Myostatin
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Encoded by the gene Mstn
Synthesized in skeletal muscle cells
Inhibits myogenesis
Myostatin mutants found in whippets,
humans, and cows
• Restricted to Skeletal muscle in
embryogenesis but can also be found in the
heart, mammary glands, and adipose tissue in
adults
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Discovery of Myostatin
• Discovered in 1997 by Geneticists Se-Jin Lee
and McPherron when they created “Mighty
Mice”
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The myosatin prepropeptide is made up of 3 subunits:
•C-terminal peptide
•Signal Sequence
•Cleaved by a proprotein convertase called Furin in the Golgi Apparatus
•N-terminal peptide
•Inhibitory role and necessary for C-peptide to fold into a cysteine knot
structure
•Needs to be destroyed in order for the C-peptide to be active. Cleavage
is done by Bone morphogenetic protein (BMP-1) and other proteins of the
tolloid family
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For myostatin to be in its active form:
•It needs to cleaved free from the
propeptide complex:
•PACE
•metalloproteinases
•Needs to fold into a cysteine knot
structure
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Components of the Myostatin Signal
transduction pathway
• Not much is known about the Myostatin signal pathway but
it is assumed that it is similar other TGF-β proteins
• Components:
– Type-II Receptors: ActRIIB
• serine/threonine kinases
– Type-I recptors: ALK-4 amd ALK-5
• GS Domain: TTSGSGSG
• GS Domain needs to by phosphorylated
• Tyrosine kinase
– SmaD Proteins – SmaD 2, 3, 6, and 7
• Needed for the inhibition of myogenesis associated genes
• Phosphorylated by Type-I receptors
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Myostatin Signal Pathway
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AKT(Protein Kinase B)
• When phosphorylated it activates TORC1 and
TORC2
• TORC1
– Activates p70s6k which leads to the expression of the
proteins MyoD, myogenin, and myf5
– Inhibits SmaD2/3
• Inhibits FoxO transcription factors
– family of proteins that regulates genes involved in cell
growth, proliferation, and differentiation
– MurF1, MAFbx, and Antrogin-1
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Myostatin Signal Pathway
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Inhibitors of Myostatin
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• Goal:
– To determine the effects of propetide and
follistatin on muscle mass and the receptor
binding of myostatin
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Effects of Follistatin and Propeptide on the binding of Myostatin
to ActRIIB
•Generated COS-7 cells expressing the
ActRIIB receptor on their cell surface
•Incubated the cells with myostatin labeled
with radioactive iodine
•The cells were also incubated with
unlabeled follistatin, propeptide, or
myostatin
•Cells were lysed and counted with a gamma
counter
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Generation of Transgenic Mice
Vector is created containing genes
for AktRIIB or follistatin
Inject recombinant DNA construct into male pronucleus
Insert the oocyte containing the construct into a mouse
embryo
Transplant the embryo into a pseudo-pregnant female mouse
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Effects of Follistatin and mutated
ActRIIB on muscle mass
•Used transgenic mice expressing follistatin or the ActRIIB
receptor missing the kinase domain
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Follistatin and ActRIIB Continued
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Why should we learn about
Myostatin?
• Cachexia
• Muscular Dystrophy
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Cachexia
• Loss of weight, muscle mass, appetite, and
feelings of weakness and fatigue that cannot
be reversed by the uptake of nutrients
• Seen in patients of: AIDS, cancer, tuberculosis,
COPD, and congestive heart failure
• Increases the mortality rate of any underlying
illness
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Current Treatments
• Treatments currently aim to increase the
anabolic processes while simultaneously
reducing catabolic ones
– Making sure the patient has a healthy diet despite
not having an appetite
– Nutritional supplements like fish oils
– Exercise
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Pathogenesis of Cachexia
• The main contributor to Cachexia is the over
abundance of cytokines produced in systemic
inflammation
• The cytokines that are important to the
pathogenesis of cachexia are; IL-1, IL-2,
Interferon-γ, and TNF-α are produced
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These Cytokines lead to….
• Muscle Atrophy
– Activation of FoxO, Smad proteins, increased levels of
myostatin, and NF-κB
• Upregulation of Ubiquitin mediated proteolytic system
– Proteins are labeled with ubiquitin and degraded by a
proteosome
– Used to create C-reactive peptide and serum amyloid peptide
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Release of cortisol and catecholamines
Decrease in the activity of fat and liver lipoprotein lipase
Inhibition of MyoD
Nervous system component
– listlessness, malaise, and anhedonia
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• Goal:
– To determine if inhibiting ActRIIB could reverse
the muscle wasting effects of Cachexia
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Experimental Design
• Used colon-26 tumor-bearing mice and
C57B/7 mice as the control
• Injected the sActRIIB decoy receptor into mice
bloodstream
• Measured muscle mass, cytokine levels, and
tumor size
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•Introduced a decoy ActRIIB
receptor called sActRIIB
•At different stages of
Cachexia (onset or when 10%
of body mass was lost) the
decoy receptor was injected
and then further administered
every week
•Used colon-26 tumor-bearing
mice
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sActRIIB affect on tumor, fat and
muscle Mass
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Measured muscle and fat mass using Nuclear Magnetic Resonance
– extensor digitalis longus (EDL)
– soleus
– calf and tibialis anterior muscles
• Tumor mass was measured using an electronic caliper
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Effect of ActRIIB on cytokine levels
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Since cytokines are thought to be the main factor in the development in cachexia
cytokine levels were measured to see if sActRIIB altered them in order to restore
muscle mass
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Measured cytokine levels in control mice, colon-26 tumor-bearing mice without
the sActRIIB injections, and colon-26 tumor-bearing mice with ActRIIB
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Cytokine levels were measured using MAP mouse cytokine kit
– IL-1, IL-2, Interferon-γ, and TNF-α
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Muscular Dystrophy
• Recessive mutation in the dystrophin gene in
the X chromosome
• One of largest genes in the human genome
• Dystrophin maintains muscle cell structure,
allows the muscle cell to return to its natural
shape after deformation, and maintains
muscle integrity
• Mutation can result in the loss of dystrophin
or a truncated version
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Muscular Dystrophy
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• Goal:
– To determine the effects of the loss of myostin on
mice with muscular dystrophy
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The effects of Myostatin null mutants
on Muscular Dystrophy
• Used Mdx mice
– Nonsense mutation in the dystrophin gene
• In these mice they introduced a mstn
mutation
• At 6 to 9 months of age the mice were
sacrificed
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Test Parameters
• Measured the mass of the pectorals, triceps,
quadriceps, and gastrocnemius muscles
• Measured the diameter of muscle fibers
• Measured the forearm grip strength
– automated grip strength reader
• Histological examination of the diaphragm
– hydroxyproline content
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Results
Muscle Mass
Forearm Grip Strength
Hydroxyproline Conent
= Mstn-/=C57BL/6 mice
= Mstn-/-/mdx
= Mstn+/+/mdx
•The myostatin mutation increased grip strength in mdx mice with the myostatin mutation
•Muscle mass increased from mdx mice to mdx mice with the myostatin mutation
•Reduction in Hydroxyproline content in mdx mice null for myostatin
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However….
• Despite the promising results the muscle
fibers of MDX/myostatin null mice still
displayed irregularities:
– irregular muscle fiber size,
– fiber splitting
– nuclei located at the center of the fiber
– fiber necrosis and inflammation
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Works Cited
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Bentzinger, Florian. "Building Muscle: Molecular Regulation of Myogenesis." Cold Spring Harbor
Perspectives inBiology. (2010): n. page. Print.
<http://cshperspectives.cshlp.org/content/4/2/a008342.full>.
Lee, Se-jin. "Myostatin and the control of skeletal muscle mass." Current Opinion in Genetics &
Development. 9. (1999): 604–607. Print.
Massague, John. "TGF-¯ SIGNAL TRANSDUCTION."Annual Review of Biochemistry. 67. (1998): 753-791. Print.
Morely, John. "Cachexia: pathophysiology and clinical relevance." American Journal of Clinical Nutrition.
(2006): 735-743. Print. <http://ajcn.nutrition.org/content/83/4/735.full>.
"Myogenesis: The Development of Muscle." Developmental Biology. (2000): n. page. Print.
<http://www.ncbi.nlm.nih.gov/books/NBK10006/>.
Se-jin, Lee. "REGULATION OF MUSCLE MASS BY MYOSTATIN." Annual Review of Cellular Development. (2004): 6186. Print.
Se-Jin, Lee. "Regulation of myostatin activity and muscle growth." PNAS. 98.16 (2001): n. page. Print.
<www.pnas.orgycgiydoiy10.1073ypnas.151270098>.
Trendelen, Anne. "Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and
myotube size." American Physiological Association. 296.1258-1270 (2009): n. page. Print.
<http://ajpcell.physiology.org/content/296/6/C1258>.
Wagner, Kathryn. "Loss of Myostatin Attenuates Severity of Muscular Dystrophy in mdx Mice." Annual
Nuerology. 52 (2002): 832-836. Print.
Zhou, Xiaolan. "Reversal of Cancer Cachexia and Muscle Wasting by ActRIIB Antagonism Leads to Prolonged
Survival." Cell. (2010): 531-544. Print. <http://rfi.fmrp.usp.br/pg/fisio/cursao2012/Semin5B.pdf>.
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