A Role for Calcium Calmodulin-Dependent Protein Kinase II in the Early Development of Atherosclerosis

Atherosclerosis is the leading cause of cardiovascular death in the developed world. Endothelial dysfunction is an early event in the development of atherosclerosis and is characterised by increased reactive oxygen species (ROS), inflammation and a reduction in nitric oxide (NO) bioavailability. These changes culminate to drive early atherosclerotic lesion development. Endothelial nitric oxide synthase (eNOS) is the enzyme responsible for NO production in endothelial cells. During endothelial dysfunction, eNOS undergoes uncoupling where it is transformed from a NO to a superoxide (O2-)-producing enzyme. eNOS is regulated by phosphorylation at Ser1177 (P1177-eNOS) that drives accelerated activity. The presence of P1177-eNOS in the context of uncoupling augments O2- production exacerbating a highly oxidant environment. In physiological conditions, calcium/calmodulin-dependent protein kinase II (CaMKII) drives phosphorylation of eNOS at Ser1177 to promote NO production. In an oxidant environment, CaMKII becomes oxidised, causing inter-subunit auto- phosphorylation at threonine 287 (Thr287-CaMKII) leading to chronic activity. Chronically active ox-CaMKII has an established role in driving cardiac disease. An association between ox-CaMKII and uncoupled P1177-eNOS in the early development of atherosclerosis has not been explored. The aim of the study was to identify whether chronic activation of CaMKII plays a critical role in mediating early atherosclerotic lesion development. To address this ApoE-/- mice were subject to CaMKII inhibition from 16- to 20-weeks using KN-93 or the inactive analogue KN-92 as a control. The brachiocephalic artery underwent longitudinal sectioning and was used for lesion area and immunohistochemistry analysis. The relative levels of P1177-eNOS and the post-translationally modified ox- CaMKII and P287-CaMKII were measured. CaMKII inhibition led to a dramatic 10.9% reduction in foam cell lesion development throughout the brachiocephalic artery of ApoE-/- mice (P