Transcript The involvement of oxidative stress and NF

Molecular Mechanisms and Signaling Pathways in
Muscle Atrophy in Immobilization and Aging
Marina Bar- Shai
Abraham Z. Reznick
Department of Anatomy and Cell Biology
The Bruce Rappaport Faculty of Medicine
Technion – Israel Institute of Technology
Haifa, Israel
Summary of the main topics
1. Introduction to aging and muscle protein
degradation
2. In vivo model of immobilization and the
stages of skeletal muscle breakdown
3. In vitro model of the involvement of RNS in
activation of NF- κB in muscle cells
Factors in aging of skeletal muscle
Environmental
and external
influences
*Decrease in physical
activity
* Disuse and
immobilization
*Nutrition and food
restriction
*se sae siD
*Injuries
*sn oi taci de M
n i se gn ah C
the
muscle apparatus
with aging
Intrinsic
molecular and
cellular changes
n i se gn ah C
the
neural system and
stimuli
Neural
molecular and
cellular changes
Atrophy and loss of
muscle fibers
Changes
in CNS
*sti n u rotom fo ssoL
*Changes in oru e n e h t
n oi tcn u j ralu csu m
Decline in muscle mass (Sarcopenia)
Loss of force generating
capacity and functional
decline
The fast phase of muscle breakdown due to
immobilization
Immobilization (first 24 -48h)
Ca+2 influx
Increased Ca+2 dependent proteolysis by calpains
Initiation of myofibrillar proteins degradation and
Z- disk disintegration
The slow phase of muscle breakdown due to immobilization
(2-30 days)
Infiltration of monocytes and differentiation
into macrophages
Macrophages activation
Synthesis of cytokines
IL-1, IL-6, TNF- α by the macrophages
Oxidative stress
Activation of NF-kB and
AP-1 (?) transcription factors
Biphasic regulation of the
:transcription factors by NO
Low levels activate, high
levels shut down
Upregulation of stress and inflammation
genes including iNOS
NO, ONOO-
RNS
Ubiquitin- proteasome- dependent
proteolysis
Lysosomal proteolysis
Ca+2 dependent proteolysis
Increased muscle wasting
Mobilization
Excessive mobilization
(strenuous exercise)
Immobilization
In vivo model:
Immobilized young and old rats
The external fixation model of immobilization
The external fixation model of immobilization (contd.)
Experimental design
 6-8 months old female Wistar rats
(250-300gr) and 24 months old
female Wistar rats (300-350gr)
 Immobilization periods : one, two,
three and four weeks
 Right limbs were immobilized, left
limbs served as controls
 At the end of each immobilization
period the muscles were removed
for biochemical and histological
studies
Normal vs. immobilized skeletal muscle of an old animal
after 4 weeks of immobilization
The activation of various
muscle protein degradation
systems in immobilized animals
Muscle proteolytic systems
Intracellular:
 Ca+2 – dependent proteases (calpains)
 Ubiquitin- proteasome system
 Intracellular lysosomal proteases (Cathepsins D, H, L, B., nucleases, lipases,
glycosidases, ACP)
Extracellular:
 Macrophage lysosomal proteases
 Matrix Metalloproteases (MMPs): MMP-2, MMP-9
Ubiquitination of muscle proteins following immobilization of
young rats
Protein staining
Immunostaining (anti- Ubiquitin AB.)
L-control leg
R- immobolized leg
*
Acid phosphatase activity in normal vs. immobilized (30 days of E.F)
muscle of young animals (histochemical staining)
Zymography of gastrocnemius muscles of five
young rats after 21 and 30 days of immobilization
L-control leg
R- immobolized leg
Observations
In the slow phase of muscle atrophy due to limb
immobilization, the kinetics of activation of the
extracellular and the intracellular degradation
systems are very similar.
Conclusion
There appears to be a link
between the activation of the
extracellular and intracellular
proteolytic systems
phase
The slow phase of muscle breakdown due to immobilization
(2-30 days)
Infiltration of monocytes and differentiation
into macrophages
Macrophages activation
Synthesis of cytokines
IL-1, IL-6, TNF- α by the macrophages
Oxidative stress
Activation of NF-kB and
AP-1 (?) transcription factors
Biphasic regulation of the
:transcription factors by NO
Low levels activate, high
levels shut down
Upregulation of stress and inflammation
genes including iNOS
NO, ONOORNS
Ubiquitin- proteasome- dependent
proteolysis
Lysosomal proteolysis
Ca+2 dependent proteolysis
Increased muscle wasting
9th Annual Meeting of The Oxygen Society
San Antonio , TX, U.S.A
Nov. 20-24, 2002
Acknowledgements
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Eli Carmeli, PhD
Raymond Coleman, PhD
Ophir Menashe, MSc
Marina Bar Shai, BSc
Erez Hasnis, BSc
Pessia Shantzer
Bilha Pinkhasi
Shoshan Perek
Yotam Shkedi