In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

Sprenger, Julia; Perera, Ruwan K.; Steinbrecher, Julia H.; Lehnart, Stephan E.; Maier, Lars S.; Hasenfuß, Gerd; Nikolaev, Viacheslav O.

doi:10.1038/ncomms7965

Cited by 114 publications

(136 citation statements)

References 43 publications

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“…21,22 A multiplicity of infection of 30 to 50 was used to infect HUVECs 40 to 48 hours before live-cell fluorescence resonance energy transfer (FRET) measurements. 21 Twenty-four hours before measurements, cells were incubated with TNF-α or vehicle and then subsequently with ANP, adenosine (Sigma), and isoprenaline (Sigma).…”

Section: Fluorescence Resonance Energy Transfer To Monitor Subsarcolementioning

confidence: 99%

Endothelial Actions of ANP Enhance Myocardial Inflammatory Infiltration in the Early Phase After Acute Infarction

Chen

Spitzl

Mathes

et al. 2016

Circulation Research

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Rationale: In patients after acute myocardial infarction (AMI), the initial extent of necrosis and inflammation determine clinical outcome. One early event in AMI is the increased cardiac expression of atrial natriuretic peptide (NP) and B-type NP, with their plasma levels correlating with severity of ischemia. It was shown that NPs, via their cGMP-forming guanylyl cyclase-A (GC-A) receptor and cGMP-dependent kinase I (cGKI), strengthen systemic endothelial barrier properties in acute inflammation. Objective: We studied whether endothelial actions of local NPs modulate myocardial injury and early inflammation after AMI. Methods and Results: Necrosis and inflammation after experimental AMI were compared between control mice and littermates with endothelial-restricted inactivation of GC-A (knockout mice with endothelial GC-A deletion) or cGKI (knockout mice with endothelial cGKI deletion). Unexpectedly, myocardial infarct size and neutrophil infiltration/activity 2 days after AMI were attenuated in knockout mice with endothelial GC-A deletion and unaltered in knockout mice with endothelial cGKI deletion. Molecular studies revealed that hypoxia and tumor necrosis factor-α, conditions accompanying AMI, reduce the endothelial expression of cGKI and enhance cGMP-stimulated phosphodiesterase 2A (PDE2A) levels. Real-time cAMP measurements in endothelial microdomains using a novel fluorescence resonance energy transfer biosensor revealed that PDE2 mediates NP/cGMP-driven decreases of submembrane cAMP levels. Finally, intravital microscopy studies of the mouse cremaster microcirculation showed that tumor necrosis factor-α–induced endothelial NP/GC-A/cGMP/PDE2 signaling impairs endothelial barrier functions. Conclusions: Hypoxia and cytokines, such as tumor necrosis factor-α, modify the endothelial postreceptor signaling pathways of NPs, with downregulation of cGKI, induction of PDE2A, and altered cGMP/cAMP cross talk. Increased expression of PDE2 can mediate hyperpermeability effects of paracrine endothelial NP/GC-A/cGMP signaling and facilitate neutrophil extravasation during the early phase after MI.

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Section: Fluorescence Resonance Energy Transfer To Monitor Subsarcolementioning

confidence: 99%

Endothelial Actions of ANP Enhance Myocardial Inflammatory Infiltration in the Early Phase After Acute Infarction

Chen

Spitzl

Mathes

et al. 2016

Circulation Research

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“…Therefore, biophysical methods, including FRET‐based biosensors, have been developed to facilitate real‐time measurement. FRET allows the visualization of cAMP fluctuations in living cells with high temporal and spatial resolution (Adams et al ., 1991; DiPilato et al ., 2004; Nikolaev et al ., 2004; Violin et al ., 2008; Sprenger et al ., 2015). Although FRET has previously been used to estimate cAMP levels in human airway epithelial cells (Schmid et al ., 2006, 2015), airway smooth muscle cells (Billington and Hall, 2011) and endothelial cells (Yañez‐Mó et al ., 2008), real‐time monitoring cAMP levels in the lung tissue has not been reported so far.…”

Section: Discussionmentioning

confidence: 99%

“…(2011), with minor modifications (Sprenger et al ., 2015). A lung slice was placed in the Attofluor cell chamber (Invitrogen, Landsmeer, Netherlands).…”

Section: Methodsmentioning

confidence: 99%

Cigarette smoke up‐regulates PDE3 and PDE4 to decrease cAMP in airway cells

Zuo

Han

Poppinga

et al. 2018

British J Pharmacology

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Background and PurposecAMP is a central second messenger that broadly regulates cell function and can underpin pathophysiology. In chronic obstructive pulmonary disease, a lung disease primarily provoked by cigarette smoke (CS), the activation of cAMP‐dependent pathways, via inhibition of hydrolyzing PDEs, is a major therapeutic strategy. Mechanisms that disrupt cAMP signalling in airway cells, in particular regulation of endogenous PDEs, are poorly understood.Experimental ApproachWe used a novel Förster resonance energy transfer (FRET) based cAMP biosensor in mice in vivo, ex vivo precision cut lung slices (PCLS) and in human cell models, in vitro, to track the effects of CS exposure.Key ResultsUnder fenoterol stimulation, FRET responses to cilostamide were significantly increased in in vivo, ex vivo PCLS exposed to CS and in human airway smooth muscle cells exposed to CS extract. FRET signals to rolipram were only increased in the in vivo CS model. Under basal conditions, FRET responses to cilostamide and rolipram were significantly increased in in vivo, ex vivo PCLS exposed to CS. Elevated FRET signals to rolipram correlated with a protein up‐regulation of PDE4 subtypes. In ex vivo PCLS exposed to CS extract, rolipram reversed down‐regulation of ciliary beating frequency, whereas only cilostamide significantly increased airway relaxation of methacholine pre‐contracted airways.Conclusion and ImplicationsExposure to CS, in vitro or in vivo, up‐regulated expression and activity of both PDE3 and PDE4, which affected real‐time cAMP dynamics. These mechanisms determine the availability of cAMP and can contribute to CS‐induced pulmonary pathophysiology.

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“…However, in heart failure, the influence of the local PDE4 is decreased, whereas the influence of PDE3 was not affected [176]. Both PDEs interact with AKAP18 [167,168,177] and PDE4D3 with mAKAP (see above); it remains to be determined whether the observed effects involve tethering of the PDEs to the SERCA2 microdomain by one or both of these or yet another AKAP.…”

Section: Akaps and Pde Interactionsmentioning

confidence: 99%

Pharmacological targeting of AKAP-directed compartmentalized cAMP signalling

Dema

Perets

Schulz

et al. 2015

Cellular Signalling

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The second messenger cyclic adenosine monophosphate (cAMP) can bind and activate protein kinase A (PKA). The cAMP/PKA system is ubiquitous and involved in a wide array of biological processes and therefore requires tight spatial and temporal regulation. Important components of the safeguard system are the A-kinase anchoring proteins (AKAPs), a heterogeneous family of scaffolding proteins defined by its ability to directly bind PKA. AKAPs tether PKA to specific subcellular compartments, and they bind further interaction partners to create local signalling hubs. The recent discovery of new AKAPs and advances in the field that shed light on the relevance of these hubs for human disease highlight unique opportunities for pharmacological modulation. This review exemplifies how interference with signalling, particularly cAMP signalling, at such hubs can reshape signalling responses and discusses how this could lead to novel pharmacological concepts for the treatment of disease with an unmet medical need such as cardiovascular disease and cancer.4

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In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

Cited by 114 publications

References 43 publications

Endothelial Actions of ANP Enhance Myocardial Inflammatory Infiltration in the Early Phase After Acute Infarction

Endothelial Actions of ANP Enhance Myocardial Inflammatory Infiltration in the Early Phase After Acute Infarction

Cigarette smoke up‐regulates PDE3 and PDE4 to decrease cAMP in airway cells

Pharmacological targeting of AKAP-directed compartmentalized cAMP signalling

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