PONE-D-19-34630

TBX5 R264K induces chronic heart failure not via T-box pathway

PLOS ONE

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PLOS ONE

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https://academic.oup.com/eurheartj/article/29/2/270/433902

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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

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Reviewer #1: No

Reviewer #2: No

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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: Minor Comments

1. Authors are suggested to change the title from “TBX5 R264K induces chronic heart failure not via T-box pathway” to “TBX5 R264K induces chronic cardiomyopathy independently of T-box pathway”, so that it is more appealing to the reader.

2. Please avoid the use of the term heart failure in your in vivo experiments, as a NYHA scoring should be performed to claim it. Please change the term to cardiomyopathy, when referring to the murine models.

3. Table 2B: Please correct Ds to LVDs.

4. Figure 5, considering the Western Blot analysis of ACTA1 is not self explanatory. Proper labelling of the WB images is missing, as well as the molecular weight of the identified targets and the labelling of the samples. The authors are suggested to change it accordingly.

5. The resolution of the figures is poor, which creates an inconvenience for the reader. Authors are suggested to provide a higher image resolution to the given figures. Moreover, concerning the column plots, authors are suggested to use another software, rather than Microsoft Office Excel, in order to export more appealing plots and figures.

Major Comments

1. Authors state to have used transgenic mice of 6-9 months old as an aging model. However, according the literature, mice of that age correspond to human adults around 40 years of age. In order to consider a mouse aged, the minimum age of 18 months should be reached. Authors are advised either to correct the statement, or to present mortality data justifying the selection of the specific age for their murine model.

2. Hematoxylin-Eosin staining seems to be poorly performed as nuclei (blue) are not visible in the histology images. Please refine your staining, by improving your H&E protocol and by washing your specimens with PBS for the removal of blood (as visible in Fig. 1A lower panel). Moreover, considering that authors are commenting on intracardiac fibrosis in their transgenic mice, maybe another more specific staining should be applied such as Sirius Red or Masson Trichrome. By the use of these staining techniques the visualization of collagen deposition will be more straight-forward.

3. Column graphs are not presented in a sufficient manner. Individual values, showing the distribution and the number of the biological replicates should be present in the graphs (i.e. scatter plotted column graphs) and statistical analysis (i.e. asterisks) should be presented, deducing the statistically significantly differences between the groups.

4. The use of isoproterenol is not sufficiently justified. Transgenic mice, already present cardiac contractility deficits (e.g. decreased FS%) in the absence of the β-agonist. Therefore, the use of isoproterenol should be better discussed in the Discussion section, considering its transcriptional value in the experiments.

5. Authors shown that the given SNP in the TBX5 leads to a downregulation of ACTA-1 in the myocardium. Concerning that ACTA-1 is a highly important protein in the cellular cytoskeleton, authors are suggested to perform electron microscopical evaluation of the cardiomyocytes’ structure in the transgenic mice. Therefore, a more detailed representation of the effect of TBX5 SNP on cardiomyocytes’ architecture will be available.

6. If applicable, it would be interesting if the authors could confirm the results from their transgenic murine model in human cardiac biopsies, following simple RT-PCR or Western Blot experiments. Thus, the translational value of the current manuscript will be increased.

Reviewer #2: The manuscript describes a new variant of the TBX5 gene located outside the T-box regulatory domain. The variant is over-represented in cardiomyopathy patients and leads to compensated dilated cardiomyopathy in a knock-in animal model. The study is well conducted and organized. Although the new SNP does not seem to be sufficient to induce de-compensated heart disease by itself, the results indicate that it may work as an important phenotype modifier during the evolution of adverse cardiac remodeling in patients. However, the following points must be addressed.

Major points

-The authors focused part of the study on the contribution of differentially expressed mRNAs in the observed phenotypes, have the authors performed a functional enrichment analysis of the differentially expressed transcripts to explore putative underlying biological processes?

-The majority of the differentially expressed transcripts are still uncharacterized non coding RNAs. To foster future investigation, it should be interesting if the authors could briefly discuss their putative role in the observed phenotype.

-According to the results shown on page 13 lines 362-369, the differential expression of Myot between wt and mutant mice was not confirmed by PCR. However the authors seem to disregard this finding when discussing the putative role of Myot in disease evolution. Please clarify.

-Why only 2 out of 8 differentially expressed mRNAs were validated by rt-PCR? To confirm the microarray data all the 8 differentially expressed mRNAs must be validated by real time PCR.

-In some instances the authors report the findings as data not shown (see results lines 330, 334, 368). Although these results refer to not statistically significant differences, all the data must be fully available for the reader. Please amend.

-The authors observed a milder phenotype in the aged mutants with respect to the young-adult ones. How do they explain this finding?

- In the light of the limitations acknowledged by the authors themselves (see discussion 457-462), the title should be rephrased given more emphasis to the role of the mutation as a phenotype modifier rather that as an indipendent pathogenic mechanism.

-The quantification of the histological results shown in fig 1 is lacking and must be added.

-The quantification of the WB data shown in figure 5 is lacking and must be added.

-The plot shown in figure 6 is reported without indication of the statistical analysis. Although evident, the statistically significant differences must be indicated.

Minor points

-Abstract, line 38 the authors state: “there were minor differences in activation of the ANF promoter”, however the results show no differences at all, please amend.

-Not standard abbreviations should be listed in alphabetical order.

-Material and methods, lines 176-177: the descriptions of the young-adult and aged rats seem to be inverted, please amend.

-Discussion, lines 443-444: the sentence “Our results support this speculation by (Fig5).” Is incorrect, please amend.

-In some instances the SNP is referred to as p.Arg264Lys in some others as R264K, to avoid confusion a unique acronym should be used throughout the manuscript.

-In fig.2 caption all the transcripts are referred to as mRNAs, however also non coding RNAs are included in the image, please amend.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PONE-D-19-34630_reviewer-1 comments.docx

TBX5 R264K acts as a modifier to develop dilated cardiomyopathy in mice independently of T-box pathway

PONE-D-19-34630R1

Dear Dr. Hirono,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Vincenzo Lionetti, M.D., PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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

**********

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 provided a satisfactory reply to my comments and the manuscript appears to be quite interesting for the scope of the journal.

Reviewer #2: The authors have adequately addressed the comments raised in my previous review and I feel that the manuscript is now acceptable for publication.

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Reviewer #1: No

Reviewer #2: No