Lack of desmin in mice causes structural and functional disorders of neuromuscular junctions

Desmin, the major intermediate filament (IF) protein in muscle cells, interlinks neighboring myofibrils and connects the whole myofibrillar apparatus to myonuclei, mitochondria, and the sarcolemma. However, desmin is also known to be enriched at postsynaptic membranes of neuromuscular junctions (NMJs). The pivotal role of the desmin IF cytoskeletal network is underscored by the fact that over 100 mutations of the human DES gene cause hereditary and sporadic myopathies and cardiomyopathies. A subgroup of human desminopathies comprises autosomal recessive cases resulting in complete abolition of desmin protein. In these patients, who display a more severe phenotype than the autosomal dominant cases, it has been reported that some individuals also suffer from a myasthenic syndrome in addition to the classical occurrence of myopathy and cardiomyopathy. Since further studies on the NMJ pathology is hampered by the lack of available human striated muscle biopsy specimens, we exploited homozygous desmin knock-out mice which closely mirror the striated muscle pathology of human patients lacking desmin protein. Here, we report on the impact of the lack of desmin on the structure and function of NMJs and on the transcription of genes coding for postsynaptic proteins. Desmin knock-out mice display a fragmentation of NMJs in soleus, but not in extensor digitorum longus muscle. Moreover, soleus muscle fibers show larger NMJs. Further, transcription levels of acetylcholine receptor (AChR) genes are increased in muscles from desmin knock-out mice, especially of the AChRgamma subunit, which is known as a marker of muscle fiber regeneration. Electrophysiological recordings depicted a pathological decrement of nerve-dependent endplate potentials and a faster rise time of the nerve-independent miniature endplate potentials. The latter is indicating an enhanced opening time of the AChR channels. Our study highlights the essential role of desmin for the structural and functional integrity of mammalian neuromuscular junctions. Differential gene expression analysis of striated muscle in wildtype vs. Desmin knock out mice was performed on 5 biological replicates of each genotype.结蛋白（Desmin）是肌细胞内主要的中间丝（intermediate filament, IF）蛋白，可将相邻肌原纤维相互连接，并将完整的肌原纤维装置锚定至肌核、线粒体与肌膜。既往研究证实，结蛋白同时富集于神经肌肉接头（neuromuscular junctions, NMJs）的突触后膜。人类DES基因存在超过100种突变，可引发遗传性与散发性肌病及心肌病，这充分凸显了结蛋白中间丝细胞骨架网络的关键作用。一类结蛋白病患者为常染色体隐性遗传，其体内结蛋白蛋白完全缺失；相较于常染色体显性遗传患者，这类患者表型更为严重，且有报道显示部分患者除典型的肌病与心肌病外，还并发肌无力综合征。由于缺乏可用的人类横纹肌活检标本，相关神经肌肉接头病理的后续研究受到极大限制。为此，我们采用了可精准模拟人类结蛋白缺失患者横纹肌病理表型的纯合结蛋白敲除小鼠作为模型。本研究探讨了结蛋白缺失对哺乳动物神经肌肉接头结构与功能的影响，以及突触后蛋白编码基因的转录调控变化。实验结果显示，结蛋白敲除小鼠的比目鱼肌中存在神经肌肉接头碎片化现象，但趾长伸肌未出现该改变；同时，比目鱼肌肌纤维的神经肌肉接头面积更大。此外，结蛋白敲除小鼠肌肉内的乙酰胆碱受体（acetylcholine receptor, AChR）基因转录水平上调，其中以作为肌纤维再生标志物的AChRγ亚基基因上调最为显著。电生理记录结果表明，神经依赖性终板电位出现病理性衰减，而神经非依赖性微小终板电位的上升时程加快，后者提示乙酰胆碱受体通道的开放时长增加。本研究证实了结蛋白对维持哺乳动物神经肌肉接头结构与功能完整性的核心作用。本研究针对野生型与结蛋白敲除小鼠的横纹肌，采用每组5个生物学重复样本完成了差异基因表达分析。