Lipid peroxidation in exercise myopathy

EXPERIMENTAL AND MOLECULAR PATHOLOGY 38, 380-388 (1983)

Lipid Peroxidation in Exercise Myopathy A. SALMINEN AND V. VIHKO

Division of Muscle Research, Department of Cell Biology, University of Jyviiskyki, SF-40100 Jyviishylii 10. Finland

Received October 6, 1982, and in revised form December 14, 1982

Selected estimates of lipid peroxidation were analyzed in mouse quadriceps femoris muscle immediately after submaximal prolonged (9 hr) and exhaustive maximal running (2-3 hr), and at intervals l-10 days afterward during the exercise-induced myopathy. Immediately after the two types of exertion no significant changes were observed in the concentrations of lipid peroxidation products (thiobarbituric acid (TBA) reactive substances, and lipofuscin) or in the estimates of autoxidation (spontaneous and Fe*+-induced autoxidations) and antioxidant (catalase, glutathione peroxidase, and vitamin E) capacities. The enzymatic estimate of exercise myopathy (B-glucuronidase) increased considerably (2-6 days) after both types of exertion. Simultaneously, the lipid peroxidation rate of muscle homogenates in vitro increased markedly and in highly significant correlation with the activity of B- glucuronidase. The concentrations of TBA reactants and lipofuscin as well as Fe*+-induced lipid peroxidation were not affected during exercise myopathy. The activities of catalase and glutathione peroxidase increased significantly after both exertions, while the concentration of vitamin E was unchanged. Exhaustive running of endurance-trained mice caused only slight signs of myopathy and no increase in the rate of lipid peroxidation in vitro.

INTRODUCTION

Heavy exercise may induce acute necrotic myopathy in skeletal muscles (see Reneman, 1968; Hecht ef al., 1975; Vihko et al., 1978a). Exercise myopathy, e.g., in the muscles of inexpansible compartments, is considered as an ischemic com- pression syndrome caused by fluid accumulation in muscular tissue after strenuous exercise (Getzen and Cat-r, 1967; Reneman, 1968). Focal necrosis and inflammation occur on the second postexercise day (Vihko et al., 1978a). The activities of lysosomal acid hydrolases increase considerably in myopathic skeletal muscles (Vihko et al., 1978b; Salminen and Vihko, 1980). This increase is due to invasion of inflammatory phagocytes and especially to increase of lysosomal enzymes in surviving skeletal muscle fibers (Vihko et al., 1978a). The increase of lysosomal enzyme activities establishes a good biochemical estimate of cell injuries in necrotic myopathies (Shannon et al., 1974; Vihko et al., 1978a; Meijer and Israel, 1979).

Lipid peroxidation induced by free radical reactions results in deteriorative changes in subcellular membranes, e.g., in association with hypo- or hyperox- ygenation syndromes, radiation, or several chemicals (see Del Maestro, 1980). The univalent reduction of oxygen by mitochondria produces superoxide anion and other free radicals of oxygen (Boveris, 1977; Del Maestro, 1980), which may initiate lipid peroxidation. During heavy prolonged exertion the energy for increased demand is produced by oxidative metabolism. Hence, the increased mitochondrial respiration could expose muscle fibers to oxidative injuries. Dillard et al. (1978) have shown that exercise increases the amount of expired pentane, a product of lipid peroxidation. Strenuous swimming increases the production of thiobarbituric acid-reactive substances in rat skeletal muscles (Brady et al., 1979;

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LIPID PEROXIDATION IN EXERCISE MYOPATHY 381

Wilhelm and Sonka, 1980), indicating an increase in lipid peroxidation. The present study was designed to evaluate the role of lipid peroxidation in the pathogenesis of exercise myopathy. Autoxidative properties in vitro and the levels of certain antioxidants and peroxidation products were analyzed.

METHODS

Experimental procedures. Lipid peroxidation in mouse skeletal muscle was studied after prolonged submaximal running and after exhaustive running. In the experiment involving prolonged running (experiment I), male NMRI mice, aged 6.5 months, were made to run for 9 hr on a motor-driven treadmill with 6” uphill tracks. The running speed was 13.5 m min-I. After 3 and 6 hr running, there was a lo- to 15min pause, during which the mice had free access to drinking water. The mice were killed by cervical dislocation either immediately after the exertion or 2, 4, and 6 days later.

In the exhaustive exercise (experiment II), male NMRI mice, aged 4.5 months, were first familiarized with the running situation at a speed of 18 m min-1 for 30 min. The speed was then increased to 25 m min- 1 for 10 min and to 28 m min- 1 for 5 min. Thereafter the speed was decreased to 18 m min-’ for 15 min. This program was repeated three times. Some mice were not able to follow the highest speed and were allowed to rest during these periods. After this intermittent phase the mice were exhausted by gradually increasing the speed from 18 to 28 m min-l. This extended running lasted from 30 min to 2 hr until the mice were unable to continue. The mice were killed either immediately after the exhaustion or 1, 2, 5, and 10 days later.

In the third experiment (experiment III), prior endurance-trained NMRI mice, aged 4.5 months, were exhausted to determine whether the previously reported training-induced resistance to exercise myopathy (Vihko et al., 1979) could also be detected as a change in lipid peroxidative properties. Endurance training con- sisted of running on the motor-driven treadmill for 1 h day-’ over 3 weeks (5 days week- I). During the first week the speed was increased to 25 m min- *. Thereafter, the speed was maintained at 25 min-1 for the second week and increased to 28 m min- 1 for the third week. The next day after this program, the mice were exhausted as the untrained mice except that the lowest speed was 25 m min-l and the speed increment was to 31 m min-1 for 10 min. After three repetitions, the mice were exhausted by gradually increasing the speed of the treadmill. The overall duration of the program was approximately 3 hr. The exhausted endurance-trained mice lived for 3 days under normal cage conditions before being killed.

Muscle samples. The skeletal muscle samples were detached from the quadriceps femoris muscle. The red part of quadriceps muscle was used in the experiments because it is more susceptible to exercise myopathy than the white part (Vihko et al., 1978b; Salminen and Vihko, 1980). The sample was composed of the red parts of the proximal heads of the vastus lateralis, vastus medialis, and rectus femoris muscles and the red fibers of vastus intermedius. The samples in experiments I and III were prepared as quickly as possible after killing of the mice. The samples were weighed, frozen in liquid nitrogen, and stored at - 80°C until being analyzed within 2-3 weeks. In experiment II, the hindlegs of the mice were cut off and frozen immediately after the kilhng. For the assays, the muscle

382 SALMINEN AND VIHKO

samples were prepared in the cold after thawing in an ice bath. The samples for the fluorometric measurements were prepared from the right leg and those for the other measurements from the left leg. The sample for thiobarbituric acid reactants in experiment II was the proximal part of the vastus medialis muscle.

Assay methods. The muscle samples for the fluorometric measurements were homogenized in ice-cold 0.25 M sodium phosphate buffer, pH 8.0, using an all- glass Potter-Elvehjem homogenizer. Homogenates were made to 4.0% (w/v). The lipofuscin concentration was analyzed as described by Tappel (1975). Vitamin E concentration was assayed according to the method of Taylor et al. (1976), eli- minating vitamin A interference with H,SO,.

The muscle sample for the other measurements was homogenized (3%, w/v) in ice-cold 0.1 M potassium phosphate buffer, pH 7.4, using an all-glass Potter- Elvehjem homogenizer. The rate of lipid peroxidation in vitro was estimated by the method of Placer et al. (1966). The homogenates were incubated for 30 min in 0.2 M Tris-maleate buffer, pH 5.9, at 37°C. Spontaneous autoxidation was measured as described by McMurray and Dormandy (1974) without any stimulation. Sodium azide was used to inhibit the enzymatic breakdown of lipid peroxides (McMurray and Dormandy, 1974). Thiobarbituric acid (TBA)-reactive substances produced by spontaneous autoxidation in 18 hr were assayed according to the method of Kombrust and Mavis (1980) with minor modifications. Fez+- activated lipid peroxidation was measured as described by Kombrust and Mavis (1980), using a stimulation medium consisting of FeSO, (60 CLM), sodium ascor- bate (1 .O mM), and EDTA (50 PM). The content of peroxidizable lipids was estimated by measuring the TBA-reactive substances produced in 1 hr by the Fe2+-stimulation of muscle homogenates in an ice-bath (Kornbrust and Mavis, 1980). The sensitivity of muscle homogenates to lipid peroxidation was estimated by the ratio 8 mm/60 min of the peroxidation products.

In experiment II, the TBA-reactive substances in skeletal muscle were assayed by the method of Uchiyama and Mihara (1978). Muscle samples were homogenized in a mixture containing 1.5 ml 1 .O% H3P04 and 0.5 ml 0.8% TBA. The TBA value was calculated from the difference in absorbance at 535 and 520 nm.

The catalase activity of homogenates was assayed and calculated as described by Leighton ef ~1. (1968). Muscle homogenates were incubated at 27°C for 7 min. The activity of glutathione peroxidase was analyzed by slightly modifying the method of Paglia and Valentine (1967). Muscle homogenates were stored at - 18°C until being analyzed within 3 days. Before the assay, the homogenates were made 0.5% with respect to Triton X-100 and centrifuged for 10 min at 6OOg. The supematant was used in the glutathione peroxidase assay. The activity of p-glucuronidase and the protein concentrations were assayed as described earlier (Vihko et al., 1978b).

Statistical methods. Standard procedures were used to calculate means and standard errors. The significance of the differences between the means was tested by Student’s t test. The correlation between the lipid peroxidation rate and the activity of B-glucuronidase was calculated by the least square method.

RESULTS

Lipid peroxidation estimates immediately after strenuous exertion. Immedi- ately after the exertions, no statistically significant changes were observed in


TABLE I Effects of Prolonged Running (9 hr) on Lipid Peroxidation Estimates in Mouse Skeletal Muscle

Time after exercise (days)

Control (n = 14) (n =” 13) (n =’ 13) (n =” 14) (n =” 14)

Rroxidation in muscle Lipofuscin (fluorescence) 89 -c 15 87 k 13 80-c 11 88 5 22 94 f 16

Peroxidation in vitro Peroxidation rate 1462 I1 136 & 15 254 2 23*** 286 -t 32*** 273 k 25*** Spontaneous autoxidation 530 e 17 488 2 19 518 ? 18 553 2 18 556 + 18 Fez+-activated autoxi-

dation Total 2680 2 80 2650 f 100 2520 k 90 2500 e 90 2650 k 70 Sensitivity 11.2 f 1.6 11.1 -c 1.5 11.2 * 1.7 9.8 ~fr 1.6 11.9 + 2.0

Antioxidants Catalase 1.42 f 0.06 1.33 + 0.05 1.55 + 0.06 1.83 f 0.07*** 1.90 f O.lO*** Glutathione peroxidase 290 f 10 332 k 14* 346 f 13** 356 f 19** 377 k 21** Vitamin E 8.49 f 0.46 8.28 + 0.62 7.59 k 0.37 8.31 r 0.45 7.55 k 0.36

Lysosomal system S-Glucuronidase 0.16 k 0.01 0.16 * 0.01 0.49 f 0.07*** 0.75 + O.ll*** 0.63 f 0.07***

Protein concentration 171 + 4 172 e 4 162 k 5 162 t 4 168 r 5

Note. Lipotuscin concentration is given as fluorescence units kg-r muscle. One fluorescence unit is equivalent to the fluorescence produced by 1 pg quinine sulfate/ml 0.1 N H2S04 in the assay system. Vitamin E concentration is given as mg a-tocopherol kg-1 muscle and protein concentration as g protein kg-r muscle. Lipid peroxidations in vitro are given as follows: peroxidation rate as pmol MDA kg1 during a 30-min incubation, spontaneous autoxidation (with azide) as pmol MDA kg-r muscle produced during 18 hr incubation, total Fe’+-activated autoxidation as umol MDA kg-l muscle produced in 1 hr, and sensitivity as percentage of total peroxidative lipids autoxidated during the first 8 min of incubation. Enzyme activities are as follows: S-glucuronidase as pmol reaction products set-r kg-r muscle, ghttathione peroxidase as umol oxidized NADPH set-r kg-r supematant protein, and catalase as arbitrary units g-r muscle (see Materials and Methods). Values are means f SE.

* P < 0.05. ** P < 0.01.

*** P i 0.001.

lipid peroxidation products or in the estimates of autoxidation and antioxidant capacities (Tables I and II).

Lipid peroxidation estimates during exercise myopathy. The development and the degree of exercise myopathy were estimated by assaying the activity of p- glucuronidase (Vihko et al., 1978a, b). @Glucuronidase activity increased highly significantly after both types of exertion (Tables I and II). The content of lipofuscin was unaffected in skeletal muscle during exercise myopathy, as was the concentration of TBA reactants in experiment II (Tables I and II).

The rate of lipid peroxidation in vitro was already increased on the first day after the exhaustive exercise (Table II). The highest rates were observed 2-6 days after both types of exertion. A highly significant correlation was observed between the rate of lipid peroxidation and the activity of l%glucuronidase, both in the prolonged running (r = 0.737, n = 71) and the exhaustive running (r = 0.625, n = 81) experiments. The prolonged exercise caused no changes in the capacity of spontaneous autoxidation (Table I). The concentration of lipids susceptible to Fe2+-induced lipid peroxidation and the sensitivity to Fez+-stimulated lipid peroxidation were unaffected during exercise myopathy. The capacity for spontaneous autoxidation slightly decreased after the exhaustive exercise (Table II). Simultaneously, the protein concentration also slightly decreased, although it was unchanged after the prolonged exercise.

The activities of catalase and glutathione peroxidase increased in the skeletal

TABL

E II

Effe

cts

of E

xhau

stiv

e Ru

nnin

g (2

-3

hr)

on L

ipid

Pe

roxid

atio

n Es

timat

es

in M

ouse

Sk

elet

al

Mus

cle

Varia

ble

Con

trol

(n

= 15

) (n

”

18)

Tim

e af

ter

exer

cise

(day

s)

I 2

5 10

02

=

14)

(n

= 14

) (n

=

II)

(n

= 9)

Pero

xidat

ion

in m

uscl

e TB

A re

acta

nts

Lipo

fusc

in

(fluo

resc

ence

)

Pero

xidat

ion

in v

itro

Pero

xidat

ion

rate

Sp

onta

neou

s au

toxid

atio

n Fe

’+-a

ctiv

ated

au

toxid

atio

n To

tal

(60

min

)

Antio

xidan

ts

Cat

alas

e G

luta

thio

ne

pero

xidas

e Vi

tam

in

E

Lyso

som

al

syst

em

B-G

lucu

roni

dase

Prot

ein

conc

entra

tion

22.4

t

1.6

94 k

8

26.8

k

2.0

116

_’ 1

4 20

.8

2 1.

6 10

6 h

17

18.8

2

1.2

21.6

2

1.6

83 _

’ 8

76 2

8

20.0

r

2.3

90 t

14

154

r 9

450

k 11

14

0 2

I1

431

f 18

19

5 *

15*

408

-c I

S*

265

-c 1

6***

23

8 1-

9**

* 37

2 -c

- 14*

**

443

2 27

16

2 2

7 40

2 t

26

2960

i

130

3100

-c

60

3060

k

70

3080

-c

80

2950

”

IO

2980

i

90

1.30

2

0.04

29

2 2

IO

7.93

2

0.37

1.35

5

0.05

32

1 f

12

8.38

f

0.47

1.12

”

0.03

**

332

” 12

* 8.

09 2

0.

42

1.43

k

0.06

40

8 k

23**

* 7.

32

-c 0

.23

1.78

+

O.lO

***

377

t 23

*+

7.61

-c

0.2

6

1.56

2

0.08

**

315

k 17

***

8.16

*

0.50

0.16

k

0.01

182

‘- 2

0.16

f

0.01

181

? 3

0.22

-t

0.01

***

168

t 3*

**

0.63

t

0.07

***

0.58

t

0.06

***

170

t 3*

* 17

3 k

3*

0.36

r

0.02

***

180

-t 3

Not

e.

Thio

barb

ituric

ac

id r

eacta

nts

are

expr

esse

d as

km

ol

MDA

kg

-l m

uscl

e.

Oth

er

lege

nds

are

as

in T

able

1

* P

< 0.

05.

** P

<

0.01

. **

* P

< 0.

001.


muscle during exercise myopathy (Tables I and II). After the exertion, the activity of glutathione peroxidase increased more rapidly than that of catalase, being already significantly increased on the second day. The concentration of vitamin E was unchanged.

Lipid peroxidation estimates after exertion in endurance-trained mice. Endur- ance training induced a protection against exercise myopathy as estimated by the activity of @glucuronidase (Table III), as also was observed in an earlier study (Vihko et al., 1979). The endurance training in itself slightly increased the activity of P-glucuronidase and decreased the rate of lipid peroxidation in muscle homogenates. The rate of lipid peroxidation was unaffected by the exhaustive running in the endurance-trained mice (Table III).

DISCUSSION

Several observations have indicated that intensive activity of skeletal muscles induces lipid peroxidation (Blokha et al., 1972; Dillard et al., 1978; Brady et aZ., 1979; Wilhelm and Sonka, 1980). Blokha et al. (1972) showed that electrical stimulation of isolated muscles considerably increases the concentration of free radicals and peroxides in the contracting muscles. Brady et al. (1979) and Wilhelm and Sonka (1980) reported the accumulation of thiobarbituric acid-reactive substances, most probably malondialdehyde, in rat skeletal muscles subsequent to exhaustive swimming. In our study, the highest value of TBA reactants in mouse skeletal muscle was also observed immediately after the exhaustive running. However, the increase was slight and statistically insignificant. The content of lipofuscin, a fluorescent product of lipid peroxidation, was not increased in mice after exhaustive running or in rats after exhaustive swimming (Wilhelm and Sonka, 1980). Similarly the antioxidant variables and the autoxidation properties of muscle homogenates were unchanged immediately after both exertions.

The myopathic phase after strenuous exercise is first observed on the second postexercise day (Hecht et al., 1975; Vihko et al., 1978a). Histochemical studies have shown focal necrosis and intlammation as well as an increase in acid hydrolase activities in surviving muscle fibers (Vihko et al., 1978a). The regenerating muscle fibers appear on the seventh postexercise day. The changes in p-glucuronidase activity during the myopathic period after both types of exertion were similar in this study to those observed in earlier experiments (Vihko et al., 1978b,

TABLE III Lipid Peroxidation Rate and P-Glucuronidase Activity in Skeletal Muscles of Untrained and

Endurance-Trained Mice on the Third Day after Exhaustive Exercise

Untrained Endurance trained

Control Exhausted Control Exhausted (n = 15) (n = 13) (n = 1.5) (n = 13)

Lipid peroxidation rate

B-Glucuronidase 201 f 8 288 + 28* 15.5 + 80 134 + 8

0.16 f 0.01 0.75 f 0.14** 0.21 + 0.010 0.26 f O.Ol**

Note. Other legends are as in Tables I and II. * P < 0.01 (effect of exhaustion).

** P < 0.001 (effect of exhaustion). 8 P < 0.001 (effect of endurance training).


1979). The estimates of lipid peroxidation in viva, TBA reactants, and lipofuscin, were unaffected during exercise myopathy. Kar and Pearson (1979) have observed that the concentration of TBA-reactive products is considerably higher in the muscles of patients with muscular dystrophies than in control subjects. Thus, the injurious processes are probably different in exercise and hereditary myopathies.

The rate of lipid peroxidation in vitro was markedly increased in muscle homogenates for 2-6 days after both types of exertion. The increase in lipid peroxidation occurred simultaneously with that in @glucuronidase activity. These results suggest that either the membranes of myopathic muscles are more susceptible to lipid peroxidation or that inflammatory cells may enhance the rate of lipid peroxidation in muscle samples. Superoxide production by activated polymorphonuclear leukocytes is well documented (McCord and Fridovich, 1978). On exposure to appropriate stimuli, neutrophils release superoxide anion by the ac- tion of NADPH oxidase. Hence, the generation of superoxide radicals by the enzymes of neutrophils could give rise to lipid peroxidation in muscle homogenates. This suggestion is in accordance with the observation that lipid peroxidation in vitro is enhanced in exhausted control mice but not in exhausted endurance-trained mice, because fiber necrosis and inflammation are not observed in trained mice after exhaustive exercise (Vihko ef al., 1979). On the other hand, the increase in lipid peroxidation could be an enzymatic process, because the Fe2+-activated peroxidation, carried out in an ice bath, was not affected during exercise myopathy. The myeloperoxidase of polymorphonuclear leukocytes may also participate in the lipid peroxidation of myopathic muscles in vitro because this peroxidase is inhibited by azide (Klebanoff and Rosen, 1979) and we could find no increase in the rate of azide-inhibited spontaneous autoxidation. However, the sensitivity of muscle fibers to lipid peroxidation could also be increased in exercise myopathy. By comparison, the formation of lipoperoxides by hepatic microsomes in vitro is markedly increased in the prenecrotic stages of dietary vitamin E and selenium deficiencies (Porta et al., 1977).

The activities of catalase and glutathione peroxidase, both enzymatic antioxidants, but not the concentration of vitamin E, a nonenzymatic antioxidant, increased during exercise myopathy. Stauber et al. (1977) have proposed that the activity of catalase may be a valid indicator of the degree of muscle wasting. Chloroquine administration and starvation were especially effective in increasing the activity of catalase in muscles (Stauber et al., 1977). In this study, the increase in the catalase activity of skeletal muscle was not observed until the fourth day after exertion, whereas the increase in glutathione peroxidase activity occurred at an early stage of the myopathy. Noncorrelated changes in the activities of these hydrogen peroxide scavengers have also been observed in several tissues of rats receiving a dilute aqueous solution of hydrogen peroxide (Matkovics and Novak, 1977).

Our results do not support the hypothesis that lipid peroxidation could be the cause of exercise myopathy in mice. It must, however, be emphasized that the concentrations of malondialdehyde and lipofuscin may be unreliable indicators of peroxidative damages in vivo. Demopoulos et al. (1980) preferred the assaying of polyunsaturated fatty acids, ascorbic acid, and cholesterol in order to verify free radical reactions in cerebral ischemia and acute spinal cord trauma. On the other hand, skeletal muscle fibers strongly increase their oxygen consumption during


strenuous exercise and may therefore have an unusually effective defence against mitochondrial hydrogen peroxide production. Such an effective defence might also explain the resistance of muscle tissues to ionizing radiation (Wilhelm and Sonka, 1980).

ACKNOWLEDGMENTS This study was supported by the Academy of Finland and the Research Council for Physical

Education and Sport (Ministry of Education, Finland). We thank Mrs. Irene Helkala and Mr. Matti Virtanen for skillful technical assistance.

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Lipid peroxidation in exercise myopathy