Degree of Suppression of Mouse Myoblast Cell Line C2C12 Differentiation Varies According to Chondroitin Sulfate Subtype

Abstract

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a factor involved in the suppression of myogenic differentiation. CS comprises two repeating sugars and has different subtypes depending on the position and number of bonded sulfate groups. However, the effect of each subtype on myogenic differentiation remains unclear. In this study, we spiked cultures of C2C12 myoblasts, cells which are capable of undergoing skeletal muscle differentiation, with one of five types of CS (CS-A, -B, -C, -D, or -E) and induced differentiation over a fixed time. After immunostaining of the formed myotubes with an anti-MHC antibody, we counted the number of nuclei in the myotubes and then calculated the fusion index (FI) as a measure of myotube differentiation. The FI values of all the CS-treated groups were lower than the FI value of the control group, especially the group treated with CS-E, which displayed notable suppression of myotube formation. To confirm that the sugar chain in CS-E is important in the suppression of differentiation, chondroitinase ABC (ChABC), which catabolizes CS, was added to the media. The addition of ChABC led to the degradation of CS-E, and neutralized the suppression of myotube formation by CS-E. Collectively, it can be concluded that the degree of suppression of differentiation depends on the subtype of CS and that CS-E strongly suppresses myogenic differentiation. We conclude that the CS sugar chain has inhibitory action against myoblast cell fusion.

Sulfated glycosaminoglycans and non-classically secreted proteins, basic FGF and epimorphin, coordinately regulate TGF-β-induced cell behaviors of human scar dermal fibroblasts

“…These GAGs were highly soluble in physiological buffers and 1.0 mg/ml of them did not show any toxic effect on HSF cells. In accordance with a recent study [31], these GAGs were added to medium at a concentration of 0.2 mg/ml, unless otherwise noted. TGF-b (2 ng/ ml) and bFGF (10 ng/ml) (to elicit cell responses) were from Avnova and PeproTech, respectively.…”

Chondroitin sulfate E downregulates N-cadherin and suppresses myotube formation

“…Compared to monosulfated CSs, such as CS-A, -B, and -C, the highly sulfated CSs, such as CS-E and -H, are known to be highly involved in the regulation of cell differentiation by acting on proteins, such as growth factors, and the ECM [ 9 , 10 ]. In fact, previous studies have shown that CS-E has a stronger effect on various cell physiological processes, such as cell differentiation and cell division, during development than other monosulfated CSs [ 23 , 26 , 27 , 33 ].…”

“…We had previously reported that CS-E, a hypersulfated CS chain, has the strongest inhibitory effect on myotube formation among CS-A, -B, -C, -D, and -E subtypes [ 33 ]. In this study, we hypothesized that the effects of CS-E on cell adhesion, via N-cadherin, and cytoskeletal reorganization, via cofilin 2, may be one of the mechanisms by which CS-E suppresses myogenesis.…”

Preliminary study of the gene expression of sulfation and degradation enzymes for chondroitin sulfate in glycerol-treated C2C12 myoblast cells

“…CS-treated myoblasts fused, but myotube formation (elongation) was inhibited, and the cells showed a similar circular shape [17]. On the other hand, reducing CS from a culture environment smoothly promotes myodifferentiation [11,19].…”

“…CS-treated myoblasts fused, but myotube formation (elongation) was inhibited, and the cells showed a similar circular shape [ 17 ]. On the other hand, reducing CS from a culture environment smoothly promotes myodifferentiation [ 11 , 19 ]. Although CS is being recognized as a regulator of the processes of myodifferentiation and myoregeneration, there is little information on CS sulfation and degradation enzymes.…”