Uterine Arterial Spontaneous Transient Outward Currents in Pregnancy

项目摘要 妊娠期子宫血流显著增加的子宫血管适应性变化对子宫内膜异位症的发生至关重要。 胎儿发育以及母亲的心血管健康。虽然血管舒张是 子宫循环对妊娠的适应至关重要，但其潜在机制并不 完全理解自发瞬时外向电流（STOCs）在生理膜电位（EMPs） 30-40 mV）从根本上调节血管肌源性张力和血流 以及动脉压。我们对绵羊的初步研究表明， 显著增加子宫动脉平滑肌细胞中的STOCs，确定了一个令人兴奋的调节靶点， 在妊娠期子宫血管适应中的作用。STOC主要由大电导Ca ~（2+）- 激活K（BKca）通道。血管平滑肌中的BKca通道含有通道形成蛋白， + 亚基和最多四个辅助β1亚基，通过ryanodine的开放感测Ca 2+火花 受体。我们以前的研究表明，BKβ1亚基表达显著上调， 导致妊娠动物子宫动脉中β1：β 2亚基化学计量增加。这是由于 子宫动脉中BKβ1亚基基因启动子甲基化降低。动态调节 DNA甲基化和去甲基化是基因表达表观遗传修饰的主要机制 模式和生物功能。最近的研究表明，10 - 11的强大机制 易位1-3（TET 1 -3）蛋白通过将5-甲基胞嘧啶（5 mC）转化为 5-羟甲基胞嘧啶（5 hmC）。我们的初步研究表明怀孕和类固醇激素 子宫内膜BKβ1启动子区TET 1和TET 2表达及5 hmC丰度显著增加 动脉，提示子宫内膜异位症介导的主动DNA去甲基化的一种非常新的机制， 血管对妊娠的适应此外，初步数据表明，怀孕导致 子宫动脉中Ryanodine受体介导的Ca 2+火花显著增加。有了这些令人兴奋的 调查结果和许多非常新颖的线索，我们现在定位于推动该领域向前发展， 通过启动一个新的研究重点，旨在了解分子和表观遗传的方式， 在子宫动脉血管舒张中具有根本重要性的STOCs调节机制 在怀孕期间因此，本研究将验证妊娠上调BKβ1的假设 通过TET介导的DNA去甲基化亚基表达并增加ryanodine受体诱导的Ca 2 + 火花，导致子宫血管适应妊娠的STOC升高。的结果 拟议的研究将提供新的见解，子宫胎盘适应的分子机制， 怀孕，并将有重大影响，在我们的理解病理生理机制的基础上， 妊娠并发症，包括先兆子痫和胎儿生长受限，由适应不良引起的 子宫胎盘循环考虑到STOC在调节血管张力方面至关重要， 几乎所有血管床的压力，揭示了在血管平滑肌细胞中STOCs功能的表观遗传调节。 肌肉确实将在全面了解分子生物学方面产生更广泛的影响， 血管生理学和病理生理学机制。

PROJECT SUMMARY Uterine vascular adaptation with the striking increase of uterine blood flow during gestation is essential for fetal development as well as for cardiovascular well-being of the mother. Although vasodilatation is of critical importance in the adaptation of uterine circulation to pregnancy, the underlying mechanisms are not fully understood. Spontaneous transient outward currents (STOCs) at physiological membrane potentials ( 30-40 mV) of vascular smooth muscle cells fundamentally regulate vascular myogenic tone and blood flow in an organ, as well as arterial pressure. Our preliminary studies in sheep revealed that pregnancy significantly increased STOCs in uterine arterial smooth muscle cells, identifing an exciting regulatory target in uterine vascular adaptation in gestation. STOCs are mainly mediated by large-conductance Ca2+- activated K (BKca) channels. The BKca channel in vascular smooth muscle contains the channel-forming  + subunit and up to four accessory β1 subunits that sense Ca2+ sparks via the opening of ryanodine receptors. Our previous studies demonstrated a significant upregulation of BKβ1 subunit expression, resulting in increased β1: subunit stoichiometry in uterine arteries of pregnant animals. This resulted from a decrease in promoter methylation of the BKβ1 subunit gene in uterine arteries. Dynamic regulation of DNA methylation and demethylation is a chief mechanism in epigenetic modifications of gene expression pattern and biological function. Recent studies have suggested a robust mechanism of ten-eleven translocation 1-3 (TET1-3) proteins in active DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Our preliminary studies showed that pregnancy and steroid hormones significantly increased TET1 and TET2 expression and 5hmC abundance at the BKβ1 promoter in uterine arteries, suggesting a highly novel mechanism of hormone-mediated active DNA demethylation in uterine vascular adaptation to pregnancy. In addition, the preliminary data suggested that pregnancy resulted in a significant increase in Ca2+ sparks mediated by ryanodine receptors in uterine arteries. With these exciting findings and many highly novel leads, we are now positioned to move the field forward significantly in a manner by launching a new focus of research aiming at the understanding of molecular and epigenetic mechanisms in the regulation of STOCs that are of fundamental importance in uterine arterial vasodilatation during gestation. Thus, the proposed study will test the hypothesis that pregnancy upregulates BKβ1 subunit expression via TET-mediated DNA demethylation and increases ryanodine receptor-induced Ca2+ sparks, resulting in heightened STOCs in uterine vascular adaptation to pregnancy. The outcome of proposed study will provide novel insights in molecular mechanisms of uteroplacental adaptation to pregnancy, and will have a major impact in our understanding of pathophysiologic mechanisms underlying pregnancy complications including preeclampsia and fetal growth restriction, caused by maladaptation of uteroplacental circulation. Given that STOCs are fundamentally important in regulating vascular tone and pressure in virtually all vascular beds, revealing epigenetic regulation of STOCs function in vascular smooth muscle will indeed have a much broader impact in the comprehensive understanding of molecular mechanisms in vascular physiology and pathophysiology.