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

Changes in the neural system and

stimuli

Changes in the muscle apparatus

with aging

Environmental and external

influences

Loss of motor units*

*Changes in the neuromuscular junction

Atrophy and loss of

muscle fibers

*Decrease in physical activity

* Disuse and immobilization

*Nutrition and food restriction

Diseases* *Injuries

Medications*

Decline in muscle mass (Sarcopenia)

Loss of force generating capacity and functional

decline

Changes in CNS

Neural molecular and cellular changes

Intrinsic molecular and

cellular changes

Factors in aging of skeletal muscle

Immobilization (first 24 -48h)

CaCa+2+2 influx influx

Increased CaIncreased Ca+2+2 dependent proteolysis by calpains dependent proteolysis by calpains

Initiation of myofibrillar proteins degradation and

Z- disk disintegration

The fast phase of muscle breakdown due to immobilization

Infiltration of monocytes and differentiation into Infiltration of monocytes and differentiation into macrophagesmacrophages

Macrophages activationMacrophages activation

Synthesis of cytokinesSynthesis of cytokines

IL-1, IL-6, TNF- IL-1, IL-6, TNF- αα by the macrophages by the macrophages

Oxidative stressOxidative stress

Activation of NF-kB andActivation of NF-kB and

AP-1 (?) transcription factorsAP-1 (?) transcription factors

Upregulation of stress and inflammation Upregulation of stress and inflammation genes including iNOSgenes including iNOS

NO, ONOONO, ONOO--

RNSRNS

Increased muscle wastingIncreased muscle wasting

Biphasic regulation of the Biphasic regulation of the transcription factors by NOtranscription factors by NO::

Low levels activate, high Low levels activate, high levels shut downlevels shut down

Ubiquitin- proteasome- dependent Ubiquitin- proteasome- dependent proteolysisproteolysis

Lysosomal proteolysis Lysosomal proteolysis

CaCa+2+2 dependent proteolysis dependent proteolysis

The slow phase of muscle breakdown due to immobilization (2-30 days)

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:

CaCa+2+2 – dependent proteases (calpains) – dependent proteases (calpains)

Ubiquitin- proteasome systemUbiquitin- proteasome system

Intracellular lysosomal proteases (Cathepsins D, H, L, B., nucleases, lipases, Intracellular lysosomal proteases (Cathepsins D, H, L, B., nucleases, lipases, glycosidases, ACP)glycosidases, ACP)

Extracellular:Extracellular:

Macrophage lysosomal proteasesMacrophage lysosomal proteases

Matrix Metalloproteases (MMPs): MMP-2, MMP-9Matrix 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

ObservationsIn 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.

ConclusionConclusion

There appears to be a link There appears to be a link between the activation of the between the activation of the extracellular and intracellular extracellular and intracellular proteolytic systemsproteolytic systems

phase

Infiltration of monocytes and differentiation into Infiltration of monocytes and differentiation into macrophagesmacrophages

Macrophages activationMacrophages activation

Synthesis of cytokinesSynthesis of cytokines

IL-1, IL-6, TNF- IL-1, IL-6, TNF- αα by the macrophages by the macrophages

Oxidative stressOxidative stress

Activation of NF-kB andActivation of NF-kB and

AP-1 (?) transcription factorsAP-1 (?) transcription factors

Upregulation of stress and inflammation Upregulation of stress and inflammation genes including iNOSgenes including iNOS

NO, ONOONO, ONOO--

RNSRNS

Increased muscle wastingIncreased muscle wasting

Biphasic regulation of the Biphasic regulation of the transcription factors by NOtranscription factors by NO::

Low levels activate, high Low levels activate, high levels shut downlevels shut down

Ubiquitin- proteasome- dependent Ubiquitin- proteasome- dependent proteolysisproteolysis

Lysosomal proteolysis Lysosomal proteolysis

CaCa+2+2 dependent proteolysis dependent proteolysis

The slow phase of muscle breakdown due to immobilization (2-30 days)

9th Annual Meeting of The Oxygen Society San Antonio , TX, U.S.A Nov. 20-24, 2002

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