טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTafesh Hanan
SubjectElucidating the Roles of Lysyl Oxidase in Myofibers During
Muscle Regeneration
DepartmentDepartment of Medicine
Supervisor Professor Peleg Hasson


Abstract

Skeletal muscles are the largest tissues in the human body, constituting up to 50% of total body mass, and facilitate the movement of our body. Upon injury the skeletal muscles are affected and go through a regeneration process. This regeneration segment occurs efficiently and is rapidly mediated by a specific genetic program, where both the myofibers and the connective tissues (CT) surrounding them are rebuilt. The crosstalk formed between them is crucial for proper regeneration. We set to test the role of Lysyl Oxidase (Lox), a key enzyme in the CT, during muscle regeneration.

Lox is the primary member of an enzyme family consisting of five members?The Lysyl oxidases?known for their essential function in extra cellular matrix (ECM) maturation, where Lox-dependent oxidation of lysine residues in collagen and elastin, gives rise to the cross-linked fibers.

Previous experiments conducted in our lab, showed that Lox is primarily expressed in myofibers during embryonic development, and the Lox mutants develop defective muscles. When followed during adult muscle regeneration, Lox was found to be primarily upregulated in regenerating myofibers and their ECM, where it significantly delayed the regeneration when inhibited at the onset of the injury.

We followed the dynamics of Lox roles in myofiber maturation at different timepoints in the myogenic program by neutralizing Lox’s enzymatic activity. Consequently, we asked the following questions: 1. What will the specific deletion of Lox, in the myofibers during muscle regeneration at different time points, cause to the regeneration process in vivo? 2. What are the activities of Lox in myofibers in vitro? Would the deletion of Lox prior to, during and following myogenic differentiation affect the myogenic program? And, if so, then how?

The persistent Lox expression during the regeneration process led us to hypothesize that Lox is required also for the later stage of myogenic regeneration.

In this study we used inhibition and genetic deletion methods. In vivo analyses showed that there was no effect on the regeneration process in both cases: when Lox was deleted prior to regeneration, or when Lox was inhibited post regeneration. This was inconsistent with preliminary results found in our lab. Thus, we turned to in vitro analyses of Lox deletion during different stages of myofiber maturation: It showed that deletion prior to and after a short period myofiber differentiation led to significant difference; however, when deletion was carried out after a longer duration of differentiation no significance was observed.

Overall, deletion of Lox in the early stages of myogenesis did not block their commitment towards the myogenic program but significantly inhibited myofiber maturation. We found that Lox has major role in the first stages of myogenic differentiation, presumably during the fusion stage, but has no effect on the maintenance of the mature myofiber.

In conclusion, this study provides a new insight into Lox requirement during the different stages of myofiber maturation. Further studies are necessary for the understanding of its role. These studies could eventually open doors to possible avenues for improving Lox based illnesses therapies.