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J Physiol (2003), 550.2, pp. 413-418
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.042846

pH-dependent and -independent effects inhibit Ca²⁺-induced Ca²⁺ release during metabolic blockade in rat ventricular myocytes

S. C. O'Neill and D. A. Eisner

	ABSTRACT

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We have investigated the role of changes of intracellular pH (pH_i) in the effects of metabolic blockade (cyanide plus 2-deoxyglucose) on Ca²⁺ release from the sarcoplasmic reticulum (SR) in rat ventricular myocytes. pH_i and cell length were measured simultaneously. Metabolic blockade decreased the frequency of Ca²⁺ waves, an effect previously shown to be due to inhibition of Ca²⁺ release from the SR. This was accompanied by an intracellular acidification. Intracellular acidification was produced in the absence of metabolic inhibition by application of sodium butyrate. A maintained intracellular acidosis produced a decrease of wave frequency. A hysteresis between pH_i and wave frequency was observed such that during the onset of the acidification the wave frequency decreased more than in the steady state. Comparison of the steady state relationship between pH_i and wave frequency showed that the decrease of wave frequency produced by metabolic blockade was greater than could be accounted for simply by the accompanying decrease of pH_i. In other experiments the buffering power of the solution was increased. Under these conditions, metabolic blockade produced no change of pH_i but the decrease of wave frequency persisted. We conclude that, although intracellular acidification occurs during metabolic blockade, it is not responsible for most of the inhibition of Ca²⁺ release from the SR.

(Resubmitted 11 March 2003; accepted after revision 28 April 2003; first published online 23 May 2003)
Corresponding author S. C. O'Neill: Unit of Cardiac Physiology, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK. Email: stephen.c.o'neill{at}man.ac.uk

	INTRODUCTION

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During systole Ca²⁺ is released from the sarcoplasmic reticulum (SR) into the cytoplasm through a release channel known as the ryanodine receptor (RyR). The probability that the RyR is open is increased by an increase of cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and this results in calcium-induced calcium release (CICR) as Ca²⁺ entry into the cell triggers release from the SR. However, the open probability of the RyR is also influenced by many intracellular metabolic components that are known to change during myocardial ischaemia. For instance, lowering [ATP], raising free [Mg²⁺] and lowering intracellular pH are all known to lower the RyR open probability (Xu et al. 1996) and all three occur during ischaemia ( Headrick & Willis, 1991; Elliott et al. 1992). We have recently demonstrated in single cardiac myocytes that the Ca²⁺-induced Ca²⁺ release mechanism is inhibited during metabolic blockade when both aerobic and anaerobic metabolism are inhibited as in ischaemic tissue. We found that the frequency of spontaneous waves of Ca²⁺-induced Ca²⁺ release was reduced along with an increase of SR Ca²⁺ content (Overend et al. 2001). This combination of effects also occurs when the open probability of the RyR is decreased by application of the local anaesthetic tetracaine indicating the inhibition of CICR in metabolic blockade (Overend et al. 1997).

Although inhibition of CICR would not contribute to the failure of systolic Ca²⁺ release that occurs during ischaemia (see Eisner et al. 2000 for review), it might be profoundly important for the outcome of re-perfusion of previously ischaemic tissue. It is well known that, paradoxically, re-perfusion of ischaemic tissue can worsen damage to the tissue, partly as the result of a large influx of Ca²⁺ (Piper et al. 1993). One consequence of this influx is Ca²⁺ overload of the cardiac cells, resulting in spontaneous release of Ca²⁺ from the sarcoplasmic reticulum and the risk of generating arrhythmias. The SR by virtue of its role as a store of intracellular Ca²⁺ could reduce the impact of this influx of Ca²⁺ if spare capacity for Ca²⁺ storage were available. The higher than normal SR Ca²⁺ content during metabolic blockade, however, coupled with the removal of the inhibition of CICR on re-perfusion means that the SR is, at best, poorly placed to deal with this influx and may already be set to increase spontaneous activity due to its high load. Inhibition of CICR during metabolic blockade may, in fact, worsen the effects of re-perfusion. It is important, therefore, to understand the factors involved in the reduced open probability of the RyR during ischaemia.

The goal of the present paper is to understand which of the cellular changes occurring during metabolic blockade is responsible for the decreased frequency of spontaneous waves and increased SR Ca²⁺ content. One candidate is the intracellular acidification. This will decrease the open probability of the RyR (Xu et al. 1996). Consistent with this, a decrease in the frequency of Ca²⁺ 'sparks' is observed (Balnave & Vaughan-Jones, 2000) and there is a transient decrease in the amplitude of the systolic Ca²⁺ transient during acidification (Choi et al. 2000). It is not, however, known what the effects of acidosis are on spontaneous Ca²⁺ waves as previous work showing effects of pH on spontaneous Ca²⁺ release could not distinguish between direct effects of pH and those secondary to changes of intracellular Ca²⁺ (Orchard et al. 1987). In this paper we therefore address two questions. (1) What are the effects of intracellular acidification on Ca²⁺ wave frequency? (2) Can these effects of acidification account for all of the inhibitory effects of metabolic blockade on the Ca²⁺ release mechanism? Our results show that, although acidification has qualitatively similar effects to metabolic blockade, only part of the inhibitory effect of metabolic blockade is due to the resultant acidification and that, during the latter stages especially, some other powerful inhibitory influence develops.

	METHODS

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Rat ventricular myocytes were isolated using a collagenase and protease technique as previously described (Eisner et al. 1989). Rats were killed by stunning and cervical dislocation. All experiments and animal care were in accordance with the provisions of the Animal Procedures Act (1986). For intracellular pH measurements, cells were loaded with the membrane-permeant form of carboxy SNARF-1 at 5 µM for 30 min and 20 min were allowed for de-esterification. Cells were placed in a superfusion chamber on the stage of an inverted microscope. Fluorescence was excited at 530 nm and recorded at 580 and 650 nm (Choi et al. 2000) using epi-fluorescence optics. An in vitro calibration was carried out on the microscope using 2 µM of the free acid of carboxy SNARF set to pH 4.0, 7.0 and 10.0.

The bathing solution was as follows (mM): NaCl, 135; KCl, 4; Hepes, 10; glucose, 11; MgCl₂, 1; titrated to pH 7.4 with NaOH. Initially cells were bathed in the above solution at 1 mM CaCl₂; this level was raised to 2 mM and ouabain (0.5 mM) was added to induce spontaneous waves of Ca²⁺ release. Metabolic blockade was induced by addition of 2 mM CN^- and replacement of glucose with 2-deoxyglucose (2-DOG) to block aerobic and anaerobic metabolism.

In experiments where it was required that changes in intracellular pH be prevented, the bathing solution was altered to one with a higher buffering power produced by bicarbonate (mM): NaHCO₃, 130; NaCl, 10; KCl, 4; MgCl₂, 1; CaCl₂, 1. This solution was gassed with 20 % CO₂ to set the pH to 7.2. As explained above spontaneous waves were induced by raising external Ca²⁺ to 2 mM and addition of 0.5 mM ouabain.

All experiments were carried out at room temperature (25 °C).

Data are presented as means ± S.E.M.

	RESULTS

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A typical effect of metabolic blockade on spontaneous waves of CICR is shown in Fig. 1. Ca²⁺ overload was induced as described in Methods; each wave of CICR is indicated by a decrease of the cell length. The interval between waves is shown in the lower trace. When metabolic blockade is applied the frequency of waves decreases (interval increases) until after several minutes they are abolished. We have previously established that at this point SR Ca²⁺ content is elevated, i.e. CICR has been inhibited (Overend et al. 2001). One possible cause of this inhibition is the acidification known to take place during metabolic blockade (Eisner et al. 1989). Therefore, in the remainder of this paper we have investigated the hypothesis that intracellular acidification is responsible for the abolition of waves in metabolic blockade.

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Figure 1. Effects of metabolic blockade on the frequency of spontaneous waves Upper trace, percentage change of cell length; lower trace, the interval between successive waves. Metabolic blockade was induced for the period shown (see Methods for details of solutions).

To study the effects of a pure acidification on CICR we have used application of the weak acid butyrate to lower intracellular pH (pH_i) without changing external pH. The effects of this on pH_i and spontaneous waves of CICR are shown in Fig. 2A. On application of 20 mM butyrate, pH_i went acid by about 0.4 of a pH unit and the frequency of waves was reduced. On removal of butyrate pH_i returned to control levels and wave frequency recovered. Close examination of the plot of interval between waves against time in the lower part of Fig. 2A shows that the wave frequency is transiently lower at first on application of butyrate before reaching a steady state and transiently rises above control on its removal. These transient effects, as well as the maintained slowing, are similar to the effects of adding the RyR inhibitor tetracaine and suggest a similar mechanism for acidosis and tetracaine. Figure 2B plots the relationship between wave interval and pH_i. The instantaneous relationship (filled symbols) shows that at a given pH_i value during acidification, the wave interval is longer than when pH_i is recovering following removal of butyrate. This probably reflects the changes of SR Ca²⁺ content taking place as the SR load first rises during acidification and then falls again on removal of the acid. As the pH changes in metabolic blockade are relatively slow, the steady state relationship (open symbols) is probably more appropriate as a comparison with metabolic blockade. This is derived from the application of several different concentrations of butyrate (5, 10 and 20 mM) to produce different levels of acidification. Each steady state interval achieved is plotted as a function of the mean pH_i over the same time period. The steady state relationship differs from the initial state in butyrate as SR Ca²⁺ content has risen and from the recovery phase as the inhibitory effect of the acidification is still present. The steady state data, therefore, lie between the two limbs of the instantaneous relationship.

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Figure 2. The effects of changes of intracellular pH on the frequency of spontaneous waves A, original data. Upper trace, pHi; middle trace, cell length; lower trace, interval between waves. Sodium butyrate (But; 20 mM) was applied for the period shown (constant external pH). B, relationship between pHi (abscissa) and interval between waves (ordinate). Filled circles represent values obtained during the application and removal of 20 mM butyrate shown in A. The arrows show the sequence of changes with time during the application (on) and removal (off) of butyrate. The three left hand open circles were obtained after applying butyrate at concentrations of 5, 10 and 20 mM. The four right hand open circles are control points obtained before and after adding each of the concentrations of butyrate and show an acid drift due to incomplete recovery from each of the butyrate concentrations. Measurements in butyrate were obtained when a steady state pHi was reached (typically between 30 s and 1 min in butyrate).

The steady state curve shown in Fig. 2B should allow us to determine whether the inhibitory effect of metabolic blockade on CICR is due to acidification. This comparison is made in Fig. 3; the top two traces in panel A show pH_i and cell length. On application of metabolic blockade (CN^- to inhibit oxidative phosphorylation and 2-DOG to inhibit glycolysis) both intracellular pH and the frequency of spontaneous waves fall. The lower record in Fig. 3A shows the interval between waves during the control period (filled circles) and during metabolic blockade (open circles). There is an initial, abrupt increase of oscillation interval on commencing metabolic blockade, followed by a gradual increase with time until waves were abolished and the cell entered a rigor contracture, indicating that reserves of ATP had been completely depleted. In Fig. 3B we can directly compare the effect of metabolic blockade with the steady state relationship between intracellular pH and the interval between waves (triangles). The control data from Fig. 3A (filled circles) lie on this steady state relationship (from Fig. 2) and, leaving aside the first two data points, so too do the earlier points during metabolic blockade. Initially, therefore, in metabolic blockade, CICR appears to be inhibited by acidification alone. Once the pH has reached about 6.9 during metabolic blockade, however, the interval between waves is increased much more than would be expected from the changes in pH measured. This suggests that another inhibitory influence is developing alongside that of acidification; similar results were seen in another three cells studied. The first two points in metabolic blockade in Fig. 3B also deviate from the steady state pH effect. This may be due to the rather rapid acidification that develops at this time and can be explained in the same manner as the difference between the instantaneous and steady state relationships shown in Fig. 2.

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Figure 3. Comparison of the relationship between pHi and wave frequency during metabolic blockade and direct intracellular acidification A, the effects of metabolic blockade. Traces show (from top to bottom): pHi; cell length; interval between waves. Metabolic blockade was induced for the period shown. B, the relationship between pHi (abscissa) and interval between waves (ordinate). Filled circles represent values obtained in control conditions and open circles represent values obtained during metabolic blockade as shown in A. The open triangles represent values obtained following application of various concentrations of butyrate when pHi had reached a steady state (cf. Fig. 2).

The data of Fig. 3 suggest that some of the effects of metabolic blockade are too large to be accounted for by the measured change of pH_i. This raises the possibility that another factor also contributes. In the final series of experiments we have taken steps to minimize the change of pH_i produced by metabolic blockade in order to evaluate the contribution of pH-independent factors. This was done by 'clamping' pH by raising the pH buffering capacity of the cell using higher than normal levels of HCO₃^- and CO₂ (Allue et al. 1996). Results from such an experiment are shown in Fig. 4. Again Ca²⁺ overload was induced by raising external Ca²⁺ and metabolic blockade by application of CN^- and 2-DOG. The top trace shows that pH_i is almost completely unchanged during metabolic blockade; however, the frequency of waves still falls. On average (in six cells) there was no significant change of pH_i (mean acidification 0.022 ± 0.013; paired t test, P = 0.18) at the time that waves were abolished. This fall is clearly not due to any changes in pH_i and must be due to the second factor we demonstrated in Fig. 3.

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Figure 4. The effects of metabolic blockade under conditions of increased pH buffering Traces show (from top to bottom): pHi; cell length; interval between waves. Metabolic blockade was applied for the period shown. In this experiment the solution was buffered with 130 mM bicarbonate (see Methods).

The data presented in this paper are summarized and can be compared in Fig. 5. This shows the effects on pH_i and wave frequency of butyrate application, and of metabolic blockade (with pH_i either free to change or heavily buffered, pH clamp). Each demonstrates a reduction of frequency from the control (defined as 1). The point for butyrate application shows the change in pH_i and wave frequency in the steady state in 20 mM butyrate (n = 7). At the time of maximum effect of metabolic blockade on wave frequency in the same seven cells there is clearly more reduction of wave frequency for less change of pH_i than was produced by butyrate. Finally, with essentially no change of pH_i cells (n = 6) under pH clamp undergo a reduction of frequency similar to that in cells where pH_i was free to change during metabolic blockade. This demonstrates that metabolic blockade lies between the two extremes of pure pH change and non-pH change.

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Figure 5. The relationship between wave frequency and pHi under different conditions The ordinate plots wave frequency divided by frequency in control and the abscissa is the acidification relative to control. Symbols show the response to the following manoeuvres: , 20 mM butyrate; , metabolic inhibition; , metabolic inhibition with elevated pH buffer ('pH clamp').

	DISCUSSION

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The overall conclusion of this work is that inhibition of the Ca²⁺ release mechanism of the sarcoplasmic reticulum (SR) during metabolic blockade in rat ventricular myocytes is due only in part to the accompanying acidification. We have demonstrated this by comparing in the same cells the effect on the frequency of spontaneous waves of Ca²⁺ release of a simple acidification, induced by addition of a weak acid, with that of metabolic blockade. The effect of metabolic blockade on wave frequency is greater than can be accounted for by the acidification that takes place. Furthermore, even when changes of pH_i are prevented by increased buffering, a marked decrease of wave frequency persists (Fig. 4 and Fig. 5). This suggests the presence of another factor produced by metabolic blockade that inhibits Ca²⁺ release from the SR

We have used the frequency of spontaneous SR Ca²⁺ release as a measure of the sensitivity of the Ca²⁺ release mechanism of the SR. Thus a steady state decreased frequency of waves associated with increased SR Ca²⁺ content indicates inhibition of the release mechanism as shown previously for tetracaine (Overend et al. 1997) and metabolic blockade (Overend et al. 2001). Conversely stimulation of the release mechanism with caffeine results in a steady state increase of frequency associated with a decreased SR content (Trafford et al. 2000). It should be noted that studying the systolic release of Ca²⁺ would be unsuitable in this work for two reasons: (1) modulation of CICR has no steady state effect on Ca²⁺ transient amplitude (Eisner et al. 1998), and (2) the effects of metabolic blockade on the action potential (Lederer et al. 1989) would complicate interpretation.

Our results show that acidification decreases the frequency of Ca²⁺ waves. This is not unexpected, given that acidification decreases the open probability of the RyR (Xu et al. 1996). Previous work has shown that acidification decreases the frequency of Ca²⁺ sparks (Balnave & Vaughan-Jones, 2000). The effects on Ca²⁺ waves that we now find for acidosis are very similar to those found previously for tetracaine (Overend et al. 1997). Initially, as pH_i falls, the frequency of waves is reduced (the interval between waves increases; Fig. 2) but then recovers somewhat to reach a new steady state. This recovery occurs because SR Ca²⁺ content is increasing while release is inhibited. Eventually, the higher content can provide a sufficiently high gain of CICR to overcome the inhibition by the acidic pH. When butyrate is removed and pH recovers there follows a transient rise above the control frequency as the SR can now no longer maintain the increased Ca²⁺ content once inhibition of release has been removed.

Can acidosis account for the effects of metabolic blockade on wave frequency?

The present results show that intracellular acidosis can mimic metabolic blockade in reducing the frequency of waves. However, we wished to determine whether the effects of changes of pH_i were sufficient to account for all of the effects of metabolic blockade. This required comparing the inhibitory effect on the SR Ca²⁺ release mechanism of acidification with that of metabolic blockade. To do this, one must be sure that they can be fairly compared. It is clear from Fig. 2 that the instantaneous and steady state relationships between pH_i and waves interval are very different. This is due to the changes in SR Ca²⁺ content and how these change the frequency of waves. In metabolic blockade the changes of pH_i taking place are, we believe, sufficiently slow to allow the SR Ca²⁺ content to remain in equilibrium with the changing conditions. For this reason we compare the effects of metabolic blockade with the steady state relationship obtained from butyrate applications.

The results show that for a given change of pH_i the reduction of wave frequency is greater during metabolic blockade than when pH_i alone is changed. This indicates the presence of another inhibitory factor on Ca²⁺ release. Another argument suggesting a major contribution from a pH-independent mechanism to the effects of metabolic blockade is the fact that when the changes of pH_i are greatly reduced by using a highly buffered solution, there is still a marked effect of metabolic blockade on wave frequency. These differences are illustrated by the different end points for each treatment shown in Fig. 5. Metabolic blockade clearly occupies the space between a simple pH change and the situation where pH change has been prevented.

We may now ask what this pH-independent factor inhibiting SR Ca²⁺ release might be? Two obvious possibilities are the changes in ATP and free Mg²⁺ that take place in metabolic blockade (Headrick & Willis, 1991; Elliott et al. 1992). It has been shown in studies on isolated RyR channels reconstituted into lipid bilayers that both an increase of [Mg²⁺] and a decrease of [ATP] reduce the open probability of the channel (Xu et al. 1996). In addition, in permeabilised cardiac myocytes these two factors have been shown to reduce the frequency of spontaneous sparks and/or waves of Ca²⁺ release (Yang & Steele, 2000; Lukyanenko et al. 2001; Smith & O'Neill, 2001). We could expect, therefore, that either or both of these changes known to occur in metabolic blockade might be responsible for the pH_i-independent decrease of wave frequency. ATP is the major buffer for Mg²⁺ inside the cell; therefore, loss of ATP results in an almost equimolar increase of free Mg²⁺ (Kirkels et al. 1989; Murphy et al. 1989; Headrick & Willis, 1991). For small changes of [ATP] and free Mg²⁺, the more potent inhibitory effect on the open probability of RyR is likely to be from Mg²⁺. Work in single RyR channels and permeabilised cells shows that ATP has to be lowered to less than 1 mM (from a control value of about 6 mM) before there is any effect on open probability while raising Mg²⁺ from 1 mM to 2 mM is already enough to produce a noticeable effect (Xu et al. 1996). It is likely, therefore, that most of the effect in Fig. 4 is due to inhibition by increased Mg²⁺, at least in the early stages. In the latter stages, as ATP reserves are completely depleted then inhibition of the RyR will be from a combination of low ATP and high free Mg²⁺.

The importance of the inhibitory effect on SR Ca²⁺ release exerted during metabolic blockade can be seen when we consider the end of a period of ischaemia in ventricular muscle. On re-perfusion of the tissue inhibitory factors present in the ischaemic tissue are removed, i.e. ATP levels rise and pH begins to return to normal (Eisner et al. 1989; Elliott et al. 1992). The SR has raised its Ca²⁺ content as a result of these inhibitory influences and can now no longer maintain this higher load. In addition there is a large influx of Ca²⁺ from outside the cell (Smith & Allen, 1988). This combination of removal of inhibition of release from the SR and the extra Ca²⁺ influx will very strongly favour pro-arrhythmic spontaneous waves of Ca²⁺ release from the SR.

	REFERENCES

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Acknowledgements

We thank the British Heart Foundation for grant support.

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This Article Abstract Full Text (PDF) All Versions of this Article: 550/2/413    most recent 2003.042846v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by O'Neill, S. C. Articles by Eisner, D. A. Search for Related Content PubMed PubMed Citation Articles by O'Neill, S. C. Articles by Eisner, D. A.