METTL3 deficiency resulted in accelerated decay of calcium transients in atrial myocytes. (A) Expression of METTL3 mRNA was determined by RT–qPCR in NMAMs infected with knockdown lentivirus. Data are represent as mean ± SD. Sh-Ctrl (n = 6), sh-Mettl3 (n = 4). **P < 0.01, comparisons made using unpaired Student’s t-test. (B) Expression of METTL3 protein was determined by western blotting in NMAMs infected with knockdown lentivirus. Data are represented as mean ± SD. Sh-Ctrl (n = 3), sh-Mettl3 (n = 3). **P < 0.01, comparisons made using unpaired Student’s t-test. (C) N6-methyladenosine modification levels were detected by dot blot assay in METTL3 knockdown NMAMs. (D) Calcium transients were measured in METTL3 knockdown NMAMs using confocal microscopy. Data are represent as mean ± SD. Sh-Ctrl (n = 42 cells from three technical replicates), sh-Mettl3 (n = 48 cells from three technical replicates). **P < 0.01, ****P < 0.0001, comparisons made using unpaired Student’s t-test. (E) Calcium transients in AMAMs were detected by the confocal microscopy. Data are represented as mean ± SD. WT (n = 15 cells from three mice), Mettl3+/− (n = 11 cells from three mice). **P < 0.01, comparisons made using non-parametric Mann–Whitney test (for amplitude) and unpaired Student’s t-test (for decay time). AMAMs, adult mice atrial myocytes; m6A, N6-methyladenosine; MB, methylene blue staining; METTL3, methyltransferase like 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NMAMs, neonatal mice atrial myocytes; RT–qPCR, reverse transcriptase–quantitative polymerase chain reaction; SD, standard deviation; WT, wild type.
