Molecular Exercise Physiology
Skeletal Muscle Hypertrophy
Presentation 8
Henning Wackerhage
Learning outcomes
At the end of this presentation you should be able to:
• Explain the effects of myostatin knockout on muscle growth.
• Explain how myostatin signalling may inhibit muscle growth
TGF-b signalling
The TGFb superfamily is a large group of proteins that regulate
growth, differentiation and apoptosis. One subclass within the TGFb
superfamily are growth and differentiation factors (GDFs). Myostatin
is a GDF whose knockout in mice resulted in skeletal muscle
hypertrophy and hyperplasia (McPherron et al., 1997). The muscles
of myostatin knockout mice are shown on the next slide.
Myostatin knockout mice
Wildtype
Myostatin
knockout
McPherron et al. (1997) knocked out growth and development factor 8
(gdf-8) which was later termed myostatin. The absence of myostatin
leads to muscle hypertrophy (e) compared to the wildtype (d).
Figure from Lee et al. (1999)
Myostatin also affects fibre type
Myostatin knockout (MSTN-/-) also leads to a reduction in type I
fibres
when
compared
to
the
wildtype
Girgenrath et al. (2005).
Myostatin
Discovery of myostatin (GDF-8):
• Myostatin is almost only expressed in
skeletal muscle.
• Myostatin could first be detected at day
9.5 post-coitum.
• Myostatin knock-out mice muscles
weigh 2-3 times more than those of wild
type animals.
• Hyperplasia
(more
fibres)
and
hypertrophy (larger fibres); more DNA.
(McPherron et al. 1997)
Myostatin is a negative growth factor. If
you have lots of it, you’re small. If you
don’t have it, you’re musculous.
Natural myostatin mutations occur
Natural myostatin mutations can occur as well: McPherron et al.
(1997) found in Belgian Blue and Piedmontese cattle a natural
myostatin mutation resulting in non-functional myostatin and
increased muscle size. An article reporting a toddler with a natural
myostatin mutation is on the website.
Figure: A fullblood Belgian Blue bull showing the double muscling
phenotype (McPherron et al. 1997).
Natural myostatin mutation in cattle
Belgian blue deletion
Piedmontese GA mutation
Myostatin protein structure and effect of mutation
Myostatin mutations (mut) in Belgian Blue and Piedmontese cattle
compared with wild-type (wt) Holstein cattle. These mutations lead to
a large increase in muscle mass, known as double-muscling
(McPherron et al. 1997)
Flex Wheeler: a mutant?
Flex Wheeler has a website where
someone states that Flex has a special
genotype linked to his muscle grwoth:
“Flex was one of only nine extreme
responders that had the very rare
"myostatin mutation." Myostatin is the
gene that "limits muscle growth."
Specifically, Flex had the rarest form of
myostatin mutation at the "exon 2"
position on the gene. This simply means
Flex has a much larger number of muscle
fibers compared to the other subjects or
the normal population.”
Flex Wheeler: It’s all natural!?
Task: Search for scientific literature on this SNP/mutation. Does it
exist?
Flex Wheeler: a mutant – hang on…
The gentleman who has written
the letter for Flex is Victor Conte.
This is what the BBC says
(23.10.2004): “Victor Conte is
founder and president of Bay Area
Laboratory Co-operative (Balco),
the San Francisco-based company
which the United States AntiDoping Agency says developed
the
banned
steroid
THG
(tetrahydrogestrinone)”.
So, Flex is all natural?
Victor Conte
How is myostatin expression regulated?
Myostatin expression is regulated via DNA binding elements for
glucocorticoid (GC), androgen (Tes), thyroid hormone (Thy),
myogenic regulatory factors (MRFs), MEF2, PPARg (Ma et al., 2001).
Only the glucocorticoid binding site has been experimentally verified
(Ma et al., 2003).
Resistance Other
Tes
training regulators
Myostatin
GC
Does myostatin regulate the response to exercise?
If myostatin has such a dramatic effect on muscle mass, is it involved
in mediating hypertrophy in response to resistance training?
The story is not clear at the moment: Some reports suggest that
myostatin mRNA or circulating myostatin does decrease after
resistance training (Roth et al., 2003;Zambon et al., 2003;Walker et
al., 2004) whereas others suggest it does not (Willoughby, 2004).
The jury is still out and a high-resolution time course needs to be
obtained after human resistance training in order to see whether
there is a regulatory myostatin expression change in response to
resistance training. Some data are shown on the following slide.
Myostatin response to resistance training
Figure. Myostatin expression levels (arbitrary units) for before ST (n =
15) versus after ST (n = 15) for each individual. •, males; , females. A
significant decrease in myostatin expression was observed in response
to ST, P < 0.05, with no group differences. (Roth et al. 2003)
How does myostatin inhibit growth?
Myostatin is like IGF-1 a protein that is secreted by muscle cells and
myostatin dimers bind so-called activin receptors. These receptors
then phosphorylate and activate the transcription factor Smad2/3. A
simplified version of the events is shown below.
Myostatin dimer
Activin type I and II
receptors
P
SMAD2,3
P
SMAD2,3 P
Growth
inhibition
Myostatin inhibitors (or not?)
Obviously, bodybuilders are very keen to inhibit
myostatin in order to promote muscle growth further.
“Champion Nutrition” already managed to develop a
myostatin inhibitor (or not?): “After extensive
research, Champion Nutrition found a scientist that
had isolated a naturally occurring fraction of a sea
vegetable. It binds strongly to myostatin, thereby
deactivating it. This discovery made it possible for
Champion to begin work on the first product ever
specifically designed to bind myostatin in humans”.
See: www.bodybuilding.com/store/cn/myostim.html
Myostatin inhibitors
Researchers have identified natural
the receptor binding of myostatin
proteins form so-called myostatin
are myostatin propeptide, follistatin
Myostatin
homodimer
can bind
receptor
myostatin inhibitors that regulate
in the organism. These bindingheterodimers. Inhibitor proteins
and GASP-1.
Myostatin-gasp1 or
myostatin-follistatin
heterodimers can not
bind receptor
Activin type I and II
receptors
P
Myostatin inhibition can improve muscular dystrophy
Bogdanovich et al. (2003) treated mdx mice (model for muscular
dystrophy) with a monoclonal antibody against myostatin which
inhibited myostatin. Control mdx mice (a) had significantly greater
pathological changes (arrowheads) in the diaphragm than treated
mice (b), as evidenced by a lack of cellular infiltration and fibrosis.
Myostatin is an ideal treatment target
Myostatin is probably a very good target for improving muscle mass
in various conditions for the following reasons:
a) Myostatin is secreted and thus treatments do not need to cross
membranes;
b) Myostatin needs to be inhibited which usually is easier than
activation;
c) Natural inhibitors (follistatin, gasp-1) exist which can be copied;
d) Myostatin expression is largely limited to muscle and thus side
effects should be limited.
A myostatin-related therapy via antibodies, peptides or other drugs
seems a realistic prospect.
Summary
Myostatin is a muscle-specific muscle growth inhibitor whose
expression is probably controlled by glucocorticoids and anabolic
steroids among other. Myostatin binds activin receptors as a dimer.
Myostatin binding to specific inhibitory proteins (follistatin, gasp-1)
can prevent myostatin binding to its receptor. Myostatin binding to
the receptor activates the kinase bit of the receptor which causes
phosphorylation of Smad2/3. Smad2/3 transport to the nucleus and
DNA binding can be modulated by inhibitory and co-Smads. Smad2/3
probably regulates genes that inhibit muscle growth.
The events mentioned above are shown on the next slide.
Myostatin-gasp1 or
myostatin-follistatin
heterodimer
Summary
Myostatin
homodimer
Activin type I and II
receptors
P
Myostatin
modification
Androgens,
Glucocorticoids,
thyroid hormones,
MRFs, MEF2
I-SMAD
SMAD2,3
Co-SMAD
P
SMAD2,3 P
Co-SMAD
Growth
inhibition
Enhancers and
silencers
Nucleus
Skeletal muscle fibre
The End