Skeletal muscle necrosis and regeneration after injection of Thalassophryne nattereri (niquim) fish venom in mice

Abstract

Stings by Thalassophryne nattereri are responsible for envenomation of fishermen in north-eastern Brazil. Its venom induces prominent local tissue damage, characterized by pain, oedema and necrosis. The pathogenesis of acute muscle damage induced by T. nattereri venom was studied in mice. Intramuscular injection induced myonecrosis within the first hours. Some muscle cells presented a hypercontracted morphology, but most necrotic fibres were not hypercontracted, being instead characterized by a disorganization of myofibrils, with Z line loss, mitochondrial swelling and sarcolemmal disruption. In addition, thrombosis was observed histologically in venules and veins, together with vascular congestion and stasis, evidenced by intravital microscopy. Venom induced a rapid increment in serum creatine kinase (CK) levels, concomitant with a reduction in gastrocnemius muscle CK activity, whereas no increments in muscle lactic acid were detected. A rapid cytolytic effect was induced by the venom on C2C12 murine myoblasts in culture. The inflammatory reaction in affected muscle was characterized by oedema and scarce cellular infiltrate of polymorphonuclear leucocytes and macrophages, with a consequent delay in the removal of necrotic material. Skeletal muscle regeneration was partially impaired, as evidenced by the presence of regenerating fibres of variable size and by the increase of fibrotic tissue in endomysium and perimysium. It is suggested that T. nattereri venom affects muscle fibres by a direct cytotoxic effect, and that the vascular alterations described preclude a successful regenerative process.

Figure 1

Light micrographs of sections of gastrocnemius muscle taken 3 h after intramuscular injection of T. nattereri venom. (a) Necrotic muscle fibres (n) showing hypercontraction and clumping of myofilaments. Bar represents 25 µm (b) Group of necrotic fibres in which there is disorganization of myofibrillar structure with very few areas of hypercontraction. Bar represents 25 µm.

Figure 2

Electron micrographs of sections of gastrocnemius muscle taken 6 h after intramuscular injection of T. nattereri venom. (a) Portion of a necrotic muscle fibre showing Z line loss (arrows) and swollen mitochondria. Plasma membrane has been lost and only the basal lamina (b) remains at the periphery of the fibre. Bar represents 2 µm (b) Portion of a necrotic muscle fibre showing disorganization of myofibrils associated with Z line loss (arrow). Notice the lack of hypercontraction of myofilaments. Bar represents 2 µm.

Figure 3

Light micrograph of a section of gastrocnemius muscle 3 h after injection of T. nattereri venom. There is widespread myonecrosis, with fibres showing a morphology characterized by disorganization of myofibrillar material with very few hypercontracted regions. A thrombosed venule is observed (v). Bar represents 50 µm.

Figure 4

Light micrographs of sections of gastrocnemius muscle taken at different times after intramuscular injection of T. nattereri venom. (a) 14 days; abundant regenerating fibres of varying size, some of them showing centrally located nuclei (arrow heads). Notice a calcified necrotic cell with a small regenerating fibre in its vicinity. Bar represents 50 µm (b) 28 days; portion of tissue in which regenerating fibres of varying size are observed, some of them showing fibre splitting (arrows). Prominent fibrosis (f) is observed in an area of poor muscle regeneration. Bar represents 50 µm.

Figure 5

Changes in plasma CK activity of mice injected intramuscularly in the gastrocnemius with 100 µg T. nattereri venom dissolved in 100 µL PBS. Control animals received PBS alone. Mice were bled at different time intervals and plasma CK quantified. In the case of mice injected with PBS, CK activity was determined 3 h after injection. Results are presented as mean ± SD (n = 4).

Figure 6

(a) Changes in wet weight of mouse gastrocnemius muscle after intramuscular injection of 100 µg T. nattereri venom dissolved in 100 µL PBS. At various time intervals mice were sacrificed and both injected and contralateral muscles were excised and weighed. Results are expressed as a percentage, taking as 100% the weight of contralateral, noninjected muscle. (b) CK content of mouse gastrocnemius muscle at different time intervals after intramuscular injection of 100 µg T. nattereri venom dissolved in 100 µL PBS. Both injected and contralateral gastrocnemius muscles were homogenized and, after centrifugation, CK activity of the supernatant was quantified. CK activity in envenomated muscle is expressed as a percentage, taking as 100% the CK activity of contralateral, noninjected gastrocnemius. Results are presented as mean ± SD (n = 5).

Figure 7

Light micrograph of mouse cremaster muscle examined in intravital microscopy 7 min after topical application of 20 µg of T. nattereri venom, dissolved in 20 µL of PBS. A prominent thrombus is observed in a venule (arrow), whereas blood flow was halted in a nearby smaller venule (arrow head). Notice the presence of myofibrillar hypercontraction in an affected muscle cell (M). Bar represents 100 µm.

Figure 8

Cytotoxic activity of T. nattereri venom on C2C12 myoblasts in culture. Various concentrations of venom were added to myoblasts and incubated for 3 h at 37 °C. Cytotoxicity was estimated by the release of lactic dehydrogenase to the supernatant. Cytotoxic effect was expressed as a percentage release, taking as 100% the lactic dehydrogenase activity of supernatants of wells in which cells were incubated with assay medium containing 0.1% Triton X-100. Results are presented as mean ± SD (n = 3).