rat ventricular myocytes. J Physiol. 1996;496:575–581.
Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR, Dulhunty AF. Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. J Membr Biol. 1995; 147: 7–22.
Korge P, Byrd SK, Campbell KB. Functional coupling between sarcoplasmic-reticulum-bound creatine kinase and Ca2+-ATPase. Eur J Biochem. 1993; 213: 973–980.
Meissner G, Henderson JS. Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin. J Biol Chem. 1987; 262: 3065–3073.
McGarry SJ, Williams AJ. Adenosine discriminates between the caffeine and adenine-nucleotide sites on the sheep cardiac sarcoplasmic-reticulum calcium-release channel. J Membr Biol. 1994; 137: 169–177.
Sitsapesan R, Williams AJ. Regulation of the gating of the sheep cardiac SR Ca2+ release channel by luminal Ca2+. J Membr Biol. 1994; 137: 215–226.
Feher JJ, Fabiato A. Cardiac sarcoplasmic reticulum: calcium uptake and release. In: Langer GA, ed. Calcium and the Heart. New York, NY: Raven Press Ltd; 1990.
Bassani JWM, Yuan WL, Bers DM. Fractional SR Ca2+ release is regulated by trigger Ca2+ and SR Ca2+ content in cardiac myocytes. Am J Physiol. 1995; 37: C1313–C1319.
Wit AL, Rosen MR. Afterdepolarisations and triggered activity: distinction from automaticity as an arrhythmogenic mechanism. In: Fozzard HA, ed. The Heart and the Cardiovascular System. New York, NY: Raven Press Ltd; 1992.
Cheng H, Lederer MR, Lederer WJ, Cannell MB. Ca2+ sparks and [Ca2+]i waves in cardiac myocytes. Am J Physiol. 1996; 39: C148–C159.
Overend CL, Eisner DA, O’Neill SC. Altered cardiac sarcoplasmic reticulum function of intact myocytes of rat ventricle during metabolic inhibition. Circ Res. 2001; 88: 181–187.