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Toxicology in Vitro 21 (2007) 1382–1389

Cytotoxicity induced in myotubes by a Lys49 phospholipase A2

homologue from the venom of the snake Bothrops asper: Evidenceof rapid plasma membrane damage and a dual role for

extracellular calcium

Juan Carlos Villalobos a, Rodrigo Mora a,b, Bruno Lomonte a, Jose Marıa Gutierrez a,Yamileth Angulo a,c,*

a Instituto Clodomiro Picado, Facultad de Microbiologıa, Universidad de Costa Rica, San Jose, Costa Ricab Departamento de Microbiologıa e Inmunologıa, Facultad de Microbiologıa, Universidad de Costa Rica, San Jose, Costa Rica

c Departamento de Bioquımica, Escuela de Medicina, Universidad de Costa Rica, San Jose, Costa Rica

Received 13 March 2007; accepted 4 April 2007Available online 29 April 2007

Abstract

Acute muscle tissue damage, myonecrosis, is a typical consequence of envenomations by snakes of the family Viperidae. Catalytically-inactive Lys49 phospholipase A2 homologues are abundant myotoxic components in viperid venoms, causing plasma membrane damageby a mechanism independent of phospholipid hydrolysis. However, the precise mode of action of these myotoxins remains unsolved. Inthis work, a cell culture model of C2C12 myotubes was used to assess the action of Bothrops asper myotoxin II (Mt-II), a Lys49 phos-pholipase A2 homologue. Mt-II induced a dose- and time-dependent cytotoxic effect associated with plasma membrane disruption, evi-denced by the release of the cytosolic enzyme lactate dehydrogenase and the penetration of propidium iodide. A rapid increment incytosolic Ca2+ occurred after addition of Mt-II. Such elevation was associated with hypercontraction of myotubes and blebbing ofplasma membrane. An increment in the Ca2+ signal was observed in myotube nuclei. Elimination of extracellular Ca2+ resulted inincreased cytotoxicity upon incubation with Mt-II, suggesting a membrane-protective role for extracellular Ca2+. Chelation of cytosolicCa2+ with BAPTA-AM did not modify the cytotoxic effect, probably due to the large increment induced by Mt-II in cytosolic Ca2+

which overrides the chelating capacity of BAPTA-AM. It is concluded that Mt-II induces rapid and drastic plasma membrane lesionand a prominent Ca2+ influx in myotubes. Extracellular Ca2+ plays a dual role in this model: it protects the membrane from the cytolyticaction of the toxin; at the same time, the Ca2+ influx that occurs after membrane disruption is likely to play a key role in the intracellulardegenerative events associated with Mt-II-induced myotube damage.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Myotoxic PLA2; Snake venom; Ca2+ homeostasis; Cytotoxicity; Mitochondria

1. Introduction

Muscle tissue degeneration occurs in many pathologicphenomena, including dystrophies, mechanical and cold-

0887-2333/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tiv.2007.04.010

* Corresponding author. Address: Instituto Clodomiro Picado, Facultadde Microbiologıa, Universidad de Costa Rica, San Jose, Costa Rica. Tel.:+506 2293135; fax: +506 2920485.

E-mail address: yangulo@icp.ucr.ac.cr (Y. Angulo).

induced damage, various infections, and intoxications bysynthetic or natural agents (Carpenter and Karpati, 2001).Envenomations by snakes often concur with prominentlocal and systemic muscle degeneration, owing to either thedirect action of myotoxins or to the ischemia that occursin muscle tissue as a consequence of venom-induced micro-vascular alterations (Gutierrez and Rucavado, 2000;Gutierrez and Ownby, 2003). The most abundant myo-toxic components in snake venoms correspond to

J.C. Villalobos et al. / Toxicology in Vitro 21 (2007) 1382–1389 1383

phospholipases A2 (PLA2) of classes I (elapid venoms) andII (viperid venoms) (Mebs and Ownby, 1990; Harris andCullen, 1990; Gutierrez and Ownby, 2003). MyotoxicPLA2s of class II are subdivided into catalytically-active,Asp49 variants, and catalytically-inactive homologueshaving Lys, Ser or Arg at position 49 (Lomonte et al.,2003a). Lys49 PLA2 homologues constitute highly interest-ing tools to investigate the mechanisms of muscle fiberdegeneration in conditions where an intrinsic enzymaticphospholipid hydrolysis is absent.

Lys49 PLA2 homologues are able to interact and disruptplasma membranes of various eukaryotic cell types(Lomonte et al., 1994a, 1999) as well as of bacteria (Para-mo et al., 1998). Such membrane-disruptive effects are pro-voked by a molecular region, enriched in hydrophobic andcationic residues, located near the C-terminus of the toxin(Lomonte et al., 1994c, 2003a; Chioato et al., 2002). Lys49PLA2 homologues are present in relatively high concentra-tions in the venoms of many viperid species, thus playing arelevant role in the myotoxic action induced by these ven-oms (Lomonte et al., 2003a).

In vivo observations have demonstrated that myotoxicPLA2s induce rapid plasma membrane damage, followedby a series of cellular degenerative events, such as hypercon-traction of myofilaments, mitochondrial swelling and dis-ruption, disorganization of sarcoplasmic reticulum and Ttubule membrane systems, and nuclear pycnosis (see Harrisand Cullen (1990) and Gutierrez and Ownby (2003) forreviews). Such rapid plasma membrane damage provokesan influx of extracellular Ca2+ following the prominent elec-trochemical gradient that exists for this cation across themembrane (Gutierrez and Ownby, 2003). However, suchCa2+ increment has been evidenced only by quantificationof total muscle Ca2+ levels (Gutierrez et al., 1984a, 1989).Intracellular Ca2+ homeostasis includes a highly complexsignaling toolkit, with components at the levels of plasmamembrane, mitochondria and sarcoplasmic reticulum, inaddition to several Ca2+-binding proteins in the cytosol(Berridge et al., 2003). The integrity of the plasma mem-brane therefore plays a crucial role in the regulation ofCa2+ permeability and, consequently, in controlling thecytosolic Ca2+ concentration.

Cultures of myotubes constitute a useful model of skele-tal muscle fibers. Muscle precursor cells, myoblasts, can beinduced to differentiate into multinucleated myotubes,which have many of the biochemical and physiological fea-tures of mature muscle fibers (Yaffe and Saxel, 1977). Myo-tubes have been used to study the effect of myotoxic PLA2s(Bruses et al., 1993; Bieber et al., 1994; Incerpi et al., 1995;Lomonte et al., 1999; Angulo and Lomonte, 2005). A highersusceptibility to the action of viperid myotoxic PLA2s hasbeen shown for myotubes, when compared to undifferenti-ated myoblasts and other cell types (Lomonte et al., 1999;Angulo and Lomonte, 2005), thus reproducing the predom-inant myotoxic action of these proteins in vivo (Gutierrezet al., 1984b), and suggesting the presence of an ‘acceptor’for these toxins in muscle cell plasma membrane. Owing

to the possibility of manipulating cellular processes andmechanisms in vitro, myotube cell culture allows the studyof specific aspects of myotoxin action on muscle cells.

In order to further investigate the mechanism by whichLys49 PLA2 homologues affect muscle fibers, a cell culturemodel of differentiated myotubes was used in this work toassess (1) whether a myotoxic Lys49 PLA2 homologueinduces a rapid increase in cytosolic Ca2+, associated withits plasma membrane-disruptive action, and (2) whetherthe cytotoxic effect induced by this myotoxin can be alteredby chelation of extracellular or cytosolic Ca2+.

2. Materials and methods

2.1. Myotoxin

Myotoxin II (Mt-II) was isolated by ion-exchange chro-matography on CM-Sephadex from a venom poolobtained from adult specimens of Bothrops asper collectedin the Caribbean region of Costa Rica (Lomonte andGutierrez, 1989). Homogeneity was assessed by analyticalreverse-phase HPLC on a C4 column (4.6 · 250 mm), usinga gradient of 0–60% acetonitrile in 0.1% trifluoroacetic acid(v/v).

2.2. Cell cultures

The murine skeletal muscle cell line C2C12 (CRL-1772,ATCC) was used. Myoblasts were grown in 25 cm2 bottlesusing Dulbecco’s Modified Eagle’s Medium (DMEM,Sigma–Aldrich, St. Louis, MO, USA), supplemented with15% fetal calf serum (FCS) (Sigma–Aldrich), 2 mM gluta-mine, 1 mM pyruvic acid, penicillin (100 U/mL), strepto-mycin (0.1 mg/mL), and amphotericin B (0.25 lg/mL), ina humidified atmosphere with 7% CO2, at 37 �C. Subcon-fluent monolayers were treated, for 5 min at 37 �C, withtrypsin (1500 U/mL), containing 5.3 mM EDTA. Resus-pended cells were seeded in 96-well microplates, at anapproximate initial density of 1–4 · 103 cells per well, inthe same culture medium. Differentiation of myoblasts intomyotubes was achieved by reducing the concentration ofFCS in the medium to 1% (Lomonte et al., 1999). After5–6 days, the majority of the cells (80–90%) in the wellscorresponded to myotubes.

2.3. Cytotoxicity assays

Cytolysis was assessed by determining the release of thecytosolic enzyme lactic dehydrogenase (LDH), as previ-ously described (Lomonte et al., 1994a, 1999). Briefly, myo-tube cultures were incubated with various concentrationsof Mt-II, dissolved in culture medium containing 1%FCS, at a volume of 100 lL/well. Aliquots of the superna-tant in culture wells were collected at various time intervals,and LDH activity was determined by using a commercialkit (Biocon LDH-P, Analyticon Biotechnologies AG, Ger-many). Reference controls for 0% and 100% cytolysis

Time (hr)0 10

LDH

rele

ase

(U/m

l)

0

200

400

600

50403020

Fig. 1. Cytotoxic activity of Mt-II on skeletal muscle myotubes. Thefollowing concentrations of Mt-II were added to myotubes: 30 lg/mL (d),100 lg/mL (s), 300 lg/mL (.), and incubations were carried out atvarious time intervals. Cytotoxicity was estimated by the release of lacticdehydrogenase (LDH) into supernatants, and expressed as units/mL. Eachpoint represents mean ± SD of triplicates. LDH activities of supernatantsof myotubes incubated for 48 h with medium alone or with 0.1% Triton X-100 were 24 ± 5 U/mL and 595 ± 110 U/mL, respectively.

1384 J.C. Villalobos et al. / Toxicology in Vitro 21 (2007) 1382–1389

consisted of medium alone and medium from cells incu-bated with 0.1% (v/v) of Triton X-100, respectively. Allassays were carried out in triplicates. In some experiments,cytolysis was assessed by the incorporation of propidiumiodide (PI). Briefly, myotubes were incubated with Mt-II,as described. At various time intervals, cells were treatedwith 1 lg/mL PI and 1 lg/mL Hoechst (Molecular Probes,Invitrogen, USA). PI stains nuclei of cells with disruptedplasma membrane, whereas Hoechst stains all nuclei. After30 min of incubation at 37 �C, cultures were observed in afluorescence microscope, and nuclei stained with each fluo-rochrome were counted. Analysis was performed using thesoftware IMAGE ProR (Media Cybernetics, Maryland,USA), and cytolysis was expressed as the percentage ofnuclei stained with PI. In order to assess cytotoxicity usinga parameter different from cell membrane disruption, theMTT assay (Mosmann, 1983), which detects cell viabilityon the basis of oxidative metabolism, was used in someexperiments. After incubation of myotubes with Mt-II, cul-ture medium was removed, and 100 lL of medium contain-ing 0.5 mg/mL of MTT was added to each well. After 1 hof incubation at 37 �C on a 7% CO2 atmosphere, plateswere allowed to dry for 24 h, and then 100 lL of ethanolwere added per well. Absorbances at 570 nm were recordedin a microplate reader.

2.4. Microscopic analysis of cytosolic Ca2+ increments

Myotube cultures were incubated with a solution con-taining 6 lM Fluo-3 acetomethyl ester (Fluo-3 AM, Molec-ular Probes) and 0.1% of the detergent Pluronic (MolecularProbes) for 60 min at 37 �C. Culture wells were then washedwith phosphate-buffered saline solution, pH 7.2 (PBS), fol-lowed by the addition of cell culture medium containing 1%FCS, 1 lg/mL PI and Mt-II (50 lg/mL). Cells were incu-bated at various time intervals, and observed in a fluores-cence microscope. Image capture and analysis wereperformed with the software IMAGE ProR. This method-ology allowed the simultaneous analysis of PI and ofFluo-3AM fluorescence, thus detecting plasma membranedamage and increments in cytosolic Ca2+, respectively.

2.5. Effect of Ca2+ chelators on MT-II-induced cytotoxicity

The role of extracellular Ca2+ was assessed using 2 mMEGTA (Shakhman et al., 2003). Cytosolic Ca2+ was inhib-ited by chelation with 1,2-bis(aminophenoxy)-ethane-N,N,N 0-tetraacetic acid (BAPTA-AM, 50 lM) (Shakhmanet al., 2003; Mora et al., 2006). EGTA and BAPTA-AMwere from Sigma–Aldrich. The role of extracellular Ca2+

was also investigated by varying the concentration of thiscation in the culture medium. In some cases, the concentra-tion of Mg2+ in the medium was also varied. The chelatorswere added to myotubes in culture and incubated at 37 �Cduring 1 h. Then, various concentrations of Mt-II wereadded and cytotoxicity was assessed at various time inter-vals, as described above. Before studying the effect of these

chelators on Mt-II action, the compounds alone, at theconcentrations selected, were added to myotubes, and cyto-toxicity was evaluated by the LDH assay. Moreover, thelack of effect of these chelators on the LDH assay was cor-roborated by incubating them, at the concentration used incell culture experiments, with a solution obtained from son-ication of myotube monolayers, followed by the quantifica-tion of LDH activity, compared with conditions whereEGTA and BAPTA-AM were excluded from the mixture.

2.6. Statistical analysis

The Student’s t test was used to determine the signifi-cance of the differences between the mean values of twoexperimental groups.

3. Results

3.1. Cytotoxic effect induced by Mt-II on myotubes

Mt-II induced a dose-dependent cytolysis of myotubes,evidenced by the release of LDH to supernatants. Whenusing toxin concentrations of 300 and 100 lg/mL, thetime-course of DHL release revealed a rapid membranedamage within the first hours (Fig. 1). The time requiredto reach 50% of LDH release, considering that 100%release corresponded to an activity of 595 ± 110 U/mL,was 1 h for a Mt-II concentration of 300 lg/mL, and 6 hfor a Mt-II concentration of 100 lg/mL (Fig. 1). In con-trast, at 30 lg/mL, LDH release had a delayed onset, neverreaching 50% release (Fig. 1). Plasma membrane disruptionwas corroborated by the incorporation of PI into the nuclei(Fig. 2). Cytotoxicity was also evident by a drop in oxida-tive metabolism, assessed by the MTT assay (not shown).

Prop

idiu

m io

dide

-sta

ined

nuc

lei (

%)

0

20

40

60

80

100

30 50 100 30 50 100

3 hr 24 hr

Myotoxin II (μg/ml)

Fig. 2. Cytotoxic activity of Mt-II on skeletal muscle myotubes, asdetermined by incorporation of propidium iodide. Myotube cultures wereincubated with various concentrations of Mt-II. After either 3 h or 24 h,propidium iodide (1 lg/mL) and Hoechst (1 lg/mL) were added to theculture wells, followed by an incubation period of 30 min at 37 �C.Cultures were observed in a fluorescence microscope and images werecaptured. After counting the total nuclei (stained with Hoechst) and thenuclei in affected cells (stained with propidium iodide), cytotoxicity wasexpressed as the percentage of propidium iodide-stained nuclei whencompared to total nuclei. Propidium iodide incorporation in myotubesincubated with medium alone was <1%. Results correspond to mean ± SDof triplicates.

Fig. 3. Increments in cytosolic Ca2+ in myotubes incubated with Mt-II (50propidium iodide (1 lg/mL). Then, Mt-II was added and the changes in intracafter toxin addition. Notice fluorescence along a myotube. (C) 10 s after toxin aof blebs at the periphery of the cell. (D) 5 s after toxin addition. Prominent fl

J.C. Villalobos et al. / Toxicology in Vitro 21 (2007) 1382–1389 1385

3.2. Mt-II increases the levels of cytosolic Ca2+

Myotubes were pretreated with fluo-3AM and PI, andthen Mt-II (50 lg/mL) was added. No fluorescence wasobserved in myotubes before toxin addition (Fig. 3A).Upon addition of Mt-II, there was an increase in the intra-cellular green fluorescence within the first 2–5 s, revealing arapid increment in cytosolic Ca2+ concentration (Fig. 3B).In general, fluorescence occurred throughout the extensionof myotubes and not in focalized regions. The staining ofnuclei with PI started 5–15 s after the onset of increase influo-3AM fluorescence (not shown). Some cells presenteda granular pattern of fluorescence, with highest intensityin organelles which probably correspond to mitochondria,and some cells presented evident blebbing in their plasmamembrane (Fig. 3C). A high intensity of fluorescence wasobserved in myotube nuclei (Fig. 3D), an observation thatwas performed also in cells that were not incubated withPI, thus evidencing increments in Ca2+ levels in the nuclei.Upon addition of Mt-II, myotubes sometimes showedspontaneous contractions, a phenomenon not observed incells incubated in the absence of toxin.

3.3. Effect of chelation of extracellular and cytosolic Ca2+ on

cytotoxicity

Neither EGTA nor BAPTA-AM was cytotoxic at theconcentrations utilized and did not affect the activity of

lg/mL). Myotube cultures were pretreated with fluo-3AM (6 lM) andellular green fluorescence were followed. (A) Before toxin addition. (B) 5 sddition. Intense fluorescence is observed in the cytosol; notice the presenceuorescence is observed in various nuclei. 40·.

) 200

400

600

3 hr

1386 J.C. Villalobos et al. / Toxicology in Vitro 21 (2007) 1382–1389

LDH when incubated with a cellular lysate, thus discardingthe possibility of interference of these chelators with theassays performed. When cells were pretreated with EGTA,in order to remove extracellular Ca2+, an unexpectedobservation was made. The presence of EGTA significantlyincreased the susceptibility of myotubes to Mt-II (Fig. 4),although EGTA by itself was not cytotoxic. This observa-

Myotoxin II (μg/ml)

LDH

rele

ase

(U/m

l)

0

200

400

600

10 30 50 100 EGTA

*

* *

Fig. 4. Effect of EGTA on the cytotoxic activity of Mt-II. Myotubes wereincubated for 3 h with various concentrations of Mt-II in either normalculture medium (black bars) or medium containing 2 mM EGTA (graybars). The empty bar labeled EGTA corresponds to myotubes incubatedwith 2 mM EGTA alone, which induced a LDH release that did not differfrom LDH release in cells incubated with medium alone. LDH activity ofsupernatants of myotubes incubated with 0.1% Triton X-100 was595 ± 110 U/mL. *p < 0.05 when compared with cells incubated withMt-II without EGTA.

Control

LDH

rele

ase

(U/m

l)

0

200

400

600 DMEM 1.8 mM 0.5 mM 0 mM

Mt-II Mt-II + EGTA

**

Fig. 5. Effect of varying the concentration of Ca2+ in the medium on thecytotoxic activity of Mt-II. Myotubes were incubated with either normalDMEM medium (containing 1.8 mM CaCl2), or medium containing thesame components as DMEM, but having different Ca2+ concentrations(0 mM, 0.5 mM, 1.8 mM). Myotubes were incubated with either no Mt-II(control), 50 lg/mL Mt-II (Mt-II) or 50 lg/mL Mt-II and 2 mM EGTA(Mt-II + EGTA). LDH activity in the supernatant was determined upon3 h of incubation. LDH activity of supernatants of myotubes incubatedwith 0.1% Triton X-100 was 680 ± 105 U/mL Results are presented asmean ± SD of triplicates. *p < 0.05 when compared with cells incubatedwithout EGTA at the same Ca2+ concentration.

Myotoxin II (μg/ml)LD

H re

leas

e (U

/ml

0

30 50 100 BM0

200

400

24 hr

Fig. 6. Effect of the cytosolic Ca2+ chelator BAPTA-AM on the cytotoxicactivity of Mt-II. Myotubes were pretreated with either medium (blackbars) or 50 lM BAPTA-AM (gray bars). Then, various concentrations ofMt-II were added and the release of LDH was determined at 3 and 24 h.M: myotubes incubated with medium alone, B: myotubes incubated withBAPTA-AM alone. LDH activity of supernatants of myotubes incubatedwith 0.1% Triton X-100 was 580 ± 95 U/mL. Results are presented asmean ± SD of triplicates.

tion was performed when using incubation times of 3 h(Fig. 4) and 24 h (not shown). In order to assess whetherthis effect was due to the lack of Ca2+ or instead to thepresence of EGTA, experiments were conducted in whichthe Ca2+ concentration of the culture medium was chan-ged. Results show that reduction of extracellular Ca2+ pro-moted a higher sensitivity of myotubes to the cytolyticeffect of Mt-II (Fig. 5). At 0 mM Ca2+, LDH release wassimilar to conditions where EGTA was added to a Ca2+-containing medium (Fig. 5). To determine whether thiseffect could be also reproduced by varying the concentra-tions of another divalent cation, experiments were per-formed with media having different concentrations ofMg2+. Variation of Mg2+ concentration of the mediumdid not affect the cytotoxic activity of Mt-II (results notshown), thus indicating that the increased susceptibilityeffect was selective for Ca2+. On the other hand, chelationof cytosolic Ca2+ by BAPTA-AM did not affect the cyto-toxic activity of Mt-II (Fig. 6).

4. Discussion

Myotoxic PLA2s isolated from snake venoms inducerapid skeletal muscle degeneration, which starts with thebinding of these toxins to muscle sarcolemma, followedby a rapid functional and structural disruption of the

J.C. Villalobos et al. / Toxicology in Vitro 21 (2007) 1382–1389 1387

plasma membrane (Gutierrez et al., 1984a,b; Dixon andHarris, 1996; Gutierrez and Ownby, 2003). In the case ofLys49 PLA2 homologues, such as Mt-II, which lack cata-lytic activity, a mechanism of action has been proposedwhereby the toxin binds to anionic moieties of the plasmamembrane, and subsequently alters its permeability using astretch of cationic and hydrophobic amino acid residueslocated at its C-terminal region (Lomonte et al., 1994c,2003a).

Earlier work has shown that the myogenic cell lineC2C12 constitutes a good model for the study of class IIPLA2 myotoxins (Lomonte et al., 1999; Angulo andLomonte, 2005). Our present results clearly show that therapid cytolytic effect induced by B. asper Mt-II in C2C12myotubes is associated with a very fast increment in cyto-solic Ca2+ levels, evidenced within few seconds. In agree-ment with this result, the permeabilization of bacterialcell membranes by Mt-II has also been demonstrated tooccur within seconds of exposure to toxin (Paramo et al.,1998). It has been proposed that plasma membrane disrup-tion by these toxins induces a prominent Ca2+ influx, fol-lowing the steep electrochemical gradient that existsacross the plasma membrane (Gutierrez and Ownby,2003); our observations corroborate this hypothesis. Inter-estingly, increments in cytosolic Ca2+ seem to precede, fora brief time lapse, the influx of PI, suggesting that the ear-liest perturbations in the plasma membrane may inducecytosolic Ca2+ increments without overt membrane dam-age. Such early Ca2+ rise may be also caused by the releaseof intracellular Ca2+ stores due to ‘calcium-induced cal-cium release’ (CICR) (Berridge et al., 2003) secondary tomembrane depolarization and release of Ca2+ from sarco-plasmic reticulum. Crotoxin, a potent neurotoxic and myo-toxic heterodimeric PLA2 from the venom of Crotalus

durissus terrificus, induces a very rapid membrane depolar-ization in muscle fibers before more evident signs of celldegeneration are observed (Melo et al., 2004). In the caseof Mt-II, the control of membrane permeability to PIand LDH is lost within few minutes, clearly revealing thatthe initial membrane perturbation is followed by a moredrastic effect on membrane integrity, with the consequentCa2+ influx from the extracellular fluid.

The rapid Ca2+ influx occurs concomitantly with con-traction of myotubes and with the formation of blebs atthe periphery of the cell, a phenomenon that may be relatedto myotube hypercontraction and to disruption in the con-nection between plasma membrane and the cytoskeleton,which has been associated with sarcolemmal lesions inischemia (Reimer and Jennings, 1992). The contraction ofskeletal muscle fibers within seconds after exposure toMt-II has been observed in vivo, using intravital miscros-copy techniques (Lomonte et al., 1994b). Furthermore,intracellular foci of high fluorescence were detected in smallorganelles that probably represent mitochondria, as well asin the nuclei of C2C12 myotubes. Accumulation of Ca2+ inmitochondria has been shown in many examples of musclecell pathology, including the action of myotoxic PLA2s

(Gopalakrishnakone et al., 1984). This Ca2+ influx to mito-chondria is an energy-dependent process that occursthrough the uniporter located at the inner mitochondrialmembrane (Berridge et al., 2003). The observed accumula-tion of Ca2+ in the nuclei of affected myotubes is notewor-thy. Ca2+ release into nucleoplasm may occur due to aCICR through inositol-3,4,5-trisphosphate (InsP3) andryanodine receptors present in the so-called nucleoplasmicreticulum (Marius et al., 2006). Ca2+ increments in thenucleoplasm may affect processes such as gene expression,DNA replication and activity of enzymes (Nicotera andRossi, 1994; Marius et al., 2006). In addition, myonucleimay constitute a hitherto unrecognized Ca2+ buffering sys-tem within the muscle fiber, whose role in the adaptation ofthese cells to injurious stimuli remains to be clarified.

The potential role of extracellular Ca2+ in the onset ofmyotube damage was assessed by pretreating cells withthe chelating agent EGTA. However, such treatment madethe cells more susceptible to the action of Mt-II. This effectwas caused by the removal of extracellular Ca2+ and not bya direct action of EGTA, as judged from experimentswhere Ca2+ concentration was modified in the absence ofEGTA. Ca2+ plays a stabilizing role in plasma membranein a number of cell types, and protects from the action ofa variety of cytolytic agents (Bashford et al., 1988). Inthe case of Mt-II, the mechanisms behind such protectiverole may be related with the binding of Ca2+ to nega-tively-charged lipid domains, which are likely to constitute‘acceptor’ sites for Lys49 PLA2 homologues (Dıaz et al.,2001; Lomonte et al., 2003b). Thus, extracellular Ca2+

has a dual role in this experimental model: it protects themembrane from the disruptive action of myotoxins, whileat the same time promotes a series of degenerative eventssecondary to its rapid influx to the cytosol once the plasmamembrane is disrupted by the action of Mt-II.

On the other hand, preincubation of myotubes with thecytosolic Ca2+ chelator BAPTA-AM failed to reduce cyto-toxicity. Mt-II-induced plasma membrane disruptioncauses a prominent influx of Ca2+ to the cytosol, and itis likely that such increment rapidly overrides the bufferingcapacity of BAPTA-AM, thus rendering this chelator inef-fective at controlling Ca2+ increment and its associated cel-lular damage. This finding agrees with the observations ofMora et al. (2006) on the action of Mt-II on a lymphoblas-toid cell line. When using a high toxin concentration, whichprovokes necrosis, BAPTA-AM was ineffective at prevent-ing this effect; in contrast, at lower toxin concentrations,which predominantly induce apoptosis associated withlower increments in cytosolic Ca2+, BAPTA-AM inhibitedthis effect (Mora et al., 2006).

In conclusion, Mt-II induces rapid and prominent incre-ment in cytosolic Ca2+, a process that seems to depend onthe disruptive action of this Lys49 PLA2 homologue on themyotube plasma membrane. Extracellular Ca2+ plays adual role in the cytotoxic action of Mt-II: its influx intothe cytosol contributes to acute cell damage and, on theother hand, extracellular Ca2+ protects the plasma

1388 J.C. Villalobos et al. / Toxicology in Vitro 21 (2007) 1382–1389

membrane from the cytotoxic action of Mt-II. Overall, ourresults demonstrate that Mt-II induces a prominent effecton the integrity of myotube plasma membrane, promotingincrements in cytosolic Ca2+ and rapid degenerative eventsthat bring the cell within few minutes beyond the ‘point ofno return’. These observations fully agree with experimen-tal evidence of rapid and drastic degenerative events thatoccur in skeletal muscle fibers in vivo after myotoxin-induced plasma membrane disruption (Gutierrez andOwnby, 2003).

Conflicts of interest

The authors declare that they do not have any conflict ofinterest in relation with this manuscript.

Acknowledgements

The authors thank Rodrigo Chaves for collaboration inthe laboratory work. This study was supported by Vice-rrectorıa de Investigacion, Universidad de Costa Rica(Project VI-741-A4-070) and the International Foundationfor Science (Project F/2766-3).

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