Mettl3-mediated m⁶A modification of Fgf16 restricts cardiomyocyte proliferation during heart regeneration.

Abstract

Cardiovascular disease is the leading cause of death worldwide due to the inability of adult heart to regenerate after injury. <i>N</i>⁶-methyladenosine (m⁶A) methylation catalyzed by the enzyme methyltransferase-like 3 (Mettl3) plays an important role in various physiological and pathological bioprocesses. However, the role of m⁶A in heart regeneration remains largely unclear. To study m⁶A function in heart regeneration, we modulated Mettl3 expression in vitro and in vivo. Knockdown of <i>Mettl3</i> significantly increased the proliferation of cardiomyocytes and accelerated heart regeneration following heart injury in neonatal and adult mice. However, <i>Mettl3</i> overexpression decreased cardiomyocyte proliferation and suppressed heart regeneration in postnatal mice. Conjoint analysis of methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq identified <i>Fgf16</i> as a downstream target of Mettl3-mediated m⁶A modification during postnatal heart regeneration. RIP-qPCR and luciferase reporter assays revealed that Mettl3 negatively regulates <i>Fgf16</i> mRNA expression in an m⁶A-Ythdf2-dependent manner. The silencing of <i>Fgf16</i> suppressed the proliferation of cardiomyocytes. However, the overexpression of ΔFgf16, in which the m⁶A 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 <i>Fgf16</i> mRNA levels through an m⁶A-Ythdf2-dependen pathway, thereby controlling cardiomyocyte proliferation and heart regeneration.