The PIDDosome controls cardiomyocyte polyploidization during postnatal heart development

  1. M Leone
  2. N Kinz
  3. F Eichin
  4. D Obwegs
  5. VC Sladky
  6. VZ Braun
  7. D Rizzotto
  8. L Englmaier
  9. C Manzl
  10. K Moos
  11. Julia Mergner
  12. P Giansanti
  13. MN Garcia
  14. MM Marques
  15. ED Jacotot
  16. C Savko
  17. M Boerries
  18. MA Sussman
  19. A Villunger

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Abstract

The adult mammalian heart is characterized by post-mitotic polyploid cardiomyocytes (CMs). Understanding how CMs regulate cell cycle exit and ploidy can help developing new heart regenerative therapies. Here, we uncover that the PIDDosome, a multi-protein complex activating the endopeptidase Caspase-2, helps to implement a CM-specific differentiation program that limits ploidy during postnatal heart development. DNA content analyses show that PIDDosome-loss causes a cell-autonomous increase in nuclear and cellular CM ploidy. Remarkably, increased ploidy does not affect cardiac structure nor function. PIDDosome-imposed ploidy restriction commences at postnatal day 7 (P7), reaching a plateau on P14. PIDDosome activation requires ANKRD26, targeting PIDD1 to mother centrioles. Opposite to prior observations in liver development, the PIDDosome limits CM polyploidization in a p53-independent manner but reliant on p21/Cdkn1a , a notion supported by nuclear RNA sequencing and genetic deletion experiments. Our results provide new insights how proliferation of polyploid CMs is restricted during postnatal heart development.

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    Reply to the reviewers

    Manuscript number: RC-2025-02953

    Corresponding author(s): Andreas, Villunger

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    *We would like to thank the reviewers for their constructive input and overall support. We appreciate to provide a provisional revision plan, as outlined here, and are happy to engage in additional communication with journal editors via video call, in case further clarifications are needed. *

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    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    Reviewer #1

    __Evidence, reproducibility and clarity __

    Summary: This manuscript by Leone et al describes the role of the PIDDosome in cardiomyocytes. Using a series of whole body and cardiomyocyte specific knockouts, the authors show that the PIDDosome maintains correct ploidy in these cells. It achieves this through inducing cell cycle arrest in cardiomyocytes in a p53 dependent manner. Despite this effect on ploidy, PIDDosome-deficient hearts show no structural or functional defects. Statistics and rigor appear to be adequate.

    We thank this referee for taking the time to evaluate our work and their valuable comments. We assume that this reviewer by mistake indicates that the phenomenon we describe, depends on p53. As outlined in the abstract and throughout the manuscript, the effect is independent of p53, but may additionally still involve p21, acting along or parallel to the PIDDosome.

    Major comments:

    1. Figure 1 uses fluorescent intensity of a nuclear stain to determine ploidy per nucleus and they further separate the results into mononucleated, binucleated or multinucleated cells. It is hard to know how to interpret these results without further information or controls. Is there a good positive control that can be used to help to show whether this assay is quantitative? The differences are larger with the Raidd and caspase-2 knockouts than with the Pidd knockouts but this is not addressed.

    *We appreciate this concern. Regarding a “good positive control” we can say that we follow state-of the art in the cardiomyocyte field and studies by the Evans (PMID: 36622904), Kuhn (PMID: 32109383), Bergmann (PMID: 26544945) and Patterson labs (PMID: 28783163, 36912240) all use the identical approach to discriminate 2n from 4n nuclei in microscopy images at the cellular level. The fact that the majority of rodent CM nuclei is indeed diploid (PMID: 31175264, 31585517 and 32078450) and a large number of nuclei has been evaluated to assess their mean fluorescence intensity (MFI) reduces the risk of a systematic bias in our analysis. Moreover, we have used an orthogonal approach that is indeed quantitative to define DNA content, i.e,. flow-cytometry based evaluation of DNA content in isolated CM nuclei (Fig. 1C). We hence are confident our assays are quantitative. *

    *Regarding the fact that loss of Pidd1 causes a more saddle phenotype, we can offer to discuss this in light of the fact that Pidd1 has additional functions, outside the PIDDosome (PMID: **35343572), and that we made similar observations when analyzing ploidy in hepatocytes (PMID: **31983631). Given the fact that all components of the PIDDosome show a similar phenotype, and that this phenotype is mimicked by loss of the protein that connects PIDD1 and centrosomes, ANKRD26 (Fig. 4a), we are confident that this biological variation in our analysis is not affecting our conclusions. *

    On line 459 the authors state that the increase in polyploidy in PIDDosome knockouts occurs in adult hood but this is not directly tested. In fact, in the next section the polyploidy is assessed in early postnatal development. This statement should be explained or removed.

    We see that we have made an unclear statement here. In fact, we first noted increases in ploidy in adult heart and then define the time window in development when this happens. This sentence will be rephrased.

    In Figure 4. The authors obtained RNAseq data for P1, P7 and P14 but only show the differences with and without caspase-2 at P7. Given that the differences in ploidy are more significant at P14 (Fig 3D), all the comparisons should be shown along with analysis of whether the same genes/gene families are altered in the absence of caspase-2.

    *The reason why we focus on postnatal day 7 (P7) is that data from Alkass et al (PMID: **26544945) and other labs (PMID: 31175264 ) document that on this day the initial wave of binucleation peaks. Hence, we hypothesized that the PIDDosome must be active in most CM, which aligns well with the increased mRNA levels of all of its components (Figure 3). Interestingly, it seems that its action is tightly regulated, as mRNA of PIDDosome components drop on P10, suggesting PIDDosome shut-down or downregulation. Similar findings have been noted in the liver (PMID: **31983631). Alkass and colleagues also show that very few CMs enter another round of DNA synthesis between P7 and P14, and hence possible transcriptome changes in the absence of the PIDDosome will be strongly diluted. *

    *Please note that on P1, there is no difference between genotypes to be expected as all CM are mononucleated diploids and cytokinesis competent, as previously demonstrated (PMID: **26544945). Moreover, PIDDosome expression levels are extremely low (Fig. 3A). As such, no difference between genotypes are expected on P1. In addition, on P14 the ploidy phenotype observed in PIDDosome knockout mice reaches the maximum and ploidy increases are comparable to adult tissue. Thus, at this time the trigger for PIDDosome activation (cytokinesis failure) is no longer observed as the majority of CMs are post-mitotic, (PMID: 26247711). As such the impact of PIDDosome activation on the P14 transcriptome is most likely negligible. However, if desired, we can expand our bioinformatics analysis summarizing findings made related to DEGs over time in wt animals by comparing genotypes also on day 1 and day 14. In light of the above, analysis between genotypes on P7 holds still appears as the one most meaningful. *

    Some validation of the RNAseq and/or proteomics results would be an important addition to this study

    We agree with this notion and propose to validate key candidates related to cardiomyocyte proliferation and polyploidization, some of which we found to be differentially expressed at the mRNA level on day 7in the RNAseq data (e.g., p21, Foxm1, Kif18a, Lin37 and others)

    *Regarding the proteomics results, we face the challenge that we can only try to confirm if candidate proteins are likely caspase substrates in silico using DeepCleave, and potentially pick one or two candidates linked to CM differentiation for further analysis in vitro and in heterologous cell based assays (e.g. 293T cells), as no bona-fide ventricular cardiomyocyte cell lines exist. Primary postnatal CMs are extremely difficult to transfect, nor they proliferate without drug-treatment, or fail cytokinesis ex vivo. *

    Figure 4D: the authors make the conclusion that p21 is downstream of PIDD (et p53 independent). However, this is not supported by the data because the increase in 4N cells/decrease in 2N cells, although statistically significant, is nowhere near that of caspase-2 KO and caspase-2/p21 KO. Statistics should also compare p32KO with c2KO. In the absence of any other data, the more likely conclusion is that p21 is not involved.

    *We agree that the findings related to the impact seen upon loss of p21 suggest that it is not the only effector involved in ploidy control and it may not even be an effector engaged by caspase-2, as C2/p21 DKO mice have an even higher ploidy increase, albeit not statistically significant. However, it is important to highlight that p21 (Cdkn1a) was found to be downregulated in our transcriptomic analysis suggesting an involvement in the caspace2-cascade. We are happy to highlight this when presenting the results and in the discussion. *

    *We assume that this referee refers to p73 KO data that should be compared to Casp2 KO data (could be read as p73 or p53, but the latter we compare side by side with Casp2 in Fig. 4 already). As p73 KO mice were not found to be viable beyond day 7 (our attempt to find animals on day 10 failed, in line with published literature (PMID: 24500610, 10716451)), we can only offer to compare this data set to the data presented in Figure 3C, where we have analyzed ploidy increases on day 7 from wt and PIDDosome mutant mice. This re-analysis will show that only Caspase-2 mutant mice display a significant ploidy increase on P7, when compared to wt or p73 mutant animals, while no difference are noted between wt and p73 mutant mice (to be included in new Suppl. Fig. 3C) *

    Minor comments: Suggest moving Figure 4A to Figure 3 as it seems to fit better there based on the citation of this figure in the text

    *We can see some benefit in this recommendation and included panel 4A now in an updated version of Figure 3. *

    Recommend enhancing the brightness of microscopy images in Figure 1E and 2D

    We will try to improve image quality, may have been due to PDF conversion

    Significance

    This study provides interesting information for the role of the PIDDosome in protecting from polyploidy and adds to the body of work by this same group studying this pathway in the liver.

    The main weakness in terms of significance is the lack of a phenotype in the hearts of these animals. Therefore, it is clear that ploidy (or at least PIDDosome dependent ploidy) has minimal impact on cardiac development.

    We respectfully disagree with the comment that the lack of impact on cardiac function constitutes a weakness of our findings. Several studies on ploidy control in the liver (PMID 34228992) but importantly also heart (PMID: 36622904) have failed to document a clear impact of increased ploidy on organ function. This does not infer insignificance, but maybe rather that the context where this becomes relevant has not been identified. We are happy expand on this in our discussion

    The authors mention that they have not tried giving these mice an myocardial infarct (MI) or inducing any other type of cardiac damage. Although it is understood that these experiments are likely outside of the scope of the present study, without this information the impact of this study is moderate. I recommend expanding the discussion to provide a more in-depth possible rationale as to why ploidy perturbations do not lead to structural changes like in the liver.

    Despite this, the insights to the pathway itself are interesting to investigators in the caspase-2 field if a little underdeveloped, especially concerning the role of p21.

    My expertise is in cell death and caspase biology (especially caspase-2). I have sufficient expertise to evaluate all parts of this paper.

    *As mentioned above, we will amend our conclusions on p21, in light of potential findings made when validating DEG candidates, as stated above. *

    *We hope that the changes and amendments proposed here will be satisfactory to this referee to recommend publication of a revised manuscript. *

    Reviewer #2

    __Evidence, reproducibility and clarity: __

    __Summary: __

    In this study, the authors investigated the role of the PIDDosome during cardiomyocyte polyploidization. PIDDosome is a multi-protein complex activating the endopeptidase Caspase-2, and shown to be involved in eliminating cells with extra centrosomes or in response to genotoxic stress (Burigotto & Fava, 2021, Sladky and Villunger, 2020). In both cases, the PIDDosome is recruited in a ANKRD26-dependent manner at the centrosomes leading to p53 stabilization and cell death (Burigotto & Fava, 2021; Evans et al., 2020; Burigotto et al., 2021).

    Here, by studying mouse cardiomyocyte differentiation, the authors showed that PIDDosome is imposing ploidy restriction during cardiomyocyte differentiation. Importantly, in contrast to a previous report in the liver (Sladky et al., 2020), they showed that PIDDosome acts in a p53-independent manner in cardiomyocytes. Indeed, they suggested that PIDDosome controls ploidy in cardiomyocytes through p21 activation.

    We want to thank this reviewer for the time taken to evaluate our work and provide critical feedback that will help to improve our revised manuscript.

    __Major comments: __

    In general the conclusions of the authors are well supported by the experiments. However, I would suggest the following experiments/analysis to strengthen the paper:

    The authors should improve the Figure 1 to help the readers who are not familiar with cardiomyocyte polyploidization. For instance, I would suggest to add a scheme to summarize cardiomyocyte polyploidization (in terms of nuclear size, mono vs multi and so on).

    *We agree that a visual summary of the postnatal timing of CM polyploidization will be helpful for the generalist not familiar with the topic and have added a scheme, adapted from a study by Alkass et al. (PMID: **26544945), who elegantly defined the timing of this process during postnatal mice life (now Fig. 1A). *

    Based on the images they presented in 1B, the authors should also measure the nuclear area or volume in the different conditions in which components of the PIDDosome were depleted. Indeed, these two parameters should be easier to conceptualize for the readers (instead of the fluorescence nuclear intensity). This could help to understand if the nuclear size is maintained between the different conditions and if this is comparable between mono, bi or multinucleated cardiomyocytes.

    We have acquired this data and it can be used to provide additional information on nuclear area and/or volume. We propose to focus on re-analyzing data from wt, Casp2 and XMLC2CRE/Casp2f/f mice. The additional information can be included in Figures 1 & 2, respectively.

    • In Figure 2A, the authors presented cross section of heart from animals showing that PIDDosome depletion has no effect on heart size. This is a surprising result since cardiomyocytes have higher ploidy levels and this could have an effect on their function. Since the importance of this observation, the authors should present a quantification of the heart size in the different conditions shown in Figure 2A.

    We agree with this comment. We can measure the heart vs. body weight ratio or tibia length in adult Casp2-/- vs. WT (3 month old) in order to indirectly evaluate possible increases in CM size linked to increased ploidy.

    Also, the percentage of cardiomyocytes presenting higher levels of ploidy seems quite low. The authors should discuss this point. In particular because this could explain the absence of consequences on heart size and function at steady state.

    *We agree with this conclusion and will expand on this in our discussion. It is important to note that as opposed to findings made in liver (PMID: **31983631), genetic manipulation of ploidy regulators such as E2f7/8 (PMID: 36622904), only led to modest changes in CM ploidy, suggesting that either a small band-width compatible with normal heart function exists, or that additional mechanisms exist that take control when these thresholds set by the PIDDosome or E2f7/8 are exceeded. These mechanisms could involve Cyclin G (PMID: 20360255), or TNNI3K (PMID: 31589606). Importantly, a recent publication has shown that overexpression of Plk1(T210D) and Ect2 from birth causes increased heart weight coupled with a minor decrease in CM size. These mice undergo to premature death (PMID: 39912233) suggesting that CM polyploidization is a tight regulated process regulated by several independent mechanisms during heart development. *

    In Figure 2D, the authors measured the cardiomyocyte cross-sectional area and concluded that removing PIDDosome components have no effect on cardiomyocyte cell size. Since it has been shown that ploidy increase is normally associated with an increase in cell area, the authors should measure cell area of cardiomyocytes analyzed in Figure 1B. It could be then interesting to establish a correlation with nuclear area and the mono, bi or multinucleated status. This will strengthen the results showing that ploidy increases without affecting cell area.

    *Indeed, studies in PIDDosome deficient livers suggest that tissue is containing fewer but bigger cells (PMID: **31983631). As opposed to the liver the percentage of cardiomyocytes presenting higher levels of ploidy is relatively low. Thus, a possible increase in CM size in PIDDosome deficient mice may be masked in heart cross-sections. In order to better correlate the ploidy with cell size, we propose to reanalyze our microscopy images used to extract the data displayed in Fig. 1D. We may run into the problem though that the number of cells acquired may become limiting to achieve sufficient statistical power. In this case we could pool data from different PIDDosome mutant CM to increase statistical power. Again, we propose to initially prioritize wt vs. Casp2 vs. XMLC2/Casp2f/f mice. In addition, we can offer to quantify heart to body weight ratio or tibia length as an additional read-out (see answer to a previous reviewer comment). *

    The authors should discuss the fact that PIDDosome depletion lead only to a mild increase in ploidy levels (4N) in a small percentage of cardiomyocyte. If the PIDDosome is controlling ploidy, one could expect that removing it should lead to a drastic increase in the ploidy levels. Is PIDDosome depletion leading to cell death in some cardiomyocyte? The authors should discuss this point in the discussion or if relevant show a staining with an apoptosis marker. Is another mechanism compensating to prevent higher ploidy levels in cardiomyocytes?

    *These are valid thoughts, some of which we contemplated before. In part, we have addressed them in our response to Reviewer#1, above, discussing similar findings made in E2f7/8 deficient hearts (PMID: 36622904), or Cyclin G overexpressing hearts (PMID: 20360255), where also only modest changes in ploidy were achieved. Together these observations are suggesting alternative control mechanism able to act, or limited tolerance towards larger shifts in ploidy, incompatible with proper cell function and survival. Towards this end, we can offer to test if we find increased signs of cell death in PIDDosome mutant hearts by TUNEL staining of histological sections. Of note, we did not find evidence for such a phenomenon in the liver (PMID: *31983631).

    Even if the authors presented RNAseq data suggesting that the PIDDosome is activated during cardiomyocyte differentiation, they should clearly demonstrate this point to strengthen the message of the paper. Indeed, the conclusions are based on the absence of PIDDosome components triggering higher ploidy in cardiomyocytes. However, we don't know whether (and when) the PIDDosome is activated during cardiomyocyte differentiation to control their ploidy levels. I would suggest to analyze PIDDosome activation markers by immunofluorescence in *cardiomyocytes at different developmental stages. *

    *We agree with this referee that direct proof of PIDDosome activation would be helpful and that we only infer back from loss of function phenotypes when and where the PIDDosome becomes activated. However, several technical issues prevent us from collecting more direct evidence of PIDDosome activation in the developing heart. 1) Polyploidization in heart CM appears to happen gradually in CM from day 3 on with a peak at day 7 (PMID: 26544945). Hence, this is not a synchronous process, where we could pinpoint simultaneous activation of the PIDDosome in all cells at the same time, which would facilitate biochemical analysis, e.g., by western blotting for signs of Caspase-2 activation (i.e. the loss of its pro-form, PMID: 28130345). 2) Our most reliable readout, MDM2 cleavage by caspase-2 giving rise to specific fragments detectable in western, is not applicable to mouse tissue, as the antibody we use only detects human MDM2 (PMID: 28130345) and no other MDM2 Ab we tested gave satisfactory results. Independent of that, 3) we do not see involvement of p53 in CM ploidy control (arguing against a role of MDM2). *

    *As such, we can only offer to look at extra centrosome clustering in postnatal binucleated CM (as also suggested further below), as a putative trigger for PIDDosome activation. However, this has been published by the first author of this study before (PMID 31301302). Given that we have made the significant effort to time resolve the increase in ploidy in postnatal mice (please note that several hearts needed to be pooled for each time point, analyzed in multiple biological replicates), we think that our conclusions are well-justified based on the genetic data provided. *

    Concerning the methods, the authors must add the references for each product they used and not only the origin. When relevant, the RRID should be indicated. Without this information the method and the data cannot be reproduced.

    We will update this information where relevant to reproduce our results

    Minor comments:

    In general, the text and the figures are clear. Nevertheless, I would suggest the following changes:

    • Figures 1B, 2B and 2C: the y-axis must start at 0.

    We will adopt axes accordingly

    Figure 4A: The authors should stain centrosomes in cardiomyocytes. This should strengthen the conclusion taken by the authors based on the results obtained in mice depleted for ANKRD26. Indeed, for the moment they are insufficient to conclude about the role of the centrosomes. The authors should show that centrosomes cluster in cardiomyocytes (a condition necessary for PIDDosome activation in polyploid cells) and if possible that component of the PIDDosome are recruited here.

    *This point is well taken and addressed in part above. Clustering of extra centrosomes has been documented and published by the first author of this study in rat polyploid cardiomyocytes (PIMID; cited). We can offer to show clustering of centrosomes in mouse CM isolated from day 7 hearts, but while PIDD1 can be detected well in MEF, we repeatedly failed to stain fro PIDD1 in primary CMs. *

    Figure 4F: I would suggest to modify the working model to emphasize more the differences between WT and PIDDosome KO.

    We will aim to improve this cartoon/graphical abstract

    The prior studies are referenced appropriately.

    Reviewer #2 (Significance (Required)):

    How polyploid cells control their ploidy levels during differentiation remains poorly understood. The data presented here represent thus an advance concerning this question. The actual model concerning PIDDosome activation relies on the presence of extra centrosomes that drives the ANKDR26-dependent recruitment of the PIDDosome. Then, Caspase 2 is activated leading to a p53-p21 dependent cell cycle arrest (Burigotto & Fava, 2021, Sladky and Villunger, 2020; Janssens & Tinel, 2012; Evans et al., 2020; Burigotto et al., 2021). In this study, the authors showed that similar pathway takes place during cardiomyocyte differentiation to control ploidy levels. These data are reminiscent of previous work showing PIDDosome involvement during hepatocyte polyploidization (Sladky et al. 2020). Together, these data highlight the prominent role of the PIDDosome complex in controlling ploidy levels in physiological context. Importantly, this study identified that the classical p53-dependent cell cycle arrest described after PIDDosome activation is not involved here. Instead, the data established that independently of p53, p21 contribute to control cardiomyocyte ploidy. In consequence, this study extends the initial pathway associated with PIDDosome activation and suggest that other mechanisms could take place to restrain cell proliferation upon PIDDosome activation. Overall, this makes this paper significant and of interest for the following fields: polyploidy, heart/cardiomyocyte development and PIDDosome.

    My field of expertise includes polyploidy, cell cycle and genetic instability.

    We thank this reviewer for the time taken and the positive feedback provided.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    N/A

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    • *As outlined above, limited tools are available to validate putative caspase-2 substrates, identified in proteomics analysis, in an impactful manner. *
    • *Also, as discussed above, we deem myocardial infarction experiments in mice as unsuitable to improve our work, as with all likely-hood, they will yield negative results. *
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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this study, the authors investigated the role of the PIDDosome during cardiomyocyte polyploidization. PIDDosome is a multi-protein complex activating the endopeptidase Caspase-2, and shown to be involved in eliminating cells with extra centrosomes or in response to genotoxic stress (Burigotto & Fava, 2021, Sladky and Villunger, 2020). In both cases, the PIDDosome is recruited in a ANKRD26-dependent manner at the centrosomes leading to p53 stabilization and cell death (Burigotto & Fava, 2021; Evans et al., 2020; Burigotto et al., 2021).

    Here, by studying mouse cardiomyocyte differentiation, the authors showed that PIDDosome is imposing ploidy restriction during cardiomyocyte differentiation. Importantly, in contrast to a previous report in the liver (Sladky et al., 2020), they showed that PIDDosome acts in a p53-independent manner in cardiomyocytes. Indeed, they suggested that PIDDosome controls ploidy in cardiomyocytes through p21 activation.

    Major comments:

    In general the conclusions of the authors are well supported by the experiments. However, I would suggest the following experiments/analysis to strengthen the paper:

    • The authors should improve the Figure 1 to help the readers who are not familiar with cardiomyocyte polyploidization. For instance, I would suggest to add a scheme to summarize cardiomyocyte polyploidization (in terms of nuclear size, mono vs multi and so on). Based on the images they presented in 1B, the authors should also measure the nuclear area or volume in the different conditions in which components of the PIDDosome were depleted. Indeed, these two parameters should be easier to conceptualize for the readers (instead of the fluorescence nuclear intensity). This could help to understand if the nuclear size is maintained between the different conditions and if this is comparable between mono, bi or multinucleated cardiomyocytes.
    • In Figure 2A, the authors presented cross section of heart from animals showing that PIDDosome depletion has no effect on heart size. This is a surprising results since cardiomyocytes have higher ploidy levels and this could have an effect on their function. Since the importance of this observation, the authors should present a quantification of the heart size in the different conditions shown in Figure 2A. Also, the percentage of cardiomyocytes presenting higher levels of ploidy seems quiet low. The authors should discuss this point. In particular because this could explain the absence of consequences on heart size and function at steady state.
    • In Figure 2D, the authors measured the cardiomyocyte cross-sectional area and concluded that removing PIDDosome components have no effect on cardiomyocyte cell size. Since it has been shown that ploidy increase is normally associated with an increase in cell area, the authors should measure cell area of cardiomyocytes analyzed in Figure 1B. It could be then interesting to establish a correlation with nuclear area and the mono, bi or multinucleated status. This will strengthen the results showing that ploidy increases without affecting cell area.
    • The authors should discuss the fact that PIDDosome depletion lead only to a mild increase in ploidy levels (4N) in a small percentage of cardiomyocyte. If the PIDDosome is controlling ploidy, one could expect that removing it should lead to a drastic increase in the ploidy levels. Is PIDDosome depletion leading to cell death in some cardiomyocyte? The authors should discuss this point in the discussion or if relevant show a staining with an apoptosis marker. Is another mechanism compensating to prevent higher ploidy levels in cardiomyocytes?
    • Even if the authors presented RNAseq data suggesting that the PIDDosome is activated during cardiomyocyte differentiation, they should clearly demonstrate this point to strengthen the message of the paper. Indeed, the conclusions are based on the absence of PIDDosome components triggering higher ploidy in cardiomyocytes. However, we don't know whether (and when) the PIDDosome is activated during cardiomyocyte differentiation to control their ploidy levels. I would suggest to analyze PIDDosome activation markers by immunofluorescence in cardiomyocytes at different developmental stages.

    Concerning the methods, the authors must add the references for each product they used and not only the origin. When relevant, the RRID should be indicated. Without this information the method and the data cannot be reproduced.

    The statistics are well indicated in the figures and in the figure legends.

    Minor comments:

    In general, the text and the figures are clear. Nevertheless, I would suggest the following changes:

    • Figures 1B, 2B and 2C: the y-axis must start at 0.
    • Figure 4A: The authors should stain centrosomes in cardiomyocytes. This should strengthen the conclusion taken by the authors based on the results obtained in mice depleted for ANKRD26. Indeed, for the moment they are insufficient to conclude about the role of the centrosomes. The authors should show that centrosomes cluster in cardiomyocytes (a condition necessary for PIDDosome activation in polyploid cells) and if possible that component of the PIDDosome are recruited here.
    • Figure 4F: I would suggest to modify the working model to emphasize more the differences between WT and PIDDosome KO.

    The prior studies are referenced appropriately.

    Significance

    How polyploid cells control their ploidy levels during differentiation remains poorly understood. The data presented here represent thus an advance concerning this question.

    The actual model concerning PIDDosome activation relies on the presence of extra centrosomes that drives the ANKDR26-dependent recruitment of the PIDDosome. Then, Caspase 2 is activated leading to a p53-p21 dependent cell cycle arrest (Burigotto & Fava, 2021, Sladky and Villunger, 2020; Janssens & Tinel, 2012; Evans et al., 2020; Burigotto et al., 2021). In this study, the authors showed that similar pathway takes place during cardiomyocyte differentiation to control ploidy levels. These data are reminiscent of previous work showing PIDDosome involvement during hepatocyte polyploidization (Sladky et al. 2020). Together, these data highlight the prominent role of the PIDDosome complex in controlling ploidy levels in physiological context.

    Importantly, this study identified that the classical p53-dependent cell cycle arrest described after PIDDosome activation is not involved here. Instead, the data established that independently of p53, p21 contribute to control cardiomyocyte ploidy. In consequence, this study extend the initial pathway associated with PIDDosome activation and suggest that other mechanisms could take place to restrain cell proliferation upon PIDDosome activation.

    Overall, this makes this paper significant and of interest for the following fields: polyploidy, heart/cardiomyocyte development and PIDDosome.

    My field of expertise includes polyploidy, cell cycle and genetic instability.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    This manuscript by Leone et al describes the role of the PIDDosome in cardiomyocytes. Using a series of whole body and cardiomyocyte specific knockouts, the authors show that the PIDDosome maintains correct ploidy in these cells. It achieves this through inducing cell cycle arrest in cardiomyocytes in a p53 dependent manner. Despite this effect on ploidy, PIDDosome-deficient hearts show no structural or functional defects. Statistics and rigor appears to be adequate.

    Major comments:

    1. Figure 1 uses fluorescent intensity of a nuclear stain to determine ploidy per nucleus and they further separate the results into mononucleated, binucleated or multinucleated cells. It is hard to know how to interpret these results without further information or controls. Is there a good positive control that can be used to help to show whether this assay is quantitative. The differences are larger with the Raidd and caspase-2 knockouts than with the Pidd knockouts but this is not addressed.
    2. On line 459 the authors state that the increase in polyploidy in PIDDosome knockouts occurs in adult hood but this is not directly tested. In fact in the next section the polyploidy is assessed in early postnatal development. This statement should be explained or removed
    3. In Figure 4. The authors obtained RNAseq data for P1, P7 and P14 but only show the differences with and without caspase-2 at P7. Given that the differences in ploidy are more significant at P14 (Fig 3D), all the comparisons should be shown along with analysis of whether the same genes/gene families are altered in the absence of caspase-2
    4. Some validation of the RNAseq and/or proteomics results would be an important addition to this study
    5. Figure 4D: the authors make the conclusion that p21 is downstream of PIDD (et p53 independent). However, this is not supported by the data because the increase in 4N cells/decrease in 2N cells, although statistically significant, is nowhere near that of caspase-2 KO and caspase-2/p21 KO. Statistics should also compare p32KO with c2KO. In the absence of any other data, the more likely conclusion is that p21 is not involved.

    Minor comments:

    Suggest moving Figure 4A to Figure 3 as it seems to fit better there based on the citation of this figure in the text Recommend enhancing the brightness of microscopy images in Figure 1E and 2D

    Significance

    This study provides interesting information for the role of the PIDDosome in protecting from polyploidy and adds to the body of work by this same group studying this pathway in the liver. The main weakness in terms of significance is the lack of a phenotype in the hearts of these animals. Therefore, it is clear that ploidy (or at least PIDDosome dependent ploidy) has minimal impact on cardiac development. The authors mention that they have not tried giving these mice an MI or inducing any other type of cardiac damage. Although it is understood that these experiments are likely outside of the scope of the present study, without this information the impact of this study is moderate. I recommend expanding the discussion to provide a more indepth possible rationale as to why ploidy perturbations do not lead to structural changes like in the liver. Despite this, the insights to the pathway itself are interesting to investigators in the caspase-2 field if a little underdeveloped, especially concerning the role of p21.

    My expertise is in cell death and caspase biology (especially caspase-2). I havesufficient expertise to evaluate all parts of this paper.

  4. Version published to 10.1101/2024.08.27.609375v2 on bioRxiv
  5. Version published to 10.1101/2024.08.27.609375v1 on bioRxiv