Fluorescence resonance energy transfer-based sensor camui provides new insight into mechanisms of calcium/calmodulin-dependent protein kinase II activation in intact cardiomyocytes

Jeffrey R. Erickson, Ruchi Patel, Amanda Ferguson, Julie Bossuyt, Donald M. Bers

Research output: Contribution to journalArticle

52 Scopus citations

Abstract

Rationale: 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. Methods and 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.

Original languageEnglish (US)
Pages (from-to)729-738
Number of pages10
JournalCirculation research
Volume109
Issue number7
DOIs
StatePublished - Sep 16 2011
Externally publishedYes

Keywords

  • C/calmodukin-dependent protein kinase II
  • CaMKII
  • FRET
  • biosensor
  • oxidative stress
  • phosphorylation
  • signaling pathways

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

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