Eur J Appl Physiol (1981) 47:57-64 European Journal o f

Applied Physiology and Occupational Physiology �9 Springer-Verlag 1981

Acid Hydrolase Activities in Mouse Cardiac and Skeletal Muscle Following Exhaustive Exercise*

A. Salminen and V. Vihko

Division of Muscle Research, Department of Cell Biology, University of Jyv~iskylfi, SF-40100 Jyvfiskylfi 10, Finland

Summary, Acid hydrolase activities in skeletal and cardiac muscle were studied 5, 10, and 20 days after exhaustive intermittent running by untrained and endurance-trained mice. Exhaustion increased the activities of cathepsin D, fl-glucuronidase and ribonuclease, but not that of p-nitrophenylphos- phatase in skeletal muscle of untrained mice. Activities were highest on the fifth day after exhaustion and decreased during the following two weeks. More intensive loading produced no changes in acid hydrolytic capacity in skeletal muscle of endurance-trained mice. Acid hydrolase activities in cardiac muscle of both untrained and trained mice were unaffected by exhaustive running. It is suggested that exhaustive running causes both lethal and sublethal hypoxic fiber injuries in the skeletal muscle of untrained mice but not in that of endurance-trained mice or in the cardiac muscle of animals of either group. These injuries manifest themselves as fiber necrosis (lethal) and as increased acid hydrolytic capacity in surviving fibers (sublethal).

Key ,~ords: Exertion - Muscles - Myocardium - Lysosomes

Acid hydrolase activities in skeletal muscles increase in some muscular dystrophies (Kar and Pearson 1978) and in several kinds of atrophy, e.g., during denervation (Pollack and Bird 1968; Weinstock and Iodice 1969), tenotomy (Pollack and Bird 1968), starvation (Canonico and Bird 1970) and ageing (Pennington 1977). Acute and chronic hypoxic injuries (Shannon et al. 1974; Digiesi et al. 1975) also induce an increased activity of lysosomal enzymes in skeletal muscle. Ultrastructural studies have shown an increased number of lysosomes and autophagic vacuoles in skeletal muscle fibers in connection with such muscle deterioration. The lysosomal system of cardiac muscle is activated in some idiopathic, obstructive and toxic cardiomyopathies (Ravens and Gud-

* This study was financially supported by the Academy of Finland and the Finnish Research Council for Physical Education and Sport (Ministry of Education)

Offprint requests to: Dr. A. Salminen (address see above)

0301-5548/81/0047/0057/$ 01.60

58 A. Salminen and V. Vihko

bjarnason 1969; see Bajusz and Rona 1974) and also during starvation (Smith 1977) and ageing (Wildenthal et al. 1977).

In previous biochemical and histochemical studies (Vihko et al. 1978a, b) we have shown that physical stress, especially exhaustive exercise, increases the acid hydrolytic capacity of skeletal muscle. The activities of lysosomal hydrolases are highest during the 3-7 days following exhaustive running. The activation of the lysosomal system is most pronounced in red, oxidative skeletal muscle fibers (Vihko et al. 1978a).

The present investigation was designed to provide more insight into the activation of the lysosomal system in skeletal and cardiac muscles, in relation to preceding endurance training, during the recovery period following strenuous exhaustive exercise.

Material and Methods

Mate NMRI mice, aged 9-11 weeks at the beginning of the study, were housed in Scanbur (Denmark) type IV cages, with 7 - 1 2 animals to each cage. The mice had free access to solid food pellets (R3, Astra Ewos, Sweden) and water. The cage-day was artificially divided into a 12/12 h light/dark schedule. Temperature (21-22 ~ C) and humidity (40%) were kept constant.

Animals (n = 100) were initially divided randomly into control (n = 40) and training (n = 60) groups. Mice were trained on a motor-driven treadmill by being made to run on 6 ~ uphill tracks on 5 days a week. During the first exercises the running speed was increased and after one week the mice ran for 90 min daily at a speed of 25 m/min. After one month's training the animals were randomly divided into three groups (see Fig. 1): continued training (n = 20), detraining (n = 20), and exhausted (n = 20). Control mice were also divided into exhausted (n = 20) and non-exhausted control (n = 20) groups.

Exhaustion Experimental groups o r

termination 5days 10days 20days of training after after after

I I n=5 Exhausted I n=5

(rest) In=10 Trained Terminated In=5 mice training I - - - I n =5

(rest) In=10 (n=60) I n = 5

Continued In=5 training ]n=lO

I Exhausted I Cootrol (rest) Jn=10 n=5 mice

(n=40) Non - In=5 exhousted ln=10

Fig. 1. Schematic presentation of experimental design

In=5

In=5

Acid Hydrolase Activities and Exhaustion 59

An intermittent type of loading was used for exhausting the animals on the treadmill with horizontal tracks. Control mice were first familiarized with slow running (18 m/min). Running by both trained and control mice was motivated, when necessary, by administering light electric shocks from electrode grids located in the escape gate of each track. After 30 min the speed was increased to 25 and then to 28 and 31 m/rain at 5 min intervals. Thereafter the speed was decreased to 18 m/min for 10 min. This exercise program was repeated five times. Some control mice were not able to follow the highest speed, usually during the last two or three repetitions of the program. They were allowed to rest during these periods. Some control mice, on the other hand, performed the whole program easily. These animals were exhausted by extending running at the highest speed or even faster.

Exhaustion of the trained mice was commenced at a speed of 28 m/rain for 30 rain, with subsequent increments at 5 rain intervals to 31, 36, and 42 m/rain, on the treadmill with horizontal tracks. The track speed was thereafter lowered to 28 m/rain for 10 rain and the series of increments repeated five times. Unexhausted mice were made to run at 42 m/min or at an even higher speed (50 mJmin) until exhausted.

Exhausted control and trained mice lived for 5 (n = 10), 10 (n = 5), or 20 (n = 5) days under normal cage conditions before being killed (see Fig. 1). Detraining mice terminated training 5 (n = 10), 10 (n = 5) or 20 (n = 5) days before being killed. Non-exhausted control and trained animals were also assigned into the same time groups. The different time groups of controls and their corresponding trained groups were combined in the results to form one control and one trained group, because there were no statistically significant differences between the different control or trained groups. The mean weight (+ SE) of control mice was 41.7 _+ 0.4 g and that of trained mice 39.4 _+ 0.7 g. This difference is statistically significant (p < 0.01). The mean weights of mice in the exhausted control and exhausted trained groups did not differ significantly from the values for non-exhausted control and trained groups, respectively.

Mice were killed by cervical dislocation and the heart was rapidly removed and washed free of blood in the homogenization buffer. The rectus femoris muscle was removed from the right thigh. Muscle samples were rapidly weighed and deep-frozen ( -80 ~ C) until analyzed four months later. The hearts of controls weighed 159 + 2 mg and those of trained mice 173 _+ 3 rag, showing significant (p < 0.001) hypertrophy in trained mice. No other statistically significant changes were recorded in the weights of cardiac and skeletal muscles.

Muscle samples were minced with scissors and homogenized in icecold buffer (150 mM KCI, 50 mM KHCO3, 6 mM EDTA, pH 7.4) in an all-glass Potter-Elvehjem homogenizer (670 rpm). Homogenates were made to 3% (w/v) and contained 0.1% (w/v) Triton X-100.

The following acid hydrolase activities were assayed as estimates of the lysosomal capacity of the skeletal and heart muscles: cathepsin D (E.C.3.4.23.5), fl-glucuronidase (E.C.3.2.1.31), ribonu- clease (E.C.2.7.7.16), and p-nitrophenylphosphatase (E.C.3.1.3.2). Assays were performed essentially as described by Barrett (1972). Malate dehydrogenase activity (E.C.I.1.1.37) was used to estimate the oxidative capacity of muscles in different groups. Malate dehydrogenase activity and the protein content of muscle samples were determined as described earlier (Vihko et al. 1978b).

Results

T h e t r a i n i n g p r o g r a m u s e d s ign i f ican t ly i n c r e a s e d the ac t iv i ty of m a l a t e d e h y d r o g e n a s e in rec tus f e mo r i s mu s c l e , b u t h a d n o effect o n the ox ida t i ve capac i t y of ca rd i ac m u s c l e (Tab l e s 1, 2). T r a i n i n g also i n c r e a s e d the act iv i t ies of

f l - g l u c u r o n i d a s e (p < 0.001) a n d c a t h ep s i n D (p < 0.05) in ske l e t a l m u s c l e , b u t c a u s e d n o chang es in ca rd i ac musc le . T e r m i n a t i o n of t r a i n i n g did no t , h o w e v e r , cause s ign i f i can t ch an g es in ske le ta l a n d ca rd iac musc l e s as c o m p a r e d to t he

c o r r e s p o n d i n g musc l e s of t r a i n e d mice . T h e effects of e x h a u s t i o n in rec tus f emor i s a n d ca rd iac m usc l e s of c o n t r o l

a n d t r a i n e d mice a re g i v en in T a b l e s 1 a n d 2. T h e act ivi t ies o f c a t h e p s i n D , f l - g l u c u r o n i d a s e a n d r i b o n u c l e a s e were h i g h e r t h a n n o r m a l (p < 0 .001) in the

Tab

le 1

. E

ffec

t of

exh

aust

ion

on e

nzy

me

acti

viti

es a

nd

pro

tein

con

tent

in

rect

us f

emor

is m

usc

le o

f co

ntro

l an

d tr

aine

d m

ice.

Aci

d h

ydro

lase

act

ivit

ies

are

expr

esse

d as

pm

ol.

min

-1.

mg

-~ f

resh

mu

scle

and

mal

ate

deh

yd

rog

enas

e ac

tivi

ty a

s n

mo

l-ra

in -

1.

mg

-1 f

resh

mus

cle.

P

rote

in

con

ten

t is

giv

en a

s bt

g pr

otei

n/m

g fr

esh

mus

cle.

Val

ues

are

mea

ns

• S

E

a',

Var

iabl

e C

ontr

ol

Day

s af

ter

exh

aust

ion

T

rain

ed

Day

s af

ter

exh

aust

ion

5 10

20

5

10

20

(n =

20)

(n

=

10)

(n=

5)

(n=

5)

(n =

20)

(n

=

10)

(n =

5)

(n =

5)

Cat

heps

in D

21

2 •

8 26

0 •

9 a

240

• 16

24

0 •

23

240

+ 8 b

23

8 •

12

246

-4-_

19

224

+ 15

/3

-Glu

curo

nida

se

7.8

• 0.

1 19

.5 •

4.

1 ~

10.6

•

1.24

10

.7 •

0.

59

10.3

•

0.5

d 9.

8 •

0.3

9.1

• 0.

7 8.

5 +

0.4

Rib

onuc

leas

e 31

2 +

6 42

5 +

23 a

300

+ 12

32

7 •

11

302

_+ 9

28

0 •

9 29

7 •

18

311

_+ 1

3 p-

Nit

roph

enyl

- 1,

430

+ 30

1,

410

• 50

1,

480

• 50

1,

470

+ 50

1,

420

+ 30

1,

390

• 40

1,

460

• 40

1,

460

• 30

ph

osph

atas

e

Mal

ate

dehy

drog

enas

e 38

6 •

11

361

• 15

38

6 •

19

391

+ 14

43

9 +

13 ~

448

• 14

38

5 •

21

425

• 25

P

rote

in c

onte

nt

188

+_ 3

19

0 +

4 18

6 +

6 18

7 _+

3

187

• 3

190

+ 4

180

+_ 4

17

7 •

2

a p

< 0.

001

(con

trol

/exh

aust

ed g

roup

s)

b p

< 0.

05

(con

trol

/tra

ined

gr

oups

) c

p <

0.01

(c

ontr

ol/t

rain

ed

grou

ps)

a p

< 0.

001

(con

trol

/tra

ined

gr

oups

)

Tab

le 2

. E

ffec

t of

exh

aust

ion

on e

nzy

me

acti

viti

es a

nd p

rote

in c

on

ten

t in

the

car

diac

mus

cle

of c

ontr

ol

and

trai

ned

mic

e

Var

iabl

e C

ontr

ol

Day

s af

ter

exh

aust

ion

T

rain

ed

Day

s af

ter

exh

aust

ion

5 10

20

5

10

(n =

20)

(n =

10)

(n

= 5

) (n

= 5

) (n

=20)

(n

= 1

0)

(n =

5)

20

(n =

5)

Cat

heps

in D

46

4 _+

13

fi-G

lucu

roni

dase

16

.0 •

1.

1 R

ibon

ucle

ase

584

+ 17

p-

Nit

roph

enyl

- 1,

510

• 40

ph

osph

atas

e

Mal

ate

deh

yd

rog

enas

e 1,

100_

+ 3

0 P

rote

in c

onte

nt

167

+ 3

496

__+ 1

3 43

8 +

30

432

+ 23

49

6 +

8 53

2 +

18

470

__+ 3

8 50

8 +

35

17.2

•

0.7

18.0

+

0.8

17.4

+

1.5

17.7

__

0.7

17.9

•

0.9

18.7

+

1.0

19.8

•

2.0

626

+ 26

55

7 __

+ 25

632

+ 19

54

7 +

11

560

• 20

55

7 __

+ 21

569

__+ 2

0 1,

510

+ 50

1,

570

• 50

1,

610

+ 50

1,

510

+ 20

1,

540

• 40

1,

570

• 20

1,

560

• 50

1,05

0 +

40

1,11

0 •

80

1,11

0 +_

_ 50

1,15

0 +

10

1,17

0 •

30

1,10

0 •

80

1,07

0 •

50 a

170

+ 4

175

+ 4

167

+ 8

163

__+ 3

16

8 •

3 17

5 __

+ 8

170

+ 5

< .<

�9

Enz

yme

acti

viti

es,

prot

ein

cont

ent

and

oth

er

expl

anat

ions

are

as

in T

able

1

a p

< 0.

05

Acid Hydrolase Activities and Exhaustion 61

rectus femoris muscle of untrained mice on the fifth day after exhaustion. The activities of cathepsin D and ribonuclease were normal on the tenth day after exertion./3-Glucuronidase activity increased most markedly, and was still higher than that of controls three weeks after exhaustion (p < 0.001). The activities of p-nitrophenylphosphatase and malate dehydrogenase were not influenced by the exhaustive running.

In spite of the more intensive loading of the trained mice, no changes in the acid hydrolase activities of their skeletal muscle were detected. Similarly, no changes were observed in the acid hydrolase activities of the cardiac muscle of untrained or trained mice after exhaustive exercise.

Discussion

Strenuous loading produces focal fiber necrosis in exercised muscles (Hecht et al. 1975; Vihko et al. 1978a). Our previous observations (Vihko et al. 1979) showed that exhaustion necrotized some fibers in the rectus femoris muscle of untrained mice, but not in that of trained mice. In surviving fibers acid hydrolase activities (fi-glucuronidase, fl-N-acetylglucosaminidase) increased, especially in red fibers. No activity changes were observed in the skeletal muscle of trained exhausted mice. These histochemical findings are consistent with the biochem- ical results of the present study. A similar stimulation of the lysosomal system is found after experimental ischemia of surviving fibers (Shannon et al. 1974). The induction of acid hydrolase activities was selective both after exhaustion and ischemia./3-Glucuronidase activity was most sensitive to these treatments. The recruitment of fibers during running and the capacity of fibers to sustain exhaustive stress evidently determine the extent of fiber necrosis and that of the increase in lysosomal enzymes in surviving fibers.

Activation of the lysosomal system is an evidence of sublethal cell injury (Arstila et al. 1974), i.e., of disturbances in cell function. One possible reason for lethal and sublethal muscle fiber injuries during ischemia and exhaustive exercise is hypoxia, which disturbs energy production and thereby upsets the homeostasis of muscle fibers (Trump et al. 1976). The protective effect of endurance training against these lethal and sublethal injuries might be increased capacity for energy production (Holloszy and Booth 1976). This would augment the energy supply for mechanisms such as ion pumps. Some other protective mechanisms, such as those against lipid peroxidation, may also be acti- vated.

Endurance training increased slightly the activities of cathepsin D and /3-glucuronidase in skeletal muscle. The response, however, was much less than that observed after exhaustion of untrained mice, as reported earlier (Vihko et al. 1979). Endurance training induced response may be due to either sublethal injuries in muscle fibers or increase in the amount of interstitial structures, which are a rich source of acid hydrolases (Lojda and Gutmann 1976). In the beginning of training program the activities may increase in muscle fibers due to sublethal cell injuries caused by the first exercises of untrained animals. Later in the course

62 A. Salminen and V. Vihko

of training the increase may originate in increased interstitial structures, e.g., the increased capillary density, which is a typical training effect in skeletal muscles (Zika et al. 1973).

No changes in the activities of acid hydrolases in cardiac muscle were observed. Running to exhaustion produces no myocardial lesions in healthy rabbits, but striking necrotic changes occur in the myocardium of animals with experimental atherosclerosis (Kipshidze 1966). Myocardial lesions are also very prominent after swimming in cardiomyopathic guinea pigs, whereas no lesions are found in normal animals (see Bajusz and Rona 1974). Changes of acid hydrolase activities are associated with experimental myocardial infarction (Ravens and Gudbjarnason 1969). During a week after coronary occlusion the free acid hydrolase activities are at their highest whereas the total activities slowly increase and reach their highest values on the tenth day, being then four to ten times higher than the control activities. Strenuous exercise does not produce necrotic lesions or activation of the lysosomal system in the cardiac muscle of healthy mice.

The lysosomal system of skeletal muscle fibers is activated under many pathological conditions in which the catabolic rate is increased, e.g., during denervation (Goldspink 1976) and immobilization (Goldspink 1977). Neutral and alkaline proteases also participate in the catabolic processes in skeletal and cardiac muscles (Pennington 1977; Smith 1977). The role of the lysosomal system and its interaction with other degradative mechanisms in the metabolism of cells is still unknown. A dual-pathway hypothesis of intracellular protein degradation has recently received much attention (Ballard 1977). In addition to substrate-limited degradation, which is probably very important in the normal turnover of proteins, a system-limited degradation has been described, in which the autophagic capacity is the limiting factor of degradation. In skeletal muscle fibers some autophagosomes and lysosomes have also been found during increased protein breakdown, suggesting a mechanism of enhanced proteolysis (Weinstock and Iodice 1969; Schiaffino and Hanzlikova 1972).

The lysosomal system, in association with autophagy, is one mechanism causing increased breakdown in injured muscle fibers, functioning in the bulk catabolism of mitochondria and other membranous structures (Schiaffino and Hanzlikova 1972; Schmalbruch 1980). Exhaustion and ischemia produce varying numbers of focally necrotized fibers and transient subcellular changes e.g., swelling of mitochondria and dilatation of sarcoplasmic reticulum (Gollnick and King 1969; see also Gale 1974). Inflammatory mononuclear cells containing high activities of lysosomal enzymes phagocytize necrotic material (Reznik 1973; Vihko et al. 1978a). This process is followed by muscle fiber regeneration. It is interesting that in surviving fibers there is also a highly enhanced catabolic capacity (Shannon et al. 1974; Vihko et al. 1978a), suggesting increased breakdown of cell organelles and membranes by autophagy. The factor stimulating breakdown may be damage to cell organelles or an enhanced turnover of cell constituents, which could activate the autophagic pathway. Biosynthetic reactions are most probably associated with the increased breakdown. This hypothetical series of repair of subcellular injuries might be considered as subcellular regeneration. Other possibilities also exist, such as

Acid Hydrolase Activities and Exhaustion 63

increased endocytosis as proposed in dystrophy and denervation (Libelius et al. 1978).

Acknowledgements. We thank Mrs. Aria Mansikkaviita for skilful technical assistance.

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Accepted March 3, 1981