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.

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."

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.

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?

(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.

(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.

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

Author response:

Reviewer #1:

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."

Our response regarding time points of transcriptomic analysis:

We appreciate this constructive suggestion. We fully agree that performing RNA-seq at multiple time points would provide valuable insights into the temporal dynamics of molecular pathways during cardiac regeneration. However, given that our study represents the first comprehensive characterization of cardiac regeneration in poeciliids, we deliberately focused our resources on establishing the foundational framework, including morphological, cellular, and initial transcriptomic analyses between zebrafish and platyfish. Expanding to multiple time points would constitute a substantial additional study that, while scientifically valuable, would extend beyond the scope of this initial characterization.

We will acknowledge this limitation in the Discussion and indicate that temporal transcriptomic profiling is an important direction for future investigation.

Reviewer #2:

(1) Title selection

Our response regarding the use of the term “partially regenerate” in the title and results:

We thank Reviewer 2 for this important point regarding the terminology used to describe the cardiac response in platyfish and swordtails. We agree that the term "partially regenerate" may overstate the regenerative capacity of these species, particularly given the persistence of a substantial collagenous scar at the injury site. The reviewer is correct that, based on established criteria in the field, including the landmark studies on cavefish (PMID: 30462998) and medaka (PMID: 24947076), the presence of such prominent fibrotic scarring would be more appropriately characterized as limited or minimal regenerative capacity rather than partial regeneration.

While we observe a significant reduction in wound volume at 30 dpci and some degree of tissue remodeling, we acknowledge that the persistent scarring and incomplete myocardial recovery more accurately reflect a healing or repair process rather than true regeneration. We therefore agree with the reviewer's suggestion to revise our terminology throughout the manuscript.

We will revise the title to: "The livebearers platyfish and swordtails heal their hearts with persistent scarring." We will also modify other relevant sections of the Results and Discussion to consistently describe these processes as "healing" or "repair" rather than "regeneration", while still acknowledging the biological changes that do occur (wound contraction, remodeling, limited cardiomyocyte proliferation). This revised framing better aligns our work with the established terminology in the comparative cardiac regeneration literature and more accurately represents the phenotype we observe.

We believe this change will strengthen the manuscript by providing a more precise characterization of the cardiac response in these species and facilitating clearer comparisons with other model systems.

(2) Cross-species comparisons

Our response regarding the inconsistent presentation of results for different species:

We thank the reviewer for recognizing that our conclusions regarding the regenerative capacity of livebearers are strengthened by including two poeciliid species, platyfish and swordtails. We agree that presenting results more consistently across both species will significantly improve the manuscript. We acknowledge that our current presentation creates a burden on the reader by asking them to assume similarities between species without providing supporting data. While we initially focused primarily on platyfish due to its superior genome annotation (critical for our transcriptomic analyses), we recognize that this approach left important gaps in the manuscript.

We will address this by generating comprehensive supplementary figures that present swordtail data alongside platyfish for key findings. Specifically, we will add a complete anatomical characterization of swordtail ventricle architecture, demonstrating the structural similarities to platyfish that underpin our comparative conclusions. We will also perform quantification of wound area reduction and immune response dynamics over time in swordtails, allowing direct comparison between species.

We clarify that we did perform detailed analyses of swordtail heart anatomy during our initial studies, which revealed remarkable similarity to platyfish. However, space constraints in Figures 1 and S1 (which already span full pages with zebrafish-platyfish comparisons) prevented us from including these data in the original submission. We now recognize that explicitly presenting these data is essential for the reader to evaluate our conclusions.

Our response regarding quantification and comparison with zebrafish:

We appreciate the reviewer's suggestion to move beyond qualitative observations toward rigorous quantification of the "partial regeneration" phenotype. As suggested by the reviewer for the PCNA analysis, we will provide direct quantitative comparisons with published zebrafish regeneration studies, including data from several relevant studies and our own lab's work. This comparison will delineate the extent of differences in proliferative response between complete regenerators (zebrafish) and limitted regenerators (poeciliids).

These additions will transform our descriptive observations into quantitative assessments that rigorously define the incomplete healing phenotype in poeciliids relative to complete regeneration in zebrafish. We believe these changes will substantially strengthen the manuscript and address the reviewer's concerns about comparative rigor.

(3) Lack of coronary vasculature

Our response regarding inconsistencies in vascularization data:

We thank the reviewer for his/her comment regarding our data on the absence of coronary vasculature in the platyfish heart. The reviewer noted differences between alkaline phosphatase (AP) enzymatic staining and anti-Podocalyxin-2 immunofluorescence staining. We would like to clarify that these observed differences are not inconsistencies but rather reflect the distinct specificities of these two complementary approaches.

Alkaline phosphatase staining is selective for arterial branches and capillaries in the heart (PMID: 13982613; PMID: 9477306; PMID: 8245430; PMID: 3562789; PMID: 29023576; PMID: 28632131) and revealed a typical vascular pattern in the bulbus arteriosus and ventricle in zebrafish but not in platyfish. Anti-Podocalyxin-2 staining displayed a vessel-like pattern in zebrafish but not in platyfish. However, in both species Podocalyxin staining also labeled other types of non-vascular structures. This is expected given that Podocalyxin is a cell surface sialomucin with broader expression beyond blood vessels, including the endocardium (PMID: 19142011) and certain neuronal populations, in addition to other non-cardiac tissue types (PMID: 19578008; PMID: 3511072; PMID: 34201212).

We will revise the manuscript to emphasize this distinction and clarify our rationale: we deliberately employed Podocalyxin-2 staining as a complementary, less selective approach to corroborate our alkaline phosphatase findings. In platyfish, the convergent evidence from both methods (the absence of typical vascular structures with a selective AP staining and the detection of only non-vascular patterns with the broader marker Podocalyxin-2) strengthens our conclusion that platyfish hearts lack a conventional coronary vascular network.

Our response regarding reproducibility:

The assays were performed independently by two researchers at different stages of the study using two different batches of adult platyfish. The results were consistent in both assays, and we are therefore confident in the reproducibility of our findings.

Our response regarding citations of references on revascularization:

We thank the reviewer for recommending the studies PMID: 27647901 and PMID: 31743664 that revealed the importance of rapid revascularization during heart regeneration in zebrafish. We will be pleased to integrate these works to present our data in the appropriate context of current knowledge.

Our response regarding a link to pseudoaneurysms:

We appreciate the reviewer's feedback regarding the link to pseudoaneurysm. We agree that the primary contributions of our work stand on their own merit, and we will revise the text to present the livebearer findings more cautiously without overstating their potential medical relevance. We will focus on the intrinsic biological significance of our findings.