Pharmac. Ther. Vol. 48, pp. 223-236, 1990 Printed in Great Britain.All rights reserved

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Associate Editor: A. L. HAaVEY

MYOTOXIC COMPONENTS OF SNAKE VENOMS: THEIR BIOCHEMICAL A N D BIOLOGICAL ACTIVITIES D. MEBS* a n d C. L. OWNBYt *Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt-70, F.R.G. tDepartment of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078, U.S.A. Abstract--Necrosis of skeletal muscle is produced by two types of snake venom components: single chain peptides consisting of 42-44 amino acid residues and phospholipases A2 representing either single chain proteins or existing as complexes of several enzyme subunits or combined with other nonenzymatic proteins. Vacuolation, lysis and necrosis of skeletal muscle cells are the major pathological effects of these myotoxins. Although the exact mode of action of these toxins is not clear, interactions with the plasma membrane leading to permeability changes for ions and to their complete destruction is evident. The high specificities of some venom phospholipases A2for skeletal muscle cells suggest a specificbinding to certain membrane receptors; however, an enzymatic action on membranes may also be involved.

CONTENTS 1. Introduction 2. Snake Venoms: Myotoxic Components and their Chemistry 2.1. Myotoxic peptides 2.2. Myotoxic phospholipases A2 3. Pathogenesis of Myonecrosis 3.1. Normal structure of skeletal muscle 3.2. Effects of myotoxic peptides 3.3. Effects of myotoxic phospholipases A2 4. Relationship between Protein Structure and Myotoxic Activity Acknowledgements References

1. I N T R O D U C T I O N Snake venoms produce a variety of symptoms when injected into experimental animals or in humans in cases of snake bite. Beside acute life-threatening effects such as cardiovascular failure, shock, paralysis and bleeding, local hemorrhage and necrosis affecting the skin and muscle layer is another serious manifestation causing prolonged and sometimes permanent disability. Extensive pathologic alterations are observed after the injection of various snake venoms into muscle tissue, ranging from swelling, edema and hemorrhage to degenerative events and severe myolytic processes (Homma and Tu, 1971; Ownby, 1982; Mebs et al., 1983). In the case of viperine venoms, myotoxic activity is accompanied by strong hemorrhagic activity which complicates the evaluation of the direct myotoxic activity of the venom. Dark brown urine is a common symptom in envenomations due to sea snakes (Marsden and Reid, 1961), Australian elapid snakes (Furtado and Lester, 1968; Rowlands et al., 1969; Harris et al., 1976; Hood and Johnson, 1975; Brigden and Sutherland, 1981) and the South American rattlesnake Crotalus durissus wT 48,2-n

223 224 224 225 226 226 228 232 234 234 234

terrificus (Azevedo-Marques et al., 1985, 1987). Often misdiagnosed as hemoglobinuria (cf. Rosenfeld, 1971), which is a rare event in snake bite, it is in fact myoglobinuria, which may result in renal failure. Large quantities of myoglobin and enzymes like creatine kinase (CK) are released from severely damaged muscle fibers. Although necrotic lesions occur mainly locally, at the site of venom injection, systemic effects are also common where muscles of the whole body are involved. Terms like local necrosis have often been used to describe the effects of venoms on the skin and muscle layer. However, these terms cause confusion and do not really differentiate between local hemorrhage and direct myotoxic effects. Therefore, myotoxicity, the subject of this review, is defined as a specific venom action on skeletal muscle, affecting muscle fibers only and leaving other tissue structures like connective tissue, nerves and vessels essentially unharmed. Myonecrosis is the result of the myotoxic action. Although hemorrhagins from snake venoms have sometimes been considered to be directly involved in myotoxicity (cf. Fabiano and Tu, 1981; Ownby et al., 1978; Komori and Sugihara, 1988), these venom components will not be included in this review.

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After the injection of a myotoxic venom, or more specifically of myotoxic venom components, the skeletal muscle tissue exhibits characteristic changes (as described in Section 3). Depending on the particular myotoxin injected, early (15 min to 3 hr) damage to skeletal muscle cells can be almost immediate, including lysis of the plasma membrane and hypercontraction, or it can be delayed and include dilatation of the sarcoplasmic reticulum. In either case, by 24-48 hr after injection the damaged skeletal muscle cells will appear as amorphous masses invaded by phagocytic cells, probably macrophages. In most cases the basal lamina is not disrupted and after phagocytosis of cellular debris, the muscle cells begin to regenerate. The amount and degree of regeneration depend on the type and dose of toxin injected, but in most cases the regenerated cells are as large as normal muscle cells but still have centrally located nuclei. To detect and evaluate myotoxicity of a venom component, histological examination of the muscle tissue affected is superior to methods such as assaying serum creatine kinase (CK) levels. Increase of CK-activity in serum may also occur in severe hemorrhage when vascular injuries and interrupted blood flow may cause secondary muscle damage. Significant changes in this enzyme activity may be absent in a case of rather moderate myonecrosis (Mebs et al., 1983). Since vacuolation of skeletal muscle cells, which is easily observed at the light microscopic level, is a characteristic effect of myotoxic peptides like myotoxin a, Ownby et al. (1982) used this feature to quantitate myonecrosis induced by this toxin. They obtained a vacuolation index at various times after injection of the toxin and compared these values obtained by measuring the levels of CK in plasma taken at the same time. CK levels exhibited two peaks after myotoxin injection, whereas the amount of myonecrosis measured by histological examination showed only one peak. The first peak in CK levels at 3 hr after injection did not correlate with a high vacuolation index, but did correlate with contraction of the leg induced by the toxin. There was a better correlation if vacuolation indexes from tissue taken at 6-48 hr after injection were considered, times at which the muscle cells became necrotic. Snake venoms are the major source of myotoxic compounds which are of protein or polypeptide nature. However, a myotoxic acidic phosphopholipase A 2 has recently been isolated from the epithelial tissue of the spines of the crown-of-thorn sea star, Acanthaster planci (Mebs, 1990).

2. SNAKE VENOMS: MYOTOXIC COMPONENTS A N D THEIR CHEMISTRY When histological methods for the detection of myotoxicity are applied, more and more snake venoms can be labeled 'myotoxic' (Homma and Tu, 1971; Mebs et al., 1983; Mebs, 1986; Ownby and Colberg, 1988). These include venoms from sea snakes as well as from cobras (Naja species) and coral snakes (Micrurus species), which are usually considered to be 'neurotoxic' venoms. Moreover, quite a number of viper and rattlesnake venoms produce severe muscle tissue lesions in addition to extensive

hemorrhage. In this case clear distinction between primary myonecrosis and secondary effects due to massive bleeding seems to be difficult. On the other hand, venoms like those from Bitis arietans which contain some of the most active hemorrhagins (Mebs and Panholzer, 1982) do not cause any lesions in skeletal muscle. It may therefore be assumed that myotoxic effects seen after the injection of hemorrhagic venoms are due to specific myotoxic factors rather than to secondary effects due to hemorrhagins, which primarily lead to leakage of capillaries and bleeding. It has to be emphasized that myotoxic venom components are extremely potent and are among the most active natural products. Their application in doses of even less than one microgram in experimental animals (mice or rats) often results in massive damage to skeletal muscle fibers. Two types of venom components have been found to be myotoxic: polypeptides like crotamine and myotoxin a, which are free of any enzymatic activity, and phospholipases A 2. Myotoxicity associated with other venom components such as cyto-/cardiotoxins from cobra venoms (Lai et al., 1972) could be attributed to contaminating phospholipases A2 (Mebs, 1986). Viriditoxin, a slightly acidic, but high molecular weight, myotoxic-hemorrhagic component isolated from the venom of Crotalus viridis viridis, has been claimed to exert direct myotoxicity beside its hemorrhagic effects (Fabiano and Tu, 1981). However, studies on this toxin and on a viriditoxin variant indicated that myonecrosis occurs long after the extensive destruction of blood vessels in muscle tissue, suggesting that the myotoxic action is a secondary effect of the toxin (Gleason et al., 1983). 2.1. MYOTOXICPEPTIDES From Crotalus snake venoms, basic polypeptides have been isolated which cause characteristic and immediate effects upon injection in mice: prostration with severe contracture of the hind limbs and death due to respiratory arrest. These toxins are named crotamine (from Crotalus durissus terrificus venom; Laure, 1975), myotoxin a (from Crotalus viridis viridis venom; Cameron and Tu, 1977), peptide c (from Crotalus viridis helleri venom; Maeda et al., 1978), myotoxin I and II (from Crotalus viridis concolor venom; Engle et al., 1983; Bieber et al., 1987), toxin III (from Crotalus horridus horridus venom; Mebs et al., 1983), and CAM-toxin (from Crotalus adamanteus venom; Samejima et al., 1988). They are single peptide chains consisting of 43-45 amino acid residues cross-linked by three disulfide bridges (Fox et al., 1979). Their isoelectric points are between 9 and 11. Amino acid sequences exhibit a high degree of homology (Table 1). Conformational studies on myotoxin a using circular dichroism, laser Raman spectroscopy, and predictions from the amino acid sequence revealed that the secondary structure of the toxin consists of a significant amount of fl-sheet and fl-turns, with some indications of random coil and ~-helix (Bailey et al., 1979). By ~H-NMR spectroscopy resonances in the aromatic region could be assigned to specific amino acid residues (Henderson et al., 1987). Observations

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TABLE 1. Myotoxic Peptides Inducing Contracture of Skeletal Muscle and Causing Local Myonecrosis

LDm C.d.terr., crotamine C.v.c., myotoxin I C.v.c., myotoxin II C.v.c., myotoxin II, micro. C.v.h., peptide c C.v.v., myotoxin a C.adam., CAM

YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG YKRCHKKEGHCFPKTVICLPPSSDFGKMDCRWKWKCCKKGSVN YKRCHKKGGHCFPKEKICTPPSSDFGKMDCRWKWKCCKKGSVN

1.5 (i.v.) n.a. n.a.

YKRCHKKGGHCFPKTVICLPPSSDFGKMDCRWRWKCCKKGSVN YKRCHKKGGHCFPKTVICLPPSSDFGKMDCRWKWKCCKKSVN YKQCHKKGGHCFPKEKICIPPSSDLGKMDCRWKWKCCKKGSG YKRCHKKGGHCFPKTVICLPPSSDFGKMDCRWRWKCCKKGSVNN

n.a. 1.96 (i.v.) 3.0 (i.m.) 0.9 (s.c.)

Abbreviations: C.d.terr.--Crotalus durissus terrificus; C.v.c.--Crotalus viridis concolor; C.v.h.--Crotalus viridis helleri; C.v.v.--Crotalus viridis t,iridis; C.adam.--Crotalus adamanteus; myotoxin II, micro, indicates microheterogeneity in structure, i.v.--intravenous, i.m.--intramuscular, s.c.--subcutaneous injection.

of shift perturbations depending on pH suggest a helical arrangement of the amino terminal region which places the aromatic residues in close proximity to each other. Using immunodiffusion or an ELISA-test for the detection of myotoxin a or similar toxins in snake venoms, Bober et al. (1988) found that the toxin appears to be widely distributed among Crotalus and Sistrurus species. Other viperid genera (Bothrops, Agkistrodon, Calloselasma, Bitis, Vipera) as well as cobra venom (Naja naja kaouthia) lack these components. 2,2. MYOTOXICPHOSPHOLIPASESA 2 A m o n g the numerous phospholipases A 2 isolated from snake venoms (cf. Mebs and Claus, 1990), some

were found to exhibit distinct myotoxic activity. Since most of the phospholipases A2 available in pure state have not yet been tested for their effect on skeletal muscle in vivo, the enzymes summarized in Table 2 represent only a small part of an obviously much larger group of phospholipases A 2 with this particular property. Using LDs0 values as a criterion, two major groups of myotoxic phospholipases A 2 can be distinguished: those with LDs0s less than I mg per kg and those with LDs0s higher than 1 mg per kg (although different routes of application have been used, i.e. intravenous, intraperitoneal, subcutaneous, no large differences are generally found). Enzymes of the first group are the most lethal snake venom components (Harris, 1985). Toxins like crotoxin, mojave toxin, notexin, notechis 11-5, taipoxin, and caudoxin act at the

TABLE2. Myotoxic Phospholipases A 2from Snake Venoms Name Crotoxin Mojave-toxin Notexin Notechis I1-5 Taipoxin Enh. schistosa VI : 5 Basic phospholipase A 2 Myotoxic phospholipase A 2 Caudoxin Mulgotoxin P. australis VIII-A P. colletti II P. colletti IV P. porphyriacus I-B Myotoxin Myotoxin Myotoxin II Myotoxin Bothropstoxin Myotoxin Myotoxin M yotoxin Myotoxin

Snake species Crotalus durissus terrificus Crotalus s. scutulatus Notechis scutatus scutatus Oxyuranus scutellatus scutellatus Enhydrina schistosa Naja nigricollis Naja naja naja Bitis caudalis Pseudechis australis Pseudechis colletti Pseudechis porphyriacus Micrurus n. nigrocinctus Bothrops asper Bothrops nummifer Bothrops jararacussu Agkistrodon bilineatus Agkistrodon contortrix mokeson Agkistrodon piscivorus piscivorus Trirneresurus flavoviridis

LDso (mg/kg)

Ref.

0.23 i.v. 0.06 i.v. 0.02 i.v. 0.05 i.v. 0.002 i,v. 0.10 i,v. 1.2 s.c. 2.4 i,p. 0.18 i.p. 0.20 i.p. 7.7 s.c. 4.5 s.c. 4.3 s.c. 6.4 s.c. n.a. 5.6 i.v. n.a. n.a. 4.8 i.v. n.a. n.a. n.a. n.a.

(1-3) (4) (5) (6) (7-9) (10, I 1) (12) (13) (14) (15) (16) (16) (16) (16) (17) (18, 19) (20) (21) (22) (19) (19) (19) (19)

Abbreviations: i.v.--intravenous, i.p.--intraperitoneal, s.c.--subcutaneous, n.a.---data not available. References: (I) Habermann and Breithaupt (1978); (2) Fraenkel-Conrat et al. (1980); (3) Aird et al. (1985, 1988); (4) Cate and Bieber (1978); (5) Halpert and Eaker (1975); (6) Halpert and Eaker (1976); (7) Fohlman et al. (1976, 1977); (8) Lind and Eaker (1982); (9) Lind (1982); (10) Fohlman and Eaker (1977); (11) Lind and Eaker (1981); (12) Mebs (1986); (13) Bhat and Gowda (1989); (14) Viljoen et al. (1982); (15) Leonardi et al. (1979); (16) Mebs and Samejima (1980); (17) Arroyo et al, (1987); (18) Gutierrez et al. (1984a); (19) Mebs and Samejima (1986); (20) Lomonte and Gutierrez (1989); (12) Gutierrez et al. (1989); (22) Homsi-Brandenburgo et al. (1988).

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presynaptic site of the motor nerve endplate and block transmitter release. Although their myotoxic action has been considered to be a side effect, it has to be noted that one phospholipase A 2 toxin fl-bungarotoxin, leaves the skeletal muscle tissue entirely unaffected. The lesions in skeletal muscle produced by neurotoxic phospholipases A 2 are essentially identical to those seen after application of the less lethal enzymes. On the other hand, most of these phospholipases A 2 of the second group do not cause neurotoxic symptoms. Myoglobinuria resulting in renal failure seems to be the major cause of death in mice which have been injected with high doses ( > 10 mg/kg); the animals usually die in an emaciated state after several days (Fohlman and Eaker, 1977; Mebs and Samejima, 1980). The structures and modes of action of the single chain phospholipases A 2 or the phospholipases A2 complexes crotoxin~ mojave toxin, notexin, notechis 1I-5 and taipoxin have been reviewed previously (Harris, 1984, 1985, 1990). They consist either of one polypeptide chain like notexin or its homolog notechnis I1-5 (119 amino acid residues, cross-linked by seven disulfide bridges; Halpert and Eaker, 1975, 1976), or are a complex of two subunits like crotoxin and mojave toxin. The subunits of these complexes are not covalently linked and in the case of crotoxin dissociate reversibly into a basic single chain protein of 140 amino acid residues, the phospholipase A 2 molecule (crotoxin B) and a three-chain acidic polypeptide of 88 amino acids, crotoxin A or crotapotin, which inhibits the enzyme activity of the phospholipase A 2 and enhances its toxicity (Breithaupt et al., 1974; Habermann and Breithaupt, 1978; Cate and Bieber, 1978). Taipoxin is a ternary complex of noncovalently linked subunits named ct-, fl- and 7-taipoxin, strongly basic, neutral and acidic phospholipase A2 molecules, respectively (Fohlman et al., 1976, 1977; Lind and Eaker, 1982; Lind, 1982). The less lethal, but nevertheless highly myotoxic phospholipases A 2 are single chain basic polypeptides. They consist of 114-129 amino acid residues cross-linked by seven disulfide bridges (with the exception of bothropstoxin and Pseudechis colletti II myotoxin possessing 8 or 6 disulfide bridges, respectively). For amino acid sequence information, refer to a recent compilation (Mebs and Claus, 1990). The major pathologic effect of these phospholipases A 2 is myotoxicity. However, the myotoxin from Enhydrina schistosa venom (VI:5) has weak presynaptic effects (Fohlman and Eaker, 1977). Neurotoxic-like symptoms such as respiratory distress and paralysis of hind limibs have been observed in mice after the injection of the myotoxic phospholipase A2 from Naja naja naja venom (Bhat and Gowda, 1989). On the other hand, caudoxin, a single chain toxic phospholipase A 2 from Bitis caudalis venom has strong presynaptic blocking activity, but is a rather weak myotoxin (Lee et al., 1982). The dose required to produce myotoxic lesions in mouse skeletal muscle was much higher (2 mg/kg) than that of other myotoxic phospholipases A 2 such as notexin (0.035 mg/kg). When compared with other venom phospholipases A 2, especially those with neutral or acidic isoelectric points, and using conventional substrates like egg-

yolk lecithin, most myotoxic phospholipases A2 have rather low enzyme activities. Two of them seem to be devoid of any enzyme activity at least under the assay conditions applied, i.e. egg-yolk lecithin as substrate, hemolysis test: bothropstoxin from Bothrops jararacussu venom (Homsi-Brandenburgo et al., 1988) and the myotoxin from Bothrops nummifer venom (Gutierrez et al., 1989). There exists evidence that the latter myotoxin may interact with and disorganize phospholipid bilayers, such as disrupting liposomes made of muscle phospholipids.

3. PATHOGENESIS OF MYONECROSIS 3.1. NORMAL STRUCTURE OF SKELETAL MUSCLE Muscle is composed of muscle cells surrounded by connective tissue sheaths through which blood vessels and nerves pass and in which various connective tissue cells reside. The outermost sheath, the epimysium, surrounds the entire muscle and is continuous with the surrounding connective tissue of the fascia. The perimysium is a thinner connective tissue sheath which surrounds bundles of muscle cells, i.e. fascicles. Immediately surrounding individual muscle cells is a thinner connective tissue sheath, the endomysium, containing the smallest blood vessels, the capillaries. Fig. 1 shows the histological appearance of normal mouse skeletal muscle. Skeletal muscle cells are elongated cells, slightly tapered or blunt at the ends. Like other cells, skeletal muscle cells have a cell membrane, often called sarcolemma, and cytoplasm and often called sarcoplasm. Unlike other cells, skeletal muscle cells have many nuclei, located peripherally just beneath the cell membrane, and when viewed in longitudinal section, they have transverse striations. Figure 2 shows the normal fine structural appearance of a skeletal muscle cell. The cytoplasm contains the normal cellular organelles plus myofilaments which are grouped into bundles called myofibrils. Mitochondria are numerous and occur in groups near the poles of the nuclei, beneath the cell membrane and interdigitated among the myofibrils (Fig. 2A). A small and inactive Golgi apparatus is located near many of the nuclei, glycogen is abundant between and within the myofibrils, and lipid droplets are located near large mitochondria. The striated pattern of the skeletal muscle cell is due to the fine structural organization of the myofilaments into myofibrils (Fig. 2B). Each myofibril is composed of numerous myofilaments arranged in register, resulting in the distinctive transverse banding pattern. The darker A bands separate the lighter I bands, and each A band has a clear central zone called the H band while each I band is bisected by a dense line called the Z line. The region between two Z lines is called a sarcomere, and is considered the structural and functional unit of contraction. The sarcomere is repeated along the entire length of the myofibril. There are two major types of myofilaments, a thicker myosin filament and a thinner actin filament. The actin filaments are anchored at the Z line and are the main component of the I band and extend into the

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FIG. 1. Light micrograph of normal murine skeletal muscle. Muscle cells (M) are arranged in bundles called fascicles which are surrounded by connective tissue sheaths. The sheath immediately surrounding the muscle cells is called the endomysium which contains capillaries (arrows). The endomysium blends with the perimysium (P) which contains larger blood vessels and nerves. A band between the myosin filaments. Two regulatory l~roteins, troponin and tropomyosin, are closely associated with the actin filament. The regulation of contraction by these proteins is based on calcium conct:ntrations in the cytoplasm. When calcium levels are low, tropomyosin is located more peripherally alon~; the actin filament and blocks G actin monomers from interacting with myosin. When calcium leveL, are sufficiently high, calcium binds to troponin, and :his causes the tropomyosin molecule to move into Lhe groove and previously blocked sites on the actin filament are made available for contact with myo, in heads. In skeletal muscle cells the elements of the smooth endoplasmic reticulum are highly organized and specialized and are referred to as sarcoplasmic reticulum.The membranes of the sarcoplasmic reticulum form an er:tensive network of cisterns and tubules around the tayofibrils (Fig. 2A and B). They are associated with a transverse membrane system called T tubules origi aatingatthesurfaceofthecellasanextensionof the cell membrane into the cell (Fig. 2B). The arrangemen1 of these membranes is critical to the conduction of the excitation signal from the outside of the muscle cell ~o the interior where contraction occurs. TILe primary function of skeletal muscle cells is that of contraction. Although the myofilaments are responsible for contraction of the cell, the membrane systems of the cell also play a vital role. Contraction is initiated at the motor endplate where an action poteatial is triggered in the muscle cell plasma membrane. The action potential spreads along the cell merr brane and into the interior of the muscle cell via the Lransverse tubular system. This depolarization causes the release of calcium from the sarcoplasmic

reticulum which results in an increase in the concentration of sarcoplasmic calcium. Calcium is available to the elements of the myofilaments and induces changes in them which lead to contraction. According to the sliding filament hypothesis, the thick and thin filaments themselves do not shorten, they merely slide past each other to effect an overall shortening of the muscle fiber. Contraction of skeletal muscle cells depends on ATP as an energy source, but the supply of ATP in a muscle cell is not sufficient to sustain contraction for long periods of time. Thus, for the cell to undergo active and continual contraction, ATP must be continually replenished. This is done through glycolysis and the tricarboxylic acid (TCA) cycle. As long as adequate oxygen is available, muscle cells also maintain a reserve of high energy phosphate bonds as creatine phosphate. However, when the oxygen level is inadequate, the TCA cycle does not produce ATP and the cell must switch to anaerobic respiration. Pyruvic acid is converted to lactic acid, which may accumulate. Skeletal muscle cells adapt their morphology to the functional demands made upon them. Increased use results in an increase in the size of the muscle organ because of hypertrophy of individual muscle cells. This is due to an increase in both cytoplasm and the number of myofibrils. Likewise, lack of use results in atrophy in which the size of individual fibers is decreased, and there is a loss of the contractile proteins, actin and myosin. Atrophy can result from disuse due to immobilization, confinement or loss of innervation. Muscular contraction, i.e. exercise and nervous stimulation are essential for maintenance of the normal muscle mass.

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FIG. 2. Electron micrographs of normal murine skeletal muscle. (A) Note the basal lamina (BL), plasma membrane (PM), mitochondria (Mi) and parts of the sarcoplasmic reticulum (SR). (B) The myofilaments are arranged into bundles called myofibrils (Mf) between which are located the membranes of the sarcoplasmic reticulum (arrows) and T-tubules (T). The striated pattern of the myofibrils and hence the muscle cell is due to the arrangement in register of the myofilaments which can be distinguished as a dark A band (A) and a light I (I) band. In the middle of the A band is a clear zone called the H zone (H), and in the middle of the I band is a dark line called the Z line (Z). The functional unit of the muscle cell is the sarcomere (S), defined as the myofilaments extending from one Z line to another. 3.2. EFFECTS OF MYOTOXIC PEPTIDES Biological activities of these toxins include induction o f c o n t r a c t u r e o f skeletal muscle u p o n i.m. injection o f the toxin in vivo, which m a y last for

1-3 hr after injection ( O w n b y et al., 1982, 1988); v a c u o l a t i o n o f skeletal muscle cells a n d subsequent necrosis (Ownby et al., 1976, 1982, 1988); a n d hemolysis o f erythrocytes in vitro ( C a m e r o n a n d Tu, 1977). Thus, these toxins a p p e a r to be fairly specific to

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FIG. 3. Light micrographs of skeletal muscle taken from a mouse after the i.m. injection of 1.0 #g/g crotamine. (A) 12 hr. Affected muscle cells contain numerous vacuoles (V) while unaffected muscle cells (M) have normal morphology. (B) 72 hr. Some of the affected muscle cells appear to be necrotic (arrow) while some are still in the vacuolated state (V). Unaffected cells have normal morphology (M). skeletal muscle cells, but not exclusively since they do have slight activity on the membranes of erythrocytes. Contracture of muscle upon i.m. injection is usually observed for myotoxin a, and this muscle contracture has also been observed in vitro (Chang et al., 1983). Ownby et al. (1976) described the changes in skeletal muscle cells at 3, 6, 12, 24, 48 and 72 hr after the i.m. injection to mice of 1.5 pg/g myotoxin a. At

the light microscopic level, vacuolation of muscle cells is seen as early as 3 hr after injection. Vacuolation also results from the injection of crotamine (Cameron and Tu, 1977), as well as a homologous myotoxin isolated from the midget faded rattlesnake (Crotalus viridis concolor) venom (Ownby et al., 1988). Thus, the biological activities of these homologous toxins appear to be very similar, if not identical. Figure 3 shows changes visible at the light

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FtG. 4. Electron micrographs of skeletal muscle taken from a mouse after the i.m. injection of 1.5/~g/g myotoxin a. (A) 12hr. The sarcoplasmic reticulum (SR) is dilated, but the T tubules (arrows) are morphologically normal. (B) 72 hr. The normal fine structure of the cell is disrupted; the sarcoplasmic reticulum (SR) is dilatated and the myofilaments are disrupted (arrows). microscopic level induced in skeletal muscle cells by these toxins. Early damage (12 hr) induced by crotamine, for example, is visible as a vacuolation of the muscle cells (Fig. 3A). Later (72 hr) the damaged cells appear to be necrotic (Fig. 3B). The presence of muscle cells appearing as an amorphous mass containing phagocytic cells is evidence for necrosis induced by myotoxin a (Ownby et al., 1976, 1982). There may be some differences in the potency of these

toxins in causing death of animals (Ownby et aL, 1988) as well as necrosis of skeletal muscle cells (Ownby, unpublished observations). Electron microscopic examination of tissue affected by these toxic peptides shows that vacuolation is due to dilatation of the sarcoplasmic reticulum. In a study on myotoxin a (Ownby et al., 1976), such dilatation of the sarcoplasmic reticulum as well as the perinuclear space was observed as early as 3 hr,

Myotoxic components of snake venoms and by 24 hr the sarcoplasmic reticulum was severely and extensively dilatated (Fig. 4A). By 48 hr mitochondria were swollen and the myofilaments were undergoing degeneration, and by 72 hr the myofibrils were completely disorganized (Fig. 4B), and the cells appeared to be necrotic. No morphological changes were detected in the plasma membrane or the T tubules. Engle et al. (1983) also reported that dilatation of sarcoplasmic reticulum resulted from the injection of two small, basic myotoxins isolated from the venom of Crotalus viridis concolor; this dilatation was comparable to that described for homologous myotoxins. By 72 hr after injection, the muscle cells affected by myotoxin I appeared to be relatively intact, but the cells affected by myotoxin II appeared to be completely disrupted. These studies show that there may be differences in the extent of muscle cell damage induced by similar toxins. Although there have been numerous investigations into the pharmacological and pathophysiological actions of crotamine and myotoxin a, the exact mechanism by which these myotoxins cause the contracture and degeneration of skeletal muscle cells is still not clear. From microscopic studies it appears that the initial visible change in the cells is dilatation or enlarging of the elements of the sarcoplasmic reticulum (SR). This could be due to an indirect or direct effect of the toxin on SR membranes. For example, if the toxin acted at the levels of the plasma membrane of the cell to open the Na ÷ channels and allow unrestricted influx of N a + into the cell, water would follow down the osmotic gradient and begin to accumulate in the cell. It is known that the endoplasmic reticulum, SR in this case, serves as a reservoir to take up water from the cytosol. Thus, as the SR fills with water, it becomes swollen. This process is known to occur in other mammalian cells (Ginn et al., 1968). However, it might be possible that the toxin enters the cell and interacts with the membranes of the SR directly. This might also cause swelling, although the mechanism by which this could happen is not clear. Recent work into the mechanism of action has been primarily along two lines: the effect of the toxin on ion transport across membranes and the interaction of the toxin with lipids. The results from studies on ion transport in membrane systems seem to be contradictory. Work by Chang and Hong and coworkers (Chang and Tseng, 1978; Chang et al., 1983; Hong and Chang, 1985) supports the hypothesis that these toxins act at the level of the plasma membrane. Chang and Tseng (1978) presented substantial evidence indicating that crotamine acts at the level of the plasma membrane to increase the influx of Na ÷ into the cell. They showed that crotamine at high doses of 10-50#g/ml induced contracture and spontaneous fibrillation in isolated rat and mouse diaphragm preparations. They also reported rapid depolarization of the resting membrane potential to about - 5 0 m V within 5min of toxin application. Crotamine increased the influx of 24Na into cells in the rat diaphragm. It also appears to act primarily on mammalian skeletal muscle and does not affect other excitable organs, nerve or heart. Additional work by

231

Chang et al. (1983) using crotamine as well as scorpion toxin and a sea anemone toxin (toxin II from Anemonia sulcata) indicated also that crotamine acts at sites on the voltage-dependent sodium channel in the plasma membrane. More recent work by Hong and Chang (1985) using myotoxin a indicates that myotoxin a acts specifically on the Na+-channel of the sarcolemma or T-tubule, like crotamine. The site of action of myotoxin a on the Na+-channel is different from the site of action of tetrodotoxin, veratridine or sea anemone toxin II. All of these data indicate that these small basic toxins act on the Na ÷-channel of the plasma membrane of mammalian skeletal muscle cells to increase the resting membrane permeability to sodium. Thus, such action could be the initial step which leads to swelling of the sarcoplasmic reticulum and eventually to necrosis of the skeletal muscle cell. Other investigators have focused on the interaction of myotoxin a with the sarcoplasmic reticulum. Tu and Morita (1983) examined the interaction of a peroxidase-conjugated myotoxin a with frozen sections of a human skeletal muscle biopsy. The labeled toxin was incubated in vitro with the frozen sections for 1 hr, and then the sections were processed for electron microscopic examination. They reported that the labeled toxin bound specifically to the sarcoplasmic reticulum, and none was observed on the plasma membrane, T-tubules or mitochondria. They also found some labeled myotoxin in the cisternae of the SR. Although the toxin could be shown to 'bind' to SR membranes in vitro after the tissue is sliced open to expose all membrane receptors, this does not explain how the toxin could gain access to these membranes in vivo. In the cell, the toxin must either penetrate the plasma membrane or gain access to the SR via the T-tubule, in which case it still has to pass through the membrane of the T-tubule, unless these membranes fuse with those of the SR. Volpe et al. (1986) investigated the interaction of myotoxin a with the Ca2+-ATPase of SR vesicles isolated from rabbit skeletal muscle. They concluded that the myotoxin attaches to the SR Ca2+-ATPase and uncouples Ca 2+ uptake from Ca2÷-dependent ATP hydrolysis. Their evidence is that the toxin inhibited Ca ~+ loading and stimulated Ca2+-depen dent ATPase without affecting unidirectional efflux of Ca 2+. The toxin also partially blocked the binding of a specific anti-(rabbit SR Ca2+-ATPase) antibody and it prevented the formation of decavanadate-induced two dimensional crystalline arrays of the SR Ca:+-ATPase. However, studies with two homologous myotoxins from Crotalus viridis concolor venom found that they do not cause any significant changes in Ca:+-Mg2+-ATPase or in the uptake or release of Ca 2÷ from isolated SR vesicles (Engle et al., 1983). These studies raise questions about the interaction of the myotoxic peptides with the calcium transport mechanisms in muscle cells or their isolated membrane systems. Also, there is no morphological evidence that abnormal increases in calcium levels in the cytosol are involved in the pathogenesis of myonecrosis induced by these toxins. The other line of investigation of the mechanism of action of these toxins has been into their interaction with artificial membranes, lipids and phospholipids.

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Liddle and Tu (1985) used Raman spectroscopy to look at the interaction of myotoxin a with artificial membranes containing dimyristoyl phosphatidylcholine (DMPC) and dimyristoyiphosphatidylserine (DMPS). They found that the myotoxin destabilized the ordered structure of the gel-phase of the phospholipid bilayers, and they concluded that the toxin alters the phase behavior of DMPS, but was more active on DMPC than on DMPS. Since DMPS naturally occurs in SR, these investigatiors conclude that the toxin could be destabilizing the SR membranes in vitro. However, since these studies were done in vitro, it is difficult to know whether the same events occur in vivo.

Liddle et al. (1987) also used differential scanning calorimetry to examine the effect of myotoxin a on the thermotropic phase behavior of model lipid membranes. They reported that the toxin significantly changed the normal phase behavior of DMPC, and that the effect was concentration dependent. Dufourcq et al. (1987) used the intrinsic fluorescence of myotoxin a to study its interaction with phospholipids. They found that the interaction is electrostatic amd thus the toxin has some specificity for negatively charged interfaces. The stability of the toxin-phospholipid complexes increased in the following sequence: phosphatidylinositol < phosphatidylserine < phosphatidylglycerol < phosphatidic acid < cardiolipin. They propose that the toxin lies at the interface, does not penetrate the membrane, and does not severely change the structure of the lipid chain order nor the permeability of the lipid vesicles. Thus, the damage to the lipid bilayer induced by the toxin is weak and not drastic when compared to other toxins such as the cardiotoxins. 3.3. EFFECTS OF MYOTOXIC PHOSPHOLIPASES A 2 Detailed studies of the pathogenesis of myonecrosis induced by these toxins have been limited to a few toxins. However, the results with these few are very similar, indicating that they may all have essentially the same mechanism of action on skeletal muscle cells. Figure 5 shows an example of the pathogenesis of myonecrosis induced by one of the myotoxic phospholipases A 2. In general, upon experimental injection these toxins act very rapidly to cause necrosis. Light microscopic examination shows that by 1-3 hr many cells are undergoing degeneration, there is intense interstitial edema and substantial infiltration by phagocytic cells. By 24-48 hr after injection, most of the necrotic cells have been infiltrated by phagocytic cells, which are removing the cellular debris. By 72 hr small myogenic cells may be seen in the sarcolemmal tubes indicating that regeneration is beginning. Regeneration typically occurs fairly rapidly and completely, so that by 3-4 weeks mature cells can be observed. However, these cells retain their centrally-located nuclei for many weeks and perhaps even as long as six months (Harris and Johnson, 1978). These studies have been done using notexin (Harris et al., 1975; Harris and Johnson, 1978), taipoxin (Harris and Maltin, 1982), crotoxin (Gopalakrishnakone et al., 1984), and Bothrops asper myotoxin (Gutierrez et al., 1984b and c).

Electron microscopic studies indicate that taipoxin (Harris and Maltin, 1982), crotoxin (Gopalakrishakone et al., 1984) and Bothrops asper myotoxin (Gutierrez et al., 1984b) all induce myonecrosis having a similar pathogenesis. Early changes (30 min-3 hr) include lysis of the plasma membrane, presence of 'delta lesions' (wedge-shaped clear areas within the muscle clear of organelles) and cells with hypercontracted myofibrils which appear densely clumped. Mitochondria might be swollen, contain flocculent densities and dense cristae, or might be disrupted with lysed membranes and vesiculated cristae. There is evidence for accumulation of calcium in the mitochondria of damaged cells (Gopalakrishnakone et al., 1984). At later time periods (6--12 hr), the dense clumps become less dense and the cells have the more amorphous and hyaline appearance of necrotic cells. By 24 hr after injection the cells are amorphous and contain many phagocytic cells. Throughout this process, the basal lamina remains intact and usually by 3-4 days after injection small myotubes are present. The size of these regenerating muscle cells increases at 1 and 2 weeks after injection, and by about 4 weeks the cells are as large as normal muscle cells but still retain the central nucleus. Myotoxic phospholipases A2 may exhibit some striking selectivity in terms of susceptible muscles (Harris, 1985). Notexin, for example, is very potent when applied to rat or chicken muscle, but is much less toxic to mouse muscle. Crotoxin affects mouse muscle as easily as rat muscle; but Enhydrina schistosa myotoxin is more toxic against mouse than against rat muscle. Generally, oxidative and oxidative/glycolytic muscles seem to be more susceptible to damage caused by myotoxic phospholipases A2 than glycolytic muscles. On the other hand, immature muscle cells are highly resistant to these myotoxins (Harris et al., 1975; Harris and Johnson, 1978). It is an interesting phenomenon that the satellite cells, which are considered to be myogenic stem cells, survive even widespread muscle cell breakdown (Klein-Ogus and Harris, 1983). This is an important observation, because the myotoxins seem to act primarily on plasma membranes. The fact that satellite cells are resistant to the toxin attack may suggest that at some time during their transformation to muscle cells, the plasma membrane of myogenic cells undergoes significant changes in constitution or configuration. The survival of satellite cells in necrotic muscle appears to be the key to the rapid regeneration of the muscle (Maltin et al., 1983). Satellite cells in a highly activated state are regularly found surrounding the damaged muscle areas (Klein-Ogus and Harris, 1983). They exhibit an increased cytoplasmic volume and prominent organelles including rough endoplasmic reticulum and free ribosomes. Many satellite cells have cytoplasmic processes and appear to be leaving the parent muscle through the basal lamina (Maltin et al., 1983). But the destination of their movement is obscure. Whether they enter the necrotic muscle segment and participate in the repair or form a new muscle in the interstitial space is still a matter of speculation and worth further investigation.

Myotoxic components of snake venoms

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FIG. 5. Light micrographs of skeletal muscle taken from a mouse at various times after injection of 0.15/~g/g crotoxin. Note the presence of some normal muscle cells (M) at all time periods. (A) 6 hr. Affected cells (arrows) have densely clumped myofibrils. (B) 24 hr. Affected cells (arrows) appear as an amorphous mass of disrupted myofilbrillar material. (C) 72 hr. Regenerating cells (arrows) or myotubes with rows of centrally-located nuclei are prominent. (D) 2 weeks. Regenerated cells are as large as normal cells but retain their centrally-located nuclei (arrows).

The general consensus on the mechanism of action of these toxins is that the plasma membrane is the primary site of action. Evidence for early damage to the plasma membrane comes from direct observation of broken membranes as well as from data showing an increase in the intracellular

levels of calcium, indicating a change in the permeability of the plasma membrane to this ion. However, there is no consensus about whether or not the hydrolytic activity of these phospholipases A2 is required for their action on skeletal muscle cells.

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4. R E L A T I O N S H I P BETWEEN PROTEIN S T R U C T U R E A N D MYOTOXIC ACTIVITY

The polypeptide myotoxins (nonphospholipase A: group) have no sequence homology with any other snake venom proteins, such as phospholipases A2, postsynaptic neurotoxins or cardiotoxins (Fox et al., 1979; Bieber et al., 1987). Analyzing the hydropathy profile of the amino acid sequences, Kini and Iwanaga (1986) found that these myotoxins possess a cationic region at residues 2-10 which is followed by a hydrophobic region. Both these regions are probably located on the outer surface of the molecule. However, exact data on the tertiary structure of these proteins are not available. A similar combination of both cationic and hydrophobic regions is present in the sequences of myotoxic phospholipases A2. Assuming that common pharmacological properties such as myotoxicity are linked to a similar amino acid sequence in certain regions of a molecule, Kini and Iwanaga (1986) investigated also a number of primary structures of myotoxic phospholipases A2, deriving hydropathic profiles. In a three-dimensional model, a sequence of about 15 amino acid residues having cationic properties is located before a hydrophobic helical part of the molecule (helix E; Dijkstra et al., 1978). This segment, amphiphilic in nature, is on the outer surface of the molecule and thus in a good position for interaction with membranes. This seems to support the hypothesis that cationic and hydrophobic sites in these molecules, phospholipases A2 as well as myotoxins, are essential for the determination of myotoxicity. Such 'cytolytic regions' are a common feature in the amino acid sequence of various cytolysins such as hemolysins of bacterial origin and in bee venom (melittin), of antibacterial cytolysins (cecropin and related peptides) and of snake venom cardiotoxins (Kini and Evans, 1989). However, the authors admit that the proposed cytolytic regions do not provide a comprehensive explanation of cell lysis, but they suggest that these structural peculiarities are important determinants of the lytic effect. A direct involvement of phospholipase A2 activity of notexin in its myotoxicity (and neurotoxicity as well) has been assumed by Harris and Macdonell (1981), because acetylated notexin derivatives have reduced myotoxic as well as enzyme activity when egg-yolk lecithin is used as substrates (Harris and Johnson, 1978). Moreover, lysophosphatides have been extracted from homogenates of necrotic muscle tissue. However, these experiments do not rule out the possibility that minor changes in the muscle cell membrane, either due to phospholipid hydrolysis or to binding to certain membrane acceptors causing conformational changes, may primarily produce the myotoxic effects. The recent discovery of two phospholipases A2 homologs, bothropstoxin and myotoxin from two Bothrops venoms (almost) free of enzyme activity (or at least exhibiting no activity on conventional substrates), is of considerable interest and may lead to an answer to the question of which part of the molecule is responsible for myotoxicity. Since the latter myotoxin behaves as an amphiphilic protein in electrophoresis, it has been suggested that the protein may

bind to and penetrate skeletal muscle plasma membrane by means of hydrophobic interactions leading to disorganization and disruption of the supramolecular organization of the membrane (Gutierrez et al., 1989). An increasing number of amino acid sequences of snake venom phospholipases A2, which are suspected to produce myotoxic lesions, will become available (cf. Takasaki et al., 1990). Studies on correlation between variations in structural characteristics and their myotoxic activity may enable more convincing conclusions. Acknowledgements--Part of the work done for this article

was supported by Public Health Service Grant 5 R01 AI26923-03 to CLO from the National Institute of Allergy and Infectious Diseases, the National Institutes of Health, U.S.A. CLO thanks Dr Sandra Reisbeck for performing the technical work on the experiments with crotamine and crotoxin.

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