Research ArticleCELL BIOLOGY

Nebulin and Lmod2 are critical for specifying thin-filament length in skeletal muscle

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Science Advances  11 Nov 2020:
Vol. 6, no. 46, eabc1992
DOI: 10.1126/sciadv.abc1992
  • Fig. 1 Basic characterization of mouse models expressing shortened and lengthened nebulin, the NebΔS9–11, and NebDupS9–11 models.

    (A) Schematic of skeletal muscle sarcomere. Nebulin’s C terminus is anchored in the Z-disk, and its N terminus is near the thin-filament pointed-end (capped by Tmod and interacting with leiomodin Lmod). Nebulin super-repeats 9 to 11 and the antibody against its N-terminal modules are highlighted. In the NebΔS9–11 and NebDupS9–11 nebulin models, S9 to 11 have been deleted and duplicated, respectively. (B) Nebulin expression in EDL, diaphragm, and soleus muscles of Hom mice, normalized to myosin heavy chain (MHC). Nebulin expression is not altered in the gene targeted mice. (C) Nebulin expression in EDL muscle of WT, Hom NebΔS9–11, Hom NebDupS9–11, and a compound heterozygous cross of these models (referred to as compound-Het). Dotted line: location of WT nebulin. Total nebulin expression in the compound-Het EDL muscle is the same as in WT muscle. (D) IEM with the nebulin N-terminal antibody resulted in clear labeling (arrows) that revealed the sarcomeric incorporation of shorter and longer nebulin, compared to WT, in Hom NebΔS9–11 and Hom NebDupS9–11 EDL, respectively. (E) Analyzed nebulin length results. (F) Results of Het NebΔS9–11 EDL labeled by the nebulin N-terminal antibody. A doublet is detected in the sarcomere with 116-nm separation. Scale bars, 500 nm. [Graphs, means ± SEM; (B) and (E), comparisons based on analysis of variance (ANOVA); NS, nonsignificant. ****P < 0.0001 in comparison versus WT; ####P < 0.0001 in comparison versus NebΔS9–11. (C), comparison based on unpaired two-tailed t test.].

  • Fig. 2 Nebulin exon expression in WT, Hom NebΔS9–11, and Hom NebDupS9–11 mice, shown as PSI.

    (A) Expression analysis in soleus (Sol), EDL, and diaphragm (Dia) muscle in WT (top), NebΔS9–11 (middle), and NebDupS9–11 (bottom) mice. Indicated are the various regions of the gene, corresponding to nebulin’s N terminus (pink), super-repeat region (gray), and Z-disk region (yellow). Shown are also the targeted exons 53 to 64 that were deleted in the NebΔS9–11 (middle) and duplicated in the NebDupS9–11 model (bottom). Because the added exons 53 to 64 cannot be distinguished from the native exons, the normalized read density of exons 53 to 64 were determined (bottom, right scale), revealing a higher read density for exons 53 to 64. Note that in the gene-targeted mice, there are minimal adaptations in PSI outside the targeted region. (B) Top: Difference in PSI for Hom NebΔS9–11 and Hom NebDupS9–11 exons. Bottom: P values for NebΔS9–11 - NebDupS9–11 PSI comparison (Shown values are the mean of 12 mice. For additional details, see fig. S3, A to D, and table S1.).

  • Fig. 3 PSI of Z-disk exons (Neb exons 140 to 166) in soleus and EDL muscle.

    (A) PSI values in WT (circles), NebΔS9–11 (diamond), and NebDupS9–11 (squares) genotypes. (B) P values for PSI difference between soleus and EDL in WT (circles), NebΔS9–11 (diamond), and NebDupS9–11 (squares) genotypes. Exons 152 to 160 expression varies greatly between soleus and EDL muscles, but genotype has little effect.

  • Fig. 4 Nebulin length, thin-filament length, and force-sarcomere length relation in EDL muscle.

    (A) Examples SR-SIM images on skinned NebDupS9–11 Hom EDL muscle. Left: merged, middle: individual channels; red: N terminus of nebulin, orange: Tmod1, green: phalloidin, blue: A-band (Ti102 antibody). Right: Neb-N and Tmod1 epitope distance together with TFL were measured on intensity plot profiles across the Z-disk. (The Ti102 epitope distance was measured across the A-band.). (B) SL dependence of the TFL and nebulin epitope to mid–Z-disk distance. The numbers represent the mean results averaged in the 2.6- to 3.0-μm SL range. (C) Nebulin length (average ± SEM). The shown length difference at the top is compared to WT. (D) Left: F-SL relationship for Hom NebΔS9–11 and NebDupS9–11 EDL muscle fibers. The descending limbs of these relationships are offset along the SL axis. (D) Right: Specific force at optimal SL; SL50: SL at half-maximal active force. (E) SL dependence of the short and long nebulin isoforms and proximal and distal Tmod epitopes (see also fig. S7A) in EDL muscle of compound Het NebΔS9–11/DupS9–11. (F) Results in the 2.6- to 3.0-μm SL range [gray in (E)]. [Graphs: means ± SEM. (C) ANOVA. ****P < 0.0001 in comparison versus WT. (D, right) and (F): unpaired two-tailed t test. ***P < 0.001; ****P < 0.0001; ns, not significant)]

  • Fig. 5 Nebulin length and TFL measured by TEM and IEM on EDL muscle.

    (A) Top: Example TEM micrographs of skinned muscles from WT and Hom NebΔS9–11 and Hom NebDupS9–11 mice. Bottom: TFL was measured using densitometry and determining the distance between the middle of the Z-disk and the H-zone. (B) Top: Example IEM micrographs using an N-terminal nebulin antibody. Bottom: Nebulin length was determined using densitometry and determining the distance between the middle of the Z-disk and the nebulin N-terminal epitope in WT, Hom NebΔS9–11, and Hom NebDupS9–11 skinned fiber bundles. (C) SL dependence of length of nebulin and TFL. (D) Mean TFL and nebulin length in the 2.6- to 3.0-μm SL range. The dashed horizontal lines correspond to the average TFL of each genotype. (measurements were corrected for shrinkage; see Materials and Methods for additional). No significant differences between TFL and nebulin length were found (one-way ANOVA with Sidak multiple testing correction).

  • Fig. 6 Thin-filament and nebulin lengths in diaphragm (DIA) muscle.

    (A) Example images showing the same area of a Hom NebDupS9–11 diaphragm muscle. Top: Ti102 (A-band, blue), phalloidin (green), and nebulin N-terminal antibody (red); bottom: Ti102 (A-band, blue), phalloidin (green), and Tmod4 (orange). White arrowheads mark the N terminus of nebulin. Scale bars, 2 μm, Z, Z-disk. (B) Nebulin length (average ± SEM in SL 2.6- to 3.0-μm range). The numbers show the difference compared to WT. (C) SL dependence of the TFL distance or nebulin N terminus to middle of Z-disk. The numbers represent the mean epitope distance in nanometers, averaged in the 2.6- to 3.0-μm SL range (gray background). (D) TFLs in diaphragm undergoing unilateral diaphragm denervation (UDD). In this model, the right diaphragm is denervated and as a result is passively stretched by ~25%. The left diaphragm remains innervated and continues to actively shorten. Left: Example SR-SIM images of diaphragm muscle sections. Red arrowheads mark the N terminus of nebulin. Scale bars, 2 μm. Middle and right: SL dependence of the thin-filament and nebulin length in the innervated side (middle) and denervated side (right) of the diaphragm. The numbers represent the mean values in nanometers, averaged in the 2.6- to 3.0-μm SL range (gray background). The proximal nebulin-containing thin-filament segment is largely unchanged (as reflected by the similar nebulin epitope distances), but the distal nebulin-free segment is longer in the denervated side (Tmod4 and phalloidin distances increased by ~150 nm) [(B) Graph is means ± SEM. ANOVA. ****P < 0.0001 in comparison versus WT].

  • Fig. 7 Thin-filament and nebulin lengths in soleus muscle.

    (A) Example SR-SIM images. Top: Hom NebDupS9–11; Ti102 (A-band, blue), phalloidin (green), and nebulin N-terminal antibody (red). Gray arrowheads mark the N terminus of nebulin. Bottom: Hom NebΔS9–11; α-actinin (Z-disk, green), nebulin N-terminal antibody (red), and Tmod4 (orange). Tmod4 (white arrow) is localized distal from Neb-N (gray arrowhead). Scale bars, 2 μm, Z, Z-disk. Inset: enlarged sarcomere with epitope localizations emphasizing the proximal nebulin-containing and distal nebulin-free segment of the thin filament. (B) Nebulin length (average ± SEM). (C) SL dependence of the TFL and nebulin epitope distances. The numbers represent the mean epitope distance in nanometers, averaged in the 2.6- to 3.0-μm SL range (gray background). The numbers show the difference compared to WT. (D) TFL versus nebulin length. The distal thin-filament segment (nebulin free) varies in size in the different muscle types. It is large and responds to the length of nebulin in the soleus, it is small and does not respond to the length of nebulin in EDL and is intermediate in the diaphragm. Note that the SEM bars are merged into the actual data points. (E) Relation between distal thin-filament segment (nebulin-free) and proximal thin-filament segment (nebulin-containing). [(B) Graph is means ± SEM. ANOVA. ****P < 0.0001 in comparison versus WT].

  • Fig. 8 Expression levels of thin-filament proteins Lmod2, Lmod3, Tmod1, and Tmod4.

    (A) Analysis in EDL, soleus, and diaphragm muscle of WT, Hom NebDupS9–11, and Hom NebΔS9–11 mice. Left: Example Western blots and bottom: expression levels [normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. The expression level of Lmod2 is very low in EDL muscle, ~50-fold higher in soleus and intermediate in diaphragm. In addition, Lmod2 expression is lower in NebDupS9–11 than in NebΔS9–11 mice. (B) Expression levels of Lmod2/3 and Tmod1/4 in soleus muscle in the three genotypes plotted against the length of the distal thin-filament segment (nebulin-free). The slopes of the regression lines are significant for Lmod2 and Tmod4). (C) Expression analysis in left and right sided diaphragm muscle of sham-operated mice and UDD mice where the left side continues to contract but the right side is denervated for 30 hours. Lmod2 is ~three-fold up-regulated in the denervated side (P < 0.0001). [(A) regular two-way ANOVA with multiple comparison testing using Tukey: *P < 0.05 and ****P < 0.0001. (B) linear regression analysis. (C) One-way ANOVA with multiple comparison testing with Tukey correction. ****P < 0.0001 in comparison to left side, left. Graphs show means ± SEM). a.u., arbitrary units.

  • Fig. 9 Effect of deleting Lmod2 on TFL in soleus muscle.

    Conditional Lmod2 KO mice were injected with an adeno-associated virus (AAV)–Cre virus, and 3 weeks later, the soleus muscles were harvested. Muscles were analyzed for protein expression and TFL measurement. (A) Example Western blots (left) and expression levels (right, values normalized to GAPDH and the mean value in control mice) in soleus muscle reveal a ~95% reduction in Lmod2 levels in soleus muscle of AAV-Cre–treated Lmod2 conditonal KO (cKO) mice, without affecting Lmod2, Tmod1, and Tmod4 expression levels. (B) Left: Length of nebulin and lengths of thin filaments in soleus muscle, based on Tmod4 and phalloidin localization, as a function of SL. In the 2600- to 3000-nm SL range, the nebulin length and TFL are ~1100 nm. (B) Right: Comparison between WT (same data as in Fig. 7C) and Lmod2 cKO soleus treated with AAV-Cre shows a highly significant reduction in TFL in Lmod2 cKO soleus muscle both when measured with Tmod4 antibody and phalloidin [analyzed with one-way ANOVA with multiple testing (Sidak) correction; *P < 0.05; ****P < 0.0001. Graphs are means ± SEM).

  • Fig. 10 Effect of deleting Lmod2 on TFL in denervated diaphragm muscle.

    Conditional Lmod2 KO mice were injected with an AAV-Cre virus; 3 weeks later, UDD surgery was performed; and 30 hours later, the innervated and denervated hemi-diaphragms were collected. UDD was also performed on WT mice. The innervated (Inn.) and denervated (Den.) hemi-diaphragms were both analyzed for protein expression; the denervated muscles were also used for nebulin length and TFL measurements. (A) Example Western blots (left) and expression levels (right, values normalized to GAPDH and the mean value in control mice) reveal a >95% reduction in Lmod2 levels in both innervated and denervated diaphragm muscles of AAV-Cre–treated Lmod2 cKO mice, without affecting Lmod3 and Tmod4 expression levels. Tmod1 was slightly increased in the denervated diaphragm of the cLmod2 KO mice compared to the innervated side but was not different when compared to WT mice. (B) Length of nebulin and length of thin filaments, based on Tmod4 and phalloidin localization, as a function of SL. The shown values are mean values in the 2600- to 3000-nm SL range. In WT denervated diaphragm (left), the thin-filament pointed-end extends far beyond nebulin and in cLmod2 KO denervated muscle (right), this is not the case. (C) Comparison between WT and Lmod2 cKO denervated diaphragm. Lmod2 deficiency results in much shorter thin filaments (~12%) in denervated diaphragm (analyzed with one-way ANOVA with multiple testing (Sidak) correction; *P < 0.05 and ****P < 0.0001. Graphs are means ± SEM).

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