Please cite this article in press as: Fuentes, E.N., et al., Transient inactivation of myostatin induces muscle hypertrophy and overcompensatory growth in zebraﬁsh via inactivation of the SMAD signaling pathway. J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.10.028 ARTICLE IN PRESS G Model BIOTEC 6501 1–8 Journal of Biotechnology xxx (2013) xxx–xxx Contents lists available at ScienceDirect Journal of Biotechnology jo u r n al homep age: www.elsevier.com/locate/jbiotec Transient inactivation of myostatin induces muscle hypertrophy and overcompensatory growth in zebraﬁsh via inactivation of the SMAD signaling pathway Eduardo N. Fuentes a,b,∗ , Katherine Pino a , Cristina Navarro a , Iselys Delgado a , Q1 Juan Antonio Valdés a,b , Alfredo Molina a,b,∗ a Universidad Andres Bello, Departmento de Ciencias Biologicas, Facultad de Ciencias Biologicas, Av. Republica 217, Santiago, Chile b FONDAP, Interdisciplinary Center for Aquaculture Research (INCAR), Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile a r t i c l e i n f o Article history: Received 11 July 2013 Received in revised form 30 September 2013 Accepted 21 October 2013 Available online xxx Keywords: Skeletal muscle growth SMAD signaling pathways Nutritional status Zebraﬁsh a b s t r a c t Myostatin (MSTN) is the main negative regulator of muscle growth and development in vertebrates. In ﬁsh, little is known about the molecular mechanisms behind how MSTN inactivation triggers skeletal muscle enhancement, particularly regarding the signaling pathways involved in this process. Moreover, there have not been reports on the biotechnological applications of MSTN and its signal transduction. In this context, zebraﬁsh underwent compensatory growth using fasting and refeeding trials, and MSTN activity was inactivated with dominant negative LAPD76A recombinant proteins during the refeeding period, when a rapid, compensatory muscle growth was observed. Treated ﬁsh displayed an overcompen- sation of growth characterized by higher muscle hypertrophy and growth performance than constantly fed, control ﬁsh. Treatment with LAPD76A recombinant proteins triggered inactivation of the SMAD signaling pathway in skeletal muscle, the main signal transduction used by MSTN to achieve its bio- logical actions. Therefore, transient inactivation of MSTN during the compensatory growth of zebraﬁsh led to a decrease in the SMAD signaling pathway in muscle, triggering muscle hypertrophy and ﬁnally improving growth performance, thus, zebraﬁsh achieved an overcompensation of growth. The present study shows an attractive strategy for improving muscle growth in a ﬁsh species by mixing a classical strategy, such as compensatory growth, and a biotechnological approach, such as the use of recombinant proteins for inhibiting the biological actions of MSTN. The mix of both strategies may represent a method that could be applied in order to improve growth in commercial ﬁsh of interest for aquaculture. © 2013 Published by Elsevier B.V. 1. Introduction Several technologies have been developed to increase com- petitiveness in ﬁnﬁsh aquaculture; however, very few successful biotechnological applications have been used to improve the pro- ductivity of this industry. In ﬁnﬁsh aquaculture, where a key goal is to improve the quality and quantity of myotomal muscle, an upgraded understanding of the key molecules controlling ﬁsh mus- cle growth is clearly of major importance. A pivotal molecule regulating muscle mass in ﬁsh and in other vertebrates is myo- statin (MSTN). MSTN, also called growth differentiation factor-8 ∗ Corresponding authors at: Laboratorio de Biotecnología Molecular and FON- DAP, Interdisciplinary Center for Aquaculture Research (INCAR), Departamento de Ciencias Biologicas, Facultad Ciencias Biologicas, Universidad Andres Bello, 8370146 Santiago, Chile. E-mail addresses: edua.fuentes@uandresbello.edu, edua.fuentes@gmail.com (E.N. Fuentes), amolina@unab.cl (A. Molina). (GDF-8), is a member of the superfamily of transformation and growth factors-beta (TGF-), and it negatively regulates the development and growth of skeletal muscle mass in vertebrates (McPherron and Lee, 1997; Rodgers and Garikipati, 2008). As a member of this superfamily, MSTN is synthesized as a prepropep- tide that undergoes several post-translational modiﬁcations (Lee, 2004). In mammals, MSTN undergoes two proteolytic processing events. One removes the N-terminal signal sequence, and the sec- ond cleavages the MSTN peptide in a RXXR conserved region, giving rise to the latency-associated peptide (LAP) and the active peptide (AP) (Lee, 2004). These two peptides remain associated by noncova- lent interactions, forming a heterotetrameric structure that inhibits the ability of AP to bind to its receptor (Gonzalez-Cadavid et al., 1998; Thies et al., 2001; Wolfman et al., 2003). The activation of latent MSTN occurs through the proteolytic cleavage of the LAP dimer by BMP-1/tolloid family members metalloproteases, which cleavage the aspartic amino acid residue at position 76 (Asp, D76), causing latent complex dissociation and the release of the AP (Lee, 2004; Wolfman et al., 2003). 0168-1656/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jbiotec.2013.10.028 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51