目的 探讨甲基化转移酶样蛋白3(methyltransferase-like protein 3,METTL3)介导的 N-6 甲基腺苷(N6-methylade-nosine,m6A)修饰对高静水压下心房肌细胞L型钙通道的调控作用.方法 C57BL/6J小鼠随机分为对照组和高血压组(血管紧张素持续给药4周).采用Masson染色观察小鼠心房组织纤维化情况,斑点印迹实验检测小鼠心房组织中m6A,Western blot 检测 METTL3 和 Cav1.2 表达情况.体外培养的小鼠心房细胞株HL-1细胞,予加压构建高静水压模型及干预METTL3,观察细胞中m6A表达含量、METTL3和Cav1.2水平的改变,全细胞膜片钳检测HL-1动作电位时程(action potential duration,APD)及 L 型钙电流(L-type calcium current,ICa L).结果 与对照组相比,高血压组小鼠心房肌细胞形态紊乱,间质纤维化增加,且心房组织中m6A含量及METTL3表达水平也明显增加,离子通道蛋白Cav1.2表达减少.体外培养的HL-1细胞,施予不同静水压(0、20、40 mmHg)干预后,随着静水压的升高,细胞中m6A、METTL3上调,Cav1.2下调.STM2457(特异性METTL3抑制剂)和si-METTL3可逆转上述变化,同时STM2457逆转高静水压所致的HL-1细胞的APD缩短和ICa,L峰值密度降低,METTL3可直接与CACNA1C(Cav1.2 mRNA)发生结合.结论 高静水压下METTL3介导的m6A修饰可直接调控CACNA1C参与心房肌细胞电重塑.
Aim To investigate the regulation of N6-methyladenosine(m6A)modification on L-type calci-um channels in atrial myocytes under high hydrostatic pressure,mediated by methyltransferase-like protein 3(METTL3).Methods C57BL/6J mice were ran-domly assigned to the control group and the hyperten-sion group(treated with continuous administration of angiotensin for four weeks).Masson staining was used to observe the fibrosis of mouse atrial tissue,while dot blot assay and Western blot were used to detect the lev-els of m6A,METTL3,and Cav1.2 in the atrial tissue.A high hydrostatic pressure model was constructed u-sing the HL-1 cell line cultured in vitro,and METTL3 was intervened to observe changes in m6A expression levels,METTL3 and Cav1.2 levels in cells,and action potential duration(APD)and L-type calcium current(ICa,L)were detected using whole-cell patch clamp.Results Compared to the control group,the hyperten-sion group showed disordered atrial myocyte morpholo-gy,increased interstitial fibrosis,significant increased m6A content and METTL3 expression levels in the atri-al tissue,and decreased expression of ion channel pro-tein Cav1.2.In HL-1 cells cultured in vitro,increasing hydrostatic pressure(0,20,40 mmHg)up-regulated m6 A and METTL3 expression levels,down-regulated Cav1.2,and these changes were reversed with STM2457 and si-METTL3.Furthermore,specific MET-TL3 inhibitor STM2457 reversed the shortening of APD and the decrease of ICa L peak density in HL-1 cells caused by high hydrostatic pressure,while METTL3 di-rectly bound to CACNA1C.Conclusion METTL3-mediated m6A modification might directly regulate CACNA1C to participate in electrical remodeling of at-rial myocytes under high hydrostatic pressure.