There are a number of ways to use the C2C12 cell line to model skeletal muscle atrophy, one of the easiest is starvation atrophy. This protocol is best used on differentiated myotubes, and results in clear protein degradation and upregulation of the E3 ubiquitin ligases MuRF1 and MAFbx (Atrogin1), as well as the NAD+ dependent histone/protein deacetylase SirT1. I use DMEM that lacks both glucose and sodium-pyruvate, and then I add a low concentration of glucose back into the media (2.5mM, or 1/10th of the concentration used in most C2C12 growth media).
- Use an appropriately sized plate containing differentiated myotubes (~2-3days in differentiation media)
- Wash cells in PBS
- Add starvtaion media (DMEM [no glucose, no sodium-pyruvate], 0.1% horse serum, 2.5mM glucose, and penicillin/streptomycin)
- Cells can then be incubated for 1-24hrs (cell death will occur at ~16-24hrs)
- It is also possible to induce rapid starvation, via incubation in PBS, this should only be used for 3-6hrs as the majority of the cells will die beyond 6hrs
Figure. qPCR measurements of the E3 ubiquitin ligases MuRF1 and MAFbx, the forkhead box O3 transcription factor, and the NAD+ dependent histone/protein deacetylase SirT1, following ~20hrs in different starvation media.