Additional file 1: Table S1. of Investigating mechanisms underpinning the detrimental impact of a high-fat diet in the developing and adult hypermuscular myostatin null mouse

Blood parameters. Values from minimum of six male mice. (DOCX 13 kb

Additional file 3: Figure S2. of Investigating mechanisms underpinning the detrimental impact of a high-fat diet in the developing and adult hypermuscular myostatin null mouse

Effect of high fat diet on Gastrocnemius lipid content. All values are fraction of total lipid content. ANOVA; (*) P<0.05 vs. WT ND; WT HF; (**) P<0.05 vs. Mstn-/- ND. N=6 male mice per group

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

Molecular and protein analysis of the Tibialis Anterior (TA) for catabolic and anabolic markers in response to acute starvation.

<p>(A) qPCR analysis of changes in the expression of master regulator of catabolism, <i>Foxo1</i>. (B-C) E3 class of ubiquitin ligases, <i>MuRF1</i> and <i>atrogin</i>. Regulators of autophagy (D) <i>Bnip3</i>, (E) <i>Beclin</i>, ((F) <i>LC3b</i>, (G) <i>ATF4</i>, (H) <i>CathepsinL</i> (I) <i>Vsp34</i> (J) <i>p62</i>, (K) <i>Gadd34</i>. (L) mRNA expression of <i>Mul1</i> a mitophagy regulator. All qPCR levels normalised to levels of G6PDH. (M) Quantification of LC3-II in relation to LC3 at protein level. (N) Quantification of pAkt at residue S473 in relation total Akt at protein level. (O) Western blot analysis of regulators of autophagy (LC3 and p62), and protein synthesis (Akt-total and phosphorylated versions at S473 and T308, 4ebp1 and S6) with tubulin serving as loading control. (Two-way ANOVA; *p<0.05).</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

Expression of SGK1 in wild-type and <i>Mstn</i>^{<i>−/−</i>} Tibialis Anterior (TA) before and after acute 24 starvation.

<p>(A) Western blot and scanning densitometry analysis of 54kD band. (B) (D) Altered profile of SGK1 expression in IIB fibres of <i>Mstn</i>^{<i>−/−</i>} TA muscle. (C) Immunochemistry for the expression of MCH IIB and SGK1 (Scale bar = 100μm). (E) Western blot analysis of phosphorylated FoxO3a at residues T32 and S253 from the TA muscles and the influence of starvation. Phosphorylation at T32 indicated by arrow marking the upper band. (F) Quantification of pFoxO3a-T32/ FoxO3a (Two-way ANOVA; *p<0.05).</p

Intra-muscular bodies and muscle function.

<p>(A) Immunohistochemical analysis of p62 expression in the EDL of wild-type and <i>Mstn</i>^{<i>−/−</i>} mice. (AI) Wild-type mice on normal diet show very few punctate p62 positive bodies in any fibre whereas many appear after starvation (AII). (AIII) Normal diet <i>Mstn</i>^{<i>−/−</i>} EDL fibres show large number of punctate p62 bodies predominantly in the large fibres which almost all disappear after starvation (IV) (Scale bar 75μm). Inset images shows high magnification images of p62 staining (scale bar 10μm). (B) Quantification of (B) twitch tension and (C) specific tension of the EDL from wild-type and <i>Mstn</i>^{<i>−/−</i>} before and after starvation. (Two-way ANOVA; *p<0.05).</p

Changes in body mass in wild-type (WT) and <i>Mstn</i>^{<i>−/−</i>} (Mstn) at 12 and 24 hours after complete food withdrawal (acute starvation).

<p>(A) Wild-type mice showed a significant decrease after 24h starvation. In contrast <i>Mstn</i>^{<i>−/−</i>} lost mass in the first and second 12h of starvation. (B) Changes in body weight expressed as a % of normal body weight. Weight changes of (C) EDL, (D) soleus, (E) TA and (F) Gastrocnemius at 12 and 24h after acute starvation. Wild-type animals showed no significant muscle mass reduction in contrast to all muscles examined from <i>Mstn</i>^{<i>−/−</i>}. (Two-way ANOVA; *p<0.05)</p