{"paper_id":"19a463ef-6488-43e9-bda3-5f8673f4ae95","body_text":"The titin N2A-MARP signalosome constrains muscle longitudinal hypertrophy in 1 \nresponse to stretch 2 \nRobbert van der Pijl 1,2, Jochen Gohlke 1, Josh ua Strom 1, Eva Peters 1, Shengyi Shen 1, Stefan 3 \nConijn2, Zaynab Hourani 1, Stephan Lange 3,4, Ju Chen 3, Paul Langlais 5, Siegfried Labeit 6, Henk 4 \nGranzier1, Coen Ottenheijm1,2. 5 \n 6 \n1 Department of Cellular and Molecular Medicine, University of Arizona, Tucson, United States   7 \n2 Department of Physiology, Amsterdam University medical center, location VUMC, Amsterdam, 8 \nthe Netherlands 9 \n3 School of Medicine, University of California San Diego, San Diego, United States 10 \n4 Department of Biomedicine, Aarhus University, Aarhus, Denmark 11 \n5 Department of Endocrinology, University of Arizona, Tucson, United States 12 \n6 Department of Integrative Pathophysiology, Medical Faculty Mannheim, Mannheim, Germany 13 \n 14 \n 15 \n 16 \nCorresponding author: 17 \nCoen Ottenheijm, PhD 18 \nCellular and Molecular Medicine 19 \nUniversity of Arizona, Tucson, USA  20 \nPhone: 520-626-4198 21 \ncoeno@arizona.edu  22 \nc.ottenheijm@amsterdamumc.nl 23 \n 24 \n 25 \n  26 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nABSTRACT 27 \nTitin-based mechanosensing is a key driver of trophic signaling in muscle, yet the downstream 28 \npathways linking titin sensing to muscle remodeling remain poorly understood. To investigate 29 \nthese signaling mechanisms, we utilized unilateral diaphragm denervation (UDD), an in vivo 30 \nmodel that induces titin-stiffness-dependent hypertrophy via mechanical stretch. Using UDD in 31 \nrats and mice, we characterized the longitudinal hypertrophic response and distinguished 32 \nstretch-induced signaling from denervation effects by performing global transcriptomic and 33 \nproteomic analyses following UDD and bilateral diaphragm denervation (BDD) in rats. Our 34 \nfindings identified upregulation of titin-associated muscle ankyrin repeat proteins (MARPs). 35 \nSubsequent phosphorylation enrichment mass spectrometry in mouse diaphragm highlighted 36 \nthe involvement of the N2A-element. UDD in MARP knockout (KO) mice resulted in enhanced 37 \nlongitudinal hypertrophy, with Western blot analysis revealing activation of the mTOR pathway. 38 \nFurthermore, pharmaco logical inhibition of mTORC1 with rapamycin suppressed longitudinal 39 \nhypertrophy, demonstrating that mTOR signaling regulates titin-mediated hypertrophic growth in 40 \na MARP-dependent manner. These findings establish MARPs as key modulators of titin-based 41 \nmechanotransduction and highlight mTORC1 as a central regulator of longitudinal muscle 42 \nhypertrophy. 43 \n 44 \nKEYWORDS 45 \nHypertrophy, Muscle, Signaling, Mechanosensing, Titin, MARP, mTOR 46 \n  47 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nINTRODUCTION [401 WORDS] 48 \nTitin is a giant protein in striated muscle and forms an elastic filament in sarcomeres 1, the 49 \nsmallest contractile unit in muscle. One of the functions attributed to titin is to generate passive 50 \ntension in muscle 2,3, ensuring optimal overlap of the actin-based thin filaments and myosin-51 \nbased thick filaments for muscle contraction. The elastic properties of titin combined with its 52 \narrangement in thesarcomeres, spanning the half-sarcomer, make titin an ideal stress sensor. 53 \nThe mechanosensory properties of titin have been extensively described 4–6 and appear to 54 \nlocalize to several signaling hotspots on titin.  55 \nIn skeletal muscle, the N2A element is the prevalent titin region associated with hypertrophy 56 \nsignaling. The N2A element is known for its interaction with the muscle ankyrin repeat proteins 57 \n(MARP1-3)7,8 and calpain3 9. All MARPs have been implicated in trophicity signaling, with 58 \nredundancy between the MARP proteins 7,10. MARP1 tethers titin to the thin filament, forming a 59 \nmechanism for increasing passive tension 11,12. In the heart, MARP1 also interacts with MLP and 60 \nprotein kinase C alpha in intercalated disks, resulting in a maladaptive trophic response in 61 \ndilated cardiomyopathy 10. MARP2 can be phosphorylated at serine-99 (S69 in mice) by Akt, 62 \nresulting in reduced differentiation potential of myoblasts 13. MARP2 has also been linked to the 63 \nNFkB-pathway in inflammatory responses 14, suggesting MARP2 may have roles in atrophy 64 \nsignaling. MARP3  is the least studied member of the MARPs but appears to play a role in 65 \nglucose uptake and vascular remodeling in skeletal muscle 15,16. Titin has previously been shown 66 \nto activate signaling in response to stretch 17,18, providing a potential activation mechanism for 67 \nN2A to activate trophic signaling.  68 \nMuscle hypertrophy is directly associated with titin-based stiffness. High stiffness, as observed 69 \nin the Ttn Δex112-158 and TtnΔex219-225 mouse models 19,20, results in longitudinal hypertrophy, i.e. an 70 \nincrease in serial-linked sarcomeres, to reduce sarcomere length and normalize passive 71 \ntension. We previously used a surgical model in which we denervated one hemi-diaphragm 72 \n(unilateral diaphragm denervation, UDD) 21, inducing passive cyclic stretch of the denervated 73 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nhemi-diaphragm by the innervated costal to induce (longitudinal) hypertrophy. This hypertrophy 74 \nwas dependent on titin-based stiffness, as higher titin stiffness resulted in exaggerated 75 \nhypertrophy and lower titin stiffness resulted in attenuated hypertrophy 21. UDD is a unique 76 \nmodel as it allows the in vivo study of passive stretch-induced muscle hypertrophy.  77 \n 78 \nWe used UDD as a model for studying titin-mechanosensing and examined the signaling that 79 \nunderlies the hypertrophy response. Our findings support the N2A-MARP signalosome inhibiting 80 \nlongitudinal hypertrophy and show mTOR signaling to be a prominent contributor to longitudinal 81 \nhypertrophy.  82 \n  83 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nRESULTS 84 \nLongitudinal hypertrophy regulates transient trophicity in UDD. 85 \nA unique aspect of unilateral diaphragm denervation (UDD) is the transient nature of the 86 \nhypertrophy. This transient nature is likely the result from changes in fractional extension of 87 \nsarcomeres. Sarcomere length (SL) at end-expiration length was 2.9±0.1 µm in sham, while 88 \nstretch lengths at end-inspiration were predicted to reach 3.7 µm directly after denervation of the 89 \nright costal diaphragm (FIG.1A, previously published 21). Such sarcomere lengths put high strain 90 \non titin (FIG.1A, right panel), providing a potent trigger for mechanosensing and subsequent 91 \nhypertrophy signaling. Our results show that during 6-days of UDD the denervated costal rapidly 92 \nhypertrophies, followed by slow onset of atrophy (FIG.1B). The mass increase coincides with 93 \nlongitudinal hypertrophy of muscle fibers, addition of serial-linked sarcomeres, adding 952±81 94 \nsarcomeres (30.2% increase in total fiber length; p<0.0001; FIG.1C ) following 6-days of UDD. 95 \nThis increase in sarcomere number appeared to stabilize by 6-days UDD, as at 12-day  UDD 96 \nsarcomere addition was measured to be 778±87 sarcomeres versus sham levels (not 97 \nsignificantly different compared to 6-day UDD). Assuming that fractional extension of 98 \nsarcomeres during inspiration decreases with longitudinal hypertrophy, 30.2% increase in 99 \nsarcomere number would reduce the sarcomere length at end inspiration from 3.7 µm to ~2.8 100 \nµm, in close agreement with whole body formaldehyde perfusion experiments which showed 101 \nsarcomere lengths of 2.7±0.1 µm 21. As a SL of 2.8 µm falls below the end-expiration SL, the 102 \ndenervated costal effectively no longer experiences “stretch”, reducing titin’s mechanosensing 103 \ntrigger and thus explaining the transient nature of the hypertrophy seen in UDD. 104 \n 105 \nTo confirm that stretch is the trigger for hypertrophy, rather than denervation, we performed 106 \nbilateral diaphragm denervation in rats (BDD), resulting in complete inactivity of the diaphragm . 107 \nNote that we attempted BDD in mice but experienced high mortality rates, while survival in rat 108 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nwas >75%. All rats were of similar body weight pre-surgery and showed a slight decrease in 109 \nbody weight after 3-day BDD (Supplemental FIG.1A; p=0.036), and comparable tibial length and 110 \nsoleus weights (Supplemental FIG.1B & C). Similar to mice, rats present with increased 111 \ndenervated costal mass after 3-day  UDD: 14.68±0.64 mg/mm versus sham rats 10.82±0.39 112 \nmg/mm (FIG.1D; p>0.0001; muscle weight normalized to tibial length). However, 3-day  BDD 113 \nrats did not develop increased mass 11.92±0.89 mg/mm compared to sham rats 10.82±0.39 114 \nmg/mm. The increase in costal mass at 3-day UDD result s in part from an increase in 115 \nlongitudinal hypertrophy, with denervated costal diaphragm adding 933±301 serial sarcomeres 116 \ncompared to sham animals (FIG.1E; p=0.018), whereas 3-day BDD rats showed no difference 117 \nin serial sarcomere number (7559±313 versus sham 7740±111). Thus, stretch and not 118 \ndenervation triggers longitudinal hypertrophy. 119 \n 120 \nTo support titin ’s role as mechanosensor of stretch driven hypertrophy, we performed UDD on 121 \nRbm20ΔRRM mice and Rbm20 ko rats. Rbm20 is an RNA splicing-factor for titin and both the 122 \nmouse and rat model generate larger, more compliant titin isoforms and should thus be less 123 \nsensitive to stretch. Rbm20 ΔRRM mice showed relatively less tissue mass increase compared to 124 \nwildtype (WT) 3, 6, or 12-days UDD (Supplemental FIG.2A), without changes in longitudinal 125 \nhypertrophy at 6-days UDD (Supplemental FIG.2B) suggesting the difference in mass is a result 126 \nfrom radial fiber growth, in agreement with our previous findings 21. Finally, we compared 127 \ndenervated costal mass from Sprague Dawley (SD) and Rbm20 ko rats 3-day  UDD, showing a 128 \ndifference in mass increase of 24.4±3.8% in Rbm20 ko and 35.5±5.9% in SD rats ( p=0.02; 129 \nSupplemental FIG.2C ), supporting that stretch evokes hypertrophy in muscle and that titin 130 \ncompliance modulates the extent of hypertrophy across species . Thus, muscle stretch in the 131 \ncostal diaphragm is a potent trigger for longitudinal muscle hypertrophy, with titin being a 132 \nprimary sensor. 133 \n 134 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nGlobal transcriptome and proteome level studies reveal a distinct subset of genes and 135 \nproteins involved in UDD stretch hypertrophy. 136 \nTo identify the cellular processes involved in stretch-based hypertrophy we performed both 137 \ntranscriptome wide studies by RNA sequencing (RNAseq) as well as proteome studies by global 138 \nmass spectrometry (MS) on our rat 3-day sham, UDD and BDD samples. Initial studies focused 139 \non separating the effect of denervation versus stretch. Principal component analysis of the top 140 \n500 genes showed close clustering of samples by group (Supplemental FIG.3A), suggesting 141 \ndistinct gene programs are active between sham, UDD and BDD. At the protein level, principal 142 \ncomponent analysis of the top 250 proteins suggests narrow separation of groups, predicting 143 \noverlap of the BDD and UDD samples (Supplemental FIG.3B). Gene expression studies 144 \nrevealed very close overlap of BDD and UDD profiles with 77.15% overlap in differentially 145 \nexpressed genes (DEG’s: padj<0.05) and just 9.4% of DEG’s being specific for UDD (FIG.2A). 146 \nVolcano plots of UDD>Sham (FIG.2B, left panel) and BDD>Sham (FIG.2B; middle panel) 147 \nshowed the ~11.000 DEG’s (Supplemental data Tables 1-6) to be nearly equally distributed 148 \nbetween up and downregulated DEG’s . Direct comparison of UDD>BDD (FIG.2B, right panel) 149 \nrevealed 850 DEG’s to be uniquely associated with UDD, of which 581 were upregulated 150 \n(Supplemental data Table 3). MS studies broadly agreed with the RNAseq data, showing UDD 151 \nand BDD share overlapping expression programs (FIG. 2A & C). Twenty percent of the 152 \ndifferentially expressed proteins (DEP’s ; P<0.05) detected are unique to UDD (FIG.2C) . 153 \nVolcano plots of UDD>Sham (FIG.2D, left panel) and BDD>Sham (FIG.2D, middle panel ) 154 \nshowed 889 and 789 DEP’s, respectively, (Supplemental data Tables 7-12) which were 155 \nprimarily increased. Comparing UDD>BDD revealed 173 DEP’s (FIG.2D, right panel) that are 156 \nunique to UDD. Most of DEP’s (and DEG’s) found are related to transcription, translation, 157 \nenergetics and folding, with the highest fold DEP’s in rat UDD>BDD being: Med15 (15.5-fold), 158 \nEif3i (6.0-fold) and Gtpbp3 (3.9-fold) (Supplemental data  Table 9). GOterm enrichment of 159 \nUDD>BDD, separated by downregulated and upregulated DEG’s (Supplemental FIG.3C, left top 160 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nand bottom graph, respectively) , and DEP’s (Supplemental FIG.3C, right top and bottom graph, 161 \nrespectively), indicated that upregulated DEG/DEP’s are primarily related to muscle 162 \ndevelopment and function consistent with an active hypertrophy program and downregulated 163 \ntargets with metabolism and cellular respiration. To identify the pathways involved in 164 \nUDD>BDD, we searched the DEG’s against the KEGG PATHWAY database (Supplemental 165 \ndata Table 6), confirm ing GOterm results and indicating that the DEG’s are involved in muscle 166 \nremodeling. Thus, UDD has a distinct expression profile from denervation (BDD), focused on 167 \nremodeling of muscle.  168 \n 169 \nExploring if titin-mechanosensing was active in either UDD or BDD we generated heatmaps of 170 \ntitin-binding proteins based on RNAseq (Supplemental FIG.4) and global MS data (FIG.2E). To 171 \nnarrow down proteins that were differential between UDD and BDD we tested (2-way ANOVA 172 \npint<0.1) the z-score and found : MARP2 (Ankrd2; p=0.003), Cryab (p= 0.026), Csrp3 (p= 0.002), 173 \nHsp90ab1 (p=  0.09), Hspb1 (p=  0.008) and Smyd2 (p=  0.004), to be unique to UDD . 174 \nInterestingly, the se DEP’s (MARP2, Smyd2, Hsp1b, Hsp90ab and Cryab) are established 175 \nbinding partners of the N2A element of titin.22 176 \n 177 \nTitin phosphorylation in UDD 178 \nWith the rat data indicating changes in titin-associated signaling and titin stiffness modifying the 179 \nhypertrophy response in mice, we evaluated how UDD affects titin post-translationally. W e 180 \nperformed MS on phosphorylation enriched peptides from 24-hour UDD diaphragm samples. 181 \n24-hour UDD was selected to study the early phosphorylation events . MS revealed 2870 182 \nphosphorylated peptides (p <0.05), of which 142-sites were in titin (~700-sites identified 183 \nincluding non -significant sites; Supplemental data Table 13). We opted to define titin 184 \nphosphorylation by analyzing the phosphorylation at the domain level to study if titin show ed 185 \nregional changes in phosphorylation (FIG.3A and Supplemental data Table 14), as total titin 186 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nphosphorylation (FIG.3B) was unchanged. Domain level analysis indicated titin phosphorylation 187 \nwas primarily changing in the I-band. This motivated quantifying titin phosphorylation by 188 \nsegment (FIG.3C and Supplemental data Table 15; following established naming conventions in 189 \nKolmerer and Labeit1, and Bang et al23). Segmental analysis of titin (FIG.3C) revealed a marked 190 \nincrease in phosphorylation of the N2A-element and I/A-junction, and a decrease in the Z/I-191 \njunction. Focusing on the increase in phosphorylation of the N2A-element, a known trophicity 192 \nsignaling hub (reviewed in 22), we assessed the specific domain phosphorylation of the N2A-193 \nelement (FIG.3C, highlight). In the N2A-element we found 7 phosphorylation sites (FIG.3D -E) of 194 \nwhich S9346 & S9350 are located in a linker sequence between I79 and I80, S9483 in I80, 195 \nS9459 & S9520 in the N2A unique sequence, S9654 in I81 and S9643 in I82). The 2 sites 196 \nlocated in the N2A unique sequence were significantly upregulated following UDD (FIG.3D-E). 197 \npS9459 and pS9520 currently have no known function but could serve as a recruitment signal 198 \n(see discussion).  199 \n 200 \nThe MARP proteins regulate hypertrophy in UDD 201 \nThe MARP1 and MARP2 proteins were strongly expressed both at the RNA level and protein 202 \nlevel following UDD (FIG.2E). MARPs have been shown to be important regulators of trophicity 203 \nand have been proposed as intermediaries between titin and trophic signaling pathways (see 204 \ndiscussion). As the MARPs share high homology and possible redundancy 7, we performed 6-205 \nday UDD on both single- and multi-KO combinations of the MARPs. We initially performed UDD 206 \non the MARP triple KO mice (MARP t KO; knockout of Ankrd1, -2 and -23 genes) and found a 207 \n12% reduction in the hypertrophy response compared to WT (FIG.4A; p =0.0099). This reduction 208 \nin hypertrophy supports a potential link between titin-mechanosensing and MARP-based trophic 209 \nsignaling. To test if one or a combination of MARPs caused the reduction in hypertrophy, we 210 \nperformed 6-day UDD on MARP1 (Ankrd1), MARP2 (Ankrd2) and MARP3 (Ankrd23) KO mice 211 \n(FIG.4B-D) and double KO for MARP1/2, MARP1/3 and MARP2/3 (Supplemental FIG.6 ). 212 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nWhereas MARP1 KO mice did not show a difference in denervated costal diaphragm mass, 213 \nMARP2 KO mice showed a 14% increase (p=0.003) in mass and MARP3 KO mice showed a 214 \n13% reduction (p =0.004) in mass gain. These data suggest that MARP3 attenuat es 215 \nhypertrophy. However, all MARP double KO mice also presented with attenuated hypertrophy 216 \n(Supplemental FIG.6). This suggests that the separate MARP proteins could have distinct 217 \nfunctions in UDD, and there may be a dependency for two MARPs in regulating trophic 218 \nsignaling, further discussed in the discussion section.  219 \n 220 \nMARPs negatively regulate longitudinal hypertrophy 221 \nPrompted by the reduction in diaphragm mass gain during UDD in MARP tKO mice and by the 222 \ncomplexity of targeting all the single MARP KOs, we assessed longitudinal hypertrophy in 6-day 223 \nUDD MARP tKO samples . MARP tKO mice at baseline have fewer serial sarcomeres than WT 224 \n(2476±55 vs. 3157±69, respectively; p<0.0001), however 6-days UDD MARP t KO had similar 225 \nnumbers of sarcomeres to WT, 4081±44 versus 4109±59 (FIG.5 A). These data show MARP 226 \ntKO mice add 653 ± 91. 6 more sarcomeres than wildtype mice (FIG.5B; 1605± 71 vs. 952±59 , 227 \nrespectively; p<0.0001). To discern a possible mechanism of the MARPs inhibiting longitudinal 228 \nhypertrophy, we probed several candidate proteins of hypertrophy pathways that were 229 \nprominent in the MS datasets. Western blots for M apk1/3, Calcineurin, mT or, P70 s6k and 4E-230 \nbp1 were performed on costal diaphragms of WT and MARP tKO, 6-day Sham and UDD 231 \nanimals. All samples were normalized to Gapdh and data were presented as relative to WT 232 \nsham (FIG.5C ; representative western blot images in FIG.5D). Mapk1 showed increases in 233 \nprotein level that were comparable between WT and MARP tKO, whereas MAPK3 showed 234 \npreferential upregulation in MARP tKO (p=  0.0099). Calcineurin was unchanged following UDD , 235 \nnote that in a t-test the WT mice show ed a significant increase in UDD . In WT mice, mTor 236 \nshowed a striking increase in expression following 6-day UDD (p=  0.0006), whereas in MARP 237 \ntKO this was trending (P=  0.08). Downstream proteins of mTORC1; P70 s6k and 4E-bp 1 both 238 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nshowed altered regulation, with P70 s6k being similarly upregulated in WT (p=  0.0005) versus 239 \nMARP tKO (p= 0.001), while 4E-bp1 was only significantly upregulated in MARP tKO (p= 0.033). 240 \n2way-ANOVA for the signaling proteins did not indicate specific pathway changes between WT 241 \nand MARP tKO.  242 \n 243 \nmTor signaling regulates longitudinal hypertrophy in UDD 244 \nMotivated by the data from the mTor western blots in the MARP tKO mice and mT or being a 245 \nprominent regulator of skeletal muscle hypertrophy, we performed UDD in WT mice treated with 246 \nrapamycin, an inhibitor of mTORC1 signaling and skeletal muscle hypertrophy . The rat 247 \ntranscriptome studies also indicated calcium signaling (Supplemental data Table 6) to be a 248 \nprominent pathway in UDD. To test the role of calcium signaling we included a second inhibitor; 249 \ncyclosporin A, an inhibitor of the calcineurin-NFAT pathway, another hypertrophy regulating 250 \npathway. Mice received twice daily intraperitoneal injections of either rapamycin (2.5 251 \nmg/kg/day), cyclosporin A (25 mg/kg/day) or vehicle (DMSO), starting 3-days prior to surgery 252 \nuntil sacrifice of the mice (schematic in FIG.6A). Cyclosporin A treated mic e did not show a 253 \nchange in hypertrophy (FIG.6 B) or serial sarcomere addition (FIG.6 C), indicating that the 254 \ncalcineurin-NFAT pathway is not a primary mechanism for hypertrophy in UDD. Treatment did 255 \nnot affect the innervated left costal diaphragm (FIG.6D ), body weight (FIG.6E) or tibia length 256 \n(FIG.6F). Interestingly, mice that received rapamycin displayed less hypertrophy of the 257 \ndenervated costal diaphragm (FIG.6B; - 11.2%; p<0.001) compared to vehicle treated mice. 258 \nThis attenuated hypertrophy response coincided with a reduction in serial sarcomere addition 259 \n(FIG.5C; - 8.5%; p<0.001) compared to vehicle. Note that following 3-day UDD, rapamycin 260 \ntreated mice did not show significant increases in serial sarcomer es compared to untreated 261 \nsham animals (Δ67 ± 81 sarcomeres, versus vehicle treated mice Δ363 ± 79 sarcomeres). This 262 \nsuggested rapamycin treatment almost completely inhibited longitudinal hypertrophy and that 263 \nmTORC1 signaling plays a vital role in longitudinal hypertrophy development. We thus propose 264 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nthat mTORC1 signaling positively regulates longitudinal hypertrophy in skeletal muscle and that 265 \ntitin’s N2A-element tunes the extent of longitudinal hypertrophy through the MARPs to prevent 266 \nexcess hypertrophy (graphic summary in FIG. 6G). 267 \n 268 \nDISCUSSION 269 \n 270 \nLongitudinal hypertrophy as a mechanism for reducing stretch-induced mechanosensing 271 \nOne of the most striking aspects of the UDD surgical model is the early transient hypertrophy. 272 \nNo other muscle denervation model induces hypertrophy, supporting the notion that stretch, 273 \neven in inactive muscle, is a potent driver of hypertrophic growth. The extreme nature of the 274 \nstretch at work in UDD, ~25% stretch of the denervated costal by the innervated costal at a 275 \nfrequency of 120-230 times a minute (respiration rate) 21, forms a potent trigger for muscle 276 \nhypertrophy. Following 6-days UDD the denervated costal diaphragm develops 49.7±10.0% 277 \n(FIG.1B) increase in mass , which is primarily caused by addition of 952±81 sarcomeres 278 \n(FIG.1C) and to a lesser extent by radial fiber growth 21. The transient nature of the hypertrophy 279 \ncan be explained by the reduction in sarcomere strain due to the addition of sarcomeres  in 280 \nseries (longitudinal hypertrophy), removing the “trigger” that underlies the hypertrophy signaling. 281 \nThis hypothesis fits the data (FIG.1B -C), where before remodeling UDD costal width (i.e., fiber 282 \nlength) is ~8.7 mm (3000 sarcomeres x SL 2.9 µm), and when stretched 25% 21 equals a width 283 \nof ~10.8 mm. Following 6-days hypertrophic remodeling (UDD), costal width is ~10.8 mm (4000 284 \nsarcomeres x SL 2.7 µm). This suggests that the costal width increase attenuates the 285 \nhypertrophy trigger caused by stretching, finally resulting in atrophy (FIG.1B; 35-day UDD). 286 \nDenervation itself does not appear to induce hypertrophy of the diaphragm, as 3-day BDD in rat 287 \nshowed no hypertrophy (FIG.1D -E). UDD being a denervation model shows that hypertrophy is 288 \nnot necessarily dependent on a muscle’s ability to contract . This indicates that skeletal muscle 289 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nmay derive its signals for hypertrophy during the muscle relaxation-phase when the muscle is at 290 \nits longest state. Muscle stretch in human is known to benefit muscle growth and is a vital part 291 \nof exercise routines (reviewed in 24,25). Perhaps muscle antagonism may provide sufficient 292 \nstretch to induce hypertrophy . As contracting muscles inadvertently stretch their relaxed 293 \nantagonists, they create an elegant feedback loop that promotes both muscle growth and the 294 \nmaintenance of muscle mass.  295 \n 296 \nTitin’s response to stretch 297 \nPosttranslational changes in titin, particularly phosphorylation, have been studied mostly in 298 \nrelation to passive tension development 26–28. Specific PTMs for titin have not been widely 299 \nstudied and only recently has ubiquitination been shown to recruit autophagic receptors to the 300 \nkinase domain of titin 29,30. Autophosphorylation of titin kinase at Y170 is considered one of the 301 \nclassic mechanosensing responses in titin resulting in phosphorylation of the autophagic 302 \nreceptor Nbr1 at S115/116, activating autophagy signaling in vitro 31. We did not observe any 303 \nsigns following UDD that titin kinase was autophosphorylated at Y170. This could be related to 304 \nphospho-peptide abundance being below the detection limit, or that the titin kinase is inactive 32. 305 \nIn total we identified ~700 phosphorylation sites in titin of which 1 42 were significantly affected 306 \nby stretch. These sites were distributed along the entire length of titin with several hot spots in 307 \nthe PEVK region, likely involved in stiffness regulation, and several in the Z-disk, which could be 308 \nrelated to signaling or structural interactions. The sites in the N2A-element (FIG.3) were of 309 \nparticular interest as previously it has been suggested that S9540 (S9895 according to 310 \ndiaphragm RNAseq by Brynnel et al 20) could serve as a recruitment signal for MARP1 33, a 311 \nprotein that is highly upregulated following UDD (FIG.2E). MARPs have been shown to quench 312 \nN2A phosphorylation33,34, which make the phosphorylation sites in the N2A segment tantalizing 313 \ntargets for studying titin-MARP binding. If S9459 and S9520 (FIG.3D-E)  play such a role 314 \nremains to be determined, but such insights could provide future avenues for manipulating titin-315 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nMARP interactions. Similarly, we found many titin-associated proteins to show differential 316 \nphosphorylation in UDD, including 3 sites in the MARPs (Supplemental FIG.5). How these sites 317 \ncontribute to signaling or recruitment to the N2A-element remains to be seen but provide 318 \ntantalizing targets for follow-up study. 319 \n 320 \nThe MARP proteins and muscle trophicity 321 \nGlobal transcriptome and proteome studies from 3-day UDD show ed multiple titin-associated 322 \nproteins being upregulated (FIG.2E). W e focused our efforts on the MARP proteins, as they are 323 \nknown interacting partners of titin’s N2A -element and have been shown to be important for 324 \nhypertrophy regulation in the heart 35,36. We used UDD on MARP KO mice, focusing on the triple 325 \nKO for MARP1-3 to account for expected redundancy 7,34. MARP tKO showed a 12% reduction 326 \nin hypertrophy following 6-day UDD (FIG.4A). This suggested stretch-mechanosensing 327 \noperated through the MARP proteins. In an attempt to isolate a single MARP protein as the 328 \nmain effector, we performed UDD on single MARP knock-out mice (FIG.4B-D) . MARP1 KO did 329 \nnot reveal changes in hypertrophy. However, we previously established that MARP1 localizes to 330 \nthe N2A-segment of titin following UDD 21. We also independently determined that MARP1 331 \ncross-links titin to the thin filament to increase passive tension 37,38, suggesting MARP1 plays 332 \nmechanical roles over trophic regulation in skeletal muscle. MARP2 KO developed an 333 \nexaggerated hypertrophy and lastly MARP3 KO showed an attenuated hypertrophy response to 334 \nUDD, suggesting MARP2 and 3 play opposing roles in hypertrophy regulation. MARP2 interacts 335 \nwith Akt2 13, providing a tentative link with the mT or pathway, discussed below. Additionally, 336 \nMARP2 interaction with P50-NFκB acting as an analogue for IκB 14 suggests a possible role in 337 \ninhibiting NFκB atrophy signaling. MARP3 is poorly understood but has been linked to glucose 338 \nmetabolism15, forming another tentative link to mT or signaling39. To specifically determine if the 339 \nMARPs affected longitudinal hypertrophy we measured the number of serial sarcomeres across 340 \nthe width of the costal diaphragm in MARP tKO and found that the tKO mice added 653.2 ± 341 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\n91.55 more sarcomeres than WT following 6-day UDD (FIG.5B). This suggest s that the MARP 342 \nproteins inhibit longitudinal hypertrophy. A possible mechanism could be that following stretch 343 \nthe three MARPs bind and compete for the N2A- binding site7,34 resulting in translocation of 344 \nspecific MARPs for signaling purposes. The benefit of MARP deletion in cardiomyopathy was 345 \nshown by Lange et al 10, where deletion of MARP1/2 ameliorated MLP KO induced DCM. MARP 346 \nKO has not proven detrimental in mice 40, making this family an attractive target for therapeutic 347 \nintervention.  348 \n 349 \nmTor and longitudinal hypertrophy 350 \nMuscle hypertrophy is r egulated through a number of pathways, with the insulin -insulin growth 351 \nfactor (IGF) mediated pathway 41–43 being the most well understood in skeletal muscle and the 352 \ncalcineurin-NFAT pathway in cardiac muscle 44,45. With most studies focused on radial (cross-353 \nsectional) hypertrophy, we aimed to gain insight into the regulatory mechanism underlying 354 \nlongitudinal hypertrophy, two types of hypertrophy that are not necessarily mutually exclusive. 355 \nThe mT or signaling pathway was a prime candidate as UDD in MARP tKO mice suggested 356 \naltered mTor activity (FIG.5C-D). This prompted us to test inhibition of mTor signaling and see if 357 \nmTor regulates longitudinal hypertrophy. Using rapamycin 46, an inhibitor that targets the 358 \nmTORC1 (protein synthesis regulation) complex, we found a reduction in longitudinal 359 \nhypertrophy following 3-days UDD compared to vehicle treated mice (FIG.6B). This strongly 360 \nsuggests that the mTORC1- pathway is in-part responsible for longitudinal hypertrophy following 361 \nUDD.  Although we focused on mTor signaling in longitudinal hypertrophy development, we do 362 \nnot exclude other pathways being important. mTOR formed an attractive target as previous 363 \nwork showed that longitudinal stretch phosphorylates Akt and upregulates MARP2 47, forming a 364 \ntentative link between longitudinal stretch, MARPs and the mTor pathway.   Further studies are 365 \nneeded to establish the roles of the various pathways in stretch hypertrophy.  366 \n 367 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nIn conclusion, we found that the transient hypertrophy induced by UDD is dependent on muscle 368 \nfiber length, with longitudinal hypertrophy reducing the trigger for hypertrophy. The hypertrophy 369 \ncoincides with increased phosphorylation of the N2A element . The N2A-associated MARP 370 \nproteins are strongly upregulated following UDD and deletion of the MARPs increases the 371 \nextent of longitudinal hypertrophy following UDD, indicating the MARPs serve roles in inhibiting 372 \nlongitudinal hypertrophy. MARP tKO mice show altered regulation in the mTORC1 pathway 373 \nfollowing UDD and inhibition of mTORC1 by rapamycin shows mTor is a main regulator of 374 \nlongitudinal hypertrophy.  375 \n 376 \nFunding 377 \nThis work was financially supported by National Institutes of Health grants R01HL121500 (CO), 378 \nR01AR083233 (HG) and R35HL144998 (HG) 379 \n 380 \nReferences 381 \n 382 \n1. Labeit S, Kolmerer B. Titins: giant proteins in charge of muscle ultrastructure and elasticity. 383 \nScience. 1995;270(5234):293-296. doi:10.1126/science.270.5234.293 384 \n2. Horowits R, Kempner ES, Bisher ME, Podolsky RJ. A physiological role for titin and nebulin in 385 \nskeletal muscle. Nature. 1986;323(6084):160-164. doi:10.1038/323160a0 386 \n3. Swist S, Unger A, Li Y, et al. Maintenance of sarcomeric integrity in adult muscle cells cruciall y 387 \ndepends on  Z-disc anchored titin. Nat Commun. 2020;11(1):4479. doi:10.1038/s41467-020-388 \n18131-2 389 \n4. Ottenheijm CAC, van Hees HWH, Heunks LMA, Granzier H. Titin-based mechanosensing and 390 \nsignaling: role in diaphragm atrophy during  unloading? Am J Physiol Lung Cell Mol Physiol. 391 \n2011;300(2):L161-6. doi:10.1152/ajplung.00288.2010 392 \n5. van der Pijl RJ, Granzier HL, Ottenheijm CAC. Diaphragm contractile weakness due to reduced 393 \nmechanical loading: role of titin. Am J Physiol Cell Physiol. 2019;317(2):C167-C176. 394 \ndoi:10.1152/ajpcell.00509.2018 395 \n6. Voelkel T, Linke WA. Conformation-regulated mechanosensory control via titin domains in 396 \ncardiac muscle. Pflugers Arch. 2011;462(1):143-154. doi:10.1007/s00424-011-0938-1 397 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\n7. Miller MK, Bang ML, Witt CC, et al. The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and 398 \nDARP as a family of titin filament-based stress response molecules. J Mol Biol. 2003;333(5):951-399 \n964. doi:10.1016/j.jmb.2003.09.012 400 \n8. Wette SG, Smith HK, Lamb GD, Murphy RM. Characterization of muscle ankyrin repeat proteins in 401 \nhuman skeletal muscle. Am J Physiol Cell Physiol. 2017;313(3):C327-C339. 402 \ndoi:10.1152/ajpcell.00077.2017 403 \n9. Hayashi C, Ono Y, Doi N, et al. Multiple molecular interactions implicate the connectin/titin N2A 404 \nregion as a modulating scaffold for p94/calpain 3 activity in skeletal muscle. Journal of Biological 405 \nChemistry. 2008;283(21):14801-14814. doi:10.1074/jbc.M708262200 406 \n10. Lange S, Gehmlich K, Lun AS, et al. MLP and CARP are linked to chronic PKCalpha signalling in 407 \ndilated cardiomyopathy. Nat Commun. 2016;7:12120. doi:10.1038/ncomms12120 408 \n11. van der Pijl RJ, van den Berg M, van de Locht M, et al. Muscle ankyrin repeat protein 1 (MARP1) 409 \nlocks titin to the sarcomeric thin filament and is a passive force regulator. Journal of General 410 \nPhysiology. 2021;153(7). doi:10.1085/jgp.202112925 411 \n12. Zhou T, Fleming JR, Lange S, et al. Molecular Characterisation of Titin N2A and Its Binding of CARP 412 \nReveals a Titin/Actin Cross-linking Mechanism. J Mol Biol. 2021;433(9). 413 \ndoi:10.1016/j.jmb.2021.166901 414 \n13. Cenni V, Bavelloni A, Beretti F, et al. Ankrd2/ARPP is a novel Akt2 specific substrate and regulates 415 \nmyogenic  differentiation upon cellular exposure to H(2)O(2). Mol Biol Cell. 2011;22(16):2946-416 \n2956. doi:10.1091/mbc.E10-11-0928 417 \n14. Bean C, Verma NK, Yamamoto DL, et al. Ankrd2 is a modulator of NF-κB-mediated inflammatory 418 \nresponses during muscle  differentiation. Cell Death Dis. 2014;5(1):e1002. 419 \ndoi:10.1038/cddis.2013.525 420 \n15. Shimoda Y, Matsuo K, Kitamura Y, et al. Diabetes-Related Ankyrin Repeat Protein 421 \n(DARP/Ankrd23) Modifies Glucose Homeostasis  by Modulating AMPK Activity in Skeletal Muscle. 422 \nPLoS One. 2015;10(9):e0138624. doi:10.1371/journal.pone.0138624 423 \n16. Laughlin MH, Yang HT, Tharp DL, Rector RS, Padilla J, Bowles DK. Vascular cell transcriptomic 424 \nchanges to exercise training differ directionally along  and between skeletal muscle arteriolar 425 \ntrees. Microcirculation. 2017;24(2). doi:10.1111/micc.12336 426 \n17. Mayans O, van der Ven PF, Wilm M, et al. Structural basis for activation of the titin kinase domain 427 \nduring  myofibrillogenesis. Nature. 1998;395(6705):863-869. doi:10.1038/27603 428 \n18. Bertz M, Wilmanns M, Rief M. The titin-telethonin complex is a directed, superstable molecular 429 \nbond in the muscle  Z-disk. Proc Natl Acad Sci U S A. 2009;106(32):13307-133310. 430 \ndoi:10.1073/pnas.0902312106 431 \n19. van der Pijl RJ, Hudson B, Granzier-Nakajima T, et al. Deleting Titin’s C-Terminal PEVK Exons 432 \nIncreases Passive Stiffness, Alters Splicing,  and Induces Cross-Sectional and Longitudinal 433 \nHypertrophy in Skeletal Muscle. Front Physiol. 2020;11:494. doi:10.3389/fphys.2020.00494 434 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\n20. Brynnel A, Hernandez Y, Kiss B, et al. Downsizing the molecular spring of the giant protein titin 435 \nreveals that skeletal  muscle titin determines passive stiffness and drives longitudinal 436 \nhypertrophy. Elife. 2018;7:e40532. doi:10.7554/eLife.40532 437 \n21. van der Pijl R, Strom J, Conijn S, et al. Titin-based mechanosensing modulates muscle 438 \nhypertrophy. J Cachexia Sarcopenia Muscle. 2018;9(5):947-961. doi:10.1002/jcsm.12319 439 \n22. van der Pijl RJ, Domenighetti AA, Sheikh F, Ehler E, Ottenheijm CAC, Lange S. The titin N2B and 440 \nN2A regions: biomechanical and metabolic signaling hubs in cross-striated muscles. Biophys Rev. 441 \n2021;13(5):653-677. doi:10.1007/s12551-021-00836-3 442 \n23. Bang ML, Centner T, Fornoff F, et al. The complete gene sequence of titin, expression of an 443 \nunusual approximately 700-kDa  titin isoform, and its interaction with obscurin identify a novel Z-444 \nline to I-band linking system. Circ Res. 2001;89(11):1065-1072. doi:10.1161/hh2301.100981 445 \n24. Nunes JP, Schoenfeld BJ, Nakamura M, Ribeiro AS, Cunha PM, Cyrino ES. Does stretch training 446 \ninduce muscle hypertrophy in humans? A review of the literature. Clin Physiol Funct Imaging. 447 \n2020;40(3):148-156. doi:10.1111/cpf.12622 448 \n25. Warneke K, Lohmann LH, Lima CD, et al. Physiology of Stretch-Mediated Hypertrophy and 449 \nStrength Increases: A Narrative Review. Sports Medicine. 2023;53(11):2055-2075. 450 \ndoi:10.1007/s40279-023-01898-x 451 \n26. Hidalgo C, Granzier H. Tuning the molecular giant titin through phosphorylation: Role in health 452 \nand disease. Trends Cardiovasc Med. 2013;23(5):165-171. doi:10.1016/j.tcm.2012.10.005 453 \n27. Krüger M, Kötter S, Grützner A, et al. Protein kinase G modulates human myocardial passive 454 \nstiffness by phosphorylation of  the titin springs. Circ Res. 2009;104(1):87-94. 455 \ndoi:10.1161/CIRCRESAHA.108.184408 456 \n28. Loescher CM, Hobbach AJ, Linke WA. Titin (TTN): from molecule to modifications, mechanics, and 457 \nmedical significance. Cardiovasc Res. 2022;118(14). doi:10.1093/cvr/cvab328 458 \n29. Müller E, Salcan S, Bongardt S, Barbosa DM, Krüger M, Kötter S. E3-ligase knock down revealed 459 \ndifferential titin degradation by autophagy and the ubiquitin proteasome system. Sci Rep. 460 \n2021;11(1). doi:10.1038/s41598-021-00618-7 461 \n30. Bogomolovas J, Fleming JR, Franke B, et al. Titin kinase ubiquitination aligns autophagy receptors 462 \nwith mechanical signals in the sarcomere. EMBO Rep. 2021;22(10). 463 \ndoi:10.15252/embr.201948018 464 \n31. Lange S, Xiang F, Yakovenko a, et al. The kinase domain of titin controls muscle gene expression 465 \nand protein turnover. Science …. 2005;308(June):1599-1603. doi:10.1126/science.1110463 466 \n32. Bogomolovas J, Gasch A, Simkovic F, Rigden DJ, Labeit S, Mayans O. Titin kinase is an inactive 467 \npseudokinase scaffold that supports MuRF1 recruitment to the sarcomeric M-line. Open Biol. 468 \n2014;4(5):140041. doi:10.1098/rsob.140041 469 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\n33. Lanzicher T, Zhou T, Saripalli C, et al. Single-Molecule Force Spectroscopy on the N2A Element of 470 \nTitin: Effects of  Phosphorylation and CARP. Front Physiol. 2020;11:173. 471 \ndoi:10.3389/fphys.2020.00173 472 \n34. Lun AS, Chen J, Lange S. Probing Muscle Ankyrin-Repeat Protein (MARP) structure and function. 473 \nAnatomical Record. 2014;297(9):1615-1629. doi:10.1002/ar.22968 474 \n35. Zhong L, Chiusa M, Cadar AG, et al. Targeted inhibition of ANKRD1 disrupts sarcomeric ERK-475 \nGATA4 signal transduction and abrogates phenylephrine-induced cardiomyocyte hypertrophy. 476 \nCardiovasc Res. 2015;106(2):261-271. doi:10.1093/cvr/cvv108 477 \n36. Lange S, Gehmlich K, Lun AS, et al. MLP and CARP are linked to chronic PKCα signalling in dilated 478 \ncardiomyopathy. Nat Commun. 2016;7:12120. doi:10.1038/ncomms12120 479 \n37. van der Pijl RJ, van den Berg M, van de Locht M, et al. Muscle ankyrin repeat protein 1 (MARP1) 480 \nlocks titin to the sarcomeric thin filament and is a passive force regulator. Journal of General 481 \nPhysiology. 2021;153(7). doi:10.1085/jgp.202112925 482 \n38. Zhou T, Fleming JR, Lange S, et al. Molecular Characterisation of Titin N2A and Its Binding of CARP 483 \nReveals a Titin/Actin Cross-linking Mechanism. J Mol Biol. 2021;433(9). 484 \ndoi:10.1016/j.jmb.2021.166901 485 \n39. Szwed A, Kim E, Jacinto E. Regulation and metabolic functions of mTORC1 and mTORC2. Physiol 486 \nRev. 2021;101(3):1371-1426. doi:10.1152/physrev.00026.2020 487 \n40. Barash IA, Bang ML, Mathew L, Greaser ML, Chen J, Lieber RL. Structural and regulatory roles of 488 \nmuscle ankyrin repeat protein family in skeletal muscle. Am J Physiol Cell Physiol. 489 \n2007;293(1):C218-C227. 490 \n41. Schiaffino S, Mammucari C. Regulation of skeletal muscle growth by the IGF1-Akt/PKB pathway: 491 \ninsights from  genetic models. Skelet Muscle. 2011;1(1):4. doi:10.1186/2044-5040-1-4 492 \n42. Goodman CA. Role of mTORC1 in mechanically induced increases in translation and skeletal 493 \nmuscle  mass. J Appl Physiol (1985). 2019;127(2):581-590. doi:10.1152/japplphysiol.01011.2018 494 \n43. Yoon MS. mTOR as a Key Regulator in Maintaining Skeletal Muscle Mass. Front Physiol. 495 \n2017;8:788. doi:10.3389/fphys.2017.00788 496 \n44. Molkentin JD. Calcineurin-NFAT signaling regulates the cardiac hypertrophic response 497 \nin  coordination with the MAPKs. Cardiovasc Res. 2004;63(3):467-475. 498 \ndoi:10.1016/j.cardiores.2004.01.021 499 \n45. Molkentin JD. Parsing good versus bad signaling pathways in the heart: role of calcineurin-500 \nnuclear  factor of activated T-cells. Circ Res. 2013;113(1):16-19. 501 \ndoi:10.1161/CIRCRESAHA.113.301667 502 \n46. Li J, Kim SG, Blenis J. Rapamycin: one drug, many effects. Cell Metab. 2014;19(3):373-379. 503 \ndoi:10.1016/j.cmet.2014.01.001 504 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\n47. Mohamed JS, Lopez MA, Cox GA, Boriek AM. Anisotropic regulation of Ankrd2 gene expression in 505 \nskeletal muscle by mechanical stretch. The FASEB Journal. 2010;24(9):3330-3340. 506 \ndoi:10.1096/fj.10-158386 507 \n48. Bang ML, Gu Y, Dalton ND, Peterson KL, Chien KR, Chen J. The muscle ankyrin repeat proteins 508 \nCARP, Ankrd2, and DARP are not essential for  normal cardiac development and function at basal 509 \nconditions and in response to pressure overload. PLoS One. 2014;9(4):e93638. 510 \ndoi:10.1371/journal.pone.0093638 511 \n49. Methawasin M, Hutchinson KR, Lee EJ, et al. Experimentally increasing titin compliance in a novel 512 \nmouse model attenuates the  Frank-Starling mechanism but has a beneficial effect on diastole. 513 \nCirculation. 2014;129(19):1924-1936. doi:10.1161/CIRCULATIONAHA.113.005610 514 \n50. Li S, Guo W, Dewey CN, Greaser ML. Rbm20 regulates titin alternative splicing as a splicing 515 \nrepressor. Nucleic Acids Res. 2013;41(4):2659-2672. doi:10.1093/nar/gks1362 516 \n51. Lindqvist J, van den Berg M, van der Pijl R, et al. Positive End-Expiratory Pressure Ventilation 517 \nInduces Longitudinal Atrophy in  Diaphragm Fibers. Am J Respir Crit Care Med. 2018;198(4):472-518 \n485. doi:10.1164/rccm.201709-1917OC 519 \n52. Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 520 \n2013;29(1):15-21. doi:10.1093/bioinformatics/bts635 521 \n53. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq 522 \ndata with DESeq2. Genome Biol. 2014;15(12):550. doi:10.1186/s13059-014-0550-8 523 \n54. Bang ML, Centner T, Fornoff F, et al. The Complete Gene Sequence of Titin, Expression of an 524 \nUnusual  700-kDa Titin Isoform, and Its Interaction With Obscurin Identify a Novel Z-Line to I-525 \nBand Linking System. Circ Res. 2001;89(11):1065-1072. doi:10.1161/hh2301.100981 526 \n55. Levine AA, Liktor-Busa E, Balasubramanian S, et al. Depletion of Endothelial-Derived 2-AG 527 \nReduces Blood-Endothelial Barrier Integrity via Alteration of VE-Cadherin and the Phospho-528 \nProteome. Int J Mol Sci. 2023;25(1):531. doi:10.3390/ijms25010531 529 \n56. Keresztes A, Olson K, Nguyen P, et al. Antagonism of the mu-delta opioid receptor heterodimer 530 \nenhances opioid antinociception by activating Src and calcium/calmodulin-dependent protein 531 \nkinase II signaling. Pain. 2022;163(1):146-158. doi:10.1097/j.pain.0000000000002320 532 \n57. Parker SS, Krantz J, Kwak EA, et al. Insulin Induces Microtubule Stabilization and Regulates the 533 \nMicrotubule Plus-end  Tracking Protein Network in Adipocytes. Mol Cell Proteomics. 534 \n2019;18(7):1363-1381. doi:10.1074/mcp.RA119.001450 535 \n58. Hulsen T, de Vlieg J, Alkema W. BioVenn – a web application for the comparison and visualization 536 \nof biological lists using area-proportional Venn diagrams. BMC Genomics. 2008;9(1). 537 \ndoi:10.1186/1471-2164-9-488 538 \n59. Ge SX, Jung D, Yao R. ShinyGO: a graphical gene-set enrichment tool for animals and plants. 539 \nBioinformatics. 2020;36(8). doi:10.1093/bioinformatics/btz931 540 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\n60. Tyanova S, Temu T, Sinitcyn P, et al. The Perseus computational platform for comprehensive 541 \nanalysis of (prote)omics data. Nat Methods. 2016;13(9). doi:10.1038/nmeth.3901 542 \n61. Babicki S, Arndt D, Marcu A, et al. Heatmapper: web-enabled heat mapping for all. Nucleic Acids 543 \nRes. 2016;44(W1). doi:10.1093/nar/gkw419 544 \n  545 \n  546 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nMETHODS 547 \n 548 \nAnimal studies 549 \nAll experiments were done in accordance with the University of Arizona Institutional Animal 550 \nCare and Use Committee and followed the US National Institutes of Health Using Animals in 551 \nIntramural Research guidelines for animal use. We used 3-month-old C57BL/6J mice referred to 552 \nas wildtype (WT), and 6-month-old Sprague Dawley rats (SD). Knockout models for titin binding 553 \nproteins MARP1-3 (Ankrd1, Ankrd2 and Ankrd23)10,40,48 were kindly provided by Dr Ju Chen and 554 \nDr Stephan Lange. Homozygous Rbm20 ΔRRM mice 21,49 and Rbm20 ko rats 50,51, have previously 555 \nbeen described. Mice were maintained on a C57BL/6J background, with the data from the 556 \nMARP mice being on a black swiss background.  557 \n 558 \nSurgical procedure 559 \nFor unilateral diaphragm surgery (UDD) 21 or bilateral diaphragm denervation (BDD) studies, 560 \nmice or rats were anaesthetized with 2-3% isoflurane and a small incision was made in the neck 561 \narea just above the clavicle. The right phrenic nerve was isolated behind the sternohyoid 562 \nmuscle, and a 3–4 mm section was transected at the height of the supraclavicular nerve branch. 563 \nFor BDD surgery both left, and right phrenic nerves being transected. Sham operated animals 564 \nunderwent the same procedure, except the phrenic nerve was left intact. Animals were 565 \nsacrificed 1, 3, 6, 12 or 35-days after surgery for morphometric analysis and tissue harvest. 566 \n 567 \nPharmacological inhibition of hypertrophy 568 \nInhibitor studies with rapamycin (mTOR inhibitor) and cyclosporin A (Calcineurin inhibitor) were 569 \nperformed by injecting mice, twice daily intra-peritoneal (IP), with 2.5 or 25 mg/kg/day, 570 \nrespectively. Each inhibitor was dissolved in Dimethylsulfoxide (DMSO) and diluted to 20% 571 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nDMSO with saline solution just before injection. Mice received inhibitors or vehicle (20% DMSO 572 \nin saline) starting 3-days prior to UDD surgery to prime the mice and continued following UDD to 573 \ninhibit hypertrophy growth (FIG.4C).  Mice were sacrificed after 3-days UDD and processed for 574 \nserial sarcomere measurements as described below.  575 \n 576 \nSerial sarcomere measurements 577 \nPreviously described in van der Pijl et al 21. Briefly, mice were anaesthetized with a 140/10 578 \nmg/kg ketamine/xylazine solution, a small incision to visualize the jugular vein, which was 579 \nsubsequently cannulated for perfusion. Mice were perfused with a solution consisting of 4% 580 \nformaldehyde, with 70 U/mL of heparin in phosphate buffered saline (PBS), after which the 581 \ndiaphragm was removed and stored in 4% formaldehyde in PBS overnight for complete fixation. 582 \nFull length diaphragm midcostal strips were gently dissected and flattened between glass slides, 583 \ncostal width was measured using a caliper and sarcomere lengths were measured using a 584 \nHe/Ne laser diffraction system. 585 \nAlternatively, muscle fiber bundels from chemically demembranated full length 586 \ndiaphragm midcostal strips of 3-day mice were flattened between glass slides. Costal width and 587 \nsarcomere lengths were measured using a Zeiss Axio Imager M1 microscope (Zeiss), at ×50 588 \nmagnification to measure the length of the muscle bundles and ×640 for sarcomere length along 589 \nfour points of the fiber bundles. Images were captured using AxioCam MRc with Axiovision 590 \nsoftware (Zeiss) and images were calibrated using a 0.01 mm stage micrometre (Edmund 591 \nOptics). To determine the number of serial sarcomeres the muscle bundle length (costal width) 592 \nwas divided by the sarcomere length.  593 \nDemembranating solution consisted of a relaxing solution (in mM; 20 BES, 10 EGTA, 594 \n6.56 MgCl2, 5.88 NaATP, 1 DTT, 46.35 K ‐propionate, 15 creatine phosphate, pH 7.0), with 1% 595 \nTriton‐X‐100 at 4°C, and protease inhibitors (phenylmethylsulfonyl fluoride (PMSF), 0.25 mM; 596 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nleupeptin, 0.04 mM; E64, 0.01 mM, ), after demembranation, samples were stored in just 597 \nrelaxing solution plus inhibitors (without triton X-100) at 4°C. 598 \n 599 \nTranscriptome studies 600 \nRNA sequencing (RNAseq) was performed on right costal diaphragm samples collected from 3-601 \nday sham and UDD animals and flash frozen in liquid nitrogen until further processing. For RNA 602 \nextraction, samples were incubated overnight in RNAlater-ICE (Thermo Scientific) and 603 \nsubsequently transferred to RLT buffer for extraction according to the RNeasy Fibrous Tissue 604 \nMini Kit (Qiagen). Tissue disruption was achieved using a Bullet Blender (Next Advance) and 605 \nGreen Eppendorf lysis kit tubes (Next Advance), by grinding samples for 4 minutes at setting 606 \n10. Thereafter, total RNA extraction was performed following the RNeasy Fibrous Tissue Mini 607 \nKit’s instr uctions and quantified using a Nanodrop ND-1000 spectrophotometer (Thermo 608 \nScientific). Each sample consisted of 3 biological replicate sham or UDD samples. Both Library 609 \npreparation and sequencing was performed by the University of Chicago Genomics Facility , 610 \nChicago, USA. Briefly, library preparation: rRNA was depleted from RNA preparations from 1 µg 611 \ntotal RNA. Libraries were prepared using an RNA Library Prep Kit from Illumina following the 612 \nmanufacturer’s instructions. Sequencing was performed on an Illumin a Hiseq2500 sequencer 613 \nusing 100 bp paired-end sequencing. For RNAseq analysis see 20. Briefly, Adapters and low-614 \nquality reads were removed with Trim Galore and reads were mapped to the rat genome 615 \n(Release mRatBN7.2) using STAR 52 with default settings. Differentially expressed genes were 616 \ndetermined with DESeq2 53. Genes with population adjusted p-values (p adj) <0.05 were 617 \nconsidered differentially expressed. For titin splicing, percent spliced in index (PSI) was 618 \ncalculated as a measure for determining if an exon is spliced in, following the titin exon 619 \nannotation by Bang et al.54 620 \n 621 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nProteome studies 622 \nPreparation of muscle for mass spectrometry analysis 623 \nDiaphragm muscle was ground to a fine powder using Dounce homogenizers cooled in liquid 624 \nnitrogen and acclimated to –20°C for 30 min before continuing. Tissue powder was 625 \nresuspended at a concentration of 50 mg/ml in a Urea buffer (4 M urea, 1 M thiourea, 25 m M 626 \nTris–HCl, 75 mM dithiothreitol, 1.5 % SDS, 25 % glycerol, pH 6.8) with protease inhibitors (0.04 627 \nmM E‐64, 0.16 mM leupeptin, and 0.2 mM PMSF). The solution was mixed for 4 min, followed 628 \nby 10 min of incubation at 60°C. Samples were centrifuged at 12.000 rpm and the supernatant 629 \nflash frozen for storage at −80°C. 630 \n 631 \nIn-solution Tryptic Digestion 632 \n50 µg of rat costal diaphragm lysate was subjected to acetone precipitation by adding six times 633 \nthe sample volume of pre-chilled 100 % acetone and incubated one hour at -20°C. The 634 \nprecipitates were centrifuged at 16,000 x g for 10 minutes at 4°C and the acetone was removed. 635 \n400 µL of pre-chilled 90% acetone was added to the protein pellet, briefly vortexed and 636 \ncentrifuged at 16,000 x g for 5 minutes at 4°C. The remaining acetone was removed, the protein 637 \npellets were air dried for 3 minutes, resuspended in 100 µL of 50 mM NH 4HCO3 and sonicated 638 \nfor 5 minutes. The samples were supplemented with dithiothreitol (DTT) at a final concentration 639 \nof 5 mM and incubated at 70°C for 30 minutes. Samples were cooled to room temperature for 640 \n10 minutes and incubated with 15 mM acrylamide for 30 minutes at room temperature while 641 \nprotected from light. The reaction was quenched with DTT with a final concentration of 5 mM 642 \nand incubated in the dark for 15 minutes.  One µg of Lys-C was added to each sample and 643 \nincubated at 37° C for 2-3 hours while shaking at 300 rpm followed by the addition of 50 µL of 644 \n50 mM ammonium bicarbonate and 2 µg of trypsin and incubation overnight at 37°C while 645 \nshaking at 300 rpm. 14.7 µL of 40 % FA/1 % HFBA was added to each sample and incubated 646 \nfor 10 minutes (final concentration is 4 % FA/0.1 % HFBA) to stop trypsin digestion. The 647 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nsamples were desalted with Pierce Peptide Desalting Spin Columns per the manufacturer’s 648 \nprotocol (ThermoFisher Scientific, cat no. 89852) and the peptides were dried by vacuum 649 \ncentrifugation. The dried peptides were resuspended in 20 µL of 0.1 % FA (v/v) and the peptide 650 \nconcentration was determined with the Pierce Quantitative Colorimetric Peptide Assay Kit per 651 \nthe manufacturer’s protocol (ThermoFisher Scienti fic, cat no. 23275). 350 ng of the final sample 652 \nwas analyzed by mass spectrometry. 653 \n 654 \nPhosphoproteomics 655 \nTo determine global differences in protein phosphorylation abundance between sham or UDD, 1 656 \nmL of protein lysate corresponding to 50 mg diaphragm per sample (pooled costal diaphragm of 657 \n2 mice) was subjected to in-solution tryptic digestion and phosphopeptide enrichment using 658 \nsequential enrichment from metal oxide affinity chromatography per manufacturer’s protocol 659 \n(Thermo Scientific, cat no. A32993 & A32992) similar to as previously described 55,56. The dried 660 \npeptides were resuspended in 20 µL of 0.1 % FA (v/v) and the peptide concentration was 661 \ndetermined with the Pierce Quantitative Colorimetric Peptide Assay Kit per the manufacturer’s 662 \nprotocol. 350 ng of the final sample was then analyzed by mass spectrometry. 663 \n 664 \nMass Spectrometry 665 \nHPLC-ESI-MS/MS was performed in positive ion mode on a Thermo Scientific Orbitrap Fusion 666 \nLumos tribrid mass spectrometer fitted with an EASY-Spray Source (Thermo Scientific, San 667 \nJose, CA). NanoLC was performed using a Thermo Scientific UltiMate 3000 RSLCnano System 668 \nwith an EASY Spr ay C18 LC column (Thermo Scientific, 50cm x 75 μm inner diameter, packed 669 \nwith PepMap RSLC C18 material, 2 µm, cat. # ES803); loading phase for 15 min at 0.300 670 \nµL/min; mobile phase, linear gradient of 1 –34 \n671 \nby 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 \nstep to 1 % Buffer B over 5 min at 0.250 µL/min and a final hold for 10 min (total run 159 min); 673 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nBuffer A = 0.1 % FA/H 2O; Buffer B = 0.1 % FA in 80 % ACN. All solvents were liquid 674 \nchromatography mass spectrometry grade. Spectra were acquired using XCalibur, version 2.3 675 \n(ThermoFisher Scientific). A “TopSpeed” data -dependent MS/MS analysis was performed 676 \n(acquisition of a full scan spectrum followed by collision-induced dissociation mass spectra of 677 \nthe Top N most intense precursor ions within the 3 second cycle time). Dynamic exclusion was 678 \nenabled with a repeat count of 1, a repeat duration of 30 seconds, an exclusion list size of 500, 679 \nand an exclusion duration of 40 seconds. 680 \n 681 \nLabel-free Quantitative Proteomics  682 \nProgenesis QI for proteomics software (version 2.4, Nonlinear Dynamics Ltd., Newcastle upon 683 \nTyne, UK) was used to perform ion-intensity based label-free quantification as previously 684 \ndescribed57. In brief, in an automated format, raw files were imported and converted into two-685 \ndimensional maps (y-axis = time, x-axis =m/z) followed by selection of a reference run for 686 \nalignment purposes. An aggregate data set containing all peak information from all samples was 687 \ncreated from the aligned runs, which was then further narrowed down by selecting only +2, +3, 688 \nand +4 charged ions for further analysis. The samples were then grouped according to 689 \ntreatment. Peak lists of the top ten fragment ion spectra were exported in Mascot generic file 690 \n(mgf) format and searched against either the 2020_06 Swiss-Prot Rattus norvegicus database 691 \n(8128 entries) , the 2018_11 Swiss-Prot Mus musculus  database (17008 entries), or species 692 \nrespective TrEMBL databases using Mascot (Matrix Science, London, UK; version 2.6.0). The 693 \nsearch variables that were used were: 10 ppm mass tolerance for precursor ion masses and 0.5 694 \nDa for product ion masses; digestion with trypsin; a maximum of two missed tryptic cleavages; 695 \nvariable modifications of oxidation of methionine, phosphorylation of serine, threonine, and 696 \ntyrosine, and carbamidomethylation of cysteine;  13C = 1. The resulting Mascot .xml file was 697 \nthen imported into Progenesis, allowing for peptide/protein assignment, while peptides with a 698 \nMascot Ion Score of <25 were not considered for further analysis. Abundances were normalized 699 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nto the total ion current (TIC) to correct for differences in sample loading and instrument 700 \nresponse. For quantification, proteins must have possessed at least one or more unique, 701 \nidentifying peptide. 702 \n 703 \nVisualization and analysis of transcriptome and proteome data 704 \nFigures were generated using online resources, briefly, quantitative Venn diagrams were made 705 \nusing https://www.biovenn.nl/58. Lolipop graphs of gene onthology (GO) enritchment analysis by 706 \nShinyGO v0.75 http://bioinformatics.sdstate.edu/go/ 59. Heatmaps were generated using a 707 \ncombination of Graphpad Prism v9.1, Perseus v2.0.3.1 60 and Heatmapper 708 \n(http://heatmapper.ca/)61. Perseus v2.0.3.1 was also used to analyze principal components and 709 \nvisualized using Graphpad Prism v9.1. 710 \n 711 \nWestern blot 712 \nWestern blot experiments previously described in van der Pijl et al 21. Proteins were transferred 713 \nonto Immobilon-P PVDF 0.45 μm  membranes (Millipore) using semi dry transfer (Bio-Rad). 714 \nMembranes were blocked with Odyssey blocking buffer (Li-Cor Biosciences) for 1 hour, and 715 \nsubsequently probed with primary antibodies at 4°C overnight (See Table 1). Near Infra-Red 716 \ndyes were used as secondary antibodies for detection with Odyssey CLx Imaging System (Li-717 \nCor Biosciences, United states). 718 \n  719 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nAntibody Source/isotype Dilution Company Catalog# \nMAPK1/3 (ERK2/1) Mouse IgG1 1:200 Cell Signaling #4696 \nCalcineurin Mouse IgG2a  1:750 BD biosciences 610260 \nmTOR Rabbit IgG 1:750 Cell Signaling #2983 \nP70S6K Rabbit IgG 1:500 Cell Signaling #2708 \n4EBP1 Rabbit IgG 1:750 Cell Signaling #9452 \nGapdh Rabbit IgG 1:5000 Cell Signaling #2112 \nGapdh Mouse IgG1 1:3000 Thermo Fisher Sci. \nMA5-\n15738 \nCF790 Goat anti Mouse IgG Goat IgG 1:10.000 Biotium 20342 \nCF680 Goat anti Mouse IgG Goat IgG 1:10.000 Biotium 20065 \nCF680 Goat anti Rabbit IgG Goat IgG 1:10.000 Biotium 20067 \nCF680R Goat anti Mouse \nIgG2a Goat IgG 1:10.000 Biotium 20842 \nTable 1 Antibodies used in this study  720 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nFIGURE 1.  Transient hypertrophy following UDD in the denervated costal diaphragm. (A) 721 \nschematic of how UDD affects sarcomere length in the denervated costal (left panel) and how 722 \nstretch extends titin for mechanosensing (right panel, generated through BioRender).  (B ) 723 \nMouse diaphragm costal weight normalized to tibial length following 1, 3, 6, 12 and 35-days of 724 \nUDD, showing the hypertrophy phase peaking at 6-days and progressing to the atrophy phase 725 \nat 12-days post-UDD (n=6-22, shams grouped for simplicity). A substantial part of the 726 \nhypertrophy encompasses longitudinal hypertrophy (C), lengthening of the muscle fibers by 727 \naddition of serial sarcomeres (n=10 -13). The increased fiber length likely reduces the stretch-728 \nbased hypertrophy signaling and thus explains the transient nature of hypertrophy. (D) 3-day 729 \nBDD in rats (n=6-9) confirms stretch is the trigger for inducing hypertrophy in UDD at the tissue 730 \nmass level (D; diaphragm right costal normalized to tibial length, denervated in UDD) and serial 731 \nsarcomeres level (E). One-way ANOVA, with Tukey post-hoc testing.  732 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nFIGURE 2. Global transcriptomics and proteomics following 3-days UDD and BDD in rats. 733 \nGlobal transcript studies by RNAseq of sham, UDD and BDD right costal diaphragm 734 \n(n=5/group). Same parameters apply to the global proteome studies (C-D) with mass 735 \nspectrometry. Quantitative Venn diagrams of the transcriptome (A) showing overlap gene 736 \nregulation between UDD and BDD. Vulcano plots of UDD (B, left) and BDD (B, middle) showed 737 \nsimilar gene regulation. Comparing UDD to BDD directly revealed just 850 differentially 738 \nregulated genes (B, right) indicating a small subset being responsible for hypertrophy regulation. 739 \nQuantitative Venn diagrams of the proteome (C) showed similar regulation compared to 740 \ntranscriptome. Volcano plots of UDD (D, left) and BDD (D, middle) showed primarily 741 \nupregulation of proteins. Comparing UDD to BDD directly revealed just 173 differentially 742 \nregulated proteins (D, right). Green-dots: upregulated genes/proteins, red-dots: downregulated 743 \ngenes/proteins. Titin-associated proteins in heatmap of proteome (E; Z-score: red= upregulated, 744 \nblue= downregulated) and violin plots (right panel) of differential proteins between UDD (red) 745 \nand BDD (blue), indicating upregulation of titin-associate proteins following stretch. 2-way 746 \nANOVA pint: *p<0.05, **P<0.01. 747 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nFIGURE 3.  Phosphorylation of titin following 24-hours of UDD by mass spectrometry 748 \n(n=4/group). Phosphorylation of individual titin domains (A) relative Z-score (log2) of titin 749 \nphosphorylation showing domain specific changes in phosphorylation. Total phosphorylation of 750 \ntitin (B) is not affected by UDD, however titin showed regional changes in phosphorylation (C), 751 \nnotably increased phosphorylation of the N2A-element (boxed). Quantitation of the 752 \nphosphorylation signal for the 5 main sites found in the N2A-element (D). (E) Schematic of the 753 \nN2A element with the 2 pSer found in the N2Aus (Transcript: ENSMUST00000099981.10 Ttn-754 \n203). Red: Ig domain coding and blue: unique sequence coding. Mouse titin phosphorylation 755 \nand global mass spectrometry was analyzed by t-test, Kolmogorov-Smirnov test or multiple t-756 \ntest with a cut-off at P<0.05.  757 \n  758 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nFIGURE 4.  6-day UDD on KO mice of MARP proteins. MARP triple KOs show a reduced 759 \nresponse to UDD (A; p<0.01). No effect of single MARP1 KO (B) on UDD, increased 760 \nhypertrophy following MARP2 KO (B; p<0.01; t-test), indicating possible roles in hypertrophy 761 \nsuppression or atrophy signaling and MARP3 KO (C) showed baseline hypertrophy in costal 762 \ndiaphragm in addition to less hypertrophy development in UDD (p<0.01; t-test) compared to 763 \nWT, implying roles as a suppressor of hypertrophy. Left panel, diaphragm right costal mass 764 \nnormalized to tibial length and right panel, percentual increase in right costal mass relative to 765 \nsham. S= Sham, U= UDD (n=10-12). Statistical testing by t-test or two-way ANOVA with Tukey 766 \npost-hoc testing.  767 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nFIGURE 5. MARPs inhibits longitudinal hypertrophy. Longitudinal hypertrophy measured in right 768 \ncostal strips of WT and MARP tKO mice in 6-da y sham and UDD mice (A). Numerical increase 769 \nin serial sarcomeres is higher in MARP tKO (P<0.0001) mice compared to WT mice (B; n=7-12), 770 \nsuggesting that the MARPs inhibits longitudinal growth. Probing hypertrophy signaling by 771 \nwestern blot, normalized to Gapdh, with expression set relative to WT sham levels (C; n=8-12). 772 \nDifferential mTor response suggests role in regulating longitudinal hypertrophy. (D) 773 \nRepresentative blot images of the signaling proteins. Statistical testing by one-way or two-way 774 \nANOVA with Tukey post-hoc testing.  775 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nFIGURE 6. Pharmacological inhibition of the mTor (rapamycin) and calcium (cyclosporin A) 776 \nbased hypertrophy pathways revealed mTor to be involved in longitudinal hypertrophy. (A) 777 \nSchematic of the inhibition protocol, showing mice were injected with inhibitors for 3-days prior 778 \nto receiving UDD surgery, with continued twice daily dosing of inhibitors until sacrifice at day 3 779 \npost-UDD. Rapamycin inhibited hypertrophy development both at the costal diaphragm mass 780 \nlevel (B; p<0.001) and at the longitudinal hypertrophy level (C; p<0.001), whereas cyclosporine 781 \nA had no effect. Neither cyclosporine A or rapamycin affected the innervated costal diaphragm 782 \n(D), or body mass (E) and all mice used were of approximately the same size based on skeletal 783 \nsize, as measured by tibia length (F). (G) Hypothetical mechanism for longitudinal hypertrophy 784 \nfollowing muscle stretch. The mTorc1 pathway is activated by stretch and initiates longitudinal 785 \nmuscle hypertrophy. MARP proteins sequestered by titin’s N2A element are released upon 786 \nstretch and tunes the longitudinal hypertrophy, thus preventing excessive longitudinal 787 \nhypertrophy (Image was generated through BioRender). N=8-10/group, statistical testing by 1-788 \nway-ANOVA and Dunnett's multiple comparisons test.  789 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\n 790 \n 791 \n 792 \n 793 \n 794 \n 795 \n 796 \n 797 \nSupplemental figures 798 \n  799 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nS FIGURE 1.  3-days bilateral diaphragm denervation in rats showed similar body weights 800 \ncompared to sham animals (A; n=6-9/group) and were of similar size based on tibia length (B) 801 \nand soleus muscle weights (C). Statistical testing by one-way ANOVA and Dunnett's multiple 802 \ncomparisons test.  803 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nS FIGURE 2.  Role of titin stiffness on hypertrophy following UDD. (A) Transient hypertrophy 804 \nresponse in Rbm20 ΔRRM mice (more compliant titin) showing a blunted hypertrophy response 805 \ncompared to WT mice, based on percent increase of diaphragm right costal mass relative to 806 \nsham (n=10-12). (B) Titin-based stiffness does not alter longitudinal hypertrophy response, as 807 \nboth WT and Rbm20 ΔRRM mice show a similar increase in serial sarcomeres following 6-days 808 \nUDD. Rbm20-KO rat response to 3-days UDD, based on percent increase of diaphragm right 809 \ncostal mass relative to sham (mouse n=10-11, rat n=8) supporting titin-based stiffness 810 \nregulating muscle hypertrophy similarly across species. Statistical testing by t-test.  811 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nS FIGURE 3.  Principal component analysis of the rat 3-day UDD and BDD transcriptome (A) 812 \nand proteome (B), showing clear separation of groups at the transcript level and overlap of BDD 813 \nand UDD samples at the protein level. GOterm enrichment of UDD>BDD separated by up- or 814 \ndown-regulated transcriptomes and proteome (C, left and right, respectively) show distinct, yet 815 \noverlapping cellular processes. Global mass spectrometry was analyzed by ANOVA and 816 \ncorrected for multiple comparisons with false discovery rate with a cut-off at p<0.05.  817 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nS FIGURE 4. Transcriptome regulation of titin-associated and myofilament genes by RNAseq in 818 \nrats following 3-days of UDD/BDD. Heatmaps showing similar regulation between UDD and 819 \nBDD samples (n=4-5; Z-score: red= upregulated, blue= downregulated) at the transcript level 820 \nfor titin-associated and myofilament genes, based on hierarchal clustering.  821 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\n822 \nS FIGURE 5. Titin N2A associated protein phosphorylation events at 24-hour UDD. Violin plots 823 \nof phosphorylation events in N2A-associated proteins following UDD: MARP1 (Transcript: 824 \nENSMUST00000237142.2 Ankrd1-205), MARP2 (Transcript: ENSMUST00000026172.3 825 \nAnkrd2-201), Smyd2 (Transcript: ENSMUST00000027897.8 Smyd2-201), Capn3 ( Transcript: 826 \nENSMUST00000028749.15 Capn3-202), Hsp90ab (Transcript: ENSMUST00000024739.14 827 \nHsp90ab1-201), Mypn (Transcript: ENSMUST00000095580.3 Mypn-201), Hspb1 (Transcript: 828 \nENSMUST00000005077.7 Hspb1-201), Cryab (Transcript: ENSMUST00000217475.2 Cryab-829 \n206) and Prkca/PKA (Transcript: ENSMUST00000005606.8 Prkaca-201 ). Data represented as 830 \nLog2 of the normalized abundance with significance determined by Kolmogorov-Smirnov test.  831 \n-2\n-1\n0\n1\n2\n3\nMARP1 S16\nNormalized abundance\n(Z-score)\n✱✱✱✱\n-3\n-2\n-1\n0\n1\n2\n3\nMARP2 S29\n✱\n-3\n-2\n-1\n0\n1\n2\n3\nMARP2 S36\n✱\n-3\n-2\n-1\n0\n1\n2\nSmyd2 S283\n✱✱✱\n-3\n-2\n-1\n0\n1\n2\nSmyd2 S284\n✱✱✱\n-2\n-1\n0\n1\n2\nCapn3 S6\n✱✱✱\n-2\n-1\n0\n1\n2\n3\nCapn3 T8\n✱\n-2\n-1\n0\n1\n2\nCapn3 S19\n✱✱✱\n-2\n0\n2\n4\nHsp90ab S226\nNormalized abundance\n(Z-score)\n✱\n-2\n-1\n0\n1\n2\n3\n4\nHsp90ab S255\n✱✱\n-2\n-1\n0\n1\n2\n3\nHspb1 S13\n✱✱✱✱\n-2\n-1\n0\n1\n2\n3\n4\nHspb1 S15\n✱✱✱✱\n-2\n-1\n0\n1\n2\n3\nHspb1 S86\n✱✱✱\n-2\n-1\n0\n1\n2\n3\nHspb1 S102\n✱\n-2\n-1\n0\n1\n2\n3\nHspb1 T155\n✱\n-2\n-1\n0\n1\n2\n3\nHspb1 S158\nNormalized abundance\n(Z-score)\n✱\n-2\n-1\n0\n1\n2\n3\nHspb1 S159\n✱\n-2\n-1\n0\n1\n2\n3\nHspb1 S160\n✱\n-2\n-1\n0\n1\n2\n3\nHspb1 S162\n✱\n-2\n-1\n0\n1\n2\n3\nHspb1 S180\n✱✱✱✱\n-2\n-1\n0\n1\n2\n3\nCryab S59\n✱✱✱✱\n-2\n-1\n0\n1\n2\n3\nCryab T63\n✱✱✱\n-1\n0\n1\n2\n3\n4\nCryab S66\n✱✱✱\n-2\n-1\n0\n1\n2\n3\nMypn S194\n✱✱✱\nSham UDD\n-2\n-1\n0\n1\n2\n3\n4\nMypn S248\nNormalized abundance\n(Z-score)\n✱✱✱✱\nSham UDD\n-2\n-1\n0\n1\n2\n3\nMypn T249\n✱✱✱✱\nSham UDD\n-2\n-1\n0\n1\n2\nMypn S252\n✱\nSham UDD\n-2\n-1\n0\n1\n2\n3\n4\nMypn S253\n✱✱✱✱\nSham UDD\n-2\n-1\n0\n1\n2\n3\nMypn S255\n✱✱✱✱\nSham UDD\n-2\n-1\n0\n1\n2\n3\nMypn S256\n✱✱✱✱\nSham UDD\n-2\n-1\n0\n1\n2\nMypn S903\n✱\nSham UDD\n-4\n-2\n0\n2\n4\nPKA S339\n✱✱✱\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint \n\nS FIGURE 6.  6-day UDD on double KO mice of MARPs. Double KO of MARP1/2 (A), MARP1/3 832 \n(B) and MARP2/3 (C) all showed a reduction in hypertrophy following UDD, suggesting 833 \nredundancy between the MARPs. Left panel, diaphragm right costal mass normalized to tibial 834 \nlength and right panel, percentual increase in right costal mass relative to sham. S= Sham, U= 835 \nUDD (n=10-12). 836 \n 837 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted June 25, 2025. ; https://doi.org/10.1101/2025.06.19.660595doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}