Anabolic Characterization Of Immortalized Murine L6, And Canine Myok9 Skeletal Myotubes

Abstract

Healthy skeletal muscle is an important site of metabolic regulation. Metabolic dysregulation of skeletal muscle contributes to the development of obesity, the metabolic syndrome, and muscle degeneration. Interest in developing an in vitro canine model has been increasing as canine skeletal muscle is more pathologically similar to human skeletal muscle than rodent. In vitro models are useful to control variables, reduce clinical costs, and assess molecular characteristics of tissue. Therefore, characterization of Myok9 myotube anabolic function and comparison to an established cell line is necessary to understand metabolic function of this novel cell line for its use in the study of skeletal muscle dysfunction and disease. PURPOSE: To describe anabolic signaling and determine fractional synthesis rates of novel Myok9 myotubes when compared to established murine L6 myotubes. METHODS: L6 and Myok9 myoblasts were grown in vitro in standard culture medium until 70% confluent. Differentiation was induced by switching to a low serum medium. Media containing 4% deuterium oxide (stable isotope label) supplemented with the appropriate pharmacological treatment (DMSO, rapamycin, and torin1) was added to myotubes during the final 24-hours of the 7-day differentiation period. Cells and media samples were harvested for fractional synthesis rates, and protein content of key anabolic markers using Western blotting techniques. RESULTS: Myofibrillar protein synthesis rates (MPS) were similar between species, but slightly lower in canine versus rat cells. Both cell lines exhibited significantly lower MPS in the myotubes treated with torin1. The phosphorylated to total protein ratios of 4EBP1 were significantly lower in both L6 and Myok9 myotubes treated with torin1. The phosphorylated to total protein ratios of p70S6K were significantly lower in L6 and Myok9 myotubes treated with rapamycin or torin1. CONCLUSION: Similar anabolic responses to pharmacologic inhibition occurred in both L6 and Myok9 myotubes. Interestingly, MPS rates in Myok9 myotubes were slightly lower compared to L6 myotubes. These results suggest Myok9 cell line could be used in the development of pharmacologic treatments of skeletal muscle dysfunction or disease while remaining cognizant of lower overall protein synthesis rates.