Mitochondrial oxidative phosphorylation in low-flow hypoxia: Role of free fatty acids

3ownal of Molecuiar andCellular Cardiology (1978) 10,857-875

Mitochondrial Oxidative Phosphorylation in Low-flow Hypoxia: Role of Free Fatty Acids AMANDA

LOCHNER,

JOHANNES AND

C. N. KOTZ&,

WIELAND

AMBROSE

J. S. BENADE

GEVERS

M.R.C. Molecular and Cellular Cardiology Unit, Dehrtment of Medical Bi&emistry, University of Stellenbosch Medical Schvol, M.R.C. National Ruenrch Institute for Nutritional Diseases, Tygerberg 7505 Republic of South Africa (Received 8 August 1977, accepted in revisedform 25 JVovember 1977) A. L~CHNER, Phosphorylation

J. C. N. KOTZI?, A. J. S. BENADE AND W. GEVERS. Mitochondrial Oxidative in Low-flow Hypoxia: Role of Free Fatty Acids. 3oumal of Molecular and Cellular Cardiology (1978) 10, 857-875. The mechanism of mitochondrial damage was investigated in hypoxic hearts, perfused at low pressure without exogenous substrate, as a model of myocardial ischaemia. Mitochondrial and tissue free fatty acid (FFA) contents were determined in control hearts (perfused aerobically at a higher pressure, without exogenous substrate), and in the hypoxic hearts; the functional capacities of mitochondria isolated from the two types of tissue were also compared. Mitochondrial FFA contents were found to be elevated, relative to the controls, after 20 min of low-flow hypoxic perfusion. However, mitochondrial FFA contents were not different in control and hypoxic hearts after 70 min of perfusion. Low-flow hypoxic perfusion for 70 min causeda significant elevation of tissue C16:O. C18:O and C18: 1 FFA fractions. while total FFAand trinlvceride contents were also increased. Accumulation of FFA in whole tissue was accompanied by a depression in mitochondrial function. Thus, after 20 min, both tissue FFA contents and ADP/O and RCI values of mitochondria isolated from control and hypoxic hearts were not different, whereas after 70 min, tissue FFA levels were significantly elevated in hypoxic hearts, with an equally significant depression in the function of isolated mitochondria. KEY

WORDS:

chondrial FFA Mitochondrial phosphorylation;

Mitochondria; Ischaemia; Hypoxia; Isolated perfused rat hearts; Mitocontents; Tissue FFA content; Tissue triglyceride content; Acyl CoA; oxygen uptake; Respiratory control index; Uncoupling of oxidative Albumin.

1. Introduction Myocardial ischaemia produced by coronary artery ligation in the dog or baboon [S, 25, 3fJ and anoxia in isolated rat hearts [29, 301 are characterized by a significant depression in the function of mitochondria isolated from the affected heart muscle. The precise mechanismsinvolved are not yet understood in detail but have been reviewed [29, 301. Beneficial effects of albumin addition to such defective mitochondria [29] raise the possibility that accumulation of free fatty acids might be responsible for the observed effects: it is well-known that albumin is an avid binder of long-chain FFA [19, 51, 521, while these compounds have a profound effect on mitochondrial oxidative phosphorylation in vitro [3,24,29,40]. 002%2828/78/090857+

19 $02.00/O

0

1978 Academic

Press Inc.

(London)

Limited

858

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ET AL.

To ascertain whether long-chain FFA might be responsible for damage to mitochondrial membranes in the above-mentioned conditions, the FFA and triglyceride contents of isolated rat hearts, perfused at low pressures and oxygen tensions, were compared with those of control hearts perfused at normal pressures in the presence of oxygen. The FFA contents of mitochondria isolated from such tissues were also measured. This convenient small animal model for global “ischaemia” was chosen for detailed study, because the properties of mitochondria prepared from such hearts are similar in very many respects to those of particles isolated from ischaemic muscle areas after coronary artery ligation in dogs and baboons [29,3ll. Severe depletion of ATP, creatine phosphate and glycogen as well as intracellular accumulation of lactate is characteristic of these hypoxic hearts [de Kock, A. and Lochner, A., to be published elsewhere]. For convenience, this system is called low-flow hypoxia. 2. Materials

and Methods Animals

Female albino The rats were 30 min before mitochondrial mentation.

rats, weight 150 to 250 g, were used in all perfusion experiments. anaesthetized by intraperitoneal injection of Nembutal (30 mg/kg) removal of the hearts. (Decapitation was found to increase total free fatty acid contents.) Animals were fed ad libitum before experi-

Techniques Production of ischaemic tissue After removal of the rat hearts, they were placed in ice-cold saline and perfused in a modified recirculating Langendorif perfusion system for 70 min, using 30 ml Krebs-Ringer bicarbonate medium [29, 301. Both control and low-flow hypoxic hearts were perfused without exogenous substrate. Control hearts were perfused at a perfusion pressure of 80 mmHg, with 95% 0s : 5% CO2 as gas phase (mean coronary flow rate 8.5 f 0.6 ml/min). L ow-flow hypoxic hearts were obtained by perfusion at a suboptimal pressure of 40 mmHg, the medium being gassed with 95% Nz : 5% COs. The mean coronary flow rate was 5.4 & 0.7 ml/mm under these conditions, and the aortic POS was 90 mmHg, compared with 420 mmHg in control perfusions. Determination of mitochondrial FFA contents After perfusion, the hearts were plunged into ice-cold mitochondrial isolation medium (either KE : 0.18 M KCI, 10 mM EDTA; pH adjusted to 7.4 with Tris base

OXIDATIVJS

PHOSPHORYLATION

AND

FFA

IN

HYPOXIA

859

at PC, or KEA : 0.18 M KCl, 10 mu EDTA, 0.5% bovine serum albumin (Miles, Cape Town), adjusted to pH 7.4 with Tris base at 4”C), and mitochondria were isolated as previously described [29,30]. Mitochondrial pellets were suspended in isolation medium at a final concentration of 6 to 10 mg protein/ml. The protein concentrations were measured by the method of Lowry et al. [32]. Immediately after preparation of mitochondria, FFA were extracted according to the method of Dole and Meinertz [I4 and analyzed gas chromatographically as described by Hagenfeldt [ZO] . For this, a Beckman model GC4 gas chromatograph with flame ionization detectors was used, together with a 1 mV potentiometric recorder (Beckman Instruments Inc., Fullerton, California). Glass U-columns, 2000 mm long and with 3 mm internal diameter, were packed with 20% (by weight) DEGS on 80 to 100 mesh Chromosorb WHMDS. The following conditions were employed : oven temperature 170”, flow rate of carrier gas (nitrogen) 30 ml x min-1, and flow rate of hydrogen 60 ml x min-1. Heptadecanoic acid (Applied Science Laboratories, Inc.) was used as an internal standard. Peak areas were measured by triangulation and compared with the peak area of the internal standard. In some experiments the total mitochondrial FFA contents were determined by the Duncombe method [12]. Tissue contents of mutral gbcerides and FFA After perfusion, the ventricles were freeze-clamped with Wohenberger tongs prechilled in liquid nitrogen, and were then plunged into liquid nitrogen. Care was taken not to include the atria and visible epicardial fat. Lipids were extracted by homogenizing a portion of the frozen tissue (Jr 0.3 g) with 20 vols of the Dole extraction mixture [II]. After separation of the phase containing neutral lipid, the FFA in the other phase were converted to methylesters by diazomethane at 0°C and analyzed by gas chromatography, as described above in the case of mitochondrial FFA. The lipid-containing heptane layer was evaporated to dryness in a water bath at 70°C under a stream of nitrogen. The lipid residue was finally dissolved in 100 4 chloroform and the component triglycerides, diglycerides, and monoglycerides separated by one dimensional, two-step thin-layer chromatography on silica gel (Kieselgel H, Merck). In the first step of development of the chromatograms, isopropyl ether-acetic acid (96 : 4, v/v) was used, and in the second step, light petroleum-ethyl-acetic acid (90 : 10 : 1, v/v). Lipids were located by exposure to a 2’,7’-dichlorofluoroescein spray (0.2% in ethanol). The lipid zones were removed from the plates by suction and extracted with methanol. Spots containing triglycerides from one heart were extracted twice with 4 ml methanol, while spots containing di- and monoglycerides from two to four animals were combined and extracted twice with 4 ml of methanol. Mono-, di- and triglycerides were then assayed as glycerol after saponification. A suitable volume (8 ml) of the lipid-containing methanol solution was evaporated to dryness at 70°C in a water-

860

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LOCHNER

ETAL.

bath. The glycerides were saponified for 30 min at 70°C in a waterbath by addition of 0.5 ml ethanolic KOH (0.5 N). After addition of 1 ml 0.15 M MgSO4, the samples were centrifuged and 0.2 ml of the clear supernatant usedfor the glycerol determination (Biochemica Test Combination for glycerol, Boehringer Mannheim GmbH). Measurement of mitochondrialfutution Mitochondrial oxidative phosphorylation was determined polarographically as described by Sordahl et al. [49, 501 using L-glutamate (5 mM) as substrate. The following indices were determined: ADP/O ratio (nmol ATP produced/natom oxygen uptake) ; mitochondrial oxygen uptake (natom/mg protein/mm) ; respiratory control index (ratio of oxygen consumed in the presenceof ADP to that after phosphorylation of ADP) .

Statisticalevaluation All results were expressed as means f S.&M. (number of observations). P values were calculated from Student’s t-test, using standard procedures for paired or unpaired comparisons.

3. Results

Mitochondrialfree fatty acid contentsin perfued rat hearts In the study of mitochondrial FFA contents, care was taken to eliminate possible contamination of the particles with cytoplasmic FFA. Since repeated washing of mitochondrial pellets with KE had no effect on mitochondrial FFA contents, such a source of error appeared to be largely absent. Mitochondrial contents of individual as well as total FFA were similar after 20 and 70 min of control perfusion, with the exception of two species, Cl 8:2 and C20 ~4, which were significantly increased after 70 min of perfusion (Table 1). Twenty min after the beginning of a low-flow hypoxic perfusion, a significant increase in all mitochondrial FFA &actions was observed (ranging from 733% in the caseof C14:O to 50% in that of C 16:I), when a comparison was made with mitochondria prepared at this time after control perfusions. The total mitochondrial FFA content was increased by 194% (P
Pl

time

(6)

(4)

12 :o

acid

0

0

14:l

N.S.

< 0.001

< 0.001

1.15

4.10

f

0.25 N.S.

f0.02

12.8

Content 16:0

*

1.9

21.0

- -

perfusion

f 3.4

after

< 0.005

N.S.

=I= 1.24

9.87

f 0.79

9.1

0.05 < P < 0.1

--__-~

contents

5.0 0.67 f 0.72 f 0.35

0.08 -&to.05 < 0.05

0.58 & 0.21

27.5

& 1.4 < 0.001

1.5

3.3

1.70

f

14:o

free fatty

&O.lO < 0.001

0.30 & 0.20

I. Mitochondrial

N.S.

N.S.

f 0.19

1.70

2.5 f 0.7

N.S.

3.3 f 1.2 11.1

f1.4

6.6

species 18:O

rat hearts

0.49

7.0

f

12.6 1.0

18:2

f

fl.O

0.91

7.30

6.8

17.63

19.8 1.9

< El01

& 1.27

f

35.8 f 1.6 < 0.005 < 0.001 17.2 & 1.1

rt1.5

8.6

18:l

< pd:;os <~:siol

f

7.3 f0.6

f 0.7 < 0.05

acid

2.2 + 0.40

of fatty 16:l

of isolated

0

0

0

0

18:3

Results expressed as nmol/mg mitochondrial protein. Mitochondrial isolation medium: KE. Numbers in parenthesis indicate number of samples. Each mitochondrial sample represents one or two hearts. Pl indicates significance of difference between control and low-flow hypoxic hearts after 20 min perfusion. P2 indicates significance of difference between control and low-flow hypoxic hearts after 70 min perfusion. P3 indicates significance of difference between low-flew hypoxic hearts, perfused for 20 and 70 min respectively.

70 min Control

20 min Control

Perfusion

TABLE

N.S.

N.S.

4.15 ho.18

4.5 f 0.64

4.5 & 0.2 < 0.001

0.6 & 0.5

20 :4

N.S.

4.13

54.57 < 0.001

f

58.97 & 6.04

143.49 f 8.63 < 0.001

48.87 f 5.89

Total fatty acid content

a62

A. LOCHNER

FFA contents of mitochondria,

ET AL.

isolated in the presence of albumin, low-Jaw hypoxia

after 70 min of

Hearts were perfused for 70 min under low-flow hypoxic conditions and subsequently homogenized either in an albumin-containing medium (KEA) or in KE. The mitochondrial pellets were then thoroughly washed with KE for at least 3 times to remove asmuch albumin as possibleand the mitochondrial FFA contents were determined (Table 2). Despite the thorough washing procedure, the pellets of albumin-treated mitochondria still retained a significant amount of FFA. This increase in FFA contents might be attributed to FFA bound to albumin still adhering to the mitochondrial pellet. The source of this additional FFA is not solely the FFA pre-bound to the albumin preparation used (Table 3), but predominantly tissue FFA newly bound to albumin during the homogenization procedure. Taking into account the FFA contents (individual aswell as total) of tissue samplesperfused hypoxically for 70 min (Table 5), as well as the FFA contents of the albumin-containing isolation medium (15 ml/g tissue), a rough estimation could be made of the amounts of FFA which would be bound to albumin in a homogeneoussuspensionsuch as a homogenate. When the concentration of each individual FFA specieswas calculated as a percentage of the calculated total FFA content of the homogenate, the values obtained closely resembled those obtained in the albumin-treated mitochondrial pellet. For this reason it can be concluded that the increased FFA content of albumin-treated particles arose from albumin contamination, containing bound FFA. Furthermore, analysis of the FFA content of KEA before and after homogenization of tissue, showed that albumin was indeed capable of removing large amounts of FFA from tissue (Table 3). The post-mitochondrial supernatants of tissuehomogenized in KEA, were significantly enriched with respect to C16:0, 16: 1, 18:0, 18:1, 18:2 and 20 :4 species.Since this increase in post-mitochondrial supernatant FFA represents both cytoplasmic and mitochondrial FFA, an experiment was performed in which mitochondria were isolated in KE, followed by washing with KEA. An increase in post-mitochondrial FFA contents was obtained, indicating that albumin can also bind mitochondrial FFA. .Neutral glyceride and FFA contents of perfusedrat hearts

Gas chromatographic analysis of tissue FFA speciesappearing immediately preceding Cl6 :O and Cl8 :O, showed dimethylacetals as reported by Harris and Gloster [18]. Perfusion of isolated rat hearts under control conditions (adequate oxygen and coronary flow) was associatedwith a progressive increase in all the FFA fractions measured (Table 4). After 20 min of perfusion, C12:0, C16: 1 and C18:2 speciesas well as total tissue FFA contents, were significantly increased. A significant fall in tissue triglyceride contents occurred concurrently, without

4.10 1.15 15.24 & 2.31 < 0.001

f

14:o

medium

f

0.25 0.02 0

14:l

of FFA

16:O

N.S.

acid 16:l

1.70 0.19 8.08 f2.31 < 0.02 f

Fatty

of mitochondria

9.87 & 1.24 13.04 & 1.35

content

7.0 -&0.49 9.46 f 0.74 < 0.02

content 18:O

prepared

3. FFA

* Post-mitochondrial supematant. KEA: KC1 (0.18 M), EDTA (10 mu), Albumin Results are expressed as nmol/ml mitochondrial

21.38 2.25

f

8.48 0.81

(4)

14:o

and after

20.15

12:o

before

5.6

f

of KEA

(2)

content

0

0

14:l

f

acid

13.95 5.0

1.06

f

0.05

after

wet wt.

10.68 1.20

2.0

18:0 -

18:I

70 min

23.38 & 5.9

4.3

18:l

washed

f

I8:2

22.68 5.5

3.9

18:2

3 times

0

0

4.15 0.18 5.66 f 0.57 < 0.05 f

20:4

f

---

3.3 0.19

0

20:4

KE to remove

0

0

18:3

18:3

with

hypoxia

17.63 1.27 43.95 5 3.66 < 0.001 f

low-flow

< P < 0.1

7.30 rf; 0.91 9.81 & 0.81

thoroughly analysis.

tissue.

used/g

content 16:l

of heart

(0.5%). 15 ml KEA isolation medium.

29.18 & 5.97

6.15

Fatty 16:0

homogenization

* These hearts were homogenized in KEA and the mitochondrial pellets albumin. The pellets were Anally suspended in KE for gas chromatographic Results are expressed as nmol/mg mitochondrial protein. P values indicate significance of difference between means.

Before homogenization After* homogenization

ABLE

(8)

KEA*

P

0.08 0.05 2.84 & 0.69 < 0.005

(6)

KE

f

12:o

medium

Isolation

ofisolation

2. Effect

TABLE

f

142.65 26.76

52.50

Total

all traces

54.57 & 4.13 119.60 & 12.24 < 0.001

Total

of

864

A.

LOCHNER

El

AL.

changes in the diglyceride and monoglyceride contents. This caused a decrease in the calculated total FA content (P
E$ccts of low-jlow hyfloxia on neutral glyceride and FFA contents of @&.red rat hearts After 20 min of perfkion, no differences were observed between the different neutral glycerides and FFA contents of control and low-flow hypoxic rat hearts (Table 5). However, after 70 min of low-flow hypoxic perfusion, tissue Cl6 :0, C 18 :0 and C 18 : 1 fractions, as well as the total FFA and triglyceride contents were significantly elevated. The calculated total tissue FA content was significantly (P
Effect of @erfm*on on o&dative ~ho@wrylation

in isolated mitochondria

To determine whether any correlation existed between the FFA contents of hearts and the functional status of mitochondria isolated from them, oxidative phosphorylation was studied at two time intervals viz. 20 min and 70 min, in the case both of control and low-flow hypoxic perfusions. Aerobic perfusion without substrate for a period of 20 min yielded mitochondria which were significantly depressed with respect to RCI and QOs values, when compared with particles prepared from (unperfused hearts (Table 6). Prolongation of the perfusion period to 70 min had little further effects on the QOs and RCI values. Comparison of mitochondria prepared from control and low-flow hypoxic hearts, both perfused for 20 min, showed no differences between mitochondrial ADP/O and RCI values, while the oxygen uptake rates of mitochondria isolated from low-flow hypoxic tissue were significantly depressed. Low-flow hypoxic perfusion for 70 min was associated with a significant depression of all parameters of mitochondrial function studied. Effects of acyl-CoA on mitochondrial function: role of albumin in incubation medium The results obtained in Tables 1, 5 and 6 suggest a possible inverse correlation between tissue FFA contents and mitochondrial function in vitro, whereas the level of mitochondrial FFA did not seem to correlate with the characteristic depression in mitochondrial function caused by low-flow hypoxia. Increased tissue acyl-CoA

P2

70min

75.65 f 31.76 < 0.05

22.46 f 9.30 314.59 f 91.63 < 0.02

12:o

N.S.

298.48 f 67.88

N.S.

164.54 f 6.77 213.99 f 42.29

14:O

fat and FFA

0

0

0

14:l

contents

16:l

N.S.

1081.37 f 92.52 1222.08 & 75.35

N.S.

f

883.31 125.15 < 0.005

N.S.

304.01 14.96 360.95 & 36.68

f

acid content 18:O 18:l

1509.42 -& 155.01

Fatty

rat hearts*

f432.17 0 f 29.47 517.90 51.28 f 30.41 f 20.51 0.05 < 0.05 < P< 0.1 999.60 131.35 f 116.66 f 59.47 <0.m5 N.S.

16:O

of perfused

3438.66 f 436.05
939.64 f 74.53 1758.23 f 277.73 < 0.02

18:2

0

0

0

18:3

1444.03 & 177.84 f < 0.02

N.S.

1037.49 f 80.81 917.86 & 47.51

20:4

N.S.

7457.51 1083.89

3978.77 f 225.94 5357.42 f 373.16 < 0.02

Total FFA measured

* Perfusion pressure 80 mmHg: gas phase 95% Os,5% COs. Results expressed as nmol/g dry wt. Pl indicates significance of difference between unperfused hearts and hearts perfused for 20 min. P2 indicates significance of difference between hearts perfused for 20 and 70 min. Total tissue FA content was calculated from FFA, triglyceride, diglyceride and monoglyceride contents.

8

8

2Omin

Pl

5

No. of hearts

Perfusion time

Omin

4. Neutral

TABLE

f

N.S.

7054.29 1028.19

11994.26 + 320.42 7953.32 f 1647.39 < 0.05

Triglyceride

478.79 f 67.65

309.31

128.56

135.38

292.52 245.56

Monoglyceride

Diglyceride

< 0.01

f2040

31018

40685 f~l 29836 f 4309 < 0.05

Total FA content

hearts

8

l1

2omin Control

Low-flow hypoxia

12:o

N.S.

261.30 72.12

N.S.

168.25 &- 79.20

75.65 f. 31.76

f

N.S.

347.76 f 51.82

298.48 f 67.88

N.S.

270.66 f 65.04

213.99 & 42.29

14:o

0

0

0

0

14:l

hypoxia

f

131.35 f 59.47

N.S.

48.64 & 19.53

51.28 f 20.51

Fatty acid 16:l

1509.42 155.01

N.S.

1071.08 124.77

1222.08 f 75.35

2061.80 + 191.93 < 0.05

f

f

f

f

contents

contents 18:0

fat and FFA

1678.87 304.39 139.63 & 86.41 < 0.005 N.5.

999.60 A 116.66

N.S.

555.81 + 41.72

517.90 & 30.41

16:O

on the neutral

f

f

1299.75 122.0 < 0.05

883.31 125.15

N.S.

387.12 30.28

360.95 36.68

18:l

ofperfiicd

N.S.

4160.45 & 426.84

3438.66 f 436.05

N.S.

1409.28 & 147.30

1758.23 j, 277.73

18:2

1444.03 177.64

N.S.

1814.68 h 143.84

f

N.S.

820.19 -j= 87.95

917.86 -j= 47.51

20:4

contents.

0

0

0

18:3

rat hearts

Control hearts: perfusion pressure 80 mmHg; gas phase 95% Oa,5% COz. Law-flow hypoxic hearts: perfusion pressure 40 mmHg; gas phase 95% Na, 5% COz. Results expressed as nmol/g dry wt. Pvalues indicates significance of diklkence between control and low-flow hypoxic hearts + Total FA content was calculated from FFA, triglyceride, diglyceride and monoglyceride

1o

Low-flow hypoxia

P

8

70 mill Control

P

NO. Of

Perfusion time

oflow-flow

314.59 & 91.63

5. Effects

TABLE

f

f

10685.88 963.50 < 0.05

7457.51 1083.89

N.S.

4967.46 & 457.95

5357.42 & 373.16

Total FFA measured

f

478.79 f 67.65

246.10

245.16

Diglyceride

11405.23 413.97 1676.66 & 48.38 < 0.05 N.S.

7054.29 -& 1028.17

N.S.

9010.39 f 576.04

7953.32 + 1647.39

Triglyceride

94.45

309.31

114.25

128.56

Monoglyceride

46898 *4093 < 0.005

31018 -I- 2040

N.S.

32523 + 1581

29836 f 4309

Total* FA content

OXIDATIVE

TABLE

PHOSPHORYLATION

AND

6. Effect of perfusion time on oxidative

Length of perfusion 0 min 20 min

IN

phosphorylation

ADP/O

(7)

FFA

in isolated mitochondria

QOz

RCI

3.13 & 0.11

867

HYPOXIA

10.77

5 1.34

71.66 f 4.15

4.95

& 0.39

51.02 & 3.98

Control

(8)

3.15

;;;;;”

(8)

3.20 f 0.06

4.68 -& 0.35

38.07 f 1.61

N.S.

N.S.

< 0.01

(10)

2.64 f 0.06

4.48 & 0.34

51.96 * 7.83

(29)

2.17 f 0.09

1.99

17.14 & 0.76

P 70 min Control

g;z;w P

f

0.06

< 0.001

Numbers in parentheses indicate number P values indicate significance of difference Substrate: Glutamate 5 mM. Polarographic : Rate of oxygen

uptake

in presence

0.13

< 0.001

of samples. Each sample from control. experiment.

QOs: natom oxygen uptake/mg mitochondrial RCI

*

4 0.001 represents

two pooled

hearts.

protein/min.

of ADP/rate

of oxygen

uptake

in absence of ADP.

levels have been demonstrated in ischaemia [35, 471, and the possibility exists that increased tissue FFA concentrations affect mitochondrial function by increasing the intra-mitochondrial content of acyl-CoA. Since the presence of albumin is known to reverse the effects of added FFA on mitochondria [3, 291, and also to improve the function of mitochondria prepared from anoxic tissue [29] it was necessaryto test whether albumin would reverse acyl-CoA-mediated inhibition of mitochondrial oxidative phosphorylation in vitro. Both palmitoyl-CoA and oleoylCoA significantly depressedthe QO2 and RCI of mitochondria isolated from unperfused rat hearts (Figure 1). This depressionwas dependent both on dosageand on the mitochondrial protein contents. Addition of albumin (2 mg/ml) to the incubation medium resulted in a marked improvement at both concentrations of acyl CoA studied: RCI and QO2 values were raised to control levels in the presence of 40 PM palmitoyl-CoA, and significant improvement was also seen at 80 @ palmitoyl-CoA. In a similar manner, albumin improved mitochondrial function in the presenceof oleoyl-CoA. 4. Discussion

To elucidate the mechanisms responsible for mitochondrial damage in low-flow hypoxia, we have attempted to show a relationship between the function of isolated particles in vitro and their FFA contents. Precautions were taken to avoid contamination of isolated mitochondria with cytoplasmic or microsomal FFA. To avoid

868

A.

LOCHNER

El-U.

(b)

(0)

40

80

40

80

FM palmitayi-CaA FIGURE 1. Effects of pahnitoyl-CoA and albumin (a) QOa; (b) RCI. (- - - -) + albumin; ( -) 480 nmol. Mitochondrial protein: 4 mg. Palmitoyl-CoA mitochondrial isolation medium.

on mitochondrial oxidative phosphorylation. albumin. Substrate L-Glutamate, 5 mu. ADP: and albumin (2 mg/ml) was added to the

phospholipid breakdown, mitochondrial pellets were extracted immediately after preparation. Pellets kept for 24 h at 2 to 4°C showed very little increase in FFA content, indicating low continuing phospholipase activity. Homogenization of tissue with an albumin-containing medium resulted in a significant increase in mitochondrial FFA contents (Table 2). However, mitochondria isolated from hypoxic tissue in this manner always exhibited improved function [29]. It would appear that the increased mitochondrial FFA content of albumin-treated mitochondria was due to FFA bound to contaminating albumin present in the pellet, such FFA not being capable of exerting effects on mitochondrial function. The binding capacity of albumin for FFA makes it unlikely that albumin will facilitate FFA exchange between the cell fractions during the isolation procedures, as occurred in the studies of Fields et al. [15j. FFA contents of mitochondria, when isolated in KE, showed no correlation with mitochondrial function. For example, mitochondrial FFA contents were elevated after low-flow hypoxic perfusion, at a time (20 min) when the ADP/O ratios and respiratory control indices of mitochondria isolated from control and low-flow hypoxic tissue were similar. Conversely, the function of mitochondria isolated from low-flow hypoxic tissue was severely depressed at a time when no change was

OXIDATIVE

PHOSPHORYLATION

AND

FFA

IN

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observed in mitochondrial FFA contents (70 min) (Table 1). These results are in contrast with the results reported by Boime et al. [2], with reference to mitochondria from ischaemic liver tissue. Liver mitochondrial FFA content showed a progressive increase between 0 and 180 min of ischaemia at 38°C correlating with a reduction of function. The total FFA content of liver mitochondria (2 nmol/mg mitochondrial protein) reported by Boime and co-workers is much less than that found by us in heart mitochondria (25 nmol/mg mitochondrial protein). Possible explanations for this discrepancy might have been that these authors [2] used liver tissue kept for different periods in a moist atmosphere at 38”, while we studied isolated rat hearts subjected to anoxic, low-flow perfusion. Furthermore, different methods for fatty acid extraction were used: Boime et al. [2] used a modification of the Bligh and Dyer method [A and subsequently separated lipid classes by thin-layer chromatography, whereas in our work the FFA-containing heptane phase (Dole extraction) was used directly for FFA analysis. Our values for the FFA content of heart muscle mitochondria are, however, in agreement with those reported by Gloster and Harris [ 183. Since mitochondrial FFA levels showed no correlation with mitochondrial function in low-flow hypoxia, it was decided to study tissue FFA contents. Values obtained in control unperfused rat hearts were similar to those reported by Olson and Hoeschen [363, but much lower than those reported for rat hearts in vivo [27, 441. Tissue FFA contents appeared to be quite well correlated with changes in mitochondrial function (Tables 4 and 6). Analysis of FFA content during prolonged perfusion of control hearts showed a progressive increase of all fractions with time (except Cl2 :0), the most significant changes occurring in Cl6 :O, C 18 : 1, C 18 :2, which have been shown to be detrimental to mitochondrial function [3]. This increased tissue FFA content may be the cause of the depression in mitochondrial function occurring during prolonged control perfusions without exogenous substrate (Table 6). Increased tissue FFA levels probably resulted from triglyceride breakdown, since fat levels were significantly depressed at 70 min of control perfusions. Reduction, but not exhaustion, of neutral glyceride levels of control hearts, perfused without exogenous substrate, has also been shown by several other co-workers [IO, 17, 34, 3s]. The possibility also exists that membrane damage, occurring during prolonged perfusion, might be associated with phospholipid breakdown causing or compounding the accumulation of FFA in the cytoplasm. Hypoxic, low-flow perfusion of isolated rat hearts for 70 min increased both tissue triglyceride and FFA levels (Table 5), causing an overall increase in tissue fatty acid contents. Crass and Pieper [a also reported that hypoxic perfusion of the isolated heart resulted in a small increase in tissue W-FFA, with inhibition of ‘%-triglyceride fatty acid oxidation. In ischaemic hearts, inhibition of fatty acid oxidation appears to develop at the level of p-oxidation, as shown by accumulation of high levels of long-chain acyl CoA and acyl carnitine derivatives, associated with

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a large decrease in the levels of both acetyl CoA and acetyl carnitine [35j. Hearts perfused with media containing exogenous FFA, show increased esterification in hypoxia or ischaemia [14, 451, providing an explanation for the lipid droplets seen histologically in oxygen-deficient muscle [4, 531. In the present study, hearts perfused without exogenous substrate showed triglyceride levels similar to those of unperfused hearts (Tables 4 and 5). This increase in triglyceride levels occurred despite the fact that there could have been an increase in hormone-sensitive lipase activity; this has been demonstrated in isolated rat hearts, perfused at low pressure [23]. No reason for the net accumulation of triglycerides can yet be given. The possibility exists that accumulation of glycerol-3-phosphate [3O, 37, 5s] arising from glycogen breakdown [.%‘I, may promote the re-esterification of hydrolyzed triglyceride esters. Wood et al. [56J have demonstrated the potentiating effect of glycerol-3-phosphate on myocardial lipid biosynthesis in oxygen-deficient cardiac muscle. Despite re-esterScation of hydrolyzed triglyceride esters and increased triglyceride levels, accumulation of tissue FFA was found to occur in low-flow hypoxic hearts in the present study (Table 5). This could be due wholly or partly to phospholipid breakdown. Ekholm [13] reported a significant reduction in myocardial cardiolipin and lysolecithin contents after 20 min of tissue ischaemia in dogs. However, phospholipid changes have not yet been studied in the particular model used in the present study. Another possibility which has to be considered is breakdown of muscle proteins during prolonged low-flow hypoxic perfusion without exogenous substrate, with endogenous lipogenesis from the carbon skeletons generated from this source. However, Rannals et al. [4.?] showed protein breakdown to be inhibited in ischaemia. In the study of mitochondrial function of isolated perfused hearts, it was noted that aerobic substrate-free perfusion resulted within 20 min in a significant depression of mitochondrial QOs and RCI values, when compared with those obtained when the tissue was processed immediately. Prolongation of the perfusion period to 70 min had little further effect on these parameters (Table 6). Dhalla et al. [9] showed that aerobic perfusion without exogenous substrates had profound effects on myocardial function and metabolism, concomitant with marked mitochondrial ultrastructure changes. Kako [26’j also found that prolonged aerobic perfusion (>60 min) without substrates resulted in a significant depression of mitochondrial QOs and RCI values. According to Dhalla [9], absence ofexogenous substrates might be responsible for the changes observed, but addition of glucose (10 mM) to the perfusates of aerobic control hearts had no effect in our hands on various parameters of mitochondrial function measured [30]. Another possibility to be considered is the significant increase in tissue FFA which occurred in control hearts within 20 min of perfusion (Table 4). Tissue FFA levels in low-flow hypoxic perfused hearts correlated with the depression obtained in mitochondrial function (Tables 5 and 6). Tissue FFA

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contents were similar in control and hypoxic hearts perfused for 20 min; mitochondria isolated from these hearts had similar ADP/O and RCI values, with a slight depression in oxygen uptake. Seventy min of low-flow hypoxic perfusion caused an increase of all FFA fractions, especially C 16 :0, C 18 :0 and C 18 : 1, corresponding with the significant depression in mitochondrial function. The effects of FFA on mitochondrial function are well-established: FFA uncouple electron transport from oxidative phosphorylation, decrease P/O ratios, inhibit ATP-PI exchange, stimulate latent ATPase [3, 24, 551 and inhibit mitochondrial oxidation [40]. It is also ofinterest that mitochondriaisolated from brown adipose tissue are usually uncoupled or loosely coupled [I6j with inhibition of respiration [5] and adenine nucleotide transport [S]. Recoupling can occur with albumin [41] and appears to involve removal or transfer of a small proportion of FFA bound to the mitochondria [22]. Further evidence in favour of FFA toxicity is the finding that albumin is capable of binding significant amounts of FFA present in tissue homogenates, as well as in mitochondrial pellets (Table 3) bringing about a significant improvement in function [29]. The finding that mitochondrial FFA levels are unaltered in mitochondria with depressed function (Table 1) raises the question as to whether FFA per se are responsible for the changes observed. Increased tissue FFA levels could cause an increase in mitochondrial acyl-CoA content which, in its turn, could seriously affect mitochondrial function. It is well-established that long-chain acyl CoAesters inhibit adenine nucleotide translocation [46, 471, and also depress mitochondrial oxygen uptake and oxidative phosphorylation [21, 391. Furthermore, increased tissue acyl-CoA contents in ischaemia have been found by Shug and Shrago [47] as well as by Neely [35]. Since approximately 90% of the total tissue CoA is located in the mitochondrial matrix [3&j, it can be assumed that a significant portion of the tissue acyl-CoA in ischaemic tissue is actually present in the mitochondria. It is evident that mitochondrial acyl-CoA contents in low-flow hypoxia need to be determined. The finding that albumin can also, to a very large extent, reverse the deleterious effect of palmitoyl-CoA and oleoyl-CoA on mitochondrial oxidative phosphorylation (Figure 1) is additional evidence that mitochondrial acyl-CoA accumulation could also be responsible for the depression in mitochondrial function in low-flow hypoxia. At this stage, however, toxic effects of FFA on mitochondrial function in low-flow hypoxia cannot be ruled out. Although Shug and Shrago [47] reported that addition of r.,-carnitine to the incubation medium of mitochondria isolated from ischaemic dog hearts, caused partial restoration of oxygen uptake, we found that carnitine was without effect on mitochondrial function when compared with the improvement obtained by albumin [29]. Thus, the observation that albumin was able to reverse the deleterious effects of both FFA [29] and acyl CoA (Figure 1) on mitochondria, while carnitine reversed acyl-CoA effects only, could suggest that tissue FFA and acyl CoA species both might be responsible for the depression in mito-

872

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ETAL.

chondrial function observed in the particular model used in this study. FFA and acyl CoA also appear to have similar effects on several other mitochondrial functions e.g. (i) it has been shown that high concentrations of FFA and their acyl CoA derivatives have an inhibitory effect on phosphate transport in mitochondria [Is] ; (ii) recent studies by Wojtczak [Sl/l revealed that both FFA and acyl CoA stimulate the mitochondrial efflux of K+ in a medium of low concentration of this cation as well as increase the energy-dependent uptake in K+-supplement media. With regard to the protective effect of albumin on mitochondria isolated from low-flow hypoxic tissue, it also has to be considered that albumin might bind other lipids e.g. lysolecithin [43]. Albumin is also capable of reversing mitochondrial swelling associated with fatty acid oxidation [28, 331. The significance of these factors in the production of defective mitochondrial oxidative phosphorylation in low-flow hypoxia remains to be determined. In a sense it is disappointing that the functional defects of mitochondria in ischaemic heart muscle cannot be ascribed in a simple manner to an accumulation of FFA in the membrane system. On the other hand, the importance of mitochondrial dysfunction in the pathophysiology of ischaemia is very evident and demands detailed elucidation of all contributing primary and secondary factors and processes. Acknowledgements The South African Medical Research thanked for financial support.

Council and the Harry

Crossley Fund are

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