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Improving the quality of life and reducing sudden cardiac death by limiting the impact of heart rhythm disturbances.
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OUR MISSION: TO REDUCE THE BURDEN OF CARDIOVASCULAR DISEASE
Shokoufeh GHEZELBASH for the presentation on ''Enhanced Expression and PKCdelta-mediated Hyperphosphorylation Underlie the Proarrythmic Increase in Sodium-Calcium-Exchanger Activity in Patients with Chronic Atrial Fibrillation''
Ghezelbash S, Molina CE, Badimon L, Kamler M, Heijman J, Dobrev D. Centre of affiliation: Institute of Pharmacology, University Duisburg Duisburg-Essen, Essen-Germany
Marie-Cécile KIENITZ for the presentation on ''Receptor-species dependent desensitization controls IKs as a downstream effector of Gq protein-coupled receptors''
Marie-Cécile Kienitz, Dilyana Vladimirova, Christian Müller, Lutz Pott and Andreas Rinne. Centre of affiliation: Department of Cellular Physiology, Ruhr-University, Bochum-Germany
Activation of Gq protein-coupled receptors (GqPCRs) has been shown to induce divergent cellular responses, which might be attributed to receptor-specific activation of different branches of the Gq signalling pathway or different efficiencies in activating downstream Gq effectors. Receptor-specific desensitization might provide a mechanism of effector modulation by restricting the spatiotemporal activation of signalling components downstream of Gq.
To adress this hypothesis, we expressed biosensors that rely on Förster Resonance Energy Transfer (FRET) in HEK 293 cells or in a KCNQ1/KCNE1 CHO cell line to quantify desensitization of GqPCR signalling on the level of Gq protein activation and downstream effectors, such as phospholipase C (PLC). Simultaneous measurements of the ionic current IKs of KCNQ1/KCNE1 channels as a functional readout for GqPCR signalling allowed us to precisely relate the degree of receptor desensitization with the time course of IKs modulation.
Activation of the M1 receptor for acetylcholine (M1-R) caused robust inhibition of IKs with kinetics that resembled the time course of PIP2-depletion and recovery of IKs upon removal of the agonist. In contrast, the a1B-adrenergic receptor (a1B-AR) induced an inhibition of IKs that was transient and turned into activation after agonist withdrawal. This delayed response exceeded basal activation levels and occurred when the PIP2 pool was completely restored.
Furthermore, the a1B-AR, but not the M1-R, displayed substantial desensitization in the presence of agonist that was evident on the levels of Gq and PLC activation.
This suggests a feedback modulation by kinases. Indeed, global inhibition of all protein kinase C (PKC) isoforms with staurosporine removed desensitization of a1B-ARs and abolished the delayed facilitation of IKs. A more specific inhibition of Ca2+-sensitive PKCs with Gö6976 reduced a1B-AR desensitization but not facilitation of IKs, which was rather mediated by diacylglycerol-activated kinases. Notably, activation of M1-R and its influence on IKs were not dependent on PKC activation.
We conclude from these data that fine-tuning of IKs activity via Gq proteins is controlled by recruitment of kinases downstream of PIP2 depletion in a receptor species-specific fashion.
Cristina MORENO VADILLO for the presentation on ''Priming for Action: Interventions That Promote IKs-Channel Opening Restore Defective cAMP-Dependent Upregulation in Long-QT1 Syndrome''
Cristina Moreno, Roel LHMG Spätjens, Sandrine. Seyen, Gabriele. Menini, Paul G.A. Volders
Patients with the KCNQ1 mutation A341V are at risk of sympathetically-triggered ventricular tachyarrhythmias and sudden cardiac death. A341V decreases basal Iks and disrupts cAMP-dependent upregulation due to impaired phosphorylation at S27, which can be rescued by the phosphomimetic substitution KCNQ1-S27D. We hypothesize that A341V imposes a different conformational state of the Iks channel, which prevents full phosphorylation of KCNQ1-S27 and therefore loss of upregulation.Here, we examine whether strategies promoting channel opening can rescue cAMP dependent upregulation.
Chinese hamster ovary cells were transfected with wildtype KCNQ1 (WT) or A341V-KCNQ1 in heterozygosis (WT+A341V-KCNQ1), together with KCNE1 and Yotiao. In the absence and presence of cAMP/okadaic acid (OA), we examined IKs responses to (1) the KCNQ1 I346-K358 short peptide (shown to unlock the closed state of IKs by binding to the endogenous S4-S5-linker of KCNQ1), (2) the double mutant A341V/L353K (L353K was previously shown to promote constitutively open channels), and (3) the pharmacological channel activator ML277 (1 μmol/L).
As previously shown, WT but not A341V IKs was upregulated by intrapipette cAMP/OA. The unlocking effect of the I346-K358 peptide and the constitutive conduction produced by the co-presence of L353K facilitated A341V IKs enhancement by cAMP/OA (+61% and +71%, respectively). Similar results were obtained for the IKs activator ML277. Under baseline conditions, ML277 increased both WT and A341V IKs by approximately 60%. The IKs response to ML277 in cells stimulated with cAMP/OA was amplified more than twofold in WT, but strikingly also in A341V IKs. This larger ML277 effect is likely explained by an augmented cAMP-dependent regulation of A341V IKs.
Pharmacological and molecular interventions promoting IKs-channel opening rescue from defective cAMP-dependent upregulation by the hotspot mutation A341V. Our results suggest novel therapeutic strategies to restore the beta-adrenergic control of repolarization in long-QT1 syndrome.
Angelo G. TORRENTE for the presentation on ''L-type Cav1.3 Channels facilitate Ryanodine Receptor-mediated Sarcoplasmic Ca2+ Release during Sino-Atrial Node Pacemaker Activity''
Angelo G. Torrente, Pietro Mesirca, Patricia Neco, Riccardo Rizzetto, Christian Barrere, Joerg Striessnig, Sylvain Richard, Joël Nargeot, Ana Maria Gomez, Matteo E. Mangoni. Centre of affiliation: IGF – CNRS, Montpellier-France
The Sino-atrial node (SAN) is the main pacemaker tissue that in normal condition control heart rate generation. Despite its essential function, its working mechanism is still not completely understood. Several studies proposed the dynamic of intracellular Ca2+ ([Ca2+]i) as an essential process for the generation and regulation of SAN pacemaker activity, but they misconceived the implication of voltage-dependent L-type Ca2+ channels (LTCCs) in this mechanism. Cav1.3 is the predominant LTCC channel isoform in SAN cells, and could constitutes an important influx of Ca2+ already during the diastolic depolarization phase (DDP) characterizing SAN cells. Therefore, we used isolated cells and ex vivo SAN preparations obtained from wild-type (WT) and Cav1.3 knockout (KO) mice (Cav1.3-/-), to understand how Cav1.3 intervene to the intracellular Ca2+ dynamics of these systems.
Methods and results:
We used action potentials recorded in WT SAN cells as voltage-clamp commands for Cav1.3-/- cells. These experiments confirmed the presence of Cav1.3 mediated-Ca2+ influx during DDP of WT, and showed abolished Ca2+ entry in the same range of KO SAN cells. Nevertheless, the quantification of sarcoplasmic Ca2+ load using Fura-2 indicated equivalent charge in both genotypes. Consequently to Cav1.3 ablation, we observed reduction of local [Ca2+]i release events frequency and synchronization. This impairment inhibited the generation of Ca2+ transients and delayed spontaneous activity, indicating a global deficiency of [Ca2+]i dynamics in KO cells. Pacemaker activity in both genotypes responded positively to the β-adrenergic stimulation, nevertheless Cav1.3-/- SAN cells remained slower than WT, indicating that the activation of this pathway cannot compensate the absence of the nodal LTCC. Instead, we rescued automaticity in SAN cells and intact tissue from Cav1.3-/-, through direct caffeine stimulation of Ryanodine Receptor (RyR) mediated Ca2+-induced Ca2+-release..
Cav1.3 channels play a critical role in the regulation of [Ca2+]i dynamics, facilitating RyR activation and providing an unanticipated mechanism for triggering local [Ca2+]i releases and thereby controlling pacemaker activity.
Our study also provides an additional pathophysiologic mechanism for congenital SAN dysfunction and heart block linked to Cav1.3 loss of function in humans.
Alexandra ZAHRADNÍKOVÁ for the presentation on ''Development of excitation-contraction coupling in young rat hearts''
Zahradníková jr., A., Macková, K., Zahradník I., Zahradníková A.. Centre of affiliation: Institute of Molecular Physiology and Genetics, Bratislava-Slovakia
In mammalian ventricular myocytes, excitation-contraction coupling is dependent on spatially distributed and synchronized calcium release. This takes place in dyadic junctions, where calcium release channels (RyRs) come into close contact with voltage sensitive DHPR channels of the sarcolemma. To spread the excitation signal evenly throughout the ventricular myocyte, sarcolemma forms a network of t-tubules. This network develops during maturation of ventricular myocytes, which in rats takes place during the early postnatal period. In the process of excitation-contraction coupling, RyRs are activated by calcium ions injected during the action potential through DHPRs. Subsequently, calcium ions released from the sarcoplasmic reticulum by RyRs rapidly inactivates DHPR channels.
In this work we have compared the changes in morphology and growth of t-tubules with the development of calcium signalling. We have used ventricular myocytes isolated from 1-21 days old and adult rats (> 60 days).
To quantify the development of t-tubular network, we have stained isolated myocytes with the lipophilic dye FM 4-64 and imaged the cells using laser-scanning confocal microscopy. We used the stereological method of vertical sections to estimate the surface density of sarcolemma and its components – free plasmalemma, transversal tubules and longitudinal tubules. To characterize the development of calcium signalling, we used whole-cell patch-clamp to record calcium currents using 80-ms depolarization pulses from ‑50 to 0 mV. We have analysed the maximum amplitude, kinetics of calcium current inactivation, and the fraction of the fast (calcium release-dependent) component of inactivation.
We observed initial formation of sarcolemmal invaginations around day 9. The surface density of sarcolemma increased proportionally to the increase of cell capacitance, while the fraction of free plasmalemma decreased. The amplitude of calcium current increased with ICa density saturating at adult values after day 11. The fast component of ICa inactivation was first observed at day 4. Its fraction increased with age, reaching adult values at around day 19. When present, the time constant of fast ICa inactivation in neonatal myocytes was the same as in adult ventricular myocytes.
These data suggest that functional local calcium signalling, that is formation of dyadic junctions, precedes development of t-tubules.
Support: APVV-15-0302-CAMYS, VEGA 2/0095/15, VEGA 2/0147/14
Susanne Scherübel for the presentation on: "The Contribution of If and Ik1 to Focal Activity in Atrial Fibrillation"S. Scherübel(a), C. Koyani(b), P. Lang(a), E. Bernhart(b), S. Hallström(c), H. Mächler(d), G. Plank(a), K. Zorn-Pauly(a), B. Pelzmann(a)(a) Institute of Biophysics, Medical University of Graz(b) Institute of Molecular Biology and Biochemistry, Medical University of Graz(c) Institute of Physiological Chemistry, Medical University of Graz
Nadjet BELBACHIR for the presentation on ''Rad GTPase as a new potential actor in Brugada syndrome''
Nadjet Belbachir, Vincent Portero, Eva LePogam, Nathalie Gaborit, Solena Le Scouarnec, Isabelle Baró, Christophe Guilluy, Celine Marionneau, Vincent Probst, Jean-Jacques Schott, Richard Redon, Flavien Charpentier. Centre of affiliation: l'institut du thorax, Nantes-France
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