The titin N2A-MARP signalosome constrains muscle longitudinal hypertrophy in response to stretch
- Department of Cellular and Molecular Medicine, University of Arizona, United States
- Department of Physiology, Amsterdam University Medical Center, VUMC, Netherlands
- School of Medicine, University of California San Diego, United States
- Department of Biomedicine, Aarhus University, Denmark
- Department of Endocrinology, University of Arizona, United States
- Department of Integrative Pathophysiology, Medical Faculty Mannheim, Germany
Figures
Figure 1 with 3 supplements
Transient hypertrophy following unilateral diaphragm denervation (UDD) in the denervated costal diaphragm.
(A) Schematic of how UDD affects sarcomere length in the denervated costal (left panel) and how stretch extends titin for mechanosensing (right panel). (B) Mouse diaphragm costal weight normalized to tibial length following 1, 3, 6, 12, and 35 days of UDD, showing the hypertrophy phase peaking at 6 days and progressing to the atrophy phase at 12 days post-UDD (n=6–22, shams grouped for simplicity). A substantial part of the hypertrophy seen in mice encompasses longitudinal hypertrophy (C), lengthening of the muscle fibers by addition of serial sarcomeres (n=10–13). The increased fiber length likely reduces the stretch-based hypertrophy signaling and thus explains the transient nature of hypertrophy. (D) Three-day bilateral diaphragm denervation (BDD) in rats (n=6–9) confirms stretch is the trigger for inducing hypertrophy in UDD at the tissue mass level (D; rat diaphragm right costal normalized to tibial length, denervated in UDD) and serial sarcomeres level (E). One-way ANOVA, with Tukey post-hoc testing. Bar graphs denote mean ± SEM. Figure 1A was created with BioRender.com.
Figure 1—figure supplement 1
Three days bilateral diaphragm denervation in rats showed similar body weights compared to sham animals (A; n=6–9/group) and were of similar size based on tibia length (B) and soleus muscle weights (C).
Statistical testing by one-way ANOVA and Dunnett’s multiple comparisons test. Bar graphs denote mean ± SEM.
Figure 1—figure supplement 2
Titin splicing in 3 day unilateral and bilateral diaphragm denervation in rats showed similar levels of splicing between unilateral diaphragm denervation (UDD) (red line) and sham (black line) animals, while bilateral diaphragm denervation (BDD) (blue line) animals showed a decrease in exons coding for the elastic PEVK element.
Statistical testing by Multiple t-testing, comparing sham to UDD and sham to BDD (n=5/group). Bar graphs denote mean ± SEM.
Figure 1—figure supplement 3
Role of titin stiffness on hypertrophy following unilateral diaphragm denervation (UDD).
(A) Transient hypertrophy response in Rbm20ΔRRM mice (more compliant titin) showing a blunted hypertrophy response compared to wild-type (WT) mice, based on percent increase of diaphragm right costal mass relative to sham (n=10–12). (B) Titin-based stiffness does not alter longitudinal hypertrophy response, as both WT and Rbm20ΔRRM mice show a similar increase in serial sarcomeres following 6 days UDD. Rbm20 knockout (KO) rat response to 3 days UDD, based on percent increase of diaphragm right costal mass relative to sham (mouse n=10–11, rat n=8) supporting titin-based stiffness regulating muscle hypertrophy similarly across species. Statistical testing by t-test. Bar graphs denote mean ± SEM.
Figure 2 with 2 supplements
Global transcriptomics and proteomics following 3 days unilateral diaphragm denervation (UDD) and bilateral diaphragm denervation (BDD) in rats.
Global transcript studies by RNA-seq of sham, UDD, and BDD right costal diaphragm (n=5/group). Same parameters apply to the global proteome studies (C–D) with mass spectrometry. Quantitative Venn diagrams of the transcriptome (A) showing overlap gene regulation between UDD and BDD. Vulcano plots of UDD (B, left) and BDD (B, middle) showed similar gene regulation. Comparing UDD to BDD directly revealed just 850 differentially regulated genes (B, right) indicating a small subset being responsible for hypertrophy regulation. Quantitative Venn diagrams of the proteome (C) showed similar regulation compared to transcriptome. Volcano plots of UDD (D, left) and BDD (D, middle) showed primarily upregulation of proteins. Comparing UDD to BDD directly revealed just 173 differentially regulated proteins (D, right). Green-dots: upregulated genes/proteins, red-dots: downregulated genes/proteins. Titin-associated proteins in heatmap of proteome (E; Z-score: red = upregulated, blue = downregulated) and violin plots (right panel) of differential proteins between UDD (red) and BDD (blue), indicating upregulation of titin-associate proteins following stretch. Two-way ANOVA (sham vs UDD and sham vs BDD) pint: *p<0.05, **p<0.01. Violin plot denotes median ± quartiles.
Figure 2—figure supplement 1
Principal component analysis of the rat 3 day unilateral diaphragm denervation (UDD) and bilateral diaphragm denervation (BDD) transcriptome (A) and proteome (B), showing clear separation of groups (n=5/group) at the transcript level and overlap of BDD and UDD samples at the protein level.
Gene ontology (GO) term enrichment of UDD>BDD separated by up- or down-regulated transcriptomes and proteome (C, left and right, respectively) show distinct, yet overlapping cellular processes. Global mass spectrometry was analyzed by ANOVA and corrected for multiple comparisons with false discovery rate with a cut-off at p<0.05.
Figure 2—figure supplement 2
Transcriptome regulation of titin-associated and myofilament genes by RNA-seq in rats following 3 days of unilateral diaphragm denervation (UDD)/bilateral diaphragm denervation (BDD).
Heatmaps showing similar regulation between UDD and BDD samples (n=5/group; Z-score: red = upregulated, blue = downregulated) at the transcript level for titin-associated and myofilament genes, based on hierarchical clustering.
Figure 3 with 1 supplement
Phosphorylation of titin following 24 hr of unilateral diaphragm denervation (UDD) by mass spectrometry (n=4/group).
Phosphorylation of individual titin domains (A) relative Z-score (log2) of titin phosphorylation showing domain specific changes in phosphorylation. Total phosphorylation of titin (B) is not affected by UDD, however, titin showed regional changes in phosphorylation (C), notably increased phosphorylation of the N2A-element (boxed). Fold-change of the phosphorylation signal for the five main sites found in the N2A-element (D). (E) Schematic of the N2A element with the 2 pSer found in the N2Aus (Transcript: ENSMUST00000099981.10 Ttn-203). Red: Ig domain coding and blue: unique sequence coding. Mouse titin phosphorylation and global mass spectrometry was analyzed by t-test, Kolmogorov-Smirnov test or multiple t-test with a cut-off at p<0.05. Bar graph denotes mean ± SEM and violin plots denote median ± quartiles.
Figure 3—figure supplement 1
Titin N2A associated protein phosphorylation events at 24 hr unilateral diaphragm denervation (UDD).
Violin plots of phosphorylation events in N2A-associated proteins following UDD: MARP1 (Transcript: ENSMUST00000237142.2 Ankrd1-205), MARP2 (Transcript: ENSMUST00000026172.3 Ankrd2-201), Smyd2 (Transcript: ENSMUST00000027897.8 Smyd2-201), Capn3 (Transcript: ENSMUST00000028749.15 Capn3-202), Hsp90ab (Transcript: ENSMUST00000024739.14 Hsp90ab1-201), Mypn (Transcript: ENSMUST00000095580.3 Mypn-201), Hspb1 (Transcript: ENSMUST00000005077.7 Hspb1-201), Cryab (Transcript: ENSMUST00000217475.2 Cryab-206), and Prkca/PKA (Transcript: ENSMUST00000005606.8 Prkaca-201). Data n=4/group represented as Log2 of the normalized abundance with significance determined by Kolmogorov-Smirnov test. Violin plot denotes median ± quartiles.
Figure 4 with 1 supplement
Six-day unilateral diaphragm denervation (UDD) on knockout (KO) mice of muscle ankyrin repeat proteins (MARPs).
MARP triple knockouts (KOs) show a reduced response to UDD (A; p<0.01). No effect of single MARP1 KO (B) on UDD, increased hypertrophy following MARP2 KO (B; p<0.01; t-test), indicating possible roles in hypertrophy suppression or atrophy signaling and MARP3 KO (C) showed baseline hypertrophy in costal diaphragm in addition to less hypertrophy development in UDD (p<0.01; t-test) compared to wild-type (WT), implying roles as a suppressor of hypertrophy. Left panel, diaphragm right costal mass normalized to tibial length and right panel, percentual increase in right costal mass relative to sham. S=Sham, U=UDD (n=10–12). Statistical testing by t-test or two-way ANOVA with Tukey post-hoc testing. Bar graphs denote mean ± SEM.
Figure 4—figure supplement 1
Six-day unilateral diaphragm denervation (UDD) on double knockout (KO) mice of muscle ankyrin repeat proteins (MARPs).
Double KO of MARP1/2 (A), MARP1/3 (B), and MARP2/3 (C) all showed a reduction in hypertrophy following UDD, suggesting redundancy between the MARPs. Left panel, diaphragm right costal mass normalized to tibial length and right panel, percentual increase in right costal mass relative to sham. S=Sham, U=UDD (n=10–12). Statistical testing by t-test or two-way ANOVA with Tukey post-hoc testing. Bar graphs denote mean ± SEM.
Figure 5
Muscle ankyrin repeat proteins (MARPs) inhibit longitudinal hypertrophy.
Longitudinal hypertrophy measured in right costal strips of wild-type (WT) and MARP triple knockout (tKO) mice in 6 day sham and UDD mice (A). Numerical increase in serial sarcomeres is higher in MARP tKO (p<0.0001) mice compared to WT mice (B; n=11–13), suggesting that the MARPs inhibit longitudinal growth. Probing hypertrophy signaling by western blot, normalized to Gapdh, with expression set relative to WT sham levels (C; n=10–12). Differential mTOR response suggests role in regulating longitudinal hypertrophy. (D) Representative blot images of the signaling proteins. Statistical testing by one-way or two-way ANOVA with Tukey post-hoc testing. Bar graphs denote mean ± SEM.
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Figure 5—source data 1
Unedited western blots.
- https://cdn.elifesciences.org/articles/107597/elife-107597-fig5-data1-v1.pptx
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Figure 5—source data 2
Unedited Western blots.
- https://cdn.elifesciences.org/articles/107597/elife-107597-fig5-data2-v1.zip
Figure 6
Pharmacological inhibition of the mTOR (rapamycin) and calcium (cyclosporin A) based hypertrophy pathways revealed mTOR to be involved in longitudinal hypertrophy.
(A) Schematic of the inhibition protocol, showing mice were injected with inhibitors for 3 days prior to receiving unilateral diaphragm denervation (UDD) surgery, with continued twice daily dosing of inhibitors until sacrifice at day 3 post-UDD. Rapamycin inhibited hypertrophy development both at the costal diaphragm mass level (B; p<0.001) and at the longitudinal hypertrophy level (C; p<0.001), whereas cyclosporin A had no effect. Neither cyclosporin A or rapamycin affected the innervated costal diaphragm (D), or body mass (E) and all mice used were of approximately the same size based on skeletal size, as measured by tibia length (F). (G) Hypothetical mechanism for longitudinal hypertrophy following muscle stretch. The mTORC1 pathway is activated by stretch and initiates longitudinal muscle hypertrophy. Muscle ankyrin repeat proteins (MARPs) sequestered by titin’s N2A element are released upon stretch and tune the longitudinal hypertrophy, thus preventing excessive longitudinal hypertrophy. N=8–10/group, statistical testing by one-way-ANOVA and Dunnett’s multiple comparisons test. Bar graphs denote mean ± SEM. Figure 6G was created with BioRender.com.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Ttn | NCBI Gene | Gene ID: 22138 | Titin |
| Gene (Mus musculus) | Ankrd1 | NCBI Gene | Gene ID: 107765 | MARP1 |
| Gene (Mus musculus) | Ankrd2 | NCBI Gene | Gene ID: 107766 | MARP2 |
| Gene (Mus musculus) | Ankrd23 | NCBI Gene | Gene ID: 64009 | MARP3 /DARP |
| Genetic reagent (Mus musculus) | MARP1 KO (Ankrd1 KO) | Bang et al., 2014 | https://doi.org/10.1371/journal.pone.0093638 | Used for UDD studies |
| Genetic reagent (Mus musculus) | MARP2 KO (Ankrd2 KO) | Bang et al., 2014 | https://doi.org/10.1371/journal.pone.0093638 | Used for UDD studies |
| Genetic reagent (Mus musculus) | MARP3 KO (Ankrd23 KO) | Bang et al., 2014 | https://doi.org/10.1371/journal.pone.0093638 | Used for UDD studies |
| Genetic reagent (Mus musculus) | MARP Triple KO | Barash et al., 2007 | https://doi.org/10.1152/ajpcell.00055.2007 | Used for UDD studies |
| Genetic reagent (Mus musculus) | Rbm20ΔRRM | Methawasin et al., 2014 | https://doi.org/10.1161/CIRCULATIONAHA.113.005610 | Used for UDD studies |
| Genetic reagent (Rattus norvegicus) | Sprague Dawley | Charles River | Crl:CD(SD) | Used for BDD/UDD studies |
| Genetic reagent (Rattus norvegicus) | Rbm20 KO | Li et al., 2013 | https://doi.org/10.1093/nar/gks1362 | Used for UDD studies |
| Commercial assay, kit | RNeasy Fibrous Tissue Mini Kit | Qiagen | Cat#74704 | RNA isolation |
| Commercial assay, kit | TruSeq RNA Library Prep Kit v2 | Illumina | RS-122–2001 | RNA-seq library preparation |
| Commercial assay, kit | Total RNA Prep, Ligation with Ribo-Zero Plus | Illumina | 20040525 | rRNA depletion before library prep |
| Commercial assay, kit | Pierce Quantitative Colorimetric Peptide Assay Kit | Thermo Fisher | Cat#23275 | Peptide quantification |
| Commercial assay, kit | High-Select Fe-NTA Phosphopeptide Enrichment Kit | Thermo Fisher | Cat#A32992 | Phosphopeptide enrichment |
| Commercial assay, kit | High-Select TiO2 Phosphopeptide Enrichment Kit | Thermo Fisher | Cat#A32993 | Phosphopeptide enrichment |
| Antibody | anti-MAPK1/3 (Mouse IgG1) | Cell Signaling | Cat#4696 | WB (1:200) |
| Antibody | anti-Calcineurin (Mouse IgG2a) | BD Biosciences | Cat#610260 | WB (1:750) |
| Antibody | anti-mTOR (Rabbit IgG) | Cell Signaling | Cat#2983 | WB (1:750) |
| Antibody | anti-P70S6K (Rabbit IgG) | Cell Signaling | Cat#2708 | WB (1:500) |
| Antibody | anti-4EBP1 (Rabbit IgG) | Cell Signaling | Cat#9452 | WB (1:750) |
| Antibody | anti-GAPDH (Rabbit IgG) | Cell Signaling | Cat#2112 | WB (1:5000) |
| Antibody | anti-GAPDH (Mouse IgG1) | Thermo Fisher | Cat#MA5-15738 | WB (1:3000) |
| Chemical compound, drug | Rapamycin | Selleck Chem | S1039 | 2.5 mg/kg/day |
| Chemical compound, drug | Cyclosporin A | Selleck Chem | S2286 | 25 mg/kg/day |
| Software, algorithm | STAR | Dobin et al. | RRID:SCR_015899 | RNA-seq alignment |
| Software, algorithm | DESeq2 | Love et al. | RRID:SCR_015687 | Differential expression |
| Software, algorithm | Progenesis QI for Proteomics | Nonlinear Dynamics | RRID:SCR_018923 | Proteomics analysis, Version 2.4 |
| Software, algorithm | Perseus | Tyanova et al., 2016 | RRID:SCR_015753 | Proteomics analysis, Version 2.0.3.1 |
| Software, algorithm | GraphPad Prism | GraphPad | RRID:SCR_002798 | Statistics, Version 9.1 |
| Software, algorithm | ShinyGO | Ge et al., 2020 | RRID:SCR_019213 | GO analysis, Version 0.75 |
| Software, algorithm | Heatmapper | Babicki et al., 2016 | RRID:SCR_016974 | Heatmap generation |
| Software, algorithm | BioVenn | Hulsen et al., 2008 | RRID:SCR_026853 | Venn analysis |
Table 1
Antibodies used in this study.
| Antibody | Source/isotype | Dilution | Company | Catalog# |
|---|---|---|---|---|
| MAPK1/3 (ERK2/1) | Mouse IgG1 | 1:200 | Cell Signaling | #4696 |
| Calcineurin | Mouse IgG2a | 1:750 | BD Biosciences | 610260 |
| mTOR | Rabbit IgG | 1:750 | Cell Signaling | #2983 |
| P70S6K | Rabbit IgG | 1:500 | Cell Signaling | #2708 |
| 4EBP1 | Rabbit IgG | 1:750 | Cell Signaling | #9452 |
| Gapdh | Rabbit IgG | 1:5000 | Cell Signaling | #2112 |
| Gapdh | Mouse IgG1 | 1:3000 | Thermo Fisher Scientific. | MA5-15738 |
| CF790 Goat anti Mouse IgG | Goat IgG | 1:10,000 | Biotium | 20342 |
| CF680 Goat anti Mouse IgG | Goat IgG | 1:10,000 | Biotium | 20065 |
| CF680 Goat anti Rabbit IgG | Goat IgG | 1:10,000 | Biotium | 20067 |
| CF680R Goat anti Mouse IgG2a | Goat IgG | 1:10,000 | Biotium | 20842 |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/107597/elife-107597-mdarchecklist1-v1.docx
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Supplementary file 1
Rat RNAseq analysis.
- https://cdn.elifesciences.org/articles/107597/elife-107597-supp1-v1.xlsx
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Supplementary file 2
Rat Global mass spectrometry analysis.
- https://cdn.elifesciences.org/articles/107597/elife-107597-supp2-v1.xlsx
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Supplementary file 3
Mouse Phosphorylation mass spectrometry analysis.
- https://cdn.elifesciences.org/articles/107597/elife-107597-supp3-v1.xlsx
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The titin N2A-MARP signalosome constrains muscle longitudinal hypertrophy in response to stretch
eLife 14:RP107597.
https://doi.org/10.7554/eLife.107597.3
