Department of Pediatrics, St. Louis, MO, 63110, USA.
J Mol Cell Cardiol. 2012 Sep;53(3):437-45. doi: 10.1016/j.yjmcc.2012.07.001. Epub 2012 Jul 13.
Transgenic mice overexpressing SUR1 and gain of function Kir6.2[∆N30, K185Q] K(ATP) channel subunits, under cardiac α-myosin heavy chain (αMHC) promoter control, demonstrate arrhythmia susceptibility and premature death. Pregnant mice, crossed to carry double transgenic progeny, which harbor high levels of both overexpressed subunits, exhibit the most extreme phenotype and do not deliver any double transgenic pups. To explore the fetal lethality and embryonic phenotype that result from K(ATP) overexpression, wild type (WT) and K(ATP) overexpressing embryonic cardiomyocytes were isolated, cultured and voltage-clamped using whole cell and excised patch clamp techniques. Whole mount embryonic imaging, Hematoxylin and Eosin (H&E) and α smooth muscle actin (αSMA) immunostaining were used to assess anatomy, histology and cardiac development in K(ATP) overexpressing and WT embryos. Double transgenic embryos developed in utero heart failure and 100% embryonic lethality by 11.5 days post conception (dpc). K(ATP) currents were detectable in both WT and K(ATP)-overexpressing embryonic cardiomyocytes, starting at early stages of cardiac development (9.5 dpc). In contrast to adult cardiomyocytes, WT and K(ATP)-overexpressing embryonic cardiomyocytes exhibit basal and spontaneous K(ATP) current, implying that these channels may be open and active under physiological conditions. At 9.5 dpc, live double transgenic embryos demonstrated normal looping pattern, although all cardiac structures were collapsed, probably representing failed, non-contractile chambers. In conclusion, K(ATP) channels are present and active in embryonic myocytes, and overexpression causes in utero heart failure and results in embryonic lethality. These results suggest that the K(ATP) channel may have an important physiological role during early cardiac development.
在心脏α肌球蛋白重链(αMHC)启动子控制下过度表达 SUR1 和功能获得性 Kir6.2[∆N30,K185Q] K(ATP)通道亚单位的转基因小鼠表现出心律失常易感性和过早死亡。携带高表达亚单位的双转基因后代的怀孕小鼠杂交,表现出最极端的表型,并且不产生任何双转基因幼崽。为了探索 K(ATP)过度表达导致的胎儿致死性和胚胎表型,分离并培养野生型(WT)和 K(ATP)过度表达的胚胎心肌细胞,并用全细胞膜片钳和膜片钳技术进行电压钳位。全胚胎成像、苏木精和伊红(H&E)和α平滑肌肌动蛋白(αSMA)免疫染色用于评估 K(ATP)过度表达和 WT 胚胎的解剖、组织学和心脏发育。双转基因胚胎在子宫内发生心力衰竭,在受精后 11.5 天(dpc)时 100%胚胎致死。WT 和 K(ATP)过度表达的胚胎心肌细胞在心脏发育早期(9.5 dpc)即可检测到 K(ATP)电流。与成年心肌细胞不同,WT 和 K(ATP)过度表达的胚胎心肌细胞表现出基础和自发性 K(ATP)电流,这表明这些通道在生理条件下可能是开放和活跃的。在 9.5 dpc,活的双转基因胚胎表现出正常的环曲模式,尽管所有的心脏结构都塌陷,可能代表失败的、非收缩的腔室。总之,K(ATP)通道存在于胚胎心肌细胞中并具有活性,过度表达导致子宫内心力衰竭并导致胚胎致死。这些结果表明,K(ATP)通道在心脏早期发育过程中可能具有重要的生理作用。