gms | German Medical Science

Objectives: According to its definition, sarcopenia is a loss of muscle mass due to aging. Sarcopenia leads to impaired walking performance and, subsequently, increased risk of falling with associated fractures. Facing an aging population, sarcopenia is becoming more and more important. Since FOXO3 promotes the atrophy related ubiquitin ligase Atrogin-1 expression, targeting Foxo3-expression should prevent an atrophying phenotype. However, a potential therapy would require long-term effects. Here we investigate a strategy for a long-term Foxo3-knockdown in vitro to treat sarcopenic patients in the future.

Methods: C2C12 myoblasts were transduced with a siRNA/GFP-AAV to post-translationally degrade Foxo3 mRNA for an isolated Foxo3-knockdown. As control, we used a GFP-AAV containing a functionless siRNA. Successfully transduced cells (GFP positive) were selected by FACS cell sorting to generate high knockdown single clones. We screened these for Foxo3 expression by RT-qPCR. Subsequently, cells were seeded and differentiated into myotubes under low serum conditions to detect Foxo3 expression during myogenic differentiation. For a morphological investigation, we stained myotubes for myosin heavy chain (MHC) as parameter of differentiation and performed a DAPI staining for nuclei localization. Fluorescence microscopy was performed and we calculated the fiber size according to the MHC signal and nuclei fusion index (DAPI) in ImageJ.

Results and conclusion: We established a stable AAV-mediated Foxo3-knockdown C2C12 cell line. The Foxo3-knockdown was sufficient and stable for at least ten passages indicating its long-term character. Surprisingly, immediately after initiation of differentiation the Foxo3-expression increased and even exceeded the expression of the control group cells from day 5 (One-way ANOVA; p<0.001). Interestingly, the expression of MyoD (early) and myogenin (later differentiation) were significantly reduced upon differentiation. These results were consistent with a smaller muscle fiber phenotype on day 9 (Student's t-test; p<0.001), indicated by a reduced MHC-positive area and a smaller nuclei fusion index (Student's t-test; p<0.001). Overall, our results showed the long-term potential and persistence of an AAV-mediated treatment. However, a long-term Foxo3-knockdown seems to cause opposing effects indicated by the reduction of fiber size in vitro. This effect is the focus of ongoing studies.