Contributions of Titin and Collagen to Passive Stress in Muscles from Mice with a Small Deletion in Titin's Molecular Spring.

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.