Associate Professor Hampden-Sydney College Hampden-Sydney, Virginia, United States
Introduction: The skeletal muscle system provides support and movement for the body, thus being an essential component of human anatomy. Major loss of skeletal muscle mass can occur from aging, trauma, atrophy, and degenerative diseases. Athletes are also susceptible to muscle damage. Frequent physical activity, breakdown of their body, and injuries are factors that promote muscle damage (Evans, 2010). A healthy lifestyle and maintenance of one’s body is essential. Metabolism, cellular reproduction, and protein synthesis all help the body function and recover. Branched-chain amino acids (BCAAs) build muscle tissue and increase protein synthesis in humans, which could be a potential regenerative method for skeletal muscle (Zhang, 2017). This project investigated the effect that each BCAA has on skeletal muscle cell maturation and development. Muscle cell maturation was determined through the proliferation, differentiation, and fusion of myoblasts. The goal of these tests was to discover which concentration of BCAA enhanced skeletal muscle growth and maturation. It was hypothesized that BCAAs will have a significant effect on cell maturation with leucine producing the most substantial result for skeletal muscle maturation.
Materials and
Methods: Each BCAA daily recommended amount (DRA) was calculated using the average weight of a college baseball player in the Old Dominion Athletic Conference (Cleveland Clinic medical, 2021). Then, each one was divided by fiver liters of blood to find the concentration (g/L) for leucine, isoleucine, and valine. The correct amount was weighed out, added to Dulbecco’s Modified Eagle’s Medium (DMEM), allowed to dissolve, and filter sterilized to make three distinct supplemented media. Wells were seeded with 10,000 C2C12 cells and DMEM growth media added. A calibration curve with known cell numbers and the absorption values was completed. On day four, the media was switched to either the isoleucine, leucine, valine, or the 2% horse serum (HS) supplemented media as a positive control for maturation. On days seven and ten, each group was assessed for cellular activity using an MTS assay and the cell number was calculated from the calibration curve (n=6/group/time point). Maturation was examined by fluorescently staining the cytoskeletal actin and nuclei to determine the myotube fusion index (n=3/group/time point).
In the second study, the concentration of each BCAA was reduced to ¼ of the DRA by diluting the three supplemented media to make ¼ concentrations. The cell study was setup as described above. On day four, the cells were fed with one of the ¼ concentration BCAA media or the 2% HS media. Cellular activity, cell number, and the myotube fusion index were determined as described above.
Results, Conclusions, and Discussions: The goal was to discover the BCAA concentration that enhanced skeletal muscle growth and maturation. Day 7 average cellular activities for isoleucine, leucine, valine, and HS were 0.89 ± 0.10, 0.83 ± 0.10, 0.84 ± 0.14, and 0.91 ± 0.13 respectively. On day 10, it was 0.89 ± 0.08 for isoleucine, 0.90 ± 0.08 for leucine, 0.92 ± 0.11 for valine, and 0.93 ± 0.08 for HS (Figure 1A). Since the average cell number was calculated based on the cellular activity values, there were no differences between the four groups. The full DRA for isoleucine, leucine, and valine had similar cellular activities and number compared to the HS maturation media. Next, a ¼ BCAA concentration was tested. The day 7 average cellular activities for isoleucine, leucine, valine, and HS were 0.62 ± 0.07, 0.58 ± 0.06, 0.60 ± 0.06, and 0.62 ± 0.07 respectively. Day 10 values were 0.65 ± 0.06 for isoleucine, 0.64 ± 0.08 for leucine, 0.66 ± 0.04 for valine, and 0.63 ± 0.06 for HS (Figure 1B). Yet again, the cellular activity values were similar, so the cell numbers for all four groups were similar. This data indicates that the ¼ DRA for each BCAA had a similar effect on cellular growth as the HS maturation media. Fluorescent image analysis to ascertain the myotube fusion index for maturation is currently ongoing for both studies.
Isoleucine, leucine, valine achieved the same effect as the HS for cell activity and number at the full and ¼ DRAs. However, determining whether leucine had a greater maturation effect since fluorescent image analysis for maturation is ongoing. Future work should focus on finding the lowest BCAA concentration necessary for cell maturation to aid skeletal muscle regeneration.
References Evans WJ. Skeletal muscle loss: cachexia, sarcopenia, and inactivity. Am J Clin Nutr. 2010 91(4):1123S-1127S. Zhang, S., Zeng, X., Ren, M. et al. Novel metabolic and physiological functions of branched-chain amino acids: a review. J Animal Sci Biotechnol 8, 10 (2017). Cleveland Clinic medical. “Amino Acid: Benefits & Food Sources.” Cleveland Clinic, my.clevelandclinic.org/health/articles/22243-amino-acids. (2021).
Acknowledgements (Optional): We would like to acknowledge Dr. Mike Wolyniak and the Undergraduate Research Program at Hampden-Sydney College for funding this project, the Biology department at Hampden-Sydney College, and Mrs. Jennie Jenkins.
