CaMKII regulates intracellular Ca²⁺ dynamics in native endothelial cells

Localized endothelial Ca(2+) signalling, such as Ca(2+) pulsars, can modulate the contractile state of the underlying vascular smooth muscle cell through specific endothelial targets. In addition to K(Ca)3.1 as a target, Ca(2+) pulsars, an IP3R-dependent pulsatile Ca(2+) release from the endoplasmic reticulum (ER) could activate a frequency-sensitive Ca(2+)-dependent kinase such as CaMKII. In the absence of extracellular Ca(2+), acetylcholine increased endothelial CaMKII phosphorylation and activation, thereby suggesting CaMKII activation independently of Ca(2+) influx. Herein, a reciprocal relation where CaMKII controls endothelial Ca(2+) dynamics has been investigated in mesenteric arteries. Both CaMKIIα and β isoforms have been identified in endothelial cells and close proximity (<40 nm) suggests their association in heteromultimers. Intracellular Ca(2+) monitoring with high speed confocal microscopy then showed that inhibition of CaMKII with KN-93 significantly increased the population of Ca(2+) pulsars active sites (+89%), suggesting CaMKII as a major regulator of Ca(2+) pulsars in native endothelium. Mechanistic insights were then sought through the elucidation of the impact of CaMKII on ER Ca(2+) store. ER Ca(2+) emptying was accelerated by CaMKII inhibition and ER Ca(2+) content was assessed using ionomycin. Exposure to KN-93 strongly diminished ER Ca(2+) content (-61%) by relieving CaMKII-dependent inhibition of IP3 receptors (IP3R). Moreover, in situ proximity ligation assay suggested CaMKII-IP3R promiscuity, essential condition for a protein-protein interaction. Interestingly, segregation of IP3R within myoendothelial projection (MEP) appears to be isoform-specific. Hence, only IP3R type 1 and type 2 are detected within fenestrations of the internal elastic lamina, sites of MEP, whilst type 3 is absent from these structures. In summary, CaMKII seems to act as a Ca(2+)-sensitive switch of a negative feedback loop regulating endothelial Ca(2+) homeostasis, including Ca(2+) pulsars.