Abstract
Myostatin is a potent negative regulator of muscle growth in mammals. Despite high structural conservation, functional conservation in nonmammalian species is only assumed. This is particularly true for fish due to the presence of several myostatin paralogs: two in most species and four in salmonids (MSTN-1a, -1b, -2a, and -2b). Rainbow trout are a rich source of primary myosatellite cells as hyperplastic muscle growth occurs even in adult fish. These cells were therefore used to determine myostatin's effects on proliferation whereas our earlier studies reported its effects on quiescent cells. As in mammals, recombinant myostatin suppressed proliferation with no changes in cell morphology. Expression of MSTN-1a was several fold higher than the other paralogs and was autoregulated by myostatin, which also upregulated the expression of key differentiation markers: Myf5, MyoD1, myogenin, and myosin light chain. Thus, myostatin-stimulated cellular growth inhibition activates rather than represses differentiation. IGF-1 stimulated proliferation but had minimal and delayed effects on differentiation and its actions were suppressed by myostatin. However, IGF-1 upregulated MSTN-2a expression and the processing of its transcript, which is normally unprocessed. Myostatin therefore appears to partly mediate IGF-stimulated myosatellite differentiation in rainbow trout. This also occurs in mammals, although the IGF-stimulated processing of MSTN-2a transcripts is highly unique and is indicative of subfunctionalization within the gene family. These studies also suggest that the myokine's actions, including its antagonistic relationship with IGF-1, are conserved and that the salmonid gene family is functionally diverging.
Introduction
Muscle growth results from the proliferation of myoblasts and their subsequent differentiation into muscle fibers (Buas & Kadesch 2010). This occurs with embryonic myoblasts and with myosatellite cells that lay just under the basal lamina of mature muscle. These quiescent stem cells are activated in response to exercise or injury and ultimately contribute to hypertrophic growth as they differentiate and fuse to mature fibers (Grounds & Yablonka-Reuveni 1993, Pavlath & Horsley 2003, Charge & Rudnicki 2004). Some of the underlying molecular mechanisms involved appear to be fairly well conserved in all vertebrates (Buckingham & Vincent 2009) as primary myosatellite cells from a variety of species spontaneously differentiate and respond similarly to growth factors (Thomas et al. 1994, Galvin et al. 2003, Castillo et al. 2004, Manceau et al. 2008, Bower & Johnston 2010a, Gabillard et al. 2010, Zhu et al. 2010). Unlike birds and mammals, however, where muscle hyperplasia stops after birth, both hyperplastic and hypertrophic growth occurs in fish muscle (Mommsen 2001). Indeed, bony fish are a rich source of primary myosatellite cells, and, in contrast to mammals, these cells are easily obtained from rainbow trout and at comparatively much higher titers (Alfei et al. 1989, Koumans et al. 1991a,b, 1993, Fauconneau & Paboeuf 2000, 2001, Mommsen 2001, Castillo et al. 2002, 2004, 2006). A better understanding of this unique phenomenon will, therefore, help explain myogenesis in fish. It may even help to develop a novel biomedical model assuming that the general mechanisms of myogenesis are conserved in humans and fish.
The process of proliferation and differentiation of myoblasts in general is regulated by several factors including myostatin, a negative regulator of muscle growth in mammals (Rodgers & Garikipati 2008), and insulin-like growth factor 1 (IGF-1), a positive regulator (Duan et al. 2010). Most bony fish species possess at least two myostatin genes (MSTN-1 and -2; Kerr et al. 2005, Rodgers et al. 2007) as a result of early genome duplication events (Amores et al. 1998, Postlethwait et al. 1998), while rainbow trout and other salmonids have four (MSTN-1a, -1b, -2a, and -2b) due to their recent tetraploidization (Postlethwait et al. 1998, Kerr et al. 2005, Garikipati et al. 2006, 2007). Although MSTN-2b is a pseudogene, functions of the other paralogs are presumably intact and each gene is differentially expressed in a tissue- and developmental-specific manner (Rodgers & Garikipati 2008). Direct evidence of myostatin function in fish, however, is seriously lacking. A few studies have overexpressed or attempted to disrupt the production and/or bioavailability of individual paralogs with mixed results (Xu et al. 2003, Amali et al. 2004, Rodgers & Garikipati 2008, Lee et al. 2009). This includes the generation of transgenic rainbow trout overexpressing follistatin, a myostatin binding protein, which developed hypermuscularity that resembled myostatin null phenotypes in mammals (Medeiros et al. 2009). These phenotypes likely result in part from myostatin inactivation, although they could also be explained by compensatory changes in paralog expression or to the functional inactivation of other factors (e.g. activin, growth, and differentiation factor 11) also recognized by myostatin-binding proteins (follistatin, the myostatin latency-associated peptide (LAP), etc.). The identity of the most ‘muscle relevant’ paralog is also unknown as any or all may contribute to different aspects of the myogenic process.
Using primary rainbow trout myosatellite cells, we recently determined that myostatin stimulates the differentiation of quiescent cells (Garikipati & Rodgers 2012), although it is unknown whether it can activate myogenic pathways in cells that are actively proliferating. These are distinctly different questions as myostatin has in fact been demonstrated to activate cell cycle withdrawal in mammalian myoblasts and consequently to stimulate quiescence rather than differentiation or apoptosis (McCroskery et al. 2003, Amthor et al. 2006, Manceau et al. 2008). Seiliez et al. (2012) recently reported that myostatin inhibits the proliferation of rainbow trout myosatellite cells but has no effect on differentiation. Their conclusions, however, are highly questionable as proliferation assays were surprisingly performed in differentiation medium and differentiation assays in proliferation medium (Garikipati & Rodgers 2012). Our goal, therefore, was to determine whether myostatin's anti-proliferative effects in mammals were conserved in fish and whether this resulted in quiescence or differentiation. Our results suggest that myostatin indeed inhibits proliferation and as a result activates differentiation. They further suggest that myostatin and IGF-1 antagonize one another's actions, that MSTN-1a and -2a are together the ‘muscle myostatins’, and that IGF-stimulated differentiation is likely influenced by the alternative processing and gene expression of MSTN-2a. These studies provide further evidence that the gene family has subfunctionalized in salmonids as the partitioned expression patterns support similarly partitioned roles during myogenesis. They also suggest that the current understanding of myostatin action, based primarily on mammalian cell lines, may not be entirely applicable to other vertebrate systems, or alternatively that the mammalian model is in need of significant revision.
Materials and Methods
Animal maintenance and myosatellite cell isolation
Rainbow trout were obtained from the Washington State University hatchery, a Center of Reproductive Biology core facility. Fish were reared and used according to protocols preapproved by the Institutional Animal Care and Use Committee. Myosatellite cells were isolated using a procedure previously described (Garikipati & Rodgers 2012). Briefly, fish were collected, weighed, and killed by a sharp blow to the head and immersed in 70% ethanol for 30 s. The skin was removed and the dorsal white muscle was carefully dissected and collected in cold extraction medium, DMEM (Sigma), containing 9 mM NaHCO3, 20 mM HEPES (pH 7.4), 15% horse serum (HS, Sigma), streptomycin (200 μg/ml), penicillin (200 U/ml), fungizone (25 μg/ml), and gentamicin (7 μg/ml). Muscle was weighed and cut into small pieces with scissors and centrifuged at 300 g for 15 min to remove floating pieces. It was then resuspended in 0.25% collagenase for 70 min at 18 °C before centrifuging for 15 min. The cell pellet was washed and incubated with 0.1% trypsin for 20 min at 18 °C and the resulting cell suspension was centrifuged for 1 min. The supernatant was diluted with four volumes of DMEM containing 15% HS before a second trypsin digestion for 20 min. Trypsin was removed by diluting cells with four volumes of extraction medium, centrifuging for 20 min, and resuspending in basal medium (DMEM with 10% fetal bovine serum (FBS), antibiotics, and fungizone). The suspension was then sequentially filtered through 70 and 40 μm nylon cell strainers, centrifuged, resuspended, and counted before plating. Cells isolated using this procedure have 90% purity for myosatellite cells (Koumans et al. 1990, Fauconneau & Paboeuf 2001, Gabillard et al. 2010).
Cell culture conditions
Culture dishes and plates were pretreated with 100 μg/ml poly-l-lysine (Sigma) for at least 3 h at 18 °C. Cells were then washed with sterile water and treated with 5 μg/ml laminin (Sigma) diluted in PBS overnight at 18 °C. The laminin solution was aspirated and cells were plated the following day. In all experiments, cells were incubated at 18 °C and the culture plates were sealed to prevent evaporation of the medium.
Proliferation assays
We previously determined that serum concentration can be altered to influence the proliferative capacity and cellular differentiation of primary rainbow trout myosatellite cells (Garikipati & Rodgers 2012). Compared to cells cultured in 5% FBS, differentiation is substantially delayed in cells cultured in 1% or 2% FBS, although 2% allows cells to proliferate while 1% produces quiescence. This is in contrast to immortalized muscle cell lines as primary myosatellites from a variety of vertebrates differentiate in response to high serum concentrations (Rando & Blau 1994, Thomas et al. 1994, Manceau et al. 2008, Beermann et al. 2010, Bower & Johnston 2010a, Gabillard et al. 2010, Zhu et al. 2010). In addition, rainbow trout cells spontaneously differentiate at low cell densities and high serum levels (Matschak & Stickland 1995, Castillo et al. 2004, Montserrat et al. 2007, Diaz et al. 2009, Bower & Johnston 2010a,b), which also differs from immortalized cell lines. Our experiments were therefore performed with 2% FBS as this allows cells to proliferate but not readily differentiate (Garikipati & Rodgers 2012).
Cells were seeded on laminin/poly-l-lysine 96-well plates at a density of 450 cells/well and were preincubated overnight. The medium was changed and cells were then cultured for 72 h in DMEM/2% FBS supplemented with different combinations of recombinant mouse myostatin (0, 25, or 50 nM; R&D Systems, Minneapolis, MN, USA) and/or human IGF-1 (0, 25, 50, or 100 nM; Diagnostic System Laboratories, Webster, TX, USA). The C-terminal mature myostatin peptides in mammals and fish, including each rainbow trout homolog, are highly conserved (88–100% identical) (Rodgers & Weber 2001, Rodgers & Garikipati 2008). The rainbow trout myostatin homologs also share receptor-binding domains that are conserved between mammalian myostatin and activin proteins (Harrison et al. 2003, 2004). We therefore used mouse myostatin as fish recombinants are not commercially available. Human IGF-1 is also well known to possess conserved bioactivity in rainbow trout (Castillo et al. 2002, 2004, 2006). Cultures were terminated and total cell number was determined using Cell Titer 96 (Promega) according to the manufacturer's protocol. Each experiment was repeated three times and data were compiled for analysis. For assessment of gene expression, cells were seeded on coated 12-well plates and treated with 50 nM MSTN and/or 50 nM IGF-1. They were then grown for 3 and 7 days, although culture medium and growth factors were replaced after 3 days in the latter group.
Gene expression assays
Total RNA was extracted from duplicate wells of three separate cell cultures using Trizol reagent (Invitrogen). RNA was treated with DNase (Promega) and its integrity was assessed by agarose gel electrophoresis. Total RNA (2 μg) was then reverse transcribed using 10 μM Oligo DT primers and 200 U Superscript transcriptase III (Invitrogen) in a 20 μl reaction volume. Subsequent quantitative RT-PCR assays were performed using the iCycler iQ Real-Time PCR detection system (Bio-Rad) and specific primer sets (Table 1). For each sample, 1 μl cDNA was combined with 7.5 μl 2× SYBR Green PCR master mix (Bio-Rad). For each reaction, 6 μl of this mixture were added to 9 μl primer mixture containing 450 nM each primer. The reactions were carried out as follows: 95 °C for 10 min, then 50 cycles of 95 °C for 15 s, 60 °C for 1 min, and 72 °C for 30 s, although typical threshold cycle (Ct) values were between 20 and 30 cycles. The cycling reaction was followed by a dissociation curve to verify amplification of a single product and the relative standard curve method was employed to quantify gene expression using the Q-gene method (Muller et al. 2002). For each primer set, a serial dilution of a mixed tissue cDNA was used to construct a standard curve for each assay plate. The standard curve was constructed by plotting the Ct vs the natural log of input RNA (ng). This curve was then used to calculate the relative abundance of each transcript. Sample values were then normalized to those of 18s to control for differences in RNA and cDNA loading. Each sample was run in duplicate and each plate was run in duplicate. Assays were repeated with different samples and all data are presented as normalized gene expression.
Real-time quantitative RT-PCR primer sequences. All primers were used at 60 °C
| Primer name | Sequence (5′–3′) | Reference |
|---|---|---|
| Myf5 | F: CCG ACA GCA TGG TTG ACT GT | AY751283.1a |
| R: TGC CAT AGC TTG TGT TCA TCT GA | ||
| MyoD1a | F: GTG CTT GGA TAA AAA CGT GCGC | Levesque et al. (2008) |
| R: CAA AGA AAT GCA TGT CGC | ||
| Myogenin | F: GTG GAG ATC CTG AGG AGT GC | Levesque et al. (2008) |
| R: GGT CCT CGT TGC TGT AGC TC | ||
| MLC | F: GGC CCC ATC AAC TTC AC | Vegusdal et al. (2004) |
| R: CTC CTC CTT TCC TCT CCG TG | ||
| 18s | F: TGC GGC TTA ATT TGA CTC AAC A | Garikipati et al. (2007) |
| R: CAA CTA AGA ACG GCC ATG CA | ||
| MSTN-1a | F: CTT CAC ATA TGC CAA TAC ATA TTA | Garikipati et al. (2006) |
| R: GCA ACC ATG AAA CTG AGA TAA A | ||
| MSTN-1b | F: TTC ACG CAA ATA CGT ATT CAC | Garikipati et al. (2006) |
| R: GAT AAA TTA GAA CCT GCA TCA GAT TC | ||
| MSTN-2a | F: AAT CTC CCC GCA TAA AAG CAA CCA C | Garikipati et al. (2007) |
| R: CAC CAG AAG CCA CAT CGA TCT T |
NCBI Accession number. MyoD1a amplicons were verified by sequencing.
MSTN-2a processing
For qualitative assessment of MSTN-2a transcript processing, total RNA from skeletal muscle and brain cells treated for 3 days with 50 nM MSTN, 50 nM IGF-1, or both and cells treated with 10, 20, 50, 100, and 200 nM IGF-1 for 3 and 7 days was extracted and reverse transcribed as described earlier. cDNA was then amplified using primers and conditions previously validated (Garikipati et al. 2007). The primers annealed to regions in the first and second exon and were therefore capable of detecting both spliced and unspliced transcripts.
Actin and nuclear staining
Myosatellite cells were grown on glass coverslips treated with poly-l-lysine and laminin as described earlier. Cells were washed twice with PBS, fixed in 2% paraformaldehyde for 15 min on ice, washed three times with PBS, permeabilized with 0.2% triton X-100, and washed three more times before staining. Rhodamine-conjugated phalloidin was then added at a concentration of 70 nM. Nuclei were stained with 5 μM Hoechst stain and cells were imaged using a Zeiss fluorescent microscope.
Statistical analysis
Three independent experiments were performed for proliferation and quantitative expression analysis. Differences between means were determined by a one-way ANOVA coupled to Tukey's post hoc test for mean comparisons when appropriate (P≤0.05).
Results
Myostatin and IGF-1 coregulation of myosatellite cell proliferation
IGF-1 is a potent regulator of skeletal muscle growth (Duan et al. 2010) and stimulates the proliferation of primary myosatellite cells from a variety of vertebrate species including rainbow trout (Adams 2002, Castillo et al. 2004, Codina et al. 2008, Duan et al. 2010). Our study verified these findings as IGF-1-stimulated proliferation in a dose-dependent manner (Fig. 1A) as total cell number increased 5–15% after 72 h, depending on the dose. These cells are cultured at 18 °C and grow considerably slower than mammalian muscle cell lines (e.g. rat L6 or mouse C2C12) (Castillo et al. 2002), which often doubles in <24 h. We therefore cocultured cells with myostatin and IGF-1 in order to determine whether myostatin inhibited myosatellite proliferation. As expected, basal proliferation was not significantly affected by myostatin as the cells proliferated slowly under these specific culture conditions. By contrast, 50 nM myostatin completely attenuated the proliferative effects of 50 nM IGF-1 (Fig. 1A). Furthermore, cells were inspected daily and no phenotypic evidence of cell death or apoptosis (e.g. rounded or floating cells) was detected. Fibroblast contamination is always a concern when culturing primary myosatellite cells and is consistently estimated to be 10% or less using our protocol. The changes described, however, are not due to contamination as myostatin stimulates rather than inhibits fibroblast proliferation (Zhu et al. 2007, Li et al. 2008).
Myostatin and IGF-1 differentially regulate myosatellite cell proliferation. Primary myosatellite cells were cultured for 72 h in DMEM/2% FBS and with the indicated combinations and concentrations (nM) of recombinant mouse myostatin (MSTN) and IGF-1. (A) Proliferation was quantified by measuring total cell number (n=24, mean±s.e.m.; Different letters signify statistical differences) as described in Materials and Methods section and (B) cellular morphology was observed after 72 h at 100× magnification.
Citation: Journal of Endocrinology 215, 1; 10.1530/JOE-12-0260
Cell morphology
A subjective assessment of myosatellite cells treated with myostatin or IGF-1 suggested that both groups were similar in appearance despite the fact that there were more cells in the IGF-treated group (Fig. 1B). To investigate further, cells were cultured on coverslips and treated with 50 nM myostatin and/or 50 nM IGF-1 for 3 and 7 days before staining for actin, a marker of myosatellite differentiation (Fig. 2). No actin staining was observed after 3 days in control and IGF-treated cells (Fig. 2A) and only light staining was detected in cells treated with myostatin alone or in combination with IGF-1. After 7 days, however, actin staining was detected in control cells and in cells treated with myostatin alone or with myostatin and IGF-1, but not in those treated with IGF-1 alone (Fig. 2B). These results suggest that IGF-1 stimulates proliferation and thereby prevents differentiation, at least under these culture conditions, and that myostatin has the opposite effect while again attenuating IGF-1.
Myostatin and IGF-1 regulation of terminal differentiation in proliferating myosatellite cells. Primary myosatellite cells were cultured for 72 h in DMEM/2% FBS and with the indicated combinations of 50 nM recombinant mouse myostatin (MSTN) and IGF-1. Cells were fixed and filamentous actin (red) was labeled with rhodamine-conjugated phalloidin and nuclei (blue) with Hoechst DNA stain after 3 (A) or 7 days (B). Representative images are shown. Note positive actin staining of muscle debris from isolation procedure, which serves as a positive staining control in light of faint or no staining in panel A.
Citation: Journal of Endocrinology 215, 1; 10.1530/JOE-12-0260
Expression of differentiation markers
To quantify the effects of myostatin and IGF-1 on the spontaneous differentiation of proliferating myosatellite cells, which is known to occur in fish primary cells (Matschak & Stickland 1995, Rescan et al. 1995, Vegusdal et al. 2004, Montserrat et al. 2007, Diaz et al. 2009, Bower & Johnston 2010a, Bower et al. 2010, Gabillard et al. 2010), the expression of markers for early (Myf5 and MyoD1), mid (myogenin), and late (MLC) differentiation was measured (Perry & Rudnick 2000, Rudnicki et al. 2008). Differences in individual marker expression were noted, although for the most part, the patterns were similar for all markers. For example, myostatin significantly increased the expression of Myf5, MyoD, and myogenin after 3 and 7 days of treatment (Fig. 3A, B and C). By contrast, IGF-1 had only minimal effects as their expression was elevated after 7, but not 3 days. This suggests that although myostatin and IGF-1 both stimulate differentiation, the effects of myostatin are more significant, which was also reflected in phalloidin staining of actin (Fig. 2B). In fact, MLC expression was only elevated in cells stimulated by myostatin and this occurred at both time points (Fig. 3D). Myostatin and IGF-1 antagonism was probably best illustrated by the fact that their independent effects were compromised when cells were cultured with both hormones. Early marker expression was only elevated after 7 days, as with IGF-treated cells, and MLC expression was elevated, albeit slightly, after 3 days, as with myostatin-treated cells.
Myostatin and IGF-1 regulation of differentiation marker expression in proliferating myosatellite cells. Primary myosatellite cells were cultured for 72 h in DMEM/2% FBS and with the indicated combinations of 50 nM recombinant mouse myostatin (MSTN) and IGF-1. Cells were terminated after 3 (white bars) and 7 (hatched bars) days and RNA was extracted for gene expression analysis. Expression of markers for early (Myf5 and MyoD1), mid (myogenin), and late (MLC) stages of differentiation was then measured by quantitative RT-PCR. Treatments with different superscripts are statistically different (P<0.05). Same letters denote no statistical difference (n=9, mean±s.e.m.).
Citation: Journal of Endocrinology 215, 1; 10.1530/JOE-12-0260
Autoregulation of myostatin gene expression
Although MSTN-2b is a pseudogene, the other three paralogs are expressed in several tissues, including skeletal muscle, to varying degrees (Rodgers & Garikipati 2008). It is therefore unknown whether any of the paralogs individually or together regulate myogenesis. Of the three active paralogs, MSTN-1a is the most highly expressed in skeletal muscle (Garikipati et al. 2006) and in myosatellite cells (Fig. 4A and D). It is therefore the most likely candidate ‘muscle myostatin’. Myostatin increased MSTN-1a expression after 3 and 7 days and MSTN-2a expression after 7 days but had no effect on MSTN-1b (Fig. 4A, B and C). Changes in MSTN-2a expression were somewhat unexpected, as although we previously demonstrated MSTN-2a expression in these cells, it occurs primarily in the brain and its transcript is rarely processed to maturity (see below) in any other tissue (Garikipati et al. 2007). The effects of IGF-1 were equally surprising as it reduced MSTN-1a (day 7) and -1b (day 3) and increased MSTN-2a expression (days 3 and 7; Fig. 4B and C). Assuming its transcript is properly processed (see below), these results suggest that MSTN-2a specifically mediates IGF-1 action. These data also indicate that myostatin and IGF-1 differentially regulate expression of the individual paralogs, although both hormones upregulate at least one (Fig. 4D). Co-stimulating cells with both myostatin and IGF-1 produced patterns similar to those of IGF-1, except for MSTN-1a where its 7-day expression value was between those of the myostatin- and IGF-stimulated values (Fig. 4A). This once again illustrates the antagonistic relationship between myostatin and IGF-1 as both can attenuate each other's actions.
Myostatin and IGF-1 differentially regulate MSTN-1a, -1b, and -2a expression in proliferating myosatellite cells. Primary myosatellite cells were cultured for 72 h in DMEM/2% FBS and with the indicated combinations of 50 nM recombinant mouse myostatin (MSTN) and IGF-1. (A, B, and C) Cells were terminated after 3 (white bars) and 7 (hatched bars) days and RNA was extracted for gene expression analysis. Expression of each paralog was then measured by quantitative RT-PCR. Treatments with different superscripts are statistically different (P<0.05). Same letters denote no statistical difference. (D) Day 3 values for each paralog are plotted to facilitate the relative comparison of changes between MSTN-1a (white bars), 1b (hatched bars), and 2a (black bars). (n=9, mean±s.e.m.).
Citation: Journal of Endocrinology 215, 1; 10.1530/JOE-12-0260
Spliced variation of MSTN-2a transcripts
We previously reported that the alternative and tissue-specific processing of MSTN-2a transcripts contributes to the gene's subfunctionalization, as full transcript processing is limited to the brain (Garikipati et al. 2007). Thus, the IGF-induced changes in MSTN-2a mRNA are presumably insignificant as it would not result in increased MSTN-2a protein. We therefore performed qualitative RT-PCR with intron-spanning primers to determine whether the MSTN-2a transcript was processed. As shown in Fig. 5A, MSTN-2a is expressed as an unspliced transcript in skeletal muscle as it retains both introns (band shown represents intron 1 inclusion), although it is fully processed in the brain. Basal expression of MSTN-2a was below detection limits in control cells and in those treated with myostatin. However, IGF-1 not only stimulated MSTN-2a expression (Figs 4C and 5A, B and C), but also the processing of mature transcripts. This effect was dose dependent after 3 and 7 days of culture (Fig. 5B and C) and was blocked by myostatin (Fig. 5A) but never resulted in complete processing of MSTN-2a transcripts.
Endocrine regulation of MSTN-2a transcript processing in proliferating myosatellite cells. (A) MSTN-2a transcripts were amplified using a nonquantitative RT-PCR assay and RNA extracted from brain (BR), skeletal muscle (SM) or proliferating myosatellite cells. The latter were treated with IGF-1 alone (I) or with myostatin and IGF-1 (M+I, 50 nM for all doses; UT, untreated control; C, RT-control). Cells were also treated with only IGF-1 for 3 (B) or 7 days (C) using the indicated doses. Larger molecular mass bands correspond to unspliced transcripts, smaller bands to spliced.
Citation: Journal of Endocrinology 215, 1; 10.1530/JOE-12-0260
Discussion
We recently demonstrated, for the first time, direct evidence for myostatin action in any fish system (Garikipati & Rodgers 2012). These studies determined that myostatin activates differentiation in quiescent rainbow trout myosatellite cells and that the expression of MSTN-1a, in particular, and that of MSTN-1b and -2a increases with differentiation (serum and myostatin stimulated), although MSTN-2a transcripts remain unprocessed. The current study indicates that myostatin suppresses the proliferation of these cells and consequently initiates differentiation. They further suggest that although IGF-1 also stimulates proliferation and differentiation, the latter effects are likely mediated by the upregulation and endocrine-controlled alternative processing of MSTN-2a. Activating differentiation, either by serum or IGF-1, therefore appears to be independently regulated by different myostatin paralogs and by different cellular processes (i.e. gene expression and transcript processing).
Recent studies suggest that myostatin may indirectly regulate fish muscle growth as it does in mammals (Medeiros et al. 2009, Lee et al. 2010, Li et al. 2011). Muscle mass and fiber number were increased in transgenic rainbow trout overexpressing follistatin (Medeiros et al. 2009) and in transgenic medaka (Sawatari et al. 2010) overexpressing a MSTN-1 LAP (a.k.a. prodomain) respectively. Both follistatin and LAP are myostatin binding proteins that sequester and inactivate the myokine. However, these phenotypes could be partly due to the sequestration of activin and/or GDF11 as both also bind follistatin and possibly LAP (Rodgers & Garikipati 2008). Nevertheless, these reports and our current findings together indicate that myostatin is indeed a negative regulator of skeletal muscle growth in fish.
Myostatin's growth inhibitory actions are well documented in mammalian systems and include cell cycle arrest of myoblasts and primary myosatellite cells (Joulia et al. 2003, McCroskery et al. 2003, Rios et al. 2004, Rodgers et al. 2009). Myostatin has also been reported to downregulate MyoD, Myf5, and myogenin and thereby inhibit muscle cell differentiation (Amthor et al. 2002, 2004, Langley et al. 2002, Rios et al. 2002, Joulia et al. 2003), although the significance of this effect is somewhat controversial as cell cycle arrest is necessary for the initiation of differentiation (Thomas et al. 2000, Rios et al. 2001). Our data directly conflict with these reports and suggest that myostatin increases the expression of several differentiation markers and may even facilitate IGF-stimulated differentiation (see below). Immortalized mammalian cell lines were used in the previous studies and differentiation was therefore ‘artificially’ induced in confluent cells by serum withdrawal. Thus, myostatin did not inhibit differentiation per se, but progression through the already activated process. By contrast, our data indicate that myostatin activates differentiation in proliferating cells and may therefore be more physiologically relevant.
Using primary rainbow trout myosatellite cells, Seiliez et al. (2012) recently reported that myostatin inhibits proliferation, although they additionally reported that the differentiation of cells was unaffected by myostatin. Their culture conditions, however, were inappropriate and were based on those used with mammalian cell lines rather than primary myosatellites. Differentiation is induced in immortalized cell lines by serum withdrawal and only after cells are confluent, whereas primary myosatellite cells from a variety of vertebrates, including rainbow trout, will spontaneously differentiate when subconfluent and in response to serum (Rando & Blau 1994, Thomas et al. 1994, Matschak & Stickland 1995, Castillo et al. 2004, Montserrat et al. 2007, Manceau et al. 2008, Diaz et al. 2009, Beermann et al. 2010, Bower & Johnston 2010a,b, Gabillard et al. 2010, Zhu et al. 2010). In fact, our recent studies clearly demonstrate this positive effect of serum on rainbow trout myosatellite cell differentiation in vitro (Garikipati & Rodgers 2012).
By contrast, Seiliez et al. performed proliferation assays in high serum, which promotes differentiation, and differentiation assays in low serum, which suppresses differentiation. They also report that MSTN-1a and -1b expression is downregulated (MSTN-2a expression and processing was not measured) as cells differentiate, which again conflicts with data presented herein and previously (Garikipati & Rodgers 2012). Keeping in mind that their culture conditions are reversed (proliferation medium is actually differentiation medium), their results are consistent with ours and support the conclusion that myostatin facilitates myosatellite cell differentiation. Myostatin also failed to increase MyoD or myogenin expression in their studies, although they cultured cells for only 48 h and our results again clearly indicate that 3–7 days are required to detect myostatin's stimulatory effects on differentiation (Fig. 3). It is important to note that our current and previous studies are consistent with Sato et al. (2006) who demonstrated that disrupting myostatin production in proliferating primary chick cells delays rather than stimulates differentiation. Describing myostatin's actions as purely inhibitory may therefore be overly simplistic, as it appears to stimulate differentiation in proliferating primary myosatellite cells and in growth arrested cells. In fact, trout myosatellite cells were far more sensitive to myostatin than IGF-1, a known muscle differentiation factor, as differentiation marker expression increased at day 3 in cells treated with myostatin and at day 7 in IGF-treated cells (Figs 2 and 3).
Muscle growth in mammals is largely regulated by the opposing effects of IGF-1 and myostatin, which together stimulate and inhibit different myogenic processes respectively. This includes the regulation of myoblast and myosatellite cell proliferation and differentiation as well as myotube protein synthesis (Rodgers & Garikipati 2008, Duan et al. 2010). This yin–yang relationship extends intracellularly as myostatin attenuates IGF receptor signaling in both myoblasts and myotubes (McFarlane et al. 2006, Morissette et al. 2009, Trendelenburg et al. 2009). We recently demonstrated that hepatic production of IGF-1 and several IGF-binding proteins is altered in myostatin null mice in a way that increases total and free IGF-1 (Williams et al. 2011). The two hormones, therefore, oppose one another's actions and optimize muscle growth under different physiological conditions, including stress (Schakman et al. 2008). This relationship appears extremely well conserved, as in the current study, myostatin inhibited IGF-stimulated myosatellite cell proliferation and MSTN-2a processing. Similarly, IGF-1 inhibited myostatin-stimulated differentiation of myosatellite cells and MSTN-1a expression.
Myostatin autoregulation of expression was demonstrated by its ability to increase MSTN-1a mRNA levels at 3 and 7 days (Fig. 4). This also occurs in mammals (Forbes et al. 2006) and may provide a potential mechanism for synchronizing cell populations through the paracrine actions of myostatin. The expression of other paralogs was unaffected by myostatin while basal expression of MSTN-1a was several fold higher, a pattern that mirrors mature rainbow trout skeletal muscle (Garikipati et al. 2006, 2007). This suggests that MSTN-1a is the ‘muscle myostatin’, although it is by no means the only paralog involved in the myogenic process as both MSTN-2a expression and transcript processing were stimulated by IGF-1 (Figs 4 and 5), a known mitogen in rainbow trout myosatellite cells (Castillo et al. 2004, 2006, Codina et al. 2008, Gabillard et al. 2010). This effect was dose dependent and is the first example of endocrine-regulated alternative processing of myostatin in any vertebrate. Together, these data also suggest that endocrine-regulated differentiation of proliferating myosatellite cells is uniquely regulated by different myostatin paralogs; MSTN-1a for autoregulation and MSTN-2a for IGF-stimulated differentiation.
IGF-1 also stimulates myostatin expression in mammalian skeletal and cardiac muscle and is a proposed chalone for both tissues (Shyu et al. 2005, Yang et al. 2007, Rodgers et al. 2009). It is not surprising, therefore, that IGF-1 similarly upregulates the expression of at least one trout MSTN paralog. However, IGF-1 also downregulated MSTN-1b expression, indicating that it differentially regulates the production of each paralog. Although the physiological significance of this is currently unknown, the high level of conservation shared between each rainbow trout homolog suggests that functional specification is determined by differences in gene expression, not protein structure, as MSTN-1a and -1b are identical and are 96% similar to MSTN-2a. Nevertheless, each paralog's role in regulating myosatellite cell proliferation and differentiation, in rainbow trout and other salmonids, has clearly subfunctionalized via mechanisms that include divergence of gene promoters and transcript processing sites.
These studies together indicate that the general understanding of myostatin action in immortalized mammalian cell lines may not reflect its actions in primary cells, especially those from fish. Specifically, they suggest that myostatin inhibition of proliferation activates, rather than inhibits, differentiation and that subfunctionalization of the salmonid myostatin gene family helped preserve these gene duplicates. The system itself is therefore similar to zebra fish, as despite widespread gene duplication, which also occurs in zebra fish, the phenotypic responses of primary trout myosatellites are similar to those from mammals, although both differ considerably from immortalized mammalian cell lines. Future studies are needed to determine the functional significance of MSTN-2a processing in differentiating cells. Nevertheless, this represents a novel and powerful tool for investigating not only myostatin's comparative actions but also fundamental networks that control transcript processing.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
These studies were supported by a grant from the National Science Foundation (0840644) to B D R.
References
Adams GR 2002 Invited review: autocrine/paracrine IGF-I and skeletal muscle adaptation. Journal of Applied Physiology 93 1159–1167. (doi:10.1152/japplphysiol.01264.2001)
Alfei L, Maggi F, Parvopassu F, Bertoncello G & De Vita R 1989 Postlarval muscle growth in fish: a DNA flow cytometric and morphometric analysis. Basic and Applied Histochemistry 33 147–158.
Amali AA, Lin CJ, Chen YH, Wang WL, Gong HY, Lee CY, Ko YL, Lu JK, Her GM & Chen TT et al. 2004 Up-regulation of muscle-specific transcription factors during embryonic somitogenesis of zebrafish (Danio rerio) by knock-down of myostatin-1. Developmental Dynamics 229 847–856. (doi:10.1002/dvdy.10454)
Amores A, Force A, Yan YL, Joly L, Amemiya C, Fritz A, Ho RK, Langeland J, Prince V & Wang YL et al. 1998 Zebrafish hox clusters and vertebrate genome evolution. Science 282 1711–1714. (doi:10.1126/science.282.5394.1711)
Amthor H, Huang R, McKinnell I, Christ B, Kambadur R, Sharma M & Patel K 2002 The regulation and action of myostatin as a negative regulator of muscle development during avian embryogenesis. Developmental Biology 251 241–257. (doi:10.1006/dbio.2002.0812)
Amthor H, Nicholas G, McKinnell I, Kemp CF, Sharma M, Kambadur R & Patel K 2004 Follistatin complexes Myostatin and antagonises Myostatin-mediated inhibition of myogenesis. Developmental Biology 270 19–30. (doi:10.1016/j.ydbio.2004.01.046)
Amthor H, Otto A, Macharia R, McKinnell I & Patel K 2006 Myostatin imposes reversible quiescence on embryonic muscle precursors. Developmental Dynamics 235 672–680. (doi:10.1002/dvdy.20680)
Beermann ML, Ardelt M, Girgenrath M & Miller JB 2010 Prdm1 (Blimp-1) and the expression of fast and slow myosin heavy chain isoforms during avian myogenesis in vitro. PLoS ONE 5 e9951 doi:10.1371/journal.pone.0009951)
Bower NI & Johnston IA 2010a Paralogs of Atlantic salmon myoblast determination factor genes are distinctly regulated in proliferating and differentiating myogenic cells. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 298 R1615–R1626. (doi:10.1152/ajpregu.00114.2010)
Bower NI & Johnston IA 2010b Transcriptional regulation of the IGF signaling pathway by amino acids and insulin-like growth factors during myogenesis in Atlantic salmon. PLoS ONE 5 e11100 doi:10.1371/journal.pone.0011100)
Bower NI, de la Serrana DG & Johnston IA 2010 Characterisation and differential regulation of MAFbx/Atrogin-1α and β transcripts in skeletal muscle of Atlantic salmon (Salmo salar). Biochemical and Biophysical Research Communications 396 265–271. (doi:10.1016/j.bbrc.2010.04.076)
Buas MF & Kadesch T 2010 Regulation of skeletal myogenesis by Notch. Experimental Cell Research 316 3028–3033. (doi:10.1016/j.yexcr.2010.05.002)
Buckingham M & Vincent SD 2009 Distinct and dynamic myogenic populations in the vertebrate embryo. Current Opinion in Genetics & Development 19 444–453. (doi:10.1016/j.gde.2009.08.001)
Castillo J, Le Bail PY, Paboeuf G, Navarro I, Weil C, Fauconneau B & Gutierrez J 2002 IGF-I binding in primary culture of muscle cells of rainbow trout: changes during in vitro development. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 283 R647–R652. (doi:10.1152/ajpregu.00121.2002)
Castillo J, Codina M, Martinez ML, Navarro I & Gutierrez J 2004 Metabolic and mitogenic effects of IGF-I and insulin on muscle cells of rainbow trout. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 286 R935–R941. (doi:10.1152/ajpregu.00459.2003)
Castillo J, Ammendrup-Johnsen I, Codina M, Navarro I & Gutierrez J 2006 IGF-I and insulin receptor signal transduction in trout muscle cells. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 290 R1683–R1690. (doi:10.1152/ajpregu.00294.2005)
Charge SB & Rudnicki MA 2004 Cellular and molecular regulation of muscle regeneration. Physiological Reviews 84 209–238. (doi:10.1152/physrev.00019.2003)
Codina M, Garcia de la serrana D, Sanchez-Gurmaches J, Montserrat N, Chistyakova O, Navarro I & Gutierrez J 2008 Metabolic and mitogenic effects of IGF-II in rainbow trout (Oncorhynchus mykiss) myocytes in culture and the role of IGF-II in the PI3K/Akt and MAPK signalling pathways. General and Comparative Endocrinology 157 116–124. (doi:10.1016/j.ygcen.2008.04.009)
Diaz M, Vraskou Y, Gutierrez J & Planas JV 2009 Expression of rainbow trout glucose transporters GLUT1 and GLUT4 during in vitro muscle cell differentiation and regulation by insulin and IGF-I. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 296 R794–R800. (doi:10.1152/ajpregu.90673.2008)
Duan C, Ren H & Gao S 2010 Insulin-like growth factors (IGFs), IGF receptors, and IGF-binding proteins: roles in skeletal muscle growth and differentiation. General and Comparative Endocrinology 167 344–351. (doi:10.1016/j.ygcen.2010.04.009)
Fauconneau B & Paboeuf G 2000 Effect of fasting and refeeding on in vitro muscle cell proliferation in rainbow trout (Oncorhynchus mykiss). Cell and Tissue Research 301 459–463. (doi:10.1007/s004419900168)
Fauconneau B & Paboeuf G 2001 Sensitivity of muscle satellite cells to pollutants: an in vitro and in vivo comparative approach. Aquatic Toxicology 53 247–263. (doi:10.1016/S0166-445X(01)00170-9)
Forbes D, Jackman M, Bishop A, Thomas M, Kambadur R & Sharma M 2006 Myostatin auto-regulates its expression by feedback loop through Smad7 dependent mechanism. Journal of Cellular Physiology 206 264–272. (doi:10.1002/jcp.20477)
Gabillard JC, Sabin N & Paboeuf G 2010 In vitro characterization of proliferation and differentiation of trout satellite cells. Cell and Tissue Research 342 471–477. (doi:10.1007/s00441-010-1071-8)
Galvin CD, Hardiman O & Nolan CM 2003 IGF-1 receptor mediates differentiation of primary cultures of mouse skeletal myoblasts. Molecular and Cellular Endocrinology 200 19–29. (doi:10.1016/S0303-7207(02)00420-3)
Garikipati DK & Rodgers BD 2012 Myostatin stimulates myosatellite cell differentiation in a novel model system: evidence for gene subfunctionalization. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 302 R1059–R1066. (doi:10.1152/ajpregu.00523.2011)
Garikipati DK, Gahr SA & Rodgers BD 2006 Identification, characterization, and quantitative expression analysis of rainbow trout myostatin-1a and myostatin-1b genes. Journal of Endocrinology 190 879–888. (doi:10.1677/joe.1.06866)
Garikipati DK, Gahr SA, Roalson EH & Rodgers BD 2007 Characterization of rainbow trout myostatin-2 genes (rtMSTN-2a and -2b): genomic organization, differential expression, and pseudogenization. Endocrinology 148 2106–2115. (doi:10.1210/en.2006-1299)
Grounds MD & Yablonka-Reuveni Z 1993 Molecular and cell biology of skeletal muscle regeneration. Molecular and Cell Biology of Human Diseases Series 3 210–256.
Harrison CA, Gray PC, Koerber SC, Fischer W & Vale W 2003 Identification of a functional binding site for activin on the type I receptor ALK4. Journal of Biological Chemistry 278 21129–21135. (doi:10.1074/jbc.M302015200)
Harrison CA, Gray PC, Fischer WH, Donaldson C, Choe S & Vale W 2004 An activin mutant with disrupted ALK4 binding blocks signaling via type II receptors. Journal of Biological Chemistry 279 28036–28044. (doi:10.1074/jbc.M402782200)
Joulia D, Bernardi H, Garandel V, Rabenoelina F, Vernus B & Cabello G 2003 Mechanisms involved in the inhibition of myoblast proliferation and differentiation by myostatin. Experimental Cell Research 286 263–275. (doi:10.1016/S0014-4827(03)00074-0)
Kerr T, Roalson EH & Rodgers BD 2005 Phylogenetic analysis of the myostatin gene sub-family and the differential expression of a novel member in zebrafish. Evolution & Development 7 390–400. (doi:10.1111/j.1525-142X.2005.05044.x)
Koumans JTM, Akster HA, Dulos GJ & Osse JWM 1990 Myosatellite cells of Cyprinus carpio (teleostei) in vitro – isolation, recognition and differentiation. Cell and Tissue Research 261 173–181. (doi:10.1007/BF00329450)
Koumans JT, Akster HA, Booms GH, Lemmens CJ & Osse JW 1991a Numbers of myosatellite cells in white axial muscle of growing fish: Cyprinus carpio L. (teleostei). American Journal of Anatomy 192 418–424. (doi:10.1002/aja.1001920409)
Koumans JTM, Akster HA, Booms GHR, Lemmens CJJ & Osse JWM 1991b Numbers of myosatellite cells in white axial muscle of growing fish (Cyprinus carpio L.). American Journal of Anatomy 192 418–424. (doi:10.1002/aja.1001920409)
Koumans JTM, Akster HA, Booms GHR & Osse JWM 1993 Growth of carp (Cyprinus carpio) white axial muscle – hyperplasia and hypertrophy in relation to the myonucleus sarcoplasm ratio and the occurrence of different subclasses of myogenic cells. Journal of Fish Biology 43 69–80. (doi:10.1111/j.1095-8649.1993.tb00411.x)
Langley B, Thomas M, Bishop A, Sharma M, Gilmour S & Kambadur R 2002 Myostatin inhibits myoblast differentiation by down regulating MyoD expression. Journal of Biological Chemistry 277 49831–49840. (doi:10.1074/jbc.M204291200)
Lee CY, Hu SY, Gong HY, Chen MH, Lu JK & Wu JL 2009 Suppression of myostatin with vector-based RNA interference causes a double-muscle effect in transgenic zebrafish. Biochemical and Biophysical Research Communications 387 766–771. (doi:10.1016/j.bbrc.2009.07.110)
Lee C, Safdie FM, Raffaghello L, Wei M, Madia F, Parrella E, Hwang D, Cohen P, Bianchi G & Longo VD 2010 Reduced levels of IGF-I mediate differential protection of normal and cancer cells in response to fasting and improve chemotherapeutic index. Cancer Research 70 1564–1572. (doi:10.1158/0008-5472.CAN-09-3228)
Levesque HM, Shears MA, Fletcher GL & Moon TW 2008 Myogenesis and muscle metabolism in juvenile Atlantic salmon (Salmo salar) made transgenic for growth hormone. Journal of Experimental Biology 211 128–137. (doi:10.1242/jeb.006890)
Li ZB, Kollias HD & Wagner KR 2008 Myostatin directly regulates skeletal muscle fibrosis. Journal of Biological Chemistry 283 19371–19378. (doi:10.1074/jbc.M802585200)
Li X, Nie F, Yin Z & He J 2011 Enhanced hyperplasia in muscles of transgenic zebrafish expressing Follistatin1. Science China. Life Sciences 54 159–165. (doi:10.1007/s11427-010-4121-2)
Manceau M, Gros J, Savage K, Thome V, McPherron A, Paterson B & Marcelle C 2008 Myostatin promotes the terminal differentiation of embryonic muscle progenitors. Genes and Development 22 668–681. (doi:10.1101/gad.454408)
Matschak TW & Stickland NC 1995 The growth of Atlantic salmon (Salmo salar L.) myosatellite cells in culture at two different temperatures. Experientia 51 260–266. (doi:10.1007/BF01931109)
McCroskery S, Thomas M, Maxwell L, Sharma M & Kambadur R 2003 Myostatin negatively regulates satellite cell activation and self-renewal. Journal of Cell Biology 162 1135–1147. (doi:10.1083/jcb.200207056)
McFarlane C, Plummer E, Thomas M, Hennebry A, Ashby M, Ling N, Smith H, Sharma M & Kambadur R 2006 Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-kappaB-independent, FoxO1-dependent mechanism. Journal of Cellular Physiology 209 501–514. (doi:10.1002/jcp.20757)
Medeiros EF, Phelps MP, Fuentes FD & Bradley TM 2009 Overexpression of follistatin in trout stimulates increased muscling. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 297 R235–R242. (doi:10.1152/ajpregu.91020.2008)
Mommsen TP 2001 Paradigms of growth in fish. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology 129 207–219. (doi:10.1016/S1096-4959(01)00312-8)
Montserrat N, Gabillard JC, Capilla E, Navarro MI & Gutierrez J 2007 Role of insulin, insulin-like growth factors, and muscle regulatory factors in the compensatory growth of the trout (Oncorhynchus mykiss). General and Comparative Endocrinology 150 462–472. (doi:10.1016/j.ygcen.2006.11.009)
Morissette MR, Cook SA, Buranasombati C, Rosenberg MA & Rosenzweig A 2009 Myostatin inhibits IGF-I-induced myotube hypertrophy through Akt. American Journal of Physiology. Cell Physiology 297 C1124–C1132. (doi:10.1152/ajpcell.00043.2009)
Muller PY, Janovjak H, Miserez AR & Dobbie Z 2002 Processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques 32 1372–1374, 1376, 1378–1379.
Pavlath GK & Horsley V 2003 Cell fusion in skeletal muscle – central role of NFATC2 in regulating muscle cell size. Cell Cycle 2 420–423. (doi:10.4161/cc.2.5.497)
Perry RL & Rudnick MA 2000 Molecular mechanisms regulating myogenic determination and differentiation. Frontiers in Bioscience 5 D750–D767. (doi:10.2741/Perry)
Postlethwait JH, Yan YL, Gates MA, Horne S, Amores A, Brownlie A, Donovan A, Egan ES, Force A & Gong Z et al. 1998 Vertebrate genome evolution and the zebrafish gene map. Nature Genetics 18 345–349. (doi:10.1038/ng0498-345)
Rando TA & Blau HM 1994 Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. Journal of Cell Biology 125 1275–1287. (doi:10.1083/jcb.125.6.1275)
Rescan PY, Gauvry L & Paboeuf G 1995 A gene with homology to myogenin is expressed in developing myotomal musculature of the rainbow trout and in vitro during the conversion of myosatellite cells to myotubes. FEBS Letters 362 89–92. (doi:10.1016/0014-5793(95)00215-U)
Rios R, Carneiro I, Arce VM & Devesa J 2001 Myostatin regulates cell survival during C2C12 myogenesis. Biochemical and Biophysical Research Communications 280 561–566. (doi:10.1006/bbrc.2000.4159)
Rios R, Carneiro I, Arce VM & Devesa J 2002 Myostatin is an inhibitor of myogenic differentiation. American Journal of Physiology. Cell Physiology 282 C993–C999. (doi:10.1152/ajpcell.000372.2001)
Rios R, Fernandez-Nocelos S, Carneiro I, Arce VM & Devesa J 2004 Differential response to exogenous and endogenous myostatin in myoblasts suggests that myostatin acts as an autocrine factor in vivo. Endocrinology 145 2795–2803. (doi:10.1210/en.2003-1166)
Rodgers BD & Garikipati DK 2008 Clinical, agricultural, and evolutionary biology of myostatin: a comparative review. Endocrine Reviews 29 513–534. (doi:10.1210/er.2008-0003)
Rodgers BD & Weber GM 2001 Sequence conservation among fish myostatin orthologues and the characterization of two additional cDNA clones from Morone saxatilis and Morone americana. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology 129 597–603. (doi:10.1016/S1096-4959(01)00350-5)
Rodgers BD, Roalson EH, Weber GM, Roberts SB & Goetz FW 2007 A proposed nomenclature consensus for the myostatin gene family. American Journal of Physiology. Endocrinology and Metabolism 292 E371–E372. (doi:10.1152/ajpendo.00395.2006)
Rodgers BD, Interlichia JP, Garikipati DK, Mamidi R, Chandra M, Nelson OL, Murry CE & Santana LF 2009 Myostatin represses physiological hypertrophy of the heart and excitation–contraction coupling. Journal of Physiology 587 4873–4886. (doi:10.1113/jphysiol.2009.172544)
Rudnicki MA, Le Grand F, McKinnell I & Kuang S 2008 The molecular regulation of muscle stem cell function. Cold Spring Harbor Symposia on Quantitative Biology 73 323–331. (doi:10.1101/sqb.2008.73.064)
Sato F, Kurokawa M, Yamauchi N & Hattori MA 2006 Gene silencing of myostatin in differentiation of chicken embryonic myoblasts by small interfering RNA. American Journal of Physiology. Cell Physiology 291 C538–C545. (doi:10.1152/ajpcell.00543.2005)
Sawatari E, Seki R, Adachi T, Hashimoto H, Uji S, Wakamatsu Y, Nakata T & Kinoshita M 2010 Overexpression of the dominant-negative form of myostatin results in doubling of muscle-fiber number in transgenic medaka (Oryzias latipes). Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology 155 183–189. (doi:10.1016/j.cbpa.2009.10.030)
Schakman O, Gilson H & Thissen JP 2008 Mechanisms of glucocorticoid-induced myopathy. Journal of Endocrinology 197 1–10. (doi:10.1677/JOE-07-0606)
Seiliez I, Sabin N & Gabillard JC 2012 Myostatin inhibits proliferation but not differentiation of trout myoblasts. Molecular and Cellular Endocrinology 351 220–226. (doi:10.1016/j.mce.2011.12.011)
Shyu KG, Ko WH, Yang WS, Wang BW & Kuan P 2005 Insulin-like growth factor-1 mediates stretch-induced upregulation of myostatin expression in neonatal rat cardiomyocytes. Cardiovascular Research 68 405–414. (doi:10.1016/j.cardiores.2005.06.028)
Thomas D, Lehmann S, Kuchler S, Marschal P & Zanetta JP 1994 Differential expression of an endogenous mannose-binding protein R1 during muscle development and regeneration delineating its role in myoblast fusion. Glycobiology 4 23–38. (doi:10.1093/glycob/4.1.23)
Thomas M, Langley B, Berry C, Sharma M, Kirk S, Bass J & Kambadur R 2000 Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. Journal of Biological Chemistry 275 40235–40243. (doi:10.1074/jbc.M004356200)
Trendelenburg AU, Meyer A, Rohner D, Boyle J, Hatakeyama S & Glass DJ 2009 Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size. American Journal of Physiology. Cell Physiology 296 C1258–C1270. (doi:10.1152/ajpcell.00105.2009)
Vegusdal A, Ostbye TK, Tran TN, Gjoen T & Ruyter B 2004 β-Oxidation, esterification, and secretion of radiolabeled fatty acids in cultivated Atlantic salmon skeletal muscle cells. Lipids 39 649–658. (doi:10.1007/s11745-004-1278-3)
Williams NG, Interlichia JP, Jackson MF, Hwang D, Cohen P & Rodgers BD 2011 Endocrine actions of myostatin: systemic regulation of the IGF and IGF binding protein axis. Endocrinology 152 172–180. (doi:10.1210/en.2010-0488)
Xu C, Wu G, Zohar Y & Du SJ 2003 Analysis of myostatin gene structure, expression and function in zebrafish. Journal of Experimental Biology 206 4067–4079. (doi:10.1242/jeb.00635)
Yang W, Zhang Y, Li Y, Wu Z & Zhu D 2007 Myostatin induces cyclin D1 degradation to cause cell cycle arrest through a phosphatidylinositol 3-kinase/AKT/GSK-3β pathway and is antagonized by insulin-like growth factor 1. Journal of Biological Chemistry 282 3799–3808. (doi:10.1074/jbc.M610185200)
Zhu J, Li Y, Shen W, Qiao C, Ambrosio F, Lavasani M, Nozaki M, Branca MF & Huard J 2007 Relationships between transforming growth factor-β1, myostatin, and decorin: implications for skeletal muscle fibrosis. Journal of Biological Chemistry 282 25852–25863. (doi:10.1074/jbc.M704146200)
Zhu W, Wang Y, Qiu G & Chen B 2010 Characterization of the purification and primary culture of adult canine myoblasts in vitro. Molecular Medicine Reports 3 463–468. (doi:10.3892/mmr_00000281)
