Isolated Guinea Pig Ventricular Myocytes Research Articles

Extracellular Perinexal Separation Is a Principal Determinant of Cardiac Conduction.

Cardiac conduction is understood to occur through gap junctions. Recent evidence supports ephaptic coupling as another mechanism of electrical communication in the heart. Conduction via gap junctions predicts a direct relationship between conduction velocity (CV) and bulk extracellular resistance. By contrast, ephaptic theory is premised on the existence of a biphasic relationship between CV and the volume of specialized extracellular clefts within intercalated discs such as the perinexus. Our objective was to determine the relationship between ventricular CV and structural changes to micro- and nanoscale extracellular spaces. Conduction and Cx43 (connexin43) protein expression were quantified from optically mapped guinea pig whole-heart preparations perfused with the osmotic agents albumin, mannitol, dextran 70 kDa, or dextran 2 MDa. Peak sodium current was quantified in isolated guinea pig ventricular myocytes. Extracellular resistance was quantified by impedance spectroscopy. Intercellular communication was assessed in a heterologous expression system with fluorescence recovery after photobleaching. Perinexal width was quantified from transmission electron micrographs. CV primarily in the transverse direction of propagation was significantly reduced by mannitol and increased by albumin and both dextrans. The combination of albumin and dextran 70 kDa decreased CV relative to albumin alone. Extracellular resistance was reduced by mannitol, unchanged by albumin, and increased by both dextrans. Cx43 expression and conductance and peak sodium currents were not significantly altered by the osmotic agents. In response to osmotic agents, perinexal width, in order of narrowest to widest, was albumin with dextran 70 kDa; albumin or dextran 2 MDa; dextran 70 kDa or no osmotic agent, and mannitol. When compared in the same order, CV was biphasically related to perinexal width. Cardiac conduction does not correlate with extracellular resistance but is biphasically related to perinexal separation, providing evidence that the relationship between CV and extracellular volume is determined by ephaptic mechanisms under conditions of normal gap junctional coupling.

Inhibition of the INa/K and the activation of peak INa contribute to the arrhythmogenic effects of aconitine and mesaconitine in guinea pigs.

Aconitine (ACO), a main active ingredient of Aconitum, is well-known for its cardiotoxicity. However, the mechanisms of toxic action of ACO remain unclear. In the current study, we investigated the cardiac effects of ACO and mesaconitine (MACO), a structurally related analog of ACO identified in Aconitum with undocumented cardiotoxicity in guinea pigs. We showed that intravenous administration of ACO or MACO (25 μg/kg) to guinea pigs caused various types of arrhythmias in electrocardiogram (ECG) recording, including ventricular premature beats (VPB), atrioventricular blockade (AVB), ventricular tachycardia (VT), and ventricular fibrillation (VF). MACO displayed more potent arrhythmogenic effect than ACO. We conducted whole-cell patch-clamp recording in isolated guinea pig ventricular myocytes, and observed that treatment with ACO (0.3, 3 μM) or MACO (0.1, 0.3 μM) depolarized the resting membrane potential (RMP) and reduced the action potential amplitude (APA) and durations (APDs) in a concentration-dependent manner. The ACO- and MACO-induced AP remodeling was largely abolished by an INa blocker tetrodotoxin (2 μM) and partly abolished by a specific Na+/K+ pump (NKP) blocker ouabain (0.1 μM). Furthermore, we observed that treatment with ACO or MACO attenuated NKP current (INa/K) and increased peak INa by accelerating the sodium channel activation with the EC50 of 8.36 ± 1.89 and 1.33 ± 0.16 μM, respectively. Incubation of ventricular myocytes with ACO or MACO concentration-dependently increased intracellular Na+ and Ca2+ concentrations. In conclusion, the current study demonstrates strong arrhythmogenic effects of ACO and MACO resulted from increasing the peak INa via accelerating sodium channel activation and inhibiting the INa/K. These results may help to improve our understanding of cardiotoxic mechanisms of ACO and MACO, and identify potential novel therapeutic targets for Aconitum poisoning.

Prevailing Effects of Ibutilide on Fast Delayed Rectifier K+ Channel.

Effects of ibutilide, a class III antiarrhythmic drug, on delayed rectifier potassium currents (IK) 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 100µM inhibited IKr in dose-dependent manner with a half maximal effective concentration of 2.03 ± 0.74µM (n = 5-10). The amplitude of tail currents activated by prepulse to + 20mV was decreased from 253 ± 52 to 130 ± 25 pA (n = 8, p < 0.01) in the presence of 1µM ibutilide. The envelope test revealed time-dependent changes in ratio of IK-tail/ΔIK during 0.2-2s pulse durations in the absence of drug. With ibutilide, regardless of pulse duration, a relatively constant ratio was estimated, indicative of predominant involvement of IKr component. The slow IKs persisted to greater extent even at 100 μM ibutilide revealing a distinguishable selectivity toward the IKr component.

Nicorandil stimulates a Na⁺/Ca²⁺ exchanger by activating guanylate cyclase in guinea pig cardiac myocytes.

Nicorandil, a hybrid of an ATP-sensitive K(+) (KATP) channel opener and a nitrate generator, is used clinically for the treatment of angina pectoris. This agent has been reported to exert antiarrhythmic actions by abolishing both triggered activity and spontaneous automaticity in an in vitro study. It is well known that delayed afterdepolarizations (DADs) are caused by the Na(+)/Ca(2+) exchange current (I NCX). In this study, we investigated the effect of nicorandil on the cardiac Na(+)/Ca(2+) exchanger (NCX1). We used the whole-cell patch clamp technique and the Fura-2/AM (Ca(2+) indicator) method to investigate the effect of nicorandil on I NCX in isolated guinea pig ventricular myocytes and CCL39 fibroblast cells transfected with dog heart NCX1. Nicorandil enhanced I NCX in a concentration-dependent manner. The EC50 (half-maximum concentration for enhancement of the drug) values were 15.0 and 8.7 μM for the outward and inward components of I NCX, respectively. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a membrane-permeable analog of guanosine 3',5'-cyclic monophosphate (cGMP), enhanced I NCX. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor (10 μM), completely abolished the nicorandil-induced I NCX increase. Nicorandil increased I NCX in CCL39 cells expressing wild-type NCX1 but did not affect mutant NCX1 without a long intracellular loop between transmembrane segments (TMSs) 5 and 6. Nicorandil at 100 μM abolished DADs induced by electrical stimulation with ouabain. Nicorandil enhanced the function of NCX1 via guanylate cyclase and thus may accelerate Ca(2+) exit via NCX1. This may partially contribute to the cardioprotection by nicorandil in addition to shortening action potential duration (APD) by activating KATP channels.

Cell-Specific Cardiac Electrophysiology Models

The traditional cardiac model-building paradigm involves constructing a composite model using data collected from many cells. Equations are derived for each relevant cellular component (e.g., ion channel, exchanger) independently. After the equations for all components are combined to form the composite model, a subset of parameters is tuned, often arbitrarily and by hand, until the model output matches a target objective, such as an action potential. Unfortunately, such models often fail to accurately simulate behavior that is dynamically dissimilar (e.g., arrhythmia) to the simple target objective to which the model was fit. In this study, we develop a new approach in which data are collected via a series of complex electrophysiology protocols from single cardiac myocytes and then used to tune model parameters via a parallel fitting method known as a genetic algorithm (GA). The dynamical complexity of the electrophysiological data, which can only be fit by an automated method such as a GA, leads to more accurately parameterized models that can simulate rich cardiac dynamics. The feasibility of the method is first validated computationally, after which it is used to develop models of isolated guinea pig ventricular myocytes that simulate the electrophysiological dynamics significantly better than does a standard guinea pig model. In addition to improving model fidelity generally, this approach can be used to generate a cell-specific model. By so doing, the approach may be useful in applications ranging from studying the implications of cell-to-cell variability to the prediction of intersubject differences in response to pharmacological treatment.

Urocortin2 prolongs action potential duration and modulates potassium currents in guinea pig myocytes and HEK293 cells

We previously reported that activation of corticotropin releasing factor receptor type 2 by urocortin2 up-regulates both L-type Ca2+ channels and intracellular Ca2+ concentration in ventricular myocytes and plays an important role in cardiac contractility and arrhythmogenesis. This study goal was to further test the hypothesis that urocortin2 may modulate action potentials as well as rapidly and slowly activating delayed rectifier potassium currents. With whole cell patch-clamp techniques, action potentials and slowly activating delayed rectifier potassium currents were recorded in isolated guinea pig ventricular myocytes, respectively. And rapidly activating delayed rectifier potassium currents were tested in hERG-HEK293 cells. Urocortin2 produced a time- and concentration-dependent prolongation of action potential duration. The EC50 values of action potential duration and action potential duration at 90% of repolarization were 14.73 and 24.3nM respectively. The prolongation of action potential duration of urocortin2 was almost completely or partly abolished by H-89 (protein kinase A inhibitor) or KB-R7943 (Na+/Ca2+ exchange inhibitor) pretreatment respectively. And urocortin2 caused reduction of rapidly activating delayed rectifier potassium currents in hERG-HEK293 cells. In addition, urocortin2 slowed the rate of slowly activating delayed rectifier potassium channel activation, and rightward shifted the threshold of slowly activating delayed rectifier potassium currents to more positive potentials. Urocortin2 prolonged action potential duration via activation of protein kinase A and Na+/ Ca2+ exchange in isolated guinea pig ventricular myocytes in a time- and concentration- dependent manner. In hERG-HEK293 cells, urocortin2 reduced rapidly activating delayed rectifier potassium current density which may contribute to action potential duration prolongation.

Acute Modulation of Sodium Channel Biophysical Properties using High-Frequency Stimulation

Voltage-gated ion channels are typically characterized by families of voltage-dependent curves, in particular, steady-state activation/inactivation (SSA/SSI) and peak current-voltage (IV) curves, which are often shifted or altered during disease. Previous studies have demonstrated that high-frequency stimulation (HFS) can be used to block electrical conduction in nerve axons, and more recently, in cardiac tissue. However, the use of HFS to acutely modulate ion channels has not been explored. We demonstrate that HFS can acutely and reversibly modulate sodium channel biophysical properties using a combined theoretical, computational, and experimental approach. Using the sodium channel kinetic description from the LR1 myocyte model and a multi-scale method based on a separation of time scales, HFS is predicted to shift and reduce the steepness of the SSA and SSI curves, and shift and reduce the peak IV curve. Myocyte simulations demonstrate HFS depolarizes the resting membrane potential, such that the steady-state inactivation is increased. In agreement with theoretical and computational predictions, whole-cell patch clamp of isolated guinea pig ventricular myocytes show that 25-kHz HFS significantly reduces peak sodium current and suggest a shift in the peak to more depolarized potentials. Importantly, upon HFS cessation, peak current returns to control levels, demonstrating that HFS is acutely reversible. Further, in whole-heart optical mapping experiments, 25-kHz HFS reversibly decreased lateral and transverse conduction velocities in a graded manner, consistent with increased sodium channel inactivation. We demonstrate a novel method for the acute modulation of sodium channel biophysics using HFS. Further, our theoretical work predicts that HFS may be a general approach to modulate all voltage-dependent ion channels, suggesting a multitude of potential applications.

Application of stochastic phenomenological modelling to cell-to-cell and beat-to-beat electrophysiological variability in cardiac tissue.

Variability in the action potential of isolated myocytes and tissue samples is observed in experimental studies. Variability is manifested as both differences in the action potential (AP) morphology between cells (extrinsic variability), and also ‘intrinsic’ or beat-to-beat variability of repolarization (BVR) in the AP duration of each cell. We studied the relative contributions of experimentally recorded intrinsic and extrinsic variability to dispersion of repolarization in tissue. We developed four cell-specific parameterizations of a phenomenological stochastic differential equation AP model exhibiting intrinsic variability using APs recorded from isolated guinea pig ventricular myocytes exhibiting BVR. We performed simulations in tissue using the four different model parameterizations in the presence and the absence of both intrinsic and extrinsic variability. We altered the coupling of the tissue to determine how inter-cellular coupling affected the dispersion of the AP duration in tissue. Both intrinsic and extrinsic variability were gradually revealed by reduction of tissue coupling. However, the recorded extrinsic variability between individual myocytes produced a greater degree of dispersion in repolarization in tissue than the intrinsic variability of each myocyte.

Nicorandil increases Na/Ca exchange current in heart (850.2)

Nicorandil is a K(ATP) channel opener, nitric oxide donor and antioxidant, and is used as a drug for the treatment of angina pectoris. This durg induces cardioprotection and vasodilatation after ischemia reperfusion. It was previously reported that nicorandil may exert antiarrhythmic actions by abolishing both spontaneous and triggered activity [early after‐depolarization (EADs), delayed after‐depolarization (DADs)] (Lathrop et al., Br J Pharmacol, 1990). The triggered activity induced by DADs is one of the mechanisms of ventricular arrhythmias associated with intracellular Ca2+ overload.In this study we examined the effect of nicorandil on Na+/Ca2+ exchange current (INCX) in isolated guinea pig ventricular myocytes using the whole‐cell patch clamp technique.Nicorandil enhanced INCX in a concentration‐dependent manner. The EC50 values of nicorandil were 15.0 μM and 8.7 μM for the outward and inward components of INCX, respectively. Eight‐Br‐cGMP, a membrane permeable analog of cGMP also enhanced the INCX in a concentration‐dependent manner. ODQ, a guanylate cyclase inhibitor (10 µM) almost completely abolished the effects of both nicorandil and Eight ‐Br‐cGMP on INCX. DADs induced by electrical stimulation with ouabain disappeared in the presence of 100 µM nicorandil. We conclude that nicorandil enhances the function of Na+/Ca2+ exchanger (NCX) via guanylate cyclase, and this may partially contribute to the cardioprotection of nicorandil by acceleratiing Ca2+ exit via NCX.

Differential Regulation of Slow and Rapid Delayed Rectifier Potassium Currents by cGMP Dependent Nitric Oxide Signalling Pathways in Isolated Adult Guinea Pig Ventricular Myocytes

Nitric oxide (NO) signalling pathways are reported to regulate cardiac repolarisation. This was investigated using BAY 60-2770, which activates soluble guanylyl cyclase (sGC) in a NO and haem independent manner to generate cGMP. Action potentials (APs) and currents were recorded from isolated guinea pig ventricular myocytes at 37oC using the perforated patch clamp technique. APs were stimulated at 2 Hz. The slow and rapid delayed rectifier potassium currents, IKs and IKr were measured as tail currents elicited by a voltage step from +40 mV to −50 mV. The following experiments were carried out in the presence of 100 µM IBMX (3-Isobutyl-1-methylxanthine) a non-selective phosphodiesterase (PDE) inhibitor. 1 µM BAY 60-2770 lengthened APD90 by 15.7 ms (p<0.05, n=7), suggesting that sGC activation and an increase in cGMP inhibits repolarising currents. Further experiments showed that IKs was inhibited by BAY 60-2770. IBMX alone enhanced IKs by 77 ± 9 % (p<0.001, n=8) and subsequent addition of BAY 60-2770 reduced the IBMX dependent enhancement of IKs by 68 ± 2 % (p<0.001, n=8). This suggests that PDE inhibition permits IKs to be inhibited by cGMP dependent NO signalling pathways. The sGC mediated inhibition of IKs by BAY 60-2770 is unlikely to be due to phosphorylation by protein kinase G (PKG) because the inhibition remained in the presence of 100 nM KT-5823, a PKG inhibitor and because PKG activation with 100 µM 8-bromo-cGMP had no effect on IKs. IKr was not regulated by PDE inhibition, sGC activation or PKG activation. Overall, these findings suggest that cGMP dependent NO signalling pathways regulate IKs, but not IKr, via a PKG independent mechanism.

The Role of CGMP Dependent Nitric Oxide Signalling on Cardiac Repolarisation in Adult Guinea Pig Ventricular Myocytes

Recently, roles for nitric oxide (NO) signalling pathways have been linked with abnormalities in cardiac repolarisation and cardiac arrhythmias. Studies from isolated heart preparations have demonstrated an antifibrillatory effect following vagus nerve stimulation, which is NO dependent. The cellular and molecular basis for this protective effect is unknown. In this study, cGMP dependent regulation of repolarisation was investigated using BAY 60-2770, a novel NO and haem independent soluble guanylyl cyclase (sGC) activator. Action potentials and currents were recorded from isolated guinea pig ventricular myocytes using the perforated patch clamp technique. Cellular cGMP was quantified by radioimmunoassay and expressed as a fold change relative to basal levels. 1 μM BAY 60-2770 increased cellular cGMP by 2.2 fold and by 7.1 fold in the presence of 100 μM IBMX (3-Isobutyl-1-methylxanthine), a non-selective phosphodiesterase (PDE) inhibitor. In vitro assays on purified sGC have shown that ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) potentiates the action of BAY 60-2770. This was corroborated in our experiments, BAY 60-2770 + 10 μM ODQ increased cGMP by 27.9 fold. Despite substantial increases in cellular cGMP, changes in times to 90% repolarisation (APD90) were modest. BAY 60-2770 shortened APD90 by 11.3 ms and adding ODQ to BAY 60-2770 caused no further APD90 shortening. In contrast, when cGMP hydrolysis was blocked by IBMX, BAY 60-2770 lengthened APD90 by 15.7 ms. Preliminary findings indicate that delayed rectifier potassium currents are modulated by these pathways. Overall, these findings demonstrate the complex interplay between cGMP and cAMP mediated effects on the ion channels regulating cardiac repolarisation. The modest effect of BAY 60-2770 + ODQ on APD90, despite a substantial increase in cellular cGMP, highlights that PDEs limit cGMP accumulation close to ion channels in the sarcolemma, thus compartmentalising cGMP signalling.

Profile of the Late Sodium Current during Cardiac Action Potential in Guinea Pig Ventricular Cardiomyocytes

The late sodium current [INa,late] can play an important role in generating arrhythmogenic afterdepolarisations in cardiac myocytes. However the profile of INa,late during the action potential [AP] has not yet been directly measured. The goal of the present study was to directly record and describe the profile of INa,late as it could be observed in situ (in a physiological milieu, during AP, with undisturbed cellular calcium homeostasis).METHODS: Our newly developed ‘Onion-Peeling’ technique (a variant of action potential voltage-clamp) was utilised to dissect out INa,late during AP by using tetrodotoxin. The experiments were done on isolated guinea pig ventricular myocytes at 36 °C. In one set of experiments no exogenous calcium buffer was used in the internal solution, which enabled intracellular calcium cycling during AP. In the other set of recordings intracellular calcium concentration was clamped with BAPTA. The intracellular calcium concentration was measured by using Fura-2.RESULTS: The tetrodotoxin-sensitive current first displayed a large peak during the AP upstroke attributable to the fast sodium current, and then showed a sustained current throughout the AP due to INa,late. With cycling calcium, INa,late showed a gradual increase during the AP plateau phase and then peaked during the fast repolarising phase. When the intracellular calcium was clamped, INa,late showed larger current density throughout the AP time course but the overall current profile remained similar.CONCLUSION: The INa,late profile directly recorded in situ by using the ‘Onion-Peeling’ method is different from what previously predicted by model simulations based on the data obtained using conventional voltage-clamp under simplified conditions (ion substitution, calcium buffering, rectangular voltage pulses). The observed high INa,late density during the AP repolarising phase underlies the arrhythmogenic role of the current.

Parameter estimation for a phenomenological model of the cardiac action potential

The action potential of a cardiac cell is made up of a complex balance of ionic currents which flow across the cell membrane in response to electrical excitation of the cell. Biophysically detailed mathematical models of the action potential have grown larger in terms of the variables and parameters required to model new findings in subcellular ionic mechanisms. The fitting of parameters to such models has seen a large degree of parameter and module re-use from earlier models. An alternative method for modelling electrically excitable cardiac tissue is a phenomenological model, which reconstructs tissue level action potential wave behaviour without subcellular details. A new parameter estimation technique to fit the morphology of the action potential in a four variable phenomenological model is presented. An approximation of a nonlinear ordinary differential equation model is established that corresponds to the given phenomenological model of the cardiac action potential. The parameter estimation problem is converted into a minimisation problem for the unknown parameters. A modified hybrid Nelder--Mead simplex search and particle swarm optimisation then solves the minimisation problem for the unknown parameters. The successful fitting of data generated from a well known biophysically detailed model is demonstrated. A successful fit to an experimental action potential recording that contains both noise and experimental artefacts is also produced. The parameter estimation method's ability to fit a complex morphology to a model with substantially more parameters than previously used is established. References E. Carmeliet and J. Vereecke, Cardiac Cellular Electrophysiology, Springer, 2002. V. Iyer, R. Mazhari and R. Winslow, A computational model of the human left--ventricular epicardial myocyte, Biophys. J., 87, 2004, 1507--1525, doi:10.1529/biophysj.104.043299 S. Niederer, M. Fink, D. Noble and N. P. Smith, A meta-analysis of cardiac electrophysiology computational models, Exp. Physiol., 94, 2009, 486--495, doi:10.1113/expphysiol.2008.044610 F. Fenton, and A. Karma, Vortex dynamics in three-dimensional continuous myocardium with fiber rotation: Filament instability and fibrillation, Chaos, 8, 1998, 20--47, doi:10.1063/1.166311 A. Bueno-Orovio, E. M. Cherry and F. H. Fenton, Minimal model for human ventricular action potentials in tissue, J. Theor. Biol., 253, 2008, 544--560, doi:10.1016/j.jtbi.2008.03.029 J. Walmsley, G. Mirams, M. Bahoshy, C. Bollensdorff, B. Rodriguez and K. Burrage, Phenomenological modeling of cell-to-cell and beat-to-beat variability in isolated Guinea Pig ventricular myocytes, Conf. Proc. IEEE Eng. Med. Biol. Soc. 2010, 2010, 1457--60, doi:10.1109/IEMBS.2010.5626858 C. Luo and Y. Rudy, A model of the ventricular cardiac action potential. Depolarization, repolarization, and their interaction, Circ. Res., 68, 1991, 1501--1526, doi:10.1161/01.RES.68.6.1501 J. Keener and J. Sneyd, Mathematical Physiology, Springer, 1998. M. M. Macconi, B. Morini and M. Porcelli, A Gauss-Newton method for solving bound-constrained underdetermined nonlinear systems, Optimization Method and Software, 24(2), 2010, 219--235, doi:10.1080/10556780902753031 J. A. Nelder and R. A. Mead, A simplex method for function minimization, Computer Journal, 7, 1965, 308--313, doi:10.1093/comjnl/7.4.308 R. C. Eberhart and J. Kennedy, A new optimizer using particle swarm theory, Proc. of the Sixth International Symposium on Micro Machine and Human Science, Nagoya, Japan, 1995, 39--43, doi:10.1109/MHS.1995.494215 F. Liu and K. Burrage, Novel techniques in parameter estimation for fractional dynamical models arising from biological systems, Computers and Mathematics with Applications, 2011, doi:10.1016/j.camwa.2011.03002

Effect of dronedarone on Na+, Ca2+ and HCN channels

Previous studies showed that amiodarone causes state-dependent inhibition of Na(+) channels thereby mediating an atrial-selective drug effect. The aim of the present study was to investigate the impact of the new antiarrhythmic compound dronedarone on Na(+), Ca(2+) and hyperpolarization-activated cyclic nucleotide-gated ion channels. Monophasic action potentials (MAP) and effective refractory period (ERP) were studied in arterially perfused left atria and ventricular wedge preparations of the pig. Fast Na(+) and Ca(2+) currents in isolated guinea pig ventricular myocytes as well as human HCN4 channels expressed in Chinese hamster ovary (CHO) cells were investigated with the patch-clamp technique. In left atrial epicardial tissue, dronedarone (3μM) had no effect on the MAP duration, but the drug caused a significant prolongation of the ERP from 145 ± 9 to 184 ± 17ms (n = 6; p < 0.05). In guinea pig ventricular myocytes, dronedarone exhibited a state-dependent inhibition of the fast Na(+) channel current with an IC(50) of 0.7 ± 0.1μM, when the holding potential (V (hold)) was -80mV. The maximal block at the highest concentration used was 77 ± 8%. In contrast, when V (hold) was -100mV, inhibition with 10μM dronedarone was only 9 ± 3% (n = 7). Dronedarone blocked Ca(2+) currents elicited by rectangular pulses at V (hold) = -40mV with an IC(50) value of 0.4 ± 0.1μM (maximal block by 10μM dronedarone, 80 ± 6%), whereas at V (hold) = -80mV, 10μM dronedarone blocked only 20 ± 6% (n = 4) of the current. Applying an action potential clamp (V (hold) = -80mV) yielded an IC(50) of 0.4 ± 0.3μM. Human HCN4 channels expressed in CHO cells were blocked by dronedarone with an IC(50) of 1.0 ± 0.1μM. Inhibition of fast Na(+) and Ca(2+) channels by dronedarone depends on the cell's resting membrane potential (state-dependent block) favouring an atrial-selective mode of action. Besides fast Na(+) and Ca(2+) channels, dronedarone also inhibits HCN4 currents. This might contribute to the clinically observed reduction in heart rate seen in patients in sinus rhythm after dronedarone treatment.

Dynamical Mechanism for Subcellular Alternans in Cardiac Myocytes

Cardiac repolarization alternans is an arrhythmogenic rhythm disturbance, manifested in individual myocytes as a beat-to-beat alternation of action potential durations and intracellular calcium transient magnitudes. Recent experimental studies have reported "subcellular alternans," in which distinct regions of an individual cell are seen to have counterphase calcium alternations, but the mechanism by which this occurs is not well understood. Although previous theoretical work has proposed a possible dynamical mechanism for subcellular alternans formation, no direct evidence for this mechanism has been reported in vitro. Rather, experimental studies have generally invoked fixed subcellular heterogeneities in calcium-cycling characteristics as the mechanism of subcellular alternans formation. In this study, we have generalized the previously proposed dynamical mechanism to predict a simple pacing algorithm by which subcellular alternans can be induced in isolated cardiac myocytes in the presence or absence of fixed subcellular heterogeneity. We aimed to verify this hypothesis using computational modeling and to confirm it experimentally in isolated cardiac myocytes. Furthermore, we hypothesized that this dynamical mechanism may account for previous reports of subcellular alternans seen in statically paced, intact tissue. Using a physiologically realistic computational model of a cardiac myocyte, we show that our predicted pacing algorithm induces subcellular alternans in a manner consistent with theoretical predictions. We then use a combination of real-time electrophysiology and fluorescent calcium imaging to implement this protocol experimentally and show that it robustly induces subcellular alternans in isolated guinea pig ventricular myocytes. Finally, we use computational modeling to demonstrate that subcellular alternans can indeed be dynamically induced during static pacing of 1D fibers of myocytes during tissue-level spatially discordant alternans. Here we provide the first direct experimental evidence that subcellular alternans can be dynamically induced in cardiac myocytes. This proposed mechanism may contribute to subcellular alternans formation in the intact heart.

ß-adrenoceptor stimulation exacerbates detrimental effects of ischemia and reperfusion in isolated guinea pig ventricular myocytes

We investigated whether ß-adrenoceptor stimulation exacerbates detrimental effects of ischemia and reperfusion on electrical and contractile function and on intracellular Ca 2+ homeostasis in isolated guinea pig ventricular myocytes. Myocytes were exposed to 20 min of simulated ischemia (37 °C) in the absence or presence of isoproterenol (10 nM, applied prior to and during ischemia) and reperfused with Tyrode's solution for 30 min. Unloaded cell shortening, Ca 2+ transients (fura-2), and cell viability were recorded at 5 min intervals in field-stimulated cells (2 Hz). In experiments using microelectrodes, membrane potentials, contractions, and transmembrane currents also were recorded at 5 min intervals. In the absence of ischemia, 10 nM isoproterenol had little effect on either contractile function or Ca 2+ homeostasis. In contrast, when cells were exposed to ischemia, isoproterenol increased the size of contractions and Ca 2+ transients and augmented the increase in diastolic Ca 2+ concentration during ischemia in field-stimulated myocytes. Exposure to isoproterenol also promoted contractile depression in reperfusion. In voltage clamp experiments, isoproterenol abolished the decrease in the magnitude of L-type Ca 2+ current caused by ischemia. Isoproterenol also increased the incidence of abnormal contractile activity and induced delayed afterdepolarizations and the arrhythmogenic transient inward current in ischemia. Additionally, the decline in cell viability in ischemia and reperfusion was exacerbated by isoproterenol. This study shows that ß-adrenoceptor stimulation strongly potentiates adverse effects of ischemia and reperfusion on electrical and contractile function. These adverse effects of isoproterenol are likely caused by an increase in intracellular Ca 2+ accumulation during ischemia.

Different Na+K+-ATPase signal pathways was involved in the increase of [Ca2+]i induced by strophanthidin in normal and failing isolated guinea pig ventricular myocytes

To determine whether different Na+/K+-ATPase signal transduction pathways have positive inotropic effects on normal ventricular myocytes (NC) and failing ventricular myocytes (FC), and are involved in an increase of [Ca2+]i induced by strophanthidin (Str). A guinea pig model of congestive heart failure was made by constricting descending aorta. The left ventricular myocytes were enzymatically isolated. The effects of 25 micromol/L Str with different signal-transducing inhibitors on contractility and the calcium transient of NC or FC from guinea pigs were simultaneously assessed and compared with those in the 25 micromol/L Str-only group by a video-based, motion-edge detection system. Str at 1, 10, and 25 micromol/L in NC and Str at 0.1, 1, 10, and 25 micromol/L) in FC elevated the calcium transient amplitude and increased the positive inotropic effects in a concentration-dependent manner, respectively. At the same concentration, the effects of Str were more potent in FC than in NC. In FC, both the mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) signal transduction pathway of Na+/K+-ATPase were involved in the increase of the calcium transient induced by Str, but only activation of the MAPK pathway increased the calcium transient in NC. However, only the ROS pathway was involved in positive inotropic effects both in NC and FC. The present study suggests that Na+/K+-ATPase signaling pathways involved in the inotropic effects of Str in NC and FC are consistent, and Na+/K+-ATPase signaling pathways involved in the increase of [Ca2+]i by Str in NC and FC are different.

Progesterone Regulates Cardiac Repolarization Through a Nongenomic Pathway

Female sex is an independent risk factor for torsade de pointes in long-QT syndrome. In women, QT interval and torsade de pointes risk fluctuate dynamically during the menstrual cycle and pregnancy. Accumulating clinical evidence suggests a role for progesterone; however, the effect of progesterone on cardiac repolarization remains undetermined. We investigated the effects of progesterone on action potential duration and membrane currents in isolated guinea pig ventricular myocytes. Progesterone rapidly shortened action potential duration, which was attributable mainly to enhancement of the slow delayed rectifier K+ current (I(Ks)) under basal conditions and inhibition of L-type Ca2+ currents (I(Ca,L)) under cAMP-stimulated conditions. The effects of progesterone were mediated by nitric oxide released via nongenomic activation of endothelial nitric oxide synthase; this signal transduction likely takes place in the caveolae because sucrose density gradient fractionation experiments showed colocalization of the progesterone receptor c-Src, phosphoinositide 3-kinase, Akt, and endothelial nitric oxide synthase with KCNQ1, KCNE1, and Ca(V)1.2 in the caveolae fraction. We used computational single-cell and coupled-tissue action potential models incorporating the effects of progesterone on I(Ks) and I(Ca,L); the model reproduces the fluctuations of cardiac repolarization during the menstrual cycle observed in women and predicts the protective effects of progesterone against rhythm disturbances in congenital and drug-induced long-QT syndrome. Our data show that progesterone modulates cardiac repolarization by nitric oxide produced via a nongenomic pathway. A combination of experimental and computational analyses of progesterone effects provides a framework to understand complex fluctuations of QT interval and torsade de pointes risks in various hormonal states in women.

Differential effects of the sodium calcium exchange inhibitor, KB-R7943, on ischemia and reperfusion injury in isolated guinea pig ventricular myocytes

Effects of the Na +–Ca 2+ exchange (NCX) inhibitor KB-R7943 on electrical and contractile function were examined in guinea pig ventricular myocytes exposed to ischemia and reperfusion. Action potentials and transmembrane currents were recorded with microelectrodes; contractions were measured with an edge detector. Cells were exposed to simulated ischemia (hypoxia, hypercapnia, hyperkalemia, acidosis, lactate accumulation, no glucose) for 20 min and reperfused with Tyrode's solution. Experiments were conducted at 37 °C in the absence or presence of KB-R7943. Low concentrations of KB-R7943 (0.1 μM) had little impact on changes in contractions, membrane potential, or Ca 2+ current induced by ischemia and reperfusion. However, higher concentrations of KB-R7943 (0.5 and 1.0 μM) reduced the magnitude of Ca 2+ current and promoted action potential abbreviation in both ischemia and reperfusion. High concentrations of KB-R7943 also promoted post-ischemic contractile dysfunction (stunning) in reperfusion. In the absence of KB-R7943, the arrhythmogenic transient inward current ( I TI) plus aftercontractions occurred upon reperfusion, and some cells exhibited irreversible cell injury (hypercontracture). Higher concentrations of KB-R7943 (0.5 and 1.0 μM) did not affect the occurrence or magnitude of I TI and aftercontractions and did not affect the occurrence of hypercontracture. In contrast, 0.1 μM KB-R7943 virtually abolished I TI, aftercontractions and hypercontracture. Thus, low concentrations of KB-R7943 protected against ischemia and reperfusion injury, but higher concentrations of drug actually exacerbated detrimental effects of ischemia and reperfusion. These results suggest that inhibition of I TI may contribute to the antiarrhythmic effects of KB-R7943 on reperfusion-induced arrhythmias.

Effect of YM-244769, a novel NCX inhibitor in guinea pig cardiac ventricular myocytes

Recently, it was reported that YM-244769 (N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide) is a new selective Na+/Ca2+ exchange (NCX) inhibitor (Iwamoto and Kita, 2006). We examined the effect of YM-244769 on the NCX current in isolated guinea pig ventricular myocytes with the whole-cell voltage-clamp technique. YM-244769 suppressed the bi-directional NCX current in a concentration-dependent manner. The IC50 values of YM-244769 were 0.12 μM and 0.1 μM for the bi-directional outward and inward NCX current, respectively, with a Hill coefficient of 0.7 and 0.8 for the bi-directional outward and inward NCX current, respectively.