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; 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疾病的发病机理中起关键作用，这使缺血性易感性 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 糖尿病的功能障碍与小（SKCA）和中间（IKCA）的新陈代谢改变和灭活有关 动物和人类冠状动脉脉管系统中的电导钙激活钾通道。但是，确切的 导致SKCA/IKCA糖尿病失活和冠状动脉内皮功能障碍的机制仍然不确定。 最近，我们证明了细胞内NADH的升高导致内皮SKCA/IKCA的显着降低， 在糖尿病和缺血/再灌注的情况下，SKCA/IKCA基因/蛋白质丰度缺乏变化（I/R） 表明这种影响是翻译后的。该项目的目的是调查代谢过程中的新陈代谢变化 糖尿病对动物/人类内皮细胞的SKCA/IKCA通道负调节 动物/人类冠状动脉微举行，并评估SKCA/IKCA激活和/或代谢调节是否保护 内皮细胞/血管针对糖尿病和缺血性损伤。我们假设反应性持续过度生产 通过NADPH氧化酶（NOX），糖尿病期间的线粒体和PKC功能障碍，氧气（ROS）将导致1） 内皮SKCA/IKCA的灭活，2）冠状动脉内皮功能/小动脉松弛的损害；那3） 抑制NOX和MRO和/或PKC SKCA/IKCA过表达可能会增强SKCA/IKCA激活剂引起的内皮 保护内皮细胞/冠状动脉动脉对模拟的心脏外痛I/R损伤。使用2型糖尿病小鼠 模型和心脏/血管/内皮细胞样品来自患者，我们将通过完成4个特定目标来检验我们的假设。 AIM 1将研究分子机制，通过该机制，nADH/NOX在此期间持续过表达/激活 糖尿病导致MROS和PKC过量/激活，导致SKCA/IKCA失活，内皮 功能障碍/血管扩张受损，AIM 2将阐明MROS持续增加MRO的机制 线粒体复合物是SKCA/IKCA糖尿病灭活所必需的，内皮功能和小动脉 血管舒张。 AIM 3将定义糖尿病期间持续性PKC激活的信号通路 修饰SKCA/IKCA，冠状动脉内皮功能和微血管松弛。这些实验还将确定 如果PKC通过直接在通道复合物上或引起通道对SKCA/IKCA通道的影响 从肌膜分离。目标4：检查NOX，MROS，PKC和/或的药理抑制/基因敲低是否 SKCA/IKCA过表达可能会增强SKCA/IKCA激活剂引起的内皮保护，以防止模拟 心脏局部I/R损伤。为了实现这些目标，将采用多种方法，例如贴片夹紧，分子 和细胞生物学，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/细胞样品。 本研究应导致新的治疗策略，以保持冠状动脉内皮功能和微血管 心脏手术期间患有缺血性心脏病的糖尿病或非糖尿病患者的放松。