Reduced Expression of Septin7 Hinders Skeletal Muscle Regeneration

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Major concerns regarding this manuscript still remain. 

H&E staining images in Fig. 1 are not of sufficient quality to assess congruence with quantitative results reported in Fig. 7.  Furthermore, in Fig. 1,  if the reader were to just count the number of dark stained structures, which the authors claim to be inflammatory cells, there are more "dark dots" in the D4, Cre- panel than the D4, Cre+ panel.  Additionally, in Fig. 1, the D14, Cre+ panel does not show an increase in potential inflammatory cell infiltration compared to D14, Cre- -- what the arrows point to are some areas of interstitial edema -- there is also significant artifact similar to cryodamage in Fig. 1, D14, Cre+, which makes it difficult to appreciate the details reported in the paper.

Concerns remain regarding the extent of Septin7 knockdown that has been achieved and linking this with altered muscle regeneration.  Even with regards to muscle regeneration markers, there is no difference in Pax7 and myogenin expression between the Cre- and Cre+.  With just the two time points that have been studied the minimal changes that are seen in Septin7 knockdown, drawing conclusions regarding a shift in the timeline for inflammation and regeneration is an over-interpretation of the results.

Author Response

We thank the reviewer for his critical comments, which we accept and have made every effort to improve. However, we have not always been fully successful and these are addressed in the responses to each point.

Major concerns regarding this manuscript still remain. 

H&E staining images in Fig. 1 are not of sufficient quality to assess congruence with quantitative results reported in Fig. 7.  Furthermore, in Fig. 1,  if the reader were to just count the number of dark stained structures, which the authors claim to be inflammatory cells, there are more "dark dots" in the D4, Cre- panel than the D4, Cre+ panel.  Additionally, in Fig. 1, the D14, Cre+ panel does not show an increase in potential inflammatory cell infiltration compared to D14, Cre- -- what the arrows point to are some areas of interstitial edema -- there is also significant artifact similar to cryodamage in Fig. 1, D14, Cre+, which makes it difficult to appreciate the details reported in the paper.

Figure 1 was replaced, new captures were taken, and representative images were chosen to better reflect the results of image analysis. For the 4-day samples, a lower magnification was chosen to detect the extent of leukocyte infiltration. For the 14-day samples, a higher magnification was used due to the visibility of the central nuclei and the better visibility of the residual, small infiltration. Arrows are also used to mark the location of infiltration and the central nuclei.

Concerns remain regarding the extent of Septin7 knockdown that has been achieved and linking this with altered muscle regeneration.  Even with regards to muscle regeneration markers, there is no difference in Pax7 and myogenin expression between the Cre- and Cre+.  With just the two time points that have been studied the minimal changes that are seen in Septin7 knockdown, drawing conclusions regarding a shift in the timeline for inflammation and regeneration is an over-interpretation of the results.

We agree with the Reviewer that the knock-down rate is not excessive in the protein level but rather substantial in the mRNA level. However, even with this small deficiency of Septin7 protein content we found significant changes in the regeneration process, which supports the importance of this protein in inducing morphological/functional changes following injury. Furthermore, the very large difference in the number of central nuclei found between fibers isolated from Cre- and Cre+ animals at the 14^th day following injection is a clear indication for the delayed regeneration in Cre+ animals in itself. Please also note the large increase in Septin7 mRNA after injury in Cre+ animals also indicative of the importance of this protein in regeneration. We are aware - and this relates to the question regarding Pax7, too - that this is partially due to the activation of satellite cells where the CrispR/Cas9 system is less active as the actin promoter in the MerCreMer construct is not fully active while the cells are dormant.

We also agree that testing samples at additional time points could have increased clarity, but unfortunately this was not done due to the already very high number of animals used and tested. The time points chosen seemed to be appropriate when the preliminary studies before the final design of the experimental protocol was done.

We have also used our previous observations to create the hypothetical graph presented in the Discussion. However, these samples did not reach the required sample size and therefore could not be presented as results. But it did provide additional information for hypothesizing the time course of the process. To further convince the Reviewer, we also planned to investigate the time course of expression of myosin heavy chains. Unfortunately, very few protein samples were available at the end of the experiments, so no statistically valuable analysis could be performed, but the trend was observed for both MYH4 and MYH3. These results are presented below:

Wes analysis of MYH3 and MYH4 protein expression in Cre- and Cre+ animals 14 days after injury. The bar graphs represents the normalized densitometry (injected relative to control). MYH3 level is elevated, while MYH4 is reduced in Cre+ as compared to Cre- animals. The former indicates the embryonic while the latter the adult myosin heavy chain isoforms, respectively. This pattern of expression is consistent with the assumption that at day 14 the regeneration in Cre+ animals is still in an earlier stage.

Taking all this into account, we believe that although the article contains indirect conclusions and assumptions, the observations support the contribution of Septin7 to muscle regeneration. In any case, we have tried to point out the limitations of the experiments.

Please, find the figure in the word file attached.

-------------------------------------------------------------------------------------------------

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

The authors have conducted an exemplary investigation on the role of Septin7 in skeletal muscle regeneration. Building upon their previous work published in eLife in 2022, this manuscript employs a Cre/Lox system to selectively downregulate and regulate the expression of Septin7 in the skeletal muscles of mice. The experiments are meticulously designed, incorporating appropriate controls, and demonstrate a high level of quality. Based on the strength of their findings, I highly recommend the publication of this manuscript.

Author Response

We thank the Reviewer for the positive comments.

Reviewer 3 Report (New Reviewer)

Thank you for the opportunity to read this well written, clear and concise study. It was interesting and a pleasure to read.

The introduction is very complete and allows the reader to know the main topic of the research, informs about the purpose and importance of the work in the clinical field, and also answers the question posed in the scientific context. It includes previous works on the subject in question and makes clear the detailed aspects of the review, which constitutes the object of the proposed investigation. It explains the general problem of the research, includes previous work on the topic in question, and specifies the objective of the study.

The "methods" section is one of the most fundamental sections of a scientific article with these characteristics and is well developed and organized. However, some aspects should be reviewed to give a higher quality to the study:

- A first subsection should be added with the study design

- All clinical trials must be previously registered so that they can be published. Please add in section 2.1. the reference of that record.

- A flowchart should be added.

- Other subsections that refer to the calculation of the sample must be incorporated.

The authors of this article have meticulously explained the results by providing relevant tables to what is explained in the text. On the other hand, they have provided a clear and complete discussion comparing their results with previous studies and arguing the existing differences. Furthermore, the limitations of this research are presented very clearly.

Likewise, the references section complies with the journal's standards.

Author Response

We thank the Reviewer for the positive comments.

A first subsection should be added with the study design

- All clinical trials must be previously registered so that they can be published. Please add in section 2.1. the reference of that record.

- A flowchart should be added.

- Other subsections that refer to the calculation of the sample must be incorporated.

Please, find our answers below:

A flow chart representing the study design has been added.

These experiments were not part of a clinical trial.

In Hungary, the ethical approval includes a section where sample size has to be determined, taking into account the minimal necessary number of experimental animals to be used. This was followed in these experiments.

In the figures presented in the manuscript numbers in brackets always represent the number of animals. This is now explicitly stated in the Materials and Methods section.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This manuscript by László Szabó et al. describes the role of septin 7 in skeletal muscle regeneration.

Although the authors took a good effort to prove the role of septin 7 in skeletal muscle regeneration using a mouse model with cre Lox system, data quality needs to be significantly improved and data is not convincing the claims of the authors.

Eg., Figure 1 H&E image quality (resolution) is not good for a publication, a high resolution and magnification images are required. Also, muscle regeneration (centronucleation) should be quantified so it will show the overall effect in the left TA muscle. Other methods for inflammation can be added like the CD68 macrophage marker.

There should be clear subtitles for figures eg., line 118 and lines 119-121 are already described in the introduction.

There is no mention of Figure 2 in section 2.2.

Figure 2 quality needs to be improved and quantification is required.

Figure 3 does not reveal the significant increase after injury in both C57BL/7 and Cre- mice. As the authors mentioned no correlation between mRNA and protein levels after injury and Western images were not quantified.

Another recommendation is, Introduction section needs to be short, especially the information on the muscle regeneration process can be short and should focus on septins more. In the introduction, references are missing in many places eg. lines 51-59.

Author Response

We thank the reviewer for his/her careful work, we will respond to his/her comments one by one.

Although the authors took a good effort to prove the role of septin 7 in skeletal muscle regeneration using a mouse model with cre Lox system, data quality needs to be significantly improved and data is not convincing the claims of the authors.

We appreciate the Reviewer’s valuable critical comments. We regret that the data did not prove to be completely convincing. We hope that the modifications made during the revision will help with this.

Eg., Figure 1 H&E image quality (resolution) is not good for a publication, a high resolution and magnification images are required. Also, muscle regeneration (centronucleation) should be quantified so it will show the overall effect in the left TA muscle. Other methods for inflammation can be added like the CD68 macrophage marker.

H&E images were taken at a resolution of 2048 X 1536 at 20x magnification, which is usually sufficient for publications. At the same time, we acknowledge that the resolution of the zoomed images presented in the inserts and, in the case of some earlier images, the contrast should be improved, which we did in the revised version. Furthermore, a series of images taken at 40X magnification was included in the Supplementary information.

The number of central nuclei was evaluated, this is shown in Figure 7. At the same time, it may have caused a misunderstanding, because we noticed this observation during the histological analyses, and we mentioned it in the paper during the presentation of these results, but their analysis took place later. Therefore, the note referring to this phenomena has been removed from the description of the histological samples, and we will only cover it with the explanation of the result.

Unfortunately, we did not have the CD68 marker available during this short time, but we performed an immunohistochemical examination on the sections made on day 14 (we performed a quantitative analysis at this time point later) with an anti-CD45 antibody, which is a receptor-bound protein tyrosine phosphatase that is expressed on all leukocytes. The representative figures of this study have also been attached to the supplementary information.

There should be clear subtitles for figures eg., line 118 and lines 119-121 are already described in the introduction.

The figure titles and captions have been modified.

There is no mention of Figure 2 in section 2.2.

We apologize for the error; we have included the reference to the figure in the text.

Figure 2 quality needs to be improved and quantification is required.

Unfortunately, we cannot improve the resolution of the images, these are very high resolution images. although we admit that the distribution pattern of Septin7 is not clearly outlined. At the same time, we added a slightly more detailed explanation to the text, and the quantification of the images was carried out by presenting the intensity distribution histogram. We hope that this makes the phenomenon presented here more understandable, namely that we saw a difference in the expression of septin7, but this was not clearly perceptible, so we did not want to draw the conclusion based on these images.

Figure 3 does not reveal the significant increase after injury in both C57BL/7 and Cre- mice. As the authors mentioned no correlation between mRNA and protein levels after injury and Western images were not quantified.

We agree with the reviewer that the increase in septin7 in CL57/B6 and Cre mice after injury is not yet strong on day 4. Physiological amounts of protein were expressed in these animals. On the other hand, in Cre+ animals, where only a reduced amount of Septin7 is present, an increased production of septin7 begins immediately, suggesting that the reduced amount is not sufficient to initiate the regeneration cascade. At this point in time, this is only visible at the mRNA level, the protein expression follows with a slight delay. By day 14, septin7 production increases in all strains. According to our interpretation, this indicates that in the later phase of regeneration, for the fusion of newly formed muscle cells, an increase in the amount of Septin7 is essential. We also agree with the reviewer that the quantification of Western Blot samples helps to confirm the protein expression variations. The western blots presented on the figures show only one representative sample, the quantitative analysis of the whole sample was carried out, and the histograms were created based on these data.

Another recommendation is, Introduction section needs to be short, especially the information on the muscle regeneration process can be short and should focus on septins more. In the introduction, references are missing in many places eg. lines 51-59.

We appreciate the reviewer's advice. Accordingly, we modified the Introduction and supplemented it with missing references.

Reviewer 2 Report

Synopsis

The paper describes a study that was conducted to ascertain whether or not septin-7 plays a role in muscle regeneration following myotoxin injury in mice.  A cre-lox system was used to knock down septin-7 in mice and assess recovery from muscle damage induced by BaCl2 intramuscular injection.

Major Concerns

The Western bot data suggest that, the extent of septin7 knockdown is minimal.  This makes it difficult to conclude if the results obtained are directly linked to septin7 levels.

There is no functional measure (e.g., muscle force measurement) to confirm that there is indeed a delay in muscle regeneration in the septin7 knockdown mice.

The central nucleation data question the level to which the BaCl2 injection was able to induce widespread and consistent levels of damage across the entire tibialis anterior muscle.  If the entire tibialis anterior muscle was damaged consistently by BaCl2, at Day 14, almost all fibers should have central nucleation, since central nucleation persists in most mouse strains for several weeks.

The quality of the histological images are not adequate to assess congruence with the quantitative data.

There is no information on how it was confirmed that cells in infiltrated areas were in fact inflammatory cells.  It would be necessary to label sections with antibodies to neurtrophils and macrophages to confirm that cells counted were indeed inflammatory cells. 

Actual p-values are not included for statistical tests

Minor concerns and/or suggestions

The introduction has superfluous information that is not directly relevant to the role of septin7 and the study that was performed.  It would help to focus on the possible role of septin7 in muscle repair and regeneration.

It would help to perform immunolabeling for developmental isoforms of myosin to demonstrate that regenerative activity is prolonged.

It would help to perform a more functional form of muscle damage, such as through eccentric contractions, to demonstrate that there is a functional consequence when septin7 is reduced/absent.  With such experiments, it is possible to blunt myogenesis with X-ray or gamma irradiation, and assess function for several weeks after injury to see if septin7 deficient mice are more dependent on myogenesis for recovery.

Author Response

Thank you for the reviewer's comments, we will respond to them one by one below.

Synopsis

The paper describes a study that was conducted to ascertain whether or not septin-7 plays a role in muscle regeneration following myotoxin injury in mice.  A cre-lox system was used to knock down septin-7 in mice and assess recovery from muscle damage induced by BaCl2 intramuscular injection.

Major Concerns

The Western bot data suggest that, the extent of septin7 knockdown is minimal.  This makes it difficult to conclude if the results obtained are directly linked to septin7 levels.

We have to agree with the reviewer that the downregulation of septin7 is not particularly high. However, the only modification we created in this mouse strain was reduced septin7 expression (mild but significant). Thus, we attribute the observed changes to this modification. We do not and cannot claim that the prolongation of regeneration is a direct consequence of the reduced septin7 expression, but this knockdown causes the differences. Presumably, the modification of the cytoskeletal structure is behind the alterations, in the formation of which septin7 plays a significant role.

There is no functional measure (e.g., muscle force measurement) to confirm that there is indeed a delay in muscle regeneration in the septin7 knockdown mice.

In our previous experiments, we found that septin7 deficiency results in reduced muscle strength in EDL muscle (Elife. 2022 Aug 5;11:e75863. doi: 10.7554/eLife.75863. PMID: 35929607; PMCID: PMC9355566.). Since the expression of septin7 is lower even on day 14, muscle strength is presumably also lower (theoretically, regardless of whether muscle regeneration has taken place or not). On the other hand, we do not have a system suitable for measuring the force of TA muscles, while the injection of EDL with BaCl2 caused technical difficulties.

The central nucleation data question the level to which the BaCl2 injection was able to induce widespread and consistent levels of damage across the entire tibialis anterior muscle.  If the entire tibialis anterior muscle was damaged consistently by BaCl2, at Day 14, almost all fibers should have central nucleation, since central nucleation persists in most mouse strains for several weeks.

In the article, we mention "mild injury" we wanted to achieve with the BaCl2 injection, with which we wanted to simulate injuries that occur physiologically during strenuous muscle work (in contrast to the widely used cardiotoxins, which cause complete muscle destruction). We are sorry that this is not clear in the article, so we have corrected the relevant parts.

The quality of the histological images are not adequate to assess congruence with the quantitative data.

The histological sections were made in high resolution (2048 X 1536). In the revised version, we tried to improve the representation of the images.

There is no information on how it was confirmed that cells in infiltrated areas were in fact inflammatory cells.  It would be necessary to label sections with antibodies to neurtrophils and macrophages to confirm that cells counted were indeed inflammatory cells. 

We performed immunohistochemical examination on the sections made on day 14 with an anti-CD45 antibody, which is a receptor-bound protein tyrosine phosphatase that is expressed on all leukocytes. The representative figures of this study have also been attached to the supplementary information.

Actual p-values are not included for statistical tests

Minor concerns and/or suggestions

The introduction has superfluous information that is not directly relevant to the role of septin7 and the study that was performed.  It would help to focus on the possible role of septin7 in muscle repair and regeneration.

The Introduction was modified according tot he Reviewer’s suggestions.

It would help to perform immunolabeling for developmental isoforms of myosin to demonstrate that regenerative activity is prolonged.

We agree with the reviewer that the study of isoforms of myosin can help provide additional evidence for the prolongation of regeneration, but we cannot do this in the short time available.

It would help to perform a more functional form of muscle damage, such as through eccentric contractions, to demonstrate that there is a functional consequence when septin7 is reduced/absent.  With such experiments, it is possible to blunt myogenesis with X-ray or gamma irradiation, and assess function for several weeks after injury to see if septin7 deficient mice are more dependent on myogenesis for recovery.

We also agree that the above-mentioned experiments can support our observations from a new perspective, but this faces several difficulties. The main reason for this is that it requires interventions and treatments (irradiation, eccentric muscle damage) that cause discomfort/pain for the animals. The authorization of such tests is carried out according to very strict rules. We do not have the necessary ethical approval to carry out such tests, and obtaining it is very long process (it lasts at least half a year).

Reviewer 3 Report

The authors studied the role of septin7 in skeletal muscle regeneration. The manuscript is interesting and has merit, however, I have only a few comments.

1.     There are many repetitions in the text especially in introduction section, introductory sentences in result section and discussion, please make the text more concise.

2.     Why was TA muscle used? In mice, this muscle is heterogenous in fibre type composition (some parts having more fast and other more slow fibre types).

3.     It is stated that student’s t test was performed when testing differences between control leg and injected leg. Was paired t test used, since these are dependent data coming from same animals? How was the normality of data tested for?

4.     Please indicate the number of animals used for every group and every test. Why was different animals used for every group and time point of the experiment?

Author Response

Thank you for the reviewer's comments, we will respond to them one by one below.

The authors studied the role of septin7 in skeletal muscle regeneration. The manuscript is interesting and has merit, however, I have only a few comments.

We are very grateful for the reviewer's appreciation, and we will respond to the comments below.

1. There are many repetitions in the text especially in introduction section, introductory sentences in result section and discussion, please make the text more concise.

We regret these errors and omissions. In the revised version, we tried to improve on these and create a more concise text.

2. Why was TA muscle used? In mice, this muscle is heterogenous in fibre type composition (some parts having more fast and other more slow fibre types).

The choice of the muscle was justified by technical/anatomical reasons. The TA is a well-accessible, near-superior muscle bundle. Thus, the place of the injection can be well determined, and it affects the desired muscle with great certainty. We wanted to use EDL and Soleus muscles, but injecting them resulted in serious technical difficulties and great uncertainty, so we decided in favor of TA.

3. It is stated that student’s t test was performed when testing differences between control leg and injected leg. Was paired t test used, since these are dependent data coming from same animals? How was the normality of data tested for?

Yes, during the statistical tests we performed a paired t-test to compare the control and injected legs. Normality was checked with the Shipiro-Wilk test, which is now included in the Methods section

4. Please indicate the number of animals used for every group and every test. Why was different animals used for every group and time point of the experiment?

The 4th day showed a relatively high individual susceptibility to the development of inflammation in the control mice, so we worked with a larger number of elements at this time. In the case of the 14th day, we used a slightly smaller number of mice due to the minimization of the number of experimental animals, as this was made possible by greater homogeneity. There was a technical difference between the different sample numbers of the same time points. The number of elements was occasionally reduced due to errors made during sample preparation. Furthermore, we did not use the animal for experiments even if we saw signs of external damage, or if it died during the experimental period.

Round 2

Reviewer 1 Report

Authors addressed the reviewers comments but still the revised mansucript lacks significant improvement in the results section especially Figures 1 & 2. Introduction was improved but results sections needs further improvement.

Reviewer 2 Report

The response to the review and the edits to the text are appreciated.  However, the major concerns that I had indicated have still not been addressed.