CaMKII and Endothelial SK Channel Function in Diabetic Coronary Microcirculation

Summary/Abstract: we do not anticipate any changes as a result of this change of institution Patients with diabetes mellitus (DM) manifest coronary microvascular endothelial dysfunction, characterized by impaired endothelium-dependent relaxation. Impaired endothelium-dependent vasodilation decreases coronary blood flow and myocardial perfusion, resulting in cardiac dysfunction/diabetic cardiomyopathy, and myocardial ischemia with no obstructive arteries (INOCA), which increase morbidity and mortality in the diabetic population. Our recent studies indicate that dysfunction of small-conductance calcium-activated potassium channels (SK channels) play a key role in DM-induced endothelial dysfunction in animal and human coronary microcirculation. However, the mechanisms responsible for diabetic dysregulation of endothelial SK channels and coronary microvasculature remain undefined. Our pilot data indicate that DM causes excessive phosphorylation of CaMKII (p-CaMKII), O- GlcNAcylation of CaMKII (OG-CaMKII), and oxidation of CaMKII (ox-CaMKII), along with increased mitochondrial ROS (mROS) production in the heart and endothelial cells. Moreover, our preliminary studies demonstrate that chronic p-CaMKII, ox-CaMKII and O-GlcNAcylation during DM reduce endothelial SK channel activity, or coronary microvascular relaxation, and/or myocardial blow flow in both male and female mice. These results suggest that CaMKII posttranslational modification plays an important role in diabetic dysregulation of endothelial SK channels, coronary microvascular function and myocardial perfusion. Thus, the overall goal of this project is to investigate how CaMKII posttranslational modifications during chronic DM alter endothelial SK channel activity, coronary microvascular endothelial-dependent relaxation, and myocardial blow flow. Our central hypothesis is that sustained and excessive posttranslational modification of CaMKII during chronic DM dysregulates endothelial SK3/SK4(IK) channel activity, eNOS signaling, and endothelial function, resulting in coronary microvascular dysfunction and myocardial hypo-perfusion. We will test our hypothesis by employing multiple innovative approaches from cellular/molecular analyses and microvascular biology to physiology and pathophysiology. These approaches include type-1 and type-2 diabetic mouse models in combination with knock-in mouse models of oxidation-resistant CaMKII (MMVV), O-GlcNAcylation-resistant CaMKIIδ (S280) and a transgenic mouse model of endothelial cells expressing synthetic CaMKII inhibitory peptide (AC3-I) in both male and female animals, as well as endothelial ion channel recordings by patch clamp methods, measurement of microvascular reactivity in-vitro by a vessel myograph, contrast echocardiography to measure myocardial blood flow reserve in-vivo, immunoprecipitation (IP), LC/MS-MS, and site-directed mutagenesis, amongst other methods. The impact of gender differences on diabetes induced CaMKII posttranslational modification and related coronary microvascular dysfunction will also be analyzed. This integrated proposal will result in the identification of novel pathways, molecular targets, and novel therapeutics strategies for improving coronary microvascular function in diabetic patients with coronary heart disease.