Macrostructure of tibialis anterior muscle after CAMP treatment.

A, representative images of muscles treated with either CTX or CAMP for 5 or 10 days. Quantification of TA weight at 5 (B) and 10 (C) days post administration. Scale bar represents 5 mm. Data represent mean ± S.D. (n = 5 for each cohort). The p values shown are as calculated by One-way ANOVA followed by post hoc Tukey's test using GraphPad Prism (*p<0.05, **p<0.01 and ***p<0.001).</p

Analysis of tibialis anterior muscle regeneration after administration of CAMP or CTX.

A, H and E staining of muscle identifying centrally located fibre nuclei (CLN) (arrows) and (B) quantification of centrally located muscle fibre size 5 days post administration. C, H and E staining of muscle (arrows) and (D) quantification of centrally located muscle fibre size 10 days post administration. E, intra-fibre IgG localisation for necrotic muscle fibres (arrows) and quantification of necrotic fibre density (F) and size (G) 5 days post administration. H, intra-fibre IgG localisation for necrotic fibres (arrows) and (I) quantification of necrotic fibre density 10 days post administration. J, identification of regenerating muscle fibres through the expression of MYH3 (arrows) and quantification of regenerating muscle fibre density (K) and size (L) 5 days post administration. M, identification of regenerating muscle fibres through the expression of MYH3 (arrows) and quantification of regenerating muscle fibre density (N) and size (O) 10 days post administration. P, Localisation of endothelial marker CD31 and (Q) quantification of capillaries per regenerating muscle fibre 5 days post administration. R, localisation of endothelial marker CD31 and (S) quantification of capillaries per regenerating muscle fibre 10 days post administration. T, immunostaining with antibody F4/80 and (U) its density quantification in damaged region 5 days post administration. V, immunostaining with antibody F4/80 and (W) its density quantification in damaged region 10 days post administration. Data represent mean ± S.D. (n = 5 for each cohort). The p values shown are as calculated by two-tailed Student’s T test for independent variables using GraphPad Prism (*p<0.05, **p<0.01 and ***p<0.001).</p

Purification of CAMP from the venom of C. atrox.

A, A chromatogram demonstrates the purification profile of 10mg of whole C. atrox venom by anion exchange chromatography. B, a Coomassie stained gel displays the protein profile of whole C. atrox venom and fractions 11–18 of anion exchange chromatography. A chromatogram (C) and Coomassie stained gel (D) show the purification profile of fractions 14–18 of anion exchange chromatography by gel filtration. E, a chromatogram of the second step of gel filtration for fractions 62–67 from the previous step and (F) a Coomassie stained gel shows the purified protein at approximately 50kDa. G, the tryptic digested samples of the purified protein were analysed by mass spectrometry and the identified peptide sequences match (via Mascot search) with the known sequence of VAP2A (coverage 43%; the mass spectrometry-identified peptide sequences of the purified protein are shown in red) and confirms that the purified protein is most likely to be VAP, a group III metalloprotease. The purified protein was named as CAMP to represent C. atrox metalloprotease.</p

The functional characterisation of CAMP.

A, the serine protease activity of 10μg/mL whole venom or CAMP was analysed using a fluorogenic substrate, Nα-Benzoyl-L-Arginine-7-Amido-4-methylcoumarin hydrochloride (BAAMC) by spectrofluorimetry. Similarly, (B) the metalloprotease activity of 10μg/mL whole venom or CAMP was analysed using DQ-gelatin, a specific fluorogenic substrate for collagenolytic enzymes and the level of fluorescence was measured by spectrofluorimetry. C, a Coomassie stained gel demonstrates the fibrinogenolytic activity of CAMP in comparison with whole C. atrox venom. Lanes, 1—undigested fibrinogen, 2—fibrinogen incubated with whole venom (100μg/mL), fibrinogen incubated with CAMP (100μg/mL) after 30 (3), 60 (4) and 90 (5) minutes, fibrinogen incubated with CAMP (50μg/mL) after 30 (6), 60 (7) and 90 (8) minutes, and CAMP alone (9). Representative aggregation traces (D) and data (E) demonstrate the impact of CAMP on cross-linked collagen related peptide (CRP-XL)-induced human platelet (PRP) aggregation. Data represent mean ± S.D. (n = 3). The p values shown are as calculated by One-way ANOVA followed by post hoc Tukey's test using GraphPad Prism (**ppp<0.0001).</p

CAMP treatment affects proliferation and migration of satellite cells.

(A-C) Increasing concentrations of CAMP change the distribution of collagen IV. Satellite cell proliferative characteristics following CAMP treatment were analysed. For example, satellite cell number per fibre (D), satellite cell clusters per fibre (E) and cluster size per fibre (F) were quantified. G, myogenic characteristics of satellite cells following CAMP treatment. H, migration rate of satellite cells. Scale bar represents 20μm. Data represent mean ± S.D. (n = 20 fibres for each cohort). The p values shown are as calculated by One-way ANOVA followed by post hoc Tukey's test using GraphPad Prism (*p<0.05, **p<0.01 and ***p<0.001). CNT represents control.</p

Immunohistochemical analysis of muscle extracellular matrix and associated proteins after tibialis anterior damage with CAMP or CTX.

Localisation (arrows) (A) and thickness (B) of collagen IV 5 days post administration. Similarly, localisation (arrows) (C) and thickness (D) of collagen IV 10 days post injury. Note: CTX damaged tissues show circumferential collagen compared to foci in CAMP treatment. Localisation (arrows) (E) and thickness (F) of laminin 5 days post injury. Localisation (arrows) (G) and thickness (H) of laminin 10 days post injury. I, localisation of dystrophin 5 days post injury (arrows). Note: dystrophin around centrally located nuclei in CTX-treated muscle. In contrast, incomplete dystrophin domain around CAMP-damaged muscle. J, intensity of dystrophin 5 days post injury. Localisation (arrows) (K) and intensity (L) of dystrophin 10 days post injury. M, localisation of nNOS 5 days post injury (no circumferential nNOS was detectable at day 5) and (N) thickness of nNOS 5 days post injury. O, localisation of nNOS 10 days post administration (arrows). Note: circumferential nNOS was only detectable in CTX-treated muscle and (P) intensity of nNOS 10 days post injury. Localisation of CAMP in damaged region at day 5 (Q) and 10 (R) (arrows). Data represent mean ± S.D. (n = 5 for each cohort). The p values shown are as calculated by One-way ANOVA followed by post hoc Tukey's test using GraphPad Prism (*p<0.05, **p<0.01 and ***p<0.001). CNT represents control.</p