INFECTION AND IMMUNITY, June 2004, p. 3120–3128 Vol. 72, No. 60019-9567/04/$08.00�0 DOI: 10.1128/IAI.72.6.3120–3128.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Degeneration and Regeneration of Murine Skeletal NeuromuscularJunctions after Intramuscular Injection with a Sublethal Dose of

Clostridium sordellii Lethal ToxinJulien Barbier,1 Michel R. Popoff,2 and Jordi Molgo1*

Laboratoire de Neurobiologie Cellulaire et Moleculaire, U. P. R. 9040, Centre National de la RechercheScientifique, Gif-sur-Yvette,1 and Centre National de Reference Anaerobies,

Institut Pasteur, Paris,2 France

Received 10 September 2003/Returned for modification 14 October 2003/Accepted 31 January 2004

Clostridium sordellii lethal toxin (LT), a 250-kDa protein which is the bacteria’s major virulence factor,belongs to a family of large clostridial cytotoxins which glucosylate small GTP-binding proteins. Here, wereport the results of our ex vivo analysis of the structure and function of skeletal neuromuscular tissueobtained from mice at various times after intramuscular injection of a sublethal dose of LT (0.25 ng/g of bodywt). The toxin caused, within 24 h, pronounced localized edema, inflammation, myofibril disassembly, anddegeneration of skeletal muscle fibers in the injected area, and it glucosylated the muscle tissue’s smallGTPases. Regeneration of the damaged fibers was evident 6 to 9 days postinjury and was completed by 60 days.The expression of dystrophin, laminin, and fast and neonatal myosin in regenerating fibers, detected byimmunofluorescence microscopy, confirmed that LT does not impair the high regenerative capacity of murineskeletal muscle fibers. Functional studies revealed that LT affects muscle contractility and neuromusculartransmission. However, partial recovery of nerve-evoked muscle twitches and tetanic contractions was observedby day 15 postinjection, and extensive remodeling of the neuromuscular junction’s nerve terminals and clustersof muscle acetylcholine receptors was still evident 30 days postinjection. In conclusion, to the best of ourknowledge, this is the first report to characterize the degeneration and regeneration of skeletal neuromusculartissue after in vivo exposure to a large clostridial cytotoxin. In addition, our data may provide an explanationfor the severe neuromuscular alterations accompanying wound infections caused by C. sordellii.

The genus Clostridium includes anaerobic species that causesoft tissue-necrotizing infections (i.e., cellulitis and gas gan-grene) and toxic shock by virtue of their ability to producenumerous tissue-damaging enzymes and protein toxins (37, 38,41). Infections with Clostridium sordellii often result in toxicshock and multiorgan failure (1, 4) because of the toxin-in-duced necrosis of various cell types. The bacteria’s major vir-ulence factor is a high-molecular-weight (Mr, ca. 250,000) le-thal toxin (LT) that belongs to a family of large clostridialcytotoxins (5, 6, 23) and is antigenically related to toxin B ofClostridium difficile (28, 32), a major cause of pseudomembra-nous colitis after oral clindamycin therapy (14, 21).

The C. sordellii LT is a glucosyltransferase which uses aUDP-glucose cofactor (9) to glucosylate and inactivate small-molecular-mass, GTP-binding proteins (9, 22), i.e., the Rassuperfamily (33). The toxin also inhibits exocytosis in chromaf-fin cells (15) and neurotransmitter release when injected intopresynaptic neurons at identified synapses of the Aplysia cen-tral nervous system, with a potency comparable to that of theClostridium botulinum neurotoxins (11). The observed inhibi-tion of neurotransmitter release has been proposed (19) to becaused by the inactivation of the Rac GTPase associated withsynaptic vesicles in nerve terminals. Interestingly, LT has beenreported (12) to inhibit phospholipase D1, and the introduc-

tion of inactive phospholipase D1 into neurons has recentlybeen found (20) to reproduce the exocytosis blockade causedby the toxin.

The present ex vivo study was designed to characterize theeffects of the C. sordellii LT on skeletal muscle and neuromus-cular transmission in order to improve our understanding of itsmode of action. To the best of our knowledge, this is the firstpublished study to describe the degeneration and regenerationof skeletal muscle tissue and the remodeling of the neuromus-cular junction after in vivo exposure to a potent clostridialcytotoxin.

MATERIALS AND METHODS

C. sordellii LT, antibodies, and reagents. C. sordellii LT produced by strainIP82 was purified as previously described (32). Our immunofluorescence studiesused monoclonal antibodies directed against (i) the C-terminal domain of dys-trophin (1:500 dilution; Novocastra laboratories Ltd., Newcastle upon Tyne,United Kingdom), (ii) sarcomeric �-actinin (1:25 dilution; Sigma, Saint-QuentinFallavier, France), (iii) the heavy chain of fast myosin (1:10 dilution; Novocas-tra), (iv) troponin T (clone JLT-12; 1:100 dilution; Sigma), and (v) the 160-kDaisoform of neurofilaments (1:75 dilution; Sigma). Rabbit polyclonal antibodiesagainst neonatal-myosin (1:50 dilution) were kindly provided by C. Cifuentes-Diaz (Laboratoire de Neurogenetique Moleculaire, INSERM, Evry, France),and antibodies directed against laminin (1:500 dilution) were purchased fromSigma. Alexa 488- and Alexa 633-conjugated anti-mouse immunoglobulin G(1:1,500 and 1:1,000 dilutions, respectively), Alexa 488-conjugated anti-rabbitimmunoglobulin G (1:1,000 dilution), fluorescein isothiocyanate (FITC)- andtetramethylrhodamine isothiocyanate (TRITC)-conjugated �-bungarotoxin (�-BTX; 1:1,000 dilution), and TOTO-3 dye (1:500 dilution) were from MolecularProbes Europe BV, (Leiden, The Netherlands). TRITC-conjugated fasciculin-2(1:500 dilution; in phosphate-buffered saline [PBS]) was prepared by using theFluorReporter protein labeling kit (Molecular Probes) and was kindly provided

* Corresponding author. Mailing address: Laboratoire de Neurobi-ologie Cellulaire et Moleculaire, U. P. R. 9040, C. N. R. S., 1 Avenuede la Terrasse, 91198 Gif-sur-Yvette CEDEX, France. Phone: 33 1 6982 36 42. Fax: 33 1 69 82 94 66. E-mail: Jordi.Molgo@nbcm.cnrs-gif.fr.

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by Eric Krejci (Ecole Normale Superieure, Paris, France). Paraformaldehyde,Procion Orange, and Evans blue dye were from Sigma. Antifading mountingmedia (Vectashield and Vectashield with 4�,6�-diamidino-2-phenylindole[DAPI]) were from Vector Laboratories Inc. (Burlingame, Calif.). Other chem-icals were of the highest purity available.

Injection of mice with C. sordellii LT. All experiments were performed inaccordance with French and European Community guidelines for laboratoryanimal handling. The anterolateral region of the left hindlimb or the immediatevicinity of the levator auris longus (LAL) muscle (3) of 62 adult female Swiss-Webster mice (20 to 25 g) lightly anesthetized with ether was injected intramus-cularly with C. sordellii LT (0.25 ng/g body weight; in 50 �l of PBS), and controlmice were injected with PBS. Several doses of the toxin initially were tested, andwe chose the above-mentioned dose for the studies described in this reportbecause it was not lethal for mice and because its local effects were reproducibleand moderate. Control mice were similarly injected with PBS. The sublethal doseof LT injected intramuscularly produced pronounced localized edema and in-flammation in the area of injection but little or no disturbance in the generalcondition of the animals. Food and water were provided ad libitum, and theanimals were housed in the animal care facility, under standard conditions at aconstant temperature of 22°C with a cycle of 12 h of darkness and 12 h ofdaylight. At various times postinjection, mice were killed by dislocation of theircervical vertebrae, and the tibialis anterior (TA) and extensor digitorum longus(EDL) muscles were removed from the injected legs, together with their asso-ciated motor nerves. A similar procedure was used for ex vivo analysis of theactions of LT on the LAL muscles.

Detection of in vivo glucosylation of small GTPases in skeletal muscle tissue.Control and LT-injected EDL muscles were harvested at various times postin-jection, frozen with liquid nitrogen, and homogenized with a tissue grinder, andthe preparations’ proteins were examined for C. sordellii LT-mediated glucosy-lation as previously described (33). Briefly, samples of the muscle preparationswere dissolved in glucosylation buffer (50 mM triethanolamine [pH 7.5], 2 mMMgCl2, 1 mM dithiothreitol, 0.3 mM GDP) containing 10 �g of leupeptin/ml, 1�M pepstatin, 0.1 mM phenylmethylsulfonyl fluoride, and 0.5% Triton X-100.After centrifugation (1,000 � g for 5 min), the supernatant fluids were collected,and the amount of protein in each cell lysate was estimated by Coomassie bluestaining (Bio-Rad, Verrieres-Le-Buisson, France). Samples containing 50 �g oftotal protein were used for in vitro glucosylation assays with 7 �M UDP-[14C]glu-cose (DuPont NEN, Les Ulis, France) and 1 �g of LT in a final volume of 20 �l.After incubation (1 h at 37°C), the mixtures were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (10% monomer concentration), andthe gel was stained, destained, dried, and analyzed for radioactivity by autora-diography. The relative intensities of the radiolabeled bands were quantified byusing Densylab 2.5.2 software (Microvision Instruments, Evry, France).

Twitch tension measurements. Isolated mouse EDL nerve-muscle prepara-tions were mounted in Rhodorsil-lined organ baths superfused with an oxygen-ated standard solution containing 154 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mMMgCl2, 5 mM HEPES (pH 7.4), and 11 mM D-glucose. For mechanical record-ings, one tendon of the muscle was tied with silk thread via an adjustable stainlesssteel hook to an FT03 isometric transducer (Grass Instruments, West Warwick,R.I.), and the other tendon was pinned to the silicone-lined chamber. Twitcheswere evoked by stimulating the motor nerve via a suction electrode, with supra-maximal current pulses of 0.15-ms duration at 0.1 Hz or 60 Hz, or via anelectrode assembly placed along the length of the muscle. The resting tensionwas adjusted for each preparation to obtain maximal contractile responses uponindirect- and direct-muscle stimulation. Signals from the isometric transducerwere amplified, collected, and digitized with a computer equipped with a DT2821analogue-to-digital interface board (Data Translation, Marlboro, Mass.). Com-puterized data acquisition and analysis were performed with a program kindlyprovided by John Dempster (University of Strathclyde, Glasgow, United King-dom).

Electrophysiological recordings to measure the membrane potential of musclefibers and synaptic potentials were made at room temperature (22 to 24°C) withintracellular microelectrodes filled with 3 M KCl (8 to 12 M� resistance) byusing conventional techniques and an Axoclamp-2A system (Axon Instruments,Union City, Calif.). Electrical signals after amplification were collected anddigitized, at a sampling rate of 25 kHz, with a computer equipped with ananalogue-to-digital interface board.

Morphological and immunofluorescence experiments. A histological exami-nation was performed and the histochemistry of skeletal muscles was determinedwith unfixed tissue frozen in isopentane precooled in liquid nitrogen. Cryosec-tions (7 to 10 �m thick) cut in the middle part of the muscle (in order to obtainthe largest sectional area) were placed on slides, air dried, and stained withhematoxylin and eosin. For immunofluorescence analysis, cryosections were

fixed in cold methanol (10 min at 4°C) and rinsed (10 min) with PBS (0.1 M, pH7.4) supplemented with Triton X-100 (0.03%). After washing and incubation (30min) in blocking solution (PBS containing 3% bovine serum albumin and 3%goat serum), the sections were incubated (2 h at room temperature) with primaryantibodies diluted with blocking solution, washed three times (each washing wasfor 10 min) with PBS supplemented with Tween-20 (0.1%), incubated (1 h atroom temperature) with fluorescent secondary antibodies, rinsed with PBS-Tween-20 (0.1%), and examined by fluorescence microscopy.

The sarcolemmal integrity of the skeletal muscle fibers was determined byharvesting the muscles from toxin-injected and control mice 12 h after theanimals were injected intraperitoneally with a solution (0.1 ml/10 g body weight)of Evans blue dye (10 mg/ml of PBS), followed by examining the tissues accord-ing a method previously described (39). Sarcolemmal integrity also was deter-mined by incubating (15 min) the muscles in standard Krebs-Ringer solutioncontaining 1% Procion Orange, freezing them in isopentane precooled in liquidnitrogen, and examining their cryostat tissue sections by fluorescence microscopy(40). The presence of the fluorescent tracer in the cytoplasm of the cells in thecryostat sections indicated membrane damage.

Immunocytochemical studies of whole-mount preparations utilized the thin,flat LAL muscle (3). The muscle was fixed (25 min at room temperature) with4% paraformaldehyde in 0.1 M PBS (pH 7.4), treated (2 h) with 0.5% TritonX-100 in PBS, rinsed three times for 10 min, and incubated (at 4°C overnight)with the primary antibody in a blocking solution containing 3% bovine serumalbumin. After washing, the muscle was incubated (2 h at room temperature)with secondary antibodies, rinsed in PBS, and mounted on a glass slide withVectashield antifading mounting medium. In all experiments, the specificity ofthe immunostaining was determined with preparations that were not incubatedwith the primary antibody.

Preparations were observed either with a Leica DM-RXA2 upright micro-scope equipped with a CDD camera (CoolSNAP FX; Princeton Scientific In-struments, Monmouth Junction, N.J.) and OpenLab software (Improvision, Con-ventry, United Kingdom) or with a TCS SP2 confocal laser scanning system(Leica Microsystems, Mannheim, Germany) mounted on an upright microscopeand controlled through the manufacturer-supplied software and workstation.Images were collected by using either a 10� or 40� oil-immersion lens (numer-ical aperture, 0.75 and 1.25, respectively). The 488-nm wavelength line of anArgon-ion laser and the 543- and 633-nm wavelength lines of a HeNe laser wereused for FITC, TRITC, and TOTO-3 excitation, respectively. Images were dig-itized into a maximal array of 1024 by 1024 pixels.

Measurement of muscle fibers. ImageJ, a public domain image analysis soft-ware package (http://rsb.info.nih.gov/ij/download/), was used to measure thecross-sectional area of fibers in unfrozen sections stained with hematoxylin andeosin.

Statistical analysis. Data were compared by using Student’s t test, and a Pvalue of �0.05 was considered statistically significant. Unless otherwise stated,the values in the text are expressed as means � standard deviations (SD).

RESULTS

Degeneration and regeneration of skeletal muscle fibers af-ter in vivo exposure to C. sordellii LT. A single sublethal injec-tion of C. sordellii LT (0.25 ng/g of body weight) into theimmediate vicinity of the TA, LAL, or EDL muscles induced,within 24 h, pronounced localized edema and inflammation inthe injected area, which was not observed in control animalsinjected with PBS. A histological examination of muscle sec-tions prepared from the toxin-injected animals revealed degen-eration and necrosis of muscle fibers (Fig. 1A). In addition, thepresence of many mononuclear cells and phagocytes in theinterstitial connective tissue indicated an active phagocyticprocess. One day after LT injection, immunostaining of theskeletal muscle fibers with �-actinin antibody revealed disor-ganization of the muscle Z line (Fig. 2A and B). Also, immu-nostaining of troponin T (a specific marker of thin filaments ofthe striated muscle sarcomere) indicated myofibril disassem-bly; that is, the well-organized parallel bands observed in con-trol fibers (Fig. 2C) were not seen in the LT-treated fibers (Fig.2D). These findings were confirmed by electron microscopy

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FIG. 1. Representative transverse TA muscle sections stained with hematoxylin and eosin (A) and distribution of myofiber cross-sectional area(B) from control mice and at various times after mice received a sublethal intramuscular injection of C. sordellii LT. In panel A, note the presence of onlynecrotic fibers (asterisks) and abundant inflammatory infiltrates (arrows) by 3 days postinjection. Regenerating small myocytes with central nuclei areevident (arrowheads) by 9 to 17 days postinjection, but necrotic fibers and connective tissue are not present, and skeletal muscles possess largeregenerating fibers with central nuclei by 17 to 30 days postinjection. Muscle structure and fiber size are almost completely restored by 30 dayspostinjection, but myofibers still contain central nuclei. Bar 50 �m. In panel B, the mean value and coefficient of variation (CV SD/mean) areindicated for each distribution of myofiber cross-sectional area based on data obtained from three independent experiments (n 250 in each histogram).

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observation of the vacuolization of the necrotic muscle fibersassociated with sarcolemmal disruption (data not shown).

The beginning of regeneration of the LT-damaged skeletalmuscle fibers was evident 6 to 9 days postinjection of the toxin,as demonstrated by the appearance of many small muscle cells(cross-sectional area of �500 �m2) exhibiting central nuclei(Fig. 1). During the development of the regeneration process(12 to 30 days postinjection), the number of newly formed cellsgrew over time and increased in diameter (Fig. 1). Interstitialfibrosis was not observed, but persistent central myonucleiwere still evident 30 and 60 days after LT-induced injury.These results indicate that LT does not impair skeletal muscleregeneration.

In vivo glucosylation of skeletal muscle GTPases by C. sor-dellii LT. In order to monitor the toxin’s possible in vivo abilityto glucosylate small GTPases in skeletal muscle tissue, lysatesof control and LT-injected EDL muscles were incubated invitro with LT in the presence of UDP-[14C]glucose. The con-trol muscle preparations (four different experiments) exhibitedthe radiolabeled glucosylation of proteins with a mass in therange of 20 to 22 kDa, which is in the molecular mass range forlow-molecular-weight GTPases (other proteins were not la-beled).

In contrast, only weak glucosylation of proteins was ob-served in muscles previously injected in vivo with the LT andremoved 1, 3, 6, 9, and 24 h postinjection (Fig. 3). These resultsindicated that the C. sordellii LT glucosylated the muscle’ssmall GTPases in vivo, thus inhibiting incorporation of[14C]glucose into those GTP-binding proteins during the sub-sequent in vitro incubation with the toxin.

Changes in the expression of muscle proteins after in vivoexposure to C. sordellii LT. The kinetics of expression of vari-ous muscle proteins was followed by immunofluorescence mi-croscopy during the above-described regeneration process.The proteins included (i) dystrophin, a cytoskeletal proteinlocated in the inner face of the plasma membrane of musclefibers, (ii) the extracellular matrix protein laminin, and (iii) fastand neonatal myosin in degenerating and regenerating skeletalmuscle fiber sections.

Several observations confirmed that, under our experimental

conditions, the C. sordellii LT did not impair the high regen-erative capacity of murine skeletal muscle fibers. First, dystro-phin immunostaining was absent on day 3 postinjection of LTbut was detected in small-diameter muscle cells on day 9postinjection (Fig. 4). The intensity of the immunostainingthereafter increased and was homogeneously distributedamong the fibers by day 9 to day 12 postinjection. Second,immunolabeling of the extracellular matrix protein �-laminin,which was markedly reduced at day 3 postinjection, was faintlyobserved in small regenerating fibers on day 9 but was in-creased by day 12 postinjection. Third, although immunolabel-ing of nicotinic acetylcholine receptors (AChRs) with FITC-conjugated �-BTX was diffuse in necrotic fibers on day 3postinjection and was not detected on day 9, it was observed onday 12 (Fig. 4, arrows). Fourth, immunostaining of fast myosin

FIG. 2. Disorganization of muscle fiber cytoskeleton observed 1 day after mice received a sublethal intramuscular injection of C. sordellii LT.Single-teased fibers from control TA muscles (panels A and C) and from LT-injected muscles (panels B and D) were immunostained withanti-�-actinin (panels A and B) and anti-troponin T (panels C and D) antibodies. Note the striated and regular parallel lines of the twoimmunostained muscle proteins in the control muscles (panels A and C) and the loss of the striated appearance associated with a disorientedmeshwork of myofilaments in LT-injected muscles (panels B and D). Bar 20 �m.

FIG. 3. LT injection in skeletal muscle induces GTPase glucosyla-tion in vivo. Samples of EDL muscle tissue, harvested at various timesafter in vivo injection of LT and containing 50 �g of protein, wereincubated with LT (50 ng/�l) and UDP-[14C]glucose, subjected tosodium dodecyl sulfate-polyacrylamide gel electrophoresis, and auto-radiographed. The intensities of the bands estimated by densitometryare plotted on the graph. The value of the control was assigned to100%. Notice the decrease in the intensities at 1 to 24 h after LTinjection, indicating an in vivo modification of the LT substrates. Datashown are representative of four independent determinations made atthe times indicated.

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(only the fast myosin heavy chain isoforms are present in adultmouse TA muscles [31]) revealed homogeneous labeling ofregenerating cells (characterized by their unstained centralnuclei) at 9 and 12 days after the LT injection. Fifth, perme-abilization of necrotic muscle fibers was detected by a brightred Evans blue dye staining of fibers during the first few days

after toxin injection. However, muscle fiber permeabilizationwas not evident on day 9 postinjection, and staining was onlyobserved in some intramuscular blood vessels (Fig. 4). Sixth,immunostaining to determine the distribution of neonatal my-osin, which is expressed in developing but not in adult skeletalmuscle tissue (7, 27), revealed regenerating muscle fibers ex-

FIG. 4. Changes in protein expression patterns (revealed by immunostaining of cryosections) during degeneration and regeneration after micereceived a sublethal injection of C. sordellii LT into their TA muscles. The immunolabeling of dystrophin, laminin, fast myosin, and neonatal myosinwas followed at the times indicated post-LT injection. Staining of acetylcholine receptors (AChRs) was performed with FITC-conjugated �-BTX,and staining with Evans blue dye (EBD) was used to assess muscle membrane injury (red). For details, see Materials and Methods. Note thetransient expression of neonatal myosin at day 9 postinjection. Bar 50 �m.

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pressing the neonatal myosin isoform on day 9 postinjection oftoxin but not on day 3. At 12 days postinjection there is noabnormal persistence of neonatal myosin (Fig. 4).

Blockade of indirectly and directly elicited skeletal musclecontractions after in vivo exposure to C. sordellii LT. Singleand repetitive nerve stimulation of isolated EDL nerve-musclepreparations removed from control animals evoked typicaltwitch and tetanic contractions (Fig. 5). In contrast, EDL mus-cles (n 3) isolated from animals exposed to LT exhibitedabout a 60% blockade of nerve-evoked muscle twitch (Fig. 5A)and tetanic responses (Fig. 5B) at 1 day postinjection, and acomplete block was observed between 3 to 5 days after localLT injection (Fig. 5). In addition to the blockade of indirectlyelicited muscle twitches, directly elicited twitches also wereblocked in LT-treated EDL muscles (data not shown). Ourresults indicated that the C. sordellii LT affects muscle contrac-tility as well as neuromuscular transmission. Thus, we subse-quently performed studies to determine whether the effects weobserved were a function of LT-induced changes in musclemembrane permeability.

Intracellular recordings performed with EDL muscles re-

moved from animals 1 and 5 days postinjection of LT revealeda significant (P � 0.01) decrease in the resting membranepotential of muscle fibers (Table 1). This membrane depolar-ization, which was more pronounced at 5 days than at 1 daypostinjection, indicates that LT significantly alters the musclefibers’ membrane permeability. Electrophysiological record-ings performed with EDL neuromuscular junctions 5 days afterLT injection revealed that nerve stimulation was unable totrigger endplate potentials and action potentials in the depo-larized fibers (n 4) and that miniature endplate potentialscould not be recorded in the depolarized fibers (data notshown).

Muscle membrane damage induced by LT was further con-firmed by assessing the uptake of Procion Orange dye by thetoxin-exposed muscle fibers. One day after LT injection, ca.50% of the fibers in the cryosections contained the dye (datanot shown), and almost all of the fibers contained the dye by 5days postinjection. These results are consistent with themarked depolarization of the muscle membrane detected inour electrophysiological recordings.

The observed LT-induced blockade of nerve-evoked muscletwitches and tetanic contractions partially recovered by day 15postinjection of toxin. Interestingly, although tetanic stimula-tion at 60 Hz elicited a maximal tetanic force that was 73% �10.8% (n 3) of that recorded in control muscles (P � 0.01),twitches evoked by nerve stimulation at 0.1 Hz developed apeak force that was only about 33% � 4.7% (n 3) of thatrecorded in the control preparations (P � 0.01).

Neuromuscular junction remodeling after in vivo exposureto C. sordellii LT. FITC-conjugated �-BTX was used to char-acterize the distribution of AChRs in skeletal muscle endplatesafter in vivo exposure to a sublethal dose of LT. We found thatAChRs were distributed in continuous and dense clusters incontrol muscle fibers (Fig. 6A) and that their distribution wasmore diffuse and less dense, as revealed by the intensity pro-files, in LT-exposed muscle fibers (Fig. 6B). The LT-induceddistribution pattern persisted for several days, until the end-plates were barely detectable by �-BTX staining. Staining with�-BTX (revealing the presence of AChRs) was again evidentin regenerating muscle fibers ca. 12 days postinjection of thetoxin (data not shown). At 30 days postinjection, muscle fibersstill exhibited central myonuclei (Fig. 6D), and their distribu-tion of AChRs was either similar to that of the controls (Fig.6C and D, asterisks) or was observed in small, closely spaced

FIG. 5. Nerve-evoked muscle twitches and tetanic responses ofEDL nerve-muscle preparations isolated from controls and from micethat received a sublethal intramuscular injection of C. sordellii LT.(A) Typical muscle twitches evoked by nerve stimulation of controlmuscle and of muscles injected with LT 1, 5, and 15 days previously.(B) Tetanic responses evoked by nerve stimulation (60 Hz) undercontrol conditions and 1, 5, and 15 days postinjection of LT. Note theblockade of muscle twitching and tetanic responses at day 5 after LTinjection and the partial recovery of the contractile responses at day 15postinjection.

TABLE 1. Resting membrane potential of skeletal muscle fibersfrom control and LT-injected EDL muscles at various times

postinjection of LT

Day(s)postinjection

Membrane potential (mV)a

Controlb LT-injected

1 �72.1 � 2.4 �56.5 � 2.1c

5 �70.3 � 1.4 �16.8 � 2.4c

15 �71.5 � 3.4 �62.7 � 2.9c

a Values represent the mean � SD of 18 measurements performed under eachcondition (in triplicate).

b Controlateral EDL muscles were used as controls.c Values is statistically significant (P � 0.01) compared to control.

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membrane patches (Fig. 6C and E), thus suggesting the for-mation of new endplate regions. A strong similarity was ob-served in the distribution pattern of AChRs and of acetylcho-linesterase staining with TRITC-conjugated fasciculin-2, aspecific inhibitor of the enzyme (13, 24). The distribution ofacetylcholinesterase in neuromuscular junctions 30 days afterin vivo exposure to LT is shown in Fig. 6F.

Morphological examination of the motor innervation ofmuscles 30 days postinjection with LT revealed many thinfilaments emerging from the nodes of Ranvier of the intra-

muscular myelinated axons (Fig. 6C and E, arrowheads).The collateral sprouting (revealed by neurofilament immu-nostaining) abutted clusters of AChRs labeled with TRITC-conjugated �-BTX (Fig. 6C and E), at a time when acetyl-cholinesterase was present in the neuromuscular junctions(Fig. 6F). Our results indicate that the recovery of neuro-muscular transmission after in vivo exposure to a sublethaldose of C. sordellii LT injected intramuscularly correlateswith the significant remodeling of the neuromuscular junc-tion’s constitutive elements.

FIG. 6. Remodeling of the neuromuscular junction in whole-mount LAL muscles obtained from mice at various times after they received asublethal injection of C. sordellii LT. (A) Control neuromuscular junction in which AChRs are stained with TRITC-conjugated �-BTX (red) andsubsynaptic nuclei are stained with DAPI (blue). (B) Staining of AChRs and subsynaptic nuclei (as in panel A) in a junction from a muscle 1 dayafter LT injection. Note the different intensity profiles for the AChR staining shown in panels A and B, with fluorescence intensity presented asan arbitrary unit of values between 0 and 255 color levels. (C and E) Nodal nerve sprouting (arrowheads) revealed by immunostaining with anantineurofilament antibody (green), and AChRs labeled with TRITC-conjugated �-BTX (red) 30 days post-LT injection. Note the presence ofneoformed (arrows) and mature-like AChR clusters (asterisk). (D) Staining of AChRs with FITC-conjugated �-BTX (green), and TOTO-3 dye(blue) staining of nuclei in a regenerated muscle 30 days post-LT injection. Note the presence of large, aligned central nuclei in various fibers.(F) Staining of acetylcholinesterase with TRITC-conjugated fasciculin-2 and axons labeled with an antineurofilament antibody 30 days post-LTinjection. Note the similarity between the staining of AChRs in panel C and of acetylcholinesterase in panel F. Bars 4 �m (A and B), 20 �m(C and D), and 15 �m (E and F).

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DISCUSSION

The present study demonstrates that the intramuscular in-jection of a single sublethal dose of C. sordellii LT causessignificant damage to skeletal muscle fibers. The mechanism bywhich LT elicits necrosis of skeletal muscle fibers has not beencompletely elucidated, but it appears to be related to its abilityto glucosylate small GTP-binding proteins. Data obtained inthe present study indicate that the LT-induced degeneration ofmuscle fibers in vivo correlates with the toxin’s effect on smallGTP-binding proteins in skeletal muscle. More specifically, weobserved (Fig. 3) that LT glucosylates these skeletal muscleproteins in vivo, as previously reported for other eukaryoticcells (9, 22, 33). Therefore, our results support the idea that,within 24 h after the toxin’s internalization into skeletal musclefibers, it exerts an action on small GTPases in the fibers. Also,to the best of our knowledge, this report is the first time that alarge clostridial cytotoxin or LT has been shown to producesimilar in vivo and in vitro effects in skeletal muscle fibers.However, further studies are needed to identify precisely thesmall GTP-binding proteins and GTPases targeted by LT inskeletal muscle. Also, it is likely that the toxin has an effect onendothelial cells located at the interface between blood andskeletal muscle, leading to the expression of proinflammatorymediators, vascular leakage, and subsequent ischemia of skel-etal muscle tissue. Indeed, C. sordellii LT has been shown (42)to disrupt the actin microfilament system and the monolayerintegrity of primary cultured human endothelial cells. LT-in-duced endothelial dysfunction and vascular injury could (i)cause electrolytes and water to be released into the interstitialspace, thus contributing to the marked localized edema weobserved, and (ii) impair oxygen delivery to and alter the pH ofmuscle tissue. Thus, a combination of events triggered by LTcould lead to rapid anoxia and the necrosis of skeletal muscle.

A few days after the injection of LT, some muscle fiberswere necrotic, while others exhibited various degrees of myo-fibrillar degeneration. Immunostaining of troponin T, �-acti-nin, and dystrophin in the control and toxin-treated musclesdetected myofibril disorganization 1 day after exposure to LT,and it revealed that dystrophin was absent in necrotic fibers byday 3 postinjection of LT. The severity of the LT-inducedmuscle membrane injury was indicated by the high percentageof fibers with a fluorescent cytoplasm as revealed by inwardleakage of Procion Orange dye or Evans blue dye (17) and bythe marked muscle fiber depolarization (Table 1) detected byour electrophysiological recordings. Five days postinjection ofLT, the large number of fibers exhibiting membrane injuryresulted in the loss of synaptic responses and a complete block-ade of neuromuscular transmission and muscle force evokedby nerve stimulation.

We observed a relatively rapid (by ca. 30 days) regenerationof neuromuscular components after the initial LT-induced ne-crosis of muscle fibers. This observation is not surprising, sinceskeletal muscle is known to have a remarkable capacity toregenerate after being subjected to a variety of injuries result-ing in either partial damage to or necrosis of its muscle fibers.The events that must occur in order to achieve the completeregeneration of skeletal muscle tissue include the phagocytosisof necrotic muscle debris, revascularization, activation, prolif-eration and differentiation of muscle precursor cells, and rein-

nervation (8, 25). The onset of muscle regeneration is triggeredby events that occur during muscle degeneration, and it isdependent on the proliferation and differentiation of myogenicprogenitors called satellite cells and on muscle-derived stemcells that have been suggested to be pluripotent (18, 35, 36).Satellite mononucleated cells are usually quiescent. However,after skeletal muscle damage they must be activated in order toproliferate and differentiate into myotubes and myofibers.Thus, despite the muscle and vascular alterations that may becaused by LT (42), we found that muscle satellite cells wereable to initiate an effective and successful myogenic programallowing muscle repair. The regeneration is rapidly completedin LT-treated muscles, in contrast to the poor regenerativeresponses of muscles exposed to the blood vessel-damagingmetalloproteases present in some snake venoms (reviewed inreference 16). Also, although the recovery of injured skeletalmuscle often is hindered by the development of interstitialfibrosis (26), we found that the regeneration of LT-treatedmuscles occurs without fibrosis.

The regeneration of adult mouse skeletal muscles is a well-described phenomenon (2), and it has been studied after mus-cle fiber necrosis produced by the most potent Elapidae snaketoxins known, e.g., notexin from Notechis scutatus venom (10).However, since the molecular steps required to recruit thesatellite cells needed to regenerate damaged muscle tissue arestill poorly characterized, the C. sordellii LT may be a usefulreagent in studies to elucidate the factors responsible for themigration, proliferation, and differentiation of the precursorcells.

We deduced the remodeling of the neuromuscular junctionat 30 days postinjection of C. sordellii LT from observations ofwhole-mount LAL muscles (Fig. 6). Presynaptic and postsyn-aptic elements underwent a remodeling process characterizedby a profuse nodal sprouting that restored synaptic connec-tions and by a normalization of the distribution of nicotinicAChRs and acetylcholinesterase. The results of our functionalstudies (Fig. 5) also demonstrated that motor nerve terminalsrapidly regenerated after exposure to C. sordellii LT. Thisremodeling is quite different from that observed after exposureto various serotypes of C. botulinum neurotoxins (BoNTs),which are highly specific Zn2�-dependent endoproteases thatcleave single peptide bonds (except for BoNT/C1) in one of thethree proteins (the 25-kDa synaptosomal-associated protein,syntaxin 1, and synaptobrevin) constituting the essential com-ponents for synaptic vesicle docking and fusion during regu-lated exocytosis (reviewed in references 30 and 34). Thus,these neurotoxins block acetylcholine release and produce pro-found but transient skeletal muscle paralysis (29) and atrophyin vivo, while leaving the viability of nerve endings unaltered(reviewed in reference 30). In contrast, the C. sordellii LT,which also blocks the regulated exocytosis of neurotransmitters(11, 15, 19), causes microscopically observable skeletal myone-crosis and degenerative changes in the neuromuscular junc-tion.

In conclusion, to the best of our knowledge, this is the firstpublished study to describe the degeneration and regenerationof skeletal muscle tissue and the remodeling of the neuromus-cular junction after in vivo exposure to a large clostridial cy-totoxin, i.e., the C. sordellii LT. Our data may provide anexplanation for the severe myonecrosis and neuromuscular

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alterations accompanying wound infections caused by C. sor-dellii.

ACKNOWLEDGMENTS

We thank Sabine De la Porte (C.N.R.S. Gif-sur-Yvette) for sharingequipment during our studies and Arnold Kreger (University of Mary-land School of Medicine) for helpful suggestions during the revisionsof the manuscript.

Our studies were supported by the Direction des Systemes de Forceset de la Prospective (grant 026065093 to J.M.), the Programme deRecherche Fondamentale en Microbiologie from the Ministere Dele-gue a la Recherche et aux Nouvelles Technologies (to M.R.P. andJ.M.), and the Association Francaise contre les Myopathies (to J.M.).J.B. was supported by a fellowship from the DGA-CNRS.

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