טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMenashe Ofir
SubjectMolecular Mechanisms of Muscle Degragation Processes Due to
Immobilization
DepartmentDepartment of Medicine
Supervisor Professor Abraham Reznick


Abstract

Limb immobilization results in a fairly rapid loss of muscle mass. This muscle disuse atrophy is seen in fractured limbs set in plaster casts or after external fixation techniques. We used an external fixation technique to immobilize movement in the right legs of 6-8 month old Wistar rats. The contralateral (left) legs served as controls. In addition untreated age-matched animals served as further controls. After 5, 10, 21 and 30 days of hindlimb immobilization the animals were sacrificed and the hindlimb muscles (gastrocnemius, quadriceps, soleus and plantaris) removed for analysis. The aims of the study were: (1) to follow the dynamics of muscle weight loss in the immobilized limbs for a period of 30 days (2) to study and characterize the extracellular degradation systems (using acid phosphatase activities as an indication of macrophage activities) and matrix metalloproteinases (MMPs) activated by immobilization (3) to study and characterize the intracellular degradation systems with emphasis on the ubiquitin pathway (4) to examine the progressive histological changes in the immobilized limbs (5) to improve our understanding of protein breakdown in muscles resulting from disuse due to leg immobilization.

     Results showed a progressive weight loss in the hindlimb muscles reaching 41-47% at the end of the experimental period. Acid phosphatase activities increased by 54-55% in the gastrocnemius and quadriceps muscles in the immobilized legs. The histochemical localization of acid phosphatase activities showed increased localization in the extracellular tissue surrounding the myofibers and also in atrophying myofibers in the affected muscles. By means of gelatin zymography and Western blot analysis it was shown that the metalloproteinases MMP-2 and MMP-9 and their inhibitor (TIMP-1) increased in immobilized muscle mainly in the fourth week after external fixation. Using Western blot analysis it was shown that the ubiquitin degradation system was also activated mainly in the fourth week after the external fixation.

      Our results indicate that the slow phase of muscle protein breakdown begins with the secretion of cytokines such as TNF-a by invading macrophages followed by an increased intracellular oxidative state and the activation of iNOS enzyme. Using an in vitro culture of L-8 muscle cells, it was shown that TNF-a induced an increase in protein oxidation concomitant with the activation of protein ubiquitination. Some evidence is also presented indicating that increased NO may also change ubiquitination of some muscle cell proteins. We conclude that in our rat model of hindlimb external fixation, the fourth week appears to be the crucial period for significant expression of both intracellular and extracellular degradation systems, which may connected.