CaMKII and Endothelial SK Channel Function in Diabetic Coronary Microcirculation

内皮功能障碍在糖尿病（DM）微血管病变的发病机制中起关键作用， 发病率和死亡率。小电导钙激活钾（SK）通道功能障碍有助于 DM诱导的冠状动脉微循环内皮功能障碍。新出现的证据表明， 表明DM引起CaMKII的过度磷酸化（p-CaMKII）、CaMKII的O-GlcNAc酰化（OG- CaMKII）和/或CaMKII氧化（ox-CaMKII），沿着增强的线粒体ROS（mROS）产生。 心脏和内皮细胞（EC）。然而，CaMK II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仍然在很大程度上不明确。值得注意的是， 我们最近发现，糖尿病期间CaMKII和O-GlcNAc酰化的慢性活化/氧化降低了内皮细胞 SK通道活性和冠状动脉微血管舒张，表明CaMKII在DM中起关键作用 内皮SK通道的失调。因此，本项目的总体目标是研究CaMKII如何 慢性DM期间的翻译后修饰改变内皮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/过量的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失调，导致冠状动脉粥样硬化， 微血管功能障碍我们将通过完成4个具体目标来测试我们的假设： DM诱导的持久性p-CaMKII（Aim 1）、OG-CaMKII（Aim 2）和ox-CaMKII（Aim 3）的机制均 导致SK通道和冠状动脉内皮功能障碍，并探讨抑制/阻断糖尿病 CaMKII的翻译后修饰改善冠状动脉微血管舒张（Aim 4）。目标1将研究 使用表达合成CaMK Ⅱ的内皮细胞转基因小鼠模型抑制p-CaMK Ⅱ的作用 抑制肽（AC 3-I）或CaMKII抑制剂对T1 DM/T2 DM背景下内皮SK通道活性的影响;和 还通过使用定点突变对CaMKII磷酸化的影响来检查SK通道突变 目的2将检测基因突变（CaMK II δ S280敲入）或 用特异性O-GlcNAc抑制剂对OG-CaMKII的药理学抑制影响内皮SK电流密度， 在T1 DM和/或T2 DM小鼠中的OG-CaMK II和OG-CaMK II-SK相互作用。目的3将检查对ox的抑制作用。 使用抗氧化CaMKII抑制剂（MM-VV）和SK激活剂诱导的CaMKII抑制剂的敲入小鼠模型， 以及进一步确定mROS的慢性抑制是否影响内皮SK DM小鼠中的电流密度、ox-CaMK II、p-CaMK II和OG-CaMK II。目标4将研究 糖尿病期间CaMKII翻译后修饰对冠状动脉微血管舒张的抑制/阻断。这 该提案将通过研究新的机制来提高我们对糖尿病心脏/血管疾病的理解， CaMKII失调影响内皮SK通道功能。这些工作将导致新的方法， 改善糖尿病合并冠心病患者冠状动脉微血管功能

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.