Title: Pathways of Skeletal Muscle Atrophy: HIV as a Model System?
1Pathways 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
2Presentation Pathway
- Introduction
- Diseases
- HIV
- Cachexia
- Oxidative Stress
- Diabetes
- Discussion
3Paradigm of SMA
Introduction
Hypertrophy vs. Atrophy
http//www.nndb.com
4Most Studied Pathway
Introduction
Stress
PI3K
mTOR
Protein synthesis
AKT
S6K
FOXO
p
p
(Nucleus)
Atrogin-1
Protein degradation
5?
?
Oxidative Stress
?
?
?
?
?
?
Skeletal Muscle Atrophy
6The Past of HIV
Introduction
- Vpr protein stops the cell cycle
- Prevents programmed cell death
- Increased replication of HIV
- AIDS muscle wasting symptom
7HIV to SMA?
Introduction
- Vpr secreted like a hormone
- Infects other cells and organs
- AIDS wasting syndrome in skeletal muscle
8Cancer Cachexia
Introduction
- Cachexia is a syndrome in cancer patients
- Progressive muscle wasting, weight loss,
weakness and fatigue
- http//www2.msstate.edu/shbryd/Disorders.html
9The Past of Cachexia
Introduction
- Pathway unknown
- Cytokines induced muscle wasting
- One hypothesis Muscle wasting in cancer due to
increased energy consumption
10What is Oxidative Stress?
Introduction
- Cancer, Parkinsons, Diabetes, and SMA
- Free Radicals or Reactive Oxygen Species
- Steal electrons to restore valence stability
11The 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.
12Diabetes
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
13The 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
14Presentation Pathway
- Introduction
- Diseases
- HIV
- Cachexia
- Oxidative Stress
- Diabetes
- Therapies
- Discussion
15HIV
?
Skeletal Muscle Atrophy
16HIVs Vpr protein
HIV
- Two Direct Pathways to SMA
- 1. Insulin Resistance
- 2. Glucocorticoid Hypersensitivity
17Effects of Vpr binding
HIV
Serine/Threonine residues
Vpr
Vpr
14-3-3
14-3-3
Cdc25
Cdc25
Vpr
14-3-3
Cdc25
18Vpr Inhibits Cell Cycle
HIV
Triggers mitosis machinery
Alberts et al 2004
19Stopped Cell Cycle
HIV
G2/M
Dividing
G2/M
Dividing
- Vpr stops the cell cycle at G2/M
He et al 1995
20Vpr inhibits insulin effects on FOXO
HIV
Serine/Threonine residues on FOXO
Vpr
Vpr
14-3-3
14-3-3
Cdc25
21Effect of Vpr on FOXO
HIV
Kino et al 2005
22What 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
23Vpr wont let insulin work!
HIV
FOXO
No FOXO
Kino et al 2005
24How 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
25Vpr as a Co-regulator
Kino et al 2000
26Vpr 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
27Summary of Vpr SMA
Vpr
GRE
Vpr
14-3-3
Glucocorticoid
FOXO
nucleus
Atrogin-1
Skeletal Muscle Atrophy
28Therapies for HIV muscle wasting
- Steroid hormone receptor antagonists (RU 486)
- Vpr antagonists
- Current antiretroviral therapies
29Cancer Cachexia
?
Skeletal Muscle Atrophy
30NF-?B
Cachexia
I?B
Nucleus
31Cai et al. Study
Cachexia
MISR Mouse
MIKK Mouse
Constitutively active I?Ba
Constitutively active I?B
Always active NF-?B
Inactive NF-?B
32NF-?B Activity
Cachexia
- NF-?B activity is high in MIKK mice
33Cachexia
MIKK Mice vs. MISR Mice
- MIKK mice have a much lower body mass
34Tumor Activity
Cachexia
- Presence of a tumor increases the level of NF-?B
activity in wild type mice
35Tumor Necrosis Factor - TNFa
Cachexia
- In the presence of I?Ba, activity decreases
- Without I?Ba, inhibitor does not stop production
36What does NF-?B affect?
Cachexia
- MURF1 mRNA is much higher in MIKK mice than in
MISR mice
37What else does NF-?B affect?
Cachexia
- TNF activates NF-?B which causes a decrease
in MyoD production
38Troponin in Cardiac Muscle
Cachexia
- Troponin-1 is degraded in the presence of MURF1
39Cachexia Pathway
activates
decreases
increases
40Therapy for Cachexia
Cachexia
- Salicylate inhibits the NF-?B pathway,
preventing muscle loss
41Oxidative Stress
?
Skeletal Muscle Atrophy
42Oxidative Stress
Oxidative Stress
Reactive Oxygen Species
Caspase-3
Calpain
Calpastatin
Sarcomere Unit of Myofibril
20S/26S Proteasome
43Effect of Disuse
Oxidative Stress
- Disuse increases oxidative stress
Tidball et al, 2002
44Effect of Oxidative Stress
Oxidative Stress
Laser Densitometry
- Increase of oxidative stress increases
calsequesterin - Calsequestrin sequesters intracellular calcium
Hunter et al, 2001
45Effect of Increased Calcium
Oxidative Stress
Type II Diaphragm Muscle Fibers by
Immunohistochemistry
- Disuse increases calpain and 20S proteasome
activity
Tidball et al, 2002
46Calpain Proteolysis
Oxidative Stress
- Calcium treatment increases protein cleavage
- Protein cleavage can be inhibited by nitric
oxide and calpastatin
Koh et al, 2000
47Caspase Activity
Oxidative Stress
- Caspases inhibit calpastatin
- Calpastatin is a calpain inhibitor
Wang et al, 1998
48Summary
Oxidative Stress
Increase Ca2
Calpain Caspase 3 activity increases
Releases Actomyosin to be degraded in proteasome
Skeletal Muscle Atrophy
49Therapies for Oxidative Stress
Oxidative Stress
ROS
Cell
Vitamin E
- NO and Calpastatin Transgene
- Vitamin E protects against ROS
- Vitamin C restores Vitamin E activity
50Diabetes
?
Skeletal Muscle Atrophy
51Diabetes
Insulin levels
Ub-ligase E3-a
26 S proteasome
52What 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
53Effects 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
54Results of Pathway Activation
Diabetes
- Levels of Ub-ligase and its coenzyme increase
- Amount of Ub-conjugation by these increases
Goldberg et al., 1999
55Proteasome Formation
Diabetes
- mRNA for 19 S and 20 S subunits increases
- Formation of 26 S proteasome increases
Attaix et al., 2004
56Proteasome Activity
Diabetes
- Flourescence in atrophied muscles higher than
control muscles - Flourescence is analogous to amount of 26 S
proteasome activity
Attaix et al., 2004
57Summary of Diabetes and SMA
Diabetes
Insulin decrease/ glucocorticoid increase
E3-alpha ubiquitin ligase increases
26 S Proteasome activity increases
Skeletal Muscle Atrophy
58Therapy 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
59Presentation Pathway
- Introduction
- Diseases
- HIV
- Cachexia
- Oxidative Stress
- Diabetes
- Discussion
60?
?
Oxidative Stress
?
Skeletal Muscle Atrophy
61Vpr
Co-activates glucocorticoid receptor
Inhibit insulin effects on FOXO
Glucocorticoid hypersensitivity
Atrogin-1 induction
Skeletal Muscle Atrophy
62Cachexia
IKK/NF-kappa B pathway
Murf-1 increase
MyoD mRNA decrease
Skeletal Muscle Atrophy
63Oxidative Stress
Oxidative Stress
Increase Ca2
Calpain Caspase 3 activity increases
Releases Actomyosin to be degraded in proteasome
Skeletal Muscle Atrophy
64Diabetes
Diabetes
Insulin decrease/ glucocorticoid increase
E3-alpha ubiquitin ligase increases
26 S Proteasome activity increases
Skeletal Muscle Atrophy
65IKK/NF-kappa B pathway
Murf-1 increase
MyoD mRNA decrease
Skeletal Muscle Atrophy
66Acknowledgements
- Thanks to Dr. D, Sara Herrera, Tammy
- Hibler, Arun Paul, and Chris Prater