Acta Physiol Scand 1984, 120: 15-19

Lysosomal changes related to exercise injuries and training-induced protection in mouse skeletal muscle

A. SALMINEN, K. HONGISTO and V. VIHKO

Division of Muscle Research, Department of Cell Biology, University of Jyvaskyla, SF-40100 Jyvaskyla 10, Finland

SALMINEN, A, , HONGISTO, K. & VIHKO, V. Lysosomal changes related to exercise injuries and training-induced protection in mouse skeletal muscle. Acta Physiol Scand 1984, 120: 15-19. Received 14 March 1983. ISSN 0001-6772. Division of Muscle Research, Department of Cell Biology, University of Jyvaskyla, Finland. Three experiments were designed to study the lysosomal changes associated with the development and maintenance of the endurance training induced resistance against exer- cise injuries in mouse skeletal muscles. The activities of arylsulphatase, cathepsin C, cathepsin D, and P-glucuronidase were assayed from the red part of mouse quadriceps femoris muscle 4 days after prolonged strenuous running of 4-9 h duration. Exercise injuries were characterized by necrotic fibers and focal inflammation. Strenuous running of untrained mice induced necrotic lesions and a 4-5 fold increase in the activities of lysosomal enzymes. This lysosomal response was considerably reduced already by daily training bouts on the 3 days preceding the strenuous exertion. Simultaneously exercise injuries were markedly reduced. Extending the endurance training program increased the running ability of mice and further reduced the necrotic lesions and lysosomal changes induced by the strenuous exercise. The detraining of 1 week after the termination of regular endurance training considerably increased the degree of exercise induced lysoso- ma1 response. The detraining of longer durations further increased the lysosomal response and no effect of prior endurance training existed after 1 month detraining. Our observa- tions suggest that the severity of exercise injuries is related to the strength of the exercise stimulus and the level of preceding physical activity and can be characterized by the lysosomal changes. Key words: Exertion; muscle (enzymology, pathology), lysosomes (enzymology. patho- logy), mouse

Acute necrotic lesions in skeletal muscles may be caused by ischemia (Shannon et al. 1974), drugs (MacDonald & Engel 1970, Meijer & Israel 1979), and heavy exercise (Highman & Altland 1963, Schumann 1972, Vihko et al. 1978~) . Exertional rhabdomyolysis has been characterized in several muscles of man and many animal species (Green- berg & Arneson 1967, Reneman 1968, Schumann 1972). The clinical indications of exercise injuries include muscle soreness, elevated activities of se- rum enzymes and myoglobinemia (Greenberg & Arneson 1967, Nuttall & Jones 1968, Ritter et at. 1979). Highly increased biochemical activities of acid hydrolases in skeletal muscle are also associat- ed with repair of exercise injuries (Vihko et al.

1978b, Salminen & Vihko 1980). Histological stud- ies on injured muscles have shown focal necrosis and inflammation (Schumann 1972, Vihko et al. 1978 a). Inflammation contributes to the increase of biochemical acid hydrolase activities because of the high activities of lysosomal enzymes in inflamma- tory phagocytes.

Necrotic muscle fibers after heavy exercise con- stitute only a minute proportion, 1-2%, of all the muscle fibers. The majority of the fibers are histo- logically normal (Highmann & Altland 1963, Vihko et al. 1979~) . We have, however, observed that in surviving fibers the activities of certain lysosomal enzymes are strongly increased after the exertion (Vihko et al. 1978~) . The lysosomal response is

Acta Physiol Scand I20

16 A . Salminen et al.

most prominent in red oxidative muscle fibers, ex- ceeding that in white fibers and in non-muscle cells. The lysosomal stimulation as well as the necrotic changes are related to the duration of the exertion (Salminen & Vihko 1980). A 9 h running produces prominent necrotic changes in mouse quadriceps femoris muscle. After 3 h exertion the lysosomal enzyme activities in the same muscle are increased, but without necrotic changes (Salminen & Vihko 1980).

Several observations have indicated that physical training decreases the susceptibility of skeletal muscles to exercise injuries (Highman & Altland 1963, Garbus et al. 1964, Ritter et al. 1979, Vihko et al. 1979). Exhaustive exercise of endurance-trained mice does not cause fiber necrosis or stimulation of the lysosomal system in their skeletal muscle (Vihko et al. 1979). 'Raining evidently induces a resistance against exercise injuries. The present ex- periments were designed to study strenuous exer- cise induced lysosomal changes during the develop- ment of this resistance by daily training and the disappearance of the resistance after the termina- tion of regular endurance training.

METHODS Experimental procedures. Male NMRI mice, aged 3 4 months, were used in the experiments. Animal care was undertaken as described previously (Vihko et al. 1979). Two experiments were performed to study changes in lysosomal responses to strenuous exercise during the de- velopment of protection by daily training. Mice were trained on a motor-driven treadmill with 6" uphill tracks. In Experiment I (Expt. I) the mice trained once a day at a speed of 18 dmin . On the first two days of training the running time was 30 min and 45 min, after which it was increased to I h/day. A prolonged submaximal exertion was executed on the day after 3, 6, 9, and 15 training exercises had been completed. The exertion involved 4 separate running periods of 50 min at a speed of 20 d m i n intervening with 10 min pauses. The untrained control animals performed the same exertion. The mice were killed 4 days after the exertion, because the highest acid hydrolase activities have been observed 3-5 days after strenuous exercise (Vihko et al. 1978 b).

In Expt. I1 the endurance training program begap with a 1 h daily exercise period for 3 days at a speed of I5 d m i n and continued with daily 1 h bouts at 18 d m i n for 4 days and 20 d m i n for the next 8 days. The prolonged exertion was undertaken on the day after 3 , 7, and 15 training sessions. The mice trained 3 times ran for 9 h at a speed of 14 dmin , while those trained 7 or I5 times ran for 9 h at speeds of 16 d m i n or 18 dmin , respectively. The un- trained control mice ran for 8 h at 13 dmin . Short 10 min pauses were allowed at 3 and 6 h running. The mice were killed 4 days after the exertion.

In Expt. 111 the maintenance of the training induced protection was studied after the termination of the regular training. The training program involved 1 h exercise/day over 24 days. The running speed was successively in- creased to a maximum of 25 d m i n at the end of the training program. After the termination of training the mice lived under normal cage conditions before the pro- longed exertion was imposed 7, 14,30, or 60 days after the last exercise of the training program. The trained mice were exposed to a 6 h running period at a speed of 20 d m i n the day after the completed training program. The mice detrained for 7 or 14 days ran for 6 h at a speed of 18 d m i n and those detrained 30 or 60 days ran for 6 h at 15 dmin . The untrained control mice ran for 6 h at 13.5 dmin . The mice were killed 4 days after the prolonged exertion.

The control mice of Expts I, I1 and 111 weighed 34.2f1.3 g (SE). There were no statistically significant differences between the exercised and corresponding con- trol groups.

Muscle samples. Mice were killed by cervical disloca- tion. Skeletal muscle sample was detached from the m. quadriceps femoris (MQF). The red part of MQF was used in the studies, because it is more sensitive to exer- cise injuries than the white part (Vihko et al. 1978b, Salminen & Vihko 1980). The muscle sample was com- posed of the red parts of the proximal heads of m. vastus lateralis and m. vastus medialis and the red fibers of m. vastus intermedius. The weights of muscle samples were in Expt. I 28.7f2.3, in Expt. I1 49.8f1.6 (the muscle sample was detached from both MQF) and in Expt. 111 19.8f0.9 mg. There were no statistically significant differ- ences between the exercised and corresponding control groups.

The muscle samples were quickly prepared, weighed, frozen and kept at -80°C until analyzed. After thawing, muscle samples were cut into small pieces and homo- genized in ice-cold distilled water using an all-glass Potter- Elvehjem microhomogenizer. The homogenates were made up to 3 % (wthol).

Assay of enzyme activities. The activity of citrate (sib synthase (EC 4.1.3.7) was used to estimate the oxidative capacity of muscle samples. The following acid hydrolase activities were assayed as estimates of the lysosomal ca- pacity: arylsulphatase (EC 3.1.6.1). cathepsin C (EC 3.4.14.1), cathepsin D (EC 3.4.233, and /3-glucuronidase (EC 3.2.1.31). The enzyme assays and protein determina- tion were performed as earlier (Vihko et al. 1978b. Sal- minen & Vihko 1980). The protein content of the control samples was 198f4 g proteinkg muscle. The only signifi- cant change from this figure was a decrease of the muscle protein content in untrained exercised mice in Expt. 11. In these mice the protein content was 181f4 g proteidkg muscle on the 4th day after exercise (p<O.OOI).

Histological methods. Cryostate sections for hematox- ylin-eosin staining were cut from the proximal part of m. rectus femoris. Histological investigation was undertaken in Expt. I1 to evaluate the severity of exercise injuries (Vihko et al. 1978~) .

Statistical methods. Means and standard errors (SE) were calculated. The significance of the differences be- tween means were tested by the Student's t-test.

Acta Physiol Scand 120

Protection aguinst exercise injuries 17

- DAILY T R A I N I N G

E X P I EXPI I 0 p - G l u c u r o n i d o s e

n~ Cot h e p si n C

15 Days :on t r o l 3 6 1 9

D A I L Y T R A I N I E ! G

Fig. 1 . Changes induced in the lysosomal response of skeletal muscle to strenuous exertion by prior daily training. Results are plotted from Expts I and 11. The effect of endurance training on the acid hydrolase activities of skeletal muscle is represented in the insert. Statistical significances as in Table 1 .

RESULTS

Lysosomal responses during the development of protection Histological examination in Expt. I1 showed an increased number of necrotic fibers and inflamma- tion in the rectus femoris muscle of untrained exer- cised mice, especially in the area containing red fibers. Endurance training prior to the strenuous exercise reduced necrotic changes. Necrotic fibers and focal inflammation were infrequent in trained mice after exhaustive exercise. Daily training on only three successive days considerably reduced the strenuous exercise induced lysosomal response (Fig. 1, Table I ) . The heavy exercise in Expt. 11, however, increased the activities of arylsulphatase, cathepsin C, and /I-glucuronidase also in the skel- etal muscle of the trained mice (Fig. 1 , Table I). After slight exertion of Expt. I the lysosomal changes were minimal in the trained mice (Fig. 1) . Citrate synthase activity decreased in the skeletal muscle of exercised control mice (Table l ) , reflect- ing the decrease in protein content (see METH- ODS). Statistically significant changes were not ob- served in the citrate synthase activity of the trained mice after exertion (Table 1).

Lysosomal responses during the disappearance

of protection. Raining-induced reduction of lysoso- ma1 changes after heavy exercise disappeared after the termination of the daily training (Table 2). One week's detraining already increased the lysosomal response after strenuous exertion. The degree of exercise-induced lysosomal response further in- creased during the second week after cessation of the daily training and no effect of prior endurance training existed after one month detraining.

DISCUSSION

The activities of acid hydrolases increase in skeletal muscles in association with hereditary dystrophies (Kar & Pearson 1978) and several kinds of experi- mental injuries, such as ischemia (Shannon et al. 1974) and toxic injuries (Meijer & Israel 1979). The increase in acid hydrolase activities is especially high in necrotic myopathies. For instance, more than 10 fold increase in B-glucuronidase activity is observed in necrotic myopathies induced by vita- min E deficiency (Meijer & Israel 1978) or acute ischemia (Shannon et al. 1974). We have earlier observed 5-7 fold increase in P-glucuronidase activ- ity in exercise induced skeletal muscle injuries in mice (Vihko et al. 1978b, Salminen & Vihko 1980

2-848041 Acta Physiol Scand I20

18 A. Salminen et al.

Table 1. Effects of prior daily training on the acid hydrolase response induced by strenuous exercise Acid hydrolase activities are expressed as pmol hydrolyzed substrate X s-' X kg-' muscle. Citrate synthase activity is given as mmol x s-' x kg-' muscle. Values are means f SE

Citrate Group (n) Arylsulphatase Cathepsin C @-Glucuronidase synthase

Untrained Control Exercised

Trained 3x Control Exercised

Trained 7 x Control Exercised

Trained 15x Control Exercised

0.22f0.01 1.14+0.10***

0.27f0.02 0.51f0.02***

0.23f0.01 0.49f0.02***

0.24f0.01 0.45f0.02***

0.98f0.04 0.72+0.04***

0.90f0.04 0.91 f0.02

0.98f0.03 0.97 f0.03

1.04f0.05 0.99f0.03

0.53f0.03 1.98+0.16***

0.62f0.03 0.%+0.04***

0.57f0.02 0.85+0.04***

0.57f0.03 0.79+0.04***

0.66 f 0.04 3.54+0.34***

0.79f0.05 I .21+0.07***

0.70 f 0.03 I .22+0.08***

0.71 f0.04 I .08+0.06***

Statistical significance: *p<0.05, **p<O.OI and ***p<O.OOI (controYexercised).

& 1982). The results of this study are consistent with our earlier observations. A considerably high- er increase in lysosomal enzymes was recorded after the heavier exertion of Expt. I1 than after the light exertion in Expt. I or the moderate exertion in Expt. 111. Furthermore, the acid hydrolase re- sponses were not of equal magnitude, being the

highest in the B-glucuronidase activity and the smallest in the cathepsin D activity. Histochemical studies have shown that the increase in lysosomal enzymes originate in invaded phagocytes and in muscle fibers adjacent to necrotic fibers (Shannon et al. 1974, Vihko et al. 1978a, Meijer & Israel 1979). The causes both for necrotic lesions after

Table 2. Effects of the termination of prior daily training on the acid hydrolase response induced by strenuous exercise Explanations as for Table I

Citrate Group (n) Cathepsin C Cathepsin D @-Glucuronidase synthase

Untrained Control Exercised

Control Exercised

Control Exercised

Control Exercised

Control Exercised

Control Exercised

Trained

Detrained 7 days

Detrained 14 days

Detrained 30 days

Detrained 60 days

0.89f0.05 2.24+0.23***

17.0f0.8 21.1 +0.6***

0.25f0.03 0.72+0.09***

0.81f0.05 0.70f0.03*

0.24f0.02 0.29+0.02*

0.92 f O . 0 6 0.88f0.03

0.88 f 0.07 0.97f0.06

16.2f0.8 18.7f0.4**

0.90f0.07 I .22+0.08**

16.3f0.5 18.9+0.7**

0.24f0.01 0.41 f0.02***

0.80f0.03 0.75f0.06

1.05f0.05 1.81 +0.17***

16.4f0.6 19,1f0.8**

0.23f0.01 0.47f0.05***

0.81f0.03 0.76f0.04

16.1 f0.6 21.7f1 .O***

0.23f0.01 0.79k0.08***

0.80f0.02 0.68+0.02***

1.02f0.06 2.49+0.19***

0.79f0.03 2.31+0.23***

14.5f0.5 19.92 1.1***

0.23f0.01 0.81+0.09***

0.83 f 0.03 0.75f0.02

Acto Physiol Scand 120

Protection against exercise injuries 19

exertion and for the stimulation of lysosomal sys- tem in surviving muscle fibers are still unknown.

Several studies (Highman & Altland 1963, Gar- bus et al. 1964, Ritter e t al. 1979, Vihko et al. 1979) imply that physical training induces a resistance against exercise injuries. We have earlier observed (Vihko et al. 1979) that exhaustive prolonged run- ning does not cause fiber necrosis or the stimulation of lysosomal system in the quadriceps femoris mus- cle of trained mice. Our present results show fur- ther that the development of the protection is sur- prisingly fast. A training of only three days prior t o the strenuous exertion caused a considerable pro- tection against exercise injuries. The protection ap- peared as a lack of necrosis in the rectus femoris muscle of the trained mice after strenuous exercise. The adaptations of endocrine and muscular systems most probably contribute to the development of the resistance against exercise injuries.

Interestingly, the lysosomal response to strenu- ous exercise decreased simultaneously as the pro- tection against necrotic lesions appeared. Howev- er , a small response existed in the trained mice in Expt. I1 in which the mice were exposed to vigor- ous exertion. Lysosomal responses to strenuous exercise rapidly increased after the termination of daily training. The detraining of already one week considerably increased the degree of exercise-in- duced lysosomal response. After one month no effect of preceding endurance training existed. Ne- crotic lesions were not traced in this experiment. This study and our earlier studies (Vihko et al. 1978a, h, Vihko et al. 1979, Salminen & Vihko 1980, 1982) suggest that lysosomal enzymes render a good estimate of exercise-induced skeletal muscle injuries, although the quantitative connection be- tween the lysosomal changes and the degree of cell injuries has not been demonstrated.

This study was supported by the Academy of Finland and the Research Council for Physical Education and Sport (Ministry of Education, Finland).

REFERENCES GARBUS, J . , HIGHMAN, B. & ALTLAND, P. D. 1964.

Serum enzymes and lactic dehydrogenase isoenzymes after exercise and training in rats. Am J Physiol 207: 467-472.

GREENBERG, J. & ARNESON, L. 1967. Exertional rhabdomyolysis with myoglobinuria in a large group of military trainees. Neurology 17: 216-222.

HIGHMAN, B. & ALTLAND. P. D. 1963. Effects of exercise and training on serum enzyme and tissue changes in rats. Am J Physiol 205: 162-166.

KAR, N. C. & PEARSON, C. M. 1978. Muscular dystro- phy and activation of proteinases. Muscle Nerve 1: 301313.

MACDONALD, R. D. & ENGEL. A. G. 1970. Experi- mental chloroquine myopathy . J Neuropathol Exp Neurol 29: 479499.

MEIJER, A. E. F. H. & ISRAEL, D. E. 1978. Evaluation of histochemical observations of activity of acid hy- drolases obtained with semipermeable membrane techniques: A combined histochemical and biochemi- cal investigation. 2. The biochemical investigation and comparison with the histochemical observations. His- tochemistry 57: 23-3 I .

MEIJER, A. E. F. H. & ISRAEL, D. E. 1979. The increase in activity of acid hydrolases in muscles of rats after subcutaneous administration of dimethyl- para-phenylene diamine. A combined histochemical and biochemical investigation. 1. The histochemical investigation. Histochemistry 61: 81-91.

NUTTALL, F. Q. & JONES, B. 1968. Creatine kinase and glutamic oxalacetic transaminase activity in se- rum: Kinetics of change with exercise and effect of physical conditioning. J Lab Clin Med 71: 847-854.

RENEMAN, R. S. 1968. The anterior and the lateral compartment syndrome of the leg. Mouton & Co., Hague.

RITTER, W. S., STONE, M. J. & WILLERSON, J. T. 1979. Reduction in exertional myoglobinemia after physical conditioning. Arch Intern Med 139: 644-647.

SALMINEN, A. & VIHKO, V. 1980. Acid proteolytic capacity in mouse cardiac and skeletal muscles after prolonged submaximal exercise. Pflugers Arch

SALMINEN, A. & VIHKO, V. 1982. Changes of dipepti- dyl aminopeptidase activities in mouse skeletak mus- cle following prolonged running. Comp Biochem Phy- siol 71B: 23-27.

SCHUMANN. H.-J. 1972. Uberlastungsnekrosen der Skelettmuskulatur nach experimentellem Laufzwang. Zbl Allg Path 116: 181-190.

SHANNON, A. D., ADAMS, E. P. & COURTICE, F. C. 1974. The lysosomal enzymes acid phosphatase and B- glucuronidase in muscle following a period of ischae- mia. Aust J Exp Biol Med Sci 52: 157-171.

VIHKO, V., RANTAMAKI, J. & SALMINEN, A. 1978 u. Exhaustive physical exercise and acid hydro- lase activity in mouse skeletal muscle. A histochemi- cal study. Histochemistry 57: 237-249.

VIHKO, V., SALMINEN, A. & RANTAMAKI, J. 19786. Acid hydrolase activity in red and white skel- etal muscle of mice during a two-week period follow- ing exhausting exercise. Pfliigers Arch 378: 99-106.

VIHKO, V., SALMINEN, A. & RANTAMAKI. J. 1979. Exhaustive exercise, endurance training, and acid hy- drolase activity in skeletal muscle. J Appl Physiol 47: 43-50.

389: 17-20.

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