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(Circulation Research. 2001;88:181.)
© 2001 American Heart Association, Inc.

Cellular Biology

Altered Cardiac Sarcoplasmic Reticulum Function of Intact Myocytes of Rat Ventricle During Metabolic Inhibition

C. L. Overend, D. A. Eisner, S. C. O’Neill

From the Department of Medicine, University of Manchester, Manchester, UK.

Correspondence to Stephen C. O’Neill, Department of Medicine, 1.525 Stopford Building, Oxford Rd, University of Manchester, Manchester M13 9PT, UK. E-mail stephen.c.o'neill{at}man.ac.uk

	Abstract

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Abstract—Changes in the behavior of the sarcoplasmic reticulum (SR) in rat ventricular myocytes were investigated under conditions of metabolic inhibition using laser-scanning confocal microscopy to measure intracellular Ca²⁺ and the perforated patch-clamp technique to measure SR Ca²⁺ content. Metabolic inhibition had several effects on SR function, including reduced frequency of spontaneous releases of Ca²⁺ (sparks and waves of Ca²⁺-induced Ca²⁺ release), increased SR Ca²⁺ content (79.4±5.7 to 115.2±6.6 µmol/L cell volume [mean±SEM; P<0.001]), and, after a wave of Ca²⁺ release, slower reuptake of Ca²⁺ into the SR (rate constant of fall of Ca²⁺ reduced from 8.5±1.1 s^-1 in control to 5.2±0.4 s^-1 in metabolic inhibition [P<0.01]). Inhibition of L-type Ca²⁺ channels with Cd²⁺ (100 µmol/L) did not reproduce the effects of metabolic inhibition on spontaneous Ca²⁺ sparks. These results are evidence of inhibition of both Ca²⁺ release and reuptake mechanisms. Reduced frequency of release could be attributable to either of these effects, but the increased SR Ca²⁺ content at the time of reduced frequency of spontaneous release of Ca²⁺ shows that the dominant effect of metabolic inhibition is to inhibit release of Ca²⁺ from the SR, allowing the accumulation of greater than normal amounts of Ca²⁺. In the context of ischemia, this extra accumulation of Ca²⁺ would present a risk of potentially arrhythmogenic, spontaneous release of Ca²⁺ on reperfusion of the tissue.

Key Words: cardiac • sarcoplasmic reticulum • calcium • metabolic inhibition

	Introduction

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During myocardial ischemia, there are profound changes in many metabolite concentrations; for example, the tissue grows progressively more acid, ATP levels fall,¹ and inorganic phosphate² and free magnesium rise.³ Once blood supply is renewed, there follows a large rise of intracellular Ca²⁺ concentration ([Ca²⁺]_i).^{4 5} This may be the result of reactivation of the Na⁺-Ca²⁺ exchanger⁶ under conditions of elevated intracellular Na⁺ concentration, favoring influx of Ca²⁺ and efflux of Na⁺. As a consequence, the myocytes and, in particular, the sarcoplasmic reticulum (SR) become overloaded with Ca²⁺, a state that allows spontaneous release of Ca²⁺ that propagates along cells. This is of major importance, because spontaneous release of Ca²⁺ from the SR can lead to arrhythmias.⁷

The conditions after reperfusion clearly favor loading of the SR with Ca²⁺; however, relatively little is known of the loading of the SR during ischemic conditions. Many of the metabolic changes that occur during ischemia are known to inhibit both the Ca²⁺ ATPase and the Ca²⁺ release channel (ryanodine receptor [RyR]) of cardiac SR. For example, reducing [ATP] and increasing [ADP] will decrease both the activity of the SR Ca²⁺ ATPase and the open probability of the RyR.^{8 9} Intracellular acidification will also affect both.^{9 10} Therefore, the ability of the SR to accumulate Ca²⁺ may be compromised during ischemia, but its ability to retain Ca²⁺ may be improved. Therefore, it is difficult to say whether the SR Ca²⁺ content would increase or decrease. Indeed, examples in the literature can be found to demonstrate an increase, decrease, or no change in SR content.^{11 12 13}

Such considerations are important from 2 points of view. First, the loading of the SR before reperfusion will influence the likelihood of spontaneous release of Ca²⁺ on reperfusion. Second, if the SR Ca²⁺ store is being depleted during the onset of ischemia, this will accelerate the failure of contraction.

In the experiments reported here, we have attempted to determine what effects metabolic inhibition, as a means of imitating ischemia, has on the behavior and Ca²⁺ content of the SR in myocytes isolated from rat ventricular muscle. We have looked at changes in the frequency both of Ca²⁺ sparks and spontaneous waves of Ca²⁺ release and measured SR Ca²⁺ content. Our results show that the dominant effect of metabolic inhibition is to inhibit Ca²⁺ release through the RyR, thus favoring Ca²⁺ accumulation by the SR.

	Materials and Methods

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Care and use of animals were in accordance with the Animals (Scientific Procedures) Act 1986. Animals were supplied by Charles River UK. Rat myocytes were isolated using a collagenase and protease technique, as previously described.¹⁴ Rats were killed by stunning and cervical dislocation. For intracellular calcium measurements, cells were loaded with the membrane permeant form of Fluo-3, 5 µmol/L for 5 minutes, and 20 minutes were allowed for deesterification. Cells were placed in a superperfusion chamber on the stage of an inverted microscope, and Fluo-3 fluorescence was excited at 488 nm and measured at 515 nm using the Bio-Rad MRC 1024 confocal microscope. All voltage-clamp experiments (eg, Figure 4) were carried out using the perforated patch-clamp technique¹⁵ using the switch-clamp mode of the Axoclamp 2B amplifier (Axon Instruments). Pipettes were filled with the following solution (in mmol/L): KCH₃O₃S 125, KCl 20, NaCl 10, HEPES 10, and MgCl₂ 5, titrated to pH 7.2 with KOH and a final concentration of amphotericin B of 240 µg/mL.

View larger version (25K): [in this window] [in a new window]   Figure 4. Increased SR Ca2+ content during metabolic inhibition. A, Measurements of SR Ca2+ content in control (left) and late in metabolic inhibition (right). Upper traces show Na+-Ca2+ exchange currents after application of 20 mmol/L caffeine (indicated by the bars) to a voltage-clamped ventricular myocyte held at -80 mV. Lower traces are the integrals of these currents converted into concentrations of Ca2+. These measurements were carried out in cells that had been exposed to 20 µmol/L carboxyeosin to inhibit the surface membrane Ca2+ ATPase. B, Mean data for 7 cells.

The bathing solution was as follows (in mmol/L): NaCl 135, KCl 4, HEPES 10, glucose 11, and MgCl₂ 1, titrated to pH 7.4 with NaOH. Initially, cells were bathed in the above solution at 1 mmol/L CaCl₂. This level was altered to between 2 and 8 mmol/L, as indicated in the figure legends, to induce spontaneous waves of Ca²⁺ release. In voltage-clamp experiments, the above solution was modified to contain 5 mmol/L 4-aminopyridine (4-AP) and 0.1 mmol/L BaCl₂ to decrease outward currents. Cell length was measured using a video-based edge-detection system modified from a published circuit.¹⁶

Cells under voltage clamp were electrically stimulated by 100-ms depolarizations from -40 to 0 mV. Ca²⁺ content of the SR was measured from the integral of the caffeine-induced Na⁺-Ca²⁺ exchange inward current, as previously reported.^{17 18} One problem with this technique is that some of the Ca²⁺ is pumped out of the cell by the electroneutral Ca²⁺ ATPase and must be corrected for. To avoid uncertainties in this correction (see Results), we preincubated the cells for 15 minutes in 20 µmol/L 5- (and 6-) carboxyeosin diacetate (succinimidyl ester) from Molecular Probes to inhibit the sarcolemmal Ca²⁺ ATPase.¹⁹ The integrals are expressed with respect to the volume of the cell. Caffeine (20 mmol/L) applications were performed under control conditions and, when the current records indicated the later stages of metabolic inhibition by the development of a large outward current.

All experiments were carried out at room temperature (25°C). All statistics quoted are mean±SEM; Student’s paired t tests were used throughout to test significance.

	Results

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Effects of Metabolic Inhibition on the Frequency of Ca²⁺ Waves and Sparks
To determine how metabolic inhibition alters the behavior of cardiac SR, we have examined spontaneous release of Ca²⁺ during both waves and sparks. The trace of cell length in Figure 1 represents waves of Ca²⁺ release passing along the cell as a deflection of the trace (shortening of the cell). These waves of Ca²⁺ release were induced by bathing the cell in elevated external Ca²⁺ (5 mmol/L). When metabolic inhibition begins, there is a gradual reduction in the frequency of spontaneous waves until the cell begins to develop a contracture. At this stage, cyanide (CN) was removed, and the cell relaxed and resumed spontaneous waves.

View larger version (25K): [in this window] [in a new window]   Figure 1. Metabolic inhibition decreases the frequency of spontaneous waves of contraction. Metabolic inhibition in a single ventricular myocyte was achieved by addition of 2 mmol/L CN- and 10 mmol/L 2-deoxyglucose to the bathing solution.

It is known that spontaneous waves are initiated by Ca²⁺ sparks.²⁰ Therefore, we have examined the effects of metabolic inhibition on sparks. In Figure 2, we have measured Fluo-3 fluorescence using laser-scanning confocal microscopy in the line scan mode. The trace of Figure 2A is an average of the fluorescence from the central 20 pixels of the line scans. During the breaks in the trace, the laser scan was stopped to allow, respectively, the effects of metabolic inhibition to develop and recovery to be completed. It shows that, as in Figure 1, in metabolic inhibition, waves of spontaneous release of Ca²⁺ are abolished. Furthermore, in agreement with previous reports,^{4 5 14} it also shows an increase in baseline [Ca²⁺]_i. The effect of metabolic inhibition on spark activity can be seen in Figure 2B. Line scan a shows the occurrence of Ca²⁺ sparks under control conditions. No sparks are evident in line scan b, which was obtained during metabolic inhibition. There is also an increase of resting [Ca²⁺]_i. It might be argued that the increase of resting [Ca²⁺]_i increases the noise in the line scan and makes it harder to see sparks. In other words, the apparent disappearance of sparks in line scan b could simply be an artifact of raised resting [Ca²⁺]_i. To investigate this, we have added the sparks in line scan a to the image in line scan b and displayed the result in line scan d. Specifically, d=b+(a-56), where 56 is the mean signal level of a in the absence of sparks. Sparks are still visible against this elevated background; ie, if sparks similar to those in control had been present in metabolic inhibition, the elevated baseline fluorescence would not have obscured them. From our line scan data, on average, spark frequency fell from 2.8±0.7 s^-1 in control to 0.2±0.04 s^-1 (n=9, P<0.01) just before contracture in metabolic inhibition.

View larger version (73K): [in this window] [in a new window]   Figure 2. Metabolic inhibition abolishes spontaneous waves and sparks. A, Fluo-3 fluorescence records showing the effects of metabolic inhibition on intracellular Ca2+ in a single ventricular myocyte. The trace is an extended wave profile and represents the mean value of the central 10 pixels in each line scan. B, Line scans from the same cell as in panel A. a, control sparks; b, late in metabolic inhibition; c, recontrol; d, line scans a and b added with the mean value of background fluorescence in line scan a subtracted.

Does Metabolic Inhibition Affect Spark Frequency by Inhibiting L-Type Ca²⁺ Channels?
During metabolic inhibition, the L-type Ca²⁺ current is decreased.^{11 21} In voltage-clamp experiments (see later) at the time of onset of metabolic inhibition, as judged by the increase of outward current, the L-type Ca²⁺ current was reduced in amplitude by 16.8±5.7% (n=6, P<0.05; not shown). One theory of the origin of sparks is that they represent SR Ca²⁺ release triggered by random openings of L-type channels. It is possible, therefore, that inhibition of L-type channels might account for the disappearance of sparks in metabolic inhibition. To test this, we have applied 100 µmol/L Cd²⁺ to completely inhibit L-type Ca²⁺ current while measuring spark activity. As shown in Figure 3, after 20 seconds in Cd²⁺, spark activity persists (parallel experiments showed that 20-second exposure to 100 µmol/L Cd²⁺ completely abolished contraction). The elevated background fluorescence in the right panel is probably attributable to interaction between Fluo 3 and Cd²⁺.²² Similar results were seen in another 7 cells. The relatively modest degree of L-type current inhibition during metabolic inhibition and the lack of effect of Cd²⁺ on sparks means that complete abolition of sparks, as shown in Figure 2, is unlikely to be attributable to inhibition of L-type Ca²⁺ channels alone.

View larger version (113K): [in this window] [in a new window]   Figure 3. The effect of Cd2+ on spark activity. Line scan records showing spark activity in control (left) and 20 seconds after application of 100 µmol/L Cd2+. Although background fluorescence is increased in Cd2+, Ca2+ sparks are still clearly present.

Effects of Metabolic Inhibition on SR Ca²⁺ Content
There remain 2 possible explanations for the abolition of waves and sparks in metabolic inhibition: (1) inhibition of the Ca²⁺ release mechanism or (2) inhibition of the SR Ca²⁺ ATPase. These hypotheses can be distinguished, because they predict opposite changes of SR Ca²⁺ content before the development of a contracture, in (1) an increase and in (2) a decrease. This question was addressed in Figure 4. The SR Ca²⁺ content can be measured under voltage-clamp conditions by integrating the Na⁺-Ca²⁺ exchange current after application of a high concentration of caffeine to release the SR Ca²⁺ store.¹⁷ However, this method requires correction for the fraction of Ca²⁺ release from the SR pumped out of the cell, not by the Na⁺-Ca²⁺ exchange but by the electroneutral sarcolemmal Ca²⁺ ATPase. This can be done if the relative activities of the Na⁺-Ca²⁺ exchanger and Ca²⁺ ATPase are known. Use of this method in the present study is complicated by changes in, for example, [Na⁺] and [ATP] taking place during metabolic inhibition,^{2 23} which would be expected to alter the relative contributions of the exchanger and pump to Ca²⁺ efflux. To avoid this potential problem, voltage-clamp experiments were carried out after inhibition of the sarcolemmal Ca²⁺ ATPase by carboxyeosin.^{19 24 25} The 2 upper traces in Figure 4A show representative records of caffeine-induced Na⁺-Ca²⁺ exchange current in control (left) and after 4 minutes of metabolic inhibition (right). The integrals below each current show that there has been an increase of SR Ca²⁺ content during metabolic inhibition. Figure 4B shows that in a total of 7 cells exposed to metabolic inhibition for between 1 and 4 minutes, SR Ca²⁺ content increased from 79.4±5.7 to 115.2±6.6 µmol/L cell volume (mean±SEM; P<0.001).

The above changes in the SR Ca²⁺ content and frequency of spontaneous waves are consistent with inhibition of the Ca²⁺ release mechanism. We have also investigated whether the characteristics of spontaneous waves of Ca²⁺ release are altered in metabolic inhibition. A slow time-base record of spontaneous, propagating waves of Ca²⁺-induced Ca²⁺ release is shown in Figure 5A. One problem with this type of experiment is that prolonged exposure to the laser light causes the fluorescence intensity to decline as dye is bleached. Before conversion to Ca²⁺ concentration in Figure 5A, the fluorescence trace was corrected for bleaching by the laser light. The bleach rate was calculated by fitting an exponential to the decline in wave amplitude in the control period, and compensation was applied throughout the record. This shows the typical reduction in wave frequency during metabolic inhibition. In addition, 2 other effects are obvious: an increase in the amplitude of waves and a burst of high-frequency waves after removal of CN. The line scans below in Figure 5B show the individual waves marked in Figure 5A. Ca²⁺ remains elevated longer during the wave in metabolic inhibition. The wave profiles below show how the [Ca²⁺] changed with time at one point in the cell as the wave propagated. The fall of the Ca²⁺ transient is clearly slower in metabolic inhibition. On average (n=6), the rate constant of decay of Ca²⁺ fell from 8.5±1.1 s^-1 in control to 5.2±0.4 s^-1 in metabolic inhibition (P<0.01, Student’s paired t test). One explanation of such a slowing of the fall of Ca²⁺ would be an inhibition of the SR Ca²⁺ ATPase. Changes of wave amplitude similar to those in Figure 5 were seen in another 5 cells after correction for bleaching of the dye. Metabolic inhibition had little effect on propagation velocity (a nonsignificant increase of 11.3±5.8%; P>0.1).

View larger version (53K): [in this window] [in a new window]   Figure 5. Increased amplitude and slowed recovery of propagating waves in metabolic inhibition. A, Inhibition of spontaneous waves of Ca2+ release by metabolic inhibition in a single ventricular myocyte (Fluo-3 fluorescence has been corrected for bleaching). The trace is an extended-wave profile and represents the mean value of the central 10 pixels in each line scan. B, Sample line scans showing the waves indicated by the arrows in panel A. Line scans and wave profiles below show slowing of the recovery phase of the wave in metabolic inhibition. Wave profile in metabolic inhibition is shown in red; control wave (shown in black) has been normalized for amplitude and superimposed.

As mentioned above, the reduced rate of fall of the wave profile in Figure 5 may be attributable to inhibition of the SR Ca²⁺ ATPase. If so, we would expect to see similar effects from the SR Ca²⁺-ATPase inhibitor thapsigargin. Figure 6A shows that inhibition of the SR Ca²⁺ ATPase reduces both frequency and amplitude of spontaneous waves. In the line scans and wave profiles shown in Figure 6B, it is clear that there is also a slower recovery of Ca²⁺ during the wave. Similar results were seen in another 8 cells. The similar slowing of recovery of the Ca²⁺ wave in metabolic inhibition, therefore, is consistent with inhibition of the SR Ca²⁺ pump.

View larger version (49K): [in this window] [in a new window]   Figure 6. Inhibition of the SR Ca2+ ATPase slows the recovery of spontaneous waves of Ca2+ release. A, Effect of thapsigargin (1 µmol/L) on spontaneous waves of Ca2+ release in a single ventricular myocyte. The trace is an extended wave profile and represents the mean value of the central 10 pixels in each line scan. B, Sample line scans showing the waves indicated in panel A. Wave profile in thapsigargin is shown in red; the superimposed profile in black is from the control wave and has been normalized for amplitude.

	Discussion

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The immediate consequences of metabolic inhibition involve a dramatic reduction in the action potential duration and, as a result, loss of contractile function.²¹ With the failure of the action potential, there is no trigger Ca²⁺ current to activate release of Ca²⁺ from the SR, and so contraction fails. However, little is known of what happens to the SR itself at this time. Measurements of the Ca²⁺ transient produced by application of caffeine in whole-cell voltage-clamped guinea pig ventricular myocytes have shown that SR Ca²⁺ content is at least maintained¹² and may in fact increase during metabolic inhibition.¹¹ There is also evidence from whole ferret hearts that SR Ca²⁺ content is at least maintained in ischemia.²⁶ However, experiments using anoxic ventricular myocytes from guinea pigs have shown a reduced content.¹³ During ischemia, the concentrations of many metabolites change dramatically; for example, intracellular pH falls by as much as one pH unit, and ATP levels may fall from 5 to 10 mmol/L to <100 µmol/L.² These particular changes are known to reduce the open probability of the Ca²⁺ release channel in the SR membrane. However, these same changes would also have powerful effects on the SR Ca²⁺ ATPase and would most likely reduce its ability to sequester Ca²⁺. Therefore, on the one hand, the pumping of Ca²⁺ into the SR may be compromised during ischemia, but the ease with which Ca²⁺ could leave the SR may also be reduced. Hence, it is unclear what changes in SR Ca²⁺ content, if any, to expect in ischemia. Therefore, we have investigated the consequences of ischemia, simulated by metabolic inhibition, on SR function in single, isolated ventricular myocytes from rat hearts.

Does Inhibition of L-Type Channels Explain the Disappearance of Sparks?
A consistent finding in this study is that metabolic inhibition abolishes spontaneous release of Ca²⁺ from the SR in the form of Ca²⁺ sparks and waves. In our experiments, we also find a reduction of L-type Ca²⁺ current amplitude during metabolic inhibition (not shown). This is in agreement with previous reports.^{11 21} Because L-type Ca²⁺ channel openings are thought to trigger Ca²⁺ sparks,²⁷ inhibition of Ca²⁺ current could be at least partly responsible for the loss of sparks we report. We conclude that this is unlikely to explain the complete abolition of sparks because of the modest degree of inhibition of the L-type current in our experiments and the data shown in Figure 3. Sparks (presumably attributable to spontaneous openings of RyRs) are clearly present, even when L-type Ca²⁺ current is completely inhibited by Cd²⁺. That spontaneous sparks do not rely entirely on L-type Ca²⁺ current has been previously reported.²⁸ Complete abolition of sparks in metabolic inhibition cannot, therefore, be attributable solely to inhibition of L-type channel openings.

Pump or RyR Inhibition?
In Ca²⁺-overloaded cardiac cells, spontaneous waves of Ca²⁺-induced Ca²⁺ release occur once a certain threshold level of SR content is reached.²⁹ This threshold level of Ca²⁺ content may allow waves to propagate as the gain of Ca²⁺-induced Ca²⁺ release is enhanced, or it may actually initiate release. Either way, the loss of Ca²⁺ from the cell activated by release of Ca²⁺ from the SR serves to limit the amount of Ca²⁺ the SR can hold. The two possible effects of metabolic inhibition on the SR can be distinguished by their effects on SR Ca²⁺ content. Inhibition of the SR Ca²⁺ ATPase under conditions of Ca²⁺ overload would have one of two effects: either Ca²⁺ waves would stop and SR Ca²⁺ content would fall (failure to reach threshold) or wave frequency would fall with no change in SR Ca²⁺ content (threshold reached more slowly). One would expect the effects of inhibition of the RyR to be similar to those produced by substances that also inhibit the RyR,³⁰ such as tetracaine; ie, a decrease in the frequency of spontaneous release accompanied by an increase of SR Ca²⁺ content.³¹ As shown in Figure 4, SR Ca²⁺ content is increased on average by 50% in the period before onset of contracture that marks the final fall of ATP below the level that initiates rigor. Thus, an increase in SR Ca²⁺ content accompanies a decrease in the frequency of SR spontaneous-release events. We conclude, therefore, that the dominant effect on the SR of changes in metabolite levels during metabolic inhibition is reduced RyR sensitivity and not pump inhibition. Previous studies that have examined the effect of acidification in skinned cardiac muscle have found that SR Ca²⁺ content is reduced.^{32 33} One possibility to explain the difference from our results may be that in the skinned preparation, the relative effects of acid pH on the RyR and the SR Ca²⁺ ATPase are different. However, it is also possible that by allowing the cell to oscillate spontaneously rather than using a predetermined time at which to measure SR Ca²⁺ content, we allow the SR to accumulate more Ca²⁺ than in the control steady state; ie, the rate of Ca²⁺ accumulation in the more-acid conditions may be slowed, but the total amount stored may be greater.

The present effects of metabolic inhibition are similar to those of tetracaine in that the frequency of spontaneous waves of Ca²⁺ release is reduced whereas SR Ca²⁺ content is increased. In addition, as with tetracaine, the magnitude of each Ca²⁺ wave is increased (Figure 5). This may be a direct result of the increase of SR Ca²⁺ content. With tetracaine, the combination of decreased frequency and increased magnitude means that Ca²⁺ efflux per unit time activated by waves is unaffected by tetracaine, a result that is required to balance constant influx of Ca²⁺ into the cell. A similar argument can be applied to metabolic inhibition, but it is also possible that prolongation of the Ca²⁺ elevation in the wave (Figure 5), which would activate sarcolemmal Ca²⁺ efflux pathways for longer, assists in maintaining Ca²⁺ flux balance. Another similarity with tetracaine is the presence of the high frequency burst on removal of CN (Figure 5). This is not seen in all cases but does suggest removal of inhibition in the face of increased SR Ca²⁺ content. That it is not always seen may be attributable to the relatively slower reversal of inhibition than is possible with tetracaine.

Evidence of SR Ca²⁺-ATPase Inhibition
The wave profiles in Figure 5 show slowing of the recovery of the Ca²⁺ transient at any given point in the cell as it participates in the wave. This might reflect impaired pumping of Ca²⁺ into the SR, a possibility supported by the similar effect of the SR Ca²⁺-ATPase inhibitor thapsigargin (Figure 6). The SR Ca²⁺ ATPase might be inhibited by either or both of the profound intracellular acidification and changes in high-energy phosphate compounds. Despite this evidence of pump inhibition, SR Ca²⁺ content is increased, indicating that inhibition of the RyR is the more powerful effect on SR function. The question remains of which of the many changes taking place within the cell during metabolic inhibition are responsible for the change in RyR sensitivity. The record shown in Figure 1 may give some indication. The fall in frequency seems a very early consequence of metabolic inhibition and is associated with an increase in the resting cell length. One event likely to produce these effects is a fall of intracellular pH. It is known that pH begins shifting acid relatively early in metabolic inhibition,^{14 34} whereas ATP (and, therefore, also free Mg²⁺) remains more or less constant for a substantial period, being buffered by breakdown of creatine phosphate. The increase in resting cell length (Figure 1) can also be attributed to falling pH. In the Ca²⁺-overload conditions of the experiment, it is likely that resting intracellular Ca²⁺ is high enough to activate some contraction. This would be inhibited by the progressively more acid conditions,³² and the cell would relax as a result. Therefore, it may be that the early effects of metabolic inhibition on the Ca²⁺ release mechanism are attributable to the fall of intracellular pH. It is known that the resting level of intracellular Ca²⁺ rises in metabolic inhibition before the onset of the contracture (Figure 2 and Reference 14¹⁴ ). Might this explain the increase of SR Ca²⁺ content we report without recourse to any inhibition of the RyR? Certainly elevated cytoplasmic [Ca²⁺] would allow sequestration of more Ca²⁺ within the SR, but associated with this, one would expect an increased frequency of spontaneous release of Ca²⁺. In fact, the frequency of spontaneous release falls, and so we conclude that raised cytoplasmic [Ca²⁺] is a less important influence on SR Ca²⁺ content than inhibition of the RyR.

Inhibition of RyR and Contractile Failure
In intact cardiac muscle following normal sinus rhythm, ischemia leads to contractile failure as the action potential fails to propagate into the ischemic area. What effect would a progressively greater inhibition of the SR Ca²⁺ release mechanism have on contractile function? Perhaps surprisingly, there would probably be little or no effect.³⁵ It has been shown that rapid application of tetracaine, an inhibitor of the RyR, causes only transient inhibition of systolic release of Ca²⁺ from the SR.³⁶ This is because SR Ca²⁺ content increases, compensating for the reduced sensitivity of the release channel. In this way, the same level of contraction is achieved even though RyR sensitivity is reduced. Therefore, during ischemia, as inhibition of release becomes progressively greater, so too would the compensating increase of SR Ca²⁺ content. This will continue until impairment of the SR Ca²⁺ ATPase means the compensatory increase can no longer continue. From that point, the SR may only be able to maintain its load (as leak will be very low) without any additional increase. This compensatory increase of SR Ca²⁺ content will only compensate for the effects of inhibition of the RyR; it will not affect the inhibition of contraction produced by shortening of the action potential. The progressive loss of contractile function in ischemia is not, therefore, attributable to inhibition of the RyR by changes in metabolite concentrations. If the main influence of the metabolite changes in ischemia were to impair the function of the pump, the failure of contraction would be accelerated.

Functional Importance During Reperfusion
The status of the SR Ca²⁺ store may seem of little importance as the cell enters a terminal contracture in the final stages of ischemia. It would be much more important, however, if the tissue is saved from this fate by reperfusion. The two possible consequences of ischemia for SR Ca²⁺ content (depleted because of pump inhibition or overfilled because of RyR inhibition) might lead to quite different outcomes on reperfusion. During metabolic inhibition, intracellular Na⁺ rises^{23 37} ; on reperfusion, the Na⁺-Ca²⁺ exchanger is reactivated,⁶ and Ca²⁺ influx on the exchanger results. An empty SR would be able to take some of this influx with less likelihood of spontaneous, arrhythmogenic waves of Ca²⁺ release. However, the SR is more than usually full as ischemia progresses and, therefore, is probably predisposed to spontaneous release of Ca²⁺ on removal of the inhibitory effect on the RyR, even without additional influx. Therefore, the changes of metabolite concentrations taking place in metabolic inhibition serve to increase the likelihood of spontaneous release on reperfusion.

	Acknowledgments

 
This work was supported by grants from The British Heart Foundation and The Wellcome Trust.

	Footnotes

 
Original received September 13, 2000; revision received November 28, 2000; accepted November 28, 2000.

	References

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