The livebearers platyfish and swordtails partially regenerate their hearts with persistent scarring

Reviewer #1 (Public review):

Summary:

How the regenerative capacity of the heart varies among different species has been a long-standing question. Within teleosts, zebrafish can regenerate their hearts, while medaka and cavefish cannot. The authors examined heart regeneration in two livebearers, platyfish and swordtails. Interestingly, they found that these two fish species lack the compact myocardium layer that contains coronary vessels. Furthermore, these fish form a "pseudoaneurysm" after cryoinjury without initial deposition of fibrotic tissues. However, delayed leukocyte infiltration and prolonged inflammation lead to permanent scar tissue in the injured heart. Although their cardiomyocytes can also proliferate, platyfish and swordtails can only regenerate partially. The authors argue that the restorative mechanism of platyfish and swordtails likely reflects "evolutionary innovations in the ventricle type and the immune system".

Strengths:

The authors took advantage of the annotated genome of platyfish to perform transcriptomic analyses. The histological analyses and immunostaining are beautifully done.

Comments on revised version:

The authors have addressed all my previous concerns. I don't have further questions.

Reviewer #2 (Public review):

This manuscript by Hisler, Rees, and colleagues examines the cardiac regenerative ability of two livebearer species, the platyfish and swordtail. Unlike zebrafish, these species lack cortical myocardium and coronary vasculature. Cryoinjury to their hearts caused persistent scarring at 60 and 90 days post-injury and prevented most of the myocardium from regenerating. Although the wound size progressively shrinks and fibronectin content decreases, the myocardial wall does not recover. Transcriptomic profiling at 7 dpi revealed significant differences between zebrafish and platyfish, including alterations in ECM deposition, immune regulation, and signaling pathways involved in regeneration, such as TGFβ, mTOR, and Erbb2. Platyfish exhibit a delayed but chronic immune response, and although some cardiomyocyte proliferation is observed, it does not appear to contribute to myocardial recovery significantly.

Overall, this is an excellent manuscript that tackles a crucial question: do different fish lineages have the ability to regenerate hearts, or is this capability limited to a few groups? Therefore, this work is relevant to the fields of cardiac regeneration and comparative regenerative biology for a broad audience. I am very enthusiastic about expanding the list of species tested for their heart regeneration abilities, and this study is detailed and rigorous, providing a solid foundation for future comparative research. However, there are several aspects where additional work could significantly strengthen the manuscript.

Comments on revised version:

The authors have done a fantastic job addressing all my comments, covering both the experimental and the more conceptual aspects of my critique. I am fully satisfied with their response.

Author response:

The following is the authors’ response to the original reviews.

We sincerely thank the editors and reviewers for their thorough evaluation of our manuscript. We are grateful for their enthusiastic support of our comparative approach to understanding cardiac regeneration across fish lineages, and we appreciate the care with which they captured and summarized the key findings of our study. Their constructive suggestions have been invaluable in strengthening the work, and we are pleased to submit the revised manuscript enclosed herewith.

The revised manuscript contains 7 main figures and 8 supplementary figures, representing the addition of two figures relative to the previous version. Specifically, we have included additional Podocalyxin immunostaining to clarify our characterization of ventricular vascularization, and Picrosirius Red staining to improve the characterization of fibrillar collagen in uninjured Xiphophorus hearts relative to zebrafish. We have also refined our RNA-seq analysis by manually curating well-defined markers of cardiomyocyte cell fate change and immune system activation, such that the manuscript no longer relies on a single-cell RNA-seq dataset. All remaining revisions have been made in direct response to the reviewers' comments, as detailed below.

Reviewer #1 (Public review):

Minor Weaknesses:

Transcriptomic analysis was only done for one time point. Different time points could be included to validate whether some processes occur at different time points. But this can be done in the future for more detailed studies.

We agree with this valuable suggestion. While RNA-seq at multiple time points would undoubtedly enrich our understanding of the temporal dynamics of Xiphophorus heart regeneration, such an expansion would constitute a substantial independent study extending well beyond the scope of this initial characterization. We agree that this represents a compelling avenue for future investigation, and we have acknowledged this limitation explicitly in the Discussion section of the revised manuscript (lines 357-363).

Reviewer #1 (Recommendations for the authors):

(1) Figure 5B and quantification in Figure 5D seem inconsistent. The numbers of Mpx neutrophils at Day 7 seem the same as Day 14 and 30, but overall, there are very few Mpx+ cells. The authors should examine earlier time points, such as Day 1 and 3, to determine if neutrophil and macrophage infiltration is different in zebrafish.

We thank the reviewer for this careful observation. We agree that the apparent similarity in Mpx⁺ cell numbers across Day 7, 14, and 30 in the original figures warranted closer examination of earlier time points. We have therefore performed new Mpx and L-plastin immunostaining at 1 and 3 dpci in Xiphophorus hearts. These analyses confirm low immune cell infiltration at these early time points, with Mpx⁺ cell numbers peaking at 14 dpci, which is a pattern strikingly different from the rapid and robust neutrophil infiltration observed in zebrafish within the first few days post-injury. These results reinforce our conclusion that the inflammatory response in Xiphophorus is both delayed and attenuated relative to zebrafish, and the updated data are now presented in Figure 6.

(2) Their transcriptomic data at 7 days post injury suggest that TGFß signaling was not activated after injury, and tenascin C was not expressed in platyfish. The authors might check whether TGFB signaling is activated and tenascin is expressed at later time points, since platyfish show persistent scarring. This may help determine whether the molecular mechanisms of scarring in platyfish are the same as in zebrafish, and it just happens late.

We think it is a good suggestion. Unfortunately, our anti-TnC antibodies do not produce a clear or specific signal in platyfish tissue, precluding immunofluorescence-based analysis of TnC expression at later regenerative stages. To partially address this limitation, we have included transcript abundance data for several genes associated with TGF-β signaling in the new Figure 5, which provides an initial view of this pathway's activity during Xiphophorus heart regeneration. Nevertheless, a thorough characterization of TGF-β activity and TnC expression, especially including protein-level validation and temporal profiling, would require dedicated methodological development and constitutes a new study. We therefore consider this to be an important avenue for future investigation rather than a component of the present initial characterization.

(3) The PCNA staining BrdU labeling experiments suggest that proliferating cardiomyocytes are not maintained even though they re-enter the cell cycle. Since the platyfish hearts lack the proliferative compact cardiomyocytes that account for myocardial regeneration in zebrafish, do the authors suggest that their trabecular cardiomyocytes proliferate or that they only undergo DNA synthesis?

This question touches on a central unresolved aspect of our findings. Our results indicate that a subset of cardiomyocytes re-enter the cell cycle and undergo DNA synthesis at 7 and 14 dpci, as evidenced by PCNA staining (new Figure 7D). However, BrdU incorporation combined with Tropomyosin immunostaining reveals an absence of BrdU⁺/Tropomyosin⁺ cardiomyocytes within the border zone myocardium (new Figure 7F), indicating that newly synthesized DNA does not translate into efficient cardiomyocyte repopulation of the injured area. Whether the detected S-phase entry is followed by mitosis, or whether these cardiomyocytes undergo DNA synthesis without completing cell division, a phenomenon known as endoreplication, which has been described in other cardiac contexts, remains to be determined. Resolving this question would require live imaging or mitotic marker analyses beyond the scope of the present study, and we have highlighted this as an important open question in the revised Discussion.

(4) Line 136-137. The authors might clarify what they meant by "N2.261 antibody recognizes different myosin types in zebrafish versus platyfish". Are these N2.261+ cardiomyocytes in the atria of platyfish also immature cardiomyocytes, but are there more immature cardiomyocytes in platyfish than in zebrafish?

We thank the reviewer for this good question. In zebrafish, N2.261 has been established as a marker of immature cardiomyocytes in larvae and regenerating myocardium; however, we cannot currently conclude that N2.261-positive atrial cardiomyocytes in platyfish represent an analogous immature population. Rather, the broad atrial immunoreactivity in platyfish most likely reflects the presence of the N2.261 epitope within the dominant atrial myosin heavy chain isoform of this species. Thus, platyfish atrial myosin shares greater sequence similarity with the antibody's target epitope than the corresponding zebrafish atrial myosin does. In other words, the differential labeling pattern between species is more likely attributable to evolutionary divergence in myosin heavy chain sequences than to differences in cardiomyocyte maturation state. To directly identify which amino acid residues are essential for N2.261 immunoreactivity and to resolve these evolutionary differences, we have initiated a dedicated epitope-mapping study.

To clarify this interpretation in the manuscript, we have replaced the previous concluding statement with the following: "Together, these findings indicate that N2.261 recognizes distinct myosin heavy chain isoforms in zebrafish and Xiphophorus: an embryonic ventricular isoform in the former and an atrial isoform in the latter. The differential labeling pattern between species is more likely attributable to evolutionary divergence in myosin heavy chain sequences than to differences in cardiomyocyte maturation state, reflecting the substantial lineage-specific reshaping of cardiac myosin repertoires that has occurred between cyprinids and poeciliids."

(5) Line 212-"Interspecies comparison revealed that upon cryoinjury, 199 and 268 orthologous gene transcripts were more abundant in zebrafish than in platyfish, respectively". Does the author mean that "more abundant in zebrafish than in platyfish and vice versa"?

We thank the reviewer for flagging this ambiguity. The original sentence was indeed unclear, and we have revised it to read: "Interspecies comparison revealed that upon cryoinjury, 199 orthologous gene transcripts showed a higher log₂FC in zebrafish than in platyfish, while 268 showed the opposite pattern, indicating that the two species mount distinct transcriptional responses to cardiac injury." We believe this phrasing now unambiguously conveys that the comparison is bidirectional.

Reviewer #2 (Public review):

Major comments

(1) Title selection

The title the authors chose suggests that platyfish and swordtails "partially regenerate," but I do wonder how much these animals truly regenerate. This may be a semantic discussion and a matter of personal preference. Still, based on other significant work on regenerative capacity (see, for example, the landmark cavefish regeneration paper PMID: 30462998 or work on medaka PMID: 24947076), the persistence of such a prominent fibrotic scar would be considered a minimal regenerative capacity. Measuring this "partial regeneration" more precisely by comparing zebrafish with platyfish and swordtails would also greatly strengthen the comparisons made here - see below.

The same can be said about line 152-153 - do these hearts "regenerate" with deformation and partial scarring, or would it be more fair to say that they are "healed" or "repaired" with a process that involves fibrosis?

We thank the reviewer for raising this conceptual point, and we appreciate the references to the cavefish and medaka literature. We acknowledge that the term "partially regenerate" requires careful justification given the persistence of a substantial collagenous scar at the injury site. We have retained this terminology for the following reasons:

(1) The bulging wound undergoes resorption over time, suggesting that the initial structural deformation is transient rather than permanent.

(2) Wound size is significantly reduced and the proportion of hearts retaining visible injury decreases over the course of the experiment.

(3) Cardiomyocyte proliferation is detectably elevated in the myocardium at 7 and 14 dpci, indicating that some regenerative machinery is engaged.

We would also note that partial heart regeneration accompanied by residual scarring has been described in newts, a classically regenerative vertebrate. This suggests that the boundary between regeneration and fibrotic repair is not always clear-cut. Rather than a binary distinction, the cardiac injury response may exist on a continuum, where hallmarks of regeneration, such as wound resorption, reduced scar size, and cardiomyocyte proliferation, can coexist with persistent fibrosis. We therefore believe that "partial regeneration with persistent scarring" accurately and honestly reflects our findings, and we have refined the relevant passages in the manuscript, including the indicated lines, to make this nuance explicit.

(2) Cross-species comparisons

Having two species of livebearers strengthens the findings of this paper, but the presentation of results from both species is inconsistent. For example, the reader should not be asked to assume that the architecture of the swordtail ventricle is similar to that of the platyfish (line 125). The same applies to the presence or absence of coronary vessels (Figure 1), the reduction in wound area over time (Figure 3), and the immune system's response (Figure 5). Most importantly, the authors miss an opportunity to move from qualitative observations to quantifying the "partial regeneration" phenotype they observe. Specifically, providing a side-by-side comparison between these new species and zebrafish would help define the extent of differences in regeneration potential. For instance, in Figure 6, while the authors provide excellent quantification of PCNA staining in platyfish, these data are less meaningful without a direct comparison with zebrafish results. The same applies to Figures 6E and 6F - although differences are noted, quantifying these results would enable a more rigorous assessment of the process.

We thank the reviewer for this constructive critique. We agree that greater consistency in the cross-species presentation strengthens the comparative framework of the paper, and we have made several additions to address this.

To document the cardiac architecture of swordtails explicitly, rather than asking the reader to assume similarity with platyfish, we have added the following data:

(1) New supplementary figure (Figure S1) dedicated to the swordtail ventricle, incorporating 1) AFOG, 2) Picrosirius Red, 3) Alkaline Phosphatase Assay for the vasculature, 4) and immunostaining against Fibronectin, N2.261, and F-actin.

(2) The dynamics of swordtail heart regeneration across 7, 14, 30, 60, and 90 dpci are presented in a separate supplementary figure (Figure S5), which also includes quantification of wound size over time. These data show that the wound, representing approximately 20% of ventricular area at 7 dpci, is reduced to approximately 2.5% by 60–90 dpci, providing quantitative support for our characterization of partial regeneration in this species.

We appreciate the reviewer's point regarding side-by-side quantitative comparisons with zebrafish for markers such as PCNA. We respectfully note, however, that cardiomyocyte proliferation in zebrafish following cryoinjury is extensively documented across multiple independent studies, and we consider this body of evidence sufficient to contextualize our platyfish findings without requiring full parallel quantification. Nevertheless, in response to this comment, we have included side-by-side zebrafish and platyfish data for BrdU staining in the new Figure 7, with zebrafish quantification drawn from our previously published dataset (Sallin et al., 2015). We believe this addition meaningfully strengthens the cross-species comparison at this key figure while remaining within the scope of the present study.

(3) Lack of coronary vasculature

There is a growing body of evidence highlighting the importance of the coronary vessels during zebrafish heart regeneration (PMIDs: 27647901, 31743664). Surprisingly, this finding has not been integrated or discussed in the context of this literature.

The results of the alkaline phosphatase assay and anti-podocalyxin-2 staining appear inconsistent. Specifically, in Supplementary Figure 1L-M, we can see some vessels covering the bulbus arteriosus and also what appears to be a signal in the ventricle. However, in Figures 1 K and 1L, we cannot see any vessels, even in the bulbus. The authors should also be more rigorous and add a description of how many animals were analyzed, their ages, and sizes. In zebrafish, the formation of the coronary arteries appears to depend on animal size and age. With the data provided, we cannot say whether this is a one-time observation or a consistent finding across many animals at different ages and across both species.

We thank the reviewer for raising this point and for directing us to the relevant literature. We agree that the role of coronary vasculature in zebrafish heart regeneration is an important and growing area of research, and we have now integrated a discussion of this evidence into the manuscript, highlighting the contrast with the avascular Xiphophorus ventricle and its potential implications for regenerative capacity.

Regarding the apparent inconsistency between the alkaline phosphatase assay and the anti-Podocalyxin (anti-Podxl) staining, we thank the reviewer for this careful observation. We have performed additional staining to resolve this discrepancy and conclude that the two approaches, rather than being inconsistent, reflect distinct and complementary aspects of ventricular organization.

To better characterize the anti-Podxl signal, we performed immunofluorescence on thick (50 µm) sections of both platyfish and zebrafish hearts. In contrast to zebrafish, anti-Podxl immunoreactivity in platyfish is confined to a very thin outer layer of the myocardium. This subtle signal accounts for the weak staining previously observed in whole-ventricle preparations and was already visible, though not highlighted, in the original figures (see white arrow in new Supplementary Figure S3C, H, O–R). A direct comparison of anti-Podxl staining across species (Supplementary Figure S3A, D, N, R) clearly demonstrates the absence of coronary vascularization in the platyfish ventricle. Consistent with this, no vascular signal is detected in the platyfish bulbus arteriosus (Supplementary Figure S3I).

To further clarify the nature of the outer myocardial layer in Xiphophorus, we performed Picrosirius Red (PSR) staining, which selectively labels fibrillar collagen, across all three species. Whereas the compact outer myocardium of zebrafish is PSR-negative, a thin but distinct PSR-positive layer is present at the ventricular surface of both platyfish and swordtails, closely mirroring the anti-Podx1 staining pattern.

Taken together, these findings indicate that the outer ventricular layer of Xiphophorus fish is composed of a thin collagen- and Podxl-immunoreactive matrix, which is structurally distinct from the vascularized compact myocardium of zebrafish and is consistent with the absence of coronary vessels in these species.

Finally, we acknowledge the reviewer's request for greater rigor regarding sample sizes, animal ages, and body sizes. These details have now been added to the Methods section. We note that, in line with the reviewer's observation regarding zebrafish coronary development, we have ensured that animals of comparable size and age ranges are described for each species to allow meaningful cross-species interpretation.

(4) The link between livebearers' responses and pseudoaneurysms is overstated. This work is already extremely relevant without trying to make it medically oriented.

We agree that the clinical parallel with pseudoaneurysms was overstated in the original manuscript, and that the comparative and evolutionary relevance of our findings stands on its own merits without requiring a medical framing. We have accordingly removed the term from the Results section and now invoke it only briefly at the close of the Discussion, where a concise note on broader medical relevance is appropriate without overshaping the narrative of the paper.

Reviewer #2 (Recommendations for the authors):

(1) The description of the N2.261 staining (Figure 1 and Supplementary Figure 1) is entirely irrelevant for the rest of the manuscript. One wonders why the authors have not used this antibody to characterize the presence or absence of "dedifferentiated" muscle after injury. Given that they make a point later about potential differences in dedifferentiation in livebearers, this is a missed opportunity to address it using tools the authors have characterized extensively in zebrafish.

This is a valuable suggestion that we have now addressed. We performed N2.261 immunostaining on injured platyfish and swordtail hearts at 7 dpci, and included the results as an additional supplementary figure (Suppl. Fig. S4C). Importantly, N2.261 immunoreactivity was not detected in the peri-injury zone of the myocardium, suggesting that, unlike in regenerating zebrafish hearts, the embryonic cardiac myosin heavy chain isoform is not upregulated at the injury site in platyfish. This finding is particularly informative given the broad atrial N2.261 reactivity observed in intact platyfish hearts: the absence of enhanced staining in the injury zone argues against a dedifferentiation-associated upregulation of this isoform and instead suggests that platyfish cardiomyocytes may not undergo the same embryonic gene re-expression program that characterizes zebrafish cardiac regeneration. This result therefore directly informs our later discussion of potential differences in dedifferentiation between zebrafish and livebearers, and we have updated the relevant section of the manuscript accordingly.

(2) Many of the markers highlighted here as part of the differential gene expression analysis are not the most canonical ones, and it is unclear how the authors selected them. For example, in Figure 5, anxa2a, hlx1, and nup153 are presented as macrophage markers. However, anxa2a appears to be expressed predominantly in the endocardium in response to injury (see PMID: 32341028), and other markers (mpeg, mfap4, etc) would have been more consistent as macrophage markers according to other literature. The same is true for cardiomyocyte proliferation markers and dedifferentiation markers in Figure 6. In this last case, N2.261 could have been used as reported by the authors before. L-plastin is a pan-leukocyte marker, not a macrophage marker. This should be corrected (Figure 5 and lines 258-279).

We agree with this critique that several of the markers used in the original analysis were neither sufficiently canonical nor specific for the cell populations we intended to characterize. We apologize for this oversight. In response, we have moved away from scRNA-seq-derived marker sets and now rely entirely on manually curated markers drawn from the established literature, as detailed below.

(1) Cardiomyocyte cell fate change (new Figure 7, Supplementary Figure S8): We now report the transcript abundance of genes encoding proteins with well-documented roles in the transcriptional reprogramming associated with cardiomyocyte dedifferentiation and redifferentiation. These include members of the Activator Protein-1 (AP-1) complex, the SWI/SNF chromatin remodeling complex, and the transcriptional coactivator cited4a, alongside established markers of cardiomyocyte proliferation (cx43) and sarcomere reassembly (the Rbfox family). We also include myh7 transcript abundance, which in zebrafish is upregulated in dedifferentiated cardiomyocytes.

(2) Immune response (new Figure 6, Supplementary Figure S7): New Figure 6 focuses on pan-leukocyte markers, while Supplementary Figure S7 presents genes involved in innate myeloid activation and inflammation, including components of the NF-κB/TNF-α pathway, TLR signaling, and inflammatory regulation, as well as specific markers of neutrophils (mpx) and macrophages (mpeg1, mfap4), in line with the markers recommended by the reviewer.

Regarding L-plastin, we thank the reviewer for this correction. The text has been amended throughout to describe L-plastin accurately as a pan-leukocyte marker rather than a macrophage/phagocyte marker.

(3) In several instances, the authors reference papers without citing the original source. In many other instances, there are some oversights regarding citations. I would advise revising many of these:

We thank the reviewer for drawing our attention to these citation oversights. We have carefully revised the reference list throughout the manuscript to ensure that original discovery papers are cited alongside, or in place of, review articles wherever appropriate.

(4) Lines 49-51 - several very interesting reviews of cardiac regeneration are listed here, but referencing the source of the discovery is always most rigorous.

For lines 49–51, we have replaced the previous review citations with three original research papers reporting the discovery of cardiac regeneration in zebrafish and three reporting it in axolotl. We have applied the same principle of prioritizing primary sources across all other instances flagged by the reviewer.

(5) Line 76. When discussing the recovery of the muscle after cryoinjury, Poss et al. 2002 shouldn't be referenced. Sánchez-Iranzo et al 2018 should be replaced by González-Rosa et al. 2011, which is the contemporary manuscript to those of Chablais 2011 and Schnabel 2011.

Done

(6) Line 284 - when discussing cardiomyocyte dedifferentiation, it would be fair to reference also Kikuchi et al 2010.

Done