Rainbow trout Oncorhynchus mykiss Walbaum were exercised at 0.8 and 1.6·body lengths·s–1 for 18·h a day over a 30·day period. Exercise resulted in a 24–30% increase in the average cross-sectional area of fast muscle

Calcineurin is a heterodimeric Ca2+/calmodulin dependent protein phosphatase that has been implicated in the regulation of skeletal muscle hypertrophy in mammals (Dunn et al., 1999; Musaro et al., 1999; Semsarian et al., 1999). Calcineurin is widely expressed in eukaryotes and highly conserved (reviewed by Guerini, 1997; Sugiura et al., 2001). Prolonged increases in intracellular Ca2+ levels selectively activate calcineurin, which in skeletal muscle leads to dephosphorylation of nuclear factor of T-cells 2 (NFAT2) and nuclear localisation of the calcineurin/NFAT2 complex (Shaw et al., 1995; Dolmetsch et al., 1997). The nuclear association of calcineurin and NFAT2 synergistically initiates a programme of gene expression leading to hypertrophy (Musaro et al., 1999; Semsarian et al., 1999). mRNA expression of calcineurin signaling pathway associated genes is increased in response to moderate endurance exercise training (Hitomi et al., 2003; Norrbom et al., 2004). Myostatin or growth/differentiation factor·8 (GDF8), a member of the transforming growth factor-β (TGF-β) superfamily, is a negative regulator of skeletal muscle mass in higher vertebrates (McPherron et al., 1997). In myostatin null mice and three breeds of cattle lacking a functional myostatin protein, a hypermuscular phenotype was observed, the increase in muscle mass occurring through increased fibre number and muscle fibre hypertrophy (Kambadur et al., 1997; McPherron and Lee, 1997; Bass et al., 1999; Thomas et al., 2000; reviewed by Kocamis and Killefer, 2002). In studies on humans, myostatin mRNA expression was downregulated by 37% (Roth et al., 2003) and protein concentration was reduced by 20% (Walker et al., 2004), in response to periods of resistance training. Forced exercise is a powerful stimulus for skeletal muscle hypertrophy in teleosts (Walker and Pull, 1973; Johnston and Moon, 1980a,b; Totland et al., 1987). Comparative studies of the structure of myostatin in teleosts and mammals indicate a high degree of sequence identity (McPherron and Lee, 1997; Roberts and Goetz, 2003). Inhibition of myostatin in transgenic zebrafish Danio rerio Hamilton resulted in a 20% increase in fast muscle fibre number (Xu et al., 2003). Zebrafish D. rerio embryos treated with a myostatin specific morpholino exhibited a marked upregulation of myogenic regulatory transcription factors MyoD and myogenin (Amali et al., 2004). These findings imply a pivotal role for myostatin during embryonic myogenesis in teleosts. In addition, myostatin mRNA expression is affected by fasting in larval and adult stages of tilapia Oreochromis mossambicus Peters (Rodgers et al., 2003). However, the role of myostatin in regulating muscle mass during exercise in adult fish has not been investigated. The main aim of the present study was to use exercise training to investigate the molecular signalling pathways regulating muscle fibre hypertrophy. Specifically, we wanted to test the hypothesis that myostatin and proteins associated The Journal of Experimental Biology 208, 2083-2090 Published by The Company of Biologists 2005 doi:10.1242/jeb.01605