由3个外显子编码的肌生发育抑制素(MSTN)基因编码的肌生发育抑制素是骨骼肌生长的强大负调节因子。虽然已经发明了各种肌生发育抑制素抑制剂以增加肌肉质量治疗肌肉消耗性疾病，但目前还没有有效的抑制剂可供临床使用。已经报道了几种动物中的肌生发育抑制素亚型可以抑制肌生发育抑制素，但人类MSTN基因中的亚型从未被鉴定出来，这是一种在动物中具有保守性的基因。在这里，研究了人类MSTN基因的剪接变体。转录本和蛋白质通过逆转录聚合酶链式反应扩增和免疫印迹分析。蛋白质通过表达质粒来表达。肌生发育抑制素信号通过SMAD响应性荧光素酶活性来检测。细胞增殖通过细胞计数试剂盒-8(CCK-8)试验和细胞计数试验来检测。细胞周期通过FastFUCCI系统进行分析。在CRL-2061横纹肌肉瘤细胞的全长MSTN转录本的逆转录聚合酶链式反应扩增中发现了两个带有预期较大产物的厚带和一个带有额外小尺寸产物的薄带。小尺寸产物的测序显示在外显子3的5'端有一个963 bp的缺失，形成一个包含不寻常剪接受体TG dinucleotides的外显子3s。这个新变体在其他人类细胞系中鉴定出来，尽管在骨骼肌中并未鉴定出来。由新变体编码的251氨基酸亚型(myostatin-b)在CRL-2061横纹肌肉瘤细胞中得到鉴定。将myostatin-b表达质粒转染到CRL-2061和肌母细胞中抑制内源性肌生发育抑制素信号(分别为44%，P < 0.001和63%，P < 0.001)。此外，myostatin-b抑制重组肌生发育抑制素诱导的肌生发育抑制素信号(68.8%，P < 0.001)。相比之下，myostatin-b不抑制重组生长分化因子11诱导的肌生发育抑制素信号(9.2%，P = 0.70)，转化生长因子β(+3.1%，P = 0.83)或活化素A(+1.1%，P = 0.96)。这些结果表明了myostatin-b的肌生发育抑制素特异性抑制作用。值得注意的是，myostatin-b在肌母细胞中的表达显著增强了细胞增殖，通过CCK-8和直接细胞计数试验分别高于模拟转染细胞组(60%，P<0.05和39%，P<0.05)。与模拟转染细胞组相比，myostatin-b显著增加了S期细胞的百分比(53% vs. 80%，P<0.05)。我们克隆了一种由非正常剪接产生的新型人类MSTN变体。这个变体编码了一种新型肌生发育抑制素亚型myostatin-b，它通过特异性肌生发育抑制素形式抑制肌生发育抑制素信号，并通过改变细胞周期增强肌母细胞增殖。myostatin-b具有肌生发育抑制素特异性抑制活性，可以作为天然肌生发育抑制素抑制剂的开发候选物。© 2023 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.

Myostatin, encoded by the MSTN gene comprising 3 exons, is a potent negative regulator of skeletal muscle growth. Although a variety of myostatin inhibitors have been invented for increasing muscle mass in muscle wasting diseases, no effective inhibitor is currently available for clinical use. Myostatin isoforms in several animals have been reported to inhibit myostatin, but an isoform has never been identified for the human MSTN gene, a conserved gene among animals. Here, a splice variant of the human MSTN gene was explored.Transcripts and proteins were analysed by reverse transcription-PCR amplification and western blotting, respectively. Proteins were expressed from expression plasmid. Myostatin signalling was assayed by the SMAD-responsive luciferase activity. Cell proliferation was assayed by the Cell Counting Kit-8 (CCK-8) assay and cell counting. Cell cycle was analysed by the FastFUCCI system.Reverse transcription-PCR amplification of the full-length MSTN transcript in CRL-2061 rhabdomyosarcoma cells revealed two bands consisting of a thick expected-size product and a thin additional small-size product. Sequencing of the small-size product showed a 963-bp deletion in the 5' end of exon 3, creating exon 3s, which contained unusual splice acceptor TG dinucleotides. The novel variant was identified in other human cell lines, although it was not identified in skeletal muscle. The 251-amino acid isoform encoded by the novel variant (myostatin-b) was identified in CRL-2061 rhabdomyosarcoma cells. Transfection of a myostatin-b expression plasmid into CRL-2061 and myoblast cells inhibited endogenous myostatin signalling (44%, P < 0.001 and 63%, P < 0.001, respectively). Furthermore, myostatin-b inhibited myostatin signalling induced by recombinant myostatin (68.8%, P < 0.001). In remarkable contrast, myostatin-b did not inhibit the myostatin signalling induced by recombinant growth differentiation factor 11 (9.2%, P = 0.70), transforming growth factor β (+3.1%, P = 0.83) or activin A (+1.1%, P = 0.96). These results indicate the myostatin-specific inhibitory effect of myostatin-b. Notably, the expression of myostatin-b in myoblasts significantly enhanced cell proliferation higher than the mock-transfected cells by the CCK-8 and direct cell counting assays (60%, P < 0.05 and 39%, P < 0.05, respectively). Myostatin-b increased the percentage of S-phase cells significantly higher than that of the mock-transfected cells (53% vs. 80%, P < 0.05).We cloned a novel human MSTN variant produced by unorthodox splicing. The variant encoded a novel myostatin isoform, myostatin-b, that inhibited myostatin signalling by myostatin-specific manner and enhanced myoblast proliferation by shifting cell cycle. Myostatin-b, which has myostatin-specific inhibitory activity, could be developed as a natural myostatin inhibitor.© 2023 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.