Improved Electrical Stimulation-Based Exercise Model to Induce Mice Tibialis Anterior Muscle Hypertrophy and Function

Round 1

Reviewer 1 Report

“Optimized Resistance Exercise Model to Induce Mice Tibialis Anterior Muscle Hypertrophy and Function,” submitted by Alves and colleagues, presents data to support the idea, advanced in a number of earlier papers, that skeletal muscle training via electrical stimulation leads to hypertrophy. A variety of different electrical stimulation protocols have been shown to achieve muscle hypertrophy, and the authors present yet another protocol variation to induce hypertrophy following a common set, repetition, and rest interval scheme employed in human resistance training. Weaknesses lie in the fact that the results are largely confirmatory of previous work, and incompleteness or errors are throughout the manuscript. Although the authors attempt to present their approach as an “optimized” protocol, that is a subjective point of view and depends on the experimenter's goals. Furthermore, vetting of the current protocols was insufficient to make such a claim. Enthusiasm for the paper would be greater if additional evidence were presented to support the claim of an “optimized” protocol for inducing TA hypertrophy, along with evidence indicating that the rapid and extensive hypertrophy is translatable to human resistance exercise outcomes.

Major Concerns:

1. Assessment of hypertrophy was insufficient. Skeletal muscle hypertrophy is often determined by an increase in overall muscle mass, yet the authors did not include the TA muscle mass in the manuscript. Hypertrophy is also frequently determined by an increase in muscle fiber cross-sectional area (CSA), which the authors did include; however, according to the Methods subsection 2.3 and Figure 1’s caption, experimenters measured “~1,200 fibers per group,” and there were “n=5-6” mice per group, which means only 200-240 fibers were analyzed per mouse. This is insufficient analysis of muscle fiber CSA. Overall, the assessment of hypertrophy is incomplete.

2. The rapid and excessive hypertrophy may have unintended consequences due to aberrant remodeling, but since the authors only chose to examine satellite cell markers, evidence of the remodeling process is lacking. In Figure 1B, the ES2 image is the only image that shows multiple fibers with internally-localized nuclei. This data can be quantified from existing images. Other methods to evaluate remodeling that could easily be performed without the need for additional mice would include: immunofluorescent staining of inflammatory markers, immunofluorescent staining with anti-mouse IgG to detect damaged fibers, and immunofluorescent staining of dystrophin to detect fibers lacking sarcolemmal-localized dystrophin.

Minor Concerns:

1. The use of “resistance exercise” in the title and elsewhere is misleading. Skeletal muscle training was induced by electrical stimulation. Resistance training or exercise should not be used to describe high-frequency electrical stimulation of skeletal muscle. If necessary, alternative phrasing could include “resistance-like exercise” or “resistance-type exercise.”

2. Throughout the manuscript, comparisons are repeatedly made to hypertrophic results from different rodent species, gender, and age. However, there is no mention of the differences in hypertrophic response between rats and mice, males and females, or the influence of age. Please address.

3. Include a comparison to papers that employed a stretch-shortening contraction (SSC) training protocol. The SSC training protocols may be the most appropriate comparison to the protocols used in the current manuscript. Oddly, this comparison was never made in the manuscript draft.

4. Methods section 2.1 states, “Forty male C57BL/6 mice (8-12 weeks….) were randomly divided into four groups:”… CTR one week (n=5), CTR two weeks (n=5), ES1 (n=5), ES2 (n=5). That is 20 mice, not 40.

5. In Methods section 2.2, include the range of motion (degrees) and the ankle rotation speed (degrees/second).

6. In Methods section 2.2, include the average or estimated peak torque values produced during the movement's concentric and eccentric phases.

7. Figure 1 describes data from “n=5-6” mice. Figure 2 describes data from "n=6' mice. Methods section 2.1 states that only 5 mice were used per group. Which is correct?

8. Include individual data points on all histograms.

9. Include the mean and SD or SEM in the results.

10. Include a description of Figures 1D, E, and F in the text of the results.

11. In Figure 2, the force-frequency curves (2C and 2F) show very different results for the Control groups. This is concerning. Explain the variability.

12. The Introduction is unnecessarily long. Nine paragraphs and 77 lines. Multiple paragraphs describing rodent resistance training protocols should be narrowed down to 1-3 sentences in total.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Resistance exercise (RE) is commonly accepted method for inducing a hypertrophy of muscular tissue, yet having its limitations. The Authors propose a faster and efficient method for achieving the same result. The results using mice as an animal model are promising. However, I have some comments I’d like to express.

·         My main concern regards the statement “optimized response”. The Authors claim that “an optimized response was found at two weeks of RE training” (line 286). However, there is no rationale for such statement. Although the results provided with the manuscript showing better response after two weeks of RE training, you cannot say that it is optimized response. Did you examine longer times of RE training? Were the results worse than after 2 weeks? If not, I suggest change the word “optimized” for “better” because you did not optimized the timing of RE training. Otherwise you should provide some more data evidencing your statement (at least in supplement).

·         Taking the above into account, the conclusion seems to be not fully supported by the results provided. You should either add more data, or re-thing this part of the manuscript. How did you verify the resilience of analyzed muscle hypertrophy?

·         There is a repeating text in brackets in abstract (line 15); similarly, in the Materials and Methods section – line 114.

·         Please, provide the explanation of the CSA abbreviation in Abstract, since it is the part of the article that should be self-explanatory and easy understandable for the reader without reading whole article.

·         P vales in Abstract (lines 17, 18) are different from those in Results (lines 235, 237, respectively). Also, presenting p < 0.005 seem to be a typo mistake.

·         Reference numbering style should be corrected to be in accordance with instructions to authors (e.g. [6,7] instead of [6], [7], or [10-12] instead of [10]-[12].

·         Providing information regarding any equipment, reagents, etc. there should be added full information about the producer/developer including city and country.

·         Please, correct the information regarding DAPI in line 195. I’m sure that “(v. 1.45s, National Institutes of Health)” does not apply to DAPI.

·         I’m not sure if 5-6 mice in group is enough to use parametric statistics. How did you verified normality of data distribution?

·         In Figure 1 (D-E) caption you should be more precise. Those graphs present the distribution of fiber cross-sectional area frequencies, not the distribution of fiber frequencies. It makes difference.

·         In Figure 4 caption there lacks the explanation for #.

·         The style of reference list also needs to be corrected to be in accordance with instructions to authors (the number of authors mentioned, using bolds etc.).

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Animal models have been well documented as to the need for improvements, particularly as it relates to the study of RE training, and thus, this manuscript offers potential relevance to the reader. Nevertheless, to improve upon impact of this manuscript, further revisions are warranted. To begin with, how does the authors define “optimized”. This term is used in the title and in the conclusions within the abstract and at the end of the manuscript, however, objective measures of what is considered “optimized” is not given, and although some limitations are noted by the authors concerning the model, the authors are advised to select another term besides “optimized” that is more appropriate for reflecting the researched model studied within this manuscript. A more objective term should be considered. Similarly, while the authors are restricted in the length of the abstract, the terms “lengthy” and “variability difficulties” within line 11 are difficult to interpret and require further clarification within the abstract.

As for the introduction, while animal models are needing improvement, and thus, remain the focus of the study, it’s important to relate back the relevance of understanding RE training for humans. Why is there a need for studying RE training? What is the value of this research beyond just the sake of creating a better model? This is touched on very briefly on line 46, but needs to be expanded within the introduction, and then, followed up in the discussion. At the end of the introduction, although aims of the study are given, it would help to see a clearer, more specific objective statement along with a hypothesis statement.

Overall, the most substantial limitation for this study would be the low sample population. Mice are used as a model due to the ability to utilize a larger sample size than other animal models. While 40 seems like a fairly adequate sample size, the “n” for each group is quite small. What is the justification of such a small sample size per group. It also appears that each group had varying numbers, but it is unclear why this was the case for this study. In addition, for animal housing, the mice were kept in a “normal” cage, but what defines “normal” utilizing objective measures. Furthermore, while Table 1 is useful to clearly outline the RE protocol, it is repetitive compared to what is given within the text. Authors should remove descriptions of the protocol within the text and refer the reader to the table instead. However, along with the title of the table, give a short description of the content of the table within the table heading. It would also help to have photos of the experimental procedure, if available, of the RE training.

As for the results, with a low sample size, it is essential that specific p-values for each statistical analysis are given, rather than indication that a value is above a set number. For figures one and two, the line graphs are difficult to see. The information within these figures should be divided up so that what was presented can be enlarged. Same is true for figure five.

Finally, for the discussion section, again, it’s important for the reader to understand the value of this model specific to the human and how this will benefit humans, more so than just the development of a new model to use for research. How is research using this new and improved model going to impact the human subject? Furthermore, authors should expand more on the limitations of this model and the study itself such as the small sample size, and again, it’s important to clearly define within the conclusions what “optimizations” have been made.

Author Response

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Author Response File: Author Response.docx

Reviewer 4 Report

A review of the manuscript titled “ Optimized Resistance Exercise Model to Induce Mice Tibialis Anterior Muscle Hypertrophy and Function” by Paula Ketilly Nascimento Alves; João G. Silvestre; Wenddy Wyllie Damascena Sougey; André Cruz and Anselmo Sigari Moriscot, submitted to Applied Sciences.

In the manuscript, the authors developed an optimized RE training for mice by directly stimulating the tibialis anterior muscle motor point using electrical stimulation. The authors showed observed an increase in the number of Pax-7-positive nuclei after one and two weeks of RE training, the number of positive nuclei for MyoD after two weeks as well as phosphorylation of mTOR and p70 following two weeks of RE training.

The authors indicated that this optimized RE training strategy is appropriate for promoting quick and intense hypertrophy that can be longitudinally evaluated.

The work is well thought out, each study is well reasoned. This undoubtedly shows the scientific maturity of the researchers. 

Please reconsider the following:

1.       Why were the animals fasted only for 3 hours before euthanasia and not, for example, overnight?

2.       How can the authors explain the lack of changes in total and phospho AKT levels between the RE training group and the control group?

3.       Were the samples used for the Western blot normalized? The distribution of the GAPDH bands appears to vary in size.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript has been improved, and most of my concerns have been adequately addressed. Only minor concerns remain.

1) Refer to Major Concern Point #2 -- "In Figure 1B, the ES2 image is the only image that shows multiple fibers with internally-localized nuclei. This data can be quantified from existing images." Assuming the ES2 dystrophin+DAPI image is a representative image for the group, readers can assume that the percentage of internal/central nuclei is over 5% (2 fibers with internal nuclei out of 27 fibers that were fully visible within the image = 7.4%). Such a high percentage of internal/central nuclei would indicate muscle fiber remodeling, and, based on the newly added IgG data, the remodeling fibers have an maintained an intact sarcolemma or repaired the sarcolemma. Knowing the extent of muscle remodeling caused by the electrical stimulation protocol could influence readers on whether or not to follow the protocol for their studies.

2) I made an error in the original review. Refer to Point #8 -- "Include individual data points on all histograms." I meant to request individual data points on all bar graphs. If possible, adjust the bar graphs to include individual data points.

Author Response

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Author Response File: Author Response.docx