Insights into Cardiomyocyte Regeneration from Screening and Transcriptomics Approaches

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study Dr. Fuller et al. have provided a comprehensive review of current in vitro and in vivo models for high-throughput screening to address the challenge of adult cardiomyocyte regeneration and identify novel targets for cardiomyocyte proliferation. The review is well-structured, and comprehensive and up-to-date. The manuscript is clearly written and covers properly various models, platforms and emerging technologies, and limitations of each models and technologies are appropriately highlighted. Other specific comments:

1. Cardiomyocyte proliferation is the key for cardiac regeneration and one of major focus of this review. The review effectively covers key genetic reporters (e.g., FUCCI, MADM). To further strengthen this section, consider mentioning the ProTracer (Proliferation Tracer) mouse model. This innovative system, which combines Dre and Cre recombinases for continuous, cumulative lineage tracing of proliferating CMs, represents a significant advance for in vivo quantification of CM turnover and could be included.
2. The discussion of 3D human cardiac organoids (hCOs) and recent assembloids incorporating neural crest or macrophages is appreciated. To enhance comprehensiveness, it would be valuable to also acknowledge other seminal studies that have generated multi-lineage organoids or assembloids containing such as endoderm-derived tissues or vascular endothelial networks. These models better recapitulate physiological cell-cell interactions, polarity, and compartmentalization, offering more nuanced platforms for functional screening.
3. While translational challenges are discussed, it is desirable to emphasize more on clinical relevance and include a table or paragraph on clinical/preclinical advancements related to discussed targets, that would strengthen the review’s impact.
4. On Page 3, line ~122, the phrase “…the downside is that they lack multicellular heterogeneity and…” could be clarified.

Overall, this is a high-quality, insightful review and suitable for publication in International Journal of Molecular Sciences after minor revisions to enhance its completeness and translational perspective.

Author Response

Comments 1: Cardiomyocyte proliferation is the key for cardiac regeneration and one of major focus of this review. The review effectively covers key genetic reporters (e.g., FUCCI, MADM). To further strengthen this section, consider mentioning the ProTracer (Proliferation Tracer) mouse model. This innovative system, which combines Dre and Cre recombinases for continuous, cumulative lineage tracing of proliferating CMs, represents a significant advance for in vivo quantification of CM turnover and could be included.
Response 1: We appreciate the reviewer’s suggestion and have added a description of the ProTracer method to the paragraph describing FUCCI and MADM readouts (lines 345).

 

Comments 2: The discussion of 3D human cardiac organoids (hCOs) and recent assembloids incorporating neural crest or macrophages is appreciated. To enhance comprehensiveness, it would be valuable to also acknowledge other seminal studies that have generated multi-lineage organoids or assembloids containing such as endoderm-derived tissues or vascular endothelial networks. These models better recapitulate physiological cell-cell interactions, polarity, and compartmentalization, offering more nuanced platforms for functional screening.
Response 2: We agree these advances are important and have added references to several studies that showcase recent advances in hCO technology, with a particular focus on vascularization (Section 2.2, line 162).

 

Comments 3: While translational challenges are discussed, it is desirable to emphasize more on clinical relevance and include a table or paragraph on clinical/preclinical advancements related to discussed targets, that would strengthen the review’s impact.
Response 3: We have addressed this comment with a new subsection (8.3), which covers several potential therapies that have progressed through advanced preclinical trials and one potential therapy (propranolol) that is currently in phase I clinical trial.

 

Comments 4: On Page 3, line ~122, the phrase “…the downside is that they lack multicellular heterogeneity and…” could be clarified.
Response 4: We have revised this statement to increase readability (lines 137).

Reviewer 2 Report

Comments and Suggestions for Authors

Despite being rich in data, the review is very descriptive overall. It often lacks a summary in which the authors explain:
Which screening approaches are more robust, and which are less so?
which results are reproducible and confirmed across multiple models, and which remain preliminary.
Which pathways emerge as real convergences (e.g. Hippo/YAP, metabolism and mevalonate signalling) and which are unique to individual studies?

It is unclear whether this is a purely narrative review or if a minimum level of systematic selection was performed in terms of the search strategy, keywords, time frame and inclusion/exclusion criteria.
Currently, the text gives the impression of an ad hoc selection of studies, particularly the most 'visible' ones (Nature, Nature Cardiovascular Research, etc.), which carries the risk of citation bias.

The authors rightly emphasise the difficulties of translating proliferative strategies into human therapy (oncogenicity, off-target effects and safety).
However, this point is only addressed briefly in the final few sentences and is not critically discussed in the previous sections. For example:
Many of the cited molecules and strategies (e.g. CDK/cyclin cocktails, robust YAP activation and modulation of pro-proliferative miRNAs) carry an inherently high oncogenic risk, which merits more concrete discussion of the following:
- duration of activation;
spatial targeting (border zone vs. global myocardium);
'off-switch' and reversibility.
It would be useful to make a more explicit comparison between what has been demonstrated only in zebrafish and neonatal models, and what has shown at least minimal efficacy in preclinical adult models that are closer to humans, such as swine and IR models.

 

While some sections are very detailed (e.g. the classification of CM clusters in various single-cell datasets with gene lists), other key concepts are covered quickly.
There is a brief mention of the distinction between proliferation and 'pathological detachment' (e.g. dilated cardiomyopathy in cases of chronic sarcomeric structure loss), but the potential risk of chronically active pro-proliferative interventions promoting heart failure rather than regeneration is not truly explored.
There is only a brief mention of the use of large animal models (swine), and virtually nothing about the regulatory pipeline and safety requirements needed to advance these approaches to Phase I/II.

Overall, the manuscript addresses a highly relevant topic and makes a significant effort to summarise recent literature on cardiac regeneration, screening and omics. However, to become a truly useful and authoritative review for the community, it requires:
- a greater critical/comparative dimension;
better conceptual integration between the different sections (screening, omics, microenvironment and epigenetics);
, and a more in-depth discussion of translational and safety aspects.
I therefore recommend major revisions before considering publication.

Author Response

Comments 1: Despite being rich in data, the review is very descriptive overall. It often lacks a summary in which the authors explain: Which screening approaches are more robust, and which are less so? which results are reproducible and confirmed across multiple models, and which remain preliminary. Which pathways emerge as real convergences (e.g. Hippo/YAP, metabolism and mevalonate signalling) and which are unique to individual studies?
Response 1:
- We agree that a comparative summary of screening approaches was needed. We have added Section 3.3, which outlines the relative strengths and limitations of common strategies. In particular, we discuss that high-throughput chemical and in silico screens offer scalability but may lack mechanistic clarity, whereas target-directed and organoid/adult CM models provide more robust and physiologically relevant readouts at higher cost. We also highlight the value of sequential or parallel screening across complementary platforms to identify candidates with conserved pro-proliferative effects.
- We have added sentences to the final paragraph of the discussion summarizing reproducible versus preliminary results, highlighting consistent pathways such as Hippo/YAP, metabolic, and mevalonate signaling.

Comments 2: It is unclear whether this is a purely narrative review or if a minimum level of systematic selection was performed in terms of the search strategy, keywords, time frame and inclusion/exclusion criteria. Currently, the text gives the impression of an ad hoc selection of studies, particularly the most 'visible' ones (Nature, Nature Cardiovascular Research, etc.), which carries the risk of citation bias.
Response 2: We thank the reviewer for this thoughtful comment. Our intention was to provide a narrative, mechanistically focused review, rather than a formal systematic review. We therefore did not follow a PRISMA-style protocol, but we did use structured literature searches (primarily PubMed and Google) and selected studies based on biological relevance to CM proliferation, breadth of experimental models (2D cells, organoids, zebrafish, rodent and large-animal injury models), and the incorporation of screening and/or single-cell/omics approaches.
We agree that reliance on only the most “visible” papers would introduce citation bias. To minimize this, we deliberately included studies across a range of journals and years, including earlier or less widely cited work when it provided unique mechanistic or translational insight. Due to space constraints, the review is not exhaustive, and we fully acknowledge that some relevant studies may not have been included; our goal was to highlight representative and influential examples rather than catalogue all available data.

Comments 3: The authors rightly emphasise the difficulties of translating proliferative strategies into human therapy (oncogenicity, off-target effects and safety). However, this point is only addressed briefly in the final few sentences and is not critically discussed in the previous sections. For example: Many of the cited molecules and strategies (e.g. CDK/cyclin cocktails, robust YAP activation and modulation of pro-proliferative miRNAs) carry an inherently high oncogenic risk, which merits more concrete discussion of the following: - duration of activation;
spatial targeting (border zone vs. global myocardium); 'off-switch' and reversibility. It would be useful to make a more explicit comparison between what has been demonstrated only in zebrafish and neonatal models, and what has shown at least minimal efficacy in preclinical adult models that are closer to humans, such as swine and IR models.
Response 3:
- We thank the reviewer for this important suggestion. We have expanded the discussion of translational and safety challenges in newly added Section 8.1 and part of Section 8.2, with a focused critique of oncogenicity and off-target risks associated with CDK/cyclin activation, sustained YAP signaling, and pro-proliferative miRNA strategies. In particular, we now emphasize the importance of controlling activation duration, spatial targeting, and reversibility through effective “off-switches” for clinical translation.
- We agree with the reviewer regarding the importance of this distinction and have added a paragraph to Section 4.2 that mentions specific CM proliferation targets and the models in which they have been validated (including 2D culture, 3D culture, zebrafish, neonatal mice, adult mice, and swine).

Comments 4: While some sections are very detailed (e.g. the classification of CM clusters in various single-cell datasets with gene lists), other key concepts are covered quickly. There is a brief mention of the distinction between proliferation and 'pathological detachment' (e.g. dilated cardiomyopathy in cases of chronic sarcomeric structure loss), but the potential risk of chronically active pro-proliferative interventions promoting heart failure rather than regeneration is not truly explored. There is only a brief mention of the use of large animal models (swine), and virtually nothing about the regulatory pipeline and safety requirements needed to advance these approaches to Phase I/II.
Response 4:
- We thank the reviewer for raising this important point. We have added Section 8.2 to explicitly discuss the risk that chronically active pro-proliferative interventions may promote pathological remodeling and heart failure rather than regeneration, including the distinction between productive CM proliferation and maladaptive states associated with sarcomeric disassembly.
- We have also discussed the importance of using multiple in vitro, in vivo, and in silico models for validation.

Comments 5: Overall, the manuscript addresses a highly relevant topic and makes a significant effort to summarise recent literature on cardiac regeneration, screening and omics. However, to become a truly useful and authoritative review for the community, it requires: - a greater critical/comparative dimension; better conceptual integration between the different sections (screening, omics, microenvironment and epigenetics), and a more in-depth discussion of translational and safety aspects.
Response 5:
- We have addressed this comment with a new subsection (8.3), which covers regulatory and safety considerations that must be taken into account to advance preclinical findings to clinical trials.
- We added a graphic abstract to help with conceptual integration (page 2).