P2869Role of PDE8 in cAMP dynamics in human atrial fibrillation

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
N Grammatika Pavlidou ◽  
S Pecha ◽  
H Reichenspurner ◽  
T Christ ◽  
V O Nikolaev ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Receptors ◽  
Intracellular Camp ◽  
Atrial Myocytes ◽  
Human Atrial Myocytes ◽  
Intracellular Compartments ◽  
Camp Levels

Abstract Background Cardiac arrhythmias, such as atrial fibrillation (AF), are often related to remodeling of membrane receptors and alterations in cAMP-dependent regulation of Ca2+ handling mechanisms. For instance, decreased L-type calcium current (ICa,L) density but upregulated RyR2 are major hallmarks of AF. These inhomogeneous AF-associated changes of protein phosphorylation point to a local regulation of PKA activity within these intracellular compartments. Local cAMP compartmentation and the role of phosphodiesterase (PDEs) have ben extensively studied in ventricular myocytes from animals. However, only a few studies have evaluated the contribution of PDEs to the pathophysiology of AF and the reason for the persistent AF-associated hypophosphorylation of the L-type calcium channel (LTCC) is currently unknown. The aim of this study was to investigate whether a change in the expression level of PDE8 in human atrium may affects cAMP nearby LTCC promoting the reduction of the ICa,L observed in persistent AF. Methods Atrial myocytes were isolated from tissue of 47 patients in sinus rhythm (SR) and with AF. Cells were then transfect with an adenovirus (Epac1-camps or pm-Epac1-camps) in order to express the (cytosolic or membrane, respectively) FRET-based cAMP sensor and cultured during 48 hours. Föster-resonance energy transfer (FRET) was used to measure cAMP in 232 isolated human atrial myocytes. Ro-20-1724 (10 μM), Cilostamide (1 μM) and PF-04957325 (30 nM) and IBMX (100 μM) were used as PDE4, PDE3, PDE8 and non-selective phosphodiesterases (PDEs) inhibitor respectively. Results Effects of PDE4 and especially PDE3 inhibition on cytosolic [cAMP] are reduced in AF. Pharmacological PDE8 inhibition induces only a small increase in basal intracellular [cAMP] in AF but it showed a big synergic effect when PDE4 was inhibit at the same time. By contrast, PDE8 inhibition dramatically increased basal [cAMP] in the subsarcolemmal compartment in AF while PDE3 or PDE4 inhibition had a smaller effect that didn't change between SR and AF. Conclusions PDE8 controls basal cytosolic cAMP levels in human atrial myocytes from patients with persistent AF while PDE3 effects tends to be reduced in these patients. Furthermore, PDE8 is the main PDE in controlling cAMP levels at the membrane in persistent AF. Thus, our study may provide a clue for the reported reduction of the ICa,L in persistent AF.

scholarly journals GPCR signaling is highly compartmentalized in human cardiomyocytes and severely remodeled in atrial fibrillation

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Kira Beneke ◽  
Nefeli Grammatika Pavlidou ◽  
Andreas Schäfer ◽  
Viacheslav O. Nikolaev ◽  
Cristina E. Molina
Keyword(s):  
Atrial Fibrillation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Receptors ◽  
Human Cardiomyocytes ◽  
Specific Effects ◽  
And Control ◽  
G Protein Coupled ◽  
Camp Levels ◽  
A2ar Agonist

Atrial fibrillation (AF) has been linked to the remodeling of membrane receptors and alterations in downstream cAMP-dependent regulation. However, to date, no study has elucidated how the increase on cAMP upon different G-protein-coupled receptors (GPCRs) can lead to different physiological compartmentalized responses. The aim of this study was to investigate the compartmentally specific effects of GPCRs on cAMP levels in human atrial myocytes (HAMs) from patients with AF and control patients without AF (Ctl), and how these compartmentalized effects are altered in AF. HAMs were isolated from 60 AF and 76 Ctl patient tissues. Cells were transduced with adenoviruses (Epac1-camps, pm-Epac1-camps and Epac1-JNC) and cultured for 48 hours to express the FRET-based cAMP sensor in the cytosolic, membrane, and RYR2 nanodomains. Förster-resonance energy transfer (FRET) was used to measure cAMP levels in 525 HAMs stimulated with isoprenaline (100 µM), serotonin (100 µM), or the A2AR agonist CGS (200 nM). A desensitization to β-adrenergic receptor stimulation was exclusively found in the cytosol of AF myocytes, while no difference was seen in the RYR2 or LTCC compartment. Similar effects were observed upon serotonin stimulation with a significant desensitization in the cytosol, and no difference in the RYR2 compartment. In response to A2ARs stimulation AF myocytes displayed a significantly higher cytosolic increase in cAMP levels. However, no response was seen in the LTCC compartment in response to serotonin or A2AR stimulation. Collectively, our data show that cAMP levels are highly compartmentalized and differentially regulated by GPCRs. Furthermore, these results provide a mechanistic insight for the previously reported functional effects seen upon stimulation of these three receptors.

P2863Regional specific remodeling of cGMP pathway in human atrial fibrillation

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
N I Bork ◽  
N G Pavlidou ◽  
B Reiter ◽  
H Reichenspurner ◽  
T Christ ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Sinus Rhythm ◽  
Ryanodine Receptors ◽  
Membrane Receptors ◽  
Mrna Levels ◽  
Atrial Myocytes ◽  
Human Atrial Myocytes ◽  
Cgmp Pathway ◽  
The Right

Abstract Background Atrial fibrillation (AF) is accompanied by a profound remodeling of membrane receptors and alterations in cyclic nucleotides-dependent regulation of Ca2+-handling. Thus, while basal ryanodine receptors activity is upregulated, L-type calcium current (ICa,L) density is diminish in AF, due to local microdomain-specific cAMP dynamics. The same seems true for cGMP regulation in AF. In AF cGMP-mediated increase in ICa,L is blunted but NO-mediated attenuation of β-adrenoceptors stimulation-mediated increase is preserved. However, although the role of cGMP in controling atrial function and pathophysiology is controversial, no study has been ever performed in human myocytes to measure cGMP directly. Methods We isolated myocytes from the right and/or left atrium of 27 patients in sinus rhythm (SR), and with AF. Cells were then transfected with adenovirus to express the cytosolic FRET-based cGMP sensor red-cGES-DE5 and cultured for 48 hours. Förster resonance energy transfer (FRET) was used to measure cGMP in 61 living human atrial myocytes. We stimulated cells with the C-type natriuretic peptide CNP (100 nM and 1 μM), and the non-selective phosphodiesterases (PDEs) inhibitor IBMX (100 μM). Additionally, PDE specific inhibitors for PDE2 (Bay 60–7550, 100 nM) and PDE3 (Cilostamide, 10 μM) as well as inhibitor of the soluble guanylyl cyclase (ODQ, 50 μM) were used. We also measured PDE2 and PDE3 mRNA levels in atrial tissue samples from both groups of patients using RT-qPCR. Results We could show that stimulation with CNP increased cGMP levels in human atrial myocytes. However, in myocytes from patients with AF global cGMP responses to CNP and to IBMX was reduced compared to SR. Additionally, there was a difference in response to CNP and IBMX in patients with AF between the right and the left atria. Whereas in the right atria IBMX could further increase cGMP levels in the cell, in the left atria leaded to a reduction in cGMP levels. RT-qPCR showed a tendency of PDE3 to be reduced in AF. On the other hand, PDE2A gene expression was upregulated in the left atria. Conclusions We have shown that PDEs contributes cGMP signaling in the human atria and that they are involved in atrial pathophysiology. Now our data clearly show differences in cGMP regulation in cardiomyocytes isolated from left and right atrium from patients in atrial fibrillation and sinus rhythm. We observe a major role of PDEs, regulating cGMP pathway promoted by the reduced responses in AF, especially PDE2 in the left atria.

scholarly journals Cardiac Hypertrophy Changes Compartmentation of cAMP in Non-Raft Membrane Microdomains

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 535
Author(s):  
Nikoleta Pavlaki ◽  
Kirstie A. De Jong ◽  
Birgit Geertz ◽  
Viacheslav O. Nikolaev ◽  
Alexander Froese
Keyword(s):  
Cardiac Hypertrophy ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Pressure Overload ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Differential Regulation ◽  
Membrane Microdomains ◽  
Transgenic Mouse Line

3′,5′-Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which plays critical roles in cardiac function and disease. In adult mouse ventricular myocytes (AMVMs), several distinct functionally relevant microdomains with tightly compartmentalized cAMP signaling have been described. At least two types of microdomains reside in AMVM plasma membrane which are associated with caveolin-rich raft and non-raft sarcolemma, each with distinct cAMP dynamics and their differential regulation by receptors and cAMP degrading enzymes phosphodiesterases (PDEs). However, it is still unclear how cardiac disease such as hypertrophy leading to heart failure affects cAMP signals specifically in the non-raft membrane microdomains. To answer this question, we generated a novel transgenic mouse line expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor E1-CAAX targeted to non-lipid raft membrane microdomains of AMVMs and subjected these mice to pressure overload induced cardiac hypertrophy. We could detect specific changes in PDE3-dependent compartmentation of β-adrenergic receptor induced cAMP in non-raft membrane microdomains which were clearly different from those occurring in caveolin-rich sarcolemma. This indicates differential regulation and distinct responses of these membrane microdomains to cardiac remodeling.

scholarly journals Heart failure leads to altered β2-adrenoceptor/cyclic adenosine monophosphate dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain

2018 ◽  
Vol 115 (3) ◽  
pp. 546-555 ◽  
Author(s):  
Zeynep Bastug-Özel ◽  
Peter T Wright ◽  
Axel E Kraft ◽  
Davor Pavlovic ◽  
Jacqueline Howie ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adult Rat ◽  
C Terminus ◽  
Complex Forms ◽  
Cardiac Excitation

Abstract Aims Cyclic adenosine monophosphate (cAMP) regulates cardiac excitation–contraction coupling by acting in microdomains associated with sarcolemmal ion channels. However, local real time cAMP dynamics in such microdomains has not been visualized before. We sought to directly monitor cAMP in a microdomain formed around sodium–potassium ATPase (NKA) in healthy and failing cardiomyocytes and to better understand alterations of cAMP compartmentation in heart failure. Methods and results A novel Förster resonance energy transfer (FRET)-based biosensor termed phospholemman (PLM)-Epac1 was developed by fusing a highly sensitive cAMP sensor Epac1-camps to the C-terminus of PLM. Live cell imaging in PLM-Epac1 and Epac1-camps expressing adult rat ventricular myocytes revealed extensive regulation of NKA/PLM microdomain-associated cAMP levels by β2-adrenoceptors (β2-ARs). Local cAMP pools stimulated by these receptors were tightly controlled by phosphodiesterase (PDE) type 3. In chronic heart failure following myocardial infarction, dramatic reduction of the microdomain-specific β2-AR/cAMP signals and β2-AR dependent PLM phosphorylation was accompanied by a pronounced loss of local PDE3 and an increase in PDE2 effects. Conclusions NKA/PLM complex forms a distinct cAMP microdomain which is directly regulated by β2-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted β2-AR signalling to cAMP in the vicinity of PLM.

scholarly journals Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability

2012 ◽  
Vol 198 (2) ◽  
pp. 251-263 ◽  
Author(s):  
Enora Moutin ◽  
Fabrice Raynaud ◽  
Jonathan Roger ◽  
Emilie Pellegrino ◽  
Vincent Homburger ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Receptors ◽  
Physical Interaction ◽  
Scaffolding Proteins ◽  
Cellular Functions ◽  
Electrophysiological Recordings ◽  
Living Neurons ◽  
Activity Dependent

Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor–mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.

Abstract 311: Measurements of cAMP Dynamics in the SERCA2 Microdomain in Adult Mouse Cardiomyocytes using a Targeted FRET Biosensor

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Julia U Sprenger ◽  
Viacheslav O Nikolaev
Keyword(s):  
Transgenic Mice ◽  
Molecular Mechanisms ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Heart Weight ◽  
Cell Fractionation ◽  
Adrenergic Stimulation ◽  
Cardiac Phenotype ◽  
Fractionation Analysis

PURPOSE: cAMP is a central regulator of cardiac function and disease. This global second messenger acts in a compartmentalized fashion, and changes in cAMP dynamics are linked to cardiac diseases. In this project, we visualized cAMP signals directly in such microdomains to gain insights into the molecular mechanisms involved in cAMP compartmentation and its alterations in hypertrophy. Methods: We generated transgenic mice expressing a new Förster resonance energy transfer (FRET)-based cAMP sensor Epac1-camps-PLN to measure cAMP dynamics in the microdomain around the sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2). This sensor is targeted to SERCA2 via phospholamban (PLN). Results: Colocalization and cell fractionation analysis confirmed proper localization of the sensor in transgenic mouse hearts. qPCR analysis revealed a two-fold overexpression of PLN. However, no adverse cardiac phenotype could be detected by histological analysis and heart weight to body weight ratios. Local cAMP dynamics were measured using freshly isolated adult ventricular myocytes and compared to cAMP signals in the bulk cytosol using cardiomyocytes from Epac1-camps mice. We detected the predominant role of phosphodiesterases (PDEs) 4 and 3 in the SERCA2 compartment under basal conditions. These PDEs were responsible for shaping the microdomain and its segregation from the cytosolic compartment. Interestingly, beta1-adrenergic stimulation led to a stronger increase of local cAMP in the SERCA2 compartment compared to the bulk cytosol. 8 weeks after transverse aortic constriction (TAC), PDE4 activity was downregulated in the SERCA2 microdomain compared to sham cardiomyocytes. Conclusion: We successfully generated transgenic mice expressing the targeted Epac1-camps-PLN biosensor to visualize cAMP dynamics in the SERCA2 compartment. We could show distinct cAMP dynamics around the SERCA2 compartment compared to the bulk cytosol and uncovered its alterations in hypertrophied cardiomyocytes

scholarly journals Illuminating the allosteric modulation of the calcium-sensing receptor

2020 ◽  
Vol 117 (35) ◽  
pp. 21711-21722
Author(s):  
Hongkang Liu ◽  
Ping Yi ◽  
Wenjing Zhao ◽  
Yuling Wu ◽  
Francine Acher ◽  
...  
Keyword(s):  
Amino Acids ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Receptors ◽  
Allosteric Modulators ◽  
Active Conformation ◽  
Calcium Sensing Receptor ◽  
Single Receptor ◽  
Calcium Sensing

Many membrane receptors are regulated by nutrients. However, how these nutrients control a single receptor remains unknown, even in the case of the well-studied calcium-sensing receptor CaSR, which is regulated by multiple factors, including ions and amino acids. Here, we developed an innovative cell-free Förster resonance energy transfer (FRET)-based conformational CaSR biosensor to clarify the main conformational changes associated with activation. By allowing a perfect control of ambient nutrients, this assay revealed that Ca2+alone fully stabilizes the active conformation, while amino acids behave as pure positive allosteric modulators. Based on the identification of Ca2+activation sites, we propose a molecular basis for how these different ligands cooperate to control CaSR activation. Our results provide important information on CaSR function and improve our understanding of the effects of genetic mutations responsible for human diseases. They also provide insights into how a receptor can integrate signals from various nutrients to better adapt to the cell response.

scholarly journals Temporal cAMP Signaling Selectivity by Natural and Synthetic MC4R Agonists

2015 ◽  
Vol 29 (11) ◽  
pp. 1619-1633 ◽  
Author(s):  
Brent M. Molden ◽  
Kimberly A. Cooney ◽  
Kirk West ◽  
Lex H. T. Van Der Ploeg ◽  
Giulia Baldini
Keyword(s):  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Camp Signaling ◽  
Intracellular Camp ◽  
Specific Cell ◽  
Melanocortin 4 Receptor ◽  
Green Fluorescent ◽  
Camp Induction ◽  
Agouti Related Protein

Abstract The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain, where it controls energy balance through pathways including α-melanocyte-stimulating hormone (α-MSH)-dependent signaling. We have reported that the MC4R can exist in an active conformation that signals constitutively by increasing cAMP levels in the absence of receptor desensitization. We asked whether synthetic MC4R agonists differ in their ability to increase intracellular cAMP over time in Neuro2A cells expressing endogenous MC4R and exogenous, epitope-tagged hemagglutinin-MC4R-green fluorescent protein. By analyzing intracellular cAMP in a temporally resolved Förster resonance energy transfer assay, we show that withdrawal of α-MSH leads to a quick reversal of cAMP induction. By contrast, the synthetic agonist melanotan II (MTII) induces a cAMP signal that persists for at least 1 hour after removal of MTII from the medium and cannot be antagonized by agouti related protein. Similarly, in mHypoE-42 immortalized hypothalamic neurons, MTII, but not α-MSH, induced persistent AMP kinase signal, which occurs downstream of increased cAMP. By using a fluorescence recovery after photobleaching assay, it appears that the receptor exposed to MTII continues to signal after being internalized. Similar to MTII, the synthetic MC4R agonists, THIQ and BIM-22511, but not LY2112688, induced prolonged cAMP signaling after agonist withdrawal. However, agonist-exposed MC4R desensitized to the same extent, regardless of the ligand used and regardless of differences in receptor intracellular retention kinetics. In conclusion, α-MSH and LY2112688, when compared with MTII, THIQ, and BIM-22511, vary in the duration of the acute cAMP response, showing distinct temporal signaling selectivity, possibly linked to specific cell compartments from which cAMP signals may originate.

scholarly journals Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain.

2021 ◽  
Author(s):  
Maria Kowalski-Jahn ◽  
Hannes Schihada ◽  
Ainoleena Turku ◽  
Thomas Huber ◽  
Thomas P. Sakmar ◽  
...  
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Membrane Receptors ◽  
Rich Domain ◽  
Linker Domain ◽  
Cysteine Rich Domain

Frizzleds (FZD1-10) comprise a class of G protein-coupled receptors containing an extracellular cysteine-rich domain (CRD) that binds lipoglycoproteins of the Wingless/Int-1 family (WNTs). Despite the prominent role of the WNT/FZD system in health and disease, our understanding of how WNT binding to the FZD CRD is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics and the involvement of the linker domain connecting the CRD with the seven-helical transmembrane core of FZD. To clarify the mechanism of WNT binding to FZD and to elucidate how WNT/FZD complexes achieve signaling pathway specificity, we devised conformational FZD-CRD biosensors based on bioluminescence-resonance-energy-transfer (BRET). Using FZD engineered with N-terminal nanoluciferase and fluorescently-labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct downstream signaling pathways, and that CRD dynamics are not required for WNT/β-catenin signaling. Thus, the novel FZD-CRD biosensors we report provide insights into the stepwise binding, activation and signaling processes in FZDs. The sensor design is broadly applicable to explore fundamental events in signal transduction mediated by other membrane receptors.

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