CaMKII and Endothelial SK Channel Function in Diabetic Coronary Microcirculation

内皮功能障碍在糖尿病 (DM) 微血管疾病的发病机制中起着关键作用，增加了 发病率和死亡率。小电导钙激活钾 (SK) 通道功能障碍导致 对 DM 引起的冠状动脉微循环内皮功能障碍有强烈影响。新出现的证据表明 研究表明，DM 会导致 CaMKII 过度磷酸化 (p-CaMKII)、CaMKII 的 O-GlcNAc 酰化 (OG- CaMKII)，和/或 CaMKII (ox-CaMKII) 的氧化，以及线粒体 ROS (mROS) 产生的增强 心脏和内皮细胞（EC）。然而，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在很大程度上仍不清楚。值得注意的是， 我们最近发现，DM 期间 CaMKII 和 O-GlcNAcNAc 的慢性激活/氧化减少了内皮细胞 SK 通道活性和冠状动脉微血管舒张，表明 CaMKII 在 DM 中发挥关键作用 内皮 SK 通道失调。因此，该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 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 微血管功能障碍。我们将通过完成 4 个具体目标来检验我们的假设： DM 诱导持久性 p-CaMKII（目标 1）、OG-CaMKII（目标 2）和 ox-CaMKII（目标 3）的机制 导致 SK 通道和冠状动脉内皮功能障碍，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 使用表达合成 CaMKII 的内皮细胞转基因小鼠模型抑制 p-CaMKII 的效果 抑制肽 (AC3-I) 或 CaMKII 抑制剂，对 T1DM/T2DM 情况下内皮 SK 通道活性的影响；和 还通过定点诱变检查 SK 通道突变对 CaMKII 磷酸化的影响 位点与 LC/MS-MS 相结合。目标 2 将测试是否存在基因突变（CaMKIIδ S280 敲入）或 使用特定的 O-GlcNAc 抑制剂对 OG-CaMKII 进行药理学抑制会影响内皮 SK 电流密度， T1DM 和/或 T2DM 小鼠中 OG-CaMKII 和 OG-CaMKII-SK 相互作用。目标 3 将检查对 ox- 的抑制 CaMKII 使用抗氧化 CaMKII (MM-VV) 和 SK 激活剂诱导的敲入小鼠模型 DM 存在时的松弛；并进一步确定mROS的慢性抑制是否影响内皮SK DM 小鼠中的电流密度、ox-CaMKII、p-CaMKII 和 OG-CaMKII。目标 4 将调查以下因素的影响 DM 期间 CaMKII 翻译后修饰的抑制/阻断对冠状动脉微血管舒张的影响。这个 该提案将通过研究新的机制来提高我们对 DM 心脏/血管疾病的理解 CaMKII 失调影响内皮 SK 通道功能。此类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.