טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBar-Shai Marina
SubjectThe Involvement of Oxidative Stress and NF-kB Activation in
the Degradation of Skeletal Muscle Proteins in
Immobilization and Aging
DepartmentDepartment of Medicine
Supervisors Professor Abraham Reznick
Professor Raymond Coleman
Full Thesis textFull thesis text - English Version


Abstract

The role of Reactive Nitrogen Species (RNS) in NF-kB activation and protein degradation remains controversial. This study investigated NF-kB activation by RNS in skeletal myoblasts. Furthermore, this study investigated RNS and NF-kB- dependent proteolysis in skeletal myocytes. Additionally, the molecular mechanisms of muscle protein breakdown during immobilization in aging were studied in a model of young and old rats.

We found that both NO and peroxynitrite caused NF-kB activation in skeletal myocytes. NO donors- induced NF-kB activation was transient and dependent on I-kB a degradation. Conversely, peroxynitrite donors- induced NF- kB activation was  dependent on tyrosine nitration of I-kB a, but independent of its  serine phosphorylation and degradation. Prolonged exposure to peroxynitrite resulted in non-transient NF-kB activation and high iNOS expression.

 Further, myoblasts were exposed to NO and peroxynitrite donors in combination with various chemicals that inhibit either extracellular or intracellular formation of RNS. It was demonstrated that both extracellular and intracellular NO had to be intracellularly converted into peroxynitrite to activate NF- κB. Conversely, high extracellular peroxynitrite may induce continuous activation of NF- κB, which is independent of intracellular NO and superoxide formation.

 Fully differentiated myotubes were treated with NO and peroxynitrite donors.  NF-kB activation, the activation of ubiquitin- proteasome pathway and matrix metalloproteinases and the levels of muscle- specific proteins were investigated. RNS donors caused  NF-kB  activation, and increased activation of proteolytic systems, as well as the degradation of muscle- specific proteins. Antioxidant treatment, tyrosine nitration inhibition and NF-kB molecular inhibition were effective in downregulation of  NF-kB  and slowing down the degradation of muscle- specific proteins.

In immobilized hindlimbs of young and old rats after 4 weeks of External Fixation, acid phosphatase, matrix metalloproteinases and NF-κB were differentially activated  at various stages of muscle disuse atrophy in young versus old animals.

This research has demonstrated the direct link between NF-kB and RNS in skeletal muscle cells, and has delineated a novel pathway of NF-kB activation, which is self- amplifying under the conditions of chronic inflammation. Moreover, this study has shown that increased muscle proteolysis may be induced by RNS through direct mediation of NF-kB, and has demonstrated that in both young and old animals muscle breakdown following immobilization is associated with increased NF-kB activity, with different kinetics of activation of muscle proteolytic systems in young and old animals.