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
Paradigm of SMA
Introduction
Hypertrophy vs. Atrophy
http//www.nndb.com
4
Most Studied Pathway
Introduction
Stress
PI3K
mTOR
Protein synthesis
AKT
S6K
FOXO
p
p
(Nucleus)
Atrogin-1
Protein degradation
5
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Oxidative Stress
?
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?
?
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Skeletal Muscle Atrophy
6
The Past of HIV
Introduction

• Vpr protein stops the cell cycle
• Prevents programmed cell death
• Increased replication of HIV
• AIDS muscle wasting symptom

7
HIV to SMA?
Introduction

• Vpr secreted like a hormone
• Infects other cells and organs
• AIDS wasting syndrome in skeletal muscle

8
Cancer Cachexia
Introduction

• Cachexia is a syndrome in cancer patients
• Progressive muscle wasting, weight loss,
  weakness and fatigue

• http//www2.msstate.edu/shbryd/Disorders.html

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The Past of Cachexia
Introduction

• Pathway unknown
• Cytokines induced muscle wasting
• One hypothesis Muscle wasting in cancer due to
  increased energy consumption

10
What is Oxidative Stress?
Introduction

• Cancer, Parkinsons, Diabetes, and SMA
• Free Radicals or Reactive Oxygen Species
• Steal electrons to restore valence stability

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The Past of Oxidative Stress
Introduction

• 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
Diabetes
Introduction

• 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

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The Past of Diabetes
Introduction

• 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

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Presentation Pathway

• Introduction
• Diseases
• HIV
• Cachexia
• Oxidative Stress
• Diabetes
• Therapies
• Discussion

15
HIV
?
Skeletal Muscle Atrophy
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HIVs Vpr protein
HIV

• Two Direct Pathways to SMA
• 1. Insulin Resistance
• 2. Glucocorticoid Hypersensitivity

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Effects of Vpr binding
HIV
Serine/Threonine residues
Vpr
Vpr
14-3-3
14-3-3
Cdc25
Cdc25
Vpr
14-3-3
Cdc25
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Vpr Inhibits Cell Cycle
HIV
Triggers mitosis machinery
Alberts et al 2004
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Stopped Cell Cycle
HIV
G2/M
Dividing
G2/M
Dividing

• Vpr stops the cell cycle at G2/M

He et al 1995
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Vpr inhibits insulin effects on FOXO
HIV
Serine/Threonine residues on FOXO
Vpr
Vpr
14-3-3
14-3-3
Cdc25
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Effect of Vpr on FOXO
HIV

• Vpr doesnt bind FOXO

Kino et al 2005
22
What is insulin supposed to do?
HIV
Insulin
Insulin
p
P
FOXO
FOXO
14-3-3
FOXO
p
14-3-3
FOXO
p
14-3-3
(Nucleus)
FOXO
p
14-3-3
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Vpr wont let insulin work!
HIV
FOXO
No FOXO
Kino et al 2005
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How does Vpr affect glucocorticoid receptors?
HIV
Vpr
LQQLL
Kino et al 2000

• Specific LXXLL motif binds GRE
• Completely different from ability to arrest cell
  cyle

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Vpr as a Co-regulator
Kino et al 2000
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Vpr as a Co-regulator of GR
HIV
Vpr
G R
GRE
TFIIB
RNA polymerase II
TFIID
Transcription enhancing glucocorticoid signal
TATA
Kino et al 2000
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Summary of Vpr SMA
Vpr
GRE
Vpr
14-3-3
Glucocorticoid
FOXO
nucleus
Atrogin-1
Skeletal Muscle Atrophy
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Therapies for HIV muscle wasting

• Steroid hormone receptor antagonists (RU 486)
• Vpr antagonists
• Current antiretroviral therapies

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Cancer Cachexia
?
Skeletal Muscle Atrophy
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NF-?B
Cachexia
I?B
Nucleus
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Cai et al. Study
Cachexia
MISR Mouse
MIKK Mouse
Constitutively active I?Ba
Constitutively active I?B
Always active NF-?B
Inactive NF-?B
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NF-?B Activity
Cachexia

• NF-?B activity is high in MIKK mice

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Cachexia
MIKK Mice vs. MISR Mice

• MIKK mice have a much lower body mass

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Tumor Activity
Cachexia

• Presence of a tumor increases the level of NF-?B
  activity in wild type mice

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Tumor Necrosis Factor - TNFa
Cachexia

• In the presence of I?Ba, activity decreases
• Without I?Ba, inhibitor does not stop production

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What does NF-?B affect?
Cachexia

• MURF1 mRNA is much higher in MIKK mice than in
  MISR mice

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What else does NF-?B affect?
Cachexia

• TNF activates NF-?B which causes a decrease
  in MyoD production

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Troponin in Cardiac Muscle
Cachexia

• Troponin-1 is degraded in the presence of MURF1

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Cachexia Pathway
activates
decreases
increases
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Therapy for Cachexia
Cachexia

• Salicylate inhibits the NF-?B pathway,
  preventing muscle loss

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Oxidative Stress
?
Skeletal Muscle Atrophy
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Oxidative Stress
Oxidative Stress
Reactive Oxygen Species
Caspase-3
Calpain
Calpastatin
Sarcomere Unit of Myofibril
20S/26S Proteasome
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Effect of Disuse
Oxidative Stress

• Disuse increases oxidative stress

Tidball et al, 2002
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Effect of Oxidative Stress
Oxidative Stress
Laser Densitometry

• Increase of oxidative stress increases
  calsequesterin
• Calsequestrin sequesters intracellular calcium

Hunter et al, 2001
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Effect of Increased Calcium
Oxidative Stress
Type II Diaphragm Muscle Fibers by
Immunohistochemistry

• Disuse increases calpain and 20S proteasome
  activity

Tidball et al, 2002
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Calpain Proteolysis
Oxidative Stress

• Calcium treatment increases protein cleavage
• Protein cleavage can be inhibited by nitric
  oxide and calpastatin

Koh et al, 2000
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Caspase Activity
Oxidative Stress

• Caspases inhibit calpastatin
• Calpastatin is a calpain inhibitor

Wang et al, 1998
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Summary
Oxidative Stress
Increase Ca2
Calpain Caspase 3 activity increases
Releases Actomyosin to be degraded in proteasome
Skeletal Muscle Atrophy
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Therapies for Oxidative Stress
Oxidative Stress
ROS
Cell
Vitamin E

• NO and Calpastatin Transgene
• Vitamin E protects against ROS
• Vitamin C restores Vitamin E activity

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Diabetes
?
Skeletal Muscle Atrophy
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Diabetes
Insulin levels
Ub-ligase E3-a
26 S proteasome
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What activates the Ubiquitin-proteasome pathway?
Diabetes

• No difference in diabetics without vs. diabetics
  with acidosis
• Acidosis is not a stimulus of ubiquitin dependent
  atrophy

Price et al. 1999
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Effects of Insulin on Ub-proteasome pathway
Diabetes

• Less protein degradation in insulin treated
  muscles
• Lower insulin levels leads to activation of
  Ub-proteasome pathway

Price et al., 1999
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Results of Pathway Activation
Diabetes

• Levels of Ub-ligase and its coenzyme increase
• Amount of Ub-conjugation by these increases

Goldberg et al., 1999
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Proteasome Formation
Diabetes

• mRNA for 19 S and 20 S subunits increases
• Formation of 26 S proteasome increases

Attaix et al., 2004
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Proteasome Activity
Diabetes

• Flourescence in atrophied muscles higher than
  control muscles
• Flourescence is analogous to amount of 26 S
  proteasome activity

Attaix et al., 2004
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Summary of Diabetes and SMA
Diabetes
Insulin decrease/ glucocorticoid increase
E3-alpha ubiquitin ligase increases
26 S Proteasome activity increases
Skeletal Muscle Atrophy
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Therapy for Diabetes
Diabetes

• Treatments for diabetes generally focus on
  maintaining available insulin levels NOT SMA
• Side effect of the insulin treatment, however, is
  associated with the reverse pathway of atrophy,
  i.e. hypertrophy

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Presentation Pathway

• Introduction
• Diseases
• HIV
• Cachexia
• Oxidative Stress
• Diabetes
• Discussion

60
?
?
Oxidative Stress
?
Skeletal Muscle Atrophy
61
Vpr
Co-activates glucocorticoid receptor
Inhibit insulin effects on FOXO
Glucocorticoid hypersensitivity
Atrogin-1 induction
Skeletal Muscle Atrophy
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Cachexia
IKK/NF-kappa B pathway
Murf-1 increase
MyoD mRNA decrease
Skeletal Muscle Atrophy
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Oxidative Stress
Oxidative Stress
Increase Ca2
Calpain Caspase 3 activity increases
Releases Actomyosin to be degraded in proteasome
Skeletal Muscle Atrophy
64
Diabetes
Diabetes
Insulin decrease/ glucocorticoid increase
E3-alpha ubiquitin ligase increases
26 S Proteasome activity increases
Skeletal Muscle Atrophy
65
IKK/NF-kappa B pathway
Murf-1 increase
MyoD mRNA decrease
Skeletal Muscle Atrophy
66
Acknowledgements

• Thanks to Dr. D, Sara Herrera, Tammy
• Hibler, Arun Paul, and Chris Prater