Scn5a互斥型剪接异构体介导失神经后肌类型特异性萎缩的机制研究

失神经骨骼肌萎缩过程中慢肌比快肌萎缩更为严重，其深入的分子机制尚不清楚。本课题前期发现，钠离子通道编码基因Scn5a在失神经后的快肌和慢肌之间无表达差异但发生了不同程度的互斥外显子型可变剪接，慢肌较快肌产生了更多的Scn5a-6A异构体，该异构体由于第211位氨基酸D-K转换导致了骨骼肌萎缩。以此提出假说：Scn5a的互斥外显子型可变剪接差异介导了快慢肌的特异性萎缩。本项目拟采用病毒介导的敲低、过表达和电生理技术明确Scn5a-6A异构体对骨骼肌萎缩的作用；通过生物信息学、小基因构建和功能验证探索Scn5a-6A异构体引起骨骼肌萎缩的分子机制和RNA结合蛋白肌类型特异性调控Scn5a可变剪接的分子机制；最后设计反义寡核苷酸药物，通过纠正剪接治疗失神经肌萎缩。本项目的完成不仅能解释骨骼肌特异性萎缩现象，也将提出一种新的治疗策略，具理论和应用价值。

The molecular mechanism underlying the more severe atrophy of slow twitch muscle than fast twitch muscle during denervated skeletal muscle atrophy remains unclear. In our present work, we found that the sodium channels encoding genes Scn5a was not differentially expressed between fast and slow twitch muscle after denervation but underwent different levels of alternative splicing of mutually exclusive exons. Compared with fast twitch muscle, slow twitch muscle produced more Scn5a-6A variant, which led to skeletal muscle atrophy due to amino acid D-K transition at position 211. Based on those, we propose that the difference in alternative splicing of mutually exclusive exons in Scn5a mediates the specific atrophy of fast and slow twitch muscles. This project will characterize the effects of Scn5a-6A variant on skeletal muscle atrophy by employing virus-mediated gene knockdown, gene overexpression, and electrophysiology, explore the molecular mechanism of Scn5a-6A variant-induced skeletal muscle atrophy and the molecular mechanism of RNA-binding protein muscle type-specific regulation of Scn5a alternative splicing through bioinformatics, minigene construction, and functional validation, and design antisense oligonucleotide drugs for the treatment of denervated muscle atrophy by correcting splicing. This study will not only provide an explanation for skeletal muscle-specific atrophy, but will also offer a new therapeutic strategy with extensive theoretical and application value.