-->PONE-D-25-27545-->-->IRX3 Depletion Promotes Early Cardiac Commitment of hiPSC-Derived Cardiomyocytes-->-->PLOS ONE

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Reviewers' comments:

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-->5. Review Comments to the Author

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Reviewer #1: This study by da Silva et al. tests whether suppressing IRX3 enhances human iPSC-cardiomyocyte differentiation. Using CRISPR/Cas9 IRX3-null hiPSC lines, the authors show that IRX3 depletion (knockdown and knockout) increased cardiomyocyte yield, higher expression of ventricular markers (TNNI1, MLC2V), elevated CX43, and reduced SCN5A. Functional assessments indicate that IRX3-deficient cells exhibit improved calcium handling, shorter APDs, greater contraction/relaxation velocities, broader mitochondrial networks, and more ordered sarcomeres. Temporal analysis shows IRX3 expression peaks at day 4 of iPSC-CM differentiation. Its loss upregulates early cardiogenic TFs (GATA4, NKX2-5, TBX5, MEF2, HAND), expands progenitors, and activates Wnt-related programs. The authors utilized publicly available ATAC and ChIP data for integrative analysis and suggested IRX3 co-regulates target genes with GATA4/NKX2-5/TBX5. Together, the authors propose that IRX3 acts as a brake on early cardiogenesis.

Overall, this manuscript presented potentially impactful findings. However, there are several concerns regarding citation accuracy, strength of genetic perturbation, and interpretation of integrative in silico analyses. Clarification and further validation are needed to strengthen the conclusions.

Major comments:

1. While the authors targeted IRX3 for its depletion in hiPSC cells, mRNA and protein expression data in all three different clones showed moderately decreased IRX3 protein level (~40%). Given that IRX3 is not known to be haploinsufficient, it remains unclear whether the observed phenotypes are directly attributable to functional IRX3 loss. The authors should clarify and discuss this limitation.

2. The authors cited previous papers investigating Irx3 and Irx5, such as Development (2012) and Scientific Reports (2016) and mentioned as follows:

Line 74: Given the repressive role of Irx3 in the differentiation of working CMs in mouse..

Line 91: Given the established role of Irx3 in mouse CM differentiation and maturation in vivo..

Line 125: IRX3 depletion promotes CM differentiation and may bias subtype specification toward ventricular working CMs, consistent with in vivo mouse data

However, previous studies have primarily focused on the role of this factor in the development and maturation of the ventricular conduction system, rather than its repressive effect on working CM differentiation. Therefore, these citations seem not to be correctly used to build rationale and justify their experiments and findings on Irx3, respectively. It should be revised accordingly.

3. IRX3 is also implicated in the development of multiple lineages, including neurogenesis, adipogenesis, and osteogenesis, etc. This broad expression pattern suggests that IRX3 may influence the early specification of diverse stem cell lineages. Therefore, without temporal depletion experiments that selectively target IRX3 during defined windows of cardiac differentiation, it is an overstatement to conclude that IRX3 functions specifically as a molecular barrier to cardiomyocyte maturation.

4. In Figure 5, the authors utilized publicly available ATAC-seq and ChIP-seq data, and their bioinformatic analysis suggested that IRX3 potentially interacted with NKX2.5, TBX5 and GATA4 and regulated genes involved in cardiac differentiation in cardiac progenitor cells. This is an interesting approach; however, a few issues and limitations were noted below.

- This type of in silico approach does not directly test the causal role of IRX3. Thus, the conclusion of this analysis is association rather than causality.

- The authors mentioned that they used ATAC-seq data from GSE181346, CMs derived from induced pluripotent stem cells (iPSCs) after 5 days of differentiation. This data is scATAC-seq from Day 5 cardiac progenitor cells. However, the method section did not describe how the author analyzed scATAC-seq.

- The authors used ChIP-seq data (GSE159411) which was generated on day 6, hiPSC-derived cardiac progenitors. However, it is unclear whether this day 6 genetic information is comparable to day 5 ATAC-seq data and day 4 RNA-seq data. As transcriptional dynamics change rapidly during early differentiation, without clear justification and through marker gene analysis, temporal mismatches could lead to misleading interpretations.

- The original study that performed ChIP-seq of NKX2.5, GATA4, and TBX5, also conducted ChIP-seq for H3K27ac, H3K4me3, H3K4me1, H3k27me3 and MED1. It is unclear why the author chose these data over ATAC-seq data.

- Figure 5A, the authors described that “IRX3 binding sequences in open chromatin near each of the three TF loci” -> It is not clear whether IRX3 motifs are located and whether they overlap with the binding sites of NKX2.5, TBX5 or GATA4.

- Line 243-246: The authors wrote “Motif scanning identified IRX3 binding sequences in open chromatin near each of the three TF loci”. It is not indicated how the authors defined a gene as being regulated by a TF. Is it the proximity of the motif to the gene for Irx3? Please define ‘near’ (e.g., +/- 500 bp?)

- IRX3 motifs were identified in silico. It would be more informative if predicted IRX3 motif sequences were presented.

- Please deposit the source code used in the bioinformatic analysis.

Minor points

- Figure 3A: Typo for ACNT2 (supposed to be ACTN2)

- Figure 4A: It is not clear what gene/protein expression is scaled to. Please consider adding raw data.

- Figure S2A: In the flow cytometry, the cell population positive for certain undifferentiated markers may not be high enough (aim for 90-95% positive?).

- Figure S2C and S4F: No details in the figure caption or legend to describe what the coloured boxes show in the H&E teratoma images.

- Figure S3A-D: It only showed the data of clone 1.1.

Reviewer #2: In this manuscript, the authors hypothesize that invalidation of IRX3 expression improves the maturation of cardiomyocytes differentiated from induced pluripotent stem cells.

To do this, they used the CRISPR-Cas9 technique to generate hIPS cells deficient in IRX3.

They then studied various parameters defined as representative of the level of cardiomyocyte maturation in cardiomyocytes differentiated from these hIPSCs.

In a second part fully In Silico part of the study, they looked at the interactions of IRX3 with transcription factors NKX2-5, GATA4 and TBX5 and question the putative role of these 4 TFs on expression regulation of early cardiac differentiation of hIPSCs, specifically trying to dissect the involvement of IRX3 and the impact of reduction of tits expression.

While the hypothesis is interesting, this manuscript does not fully demonstrate the initial hypothesis because:

(1) some methodological issues exist regarding IRX-/- cells generation and also some of the experiment to demonstrate enhanced maturation

(2) Interpretation of some of the experimental data is not completely convincing and, to my opinion, presented data sometimes lead to divergent interpretations.

The in silico part in interesting but it seems that most of this is not new and, more importantly, it remains at most hypothesis-generating as no confirmation experiment is performed on the lines used in this study.

I also consider that the graphical abstract is an overstatement, as it seems more a hypothetic model as the present study only partially demonstrate the depicted mechanisms.

Major points:

• Generation of pluripotent stem cells induced deleted for IRX3:

How was the target zone chosen on the IRX3 gene?

Where is this zone located on the IRX3 gene, particularly in relation to the functional domains of the protein?

Where are located the deletions obtained, in relation to the initial target zone?

Is there really a stop codon as initially designed?

The four obtained deletions are different (zone and length) also it seems that the target zome was unique. Can authors explain this result?

Furthermore, the use of a karyotype is not sufficient to identify genomic abnormalities related to the generation of hIPS clones or to the CRISPR technique.

Molecular karyotyping such as with SNP array is necessary to validate the lines

Also, it is not clear from panel S4C if IRX3-/-cl2 line if both IRX3 alleles have been modified. Panel S4B also suggest modification of a single allele.

In panel 1C, staining of TnI seems unusual with most of the protein at the membrane for all 3 clones. Can author provide a higher magnification picture (as in fig 2J for the other lines)?

In figure S1:

- full- length protein is expressed whereas deletion was supposed to create a stop codon.

- change in protein expression between -/- and +/+ lines does not correspond to what is expected with the aim of a bi-allelic deletion

- IRX3 protein is clearly present in the cells at day 6 of the differentiation

Therefore it is at least necessary to know if the produced IRX3 protein functionally active in the -/- cells

• Gene expression of IRX3-/- vs IRX3+/+ cells (in figure S3)

Overall the data does not seem very meaningful as TNNI1 is a fetal isoform, as CX43 is anyway supposed to be express in all cardiomyocytes, as Cx40 is more specifically expressed on conduction system, and as SCN5A is key to mature cardiomyocyte contractile activity.

Instead, comparing the relative expression of fetal vs mature forms of some cardiomyocyte key proteins would be more relevant (such as TNNI3 and TNNI1, MYL2 and MYL7, or MYH6 and MYH7)

Indeed, it would be interesting to have the expression data for lines derived from cl1 and cl2

• Electrical activity

Can authors provides representatives traces of action potential for all clones to help appreciate le level of maturity? For instance, observing an overshoot or a notch would be very important.

Some patch-clamp traces would also be more relevant given the importance of these parameters to assess cell maturation state.

• Figure 4

It seems critical to assess IRX expression in all the IRX3 +/+ and IRX3-/- lines used in this paper as well, especially at day 4

Could authors please provide the microarray expression data for IRX3, IRX5, GATA4, NKX2-5?

• Figure 5

Can the authors precisely indicated which samples were used from GSE181346 and GSE159411 data sets? How were, if any, the replicates handled?

It seems that ATAC-seq data are from single-seq data, how was data from each single cell consolidated into a unique result? What was the observed variability?

Minor points:

• In panel S4C, it seems that legends are misplaced.

• I understand that panel 1D and 1E have been obtained by counting cells from panel 1C images, is it true? (I have not been able to find this information). If yes, % of positive cells in panel 1D/E does not match with what can be observed in 1C especially for MLC2v

• Mitochondrial organization

Can authors provide references indicating that parameters measured in figures 2 K-N are indeed related to a better maturation of hIPS derived cardiomyocytes

Alternatively, functional data showing an effective metabolic switch would be more convincing.

• Figure 3

It seem difficult to obtain accurate data on cell area and sarcomere organization if the analysis was performed when cell overlap as shown in 3A and when there is no staining for cell membrane. Can the authors comment on that?

How was done the quantification in 3F. Has this methodology already been validated?

• Figure 4

For panel E, I am not sure that the quantification method used for GATA4 and NKX2-5 is valid. Could authors provide a reference publication for this methodology?

Also, I suggest % of Ki67+ cells should calculated on GATA4 or NKX2-5+ to indicate that this enhanced proliferation is related to cardiac lineage cells rather than another non relevant cell population

• Panel 5B: It seems that numbers in black correspond to typos

• Panel S6A: It would be interesting to define how many gene containing IRX3 binding sequences in open chromatin does not contain binding sequence for any of the 3 other genes (NKX2-5, GATA4 or TBX5). This would give an indication of the importance of the co-regulation of these 4 TFs.

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Reviewer #1: No

Reviewer #2: Yes:  Pr Guillaume Lamirault

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-->PONE-D-25-27545R1-->-->IRX3 Depletion Promotes Early Cardiac Commitment of hiPSC-Derived Cardiomyocytes-->-->PLOS One

Dear Dr. Krieger,

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.-->

• Note that, while both reviewers acknowledged that the revised version of the manuscript represents certain improvements, those did not go far enough to render the experimental evidence of high quality, a prerequisite for publication in PLOS ONE. It is thus of crucial importance to address the remaining comments in full in order to render your submission acceptable for publication in PLOS ONE.

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Reviewers' comments:

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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-->6. Review Comments to the Author

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Reviewer #1:   The authors have adequately addressed my major concerns. The revised manuscript corrects earlier overstatements in citation and interpretation, particularly regarding IRX3’s role in working cardiomyocyte differentiation. In silico analyses are now appropriately framed as associative rather than causal, with clearer methodological description and acknowledgment of limitations. Overall, the revisions strengthen the clarity of the study.

Minor point:

The genotyping labels in the heatmap of Figure 4D (IRX3-/- 1, IRX3-/- 2, IRX3+/+ 2, IRX3+/+ 1) may be reversed. Genes such as TBX5 and others are described in the text as being upregulated in IRX3-/- cells, yet the heatmap visually suggests reduced expression in these samples. Please confirm whether the sample labels are correct and revise the figure accordingly if an error is present.

Reviewer #2:   I read with interest the revised version of the manuscript submitted by Silva et al.

Some comments have been taken into account and addressed, but there are still major limitations to this study.

It is still unclear whether the invalidation of both alleles of the IRX3 gene is effective or not in the different hIPSC lines produced. Sequencing data are the only data suggesting this. No other experimental confirmation is provided. This is a crucial prerequisite for all experiments and therefore casts major doubt on the validity of this model, the scientific hypothesis associated with this paper, and the associated results.

For example, considering the sequencing data, there is no reason to observe a decrease of the amount of IRX3 RNA for most of the mutated clones. Most of the deletions does not include the TSS and the primers used are outside the deleted sequence.

Regarding anti-IRX3 antibodies, how many antibodies have been tested? Human anti-IRX3 antibodies have already been used successfully (see publications by Benoît Bruneau's team, for instance doi: 10.1242/dev.081703).

To move forward, other technical options appear feasible:

- Validation of sequencing by PCR (on IRX3 DNA) and RT-PCR (on IRX3 RNA) by framing the targeted zone and measuring the size of the amplicons on gel after migration.

- Targeted IRX3 proteomic analyses

For example, can the authors show the agarose gel used for electrophoresis of the PCR products used for the Sanger sequencing? This may be informative.

The markers used to define the level of cardiomyocyte maturation remain mostly outside of what is conventionally accepted in the literature. The authors may refer to the following publication for an overview of commonly recognized markers of maturation. doi: 10.1161/CIRCRESAHA.119.315862

The increase in both MYH6 and MYH7 expression in IRX3-deleted cells is not a sign of maturation by itself. The use of MYH6/7 expression ratio as well as TNNI3/1 and MYL2/7, as previously indicated, are validated markers.

Electrophysiological data remains focused on calcium. This is insufficient to truly understand the electrophysiological maturity of cells. In addition, no representative singles traces (not averaged data) of the obtained signal is provided.

Based on analysis using Fluovolt, control cells appear to have a more pronounced plateau phase than mutated cells, which is a sign of greater maturity in cells that are not IRX3-invalidated. The time-to-peak ratio and amplitude can be quantified using this data.

Measuring the diastolic membrane potential using patch clamp or proof of increase INa current could also be very convincing markers.

Regarding the analysis of the genome integrity of the produced lines, a global analysis of the genome is not sufficient. It is necessary to precisely identify the areas in which chromosomal alterations have occurred (deletion/duplication, size, location, and genes involved) in order to determine whether they are likely to modify the results on maturation and proliferation.

The bioinformatics section of the manuscript remains less innovative, but the methodology and presentation of the results have been significantly improved, making the data more explicit.

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Reviewer #1: No

Reviewer #2: No

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-->PONE-D-25-27545R2-->-->IRX3 Depletion Promotes Early Cardiac Commitment of hiPSC-Derived Cardiomyocytes-->-->PLOS One

Dear Dr. Krieger,

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 remaining point raised during the review process. The manuscript should thus be re-worked carefully to remove grammatical errors and typos.

Please submit your revised manuscript by Jun 27 2026 11:59PM. 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.

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Michael Schubert

Academic Editor

PLOS One

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Reviewer #1: All comments have been addressed

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: The authors have adequately addressed my major comment.

There are a number of grammatical and typo issues throughout the text. Here are a few examples.

- Line 243: ‘Data are presented’ repeated

- Line 823: scTAC-seq -> scATAC-seq

- Line 874: Student’s t-test test

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Reviewer #1: No

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To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

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IRX3 Depletion Promotes Early Cardiac Commitment of hiPSC-Derived Cardiomyocytes

PONE-D-25-27545R3

Dear Dr. Krieger,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Michael Schubert

Academic Editor

PLOS One