Loss of the Nuclear Envelope Protein LAP1B Disrupts Myogenic Differentiation of Patient-Derived Fibroblasts. Loss of the Nuclear Envelope Protein LAP1B Disrupts Myogenic Differentiation of Patient-Derived Fibroblasts

Lamina-associated polypeptide 1 (LAP1) is a ubiquitously expressed inner nuclear membrane protein encoded by TOR1AIP1, and present as two isoforms in humans, LAP1B and LAP1C. While loss of both isoforms results in a multisystemic progeroid-like syndrome, specific loss of LAP1B causes muscular dystrophy and cardiomyopathy, suggesting that LAP1B has a critical role in striated muscle. To gain more insight into the molecular pathophysiology underlying muscular dystrophy caused by LAP1B, we established a patient-derived fibroblast line transdifferentiated into myogenic cells with inducible MyoD expression. Compared to controls, we observed strikingly reduced myogenic differentiation and fusion potentials. Similar defects were observed in C2C12 myoblasts carrying loss-of-function LAP1A/B mutations. Using RNA sequencing, we showed that despite MyoD overexpression and efficient cell cycle exit, transcriptional reprogramming of LAP1B-deficient cells into the myogenic lineage was impaired with delayed activation of myogenin and muscle-specific downstream genes. Gene set enrichment analysis suggested dysregulation of protein metabolism, extracellular matrix and chromosome organization. Finally, we found that LAP1B-deficient cells exhibit nuclear deformations such as increased micronuclei and altered morphometric parameters. This study reports the phenotypic and transcriptomic changes during myoconversion of patient-derived fibroblasts and provides a useful resource to gain new insights into the mechanisms implicated in LAP1B-associated nuclear envelopathies. Overall design: Comparative gene expression profiling analysis of RNA-seq data for control and LGMD2Y patient-derived immortalized myoconverted fibroblasts.

核纤层相关多肽1（Lamina-associated polypeptide 1, LAP1）是由TOR1AIP1编码的广泛表达的内核膜蛋白，在人类中以LAP1B和LAP1C两种同工型存在。同时缺失两种同工型会引发多系统早老症样综合征，而特异性缺失LAP1B则会导致肌营养不良与心肌病，提示LAP1B在横纹肌中发挥关键作用。为深入探究LAP1B缺失所致肌营养不良的分子病理生理机制，我们构建了可诱导MyoD表达、将患者来源成纤维细胞转分化为肌源性细胞的细胞系。与对照组相比，我们观察到肌源性分化与融合能力显著受损。在携带功能缺失型LAP1A/B突变的C2C12成肌细胞中，也可观测到类似缺陷。通过RNA测序（RNA Sequencing, RNA-seq），我们证实尽管MyoD过表达且细胞周期退出效率正常，但LAP1B缺陷细胞向肌源性谱系的转录重编程过程受损，肌细胞生成素（myogenin）及肌肉特异性下游基因的激活出现延迟。基因集富集分析（Gene Set Enrichment Analysis, GSEA）结果显示，蛋白质代谢、细胞外基质与染色体组织的调控出现异常。最后，我们发现LAP1B缺陷细胞存在核形态异常，如微核数量增多以及形态计量参数改变。本研究报道了患者来源成纤维细胞肌转化过程中的表型与转录组变化，为阐明LAP1B相关核包膜病的致病机制提供了极具价值的研究资源。实验设计概况：对对照组与肢带型肌营养不良2Y型（LGMD2Y）患者来源的永生化肌转化成纤维细胞的RNA-seq数据开展比较基因表达谱分析。