PONE-D-24-02730Sporadic inclusion body myositis-derived myotube culture revealed muscle cell-autonomous expression profilesPLOS ONE

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This research was partially supported by Intramural Research Grants 29-4 and 2-5 for Neurological and Psychiatric Disorders provided to M.A. from the National Center of Neurology and Psychiatry of Japan; the Practical Research Project for Rare/Diseases (20dk0310086) provided to M.A. and Moonshot R&D Program (JPMJMS 23zf0127001h0003) to T.A. from the Japan Agency for Medical Research and Development (AMED); Grants-in-Aid for Research on Rare and Intractable Diseases (H29-nanchitou(nan)-ippan-030 and 20CF1036) provided to M.A. from the Ministry of Health, Labor and Welfare of Japan; a Grant-in-Aid for Challenging Exploratory Research (20K21563) provided to M.A. and N.S., Scientific Research C (18K07519) provided to N.S., from the Japanese Ministry of Education, Culture, Sports, Science and Technology. This research was also supported by the Cooperative Research Project Program of the Joint Usage/Research Center at the Institute of Development, Aging and Cancer, Tohoku University.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: I Don't Know

Reviewer #2: 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: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: 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: A straightforward study examining the similarities between myotubes derived from myoblasts from inclusion body myositis patients and age matched controls. These samples are assessed by RNA-seq and compared to RNA-seq data from whole muscle biopsies. Overall, the study is interesting but several areas would strengthen the conclusions and allows for more complete interpretation of the data.

Methods: Central to this study is the isolation of myogenic precursor cells from human muscle biopsies. The protocol used (FACS sorting) relies heavily on negative sorts to isolate myoblasts (MuSCs?) and only CD56 is used as a positive sort. To increase confidence, the isolated cells should also be subjected to a positive sort, or in this case, since the RNA-seq analysis is complete, that validation of the purity of the cells used for differentiation be used with accepted markers for human MuScs (eg. Integrin alpha 7, m-cadherin, CD82).

Figure 1B-E. Overlay of the DAPI and desmin stain is critical to appreciate the % of cells that have not differentiated. While this appears to be high for both conditions, a calculation of the differentiation index would also help appreciate the level of contamination from reserve cells and the expected variability from sample to sample and experiment to experiment. This has an impact on the downstream analysis and the interpretation of the findings.

Figure 2. For the RNA-seq analysis, it would be beneficial to include the FDR cut-offs used for significance in the text. Changes in the expression of factors associated with differentiation and myofiber maturity/contraction can be related to differences in the efficiency of differentiation between samples.

For the comparative analysis, it is not surprising that genes that are uniquely expressed in myofibers are also commonly regulated in whole muscle samples. More interesting however, is the massive number of genes oppositely regulated. No further analysis is provided here, but would be very useful in terms of understanding the origins. Was a similar analysis done for controls (or were the DEGs determined as relative to controls?). It would be interesting to see how divergent the control myotubes are from the healthy whole muscle, as a way to infer differences that the microenvironment contributes.

For the comparison between myotubes generated in culture and whole muscle biopsy samples, The gene name for Tmem8c is actually MYMK and this should be changed throughout.

Figure 3. The text says that the levels were verified at the protein level, however FPKM are shown and no quantification over the patient samples are provided for the immunostained images. Quantifications are essential to validate the conclusions.

Figure 4. Similar comments to Figure 3.

Discussion:

Line 323. I do not think the word pathogenesis applied to the myotubes in culture. I appreciate that you mean a myofiber-intrinsic contribution to the pathogenesis of the disease, but this is not clear as written.

This statement (lines 332-336) “The examination of muscle biopsies suggested altered gene expression in lymphocytes and other immunopathological conditions, phenomena not mirrored in myotube samples.” Is not supported by the data shown.

The statement in lines 337-340 is also unclear as to where the findings come from (as related to SASP). Please clarify.

Minor comments: Care should be taken to use the appropriate gene nomenclature for humans throughout to avoid confusion with mouse genes or protein names.

Reviewer #2: Suzuki et al. present an interesting finding regarding the pathological mechanism(s) involved in sIBM. Previous findings have failed to define the pathology as autoimmune or degenerative, not having determined whether the pathology observed is primarily due to intrinsic or extrinsic forces. In their study the authors confront the gene expression profile using RNA-seq between sIBM biopsy samples and controls as well as with myotubes derived from myoblasts isolated from control and sIBM biopsies. In such a manner, the study is able to differentiate between gene expression profiles of sIBM biopsies (due both to intrinsic and extrinsic forces), and gene expression profiles of in vitro differentiated sIBM-derived myotubes (due to intrinsic forces). The data are interesting and find similar results to previous reports. What is missing is a full consideration of the findings.

Major comments:

1. Figure 1, please show the merged image of DAPI and desmin.

2. As sIBM leads to muscle death and atrophy, it is important to known if any difference was observed in the differentiation potential/rate and viability between controls and sIBM biopsy-derived myoblasts.

3. Data from myotubes suggests a potential alteration of the insulin growth factor response and cellular adhesion in sIBM. How does this fit in with previous findings in the field?

4. In discussing differences between data in Supplementary Table 2 and Supplementary Table 3, the authors observe the absence of immunopathological gene expression (lines 333-341) in myotubes and suggest that some phenotypic findings may be linked to STING and non-cell-autonomous mechanisms. The authors should be careful here with this interpretation. First, there is more than STING to consider; second, data in Supplemental Table 2 could suggest a defective type I interferon response; and third, the authors used a cut-off for choosing which genes to include in their analysis, but it should be noted that in almost all cases, the ratio of gene expression of common genes between sIBM-derived myotubes/control versus sIGM biopsy/control is always decreased. therefore some changes seen in the biopsy samples may have been below the threshold for consideration in the myotubes, but none-the-less still present.

5. The authors focus then on ApoE, TMEM8C and myogenin. The pathophysiological role that upregulation of these proteins has in sIBM is difficult to determine. TMEM8C and myogenin should, at face value, favor muscle development. Likewise, the role of ApoE is difficult to determine due to its varying immunomodulatory roles, including T cell suppression.

6. From the RNA-seq data, the authors should state the number of reads that could not be matched for each experiment. Additionally, in regard to the 53 genes common to myotubes and biopsies, the authors should look at the sequence and exon usage to determine if any of these common genes contain mutations or are potentially alternatively spliced between control and sIBM, including ApoE and TMEM8C (use of a monoclonal antibody may not necessarily catch all isoforms).

Minor comments:

1. Please define the supplier DBS, line 221.

2. For the lysis buffer on line 233, add "(RIPA)" between "radio-immunoprecipitation" and "buffer".

3. In Supplemental Table 3 it might be beneficial to organize the table as those genes that commonly increase in sIBM versus those that increase in biopsy but decrease in myotubes or vice versa.

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Reviewer #1: No

Reviewer #2: No

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Sporadic inclusion body myositis-derived myotube culture revealed muscle cell-autonomous expression profiles

PONE-D-24-02730R1

Dear Dr. Suzuki,

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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Keisuke Hitachi

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have carefully and completely addressed my comments from the first round of review. The revised manuscript better discusses limitations of the experimental approach and also provides additional information to the reader which helps interpretation of the data sets.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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