P38α-MAPK Signaling Inhibition Attenuates Soleus Atrophy during Early Stages of Muscle Unloading

Original manuscript: ijms-710486

Reviewer comments

Belova S et al. analyzed the effects of VX-745, an inhibitor for P38 MAPK, on the unloading-induced muscle atrophy. They showed that treatment with VX-745 prevented the unloading-induced decrease of muscle weight and the increase of atrogenes. 

Major points.

1) As the authors described, the basic concepts regarding the role of p38MAPK on muscle atrophy have already been reported (Yuasa K et al., Sci Rep, 2018). Thus, the novelty of this study was quite low. Why did the authors use VX-745? What were the differences between SB203580 and VX-745?  If VX-745 does not have anything better than SB203580, what is the novel point of this study? The authors should mention this point.

2) In Fig.1, the authors analyzed the phosphorylation of p38 MAPK. The authors should show the phosphorylation of other MAPK, such as p44/42 MAPK and JNK, to show the specificity of VX-745. In addition, the authors should show which lanes indicate control, HS and VX.

3) In Fig. 2, the authors only analyze the muscle weight. The muscle weight should be normalized by body weight. This is important because hindlimb-suspension causes a decrease in body weight. The authors should show that the decrease in muscle weight was not due to the decrease in body weight. Furthermore, the authors should analyze the cross-sectional area of muscle fibers to show that these rats cause muscle fiber atrophy.

4) Although the authors analyzed several atrophy-related genes, the authors did not show any relationship between those genes and the activity of p38 MAPK. The authors should show that calpain-1, ubiquitin, atrogin-1, MuRF1, PGC-1a, IL-6, and IL-6R were directly regulated by p38 MAPK. Overexpression or suppression of p38 MAPK without hindlimb-suspension should be required.

Minor points

5) In all figures, VX should be HSVX.

6) In line 328, "P 0.05" should be "P<0.05".

Reviewer comments

In this manuscript, Belova et al., studied the p38α MAPK inhibition in soleus atrophy during early stages (3days) of skeletal muscle unloading. Inhibition of p38α MAPK by VX-745 inhibitor in vivo situation by food supplementation, attenuates muscle atrophy by unloading in early stages. Actication of MuRF1 pathway, but not MAFbx, is suppressed by hindlimb suspension (HS).

Suppression of pFoxO3, but not pAKT, by HS is attenuated by VX-745, suggesting FOX pathway independent of Akt pathway is involved in MuRF1 regulation in this study. Expression levels of calpain, ubiquitin, PGC-1α, and IL-6 but not IL-6Rc are regulated between HS and VX treatment.

This study is based on previous studies of authors and others. Current study has some progresses, however the study seems descriptive rather than mechanistic, and most of the concept depends on the published works and lacks novel molecular mechanisms, and should be revised.

The manuscript in the current version contains several errors and need corrections before consideration in the journal. I hope the comments will be of help.

Comments

In many cases even in the situation of stating p38α MAPK is adequate, the authors does not specify p38α MAPK and write simply as p38 MAPK. Specify p38α MAPK when appropriate. The reason why the authors study soleus muscle is not clear. Muscle-specific ablation of p38α MAPK, MuRF1 and MAFbx in fast muscle is reported in ref 10. Authors should discuss the differential regulation of Murf1 and MAFbx in more depth, cosidering time course, species difference, and muscle fiber types. Histological analyses are lacking.

Other comments

In Fig. 1 and 4C, 5, 6, 8, define lanes with C, HS and VX. In Fig.4, western blot for MAFbx is not provided. Is the antibody suitable for protein blotting available? In method section 4.3, total and phosphorylated pP38 MAPK should be total and phosphorylated p38 MAPK, respectively. Some errors in English are found.

Line 166, [picks] should be corrected.

Line 227, correct [we think]. [it is possible] might be more appropriate.

Line 228, [in in muscle atrophy] should be corrected.

Line 256, 261, bread is not good for rat.

Line 305, [1 μl of cDNA] should read [One μl of cDNA].

Line 325, < is missing. P < 0.05.

In references, unify description of page numbers such as 2181-2188 and R1546-57. In some references (ref 7, 17, 23, 33), page numbers and/or identification numbers is missing. Although the authors cite ref 43 for VX-745 dose determination, 3 weeks of administration of VX-745 (50 mg/kg) was reported in ref 43. By contrast, only 3 day administration is performed in this study. Therefore, it is not clear how the authors determine the dose of the VX-745. What is the status of clinical trial for VX-745 (Neflamapimod)? Is the dose for human available? If available, the authors can compare the dose of the current study with that of clinical trial. These informations would be of help understand the determination of dose.

New submission: ijms-735477

Authors responses to the reviewer

We would like to thank the reviewers for taking time to thoroughly evaluate the manuscript. 

Reviewer #1: Critique 1. As the authors described, the basic concepts regarding the role of p38MAPK on muscle atrophy have already been reported (Yuasa et al., Sci Rep, 2018).  Thus, the novelty of this study was quite low.  Why did the authors use VX-745?  What were the differences between SB203580 and VX-745?  If VX-745 does not have anything better than SB203580, what is the novel point of this study?  The authors should mention this point.

Answer: Current study is novel and it is significantly different from the study previously published by Yuasa and colleagues (2018). We are very familiar with the study published by Yuasa and colleagues and we referenced it in our manuscript. There are several principal differences between our study and the study published by Yuasa and colleagues: 

1. Yuasa and colleagues used a denervation model to study skeletal muscle atrophy. In our study, skeletal muscle atrophy was induced by the unloading with preserved muscle innervation. Previous studies reported no abnormalities in nerve conduction in rat models during unloading. Although both denervation and unloading cause skeletal muscle atrophy, the signaling mechanisms involved in the atrophy in these two models might be completely different. Skeletal muscle unloading is a well-defined model. Nevertheless, role of p38MAP kinase signaling in muscle atrophy in this model was not previously described. Current study for the first time shows the importance of p38α MAP kinase in the regulation of skeletal muscle atrophy during unloading.
2. Yuasa and colleagues used p38α MAP kinase knockout mice in their study. Numerous studies showed that during embryonic/fetal development knockout mice adapt to the lack of specific genes by increased/modified expression of other genes. It was previously reported that p38 MAP kinase knockout mice have increased embryonic/fetal lethality. In addition, Yuasa and colleagues reported significantly reduced total body weight in adult p38α MAP kinase knockout mice. It is evident, that lack of p38 MAP kinase causes significant changes during embryonic/fetal development and post-natal growth. The nature of these changes is not well characterized. Due to the presence of these uncharacterized adaptations in p38α MAP kinase knockout mice, it is unclear what effect these adaptations could have had on the denervationinduced atrophy. 3. Current study used clinically approved VX-745 inhibitor. This inhibitor (Neflamapimod) is recommended for the treatment of dementia and Alzheimer's disease in human patients. We for the first time used VX-745 inhibitor in the unloading-induced skeletal muscle atrophy model. 4. VX-745 inhibitor is significantly different from the inhibitor used in previous studies (SB203580 cell-permeable p38 MAPK inhibitor) due to the specificity of its action on the p38α MAPK. SB203580, in addition to inhibiting p38 MAPK, also asserts some inhibitory activity on GAK, CK1, RIP2, c-Raf and GSK3. VX-745 is a potent and selective p38 alpha MAPK inhibitor, with 20-fold selectivity over p38 beta and 1000-fold selectivity over closely related kinases ERK1 and JNK1-3.

Critique 2. In Fig.1, the authors analyzed the phosphorylation of p38 MAPK.  The authors should show the phosphorylation of other MAPK, such as p44/42 MAPK and JNK, to show the specificity of VX-745.  In addition, the authors should show which lanes indicate control, HS and VX.

Answer: VX-745 inhibitor was selected for our study based on it’s high specificity of action on p38 alpha MAPK. It was reported in the previously published studies that VX-745 has 1000-fold higher selectivity for p38 alpha MAPK when compared with closely related kinases ERK1 and JNK1-3. 

Critique 3. In Fig. 2, the authors only analyze the muscle weight.  The muscle weight should be normalized by body weight.  This is important because hindlimb-suspension causes a decrease in body weight.  The authors should show that the decrease in muscle weight was not due to the decrease in body weight.  Furthermore, the authors should analyze the cross-sectional area of muscle fibers to show that these rats cause muscle fiber atrophy.

Answer: There was no statistically significant decrease in the total body weight due to unloading in any experimental group in our study. We provided data on the body weight in the revised version of the manuscript. Therefore, changes in skeletal muscle weight in our study are unrelated to the changes in total body weight. The goal of our study was to evaluate changes in the signaling pathways due to the early stages of unloading. It is well-documented that unloading causes atrophy of skeletal muscle fibers. Therefore, in our opinion, unloading-induced decrease in skeletal muscle mass in our study is a result of the previously well-documented skeletal muscle fiber atrophy. 

Critique 4. Although the authors analyzed several atrophy-related genes, the authors did not show any relationship between those genes and the activity of p38 MAPK.  The authors should show that calpain-1, ubiquitin, atrogin-1, MuRF1, PGC-1a, IL-6, and IL-6R were directly regulated by p38 MAPK.  Overexpression or suppression of p38 MAPK without hindlimbsuspension should be required.

Answer: The goal of our study was to evaluate whether p38 alpha MAPK signaling has a critical role in unloading-induced skeletal muscle atrophy. We evaluated the effect of p38 alpha MAPK inhibition on the activity/expression of atrophy-related genes. We were not evaluating whether p38 alpha MAPK has direct effect on these genes or it works indirectly via some other signaling mechanisms. This type of evaluations would be very difficult to perform using rat model. The most appropriate model would be cell culture and it can be a topic for a different study. We thank the reviewer for the suggestion. It might be a focus of our future studies. Yuasa and colleagues (2018) showed that the levels of expression of E3 ubiquitin ligases and  FOXO3 in skeletal muscles, as well as muscle mass/total body mass ratio in p38MAP kinase knockout mice was the same as in the wild type mice. p38MAP kinase is involved in cellular response to stress and cytokines, which are significantly upregulated during muscle unloading. Moreover, inhibiting p38MAP kinase in the wild type mice can have an effect unrelated to the response to unloading. For example, p38MAP kinase knockout mice have significantly smaller

than the wild type mice, and the signaling pathways involved in this differences are not elucidated (growth hormone?, IGFs?). 

Critique 5. In all figures, VX should be HSVX.

Answer: Thank you for the suggestion. We modified the manuscript.  

Critique 6. In line 328, "P 0.05" should be "P<0.05".

Answer: Thank you for the suggestion. We modified the manuscript.

Authors responses to the reviewer

We would like to thank again the reviewers for taking time to thoroughly evaluate the resubmission of our manuscript.

Critique 1. In many cases even in the situation of stating p38α MAPK is adequate, the authors does not specify p38α MAPK and write simply as p38 MAPK. Specify p38α MAPK when appropriate. Answer: Thank you for the suggestion. We modified the manuscript stating that the effect was specific for the p38α MAPK.

Critique 2. The reason why the authors study soleus muscle is not clear. Muscle-specific ablation of p38α MAPK, MuRF1 and MAFbx in fast muscle is reported in ref 10.

Answer: During skeletal muscle unloading rat soleus muscle undergo atrophy significantly faster than the rest of the highndlimb muscles. Soleus is the best muscle to evaluate early changes in the response to unloading. For example, the highest levels of expression of E3 ubiquitin ligases are reached in soleus muscle after three days of unloading. Detectable atrophy in fast muscle might require 6-7 days of unloading.

Critique 3. Authors should discuss the differential regulation of Murf1 and MAFbx in more depth, cosidering time course, species difference, and muscle fiber types. Histological analyses are lacking.

Answer: Thank you for the suggestion. We expanded the discussion of differential regulation of Murf1 and MAFbx E3 ubiquitin ligases in the manuscript. The difference between the results of our study and previously published data by Yuasa and colleagues (2018) could be related to the distinct signaling pathways/ transcription factors regulating denervation and unloading in skeletal muscle. Expression of E3 ubiquitin ligases during skeletal muscle unloading can be regulated by several different mechanisms (Mochalova EP, Belova SP, Mirzoev TM, Shenkman BS, Nemirovskaya TL. Atrogin-1/MAFbx mRNA expression is regulated by histone deacetylase 1 in rat soleus muscle under hindlimb unloading. Sci Rep. 2019 Jul 16;9(1):10263. doi: 10.1038/s41598-019-46753-0).

The goal of our study was to evaluate changes in p38α MAPK signaling pathways during unloading. It is known from previous publications that unloading causes atrophy of skeletal muscle fibers. Therefore, we have not performed histological analyses in our study. 

Critique 4. In Fig. 1 and 4C, 5, 6, 8, define lanes with C, HS and VX. In Fig.4, western blot for MAFbx is not provided. Is the antibody suitable for protein blotting available? In method section 4.3, total and phosphorylated pP38 MAPK should be total and phosphorylated p38 MAPK, respectively. Some errors in English are found.

Answer: Thank you for the suggestions. We modified the manuscript.

Critique 6. Line 227, correct [we think]. [it is possible] might be more appropriate.

Answer: Thank you for the suggestions. We modified the manuscript.

Critique 7. Line 256, 261, bread is not good for rat.

Answer: Thank you for the suggestions. The amount of bread administered to rats was very small. VX-745 inhibitor was homogeneously distributed in the bread. We do not think that small amount of bread adversely affected the health of rats in the current study. 

Critique 8. Line 305, [1 μl of cDNA] should read [One μl of cDNA].

Answer: Thank you for the suggestion. We modified the manuscript.

Critique 9. Line 325, < is missing. P < 0.05.

Answer: Thank you for the suggestion. We modified the manuscript.

Critique 10. In references, unify description of page numbers such as 2181-2188 and R154657. In some references (ref 7, 17, 23, 33), page numbers and/or identification numbers is missing. Although the authors cite ref 43 for VX-745 dose determination, 3 weeks of administration of VX-745 (50 mg/kg) was reported in ref 43. By contrast, only 3 day administration is performed in this study. Therefore, it is not clear how the authors determine the dose of the VX-745. What is the status of clinical trial for VX-745 (Neflamapimod)? Is the dose for human available? If available, the authors can compare the dose of the current study with that of clinical trial. These information would be of help understand the determination of dose.

Answer:  Thank you for noticing inconsistency in the list of references. We used EndNote program to generate list of references for the current manuscript using Int. J. Molecular Sciences formatting style. EndNote takes information for references in the PubMed and Web of Science Core Collection. We checked all of the references at PubMed and we were not able to find page numbers for the references that you listed. Nevertheless, all of the references were easily found in PubMed. We think that the readers will have no difficulties in finding any of the listed references in the PubMed. In the clinical trial, 40 mg capsules were administered orally, BID or TID with food for 16 weeks; subjects followed the BID regimen if weighing <80 kg or the TID regimen if weighing ≥80 kg (ClinicalTrials.gov). In our study, rats received 10 mg/kg/day. This correlates with the dose administered to the patients in the clinical trial.

Reviewer comments:

Overall, the authors did not address my experimental concern.

Critique 1. Äb0As the authors described, the basic concepts regarding the role of p38MAPK on muscle atrophy have already been reported (Yuasa et al., Sci Rep, 2018). Thus, the novelty of this study was quite low. Why did the authors use VX-745? What were the differences between SB203580 and VX-745? If VX-745 does not have anything better than SB203580, what is the novel point of this study? The authors should mention this point.

Answer: Äb0Current study is novel and it is significantly different from the study previously published by Yuasa and colleagues (2018). We are very familiar with the study published by Yuasa and colleagues and we referenced it in our manuscript.

There are several principal differences between our study and the study published by Yuasa and colleagues:

1. Yuasa and colleagues used a denervation model to study skeletal muscle atrophy. In our study, skeletal muscle atrophy was induced by the unloading with preserved muscle innervation. Previous studies reported no abnormalities in nerve conduction in rat models during unloading. Although both denervation and unloading cause skeletal muscle atrophy, the signaling mechanisms involved in the atrophy in these two models might be completely different. Skeletal muscle unloading is a well-defined model. Nevertheless, role of p38MAP kinase signaling in muscle atrophy in this model was not previously described. Current study for the first time shows the importance of p38α MAP kinase in the regulation of skeletal muscle atrophy during unloading.
2. Yuasa and colleagues used p38α MAP kinase knockout mice in their study. Numerous studies showed that during embryonic/fetal development knockout mice adapt to the lack of specific genes by increased/modified expression of other genes. It was previously reported that p38 MAP kinase knockout mice have increased embryonic/fetal lethality. In addition, Yuasa and colleagues reported significantly reduced total body weight in adult p38α MAP kinase knockout mice. It is evident, that lack of p38 MAP kinase causes significant changes during embryonic/fetal development and post-natal growth. The nature of these changes is not well characterized. Due to the presence of these uncharacterized adaptations in p38α MAP kinase knockout mice, it is unclear what effect these adaptations could have had on the denervation-induced atrophy.
3. Current study used clinically approved VX-745 inhibitor. This inhibitor (Neflamapimod) is recommended for the treatment of dementia and Alzheimer's disease in human patients. We for the first time used VX-745 inhibitor in the unloading-induced skeletal muscle atrophy model.
4. VX-745 inhibitor is significantly different from the inhibitor used in previous studies (SB203580 cell-permeable p38 MAPK inhibitor) due to the specificity of its action on the p38α MAPK. SB203580, in addition to inhibiting p38 MAPK, also asserts some inhibitory activity on GAK, CK1, RIP2, c-Raf and GSK3. VX-745 is a potent and selective p38 alpha MAPK inhibitor, with 20-fold selectivity over p38 beta and 1000-fold selectivity over closely related kinases ERK1 and JNK1-3.

The authors should mention these points in the manuscript. In addition, the authors should note that the role of p38 MAPK for muscle atrophy was evaluated by several experimental models including fasting (Ryu Y et al., Int J Mol Sci. 2019), denervation (Yuasa et al., Sci Rep, 2018, using similar inhibitor), casting (Kim J et al., Korean J Physiol Pharmacol. 2009) which is very similar to unloading model, and hind-limb suspension at late stage (Hilder TL et al., J Appl Physiol, 2005).

Thus, the novelty of this study is that the authors focused on the early stage of unloading-induced muscle atrophy. Nevertheless, the analysis of this study was preliminary and did not show any molecular and pharmacological advantage of VX-745.

Critique 2. Äb0In Fig.1, the authors analyzed the phosphorylation of p38 MAPK. The authors should show the phosphorylation of other MAPK, such as p44/42 MAPK and JNK, to show the specificity of VX-745. In addition, the authors should show which lanes indicate control, HS and VX.

Answer: Äb0VX-745 inhibitor was selected for our study based on it’s high specificity of action on p38 alpha MAPK. It was reported in the previously published studies that VX-745 has 1000-fold higher selectivity for p38 alpha MAPK when compared with closely related kinases ERK1 and JNK1-3.

Did the authors evaluate the specificity of VX-745 in the muscle atrophy models?

If the authors' conclusion is "P38α-MAPK signaling inhibition attenuate soleus atrophy during early stages of muscle unloading.", the authors should show the specificity of VX-745 in vivo muscle atrophy model.

CÄb0ritique 3. Äb0In Fig. 2, the authors only analyze the muscle weight. The muscle weight should be normalized by body weight. This is important because hindlimb-suspension causes a decrease in body weight. The authors should show that the decrease in muscle weight was not due to the decrease in body weight. Furthermore, the authors should analyze the cross-sectional area of muscle fibers to show that these rats cause muscle fiber atrophy.

Answer: Äb0There was no statistically significant decrease in the total body weight due to unloading in any experimental group in our study. We provided data on the body weight in the revised version of the manuscript. Therefore, changes in skeletal muscle weight in our study are unrelated to the changes in total body weight. The goal of our study was to evaluate changes in the signaling pathways due to the early stages of unloading. It is well-documented that unloading causes atrophy of skeletal muscle fibers. Therefore, in our opinion, unloading-induced decrease in skeletal muscle mass in our study is a result of the previously well-documented skeletal muscle fiber atrophy.

Again, the authors did not address my experimental concern. The authors should note that muscle weight is affected by several factors including inflammation. How did the authors conclude the effects of VX-745 on muscle atrophy without analyzing any histological aspects?

Critique 4. Äb0Although the authors analyzed several atrophy-related genes, the authors did not show any relationship between those genes and the activity of p38 MAPK. The authors should show that calpain-1, ubiquitin, atrogin-1, MuRF1, PGC-1a, IL-6, and IL-6R were directly regulated by p38 MAPK. Overexpression or suppression of p38 MAPK without hindlimb-suspension should be required.

Answer: Äb0The goal of our study was to evaluate whether p38 alpha MAPK signaling has a critical role in unloading-induced skeletal muscle atrophy. We evaluated the effect of p38 alpha MAPK inhibition on the activity/expression of atrophy-related genes. We were not evaluating whether p38 alpha MAPK has direct effect on these genes or it works indirectly via some other signaling mechanisms. This type of evaluations would be very difficult to perform using rat model. The most appropriate model would be cell culture and it can be a topic for a different study. We thank the reviewer for the suggestion. It might be a focus of our future studies.

Yuasa and colleagues (2018) showed that the levels of expression of E3 ubiquitin ligases and FOXO3 in skeletal muscles, as well as muscle mass/total body mass ratio in p38MAP kinase knockout mice was the same as in the wild type mice. p38MAP kinase is involved in cellular response to stress and cytokines, which are significantly upregulated during muscle unloading. Moreover, inhibiting p38MAP kinase in the wild type mice can have an effect unrelated to the response to unloading. For example, p38MAP kinase knockout mice have significantly smaller

than the wild type mice, and the signaling pathways involved in this differences are not elucidated (growth hormone?, IGFs?).

The authors did not show any experimental evidence that VX-745 ameliorate muscle atrophy by inhibiting p38 MAPK, and that p38 MAPK regulated its suggested downstream targets in muscle atrophy models. Thus, this manuscript is rather descriptive since many molecular relationships between p38 MAPK and its suggested downstream target was not analyzed.

The authors should show the molecular link between p38 MAPK and suggested downstream targets in muscle atrophy models.

Reviewer comments

Some of the previous comments have been amended in the revision.

However, some criticisms should be properly replied and corrected in the manuscript.

Critique 1

Line 40 and 290, p38α MAP kinase would be proper.

Line 273, inhibition of p38α MAP kinase would be proper.

Critique 3

It is better to add histological analyses including measurement of cross-sectional area of muscle fibers to show muscle fiber atrophy in this study.

Critique 4

The reason why western blotting for Mafbx is not performed is not replied.

Critique 7

The reviewer think that the term ‘bread’ is not suitable for experimental animals. Use ‘food’ or other appropriate wording

Critique 10

The page numbers in the references are not unified yet.

The reviewer means that page numbers of ref 2 is R1546-57, however, in ref 14, the page numbers are written as R408-R417. So, unify the description of page numbers. Also, in ref 26, page numbers are written as 225-9, and in ref 45, page numbers are written as 402-8. By contrast, in ref 42, page numbers are written as 1299-1317, and in ref 43, page numbers are written as 549-556. So, unify the description of the pages throughout the references.

As references, following addition might be informative for the readers.

Ref 25, Sci Rep 2019, 9(1), 10263. (Remove uk , and add 10263)

Ref 7, Physiol. Rep. 2017, 5(16), pii: e13291.

Ref 17, Physiol. Rep. 2016, 4(15), pii: e12876.

Ref 34, Int J Mol Sci 2018, 19(9). pii: E2804.

Figures need corrections.

In Figure 1, the words (C, HS, HSVX) near the bottom line are not shown properly.

Font sizes in different figures are not uniform. For example compare between Fig 1 and Fig 8. The font size is different among the figures. It is not good for the readers.

In Figure 8, the box surrounding the figure is not properly shown. The font size is too small to read.

Correct following typo errors.

Line 36, out previous studies should read our previous studies.

Line 197, is picks right? Is it peaks?

Reviewer comments

Manuscript ID: ijms-735477

 P38α-MAPK signaling inhibition attenuate soleus atrophy during early

stages of muscle unloading

WE should know if HSVX produced any adverse reactions

The method how the weight of soleus muscle was determined should be described

MURF1 and MAFbx should be written in full also in the abstract t first occurrence

3/20

Round 1

Authors responses to the reviewer

We would like to thank again the reviewers for taking time to thoroughly evaluate the resubmission of our manuscript.

Reviewer #2:

Critique 1. The authors should mention these points in the manuscript. In addition, the authors should note that the role of p38 MAPK for muscle atrophy was evaluated by several experimental models including fasting (Ryu Y et al., Int J Mol Sci. 2019), denervation (Yuasa et al., Sci Rep, 2018, using similar inhibitor), casting (Kim J et al., Korean J Physiol Pharmacol. 2009) which is very similar to unloading model, and hind-limb suspension at late stage (Hilder TL et al., J Appl Physiol, 2005).

Thus, the novelty of this study is that the authors focused on the early stage of unloading-induced muscle atrophy. Nevertheless, the analysis of this study was preliminary and did not show any molecular and pharmacological advantage of VX-745.

Answer: Thank you for pointing out the other published studies on the role of p38 MAPK for muscle atrophy during denervation, fasting, casting, and late stage unloading. We are well aware about these and similar publications and referenced several of them in the current manuscript.

For example, we referenced Yuasa et al., Sci Rep, 2018 (reference 10), Hilder TL et al., J Appl Physiol, 2005 (reference 13).

The originality of our study is that 1) we focused on the early stage of the unloading-induced muscle atrophy and 2) we used p38α – specific inhibitor VX-745. The previous publications, including those listed by the reviewer, used SB202190 p38 MAPK inhibitor. It was recently reported that SB202190 should not be used in research studies as an inhibitor of p38 MAPK due to its action on the autophagy-lysosomal axis. (A stress response p38 MAP kinase inhibitor SB202190 promoted TFEB/TFE3-dependent autophagy and lysosomal biogenesis independent of p38. Yang C, Zhu Z, Tong BC, Iyaswamy A, Xie WJ, Zhu Y, Sreenivasmurthy SG, Senthilkumar K, Cheung KH, Song JX, Zhang HJ, Li M. Redox Biol. 2020 Jan 28:101445. doi: 10.1016/j.redox.2020.101445; Differential effect of p38 and MK2 kinase inhibitors on the inflammatory and toxicity biomarkers in vitro. Singh RK, Diwan M, Dastidar SG, Najmi AK. Hum Exp Toxicol. 2018 May;37(5):521-531. doi: 10.1177/0960327117715901 ).

Used in our study p38α MAPK inhibitor VX-745 is highly specific for the p38α MAPK and has minimal effects on other kinases. The results obtained in our study for the first time showed how targeted inhibition of p38α MAPK affects skeletal muscle atrophy during unloading.

Critique 2. Did the authors evaluate the specificity of VX-745 in the muscle atrophy models?

If the authors' conclusion is "P38α-MAPK signaling inhibition attenuate soleus atrophy during early stages of muscle unloading", the authors should show the specificity of VX-745 in vivo muscle atrophy model.

Answer: VX-745 (5-(2,6-Dichlorophenyl)-2-(2,4-difluorophenylthio)-6H-pyrimido[1,6-b] pyridazin-6-one) is a highly potent (IC50 = 10 nM) inhibitor of p38α. VX-745 is also highly selective for p38α MAPK, with 20-fold selectivity over p38beta and 1000-fold selectivity over the closely related kinases ERK1 and JNK1-3. VX-745 was used in the previous studies and its specificity as p38α MAPK inhibitor was documented. Moreover, VX-745 is currently used in human clinical trials for the dementia and Alzheimer's disease (ClinicalTrials.gov).

Critique 3. Again, the authors did not address my experimental concern. The authors should note that muscle weight is affected by several factors including inflammation. How did the authors conclude the effects of VX-745 on muscle atrophy without analyzing any histological aspects?

Answer: VX-745 was delivered systemically with food in our study. It was not injected into the muscle, therefore inflammation was unlikely. Moreover, VX-745 is currently used in human clinical trials for the dementia and Alzheimer's disease (ClinicalTrials.gov) and it does not cause inflammation. We have stained with H&E sections of soleus muscle form two HSVX rats and there was no inflammation present (see representative picture). Previously published studies on muscle unloading also have not observed any skeletal muscle inflammation. 

Critique 4. The authors did not show any experimental evidence that VX-745 ameliorate muscle atrophy by inhibiting p38 MAPK, and that p38 MAPK regulated its suggested downstream targets in muscle atrophy models. Thus, this manuscript is rather descriptive since many molecular relationships between p38 MAPK and its suggested downstream target was not analyzed.

The authors should show the molecular link between p38 MAPK and suggested downstream targets in muscle atrophy models.

Answer: The goal of our study was to evaluate whether p38α MAPK signaling has a critical role in unloading-induced skeletal muscle atrophy. Evaluations of the molecular link between p38α MAPK and suggested downstream targets in muscle atrophy models will require a completely different study. We thank the reviewer for the suggestion. It might be a focus of our future investigations.

Authors responses to the reviewer

We would like to thank again the reviewers for taking time to thoroughly evaluate the resubmission of our manuscript.

Reviewer #1:

Critique 1. Line 40 and 290, p38α MAP kinase would be proper.

Line 273, inhibition of p38α MAP kinase would be proper.

Answer: Thank you for the suggestion. We modified the manuscript.

Critique 3. It is better to add histological analyses including measurement of cross-sectional area of muscle fibers to show muscle fiber atrophy in this study.

Answer: According to our prior experience, after three days of unloading the muscle fiber atrophy is relatively small and measurements of the cross-sectional area of muscle fibers might not produce statistically significant results between C, HS, and HSVX groups.

Critique 4. The reason why western blotting for Mafbx is not performed is not replied.

Answer: Our study was focused on the early stages of skeletal muscle unloading. Usually, changes in gene expression precede the changes in protein expression. Therefore, we concentrated on the evaluation of the gene expression in this study. Moreover, it was previously reported that MuRF1plays larger role in the degradation of the muscle contractile proteins than MAFbx. Therefore, we focused on the evaluation of the MuRF1 gene and protein expression in C, HS, and HSVX groups.

Critique 7. The reviewer think that the term ‘bread’ is not suitable for experimental animals. Use ‘food’ or other appropriate wording

Answer: Thank you for the suggestion. We modified the manuscript.

Critique 10. The page numbers in the references are not unified yet.

The reviewer means that page numbers of ref 2 is R1546-57, however, in ref 14, the page numbers are written as R408-R417. So, unify the description of page numbers. Also, in ref 26, page numbers are written as 225-9, and in ref 45, page numbers are written as 402-8. By contrast, in ref 42, page numbers are written as 1299-1317, and in ref 43, page numbers are written as 549-556. So, unify the description of the pages throughout the references.

As references, following addition might be informative for the readers.

Ref 25, Sci Rep 2019, 9(1), 10263. (Remove uk , and add 10263)

Ref 7, Physiol. Rep. 2017, 5(16), pii: e13291.

Ref 17, Physiol. Rep. 2016, 4(15), pii: e12876.

Ref 34, Int J Mol Sci 2018, 19(9). pii: E2804.

Figures need corrections.

In Figure 1, the words (C, HS, HSVX) near the bottom line are not shown properly.

Font sizes in different figures are not uniform. For example compare between Fig 1 and Fig 8. The font size is different among the figures. It is not good for the readers.

In Figure 8, the box surrounding the figure is not properly shown. The font size is too small to read.

Correct following typo errors.

Line 36, out previous studies should read our previous studies.

Line 197, is picks right? Is it peaks?

Answer: Thank you for the suggestion. We modified the manuscript.

Authors responses to the reviewer

We would like to thank again the reviewers for taking time to thoroughly evaluate the resubmission of our manuscript.

Reviewer #3:

Critique 1. We should know if HSVX produced any adverse reactions.

Answer: We have not detected any adverse effects in rats when administering VX-745 with food. VX-745 is currently used in human clinical trials for the dementia and Alzheimer's disease (ClinicalTrials.gov) and no adverse effects have been reported so far.

Critique 2. The method how the weight of soleus muscle was determined should be described.

Answer: Soleus muscle was carefully dissected, lightly blotted with a filter paper to remove access of blood, and weighted on a very sensitive scale.

Critique 3. MURF1 and MAFbx should be written in full also in the abstract at first occurrence.

Answer: MURF1 and MAFbx are the terms well known in skeletal muscle area of research. Providing the description of the full names for these abbreviations will require a lot of space. The abstract has extremely limited number of words allowed. If we describe the full names for the MURF1 and MAFbx in the abstract, it will take ~5% of the space. Therefore, we provided the full names for these abbreviations when they were mentioned for the first time in the Introduction.

Round 2

Reviewer coments

Critique 2. Did the authors evaluate the specificity of VX-745 in the muscle atrophy models?

If the authors' conclusion is "P38α-MAPK signaling inhibition attenuate soleus atrophy during early stages of muscle unloading", the authors should show the specificity of VX-745 in vivo muscle atrophy model.

Answer: VX-745 (5-(2,6-Dichlorophenyl)-2-(2,4-difluorophenylthio)-6H-pyrimido[1,6-b] pyridazin-6-one) is a highly potent (IC50 = 10 nM) inhibitor of p38α. VX-745 is also highly selective for p38α MAPK, with 20-fold selectivity over p38beta and 1000-fold selectivity over the closely related kinases ERK1 and JNK1-3. VX-745 was used in the previous studies and its specificity as p38α MAPK inhibitor was documented. Moreover, VX-745 is currently used in human clinical trials for the dementia and Alzheimer's disease (ClinicalTrials.gov).

The authors did not address my "experimental" concern. Did the authors evaluate the specificity of VX-745 "in the muscle atrophy models"? Without analyzing other MAPKs, how did the author concluded "P38α-MAPK signaling" inhibition attenuate soleus atrophy during early stages of muscle unloading?

Critique 3. Again, the authors did not address my experimental concern. The authors should note that muscle weight is affected by several factors including inflammation. How did the authors conclude the effects of VX-745 on muscle atrophy without analyzing any histological aspects?

Answer: VX-745 was delivered systemically with food in our study. It was not injected into the muscle, therefore inflammation was unlikely. Moreover, VX-745 is currently used in human clinical trials for the dementia and Alzheimer's disease (ClinicalTrials.gov) and it does not cause inflammation. We have stained with H&E sections of soleus muscle form two HSVX rats and there was no inflammation present (see representative picture). Previously published studies on muscle unloading also have not observed any skeletal muscle inflammation.

The authors did not address my "experimental" concern. The authors should perform histological analysis. From the revised manuscript, the authors described that "The average total body weight was 200 ± 13.5, 186 ±9.97, and 195 ±12.9 grams for C, HS and HSVX rats, respectively". Because the decrease rate of soleus muscle weight was approximately 10% (Fig.2), when muscle weight is corrected for body weight, no significant loss may be observed. If this is true, it means that the authors used the starvation model, not the unloading model. Soleus muscle weight should be normalized by body weight, and the authors should show direct evidence for fiber atrophy by evaluating the cross-sectional area of each muscle fibers.

Critique 4. The authors did not show any experimental evidence that VX-745 ameliorate muscle atrophy by inhibiting p38 MAPK, and that p38 MAPK regulated its suggested downstream targets in muscle atrophy models. Thus, this manuscript is rather descriptive since many molecular relationships between p38 MAPK and its suggested downstream target was not analyzed.

The authors should show the molecular link between p38 MAPK and suggested downstream targets in muscle atrophy models.

Answer: The goal of our study was to evaluate whether p38α MAPK signaling has a critical role in unloading-induced skeletal muscle atrophy. Evaluations of the molecular link between p38α MAPK and suggested downstream targets in muscle atrophy models will require a completely different study. We thank the reviewer for the suggestion. It might be a focus of our future investigations.

The authors did not address my experimental concern. The authors should show the molecular link between p38 MAPK and suggested downstream targets in muscle atrophy models in this study.