PONE-D-22-33716Fortetropin® supplementation prevents the rise in circulating myostatin but not disuse-induced muscle atrophy in young men during immobilization: a randomized controlled trialPLOS ONE

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Reviewers' comments:

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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: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: No

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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: This study aimed to investigate the impact of Fortetropin® (FOR) supplementation on immobilization-induced muscle atrophy and weakness. The research question is relevant due to the increasing interest in the scientific literature on disuse countermeasures and the promising findings of FOR supplementation in previous training studies. The authors employed an appropriate disuse model (2-week knee brace immobilization) with subsequent recovery, controlled for/assessed potential confounding factors (diet and physical activity levels) and evaluated alterations in molecular markers involved in muscle atrophy. Overall, the manuscript is clear and easy to follow. However, there are some crucial matters to consider regarding plasma myostatin concentration evaluation.

First, blood samples were collected only on day 1 (onset of the run-in phase) and day 42 (end of the recovery). Due to this limitation in study design, the authors cannot determine with certainty whether the increased myostatin concentration occurred during the immobilization phase or the recovery phase, or both. This is particularly relevant in light of a recent 10-day bed rest study that found an unexpected increase in circulating myostatin after two days of recovery, but not during the unloading period (Oranger et al., 2022). Why did the authors decide to not collect blood samples on day 14 (onset of the immobilization phase) and day 28 (end of the immobilization phase)? Blood sampling is a relatively fast procedure and less invasive compared to muscle biopsies that were obtained also at these two additional time points.

Second, caution is needed in the interpretation of findings exclusively drawn from circulating biomarkers when employing “local” disuse models, such as knee brace immobilization. Indeed, disuse effects are expected mostly at a local muscle level and results obtained from systemic biomarkers may be affected by the relatively low muscle mass undergoing unloading/immobilization (one leg only).

To overcome these issues, have the authors considered evaluating myostatin transcript and protein levels from muscle biopsies? I strongly recommend performing these additional analyses, if possible.

Other comments:

L1: In the title, the authors suggest that the rise in myostatin occurred during the immobilization phase. However, since blood samples were not collected on days 14 and 28 the authors cannot be completely sure of this (see my main comment).

L28: If word count allows it, I suggest the authors specify that the recovery phase in this study consisted just of the resumption of habitual physical activities.

L34-39: The authors reported just one percentage of reduction and p-value for each muscle force and mass parameter. To which group are they referred (FOR-SUPP or PLA-SUPP)? Or is it an average?

L39-40: As for the title, I believe the authors cannot affirm that the rise in myostatin is due to the immobilization period.

L59: I feel some background on the role of myostatin in disuse atrophy is needed here in the introduction. Is increased myostatin an established finding with muscle disuse/immobilization? To the best of my knowledge, there is conflicting evidence on the effect of human limb disuse/immobilization on myostatin levels. Previous studies employing cast immobilization found unchanged (Jones et al., 2004; Chen et al., 2007) or increased (Dirks et al., 2014) MSNT expression, or even decreased myostatin protein content (Snijders et al., 2014). Increased myostatin gene and protein levels were observed in response to 3 days of unilateral lower limb suspension (ULLS) (Gustafsson et al., 2010), but no changes were found in a longer 14-day ULLS (de Boer et al., 2007). Finally, 25 days (Zachwieja et al., 1999), but not 10 days (Oranger et al., 2022) of bed rest increased circulating myostatin levels. Results in animal disuse studies seem also ambiguous (Carlson et al., 1999; Wehling et al., 2000; Allen et al., 2009). I believe the authors should cite and discuss at least part of this previous literature in the manuscript to give to the reader a broader background.

L82: Authors should add DXA procedures to the Methods section or at least refer the reader to previous studies using the same procedures.

L145: How was the amount of daily FOR supplementation (19.8g/d) determined?

L168: Were the subjects asked to refrain from intense physical activity during the whole protocol (run-in + immobilization + recovery) or just during the immobilization phase?

L180-181: Are the ultrasound-derived CSA values presented in the article the average of these 7-9 images? Why did you choose to collect the images at 2cm intervals, instead of capturing different muscle regions (e.g. 70%, 60%, 50%, 40% 30%, 20% of femur length) to take into account potential regional differences?

L210: Could you please provide more information regarding muscle biopsies exact location and precautions to avoid the effects of pre-sampling?

L313: How was participants’ compliance evaluated?

L332: Have the authors collected any functional or morphological data on the control leg? I think it would be a nice addition to the manuscript.

L364: I suggest the authors state here that no differences were observed in absolute CSA values of Type I fibres, as reported in Figure 3A.

L454: The authors are invited to report also in the main text that WB was performed only on a subgroup of participants (N=9 for FOR-SUPP and n=6 for PLA-SUPP) due to shortage of muscle tissue.

L455: Add “(data not shown)” after “E3 ubiquitin ligases”, as this result is not reported in any figures or tables.

L480: Did E3 ubiquitin ligases change their expression with immobilization? In the results section (L455), the authors reported only that no interactions between group and time for E3 ubiquitin ligases were found, but here in the discussion, the authors suggest that is one of the proved molecular mechanisms involved in muscle atrophy with disuse.

L486: I suggest the authors discuss here that the literature however is not always in agreement and different studies found unchanged or decreased myostatin levels in disuse scenarios (Jones et al., 2004; de Boer et al., 2007; Snijders et al., 2014; Oranger et al., 2022).

L507: While MuRF1 and Atrogin-1 expressions did not differ between conditions, they were also unaltered by immobilization, thus I am not sure these results are in line with the reduced muscle size and LM observed.

L510-511: Did the authors expect this increase in myostatin-positive SC with immobilization? I believe this novel finding should be further discussed.

L543: The authors forgot to add “complexity” after “motor unit potential”. In addition, I suggest specifying that the retraining period of the cited study was based on resistance training, differently from the present manuscript.

L614: please fix reference number 12, probably an error with the reference manager occurred.

References:

- Allen DL, Bandstra ER, Harrison BC, Thorng S, Stodieck LS, Kostenuik PJ, Morony S, Lacey DL, Hammond TG, Leinwand LL, Argraves WS, Bateman TA & Barth JL (2009). Effects of spaceflight on murine skeletal muscle gene expression. J Appl Physiol 106, 582–592.

- de Boer MD, Selby A, Atherton P, Smith K, Seynnes OR, Maganaris CN, Maffulli N, Movin T, Narici M V. & Rennie MJ (2007). The temporal responses of protein synthesis, gene expression and cell signalling in human quadriceps muscle and patellar tendon to disuse. J Physiol 585, 241–251.

- Carlson CJ, Booth FW & Gordon SE (1999). Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading. Am J Physiol - Regul Integr Comp Physiol 277, 601–606.

- Chen YW, Gregory CM, Scarborough MT, Shi R, Walter GA & Vandenborne K (2007). Transcriptional pathways associated with skeletal muscle disuse atrophy in humans. Physiol Genomics 31, 510–520.

- Dirks ML, Wall BT, Snijders T, Ottenbros CLP, Verdijk LB & Van Loon LJC (2014). Neuromuscular electrical stimulation prevents muscle disuse atrophy during leg immobilization in humans. Acta Physiol 210, 628–641.

- Gustafsson T, Osterlund T, Flanagan JN, Von Waldén F, Trappe TA, Linnehan RM & Tesch PA (2010). Effects of 3 days unloading on molecular regulators of muscle size in humans. J Appl Physiol 109, 721–727.

- Jones SW, Hill RJ, Krasney PA, O’Conner B, Peirce N & Greenhaff PL (2004). Regulation of skeletal muscle mass and adipose tissue mass by follistatin and follistatin-related gene (FLRG) and development of novel polypeptides as medical drugs. FASEB J; DOI: 10.1096/fj.03-1228fje.

- Oranger A, Storlino G, Dicarlo M, Zerlotin R, Pignataro P, Sanesi L, Narici M, Pišot R, Simunič B, Colaianni G, Grano M & Colucci S (2022). Impact of 10-day bed rest on serum levels of irisin and markers of musculoskeletal metabolism. FASEB J 37, e22668.

- Snijders T, Wall BT, Dirks ML, Senden JMG, Hartgens F, Dolmans J, Losen M, Verdijk LB & Van Loon LJC (2014). Muscle disuse atrophy is not accompanied by changes in skeletal muscle satellite cell content. Clin Sci 126, 557–566.

- Wehling M, Cai B & TidballL J (2000). Modulation of myostatin expression during modified muscle use. FASEB J 14, 103–110.

- Zachwieja JJ, Smith SR, Sinha-Hikim I, Gonzalez-Cadavid N & Bhasin S (1999). Plasma myostatin-immunoreactive protein is increased after prolonged bed rest with low-dose T3 administration. J Gravitational Physiol.

Reviewer #2: Major revisions

1. Sample size determination:

a) Line 104: please provide the references for the effect size interpretation;

b) Line 105: it is indicated that total calculated sample size is 24 (12 participants per each group). While, the final statistical analysis covered 11 participants is FOR-SUPP group and 9 participants in PLA-SUPP groups. In this circumferences, what were the actual effect size, alpha and power? Was the final sample size sufficient? Why Authors did not accounted for possible drop outs at the stage of a priori sample size determination? Western blot analysis was performed for even lower number of participants.

2. What was the reason for performing blood evaluations ‘solely’ at days 1 and 42? Why were they not performed at days 14 and 28?

3. Lines 143-145: how was the nutritional value of Fortetropin supplement evaluated? Please provide methodology and/or relevant references.

4. Line 161: With respect to protein intake during the immobilization phase (1.2 g protein per kg of body mass), Authors are referring to the paper published in 2022 (doi: 10.1002/jcsm.12922), while the study on Fortetropin supplementation started in 2020. Please provide the references/guidelines that actually were implemented when establishing protein intake during immobilization phase.

5. It was mentioned that energy requirements during immobilization phase were calculated by multiplying basal metabolic rate (as calculated with Harris-Benedict equation) by physical activity level. Further Authors indicated that energy expenditures were monitored using Actigraph wGT2X-BT activity monitor. Thus, is it possible to compare the calculated/pre-assumed energy expenditures and the actual/established with Actigraph energy expenditures? What was the actual energy balance in participants during immobilization phase?

6. Lines 337-352: It is not clear to which group (FOR-SUPP or PLA-SUPP) the provided results (CSA, LM, and changes in these indices) refer to? It must be clarified and specified. Are the values means or medians?

7. Lines 362-374: It is not clear to which group (FOR-SUPP or PLA-SUPP) the provided results (muscle fiber CSA) refer to? It must be clarified and specified. Although, there were no significant differences between groups, still it is impossible that the results are exactly the same in both groups. Maybe be it would be reasonable to decline from providing the exact mean results/values in the text (leave ‘P’ values only) – while the exact results are provided in the corresponding figures. The figures are very clear and highly-informative.

8. Please refer point 7 to results on isometric knee-extensor torque.

Minor revisions

1. Initial concentrations of plasma myostatin were 5487 ± 489 pg/mL in FOR-SUPP group and 4221 ± 541 pg/mL in PLA-SUPP group. Thus, was there a significant difference in baseline plasma myostation between groups? Please correct decimal places in lines 409-410 (results section)

2. Please standardize the way of recording of ‘Fortetropin’ name throughout the manuscript – standardize to use or not to use ‘®’ sign.

3. Line 40: the following sing ‘-‘ is lacking the writing of ‘2-wk’.

4. Please standardize using or not to using spaces between special signs such as ‘=/±/>/<!--≤/≥’ throughout the manuscript. Correct the using/not using the spaces between values and units throughout the manuscript.<br /-->5. Please standardize the way of recording of ‘vs.’ or ‘vs’ throughout the manuscript.

6. Line 108-111: In general, this part of the text is unclear and needs to be reworded.

7. Line 119, 471: Consider not using first person expressions.

8. Line 126: Precise the writing of the hours of arriving to the laboratory.

9. Line 166: Correct the writing of word ‘exercise’.

10. Standardize the writing of the names of study phases throughout the manuscript – sometimes capitals are used as a first letter of the name of the phases, and in the other parts of the manuscript lowercase is used.

11. Lines 168-169: Correct and standardize the writing of units (i.e., ‘steps/d-1; energy expenditure, kcal/d; metabolic equivalents of task [METs/d-1]’). It also refers to Table 2.

12. Line 194: Maybe it is better to write ‘3 trials’ if you are using the numbers in the earlier part of the paragraph (i.e., 4 repetitions).

13. Lines 204-205: What is the rational to use capitals for ‘Creatinine’, ‘Glucose’ etc.?

14. Line 306-307: Part of results are expressed as median and percentile ranges.

15. The references list it messy. It must be corrected according to PLoS One guidelines.

16. Please consider emergence ‘Conclusions’ from ‘Discussion section’.

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Reviewer #1: Yes: Fabio Sarto

Reviewer #2: No

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PONE-D-22-33716R1Fortetropin® supplementation prevents the rise in circulating myostatin but not disuse-induced muscle atrophy in young men with limb immobilization: a randomized controlled trialPLOS ONE

Dear Dr. Phillips,

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.

Please submit your revised manuscript by Jun 10 2023 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. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Krzysztof Durkalec-Michalski, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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: (No Response)

Reviewer #2: (No Response)

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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

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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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

Reviewer #2: Yes

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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

Reviewer #2: Yes

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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 addressed most of my concerns.

In my opinion, the collection of blood samples only on days 14 and 28 should be clearly stated as a limitation of the manuscript.

Other minors comments:

L62: it's "Jones et al"

L537: add please the reference

Reviewer #2: Minor revisions

1. The reference list is still in mess. It must be improved.

2. The writing of values and units still require correction within the whole manuscript. Apart from ‘%’, Authors should use put space between the value and the unit. Unify the way of writing of units within the whole manuscript and in the figures/tables.

3. Line 62: Put ‘.’ after ‘et al.’.

4. What is the rational to use capitals for ‘Creatinine’, ‘Glucose’ etc.? – it should be corrected within the whole manuscript.

5. Lines 140-141: ‘All data were stored in a locked filing cabinet and a computer protected 141 by a password’ the sentence seems to be not necessary. It can be removed.

6. Line 163: Replace ‘consumed’ with ‘intake’.

7. Line 225: Replace ‘tissues’ with ‘samples’.

8. Replace ‘minutes’ with ‘min’ within the whole manuscript.

9. Lines 300 – 301: The space between these lines is wider compared to other lines.

10. Lines 333 and Table 2: I suggest replacing ‘activity’ with ‘energy expenditure related to/arising from physical activity’ or with similar expression. The expression ‘activity’ is misleading.

11. Line 355: ‘The value of immobilized leg LM showed a similar trend as vastus lateralis’ the sentence needs to be reformulated and improved. In the current version it is not clear.

12. Line 387: Pleas put the word ‘between’ before ‘groups’.

13. Regarding the remark from the first round of the review:

“Lines 337-352: It is not clear to which group (FOR-SUPP or PLA-SUPP) the provided results (CSA, LM, and changes in these indices) refer to? It must be clarified and specified. Are the values means or medians? Thank you. Because there was no time-by-group interaction, we described the data of the main effect for time using the values combined in both groups, with the saying ‘no difference between groups.’ We have revised the sentences to make them clearer for readers on lines 350-362. All data are presented as mean +/- SD.”

Please provide the information that you used mean value from both groups at the beginning of each paragraph within which the procedure was implemented.

14. Please verify if all the units provided in the Table 3 are correct.

15. Line 505: Remove ‘There were” from the beginning of the sentence.

16. Lines 541, 544: Authors can use ‘MPS’ instead of ‘muscle protein synthesis’.

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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: Yes: Fabio Sarto

Reviewer #2: No

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Fortetropin® supplementation prevents the rise in circulating myostatin but not disuse-induced muscle atrophy in young men with limb immobilization: a randomized controlled trial

PONE-D-22-33716R2

Dear Prof. Philips,

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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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,

Krzysztof Durkalec-Michalski, Ph.D

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

Reviewer #2: All comments have been addressed

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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

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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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

Reviewer #2: Yes

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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

Reviewer #2: Yes

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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 addressed my all of my concerns and added the limitation requested.

I have no further comments.

Reviewer #2: (No Response)

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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: Yes: Fabio Sarto

Reviewer #2: No

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