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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. Presentation Pathway. Introduction Diseases HIV Cachexia Oxidative Stress
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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
Presentation Pathway Introduction Diseases HIV Cachexia Oxidative Stress Diabetes Discussion
Introduction Paradigm of SMA Hypertrophy vs. Atrophy http://www.nndb.com
Introduction Most Studied Pathway Stress PI3K mTOR Protein synthesis AKT S6K FOXO p p (Nucleus) Atrogin-1 Protein degradation
Diabetes HIV VPR Protein Cachexia ? ? Oxidative Stress ? ? ? ? ? ? Skeletal Muscle Atrophy
Introduction The Past of HIV • Vpr protein stops the cell cycle • Prevents programmed cell death • Increased replication of HIV • AIDS muscle wasting symptom
Introduction HIV to SMA? • Vpr secreted like a hormone • Infects other cells and organs • AIDS wasting syndrome in skeletal muscle
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
Introduction The Past of Cachexia • Pathway unknown • Cytokines induced muscle wasting • One hypothesis: Muscle wasting in cancer due to increased energy consumption
Introduction What is Oxidative Stress? • Cancer, Parkinson’s, Diabetes, and SMA • Free Radicals or Reactive Oxygen Species • Steal electrons to restore valence stability
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.
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
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
Presentation Pathway Introduction Diseases HIV Cachexia Oxidative Stress Diabetes Therapies Discussion
HIV ? Skeletal Muscle Atrophy
HIV HIV’s Vpr protein • Two Direct Pathways to SMA 1. Insulin Resistance 2. Glucocorticoid Hypersensitivity
HIV Effects of Vpr binding Serine/Threonine residues Vpr Vpr 14-3-3 14-3-3 Cdc25 Cdc25 Vpr 14-3-3 Cdc25
HIV Vpr Inhibits Cell Cycle Triggers mitosis machinery Alberts et al 2004
HIV Stopped Cell Cycle Dividing G2/M Dividing G2/M • Vpr stops the cell cycle at G2/M He et al 1995
HIV Cdc25 Vpr inhibits insulin effects on FOXO Serine/Threonine residues on FOXO Vpr Vpr 14-3-3 14-3-3 Cdc25
HIV Effect of Vpr on FOXO • Vpr doesn’t bind FOXO Kino et al 2005
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
HIV Vpr won’t let insulin work! FOXO No FOXO Kino et al 2005
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
Vpr as a Co-regulator Kino et al 2000
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
VPR Protein Summary of Vpr & SMA Vpr GRE Vpr 14-3-3 Glucocorticoid FOXO nucleus Atrogin-1 Skeletal Muscle Atrophy
Therapies for HIV muscle wasting • Steroid hormone receptor antagonists (RU 486) • Vpr antagonists • Current antiretroviral therapies
Cancer Cachexia ? Skeletal Muscle Atrophy
Cachexia P IκBα NF- κB NF-κB IκB Nucleus
Cachexia Cai et al. Study MISR Mouse MIKK Mouse Constitutively active IκBα Constitutively active IκB Always active NF-κB Inactive NF-κB
Cachexia NF-κB Activity • NF-κB activity is high in MIKK mice
Cachexia MIKK Mice vs. MISR Mice • MIKK mice have a much lower body mass
Cachexia Tumor Activity • Presence of a tumor increases the level of NF-κB activity in wild type mice
Cachexia Tumor Necrosis Factor - TNFα • In the presence of IκBα, activity decreases • Without IκBα, inhibitor does not stop production
Cachexia What does NF-κB affect? • MURF1 mRNA is much higher in MIKK mice than in MISR mice
Cachexia What else does NF-κB affect? • TNF activates NF-κB which causes a decrease in MyoD production
Cachexia Troponin in Cardiac Muscle • Troponin-1 is degraded in the presence of MURF1
TNF-α NF-κB MURF1 MyoD Cachexia Pathway activates decreases increases
Cachexia Therapy for Cachexia • Salicylate inhibits the NF-κB pathway, preventing muscle loss
Oxidative Stress ? Skeletal Muscle Atrophy
Oxidative Stress Calcium Mitochondria Cytochrome c Calpastatin NO Oxidative Stress Reactive Oxygen Species Calpain Caspase-3 Calpastatin Sarcomere Unit of Myofibril 20S/26S Proteasome
Oxidative Stress Effect of Disuse • Disuse increases oxidative stress Tidball et al, 2002
Oxidative Stress Effect of Oxidative Stress Laser Densitometry • Increase of oxidative stress increases calsequesterin • Calsequestrin sequesters intracellular calcium Hunter et al, 2001
Oxidative Stress Effect of Increased Calcium Type II Diaphragm Muscle Fibers by Immunohistochemistry • Disuse increases calpain and 20S proteasome activity Tidball et al, 2002
Oxidative Stress Calpain Proteolysis • Calcium treatment increases protein cleavage • Protein cleavage can be inhibited by nitric oxide and calpastatin Koh et al, 2000
Oxidative Stress Caspase Activity • Caspases inhibit calpastatin • Calpastatin is a calpain inhibitor Wang et al, 1998
Summary Oxidative Stress Increase Ca2+ Calpain & Caspase 3 activity increases Releases Actomyosin to be degraded in proteasome Skeletal Muscle Atrophy
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
Diabetes ? Skeletal Muscle Atrophy