Transient inactivation of myostatin induces muscle hypertrophy and overcompensatory growth in zebrafish via inactivation of the SMAD signaling pathway.

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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Transient inactivation of myostatin induces muscle hypertrophy and overcompensatory growth in zebrafish via inactivation of the SMAD signaling pathway

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Eduardo N. Fuentes a,b,∗ , Katherine Pino a , Cristina Navarro a , Iselys Delgado a , 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 FONDAP, Interdisciplinary Center for Aquaculture Research (INCAR), Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile b

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Article history: Received 11 July 2013 Received in revised form 30 September 2013 Accepted 21 October 2013 Available online xxx

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Keywords: Skeletal muscle growth SMAD signaling pathways Nutritional status Zebrafish

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1. Introduction

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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 overcompensation 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 biological 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.

Several technologies have been developed to increase competitiveness in finfish aquaculture; however, very few successful biotechnological applications have been used to improve the productivity 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 muscle growth is clearly of major importance. A pivotal molecule regulating muscle mass in fish and in other vertebrates is myostatin (MSTN). MSTN, also called growth differentiation factor-8

∗ Corresponding authors at: Laboratorio de Biotecnología Molecular and FONDAP, Interdisciplinary Center for Aquaculture Research (INCAR), Departamento de Ciencias Biologicas, Facultad Ciencias Biologicas, Universidad Andres Bello, 8370146 Santiago, Chile. E-mail addresses: [email protected], [email protected] (E.N. Fuentes), [email protected] (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 prepropeptide 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 second 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 noncovalent 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

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

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In mammals, MSTN is expressed primarily in skeletal muscle and acts in an autocrine/paracrine fashion to inhibit myoblast proliferation (Taylor et al., 2001; Thomas et al., 2000), differentiation (Joulia et al., 2003), and protein synthesis (Taylor et al., 2001). Also, MSTN has metabolic functions in other tissue different than skeletal muscle, as was shown in mice without MSTN which have reduced adiposity (McPherron, 2010). The biological effects of MSTN on muscle cells are mediated through specific binding with the Activin type II receptors (ACTRII), subsequently activating, by phosphorylation, the SMAD signaling pathway, and particularly Smad2/Smad3, which act as transcription factors that ultimately suppress myogenesis (Zhu et al., 2004). In fish, a number of functional studies have investigated MSTN action on muscle growth. Mutant medaka C315Y MSTN shows an increase in muscle mass by both hyperplasia and hypertrophy, which is directly correlated with an enhancement in growth performance (e.g. weight, length, condition factor) (Chisada et al., 2011). Transgenic zebrafish show an increase in body weight and muscle mass, associated with hypertrophy, without hyperplasia, but not body length (Lee et al., 2009). C315Y MSTN dominant negative medaka and transgenic zebrafish that overexpress the prodomain of MSTN shows an increase in the number of fibers in skeletal muscle in comparison with wild-type individuals, but no difference are observed in the size of these fibers and growth performance (Sawatari et al., 2010; Xu et al., 2003). These results show the wide diversity of effects that MSTN has on muscle growth and growth performance. However, little is known about the molecular mechanisms behind how myostatin inactivation triggers skeletal muscle enhancement in fish, 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 with the purpose of improving productivity in the aquaculture industry. In aquaculture, the establishment of a growth–food ratio relationship is highly important in order to determine the optimum feeding strategies that may enhance growth (Sun et al., 2006; Nicieza and Metcalfe, 1997). In this context, “compensatory growth” may be used to alter the growth–food ratio relationship and has been suggested as method for optimizing body mass in fish (Jobling et al., 1994; Hayward et al., 1997; Ali et al., 2003; Jobling, 2010), because it affects muscle mass and flesh composition (Bugeon et al., 2004; Heide et al., 2006; Young et al., 2005). Compensatory growth is a phenomenon characterized by a stage of rapid growth when favorable conditions are restored after a period of growth depression (Jobling et al., 1994; Jobling, 2010; Won and Borski, 2013). This phenomenon is observed, for example, during annual cycles in the aquatic environment where fish have to alternate fasting and refeeding periods. Fasting leads to decreased body weight and growth rates (Jobling et al., 1994; Nicieza and Metcalfe, 1997; Ali et al., 2003; Picha et al., 2006, 2008; Jobling, 2010; Fuentes et al., 2011, 2012a; Won and Borski, 2013), affecting negatively skeletal muscle growth by increasing protein degradation, catabolism, and atrophy (Seiliez et al., 2008; Cleveland et al., 2009; Fuentes et al., 2012c). Subsequently, after this fasting period, if an abundant food supply becomes available, the growth retardation is overcome, or at least reduced, and higher growth rates and an increased body size are observed demonstrating that fish are able to “catch-up” growth (Jobling et al., 1994; Ali et al., 2003; Picha et al., 2006, 2008; Jobling, 2010; Fuentes et al., 2011, 2012a; Won and Borski, 2013). Skeletal muscle is also affected during this period through increasing protein synthesis and anabolism, triggering hypertrophy (Beardall and Johnston, 1985; Garcia de la Serrana et al., 2012). The aim of this study was to use a biotechnological approach involving the manipulation of MSTN to increase muscle mass in fish as well as to gain insight on the role of MSTN and its signaling pathway on muscle growth in this group of vertebrates.

Fasting and refeeding trials that trigger compensatory growth were implemented using different groups of juvenile fish. Transient inactivation of the biological actions of MSTN was performed by using LAPD76A recombinant proteins during the phase of rapid compensatory growth in one group of fish with the purpose of increasing muscle mass and growth performance. 2. Materials and methods 2.1. Production of dominant negative LAPD76A recombinant protein Total RNA was extracted from one year old adult zebrafish muscle tissue using the TRIzol® reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. 2 ␮g of total RNA were used to synthesize cDNA. The LAP region of myostatin (GenBank Accession Number: AY323521) was amplified by PCR using the cDNA previously synthesized. PCR was performed using a 1 ␮L cDNA template, 5 ␮L of PCR buffer 10X, 200 mM of each dNTP, 500 nM of each primer, 0.3 ␮L of Taq DNA polymerase (12 U/mL) (Promega, Madison, USA), and RNAse-free water to a final volume of 50 mL. Thermal cycling conditions were the following: initial activation of 10 min at 95 ◦ C, followed by 40 cycles of 30 s at 95 ◦ C; 30 s at 55 ◦ C; and 30 s at 72 ◦ C with a final extension of 10 min at 72 ◦ C. Primers used for obtaining the LAP region of myostatin were the following: forward 5 -CTCGAGTGTGGTCCAGTGGGTTATGGA-3 and reverse 5 -CTCGAGCTACCTCCGGATTCGTTTTGGGCCCT-3 . The PCR product was cloned into pGEM-T-Easy (Promega, Madison, USA), and subsequently, site-directed mutagenesis was performed using the QuikChange® kit (Stratagene, La Jolla, USA) inducing a specific mutation in the amino acid in position 76, thus replacing aspartic acid (D) for alanine (A) (Fig. 1A). Zebrafish contain two copies of mstn genes, mstn1 and mstn2 (Biga et al., 2005), and for both MSTN, the amino acid located in position 76 is aspartic acid (D). Therefore, MSTN1 and MSTN2 are affected by this mutation. This vector was sequenced in order to corroborate the specific mutation. This mutated vector containing the LAP region of myostatin was digested with XhoI restriction enzyme and then ligated to the XhoI linearized and CIAP fosfatase treated pET15b vector (Novagen, Darmstadt, Germany). This new vector was then sequenced. Escherichia coli BL21 (DE3) competent cells were transformed with the aforementioned vector. Bacteria containing this clone were grown at 37 ◦ C with agitation in a LB medium plus 50 mg/mL ampicilllin until the log-phase and subsequently induced with 0.3 mM IPTG (isopropyl-1-thio-␤-d-galactopyranoside) for 4 h. The recombinant protein was purified under denaturing conditions by Ni-NTA according to manufacturer’s instructions (Qiagen Austin, USA). After this, the recombinant protein was renaturalized in the dialysis membrane, concentrated, and quantified using a Pierce BCA Protein Assay Kit (Thermo Scientific, IL, USA). This was finally analyzed in a 10% SDS-PAGE gel. 2.2. Fish husbandry, experimental design, and sampling Juvenile zebrafish (Danio rerio) (0.04 ± 0.002 g; 1.3 ± 0.03 cm) were maintained according to standard procedures (Westerfield, 2000). The fish were randomly divided among 3 tanks (150 fish per tank), acclimatized for two weeks before the start of the trial, and all groups were fed to satiation. The experimental design is shown in Fig. 1B. At the start of the experiment (week 0), one group was fed until sated (control (C) group; continuously fed). The second group of fish was fasted (F) for 14 days and then refed (R) for 56 days (FR group). Finally, the last group was fasted (F) for 14 days, refed (R) for 56 days, and treated with dominant negative LAPD76A


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Fig. 1. Schematic representation of the mutation in LAP sequence of zebrafish MSTN and experimental design of the fasting and refeeding trial. (A) MSTN precursor scheme where the arrow indicates the sites of the dominant negative mutation in the aspartic 76 of the LAP (LAPD76A). (B) Experimental design in which the black bar represents the control (C) group which was constantly fed throughout the trial. The gray bar represents the group of fish fasted (F) for 14 days and refed (R) for 56 days (FR group). The white bar represent the group of fish fasted (F) for 14 days, refed (R) for 56 day, and treated with dominant negative LAPD76A (L) during the first 14 days of the refeeding period (RL) (FRL group). Arrows represent sampling points throughout the trial (0, 14, 28, and 70 days). Abbreviations: SP, Signal peptide; LAP, latency associated peptide; AP, Active peptide; Wt, wild-type.

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(L) throughout the first 14 days of the refeeding period (FRL group). Particularly for this group, 150 fish were incubated with LAPD76A recombinant protein (0.1 mg/L). Fish were immersed in a bath with dominant negative LAPD76A recombinant proteins over 2 weeks, 3 times per week for 2 h. After 2 weeks of LAPD76A recombinant protein treatment, juvenile zebrafish were allowed to grow normally. Fish were sampled at 0, 14, 28, and 70 days of the trial (indicated as ↓ in Fig. 1). Fish were sacrificed using anesthesia (3-aminobenzoic acid ethyl ester, 100 mg/L) and white myotomal muscle was collected. For protein extraction and Western blot analyses, muscle samples were frozen immediately in liquid nitrogen and subsequently stored at −80 ◦ C. For muscle histology, samples were immediately frozen in isopentane. The study adhered to animal welfare procedures and was approved by the bioethical committees of the Universidad Andres Bello and the National Commission for Scientific and Technological Research of the Chilean government.

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Fish from each group and sampling point were immersed in frozen isopentane during 20 min, followed by a cryostat cross-section of OCT-embedded muscle at 0.50 length. Subsequently, samples were stained with hematoxylin/eosin, and the images were digitalized. The muscle fiber area was determined using the Image J program (National Institutes of Health, USA).

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2.5. Western blotting Total proteins were extracted and prepared according to Fuentes et al. (2011, 2012a). Protein concentration was determined by Pierce® BCA Protein Assay Kit (Thermo Scientific, IL, USA). Western blot assays were performed according Fuentes et al. (2011, 2012a). Briefly, proteins were transferred to polyvinylidene diflouride membranes (PVDF) (Millipore, Bedford, MA, USA) and blocked for 1 h at room temperature in 2% ECL AdvanceTM blocking agent (GE Healthcare, Buckinghamshire, UK) dissolved in Tris-buffered saline (TBS 1×). Primary antibody incubations were performed at 4 ◦ C overnight. Phosphorylated (p) SMAD3 (S423/425) (#9520) was purchased from Cell Signaling (Beverly, USA). Membranes were visualized by a high sensitivity enhanced chemiluminescence kit, the ECL AdvanceTM Western Blotting Detection Kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. Subsequently, membranes were stripped off and blotted for total SMAD3 (#5678) (Cell Signaling, Beverly, USA), therefore obtaining the ratio between the phosphorylated and total protein. Densitometric analysis was performed with the Image J program (National Institute of Health, USA). 2.6. Statistical analyses Statistical analysis used for studying differences in gene expression was based on an advanced, general linear model (GLM) followed by Tukey’s post hoc test. All statistical analyses were performed using the STATISTICA 7 software (Tulsa, OK, USA). 3. Results

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Length and total body weight were measured during all sampling points and in all groups. With these measurements, and to evaluate growth performance in the zebrafish, the condition factor (CF) was calculated. The condition factor was calculated as CF = (W/L3 ) × 100, where W is the weight and L is the length.

3.1. Effects of fasting, refeeding, and dominant negative LAP76A on skeletal muscle growth in zebrafish Muscle cross-section (Fig. 2) and mean fiber area (Fig. 3) increased steadily in the C group throughout the whole trial (70 days). In both FR and FRL groups, muscle cross-section (Fig. 2)


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Fig. 2. Muscle growth in zebrafish throughout the fasting and refeeding trial in the C, FR, and FRL groups. Cross sectional muscle histology showing a representative image of each group at each sampling point.

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and mean fiber area (Fig. 3) decreased at the end of fasting and increased during the whole refeeding period (56 days), where full-compensating muscle mass loss was observed during fasting. However, it is possible to observe that at the end of the refeeding period (day 70), the FRL group showed a larger muscle

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Time (days) Fig. 3. Mean fiber area in zebrafish throughout the fasting and refeeding trial in the C (black bars), FR (gray bars), and FRL (white bars) groups. Downward arrows show the end of fasting for the FR and FRL groups. Different letters indicate a significant difference (P < 0.05). Results are expressed as means ± SEM (n = 4).

cross-section (Fig. 2) and mean fiber area (Fig. 3) than the C and FR groups. 3.2. Effects of fasting, refeeding, and dominant negative LAP76A on growth performance in zebrafish Weight, length, and CF increased significantly throughout the whole trial in the C group (Fig. 4). In the FR and FRL groups, total weight and CF decreased significantly during fasting and were subsequently compensated during refeeding (Fig. 4). Length was not affected in the FR and FRL groups during fasting, however, this parameter increased significantly during refeeding (Fig. 4). Significant differences among groups were also observed. At the end of fasting (day 14) and the first week of refeeding (day 21) there were significant differences in weight, length, and CF between the C group and the FR and FRL groups (Fig. 4). At the end of refeeding (day 70), there were significant differences between the FLR group and the C and FR groups in total weight and CF (Fig. 4). There were no significant differences in length among the groups at the end of refeeding (Fig. 4). 3.3. Effects of fasting, refeeding, and dominant negative LAP76A on SMAD signaling pathway activation in zebrafish muscle SMAD signaling pathway activation was assessed by using the ratio between pSMAD3/SMAD3. In the C group, SMAD3 activation


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Time (days) Fig. 4. Growth performance in zebrafish throughout the fasting and refeeding trial in the C (black square), FR (gray diamond), and FRL (white circle) groups. (A) Weight, (B) length, and (C) CF in the C, FR, FRL groups during the fasting and refeeding trial. Asterisk indicates a significant difference (P < 0.05) between the FRL and C groups. Upward arrows show the end of fasting and beginning of refeeding for the FR and FRL groups. Dashed lines show the period of time in which zebrafish belonging to the FRL group were incubated with recombinant LAPD76A protein. Results are expressed as means ± SEM (n = 35).

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did not change throughout the trial. In the FR and FRL groups, SMAD3 activation increased significantly at the end of fasting. In the FR group after 2 weeks of refeeding (day 28), SMAD3 showed the same activation levels as those at the end of fasting. Conversely, in the FRL group after 2 weeks of refeeding, SMAD3 activation decreased significantly in comparison with the FR group, reaching the same levels as the C group. At the end of refeeding (day 70), all groups showed basal levels of activation for SMAD3.

4. Discussion MSTN is a potent muscle growth inhibitor, with LAP being the most potent inhibitor of MSTN biological actions. In fish, the use of mutations involved in the proteolytic cleavage sites (D76), which prevents the release of LAP from the AP and thus inhibit MSTN biological actions, has not been described. The present study induces compensatory growth in fish by administrating LAPD76A MSTN dominant negative recombinant proteins during the refeeding period, when a rapid, compensatory muscle growth is observed with the purpose of increasing muscle mass and growth performance in zebrafish.

4.1. Dominant negative LAP76A effects on skeletal muscle growth in zebrafish In the FR and FRL groups during fasting, a reduction of cross sectional and mean fiber area was observed, suggesting that muscle atrophy was induced. In other fish species during fasting, the same phenomenon has been observed. In the fine flounder, an atrophic phenotype characterized by a large number of small fibers has been observed during fasting (Fuentes EN, Björnsson BT, Molina A, unpublished results). During refeeding, the FR and FRL groups displayed hypertrophic growth. Hypertrophy rather than hyperplasia has been reported as the main mechanism of muscle mass recovery during compensatory growth (Beardall and Johnston, 1985; Garcia de la Serrana et al., 2012; Fuentes EN, Björnsson BT, Molina A, unpublished results). The FRL group displayed higher muscle hypertrophy than the C and FR groups. In zebrafish, the inactivation of MSTN leads to an increase muscle mass associated with hypertrophy (Lee et al., 2009). However, other studies have shown that inactivation of MSTN in fish leads to both hyperplasia and hypertrophy (Chisada et al., 2011), or just hyperplasia (Sawatari et al., 2010; Xu et al., 2003). The present results suggest that LAP treatments in zebrafish during refeeding after fasting enhance the inherent capacity of muscle growth by hypertrophy during this period.


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Fasting and refeeding trials trigger compensatory growth, which is a complex phenomenon that shows different degrees of com314 pensation depending on feeding protocols, including length and 315 intensity of deprivation (Nicieza and Metcalfe, 1997; Ali et al., 2003; 316 Won and Borski, 2013). In the FR and FRL groups, weight, length, 317 and CF decreased during fasting, as has been shown in several other 318 fish species (Gabillard et al., 2006; Fox et al., 2006, 2010; Picha et al., 319 2006, 2008; Fuentes et al., 2011, 2012a). During refeeding, partic320 ularly at the end of this period, the FR group partially catches-up 321 growth, whereas the FRL passed the C group in growth. There are 322 four different degrees of compensatory growth in fish as follows: 323 no compensation (e.g. fish are not able to recover growth), partial 324 compensation (e.g. fish fail to achieve the same growth as the C 325 group), full compensation (e.g. fish achieve the same growth as the 326 C group), and overcompensation (e.g. fish achieve greater growth 327 than C fish) (Jobling et al., 1994; Jobling, 2010; Ali et al., 2003). In fish 328 displaying partial, full, and overcompensation, growth rates during 329 part of the refeeding are higher than constantly fed fish (the C group 330 in this study) (Jobling et al., 1994; Ali et al., 2003; Gabillard et al., 331 2006; Fox et al., 2006, 2010; Picha et al., 2006, 2008; Jobling, 2010; 332 Fuentes et al., 2011, 2012a). Particularly, partial compensation is 333 the most common phenomenon observed in fish, with several fish 334 species displaying this type of growth (Ali et al., 2003; Gabillard 335 Q2 et al., 2006; Picha et al., 2006, 2008; Fox et al., 2009). On the other 336 hand, full compensation is rather infrequent but has been observed 337 in fish species such as the fine flounder (Fuentes et al., 2011, 2012a). 338 Finally, overcompensation is the rarest outcome and is very diffi339 cult to achieve. The only study to show this phenomenon is that 340 of Hayward et al. (1997) which showed that 1 year-old juvenile 341 hybrid sunfish reached masses of up to twice those of controls in 342 a 105-day study. The present study shows for the first time over343 compensation of growth in a teleost species during refeeding after 344 a fasting period using a biotechnological approach such as that of 345 recombinant proteins. Compensatory growth has been suggested 346 as a method for optimizing body mass in fish (Jobling et al., 1994; 347 Hayward et al., 1997; Ali et al., 2003; Jobling, 2010), which affects 348 muscle mass and flesh composition (Bugeon et al., 2004; Heide 349 et al., 2006; Young et al., 2005). Therefore, the present results sug350 gest that the use of recombinant proteins that transiently inhibit 351 the biological activity of MSTN will trigger overcompensation of 352 growth due to an enhancement of muscle hypertrophy. For fish, 353 body mass is strongly related to muscle mass (Weatherley et al., 354 1988) because skeletal muscle in fish comprises the largest single 355 tissue compartment, representing up to 70% of total body mass, 356 a relatively larger component than that in mammals (Weatherley 357 et al., 1988). Therefore, these results highlight the fact that manipu358 lating molecules that control muscle mass in fish, such as MSTN, will 359 directly lead to improved growth rates. Considering that zefrafish 360 display the same type of muscle growth (hypertrophy) during 361 compensatory growth as other fish species of commercial inter362 est, it is likely that these fish species will respond similarly to the 363 treatment. 312 313

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4.3. Dominant negative LAP76A effects on SMAD signaling pathway activation in zebrafish muscle Several studies have used fasting and refeeding trials as a model for studying MSTN biology in fish muscle growth. However, these studies have only evaluated mRNA contents of mstn and have found very contrasting results (Chauvigné et al., 2003; De Santis and Jerry, 2011; Terova et al., 2006; Montserrat et al., 2007; Rodgers et al., 2003; Meyer et al., 2013). In this context, the main signaling pathway activated by MSTN, the SMAD signal transduction, was

Fig. 5. Activation of SMAD signaling pathway in the muscle of zebrafish during the fasting and refeeding trial in the C (black square), FR (gray diamond), and FRL (white circle) groups. Downward arrows show the end of fasting for the FR and FRL groups. Different letters indicate a significant difference (P < 0.05). Results are expressed as means ± SEM (n = 4).

assessed during fasting and refeeding in zebrafish muscle. During fasting, the SMAD signaling pathway was strongly activated in the FR and FRL groups. Other TGF-␤ members can also activate the SMAD signaling pathway and may have strong effects on skeletal muscle growth (Kollias and McDermott, 2008). The SMAD signaling pathway induces muscle atrophy in mammals, and the inhibition of this pathway promotes muscle hypertrophy (Sartori et al., 2009), thus suggesting that MSTN and other TGF-␤ members may be playing an important role in the muscleinduced atrophy observed in this stage for zebrafish. In fish, the PI3K/AKT/FOXO and NF␬B/I␬B␣ signaling pathways contribute to muscle atrophy (Fuentes et al., 2012c). The present study shows the first evidence that suggests that the SMAD signaling pathway is also contributing to muscle catabolism during fasting, and that this activation could be mediated by MSTN or other TGF-␤ members. Interestingly, during the first two weeks of refeeding, the SMAD signaling pathway in the FR group was still activated but not in the FRL group, indicating that treatments with LAPD76A inhibit MSTN actions. Multiple alignments analyses with zebrafish MSTN and other TGF-␤ members show no evidence for sequence conservation of the cleavage site at the aspartic acid residue at position 76 (Asp, D76) in other TGF-␤ members (data not shown). Therefore, it is plausible to suggest that the LAPD76A treatment transiently inactivates just MSTN but no other TGF-␤ members, thus triggering the inactivation of SMAD3. A complete scenario regarding the molecular regulation of muscle growth during refeeding in fish has been previously shown (Fuentes et al., 2011, 2012a,b,c, 2013a,b; Safian et al., 2012). During the first stages of refeeding (e.g. hours of refeeding), positive regulators of muscle growth largely exceed negative signals, thus laying the foundation for the subsequent promotion of strong, catch-up growth (Fuentes et al., 2011, 2012a,b,c, 2013a,b; Safian et al., 2012). Conversely, during more advanced stages of refeeding (weeks of refeeding), positive and negative signals start to become balanced, and the majority of components returning to basal levels and reestablish homeostatic growth of the muscle


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Fig. 6. Schematic diagram summarizing the effects of fasting, refeeding, and LAPD76A treatment on SMAD signaling pathway activation, muscle cellularity, and growth performance in zebrafish. The schematic diagram illustrates the events occurring in (A) Fasted zebrafish, (B) refed zebrafish, and (C) refed zebrafish treated with LAPD76A for 2 weeks.

(Fuentes et al., 2011, 2012a,b,c, 2013a,b; Safian et al., 2012). Therefore, altogether these results suggest that treatment with LAPD76A 411 recombinant proteins during the first two weeks of refeeding in 412 zebrafish disturbs the balance between positive and negative sig413 nals, allowing for muscle growth without important inhibitors such 414 Q3 as MSTN and the SMAD signaling pathway (Fig. 5). 409

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This work was supported by Fondo Nacional de Desarrollo Científico y Tecnologico (FONDECYT) Grants 1090416 and 1130545 (to A. Molina); Universidad Andres Bello fund DI-14-11/I (to EN Fuentes); and CONICYT/FONDAP/15110027 (to EN Fuentes, A Molina, JA Valdes).

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4.4. Conclusions and perspectives Uncited references

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The events triggered by the effects of fasting, refeeding, and LAPD76A treatment on the SMAD signaling pathway activation, muscle cellularity, and growth performance in zebrafish are summarized in Fig. 6. The biological action of MSTN, and probably of other TGF-␤ members, during fasting is more pronounced, triggering SMAD signaling pathway activation in muscle and leading to muscle atrophy. This directly affects growth performance by decreasing total weight and condition factor (Fig. 6A). During the first week of refeeding, the SMAD signaling pathway is still activated, indicating that the actions of MSTN, and probably of other TGF-␤ members, are present during this period. Thus, this negative regulator of growth could be competing with positive modulators of growth, allowing controlled muscle hypertrophy which finally triggers a partial compensation of growth (Fig. 6B). Transient inactivation of MSTN during the first stage of refeeding triggers the inactivation of the SMAD signaling pathway, leading to an increase in muscle hypertrophy, and, finally, overcompensatory growth (Fig. 6C). The present study shows an attractive strategy for improving muscle growth in fish species, mixing a classical strategy for manipulating body mass, such as compensatory growth, and a biotechnological approach, such as the use of recombinant proteins that inhibit the biological actions of MSTN. The mix of both strategies significantly improves growth rates in zebrafish. This could represent a useful strategy that could be applied in order to improve growth in commercial fish of interest for aquaculture. Therefore, this approach could increase the competitiveness and productivity of this industry, which is mainly focused on the quality and quantity of myotomal muscle.

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Smad signaling pathway induces the differentiation of mouse spermatogonial stem cells via upregulation of Sohlh2.

Epigallocatechin gallate inhibits the growth of MDA-MB-231 breast cancer cells via inactivation of the β-catenin signaling pathway.

Serenoa Repens Induces Growth Arrest, Apoptosis and Inactivation of STAT3 Signaling in Human Glioma Cells.

Smad signaling pathway.

Down-regulation of RPS9 Inhibits Osteosarcoma Cell Growth through Inactivation of MAPK Signaling Pathway.

Silencing Myostatin Using Cholesterol-conjugated siRNAs Induces Muscle Growth.

Silencing Myostatin Using Cholesterol-conjugated siRNAs Induces Muscle Growth.

Retraction: Guggulsterone inhibits prostate cancer growth via inactivation of Akt regulated by ATP citrate lyase signaling.

Cobalamin inactivation induces formyltetrahydrofolate synthetase.

AKT Signaling Pathway.

Smad Signaling Pathway.

STAT3 signaling pathway in human renal carcinoma Caki cells.

SMAD signaling pathway in CRC.

Upregulation of DACT2 suppresses proliferation and enhances apoptosis of glioma cell via inactivation of YAP signaling pathway.

CXCR4 signaling inhibits intrahepatic cholangiocarcinoma progression and metastasis via inactivation of canonical Wnt pathway.

Smad signaling pathway.

Tumor-specific signaling to p53 is mimicked by Mdm2 inactivation in zebrafish: insights from mdm2 and mdm4 mutant zebrafish.

Smad signaling pathway in pathogenesis and development of gluteal muscle contracture.

Sip1 signaling pathway in CRC.

Smad signaling pathway.

Dystrophic muscle improvement in zebrafish via increased heme oxygenase signaling.

Combined Strategies for Maintaining Skeletal Muscle Mass and Function in Aging: Myostatin Inactivation and AICAR-Associated Oxidative Metabolism Induction.

Smad signaling pathway.

Dystrophic muscle improvement in zebrafish via increased heme oxygenase signaling.