Irene Amorós

Nitric Oxide Increases Cardiac IK1 by Nitrosylation of Cysteine 76 of Kir2.1 Channels

Rationale: The cardiac inwardly rectifying K+ current (IK1) plays a critical role in modulating excitability by setting the resting membrane potential and shaping phase 3 of the cardiac action potential. Objective: This study aims to analyze the effects of nitric oxide (NO) on human atrial IK1 and on Kir2.1 channels, the major isoform of inwardly rectifying channels present in the human heart. Methods and Results: Currents were recorded in enzymatically isolated myocytes and in transiently transfected CHO cells, respectively. NO at myocardial physiological concentrations (25 to 500 nmol/L) increased inward and outward IK1 and IKir2.1. These effects were accompanied by hyperpolarization of the resting membrane potential and a shortening of the duration of phase 3 of the human atrial action potential. The IKir2.1 increase was attributable to an increase in the open probability of the channel. Site-directed mutagenesis analysis demonstrated that NO effects were mediated by the selective S-nitrosylation of Kir2.1 Cys76 residue. Single ion monitoring experiments performed by liquid chromatography/tandem mass spectrometry suggested that the primary sequence that surrounds Cys76 determines its selective S-nitrosylation. Chronic atrial fibrillation, which produces a decrease in NO bioavailability, decreased the S-nitrosylation of Kir2.1 channels in human atrial samples as demonstrated by a biotin-switch assay, followed by Western blot. Conclusions: The results demonstrated that, under physiological conditions, NO regulates human cardiac IK1 through a redox-related process.

Flecainide increases Kir2.1 currents by interacting with cysteine 311, decreasing the polyamine-induced rectification

Both increase and decrease of cardiac inward rectifier current (IK1) are associated with severe cardiac arrhythmias. Flecainide, a widely used antiarrhythmic drug, exhibits ventricular proarrhythmic effects while effectively controlling ventricular arrhythmias associated with mutations in the gene encoding Kir2.1 channels that decrease IK1 (Andersen syndrome). Here we characterize the electrophysiological and molecular basis of the flecainide-induced increase of the current generated by Kir2.1 channels (IKir2.1) and IK1 recorded in ventricular myocytes. Flecainide increases outward IKir2.1 generated by homotetrameric Kir2.1 channels by decreasing their affinity for intracellular polyamines, which reduces the inward rectification of the current. Flecainide interacts with the HI loop of the cytoplasmic domain of the channel, Cys311 being critical for the effect. This explains why flecainide does not increase IKir2.2 and IKir2.3, because Kir2.2 and Kir2.3 channels do not exhibit a Cys residue at the equivalent position. We further show that incubation with flecainide increases expression of functional Kir2.1 channels in the membrane, an effect also determined by Cys311. Indeed, flecainide pharmacologically rescues R67W, but not R218W, channel mutations found in Andersen syndrome patients. Moreover, our findings provide noteworthy clues about the structural determinants of the C terminus cytoplasmic domain of Kir2.1 channels involved in the control of gating and rectification.

Nitric oxide inhibits Kv4.3 and human cardiac transient outward potassium current (Ito1).

AIMS Chronic atrial fibrillation (CAF) is characterized by a shortening of the plateau phase of the action potentials (AP) and a decrease in the bioavailability of nitric oxide (NO). In this study, we analysed the effects of NO on Kv4.3 (I(Kv4.3)) and on human transient outward K(+) (I(to1)) currents as well as the signalling pathways responsible for them. We also analysed the expression of NO synthase 3 (NOS3) in patients with CAF. METHODS AND RESULTS I(Kv4.3) and I(to1) currents were recorded in Chinese hamster ovary cells and in human atrial and mouse ventricular dissociated myocytes using the whole-cell patch clamp. The expression of NOS3 was analysed by western blotting. AP were recorded using conventional microelectrode techniques in mouse atrial preparations. NO and NO donors inhibited I(Kv4.3) and human I(to1) in a concentration- and voltage-dependent manner (IC(50) for NO: 375.0 +/- 48 nM) as a consequence of the activation of adenylate cyclase and the subsequent activation of the cAMP-dependent protein kinase and the serine-threonine phosphatase 2A. The density of the I(to1) recorded in ventricular myocytes from wild-type (WT) and NOS3-deficient mice (NOS3(-/-)) was not significantly different. Furthermore, the duration of atrial AP repolarization in WT and NOS3(-/-) mice was not different. The increase in NO levels to 200 nM prolonged the plateau phase of the mouse atrial AP and lengthened the AP duration measured at 20 and 50% of repolarization of the human atrial CAF-remodelled AP as determined using a mathematical model. However, the expression of NOS3 was not modified in left atrial appendages from CAF patients. CONCLUSION Our results suggested that the increase in the atrial NO bioavailability could partially restore the duration of the plateau phase of CAF-remodelled AP by inhibiting the I(to1) as a result of the activation of non-canonical enzymatic pathways.

Chronic Atrial Fibrillation Increases MicroRNA-21 in Human Atrial Myocytes Decreasing L-Type Calcium Current

Background—Atrial fibrillation is characterized by progressive atrial structural and electrical changes (atrial remodeling) that favor arrhythmia recurrence and maintenance. Reduction of L-type Ca2+ current (ICa,L) density is a hallmark of the electrical remodeling. Alterations in atrial microRNAs could contribute to the protein changes underlying atrial fibrillation–induced atrial electrical remodeling. This study was undertaken to compare miR-21 levels in isolated myocytes from atrial appendages obtained from patients in sinus rhythm and with chronic atrial fibrillation (CAF) and to determine whether L-type Ca2+ channel subunits are targets for miR-21. Methods and Results—Quantitative polymerase chain reaction analysis showed that miR-21 was expressed in human atrial myocytes from patients in sinus rhythm and that its expression was significantly greater in CAF myocytes. There was an inverse correlation between miR-21 and the mRNA of the &agr;1c subunit of the calcium channel (CACNA1C) expression and ICa,L density. Computational analyses predicted that CACNA1C and the mRNA of the &bgr;2 subunit of the calcium channel (CACNB2) could be potential targets for miR-21. Luciferase reporter assays demonstrated that miR-21 produced a concentration-dependent decrease in the luciferase activity in Chinese Hamster Ovary cells transfected with CACNA1C and CACNB2 3′ untranslated region regions. miR-21 transfection in HL-1 cells produced changes in ICa,L properties qualitatively similar to those produced by CAF (ie, a marked reduction of ICa,L density and shift of the inactivation curves to more depolarized potentials). Conclusions—Our results demonstrated that CAF increases miR-21 expression in enzymatically isolated human atrial myocytes. Moreover, it decreases ICa,L density by downregulating Ca2+ channel subunits expression. These results suggested that this microRNA could participate in the CAF-induced ICa,L downregulation and in the action potential duration shortening that maintains the arrhythmia.

Endocannabinoids and cannabinoid analogues block human cardiac Kv4.3 channels in a receptor-independent manner

Endocannabinoids are amides and esters of long chain fatty acids that can modulate ion channels through both receptor-dependent and receptor-independent effects. Nowadays, their effects on cardiac K(+) channels are unknown even when they can be synthesized within the heart. We have analyzed the direct effects of endocannabinoids, such as anandamide (AEA), 2-arachidonoylglycerol (2-AG), the endogenous lipid lysophosphatidylinositol, and cannabinoid analogues such as palmitoylethanolamide (PEA), and oleoylethanolamide, as well as the fatty acids from which they are endogenously synthesized, on human cardiac Kv4.3 channels, which generate the transient outward K(+) current (I(to1)). Currents were recorded in Chinese hamster ovary cells, which do not express cannabinoid receptors, by using the whole-cell patch-clamp. All these compounds inhibited I(Kv4.3) in a concentration-dependent manner, AEA and 2-AG being the most potent (IC(50) approximately 0.3-0.4 microM), while PEA was the least potent. The potency of block increased as the complexity and the number of C atoms in the fatty acyl chain increased. The effects were not mediated by modifications in the lipid order and microviscosity of the membrane and were independent of the presence of MiRP2 or DPP6 subunits in the channel complex. Indeed, effects produced by AEA were reproduced in human atrial I(to1) recorded in isolated myocytes. Moreover, AEA effects were exclusively apparent when it was applied to the external surface of the cell membrane. These results indicate that at low micromolar concentrations the endocannabinoids AEA and 2-AG directly block human cardiac Kv4.3 channels, which represent a novel molecular target for these compounds.

Chronic Atrial Fibrillation Up-regulates β1-adrenoceptors Affecting Repolarizing Currents and Action Potential Duration

AIMS β-adrenergic stimulation has profound influence in the genesis and maintenance of atrial fibrillation (AF). However, the effects of β-Adrenoceptor stimulation on repolarizing currents and action potential (AP) characteristics in human atrial myocytes from left (LAA) and right atrial appendages (RAA) obtained from sinus rhythm (SR) and chronic atrial fibrillation (CAF) patients have not been compared yet. METHODS AND RESULTS Currents and APs were recorded using whole-cell patch-clamp in RAA and LAA myocytes from SR and CAF patients. Isoproterenol concentration-dependently decreased the Ca(2+)-independent 4-aminopyridine-sensitive component of the transient outward current (I(to1)) and the inward rectifying current (I(K1)). CAF significantly enhanced this inhibition, this effect being more marked in the left than in the right atria. CAF dramatically enhanced β-Adrenoceptor-mediated increase in the slow component of the delayed rectifier current (I(Ks)), whose density was already markedly increased by CAF. Conversely, the ultrarapid component of the delayed rectifier current (I(Kur)) of both SR and CAF myocytes was insensitive to low isoproterenol concentrations. As a consequence, stimulation of β1-Adrenoceptors in SR myocytes lengthened, whereas in CAF myocytes shortened, the AP duration. Quantitative PCR revealed that CAF up-regulated β1-Adrenoceptor expression, preferentially in the left atria. CONCLUSION The present results demonstrate that CAF increases the effects of β1-Adrenoceptor stimulation on repolarizing currents by means of a chamber-specific up-regulation of the receptors. This, together with the ion channel derangements produced by CAF, could contribute to the long-term stabilization of the arrhythmia by shortening the AP duration.

Regulation of SCN5A by microRNAs: miR-219 modulates SCN5A transcript expression and the effects of flecainide intoxication in mice

BACKGROUND The human cardiac action potential in atrial and ventricular cells is initiated by a fast-activating, fast-inactivating sodium current generated by the SCN5A/Nav1.5 channel in association with its β1/SCN1B subunit. The role of Nav1.5 in the etiology of many cardiac diseases strongly suggests that proper regulation of cell biology and function of the channel is critical for normal cardiac function. Hence, numerous recent studies have focused on the regulatory mechanisms of Nav1.5 biosynthetic and degradation processes as well as its subcellular localization. OBJECTIVE The purpose of this study was to investigate the role of microRNAs in the Scn5a/Nav1.5 posttranscriptional regulation. METHODS Quantitative polymerase chain reaction, immunohistochemical and electrophysiological measurements of distinct microRNA gain-of-function experiments in cardiomyocytes for the assessment of Scn5a expression. RESULTS Functional studies of HL-1 cardiomyocytes and luciferase assays in fibroblasts demonstrate that Scn5a is directly (miR-98, miR-106, miR-200, and miR-219) and indirectly (miR-125 and miR-153) regulated by multiple microRNAs displaying distinct time-dependent profiles. Cotransfection experiments demonstrated that miR-219 and miR-200 have independent opposite effects on Scn5a expression modulation. Of all the microRNAs studied, only miR-219 increases Scn5a expression levels, leading to altered contraction rhythm of HL-1 cardiomyocytes. Electrophysiological analyses in HL-1 cells revealed that miR-219 increases the sodium current. In vivo administration of miR-219 does not alter normal cardiac rhythm, but abolishes some of the effects of flecainide intoxication in mice, particularly QRS prolongation. CONCLUSION This study demonstrates the involvement of multiple microRNAs in the regulation of Scn5a. Particularly, miR-219 increases Scn5a/Nav1.5 transcript and protein expression. Our data suggest that microRNAs, such as miR-219, constitute a promising therapeutical tool to treat sodium cardiac arrhythmias.

New Investigational Drugs for the Management of Acute Heart Failure Syndromes

Acute heart failure syndromes (AHFS) enclose a broad spectrum of conditions with different clinical presentations, heart failure history, pathophysiology, prognosis and treatment. AHFS represent a major public health problem because of their high prevalence, high rates of mortality and readmissions and significant healthcare costs, and a therapeutic challenge for the clinicians because management strategies vary markedly. Traditionally used drugs for the treatment of AHFS, including diuretics, vasodilators and positive inotropics, improve clinical signs and symptoms as well as hemodynamics, but present important limitations, as they fail to reduce and may even increase in-hospital and postdischarge mortality, especially in patients with coronary artery disease. Thus, we need new pharmacological agents to not only improve signs and symptoms and cardiac performance, but also improve both short- and long-term outcomes (hospitalizations/survival). In the last decade, significant efforts have been made to identify new therapeutic targets involved in the genesis/progression of AHFS and to develop new therapeutic strategies that may safely improve outcomes. As a result, several new families of drugs have been developed and are currently studied in experimental models and in Phase II and III clinical trials, in an attempt to define their efficacy and safety profiles as well as their precise role in the treatment of AHFS patients. This review firstly analyzes the main clinical applications and limitations of conventional drugs, and then focuses on the mechanisms of action and effects of recently approved drugs and of new investigational agents on signs, symptoms, hemodynamics and outcomes in AHFS patients.

Pitx2c increases in atrial myocytes from chronic atrial fibrillation patients enhancing IKs and decreasing ICa,L

AIMS Atrial fibrillation (AF) produces rapid changes in the electrical properties of the atria (electrical remodelling) that promote its own recurrence. In chronic AF (CAF) patients, up-regulation of the slow delayed rectifier K(+) current (IKs) and down-regulation of the voltage-gated Ca(2+) current (ICa,L) are hallmarks of electrical remodelling and critically contribute to the abbreviation of action potential duration and atrial refractory period. Recent evidences suggested that Pitx2c, a bicoid-related homeodomain transcription factor involved in directing cardiac asymmetric morphogenesis, could play a role in atrial remodelling. However, its effects on IKs and ICa,L are unknown. METHODS AND RESULTS Real-time quantitative polymerase chain reaction analysis showed that Pitx2c mRNA expression was significantly higher in human atrial myocytes from CAF patients than those from sinus rhythm patients. The expression of Pitx2c was positively and negatively correlated with IKs and ICa,L densities, respectively. Expression of Pitx2c in HL-1 cells increased IKs density and reduced ICa,L density. Luciferase assays demonstrated that Pitx2c increased transcriptional activity of KCNQ1 and KCNE1 genes. Conversely, its effects on ICa,L could be mediated by the atrial natriuretic peptide. CONCLUSION Our results demonstrated for the first time that CAF increases Pitx2c expression in isolated human atrial myocytes and suggested that this transcription factor could contribute to the CAF-induced IKs increase and ICa,L reduction observed in humans.

Investigational Positive Inotropic Agents for Acute Heart Failure

Acute heart failure represents a major public health problem due to its high prevalence, high rates of mortality and readmissions and significant healthcare costs. Patients with AHF and low cardiac output represent a small subgroup of patients with very high mortality rates that require inotropic support to improve cardiac systolic function. Classical inotropic agents, such as beta1-adrenergic agonists (dobutamine, dopamine) and phosphodiesterase III inhibitors (milrinone, enoximone) improve symptoms and hemodynamics by increasing free intracellular Ca(2+) levels, but also increase myocardial O(2) demands and exert arrhythmogenic effects. These actions explain why these drugs increase both short- and long-term mortality, particularly in patients with AHF and coronary artery disease. Thus, we need new inotropic agents that do not increase cytosolic Ca(2+) or myocardial oxygen demands or produce arrhythmogenesis for the treatment of high-risk patients with acute heart failure and low cardiac output. This review describes three new classes of investigational agents: levosimendan, a calcium sensitizer and potassium channel opener, istaroxime, the first new luso-inotropic agent and cardiac myosin activators.