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

Figures

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  Figure 1. Fast mdm muscles (red) expressed more Z-repeats compared to WT muscles (gray). (A) Percent spliced-in (PSI) indexes calculated for exons 10–14 using RNA-Seq data (top row), and agarose gel images for amplification of exons 11–13 using primers annealed on flanking exons (bottom row). Expression of exons 12 and 13 encoding Z-repeats 5 and 6 was upregulated in mdm EDL and psoas muscles compared to WT muscles (two-way ANOVA, all p < 0.0001). PSI index varies from 0 = complete exclusion to 1 = complete inclusion of exons in the transcript pool. Exon labels are suffixed with a period followed by a number if the region was split into separate segments based on the variable distribution of reads in the alignments. Segments flanking annotated exon boundaries are suffixed with “_hang”. The shaded areas show the standard deviation of mean PSI values. Illustration shows the primer locations with arrows and exon arrangement in the region of interest. Agarose gel images show four variants: (a) expression of exons 11–13 (599 bp); (b) exclusion of one exon (461 bp); (c) exclusion of two exons (323 bp); and (d) complete exclusion of exons 11–13 (185 bp). (B) Expression of α-actinin1–4 in mdm…

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  Figure 2. PEVKI and PEVKII exons have significantly lower expression levels in mdm muscles. Expression of PEVK exons in mdm muscles (red) and WT muscles (grey) varied among regions, with the most variations occurring in PEVKI and PEVKII. PSI index varies from 0 = complete exclusion to 1 = complete inclusion in the transcript pool. The shaded area shows the standard deviation of mean PSI values. Exons undergoing an expression drop in more than one muscle type are shown at the top. Exon regions specific to each muscle are shown in the respective rows. Two-way ANOVA demonstrated significant downregulation of exons 124–127, 133–134, 138–145, and 194 in mdm compared to WT, all p < 0.0029).

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  Figure 3. ∆PSI plots for EDL (purple), psoas (blue), and soleus (green) highlighting that the major splice variation occurs in the PEVK region. The PSI values of the mdm titin exons were subtracted from the WT PSI values to determine the PSI difference (dPSI). Negative PSI values indicate a higher rate of exon inclusion in WT titin.

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  Figure 4. Expression of MEx5 (exon 346) is upregulated in fast mdm muscles (two-way ANOVA, p < 0.0001). Mean PSI values calculated for MEx1-6 encoding exons are shown. PSI = 0—complete exclusion and PSI = 1–100% inclusion of the exon/exon segment into the expressed transcript pool. The shaded area shows the standard deviation of mean PSI values. Exon labels are suffixed with a period followed by a number if the region was split into separate segments based on the variable distribution of reads in the alignments.

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  Figure 5. Gene expression changes in ECM-associated genes in mdm skeletal muscles compared to WT muscles. The expression heatmap was generated using ECM-associated differentially expressed genes identified from the RNA sequencing data. The heatmap was generated using Z-scores calculated for each gene in each muscle using variance-stabilized transformed expression levels. Regions containing downregulated genes in mdm EDL muscles compared to WT EDL are highlighted with dotted lines.

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  Figure 6. (A) Passive tension of mdm fiber bundles was significantly higher than WT for untreated control (ANOVA; F = 131.6; p < 0.0001), trypsin-treated (ANOVA; F = 45.5; p < 0.0001), and trypsin + KCl + KI-treated (ANOVA; F = 22.7; p = 0.0014) fiber bundles. Passive tension decreased significantly from control to trypsin-treated fiber bundles in both WT and mdm (ANOVA; F = 419.7; p < 0.0001). In addition, there was a significant decrease in passive tension after trypsin + KCl + KI in both WT (ANOVA; F = 142.82; p < 0.0001) and mdm fiber bundles (ANOVA; F = 29.45; p = 0.0006). All six groups differed significantly from each other (Tukey’s HSD, p < 0.05). (B) Reduction (%) in passive stress after trypsin + KCl + KI treatment was significantly greater in mdm soleus fiber bundles compared to WT (ANOVA; F = 64.1; p < 0.0001), suggesting a higher titin-based passive tension in mdm muscle.