[An experimental study of the role of myosin light chain in myogenesis in vitro].
OBJECTIVE
To investigate the role of myosin light chain (Myl) in myogenesis in vitro.


METHODS
The extraocular muscle, diaphragm and gastrocnemius muscle myoblasts (eMb, dMb and gMb) were isolated and purified from 12 3-week-old C57BL/6 mice by using the enzyme digestion and Preplate technique, and then were subcultivated. The Myl expression in Mb was detected by RT-PCR and Western blot analysis; the Mb proliferation activity was tested by methylene blue assay, and the myotube formation was observed. After anti-Myl antibody (1, 2, 3, 8, 16 ng/mL) was induced in the Mb culture (experimental group), the ability of proliferation of myoblasts and the myotube formation were identified. Meanwhile, the Mb which was cultured without anti-Myl antibody was identified as the control group.


RESULTS
The results of RT-PCR and Western blot analysis showed that Myl1 and Myl4 mRNA and Myl protein were expressed in eMb, dMb and gMb at 24 hours after seeding, and their expression level were lower in eMb than in dMb and gMb (P < 0.01), and the latter two did not show any significant difference (P > 0.05). Myl2 and Myl3 mRNA was not detected in these three myoblasts. The proliferation assay showed that the eMb proliferated faster as compared with dMb and gMb (P < 0.01). eMb began to yield myotubes at 40 hours after seeding and dMb and gMb at 16 hours after seeding. At 6 days, the number of myotubes derived from eMb was (137.2 +/- 24.5)/field, which was significantly larger than that of myotubes from dMb [(47.6 +/- 15.5)/field ] and gMb [(39.8 +/- 5.1)/ field ] (P < 0.01). There was not statistically significant difference between the latter two groups (P > 0.05). After the antibody treatment, the absorbency values of the eMb, dMb and gMb in the experimental groups at each antibody concentration point were significantly higher than those in the corresponding control groups (P < 0.05), and the dose-dependent way was performed. The numbers of myotubes from dMb at 16 hours were (48.2 +/- 7.1)/ well in the experimental group and (23.4 +/- 4.9)/ well in the control group, and at 6 days were (40.6 +/- 10.2)/ field in the experimental group and (63.1 +/- 6.1)/ field in the control group. There was statistically significant difference between the experimental and control groups (P < 0.01).


CONCLUSIONS
Myl may play a role in myogenesis through the negative effect on the myoblast proliferation.
Zhang
Suzhen
S
School of Life Science, Sichuan University, Chengdu Sichuan, 610041, P.R.China.
Xie
Huiqi
H
Xu
Yong
Y
Li
Xiuqun
X
Deng
Li
L
Chen
Xiaohe
X
Qiu
Lin
L
Yang
Zhiming
Z
D000818:Animals; D049109:Cell Proliferation; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D024510:Muscle Development; D032446:Myoblasts / Q000166:cytology*; D018994:Myosin Light Chains / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics
D018994:Myosin Light Chains; D012333:RNA, Messenger