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Myostatin and MyoD family expression in skeletal muscle of IGF-1 knockout mice
Cell Biology International 31 (2007) 1274e1279 www.elsevier.com/locate/cellbi
Myostatin and MyoD family expression in skeletal muscle of IGF-1 knockout mice Masato Miyake a, Shinichiro Hayashi a, Tomomi Sato b, Yoshikazu Taketa a, Kouichi Watanabe a, Shinji Hayashi b, Sachi Tanaka a, Shyuichi Ohwada a, Hisashi Aso a, Takahiro Yamaguchi a,* a
Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan b Graduate School of Integrated Science, Yokohama City University, 22-2 Seto, Yokohama 236-0027, Japan Received 25 February 2007; revised 5 April 2007; accepted 12 May 2007
Abstract Insulin-like growth factor-1 (IGF-1) is a positive regulator in proliferation and differentiation of skeletal muscle cells, while myostatin (MSTN) is a member of transforming growth factor b superfamily that acts as a negative regulator of skeletal muscle mass. The present study was performed to detail whether a correlation exists between MSTN and IGF-1 in skeletal muscle of IGF-1 knockout mice (IGF-1/) and their wild type (WT; i.e., IGF-1þ/þ) littermates. The body weight of IGF-1/ animals was 32% that of WT littermates. The fiber cross-sectional areas (CSA) and number of fibers in M. rectus femoris of IGF-1/ animals were 49 and 59% those of WT animals, respectively. Thus, muscle hypoplasia of IGF-1/ undoubtedly was confirmed. Myostatin mRNA levels and protein levels were similar between M. gastrocnemius of IGF-1/ and WT animals. Myostatin immunoreactivity was similarly localized in muscle fibers of both IGF-1/ and WT M. rectus femoris. The mRNA levels of MyoD family (Myf5, MyoD, MRF4, myogenin) were differentially expressed in IGF-1/ M. gastrocnemius, in which the mRNA expression of MRF4 and myogenin was significantly lower, whereas there were no changes in the mRNA expression of Myf5 and MyoD. These findings first describe that myostatin expression is not influenced by intrinsic failure of IGF-1, although MRF4 and myogenin are downregulated. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Skeletal muscle; Insulin-like growth factor-1; Myostatin; MRF4; Myogenin
1. Introduction Insulin-like growth factor 1 (IGF-1) plays key roles in the development and the growth of normal muscle (Liu et al., 1993; Powell-Braxton et al., 1993). In addition, IGF-1 also functions on muscle hypertrophy (Coleman et al., 1995) and the muscle regeneration following injury (Caroni and Schneider, 1994). In vitro, IGF-1 promotes both proliferation and differentiation of skeletal muscle cells, and myotube hypertrophy (Hawke and Garry, 2001). In addition, functional inactivation of endogenous IGF-1 receptors results in the * Corresponding author. Tel.: þ81 (0)22 717 8702; fax: þ81 (0)22 717 8880. E-mail address: [email protected] (T. Yamaguchi).
marked delay in the induction of muscle differentiation (Cheng et al., 2000). When IGF-1 levels are enhanced using a muscle-specific promoter in transgenic mice, skeletal muscle increase in size (Coleman et al., 1995). The hypertrophy in adult skeletal muscle by increased load is accompanied by the increased expression of IGF-1 (Coleman et al., 1995). These reports have shown that IGF-1 plays an important role as a positive regulator in proliferation and differentiation of skeletal muscle cells. Myostatin (MSTN) is identified as a member of TGF-beta family (McPherron et al., 1997). MSTN is produced as a 52 kDa precursor protein, and then processed to N-terminal Latency Associated peptide (LAP) and C-terminal mature MSTN peptide, 26 kDa homodimer protein before secretion
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.05.007
M. Miyake et al. / Cell Biology International 31 (2007) 1274e1279 Table 1 Primer sequence used in semi-quantitative RT-PCR Gene Myostatin Myf5 MyoD MRF4 Myogenin G3PDH
Primer sequence 0
Product size (bp) 0
5 - CAGCCTGAATCCAACTTAGG-3 50 - TCGCAGTCAAGCCCAAAGTC-30 50 -TGTATCCCCTCACCAGAGGAT-30 50 -GGCTGTAATAGTTCTCCACCTGTT-30 50 -GCCCGCGCTCCAACTGCTCTGAT-30 50 -CCTACGGTGGTGCGCCCTCTG-30 50 -CTACATTGAGCGTCTACAGGACC-3 50 -CTGAAGACTGCTGGAGGCTG-30 50 -TGGAGCTGTATGAGACATCCC-3 50 -TGGACAATGCTCAGGGGTCCC-30 50 -TCCACCACCCTGTTGCTGTA-30 50 -ACCACAGTCCATGCCATCAC-30
167 379 408 235 184 451
(Thomas et al., 2000; Lee and McPherron, 2001; McFarlane et al., 2005). When MSTN gene is disrupted in mice, skeletal muscle mass significantly increases up to three times the normal size (McPherron et al., 1997). In addition, inactivating mutations in the mstn gene on cattle, referred to double-muscled cattle breeds such as Belgian blue and Piedmontese, induce increased muscle mass (McPherron and Lee, 1997). In vitro, an addition of MSTN to muscle cell culture inhibits cell proliferation, differentiation of myoblasts into myotubes and myotube
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hypertrophy (Thomas et al., 2000; Rios et al., 2001; Joulia et al., 2003). Therefore, it has been proposed that MSTN acts as a negative regulator of skeletal muscle growth. MyoD family (Myf5, MyoD, MRF4, myogenin), the basic helix-loop-helix (bHLH) transcription factor, plays an essential role as central regulators of myogenesis (Perry and Rudnick, 2000). Myf5 and MyoD are expressed in myoblasts and myotubes, and are required for myogenesis (Perry and Rudnick, 2000). In comparison, MRF4 and myogenin are critical for myotube formation and terminal myogenic differentiation events (Perry and Rudnick, 2000) since they are highly expressed when myoblasts commit to differentiation state to form myotubes. In vitro, MyoD and myogenin expression are increased by an addition of IGF-1 (Coleman et al., 1995). Conversely, myostatin down-regulates MyoD expression (Langley et al., 2002). Therefore, MyoD family controls on skeletal muscle growth could be achieved by the differential expression that is regulated by factors such as IGF-1 and myostatin. As described above, IGF-1 and myostatin have an opposite function on skeletal muscle differentiation. IGF-1 gene expression is similar between control and myostatin knockout mice (Kocamis et al., 2002). Recent evidence indicates that myostatin expression in C2C12 cells is induced by IGF-1 (Yang et al., 2007). Thus, there are some discrepancies on the mutual
Fig. 1. Photomicrographs of standard hematoxylin and eosin stain of M. rectus femoris sections from WT (A, C) and IGF-1/ (B, D) mice. Scale bars ¼ 400 mm in A, B; 10 mm in C, D.
M. Miyake et al. / Cell Biology International 31 (2007) 1274e1279
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relationship between IGF-1 and myostatin actions during skeletal muscle growth. We reasoned therefore that myostatin and MyoD family expression in IGF-1 knockout mice (IGF-1/) may explain in part whether a correlation exists between MSTN and IGF-1 in skeletal muscle. 2. Materials and methods
10 min. Twenty mg of the clarified lysates were loaded and separated by SDSPAGE in PAGEL (AE-6000, Atto, Tokyo, JP) and transfer to an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was blocking with 0.05% Tween20/PBS (PBS-T) containing 3% normal rabbit serum overnight at 4 C and incubated in goat polyclonal anti myostatin antibody (1 ng/ml R&D systems for 2 h. The membrane was incubated in Histofine simple stain MAX-PO (G) (1:20 Nichilei) for 2 h and the immunoreactive protein was finally visualized using DAB detection kit (Vector laboratories, Burlingam, CA).
2.1. Animals Male IGF-1 knockout mice (IGF-1/: n ¼ 3) and their wild type (WT; i.e., IGF-1þ/þ: m ¼ 3) littermates were provided by Tomomi Sato (Yokohama City University) (Baker et al., 1996).
2.2. Histological and immunohistochemical analysis /
M. rectus femoris from IGF-1 and WT mice were removed immediately after sacrifice and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4). The tissues were dehydrated through a series of graded ethanol and embedded in paraffin. Sections were cut at 5 mm thickness and stained hematoxylin and eosin (H&E) to assess the muscle fiber cross-sectional area (CSA) and number of muscle fiber. CSA was determined from the sections using NIH image computerized densitometry program (Wayne Rasbadn, NIH, Bethesda, MD) at least 100 fibers in M. rectus femoris of each mice. Fiber number was analyzed on a microscope in M. rectus femoris. The data were statistically analyzed by Tukey’s multiple comparison methods. The differences between means were considered significant at P < 0.05. Myostatin localization in muscle fibers was detected by immunohistochemistry as reported previously (Hayashi et al., 2004). In brief, the sections were treated with 90% methanol containing 3% H2O2 for 10 min, with 3% normal rabbit serum for 30 min and then incubated in goat polyclonal anti myostatin antibody (20 ng/ml; R&D systems, Minneapolis, MN) in PBS overnight at 4 C. After the incubation, the sections were in Histofine simple stain MAX-PO (G) (Nichilei, Tokyo JP) for 60 min. The reaction products were visualized by 0.025% 3,30 -diaminobenzidine tetrahydrochloride (DAB) and 0.01% H2O2 in 0.05 M tris buffer. The sections were counterstained hematoxyline. Negative control was run without the primary antibody.
3. Results 3.1. Influence of IGF-1 gene deletion on skeletal muscle formation The body weight of IGF-1/ mice was 9.8 1.2 g which was 32% that of WT littermates (36.4 1.1 g) (P < 0.01). The histology showed that the size of M. rectus femoris in IGF-1/ animals was extremely small compared with WT littermates (Fig. 1A,B). In addition, the myofiber size of IGF-1/ animals was smaller than that of WT animals (Fig. 1C,D). The CSA (2434.5 130.5 mm2) of M. rectus femoris in IGF-1/ animals was significantly smaller than that (1195.9 70.5 mm2) in WT animals and was reduced by 49% that of WT littermates (P < 0.01; Fig. 2A). The myofiber number per M. rectus femoris was 2202.0 210.2 in IGF-1/ mice and 3756.0 191.4 in WT mice, and the former was significantly decreased by 59% that of the latter (P < 0.05; Fig. 2B).
2.3. RT-PCR To detect myostatin and MyoD family mRNA, total RNA was extracted from M. gastrocnemius of WT and IGF-1/ mice using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) (Hayashi et al., 2004). For myostatin mRNA, first strand cDNA were synthesized using 1 mg total RNA with the oligo (dT) primer and Superscript III reverse transcriptase (invitrogen). PCR was performed with 1 mg of the RT reaction using the primers specific for myostatin, Myf5, MyoD, MRF4 and myogenin (Table 1). The annealing temperatures were 62 C for MSTN, Myf5, MRF4, myogenin and 55 C for MyoD. The numbers of cycles of PCR was 30 for MSTN and Myf5, 25 for MRF4, and 35 for MyoD and myogenin. G3PDH specific primers (Table 1) were used as internal controls. PCR products were then separated by electrophoresis in 2% agarose gels and were stained with ethidium bromide. For MyoD family mRNA, the image of the gel was digitized using NIH image computerized densitometry program (Wayne Rasbadn, NIH, Bethesda, MD) and the results were normalized to G3PDH. One-way ANOVA and Tukey’s multiple comparison method were used for statistical analysis. The differences between means were considered significant at P < 0.05.
2.4. Western blot The expression of myostatin protein in M. gastrocnemius of IGF-1/ and WT mice was determined by Western blot analysis. The muscles were lysed in a buffer containing 10 mM TriseHCl, pH 7.5, 150 mM NaCl, 0.5% Triton X100 and 0.2 mM PMSF. This was followed by centrifugation at 14,000 g for
Fig. 2. Data for myofiber cross-sectional areas (CSA) (A) and total number of myofibers (B) in M. rectus femoris from three WT and IGF-1/ mice. Data are mean SD. Means with asterisks were significantly different (*: P < 0.05, **: P < 0.01).
M. Miyake et al. / Cell Biology International 31 (2007) 1274e1279
3.2. Expression of MSTN in IGF-1/ mice The expression of myostatin mRNA in M. gastrocnemius of IGF-1/ animals was compared with that of WT animals by RT-PCR. Myostatin mRNA was detected at the expected size of 167 bp. The bands of myostatin mRNA was similarly detected in both in M. gastrocnemius of IGF-1/ and WT animals (Fig. 3A). Western blot analysis showed that myostatin precursor protein at 52 kDa was predominantly detected but mature myostatin at 26 kDa was hardly detected in both IGF1/ and WT animals (Fig. 3B). The expression of myostatin protein levels was not different between IGF-1/ and WT animals. The immunochemistry of myostatin revealed that myostatin immunostaining was contained in relatively smaller myofibers of IGF-1/ and IGF-1/ and WT M. rectus femoris (Fig. 3C). There were no differences in the localization and intensity of myostatin immunostaining between WT and IGF1/ animals. 3.3. Expression of MyoD family mRNA in IGF-1/ mice It has been reported that IGF-1 promotes cell proliferation and differentiation through MyoD expression in vitro (Coleman et al., 1995). RT-PCR analysis of MyoD family in M. gastrocnemius (Fig. 4A,B) showed that Myf5, MyoD, MRF4 and myogenin mRNA were detected with their expected
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size. The expression of MRF4 and myogenin mRNA in IGF1/ animals was significantly lower than that of WT animals (P < 0.01) (Fig. 4B). However, there were no changes in the expression of Myf5 and MyoD mRNA between IGF-1/ and WT animals. 4. Discussion We have shown that the mass of M. rectus femoris in IGF1/ mice significantly decreased up to about one third of WT littermates. Additionally the decreased CSA and myofiber number was linked to IGF-1/ mice. These findings confirmed previous studies that IGF-1/ mice result in skeletal muscle hypoplasia by reduced cell number and size (Fournier and Lewis, 2000). The increased expression of myostatin protein in skeletal muscle shows the hypoplasia phenotype in male mice (Reisz-Porszasz et al., 2003). Additionally, IGF-1 deficient mice also occurs the hypoplasia phenotype as described above. Therefore, it is proposed that myostatin expression in IGF-1/ mice is higher than that in WT mice. However, in the present study, there were no differences in the expressions of myostatin mRNA and protein between IGF-1/ and WT mice and the localization of myostatin in myofibers was similar in IGF-1/ and WT mice. Kocamis et al. (2002) have reported that expression of IGF-1 mRNA in myostatin knockout was
Fig. 3. Expression of MSTN in M. rectus femoris of WT and IGF-1/ mice. PCR products of MSTN are amplified at 167-bp by primers specific for mouse MSTN (A). The housekeeping gene, G3PDH, served as the internal standard. Immunoblot analysis shows precursor and processed forms of MSTN are indicated at 52 kDa and 26 kDa, respectively (B). The sections of M. rectus femoris from WT and IGF-1/ mice were immunostained with anti-myostatin antibody (C). Scale bars ¼ 10 mm.
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M. Miyake et al. / Cell Biology International 31 (2007) 1274e1279
gene cause a severe reduction of skeletal muscle mass (Knapp et al., 2006). Furthermore, it has been reported that MRF4 acts upstream at MyoD and directly on the differentiation of embryonic multipotent cells into myogenic lineage (Kassar-Duchossoy et al., 2004). The present study showed that the severe reduction of skeletal mass in IGF-1/ mice was caused by the decrease of CSA and number of myofiber. The results are supported by the decrease of myogenin and MRF4. In conclusion, the present study first demonstrated that myostatin expression was not affected by IGF-1 deletion, but the mRNA expression of MRF4 and myogenin, muscle specific transcription factors, was downregulated in IGF-1/ mice. However, further research is required to understand how MyoD family is involved in skeletal muscle hypoplasia of IGF-1/ mice. Acknowledgments We thank Drs. Argiris Efstratiadis (Columbia University) and Matthew Hardy (Population Council) for supplying the igf1 knockout mice. This study was supported by a Grantin-Aid for Scientific Research (A) (No. 17208024) from the Ministry of Education, Culture, Sports, Science and Technology and a Research project for utilizing advanced technologies in agriculture, forestry and fisheries (number 1523) from the Ministry of Agriculture, Forestry and Fisheries, Japan. Fig. 4. Expression of MyoD family mRNA in M. gastrocnemius of WT and IGF-1/ mice. First strand cDNAs were synthesized from 1 mg total RNA from M. gastrocnemius of WT and IGF-1/ mice, and then amplified by PCR using specific primers for Myf5 (379-bp), MyoD (408-bp), MRF4 (235-bp) and Myogenin (184-bp). G3PDH served as the internal standard (A). The mRNA expression of MyoD family was semi-quantified using a densitometric method (B). Means with asterisks were significantly different (**: P < 0.01). Results are representative of three identical experiments.
the same as that in their WT littermates. These findings strongly support that IGF-1 and myostatin, reciprocal regulators on muscle differentiation, independently act during myogenesis. The mRNA expression of MyoD and myogenin is enhanced in skeletal muscle cell culture by an addition of IGF-1 (Coleman et al., 1995). The present study showed that the mRNA of MyoD and Myf5 did not change although the mRNA expression of myogenin and MRF4 significantly decreased in IGF1/ mice compared with WT littermates. These data indicate that IGF-1 does not regulate intrinsically MyoD mRNA expression but possibly controls MyoD expression by an indirect manner through some factors that are induced by its action. MyoD and Myf5 are expressed in skeletal muscle cells during proliferation period in vitro and act as master regulators of the differentiation from stem cells to myogenic cells and the activation of satellite cell in adult skeletal muscle (Perry and Rudnick, 2000). However, the mutation in MyoD or Myf5 of mice shows normal skeletal muscle phenotype in vivo (Sabourin and Rudnicki, 2000). In the other hand, myogenin and MRF4 are mainly expressed in differentiated skeletal muscle cells in vitro (Perry and Rudnick, 2000). The null mutations of myogenin
References Baker J, Hardy MP, Zhou J, Bondy C, Lupu F, Bellve AR, et al. Effects of an Igf1 gene null mutation on mouse reproduction. Mol Endocrinol 1996;10:903e18. Caroni P, Schneider C. Signaling by insulin-like growth factors in paralyzed skeletal muscle: rapid induction of IGF1 expression in muscle fibers and prevention of interstitial cell proliferation by IGF-BP5 and IGF-BP4. J Neurosci 1994;14:3378e88. Cheng ZQ, Adi S, Wu NY, Hsiao D, Woo EJ, Filvaroff EH, et al. Functional inactivation of the IGF-I receptor delays differentiation of skeletal muscle cells. J Endocrinol 2000;167:175e82. Coleman ME, DeMayo F, Yin KC, Lee HM, Geske R, Montgomery C, et al. Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J Biol Chem 1995;270:12109e16. Fournier M, Lewis MI. Influences of IGF-I gene disruption on the cellular profile of the diaphragm. Am J Physiol Endocrinol Metab 2000;278:E707e15. Hawke TJ, Garry DJ. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 2001;91:534e51. Hayashi S, Aso H, Watanabe K, Nara H, Rose MT, Ohwada S, et al. Sequence of IGF-I, IGF-II, and HGF expression in regenerating skeletal muscle. Histochem Cell Biol 2004;122:427e34. Joulia D, Bernardi H, Garandel V, Rabenoelina F, Vernus B, Cabello G. Mechanisms involved in the inhibition of myoblast proliferation and differentiation by myostatin. Exp Cell Res 2003;286:263e75. Kassar-Duchossoy L, Gayraud-Morel B, Gomes D, Rocancourt D, Buckingham M, Shinin V, et al. Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice. Nature 2004;431:466e71. Knapp JR, Davie JK, Myer A, Meadows E, Olson EN, Klein WH. Loss of myogenin in postnatal life leads to normal skeletal muscle but reduced body size. Development 2006;133:601e10. Kocamis H, Gahr SA, Batelli L, Hubbs AF, Killefer J. IGF-I, IGF-II, and IGFreceptor-1 transcript and IGF-II protein expression in myostatin knockout mice tissues. Muscle Nerve 2002;26:55e63.
M. Miyake et al. / Cell Biology International 31 (2007) 1274e1279 Langley B, Thomas M, Bishop A, Sharma M, Gilmour S, Kambadur R. Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. J Biol Chem 2002;277:49831e40. Lee SJ, McPherron AC. Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci U S A 2001;98:9306e11. Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 1993;75:59e72. McFarlane C, Langley B, Thomas M, Hennebry A, Plummer E, Nicholas G, et al. Proteolytic processing of myostatin is auto-regulated during myogenesis. Dev Biol 2005;283:58e69. McPherron AC, Lee SJ. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci U S A 1997;94:12457e61. McPherron AC, Lawler AM, Lee SJ. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 1997;387:83e90. Perry RL, Rudnick MA. Molecular mechanisms regulating myogenic determination and differentiation. Front Biosci 2000;5:D750e67.
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Powell-Braxton L, Hollingshead P, Warburton C, Dowd M, Pitts-Meek S, Dalton D, et al. IGF-I is required for normal embryonic growth in mice. Genes Dev 1993;7:2609e17. Reisz-Porszasz S, Bhasin S, Artaza JN, Shen R, Sinha-Hikim I, Hogue A, et al. Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin. Am J Physiol Endocrinol Metab 2003;285:E876e88. Rios R, Carneiro I, Arce VM, Devesa J. Myostatin regulates cell survival during C2C12 myogenesis. Biochem Biophys Res Commun 2001;280:561e6. Sabourin LA, Rudnicki MA. The molecular regulation of myogenesis. Clin Genet 2000;57:16e25. Thomas M, Langley B, Berry C, Sharma M, Kirk S, Bass J, et al. Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J Biol Chem 2000;275:40235e43. Yang W, Zhang Y, Li Y, Wu Z, Zhu D. Myostatin induces cyclin D1 degradation to cause cell cycle arrest through a phosphatidylinositol 3-kinase/ AKT/GSK-3beta pathway and is antagonized by insulin-like growth factor 1. J Biol Chem 2007;282:3799e808.
Myostatin and MyoD family expression in skeletal muscle of IGF-1 knockout mice Masato Miyake a, Shinichiro Hayashi a, Tomomi Sato b, Yoshikazu Taketa a, Kouichi Watanabe a, Shinji Hayashi b, Sachi Tanaka a, Shyuichi Ohwada a, Hisashi Aso a, Takahiro Yamaguchi a,* a
Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan b Graduate School of Integrated Science, Yokohama City University, 22-2 Seto, Yokohama 236-0027, Japan Received 25 February 2007; revised 5 April 2007; accepted 12 May 2007
Abstract Insulin-like growth factor-1 (IGF-1) is a positive regulator in proliferation and differentiation of skeletal muscle cells, while myostatin (MSTN) is a member of transforming growth factor b superfamily that acts as a negative regulator of skeletal muscle mass. The present study was performed to detail whether a correlation exists between MSTN and IGF-1 in skeletal muscle of IGF-1 knockout mice (IGF-1/) and their wild type (WT; i.e., IGF-1þ/þ) littermates. The body weight of IGF-1/ animals was 32% that of WT littermates. The fiber cross-sectional areas (CSA) and number of fibers in M. rectus femoris of IGF-1/ animals were 49 and 59% those of WT animals, respectively. Thus, muscle hypoplasia of IGF-1/ undoubtedly was confirmed. Myostatin mRNA levels and protein levels were similar between M. gastrocnemius of IGF-1/ and WT animals. Myostatin immunoreactivity was similarly localized in muscle fibers of both IGF-1/ and WT M. rectus femoris. The mRNA levels of MyoD family (Myf5, MyoD, MRF4, myogenin) were differentially expressed in IGF-1/ M. gastrocnemius, in which the mRNA expression of MRF4 and myogenin was significantly lower, whereas there were no changes in the mRNA expression of Myf5 and MyoD. These findings first describe that myostatin expression is not influenced by intrinsic failure of IGF-1, although MRF4 and myogenin are downregulated. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Skeletal muscle; Insulin-like growth factor-1; Myostatin; MRF4; Myogenin
1. Introduction Insulin-like growth factor 1 (IGF-1) plays key roles in the development and the growth of normal muscle (Liu et al., 1993; Powell-Braxton et al., 1993). In addition, IGF-1 also functions on muscle hypertrophy (Coleman et al., 1995) and the muscle regeneration following injury (Caroni and Schneider, 1994). In vitro, IGF-1 promotes both proliferation and differentiation of skeletal muscle cells, and myotube hypertrophy (Hawke and Garry, 2001). In addition, functional inactivation of endogenous IGF-1 receptors results in the * Corresponding author. Tel.: þ81 (0)22 717 8702; fax: þ81 (0)22 717 8880. E-mail address: [email protected] (T. Yamaguchi).
marked delay in the induction of muscle differentiation (Cheng et al., 2000). When IGF-1 levels are enhanced using a muscle-specific promoter in transgenic mice, skeletal muscle increase in size (Coleman et al., 1995). The hypertrophy in adult skeletal muscle by increased load is accompanied by the increased expression of IGF-1 (Coleman et al., 1995). These reports have shown that IGF-1 plays an important role as a positive regulator in proliferation and differentiation of skeletal muscle cells. Myostatin (MSTN) is identified as a member of TGF-beta family (McPherron et al., 1997). MSTN is produced as a 52 kDa precursor protein, and then processed to N-terminal Latency Associated peptide (LAP) and C-terminal mature MSTN peptide, 26 kDa homodimer protein before secretion
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.05.007
M. Miyake et al. / Cell Biology International 31 (2007) 1274e1279 Table 1 Primer sequence used in semi-quantitative RT-PCR Gene Myostatin Myf5 MyoD MRF4 Myogenin G3PDH
Primer sequence 0
Product size (bp) 0
5 - CAGCCTGAATCCAACTTAGG-3 50 - TCGCAGTCAAGCCCAAAGTC-30 50 -TGTATCCCCTCACCAGAGGAT-30 50 -GGCTGTAATAGTTCTCCACCTGTT-30 50 -GCCCGCGCTCCAACTGCTCTGAT-30 50 -CCTACGGTGGTGCGCCCTCTG-30 50 -CTACATTGAGCGTCTACAGGACC-3 50 -CTGAAGACTGCTGGAGGCTG-30 50 -TGGAGCTGTATGAGACATCCC-3 50 -TGGACAATGCTCAGGGGTCCC-30 50 -TCCACCACCCTGTTGCTGTA-30 50 -ACCACAGTCCATGCCATCAC-30
167 379 408 235 184 451
(Thomas et al., 2000; Lee and McPherron, 2001; McFarlane et al., 2005). When MSTN gene is disrupted in mice, skeletal muscle mass significantly increases up to three times the normal size (McPherron et al., 1997). In addition, inactivating mutations in the mstn gene on cattle, referred to double-muscled cattle breeds such as Belgian blue and Piedmontese, induce increased muscle mass (McPherron and Lee, 1997). In vitro, an addition of MSTN to muscle cell culture inhibits cell proliferation, differentiation of myoblasts into myotubes and myotube
1275
hypertrophy (Thomas et al., 2000; Rios et al., 2001; Joulia et al., 2003). Therefore, it has been proposed that MSTN acts as a negative regulator of skeletal muscle growth. MyoD family (Myf5, MyoD, MRF4, myogenin), the basic helix-loop-helix (bHLH) transcription factor, plays an essential role as central regulators of myogenesis (Perry and Rudnick, 2000). Myf5 and MyoD are expressed in myoblasts and myotubes, and are required for myogenesis (Perry and Rudnick, 2000). In comparison, MRF4 and myogenin are critical for myotube formation and terminal myogenic differentiation events (Perry and Rudnick, 2000) since they are highly expressed when myoblasts commit to differentiation state to form myotubes. In vitro, MyoD and myogenin expression are increased by an addition of IGF-1 (Coleman et al., 1995). Conversely, myostatin down-regulates MyoD expression (Langley et al., 2002). Therefore, MyoD family controls on skeletal muscle growth could be achieved by the differential expression that is regulated by factors such as IGF-1 and myostatin. As described above, IGF-1 and myostatin have an opposite function on skeletal muscle differentiation. IGF-1 gene expression is similar between control and myostatin knockout mice (Kocamis et al., 2002). Recent evidence indicates that myostatin expression in C2C12 cells is induced by IGF-1 (Yang et al., 2007). Thus, there are some discrepancies on the mutual
Fig. 1. Photomicrographs of standard hematoxylin and eosin stain of M. rectus femoris sections from WT (A, C) and IGF-1/ (B, D) mice. Scale bars ¼ 400 mm in A, B; 10 mm in C, D.
M. Miyake et al. / Cell Biology International 31 (2007) 1274e1279
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relationship between IGF-1 and myostatin actions during skeletal muscle growth. We reasoned therefore that myostatin and MyoD family expression in IGF-1 knockout mice (IGF-1/) may explain in part whether a correlation exists between MSTN and IGF-1 in skeletal muscle. 2. Materials and methods
10 min. Twenty mg of the clarified lysates were loaded and separated by SDSPAGE in PAGEL (AE-6000, Atto, Tokyo, JP) and transfer to an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was blocking with 0.05% Tween20/PBS (PBS-T) containing 3% normal rabbit serum overnight at 4 C and incubated in goat polyclonal anti myostatin antibody (1 ng/ml R&D systems for 2 h. The membrane was incubated in Histofine simple stain MAX-PO (G) (1:20 Nichilei) for 2 h and the immunoreactive protein was finally visualized using DAB detection kit (Vector laboratories, Burlingam, CA).
2.1. Animals Male IGF-1 knockout mice (IGF-1/: n ¼ 3) and their wild type (WT; i.e., IGF-1þ/þ: m ¼ 3) littermates were provided by Tomomi Sato (Yokohama City University) (Baker et al., 1996).
2.2. Histological and immunohistochemical analysis /
M. rectus femoris from IGF-1 and WT mice were removed immediately after sacrifice and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4). The tissues were dehydrated through a series of graded ethanol and embedded in paraffin. Sections were cut at 5 mm thickness and stained hematoxylin and eosin (H&E) to assess the muscle fiber cross-sectional area (CSA) and number of muscle fiber. CSA was determined from the sections using NIH image computerized densitometry program (Wayne Rasbadn, NIH, Bethesda, MD) at least 100 fibers in M. rectus femoris of each mice. Fiber number was analyzed on a microscope in M. rectus femoris. The data were statistically analyzed by Tukey’s multiple comparison methods. The differences between means were considered significant at P < 0.05. Myostatin localization in muscle fibers was detected by immunohistochemistry as reported previously (Hayashi et al., 2004). In brief, the sections were treated with 90% methanol containing 3% H2O2 for 10 min, with 3% normal rabbit serum for 30 min and then incubated in goat polyclonal anti myostatin antibody (20 ng/ml; R&D systems, Minneapolis, MN) in PBS overnight at 4 C. After the incubation, the sections were in Histofine simple stain MAX-PO (G) (Nichilei, Tokyo JP) for 60 min. The reaction products were visualized by 0.025% 3,30 -diaminobenzidine tetrahydrochloride (DAB) and 0.01% H2O2 in 0.05 M tris buffer. The sections were counterstained hematoxyline. Negative control was run without the primary antibody.
3. Results 3.1. Influence of IGF-1 gene deletion on skeletal muscle formation The body weight of IGF-1/ mice was 9.8 1.2 g which was 32% that of WT littermates (36.4 1.1 g) (P < 0.01). The histology showed that the size of M. rectus femoris in IGF-1/ animals was extremely small compared with WT littermates (Fig. 1A,B). In addition, the myofiber size of IGF-1/ animals was smaller than that of WT animals (Fig. 1C,D). The CSA (2434.5 130.5 mm2) of M. rectus femoris in IGF-1/ animals was significantly smaller than that (1195.9 70.5 mm2) in WT animals and was reduced by 49% that of WT littermates (P < 0.01; Fig. 2A). The myofiber number per M. rectus femoris was 2202.0 210.2 in IGF-1/ mice and 3756.0 191.4 in WT mice, and the former was significantly decreased by 59% that of the latter (P < 0.05; Fig. 2B).
2.3. RT-PCR To detect myostatin and MyoD family mRNA, total RNA was extracted from M. gastrocnemius of WT and IGF-1/ mice using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) (Hayashi et al., 2004). For myostatin mRNA, first strand cDNA were synthesized using 1 mg total RNA with the oligo (dT) primer and Superscript III reverse transcriptase (invitrogen). PCR was performed with 1 mg of the RT reaction using the primers specific for myostatin, Myf5, MyoD, MRF4 and myogenin (Table 1). The annealing temperatures were 62 C for MSTN, Myf5, MRF4, myogenin and 55 C for MyoD. The numbers of cycles of PCR was 30 for MSTN and Myf5, 25 for MRF4, and 35 for MyoD and myogenin. G3PDH specific primers (Table 1) were used as internal controls. PCR products were then separated by electrophoresis in 2% agarose gels and were stained with ethidium bromide. For MyoD family mRNA, the image of the gel was digitized using NIH image computerized densitometry program (Wayne Rasbadn, NIH, Bethesda, MD) and the results were normalized to G3PDH. One-way ANOVA and Tukey’s multiple comparison method were used for statistical analysis. The differences between means were considered significant at P < 0.05.
2.4. Western blot The expression of myostatin protein in M. gastrocnemius of IGF-1/ and WT mice was determined by Western blot analysis. The muscles were lysed in a buffer containing 10 mM TriseHCl, pH 7.5, 150 mM NaCl, 0.5% Triton X100 and 0.2 mM PMSF. This was followed by centrifugation at 14,000 g for
Fig. 2. Data for myofiber cross-sectional areas (CSA) (A) and total number of myofibers (B) in M. rectus femoris from three WT and IGF-1/ mice. Data are mean SD. Means with asterisks were significantly different (*: P < 0.05, **: P < 0.01).
M. Miyake et al. / Cell Biology International 31 (2007) 1274e1279
3.2. Expression of MSTN in IGF-1/ mice The expression of myostatin mRNA in M. gastrocnemius of IGF-1/ animals was compared with that of WT animals by RT-PCR. Myostatin mRNA was detected at the expected size of 167 bp. The bands of myostatin mRNA was similarly detected in both in M. gastrocnemius of IGF-1/ and WT animals (Fig. 3A). Western blot analysis showed that myostatin precursor protein at 52 kDa was predominantly detected but mature myostatin at 26 kDa was hardly detected in both IGF1/ and WT animals (Fig. 3B). The expression of myostatin protein levels was not different between IGF-1/ and WT animals. The immunochemistry of myostatin revealed that myostatin immunostaining was contained in relatively smaller myofibers of IGF-1/ and IGF-1/ and WT M. rectus femoris (Fig. 3C). There were no differences in the localization and intensity of myostatin immunostaining between WT and IGF1/ animals. 3.3. Expression of MyoD family mRNA in IGF-1/ mice It has been reported that IGF-1 promotes cell proliferation and differentiation through MyoD expression in vitro (Coleman et al., 1995). RT-PCR analysis of MyoD family in M. gastrocnemius (Fig. 4A,B) showed that Myf5, MyoD, MRF4 and myogenin mRNA were detected with their expected
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size. The expression of MRF4 and myogenin mRNA in IGF1/ animals was significantly lower than that of WT animals (P < 0.01) (Fig. 4B). However, there were no changes in the expression of Myf5 and MyoD mRNA between IGF-1/ and WT animals. 4. Discussion We have shown that the mass of M. rectus femoris in IGF1/ mice significantly decreased up to about one third of WT littermates. Additionally the decreased CSA and myofiber number was linked to IGF-1/ mice. These findings confirmed previous studies that IGF-1/ mice result in skeletal muscle hypoplasia by reduced cell number and size (Fournier and Lewis, 2000). The increased expression of myostatin protein in skeletal muscle shows the hypoplasia phenotype in male mice (Reisz-Porszasz et al., 2003). Additionally, IGF-1 deficient mice also occurs the hypoplasia phenotype as described above. Therefore, it is proposed that myostatin expression in IGF-1/ mice is higher than that in WT mice. However, in the present study, there were no differences in the expressions of myostatin mRNA and protein between IGF-1/ and WT mice and the localization of myostatin in myofibers was similar in IGF-1/ and WT mice. Kocamis et al. (2002) have reported that expression of IGF-1 mRNA in myostatin knockout was
Fig. 3. Expression of MSTN in M. rectus femoris of WT and IGF-1/ mice. PCR products of MSTN are amplified at 167-bp by primers specific for mouse MSTN (A). The housekeeping gene, G3PDH, served as the internal standard. Immunoblot analysis shows precursor and processed forms of MSTN are indicated at 52 kDa and 26 kDa, respectively (B). The sections of M. rectus femoris from WT and IGF-1/ mice were immunostained with anti-myostatin antibody (C). Scale bars ¼ 10 mm.
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gene cause a severe reduction of skeletal muscle mass (Knapp et al., 2006). Furthermore, it has been reported that MRF4 acts upstream at MyoD and directly on the differentiation of embryonic multipotent cells into myogenic lineage (Kassar-Duchossoy et al., 2004). The present study showed that the severe reduction of skeletal mass in IGF-1/ mice was caused by the decrease of CSA and number of myofiber. The results are supported by the decrease of myogenin and MRF4. In conclusion, the present study first demonstrated that myostatin expression was not affected by IGF-1 deletion, but the mRNA expression of MRF4 and myogenin, muscle specific transcription factors, was downregulated in IGF-1/ mice. However, further research is required to understand how MyoD family is involved in skeletal muscle hypoplasia of IGF-1/ mice. Acknowledgments We thank Drs. Argiris Efstratiadis (Columbia University) and Matthew Hardy (Population Council) for supplying the igf1 knockout mice. This study was supported by a Grantin-Aid for Scientific Research (A) (No. 17208024) from the Ministry of Education, Culture, Sports, Science and Technology and a Research project for utilizing advanced technologies in agriculture, forestry and fisheries (number 1523) from the Ministry of Agriculture, Forestry and Fisheries, Japan. Fig. 4. Expression of MyoD family mRNA in M. gastrocnemius of WT and IGF-1/ mice. First strand cDNAs were synthesized from 1 mg total RNA from M. gastrocnemius of WT and IGF-1/ mice, and then amplified by PCR using specific primers for Myf5 (379-bp), MyoD (408-bp), MRF4 (235-bp) and Myogenin (184-bp). G3PDH served as the internal standard (A). The mRNA expression of MyoD family was semi-quantified using a densitometric method (B). Means with asterisks were significantly different (**: P < 0.01). Results are representative of three identical experiments.
the same as that in their WT littermates. These findings strongly support that IGF-1 and myostatin, reciprocal regulators on muscle differentiation, independently act during myogenesis. The mRNA expression of MyoD and myogenin is enhanced in skeletal muscle cell culture by an addition of IGF-1 (Coleman et al., 1995). The present study showed that the mRNA of MyoD and Myf5 did not change although the mRNA expression of myogenin and MRF4 significantly decreased in IGF1/ mice compared with WT littermates. These data indicate that IGF-1 does not regulate intrinsically MyoD mRNA expression but possibly controls MyoD expression by an indirect manner through some factors that are induced by its action. MyoD and Myf5 are expressed in skeletal muscle cells during proliferation period in vitro and act as master regulators of the differentiation from stem cells to myogenic cells and the activation of satellite cell in adult skeletal muscle (Perry and Rudnick, 2000). However, the mutation in MyoD or Myf5 of mice shows normal skeletal muscle phenotype in vivo (Sabourin and Rudnicki, 2000). In the other hand, myogenin and MRF4 are mainly expressed in differentiated skeletal muscle cells in vitro (Perry and Rudnick, 2000). The null mutations of myogenin
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