Karimi Esmat, van der Borgh Mila, Lindqvist Johan, Gohlke Jochen, Hourani Zaynab, Kolb Justin, Cossette Stacy, Lawlor Michael W, Ottenheijm Coen, Granzier Henk
bioRxiv. 2023 Dec 21:2023.12.20.572678. doi: 10.1101/2023.12.20.572678.
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. Mutations in the nebulin gene ( ) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking therapies targeting the underlying pathological mechanisms. In this study, we examined a cohort of ten NEM2 patients, each with unique mutations, aiming to understand their impact on mRNA, protein, and functional levels. Results show that truncation mutations affect 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 splicing mutations which is expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with relatively normal nebulin levels and others with markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, and a positive relation between the reduction in nebulin level and the reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a duplication mutation in nebulin that resulted in a 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 I muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87-318%, 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 splicing mutations. 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.
伴肌动蛋白是骨骼肌细肌丝的一种关键蛋白，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。伴肌动蛋白基因（ ）的突变会导致基于伴肌动蛋白的杆状体肌病（NEM2），这是一种遗传异质性疾病，其特征为肌张力减退和肌肉无力，目前缺乏针对潜在病理机制的治疗方法。在本研究中，我们检查了一组10名NEM2患者，每名患者都有独特的突变，旨在了解这些突变对mRNA、蛋白质和功能水平的影响。结果表明，截短突变会影响mRNA稳定性，并导致突变转录本的无义介导衰变。此外，在剪接突变患者中发现了隐匿性剪接位点激活的高发生率，这预计会破坏伴肌动蛋白的肌动蛋白结合位点。蛋白质水平的测定显示，有些患者的伴肌动蛋白水平相对正常，而另一些患者的伴肌动蛋白水平则显著降低。我们观察到伴肌动蛋白减少与TFL减少之间呈正相关，伴肌动蛋白水平降低与张力降低（最大张力和次最大张力）之间呈正相关。有趣的是，我们的研究发现伴肌动蛋白中的一个重复突变导致了更大的伴肌动蛋白和更长的TFL。此外，我们研究了心肌肌球蛋白小分子激活剂奥米卡替麦卡比（OM）对NEM2患者I型肌纤维力量产生的影响。OM治疗使所有NEM2患者的次最大张力大幅增加，增幅在87%至318%之间，对伴肌动蛋白水平最低的患者影响最大。总之，本研究表明转录后或翻译后机制调节伴肌动蛋白表达。此外，我们提出NEM2的发病机制不仅涉及细肌丝缩短，还涉及细肌丝延长，以及剪接突变导致的肌动蛋白结合位点破坏。重要的是，我们的研究结果突出了OM治疗改善NEM2患者骨骼肌功能的潜力，尤其是那些伴肌动蛋白水平大幅降低的患者。
