Direct monitoring of cAMP at the cardiac ryanodine receptor using a novel targeted fluorescence biosensor mouse

Rationale: Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which, upon beta-adrenergic receptor (beta-AR) stimulation, acts in microdomains to regulate cardiac excitation-contraction coupling by activating the cAMP-dependent protein kinase (PKA) 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 upon RyR2 hyperphosphorylation by PKA and calcium/calmodulin-dependent kinase.Objective: We sought to establish a real time approach capable of directly visualizing cAMP and its pathological changes in the vicinity of RyR2 by generating a proper targeted biosensor and transgenic mouse model to express it in adult cardiomyocytes.Methods and Results: We generated transgenic mice expressing a novel targeted fluorescent biosensor for RyR2-associated cAMP in adult mouse cardiomyocytes. In healthy cardiomyocytes, beta1-AR but not beta2-AR stimulation strongly increased local RyR2-associated cAMP levels. However, 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 measurable beta2-AR-induced cAMP signals, increased SR calcium leak and arrhythmia susceptibility.Conclusions: Our new targeted biosensor expressed in transgenic mice can visualize cAMP levels in the vicinity of cardiac RyR2 in healthy and diseased cardiomyocytes. In the future, this novel biosensor can be used to better understand alterations of RyR2-associated cAMP in cardiovascular diseases and local actions of new therapies.