1. 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

2. 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

3. Factors in aging of skeletal muscle

5. Immobilization (first 24 -48h) Ca +2 influx Increased Ca +2 dependent proteolysis by calpains Initiation of myofibrillar proteins degradation and Z- disk disintegration The fast phase of muscle breakdown due to immobilization

6. 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 AP-1 (?) transcription factors Upregulation of stress and inflammation genes including iNOS NO, ONOO - RNS Increased muscle wasting Biphasic regulation of the transcription factors by NO: Low levels activate, high levels shut down Ubiquitin- proteasome- dependent proteolysis Lysosomal proteolysis Ca +2 dependent proteolysis The slow phase of muscle breakdown due to immobilization (2-30 days)

7. Mobilization Excessive mobilization (strenuous exercise) Immobilization

8. In vivo model: Immobilized young and old rats

9. The external fixation model of immobilization

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

11. 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

12. Normal vs. immobilized skeletal muscle of an old animal after 4 weeks of immobilization

13. The activation of various muscle protein degradation systems in immobilized animals

14. 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

15. Ubiquitination of muscle proteins following immobilization of young rats Protein stainingImmunostaining (anti- Ubiquitin AB.) L-control leg R- immobolized leg

16. *

18. Acid phosphatase activity in normal vs. immobilized (30 days of E.F) muscle of young animals (histochemical staining)

19. Zymography of gastrocnemius muscles of five young rats after 21 and 30 days of immobilization L-control leg R- immobolized leg

20. 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.

21. Conclusion There appears to be a link between the activation of the extracellular and intracellular proteolytic systems

22. phase

23. 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 AP-1 (?) transcription factors Upregulation of stress and inflammation genes including iNOS NO, ONOO - RNS RNS Increased muscle wasting Biphasic regulation of the transcription factors by NO: Low levels activate, high levels shut down Ubiquitin- proteasome- dependent proteolysis Lysosomal proteolysis Ca +2 dependent proteolysis The slow phase of muscle breakdown due to immobilization (2-30 days)

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

25. 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