Conditional immortalization of human atrial myocytes for the generation of in vitro models of atrial fibrillation

Rhythm disturbances are life-threatening cardiovascular diseases, accounting for many deaths annually worldwide. Abnormal electrical activity might arise in a structurally normal heart in response to specific triggers or as a consequence of cardiac tissue alterations, in both cases with catastrophic consequences on heart global functioning. Preclinical modeling by recapitulating human pathophysiology of rhythm disturbances is fundamental to increase the comprehension of these diseases and propose effective strategies for their prevention, diagnosis, and clinical management. In silico, in vivo, and in vitro models found variable application to dissect many congenital and acquired rhythm disturbances. In the copious list of rhythm disturbances, diseases of the conduction system, as sick sinus syndrome, Brugada syndrome, and atrial fibrillation, have found extensive preclinical modeling. In addition, the electrical remodeling as a result of other cardiovascular diseases has also been investigated in models of hypertrophic cardiomyopathy, cardiac fibrosis, as well as arrhythmias induced by other non-cardiac pathologies, stress, and drug cardiotoxicity. This review aims to offer a critical overview on the effective ability of in silico bioinformatic tools, in vivo animal studies, in vitro models to provide insights on human heart rhythm pathophysiology in case of sick sinus syndrome, Brugada syndrome, and atrial fibrillation and advance their safe and successful translation into the cardiology arena.

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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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Differential regulation of voltage-activated potassium currents in cultured human atrial myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1996.271.4.h1609 ◽

1996 ◽

Vol 271 (4) ◽

pp. H1609-H1619 ◽

Cited By ~ 1

Author(s):

S. N. Hatem ◽

A. Benardeau ◽

C. Rucker-Martin ◽

J. L. Samuel ◽

E. Coraboeuf ◽

...

Keyword(s):

Reversal Potential ◽

Maximal Response ◽

Atrial Myocytes ◽

Human Atrial Myocytes ◽

Kinase C ◽

High K ◽

Highly Sensitive ◽

K Current ◽

Current Reversal

To examine whether the two components of the voltage-activated outward K+ current, an initially rapidly inactivating component (Ito,1) and a slowly inactivating sustained component (Isus), in human atrial myocytes are distinct currents differentially regulated, we studied their behavior during serum-induced growth of cultured myocytes. Currents were recorded in whole cell patch clamped myocytes. After 1 wk of culture (day 8), membrane capacitance was twice the value in freshly dissociated myocytes (178.7 +/- 23 vs. 83.1 +/- 5.5 pF; P < 0.001). Ito,1 density did not differ from that in freshly dissociated myocytes (at +40 mV: 4.38 +/- 0.8 vs. 3.71 +/- 0.6 pA/pF), whereas that of Isus was markedly increased (at +40 mV: 9.76 +/- 2 vs. 2.21 +/- 0.29 pA/pF; P < 0.001). After inactivation of Ito,1 by a prepulse, sustained depolarization elicited in cultured myocytes an Isus with a density of 10.22 +/- 1.18 pA/pF and an apparent tail current reversal potential of -73.5 +/- 3.2 mV, indicating high K+ selectivity. Isus was highly sensitive to 4-aminopyridine (55.4 +/- 4.4% inhibition in 50 microM) and to D-600 (with a concentration inhibiting 50% of maximal response of 34.2 x 10(-6) M). Addition of 5-10 nM staurosporine at day 3 prevented cell growth and reduced Ito,1 density but not the increase in Isus density, which was inhibited by 10 microM staurosporine. Our results indicate that Ito,1 and Isus are regulated independently during in vitro myocyte growth in human atrial myocytes and that the increase in Isus density is not mediated by a protein kinase C-dependent pathway.

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