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doi: 10.1242/10.1242/jcs.00358

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The block of ryanodine receptors selectively inhibits fetal myoblast differentiation

¹ Department of Histology and Medical Embryology, University of Rome `La Sapienza', 00161 Rome, Italy
² Section of Molecular Medicine, Department of Neuroscience, University of Siena, I-53100 Siena, Italy

View larger version (58K): [in a new window]   Fig. 1. Expression of RyRs during limb development. (A) Western blot analysis of microsomal fractions prepared from hindlimbs from E11, 13, 15 and 17 reacted with the antisera specific to type 1 (top) or type 3 (bottom) RyRs, as indicated. (B) Western blot analysis of microsomal fractions prepared from HEK293 cells transfected with RyR1 or RyR3 expression vectors and incubated with the anti-RyR1 or RyR3 antibody, as indicated.

View larger version (76K): [in a new window]   Fig. 2. Immunolocalization of RyRs in E13 limb muscle. Double immunofluorescence analysis of hind limb cryosections from E13 embryos; the same section was incubated with anti-RyR1 antiserum (A) and anti-MyHC antibody MF20 (B), or with anti-RyR3 antiserum (D) and MF20 (E). Phase contrast micrographs are also shown for each section (C,F). Bar, 10 µm.

View larger version (86K): [in a new window]   Fig. 3. Immunolocalization of RyRs in E17 limb muscle. Double immunofluorescence analysis of hind limb cryosections from E17 embryos; the same section was incubated with anti-RyR1 antiserum (A) and anti-MyHC antibody MF20 (B) or with anti-RyR3 antiserum (D) and MF20 (E). Phase contrast micrographs are also shown for each section (C,F). Bar, 10 µm.

View larger version (118K): [in a new window]   Fig. 4. Immunolocalization of RyR1 in cultured skeletal muscle cells. Double immunofluorescence analysis of primary cultures prepared from E9.5 somites (somitic cells, A-C), E11 hind limbs (embryonic myoblasts, D-F), E16 hind limbs (fetal myoblasts, G-I) and 2-week-old mice (satellite cells, L-N). After differentiation (72 hours), the cells were fixed and reacted with an anti-RyR1-specific anti-serum (A,D,G,L) together with the anti-MyHC antibody MF20 (B,E,H,M). Phase contrast micrographs of each field are also shown (C,F,I,N). Bar, 10 µm.

View larger version (118K): [in a new window]   Fig. 5. Immunolocalization of RyR3 in cultured skeletal muscle cells. Double immunofluorescence analysis of primary cultures prepared from E9.5 somites (somitic cells, A-C), E11 hind limbs (embryonic myoblasts, D-F), E16 hind limbs (fetal myoblasts, G-I) and 2-week-old mice (satellite cells, L-N). After differentiation (72 hours) the cells were fixed and reacted with anti-RyR3-specific anti-serum (A,D,G,L) together with the anti-MyHC antibody MF20 (B,E,H,M). Phase contrast micrographs of each field are also shown (C,F,I,N). Bar, 10 µm.

View larger version (16K): [in a new window]   Fig. 6. Kinetics of expression of RyRs during differentiation of embryonic and fetal myoblasts in culture. The number of nuclei in RyR1- and RyR3-positive cells (plotted together, since both curves overlap) or in MyHC-positive cells in embryonic and fetal myoblasts in primary cultures were counted at each time point and expressed as a percentage of the total number of nuclei. At least 20 random microscopic fields were counted for each sample in three independent experiments. Triangles: embryonic myoblasts; circles: fetal myoblasts; filled simbols: RyRs; empty simbols: MyHC. Bars indicate s.d.

View larger version (20K): [in a new window]   Fig. 7. Calcium release experiments in embryonic and fetal myoblasts. Embryonic (a-b) and fetal (c-d) myoblasts after 4 days of culture under differentiating conditions were loaded with Fluo 3 AM and analyzed by a confocal laser microscope. Stimulation with either 10 µM carbachol and 40 mM caffeine exerted a transient increase on the intracellular calcium concentration in both embryonic (a) and fetal (c) myoblasts. Treatment with 300 µM ryanodine abolished caffeine- but not carbachol-induced calcium release in both embryonal (b) and fetal (d) myoblasts.

View larger version (46K): [in a new window]   Fig. 8. Expression of muscle-specific markers in ryanodine-treated primary cultures. (A) Western blot analysis of protein extracts prepared from embryonic, fetal and satellite muscle cells, cultured for 72 hours in the absence of ryanodine (C) or treated with ryanodine (Ry) for the indicated time periods in culture. At the end of 0-24 hours and of 24-48 hours of treatment, ryanodine was removed and the cells cultured for an additional 48 and 24 hours, respectively. The blots were reacted with the anti-MyHC mAb MF20 and with the anti-myogenin mAb F5D. An anti-tubulin antibody was used to normalize the blot. (B) RT-PCR analysis of total RNA isolated from fetal muscle cells cultured for 72 hours in the absence of ryanodine (C) or treated with ryanodine in the 24-48 hours time period in culture, after which ryanodine was removed and the cells cultured for the additional 24 hours. 100 ng of total RNA were reverse-transcribed and PCR-amplified using primers specific for MyHC, MyoD, myogenin and myf5, as indicated; by increasing the PCR cycles (over 30 cycles), an amplified product for MyHC becomes detectable also in ryanodine-treated cells (data not shown); primers specific for ß-actin were used to normalize the reaction. The experiments shown here were performed using 100 µM ryanodine; same results were obtained using 300 µM ryanodine (data not shown).

View larger version (169K): [in a new window]   Fig. 9. Effect of ryanodine treatment on differentiation of fetal myoblasts in culture. Fetal myoblasts were prepared from MLC3F-LacZ E16 embryos and cultured for 72 hours. Ryanodine was added in culture during the 24-48 hour time period and then removed (C,D) or during the 48-72 hours time period (E,F). All cells were fixed at 72 hours in culture and stained for ß-galactosidase activity. Untreated cells (A,B); cells treated with ryanodine during the 24-48 hour time period in culture (C,D); cells treated with ryanodine during the 48-72 hours time period in culture (E,F); ß-galactosidase staining (A,C,E); phase contrast micrographs (B,D,F). Bar, 10 µm. The experiments shown here were performed using 100 µM ryanodine; the same results were obtained using 300 µM ryanodine (data not shown).

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