Follistatin induction by nitric oxide through cyclic GMP: a tightly regulated signaling pathway that controls myoblast fusion

Abstract

The mechanism of skeletal myoblast fusion is not well understood. We show that endogenous nitric oxide (NO) generation is required for myoblast fusion both in embryonic myoblasts and in satellite cells. The effect of NO is concentration and time dependent, being evident only at the onset of differentiation, and direct on the fusion process itself. The action of NO is mediated through a tightly regulated activation of guanylate cyclase and generation of cyclic guanosine monophosphate (cGMP), so much so that deregulation of cGMP signaling leads to a fusion-induced hypertrophy of satellite-derived myotubes and embryonic muscles, and to the acquisition of fusion competence by myogenic precursors in the presomitic mesoderm. NO and cGMP induce expression of follistatin, and this secreted protein mediates their action in myogenesis. These results establish a hitherto unappreciated role of NO and cGMP in regulating myoblast fusion and elucidate their mechanism of action, providing a direct link with follistatin, which is a key player in myogenesis.

Figure 1.

NO induces fusion of satellite cells. (A) Satellite cells from newborn mice were maintained for 48 h in growth medium and then switched to the differentiation medium in the presence or absence of increasing concentrations of DETA-NO or L-NAME. Cells were stained after 72 h with the anti–sarcomeric myosin MF20 mAb and the nuclear dye Hoechst. The fusion index indicates the number of nuclei in myosin-expressing cells with more than two nuclei versus the total number of nuclei measured. (B) Same as A, except that satellite cells were exposed or not (NT) to single concentrations of DETA-NO (50 μM) and L-NAME (5 mM). The fusion index was measured after 24, 48, or 72 h. (C) Representative images of B showing the effects of 50 μM DETA-NO and 5 mM L-NAME on satellite cell fusion after 72 h with respect to those of untreated controls. The staining for myosin is in green (FITC). (D) 5 mM L-NAME and 50 μM DETA-NO were added to differentiating satellite cells at the indicated time points. The fusion index was assessed after 72 h (n = 6). (E and F) NOS activity, measured as conversion of l-arginine into l-citrulline (E; n = 4), and NOS Iμ and III expression, measured by Western blotting with specific mAbs (F), in the course of satellite cell differentiation in vitro as described in A. The Western blot shown here is representative of four reproducible experiments. (G) MyoD, PCNA, and GAPDH expression measured by Western blotting with specific mAbs in satellite cells exposed or not for 24 h in differentiation medium to either 50 μM DETA-NO or 5 mM L-NAME. Results are representative of four reproducible experiments. (H) Percentage of mononucleated cells, binucleated cells, and multinucleated myotubes expressing sarcomeric myosin (myo) in satellite cells exposed or not for 72 h in differentiation medium to either 50 μM DETA-NO or 5 mM L-NAME as described in A. Values are expressed as percentage over those observed in NT. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus NT. Bar, 250 μm.

Figure 2.

The effect of NO on satellite cell fusion depends on cGMP generation. (A and B) Satellite cells were maintained for 48 h in growth medium and then switched to the differentiation medium for up to 72 h. (A) Guanylate cyclase activity, measured as formation of cGMP after stimulation with or without (NT) 50 μM DETA-NO or 5 mM L-NAME administered for 30 min at the indicated time points (n = 4). (B) Expression of soluble guanylate cyclase and GAPDH (loading control), measured by Western blotting with mAbs specific for the α and β subunits of the enzyme. Results are representative of four reproducible experiments. (C) Satellite cell differentiation was performed as described in A for 72 h in the presence of an increasing concentration of 8 Br-cGMP. The fusion index was measured as described in Fig. 1(n = 4). (D and E) Same as in C except that satellite cell differentiation was performed for 72 h in the presence or absence of 5 mM L-NAME, 50 μM DETA-NO, 3 μM ODQ, or3 mM 8 Br-cGMP in various combinations as detailed in the key to the panels. Fusion index was measured as described in Fig. 1. Representative images are shown in E.Anti–sarcomeric myosin MF20 mAb is in green (FITC), and the nuclear dye Hoechst in blue. **, P < 0.01 versus NT; ***, P < 0.001 versus NT; †, P < 0.001 versus8 Br-cGMP–treated cells (n = 6). Error bars represent SEM. Bar, 200 μm.

Figure 3.

Persistence of cGMP leads to fusion-induced muscle hypertophy at different stages of muscle development. (A–C) Satellite cells from newborn mice were plated at high density (3 × 10⁴ cells/cm²), maintained for 48 h in growth medium, and switched to the differentiation medium in the presence or absence of 50 or 150 μM DETA-NO or 0.3, 1.0, or 3.0 mM 8 Br-cGMP. After 48 h, cells were either stained with the anti–sarcomeric myosin MF20 mAb (green, FITC) and the nuclear dye Hoechst (blue) or processed for Western blot analysis. (A) Representative images of four reproducible experiments (3 mM 8 Br-cGMP and 50 μM DETA-NO). 10× magnification. (B) Percentage of nuclei containing >12 nuclei, calculated as depicted in Fig. 1. *, P < 0.05; ***, P < 0.001 versus untreated cells (NT; n = 4). (C) Sarcomeric myosin (myosin) and GAPDH expression, determined by Western blotting (one out of four reproducible experiments). (D and E) PSM explants from MLC1/3F-nLacZ E9.5 embryos were differentiated for 4 d in the presence or absence of 50 or 150 μM DETA-NO, 5 mM l-NAME, or 1 or 3 mM 8 Br-cGMP. (D, left) Immunofluorescence using the anti–sarcomeric myosin MF20 mAb (green, FITC). The myogenic nuclei are labeled by X-Gal staining (dark blue). (right) Same fields as in the left panels, but the X-Gal staining is merged with Hoechst staining (light blue), labeling all nuclei. Arrows demarcate multinucleated myotubes, which are absent in untreated explant cultures and frequent in cultures treated with 3 mM 8 Br-cGMP. Results shown are representative of four reproducible experiments. (E) Fusion index, measured in the same conditions as detailed in Fig. 1. *, P < 0.05; ***, P < 0.001 versus NT (n = 4). (F) X-Gal staining of MLC1/3F-nLacZ embryos, developed in the absence (left) or presence (right) of 8 Br-cGMP (3 g/kg body weight) administered through the pregnant mother from E10.5 to either E12.5 (top and middle) or E15.5 (bottom). The areas selected in red in the top panels are enlarged in the middle panels. Arrows show larger and more elongated fibers, indicating an increased level of myogenesis. Results are representative of three reproducible experiments. Error bars represent SEM. Bars: (A) 150 μm; (D) 125 μm; (F, top) 1.5 mm; (F, middle) 500 μm; (F, bottom) 1 mm.

Figure 4.

Effects of NO/cGMP on gene expression at different stages of muscle development. (A) RT-PCR using primers specific for MLC3, IGF-I, myostatin, follistatin, and GAPDH on mRNA obtained from satellite cells, E9.5 PSM explants, muscle tissues dissected from E12.5 and E15.5 mouse embryos, differentiated in presence of L-NAME, 50 μM DETA-NO, or 8 Br-cGMP (3 mM or 3 g/kg body weight). Results are representative of three reproducible experiments. (B) Expression of sarcomeric myosin, IGF-I, follistatin, myostatin, and GAPDH determined by Western blotting using specific antibodies on satellite cells and muscle tissue from E15.5 embryos, differentiated in the presence of 5 mM L-NAME, 50 μM DETA-NO, or 8 Br-cGMP (3 mM or 3 g/kg body weight). Results are representative of three reproducible experiments.

Figure 5.

NO/cGMP increase expression of follistatin. Satellite (A, E, and F) and C2C12 cells (B–D) were differentiated for 48 h in the presence or absence (NT) of 8 Br-cGMP (3 mM, where not otherwise indicated), 4 μg/ml anti-follistatin antibody (αFs), 5 μM LY294002, or 10 ng/ml IGF-I in various combinations as indicated. (A and B) Representative images of four reproducible experiments after staining with the anti-myosin MF20 mAb (green, FITC) and the nuclear dye Hoechst (blue). (C) Same as in B, except that C2C12 cells were lysed and mRNA analyzed by real-time PCR. (D) Same as in B except that C2C12 cells were previously transfected with Fs-Luc and transfected with or without Id1, A-CREB, or VIVIT before differentiation or treated with 3 μM KT5823, 3 μM KT5720, 3 μM (Rp)-8-pCPT-cGMPS, or 3 μM (Rp)-8-pCPT-cAMPS. Expression of Fs-Luc was quantified by chemiluminescence. (E and F) Satellite cell fusion index in the various conditions. ***, P < 0.001 versus controls; †, P < 0.001 versus 8 Br-cGMP–treated cells (n = 3). Error bars represent SEM. Bars: (A) 100 μm; (B) 200 μm.