CaMKII and Endothelial SK Channel Function in Diabetic Coronary Microcirculation

CaMKII 和内皮 SK 通道在糖尿病冠状动脉微循环中的功能

• 批准号: 10930197
• 负责人: Jun Feng
• 金额: $ 53.33万
• 依托单位: RHODE ISLAND HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-22 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10930197
• 关键词: Acute; Affect; Animals; Arrhythmia; Binding Sites; Blood Vessels; Calcium; Calmodulin; Cardiac; Cardiac Myocytes; Chronic; Clinical; Coronary; Coronary heart disease; DNA Sequence Alteration; Data; Development; Diabetes Mellitus; Disease; Endothelial Cells; Endothelium; Functional disorder; Genetic; Goals; Heart; Heart Hypertrophy; Heart failure; Human; Hyperglycemia; Immunoprecipitation; Impairment; Insulin-Dependent Diabetes Mellitus; Ion Channel; Knock-in; Knock-in Mouse; Lead; Lentivirus; Link; Maps; Mass Spectrum Analysis; Microcirculation; Microvascular Dysfunction; Mitochondria; Molecular; Morbidity - disease rate; Mus; Mutation; Myocardial Infarction; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organ; Oxidative Stress; Pathogenesis; Pathologic; Patients; Peptides; Phosphorylation; Phosphorylation Inhibition; Phosphorylation Site; Phosphotransferases; Physiological; Play; Post-Translational Protein Processing; Potassium; Prevention; Production; Relaxation; Reporting; Research; Resistance; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Streptozocin; Testing; Tissues; Transgenic Mice; Vascular Endothelium; Vascular Endothelium-Dependent Relaxation; Vasodilation; Vasomotor; Work; calmodulin-dependent protein kinase II; coronary perfusion; density; diabetic; diabetic cardiomyopathy; diabetic patient; endothelial dysfunction; glycosylation; heart cell; heart function; improved; inhibitor; mortality; mouse model; mutant; novel; novel strategies; novel therapeutic intervention; overexpression; oxidation; pharmacologic; prevent

Endothelial dysfunction plays a key role in the pathogenesis of diabetic (DM) microvascular disease, increasing morbidity and mortality. Dysfunction of small conductance calcium-activated-potassium (SK) channels contributes strongly to DM-induced endothelial dysfunction in the coronary microcirculation. Emerging evidence has demonstrated that DM causes excessive phosphorylation of CaMKII (p-CaMKII), O-GlcNAcylation of CaMKII (OG- CaMKII), and/or oxidation of CaMKII (ox-CaMKII), along with enhanced mitochondrial ROS (mROS) production in the heart and endothelial cells (EC). However, the role of CaMKII posttranslational modifications in DM dysregulation of endothelial SK channels and coronary microvascular function remains largely undefined. Of note, we recently found that chronic activation/oxidation of CaMKII and O-GlcNAcylation during DM reduced endothelial SK channel activity and coronary microvascular relaxation, suggesting that CaMKII plays a key role in DM dysregulation of endothelial SK channels. Thus, the overall goal of this project is to investigate how CaMKII posttranslational modifications during chronic DM alters endothelial SK channel activity and coronary microvascular endothelial function. Our central hypothesis is that sustained/excessive p-CaMKII, OG-CaMKII, and ox-CaMKII during chronic DM dysregulates endothelial SK channel activity and endothelial function, resulting in coronary microvascular dysfunction. We will test our hypothesis by completing 4 specific aims: To investigate the molecular mechanisms by which DM-induced persistent p-CaMKII (Aim 1), OG-CaMKII (Aim 2) and ox-CaMKII (Aim 3) all lead to SK channel and coronary endothelial dysfunction, and to explore if inhibition/blockade of diabetic posttranslational modifications of CaMKII improves coronary microvascular relaxation (Aim 4). Aim 1 will study the effects of inhibiting p-CaMKII using a transgenic mouse model of endothelial cells expressing synthetic CaMKII inhibitory peptide (AC3-I) or CaMKII inhibitors, on endothelial SK channel activity in the setting of T1DM/T2DM; and also examine the effects of SK-channel mutation by using site-directed mutagenesis on CaMKII phosphorylation sites combined with LC/MS-MS. Aim 2 will test whether genetic mutation (CaMKIIδ S280 knock-in) or pharmacologic inhibition of OG-CaMKII with specific O-GlcNAc inhibitors affects endothelial SK current density, OG-CaMKII, and OG-CaMKII-SK interactions in T1DM and/or T2DM mice. Aim 3 will examine inhibition of ox- CaMKII using a knock-in mouse model of oxidation-resistant CaMKII (MM-VV) and SK-activator-induced relaxation in the presence of DM; and further determine whether chronic inhibition of mROS affects endothelial SK current density, ox-CaMKII, p-CaMKII and OG-CaMKII in DM mice. Aim 4 will investigate the effects of inhibition/blockade of CaMKII posttranslational modification during DM on coronary microvascular relaxation. This proposal will improve our understanding of DM heart/vessel disease by studying novel mechanisms by which CaMKII dysregulation affects endothelial SK channel function. Such work will lead to novel approaches for improving coronary microvascular function in DM patients with coronary heart disease.

内皮功能障碍在糖尿病（DM）微血管疾病的发病机理中起关键作用，增加 发病率和死亡率。小电导的功能障碍钙激活钾（SK）通道有助于 在冠状动脉微循环中，强烈对DM诱导的内皮功能障碍。新兴证据有 证明DM会导致CAMKII（P-CAMKII）的过度磷酸化，CAMKII的O-Glcnacylation（OG-- CAMKII）和/或CAMKII（OX-CAMKII）的氧化，以及增强的线粒体ROS（MROS）的产生 心脏和内皮细胞（EC）。但是，CAMKII翻译后修饰在DM中的作用 内皮SK通道和冠状动脉微血管功能的失调基本上不确定。值得注意的是， 我们最近发现，在DM减少内皮的过程中，CAMKII和O-Glcnacylation的慢性激活/氧化 SK通道活性和冠状动脉微血管松弛，表明CaMKII在DM中起关键作用 内皮SK通道的失调。这是该项目的总体目标是调查CAMKII 慢性DM期间的翻译后修饰改变了内皮SK通道活性和冠状动脉微血管 内皮功能。我们的中心假设是持续/过多的P-Camkii，OG-Camkii和Ox-Camkii 在慢性DM期间，内皮SK通道活性和内皮功能，导致冠状动脉 微血管功能障碍。我们将通过完成4个特定目的来检验我们的假设：研究分子 DM诱导持续的P-Camkii（AIM 1），OG-Camkii（AIM 2）和Ox-Camkii（AIM 3）的机制 导致SK通道和冠状动脉内皮功能障碍，并探讨糖尿病的抑制/阻滞 CAMKII的翻译后修饰可改善冠状动脉微血管松弛（AIM 4）。 AIM 1将研究 使用表达合成CAMKII的内皮细胞的转基因小鼠模型抑制P-CAMKII的效果 在T1DM/T2DM的情况下，在内皮SK通道活性上抑制性肽（AC3-I）或CAMKII抑制剂；和 还可以通过使用位置定向诱变对CAMKII磷酸化的SK通道突变的影响 站点与LC/MS-MS结合。 AIM 2将测试遗传突变（CAMKIUδS280敲门）还是 具有特定O-GLCNAC抑制剂的OG-CAMKII的药理学抑制会影响内皮SK电流密度， OG-CAMKII和T1DM和/或T2DM小鼠中的OG-Camkii-SK相互作用。 AIM 3将检查对牛的抑制作用 使用抗氧化抗氧化的CAMKII（MM-VV）和SK-激活剂诱导的CAMKII 在存在DM的情况下放松；并进一步确定MRO的慢性抑制是否会影响内皮SK DM小鼠中电流密度，OX-CAMKII，P-CAMKII和OG-CAMKII。 AIM 4将调查 冠状动脉微血管松弛时，DM期间CAMKII翻译后修饰的抑制/封锁。这 提案将通过研究新机制来提高我们对DM心脏/血管疾病的理解 CAMKII失调会影响内皮SK通道功能。这样的工作将导致新颖的方法 DM冠心病患者的冠状动脉微血管功能改善。