Fibroblast growth factor 9 (FGF9) inhibits myogenic differentiation of C2C12 and human muscle cells

Abstract

Osteoporosis and sarcopenia (osteosarcopenia (OS)) are twin-aging diseases. The biochemical crosstalk between muscle and bone seems to play a role in OS. We have previously shown that osteocytes produce soluble factors with beneficial effects on muscle and vice versa. Recently, enhanced FGF9 production was observed in the OmGFP66 osteogenic cell line. To test its role in myogenic differentiation, C2C12 myoblasts were treated with recombinant FGF9. FGF9 as low as 10 ng/mL inhibited myogenic differentiation, suggesting that FGF9 might be a potential inhibitory factor produced from bone cells with effects on muscle cells. FGF9 (10–50 ng/mL) significantly decreased mRNA expression of MyoG and Mhc while increasing the expression of Myostatin. Consistent with the phenotype, RT-qPCR array revealed that FGF9 (10 ng/mL) increased the expression of Icam1 while decreased the expression of Wnt1 and Wnt6 decreased, respectively. FGF9 decreased caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and reduced the expression of genes (i.e. Cacna1s, RyR2, Naftc3) directly associated with intracellular Ca2+ homeostasis. Myogenic differentiation in human skeletal muscle cells was similarly inhibited by FGF9 but required higher doses of 200 ng/mL FGF9. FGF9 was also shown to stimulate C2C12 myoblast proliferation. FGF2 and the FGF9 subfamily members FGF16 and FGF20 also inhibited C2C12 myoblast differentiation and enhanced proliferation. Intriguingly, the differentiation inhibition was independent of proliferation enhancement. These findings suggest that FGF9 may modulate myogenesis via a complex signaling mechanism.

Deletion ofFibroblast growth factor 9globally and in skeletal muscle results in enlarged tuberosities at sites of deltoid tendon attachments

“…Fgf9 is expressed in muscle during embryonic stages, which we and others have observed using ISH (Colvin et al, 1999; Garofalo et al, 1999; Hung et al, 2007; Yang and Kozin, 2009). Previous work has established a connection between Fgf9 and muscle, as treatment of muscle and muscle progenitor cells with FGF9 slows maturation, enhances proliferation, and decreases expression of various myogenic genes (Huang et al, 2019). This study found supporting evidence that Fgf9 expression in muscle may be a limiting factor in tuberosity growth.…”

Ionic Silicon Protects Oxidative Damage and Promotes Skeletal Muscle Cell Regeneration

“…Using C2C12 myoblasts provides a more stable environment with fewer confounding variables than the normal physiologic environment of human primary cells [ 70 ]. Thus, C2C12 myoblasts were cultured using well-established and previously published protocols [ 71 , 72 , 73 ]. Briefly, C2C12 cells were cultured in growth medium (GM), GM consisting of DMEM/high glucose + 10% FBS + 100 U/mL P/S, in 75-cm 2 Corning cell culture flasks with canted neck and vented caps under normal conditions (37 °C, 5% CO 2 ) maintaining cell density at 40–70% confluency.…”

“…Fusion index [ 73 ] (FI), area covered by myotubes (%), and total number of nuclei per fluorescent image were counted to quantify myogenic differentiation of C2C12 cells. FI is defined as: the total number of nuclei within the MHC-expressing multinucleated myotubes divided by the total number of nuclei in the FOV × 100 [ 73 ]. For all experiments, three FOVs in each well were randomly selected for imaging, then ImageJ was used for cell counting and myotube area calculation.…”

“…Five concentrations of H 2 O 2 (0.2, 0.4, 0.6, 0.8, and 1.0 mM) were used to compare the effect of H 2 O 2 to the normal GM as control. C2C12 cell viability experiment was performed by seeding 2.5 × 10 4 cells/well in a 24-well plate [ 73 , 74 ]. Then, cells were provided with 500 µL of GM as control, and GM + 0.2 mM H 2 O 2 , GM + 0.4 mM H 2 O 2 , GM + 0.6 mM H 2 O 2 , GM + 0.8 mM H 2 O 2 , and GM + 1.0 mM H 2 O 2 as the experimental H 2 O 2 groups.…”

Expression and Function of WNT6: From Development to Disease

“…For example, both WNT6 and WNT10a inhibit adipogenesis and stimulate osteoblastogenesis (Cawthorn et al, 2012). However, among the WNT proteins, WNT1 is the most homologous to WNT6 in the human genome (Kirikoshi et al, 2001); They have synergistic effects in mice (Otto et al, 2008;Hitchins et al, 2013;Huang et al, 2019) and antagonistic effects in humans and mice (Otto et al, 2008;Scheller et al, 2008;Hitchins et al, 2013; at different time points.…”