Chemistry Department, University of Massachusetts Lowell, Lowell, MA 01854, USA.
Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011-5640, USA.
Int J Mol Sci. 2022 Aug 9;23(16):8858. doi: 10.3390/ijms23168858.
Muscular dystrophy with myositis () is a naturally occurring mutation in the mouse gene that results in higher passive stress in muscle fibers and intact muscles compared to wild-type (WT). The goal of this study was to test whether alternative splicing of titin exons occurs in muscles, which contain a small deletion in the N2A-PEVK regions of titin, and to test whether splicing changes are associated with an increase in titin-based passive tension. Although higher levels of collagen have been reported previously in muscles, here we demonstrate alternative splicing of titin in skeletal muscle fibers. We identified Z-band, PEVK, and C-terminus Mex5 exons as splicing hotspots in titin using RNA sequencing data and further reported upregulation in ECM-associated genes. We also treated skinned soleus fiber bundles with trypsin, trypsin + KCl, and trypsin + KCL + KI to degrade titin. The results showed that passive stress dropped significantly more after trypsin treatment in fibers (11 ± 1.6 mN/mm) than in WT fibers (4.8 ± 1 mN/mm; = 0.0004). The finding that treatment with trypsin reduces titin-based passive tension more in than in WT fibers supports the hypothesis that exon splicing leads to the expression of a stiffer and shorter titin isoform in fibers. After titin extraction by trypsin + KCl + KI, fibers (6.7 ± 1.27 mN/mm) had significantly higher collagen-based passive stress remaining than WT fibers (2.6 ± 1.3 mN/mm; = 0.0014). We conclude that both titin and collagen contribute to higher passive tension of muscles.
肌营养不良伴肌炎（）是一种在小鼠基因中自然发生的突变，与野生型（WT）相比，它导致肌肉纤维和完整肌肉的被动张力更高。本研究的目的是测试在含有肌联蛋白 N2A-PEVK 区域小缺失的 肌肉中是否存在肌联蛋白外显子的选择性剪接，并测试剪接变化是否与肌联蛋白的被动张力增加有关。尽管以前有报道称 肌肉中的胶原水平较高，但在这里我们证明了 骨骼肌纤维中的肌联蛋白发生了选择性剪接。我们使用 RNA 测序数据鉴定了 Z 带、PEVK 和 C 末端 Mex5 外显子作为 肌联蛋白的剪接热点，并进一步报告了细胞外基质相关基因的上调。我们还使用胰蛋白酶、胰蛋白酶+KCl 和胰蛋白酶+KCl+KI 处理去皮的 比目鱼肌纤维束，以降解肌联蛋白。结果表明，在 纤维中，经胰蛋白酶处理后，被动张力显著下降（11±1.6 mN/mm），而在 WT 纤维中，被动张力下降（4.8±1 mN/mm；=0.0004）。用胰蛋白酶处理后， 纤维中的肌联蛋白依赖的被动张力降低幅度明显大于 WT 纤维，这一发现支持了这样一种假说，即外显子剪接导致 纤维中表达一种更硬和更短的肌联蛋白同工型。用胰蛋白酶+KCl+KI 提取肌联蛋白后， 纤维（6.7±1.27 mN/mm）的胶原依赖的剩余被动张力明显高于 WT 纤维（2.6±1.3 mN/mm；=0.0014）。我们的结论是，肌联蛋白和胶原都有助于 肌肉的高被动张力。
