Two-step mechanism of myofibrillar protein degradation in acute plasmocid-induced muscle necrosis

Biochimica et Biophysica Acta, 798 (1984) 333-342 Elsevier

333

BBA 21725

TWO-STEP MECHANISM OF MYOFIBRILLAR PROTEIN DEGRADATION IN ACUTE PLASMOCID-INDUCED MUSCLE NECROSIS SHOICHI ISHIURA, IKUYA NONAKA, HIROFUMI NAKASE, AIKO TADA and HIDEO SUGITA

National Center for Neroous, Mental and Muscular Disorders, Kodaira, Tokyo 187 (Japan) (Received December 29th, 1982) (Revised manuscript received January 2nd, 1984)

Key words: Protein degradation; Plasmocid; Necrosis," (Muscle)

Acute muscle necrosis was induced in rats by intramuscular injection of plasmocid, a known myotoxic agent. A single injection of 5 mg/mi plasmocid produced massive fiber necrosis with extensive phagocytosis. Plasmocid administration led to a preferential decrease of a-actinin with preservation of other structural proteins within 3 h after injection, and large increases (2-7-fold) in the activities of acid hydrolases, cathepsins B and L, cathepsin D and a-galactosidase within 48 h after injection. The plasmocid-induced stimulation of a-actinin loss seen at 3 h, when no increases of acid hydrolases occurred, could be inhibited by a cysteine protease inhibitor, Ep-475 (E764-c), and EGTA. On the other hand, increased lysosomal enzyme activity seemed to have a close correlation with the appearance of invading mononuclear cells, probably macrophages, and not muscle iysosomes. These observations suggest that a two step mechanism of protein degradation (nonlysosomal and lysosomal processes) possibly occurs in plasmocid-induced muscle degradation and macrophages can serve as a main endogenous reservoir of proteases in pathological states.

Introduction The enhancement of proteolysis in degenerating muscle has been known for many years, but its importance has only been realized since the discovery of muscle proteases (for review see Ref. 1). Over the past ten years, much effort has been directed towards the characterization of these proteases including nonlysosomal Ca2÷-activated protease [2-4], trypsin-like neutral protease [5], lysosomal cathepsins [6-10] and other interstitial proteases such as a serine protease of mast cell origin [11,12]. Almost all these proteases have the ability to degrade muscle structural proteins i.e., cathepsins B and D degrade contractile proteins, myosin and actin [13,14], and Ca2÷-activated protease specifically removes Z-lines [15] and digests cytoskeletal proteins, desmin [16] and vimentin [17,18]. There is still much controversy concerning the 0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V.

relative importance of these proteases in muscle protein degradation. Recent results suggest that two mechanisms, lysosomal and nonlysosomal, may be involved in protein degradation in mammalian skeletal muscle. Lysosomes in skeletal muscle, as well as in other tissues, play a major role under nutritional deprivation. However, there is no evidence that the lysosomal apparatus in muscle participates in pathological protein turnover in atrophying muscles. In such cases, slow degeneration accompanies active muscle regeneration. The muscle in Duchenne muscular dystrophy is a typical case. We cannot, therefore, determine the pathway of protein degradation using a muscle homogenate because of contamination by regenerating muscle fibers. Acute muscle necrosis induced by chemical toxins presented a unique model for protein degradation. We used a protoplasmic toxin,

334 plasmocid, for this purpose. If a large dose was administered intraperitoneally (50 m g / k g body weight), the rats became ill within an hour and died. A very small amount of intraperitoneally-administered plasmocid (10 mg/kg) caused destruction of striated muscle (diaphragm, tongue) and cardiac muscle, and some of the rats became weak [19,20]. To avoid sudden death we injected the drug directly into the soleus muscle. Comparison of direct injection and intraperitoneal injection will be reported elsewhere. The effect of a single intramuscular injection of another myotoxic drug, bupivacaine, has been reported [21]. Plasmocid caused removal of the Z-lines within 3 h after injection and massive dissolution of myofilaments [20] by 48 h. So we can determine the distinct pathway of protein degradation in vivo. By determining the activity of lysosomal enzymes, a two step mechanism of myofibrillar protein degradation has been discovered in affected muscles. A preliminary report of the results has been presented in abstract form [22]. Materials and Methods

Materials. A protease inhibitor, Ep-475 (E-64-c), was provided by Taisho Pharmaceutical Co. Ltd. (Saitama, Japan). Plasmocid-induced myopathy. A fresh solution of plasmocid (8-(3-diethylamino-propylamino)-6methoxyquinoline) in saline was prepared daily (5 mg/ml). To neutralize it, a few drops of 1 N N a O H were added because an acidified solution sometimes caused nonspecific muscle necrosis without regeneration. Stability of the neutralized solution was checked by thin-layer chromatography, the agent was fairly stable at 4°C for 1 week. All experiments were performed on soleus muscle from male Wistar rats, 200-250 g. Animals were injected intramuscularly with 2 mg plasmocid under pentobarbital anesthesia. The animals received a 0.4 ml injection. The contralateral soleus muscle injected with saline was used as a control. Control muscles returned to a normal appearance in 1 h, whereas swelling persisted for 2 days in plasmocid-treated muscles. Enzyme assays. Animals were killed by decapitation. The injured soleus and the contralateral one were excised, chilled and rinsed in phosphate-

buffered saline. A part of each was homogenized with 10 v o l u m e s of p h o s p h a t e - b u f f e r e d saline/0.1% Triton X-100 solution in a glass homogenizer, twice for 30 s. This was sufficient to break up the fibers completely. Preliminary experiments indicated that inclusion of Triton X-100 in association with phosphate-buffered saline caused a high yield ( > 95%) of lysosomal enzymes. Similarly, homogenates from soleus muscles were obtained from control animals of the same age. The contralateral soleus muscle was not affected by plasmocid treatment. The homogenate was centrifuged at 10000 × g for 20 min and the supernatant retained. The activity of thiol-dependent cathepsin was determined by using a new synthetic substrate, succinyl-Tyr-Met-naphthylamide (kindly provided by Prof. N. Katunuma, Tokushima Univ., Japan). The activity of cathepsins in muscle homogenates towards this substrate was 8-times higher than that towards benzoyl-Arg-naphthylamide and completely inhibited by cysteine protease inhibitors, Ep-475 (10 -6 M) and leupeptin (10 -6 M), but not bestatin (10-3 M), an aminopeptidase inhibitor, or pepstatin (10 -3 M), a carboxyl protease inhibitor (these inhibitors were generous gifts from Profs. H. Umezawa and T. Aoyagi, Inst. Microbial Chem., Tokyo, Japan). Previous studies by Katunuma et al. [23] indicated that purified cathepsins B and L hydrolyzed succinyl-Tyr-Metnaphthylamide at relative rates of 3 : 2. Cathepsin H did not hydrolyze it [23,24]. Accordingly, we tentatively assigned the activity towards succinylTyr-Met-naphthylamide as due to cathepsins B and L. The hydrolysis of this substrate was measured in a medium (1 ml) containing substrate (2 mM), 2 mM cysteine, 1 mM EDTA, 10% dimethylsulfoxide, 0.3% Triton X-100, and 0.1 M acetate buffer, pH 5.0. Incubation was for 1 h at 40°C, and the reaction was stopped by the addition of the color reagent, Fast Garnet, of Barrett [7]. After color development, n-butanol was added and the tubes were vigorously shaken. After standing for 5 min in a rack, an aliquot of the butanol layer was withdrawn and the absorbance at 520 nm was read with 2-naphthylamine as the standard. 1 unit of cathepsins B and L was defined as nmol naphthylamine liberated/h per mg protien. Cathepsin D was estimated by the method of Barrett [8].

335 Acid phosphatase was assayed with pnitrophenylphosphate as the substrate [25]. Creatine kinase was determined with a Boehringer assay kit. a-Glucosidase, a-galactosidase and flglucuronidase were assayed by using the corresponding 4-methylumbelliferyl substrates (KockLight Lab. Ltd., U.K.) at pH 4.0 [26]. Ca2+activated protease was measured by our previous method [2]. Protein concentrations were determined by the method of Lowry et al. [27] using bovine serum albumin as a standard. Separation of macrophages from soleus muscle. Separation of mononuclear cells from soleus muscle was performed according to the method of Maskrey et al. [28] with slight modification. Soleus muscles were removed and chopped with scissors. The chopped muscle was weighed and transferred to a 15 ml culture tube containing RPMI-1640 medium (Flow Lab., VA, U.S.A., 10 ml/soleus). The tissue suspension was incubated for 30 rain at 37°C with continuous shaking. After incubation, the suspension was filtered through lens paper to separate the cell suspension from muscle residue. The cell suspension was placed in 60 mm petri dishes and incubated at 37°C for 2 h. Nonadherent cells were then removed by washing with RPMI-1640 medium. The adherent cells were scraped off with phosphate-buffered saline containing 0.1% Triton X-100 for the enzyme assay. The muscle residue was then homogenized with the same buffer as described before. The mononuclear cells retained 90% viability as judged by Trypan blue exclusion. UltrastructuraUy, these cells were shown to be macrophages. Ca 2+-activated protease and cathepsin B from rat tissues. A Ca2+-activated protease which require mM order Ca 2÷ for its activity was purified from rat skeletal muscle through DEAE-cellulose, Ultrogel AcA 34 and hydroxyapatite column chromatographs as previously reported briefly [22]. This yielded an electrophoretically single protein, which migrated as a single polypeptide of a molecular weight of 73000 on a SDS-polyacrylamide gel. Purified cathepsin B from rat liver was a generous gift from Dr. T. Noda, Tokushima University, Japan. SDS-gel electrophoresis. The remaining parts of soleus muscle were preserved in 20 mM Tris-HC1, pH 7.5, 0.1 M KC1, 5 mM EGTA and 50% glycerol

at - 2 0 ° C for 5 days to remove excess soluble proteins which interfered with quantification of each structural protein on SDS-gels by tracing. We have ascertained that the content of myofibrillar proteins did not change during storage [29]. Then the muscles were homogenized with a solution containing 1% SDS and 5% 2-mercaptoethanol, and subjected to 12% SDS gel electrophoresis according to the method of Laemmli [30]. Gels were stained overnight in 0.1% Coomassie blue R-250, 45% methanol and 7% acetic acid and destained by shaking in 7% acetic acid. Each slab gel contained authentic a-actinin purified from 20 rat soleus muscles [31] to minimize the difference between experiments. The gels were traced at 600 nm with Gilford gel scanner. Histochemistry. The muscles were frozen immediately after dissection in isopentane cooled with liquid nitrogen. Serial frozen sections were stained with hematoxylin and eosin and acid phosphatase [32]. Electron microscopy. The removed soleus muscles were subdivided along the longitudinal axes. The strips were fixed in cacodylate-buffered 2.8% glutaraldehyde for 1 h. The specimens were post-fixed in the same buffer containing 1.3% OsO4 and 1.5% lanthanum nitrate for 1 h and then embedded in epoxy resin. The samples were examined with a Hitachi H-300 electron microscope. Results

Plasmocid-induced alteration of myofilaments The intramuscular administration of plasmocid caused the massive degeneration of myofilaments in the first 48 h. 3 h after plasmocid injection changes were less discernible histochemically although some of the muscle fibers were seen to have disappeared in the cross-sectioned area (Fig. la). The necrosis of muscle fibers was segmental, with abrupt transition from normal to empty (data not shown). Acid phosphatase activity showed that no activation or proliferation of the endogenous lysosomes occurred in muscle fibers (Fig. lb). Note that no phagocytosis was observed at this stage. Fully developed lesions were seen in the plasmocid-injected solei of all animals killed between 24 and 72 h. As shown in Fig. lc (48 h after injection), all the muscle fibers underwent phagocytosis. The

336

Fig, 1. Serial sections of soleus muscle 3 h (a, b) and 48 h (c, d) after plasmocid injection. The muscle fibers appear to be either hypercontracted (single arrowheads) or empty (double arrowheads) 3 h after plasmocid injection with few phagocytosis. Note numerous necrotic fibers showing active phagocytosis at 48 h after injection, a, c: hematoxylin and eosin; b. d: acid phosphatase staining, x 200.

phagocytic macrophages in the necrotic muscle fibers are responsible for the heavy acid phosphatase activity (Fig. ld). In order to clarify the pathway of muscle protein degradation, soleus muscles were removed every 3 h after injection. 3 h after administration almost all the Z-lines had disappeared with preservation of other structural elements (Fig. 2). By 3 h, a significant decrease in a-actinin was evident. Fig. 3 shows the SDS-gel electrophoretic pattern of myofibrillar proteins of 8 individual rats. Plasmocid caused preferential removal of a-actinin from myofibrils (Fig. 3, lanes 3 and 4) as compared with the untreated control (Fig. 3, lanes 1 and 2). Z-lines and a-actinin from saline-treated muscle were fairly well preserved and indis-

Fig. 2. Electron micrograph of plasmocid-treated soleus muscle at 3 h after injection. Note selective loss of the Z-lines and disarrangement of adjacent thin filaments. Mitochondria and thick filaments appear unaffected.

337

both Ca2+-dependent and cysteine-protease specific processes, possibly by Ca 2+-activated protease in muscle cells. Table I summarizes the results of these experiments. By tracing the gels, the residual amounts of a-actinin could be calculated. The data were expressed as relative amounts of a-actinin to actin because the actin content had not changed by the later stage of muscle necrosis. As shown in Table I, EGTA and Ep-475 significantly abolished the effect of plasmocid. EGTA or Ep-475 alone did not induce any changes in relative protein content.

l

"|1 ~ t

w o

w s

~11P

Fig. 3. SDS-gel electrophoresis of myofibrillar proteins of rat soleus muscle 3 h after plasmocid injection. Lanes 1 and 2, control, muscles without treatment; lanes 3 and 4, plasmocidinjected muscles; lanes 5 and 6, muscles with 5 m M EGTA and plasmocid; lanes 7 and 8, muscles with 1 m M Ep-475 and plasmocid. M: myosin heavy chain, A: actin, a-actinin, identified by immunoreplication, is indicated by an arrow.

tinguishable from those of the untreated control (data not shown). To determine whether this change was a nonspecific effect of the agent or whether it was related to some proteolytic activation as suggested in vitro [4,33], we investigated the effect of protease inhibitors or other compounds on the removal of a-actinin in soleus muscle. At all concentrations tested (1 mM, 5 mM), EGTA and Ep-475 coinjected with plasmocid reduced the plasmocid-induced stimulation of a-actinin release (Fig. 3, lanes 5-8). Thus, the data presented above suggest that the loss of a-actinin is mediated by

Inhibition by Ep-475 of intracellularproteinases. Ep-475 (L-trans-epoxysuccinyl-leucylamido(3methyl)-butane) has proved to be the most reactive analogue of L-trans-epoxysuccinyl leucyl agmatine (E-64) derivatives with cysteine proteinases such as cathepsins B and L [34], Ca2+-activated protease [35] and papain [36]. We tried to inhibit typical purified intracellular proteinases of rat origin, cathepsin B and Ca2+-activated protease, in vitro to determine whether Ep-475 could inhibit these proteinases at the concentrations used in this experiment. On ultrastructural observation, the disruption of plasma membranes was widely detected soon after injection of plasmocid with or without Ep-475, the inhibitor could easily penetrate into muscle cells [20]. Fig. 4 shows in vitro inhibition of caseinolytic activity by Ep-475. From these results, we calculated the half maximal concentration of Ep-475 for inactivation of these enzymes to be 0.2 t~M for cathepsin B, 1.7 btM for rat Ca2+-activated protease and 3.0 /LM for chicken Ca2+-activated protease. All these enzymes could be inhibited by

TABLE I SELECTIVE INHIBITION O F a - A C T I N I N LOSS BY EGTA (5 mM) O R Ep-475 (1 m M ) C O I N J E C T E D W I T H P L A S M O C I D 3 h after injection of the agents, muscles were removed and glycerinated. The data for each tracing are the means of 3 gels. Plasmocid is abbreviated as Pls. a p < 0.01, b p < 0.01. Mean + S.D. Additions

Control (uninjected) Pls. Pls. + EGTA PIs. + Ep-475

(n (n (n (n

= = = =

8) 6) 6) 6)

a-Actinin

Myosin

Actin

Actin

0.160 + 0.031 0.035 + 0.015 a 0.056 + 0.028 b 0.108 + 0.016 a

0.93 + 0.20 0.95 + 0.22 0.82 ± 0.26 0.85 + 0.14

338

100

°

\\:,

50

0

~X'c~" -.~ ~ 9

8

7

6

5

4

3

p(Ep-475) Fig. 4. Inhibitory effect of Ep-475 on two typical intracellular proteinases, cathepsin B (open circles) and Ca2+-activated protease (closed circles) of rat. The data for chicken skeletal muscle Ca2+-activated protease (open circles, dashed line) are also shown in this figure. Inhibitory activity of Ep-475 was determined using casein as protein substrate. The proteinase activity of cathepsin B was measured in sodium acetate buffer (0.1 M, pH 5.5). 1 unit of the proteinase activity was defined as the amount of the enzyme which catalyzed an increase Of 1.0 absorbance unit at 280 nm under these conditions. Approximately 0.2 units of cathepsin B and Ca2+-activated protease were used in this experiment.

10 -4 M Ep-475. Therefore, Ep-475 at a concentrations of 10 -3 M is adequate for inactivation of these cyste{ne proteinases. The recent finding that Ca 2÷ is essential for the inhibition of CA 2+ activated protease by Ep-475 [35,37] is quite important in assessing the effect of Ep-475 in vivo. The intracellular concentration of

Ca 2+ in plasmocid-treated muscle was shown to be higher than 10 - 4 M by a histochemical technique (Ref. 20 and H. Nakase, unpublished data), whereas in normal muscle cells the concentration never exceeds 10 -5 M. Together with the finding that an unusual feature of the plasma membrane is observed on electron microscopy (Ref. 20 and H. Nakase, unpublished data), we concluded that Ca2+-activated protease could be inhibited by Ep475 in vivo, if the enzyme digested Z-lines in muscle cells. However, the activity of Ca 2+ activated protease in plasmocid-treated muscle (enzyme source: muscle homogenate; substrate: [14C]casein; pH 7.5) was almost the same as that in normal soleus. This result suggests that Ca 2+activated protease in muscle cells did not increase in content upon plasmocid treatment but was activated by influxed Ca 2+ in vivo.

Lysosomal enzyme activities in plasmocid-treated muscle To determine whether lysosomal cathepsins are involved in these plasmocid-induced changes, we determined various lysosomal enzyme activities at various stages of muscle degradation. Two typical results are presented in Table II, one of them is the result at 3 h after injection when only the Z-lines had disappeared without any alteration in myofilaments, there being no phagocytic cells (lst step, see Fig. la), and the other is at 48 h after injection when all the components of myofibrils had disappeared microscopically with large amounts of invading phagocytes (2nd step, see Fig. lc). As shown

TABLE II VARIOUS ENZYME ACTIVITIES IN PLASMOCID-TREATED SOLEUS MUSCLE Plasmocid-injected muscles (n = 23) and safine-injected muscles (n = 19) were removed and subjected to enzyme analysis. Muscles were excised from rats both 3 h (n = 14) and 48 h (n = 9) after injection of plasmocid. Data from uninjected controls and from saline-injected at 3 h were almost the same as those from saline-injected at 48 h. Results are presented as means-+S.D, with the number of observations. Statistical significance: a p < 0.01. Enzymes

Cathepsins B and L Cathepsin D a-Glucosidase(pH4) a-Galactosidase CreatineKinase

Enzyme activity(U/mg) 3 h after injection

48 h after injection

Control muscle

5.71 0.039 0.38 0.24 1.29

137.5 0.147 0.91 2.21 0.17

20.9 0.067 2.20 0.99 20.9

-+ 1.66 a + 0.014 a 5:0.30 a +_0.05 a _+0.71 ~

_+70,3 a + 0.016 ~ + 0.40 ~ -+ 0.34 ~ -+ 0.09 ~

___5.2 + 0.016 +0.26 -+0.13 _+5.2

339 in Table II, the content of soluble proteins had increased slightly but not significantly by 3 h after injection and then appeared to return to the control level. Two-dimensional gel electrophoresis revealed that a large amount of serum albumin contaminated the soluble fraction of the injected muscle, especially at 3 h after injection (data not shown). Therefore we concluded that the transient increase of cytosolic proteins within 3 h was due to influx of serum proteins into muscle cells. Cytosolic creatine kinase decreased to 6% of the contralateral soleus value at 3 h after plasmocid treatment, suggesting that massive disruption of muscle cells had occurred and cytosolic enzymes as well as lysosomal enzymes had leaked into the serum. We assumed leakage of creatine kinase because of the following: when the soleus muscle was incubated in Krebs-Ringer solution with plasmocid, considerable amounts of creatine kinase were released into the medium, whereas the total amounts of creatine kinase (both in muscle and medium) had not altered (Ishiura, S., unpublished data), indicating the leakages of creatine kinase and not intracellular proteolysis. The fact that the decreases in lysosomal enzymes are lower than that of cytosolic creatine kinase implies the organelle-bound nature of lysosomal enzymes. The most prominent feature of plasmocidtreated muscle is the drastic increase in cathepsins B and L within 48 h after injection as shown in Table II. A time lag was strikingly noticeable in the onset of cathepsins B and L burst. The activity was rather lower than the control (untreated or

saline-injected) at 3 h when the muscle already lost the Z-lines. The above result also cast doubt on the role of lysosomal cathepsins in the initial step of myofibrillar degeneration. At the later stage of muscle degeneration, i.e., 48 h after injection, great enhancement of other lysosomal enzymes, such as cathepsin D and ~t-galactosidase was observed. In contrast, the activity of acid a-glucosidase did not increase even after 48 h. The enzyme pattern mimicked those of cultured peritoneal macrophages, namely high contents of cathepsin B and L and ct-galactosidase in comparison with a low level of acid ot-glucosidase. To further confirm the above, macrophages were collected from the injured soleus muscle. As shown in Table III, cathepsins B and L, cathepsin D and a-galactosidase were fairly high in activity in the lysate from plasmocid-injected muscles. In addition, cathepsin B was shown to be specific for macrophages in degenerating skeletal muscle [21]. Therefore, we assumed that infiltrating macrophages contributed to the second step of muscle degradation.

Table Ill RECOVERY OF LYSOSOMAL ENZYME ACTIVITY IN THE LYSATE OF MONONUCLEAR CELLS Mononuclear cells were harvested from both the plasmocid-injected and the contralateral soleus muscles48 h after treatment. Data are averages of 5 animal experiments and represented as the proportion of those for injured muscle relative to the control. All values were statistically significant (P < 0.001)

TABLE IV

Effect of Ep-475 on lysosomal enzyme activity in vivo From a therapeutic viewpoint, it is important to clarify if the second step of muscle degeneration can be inhibited by Ep-475, because Ep-475 suppresses the first step of degeneration to some extent (Table IV). The decrease of creatine kinase was not inhibited by Ep-475, which was the expected result because the disappearance of creatine

EFFECT OF Ep-475 ON MUSCLE ENZYMES 48 h AFTER PLASMOCID INJECTION Data are from 9 experiments, as means+ S.D. Control shows the normal levels of the enzymes. Addition

Time Enzymeactivity(U/rag) (h)

Enzymes Cathepsins B and L Cathepsin D a-Galactosidase ~-Glucosidase(pH 4) /3-Glucuronidase

Plasmocid/control 24.7 8.0 5.5 1.8 10.0

None 1 mM Ep-475

Creatinekinase Cathepsins B & L

3

1.525:0.80 0,925:0.51

5.95:1.0 5.8+ 0.8

None 48 1 mM Ep-475

0.17 + 0,03 0.16+0.02

140.5 5:60.2 148.35:70.5

Control

10.2 +3.2

20.9+ 5.2

340 kinase was thought to be only a diffusion process. On the other hand, the addition of Ep-475 also failed to reduce the increasing activity of cathepsins B and L, although this agent certainly inhibited the first step of muscle degeneration, namely Z-line removal. These results strongly suggest that the removal of the Z-lines is not indispensable for acute plasmocid-induced muscle degradation. Discussion

Many reports have been concerned with the Ca2+-activated protease, the in vivo effect of the enzyme is controversial. Since two factors affect the activity of this enzyme, Ca z + [37] and endogenous inhibitor [38], careful investigation is necessary for understanding its role in living cells. The above results indicate that Ca z +-activated protease is the most plausible candidate for removal of the Z-lines in vivo. We agree with the result that the protease is not concerned in overall protein degradation as shown by the release of amino acid from isolated muscle in vitro [39]. Acute plasmocid-induced degeneration of skeletal muscle is apparently different from that in normal conditions. The intracellular concentration of Ca 2+ has been found to be high enough to activate neutral protease. It should be specifically noted that all lysosomal enzyme tested decreased at the initial stage of muscle degeneration (3 h after injection). Thus, we concluded that Ca2+-activated protease digested and removed the Z-lines prior to massive degradation of myofilaments. At the second stage of muscle degradation, the activity of lysosomal cathepsins dramatically increased in contrast to that of cytosolic creatine kinase. There is an intimate correlation between the presence of lysosomal cathepsins and the degradation of residual proteins. Despite the lack of direct evidence that cathepsins actually degrade myofibrillar proteins in vivo, it seems reasonable to conclude that the degradation of muscle proteins is caused by the enhancement of cathepsin activity. Surprisingly, the localization of one of these enzymes, cathepsin B, was limited to invading phagocytes [21]. The number of phagocytes increase in proportion to the cathepsin activity, suggesting that the activity of cathepsins B and L

in the muscle homogenate at the second step of muscle degeneration is perhaps derived from invading cells. The results that macrophages extracted from the plasmocid-treated soleus muscle contained large amounts of cathepsins B and L confirmed these possibilities. Of many interstitial cells, the invasion of macrophages is prominent on electron microscopy (data not shown). The enzyme activities in cultured macrophages are remarkably characteristic, i.e., high specific activities of cathepsins, especially cathepsins B and L, a-galactosidase and neutral a-glucosidase. In particular, the activity of c~galactosidase is 2- or 3-fold higher than that of a-glucosidase at pH 4.0 with the same assay conditions except for the substrate. Although the activity of a-galactosidase in muscle homogenates is always lower than that of a-glucosidase the situation is reversed only after cell infiltration. Moreover, there is increased neutral o~-glucosidase activity at the later stage, which is contained in macrophages in large amounts [40]. Thus, the burst of some lysosomal enzymes at 48 h seemed to be derived from macrophages. As a matter of course, investigation of other interstitial cells is necessary. Mast cells, which contain large amounts of serine protease of unknown biological function, have been known to exist in large quantities in rat skeletal muscle. In pathological conditions such as muscular dystrophy, increasing activity of the enzyme has been reported by Katunuma et al. [41]. But in our case, mast cells were not found to be increased by a histochelnical method after 48 h. Another interesting result in our experiment is that the massive degeneration of myofilaments, which means both degeneration of main structural proteins to lower molecular weights and invasion of macrophages, occurred as well when the removal of the Z-lines and decrease of a-actinin were inhibited by coadministration of a protease inhibitor, Ep-475. The following possibilities can be considered to explain the above results. The first is that the intramuscular concentration of Ep-475 after 48 h of injection is too low to inhibit cathepsins B and L. According to Ohzeki et al. [42] the serum level of Ep-475 reached the maximum within 1 h on subcutaneous injection and the agent was then secreted immediately into the urine. The second is that exogenously added Ep-475 does not

341

inhibit macrophage enzymes. When the macrophages were cultured in the presence of Ep-475 (10-3-10 -9 M) for 48 h, a specific decrease in cathepsins B and L activity was observed (K i = 1.0 /xM). However, the Ki value was significantly higher than that observed in vitro ( K i = 0.2 /.tM) (S. Ishiura, unpublished data). The results suggest that a relatively higher concentration of Ep-475 may be necessary to inhibit cathepsins B and L in ~macrophages than theoretically predicted. It is interesting that Z-line loss is not necessary for the subsequent step of myofibrillar degeneration. In many pathological states, including human muscular dystrophy [43-45], drug-induced degeneration of skeletal muscle [46] and ischemia [47], Z-line loss is a common phenomenon, suggesting that the preferential removal of the Z-lines would cause fragmentation of the myofibrils and the following myofibrillar degeneration. In plasmocid-induced myopathy, fragmentation of the myofibrils was observed on electron microscopy whereas the Z-lines were fairly preserved when Ep-475 was coinjected. So we assume that digestion of the Z-lines is not necessarily the trigger of macrophage invasion. In conclusion, a two-step mechanism of myofibrillar protein degradation was discovered in acute plasmocid-induced muscle degeneration in vivo. Step 1 (0-12 h): damage occurs in the plasma membrane, which causes Ca2+ influx into the muscle cells. Direct fragmentation of the myofibrils can occur, or this can be preceded by the dissolution of Z-lines by a Ca2+-activated proteinase. Step 2 (12-72 h); invasion of phagocytes is followed by an increase in lysosomal cathepsins (mainly derived from macrophages). The end result is degradation of the structural proteins. Lysosomes in affected muscle seem not to be involved in protein breakdown as in normal muscle cells [48,49]. The decrease of lysosomal enzyme activities in the early stage of necrosis strongly implies that the degradation of structural proteins is accomplished via lysosomal enzymes of nonmuscle origin. In addition, we have recently demonstrated that inhibition of macrophage proliferation by cycloheximide prevented acute drug-induced degradation of structural proteins [50]. All of the results suggested that the crucial step of the degradation of myofibrillar proteins is the lysosomal process. Although we cannot say at

present whether this mechanism occurs in all kinds of acute muscle necrosis, the evidence that different proteases of different origins are involved in myofibrillar degeneration may have some therapeutic implications.

Acknowledgments We thank Professor N. Katunuma and Drs. E. Kominami and K. Hanada for providing synthetic substrates. We also thank Misses Kikue Tsuchiya, Satomi Okada and Harumi Anraku for their excellent technical assistance. This work was supported by a Grant in Aid for New Drug Development from the Ministry of Health and Welfare, Japan (S.I.) and by Grant 82-04 from the National Center for Nervous, Mental and Muscular Disorders of the Ministry of Health and Welfare, Japan (H.S.).

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