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Contents lists available at ScienceDirect

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

1 Transient inactivation of myostatin induces muscle hypertrophy and

2 overcompensatory growth in zebrafish via inactivation of the SMAD
3 signaling pathway
4 Q1 Eduardo N. Fuentes a,b,∗ , Katherine Pino a , Cristina Navarro a , Iselys Delgado a ,
5 Juan Antonio Valdés a,b , Alfredo Molina a,b,∗
a
6 Universidad Andres Bello, Departmento de Ciencias Biologicas, Facultad de Ciencias Biologicas, Av. Republica 217, Santiago, Chile
b
7 FONDAP, Interdisciplinary Center for Aquaculture Research (INCAR), Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, Universidad
8 Andrés Bello, Santiago, Chile
9

10 a r t i c l e i n f o a b s t r a c t
11
12 Article history: Myostatin (MSTN) is the main negative regulator of muscle growth and development in vertebrates. In
13 Received 11 July 2013 fish, little is known about the molecular mechanisms behind how MSTN inactivation triggers skeletal
14 Received in revised form muscle enhancement, particularly regarding the signaling pathways involved in this process. Moreover,
15 30 September 2013
there have not been reports on the biotechnological applications of MSTN and its signal transduction.
16 Accepted 21 October 2013
In this context, zebrafish underwent compensatory growth using fasting and refeeding trials, and MSTN
Available online xxx
activity was inactivated with dominant negative LAPD76A recombinant proteins during the refeeding
17
period, when a rapid, compensatory muscle growth was observed. Treated fish displayed an overcompen-
18 Keywords:
19 Skeletal muscle growth
sation of growth characterized by higher muscle hypertrophy and growth performance than constantly
20 SMAD signaling pathways fed, control fish. Treatment with LAPD76A recombinant proteins triggered inactivation of the SMAD
21 Nutritional status signaling pathway in skeletal muscle, the main signal transduction used by MSTN to achieve its bio-
22 Zebrafish 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.

23 1. Introduction (GDF-8), is a member of the superfamily of transformation and 33

growth factors-beta (TGF-␤), and it negatively regulates the 34

24 Several technologies have been developed to increase com- development and growth of skeletal muscle mass in vertebrates 35

25 petitiveness in finfish aquaculture; however, very few successful (McPherron and Lee, 1997; Rodgers and Garikipati, 2008). As a 36

26 biotechnological applications have been used to improve the pro- member of this superfamily, MSTN is synthesized as a prepropep- 37

27 ductivity of this industry. In finfish aquaculture, where a key goal tide that undergoes several post-translational modifications (Lee, 38

28 is to improve the quality and quantity of myotomal muscle, an 2004). In mammals, MSTN undergoes two proteolytic processing 39

29 upgraded understanding of the key molecules controlling fish mus- events. One removes the N-terminal signal sequence, and the sec- 40

30 cle growth is clearly of major importance. A pivotal molecule ond cleavages the MSTN peptide in a RXXR conserved region, giving 41

31 regulating muscle mass in fish and in other vertebrates is myo- rise to the latency-associated peptide (LAP) and the active peptide 42

32 statin (MSTN). MSTN, also called growth differentiation factor-8 (AP) (Lee, 2004). These two peptides remain associated by noncova- 43

lent interactions, forming a heterotetrameric structure that inhibits 44

the ability of AP to bind to its receptor (Gonzalez-Cadavid et al., 45

1998; Thies et al., 2001; Wolfman et al., 2003). The activation of 46

∗ Corresponding authors at: Laboratorio de Biotecnología Molecular and FON- latent MSTN occurs through the proteolytic cleavage of the LAP 47
DAP, Interdisciplinary Center for Aquaculture Research (INCAR), Departamento de dimer by BMP-1/tolloid family members metalloproteases, which 48
Ciencias Biologicas, Facultad Ciencias Biologicas, Universidad Andres Bello, 8370146
cleavage the aspartic amino acid residue at position 76 (Asp, D76), 49
Santiago, Chile.
E-mail addresses: edua.fuentes@uandresbello.edu, edua.fuentes@gmail.com causing latent complex dissociation and the release of the AP (Lee, 50

(E.N. Fuentes), amolina@unab.cl (A. Molina). 2004; Wolfman et al., 2003). 51

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
 ARTICLE IN PRESS
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2 E.N. Fuentes et al. / Journal of Biotechnology xxx (2013) xxx–xxx

52 In mammals, MSTN is expressed primarily in skeletal mus- Fasting and refeeding trials that trigger compensatory growth were 118

53 cle and acts in an autocrine/paracrine fashion to inhibit myoblast implemented using different groups of juvenile fish. Transient inac- 119

54 proliferation (Taylor et al., 2001; Thomas et al., 2000), differen- tivation of the biological actions of MSTN was performed by using 120

55 tiation (Joulia et al., 2003), and protein synthesis (Taylor et al., LAPD76A recombinant proteins during the phase of rapid compen- 121

56 2001). Also, MSTN has metabolic functions in other tissue differ- satory growth in one group of fish with the purpose of increasing 122

57 ent than skeletal muscle, as was shown in mice without MSTN muscle mass and growth performance. 123

58 which have reduced adiposity (McPherron, 2010). The biological

59 effects of MSTN on muscle cells are mediated through specific
2. Materials and methods 124
60 binding with the Activin type II receptors (ACTRII), subsequently
61 activating, by phosphorylation, the SMAD signaling pathway, and
2.1. Production of dominant negative LAPD76A recombinant 125
62 particularly Smad2/Smad3, which act as transcription factors that
protein 126
63 ultimately suppress myogenesis (Zhu et al., 2004). In fish, a num-
64 ber of functional studies have investigated MSTN action on muscle
Total RNA was extracted from one year old adult zebrafish mus- 127
65 growth. Mutant medaka C315Y MSTN shows an increase in muscle
cle tissue using the TRIzol® reagent (Invitrogen, Carlsbad, USA) 128
66 mass by both hyperplasia and hypertrophy, which is directly cor-
according to the manufacturer’s instructions. 2 ␮g of total RNA 129
67 related with an enhancement in growth performance (e.g. weight,
were used to synthesize cDNA. The LAP region of myostatin (Gen- 130
68 length, condition factor) (Chisada et al., 2011). Transgenic zebrafish
Bank Accession Number: AY323521) was amplified by PCR using 131
69 show an increase in body weight and muscle mass, associated
the cDNA previously synthesized. PCR was performed using a 1 ␮L 132
70 with hypertrophy, without hyperplasia, but not body length (Lee
cDNA template, 5 ␮L of PCR buffer 10X, 200 mM of each dNTP, 133
71 et al., 2009). C315Y MSTN dominant negative medaka and trans-
500 nM of each primer, 0.3 ␮L of Taq DNA polymerase (12 U/mL) 134
72 genic zebrafish that overexpress the prodomain of MSTN shows an
(Promega, Madison, USA), and RNAse-free water to a final volume of 135
73 increase in the number of fibers in skeletal muscle in comparison
50 mL. Thermal cycling conditions were the following: initial acti- 136
74 with wild-type individuals, but no difference are observed in the
vation of 10 min at 95 ◦ C, followed by 40 cycles of 30 s at 95 ◦ C; 137
75 size of these fibers and growth performance (Sawatari et al., 2010;
30 s at 55 ◦ C; and 30 s at 72 ◦ C with a final extension of 10 min at 138
76 Xu et al., 2003). These results show the wide diversity of effects that
72 ◦ C. Primers used for obtaining the LAP region of myostatin were 139
77 MSTN has on muscle growth and growth performance. However,
the following: forward 5 -CTCGAGTGTGGTCCAGTGGGTTATGGA-3 140
78 little is known about the molecular mechanisms behind how myo-
and reverse 5 -CTCGAGCTACCTCCGGATTCGTTTTGGGCCCT-3 . The 141
79 statin inactivation triggers skeletal muscle enhancement in fish,
PCR product was cloned into pGEM-T-Easy (Promega, Madison, 142
80 particularly regarding the signaling pathways involved in this pro-
USA), and subsequently, site-directed mutagenesis was performed 143
81 cess. Moreover, there have not been reports on the biotechnological
using the QuikChange® kit (Stratagene, La Jolla, USA) inducing a 144
82 applications of MSTN and its signal transduction with the purpose
specific mutation in the amino acid in position 76, thus replac- 145
83 of improving productivity in the aquaculture industry.
ing aspartic acid (D) for alanine (A) (Fig. 1A). Zebrafish contain 146
84 In aquaculture, the establishment of a growth–food ratio
two copies of mstn genes, mstn1 and mstn2 (Biga et al., 2005), and 147
85 relationship is highly important in order to determine the opti-
for both MSTN, the amino acid located in position 76 is aspar- 148
86 mum feeding strategies that may enhance growth (Sun et al.,
tic acid (D). Therefore, MSTN1 and MSTN2 are affected by this 149
87 2006; Nicieza and Metcalfe, 1997). In this context, “compensatory
mutation. This vector was sequenced in order to corroborate the 150
88 growth” may be used to alter the growth–food ratio relationship
specific mutation. This mutated vector containing the LAP region 151
89 and has been suggested as method for optimizing body mass in
of myostatin was digested with XhoI restriction enzyme and then 152
90 fish (Jobling et al., 1994; Hayward et al., 1997; Ali et al., 2003;
ligated to the XhoI linearized and CIAP fosfatase treated pET15b 153
91 Jobling, 2010), because it affects muscle mass and flesh compo-
vector (Novagen, Darmstadt, Germany). This new vector was then 154
92 sition (Bugeon et al., 2004; Heide et al., 2006; Young et al., 2005).
sequenced. Escherichia coli BL21 (DE3) competent cells were trans- 155
93 Compensatory growth is a phenomenon characterized by a stage of
formed with the aforementioned vector. Bacteria containing this 156
94 rapid growth when favorable conditions are restored after a period
clone were grown at 37 ◦ C with agitation in a LB medium plus 157
95 of growth depression (Jobling et al., 1994; Jobling, 2010; Won and
50 mg/mL ampicilllin until the log-phase and subsequently induced 158
96 Borski, 2013). This phenomenon is observed, for example, dur-
with 0.3 mM IPTG (isopropyl-1-thio-␤-d-galactopyranoside) for 159
97 ing annual cycles in the aquatic environment where fish have to
4 h. The recombinant protein was purified under denaturing 160
98 alternate fasting and refeeding periods. Fasting leads to decreased
conditions by Ni-NTA according to manufacturer’s instruc- 161
99 body weight and growth rates (Jobling et al., 1994; Nicieza and
tions (Qiagen Austin, USA). After this, the recombinant protein 162
100 Metcalfe, 1997; Ali et al., 2003; Picha et al., 2006, 2008; Jobling,
was renaturalized in the dialysis membrane, concentrated, and 163
101 2010; Fuentes et al., 2011, 2012a; Won and Borski, 2013), affecting
quantified using a Pierce BCA Protein Assay Kit (Thermo Sci- 164
102 negatively skeletal muscle growth by increasing protein degrada-
entific, IL, USA). This was finally analyzed in a 10% SDS-PAGE 165
103 tion, catabolism, and atrophy (Seiliez et al., 2008; Cleveland et al.,
gel. 166
104 2009; Fuentes et al., 2012c). Subsequently, after this fasting period,
105 if an abundant food supply becomes available, the growth retar-
106 dation is overcome, or at least reduced, and higher growth rates 2.2. Fish husbandry, experimental design, and sampling 167

107 and an increased body size are observed demonstrating that fish
108 are able to “catch-up” growth (Jobling et al., 1994; Ali et al., 2003; Juvenile zebrafish (Danio rerio) (0.04 ± 0.002 g; 1.3 ± 0.03 cm) 168

109 Picha et al., 2006, 2008; Jobling, 2010; Fuentes et al., 2011, 2012a; were maintained according to standard procedures (Westerfield, 169

110 Won and Borski, 2013). Skeletal muscle is also affected during this 2000). The fish were randomly divided among 3 tanks (150 fish per 170

111 period through increasing protein synthesis and anabolism, trigger- tank), acclimatized for two weeks before the start of the trial, and 171

112 ing hypertrophy (Beardall and Johnston, 1985; Garcia de la Serrana all groups were fed to satiation. The experimental design is shown 172

113 et al., 2012). in Fig. 1B. At the start of the experiment (week 0), one group was 173

114 The aim of this study was to use a biotechnological approach fed until sated (control (C) group; continuously fed). The second 174

115 involving the manipulation of MSTN to increase muscle mass group of fish was fasted (F) for 14 days and then refed (R) for 56 175

116 in fish as well as to gain insight on the role of MSTN and its days (FR group). Finally, the last group was fasted (F) for 14 days, 176

117 signaling pathway on muscle growth in this group of vertebrates. refed (R) for 56 days, and treated with dominant negative LAPD76A 177

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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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.

178 (L) throughout the first 14 days of the refeeding period (FRL group). 2.5. Western blotting 210

179 Particularly for this group, 150 fish were incubated with LAPD76A
180 recombinant protein (0.1 mg/L). Fish were immersed in a bath with Total proteins were extracted and prepared according to Fuentes 211

181 dominant negative LAPD76A recombinant proteins over 2 weeks, et al. (2011, 2012a). Protein concentration was determined by 212

182 3 times per week for 2 h. After 2 weeks of LAPD76A recombinant Pierce® BCA Protein Assay Kit (Thermo Scientific, IL, USA). 213

183 protein treatment, juvenile zebrafish were allowed to grow nor- Western blot assays were performed according Fuentes et al. 214

184 mally. (2011, 2012a). Briefly, proteins were transferred to polyvinyli- 215

185 Fish were sampled at 0, 14, 28, and 70 days of the trial dene diflouride membranes (PVDF) (Millipore, Bedford, MA, USA) 216

186 (indicated as ↓ in Fig. 1). Fish were sacrificed using anesthesia and blocked for 1 h at room temperature in 2% ECL AdvanceTM 217

187 (3-aminobenzoic acid ethyl ester, 100 mg/L) and white myotomal blocking agent (GE Healthcare, Buckinghamshire, UK) dissolved 218

188 muscle was collected. For protein extraction and Western blot anal- in Tris-buffered saline (TBS 1×). Primary antibody incubations 219

189 yses, muscle samples were frozen immediately in liquid nitrogen were performed at 4 ◦ C overnight. Phosphorylated (p) SMAD3 220

190 and subsequently stored at −80 ◦ C. For muscle histology, sam- (S423/425) (#9520) was purchased from Cell Signaling (Beverly, 221

191 ples were immediately frozen in isopentane. The study adhered USA). Membranes were visualized by a high sensitivity enhanced 222

192 to animal welfare procedures and was approved by the bioethical chemiluminescence kit, the ECL AdvanceTM Western Blotting 223

193 committees of the Universidad Andres Bello and the National Com- Detection Kit (GE Healthcare, Buckinghamshire, UK) according to 224

194 mission for Scientific and Technological Research of the Chilean the manufacturer’s instructions. Subsequently, membranes were 225

195 government. stripped off and blotted for total SMAD3 (#5678) (Cell Signaling, 226

Beverly, USA), therefore obtaining the ratio between the phospho- 227

rylated and total protein. Densitometric analysis was performed 228

196 2.3. Muscle histology analyses with the Image J program (National Institute of Health, USA). 229

197 Fish from each group and sampling point were immersed
2.6. Statistical analyses 230
198 in frozen isopentane during 20 min, followed by a cryostat
199 cross-section of OCT-embedded muscle at 0.50 length. Sub-
Statistical analysis used for studying differences in gene expres- 231
200 sequently, samples were stained with hematoxylin/eosin, and
sion was based on an advanced, general linear model (GLM) 232
201 the images were digitalized. The muscle fiber area was deter-
followed by Tukey’s post hoc test. All statistical analyses were per- 233
202 mined using the Image J program (National Institutes of Health,
formed using the STATISTICA 7 software (Tulsa, OK, USA). 234
203 USA).

3. Results 235
204 2.4. Growth performance assessment
3.1. Effects of fasting, refeeding, and dominant negative LAP76A 236
205 Length and total body weight were measured during all samp- on skeletal muscle growth in zebrafish 237
206 ling points and in all groups. With these measurements, and to
207 evaluate growth performance in the zebrafish, the condition fac- Muscle cross-section (Fig. 2) and mean fiber area (Fig. 3) 238
208 tor (CF) was calculated. The condition factor was calculated as increased steadily in the C group throughout the whole trial (70 239
209 CF = (W/L3 ) × 100, where W is the weight and L is the length. days). In both FR and FRL groups, muscle cross-section (Fig. 2) 240

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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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.

241 and mean fiber area (Fig. 3) decreased at the end of fasting and cross-section (Fig. 2) and mean fiber area (Fig. 3) than the C and FR 246

242 increased during the whole refeeding period (56 days), where groups. 247

243 full-compensating muscle mass loss was observed during fas-

244 ting. However, it is possible to observe that at the end of the 3.2. Effects of fasting, refeeding, and dominant negative LAP76A 248
245 refeeding period (day 70), the FRL group showed a larger muscle on growth performance in zebrafish 249

Weight, length, and CF increased significantly throughout the 250

whole trial in the C group (Fig. 4). In the FR and FRL groups, total 251
300 C d
weight and CF decreased significantly during fasting and were sub-
Mean fiber area (µM)

252
FR sequently compensated during refeeding (Fig. 4). Length was not
c c
253
FRL affected in the FR and FRL groups during fasting, however, this 254

parameter increased significantly during refeeding (Fig. 4). Signif- 255

200
ac ac ac icant differences among groups were also observed. At the end 256
a a ac of fasting (day 14) and the first week of refeeding (day 21) there
a 257

were significant differences in weight, length, and CF between the 258

b b
100 C group and the FR and FRL groups (Fig. 4). At the end of refeeding 259

(day 70), there were significant differences between the FLR group 260

and the C and FR groups in total weight and CF (Fig. 4). There were 261

no significant differences in length among the groups at the end of 262

0 refeeding (Fig. 4). 263
0 14 28 70
Time (days) 3.3. Effects of fasting, refeeding, and dominant negative LAP76A 264

on SMAD signaling pathway activation in zebrafish muscle 265

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 SMAD signaling pathway activation was assessed by using the 266

difference (P < 0.05). Results are expressed as means ± SEM (n = 4). ratio between pSMAD3/SMAD3. In the C group, SMAD3 activation 267

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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A B
0,40 3,0
C
*
0,35
FR 2,7

0,30
FRL 2,4
Weight (grs)

Lenght (cm)
0,25 2,1

0,20 1,8
*
0,15 1,5
*
0,10 1,2

0,05
* * 0,9

0,00 0,6

0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70

Time (days) Time (days)

C
100 *
80
CF ((W/L3) X 100)

60

40

20
a * *
0

0 10 20 30 40 50 60 70

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).

268 did not change throughout the trial. In the FR and FRL groups, 4.1. Dominant negative LAP76A effects on skeletal muscle growth 288

269 SMAD3 activation increased significantly at the end of fasting. in zebrafish 289

270 In the FR group after 2 weeks of refeeding (day 28), SMAD3

271 showed the same activation levels as those at the end of fas- In the FR and FRL groups during fasting, a reduction of cross sec- 290

272 ting. Conversely, in the FRL group after 2 weeks of refeeding, tional and mean fiber area was observed, suggesting that muscle 291

273 SMAD3 activation decreased significantly in comparison with the atrophy was induced. In other fish species during fasting, the same 292

274 FR group, reaching the same levels as the C group. At the end of phenomenon has been observed. In the fine flounder, an atrophic 293

275 refeeding (day 70), all groups showed basal levels of activation for phenotype characterized by a large number of small fibers has 294

276 SMAD3. been observed during fasting (Fuentes EN, Björnsson BT, Molina A, 295

unpublished results). During refeeding, the FR and FRL groups dis- 296

played hypertrophic growth. Hypertrophy rather than hyperplasia 297

277 4. Discussion has been reported as the main mechanism of muscle mass recov- 298

ery during compensatory growth (Beardall and Johnston, 1985; 299

278 MSTN is a potent muscle growth inhibitor, with LAP being Garcia de la Serrana et al., 2012; Fuentes EN, Björnsson BT, Molina 300

279 the most potent inhibitor of MSTN biological actions. In fish, the A, unpublished results). The FRL group displayed higher muscle 301

280 use of mutations involved in the proteolytic cleavage sites (D76), hypertrophy than the C and FR groups. In zebrafish, the inactivation 302

281 which prevents the release of LAP from the AP and thus inhibit of MSTN leads to an increase muscle mass associated with hyper- 303

282 MSTN biological actions, has not been described. The present study trophy (Lee et al., 2009). However, other studies have shown that 304

283 induces compensatory growth in fish by administrating LAPD76A inactivation of MSTN in fish leads to both hyperplasia and hypertro- 305

284 MSTN dominant negative recombinant proteins during the refeed- phy (Chisada et al., 2011), or just hyperplasia (Sawatari et al., 2010; 306

285 ing period, when a rapid, compensatory muscle growth is observed Xu et al., 2003). The present results suggest that LAP treatments 307

286 with the purpose of increasing muscle mass and growth perfor- in zebrafish during refeeding after fasting enhance the inherent 308

287 mance in zebrafish. capacity of muscle growth by hypertrophy during this period. 309

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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310 4.2. Dominant negative LAP76A effects on growth performance in

311 zebrafish

312 Fasting and refeeding trials trigger compensatory growth, which

313 is a complex phenomenon that shows different degrees of com-
314 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, partic-
320 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 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
333 the most common phenomenon observed in fish, with several fish circle) groups. Downward arrows show the end of fasting for the FR and FRL groups.
334 species displaying this type of growth (Ali et al., 2003; Gabillard Different letters indicate a significant difference (P < 0.05). Results are expressed as
Q2 et al., 2006; Picha et al., 2006, 2008; Fox et al., 2009). On the other
335 means ± SEM (n = 4).
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 diffi-
339 cult to achieve. The only study to show this phenomenon is that assessed during fasting and refeeding in zebrafish muscle. Dur- 373

340 of Hayward et al. (1997) which showed that 1 year-old juvenile ing fasting, the SMAD signaling pathway was strongly activated 374

341 hybrid sunfish reached masses of up to twice those of controls in in the FR and FRL groups. Other TGF-␤ members can also acti- 375

342 a 105-day study. The present study shows for the first time over- vate the SMAD signaling pathway and may have strong effects 376

343 compensation of growth in a teleost species during refeeding after on skeletal muscle growth (Kollias and McDermott, 2008). The 377

344 a fasting period using a biotechnological approach such as that of SMAD signaling pathway induces muscle atrophy in mammals, 378

345 recombinant proteins. Compensatory growth has been suggested and the inhibition of this pathway promotes muscle hypertro- 379

346 as a method for optimizing body mass in fish (Jobling et al., 1994; phy (Sartori et al., 2009), thus suggesting that MSTN and other 380

347 Hayward et al., 1997; Ali et al., 2003; Jobling, 2010), which affects TGF-␤ members may be playing an important role in the muscle- 381

348 muscle mass and flesh composition (Bugeon et al., 2004; Heide induced atrophy observed in this stage for zebrafish. In fish, the 382

349 et al., 2006; Young et al., 2005). Therefore, the present results sug- PI3K/AKT/FOXO and NF␬B/I␬B␣ signaling pathways contribute to 383

350 gest that the use of recombinant proteins that transiently inhibit muscle atrophy (Fuentes et al., 2012c). The present study shows 384

351 the biological activity of MSTN will trigger overcompensation of the first evidence that suggests that the SMAD signaling pathway 385

352 growth due to an enhancement of muscle hypertrophy. For fish, is also contributing to muscle catabolism during fasting, and that 386

353 body mass is strongly related to muscle mass (Weatherley et al., this activation could be mediated by MSTN or other TGF-␤ mem- 387

354 1988) because skeletal muscle in fish comprises the largest single bers. 388

355 tissue compartment, representing up to 70% of total body mass, Interestingly, during the first two weeks of refeeding, the SMAD 389

356 a relatively larger component than that in mammals (Weatherley signaling pathway in the FR group was still activated but not in 390

357 et al., 1988). Therefore, these results highlight the fact that manipu- the FRL group, indicating that treatments with LAPD76A inhibit 391

358 lating molecules that control muscle mass in fish, such as MSTN, will MSTN actions. Multiple alignments analyses with zebrafish MSTN 392

359 directly lead to improved growth rates. Considering that zefrafish and other TGF-␤ members show no evidence for sequence conser- 393

360 display the same type of muscle growth (hypertrophy) during vation of the cleavage site at the aspartic acid residue at position 76 394

361 compensatory growth as other fish species of commercial inter- (Asp, D76) in other TGF-␤ members (data not shown). Therefore, it 395

362 est, it is likely that these fish species will respond similarly to the is plausible to suggest that the LAPD76A treatment transiently inac- 396

363 treatment. tivates just MSTN but no other TGF-␤ members, thus triggering the 397

inactivation of SMAD3. A complete scenario regarding the molec- 398

364 4.3. Dominant negative LAP76A effects on SMAD signaling ular regulation of muscle growth during refeeding in fish has been 399

365 pathway activation in zebrafish muscle previously shown (Fuentes et al., 2011, 2012a,b,c, 2013a,b; Safian 400

et al., 2012). During the first stages of refeeding (e.g. hours of refeed- 401

366 Several studies have used fasting and refeeding trials as a model ing), positive regulators of muscle growth largely exceed negative 402

367 for studying MSTN biology in fish muscle growth. However, these signals, thus laying the foundation for the subsequent promotion of 403

368 studies have only evaluated mRNA contents of mstn and have found strong, catch-up growth (Fuentes et al., 2011, 2012a,b,c, 2013a,b; 404

369 very contrasting results (Chauvigné et al., 2003; De Santis and Safian et al., 2012). Conversely, during more advanced stages of 405

370 Jerry, 2011; Terova et al., 2006; Montserrat et al., 2007; Rodgers refeeding (weeks of refeeding), positive and negative signals start 406

371 et al., 2003; Meyer et al., 2013). In this context, the main signaling to become balanced, and the majority of components returning 407

372 pathway activated by MSTN, the SMAD signal transduction, was to basal levels and reestablish homeostatic growth of the muscle 408

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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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.

409 (Fuentes et al., 2011, 2012a,b,c, 2013a,b; Safian et al., 2012). There- Funding 445

410 fore, altogether these results suggest that treatment with LAPD76A
411 recombinant proteins during the first two weeks of refeeding in This work was supported by Fondo Nacional de Desarrollo Cien- 446

412 zebrafish disturbs the balance between positive and negative sig- tífico y Tecnologico (FONDECYT) Grants 1090416 and 1130545 447

413 nals, allowing for muscle growth without important inhibitors such (to A. Molina); Universidad Andres Bello fund DI-14-11/I (to 448

Q3 as MSTN and the SMAD signaling pathway (Fig. 5).

414 EN Fuentes); and CONICYT/FONDAP/15110027 (to EN Fuentes, A 449

Molina, JA Valdes). 450

415 4.4. Conclusions and perspectives

Uncited references Q4 451

416 The events triggered by the effects of fasting, refeeding, and

417 LAPD76A treatment on the SMAD signaling pathway activation, Acosta et al. (2005) and Fuentes et al. (2013c). 452

418 muscle cellularity, and growth performance in zebrafish are sum-

419 marized in Fig. 6. The biological action of MSTN, and probably of Acknowledgements 453
420 other TGF-␤ members, during fasting is more pronounced, trigg-
421 ering SMAD signaling pathway activation in muscle and leading We thank Ashley VanCott, Editor in Chief of Cinder Services 454
422 to muscle atrophy. This directly affects growth performance by (http://cinderservices.wordpress.com/), for improving and correct- 455
423 decreasing total weight and condition factor (Fig. 6A). During ing the English of the manuscript. 456
424 the first week of refeeding, the SMAD signaling pathway is still
425 activated, indicating that the actions of MSTN, and probably of
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