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  1. Evaluation Summary:

    The authors identify a novel developmental role for the beta-adrenergic system in the regulation of mammalian cardiac regenerative capacity. Using genetic and pharmacological loss-of-function approaches, the authors identify a link between Yap and β-adrenergic receptor blockade. The conditional genetic loss-of-function studies are a particular strength of the manuscript and provide strong support for the Gas/Yap-dependent nature of the cardiomyocyte proliferative response to beta adrenergic blockade. Given the widespread use of beta blockers in the clinical management of heart failure, the findings are potentially very important. However, further evidence is required to substantiate the induction of bona fide cardiomyocyte proliferation and cardiac regeneration and clarify the associated mechanisms.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    The current study by Sakabe et al identifies an adrenergic signaling mechanism controlling cardiac regenerative capacity in mice. Using pharmacological and genetic loss-of-function studies, the authors demonstrate that inhibition of beta adrenergic signaling prolongs the cardiac regenerative window in neonatal mice. The study mechanistically connects several signaling pathways that are known to control cardiomyocyte proliferation including adrenergic signaling, G-proteins and the Hippo/Yap pathway. The results are potentially clinically significant given the widespread use of beta blockers in heart failure management.

    This is an impressive body of work that addresses an important and largely unresolved question in the field regarding signaling mechanisms controlling cardiac regeneration in the postnatal period in mammals. Through pharmacological and conditional genetic loss-of-function studies the authors provide several lines of evidence implicating the beta adrenergic signaling and the Hippo/Yap pathway in cardiomyocyte proliferation. The conditional genetic loss-of-function studies are a particular strength of the manuscript and provide strong support for the Gas/Yap-dependent nature of the cardiomyocyte proliferative response to beta adrenergic blockade.

    Although the study clearly implicates beta adrenergic signaling in the developmental regulation of cardiomyocyte proliferative potential, it is unclear whether the protective effects observed following myocardial infarction are due to cardiac regeneration or alternative mechanisms (e.g. immunomodulation, inhibition of cell death, angiogenesis, reduced contractile loading, improved coronary flow, etc). Induction of cardiomyocyte proliferation following administration of metoprolol in neonatal mice is fairly modest (~0.3% pH3-positive cardiomyocytes) and it seems unlikely that such a small number of proliferating cardiomyocytes could mediate such marked effects on cardiac function and fibrosis post-MI. In the absence of definitive data demonstrating that improvements in cardiac function are due to induction of cardiomyocyte proliferation (and by inference cardiac regeneration), such conclusions should be tempered. In addition, it is unclear why beta blocker studies were not conducted in adult mice (rather than P7/P14 mice) to determine whether inhibition of this pathway is sufficient to induce adult cardiomyocyte cell cycle re-entry and regeneration post-MI.

  3. Reviewer #2 (Public Review):

    The manuscript by Sabake and colleagues reports that a commonly used beta blocker drug (metoprolol) induces cardiomyocyte proliferation and, when administered after myocardial infarction, cardiac regeneration. The authors propose that this drug leads to the activation of YAP, a master regulator of cardiomyocyte proliferation. This work is not aligned with common clinical evidence, namely that patients treated with beta blockage after myocardial infarction still do not undergo clinically appreciable regeneration as measured objectively by cardiac magnetic resonance. This is despite the therapeutic usefulness of beta blockage for cardiac function.

    My main concern with this manuscript is that its striking conclusions are not sufficiently supported by the data presented. In particular, the evidence for cardiomyocyte replication is not convincing. The immunostaining for phospho-H3 in Figs 1e and 1f shows relatively limited cell proliferation to justify the claim for cardiac regeneration, while, most important, it is unclear whether the putatively proliferating cells are cardiomyocytes (at least in the images provided). For regeneration to be plausible, there should be data on cardiomyocytes entering the S phase and, most important, undergoing cytokinesis, which is not provided here. Similar considerations also apply to the Gnas KO mice (Fig. 3) and to the Ki67 stainings in Suppl. Fig. 1.

    The claim that YAP is regulated downstream of the betaAR is essentially based on the finding of a YAP gene transcriptional signature (Fig. 4). However, there is consolidated evidence that YAP is also activated during the hypertrophy response. Thus, several of the data presented are equally consistent with the possibility of a compensatory hypertrophic program being elicited by the treatments. In this respect, the authors report that Gnas KO mice do not show increased cross-sectional area. This is quite surprising, as the mechanisms for hypertrophy can also be different from those ensuing from the betaAR.

    The canonical mechanism for YAP activation foresees decreased proteasomal degradation consequent to its reduced phosphorylation on specific residues, followed by its nuclear translocation. Nuclear translocation of the factor, which is essential for its activation, is not shown here biochemically. On the contrary, the authors show that there is nuclear YAP in basal conditions by immunofluorescence, while this drops to virtually null after epinephrine treatment (bar chart in Suppl. Fig. 5f). Incidentally, the over 30% nuclear positivity shown in the graph in basal conditions is difficult to reconcile with the lack of nuclear staining in Suppl. Fig. 5b.

  4. Reviewer #3 (Public Review):

    The authors study mammalian heart regeneration and study the connection between Yap and β-adrenergic receptor (β-AR) blockade. Interestingly, metoprolol robustly enhanced cardiomyocyte proliferation and promoted cardiac regeneration post myocardial infarction, resulting in reduced scar formation and improved cardiac function. The conclusion was also supported by genetic deletion of Gnas. CMs had an immature cell state with enhanced activity of Hippo-effector YAP. They also find that increased YAP activity is modulated by RhoA.

    Overall, the data are supportive of the conclusions and this may provide new insight into treating heart disease. The final mechanisms connecting Hippo signaling to Rho activity remain incompletely defined.