The effects of various fatty acids on action potential shortening during sequential periods of ischaemia and reperfusion

Journal

of Molecular

and Cellular Cardiology

(1980)

12, 347-369

The Effects of Various Fatty Acids on Action Potential Shortening During Sequential Periods of Ischaemia and Reperfusion J. C. COWAN AND E. M. VAUGHAN WILLIAMS Department of Pharmacology, University of Oxford, Oxford (Received

2 1 June

1978,

accepted in revised form

7 August

1979)

J. C. COWAN AND E. M. VAUGHAN WILLIAMS. The Effects of Various Fatty Acids on Action Potential Shortening During Sequential Periods of Ischaemia and Reperfusion. Journal of Molecular and Cellular Cardiology (1980) 12, 347-369. Intracellular potentials were recorded from Langendorff-perfused guinea-pig hearts. All fatty acids studied (palmitate, linoleate, octanoate and acetate) potentiated action potential (AP) shortening in an experimental protocol involving sequential periods of coronary flow reduction and reperfusion. Pyruvate and acetoacetate did not share this effect. The dose relation of the AP shortening effect was studied in the case of palmitate and found to saturate at a relatively low palmitate : albumin molar ratio. Palmitate-induced potentiation of ischaemic AP shortening was less marked in sustained ischaemia than in the protocol involving periods of reperfusion. Palmitate and acetate were shown to cause an exacerbation of the decline in glycogen levels in the sequential low flow protocol. Ischaemic AP duration was closely correlated with glycogen content at low, but not at high glycogen levels. It seemed possible that part of the fatty acid induced potentiation of AP shortening in the sequential low flow protocol might be attributable to glycogen depletion. In the absence of a glycolytic substrate, acetate and octanoate potentiated ischaemic AP shortening, whereas palmitate was without effect. The degree of ischaemia studied was associated with a stimulation of exogenous glucose utilization, and fatty acids did not prevent this stimulation. KEY

WORDS:

Reperfusion;

Free fatty acids; Arrhythmias; Action potential Adenosine triphosphate; Glycogen; Glycolysis.

duration;

Ischaemia;

1. Introduction Twelve years ago Oliver et al. [29] reported an association between serum free fatty acids (FFA) and the incidence of cardiac arrhythmias after myocardial infarction, yet in spite of much further investigation it is stijl unproven that FFA are themselves arrhythmogenic. A recent approach to the problem has been to study the effects of FFA on the cardiac intracellular action potential (AP). Borbola et al. [5] observed a shortening of action potential duration (APD) in rabbit ventricle with octanoate (2.4 mM). Wasilewska-Dziubinska et al. [47] demonstrated a shortening of APD in Langendorff-perfused guinea-pig ventricle in the presence of 0.1 or 0.5 mM palmitate and 4% albumin. Liideritz et al. [20], using superfused guinea-pig ventricular muscle, observed APD shortening with 0022-2828/80/040347+23

$02.00/O

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Press Inc.

(London)

Limited

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palmitate (0.84 and 1.3 mM) or linoleate (0.25 and 0.5 mM) both in the presence of 2% albumin. In contrast, Ravens and Ravens [36] failed to observe any effect of linoleate (1.8 mM, 2% albumin) on APD in guinea-pig ventricle. We have found that palmitate (0.6 mM, 1% albumin) has no effect on APD in normoxic Langdendorff-perfused guinea-pig ventricle, but that it potentiates the AP shortening which occurs in hypoxia or upon reduction of coronary flow rate [7]. The degree of this potentiation varied greatly, depending on the precise experimental protocol of energy limitation employed. Palmitate induced shortenmodel involving sequential periods of ing was most marked in an “ischaemic” flow reduction and reperfusion. In the present study we have extended our original observations to investigate the concentration dependence of the palmitate effect in this model and the effects of other fatty acids, including the fatty acid analogue acetate. In addition the effects of various non-fatty acid substrates have been investigated. The importance of the particular protocol of successive ischaemic periods in evoking a maximal effect of palmitate has been assessed. In our previous study the time course of palmitate’s potentiation of AP shortening over the sequential periods of flow reduction was rather complex. In both control and palmitate perfused hearts the degree of AP shortening was found to increase progressively in successive ischaemic periods. However, this progressive increase of AP shortening was exacerbated in the palmitate group, so that it appeared that palmitat e potentiated ischaemic AP shortening. The potentiation of shortening was not observed during the first period of flow reduction, but was apparent in the second, became maximal in the third and diminished in the fourth and fifth periods. It seemed possible that the additional, palmitate-induced shortening might, either in whole or in part, be due to an accentuation of the factors responsible for the potentiation of shortening over successive ischaemic periods in the control group. There is evidence that glycolytically-produced ATP may reduce or prevent hypoxic AP shortening [15, 211. One possible explanation of the progressive increase in AP shortening in successive ischaemic periods would be a progressive depletion of glycogen, causing a growing limitation of the energy yield from glycolysis. By extension of this hypothesis, the additional AP shortening caused by palmitate might be due to an acceleration of glycogen depletion. In the present investigation we set out to test this possibility by measuring the effect of palmitate on glycogen levels in the sequential low flow protocol.

2. Methods Heparinized guinea-pigs were stunned, and their hearts removed and .placed in cooled, oxygenated solution. For all electrophysiological studies the hearts were perfused in a constant flow recirculating Langendorff perfusion system, as described previously [7]. The normal and reduced (“ischaemic”) flow rates were 10 and 2 ml/min respectively. Perfusion pressure and contraction were

FATTY

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AND

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ACTION

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SHORTENING

349

continuously recorded. Intracellular potentials were recorded from the epicardium by the floating microelectrode technique of Woodbury and Brady [50]. The hearts were driven at 3.5 Hz and temperature was maintained at 32°C. The perfusion medium contained (mM): NaCl, 118; KC1 4.5; CaCl,, 2.5; MgCl,, 1.0; NaH,PO,, 0.8; NaHCO,, 25, and was equilibrated with 0, 95q& CO, 50!, giving a pH of 7.4. In addition glucose (11 mM) was present, unless otherwise stated. Sodium acetate, palmitate, linoleate or octanoate were added to the perfusion medium. Sodium linoleate and palmitate were first complexed to defatted albumin, previously dialysed against basic perfusion medium [7]. The final concentration of albumin was 1 o/o (approximately 0.15 mM). Calcium activities of the final perfusates were kindly measured by Dr P. A. Poole-Wilson (Cardiothoracic Institute, London) by means of a calcium selective electrode [3]. Within the accuracy of the technique (approximately * 296), the calcium activity of basic perfusion medium was unaffected by the addition of albumin, albumin + palmitate (0.6 mM) or albumin + linoleate (0.6 mM). For the assay of metabolites the ventricles were detached from the rest of the heart at the end of the perfusion by rapidly cutting round the atrioventricular groove, and then frozen with aluminium tongs [49]. Inevitably this involved a delay of several seconds between interruption of coronary flow and freezeclamping, which could have resulted in a slight under-estimation of the more unstable intermediates [48]. The frozen hearts were stored under liquid nitrogen prior to assay. Weighed portions of the deep-frozen tissue were homogenized in perchloric acid and the homogenate divided into two parts, for glycogen and high energy phosphate estimation respectively. Glycogen was assayed by estimation of glucose release (hexokinase method) following enzymatic hydrolysis of the particulate homogenate with amyloglucosidase. High energy phosphate assays were performed by means of standard enzymatic methods [4]. In a number of experiments attempts were made to deplete glycogen by 30 min hypoxic (39/o 0,) perfusions with substrate-free solution, at a high stimulation rate (5 Hz). This procedure only partially depleted glycogen content, the mean value being 10.8 f 2.0 pmol glucose equivalents/g w. wt. following depletion, as compared with 24.9 & 1.7 pmol glucose equivalents/g w. wt. following 60 min perfusion in the presence of glucose + 95% 0,. However, the residual glycogen may not be readily available for glycogenolysis [23, 391. In this situation measurements of the rate of exogenous glucose utilization via the glycolytic pathway should give an approximate estimate of the glycolytic rate, the closeness of the approximation depending on the degree of residual glycogenolysis. Accordingly, glycolytic rate was estimated by measuring the release of 3H,0 from 5-3H-glucose [27]. Perfusions with 5-3H-glucose were carried out in a non-recirculating perfusion system with the heart suspended vertically. 5-3H-glucose was freeze-dried prior to use and redissolved to give a specific activity of 18.2 nCi/pmol (glucose concentration, 11 mM). The effluent perfusate collected from the heart was immediately

350

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WILLIAMS

placed in ice. 3H,0 and 5-3H-glucose were separated within 2 h of collection by a procedure involving the chemical binding of glucose to metaborate. A 1 ml sample of perfusate was eluted with water through 3 ion-exchange columns arranged in series (Dowex 50 x 4-200-H+ form, followed by Dowex 1 x 4-lOOOH- form, followed by Dowex 1 x 4-lOO-metaborate form). The first 2 columns were regenerated after use, but a fresh metaborate column was used for each sample. 99.798 * O.OO2o/o of the 5-3H-label was retained on the column with a 99.15 f 0.51% recovery of 3H,0. Even with this high degree of separation, a substantial, but reproducible, blank correction was necessary. A 3 ml portion of each eluted sample was added to 10 ml of cocktail (0.4% 2,5-diphenyloxazole in scintillation counting (Beckman 67% toluene, 33% TritonX-100) f or liquid LS-2000 B). Reagents used Palmitic acid, octanoic acid, sodium acetate, sodium pyruvate, sodium-DL-8hydroxybutyrate and sodium-DL-lactate (Sigma Chemical Company, Limited) ; biochemical reagents for the glycogen, high energy phosphate and metabolite assays (Boehringer Corporation, Limited) ; 5-3H-glucose (Radiochemical Centre, Amersham). Sodium acetoacetate was kindly provided by Dr D. H. Williamson (Metabolic Research Unit, Radcliffe Infirmary, Oxford). 3. Results E$ects offat@

acids of varying chain length on ischaemic AP shortening

In our previous study, palmitate’s potentiation of ischaemic or hypoxic shortening of APD was found to be most marked with a protocol involving 10 min periods of ischaemia alternating with 20 min periods of normal perfusion. In the present investigation of other fatty acids a similar protocol was employed. Thirty min after the addition of the fatty acid under study, flow rate was reduced to 2 ml/min for 10 min. Two further low flow periods followed, separated by 20 min recovery periods at 10 ml/min. In the first series of experiments, the effects of sodium acetate (5 mM) were studied in comparison with those of palmitate (0.6 mM). AP shortening was progressively greater in successive ischaemic periods, with almost complete recovery occurring during the intervening normal flow periods (Figure 1). Both acetate and palmitate groups developed a statistically significant potentiation of AP shortening with respect to the control group, in the second ischaemic period. The potentiation became more marked in the third period (acetate, P < 0.005; palmitate, P < 0.001). In a second series of experiments, the effects of the intermediate chain length fatty acid, octanoate, and the long chain unsaturated fatty acid, linoleate, were

FATTY

ACIDS

AND

ISCHAEMIC

ACTION

POTENTIAL

SHORTENING

IO

5

351

160 c

14c , -

2 E

12c I -

8 E 4 -

Control

------

Palmitate

IOC I -

Acetate

60 , -

0

5

IO

0

5

0

IO

(min)

FIGURE 1. Effects of palmitate and acetate on action potential shortening in sequential periods of ischaemia. Coronary flow rate was reduced to 2 ml/min during each of three 10 min periods. The intervening 20 min recovery periods are not shown. In this and-subsequent figures illustrating ischaemic AP shortening, the ordinate was calculated as the mean change in APD,, rather than as its absolute value. However, for clarity of graphical presentation, these mean changes have been subtracted from a normalized value of 150 ms for APD,, and plotted as absolute values. Glucose and albumin (1 Oh) were present in all experiments. Control, n = 7; palmitate (0.6 mM), n = 7; acetate (5 mu), n = 6.

studied. Both linoleate (0.6 mM + 1% albumin) and octanoate (0.5 mM, no albumin) caused a potentiation of AP shortening similar to that seen with acetate (Figure 2). The effect was maximal in the third low flow period (P < 0.005 for both linoleate and octanoate) and accompanied by a statistically significant potentiation of ischaemic contractile depression (results not presented; P < 0.05 for linoleate, P < 0.005 for octanoate). It can be concluded, therefore, that all the fatty acids which we have studied (palmitate, linoleate, octanoate and acetate) share a similar action in potentiating AP shortening in this sequential low flow protocol.

352

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155

140

2 E o

120

0” :

-

Control

------

Linoleate

100 .“..

.... Octanoate

I

‘. .

80

I

0

5

IO

I

0

5

IO

I

0

I 5

1

(min)

FIGURE 2. Effects of linoleate periods of ischaemia. Control group octanoate (0.5 mu), n = 5.

Concentration

and octanoate n = 6 (4 with

on action potential shortening in sequential albumin); albumin-linoleate (0.6 m~), n = 6;

dependence of palmitate’s potentiation shortening

of ischaemic action potential

The sequential low flow protocol was used for the study of a range of palmitate concentrations (0.15 to 1.2 mM). The shortening of APD produced by various concentrations during the third low flow period is illustrated in the upper part of Figure 3. In the lower part of the figure the maximal degree of shortening is plotted as a function of the concentration of palmitate on a logarithmic scale. With 0.15 mM palmitate the potentiation of AP shortening was approximately half that with 0.6 mM. The shortening of APD with 0.15 mM palmitate was significantly significantly greater (P < 0.05) than with albumin alone. It was not, however, less than the degree of shortening with 0.6 mM palmitate (0.1 > P > 0.05). Nevertheless, the general trend of the results suggests a concentration dependence of the palmitate effect at low concentrations, and independence at higher concentrations.

FATTY

ACIDS

AND

ISCHAEMIC

ACTION

POTENTIAL

SHORTENING

3r,3

I--

155

Polmitate 140

i 0 4 q

120

100

I

I

0

5

IO

(min)

-60

-

-50

-30.

-20

I

-

// 0

//

0.15

0.3 Palmitote

0.6

I 1.2

(mM)

FIGURE 3. Concentration dependence of palmitate’s in sequential periods of ischaemia. The upper part of third low flow period. In the lower part of the figure the function of palmitate concentration. Albumin (19~~) n = 6; 0.15 mM palmitate, n = 6; 0.3 mM palmitate, palmitate, n = 4.

potentiation of action potential shortening the figure illustrates AP shortening in the degree of shortening at 9 min is plotted a~ a was present in all experiments. Control, n = 6; 0.6 rnM palmitate, n -: 4; 1.2 rnx

354

J. C. COWAN

AND

Effects of palmitate

E. M. VAUGHAN

WILLIAMS

during sustained Jrow reduction

In our previous study [7] the maximum effects of palmitate observed in the sequentially ischaemic protocol were considerably greater than the maximum effects observed in simple sustained hypoxia. This enhancement could be due to the additional influenc of flow reduction in ischaemia, or to the particular e protocol of successive low flow periods employed. These possibilities were investigated by studying the effects of palmitate on APD during sustained flow reduction (Figure 4). Flow was reduced to 2 ml/min for 70 min, corresponding to the overall time to the end of the third low flow period in the sequential protocol. The effects of palmitate were less marked than in the sequential protocol, barely achieving statistical significance (P < 0.05 at 45 min). After 20 min recovery at 10 ml/min, flow was reduced to 2 ml/min again for 10 min. APD fell to a greater extent than

160

140

2 E

120

a 9 u

100 -

Control

------

Palmitate

80

1 0

20

1 60

40 (min)

FIGURE 4. Effects of palmitate on action potential shortening during sustained ischaemia. Coronary flow rate was maintained at 2 ml/min over a 70 min period. Two further low flow periods of 10 min duration followed. The intervening 20 min recovery periods are not shown. Albumin (1%) was present in all experiments. Control, n = 6; palminate (0.6 mM), r~ = 6.

FATTY

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355

in the 70 min low flow period, and the difference between palmitate and control groups increased. A further recovery and low flow period resulted in additional AP shortening. Palmitate had no significant effect on contraction at any stage in this series of experiments (results not presented). It seems probable, therefore, that the substantial effects of palmitate on AP shortening in the sequential ischaemic protocol are more marked than in sustained ischaemia. While this raises difficulties in interpretation, the usefulness of this particular protocol lies in the considerable magnitude of the fatty acid effect which it produces. Efects of palmitate

and acetate on glycogen levels in irchaemia

Experiments were undertaken to investigate the possibility that fatty acid potentiation of AP shortening in the sequential low flow protocol might be due to an acceleration of glycogen depletion. The effects of palmitate (0.6 mM) and acetate (5 mM) were studied. Albumin (1%) was present in all experiments in the palmitate group (n = 13), and in 11 of 16 experiments in both acetate and control groups. As the presence or absence of palmitate did not appear to effect the parameters under study, the results have been pooled for purposes of presentation. Hearts were freeze-clamped at the end of the third period of ischaemia, that is at the time of maximal fatty acid potentiation of AP shortening. The values of APD immediately prior to clamping were 137.6 & 4.0 ms in the no addition group, 99.8 + 5.1 ms in the palmitate group (P < 0.001) and 97.6 5 8.2 ms in the acetate group (P < 0.001). Glycogen, high energy phosphate and lactate values are presented in Table 1. Glycogen levels were found to be significantly lower in the presence of acetate or palmitate, in comparison with the no addition group (P < 0.05). ATP and creatine phosphate also fell to a greater extent in the presence of acetate and palmitate, the effects bordering on statistical significance and achieving it where indicated. A further group of hearts was freeze-clamped immediately prior to the first period of flow reduction (i.e. after 60 min normal flow perfusion), to eliminate the possibility that these changes might be present before the induction of ischaemia. The values of APD prior to clamping were 159.3 5 2.7 ms in the no addition group, 160.0 + 1.7 ms in the palmitate group, and 157.8 + 0.8 ms in the acetate group. There was no evidence of high energy phosphate or glycogen depletion in the presence of palmitate or acetate with respect to the no addition group. The only significant difference was an elevation of glycogen content in the presence of palmitate (P < 0.05). Elevation of glycogen content during normoxic perfusion with FFA has been observed by other authors [41] and has been attributed to FFA-induced glycolytic inhibition. It would seem therefore that the presence of acetate or palmitate during sequential periods of ischaemia causes an acceleration of glycogen depletion. An

356 TABLE

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1. Effects of acetate and palmitate on high energy phosphate at normal and reduced flow rates Non-ischaemic Control n=5

Creatine

phosphate

and glycogen levels

Ischaemic

Palmitate n=5

Acetate 7l=5

Control n= 16

Palmitate n= 13

Acetate R= 16

18.3 $2.5

l

22.6 2.4

19.7 f0.4

5.6 10.9

4.1 $0.8

3.1* f0.6

ATP

15.8 fl.O

18.8 12.2

17.6 * 1.3

11.3 &0.6

9.4* rto.7

9.7 10.8

ADP

4.88 f0.55

5.11 f0.88

3.89 f0.20

6.22 kO.26

5.65 f0.38

6.42 *0.34

AMP

0.93 f0.23

0.96 50.39

0.58 *to.05

2.11 f0.24

l

2.42 0.31

2.92 kO.35

Lactate

8.3 f0.9

5.8 52.4

6.5 f2.3

20.0 *2.5

21.8 $2.2

20.1 f1.5

Glycogen (pm01 glucose equivs.)

183.3

258.8*

185.0

134.1

100.9*

105.0*

f8.3

*9.9

*9.1

Metabolic levels are expressed after 60 min perfusion (30 min with The ischaemic hearts were clamped protocol. * Indicates P < 0.05 of the control group.

122.3

f 12.6

i

13.6

in pmol/g dry wt. The non-ischaemic hearts were clamped basic perfusion medium and 30 min with the additions shown). at the end of the third low flow period in the sequential low flow palmitate and acetate groups in comparison with the equivalent

interesting association was observed between glycogen content and APD prior to clamping. The linear correlation coefficient between the two was 0.69 (n = 45, P < 0.0001). This compared with a linear correlation coefficient of 0.52 between APD and ATP content. However, on plotting the individual glycogen and APD values, the relation between them appeared markedly non-linear [Figure 5(a)]. This is illustrated by the fact that log glycogen correlated more closely (r = 0.76) with APD than simple glycogen content. Polynomial regression lines were therefore computed (on a Hewlett Packard 9830 calculator to 3 terms in X) for each of the three groups [Figure 5(b)]. Th ese showed a marked concavity with respect to the glycogen axis in their central portions, although diverging at their ends where the number of points fitted was small. It is an important point that the acetate and palmitate regressions lay below the control group regression, since this indicates that part of their AP shortening action must be independent of any glycogen-depleting effect.

FATTY

ACIDS

AND

ISCHAEMIC

ACTION

POTENTIAL

Acetate

40 -z -E $

1.

357

SHORTENING

n

._.,__ --I*-** . .._ _... ,_,’ ,_: ._I’ __.’ + ,/’ r_.. n

160

%

(b)

~~-

_____..0 Control

Palmitote

A

Acetate

n

c 40

80

120 Glycogen

(+nai

160 glucose

200 equivs

/g

FIGURE 5. Relation between APD and glycogen content in values. (b) Polynomial regression lines. The polynomial regression (control, palmitate, acetate) are plotted. The 3 points represent APD and glycogen content for each of the groups.

240 dry

280

wt.) ischaemic hearts. (a) Individual lines for each of the 3 groups the mean values (~s.E.M.) of

The effects of non-fatty acid substrates on ischaemic AP shortening The effects of a number of non-fatty acid, AP shortening were studied to determine action to acetate and long chain fatty acids. acetoacetate (5 mM) were studied using the above. Sodium acetate (5 mM) was included

non-glycolytic substrates on ischaemic whether or not they shared a similar Sodium pyruvate (5 mM) and sodium sequential low flow protocol described in the same series of experiments for

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Is0

-+

k ‘.A.. ..*-+

140 -

2

E

a E a

120.

100 -

I 5

0

IO

-

Control

-4--

Acetate

----D---

Acetoacetate

--.-*...

pyruvate

I

1

0

5

I

IO

0

5

IO

(min)

FIGURE 6. A comparison of the effects of acetate, pyruvate and acetoacetate on action potential Control group, n = 5; acetate (5 mM), n = 5; shortening in sequential periods of ischaemia. pyruvate (5 mu), n = 5; acetoacetate (5 mM), n = 5. The shaded area in the third low flow period includes the standard errors of points in control, pyruvate and acetoacetate groups.

purposes of comparison (Figure 6). As before acetate showed a marked potentiation of the degree of AP shortening. However, neither pyruvate nor acetoacetate showed a similar effect. In their presence the degree of AP shortening did not differ significantly from the control group. Since both pyruvate and acetoacetate constitute the oxidized half of an NAD+/NADH linked oxidation/reduction couple within the cell, it seemed. possible that their reduction might retard the intracellular build-up of reducing equivalents and mask a deletrious effect otherwise similar to that observed with acetate. With this in mind, the effects of the other half of each oxidation-reduction couple (i.e. lactate and P-hydroxybutyrate) were studied. The shortening of APD with sodium DL-lactate (5 mM) or sodium DL-p-hydroxybutyrate (5 mM) did not differ significantly from control perfusions (results not presented). Efects of fat9 The finding in ischaemia

acids on ischaemic AP shortening in the absence of a g&o&k

in the present study that fatty acids potentiate glycogen suggests the possibility of a fatty acid-induced deleterious

substrate depletion metabolic

FATTY

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359

action. One possible basis for such an action would be a maintenance of fatty acid-induced glycolytic inhibition in ischaemia. A study of the effects of the various fatty acids in ischaemia in the absence of a glycolytic substrate provided a simple means of testing this hypothesis. For these experiments glucose was therefore excluded from the perfusion medium and’glycogen depleted in a preliminary 30 min hypoxic (3% 0,) perfusion at a high stimulation rate (5 Hz). Fifteen min after the restoration of normal oxygenation and stimulation rate, the substrates to be studied were added. Microelectrode recordings were made 10 to 15 min after substrate addition. At normal pe&sion rates, APD was affected LO only a slight extent by the various substrate additions (no substrate addition, 142.4 & 2.9 ms; no substrate addition, but albumin added, 139.6 f 5.8 ms; sodium pyruvate (5 mM), 153.4 & 2.6 ms; sodium acetate (5 mM) 145.6 & 2.7 ms; sodium octanoate (2 mM), 151.4 + 2.0 ms; sodium palmitate (0.6 mM, l”,, albumin), 147.0 + 2.3 ms). Coronary flow rate was then reduced to 2 ml/min and APD fell precipitously (Figure 7). A marked potentiation of AP shortening occurred in the acetate group. By contrast palmitate and pyruvate did not significantly affect AP shortening, in comparison with the no substrate addition group. This result is particularly interesting since it reveals a dissociation between the effects of acetate and palmitate. The effect of octanoate was less clear-cut. Initially APD shortened markedly, showing a highly significant potentiation of AP shortening in comparison with the no substrate addition group at 2 min (P < 0.01). However, this potentiation was temporary and APD actually lengthened during the remainder of the ischaemic period. It is unclear to what extent this apparent reversal can be regarded as a “true” effect, since in 4 of the 8 preparations persistent fibrillation developed. As fibrillation was more prone to occur in preparations with a shorter APD, the apparent reversal of AP shortening may merely be due to exclusion of these preparations. On the other hand, a lengthening of APD was apparent even within the group of non-fibrillating hearts. Changes in contraction could only be accurately followed over the first minute of the ischaemic period, since with more prolonged ischaemia a marked rise in diastolic tension occurred obscuring the contraction trace. At 1 min contraction fell by 62.0 i 5.1% in the absence of any substrate, 48.3 & 3.8qb in the pyruvate group (P < 0.05), 57.9 + 3.4% in the palmitate group, 67.5 &, lO.l’?/b in the octanoate group and 77.6 f 3.60& in the acetate group (P < 0.05). Thus acetate was the only substrate to significantly potentiate contractile depression. Ejects of FFA on glucose utilization

in ischaemia

The studies in the absence of a glycolytic substrate were noteworthy for the total lack of any effect of palmitate. This lack of effect would be consistent with the hypothesis of a fatty acid induced deleterious metabolic action-mediated through

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Control

160

.................

Pyruvate

--------

Polmttate

-.-.-.-a-.

Octanoate

-

140

-

-

Acetate

2 E t? 2 a 100

f- --

--+--------

-4

60 I 0

1 5

I IO

(min)

FIGURE 7. The effects of various substrates on action potential shortening in ischaemia, following glycogen depletion and in the absence of exogenous glucose. The absolute values (rather than the changes) in APD are plotted, for a 10 min period of flow reduction, initiated at time zero. Control (no exogenous substrate), n = 12 (7 with albumin); pyruvate (5 mM), n = 7; albumin + palmitate (0.6 mM), n = 12; octanoate (2 mM), n = 8; acetate (5 mM), n = 1 I. Fibrillation was a frequent occurrence during the course of the ischaemic period, causing the exclusion of 3 preparations from the control group, 1 from the pyruvate, 2 from the palmitate, 4 from the octanoate and 3 from the acetate groups.

glycolytic inhibition. Accordingly experiments were undertaken to test more directly the possibility of a maintenance of FFA-induced glycolytic inhibition in ischaemia. These experiments were performed in a non-recirculating perfusion system. Glycogen was depleted as described above. Following the restoration of normal oxygenation and stimulation frequency for 15 min, perfusion was switched to a medium containing glucose (11 mM 5-3H-glucose, 18.2 nCi/pmol) and albumin (-& palmitate or acetate). After a 10 min period for equilibration of label, samples of coronary effluent were collected, for measurement of 3H,0 production. At normal flow rate (9.90 * 0.07 ml/min) the “glycolytic rate” was significantly lower in the presence of palmitate (Figure 8), thus confirming that inhibition of glucose utilization by FFA was present under the experimental conditions of the present investigation. With acetate, too, a significant inhibition of “glycolytic rate” occurred but the degree of inhibition decreased markedly with time. While the reason for this decrease in inhibition is unknown, it is of interest in view of the

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361

5.0

- - - - -

Polmitate

. . . . . . . . . . ..

*&ate

I .o

I IO

I 20

I 30

I 40

(min)

FIGURE was added experiments.

8. Measurements of “glycolytic rate” at normal and reduced flow rates. 5JH-glucose at time zero. Coronary flow rate was reduced at 20 min. Albumin was present in all Control, n = 10; palmitate (0.6 mM), n = 10; acetate (5 mM), n = 5.

failure of acetate to elevate glycogen levels noted above (Table 1). Twenty minutes after the addition of labelled glucose, flow rate was reduced (to 2.31 & 0.04 ml/min). In the control group, “glycolytic rate” rose steadily throughout the

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low flow period. This is of interest in that it indicates that the degree of flow reduction used throughout the present investigation was not so great as to prevent a stimulation of glycolysis [compare ref. (28) 1. A similar but even faster rise in “glycolytic rate” occurred in the acetate and palmitate groups. In the first 2 min of ischaemia “glycolytic rate” rose by 0.35 & 0.21, 1.58 & 0.35 and 2.28 f 0.38 pmol glucose/g dry wt./min in the control, palminate and acetate groups respectively. As a consequence the inhibition seen with acetate or palmitate at normal flow rates disappeared very rapidly. In fact, in early ischaemia, “glycolytic rate” was even greater in the acetate and palmitate groups than in the control group, the difference achieving statistical significance (P < 0.01) in the former c-ase. These findings refute the idea that FFA inhibition of exogenous glucose utilization might persist at the degree of ischaemia used in the present investigation. 4. Discussion A consideration

of the. ischaemic model

Although the terms low flow and ischaemia are used interchangeably in the present study, the strict applicability of the term “ischaemia” remains in doubt. Flow rate in Langendorff preparations is much higher than in vivo, and a fall to 2 ml/min may not represent a true decrease relative to in vivo coronary flow rates. Two factors suggest that the degree of so-called ischaemia under study is mild in comparison with the ischaemic models of other investigators. Firstly, tissue lactate levels showed only a threefold increase, which is less than that observed under even the mildest degrees of ischaemia, in the ischaemic models of some other investigators [I, 281. Secondly, the degree of flow reduction was not sufficient to prevent ave demonstrated a spectrum ranging stimulation of glycolysis. Neely et al. [28] h from stimulation to inhibition of glycolysis, with increasing degrees of flow reduction. It might well be argued that the use of a more severe degree of ischaemia in the present study would have facilitated comparison with the ischaemic models of other investigators. However, in our experience, it is considerably more difficult to achieve satisfactory microelectrode penetrations in severely ischaemic muscle, and our studies are therefore confined to mild ischaemia. AP shortening was found to be considerably potentiated in successive ischaemic periods in the sequential low flow protocol. A similar potentiation of successive hypoxic periods has been demonstrated previously [45]. It was, however, particularly surprising that sustained ischaemia failed to produce as marked AP shortening as the sequential low flow protocol. This suggests that some factor linked to the initiation or termination of ischaemia must be responsible for a considerable part of the AP shortening in the sequential model. The nature of this factor remains uncertain, but adverse effects of reperfusion are well documented [for review see (13)].

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The further problem exists of the curious time course of the effects of FFA on APD with respect to successive ischaemic periods. With ail fatty acids studied there was found to be a progressive increase in potentiation of AP shortening over the first 3 low flow periods. We have previously observed with palmitate that this potentiation declines in any subsequent low flow periods. In the present study palmitate and acetate were found to potentiate the degree of glycogen depletion at the end of the third period of ischaemia, and it seems possible that this factor might account for part of the potentiation of AP shortening. However, there is probably an additional direct effect on APD, independent of changes in glycogen levels, in view of the displacement of the polynomial regression lines relating APD and glycogen content, in the presence of acetate and palmitate. The decline in the degree of palmitate potentiation of AP shortening in subsequent low flow periods might be due to a decrease in the glycogen-related effect, available glycogen becoming fully depleted in both fatty acid and control groups. Palmitate’s potentiation of ischaemic AP shortening was considerably greater in the sequential low flow protocol than in sustained ischaemia. The exact time course of glycogen depletion and the role of the recovery periods remain unknown. This difficulty inevitably raises doubts as to the relevance of results so obtained. The protocol reliably reproduces substantial effects of FFA and so, albeit empirically, provides a useful ischaemic model for investigating possible deleterious effects of FFA. It seems possible, although it remains unproven, that the same basic mechanisms might apply in other hypoxic/ischaemic models, and that the observed differences would be of quantitative rather than of qualitative significance. Whether any conclusions derived using the model are of more general applicability, in particular to ischaemic heart disease in man, is of course open to question. An adverse metabolic action of FFA? The tissue clamping experiments were originally undertaken to test the hypothesis that FFA might accelerate glycogen depletion in the sequential low flow protocol, and this hypothesis was confirmed. A similar potentiation of glycogenolysis in ischaemia is apparent in the date of Neely et al. [(Z&Z), compare Figures ‘2 and 3 in their study]. These authors demonstrated that ischaemia evoked a more rapid stimulation of exogenous glucose utilization in the presence of palmitate. They attributed the slow rise in exogenous glucose utilization to the time required for glycogen depletion. By the same token, the faster rise in glucose utilization in the presence of palmitate may be due to an acceleration of the rate of glycogenolysis. The fatty acid induced glycogen depletion of the present study was accompanied by a slight, but in some cases significant, potentiation of the ischaemic decline in high energy phosphate levels. These various changes suggest that, under the experimental conditions of the present investigation, fatty acids may have an adverse metabolic effect in ischaemia. Possible mechanisms by which FFA might

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cause an energy-wasting effect in ischaemia have been reviewed by Opie [31]. They can be broadly divided into two categories, those dependent on the long chain FFA or their acyl CoA derivatives, and those dependent on the products of FFA oxidation. In the former category possible mechanisms include the inhibition of mitochondrial adenine nucleotide translocase by acyl CoA derivatives [42] and the energy demands of triglyceride synthesis [31]. In the latter category, FFA-induced glycolytic inhibition represents one possible mechanism of an oxidation product dependent effect. The observation in the present study that the fatty acid homologue acetate shares a similar effect to palmitate in potentiating AP shortening in the sequential low flow protocol, suggests that, in this particular model, palmitate may be acting through its oxidation products. A similar finding has recently been reported by Bricknell and Opie [6], who showed that both palmitate and acetate potentiated the enzyme release occurring on reperfusion of ischaemic rat hearts. However, in the present investigation, the dissociation of palmitate and acetate effects on AP shortening in the absence of a glycolytic substrate, suggests the need for caution in attributing the effects of palmitate to its oxidation. It may be that acetate has effects over and above those of long chain fatty acids. Alternatively, the difference between palmitate and acetate in the absence of a glycolytic substrate might be due long chain fatty acids being activated to their differing sites of activation, extramitochondrially and short chain intramitochondrially [ 121. FFA inhibition of exogenous glucose utilization was considered as a possible mechanism of a FFA-induced energy limitation in ischaemia. This inhibition is thought to be mediated by FFA oxidation products [II] and consequently disappears in anoxia [35]. In moderate hypoxia, however, FFA-induced inhibition of glycolysis has been reported to be maintained [2]. The possibility of such an inhibition seemed particularly attractive in the present context, in view of the importance of glycolytic ATP in preventing hypoxic AP shortening [15, 211. However, at the degree of ischaemia of the present investigation, FFA-induced inhibition of exogenous glucose utilization was found to disappear very rapidly on flow reduction. While this finding was made during a single period of ischaemia, the degree of flow reduction was similar to that in the sequential low flow protocol. It therefore seems unlikely that an inhibition of glucose utilization could be responsible for the AP shortening and proposed metabolic defect induced by FFA in this protocol. The contrast between the action of acetate and the lack of effect of pyruvate in potentiating AP shortening in the sequential low flow protocol seems rather curious. However, other authors have demonstrated a similar contrast between the effects of acetate and pyruvate in situations of energy limitation. Reinauer and Muller-Ruchholtz [37] found that acetate (5 mM) caused a very marked ATP depletion in anoxic Langendorff-perfused rat hearts, in comparison with the total absence of exogenous substrate. Pyruvate (1 mM) did not share this ATP-depleting

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effect. The effect was absent in normally oxygenated hearts. Bricknell and Opie [6] demonstrated that acetate (3.6 mM), in comparison with the total absence of exogenous substrate, potentiated enzyme release on reperfusion of ischaemic rat hearts. Pyruvate (5 mM) failed to show a similar potentiation of enzyme release, and in fact provided a slight protection in comparison with the total absence of exogenous substrate. These various demonstrations of a distinction between the effects of pyruvate and of acetate may have important implications with regard to the mechanism of the proposed deleterious metabolic action of acetate. Dose defiendence of FFA potentiation

of AP shortening

The observed saturation of palmitate’s potentiation of ischaemic AP shortening at relatively low FFA : albumin molar ratios is consistent with the hypothesis of a FFA-induced metabolic defect. The effects of palmitate perfusion on FFA oxidation, and on acetyl CoA and long chain acyl CoA levels, have been shown to saturate at comparably low FFA : albumin ratios in rat hearts [34]. If the results of the present study are of more general applicability, this observed saturation would argue against the idea, implicit in the original hypothesis of arrhythmogenesis by FFA [29], that pro-arrhythmic effects should become progressively greater with increasing FFA levels. Recent publications have revealed a contrast between the failure of elevating FFA levels during myocardial infarction to precipitate or exacerbate arrhythmias, and the successful use of agents which lower FFA to prevent arrhythmias. The elevation of plasma FFA levels has generally been induced by the infusion of Intralipid together with heparin. Although Kurien et al. [19] did find evidence for an increased incidence of arrhythmias on infusion of Intralipid/heparin, other authors subsequently [18, 26, 32, 331 have been unable to demonstrate a similar arrhythmogenic effect. Conversely, lowering FFA levels with anti-lipolytic drugs has been shown to have a slight anti-arrhythmic effect [38, 431. Studies on ST-segment elevation further illustrate the contrast between the effects of raising and lowering FFA levels. Ischaemic ST-segment elevation has been shown to be unaffected by Intralipidlheparin [17] but to be reduced on lowering the FFA : albumin molar ratio, whether by anti-lipolytic therapy [17, 251 or by infusion of FFA-free albumin [24]. Intralipid/heparin has however been found to cause ST-segment elevation, if given after a lipolytic inhibitor [17]. It may well be that FFA levels are sufficiently elevated in ischaemia, even prior to Intralipid/heparin, to saturate possible deleterious effects. On the other hand, lipolytic inhibitors could still have a beneficial effect in this situation. In the present study the effects of palmitate became maximal at a palmitate : albumin ratio of about 2 : 1. In other models of ischaemia, with different intravascularintracellular profiles of FFA concentration, saturation might well occur at a different FFA : albumin ratio.

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A role for glycogen in AP maintenance In ischaemia glycogen levels and APD showed a highly significant correlation. However a plot of individual values of APD and glycogen content indicated that the relation was non-linear, APD falling to a greater extent at low glycogen levels than at high. This observation is of considerable interest since it seems probable that glycogenolysis itself would not be a linear function of glycogen content, being independent at high glycogen levels and dependent, due to decreased substrate availability, at lower levels. Previous reports [30] illustrate that the rate of glycogenolysis falls off even at fairly high glycogen levels. Thus the observed nonlinearity in the relation between APD and glycogen content might well reflect on underlying linear relationship between APD and rate of glycogenolysis, suggesting that glycogenolysis may play an important role in APD maintenance in ischaemia. Many authors have obtained evidence for a protective role of glycogen in hypoxia or ischaemia [8, 14, 16, 40, 441. On theoretical grounds, the overall energy provision from glycogenolysis can only partially substitute for the energy provision of oxidative metabolism [30]. It is possible that ATP from glycogenolysis is formed in a subcellular compartment, which permits priority of support for membrane functions such as maintenance of APD. This would be consistent with the observation in the present investigation of a close relation between APD and glycogen content. A relation between APD and glycogen content has not been demonstrated previously, although a contribution from glycogenolysis to APD maintenance has been suggested [ZZ], and can be thought of as an extension of the hypothesis relating glycolytic ATP to APD maintenance. However, since the results of the present study indicate that glycogen is much more effective than glucose in maintaining APD, it may well be that glycogenderived glycolytic ATP is more closely related to membrane function than glycolytic ATP derived from external glucose. In this context it is interesting to note the recent demonstration [9, 101 o f a close association between glycogen, the enzymes of glycogenolysis and the sarcoplasmic reticulum. On the other hand, the greater efficacy of glycogen in comparison with glucose may simply be a reflection of a greater ATP yield. The maximum rate of glycolysis from glycogen is approximately twice that from external glucose [30], while the yield of ATP per hexose unit is 50% greater. Hence, the maximum rate of glycolysis from glycogen could provide about three times as much ATP as the maximum rate of glycolysis from glucose. Whatever the explanation for the greater efficiency of glycogen in comparison with exogenous glucose in protecting against ischaemic AP shortening, this observation is of potential therapeutic importance. There is considerable evidence [for review see (46)] that shortening of APD increases the susceptibility to cardiac arrhythmias, and it seems probable that it is a major factor contributing to the high incidence of arrhythmias following myocardial infarction. The present

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observation of a close association between glycogen content and maintenance of APD suggests, therefore, that glycogen conservation might have a protective effect against the development of arrhythmias in myocardial infarction. The present study also indicates one possible approach to glycogen conservation, through a lowering of plasma FFA levels. Acknowledgements

We are most grateful for the helpful Newsholme and Dr C. T. Jones.

advice

of Professor

L. H. Opie,

Dr E. A.

REFERENCES 1.

2. 3. 4. 5. 6. 7.

8. 9.

10. 11. 12.

APSTEIN, C. S., DECKELBAUM, L., MUELLER, M., HAGOPIAN, L. & HOOD, W. B. Graded global ischaemia and reperfusion. Cardiac function and lactate metabolism. Circulation 55, 864-872 (1977). APSTEIN, C. S., GMEINER, R. & BRACHFELD, N. Effects of palmitate on hypoxic myocardial metabolism and contractility. Recent Advances in Studies on Cardiac Structure and Metabolism 1, 136-146 (1972). BAND, D. M., FRY, C. H. & TREASURE, T. An ion selective electrode for the determination of calcium activity. Journal of Physiology 276, 1-2P (1978). BERGMEYER, H. U. Methods of Enzymatic Analysis, 2nd edit. New York: Academic Press (1974). BORBOLA, J., PAPP, G. & SZEKERES, L. Octanoat Hat&a szivizompraeparatumok intracellularisan regisztrllt actios potenciitiara. Kiserlites Oruostudomany 26, 32-39 (1974). BRICKNELL, 0. L. & OPIE, L. H. Effects of substrates on tissue metabolic changes in the isolated rat heart during underperfusion and on release of lactate dehydrogenase and arrhythmias during reperfusion. Circulation Research 43, 102-I 15 ( 1978). COWAN, J. C. & VAUGHAN WILLIAMS, E. M. The effects of palmitate on intracellular potentials recorded from Langendorff-perfused guinea-pig hearts in normoxia and hypoxia, and during perfusion at reduced rate of flow. Journal of Molecular and Cellulur Cardiology 9, 327-342 (1977). DAWES, G. S., MOTT, J. C. & SHELLEY, M. J. The importance of cardiac glycogen for the maintenance of life in foetal lambs and new-born animals during anoxia. 3ournal of PhysioloQ 146, 5 16-538 ( 1959). ENTMAN, M. L., BORNET, E. P., VAN WINKLE, W. B., GOLDSTEIN, M. A. & SCHWARTZ. A. Association of glycogenolysis with cardiac sarcoplasmic reticulum II. Effect of glycogen depletion, deoxycholate solubilization and cardiac ischaemia. Evidence for a phosphorylase kinase membrane complex. Journal of Molecular and Cellular Cardiology 9, 515-528 (1977). ENTMAN, M. L., KANIKE, K., GOLDSTEIN, M. A., NELSON, T. E., BORNET, E. P., FUTCH, T. W. & SCHWARTZ, A. Association of glycogenolysis with cardiac sarcoplasmic reticulum. Journal of Biological Chemistry 251, 3140-3146 (1976). GARLAND, P. B., RANDLE, P. J. & NEWSHOLME, E. A. Citrate as an intermediary in the inhibition of phosphofructokinase in rat heart muscle by fatty acids, ketone bodies, pyruvate, diabetes and starvation. Nature 200, 169-170 (1963). GROOT, P. M. E., SCHOLTE, H. R. & HULSMANN, W. C. Fatty acid activation: specificity, localization and functions. Advances in Lipid Research 14, 75-126 (1976).

M.C.C.

P

368 13. 14. 15.

16.

17.

18. 19. 20.

21.

22.

23. 24.

25.

26.

27.

28.

29.

30.

J.

C.

COWAN

AND

E.

M.

VAUGHAN

WILLIAMS

HEARSE, D. J. Reperfusion of the ischaemic myocardium. Journal of Molecular and Cellular Cardiology 9,605-6 16 ( 1977). HEWITT, R., LOLLEY, D., ANDROUNY, G. & DRAPANASI, T. Protective effect of myocardial glycogen on cardiac function during anoxia. Surgery 73, 444-453 (1973). HYDE, A., CHENEVAL, J. P., BLONDEL, B. & GIRARDIER, L. Electrophysiological correlates of energy metabolism in cultured rat heart cells. Journal de Physiologie 64, 269-292 (1972). IYENGAR, S. R. K., CHARRETTE, E. J. P., IYENGAR, C. K. S. & WASAN, S. Myocardial glycogen in prevention of perioperative ischaemic injury of the heart: A preliminary report. Canadian Journal of Surgery 19, 246-251 (1976). KJEKSHUS, J. K. & MJ~s, 0. D. Effect of inhibition of lipolysis on infarct size after experimental coronary artery occlusion. Journal of Clinical Investigation 52, 1770-1778 (1973). KOSTIS, J. B. & HORSTMANN, E. FFA and ventricular fibrillation threshold. Lancet i, 961-962 (1972). KURIEN, V. A., YATES, P. A. & OLIVER, M. F. Free fatty acids, heparin and arrhythmias during experimental myocardial infarction. Lancet ii, 185-187 (1969). L~~DERITZ, B., NAUMANN D’ALNONCOURT, C. & STEINBECK, G. Effects of free fatty acids on electrophysiological properties of ventricular myocardium. Klinische Wochenschrzft 54, 309-313 (1976). MCDONALD, T. F. & MACLEOD, D. P. Anoxia-recovery cycle in ventricular muscle: action potential duration, contractility and ATP content. PJiigers Archiv. European Journal of Physiology 325, 305-322 (197 1). MACLEOD, D. P. & PRASAD, K. Influence of glucose on the transmembrane action potential of papillary muscle. Effects of concentration, phlorizin and insulin, nonmetabolizable sugars and stimulators of glycolysis. Journal of General Physiology 53, 792-815 (1969). MERRICK, A. W. & MEYER, D. K. Glycogen fractions of cardiac muscle in normal and anoxic heart. American Journal of Physiology 177,44 l-443 (1954). MILLER, N. E., MJ~s, 0. D. & OLIVER, M. F. Relationship of epicardial ST-segment elevation to the plasma free fatty acid/albumin ratio during coronary occlusion in dogs. Clinical Science and Molecular Medicine 51,209-2 13 ( 1976). MJQS, 0. D., MILLER, N. E., RIEMERSMA, R. A. & OLIVER, M. F. Effects of pchlorophenoxyisobutyrate on myocardial free fatty acid extraction, ventricular blood flow, and epicardial ST-segment elevation during coronary occlusion in dogs. Circulation 53, 494-500 ( 1976). MOST, A. S., CAPONE, R. J. & MASTROFRANCESCO, P. A. Free fatty acids and arrhythmias following acute coronary occlusion in pigs. Cardiovascular Research 10, 198-205 (1977). NEELY, J. R., DENTON, R. M., ENGLAND, P. & RANDLE, P. J. Effects of increased heart work on the tricarboxylate cycle and its interactions with glycolysis in perfused rat heart. Biochemical Journal 128, 147-159 ( 1972). NEELY, J. R., WHITMER, J. T. & ROVETTO, M. J. Effect of coronary blood flow on glycolytic flux and intracellular pH in isolated rat hearts. Circulation Research 37, 733740 (1975). OLIVER, M. F., KURIEN, V. A. & GREENWOOD, T. W. Relation between serum-free fatty-acids and arrhythmias and death after acute myocardial infarction. Lancet i, 710-715 (1968). OPIE, L. H. Substrate utilisation and glycolysis in the heart. Cardiology 56, 2-21 (1971/72).

FATTY

31. 32. 33.

34. 35.

36.

37.

38.

39. 40. 41.

42. 43.

44.

45. 46. 47.

48.

49. 50.

ACIDS

AND

ISCHAEMIC

ACTION

POTENTIAL

369

SHORTENING

OPIE, L. H. Metabolism of free fatty acids, glucose and catecholamines in acute myocardial infarction. American Journal of Cardiology 36, 938-953 (1975). OPIE, L. H. & LUBBE, W. F. Are free fatty acids arrhythmogenic? Journal of Molecular and Cellular CardioloQ 7, 155-l 59 ( 1975). OPIE, L. H., NORRIS, R. H., THOMAS, M., HOLLAND, A. J., OWEN, P. & VAN NOORDEN, S. Failure of high concentrations of circulating free fatty acids to provoke arrhythmias in experimental myocardial infarction. Lancet i, 818-822 (1971). ORAM, J. F., BENNETCH, S. L. & NEELY, J. R. Regulation of fatty acid utilization in isolated perfused rat hearts. Journal qf Biological Chemistry 248, 5299-5309 (1973). RANDLE, P. J., NEWSHOLME, E. A. 8: GARLAND, P. B. Regulation of glucose uptake by muscle. 8. Effects of fatty acids, ketone bodies and pyruvate, and of alloxandiabetes and starvation on the uptake and metabolic fate of glucose in rat heart and diaphragm muscles. Biochemical Journal 93, 652-665 ( 1964). RAVENS, K. G. & RAVENS, U. The role of linoleic acid in hypoxia-induced changes of the action potentials and the force of contraction of isolated papillary muscles of the guineapig. Recent Advances in Studies on Cardiac Structure and Metabolism 7, 289-295 (1976). REINAUER, H. & MULLER-RUCHHOLTZ, E. R. Regulation of the pyruvate dehydrogenase activity in the isolated perfused heart of guinea-pigs. Biochimica et biofihysica acta 444, 33-42 (1976). ROWE, M. J., NEILSON, ,J. M. M. & OLIVER, M. F. Control of ventricular arrhythmias during myocardial infarction by antilipolytic treatment using a nicotinic acid analogue. Lancet i, 295-300 (1975). RUSSELL, J. & BLOOM, W. Extractable and residual glycogen in tissues of the rat. American Journal of Physiology 183, 345-355 ( 1955). SCHEUER, J. & STEZOSKI, S. W. Protective role of increased myocardial glycogen stores in cardiac anoxia in the rat. Circulation Research 27, 835-849 (1970). SHIPP, J. C., OPIE, L. H. & CHALLONER, D. Fatty acid and glucose metabolism in the perfused heart. Nature 189, 1018-1019 (1961). SHUG, A. L. & SHRAGO, E. A proposed mechanism for fatty acid effects on energy metabolism of the heart. Journal of Laboratory and Clinical Medicine 81, 214-Z 18 (1973). SMITH, E. R. & DUCE, B. R. Anti-arrhythmic and serum free fatty acid lowering effects of 5-fluoro-nicotinyl alcohol following experimentally induced myocardial infarction in the dog. Cardiovascular Research 8, 550-561 (1974). SMITHEN, C., CHRISTODOULOU, J. & BRACHFELD, N. Protective role of increased myocardial glycogen stores induced by propranolol. Recent Advances in Studies on Cardiac Structure and Metabolism 8, 141-152 (1975). TRAUTWEIN, W., GOTTSTEIN, U. & DUDEL, J. Der aktionsstrom der myokardfaser im sauerstoffmangel. PJliigers Archiv 260, 40-60 (1954). VAUGHAN WILLIAMS, E. M. Classification of antidysrhythmic drugs. Pharmacolog>a and Therapeutics (B) 1, 115-138 (1975). WASILEWSKA-DZIUBINSKA, E., CZARNECKA, M., BERESEWICZ, A. & LEWARTOWSKI, B. Influence of sodium palmitate on the cellular action potentials of the left ventricle of isolated perfused guinea-pig heart. Acta Physiologica Polonica 26, l-l 1 (1975). WOLLENBERGER, A., KRAUSE, E. G. & WAHLER, B. E. Uber den tatsachlichen orthophosphatund N-phosphorylkreatingehalt des Saugetierherzens. Pjtigers Archiv 270, 413-427 (1960). WOLLENBERGER, A., RISTAU, 0. & SCHOFPA, G. Eine einfache technik der extrem schnellen abkulung grosserer gewebestucke. PJltigers Archiv 270. 399-4 12 ( 1960). WOODBURY, J. W. & BRADY, A. J. Intracellular recording from moving tissues with a flexibly mounted ultramicroelectrode. Science 123, 100-101 (1956). P2