Journal of Physiology (1992), 454. pp. 467-490 With IO figures Printed in Great Britain

467

METABOLIC CHANGES DURING ISCHAEMIA AND THEIR ROLE IN CONTRACTILE FAILURE IN ISOLATED FERRET HEARTS By A. C. ELLIOTT*, G. L. SMITHt, D. A. EISNERt AND D. G. ALLEN§ From the *Department of Physiological Sciences, University of Manchester, Manchester MJI13 9PT, the tInstitute of Physiology, University of Glasgow, Glasgow G12 8QQ, the tDepartment of Preclinical Veterinary Sciences, University of Liverpool, Brownlow Hill, PO Box 147, Liverpool L69 3BX and the § Department of Physiology (F 13), University of Sydney, New South Wales 2006, Australia

(Received 2 August 1991) SUMMARY

1. The effects of global ischaemia on phosphorus metabolites, intracellular pH (pHi) and developed pressure were measured in isolated whole ferret hearts using 31P nuclear magnetic resonance (NMR) spectroscopy. 2. Brief (10 min) periods of global ischaemia reduced left ventricular developed pressure (LVDP) to undetectable levels. This fall in LVDP was accompanied by a fall in the intracellular concentration of phosphocreatine (PCr) and increases in the concentrations of inorganic phosphate (Pi) and phosphomonoesters. There was no change in the intracellular ATP concentration ([ATP]i). pHi fell approximately linearly at a rate of 0-04 pH units min'. 3. When ferret hearts were exposed to cyanide (CN-) in the presence of a-cyano4-hydroxycinnamate (CHC), a blocker of lactate efflux, the changes in pHi and [Pi]1 which occurred were similar to those observed during global ischaemia. However, developed pressure only fell to around 15 % of the control value. 4. Removing the intracellular acidosis (by reducing the CO2 level of the gas with which the perfusate was equilibrated) during exposure to CN- and CHC caused an increase in developed pressure, consistent with the fall in pHi being responsible for a substantial fraction of the fall in developed pressure. 5. Taken together. these results suggest that most, but not all, of the fall in developed pressure during ischaemia can be explained by the effects of the changes in pHi and [Pi]i on the contractile apparatus. 6. Action potential recordings made with a suction electrode during short periods of global ischaemia showed that there was no decrease in action potential duration over the period when developed pressure was falling, eliminating action potential shortening as a possible cause of the fall in developed pressure. 7. In hearts in which the rate of glycolysis had been reduced by glycogen depletion, global ischaemia led to a marked shortening of the action potential. NMR experiments showed that under these conditions [ATP]i decreased by around 50 % * To whom all reprint requests should be sent. NIS 9624

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A. C. ELLIOTT AND OTHERS

over the first 10 min of ischaemia, while the intracellular acidosis which occurred was smaller than that in a control ischaemic period. 8. The time course of the decline of [ATP]i was examined in several hearts during long (45 min and over) ischaemic periods without prior glycogen depletion. After 45 min of ischaemia [ATP]i fell to around two-thirds of the control value, while pH, declined to approximately 641. Resting pressure did not increase. On reperfusion pHi recovered rapidly to control levels. [ATP]i, however, did not recover. 9. If ischaemia was prolonged further, [ATP]i eventually became undetectable after 70-90 min. A rise in resting pressure occurred once [ATP]i fell below 0-5-1 0 mm. 10. Our results suggest that during ischaemia glycolysis can continue to function and maintain [ATP]i to some extent. However, if glycogen stores are depleted [ATP]1 falls more rapidly, leading to action potential shortening and eventually, when [ATP]i reaches sub-millimolar levels, to a rise in resting pressure (rigor contracture). INTRODUCTION

Ischaemia in cardiac muscle leads to a complete loss of contractile function within a few minutes. The causes of this ischaemic contractile failure remain incompletely understood. In view of the technical problems involved in producing ischaemia in isolated muscle preparations, many of the studies which bear on the causes of ischaemic contractile failure have employed experimental protocols which model some (though by no means all) of the metabolic and mechanical changes in ischaemia. The most common such model condition is anoxia or exposure to CN- (see Allen & Orchard, 1987, for a review). The evidence now suggests that the partial contractile failure which accompanies anoxia is entirely explicable in terms of the direct effects of the accumulation of intracellular metabolites, primarily inorganic phosphate (Pi) and H+, on the contractile apparatus (Allen, Morris, Orchard & Pirolo, 1985; Kusuoka, Weisfeldt, Zweier, Jacobus & Marban, 1986; Eisner, Elliott & Smith, 1987b). Similar mechanisms can be expected to play a part in ischaemic contractile failure, especially since ischaemia is accompanied by an intracellular acidosis which is rather larger than that which occurs in anoxia (reviewed by Allen & Orchard, 1987). However, electrophysiological changes are also a feature of ischaemia and have been suggested to play a role in contractile failure. It is well known that there is a decline in both the amplitude and the duration of the cardiac action potential during ischaemia (Downar, Janse & Durrer, 1977; Russell, Smith & Oliver, 1979), and such changes could clearly reduce both Ca2+ influx across the plasmalemma and Ca2+ release from the sarcoplasmic reticulum. A similar mechanism is now well established under conditions of metabolic blockade (inhibition of both aerobic and anaerobic metabolism), when dramatic action potential shortening occurs (McDonald & MacLeod, 1973; Allen, Harris & Smith, 1987; Lederer, Nichols & Smith, 1989; Elliott, Smith & Allen, 1989), leading to a failure of Ca2+ release (Allen & Orchard, 1983) and therefore to mechanical failure (Stern, Silverman, Houser, Josephson, Capogrossi, Nichols, Lederer & Lakatta, 1988; Lederer et al. 1989). The metabolic changes during longer periods of ischaemia are also of interest. It is known that intracellular free calcium concentration ([Ca2+]i) rises during ischaemia (Steenbergen, Murphy, Levy & London, 1987; Marban, Kitakaze,

CA 1TSES OF ISCHAEMIC CONTRACTILE FAILURE

469

Kusuoka, Porterfield, Yue & Chacko, 1987; Allen. Lee & Smith, 1989). It is possible that ischaemic contracture and the occurrence of ischaemic damage might be related to either the increase in [Ca2+]i, or to the fall in [ATP]i that accompanies ischaemia (Jennings & Steenbergen, 1985), or both. Furthermore, the acidosis which occurs during ischaemia has also been implicated in the pathogenesis of ischaemic damage (Lazdunski, Frelin & Vigne. 1985). In the present study we have examined the metabolic and electrophysiological events accompanying total global ischaemia in isolated ferret hearts. We have also used a combination of metabolic inhibition with CN- and blockade of lactate efflux with a cinnamic acid derivative to mimic the changes in intracellular [Pi] and pH1 which accompany ischaemia. Our work suggests that in this preparation metabolic factors (increased intracellular [Pi] and [He]) can account for approximately 80-90% of the fall of developed pressure. Action potential shortening was not observed over the period of acute ischaemic contractile failure. In contrast, if the rate of glycolysis had been reduced, action potential shortening did occur early in ischaemia. Long periods of ischaemia led to a slow decline in [ATP]i and a large fall in pHi. If hearts were reperfused while [ATP]i was still relatively high, both pHi and (usually) contractile function returned to control levels, although depletion of [ATP]1 was not reversed. In longer ischaemic periods the development of the contracture correlated well with a decrease in [ATP]i to sub-millimolar levels. These results tend to suggest that the intracellular acidosis during ischaemia is not per se a crucial factor in predisposing the tissue to ischaemic and/or reperfusion damage, and that a fall in [ATP]i is the major cause of the ischaemic contracture. Preliminary accounts of parts of this work have been presented to the Physiological Society (Eisner, Elliott & Smith, 1987 a) and the International Society for Heart Research (Elliott, Smith & Allen, 1988). METHODS

The experimental methods used to measure developed pressure and 31P nuclear magnetic resonance (NAIR) spectra from Langendorff-perfused ferret hearts, and to measure metabolite levels from the NMR spectra, have been described in detail previously (Allen et al. 1985; Allen, Eisner, Morris, Pirolo & Smith, 1986; Eisner et al. 1987b). In brief, female ferrets were killed by an intravenous overdose of pentobarbitone sodium. The heart was removed and the aorta cannulated. A balloon-tipped catheter was inserted into the left ventricle and the right atrium and the atrioventricular node were destroyed to decrease the spontaneous heart rate. The hearts were then stimulated electrically at 0-8-15 Hz. All experiments were carried out at 30 'C. 31P NMIR spectra were obtained at 81 MHz on a Bruker WM-200 wide-bore NMR spectrometer. Forty-five degree radiofrequency pulses were applied at an interpulse interval of 1 s. Spectra were generally obtained (by Fourier transform) from the sum of 120 such scans, giving a time resolution of 2 min. although in some experiments spectra were collected over 4 or 8 min. Peak areas in the spectra were measured by planimetry and converted to concentrations as follows. The peak areas were first corrected for saturation by multiplying them by the following saturation factors: phosphomonoesters (PME), 1 43; Pi, 1-34; PCr, 1 7; a-ATP, 1 11; f3-ATP, 1-17; y-ATP, 1-08. The concentrations were then normalized to the [ATP]i at the start of collection of data from a heart. This was assumed to be 7 5 mm, the value determined by biochemical analysis of freeze-clamped normoxic ferret hearts (see Allen et al. 1985. for further details). The chemical shift of Pi was converted to pHi using the formula given by Eisner et al. (1987b). Action potential recordings were made as described by Elliott et al. (1989). Briefly, a Perspex suction electrode was sewed to the left ventricular wall close to the apex. The indifferent electrode was placed in the right ventricle. Action potentials were recorded by applying suction until the

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electrode became firmly attached to the ventricular wall. Action potential duration was measured at 50 % of the maximum plateau height (APD50). Further details of this method of action potential recording, and a comparison of the suction action potentials with action potentials recorded in ferret ventricular muscle with conventional microelectrodes, are given in Elliott et al. (1989). Global ischaemia was produced by stopping the roller pump perfusing the heart and clamping the inflow perfusion line. Glycogen depletion was produced by removal of glucose from the perfusate and stimulation at an increased rate (3 Hz) for 1-2 h (see Pirolo & Allen, 1986, for details). In experiments in which cyanide was used to block oxidative phosphorylation, an aliquot of 300 mM-NaCN (freshly made up each day) was mixed with half its volume of a solution of HEPES-free acid (2 M), in order to produce a solution of cyanide at near pH 7 4. This solution was then added immediately to the Tyrode solution to give a final cyanide concentration of 2 mm. The Tyrode solution contained (mM unless otherwise stated): Na', 135; K+. 5: Mg2, 1; Ca2 , 2; Cl, 124; HCO3-, 20; glucose, 10; insulin, 40 nM; and was bubbled with 950 02+50% CO2 to give a pH of 7-4. In a few experiments a modified Tyrode solution was used in which the Clconcentration was reduced to 84 mm and the HC03- concentration was increased to 60 mm by replacement of NaCl by NaHCO3. This solution was bubbled with 85 %02-15 %CO2 to give a pH of 7-4. a-Cyano-4-hydroxycinnamate (CHC, Sigma) was dissolved directly in the Tvrode solution. Results are given as means+ standard error of the mean (S.E.M.). Tests for differences between means were made using Student's t test. RESULTS

Changes in metabolite levels and developed pressure in early ischaemia The effects of a brief period of global ischaemia on an isolated ferret heart are shown in Fig. 1. Developed pressure fell rapidly and reached undetectable levels within 6 min. The NMR spectra in Fig. lB show that ischaemia increased the size of the Pi peak, and caused a corresponding decrease in the size of the phosphocreatine (PCr) peak, with no change in the size of the ATP peaks. These metabolic changes were accompanied by an intracellular acidosis of around 0 4 pH units, emphasized in the expanded Pi regions of the spectra in Fig. 1 C. On reperfusion developed pressure, pHi, PCr and Pi all recovered to control levels. Averaged results from seven experiments are shown in Fig. 2. Developed pressure fell to 40 % of control in the first minute of ischaemia and continued to decline, reaching undetectable levels after 6-7 min. Intracellular pH fell approximately linearly over the first 10 min of ischaemia. After 7 min, when developed pressure was undetectable, the size of the acidosis was 0 34 pH units. [PCr]i fell over several minutes, with [Pi]i rising to more than 10 mm within 2-3 min. There was no change in [ATP]i. The concentration of phosphomonoesters (PME) showed a small increase, which was statistically significant after 6-7 min of ischaemia (P < 0 05 by paired t test). In view of the lack of change in [ATP]i this probably represents a build-up of glycolytic phosphates rather than of ATP breakdown products such as AMP or IMP (inosine monophosphate). No ADP could be detected by subtracting the area of the /J-ATP peak from the area of the y-ATP peak (see Allen et al. 1985). Metabolic changes during exposure to CN- and CHC The contribution of the metabolic changes which occur during ischaemia to the fall in developed pressure can be estimated from results obtained on skinned muscle preparations (see e.g. Allen et al. 1989; Lee & Allen, 1991). However, this approach is complicated by the need to estimate the degree of activation of the intact preparations (see Eisner et al. 1987b, and Allen et al. 1989, for discussion). An

471 CAUSES OF ISCHAEMIC CONTRACTILE FAILURE alternative is to try to mimic the metabolic changes accompanying ischaemia in a model with continued perfusion. Exposure to CN- leads to an increase in [Pi]i, but only to a small intracellular acidosis (Allen et al. 1985; Eisner et al. 1987b). This is presumably because lactate and protons are rapidly removed from the cell. The Ischaemia LVDP A 1 00 mmHg m1

r -2

1

A

r

3

2 min C

pHi 6-4 7-2 1 6-8

B

PCr /

1

ATP

2 PME

2

3

3

a

10

0

I

.

-10 a (p.p.m.)

.

-2!0

a

7

.

I'

\I

.

.

.

6 5 4 8 (p-p-m.)

I

I

3

Fig. 1. The effects of a brief period of global ischaemia on left ventricular developed pressure (LVDP) and 31P NMR spectra. A, left ventricular developed pressure. The heart was made globally ischaemic for the period shown above the record. B, NMR spectra obtained over the periods indicated in A. a indicates the chemical shift in parts per million of the magnetic field (p.p.m.). The identities of the peaks are shown on spectrum 1, except for the phosphomonoester (PME) peak, which is indicated on spectrum 2. C, expanded section of the region of the spectrum containing the inorganic phosphate (Pi) peak. This expanded region is calibrated in terms of both chemical shift (below) and pH, (above). The vertical scales in these expanded regions have been adjusted so that the heights of the P1 peaks are approximately the same.

evidence suggests that in cardiac muscle much of this transport of lactate and protons is mediated by a lactate-proton co-transporter which can be partially blocked by cinnamate derivatives (de Hemptinne, Marrannes & Vanheel, 1983; Trosper & Philipson, 1987). Figure 3 shows an experiment in which a heart was exposed to CN- in the absence and subsequently in the presence of x-cyano-4hydroxycinnamic acid (CHC). Four millimolar CHC alone did not affect either p or phosphorus metabolite levels in this experiment. Averaged results from nine 1B

PHY 454

A. C. ELLIOTT AND OTHERS

472

hearts exposed to 4 mm-CHC similarly showed no significant changes in pHi or metabolite levels (pHi and metabolize levels measured immediately before and 5-8 min after addition of CHC). CHC did, however, always reduce developed pressure slightly (developed pressure in CHC was 91+3 % of the pre-CHC control value, Ischaemia

LVDP (arbitrary units) 0

7-1

pHi

6-9

-\

6-8 6.71

50 Total phosphorus

Concentration of phosphorus compounds (mM)

40

20

\Pi

°

10

ATP 02 S ~~

-4

-2

0

2 4 6 Time (min)

~~

~~~PCr

8

10

Fig. 2. Averaged mechanical and metabolic data from seven ferret hearts during the early stages of global ischaemia. Each point represents data averaged over a 2 min period. The bars on the first control point of each trace show 1 s.E.M. to give an indication of the variability between preparations (where no bar is shown the S.E.M. was less than the width of the symbol). The lines drawn through the points indicate the trend and have no theoretical significance. Upper panel, left ventricular developed pressure (LVDP). Middle panel, pHi obtained from NMR spectra. Lower panel, cytosolic concentrations of phosphorus metabolites and total phosphorus (note the break in the ordinate between 20 and 40 mM). These concentrations were obtained from the NMR spectra and normalized as described in Methods.

9). In the heart shown in Fig. 3 CN- produced only a small acidosis. However, subsequent application of CN- in the presence of CHC caused a large (04 pH units)

n=

intracellular acidosis. CN- caused a similar increase in [Pi]i in the presence and in the absence of CHC. The fall of developed pressure caused by CN- was greater in the presence of CHC. Figure 4 shows averaged results from four hearts which were exposed to CN- in the presence of 4 mM-CHC. The developed pressure traces have

CA USES OF ISCHAEMIC CONTRACTILE FAILURE

473

been normalized to facilitate comparison between the two treatments (and with data in ischaemia, see below). The fall of developed pressure and the intracellular acidosis evoked by CN- were both increased when CHC was present. In contrast, the increase in [Pi]i was unaffected by the presence of CHC. The levels of [ATP]i and [PCr]i following exposure to CN- were also unaffected by CHC (not shown). CHC CN-

CNLVDP 100 mmmHg]

7-2 pH p70 6-8 -.

/

-

**--.

6-6 4~mmin-20

-

(mM) 10]

_

Fig. 3. Comparison of the mechanical and metabolic effects of CN- in the absence and in the presence of a-cyano-4-hydroxycinnamate (CHC). The traces show (from top to bottom): left ventricular developed pressure (LVDP); pH1; [Pi]i. The break in the record represents 20 min. CN- (2 mM) was added as shown. a-Cyano-4-hydroxycinnamate (CHC; 4 mM) was added 8 min before the start of the second part of the record. The measurements of pH, and [P1]1 were obtained from NMR spectra.

The data on ischaemia from Fig. 2 have been replotted in Fig. 4 for comparison with the CN- plus CHC data. The changes in pHi and [Pj]1 in the early stages of ischaemia were very similar to those produced by CN- in the presence of CHC, showing that the combination of CN- and CHC mimicked the early metabolic consequences of ischaemia very successfully. However, the fall in developed pressure was clearly larger in ischaemia (reaching undetectable levels) than in the model condition (where developed pressure fell to around 15% of control). These experiments suggest that the combined effects of the increase in [Pi]i and the intracellular acidosis which occur in early ischaemia would be expected to be a fall in developed pressure to around 15 % of control (i.e. the level of developed pressure achieved in the presence of CN- and CHC). However, in true global ischaemia these same metabolic changes were associated with a fall in developed pressure to undetectable levels, suggesting that some other factor or factors must be operating to reduce developed pressure. Figure 5 shows an experiment designed to test whether the greater fall of 16-2

A. C. ELLIOTT AND OTHERS

474

developed pressure produced by CN- in the presence compared to the absence of CHC was indeed a consequence of the larger intracellular acidosis. In this experiment the intracellular acidosis was removed (in the continued presence of CHC) by changing the gas mixture with which the perfusate was equilibrated from one CN- (or ischaemia)

A-&^

10 1

LVDP

(arbitrary units)

0.5

-

1

£~,-

--4CN* CN- +CHC

~^ ~ -

0

Ischaemia

7-1-

6-9

-

678

-

6

*N

I__

_

6-6-

20

I CN-+CHC

-

[Pi] (mM)

Ischaemia

t

10-

.

0 -i---4 -2 0

I 2

4

6

8

Time (min)

Fig. 4. Averaged data comparing the mechanical and metabolic effects of CN- with and without CHC and ischaemia. Panels show (top to bottom): left ventricular developed pressure (LVDP) normalized to that prior to the application of CN- or the onset of ischaemia; pH,; [Pi],. The circles show results obtained from four ferret hearts which were exposed to CN- alone (0---Q) and subsequently to CN- in the presence of 4 mM-CHC (S *). The absolute value of developed pressure in the presence of CHC but before addition of cyanide in these four hearts was 70 + 10 % of the initial control value. Much of this reduction of developed pressure, and the slightly more acidic initial pH,, was not associated with the presence of CHC but rather resulted from a time-dependent decline in developed pressure and pH, in two hearts, perhaps due to ageing of the preparations (for the effects of addition of CHC per se see text). The ischaemia data (A---A) have been replotted from Fig. 2 and are thus derived from seven hearts. The format of this figure is broadly similar to that of Fig. 2.

containing 15 % CO2 to one containing 5 % C02, and subsequently to one which was CO2 free. pHi returned to and in fact overshot the control (pre-CN-) value. This was accompanied by an increase of developed pressure from 21 to 43 % of the control level. There was also a decrease in [Pi]i from around 14 to around 12 mm (this fall in [Pi]i may have been due to loss of phosphates from the cell during the prolonged cyanide exposure, as it was accompanied by a decrease in total phosphorus

CAUSES OF ISCHAEMIC CONTRACTILE FAILURE

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metabolites). The pHi-independent component of the fall of developed pressure presumably resulted from the effects of [Pi]i. The approximate doubling of developed pressure on changing the CO2 level will reflect the effects of both the small fall in [Pj]j and the 'removal' of the intracellular acidosis. Skinned muscle work suggests that a-CHC

CN-

%C02 5] LVDP 100 mmmg] 7-1*

70 -0

pHi

6-9-

6-76-6 -

15[Ph, (mM)

0 0-.-a-

~0

1

10 50

02 min

Fig. 5. The effects of removing the intracellular acidosis produced by CN- and CHC. Traces show (from top to bottom): left ventricular developed pressure (LVDP); pH1; [PI1j2. CHC (4 mm) was present throughout. CN- (2 mm) was added as shown. The heart was initially perfused with a solution containing 60 mm-HC03 and equilibrated with a gas mixture containing 15 % CO2 (pH 7-4, see Methods). At the time indicated this solution was changed to one bubbled with 5 % CO2 (pH 7-6) and finally to one which was nominally CO2 free (bubbled with 100% 02; pH approximately 7-8). the effects of changing [P1]1 on contractility are most marked over the range 0-10 mm (Kentish, 1986), so that the effects of the fall in [P1]1 on developed pressure would be expected to be small. Neglecting the effects of changing [P1]1, the approximate

doubling of developed pressure on removing an intracellular acidosis of 0-35 pH units is consistent with our previous work in ferret hearts (Eisner et al. 1987 b).

Action potential measurements during early ischaemia In order to test whether action potential shortening occurred during the period of acute contractile failure in our preparation we measured action potentials with a Perspex suction electrode (see Elliott et al. 1989). Figure 6A shows records of twitches and suction action potentials recorded during the first 3 min of global ischaemia in a ferret heart. Developed pressure fell to around 10 % of the control value after 3 min of ischaemia. Although action potential amplitude declined in this

476

A. C. ELLIOTT AND OTHERS A 1 min

Control

2 min

3 min

10mV [

100 [

mmHg

s- --

0-5

05s

B

Control

1 min

2 min

3 min

2 mV [

100

A

mmHg L

0-5 s

0*5 s

Fig. 6. Comparison of the effects of global ischaemia on action potential duration and left ventricular developed pressure in a ferret heart before and after glycogen depletion. In panels A and B the traces show original records of suction electrode action potentials (upper trace) and left ventricular developed pressure (lower trace) under control conditions and following the onset of global ischaemia at the times indicated above the records. The traces in panel B were obtained from the same heart as those in panel A but after the heart had been subjected to glycogen depletion (see Methods). Panel C shows records of action potentials taken from panels A (left) and B (right) on an expanded timebase. In each case the action potentials for control conditions and following 3 min of ischaemia (marked with dots for easier identification) have been scaled to the same plateau height and superimposed to facilitate comparison of the action potential duration.

CA USES OF ISCHAEMIC CONTRACTILE FAILURE 477 heart there was little change in the duration of the action potential during ischaemia, as can be seen more clearly in the superimposed fast timebase records in panel C. The decrease in the amplitude of the suction action potential during early ischaemia which was apparent in the experiment shown in Fig. 6A was not a consistent observation. Suction action potentials generally showed no marked changes in form over the first few minutes of ischaemia other than an increase in amplitude (e.g. Fig. 6B), which was observed in about half of the experiments. If electrical recording was continued for longer periods, the action potential usually began to show gradual changes in its form until it eventually resembled an electrocardiogram (ECG) trace. The most likely explanation of this change in the suction action potential appears to be that uncoupling of the cells prevents the injury potential being recorded, and that under these conditions the two electrodes in the suction electrode assembly simply act as extracellular electrodes. In order to make the measurements of action potential duration as reliable as possible, we only determined action potential duration quantitatively in those experiments in which changes in the form of the action potential were minimal. Nevertheless, the same general changes in action potential duration were observed in experiments where the action potential became ECG-like.

We have previously shown that CN- only causes action potential shortening in ferret ventricular muscle when anaerobic metabolism has been prevented, for instance by depletion of glycogen stores (Allen et al. 1987; Elliott et al. 1989). We therefore tested whether a similar effect of glycogen depletion could be observed for ischaemia. Figure 6B shows records of developed pressure and suction action potentials obtained from the same heart as in Fig. 6A but after glycogen depletion. The glycogen depletion procedure, which took approximately 1 h in this experiment, was associated with some reduction in both developed pressure and the amplitude of the suction action potential. When the heart was made ischaemic following glycogen depletion there was a fall in both developed pressure and action potential duration, shown in the superimposed fast timebase records in panel C. Figure 7 shows averaged results on developed pressure and action potential duration from hearts which were made ischaemic either before or after glycogen depletion or both. Prior to glycogen depletion action potential duration showed no significant change over the first three and a half minutes of ischaemia (n = 6, 0). Over the same period developed pressure fell to 12 + 4% of the control value. It is clear that action potential shortening does not occur in the ferret heart over the period when the major part of ischaemic contractile failure is occurring. If anything there was a slight tendency to prolongation of the action potential over this early period of ischaemia, as has been noted by others (Russell et al. 1979). However, in our preparation this may simply represent the slight cooling of the heart which inevitably occurs when perfusion stops. In marked contrast, ischaemia did lead to a substantial fall in action potential duration when hearts had been glycogen depleted (n = 4, 0). The glycogen depletion procedure per se did not cause any shortening of the action potential, although it did cause a decrease in developed pressure to around 50% of control (Fig. 7), as reported in previous studies (Allen et al. 1985; Pirolo & Allen, 1986; Elliott et al. 1989). After three and a half minutes of ischaemia developed pressure in the glycogen-depleted hearts had fallen to 8 + 3 % of the initial control (pre-glycogen rundown) value, or approximately 14 % of the developed pressure after glycogen depletion but prior to ischaemia. This fall in developed pressure was accompanied by a fall in action potential duration to approximately half (56 + 6 %) of the control value.

A. C. ELLIOTT AND OTHERS

478

Action potential 100 duration 80 (% control) 60 100 _

80 LVDP

(% control)

60

-

6 40-

20 0 1 2 3 Time after onset of ischaemia (min)

Fig. 7. Averaged data on the effects of global ischaemia on action potential duration in ferret hearts following glycogen depletion. Upper trace, action potential duration. Lower trace, left ventricular developed pressure (LVDP). Both action potential duration and developed pressure have been normalized to the values before the first ischaemic period *, control data from hearts before glycogen (i.e. before glycogen depletion). * depletion (mean+ s.E.M., n = 6). 0 O. data from glycogen-depleted hearts (mean+ s.EM_., n = 4). Action potential duration was measured at 50% repolarization. Ischaemia

Ischaemia LVDP

50

1

(mmHg) 0 _ i-----_ m 5 min

7-2 *

6-9 6

pH1

*

6-6I

30

-

20 -* . (mM) 5

i 10

[ATP]

*

, .@~~--*-. --.

10 ] .

00 *

.

.

(mm)* Fig. 8. Comparison of the metabolic effects of ischaemia in a ferret heart before and after glycogen depletion. Traces show (top to bottom): left ventricular developed pressure (LVDP); pH,; [P1]i; [ATP]i. The break in the pressure record represents 2 h, during which time the preparation was subjected to glycogen depletion. The heart was made globally ischaemic for the periods shown.

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Metabolic consequences of ischaemia in glycogen-depleted hearts It is clear that under the conditions of our experiment ischaemia did not cause action potential shortening unless the heart had been glycogen depleted, i.e. until the rate of anaerobic glycolysis had been reduced. We performed a number of experiments to attempt to examine whether the metabolic consequences of ischaemia in glycogen-depleted hearts were markedly different from those in control hearts. By analogy with the effects of glycogen depletion on the metabolic changes evoked by anoxia or CN- (Allen et al. 1985), it might be expected that following glycogen depletion ischaemia would cause a somewhat faster decline in [ATP]i and a rather smaller intracellular acidosis. Figure 8 shows the effects of ischaemia before and after glycogen depletion in a ferret heart. During an initial (control) 10 min ischaemic period pHi fell by around 0-6 pH units with little or no reduction in [ATP]i. The heart was then glycogen depleted, resulting in a decrease in developed pressure, a small fall in [ATP]i (as observed in previous studies, Allen et al. 1985; Pirolo & Allen, 1986; Elliott et al. 1989) and a substantial increase in [Pi]i. A second ischaemic period then led to a rather smaller intracellular acidosis (approximately 40% as large as the intracellular acidosis observed in the control ischaemic period), a marked fall in [ATP]i, and a small increase in resting pressure. In five experiments the glycogen depletion procedure reduced [ATP]i to 80 + 9 % of the control value, corresponding to an [ATP]i of 6-1 mm. When these hearts were made ischaemic [ATP]i fell markedly. [ATP]i measured over the seventh to tenth minutes of ischaemia was 55 + 9 % (n = 4) of the value immediately prior to ischaemia. [Pi]i in these hearts was 10-9 + 1 1 mm following glycogen depletion and increased to 28-3 + 2-9 mm after 7-10 min of ischaemia. The intracellular acidosis which had developed over the same period of ischaemia was 0 35 + 0 03 pH units. In the same five hearts prior to glycogen depletion 10 min of ischaemia caused no measurable decline in [ATP]i, and an intracellular acidosis of 0 59 + 0 04 pH units. Metabolic consequences of long periods of ischaemia We have recently shown that when ferret papillary muscles are subjected to simulated ischaemia for long (30 min and over) periods drastic shortening of the action potential eventually occurs, accompanied by an increase in resting tension (Allen et al. 1989). The increase in resting tension occurred much earlier if the preparation had been glycogen depleted prior to the ischaemic period (Allen et al. 1989). Furthermore, Koretsune & Marban (1990) have reported that if glycolysis is completely blocked with iodoacetate ischaemia causes a contracture within a few minutes of the cessation of flow. These observations suggest that the metabolic consequences of long periods of ischaemia in non-glycogen-depleted hearts may be similar to those of shorter ischaemic periods in glycogen-depleted preparations. We therefore performed several experiments to investigate the changes in [ATP]i and pHi during long periods of ischaemia. Figure 9 shows a representative experiment in which a heart was made globally ischaemic for 45 min. The ischaemic period was not accompanied by any rise in resting pressure, and on reperfusion there was a complete recovery of mechanical function (developed pressure). [ATP]i fell to around 60 % of the control (pre-ischaemia) value after 45 min of ischaemia. The absence of a

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contracture is thus consistent with recent work suggesting that a reduction in [ATP]i to less than 10% of control values is correlated with contracture development (Koretsune & Marban, 1990). The intracellular acidosis after 45 min of ischaemia in this experiment was 0W93 pH units. On reperfusion pHi recovered rapidly to control Ischaemia LVDP (arbitrary units)

1°0 0 J

7-0~~~~~~1 -i pHi

6-866 6-4-

'

6-2 6-0

LATPI (mM)

51xi-... J

Fig. 9. The metabolic effects of a moderately long period (45 min) of ischaemia in a ferret heart. Traces show (top to bottom): left ventricular developed pressure (LVDP); pH,; [ATP]i. The heart was made globally ischaemic for the period shown.

levels. There was some hint of a small recovery in the ATP level on reperfusion, but ATP did not recover to the pre-ischaemia value. In three experiments of this type [ATP]i fell to 65 + 8 % of control after 45 min of ischaemia, corresponding to an [ATP]i of around 5 mm. The y- and fi-ATP peaks declined by similar amounts, implying that ADP did not accumulate in the cells to any measurable extent. The PME peak rose to 250 % of the control level, corresponding to a total concentration of phosphomonoesters of around 5 mm, and the mean intracellular acidosis was 0f91 + 0-05 pH units. On reperfusion [PCr]i and [Pi]i recovered to control levels in all three preparations. pHi also recovered to control values. The Pi peak usually became so small as to be undetectable during recovery, which made it impossible to measure the time course of the recovery of pHi reliably. However, pHi measured 4-8 min after reperfusion was no different from the control (pre-ischaemia) pHi, implying that the recovery of pHi must have been fairly rapid. The PME peak also returned to control levels on reperfusion. There was no significant recovery in [ATP]i on reperfusion. None of the three preparations studied showed any increase in resting pressure on reperfusion, and contractile function recovered completely in two out of the three hearts (ventricular fibrillation occurred when the third heart was reperfused). In view of the evidence that reduced [ATP]i is probably the major cause of ischaemic contracture (Koretsune & Marban, 1990), we examined the relationship between [ATP]i and the ischaemic contracture in more detail in three hearts. Figure

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10A shows one such experiment. An ischaemic contracture began to develop after around 55 min of ischaemia, at which time [ATP]i had fallen to approximately 1 mM. [ATP]i finally became undetectable after approximately 70 min of ischaemia, by which time a pronounced contracture had developed and pHi had fallen to around Ischaemia

A LVP 10 i (arbitrary units) 0__

10min

7-2

pHi

6-5

-

58

[ATP

[mT)i

10 5] 5

B

025 l

Diastolic

0°20

pressure 0-15

(arbitrary units)

o

0-10 0*05

1

0-00-1,.... ..., -D 0-00 0-1

00

1-0

10-0

[ATP]i (mM) Fig. 10. The metabolic effects of a prolonged period of ischaemia in a ferret heart. Panel A, traces show (top to bottom): left ventricular pressure (LVP: developed pressure, 0; resting pressure, *); pHi; [ATP],. The heart was made globally ischaemic for the period shown. The format of this panel is similar to that of Fig. 9. Panel B, the relationship between [ATP]i and resting pressure during the late stages of ischaemia. Symbols show data from the experiment in A (@) and another similar experiment (0). The line was fitted by eye to both sets of points and describes a sigmoidal dependence of resting pressure on

[ATP],.

5-8. [ATP]i became undetectable after between 70 and 90 min of ischaemia in all three hearts, by which time pHi had fallen to 5-86 + 0-02, and an ischaemic contracture had partially developed. Complete development of the contracture was always observed a few minutes after [ATP]i became undetectable. By acquiring NMR spectra over long (8 min) periods we were able to detect levels of [ATP]i as low as 0-2-0-3 mm. The data on [ATP]i obtained at the late stages of ischaemia (i.e. during contracture development) in the experiment shown in Fig. 10A have been replotted against resting pressure in Fig. lOB (O), together with data from another similar experiment (0). The relationship between [ATP]i and resting pressure was sigmoidal in form, broadly consistent with the work of Fabiato & Fabiato (1975) on skinned ventricular muscle.

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Metabolic changes during brief periods of ischaemia The changes in metabolite levels observed during early ischaemia are in broad agreement with previous work in rat, guinea-pig and rabbit heart (see e.g. Kubler & Spieckermann, 1970; Bailey, Williams, Radda & Gadian, 1981; Brooks & Willis, 1983; Renlund, Gerstenblith, Lakatta, Jacobus, Kallman & Weisfeldt, 1984). Perhaps the most notable difference between this and other studies is that no decrease at all in [ATP]i was observed during the 10 min ischaemic period in our experiments. Most other studies show a noticeable decline in [ATP]i within 5 min of the onset of ischaemia, with one other study on ferret hearts being a notable exception (Koretsune, Corretti, Kusuoka & Marban, 1991). Much of the difference is probably attributable to the lower temperature and the relatively slow stimulation rate employed in the present study (and also in the ferret heart study of Koretsune et al. 1991). An advantage of the ferret heart in this respect is that the contribution of the increase in [P1]1 and the fall in pHi to the decline in developed pressure can be considered without the complicating factor of a decrease in [ATP]1. The changes in [PCr]i and [Pi]i at the onset of global ischaemia in the present study were strikingly similar to those observed in the same ferret heart preparation during anoxia or exposure to CN- (e.g. see Allen et al. 1985; Eisner et al. 1987 b). The changes in pHi evoked by the two treatments are, however, quite different; the large intracellular acidosis which accompanies ischaemia contrasts with the small (0-05-015 pH units) intracellular acidosis observed in the same preparation during anoxia (Allen et al. 1985; Eisner et al. 1987 b). The obvious explanation of the larger acidosis in ischaemia is the much greater accumulation of lactate inside the cell (Rovetto, Whitmer & Neely, 1973), presumably resulting from accumulation of lactic acid in the restricted extracellular space. This is supported by the finding that the acidosis which accompanies anoxia is increased in size in the presence of extracellular lactate (Ellis & Noireaud, 1987) or in the presence of a-cyano-4hydroxycinnamate. However, a further mechanism which may contribute to the intracellular acidosis is the accumulation of CO2 within the cell (see Allen et al. 1989, for a discussion of this effect). The effects of a-cyano-4-hydroxycinnamate ca-Cyano-4-hydroxycinnamate (CHC) has been reported to inhibit the transport of lactate and some other anions across the cardiac sarcolemma (de Hemptinne et al. 1983; Mann, Zlokovic & Yudilevich, 1985; Trosper & Philipson, 1987; Poole, Halestrap, Price & Levi, 1989), and experiments on rat hearts have shown that lactate efflux during exposure to CN- in rat hearts is inhibited by CHC (Eisner, Peckett & Ware, 1988). CHC had relatively little effect on developed pressure and pHi under control (aerobic) conditions, when the rate of lactate production by glycolysis will be minimal (see e.g. Allen et al. 1985; Pirolo & Allen, 1986). However, when lactate production was stimulated by CN- in the presence of CHC pHi fell substantially (Figs 3 and 4), indicating an increase in intracellular [lactate] to around 20 mm (assuming a negligible initial [lactate]i and a total CO2 plus non-CO2 intracellular buffering power of around 50 mM (pH unit)-'; Bountra, Powell &

Vaughan-Jones, 1990).

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Role of metabolic changes in early ischaemic contractile failure Acidosis reduces the force produced by cardiac muscle, as has been clearly demonstrated in both skinned and intact preparations (see Orchard & Kentish, 1990, for review). Inorganic phosphate also has a large inhibitory effect on tension (Kentish, 1986). Since both the acidosis and the rise in [Pi]i apparent in Figs 1 and 2 will reduce the force produced by the heart, it is pertinent to ask whether these effects alone can explain all of the fall in developed pressure. It should be noted that the effects of other phosphorus metabolites whose levels change during metabolic inhibition or ischaemia (ADP, AMP) on developed pressure will be negligible (Eisner et al. 1987 b). Over the period when developed pressure fell to undetectable levels [Pi]1 increased from around 4 to 16 mm, while pHi fell by 0 34 pH units (Fig. 2). Several approaches can be used to estimate the extent to which these changes would decrease developed pressure. In the first of these, the skinned muscle results are simply extrapolated to the intact preparation. Depending on the level of activation (which must be estimated), such calculations suggest that the metabolic changes can explain a fall of force to anywhere between 0 and 20 % of control (see Allen et al. 1989; Lee & Allen, 1991). A second approach is to calibrate the effects of both changes of pHi and [Pi]i on force in intact preparations, using anoxia to elevate [Pi]i and manipulating pH1 experimentally in a number of ways (Kusuoka et al. 1986; Eisner et al. 1987 b). Experiments of this type in the same ferret heart preparation showed that an increase in [Pi]i from 3 to 14 mm was associated with a 50 % depression of developed pressure, while acidosis depressed developed pressure by approximately 20 % per 0.1 pH unit, or 70 % for a 0-35 pH unit acidosis (Eisner et al. 1987 b). The experiment in Fig. 5 leads to similar conclusions. This analysis therefore predicts that the overall effect of the metabolic changes in ischaemia, obtained by multiplying together the fractional effects of [Pi]i and pHi, will be a reduction in developed pressure to around 15 % (50 % x 30 %) of the control value. Given the time course of the changes in [Pi]i and pHi during ischaemia, the effect of [Pi]i will dominate the fall of developed pressure over the first 2-3 min of ischaemia, while the effect of pHi will become more important as ischaemia progresses. A final approach to predicting the effects of the metabolic changes on developed pressure, and the one which we have taken in the present study, is to mimic the effects of ischaemia on both [Pi]i and pHi using cyanide and CHC. This has the advantage of avoiding any complications which could arise using the two approaches described above if there were significant synergism between the inhibitory effects of Pi and H+ (although such effects probably do not exist in cardiac muscle; Kentish, 1987; Eisner et al. 1987 b). Figure 4 demonstrates that the changes in [Pi]i and pH1 associated with early ischaemic contractile failure cause a decrease in developed pressure to 15% of the control value, in good agreement with the more indirect estimates derived above. Since developed pressure actually falls to undetectable levels in ischaemia, there must be one or more other factors in addition to the effects of metabolites operating to reduce developed pressure. A similar conclusion has also been reached in a recent study by Koretsune et al. (1991). These approaches generally assume that [Ca2+]1 does not change greatly during ischaemia over the period when developed pressure is falling. While it is known that [Ca2+]i does change during

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ischaemia (Steenbergen et al. 1987; Marban et al. 1987; Lee, Smith, Mohabir & Clusin, 1987; Allen et al. 1989) the size and direction of any change during the acute contractile failure is still unclear, although the most plausible interpretation of the conflicting evidence appears to be that a modest (less than twofold) increase in [Ca2+]i occurs (see Lee & Allen, 1991, for review). A small change in [Ca2+]i during contractile failure should not alter the conclusions reached above. Furthermore, the changes in [Ca2+]i in early ischaemia are probably very similar to those that occur with anoxia and/or CN-. This would mean that the latter two approaches described above using intact preparations may in fact intrinsically allow for the effects of a small rise in [Ca2+]1 (see Lee & Allen, 1991, for a detailed discussion).

Other factors which may contribute to the fall in contractility The experiments described in this study clearly imply that factors other than metabolic changes contribute to the fall in myocardial contractility during ischaemia. Although action potential shortening has been reported in many studies on ischaemia (e.g. Downar et al. 1977; Russell et al. 1979), our experiments (and those of Koretsune et al. 1991, on ferret hearts) show that it does not occur in the ferret heart over the period of contractile failure (at least in non-glycogen-depleted hearts) and can therefore be eliminated as a possible cause of the fall in developed pressure. One further possibility is a fall in the intracellular calcium transient during early ischaemia. However, as discussed above, there is no evidence for such a fall and the available studies actually suggest that the size of the transient may increase. A final possibility is that ischaemia leads to the reversal of the so-called 'garden hose effect' whereby increased pressure in the coronary circulation leads to an increase in developed pressure (Arnold, Kosche, Miessner, Neitzert & Lochner, 1968). However, recent work by Kitakaze & Marban (1989) suggest that the 'garden hose effect' may result in large part from an effect of coronary artery pressure on [Ca2+]i in ventricular tissue, and not, as previously assumed (Arnold et al. 1968), from an effect on muscle length. If this is correct, a significant 'reversed garden hose effect' in early ischaemic contractile failure should manifest itself as a decrease in [Ca21]i, and direct measurements of [Caa2+]i during this period suggest that no such decrease occurs (see above). One recent study none the less implies a definite role for the 'reversed garden hose effect' in early ischaemic contractile failure. Koretsune et al. (1991) found that developed pressure fell notably faster when ferret hearts were made globally ischaemic (with a loss of pressure in the coronary vasculature) than when 'tissue level' ischaemia was produced (with pressure in the larger vessels maintained) by perfusion with a suspension of microspheres. The metabolic changes were identical in the two cases (Koretsune et al. 1991). While this study suggests that the 'reversed garden hose effect' may well be important, it leaves obscure the question of how the effect is mediated.

Metabolic changes and contractility during ischaemia in glycogen-depteted preparations The time course and extent of the fall in contractility during ischaemia was not modified'noticeably by prior glycogen depletion (Fig. 7). However, there is some evidence to suggest that the effects of changes in metabolites might actually play a lesser role under these circumstances. Firstly, [Pi]i was usually elevated from around 3-4 mm to around 10 mm following glycogen depletion (e.g. Fig. 8; see also Allen et al. 1985). Since most of the inhibitory effects of Pi on force occur over the

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concentration range from 0 to 10 mm (Kentish, 1986), the increase in [Pi]i during ischaemia will have less effect on force in the glycogen-depleted preparations. Secondly, the intracellular acidosis produced by ischaemia was somewhat reduced by prior glycogen depletion (Fig. 8) and so should exert less inhibitory effect on contractility. However, the present study shows that in glycogen-depleted hearts ischaemia leads to rapid action potential shortening, which will reduce the size of the Ca2" transient and thus provide an additional mechanism reducing developed pressure.

Action potential duration during ischaemia Our experiments show that shortening of the action potential is not an essential element in ischaemic contractile failure, since rapid and complete contractile failure occurred in the absence of any change in action potential duration. However, we did find that action potentials shortened markedly during ischaemia if the heart had previously been glycogen depleted to reduce the rate of glycolysis. This is reminiscent of the situation during anoxia, where action potential shortening occurs if glycolysis has also been prevented (McDonald & MacLeod, 1973; Allen et al. 1987; Lederer et al. 1989; Elliott et al. 1989), and implies that all ATP production must be prevented for action potential shortening to occur. While all the available studies of action potentials in ischaemic cardiac muscle report that the action potential duration eventually decreases, there are marked quantitative differences between studies. For instance, Samson & Scher (1960) observed that the action potential had shortened by less than 10 % after 6 min of ischaemia, while Russell et al. (1979) reported a shortening of around 40 % after only 2 min. Our work in ferret hearts (this study) and papillary muscles (Allen et al. 1989) suggests that this marked variability may be related to differences in glycogen stores and hence to the ability of glycolysis to function during ischaemia (see also Lee & Allen, 1988). If glycogen stores have not been depleted, glycolysis can continue for considerable periods and action potential shortening does not occur until long after complete contractile failure has occurred. If glycogen stores are depleted, glycolysis is impaired and action potential shortening occurs much sooner; in extreme cases it can occur during the first few minutes of ischaemia and contribute to contractile failure. Presumably the rate at which ATP is being consumed at the start of ischaemia will also play a part. It is plausible, therefore, that rapid action potential shortening may occur in studies on cardiac muscle from species with small glycogen stores (e.g. rat, see van der Vusse & Reneman, 1983), especially when the preparation is consuming ATP at a high rate just prior to ischaemia (e.g. if heart rate and the level of activation are both elevated). Any depletion of glycogen stores will exacerbate this. This may be of relevance to clinical situations, since areas which become ischaemic may well have been subjected previously to a prolonged period of reduced perfusion. Such reduced perfusion would tend to activate anaerobic glycolysis and thus reduce glycogen stores, with the result that in any subsequent ischaemic episode failure of the action potential would occur earlier.

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The possible basis of action potential shortening The above discussion highlights the fact that all ATP production needs to be blocked before action potential shortening occurs. This in turn may implicate depletion of intracellular ATP in the failure of the action potential (McDonald & MacLeod, 1973). There is considerable evidence that action potential shortening during metabolic blockade occurs as a result of an increase in outward K+ current carried by ATP-sensitive K+ channels (Vleugels, Verecke & Carmeliet, 1980; Lederer et al. 1989) and recent work suggests that the same may be true for action potential shortening during ischaemia (Wilde, Escande, Schumacher, Thuringer, Mestre, Fiolet & Janse, 1990; Gasser & Vaughan-Jones, 1990). In the present study action potential duration fell by about 50 % over 3-4 min of ischaemia in glycogen-depleted hearts (Fig. 7). The average decrease in [ATP]i over this period was very small (around 5 %), corresponding to a fall in [ATP]i from around 6-1 mm to around 5-8 mm. This is still well above the level of [ATP]i where activation of the ATPsensitive K+ channel is thought to occur (see e.g. Nichols & Lederer, 1990). A similar pattern, of a small fall in [ATP]i being associated with a considerable amount of action potential shortening, exists with regard to action potential shortening during metabolic blockade (Elliott et al. 1989). The possible explanations for such an effect, including both regulation of the K+ channels by factors other than [ATP]i and intracellular compartmentation, have been discussed previously (Elliott et al. 1989; see also Nichols & Lederer, 1990; Nichols, Ripoll & Lederer, 1991).

Metabolic changes in prolonged ischaemia The most striking feature of prolonged periods of ischaemia was the development of a profound intracellular acidosis. In addition, [ATP]i fell to around two-thirds of the control value after 45 min of ischaemia. Biochemical and NMR studies on other species have generally shown a fall in [ATP]i to less than 10% of the control value after 40 min (reviewed by Jennings & Steenbergen, 1985). Many of these differences can probably be attributed to the lower temperature (30 0C) in our experiments (and in the recent work of Koretsune & Marban, 1990), compared to 38 0C in most other studies. Interestingly, when a ferret heart was made ischaemic at 38 0C the fall in [ATP]i was approximately twice as fast as in the experiments at 30 TC, while the rate at which pHi fell was approximately the same. Although ATP declined during ischaemia in our experiments, there was no concomitant increase in ADP. This suggests that ADP does not accumulate but is further degraded to AMP (Jennings & Steenbergen, 1985). It may be that some of the increased PME peak observed in prolonged periods of ischaemia arises from AMP, although it almost certainly arises in large part from glycolytic phosphates (Kubler & Spieckermann, 1970). If AMP was formed, and was retained in the cells, one might expect- a rapid resynthesis of ATP (from AMP) on reperfusion (Jennings & Steenbergen, 1985). In fact no such recovery of ATP on reperfusion was observed, suggesting that AMP too is probably further degraded to adenosine and inosine, and perhaps reflecting the irreversible loss of part of the adenine nucleotide pool from the cell (Jennings & Steenbergen, 1985). The relatively slow time course of the fall in [ATP]i, and the continued fall of pHi,

CAUSES OF ISCHAEMIC CONTRACTILE FAILURE 487 during ischaemia implies that ferret hearts continue producing ATP by anaerobic glycolysis for long periods even at very acidic values of pHi. As has previously been noted by other authors (Bailey, Radda, Seymour & Williams, 1982), this suggests that low values of pHi are not inhibitory for glycolysis in intact cells. It is also worthy of note that, although pHi fell by almost a full pH unit during 45 min of ischaemia, the hearts subjected to this duration of ischaemia recovered fully on reperfusion. This tends to indicate that a fall in pHi alone cannot be sufficient to cause ischaemic damage, or indeed to predispose the tissue to the 'reperfusion paradox', as has been argued by some authors (Lazdunski et al. 1985). Causes of ischaemic contracture In ferret hearts [ATP]i is well maintained during global ischaemia, provided that glycolysis is functional (Koretsune & Marban, 1990; this study), and the development of a contracture therefore occurs late in ischaemia. This is in contrast to the situation in cardiac muscle from many other species, where periods of ischaemia longer than about half an hour almost always lead to the development of an ischaemic contracture and incomplete recovery on reperfusion (see Jennings & Steenbergen, 1985, and Allen & Orchard, 1987, for review). The development of a contracture (implying rigor tension) in such studies is generally taken as indicating that ATP has fallen to very low levels. This is consistent with the evidence discussed above that [ATP]i falls faster during global ischaemia in these species than in the ferret. Although [Ca21]i increases in ischaemia, and this increase in [Ca2+]i has been widely implicated in the pathology of both ischaemic and reperfusion damage, it appears unlikely that an increase in [Ca21]i is directly involved in causing the ischaemic contracture in ferret hearts. Recent NMR studies in ferret hearts indicate that [Ca2+]i increases after approximately 20 min of ischaemia, well before [ATP]i is reduced to undetectable levels and before the development of a contracture (Marban, Kitakaze, Koretsune, Yue, Chacko & Pike, 1990; Koretsune & Marban, 1990). The work of Koretsune & Marban (1990), and the present study, shows that in longer episodes of ischaemia the development of a contracture is closely correlated with the fall of [ATP]i to less than approximately 1 mm (Fig. 10). The approximately sigmoidal relationship between [ATP]i and resting pressure (Fig. 10B) is essentially what would be predicted from the skinned muscle data of Fabiato & Fabiato (1975), and supports the conclusion that the ischaemic contracture represents the development of rigor at sub-millimolar levels of [ATP]1.

Conclusions Metabolic changes can explain much, but not all, of the complete contractile failure which occurs in the early stages of global ischaemia in the ferret heart. Action potential shortening does not occur in preparations where glycolysis functions normally, and this suggests that some further factor must be involved in reducing contractility. If the rate of glycolysis is reduced by glycogen depletion then ischaemia is accompanied by action potential shortening, providing a further mechanism to reduce contractility. Many of the variable results in the literature concerning action potential shortening during ischaemia may have their origin in

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differences in the functioning of glycolysis and/or the levels of tissue glycogen stores, a point which mav have relevance to clinical ischaemic states. Finally, our results support the conclusion of Koretsune & Marban (1990) that a fall in [ATP]i to submillimolar levels (rather than an increase in [Ca2+]i) is likely to be the major cause of ischaemic contracture. This work was supported by the British Heart Foundation and the Medical Research Council. Parts of the work were completed while A. C. Elliott held a Studentship from the Science and Engineering Research Council. The NMR experiments were carried out at the Medical Research Council Biomedical NMIR Centre. National Institute for Medical Research, Mill Hill, London. We thank the staff there, and in particular Drs T. Frenkiel and C. J. Bauer, for their help. REFERENCES

ALLEN. D. G.. EISNER. D. A.. MORRIS. P. G., PIROIo, J. S. & SMITH. G. L. (1986). Metabolic consequences of increasing intracellular calcium and force production in perfused ferret hearts. Journal of Physiology 376, 121-141. ALLEN, D. G., HARRIS, M. B. & SMITn, G. L. (1987). Failure of action potentials during cyanide exposure in isolated glycogen-depleted ferret ventricular muscle. Journal of Physiology 387, 65P. ALLEN, D. G., LEE, J. A. & SMITH, G. L. (1989). The consequences of simulated ischaemia on intracellular Ca2" and tension in isolated ferret ventricular muscle. Journal of Physiology 410. 297-323. AllEN, D. GE. MORRIS, P. G., ORCHARD, C. H. & PIROLO, J. 5. (1985). A nuclear magnetic resonance study of metabolism in the ferret heart during hypoxia and inhibition of glycolysis. Journal of Physiology 361, 185-204. ALLEN, D. G. & ORCHARD, C. H. (1983). Intracellular calcium concentration during hypoxia and metabolic inhibition in mammalian ventricular muscle. Journal of Physiology 339, 107-122. ALLEN, D. G. & ORCHARD, C. H. (1987). Myocardial cell function during ischemia and hypoxia. Circulation Research 60, 153- 168. ARNOLD, G., KOSCHE, F., MIESSNER. E., NEITZERT, A. & LOCHNER, WV. (1968). The importance of the perfusion pressure in the coronary arteries for the contractility and the oxygen consumption of the heart. Pflfigers Archiv 299, 339-356. BAILEY, I. A., RADDA, G. K., SEYMOUR, A.-M. L. & WIImIAMS, S. R. (1982). The effects of insulin on myocardial metabolism and acidosis in normoxia and ischaemia. Biochimica et Biophysica Acta 720, 17-27. BAILEY. I. A., WILLIAMS, S. R.. RADDA, G. K. & GADIAN. D. G. (1981). Activity of phosphorylase in total global ischaemia in the rat heart. Biochemical Journal 196, 171-178. BOUNTRA, C.. POWELL, T. & XAUGHAN-JONES. R. 1). (1990). Comparison of intracellular pH transients in single ventricular myocytes and isolated ventricular muscle of guinea-pig. Journal of Physiology 424, 343-365. BROOKS, WV. MI. & WILLIS, R. J. (1983). 31P nuclear magnetic resonance study of the recovery characteristics of high energy phosphate compounds and intracellular pH after global ischaemia in the perfused guinea-pig heart. Journal of Molecular and Cellular Cardiology 15, 495-502. DE HEMPTINNE, A., MARRANNES, R. & VANHEEL, B. (1983). Influence of organic acids on

intracellular pH. American Journal of Physiology 245, (1178-183. I)OWNAR, E., JANSE. M. J. & DURRER. D. (1977). The effect of acute coronary artery occlusion on subepicardial transmembrane potentials in the intact porcine heart. Circulation 56, 217-224. EISNER, D. A., ELLIOTT. A. C. & SMITh, G. L. (1987a). XWhy does ischaemia decrease developed pressure in isolated ferret hearts ? Journal of Physiology 390, 57P. EISN ER. D. A.. ELLIOTT. A. C. & SMITH. (I. L. (1987b). The contribution of intracellular acidosis to the decline of developed pressure in ferret hearts exposed to cyanide. Journal of Physiology 391, 99- 108. EISNER, D. A., PECKETT. WV. R. C. & XXVARE, M. J. (1988). The effects of a-cyano-4-(OH)-cinnamic a(id (CHC) on lactate efflux from isolated perfused rat heart. Journal of Physiology 407, 113P.

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