MyoD Over-Expression Rescues GST-bFGF Repressed Myogenesis

Area of Science:

• Muscle Stem Cell Biology
• Skeletal Muscle Regeneration
• Fibroblast Growth Factors

Background:

• Basic fibroblast growth factor (bFGF) is crucial for skeletal muscle stem cell (MuSC) proliferation and myogenic fate during embryogenesis.
• The precise mechanisms by which bFGF promotes MuSC proliferation and stemness, and the role of post-translational modifications (PTMs), remain incompletely understood.
• bFGF is widely used in culturing adult MuSCs (satellite cells).

Purpose of the Study:

• To investigate the role of post-translational modification (PTM) in bFGF's stemness-promoting effects on MuSCs.
• To elucidate the molecular mechanisms underlying bFGF's influence on MuSC proliferation and differentiation.
• To evaluate GST-bFGF, a non-PTM-modified form, as a potential substitute for bFGF in MuSC culture.

Main Methods:

• Expressed and purified GST-bFGF from E.coli, which lacks eukaryotic PTM systems.
• Assessed the effects of GST-bFGF and commercial bFGF on C2C12 myoblasts and primary MuSCs.
• Analyzed the expression of key myogenic regulatory factors (MRFs) including Myf5, Pax3/7, and MyoD, and Cyclin D1.
• Utilized C2C12 cells over-expressing MyoD (C2C12-tTA-MyoD) to investigate the independence of proliferation effects from MyoD regulation.

Main Results:

• Both GST-bFGF and bFGF activated the Akt-Erk pathway, significantly promoting cell proliferation in C2C12 myoblasts and MuSCs.
• GST-bFGF reversibly inhibited myogenesis, increased Myf5, Pax3/7, and Cyclin D1 expression, while strongly repressing MyoD, indicating stemness maintenance.
• The proliferative effect of GST-bFGF was independent of MyoD down-regulation and could be rescued by MyoD over-expression, suggesting MyoD repression is reversible.

Conclusions:

• GST-bFGF exhibits similar effects to bFGF on myogenic cell proliferation and differentiation, suggesting PTMs are not essential for these functions.
• GST-bFGF promotes MuSC stemness and proliferation through differential regulation of MRFs and Pax3/7, independent of MyoD repression.
• GST-bFGF serves as an effective substitute for bFGF in maintaining MuSC stemness and proliferation, offering a valuable tool for skeletal muscle research.