Pathways of Skeletal Muscle Atrophy: HIV as a Model System?

1. Pathways of Skeletal Muscle Atrophy: HIV as a Model System? Chelsea Bueter, Michelle McKinzey, Chloe Salzmann, Michael Zorniak Department of Biology, Lake Forest College, Lake Forest, Illinois 60045 USA

2. Presentation Pathway Introduction Diseases HIV Cachexia Oxidative Stress Diabetes Discussion

3. Introduction Paradigm of SMA Hypertrophy vs. Atrophy http://www.nndb.com

4. Introduction Most Studied Pathway Stress PI3K mTOR Protein synthesis AKT S6K FOXO p p (Nucleus) Atrogin-1 Protein degradation

5. Diabetes HIV VPR Protein Cachexia ? ? Oxidative Stress ? ? ? ? ? ? Skeletal Muscle Atrophy

6. Introduction The Past of HIV • Vpr protein stops the cell cycle • Prevents programmed cell death • Increased replication of HIV • AIDS muscle wasting symptom

7. Introduction HIV to SMA? • Vpr secreted like a hormone • Infects other cells and organs • AIDS wasting syndrome in skeletal muscle

8. Introduction Cancer Cachexia • Cachexia is a syndrome in cancer patients • Progressive muscle wasting, weight loss, weakness and fatigue http://www2.msstate.edu/~shbryd/Disorders.html

9. Introduction The Past of Cachexia • Pathway unknown • Cytokines induced muscle wasting • One hypothesis: Muscle wasting in cancer due to increased energy consumption

10. Introduction What is Oxidative Stress? • Cancer, Parkinson’s, Diabetes, and SMA • Free Radicals or Reactive Oxygen Species • Steal electrons to restore valence stability

11. Introduction The Past of Oxidative Stress • Goldberg et al, 1986 • Calcium activates protein degradation • Appel et al, 1997 • Vitamin E decreases calcium levels • Vitamin E is an anti-oxidant • Thus, oxidative stress calcium levels and activates protein degradation.

12. Introduction Diabetes • Characterized by insulin deficiency or insulin intolerance • Juvenile diabetes (type 1)- genetically linked but also diet linked • Type 2 - middle-aged people-low insulin levels • Leads to many other disorders

13. Introduction The Past of Diabetes • 1993- studies showed that in non-diabetics, insulin levels and activity remained high • Diabetics showed very low insulin levels or activity • Common symptom in diabetics was SMA • Hypothesis= Insulin tolerance may be linked to SMA

14. Presentation Pathway Introduction Diseases HIV Cachexia Oxidative Stress Diabetes Therapies Discussion

15. HIV ? Skeletal Muscle Atrophy

16. HIV HIV’s Vpr protein • Two Direct Pathways to SMA 1. Insulin Resistance 2. Glucocorticoid Hypersensitivity

17. HIV Effects of Vpr binding Serine/Threonine residues Vpr Vpr 14-3-3 14-3-3 Cdc25 Cdc25 Vpr 14-3-3 Cdc25

18. HIV Vpr Inhibits Cell Cycle Triggers mitosis machinery Alberts et al 2004

19. HIV Stopped Cell Cycle Dividing G2/M Dividing G2/M • Vpr stops the cell cycle at G2/M He et al 1995

20. HIV Cdc25 Vpr inhibits insulin effects on FOXO Serine/Threonine residues on FOXO Vpr Vpr 14-3-3 14-3-3 Cdc25

21. HIV Effect of Vpr on FOXO • Vpr doesn’t bind FOXO Kino et al 2005

22. HIV What is insulin supposed to do? Insulin Insulin p P FOXO FOXO 14-3-3 FOXO p 14-3-3 14-3-3 FOXO p (Nucleus) 14-3-3 FOXO p

23. HIV Vpr won’t let insulin work! FOXO No FOXO Kino et al 2005

24. HIV How does Vpr affect glucocorticoid receptors? Vpr LQQLL Kino et al 2000 • Specific LXXLL motif binds GRE • Completely different from ability to arrest cell cyle

25. Vpr as a Co-regulator Kino et al 2000

26. HIV Vpr as a Co-regulator of GR Vpr G R GRE TFIIB RNA polymerase II TFIID Transcription: enhancing glucocorticoid signal TATA Kino et al 2000

27. VPR Protein Summary of Vpr & SMA Vpr GRE Vpr 14-3-3 Glucocorticoid FOXO nucleus Atrogin-1 Skeletal Muscle Atrophy

28. Therapies for HIV muscle wasting • Steroid hormone receptor antagonists (RU 486) • Vpr antagonists • Current antiretroviral therapies

29. Cancer Cachexia ? Skeletal Muscle Atrophy

30. Cachexia P IκBα NF- κB NF-κB IκB Nucleus

31. Cachexia Cai et al. Study MISR Mouse MIKK Mouse Constitutively active IκBα Constitutively active IκB Always active NF-κB Inactive NF-κB

32. Cachexia NF-κB Activity • NF-κB activity is high in MIKK mice

33. Cachexia MIKK Mice vs. MISR Mice • MIKK mice have a much lower body mass

34. Cachexia Tumor Activity • Presence of a tumor increases the level of NF-κB activity in wild type mice

35. Cachexia Tumor Necrosis Factor - TNFα • In the presence of IκBα, activity decreases • Without IκBα, inhibitor does not stop production

36. Cachexia What does NF-κB affect? • MURF1 mRNA is much higher in MIKK mice than in MISR mice

37. Cachexia What else does NF-κB affect? • TNF activates NF-κB which causes a decrease in MyoD production

38. Cachexia Troponin in Cardiac Muscle • Troponin-1 is degraded in the presence of MURF1

39. TNF-α NF-κB MURF1 MyoD Cachexia Pathway activates decreases increases

40. Cachexia Therapy for Cachexia • Salicylate inhibits the NF-κB pathway, preventing muscle loss

41. Oxidative Stress ? Skeletal Muscle Atrophy

42. Oxidative Stress Calcium Mitochondria Cytochrome c Calpastatin NO Oxidative Stress Reactive Oxygen Species Calpain Caspase-3 Calpastatin Sarcomere Unit of Myofibril 20S/26S Proteasome

43. Oxidative Stress Effect of Disuse • Disuse increases oxidative stress Tidball et al, 2002

44. Oxidative Stress Effect of Oxidative Stress Laser Densitometry • Increase of oxidative stress increases calsequesterin • Calsequestrin sequesters intracellular calcium Hunter et al, 2001

45. Oxidative Stress Effect of Increased Calcium Type II Diaphragm Muscle Fibers by Immunohistochemistry • Disuse increases calpain and 20S proteasome activity Tidball et al, 2002

46. Oxidative Stress Calpain Proteolysis • Calcium treatment increases protein cleavage • Protein cleavage can be inhibited by nitric oxide and calpastatin Koh et al, 2000

47. Oxidative Stress Caspase Activity • Caspases inhibit calpastatin • Calpastatin is a calpain inhibitor Wang et al, 1998

48. Summary Oxidative Stress Increase Ca2+ Calpain & Caspase 3 activity increases Releases Actomyosin to be degraded in proteasome Skeletal Muscle Atrophy

49. Oxidative Stress Therapies for Oxidative Stress ROS • NO and Calpastatin Transgene • Vitamin E protects against ROS • Vitamin C restores Vitamin E activity Cell Vitamin E

50. Diabetes ? Skeletal Muscle Atrophy