Follistatin 315aa, 344
(Activin Type 2B), GHRP6,
HMP, HGH (Jintropin), Melanotan II, PT 141, IGF1-LR3, CJC-1295, PEG MGF, Ipamorelin
FST, FS, Activin-binding protein.
Follistatin (FS) was initially identified
as a follicle-stimulating hormone inhibiting substance found in ovarian
follicular fluid. FS is a high-affinity activin-binding protein that can act
as an activin antagonist. Two alternatively spliced follistatin mRNAs exist,
encoding mature FS with 288 amino acid (aa) residues (FS-288) and 315 aa
residues (FS-315). Natural FS purified from porcine ovaries is primarily a
carboxy-terminal truncated form of FS-315 composed of 300 aa residues.
Follistatin is a single-chain gonadal protein that
specifically inhibits follicle-stimulating hormone release. The single FST
gene encodes two isoforms, FST317 and FST344 containing 317 and 344 amino
acids respectively, resulting from alternative splicing of the precursor
mRNA. In a study in which 37 candidate genes were tested for linkage and
association with polycystic ovary syndrome (PCOS) or hyperandrogenemia in 150
families, evidence was found for linkage between PCOS and follistatin.
Follistatin binds directly to activin and functions as an activin antagonist.
specific inhibitor of the biosynthesis and secretion of pituitary follicle
stimulating hormone (fsh).
Follistatin Human Recombinant produced in
E.Coli is a single, non-glycosylated polypeptide chain containing 288 amino
acids and having a total molecular mass of 31.5kDa.
The FST is purified by proprietary chromatographic
Sterile Filtered White lyophilized
Lyophilized from a concentrated (1mg/ml)
solution containing no additives.
It is recommended to reconstitute the
lyophilized Follistatin in sterile 18MΩ-cm H2O not less than 100µg/ml, which
can then be further diluted to other aqueous solutions.
Lyophilized Follistatin although stable
at room temperature for 3 weeks, should be stored desiccated below -18°C.
Upon reconstitution FST should be stored at 4°C between 2-7 days and for
future use below -18°C.
For long term storage it is recommended to add a carrier
protein (0.1% HSA or BSA).
Please prevent freeze-thaw cycles.
Greater than 95.0% as determined by(a)
Analysis by RP-HPLC.
(b) Analysis by SDS-PAGE.
Amino acid sequence
The sequence of the first five N-terminal
amino acids was determined and was found to be Gly-Asn-Cys-Trp-Leu.
The activity is determined by the ability
to neutralize Activin A inhibitory effect of mouse MPC-11 cells. The expected
ED50 is 0.1-0.4 µg/ml in the presence of 7.5 ng/ml Activin A.
ProSpec's products are furnished for
LABORATORY RESEARCH USE ONLY.They may not be used as drugs,agricultural or
pesticidal products, food additives or household chemicals.
muscles for life with just one injection of follistatin gene
You can nag
your dealer until he pins you on to the fender of his SUV, but you still won’t
get any. We’re talking about the anabolic wonder stuff that researchers at Ohio
State University are doing experiments with. A single injection will change you
for the rest of your life into a hulk of the kind that Markus Ruehl [see photo
below] would say: that much muscle just isn’t aesthetic.
steroid in question is not a hormone. It’s a common-cold virus that the
researchers have made some adjustments to.
invade cells and release their genes
into them. Then the cell obeys the virus genes’ instructions, and makes the
proteins according to the genes’ blueprint. The proteins formed are the building
blocks for new viruses.
researchers managed to get their viruses to ‘programme’ the muscle cells to
make proteins that deactivated the myostatin protein. Myostatin
is a protein that muscle cells make to prevent fitness centre owners from
having to change jobs and become SUV salesmen.
Not that the
researchers had something against fitness centre owners. They are looking for a
cure for muscular dystrophy. In their study, which was published in the
prestigious PNAS, they tested three myostatin inhibiting genes: the gene for
growth and differentiation factor-associated serum protein-1 (GASP-1),
follistatin-related gene (FLRG) and the gene for follistatin-344 (FS).
various kinds of follistatin and they all have different functions. As far as
we know, only follistatin-344 is active in muscle tissue.
The mice in
the experiment were given an injection when they were four weeks old. The
photos below show their musculature two years later. AAV1 stands for the
adenovirus that the researchers used. AAV1-GFP was the control group.
injection with the gene for follistatin-344 was the most effective. This
becomes clearer if you look at the figure below, which shows the weight of the
mice two years after the injection.
beautiful of all is the graph below. This shows how the mice’s power developed
over their lifespan. The mice in the control group – the green curve – get
weaker towards the end of their life. But the mice that were injected with
AAV1-FS – the red curve – just keep on getting stronger.
researchers obtained the same success when they injected the viruses into mice
that had congenital muscle disease. "The striking ability of FS to
provide gross and functional long-term improvement to dystrophic muscles in
aged animals warrants its consideration for clinical development to treat
musculoskeletal diseases, including older DMD patients", they write.
Only a few
years, and muscular diseases will be a thing of the past. And all fitness
centres will go bust.
Proc Natl Acad Sci U S A.
2008 Mar 18;105(11):4318-22.
myostatin blockers to grow? Add clen and you'll grow more
athletes and their gurus in the doping world have high hopes for myostatin
blockers that pharmaceutical companies are testing right now on people
with cancer or muscular diseases. Their hopes are well founded – animal studies
have shown that myostatin blockers can cause massive growth of muscle mass. But
yet another animal study, soon to be published in Muscle & Nerve, suggests
that the combination of a myostatin blocker and an old-school doping substance
like clenbuterol results in even more growth.
a muscle protein that keeps muscle growth in check. Myostatin blockers
deactivate the protein and its effect. Because myostatin is active pretty much
only in the muscles and nowhere else, scientists hope that myostatin blockers
will be the long hoped-for anabolic without side effects. Time will tell whether
their hopes are justified. In animal studies blocking myostatin has reduced stamina, and weakened
don't yet agree on how best to deactivate myostatin. Pharmaceuticals companies
like Acceleron are doing tests on synthetic imitation receptors that
replace and neutralise myostatin. Another approach is to boost
follistatin synthesis. Follistatin is a protein that deactivates myostatin in
the muscle cells. Yet another approach makes use of the immune system.
agricultural researchers for example are looking at the possibility of injecting
massive quantities of myostatin. These may well lead to muscle tissue breakdown
in the short term, but in the long term the immune system would come to regard
myostatin as a foreign substance that the body needs to break down.
that molecular scientists at the University of Hawaii will publish soon is
fundamental research. The authors used mice in which the myostatin gene had been
deactivated – a bit like Mighty Mouse. The muscles of this type of mice are
shown in the lower of the two photos at the top of the page. The top photo
shows the muscles of a normal mouse. The researchers wanted to know whether
these supermice could develop even bigger muscles by giving them a doping
substance like clenbuterol [structural formula shown above]. And yes, it
myostatin-less mice, W = normal mice. 0 ppm = no clenbuterol, 20 ppm = with
experiment only lasted 14 days. The table above shows that during that period
normal mice built up extra muscle mass when they were given clenbuterol, but
the myostatin-less mice did so too. Super-muscled myostatin-less mice gain even
more muscle through old-school doping. That's promising.
forget though: the WADA is already working on a doping test for myostatin
Muscle Nerve. 2011 May;43(5):700-7.
shot for more muscle
the protein myostatin has become even easier. In the Animal
Science Journal researchers at the Chinese Sichuan Agricultural University
describe how they made pigs more muscular by giving them four injections of
myostatin. As a result of the injections, the pigs’ immune system broke down the myostatin.
produced by muscle cells to limit their own growth. The more
myostatin your muscles make, the more difficult it is to build up muscle mass and the easier it is to break it
down. That’s why researchers are studying ways to deactivate myostatin.
Pharmaceuticals companies are already testing myostatin blockers in the hope that they can find
ways of treating muscular diseases. But most myostatin research actually takes
place at agricultural universities, where researchers are
livestock farmers by developing monster-size salmon, cattle, chickens and pigs.
The cattle are there already – Belgian Blues for example – because through a
freak of nature they don’t produce myostatin, as in the photo above.
experimented with a simple technique: they got micro-organisms to produce
myostatin and injected 1 mg or 4 mg of the protein into pigs on days 1, 14, 28
and 42 of an experiment that lasted 84 days. The pigs’ immune systems regarded
the protein as alien and produced antibodies to neutralise the myostatin. The
amount of antibodies produced is shown in the figure below.
The genes in
the muscle cells that produce myostatin started to work less hard as a result
of the myostatin injection.
were predictable. The table below shows that the animals lost fat and gained muscle. While not
spectacular, the effect is significant. The 4 mg injections worked as well as
the 1 mg injections.
much media speculation about the use of new doping substances by athletes, such
as techniques that involve genetic manipulation. In practice these techniques
are often not suitable for human use. What works in a lab is often too
complicated to use in humans. But if a technique is simple enough for livestock
farmers to use, then it should be suitable for athletes. It won’t be long
before we see myostatin manipulation in the doping world.
In fact, we
wonder: is the hormone mafia already messing around with myostatin?
Sci J. 2009 Oct 1; 80(5): 585-90.
Long-term enhancement of skeletal muscle mass and strength by single gene
administration of myostatin inhibitors
*The Research Institute, Nationwide Children's
Hospital, Columbus, OH 43205; and
†Integrated Biomedical Science and
‡Neuroscience Graduate Programs, Ohio State
University, Columbus, OH 43210
M. Haidet * , † ,
Handy * , † ,
T. Martin * , † , ‡ ,
Sahenk * , † , ‡ ,
R. Mendell * , ‡ ,
K. Kaspar * , † , ‡ , §
by Joshua R. Sanes, Harvard University, Cambridge, MA, and approved February 5,
2008 (received for review September 25, 2007)
Increasing the size and strength of muscles represents a promising
therapeutic strategy for musculoskeletal disorders, and interest has focused on
myostatin, a negative regulator of muscle growth. Various myostatin inhibitor
approaches have been identified and tested in models of muscle disease with
varying efficacies, depending on the age at which myostatin inhibition occurs.
Here, we describe a one-time gene administration of
myostatin-inhibitor-proteins to enhance muscle mass and strength in normal and
dystrophic mouse models for >2 years, even when delivered in aged animals.
These results demonstrate a promising therapeutic strategy that warrants consideration
for clinical trials in human muscle diseases.
Muscle-enhancing strategies have been proposed for a number of neuromuscular
disorders, including muscular dystrophies and age-related muscle disorders, and
have shown promising results to build or regenerate stronger, healthier muscles
strategies have mainly focused on the use of trophic factors, such as
insulin-like growth factor-1 that induce myocyte precursor proliferation and
myofiber hypertrophy (2). Attention has
recently highlighted the potential benefit for inhibiting myostatin, resulting
in the doubled muscle phenotype of myostatin deficient cattle (3–5) and myostatin
knockout mice (6,
is a transforming growth factor-β (TGF-β) family member that plays a crucial
role in regulating skeletal muscle mass (8, 9). Myostatin
appears to function in two distinct roles: to regulate the number of myofibers
formed in development and to regulate the postnatal growth of muscles. The
regulation of muscle growth postnatally is being explored by various
pharmacological methods for a number of muscle disorders. Delivery of
neutralizing antibodies against myostatin has shown promise in dystrophic mdx
yet there have been varying reports on the efficacy to enhance muscles when
delivered in aged animals (11). Furthermore,
recent data demonstrated muscle mass enhancement and morphological recovery in
muscular dystrophy mice treated with deacetylase inhibitors. The resulting
muscle enhancement was attributed to an increase in the protein follistatin,
which has been shown in part to inhibit the activity of myostatin (12).
Trichostatin A (TSA) treatment required daily administration and was not
evaluated in aged animals where off target effects may exist.
The identification of myostatin binding proteins capable of regulating
myostatin activity has led to potential new approaches for postnatal muscle
enhancement and expanded the potential for gene therapy to be considered as a
method to inhibit myostatin activity. Follistatin (FS) has been shown to bind
to some TGF-β family members and can function as a potent myostatin antagonist.
Overexpression of follistatin by transgenic approaches in muscle has been shown
to increase muscle growth in vivo (13), and a lack
of follistatin results in reduced muscle mass at birth (14). Recent data
has also shown that follistatin is capable of controlling muscle mass through
pathways independent of the myostatin signaling cascade. In these studies,
myostatin knockout mice were crossed to mice carrying a follistatin transgene.
The resulting mice had a quadrupling of muscle mass compared with the doubling
of muscle mass that is observed from lack of myostatin alone, confirming a role
for follistatin in the regulation of muscle mass beyond solely myostatin
In addition to follistatin, two other proteins have been identified that are
involved in the regulation of the myostatin. Follistatin-related gene (FLRG) is
highly similar to follistatin and has been shown to inhibit activin and
multiple bone morphogenic proteins in vitro (16, 17). Growth and
differentiation factor-associated serum protein-1 (GASP-1) is a protein that
has been discovered to contain multiple domains associated with
protease-inhibitor proteins and a domain homologous to the 10-cysteine repeat
found in follistatin. GASP-1 was shown to bind directly to the mature myostatin
and myostatin propeptide and inhibits myostatin's activity (18). Although
recombinant protein injections or myostatin blocking antibodies are feasible
strategies, gene therapy to express these myostatin inhibitor genes may prove a
more efficacious therapeutic route for numerous reasons, including the lack of
potential immune response to antibody treatment and the requirement for
Here, we report that a one-time postnatal intramuscular injection of
adeno-associated virus (AAV) encoding myostatin-inhibitor-proteins resulted in
long-term improvement of muscle size and strength in wild-type animals.
Delivery of a myostatin-inhibitor-protein in dystrophic mdx
animals reversed muscle pathology and improved strength, even when administered
in 6.5-month-old animals. Specifically, we show here that follistatin-344
resulted in the greatest effects on muscle size and function and was well
tolerated with no untoward effects on cardiac pathology or reproductive
capacity in either male or female treated animals.
Results and Discussion
AAV-mediated gene delivery to muscle provides a system to generate high
levels of protein in the target tissue or by a secreted product carried to
remote sites through the circulation (19). We cloned
the known secreted myostatin-inhibiting genes, including growth and
differentiation factor-associated serum protein-1 (GASP-1) (18),
follistatin-related gene (FLRG) (17), and
follistatin-344 (FS) (13) into AAV
serotype 1, which have demonstrated high muscle transduction capabilities.
There are two isoforms of follistatin generated by alternative splicing. The
FS-344 variant undergoes peptide cleavage to generate the FS-315 isoform and
the other FS-317 variant produces the FS-288 isoform after peptide cleavage. We
used the human FS-344 variant, which exclusively generates the serum
circulating FS-315 isoform of FS and includes a C-terminal acidic region (20). We chose
FS-344 (FS), because the other FS-317 isoform, lacking the C terminus, shows
preferential localization to the ovarian follicular fluid and high tissue
binding affinity through heparin sulfate proteoglycans, which may affect
reproductive capacity and bind to other off-target sites (21). FS-288
represents the membrane-bound form of follistatin (22), is a potent
suppressor of pituitary follicle stimulating hormone (23), is found in
the follicular fluid of the ovary and in the testes, and demonstrates a high
affinity for the granulosa cells of the ovary.
We sought to determine the efficacy of these proteins to increase muscle
mass in normal and dystrophic mice. We administered 1 × 1011 AAV1
viral particles per animal encoding FS, FLRG, GASP-1, or GFP bilaterally into
the quadriceps and tibialis anterior muscles of 4-week-old wild-type C57BL/6
mice. All animals treated with the myostatin inhibitors demonstrated an
increase in body mass with an observable gross enhancement of muscles when analyzed
at 725-days of age compared with GFP-treated controls (Fig. 1 a
and b). Evaluation of individual muscle weights showed
an increase in muscle mass for all myostatin inhibitor-treated animals, with
the greatest increase in FS-treated animals. The increased muscle mass was
found in the injected hindlimb muscles and remote muscles to the injection
site, such as the triceps. Thus, these inhibitors were secreted into the
circulation from the site of muscle injection, enhancing skeletal muscle mass
at remote sites (Fig.
1 c). The enlarged muscle mass was accompanied by
functional improvement demonstrated by an increase in hindlimb grip strength (Fig. 1 d).
There was no effect on heart mass or histological appearance of cardiomyocytes,
indicating that myostatin inhibition was selective to skeletal muscle tissue
(data not shown). There has been concern that FS adversely effects gonadal
function. We found no change in reproductive capacity in mice treated with our
AAV1 carrying the FS344 transgene (AAV1-FS, Table 1)
Furthermore, we found no histological/pathological alterations in the gonadal
tissue of FS treated-mice compared with controls (data not shown).
Myostatin inhibitor proteins increase muscle mass and
strength in wild-type C57BL/6 mice. (a) Gross
hindlimb muscle mass is increased in all myostatin-inhibitor-protein treated
mice at 725 days of age compared with AAV1-GFP injected controls. (b)
Total body mass is significantly increased in AAV1-FS-injected (**, P
≤ 0.01) and AAV1-GASP-1-injected (*, P ≤ 0.05)
mice compared with AAV1-GFP controls at 725 days of age (n
= 10). (c) The mass of individual hindlimb and forelimb
muscles is increased in mice injected with AAV expressing myostatin inhibitor
proteins (n = 10). *, P
≤ 0.05. (d) Hindlimb grip strength improves >2 years in
all treated mice with the greatest differences in AAV1-FS treated animals
compared with AAV1-GFP controls (n = 10). Error bars represent
Reproduction was normal in animals treated with AAV1-FS
Given the robust effects of FS delivery, we next tested the potential for
AAV1-FS delivered postnatally in a clinically meaningful paradigm to increase
muscle mass and strength and delay muscle deterioration in the mdx
mouse model of Duchenne muscular dystrophy (DMD). DMD is an X-linked recessive
disease resulting in the wasting of skeletal muscles and cardiac function,
ultimately resulting in death. Recently, FS was investigated in mdx
animals overexpressing a duplicated domain of the follistatin gene. Results
demonstrated increased muscle mass and attenuated pathology, although the
results were only documented to 15 weeks of age (24). In our
studies, mdx animals were injected bilaterally in the
quadriceps and tibialis anterior muscles with a low (1 × 1010 viral
particles) or high dose (1 × 1011 viral particles) of AAV1-FS at 3
weeks of age and followed for 5 months before necropsy. Increased levels of
circulating FS were detected in the serum of both low and high dose treated
animals with the high dose expressing the greatest levels of serum detected FS
(high dose, 15.3 ± 2.1 ng/ml; low dose, 6.8 ± 0.4 ng/ml; GFP controls, 0 ± 0.1
ng/ml; n = 8 per group; P <
0.01). We demonstrated that AAV1-FS increased body mass compared with GFP
treated controls, with the greatest increase in the high dose FS group (data
not shown). Gross observation of AAV1-FS treated mice displayed a significant
increase in muscle size compared with AAV1-GFP treated animals (Fig. 2 a),
with the greatest individual muscle weight increase in high dose FS-treated
animals (Fig. 2 b).
Effects were not restricted to the injected muscles; they were also found at
sites remote from directly targeted muscles (Fig. 2 b).
Increased muscle mass translated to a dose-dependent improvement in muscle
strength in the hindlimbs and forelimbs of treated animals compared with GFP
treated controls (Fig.
2 c). Histological and morphometric analyses of
AAV1-FS injected muscles and at remote sites demonstrated myofiber hypertrophy,
supporting gross observations made at the time of necropsy (Fig. 3 a–c).
Furthermore, there was no shift in muscle fiber types in AAV-FS treated
animals; however, there were fewer total fibers per square millimeter of area
in the tibialis anterior muscle in animals treated with the high dose AAV-FS (Fig. 3 d
and e). Strikingly, FS-treated mice demonstrated a
significant reduction in serum creatine kinase compared with GFP-treated
controls (Fig. 4 a).
This is of interest, because FS was protective despite its lack of correction
of the underlying dystrophin deficiency. The exact mechanism is not clear, but
one might speculate that increasing the strength of individual fibers makes
them less susceptible to damage from the stress of normal activities. The
involvement of satellite cells in postnatal myostatin inhibition remains to be
fully resolved; however, we did not see a statistical change in muscle
satellite cell markers for FS-treated animals (data not shown).
Single injection of AAV1-FS increases muscle mass and
strength in young mdx mice. (a)
Gross hindlimb muscle mass is increased in AAV1-FS-injected mdx
animals at 180 days of age compared with AAV1-GFP-injected controls. (b)
The mass of individual hindlimb and forelimb muscles is increased at 180 days
of age in mice injected at 3 weeks of age with AAV1-FS compared with AAV1-GFP
controls (n = 15). *, P
≤ 0.05. (c) Grip strength is improved in a dose-dependent
manner in young mdx mice injected at 3 weeks
of age with AAV1-FS followed for 180 days (n = 15).
Red, high-dose AAV1-FS; blue, low- dose AAV1-FS; green, AAV1-GFP controls.
Error bars represent standard errors.
mdx mice treated with AAV1-FS
at 3 weeks of age and followed for 180 days demonstrate myofiber hypertrophy. (a)
H&E staining of the tibialis anterior reveals myofiber hypertrophy in
AAV1-FS injected muscle compared with AAV1-GFP control. (Original
magnification, ×40.) (b) The mean diameter of dark
(slow-twitch oxidative), intermediate (fast-twitch oxidative glycolytic), and
light (fast twitch glycolytic) myofibers in the tibialis anterior (indicated by
hatched line) is significantly increased in mice injected with AAV1-FS compared
with AAV1-GFP-injected controls. (P <
0.001; n = 5). (c) The mean
diameter of intermediate and light myofibers (indicated by hatched line) in the
triceps is significantly increased in mice injected with AAV1-FS compared with
AAV1-GFP-injected controls. (P < 0.001; n
= 5.) (d) The distribution of dark, intermediate, and light
fibers as determined by succinic dehydrogenase (SDH) staining is not changed by
treatment with high or low doses of AAV1-FS. (P > 0.05
between all groups; n = 5.) (e)
The mean number of fibers counted per an unbiased 0.14 mm2 counting
frame is decreased in the tibialis anterior of AAV1-FS-treated mice, given that
the mean diameter of myofibers is increased. (*, P <
0.01; n = 5.) Error bars represent standard errors.
mdx mice treated with AAV1-FS
show decreased markers of muscle damage and aged mdx mice
are responsive to FS treatment with functional benefit. (a)
Serum creatine kinase levels (units/liter) are decreased at 3 months after
injection with AAV1-FS compared with AAV1-GFP-injected controls. (*, P
< 0.05; n = 10.) Error bars represent
standard errors. (b) Hindlimb grip strength is
significantly increased (P ≤ 0.05) at 275 days and
beyond in aged mdx mice treated with AAV1-FS
at 210 days of age (n = 15). Red, high-dose
AAV1-FS; green, AAV1-GFP controls. (c) H&E
stain of aged gastrocnemius demonstrates reduced pathology when injected at 210
days of age with FS compared with GFP-injected controls. (Original
magnification, ×40.) (d) H&E stained diaphragm
of animals injected at 210 days of age with FS shows less fat replacement than
GFP-injected controls at late stage. (Original magnification, ×20.)
We also evaluated the potential for AAV1-FS to increase muscle strength in mdx
animals when treated at an older age. We found that AAV1-FS injection at 210
days of age increased muscle strength ≈60 days after administration and that
the increased strength persisted long-term throughout the 560 days evaluated in
this study (Fig. 4
b). As early as 180 days of age, before AAV1-FS
treatment, there was evident pathology in muscles of untreated mdx
animals, with prominent endomysial connective tissue proliferation and inflammation
(Fig. 4 c
and d). Pathological evaluation of gastrocnemius and
diaphragm muscles at 560 days of age demonstrated that AAV1-FS treated animals
had substantially fewer focal groups of necrotic muscle fibers and mononuclear
cell infiltrates. Importantly, AAV1-FS treated animals had significantly
reduced focal areas of endomysial connective tissue proliferation, which were
pronounced in GFP treated animals, demonstrating that fibrosis, a hallmark of
muscular dystrophy, was decreased in FS-treated animals (Fig. 4 c).
Pathology in the diaphragm also showed that FS-treatment reduced inflammation
and fatty replacement compared with GFP-treated animals (Fig. 4 d).
Furthermore, AAV1-FS treatment demonstrated significant increases in muscle
fiber diameters at this age compared with control GFP-treated animals (Fig. 4 c
and d). These results demonstrated that myostatin
inhibition by FS treatment was beneficial in aged mdx animals
that had undergone multiple rounds of muscle degeneration and regeneration.
Translation to a clinical parallel suggests that AAV-mediated FS gene therapy
could have potential for the older DMD patient independent of replacing the
missing gene and may have a potential role in combinational therapy similar to
that demonstrated for IGF-1 and minidystrophin gene replacement (25).
These results suggest that inhibition of myostatin by FS-344, delivered by a
single AAV1 injection can enhance muscle size and strength and is well
tolerated for >2-years. The results of FS344 may offer a more powerful
strategy than others targeting solely myostatin because of additive effects,
such as follistatin's involvement in multiple signaling pathways, and the recent
finding demonstrating a reduction in inflammation in a model of endotoxemia (15, 26). The
striking ability of FS to provide gross and functional long-term improvement to
dystrophic muscles in aged animals warrants its consideration for clinical
development to treat musculoskeletal diseases, including older DMD patients.
Materials and Methods
C57BL/6, C57BL/10, and C57BL/10ScSn-DMDmdx/J were
purchased from The Jackson Laboratory. All studies were approved by
Institutional Animal Care and Use Committee.
Cloning and AAV Production.
The cDNA for human follistatin-344 (FS) was obtained from
Origene, follistatin-related gene (FLRG) was obtained from American Type
Culture Collection, and growth and differentiation factor-associated serum
protein 1 (GASP-1) was cloned from a human cDNA library (Clontech). Recombinant
AAV serotype 1 vectors were produced by a contract manufacturing company
AAV Injections and Testing.
Mice received bilateral intramuscular injections of a total
dose of 1 × 1011 viral particles (high = 1 × 1011, low =
1 × 1010) (n = 10–15 per group) at 3–4
weeks of age or at 6.5 months of age. Muscle strength was assessed weekly,
using a grip strength meter (27). Force
measurements were recorded in three separate trials and averaged. Mouse
coordination was tested by using the accelerating rotarod (Columbus
Muscles were dissected, weighed, snap-frozen in liquid
nitrogen-cooled isopentane, cryostat sectioned, and stained by
hematoxylin-eosin (H&E) or succinic dehydrogenase (SDH) for analysis of
fiber diameters. Five animals per group were chosen randomly for muscle fiber
size morphometry. For each analysis, five representative pictures (one central
and four peripheral) were taken of muscle sections, compounding to 0.7 mm2.
Images were captured at ×20 magnification, and diameters were measured with a
calibrated micrometer, using the AxioVision 4.2 software (Zeiss). Fiber
size-distribution histograms were generated and expressed as percentage of
total fibers analyzed.
Creatine Kinase and Follistatin Assay.
Serum CK was performed by using a CK test kit (Pointe
Scientific) and expressed as units/liter. Serum was collected at 90 days after
injection and assayed by using the human follistatin quantikine ELISA kit
(R&D Systems) with normalization to controls.
All statistical analysis was performed in Graph Pad Prizm
software, using one- and two-way ANOVA with Bonferroni post hoc analysis.