Abstract
Myoblast fusion into functionally-distinct myotubes to form in vitro skeletal muscle constructs under differentiation serum-free conditions still remains a challenge. Herein, we report that our microtopographical carbohydrate substrates composed of bioactive hexa-N-acetyl-d-glucosamine (GlcNAc6) modulated the efficiency of myoblast fusion without requiring horse serum or any differentiation medium during cell culture. Promotion of the differentiation of dissociated mononucleated skeletal myoblasts (C2C12; a mouse myoblast cell line) into robust myotubes was found only on GlcNAc6 micropatterns, whereas the myoblasts on control, non-patterned GlcNAc6 substrates or GlcNAc6-free patterns exhibited an undifferentiated form. We also examined the possible role of GlcNAc6 micropatterns with various widths in the behavior of C2C12 cells in early and late stages of myogenesis through mRNA expression of myosin heavy chain (MyHC) isoforms. The spontaneous contraction of myotubes was investigated via the regulation of glucose transporter type 4 (GLUT4), which is involved in stimulating glucose uptake during cellular contraction. Narrow patterns demonstrated enhanced glucose uptake rate and generated a fast-twitch muscle fiber type, whereas the slow-twitch muscle fiber type was dominant on wider patterns. Our findings indicated that GlcNAc6-mediated integrin interactions are responsible for guiding myoblast fusion forward along with myotube formation.
Highlights
Myoblast fusion is an indispensable step in the cell maturation process during the development and regeneration of adult skeletal muscular cells
Fabricated a synergistic combination of topographical characteristics and GlcNAc6 oligomers self-assembled in two different geometries, as depicted in Figure 1, according to our previous rep2o.1rt
Our data clearly demonstrated that carbohydrate-functionalized micropatterns induced transcriptional activation of the myosin heavy chain (MyHC) and glucose transporter type 4 (GLUT4) genes, which results in the regulation of numerous downstream signaling pathways related to myoblast fusion and myotube contractions
Summary
Myoblast fusion is an indispensable step in the cell maturation process during the development and regeneration of adult skeletal muscular cells. Many studies have revealed that signaling pathways regulate various transcription factors and have provided significant information on the molecular mechanisms of cell fusion by the cooperative crosstalk of the core signal transduction machinery of each pathway [11,12,13,14]. The understanding of these pathways has deepened through biomolecular interactions, occurring at cell-surface receptors via intracellular domain-associated signaling or adaptor proteins inside the cells. Numerous types of natural bioactive molecules, which are immobilized on cell culture scaffolds, can immensely affect myoblast functions, encouraging myotube formation and contractibility [15,16,17,18]
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