Role of nitric oxide in myotoxic activity induced by crotoxin in vivo

Toxicon 43 (2004) 425–432 www.elsevier.com/locate/toxicon

Role of nitric oxide in myotoxic activity induced by crotoxin in vivo E.H. Miyabaraa, R.C. Tostesb, H.S. Selistre-de-Arau´joc, M.S. Aokia, A.S. Moriscota,* a

Department of Histology/Embriology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Av Lineu Prestes 1524, ICB I, 05508-900 Sa˜o Paulo, Brazil b Department of Pharmacology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Av Lineu Prestes 1524, ICB I, 05508-900 Sa˜o Paulo, Brazil c Department of Physiological Sciences, Federal University of Sa˜o Carlos, 13565-905 Sa˜o Carlos, Brazil Received 30 October 2003; revised 30 January 2004; accepted 11 February 2004

Abstract This study was aimed to determine the role of nitric oxide on the skeletal myotoxic activity induced by crotoxin, the major component of the venom of Crotalus durissus terrificus. Rats were treated with N G -nitro-L -arginine methyl ester (L NAME), a non-selective inhibitor of nitric oxide synthase or vehicle for 4 days, and on the 5th day received an intramuscular injection of crotoxin into the tibialis anterior muscle. Rats were also treated with aminoguanidine bicarbonate salt or 7-nitroindazole, inhibitors of the inducible and neuronal isoforms of nitric oxide synthase, respectively, for 4 days and on the 5th day injected with crotoxin. All treated groups were sacrificed 24 h after injection of crotoxin. Tibialis anterior and soleus muscles were removed, frozen and stored in liquid nitrogen. Histological sections were stained with toluidine blue and assayed for acid phosphatase. The results show that L -NAME significantly minimizes myonecrosis induced by crotoxin and both aminoguanidine and 7-nitroindazole partially prevented myonecrosis induced by crotoxin. Based on the present results we conclude that nitric oxide is a very important intracellular signaling molecule that mediates crotoxin myotoxic activity. q 2004 Elsevier Ltd. All rights reserved. Keywords: Crotoxin; Nitric oxide; Skeletal muscle injury; Myotoxicity

1. Introduction Crotoxin (CTX), the main toxic component of SouthAmerican rattlesnake Crotalus durissus terrificus, is a heterodimer with a basic and weakly toxic secreted phospholipase A2 subunit, sPLA2 (CB) and an acidic nontoxic and nonenzymatic subunit (CA) (Hendon and Fraenkel-Conrat, 1971; Ru¨bsamen et al., 1971; Aird et al., 1985, 1986). CB, also named group IIA secreted phospholipase A2 (sPLA2-IIA), is a lipolytic enzyme that catalyses the hydrolysis of the acyl ester bond at the sn-2 position of * Corresponding author. Tel./fax: þ 55-11-309-17311. E-mail address: [email protected] (A.S. Moriscot). 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2004.02.012

phospholipids (Baek et al., 2000) and has a catalytic mechanism similar to mammalian sPLA2s (Gelb et al., 1995). It is well known that CB induces neurotoxicity (Harris, 1991; Faure et al., 2000) and myotoxicity (Gopalakrishnakone et al., 1984; Kouyoumdjian et al., 1986; Mebs and Ownby, 1990) through selective membrane-binding sites that direct it to specific cellular targets, and that such binding plays a crucial role in its mechanism of action (Kini and Evans, 1989). Since the sPLA2-binding protein is most abundant in brain, this first type of binding site was called the N-type (neuronal-type) receptor (Lambeau and Lazdunski, 1999). A second type of receptor for sPLA2-IIA was initially found in skeletal muscle, and was termed the M-type (muscle type) receptor, that was characterized as a member of a family of

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transmembrane proteins with a structural organization similar to that of the macrophage mannose receptor (Lambeau et al., 1994). A common property of this protein family involves endocytosis. However, it has been proposed that the physiological role for the M-type sPLA2 receptor is to internalize and deliver sPLA2-IIA to specific compartments within the cell where the enzyme might exert its activity (Fuentes et al., 2002). sPLA2-IIA also has other signaling properties as the generation, as a result of its catalytic activity, of both unesterified fatty acid and lysophospholipid (Fourcade et al., 1995), perturbation of the cell membrane by its interfacial interaction with substrate phospholipids (Koduri et al., 1998) and binding to acceptor proteoglycans (Fuentes et al., 2002). Furthermore, the inflammatory properties of sPLA2-IIA are based on activation of certain cytokines (IL-1b and TNF-a), which can lead to the release of arachidonic acid and subsequent production of other proinflammatory mediators (prostaglandins, leukotrienes and platelet-activating factors) (Crowl et al., 1991; Kudo et al., 1993; Vadas et al., 1993). It has been recently demonstrated that the calcineurin pathway plays a critical role in the CTX-induced myotoxic activity (Miyabara et al., 2004). Since previous studies have shown that calcineurin promotes neuronal damage dependently of NO production (Dawson et al., 1993; Butcher et al., 1997), we have hypothesized that NO could be involved in myonecrosis induced by CTX in skeletal muscle. Accordingly, other authors have shown that inhibition of PLA2 simultaneously reduces NO production and superoxide generation in cerebellar granule cells in culture (Gunasekar et al., 1995) and that the Crotalus durissus terrificus venom increases hydrogen peroxide (H2O2) and NO production in macrophages (Sampaio et al., 2001). Therefore, in the present work we have aimed to investigate the role of the NO on CTX myotoxic activity. Our results show that the pre-treatment with nitric oxide synthase (NOS) inhibitors is able to minimize the myonecrosis induced by CTX.

2. Materials and methods The experimental protocols used in this study are in accordance with the ethical principles in animal research followed by the Brazilian College of Animals Experimentation (COBEA) and were approved by the Institute of Biomedical Sciences/University of Sa˜o Paulo—Ethical Committee for animals research (CEEA). 2.1. Animals Fourty-seven Wistar rats, weighing 267.5 ^ 20.5 g were used. The animals were randomly divided into nine groups (n ¼ 5; for each group) and housed in standard plastic cages

in an animal room with controlled environmental conditions. Rats received standard food (Purinaw chow) and had ad libitum access to food and water. 2.2. Treated groups Five animals received treatment with the non-selective NOS inhibitor N G -nitro-L -arginine methyl ester (L -NAME) (50 mg/kg of body weight (b.w.)/day in the drinking water) during 4 days, and on the 5th day each animal received an intramuscular injection of CTX (0.17 mg/kg of b.w.) into the tibialis anterior (TA) muscle. We have performed a series of injections using doses of CTX ranging from 0.085 to 0.34 mg/kg of b.w. (data not shown). The dose used in the present study was chosen aiming significant necrosis in the muscle injected without decrease in total body weight. Other rats were treated with the inducible NOS (iNOS) inhibitor aminoguanidine bicarbonate salt (250 mg/kg of b.w.), via oral (gavage), for 4 days and on the 5th day they were injected with CTX as previously described. Another group received the neuronal NOS (nNOS) inhibitor 7-nitroindazole (7-NI, 50 mg/kg of b.w.; in intraperitoneal administration of 0.5 ml of mineral oil/Dimethyl Sulfoxide (DMSO) 1:1) during 4 days and, on the 5th day, the CTX injection. All treated groups were sacrificed 24 h after the CTX injection. The effects of mineral oil/DMSO (vehicle) injections were also evaluated. 2.3. Control groups Animals received an intramuscular injection of saline (NaCl 0.9%) or an intramuscular injection of CTX (0.17 mg/kg of b.w.) into the right and left TA muscle and were evaluated 24 h later. Rats were also treated with the same doses of L -NAME or aminoguanidine or 7-NI alone for the same 4 days-period, but did not receive the CTX. 2.4. Crotoxin isolation CTX was purified from Crotalus durissus terrificus crude venom by gel filtration chromatography (Landucci et al., 1994), and analyzed by a tricine-SDS-polyacrilamide gel electrophoresis (Shagger and Jagow, 1987). 2.5. Histology Animals were weighed, sacrificed by decapitation and left TAs and soleus muscles were removed, immediately frozen in melting isopentane and stored in liquid nitrogen. Frozen muscles were cut through mid-portion (end-plate region) generating 10 mm cross-sections using a cryostat (Leica CM3050, Germany). Unfixed histological sections were stained with aqueous toluidine-blue-borax solution (both 1% w/v) to reveal the general morphology (Morini et al., 1998; Salvini et al.,

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Table 1 TA and soleus muscle wet weight in control and treated groups

SOL (mg) TA (mg) a

Saline ðn ¼ 5Þ

CTX ðn ¼ 5Þ

DMSO ðn ¼ 5Þ

L -NAME

Amino ðn ¼ 5Þ

7-NI ðn ¼ 5Þ

L -NAME

ðn ¼ 5Þ

þ CTX ðn ¼ 5Þ

Amino þ CTX ðn ¼ 5Þ

7-NI þ CTX ðn ¼ 5Þ

93 ^ 4 464 ^ 30

147 ^ 32* 669 ^ 30*

102 ^ 8 483 ^ 40

93 ^ 14 473 ^ 55

97.5 ^ 7 451 ^ 34

112 ^ 8 450 ^ 15

102 ^ 10a 505 ^ 46a

120 ^ 18 621 ^ 156**

135 ^ 23** 590 ^ 30

Results are mean ^ standard deviation; *p , 0:001 vs saline, **p , 0:01 vs saline (p , 0:05 are considered statistically significant). p , 0:01 vs CTX.

1999). Macrophages were visualized by the histochemical detection of lysosomal acid phosphatase (ac-phosphatase) activity according to Bancroft (1996).

evident proof of tissue necrosis and phagocitosis (Carpenter and Karpati, 1984). 2.7. Quantification of necrosed myofibers

2.6. Muscle injury The presence of muscle fiber injury was evaluated by light microscopy (Nikon Eclipse E600). Muscle damage was identified by the presence of myonecrotic muscle fibers such as hypercontracted fibers, clear areas among the muscle fibers, which indicate tissue disruption, intracellular vacuoles and cell infiltration. Presence of ac-phosphatase activity was also considered as a signal of necrosis and phagocitosis. It is well known that normal muscle fibers do not have positive ac-phosphatase reaction, which indicates high concentration of lysosomes and is considered an

The number of necrosed myofibers in the TA muscle was expressed as the mean percentage of 1000 counted fibers (total of 3000) and for the soleus muscle as the mean percentage of 500 counted fibers (total of 1500). Three cross-sections of the TA and soleus muscles from different animals were analyzed in all groups injected with CTX. 2.8. Statistical analysis Comparisons between the weights of the TA and soleus muscles and rate of necrosis from control and treated groups

Fig. 1. Cross-sections of TA (A, B) and soleus muscles (C, D). The TA and soleus muscles from animals injected with saline had normal morphology (A and C, respectively). Note the presence of hypercontracted fibers (fibers with dark region; arrows), muscle fibers with clear areas (stars), which result from the muscle fibers disruption and edema, and intense cellular infiltration (asterisks), which is associated to tissue necrosis in TA and soleus muscles from rats injected with crotoxin (B and D, respectively). Toluidine Blue staining. Bar: 50 mm.

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Fig. 2. Serial cross-sections of TA muscles obtained from animals only injected with CTX (A, B), treated with L -NAME (C, D), or aminoguanidine (E, F) or 7-NI (G, H) and injured with CTX. (A, C, E, G) Toluidine Blue staining and (B, D, F, H) acid-phosphatase reaction. The muscles from animals treated with L -NAME and injected with CTX show less necrosed myofibers (arrow, C) with positive ac-phosphatase reaction (arrow, D) than the group only injected with CTX (arrows, A and B). Observe that the groups treated with aminoguanidine or 7-NI and subsequently injected with CTX show intermediary pattern of lesion (cell infiltration; arrows, E and G; positive ac-phosphatase reaction, arrows, F and H) between the group only injected with CTX and treated with L -NAME and injured with CTX. Bar: 50 mm.

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were analyzed by one way ANOVA following Tukey’s multiple comparison test. Significant level accepted was below 5%, ðp , 0:05Þ:

3. Results The muscle wet weight from animals injected with CTX was similarly elevated in SOL and TA muscles as compared to their respective controls (58 and 44%, respectively; Table 1). All NOS inhibitors alone and DMSO (vehicle for 7-NI) did not promote significant alterations in wet weight of SOL and TA muscles (Table 1). L -NAME administered before and following CTX injection (L -NAME þ CTX group) was able to severely reduce SOL and TA wet weight gain, reaching levels similar to the respective control groups. When aminoguanidine or 7-NI was administered prior to CTX injection it was noted that muscle wet weight gain was not significantly lower than the respective CTX controls (Table 1). TA and soleus muscles from animals injected with saline had normal morphology, with no signals of lesion (Fig. 1A and C). On the other hand, TA muscle from animals injected with CTX showed abundant presence of cell infiltration, hypercontracted muscle fibers and clear areas among muscle fibers, signals that clearly indicate tissue disruption, myonecrosis and edema (Fig. 1B). Injury in TA was, as expected, notably more intense neighboring the site of CTX injection (middle belly). Other areas of TA muscle were homogeneously injured. The soleus muscle presented a similar pattern of muscle damage as described for TA and was also severely injured (Fig. 1D), yet all regions of soleus muscle were damaged without a preferential site of injury. TA muscles from animals treated with L -NAME and injured with CTX showed decreased signals of muscle damage, such as few fibers in process of necrosis and a modest infiltration (Fig. 2C and D), in comparison with muscles from rats only injected with CTX (Fig. 2A and B). The percentage of necrosed fibers assessed by ac-phosphatase reaction in the group treated with L -NAME and injected with CTX was significantly reduced as compared to the control CTX group in the TA muscle (Table 2; Fig. 2C and D). Muscles from rats treated with aminoguanidine (Fig. 2E and F), 7-NI (Fig. 2G and H) or L -NAME (Fig. 2C and D) and injected with CTX showed reduced signals of

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skeletal muscle tissue damage (as assessed by ac-phosphatase reaction) in comparison to the group only injected with CTX (Fig. 2A and B; Table 2), although the reduction of tissue damage was more effective in the group treated with L -NAME and injected with CTX (Table 2). The muscles from L -NAME – aminoguanidine – 7-NI-treated control groups had normal morphology, similar to the control group that received saline injection (data not shown). In the soleus muscles from animals treated with L NAME and injured with CTX was observed normal morphological aspect with no signals of lesion (Fig. 3C and D), in comparison with muscles only injected with CTX (Fig. 3A and B). The muscles from rats treated with aminoguanidine or 7-NI and injected with CTX appeared to have reduced signals of skeletal muscle tissue damage, as few fibers in process of necrosis and minimal infiltration (as assessed by ac-phosphatase reaction) (Fig. 3E – H), in comparison to the group injected only with CTX (Fig. 3A and B). The percentage of necrosed fibers assessed by acphosphatase reaction in the group treated with L -NAME, aminoguanidine or 7-NI and injected with CTX was significantly reduced as compared to the CTX injected group (Table 2). Muscles from L -NAME – aminoguanidine– 7-NI-treated control groups had normal morphology, similar to the control group that received saline injection (data not shown).

4. Discussion In this study we have demonstrated that inhibition of NO production strongly minimizes myolesion induced by CTX. The results showing that the non-selective NOS inhibitor L -NAME induces a more pronounced myoprotection as compared to the selective NOS inhibitors aminoguanidine and 7-NI, suggest that both neuronal and inducible isoforms of NOS contribute for myoprotection against CTX. The possibility that myoprotection induced by NOS inhibitors is due to its anti-inflammatory properties is ruled out since in a previous study we have shown that diclofenac (a nonsteroidal anti-inflammatory) does not affect myonecrosis induced by CTX (Miyabara et al., 2004). The doses of L -NAME, aminoguanidine and 7-NI used in this study have been previously tested and significantly inhibit activity of NOS in vivo (Ribeiro et al., 1992;

Table 2 Incidence of necrosed myofibers of TA and soleus muscles

SOL (%) TA (%) a b

þ CTX

CTX ðn ¼ 3Þ

L -NAME

60.1 ^ 11 43.8 ^ 5.3

0.7 ^ 0.8* 3.2 ^ 2*

Amino þ CTX ðn ¼ 3Þ

7-NI þ CTX ðn ¼ 3Þ

2.3 ^ 1.2* 16.2 ^ 1.2*a

2.8 ^ 1.8* 23 ^ 7**b

Results are mean ^ standard deviation; *p , 0:001 vs CTX, **p , 0:01 vs CTX (p , 0:05 are considered statistically significant). p , 0:05 vs L -NAME þ CTX. p , 0:01 vs L -NAME þ CTX.

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Fig. 3. Serial cross-sections of soleus muscles obtained from animals only injected with CTX (A, B), treated with L -NAME (C, D), or aminoguanidine (E, F), or 7-NI (G, H) and injured with CTX. (A, C, E, G) Toluidine Blue staining and (B, D, F, H) acid-phosphatase reaction. Note that several necrotic fibers with cell infiltration (arrow, A) are positive to ac-phosphatase reaction, which indicate presence of necrosis and macrophages in process of phagocitosis (arrow, B). The muscles from animals treated with L -NAME and injected with CTX show normal morphological aspect (C, D), i.e. with no signals of lesion. Soleus muscles from rats treated with aminoguanidine or 7-NI and injected with CTX show minimal cell infiltration (arrows, E and G) and positive ac-phosphatase reaction (arrows, F and H) as compared to the group only injected with CTX. Bar: 50 mm.

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Sannomiya et al., 1997; Moore et al., 1993; Rao et al., 2002). It has been described that the nNOS is the most abundantly expressed isoform of NOS in skeletal muscle in basal conditions (Stamler and Meissner et al., 2001), but in adverse situations such as inflammatory myopathies and in skeletal muscle cells exposed to bacterial lipopolysaccharides or inflammatory cytokines iNOS isoform expression is greatly increased, playing an important role in injury conditions (Boczkowski et a., 1996; El-Dwairi et al., 1998). All three NOS isoforms seem to contribute to various forms of skeletal muscle injury. In certain models such as experimental muscle crush (Rubinstein et al., 1998) and ischemia-reperfusion (Thiemermann et al., 1997) injury, it was observed a remarkable induction of iNOS and nNOS, respectively; therefore it is proposed that induction of these isoforms can cause devastating damage to muscle. In addition, an induction of eNOS occurs and it is associated with the increased capillary perfusion and thus identified with the extreme vasodilatation and edema that is known to occur after injury (Rubinstein et al., 1998). Although we have not excluded the possibility that the endothelial NOS (eNOS) isoform is also involved in myoprotection against CTX, previous studies have shown that this isoform is very poorly expressed in skeletal muscle (Kapur et al., 1997) and up to date there is no specific inhibitor available. It is known that in contrast to iNOs, nNOs activation depends on the interaction with calcium and calmodulin in skeletal muscle (Nathan and Xie, 1994), leading to the interpretation that Caþ þ dependent signaling is involved with NO production. Indeed it has also been described that Caþ þ activated calcineurin dephosphorylates nNOS, increasing its catalytic activity in the nervous system (Snyder et al., 1998). We have recently shown that an inhibitor of calcineurin (a Caþ þ dependent phosphatase) called cyclosporin-A induces strong myoprotection against CTX in skeletal muscle (Miyabara et al., 2004), in line with the idea that sPLA2 myotoxic activity depends upon Caþ þ signaling. A similar mechanism seems also to be operating in the nervous system since blockade of calcineurin (by cyclosporin treatment) confers neuro-protection (Dawson et al., 1993; Butcher et al., 1997). Furthermore, calcineurin was identified as an important regulator of apoptosis in cardiomyocytes, because its activation was recently shown either to induce apoptosis (Saito et al., 2000) or to antagonize apoptosis depending on the status of p38 MAPK activation (Lotem et al., 1999). Other intracellular pathways may also play a role in the myotoxic effect of CTX. Accordingly, cytokines are known to strongly activate iNOS activity in skeletal muscle C2C12 cells, involving a thyrosine-kinase dependent pathway (Okuda et al., 1997). In conclusion, the results of the present study show a myoprotective effect of non-selective and selective

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inhibitors of neuronal and inducible NOS isoforms against CTX, indicating that NO is an important intracellular signaling molecule that mediates CTX myotoxic activity.

Acknowledgements This work was supported by FAPESP (Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo, Brazil). We thank A.G. Soares Jr. and for excellent technical assistance and J.U. Ribeiro for isolating the CTX.

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