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Skeletal Muscle Degeneration and Regeneration after Injection of Bothropstoxin-II, a Phospholipase A, Isolated from the Venom of the Snake Bothrops jararacussu J. M. GUT&RREZ,* J. NI%IEZ,* C. DfAz,*,t A. C. 0. CINTRA,$ M. I. HOMSI-BRANDEBURG~,S~ AND J. R. GIGLIOS *Institute Clodomiro Picado, Facultad de Microbiologia, and ?Departamento de Fisiologia, Facultad de Medicinn, Vniversidad de Costa Rica, San Josh, Costa Rica; #Departamento de Bioquimica, Faculdade de Medicina VSP, Ribeirrio Preto-SP, Brazil; and IDepartamento de Qut’mica e Geockkcias- IBILCE-VNESP, Sao Josh do Rio Preto-SP, Brazil Received April 17, 1991, and in revised form July 15, 1991 A myotoxic phospholipase A,, named bothropstoxin II (BthTX-II), was isolated from the venom of the South American snake Bothrops jararacussu and the pathogenesis of myonecrosis induced by this toxin was studied in mice. BthTX-II induced a rapid increase in plasma creatine kinase levels. Histological and ultrastructural observations demonstrate that this toxin affects muscle fibers by first disrupting the integrity of plasma membrane, as “delta lesions” were the earliest morphological alteration and since the plasma membrane was interrupted or absent in many portions. In agreement with this hypothesis, BthTX-II released peroxidase entrapped in negatively charged multilamellar liposomes and behaved as an amphiphilic protein in charge shift electrophoresis, an indication that its mechanism of action might be based on the interaction and disorganization of plasma membrane phospholipids. Membrane damage was followed by a complex series of morphological alterations in intracellular structures, most of which are probably related to an increase in cytosolic calcium levels. Myofilaments became hypercontracted into dense clumps which alternated with cellular spaces devoid of myofibrillar material. Later on, myofilaments changed to a hyaline appearance with a more uniform distribution. Mitochondria were drastically affected, showing high amplitude swelling, vesiculation of cristae, formation of flocculent densities, and membrane disruption. By 24 hr, abundant polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic fibers. Muscle regeneration proceeded normally, as abundant myotubes and regenerating myofibers were observed 7 days after BthTX-II injection. By 28 days regenerating fibers had a diameter similar to that of adult muscle fibers, although they presented two distinctive features: central location of nuclei and some fiber splitting. This good regenerative response may be explained by the observation that BthTX-II does not affect blood vessels, nerves, or basal laminae. 0 1991 Academic Press, Inc.
INTRODUCTION The large majority of snake bites in Brazil are caused by species of the genus Bothrops (Rosenfeld, 1971; Amaral et al., 1987). B. jurarucu~~u is a large snake distributed in the southern regions of this country (Campbell and Lamar, 1989). It induces envenomations characterized by pronounced local tissue damage as well as by systemic effects (Rosenfeld, 1971; Amaral et al., 1985). Local myonecrosis induced by B. jururucussu venom has been studied experimentally in viva (Mebs et al., 1983; Queiroz et al., 1984) and in vitro (RodriguesSimioni ef al., 1983). Moreover, two basic myotoxins have been purified to homogeneity from this venom. One of them, named bothropstoxin, is devoid of phospholipase A, activity although biochemically it resembles phospholipases in amino acid composition as well as in the sequence of its N-terminal region (Hornsi-Brandeburgo ef al., 1988). Another myotoxin having phospholipase A2 217 0014-4800191 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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activity was also isolated from this venom. In this work we investigated the pathogenesis of skeletal muscle damage induced by experimental injections of this toxin, here named bothropstoxin II (BthTX-II). MATERIALS
AND METHODS
Toxin. BthTX-II was purified from B. jararacussu venom by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on SPSephadex C-25, as described by Homsi-Brandeburgo ef al. (1988). BthTX-II corresponds to the fraction designated by these authors as Sm-SPrv. Homogeneity was demonstrated by polyacrylamide gel electrophoresis using a B-alanine-acetic acid buffer, pH 4.5 (Reisfeld et al., 1%2), as well as by isoelectric focusing and immunoelectrophoresis (Horns&Brandeburgo ef al., 1988). Biological activities ofthe toxin. Fifty micrograms of BthTX-II, dissolved in 50 l,~l of phosphate-buffered saline solution, pH 7.2 (PBS), was injected intramuscularly in the right gastrocnemius muscle of five white mice (18-20 g). Control animals were injected with 50 l.~l of PBS. At 1,3,6, and 24 hr blood samples were collected by cutting the tip of the tail and placing the blood in heparinized capillary tubes. Plasma was separated and creatine kinase (EC 2.7.3.2) activity was quantitated using the Sigma kit No. 520 (Sigma Chemical Co., St. Louis, MO). Enzyme activity was expressed in units per milliliter, 1 unit resulting in the phosphorylation of 1 nanomole of creatine per minute at 25°C. Anticoagulant activity on sheep platelet-poor plasma and phospholipase A2 activity on egg yolk were studied as described by Lomonte et al. (1990). Effect of myotoxin on liposomes. The release of peroxidase entrapped in negatively charged multilamellar liposomes by BthTX-II was studied as described by Diaz et al. (1991). Liposomes were made of phosphatidylcholine:dicetyl phosphate:cholesterol (at molar ratio of 7:2:1). Peroxidase release was expressed as percentage, taking as 100% the absorbance of samples in which liposomes were incubated with 0.2% Triton X-100 instead of toxin. To correct for spontaneous release, controls in which liposomes were incubated with PBS were run and absorbances were subtracted from sample readings. Charge shift electrophoresis. The procedure described by Helenius and Simons (1977) was used with some modifications. Myotoxin was separated electrophoretically in 1% agarose gels with a buffer containing three different combinations of detergents: (a) 0.05 M glycine-NaOH, 0.1 M NaCl, pH 9.0, containing 0.5% Triton X-100; (b) the same buffer but containing in addition 0.05 g% of the cationic detergent cetyltrimethylammonium bromide (CTAB); and (c) the same buffer as in (a), but containing in addition 0.25% of the anionic detergent deoxycholate. Electrophoreses were run at 48 mA for 20 Vhr. Then, gels were fixed with methanol: acetic acid:water (50:7:43), stained with Coomassie Blue R-250 and destained with methanohethanohacetic acid:water (20: 10:5:65). Experiments were performed three times in each buffer system and the migration of the toxin was measured. Turkey ovalbumin (Sigma) was also run as a nonamphiphilic protein (Helenius and Simons, 1977). Histological and ultrastructural studies. Groups of four white mice (18-20 g) were injected intramuscularly in the right gastrocnemius with 50 pg of BthTX-II dissolved in 50 ~1 of PBS. Controls were injected with 50 t.~l PBS. At various time intervals (15 min, 1, 3, 6, and 24 hr, and 7 and 28 days) mice were sacrificed by
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cervical dislocation and a sample of injected muscle was obtained. The tissue was immediately fixed in Karnovsky’s fixative (2.5% glutaraldehyde, 2% paraformaldehyde, 0.1 M phosphate buffer, pH 7.4) for 2 hr, washed in phosphate buffer, pH 7.2, and postfixed in 1% osmium tetroxide. Then, samples were dehydrated in ethanol and embedded in Spurr resin. Thick (1.5 pm) sections were stained with toluidine blue, and thin (silver to light gold) sections were stained with uranyl acetate and lead citrate and examined in Hitachi HU-12 and H-7000 electron microscopes. RESULTS Properties of the toxin. BthTX-II had a phospholipase A2 activity of 96 FEq/ mg.min and prolonged recalcification time of sheep platelet-poor plasma. Toxin doses of 1.25 pg and higher rendered plasma uncoagulable. In addition, intramuscular injections of BthTX-II in mice induced an increase in plasma creatine kinase levels (Fig. 1). Creatine kinase was elevated rapidly after injection, reaching its highest values at 3 hr and decreasing afterwards. When charge shift electrophoreses were run, BthTX-II behaved as an amphiphilic protein. The toxin migrated 3 mm toward the cathode in gels containing only Triton X-100 as detergent. When CTAB was included in the gels, myotoxin migrated 8 mm toward the cathode, whereas when deoxycholate was included in the gels the toxin had an anodic migration of 12 mm. In contrast, ovalbumin had the same migration (15 mm toward the anode) in the three systems tested, behaving as a nonamphiphilic protein. Effects ofBthTX-ZZ on liposomes. BthTX-II induced a dose-dependent release of peroxidase entrapped in large multilamellar liposomes (Fig. 2). A toxin concentration of 80 pg/ml induced 50% peroxidase release. Pathological Changes Induced by Bothropstoxin-ZZ Macroscopic observations. Mice had difhculties in mobilizing their right hind leg 2 to 3 min after toxin injection. After approximately 10 min there was a moderate swelling of the injected muscle mass, lasting for about 6 hr. Mice injected with PBS did not show locomotion difficulties nor swelling. Five days after toxin injection mice lOooz
aoo-
I 1
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? :” 5
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TIME (hr) FIG. 1. Changes in plasma creatine kinase levels after intramuscular injection of 50 pg of BthTX-II or PBS (0) in mouse gastrocnemius muscle. Results are expressed as means f SEM (n = 5 in toxin-injected mice and n = 3 in PBS-injected mice). For definition of CK units, see Materials and Methods.
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TOXIN (w/ml)
FIG. 2. Release of peroxidase entrapped in negatively charged multilamellar liposomes incubated with various concentrations of BthTX-II. Release is expressedas percentage, taking as 100% the peroxidase release from liposomes incubated with 0.2% Triton X-100. Results are expressed as mean + SEM (n = 4).
mobilized their right leg normally. Gross examination of injected muscle revealed the absence of hemorrhagic lesions at all times studied. Histological observations. Muscle samples from mice injected with PBS had the typical histological pattern of normal skeletal muscle, with no signs of alterations in muscle fibers, blood vessels, or nerves. In contrast, samples collected from mice injected with BthTX-II had evidence of muscle fiber damage as early as 15 min. At this time interval most affected cells had focal lesions in their periphery, identical to the “delta lesions” that have been described in a variety of muscle pathologies (Mokri and Engel, 1975; Pestronk et al., 1982; GutiCrrez et al., 1984b, 1989). At 15 min, but especially at 1 and 3 hr, a large number of muscle fibers were drastically affected, with the formation of dense clumps of hypercontracted myofibrils alternating with cytoplasmic areas apparently devoid of myofibrils (Fig. 3). At 6 and 24 hr the appearance of necrotic cells shifted from the clumped pattern to a more hyaline pattern, as myofibrillar material had a lighter staining and was more uniformly distributed in the cellular space (Fig. 4). BthTXII did not affect intramuscular nerves nor blood vessels at any time period examined. Inflammatory infiltrate in necrotic muscle was mild at 6 hr and increased prominently by 24 hr. Both polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic muscle fibers (Fig. 4). Samples obtained 7 days after toxin injection presented a widespread process of muscle regeneration, with abundant myotubes and myofibers. This process was well advanced at 28 days, when the diameter of regenerating fibers was similar to that of normal adult muscle fibers. Throughout this process, regenerating muscle fibers had centrally located nuclei and some of them were split. There was no fibrosis or adipose tissue proliferation and regenerating fibers filled most of the tissue. Ultrustructurul observations. Focal, wedge-shaped lesions located in the periphery of muscle fibers seemed to be the earliest morphological alterations in muscle samples collected 15 min and 1 hr after BthTX-II injection (Fig. 5). In these areas of degeneration myofdaments were hypercontracted, becoming amorphous dense clumps. The plasma membrane in these areas was typically interrupted or absent (Fig. 6) and mitochondria were swollen. Other fibers were in an
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FIG. 3. Light micrograph of skeletal muscle taken 3 hr after BthTX-II injection. Abundant necrotic fibers (n) with clumped myofibrils are observed. A mild inflammatory infiltrate is present (1500x).
apparently more advanced stage of degeneration, with most of their myofilaments forming clumped, hypercontracted masses (Fig. 7). In these cells there were large interruptions in the continuity of the plasma membrane, although the basal lamina remained morphologically intact at the periphery of the fibers (Figs. 6 and 7). At
FIG. 4. Light micrograph of skeletal muscle 24 hr after toxin injection. Necrotic fibers (n) have a hyaline appearance with myofibrillar material uniformly distributed in the cellular space. Some normal muscle fibers (m) are observed. Note the abundant phagocytic cells in the interstitial space and inside necrotic fibers (1500~).
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FIG. 5. Electron micrograph of muscle 1 hr after toxin injection. A wedge-shaped lesion (“delta lesion,” D) is observed in the periphery of one fiber. Notice the hypercontraction of myofdaments which leaves a space devoid of myofibrils (5250x).
6 hr, and more evidently at 24 hr, myofibrillar material had a more hyaline pattern and were uniformly distributed in the cellular space. Mitochondrial alterations were prominent at all time periods, being characterized by high amplitude swelling (Fig. 8), formation of flocculent densities, vesiculated cristae, and rupture of their membranes (Figs. 8 and 9). The ultrastructure of sarcoplasmic reticulum and T tubules was drastically at&ted in necrotic muscle fibers, since only a population of small vesicles distributed in the cytoplasm was observed. Necrotic fibers had myonuclei with clumped chromatin and interrupted nuclear membranes. Polymorphonuclear leucocytes and macrophages were observed in the vicinity as well as inside necrotic fibers by 24 hr. These phagocytic cells had vacuoles containing myofilaments and mitochondria, evidence that they were involved in phagocytosis of necrotic material. Observations at 7 days showed regenerating myotubes and myofibers, with centrally located nuclei (Fig. 10). In some myotubes myofibrils were in the process of assembly. By 28 days muscle regeneration was well advanced, since regenerating fibers had an ultrastructure similar to that of adult muscle fibers, with the exception of the central position of the nuclei (Fig. 11). DISCUSSION Two myotoxins have been isolated from the venom of the South American pit is a basic myoviper Bofhrops jararacussu. One of them, named bothropstoxin, toxic protein lacking phospholipase A, activity, although it has structural features that indicate a close relationship with phospholipases (Homsi-Brandeburgo et al., 1988). A second myotoxin, named bothropstoxin II and used in this work, is a
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FIG. 6. Electron micrograph of a peripheral portion of a necrotic muscle fiber 1 hr after BthTX-II injection. The basal lamina (BL) appears intact and the plasma membrane is interrupted (arrows) in some portions and absent in others (25,000~).
phospholipase A, which induces acute myotoxicity and has, in addition, anticoagulant activity. Biochemically BthTX-II has been characterized in terms of amino acid composition, molecular weight, and isoelectric point (HomsiBrandeburgo ef al., 1988). A similar situation has been described in the case of B.
FIG. 7. Electron micrograph showing two stages in the hypercontraction of myofdaments in two fibers affected by BthTX-II 15 min after injection. Basal laminae remain apparently intact in the periphery of the cells (arrows) whereas plasma membranes are interropted or absent (6400x).
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FIG. 8. Electron micrograph showing a group of affected mitochondria 15 min after BthTX-II injection. Mitochondrial swelling is observed, together with the presence of only one membrane in some portions. Abundant small vesicles are dispersed in the cytosol (17,500~).
venom, from which three myotoxins have been purified. Two of them have high phospholipase A2 activity and are strong anticoagulants when tested on platelet-poor plasma (Gutikrrez et al., 1984a, 1986a; Kaiser et al., 1990), whereas a third one has an extremely low phospholipolytic activity and is not anticoagulant
asper
FIG. 9. Electron micrograph showing a portion of a necrotic fiber 24 hr after BthTX-II injection. Several mitochondria (Mi) present rupture of their membranes and vesiculation of the cristae (28,oooX).
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FIG. 10. Electron micrograph of a small regenerating muscle fiber 7 days after BthTX-II injection. Myofibrils (My) appear in an advanced stage of organization. Notice the presence of abundant mitochondria and a nucleus (Nu) (5100x).
(Lomonte and GutiCrrez, 1989; Francis et al., 1991). Thus, it is likely that Bothraps venoms contain a variety of myotoxins having phospholipase A2 structure but with very different catalytic activities. Our observations indicate that BthTX-II affects skeletal muscle fibers by first
FIG. 11. Electron micrograph of a fully regenerated muscle fiber 28 days after BthTX-II injection. The ultrastructure of this fiber closely resembles that of adult normal fibers, with the exception of the central location of nuclei (Nu) (10,000x).
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altering the plasma membrane. This hypothesis is supported by several lines of evidence: (a) Plasma membrane was interrupted and, in some cases absent, in large portions of the periphery of affected fibers. Since this change was observed 15 min after toxin injection, it is evidence of early membrane damage. (b) Wedgeshaped lesions, “delta lesions” (Mokri and Engel, 1975), were the most abundant early morphological alterations in affected fibers. Similar lesions have been described in other muscle pathologies characterized by an initial disruption of the plasma membrane. Some examples are Duchenne muscular dystrophy (Mokri and Engel, 1975) and myotoxicity induced by several membrane-active agents (Pestronk et al., 1982), bupivacaine (Nonaka et al., 1983), Bothrops asper myotoxin I (Gutierrez et al., 1984b), crotoxin (Gopalakrishnakone et al., 1984), Micrurus nigrocinctus venom (Gutierrez et al., 1986b), and Bothrops nummifer myotoxin (Gutierrez et al., 1989). It was postulated that these lesions correspond to focal areas of degeneration underlying portions of the fiber where the plasma membrane has been lost (Mokri and Engel, 1975). (c) There was a rapid increase in plasma creatine kinase levels after BthTX-II injection. Since this enzyme is abundant in the cytosol of skeletal muscle fibers, this finding suggeststhat the plasma membrane of these cells has rapidly lost its ability to regulate the permeability to this enzyme. Similar observations have been made with other myotoxins from Bothrops venoms, such as B. asper myotoxin I (Gutierrez et al., 1984a,b, 1986a) and B. nummifer myotoxin (Gutierrez et al., 1989). Moreover, morphological alterations in muscle affected by the neurotoxic phospholipases A, notexin, taipoxin, and crotoxin can be viewed in the light of the hypothesis that the plasma membrane is the first site of action (Harris et al., 1975, 1980; Harris and Maltin, 1982; Gopalakrishnakone et al., 1984). The mechanism of action of Bothrops myotoxins on muscle plasma membrane remains elusive. Our data on liposomes and charge shift electrophoresis indicate that BthTX-II behaves as an amphiphilic protein and disrupts negatively charged phospholipid bilayers, a finding that has been also observed with other myotoxins from the venoms of B. asper, B. nummifer, B. atrox, and B. moojeni (Gutierrez et al., 1989; Dlaz et al., 1991; Gene et al., in preparation). Thus, it is likely that BthTX-II interacts and disrupts the phospholipid bilayer of skeletal muscle plasma membrane. However, more experimental work is necessary in order to define the precise mechanism of action. The observed disruption in the integrity of the plasma membrane rapidly after toxin injection has several immediate deleterious effects. One of them is a prominent calcium influx, following a steep electrochemical gradient that normally exists across the plasma membrane (Carafoli, 1982). Such increase in cytosolic calcium levels causes hypercontraction of myofilaments (Gutietrez et al., 1990), mitochondrial alterations (Wrogeman and Pena, 1976; Trump et al., 1982), and activation of calcium-dependent proteases and phospholipases (Trump et al., 1982; Pestronk et al., 1982). Initially, hypercontraction causes clumping of myofilaments into dense masses.Then, after 6 hr, myofilaments present a more uniform distribution in the cellular space. These findings are very similar to those described in myonecrosis induced by B. asper myotoxin I (Gutierrez et al., 1990), an indication that plasma membrane damage by these toxins causes a similar series of degenerative changes in myofibrils. On the other hand, necrotic fibers had mitochondria with high amplitude swelling, vesiculated cristae, and flocculent
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densities, typical features of mitochondrial damage in acute cell injury (Trump et al., 1982). Removal of necrotic material by phagocytes was under way 24 hr after injection of BthTX-II. Then, a normal and successful regenerative process followed. Regenerative fibers reached the diameter and morphology of adult normal fibers, with the exception of central location of the nuclei and some fiber splitting. Both of these phenomena have been described in regenerating muscle in a variety of muscle pathologies (Mastaglia ef al., 1970; Bradley, 1979; Harris and Maltin, 1982; Gutierrez et al. 1984~). Since intact vasculature and nerve supply are basic requirements for regeneration (Allbrook, 1981; Plaghki, 1985), the basis for the good regenerative response observed might be due to the fact that BthTX-II, similarly to bothropstoxin, does not affect blood vessels nor nerves. In addition, this toxin does not seem to affect the integrity of the basal lamina which surrounds muscle fibers. This is another element that might contribute to good regeneration, as basal lamina plays a critical role in orderly cell replacement (Vracko and Benditt, 1972; Grounds, 1990). In agreement with these observations, regeneration following myonecrosis induced by purified myotoxins and local anesthetics is adequate (Harris and Maltin, 1982; Nonaka et al., 1983; Gutierrez et al., 1984c, 1989). This contrasts with regeneration after injection of venoms containing both myotoxic and hemorrhagic components, where regenerative response is poor, probably due to venom-induced damage to microvasculature, nerves, and extracellular matrix (Queiroz et al., 1984; Queiroz and Petta, 1984; Gutitrrez et al., 1984~; Arce et al., 1991). ACKNOWLEDGMENTS The authors thank Dr. Francisco Hem&ndez, Dr. Olga Arroyo and Lit Lisela Moreira, as well as the staff of the Electron Microscopy Unit and Institute Clodomiro Picado, University of Costa Rica, for their collaboration. This project was supported by Vicerrectorta de Investigaci6n, University of Costa Rica, the Third World Academy of Sciences (Grant BC 8&54), the Japanese International Cooperation Agency (JICA), Conselho National de Desenvolvimiento Cientifico e Tecnol6gico (CNPq) and Fundacao de Amparo a Pesquisa do E&ado de S&o Paul0 (FAPESP). J. M. Gutidrrez is a recipient of a research career award from the Costa Rican National Scientific and Technological Research Council (CONICIT).
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from Bothrops snake venoms on multilamellar liposomes: Relationship to phospholipase A2, anticoagulant and myotoxic activities. Biochim. Biophys. Actu, in press. FRANCIS, B., Gur~Faanz, J. M., LOMONTE, B., and KAISER, I. I. (1991). Myotoxin II from Bothrops asper (terciopelo) venom is a lysine-49 phospholipase A,. Arch. Biochem. Biophys. 284, 352359. GOPALAKRISHNAKONE, P., DEMPSTER, D. W., HAWG~~D, B. J., and ELDER, H. Y. (1984). Cellular and mitochondrial changes induced in the structure of murine skeletal muscle by crotoxin, a neurotoxic phospholipase A, complex. Toxicon 22, 85-98. GROUNDS, M. (1990). Factors controlling skeletal muscle regeneration in vivo. In “Pathogenesis and Therapy of Duchenne and Becker muscular dystrophy” (B. A. Kakulas and F. L. Mastaglia, Eds.), pp. 171-180. Raven Press, New York. GUTIBRREZ, J. M., OWNBY, C. L., and ODELL, G. V. (1984a). Isolation of a myotoxin from Bothrops asper venom: Partial characterization and action on skeletal muscle. Toxicon 22, 115-128. GUTI~RREZ, J. M., OWNBY, C. L., and ODELL, G. V. (1984b). Pathogenesis of myonecrosis induced by crude venom and a myotoxin of Bothrops asper. Bxp. Mol. Pathol. 40, 367-379. GUTII~RREZ, J. M., OWNBY, C. L., and ODELL, G. V. (1984c). Skeletal muscle regeneration after myonecrosis induced by crude venom and a myotoxin from the snake Bothrops asper (Fer-deLance). Toxicon 22, 719-731. GUTIERREZ, J. M., LOMONTE, B., CHAVES, F., MORENO, E., and CERDAS, L. (1986a). Pharmacological activities of a toxic phospholipase A isolated from the venom of the snake Bothrops asper. Comp. Biochem. Physiol. 84C, 159-164. GUTII~RREZ, J. M., ARROYO, O., CHAVES, F., LOMONTE, B., and CERDAS, L. (1986b). Pathogenesis of myonecrosis induced by coral snake (Micrurus nigrocinctus) venom in mice. Br. .I. Exp. Pathol. 67, 1-12. GUTIBRREZ, J. M., CHAVES, F., GENB, J. A., LOMONTE, B., CAMACHO, Z., and SCHOSINSKY, K. (1989). Myonecrosis induced in mice by a basic myotoxin isolated from the venom of the snake Bothrops nummifer (jumping viper) from Costa Rica. Toxicon 27, 735-745. GUTIERREZ, J. M., ARCE, V., BRENES, F., and CHAVES, F. (1990). Changes in myotibrillar components after skeletal muscle necrosis induced by a myotoxin isolated from the venom of the snake Bothrops
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HARRIS, J. B., and MALTIN, C. A. (1982). Myotoxic activity of the crude venom and the principal neurotoxin, taipoxin, of the Australian taipan, Oxyuranus scutellatus. Br. J. Pharmacol. 76,61-75. HARRIS, J. B., JOHNSON, M. A., and KARLSSON, E. (1975). Pathological responses of rat skeletal muscle to a single subcutaneous injection of a toxin isolated from the venom of the Australian tiger snake, Notechis scutatus scutatus. Clin. Exp. Pharmacol. Physiol. 2, 383-W. HARRIS, J. B., JOHNSON, M. A., and MACDONELL, C. A. (1980). Muscle necrosis induced by some presynaptically active neurotoxins. In “Natural Toxins” (D. Eaker and T. Wadstrom, Eds.), pp. 569-578. Pergamon Press, Oxford. HELENIUS, A., and SIMONS, K. (1977). Charge shift electrophoresis: Simple method for distinguishing between amphiphilic and hydrophilic proteins in detergent solution. Proc. Natl. Acad. Sci. USA 74, 529-532. HOMSI-BRANDEBURGO, M. I., QUEIROZ, L. S., SANTO-NETO, H., RODRIGUES-SIMIONI, L., and GIGLIO, J. R. (1988). Fractionation of Borhrops jararacussu snake venom: Partial chemical characterization and biological activity of bothropstoxin. Toxicon 26, 605-627. KAISER, I. I., GUTI~RREZ, J. M., PLUMMER, D., AIRD, S. D., and ODELL, G. V. (1998). The amino acid sequence of a myotoxic phospholipase from the venom of Bothrops asper. Arch. Biochem. Biophys. 278, 319-325. LOMONTE, B., and GUTI~XREZ, J. M. (1989) A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo). Toxicon 27, 725-733. LOMONTE, B., GUTIBRREZ, J. M., FURTADO, M. F., OTERO, R., Rosso, J. P., VARGAS, O., CARMONA, E., and ROVIRA, M. E. (1990). Isolation of basic myotoxins from Bothrops moojeni and Bothrops atrox snake venoms. Toxicon 28, 1137-l 146. MASTAGLIA, F. L., PAPADIMITRIOU, J. M., and KAKULAS, B. A. (1970). Regeneration of muscle in Duchenne muscular dystrophy: An electron microscopic study. J. Neural. Sci. 11, 4254&t. MEBS, D., EHRENFELD, M., and SAMEJIMA, Y. (1983). Local necrotizing effect of snake venoms on skin and muscle: Relationship to serum creature kinase. Toxicon 21, 39m. MOKRI, B., and ENGEL, A. G. (1975). Duchenne dystrophy: Electron microscopic findings pointing to a basic or early abnormality in the plasma membrane of the muscle fiber. Neurology 25, 111 l-l 120.
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NONAKA, I., TAKAGI, A., ISHIURA, S., NAKASE, H., and SUGITA, H. (1983). Pathophysiology of muscle fiber necrosis induced by bupivacaine-hydrochloride. Acta Neuropathof. 60, 167-174. PESTRONK, A., PARHAD, I. M., DRACHMAN, D. B., and PRICE, D. L. (1982). Membrane myopathy: Morphological similarities to Duchenne muscular dystrophy. Muscle Nerve 5, 209-214. PLAGHKI, L. (1985). Regeneration et myogenese du muscle strib. .Z. Physiol Paris 80, 51-110. QUEIROZ, L. S., and PETTA, C. A. (1984). Histopathological changes caused by venom of urutu snake (Bothrops alternatus) in mouse skeletal muscle. Rev. Inst. Med. Trap. Sao Paul0 26, 247-253. QUEIROZ, L. S., SANTO-NETO, H., RODRIGUES-SIMIONI, L., andPRADo-F~ANCESCHI, J. (1984). Muscle necrosis and regeneration after envenomation by Bothrops jararacussu snake venom. Toxicon 22,339346.
REISFELD, R. A., LEWIS, U. J., and WILLIAMS, D. E. (1962). Disk electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature (London) 195,281~283. RODRIGUES-SIMIONI, L., BORGESE, N., and CECCARELLI, B. (1983). The effects of Bothrops jararacussu venom and its components on frog nerve-muscle preparation. Neuroscience 10. 475-489. ROSENFELD, G. (1971). Symptomatology, pathology and treatment of snake bites in South America. In “Venomous Animals and Their Venoms. Vol II. Venomous Vertebrates” (W. Bucherl and E. Buckley, Eds.), pp. 345-384. Academic Press, New York. TRUMP, B. F., BEREZSKY, I. K. and COWLEY, R. A. (1982). The cellular and subcellular characteristics of acute and chronic injury with emphasis on the role of calcium. In “Pathophysiology of Shock, Anoxia and Ischemia” (R. A. Cowley and B. F. Trump, Eds.), pp. 6-l6. Williams and Wilkins, Baltimore. VRACKO, R., and BENDITT, E. P. (1972). Basal lamina: The scaffold for orderly cell replacement. Observations on regeneration of injured skeletal muscle fibers and capillaries. .Z. Cell Biol. 55, 406419. WROGEMAN, K., and PENA, S. D. J. (1976). Mitochondrial calcium overload: A general mechanism for cell necrosis in muscle diseases. Lancet i, 672-673.
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(1991)
Skeletal Muscle Degeneration and Regeneration after Injection of Bothropstoxin-II, a Phospholipase A, Isolated from the Venom of the Snake Bothrops jararacussu J. M. GUT&RREZ,* J. NI%IEZ,* C. DfAz,*,t A. C. 0. CINTRA,$ M. I. HOMSI-BRANDEBURG~,S~ AND J. R. GIGLIOS *Institute Clodomiro Picado, Facultad de Microbiologia, and ?Departamento de Fisiologia, Facultad de Medicinn, Vniversidad de Costa Rica, San Josh, Costa Rica; #Departamento de Bioquimica, Faculdade de Medicina VSP, Ribeirrio Preto-SP, Brazil; and IDepartamento de Qut’mica e Geockkcias- IBILCE-VNESP, Sao Josh do Rio Preto-SP, Brazil Received April 17, 1991, and in revised form July 15, 1991 A myotoxic phospholipase A,, named bothropstoxin II (BthTX-II), was isolated from the venom of the South American snake Bothrops jararacussu and the pathogenesis of myonecrosis induced by this toxin was studied in mice. BthTX-II induced a rapid increase in plasma creatine kinase levels. Histological and ultrastructural observations demonstrate that this toxin affects muscle fibers by first disrupting the integrity of plasma membrane, as “delta lesions” were the earliest morphological alteration and since the plasma membrane was interrupted or absent in many portions. In agreement with this hypothesis, BthTX-II released peroxidase entrapped in negatively charged multilamellar liposomes and behaved as an amphiphilic protein in charge shift electrophoresis, an indication that its mechanism of action might be based on the interaction and disorganization of plasma membrane phospholipids. Membrane damage was followed by a complex series of morphological alterations in intracellular structures, most of which are probably related to an increase in cytosolic calcium levels. Myofilaments became hypercontracted into dense clumps which alternated with cellular spaces devoid of myofibrillar material. Later on, myofilaments changed to a hyaline appearance with a more uniform distribution. Mitochondria were drastically affected, showing high amplitude swelling, vesiculation of cristae, formation of flocculent densities, and membrane disruption. By 24 hr, abundant polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic fibers. Muscle regeneration proceeded normally, as abundant myotubes and regenerating myofibers were observed 7 days after BthTX-II injection. By 28 days regenerating fibers had a diameter similar to that of adult muscle fibers, although they presented two distinctive features: central location of nuclei and some fiber splitting. This good regenerative response may be explained by the observation that BthTX-II does not affect blood vessels, nerves, or basal laminae. 0 1991 Academic Press, Inc.
INTRODUCTION The large majority of snake bites in Brazil are caused by species of the genus Bothrops (Rosenfeld, 1971; Amaral et al., 1987). B. jurarucu~~u is a large snake distributed in the southern regions of this country (Campbell and Lamar, 1989). It induces envenomations characterized by pronounced local tissue damage as well as by systemic effects (Rosenfeld, 1971; Amaral et al., 1985). Local myonecrosis induced by B. jururucussu venom has been studied experimentally in viva (Mebs et al., 1983; Queiroz et al., 1984) and in vitro (RodriguesSimioni ef al., 1983). Moreover, two basic myotoxins have been purified to homogeneity from this venom. One of them, named bothropstoxin, is devoid of phospholipase A, activity although biochemically it resembles phospholipases in amino acid composition as well as in the sequence of its N-terminal region (Hornsi-Brandeburgo ef al., 1988). Another myotoxin having phospholipase A2 217 0014-4800191 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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activity was also isolated from this venom. In this work we investigated the pathogenesis of skeletal muscle damage induced by experimental injections of this toxin, here named bothropstoxin II (BthTX-II). MATERIALS
AND METHODS
Toxin. BthTX-II was purified from B. jararacussu venom by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on SPSephadex C-25, as described by Homsi-Brandeburgo ef al. (1988). BthTX-II corresponds to the fraction designated by these authors as Sm-SPrv. Homogeneity was demonstrated by polyacrylamide gel electrophoresis using a B-alanine-acetic acid buffer, pH 4.5 (Reisfeld et al., 1%2), as well as by isoelectric focusing and immunoelectrophoresis (Horns&Brandeburgo ef al., 1988). Biological activities ofthe toxin. Fifty micrograms of BthTX-II, dissolved in 50 l,~l of phosphate-buffered saline solution, pH 7.2 (PBS), was injected intramuscularly in the right gastrocnemius muscle of five white mice (18-20 g). Control animals were injected with 50 l.~l of PBS. At 1,3,6, and 24 hr blood samples were collected by cutting the tip of the tail and placing the blood in heparinized capillary tubes. Plasma was separated and creatine kinase (EC 2.7.3.2) activity was quantitated using the Sigma kit No. 520 (Sigma Chemical Co., St. Louis, MO). Enzyme activity was expressed in units per milliliter, 1 unit resulting in the phosphorylation of 1 nanomole of creatine per minute at 25°C. Anticoagulant activity on sheep platelet-poor plasma and phospholipase A2 activity on egg yolk were studied as described by Lomonte et al. (1990). Effect of myotoxin on liposomes. The release of peroxidase entrapped in negatively charged multilamellar liposomes by BthTX-II was studied as described by Diaz et al. (1991). Liposomes were made of phosphatidylcholine:dicetyl phosphate:cholesterol (at molar ratio of 7:2:1). Peroxidase release was expressed as percentage, taking as 100% the absorbance of samples in which liposomes were incubated with 0.2% Triton X-100 instead of toxin. To correct for spontaneous release, controls in which liposomes were incubated with PBS were run and absorbances were subtracted from sample readings. Charge shift electrophoresis. The procedure described by Helenius and Simons (1977) was used with some modifications. Myotoxin was separated electrophoretically in 1% agarose gels with a buffer containing three different combinations of detergents: (a) 0.05 M glycine-NaOH, 0.1 M NaCl, pH 9.0, containing 0.5% Triton X-100; (b) the same buffer but containing in addition 0.05 g% of the cationic detergent cetyltrimethylammonium bromide (CTAB); and (c) the same buffer as in (a), but containing in addition 0.25% of the anionic detergent deoxycholate. Electrophoreses were run at 48 mA for 20 Vhr. Then, gels were fixed with methanol: acetic acid:water (50:7:43), stained with Coomassie Blue R-250 and destained with methanohethanohacetic acid:water (20: 10:5:65). Experiments were performed three times in each buffer system and the migration of the toxin was measured. Turkey ovalbumin (Sigma) was also run as a nonamphiphilic protein (Helenius and Simons, 1977). Histological and ultrastructural studies. Groups of four white mice (18-20 g) were injected intramuscularly in the right gastrocnemius with 50 pg of BthTX-II dissolved in 50 ~1 of PBS. Controls were injected with 50 t.~l PBS. At various time intervals (15 min, 1, 3, 6, and 24 hr, and 7 and 28 days) mice were sacrificed by
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II
cervical dislocation and a sample of injected muscle was obtained. The tissue was immediately fixed in Karnovsky’s fixative (2.5% glutaraldehyde, 2% paraformaldehyde, 0.1 M phosphate buffer, pH 7.4) for 2 hr, washed in phosphate buffer, pH 7.2, and postfixed in 1% osmium tetroxide. Then, samples were dehydrated in ethanol and embedded in Spurr resin. Thick (1.5 pm) sections were stained with toluidine blue, and thin (silver to light gold) sections were stained with uranyl acetate and lead citrate and examined in Hitachi HU-12 and H-7000 electron microscopes. RESULTS Properties of the toxin. BthTX-II had a phospholipase A2 activity of 96 FEq/ mg.min and prolonged recalcification time of sheep platelet-poor plasma. Toxin doses of 1.25 pg and higher rendered plasma uncoagulable. In addition, intramuscular injections of BthTX-II in mice induced an increase in plasma creatine kinase levels (Fig. 1). Creatine kinase was elevated rapidly after injection, reaching its highest values at 3 hr and decreasing afterwards. When charge shift electrophoreses were run, BthTX-II behaved as an amphiphilic protein. The toxin migrated 3 mm toward the cathode in gels containing only Triton X-100 as detergent. When CTAB was included in the gels, myotoxin migrated 8 mm toward the cathode, whereas when deoxycholate was included in the gels the toxin had an anodic migration of 12 mm. In contrast, ovalbumin had the same migration (15 mm toward the anode) in the three systems tested, behaving as a nonamphiphilic protein. Effects ofBthTX-ZZ on liposomes. BthTX-II induced a dose-dependent release of peroxidase entrapped in large multilamellar liposomes (Fig. 2). A toxin concentration of 80 pg/ml induced 50% peroxidase release. Pathological Changes Induced by Bothropstoxin-ZZ Macroscopic observations. Mice had difhculties in mobilizing their right hind leg 2 to 3 min after toxin injection. After approximately 10 min there was a moderate swelling of the injected muscle mass, lasting for about 6 hr. Mice injected with PBS did not show locomotion difficulties nor swelling. Five days after toxin injection mice lOooz
aoo-
I 1
w sooI 3Y %’ 2I= l -• \ is 8 200 -/ o&--O 0
? :” 5
10
15
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5
TIME (hr) FIG. 1. Changes in plasma creatine kinase levels after intramuscular injection of 50 pg of BthTX-II or PBS (0) in mouse gastrocnemius muscle. Results are expressed as means f SEM (n = 5 in toxin-injected mice and n = 3 in PBS-injected mice). For definition of CK units, see Materials and Methods.
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TOXIN (w/ml)
FIG. 2. Release of peroxidase entrapped in negatively charged multilamellar liposomes incubated with various concentrations of BthTX-II. Release is expressedas percentage, taking as 100% the peroxidase release from liposomes incubated with 0.2% Triton X-100. Results are expressed as mean + SEM (n = 4).
mobilized their right leg normally. Gross examination of injected muscle revealed the absence of hemorrhagic lesions at all times studied. Histological observations. Muscle samples from mice injected with PBS had the typical histological pattern of normal skeletal muscle, with no signs of alterations in muscle fibers, blood vessels, or nerves. In contrast, samples collected from mice injected with BthTX-II had evidence of muscle fiber damage as early as 15 min. At this time interval most affected cells had focal lesions in their periphery, identical to the “delta lesions” that have been described in a variety of muscle pathologies (Mokri and Engel, 1975; Pestronk et al., 1982; GutiCrrez et al., 1984b, 1989). At 15 min, but especially at 1 and 3 hr, a large number of muscle fibers were drastically affected, with the formation of dense clumps of hypercontracted myofibrils alternating with cytoplasmic areas apparently devoid of myofibrils (Fig. 3). At 6 and 24 hr the appearance of necrotic cells shifted from the clumped pattern to a more hyaline pattern, as myofibrillar material had a lighter staining and was more uniformly distributed in the cellular space (Fig. 4). BthTXII did not affect intramuscular nerves nor blood vessels at any time period examined. Inflammatory infiltrate in necrotic muscle was mild at 6 hr and increased prominently by 24 hr. Both polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic muscle fibers (Fig. 4). Samples obtained 7 days after toxin injection presented a widespread process of muscle regeneration, with abundant myotubes and myofibers. This process was well advanced at 28 days, when the diameter of regenerating fibers was similar to that of normal adult muscle fibers. Throughout this process, regenerating muscle fibers had centrally located nuclei and some of them were split. There was no fibrosis or adipose tissue proliferation and regenerating fibers filled most of the tissue. Ultrustructurul observations. Focal, wedge-shaped lesions located in the periphery of muscle fibers seemed to be the earliest morphological alterations in muscle samples collected 15 min and 1 hr after BthTX-II injection (Fig. 5). In these areas of degeneration myofdaments were hypercontracted, becoming amorphous dense clumps. The plasma membrane in these areas was typically interrupted or absent (Fig. 6) and mitochondria were swollen. Other fibers were in an
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FIG. 3. Light micrograph of skeletal muscle taken 3 hr after BthTX-II injection. Abundant necrotic fibers (n) with clumped myofibrils are observed. A mild inflammatory infiltrate is present (1500x).
apparently more advanced stage of degeneration, with most of their myofilaments forming clumped, hypercontracted masses (Fig. 7). In these cells there were large interruptions in the continuity of the plasma membrane, although the basal lamina remained morphologically intact at the periphery of the fibers (Figs. 6 and 7). At
FIG. 4. Light micrograph of skeletal muscle 24 hr after toxin injection. Necrotic fibers (n) have a hyaline appearance with myofibrillar material uniformly distributed in the cellular space. Some normal muscle fibers (m) are observed. Note the abundant phagocytic cells in the interstitial space and inside necrotic fibers (1500~).
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FIG. 5. Electron micrograph of muscle 1 hr after toxin injection. A wedge-shaped lesion (“delta lesion,” D) is observed in the periphery of one fiber. Notice the hypercontraction of myofdaments which leaves a space devoid of myofibrils (5250x).
6 hr, and more evidently at 24 hr, myofibrillar material had a more hyaline pattern and were uniformly distributed in the cellular space. Mitochondrial alterations were prominent at all time periods, being characterized by high amplitude swelling (Fig. 8), formation of flocculent densities, vesiculated cristae, and rupture of their membranes (Figs. 8 and 9). The ultrastructure of sarcoplasmic reticulum and T tubules was drastically at&ted in necrotic muscle fibers, since only a population of small vesicles distributed in the cytoplasm was observed. Necrotic fibers had myonuclei with clumped chromatin and interrupted nuclear membranes. Polymorphonuclear leucocytes and macrophages were observed in the vicinity as well as inside necrotic fibers by 24 hr. These phagocytic cells had vacuoles containing myofilaments and mitochondria, evidence that they were involved in phagocytosis of necrotic material. Observations at 7 days showed regenerating myotubes and myofibers, with centrally located nuclei (Fig. 10). In some myotubes myofibrils were in the process of assembly. By 28 days muscle regeneration was well advanced, since regenerating fibers had an ultrastructure similar to that of adult muscle fibers, with the exception of the central position of the nuclei (Fig. 11). DISCUSSION Two myotoxins have been isolated from the venom of the South American pit is a basic myoviper Bofhrops jararacussu. One of them, named bothropstoxin, toxic protein lacking phospholipase A, activity, although it has structural features that indicate a close relationship with phospholipases (Homsi-Brandeburgo et al., 1988). A second myotoxin, named bothropstoxin II and used in this work, is a
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FIG. 6. Electron micrograph of a peripheral portion of a necrotic muscle fiber 1 hr after BthTX-II injection. The basal lamina (BL) appears intact and the plasma membrane is interrupted (arrows) in some portions and absent in others (25,000~).
phospholipase A, which induces acute myotoxicity and has, in addition, anticoagulant activity. Biochemically BthTX-II has been characterized in terms of amino acid composition, molecular weight, and isoelectric point (HomsiBrandeburgo ef al., 1988). A similar situation has been described in the case of B.
FIG. 7. Electron micrograph showing two stages in the hypercontraction of myofdaments in two fibers affected by BthTX-II 15 min after injection. Basal laminae remain apparently intact in the periphery of the cells (arrows) whereas plasma membranes are interropted or absent (6400x).
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FIG. 8. Electron micrograph showing a group of affected mitochondria 15 min after BthTX-II injection. Mitochondrial swelling is observed, together with the presence of only one membrane in some portions. Abundant small vesicles are dispersed in the cytosol (17,500~).
venom, from which three myotoxins have been purified. Two of them have high phospholipase A2 activity and are strong anticoagulants when tested on platelet-poor plasma (Gutikrrez et al., 1984a, 1986a; Kaiser et al., 1990), whereas a third one has an extremely low phospholipolytic activity and is not anticoagulant
asper
FIG. 9. Electron micrograph showing a portion of a necrotic fiber 24 hr after BthTX-II injection. Several mitochondria (Mi) present rupture of their membranes and vesiculation of the cristae (28,oooX).
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FIG. 10. Electron micrograph of a small regenerating muscle fiber 7 days after BthTX-II injection. Myofibrils (My) appear in an advanced stage of organization. Notice the presence of abundant mitochondria and a nucleus (Nu) (5100x).
(Lomonte and GutiCrrez, 1989; Francis et al., 1991). Thus, it is likely that Bothraps venoms contain a variety of myotoxins having phospholipase A2 structure but with very different catalytic activities. Our observations indicate that BthTX-II affects skeletal muscle fibers by first
FIG. 11. Electron micrograph of a fully regenerated muscle fiber 28 days after BthTX-II injection. The ultrastructure of this fiber closely resembles that of adult normal fibers, with the exception of the central location of nuclei (Nu) (10,000x).
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altering the plasma membrane. This hypothesis is supported by several lines of evidence: (a) Plasma membrane was interrupted and, in some cases absent, in large portions of the periphery of affected fibers. Since this change was observed 15 min after toxin injection, it is evidence of early membrane damage. (b) Wedgeshaped lesions, “delta lesions” (Mokri and Engel, 1975), were the most abundant early morphological alterations in affected fibers. Similar lesions have been described in other muscle pathologies characterized by an initial disruption of the plasma membrane. Some examples are Duchenne muscular dystrophy (Mokri and Engel, 1975) and myotoxicity induced by several membrane-active agents (Pestronk et al., 1982), bupivacaine (Nonaka et al., 1983), Bothrops asper myotoxin I (Gutierrez et al., 1984b), crotoxin (Gopalakrishnakone et al., 1984), Micrurus nigrocinctus venom (Gutierrez et al., 1986b), and Bothrops nummifer myotoxin (Gutierrez et al., 1989). It was postulated that these lesions correspond to focal areas of degeneration underlying portions of the fiber where the plasma membrane has been lost (Mokri and Engel, 1975). (c) There was a rapid increase in plasma creatine kinase levels after BthTX-II injection. Since this enzyme is abundant in the cytosol of skeletal muscle fibers, this finding suggeststhat the plasma membrane of these cells has rapidly lost its ability to regulate the permeability to this enzyme. Similar observations have been made with other myotoxins from Bothrops venoms, such as B. asper myotoxin I (Gutierrez et al., 1984a,b, 1986a) and B. nummifer myotoxin (Gutierrez et al., 1989). Moreover, morphological alterations in muscle affected by the neurotoxic phospholipases A, notexin, taipoxin, and crotoxin can be viewed in the light of the hypothesis that the plasma membrane is the first site of action (Harris et al., 1975, 1980; Harris and Maltin, 1982; Gopalakrishnakone et al., 1984). The mechanism of action of Bothrops myotoxins on muscle plasma membrane remains elusive. Our data on liposomes and charge shift electrophoresis indicate that BthTX-II behaves as an amphiphilic protein and disrupts negatively charged phospholipid bilayers, a finding that has been also observed with other myotoxins from the venoms of B. asper, B. nummifer, B. atrox, and B. moojeni (Gutierrez et al., 1989; Dlaz et al., 1991; Gene et al., in preparation). Thus, it is likely that BthTX-II interacts and disrupts the phospholipid bilayer of skeletal muscle plasma membrane. However, more experimental work is necessary in order to define the precise mechanism of action. The observed disruption in the integrity of the plasma membrane rapidly after toxin injection has several immediate deleterious effects. One of them is a prominent calcium influx, following a steep electrochemical gradient that normally exists across the plasma membrane (Carafoli, 1982). Such increase in cytosolic calcium levels causes hypercontraction of myofilaments (Gutietrez et al., 1990), mitochondrial alterations (Wrogeman and Pena, 1976; Trump et al., 1982), and activation of calcium-dependent proteases and phospholipases (Trump et al., 1982; Pestronk et al., 1982). Initially, hypercontraction causes clumping of myofilaments into dense masses.Then, after 6 hr, myofilaments present a more uniform distribution in the cellular space. These findings are very similar to those described in myonecrosis induced by B. asper myotoxin I (Gutierrez et al., 1990), an indication that plasma membrane damage by these toxins causes a similar series of degenerative changes in myofibrils. On the other hand, necrotic fibers had mitochondria with high amplitude swelling, vesiculated cristae, and flocculent
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densities, typical features of mitochondrial damage in acute cell injury (Trump et al., 1982). Removal of necrotic material by phagocytes was under way 24 hr after injection of BthTX-II. Then, a normal and successful regenerative process followed. Regenerative fibers reached the diameter and morphology of adult normal fibers, with the exception of central location of the nuclei and some fiber splitting. Both of these phenomena have been described in regenerating muscle in a variety of muscle pathologies (Mastaglia ef al., 1970; Bradley, 1979; Harris and Maltin, 1982; Gutierrez et al. 1984~). Since intact vasculature and nerve supply are basic requirements for regeneration (Allbrook, 1981; Plaghki, 1985), the basis for the good regenerative response observed might be due to the fact that BthTX-II, similarly to bothropstoxin, does not affect blood vessels nor nerves. In addition, this toxin does not seem to affect the integrity of the basal lamina which surrounds muscle fibers. This is another element that might contribute to good regeneration, as basal lamina plays a critical role in orderly cell replacement (Vracko and Benditt, 1972; Grounds, 1990). In agreement with these observations, regeneration following myonecrosis induced by purified myotoxins and local anesthetics is adequate (Harris and Maltin, 1982; Nonaka et al., 1983; Gutierrez et al., 1984c, 1989). This contrasts with regeneration after injection of venoms containing both myotoxic and hemorrhagic components, where regenerative response is poor, probably due to venom-induced damage to microvasculature, nerves, and extracellular matrix (Queiroz et al., 1984; Queiroz and Petta, 1984; Gutitrrez et al., 1984~; Arce et al., 1991). ACKNOWLEDGMENTS The authors thank Dr. Francisco Hem&ndez, Dr. Olga Arroyo and Lit Lisela Moreira, as well as the staff of the Electron Microscopy Unit and Institute Clodomiro Picado, University of Costa Rica, for their collaboration. This project was supported by Vicerrectorta de Investigaci6n, University of Costa Rica, the Third World Academy of Sciences (Grant BC 8&54), the Japanese International Cooperation Agency (JICA), Conselho National de Desenvolvimiento Cientifico e Tecnol6gico (CNPq) and Fundacao de Amparo a Pesquisa do E&ado de S&o Paul0 (FAPESP). J. M. Gutidrrez is a recipient of a research career award from the Costa Rican National Scientific and Technological Research Council (CONICIT).
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from Bothrops snake venoms on multilamellar liposomes: Relationship to phospholipase A2, anticoagulant and myotoxic activities. Biochim. Biophys. Actu, in press. FRANCIS, B., Gur~Faanz, J. M., LOMONTE, B., and KAISER, I. I. (1991). Myotoxin II from Bothrops asper (terciopelo) venom is a lysine-49 phospholipase A,. Arch. Biochem. Biophys. 284, 352359. GOPALAKRISHNAKONE, P., DEMPSTER, D. W., HAWG~~D, B. J., and ELDER, H. Y. (1984). Cellular and mitochondrial changes induced in the structure of murine skeletal muscle by crotoxin, a neurotoxic phospholipase A, complex. Toxicon 22, 85-98. GROUNDS, M. (1990). Factors controlling skeletal muscle regeneration in vivo. In “Pathogenesis and Therapy of Duchenne and Becker muscular dystrophy” (B. A. Kakulas and F. L. Mastaglia, Eds.), pp. 171-180. Raven Press, New York. GUTIBRREZ, J. M., OWNBY, C. L., and ODELL, G. V. (1984a). Isolation of a myotoxin from Bothrops asper venom: Partial characterization and action on skeletal muscle. Toxicon 22, 115-128. GUTI~RREZ, J. M., OWNBY, C. L., and ODELL, G. V. (1984b). Pathogenesis of myonecrosis induced by crude venom and a myotoxin of Bothrops asper. Bxp. Mol. Pathol. 40, 367-379. GUTII~RREZ, J. M., OWNBY, C. L., and ODELL, G. V. (1984c). Skeletal muscle regeneration after myonecrosis induced by crude venom and a myotoxin from the snake Bothrops asper (Fer-deLance). Toxicon 22, 719-731. GUTIERREZ, J. M., LOMONTE, B., CHAVES, F., MORENO, E., and CERDAS, L. (1986a). Pharmacological activities of a toxic phospholipase A isolated from the venom of the snake Bothrops asper. Comp. Biochem. Physiol. 84C, 159-164. GUTII~RREZ, J. M., ARROYO, O., CHAVES, F., LOMONTE, B., and CERDAS, L. (1986b). Pathogenesis of myonecrosis induced by coral snake (Micrurus nigrocinctus) venom in mice. Br. .I. Exp. Pathol. 67, 1-12. GUTIBRREZ, J. M., CHAVES, F., GENB, J. A., LOMONTE, B., CAMACHO, Z., and SCHOSINSKY, K. (1989). Myonecrosis induced in mice by a basic myotoxin isolated from the venom of the snake Bothrops nummifer (jumping viper) from Costa Rica. Toxicon 27, 735-745. GUTIERREZ, J. M., ARCE, V., BRENES, F., and CHAVES, F. (1990). Changes in myotibrillar components after skeletal muscle necrosis induced by a myotoxin isolated from the venom of the snake Bothrops
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