Carbon metabolism-mediated myogenic differentiation

Abstract

The role of nutrients and metabolism in cellular differentiation is poorly understood. Using RNAi screening, metabolic profiling and small-molecule probes, we discovered that the knockdown of three metabolic enzymes-phosphoglycerate kinase (Pgk1), hexose-6-phosphate dehydrogenase (H6pd) and ATP citrate lyase (Acl)-induces differentiation of mouse C2C12 myoblasts even in the presence of mitogens. These enzymes and the pathways they regulate provide new targets for the control of myogenic differentiation in myoblasts and rhabdomyosarcoma cells.

Figure 1

Figure 1a–e. RNAi screen and effects of cyclosporin (CsA) and trichostatin (TSA) on C2C12 differentiation. Knockdown of Pgk1, Acl and H6pd causes myoblast differentiation (a) Immunofluorescence of myosin heavy chain expression. The myosin heavy chain fluorescence intensity was normalized to the integrated fluorescence intensity of Hoechst nuclear stain. Significant changes in levels of myosin heavy chain expression relative to control hairpins were calculated as Z scores (* Z score > 2.6). The entire screen was performed three independent times. Error bars represent standard error of the mean. (b) Images show formation of multi-nucleated myotubes and increased myosin heavy chain intensity upon shRNA knockdown. Cells cultured in 2% horse serum served as positive controls for differentiation. (c) CsA treatment inhibits C2C12 differentiation induced by shH6pd. Cells were infected with shAcl, shH6pd or shPgk1. Following gene knockdown, cells were cultured for three days in 0.1 ug/ml cyclosporin (CsA). Data represent three independent experiments performed in triplicate. Error bars represent standard error of the mean, P-value < 0.05. (d) TSA treatment inhibits C2C12 differentiation. 100nM TSA is sufficient to block differentiation initiated by shAcl. (e) Western blot analysis depicts changes in histone acetylation upon overnight treatment with TSA (500 nM), positive control, DM cells were cultured for three days in differentiation medium, and hairpin-infected cells were harvested 48 hours post-infection, shAcl and shGFP.

Figure 2

Figure 2a–c. Fluvastatin treatment causes differentiation of human rhabdomyo-sarcoma cells and inhibits their proliferation and growth on soft agar. (a) RD human rhabdomyosarcoma cells were treated with fluvastatin in normal growth media for four days. Cells were fixed and stained for troponin T expression. Immunofluorescence was normalized to Hoechst nuclear stain. (*P-value < 0.05 t-test) (b) Fluvastatin inhibits human rhabdomyosarcoma cell proliferation. RD rhabdomyosarcoma cells were plated in triplicate. Cells were treated with increasing doses of fluvastatin 2.5–20 μM for four days. Cells were fixed and nuclei were stained with Hoechst dye. Total nuclear fluorescence intensity was quantitated as a measure of cell proliferation. (c) Fluvastatin prevents rhabdomyosarcoma growth on soft agar. Cells were grown in triplicate on soft agar and treated with 10 μM fluvastatin. Images were captured 14 days post-seeding.

Figure 3