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,1
* Division for Therapies Against Intractable Diseases, Institute for Comprehensive Medical Sciences, Fujita Health University, Toyoake, Aichi, Japan;
The Institute for Enzyme Research, The University of Tokushima, Tokushima, Japan;
Laboratories of Experimental Animal Science, Kitasato University School of Veterinary Medicine and Animal Sciences, Towada, Aomori, Japan;
Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan;
|| Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima, Japan; and
Division of Neurology, Department of Internal Medicine, Kawasaki Medical School, Kurashiki, Okayama, Japan
2Correspondence: Division for Therapies Against Intractable Diseases, Institute for Comprehensive Medical Sciences (ICMS), Fujita Health University, Toyoake, Aichi 470-1192, Japan. E-mail: tsuchida{at}fujita-hu.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Duchenne muscular dystrophy • activin • therapy • myostatin blockade
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Mice, cattle, sheep, and humans with genetic mutations in the myostatin gene show marked increases in skeletal muscle mass, due to hypertrophy and/or hyperplasia (3
4
5
6)
. Therefore, myostatin is a rational candidate for a muscle chalone, defined as a molecule that regulates the size of a particular tissue (1)
. Myostatin is predominantly expressed in skeletal muscles and to a lesser extent in adipose tissues, and circulates in the serum. Intriguingly, myostatin forms a complex with a number of other proteins, such as myostatin propeptide and follistatin-related gene product (FLRG) (7)
.
Interestingly, inhibition of myostatin activity is capable of increasing muscle mass and strength in the postnatal period and even in adults. These observations suggest that targeting of myostatin would be suitable as a therapy for degenerating muscle diseases, such as muscular dystrophy and cachexia, as well as for preventing muscle wasting due to aging (8
9
10)
. In fact, antibody-mediated myostatin blockade in mdx mice, a model for Duchenne muscular dystrophy, was found to ameliorate the pathophysiology and muscle weakness (11)
. Myostatin propeptide-mediated amelioration of the symptoms in mdx mice has also been reported (12)
. However, elimination of myostatin did not improve the phenotype in a laminin-
2-deficient dyw mouse (13)
. Thus, myostatin inhibition may show disease-specific effects. It is also of note that one report showed that lack of myostatin results in excessive muscle growth but impaired force generation (14)
. In addition to myostatin propeptide and myostatin antibodies, follistatin (FS) and FS domain-containing proteins can bind to myostatin in vivo and act as effective myostatin inhibitors (7
, 15
, 16)
. FS was originally identified as a single-chain polypeptide with a weak inhibitory activity toward follicle-stimulating hormone secretion from anterior pituitary cells. Later, FS was found to be an activin-binding protein (16
17
18)
. Gene knockout analyses revealed that FS gene ablation causes multiple effects, including skeletal and cutaneous abnormalities, suggesting that FS may have additional functions other than activin inhibition (19)
. Recently, FS was shown to bind to myostatin and inhibit its activity (20)
. Similar to myostatin, activins belong to the TGF-β superfamily and have pleiotrophic effects on numerous tissues. Because activins have a variety of functions in tissues other than skeletal muscles, and their inhibition by FS is very efficient, FS has multiple effects on not only skeletal muscles but also other tissues. In fact, transgenic expression of the FS gene under the control of the metallothionein promoter has profound effects on reproductive performance and fertility (21)
. Recent crystallographic analyses have revealed that the minimal activin-inhibiting fragment of FS comprises the FS I and FS II domains, and each FS domain may have different ligand binding activity (22
, 23)
.
In the present study, we developed and characterized a myostatin inhibitor derived from FS, designated FS I-I, and further investigated its effects on muscle mass and strength in mdx mice. Because myostatin blockade is one of the most promising therapies for muscular dystrophy, the results of our study should provide an additional rational therapeutic strategy for intractable muscular diseases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A cDNA fragment containing the N-terminal region and FS I domain (FS-A) was amplified using the sense primer 5'-CGGAATTCATGGTCCGCGCGAGGCACCAG-3' and antisense primer 5'-CGAAGCTTTTTACATCTGCCTTGG-3'. A cDNA fragment containing only the FS I domain (FS-B) was amplified using the sense primer 5'-GCAAGCTTTGTGAGAACGTGGACTGTG-3' and antisense primer 5'-GCCTCGAGATATCTTCACAAGTCTTTTTACATCTGCC-3'. The FS-A and FS-B cDNA fragments were subcloned into the pGEM-T Easy cloning vector (Promega, Tokyo, Japan), and the EcoRI-HindIII fragment of FS-A and HindIII-SalI fragment of FS-B were then inserted into the pBluescript cloning vector. The resultant cDNA contained the N-terminal region and the two FS I domains in tandem (FS I-I in pBluescript). The EcoRI-SpeI fragment of FS I-I in pBluescript was subcloned into the pcDNA3 expression vector (Invitrogen, Tokyo, Japan). To create an FS I-I-Fc cDNA, a BamHI-XbaI fragment of human Fc was subcloned into pcDNA3 (25)
, and digested with KpnI and BamHI. Next, a KpnI-BamHI fragment covering the whole FS I-I sequence was subcloned into the KpnI-BamHI-digested human Fc in pcDNA3 (FS I-I-Fc in pcDNA3). Coding sequences of FS I-I-Fc consist of nucleotide 1–501 and 283–501 of GenBank accession number CR541813, fused to BamHI-XbaI fragment of human IgG Fc portion (25)
.
GST-FS I-I enzyme-linked immunosorbent assay analysis
The PstI-EcoRV fragment of FS I-I in pcDNA3 was subcloned into pBluescript, and the SmaI-XhoI fragment was then subcloned into the bacterial expression vector pGEX6P-1 (GE Healthcare Bioscience, Tokyo, Japan). pGEX6P-1-FS I-I cDNA or empty pGEX6P-1 vector cDNA was transformed in Origami competent cells (Takara Bio, Tokyo, Japan). A GST-FS I-I fusion protein and naive GST protein were expressed and purified using glutathione-Sepharose beads, as described previously (26)
. Enzyme-linked immunosorbent assay (ELISA) plates (Sumilon Bakelite Co., Ltd., Tokyo, Japan) were coated with either 100 µl of 1 µg/ml bovine activin A (WAKO Chemicals, Osaka, Japan) or human myostatin in carbonate buffer (pH 9.6) at 4°C overnight, and then washed twice with washing buffer (0.05% Tween-20 in 25 mM Tris-buffered saline, pH 7.5). After blocking with Blocking One solution (Nakalai Tesque, Kyoto, Japan) for 45 min at room temperature, the wells were washed three times with washing buffer, and incubated with various amounts of GST or GST-FS I-I proteins at 37°C for 60 min in PBS containing Blocking One solution and 0.01% Tween-20, according to the manufacturer’s protocol. After three washes with washing buffer, the wells were incubated with a rabbit polyclonal anti-GST antibody at a dilution of 1:750 in Blocking One solution at 37°C for 50 min, washed, incubated with a horseradish peroxidase-conjugated secondary antibody at a dilution of 1:1000, and then incubated with o-phenylenediamine dihydrochloride for color development. The optical densities at 450 nm were measured.
Purification of human FS I-I-Fc protein
Chinese hamster ovary (CHO-K1) cells were transfected with the human FS I-I-Fc cDNA in pcDNA3 by electroporation, and clonal cells were established by limiting dilution (27)
. CHO-K1 cells stably expressing the FS I-I-Fc cDNA were grown in -modified essential medium (MEM)-10% fetal calf serum (FCS) until they reached confluency, and then conditioned by EX-CELL301 medium (Nichirei Biosciences, Tokyo, Japan). The conditioned medium was passed through a heparin-Sepharose 6 Fast Flow column, followed by protein A-Sepharose Fast Flow chromatography (GE Healthcare Biosciences, Bucks, UK). The FS I-I-Fc protein was eluted with 5 ml of 100 mM glycine-HCl (pH 3.0), and immediately neutralized with 500 µl of 1 M Tris-HCl (pH 8.0). A total of 850 µg of FS I-I-Fc protein was obtained from 4 liters of conditioned medium.
Surface plasmon resonance biosensor analysis
All surface plasmon resonance (SPR) measurements were taken using a BIAcore X system (Biacore AB, Tokyo, Japan), as described previously (28)
. For immobilization of samples, proteins were dissolved in 20 mM sodium acetate (pH 4.5) at a concentration of 10 µg/ml and immobilized on a CM5 sensor chip at a flow rate of 5 µl/min at 25°C using an amine-coupling method. Kinetic measurements were performed by injection of each analyte for 240 s followed by dissociation in buffer flow for 240 s at a flow rate of 20 µl/min at 25°C. The immobilized ligands were regenerated after each cycle using a 40 µl injection of 10 mM HEPES (pH 7.4)/2 M guanidine-HCl. The kinetic parameters, association rate constant (kon) and dissociation rate constant (koff) were determined using BIAevaluation software version 3.0 (Biacore AB). Activin A was purified from bovine follicular fluid, as described previously (29)
. Human FS and mouse myostatin were obtained from R&D Systems (Minneapolis, MN, USA).
Biological assay for myostatin signaling
A204 rhabdomyosarcoma cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich Japan, Tokyo, Japan) supplemented with 10% FCS. Cells were plated at a density of 8 x 104 cells/well in 24-well dishes and were transfected with 0.5 ng of CAGA-lux and 0.1 ng of a cytomegalovirus promoter-driven β-galactosidase (CMV-β-gal) DNA using the GeneJammer transfection reagent (Stratagene, La Jolla, CA, USA), according to the manufacturer’s protocol. At 24 h after transfection, the cells were stimulated with either myostatin or activin A in DMEM containing 1% FCS for 24 h. To examine whether FS or FS I-I-Fc had inhibitory effects on myostatin or activin, FS or FS I-I-Fc was simultaneously added with either myostatin or activin. The luciferase activities were measured and normalized by the corresponding β-galactosidase activities, as described previously (30)
.
CHO-K1 cells were transfected with 0.2 ng each of ActRIIB and wild-type ALK5 or ALK4 expression plasmids, 0.3 ng of a CAGA-lux reporter plasmid and 0.1 ng of the CMV-β-gal plasmid per well of 12-well dishes using the Transfast liposome reagent (Promega K.K. Japan, Tokyo, Japan) (30)
. 10T1/2 mouse fibroblastic cells were transfected with 0.2 ng each of a MyoD plasmid, Smad3 plasmid, and wild-type ALK5 or ALK4 expression plasmid, 0.3 ng of a MyoD-responsive 6R-lux reporter plasmid and 0.1 ng of the CMV-β-gal plasmid per well of 12-well dishes using the Effectene reagent (Qiagen in Japan, Tokyo, Japan) (31)
. At 24 h after transfection, the cells were stimulated with 20 ng/ml of myostatin either alone or in the presence of increasing amounts of FS I-I-Fc.
Generation and characterization of FS I-I transgenic mice
An EcoRI-XhoI fragment containing the whole FS I-I cDNA was subcloned into the CAG-GS vector (CAGGS-FS I-I) (32)
. Next, an EcoRI-SmaI fragment covering the whole coding sequence of FS I-I from CAGGS-FS I-I was subcloned into the MDAF2 vector containing the myosin light chain promoter, SV40 processing sites and MLC1/3 enhancer (33)
. The FS I-I transgene with the skeletal muscle-specific promoter consisting of 3.9 kb was recovered by ClaI digestion, and transgenic mice were generated by standard pronuclear microinjection techniques (21)
. The presence of the transgene was determined by PCR amplification of tail genomic DNA. The PCR was performed using the sense primer 5'-CACCACTGCTCTTCCAAGTGTC-3' (MDAF2S) and antisense primer 5'-GTCACACCACAGAAGTAAGGTC-3' (MDAF2AS) with a program of 35 cycles of denaturation for 30 s at 94°C, annealing for 15 s and extension for 30 s at 72°C. The annealing temperature was altered from 74°C to 54°C in five steps of 4°C intervals at 3 cycles per step, followed by 20 additional cycles at 54°C. The final extension reaction was carried out for 10 min at 72°C. The PCR products were separated in a 2% agarose gel and visualized by ethidium bromide staining.
Transgenic founders were mated to wild-type C57BL/6 mice, and the offspring were analyzed. Individual muscles were manually dissected from five mice and weighed. To obtain transgenic mdx mice expressing FS I-I, female mdx mice were crossed with male FS I-I transgenic mice, and the male F1 generation was screened for the presence of the FS I-I transgene as described above and used for analyses.
Immunoblot analysis
Sections of the quadriceps femoris muscles from FS I-I transgenic mice and age-matched control mice were homogenized in a buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM NaF, 5 mM β-glycerophosphate, 1 mM Na vanadate, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 4 µg/ml leupeptin, and 1 µg/ml aprotinin) and centrifuged at 15,000 rpm for 10 min at 4°C. The supernatants were recovered as cell lysates. Aliquots of the lysates containing 250 µg of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (27)
. The membranes were probed with antiphosphorylated Smad2, anti-Smad2, antiphosphorylated Erk1/2 and anti-Erk1/2 antibodies (all obtained from Cell Signaling Technology, Beverly, MA, USA). After incubation with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence reactions, images of the developed immunoblots were captured using a cooled CCD camera system (Light-Capture; ATTO, Tokyo, Japan).
Immunohistochemical analysis and determination of muscle fiber size and number
Mice were subjected to histological analysis at 5, 9, and 13 wk of age. The quadriceps femoris and diaphragm muscles were snap-frozen in liquid nitrogen-cooled isopentane and sectioned using a cryostat (Leica Microsystems Japan, Tokyo, Japan). The sections were stained with hematoxylin and eosin. The myofiber size and total myofiber number in individual skeletal muscles were measured from fluorescence images of antilaminin-2-stained sections using WinROOF software (Mitani Corporation, Fukui, Japan). Infiltration of macrophages into the skeletal muscles was detected by staining with an anti-Mac1 (CD11b/18) antibody (COSMO BIO, Tokyo, Japan).
Rotarod analysis and grip strength test
A rotarod RRAC-3002 (O’Hara & Co. Ltd., Tokyo, Japan) was used. Mice were subjected to the rotarod analysis in the acceleration mode (3–30 rpm/min) over 5 min. In total, 21 wild-type male mice and 20 FS I-I male mice were analyzed.
The peak grip force was measured using an MK-380S grip strength meter (Muromachi Kikai Co. Ltd., Tokyo, Japan), according to the manufacturer’s protocol. Male mice (9–13 wk of age) were allowed to grasp a wire mesh with their forelimbs and hindlimbs, and then pulled steadily by their tails horizontally until they lost their grip. The maximal force values were recorded. Measurements were performed 8 times using wild-type mice, FS I-I transgenic male mice, mdx mice and mdx/FS I-I mice (n=6–8).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). Since the two mutants were found to have similar activities and FS I-I is shorter than FS I-I-III, we characterized FS I-I in the subsequent experiments. Interestingly, FS I-I retained its myostatin binding but showed a significantly weaker activin-binding activity. The results of solid-phase binding assays of GST-FS I-I with myostatin and activin A are shown in Fig. 1B
. GST alone did not show any binding to either myostatin or activin. In contrast, GST-FS I-I lost its binding activity toward activin but bound to myostatin in a dose-dependent manner (Fig. 1B
). To determine the affinities of FS I-I for myostatin and activin, we prepared a highly purified FS I-I recombinant protein. Since the naive FS I-I protein showed weak heparin-binding activity and was difficult to purify, we used an FS I-I-Fc fusion protein and characterized its binding activities toward myostatin and activin by SPR biosensor analysis. First, we analyzed the affinities of FS for myostatin and activin A. The association constant (kon) of FS for activin A was slightly faster than that for myostatin, while the dissociation constants (koff) of FS for myostatin and activin A were comparable. We further calculated the affinities (Kd) of FS for myostatin and activin A to be 12.3 and 1.72 nM, respectively (Table 1
). Next, we analyzed the binding activities of FS I-I-Fc to myostatin and activin. Myostatin and activin A were separately immobilized on sensor chips, and various concentrations of FS I-I-Fc were passed over these sensor chips as analytes. As shown in Table 1
, the association constant (kon) of FS I-I-Fc for myostatin was much faster than that for activin A, while the dissociation constants (koff) of FS I-I-Fc for myostatin and activin A were comparable. On the basis of the association and dissociation rate constants obtained through the SPR analyses, we calculated the affinities (Kd) of FS I-I-Fc for myostatin and activin A to be 46.8 nM and 64.3 µM, respectively (Table 1)
. Thus, FS showed high-affinity binding activities toward both myostatin and activin, whereas FS I-I retained the high-affinity myostatin-binding activity but lost the high-affinity activin-binding activity.
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Next, we studied the actions of FS I-I on the myostatin- and activin-signaling pathways. The intracellular signaling pathway of myostatin is similar to those of activin and TGF-β. Myostatin signals through a combination of activin type II receptors (ActRIIB and ActRIIA) and activin receptor-like kinases 5 and 4 (ALK5 and ALK4) (34)
. The activated receptors phosphorylate Smad2/3, which then associate with the common Smad, Smad4, and translocate into the nucleus to activate gene transcription (1
, 2
, 16)
. Myostatin signaling can be monitored by a conventional reporter assay, called the CAGA-lux assay, in human A204 rhabdomyosarcoma cells, which are responsive to both myostatin and activin. The reporter activity stimulated by myostatin was repressed by coincubation with FS I-I-Fc in a dose-dependent manner (Fig. 2
A, left panel). The IC50 (median inhibitory dose) of FS I-I-Fc for inhibition of 20 ng/ml of myostatin was estimated to be 60 ng/ml. In contrast, the reporter activity stimulated by activin was not repressed by coincubation with FS I-I-Fc, even at 1 µg/ml (Fig. 2A
, right panel). As expected, FS efficiently inhibited both the myostatin and activin activities. The myostatin-inhibiting activity of FS I-I was further studied in another cell line, CHO-K1 cells (Fig. 2B
). In this experiment, the myostatin signaling was augmented by exogenous transfection of ActRIIB expression plasmid either with wild-type ALK5 or ALK4 expression plasmids. Similar to the A204 cells, the myostatin activity was efficiently blocked by FS I-I. We also studied the actions of FS I-I using a different reporter construct. 10T1/2 cells differentiate into myoblastic cells on forced expression of a MyoD cDNA, and myostatin is capable of repressing the activity of a MyoD-dependent reporter, 6R-lux. In this experiment, Smad3 and wild-type ALK5 or ALK4 plasmids were cotransfected to evaluate the actions of myostatin. As shown in Fig. 2C
, the inhibition of the MyoD-dependent reporter activity by myostatin was recovered by coincubation with FS I-I-Fc. In summary, FS I-I was capable of inhibiting the actions of myostatin in multiple assays but hardly affected the activin activity. These observations are consistent with the low binding activity of FS I-I toward activin (Fig. 1B
and Table 1
).
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Analysis of transgenic mice overexpressing FS I-I in skeletal muscles
To evaluate the actions of FS I-I in vivo, we overexpressed FS I-I in mice. FS I-I was placed downstream of a skeletal muscle-specific myosin light chain promoter (Fig. 3
A) (33)
. Four founders positive for the transgene by PCR genotyping were obtained (Fig. 3B
). Protein expression of FS I-I was also verified by Western blot analysis (Fig. 3C
). We selected one high-expressing line that showed significant increases in skeletal muscle mass for further characterization. Lysates from the quadriceps femoris muscles of male FS I-I transgenic mice and matched wild-type littermates were prepared and subjected to immunoblot analysis for phosphorylated and total Smad2 (Fig. 4
A). Although the total Smad2 levels were comparable between wild-type and transgenic mice, phosphorylated Smad2 was reduced in the transgenic mice compared with the level in wild-type mice. Since FS I-I inhibits myostatin in vitro, and myostatin signals through phosphorylation of Smad2 and Smad3, it is reasonable that the FS I-I expressed in skeletal muscles suppressed myostatin signaling in FS I-I transgenic mice. Interestingly, we found that phosphorylation of Erk1/2, a MAP kinase family member, was also reduced in FS I-I transgenic mice. Next, changes in the body weight and skeletal muscle mass were studied (Fig. 4B
). No difference in the weights was detected until 5 wk of age. From 6 to 14 wk of age, the weight of the transgenic male mice was greater than that of wild-type littermates. However, after 15 wk, the weight of the wild-type mice became larger than that of FS I-I transgenic mice. This is likely to be due to an increase in the adipose tissue mass in wild-type mice. Transgenic mice exhibited pronounced muscles, especially the pectoralis major, triceps brachii, gluteus, and quadriceps femoris muscles (Fig. 4C
). Representative skeletal muscles were dissected out and weighed. The weight of the tibialis anterior muscle of transgenic mice showed 1.33-fold increase compared with that of wild-type mice (Fig. 4D
and Table 2
). Other individual muscles also showed increases (Table 2)
. To investigate whether the increases in skeletal muscle mass and weight arose from hypertrophy and/or hyperplasia, we measured the myofiber size and number (Table 2)
. As shown in Table 2
, FS I-I transgenic mice and their wild-type littermates had 3,372 ± 467 and 2,707 ± 284 fibers in their tibialis anterior muscles, respectively, indicating that the fiber number in the transgenic mice showed 1.25-fold increase. In other skeletal muscles such as the extensor digitorum longus, the fiber numbers were increased by 1.2-fold. Next, we quantified the myofiber size by staining each fiber of the quadriceps femoris muscle with an antilaminin-2 antibody (Fig. 4E
). Enlargement of the myofibers in FS I-I transgenic mice was noted. Quantification of the fiber areas of individual myofibers is shown in Fig. 4F
. The mean fiber areas were increased in FS I-I transgenic mice. The estimated mean fiber sizes in wild-type littermates and FS I-I transgenic mice were 1924 and 2504 µm2, respectively. These results indicate that both hypertrophy and hyperplasia were responsible for the increase in muscle mass in FS I-I transgenic mice.
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FS I-I transgenic mice did not show any behavioral abnormalities and reproduced normally. To assess the functionality of the increased muscle mass, we used two independent methods, namely the rotarod test and grip strength test. In the rotarod test, FS I-I transgenic mice performed better than control mice (Fig. 5
A). The endurance times for wild-type and FS I-I transgenic mice were 160.8 ± 48.7 and 211.8 ± 76.6 s, respectively. We further studied the muscle function by the grip strength test (Fig. 5B
). In the grip test, the mice grasped a mesh with both their forelimbs and hindlimbs, and the horizontal force required to dislodge their grip was measured. The mean values for wild-type and FS I-I transgenic mice were 218.4 ± 17.0 and 297.9 ± 32.7 g, respectively. Thus, these two independent tests for assessing muscle strength both indicated that the skeletal muscles of FS I-I transgenic mice were functional and had increased strength.
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Amelioration of the pathophysiology of mdx mice by crossing with FS I-I transgenic mice
Myostatin inhibition is one of the most promising therapeutic approaches for muscular dystrophy. We analyzed the effects of FS I-I on the mdx mouse model of Duchenne muscular dystrophy. mdx mice are deficient in the dystrophin gene carried on the X chromosome. Male FS I-I mice were crossed with female mdx mice to generate mdx/FS I-I mice. The skeletal muscles of mdx/FS I-I mice were larger than those of mdx mice (Fig. 6
A). One of the characteristics of mdx mice was irregularity of myofiber size. The diameters of the myofibers in the quadriceps femoris of mdx/FS I-I mice became larger than those of mdx mice (Fig. 6A
). More importantly, the sizes of the myofibers in mdx/FS I-I mice were homogeneous (Fig. 6A
). Massive cell infiltration and tissue damage were evident in the skeletal muscles of mdx mice (Fig. 6B
). Interestingly, at both early (5 wk, Fig. 6B
; see also ref. 1
) and late (9 wk, Fig. 6B
; see also ref. 2
) phases, the cell infiltration and fibrous changes observed in the diaphragm of mdx mice were significantly reduced in mdx/FS I-I mice. At 9 wk of age, several sections without cell infiltration were observed histologically (Fig. 6B
; see also ref. 2
). Similarly, recovery from cell infiltration was observed in the quadriceps femoris muscle in mdx/FS I-I mice (Fig. 6C
). We quantified the cell infiltration in the skeletal muscles by counting Mac1-positive macrophages in tissue sections of the diaphragm. In mdx mice, 8.45% of the total surface area of the diaphragm was positive for Mac1, compared to only 4.03% in mdx/FS I-I mice, indicating that macrophage infiltration was significantly reduced in mdx/FS I-I mice. In addition, a grip strength test showed that the muscle strength in mdx/FS I-I mice was increased compared with that in mdx mice and became comparable with that of wild-type mice (Fig. 6E
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Inhibition of myostatin is capable of increasing skeletal muscle mass, even in adults, and myostatin blockade could increase skeletal muscle mass regardless of the type of muscular dystrophy. Experimentally, the pathology of the mdx mouse model of Duchenne muscular dystrophy can be recovered by myostatin inhibition (11
, 12
, 36)
. In addition, three models of limb-girdle muscular dystrophy, including 
-sarcoglycan deficiency, caveolin-3 mutation, and calpain-3 deficiency, are also ameliorated by myostatin blockade (37
38
39)
. Even the muscle atrophy associated with amyotrophic lateral sclerosis and aging can be ameliorated by myostatin inhibition (40)
. It is also noteworthy that elimination of myostatin did not combat a laminin-2-deficient dyw mouse model but did increase postnatal lethality due to fat loss in one study (13)
.
There are multiple ways to inhibit myostatin (1
, 2)
. Myostatin propeptide, FS, FLRG, and antimyostatin antibodies bind to myostatin and inhibit its activities. FS is a naturally occurring hormone that shows potent myostatin inhibition (2)
. It is also an activin-binding protein that causes activin inhibition (2
, 16
, 20)
. Myostatin is primarily produced in skeletal muscles and acts specifically on skeletal muscles. In contrast, activins are produced by various organs, including the gonads, pituitary, brain, liver, and gastrointestinal tract, and have pleiotrophic effects on various cell types.
In the present study, we developed a myostatin-specific inhibitor derived from FS, which did not affect the activin bioactivity. This molecule, designated FS I-I, consists of the FS N-terminal domain and two consecutive FS I domains. The precise mechanisms for how FS I-I discriminates between myostatin and activins remain to be determined. Recent crystallographic analyses have revealed that the minimal activin-inhibiting fragment of FS comprises the FS I and FS II domains. Two FS I-II molecules encircle and wrap around the back of the ‘wings’ of activin, thereby blocking its type II receptor-binding site. In particular, arginine residue 192 in the FS II domain is a key player in this interaction and inserts itself into the activin finger (22
, 23)
. It is also worthwhile to note that an analogous arginine residue is conserved in the COOH-terminal activin-binding region of FLRG (22
, 26)
. Although myostatin and activins are structurally similar among the TGF-β superfamily members, several key residues are different (1)
. Our findings that FS I-I binds and inhibits myostatin much more effectively than its effects on activin argue that the FS II domain, which is important for binding to activin, could be dispensable for the interaction with myostatin, although the precise mechanism remains to be determined.
We established transgenic mouse lines stably expressing FS I-I under the control of a skeletal muscle-specific promoter. The FS I-I overexpressing mice showed increased skeletal muscle mass. The mice were fertile and did not show any abnormalities except for increased muscle mass. A lack of reproductive phenotype in FS I-I overexpressor could be due to low-affinity binding of FS I-I to activin in vivo. However, since a skeletal muscle-specific promoter was used, it is likely that FS I-I did not express at high levels in tissues other than skeletal muscle where activins were expressed. The individual weights of the tibialis anterior, quadriceps femoris, and extensor digitorum longus muscles were apparently increased. The masses of other skeletal muscles were also increased, whereas the sizes of other internal organs, including the heart, did not differ from those of control mice (data not shown). Both hypertrophy and hyperplasia of the myofibers were found to be responsible for the increased muscle weight.
Similar to our FS I-I transgenic mice, both hypertrophy and hyperplasia are observed in skeletal muscles in myostatin knockout mice (3)
. The myosin light chain promoter used in the present study is activated weakly during the prenatal period and becomes active in the postnatal period (41)
. Thus, myostatin is inhibited after the postnatal period in FS I-I transgenic mice, whereas myostatin is missing throughout embryonic development and the postnatal period in myostatin knockout mice. Our findings argue that inhibition of myostatin, even in the postnatal period, could regulate both the number and size of the myofibers. Interestingly, the increased skeletal muscle mass induced by myostatin inhibition can be caused by hyperplasia without hypertrophy (42
, 43)
, hypertrophy without hyperplasia (44
, 45)
or both hyperplasia and hypertrophy (3
, 20
, present study). The question of whether these differences are caused by species differences and/or the extents of myostatin inhibition remains to be determined.
The functionality of the skeletal muscles of FS I-I transgenic mice was studied by two independent approaches, namely the rotarod test and grip strength test. Both of these analyses revealed that the skeletal muscles of FS I-I transgenic mice were functional and had increased strength. The possibility that FS I-I could ameliorate the pathophysiology of mdx Duchenne muscular dystrophy model mice was studied by crossing FS I-I transgenic mice with mdx mice. The myofibers of the skeletal muscles in mdx/FS I-I mice became homogeneous and were larger than those in mdx mice. Furthermore, the fibrosis and cell infiltration normally observed in the diaphragm and quadriceps femoris muscle of mdx mice were significantly ameliorated. Muscle strength was also recovered in mdx/FS I-I mice.
In summary, we have developed and characterized a myostatin inhibitor, FS I-I, derived from FS. The myostatin inhibition by FS I-I is capable of promoting muscle regeneration and preventing muscle fibrosis in mdx mice without any adverse effects. An antimyostatin antibody is currently under investigation in clinical trials for the treatment of several forms of muscular dystrophy. Thus, similar to other myostatin inhibitors, FS I-I could be a candidate for a lead compound for the development of drugs for muscular dystrophy. Although myostatin inhibition by FS I-I did not restore the dystrophin expression in mdx mice, it could offer effective amelioration of the symptoms of muscular dystrophy caused by the primary loss of dystrophin.
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ACKNOWLEDGMENTS |
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Received for publication April 1, 2007. Accepted for publication August 16, 2007.
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) toward myostatin and activin A. ELISA plates coated with myostatin (1) or activin (2) were incubated with various amounts of GST fusion proteins. The relative binding activities were quantified by a colorimetric assay. GST does not bind to either myostatin or activin, whereas GST-FS I-I shows myostatin-binding activity in a dose-dependent manner.
) and FS I-I transgenic mice (FS I-I, 
). Mice were weighed at the indicated weeks (n=5). *P < 0.001, Student’s t test. C) Phenotypes of FS I-I transgenic mice (left) and wild-type littermates (right). Pictures of the pectoral regions (top panels, 1), quadriceps femoris muscles (middle panels, 2) and upper and lower limbs (bottom panels, 3) of skinned transgenic mice and wild-type littermates are shown. D) Pictures of dissected skeletal muscles from FS I-I transgenic and wild-type littermates. TA, tibialis anterior muscle; EDL, extensor digitorum longus muscle; Qf, quadriceps femoris muscle. E) Immunohistochemical analysis of the quadriceps femoris muscles from FS I-I transgenic mice and wild-type littermates using an antilaminin-
