The titin N2A-MARP signalosome constrains muscle longitudinal hypertrophy in response to stretch

27 Titin-based mechanosensing is a key driver of trophic signaling in muscle, yet the downstream 28 pathways linking titin sensing to muscle remodeling remain poorly understood. To investigate 29 these signaling mechanisms, we utilized unilateral diaphragm denervation (UDD), an in vivo 30 model that induces titin-stiffness-dependent hypertrophy via mechanical stretch. Using UDD in 31 rats and mice, we characterized the longitudinal hypertrophic response and distinguished 32 stretch-induced signaling from denervation effects by performing global transcriptomic and 33 proteomic analyses following UDD and bilateral diaphragm denervation (BDD) in rats. Our 34 findings identified upregulation of titin-associated muscle ankyrin repeat proteins (MARPs). 35 Subsequent phosphorylation enrichment mass spectrometry in mouse diaphragm highlighted 36 the involvement of the N2A-element. UDD in MARP knockout (KO) mice resulted in enhanced 37 longitudinal hypertrophy, with Western blot analysis revealing activation of the mTOR pathway. 38 Furthermore, pharmaco logical inhibition of mTORC1 with rapamycin suppressed longitudinal 39 hypertrophy, demonstrating that mTOR signaling regulates titin-mediated hypertrophic growth in 40 a MARP-dependent manner. These findings establish MARPs as key modulators of titin-based 41 mechanotransduction and highlight mTORC1 as a central regulator of longitudinal muscle 42 hypertrophy. 43 44

45 Hypertrophy, Muscle, Signaling, Mechanosensing, Titin, MARP, mTOR 46 47 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint

[401 WORDS] 48 Titin is a giant protein in striated muscle and forms an elastic filament in sarcomeres 1, the 49 smallest contractile unit in muscle. One of the functions attributed to titin is to generate passive 50 tension in muscle 2,3, ensuring optimal overlap of the actin-based thin filaments and myosin-51 based thick filaments for muscle contraction. The elastic properties of titin combined with its 52 arrangement in thesarcomeres, spanning the half-sarcomer, make titin an ideal stress sensor. 53 The mechanosensory properties of titin have been extensively described 4–6 and appear to 54 localize to several signaling hotspots on titin. 55 In skeletal muscle, the N2A element is the prevalent titin region associated with hypertrophy 56 signaling. The N2A element is known for its interaction with the muscle ankyrin repeat proteins 57 (MARP1-3)7,8 and calpain3 9. All MARPs have been implicated in trophicity signaling, with 58 redundancy between the MARP proteins 7,10. MARP1 tethers titin to the thin filament, forming a 59 mechanism for increasing passive tension 11,12. In the heart, MARP1 also interacts with MLP and 60 protein kinase C alpha in intercalated disks, resulting in a maladaptive trophic response in 61 dilated cardiomyopathy 10. MARP2 can be phosphorylated at serine-99 (S69 in mice) by Akt, 62 resulting in reduced differentiation potential of myoblasts 13. MARP2 has also been linked to the 63 NFkB-pathway in inflammatory responses 14, suggesting MARP2 may have roles in atrophy 64 signaling. MARP3 is the least studied member of the MARPs but appears to play a role in 65 glucose uptake and vascular remodeling in skeletal muscle 15,16. Titin has previously been shown 66 to activate signaling in response to stretch 17,18, providing a potential activation mechanism for 67 N2A to activate trophic signaling. 68 Muscle hypertrophy is directly associated with titin-based stiffness. High stiffness, as observed 69 in the Ttn Δex112-158 and TtnΔex219-225 mouse models 19,20, results in longitudinal hypertrophy, i.e. an 70 increase in serial-linked sarcomeres, to reduce sarcomere length and normalize passive 71 tension. We previously used a surgical model in which we denervated one hemi-diaphragm 72 (unilateral diaphragm denervation, UDD) 21, inducing passive cyclic stretch of the denervated 73 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint hemi-diaphragm by the innervated costal to induce (longitudinal) hypertrophy. This hypertrophy 74 was dependent on titin-based stiffness, as higher titin stiffness resulted in exaggerated 75 hypertrophy and lower titin stiffness resulted in attenuated hypertrophy 21. UDD is a unique 76 model as it allows the in vivo study of passive stretch-induced muscle hypertrophy. 77 78 We used UDD as a model for studying titin-mechanosensing and examined the signaling that 79 underlies the hypertrophy response. Our findings support the N2A-MARP signalosome inhibiting 80 longitudinal hypertrophy and show mTOR signaling to be a prominent contributor to longitudinal 81 hypertrophy. 82 83 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint

84 Longitudinal hypertrophy regulates transient trophicity in UDD. 85 A unique aspect of unilateral diaphragm denervation (UDD) is the transient nature of the 86 hypertrophy. This transient nature is likely the result from changes in fractional extension of 87 sarcomeres. Sarcomere length (SL) at end-expiration length was 2.9±0.1 µm in sham, while 88 stretch lengths at end-inspiration were predicted to reach 3.7 µm directly after denervation of the 89 right costal diaphragm (FIG.1A, previously published 21). Such sarcomere lengths put high strain 90 on titin (FIG.1A, right panel), providing a potent trigger for mechanosensing and subsequent 91 hypertrophy signaling. Our results show that during 6-days of UDD the denervated costal rapidly 92 hypertrophies, followed by slow onset of atrophy (FIG.1B). The mass increase coincides with 93 longitudinal hypertrophy of muscle fibers, addition of serial-linked sarcomeres, adding 952±81 94 sarcomeres (30.2% increase in total fiber length; p<0.0001; FIG.1C ) following 6-days of UDD. 95 This increase in sarcomere number appeared to stabilize by 6-days UDD, as at 12-day UDD 96 sarcomere addition was measured to be 778±87 sarcomeres versus sham levels (not 97 significantly different compared to 6-day UDD). Assuming that fractional extension of 98 sarcomeres during inspiration decreases with longitudinal hypertrophy, 30.2% increase in 99 sarcomere number would reduce the sarcomere length at end inspiration from 3.7 µm to ~2.8 100 µm, in close agreement with whole body formaldehyde perfusion experiments which showed 101 sarcomere lengths of 2.7±0.1 µm 21. As a SL of 2.8 µm falls below the end-expiration SL, the 102 denervated costal effectively no longer experiences “stretch”, reducing titin’s mechanosensing 103 trigger and thus explaining the transient nature of the hypertrophy seen in UDD. 104 105 To confirm that stretch is the trigger for hypertrophy, rather than denervation, we performed 106 bilateral diaphragm denervation in rats (BDD), resulting in complete inactivity of the diaphragm . 107 Note that we attempted BDD in mice but experienced high mortality rates, while survival in rat 108 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint was >75%. All rats were of similar body weight pre-surgery and showed a slight decrease in 109 body weight after 3-day BDD (Supplemental FIG.1A; p=0.036), and comparable tibial length and 110 soleus weights (Supplemental FIG.1B & C). Similar to mice, rats present with increased 111 denervated costal mass after 3-day UDD: 14.68±0.64 mg/mm versus sham rats 10.82±0.39 112 mg/mm (FIG.1D; p>0.0001; muscle weight normalized to tibial length). However, 3-day BDD 113 rats did not develop increased mass 11.92±0.89 mg/mm compared to sham rats 10.82±0.39 114 mg/mm. The increase in costal mass at 3-day UDD result s in part from an increase in 115 longitudinal hypertrophy, with denervated costal diaphragm adding 933±301 serial sarcomeres 116 compared to sham animals (FIG.1E; p=0.018), whereas 3-day BDD rats showed no difference 117 in serial sarcomere number (7559±313 versus sham 7740±111). Thus, stretch and not 118 denervation triggers longitudinal hypertrophy. 119 120 To support titin ’s role as mechanosensor of stretch driven hypertrophy, we performed UDD on 121 Rbm20ΔRRM mice and Rbm20 ko rats. Rbm20 is an RNA splicing-factor for titin and both the 122 mouse and rat model generate larger, more compliant titin isoforms and should thus be less 123 sensitive to stretch. Rbm20 ΔRRM mice showed relatively less tissue mass increase compared to 124 wildtype (WT) 3, 6, or 12-days UDD (Supplemental FIG.2A), without changes in longitudinal 125 hypertrophy at 6-days UDD (Supplemental FIG.2B) suggesting the difference in mass is a result 126 from radial fiber growth, in agreement with our previous findings 21. Finally, we compared 127 denervated costal mass from Sprague Dawley (SD) and Rbm20 ko rats 3-day UDD, showing a 128 difference in mass increase of 24.4±3.8% in Rbm20 ko and 35.5±5.9% in SD rats ( p=0.02; 129 Supplemental FIG.2C ), supporting that stretch evokes hypertrophy in muscle and that titin 130 compliance modulates the extent of hypertrophy across species . Thus, muscle stretch in the 131 costal diaphragm is a potent trigger for longitudinal muscle hypertrophy, with titin being a 132 primary sensor. 133 134 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint Global transcriptome and proteome level studies reveal a distinct subset of genes and 135 proteins involved in UDD stretch hypertrophy. 136 To identify the cellular processes involved in stretch-based hypertrophy we performed both 137 transcriptome wide studies by RNA sequencing (RNAseq) as well as proteome studies by global 138 mass spectrometry (MS) on our rat 3-day sham, UDD and BDD samples. Initial studies focused 139 on separating the effect of denervation versus stretch. Principal component analysis of the top 140 500 genes showed close clustering of samples by group (Supplemental FIG.3A), suggesting 141 distinct gene programs are active between sham, UDD and BDD. At the protein level, principal 142 component analysis of the top 250 proteins suggests narrow separation of groups, predicting 143 overlap of the BDD and UDD samples (Supplemental FIG.3B). Gene expression studies 144 revealed very close overlap of BDD and UDD profiles with 77.15% overlap in differentially 145 expressed genes (DEG’s: padjSham (FIG.2B, left panel) and BDD>Sham (FIG.2B; middle panel) 147 showed the ~11.000 DEG’s (Supplemental data Tables 1-6) to be nearly equally distributed 148 between up and downregulated DEG’s . Direct comparison of UDD>BDD (FIG.2B, right panel) 149 revealed 850 DEG’s to be uniquely associated with UDD, of which 581 were upregulated 150 (Supplemental data Table 3). MS studies broadly agreed with the RNAseq data, showing UDD 151 and BDD share overlapping expression programs (FIG. 2A & C). Twenty percent of the 152 differentially expressed proteins (DEP’s ; PSham (FIG.2D, left panel) and BDD>Sham (FIG.2D, middle panel ) 154 showed 889 and 789 DEP’s, respectively, (Supplemental data Tables 7-12) which were 155 primarily increased. Comparing UDD>BDD revealed 173 DEP’s (FIG.2D, right panel) that are 156 unique to UDD. Most of DEP’s (and DEG’s) found are related to transcription, translation, 157 energetics and folding, with the highest fold DEP’s in rat UDD>BDD being: Med15 (15.5-fold), 158 Eif3i (6.0-fold) and Gtpbp3 (3.9-fold) (Supplemental data Table 9). GOterm enrichment of 159 UDD>BDD, separated by downregulated and upregulated DEG’s (Supplemental FIG.3C, left top 160 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint and bottom graph, respectively) , and DEP’s (Supplemental FIG.3C, right top and bottom graph, 161 respectively), indicated that upregulated DEG/DEP’s are primarily related to muscle 162 development and function consistent with an active hypertrophy program and downregulated 163 targets with metabolism and cellular respiration. To identify the pathways involved in 164 UDD>BDD, we searched the DEG’s against the KEGG PATHWAY database (Supplemental 165 data Table 6), confirm ing GOterm results and indicating that the DEG’s are involved in muscle 166 remodeling. Thus, UDD has a distinct expression profile from denervation (BDD), focused on 167 remodeling of muscle. 168 169 Exploring if titin-mechanosensing was active in either UDD or BDD we generated heatmaps of 170 titin-binding proteins based on RNAseq (Supplemental FIG.4) and global MS data (FIG.2E). To 171 narrow down proteins that were differential between UDD and BDD we tested (2-way ANOVA 172 pint<0.1) the z-score and found : MARP2 (Ankrd2; p=0.003), Cryab (p= 0.026), Csrp3 (p= 0.002), 173 Hsp90ab1 (p= 0.09), Hspb1 (p= 0.008) and Smyd2 (p= 0.004), to be unique to UDD . 174 Interestingly, the se DEP’s (MARP2, Smyd2, Hsp1b, Hsp90ab and Cryab) are established 175 binding partners of the N2A element of titin.22 176 177 Titin phosphorylation in UDD 178 With the rat data indicating changes in titin-associated signaling and titin stiffness modifying the 179 hypertrophy response in mice, we evaluated how UDD affects titin post-translationally. W e 180 performed MS on phosphorylation enriched peptides from 24-hour UDD diaphragm samples. 181 24-hour UDD was selected to study the early phosphorylation events . MS revealed 2870 182 phosphorylated peptides (p <0.05), of which 142-sites were in titin (~700-sites identified 183 including non -significant sites; Supplemental data Table 13). We opted to define titin 184 phosphorylation by analyzing the phosphorylation at the domain level to study if titin show ed 185 regional changes in phosphorylation (FIG.3A and Supplemental data Table 14), as total titin 186 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint phosphorylation (FIG.3B) was unchanged. Domain level analysis indicated titin phosphorylation 187 was primarily changing in the I-band. This motivated quantifying titin phosphorylation by 188 segment (FIG.3C and Supplemental data Table 15; following established naming conventions in 189 Kolmerer and Labeit1, and Bang et al23). Segmental analysis of titin (FIG.3C) revealed a marked 190 increase in phosphorylation of the N2A-element and I/A-junction, and a decrease in the Z/I-191 junction. Focusing on the increase in phosphorylation of the N2A-element, a known trophicity 192 signaling hub (reviewed in 22), we assessed the specific domain phosphorylation of the N2A-193 element (FIG.3C, highlight). In the N2A-element we found 7 phosphorylation sites (FIG.3D -E) of 194 which S9346 & S9350 are located in a linker sequence between I79 and I80, S9483 in I80, 195 S9459 & S9520 in the N2A unique sequence, S9654 in I81 and S9643 in I82). The 2 sites 196 located in the N2A unique sequence were significantly upregulated following UDD (FIG.3D-E). 197 pS9459 and pS9520 currently have no known function but could serve as a recruitment signal 198 (see discussion). 199 200 The MARP proteins regulate hypertrophy in UDD 201 The MARP1 and MARP2 proteins were strongly expressed both at the RNA level and protein 202 level following UDD (FIG.2E). MARPs have been shown to be important regulators of trophicity 203 and have been proposed as intermediaries between titin and trophic signaling pathways (see 204 discussion). As the MARPs share high homology and possible redundancy 7, we performed 6-205 day UDD on both single- and multi-KO combinations of the MARPs. We initially performed UDD 206 on the MARP triple KO mice (MARP t KO; knockout of Ankrd1, -2 and -23 genes) and found a 207 12% reduction in the hypertrophy response compared to WT (FIG.4A; p =0.0099). This reduction 208 in hypertrophy supports a potential link between titin-mechanosensing and MARP-based trophic 209 signaling. To test if one or a combination of MARPs caused the reduction in hypertrophy, we 210 performed 6-day UDD on MARP1 (Ankrd1), MARP2 (Ankrd2) and MARP3 (Ankrd23) KO mice 211 (FIG.4B-D) and double KO for MARP1/2, MARP1/3 and MARP2/3 (Supplemental FIG.6 ). 212 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint Whereas MARP1 KO mice did not show a difference in denervated costal diaphragm mass, 213 MARP2 KO mice showed a 14% increase (p=0.003) in mass and MARP3 KO mice showed a 214 13% reduction (p =0.004) in mass gain. These data suggest that MARP3 attenuat es 215 hypertrophy. However, all MARP double KO mice also presented with attenuated hypertrophy 216 (Supplemental FIG.6). This suggests that the separate MARP proteins could have distinct 217 functions in UDD, and there may be a dependency for two MARPs in regulating trophic 218 signaling, further discussed in the discussion section. 219 220 MARPs negatively regulate longitudinal hypertrophy 221 Prompted by the reduction in diaphragm mass gain during UDD in MARP tKO mice and by the 222 complexity of targeting all the single MARP KOs, we assessed longitudinal hypertrophy in 6-day 223 UDD MARP tKO samples . MARP tKO mice at baseline have fewer serial sarcomeres than WT 224 (2476±55 vs. 3157±69, respectively; p<0.0001), however 6-days UDD MARP t KO had similar 225 numbers of sarcomeres to WT, 4081±44 versus 4109±59 (FIG.5 A). These data show MARP 226 tKO mice add 653 ± 91. 6 more sarcomeres than wildtype mice (FIG.5B; 1605± 71 vs. 952±59 , 227 respectively; p<0.0001). To discern a possible mechanism of the MARPs inhibiting longitudinal 228 hypertrophy, we probed several candidate proteins of hypertrophy pathways that were 229 prominent in the MS datasets. Western blots for M apk1/3, Calcineurin, mT or, P70 s6k and 4E-230 bp1 were performed on costal diaphragms of WT and MARP tKO, 6-day Sham and UDD 231 animals. All samples were normalized to Gapdh and data were presented as relative to WT 232 sham (FIG.5C ; representative western blot images in FIG.5D). Mapk1 showed increases in 233 protein level that were comparable between WT and MARP tKO, whereas MAPK3 showed 234 preferential upregulation in MARP tKO (p= 0.0099). Calcineurin was unchanged following UDD , 235 note that in a t-test the WT mice show ed a significant increase in UDD . In WT mice, mTor 236 showed a striking increase in expression following 6-day UDD (p= 0.0006), whereas in MARP 237 tKO this was trending (P= 0.08). Downstream proteins of mTORC1; P70 s6k and 4E-bp 1 both 238 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint showed altered regulation, with P70 s6k being similarly upregulated in WT (p= 0.0005) versus 239 MARP tKO (p= 0.001), while 4E-bp1 was only significantly upregulated in MARP tKO (p= 0.033). 240 2way-ANOVA for the signaling proteins did not indicate specific pathway changes between WT 241 and MARP tKO. 242 243 mTor signaling regulates longitudinal hypertrophy in UDD 244 Motivated by the data from the mTor western blots in the MARP tKO mice and mT or being a 245 prominent regulator of skeletal muscle hypertrophy, we performed UDD in WT mice treated with 246 rapamycin, an inhibitor of mTORC1 signaling and skeletal muscle hypertrophy . The rat 247 transcriptome studies also indicated calcium signaling (Supplemental data Table 6) to be a 248 prominent pathway in UDD. To test the role of calcium signaling we included a second inhibitor; 249 cyclosporin A, an inhibitor of the calcineurin-NFAT pathway, another hypertrophy regulating 250 pathway. Mice received twice daily intraperitoneal injections of either rapamycin (2.5 251 mg/kg/day), cyclosporin A (25 mg/kg/day) or vehicle (DMSO), starting 3-days prior to surgery 252 until sacrifice of the mice (schematic in FIG.6A). Cyclosporin A treated mic e did not show a 253 change in hypertrophy (FIG.6 B) or serial sarcomere addition (FIG.6 C), indicating that the 254 calcineurin-NFAT pathway is not a primary mechanism for hypertrophy in UDD. Treatment did 255 not affect the innervated left costal diaphragm (FIG.6D ), body weight (FIG.6E) or tibia length 256 (FIG.6F). Interestingly, mice that received rapamycin displayed less hypertrophy of the 257 denervated costal diaphragm (FIG.6B; - 11.2%; p<0.001) compared to vehicle treated mice. 258 This attenuated hypertrophy response coincided with a reduction in serial sarcomere addition 259 (FIG.5C; - 8.5%; p<0.001) compared to vehicle. Note that following 3-day UDD, rapamycin 260 treated mice did not show significant increases in serial sarcomer es compared to untreated 261 sham animals (Δ67 ± 81 sarcomeres, versus vehicle treated mice Δ363 ± 79 sarcomeres). This 262 suggested rapamycin treatment almost completely inhibited longitudinal hypertrophy and that 263 mTORC1 signaling plays a vital role in longitudinal hypertrophy development. We thus propose 264 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint that mTORC1 signaling positively regulates longitudinal hypertrophy in skeletal muscle and that 265 titin’s N2A-element tunes the extent of longitudinal hypertrophy through the MARPs to prevent 266 excess hypertrophy (graphic summary in FIG. 6G). 267 268

269 270 Longitudinal hypertrophy as a mechanism for reducing stretch-induced mechanosensing 271 One of the most striking aspects of the UDD surgical model is the early transient hypertrophy. 272 No other muscle denervation model induces hypertrophy, supporting the notion that stretch, 273 even in inactive muscle, is a potent driver of hypertrophic growth. The extreme nature of the 274 stretch at work in UDD, ~25% stretch of the denervated costal by the innervated costal at a 275 frequency of 120-230 times a minute (respiration rate) 21, forms a potent trigger for muscle 276 hypertrophy. Following 6-days UDD the denervated costal diaphragm develops 49.7±10.0% 277 (FIG.1B) increase in mass , which is primarily caused by addition of 952±81 sarcomeres 278 (FIG.1C) and to a lesser extent by radial fiber growth 21. The transient nature of the hypertrophy 279 can be explained by the reduction in sarcomere strain due to the addition of sarcomeres in 280 series (longitudinal hypertrophy), removing the “trigger” that underlies the hypertrophy signaling. 281 This hypothesis fits the data (FIG.1B -C), where before remodeling UDD costal width (i.e., fiber 282 length) is ~8.7 mm (3000 sarcomeres x SL 2.9 µm), and when stretched 25% 21 equals a width 283 of ~10.8 mm. Following 6-days hypertrophic remodeling (UDD), costal width is ~10.8 mm (4000 284 sarcomeres x SL 2.7 µm). This suggests that the costal width increase attenuates the 285 hypertrophy trigger caused by stretching, finally resulting in atrophy (FIG.1B; 35-day UDD). 286 Denervation itself does not appear to induce hypertrophy of the diaphragm, as 3-day BDD in rat 287 showed no hypertrophy (FIG.1D -E). UDD being a denervation model shows that hypertrophy is 288 not necessarily dependent on a muscle’s ability to contract . This indicates that skeletal muscle 289 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint may derive its signals for hypertrophy during the muscle relaxation-phase when the muscle is at 290 its longest state. Muscle stretch in human is known to benefit muscle growth and is a vital part 291 of exercise routines (reviewed in 24,25). Perhaps muscle antagonism may provide sufficient 292 stretch to induce hypertrophy . As contracting muscles inadvertently stretch their relaxed 293 antagonists, they create an elegant feedback loop that promotes both muscle growth and the 294 maintenance of muscle mass. 295 296 Titin’s response to stretch 297 Posttranslational changes in titin, particularly phosphorylation, have been studied mostly in 298 relation to passive tension development 26–28. Specific PTMs for titin have not been widely 299 studied and only recently has ubiquitination been shown to recruit autophagic receptors to the 300 kinase domain of titin 29,30. Autophosphorylation of titin kinase at Y170 is considered one of the 301 classic mechanosensing responses in titin resulting in phosphorylation of the autophagic 302 receptor Nbr1 at S115/116, activating autophagy signaling in vitro 31. We did not observe any 303 signs following UDD that titin kinase was autophosphorylated at Y170. This could be related to 304 phospho-peptide abundance being below the detection limit, or that the titin kinase is inactive 32. 305 In total we identified ~700 phosphorylation sites in titin of which 1 42 were significantly affected 306 by stretch. These sites were distributed along the entire length of titin with several hot spots in 307 the PEVK region, likely involved in stiffness regulation, and several in the Z-disk, which could be 308 related to signaling or structural interactions. The sites in the N2A-element (FIG.3) were of 309 particular interest as previously it has been suggested that S9540 (S9895 according to 310 diaphragm RNAseq by Brynnel et al 20) could serve as a recruitment signal for MARP1 33, a 311 protein that is highly upregulated following UDD (FIG.2E). MARPs have been shown to quench 312 N2A phosphorylation33,34, which make the phosphorylation sites in the N2A segment tantalizing 313 targets for studying titin-MARP binding. If S9459 and S9520 (FIG.3D-E) play such a role 314 remains to be determined, but such insights could provide future avenues for manipulating titin-315 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint MARP interactions. Similarly, we found many titin-associated proteins to show differential 316 phosphorylation in UDD, including 3 sites in the MARPs (Supplemental FIG.5). How these sites 317 contribute to signaling or recruitment to the N2A-element remains to be seen but provide 318 tantalizing targets for follow-up study. 319 320 The MARP proteins and muscle trophicity 321 Global transcriptome and proteome studies from 3-day UDD show ed multiple titin-associated 322 proteins being upregulated (FIG.2E). W e focused our efforts on the MARP proteins, as they are 323 known interacting partners of titin’s N2A -element and have been shown to be important for 324 hypertrophy regulation in the heart 35,36. We used UDD on MARP KO mice, focusing on the triple 325 KO for MARP1-3 to account for expected redundancy 7,34. MARP tKO showed a 12% reduction 326 in hypertrophy following 6-day UDD (FIG.4A). This suggested stretch-mechanosensing 327 operated through the MARP proteins. In an attempt to isolate a single MARP protein as the 328 main effector, we performed UDD on single MARP knock-out mice (FIG.4B-D) . MARP1 KO did 329 not reveal changes in hypertrophy. However, we previously established that MARP1 localizes to 330 the N2A-segment of titin following UDD 21. We also independently determined that MARP1 331 cross-links titin to the thin filament to increase passive tension 37,38, suggesting MARP1 plays 332 mechanical roles over trophic regulation in skeletal muscle. MARP2 KO developed an 333 exaggerated hypertrophy and lastly MARP3 KO showed an attenuated hypertrophy response to 334 UDD, suggesting MARP2 and 3 play opposing roles in hypertrophy regulation. MARP2 interacts 335 with Akt2 13, providing a tentative link with the mT or pathway, discussed below. Additionally, 336 MARP2 interaction with P50-NFκB acting as an analogue for IκB 14 suggests a possible role in 337 inhibiting NFκB atrophy signaling. MARP3 is poorly understood but has been linked to glucose 338 metabolism15, forming another tentative link to mT or signaling39. To specifically determine if the 339 MARPs affected longitudinal hypertrophy we measured the number of serial sarcomeres across 340 the width of the costal diaphragm in MARP tKO and found that the tKO mice added 653.2 ± 341 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint 91.55 more sarcomeres than WT following 6-day UDD (FIG.5B). This suggest s that the MARP 342 proteins inhibit longitudinal hypertrophy. A possible mechanism could be that following stretch 343 the three MARPs bind and compete for the N2A- binding site7,34 resulting in translocation of 344 specific MARPs for signaling purposes. The benefit of MARP deletion in cardiomyopathy was 345 shown by Lange et al 10, where deletion of MARP1/2 ameliorated MLP KO induced DCM. MARP 346 KO has not proven detrimental in mice 40, making this family an attractive target for therapeutic 347 intervention. 348 349 mTor and longitudinal hypertrophy 350 Muscle hypertrophy is r egulated through a number of pathways, with the insulin -insulin growth 351 factor (IGF) mediated pathway 41–43 being the most well understood in skeletal muscle and the 352 calcineurin-NFAT pathway in cardiac muscle 44,45. With most studies focused on radial (cross-353 sectional) hypertrophy, we aimed to gain insight into the regulatory mechanism underlying 354 longitudinal hypertrophy, two types of hypertrophy that are not necessarily mutually exclusive. 355 The mT or signaling pathway was a prime candidate as UDD in MARP tKO mice suggested 356 altered mTor activity (FIG.5C-D). This prompted us to test inhibition of mTor signaling and see if 357 mTor regulates longitudinal hypertrophy. Using rapamycin 46, an inhibitor that targets the 358 mTORC1 (protein synthesis regulation) complex, we found a reduction in longitudinal 359 hypertrophy following 3-days UDD compared to vehicle treated mice (FIG.6B). This strongly 360 suggests that the mTORC1- pathway is in-part responsible for longitudinal hypertrophy following 361 UDD. Although we focused on mTor signaling in longitudinal hypertrophy development, we do 362 not exclude other pathways being important. mTOR formed an attractive target as previous 363 work showed that longitudinal stretch phosphorylates Akt and upregulates MARP2 47, forming a 364 tentative link between longitudinal stretch, MARPs and the mTor pathway. Further studies are 365 needed to establish the roles of the various pathways in stretch hypertrophy. 366 367 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint In conclusion, we found that the transient hypertrophy induced by UDD is dependent on muscle 368 fiber length, with longitudinal hypertrophy reducing the trigger for hypertrophy. The hypertrophy 369 coincides with increased phosphorylation of the N2A element . The N2A-associated MARP 370 proteins are strongly upregulated following UDD and deletion of the MARPs increases the 371 extent of longitudinal hypertrophy following UDD, indicating the MARPs serve roles in inhibiting 372 longitudinal hypertrophy. MARP tKO mice show altered regulation in the mTORC1 pathway 373 following UDD and inhibition of mTORC1 by rapamycin shows mTor is a main regulator of 374 longitudinal hypertrophy. 375 376 Funding 377 This work was financially supported by National Institutes of Health grants R01HL121500 (CO), 378 R01AR083233 (HG) and R35HL144998 (HG) 379 380

381 382 1. Labeit S, Kolmerer B. Titins: giant proteins in charge of muscle ultrastructure and elasticity. 383 Science. 1995;270(5234):293-296. doi:10.1126/science.270.5234.293 384 2. Horowits R, Kempner ES, Bisher ME, Podolsky RJ. A physiological role for titin and nebulin in 385 skeletal muscle. Nature. 1986;323(6084):160-164. doi:10.1038/323160a0 386 3. Swist S, Unger A, Li Y, et al. Maintenance of sarcomeric integrity in adult muscle cells cruciall y 387 depends on Z-disc anchored titin. Nat Commun. 2020;11(1):4479. doi:10.1038/s41467-020-388 18131-2 389 4. Ottenheijm CAC, van Hees HWH, Heunks LMA, Granzier H. Titin-based mechanosensing and 390 signaling: role in diaphragm atrophy during unloading? Am J Physiol Lung Cell Mol Physiol. 391 2011;300(2):L161-6. doi:10.1152/ajplung.00288.2010 392 5. van der Pijl RJ, Granzier HL, Ottenheijm CAC. Diaphragm contractile weakness due to reduced 393 mechanical loading: role of titin. Am J Physiol Cell Physiol. 2019;317(2):C167-C176. 394 doi:10.1152/ajpcell.00509.2018 395 6. Voelkel T, Linke WA. Conformation-regulated mechanosensory control via titin domains in 396 cardiac muscle. Pflugers Arch. 2011;462(1):143-154. doi:10.1007/s00424-011-0938-1 397 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint 7. Miller MK, Bang ML, Witt CC, et al. The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and 398 DARP as a family of titin filament-based stress response molecules. J Mol Biol. 2003;333(5):951-399 964. doi:10.1016/j.jmb.2003.09.012 400 8. Wette SG, Smith HK, Lamb GD, Murphy RM. Characterization of muscle ankyrin repeat proteins in 401 human skeletal muscle. Am J Physiol Cell Physiol. 2017;313(3):C327-C339. 402 doi:10.1152/ajpcell.00077.2017 403 9. Hayashi C, Ono Y, Doi N, et al. Multiple molecular interactions implicate the connectin/titin N2A 404 region as a modulating scaffold for p94/calpain 3 activity in skeletal muscle. Journal of Biological 405 Chemistry. 2008;283(21):14801-14814. doi:10.1074/jbc.M708262200 406 10. Lange S, Gehmlich K, Lun AS, et al. MLP and CARP are linked to chronic PKCalpha signalling in 407 dilated cardiomyopathy. Nat Commun. 2016;7:12120. doi:10.1038/ncomms12120 408 11. van der Pijl RJ, van den Berg M, van de Locht M, et al. Muscle ankyrin repeat protein 1 (MARP1) 409 locks titin to the sarcomeric thin filament and is a passive force regulator. Journal of General 410 Physiology. 2021;153(7). doi:10.1085/jgp.202112925 411 12. Zhou T, Fleming JR, Lange S, et al. Molecular Characterisation of Titin N2A and Its Binding of CARP 412 Reveals a Titin/Actin Cross-linking Mechanism. J Mol Biol. 2021;433(9). 413 doi:10.1016/j.jmb.2021.166901 414 13. Cenni V, Bavelloni A, Beretti F, et al. Ankrd2/ARPP is a novel Akt2 specific substrate and regulates 415 myogenic differentiation upon cellular exposure to H(2)O(2). Mol Biol Cell. 2011;22(16):2946-416 2956. doi:10.1091/mbc.E10-11-0928 417 14. Bean C, Verma NK, Yamamoto DL, et al. Ankrd2 is a modulator of NF-κB-mediated inflammatory 418 responses during muscle differentiation. Cell Death Dis. 2014;5(1):e1002. 419 doi:10.1038/cddis.2013.525 420 15. Shimoda Y, Matsuo K, Kitamura Y, et al. Diabetes-Related Ankyrin Repeat Protein 421 (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle. 422 PLoS One. 2015;10(9):e0138624. doi:10.1371/journal.pone.0138624 423 16. Laughlin MH, Yang HT, Tharp DL, Rector RS, Padilla J, Bowles DK. Vascular cell transcriptomic 424 changes to exercise training differ directionally along and between skeletal muscle arteriolar 425 trees. Microcirculation. 2017;24(2). doi:10.1111/micc.12336 426 17. Mayans O, van der Ven PF, Wilm M, et al. Structural basis for activation of the titin kinase domain 427 during myofibrillogenesis. Nature. 1998;395(6705):863-869. doi:10.1038/27603 428 18. Bertz M, Wilmanns M, Rief M. The titin-telethonin complex is a directed, superstable molecular 429 bond in the muscle Z-disk. Proc Natl Acad Sci U S A. 2009;106(32):13307-133310. 430 doi:10.1073/pnas.0902312106 431 19. van der Pijl RJ, Hudson B, Granzier-Nakajima T, et al. Deleting Titin’s C-Terminal PEVK Exons 432 Increases Passive Stiffness, Alters Splicing, and Induces Cross-Sectional and Longitudinal 433 Hypertrophy in Skeletal Muscle. Front Physiol. 2020;11:494. doi:10.3389/fphys.2020.00494 434 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint 20. Brynnel A, Hernandez Y, Kiss B, et al. Downsizing the molecular spring of the giant protein titin 435 reveals that skeletal muscle titin determines passive stiffness and drives longitudinal 436 hypertrophy. Elife. 2018;7:e40532. doi:10.7554/eLife.40532 437 21. van der Pijl R, Strom J, Conijn S, et al. Titin-based mechanosensing modulates muscle 438 hypertrophy. J Cachexia Sarcopenia Muscle. 2018;9(5):947-961. doi:10.1002/jcsm.12319 439 22. van der Pijl RJ, Domenighetti AA, Sheikh F, Ehler E, Ottenheijm CAC, Lange S. The titin N2B and 440 N2A regions: biomechanical and metabolic signaling hubs in cross-striated muscles. Biophys Rev. 441 2021;13(5):653-677. doi:10.1007/s12551-021-00836-3 442 23. Bang ML, Centner T, Fornoff F, et al. The complete gene sequence of titin, expression of an 443 unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-444 line to I-band linking system. Circ Res. 2001;89(11):1065-1072. doi:10.1161/hh2301.100981 445 24. Nunes JP, Schoenfeld BJ, Nakamura M, Ribeiro AS, Cunha PM, Cyrino ES. Does stretch training 446 induce muscle hypertrophy in humans? A review of the literature. Clin Physiol Funct Imaging. 447 2020;40(3):148-156. doi:10.1111/cpf.12622 448 25. Warneke K, Lohmann LH, Lima CD, et al. Physiology of Stretch-Mediated Hypertrophy and 449 Strength Increases: A Narrative Review. Sports Medicine. 2023;53(11):2055-2075. 450 doi:10.1007/s40279-023-01898-x 451 26. Hidalgo C, Granzier H. Tuning the molecular giant titin through phosphorylation: Role in health 452 and disease. Trends Cardiovasc Med. 2013;23(5):165-171. doi:10.1016/j.tcm.2012.10.005 453 27. Krüger M, Kötter S, Grützner A, et al. Protein kinase G modulates human myocardial passive 454 stiffness by phosphorylation of the titin springs. Circ Res. 2009;104(1):87-94. 455 doi:10.1161/CIRCRESAHA.108.184408 456 28. Loescher CM, Hobbach AJ, Linke WA. Titin (TTN): from molecule to modifications, mechanics, and 457 medical significance. Cardiovasc Res. 2022;118(14). doi:10.1093/cvr/cvab328 458 29. Müller E, Salcan S, Bongardt S, Barbosa DM, Krüger M, Kötter S. E3-ligase knock down revealed 459 differential titin degradation by autophagy and the ubiquitin proteasome system. Sci Rep. 460 2021;11(1). doi:10.1038/s41598-021-00618-7 461 30. Bogomolovas J, Fleming JR, Franke B, et al. Titin kinase ubiquitination aligns autophagy receptors 462 with mechanical signals in the sarcomere. EMBO Rep. 2021;22(10). 463 doi:10.15252/embr.201948018 464 31. Lange S, Xiang F, Yakovenko a, et al. The kinase domain of titin controls muscle gene expression 465 and protein turnover. Science …. 2005;308(June):1599-1603. doi:10.1126/science.1110463 466 32. Bogomolovas J, Gasch A, Simkovic F, Rigden DJ, Labeit S, Mayans O. Titin kinase is an inactive 467 pseudokinase scaffold that supports MuRF1 recruitment to the sarcomeric M-line. Open Biol. 468 2014;4(5):140041. doi:10.1098/rsob.140041 469 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint 33. Lanzicher T, Zhou T, Saripalli C, et al. Single-Molecule Force Spectroscopy on the N2A Element of 470 Titin: Effects of Phosphorylation and CARP. Front Physiol. 2020;11:173. 471 doi:10.3389/fphys.2020.00173 472 34. Lun AS, Chen J, Lange S. Probing Muscle Ankyrin-Repeat Protein (MARP) structure and function. 473 Anatomical Record. 2014;297(9):1615-1629. doi:10.1002/ar.22968 474 35. Zhong L, Chiusa M, Cadar AG, et al. Targeted inhibition of ANKRD1 disrupts sarcomeric ERK-475 GATA4 signal transduction and abrogates phenylephrine-induced cardiomyocyte hypertrophy. 476 Cardiovasc Res. 2015;106(2):261-271. doi:10.1093/cvr/cvv108 477 36. Lange S, Gehmlich K, Lun AS, et al. MLP and CARP are linked to chronic PKCα signalling in dilated 478 cardiomyopathy. Nat Commun. 2016;7:12120. doi:10.1038/ncomms12120 479 37. van der Pijl RJ, van den Berg M, van de Locht M, et al. Muscle ankyrin repeat protein 1 (MARP1) 480 locks titin to the sarcomeric thin filament and is a passive force regulator. Journal of General 481 Physiology. 2021;153(7). doi:10.1085/jgp.202112925 482 38. Zhou T, Fleming JR, Lange S, et al. Molecular Characterisation of Titin N2A and Its Binding of CARP 483 Reveals a Titin/Actin Cross-linking Mechanism. J Mol Biol. 2021;433(9). 484 doi:10.1016/j.jmb.2021.166901 485 39. Szwed A, Kim E, Jacinto E. Regulation and metabolic functions of mTORC1 and mTORC2. Physiol 486 Rev. 2021;101(3):1371-1426. doi:10.1152/physrev.00026.2020 487 40. Barash IA, Bang ML, Mathew L, Greaser ML, Chen J, Lieber RL. Structural and regulatory roles of 488 muscle ankyrin repeat protein family in skeletal muscle. Am J Physiol Cell Physiol. 489 2007;293(1):C218-C227. 490 41. Schiaffino S, Mammucari C. Regulation of skeletal muscle growth by the IGF1-Akt/PKB pathway: 491 insights from genetic models. Skelet Muscle. 2011;1(1):4. doi:10.1186/2044-5040-1-4 492 42. Goodman CA. Role of mTORC1 in mechanically induced increases in translation and skeletal 493 muscle mass. J Appl Physiol (1985). 2019;127(2):581-590. doi:10.1152/japplphysiol.01011.2018 494 43. Yoon MS. mTOR as a Key Regulator in Maintaining Skeletal Muscle Mass. Front Physiol. 495 2017;8:788. doi:10.3389/fphys.2017.00788 496 44. Molkentin JD. Calcineurin-NFAT signaling regulates the cardiac hypertrophic response 497 in coordination with the MAPKs. Cardiovasc Res. 2004;63(3):467-475. 498 doi:10.1016/j.cardiores.2004.01.021 499 45. Molkentin JD. Parsing good versus bad signaling pathways in the heart: role of calcineurin-500 nuclear factor of activated T-cells. Circ Res. 2013;113(1):16-19. 501 doi:10.1161/CIRCRESAHA.113.301667 502 46. Li J, Kim SG, Blenis J. Rapamycin: one drug, many effects. Cell Metab. 2014;19(3):373-379. 503 doi:10.1016/j.cmet.2014.01.001 504 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint 47. Mohamed JS, Lopez MA, Cox GA, Boriek AM. Anisotropic regulation of Ankrd2 gene expression in 505 skeletal muscle by mechanical stretch. The FASEB Journal. 2010;24(9):3330-3340. 506 doi:10.1096/fj.10-158386 507 48. Bang ML, Gu Y, Dalton ND, Peterson KL, Chien KR, Chen J. The muscle ankyrin repeat proteins 508 CARP, Ankrd2, and DARP are not essential for normal cardiac development and function at basal 509 conditions and in response to pressure overload. PLoS One. 2014;9(4):e93638. 510 doi:10.1371/journal.pone.0093638 511 49. Methawasin M, Hutchinson KR, Lee EJ, et al. Experimentally increasing titin compliance in a novel 512 mouse model attenuates the Frank-Starling mechanism but has a beneficial effect on diastole. 513 Circulation. 2014;129(19):1924-1936. doi:10.1161/CIRCULATIONAHA.113.005610 514 50. Li S, Guo W, Dewey CN, Greaser ML. Rbm20 regulates titin alternative splicing as a splicing 515 repressor. Nucleic Acids Res. 2013;41(4):2659-2672. doi:10.1093/nar/gks1362 516 51. Lindqvist J, van den Berg M, van der Pijl R, et al. Positive End-Expiratory Pressure Ventilation 517 Induces Longitudinal Atrophy in Diaphragm Fibers. Am J Respir Crit Care Med. 2018;198(4):472-518 485. doi:10.1164/rccm.201709-1917OC 519 52. Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 520 2013;29(1):15-21. doi:10.1093/bioinformatics/bts635 521 53. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq 522 data with DESeq2. Genome Biol. 2014;15(12):550. doi:10.1186/s13059-014-0550-8 523 54. Bang ML, Centner T, Fornoff F, et al. The Complete Gene Sequence of Titin, Expression of an 524 Unusual 700-kDa Titin Isoform, and Its Interaction With Obscurin Identify a Novel Z-Line to I-525 Band Linking System. Circ Res. 2001;89(11):1065-1072. doi:10.1161/hh2301.100981 526 55. Levine AA, Liktor-Busa E, Balasubramanian S, et al. Depletion of Endothelial-Derived 2-AG 527 Reduces Blood-Endothelial Barrier Integrity via Alteration of VE-Cadherin and the Phospho-528 Proteome. Int J Mol Sci. 2023;25(1):531. doi:10.3390/ijms25010531 529 56. Keresztes A, Olson K, Nguyen P, et al. Antagonism of the mu-delta opioid receptor heterodimer 530 enhances opioid antinociception by activating Src and calcium/calmodulin-dependent protein 531 kinase II signaling. Pain. 2022;163(1):146-158. doi:10.1097/j.pain.0000000000002320 532 57. Parker SS, Krantz J, Kwak EA, et al. Insulin Induces Microtubule Stabilization and Regulates the 533 Microtubule Plus-end Tracking Protein Network in Adipocytes. Mol Cell Proteomics. 534 2019;18(7):1363-1381. doi:10.1074/mcp.RA119.001450 535 58. Hulsen T, de Vlieg J, Alkema W. BioVenn – a web application for the comparison and visualization 536 of biological lists using area-proportional Venn diagrams. BMC Genomics. 2008;9(1). 537 doi:10.1186/1471-2164-9-488 538 59. Ge SX, Jung D, Yao R. ShinyGO: a graphical gene-set enrichment tool for animals and plants. 539 Bioinformatics. 2020;36(8). doi:10.1093/bioinformatics/btz931 540 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint 60. Tyanova S, Temu T, Sinitcyn P, et al. The Perseus computational platform for comprehensive 541 analysis of (prote)omics data. Nat Methods. 2016;13(9). doi:10.1038/nmeth.3901 542 61. Babicki S, Arndt D, Marcu A, et al. Heatmapper: web-enabled heat mapping for all. Nucleic Acids 543 Res. 2016;44(W1). doi:10.1093/nar/gkw419 544 545 546 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint

547 548 Animal studies 549 All experiments were done in accordance with the University of Arizona Institutional Animal 550 Care and Use Committee and followed the US National Institutes of Health Using Animals in 551 Intramural Research guidelines for animal use. We used 3-month-old C57BL/6J mice referred to 552 as wildtype (WT), and 6-month-old Sprague Dawley rats (SD). Knockout models for titin binding 553 proteins MARP1-3 (Ankrd1, Ankrd2 and Ankrd23)10,40,48 were kindly provided by Dr Ju Chen and 554 Dr Stephan Lange. Homozygous Rbm20 ΔRRM mice 21,49 and Rbm20 ko rats 50,51, have previously 555 been described. Mice were maintained on a C57BL/6J background, with the data from the 556 MARP mice being on a black swiss background. 557 558 Surgical procedure 559 For unilateral diaphragm surgery (UDD) 21 or bilateral diaphragm denervation (BDD) studies, 560 mice or rats were anaesthetized with 2-3% isoflurane and a small incision was made in the neck 561 area just above the clavicle. The right phrenic nerve was isolated behind the sternohyoid 562 muscle, and a 3–4 mm section was transected at the height of the supraclavicular nerve branch. 563 For BDD surgery both left, and right phrenic nerves being transected. Sham operated animals 564 underwent the same procedure, except the phrenic nerve was left intact. Animals were 565 sacrificed 1, 3, 6, 12 or 35-days after surgery for morphometric analysis and tissue harvest. 566 567 Pharmacological inhibition of hypertrophy 568 Inhibitor studies with rapamycin (mTOR inhibitor) and cyclosporin A (Calcineurin inhibitor) were 569 performed by injecting mice, twice daily intra-peritoneal (IP), with 2.5 or 25 mg/kg/day, 570 respectively. Each inhibitor was dissolved in Dimethylsulfoxide (DMSO) and diluted to 20% 571 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint DMSO with saline solution just before injection. Mice received inhibitors or vehicle (20% DMSO 572 in saline) starting 3-days prior to UDD surgery to prime the mice and continued following UDD to 573 inhibit hypertrophy growth (FIG.4C). Mice were sacrificed after 3-days UDD and processed for 574 serial sarcomere measurements as described below. 575 576 Serial sarcomere measurements 577 Previously described in van der Pijl et al 21. Briefly, mice were anaesthetized with a 140/10 578 mg/kg ketamine/xylazine solution, a small incision to visualize the jugular vein, which was 579 subsequently cannulated for perfusion. Mice were perfused with a solution consisting of 4% 580 formaldehyde, with 70 U/mL of heparin in phosphate buffered saline (PBS), after which the 581 diaphragm was removed and stored in 4% formaldehyde in PBS overnight for complete fixation. 582 Full length diaphragm midcostal strips were gently dissected and flattened between glass slides, 583 costal width was measured using a caliper and sarcomere lengths were measured using a 584 He/Ne laser diffraction system. 585 Alternatively, muscle fiber bundels from chemically demembranated full length 586 diaphragm midcostal strips of 3-day mice were flattened between glass slides. Costal width and 587 sarcomere lengths were measured using a Zeiss Axio Imager M1 microscope (Zeiss), at ×50 588 magnification to measure the length of the muscle bundles and ×640 for sarcomere length along 589 four points of the fiber bundles. Images were captured using AxioCam MRc with Axiovision 590 software (Zeiss) and images were calibrated using a 0.01 mm stage micrometre (Edmund 591 Optics). To determine the number of serial sarcomeres the muscle bundle length (costal width) 592 was divided by the sarcomere length. 593 Demembranating solution consisted of a relaxing solution (in mM; 20 BES, 10 EGTA, 594 6.56 MgCl2, 5.88 NaATP, 1 DTT, 46.35 K ‐propionate, 15 creatine phosphate, pH 7.0), with 1% 595 Triton‐X‐100 at 4°C, and protease inhibitors (phenylmethylsulfonyl fluoride (PMSF), 0.25 mM; 596 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint leupeptin, 0.04 mM; E64, 0.01 mM, ), after demembranation, samples were stored in just 597 relaxing solution plus inhibitors (without triton X-100) at 4°C. 598 599 Transcriptome studies 600 RNA sequencing (RNAseq) was performed on right costal diaphragm samples collected from 3-601 day sham and UDD animals and flash frozen in liquid nitrogen until further processing. For RNA 602 extraction, samples were incubated overnight in RNAlater-ICE (Thermo Scientific) and 603 subsequently transferred to RLT buffer for extraction according to the RNeasy Fibrous Tissue 604 Mini Kit (Qiagen). Tissue disruption was achieved using a Bullet Blender (Next Advance) and 605 Green Eppendorf lysis kit tubes (Next Advance), by grinding samples for 4 minutes at setting 606 10. Thereafter, total RNA extraction was performed following the RNeasy Fibrous Tissue Mini 607 Kit’s instr uctions and quantified using a Nanodrop ND-1000 spectrophotometer (Thermo 608 Scientific). Each sample consisted of 3 biological replicate sham or UDD samples. Both Library 609 preparation and sequencing was performed by the University of Chicago Genomics Facility , 610 Chicago, USA. Briefly, library preparation: rRNA was depleted from RNA preparations from 1 µg 611 total RNA. Libraries were prepared using an RNA Library Prep Kit from Illumina following the 612 manufacturer’s instructions. Sequencing was performed on an Illumin a Hiseq2500 sequencer 613 using 100 bp paired-end sequencing. For RNAseq analysis see 20. Briefly, Adapters and low-614 quality reads were removed with Trim Galore and reads were mapped to the rat genome 615 (Release mRatBN7.2) using STAR 52 with default settings. Differentially expressed genes were 616 determined with DESeq2 53. Genes with population adjusted p-values (p adj) <0.05 were 617 considered differentially expressed. For titin splicing, percent spliced in index (PSI) was 618 calculated as a measure for determining if an exon is spliced in, following the titin exon 619 annotation by Bang et al.54 620 621 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint Proteome studies 622 Preparation of muscle for mass spectrometry analysis 623 Diaphragm muscle was ground to a fine powder using Dounce homogenizers cooled in liquid 624 nitrogen and acclimated to –20°C for 30 min before continuing. Tissue powder was 625 resuspended at a concentration of 50 mg/ml in a Urea buffer (4 M urea, 1 M thiourea, 25 m M 626 Tris–HCl, 75 mM dithiothreitol, 1.5 % SDS, 25 % glycerol, pH 6.8) with protease inhibitors (0.04 627 mM E‐64, 0.16 mM leupeptin, and 0.2 mM PMSF). The solution was mixed for 4 min, followed 628 by 10 min of incubation at 60°C. Samples were centrifuged at 12.000 rpm and the supernatant 629 flash frozen for storage at −80°C. 630 631 In-solution Tryptic Digestion 632 50 µg of rat costal diaphragm lysate was subjected to acetone precipitation by adding six times 633 the sample volume of pre-chilled 100 % acetone and incubated one hour at -20°C. The 634 precipitates were centrifuged at 16,000 x g for 10 minutes at 4°C and the acetone was removed. 635 400 µL of pre-chilled 90% acetone was added to the protein pellet, briefly vortexed and 636 centrifuged at 16,000 x g for 5 minutes at 4°C. The remaining acetone was removed, the protein 637 pellets were air dried for 3 minutes, resuspended in 100 µL of 50 mM NH 4HCO3 and sonicated 638 for 5 minutes. The samples were supplemented with dithiothreitol (DTT) at a final concentration 639 of 5 mM and incubated at 70°C for 30 minutes. Samples were cooled to room temperature for 640 10 minutes and incubated with 15 mM acrylamide for 30 minutes at room temperature while 641 protected from light. The reaction was quenched with DTT with a final concentration of 5 mM 642 and incubated in the dark for 15 minutes. One µg of Lys-C was added to each sample and 643 incubated at 37° C for 2-3 hours while shaking at 300 rpm followed by the addition of 50 µL of 644 50 mM ammonium bicarbonate and 2 µg of trypsin and incubation overnight at 37°C while 645 shaking at 300 rpm. 14.7 µL of 40 % FA/1 % HFBA was added to each sample and incubated 646 for 10 minutes (final concentration is 4 % FA/0.1 % HFBA) to stop trypsin digestion. The 647 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint samples were desalted with Pierce Peptide Desalting Spin Columns per the manufacturer’s 648 protocol (ThermoFisher Scientific, cat no. 89852) and the peptides were dried by vacuum 649 centrifugation. The dried peptides were resuspended in 20 µL of 0.1 % FA (v/v) and the peptide 650 concentration was determined with the Pierce Quantitative Colorimetric Peptide Assay Kit per 651 the manufacturer’s protocol (ThermoFisher Scienti fic, cat no. 23275). 350 ng of the final sample 652 was analyzed by mass spectrometry. 653 654 Phosphoproteomics 655 To determine global differences in protein phosphorylation abundance between sham or UDD, 1 656 mL of protein lysate corresponding to 50 mg diaphragm per sample (pooled costal diaphragm of 657 2 mice) was subjected to in-solution tryptic digestion and phosphopeptide enrichment using 658 sequential enrichment from metal oxide affinity chromatography per manufacturer’s protocol 659 (Thermo Scientific, cat no. A32993 & A32992) similar to as previously described 55,56. The dried 660 peptides were resuspended in 20 µL of 0.1 % FA (v/v) and the peptide concentration was 661 determined with the Pierce Quantitative Colorimetric Peptide Assay Kit per the manufacturer’s 662 protocol. 350 ng of the final sample was then analyzed by mass spectrometry. 663 664 Mass Spectrometry 665 HPLC-ESI-MS/MS was performed in positive ion mode on a Thermo Scientific Orbitrap Fusion 666 Lumos tribrid mass spectrometer fitted with an EASY-Spray Source (Thermo Scientific, San 667 Jose, CA). NanoLC was performed using a Thermo Scientific UltiMate 3000 RSLCnano System 668 with an EASY Spr ay C18 LC column (Thermo Scientific, 50cm x 75 μm inner diameter, packed 669 with PepMap RSLC C18 material, 2 µm, cat. # ES803); loading phase for 15 min at 0.300 670 µL/min; mobile phase, linear gradient of 1 –34 671 by a step to 95% Buffer B over 4 min at 0.220 µL/min, hold 5 min at 0.250 µL/min, and then a 672 step to 1 % Buffer B over 5 min at 0.250 µL/min and a final hold for 10 min (total run 159 min); 673 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint Buffer A = 0.1 % FA/H 2O; Buffer B = 0.1 % FA in 80 % ACN. All solvents were liquid 674 chromatography mass spectrometry grade. Spectra were acquired using XCalibur, version 2.3 675 (ThermoFisher Scientific). A “TopSpeed” data -dependent MS/MS analysis was performed 676 (acquisition of a full scan spectrum followed by collision-induced dissociation mass spectra of 677 the Top N most intense precursor ions within the 3 second cycle time). Dynamic exclusion was 678 enabled with a repeat count of 1, a repeat duration of 30 seconds, an exclusion list size of 500, 679 and an exclusion duration of 40 seconds. 680 681 Label-free Quantitative Proteomics 682 Progenesis QI for proteomics software (version 2.4, Nonlinear Dynamics Ltd., Newcastle upon 683 Tyne, UK) was used to perform ion-intensity based label-free quantification as previously 684 described57. In brief, in an automated format, raw files were imported and converted into two-685 dimensional maps (y-axis = time, x-axis =m/z) followed by selection of a reference run for 686 alignment purposes. An aggregate data set containing all peak information from all samples was 687 created from the aligned runs, which was then further narrowed down by selecting only +2, +3, 688 and +4 charged ions for further analysis. The samples were then grouped according to 689 treatment. Peak lists of the top ten fragment ion spectra were exported in Mascot generic file 690 (mgf) format and searched against either the 2020_06 Swiss-Prot Rattus norvegicus database 691 (8128 entries) , the 2018_11 Swiss-Prot Mus musculus database (17008 entries), or species 692 respective TrEMBL databases using Mascot (Matrix Science, London, UK; version 2.6.0). The 693 search variables that were used were: 10 ppm mass tolerance for precursor ion masses and 0.5 694 Da for product ion masses; digestion with trypsin; a maximum of two missed tryptic cleavages; 695 variable modifications of oxidation of methionine, phosphorylation of serine, threonine, and 696 tyrosine, and carbamidomethylation of cysteine; 13C = 1. The resulting Mascot .xml file was 697 then imported into Progenesis, allowing for peptide/protein assignment, while peptides with a 698 Mascot Ion Score of <25 were not considered for further analysis. Abundances were normalized 699 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint to the total ion current (TIC) to correct for differences in sample loading and instrument 700 response. For quantification, proteins must have possessed at least one or more unique, 701 identifying peptide. 702 703 Visualization and analysis of transcriptome and proteome data 704 Figures were generated using online resources, briefly, quantitative Venn diagrams were made 705 using https://www.biovenn.nl/58. Lolipop graphs of gene onthology (GO) enritchment analysis by 706 ShinyGO v0.75 http://bioinformatics.sdstate.edu/go/ 59. Heatmaps were generated using a 707 combination of Graphpad Prism v9.1, Perseus v2.0.3.1 60 and Heatmapper 708 (http://heatmapper.ca/)61. Perseus v2.0.3.1 was also used to analyze principal components and 709 visualized using Graphpad Prism v9.1. 710 711 Western blot 712 Western blot experiments previously described in van der Pijl et al 21. Proteins were transferred 713 onto Immobilon-P PVDF 0.45 μm membranes (Millipore) using semi dry transfer (Bio-Rad). 714 Membranes were blocked with Odyssey blocking buffer (Li-Cor Biosciences) for 1 hour, and 715 subsequently probed with primary antibodies at 4°C overnight (See Table 1). Near Infra-Red 716 dyes were used as secondary antibodies for detection with Odyssey CLx Imaging System (Li-717 Cor Biosciences, United states). 718 719 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint 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 Sci. 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 Table 1 Antibodies used in this study 720 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint FIGURE 1. Transient hypertrophy following UDD in the denervated costal diaphragm. (A) 721 schematic of how UDD affects sarcomere length in the denervated costal (left panel) and how 722 stretch extends titin for mechanosensing (right panel, generated through BioRender). (B ) 723 Mouse diaphragm costal weight normalized to tibial length following 1, 3, 6, 12 and 35-days of 724 UDD, showing the hypertrophy phase peaking at 6-days and progressing to the atrophy phase 725 at 12-days post-UDD (n=6-22, shams grouped for simplicity). A substantial part of the 726 hypertrophy encompasses longitudinal hypertrophy (C), lengthening of the muscle fibers by 727 addition of serial sarcomeres (n=10 -13). The increased fiber length likely reduces the stretch-728 based hypertrophy signaling and thus explains the transient nature of hypertrophy. (D) 3-day 729 BDD in rats (n=6-9) confirms stretch is the trigger for inducing hypertrophy in UDD at the tissue 730 mass level (D; diaphragm right costal normalized to tibial length, denervated in UDD) and serial 731 sarcomeres level (E). One-way ANOVA, with Tukey post-hoc testing. 732 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint FIGURE 2. Global transcriptomics and proteomics following 3-days UDD and BDD in rats. 733 Global transcript studies by RNAseq of sham, UDD and BDD right costal diaphragm 734 (n=5/group). Same parameters apply to the global proteome studies (C-D) with mass 735 spectrometry. Quantitative Venn diagrams of the transcriptome (A) showing overlap gene 736 regulation between UDD and BDD. Vulcano plots of UDD (B, left) and BDD (B, middle) showed 737 similar gene regulation. Comparing UDD to BDD directly revealed just 850 differentially 738 regulated genes (B, right) indicating a small subset being responsible for hypertrophy regulation. 739 Quantitative Venn diagrams of the proteome (C) showed similar regulation compared to 740 transcriptome. Volcano plots of UDD (D, left) and BDD (D, middle) showed primarily 741 upregulation of proteins. Comparing UDD to BDD directly revealed just 173 differentially 742 regulated proteins (D, right). Green-dots: upregulated genes/proteins, red-dots: downregulated 743 genes/proteins. Titin-associated proteins in heatmap of proteome (E; Z-score: red= upregulated, 744 blue= downregulated) and violin plots (right panel) of differential proteins between UDD (red) 745 and BDD (blue), indicating upregulation of titin-associate proteins following stretch. 2-way 746 ANOVA pint: *p<0.05, **P<0.01. 747 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint FIGURE 3. Phosphorylation of titin following 24-hours of UDD by mass spectrometry 748 (n=4/group). Phosphorylation of individual titin domains (A) relative Z-score (log2) of titin 749 phosphorylation showing domain specific changes in phosphorylation. Total phosphorylation of 750 titin (B) is not affected by UDD, however titin showed regional changes in phosphorylation (C), 751 notably increased phosphorylation of the N2A-element (boxed). Quantitation of the 752 phosphorylation signal for the 5 main sites found in the N2A-element (D). (E) Schematic of the 753 N2A element with the 2 pSer found in the N2Aus (Transcript: ENSMUST00000099981.10 Ttn-754 203). Red: Ig domain coding and blue: unique sequence coding. Mouse titin phosphorylation 755 and global mass spectrometry was analyzed by t-test, Kolmogorov-Smirnov test or multiple t-756 test with a cut-off at P<0.05. 757 758 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint FIGURE 4. 6-day UDD on KO mice of MARP proteins. MARP triple KOs show a reduced 759 response to UDD (A; p<0.01). No effect of single MARP1 KO (B) on UDD, increased 760 hypertrophy following MARP2 KO (B; p<0.01; t-test), indicating possible roles in hypertrophy 761 suppression or atrophy signaling and MARP3 KO (C) showed baseline hypertrophy in costal 762 diaphragm in addition to less hypertrophy development in UDD (p<0.01; t-test) compared to 763 WT, implying roles as a suppressor of hypertrophy. Left panel, diaphragm right costal mass 764 normalized to tibial length and right panel, percentual increase in right costal mass relative to 765 sham. S= Sham, U= UDD (n=10-12). Statistical testing by t-test or two-way ANOVA with Tukey 766 post-hoc testing. 767 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint FIGURE 5. MARPs inhibits longitudinal hypertrophy. Longitudinal hypertrophy measured in right 768 costal strips of WT and MARP tKO mice in 6-da y sham and UDD mice (A). Numerical increase 769 in serial sarcomeres is higher in MARP tKO (P<0.0001) mice compared to WT mice (B; n=7-12), 770 suggesting that the MARPs inhibits longitudinal growth. Probing hypertrophy signaling by 771 western blot, normalized to Gapdh, with expression set relative to WT sham levels (C; n=8-12). 772 Differential mTor response suggests role in regulating longitudinal hypertrophy. (D) 773 Representative blot images of the signaling proteins. Statistical testing by one-way or two-way 774 ANOVA with Tukey post-hoc testing. 775 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint FIGURE 6. Pharmacological inhibition of the mTor (rapamycin) and calcium (cyclosporin A) 776 based hypertrophy pathways revealed mTor to be involved in longitudinal hypertrophy. (A) 777 Schematic of the inhibition protocol, showing mice were injected with inhibitors for 3-days prior 778 to receiving UDD surgery, with continued twice daily dosing of inhibitors until sacrifice at day 3 779 post-UDD. Rapamycin inhibited hypertrophy development both at the costal diaphragm mass 780 level (B; p<0.001) and at the longitudinal hypertrophy level (C; p<0.001), whereas cyclosporine 781 A had no effect. Neither cyclosporine A or rapamycin affected the innervated costal diaphragm 782 (D), or body mass (E) and all mice used were of approximately the same size based on skeletal 783 size, as measured by tibia length (F). (G) Hypothetical mechanism for longitudinal hypertrophy 784 following muscle stretch. The mTorc1 pathway is activated by stretch and initiates longitudinal 785 muscle hypertrophy. MARP proteins sequestered by titin’s N2A element are released upon 786 stretch and tunes the longitudinal hypertrophy, thus preventing excessive longitudinal 787 hypertrophy (Image was generated through BioRender). N=8-10/group, statistical testing by 1-788 way-ANOVA and Dunnett's multiple comparisons test. 789 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint 790 791 792 793 794 795 796 797 Supplemental figures 798 799 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint S FIGURE 1. 3-days bilateral diaphragm denervation in rats showed similar body weights 800 compared to sham animals (A; n=6-9/group) and were of similar size based on tibia length (B) 801 and soleus muscle weights (C). Statistical testing by one-way ANOVA and Dunnett's multiple 802 comparisons test. 803 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint S FIGURE 2. Role of titin stiffness on hypertrophy following UDD. (A) Transient hypertrophy 804 response in Rbm20 ΔRRM mice (more compliant titin) showing a blunted hypertrophy response 805 compared to WT mice, based on percent increase of diaphragm right costal mass relative to 806 sham (n=10-12). (B) Titin-based stiffness does not alter longitudinal hypertrophy response, as 807 both WT and Rbm20 ΔRRM mice show a similar increase in serial sarcomeres following 6-days 808 UDD. Rbm20-KO rat response to 3-days UDD, based on percent increase of diaphragm right 809 costal mass relative to sham (mouse n=10-11, rat n=8) supporting titin-based stiffness 810 regulating muscle hypertrophy similarly across species. Statistical testing by t-test. 811 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint S FIGURE 3. Principal component analysis of the rat 3-day UDD and BDD transcriptome (A) 812 and proteome (B), showing clear separation of groups at the transcript level and overlap of BDD 813 and UDD samples at the protein level. GOterm enrichment of UDD>BDD separated by up- or 814 down-regulated transcriptomes and proteome (C, left and right, respectively) show distinct, yet 815 overlapping cellular processes. Global mass spectrometry was analyzed by ANOVA and 816 corrected for multiple comparisons with false discovery rate with a cut-off at p<0.05. 817 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint S FIGURE 4. Transcriptome regulation of titin-associated and myofilament genes by RNAseq in 818 rats following 3-days of UDD/BDD. Heatmaps showing similar regulation between UDD and 819 BDD samples (n=4-5; Z-score: red= upregulated, blue= downregulated) at the transcript level 820 for titin-associated and myofilament genes, based on hierarchal clustering. 821 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint 822 S FIGURE 5. Titin N2A associated protein phosphorylation events at 24-hour UDD. Violin plots 823 of phosphorylation events in N2A-associated proteins following UDD: MARP1 (Transcript: 824 ENSMUST00000237142.2 Ankrd1-205), MARP2 (Transcript: ENSMUST00000026172.3 825 Ankrd2-201), Smyd2 (Transcript: ENSMUST00000027897.8 Smyd2-201), Capn3 ( Transcript: 826 ENSMUST00000028749.15 Capn3-202), Hsp90ab (Transcript: ENSMUST00000024739.14 827 Hsp90ab1-201), Mypn (Transcript: ENSMUST00000095580.3 Mypn-201), Hspb1 (Transcript: 828 ENSMUST00000005077.7 Hspb1-201), Cryab (Transcript: ENSMUST00000217475.2 Cryab-829 206) and Prkca/PKA (Transcript: ENSMUST00000005606.8 Prkaca-201 ). Data represented as 830 Log2 of the normalized abundance with significance determined by Kolmogorov-Smirnov test. 831 -2 -1 0 1 2 3 MARP1 S16 Normalized abundance (Z-score) ✱✱✱✱ -3 -2 -1 0 1 2 3 MARP2 S29 ✱ -3 -2 -1 0 1 2 3 MARP2 S36 ✱ -3 -2 -1 0 1 2 Smyd2 S283 ✱✱✱ -3 -2 -1 0 1 2 Smyd2 S284 ✱✱✱ -2 -1 0 1 2 Capn3 S6 ✱✱✱ -2 -1 0 1 2 3 Capn3 T8 ✱ -2 -1 0 1 2 Capn3 S19 ✱✱✱ -2 0 2 4 Hsp90ab S226 Normalized abundance (Z-score) ✱ -2 -1 0 1 2 3 4 Hsp90ab S255 ✱✱ -2 -1 0 1 2 3 Hspb1 S13 ✱✱✱✱ -2 -1 0 1 2 3 4 Hspb1 S15 ✱✱✱✱ -2 -1 0 1 2 3 Hspb1 S86 ✱✱✱ -2 -1 0 1 2 3 Hspb1 S102 ✱ -2 -1 0 1 2 3 Hspb1 T155 ✱ -2 -1 0 1 2 3 Hspb1 S158 Normalized abundance (Z-score) ✱ -2 -1 0 1 2 3 Hspb1 S159 ✱ -2 -1 0 1 2 3 Hspb1 S160 ✱ -2 -1 0 1 2 3 Hspb1 S162 ✱ -2 -1 0 1 2 3 Hspb1 S180 ✱✱✱✱ -2 -1 0 1 2 3 Cryab S59 ✱✱✱✱ -2 -1 0 1 2 3 Cryab T63 ✱✱✱ -1 0 1 2 3 4 Cryab S66 ✱✱✱ -2 -1 0 1 2 3 Mypn S194 ✱✱✱ Sham UDD -2 -1 0 1 2 3 4 Mypn S248 Normalized abundance (Z-score) ✱✱✱✱ Sham UDD -2 -1 0 1 2 3 Mypn T249 ✱✱✱✱ Sham UDD -2 -1 0 1 2 Mypn S252 ✱ Sham UDD -2 -1 0 1 2 3 4 Mypn S253 ✱✱✱✱ Sham UDD -2 -1 0 1 2 3 Mypn S255 ✱✱✱✱ Sham UDD -2 -1 0 1 2 3 Mypn S256 ✱✱✱✱ Sham UDD -2 -1 0 1 2 Mypn S903 ✱ Sham UDD -4 -2 0 2 4 PKA S339 ✱✱✱ .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint S FIGURE 6. 6-day UDD on double KO mice of MARPs. Double KO of MARP1/2 (A), MARP1/3 832 (B) and MARP2/3 (C) all showed a reduction in hypertrophy following UDD, suggesting 833 redundancy between the MARPs. Left panel, diaphragm right costal mass normalized to tibial 834 length and right panel, percentual increase in right costal mass relative to sham. S= Sham, U= 835 UDD (n=10-12). 836 837 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint