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.

The goal of the proposed research is to investigate how CaMKII posttranslational modifications during diabetes mellitus dysregulates the small conductance calcium-activated-potassium (SK) channels, coronary endothelial function and coronary microvascular reactivity. The present study will lead to novel therapeutic approaches for protecting coronary endothelium and coronary microcirculation in diabetic patients with coronary heart disease.