976Adenosine A2A receptor activation induces afterdepolarizations in human atrial myocytes by selectively increasing the calcium spark frequency near the sarcolemma

Background: Hypoxic-ischemic encephalopathy (HIE) is a major cause of pediatric and adult mortality and morbidity. Unfortunately, to date, no effective treatment has been identified. In the striatum, neuronal injury is analogous to the cellular mechanism of necrosis observed during NMethyl- D-Aspartate (NMDA) excitotoxicity. Adenosine acts as a neuromodulator in the central nervous system, the role of which relies mostly on controlling excitatory glutamatergic synapses. Objective: To examine the effect of pretreatment of SCH58261, an adenosine 2A (A2A) receptor antagonist and modulator of NMDA receptor function, following hypoxic-ischemia (HI) on sodium- potassium ATPase (Na+, K+-ATPase) activity and oxidative stress. Methods: Piglets (4-7 days old) were subjected to 30 min hypoxia and 7 min of airway occlusion producing asphyxic cardiac arrest. Groups were divided into four categories: HI samples were divided into HI-vehicle group (n = 5) and HI-A2A group (n = 5). Sham controls were divided into Sham vehicle (n = 5) and Sham A2A (n = 5) groups. Vehicle groups were pretreated with 0.9% saline, whereas A2A animals were pretreated with SCH58261 10 min prior to intervention. Striatum samples were collected 3 h post-arrest. Sodium-potassium ATPase (Na+, K+-ATPase) activity, malondialdehyde (MDA) + 4-hydroxyalkenals (4-HDA) and glutathione (GSH) levels were compared. Results: Pretreatment with SCH58261 significantly attenuated the decrease in Na+, K+-ATPase, decreased MDA+4-HDA levels and increased GSH in the HI-A2A group when compared to HIvehicle. Conclusion: A2A receptor activation may contribute to neuronal injury in newborn striatum after HI in association with decreased Na+, K+-ATPase activity and increased oxidative stress.

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A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators

International Journal of Molecular Sciences ◽

10.3390/ijms22031417 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1417

Author(s):

Azeem Danish ◽

Robin Gedschold ◽

Sonja Hinz ◽

Anke C. Schiedel ◽

Dominik Thimm ◽

...

Keyword(s):

Gap Junctions ◽

Small Molecules ◽

High Throughput Screening ◽

Cell Communication ◽

Receptor Activation ◽

Adenosine A2a Receptor ◽

Intracellular Camp ◽

Donor Cells ◽

A2a Receptor ◽

Adenosine A2a

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

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Treatment with beta-blockers normalizes RyR2 phosphorylation and calcium spark activity in atrial myocytes from patients with atrial fibrillation

European Heart Journal ◽

10.1093/ehjci/ehaa946.3691 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

V Jimenez-Sabado ◽

S Casabella ◽

P Izquierdo ◽

C Tarifa ◽

A Llach ◽

...

Keyword(s):

Atrial Fibrillation ◽

Calcium Imaging ◽

Adrenergic Receptors ◽

Beta Blockers ◽

Funding Source ◽

Atrial Myocytes ◽

Calcium Sparks ◽

Beta Adrenergic ◽

Calcium Spark ◽

Confocal Calcium Imaging

Abstract Background Atrial fibrillation has been associated with an increase in ryanodine receptor (RyR2) phosphorylation and local calcium release (calcium sparks). Carvedilol, a nonselective beta-adrenergic receptor blocker also inhibits the cardiac ryanodine receptor (RyR2), but it has been suggested that the enantiomer R-carvedilol only inhibits RyR2 activity and hence has the potential to inhibit calcium sparks without affecting RyR2 phosphorylation. Purpose This study aimed to determine the ability of the enantiomers R- and S-carvedilol to reverse RyR2 phosphorylation at s2808 and calcium sparks induced by the β2-adrenergic agonist fenoterol, in order to determine the relationship between RyR2 phosphorylation at s2808 and calcium spark frequency, and to assess the efficacy of R- and S-carvedilol. Methods Human right atrial myocytes were isolated and subjected to immunofluorescent labelling of total and s2808 phosphorylated RyR2, or loaded with fluo-4 and subjected to confocal calcium imaging. Beta-adrenergic receptors were first activated with 3μM fenoterol and then inhibited by different concentrations of carvedilol R- or S-enantiomers. Results Incubation of myocytes with fenoterol increased the s2808/RyR2 ratio from 0.32±0.03 to 0.66±0.05 (n=18, p<0.001). Incubation with 0.1, 0.3, 1 or 3μM R-carvedilol in the presence of fenoterol changed the s2808/RyR2 ratio to 0.64±0.05, 0.44±0.04, 0.34±0.07 and 0.28±0.05 (p<0.01) respectively. For comparison 3μM S-carvedilol reduced the s2808/RyR2 ratio to 0.23±0.06 in myocytes from 5 patients (p<0.01). Confocal calcium imaging revealed that fenoterol increased the spark density from 0.28±0.04 to 1.24±0.25 events/s/1000μm2 (n=9, p<0.01) and addition of 0.1, 0.3, or 1μM R-carvedilol changed the frequency to 1.32±0.52, 0.38±0.05, and 0.15±0.05 events/s/1000μm2 (p<0.01) respectively. Analysis of atrial myocytes from patients without atrial fibrillation revealed that the s2808/RyR2 ratio was similar in 25 patients treated with beta-blockers (0.39±0.04) and 57 that did not receive beta-blockers (0.44±0.03, p=0.33) while the s2808/RyR2 ratio was significantly smaller in 16 patients with atrial fibrillation receiving beta-blockers (0.43±0.08) than in 5 patients that did not (0.80±0.19, p<0.05). Conclusions R-carvedilol reverses the effects of beta-adrenergic stimulation on s2808 phosphorylation and calcium sparks in human atrial myocytes, and treatment with beta-blockers reduces excessive RyR2 phosphorylation at s2808 in patients with atrial fibrillation to levels observed in those without the arrhythmia, pointing to beta-adrenergic receptors as a target for controlling RyR2 phophorylation and activity in atrial fibrillation. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Science and Innovation & Spanish Ministry of Health and Consume

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