The European Network for Translational Research in Atrial Fibrillation (EUTRAF): objectives and initial results.

Atrial fibrillation (AF) is the most common sustained arrhythmia in the general population. As an age-related arrhythmia AF is becoming a huge socio-economic burden for European healthcare systems. Despite significant progress in our understanding of the pathophysiology of AF, therapeutic strategies for AF have not changed substantially and the major challenges in the management of AF are still unmet. This lack of progress may be related to the multifactorial pathogenesis of atrial remodelling and AF that hampers the identification of causative pathophysiological alterations in individual patients. Also, again new mechanisms have been identified and the relative contribution of these mechanisms still has to be established. In November 2010, the European Union launched the large collaborative project EUTRAF (European Network of Translational Research in Atrial Fibrillation) to address these challenges. The main aims of EUTRAF are to study the main mechanisms of initiation and perpetuation of AF, to identify the molecular alterations underlying atrial remodelling, to develop markers allowing to monitor this processes, and suggest strategies to treat AF based on insights in newly defined disease mechanisms. This article reports on the objectives, the structure, and initial results of this network

Activation of the cAMP–protein kinase A pathway facilitates Na+ translocation by the Na+–K+ pump in guinea-pig ventricular myocytes

The effects of the adenylyl cyclase activator forskolin on steady-state and transient currents generated by the Na+-K+ pump were studied in guinea-pig ventricular myocytes by means of whole-cell voltage clamp at 30 °C.In external solution containing 144 mM Na+ (Nao+) and 10 mM K+ (Ko+), steady-state Na+-K+ pump current (Ip) activated by 5 mM pipette Na+ ( Napip+) at -20 mV was reversibly augmented by forskolin (4 μM) to 133 ± 4 % of the control current (n = 15). The forskolin analogue 1,9-dideoxyforskolin (10 μM), which does not activate adenylyl cyclases, did not increase Ip (n = 2). Application of the protein kinase A (PKA) inhibitor H-89 (10 μM) in the continued presence of forskolin reversed the forskolin-induced elevation of Ip (n = 3).The forskolin effect on Ip persisted in the presence of 50 mM Napip+ which ensured that the internal Na+-binding sites of the Na+-K+ pump were nearly saturated. Under these conditions, the drug increased Ip to 142 ± 3 % of the control Ip when the pipette free Ca2+ concentration ([Ca2+]pip) was 0·013 nM (n = 5) and to 138 ± 4 % of the control Ip when free [Ca2+]pip was 15 nM (n = 9).In Na+-free external solution, Ip activated by 50 mM Napip+ and 1·5 mM Ko+ was likewise increased by forskolin but to a lesser extent than in Na+-containing medium (116 ± 3 % of control, n = 10).In order to investigate exclusively partial reactions in the Na+ limb of the pump cycle, transient pump currents under conditions of electroneutral Na+-Na+ exchange were studied. Transient pump currents elicited by voltage jumps displayed an initial peak and then decayed monoexponentially. Moved charge (Q) and the rate constant of current decay varied with membrane potential (V). The Q-V relationship followed a Boltzmann distribution characterized by the midpoint voltage (V0·5) and the maximum amount of movable charge (ΔQmax). Forskolin (2-10 μM) shifted V0·5 to more negative values while ΔQmax was not affected (n = 11). The effects of forskolin on transient pump currents were mimicked by 8-bromo-cAMP (500 μM; n = 2) and abolished by a peptide inhibitor of PKA (PKI, 10 μM; n = 5).We conclude that activation of the cAMP-PKA pathway in guinea-pig ventricular myocytes increases Na+-K+ pump current at least in part by modulating partial reactions in the Na+ limb of the pump cycle. Under physiological conditions, the observed stimulation of the cardiac Na+-K+ pump may serve to shorten the action potential duration and to counteract the increased passive sarcolemmal Na+ and K+ fluxes during sympathetic stimulation of the heart

Anti-inflammatory effects of endothelin receptor blockade in left atrial tissue of spontaneously hypertensive rats

Objective: In spontaneously hypertensive rats (SHR) atrial remodeling has been shown to involve increase in endothelin (ET) signaling. Furthermore, inflammatory processes may further contribute to tissue remodeling. The aimed of this study was to investigate whether an endothelin receptor antagonist, macitentan, could reduce left atrial (LA) remodeling in arterial hypertension. Methods: Molecular characterization of atria was performed in SHR at the age of 8 months and their age-matched normotensive control rats (WKY). SHR were treated with macitentan and, for comparison with a blood pressure reducing drug, with doxazosin. After two months of treatment, molecules involved in endocardial inflammation and atrial calcium handling were assessed. The molecular changes provoked by rapid-pacing (RAP) were analyzed in atrial tissue slices. Results: Doxazosin reduced the systolic blood pressure compared with the untreated SHR (159 ± 26 vs. 176 ± 17; P < 0.05) or macitentan (vs. 189 ± 21; P < 0.05). Macitentan lowered the increased levels of atrial ET-1 and abrogated the pacing-induced upregulation of preproET-1-mRNA in atrial slices from SHR. Macitentan reduced the elevated levels of atrial 8-isoprostanes, the increased expression of pro-inflammatory ICAM-1 and IL-8, the phosphorylation of MAP kinases, ERK and p38, the phosphorylation of NF-κB and the expression of VCAM-mRNA. Major Ca2+-regulating proteins and markers of hypertrophy and fibrosis, however, were not affected. Doxazosin elicited similar changes, except for the alterations in ET-1 levels, NF-κB phosphorylation and VCAM-mRNA. Conclusion: Macitentan reversed pro-inflammatory remodeling in hypertensive atria in a blood pressure-independent manner, which might prevent endocardial dysfunction and thereby, thrombogenesis in arterial hypertension

Urocortin II enhances contractility in rabbit ventricular myocytes via CRF2 receptor-mediated stimulation of protein kinase A

Objective: Urocortin II (UcnII), a peptide of the corticotropin-releasing factor (CRF) family, exerts profound actions on the cardiovascular system. Direct effects of UcnII on adult cardiomyocytes have not been evaluated before. Our aim was to characterize functional effects of UcnII on cardiomyocytes and to elucidate the underlying signaling pathway(s) and cellular mechanisms. Methods: Rabbit ventricular cardiomyocytes were stimulated at 0.5 Hz (22-25 degrees C). Unloaded cell shortening (FS, edge detection), [Ca2+](i) transients (Fluo-4), and L-type Ca2+ currents (I-Ca, whole-cell patch clamping) were measured. Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid application of caffeine (20 mmol/L). Results: UcnII increased cell shortening and accelerated relaxation in a time- and concentration-dependent manner (EC50: 10.7 nmol/L). The inotropic effect of UcnII was maximal at 100 nmol/L (35% +/- 11% increase in FS, n=8, P<0.05). The inotropic and lusitropic actions of UcnII were largely eliminated by inhibition of CRF2 receptors (10 nmol/L antisauvagine-30, n = 5) or protein kinase A (PKA, 500 nmol/L H-89, n=5). UcnII increased [Ca2+](i) transient amplitude (by 63% +/- 35%, n=7, P<0.05) and decreased the time constant for decay (from 800 +/- 63 to 218 +/- 27 ms, n=7,P<0.001). UcnII also increased SR Ca2+ load (by 19% +/- 7%, n=7,P<0.05) and fractional Ca2+ release (from 57% +/- 7% to 98% +/- 2%, n=7, P<0.01). I-Ca was augmented by 32.7% +/- 10.0% (n=9, P<0.05) and the I-Ca-V relationship was shifted by - 15 mV during UcnII treatment. Conclusion: UcnII exerts positive inotropic and lusitropic effects in cardiomyocytes via CRF2 receptor-mediated stimulation of PKA which augments I-Ca and SR Ca2+ load to increase SR Ca2+ release and [Ca2+](i) transients. (C) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved

Activation and Propagation of Ca2+ Release during Excitation-Contraction Coupling in Atrial Myocytes

AbstractFast two-dimensional confocal microscopy and the Ca2+ indicator fluo-4 were used to study excitation-contraction (E-C) coupling in cat atrial myocytes which lack transverse tubules and contain both subsarcolemmal junctional (j-SR) and central nonjunctional (nj-SR) sarcoplasmic reticulum. Action potentials elicited by field stimulation induced transient increases of intracellular Ca2+ concentration ([Ca2+]i) that were highly inhomogeneous. Increases started at distinct subsarcolemmal release sites spaced ∼2μm apart. The amplitude and the latency of Ca2+ release from these sites varied from beat to beat. Subsarcolemmal release fused to build a peripheral ring of elevated [Ca2+]i, which actively propagated to the center of the cells via Ca2+-induced Ca2+ release. Resting myocytes exhibited spontaneous Ca2+ release events, including Ca2+ sparks and local (microscopic) or global (macroscopic) [Ca2+]i waves. The microscopic [Ca2+]i waves propagated in a saltatory fashion along the sarcolemma (“coupled” Ca2+ sparks) revealing the sequential activation of Ca2+ release sites of the j-SR. Moreover, during global [Ca2+]i waves, Ca2+ release was evident from individual nj-SR sites. Ca2+ release sites were arranged in a regular three-dimensional grid as deduced from the functional data and shown by immunostaining of ryanodine receptor Ca2+ release channels. The longitudinal and transverse distances between individual Ca2+ release sites were both ∼2μm. Furthermore, electron microscopy revealed a continuous sarcotubular network and one peripheral coupling of j-SR with the sarcolemma per sarcomere. The results demonstrate directly that, in cat atrial myocytes, the action potential-induced whole-cell [Ca2+]i transient is the spatio-temporal summation of Ca2+ release from subsarcolemmal and central sites. First, j-SR sites are activated in a stochastic fashion by the opening of voltage-dependent sarcolemmal Ca2+ channels. Subsequently, nj-SR sites are activated by Ca2+-induced Ca2+ release propagating from the periphery

cAMP- and Ca²⁺/calmodulin-dependent protein kinases mediate inotropic, lusitropic and arrhythmogenic effects of urocortin 2 in mouse ventricular myocytes

BACKGROUND AND PURPOSE: Urocortin 2 is beneficial in heart failure, but the underlying cellular mechanisms are not completely understood. Here we have characterized the functional effects of urocortin 2 on mouse cardiomyocytes and elucidated the underlying signalling pathways and mechanisms. EXPERIMENTAL APPROACH: Mouse ventricular myocytes were field-stimulated at 0.5 Hz at room temperature. Fractional shortening and [Ca(2+)](i) transients were measured by an edge detection and epifluorescence system respectively. Western blots were carried out on myocyte extracts with antibodies against total phospholamban (PLN) and PLN phosphorylated at serine-16. KEY RESULTS: Urocortin 2 elicited time- and concentration-dependent positive inotropic and lusitropic effects (EC(50): 19 nM) that were abolished by antisauvagine-30 (10 nM, n = 6), a specific antagonist of corticotrophin releasing factor (CRF) CRF(2) receptors. Urocortin 2 (100 nM) increased the amplitude and decreased the time constant of decay of the underlying [Ca(2+)](i) transients. Urocortin 2 also increased PLN phosphorylation at serine-16. H89 (2 µM) or KT5720 (1 µM), two inhibitors of protein kinase A (PKA), as well as KN93 (1 µM), an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), suppressed the urocortin 2 effects on shortening and [Ca(2+)](i) transients. In addition, urocortin 2 also elicited arrhythmogenic events consisting of extra cell shortenings and extra [Ca(2+)](i) increases in diastole. Urocortin 2-induced arrhythmogenic events were significantly reduced in cells pretreated with KT5720 or KN93. CONCLUSIONS AND IMPLICATIONS: Urocortin 2 enhanced contractility in mouse ventricular myocytes via activation of CRF(2) receptors in a cAMP/PKA- and Ca(2+)/CaMKII-dependent manner. This enhancement was accompanied by Ca(2+)-dependent arrhythmogenic effects mediated by PKA and CaMKII

Overexpression of CaMKIIδc in RyR2(R4496C+/-) Knock-In Mice Leads to Altered Intracellular Ca(2+) Handling and Increased Mortality

OBJECTIVES: We investigated whether increased Ca(2+)/calmodulin-dependent kinase II (CaMKII) activity aggravates defective excitation-contraction coupling and proarrhythmic activity in mice expressing R4496C mutated cardiac ryanodine receptors (RyR2). BACKGROUND: RyR2 dysfunction is associated with arrhythmic events in inherited and acquired cardiac disease. METHODS: CaMKIIδc transgenic mice were crossbred with RyR2(R4496C+/-) knock-in mice. RESULTS: Heart weight-to-body weight ratio in CaMKIIδc/RyR2(R4496C) and CaMKIIδc mice was similarly increased approximately 3-fold versus wild-type mice (p < 0.05). Echocardiographic data showed comparable cardiac dilation and impaired contractility in CaMKIIδc/RyR2(R4496C) and CaMKIIδc mice. Sarcoplasmic reticulum Ca(2+) content in isolated myocytes was decreased to a similar extent in CaMKIIδc/RyR2(R4496C) and CaMKIIδc mice. However, relaxation parameters and Ca(2+) decay at 1 Hz were prolonged significantly in CaMKIIδc mice versus CaMKIIδc/RyR2(R4496C) mice. Sarcoplasmic reticulum Ca(2+) spark frequency and characteristics indicated increased sarcoplasmic reticulum Ca(2+) leak in CaMKIIδc/RyR2(R4496C) versus CaMKIIδc myocytes (p < 0.05), most likely because of increased RyR2 phosphorylation. Delayed afterdepolarizations were significantly more frequent with increased amplitudes in CaMKIIδc/RyR2(R4496C) versus CaMKIIδc mice. Increased arrhythmias in vivo (67% vs. 25%; p < 0.05) may explain the increased mortality in CaMKIIδc/RyR2(R4496C) mice, which died prematurely with only 30% alive (vs. 60% for CaMKIIδc, p < 0.05) after 14 weeks. CONCLUSIONS: CaMKIIδc overexpression in RyR2(R4496C+/-) knock-in mice increases the propensity toward triggered arrhythmias, which may impair survival. CaMKII contributes to further destabilization of a mutated RyR2 receptor