Dissertation committee:
Αναστάσιος Φιλίππου, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Κλειώ Μαυραγάνη: Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ.
Μιχάλης Κουτσιλιέρης: Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ.
Ευανθία Κασσή: Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ.
Θεόδωρος Τρουπής: Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ.
Παναγούλα Αγγελογιάννη: Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ.
Αντώνιος Χατζηγεωργίου: Επίκουρος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ.
Summary:
Background: During aging the process of myogenesis is gradually declined and cell apoptosis increases over time. It has been shown that mechanical loading of aged skeletal muscle can ameliorate its impaired myogenic and survival potential. Yet, the molecular responses of aged muscle cells to mechanical loading remain unclear. This study examined the impact of mechanical loading of aged myoblasts on the signaling and gene expression responses associated with the progression of their myogenic lineage and survival. Methods: A model of multiple cell divisions of C2C12 myoblasts was used to replicate cell senescence. Control and aged myoblasts were investigated during myogenesis, i.e., at days 0, 2, 6 and 10 of differentiation. SA-β-gal activity and comet assay were used as markers of aging and DNA damage. Flow cytometry was performed to characterize potential differences in cell cycle between control and aged cells. Alterations in the mRNA and/or protein expression of myogenic regulatory factors (MRFs), IGF-1 isoforms, apoptotic, atrophy, inflammatory, metabolic and aging-related factors were also evaluated. Moreover, control and aged C2C12 cells were cultured on elastic membranes and underwent passive, cyclic stretching on day 0 and day 10 of differentiation. Activation of p-ERK1/2 and p-Akt, and the protein expression of FAK and the myogenic factors MyoD and Myogenin were determined by immunoblotting both in stretched and non-stretched (control) myoblasts. Quantitative RT-PCR was used to measure changes in the expression levels of the MRFs, as well as of growth, atrophy and apoptotic factors in response to mechanical loading of the C2C12 cells. Results: Compared to control cells, aged myoblasts exhibited G0/G1 cell cycle arrest, DNA damage, increased SA-β-gal activity, and increased expression of aging-related factors p16 and p21 during differentiation. Moreover, aged myoblasts showed a reduction in the expression of MRFs and metabolic/anabolic factors, along with an increased expression of apoptotic, atrophy and inflammatory factors. On the other hand, mechanical loading protocols of the aged C2C12 cells on day 0 and day 10 of their differentiation resulted in significant upregulation of protein synthesis processes. Moreover, both control and aged myoblasts exhibited a significant upregulation of of IGF-1 isoforms and MRFs, along with the downregulation of atrophy and apoptotic factors. Conclusions: A diminished differentiation capacity characterized the aged myoblasts which, in combination with the induction of apoptotic and atrophy factors, indicated a disrupted myogenic lineage in the senescent muscle cells. However, mechanical loading was beneficial for the aged myoblasts; The induction of anabolic signaling responses and the upregulation of myogenic and anabolic factors, along with the downregulation of apoptotic and atrophy factors by mechanical loading suggest an amelioration of myogenic and survival ability of the aged myotubes following mechanical stretching.
Keywords:
Myoblasts, Cell senescence, Mechanical loading, Myogenesis, Atrophy
