Myostatin

1. 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

3. Discovery of Myostatin • Discovered in 1997 by Geneticists Se-Jin Lee and McPherron when they created “Mighty Mice”

4. The myosatinprepropeptide is made up of 3 subunits: • C-terminal peptide • Signal Sequence • Cleaved by a proproteinconvertase 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

5. 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

6. 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/threoninekinases • 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

7. Myostatin Signal Pathway

8. 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

9. Myostatin Signal Pathway

10. Inhibitors of Myostatin

11. Goal: • To determine the effects of propetide and follistatin on muscle mass and the receptor binding of myostatin

12. 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

13. 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

14. Effects of Follistatin and mutated ActRIIB on muscle mass • Used transgenic mice expressing follistatin or the ActRIIB receptor missing the kinase domain

15. Follistatin and ActRIIB Continued

16. Why should we learn about Myostatin? • Cachexia • Muscular Dystrophy

17. 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

18. 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

19. 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

20. 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

21. Goal: • To determine if inhibiting ActRIIB could reverse the muscle wasting effects of Cachexia

22. 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

23. 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

24. 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

25. 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

26. 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

27. Muscular Dystrophy

28. Goal: • To determine the effects of the loss of myostin on mice with muscular dystrophy

29. The effects of Myostatin null mutants on Muscular Dystrophy • Used Mdx mice • Nonsense mutation in the dystrophin gene • In these mice they introduced a mstnmutation • At 6 to 9 months of age the mice were sacrificed

30. 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

31. Results Muscle Mass Forearm Grip Strength HydroxyprolineConent = Mstn-/-=C57BL/6 mice = Mstn-/-/mdx = Mstn+/+/mdx • The myostatin mutation increased grip strength in mdxmice with the myostatin mutation • Muscle mass increased from mdx mice to mdxmice with the myostatin mutation • Reduction in Hydroxyproline content in mdx mice null for myostatin

32. 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

33. 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 myotubesize." 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>.