SKca/IKca Channel Activation and Endothelial Protection During Cardiac Surgery

内皮功能障碍在糖尿病血管病变的发病机制中起着关键作用，糖尿病血管病变易发生缺血 心血管事件。这些血管紊乱可能会增加糖尿病患者的发病率和死亡率。内皮细胞 糖尿病的功能障碍与小细胞(SKCa)和中细胞(IKCa)代谢改变和失活有关 动物和人冠状动脉血管中的电导钙激活钾通道。然而，准确的 糖尿病患者SKCa/IKCa失活和冠状动脉内皮细胞功能障碍的机制尚不清楚。 最近，我们证明了细胞内NADH的升高导致内皮细胞SKCa/IKCa显著降低， 糖尿病和缺血/再灌注(I/R)时SKCa/IKCa基因/蛋白丰度缺乏变化 这表明这种影响是后翻译的。这个项目的目标是研究新陈代谢是如何在 糖尿病对动物/人血管内皮细胞SKCa/IKCa通道及内皮功能的负性调节 动物/人冠状动脉微血管系统及评价SKCa/IKCa激活和/或代谢调节是否具有保护作用 抗糖尿病和缺血损伤的内皮细胞/血管。我们假设反应性的持续生产过剩 糖尿病期间通过NADPH氧化酶(NOx)、功能障碍的线粒体和PKC产生的氧物种(ROS)将导致1) 内皮细胞SKCa/IKCa失活，2)冠状动脉内皮功能受损/小动脉松弛；3) 抑制NOx和MRos和/或PKC SKCa/IKCa的过度表达可能增强SKCa/IKCa激活剂诱导的内皮细胞 内皮细胞/冠状动脉对模拟心脏停搏液I/R损伤的保护作用。使用一只2型糖尿病小鼠 模型和患者的心脏/血管/内皮细胞样本，我们将通过完成4个特定目标来验证我们的假设。 目的1研究NADH/NOx持续过度表达/激活的分子机制。 糖尿病导致MRO和PKC过度生产/激活，导致SKCa/IKCa失活，内皮 功能障碍/血管扩张受损，目标2将阐明MRO持续增加的机制 线粒体复合体是糖尿病患者SKCa/IKCa失活所必需的，也是内皮功能和微动脉失活所必需的 血管扩张。目标3将定义糖尿病期间持续的PKC激活的信号通路 改善SKCa/IKCa，以及冠脉内皮功能和微血管松弛。这些实验还将确定 如果PKC通过直接作用于通道复合体或通过引起通道，介导其对SKCa/IKCa通道的影响 与肌膜隔离。目的4：检测NOx、MROS、PKC和/或 SKCa/IKCa过表达可能增强SKCa/IKCa激活剂诱导的内皮保护作用 心脏停搏性I/R损伤。为了实现这些目标，将采用多种方法，如膜片钳、分子 以及细胞生物学、生物化学、血管生理学、糖尿病小鼠模型和人类心脏组织/血管/细胞样本。 目前的研究应该导致新的治疗策略来保护冠状动脉内皮功能和微血管。 心脏手术期间糖尿病或非糖尿病合并缺血性心脏病患者的放松。

Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease, which predisposes to ischemic cardiovascular events. These vascular disturbances may increase morbidity and mortality in diabetic patients. Endothelial dysfunction from diabetes is associated with altered metabolism and inactivation of small (SKCa) and intermediate (IKCa) conductance calcium-activated-potassium channels in the animal and human coronary vasculature. However, the precise mechanisms responsible for diabetic inactivation of SKCa/IKCa and coronary endothelial dysfunction are still undefined. Recently, we demonstrated that elevation in intracellular NADH results in a significant decrease in endothelial SKCa/IKCa, and the lack of changes in SKCa/IKCa gene/protein abundances in the setting of diabetes and ischemia/reperfusion (I/R) suggests that the effect is post-translational. The goal of this project is to investigate how metabolic changes during diabetes negatively regulate SKCa/IKCa channels of animal/human endothelial cells and endothelial function in the animal/human coronary microvasculature and to evaluate if SKCa/IKCa activation and/or metabolic modulation protect endothelial cells/vessels against diabetes and ischemic insults. We hypothesize that persistent overproduction of reactive oxygen species (ROS) via NADPH oxidase (Nox), dysfunctional mitochondria and PKC during diabetes will result in 1) inactivation of endothelial SKCa/IKCa, 2) impairment of coronary endothelial function/arteriolar relaxation; and that 3) inhibition of Nox and mROS and/or PKC SKCa/IKCa overexpression may potentiate SKCa/IKCa activator-induced endothelial protection of endothelial cells/coronary arterioles against a simulated cardioplegia I/R injury. Using a type-2 diabetic mice model and heart/vessels/endothelial cell samples from patients, we will test our hypothesis by completing 4 specific aims. Aim 1 will investigate the molecular mechanisms by which persistent over-expression/activation of NADH/Nox during diabetes results in mROS and PKC overproduction/activation, leading to SKCa/IKCa inactivation, endothelial dysfunction/impaired vasodilatation, Aim 2 will elucidate the mechanisms by which persistent increases in mROS from the mitochondrial complex are required for diabetic inactivation of SKCa/IKCa, and endothelial function and arteriolar vasodilatation. Aim 3 will define the signaling pathways by which persistent PKC activation during diabetes negatively modifies SKCa/IKCa, and coronary endothelial function and microvascular relaxation. These experiments will also determine if PKC mediates its effects on the SKCa/IKCa channel either by direct action on the channel complex or by causing channel isolation from the sarcolemma. Aim 4: To examine if pharmacologic inhibition/gene knockdown of Nox, mROS, PKC and/or SKCa/IKCa overexpression may potentiate SKCa/IKCa activator-induced endothelial protection against a simulated cardioplegic I/R injury. To achieve these goals, multiple approaches will be employed such as patch clamping, molecular and cellular biology, biochemistry, vascular physiology, diabetic mouse model and human heart tissue/vessel/cell samples. The present study should lead to novel therapeutic strategies to preserve coronary endothelial function and microvascular relaxation for diabetic or non-diabetic patients with ischemic heart disease during cardiac surgery.