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PONE-D-21-21284

The effect of regular exercise and apolipoprotein B knockdown on abnormal cardiac rhythm induced by HFD

PLOS ONE

Dear Dr. Zheng,

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PLOS ONE

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This study was funded by the Instituto de Salud Carlos III (www.isciii.es) PI15CIII/00047 to TBH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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I have received assessments your manuscript from two external reviewers and I completely agree with their assessment. Therefore, your manuscript will require a major revision. The subject area is quote interesting however, the reviewers has raised reasonable questions/concerns. All these questions/concern should be addressed in the revised version for the consideration in PLoS ONE. In addition, the authors are re-using the same images for one than one panel (Fig. 1 A and B, HC and Fig. 2 A and B HC). They should use another representative image in each figure panel.

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

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: Manuscript compares the effect of heart-specific apoLpp knockdown and the effect of exercise on consequences of a high fat diet. Although the effects of HFD, apoLpp and exercise on heart performance have all been examined previously, it is of some interest to look at the combination. There are several significant problems with the study that reduce enthusiasm, including genetic background issues, lack of controls for female-specific dietary responses to egg laying, and inappropriate use of Oil Red O as a lipid quantitation assay.

Major:

1. Not enough attention has been paid to rigor in the control of genetic background. Comparisons are made between outcrossed animals and isogenic control animals without recognizing possible effects of hybrid vigor on phenotypes.

2. Rescue experiments are compared to each other (e.g. exercise to apoLpp RNAi) without including comparisons to wild type. This makes it impossible to quantitate the extent of rescue.

3. No attention has been given to possible effects of any of the treatments on feeding rate.

4. Although experiments are done in females, no attention has been given to effects of reproduction on lipid accumulation. This is an essential point to address, because eggs are a substantial subset of the lipid accumulation in any female.

5. No rationale is given for limiting experiments to females.

6. Figures showing representative flies are clearly of different lengths and overall sizes, which surely must complicate assessment of whether they have "flatter abdomens". Abdominal bulging may result from reduced egg deposition and must be controlled for.

7. Oil Red O staining is not a quantitative assay for lipid content.

8. It is a major stretch to say that apoLpp RNAi phenotypes prove that effects of HFD are because of abnormal lipoprotein concentration.

9. Discussion section does not effectively place the work in context, and basically restates the Introduction. Also, it is labelled "Discuss".

Minor:

1. ApoLpp should be spelled out and defined at first mention

2. Manuscript should be heavily edited for grammar.

3. methods descriptions should be in past tense, and some of the statistical methods do not appear quite right. For example, pairwise comparisons after a 2-way ANOVA should use post-hoc adjustment, such as a Tukey test. All the comparisons in the paper appear to be pairwise, so it is not clear why the stats methods describe something different?

4. What is interception length?

5. does "half-exposure surgery" mean semi-intact preparation?

6. Description of Figure 1 refers incorrectly to figure 2 in several places, which is very confusing

7. meaning of "superimposing effect" is not clear

Reviewer #2: Ding et al present an interesting group of studies showing the role of exercise and the apoLpp protein in high-fat-induced cardiac dysfunction. This is a nice short paper with high quality data and will be of interest to those studying the roles of diet and lipid transport on cardiac function. However, a few changes need to be made before I can recommend it for publication in PLoS One.

The triglyceride (TG) assay method is not described correctly- there are no reagents listed and an antibody/substrate approach makes no sense in this protocol because triglycerides are not typical protein antigens. A standard curve should also be used in this assay so the ug TG/mg body weight can be reported. Without the proper methods, the data on TG cannot be evaluated.

It would also be helpful if you described briefly how you isolated hearts; on line 210, it says that qPCR was used to quantify mRNA in cardiomyocytes, but the heart has nephrocytes and aliary muscles that are stuck to it and are sometimes included in the prep.

The heart rate of ~ 2 sec is surprising to me because most research papers have a rate of 2-3 beats per second. Perhaps the units should be Hz instead of seconds? Please double check this.

For Fig 2B’s legend, “there is no accumulation of fat in the abdomen of flies in the HC-KD group” is not supported by the image. Reduced accumulation even looks like a stretch, but seems to be corroborated by the quantitative data in 2H provided that a legitimate TG assay was done.

Figure 3’s title text “Regular exercise improves abnormal heart rhythm by reducing the expression of apoLpp mRNA in cardiomyocytes” is not supported by the data. The authors have only shown that apoLpp is reduced by exercise, not that this is the mechanism by which heart function is improved. For that, I think you’d have to overexpress apoLpp in the heart during exercise and show that heart function differs from the exercised control genotype. This experiment is one I would request if this were a different journal, but isn’t necessary to publish in PLoS One.

Fig 3G is TG and Fig 3H is apoLpp mRNA, but the legend has it the other way around.

With respect to the figure legends for panel A in Figs 1-4, I think it would be more clear if the text read simply “the abdomen is larger” (or smaller) rather than more prominent or flatter. It’s harder to think about how prominence and flatness are being measured and these might mean different things to different people. Large/small is straightforward- it would be even better if you graphed their wet or dry weights, data you might already have from the triglyceride assays. For 4A, remove significant; significant is reserved for statistical significance in my mind. You might say “no dramatic difference” instead. Again in 4A, I would focus on the size (smaller or larger), rather than “morphology,” which is not as easily ascertained from the images provided. There is only one view shown (perhaps the dorsal abdomen is spotted!) and the size is the most apparent difference in all of these panels.

It would be of interest to directly compare control and apoLpp RNAi responses to exercise. This is difficult considering the way the figures are currently set up; perhaps it could be helpful to add a table or expand the Results or Discussion to mention these. I found the term “superimposed” difficult to understand. It could be rephrased as “Cardiac apoLpp is required for (some of) the benefits of exercise” – then you might consider why there is no longer a benefit from exercise. It may be because apoLpp RNAi hearts are more resilient during HF feeding. So cardiac apoLpp knockdown could be metabolically equivalent to exercising… this would be great, like an exercise mimetic. When I compare 3D and 4D, it looks like the heart rate is already an “exercised” value in the RNAi flies without exercise; the same seems to be true for fibrillation rates in 3F vs 4 F.

There are several small mistakes in writing that should be addressed- I will only mention a few here. Genes and mRNAs should be italicized. Line 110 should say reagent and line 111 and elsewhere should say H2O with a subscript #2. The use of female flies appears four times in the Fig 1 legend and three times in Figure 4’s legend and should be compressed to be as concise as possible. You can mention all flies are female early in the methods and results- then maybe in the Discussion (not Discuss, as on line 336) when comparing your findings to other high fat diet studies.

Finally, I am sleep deprived but I found it hard to keep it in my mind that HFD=HC. Why not use HFD throughout all of the text and figures? It would be easier for some people.

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Reviewer #2: No

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PONE-D-21-21284R1Effects of Drosophila melanogaster  regular exercise and apolipoprotein B knockdown on abnormal heart rhythm induced by a high-fat dietPLOS ONE

Dear Dr.Zheng,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers agreed that this is much improved version of the manuscript compared to the original submission. The authors did good job in responding my comments as well as questions raised by both reviewers. However, both reviewers still had some minor comments and need additional clarification. I agree with comments made by the reviewers. I also believe that addressing these minor comments should not take that long and authors should submit their revised version with in 30-days. 

Please submit your revised manuscript by 30-days. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Girish C. Melkani, Ph.D

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Both reviewers agreed that this is much improved version of the manuscript compared to the original submission. The authors did good job in responding my comments as well as questions raised by both reviewers. However, both reviewers still had some minor comments and need additional clarification. I agree with comments made by the reviewers. I also believe that addressing these minor comments should not take that long and authors should submit their revised version with in 30-days.

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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: Authors have done an excellent job of addressing most of my concerns, I only have a few small comments remaining:

1. Authors have addressed my concerns about genetic background by clarifying that control flies are outcrossed RNAi lines, which is acceptable.

2. I am still a bit skeptical about the accuracy of Oil Red O intensity as a quantitation, and would recommend to authors that they only use this in cases where there is a clear and obvious qualitative difference in fat levels. However, since the authors have modified their methods to match those in published work, and since the results agree with the TAG assay, I think it's ok here.

3. I appreciate the addition of normal food groups in some figures to make it easier to see the degree of rescue.

4. My concern about feeding rate was not a concern about how the different groups were housed and fed, it was a concern about whether HFD or exercise themselves would alter feeding rate, which was not tested here, and would be a good control to add in future. Authors could at least reference other studies that have shown those controls, which may be good enough here.

The only change that I would insist on at this point is that the authors need to write their conclusions without stating that exercise acts through apoLpp to exert its effects on heart function and fat storage. All of these experiments are also consistent with these two things acting in parallel, and this is a key point. Just because exercise reduces apoLpp levels does not mean that this is the key mechanism of exercise effects. Authors should say something more like "apoLpp reduction may be involved in exercise-induced protection against HFD".

Reviewer #2: The authors have taken a number of steps to improve the manuscript, both with respect to the data and the writing. The data analysis and Discussion are significantly improved and I found most of the data compelling. The authors addressed most of my concerns and I still have significant concerns about the triglyceride assay, below. The other reviewer raised some very good points about the potential roles of feeding and egg laying that cannot be easily dismissed; however, these may fall outside the scope of the current manuscript.

Line 68 “sufficiently reduced to resist HFD-induced cardiac function impairment” is confusing. I think instead of resist they might mean elicit or potentiate.

line 87: w1118 has a lower case w.

Line 146. This TG assay is still of concern and my previous concern was not addressed. TG ELISAs seem to measure thyroglobulin, not triglycerides. There are not enough details to ascertain what was done. I do not believe triglycerides can be measured by an ELISA and the authors have given me no information to believe otherwise. A Google search for the kit listed produces nothing. What was used as the standard? Figs 1H and 2H should have a unit (ug TG/mg fly or something like this) not OD. OD isn’t as informative.

Line 164: How was Photoshop used to quantify the intensity of staining? Was a thresholding done to identify the red areas of the fat body cells? Were these dissociated from the cuticle before mounting and imaging? Some description would be helpful here.

Line 206: Figure 1: was this data generated in the control, transheterozygous genotype (UAS-Lpp RNAi x w1118 offspring)?

Line 288: The figure title is not fixed as the authors say in the response. The old, bad title is retained in the manuscript. Please use the new, improved title that you wrote in the reviewer response.

Line 298-300: the G and H descriptions need to be switched to match the figure.

Line 331- Figure 4: it doesn’t make sense to me to exclusively compare the normal diet control genotype to the HFD knockdown genotype, although I appreciate the fact that they are so close to each other. It seems as though the HFD control genotype should be included so the reader can see what knockdown and exercise does to each genotype. I realize this may be duplicative of the data shown in other panels, but if possible, it’s easiest to see differences when data are compared side-by-side.

Line 333: superimposed effects could read “combined effect”

Line 393: it reads “Knockdown of apoLpp in cardiomyocytes… does not directly affect the heart” I do not think this conclusion can be made given the data shown.

Line 400: reads “Flies allowed to exercise regularly show similar results to mammals with regard to high-fat- or high-sugar-induced heart damage.” The references provided do not seem to show these results.

The tracked changes would be easier for me to read without the old text in place; it’s easiest if the replacement text is highlighted.

Effects of Drosophila melanogaster  regular exercise and apolipoprotein B knockdown on abnormal heart rhythm induced by a high-fat diet

PONE-D-21-21284R2

Dear Dr. Zheng,

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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Kind regards,

Girish C. Melkani, Ph.D

Academic Editor

PLOS ONE

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