Fluorescence Resonance Energy Transfer–Based Sensor Camui Provides New Insight Into Mechanisms of Calcium/Calmodulin-Dependent Protein Kinase II Activation in Intact Cardiomyocytes

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 Here 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 which lack the sites of CaMKII autophosphorylation (T286A) and oxidative regulation (CM280/1VV) serve as useful biosensors for CaMKIIδ activation state. Camui (WT 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 amplitude Ca transients. Camui was also activated by four neurohormonal agonists. Angiotensin II and endothelin-1 activated Camui, largely via an oxidation-dependent mechanism, while isoproterenol and phenylephrine mediated mechanisms had a significant autophosphorylation-dependent component. Conclusions Camui is a novel, non-destructive tool that allows spatio-temporally 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.

S-Nitrosylation Induces Both Autonomous Activation and Inhibition of Calcium/Calmodulin-dependent Protein Kinase II δ

“…NO Induces Autonomous CaMKII␦ Activation-Camui, our FRET-based CaMKII activation state sensor, assesses changes in CaMKII conformation associated with direct or autonomous activation of the kinase (21). Therefore, the activation state of CaMKII can be assessed dynamically in real time for intact cells expressing Camui.…”

“…Each replicate was from a separate HEK cell lysate, and data at each time point were averaged over replicates (n ϭ 6) to provide error bars on these traces. Decreases of Camui FRET (increases of F CFP /F YFP ) were con-sidered to be indicative of the CaMKII conformational or activation state (21).…”

“…Generation of the Camui Constructs and Sequence Alignment-The wild-type Camui␦C construct was generated as described previously (21). In brief, mutagenesis of the CaMKII␦C sequence was done to introduce a BglII site adjacent to the start codon and to replace the stop codon with a SalI site.…”

Selective Modulation of Coupled Ryanodine Receptors During Microdomain Activation of Calcium/Calmodulin-Dependent Kinase II in the Dyadic Cleft

“…16 More recently, an in situ fluorescence resonance energy transfer reporter has been used to follow the activation of CaMKII during stimulation. 17 The process takes tens of seconds to build up and also has a slow recovery. Modeling and experimental data have shown how the N-terminus of calmodulin is thought to serve as a low-affinity Ca 2+ sensor, 18 transducing Ca 2+ in the dyad, allowing activation in response to the high Ca 2+ local to LTCC and RyR in the dyad in a frequency-dependent manner.…”

Adrenergic Signaling Regulates Mitochondrial Ca2+ Uptake Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca2+ Uniporter

“…Recently, G q PCR stimulation, including via a 1 -AR, was shown to increase ROS in cardiomyocytes that activate ROS-sensitive downstream signaling processes (e.g., Pyk2 and CaMKII) (21,27). However, the detailed mechanism by which a 1 -ARS induces oxidative stress is unclear.…”