E-34 Free Communication/Poster - Muscle Adaptations, Atrophy, and Hypertrophy Friday, June 2, 2017, 7: 30 AM - 12: 30 PM Room: Hall F
Characterization of Protein Metabolism in Undifferentiated and Differentiated Murine Muscle Tissue
2692 Board #212 June 2 11
00 AM - 12
The emergence of cell culture experiments have greatly expanded the understanding of skeletal muscle physiology. However, there is a paucity of data regarding the behaviors of cells grown in culture at various stages versus in vivo. This preliminary set of studies was designed to assess alterations of anabolic responses between undifferentiated and differentiated muscle tissue.
PURPOSE: Determine if there is a disparity in fractional synthesis rates (FSR) between C2C12 myoblasts and myotubes. * 100
METHODS: C2C12 cells were plated at 200,000 cells per T25 flask and 600,000 cells per T75 flask with 5 mL and 12 mL DMEM (respectively) supplemented with 20% Fetal Bovine Serum and 1% gentamycin. Cells were cultured in an environment at held at a constant 37°C and 5% CO2. Once cells reached confluence, media was changed to DMEM supplemented with 2% horse serum 1% gentamycin, 5% HEPES, 0.75% transferrin, and 0.75% insulin. Myoblasts were plated after the 4th passage. Deuterium oxide was applied 24 hours prior to harvest of the cells at a level of 4%. Media containing deuterium oxide was reserved for analysis. Cells were washed with multiple applications of PBS. Norris buffer was then applied to the flasks at 100 uL for T-25’s and 300 uL for T-75’s. Flasks were then placed on ice for 5 minutes. Cells were harvested and deposited into centrifuge vials. Vials were spun at 14,000 G for 30 minutes to separate cytosolic and myofibrallar fractions. The supernatant (containing the cytosolic fraction) from the vial was decanted into another vial and saved for analysis. 2H-alanine and plasma enrichment was determined by GC-MS and FSR was calculated by:
RESULTS: Preliminary data demonstrates that differentiated murine myotubes have ~76% FSR of the undifferentiated murine myoblasts (P < 0.005).
CONCLUSION: Future investigators must be aware of the ratio of undifferentiated cells and differentiated myotubes as this ratio could confound results as myoblasts are still present even at later stages of differentiation.© 2017 American College of Sports Medicine