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First published online May 24, 2005
Journal of Experimental Biology 208, 2083-2090 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01605

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The role of myostatin and the calcineurin-signalling pathway in regulating muscle mass in response to exercise training in the rainbow trout Oncorhynchus mykiss Walbaum

C. I. Martin and I. A. Johnston^*

Gatty Marine Laboratory, School of Biology, University of St Andrews, St Andrews, Scotland, KY16 8LB, UK

View larger version (23K): [in a new window]   Fig. 1. Antibody screening. (A-C) The Santa Cruz human-specific CnA (sc-9070), Calbiochem mouse-specific CnB (PC-359) and Santa Cruz human-specific NFAT2 antibodies (sc-1149-R, K18-R) were screened against nuclear protein extracts. The CnA, CnB and NFAT2 antibodies each positively detected a single protein of the expected molecular mass (55 kDa, 19 kDa and 103 kDa, respectively) in the mammalian positive controls (M, mouse and R, rat) and rainbow trout (T) nuclear protein extracts. (D) Three forms of the myostatin protein were detected in total fast muscle tissue cellular extracts of the rainbow trout (T) and Atlantic salmon positive control (S): the precursor protein (PC, 53 kDa), latency-associated peptide (LAP, 40 kDa) and the mature peptide (M, 17 kDa). (A,B,D) Ma, Santa Cruz markers (sc-2035); (C) Ma, Bio-Rad high molecular mass markers (161-0311).

View larger version (15K): [in a new window]   Fig. 2. The effect of tank rest (TR) or exercise training (E15 and E30) on the distribution of fast muscle fibre diameters. The mean smooth probability density functions (pdf) of fast fibre diameter distributions for each experimental group are represented by the solid and broken lines. In each statistical comparison, the shaded area represents 100 bootstrap estimates of the fast muscle fibre diameter distribution for the two experimental groups combined. The dotted line shows the average muscle fibre distribution of the combined groups. (A) Several regions of the average probability density functions of each experimental group lay outside the grey shaded area of 100 bootstrap estimates of the total population. This suggested that there was a significant difference between the fast muscle fibre diameter distributions of the TR (solid line) and E15 (broken line) experimental groups (P<0.05, Kolmogorov-Smirnov). (B) Several regions of the average probability density functions of each experimental group lay outside the grey shaded area of 100 bootstrap estimates of the total population. This suggested that there was a significant difference between the fast muscle fibre diameter distributions of the TR (solid line) and E30 (broken line) experimental groups (P<0.05, Kolmogorov-Smirnov). (C) The average probability density functions of each experimental group lay within the grey shaded areas of 100 bootstrap estimates of the total population. This suggested that there was no significant difference in the distribution of fast muscle fibre diameters between the exercised groups (E15, solid line; E30, broken line). The arrows represent the apparent left-to-right shift of the distribution of fast muscle fibre diameters in E15 and E30 groups relative to the TR group. TR and E15, N=9; E30, N=6.

View larger version (48K): [in a new window]   Fig. 3. The effect of tank rest or exercise training (E15, E30) on fast muscle tissue nuclear localisation and total cellular concentration of the calcineurin catalytic (CnA) and regulatory (CnB) subunits and associated transcription factor NFAT2. Nuclear and total cellular protein extracts were Coomassie stained to demonstrate equal sample loading. TR and E15, N=9; E30, N=6.

View larger version (16K): [in a new window]   Fig. 4. (A) CnA nuclear localisation was significantly higher in the E15 and E30 groups relative to the TR group (P<0.001, one-way ANOVA; **P<0.01, ***P<0.001, Tukey's test). Overall CnA concentration in fast muscle tissue cellular extracts did not vary between the three experimental groups. (B) CnB nuclear localisation was significantly higher in the E15 and E30 groups relative to the TR group (P<0.01, Kruskal Wallis; *P<0.05, aP<0.1, post-hoc multiple comparisons). Overall CnB concentration in fast muscle tissue cellular extracts was invariant between the three experimental groups. (C) NFAT2 nuclear localisation was significantly reduced in the E15 and E30 groups relative to the TR group (P<0.001, one-way ANOVA; **P<0.01, ***P<0.001, Tukey's test). Overall NFAT2 concentration was significantly lower in the E15 and E30 groups relative to the TR group (P<0.001, one-way ANOVA; ***P<0.001, Tukey's test). Data are mean group optical density (± S.E.M.) expressed as a percentage of the TR group. TR and E15, N=9; E30, N=6.

View larger version (28K): [in a new window]   Fig. 5. (A) The effect of tank rest or exercise training (E15 and E30) on overall myostatin concentration in fast muscle tissue extracts. (B) The myostatin PC was downregulated in the E15 and E30 groups relative to the TR group, but this was not significant. LAP concentration was also invariant between the three experimental groups. The myostatin mature peptide concentration was significantly lower in the E15 and E30 groups relative to the TR group (P<0.05, one-way ANOVA; *P<0.05, aP<0.1, Tukey's test). Fast muscle tissue total cellular protein extracts were Coomassie stained to demonstrate equal sample loading. Data are mean group optical density (± S.E.M.) expressed as a percentage of the TR group. TR and E15, N=9; E30, N=6.