Connexin 43 is involved in the sympathetic atrial fibrillation in canine and canine atrial myocytes

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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Connexin-43-Hemichannel-Mediated ATP Efflux Triggers Arrhythmogenic CA2+ Waves via P2X Purinoceptor Current in Atrial Myocytes

Biophysical Journal ◽

10.1016/j.bpj.2017.11.1662 ◽

2018 ◽

Vol 114 (3) ◽

pp. 290a

Author(s):

Joon-Chul Kim ◽

Min-Jeong Son ◽

Qui Anh Le ◽

Sun-Hee Woo

Keyword(s):

Connexin 43 ◽

Atrial Myocytes ◽

P2x Purinoceptor ◽

Atp Efflux

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Abstract 84: Atrial Fibrillation Causes a Progressive, Time-Dependent Reduction in Myocardial Neuronal NOS in Humans and Goats: Implications for AF-Induced Electrical Remodeling

Circulation Research ◽

10.1161/res.111.suppl_1.a84 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Svetlana Reilly ◽

Xing Liu ◽

Raja Jayaram ◽

Sunder Verheule ◽

Uli Schotten ◽

...

Keyword(s):

Atrial Fibrillation ◽

Electrical Properties ◽

Tissue Concentration ◽

Specific Effect ◽

Right Atrial ◽

Early Event ◽

Atrial Myocytes ◽

History Of ◽

Neuronal Nos ◽

The Difference

Rationale: Nitric oxide (NO) plays a key role in the regulation of cardiac and endothelial function and thrombogenesis. Atrial fibrillation (AF) has been associated with reduced NO availability but the mechanisms and implications of this finding remain to be fully investigated. Methods and Results: We evaluated NO synthase (NOS) activity and localization in right atrial (RA) tissue from 30 patients with permanent AF (vs. 65 controls in sinus rhythm, SR), and in the RA and left atrial (LA) tissue of 48 goats after 2 weeks (2W) and 6 months (6M) of pacing-induced AF. NOS activity was uncoupled in RA tissue from patients and goats in 6M-AF, and was caused by a reduction in BH4 tissue concentration and by an increase in arginase activity (HPLC). Although BH4 and arginine supplementation re-coupled NOS, it did not abolish the difference in NOS activity between AF and SR. Immunoblotting and immunolocalization revealed a progressive reduction in bi-atrial neuronal NOS (nNOS) protein with the duration of AF (by 65% at 2W, 86% at 6M in goats and by 62% in patients with AF) and a reduction in eNOS in long-standing AF. nNOS was reduced in atrial myocytes but not in neuronal tissue. The mRNA expression of NOS (qRT-PCR) was unaltered; however, the reduction in nNOS protein in AF was associated with an increase in nNOS ubiquitination which was partially reversed by inhibition of proteosomal activity with MG132; inhibition of the autophagy-lysosomal pathway with bafilomycin A1 did not restore nNOS protein. To investigate the electrophysiological consequences of a reduced nNOS in LA and RA myocytes, we compared electrical properties of the isolated atrial myocytes from nNOS-/- mice (n=18) and their wild type (WT) littermates after nNOS inhibition with SMTC. Both nNOS gene deletion and inhibition impaired myocytes' relaxation in both RA and LA, and result in a slower rate of decay of [Ca2+]i transient in the LA myocytes only. Conclusions: A reduction in bi-atrial nNOS activity and protein level is an early event in the natural history of AF that results in a chamber-specific effect on electrical properties of the myocytes.

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Abstract 3490: Mechanistic Involvement of Myeloperoxidase in the Pathogenesis of Atrial Fibrillation

Circulation ◽

10.1161/circ.118.suppl_18.s_434-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Volker Rudolph ◽

Rene Andrie ◽

Kai Friedrichs ◽

Tanja K Rudolph ◽

Anna Klinke ◽

...

Keyword(s):

Atrial Fibrillation ◽

Ex Vivo ◽

Strong Association ◽

Neutrophil Activation ◽

Right Atrial ◽

Atrial Myocytes ◽

Knock Out ◽

Protein Residues ◽

Heme Enzyme ◽

Knock Out Mice

Background: Observational clinical and ex-vivo studies have established a strong association between atrial fibrillation (AF) and inflammation. However, whether inflammation is cause or consequence of AF and which specific inflammatory mediators increase atrial susceptibility to fibrillate remain elusive. Herein, we provide evidence for mechanistic involvement of myeloperoxidase (MPO), a heme enzyme abundantly expressed by neutrophils, in the pathophysiology of AF. Methods and Results: Patients with AF assessed by pacemaker interrogation not only exhibited higher circulating plasma levels of MPO (503.1 [IR:404.6 –880.7] vs. 437.8 [IR:348.9 – 488.0 pmol/l; p=0.03; n=42), they also revealed an increased MPO burden in explanted left atrial tissue as compared to patients devoid of AF. In AF-patients MPO co-localized with markedly increased formation of 3-nitro and 3-chlorotyrosin, protein oxidations known to be catalyzed by MPO. Myeloperoxidase knock-out mice, pretreated with angiotensin II infusion for 2 weeks yielding increased neutrophil activation, revealed strikingly attenuated vulnerability for AF during right atrial electrophysiological stimulation as compared to wild type mice (probability of AF-induction: 3.0 vs. 12.7%; p<0.01). Whereas the electrical homogeneity of the atrial myocytes was not altered between the groups, atria of MPO knock out mice were indicative of significantly reduced atrial fibrosis and markedly reduced formation of 3-chloro- and 3-nitrotyrosine. Conclusion: In conclusion, the current findings not only underscore the significance of neutrophil activation as a critical pathophysiological prerequisite of AF, but reveal that MPO - by oxidatively modifying protein residues and increasing fibrosis of atrial myocytes - is causally linked to the initiation and perpetuation of AF.

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P2863Regional specific remodeling of cGMP pathway in human atrial fibrillation

European Heart Journal ◽

10.1093/eurheartj/ehz748.1172 ◽

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.

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P2869Role of PDE8 in cAMP dynamics in human atrial fibrillation

European Heart Journal ◽

10.1093/eurheartj/ehz748.1177 ◽

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.

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