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Rationale: It has been reported that patients with major depressive disorder (MDD) are prone to developing ventricular arrhythmias. Moreover, the Sigma-1 receptor not only plays a crucial role in MDD but has also been shown to have antiarrhythmic properties. The Sigma-1 receptor is a common receptor related to depression and ventricular arrhythmias., Objective: We analyzed the effects of the Sigma-1 receptor on depression and ventricular repolarization-related ion remodeling in MDD rats., Methods: MDD was induced in rats by chronic unpredictable mild stress (CUMS), and 28 days later, the rats were subjected to behavior tests. Protein expression was measured by western blotting, and cardiac morphological changes were observed by Masson staining. Electrophysiological measurement of the myocardium was performed with the whole-cell patch-clamp technique., Results: Compared with the control rats, the MDD rats exhibited lower transient outward potassium current (Ito) and L-type calcium current (I Ca-L ) amplitudes. On the other hand, a trend of depolarization of I to and hyperpolarization of I Ca-L was observed in the MDD rats. Thus, we investigated the effect of fluvoxamine, a Sigma-1 receptor agonist, on Ito and I Ca-L . Fluvoxamine enhanced Ito and altered its current kinetics, as shown by acceleration of activation and recovery from inactivation. In contrast, fluvoxamine inhibited the Ca 2+ by hyperpolarizing the steady-state activation of I Ca-L . All these effects were blocked by BD1047., Conclusion: Taken together, our results indicate that Sigma-1 receptor modulates the functions of Ito and I Ca-L to possibly exert antiarrhythmic effects.

Effects of ibutilide, a class III antiarrhythmic drug, on delayed rectifier potassium currents (I K ) in freshly isolated guinea pig ventricular myocytes were studied. Experiments were performed using the whole-cell configuration of patch-clamp technique under blockade of L-type calcium currents (Cav1). Ibutilide at concentrations ranging between 10 nM and 100 µM inhibited I Kr in dose-dependent manner with a half maximal effective concentration of 2.03 ± 0.74 µM (n = 5-10). The amplitude of tail currents activated by prepulse to + 20 mV was decreased from 253 ± 52 to 130 ± 25 pA (n = 8, p < 0.01) in the presence of 1 µM ibutilide. The envelope test revealed time-dependent changes in ratio of I K-tail /ΔI K during 0.2-2 s pulse durations in the absence of drug. With ibutilide, regardless of pulse duration, a relatively constant ratio was estimated, indicative of predominant involvement of I Kr component. The slow I Ks persisted to greater extent even at 100 μM ibutilide revealing a distinguishable selectivity toward the I Kr component.

Several studies have been reported in various domains from induction methods to utilities of somatic cell pluripotent reprogramming. However, one of the major struggles facing the research field of induced pluripotent stem cell (iPSC)-derived target cells is the lack of consistency in observations. This could be due to variety of reasons including varied culture periods post-differentiation. The cardiomyocytes (CMs) derived from iPSCs are commonly studied and proposed to be utilized in the comprehensive in vitro proarrhythmia initiative for drug safety screening. As the influence of varied culture periods on the electrophysiological properties of iPSC-CMs is not clearly known, using whole-cell patch clamp technique, we compared two groups of differentiated ventricular-like iPSC-CMs that are cultured for 10 to 15 days (D10-15) and more than 30 days (≥ D30) both under current and voltage clamps. The prolonged culture imparts increased excitability with high-frequency spontaneous action potentials, robust increase in the magnitude of peak Na + current density, relatively shallow inactivation kinetics of Na + channels, faster recovery from inactivation, and augmented Ca 2+ current density. Quantitative real-time PCR studies of α-subunit transcripts showed enhanced mRNA expression of SCN1A, SCN5A Na + channel subtypes, and CACNA1C, CACNA1G, and CACNA1I Ca 2+ channel subtypes, in ≥ D30 group. Conclusively, the prolonged culture of differentiated iPSC-CMs affects the excitability, single-cell electrophysiological properties, and ion channel expressions. Therefore, following standard periods of culture across research studies while utilizing ventricular-like iPSC-CMs for in vitro health/disease modeling to study cellular functional mechanisms or test high-throughput drugs' efficacy and toxicity becomes crucial.

To investigate the effects of isovitexin Ⅳ on transient outward potassium current in rat ventricular myocytes. In this study, MTT assay was used to investigate the safe range of isovitexin. The results showed that the IC₅₀ of the drug was in the range of 10-30 μmol•L⁻¹, and the drug concentration of 1-3 μmol•L⁻¹ for the patch clamp test was within the safe range. In addition, the single ventricular myocytes were obtained by single-enzymatic hydrolysis through aortic retrograde perfusion. The transient outward potassium current (Ito) of rat ventricular myocytes was guided and measured by whole-cell patch-clamp technique and the changes of current characteristics were recorded after isovite was applied. When the concentration of IV was less than 0.1 μmol•L⁻¹, there was no significant effect on Ito. However, with the increase in the concentration of IV (≥0.3 μmol•L⁻¹), the peak of Ito was decreased gradually, from (32.32±2.9) pA/pF to （25.83±4.3） pA/pF, 1 μmol•L⁻¹ IV and (19.51±3.5) pA/pF, 3 μmol•L⁻¹ IV respectively, with an inhibition effect in a concentration-dependent manner. In the range of 1-3 μmol•L⁻¹, IV down-regulated the I-V curve of Ito significantly. The activation curve showed that IV can enable the maximum half activation potential (V1/2) to move to the positive direction, and the V1/2 was increased from (19.59±1.6) mV to (22.81±1.7) mV and (28.86±1.4) mV at concentration of 1, 3 μmol•L⁻¹, meanwhile the activation curve moved to the right. However, the maximum half inactivating potential (V1/2) of the steady-state inactivation curve of Ito was significantly decreased from (－51.43±0.99) mV to (－61.81±1.3) mV with concentration of 1 μmol•L⁻¹ and (－71.50±1.4) mV with concentration of 3 μmol•L⁻¹. The inactivation time constant of recovery from inactivation (τ) was up-regulated significantly from (94.89±0.73) ms to (118.5±1.5) ms and (162.4±1.4) ms at concentration of 1, 3 μmol•L⁻¹ respectively. Meanwhile IV could enable the inactivation recovery curve to move to the right, which suggested that it can prolong the recovery time from inactivation of the transient outward potassium channel. In conclusion, isovitexin had a high inhibitory effect on Ito in rat ventricular myocytes., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Background: A loss of repolarization reserve due to downregulation of K(+) currents has been observed in cultured ventricular myocytes. A similar reduction of K(+) currents is well documented under numerous pathophysiological conditions. We examined the extent of K(+) current downregulation in cultured canine cardiac myocytes and determined whether a dual K(+) current activator can normalize K(+) currents and restore action potential (AP) configuration., Methods and Results: Ventricular myocytes were isolated and cultured for up to 48 h. Current and voltage clamp recordings were made using patch electrodes. Application of NS3623 to coronary-perfused left ventricular wedges resulted in increased phase 1 magnitude, epicardial AP notch and J wave amplitude. Patch clamp measurements of IKr and Ito revealed an increase in the magnitude of both currents. Culturing of Mid ventricular cells resulted in a significant decrease in Ito and IKr density. NS3623 increased Ito from 16.4 ± 2.23 to 31.8 ± 4.5 pA/pF, and IKr from 0.28 ± 0.06 to 0.47 ± 0.09 pA/pF after 2 days in culture. AP recordings from 2 day cultured cells exhibited a reduced phase 1 repolarization, AP prolongation, and early afterdepolarizations (EADs). NS3623 restored the AP notch and was able to suppress EADs., Conclusions: NS3623 is a dual Ito and IKr activator. Application of this compound to cells with a reduced repolarization reserve resulted in an increase in these currents and a shortening of AP duration, increase in phase 1 repolarization and suppression of EADs. Our results suggest a potential benefit of K(+) current activators under conditions of reduced repolarization reserve including heart failure., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Several freshwater fishes of north-temperate latitudes exhibit marked seasonal changes in cardiac action potential (AP) waveform as an outcome of temperature-dependent changes in the density of delayed rectifiers (IKr, IKs) and inward rectifier (IK1) potassium currents. Thus far, ionic mechanisms of cardiac excitability in arctic marine fishes have not been examined. To this end we examined ventricular AP and the role of two major potassium currents (IK1, IKr) in repolarization of cardiac AP in winter-acclimatized (WA, caught in March) and summer-acclimatized (SA, caught in September) navaga cod (Eleginus navaga) of the White Sea. The duration of ventricular AP of WA navaga at 3 °C (APD50 = 659.5 ± 32.8 ms) was similar to the AP duration of SA navaga at 12 °C (APD50 = 543.9 ± 14.6 ms) (p > 0.05) indicating complete thermal compensation of AP duration. This acclimation effect was associated with strong up-regulation of the cardiac potassium currents in winter. Densities of ventricular IK1 (at -120 mV) and IKr (at +50 mV) of the WA navaga at 3 °C were 2.9 times and 2.8 times, respectively, higher than those of the SA navaga at 12 °C, thus indicating marked thermal overcompensation. Qualitatively similar results were obtained from atrial myocytes. Seasonal changes in IK1 and IKr are more than sufficient to explain the complete thermal compensation of ventricular AP duration. The excellent acclimation capacity of cardiac excitability of the navaga cod is probably needed to maintain high cardiac performance at subzero temperatures in winter and to increase thermal resilience of cardiac function under seasonally variable arctic temperature conditions.

Background: Epinephrine is a first-line drug for cardiopulmonary resuscitation, but its efficacy in the treatment of bupivacaine-induced cardiac toxicity is still in question. We hypothesized that epinephrine can reverse cardiac inhibition of bupivacaine by modulating ion flows through the ventricular myocyte membrane channels of rats. The aim of this study was to observe and report the effects of epinephrine on high-concentration bupivacaine-induced inhibition of sodium (INa), L-type calcium (ICa-L), and transient outward potassium (Ito) currents in the ventricular myocytes of rats., Methods: The ventricular myocytes were isolated from Sprague-Dawley rats (250-300 g) by acute enzymatic dissociation. The whole-cell patch clamp technique was used to record the ion channel currents in single ventricular myocytes both before and after administration of medications., Result: Administration of bupivacaine 100 μmol/L significantly reduced INa, (P < 0.05). However, administration of bupivacaine 100 μmol/L in conjunction with epinephrine 0.15 μg/ml had no effect in restoring INa to its previous state. Similarly, a sharp decline of ICa-L and Ito was observed after administration of bupivacaine 100 μmol/L (P < 0.05). In contrast to INa, ICa-L and Ito were significantly improved after the administration of the aforementioned combination of bupivacaine and epinephrine (P < 0.05)., Conclusion: Epinephrine can reverse high-concentration bupivacaine induced inhibition of ICa-L and Ito, but not INa. Thus, epinephrine's effectiveness in reversal of bupivacaine-induced cardiac toxicity secondary to sodium channel inhibition may be limited.

Objective: To test the proof-of-principle that genetically-engineered mesenchymal stem cells (MSCs) transfected with the human hyperpolarization-activated cyclic nucleotide-gated channel 1 (hHCN1) gene can be modified to become cardiac pacemaker cells., Methods: MSCs were transfected with the hHCN1 gene using lentiviral-based transfection. The expressed pacemaker current (I(f)) in hHCN1-transfected MSCs was recorded using whole-cell patch-clamp analysis. The effect of the hHCN1-transfected MSCs on cardiomyocyte excitability was determined by coculturing the MSCs with neonatal rabbit ventricular myocytes (NRVM). The spontaneous action potentials of the NRVM were recorded by whole-cell current-clamp analysis., Results: A high level time- and voltage-dependent inward hyperpolarization current that was inhibited by 4 mM caesium chloride was detected in hHCN1-transfected MSCs, suggesting that the HCN1 proteins acted as I(f) channels in MSCs. The mean ± SE beating frequency in NRVMs cocultured with control MSCs transfected with the pcDNA3 plasmid control was 82 ± 8 beats/min (n = 5) compared with 129 ± 11 beats/min (n = 5) in NRVMs cocultured with hHCN1-transfected MSCs., Conclusions: Genetically-engineered MSCs transfected with the hHCN1 gene can be modified to become cardiac pacemaker cells.

The study aimed to investigate the age-related changes and drug reactions of transient outward potassium current (Ito) of ventricular myocytes. Twenty-eight Sprague Dawley rats were divided into young (3-5 months), adult (13-15 months) and aged (22-24 months) groups, and Ito currents of isolated myocytes from each group were recorded respectively by patch-clamp. The perfusion of 2.0 mmol/L 4-AP or 1.0 μmol/L isoproterenol was added respectively in each group, and the changes of Ito were observed. In comparison with young and adult groups, Ito densities of ventricular myocytes in aged group was significantly increased, the curve of steady-state activation of Ito shifted to the left, the close-state inactivation rate significantly decreased, and recovery rate from the steady-state inactivation became quicker. However, no significant changes could be detected for the Ito steady-state inactivation of ventricular myocytes in aged group. The similar responsiveness to 4-AP was observed in all three groups, but the responsiveness to isoproterenol was weaker in the aged group (55.9%) than in the other two groups (127.5% and 125.8%). In conclusion, the results show that Ito of rat ventricular myocyte of aging heart has increased current density and decreased response to isoproterenol.

Astragaloside IV (AS-IV) is one of the main active constituents of Astragalus membranaceus, which has various actions on the cardiovascular system. However, its electrophysiological mechanisms are not clear. In the present study, we investigated the effects of AS-IV on action potentials and membrane currents using the whole-cell patch clamp technique in isolated guinea-pig ventricular myocytes. AS-IV prolonged the action potential duration (APD) at all three tested concentrations. The peak effect was achieved with 1×10(-6) M, at which concentration AS-IV significantly prolonged the APD at 95% repolarization from 313.1±38.9 to 785.3±83.7 ms. AS-IV at 1×10(-6) M also enhanced the inward rectifier K(+) currents (I(K1)) and inhibited the delayed rectifier K(+) currents (I(K)). AS-IV (1×10(-6) M) strongly depressed the peak of voltage-dependent Ca(2+) channel current (I(CaL)) from -607.3±37.5 to -321.1±38.3 pA. However, AS-IV was not found to affect the Na(+) currents. Taken together, AS-IV prolonged APD of guinea-pig ventricular myocytes, which might be explained by its inhibition of I(K). AS-IV also influences Ca(2+) signaling through suppressing ICaL.

Chaotic rhythms in deterministic models can arise as a consequence of changes in model parameters. We carried out experimental studies in which we induced a variety of complex rhythms in aggregates of embryonic chick cardiac cells using E-4031 (1.0-2.5 μM), a drug that blocks the hERG potassium channel. Following the addition of the drug, the regular rhythm evolved to display a spectrum of complex dynamics: irregular rhythms, bursting oscillations, doublets, and accelerated rhythms. The interbeat intervals of the irregular rhythms can be described by one-dimensional return maps consistent with chaotic dynamics. A Hodgkin-Huxley-style cardiac ionic model captured the different types of complex dynamics following blockage of the hERG mediated potassium current.

Expression and function of cardiac ion channels exhibit postnatal developmental changes, which, however, has not yet been proven in ventricular myocytes isolated using similar techniques. In this study, ventricular myocytes were enzymatically dissociated from mouse heart at different postnatal ages (including postnatal day 0) by similar techniques using Langendorff perfusion. Whole-cell patch-clamp experiments were performed to record action potentials, I (K1), I (Kr), I (Kur), I (ss), and I (Ca,L), in ventricular myocytes freshly isolated from postnatal days 0, 7, and 14 and adult mice. Viable ventricular myocytes of day-0 mouse heart exhibited spindle-shaped appearance having cell length of approximately 50 μm, which gradually developed to a rod-shaped one having clear cross striation with cell length of approximately 120 μm (adult). The action potential duration markedly shortened, while the resting membrane potential depolarized to a small but significant extent during postnatal development. I (K1) density was maximal in postnatal day-0 ventricular myocytes and gradually decreased during development, which was accompanied by postnatal depolarization of resting membrane potential. However, I (K1) density was markedly decreased by approximately 80% in postnatal day-0 ventricular myocytes, when isolated by the chunk method. Quantitative real-time polymerase chain reaction (PCR) and western blot analyses demonstrated higher Kir2.3 expression but lower expression levels of Kir2.1 and Kir2.2 in day-0 mouse ventricles, compared with those of day-14 and adult mouse ventricles. Whereas I (Kr) exhibited marked decrease during postnatal development, I (Kur), I (ss), and I (Ca,L) exhibited postnatal developmental increase. The present cell isolation method using the Langendorff perfusion thus found that, in mouse ventricles, I (K1) exhibited postnatal developmental decrease, associated with depolarization of resting potential.

Some azole antifungal agents induce long QT syndrome and arrhythmias. Although composite functions of ion channels in cardiomyocytes contribute to the shaping of action potentials, information on the effects of azole antifungal agents on ion currents, except human-ether-a-go-go-related gene (HERG) K+ currents, is largely lacking. Using the whole cell patch-clamp technique, we investigated the effects of four azole agents (miconazole, ketoconazole, fluconazole, and itraconazole) on inward rectifying K+ currents (IKir), voltage-gated L-type Ca2+ currents (ICaL), and delayed rectifier K+ currents (IKdr) in rat neonate ventricular myocytes. Strikingly, miconazole and ketoconazole strongly inhibited IKir, IKdr, and ICaL at clinically relevant concentrations. The IC50 values of miconazole for IKdr, IKir, and ICaL inhibition were 2.5, 10.4, and 3.0 µM, respectively. The IC50 values of ketoconazole for IKdr, IKir and ICaL inhibition were 3.2, 20.8, and 3.5 µM, respectively. Fluconazole and itraconazole had relatively little effect on ion currents. These findings indicate that miconazole and ketoconazole are multiple ion channel inhibitors in cardiomyocytes. We suggest that it is necessary to consider this inhibition of ion channels by azole agents when assessing cardiovascular side effects.

Aim: Hydrogen sulfide (H₂S) is a promising cardioprotective agent and a potential modulator of cardiac ion currents. Yet its cardiac effects on humans are poorly understood due to lack of functional cardiomyocytes. This study investigates electrophysiological responses of human pluripotent stem cells (hPSCs) derived cardiomyocytes towards H₂S., Methods and Results: Cardiomyocytes of ventricular, atrial and nodal subtypes differentiated from H9 embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were electrophysiologically characterized. The effect of NaHS, a donor of H₂S, on action potential (AP), outward rectifier potassium currents (I(Ks) and I(Kr)), L-type Ca²⁺ currents (I(CaL)) and hyperpolarization-activated inward current (I(f)) were determined by patch-clamp electrophysiology and confocal calcium imaging. In a concentration-dependent manner, NaHS (100 to 300 µM) consistently altered the action potential properties including prolonging action potential duration (APD) and slowing down contracting rates of ventricular-and atrial-like cardiomyocytes derived from both hESCs and hiPSCs. Moreover, inhibitions of slow and rapid I(K) (I(Ks) and I(Kr)), I(CaL) and I(f) were found in NaHS treated cardiomyocytes and it could collectively contribute to the remodeling of AP properties., Conclusions: This is the first demonstration of effects of H₂S on cardiac electrophysiology of human ventricular-like, atrial-like and nodal-like cardiomyocytes. It reaffirmed the inhibitory effect of H₂S on I(CaL) and revealed additional novel inhibitory effects on I(f), I(Ks) and I(Kr) currents in human cardiomyocytes.

Introduction: NS5806 activates the transient outward potassium current (I(to) ) in canine ventricular cells. We compared the effects of NS5806 on canine atrial versus ventricular tissues and myocytes., Methods and Results: NS5806 (10 μM) was evaluated in arterially perfused canine right atrial and right ventricular wedge preparations. In ventricular wedges NS5806 (10 μM) accentuated phase 1 in epicardium (Epi), with little effect in endocardium (Endo), resulting in augmented J-waves on the ECG. In contrast, application of NS5806 (10 μM) to atrial preparations had no effect on phase 1 repolarization but significantly decreased upstroke velocity (dV/dt) and depressed excitability, consistent with sodium channel block. Current and voltage-clamp recordings were made in the absence and presence of NS5806 in (10 μM) enzymatically dissociated atrial and ventricular myocytes. In ventricular myocytes, NS5806 increased I(to) magnitude by 80% and 16% in Epi and Endo, respectively (at +40 mV). In atrial myocytes, NS5806 increased peak I(to) by 25% and had no effect on the sustained current, I(Kur) . Under control conditions, I(Na) density in atrial myocytes was nearly double that in ventricular myocytes. NS5806 caused a shift in steady-state mid-inactivation (V(1/2)) from -73.9 ± 0.27 to -77.3 ± 0.21 mV in ventricular and from -82.6 ± 0.12 to -85.1 ± 0.11 mV in atrial cells, resulting in reduction of I(Na) in both cell types. Expression of mRNA encoding putative I(Na) and I(to) channel subunits was evaluated by qPCR., Conclusion: NS5806 produces a prominent augmentation of I(to) with little effect on I(Na) in the ventricles, but a potent inhibition of I(Na) with little augmentation of I(to) in atria., (© 2011 Wiley Periodicals, Inc.)

Objective: To investigate the effects of docosahexaenoic acid (DHA) on sodium channel current (I(Na)) and transient outward potassium channel current (I(to)) in rat ventricular myocytes and to evaluate potential anti-arrhythmic mechanisms of DHA., Methods: I(Na) and I(to) of individual ventricular myocytes were recorded by patch-clamp technique in whole-cell configuration at room temperature. Effects of DHA at various concentrations (0, 20, 40, 60, 80, 100 and 120 micromol/L) on I(Na) and I(to) were observed., Results: (1) I(Na) was blocked in a concentration-dependent manner by DHA, stably inactivated curves were shifted to the left, and recover time from inactivation was prolonged while stably activated curves were not affected by DHA. At -30 mV, I(Na) was blocked to (1.51 ± 1.32)%, (21.13 ± 4.62)%, (51.61 ± 5.73)%, (67.62 ± 6.52)%, (73.49 ± 7.59)% and (79.95 ± 7.62)% in the presence of above DHA concentrations (all P < 0.05, n = 20), and half-effect concentration (EC(50)) of DHA on I(Na) was (47.91 ± 1.57)micromol/L. (2) I(to) were also blocked in a concentration-dependent manner by DHA, stably inactivated curves were shifted to the left, and recover time from inactivation was prolonged with increasing concentrations of DHA, and stably activated curves were not affected by DHA. At +70 mV, I(to) was blocked to (2.61 ± 0.26)%, (21.79 ± 4.85)%, (63.11 ± 6.57)%, (75.52 ± 7.26)%, (81.82 ± 7.63)% and (84.33 ± 8.25)%, respectively, in the presence of above DHA concentrations (all P < 0.05, n = 20), and the EC(50) of DHA on I(to) was (49.11 ± 2.68)micromol/L., Conclusion: The blocking effects of DHA on APD and I(to) may serve as one of the anti-arrhythmia mechanisms of DHA.

The mechanisms of cardiac toxicity caused by methamphetamine (MA) are poorly understood at present. This study was designed to investigate the effects of MA on ionic currents in myocardial cells. The effects of MA on transient outward potassium current (I(to)), inward rectifying potassium current (I(K1)), and L-type calcium current (I(Ca-L)) in isolated rat ventricular myocytes were studied using the whole-cell patch clamp technique. It was demonstrated that MA inhibited the I(to), I(K1), and I(Ca-L) in the rat ventricular myocytes concentration-dependently. MA shifted left the I(to) steady-state inactivation curve and shifted down the recovery curve, but had no influence on the steady-state activation curve. MA did not affect the I(Ca-L) steady-state activation curve or steady-state inactivation curve, but shifted down the recovery curve. We concluded that MA had inhibitory effects on the I(to), I(K1), and I(Ca-L) in ventricular myocytes, which might be one of the possible electrophysiological mechanisms of cardiac damage caused by MA.

There is considerable evidence to support a role for lipotoxicity in the development of diabetic cardiomyopathy, although the molecular links between enhanced saturated fatty acid uptake/metabolism and impaired cardiac function are poorly understood. In the present study, the effects of acute exposure to the saturated fatty acid, palmitate, on myocardial contractility and excitability were examined directly. Exposure of isolated (adult mouse) ventricular myocytes to palmitate, complexed to bovine serum albumin (palmitate:BSA) as in blood, rapidly reduced (by 54+/-4%) mean (+/-SEM) unloaded fractional cell shortening. The amplitudes of intracellular Ca(2+) transients decreased in parallel. Current-clamp recordings revealed that exposure to palmitate:BSA markedly shortened action potential durations at 20%, 50%, and 90% repolarization. These effects were reversible and were occluded when the K(+) in the recording pipettes was replaced with Cs(+), suggesting a direct effect on repolarizing K(+) currents. Indeed, voltage-clamp recordings revealed that palmitate:BSA reversibly and selectively increased peak outward voltage-gated K(+) (Kv) current amplitudes by 20+/-2%, whereas inwardly rectifying K(+) (Kir) currents and voltage-gated Ca(2+) currents were unaffected. Further analyses revealed that the individual Kv current components I(to,f), I(K,slow) and I(ss), were all increased (by 12+/-2%, 37+/-4%, and 34+/-4%, respectively) in cells exposed to palmitate:BSA. Consistent with effects on both components of I(K,slow) (I(K,slow1) and I(K,slow)(2)) the magnitude of the palmitate-induced increase was attenuated in ventricular myocytes isolated from animals in which the Kv1.5 (I(K,slow)(1)) or the Kv2.1 (I(K,slow)(2)) locus was disrupted and I(K,slow)(1) or I(K,slow2) is eliminated. Both the enhancement of I(K,slow) and the negative inotropic effect of palmitate:BSA were reduced in the presence of the Kv1.5 selective channel blocker, diphenyl phosphine oxide-1 (DPO-1).Taken together, these results suggest that elevations in circulating saturated free fatty acids, as occurs in diabetes, can directly augment repolarizing myocardial Kv currents and impair excitation-contraction coupling., (Copyright 2009 Elsevier Ltd. All rights reserved.)

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.

Antipsychotic drug perphenazine belongs to the phenothiazine group commonly reported to induce ECG changes and tachyarrhythmias. Data about its effect on ionic membrane currents in cardiomyocytes are missing. We analyzed the effect of perphenazine (0.1-100 microM) on fast sodium current I (Na) and transient outward potassium current I (to) in enzymatically isolated rat right ventricular myocytes by the whole-cell patch-clamp technique at room temperature. Perphenazine reversibly blocked I (Na) (reducing its amplitude; IC(50) = 1.24 +/- 0.10 microM) and I (to) (accelerating its apparent inactivation with a slight decrease of its amplitude; IC(50) = 38.2 +/- 3.5 microM, evaluated from changes of the time integral). The fast time constant of I (to) inactivation was significantly decreased in a concentration-dependent manner (IC(50) = 30.0 +/- 6.6 microM). Both blocks were use and frequency dependent at 3.3 Hz. We conclude that perphenazine causes concentration-, use-, and frequency-dependent block of I (Na) and I (to) . Computer simulations suggest that perphenazine interacts preferentially with I (Na) channels in inactivated states and with I (to) channels in both open and open-inactivated states.

Aim: To study the effect of Sphingosine-1-phosphate on the delayed rectifier potassium current (IK) and the inward rectifier potassium current (IK1) of guinea pig isolated ventricular myocytes., Methods: Whole cell patch clamp technique was applied to record the delayed rectifier potassium current and the delayed rectifier potassium current of guinea pig isolated ventricular myocytes., Results: (1) IK of S1P (1.1 micromol/L) decreased from (1.2 +/- 0.26)nA to (0.95 +/- 0.23)nA. While IK of S1P (2.2 micromol/L) decreased from (1.43 +/- 0.31)nA to (1.02 +/- 0.28)nA. There was significant difference compared to control group (P < 0.01, n = 6). The IK peak value was decreased from (1.29 +/- 0.26) nA to (1.26 +/- 0.37)nA at the group of S1P (1.1 micromol/L) plus suramin (200 micromol/L) and showed no significant difference compared to control group (P > 0.05, n = 6). (2) IK1 of S1P (1.1 micromol/L) decreased from (-8.94 +/- 2.01)nA to (-8.81 = 1.55)nA. While IK of S1P (2.2 micromol/L) decreased from (-8.86 +/- 1.59)nA to (-8.55 +/- 1.39)nA. There was no significant difference compared to control group (P > 0.05, n = 6)., Conclusion: S1P inhibits delayed rectifier potassium current of ventricular myocyte in guinea pig remarkably, S1P shows no effects on delayed rectifier potassium current of ventricular myocyte in guinea pig.

Aconitine (ACO), a highly toxic diterpenoid alkaloid, is recognized to have effects on cardiac voltage-gated Na(+) channels. However, it remains unknown whether it has any effects on K(+) currents. The effects of ACO on ion currents in differentiated clonal cardiac (H9c2) cells and in cultured neonatal rat ventricular myocytes were investigated in this study. In H9c2 cells, ACO suppressed ultrarapid-delayed rectifier K(+) current (I(Kur)) in a time- and concentration-dependent fashion. The IC(50) value for ACO-induced inhibition of I(Kur) was 1.4 microM. ACO could accelerate the inactivation of I(Kur) with no change in the activation time constant of this current. Steady-state inactivation curve of I(Kur) during exposure to ACO could be demonstrated. Recovery from block by ACO was fitted by a single-exponential function. The inhibition of I(Kur) by ACO could still be observed in H9c2 cells preincubated with ruthenium red (30 microM). Intracellular dialysis with ACO (30 microM) had no effects on I(Kur). I(Kur) elicited by simulated action potential (AP) waveforms was sensitive to block by ACO. Single-cell Ca(2+) imaging revealed that ACO (10 microM) alone did not affect intracellular Ca(2+) in H9c2 cells. In cultured neonatal rat ventricular myocytes, ACO also blocked I(Kur) and prolonged AP along with appearance of early afterdepolarizations. Multielectrode recordings on neonatal rat ventricular tissues also suggested that ACO-induced electrocardiographic changes could be associated with inhibition of I(Kur). This study provides the evidence that ACO can produce a depressant action on I(Kur) in cardiac myocytes.

Aim: To record funny currents (If) of ventricular myocytes and to analysize hyperpolarization-activated cation channel(HCN) expression in the rats of different ages., Methods: Fresh ventricular myocytes were isolated from 3 days rats and adult rats.HCN expressions were measured by real-time quantitative polymerase chain reaction(real-time PCR). It was recorded through whole-cell patch clamp., Results: HCN1, HCN2, HCN3, HCN4 mRNA represented 0.23% +/- 0.01%, 83.58% +/- 0.04%, 0.79% +/- 0.01%, 15.44% +/- 0.01% of total HCN mRNA in the neonatal rats, respectively. If was recorded and the threshold for activation was -75 mV. In the adult rat, HCN1, HCN2, HCN3, HCN4 mRNA represented 0.72% +/- 0.02%, 91.58% +/- 0.08%, 0.27% +/- 0.02%, 7.12% +/- 0.02% of total HCN mRNA. The ratio of HCN2 to HCN4 was approximately (13.06 +/- 0.21):1. The threshold for activation of If was approximately -115 mV in the adult rats., Conclusion: With the development of rats, the value of If is smaller. The threshold for activation of If is more negative. The ratio of HCN2 to HCN4 is bigger.

Background: Current strategies of engineering bioartificial pacemakers from otherwise silent yet excitable adult atrial and ventricular cardiomyocytes primarily rely on either maximizing the hyperpolarization-activated I(f) or on minimizing its presumptive opponent, the inwardly rectifying potassium current I(K1)., Objective: The purpose of this study was to determine quantitatively the relative current densities of I(f) and I(K1) necessary to induce automaticity in adult atrial cardiomyocytes., Methods: Automaticity of adult guinea pig atrial cardiomyocytes was induced by adenovirus (Ad)-mediated overexpression of the gating-engineered HCN1 construct HCN1-DeltaDeltaDelta with the S3-S4 linker residues EVY235-7 deleted to favor channel opening., Results: Whereas control atrial cardiomyocytes remained electrically quiescent and had no I(f), 18% of Ad-CMV-GFP-IRES-HCN1-DeltaDeltaDelta (Ad-CGI-HCN1-DeltaDeltaDelta)-transduced cells demonstrated automaticity (240 +/- 14 bpm) with gradual phase 4 depolarization (143 +/- 28 mV/s), a depolarized maximal diastolic potential (-45.3 +/- 2.2 mV), and substantial I(f) at -140 mV (I(f,-140 mV) = -9.32 +/- 1.84 pA/pF). In the remaining quiescent Ad-CGI-HCN1-DeltaDeltaDelta-transduced atrial cardiomyocytes, two distinct immediate phenotypes were observed: (1) 13% had a hyperpolarized resting membrane potential (-56.7 +/- 1.3 mV) with I(f,-140 mV) of -4.85 +/- 0.97 pA/pF; and (2) the remaining 69% displayed a depolarized resting membrane potential (-27.6 +/- 1.3 mV) with I(f,-140 mV) of -23.0 +/- 3.71 pA/pF. Upon electrical stimulation, both quiescent groups elicited a single action potential with incomplete phase 4 depolarization that was never seen in controls. Further electrophysiologic analysis indicates that an intricate balance of I(K1) and I(f) is necessary for induction of atrial automaticity., Conclusion: Optimized pacing induction and modulation can be better achieved by engineering the I(f)/I(K1) ratio rather than the individual currents.

In the present study, whole-cell patch-clamp technique was used to observe the effects of SNC162, a selective agonist of δ-opioid receptors, on L-type Ca(2+) current (I(Ca-L)) and transient outward K(+) current (I(to)) in rat ventricular myocytes. The results showed that SNC162 significantly inhibited I(Ca-L) and I(to) in rat ventricular myocytes. The maximal inhibition rate of I(Ca-L) and I(to) reached (46.13±4.12)% and (36.53±10.57)%, respectively. SNC162 at 1×10(-4) mol/L inhibited the current density of I(Ca-L) from (8.98±0.40) pA/pF to (4.84±0.44) pA/pF (P<0.01, n=5) and inhibited that of I(to) from (18.69±2.42) pA/pF to (11.73±1.67) pA/pF (P<0.01, n=5). Furthermore, the effects of naltrindole, a highly selective antagonist of δ-opioid receptors, on I(Ca-L) and I(to) were also observed. The results showed that naltrindole alone had no effects on I(Ca-L) and I(to), while it abolished the inhibitory effects of SNC162 on I(Ca-L) and I(to). In conclusion, SNC162 concentration-dependently inhibited I(Ca-L) and I(to) in rat ventricular myocytes via activation of the δ-opioid receptors, which may be a fundamental mechanism underlying the antiarrhythmic effect of activating δ-opioid receptors.

Recent evidence has suggested microRNAs as viable therapeutic targets for a wide range of human disease. However, lack of gene-specificity of microRNA actions may hinder this application. Here we developed two new approaches, the gene-specific microRNA mimic and microRNA-masking antisense approaches, to explore the possibility of using microRNA's principle of actions in a gene-specific manner. We examined the value of these strategies as rational approaches to develop heart rate-reducing agents and "biological pacemakers" by manipulating the expression of the cardiac pacemaker channel genes HCN2 and HCN4. We showed that the gene-specific microRNA mimics, 22-nt RNAs designed to target the 3'untranslated regions (3'UTRs) of HCN2 and HCN4, respectively, were efficient in abrogating expression and function of HCN2 and HCN4. The gene-specific microRNA mimics repressed protein levels, accompanied by depressed f-channel conductance and the associated rhythmic activity, without affecting mRNA levels of HCN2 and HCN4. Meanwhile, we also designed the microRNA-masking antisense based on the miR-1 and miR-133 target sites in the 3'UTRs of HCN2 and HCN4 and found that these antisense oligodeoxynucleotides markedly enhanced HCN2/HCN4 expression and function, as reflected by increased protein levels of HCN2/HCN4 and If conductance, by removing the repression of HCN2/HCN4 expression induced by endogenous miR-1/miR-133. The experimental examination of these techniques and the resultant findings not only indicate feasibility of interfering miRNA action in a gene-specific fashion but also may provide a new research tool for studying function of miRNAs. The new approaches also have the potential of becoming alternative gene therapy strategies.

Verrucotoxin is the major component of venom from the stonefish (Synanceia verrucosa). Stings from the dorsal spines of the stonefish produce intensive pain, convulsions, hypotension, paralysis, respiratory weakness and collapse of the cardiovascular system, occasionally leading to death. It has been reported that verrucotoxin might modulate ATP-sensitive K+ (KATP) current in frog atrial fibers. However, the mechanism by which verrucotoxin acts on KATP current remains unclear. In this study, we examined whether verrucotoxin inhibited KATP current in guinea pig ventricular myocytes, using the patch clamp method. Verrucotoxin suppressed KATP current induced by pinacidil (KATP channel opener) in a concentration-dependent manner, with a half maximum concentration of 16.3 microg/ml. The effect of verrucotoxin on KATP current was suppressed by atropine (1 microM), a muscarinic receptor antagonist, or by 4-diphenylacetoxy-N-methylpiperidine (100 nM), a muscarinic M3 receptor antagonist. Furthermore, the effect of verrucotoxin on KATP current was attenuated by the protein kinase C (PKC) inhibitor chelerythrine (10 microM) and calphostin C (10 microM), yet not by the cAMP-dependent protein kinase (PKA) inhibitor H-89 (0.5 microM). These results suggest that verrucotoxin inhibits KATP current through the muscarinic M3 receptor-PKC pathway. These findings enhance our understanding of the toxic effects of verrucotoxin from the stonefish.

Background: Although I(f), encoded by the hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channel gene family, is known to be functionally important in pacing, its mechanistic action is largely inferential and indeed somewhat controversial. To dissect in detail the role of I(f), we investigated the functional consequences of overexpressing in adult guinea pig left ventricular cardiomyocytes (LVCMs) various HCN1 constructs that have been engineered to exhibit different gating properties., Methods and Results: We created the recombinant adenoviruses Ad-CMV-GFP-IRES (CGI), Ad-CGI-HCN1, Ad-CGI-HCN1-delta delta delta, and Ad-CGI-HCN1-Ins, which mediate ectopic expression of GFP alone, WT, EVY235-7delta delta delta, and Ins HCN1 channels, respectively; EVY235-7delta delta delta and Ins encode channels in which the S3-S4 linkers have been shortened and lengthened to favor and inhibit opening, respectively. Ad-CGI-HCN1, Ad-CGI-HCN1-delta delta delta, and Ad-CGI-HCN1-Ins, but not control Ad-CGI, transduction of LVCMs led to robust expression of I(f) with comparable densities when fully open (approximately = -22 pA/pF at -140 mV; P>0.05) but distinctive activation profiles (V(1/2) = -70.8+/-0.6, -60.4+/-0.7, and -87.7+/-0.7 mV; P<0.01, respectively). Whereas control (nontransduced or Ad-CGI-transduced) LVCMs were electrically quiescent, automaticity (206+/-16 bpm) was observed exclusively in 61% of Ad-HCN1-delta delta delta-transduced cells that displayed depolarized maximum diastolic potential (-60.6+/-0.5 versus -70.6+/-0.6 mV of resting membrane potential of control cells; P<0.01) and gradual phase 4 depolarization (306+/-32 mV/s) that were typical of genuine nodal cells. Furthermore, spontaneously firing Ad-HCN1-delta delta delta-transduced LVCMs responded positively to adrenergic stimulation (P<0.05) but exhibited neither overdrive excitation nor suppression. In contrast, the remaining 39% of Ad-HCN1-delta delta delta-transduced cells exhibited no spontaneous action potentials; however, a single ventricular action potential associated with a depolarized resting membrane potential and a unique, incomplete "phase 4-like" depolarization that did not lead to subsequent firing could be elicited on simulation. Such an intermediate phenotype, similarly observed in 100% of Ad-CGI-HCN- and Ad-CGI-HCN1-Ins-transduced LVCMs, could be readily reversed by ZD7288, hinting at a direct role of I(f). Correlation analysis revealed the specific biophysical parameters required for I(f) to function as an active membrane potential oscillator., Conclusions: Our results not only contribute to a better understanding of cardiac pacing but also may advance current efforts that focus primarily on automaticity induction to the next level by enabling bioengineering of central and peripheral cells that make up the native sinoatrial node.

Application of the current-clamp technique in rainbow trout atrial myocytes has yielded resting membrane potentials that are incompatible with normal atrial function. To investigate this paradox, we recorded the whole membrane current (I(m)) and compared membrane potentials recorded in isolated cardiac myocytes and multicellular preparations. Atrial tissue and ventricular myocytes had stable resting potentials of -87 +/- 2 mV and -83.9 +/- 0.4 mV, respectively. In contrast, 50 out of 59 atrial myocytes had unstable depolarized membrane potentials that were sensitive to the holding current. We hypothesized that this is at least partly due to a small slope conductance of I(m) around the resting membrane potential in atrial myocytes. In accordance with this hypothesis, the slope conductance of I(m) was about sevenfold smaller in atrial than in ventricular myocytes. Interestingly, ACh increased I(m) at -120 mV from 4.3 pA/pF to 27 pA/pF with an EC(50) of 45 nM in atrial myocytes. Moreover, 3 nM ACh increased the slope conductance of I(m) fourfold, shifted its reversal potential from -78 +/- 3 to -84 +/- 3 mV, and stabilized the resting membrane potential at -92 +/- 4 mV. ACh also shortened the action potential in both atrial myocytes and tissue, and this effect was antagonized by atropine. When applied alone, atropine prolonged the action potential in atrial tissue but had no effect on membrane potential, action potential, or I(m) in isolated atrial myocytes. This suggests that ACh-mediated activation of an inwardly rectifying K(+) current can modulate the membrane potential in the trout atrial myocytes and stabilize the resting membrane potential.

Although sigma ligand haloperidol is known to affect repolarization in heart, its effect on potassium currents in cardiomyocytes has not yet been studied. We analyzed the effect of 1 micromol/l haloperidol on transient outward K(+) current (I(to)) in enzymatically isolated rat right ventricular cardiomyocytes using the whole-cell patch-clamp technique at room temperature. Haloperidol induced a decrease of amplitude and an acceleration of apparent inactivation of I(to), both in a voltage-independent manner. The averaged inhibition of I(to), evaluated as a change of its time integral, was 23.0+/-3.2% at stimulation frequency of 0.1 Hz. As a consequence of slow recovery of I(to) from the haloperidol-induced block (time constant 1482+/-783 ms), a cumulation of the block up to about 40% appeared at 3.3 Hz. We conclude that haloperidol causes a voltage-independent block of I(to) that cumulates at higher stimulation frequencies. Based on the computer reconstruction of experimental data, a block of I(to)-channels in both open and open-inactivated states appears to be likely mechanism of haloperidol-induced inhibition of I(to).

The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is activated by elevated intracellular Ca(2+) (Ca(2+)(i)), and mice with chronic myocardial CaMKII inhibition (Inh) resulting from transgenic expression of a CaMKII inhibitory peptide (AC3-I) unexpectedly showed action potential duration (APD) shortening. Inh mice exhibit increased L-type Ca(2+) current (I(Ca)), because of upregulation of protein kinase A (PKA) activity, and decreased CaMKII-dependent phosphorylation of phospholamban (PLN). We hypothesized that CaMKII is a molecular signal linking Ca(2+)(i) to repolarization. Whole cell voltage-clamp recordings revealed that the fast transient outward current (I(to,f)) and the inward rectifier current (I(K1)) were selectively upregulated in Inh, compared with wild-type (WT) and transgenic control, mice. Breeding Inh mice with mice lacking PLN returned I(to,f) and I(K1) to control levels and equalized the APD and QT intervals in Inh mice to control and WT levels. Dialysis of AC3-I into WT cells did not result in increased I(to,f) or I(K1), suggesting that enhanced cardiac repolarization in Inh mice is an adaptive response to chronic CaMKII inhibition rather than an acute effect of reduced CaMKII activity. Increasing PKA activity, by cell dialysis with cAMP, or inhibition of PKA did not affect I(K1) in WT cells. Dialysis of WT cells with cAMP also reduced I(to,f), suggesting that PKA upregulation does not increase repolarizing K(+) currents in Inh mice. These findings provide novel in vivo and cellular evidence that CaMKII links Ca(2+)(i) to cardiac repolarization and suggest that PLN may be a critical CaMKII target for feedback regulation of APD in ventricular myocytes.

Objective: To investigate the effect of Zhigancao Decoction (ZD) containing serum on transient outward potassium current (I(to)) in isolated single ventricular myocyte of rabbits., Methods: Single rabbit ventricular myocytes were isolated and divided into 6 groups, the normal group, the non-drug-containing serum group, and the ZD groups, they were treated with normal extracellular fluid alone or with ZD containing serum recording technique was used to observe the changes of I(to)., Results: After treated with 5%, 10%, 20% and 40% ZD containing serum, the I(to) was inhibited and its peak value dropped from (16.1 +/- 1.4) PA/PF to (13.9 +/-1.5) PA/PF, (11.8 +/- 1.9) PA/PF, (8.3 +/- 1.5) PA/PF and (8.2 +/- 1.2) PA/PF respectively, and the effect could be eliminated by elution., Conclusion: ZD containing serum could inhibit the I(to) in isolated ventricular myocytes of rabbits in a concentration-dependent manner, which may be the mechanism of antiarrhythmic action of ZI).

Aim: To study the effect of AMP579 and adenosine on potassium ionic (K+) or sodium ionic (Na+) channels and to elucidate ionic mechanisms underlying negative inotropic and antiarrhythmic effects of AMP579 and adenosine., Methods: Ionic channel currents of rat and guinea pig ventricular myocytes were recorded by patch clamp technique in whole-cell configuration., Results: Adenosine showed a stronger activating effect on transient outward K+ current (I(to)) than AMP579, EC50 of adenosine and AMP579 were 2.33 and 8. 32 micromol x L(-1), respectively (P < 0.05). An adenosine A1 receptor blocker, 1,3-dipropyl-8-cyclopentylxanthine (PD116948), can abolish the effects of AMP579 and adenosine on I(to), demonstrating that the effect is mediated by adenosine A1 receptor. Adenosine exerted a more obvious inhibitory effect on delayed rectifier K+ current (IK) than AMP579. IC50 of adenosine and AMP579 were 1.21 and 2.31 micromol x L(-1), respectively (P < 0.05). AMP579 had a more powerful inhibitory effect on inward rectifier K+ current (IK1) than adenosine. IC50 of AMP579 and adenosine were 4.15 and 20.7 micromol x L(-1), repectively (P < 0.01). AMP579 and adenosine exerted a similar inhibitory effect on fast inward Na+ current (INA), IC50 of AMP579 and adenosine were 9.46 and 6.23 micromol x L(-1), respectively (P > 0.05)., Conclusion: Adenosine showed a stronger activating effect on I(to) than AMP579, however, the mechanism of AMP579 and adenosine activating I(to) was mediated by adenosine A1 receptor. AMP579 has a more powerful inhibitory effect on IK1, and less inhibitory effect on IK than adenosine. Both drugs have a similar inhibitory effect on INa. The negative inotropic and antiarrhythmic effects are related to these ionic mechanisms.

Cocaine use may cause coronary artery spasm and acute myocardial ischaemia/infarction. However, its effects on ATP-sensitive K+ (KATP) channel, an ion channel responsible for ischaemic preconditioning, remain unknown. In isolated rat ventricular myocytes with whole-cell experiments, cocaine can reverse action potential shortening and increased K+ current caused by the openers of ATP-sensitive K+ (KATP) channels. In inside-out patches, cocaine applied to intracellular surface suppressed KATP-channel activity in a concentration-dependent manner with an IC50 value of 9.2 microM; however, it did not modify the single-channel conductance of this channel. The change in the kinetic behaviour of KATP channels caused by cocaine is primarily the result of an increase in mean closed time and a decrease in mean open time. Cocaine-induced inhibition of KATP channels is independent of change in intracellular ATP concentrations. In heart-derived H9c2 cells, cocaine is also capable of suppressing KATP-channel activity. The present study provides evidence that cocaine can produce a depressant action on KATP channels in cardiac myocytes, and thus disturb ischaemic preconditioning in clinical settings.

P-1075 was reported to be selective for sarcolemmal ATP-sensitive K (sarcKATP) channels because of its lack of mitochondria-oxidizing effect in isolated adult rabbit myocytes. Conflicting results however have been reported on the action of P-1075 on mitochondrial KATP (mitoKATP) channels. The aim of the present study was to reevaluate the effect of P-1075 on mitoKATP channels. Endogenous flavoprotein autofluorescence in isolated rabbit ventricular myocytes was measured to assay mitoKATP channel activity. The mitoKATP channel opener diazoxide (100 microM) oxidized the flavoprotein. P-1075 at a concentration of 30 microM significantly inhibited the oxidative effect of diazoxide from 48.6 +/- 7.1% to 28.2 +/- 5.2% of the maximum value induced by 2,4-dinitrophenol (DNP, 100 microM) (n = 4, P < 0.05). Additionally, P-1075 (30 microM) significantly inhibited the pinacidil (100 microM)-induced flavoprotein oxidation by 48.4 +/- 3.9% (n = 4, P < 0.05). The inhibitory effects of P-1075 on diazoxide-induced flavoprotein oxidation were observed in the micromolar range in a concentration dependent manner. P-1075 alone oxidized flavoprotein in the range 30 nM to 5 microM, and was nearly maximal at 150 nM (11.1 +/- 1.9% of the DN P value, n = 5). These results indicate that P-1075 exerts diverse effects on mitoKATP channel in rabbit ventricular myocytes: P-1075 has a partial agonist effect and weakly activates mitoKATP channels at low concentrations, whereas P-1075 rather inhibits the opening of mitoKATP channels in the micromolar concentration range.

Metabolic oscillations and the concomitant periodic activations of sarcolemmal ATP-sensitive K(+) channels (sarcK(ATP)) have recently been proposed as one mechanism underlying ischemia-related arrhythmia. In this study, we investigated the role of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)) and ATP synthase in the generation of metabolic oscillations during simulated ischemia from rat ventricular myocytes using patch-clamp technique and fluorescence microscopy. We have found that the combined application of creatine kinase (CK) inhibitor, 2,4-dinitrofluorobenzene, with cyanide, electron-transport-chain inhibitor causes oscillatory activations of sarcK(ATP). The oscillatory activations of sarcK(ATP) were accompanied by large periodic depolarizations in mitochondrial membrane potential (Psi(m)). 5-Hydroxydecanoate, an inhibitor of mitoK(ATP), halted the oscillations in Psi(m) at repolarized state, whereas oligomycin, an inhibitor of ATP synthase, halted them at depolarized state. In both conditions, oscillatory activations of sarcK(ATP) were abolished. Inhibitors of adenine nucleotide translocator and permeability transition pore had no effect on the oscillations in Psi(m) and sarcK(ATP). 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, an inhibitor of mitochondrial inner-membrane anion channel (IMAC), caused a full depolarization in Psi(m) and activation of sarcK(ATP), finally resulting in irreversible hypercontracture. Taken together, oscillations in Psi(m) can be explained by balance between depolarizing power of mitoK(ATP) and repolarizing power of the reverse activity of ATP synthase. ATP consumption by ATP synthase in reverse mode links periodic depolarizations in Psi(m) to oscillatory activation of sarcK(ATP). Considering that such oscillations were not induced by cyanide alone, CK system may act as an important buffer, inhibiting arrhythmia during ischemia.

Gender disparities in ECG variables and susceptibility to arrhythmia exist. The basis of these sex-related distinctions in cardiac electrophysiology has been extensively studied in various species, but is virtually unexplored in humans. The aim of this study was to clarify the cellular basis of electrophysiological gender disparities in human cardiac myocytes. Human midmyocardial left ventricular myocytes were isolated from explanted hearts of male and female patients in end-stage heart failure at the time of cardiac transplantation. The action potentials, sarcolemmal ion currents, and susceptibility to the generation of early afterdepolarizations were studied using whole-cell patch-clamp methodology. The functional effects of gender disparities in sarcolemmal ion currents were assessed by computer simulations using the Priebe-Beuckelmann or the ten Tusscher-Noble-Noble-Panfilov human ventricular cell models. Female myocytes had significantly longer action potentials and greater susceptibility to early afterdepolarizations than male myocytes. All other action potential parameters (resting membrane potential, amplitude, plateau level, upstroke velocity, maximal velocity of phase-1 and phase-3 repolarization) had similar values for both genders. In female myocytes, the transient outward potassium current (I(to1)) tended to be smaller, while the L-type calcium current (I(Ca,L)) and quasi-steady state current (I(QSS)) tended to be larger. Computer simulations showed that these subtle differences in sarcolemmal ion currents may conspire to cause the observed gender disparities in action potential properties. Female failing myocytes have longer action potentials and a greater susceptibility to early afterdepolarizations than male failing myocytes. These gender disparities may be due to slightly larger depolarizing I(Ca,L) in conjunction with slightly smaller repolarizing I(QSS) and I(to1) in female myocytes.

Background: Although pharmacological block of the slow, delayed rectifier potassium current (IKs) by chromanol 293B, L-735,821, or HMR-1556 produces little effect on action potential duration (APD) in isolated rabbit and dog ventricular myocytes, the effect of IKs block on normal human ventricular muscle APD is not known. Therefore, studies were conducted to elucidate the role of IKs in normal human ventricular muscle and in preparations in which both repolarization reserve was attenuated and sympathetic activation was increased by exogenous dofetilide and adrenaline., Methods and Results: Preparations were obtained from undiseased organ donors. Action potentials were measured in ventricular trabeculae and papillary muscles using conventional microelectrode techniques; membrane currents were measured in ventricular myocytes using voltage-clamp techniques. Chromanol 293B (10 micromol/L), L-735,821 (100 nmol/L), and HMR-1556 (100 nmol/L and 1 micromol/L) produced a <12-ms change in APD while pacing at cycle lengths ranging from 300 to 5000 ms, whereas the IKr blockers sotalol and E-4031 markedly lengthened APD. In voltage-clamp experiments, L-735,821 and chromanol 293B each blocked IKs in the presence of E-4031 to block IKr. The E-4031-sensitive current (IKr) at the end of a 150-ms-long test pulse to 30 mV was 32.9+/-6.7 pA (n=8); the L-735,821-sensitive current (IKs) magnitude was 17.8+/-2.94 pA (n=10). During a longer 500-ms test pulse, IKr was not substantially changed (33.6+/-6.1 pA; n=8), and IKs was significantly increased (49.6+/-7.24 pA; n=10). On application of an "action potential-like" test pulse, IKr increased as voltage became more negative, whereas IKs remained small throughout all phases of the action potential-like test pulse. In experiments in which APD was first lengthened by 50 nmol/L dofetilide and sympathetic activation was increased by 1 micromol/L adrenaline, 1 micromol/L HMR-1556 significantly increased APD by 14.7+/-3.2% (P<0.05; n=3)., Conclusions: Pharmacological IKs block in the absence of sympathetic stimulation plays little role in increasing normal human ventricular muscle APD. However, when human ventricular muscle repolarization reserve is attenuated, IKs plays an increasingly important role in limiting action potential prolongation.

Background: Hypothermic hyperkalemic cardioplegia results in significant myocyte swelling and impaired contractility. These detrimental effects may be eliminated by the addition of an adenosine triphosphate-sensitive potassium (KATP) channel opener. This study evaluated the hypothesis that a KATP channel opener (diazoxide) would benefit volume homeostasis by limiting volume and subsequent contractility changes during osmotic stress., Methods and Results: Isolated rabbit ventricular myocyte volume and contractility were evaluated using video microscopy and field stimulation after exposure to osmotic stress at 37 degrees C. Myocytes were exposed to Tyrode's physiological solution for 20 minutes and test solution for 20 minutes, and then reexposed to Tyrode's for 20 minutes. Test solutions included control Tyrode's (1T) and osmotically altered Tyrode's (2.6T, 0.9T, and 0.6T) solutions with or without the KATP channel opener diazoxide. Severe osmotic stress (2.6T and 0.6T) resulted in significant cell shrinkage and swelling, respectively. This was unchanged by the addition of diazoxide. Mild hyposmotic stress (0.9T) resulted in significant cell swelling that was eliminated by the addition of diazoxide. Cell swelling was associated with reduced contractility., Conclusions: Cell swelling, but not shrinkage, was detrimental to myocyte contractility. Diazoxide eliminated volume change due to mild hyposmotic stress, similar to that previously noted with hyperkalemic cardioplegia, but did not alter volume change secondary to severe osmotic stress.

Diabetes induces oxidative stress and leads to attenuation of cardiac K+ currents. We investigated the role of superoxide ions and angiotensin II (ANG II) in generating and linking oxidative stress to the modulation of K+ currents under diabetic conditions. K+ currents were measured using patch-clamp methods in ventricular myocytes from streptozotocin (STZ)-induced diabetic rats. Superoxide ion levels, indicating oxidative stress, were measured by fluorescent labelling with dihydroethidium (DHE). ANG II content was measured using enzyme-linked immunosorbent asssay (ELISA). The results showed DHE fluorescence to be significantly higher in cells from diabetic males, compared to controls. Relief of stress by the NADPH oxidase inhibitor apocynin or by superoxide dismutase (SOD) but not by catalase reversed the attenuation of K+ currents and reduced DHE fluorescence. In cells from diabetic females, neither apocynin nor SOD augmented K+ currents, ANG II was not elevated and DHE fluorescence was significantly weaker than in cells from males. Reduced glutathione (GSH) also augmented K+ currents in cells from diabetic males but not females. In ovariectomized diabetic females K+ currents were augmented by GSH and apocynin. Current augmentation and the attenuation of DHE fluorescence by apocynin were significantly blunted by excess ANG II (300 nm). Diabetic male rats pretreated with the angiotensin-converting enzyme (ACE) inhibitor quinapril were hyperglycaemic, but their cellular ANG II levels and DHE fluorescence were significantly decreased. In cells from these rats, K+ currents were insensitive to apocynin. In conclusion, diabetes-related oxidative stress attenuates K+ currents through ANG II-generated increased superoxide ion levels. When ANG II levels are lower, as in diabetic females or following ACE inhibition in males, oxidative stress is reduced, with blunted alterations in K+ currents.

The Kv channel interacting proteins (KChIPs) were identified in a yeast two hybrid screen using the N terminus of Kv4.3 as bait. Previous studies have demonstrated that KChIP2 associates with voltage-gated K(+) (Kv) pore-forming (alpha) subunits of the Kv4 subfamily and contributes to the formation of the rapidly inactivating and recovering Kv4-encoded cardiac transient outward K(+) channels, I(to,f). Here, we report that co-expression of KChIP2 (or KChIP1) also modulates the functional cell surface expression of Kv1.5-encoded K(+) channels in transiently transfected HEK-293 cells. In contrast to the effects of KChIP2 on Kv4 channels, however, co-expression of KChIP2 (or KChIP1) decreases Kv1.5-encoded K(+) currents. Although current densities are reduced, KChIP2 (or KChIP1) co-expression does not affect the time- or voltage-dependent properties of heterologously expressed Kv1.5-encoded K(+) currents. Immunohistochemical and cell surface biotinylation experiments demonstrate that KChIP2 reduces the cell surface expression of Kv1.5, likely by inhibiting forward trafficking from the endoplasmic reticulum. In addition, biochemical experiments reveal that KChIP2 co-immunoprecipitates with Kv1.5 (as well as Kv4.2/Kv4.3) from adult mouse ventricles, demonstrating that, similar to other Kv accessory subunits, KChIP2 is a multifunctional Kv channel accessory subunit. Taken together, the results here suggest that KChIP2 contributes to the formation of functional mouse ventricular (Kv1.5-encoded) I(K,slow1) channels as well, perhaps, as other Kv1.5-encoded K(+) currents, including I(Kur) (I(K,ultrarapid)), in human atria.

K(+) currents expressed in freshly dispersed rat ventricular fibroblasts have been studied using whole-cell patch-clamp recordings. Depolarizing voltage steps from a holding potential of -90 mV activated time- and voltage-dependent outward currents at membrane potentials positive to approximately -30 mV. The relatively slow activation kinetics exhibited strong dependence on the membrane potential. Selected changes in extracellular K(+) concentration ([K(+)](o)) revealed that the reversal potentials of the tail currents changed as expected for a K(+) equilibrium potential. The activation and inactivation kinetics of this K(+) current, as well as its recovery from inactivation, were well-fitted by single exponential functions. The steady-state inactivation was well described by a Boltzmann function with a half-maximal inactivation potential (V(0.5)) of -24 mV. Increasing [K(+)](o) (from 5 to 100 mM) shifted this V(0.5) in the hyperpolarizing direction by -11 mV. Inactivation was slowed by increasing [K(+)](o) to 100 mM, and the rate of recovery from inactivation was decreased after increasing [K(+)](o). Block of this K(+) current by extracellular tetraethylammonium also slowed inactivation. These [K(+)](o)-induced changes and tetraethylammonium effects suggest an important role for a C-type inactivation mechanism. This K(+) current was sensitive to dendrotoxin-I (100 nM) and rTityustoxin Kalpha (50 nM).

The mechanism of ajmaline-induced inhibition of the transient outward current (I(to)) has been investigated in right ventricular myocytes of rat using the whole cell patch clamp technique. Ajmaline decreased the amplitude and the time integral of I(to) in a concentration-dependent, but frequency- and use-independent manner. In contrast to the single exponential time course of I(to)-inactivation in control conditions (tau(i) = 37.1 +/- 2.7 ms), the apparent inactivation was fitted by a sum of two exponentials under the effect of ajmaline with concentration-dependent fast and slow components (tau(f) = 11.7 +/- 0.8 ms, tau(s) = 57.6 +/- 2.7 ms at 10 micromol/l) suggesting block development primarily in the open channel state. An improved expression enabling to calculate the association and dissociation rate constants from the concentration dependence of tau(f) and tau(s) was derived and resulted in k(on) = 4.57 x 10(6) +/- 0.32 x 10(6) mol(-1).l.s(-1) and k(off) = 20.12 +/- 5.99 s(-1). The value of K(d) = 4.4 micromol/l calculated as k(off) / k(on) was considerably lower than IC(50) = 25.9 +/- 2.9 micromol/l evaluated from the concentration dependence of the integrals of I(to). Simulations on a simple model combining Hodgkin-Huxley type gating kinetics and drug-channel interaction entirely in open channel state agreed well with the experimental data including the difference between the K(d) and IC(50). According to the model, the fraction of blocked channels increases upon depolarization and declines if depolarization is prolonged. The repolarizing step induces recovery from block with time constant of 52 ms. We conclude that in the rat right ventricular myocytes, ajmaline is an open channel blocker with fast recovery from the block at resting voltage.

Halothane, isoflurane, and sevoflurane abbreviate ventricular action potential duration (APD), and for halothane this effect is greater in the subendocardium than in the subepicardium. In this study we investigated mechanisms underlying the regional effects of these anesthetics on APD. The effect of 0.6 mM halothane, isoflurane, and sevoflurane on the action potential, L-type Ca(2+) current, transient outward K(+) current (I(to)), and steady-state current was recorded in rat left ventricular subendocardial and subepicardial myocytes. Halothane and isoflurane (but not sevoflurane) reduced APD significantly (P < 0.05), more in subendocardial than subepicardial myocytes. Peak L-type Ca(2+) current did not differ between regions and, compared with control, was reduced significantly in both regions by 40% (P < 0.001), 20% (P < 0.001), and 12% (P < 0.01) by halothane, isoflurane, and sevoflurane, respectively. I(to) was greater in subepicardial (3.95 +/- 0.29 nA) than subendocardial (1.12 +/- 0.05 nA) myocytes. In subepicardial myocytes, peak I(to) was reduced significantly by halothane (P < 0.01) and isoflurane (P < 0.05) (by 8% and 7%, respectively) but was unaffected by sevoflurane. No significant reduction of I(to) was observed in subendocardial myocytes with the three anesthetics. The steady-state current was increased significantly (P < 0.05), but the extent of this increase did not differ between the two regions or among the three anesthetics. Therefore, greater inhibition of I(to) in subepicardial than subendocardial myocytes by halothane and isoflurane could underlie their transmural effects on APD.

Activation of the platelet-activating factor (PAF) receptor leads to a decrease in outward current in murine ventricular myocytes by inhibiting the TASK-1 channel. TASK-1 carries a background or "leak" current and is a member of the two-pore domain potassium channel family. Its inhibition is sufficient to delay repolarization, causing prolongation of the action potential duration, and in some cases, early after depolarizations. We set out to determine the cellular mechanisms that control regulation of TASK-1 by PAF. Inhibition of TASK-1 via activation of the PAF receptor is protein kinase C (PKC)-dependent. Using isoform-specific PKC inhibitor or activator peptides in patch clamp experiments, we now demonstrate that activation of PKCepsilon is both necessary and sufficient to regulate murine TASK-1 current in a heterologous expression system and to induce repolarization abnormalities in isolated myocytes. Furthermore, site-directed mutagenesis studies have identified threonine 381, in the C-terminal tail of murine TASK-1, as a critical residue in this regulation.

Arsenic trioxide (As(2)O(3)) produces dramatic remissions in patients with relapsed or refractory acute promyelocytic leukemia. Its clinical use is burdened by QT prolongation, torsade de pointes, and sudden cardiac death. In the present study, we analyzed the molecular mechanisms leading to As(2)O(3)-induced abnormalities of cardiac electrophysiology. Using biochemical and electrophysiological methods, we show that long-term exposure to As(2)O(3) increases cardiac calcium currents and reduces surface expression of the cardiac potassium channel human ether-a-go-go-related gene (HERG) at clinically relevant concentrations of 0.1 to 1.5 microM. In ventricular myocytes, As(2)O(3) increases action potential duration measured at 30 and 90% of repolarization. As(2)O(3) interferes with hERG trafficking by inhibition of hERG-chaperone complexes and increases calcium currents by a faster cellular process. We propose that an increase in cardiac calcium current and reduced trafficking of hERG channels to the cell surface cause QT prolongation and torsade de pointes in patients treated with As(2)O(3). Our results suggest that calcium-channel antagonists will be useful in normalizing QT prolongation during As(2)O(3) therapy. As(2)O(3) is the first example of a drug that produces hERG liability by inhibition of ion-channel trafficking. Other drugs that interfere with proteins in the processing pathway of cardiac ion channels may be proarrhythmic for similar reasons.

This study was designed to investigate the effects of platelet activating factor (PAF) on the action potential and potassium currents in guinea-pig ventricular myocytes. Whole cell patch clamp techniques were used. With 5 mmol/L ATP in the pipette electrode(mimic normal condition), 1 micromol/L PAF increased APD(90) from 225.8+/-23.3 to 352.8+/-29.8 ms (n=5, P<0.05), decreased I(K1) and I(K) tail currents from -6.1+/-1.3 to -5.6+/-1.1 nA (n=5, P<0.05) at -120 mV and from 173.5+/-16.7 to 152.1+/-11.5 pA (P<0.05, n=4) at +30 mV, respectively. But PAF had no effect on I(K1) at potentials within the normal range of membrane potentials (between -90 mV and +20 mV). In the contrary, without ATP in the pipette electrode by which I(K.ATP) was activated (mimic ischemic condition), 1 micro mol/L PAF shortened APD(90) from 153+/-24.6 to 88.2+/-19.4 ms (n=5, P<0.01). Incubation of myocytes with 1 micro mol/L glibenclamide, a blocker of I(K.ATP ) could restore prolongation of APD induced by PAF. In conclusion, in guinea-pig ventricular myocytes, with 5 mmol/L ATP in the pipette PAF could prolong APD partly due to the inhibition of I(K); while with 0 mmol/L ATP in the pipette, PAF could induce an activation of I(K.ATP), hence a decrease in APD. It is suggested that PAF may amplify the heterogeneity between ischemic and normal cardiac myocytes during ischemia /reperfusion, which may play a vital role in the pathogenesis of the arrhythmias induced by ischemia /reperfusion.

The rapidly delayed rectifier current (I(Kr)) has been described in ventricular myocytes isolated from many species, as well as from neonatal mice. However, whether I(Kr) is present in the adult mouse heart remains controversial. We used cell-attached patch-clamp recording in symmetrical K(+) solutions to assess the presence and behaviour of single I(Kr) channels in adult mouse cardiomyocytes (mI(Kr)). Of 314 patches, 158 (50.1%) demonstrated mI(Kr) currents as compared with 131 (42.3%) for the I(K1) channel. Single mI(Kr) channel activity was rarely observed at potentials positive to -10 mV. The slope conductance at negative potentials was 12 pS. Upon repolarization, ensemble-averaged mI(Kr) showed slow deactivation with a biexponential time course. A selective I(Kr) blocker, E-4031 (1 microm), completely blocked mI(Kr) channel activity. Extracellular Ca(2+) and Mg(2+) at physiological concentrations shifted the activation by approximately 30 mV, accelerated deactivation kinetics, prolonged long-closed time, and reduced open probability without affecting single-channel conductance, suggesting a direct channel-blocking effect in addition to well-recognized voltage shifts. HERG subunits expressed in Chinese hamster ovary cells produced channels with properties similar to those of mI(Kr), except for the more-negative activation of the HERG channels. Despite the abundant expression of mI(Kr), single-channel events were rarely observed during action-potential clamp and 5 microm E-4031 had no detectable effect on the action potential parameters, confirming that mI(Kr) plays at best a minor role in repolarization of adult mouse cardiomyocytes, probably because the modulatory effects of divalent cations prevent significant mI(Kr) opening under physiological conditions.