Degeneration and regeneration of murine skeletal neuromuscular junctions after intramuscular injection with a sublethal dose of Clostridium sordellii lethal toxin

Abstract

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, we report the results of our ex vivo analysis of the structure and function of skeletal neuromuscular tissue obtained from mice at various times after intramuscular injection of a sublethal dose of LT (0.25 ng/g of body wt). The toxin caused, within 24 h, pronounced localized edema, inflammation, myofibril disassembly, and degeneration of skeletal muscle fibers in the injected area, and it glucosylated the muscle tissue's small GTPases. 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 by immunofluorescence microscopy, confirmed that LT does not impair the high regenerative capacity of murine skeletal muscle fibers. Functional studies revealed that LT affects muscle contractility and neuromuscular transmission. However, partial recovery of nerve-evoked muscle twitches and tetanic contractions was observed by day 15 postinjection, and extensive remodeling of the neuromuscular junction's nerve terminals and clusters of muscle acetylcholine receptors was still evident 30 days postinjection. In conclusion, to the best of our knowledge, this is the first report to characterize the degeneration and regeneration of skeletal neuromuscular tissue after in vivo exposure to a large clostridial cytotoxin. In addition, our data may provide an explanation for the severe neuromuscular alterations accompanying wound infections caused by C. sordellii.

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 only necrotic fibers (asterisks) and abundant inflammatory infiltrates (arrows) by 3 days postinjection. Regenerating small myocytes with central nuclei are evident (arrowheads) by 9 to 17 days postinjection, but necrotic fibers and connective tissue are not present, and skeletal muscles possess large regenerating fibers with central nuclei by 17 to 30 days postinjection. Muscle structure and fiber size are almost completely restored by 30 days postinjection, but myofibers still contain central nuclei. Bar = 50 μm. In panel B, the mean value and coefficient of variation (CV = SD/mean) are indicated for each distribution of myofiber cross-sectional area based on data obtained from three independent experiments (n > 250 in each histogram).

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 with anti-α-actinin (panels A and B) and anti-troponin T (panels C and D) antibodies. Note the striated and regular parallel lines of the two immunostained muscle proteins in the control muscles (panels A and C) and the loss of the striated appearance associated with a disoriented meshwork of myofilaments in LT-injected muscles (panels B and D). Bar = 20 μm.

FIG. 3.

LT injection in skeletal muscle induces GTPase glucosylation in vivo. Samples of EDL muscle tissue, harvested at various times after in vivo injection of LT and containing 50 μg of protein, were incubated with LT (50 ng/μl) and UDP-[¹⁴C]glucose, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiographed. The intensities of the bands estimated by densitometry are plotted on the graph. The value of the control was assigned to 100%. Notice the decrease in the intensities at 1 to 24 h after LT injection, indicating an in vivo modification of the LT substrates. Data shown are representative of four independent determinations made at the times indicated.

FIG. 4.

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

FIG. 5.

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

FIG. 6.

Remodeling of the neuromuscular junction in whole-mount LAL muscles obtained from mice at various times after they received a sublethal injection of C. sordellii LT. (A) Control neuromuscular junction in which AChRs are stained with TRITC-conjugated α-BTX (red) and subsynaptic nuclei are stained with DAPI (blue). (B) Staining of AChRs and subsynaptic nuclei (as in panel A) in a junction from a muscle 1 day after LT injection. Note the different intensity profiles for the AChR staining shown in panels A and B, with fluorescence intensity presented as an arbitrary unit of values between 0 and 255 color levels. (C and E) Nodal nerve sprouting (arrowheads) revealed by immunostaining with an antineurofilament antibody (green), and AChRs labeled with TRITC-conjugated α-BTX (red) 30 days post-LT injection. Note the presence of neoformed (arrows) and mature-like AChR clusters (asterisk). (D) Staining of AChRs with FITC-conjugated α-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-LT injection. Note the similarity between the staining of AChRs in panel C and of acetylcholinesterase in panel F. Bars = 4 μm (A and B), 20 μm (C and D), and 15 μm (E and F).