In vitro ion channel profile and ex vivo cardiac electrophysiology properties of the R(-) and S(+) enantiomers of hydroxychloroquine.

Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of corona virus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human Kir2.1 and hERG potassium channels in the 1 µM-100 µM range with a 2-4 fold enantiomeric separation. NaV1.5 sodium currents and CaV1.2 calcium currents, as well as KV4.3 and KV7.1 potassium currents remained unaffected at up to 90 µM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 µM and 30 µM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro.

Muscarinic agonists inhibit the ATP-dependent potassium current and suppress the ventricle-Purkinje action potential dispersion.

Activation of the parasympathetic nervous system has been reported to have an antiarrhythmic role during ischemia-reperfusion injury by decreasing the arrhythmia triggers. Furthermore, it was reported that the parasympathetic neurotransmitter acetylcholine is able to modulate the ATP-dependent potassium current (I K-ATP), a crucial current activated during hypoxia. However, the possible significance of this current modulation in the antiarrhythmic mechanism is not fully clarified. Action potentials were measured using the conventional microelectrode technique from canine left ventricular papillary muscle and free-running Purkinje fibers, under normal and hypoxic conditions. Ionic currents were measured using the whole-cell configuration of the patch-clamp method. Acetylcholine at 5 µmol/L did not influence the action potential duration (APD) either in Purkinje fibers or in papillary muscle preparations. In contrast, it significantly lengthened the APD and suppressed the Purkinje-ventricle APD dispersion when it was administered after 5 µmol/L pinacidil application. Carbachol at 3 µmol/L reduced the pinacidil-activated I K-ATP under voltage-clamp conditions. Acetylcholine lengthened the ventricular action potential under simulated ischemia condition. In this study, we found that acetylcholine inhibits the I K-ATP and thus suppresses the ventricle-Purkinje APD dispersion. We conclude that parasympathetic tone may reduce the arrhythmogenic substrate exerting a complex antiarrhythmic mechanism during hypoxic conditions.

Cardiac electrophysiological effects of ibuprofen in dog and rabbit ventricular preparations: possible implication to enhanced proarrhythmic risk.

Ibuprofen is a widely used nonsteroidal anti-inflammatory drug, which has recently been associated with increased cardiovascular risk, but its electrophysiological effects have not yet been properly studied in isolated cardiac preparations. We studied the effects of ibuprofen on action potential characteristics and several transmembrane ionic currents using the conventional microelectrode technique and the whole-cell configuration of the patch-clamp technique on cardiac preparations and enzymatically isolated ventricular myocytes. In dog (200 µM; n = 6) and rabbit (100 µM; n = 7) papillary muscles, ibuprofen moderately but significantly prolonged repolarization at 1 Hz stimulation frequency. In dog Purkinje fibers, repolarization was abbreviated and maximal rate of depolarization was depressed in a frequency-dependent manner. Levofloxacin (40 µM) alone did not alter repolarization, but augmented the ibuprofen-evoked repolarization lengthening in rabbit preparations (n = 7). In dog myocytes, ibuprofen (250 µM) did not significantly influence IK1, but decreased the amplitude of Ito and IKr potassium currents by 28.2% (60 mV) and 15.2% (20 mV), respectively. Ibuprofen also depressed INaL and ICa currents by 19.9% and 16.4%, respectively. We conclude that ibuprofen seems to be free from effects on action potential parameters at lower concentrations. However, at higher concentrations it may alter repolarization reserve, contributing to the observed proarrhythmic risk in patients.

Different effects of amiodarone and dofetilide on the dispersion of repolarization between well-coupled ventricular and Purkinje fibers1.

Increased transmural dispersion of repolarization is an established contributing factor to ventricular tachyarrhythmias. In this study, we evaluated the effect of chronic amiodarone treatment and acute administration of dofetilide in canine cardiac preparations containing electrotonically coupled Purkinje fibers (PFs) and ventricular muscle (VM) and compared the effects to those in uncoupled PF and VM preparations using the conventional microelectrode technique. Dispersion between PFs and VM was inferred from the difference in the respective action potential durations (APDs). In coupled preparations, amiodarone decreased the difference in APDs between PFs and VM, thus decreasing dispersion. In the same preparations, dofetilide increased the dispersion by causing a more pronounced prolongation in PFs. This prolongation was even more emphasized in uncoupled PF preparations, while the effect in VM was the same. In uncoupled preparations, amiodarone elicited no change on the difference in APDs. In conclusion, amiodarone decreased the dispersion between PFs and VM, while dofetilide increased it. The measured difference in APD between cardiac regions may be the affected by electrotonic coupling; thus, studying PFs and VM separately may lead to an over- or underestimation of dispersion.

Cannabidiol inhibits multiple cardiac ion channels and shortens ventricular action potential duration in vitro.

Cannabidiol (CBD) is a non-psychoactive component of Cannabis which has recently received regulatory consideration for the treatment of intractable forms of epilepsy such as the Dravet and the Lennox-Gastaut syndromes. The mechanisms of the antiepileptic effects of CBD are unclear, but several pre-clinical studies suggest the involvement of ion channels. Therefore, we have evaluated the effects of CBD on seven major cardiac currents shaping the human ventricular action potential and on Purkinje fibers isolated from rabbit hearts to assess the in vitro cardiac safety profile of CBD. We found that CBD inhibits with comparable micromolar potencies the peak and late components of the NaV1.5 sodium current, the CaV1.2 mediated L-type calcium current, as well as all the repolarizing potassium currents examined except Kir2.1. The most sensitive channels were KV7.1 and the least sensitive were KV11.1 (hERG), which underly the slow (IKs) and rapid (IKr) components, respectively, of the cardiac delayed-rectifier current. In the Purkinje fibers, CBD decreased the action potential (AP) duration more potently at half-maximal than at near complete repolarization, and slightly decreased the AP amplitude and its maximal upstroke velocity. CBD had no significant effects on the membrane resting potential except at the highest concentration tested under fast pacing rate. These data show that CBD impacts cardiac electrophysiology and suggest that caution should be exercised when prescribing CBD to carriers of cardiac channelopathies or in conjunction with other drugs known to affect heart rhythm or contractility.

Transmural and rate-dependent profiling of drug-induced arrhythmogenic risks through in silico simulations of multichannel pharmacology.

In vitro human ether-à-go-go related gene (hERG) inhibition assay alone might provide insufficient information to discriminate "safe" from "dangerous" drugs. Here, effects of multichannel inhibition on cardiac electrophysiology were investigated using a family of cardiac cell models (Purkinje (P), endocardial (Endo), mid-myocardial (M) and epicardial (Epi)). We found that: (1) QT prolongation alone might not necessarily lead to early afterdepolarization (EAD) events, and it might be insufficient to predict arrhythmogenic liability; (2) the occurrence and onset of EAD events could be a candidate biomarker of drug-induced arrhythmogenicity; (3) M cells are more vulnerable to drug-induced arrhythmias, and can develop early afterdepolarization (EAD) at slower pacing rates; (4) the application of quinidine can cause EADs in all cell types, while INaL is the major depolarizing current during the generation of drug-induced EAD in P cells, ICaL is mostly responsible in other cell types; (5) drug-induced action potential (AP) alternans with beat-to-beat variations occur at high pacing rates in P cells. These results suggested that quantitative profiling of transmural and rate-dependent properties can be essential to evaluate drug-induced arrhythmogenic risks, and may provide mechanistic insights into drug-induced arrhythmias.

Transient receptor potential channels in cardiac health and disease.

Transient receptor potential (TRP) channels are nonselective cationic channels that are generally Ca2+ permeable and have a heterogeneous expression in the heart. In the myocardium, TRP channels participate in several physiological functions, such as modulation of action potential waveform, pacemaking, conduction, inotropy, lusitropy, Ca2+ and Mg2+ handling, store-operated Ca2+ entry, embryonic development, mitochondrial function and adaptive remodelling. Moreover, TRP channels are also involved in various pathological mechanisms, such as arrhythmias, ischaemia-reperfusion injuries, Ca2+-handling defects, fibrosis, maladaptive remodelling, inherited cardiopathies and cell death. In this Review, we present the current knowledge of the roles of TRP channels in different cardiac regions (sinus node, atria, ventricles and Purkinje fibres) and cells types (cardiomyocytes and fibroblasts) and discuss their contribution to pathophysiological mechanisms, which will help to identify the best candidates for new therapeutic targets among the cardiac TRP family.

Electrophysiological mechanisms of vandetanib-induced cardiotoxicity: Comparison of action potentials in rabbit Purkinje fibers and pluripotent stem cell-derived cardiomyocytes.

Vandetanib, a multi-kinase inhibitor used for the treatment of various cancers, has been reported to induce several adverse cardiac effects. However, the underlying mechanisms of vandetanib-induced cardiotoxicity are unclear. This study aimed to investigate the mechanism of vandetanib-induced cardiotoxicity using intracellular electrophysiological recordings on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), rabbit Purkinje fibers, and HEK293 cells transiently expressing human ether-a-go-go-related gene (hERG; the rapidly activating delayed rectifier K+ channel, IKr), KCNQ1/KCNE1 (the slowly activating delayed rectifier K+ current, IKs), KCNJ2 (the inwardly rectifying K+ current, IK1) or SCN5A (the inward Na+ current, INa). Purkinje fiber assays and ion channel studies showed that vandetanib at concentrations of 1 and 3 µM inhibited the hERG currents and prolonged the action potential duration. Alanine scanning and in silico hERG docking studies demonstrated that Y652 and F656 in the hERG S6 domain play critical roles in vandetanib binding. In hiPSC-CMs, vandetanib markedly reduced the maximum rate of depolarization during the AP upstroke. Ion channel studies revealed that hiPSC-CMs were more sensitive to inhibition of the INa by vandetanib than in a heterogeneously expressed HEK293 cell model, consistent with the changes in the AP parameters of hiPSC-CMs. The subclasses of Class I antiarrhythmic drugs inhibited INa currents in a dose-dependent manner in hiPSC-CMs and SCN5A-encoded HEK293 cells. The inhibitory potency of vandetanib for INa was much higher in hiPSC-CMs (IC50: 2.72 µM) than in HEK293 cells (IC50: 36.63 µM). These data suggest that AP and INa assays using hiPSC-CMs are useful electrophysiological models for prediction of drug-induced cardiotoxicity.

Nonclinical cardiovascular safety of pitolisant: comparing International Conference on Harmonization S7B and Comprehensive in vitro Pro-arrhythmia Assay initiative studies.

BACKGROUND AND PURPOSE: We evaluated the concordance of results from two sets of nonclinical cardiovascular safety studies on pitolisant. EXPERIMENTAL APPROACH: Nonclinical studies envisaged both in the International Conference on Harmonization (ICH) S7B guideline and Comprehensive in vitro Pro-arrhythmia Assay (CiPA) initiative were undertaken. The CiPA initiative included in vitro ion channels, stem cell-derived human ventricular myocytes, and in silico modelling to simulate human ventricular electrophysiology. ICH S7B-recommended assays included in vitro hERG (KV 11.1) channels, in vivo dog studies with follow-up investigations in rabbit Purkinje fibres and the in vivo Carlsson rabbit pro-arrhythmia model. KEY RESULTS: Both sets of nonclinical data consistently excluded pitolisant from having clinically relevant QT-liability or pro-arrhythmic potential. CiPA studies revealed pitolisant to have modest calcium channel blocking and late INa reducing activities at high concentrations, which resulted in pitolisant reducing dofetilide-induced early after-depolarizations (EADs) in the ICH S7B studies. Studies in stem cell-derived human cardiomyocytes with dofetilide or E-4031 given alone and in combination with pitolisant confirmed these properties. In silico modelling confirmed that the ion channel effects measured are consistent with results from both the stem cell-derived cardiomyocytes and rabbit Purkinje fibres and categorized pitolisant as a drug with low torsadogenic potential. Results from the two sets of nonclinical studies correlated well with those from two clinical QT studies. CONCLUSIONS AND IMPLICATIONS: Our findings support the CiPA initiative but suggest that sponsors should consider investigating drug effects on EADs and the use of pro-arrhythmia models when the results from CiPA studies are ambiguous.

Small-Conductance Calcium-Activated Potassium Current in Normal Rabbit Cardiac Purkinje Cells.

BACKGROUND: Purkinje cells (PCs) are important in cardiac arrhythmogenesis. Whether small-conductance calcium-activated potassium (SK) channels are present in PCs remains unclear. We tested the hypotheses that subtype 2 SK (SK2) channel proteins and apamin-sensitive SK currents are abundantly present in PCs. METHODS AND RESULTS: We studied 25 normal rabbit ventricles, including 13 patch-clamp studies, 4 for Western blotting, and 8 for immunohistochemical staining. Transmembrane action potentials were recorded in current-clamp mode using the perforated-patch technique. For PCs, the apamin (100 nmol/L) significantly prolonged action potential duration measured to 80% repolarization by an average of 10.4 ms (95% CI, 0.11-20.72) (n=9, P=0.047). Voltage-clamp study showed that apamin-sensitive SK current density was significantly larger in PCs compared with ventricular myocytes at potentials ≥0 mV. Western blotting of SK2 expression showed that the SK2 protein expression in the midmyocardium was 58% (P=0.028) and the epicardium was 50% (P=0.018) of that in the pseudotendons. Immunostaining of SK2 protein showed that PCs stained stronger than ventricular myocytes. Confocal microscope study showed SK2 protein was distributed to the periphery of the PCs. CONCLUSIONS: SK2 proteins are more abundantly present in the PCs than in the ventricular myocytes of normal rabbit ventricles. Apamin-sensitive SK current is important in ventricular repolarization of normal PCs.

Effects of new antiarrhythmic agent SS-68 on excitation conduction, electrical activity in Purkinje fibers and pulmonary veins: Assessment of safety and side effects risk.

The compound SS-68 has been selected among numerous new derivatives of indole and demonstrated antiarrhythmic effects in animal models. The present study concerns several aspects of SS-68 safety and efficacy as a potential antiarrhythmic drug. The first estimation of atrioventricular conduction in mammalian heart under SS-68 has been carried out; effects of SS-68 in Purkinje fibers and myocardium of pulmonary veins have been investigated. The drug weakly affects cardiac atrioventricular conduction: only high concentrations of SS-68 (≥10 µmol/L) significantly decrease this parameter. Also, the drug weakly affects Purkinje fibers automaticity, but effectively alters action potential waveform in Purkinje fibers in a concentration-dependent manner. SS-68 (0.1-100 µmol/L) failed to induce any early or delayed afterdepolarizations in Purkinje fibers both in basal conditions and under provocation of proarrhythmic activity by norepinephrine (NE). Moreover, 10 µmol/L SS-68 suppressed NE-induced extra-beats and rapid firing in Purkinje fibers. In pulmonary veins only high concentrations of SS-68 significantly increased action potential duration, while lower concentrations (0.1-1 µmol/L) were ineffective. Also, 0.1-100 µmol/L SS-68 was unable to elicit arrhythmogenic alternations of action potential waveform in pulmonary veins. In conclusion, SS-68 has no proarrhythmic effects, such as afterdepolarizations or abnormal automaticity in used experimental models.

Effect of Exogenous Extracellular Nicotinamide Adenine Dinucleotide (NAD⁺) on Bioelectric Activity of the Pacemaker and Conduction System of the Heart.

In rat sinoatrial node, NAD(+) (10 µM) reduced the rate of spontaneous action potentials, duration of action potentials, and the velocity of slow diastolic depolarization, but the rate of action potential front propagation increases. In passed rabbit Purkinje fibers, NAD(+) (10 µM) reduced the duration of action potentials. Under conditions of spontaneous activity of Purkinje fibers, NAD(+) reduced the fi ring rate and the rate of slow diastolic depolarization. The effects of extracellular NAD(+) on bioelectric activity of the pacemaker (sinoatrial node) and conduction system of the heart (Purkinje fibers) are probably related to activation of P1 and P2 purinoceptors.

Cardiovascular safety pharmacology profile of etamicastat, a novel peripheral selective dopamine-ß-hydroxylase inhibitor.

Etamicastat, a peripheral reversible dopamine-ß-hydroxylase inhibitor, blocked the hERG current amplitude with an IC50 value of 44.0µg/ml in HEK 293 cells. At 0.3 and 3µg/ml, etamicastat had no effects on the action potential (AP) in male dog Purkinje fibers. At 30µg/ml, etamicastat significantly affected resting membrane potential (+4%), AP amplitude (-4%), AP duration at 60% (-14%) and AP duration at 90% (+5%) repolarization, and AP triangulation (+79%). In the telemetered conscious male dog, etamicastat (up to 20mg/kg) had no effects on arterial blood pressure, heart rate and the PR interval. At 10 and 20mg/kg, the QTc interval was slightly prolonged (8-9% max, P<0.05). No arrhythmia or other changes in the morphology of the ECG were observed. The maximum observed plasma concentrations (Cmax) of etamicastat (i.e. 3h post-administration) were 1.4 and 3.7µg/ml at 10 and 20mg/kg, respectively. No deleterious effects, including ECG disturbance were observed in male and female dogs dosed by gavage with etamicastat (up to 20mg/kg/day) for 28 days. Mean plasma Cmax etamicastat levels ranged between 2.4 and 6.3µg/ml on Day 1 and Day 28 of treatment, respectively. It is concluded that the blockade of the delayed rectifier potassium channels by etamicastat together with the QTc interval prolongation observed in conscious dogs can be considered as modest with respect to the measured plasmatic concentrations. These findings suggest that etamicastat is not likely to prolong the QT interval at therapeutic doses (~0.2µg/ml).

Assessment of anti-arrhythmic activity of antipsychotic drugs in an animal model: influence of non-cardiac α1-adrenergic receptors.

Torsades de Pointes (TdP) is a potentially lethal cardiac arrhythmia and a known adverse effect of many drugs secondary to block of the rapidly activating delayed rectifier potassium current (IKr). In animal models antipsychotic drugs have shown reduced pro-arrhythmic potential compared to drugs with comparable IKr-blocking characteristics. The reduced pro-arrhythmic properties of antipsychotic drugs has been attributed to a variety of different causes e.g., effects on α1-adrenergic receptors, ß-adrenergic receptors, muscarinic receptors or cardiac ion channels like Ca(2+)- and Na(+)-channels. Since only limited experimental information exists about the effects of α1-adrenergic receptor activity of antipsychotic drugs in pro-arrhythmic models, we have decided to investigate this. In this study we show that four antipsychotic drugs all have high affinity for α1-adrenergic receptor (sertindole>risperidone>haloperidol>olanzapine) and all block IKr (sertindole>haloperidol>risperidone>olanzapine). In canine Purkinje fibres, α1-adrenergic stimulation prolonged action potential duration; however, the stimulation does not cause afterdepolarizations, even in the presence of dofetilide-induced delayed repolarization. We showed for the first time in an in vivo pro-arrhythmic rabbit model that several antipsychotic drugs in accordance with their known α1-adrenergic receptor blocking properties reduced the incidence of drug-induced TdP and that the overall ability of the antipsychotic drugs to prevent TdP was associated with prevention of methoxamine induced increase in blood pressure. Further investigations are required to clarify the relative importance of α1-adrenergic receptor antagonism in conjunction with the additional effects of antipsychotic drugs on various receptors and ion channels.

Selective Na(+) /Ca(2+) exchanger inhibition prevents Ca(2+) overload-induced triggered arrhythmias.

BACKGROUND AND PURPOSE: Augmented Na(+) /Ca(2+) exchanger (NCX) activity may play a crucial role in cardiac arrhythmogenesis; however, data regarding the anti-arrhythmic efficacy of NCX inhibition are debatable. Feasible explanations could be the unsatisfactory selectivity of NCX inhibitors and/or the dependence of the experimental model on the degree of Ca(2+) i overload. Hence, we used NCX inhibitors SEA0400 and the more selective ORM10103 to evaluate the efficacy of NCX inhibition against arrhythmogenic Ca(2+) i rise in conditions when [Ca(2+) ]i was augmented via activation of the late sodium current (INaL ) or inhibition of the Na(+) /K(+) pump. EXPERIMENTAL APPROACH: Action potentials (APs) were recorded from canine papillary muscles and Purkinje fibres by microelectrodes. NCX current (INCX ) was determined in ventricular cardiomyocytes utilizing the whole-cell patch clamp technique. Ca(2+) i transients (CaTs) were monitored with a Ca(2+) -sensitive fluorescent dye, Fluo-4. KEY RESULTS: Enhanced INaL increased the Ca(2+) load and AP duration (APD). SEA0400 and ORM10103 suppressed INCX and prevented/reversed the anemone toxin II (ATX-II)-induced [Ca(2+) ]i rise without influencing APD, CaT or cell shortening, or affecting the ATX-II-induced increased APD. ORM10103 significantly decreased the number of strophanthidin-induced spontaneous diastolic Ca(2+) release events; however, SEA0400 failed to restrict the veratridine-induced augmentation in Purkinje-ventricle APD dispersion. CONCLUSIONS AND IMPLICATIONS: Selective NCX inhibition - presumably by blocking rev INCX (reverse mode NCX current) - is effective against arrhythmogenesis caused by [Na(+) ]i -induced [Ca(2+) ]i elevation, without influencing the AP waveform. Therefore, selective INCX inhibition, by significantly reducing the arrhythmogenic trigger activity caused by the perturbed Ca(2+) i handling, should be considered as a promising anti-arrhythmic therapeutic strategy.

Dantrolene improves survival after ventricular fibrillation by mitigating impaired calcium handling in animal models.

BACKGROUND: Resistant ventricular fibrillation, refibrillation. and diminished myocardial contractility are important factors leading to poor survival after cardiac arrest. We hypothesized that dantrolene improves survival after ventricular fibrillation (VF) by rectifying the calcium dysregulation caused by VF. METHODS AND RESULTS: VF was induced in 26 Yorkshire pigs for 4 minutes. Cardiopulmonary resuscitation was then commenced for 3 minutes, and dantrolene or isotonic saline was infused at the onset of cardiopulmonary resuscitation. Animals were defibrillated and observed for 30 minutes. To study the effect of VF on calcium handling and its modulation by dantrolene, hearts from 14 New Zealand rabbits were Langendorff-perfused. The inducibility of VF after dantrolene administration was documented. Optical mapping was performed to evaluate diastolic spontaneous calcium elevations as a measure of cytosolic calcium leak. The sustained return of spontaneous circulation (systolic blood pressure ≥60 mm Hg) was achieved in 85% of the dantrolene group in comparison with 39% of controls (P=0.02). return of spontaneous circulation was achieved earlier in dantrolene-treated pigs after successful defibrillation (21 ± 6 s versus 181 ± 57 s in controls, P=0.005). The median number of refibrillation episodes was lower in the dantrolene group (0 versus 1, P=0.04). In isolated rabbit hearts, the successful induction of VF was achieved in 83% of attempts in controls versus 41% in dantrolene-treated hearts (P=0.007). VF caused diastolic calcium leaks in the form of spontaneous calcium elevations. Administration of 20 µmol/L dantrolene significantly decreased spontaneous calcium elevation amplitude versus controls. (0.024 ± 0.013 versus 0.12 ± 0.02 arbitrary unit [200-ms cycle length], P=0.001). CONCLUSIONS: Dantrolene infusion during cardiopulmonary resuscitation facilitates successful defibrillation, improves hemodynamics postdefibrillation, decreases refibrillation, and thus improves survival after cardiac arrest. The effects are mediated through normalizing VF-induced dysfunctional calcium cycling.

Type 1 metabotropic glutamate receptors (mGlu1) trigger the gating of GluD2 delta glutamate receptors.

The orphan GluD2 receptor belongs to the ionotropic glutamate receptor family but does not bind glutamate. Ligand-gated GluD2 currents have never been evidenced, and whether GluD2 operates as an ion channel has been a long-standing question. Here, we show that GluD2 gating is triggered by type 1 metabotropic glutamate receptors, both in a heterologous expression system and in Purkinje cells. Thus, GluD2 is not only an adhesion molecule at synapses but also works as a channel. This gating mechanism reveals new properties of glutamate receptors that emerge from their interaction and opens unexpected perspectives regarding synaptic transmission and plasticity.

Cortisol stimulates proliferation and apoptosis in the late gestation fetal heart: differential effects of mineralocorticoid and glucocorticoid receptors.

We have previously found that modest chronic increases in maternal cortisol result in an enlarged fetal heart. To explore the mechanisms of this effect, we used intrapericardial infusions of a mineralocorticoid receptor (MR) antagonist (canrenoate) or of a glucocorticoid receptor (GR) antagonist (mifepristone) in the fetus during maternal infusion of cortisol (1 mg·kg⁻¹·day⁻¹). We have shown that the MR antagonist blocked the increase in fetal heart weight and in wall thickness resulting from maternal cortisol infusion. In the current study we extended those studies and found that cortisol increased Ki67 staining in both ventricles, indicating cell proliferation, but also increased active caspase-3 staining in cells of the conduction pathway in the septum and subendocardial layers of the left ventricle, suggesting increased apoptosis in Purkinje fibers. The MR antagonist blocked the increase in cell proliferation, whereas the GR antagonist blocked the increased apoptosis in Purkinje fibers. We also found evidence of activation of caspase-3 in c-kit-positive cells, suggesting apoptosis in stem cell populations in the ventricle. These studies suggest a potentially important role of corticosteroids in the terminal remodeling of the late gestation fetal heart and suggest a mechanism for the cardiac enlargement with excess corticosteroid exposure.

ORM-10103, a novel specific inhibitor of the Na+/Ca2+ exchanger, decreases early and delayed afterdepolarizations in the canine heart.

BACKGROUND AND PURPOSE: At present there are no small molecule inhibitors that show strong selectivity for the Na(+) /Ca(2+) exchanger (NCX). Hence, we studied the electrophysiological effects of acute administration of ORM-10103, a new NCX inhibitor, on the NCX and L-type Ca(2+) currents and on the formation of early and delayed afterdepolarizations. EXPERIMENTAL APPROACH: Ion currents were recorded by using a voltage clamp technique in canine single ventricular cells, and action potentials were obtained from canine and guinea pig ventricular preparations with the use of microelectrodes. KEY RESULTS: ORM-10103 significantly reduced both the inward and outward NCX currents. Even at a high concentration (10 µM), ORM-10103 did not significantly change the L-type Ca(2+) current or the maximum rate of depolarization (dV/dtmax ), indicative of the fast inward Na(+) current. At 10 µM ORM-10103 did not affect the amplitude or the dV/dtmax of the slow response action potentials recorded from guinea pig papillary muscles, which suggests it had no effect on the L-type Ca(2+) current. ORM-10103 did not influence the Na(+) /K(+) pump or the main K(+) currents of canine ventricular myocytes, except the rapid delayed rectifier K(+) current, which was slightly diminished by the drug at 3 µM. The amplitudes of pharmacologically- induced early and delayed afterdepolarizations were significantly decreased by ORM-10103 (3 and 10 µM) in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: ORM-10103 is a selective inhibitor of the NCX current and can abolish triggered arrhythmias. Hence, it has the potential to be used to prevent arrhythmogenic events.