FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Sprenger, Julia; Perera, Ruwan K.; Götz, Konrad R.; Nikolaev, Viacheslav O.

doi:10.3791/4081-v

Cited by 4 publications

(1 citation statement)

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“…Adult mouse ventricular cardiomyocytes were isolated by enzymatic digestion during Langendorff perfusion, plated on laminin-coated round glass coverslides, and used for Förster resonance energy transfer (FRET) or Ca 2+ measurements as previously described. 4,17,18 Human ventricular myocytes were isolated as previously described 19 from surgical samples derived from nonfailing hearts and patients with heart failure with reduced ejection fraction (Table I in the Data Supplement) at the time of transplantation, valvular surgeries, or myectomy, followed by adenoviral transduction to express Epac1-JNC biosensor for 40 to 48 hours. All samples were taken with informed consent of the donors.…”

Section: Methodsmentioning

confidence: 99%

cAMP Imaging at Ryanodine Receptors Reveals β ₂ -Adrenoceptor Driven Arrhythmias

Berisha

Götz

Wegener

et al. 2021

Circulation Research

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Rationale: 3',5'-cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which, upon β-adrenergic receptor (β-AR) stimulation, acts in microdomains to regulate cardiac excitation-contraction coupling by activating phosphorylation of calcium handling proteins. One crucial microdomain is in vicinity of the cardiac ryanodine receptor type 2 (RyR2) which is associated with arrhythmogenic diastolic calcium leak from the sarcoplasmic reticulum (SR) often occurring in heart failure. Objective: We sought to establish a real time live cell imaging approach capable of directly visualizing cAMP in the vicinity of mouse and human RyR2 and to analyze its pathological changes in failing cardiomyocytes under β-AR stimulation. Methods and Results: We generated a novel targeted fluorescent biosensor Epac1-JNC for RyR2-associated cAMP and expressed it in transgenic mouse hearts as well in human ventricular myocytes using adenoviral gene transfer. In healthy cardiomyocytes, β 1 -AR but not β 2 -AR stimulation strongly increased local RyR2-associated cAMP levels. However, already in cardiac hypertrophy induced by aortic banding, there was a marked subcellular redistribution of phosphodiesterases (PDEs) 2, 3 and 4, which included a dramatic loss of the local pool of PDE4. This was also accompanied by measurableβ2-AR/AMP signals in the vicinity of RyR2 in failing mouse and human myocytes, increased β2-AR-dependent RyR2 phosphorylation, SR calcium leak and arrhythmia susceptibility. Conclusions: Our new imaging approach could visualize cAMP levels in the direct vicinity of cardiac RyR2. Unexpectedly, in mouse and human failing myocytes, it could uncover functionally relevant local arrhythmogenic β2-AR/cAMP signals which might be an interesting antiarrhythmic target for heart failure.

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Section: Methodsmentioning

confidence: 99%

cAMP Imaging at Ryanodine Receptors Reveals β ₂ -Adrenoceptor Driven Arrhythmias

Berisha

Götz

Wegener

et al. 2021

Circulation Research

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Regulation of basal and norepinephrine-induced cAMP and ICa in hiPSC-cardiomyocytes: Effects of culture conditions and comparison to adult human atrial cardiomyocytes

Iqbal

Ismaili

Dolce

et al. 2021

Cellular Signalling

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Live-cell imaging identifies cAMP microdomains regulating β-adrenoceptor-subtype-specific lipolytic responses in human white adipocytes

Jong¹,

Ehret²,

Heeren³

et al. 2023

Cell Reports

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FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Cited by 4 publications

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cAMP Imaging at Ryanodine Receptors Reveals β ₂ -Adrenoceptor Driven Arrhythmias

cAMP Imaging at Ryanodine Receptors Reveals β ₂ -Adrenoceptor Driven Arrhythmias

Regulation of basal and norepinephrine-induced cAMP and ICa in hiPSC-cardiomyocytes: Effects of culture conditions and comparison to adult human atrial cardiomyocytes

Live-cell imaging identifies cAMP microdomains regulating β-adrenoceptor-subtype-specific lipolytic responses in human white adipocytes

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FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Cited by 4 publications

References 0 publications

cAMP Imaging at Ryanodine Receptors Reveals β 2 -Adrenoceptor Driven Arrhythmias

cAMP Imaging at Ryanodine Receptors Reveals β 2 -Adrenoceptor Driven Arrhythmias

Regulation of basal and norepinephrine-induced cAMP and ICa in hiPSC-cardiomyocytes: Effects of culture conditions and comparison to adult human atrial cardiomyocytes

Live-cell imaging identifies cAMP microdomains regulating β-adrenoceptor-subtype-specific lipolytic responses in human white adipocytes

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Product

Resources

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cAMP Imaging at Ryanodine Receptors Reveals β ₂ -Adrenoceptor Driven Arrhythmias

cAMP Imaging at Ryanodine Receptors Reveals β ₂ -Adrenoceptor Driven Arrhythmias