Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA.
Department of Pathology, Medical College of Wisconsin, Milwaukee, WI, USA.
Acta Neuropathol. 2024 Apr 18;147(1):72. doi: 10.1007/s00401-024-02726-w.
Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.
肌联蛋白是骨骼肌细肌丝的关键蛋白，在调节细肌丝长度（TFL）、横桥循环和肌原纤维排列等生理过程中发挥重要作用。肌联蛋白基因（NEB）的致病变异导致基于肌联蛋白的杆状体肌病（NEM2），这是一种遗传异质性疾病，表现为张力减退和肌肉无力，目前尚无治愈疗法。在这项研究中，我们检查了 10 名 NEM2 患者的队列，每个患者都有独特的致病变异，旨在了解它们对 mRNA、蛋白质和功能水平的影响。结果表明，致病截断变异会影响肌联蛋白 mRNA 的稳定性，并导致突变转录本的无意义介导衰变。此外，我们还发现，在具有致病剪接变异的患者中，存在高发生率的隐匿剪接位点激活，预计这会破坏肌联蛋白的肌动蛋白结合位点。蛋白质水平的测定显示，患者的肌联蛋白水平要么相对正常，要么明显降低。我们观察到肌联蛋白减少与 TFL 减少或张力减少（最大和次最大张力）之间存在正相关。有趣的是，我们的研究在肌联蛋白中发现了一个致病的重复变异，导致 NEB 的三重复区域出现四倍的增益，以及更大的肌联蛋白和更长的 TFL。此外，我们还研究了小分子肌球蛋白激活剂 Omecamtiv mecarbil（OM）对 NEM2 患者 1 型肌纤维产生力的影响。OM 治疗使所有 NEM2 患者的次最大张力都显著增加，范围为 87%至 318%，在肌联蛋白水平最低的患者中效果最大。总之，这项研究表明，转录后或翻译后机制调节肌联蛋白的表达。此外，我们提出 NEM2 的发病机制不仅涉及缩短的细肌丝，还涉及伸长的细肌丝，以及致病剪接变异导致的肌动蛋白结合位点破坏。重要的是，我们的发现强调了 OM 治疗改善 NEM2 患者骨骼肌功能的潜力，尤其是那些肌联蛋白水平大幅降低的患者。
