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

Paradigm of SMAHypertrophy vs. Atrophy

http://www.nndb.com

Introduction

Most Studied Pathway

Stress

PI3K

AKT

FOXO

mTOR

Atrogin-1

Protein degradation

Protein synthesis

S6K

(Nucleus)

pp

Introduction

HIV

Skeletal Muscle Atrophy

Diabetes

VPR Protein

Oxidative Stress

Cachexia

??

?

? ?

?

?

?

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

http://www2.msstate.edu/~shbryd/Disorders.html

• Cachexia is a syndrome in cancer patients

• Progressive muscle wasting, weight loss, weakness and fatigue

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

Introduction

Presentation Pathway

IntroductionDiseases

HIVCachexiaOxidative StressDiabetes

Therapies Discussion

HIV

?

Skeletal Muscle Atrophy

HIV’s Vpr protein

• Two Direct Pathways to SMA

1. Insulin Resistance

2. Glucocorticoid Hypersensitivity

HIV

Effects of Vpr binding

Vpr

Cdc25

Serine/Threonine residues

Cdc25

14-3-3

14-3-3

Vpr

14-3-3

Vpr

Cdc25

HIV

Vpr Inhibits Cell Cycle

Triggers mitosis machinery

Alberts et al 2004

HIV

Stopped Cell Cycle

G2/MDividing G2/MDividing

HIV

•Vpr stops the cell cycle at G2/M

He et al 1995

14-3-3

Vpr

Vpr inhibits insulin effects on FOXO

Vpr

Cdc25

14-3-3

Cdc25

Serine/Threonine residues on FOXO

HIV

Effect of Vpr on FOXO

HIV

•Vpr doesn’t bind FOXO

Kino et al 2005

What is insulin supposed to do?

FOXO

(Nucleus)

p

Insulin

FOXO p14-3-3

FOXO

Insulin

14-3-3

FOXO p14-3-3

FOXO p

14-3-3

HIV

Vpr won’t let insulin work!

FOXO

No FOXO

HIV

Kino et al 2005

How does Vpr affect glucocorticoid receptors?

Vpr

LQQLL

HIV

•Specific LXXLL motif binds GRE

•Completely different from ability to arrest cell cyle

Kino et al 2000

Vpr as a Co-regulator

Kino et al 2000

Vpr as a Co-regulator of GR

Vpr

TATA

Transcription: enhancing glucocorticoid signal

G R

TFIIB

TFIID

RNA polymerase II

GRE

HIV

Kino et al 2000

Summary of Vpr & SMAVPR Protein

Skeletal Muscle Atrophy

VprGRE

Glucocorticoid

14-3-3

Vpr

FOXOnucleus

Atrogin-1

Therapies for HIV muscle wasting

• Steroid hormone receptor antagonists (RU 486)

• Vpr antagonists

• Current antiretroviral therapies

Cancer Cachexia

Skeletal Muscle Atrophy

?

NF-κB

Nucleus

IκB

NF- κB

IκBα

P

Cachexia

Cai et al. Study

MISR Mouse MIKK Mouse

Constitutively active IκBα

Constitutively active IκB

Cachexia

Inactive NF-κB Always active NF-κB

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

Cachexia

Cachexia Pathway

TNF-α

NF-κB

MURF1 MyoD

activates

decreasesincreases

Therapy for Cachexia

• Salicylate inhibits the NF-κB pathway, preventing muscle loss

Cachexia

Oxidative Stress

Skeletal Muscle Atrophy

?

Oxidative StressReactive Oxygen Species

Mitochondria

Caspase-3Calpain

Sarcomere

Unit of Myofibril

20S/26S Proteasome

Calcium

Cytochrome c

CalpastatinCalpastatin

NO

Oxidative Stress

Effect of Disuse

• Disuse increases oxidative stress

Tidball et al, 2002

Oxidative Stress

Effect of Oxidative Stress

• Increase of oxidative stress increases calsequesterin

• Calsequestrin sequesters intracellular calciumHunter et al, 2001

Laser Densitometry

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

Koh et al, 2000

• Calcium treatment increases protein cleavage

• Protein cleavage can be inhibited by nitric oxide and calpastatin

Oxidative Stress

Caspase Activity

• Caspases inhibit calpastatin

• Calpastatin is a calpain inhibitor

Wang et al, 1998

Oxidative Stress

SummaryOxidative Stress

Increase Ca2+

Calpain & Caspase 3 activity increases

Releases Actomyosin to be degraded in proteasome

Skeletal Muscle Atrophy

Therapies for Oxidative Stress

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

Cell

Vitamin E

ROS

Oxidative Stress

Diabetes

?

Skeletal Muscle Atrophy

Diabetes

Insulin levels

Ub-ligase E3-α

26 S proteasome

What activates the Ubiquitin-proteasome pathway?

• No difference in diabetics without vs. diabetics with acidosis

• Acidosis is not a stimulus of ubiquitin dependent atrophy

Diabetes

Price et al. 1999

Effects of Insulin on Ub-proteasome pathway

• Less protein degradation in insulin treated muscles

• Lower insulin levels leads to activation of Ub-proteasome pathway

Diabetes

Price et al., 1999

Results of Pathway Activation

• Levels of Ub-ligase and its coenzyme increase

• Amount of Ub-conjugation by these increases

Diabetes

Goldberg et al., 1999

Proteasome Formation

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

Diabetes

Attaix et al., 2004

Proteasome Activity

• Flourescence in atrophied muscles higher than control muscles

• Flourescence is analogous to amount of 26 S proteasome activity

Diabetes

Attaix et al., 2004

Summary of Diabetes and SMADiabetes

Insulin decrease/ glucocorticoid increase

E3-alpha ubiquitin ligase increases

26 S Proteasome activity increases

Skeletal Muscle Atrophy

Therapy for 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

Diabetes

Presentation Pathway

Introduction

Diseases

HIV

Cachexia

Oxidative Stress

Diabetes

Discussion

HIV

Skeletal Muscle Atrophy

Diabetes

VPR Protein

Oxidative Stress

Cachexia

?

??

Vpr

VPR Protein

Inhibit insulin effects on FOXO

Glucocorticoid hypersensitivity

Co-activates glucocorticoid receptor

Atrogin-1 induction

Skeletal Muscle Atrophy

Cachexia

Cachexia

Murf-1 increase

IKK/NF-kappa B pathway

MyoD mRNA decrease

Skeletal Muscle Atrophy

Oxidative StressOxidative Stress

Increase Ca2+

Calpain & Caspase 3 activity increases

Releases Actomyosin to be degraded in proteasome

Skeletal Muscle Atrophy

DiabetesDiabetes

Insulin decrease/ glucocorticoid increase

E3-alpha ubiquitin ligase increases

26 S Proteasome activity increases

Skeletal Muscle Atrophy

HIV

Skeletal Muscle Atrophy

VPR Protein

Inhibit insulin effects on FOXO

Glucocorticoid hypersensitivity

Co-activates glucocorticoid receptor

Atrogin-1 induction

Oxidative Stress

Increase Ca2+

Calpain & Caspase 3

activity increases

Releases Actomyosin

to be degraded in proteasome

Cachexia

Murf-1 increase

IKK/NF-kappa B pathway

MyoD mRNA decrease

Diabetes

Insulin decrease/ glucocorticoid

increase

E3-alpha ubiquitin increases

26 S Proteasome activity increases

Acknowledgements

Thanks to Dr. D, Sara Herrera, Tammy

Hibler, Arun Paul, and Chris Prater