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Projects / Programmes source: ARIS

MOLECULAR MECHANISMS UNDERLYING IN VITRO REGENERATION OF HUMAN SKELETAL MUSCLE UNDER NORMAL AND HYPOXIC CONDITIONS.

Research activity

Code Science Field Subfield
3.03.00  Medical sciences  Neurobiology   

Code Science Field
B640  Biomedical sciences  Neurology, neuropsychology, neurophysiology 

Code Science Field
3.01  Medical and Health Sciences  Basic medicine 
Keywords
human skeletal muscle, muscle regeneration, myoblast, myotube, satellite cells, proliferation, hypoxia, HIF-1, IL-6, gene silencing, BrdU
Evaluation (rules)
source: COBISS
Researchers (1)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  31530  PhD Urška Matkovič  Medical sciences  Head  2011 - 2013  71 
Organisations (1)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0381  University of Ljubljana, Faculty of Medicine  Ljubljana  1627066  46,263 
Abstract
Skeletal muscle injuries are among the most prevalent conditions in clinical practice. Hypoxic injuries are especially frequent and can have various causes like impaired blood perfusion due to aterosclerosis, strenuous muscular exercise, high altitude environment or severe shock conditions; frequently such causes are combined. Skeletal muscle responds to its damage by efficient regeneration process aimed to replace the damaged part by new muscle tissue. The final outcome of muscle injury therefore depends not only on the lesion itself but also on the extent of muscle regeneration, which is therefore a potential therapeutic target at muscle injuries. However, rational approach to such therapy necessitates identification of the molecular mechanisms underlying regeneration process and these are still largely unknown. The process of muscle regeneration is stereotyped and does not depend on the cause of the damage. It starts with the activation and proliferation of the satelllite cells that reside beneath the basal lamina of the adult muscle fiber. After proliferation newly formed mononuclear myoblasts fuse and form polynuclear myotubes. Following innervation by motor neurons myotubes mature and become normal functional muscle fibers. The final amount of regenerated muscle mass critically depends on the extent of myoblast proliferation, as  it is the stage where the number of new nuclei, a source of new proteins, is determined. The mechanism of myoblast proliferation is complex. In addition to growth factors secreted from blood cells invading the site of injury, cytokine signaling is importantly involved in this process. Especially important with this regard is  IL-6, which is the main response of skeletal muscle to various environmental stimuli. The suggested physiological meaning of IL-6 secretion from myoblasts is auto- and paracrine stimulation of their proliferation and the proposed project is focused particularly on this IL-6 role. Proliferative effects of IL-6 are promoted by invading blood cells, however, the stimulatory effect of IL-6 on myoblast proliferation at muscle hypertrophy, where these cells are absent, speaks in favor of autonomous role of IL-6 in this process. I plan to investigate the effects of IL-6 on the in vitro experimental model which allows observation of all stages of muscle regeneration, from myoblast proliferation to myotube innervation and which is routinely used in the laboratory where I intend to carry out this project. In case of human muscle this is practically the only model in which proliferating myoblasts can be manipulated and where their response can be studied under controlled conditions. A robust constitutive secretion of IL-6 has been demonstrated in this model. It was additionally enhanced by pro-immflamatory factors, such as TNF-? and LPS. In the ten month period of my cooperation with the research group, I have indepedently introduced a method to measure myoblast proliferation on the basis of BrdU insertion into their DNA. I found that the synthetic IL-6 has no effect on the in vitro proliferation of human myoblasts under our conditions, but that myoblast proliferation is significantly increased after the treatment with the conditioned medium into which endogenous IL-6 is secreted after LPS stimulation of myoblasts. In this project I intend to elucidate further the IL-6 stimulatory role on the myoblast proliferation. Since IL-6 actions depend on complex interactions with membrane-bound or soluble forms of its receptor subunits and since infiltrating macrophages also have an important role in muscle regeneration, I plan to study also these effects on myoblast proliferation. Muscle regeneration is often accompannied by hypoxia and therefore I intend to study IL-6 role also under hypoxic conditions. I will test the hypothesis that muscle response to hypoxia, regulated by HIF-1, influence the mechanisms underlying proliferation of human myoblasts.
Significance for science
Investigation of the mechanisms that are responsible for the activation of muscle stem cells (so-called satellite cells), which consequently trigger the proliferation and migration of mononuclear myoblasts, is scientific field of emerging research. The muscle mass formed anew in the regeneration process critically depends on the extent of myoblast proliferation. The ability to produce sufficient number of myoblasts from primary muscle cultures, necessary for the effective treatment of damaged muscles of individual patients, is one of the major limitations of potential myoblast transfer therapy. Hence, the identification of factors promoting the early stages of muscle regeneration is of great importance, since they could be employed to increase the myoblast yield from the donor muscle under in vitro conditions. Muscle injuries often occur under hypoxic conditions. Therefore, understanding the stimulatory effects on muscle regeneration in these conditions is an important contribution to the identification of new potential targets for improvement of muscle regeneration and therapeutic approaches to muscle deseases.
Significance for the country
Through the postdoc project I become more involved in the field of neurobiology at the international level. Attending national and international scientific meetings I contributed to the creation of new opportunities for collaboration and knowledge sharing among related research groups. In this way, I introduced new approaches and techniques to our laboratory. Moreover, publishing scientific papers helped to integrate Slovenia into a group of scientifically developed countries. In addition, our latest results obtained in collaboration with prof. Paola Lorenzon from University of Trieste have strenghten the existing bilateral bonds. Beside research work, I got some experience with tutoring graduate and postgraduate students that were also included in the project, which represents an important contribution at the education level as well.
Most important scientific results Annual report 2012, final report, complete report on dLib.si
Most important socioeconomically and culturally relevant results Annual report 2011, 2012, final report, complete report on dLib.si
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