Silencing of circCacna1c Inhibits ISO-Induced Cardiac Hypertrophy through miR-29b-2-5p/NFATc1 Axis

Round 1

Reviewer 1 Report

Lu et al. reported that the silencing of circCacna1c inhibits ISO induced myocardial hypertrophy through the miR-29b-2-5p/NFATc1 axis. This is an interesting study, but there are still many problems. The main drawback is that the authors used mouse heart tissue for circRNA microarray analysis, while cell experiments used H9C2 cells (rat cell line).

Main

1. The histological characteristics of the mouse heart after ISO treatment should be presented and described. 

2. The conservation of key regulatory factors (circCacna1c, miR-29b-2-5p, NFATc1) involved in the manuscript between species needs to be analyzed and described.

3. The material method is not clear enough. Is it sufficient for each group of 3 mice, and which part (atrium, ventricle, or other) is the sample used for fixation and frozen storage. Furthermore, based on our past experience, the degree of myocardial hypertrophy varies in each mouse. From the author's circRNA heat map, it can also be seen that the sample uniformity is poor.

4. CircRNA microarray data needs to be uploaded to a public database.

5. The discussion section is too thin and needs further improvement

Minor

1. The construction of myocardial hypertrophy models requires reference to previous research or pre experiments.

2. According to limited RNA FISH's results, it seems that myocardial cells did not become larger after ISO treatment in this study. Meanwhile, the image resolution is too low.

3. There seems to be differences in the criteria for determining whether circRNA expression is different in the article. (Line 78 “Genes with an adjusted P < 0.01 and an absolute log 2 (fold change) > 1”; Line 195 “fold change > 1.5, P < 0.05”)

4. circCacna1c should be marked in Figure 1b and c.

5. Line 257 “ n ≥ 3” . It is necessary to specify the number of samples for each experiment.

6. What do the yellow and gray colors in the circRNA model in Figure 4A represent? The binding position of the miRNA that binds to it should be labeled in the diagram.

7. From Figure 4C, it can be seen that the miRNA seed sequence only has 6 binding sites, which do not seem to be tight. What are the binding free energies of these two positions?

8. Are there any other target genes for miR-29b-2-5p? Why only NFATc1 was selected.

9. In the presented WB results, it seems that the number of samples is not enough for 3.

10. The binding relationship between NFATC1 and miR-29b-2-5p has not been validated.

11. The functionality of NFATC1 has not been verified.

12. References need further standardization.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

1. In the abstract the authors should indicate the objectives of the present investigation clearly for better understanding.

2. Check the abbreviations throughout the manuscript and introduce the abbreviation when the full word appears the first time in the abstract and the remaining for the text and then use only the abbreviation (For example, nuclear factor of activated T cells (NFAT), Fluorescence in situ hybridization (FISH), etc.,). Make a word abbreviated in the article that is repeated at least three times in the text, not all words to be abbreviated. The usage of abbreviations may be avoided in the title and sub-titles.

3. The introduction part appears less informative about cardiac hypertrophy, thus this section should be indicated as detailed to understand the manuscript in clear. The authors may cite recent prevalence or incidence data about cardiac hypertrophy.

 

4. In the materials and methods, the authors may cite references for standard protocol, instead of mentioning kid or manufacture instructions, if reference is given with it and the same should be added in the reference section.

 

5. When referring to SPSS versions beginning from 19, authors should cite ‘IBM SPSS Statistics for Windows, version 25 (IBM Corp., Armonk, N.Y., USA)'.

 

6. The table legends should be improved and a proper footnote should be given. All legends should have enough description for a reader to understand the tables without having to refer back to the main text of the manuscript. For example, the necessary abbreviations should be given (For example, ISO).

 

7. The authors may improve the discussion of their results by focusing on the present findings and introducing data from other authors who also worked with the same or other studies with recent references since it is lack of sufficient references.

 

8. The limitation of the present investigation may be given along with conclusion or under separate heading for understanding the concepts clearly.

1. The English need improvement since there are some grammatical and syntax errors in the manuscript. For example,

·         in line number 40, the words “and are” may be as “and is”;

·         in line number 43, “acting” as “and acting”;

·         in line number 51, “physiological” as “the physiological”;

·         in line number 59, 211 and  246, “control” as “the control”;

·         in line number 59, “model” as “the model”;

·         in line number 93, “ISO-induced” as “the ISO-induced”;

·         in line number 94, “serum-free” as “a serum-free”;

·         in line number 162, “intensity” as “the intensity”;

·         in line number 165, “extracellular” as “the extracellular”;

·         in line number 166, “cells was” as “cells were”;

·         in line number 188, “Significant” as “A significant”;

·         in line number 211 and 212, “cardiac” as “the cardiac”;

·         in line number 219, “was upregulated” as “were upregulated”;

·         in line number 254, “Morphology” as “The morphology.

The grammar mistakes which are not mentioned here are also to be checked and corrected properly.

 

2. There are some typing mistakes as well, and authors are advised to carefully proof-read the text. For example,

·         in line number 30, the word “leads” may be as “leading”;

·         in line number 307, “calcineurin dependent” as “calcineurin-dependent”;

·         in line number 333, “participates” as “participate”;

·         in line number 347, “miRNA targeted” as “miRNA-targeted”;

·         in line number 351, “ameliorated” as “ameliorate”;

·         in line number 355 and 362, “show” as “shows”. 

The typos not mentioned here are also to be checked and corrected properly.

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

This study explores the role of circular RNA (circRNA) circCacna1c in cardiac hypertrophy and its regulatory relationship with miR-29b-2-5p and NFATc1. The researchers identify circCacna1c as a novel circRNA upregulated in ISO-induced hypertrophic heart tissues and H9c2 cells. Silencing circCacna1c inhibits hypertrophic growth and the expression of hypertrophic genes ANP and BNP. CircCacna1c is predominantly localized in the cytoplasm, suggesting its potential as a miRNA sponge. The bioinformatics analysis predicts that circCacna1c could bind to miR-29b-2-5p, which is downregulated in hypertrophic mouse heart tissues. The direct interaction between circCacna1c and miR-29b-2-5p is confirmed through a dual-luciferase reporter assay. NFATc1, a key player in cardiac hypertrophy, is identified as a target gene of miR-29b-2-5p. Overexpression of miR-29b-2-5p reduces NFATc1 expression at both mRNA and protein levels. Silencing circCacna1c increases miR-29b-2-5p expression and decreases NFATc1 expression, suggesting the involvement of the circCacna1c/miR-29b-2-5p/NFATc1 axis in regulating hypertrophic gene expression. Overall, this study sheds light on the regulatory mechanisms underlying cardiac hypertrophy. 

However, before accepting the study for publication, there are a few concerns that need to be addressed:

 

1. Image: 3B. si-circCacna1c+ISO cells are smaller than si-NC, but it does not look healthy. Even the actin polymerization is very high compared to any other treatment group. I am wondering if circCacna1c is truly modulating any phenotype or if it is showing a different phenotype altogether. Could there be some off-target effects? Additionally, is there any link between actin polymerization and cardiac hypertrophy that could explain these observations?
2. Line 262-264: ".....which predicted five miRNAs that are targeted by circCacna1c, including miR-135a-2-3p, miR-6914-5p, miR-5126, miR-29b-2-5p, and miR-669a-5p (Figure 4a). The expression of miR-29b-2-5p and miR-669a-5p were examined in hypertrophic mouse heart tissues." Why were the expressions of miR-135a-2-3p, miR-6914-5p, and miR-5126 not tested? What are their expressions in cardiac hypertrophy? How did the authors determine that these miRNAs cannot be targeted by circCacna1c?
3. "Knocked down circCacna1c increased the expression of miR-29b-2-5p in H9c2 cells (Figure 4d)." How does knocking down circCacna1c directly regulate miR-29b-2-5p expression? Please provide a clear explanation of this mechanism.
4. Fig 5B: The morphology of ISO-induced H9c2 cells with miR-29b-2-5p overexpression appears different from that in Fig. 3B (circCacna1c+ISO). According to the authors' hypothesis, shouldn't miR-29b-2-5p lead to similar morphology? The differences in morphology and actin polymerization pattern need to be discussed and addressed.
5. Not all control qPCR values are set to one. How were the qPCR values calculated, and which value was considered as one for the calculation of relative fold-change? This process needs to be clarified.
6. The authors used ANOVA in almost every scenario. However, when comparing NC-siRNA vs. si-circCacna1c or NC-mimic vs. miR-mimic, a t-test (or non-parametric Mann-Whitney test would be more appropriate) should be used. Please address this concern.
7. Fig. 6d: The level appears to be incorrect. Please correct this.
8. In 6e and f, the RNA and protein levels do not match. I could not observe any difference between NC-mimic and miR-mimic. The use of ANOVA indicates significance, but it may actually be insignificant. I suggest performing a t-test to determine if the difference is indeed insignificant.
9. The study would be more comprehensive if the authors also examined the effect of NFATc1 knock-down on ISO-treated cell morphology and the expression of ANP/BNP.

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 4 Report

The manuscript by Peipei Lu, et al elucidated the regulatory function of circCacna1c in cardiac hypertrophy, circCacna1c works as a sponge of miR-29b-2-3p modulating the expression of the transcription factor, NFATc1, in H9C2 cells under hypertrophic stress condition. The function of this circRNA is novel under cardiac hypertrophy, but lacks some critical experiments make it insufficient to support the final conclusions.

Major points:

-H9C2 cell is a good cell model to study cardiac cell-related molecular mechanisms. However, it cannot fully recapitulate the feature of primary cardiomyocyte cells. So, some critical experiments need to be repeated in primary cells.

-The authors provided comprehensive evidence to prove the functions of miR-29-2-3p and circCacna1c in regulating the expression of NFATc1 separately in H9C2 cells, but lack the evidence to support circCacna1c/miR-29-2-3p axis, which is the major conclusion of the current manuscript. The rescue experiments should be performed in this scenario. 

-Meanwhile, the miR inhibitor or overexpression of circRNA should be used to prove the regulatory function in cardiac hypertrophy.

-In most of cases, the si-NC or NC-mimics shows much stronger effects, so it's not sure wether the effects of sicircRNA or miR-mimics really come from si or miR or not, or simply from the effects of NC. It will be better to change a NC sequence.

-The rationales why the authors choose cardiomyocyte for further studies but not other cardiac cell types should be adjusted in the main text.

-Several problems with the images: 1) the resolution of FISH assay is low, using confocal to capture the stacking of multiple images may improve it; 2) Too much difference at the cell density of Fig 3b. It's better to use the images with similar cell density. Based on Fig 3b and 5b, it looks more like CM atrophy. Did you observe any cell death? 3) In Fig 6, what's the expression of NFATc1 in hypertrophic mouse hearts?

 

Minor issues:

-The dots should be added in all the bar figures to show the distribution of all the data.

-Regarding the expression change of miR-669a-5p, the result from Fig 4b shows miR-669a-5p decreased by ~2-fold, so the conclusion in the main text saying that it is not changed is unproper. The author should modify the conclusion and discussion part.

-The cutoff values for the microarray of circRNA to define significantly changed genes are different between the material part and description in the main text. Please check carefully and correct it.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The author has revised the manuscript as required.

Reviewer 2 Report

1. There are some grammatical, alignment and typographical errors are noted in the manuscript and it should be thoroughly checked and corrected throughout the manuscript. For example, in line number 166, the words “the the corresponding” may be as “the corresponding”; in line number 167, “the the ΔCT” as “the ΔCT”; in line number 170, “cell were” as “cell was”; in line number 445, “ISO induced” as “ISO-induced”.

2. The usage of abbreviations may be avoided in the main title. The ISO should be replaced with full form “isoprenaline hydrochloride”.

3. The technical terms (Latin Phrase) “in vitro” and “in vivo” should be italic and it should be checked all over the manuscript.

Reviewer 3 Report

The concerns I previously expressed have been acknowledged by the authors, who made efforts to address them. However, they did not conduct the additional experiments I recommended to enhance the comprehensiveness of the study. Instead, they proposed these experiments as potential future studies. Nevertheless, they have addressed the other comments I raised. While I'm not completely satisfied, I believe it is acceptable to recommend accepting the article.

Reviewer 4 Report

The revised version of the manuscript answered most of my minor questions but didn't contain any new experiments to answer most of the major questions. The evidence in the revised manuscript couldn't fully support the major conclusion that circCacna1c/miR-29-2-3p axis regulates cardiac hypertrophy, without any critical experiments using primary cardiac cells and rescue assays. Although the authors proposed to do these experiments in the next step, it's not efficient to support its conclusions to be published in the current manuscript.