title: Fluorescence resonance energy transfer-based sensor Camui provides new insight into mechanisms of calcium/calmodulin-dependent protein kinase II activation in intact cardiomyocytes.
abstract: BACKGROUND
  Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of intracellular signaling in the heart. However, the tools currently available for assessing dynamic changes in CaMKII localization and activation in living myocytes are limited.
  
  
  OBJECTIVE
  We use Camui, a novel FRET-based biosensor in which full-length CaMKII is flanked by CFP and YFP, to measure CaMKII activation state in living rabbit myocytes.
  
  
  RESULTS
  We show that Camui and mutant variants that lack the sites of CaMKII autophosphorylation (T286A) and oxidative regulation (CM280/1VV) serve as useful biosensors for CaMKIIδ activation state. Camui (wild-type or mutant) was expressed in isolated adult cardiac myocytes, and localization and CaMKII activation state were determined using confocal microscopy. Camui, like CaMKIIδ, is concentrated at the z-lines, with low baseline activation state. Camui activation increased directly with pacing frequency, but the maximal effect was blunted with the T286A, consistent with frequency-dependent phosphorylation of CaMKII at T286 mainly at high-frequency and high-amplitude Ca transients. Camui was also activated by 4 neurohormonal agonists. Angiotensin II and endothelin-1 activated Camui, largely through an oxidation-dependent mechanism, whereas isoproterenol- and phenylephrine-mediated mechanisms had a significant autophosphorylation-dependent component.
  
  
  CONCLUSIONS
  Camui is a novel, nondestructive tool that allows spatiotemporally resolved measurement of CaMKII activation state in physiologically functioning myocytes. This represents a first step in using Camui to elucidate key mechanistic details of CaMKII signaling in live hearts and myocytes.
authors.affiliation: Department of Pharmacology, University of California, Davis, CA 95616-8636, USA.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Erickson
authors.forename: Ruchi
authors.initials: R
authors.lastname: Patel
authors.forename: Amanda
authors.initials: A
authors.lastname: Ferguson
authors.forename: Julie
authors.initials: J
authors.lastname: Bossuyt
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D015374:Biosensing Techniques; D020013:Calcium Signaling; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002304:Cardiac Pacing, Artificial; D019332:Endothelin-1 / Q000378:metabolism; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D010084:Oxidation-Reduction; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D000316:Adrenergic alpha-Agonists; D000318:Adrenergic beta-Agonists; D019332:Endothelin-1; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000804:Angiotensin II; D010656:Phenylephrine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D007545:Isoproterenol