Transient hypertrophy following 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, generated through BioRender.com). (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 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) 3-day BDD in rats (n=6-9) confirms stretch is the trigger for inducing hypertrophy in UDD at the tissue mass level (D; diaphragm right costal normalized to tibial length, denervated in UDD) and serial sarcomeres level (E). One-way ANOVA, with Tukey post-hoc testing.

Global transcriptomics and proteomics following 3-days UDD and BDD in rats.

Global transcript studies by RNAseq 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. 2-way ANOVA pint: *p<0.05, **P<0.01.

Phosphorylation of titin following 24-hours of 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). Quantitation of the phosphorylation signal for the 5 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.

6-day UDD on KO mice of MARP proteins.

MARP triple 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 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.

MARPs inhibits longitudinal hypertrophy.

Longitudinal hypertrophy measured in right costal strips of WT and MARP 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=7-12), suggesting that the MARPs inhibits longitudinal growth. Probing hypertrophy signaling by western blot, normalized to Gapdh, with expression set relative to WT sham levels (C; n=8-12). 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.

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 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 cyclosporine A had no effect. Neither cyclosporine 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. MARP proteins sequestered by titin’s N2A element are released upon stretch and tunes the longitudinal hypertrophy, thus preventing excessive longitudinal hypertrophy (Image was generated through BioRender.com.). N=8-10/group, statistical testing by 1-way-ANOVA and Dunnett’s multiple comparisons test.

Antibodies used in this study.

3-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.

Role of titin stiffness on hypertrophy following UDD.

(A) Transient hypertrophy response in Rbm20ΔRRM mice (more compliant titin) showing a blunted hypertrophy response compared to 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-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.

Principal component analysis of the rat 3-day UDD and BDD transcriptome (A) and proteome (B), showing clear separation of groups at the transcript level and overlap of BDD and UDD samples at the protein level. GOterm 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.

Transcriptome regulation of titin-associated and myofilament genes by RNAseq in rats following 3-days of UDD/BDD.

Heatmaps showing similar regulation between UDD and BDD samples (n=4-5; Z-score: red= upregulated, blue= downregulated) at the transcript level for titin-associated and myofilament genes, based on hierarchal clustering.

Titin N2A associated protein phosphorylation events at 24-hour 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 represented as Log2 of the normalized abundance with significance determined by Kolmogorov-Smirnov test.

6-day UDD on double KO mice of 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).