title: CaMKII regulates intracellular Ca²⁺ dynamics in native endothelial cells.
abstract: 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.
authors.affiliation: Research Center, Montreal Heart Institute, Montréal, Québec, Canada; Department of Physiology, Université de Montréal, Québec, Canada.
authors.forename: Fanny
authors.initials: F
authors.lastname: Toussaint
authors.affiliation: Research Center, Montreal Heart Institute, Montréal, Québec, Canada; Department of Pharmacology, Université de Montréal, Québec, Canada.
authors.forename: Chimène
authors.initials: C
authors.lastname: Charbel
authors.affiliation: Research Center, Montreal Heart Institute, Montréal, Québec, Canada.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Blanchette
authors.affiliation: Research Center, Montreal Heart Institute, Montréal, Québec, Canada; Department of Physiology, Université de Montréal, Québec, Canada; Department of Pharmacology, Université de Montréal, Québec, Canada; Department of Medicine, Université de Montréal, Québec, Canada. Electronic address: jonathan.ledoux@umontreal.ca.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Ledoux
mesh_terms: D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000502:physiology*; D042783:Endothelial Cells / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D008638:Mesenteric Arteries / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D000096926:Benzenesulfonamides
chemical_list: D001596:Benzylamines; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D015544:Inositol 1,4,5-Trisphosphate; D053496:Inositol 1,4,5-Trisphosphate Receptors; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; C072105:KN 93; D000096926:Benzenesulfonamides
keywords: CaMKII; Calcium; Calcium pulsars; IP(3)R; Vascular endothelium