cAMP Imaging at Ryanodine Receptors Reveals β2-Adrenoceptor Driven Arrhythmias

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.

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

Cardiovascular Research ◽

10.1093/cvr/cvy221 ◽

2018 ◽

Vol 115 (3) ◽

pp. 546-555 ◽

Cited By ~ 12

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.

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

10.1101/623934 ◽

2019 ◽

Cited By ~ 1

Author(s):

Filip Berisha ◽

Konrad R. Götz ◽

Jörg W. Wegener ◽

Christiane Jungen ◽

Ulrike Pape ◽

...

Keyword(s):

Transgenic Mice ◽

Ryanodine Receptor ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Calcium Handling ◽

Calcium Calmodulin Dependent ◽

Fluorescence Biosensor ◽

Cardiac Excitation ◽

Camp Levels ◽

Cardiac Ryanodine Receptor

AbstractRationaleCyclic 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 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.ObjectiveWe 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 ResultsWe generated transgenic mice expressing a novel targeted fluorescent biosensor for RyR2-associated cAMP in adult mouse cardiomyocytes. In healthy cardiomyocytes, β1-AR but not β2-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 β2-AR-induced cAMP signals, increased SR calcium leak and arrhythmia susceptibility.ConclusionsOur 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.

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

Cells ◽

10.3390/cells10030535 ◽

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.

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Anti-Allergic and Anti-Inflammatory Effects of Undecane on Mast Cells and Keratinocytes

Molecules ◽

10.3390/molecules25071554 ◽

2020 ◽

Vol 25 (7) ◽

pp. 1554

Author(s):

Dabin Choi ◽

Wesuk Kang ◽

Taesun Park

Keyword(s):

Mast Cells ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Allergic Diseases ◽

Intracellular Camp ◽

Tnf Α ◽

Anti Inflammatory ◽

Skin Conditions ◽

Factor Α ◽

Camp Levels

The critical roles of keratinocytes and resident mast cells in skin allergy and inflammation have been highlighted in many studies. Cyclic adenosine monophosphate (cAMP), the intracellular second messenger, has also recently emerged as a target molecule in the immune reaction underlying inflammatory skin conditions. Here, we investigated whether undecane, a naturally occurring plant compound, has anti-allergic and anti-inflammatory activities on sensitized rat basophilic leukemia (RBL-2H3) mast cells and HaCaT keratinocytes and we further explored the potential involvement of the cAMP as a molecular target for undecane. We confirmed that undecane increased intracellular cAMP levels in mast cells and keratinocytes. In sensitized mast cells, undecane inhibited degranulation and the secretion of histamine and tumor necrosis factor α (TNF-α). In addition, in sensitized keratinocytes, undecane reversed the increased levels of p38 phosphorylation, nuclear factor kappaB (NF-κB) transcriptional activity and target cytokine/chemokine genes, including thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and interleukin-8 (IL-8). These results suggest that undecane may be useful for the prevention or treatment of skin inflammatory disorders, such as atopic dermatitis, and other allergic diseases.

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Integration of Rap1 and Calcium Signaling

International Journal of Molecular Sciences ◽

10.3390/ijms21051616 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1616 ◽

Cited By ~ 1

Author(s):

Ramoji Kosuru ◽

Magdalena Chrzanowska

Keyword(s):

Intracellular Signaling ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Nucleotide Exchange ◽

Cellular Functions ◽

Cellular Processes ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors ◽

Gtpase Activating Proteins ◽

Cardiac Excitation

Ca2+ is a universal intracellular signal. The modulation of cytoplasmic Ca2+ concentration regulates a plethora of cellular processes, such as: synaptic plasticity, neuronal survival, chemotaxis of immune cells, platelet aggregation, vasodilation, and cardiac excitation–contraction coupling. Rap1 GTPases are ubiquitously expressed binary switches that alternate between active and inactive states and are regulated by diverse families of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Active Rap1 couples extracellular stimulation with intracellular signaling through secondary messengers—cyclic adenosine monophosphate (cAMP), Ca2+, and diacylglycerol (DAG). Much evidence indicates that Rap1 signaling intersects with Ca2+ signaling pathways to control the important cellular functions of platelet activation or neuronal plasticity. Rap1 acts as an effector of Ca2+ signaling when activated by mechanisms involving Ca2+ and DAG-activated (CalDAG-) GEFs. Conversely, activated by other GEFs, such as cAMP-dependent GEF Epac, Rap1 controls cytoplasmic Ca2+ levels. It does so by regulating the activity of Ca2+ signaling proteins such as sarcoendoplasmic reticulum Ca2+-ATPase (SERCA). In this review, we focus on the physiological significance of the links between Rap1 and Ca2+ signaling and emphasize the molecular interactions that may offer new targets for the therapy of Alzheimer’s disease, hypertension, and atherosclerosis, among other diseases.

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Comparison of Two Nasal Cell Collection Methods in Determining Cyclic Adenosine Monophosphate Levels and its Association with Olfaction: A Feasibility Study

Allergy & Rhinology ◽

10.2500/ar.2014.5.0079 ◽

2014 ◽

Vol 5 (1) ◽

pp. ar.2014.5.0079

Author(s):

Jack J. Liu ◽

Guy C. Chan ◽

Avram S. Hecht ◽

Daniel R. Storm ◽

Greg E. Davis

Keyword(s):

Intraclass Correlation ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Olfactory Function ◽

Secondary Outcome ◽

Initial Visit ◽

Two Samples ◽

Camp Levels ◽

Collection Methods

Cyclic adenosine monophosphate (cAMP) is a second messenger that may be associated with olfactory function. No known studies have compared existing collection methods for determining nasal cAMP levels. This is a prospective study comparing the comfort and reliability of the nasal curette and cytobrush. A secondary outcome collected for feasibility testing was characterizing the association between cAMP and olfactory function. We enrolled 19 normal olfaction and 10 olfactory dysfunction subjects. Olfaction was measured by the University of Pennsylvania Smell Identification Test. Two samples were obtained from each nasal cavity at the initial visit and at 1 week follow-up. Comfort was measured by a visual analog scale (VAS). cAMP levels were determined by an enzyme immunoassay. For the curette and cytobrush, mean VAS scores were 03 and 0.7 cm (p = 0.48). Intraclass correlation coefficients were 0.81 (curette) and 0.65 (cytobrush) for the initial visit and 0.64 and 0.54 between the initial and follow-up visit. Using the curette, mean cAMP was 537 and 480 fmol/(mg/mL) for the normal and dysfunction cohorts (p = 0.18). Using the cytobrush, cAMP was 505 and 477, respectively (p = 0.65). The curette and cytobrush are both comfortable and reliable collection methods for determining nasal cAMP levels.

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Intracellular calcium handling in ventricular myocytes from mdx mice

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00688.2006 ◽

2007 ◽

Vol 292 (2) ◽

pp. H846-H855 ◽

Cited By ~ 109

Author(s):

Iwan A. Williams ◽

David G. Allen

Keyword(s):

Heart Failure ◽

Protein Expression ◽

Transient Receptor Potential ◽

Ventricular Myocytes ◽

Clinical Symptoms ◽

Disease Process ◽

Calcium Handling ◽

Mdx Mouse ◽

Mdx Mice ◽

Age Related

Duchenne muscular dystrophy (DMD) is a lethal degenerative disease of skeletal muscle, characterized by the absence of the cytoskeletal protein dystrophin. Some DMD patients show a dilated cardiomyopathy leading to heart failure. This study explores the possibility that dystrophin is involved in the regulation of a stretch-activated channel (SAC), which in the absence of dystrophin has increased activity and allows greater Ca2+ into cardiomyocytes. Because cardiac failure only appears late in the progression of DMD, we examined age-related effects in the mdx mouse, an animal model of DMD. Ca2+ measurements using a fluorescent Ca2+-sensitive dye fluo-4 were performed on single ventricular myocytes from mdx and wild-type mice. Immunoblotting and immunohistochemistry were performed on whole hearts to determine expression levels of key proteins involved in excitation-contraction coupling. Old mdx mice had raised resting intracellular Ca2+ concentration ([Ca2+]i). Isolated ventricular myocytes from young and old mdx mice displayed abnormal Ca2+ transients, increased protein expression of the ryanodine receptor, and decreased protein expression of serine-16-phosphorylated phospholamban. Caffeine-induced Ca2+ transients showed that the Na+/Ca2+ exchanger function was increased in old mdx mice. Two SAC inhibitors streptomycin and GsMTx-4 both reduced resting [Ca2+]i in old mdx mice, suggesting that SACs may be involved in the Ca2+-handling abnormalities in these animals. This finding was supported by immunoblotting data, which demonstrated that old mdx mice had increased protein expression of canonical transient receptor potential channel 1, a likely candidate protein for SACs. SACs may play a role in the pathogenesis of the heart failure associated with DMD. Early in the disease process and before the onset of clinical symptoms increased, SAC activity may underlie the abnormal Ca2+ handling in young mdx mice.

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Inhibitory action of somatostatin-14 on hormone-stimulated cyclic adenosine monophosphate induction in porcine granulosa and luteal cells

Journal of Endocrinology ◽

10.1677/joe.0.1340297 ◽

1992 ◽

Vol 134 (2) ◽

pp. 297-306 ◽

Cited By ~ 12

Author(s):

K. Rajkumar ◽

D. E. Kerr ◽

R. N. Kirkwood ◽

B. Laarveld

Keyword(s):

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Inhibitory Effect ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Luteal Cells ◽

Camp Generation ◽

Camp Induction ◽

Camp Levels ◽

Somatostatin 14

ABSTRACT Somatostatin-14 (SRIF-14) inhibited, in a concentration-dependent manner, LH- and forskolin-stimulated cyclic adenosine monophosphate (cAMP) induction in porcine granulosa and luteal cells. The inhibitory effect of SRIF-14 on hormone-induced cAMP generation was more potent in porcine ovarian cells than in the GH-3 pituitary cell line. The inhibitory effect of SRIF-14 was impeded by neutralizing its biological activity with specific antiserum. Preincubation of luteal and granulosa cells with phorbol 12-myristate 13-acetate (PMA) enhanced LH- and forskolin-stimulated cAMP levels. SRIF-14 failed to inhibit LH- or forskolin-stimulated cAMP levels in cells preincubated with PMA. It is concluded that SRIF-14 inhibits hormone-stimulated cAMP induction in the porcine ovary. LH-induced protein kinase C activation may be physiologically important to alleviate the inhibitory effects of SRIF-14. Journal of Endocrinology (1992) 134, 297–306

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Cyclic adenosine monophosphate and cyclic guanosine monophosphate variations during isometric tetanus in frog sartorius muscle

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y82-011 ◽

1982 ◽

Vol 60 (1) ◽

pp. 79-83

Author(s):

Giorgio Fang ◽

Giovanni Menchetti ◽

Giovannella Della Torre ◽

Lino Volpi ◽

Teresa Secca ◽

...

Keyword(s):

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Sartorius Muscle ◽

Tetanic Stimulation ◽

Frog Sartorius Muscle ◽

Different Temperatures ◽

Camp Levels ◽

Frog Sartorius

Tetanic stimulation at different temperatures (5 and 20 °C) of the frog sartorius muscle results in an increase of cyclic guanosine monophosphate (cGMP) directly correlated to the tension developed. Cyclic adenosine monophosphate (cAMP) levels change differently for different temperature values. The variations could be explained by the interaction between Ca2+ and the enzymes which control cyclic nucleotide levels, namely, adenylate cyclase, guanylate cyclase, and phosphodiesterase.

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Systemic effects of cAMP in chemotherapy for Heren’s carcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e14052 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e14052-e14052

Author(s):

Alla Ivanovna Shikhlyarova ◽

Elena Mikhaylovna Frantsiyants ◽

Galina Vitalyevna Zhukova ◽

Natalia D. Cheryarina ◽

Tatiana Albertovna Barteneva ◽

...

Keyword(s):

Tumor Regression ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Regenerative Processes ◽

Metabolic Substrates ◽

Response To Chemotherapy ◽

Male Wistar Rats ◽

Camp Levels ◽

The Brain ◽

Body Response

e14052 Background: Along with tumor itself, mechanisms of regulation of homeostasis are the target for tumor progression inhibition. The brain, various organs and tumor have different resources of energetic and metabolic substrates. Involvement of cyclic adenosine monophosphate (cAMP) into intimate mechanisms of proliferation, hormonal and energetic homeostasis indicates the possibility to use this factor in chemotherapy of tumors to improve the resistance of the organism. The purpose of the study was to analyze levels of endogenous cAMP in tumor and in organs as a criterium of systemic body response to chemotherapy with cAMP application. Methods: The study included 56 male Wistar rats with Heren’s carcinoma receiving peritumoral injections of cyclophosphan (CP) 50 mcg/kg (Baxter Oncology GmbH, Germany) alone and in combination with cAMP (Sigma-Aldrich, USA), P.O. at a concentration of 0.01%. cAMP levels in homogenates of organs and tumors were measured by immunoradiometric assay (Immunotech, Czech Republic) using Arian radiometer (Vitaco, Russia). Data were processed using Statistica 6. Results: cAMP levels in growing tumors in rats without treatment (the control) were maximal (7.03±1.5 nmol/L). CP injections alone during inhibition of carcinoma growth allowed the reduction of tumor cAMP level by 3.3 times. Combination of CP and cAMP resulted in tumor regression, and endogenous cAMP levels in tumor decreased by 10 times compared with the control. Similar dynamics of cAMP reductions was noted in the adrenal glands. The lungs, thymus, lymph nodes and especially the testes and the brain, on the contrary, showed accumulation of cAMP to the normal levels and higher. Conclusions: The range of cAMP levels in organs and tumors of rats receiving combination of CP and cAMP demonstrated the development of adaptive and regenerative processes in organs responsible for the neuroendocrine regulation, suppression of stimulation of stress-realizing systems and metabolic support of the processes of increasing non-specific antitumor resistance along with inhibited proliferative activity of tumors.

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