Soluble RAGE enhances muscle regeneration after cryoinjury in aged and diseased mice

Naftali Horwitz; Michael Florea; K. C. Medha; Tina Liu; Vivian Garcia; Rebekah Kim; Amy Lam; Kathleen Messemer; Christopher Rios; Albert E. Almada; Amy J. Wagers

doi:10.1371/journal.pone.0318754

Peer Review History

Original SubmissionNovember 4, 2024
4 Nov 2024 Author Response https://doi.org/10.1371/journal.pone.0318754.r001
4 Dec 2024 Decision Letter - Ram Nagaraj, Editor PONE-D-24-50348Soluble RAGE enhances muscle regeneration after cryoinjury in aged and diseased micePLOS ONE Dear Dr. Horwitz, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== ACADEMIC EDITOR:Reviewers have raised concerns about sRAGE levels, the age of the animals, and the areas of muscle examined. Please address all these issues carefully before resubmitting. ============================== Please submit your revised manuscript by Jan 18 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . 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Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors investigate the impact of soluble receptor for advanced glycation end-products (sRAGE) on the regeneration of skeletal muscle damaged by cryoinjury. They demonstrate that the expression of sRAGE improves muscle regeneration in aged mice and in atherosclerotic mouse models. Although this article contains some interesting aspects in the field of glycation, there are several issues that need to be clarified. Critique 1. In Figure 1D and G, was there a difference in the serum sRAGE concentration between the Vehicle FFB groups of 7-8-month-old and 18-month-old mice without sRAGE? Does the expression levels of sRAGE vary with age? 2. In Figure 3, why did the authors use 7-8-month-old mice, which did not show differences in CSA (cross-sectional area) as shown in Figure 1? 3. Regarding the labeling of the transgenic mice used in Figure 3A, how about providing an additional explanation in the text or the figure legend? 4. In the Discussion, the authors mention that "Earlier work has shown that RAGE ligands are chronically activated in aged MuSCs—which display reduced proliferation and differentiation [36-38]." Does this imply that sRAGE is released from MuSCs in aged individuals, and a reduction of sRAGE within MuSCs leads to insufficient sequestration of RAGE ligands, thereby negatively affecting muscle repair? Is this consistent with the statement in the Introduction that sRAGE is present in the blood of centenarians? Minor comments 1. In discussion: “In our study, our AGERs/s mice (which also lack endogenous RAGE) displayS enhanced …”. Is displayS a typo for displays? 2. sRAGE is considered to act as a decoy receptor, preventing ligands bind to RAGE and thereby inhibiting the negative effects mediated by RAGE. Would it be possible to discuss the name of AGEs that bind to RAGE or sRAGE in the main text? Reviewer #2: Horwitz et al have aimed to study the role of sRAGE in muscle regeneration after cryo/barium chloride injury in healthy, aged and diseased mice models. They have used different mouse strains to design and execute different experiments to address their objective. Overall, this is a well-designed study which provides novel insights. However, there are some concerns in the paper which needs to be addressed 1. In figure 1 (H & I), the authors state that the mean cross-sectional area (CSA) of centrally nucleated fibers increase in aged mice treated with AAV9-sRAGE relative to vehicle. Can it be corroborated whether this is a significant increase? If so, can it be related to sRAGE alone? 2. In figure 3 (I & J), the data suggest that the number of donor MuSC-derived dystropin+ fibers does not differ across different donor lines. The authors do not elaborate about this in the discussion session. Other clarifications in Materials and methods 3. Western blotting – Dilutions of Both primary and secondary antibody may be mentioned. Also mention the secondary antibody used. 4. A short description of the ELISA protocol may be provided. 5. The make of the restriction enzymes used may be provided 6. Catalog numbers and brands of all chemicals/kits/buffers may be provided ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). 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Revision 1
22 Dec 2024 Author Response Reviewer #1: 1. In Figure 1D and G, was there a difference in the serum sRAGE concentration between the Vehicle FFB groups of 7-8-month-old and 18-month-old mice without sRAGE? Does the expression levels of sRAGE vary with age? We thank the reviewer for the opportunity to further explain this point. Similar to the results in Figure1A showing no difference in endogenous sRAGE between 4- to 18-month-old mice (i.e., adult and aged animals), when we plot the FFB injected animals from young and old mice shown in Figure 1D and G on the same plot, we also observe no significant difference in serum sRAGE using a two-way ANOVA with a Šídák's multiple comparisons test. Therefore, we conclude that in sedentary C57BL/6J mice, sRAGE levels in the blood do not naturally change (up or down) during chronological aging. Please refer to the "Response to Reviewer" file for the relevant plot. 2. In Figure 3, why did the authors use 7-8-month-old mice, which did not show differences in CSA (cross-sectional area) as shown in Figure 1? We thank the reviewer for this comment. Although we didn’t find significant differences in CSA of skeletal muscle after Freeze injury between adult animals treated with AAV-sRAGE and vehicle control (Figure 1E-F), we did appreciate a trend which we anticipate may reach statistical significance in a model where there is lifelong, endogenous overexpression as seen in the sRAGE transgenic animals (Figure 3). For practical reasons, given the diverse array of mouse lines and breeding schemes required to generate the 6 different lines in Figure 3 (i.e. sRAGE transgenic x wild-type, RAGE KO x wild-type, etc.), we were limited in the scope of our colony, especially once the pandemic forced us to pause our breeding. Although we agree that investigating the older sRAGE transgenic animals would bolster our findings, we nonetheless found a significant increase in muscle CSA following Freeze injury in the adult sRAGE transgenic mice. These data ultimately support our overall hypothesis: that lifelong, endogenous overexpression of sRAGE is beneficial to skeletal muscle regeneration. 3. Regarding the labeling of the transgenic mice used in Figure 3A, how about providing an additional explanation in the text or the figure legend? We added the following sentence into the text on Pg 12, figure 3 legend, shown below: “AGER KO animals were previously generated via a cre-mediated loxP recombination of essential elements (exons 2 to 7) of the murine RAGE gene. sRAGE transgenic animals were previously generated by replacing exons 10−11 of the Ager gene, which encode the transmembrane and cytosolic signaling domains of the murine RAGE, with a bicistronic control element 2A-linked EGFP expression cassette. Therefore, this animal is a full-length RAGE-null and the resulting sRAGE transgene is expressed in RAGE-expressing cell types using its normal gene regulatory elements.” 4. In the Discussion, the authors mention that "Earlier work has shown that RAGE ligands are chronically activated in aged MuSCs—which display reduced proliferation and differentiation [36-38]." Does this imply that sRAGE is released from MuSCs in aged individuals, and a reduction of sRAGE within MuSCs leads to insufficient sequestration of RAGE ligands, thereby negatively affecting muscle repair? Is this consistent with the statement in the Introduction that sRAGE is present in the blood of centenarians? We thank the reviewer for the opportunity to clarify this point further. We believe that the elevation in blood serum concentrations of sRAGE (as seen in centenarians) is likely a compensatory mechanism by the body to sequester RAGE ligands that are chronically activated in aged skeletal muscle (including MuSCs). Whether the “higher amount of sRAGE” that centenarians have is enough to sequester all the harmful RAGE ligands and promote a regenerative benefit is unknown. Regardless, our mouse studies clearly demonstrate that the elderly bodies’ natural amount of sRAGE may not be sufficient, and indeed, boosting sRAGE in aged mammalian skeletal muscle gives the benefit of enhancing the muscle healing process. 5. In discussion: “In our study, our AGERs/s mice (which also lack endogenous RAGE) displayS enhanced …”. Is displayS a typo for displays? We thank the reviewer for catching this typo. We made the correction in the revised version of manuscript. 6. sRAGE is considered to act as a decoy receptor, preventing ligands bind to RAGE and thereby inhibiting the negative effects mediated by RAGE. Would it be possible to discuss the name of AGEs that bind to RAGE or sRAGE in the main text? We thank the reviewer for making this suggestion. We added a sentence to the discussion regarding AGE targets of RAGE/sRAGE in the revised version of the manuscript on pg 13. “While recent work suggests that RAGE/sRAGE directly interacts with various ligands known to impact muscle regeneration such as s100b, HMGB1, C1q [10,12,36,39], the role of AGEs known to interact with RAGE such as Carboxymethyl-lysine, Carboxyethyl-lysine, Methylglyoxal, Glyoxal, Pyrraline, and Pentosidine [40-45] are not as well studied in the context of skeletal muscle regeneration. Advances in technology will be needed to accurately measure AGE variants and determine (1) their abundance across lifespan and (2) determine which are most relevant for skeletal muscle pathology.” Reviewer #2 1. In figure 1 (H & I), the authors state that the mean cross-sectional area (CSA) of centrally nucleated fibers increase in aged mice treated with AAV9-sRAGE relative to vehicle. Can it be corroborated whether this is a significant increase? If so, can it be related to sRAGE alone? We welcome the opportunity to further clarify this point. Our studies found that the (1) mean cross sectional area (CSA) of myofibers (Two-Tailed, unpaired, T-test) and (2) the distribution of all centrally nucleated myofibers (CNFs) (using Mann-Whitney) are larger in the AAV9-sRAGE aged mice when compared to FFB aged control mice. These data are consistent with the idea that sRAGE supplementation enhanced muscle healing after a freeze injury. 2. In figure 3 (I & J), the data suggest that the number of donor MuSC-derived dystropin+ fibers does not differ across different donor lines. The authors do not elaborate about this in the discussion session. We thank the reviewer for pointing this out. We have included more discussion in the discussion section of our revised manuscript on pg 13. The included text is reproduced below: “Given their central role in the repair of injured muscle, we also evaluated the cell-intrinsic role of sRAGE expression on MuSCs through cell transplantations, which test the ability of these cells to engraft and contribute to the repair of the recipient muscle. Our results showed that regardless of cell number, MuSCs expressing sRAGE engrafted muscle in a similar way as wild-type MuSCs. These data suggest that sRAGE impact on MuSC activity may be through modulation of the extracellular environment that works to impede muscle repair in aging skeletal muscle.” 3. Western blotting – Dilutions of Both primary and secondary antibody may be mentioned. Also mention the secondary antibody used. We thank the reviewer for bringing this to our attention. We revised the methods section under “Western Blots” to include this information in our revised manuscript. 4. A short description of the ELISA protocol may be provided. We thank the reviewer for making this suggestions. We revised the methods section under “ELISA” to include this information in our revised manuscript. 5. The make of the restriction enzymes used may be provided We thank the reviewer for making this request. We revised the methods section under “Construction of AAV9-sRAGE” to include this information in our revised manuscript. 6. Catalog numbers and brands of all chemicals/kits/buffers may be provided We thank the reviewer for making this request. We revised the methods section under “Construction of AAV9-sRAGE” to include this information in our revised manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0318754.r003
22 Jan 2025 Decision Letter - Ram Nagaraj, Editor Soluble RAGE enhances muscle regeneration after cryoinjury in aged and diseased mice PONE-D-24-50348R1 Dear Dr. Horwitz, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Ram Nagaraj Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have addressed all of the reviewer’s questions, and this paper is now suitable for publication. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: No ******** https://doi.org/10.1371/journal.pone.0318754.r004
Formally Accepted
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