(B-68) Growth Factor-Mimetic Peptide Nanofibers Induce Localized Myogenic Cell Signaling

Introduction:: The Growth Hormone/IGF-1 signaling axis is crucial for physiological growth and regulation, and the upregulation of the Insulin-like Growth Factor 1 (IGF-1) signaling pathway has proven to be a platform for inducing therapeutically promising levels of myogenesis. However, whole growth factor supplementation is costly and has risks for ectopic side effects like type-2 diabetes mellitus, hyperglycemia, and several cancers. While growth factor mimicry supposedly induces IGF-1 receptor (IGF-1R) activation while minimizing side effect risk and steric hindrance, small peptide growth factor mimics dissociate quickly from the binding area of IGF-1R, thus upregulating the signaling pathway too little for apparent therapeutic significance and still posing a risk for off-target activity. To engineer an IGF-1-axial therapeutic model with optimal signaling characteristics, opportunity for localized administration, and stabilized signaling effects, the 13 amino acid IGF-1 peptide mimic IGF-1c (seq: GYGSSSRRAPQT) is conjugated to a self-assembling nanofiber complex (seq: KSLSLSLSLSLSLK) which assembles into fibrous scaffolds which have superior structural stability and similar signal upregulation. Additionally, these nanofibers, upon preparation, form a hydrogel that can be aspirated and injected locally for targeted therapeutic effect.

Materials and Methods:: For all involved studies, C2C12 myoblasts are used to model both the downstream IGF-1 PI3K/Akt signaling pathway and the differentiation of cells from myoblasts to myotubes.
To interrogate the PI3K/Akt signaling pathway, a Western Blot is performed on cell lysate collected from C2C12 myoblasts cultured in 6 well plates. The band density of total Akt, phosphorylated Akt, and the housekeeping protein GAPDH is quantified to analyze the total occurrence of signaling pathway activation.
To both probe the ability of these peptide constructs to encourage targeted cell differentiation and qualify the relationship between Akt signaling increase and myotube formation, a 5 day myoblast fusion assay is performed. Standard immunofluorescence is performed to illuminate Myosin Heavy Chain fibers (indicative of myotubes). Fluorescence microscopy pictures are quantified to determine the fusion index, a measure for myoblast differentiation.

Results, Conclusions, and Discussions:: As determined by Western Blot, these nanofiber constructs can increase Akt pathway signaling significantly more than vehicle control and in a similar manner to IGF-1c mimics. This verifies that Growth Factor-mimetic peptides have unchanged signaling capability while still possessing superior structural characteristics for adaptive and localized administration, thus widening the breadth of pathologies that could be treated with small peptide Growth Factor mimics.
The differentiation of myoblasts into myotubes is also significantly increased by these nanofibrous constructs, indicating promising overall capability for localized administration to provide better therapeutic effect than small mimics themselves.

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