STAT5b is a key effector of NRG-1/ERBB4-mediated cardiomyocyte growth

  1. Katri Vaparanta
  2. Anne Jokilammi
  3. Ilkka Paatero
  4. Johannes A. Merilahti
  5. Juho Heliste
  6. Karthik Amudhala Hemanthakumar
  7. Riikka Kivelä
  8. Kari Alitalo
  9. Pekka Taimen
  10. Klaus Elenius

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Abstract

The growth factor neuregulin-1 (NRG-1) regulates hypertrophic and hyperplastic myocardial growth and is currently under clinical investigation as a treatment for heart failure. We have previously demonstrated that an isoform of the NRG-1 receptor ERBB4 (ERBB4 JM-b) expressed in cardiomyocytes selectively regulates the activation of STAT5b. To explore the role of STAT5b in NRG-1/EBBB4 mediated cardiomyocyte growth, several in vitro and in vivo models were utilized. The downregulation of NRG-1/ERBB4 signaling consistently reduced STAT5b activation and transcription of STAT5b target genes Igf1, Myc and Cdkn1a in murine in vitro and in vivo models of myocardial growth. Stat5b knock-down in primary cardiomyocytes ablated NRG-1-induced cardiomyocyte hypertrophy. Stat5b was activated during NRG-1-induced hyperplastic myocardial growth and chemical inhibition of the Nrg-1/Erbb4 pathway led to the loss of myocardial growth and Stat5 activation in zebrafish embryos. Moreover, CRISPR/Cas9-mediated knock-down of stat5b in zebrafish embryos resulted in reduced myocardial growth and heart failure as indicated by reduced ventricular ejection fraction. Dynamin-2 was discovered to control the cell surface localization of ERBB4 and the chemical inhibition of dynamin-2 downregulated NRG-1/ERBB4/STAT5b signaling in models of hypertrophic and hyperplastic myocardial growth. Finally, the activation of the NRG-1/ERBB4/STAT5b signaling pathway was explored in clinical samples representing pathological cardiac hypertrophy. The NRG-1/ERBB4/STAT5b signaling pathway was differentially regulated both at the mRNA and protein levels in the myocardium of patients with pathological cardiac hypertrophy as compared to myocardium of control subjects. These results establish the role for STAT5b, and dynamin-2 in NRG-1/ERBB4-mediated myocardial growth.

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  1. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    Comments received from the Reviewers on 18th of November 2022 are included in plain font, followed point-by-point by the authors’ comments in bold.

    Reviewer #1 (Evidence, reproducibility and clarity):

    The endothelial release of NRG (neuregulin)-1 is a paracrine growth factor activating ErbB (erythroblastic leukaemia viral oncogene) receptor tyrosine kinases on various targets cells epicardial, endocardial/endothelial (autocrine stimulation) and myocardiocytes. It is well known, after the manuscript of the K.D.Poss group that Neuregulin1 induced perivascular cells after injury to the adult zebrafish heart as well as mammalian cells. Inhibition of the Erbb2 co-receptor, disrupts cardiomyocyte proliferation in response to injury, whereas myocardial NRG1 overexpression enhances this proliferation. Thus, seems to be clear, that NRG-1 could stimulate regenerative, inflammatory, fibrotic, and metabolic processes In uninjured zebrafish, the reactivation of Nrg1 expression induces cardiomyocyte dedifferentiation, overt muscle hyperplasia, epicardial activation, increased vascularization, and causes cardiomegaly through persistent addition of wall myocardium (review cited by authors in this MS). In light of the large amount of these research deals with NRG1, the molecular mechanisms linked and focused to understand the multivariate effects of the NRG1 cascade are welcome. The authors have focused the manuscript on a comparative and translational demonstration of STAT5b involvement in the signal transduction of NRG1.

    Although the authors have done several experiments they have presented these in a chaotic way among mice, zebrafish and human biopsies. I can suggest rewriting partially the results section in a way more ordered and readable. Even the discussion is a little bit chaotic and lacks some aspects. For example, who is the stimulator of NRG1 release? Moreover, the literature cited is partially or not correctly reported (in the references section). In my opinion, the authors should revise the manuscript in the light of following suggestions.

    MAJOR:

    Title: line 1. The title does not explain the translational aspects of the manuscript. I suggest something like: "STAT5b is a key effector of NRG1/ERBB4-mediated cardiomyocyte growth: a translational approach".

    We have now modified the title as suggested by the Reviewer.

    Abstract. Lines 20-25. The authors should rewrite this section by removing their previous finding ("we") reporting. In lines 25-36 the reported data is not well explained and is not clear who did that. The authors, previously? It is not well explained.

    Lines 35-37 Please, specify in which type of hypertrophic heart samples (is it from humans?) have been observed the NRG1 pathway.

    We have now rewritten this section of the abstract and changed the tense to present to better indicate which observations are presented in the manuscript.

    Introduction. Lines 44-46. All the cited papers consist of the preprint, thus they could be inserted in the discussion and not in the introduction. In the alternative, they could be inserted here following a sentence "Preliminary study seems to indicate...."

    We apologize for the mistake, the references are not preprints but articles published in Nature. We have amended the reference section so this is now indicated more clearly.

    Lines 50-51. The references cited are relative only to murine research and not to other animals. Thus, only murine models have been demonstrated. Thus, the assertion could be not valid for zebrafish or humans, thus the authors should point out this.

    The reference number 8 in the original submission refers to experiments conducted in embryonic zebrafish. We have now rewritten this section by separating the previous observations in mice and in zebrafish into separate sentences.

    Material and Methods.

    Lines 148-150. The authors should explain in which way they have treated the embryos. For example, have they treated the embryos in an immersion way of what?

    We have now added the requested information in the Materials and Methods section.

    Lines 159-160. Vanadates serve as structural mimics of phosphates. Thus it acts as a competitive inhibitor of ATPases, alkaline and acid phosphatases, and protein-phosphotyrosine phosphatases. The authors should explain the reason for the use of this chemical as a control.

    This chemical was not used as a control, but included in both the NRG-1 and control injections. This is now more clearly indicated in the Materials and Methods section. Pervanadate was added to the injections to ensure that pSTAT5 signal is not lost during sample preparation during which the larvae were kept in room temperature for 20 minutes after injection. The peak for pSTAT5 signal according to our previous research after NRG-1 stimulation is already at around 1-2 minutes and after 15 minutes the signal is already fading. We did attempt the experiment without the pervanadate and the results indeed were similar although the difference between the control and NRG-1 injections was less pronounced.

    Lines 179-181. The fish cells are partially autofluorescent. The authors did not use any system to remove the autofluorescence or, perhaps they lack to indicate in the text (i-e- pre-exposure under UV or Sudan black treatment).

    __While the authors agree on the potential contribution of autofluorescence on the background signal, this was not considered a significant confounding factor in the experimental conditions described in the manuscript. Indeed, the myosin heavy chain antibody gave a clear bright signal and the STAT5 stainings were validated with the loss of signal in the Stat5b targeting CRISPR/Cas9 treated zebrafish. __

    Results. This section should be rewritten in order by differentiating the data from zebrafish, murine and humans. For example, I can guess that the title on line 330 is referred to zebrafish, but it is not indicated in the title and the text.

    We have now more clearly separated the results from mice and zebrafish.

    Discussion. This section should be revised in light of the previous suggestion because not bring the reader to have a clear idea of the importance of this research. Thus, I suggest preparing a clear discussion on 1) who or what can stimulate the NRG1 release by endothelial cells (or also from other activated cells, i.e. endocardial); 2) if this release is similar in vertebrates studied models; 3) If the pathway studied is similar and when it is different. All these points should be documented by references. Moreover, the authors could correlate the manuscript with a draw that explains the signalling pathway that they suggest.

    We have now added the suggested information on NRG-1 release in the Introduction and added a new paragraph to the Discussion where we compare the reported differences of the NRG-1/ERBB4 pathway in mice and in zebrafish. In addition, we have included a new figure (Figure 7) that explains the signaling pathway, as recommended by the Reviewer.

    References:

    The molecular pathways that direct the process of reactivation of proliferation processes and hypertrophy are beginning to be elucidated with evidence that fibroblast growth factors, and microRNAs involvement that can be the starting time before NRG1 expression. Thus, in Mammals as well as fish the authors should read and mention some of the linked previous research (J Physiol 596.23 (2018) pp 5625-5640; Nat Med. 2007 May;13(5):613-8. doi: 10.1038/nm1582; Cardiovasc Res (2015) 107, 487-498 doi:10.1093/CVR/cvv190; Cell Death Discovery 4, 41 (2018) https://doi.org/10.1038/s41420-018-0041-x)

    We have added a new paragraph to the discussion section where we discuss the interplay of the NRG-1 signaling pathway with other pathways reported to induce cardiomyocyte growth.

    MINOR:

    References 4 and 5 are pre-print, please indicate the correct final reference

    We again apologize for the mistake. The references are now amended to refer to the correct articles published in Nature.

    Reference 14 resulted in an invalid URL address. The manuscript resulted non-existent

    __We apologize for the mistake. The URL had accidentally doubled in the reference. We have now amended the URL. __

    Reference 19 is incomplete

    Reference 20 not exhaustive is a personal communication, thus should be removed from here and reported in the text as "personal communication"

    Reference 27 is incomplete

    Reference 42 is incomplete

    Reference 69 is incomplete

    We apologize for the mistakes. We have amended the shortcomings in the reference section.

    Reviewer #1 (Significance):

    The Manuscript could be interesting for the readers, but need a deeper revision in the presentation and mainly in the Result-Discussion section.

    The Results and Discussion sections have now been revised as recommended by the Reviewer.

    After the revision, the manuscript will be of marked interest to cardiologists.

    The authors agree and wish to thank the Reviewer for valuable comments.

    Reviewer #2 (Evidence, reproducibility and clarity):

    RC-2022-01698 Review

    The authors report that NRG-1/ERBB4 signaling regulates activation of STAT5b and its target genes Igf1, Myc and Cdkn1a in murine cardiomyocytes, both in vitro and in vivo. STAT5b is a key activator in mediating NRG-1-induced cardiac hypertrophy in rodents. The NRG-1-ERRB4-STAT5 signaling axis is conserved in vertebrates since it regulates cardiomyocyte hyperplasia in zebrafish and is active in human hearts with pathological hypertrophy. Mechanistically, dynamin-2 was shown to control the cell surface localization of ERBB4 and its inhibition downregulates the NRG-1-ERRB4-STAT5 signaling pathway in hypertrophic and hyperplastic cardiomyocyte growth.

    The study is reasonably well conducted, experiments are controlled and quantified and most conclusions drawn by the authors are supported by their own data.

    This work is of high significance since it uncovers a mechanism responsible for cardiomyocyte hypertrophy which is perturbed in pathological cardiac hypertrophy. This finding opens the possibility that targeting STAT5 activation in patients with cardiomyopathy and heart failure might ameliorate the disease. It is of special note that NRG-1-ERRB4-STAT5 signaling promotes cardiomyocyte proliferation in zebrafish, a species known for its high cardiac repair capacity. This suggests the intriguing possibility that Stat5 could play an important role also in heart regeneration.

    A major shortcoming is the presentation of the scientific questions and what this paper is abou could be improved.

    As noted also above, the Introduction, Results and Discussion sections have now been revised as recommended by the Reviewers.

    Major comments:

    1. The use of the NRG1 scavenger should be validated. The provided reference #28 does not validate it. In the reference ____#28, it is reported that ERBB4 phosphorylation is downregulated with the scavenger in mice treated with AAV-VEGFB. The effect of the expression of the NRG-1 scavenger is detectable by western analysis in the mice treated with the AAV-VEGFB (Figure 6A,E) since the treatment induces prolonged activation of ERBB4 by upregulating the release and synthesis of ERBB4 ligands. Additionally, in a control pull-down experiment, the NRG-1 scavenger did bind NRG-1 from mouse serum (please see Rebuttal Figure 1).

    It is conceivable that direct manipulation of STAT5 might have effects independent from NRG-1/ERBB4 signaling and therefore might affect also hyperplasia in addition to hypertrophy. This study would benefit from additional experiments showing the proliferation rate (quantified with e.g. H3P or Aurora B) upon Stat5 knockdown or ERBB inhibition in murine cardiomyocytes.

    The proliferation rate of adult cardiomyocytes in uninjured models has been reported to be very low, less than 1% (Bergmann et al., 2009. Science. 324:98-102). For this reason we employed an immortalized dividing murine adult cardiomyocyte cell line, HL-1 Claycomb et al., 1998. Proc Natl Acad Sci U S A. 17:95:2979-84.), to address the question raised by the Reviewer. HL-1 cells were transduced with control and Stat5b-targeting shRNAs and cultured on 24-wells in the presence of NRG-1. The amount of HL-1 cells treated with Stat5b-targeting shRNAs was significantly smaller as compared to cells treated with control shRNA 72 hours after transduction (Rebuttal Figure 2A). However, it seems that increased cell death significantly contributed to the observed decrease in cell number as an increase of dead cells was observed in wells treated with Stat5b shRNAs when the cells were stained with a cell permeable (blue) and cell-impermeable (green) nuclear stain (Rebuttal Figure 2B-C). In accordance, the expression of the proliferation marker PCNA was unaltered by the Stat5b shRNA treatment in western analyses (Rebuttal Figure 2D). Thus, it seems that STAT5b does not control the proliferation rate but the viability of dividing adult mouse cardiomyocytes. The observation is not surprising since STAT5b has been reported to control the expression of the anti-apoptotic BCL2 and BCL-xL in adult cardiomyocytes (Chen et al. 2018. Cardiovasc. Res. 114:679-689).

    In lines 504-507, the authors write: "In accordance, the target genes of STAT5b IGF1 and MYC have been associated with hypertrophic and hyperplastic growth, respectively, implying that the STAT5b mediated hypertrophic growth may be mediated by the expression of MYC and the hyperplastic growth by the expression of IGF1". The authors cannot make this conclusion because both MYC and IGF1 are downregulated in murine cardiomyocytes. If hyperplastic growth is not regulated by NRG-1-ERRB4-STAT5 signaling in mice, then IGF1 expression should be unaffected by any manipulation of the pathway. It is suggested that the authors modify this statement to accurately reflect their data.

    We have decided to remove this conjecture from the discussion as suggested by the Reviewer.

    In lines 564-566, the authors write: "In accordance with clinical applicability, administration of NRG-1 and the protein product of the STAT5b target gene IGF1 has demonstrated success in attenuating dilated cardiomyopathy in clinical trials". STAT5 is already activated in pathological cardiac hypertrophy as shown in Figure 6C-D. How can the authors explain how administration of the STAT5 target gene IGF1, thus potentiating the effect of STAT5 activation, could reduce hypertrophy and thus ameliorate the disease?

    Left-ventricular hypertrophy is considered a compensatory response to increased pressure-overload and only if left unattended can lead to dilated cardiomyopathy. There is growing evidence that NRG-1/ERBB4 signaling is lost when the compensatory hypertrophic growth advances to dilated cardiomyopathy (Rohrbach et al., 2005. Basic Res. Cardiol. 100:240-9; Rohrbach et al., 1999. Circulation. 100:407-12.) which may suggest that the loss of the NRG-1/ERBB4 signaling is one of the factors that contributes to the progression of the disease. Therefore it would be feasible that the reactivation of the signaling pathway that was active during the compensatory response could ameliorate the disease. As we also indicate that the NRG-1/ERBB4/STAT5 pathway is involved in the proliferative growth of cardiomyocytes at embryonic stage in zebrafish, an organism known for its high cardiac regenerative capability (Poss et al., 2002. Science. 298:2188-90.), it is also feasible that the NRG-1/ERBB4/STAT5 pathway is involved in cardiac regeneration. Lastly, there is growing evidence that NRG-1, IGF-1 and STAT5b are also involved in cardiomyocyte survival (Mehrhof et al., 2001. Circulation. 104:2088-94; De Keulenaer et al., 2019. Circ. Heart Fail. 12:e006288; Chen et al., 2018. Cardiovasc. Res. 114:679-689) and therefore could alleviate the symptoms of dilated cardiomyopathy by compelling the cardiomyocytes more resistant to cell death. There are reports that suggest that cardiomyocyte apoptosis is increased and putatively even has a causal role in dilated cardiomyopathy (Yamamoto et al., 2003. J. Clin. Invest. 111:1463-74; Wencker et al., 2003. J. Clin. Invest. 111:1497-504.).

    Minor comments:

    1. Increase in cardiomyocyte numbers is better evidence for NRG-1-induced hyperplasia in zebrafish compared to increase in ventricle area. It is recommended that the authors swap Figure 3A-B with Supplementary Figure 3 in order to show more clearly the different role of NRG-1 in zebrafish compared to rodents. __We have now moved original Supplementary Figure 3 into new Figure 3A as recommended by the Reviewer. __

    The authors should report the p-value for all experiments including those considered not statistically significant.

    __The non-significant P-values have now been added to the figures. __

    Reviewer #2 (Significance):

    General significance of the reserach ms is high.

    The authors wish to thank the Reviewer for valuable comments.

    However, the ms is written and the study is conducted with little direction. Perhaps the authors could spend more effort on clearly explaining what the direction of their paper is.

    We have now reconstructed the manuscript and paid extra attention into explaining the direction and aim of the research.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The authors report that NRG-1/ERBB4 signaling regulates activation of STAT5b and its target genes Igf1, Myc and Cdkn1a in murine cardiomyocytes, both in vitro and in vivo. STAT5b is a key activator in mediating NRG-1-induced cardiac hypertrophy in rodents. The NRG-1-ERRB4-STAT5 signaling axis is conserved in vertebrates since it regulates cardiomyocyte hyperplasia in zebrafish and is active in human hearts with pathological hypertrophy. Mechanistically, dynamin-2 was shown to control the cell surface localization of ERBB4 and its inhibition downregulates the NRG-1-ERRB4-STAT5 signaling pathway in hypertrophic and hyperplastic cardiomyocyte growth.

    The study is reasonably well conducted, experiments are controlled and quantified and most conclusions drawn by the authors are supported by their own data.

    This work is of high significance since it uncovers a mechanism responsible for cardiomyocyte hypertrophy which is perturbed in pathological cardiac hypertrophy. This finding opens the possibility that targeting STAT5 activation in patients with cardiomyopathy and heart failure might ameliorate the disease. It is of special note that NRG-1-ERRB4-STAT5 signaling promotes cardiomyocyte proliferation in zebrafish, a species known for its high cardiac repair capacity. This suggests the intriguing possibility that Stat5 could play an important role also in heart regeneration.

    A major shortcoming is the presentation of the scientific questions and what this paper is abou could be improved. Major comments:

    1. The use of the NRG1 scavenger should be validated. The provided reference #28 does not validate it.
    2. It is conceivable that direct manipulation of STAT5 might have effects independent from NRG-1/ERBB4 signaling and therefore might affect also hyperplasia in addition to hypertrophy. This study would benefit from additional experiments showing the proliferation rate (quantified with e.g. H3P or Aurora B) upon Stat5 knockdown or ERBB inhibition in murine cardiomyocytes.
    3. In lines 504-507, the authors write: "In accordance, the target genes of STAT5b IGF1 and MYC have been associated with hypertrophic and hyperplastic growth, respectively, implying that the STAT5b mediated hypertrophic growth may be mediated by the expression of MYC and the hyperplastic growth by the expression of IGF1". The authors cannot make this conclusion because both MYC and IGF1 are downregulated in murine cardiomyocytes. If hyperplastic growth is not regulated by NRG-1-ERRB4-STAT5 signaling in mice, then IGF1 expression should be unaffected by any manipulation of the pathway. It is suggested that the authors modify this statement to accurately reflect their data.
    4. In lines 564-566, the authors write: "In accordance with clinical applicability, administration of NRG-1 and the protein product of the STAT5b target gene IGF1 has demonstrated success in attenuating dilated cardiomyopathy in clinical trials". STAT5 is already activated in pathological cardiac hypertrophy as shown in Figure 6C-D. How can the authors explain how administration of the STAT5 target gene IGF1, thus potentiating the effect of STAT5 activation, could reduce hypertrophy and thus ameliorate the disease?

    Minor comments:

    1. Increase in cardiomyocyte numbers is better evidence for NRG-1-induced hyperplasia in zebrafish compared to increase in ventricle area. It is recommended that the authors swap Figure 3A-B with Supplementary Figure 3 in order to show more clearly the different role of NRG-1 in zebrafish compared to rodents.
    2. The authors should report the p-value for all experiments including those considered not statistically significant.

    Significance

    General significance of the reserach ms is high. However, the ms is written and the study is conducted with little direction. Perhaps the authors could spend more effort on clearly explaining what the direction of their paper is.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The endothelial release of NRG (neuregulin)-1 is a paracrine growth factor activating ErbB (erythroblastic leukaemia viral oncogene) receptor tyrosine kinases on various targets cells epicardial, endocardial/endothelial (autocrine stimulation) and myocardiocytes. It is well known, after the manuscript of the K.D.Poss group that Neuregulin1 induced perivascular cells after injury to the adult zebrafish heart as well as mammalian cells. Inhibition of the Erbb2 co-receptor, disrupts cardiomyocyte proliferation in response to injury, whereas myocardial NRG1 overexpression enhances this proliferation. Thus, seems to be clear, that NRG-1 could stimulate regenerative, inflammatory, fibrotic, and metabolic processes In uninjured zebrafish, the reactivation of Nrg1 expression induces cardiomyocyte dedifferentiation, overt muscle hyperplasia, epicardial activation, increased vascularization, and causes cardiomegaly through persistent addition of wall myocardium (review cited by authors in this MS).

    In light of the large amount of these research deals with NRG1, the molecular mechanisms linked and focused to understand the multivariate effects of the NRG1 cascade are welcome. The authors have focused the manuscript on a comparative and translational demonstration of STAT5b involvement in the signal transduction of NRG1.

    Although the authors have done several experiments they have presented these in a chaotic way among mice, zebrafish and human biopsies. I can suggest rewriting partially the results section in a way more ordered and readable. Even the discussion is a little bit chaotic and lacks some aspects. For example, who is the stimulator of NRG1 release? Moreover, the literature cited is partially or not correctly reported (in the references section). In my opinion, the authors should revise the manuscript in the light of following suggestions. MAJOR: Title: line 1. The title does not explain the translational aspects of the manuscript. I suggest something like: "STAT5b is a key effector of NRG1/ERBB4-mediated cardiomyocyte growth: a translational approach".

    Abstract.

    Lines 20-25. The authors should rewrite this section by removing their previous finding ("we") reporting. In lines 25-36 the reported data is not well explained and is not clear who did that. The authors, previously? It is not well explained. Lines 35-37 Please, specify in which type of hypertrophic heart samples (is it from humans?) have been observed the NRG1 pathway.

    Introduction.

    Lines 44-46. All the cited papers consist of the preprint, thus they could be inserted in the discussion and not in the introduction. In the alternative, they could be inserted here following a sentence "Preliminary study seems to indicate...." Lines 50-51. The references cited are relative only to murine research and not to other animals. Thus, only murine models have been demonstrated. Thus, the assertion could be not valid for zebrafish or humans, thus the authors should point out this.

    Material and Methods.

    Lines 148-150. The authors should explain in which way they have treated the embryos. For example, have they treated the embryos in an immersion way of what? Lines 159-160. Vanadates serve as structural mimics of phosphates. Thus it acts as a competitive inhibitor of ATPases, alkaline and acid phosphatases, and protein-phosphotyrosine phosphatases. The authors should explain the reason for the use of this chemical as a control. Lines 179-181. The fish cells are partially autofluorescent. The authors did not use any system to remove the autofluorescence or, perhaps they lack to indicate in the text (i-e- pre-exposure under UV or Sudan black treatment).

    Results. This section should be rewritten in order by differentiating the data from zebrafish, murine and humans. For example, I can guess that the title on line 330 is referred to zebrafish, but it is not indicated in the title and the text.

    Discussion. This section should be revised in light of the previous suggestion because not bring the reader to have a clear idea of the importance of this research. Thus, I suggest preparing a clear discussion on 1) who or what can stimulate the NRG1 release by endothelial cells (or also from other activated cells, i.e. endocardial); 2) if this release is similar in vertebrates studied models; 3) If the pathway studied is similar and when it is different. All these points should be documented by references. Moreover, the authors could correlate the manuscript with a draw that explains the signalling pathway that they suggest.

    References:

    The molecular pathways that direct the process of reactivation of proliferation processes and hypertrophy are beginning to be elucidated with evidence that fibroblast growth factors, and microRNAs involvement that can be the starting time before NRG1 expression. Thus, in Mammals as well as fish the authors should read and mention some of the linked previous research (J Physiol 596.23 (2018) pp 5625-5640; Nat Med. 2007 May;13(5):613-8. doi: 10.1038/nm1582; Cardiovasc Res (2015) 107, 487-498 doi:10.1093/CVR/cvv190; Cell Death Discovery 4, 41 (2018) https://doi.org/10.1038/s41420-018-0041-x)

    Minor

    References 4 and 5 are pre-print, please indicate the correct final reference

    Reference 14 resulted in an invalid URL address. The manuscript resulted non-existent

    Reference 19 is incomplete

    Reference 20 not exhaustive is a personal communication, thus should be removed from here and reported in the text as "personal communication"

    Reference 27 is incomplete

    Reference 42 is incomplete

    Reference 69 is incomplete

    Significance

    The Manuscript could be interesting for the readers, but need a deeper revision in the presentation and mainly in the Result-Discussion section.

    After the revision, the manuscript will be of marked interest to cardiologists.

  4. Version published to 10.1101/2022.10.05.510958 on bioRxiv