Cardiovascular Mettl3 Deficiency Causes Congenital Cardiac Defects and Postnatal Lethality in Mice

N⁶-methyladenosine (m⁶A) is the most common epigenetic modification of RNA, but whether m⁶A RNA methylation modulates cardiovascular development or congenital heart diseases (CHDs) has not been determined. The published high-throughput sequencing data suggested that transcripts of genes related to CHDs were prone to m⁶A modification, while the expression of methyltransferase-like 3 (METTL3)-involved methyltransferase complex was downregulated in mouse embryonic hearts following prenatal alcohol exposure as a critical CHD risk factor, indicating the association of insufficient m⁶A RNA methylation with CHDs. Using cardiovascular-specific Mettl3 knockout mice (Tagln-Cre; Mettl3^flox/flox ), we observed that cardiovascular Mettl3 deficiency resulted in postnatal lethality and profound congenital cardiac defects, including left pulmonary stenosis, ventricular septal defects, and right ventricular hypoplasia. The m⁶A-specific methylated RNA-immunoprecipitation sequencing identified Sox4, Sox11, and Mef2a, the critical transcription factors involved in the right ventricle and outflow tract development, were the regulatory targets of METTL3-catalyzed m⁶A RNA methylation. Mettl3 deficiency-caused insufficient m⁶A RNA methylation downregulated the expression of SOX4, SOX11, and MEF2A in mouse embryonic hearts. In conclusion, cardiovascular Mettl3 deficiency directly led to congenital cardiac defects by downregulating the m⁶A-dependent expression of Mef2a, Sox4, and Sox11. METTL3-catalyzed m⁶A RNA methylation may become a potential target for preventing and treating CHDs.