METTL3 deletion promoted CaCl₂-ACh-induced AF and spontaneous AF in CREM mice. (A) RT–qPCR was used to detect the expression of METTL3 mRNA in the atrium of Mettl3^+/− mice. Data are presented as mean ± SD. WT (n = 4), Mettl3^+/− (n = 6). **P < 0.01, comparison made using unpaired Student’s t-test. (B) Western blotting was used to detect expression of METTL3 protein in the atrium of Mettl3^+/− mice. Data are presented as mean ± SD. WT (n = 3); Mettl3^+/− (n = 3). ***P < 0.001, comparison made using unpaired Student’s t-test. (C) Duration of induced AF in each group of mice following CaCl₂-ACh injection (left). The representative ECG of the limb leads from each group of mice (right). WT; control (n = 5): WT mice injected with saline, Mettl3^+/−; control (n = 5): Mettl3^+/− mice injected with saline, WT; AF (n = 6): WT mice injected with CaCl₂-ACh, Mettl3^+/−; AF (n = 3): Mettl3^+/− mice injected with CaCl₂-ACh. Data are presented as mean ± SD. **P < 0.01, ****P < 0.0001, comparison made using two-way ANOVA. (D) Schematic diagram illustrating the process of specific METTL3 knockout in atrial myocytes of CREM mice. Parental Mettl3^flox/flox mice were crossed with CREM mice to generate Mettl3^flox/+:CREM mice, which were then bred with Mettl3^flox/flox mice to obtain Mettl3^flox/flox:CREM mice. These mice were then injected with AAV9-Anf-Cre virus to specifically knock out METTL3 in atrial myocytes. The Mettl3^flox/flox:CREM mice injected with AAV9-Anf-Zsgreen virus served as controls. (E) Expression of METTL3 protein in the atrium, ventricle, and liver of Mettl3^flox/flox:CREM mice was evaluated by western blotting assay 4 weeks after AAV9-Anf virus injection. Data are represent as mean ± SD. AAV9-Anf-Zsgreen (n = 3), AAV9-Anf-Cre (n = 3). ***P < 0.001, comparisons made using unpaired Student’s t-test. (F) Statistics of spontaneous AF in Mettl3^flox/flox:CREM mice 4 weeks after AAV9-Anf virus injection (left). Schematic diagram of limb lead ECG (right). Data represent the number of mice with or without sAF. AAV9-Anf-Zsgreen (n = 10), AAV9-Anf-Cre (n = 9). *P < 0.05, comparison made using the Fisher’s test. AF, atrial fibrillation; ANOVA, analysis of variance; CREM, cAMP-responsive element modulator; ECG, electrocardiogram; METTL3, methyltransferase like 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, not significant; sAF, spontaneous atrial fibrillation; SD, standard deviation; WT, wild-type.