Molecular Mechanisms and Signaling Pathways in Muscle Atrophy in Immobilization and Aging

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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

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Summary of the main topics

• Introduction to aging and muscle protein
  degradation
• In vivo model of immobilization and the stages of
  skeletal muscle breakdown
• In vitro model of the involvement of RNS in
  activation of NF- ?B in muscle cells

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Factors in aging of skeletal muscle
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The fast phase of muscle breakdown due to
immobilization
Immobilization (first 24 -48h)
Ca2 influx
Increased Ca2 dependent proteolysis by calpains
Initiation of myofibrillar proteins degradation
and Z- disk disintegration
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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- a 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
Increased muscle wasting
Lysosomal proteolysis
Ca2 dependent proteolysis
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Mobilization
Excessive mobilization (strenuous exercise)
Immobilization
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In vivo model

• Immobilized young and old rats

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The external fixation model of immobilization
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The external fixation model of immobilization
(contd.)
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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

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Normal vs. immobilized skeletal muscle of an old
animal after 4 weeks of immobilization
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The activation of various muscle protein
degradation systems in immobilized animals
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Muscle proteolytic systems

• Intracellular
• Ca2 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

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Ubiquitination of muscle proteins following
immobilization of young rats
Protein staining
Immunostaining (anti- Ubiquitin AB.)
L-control leg R- immobolized leg
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Acid phosphatase activity in normal vs.
immobilized (30 days of E.F) muscle of young
animals (histochemical staining)
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Zymography of gastrocnemius muscles of five
young rats after 21 and 30 days of immobilization
L-control leg R- immobolized leg
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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.
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Conclusion There appears to be a link between the
activation of the extracellular and
intracellular proteolytic systems
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phase
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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- a 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
Increased muscle wasting
Lysosomal proteolysis
Ca2 dependent proteolysis
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9th Annual Meeting of The Oxygen Society
San Antonio , TX, U.S.A
Nov. 20-24,
2002
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Acknowledgements

• Eli Carmeli, PhD
• Raymond Coleman, PhD
• Ophir Menashe, MSc
• Marina Bar Shai, BSc
• Erez Hasnis, BSc
• Pessia Shantzer
• Bilha Pinkhasi
• Shoshan Perek
• Yotam Shkedi

Thank you for your attention!