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Morphological changes induced by a generalized myotoxin (myoglobinuria-inducing toxin) from the venom of Pseudechis australis (king brown snake) in skeletal muscle and kidney of mice
Pergamon
0041-0101(95)00091-7
Toxicon, Vol. 33, No. 11, pp. 1453-1467. 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights i'¢served 0041-0101/95 $9.50 + 0.00
M O R P H O L O G I C A L C H A N G E S I N D U C E D BY A GENERALIZED MYOTOXIN (MYOGLOBINURIA-INDUCING TOXIN) FROM THE VENOM OF PSEUDECHIS A USTRALIS (KING BROWN SNAKE) IN SKELETAL MUSCLE AND KIDNEY OF MICE D. PONRAJ and P. GOPALAKRISHNAKONE* Venom and Toxin Research Group, Department of Anatomy, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Republic of Singapore 0511
(Received 5 January 1995; accepted 25 May 1995)
D. Ponraj and P. Gopalakrishnakone. Morphological changes induced by a generalized myotoxin (myoglobinuria-inducing toxin), from the venom of Pseudechis australis (king brown snake) in skeletal muscle and kidney of mice. Toxicon 33, 1453-1467, 1995.--A myotoxin causing myoglobinuria was isolated from the venom of Pseudechis australis (PA myotoxin). Myoglobinuria was observed in mice 60 min post-injection (4.5 mg/kg i.m.) into calf muscles. Light microscopic observation revealed hypercontraction of muscle fibres with delta lesions and vacuolation. Severe necrosis was observed as early as 30 min. Infiltration of the muscle fibres with macrophages was seen by 3 hr with peak infiltration by 12-48hr. Electron microscopic study showed pathological changes in skeletal muscle as early as 5 min. Electron microscopic study showed disruption of the sarcolemma with dissolution and degeneration of the Z-band. Degeneration of the I-band was followed by degenerative changes in the A-band. Regeneration of muscle was evident by 3-5 days by the presence of many myotubes containing central nuclei. Regeneration was almost complete by 3 weeks. Contralateral soleus muscle which was not injected with toxin also showed degeneration followed by regeneration with central nuclei. Light microscopic studies of kidney showed myoglobin casts in both proximal and distal tubules, collecting ducts and loops of Henle. We conclude that this myotoxin probably acts on the Z-disc structures and also causes renal damage due to 'myoglobin cast nephropathy'. INTRODUCTION
Pseudechis australis (the mulga or king brown snake), one of the largest and most common venomous snakes in Australia, is capable of injecting considerable quantities of venom, the average being 180 mg and the largest amount obtained in a single 'milking' being 600 mg (Worrell, 1970). Mulgatoxin, a lethal myotoxin, was isolated from P. australis * Author to whom correspondence should be addressed. 1453
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D. PONRAJ and P. GOPALAKRISHNAKONE
v e n o m ( L e o n a r d et al., 1979). It has also been d e m o n s t r a t e d that several A u s t r a l i a n e l a p i d snakes, i n c l u d i n g the k i n g b r o w n snake, secrete v e n o m c o n t a i n i n g p h o s p h o l i p a s e s A2 which p r o d u c e m y o g l o b i n u r i a when injected into mice ( M e b s a n d S a m e j i m a , 1980; G o p a l a k r i s h n a k o n e a n d M e b s , 1990; C h e n et al., 1993). R e c e n t studies using O u t c h e r l o n y i m m u n o d i f f u s i o n conclusively identified m y o g l o b i n in the urine o f e x p e r i m e n t a l a n i m a l s after P. australis e n v e n o m a t i o n ( G o p a l a k r i s h n a k o n e et al., 1994). Several p h o s p h o l i p a s e s A2 (PLA2) a n d p o s t s y n a p t i c n e u r o t o x i n s have been identified in the v e n o m o f P. australis ( T a k a s a k i et al., 1990) a n d s o m e have been c h a r a c t e r i z e d ( N i s h i d a et al., 1985a, b; G e h et al., 1992). In vitro, P. australis v e n o m causes i n h i b i t i o n o f n e u r o m u s c u l a r t r a n s m i s s i o n a n d m o r p h o l o g i c a l d a m a g e to muscle fibres, m o t o r nerve terminals, a n d c y t o p l a s m i c organelles (Chen et aL, 1993). I n h u m a n s , P. australis v e n o m causes severe muscle d e s t r u c t i o n with c o n s e q u e n t m y o g l o b i n u r i a , which m a y lead to significant renal p r o b l e m s a n d even acute renal failure ( R o w l a n d s et al., 1969; H o o d a n d J o h n s o n , 1975; S u t h e r l a n d et al., 1981). T h e a i m o f the p r e s e n t w o r k was to s t u d y the sequence o f m o r p h o l o g i c a l changes i n d u c e d in skeletal muscle a n d k i d n e y by a m y o t o x i n (causing m y o g l o b i n u r i a ) isolated f r o m the v e n o m o f P. australis. MATERIALS AND METHODS The myotoxic fraction was isolated from the crude venom of P. australis by Sephadex G 50 gel filtration chromatography followed by further purification on FPLC mono S HR 5/5 using a linear salt gradient (a gift from Dr Kong Soo Khoo, Department of Biochemistry, National University of Singapore). The myotoxic fraction had a mol. wt of approximately 13,500 and was a basic PLA2, similar to the basic PLA2 fraction VIII-A (Mebs and Samejima, 1980) (see Table 1).
Intramuscular injection of PA myotoxin and myoglobinuria assay Male Swiss albino mice (approximately 20 g) were from the Laboratory Animal Holding Centre, National University of Singapore. They were anaesthetized with ether, and a single i.m. injection of one of various dilutions of the toxins from the venom of P. australis was given in 0.1 ml of 0.9% (w/v) sodium chloride, into the calf muscles. Myoglobinuria was detected by placing the animals in white filter paper which stained red or dark brown in positive cases (Mebs and Samejima, 1980; Gopalakrishnakone and Mebs, 1990). The toxin-injected mice were killed at various time intervals (n = 3) at 5 min, 15 min, 30 min, I hr, 3 hr, 7 hr, 12 hr, 24 hr, 48 hr, 72 hr, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks and 6 weeks after i.m. injection for both light microscopic and electron microscopic studies. The minimum dose of the toxin to produce myoglobinuria was 4.5 mg/kg i.m. Light microscopy (LM) Mice were anaesthetized with chloral hydrate (0.3 ml of 3.5% w/v i.p.). The injected leg was immobilized by pinning at the knee and ankle joints, and fixation commenced by perfusion through the left ventricle. Animals were perfused with 0.9% w/v sodium chloride to flush out whole blood and then with 10% formaldehyde in distilled water for 30 min. The soleus muscle, gastrocnemius muscle and kidney were removed, post-fixed overnight in 10% formaldehyde, dehydrated through a series of ethanol and finally embedded in wax. Sections of 7/~m thickness were cut and stained with haematoxylin and eosin (H&E) or Massons trichrome and examined under light microscope (Leitz Aristoplan). Areas exhibiting pathology were photographed with Kodak film. Table 1.
Toxin VIII-A PA myotoxin
Myogiobinuria ++ ++
Minimum dose to produce myoglobinuria, and LOs0 Mol. wt 5.0 mg/kg s.c. 13,400 7.7 mg/kg s.c. 4.5 mg/kg i.m. 13,500 7.25 mg/kg i.m.
PLA2 activity + +
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Electron microscopy (EM) The mice were anaesthetized with chloral hydrate and perfused through the left ventricle with 0.9% w/v sodium chloride followed by cold mixture containing 2% paraformaldehyde and 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min. The soleus muscle was dissected out and trimmed into pieces of approximately I mm in length and immersed in the same fixative for about 4 hr and then soaked in 0.1 M phosphate buffer (pH 7.4) overnight. The tissues were then postfixed in 1% osmium tetroxide in phosphate buffer for 2 hr. After dehydration through a graded series of ethanol and 100% acetone, tissues were kept in 1:6 of 100% acetone/araldite mixture overnight. Three changes of fresh Araldite were made before tissues were finally embedded in Araldite and incubated in an oven at 60°C for 24 hr. Semithin sections (1.0/lm) were cut on an ultramicrotome, stained with 1% toludine blue and examined under light microscope. Relevant areas were identified, trimmed and ultrathin gold sections were cut and mounted on copper grids. Sections were stained with uranyl acetate (10 min) and lead citrate (8 min), and then observed under electron microscope (Philips 400T) at a voltage of 80 kV.
RESULTS
General observation P r i o r to a d m i n i s t r a t i o n o f toxin, all mice were active; 15-30 m i n after injection o f 4.5 m g / k g i.m. o f P A m y o t o x i n the mice a p p e a r e d m o t i o n l e s s with stretched h i n d limb on the injected side. T h e white tissue p a p e r s in the cage were stained with red o r d a r k b r o w n urine b y 50% o f mice, a b o u t 2 h r after injection o f m y o t o x i n . The red urine was evident f r o m 60 m i n o n w a r d s . M o s t o f the a n i m a l s s h o w e d signs a n d s y m p t o m s for a p e r i o d o f 4 8 - 7 2 hr. A f t e r 3 - 5 d a y s m o s t o f the a n i m a l s recovered f r o m this illness a n d a p p e a r e d n o r m a l a n d active; a b o u t 5 % o f a n i m a l s died within 2 4 ~ 8 hr.
Light microscopy of muscle Pseudechis australis m y o t o x i n ( P A m y o t o x i n ) i n d u c e d m y o n e c r o s i s f r o m 30 m i n after i.m. injection o f the toxin. T h e early changes o b s e r v e d include (a) d i s r u p t i o n o f the m y o f i b r i l s with delta lesions (Fig. 1); (b) h y p e r c o n t r a c t i o n o f myofibrils with s h r u n k e n d a r k a r e a s o f c l u m p e d m a t e r i a l (Figs 1 a n d 2); (c) v a c u o l a t i o n o f the muscle cells (Fig. 2); (d) o e d e m a t o u s a p p e a r a n c e with mild cellular infiltration (Fig. 3); a n d (e) loss o f striations. All the a b o v e - m e n t i o n e d c h a n g e s were seen f r o m as early as 30 min to 7 hr.
Fig. 1. Thirty minutes after injection. Note hypercontracted clumped necrotic myofibrils (N) and delta lesions (arrowheads). (H&E stain, bar = 40/~m.)
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D. PONRAJ and P. GOPALAKRISHNAKONE
Fig, 2. Three hours after injection. Note vacuolation (arrows), blood vessel (V) unaffected, delta lesion (arrowheads) and necrosis (N). (Semithin section stained with 1% toludine blue, bar = 40 gin.)
Macrophages were also seen inside the necrotic muscle cells and in the interstitial connective tissues (Fig. 4). Regeneration of the muscle fibres was evident by 3-5 days because of the presence of m a n y myotubes containing central nuclei (Fig. 5). There were numerous connective tissue fibres and fibroblast in some areas as shown by Massons trichrome stain (Fig. 5). By 5-10 days regeneration was intense and in an advanced stage. By the 10th day m a n y f b r e s were completely regenerated, with a central row of nuclei, but there were still some areas of myonecrosis without regeneration, with connective tissue in the periphery. By about 3 weeks regeneration was almost complete with the appearance of striation of the muscle fibres, and occupation of the whole cytoplasm (Fig. 6).
Fig. 3. Seven hours after injection. Skeletal muscle cells in advanced stage of necrosis (N) with mild infiltration of necrotic fibres by macrophages. (H&E stain, bar = 40 gm.)
Morphology of P. australis Myotoxin Envenomation
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Fig. 4. Twelve hours after injection. Note infiltration of macrophages seen inside the necrotic cells and in the interstitial connective tissues. (H&E stain, bar = 40/~m.)
Electron microscopy of muscle Control calf muscle obtained from mice injected with 0.9% w/v sodium chloride showed normal sarcomere ultrastructure (Fig. 7). In experimental calf muscle the changes could be observed as early as 5 min after the injection. In early phases (5 min), some areas of the sarcomeres showed a tendency to fragment among the Z-lines with disruption and disappearance of Z-lines, sometimes appearing in an irregular manner (Fig. 8). The sarcomeres were fragmented, with dissolution and degeneration of the I-band (Fig. 9). Sarcoplasm between the damaged myofibrils was oedematous with separation of adjacent myofibrils (Fig. 9). There were areas of disrupted plasma membrane, but the basal
Fig. 5. Five days after injection. Note the central row of nuclei (arrowheads) with connective tissue (C) and fibrosis around the myofibre with different intensity of staining pattern due to regeneration. (Massons trichrome stain, bar = 40 pm.)
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D. PONRAJ and P. GOPALAKRISHNAKONE
Fig. 6. Three weeks after injection. Note the regenerated myofibrils with central row of nuclei (cn) with some of them showing fusion (arrowheads). Note the myofibrils occupy the whole of the cytoplasm showing almost complete regeneration. (H&E stain, bar = 20/lm.)
lamina appeared to be intact (Fig. 10). A few randomly located small vesicles were also seen in the sarcoplasm of the affected area. Mitochondria in these regions appeared swollen and contained dilated intercisternal spaces and floccular degeneration. Mitochondria in which the internal membranes were broken down, with the formation of vacuoles and a reduction in the number of cristae, were also seen after 60 min post-injection (Fig. 11).
Fig. 7. Control section showing normal sarcomeres with dark A-band and light I-band with Z-disc; mitochondria and triads (T) are also seen. (Bar = 0.5/tm.)
Morphology of P. australis Myotoxin Envenomation
Fig. 8. Five minutes after injection, The sarcomeres tend to fragment along the Z-lines. Note the dilated sarcoplasmic reticulum (S), and the three sarcomeres (asterisk) in various stages of fragmentation of I-band and Z-disc. (Bar = 0,5 tim.)
Fig. 9. One hour after injection. Note the discoid degeneration of the Z-discs (arrows), malalignment of Z-discs and oedematous sarcoplasm separating the myofilaments. (Bar = 1/~m.)
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D. PONRAJ and P. GOPALAKRISHNAKONE
Fig. I0. One hour after injection. Note the dissolution of the 1-band (asterisk) with the disruption of the plasma membrane (arrows). The basal lamina appears to be intact (arrowheads). (Bar = 0.25/~m.)
Fig. 11. One hour after injection. Note the mitochondria (M) scattered in the necrotic debris (N). Mitochondria in which internal membranes were broken down (arrow) and reduction of cristae are seen. (Bar = 0.25 #m.)
Morphology of P. australis Myotoxin Envenomation
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Fig. 12. Five days after injection. Note the central row of nuclei (Cn) with scattered myofilamentsat the periphery of the myoblast (arrowheads). The myoblasts have extensive surface membrane contacts with one another. (Bar = 1#m.) By 7-48 hr after injection there were still areas of hypercontracted myofilaments in some regions but many areas showed degeneration with necrotic debri. Many areas were devoid o f myofilaments, but cytoplasmic organelles like swollen mitochondria, sarcoplasmic reticulum, vesicles and pyknotic nuclei were seen. By the 5th day a clear central row of nuclei was seen in regenerating areas (Fig. 12). Myoblasts demonstrated specific characteristics such as a tendency to become aggregated and the ability to form a long thin strip of interdigitated cells (Fig. 12). Ultrastructural features of myofibrillogenesis were also seen in multinucleated myotubes. Both thick and thin myofilaments organized in bundles were seen (Fig. 12). These bundles initially appeared scattered in the cytoplasm of the myotube, but later the A- and I-bands were seen with Z-line formation in the middle of the I-band (Fig. 12). The development of these myofibrils was asynchronous, from one myotube to another, and even within the cytoplasm of the same multinucleated syncytium. By 10 days the myofibrils had become thicker and more numerous, and they occupied a larger portion of the cytoplasm with normal sarcomere structures such as dark and light bands. By 3 weeks most of the myofibrils showed almost a normal-looking sarcomere structure. A few areas of advanced stage of regeneration were also seen. Muscle fibre from the contralateral soleus muscle not injected locally with the toxin also showed degenerative changes followed by regeneration with a central row of nuclei (Fig. 13).
Light microscopy of kidney Control kidneys showed no lesions (Fig. 14). Experimental kidney showed myoglobin casts in the tubules from 1 hr onwards. The myoglobin casts were seen in proximal and distal convoluted tubules, loops of Henle and in the collecting ducts as a homogeneous
1462
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Fig. 13. Six weeks after injection. Section from the contralateral soleus muscle not injected with toxin showing central row of nuclei (cn) with regenerated myofilaments. (Bar = 2/~m.)
staining area occupying the entire lumen of the tubules (Figs 15 and 16). A cloudy swelling of the tubular epithelium with necrotic debris and degenerated tubular epithelium was also noticed. In some tubules the myoglobin casts obstructed the whole of the lumen (Fig. 16). The myoglobin casts and degenerative changes were observed with H&E stain as well as by Massons trichrome stain. (A detailed electron microscopic observation of the changes in the kidney is in preparation.)
Fig. 14. Section of control kidney showing normal tubular epithelium (arrowheads) and glomerulus (G). (H&E)
stain, bar =
20 # m . )
Morphology of P. australis Myotoxin Envenomation
1463
Fig. 15. Section of an experimental kidney showing numerous myoglobin casts (arrowsl and necrotic debris (N) in the lumen. (Massons trichrome stain, bar = 40 ~m.)
DISCUSSION
The myotoxic phospholipase from P. australis (PA myotoxin) is similar to myotoxic phospholipase VIII-A from P. australis (Mebs and Samejima, 1980). It produced myoglobinuria with a minimum dose of approximately 4.5 mg/kg i.m. and had a mol. wt of approximately 13,500. The changes caused by this generalized myotoxin (i.e. causing myoglobinuria) in muscle and kidney are described. This is in contrast to the previous studies where myotoxins mostly acted locally and did not produce visible myoglobinuira (Gopalakrishnakone et al., 1984; Guti~rrez et al., 1984; Harris, 1990; Ownby, 1990). The earliest changes were hypercontraction of the sarcomeres with the appearance of wedgeshaped delta lesion, and the delta lesion with disruption of the muscle fibres representing
Fig. 16. High power view of an experimental kidney showing degenerated tubular epithelium (arrowheads) and necrotic debris (N) and myoglobin casts. (Massons trichrome stain, bar ~ 20 ~m.)
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D. PONRAJ and P. GOPALAKRISHNAKONE
areas of cell degeneration, with portions of disrupted or discontinuous plasma membrane (Harris et al., 1980; Gutirrrez et al., 1984b, 1991). It is interesting to note that selective loss of the Z-line and I-band with dissolution of the Z-band and actin filaments (streaming) were seen in the present study. Similar observations have been reported with PLA2 from Pseudechis colletti (Weinstein et al., 1992) crotoxin (Gopalakrishnakone et al., 1984) and taipoxin (MacDonell, 1979). Duchen et al. (1974) made a similar report on the depolarizing fraction (a putative cardiotoxin) of Dendroaspis viridis venom. Dissolution of Z-band and actin filaments is the earliest degenerative change seen in many naturally occurring myopathies and in denervation atrophy (Hudgson and Pearce, 1969). This was also observed in experimental animals after poisoning with chloroquine (Aguagio and Hudgson, 1970), and in ischaemic limbs (Koreny Both et al., 1971). EM studies of myasthenia gravis have shown slight to marked change in Z-disc, such as streaming of Z-disc, splitting of sarcomeres centrally along the Z-band and fragmentation of myofilaments (Korenyi Both et al., 1973). Further research is needed to establish whether the sarcomere broke at the Z-disc or whether the A-band was released as the Z-disc broke down; it is likely that both these mechanisms may be involved. PA myotoxin caused destruction of the plasma membrane, which appears to be a prime target of many other myotoxic phospholipases (Harris et al., 1980; Gopalakrishnakone et al., 1984; Gutirrrez et al., 1984b; Johnson and Ownby, 1993); phospholipases, by hydrolysing key phospholipids, may cause the plasma membrane to become leaky. This would probably result in an increase in intracellular Ca + and hypercontraction of the myofibrils. The Z-disc and the interdigitating arrays of thick and thin filaments within each myofibril are held in place by an elaborate framework of cytoskeletal filaments. In the regions of sarcoplasm that border on the periphery of the Z-disc, ring-like arrangements of intermediate filaments that contain the proteins desmin, vimentin, and synemin have been described (Lazarides, 1980). In addition, the sarcomeres seem to be supported by a delicate flexible internal lattice made up partly of titin filaments and partly of another protein called nebulin, which spans the whole length of the sarcomere from one Z-line to the next (Wang, 1985). In our study the disruption and disappearance of Z-disc was probably due to the effect of the myotoxin damaging the cytoskeletal proteins such as desmin, leading to a weaker Z-disc with fragmentation, or its disappearance. Recent studies have shown that desmin completely disappeared in some fibres from as early as 3 hr after Notechis scutalus scutalus (Australian tiger snake) envenomation in experimental animals (Vater et al., 1992). Vater and co-workers also showed regeneration of desmin by 2 days, localized to the Z-discs once the myotubes have matured. They also showed that titin was rapidly hydrolysed during breakdown of tissues, but later than desmin, and suggested that the I-band breakdown is followed by A-band breakdown. Our ultrastructural studies also showed disruption of the I-band followed by the A-band. The myoglobin in skeletal muscle cells is localized mainly in the I-band, and in the Z-disc in particular (Kawai et al., 1987). It has been recently shown by Kawai et al. (1991) by immunocytochemistry that in degenerating muscle cells of patients with Duchenne's muscular dystrophy, and myotonic dystrophy, myoglobin was demonstrated in the distended lumen of the internal membrane system and in the intermyofibrillar space, through which it seemed to pass into the extracellular space. In amyotropic lateral sclerosis wavy Z-bands showed only slight staining. They have also shown that no staining was demonstrated in either the 1-band or Z-band in the hypercontracted area (Kawai et al., 1991). These findings suggest that myoglobin released from the I-band of the damaged muscle may pass out of the muscle cell into the circulation through an altered internal
Morphology of P. australis Myotoxin Envenomation
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m e m b r a n e system and intermyofibrillar space, and appear as a m y o g l o b i n cast in the kidneys. This is in agreement with our observation o f the disappearance o f the I - b a n d and Z-disc followed by m y o g l o b i n u r i a and cast in the kidneys. I m m u n o h i s t o c h e m i c a l studies are in progress to show the specific binding site o f this toxin in the muscle. M y o g l o b i n u r i a with subsequent myoglobinuric n e p h r o p a t h y was observed in clinical and experimental e n v e n o m a t i o n with the crude v e n o m o f several ophidian species (Weinstein et al., 1992; G o p a l a k r i s h n a k o n e and Mebs, 1990; Sutherland et al., 1981; H o o d and J o h n s o n , 1975). F r o m o u r results it is clear that renal epithelial cell necrosis and tubular m y o g l o b i n cast causing obstruction to urinary flow secondary to myoglobinuria m a y lead to renal ischaemia and p r o b a b l y to acute renal failure. These findings strongly support the clinical observation o f acute renal failure in h u m a n envenomation (Rowlands et al., 1969). In conclusion, it is suggested that P A m y o t o x i n causes streaming and fragmentation o f Z-disc and severe myonecrosis. This m a y cause myoglobin, mainly localized at the I-band, to leak out o f the d a m a g e d muscle cell and into the circulation, causing renal d a m a g e with m y o g l o b i n casts blocking the tubules. It is possible that P A m y o t o x i n m a y be acting initially on the sarcolemmal m e m b r a n e and, once inside, acting on the Z-disc and I-band, releasing the m y o g l o b i n into the circulation as well as disrupting the muscle fibre, which will be followed by degeneration o f muscle fibre and the subsequent changes. Thus this toxin causes severe myonecrosis followed by myoglobinuria, and renal d a m a g e with m y o g l o b i n cast n e p h r o p a t h y m a y play a vital role in P. australis envenomation.
Acknowledgements--This research work was carried out by a grant from National University of Singapore, A/C
no. 3602022. D. Ponraj is a recipient of a scholarship from the National University of Singapore. The authors thank Ms Diljit Kaur for kindly typing this manuscript.
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Gutirrrez, J. M., Ownby, C. L. and Odell, G. V. (1984b) Skeletal muscle regeneration after myonecrosis induced by crude venom and a myotoxin from the snake Bothrops asper (fer-de-lance). Toxicon 22, 719-731. Gutirrrez, J. M., Chaves, F., Gene, 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. Gutirrrez, J. M., Nune, Z. J., Diaz, C., Cintra, A. C. O., Homsi-Brandeburgo, M. E. and Giglio, J. R. (1991) Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase A 2 isolated from the venom of the snake Bothropsjajaracussu. Expl. mol. Path. 55, 217-229. Harris, J. B. and Cullen, M. J. (1990) Muscle necrosis caused by snake venoms and toxins. Electron Microsc. Rev. 3, 183-211. Harris, J. B. and MacDonell, C. A. (1981) Phospholipase A 2 activity of notexin and its role in muscle damage. Toxicon 19, 419-430. 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. Pharmac. 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 Australian tiger snake, Notechis scutatus scutatus. Clin. exp. Pharmac. Physiol. 2, 615-627. Harvey, A. L., Marshall, R. J. and Karlsson, E. (1982) Effects of purified cardiotoxins from the Thailand cobra (Naja naja siamensis) on isolated skeletal and cardiac muscle preparations. Toxicon 20, 379-396. Hood, V. L. and Johnson, J. R. (1975) Acute renal failure with myoglobinuria after tiger snake bite. Med. J. Aust. 2, 638-641. Hudgson, P. and Field, E. J. (1973) Regeneration of muscle. In: Structure and Function of Muscle, Vol. 11, pp. 312-359 (Bourne, G. H., Ed.). New York: Academic Press. Hudgson, P. and Pearce, G. W. (1969) In: Disorders of Voluntary Muscle, 2nd Edn, p. 277 (Walton, J. N., Ed.). London: Churchill. Johnson, E. K. and Ownby, C. L. (1993) Isolation of a myotoxin from the venom of Agkistrodon contortrix laticintus (broad-banded copperhead) and pathogenesis of myonecrosis induced by it in mice. Toxicon 31, 243-255. Johnson, E. K. and Ownby, C. L. (1994) The role of extracellular ion in the pathogenesis of myonecrosis induced by a myotoxin isolated from broad-banded copperhead (Agkistrodon contortrix laticinctus) venom. Comp. Biochem. Physiol. 107C, 359-366. Kawai, H., Nishida, Y., Masuda, K. and Saito, S. (1987) Localisation of myoglobin in human muscle cells by immunoelectron microscopy. Muscle Nerve 10, 144-149. Kawai, H., Sebe, T., Nishano, H., Nishida, Y. and Saito, S. (1991) Light and electron microscopic studies on localization of myoglobin in skeletal muscle cells in neuromuscular diseases. Muscle Nerve 14, 342-347. Korenyi-Both, A. (1983) Pattern of necrosis and autoimmune disease of skeletal muscle. In: Muscle Pathology in Neuromuscular Disease, pp. 45-56, 397 (Korenyi-Both, A. L., Ed.). Springfield: Charles C. Thomas. Lazarides, E. (1981) Molecular morphogenesis of Z disc in muscle cells. In: International Cell Biology 1980-1981, p. 392 (Schweiger, H. G., Ed.). Berlin: Springer. Leonarde, T. M., Howsen, E. H. and Spence, I. (1979) A lethal myotoxin isolated from the venom of the Australian king brown snake. Toxicon 17, 544-555. Lomonte, B. and Gutirrrez, J. M. (1989) A new muscle damaging toxin, myotoxin II, from the venoms of snake Bothrops asper (terciopelo). Toxicon 27, 725-733. Mebs, D. and Ownby, C. L. (1990) Myotoxic components of snake venom: their biochemical and biological activities. Pharmac. Ther. 48, 223-236. Mebs, D. and Samejima, Y. (1980) Purification, from Australian elapid venoms, and properties of phospholipase A which cause myoglobinuria in mice. Toxicon 18, 161-168. Nishida, S., Terashima, M., Shimazu, T., Takasaki, C. and Tamiya, N. (1985) Isolation and properties of two phospholipase A 2 from the venom of an Australian elapid snake (Pseudechis australis). Toxicon 23, 73-85. Ownby, C. L. (1990) Locally acting agents myotoxins, haemorrhagic toxins and dermonecrotic factors. In: Handbook of Toxinology, pp. 601-654 (Shier, W. T. and Mebs, D., Eds). New York: Marcel Dekker. Ownby, C. L. and Colberg, T. R. (1988) Classification of myonecrosis induced by snake venoms: venom from the prairie rattle snake (Crotalus viridis viridis), western diamondback rattlesnake (Crotalus atrox), and the Indian cobra (Naja naja naja). Toxicon 26, 459-474. Ownby, C. L., Cameron, D. and Tu, A. T. (1976) Isolation o f a myotoxic component from rattle snake (Crotalus viridis viridis) venom. Am. ,L Path. 85, 144--158. Ponraj, D. and Gopalakrishnakone, P. (1994) Ultrastructural changes induced by a myotoxin from Pseudechis australis in murine skeletal muscle and nerve. First Asia-Pacific Colloquium in Neuroscience, December 1994, Singapore, Abstract p. 194. Ponraj, D., Gopalakrishnakone, P. and Khoo, H. S. (1995) Isolation of a myotoxin, causing myoglobinuria from Pseudechis australis venom and immunohistochemical localisation of myoglobin in mice kidney. Third International Union of Biochemistry and Molecular Biology, April 1995, Singapore, Abstract p. 199. Reznik, M. (1970) Satellite cells, myoblast and skeletal muscle regeneration. In: Regeneration of Striated Muscle and Myogenesis, pp. 133-156 (Mauro, A. and Shafiq, S. A., Eds). Amsterdam: Excerpta Medica.
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Rowan, E. G., Harvey, A. L., Takasaki, C. and Tamiya, N. (1989) Neuromuscular effects of three phospholipases A 2 from the venom of the Australian king brown snake Pseudechis australis. Toxicon 27, 551-560. Rowlands, J. B., Mastaglia, F. L., Kakulas, B. A. and Hainsworth, D. (1969) Clinical and pathological aspects of a fatal case of mulga (Pseudechis australis) snakebite. Med. J. Aust. 1, 226-230. Sutherland, S. K., Campbell, D. G. and Stubbs, A. R. (1981) A study of major Australian snake venom in the monkey (Macaca fascicularis) II. Myolytic and haematological effects of venoms. Pathology 13, 705 715. Takasaki, V., Suzuki, J. and Tamiya, N. B. (1990) Purification and properties of several phospholipase A~ from the venom of Australian king brown snake (Pseudechis australis). Toxicon 17, 319-329. Tokuyasu, K. T., Dutton, A. H. and Singer, S. J. (1983) Immunoelectron microscopic studies of desmin (skeletin) localisation and intermediate filament organisation in chicken skeletal muscle. J. cell. Biol. 96, 1727-1735. Vater, R., Cullen, M. J. and Harris, J. B. (1992) The fate of desmin and titin during the degeneration and regeneration of the soleus muscle of the rat. Acta Neuropath. 84, 278-288. Wang, K. (1985) Sarcomere-associated cytoskeletal lattices in striated muscle, Review and Hypothesis. In: Cell and Muscle Motility, Vol. 6, p. 315 (Shay, J. W., Ed.). New York: Plenum Press. Weinstein, S. A., Bell, R. C., Fletcher, J. E. and Smith, L. A. (1992) Pathologic changes induced by phospholipase A 2 isolated from the venom of Collett's snake, Pseudechis colletti: a light and electron microscopic study. Toxicon 30, 171-185. White, J. (1990) The toxins of Pseudechis (black snakes). In: Poisons Information Monograph, p. 8. International Programme on Chemical Safety, World Health Organization.
0041-0101(95)00091-7
Toxicon, Vol. 33, No. 11, pp. 1453-1467. 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights i'¢served 0041-0101/95 $9.50 + 0.00
M O R P H O L O G I C A L C H A N G E S I N D U C E D BY A GENERALIZED MYOTOXIN (MYOGLOBINURIA-INDUCING TOXIN) FROM THE VENOM OF PSEUDECHIS A USTRALIS (KING BROWN SNAKE) IN SKELETAL MUSCLE AND KIDNEY OF MICE D. PONRAJ and P. GOPALAKRISHNAKONE* Venom and Toxin Research Group, Department of Anatomy, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Republic of Singapore 0511
(Received 5 January 1995; accepted 25 May 1995)
D. Ponraj and P. Gopalakrishnakone. Morphological changes induced by a generalized myotoxin (myoglobinuria-inducing toxin), from the venom of Pseudechis australis (king brown snake) in skeletal muscle and kidney of mice. Toxicon 33, 1453-1467, 1995.--A myotoxin causing myoglobinuria was isolated from the venom of Pseudechis australis (PA myotoxin). Myoglobinuria was observed in mice 60 min post-injection (4.5 mg/kg i.m.) into calf muscles. Light microscopic observation revealed hypercontraction of muscle fibres with delta lesions and vacuolation. Severe necrosis was observed as early as 30 min. Infiltration of the muscle fibres with macrophages was seen by 3 hr with peak infiltration by 12-48hr. Electron microscopic study showed pathological changes in skeletal muscle as early as 5 min. Electron microscopic study showed disruption of the sarcolemma with dissolution and degeneration of the Z-band. Degeneration of the I-band was followed by degenerative changes in the A-band. Regeneration of muscle was evident by 3-5 days by the presence of many myotubes containing central nuclei. Regeneration was almost complete by 3 weeks. Contralateral soleus muscle which was not injected with toxin also showed degeneration followed by regeneration with central nuclei. Light microscopic studies of kidney showed myoglobin casts in both proximal and distal tubules, collecting ducts and loops of Henle. We conclude that this myotoxin probably acts on the Z-disc structures and also causes renal damage due to 'myoglobin cast nephropathy'. INTRODUCTION
Pseudechis australis (the mulga or king brown snake), one of the largest and most common venomous snakes in Australia, is capable of injecting considerable quantities of venom, the average being 180 mg and the largest amount obtained in a single 'milking' being 600 mg (Worrell, 1970). Mulgatoxin, a lethal myotoxin, was isolated from P. australis * Author to whom correspondence should be addressed. 1453
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D. PONRAJ and P. GOPALAKRISHNAKONE
v e n o m ( L e o n a r d et al., 1979). It has also been d e m o n s t r a t e d that several A u s t r a l i a n e l a p i d snakes, i n c l u d i n g the k i n g b r o w n snake, secrete v e n o m c o n t a i n i n g p h o s p h o l i p a s e s A2 which p r o d u c e m y o g l o b i n u r i a when injected into mice ( M e b s a n d S a m e j i m a , 1980; G o p a l a k r i s h n a k o n e a n d M e b s , 1990; C h e n et al., 1993). R e c e n t studies using O u t c h e r l o n y i m m u n o d i f f u s i o n conclusively identified m y o g l o b i n in the urine o f e x p e r i m e n t a l a n i m a l s after P. australis e n v e n o m a t i o n ( G o p a l a k r i s h n a k o n e et al., 1994). Several p h o s p h o l i p a s e s A2 (PLA2) a n d p o s t s y n a p t i c n e u r o t o x i n s have been identified in the v e n o m o f P. australis ( T a k a s a k i et al., 1990) a n d s o m e have been c h a r a c t e r i z e d ( N i s h i d a et al., 1985a, b; G e h et al., 1992). In vitro, P. australis v e n o m causes i n h i b i t i o n o f n e u r o m u s c u l a r t r a n s m i s s i o n a n d m o r p h o l o g i c a l d a m a g e to muscle fibres, m o t o r nerve terminals, a n d c y t o p l a s m i c organelles (Chen et aL, 1993). I n h u m a n s , P. australis v e n o m causes severe muscle d e s t r u c t i o n with c o n s e q u e n t m y o g l o b i n u r i a , which m a y lead to significant renal p r o b l e m s a n d even acute renal failure ( R o w l a n d s et al., 1969; H o o d a n d J o h n s o n , 1975; S u t h e r l a n d et al., 1981). T h e a i m o f the p r e s e n t w o r k was to s t u d y the sequence o f m o r p h o l o g i c a l changes i n d u c e d in skeletal muscle a n d k i d n e y by a m y o t o x i n (causing m y o g l o b i n u r i a ) isolated f r o m the v e n o m o f P. australis. MATERIALS AND METHODS The myotoxic fraction was isolated from the crude venom of P. australis by Sephadex G 50 gel filtration chromatography followed by further purification on FPLC mono S HR 5/5 using a linear salt gradient (a gift from Dr Kong Soo Khoo, Department of Biochemistry, National University of Singapore). The myotoxic fraction had a mol. wt of approximately 13,500 and was a basic PLA2, similar to the basic PLA2 fraction VIII-A (Mebs and Samejima, 1980) (see Table 1).
Intramuscular injection of PA myotoxin and myoglobinuria assay Male Swiss albino mice (approximately 20 g) were from the Laboratory Animal Holding Centre, National University of Singapore. They were anaesthetized with ether, and a single i.m. injection of one of various dilutions of the toxins from the venom of P. australis was given in 0.1 ml of 0.9% (w/v) sodium chloride, into the calf muscles. Myoglobinuria was detected by placing the animals in white filter paper which stained red or dark brown in positive cases (Mebs and Samejima, 1980; Gopalakrishnakone and Mebs, 1990). The toxin-injected mice were killed at various time intervals (n = 3) at 5 min, 15 min, 30 min, I hr, 3 hr, 7 hr, 12 hr, 24 hr, 48 hr, 72 hr, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks and 6 weeks after i.m. injection for both light microscopic and electron microscopic studies. The minimum dose of the toxin to produce myoglobinuria was 4.5 mg/kg i.m. Light microscopy (LM) Mice were anaesthetized with chloral hydrate (0.3 ml of 3.5% w/v i.p.). The injected leg was immobilized by pinning at the knee and ankle joints, and fixation commenced by perfusion through the left ventricle. Animals were perfused with 0.9% w/v sodium chloride to flush out whole blood and then with 10% formaldehyde in distilled water for 30 min. The soleus muscle, gastrocnemius muscle and kidney were removed, post-fixed overnight in 10% formaldehyde, dehydrated through a series of ethanol and finally embedded in wax. Sections of 7/~m thickness were cut and stained with haematoxylin and eosin (H&E) or Massons trichrome and examined under light microscope (Leitz Aristoplan). Areas exhibiting pathology were photographed with Kodak film. Table 1.
Toxin VIII-A PA myotoxin
Myogiobinuria ++ ++
Minimum dose to produce myoglobinuria, and LOs0 Mol. wt 5.0 mg/kg s.c. 13,400 7.7 mg/kg s.c. 4.5 mg/kg i.m. 13,500 7.25 mg/kg i.m.
PLA2 activity + +
Morphology of P. australis Myotoxin Envenomation
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Electron microscopy (EM) The mice were anaesthetized with chloral hydrate and perfused through the left ventricle with 0.9% w/v sodium chloride followed by cold mixture containing 2% paraformaldehyde and 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min. The soleus muscle was dissected out and trimmed into pieces of approximately I mm in length and immersed in the same fixative for about 4 hr and then soaked in 0.1 M phosphate buffer (pH 7.4) overnight. The tissues were then postfixed in 1% osmium tetroxide in phosphate buffer for 2 hr. After dehydration through a graded series of ethanol and 100% acetone, tissues were kept in 1:6 of 100% acetone/araldite mixture overnight. Three changes of fresh Araldite were made before tissues were finally embedded in Araldite and incubated in an oven at 60°C for 24 hr. Semithin sections (1.0/lm) were cut on an ultramicrotome, stained with 1% toludine blue and examined under light microscope. Relevant areas were identified, trimmed and ultrathin gold sections were cut and mounted on copper grids. Sections were stained with uranyl acetate (10 min) and lead citrate (8 min), and then observed under electron microscope (Philips 400T) at a voltage of 80 kV.
RESULTS
General observation P r i o r to a d m i n i s t r a t i o n o f toxin, all mice were active; 15-30 m i n after injection o f 4.5 m g / k g i.m. o f P A m y o t o x i n the mice a p p e a r e d m o t i o n l e s s with stretched h i n d limb on the injected side. T h e white tissue p a p e r s in the cage were stained with red o r d a r k b r o w n urine b y 50% o f mice, a b o u t 2 h r after injection o f m y o t o x i n . The red urine was evident f r o m 60 m i n o n w a r d s . M o s t o f the a n i m a l s s h o w e d signs a n d s y m p t o m s for a p e r i o d o f 4 8 - 7 2 hr. A f t e r 3 - 5 d a y s m o s t o f the a n i m a l s recovered f r o m this illness a n d a p p e a r e d n o r m a l a n d active; a b o u t 5 % o f a n i m a l s died within 2 4 ~ 8 hr.
Light microscopy of muscle Pseudechis australis m y o t o x i n ( P A m y o t o x i n ) i n d u c e d m y o n e c r o s i s f r o m 30 m i n after i.m. injection o f the toxin. T h e early changes o b s e r v e d include (a) d i s r u p t i o n o f the m y o f i b r i l s with delta lesions (Fig. 1); (b) h y p e r c o n t r a c t i o n o f myofibrils with s h r u n k e n d a r k a r e a s o f c l u m p e d m a t e r i a l (Figs 1 a n d 2); (c) v a c u o l a t i o n o f the muscle cells (Fig. 2); (d) o e d e m a t o u s a p p e a r a n c e with mild cellular infiltration (Fig. 3); a n d (e) loss o f striations. All the a b o v e - m e n t i o n e d c h a n g e s were seen f r o m as early as 30 min to 7 hr.
Fig. 1. Thirty minutes after injection. Note hypercontracted clumped necrotic myofibrils (N) and delta lesions (arrowheads). (H&E stain, bar = 40/~m.)
1456
D. PONRAJ and P. GOPALAKRISHNAKONE
Fig, 2. Three hours after injection. Note vacuolation (arrows), blood vessel (V) unaffected, delta lesion (arrowheads) and necrosis (N). (Semithin section stained with 1% toludine blue, bar = 40 gin.)
Macrophages were also seen inside the necrotic muscle cells and in the interstitial connective tissues (Fig. 4). Regeneration of the muscle fibres was evident by 3-5 days because of the presence of m a n y myotubes containing central nuclei (Fig. 5). There were numerous connective tissue fibres and fibroblast in some areas as shown by Massons trichrome stain (Fig. 5). By 5-10 days regeneration was intense and in an advanced stage. By the 10th day m a n y f b r e s were completely regenerated, with a central row of nuclei, but there were still some areas of myonecrosis without regeneration, with connective tissue in the periphery. By about 3 weeks regeneration was almost complete with the appearance of striation of the muscle fibres, and occupation of the whole cytoplasm (Fig. 6).
Fig. 3. Seven hours after injection. Skeletal muscle cells in advanced stage of necrosis (N) with mild infiltration of necrotic fibres by macrophages. (H&E stain, bar = 40 gm.)
Morphology of P. australis Myotoxin Envenomation
1457
Fig. 4. Twelve hours after injection. Note infiltration of macrophages seen inside the necrotic cells and in the interstitial connective tissues. (H&E stain, bar = 40/~m.)
Electron microscopy of muscle Control calf muscle obtained from mice injected with 0.9% w/v sodium chloride showed normal sarcomere ultrastructure (Fig. 7). In experimental calf muscle the changes could be observed as early as 5 min after the injection. In early phases (5 min), some areas of the sarcomeres showed a tendency to fragment among the Z-lines with disruption and disappearance of Z-lines, sometimes appearing in an irregular manner (Fig. 8). The sarcomeres were fragmented, with dissolution and degeneration of the I-band (Fig. 9). Sarcoplasm between the damaged myofibrils was oedematous with separation of adjacent myofibrils (Fig. 9). There were areas of disrupted plasma membrane, but the basal
Fig. 5. Five days after injection. Note the central row of nuclei (arrowheads) with connective tissue (C) and fibrosis around the myofibre with different intensity of staining pattern due to regeneration. (Massons trichrome stain, bar = 40 pm.)
1458
D. PONRAJ and P. GOPALAKRISHNAKONE
Fig. 6. Three weeks after injection. Note the regenerated myofibrils with central row of nuclei (cn) with some of them showing fusion (arrowheads). Note the myofibrils occupy the whole of the cytoplasm showing almost complete regeneration. (H&E stain, bar = 20/lm.)
lamina appeared to be intact (Fig. 10). A few randomly located small vesicles were also seen in the sarcoplasm of the affected area. Mitochondria in these regions appeared swollen and contained dilated intercisternal spaces and floccular degeneration. Mitochondria in which the internal membranes were broken down, with the formation of vacuoles and a reduction in the number of cristae, were also seen after 60 min post-injection (Fig. 11).
Fig. 7. Control section showing normal sarcomeres with dark A-band and light I-band with Z-disc; mitochondria and triads (T) are also seen. (Bar = 0.5/tm.)
Morphology of P. australis Myotoxin Envenomation
Fig. 8. Five minutes after injection, The sarcomeres tend to fragment along the Z-lines. Note the dilated sarcoplasmic reticulum (S), and the three sarcomeres (asterisk) in various stages of fragmentation of I-band and Z-disc. (Bar = 0,5 tim.)
Fig. 9. One hour after injection. Note the discoid degeneration of the Z-discs (arrows), malalignment of Z-discs and oedematous sarcoplasm separating the myofilaments. (Bar = 1/~m.)
1459
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D. PONRAJ and P. GOPALAKRISHNAKONE
Fig. I0. One hour after injection. Note the dissolution of the 1-band (asterisk) with the disruption of the plasma membrane (arrows). The basal lamina appears to be intact (arrowheads). (Bar = 0.25/~m.)
Fig. 11. One hour after injection. Note the mitochondria (M) scattered in the necrotic debris (N). Mitochondria in which internal membranes were broken down (arrow) and reduction of cristae are seen. (Bar = 0.25 #m.)
Morphology of P. australis Myotoxin Envenomation
1461
Fig. 12. Five days after injection. Note the central row of nuclei (Cn) with scattered myofilamentsat the periphery of the myoblast (arrowheads). The myoblasts have extensive surface membrane contacts with one another. (Bar = 1#m.) By 7-48 hr after injection there were still areas of hypercontracted myofilaments in some regions but many areas showed degeneration with necrotic debri. Many areas were devoid o f myofilaments, but cytoplasmic organelles like swollen mitochondria, sarcoplasmic reticulum, vesicles and pyknotic nuclei were seen. By the 5th day a clear central row of nuclei was seen in regenerating areas (Fig. 12). Myoblasts demonstrated specific characteristics such as a tendency to become aggregated and the ability to form a long thin strip of interdigitated cells (Fig. 12). Ultrastructural features of myofibrillogenesis were also seen in multinucleated myotubes. Both thick and thin myofilaments organized in bundles were seen (Fig. 12). These bundles initially appeared scattered in the cytoplasm of the myotube, but later the A- and I-bands were seen with Z-line formation in the middle of the I-band (Fig. 12). The development of these myofibrils was asynchronous, from one myotube to another, and even within the cytoplasm of the same multinucleated syncytium. By 10 days the myofibrils had become thicker and more numerous, and they occupied a larger portion of the cytoplasm with normal sarcomere structures such as dark and light bands. By 3 weeks most of the myofibrils showed almost a normal-looking sarcomere structure. A few areas of advanced stage of regeneration were also seen. Muscle fibre from the contralateral soleus muscle not injected locally with the toxin also showed degenerative changes followed by regeneration with a central row of nuclei (Fig. 13).
Light microscopy of kidney Control kidneys showed no lesions (Fig. 14). Experimental kidney showed myoglobin casts in the tubules from 1 hr onwards. The myoglobin casts were seen in proximal and distal convoluted tubules, loops of Henle and in the collecting ducts as a homogeneous
1462
D. P O N R A J ',,L.'d~ll
t
i •,
~
a n d P. G O P A L A K R I S H N A K O N E " ~
:
~
'!l
Fig. 13. Six weeks after injection. Section from the contralateral soleus muscle not injected with toxin showing central row of nuclei (cn) with regenerated myofilaments. (Bar = 2/~m.)
staining area occupying the entire lumen of the tubules (Figs 15 and 16). A cloudy swelling of the tubular epithelium with necrotic debris and degenerated tubular epithelium was also noticed. In some tubules the myoglobin casts obstructed the whole of the lumen (Fig. 16). The myoglobin casts and degenerative changes were observed with H&E stain as well as by Massons trichrome stain. (A detailed electron microscopic observation of the changes in the kidney is in preparation.)
Fig. 14. Section of control kidney showing normal tubular epithelium (arrowheads) and glomerulus (G). (H&E)
stain, bar =
20 # m . )
Morphology of P. australis Myotoxin Envenomation
1463
Fig. 15. Section of an experimental kidney showing numerous myoglobin casts (arrowsl and necrotic debris (N) in the lumen. (Massons trichrome stain, bar = 40 ~m.)
DISCUSSION
The myotoxic phospholipase from P. australis (PA myotoxin) is similar to myotoxic phospholipase VIII-A from P. australis (Mebs and Samejima, 1980). It produced myoglobinuria with a minimum dose of approximately 4.5 mg/kg i.m. and had a mol. wt of approximately 13,500. The changes caused by this generalized myotoxin (i.e. causing myoglobinuria) in muscle and kidney are described. This is in contrast to the previous studies where myotoxins mostly acted locally and did not produce visible myoglobinuira (Gopalakrishnakone et al., 1984; Guti~rrez et al., 1984; Harris, 1990; Ownby, 1990). The earliest changes were hypercontraction of the sarcomeres with the appearance of wedgeshaped delta lesion, and the delta lesion with disruption of the muscle fibres representing
Fig. 16. High power view of an experimental kidney showing degenerated tubular epithelium (arrowheads) and necrotic debris (N) and myoglobin casts. (Massons trichrome stain, bar ~ 20 ~m.)
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D. PONRAJ and P. GOPALAKRISHNAKONE
areas of cell degeneration, with portions of disrupted or discontinuous plasma membrane (Harris et al., 1980; Gutirrrez et al., 1984b, 1991). It is interesting to note that selective loss of the Z-line and I-band with dissolution of the Z-band and actin filaments (streaming) were seen in the present study. Similar observations have been reported with PLA2 from Pseudechis colletti (Weinstein et al., 1992) crotoxin (Gopalakrishnakone et al., 1984) and taipoxin (MacDonell, 1979). Duchen et al. (1974) made a similar report on the depolarizing fraction (a putative cardiotoxin) of Dendroaspis viridis venom. Dissolution of Z-band and actin filaments is the earliest degenerative change seen in many naturally occurring myopathies and in denervation atrophy (Hudgson and Pearce, 1969). This was also observed in experimental animals after poisoning with chloroquine (Aguagio and Hudgson, 1970), and in ischaemic limbs (Koreny Both et al., 1971). EM studies of myasthenia gravis have shown slight to marked change in Z-disc, such as streaming of Z-disc, splitting of sarcomeres centrally along the Z-band and fragmentation of myofilaments (Korenyi Both et al., 1973). Further research is needed to establish whether the sarcomere broke at the Z-disc or whether the A-band was released as the Z-disc broke down; it is likely that both these mechanisms may be involved. PA myotoxin caused destruction of the plasma membrane, which appears to be a prime target of many other myotoxic phospholipases (Harris et al., 1980; Gopalakrishnakone et al., 1984; Gutirrrez et al., 1984b; Johnson and Ownby, 1993); phospholipases, by hydrolysing key phospholipids, may cause the plasma membrane to become leaky. This would probably result in an increase in intracellular Ca + and hypercontraction of the myofibrils. The Z-disc and the interdigitating arrays of thick and thin filaments within each myofibril are held in place by an elaborate framework of cytoskeletal filaments. In the regions of sarcoplasm that border on the periphery of the Z-disc, ring-like arrangements of intermediate filaments that contain the proteins desmin, vimentin, and synemin have been described (Lazarides, 1980). In addition, the sarcomeres seem to be supported by a delicate flexible internal lattice made up partly of titin filaments and partly of another protein called nebulin, which spans the whole length of the sarcomere from one Z-line to the next (Wang, 1985). In our study the disruption and disappearance of Z-disc was probably due to the effect of the myotoxin damaging the cytoskeletal proteins such as desmin, leading to a weaker Z-disc with fragmentation, or its disappearance. Recent studies have shown that desmin completely disappeared in some fibres from as early as 3 hr after Notechis scutalus scutalus (Australian tiger snake) envenomation in experimental animals (Vater et al., 1992). Vater and co-workers also showed regeneration of desmin by 2 days, localized to the Z-discs once the myotubes have matured. They also showed that titin was rapidly hydrolysed during breakdown of tissues, but later than desmin, and suggested that the I-band breakdown is followed by A-band breakdown. Our ultrastructural studies also showed disruption of the I-band followed by the A-band. The myoglobin in skeletal muscle cells is localized mainly in the I-band, and in the Z-disc in particular (Kawai et al., 1987). It has been recently shown by Kawai et al. (1991) by immunocytochemistry that in degenerating muscle cells of patients with Duchenne's muscular dystrophy, and myotonic dystrophy, myoglobin was demonstrated in the distended lumen of the internal membrane system and in the intermyofibrillar space, through which it seemed to pass into the extracellular space. In amyotropic lateral sclerosis wavy Z-bands showed only slight staining. They have also shown that no staining was demonstrated in either the 1-band or Z-band in the hypercontracted area (Kawai et al., 1991). These findings suggest that myoglobin released from the I-band of the damaged muscle may pass out of the muscle cell into the circulation through an altered internal
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m e m b r a n e system and intermyofibrillar space, and appear as a m y o g l o b i n cast in the kidneys. This is in agreement with our observation o f the disappearance o f the I - b a n d and Z-disc followed by m y o g l o b i n u r i a and cast in the kidneys. I m m u n o h i s t o c h e m i c a l studies are in progress to show the specific binding site o f this toxin in the muscle. M y o g l o b i n u r i a with subsequent myoglobinuric n e p h r o p a t h y was observed in clinical and experimental e n v e n o m a t i o n with the crude v e n o m o f several ophidian species (Weinstein et al., 1992; G o p a l a k r i s h n a k o n e and Mebs, 1990; Sutherland et al., 1981; H o o d and J o h n s o n , 1975). F r o m o u r results it is clear that renal epithelial cell necrosis and tubular m y o g l o b i n cast causing obstruction to urinary flow secondary to myoglobinuria m a y lead to renal ischaemia and p r o b a b l y to acute renal failure. These findings strongly support the clinical observation o f acute renal failure in h u m a n envenomation (Rowlands et al., 1969). In conclusion, it is suggested that P A m y o t o x i n causes streaming and fragmentation o f Z-disc and severe myonecrosis. This m a y cause myoglobin, mainly localized at the I-band, to leak out o f the d a m a g e d muscle cell and into the circulation, causing renal d a m a g e with m y o g l o b i n casts blocking the tubules. It is possible that P A m y o t o x i n m a y be acting initially on the sarcolemmal m e m b r a n e and, once inside, acting on the Z-disc and I-band, releasing the m y o g l o b i n into the circulation as well as disrupting the muscle fibre, which will be followed by degeneration o f muscle fibre and the subsequent changes. Thus this toxin causes severe myonecrosis followed by myoglobinuria, and renal d a m a g e with m y o g l o b i n cast n e p h r o p a t h y m a y play a vital role in P. australis envenomation.
Acknowledgements--This research work was carried out by a grant from National University of Singapore, A/C
no. 3602022. D. Ponraj is a recipient of a scholarship from the National University of Singapore. The authors thank Ms Diljit Kaur for kindly typing this manuscript.
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