Exercise myopathy: Selectively enhanced proteolytic capacity in rat skeletal muscle after prolonged running

EXPERIMENTAL AND MOLECULAR PATHOLOGY 38, 61-68 (1983)

Exercise Myopathy: Selectively Enhanced Proteolytic Capacity in Rat Skeletal Muscle after Prolonged Running’

A. SALMINEN AND V. VIHKO~

Division of Muscle Research, Department of Cell Biology, University of Jyvaskylii, SF-40100 JyvU$ii 10, Finland

Received May 20, 1982, and in revised form August 17, 1982

The proteolytic capacity of rat skeletal muscle was analyzed during the repair of fiber injuries after strenuous exercise. A single bout of prolonged exercise (8 hr running at a speed of 17 m x min-‘) caused a slight fiber necrosis and a selective response in the proteolytic activity of rat skeletal muscle. Acid proteolytic capacity (cathepsin D and acid autolysis) was considerably increased on the 4th day after exertion and partially decreased by the 10th day. The acid hydrolytic response was more prominent in red than in white skeletal muscle. Alkaline proteolytic capacity (alkaline and myotibrillar proteases), increased in several atrophic myopathies, was not affected in exercise myopathy. The rate of neutral autolysis slightly increased after exertion. The protein content of skeletal muscle was decreased on the 4th day after exertion. We suggest that the proteolytic responses to acute injuries, as well as to chronic atrophies, are highly selective in skeletal muscle fibers.

INTRODUCTION

Prolonged physical exercise causes fatigue which involves, e.g., the depletion of energy-yielding substrates together with certain physicochemical and morpho- logical changes in skeletal muscle (Simonson, 1971). These alterations disturb the maintenance of the homeostasis of muscle fibers and may cause reversible and irreversible cell injuries (Gollnick and King, 1969; Vihko et al., 1978a). Lethal injuries appear histologically as muscle fiber necrosis and focal inflammation which are most abundant 2-3 days after exercise (Vihko et al., 1978a). After this necrotic phase muscle fibers are regenerated. Exercise myopathy has been described in the leg muscles of several animal species (Schumann, 1972). Anterior tibia1 compartment syndrome, or march gangrene, is a well-known example of human exercise myopathy (Getzen and Cart-, 1967). The etiology of this syndrome consists of the edema-caused ischemic compression of muscles in the anterior compartment. Exercise and ischemic myopathies resemble each other in their pathological phenomena (Shannon et al., 1974; Vihko et al., 1978a).

Exercise myopathy also includes enzymatic and structural changes in surviving skeletal muscle fibers (Gollnick and King, 1969; Vihko et al., 1978a). We have biochemically observed that the lysosomal system is stimulated after exercise in skeletal muscle (Vihko et al., 1978b; Salminen and Vihko, 1980). Histochemical studies of certain acid hydrolases indicated that the increase in total acid hydrolase activities originated mainly in muscle fibers and to a lesser extent in, e.g., inflammatory cells (Vihko et al., 1978a). The lysosomal stimulation was most prominent in red oxidative fibers 3 -7 days after exhaustive exercise. The activation of the lysosomal system is considered as a general sign of sublethal cell injury

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

’ To whom correspondence should be sent.

61

0014-4800/83/010061-08$03.00/O Copyright @ 1983 by Academic Press, Inc. AU rights of reproduction in any form reserved.

62 SALMINEN AND VIHKO

(Arstila et al., 1974). The exercise-induced temporary stimulation of the lysosomal system suggests that proteolytic processes contribute to the repair of the reversi- bly injured muscle fibers.

A dual-pathway hypothesis has been proposed for intracellular protein breakdown (Ballard, 1977). The lysosomal system, in association with autophagy, rep- resents the acid proteolytic pathway while the other cytoplasmic pathway involves the proteases with neutral and alkaline pH optima. Skeletal muscles contain several acid, neutral, and alkaline proteolytic enzymes, both endopeptidases and exopeptidases. The activities of proteolytic enzymes increase during a variety of experimental myopathies and hereditary dystrophies (Kar and Pearson, 1978: Rothig et al., 1978; Dahlmann et al., 1979). In several myopathies, the changes in proteolytic activities coincide with enhanced protein breakdown. The activities of proteolytic enzymes are, however, not equally affected, suggesting a selective stimulation of protein breakdown. This study was aimed at clarifying whether proteolytic responses are selective in skeletal muscle during exercise-induced acute myopathy.

METHODS

Prolonged running. Male Wistar rats, aged 2-3 months, were made to run for 8 hr with two 15min pauses on a motor-driven treadmill with 6” uphill tracks. The running speed was 17 m x min-‘. After the exertion the rats lived for 4 or 10 days under cage conditions before being killed. These time periods were selected by the results of earlier studies with mice showing that the highest stimulation of the lysosomal system occurs between 3 and 5 days after exercise and decreases by the 10th day (Vihko et al., 1978a, b). The control rats weighed 262 2 11 g (*SE). No significant differences were observed between the weights of the controls and the exercised groups.

Tissue preparation and assay methods. The rats were killed by cervical dislo- cation. The proteolytic activities were analyzed both in red oxidative and in white glycolytic muscle types. Muscle samples were excised from the quadriceps femoris muscle (MQF). The predominantly white muscle sample of MQF was composed of the distal head of vastus lateralis muscle, while the predominantly red sample consisted of the red parts of the proximal heads of vastus lateralis and vastus medialis muscles. Histopathological lesions were traced from the proximal part of rectus femoris muscle using hematoxylin-eosin staining. The muscle samples were quickly prepared, frozen, and stored at -80°C until analyzed within a month.

The muscle samples of left MQF were homogenized in 0.12 M Tris-HCI buffer, pH 7.2, with an all-glass Potter-Elvehjem homogenizer. Acid proteolytic capacity was estimated by the activities of cathepsin D (EC 3.4.23.5) and p- glucuronidase (EC 3.2.1.31) and the rate of acid autolysis. The enzyme activities were assayed as in our earlier study (Vihko et at., 1978b). The incubation mixture of these enzymes was made O.l%, with respect to Triton X-100 to measure the total activities (Barrett, 1972). Acid autolytic rate was assayed essentially as described by Stauber et al. (1976), using 1 mM MgCl, as the activator. Neutral autolytic rate of homogenates was analyzed at pH 7.7 as described by Okitani et al. (1974). The proteolytic products of acid and neutral autolysis were assayed by the ninhydrin and Folin-Lowry methods (Barrett, 1972). The ninhydrin method was applied to estimate exopeptidase activity while the Folin-Lowry method also

PROTEOLYTIC CHANGES IN EXERCISE MYOPATHY 63

includes the products of endopeptidase activity. Alkaline protease activity was determined as described by Pennington (1977).

The muscle samples of right MQF, identical to the samples of left MQF, were homogenized in 0.01 M potassium phosphate buffer, pH 7.7, containing 0.05 M KCl. Homogenates were made with a Potter-Elvehjem homogenizer using a Teflon pestle. Homogenates were centrifuged 10 min at the speed of SOOOg. From the supernatant the activity of trypsin inhibitor was assayed according to Noguchi et al. (1972). Casein yellow (1.5%) was used as the substrate of trypsin. The precipitate was washed twice and finally resuspended in the homogenization solu- tion. The activity of myotibrillar protease was assayed from this suspension as described by Mayer et al. (1974). The protein contents of homogenates and frac- tions were assayed as in our earlier study (Vihko et al., 1978b).

RESULTS

Prolonged exercise caused a selective response in the proteolytic capacity of rat skeletal muscle (Table I). The activities of cathepsin D and &glucuronidase and the rate of acid autolysis were strongly increased on the 4th day after the exertion. The activities decreased by the 10th day, being still significantly higher than the control values. The response in acid hydrolytic capacity was more pronounced in

TABLE I Selected Estimates of Proteolytic Capacity in Exercise Myopathy

Variable

Red skeletal muscle Acid autolysis Cathepsin D /I-Glucuronidase Neutral autolysis Myotibrillar protease Alkaline protease Trypsin inhibitor Protein content

Control (n = 12)

7.2 f 0.3a 27.5 + 0.8 1.80 f 0.12 2.30 f 0.07 35.5 f 1.7 32.7 5 1.0

89 ? 4 213 2 3

Days after prolonged exercise

4 (n = 13) lO(n = 11)

10.7 k 0.5*** 49.3 f 3.0*** 4.03 -t- 0.25*** 3.15 k 0.12*** 37.0 ?I 1.3 34.3 f 1.5 102 t6 194 k 3***

8.7 k 0.4** 33.7 f 1.5** 3.00 f 0.20*** 2.65 + O.OS** 37.2 f 1.2 35.0 f 1.3 109 * 4** 210 2 4

White skeletal muscle Acid autolysis 4.9 k 0.3 7.0 f 0.3*** 5.9 f 0.3* Cathepsin D 22.2 ” 0.7 29.5 f 1.3*** 24.3 f 0.8 P-Glucuronidase 1.20 + 0.07 1.90 2 0.13*** 1.43 2 0.08* Neutral autolysis 1.73 f 0.03 2.05 f 0.08** 1.87 f 0.07 Myofibrillar protease 30.2 f 1.5 31.5 2 1.2 29.8 2 1.5 Alkaline protease 23.3 k 1.0 25.5 + 1.3 25.2 k 1.5 Trypsin inhibitor 77 + 4 73 k 3 83 2 3 Protein content 220 f 2 210 2 3* 219 + 5

Note. Rrotease activities and autolytic rates are expressed as protease units, unit corresponding to the enzyme activity which during 1 set at 37°C liberates per kg protein TCA-soluble hydrolysis products with a color value equivalent to 1 pmole of tyrosine. TCA-soluble reaction products were determined by the Folin-Lowry method. Trypsin inhibitor activity is expressed as trypsin inhibitor units, one unit corresponding to the inhibitor activity which inhibits (AE) the hydrolysis of azocasein by trypsin during 1 set per kg supematant protein in the described procedure. P-Glucuronidase activity is expressed as pmole reaction products x set-’ x kg-’ protein. Protein contents are given as g protein/ kg muscle. Values are means f SE. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001.


TABLE II Ninhydrin Positive Autolytic Rates in Exercise Myopathy

Variable Control (?I = 12)

Days after prolonged exercise

4 (n = 13) lO(n = 11)

Red skeletal muscle Acid autolysis Neutral autolysis

White skeletal muscle Acid autolysis Neutral autolysis

12.6 ? 0.7 23.0 k 1.5*** 16.1 2 0.7** 14.7 * 0.9 16.2 ” 0.5 16.3 + 0.8

8.1 * 0.5 13.2 t 0.8*** 9.4 ” 0.5 9.8 k 0.5 11.3 + 0.5* 11.2 2 0.3*

Note. Hydrolysis products were measured by the ninhydrin method from the same TCA-soluble supernatant as the Folin-Lowry-positive products and are expressed as mmole tyrosine equivalents released x set-’ x kg-r protein. Other legends are as in Table I.

red than in white skeletal muscle. The activities of alkaline protease and alkaline myofibrillar protease were unaffected by the exercise. The rate of neutral autolysis increased when the hydrolysis products were assayed by the Lowry method, but was almost unchanged when the ninhydrin method was used (Tables I and II). The response of the acid autolytic rate was opposite to that of neutral autolysis, being greater when the ninhydrin method was applied. Trypsin inhibitor activity was slightly increased in the red muscle samples 10 days after the exercise. Muscle protein content, particularly that of red skeletal muscle, temporarily decreased after the exertion. Histological observation showed typical symptoms of exercise myopathy. Slight focal necrosis associated with inflammatory reaction was observed on the 4th day after the exercise. Fiber injuries were almost com- pletely repaired by the 10th day.

DISCUSSION

The increase of proteolytic enzyme activities is a common feature of various degenerative myopathies (e.g., Mayer et al., 1974; Pennington, 1977; Kar and Pearson, 1978; Dahlmann et al., 1979). In the atrophic myopathies neutral and alkaline proteases are usually more prominently affected than acid proteases. The degeneration of myofibrillar proteins is a prevailing phenomenon in the atrophies. Myopathies of this type include e.g., starvation atrophy (Mayer et al., 1974), hormonally induced atrophies such as those after streptozotocin or glucocorticoid treatments (Mayer et al., 1974; Dahlmann et al., 1979), and, to a lesser extent, disuse atrophies such as after denervation, tenotomy, or immobilization (Kohn, 1969; Jakubiec-Puka and Drabikowski, 1974; Banno et al., 1975). During the early phase of denervation (McLaughlin and Bosmann, 1976; Maskrey et al., 1977) the lysosomal system is considerably stimulated. The increase in protease activities in many atrophic conditions is, however, due to selective sparing of protease molecules from degradation rather than to their increased synthesis. Protein breakdown studies imply that starvation-induced degradation is nonlysosomal in origin (Jenkins et al., 1979) and is particularly concerned with myofibrillar proteins (Millward, 1970). Alkaline protease activity and myofibrillar protein degradation increase in parallel at the onset of diabetes and decrease after insulin treatment (Dahlmann et al., 1979). Both in starvation and in streptozotocin dia-


betes, the selectivity of muscle intracellular proteolysis is abolished and the breakdown of stable proteins is preferentially enhanced (Dice et al., 1978).

In the necrotic myopathies, however, the lysosomal acid hydrolase activities increase considerably. Such myopathies are induced by, e.g., vitamin E deti- ciency (Meijer and Israel, 1978), acute and chronic ischemic or toxic treatments (Shannon et al., 1974; Digiesi et al., 1975; Meijer and Israel, 1979), and heavy exercise (Vihko et al., 1978a, b), as also observed in this study. In the hereditary dystrophies the acid as well as the neutral and alkaline proteolytic activities are simultaneously increased (Kar and Pearson, 1978). Both atrophic and necrotic lesions are typical of these muscle diseases. In tissue necrosis the biochemically measured acid protease activities are affected by inflammatory phagocytes which contain high activities of lysosomal hydrolases. Histochemical observations have, however, shown that the acid hydrolytic system is particularly enhanced in surviving muscle fibers in myopathies caused by ischemia, drugs, vitamin E deli- ciency, or strenuous exercise (Shannon et al., 1974; Vihko et al., 1978a; Meijer and Israel, 1978, 1979). The increase in the acid hydrolase activities in muscle fibers corresponds to the severity of the disease (Meijer and Israel, 1978; Vihko et al., 1978a). The response of muscle fibers appreciably exceeds that of nonmuscle cells in exercise myopathy (Vihko et al., 1978a).

Ultrastructural studies imply that the stimulation of the acid hydrolytic system is associated with enhanced autophagocytosis in surviving muscle fibers during exercise, vitamin E deficiency, or denervation-induced myopathies (see Weinstock and Iodice, 1969; our unpublished observations). The number of au- tophagosomes is increased in mouse quadriceps femoris muscle 2-5 days after prolonged exercise. Ultrastructural changes coincide with the biochemically measured increase in total acid hydrolase activities (Vihko et al., 1978b). However, no response in the activities of alkaline or myofibrillar proteases occurs after the exercise (Table I). This observation implies selective enhancement of acid or alkaline proteolysis during necrotic and atrophic myopathies. The chronic nature of the myopathy may have an influence, since vitamin E deficiency also affects the neutral and alkaline proteolytic capacities (Nakamura et al., 1972; Dayton et al., 1979).

The activity of Ca2+-activated sarcoplasmic neutral protease, which removes Z lines from myofibrils, increases in dystrophic muscles (Kar and Pearson, 1978; Dayton et al., 1979). In exercise myopathy, the increase in neutral autolytic capacity may partly originate in inflammatory leukocytes, which contain, e.g., col- lagenase and elastase activity (Anderson and Irwin, 1973; Ohlsson and Olsson, 1973). Several alkaline proteases have been purified from skeletal muscles (e.g., Mayer et al., 1974; Katunuma and Kominami, 1977; Dahlmann and Reinauer, 1978). One of these, a group-specific protease inactivating apoproteins of pyridoxal enzymes, is specifically located in the mast cells of skeletal muscles (Woodbury et al., 1978). Another alkaline protease, a myosin-cleaving protease, is also a serine protease. However, these two proteases differ in certain other properties. Dahlmann and Reinauer (1978) purified from rat skeletal muscles an alkaline protease quite different from the serine type proteases. Many similarities exist between this enzyme and the myofibrillar protease studied by Mayer et al. (1974) and assayed in this study. The treatment of rats with the mast cell de- granulator, compound 48/80, decreases the serine-type alkaline proteolytic activities (Drabikowski et al., 1977: McKee et crl., 1979). Many alkaline proteases of


various sources thus contribute to muscle alkaline proteolytic activity. Our observations suggest that the nonspecific estimates of alkaline proteolytic capacity, the activities of alkaline and myolibrillar proteases, do not originate in cells invading or proliferating in skeletal muscle during acute temporary inflammation. This does not exclude the possibility that changes could occur, but they would, however, be masked by protease inhibitors.

Exercise myopathy involves simultaneous autophagy and heterophagy. Au- tophagic processes prevail in surviving fibers, while invading phagocytes remove necrotic material from lethally injured fibers by heterophagy (Vihko er al., 1978a; our unpublished observations). Autophagic activity is augmented during survival and remodeling as in starvation and metamorphosis or in response to sublethal injuries (Ericsson, 1973). Autophagy is pathologically induced in many tissues by e.g. toxic agents, radiation, or hypoxia (Ericsson, 1973; Decker and Wildenthal, 1980). Sublethally injured cells may wall off damaged organelles to initiate a repair process. Autophagocytosis is stimulated in, e.g., cardiac myocytes during hypoxia (Decker and Wildenthal, 1980). Hypoxia causes the redistribution of lysosomal enzymes from the particulate to the nonsedimentable fraction, but does not increase the total activities in the myocardium. The pathological type of autophagy is only infrequently associated with increased acid hydrolase activities in tissues other than skeletal muscle (e.g., Weinstock and Iodice, 1969; Hendy and Grasso, 1972; Ericsson, 1973; Hirsimaki et al., 1976). The lysosomal stimulation after exercise is probably important in subcellular repair of sublethally injured muscle fibers. Another possibility might be that the surviving fibers digest cellular structures by autophagy to produce materials for the regenerating fibers. In- terestingly, the lysosomal stimulation of surviving skeletal muscle fibers is more delayed than that of hypoxic cardiomyocytes and precedes and partly coincides with muscle regeneration (Vihko et al., 1978a; Decker and Wildenthal, 1980). Neutral and alkaline proteases may also enhance protein degradation in exercise myopathy, but this enhancement would, however, occur by the activation of inactive protease molecules and would not require enzyme synthesis or any mor- phological manifestation. Nonlysosomal proteolytic activity can be regulated, e.g., by modulation of substrate proteins or by intracellular protease inhibitors (Holzer and Heinrich, 1980).

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Exercise myopathy: Selectively enhanced proteolytic capacity in rat skeletal muscle after prolonged running