PONE-D-25-28377Mixed twitch and tetanus electrical stimulation via belt-electrode effectively attenuates denervation-induced muscle atrophy in multiple muscle groups.PLOS ONE

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Research funding for this study was provided by HOMER ION Co., Ltd., H.U., K.S., R.A., K.H., S.T., and M.I. are HOMER ION Co., Ltd. employees, and K.N. is a co-researcher. The other authors have no financial disclosures related to this paper.].   

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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

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: No

Reviewer #2: Yes

Reviewer #3: No

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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

Reviewer #3: Yes

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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: The authors showed that a combination of twitch and tetanus contractions was effective in suppressing muscle atrophy induced by sciatic nerve denervation. These findings may contribute to the development of new intervention strategies for muscle atrophy. Various analyses were carefully conducted, and the framework of the study is well understood. However, I have several concerns as follows.

1. The introduction is redundant; it is unclear in L55-81 whether this study is an extension of your research or whether you are trying to establish a new experiment based on previous research. The aim of the study should be clearly indicated.

2. A variety of factors can cause muscle atrophy, but the authors used the peripheral neuropathy model in this study. However, there is no research introduction on the use of this model. Please add this point to the Introduction section.

3. The Methods and Materials section describes the animals as follows: "Acute stimulation experiment, chronic stimulation experiment, n=52." Could you please provide the specific number of animals in each group? Similarly, when stating that acute stimulation experiments were performed in three groups and chronic stimulation experiments were divided into four groups, the number of rats in each group should be specified.

4. For Western blotting, is a loading control or total protein amount quantified at the same time to eliminate differences in protein expression between samples and the effects of manipulation during the experiment? The authors should add these to the Material & Methods section.

5. The authors described “Stimulation intensity was preliminarily tested with a belt electrode and set to a minimum current run value of 3.0 mA (1.2 mA for twitch), which produced maximum torque with 60 Hz stimulation.” The authors should add the results of the preliminary investigation to Material & Methods section (or supplemental files).

6. In “Acute response to electrical stimulation with belt electrodes” section, why did the authors examine the glycogen content and phosphorylated AMPK immediately after exercise and phosphorylated p70S6K in 6 h after exercise? Please provide a reason for the time to harvest the skeletal muscles. The authors did not assess the phosphorylated AMPK and p70S6K in TA. This investigation was titled “multiple muscle groups”, the lack of these results is a primary concern for this study.

7. After denervation, the sciatic nerve may sometimes reattach. To avoid this, the proximal and distal portions are often removed a few millimeters from the amputated area. Did the authors perform this procedure? Or, did they confirm that no reattachment occurred by dissection?

8. Since the total RNA concentration differs from sample to sample, the amount of RNA contained in the RNA solution (5 μL) differs for each sample. Fortunately, the authors have determined the total RNA concentration by Nanodrop; please provide the total RNA concentration of each sample used for the 18S and 28S searches and show that there is no difference between groups.

9. Regarding RT-PCR, how did the authors quantify Atrogin-1 and MuRF1 mRNA expression? Did you use the internal control? If so, the primer sequences should be listed in Table 1.

10. It is difficult to confirm inter-individual variation in the mean±standard error. Please indicate the data as mean±standard deviation (SD). The authors should also add about the post-hoc tests. Furthermore, much data was listed as folds. Please re-describe all the data in mean±SD.

11. In Figure 3, the band images of p-p70S6K and p-AMPK are too weak to confirm; please re-prepare the band images along with t-p70S6K and t-AMPK. Furthermore, two bands can be identified in p-p70S6K. Which of the two bands did you adopt?

12. In Figure 4, why did you separate the muscle wet weight into right and left sides? I suppose they should be combined. In addition to muscle wet weight, please add data on body weight and relative weight ratio.

13. In Figure 5, which color indicates which muscle fiber type? Furthermore, since the scale bar is not listed, the sizes cannot be compared. Why is this data listed as a percentage while the muscle wet weight is listed as raw data? All data should be presented as raw data, not as ratios.

14. Which muscles did the authors use for analyses of COXIV, 18S, 28S, Atrogin-1, and MuRF1? You must indicate all the data of the above parameters in TA and GAS.

15. There is confusion between the terms “Murf1” and “MuRF1” (L355, 361). Please unify the descriptions.

16. In Figure 6, did the authors use the loading control (e.g., GAPDH)

17. The contents of L385-392 are duplicated in the Introduction section and are not necessary.

18. The authors described “muscle atrophy progresses from extracellular matrix degradation due to increased MMPs associated with decreased mitochondrial content.” (L410-412) I can’t understand this cascade. The author states that the degradation of muscle component proteins is a cause of muscle atrophy, and in this context, the degradation of the extracellular matrix (ECM) is a key factor. The primary function of MMPs is to degrade the ECM, and they are not directly involved in the degradation of muscle component proteins. This point should be described in more detail, and previous studies should be cited.

19. The authors described “twitch EMS increases mitochondrial mass and enzyme activity while suppressing muscle proteolytic signaling” in the Introduction section (L57-58). However, even though COXIV was significantly higher in DEN+CS than in DEN+60, there was no significant difference in 18S and 28S between the two groups. Furthermore, surprisingly, there was no significant difference in Atrogin-1 and MuRF1 between these two groups, despite significant differences in CSA for each muscle fiber type in GAS and TA. The author must add your idea on this discrepancy in the Discussion section.

20. Considering the title of this study, the authors likely aim to demonstrate the effectiveness of twitch and tetanus contractions on multiple skeletal muscles. This is understandable given that the study selected the TA (flexor) and GAS (extensor), which have distinct functional roles, as sample muscles. Additionally, since both skeletal muscles exhibit significant differences in muscle fiber size between their deep and superficial layers, the authors may have also wanted to investigate this point.

I read the discussion section expecting these points to be addressed, but unfortunately, there was not much discussion on the above topics. I would like you to reconsider the results and discussion regarding the following points.

1. Discuss whether any characteristic differences were found in the results of the tibialis anterior and gastrocnemius muscles between the acute and chronic experiments. Regardless of whether differences were found or not, discuss your findings in relation to previous studies.

2. Please discuss the differences in effects between the superficial and deep layers of the tibialis anterior and gastrocnemius muscles (if necessary, present the results separately for the superficial and deep layers). Based on the content of above 1, please discuss the effects of twitch and tetanus in the superficial and deep layers.

Reviewer #2: 1. This is a well-designed study combining twitch and tetanus stimulation to combat muscle atrophy. The idea is novel and valuable, especially for clinical applications in populations with limited mobility. However, please more clearly differentiate how your combined approach surpasses traditional methods using only tetanic stimulation, especially in terms of clinical scalability or mechanistic superiority.

2. The acute and chronic study arms are well constructed. Still, it would help to include a schematic diagram summarizing group allocation, stimulation protocols (e.g., duration, frequency, intensity), and outcome measurements.

3. Auhors shown a good evidence that mixed stimulation enhances both mitochondrial and protein synthesis markers. Still, consider adding a discussion on possible molecular pathways that explain the additive/synergistic effects.

4. While results are promising, some comparisons (e.g., between DEN+60 and DEN+CS) show only trends, not significant differences. Please clarify which results are statistically significant and which are not.

5. The figures are informative, but some (especially Western blot and CSA images) appear low-resolution. Please upload clearer images and ensure consistent formatting of figure legends.

6. Include exact p-values wherever possible and define the number of animals (n) per group for each analysis in both the text and figure legends.

7. The manuscript reads well overall, but a few sections (especially the Results and Discussion) would benefit from language polishing for clarity and flow.

8. Author mention future use in postoperative or elderly patients. It would be great to briefly explain how this method could be implemented practically in clinical or rehabilitation settings.

9. Please discuss limitations such as the short duration (7 days) of chronic stimulation and whether longer-term studies or human trials are being considered.

10. Maintain consistent terminology (e.g., use "TA" and "GAS" uniformly, and define all abbreviations on first use) to avoid confusion for non-specialist readers.

Reviewer #3: This study presents interesting evidence suggesting that the combination of twitch and tetanus stimulation may be more effective in suppressing muscle atrophy. The findings are valuable and provide a meaningful contribution to the field of muscle stimulation research.

Introduction

Describe in detail the molecular mechanisms by which combined stimulation therapy was predicted to more effectively inhibit denervation sarcopenia compared to twitch or tetanus stimulation alone at the planning stage of the study.

Methods

Please provide more detailed information regarding the rationale for selecting the stimulation parameters used in this study, including the frequency and temporal distribution of the combined stimuli.

Results

Muscle wet weight may have been affected by each rat’s variability, such as body weight. Please provide the muscle wet weight and body weight data for each rat at the time of sacrifice.

Histological Evaluation

In the CSA graphs, the black dots appear to represent ratios; however, the definition of the comparison target is unclear. Do these values represent, within each muscle, the total cross-sectional area of each muscle fiber type, or the average cross-sectional area per fiber type?

To better visualize fiber size changes, I recommend adding muscle fiber CSA distribution plots, as shown in your previous paper.

The composition of muscle fiber types (fast-twitch and slow-twitch) has been reported to change in sarcopenia. And electrical stimulation affects the composition of muscle fiber type (Shi H, J Neuroeng Rehabil, 2023). Please provide supplemental data on fiber type composition (e.g., number and proportion of each fiber type) in each muscle examined.

Western Blotting

In the supplementary data, it is unclear which bands correspond to the same samples between p-p70S6K and t-p70S6K, as well as between p-AMPK and t-AMPK. The correspondence between these bands should be clearly indicated. Additionally, loading controls such as GAPDH or β-actin are not shown, making it difficult to confirm equal protein loading. Please include these controls.

Discussion

Although the description of the effects and mechanisms of B-SES is informative, some of this content may be more appropriately presented in the Introduction section.

The absence of interference between twitch and tetanus stimulation is a significant finding. Please expand the discussion on the molecular mechanisms that may underlie this result.

The combination of twitch and tetanus stimulation more suppressed the expression of muscle degradation markers. It would be informative if the authors could discuss the possible molecular pathways involved in this mechanism.

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Reviewer #1: No

Reviewer #2: Yes:  Dr Dinesh Kumar

Reviewer #3: No

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Submitted filename: PONE-D-25-28377.docx

Mixed twitch and tetanus electrical stimulation via belt-electrode effectively attenuates denervation-induced muscle atrophy.

PONE-D-25-28377R1

Dear Dr. Uno,

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.

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Kind regards,

Atsushi Asakura, 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

Reviewer #3: 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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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

Reviewer #3: 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: Based on my previous comments, I carefully reviewed the revised manuscript prepared by the authors. The authors have sincerely addressed my concerns, and improvements to the manuscript have been confirmed on all points. I believe this manuscript has achieved a quality worthy of publication in PLOS ONE. I hope this work will contribute to the development of new intervention strategies for denervation-induced muscle atrophy.

Reviewer #2: The manuscript “Mixed twitch and tetanus electrical stimulation via belt-electrode effectively attenuates denervation-induced muscle atrophy” presents a novel strategy where combined twitch and tetanus stimulation more effectively preserves skeletal muscle mass than tetanus alone. The study highlights that this approach enhances both mitochondrial biogenesis and protein synthesis, addressing key pathways in denervation-induced atrophy. Importantly, the authors carefully revised the manuscript to address all editorial and reviewer concerns, including clarifying methodology, improving figures, ensuring statistical rigor, and expanding discussion on mechanisms and clinical applications

Decision: Based on its novelty, translational potential, and thorough revisions, I recommend acceptance of this manuscript for publication in PLOS ONE.

Reviewer #3: Overall comment

The manuscript has been revised, and the logic is clear and easy to follow. With a few additional revisions, it could be made even clearer.

Specific comments

Line 79–83

The manuscript states that aerobic and resistance exercise have been extensively studied. However, the outcomes of these studies are not described. Please provide a brief summary of the main findings.

Line 262

In the context of RT-PCR, the use of the term “loading control” is inaccurate. A more appropriate description would be “internal control” or “reference gene.”

In addition, could you please clarify which method was used for quantification in the RT-PCR analysis (e.g., ΔCt, ΔΔCt, or standard curve method)?

Figure 5

From the figure legend, my understanding is that the black dots represent the mean cross-sectional area of muscle fibers for each rat. However, this is not certain. Please clarify whether these values indicate the mean, the median, or another definition. Simply stating “cross-sectional area (CSA) of TA and GAS” is not sufficiently clear.

Muscle wet weights

This manuscript presents relative muscle wet weights, suggesting that individual variability has been considered. However, I would appreciate it if you could also provide the individual body weights (g) and absolute muscle wet weights (mg) of each rat.

Western blot images

Is it correct to assume that the Western blot images correspond one-to-one between the upper and lower panels? If so, please indicate this clearly in the supplementary data.

Muscle fiber type composition ratio (supplementary data)

It is difficult to distinguish between type IIa and IIb due to the color similarity. In addition, the upper edge of the graph appears to be cut off. Please adjust the legend order to match the sequence displayed in the stacked bar chart. At present, the arrangement of Type I, IIa, and IIb in the legend does not correspond to their order in the chart.

Total muscle fiber number

In addition, is it correct that the total number of muscle fibers showed no statistically significant differences between the different electrical stimulation protocols?

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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: No

Reviewer #2: No

Reviewer #3: No

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