SKca/IKca Channel Activation and Endothelial Protection During Cardiac Surgery

心脏手术期间 SKca/IKca 通道激活和内皮保护

• 批准号: 9919369
• 负责人: Jun Feng
• 金额: $ 39.15万
• 依托单位: RHODE ISLAND HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9919369
• 关键词: Affect; Animal Model; Animals; Apoptosis; Biochemistry; Blood Vessels; Calcium-Activated Potassium Channel; Cardiac Surgery procedures; Cardiovascular Diseases; Cardiovascular system; Cells; Complex; Coronary; Coronary Arteriosclerosis; Coronary artery; Data; Diabetes Mellitus; Diabetic Angiopathies; Diabetic mouse; Endothelial Cells; Endothelium; Event; Gene Proteins; Genes; Goals; Heart; Human; Impairment; Induced Heart Arrest; Investigation; Ischemia; Lead; Mediating; Membrane Potentials; Metabolic; Metabolism; Microcirculation; Mitochondria; Molecular; Molecular and Cellular Biology; Morbidity - disease rate; Mus; Myocardial Ischemia; Myocardium; NADH; NADPH Oxidase; Oxidative Stress; Pathogenesis; Patients; Pharmacology; Physiology; Play; Production; Protein Isoforms; Reactive Oxygen Species; Regulation; Relaxation; Reperfusion Injury; Reperfusion Therapy; Research; Role; Sampling; Sarcolemma; Signal Pathway; Signal Transduction; Testing; Tissues; Type 2 diabetic; Vasodilation; Vasomotor; arteriole; coronary vasculature; density; diabetic; diabetic patient; endothelial dysfunction; endothelium dependent hyperpolarization factor; experimental study; improved; inhibitor/antagonist; knock-down; mitochondrial dysfunction; mortality; mouse model; non-diabetic; novel therapeutics; overexpression; patch clamp; preservation; response

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.

内皮功能障碍在糖尿病血管病变的发病机制中起关键作用， 心血管事件。这些血管障碍可能会增加糖尿病患者的发病率和死亡率。内皮 糖尿病引起的功能障碍与小分子（SKCa）和中分子（IKCa）的代谢和失活有关。 电导钙激活钾通道在动物和人类冠状血管。然而，精确 糖尿病性SKCa/IKCa失活和冠状动脉内皮功能障碍的机制尚不清楚。 最近，我们证明细胞内NADH的升高导致内皮SKCa/IKCa的显著降低， 以及在糖尿病和缺血/再灌注（I/R）背景下SKCa/IKCa基因/蛋白丰度缺乏变化 表明这种效应是翻译后的。这个项目的目标是研究在一段时间内， 糖尿病负性调节动物/人内皮细胞的SKCa/IKCa通道和内皮功能， 动物/人冠状动脉微血管系统，并评估SKCa/IKCa激活和/或代谢调节是否保护 内皮细胞/血管对抗糖尿病和缺血性损伤。我们假设，持续过量的反应性 糖尿病期间通过NADPH氧化酶（Nox）、功能失调的线粒体和PKC产生的氧物质（ROS）将导致1） 内皮SKCa/IKCa失活，2）冠状动脉内皮功能/小动脉舒张受损;和3） 抑制Nox和mROS和/或PKC SKCa/IKCa过表达可能会增强SKCa/IKCa激活剂诱导的内皮细胞 保护内皮细胞/冠状动脉免受模拟心脏停搏液I/R损伤。使用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个特定目标来测试我们的假设。 目的1将研究细胞凋亡过程中NADH/Nox持续过表达/激活的分子机制。 糖尿病导致mROS和PKC过度产生/激活，导致SKCa/IKCa失活， 功能障碍/血管舒张受损，目标2将阐明mROS持续增加的机制 线粒体复合物是糖尿病SKCa/IKCa失活以及内皮功能和小动脉 血管舒张目的3将明确糖尿病持续性PKC激活的信号通路 改变SKCa/IKCa和冠状动脉内皮功能和微血管舒张。这些实验也将决定 如果PKC通过直接作用于通道复合物或通过引起通道内的蛋白质水平升高来介导其对SKCa/IKCa通道的作用， 与肌膜分离。目的4：检查Nox、mROS、PKC和/或PKC抑制剂的药理学抑制/基因敲低是否与Nox、mROS、PKC和/或PKC抑制剂的药理学抑制/基因敲低有关。 SKCa/IKCa过表达可能增强SKCa/IKCa激活剂诱导的内皮保护作用，对抗模拟的 心脏停搏I/R损伤。为了实现这些目标，将采用多种方法，例如膜片钳、分子生物学和分子生物学。 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心脏组织/血管/细胞样品。 目前的研究将导致新的治疗策略，以保护冠状动脉内皮功能和微血管 用于患有缺血性心脏病的糖尿病或非糖尿病患者在心脏手术期间的放松。