Genetic variability significantly contributes to individual differences in skeletal muscle mass; however, the specific genes involved in that process remain elusive. In this study, we examined the role of positional candidates, Rps6ka6 and Pou3f4, of a chromosome X locus, implicated in muscle mass variability in CFW laboratory mice. Histology of hindlimb muscles was studied in CFW male mice carrying the muscle “increasing” allele C (n = 15) or “decreasing” allele T (n = 15) at the peak marker of the locus, rs31308852, and in the Pou3f4y/− and their wild-type male littermates. To study the role of the Rps6ka6 gene, we deleted exon 7 (Rps6ka6-ΔE7) using clustered regularly interspaced palindromic repeats-Cas9 based method in H2Kb myogenic cells creating a severely truncated RSK4 protein. We then tested whether that mutation affected myoblast proliferation, migration, and/or differentiation. The extensor digitorum longus muscle was 7% larger (P < 0.0001) due to 10% more muscle fibers (P = 0.0176) in the carriers of the “increasing” compared with the “decreasing” CFW allele. The number of fibers was reduced by 15% (P = 0.0268) in the slow-twitch soleus but not in the fast-twitch extensor digitorum longus (P = 0.2947) of Pou3f4y/− mice. The proliferation and migration did not differ between the Rps6ka6-ΔE7 and wild-type H2Kb myoblasts. However, indices of differentiation (myosin expression, P < 0.0001; size of myosin-expressing cells, P < 0.0001; and fusion index, P = 0.0013) were significantly reduced in Rps6ka6-ΔE7 cells. This study suggests that the effect of the X chromosome locus on muscle fiber numbers in the fast-twitch extensor digitorum longus is mediated by the Rps6ka6 gene, whereas the Pou3f4 gene affects fiber number in slow-twitch soleus.

中文翻译：

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验证与骨骼肌质量变异相关的位点候选 Rps6ka6 和 Pou3f4

遗传变异显着影响骨骼肌质量的个体差异；然而，参与该过程的特定基因仍然难以捉摸。在这项研究中，我们检查了 X 染色体基因座的候选位置 Rps6ka6 和 Pou3f4 的作用，它们与 CFW 实验室小鼠的肌肉质量变异有关。在携带肌肉“增加”等位基因 C (n = 15) 或“减少”等位基因 T (n = 15) 的 CFW 雄性小鼠中研究了后肢肌肉的组织学，该小鼠在位点 rs31308852 的峰值标记处和 Pou3f4y/−和它们的野生型雄性同窝仔。为了研究 Rps6ka6 基因的作用，我们在 H2Kb 肌原细胞中使用基于 Cas9 的成簇规则间隔回文重复序列删除了外显子 7 (Rps6ka6-ΔE7)，从而创建了严重截短的 RSK4 蛋白。然后我们测试了该突变是否影响成肌细胞增殖、迁移和/或分化。与“减少”的 CFW 等位基因相比，“增加”的 CFW 等位基因携带者的肌纤维多了 10%（P = 0.0176），因此趾长伸肌增大了 7%（P < 0.0001）。 Pou3f4y/− 小鼠的慢肌比目鱼肌纤维数量减少了 15% (P = 0.0268)，但快肌指长伸肌则没有减少 (P = 0.2947)。 Rps6ka6-ΔE7 和野生型 H2Kb 成肌细胞的增殖和迁移没有差异。然而，Rps6ka6-ΔE7细胞中的分化指数（肌球蛋白表达，P＜0.0001；肌球蛋白表达细胞的大小，P＜0.0001；和融合指数，P＝0.0013）显着降低。这项研究表明，X 染色体位点对快肌指长伸肌中肌纤维数量的影响是由 Rps6ka6 基因介导的，而 Pou3f4 基因则影响慢肌比目鱼肌中的纤维数量。