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

Abstract

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.

Abnormal Calcium Handling in Atrial Fibrillation Is Linked to Changes in Cyclic AMP Dependent Signaling

“…FRET measurements were performed on human atrial myocytes 48 h after transduction with the adenovirus encoding Epac1-camps [ 18 ], pm-Epac1 [ 19 ] and Epac1-JNC [ 20 ]. A K + -Ringer solution containing: 144 mM NaCl, 5.4 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 10 mM HEPES, adjusted to 7.4 pH with NaOH at room temperature (RT) was used to maintain cells, and images were taken every 5 s. The FRET system used consisted of a standard inverted microscope (Leica DMI3000, Leica Biosystems, Wetzlar, Germany) with a 63×/1.4 oil immersion objective, an LED light source (pE-100, CoolLED), a beam splitter (DV2, Photometrics, Birmingham, UK), and a camera (CMOS camera optiMOS, Photometrics, Birmingham, UK).…”

Aminophylline Induces Two Types of Arrhythmic Events in Human Pluripotent Stem Cell–Derived Cardiomyocytes

“…As is well-known, the accumulation of Ca 2+ in the cytoplasm may result in delayed afterdepolarizations, which can trigger arrhythmia. Considering the increased occurrence of Ca 2+ sparks in HL-1 cardiomyocytes treated with 512 µM aminophylline ( Figure 6 ), the concentration-independent “stochastic” effect of aminophylline observed in our study may be a consequence of microdomain cAMP level–related diastolic release of Ca 2+ via independent clusters of RyR2 receptors ( Berisha et al, 2021 ). Such transient Ca 2+ accumulation may induce an increased BR and, following Ca 2+ depletion, results in a pause in the investigated cardiac cell’s beating clusters.…”

Exploration of Mechanisms of Sacubitril/Valsartan in the Treatment of Cardiac Arrhythmias Using a Network Pharmacology Approach

“…The ADRB2 gene encodes the β2-adrenergic receptor. Researchers have found that ADRB2 strongly increases localized RyR2-related cAMP levels during the over-activation of the SNS associated with cardiac hypertrophy or HF and increases ventricular arrhythmias ( 30 ). The ADRB2 also increases infiltration by pro-inflammatory macrophages, induces the production of IL-18, and sequentially stimulates the expression of connexin43 (Cx43) in fibroblasts in a paracrine manner, resulting in gap junction remodeling, myocardial fibrosis, and deterioration of cardiac function ( 31 ).…”