雌激素调控心肌钠钙交换体的信号转导机制

The aim is designed to understand the mechanism of signal transduction on estrogen regulation of sodium-calcium exchanger (NCX) in cardiac myocytes.In long QT syndrom(LQT),adult women have higher risk to lethal arrhythmia, called Torsades de Pointes(TdP). A line of evidences suggest that estrogen affect female propensity to TdP seen in LQT.We showed in preliminary data that estrogen increased density of INCX and upregulated expression of NCX1 protein.Estrogen also blunted acute effect of β-adrenergic receptor on NCX. And estrogen incubation enhanced durg-induced TdP. However, how estrogen regulates NCX haven't been studied. In this study, we will identify estrogen receptors and characterize the potential singnaling pathways in estrogen regulation of NCX. Rat cardiomyocytes H9C2 cells and ovariectomized rat will be used in the study. Specific subtype of estrogen receptors will be silenced by RNAi technology. The transcriptional factors involved in this effect will be identified by luciferase reporter gene,the decoy oligonucleotide of transcriptional factor and trangenic animals. And INCX wil be measured by whole-cell patch clamp, and real-time RT-PCR and immunocytochemical study will be used to assess the expression of NCX1 . Elucidating how NCX is regulated by estrogen will not only improve our understanding on female (LQT) propensity to TdP, but also shed new light on future arrhythmia investigation and intervention.

本研究旨在研究雌激素调控心脏钠钙交换体的信号转导机制。在长QT综合征患者中，女性更易发生致死性的尖端扭转性室性心动过速。雌激素在其中起了重要作用。我们前期研究发现雌激素增加了心肌细胞INCX，上调了NCX1的表达。雌激素还降低了INCX对β-肾上腺素受体的反应性。更重要的是，雌激素促进了药物诱发的心律失常。但雌激素调控钠钙交换体的机制尚不清楚。本研究拟鉴别介导雌激素调控钠钙交换体的受体亚型，在转录水平及翻译后水平探索雌激素调控钠钙交换体的信号转导机制。采用大鼠心肌H9C2细胞及卵巢切除的大鼠，RNAi技术沉默特定亚型雌激素受体、荧光素酶报告基因,圈套寡核苷酸技术和转基因动物鉴定转录因子，膜片钳技术测定INCX、定量RT-PCR和westernblot技术测定NCX1表达水平。本研究不仅有助于揭示TdP易感性性别差异的机制，而且加深了离子通道调控机制的认识，为改善心律失常的治疗奠定基础。

在长QT综合征患者中，女性更易发生致死性的尖端扭转性室性心动过速。雌激素在其中起了重要作用。我们前期研究已经发现雌激素增加了心肌细胞INCX，上调了NCX1的表达。雌激素还降低了INCX对β-肾上腺素受体的反应性。更重要的是，雌激素促进了药物诱发的心律失常。但是雌激素调控钠钙交换体的机制尚不清楚。本研究进一步研究了雌激素受体亚型在雌激素上调NCX1表达中的作用，结果显示ERβ可能在雌激素上调NCX1表达及NCX电流密度中发挥了更大作用。我们解析了雌激素上调NCX1表达的关键信号通路，研究结果显示雌激素可能主要通过ERβ受体，激活MAPK/ERK通路，增加转录因子SRF的磷酸化水平，进而促进NCX1的表达。我们还对雌激素钝化NCX1对β-肾上腺素受体反应性的信号通路进行了探索，结果显示雌激素信号通路与β-肾上腺素受体信号通路有交互作用，且雌激素影响心肌细胞对β-肾上腺素受体激动剂异丙肾上腺素的反应性是通过快速信号通路发挥作用。此外，我们对雌激素性别差异性及部位差异性调节心脏NCX1表达有关的circRNAs进行了筛选和初步验证，结果提示circRNA_10418在其中可能发挥重要作用。本研究不仅有助于揭示TdP易感性性别差异的机制，而且加深了离子通道调控机制的认识，为改善心律失常的治疗奠定基础。