Neonatal brachial plexus injury (NBPI) causes disabling and incurable muscle contractures that result from impaired longitudinal growth of denervated muscles. This deficit in muscle growth is driven by increased proteasome-mediated protein degradation, suggesting a dysregulation of muscle proteostasis. The myostatin (MSTN) pathway, a prominent muscle-specific regulator of proteostasis, is a putative signaling mechanism by which neonatal denervation could impair longitudinal muscle growth, and thus a potential target to prevent NBPI-induced contractures. Through a mouse model of NBPI, our present study revealed that pharmacologic inhibition of MSTN signaling induces hypertrophy, restores longitudinal growth, and prevents contractures in denervated muscles of female but not male mice, despite inducing hypertrophy of normally innervated muscles in both sexes. Additionally, the MSTN-dependent impairment of longitudinal muscle growth after NBPI in female mice is associated with perturbation of 20S proteasome activity, but not through alterations in canonical MSTN signaling pathways. These findings reveal a sex dimorphism in the regulation of neonatal longitudinal muscle growth and contractures, thereby providing insights into contracture pathophysiology, identifying a potential muscle-specific therapeutic target for contracture prevention, and underscoring the importance of sex as a biological variable in the pathophysiology of neuromuscular disorders. Injury to the brachial plexus at birth  is the most common cause of upper limb paralysis in children , occurriUsing a mouse model of NBPI, we previously discovered that contractures result from impaired longitudinal muscle growth, as characterized by the overstretch/elongation of sarcomeres in denervated muscles . Such deOne such signaling mechanism specific to skeletal muscle is the myostatin (MSTN) pathway. MSTN, also known as growth differentiation factor-8 (GDF8), is a myokine and a member of the transforming growth factor-β (TGF-β) superfamily . In normHence, in the current study, we seek to elucidate the role of MSTN in the formation of denervation-induced muscle contractures. Using a soluble decoy receptor (ACVR2B-Fc) to inhibit ligand binding of MSTN to ACVR2B , we specWhile pharmacologic inhibition of MSTN signaling with the soluble ACVR2B-Fc decoy receptor has been shown to induce robust muscle growth in adult mice , it has To elucidate the contribution of the MSTN pathway in modulating contracture formation, we began by investigating the effects of ACVR2B-Fc treatment on neonatally denervated forelimb muscles. In contrast to normally innervated neonatal muscles, pharmacologic blockade of MSTN signaling reduced muscle loss of denervated brachialis and biceps muscles only in female mice . In deneDue to the differential effects in denervated muscle growth, we speculated that the MSTN signaling pathway may mediate contracture formation in a sex-dependent manner. Indeed, we discovered that ACVR2B-Fc treatment improved passive range of motion in both elbow and shoulder joints only in female mice 4 weeks post-NBPI . Close sSince our prior work revealed that impaired longitudinal muscle growth following neonatal denervation is driven by increased levels of proteasome-mediated protein degradation , we subsHaving ruled out synthesis as an underlying mechanism for the observed sex dimorphisms, we next investigated the role of degradation, the other side of proteostasis. While total levels of K48-linked polyubiquitin were not different between normally innervated and denervated biceps muscles, we observed increased polyubiquitination of proteins greater than 40 kDa , similarHaving observed a role for sex in MSTN-dependent contracture formation, we revisited our prior findings of long-term proteasome inhibition in preventing contractures . Here, wSecondary muscle contractures in pediatric neuromuscular disorders such as NBPI and cerebral palsy are major drivers of joint immobility, limb deformity, and physical dysfunction and disability . As exisIn our present study, we pharmacologically targeted MSTN, a prominent muscle-specific negative regulator of proteostasis, and identified several key findings regarding MSTN signaling in muscle growth and contracture pathophysiology. First, we discovered that pharmacologic MSTN inhibition with ACVR2B-Fc enhanced the size and protein content of normally innervated forelimb muscles in 1-month-old mice at P33, which is equivalent to the completion of neonatal muscle development in human infants . In compSkeletal muscle exhibits high levels of sex dimorphisms. Female and male muscles typically display heterogeneity in body composition and protein turnover , regenerOur finding of sex-specific reductions in proteasome activity with MSTN inhibition compelled us to thoroughly scrutinize our prior results on proteasome inhibition with bortezomib through the lens of sex differences . Here, wNevertheless, our collective findings indicate that different upstream pathways in contracture formation between sexes ultimately converge at the 20S proteasome, thereby validating the role of the proteasome as a key mediator of NBPI-induced contractures. Despite these insights, the precise mechanism(s) by which MSTN signaling modulates proteasome activity in denervated female muscles remains to be determined. While we observed altered Akt/mTOR and Smad2/3 signaling with NBPI, which are corroborated by studies from other groups using different mouse denervation models . MSTN inAdditionally, it is unclear how proteostasis dysregulation and contractures are mediated in denervated male muscles. To this end, it is conceivable to postulate a role for sex hormones in contracture pathophysiology post-NBPI. An earlier study on juvenile frogs reported a loss of laryngeal muscle fibers only in male frogs following surgical denervation, whereas androgen treatment resulted in an additive effect on total fiber numbers only in innervated female laryngeal muscles . These rThe finding of sex dimorphism in MSTN signaling itself is not without precedent. Targeted deletion of MSTN during skeletal maturity increased masseter mass and bite force in male mice only . In contOur discovery of sex dimorphisms in denervated muscle growth also calls into question prior conclusions on the role of MSTN signaling in denervation-induced atrophy from studies analyzing only male mice. In a juvenile mouse model of sciatic nerve resection, MacDonald et al. reported an inability of both rapamycin and ACVR2B-Fc treatment to prevent atrophy in male mice, and surmised that denervation atrophy is not Akt/mTOR or MSTN-dependent . These fAdditionally, we observed that the ACVR2B-Fc decoy receptor attenuated growth of other non-skeletal muscle tissues, including the heart, spleen, and bone. Indeed, the broad ligand specificity of ACVR2B-Fc increases the risk of toxicity to non-skeletal muscle tissues and cell types, ranging from epistaxis to telangiectasias . Our obsFor a more nuanced interpretation of our findings, we must also consider the specificity of the ACVR2B-Fc decoy receptor as a ligand trap, as mentioned above. Previous studies have reported that many members of the TGF-β superfamily, such as Activin A and GDF-11, are capable of binding Activin Type IIB receptors . This brIn conclusion, we established several critical insights in this current study that enhance our understanding of contracture pathophysiology and establish the framework for future investigations. First, the efficacy of MSTN inhibition at rescuing contractures, at least in female mice, reinforces the role of skeletal muscle biology in contracture formation and provides proof of concept for potential muscle-specific therapies for contracture prevention and treatment. Second, the sex-specific improvement in longitudinal muscle growth is associated with a corresponding reduction in proteasome activity, offering further evidence that the proteasome is a key regulator of muscle contractures. Third, the rescue in longitudinal growth without perturbations in the canonical signaling pathways with which MSTN regulates proteostasis suggests a potential non-canonical pathway governing muscle length. Lastly, the discovery of sex dimorphisms in our current study highlights the importance of sex in the pathophysiology of denervation atrophy, muscle growth, and pediatric muscle contractures. Moving forward, we strongly advocate the inclusion of sex as a biological variable in both mechanistic and translational studies on muscle atrophy and acquired neuromuscular diseases.To assess the role of MSTN signaling in pediatric muscle contracture formation, we utilized our surgical mouse model of postganglionic NBPI where injury to the brachial plexus at postnatal day (P)5 induces contractures within 4 weeks after denervation . This tyBeginning 1 day prior to surgery and continuing for 4 weeks after surgery, we pharmacologically inhibited the MSTN pathway by treating wildtype neonatal mice with a soluble decoy receptor fused to an Fc domain  . The bin2 asphyxiation, whereupon passive range of motion of the elbow and shoulder joints were assessed to determine contracture severity. Prior to sacrifice, mice were fasted for 4 hr and then administered 21.8 mg/kg puromycin (Sigma-Aldrich #P7255) intraperitoneally for 30 min, which gets incorporated into newly formed peptide chains (Mice were euthanized at 4 weeks post-NBPI (P33) by COe chains . Immediae chains . This prFollowing digital photography, we harvested bilateral biceps muscles (long head only), bilateral triceps muscles, hearts, and spleens. Biceps, hearts, and spleens were also weighed. Both biceps and triceps were then flash frozen and stored at −80°C for subsequent analysis of protein dynamics and signaling pathways. The remaining forelimbs  were positioned at 90° elbow flexion on cork, and imaged with digital radiographs for humerus length. Bilateral forelimbs were subsequently fixed in 10% formalin at 90° elbow flexion to avoid sarcomere relaxation with muscle removal. Following 48 hr of fixation, bilateral brachialis muscles were dissected, soaked in 25% Lugol solution (Sigma-Aldrich #32922) overnight, and imaged by micro computed tomography (MicroCT) for assessment of whole-muscle cross-sectional area and volume . BrachiaMicroCT was performed using a Siemens Inveon PET/SPECT/CT Scanner  as previously described . The conAlthough the functional length of a whole muscle is defined by its total number of sarcomeres in series, morphological constraints restrict its direct measurement . To overFollowing overnight recovery in PBS at 4°C, the brachialis muscles were digested in 15% sulfuric acid for 30 min, and then dissected into muscle bundles for imaging with differential interference contrast (DIC) microscopy at ×40 on a Nikon Ti-E SpectraX widefield microscope. Six images representing different muscle bundles of the same brachialis were acquired per muscle. Average sarcomere length of the brachialis was subsequently determined by measuring a series of 10 sarcomeres from each of the six representative DIC images with the AxioVision program as previously described , with blTo quantify humerus lengths from digital radiographs, AxioVision software was used to measure the distance between the proximal humerus physis to the distal articular surface . Whole-mg for 10 min at 4°C, the amount of protein in supernatants was quantified using Bradford protein assay. Muscle homogenates were then heated at 65°C for 30 min, separated on 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels (10–30 µg of proteins), and transferred at 4°C to Immobilon-fluorescently labeled (Immobilon-FL) polyvinylidene fluoride (PVDF) membranes. Homogenates from adult mouse plantaris muscles that had been subjected to 7 days of mechanical overload were included in each gel as positive controls  , and procontrols . Membrancontrols .To determine the effects of MSTN inhibition on proteasome activity, we assayed for 20S proteasome subunit catalytic activity with blinding to treatment groups as previously described . BrieflyRegarding the use of different muscles for analyses of protein dynamics compared to physiological endpoints, the dissection of fiber bundles for sarcomere length assessment described above prevents any subsequent processing of the brachialis. Furthermore, while the biceps long head was processed for puromycin incorporation and total levels of K48 polyubiquitin, the limited amounts of protein available in denervated biceps precluded their use for assessment of ubiquitin ligases and the 20S proteasome subunits. Consequently, triceps muscles were used to assess muscle-specific ubiquitin ligases, proteasome activity, and the various signaling pathways described below.To assess the effects of MSTN inhibition on downstream pathways, we assayed for Akt/mTOR and Smad2/3 signaling, with blinding to treatment groups. Briefly, triceps muscle homogenates were heated at 65°C for 30 min, separated on 10% SDS–PAGE gels (20 µg of proteins), and transferred at 4°C to PVDF membranes (Immobilon-FL) . Membrant-tests . For all continuous data sets that contained at least four independent samples, outliers were detected a priori by Grubbs’ test and excluded. To compare differences in values between two data sets, all data were subsequently tested for normality with the Shapiro–Wilk test. Normally distributed data were compared with unpaired two-tailed Student’s t-tests , whereas protocols (#2020-0067) of the Cincinnati Children’s Hospital Medical Center, and every effort was made to minimize suffering. This study also adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) 2.0 guidelines, and a checklist is provided with the manuscript.All data generated or analyzed during this study are included in the manuscript and supporting files. This paper will be of interest to scientists within the field of neuromuscular disorders and has potential clinical relevance. The authors test a novel targeted strategy to reduce the sequelae of neonatal brachial plexus injury in a mouse model. The key claims of the manuscript are well supported by the data, and the approaches used are thoughtful and rigorous. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Christopher Cardozo as Reviewing Editor and Reviewer #1, and the evaluation has been overseen Mone Zaidi as the Senior Editor.Thank you for submitting your article "Sex-Specific Role of Myostatin Signaling in Neonatal Muscle Growth, Denervation Atrophy, and Neuromuscular Contractures" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) The reviewers have raised a number of important points that must be addressed to improve the clarity and completeness of the manuscript. Some of these were raised by more than one reviewer. Please carefully consider each of these points as you revise the manuscript.Reviewer #1 (Recommendations for the authors):The original description of the ActRIIB-Fc ligand trap from Acceleron reported on ligands bound by the drug which included Activin A and, possibly GDF11. The specificity of the test agent should be discussed.The authors should comment on whether their data on K48 polyubiquitination and proteasomal catalytic activity are internally consistent. Generally, reductions in proteasome catalytic activity lead to the accumulation of polyubiquitinated substrates unless the substrate is one of the small number of proteins that are degraded or processed without prior ubiquitination.When interpreting the data on proteasomal activity, the authors should discuss the possible factors that might explain why caspase-like or chymotrypsin-like activities would change independently. One would expect these to change in a coordinated manner if they were simply due to changes in the total content of the 20S proteasome. However, these substrates are most likely also cleaved by subunits of the immunoproteasome though with different kinetics. Proteasomes exhibit both cooperativity and extreme sensitivity to allosteric regulators such as the protein complexes described by Rechsteiner and colleagues and sodium dodecyl sulfate.Reviewer #2 (Recommendations for the authors):In the Introduction section:– Lines 73-74: The authors state that NBPI occurs in approximately 1.5 of every 1,000 live births. Because the article focuses partly on sex dimorphism, the reviewer wonders if the authors could provide epidemiological data about girls and boys.– Lines 107-109: The authors state "In normally innervated skeletal muscles, ligand binding of MSTN to Activin A through its Type 2 receptors (ACVR and ACVR2B) activates the downstream Smad proteins 2 and 3". The reviewer thinks that there is a mistake. To its best knowledge, the reviewer thinks that Mstn does not bind to Activin A. Mstn and Activin A are able to bind to the same receptor though. The reviewer suggests revising it.– Line 109: Please replace "ACVR" with "ACVR2". The reviewer suggests adding that ACVR is also known as ActRIIA and that ACVR2B is also known as ActRIIB.– Line 114: The authors state that "it is possible that MSTN is a signaling pathway by which denervation impairs muscle growth". The reviewer wonders what the links would be between MSTN, denervation, and contractures, and how it could impede muscle growth.– Line 121: In the sentence "to inhibit ligand binding of MSTN to Activin A", the reviewer feels there is a mistake/typo and does not clearly understand. Did the authors want to write "MSTN or Activin A"? Did the authors want to write "MSTN to ACVR2B"? The reviewer suggests rephrasing it.In the Results section:– Line 143: The authors state that it "has been shown to induce robust muscle growth in adult mice". The reviewer wonders if the authors could provide an appropriate reference for this statement.– Line 153/Figure 1G: The reviewer wonders why the total protein levels in the biceps muscle were not normalized with muscle weight, because usually if the muscle is bigger, the total protein levels should be higher .– Lines 157-158: The authors state that "it facilitates greater protein anabolism and growth in female muscles.". While the data looks promising and show a sex difference, the reviewer thinks that the figures do not show an effect on protein anabolism itself. The authors should have a moderate view of the subject and revise this sentence accordingly.– Line 161/Supplemental Figure 1B and line 176/Supplemental Figure 2: The reviewer wonders how the authors could explain the same effects observed on the humerus length of the innervated side as the denervated side . The reviewer wonders also what the literature does say about the (pharmacologic or non-pharmacologic) blockade of MSTN signaling and bone growth, and what links could be made with the situation described here.– Lines 160-161: The reviewer feels that "" is a bit confusing. The reviewer suggests renaming it Supplemental Figures 1A-D.– Lines 175-176: The reviewer feels that "" is a bit confusing. The reviewer suggests renaming it as Supplemental Figure 2.– Figure 5: While the whole assessment of MSTN inhibition on muscle mass, muscle CSA, muscle volume, muscle protein content, and sarcomere length was performed mainly on the Brachialis muscle (and Biceps long head), the reviewer wonders why it was not used for the biochemical analysis of protein synthesis and its associated signaling pathway. While Brachialis and Biceps are the flexors, Triceps is an extensor, therefore could the authors explain why they choose to use the Triceps muscle instead?Also, the reviewer wonders why a part of the analysis is made on the Biceps long head and the other part on the Triceps muscle. How the authors can be sure that these muscles behave the same way in this context? The reviewer feels that for more precision and reliability the authors should have used the same muscle.– Figure 5F: It clearly shows an increase in the phosphorylation level of Akt in control females compared to control males. The reviewer wonders how the authors could explain this difference. What would be their hypothesis?– Figure 6: While the whole assessment of MSTN inhibition on muscle mass, muscle CSA, muscle volume, muscle protein content, and sarcomere length was performed mainly on the Brachialis muscle (and Biceps long head), the reviewer wonders why it was not used for the biochemical analysis of protein degradation. While Brachialis and Biceps are the flexors, Triceps is an extensor, therefore could the authors explain why they choose to use the Triceps muscle instead?Also, the reviewer wonders why a part of the analysis is made on the Biceps long head and the other part on the Triceps muscle. How the authors can be sure that these muscles behave the same way in this context? The reviewer feels that for more precision and reliability the authors should have used the same muscle.Murf1 and Atrogin-1. The reviewer suggests the authors provide this detailed information for a better comprehension of the hidden mechanisms.– Lines 220-223: The authors state "Since MSTN is known to activate the ubiquitin-proteasome pathway by upregulating the expression of upstream ubiquitin ligases,30,31 it was surprising that its inhibition did not reduce ubiquitination levels.". Based on their statement, the reviewer wonders why the authors did not assess the mRNA and protein levels of them. For example, – Figure 7: While the whole assessment of MSTN signaling inhibition on muscle mass, muscle CSA, muscle volume, muscle protein content, and sarcomere length was performed mainly on the Brachialis muscle (and Biceps long head), the reviewer wonders why it was not used for the biochemical analysis of the MSTN signaling pathway. While Brachialis and Biceps are the flexors, Triceps is an extensor, therefore could the authors explain why they choose to use the Triceps muscle instead?Also, the reviewer wonders why the analysis is made on the Triceps muscle alone. How the authors can be sure that these muscles behave the same way in this context? The reviewer feels that for more precision and reliability the authors should have used the same muscle.– Figure 7: In order to characterize the Smad2/3 pathway, the authors investigated the Smad3 phosphorylation level. Due to prior literature in the field, the reviewer wonders why the authors did not assess Smad2 phosphorylation level, in order to get a fully explained picture of the situation. The reviewer suggests the authors provide this detailed information for a better comprehension of the hidden mechanisms.TAK1 pathway and its downstream targets such as JNK or p38 are regulated. The reviewer also wonders how the Ras pathway and its downstream targets such as ERK1/2 are regulated.– Lines 232-234: The authors state "Our collective findings thus posit a sex-specific role for MSTN in muscle proteostasis dysregulation after neonatal denervation through non-canonical signaling pathways."? While the reviewer agrees based on the data provided, the reviewer wonders why the authors did not investigate further in that direction. The reviewer wonders how the – It is known that MSTN has inhibitory effects on the Wnt/β-catenin pathway, thereby blunting satellite cell proliferation. Besides Wnt/β-catenin signaling plays a crucial role in myoblast fusion. In this context of MSTN signaling inhibition, the reviewer wonders how this Wnt/β-catenin signaling is regulated and what sort of effect it can have on longitudinal muscle growth.– Lines 245: The authors wrote "", but they forgot to call Figures 8G-H in the manuscript. The reviewer suggests revising it accordingly.In the Discussion section:– Line 272: The authors state "we pharmacologically targeted MSTN". While this is true, the authors cannot forget that it targets also the signaling mediated by Activin A. The reviewer thinks that it should appear in the whole manuscript.– Line 333: Please replace "NPBI" with "NBPI".– Lines 361-362 to 372: The authors state "These discrepancies might be attributable to sex-related differences of MSTN expression itself in skeletal muscles.". The reviewer wonders how the MSTN, Activin A, ACVR2, and ACVR2B expression is, in both females and males here. Also, the reviewer wonders what is the level of expression of MSTN, Activin A, and ACVR2 and ACVR2B expression in the Brachialis, Biceps, and Triceps. Could the authors imagine that there is also a muscle-dependent regulation?In a previous study, the authors looked at the effects of bortezomib over different periods . Here, the authors studied the effects of ACVR2B-Fc treatment over a period of 4 weeks. The reviewer wonders why they did not repeat the same durations of experimentation. The reviewer thinks it would be helpful to have this detailed information for having a better comprehension of the observed effect.Also, the reviewer wonders what the effect of combining bortezomib and ACVR2B-Fc treatment would be. What would the authors hypothesize?In the Materials and methods section:– Lines 456-458: The authors state "The binding of MSTN to the Fc domain inhibits MSTN from binding to Activin A, which blocks MSTN activity in the muscle". The reviewer thinks that it should be ACVR2B instead of Activin A. The reviewer suggests revising it.– Line 495: Please replace "micro-computed tomography" with "micro-computed tomography (MicroCT)".– Line 550: Please replace "PVDF" with "polyvinylidene fluoride (PVDF)".– Line 553: Please replace "BSA" with "bovine serum albumin (BSA)".– Lines 569-570: Please replace "β-1" with "β1".– Line 570: Please replace "β-5" with "β5".– Line 580: Please replace "SDS-PAGE" with "sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)".– Line 598: Please replace "ANOVA" with "analysis of variance (ANOVA)".– Line 602: Please replace "SD" with "standard deviation (SD)".– Line 613: Please replace "ARRIVE" with "Animal Research: Reporting of in vivo Experiments (ARRIVE)".In the Figure Legends section:– Line 650: Please replace "micro-CT" with "MicroCT".– Lines 656-658: The reviewer feels that (E) and (H) are misplaced in the sentence "Despite this, (E), (H) the increases in muscle volume, muscle weight, and protein levels were larger in females than males when compared to their respective DPBS controls."– Line 663: Please replace "micro-CT" with "MicroCT". Essential revisions:1) The reviewers have raised a number of important points that must be addressed to improve the clarity and completeness of the manuscript. Some of these were raised by more than one reviewer. Please carefully consider each of these points as you revise the manuscript.Reviewer #1 (Recommendations for the authors):The original description of the ActRIIB-Fc ligand trap from Acceleron reported on ligands bound by the drug which included Activin A and, possibly GDF11. The specificity of the test agent should be discussed.The ActRIIB-Fc ligand trap is indeed capable of binding different ligands, including Activin A and GDF11. This broad specificity confers both advantages and adverse effects – the ligand trap is capable of eliciting more robust muscle growth than MSTN-specific agents while concomitantly increasing the risk of toxicity to other tissues and cell types. Given this, we have included a discussion on the specificity of the ligand trap in our revised manuscript, and provided appropriate sources for additional clarification.69”Discussion (lines #479-481): “Indeed, the broad ligand specificity of ACVR2B-Fc increases the risk of toxicity to non-skeletal muscle tissues and cell types, ranging from epistaxis to telangiectasias.74,75 This broad array of targeted ligands makes the decoy receptor a potent inhibitor of not only MSTN, but also an inhibitor of various TGF-β family members that signal downstream of Activin binding. Of note, simultaneous inhibition of MSTN and Activin A leads to more effective muscle hypertrophy and force production in rodents and primates.76 Consequently, this ligand trap putatively elicits more robust increases in skeletal muscle growth than MSTN specific-agents.19 In our current study, it is possible that Activin A is differentially expressed and/or blocked with ACVR2B-Fc between sexes, which may account for the sex dimorphisms observed in neonatally denervated muscles. Indeed, a sex-specific role for Activin A in pancreatic ductal adenocarcinoma (PDAC)induced cachexia has recently been reported by Zhong et al. (2022). The authors observed that sex-specific differences in endogenous levels of Activin A contribute to sex dimorphisms in PDAC-induced cachexia, as well as the differential outcomes of ACVR2BFc treatment in attenuating tumor-induced Activin.77 Alternatively, we also wonder whether Activin A and/or other TGF-β family members are more acutely involved in the regulation of neonatal longitudinal muscle growth and contractures than MSTN. Future work with genetic models and expression profiles for the various TGF-β superfamily members are necessary to address these limitations in our current study.”Discussion (lines #511-531): “For a more nuanced interpretation of our findings, we must also consider the specificity of the ACVR2B-Fc decoy receptor as a ligand trap, as mentioned above. Previous studies have reported that many members of the TGF-β superfamily, such as Activin A and GDF-11, are capable of binding Activin Type IIB receptors.The authors should comment on whether their data on K48 polyubiquitination and proteasomal catalytic activity are internally consistent. Generally, reductions in proteasome catalytic activity lead to the accumulation of polyubiquitinated substrates unless the substrate is one of the small number of proteins that are degraded or processed without prior ubiquitination.We agree that our data on unaltered K48 polyubiquitination and reduced proteasomal catalytic activity in female mice after treatment do appear paradoxical, but as the reviewer pointed out, they are not without precedent. The phenomenon of ubiquitin-independent proteasomal degradation has been supported by several prior studies in a small number of proteins. These proteins share common features of belonging to the Intrinsic Disordered Protein (IDP) family and possessing intrinsically disordered regions (IDR). We have expanded our discussion to include this possibility.46,47 such as p21 and p53.48-50 Since these proteins and their associated intrinsically disordered regions (IDRs) critically regulate various cellular processes, including transcriptional regulation, posttranslational modification, and molecular signaling,51 it is possible that neonatal muscle denervation induces a direct degradation of these substrates. If so, the sex-specific reduction in proteasome catalytic activity with MSTN inhibition may be due to the preservation of these IDPs, which would not lead to an accumulation of polyubiquitinated substrates. It remains unclear what specific protein substrates are polyubiquitinated with NBPI, and future studies are needed to address this question.”Discussion (lines #342-354): “Our seemingly paradoxical finding of reduced proteasomal catalytic activity in the absence of any alterations in K48 polyubiquitin is not without precedent. Indeed, the phenomenon of ubiquitin-independent proteasomal degradation has been supported by several prior studies in a subset of proteins, which share common features of belonging to the Intrinsic Disordered Protein (IDP) family,When interpreting the data on proteasomal activity, the authors should discuss the possible factors that might explain why caspase-like or chymotrypsin-like activities would change independently. One would expect these to change in a coordinated manner if they were simply due to changes in the total content of the 20S proteasome. However, these substrates are most likely also cleaved by subunits of the immunoproteasome though with different kinetics. Proteasomes exhibit both cooperativity and extreme sensitivity to allosteric regulators such as the protein complexes described by Rechsteiner and colleagues and sodium dodecyl sulfate.We thank the reviewer for raising this point about coordinated changes among the proteasome subunits. We cannot be certain whether the enzymatic changes arise due to total changes in proteasome content, or reflect changes in affinities to specific proteins involved in muscle longitudinal growth and sarcomere addition. There have also been contrasting reports on whether the subunits act independently or in synchrony. We have updated our discussion to acknowledge both possibilities, as well as the potential of different subunit affinities for specific protein substrates.52 Importantly, mutations and crystallography of yeast 20S proteasome by Groll et al. (1999) revealed that the three active β-subunits are processed autocatalytically and independently of each other, with the processing of one subunit unaffected by the inactivation of another subunit.53 The independent processing function of the individual subunits, along with their unique affinities for specific protein substrates, may explain the independent changes in enzymatic activity observed in our current and prior studies. Although, we acknowledge that our findings contradict observations of allosteric interactions among the active subunits observed by Kisselev et al. (1999), specifically between the caspase-like and chymotrypsin-like sites and vice versa, in eukaryotic proteasomes.54 Additional studies are required to reconcile the independent changes of the subunits with total changes in proteasome content, as we continue to develop insights on proteasome function in contracture pathophysiology.”Discussion (lines #375-393): “Alternatively, the independent alterations in caspase-like and chymotrypsin-like activities could potentially reflect different affinities of the two enzymatic processes for specific sarcomeric, cytoskeletal, or regulatory proteins involved in muscle growth and sarcomere addition. This notion is supported by Kisselev et al. (2006), who showed that the unique amino acid sequence of a protein substrate differentially determines the activities of the 20S proteolytic sites during protein degradation, thereby leading to variability in efficacy of the different inhibitors.Regarding the 20S immunoproteasome, we did observe an accompanying decrease in β1i activity in the absence of any changes in β5i activity, in our prior study with bortezomib treatment (data not reported). We did not assess these activities in our current study. To our knowledge, the overall abundance of the immunoproteasome is low in non-lymphoid tissues, and requires induction by cytokines. As such, we also cannot be certain whether the immunoproteasome contributes to the discrepancies, nor can we discount its role. Finally, we thank the reviewer for bringing to our attention the cooperativity and sensitivity exhibited by proteasomes to allosteric regulators, such as the proteasome activator PA200 described by Rechsteiner and colleagues (2002). We speculate that these characteristics were not observed in our current study potentially because MSTN acts upstream of the ubiquitin-proteasome system. Thus, we suspect the decoy receptor effects upstream changes in signaling that regulates proteasome subunit activity independently, rather than binding to the site of an allosteric enzyme on the subunit as an allosteric effector would. As such factors are highly speculative at this point, we would prefer not to include them in the manuscript.Reviewer #2 (Recommendations for the authors):In the Introduction section:– Lines 73-74: The authors state that NBPI occurs in approximately 1.5 of every 1,000 live births. Because the article focuses partly on sex dimorphism, the reviewer wonders if the authors could provide epidemiological data about girls and boys.This is a relevant question given our discovery of sex dimorphisms in the pathophysiology of contracture formation. The ratio of NBPI occurrence between girls and boys is approximately 1:1 . We have included this epidemiological data in our revised Introduction.1 occurring in approximately 1.5 of every 1,000 live births, in both females and males at an equal rate.2”Introduction (lines #72-74): “Injury to the brachial plexus at birth  is the most common cause of upper limb paralysis in children,– Lines 107-109: The authors state "In normally innervated skeletal muscles, ligand binding of MSTN to Activin A through its Type 2 receptors (ACVR and ACVR2B) activates the downstream Smad proteins 2 and 3". The reviewer thinks that there is a mistake. To its best knowledge, the reviewer thinks that Mstn does not bind to Activin A. Mstn and Activin A are able to bind to the same receptor though. The reviewer suggests revising it.– Line 109: Please replace "ACVR" with "ACVR2". The reviewer suggests adding that ACVR is also known as ActRIIA and that ACVR2B is also known as ActRIIB.The reviewer is correct in saying that MSTN and Activin A are capable of binding to the ACVR2/ACVR2B receptor, as they are both members of the TGF-β superfamily. We have corrected this error, replaced “ACVR” with “ACVR2”, and included the alternative names of ActRIIA and ActRIIB in our revised Introduction.16,17”Introduction (lines #109-112): “In normally innervated skeletal muscles, ligand binding of MSTN to its Type 2 receptors, ACVR2 (ActRIIA) and ACVR2B (ActRIIB), activates the downstream Smad proteins 2 and 3.– Line 114: The authors state that "it is possible that MSTN is a signaling pathway by which denervation impairs muscle growth". The reviewer wonders what the links would be between MSTN, denervation, and contractures, and how it could impede muscle growth.We have revised our Introduction to improve the clarity of the links between MSTN, denervation, and contractures.13 which leads to impaired longitudinal muscle growth that ultimately causes contractures.9-12 However, the mechanism by which neonatal denervation increases protein degradation is unknown. Given that MSTN is a potent inhibitor of muscle growth through its mediation of muscle proteostasis, we speculate that MSTN signaling may be a mechanistic pathway linking denervation with protein degradation, leading to impaired muscle growth and ultimately contractures.”Introduction (lines #117-124): “As mentioned, we previously discovered that neonatal denervation increases protein degradation in denervated muscles,– Line 121: In the sentence "to inhibit ligand binding of MSTN to Activin A", the reviewer feels there is a mistake/typo and does not clearly understand. Did the authors want to write "MSTN or Activin A"? Did the authors want to write "MSTN to ACVR2B"? The reviewer suggests rephrasing it.Thank you for bringing this to our attention. We indeed meant to write “MSTN to ACVR2B,” as noted by the reviewer.20-23 we specifically investigated whether pharmacologic inhibition of MSTN signaling preserves longitudinal muscle growth and prevents contractures after NBPI.”Introduction (lines #130): “Using a soluble decoy receptor (ACVR2B-Fc) to inhibit ligand binding of MSTN to ACVR2B,In the Results section:– Line 143: The authors state that it "has been shown to induce robust muscle growth in adult mice". The reviewer wonders if the authors could provide an appropriate reference for this statement.We have included the appropriate citation for this statement .20 it has not been validated in neonatal mice.”Results (lines #150): “While pharmacologic inhibition of MSTN signaling with the soluble ACVR2B-Fc decoy receptor has been shown to induce robust muscle growth in adult mice,– Line 153/Figure 1G: The reviewer wonders why the total protein levels in the biceps muscle were not normalized with muscle weight, because usually if the muscle is bigger, the total protein levels should be higher .To our knowledge, total muscle protein content is a fairly standard marker of muscle hypertrophy . The reviewer is correct that total protein levels should generally track with muscle weight, thus it serves as an additional readout to compare muscle growth. We have analyzed protein levels according to muscle weight as recommended, and recorded the new observations in our revised manuscript. Normalized protein levels were not changed with MSTN inhibition in either normally innervated or denervated muscles, although treatment led to higher levels in denervated female vs. male muscles.Results (lines #160-162): “There were no changes in protein levels normalized to muscle weight with treatment .”Results (lines #183-186): “Additionally, while MSTN inhibition did not elevate protein levels normalized to muscle weight in denervated muscles beyond the respective sexspecific controls, it led to higher levels in female than male muscles .”Figure legends (lines #904-906): “Figure 1—figure supplement 1: Off-target effects of pharmacologic MSTN inhibition. (A) MSTN inhibition does not alter protein levels in normally innervated biceps when normalized to muscle weight.”Figure legends (lines #913-916): “Figure 2—figure supplement 1: Effect of pharmacologic MSTN inhibition on normalized protein levels and skeletal growth in denervated forelimb. (A) MSTN inhibition does not alter protein levels in denervated biceps when normalized to muscle weight.”– Lines 157-158: The authors state that "it facilitates greater protein anabolism and growth in female muscles.". While the data looks promising and show a sex difference, the reviewer thinks that the figures do not show an effect on protein anabolism itself. The authors should have a moderate view of the subject and revise this sentence accordingly.We thank the reviewer for this cautionary advice and have revised the sentence accordingly.Results (lines #167): “These results indicate that while neonatal inhibition of MSTN signaling effectively enhances skeletal muscle growth in both sexes, it facilitates greater increases in protein content and muscle weight in female muscles.”– Line 161/Supplemental Figure 1B and line 176/Supplemental Figure 2: The reviewer wonders how the authors could explain the same effects observed on the humerus length of the innervated side as the denervated side . The reviewer wonders also what the literature does say about the (pharmacologic or non-pharmacologic) blockade of MSTN signaling and bone growth, and what links could be made with the situation described here.We thank the reviewer for bringing these observations to our attention. We interpret the collective results here to indicate that ACVR2B-Fc attenuates neonatal skeletal growth exclusively in male mice, adding to the list of sex dimorphisms reported in our study. We state this finding more explicitly in our revised manuscript.Results (lines #189-192): “Similar to the contralateral forelimbs, MSTN inhibition in male mice resulted in an attenuation of skeletal growth in the denervated humerus . These parallel observations suggest that ACVR2B-Fc attenuates neonatal skeletal growth exclusively in male mice.”Regarding prior literature, Hamrick and colleagues (2002) found that embryonic genetic deletion of MSTN affected humerus morphology but not length in adult mice. However, these authors used a mixed-sex approach that may have masked sex-specific effects of MSTN deletion. We have revised our manuscript to include this prior literature.71”Discussion (lines 497-500): “Prior studies have failed to find an effect of embryonic MSTN deletion on humerus length in adult mice, although the use of a mixed-sex approach may have masked sex-related differences.Beyond comparison with prior findings, the deficits in bone growth of male mice strengthens the sex-specific phenotype we observed with MSTN inhibition. We would like to take this opportunity to reiterate a point we had discussed in our original manuscript that, biomechanically, a shorter bone should reduce contracture severity and result in an increase of normalized muscle size. The absence of such findings in male mice thus further underscores the sex-specificity of MSTN inhibition in denervated muscles (lines #491-497).– Lines 160-161: The reviewer feels that "" is a bit confusing. The reviewer suggests renaming it Supplemental Figures 1A-D.– Lines 175-176: The reviewer feels that "" is a bit confusing. The reviewer suggests renaming it as Supplemental Figure 2.While we concur with the reviewer’s feedback, we are unable to adopt this suggestion due to specific instructions from the editorial staff on naming our supplemental figures. We thank the reviewer for his/her understanding in this matter.Results (lines #169-170): Figure Legends (line #904-905): Figure 1—figure supplement 1: Off-target effects of pharmacologic MSTN inhibition.Results (line #186): Figures Legends (line #913-915): Figure 2—figure supplement 1: Effect of pharmacologic MSTN inhibition on normalized protein levels and skeletal growth in denervated forelimb.– Figure 5: While the whole assessment of MSTN inhibition on muscle mass, muscle CSA, muscle volume, muscle protein content, and sarcomere length was performed mainly on the Brachialis muscle (and Biceps long head), the reviewer wonders why it was not used for the biochemical analysis of protein synthesis and its associated signaling pathway. While Brachialis and Biceps are the flexors, Triceps is an extensor, therefore could the authors explain why they choose to use the Triceps muscle instead?Also, the reviewer wonders why a part of the analysis is made on the Biceps long head and the other part on the Triceps muscle. How the authors can be sure that these muscles behave the same way in this context? The reviewer feels that for more precision and reliability the authors should have used the same muscle.– Figure 6: While the whole assessment of MSTN inhibition on muscle mass, muscle CSA, muscle volume, muscle protein content, and sarcomere length was performed mainly on the Brachialis muscle (and Biceps long head), the reviewer wonders why it was not used for the biochemical analysis of protein degradation. While Brachialis and Biceps are the flexors, Triceps is an extensor, therefore could the authors explain why they choose to use the Triceps muscle instead?Also, the reviewer wonders why a part of the analysis is made on the Biceps long head and the other part on the Triceps muscle. How the authors can be sure that these muscles behave the same way in this context? The reviewer feels that for more precision and reliability the authors should have used the same muscle.– Lines 220-223: The authors state "Since MSTN is known to activate the ubiquitin-proteasome pathway by upregulating the expression of upstream ubiquitin ligases,30,31 it was surprising that its inhibition did not reduce ubiquitination levels.". Based on their statement, the reviewer wonders why the authors did not assess the mRNA and protein levels of them. For example, Murf1 and Atrogin-1. The reviewer suggests the authors provide this detailed information for a better comprehension of the hidden mechanisms.– Figure 7: While the whole assessment of MSTN signaling inhibition on muscle mass, muscle CSA, muscle volume, muscle protein content, and sarcomere length was performed mainly on the Brachialis muscle (and Biceps long head), the reviewer wonders why it was not used for the biochemical analysis of the MSTN signaling pathway. While Brachialis and Biceps are the flexors, Triceps is an extensor, therefore could the authors explain why they choose to use the Triceps muscle instead?Also, the reviewer wonders why the analysis is made on the Triceps muscle alone. How the authors can be sure that these muscles behave the same way in this context? The reviewer feels that for more precision and reliability the authors should have used the same muscle.We appreciate the reviewer’s concern and thorough scrutiny of our findings. We agree that using the same muscle type for all physiological and biochemical analyses would ideally allow for greater precision. Our selection of the different forelimb muscles was ultimately based on both methodological constraints and tissue availability. As described in the Materials and methods, we digested the brachialis muscles to isolate fiber bundles for assessment of sarcomere length. Following this terminal physiological measurement, the brachialis muscle cannot be used for subsequent biochemical analyses. We therefore utilized the biceps (long head) muscle to analyze protein dynamics, specifically levels of protein synthesis and K48 polyubiquitination. However, the limited amounts of protein available in the denervated biceps precluded their use for assessment of signaling pathways and proteasome activity. We therefore used the triceps muscle for these latter analyses. Despite being an extensor as the reviewer pointed out, it is important to note that the triceps muscle is completely denervated in our surgical mouse model of postganglionic NBPI, which essentially denervates the entire forelimb. Thus, the triceps muscle is subjected to the same denervation condition as the elbow flexors. In our revised manuscript, we provide an explanation for our choice of the different forelimb muscles in the Materials and methods, and acknowledge that using the same muscles may improve the precision of our findings in the Discussion.Murf1, and Atrogin-1). We therefore acknowledge that our study may be underpowered to detect smaller effects in certain parameters of protein dynamics, specifically signaling proteins and ubiquitin ligases. We also acknowledge that the precision of our findings would be further enhanced with the use of the same muscle type across all of our morphological, physiological, and biochemical analyses.”Discussion (lines #431-470): “First, the small size of our denervated muscles precluded the use of the same muscles for all analyses, instead requiring smaller subgroup sizes as well as different muscles for certain biochemical endpoints : “This type of injury completely denervates all muscles in the forelimb, including flexors , and extensors (triceps).”Materials and methods (lines #708-715): “Regarding the use of different muscles for analyses of protein dynamics compared to physiological endpoints, the dissection of fiber bundles for sarcomere length assessment described above prevents any subsequent processing of the brachialis. Furthermore, while the biceps long head was processed for puromycin incorporation and total levels of K48 polyubiquitin, the limited amounts of protein available in denervated biceps precluded their use for assessment of ubiquitin ligases and the 20S proteasome subunits. Consequently, triceps muscles were used to assess muscle-specific ubiquitin ligases, proteasome activity, and the various signaling pathways described below.”– Figure 5F: It clearly shows an increase in the phosphorylation level of Akt in control females compared to control males. The reviewer wonders how the authors could explain this difference. What would be their hypothesis?We thank the reviewer for this astute observation. Our finding here suggests that Akt signaling in normally innervated muscles is mediated in a sex-specific manner. This sex dimorphism in Akt signaling is not without precedent. Camper-Kirby and colleagues (2001) similarly reported elevated Akt signaling in the myocardium of young women and juvenile female mice (~6w) compared to young men and juvenile male mice, respectively. However, the mechanisms for these sex-specific differences may vary between muscles. In cardiac muscles of female mice, Camper-Kirby et al. reported a 2-fold increase in phosphorylated Akt levels when corrected for loading (P<0.01). Conversely, we observed a non-statistically significant 4-fold increase in corrected total Akt levels in skeletal muscles of male mice. As increased total levels of p70s6k  is potentially indicative of enhanced overall protein translation , we speculate the increased level of Akt similarly points to increased protein production. This speculation is corroborated by the higher levels of total protein  in normally innervated skeletal muscles of neonatal male mice. In this manner, sex dimorphisms in hypertrophic signaling pathways putatively account for the greater protein content and overall mass in male skeletal muscles. As this hypothesis is untested, we prefer to exclude the preceding discussion from our manuscript unless its inclusion is specifically requested.– Lines 220-223: The authors state "Since MSTN is known to activate the ubiquitin-proteasome pathway by upregulating the expression of upstream ubiquitin ligases,30,31 it was surprising that its inhibition did not reduce ubiquitination levels.". Based on their statement, the reviewer wonders why the authors did not assess the mRNA and protein levels of them. For example, Murf1 and Atrogin-1. The reviewer suggests the authors provide this detailed information for a better comprehension of the hidden mechanisms.Murf1 and Atrogin-1, and observed MSTN inhibition does not alter MuRF-1 or Atrogin-1 protein levels in neonatal skeletal muscles. Surprisingly, four weeks of denervation led to reduced Murf1 expression but did not alter Atrogin-1 translation. Since we previously reported increased transcriptional activity of Murf1 two weeks after NBPI , it is conceivable there is a defined window for the upregulation of muscle-specific ubiquitin ligases after NBPI. Unfortunately, we do not have samples from the current experiments at earlier timepoints to assess the kinetics of these upstream ubiquitin ligases. Lastly, we have processed all available forelimb muscles for our various physiological and biochemical endpoints, and as such, do not have viable muscle samples for RNA extraction to determine the transcriptional activities of these ligases. We are unable to add these experiments to our manuscript, and have included a statement on the need for a more extensive investigation of ubiquitin ligases in future studies.We have performed western blots for both Murf1 and Atrogin-1, . Unexpectedly, denervation decreased Murf1 and did not change Atrogin-1 levels at 4 weeks post-NBPI, which differ from our earlier reports of increased transcriptional activity of Murf1 at 2 weeks post NBPI.13 While there is a need to better characterize the precise kinetics of these upstream ubiquitin ligases over time following denervation, we show that downstream levels of ubiquitination are ultimately elevated with denervation. The relationship between ubiquitin ligase expression, ubiquitination, and proteasome activity in neonatally denervated muscle must be more extensively investigated.”Results (lines #245-254): “Further investigation revealed that MSTN inhibition did not alter protein levels of the muscle-specific E3 ligases, 13 we wonder if there is a transient temporal window for the upregulation of muscle-specific ligases after NBPI. Thus, a more extensive investigation of ubiquitin ligase expression and function in future studies will provide a better of their role in neonatal denervation and muscle growth.”Discussion (lines #354-359): “Additionally, since we observe discrepancies in the temporal expression of upstream ubiquitin ligases in our current and prior studies,Murf1Materials and methods (lines #678-690): “Membranes were subsequently blocked in 5% bovine serum albumin (BSA) in TRIS-buffered saline (TBS)-Tween (pH 7.5), and incubated overnight at 4°C with an antibody against puromycin , K48-linkage-specific-polyubiquitin , Murf1 (10 µg), and Atrogin-1 protein levels (10 µg) were quantified using the Image Studio Lite program (LI-COR Biosciences), and normalized to corresponding GAPDH protein levels.13,78”, or Atrogin-1 . GAPDH  served as a control for sample loading. Membranes were then washed and incubated with IRDye 680CW anti-goat or 800CW anti-mouse IgG2a  orDyLight anti-rabbit  secondary antibodies. Following image detection on the Odyssey infrared detection system (LI-COR Biosciences), the relative abundance of puromycin incorporation (30 µg), K48-linked protein levels (30 µg), Murf1, and (C) Atrogin-1 showed MSTN inhibition does not alter protein levels in denervated (NBPI) or contralateral triceps muscles. Data are presented as mean ± SD, n = 3 independent mice. (B), (C) 3-way ANOVA for sex, treatment, and denervation (repeated measures between forelimbs) with a Bonferroni correction for multiple comparisons. *P<0.05.”Figure Legends (lines #926-932): “Figure 6—figure supplement 1: Assessment of muscle-specific ubiquitin ligases. (A) Representative western blots and quantitative analyses of (B) – Figure 7: In order to characterize the Smad2/3 pathway, the authors investigated the Smad3 phosphorylation level. Due to prior literature in the field, the reviewer wonders why the authors did not assess Smad2 phosphorylation level, in order to get a fully explained picture of the situation. The reviewer suggests the authors provide this detailed information for a better comprehension of the hidden mechanisms.We have performed western blots for Smad2 signaling, and updated the findings in our manuscript accordingly. Our results here show MSTN inhibition does not reduce Smad2 signaling in denervated muscles, adding to our speculation that MSTN targets the proteasome independent of the Smad2/3 pathway.Results (lines #261-264): “Here, we observed sex-independent increases in both Smad2 and Smad3 phosphorylation and translation with neonatal denervation, which were not further altered with MSTN inhibition .”Materials and methods (lines #721-726): “Membranes were subsequently blocked in 5% BSA/TBS-Tween, and incubated overnight at 4°C with an antibody against phosphorylated Akt (Ser473) , total Akt , phosphorylated Smad2 (Ser465/467) , total Smad2 , phosphorylated Smad3  (1:2000 Abcam #ab51451), or total Smad3 .”Materials and methods (lines #730): “Western blot signals were subsequently imaged and the relative abundance of phosphorylated and total protein levels of Akt, Smad2, and Smad3 were quantified as described above.”Figure Legends (lines #872-882): “Figure 7: Sex-specific differences in MSTNmediated proteostasis dysregulation occurs independent of Smad2/3 signaling. (A) Representative western blots and quantitative analyses of (B) pSmad2, (C) total Smad2, (E) pSmad3, and (F) total Smad3 revealed that ACVR2B-Fc treatment does not blunt the denervation-induced increase in activity and translation of Smad2 and Smad3 in triceps muscles of both sexes. Quantification of the western signal for (D) pSmad2 and (G) pSmad3 normalized to total protein levels further indicated that MSTN inhibition does not alter Smad2/3 signaling in neonatally denervated muscles. n = 3-6 independent mice. Data are presented as mean ± SD. Statistical analyses: (B), (C), (D), (E), (F), (G) 3-way ANOVA for sex, treatment, and denervation (repeated measures between forelimbs) with a Bonferroni correction for multiple comparisons. *P<0.05, **P<0.01.”– Lines 232-234: The authors state "Our collective findings thus posit a sex-specific role for MSTN in muscle proteostasis dysregulation after neonatal denervation through non-canonical signaling pathways."? While the reviewer agrees based on the data provided, the reviewer wonders why the authors did not investigate further in that direction. The reviewer wonders how the TAK1 pathway and its downstream targets such as JNK or p38 are regulated. The reviewer also wonders how the Ras pathway and its downstream targets such as ERK1/2 are regulated.We thank the reviewer for these excellent suggestions. As noted in our response to the Public Review above, we will certainly be following up on our current findings by rigorously interrogating these pathways downstream of MSTN signaling in future studies. We have included a statement in our revised discussion to acknowledge this.TAK1 and its downstream targets JNK and p38, as well as Ras and its downstream targets MEK1 and ERK1/2.”Discussion (lines #505-507): “Additional non-canonical alternatives downstream of MSTN signaling that should be further explored include – It is known that MSTN has inhibitory effects on the Wnt/β-catenin pathway, thereby blunting satellite cell proliferation. Besides Wnt/β-catenin signaling plays a crucial role in myoblast fusion. In this context of MSTN signaling inhibition, the reviewer wonders how this Wnt/β-catenin signaling is regulated and what sort of effect it can have on longitudinal muscle growth.We have previously ruled out a role for satellite cell-mediated myonuclear accretion in longitudinal muscle growth . Briefly, we found that satellite cellspecific genetic ablation of Myomaker, a muscle-specific fusogen, does not alter sarcomere length in both normally innervated and denervated muscles. Hence, we believe it is unlikely that MSTN signaling governs longitudinal muscle growth through modulation of satellite cell activity with the Wnt/β-catenin pathway. We apologize for omitting this prior finding, and have revised our Introduction to include this salient point.13”Introduction (lines #86-87): “Such deficits in muscle length are independent of satellite cell-mediated myonuclear accretion, but are instead caused by aberrant levels of muscle protein degradation associated with increased catalytic activity of the 20S proteasome.– Lines 245: The authors wrote "", but they forgot to call Figures 8G-H in the manuscript. The reviewer suggests revising it accordingly.We apologize for this oversight. We have corrected this error and properly referenced the appropriate figures.Results (lines #274-276): “Lastly, while bortezomib did not alter β5 subunit activity in either sex , it attenuated β1 subunit activity at 8 and 12w only in male mice .”In the Discussion section:– Line 272: The authors state "we pharmacologically targeted MSTN". While this is true, the authors cannot forget that it targets also the signaling mediated by Activin A. The reviewer thinks that it should appear in the whole manuscript.The reviewer is correct that the ligand trap targets Activin A signaling, as well as other members of the TGF-β superfamily. As noted in our response to item #1 from Reviewer 1, we more clearly address the limitations of the ligand trap and how its broad specificity may affect the interpretation of our findings in our revised Discussion. While the ACVR2B-Fc decoy receptor binds to many members of the TGF-β superfamily, we have kept the focus of this extended discussion on Activin A.69”Discussion (lines #479-481): “Indeed, the broad ligand specificity of ACVR2B-Fc increases the risk of toxicity to non-skeletal muscle tissues and cell types, ranging from epistaxis to telangiectasias.74,75 This broad array of targeted ligands makes the decoy receptor a potent inhibitor of not only MSTN, but also an inhibitor of various TGF-β family members that signal downstream of Activin binding. Of note, simultaneous inhibition of MSTN and Activin A leads to more effective muscle hypertrophy and force production in rodents and primates.76 Consequently, this ligand trap putatively elicits more robust increases in skeletal muscle growth than MSTN specific-agents.19 In our current study, it is possible that Activin A is differentially expressed and/or blocked with ACVR2B-Fc between sexes, which may account for the sex dimorphisms observed in neonatally denervated muscles. Indeed, a sex-specific role for Activin A in pancreatic ductal adenocarcinoma (PDAC)induced cachexia has recently been reported by Zhong et al. (2022). The authors observed that sex-specific differences in endogenous levels of Activin A contribute to sex dimorphisms in PDAC-induced cachexia, as well as the differential outcomes of ACVR2BFc treatment in attenuating tumor-induced Activin.77 Alternatively, we also wonder whether Activin A and/or other TGF-β family members are more acutely involved in the regulation of neonatal longitudinal muscle growth and contractures than MSTN. Future work with genetic models and expression profiles for the various TGF-β superfamily members are necessary to address these limitations in our current study.”Discussion (lines #511-531): “For a more nuanced interpretation of our findings, we must also consider the specificity of the ACVR2B-Fc decoy receptor as a ligand trap, as mentioned above. Previous studies have reported that many members of the TGF-β superfamily, such as Activin A and GDF-11, are capable of binding Activin Type IIB receptors.– Line 333: Please replace "NPBI" with "NBPI".We apologize for this typographical error. We have corrected this acronym in our revised manuscript.55-57 MSTN inhibition failed to impact these canonical pathways in female mice.”Discussion (line #399): “While we observed altered Akt/mTOR and Smad2/3 signaling with NBPI, which are corroborated by studies from other groups using different mouse denervation models,– Lines 361-362 to 372: The authors state "These discrepancies might be attributable to sex-related differences of MSTN expression itself in skeletal muscles.". The reviewer wonders how the MSTN, Activin A, ACVR2, and ACVR2B expression is, in both females and males here. Also, the reviewer wonders what is the level of expression of MSTN, Activin A, and ACVR2 and ACVR2B expression in the Brachialis, Biceps, and Triceps. Could the authors imagine that there is also a muscle-dependent regulation?We share the same questions regarding expression of these TGF-β superfamily members and their receptors in our neonatal muscles. In order for relevant conclusions to be drawn on potential sex-specific and muscle-dependent differences in expression, we would require an in-depth analysis of additional data from future studies. We have included a statement in our revised Discussion to reflect this point.Discussion (lines #445-448): “Future studies should perform a comprehensive analysis of the expression of MSTN and other TGF-β superfamily members (such as Activin A), as well as the associated receptors (ACVR2 and ACVR2B) across sexes and muscle groups to account for potential sex- and/or muscle-dependent regulation.”In a previous study, the authors looked at the effects of bortezomib over different periods . Here, the authors studied the effects of ACVR2B-Fc treatment over a period of 4 weeks. The reviewer wonders why they did not repeat the same durations of experimentation. The reviewer thinks it would be helpful to have this detailed information for having a better comprehension of the observed effect.Also, the reviewer wonders what the effect of combining bortezomib and ACVR2B-Fc treatment would be. What would the authors hypothesize?We thank the reviewer for these excellent suggestions. As noted in our response to the Public Review above, we will certainly be following up on our current findings by integrating these pharmacological approaches and rigorously interrogating their longterm efficacy in preventing contractures.The focus of this study is to establish a proof of concept that targeting a muscle-specific regulator of proteostasis (MSTN) can effectively prevent contractures during the critical window of neonatal muscle growth (4 weeks). As we have established that MSTN inhibition does prevent contractures in a sex-specific manner during this stage of development, ongoing studies are currently planned to build on our findings here by interrogating the efficacy of this pharmacological approach in preventing contractures at skeletal maturity and beyond (8 and 12 weeks). These studies also include combining bortezomib with ACVR2B-Fc over different periods, which conceptually, would optimize contracture prevention and truncate the window of necessary treatment in both sexes. Beyond this broad speculation, we would prefer not to discuss our specific hypotheses for these experiments in the manuscript.Discussion (lines #532-541): “Finally, we cannot yet comment on the long-term efficacy of MSTN inhibition beyond four weeks following denervation. However, the focus of this study was to establish a proof of concept that targeting a muscle-specific regulator of proteostasis (MSTN) can effectively prevent contractures during the critical window of neonatal muscle growth (4 weeks). Furthermore, we have previously established that contracture formation plateaus at 4 weeks post-NBPI, making this time point ideal for such a proof of concept investigation. As we have established that MSTN inhibition does prevent contractures in a sex-specific manner during this stage of development, ongoing studies are currently planned to build on our findings here by interrogating the efficacy of this pharmacological approach in preventing contractures at skeletal maturity and beyond.”In the Materials and methods section:– Lines 456-458: The authors state "The binding of MSTN to the Fc domain inhibits MSTN from binding to Activin A, which blocks MSTN activity in the muscle". The reviewer thinks that it should be ACVR2B instead of Activin A. The reviewer suggests revising it.We apologize for the misunderstanding and have revised this sentence accordingly.20”Materials and methods (line #581): “The binding of MSTN to the Fc domain inhibits MSTN from binding to ACVR2B, which blocks MSTN activity in the muscle, ultimately leading to increased muscle protein synthesis and robust muscle growth.– Line 495: Please replace "micro-computed tomography" with "micro-computed tomography (MicroCT)".We have made the suggested revision.Materials and methods (line #619): “micro computed tomography (MicroCT)”– Line 550: Please replace "PVDF" with "polyvinylidene fluoride (PVDF)".– Line 580: Please replace "SDS-PAGE" with "sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)".We have made the suggested revisions.Materials and methods (lines #672-676): “Muscle homogenates were then heated at 65°C for 30 minutes, separated on 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (10-30 µg of proteins), and transferred at 4°C to Immobilon-fluorescently labeled (Immobilon-FL) polyvinylidene fluoride (PVDF) membranes.”– Line 553: Please replace "BSA" with "bovine serum albumin (BSA)".We have made the suggested revision.Materials and methods (line #679): “Membranes were subsequently blocked in 5% bovine serum albumin (BSA)”– Lines 569-570: Please replace "β-1" with "β1".– Line 570: Please replace "β-5" with "β5".We have made the suggested revisions.Materials and methods (lines #698): “The caspase-like activity of the 20S proteasome β1 catalytic subunit and chymotrypsin-like activity of the β5 catalytic subunit were assayed with 10 μg total protein per muscle”– Line 598: Please replace "ANOVA" with "analysis of variance (ANOVA)".We have made the suggested revision.Materials and methods (lines #739-740): “For data sets with two independent variables (sex and treatment), a 2-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons was performed.”– Line 602: Please replace "SD" with "standard deviation (SD)".We have made the suggested revision.Materials and methods (line #743): “All data are presented as mean ± standard deviation (SD).”– Line 613: Please replace "ARRIVE" with "Animal Research: Reporting of in vivo Experiments (ARRIVE)".We have made the suggested revision.Materials and methods (lines #755-756): “This study also adhered to the Animal Research: Reporting of in vivo Experiments (ARRIVE) 2.0 guidelines, and a checklist is provided with the manuscript.”In the Figure Legends section:– Line 650: Please replace "micro-CT" with "MicroCT".– Lines 656-658: The reviewer feels that (E) and (H) are misplaced in the sentence "Despite this, (E), (H) the increases in muscle volume, muscle weight, and protein levels were larger in females than males when compared to their respective DPBS controls."We thank the reviewer for bringing this to our attention. The original placement of (E) and (H) was indeed incorrect. We have corrected the placement to improve the readability of this sentence.Figure Legends (lines #795-797): “Despite this, the increases in (E) muscle volume, and (H) muscle weight and protein levels were larger in females than males when compared to their respective DPBS controls.”


The main goal of our study was to evaluate the surgical technique, the feasibility and patient’s satisfaction of multiple surgeries: Risk-reducing salpingo-oophorectomy (RRSO) combined with mastectomy in patients with BRCA 1–2 mutation carriers. We conducted a retrospective analysis of patients with BRCA 1–2 variants who underwent RRSO combined with risk-reducing bilateral mastectomy (RRBM) or surgeries for breast cancer from January-2015 to December-2021. We collected data about surgeries, complications, and patients’ satisfaction using a questionnaire submitted 30 days after surgery. We included 54 patients. Forty-eight patients underwent RRSO, and six patients underwent RRSO + Total laparoscopic hysterectomy (LTH). The minor postoperative complications within 30 days were four: one breast seromas aspiration (1.9%), one infectious reconstructive complication treated with antibiotics therapy (1.9%), one Red-Breast-Syndrome (1.9%) and one trocar abdominal hematoma (1.9%) associated with RRSO. The major postoperative complications within 30 days were five: two evacuations of a breast hematoma (3.7%) and three infectious reconstructive complications treated with removal expander/implant (5.6%). No postoperative complications after 30 days were observed. According to the satisfaction questionnaire, more than 90% of patients were satisfied and would have combined surgery again. In conclusion, the multiple surgeries seem feasible and safety with a single anesthesia, a single surgical time, a single postoperative recovery, and a high patients’ satisfactions without increasing morbidity. Germline mutations in the BRCA1 and BRCA2 genes correlate in the development of most cases of hereditary breast and ovarian cancer ,4; whereAccording to the National Comprehensive Cancer Network (NCCN), the risk-reducing salpingo-oophorectomy (RRSO) and the risk-reducing bilateral mastectomy (RRBM) can decrease the lifetime risk of developing OC by more than 95% and BC by 85–90% in patients with BRCA1–2 mutation carriers ,11,12. SWe conducted an observational retrospective cohort study on patients with documented BRCA1–2 germline variants who had undergone RRSO combined with RRBM or therapeutic surgeries for a new or previously diagnosis of BC at Clinic of Gynecology and Obstetrics of University of Padova-Department of Women and Children’s Health and Breast Cancer Unit of Veneto Institute of Oncology IOV–IRCCS.The surgeries were performed between January 2015 to December 2021. All women enrolled in this study had a proven germline BRCA1–2 variants. All patients signed a document approved by our institution for the anonymous use of their clinical data for scientific purposes according to the European privacy law. The institutional review board approved the study (87n/AO/21).The inclusion criteria were as follows: patients with BRCA1–2 variants diagnosed through genetic testing performed by blood sampling; patients that underwent RRSO combined with breast surgery in the setting of a new diagnosis of BC and/or in the setting of RRBM combined, according to NCCN guidelines ; asymptoPatients were identified through our institution computer database initiated to collect clinical and surgical information at the point of care. For each patient the investigators reviewed the electronic hospital records and pathology reports to determine study eligibility, patients’ general features and histopathological features. In cases of missing information, investigators directly contacted the patient by telephone or email to complete the data collection.We collected data on age, menopausal status at RRSO, family history of BC and OC, surgical technique of mastectomy  and reconstructive details (one stage/two stages reconstruction), time of breast surgeries and RRSO and duration of hospitalization. We also collected data about surgical perioperative complications subdivided into intraoperative complications, early postoperative complications (in the first 30 days) and late postoperative complications . We divided the complications into a major when an added surgeries occurred  and a minor when a patient required antibiotics, local therapy, or blood transfusion.During the first follow-up visits at 30 days, we submitted to all patients a questionnaire to evaluate general subjective satisfaction with the combined surgical approach.The primary endpoint of this study was to evaluate the surgical technique, the safety and feasibility of combined surgeries: RRSO with simultaneous (i) Bilateral mastectomy: RRBM or therapeutic surgery with or without immediate reconstruction or (ii) Unilateral mastectomy: prophylactic or therapeutic surgery with or without immediate reconstruction. Other endpoints were the following: evaluate patients’ satisfaction with the combined surgical approach, postoperative pain and recovery time.The statistical analysis was performed with the SPSS Software  for Windows version 26. Continuous variables  were expressed in absolute numbers and mean ± standard deviation, Categorical variables were expressed as absolute numbers and percentages.The surgery was subdivided into two surgical times: the first performed by a gynaecologist and the second by a general surgeon and a plastic surgeon, during the same anaesthesiologic session. In particular:(I) RRSO is a minimally invasive surgical procedure performed laparoscopically following NCCN guidelines . The pat(II) RRBM or therapeutic surgery were performed as follows: we used different types of mastectomies, including skin-sparing mastectomy (SSM) and nipple-areolar sparing mastectomy (NSM) with or without immediate breast reconstruction. In SSM most of the breast skin was conserved to create a pocket that facilitates immediate breast reconstruction with implant to achieve a quality cosmetic outcome. Instead, in NSM the nipple-areola complex (NAC) was conserved. For an SSM, the incision may be a small ellipse around the NAC or a circular incision with or without a lateral extension if needed. NSM can be performed via a variety of incisions  depending on breast shaping. When necessary, sentinel node biopsy was performed through a separate axillary incision. Implant-based  reconstruction was performed in one or two stages: (i) one-stage reconstruction where a permanent implant was inserted at the time of mastectomy; (ii) two-stage reconstruction where a tissue expander was placed following the mastectomy and a permanent implant was used to replace the tissue expander at a later date. Prosthetic devices were placed under  or above  the pectoralis major muscle.We formulated a simple questionnaire based on 5-point Likert scale for question 1 and 2 and based on a binomial answer (yes/no) for the question 3, 4 and 5.Question 1: How satisfied was she with the combined surgery? .Question 2: Do you believe that the postoperative pain was increased by the combined surgery? (yes/no).Question 3: How much was the overall impact of post-operative gynecological pain? .Question 4: Do you believe that the post-operative recovery period was prolonged by having performed the combined surgery? (yes/no).Question 5: Would you undergo combined surgery again? (yes/no).A total of 54 BRCA1–2 variants women underwent combined surgery from January 2015 to December 2021 at our institution. Specifically, 32 patients (59.3%) carried the BRCA1 variant, and 22 patients (40.7%) carried the BRCA2 variant. n = 41; 76%) underwent immediate breast reconstruction with implant-based reconstruction  patients. Specifically, 22 (40%) patients underwent RRBM, and 25 (47%) patients underwent therapeutic surgery for BC. The majority of patients  patients. In particular, 4 (7.4%) patients underwent prophylactic unilateral mastectomy, and 3 (5.6%) patients underwent therapeutic surgery for BC. Six (11%) patients underwent immediate breast reconstruction with implant-based reconstruction. Regarding gynecological surgery 48 (88.9%) patients underwent only laparoscopic RRSO and 6 (11.2%) patients underwent RRSO with concomitant laparoscopic total hysterectomy (LTH) (see The median duration of the entire procedure (mastectomy + RRSO) was 231 ± 50 min and mean duration of RRSO was 46 (32–60) minutes. The mean duration of the entire procedure (mastectomy + RRSO + LTH) was 278 (200–345) minutes and mean duration of RRSO + LTH was 105 (90–140) minutes. The average inpatient hospital stay was 3 days (range 1–9 days). All combined surgeries were performed under a single anesthetic session : (i) breast seromas aspiration  (ii) infectious reconstructive complications treated with antibiotics therapy without removal expander/implant  and (iii) fever and skin infection . Only one  minor complication was associated with gynecological surgery represented by trocar abdominal hematoma treated conservatory without evacuation. Instead, we reported 5 early major postoperative complications that included: (i) evacuation of a breast hematoma ; and (ii) infectious reconstructive complications treated with removal expander/implant . No major gynecological surgery complications were observed. No patients developed clinically relevant venous thromboembolism, and no perioperative mortality was observed. No late postoperative complications >30 days were observed.Question 1. How satisfied was she with the combined surgery? Answers: 77% of patients was very satisfied and 13% was satisfied by the combined surgery; 3% of patients was neutral and 7% was unsatisfied. No patients answered very unsatisfied.Question 2. Do you believe that the postoperative pain was increased by the combined surgery? Answers: 95% of patients affirmed that the postoperative pain was not increased by the combined surgery.Question 3. How much was the overall impact of post-operative gynecological pain? Answers: 77% of patients replied Not very much and 13% Not much; 8% of patients was neutral and 2% replied very much.Question 4. Do you believe that the post-operative recovery period was prolonged by having performed the combined surgery? Answers: 88% of patients affirmed that the post-operative recovery period was not prolonged by having performed combined surgery.Question 5. Would you undergo combined surgery again? Answers: 92% of patients affirmed yes.; however, is important to underline that almost all complications were classified as minor (treated conservatory) and only 5 patients required second surgery. The only gynecologic complication was represented by an early minor post-operative complication treated conservatory. The higher complication rate after breast surgery can be explained by the longer duration of this surgery compared to RRSO and the increased infectious risk related to reconstruction ,26. On tThe total complication rate reported by literature ranged from 18–44% ,22,23,24In 2008, Batista et al. published one of the first study about 12 patients that underwent combined surgery demonstrating the safety and efficacy of a free-flap-Y based breast reconstruction with simultaneous RRSO . SimilarIn literature, two interesting case-control studies were published with a control group who underwent mastectomies without RRSO. The two studies showed the safety of performing combined procedures with perforator-flap breast reconstruction ,23 with In terms of the secondary endpoint, our study was the first in the literature to assess patient satisfaction, postoperative pain, and recovery time. In our populations, 90% of patients were satisfied with the combined surgery, 95% agreed that the combined surgery did not increase postoperative pain, and 93% said they would have it done again.The main strength of the present study is that this is one of the largest trials on this topic and it was the first trial that evaluated also patients’ satisfaction of the combined procedures. Other points of strength are certainly related to rigorous data collection methodology and strict inclusion criteria; all information were collected from our electronic hospital records, which are compiled by clinicians at each step of patient’s treatment. We included exclusively patients referred from diagnosis to treatment to our institute, excluding any sources of bias related to heterogeneous surgical choices and procedures. Despite these strengths, our results have some limitations deriving by the retrospective nature of the study and by the fact that our institution presented a long history of interdisciplinary combined surgery, therefore its applicability to other settings is unclear. Finally, another limitation is represented by the lack of a cost-efficacy analysis. However, concerning this last point it is clear that with the same complication rate and expenses related to their management, one hospitalization with two combined surgical procedures with a single anaesthesia versus two hospitalizations with two separate surgical procedures and anaesthesia, can represent a sure saving for the institution.Breast surgery combined with RRSO for BRCA1–2 mutation carriers is a feasible and safe approach that reduces the number of hospitalizations and anesthetic administrations while allowing a single patient to recover postoperatively without increasing morbidity. Considering the high patient satisfaction, combined surgery is a viable option for patients with BRCA 1–2 mutation carriers. Certainly, prospective large studies with adequate long follow-up and evaluation of the protective effect against BC with RRSO are still mandatory.


To the editor,+) as the donor. While PARylation plays an important role in DNA damage repair, the excessive PARylation generated by PARP1 will cause cell death via the pathway of parthanatos, a unique form of caspase-independent programmed cell death. Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme responsible for hydrolyzing poly(ADP-ribose) (PAR) chain which is produced by PARP1. It has been reported that PARG could be recruited to DSBs site in a PARP1 and PCNA-dependent manner in Hela cells using laser micro irradiation [Poly(ADP-ribose) polymerase 1 (PARP1), a most abundant member of the Poly(ADP‐ribose) polymerase (PARP) superfamily, autoPARylates itself and PARylates a huge number of target proteins in response to DNA single-stranded breaks (SSB) or DNA double-stranded breaks (DSBs), such as BRCA1 . Poly and prognosis highlight the urgent need to identify new therapeutic interventions for the treatment of this disease. DSBs are the most dangerous forms of DNA lesions among various types of DNA damage, such as SSB, base damage, and nucleotide mismatch, for nearly all eukaryotic organisms. DSBs can arise from the exposure to exogenous agents, such as ultraviolet (UV) light from the sun, ionizing radiation (IR), carcinogens that are inhaled or ingested, and certain genotoxic chemicals, as well as from some endogenous biological processes, including DNA replication stresses and reactive oxygen species (ROS) which is produced as a byproduct of normal cellular metabolism. Homologous recombination (HR) and nonhomologous end joining (NHEJ) are two independent pathways responsible for repairing DNA DSBs. Unrepaired or inappropriately repaired DSBs can cause gene insertions, deletions, duplications, translocations, and chromosomal rearrangements. The giant mutations in genes arising from impaired DSBs could activate oncogenes, inactivate tumor suppressors, or increase the genome instability and eventually give rise to cell death, tumorigenesis, tumor progression or aging-related disease. Like many other types of cancers, defects in DSBs repair and increased genome instability may contribute to the tumorigenesis and development of HCC. Therefore, it is of high significance for the depth study of the mechanisms of DSBs repair in HCC. This would provide us some better understanding of the tumorigenesis, development, and treatment of HCC.. showed that PARG inhibition could significantly suppress the development of HCC and PARG inhibitors could produce a synergistic antitumor efficacy with anti-PD-1 antibodies in HCC [Yu et als in HCC . In theiTo characterize the PARylated proteome upon DNA damage, we first performed an immunoprecipitation (IP) assay coupled with liquid chromatography-mass spectrometry (LC–MS) analysis to identify proteins that are PARylated by PARP1. The LC–MS results indicated that 6 peptides of DDB1 were detected (6.62% coverage) under the condition of X-Ray treatment, but no peptide was detected in the un-treatment group Fig. , Fig. 1bThe endogenous PARG levels of different HCC cell lines using quantitative RT-PCR and western blot indicated that PARG was highly expressed in Hep3B and HCCLM3 HCC cell lines . To furt. Our results suggested that the inhibition of PARG impaired DSBs repair in HCC cells based on the clonogenic assay and the clearance of γH2AX, which provided additional mechanistic explanations for the impaired liver tumorigenesis of PARG inhibition. The indispensable role of PARG in DSBs repair in HCC provide additional evidence of targeting PARG for the HCC therapy.In response to DNA damage, the degradation of PAR chain (dePARylation) is indispensable for the proper DNA repair in cells. PARG is the primary enzyme involved in the dePARylation process of DNA repair. It was reported PARG could regulate DNA damage repair in different cancer cells and mouse cells, but the role of PARG in DSBs repair in HCC still needed to be further explored. Our study found that DDB1 was PARylated in response to DNA damage by using LC–MS, and we also confirmed that the PARylation of DDB1 was dependent on DSBs in HCC cells by using IPAdditional file 1.


Formation of long-lasting memory lymphocytes is one of the foundational characteristics of adaptive immunity and the basis of many vaccination strategies. Following the rapid expansion and contraction of effector CD8 T cells, the surviving antigen (Ag)-specific cells give rise to the memory CD8 T cells that persist for a long time and are phenotypically and functionally distinct from their naïve counterparts. Significant heterogeneity exists within the memory CD8 T cell pool, as different subsets display distinct tissue localization preferences, cytotoxic ability, and proliferative capacity, but all memory CD8 T cells are equipped to mount an enhanced immune response upon Ag re-encounter. Memory CD8 T cells demonstrate numerical stability under homeostatic conditions, but sepsis causes a significant decline in the number of memory CD8 T cells and diminishes their Ag-dependent and -independent functions. Sepsis also rewires the transcriptional profile of memory CD8 T cells, which profoundly impacts memory CD8 T cell differentiation and, ultimately, the protective capacity of memory CD8 T cells upon subsequent stimulation. This review delves into different aspects of memory CD8 T cell subsets as well as the immediate and long-term impact of sepsis on memory CD8 T cell biology. Populations of memory CD8 T cells can be maintained for their entire lifetime of the host once formed, and these cells confer protection against intracellular infections and mediate antitumor immunity –5. GenerSepsis is defined as an exaggerated immune response to a systemic infection that leads to organ dysfunction . The disSepsis is a challenging health crisis affecting nearly 50 million people annually, with a mortality rate of approximately 20%. It disproportionally affects the elderly; 75% of sepsis-related mortality occurs in individuals above 65 –32. On tMEM) cells following the rapid expansion/contraction of effector CD8 T cells -specific memory CD8 T  CD8 T cells, which have been classically subdivided into two subsets of CD62Llo CCR7lo effector (TEM) and CD62Lhi CCR7hi central memory (TCM) CD8 T cells   and white pulp of spleen, whereas TEM cells are more prevalent in blood, red pulp of spleen, and non-lymphoid tissues  infections more efficiently  CD8 T cells are non-lymphoid tissue-restricted TMEM cells that patrol tissues for pathogen invasion  (+ MP cells in non-lymphoid tissues (NLTs) are thought to be the major population contributing to the TRM pool  (EM)   and/or tEC) (EM) , as theyCM population. A small subset of CD62L+ TCF1+ MP cells with restrained effector-phase proliferation and expression of inhibitory receptors have been identified to give rise to a multipotent subset of TCM cells with superior recall responses  that remain poorly defined in murine models  TCIRCM cells are generated which can give rise to higher order TCIRCM cells upon additional Ag encounter. Higher order TCIRCM cells display differential tissue localization, phenotypic, and functional characteristics than 1° TCIRCM cells. With increasing number of Ag stimulations, higher order TMEM cells become more cytolytic with greater ability in trafficking to peripheral tissues, but reduced progression to a TCM phenotype, responsiveness to homeostatic cues, and proliferative capacity  shows no progressive enrichment in TEM-associated genes in 2°, 3°, or 4° TMEM cells (EM : TCM ratio.Following pathogen re-encounter and secondary expansion of primary (1°) Tcapacity –116. ‘TE tissues , 117. AlEM cells . Hence, RM cells which recruits immune cells including TRM precursors to the site of infection to generate more TRM population. Data suggested that 1° TRM cells could also proliferate upon reinfection to give rise to 2° TRM cells  CD8 T cells are more susceptible to radiation-induced apoptosis and are lost to a greater extent than TCIRCM cells . Using a vaccinia infection model to generate TCIRCM and TRM with the same Ag specificity, we found the number of ‘IV positive’ TCIRCM cells significantly declined after moderate sepsis, but the number of ‘IV negative’ skin TRM cells were held constant  method with one or two punctures was implemented to recapitulate moderate or severe sepsis, respectively  adoptively transferred into C57/Bl6 recipients have shown that the number of both Tf sepsis , 144. Rephocytes –147. ThiN cells, the composition and phenotype of the post-sepsis TN pool is altered. Reduced primary effector responses in the post-septic host indicates an incomplete repertoire recovery and a less diverse TN pool. This is indeed the case for naïve Ag-specific CD4 T cells  of TMEM cells is key in mediating pathogen clearance, in addition to their number, tissue localization, and ability to communicate with other cells being crucial for mounting a protective immune response. Thus, we will next discuss the immediate effect of sepsis on functional capacity of different subsets of the TMEM pool and finish with a description of the long-term impact of sepsis on TMEM -mediated immunity.Increased susceptibility of sepsis survivors to previously-encountered pathogens and viral reactivation insinuates compromised protection conferred by TCIRCM on a per cell basis. Particularly, sepsis diminishes the IFN-γ production in response to cognate Ag resulting in decreased Ag sensitivity and functional avidity of TCIRCM cells. In response to the cognate antigen, the compromised cytokine production and proliferative capacity of TCIRCM render septic hosts more susceptible to homologous reinfections. Nevertheless, TMEM cells do not mediate protection only in presence of their cognate Ag. When TMEM are ‘bathed’ in a highly inflammatory environment, they are activated to produce more cytokines and cytotoxic granules such as granzyme B. This ‘bystander activation’ of TMEM is Ag-independent, but inflammation-dependent -immune mice that underwent either CLP or sham surgeries, CLP hosts showed sustained high viral load and inability to clear VacV after re-challenge  results in the reduced prevalence of TCIRCM cells with greatest cytotoxic function (TEM and higher order TCIRCM cells)  subset of cells is needed for the host to mount the most robust immune response possible. Enrichment of a subset of TMEM at the expense of other subsets may substantially affect the overall fitness of TMEM pool as each subset possesses a specialized role and function.Our discussion so far has focused on describing the functional impairments with the CD8 T cell compartment that ensue after septic insult. While they shed light on factors contributing to the increased susceptibility of septic hosts early after the insult, a noticeable percentage of sepsis survivors suffer from long-lasting immunoparalysis. Our studies on the TM cells) , 144. EnRM pools . TCM oveSepsis research has shifted focus to characterizing the factors leading to the long-lasting state of immunoparalysis that emerges following the resolution of acute phase of sepsis. Since sepsis survivors show increased susceptibility to secondary and recurring infections, these studies demand an in-depth analysis of the impact of sepsis on memory lymphocytes – the body’s most potent weapon in fighting against reinfections. Circulating memory CD8 T cells undergo substantial numerical attrition and functional impairment shortly after a septic insult deriving the host susceptible to heterologous and homologous reinfections. Additionally, tissue-resident memory CD8 T cells also display a diminished ability in recruiting effector cells in response to localized re-infections. Despite the apparent numerical recovery and per cell function, circulatory memory CD8 T cells demonstrate long-lasting changes in their transcriptional and epigenetic programs after sepsis resolution, with the most proliferative subset being overrepresented over time. Therefore, sepsis ultimately leads to altered subset composition and reduced heterogeneity in memory CD8 T cells in the circulation. Further investigation is required to delineate the long-term sepsis-induced changes in function and maintenance of tissue-resident memory CD8 T cells.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.