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. 2010 Apr 5;189(1):95-109.
doi: 10.1083/jcb.201001125.

Tropomodulin isoforms regulate thin filament pointed-end capping and skeletal muscle physiology

David S Gokhin  1 , Raymond A Lewis, Caroline R McKeown, Roberta B Nowak, Nancy E Kim, Ryan S Littlefield, Richard L Lieber, Velia M Fowler
Affiliations

Tropomodulin isoforms regulate thin filament pointed-end capping and skeletal muscle physiology

David S Gokhin et al. J Cell Biol. .

Abstract

During myofibril assembly, thin filament lengths are precisely specified to optimize skeletal muscle function. Tropomodulins (Tmods) are capping proteins that specify thin filament lengths by controlling actin dynamics at pointed ends. In this study, we use a genetic targeting approach to explore the effects of deleting Tmod1 from skeletal muscle. Myofibril assembly, skeletal muscle structure, and thin filament lengths are normal in the absence of Tmod1. Tmod4 localizes to thin filament pointed ends in Tmod1-null embryonic muscle, whereas both Tmod3 and -4 localize to pointed ends in Tmod1-null adult muscle. Substitution by Tmod3 and -4 occurs despite their weaker interactions with striated muscle tropomyosins. However, the absence of Tmod1 results in depressed isometric stress production during muscle contraction, systemic locomotor deficits, and a shift to a faster fiber type distribution. Thus, Tmod3 and -4 compensate for the absence of Tmod1 structurally but not functionally. We conclude that Tmod1 is a novel regulator of skeletal muscle physiology.

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Figures

Figure 1.
Figure 1.
Tmod1 is dispensable for myofibril assembly. (A) Western blots of E15.5 mouse embryonic leg and flank muscle tissue lysates show absence of Tmod1 in Tmod1−/−Tg+ muscles. Tubulin and actin were loading controls. Molecular mass is indicated in kilodaltons. (B–I) Skeletal muscle from Tmod1+/+ and Tmod1−/−Tg+ E15.5 mouse embryos stained for Tmod1, F-actin, and α-actinin. Arrowheads denote H-zones. Bars, 3 µm.
Figure 2.
Figure 2.
Tmod1 is dispensable for correct localization of nebulin. (A–H) Skeletal muscle from Tmod1+/+ (A–D) and Tmod1−/−Tg+ (E–H) E15.5 mouse embryos was stained for nebulin M1M2M3, F-actin, and α-actinin. Arrowheads denote nebulin M1M2M3 stripes flanking the H-zone. Bars, 3 µm.
Figure 3.
Figure 3.
Tmod4 but not Tmod3 substitutes for Tmod1 by capping thin filament pointed ends in embryonic muscle. (A–P) Skeletal muscle from Tmod1+/+ (A–D and I–L) and Tmod1−/−Tg+ (E–H and M–P) E15.5 mouse embryos stained for F-actin, α-actinin, and either Tmod3 or -4. Tmod3 is diffusely localized, whereas Tmod4 localizes to the pointed ends in the presence or absence of Tmod1. Arrowheads denote H-zones. Bars, 5 µm.
Figure 4.
Figure 4.
Tmod1 is dispensable for adult muscle structure. (A) TEM of 1-mo-old EDL muscle from Tmod1+/+ and Tmod1−/−Tg+ mice. (B–D) EDL muscle from Tmod1+/+ and Tmod1−/−Tg+ mice stained for F-actin and either α-actinin (B), the C-terminal M160–164 domain of nebulin (C), or myomesin (D). Bars: (A) 1 µm; (B–D) 3 µm.
Figure 5.
Figure 5.
Tmod3 and -4 substitute for Tmod1 by capping thin filament pointed ends in adult skeletal muscle. (A) Western blots of 1-mo-old EDL muscle tissue lysates show no apparent changes in Tmod3 or -4 levels in the absence of Tmod1. Tubulin and actin were loading controls. Molecular mass is indicated in kilodaltons. (B–D) EDL muscle from Tmod1+/+ and Tmod1−/−Tg+ mice stained for F-actin and either Tmod1 (B), -3 (C), or -4 (D). Bars, 5 µm.
Figure 6.
Figure 6.
Tmod3 and -4 bind to striated muscle TMs more weakly than does Tmod1. (A) Increasing amounts of Tmods were separated by SDS-PAGE, transferred to nitrocellulose, and overlaid with wild-type (WT) αslow-TM (first row), M9R mutant αslow-TM (second row), or β-TM (third row). (B) Increasing amounts of TMs were separated by SDS-PAGE, transferred to nitrocellulose, and overlaid with Tmod1 (first row), -4 (second row), or -3 (third row). Parallel Coomassie-stained gels show loading (fourth row). (A and B) Molecular mass is indicated in kilodaltons. (C) Relative binding strengths among Tmods and TMs were scored semiquantitatively based on visual inspection of blots. Predicted binding between Tmods and αfast-TM (parentheses) is based on sequence identity between the N-terminal regions of αfast-TM and β-TM where Tmods bind (Stone and Smillie, 1978; Helfman et al., 1986). Asterisks indicate conserved residues. The arrow shows the location of the nemaline myopathy–causing M9R mutation. (D and E) Binding avidities were quantified using densitometry by normalizing band intensities from the blots to the corresponding Coomassie-stained bands. x-axis labels refer to the protein that is overlaid, whereas data series refer to the immobilized protein. Note that relative binding avidities can only be compared within each individual blot overlay, demarcated by tick marks, and not from one TM to the next (in D) or from one Tmod isoform to the next (in E) because of variations in antibody binding and blot exposure times. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n = 4 lanes/group. Data are mean ± SEM. a.u., arbitrary unit.
Figure 7.
Figure 7.
Deletion of Tmod1 depresses skeletal muscle contractile function. (A and B) Muscle PCSA (A) and slack sarcomere length (B) are unchanged in Tmod1−/−Tg+ mice. (C and D) Isometric force (C) and isometric stress (D) produced by EDL muscle from Tmod1−/−Tg+ mice are significantly decreased. *, P < 0.05; ***, P < 0.001. n = 8 muscles/genotype. Data are mean ± SEM.
Figure 8.
Figure 8.
Deletion of Tmod1 impairs locomotor activity in mice. (A and B) Voluntary activity in 5–7-mo-old mice was monitored over three consecutive light cycles (12 h off and 12 h on). The number of wheel counts and laser beam breaks was substantially lower in Tmod1−/−Tg+ animals. (C and D) Muscle strength in mice of various ages was assessed using the hanging wire and grip strength tests. No significant differences in time before falling or grip force were found at any age. (E) Motor coordination was assessed using the rotarod test. No differences in angular speed before falling were found in 1–2-mo-old animals and >12-mo-old animals, but a slight decrease in coordination is evident in 4- and 6-mo-old Tmod1−/−Tg+ animals. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n = 6 (in A and B) or n = 8 (in C–E) mice/genotype. Data are mean ± SD.
Figure 9.
Figure 9.
Deletion of Tmod1 shifts muscle fiber types toward a faster distribution. (A) The distribution of fiber sizes in the TA was shifted rightward in Tmod1−/−Tg+ muscle. Graph shows the size distribution of n = 560 Tmod1+/+ fibers and n = 479 Tmod1−/−Tg+ fibers measured from n = 2 mice/genotype. (B) The mean fiber cross-sectional area was significantly elevated in both the TA and soleus, suggesting a faster fiber type. (C) The proportion of type 2A/X/B fibers was elevated in the TA and soleus of Tmod1−/−Tg+ mice at the expense of type 1 fibers. (D) Although there were no significant differences in the size of type 1 fibers, type 2A/X/B fibers were significantly larger in Tmod1−/−Tg+ TA and soleus. (E) TA and soleus tissue lysates were separated on 8% SDS-PAGE gels. In both TA and soleus, a shift toward faster MHC isoforms was evident in Tmod1−/−Tg+ mice. Molecular mass is indicated in kilodaltons. (F) No significant differences in centralized nuclei were observed between Tmod1+/+ and Tmod1−/−Tg+ muscle. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n = 4 (in C and D) or n = 8 (in B, E, and F) muscles/genotype. Data are mean ± SEM.
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