Using cAMP Sensors to Study Cardiac Nanodomains

Abstract

3′,5′-cyclic adenosine monophosphate (cAMP) signalling plays a major role in the cardiac myocyte response to extracellular stimulation by hormones and neurotransmitters. In recent years, evidence has accumulated demonstrating that the cAMP response to different extracellular agonists is not uniform: depending on the stimulus, cAMP signals of different amplitudes and kinetics are generated in different subcellular compartments, eliciting defined physiological effects. In this review, we focus on how real-time imaging using fluorescence resonance energy transfer (FRET)-based reporters has provided mechanistic insight into the compartmentalisation of the cAMP signalling pathway and allowed for the precise definition of the regulation and function of subcellular cAMP nanodomains.

Tuning the Consonance of Microscopic Neuro-Cardiac Interactions Allows the Heart Beats to Play Countless Genres

“…A relevant example of how spatial organization of cAMP responses reflects the stimulation of a specific receptor subtype comes from the study of Nikolaev et al showing, in murine CMs, that β1- or β2-AR agonism elicits increases in cAMP which spread throughout the entire cytoplasm, or remain localized at the level of T-tubuli, respectively ( Nikolaev et al, 2006 ). In incremental developments, the now-accepted concept of compartmentalization of cAMP has been delved into with increasing precision and detail, thanks to an extended toolkit of diverse cAMP and PKA biosensors, with enhanced sensitivity, or targeted to specific subcellular compartments, and the combination with strategies for local delivery of extracellular stimuli, subcellular uncaging of cAMP analogs or targeted Adenylyl Cyclases ( Schleicher and Zaccolo, 2018 ; Ghigo and Mika, 2019 ). It is now well appreciated that specifically distributed Adenylyl Cyclases (that make cAMP) and specific phosphodiesterase isoforms (that degrade cAMP) are selectively localized in different cellular domains ( Bers et al, 2019 ), including the plasma membrane, SR, nucleus and mitochondria ( Mongillo and Zaccolo, 2006 ; Leroy et al, 2008 ; Mika et al, 2012 ; Stangherlin and Zaccolo, 2012 ; Bedioune et al, 2018 ; Ghigo and Mika, 2019 ).…”

Mice lacking the cAMP effector protein POPDC1 show enhanced hippocampal synaptic plasticity

“…Although we did not find significant differences in gross cAMP levels in the hippocampal slices of Popdc1 KO mice either at basal activity or following FSK stimulation, we suspect that this might be due to the inability of the ELISA assay utilized here to capture local changes in cAMP nanodomains. It will be interesting to investigate the role of POPDC1 in local cAMP dynamics during synaptic activity using imaging approaches based on genetically encoded cAMP sensors (Schleicher and Zaccolo, 2018).…”

Dissection of Protein Kinase Pathways in Live Cells Using Photoluminescent Probes: Surveillance or Interrogation?

“…The cAMP-sensing probe derived from PKA are multipartite: PKA holoenzyme consists of two catalytic subunits (PKAc) and a dimer of regulatory subunits (PKAr 2 ), and dissociation of holoenzyme occurs upon binding of cAMP. The pioneering works used labeling of purified PKAc and PKAr with suitable organic dyes and microinjection of the obtained constructs into the cells [210,211]; subsequently, genetically encoded bipartite probes consisting of PKAr-CFP and PKAc-YFP were reported [65,212]. The cGMP-sensing probe, in turn, takes advantage of PKGI, where regulatory and catalytic parts of PK are incorporated into one chain [213].…”