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

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors employ diaphragm denervation in rats and mice to study titin‑based mechanosensing and longitudinal muscle hypertrophy. By integrating bulk RNA‑seq, proteomics, and phosphoproteomics, they map the stretch‑responsive signalling landscape, uncovering robust induction of the muscle‑ankyrin‑repeat proteins (MARP1‑3) together with enhanced phosphorylation of titin's N2A element. Genetic ablation of MARPs in mice amplifies longitudinal fibre growth and is accompanied by activation of the mTOR pathway, whereas systemic rapamycin treatment suppresses the hypertrophic response, highlighting mTORC1 as a key downstream effector of titin/MARP signalling.

Strengths:

The authors address a clear biological question: "how titin‑associated factors translate mechanical stretch into longitudinal fibre growth" using a unique and clinically relevant animal model of diaphragm denervation. Using a comprehensive multiomics approach, the authors identify MARPs as potential mediators of these effects and use a genetic mouse model to provide compelling evidence supporting causality. Additionally, connecting these findings to rapamycin, a drug widely used clinically, further increases the relevance and potential impact of the study.

Weaknesses:

There are several areas where the manuscript could be substantially improved.

(1) The statistical analysis of multi-omics data needs clarification. Typically, analyses across multiple experimental groups require controlling the false discovery rate (FDR) simultaneously to avoid reporting false-positive findings. It would be very helpful if the authors could specify whether adjusted p-values were calculated using a multi-factorial statistical model (e.g., ~group) or through separate pairwise contrasts.

(2) There are three separate points regarding MARP3 that could be improved. First, the authors report that MARP3-KO mice exhibit smaller increases in muscle mass after diaphragm denervation compared to wild-type mice (a -13% difference), indicating MARP3 likely promotes rather than attenuates hypertrophy. However, the manuscript currently states the opposite (lines 215-216); this interpretation should be revisited. Second, it would be valuable if the authors could provide data showing whether MARP3 transcript or protein levels change response to denervation - if they do not, discussing mechanisms behind the observed phenotype would help clarify the findings. Finally, given that some MARP-KO mice already exhibit baseline differences, employing and reporting the full two-way ANOVA ( including genotype × treatment interaction) would allow a direct statistical assessment of whether MARP deficiency modifies the muscle's response to stretch. This analysis would help clearly resolve any existing ambiguity.

(3) The current presentation of multi-omics data is somewhat difficult to follow, making it challenging to determine whether observed changes occur at the transcript or protein level due to inconsistent gene/protein naming and capitalization (e.g., proper forms are mTOR, p70 S6K, 4E-BP1). Clearly organizing and presenting transcript and protein-level changes side-by-side, especially for key molecules discussed in later experiments, would make the data more accessible and provide clearer insights into the biology of titin-mediated mechanosensing.

(4) The current analysis relies on total protein measurements downstream of mTOR, yet mTOR's primary mode of action is to change phosphorylation status. Because the authors have already generated a phosphoproteomic dataset, it would be very helpful to report - or at least comment on - whether known mTOR target phosphosites were detected and how they respond to denervation and rapamycin. Including even a brief summary of canonical sites such as S6K1 Thr389 or 4E‑BP1 Thr37/46 would make the link between mTOR activity and hypertrophy much clearer.

(5) Finally, since rapamycin blocks only a subset of mTOR signalling, a brief discussion that distinguishes rapamycin‑sensitive from rapamycin‑insensitive pathways would be valuable. Clarifying whether diaphragm stretch relies exclusively on the sensitive branch or also engages the resistant branch would place the results in a broader mTOR context and deepen the mechanistic narrative.

Reviewer #2 (Public review):

Summary:

Muscle hypertrophy is a major regulator of human health and performance. Here, van der Pilj and colleagues assess the role of the giant elastic protein, titin, in regulating the longitudinal hypertrophy of diaphragm muscles following denervation. Interestingly, the authors find an early hypertrophic response, with 30% new serial sarcomeres added within 6 days, followed by subsequent muscle atrophy. Using RBM20 mutant mice, which express a more compliant titin, the authors discovered that this longitudinal hypertrophy is mediated via titin mechanosensing. Through an omics approach, it is suggested that the Muscle ankyrin proteins may regulate this approach. Genetic ablation of MARPs 1-3 blocks the hypertrophic response, although single knockouts are more variable, suggesting extensive complementation between these titin binding proteins. Finally, it is found through the administration of rapamycin that the mTOR signalling pathway plays a role in longitudinal hypertrophic growth.

Strengths:

This paper is well written and uses an impressive suite of genetic mouse models to address this interesting question of what drives longitudinal muscle growth.

Weaknesses:

While the findings are of interest, they lack sufficient mechanistic detail in the current state to separate cross-sectional versus longitudinal hypertrophy. The authors have excellent tools such as the RBM20 model to functionally dissect mTOR signalling to these processes. It is also unclear if this process is unique to the diaphragm or is conserved across other muscle groups during eccentric contractions.