Mettl3-mediated m6A modification of Fgf16 restricts cardiomyocyte proliferation during heart regeneration
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Evaluation Summary:
The manuscript identified m6A RNA methylation (via increased m6A writer, Mettl3 expression) as a critical regulator of cardiomyocyte proliferation during the initial regenerative window that was proposed earlier in the mouse heart. The authors have comprehensively profiled Mettl3 expression and Mettl3-dependent m6A regulation during cardiac regeneration using a variety of in vivo models as well as using in vitro primary cardiomyocytes to identify Fgf16 as a key downstream mRNA transcript for m6A RNA modification by Mettl3. Furthermore, they show that m6A-dependent cytoplasmic decay of Fgf16 mRNA in a Ythdf2-dependent pathway is the key underlying mechanism regulating cardiac regeneration in these models. In sum, a well-designed study with new data that shows suppression of a developmentally induced phenomenon as a therapeutic option for inducing cardiac proliferation.
(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 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Cardiovascular disease is the leading cause of death worldwide due to the inability of adult heart to regenerate after injury. N 6 -methyladenosine (m 6 A) methylation catalyzed by the enzyme methyltransferase-like 3 (Mettl3) plays an important role in various physiological and pathological bioprocesses. However, the role of m 6 A in heart regeneration remains largely unclear. To study m 6 A function in heart regeneration, we modulated Mettl3 expression in vitro and in vivo. Knockdown of Mettl3 significantly increased the proliferation of cardiomyocytes and accelerated heart regeneration following heart injury in neonatal and adult mice. However, Mettl3 overexpression decreased cardiomyocyte proliferation and suppressed heart regeneration in postnatal mice. Conjoint analysis of methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq identified Fgf16 as a downstream target of Mettl3-mediated m 6 A modification during postnatal heart regeneration. RIP-qPCR and luciferase reporter assays revealed that Mettl3 negatively regulates Fgf16 mRNA expression in an m 6 A-Ythdf2-dependent manner. The silencing of Fgf16 suppressed the proliferation of cardiomyocytes. However, the overexpression of ΔFgf16, in which the m 6 A consensus sequence was mutated, significantly increased cardiomyocyte proliferation and accelerated heart regeneration in postnatal mice compared with wild-type Fgf16. Our data demonstrate that Mettl3 post-transcriptionally reduces Fgf16 mRNA levels through an m 6 A-Ythdf2-dependen pathway, thereby controlling cardiomyocyte proliferation and heart regeneration.
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Evaluation Summary:
The manuscript identified m6A RNA methylation (via increased m6A writer, Mettl3 expression) as a critical regulator of cardiomyocyte proliferation during the initial regenerative window that was proposed earlier in the mouse heart. The authors have comprehensively profiled Mettl3 expression and Mettl3-dependent m6A regulation during cardiac regeneration using a variety of in vivo models as well as using in vitro primary cardiomyocytes to identify Fgf16 as a key downstream mRNA transcript for m6A RNA modification by Mettl3. Furthermore, they show that m6A-dependent cytoplasmic decay of Fgf16 mRNA in a Ythdf2-dependent pathway is the key underlying mechanism regulating cardiac regeneration in these models. In sum, a well-designed study with new data that shows suppression of a developmentally induced phenomenon as a …
Evaluation Summary:
The manuscript identified m6A RNA methylation (via increased m6A writer, Mettl3 expression) as a critical regulator of cardiomyocyte proliferation during the initial regenerative window that was proposed earlier in the mouse heart. The authors have comprehensively profiled Mettl3 expression and Mettl3-dependent m6A regulation during cardiac regeneration using a variety of in vivo models as well as using in vitro primary cardiomyocytes to identify Fgf16 as a key downstream mRNA transcript for m6A RNA modification by Mettl3. Furthermore, they show that m6A-dependent cytoplasmic decay of Fgf16 mRNA in a Ythdf2-dependent pathway is the key underlying mechanism regulating cardiac regeneration in these models. In sum, a well-designed study with new data that shows suppression of a developmentally induced phenomenon as a therapeutic option for inducing cardiac proliferation.
(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 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Accumulating evidence indicates RNA N6-adenosine methylation as an important post-transcriptional RNA modification in the regulation of gene expression, organ development, and disease development. However, the role of m6A mRNA methylation in cardiomyocyte proliferation and heart regeneration in normal development and in heart injury is not known. The authors first identified increased m6A mRNA levels during heart regeneration following injury to the neonatal heart, in association with selectively up-regulated expression of Mettl3, the methyltransferase catalyzing RNA N6-adenosine methylation, a finding suggesting a potential role of m6A in heart regeneration. Using cardiomyocyte cell lines, primary cardiomyocytes, and neonatal heart regeneration models, the authors next showed that down-regulation of Mettl3 …
Reviewer #1 (Public Review):
Accumulating evidence indicates RNA N6-adenosine methylation as an important post-transcriptional RNA modification in the regulation of gene expression, organ development, and disease development. However, the role of m6A mRNA methylation in cardiomyocyte proliferation and heart regeneration in normal development and in heart injury is not known. The authors first identified increased m6A mRNA levels during heart regeneration following injury to the neonatal heart, in association with selectively up-regulated expression of Mettl3, the methyltransferase catalyzing RNA N6-adenosine methylation, a finding suggesting a potential role of m6A in heart regeneration. Using cardiomyocyte cell lines, primary cardiomyocytes, and neonatal heart regeneration models, the authors next showed that down-regulation of Mettl3 markedly increased cardiomyocyte proliferation and heart regeneration in association with expected down-regulation of m6A mRNA levels in cardiomyocytes. This effect was selective in the injured neonatal heart. Together, the data indicate an important role of Mettl3 in the regulation of cardiomyocyte proliferation and heart regeneration. The quality of the data is high and the conclusions are convincing.
Next, the authors assessed the role of Mettl3 in heart regeneration and tissue repair at non-regenerative stages in postnatal mouse models and even in adult mice. Down-regulation of Mettl3 expression improved heart regeneration and tissue repair, in association with cardiomyocyte proliferation and improved cardiac functions. The role of Mettl3 was also assessed using Mettl3 overexpression and largely opposite effects relative to the effects of down-regulation of Mettl3 expression were detected. The authors attributed the effect of down-regulated Mettl3 expression to its impact on the regulation of Fgf16 expression. This is supported by the finding that Mettl3 down-regulation was associated with decreased Fgf16 mRNA m6A methylation and increased Fgf16 mRNA levels. Finally, the authors assessed the role of Fgf16 in heart regeneration by introducing the expression of wild type vs mutant Fgf16, with the latter having m6A consensus sequence deleted. The mutant Fgf16 increased heart regeneration in neonatal heart injury models, with increased cardiomyocyte proliferation and improved cardiac function. Overall, the authors have identified a novel mechanism for regulating cardiomyocyte proliferation and heart regeneration during heart injury. There is an impressive amount of rigorous data. It is a significant contribution to bring all these mechanisms together in the context of cardiomyocyte proliferation and heart regeneration. However, in some cases, the claims are probably overstated based on the data shown. Some of the findings are inconsistent with the interpretations. There are places where additional evidence is required in order to justify the claims.
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Reviewer #2 (Public Review):
The manuscript identified m6A RNA methylation (via increased m6A writer, Mettl3 expression) as a critical regulator of cardiomyocyte proliferation during the initial regenerative window that was proposed earlier in the mouse heart. Although these processes are developmentally induced, the results of the manuscript show Mettl3-dependent m6A RNA modifications as a negative regulator of cardiomyocyte proliferation and cardiac regeneration. The authors have comprehensively profiled Mettl3 expression and Mettl3-dependent m6A regulation during cardiac regeneration using a variety of in vivo models (both neonatal heart development and post-MI injury) as well as using in vitro primary cardiomyocytes to identify Fgf16 as a key downstream mRNA transcript for m6A RNA modification by Mettl3 to further show that …
Reviewer #2 (Public Review):
The manuscript identified m6A RNA methylation (via increased m6A writer, Mettl3 expression) as a critical regulator of cardiomyocyte proliferation during the initial regenerative window that was proposed earlier in the mouse heart. Although these processes are developmentally induced, the results of the manuscript show Mettl3-dependent m6A RNA modifications as a negative regulator of cardiomyocyte proliferation and cardiac regeneration. The authors have comprehensively profiled Mettl3 expression and Mettl3-dependent m6A regulation during cardiac regeneration using a variety of in vivo models (both neonatal heart development and post-MI injury) as well as using in vitro primary cardiomyocytes to identify Fgf16 as a key downstream mRNA transcript for m6A RNA modification by Mettl3 to further show that m6A-dependent cytoplasmic decay of Fgf16 mRNA in a Ythdf2-dependent pathway as the key underlying mechanism regulating cardiac regeneration in these models. Overall, a well-thought-out study that reports exciting new data that shows suppression of a developmentally induced phenomenon as a therapeutic option for inducing cardiac regeneration.
Strengths of the manuscript:
The manuscript investigates an important topic relevant to cardiac regeneration, which carries great clinical significance given that the cardiomyocyte turnover, as well as the processes for replacement of cardiomyocyte loss following MI injury, are limited. Therefore, any major discoveries to enhance the regenerative ability of the heart is critical to treating cardiac disease and this manuscript underscores a previously unrecognized mechanism (m6A modification) as a critical regulator of cardiomyocyte proliferation and cardiac regeneration.
The manuscript's major finding is the identification of developmental induction for Mettl3/m6A within the cardiac regenerative window (p1-p7). Another critical finding is that modulation of Mettl3 proves to be a negative regulator of cardiomyocyte proliferation and cardiac regeneration.
In the therapeutic setting, using post-MI mouse models, the manuscript further shows that targeting Mettl3 can enhance cardiac regeneration by specific effects on cardiomyocyte proliferation than on non-cardiomyocytes.
At the molecular level, the authors have carefully looked at Mettl3/m6a pathways in cardiomyocytes and non-cardiomyocyte proportions of the heart to show that Mettl3 is a critical mediator of cardiomyocyte hypertrophy as well as their proliferation in non-cardiomyocytes thus strengthening their conclusion that m6A process is being regulated in cardiomyocytes thus having a direct impact on cardiac regeneration.
Furthermore, the manuscript characterizes the downstream mRNA targets of Mettl3-mediated m6A modifications and found that Fgf16 mRNA is a critical target for m6A modification by Mettl3, which in turn leads to m6A-dependent degradation for Fgf16 via YTHDF2 pathway.
Taken together, the manuscript provides critical evidence demonstrating Mettl3-dependent m6A pathways regulating cardiomyocyte proliferation and heart regeneration.
Weaknesses of the manuscript:
It would be more appreciated if the manuscript can provide more insights and rationale by discussing why a developmentally induced Mettl3/m6A phenomenon (i.e. induced during normal developmental stages as well as post apical resection injury) would turn out to be a negative regulator of cardiac regeneration, than having a positive impact on cardiac regeneration.
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