Downsizing the molecular spring of the giant protein titin reveals that skeletal muscle titin determines passive stiffness and drives longitudinal hypertrophy
- Ambjorn Brynnel
- Yaeren Hernandez
- Balazs Kiss
- Johan Lindqvist
- Maya Adler
- Justin Kolb
- Robbert Van der Pijl
- Jochen Gohlke
- Joshua Strom
- John Smith
- Coen Ottenheim
- Henk L Granzier
- University of Arizona, United States
Figures
Figure 1 with 3 supplements
The TtnΔ112-158 mouse model.
(A and B) Titin’s I-band region is shown schematically in (A) and the exon composition of the PEVK and flanking region is indicated in B). The region targeted for deletion is highlighted. (Yellow: …
Figure 1—figure supplement 1
Body weight comparison between WT and homozygous Ttn∆112-158 female mice.
Two-way ANOVA reveals a significantly reduced BW in Ttn∆112-158 with a multiple comparison analysis revealing a significant reduction at 80 days. Note that the number of mice was less at 290 d (5 …
Figure 1—figure supplement 2
Tibia length comparison between WT and homozygous Ttn∆112-158 female mice.
Two-way ANOVA reveals no significant differences in tibia length between the genotypes.
Figure 1—figure supplement 3
Titin expression analysis.
The total amount of titin, T1 + T2 (relative to myosin heavy chain, MHC) and the fractional content of T1 (relative to total titin) are unaltered in the Ttn∆112-158 mice.
Figure 2 with 3 supplements
Titin exon expression analysis.
(A) RNAseq-based titin exon expression of diaphragm muscle, expressed as percent spliced-in (PSI) in WT and TtnΔ112-158 mice (top and below), and the PSI difference between the two genotypes and the …
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Figure 2—source data 1
Percent Spliced In source data.
- https://doi.org/10.7554/eLife.40532.010
Figure 2—figure supplement 1
Titin expression analysis in EDL muscle.
RNAseq-based titin exon expression of EDL muscle, expressed as percent spliced-in (PSI) in WT and Ttn∆112-158 mice (top and below), and the PSI difference between the two genotypes and the p-value …
Figure 2—figure supplement 2
Titin expression analysis in soleus muscle.
RNAseq-based titin exon expression of EDL muscle, expressed as percent spliced-in (PSI) in WT and Ttn∆112-158 mice (top and below), and the PSI difference between the two genotypes and the p-value …
Figure 2—figure supplement 3
Titin expression analysis in diaphragm, EDL and soleus muscle, in part of Z-disk, complete PEVK and part of M-band regions.
Top: superimposed results of WT (top) and PSI difference between EDL and diaphragm and between soleus and diaphragm (bottom). Bottom: superimposed results of Ttn∆112-158 mice (top) and PSI …
Figure 3
Extension of the PEVK segment and predicted titin-based force.
(A) Example immuno-fluorescence images and densitometry of sarcomeres labeled with N2A and I84-86 antibodies that flank the PEVK (Figure 1B shows binding sites). (B) PEVK extension (z) as function …
Figure 4
Passive Muscle stiffness.
The passive tension – sarcomere length relation was determined in Diaphragm (top), EDL (middle) and soleus (bottom) intact muscle from WT and TtnΔ112-158 mice. Passive tension is highly increased in …
Figure 5
Functionally dissecting passive muscle stiffness.
(A and B) Passive tension in EDL muscle (5th toe muscle) was measured in intact muscle, then again after skinning, and a final time after KCl/KI extraction (extraction insensitive tension). The …
Figure 6 with 1 supplement
Active tension.
(A) Example results of tetanic tension vs. stimulation frequency. Results of EDL 5th toe muscle are shown. Results of WT and TtnΔ112-158 muscles closely overlap. (B) Maximal tetanic tension in …
Figure 6—figure supplement 1
Myosin Heavy Chain (MHC) isoform analysis.
Two-way ANOVA reveals that there is no significant difference in MHC isoforms in either diaphragm, EDL, or soleus Ttn∆112-158 muscles compared to WT controls. A multiple comparison analysis only …
Figure 7
Exercise capacity.
(A) Free-wheel running exercise. Left, average running speed; Right, average distance ran in a 24 hr period. There is no significant difference in running speed and running between Ttn∆112-158 and …
Figure 8 with 1 supplement
Sarcomere length working range.
(A) Motion-mode Echo image of the diaphragm in a WT mouse. The change in strain was measuring during inspiration (shortening) and then the mouse was perfusion-fixed which arrested the diaphragm in …
Figure 8—figure supplement 1
Fiber lengths and muscle lengths are increased in Ttn∆112-158 mice.
(A) Fibers were carefully dissected from chemically fixed muscle at their slack length and their end-to-end length was measured. Fibers are longer in muscles from Ttn∆112-158 mice in male (top) and …
Figure 9
Sarcomere in series per muscle fiber.
Two-way ANOVA reveals a significantly increased number of sarcomeres in single fibers in all studied muscle types in TtnΔ112-158 mice. The average increase in the number of sarcomeres in series in …
Figure 10 with 1 supplement
Muscles of TtnΔ112-158 mice are hypertrophied without an increase in cross-sectional area.
(A–C) Hypertrophy is expressed as muscle weight (mg) normalized to tibia length (mm). Two-way ANOVA reveals a significantly increased hypertrophy in all muscle types of the TtnΔ112-158 mice (male …
Figure 10—figure supplement 1
Muscles of Ttn∆112-158 mice are hypertrophied.
Hypertrophy is expressed as muscle weight (mg) normalized to tibia length (mm). Two-way ANOVA reveals a significantly increased hypertrophy in most muscle types of the Ttn∆112-158 mice. Most of the …
Figure 11 with 1 supplement
Thin filament length (TFL) and predicted force-sarcomere length (SL) relation.
(A) TFL was measured in diaphragm muscle by super-resolution optical microscopy using the TFL-pointed end capping protein Tmod4. Top shows a WT example. Bottom: analyzed results. Sarcomere length …
Figure 11—figure supplement 1
Nebulin exon expression analysis in diaphragm muscle.
RNAseq-based nebulin exon expression of diaphragm muscle, expressed as percent spliced in (PSI) in WT (top) and Ttn∆112-158 mice (middle), and the PSI difference between the two genotypes (bottom). …
Figure 12
Passive muscle stiffness at the physiological sarcomere length range.
Passive stiffness in Ttn∆112-158 and WT EDL 5th toe muscle at an identical sarcomere length range (A) and at the physiological sarcomere length of each genotype (B). Data in A represent the average …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| gene (Mus musculus) | Titin (Ttn) | NA | Ensembl: ENSMUSG00000051747 | |
| genetic reagent (Mus musculus) | Ttn∆112-158 | this paper | Deletion of (GRCm38/mm10) chr2:76,839,202–76,867,333 | |
| strain, strain background (Mus musculus) | ‘mixed C57BL/6J and 129P2/OlaHsd " | other | Homologous recombination in E14TG2a cells (129/Ola strain, PMID: 3683574). Mice backcrossed seven generations to C57BL/6J (JAX stock#664) | |
| antibody | anti-titin N2A (chicken polyclonal) | Biogenes | Biogenes: anti-X105-X106 | (5.75 μg/mL) |
| antibody | anti-titin I84-86 (rabbit polyclonal) | Biogenes | Biogenes: anti-BK283-BK284 | (1 μg/mL) |
| antibody | anti-α-actinin (mouse monoclonal) | Sigma-Aldrich | Sigma-Aldrich:A7811; RRID:AB_476766 | (1:1000) |
| antibody | anti-Tmod4 (rabbit polyclonal) | Proteintech Group Inc | Proteintech Group:11753–1-AP; RRID:AB_2205433 | (1:750) |
| antibody | AlexaFluor-488 conjugated goat anti-mouse IgG | Invitrogen | (1:500) | |
| antibody | AlexaFluor-568 conjugated goat anti-rabbit IgG | Invitrogen | (1:250) | |
| antibody | AlexaFluor-647 conjugated goat anti-mouse IgG | Invitrogen | (1:250) | |
| antibody | CF-633 conjugated donkey anti-chicken IgY | Biotium | (1:200) | |
| other | AlexaFluor-488 conjugated phalloidin | Invitrogen | (1:1000) |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.40532.025
