Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan.
Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan.
Sci Rep. 2024 Jun 4;14(1):12826. doi: 10.1038/s41598-024-63711-7.
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.
核纤层蛋白 A/C 基因（LMNA）突变导致严重的横纹肌层板病，影响心脏和骨骼肌，治疗选择有限。在这项研究中，我们深入研究了 5 种不同的 LMNA 突变，包括在肌肉层板病患者中发现的 3 种新变体和 2 种致病性变体。我们采用斑马鱼模型全面研究这些变体。表达野生型 LMNA 和每种突变的转基因斑马鱼进行广泛的形态分析、游泳行为评估、肌肉耐力评估、心跳测量和骨骼肌组织学分析。此外，这些模型还可作为药物筛选的平台。我们通过 qPCR 和 RNAseq 进行转录组学分析，揭示肌肉组织中基因表达谱的改变。与 LMNA(WT)相比，LMNA(L35P)、LMNA(E358K)和 LMNA(R453W)转基因鱼的幼虫游泳速度降低。与 LMNA(WT)相比，所有 LMNA 转基因成鱼在 T 迷宫中的游泳速度均降低。此外，除了 LMNA(E358K)外，所有 LMNA 转基因成鱼在游泳隧道中的肌肉耐力均弱于 LMNA(WT)。组织化学染色显示所有 LMNA 突变转基因鱼的纤维大小减小，除了 LMNA(WT)鱼。有趣的是，LMNA(A539V)和 LMNA(E358K)的心跳升高。我们认识到转基因过表达的潜在局限性，并进行了关联计算，以探讨其对斑马鱼表型的影响。我们的结果表明，核纤层蛋白 A/C 的过表达可能不会直接影响突变体表型，如游泳速度受损、心率升高或肌肉纤维直径减小。我们利用 LMNA 斑马鱼模型进行药物筛选，发现 L-肉碱治疗可挽救 LMNA(L35P)的肌肉耐力，肌酸治疗可逆转 LMNA(R453W)的肌肉耐力。肌酸激活 AMPK 和 mTOR 通路，改善 LMNA(R453W)鱼的肌肉耐力和游泳速度。转录组学分析揭示了导致 LMNA 突变转基因鱼运动功能障碍、心脏异常和离子通量失调的上游调节剂和受影响基因。这些发现忠实地模拟了肌肉层板病的临床表现，包括畸形、早期死亡率、纤维大小减小和肌肉功能障碍在斑马鱼中。此外，我们的药物筛选结果表明，L-肉碱和肌酸治疗可能是挽救 LMNA(L35P)和 LMNA(R453W)斑马鱼模型肌肉耐力的潜在方法。我们的研究为开发针对与 LMNA 相关的肌肉层板病的潜在治疗方法提供了有价值的见解。
