LaminA/C基因(LMNA)突变有助于严重的横纹肌层肌病,影响心肌和骨骼肌,有限的治疗选择。在这项研究中,我们深入研究了五种不同的LMNA突变,包括在肌层肌病患者中发现的三种新变异和两种致病变异。我们的方法采用斑马鱼模型来全面研究这些变异。转基因斑马鱼表达野生型LMNA和每个突变进行广泛的形态学分析,游泳行为评估,肌肉耐力评估,心跳测量,和骨骼肌的组织病理学分析。此外,这些模型可作为集中筛选药物的平台.我们通过qPCR和RNAseq探索转录组景观,以揭示肌肉组织中改变的基因表达谱。LMNA(L35P)幼虫,LMNA(E358K),与通过DanioVision测量的LMNA(WT)相比,LMNA(R453W)转基因鱼表现出降低的游泳速度。与T型迷宫中的LMNA(WT)相比,所有LMNA转基因成年鱼的游泳速度均降低。此外,所有LMNA转基因成鱼,除了LMNA(E358K),通过游泳隧道测得的肌肉耐力比LMNA(WT)弱。组织化学染色显示所有LMNA突变转基因鱼的纤维大小减少,不包括LMNA(WT)鱼。有趣的是,LMNA(A539V)和LMNA(E358K)表现出心跳升高。我们认识到转基因过表达的潜在局限性,并进行关联计算以探索其对斑马鱼表型的影响。我们的结果表明,层粘连蛋白A/C过表达可能不会直接影响突变表型,比如游泳速度受损,心率加快,或肌纤维直径减小。利用LMNA斑马鱼模型进行药物筛选,我们确定了L-肉碱治疗挽救LMNA(L35P)中的肌肉耐力和肌酸治疗逆转LMNA(R453W)斑马鱼模型中的肌肉耐力。肌酸激活AMPK和mTOR通路,提高LMNA(R453W)鱼的肌肉耐力和游泳速度。转录组学分析揭示了上游调节因子和影响运动功能障碍的基因,心脏异常,LMNA突变体转基因鱼的离子流失调。这些发现忠实地模仿肌层病变的临床表现,包括畸形,早期死亡,纤维尺寸减小,斑马鱼的肌肉功能障碍.此外,我们的药物筛选结果表明,在LMNA(L35P)和LMNA(R453W)斑马鱼模型中,L-肉碱和肌酸治疗可作为肌肉耐力的潜在拯救者.我们的研究为LMNA相关肌层蛋白病的潜在治疗的未来发展提供了有价值的见解。
Lamin A/C gene (LMNA) mutations contribute to severe striated muscle
laminopathies, affecting cardiac and skeletal muscles, with limited treatment options. In this study, we delve into the investigations of five distinct LMNA mutations, including three novel variants and two pathogenic variants identified in patients with muscular laminopathy. Our approach employs zebrafish models to comprehensively study these variants. Transgenic zebrafish expressing wild-type LMNA and each mutation undergo extensive morphological profiling, swimming behavior assessments, muscle endurance evaluations, heartbeat measurement, and histopathological analysis of skeletal muscles. Additionally, these models serve as platform for focused drug screening. We explore the transcriptomic landscape through qPCR and RNAseq to unveil altered gene expression profiles in muscle tissues. Larvae of LMNA(L35P), LMNA(E358K), and LMNA(R453W) transgenic fish exhibit reduced swim speed compared to LMNA(WT) measured by DanioVision. All LMNA transgenic adult fish exhibit reduced swim speed compared to LMNA(WT) in T-maze. Moreover, all LMNA transgenic adult fish, except LMNA(E358K), display weaker muscle endurance than LMNA(WT) measured by swimming tunnel. Histochemical staining reveals decreased fiber size in all LMNA mutations transgenic fish, excluding LMNA(WT) fish. Interestingly, LMNA(A539V) and LMNA(E358K) exhibited elevated heartbeats. We recognize potential limitations with transgene overexpression and conducted association calculations to explore its effects on zebrafish phenotypes. Our results suggest lamin A/C overexpression may not directly impact mutant phenotypes, such as impaired swim speed, increased heart rates, or decreased muscle fiber diameter. Utilizing LMNA zebrafish models for drug screening, we identify L-carnitine treatment rescuing muscle endurance in LMNA(L35P) and creatine treatment reversing muscle endurance in LMNA(R453W) zebrafish models. Creatine activates AMPK and mTOR pathways, improving muscle endurance and swim speed in LMNA(R453W) fish. Transcriptomic profiling reveals upstream regulators and affected genes contributing to motor dysfunction, cardiac anomalies, and ion flux dysregulation in LMNA mutant transgenic fish. These findings faithfully mimic clinical manifestations of muscular
laminopathies, including dysmorphism, early mortality, decreased fiber size, and muscle dysfunction in zebrafish. Furthermore, our drug screening results suggest L-carnitine and creatine treatments as potential rescuers of muscle endurance in LMNA(L35P) and LMNA(R453W) zebrafish models. Our study offers valuable insights into the future development of potential treatments for LMNA-related muscular laminopathy.