piperidines and barium-chloride

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) provide an in vitro model of the human myocardium. Complex 3D scaffolded culture methods improve the phenotypical maturity of iPSC-CMs, although typically at the expense of throughput. We have developed a novel, scalable approach that enables the use of iPSC-CM 3D spheroid models in a label-free readout system in a standard 96-well plate-based format. Spheroids were accurately positioned onto recording electrodes using a magnetic gold-iron oxide nanoparticle approach. Remarkably, both contractility (impedance) and extracellular field potentials (EFPs) could be detected from the actively beating spheroids over long durations and after automated dosing with pharmacological agents. The effects on these parameters of factors, such as co-culture (including human primary cardiac fibroblasts), extracellular buffer composition, and electrical pacing, were investigated. Beat amplitudes were increased greater than 15-fold by co-culture with fibroblasts. Optimization of extracellular Ca

Topics: Anti-Arrhythmia Agents; Barium Compounds; Biological Assay; Calcium; Cell Differentiation; Chlorides; Coculture Techniques; Ferric Compounds; Fibroblasts; Gold; Humans; Induced Pluripotent Stem Cells; Ion Transport; Metal Nanoparticles; Models, Biological; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Piperidines; Pyridines; Spheroids, Cellular

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl(2) (n = 22). Patch-clamp techniques were used to record I(Kr) and I(K1) from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl(2), at concentrations that selectively block I(K1) (50-100 µM), failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I(K1) in 53% (20/38) of the cells studied, but presence of I(Kr) in all (11/11). Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I(K1) in the majority of cells, but robust expression of I(Kr.) In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I(Kr) due to the absence of I(K1). Thus, efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications.

Topics: Action Potentials; Barium Compounds; Cell Differentiation; Chlorides; Gene Expression Regulation; Humans; Induced Pluripotent Stem Cells; Models, Biological; Myocytes, Cardiac; Piperidines; Potassium Channel Blockers; Potassium Channels, Inwardly Rectifying; Pyridines; RNA, Messenger; Time Factors

This study investigated whether KMUP-1, a xanthine-based derivative, inhibits L-type Ca(2+) currents (I(Ca,L)) in rat basilar artery smooth muscle cells (RBASMCs). We used whole cell patch-clamp recording to monitor Ba(2+) currents (I(Ba)) through L-type Ca(2+) channels (LTCCs). Under voltage-clamp conditions, holding at -40 mV, KMUP-1 (1, 3, 10 μM) inhibited I(Ba) in a concentration-dependent manner and its IC(50) value was 2.27 ± 0.45 μM. A high concentration of KMUP-1 (10 μM) showed without modifying the I(Ba) current-voltage relationship. On the other hand, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM) increase I(Ba) was inhibited by KMUP-1. Pretreatment with the PKC inhibitor chelerythrine (5 μM) intensified KMUP-1-inhibited I(Ba). However, the Rho kinase inhibitor Y-27632 (30 μM) failed to affect the I(Ba) inhibition by KMUP-1. In light of these results, we suggest that KMUP-1 inhibition of LTCCs in concentration- and voltage-dependent manners in RBASMCs may be due, at least in part, to its modulation of the PKC pathway.

Topics: Animals; Barium Compounds; Basilar Artery; Benzophenanthridines; Calcium Channel Blockers; Calcium Channels, L-Type; Chlorides; Female; Membrane Potentials; Myocytes, Smooth Muscle; Patch-Clamp Techniques; Piperidines; Protein Kinase C; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Signal Transduction; Xanthines

To sustain cardiac muscle contractility relatively independent of temperature, some fish species are capable of temporarily altering excitation-contraction coupling processes to meet the demands of their environment. The Pacific bluefin tuna, Thunnus orientalis, is a partially endothermic fish that inhabits a wide range of thermal niches. The present study examined the effects of temperature and thermal acclimation on sarcolemmal K(+) currents and their role in action potential (AP) generation in bluefin tuna cardiomyocytes. Atrial and ventricular myocytes were enzymatically isolated from cold (14 degrees C)- and warm (24 degrees C)-acclimated bluefin tuna. APs and current-voltage relations of K(+) channels were measured using the whole cell current and voltage clamp techniques, respectively. Data were collected either at the cardiomyocytes' respective acclimation temperature of 14 or 24 degrees C or at a common test temperature of 19 degrees C (to reveal the effects of acclimation). AP duration (APD) was prolonged in cold-acclimated (CA) cardiomyocytes tested at 14 degrees C compared with 19 degrees C and in warm-acclimated (WA) cardiomyocytes tested at 19 degrees C compared with 24 degrees C. This effect was mirrored by a decrease in the density of the delayed-rectifier current (I(Kr)), whereas the density of the background inward-rectifier current (I(K1)) was unchanged. When CA and WA cardiomyocytes were tested at a common temperature of 19 degrees C, no significant effects of temperature acclimation on AP shape or duration were observed, whereas I(Kr) density was markedly increased in CA cardiomyocytes. I(K1) density was unaffected in CA ventricular myocytes but was significantly reduced in CA atrial myocytes, resulting in a depolarization of atrial resting membrane potential. Our results indicate the bluefin AP is relatively short compared with other teleosts, which may allow the bluefin heart to function at cold temperatures without the necessity for thermal compensation of APD.

Topics: Acclimatization; Action Potentials; Animals; Anti-Arrhythmia Agents; Barium Compounds; Cell Enlargement; Chlorides; Electric Capacitance; Electrophysiological Phenomena; Heart; Heart Atria; Heart Ventricles; Humans; Male; Myocytes, Cardiac; Piperidines; Potassium Channels; Pyridines; Sarcolemma; Temperature; Tuna

Reduction of repolarization reserve increases the risk of arrhythmia. We hypothesized that inhibition of K(+) current (I(K)) to decrease repolarization reserve would unmask the proarrhythmic role of endogenous, physiological late Na(+) current (late I(Na)). Monophasic action potentials (MAP) and 12-lead electrocardiogram were recorded from female rabbit isolated hearts. To block I(K) and reduce repolarization reserve, E-4031, 4-aminopyridine, and BaCl(2) were used; to block endogenous late I(Na), tetrodotoxin (TTX) and ranolazine were used. E-4031 (1-60 nM) concentration-dependently prolonged MAP duration (MAPD(90)) and increased duration of the T wave from T(peak) to T(end) (T(peak)-T(end)), transmural dispersion of repolarization (TDR), and beat-to-beat variability (BVR) of MAPD(90). E-4031 caused spontaneous and pause-triggered polymorphic ventricular tachycardia [torsade de pointes (TdP)]. In the presence of 60 nM E-4031, TTX (0.6-3 muM) and ranolazine (5-10 muM) shortened MAPD(90), decreased TDR, BVR, and T(peak)-T(end) (n = 9-20, P < 0.01), and abolished episodes of TdP. In hearts treated with BaCl(2) or 4-aminopyridine plus E-4031, TTX (0.6-3 muM) shortened MAPD(90) and decreased T(peak)-T(end). Ranolazine could not reverse the effect of E-4031 to inhibit human ether-a-go-go-related gene (HERG) K(+) current; thus, the reversal by ranolazine of effects of E-4031 was likely due to inhibition of late I(Na) and not to antagonism of the HERG-blocking action of E-4031. We conclude that endogenous, physiological late I(Na) contributes to arrhythmogenesis in hearts with reduced repolarization reserve. Inhibition of this current partially reverses MAPD prolongation and abolishes arrhythmic activity caused by I(K) inhibitors.

Topics: 4-Aminopyridine; Acetanilides; Action Potentials; Animals; Anti-Arrhythmia Agents; Barium Compounds; Cell Line; Chlorides; Enzyme Inhibitors; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Female; Heart Conduction System; Humans; Kidney; Patch-Clamp Techniques; Piperazines; Piperidines; Potassium Channel Blockers; Pyridines; Rabbits; Ranolazine; Sodium; Sodium Channel Blockers; Sodium Channels; Tachycardia, Ventricular; Tetrodotoxin; Torsades de Pointes

1 Delta9-tetrahydrocannabinol (THC) produces varying effects in mesenteric arteries: vasorelaxation (third-order branches, G3), modest vasorelaxation (G2), no effect (G1) and vasoconstriction (the superior mesenteric artery, G0). 2 In G3, vasorelaxation to THC was inhibited by pertussis toxin, but was unaffected by the CB1 receptor antagonist, AM251 (1 microM), incubation with the TRPV1 receptor agonist capsaicin (10 microM, 1 h), the TRPV1 receptor antagonist capsazepine (10 microM) or de-endothelialisation. 3 In G3, vasorelaxation to THC was inhibited by high K+ buffer, and by the following K+ channel inhibitors: charybdotoxin (100 nM), apamin (500 nM) and barium chloride (30 microM), but not by 4-aminopyridine, glibenclamide or tertiapin. 4 In G3, THC (10 and 100 microM) inhibited the contractile response to Ca2+ in a Ca2+-free, high potassium buffer, indicating that THC blocks Ca2+ influx. 5 In G0, the vasoconstrictor responses to THC were inhibited by de-endothelialisation and SR141716A (100 nM), but not by the endothelin (ET(A)) receptor antagonist FR139317 (1 microM).THC (1 and 10 microM) antagonised vasorelaxation to anandamide in G3 but not G0. THC did not antagonise the noncannabinoid verapamil, capsaicin or the CB1 receptor agonist CP55,940. 6 THC (10 and 100 microM) inhibited endothelium-derived relaxing factor (EDHF)-mediated responses to carbachol in a manner similar to the gap junction inhibitor 18alpha-glycyrrhetinic acid. 7 These data show that THC causes vasorelaxation through activation of K+ channels and inhibition of Ca2+ channels, and this involves non-CB1, non-TRPV1 but G-protein-coupled receptors. In G0, THC does not cause relaxation and at high concentrations causes contractions. Importantly, THC antagonises the effects of anandamide, possibly through inhibition of EDHF activity.

Topics: Animals; Apamin; Arachidonic Acids; Azepines; Barium Compounds; Biological Factors; Calcium; Cannabinoid Receptor Modulators; Capsaicin; Charybdotoxin; Chlorides; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Endocannabinoids; Endothelium, Vascular; Female; In Vitro Techniques; Indoles; Male; Mesenteric Arteries; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Potassium Channels; Pyrazoles; Rats; Rats, Wistar; Rimonabant; Vasodilation; Verapamil

1. The aim of this study was to analyse the effects of eliprodil, a noncardiac drug with neuroprotective properties, on the cardiac repolarisation under in vitro circumstances, under normal conditions and after the attenuation of the 'repolarisation reserve' by blocking the inward rectifier potassium current (I(K1)) current with BaCl(2). 2. In canine right ventricular papillary muscle by applying the conventional microelectrode technique, under normal conditions, eliprodil (1 microm) produced a moderate reverse rate-dependent prolongation of the action potential duration (7.4+/-1.5, 8.9+/-2.1 and 9.9+/-1.8% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=9). 3. This effect was augmented in preparations where I(K1) was previously blocked by BaCl(2) (10 microm). BaCl(2) alone lengthened APD in a reverse frequency-dependent manner (7.0+/-1.3, 14.2+/-1.6 and 28.1+/-2.1% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=8). When eliprodil (1 microm) was administered to these preparations, the drug induced a marked further lengthening relative to the APD values measured after the administration of BaCl(2) (12.5+/-1.0, 17.6+/-1.5 and 20.5+/-0.9% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=8). 4. In the normal Langendorff-perfused rabbit heart, eliprodil (1 microm) produced a significant QT(c) prolongation at 1 Hz stimulation frequency (12.7+/-1.8%, n=9). After the attenuation of the 'repolarisation reserve' by the I(K1) blocker BaCl(2) (10 microm), the eliprodil-evoked QT(c) prolongation was greatly enhanced (28.5+/-7.9%, n=6). In two out of six Langendorff preparations, this QT(c) lengthening degenerated into torsade de pointes ventricular tachycardia. 5. Eliprodil significantly decreased the amplitude of rapid component of the delayed rectifier potassium current (I(Kr)), but slow component (I(Ks)), transient outward current (I(to)) and I(K1) were not considerably affected by the drug when measured in dog ventricular myocytes by applying the whole-cell configuration of the patch-clamp technique. 6. The results indicate that eliprodil, under normal conditions, moderately lengthens cardiac repolarisation by inhibition of I(Kr). However, after the attenuation of the normal 'repolarisation reserve', this drug can induce marked QT interval prolongation, which may result in proarrhythmic action.

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Barium Compounds; Chlorides; Dogs; Electrocardiography; Electrophysiology; Female; Heart; In Vitro Techniques; Male; Membrane Potentials; Microelectrodes; Neuroprotective Agents; Papillary Muscles; Patch-Clamp Techniques; Piperidines; Potassium Channel Blockers; Potassium Channels, Inwardly Rectifying; Rabbits

The area of vulnerability (AOV) to ventricular fibrillation (VF) induction by high-voltage shocks has been proposed as a measure of vulnerability to VF. Biphasic shocks spanning the T wave and ranging between 50 V and the upper limit of vulnerability (ULV) to VF were delivered before and after terikalant (1 mg/kg) and barium (1.1 mg/kg load followed by 0.05-0.10 mg/kg/min maintenance) or vehicle in dogs. The AOV decreased by 34% and 28% (p < 0.01) after terikalant and barium (n = 8 dogs each), respectively. Mean ULV, defibrillation threshold (DFT), and ventricular vulnerability period (VVP) decreased by 16%, 23%, and 31% (p < 0.01), respectively, after terikalant, and by 25%, 17% (p < 0.01), and 13% (p = 0.08), respectively, after barium. Vehicle (n = 14) did not significantly alter any of these variables. The ULV was correlated with the DFT before and after terikalant (r = 0.78, p < 0.01) and barium (r = 0.83, p < 0.01). Potassium channel blockers of the current reduce the ability to induce VF; this effect may be related to the anti-fibrillatory action of class III anti-arrhythmic drugs and their ability to decrease DFT.

Topics: Action Potentials; Animals; Anti-Arrhythmia Agents; Barium Compounds; Chlorides; Chromans; Dogs; Electric Countershock; Electrocardiography; Female; Infusions, Intravenous; Male; Piperidines; Ventricular Fibrillation; Ventricular Function

The purpose of the study was to investigate the properties of the delayed rectifier potassium current (IK) in myocytes isolated from undiseased human left ventricles.. The whole-cell configuration of the patch-clamp technique was applied in 28 left ventricular myocytes from 13 hearts at 35 degrees C.. An E-4031 sensitive tail current identified the rapid component of IK (IKr) in the myocytes, but there was no evidence for an E-4031 insensitive slow component of IK (IKs). When nifedipine (5 microM) was used to block the inward calcium current (ICa), IKr activation was fast (tau = 31.0 +/- 7.4 ms, at +30 mV, n = 5) and deactivation kinetics were biexponential and relatively slow (tau 1 = 600.0 +/- 53.9 ms and tau 2 = 6792.2 +/- 875.7 ms, at -40 mV, n = 7). Application of CdCl2 (250 microM) to block ICa altered the voltage dependence of the IKr considerably, slowing its activation (tau = 657.1 +/- 109.1 ms, at +30 mV, n = 5) and accelerating its deactivation (tau = 104.0 +/- 18.5 ms, at -40 mV, n = 8).. In undiseased human ventricle at 35 degrees C IKr exists having fast activation and slow deactivation kinetics; however, there was no evidence found for an expressed IKs. IKr probably plays an important role in the frequency dependent modulation of repolarization in undiseased human ventricle, and is a target for many Class III antiarrhythmic drugs.

Topics: Action Potentials; Adult; Anti-Arrhythmia Agents; Barium Compounds; Cadmium; Calcium Channel Blockers; Chlorides; Female; Humans; Male; Middle Aged; Myocardium; Nifedipine; Patch-Clamp Techniques; Piperidines; Pyridines; Signal Processing, Computer-Assisted; Sodium-Potassium-Exchanging ATPase