Scottish Oceans Institute, School of Biology, University of St. Andrews, St. Andrews, Fife, United Kingdom.
Am J Physiol Regul Integr Comp Physiol. 2010 Jun;298(6):R1615-26. doi: 10.1152/ajpregu.00114.2010. Epub 2010 Apr 7.
The mRNA expression of myogenic regulatory factors, including myoD1 (myoblast determination factor) gene paralogs, and their regulation by amino acids and insulin-like growth factors were investigated in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon (Salmo salar). The cell cycle and S phase were determined as 28.1 and 13.3 h, respectively, at 18 degrees C. Expression of myoD1b and myoD1c peaked at 8 days of culture in the initial proliferation phase and then declined more than sixfold as cells differentiated and was correlated with PCNA (proliferating cell nuclear antigen) expression (R = 0.88, P < 0.0001; R = 0.70, P < 0.0001). In contrast, myoD1a transcripts increased from 2 to 8 days and remained at elevated levels as myotubes were formed. mRNA levels of myoD1c were, on average, 3.1- and 5.7-fold higher than myoD1a and myoD1b, respectively. Depriving cells of amino acids and serum led to a rapid increase in pax7 and a decrease in myoD1c and PCNA expression, indicating a transition to a quiescent state. In contrast, amino acid replacement in starved cells produced significant increases in myoD1c (at 6 h), PCNA (at 12 h), and myoD1b (at 24 h) and decreases in pax7 expression as cells entered the cell cycle. Our results are consistent with temporally distinct patterns of myoD1c and myoD1b expression at the G(1) and S/G(2) phases of the cell cycle. Treatment of starved cells with insulin-like growth factor I or II did not alter expression of the myoD paralogs. It was concluded that, in vitro, amino acids alone are sufficient to stimulate expression of genes regulating myogenesis in myoblasts involving autocrine/paracrine pathways. The differential responses of myoD paralogs during myotube maturation and amino acid treatments suggest that myoD1b and myoD1c are primarily expressed in proliferating cells and myoD1a in differentiating cells, providing evidence for their subfunctionalization following whole genome and local duplications in the Atlantic salmon lineage.
从大西洋三文鱼（Salmo salar）快速肌肌节中分离出的原代细胞培养物中，研究了肌调节因子的 mRNA 表达，包括 myoD1（成肌决定因子）基因的同源物，及其受氨基酸和胰岛素样生长因子的调节。在 18°C 时，细胞周期和 S 期分别为 28.1 和 13.3 小时。在初始增殖阶段的第 8 天培养中，myoD1b 和 myoD1c 的表达达到峰值，然后随着细胞分化，表达水平下降了六倍以上，并与 PCNA（增殖细胞核抗原）表达相关（R = 0.88，P < 0.0001；R = 0.70，P < 0.0001）。相比之下，myoD1a 转录物从第 2 天到第 8 天增加，并在形成肌管时保持升高水平。myoD1c 的 mRNA 水平平均比 myoD1a 和 myoD1b 高 3.1-和 5.7 倍。剥夺细胞氨基酸和血清会导致 pax7 迅速增加，myoD1c 和 PCNA 表达减少，表明细胞进入静止状态。相反，在饥饿细胞中替换氨基酸会导致 myoD1c（在 6 小时时）、PCNA（在 12 小时时）和 myoD1b（在 24 小时时）显著增加，pax7 表达减少，因为细胞进入细胞周期。我们的结果与细胞周期 G1 和 S/G2 期 myoD1c 和 myoD1b 表达的时间上不同的模式一致。用胰岛素样生长因子 I 或 II 处理饥饿细胞不会改变 myoD 同源物的表达。因此，在体外，仅氨基酸就足以刺激参与成肌细胞自分泌/旁分泌途径的调节肌发生的基因表达。myoD 同源物在肌管成熟和氨基酸处理过程中的不同反应表明，myoD1b 和 myoD1c 主要在增殖细胞中表达，myoD1a 在分化细胞中表达，为大西洋三文鱼谱系中全基因组和局部重复后的亚功能化提供了证据。
