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着关键作用， 发病率和死亡率。小电导钙激活钾(SK)通道功能障碍 对糖尿病所致的冠脉微循环内皮功能障碍有强烈的保护作用。新出现的证据表明 研究表明，DM导致CaMKII(p-CaMKII)过度磷酸化，O-GlcN酰化CaMKII(OG- CaMKII)，和/或CaMKII(OX-CaMKII)的氧化，以及线粒体ROS(MROS)的增加 心脏和内皮细胞(EC)。然而，CaMKII翻译后修饰在糖尿病中的作用 内皮SK通道和冠脉微血管功能的失调在很大程度上仍未明确。值得注意的是， 我们最近发现，糖尿病过程中CaMKII和O-GlcN酰化的慢性激活/氧化减少了内皮细胞 SK通道活性与冠脉微血管松弛，提示CaMKII在糖尿病发病中起关键作用 血管内皮细胞SK通道调节失调。因此，这个项目的总体目标是调查CaMKII是如何 慢性糖尿病时的翻译后修饰改变内皮细胞SK通道活性和冠脉微血管 内皮功能。我们的中心假设是持续/过量的p-CaMKII、OG-CaMKII和OX-CaMKII 慢性糖尿病时血管内皮细胞SK通道活性和内皮功能紊乱，导致冠状动脉 微血管功能障碍。我们将通过完成四个具体目标来验证我们的假设：研究分子 DM诱导持续性p-CaMKII(Aim 1)、OG-CaMKII(Aim 2)和OX-CaMKII(Aim 3)的机制 导致SK通道和冠脉内皮功能障碍，并探讨是否抑制/阻断糖尿病 CaMKII的翻译后修饰可改善冠状动脉微血管松弛(目标4)。目标1将研究 利用表达合成CaMKII的转基因小鼠模型抑制p-CaMKII的作用 抑制肽(AC3-I)或CaMKII抑制剂对T1 DM/T2 DM时内皮细胞SK通道活性的影响； 用定点突变技术检测SK通道突变对CaMKII磷酸化的影响 与LC/MS-MS联用。AIM 2将检测基因突变(CaMKIIδS280敲入)或 特异性O-GlcNAc抑制剂对OG-CaMKII的药理抑制作用影响内皮SK电流密度， OG-CaMKII和OG-CaMKII-SK在T1 DM和/或T2 DM小鼠中的相互作用。目标3将研究OX-的抑制作用- 使用抗氧化CaMKII(MM-VV)和SK-激活剂诱导的敲入小鼠模型的CaMKII DM存在时的松弛；并进一步确定长期抑制MRO是否影响内皮细胞SK 糖尿病小鼠的电流密度、OX-CaMKII、p-CaMKII和OG-CaMKII。目标4将调查 糖尿病时CaMKII翻译后修饰对冠脉微血管松弛的抑制/阻断作用这 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调节失调影响内皮细胞SK通道功能。这样的工作将带来新的方法 改善糖尿病合并冠心病患者的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.