gms | German Medical Science

Objectives: Sarcopenia is a progressive and generalized muscle disorder characterized by a decline in muscle mass and strength. If left untreated, it can lead to increased falls, fractures, reduced quality of life and increased mortality. In addition, the treatment of sarcopenic patients is expensive to healthcare systems due to an increased hospitalization rate and cost of care when hospitalized.

Patients suffering from disease-related (secondary) sarcopenia have been associated with an enhanced level of the transcription factor FOXO3 in skeletal muscle.

Foxo3 is one of several transcription factors of the highly conserved Forkhead-Box-Protein family. As a downstream target of the PI3K/AKT pathway, Foxo3 plays an important role in protein turnover and muscle wasting. Foxo3 therefore could pose to be a potential target of treatment for secondary sarcopenia.

Our aim is to develop an adeno-associated-virus (AAV)-based therapy against sarcopenia. The focus of this study is to reduce the Foxo3-expression in murine myoblasts in vitro and to analyze changes in myogenic differentiation of AAV-treated skeletal muscle cells.

Methods: In order to design an effective therapeutic strategy and study the effects of Foxo3 knockdown on myogenic differentiation, we performed a short- and a long-term knockdown of Foxo3. For a short-term knockdown, murine myoblasts were transfected with siFoxo3 via lipofection. The long-term Foxo3 knockdown was achieved by AAV-transduction of shFoxo3, tagged with GFP. In both experiments, myoblasts were differentiated to myotubes for 9 consecutive days. Infected AAV-cells were sorted via FACS and analyzed by immunofluorescence-microscopy. Length and width of myotubes were measured by light microscopy. Foxo3 mRNA was analyzed with RT-qPCR.

Results and Conclusion: We were able to reach a significant Foxo3-mRNA knockdown of up to 82% after lipofection and 80% after AAV-transduction. Functional inhibition was further demonstrated by a 32% decrease of the expression of Atrogin1-mRNA, downstream of Foxo3. Foxo3-inhibited myotubes showed an increase in length and width after knockdown. Further analysis of AAV-transduced cells are in progress and will shed light upon the role of Foxo3 in myogenic differentiation and its possible role in sarcopenia.