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Myostatin

• 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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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

Generation of Transgenic Mice

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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

• 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

• 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

• 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

• Measured cytokine levels in control mice, colon-26 tumor-bearing mice without the sActRIIB injections, and colon-26 tumor-bearing mice with ActRIIB

• 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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ResultsMuscle Mass Forearm Grip Strength Hydroxyproline Conent

•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

= Mstn-/-

=C57BL/6 mice= Mstn-/-/mdx= Mstn+/+/mdx

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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• 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):

61- 86. 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>.