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The C-terminal region of a Lys49 myotoxin mediates Ca2+ influx in C2C12 myotubes
Toxicon 55 (2010) 590–596
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
The C-terminal region of a Lys49 myotoxin mediates Ca2þ influx in C2C12 myotubes Mariana Cintra-Francischinelli a, Paola Pizzo a, Yamileth Angulo b, Jose´ M. Gutie´rrez b, Cesare Montecucco a, Bruno Lomonte b, * a b
´ di Padova, Padova, Italy Dipartimento di Scienze Biomediche, Universita Instituto Clodomiro Picado, Facultad de Microbiologı´a, Universidad de Costa Rica, San Jose´, Costa Rica
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Article history: Received 19 August 2009 Received in revised form 3 October 2009 Accepted 8 October 2009 Available online 14 October 2009
Myotoxins are abundant components of snake venoms, being a significant public health problem worldwide. Among them, Lys49 phospholipase A2 homologue myotoxins cause extensive necrosis in skeletal muscle tissue. Their mechanisms of action are still poorly understood, but there is evidence that the C-terminal region is involved in membrane damage leading to myotoxicity. To investigate the effect of the C-terminal peptide 115–129 of Agkistrodon contortrix laticinctus myotoxin on the plasma membrane of myoblasts and myotubes, the entry of Ca2þ was monitored by fluorescence imaging, and the ensuing cytotoxicity was determined. The myotoxin synthetic peptide was found to act selectively on myotubes, which were rapidly overloaded with Ca2þ with ensuing necrosis. The profile of intracellular Ca2þ increase induced by the C-terminal peptide, but not by its scrambled version control, reproduces the second, prominent wave of the biphasic response documented in previous studies using whole Lys49 myotoxins. These observations provide relevant insights into the mechanism of action of this family of toxins, with implications for the understanding of their structure–function relationships. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Myotubes Calcium Myotoxin Phospholipase A2 homologue Lys49 Peptide Venom
1. Introduction Necrosis of skeletal muscle is a frequent manifestation of snakebite envenomings, which may lead to permanent tissue loss and disability (Warrell, 2004; Gutie´rrez et al., 2006). Such effect is caused by venom components that directly target muscle fibers, most notably toxins with phospholipase A2 (PLA2) structure (Gutie´rrez and Ownby, 2003; Lomonte et al., 2003a). PLA2s are common and abundant venom components which, in addition to myotoxicity, may also display a potent presynaptic neurotoxic
Abbreviations: p-Acl, C-terminal peptide 115–129 of Agkistrodon contortrix laticinctus myotoxin; p-Scr, scrambled version peptide; PLA2, phospholipase A2; [Ca2þ]c, cytosolic calcium ion concentration; EGTA, ethylene glycol tetraacetic acid. * Corresponding author. Tel.: þ506 2229 0344; fax: þ505 2292 0485. E-mail address: [email protected] (B. Lomonte). 0041-0101/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2009.10.013
action (Montecucco et al., 2008). Venom PLA2s found in snakes of the family Viperidae are classified within the structural group IIA, with subunits of 121–122 amino acid residues characterized by a specific pattern of disulfide bonds (Schaloske and Dennis, 2006). Among them, two subgroups can be discerned. One consists of enzymatically active PLA2s with a characteristic Asp49, which is a key residue for their catalytic mechanism. The other subgroup includes proteins with a conserved PLA2 fold, but presenting the replacement of Asp49 with Lys49 (or other amino acids), which lack PLA2 activity. The latter toxins have been termed, therefore, PLA2 homologues (Lomonte et al., 2003a, 2009). Despite their radical difference in catalytic ability, both Asp49 and Lys49 proteins are myotoxic, implying two distinct mechanisms of action. Asp49 PLA2 myotoxins depend on their enzymatic activity to damage skeletal muscle fibers (Gutie´rrez and Ownby, 2003). In contrast, the catalytically inactive Lys49 PLA2
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Fig. 1. Effects of peptides on [Ca ]c and viability of C2C12 myoblasts. In panels A and B cells were loaded with fura-2 as described in Section 2. The effect on [Ca2þ]c was followed as a change in the fura-2 fluorescence ratio (340/380 nm) in different cells after addition (arrows) of p-Acl (panel A) or p-Scr (panel B) at 250 mg/ml. Each trace represents the 340/380 ratio change with time of a single cell. For presentation, the ratios were normalized to the resting value. Panel C represents the cell viability, measured with the MTS assay, after treatment with peptides for 10 (empty bars) or 30 min (grey bars).
homologues utilize as major determinant of toxicity a site encompassing residues 115–129 in the C-terminal region, which includes a variable combination of cationic and hydrophobic/aromatic residues (Lomonte et al., 2003a; Chioato and Ward, 2003). There is evidence that both types of myotoxins alter the plasma membrane permeability and trigger a series of ensuing degenerative events, but the molecular mechanisms involved are still poorly characterized, and the identity of target acceptor moieties remains unknown (Montecucco et al., 2008). Previous studies have shown that Lys49 PLA2 homologues preferentially affect differentiated skeletal muscle myotubes, in comparison to their immature myoblast precursors (Angulo and Lomonte, 2005; Cintra-Francischinelli et al., 2009). Upon exposure to Lys49 myotoxins, a rapid increase in intracellular Ca2þ concentration occurs in C2C12 myotubes (Villalobos et al., 2007). This rise is characterized by a consistent biphasic pattern: an initial calcium mobilization from intracellular stores, followed by a massive influx from the extracellular milieu (Cintra-Francischinelli et al., 2009). Ultimately, such alterations in membrane permeability and intracellular Ca2þ levels bring the muscle cells to the point of irreversible damage and necrotic death. Short synthetic peptides representing the C-terminal region of Lys49 myotoxins have cytolytic and muscledamaging activities similar to their parent proteins, although they display a lower potency (Lomonte et al., 1994, 2003b; ˜ ez et al., 2001; Angulo and Lomonte, 2005; Gebrim et al., Nu´n
2009). Their precise mode of action is not known and, therefore, it is of interest to characterize in detail the effects of such kind of small molecules on muscle cell Ca2þ homeostasis. In this study we used the relevant cellular model of C2C12 myoblast and myotube cultures, and advanced Ca2þ imaging which provides time and space resolved information on live cells. The synthetic C-terminal peptide 115–129 of Agkistrodon contortrix laticinctus (ACL) myotoxin (Selistre de Araujo et al., 1996; sequence numbering according to Renetseder et al., 1985) was prepared and studied in the present work. By comparing the results obtained here with those previously recorded upon exposure to Lys49 myotoxins, relevant insights into the mechanism of action of these proteins were obtained, with implications for the understanding of their structure–function relationships. 2. Materials and methods 2.1. Synthetic peptides A 13-mer peptide (KKYKAYFKFKCKK; p-Acl), corresponding to the sequence 115–129 of ACL myotoxin, a Lys49 PLA2 homologue from the venom of the snake A. contortrix laticinctus, and a scrambled version (FKFKYKKACKKYK; p-Scr) were synthesized at the Protein Chemistry Laboratory of the University of Padova. They were purified with a C18 reverse phase NovaPak column, and were tested at a final concentration of 250 mg/ml (146 mM), selected on the basis of
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Fig. 2. Effects of peptides on [Ca2þ]c of C2C12 myotubes. Cells were loaded with fura-2 as described in Section 2. The effect on [Ca2þ]c was followed as a change in the fura-2 fluorescence ratio (340/380 nm) in different cells after addition (arrows) of p-Acl (panels A and B) or p-Scr (panels C and D) at 250 mg/ml. Each trace represents the 340/380 ratio change with time of a single cell. For presentation, the ratios were normalized to the resting value. Experiments were performed in Ca2þ-containing buffer (panels A and C), or in Ca2þ-free EGTA-containing media (panels B and D).
previous dose–response analyses (Lomonte et al., 2003b). p-Acl was chosen for the sake of comparison with previous studies; on the other hand, its sequence is almost identical to those of the C-terminal regions of Bothrops myotoxins used in calcium-imaging studies. 2.2. Cell cultures The cell model used here was the murine skeletal muscle C2C12 line, obtained from the American Type Culture Collection (CRL-1772, ATCC). Cells were maintained at subconfluent levels in growth medium consisting of Dulbecco’s modified Eagle medium (DMEM) (Gibco) supplemented with 10% foetal bovine serum (EuroClone). To induce differentiation (5–6 days), cells were grown to 80% confluence and then the medium was replaced with DMEM supplemented with 2% horse serum (Gibco) and changed every 24–48 h. For microscopy, cells were plated on cover slips (24 mm diameter) (10–20 104 cells/well) coated overnight with poly-Llysine (Sigma) and for 2 h with collagen (BD Biosciences). 2.3. Calcium measurements Cells were loaded with 3 mM fura-2/AM at 37 C for 30 min in modified Krebs–Ringer Buffer (see below) containing 0.04% pluronic (Molecular Probes, Inc., Eugene, OR). To prevent fura-2 leakage and sequestration, 250 mM sulfinpyrazone was present throughout the loading procedure and [Ca2þ]c measurements. The cover slips were washed with a modified Krebs–Ringer Buffer (mKRB, 140 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 11 mM
glucose pH 7.4), mounted on a thermostated chamber (Medical System Corp., New York, USA) at 37 C, and placed on the stage of an inverted microscope (Zeiss, Axiovert 100 TV) equipped for single cell fluorescence measurements and imaging analysis (TILL Photonics, Martinsried, Germany). Where indicated, a Ca2þ-free, EGTA (200 mM)-containing medium was used. The sample was alternatively illuminated (t ¼ 200 ms) by monochromatic light (at 340 and 380 nm wavelengths), every second for 10 min after peptide exposure, through a 40 oil immersion objective (NA ¼ 1.30; Zeiss). The emitted fluorescence was passed through a dichroic beam splitter (455DRPL), filtered at 505–530 nm (Omega Optical and Chroma Technologies, Brattleboro, VT, USA) and captured by a cooled CCD camera (Imago, TILL Photonics). For presentation, the ratios (F340/F380) of different cells were off-line normalized to the resting value measured within the first minute of the experiment. 2.4. Time lapse Experiments were performed in thermostatic chamber, 37 C, in Ca2þ containing medium as described above and the morphological alterations of cells exposed to the synthetic peptides were examined by Leica (DMIRE2) inverted microscopy equipped with a Leica DC500 CCD camera, 63 air objective. 2.5. Viability assay Myoblasts and differentiated myotubes were grown in 96-well plates and then exposed to peptides for 10 or
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30 min. Their viability was then measured with the MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl2-(4-sulfonphenyl)-2H-tetrazolium, inner salt) assay (Galluzzi et al., 2009). The CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) was used following instructions from the manufacturer.
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3. Results 3.1. Effect of peptides on C2C12 myoblasts Using the calcium-imaging technique, a slow and reversible rise in cytosolic [Ca2þ] was observed shortly after
Fig. 3. Time-lapse analysis of morphological changes induced by peptide p-Acl in C2C12 cells. Myotubes were treated with p-Acl (250 mg/ml) for 30 min in Ca2þ-containing buffer and were observed in bright field.
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10 min observation period (Fig. 1B). Under these experimental conditions, both peptides (250 mg/ml) did not induce cytotoxicity, and full viability of myoblasts was retained even after 30 min of exposure to the peptides (Fig. 1C). 3.2. Effect of peptides on C2C12 myotubes
Fig. 4. Viability of C2C12 myotubes upon incubation with peptides. Cells were incubated with p-Acl (250 mg/ml) or p-Scr (250 mg/ml) for 10 (empty bars) or 30 min (grey bars) in a Ca2þ-containing or Ca2þ-free EGTA medium. Bars represent mean values SEM estimated in three or more experiments performed in duplicates.
addition of the C-terminal peptide, p-Acl, to myoblasts (Fig. 1A). The signal was of a low magnitude and returned to basal levels, remaining stable until the end of the experiments. On the other hand, the scrambled version of this synthetic peptide used as a control, p-Scr, did not induce a significant effect on the intracellular calcium levels in these cells, basically remaining unaltered throughout the
The effects of peptides upon [Ca2þ]c in the differentiated myotubes were different from those observed in myoblasts. Exposure to p-Acl caused a rapid and prolonged [Ca2þ]c rise (Fig. 2A). The marked increase induced by p-Acl was completely prevented when cells were incubated in Ca2þfree, EGTA-containing medium (Fig. 2B), indicating the extracellular origin of the cation. However, even under such Ca2þ-free conditions, p-Acl was able to alter the plasma membrane permeability of myotubes. In fact, the addition of CaCl2 to the medium at later times (1 mM; arrow in Fig. 2B) caused an immediate [Ca2þ]c rise, indicating its influx through existing plasma membrane lesions. The inhibitor of voltage-gated Ca2þ channels, nifedipine (10 mM), did not show any effect on the activity of p-Acl; its activity on C2C12 myotubes, whose intracellular Ca2þ stores were depleted with cyclopiazonic acid (50 mM for 5 min), was the same (not shown), providing a further indication that p-Acl induces [Ca2þ]c rise by acting on the plasma membrane. In contrast to p-Acl, p-Scr caused only a very minor and transient [Ca2þ]c rise in few myotubes, whilst no effects
Fig. 5. Proposed mode of action of p-Acl on the myogenic cell line C2C12. Location of the C-terminal bioactive region of Agkistrodon contortrix laticinctus Lys49 myotoxin, encompassing amino acids 115–129, is indicated by a dotted circle. The 13-mer synthetic peptide p-Acl, corresponding to this region, reproduces not only the cytotoxic effect of the parent protein, but also its selectivity for differentiated, multinucleated myotubes, in comparison to undifferentiated myoblast cells. The higher susceptibility of myotubes is proposed to depend on the expression of plasma membrane microdomains, depicted by ‘‘X’’, still unknown. Given that p-Acl acts preferentially on myotubes, it is hypothesized that many more ‘‘X’’ sites are present on their sarcolemma. The p-Acl–‘‘X’’ interactions may involve initial electrostatic attractions between the several positively charged residues, which characterize p-Acl, and anionic charges on the lipid microdomains (step ‘‘a’’). These interactions may be stabilized by hydrophobic forces, where the amphiphilic nature of the peptide would allow a partial insertion or penetration into lipid bilayer (step ‘‘b’’), culminating with the loss of permeability control and massive entry of extracellular calcium (step ‘‘c’’), as documented in the present work.
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were observed in the majority of them, and all cells maintained their normal homeostatic functions for this ion (Fig. 2C). In the absence of extracellular calcium (Fig. 2D), the profile of [Ca2þ]c was not altered even after the addition of CaCl2 at a later time (1 mM; arrow), indicating that p-Scr was unable to affect the permeability of the plasma membrane of myotubes. Fig. 3 shows that p-Acl caused a progressive degeneration of the myotubes with loss of morphology, accumulation of aggregates in the cytosol and fragmentation, and eventual disappearance of the sarcolemma; on the other hand, p-Scr left the myotubes unaltered (not shown), morphologically identical to the untreated controls, visible in the top left panel. Similarly, myoblasts maintained their morphology in the presence of p-Acl. This is also reflected by the sensitive assessment of cell cytotoxicity performed with the MTS method, whose results are presented in Fig. 4. The viability of myotubes exposed to p-Acl decreased significantly and in a time-dependent manner, whereas cultures treated with p-Scr retained full viability. The rapid cytotoxic effect of p-Acl on myotubes was also exerted in the absence of extracellular calcium (Fig. 4). 4. Discussion The early alterations in cytosolic calcium levels induced by Lys49 PLA2 myotoxins were recently characterized in the C2C12 cellular model using live cell imaging of cytosolic Ca2þ (Cintra-Francischinelli et al., 2009). Here, we have found that the C-terminal peptide 115–129 of a well-characterized Lys49 myotoxin (Selistre de Araujo et al., 1996; Ambrosio et al., 2005) induces Ca2þ entry into C2C12 myotubes through the plasma membrane very similar to those caused by the whole myotoxins. In all cases, it is the extracellular calcium which floods into the cell cytosol, and the membrane lesions through which the cation enters can be generated by the C-terminal peptide also in the absence of extracellular calcium, confirming its calcium-independent mechanism of membrane perturbation. The comparison of the kinetics of cytosolic [Ca2þ] rise induced by the peptide in the presence and absence of extracellular calcium (panels A and B of Fig. 2) indicates that (a) few seconds are needed for the development of the membrane lesions and (b) the membrane lesions have a defined temporal stability (panel B of Fig. 2) because Ca2þ ions enter immediately when added to a calcium-free medium after 8 min from the peptide addition; in the latter case no time is required for peptide diffusion and membrane lesion. More prolonged delays between additions could not be tested because of the cytotoxic effects caused by the change of membrane permeability to other essential ions and metabolites. All these features of the action of the C-terminal peptide are very similar to those previously recorded with the full length myotoxins and provide compelling evidence in favour of the fact that the sarcolemma lesions caused by the Lys49 myotoxins are mediated by their C-terminal region. It is likely that other structural determinants of the molecule may be implicated in binding to the sarcolemma (dos Santos et al., 2009), explaining why a high concentration of C-terminal peptides, as compared to the native toxins, is needed to achieve the same cellular effects.
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In agreement with recent results obtained with myotoxins (Cintra-Francischinelli et al., 2009), a difference between myoblasts and myotubes regarding their susceptibility to calcium alterations and cell death induced by p-Acl was also found here. At variance from what was found with myotubes, myoblasts were essentially insensitive to the membrane permeabilizing action of p-Acl. This finding cannot be accounted for by the proposal that the whole toxin possesses an acceptor-binding site whilst the C-terminal peptide does not. It is temping to speculate that both the myotoxins and their C-terminal peptides do interact with specific microdomains of the sarcolemma, which are not present, or are very scarce, in myoblasts (Fig. 5). In fact, the cytosolic calcium rise induced by Lys49 myotoxins in muscle cells has a biphasic pattern, with an initial rapid mobilization from intracellular stores, soon followed by a massive Ca2þ influx from the extracellular milieu (Cintra-Francischinelli et al., 2009). As recently proposed, the initial calcium rise from intracellular sources implies the involvement of a signaling event, therefore strongly suggesting the recognition of a membrane acceptor in the mechanism of toxicity by Lys49 PLA2 homologues (Cintra-Francischinelli et al., 2009). p-Acl did not induce the initial marked calcium rise associated with its release from intracellular stores. This finding provides an important clue for understanding the structure–function relationships of these toxins, suggesting that the C-terminal region 115–129, strongly involved in the permeabilization mechanism, is probably not involved in the recognition of the membrane acceptor associated with the initial calcium signal induced by the complete toxins, which should therefore be accomplished by a different molecular site. On the other hand, the effect of p-Acl was clearly dependent on its specific sequence in the parent toxin, as inferred by the lack of activity of its scrambled version, p-Scr, and by the conservation of the C-terminal sequences among Lys49 myotoxins in terms of presence and distribution of positively charged and hydrophobic residues (Lomonte et al., 2003a,b). The weaker toxicity of the C-terminal peptide p-Acl, and its clear ability to reproduce the second, massive wave of the biphasic profile of intracellular calcium increase induced by Lys49 myotoxins in muscle cells, together suggest that these toxins bind to specific acceptors and this localizes the toxin on the cell surface. This binding is then followed by the interaction of the C-terminal region with the lipid bilayer with changes of its ion permeability. Future efforts should focus on defining the biochemical nature and identity of the membrane acceptor(s) involved and in the identification of the lipid-toxin structure which permeabilizes the membrane. However, these results show for the first time, using imaging, the essential role of the Cterminal region of Lys49 myotoxins in causing an extensive and rapid calcium influx leading to muscle cell death within few minutes. Acknowledgements MCF is supported by a Ph.D. fellowship of the Fondazione CARIPARO. We gratefully acknowledge the support of Telethon (grant GGP06133) and Fondazione Cariparo Progetto ‘‘Physiopathology of the synapse: neurotransmitters,
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neurotoxins and novel therapies’’ to CM. The work of YA, BL, and JMG is supported by Vicerrectorı´a de Investigacio´n, Universidad de Costa Rica, and the ICGEB-CRP Program (grant COS-08-03). Conflicts of interest The authors declare that there are no conflicts of interest. References Ambrosio, A.L.B., Nonato, M.C., Selistre de Araujo, H.S., Arni, R.K., Ward, R.J., Ownby, C.L., de Souza, D.H.F., Garrat, R.C., 2005. A molecular mechanism for Lys49-phospholipase A2 activity based on ligand-induced conformational change. J. Biol. Chem. 280, 7326–7335. Angulo, Y., Lomonte, B., 2005. Differential susceptibility of C2C12 myoblasts and myotubes to group II phospholipase A2 myotoxins from crotalid snake venoms. Cell Biochem. Funct. 23, 307–313. Chioato, L., Ward, R.J., 2003. Mapping structural determinants of biological activities in snake venom phospholipases A2 by sequence analysis and site directed mutagenesis. Toxicon 42, 869–883. Cintra-Francischinelli, M., Pizzo, P., Rodrigues-Simioni, L., Ponce-Soto, L.A., Rossetto, O., Lomonte, B., Gutie´rrez, J.M., Pozzan, T., Montecucco, C., 2009. Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors. Cell. Mol. Life Sci. 66, 1718–1728. Galluzzi, L., Aaronson, S.A., Abrams, J., Alnemri, E.S., Andrews, D.W., Baehrecke, E.H., Bazan, N.G., Blagosklonny, M.V., Blomgren, K., Borner, C., Bredesen, D.E., Brenner, C., Castedo, M., Cidlowski, J.A., Ciechanover, A., Cohen, G.M., De Laurenzi, V., De Maria, R., Deshmukh, M., Dynlacht, B.D., El-Deiry, W.S., Flavell, R.A., Fulda, S., Garrido, C., Golstein, P., Gougeon, M.L., Green, D.R., Gronemeyer, H., Hajno´czky, G., Hardwick, J.M., Hengartner, M.O., Ichijo, H., Ja¨a¨ttela¨, M., Kepp, O., Kimchi, A., Klionsky, D.J., Knight, R.A., Kornbluth, S., Kumar, S., Levine, B., Lipton, S.A., Lugli, E., Madeo, F., Malomi, W., Marine, J.C., Martin, S.J., Medema, J.P., Mehlen, P., Melino, G., Moll, U. ˜ ez, G., M., Morselli, E., Nagata, S., Nicholson, D.W., Nicotera, P., Nun Oren, M., Penninger, J., Pervaiz, S., Peter, M.E., Piacentini, M., Prehn, J. H., Puthalakath, H., Rabinovich, G.A., Rizzuto, R., Rodrigues, C.M., Rubinsztein, D.C., Rudel, T., Scorrano, L., Simon, H.U., Steller, H., Tschopp, J., Tsujimoto, Y., Vandenabeele, P., Vitale, I., Vousden, K.H., Youle, R.J., Yuan, J., Zhivotovsky, B., Kroemer, G., 2009. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ. 16, 1093–1107. Gebrim, L.C., Marcussi, S., Menaldo, D.L., de Menezes, C.S., Nomizo, A., Hamaguchi, A., Silveira-Lacerda, E.P., Homsi-Brandeburgo, M.I., Sampaio, S.V., Soares, A.M., Rodrigues, V.M., 2009. Antitumor effects of snake venom chemically modified Lys49 phospholipase A2-like BthTX-I and a synthetic peptide derived from its C-terminal region. Biologicals 37, 222–229.
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
The C-terminal region of a Lys49 myotoxin mediates Ca2þ influx in C2C12 myotubes Mariana Cintra-Francischinelli a, Paola Pizzo a, Yamileth Angulo b, Jose´ M. Gutie´rrez b, Cesare Montecucco a, Bruno Lomonte b, * a b
´ di Padova, Padova, Italy Dipartimento di Scienze Biomediche, Universita Instituto Clodomiro Picado, Facultad de Microbiologı´a, Universidad de Costa Rica, San Jose´, Costa Rica
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 August 2009 Received in revised form 3 October 2009 Accepted 8 October 2009 Available online 14 October 2009
Myotoxins are abundant components of snake venoms, being a significant public health problem worldwide. Among them, Lys49 phospholipase A2 homologue myotoxins cause extensive necrosis in skeletal muscle tissue. Their mechanisms of action are still poorly understood, but there is evidence that the C-terminal region is involved in membrane damage leading to myotoxicity. To investigate the effect of the C-terminal peptide 115–129 of Agkistrodon contortrix laticinctus myotoxin on the plasma membrane of myoblasts and myotubes, the entry of Ca2þ was monitored by fluorescence imaging, and the ensuing cytotoxicity was determined. The myotoxin synthetic peptide was found to act selectively on myotubes, which were rapidly overloaded with Ca2þ with ensuing necrosis. The profile of intracellular Ca2þ increase induced by the C-terminal peptide, but not by its scrambled version control, reproduces the second, prominent wave of the biphasic response documented in previous studies using whole Lys49 myotoxins. These observations provide relevant insights into the mechanism of action of this family of toxins, with implications for the understanding of their structure–function relationships. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Myotubes Calcium Myotoxin Phospholipase A2 homologue Lys49 Peptide Venom
1. Introduction Necrosis of skeletal muscle is a frequent manifestation of snakebite envenomings, which may lead to permanent tissue loss and disability (Warrell, 2004; Gutie´rrez et al., 2006). Such effect is caused by venom components that directly target muscle fibers, most notably toxins with phospholipase A2 (PLA2) structure (Gutie´rrez and Ownby, 2003; Lomonte et al., 2003a). PLA2s are common and abundant venom components which, in addition to myotoxicity, may also display a potent presynaptic neurotoxic
Abbreviations: p-Acl, C-terminal peptide 115–129 of Agkistrodon contortrix laticinctus myotoxin; p-Scr, scrambled version peptide; PLA2, phospholipase A2; [Ca2þ]c, cytosolic calcium ion concentration; EGTA, ethylene glycol tetraacetic acid. * Corresponding author. Tel.: þ506 2229 0344; fax: þ505 2292 0485. E-mail address: [email protected] (B. Lomonte). 0041-0101/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2009.10.013
action (Montecucco et al., 2008). Venom PLA2s found in snakes of the family Viperidae are classified within the structural group IIA, with subunits of 121–122 amino acid residues characterized by a specific pattern of disulfide bonds (Schaloske and Dennis, 2006). Among them, two subgroups can be discerned. One consists of enzymatically active PLA2s with a characteristic Asp49, which is a key residue for their catalytic mechanism. The other subgroup includes proteins with a conserved PLA2 fold, but presenting the replacement of Asp49 with Lys49 (or other amino acids), which lack PLA2 activity. The latter toxins have been termed, therefore, PLA2 homologues (Lomonte et al., 2003a, 2009). Despite their radical difference in catalytic ability, both Asp49 and Lys49 proteins are myotoxic, implying two distinct mechanisms of action. Asp49 PLA2 myotoxins depend on their enzymatic activity to damage skeletal muscle fibers (Gutie´rrez and Ownby, 2003). In contrast, the catalytically inactive Lys49 PLA2
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Fig. 1. Effects of peptides on [Ca ]c and viability of C2C12 myoblasts. In panels A and B cells were loaded with fura-2 as described in Section 2. The effect on [Ca2þ]c was followed as a change in the fura-2 fluorescence ratio (340/380 nm) in different cells after addition (arrows) of p-Acl (panel A) or p-Scr (panel B) at 250 mg/ml. Each trace represents the 340/380 ratio change with time of a single cell. For presentation, the ratios were normalized to the resting value. Panel C represents the cell viability, measured with the MTS assay, after treatment with peptides for 10 (empty bars) or 30 min (grey bars).
homologues utilize as major determinant of toxicity a site encompassing residues 115–129 in the C-terminal region, which includes a variable combination of cationic and hydrophobic/aromatic residues (Lomonte et al., 2003a; Chioato and Ward, 2003). There is evidence that both types of myotoxins alter the plasma membrane permeability and trigger a series of ensuing degenerative events, but the molecular mechanisms involved are still poorly characterized, and the identity of target acceptor moieties remains unknown (Montecucco et al., 2008). Previous studies have shown that Lys49 PLA2 homologues preferentially affect differentiated skeletal muscle myotubes, in comparison to their immature myoblast precursors (Angulo and Lomonte, 2005; Cintra-Francischinelli et al., 2009). Upon exposure to Lys49 myotoxins, a rapid increase in intracellular Ca2þ concentration occurs in C2C12 myotubes (Villalobos et al., 2007). This rise is characterized by a consistent biphasic pattern: an initial calcium mobilization from intracellular stores, followed by a massive influx from the extracellular milieu (Cintra-Francischinelli et al., 2009). Ultimately, such alterations in membrane permeability and intracellular Ca2þ levels bring the muscle cells to the point of irreversible damage and necrotic death. Short synthetic peptides representing the C-terminal region of Lys49 myotoxins have cytolytic and muscledamaging activities similar to their parent proteins, although they display a lower potency (Lomonte et al., 1994, 2003b; ˜ ez et al., 2001; Angulo and Lomonte, 2005; Gebrim et al., Nu´n
2009). Their precise mode of action is not known and, therefore, it is of interest to characterize in detail the effects of such kind of small molecules on muscle cell Ca2þ homeostasis. In this study we used the relevant cellular model of C2C12 myoblast and myotube cultures, and advanced Ca2þ imaging which provides time and space resolved information on live cells. The synthetic C-terminal peptide 115–129 of Agkistrodon contortrix laticinctus (ACL) myotoxin (Selistre de Araujo et al., 1996; sequence numbering according to Renetseder et al., 1985) was prepared and studied in the present work. By comparing the results obtained here with those previously recorded upon exposure to Lys49 myotoxins, relevant insights into the mechanism of action of these proteins were obtained, with implications for the understanding of their structure–function relationships. 2. Materials and methods 2.1. Synthetic peptides A 13-mer peptide (KKYKAYFKFKCKK; p-Acl), corresponding to the sequence 115–129 of ACL myotoxin, a Lys49 PLA2 homologue from the venom of the snake A. contortrix laticinctus, and a scrambled version (FKFKYKKACKKYK; p-Scr) were synthesized at the Protein Chemistry Laboratory of the University of Padova. They were purified with a C18 reverse phase NovaPak column, and were tested at a final concentration of 250 mg/ml (146 mM), selected on the basis of
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Fig. 2. Effects of peptides on [Ca2þ]c of C2C12 myotubes. Cells were loaded with fura-2 as described in Section 2. The effect on [Ca2þ]c was followed as a change in the fura-2 fluorescence ratio (340/380 nm) in different cells after addition (arrows) of p-Acl (panels A and B) or p-Scr (panels C and D) at 250 mg/ml. Each trace represents the 340/380 ratio change with time of a single cell. For presentation, the ratios were normalized to the resting value. Experiments were performed in Ca2þ-containing buffer (panels A and C), or in Ca2þ-free EGTA-containing media (panels B and D).
previous dose–response analyses (Lomonte et al., 2003b). p-Acl was chosen for the sake of comparison with previous studies; on the other hand, its sequence is almost identical to those of the C-terminal regions of Bothrops myotoxins used in calcium-imaging studies. 2.2. Cell cultures The cell model used here was the murine skeletal muscle C2C12 line, obtained from the American Type Culture Collection (CRL-1772, ATCC). Cells were maintained at subconfluent levels in growth medium consisting of Dulbecco’s modified Eagle medium (DMEM) (Gibco) supplemented with 10% foetal bovine serum (EuroClone). To induce differentiation (5–6 days), cells were grown to 80% confluence and then the medium was replaced with DMEM supplemented with 2% horse serum (Gibco) and changed every 24–48 h. For microscopy, cells were plated on cover slips (24 mm diameter) (10–20 104 cells/well) coated overnight with poly-Llysine (Sigma) and for 2 h with collagen (BD Biosciences). 2.3. Calcium measurements Cells were loaded with 3 mM fura-2/AM at 37 C for 30 min in modified Krebs–Ringer Buffer (see below) containing 0.04% pluronic (Molecular Probes, Inc., Eugene, OR). To prevent fura-2 leakage and sequestration, 250 mM sulfinpyrazone was present throughout the loading procedure and [Ca2þ]c measurements. The cover slips were washed with a modified Krebs–Ringer Buffer (mKRB, 140 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 11 mM
glucose pH 7.4), mounted on a thermostated chamber (Medical System Corp., New York, USA) at 37 C, and placed on the stage of an inverted microscope (Zeiss, Axiovert 100 TV) equipped for single cell fluorescence measurements and imaging analysis (TILL Photonics, Martinsried, Germany). Where indicated, a Ca2þ-free, EGTA (200 mM)-containing medium was used. The sample was alternatively illuminated (t ¼ 200 ms) by monochromatic light (at 340 and 380 nm wavelengths), every second for 10 min after peptide exposure, through a 40 oil immersion objective (NA ¼ 1.30; Zeiss). The emitted fluorescence was passed through a dichroic beam splitter (455DRPL), filtered at 505–530 nm (Omega Optical and Chroma Technologies, Brattleboro, VT, USA) and captured by a cooled CCD camera (Imago, TILL Photonics). For presentation, the ratios (F340/F380) of different cells were off-line normalized to the resting value measured within the first minute of the experiment. 2.4. Time lapse Experiments were performed in thermostatic chamber, 37 C, in Ca2þ containing medium as described above and the morphological alterations of cells exposed to the synthetic peptides were examined by Leica (DMIRE2) inverted microscopy equipped with a Leica DC500 CCD camera, 63 air objective. 2.5. Viability assay Myoblasts and differentiated myotubes were grown in 96-well plates and then exposed to peptides for 10 or
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30 min. Their viability was then measured with the MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl2-(4-sulfonphenyl)-2H-tetrazolium, inner salt) assay (Galluzzi et al., 2009). The CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) was used following instructions from the manufacturer.
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3. Results 3.1. Effect of peptides on C2C12 myoblasts Using the calcium-imaging technique, a slow and reversible rise in cytosolic [Ca2þ] was observed shortly after
Fig. 3. Time-lapse analysis of morphological changes induced by peptide p-Acl in C2C12 cells. Myotubes were treated with p-Acl (250 mg/ml) for 30 min in Ca2þ-containing buffer and were observed in bright field.
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10 min observation period (Fig. 1B). Under these experimental conditions, both peptides (250 mg/ml) did not induce cytotoxicity, and full viability of myoblasts was retained even after 30 min of exposure to the peptides (Fig. 1C). 3.2. Effect of peptides on C2C12 myotubes
Fig. 4. Viability of C2C12 myotubes upon incubation with peptides. Cells were incubated with p-Acl (250 mg/ml) or p-Scr (250 mg/ml) for 10 (empty bars) or 30 min (grey bars) in a Ca2þ-containing or Ca2þ-free EGTA medium. Bars represent mean values SEM estimated in three or more experiments performed in duplicates.
addition of the C-terminal peptide, p-Acl, to myoblasts (Fig. 1A). The signal was of a low magnitude and returned to basal levels, remaining stable until the end of the experiments. On the other hand, the scrambled version of this synthetic peptide used as a control, p-Scr, did not induce a significant effect on the intracellular calcium levels in these cells, basically remaining unaltered throughout the
The effects of peptides upon [Ca2þ]c in the differentiated myotubes were different from those observed in myoblasts. Exposure to p-Acl caused a rapid and prolonged [Ca2þ]c rise (Fig. 2A). The marked increase induced by p-Acl was completely prevented when cells were incubated in Ca2þfree, EGTA-containing medium (Fig. 2B), indicating the extracellular origin of the cation. However, even under such Ca2þ-free conditions, p-Acl was able to alter the plasma membrane permeability of myotubes. In fact, the addition of CaCl2 to the medium at later times (1 mM; arrow in Fig. 2B) caused an immediate [Ca2þ]c rise, indicating its influx through existing plasma membrane lesions. The inhibitor of voltage-gated Ca2þ channels, nifedipine (10 mM), did not show any effect on the activity of p-Acl; its activity on C2C12 myotubes, whose intracellular Ca2þ stores were depleted with cyclopiazonic acid (50 mM for 5 min), was the same (not shown), providing a further indication that p-Acl induces [Ca2þ]c rise by acting on the plasma membrane. In contrast to p-Acl, p-Scr caused only a very minor and transient [Ca2þ]c rise in few myotubes, whilst no effects
Fig. 5. Proposed mode of action of p-Acl on the myogenic cell line C2C12. Location of the C-terminal bioactive region of Agkistrodon contortrix laticinctus Lys49 myotoxin, encompassing amino acids 115–129, is indicated by a dotted circle. The 13-mer synthetic peptide p-Acl, corresponding to this region, reproduces not only the cytotoxic effect of the parent protein, but also its selectivity for differentiated, multinucleated myotubes, in comparison to undifferentiated myoblast cells. The higher susceptibility of myotubes is proposed to depend on the expression of plasma membrane microdomains, depicted by ‘‘X’’, still unknown. Given that p-Acl acts preferentially on myotubes, it is hypothesized that many more ‘‘X’’ sites are present on their sarcolemma. The p-Acl–‘‘X’’ interactions may involve initial electrostatic attractions between the several positively charged residues, which characterize p-Acl, and anionic charges on the lipid microdomains (step ‘‘a’’). These interactions may be stabilized by hydrophobic forces, where the amphiphilic nature of the peptide would allow a partial insertion or penetration into lipid bilayer (step ‘‘b’’), culminating with the loss of permeability control and massive entry of extracellular calcium (step ‘‘c’’), as documented in the present work.
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were observed in the majority of them, and all cells maintained their normal homeostatic functions for this ion (Fig. 2C). In the absence of extracellular calcium (Fig. 2D), the profile of [Ca2þ]c was not altered even after the addition of CaCl2 at a later time (1 mM; arrow), indicating that p-Scr was unable to affect the permeability of the plasma membrane of myotubes. Fig. 3 shows that p-Acl caused a progressive degeneration of the myotubes with loss of morphology, accumulation of aggregates in the cytosol and fragmentation, and eventual disappearance of the sarcolemma; on the other hand, p-Scr left the myotubes unaltered (not shown), morphologically identical to the untreated controls, visible in the top left panel. Similarly, myoblasts maintained their morphology in the presence of p-Acl. This is also reflected by the sensitive assessment of cell cytotoxicity performed with the MTS method, whose results are presented in Fig. 4. The viability of myotubes exposed to p-Acl decreased significantly and in a time-dependent manner, whereas cultures treated with p-Scr retained full viability. The rapid cytotoxic effect of p-Acl on myotubes was also exerted in the absence of extracellular calcium (Fig. 4). 4. Discussion The early alterations in cytosolic calcium levels induced by Lys49 PLA2 myotoxins were recently characterized in the C2C12 cellular model using live cell imaging of cytosolic Ca2þ (Cintra-Francischinelli et al., 2009). Here, we have found that the C-terminal peptide 115–129 of a well-characterized Lys49 myotoxin (Selistre de Araujo et al., 1996; Ambrosio et al., 2005) induces Ca2þ entry into C2C12 myotubes through the plasma membrane very similar to those caused by the whole myotoxins. In all cases, it is the extracellular calcium which floods into the cell cytosol, and the membrane lesions through which the cation enters can be generated by the C-terminal peptide also in the absence of extracellular calcium, confirming its calcium-independent mechanism of membrane perturbation. The comparison of the kinetics of cytosolic [Ca2þ] rise induced by the peptide in the presence and absence of extracellular calcium (panels A and B of Fig. 2) indicates that (a) few seconds are needed for the development of the membrane lesions and (b) the membrane lesions have a defined temporal stability (panel B of Fig. 2) because Ca2þ ions enter immediately when added to a calcium-free medium after 8 min from the peptide addition; in the latter case no time is required for peptide diffusion and membrane lesion. More prolonged delays between additions could not be tested because of the cytotoxic effects caused by the change of membrane permeability to other essential ions and metabolites. All these features of the action of the C-terminal peptide are very similar to those previously recorded with the full length myotoxins and provide compelling evidence in favour of the fact that the sarcolemma lesions caused by the Lys49 myotoxins are mediated by their C-terminal region. It is likely that other structural determinants of the molecule may be implicated in binding to the sarcolemma (dos Santos et al., 2009), explaining why a high concentration of C-terminal peptides, as compared to the native toxins, is needed to achieve the same cellular effects.
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In agreement with recent results obtained with myotoxins (Cintra-Francischinelli et al., 2009), a difference between myoblasts and myotubes regarding their susceptibility to calcium alterations and cell death induced by p-Acl was also found here. At variance from what was found with myotubes, myoblasts were essentially insensitive to the membrane permeabilizing action of p-Acl. This finding cannot be accounted for by the proposal that the whole toxin possesses an acceptor-binding site whilst the C-terminal peptide does not. It is temping to speculate that both the myotoxins and their C-terminal peptides do interact with specific microdomains of the sarcolemma, which are not present, or are very scarce, in myoblasts (Fig. 5). In fact, the cytosolic calcium rise induced by Lys49 myotoxins in muscle cells has a biphasic pattern, with an initial rapid mobilization from intracellular stores, soon followed by a massive Ca2þ influx from the extracellular milieu (Cintra-Francischinelli et al., 2009). As recently proposed, the initial calcium rise from intracellular sources implies the involvement of a signaling event, therefore strongly suggesting the recognition of a membrane acceptor in the mechanism of toxicity by Lys49 PLA2 homologues (Cintra-Francischinelli et al., 2009). p-Acl did not induce the initial marked calcium rise associated with its release from intracellular stores. This finding provides an important clue for understanding the structure–function relationships of these toxins, suggesting that the C-terminal region 115–129, strongly involved in the permeabilization mechanism, is probably not involved in the recognition of the membrane acceptor associated with the initial calcium signal induced by the complete toxins, which should therefore be accomplished by a different molecular site. On the other hand, the effect of p-Acl was clearly dependent on its specific sequence in the parent toxin, as inferred by the lack of activity of its scrambled version, p-Scr, and by the conservation of the C-terminal sequences among Lys49 myotoxins in terms of presence and distribution of positively charged and hydrophobic residues (Lomonte et al., 2003a,b). The weaker toxicity of the C-terminal peptide p-Acl, and its clear ability to reproduce the second, massive wave of the biphasic profile of intracellular calcium increase induced by Lys49 myotoxins in muscle cells, together suggest that these toxins bind to specific acceptors and this localizes the toxin on the cell surface. This binding is then followed by the interaction of the C-terminal region with the lipid bilayer with changes of its ion permeability. Future efforts should focus on defining the biochemical nature and identity of the membrane acceptor(s) involved and in the identification of the lipid-toxin structure which permeabilizes the membrane. However, these results show for the first time, using imaging, the essential role of the Cterminal region of Lys49 myotoxins in causing an extensive and rapid calcium influx leading to muscle cell death within few minutes. Acknowledgements MCF is supported by a Ph.D. fellowship of the Fondazione CARIPARO. We gratefully acknowledge the support of Telethon (grant GGP06133) and Fondazione Cariparo Progetto ‘‘Physiopathology of the synapse: neurotransmitters,
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