Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, F. Edward Hebert School of Medicine, Bethesda, Maryland, United States of America.
PLoS One. 2012;7(7):e41533. doi: 10.1371/journal.pone.0041533. Epub 2012 Jul 23.
Two mammalian genes encode the SURx (SUR1, Abcc8 and SUR2, Abcc9) subunits that combine with Kir6.2 (Kcnj11) subunits to form the ATP-sensitive potassium (KATP) channel in cardiac myocytes. Different isoform combinations endow the channel with distinct physiological and pharmacological properties, and we have recently reported that the molecular composition of sarcolemmal KATP channels is chamber specific in the mouse heart. KATP channel composition is determined by what subunits are expressed in a cell or tissue. In the present study, we explore the role of CpG methylation in regulating SUR1 and SUR2 expression. In HL-1 cardiomyocytes, as in atrial myocytes, SUR1 expression is markedly greater than SUR2. Consistent with CpG methylation-dependent silencing of SUR2 expression, bisulfite sequencing of genomic DNA isolated from HL-1 cells demonstrates that 57.6% of the CpGs in the promoter region of the SUR2 gene are methylated, compared with 0.14% of the the CpG residues in the SUR1 sequence. Moreover, treatment with 10 µM 5-aza-2'-deoxycytidine (Aza-dC) significantly increased both the unmethylated fraction of the SUR2 CpG island and mRNA expression. However, we cannot rule out additional mechanisms of Aza-dC action, as Aza-dC also causes a decrease in SUR1 expression and lower doses of Aza-dC do not alter the unmethylated DNA fraction but do elicit a small increase in SUR2 expression. The conclusion that DNA methylation alone is not the only regulator of SUR subunit expression is also consistent with observations in native myocytes, where the CpG islands of both SUR genes are essentially unmethylated in both atrial and ventricular myocytes. Collectively, these data demonstrate the potential for CpG methylation to regulate SURx subunit expression and raises the possibility that regulated or aberrant CpG methylation might play a role in controlling channel structure and function under different physiological conditions or different species.
两种哺乳动物基因编码 SURx（SUR1、Abcc8 和 SUR2、Abcc9）亚基，这些亚基与 Kir6.2（Kcnj11）亚基结合形成心肌细胞中的 ATP 敏感性钾 (KATP) 通道。不同的同工型组合赋予通道不同的生理和药理学特性，我们最近报道说，在小鼠心脏中，质膜 KATP 通道的分子组成是室特异性的。KATP 通道的组成取决于细胞或组织中表达的亚基。在本研究中，我们探讨了 CpG 甲基化在调节 SUR1 和 SUR2 表达中的作用。在 HL-1 心肌细胞中，与心房肌细胞一样，SUR1 的表达明显大于 SUR2。与 SUR2 表达的 CpG 甲基化依赖性沉默一致，从 HL-1 细胞分离的基因组 DNA 的亚硫酸氢盐测序表明，SUR2 基因启动子区域的 57.6%的 CpG 发生甲基化，而 SUR1 序列中的 CpG 残基为 0.14%。此外，用 10µM 5-氮杂-2'-脱氧胞苷（Aza-dC）处理可显著增加 SUR2 CpG 岛的未甲基化部分和 mRNA 表达。然而，我们不能排除 Aza-dC 作用的其他机制，因为 Aza-dC 还会降低 SUR1 的表达，而较低剂量的 Aza-dC 不会改变未甲基化的 DNA 部分，但会引起 SUR2 表达的微小增加。CpG 甲基化不是 SUR 亚基表达的唯一调节剂的结论也与在天然肌细胞中的观察结果一致，在那里，两种 SUR 基因的 CpG 岛在心房和心室肌细胞中基本上都未甲基化。总之，这些数据表明 CpG 甲基化具有调节 SURx 亚基表达的潜力，并提出了受调控或异常 CpG 甲基化可能在不同生理条件或不同物种下控制通道结构和功能中发挥作用的可能性。
