CaMKII regulates intracellular Ca2+ dynamics in native endothelial cells

Abstract

Localized endothelial Ca2+ signalling, such as Ca2+ pulsars, can modulate the contractile state of the underlying vascular smooth muscle cell through specific endothelial targets. In addition to KCa3.1 as a target, Ca2+ pulsars, an IP3R-dependent pulsatile Ca2+ release from the endoplasmic reticulum (ER) could activate a frequency-sensitive Ca2+-dependent kinase such as CaMKII. In the absence of extracellular Ca2+, acetylcholine increased endothelial CaMKII phosphorylation and activation, thereby suggesting CaMKII activation independently of Ca2+ influx. Herein, a reciprocal relation where CaMKII controls endothelial Ca2+ dynamics has been investigated in mesenteric arteries. Both CaMKIIα and β isoforms have been identified in endothelial cells and close proximity (<40nm) suggests their association in heteromultimers. Intracellular Ca2+ monitoring with high speed confocal microscopy then showed that inhibition of CaMKII with KN-93 significantly increased the population of Ca2+ pulsars active sites (+89%), suggesting CaMKII as a major regulator of Ca2+ pulsars in native endothelium. Mechanistic insights were then sought through the elucidation of the impact of CaMKII on ER Ca2+ store. ER Ca2+ emptying was accelerated by CaMKII inhibition and ER Ca2+ content was assessed using ionomycin. Exposure to KN-93 strongly diminished ER Ca2+ 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 Ca2+-sensitive switch of a negative feedback loop regulating endothelial Ca2+ homeostasis, including Ca2+ pulsars.