Guinea Pig Ventricular Myocytes Research Articles

Nonlinear Threshold Behavior in the Induction of Arrhythmias by Channels Bearing Timothy Syndrome Mutations

Timothy Syndrome (TS) is a multisystem disorder, featuring cardiac action-potential prolongation with paroxysms of life-threatening arrhythmias. The underlying defect is a single point mutation in CaV1.2 channels. Interestingly, recent clinical studies hint that overt disease manifestation might be mitigated by diminished expression of mutant channels. To gain insight, we undertook in-depth biophysical characterization of TS channels, and used this profile to refine a widely-utilized ventricular myocyte model (AJP 292:H2854). Simulations of variable levels of TS channels suggested a remarkable behavior. As expected, a modest increase in TS channels produced moderate action-potential prolongation (A1, control; A2, TS). By contrast, only a minute further increase in TS channels induced flagrant disturbances of excitability (A3), suggesting a nonlinear threshold for induction of frank arrhythmias. To explore this experimentally in cell networks, we variably expressed TS channels in monolayers of cultured guinea-pig ventricular myocytes. Indeed, conservative expression of TS channels yielded graded action-potential prolongation (B1, control; B2, TS), but small further elevation begat severe disturbances (B3). If like behavior exists in the heart, limited changes in TS expression may generate dramatic alterations in arrhythmogenic potential, an important principle for therapeutics.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Bilirubin Attenuates Bufadienolide-Induced Ventricular Arrhythmias and Cardiac Dysfunction in Guinea-Pigs by Reducing Elevated Intracellular Na+ Levels

Bufadienolides, known ligands of the sodium pump, have been shown to inhibit the proliferation of several cancer cell types. However, their development to date as anticancer agents has been impaired by a narrow therapeutic margin resulting from their potential to induce cardiotoxicity. In the present study, we examined the effects of bilirubin, an endogenous antioxidant, on the cardiotoxicity of bufadienolides (derived from toad venom) in guinea-pigs. The results showed that bufadienolides (8mg/kg) caused ventricular arrhythmias, conduction block, cardiac dysfunction and death in guinea-pigs. Pretreatment with bilirubin (75 and 150mg/kg) significantly prevented bufadienolide-induced premature ventricular complexes, ventricular tachycardia, ventricular fibrillation and death. Bilirubin also markedly improved the inhibition of cardiac contraction in bufadienolide-treated guinea-pigs as evidenced by increases in left ventricular systolic pressure and decreases in left ventricular diastolic pressure in vivo. Furthermore, bilirubin significantly reduced the intracellular sodium content ([Na(+)]( i )) in ex vivo bufadienolide-stimulated guinea-pig ventricular myocytes loaded with the sodium indicator Sodium Green. An antitumor study showed that bilirubin did not compromise the ability of bufadienolides to inhibit gastric cancer cell MGC-803 proliferation. These results suggested that bilirubin can attenuate bufadienolide-induced arrhythmias and cardiac dysfunction in guinea-pigs by reducing elevated [Na(+)]( i ) and may improve bufadienolide therapeutic index in cancer treatment.

Influence of pH on Ca2+ current and its control of electrical and Ca2+ signaling in ventricular myocytes

Modulation of L-type Ca2+ current (ICa,L) by H+ ions in cardiac myocytes is controversial, with widely discrepant responses reported. The pH sensitivity of ICa,L was investigated (whole cell voltage clamp) while measuring intracellular Ca2+ (Ca2+i) or pHi (epifluorescence microscopy) in rabbit and guinea pig ventricular myocytes. Selectively reducing extracellular or intracellular pH (pHo 6.5 and pHi 6.7) had opposite effects on ICa,L gating, shifting the steady-state activation and inactivation curves to the right and left, respectively, along the voltage axis. At low pHo, this decreased ICa,L, whereas at low pHi, it increased ICa,L at clamp potentials negative to 0 mV, although the current decreased at more positive potentials. When Ca2+i was buffered with BAPTA, the stimulatory effect of low pHi was even more marked, with essentially no inhibition. We conclude that extracellular H+ ions inhibit whereas intracellular H+ ions can stimulate ICa,L. Low pHi and pHo effects on ICa,L were additive, tending to cancel when appropriately combined. They persisted after inhibition of calmodulin kinase II (with KN-93). Effects are consistent with H+ ion screening of fixed negative charge at the sarcolemma, with additional channel block by H+o and Ca2+i. Action potential duration (APD) was also strongly H+ sensitive, being shortened by low pHo, but lengthened by low pHi, caused mainly by H+-induced changes in late Ca2+ entry through the L-type Ca2+ channel. Kinetic analyses of pH-sensitive channel gating, when combined with whole cell modeling, successfully predicted the APD changes, plus many of the accompanying changes in Ca2+ signaling. We conclude that the pHi-versus-pHo control of ICa,L will exert a major influence on electrical and Ca2+-dependent signaling during acid–base disturbances in the heart.

Platelet-activating factor stimulates sodium-hydrogen exchange in ventricular myocytes

Sodium-hydrogen exchanger (NHE), the principal sarcolemmal acid extruder in ventricular myocytes, is stimulated by a variety of autocrine/paracrine factors and contributes to myocardial injury and arrhythmias during ischemia-reperfusion. Platelet-activating factor (PAF; 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent proinflammatory phospholipid that is released in the heart in response to oxidative stress and promotes myocardial ischemia-reperfusion injury. PAF stimulates NHE in neutrophils and platelets, but its effect on cardiac NHE (NHE1) is unresolved. We utilized quiescent guinea pig ventricular myocytes bathed in bicarbonate-free solutions and epifluorescence to measure intracellular pH (pH(i)). Methylcarbamyl-PAF (C-PAF; 200 nM), a metabolically stable analog of PAF, significantly increased steady-state pH(i). The alkalosis was completely blocked by the NHE inhibitor, cariporide, and by sodium-free bathing solutions, indicating it was mediated by NHE activation. C-PAF also significantly increased the rate of acid extrusion induced by intracellular acidosis. The ability of C-PAF to increase steady-state pH(i) was completely blocked by the PAF receptor inhibitor WEB 2086 (10 μM), indicating the PAF receptor is required. A MEK inhibitor (PD98059; 25 μM) also completely blocked the rise in pH(i) induced by C-PAF, suggesting participation of the MAP kinase signaling cascade downstream of the PAF receptor. Inhibition of PKC with GF109203X (1 μM) and chelerythrine (2 μM) did not significantly affect the alkalosis induced by C-PAF. In summary, these results provide evidence that PAF stimulates cardiac NHE1, the effect occurs via the PAF receptor, and signal relay requires participation of the MAP kinase cascade.

Effects of the histamine H1 receptor antagonist hydroxyzine on hERG K+ channels and cardiac action potential duration

To investigate the effects of hydroxyzine on human ether-a-go-go-related gene (hERG) channels to determine the electrolphysiological basis for its proarrhythmic effects. hERG channels were expressed in Xenopus oocytes and HEK293 cells, and the effects of hydroxyzine on the channels were examined using two-microelectrode voltage-clamp and patch-clamp techniques, respectively. The effects of hydroxyzine on action potential duration were examined in guinea pig ventricular myocytes using current clamp. Hydroxyzine (0.2 and 2 μmol/L) significantly increased the action potential duration at 90% repolarization (APD(90)) in both concentration- and time-dependent manners. Hydroxyzine (0.03-3 μmol/L) blocked both the steady-state and tail hERG currents. The block was voltage-dependent, and the values of IC(50) for blocking the steady-state and tail currents at +20 mV was 0.18±0.02 μmol/L and 0.16±0.01 μmol/L, respectively, in HEK293 cells. Hydroxyzine (5 μmol/L) affected both the activated and the inactivated states of the channels, but not the closed state. The S6 domain mutation Y652A attenuated the blocking of hERG current by ~6-fold. The results suggest that hydroxyzine could block hERG channels and prolong APD. The tyrosine at position 652 in the channel may be responsible for the proarrhythmic effects of hydroxyzine.

Profile of L-type Ca2+ current and Na+/Ca2+ exchange current during cardiac action potential in ventricular myocytes

The L-type Ca(2+) current (I(Ca,L)) and the Na(+)/Ca(2+) exchange current (I(NCX)) are major inward currents that shape the cardiac action potential (AP). Previously, the profile of these currents during the AP was determined from voltage-clamp experiments that used Ca(2+) buffer. In this study, we aimed to obtain direct experimental measurement of these currents during cardiac AP with Ca(2+) cycling. A newly developed AP-clamp sequential dissection method was used to record ionic currents in guinea pig ventricular myocytes under a triad of conditions: using the cell's own AP as the voltage command, using internal and external solutions that mimic the cell's ionic composition, and, importantly, not using any exogenous Ca(2+) buffer. The nifedipine-sensitive current (I(NIFE)), which is composed of I(Ca,L) and I(NCX), revealed hitherto unreported features during the AP with Ca(2+) cycling in the cell. We identified 2 peaks in the current profile followed by a long residual current extending beyond the AP, coinciding with a residual depolarization. The second peak and the residual current become apparent only when Ca(2+) is not buffered. Pharmacological dissection of I(NIFE) by using SEA0400 shows that I(Ca,L) is dominant during phases 1 and 2 whereas I(NCX) contributes significantly to the inward current during phases 3 and 4 of the AP. These data provide the first direct experimental visualization of I(Ca,L) and I(NCX) during cardiac the AP and Ca(2+) cycle. The residual current reported here can serve as a potential substrate for afterdepolarizations when increased under pathologic conditions.

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

How uniform is cAMP signaling? Focus on “Systems analysis of GLP-1 receptor signaling in pancreatic β-cells”

the goal of many cell physiologists is to ascribe cellular behavior to specific molecular signaling mechanisms. However, the reductionist experimental approach commonly used to understand molecular function often makes this task harder rather than easier to accomplish because of the complexity of

Repolarization of the mammalian heart action potential is modulated by changes in osmotic strength

This editorial refers to ‘Osmotic modulation of slowly activating I Ks in guinea-pig ventricular myocytes’ by S. Missan et al ., pp. 429–436, this issue. Repolarization of the mammalian heart action potential is a very important physiological variable. It sets action-potential duration and thus strongly modulates contractility and refractoriness in both the atria and the ventricles. Repolarization is initiated and controlled by the time- and voltage-dependent onset of a variety of different outward potassium (K+) currents, some of which are highly non-linear.1 One of these currents, the so-called slow outward delayed rectifier, or I Ks, is expressed in the human ventricle. Its activation adds to the safety factor for repolarization (the so-called repolarization reserve) through its intrinsic time and voltage dependence. This is prominent following augmentation of I Ks by sympathetic nerve stimulation.2 A paper in this issue from the McDonald laboratory3 confirms and extends what is known about the modulation of I Ks in response to changes in osmotic strength of the external medium. A significant change in osmotic strength (±20–30%) may take place in the setting of ischaemia–reperfusion of the heart. This paper demonstrates relatively rapid (3–5 min to steady state) changes in I Ks in single ventricular myocytes obtained from the hearts of adult guinea pigs in response to alterations in osmotic strength: reductions result in reversible increases in I Ks and vice versa. These changes appear to be quite selective as judged by the reported lack of effect on a selected K+ current denoted HERG. The context for these findings and some aspects of their interpretation is aided …

Osmotic modulation of slowly activating IKs in guinea-pig ventricular myocytes

The aims of the study were to determine the effects of anisosmotic bathing solution on selected properties of I(Ks), the slowly activating delayed-rectifier K(+) current important for repolarization of the action potential in cardiac cells. Guinea-pig ventricular myocytes were voltage-clamped using either the ruptured-patch or perforated-patch technique, and the amplitude, time course, and voltage dependence of I(Ks) were determined before [isosmotic (1T)] and during superfusion of hyposmotic (<1T) or hyperosmotic (>1T) bathing solution. Hyposmotic solution increased the amplitude of I(Ks), and hyperosmotic solution decreased it. Anisosmotic-induced changes in I(Ks) amplitude were complete in 2-5 min, well-maintained, reversible, and not accompanied by significant changes in I(Ks) time course and voltage dependence. There was little difference in the results obtained with the ruptured-patch technique and those obtained with the perforated-patch technique. The amplitude of I(Ks) was sensitive to small (±10%) changes in osmolarity, maximally increased by hyposmotic solution with T < 0.7, and strongly decreased by hyperosmotic solution with T > 1.5. Experimental data on a plot of relative (1T = 1.0) I(Ks) amplitude vs. the reciprocal of relative osmolarity are well-described by a Hill equation that has a lower asymptote of 0.0, an upper asymptote of 2.0, and a slope factor of 1.87 ± 0.07. Modulation of I(Ks) amplitude by anisosmotic solution is independent of patch configuration, unaccompanied by changes in current gating, and well-described by a Hill dose-response relation that predicts relatively strong responses of I(Ks) to small perturbations in external osmolarity.

Adenosine A2 receptors are involved in the activation of ATP-sensitive K+ currents during metabolic inhibition in guinea pig ventricular myocytes

It has been hypothesized that an interaction among adenosine A(1) receptors, protein kinase C (PKC) activation, and ATP-sensitive potassium channels (K(ATP)) mediates ischemic preconditioning in experiments on different animal species. The purpose of this study was to determine if activation of K(ATP) is functionally coupled to A(1) receptors and (or) PKC activation during metabolic inhibition (MI) in guinea pig ventricular myocytes. Perforated-patch using nystatin and conventional whole-cell recording methods were used to observe the effects of adenosine and adenosine-receptor antagonists on the activation of K(ATP) currents during MI induced by application of 2,4-dinitrophenol (DNP) and 2-deoxyglucose (2DG) without glucose, in the presence or absence of a PKC activator, phorbol 12-myristate 13-acetate (PMA). Adenosine accelerated the time course activation of K(ATP) currents during MI under the intact intracellular condition or dialyzed condition with l mmol/L ATP in the pipette solution. The accelerated effect of adenosine activation of K(ATP) under MI was not reversed by a nonselective Al adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline (SPT), or a specific Al adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). However, the adenosine A(2) receptor antagonist alloxazine reversed the time course activation of the K(ATP) current under MI. An adenylate cyclase activator, forskolin, did not further abbreviate the time course activation of K(ATP) with or without adenosine. Application of a PKC blocker, chelerythrine, reversed the time course activation of K(ATP) by adenosine under MI. In addition, pretreatment with a PKC activator, PMA, had similar effects to adenosine, while adenosine did not further shorten the time required for activation of K(ATP) currents during MI with PMA pretreatment. There is no direct evidence of activation of K(ATP) currents by adenosine A(1) receptor during metabolic inhibition under our experimental condition. However, adenosine A(2) receptor activation is involved in the K(ATP) channel activation in the guinea pig ventricular myocytes, of which effect is not mediated through the increase in intracellular cAMP. Adenosine seems to interact with PKC activation to open K(ATP) during MI, but a possible link between the adenosine A(2) receptor and PKC activation in this process needs further elucidation.

Increased intracellular calcium concentration causes electrical turbulence in guinea pig ventricular myocytes

Dysregulation of intracellular Ca(2+) homeostasis is associated with various pathological conditions and arrhythmogenesis of the heart. The objective of this study was to investigate the effects of an acute increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) on the electrophysiology of ventricular myocytes by mimicking intracellular Ca(2+) overload. The [Ca(2+)](i) was clamped to either a controlled (65-100 nmol L(-1)) or increased (1 μmol L(-1)) level. The transmembrane action potentials and ionic currents were recorded using whole-cell patch clamp techniques. We found that the acute increase in [Ca(2+)](i) shortened the action potential duration, reduced the action potential amplitude, maximum depolarization velocity and resting membrane potential, caused delayed after-depolarizations (DADs), and triggered activity-compared with these parameters in the control. The increased [Ca(2+)](i) augmented late I (Na) in a time-dependent manner, reduced I (CaL) and I (K1), and increased I (Kr) but not I (Ks). The results of this study can be used to explain calcium overload-induced ventricular arrhythmias.

Uncoupled Inward Currents through Native Na/K Pumps in Guinea Pig Ventricular Myocytes

The Na/K pump is a P-type ATPase that maintains essential electrochemical gradients for Na+ and K+ across the plasmalemma of animal cells. Within its transmembrane domains the pump presents 3 ion-binding sites, two of which can bind Na+ or K+ (shared sites) and another that exclusively binds Na+ . The mechanisms by which different ions are selected by each site are not fully understood.Electrophysiological studies from several laboratories investigating the function of Na/K pumps in Xenopus oocytes have shown that, without external Na+ or K+, the pump passively imports protons and possibly guanidinium+ and its derivatives. It is thought that both protons and guanidinium-derivatives are transported through the Na+ -exclusive site when the shared sites are empty. Because ion-binding sites of Na/K pumps across the animal kingdom are conserved, it is puzzling that these currents have not been reported in classical preparations where native Na/K pumps have been studied under voltage clamp. Here, we describe these uncoupled inward currents through the native Na/K pumps of Guinea pig ventricular myocytes, demonstrating they are not an artifact of the oocyte system.Under whole-cell patch-clamp with internal conditions promoting maximal Na/K pump phosphorylation (50 mM Na+i, 5 mM MgATP), cardiotonic steroid-sensitive inward currents (Iunc) were not observed in 150 mM Na+o (at all pHo) or in 150 NMG+o (at pHo =7.4). In contrast, at negative voltages, large currents Iunc=-1,7 ± 0,26 pA/pF (at −180 mV, n=10) were observed in NMG+o solutions with pHo = 6. Also consistent with observations reported in oocytes, inward currents Iunc = −1,08 ± 0,14 pA/pF (n=14) were observed in 150 mM guanidinium+o (pHo =7.4). The effects of other Na/K pump ligands on Iunc are underway. Financed by TTUHSC SABR and AHA BGIA2140172.

Human ETHER-A-Go-Go-Related Gene (HERG) K + Channel Inhibition by the Antidepressant Paroxetine

Paroxetine is a selective serotonin reuptake inhibitor (SSRI) for psychiatric disorders that can induce QT prolongation, which may lead to torsades de pointes. We studied the effects of paroxetine on human ether-a-go-go-related gene (hERG) channels expressed in Xenopus oocytes and on action potential in guinea pig ventricular myocytes. The hERG encodes the pore-forming subunits of the rapidly-activating delayed rectifier K+ channel (Ikr) in the heart. Mutations in hERG reduce Ikr and cause type 2 long QT syndrome (LQT2), a disorder that predisposes individuals to life-threatening arrhythmias. Paroxetine induced concentration-dependent decreases in the current amplitude at the end of the voltage steps and hERG tail currents. The inhibition was concentration-dependent and time-dependent, but voltage-independent during each voltage pulse. The S6 domain mutation Y652A did not affect the drug-induced hERG current block. In guinea-pig ventricular myocytes held at 36°C, treatment with 0.4 μM paroxetine for 5 min decreased the action potential duration at 90% of repolarization (APD90) by 4.3%. Our results suggest that paroxetine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of paroxetine.

Possible Roles of Cannabinoid Receptors in Cardiac Myocytes

Anandamide is an endogenous ligand of G-protein coupled cannabinoid CB1 and CB2 receptors, which has been shown to have complex effects on the cardiovascular system culminating in a hypotensive response and a reduction in cardiac contractility. The aims of this study were to investigate electrophysiologically the effects of anandamide on cardiac ventricular action potential (AP) parameters and accompanying cell contractions, and to explore the involvement, if any, of CB1 and CB2 receptors. Guinea pig ventricular myocytes were stimulated to fire APs by a 2 ms depolarising current pulse applied via an intracellular microelectrode at a frequency of 1 Hz using an Axoclamp 2 (Axon Instruments) amplifier (bridge mode). Myocyte contraction was measured using an edge detection system. Anandamide (1, 3, 10 µM) caused a concentration-dependent reduction in AP duration (APD) at 90, 50 and 20 % repolarisation, which was accompanied by a reduction in the amplitude of myocyte contraction. At a concentration of 10 µM, anandamide reduced APD 90 by 38 ± 8 % (n = 7, p < 0.001). In addition, 10 µM R-(+)-methanandamide (a non-hydrolysable analogue of anandamide) similarly reduced APD and amplitude of contraction. In the presence of the CB2 receptor antagonist AM 630, but not the CB1 receptor antagonist AM 281, the magnitudes of these reductions in response to 10 µM anandamide were partially but significantly reduced. This study confirms that anandamide causes a reduction in ventricular myocyte contractility and may modulate ventricular cell Ca2+ handling. Since these effects appear only to be partially inhibited by CB2 receptor blockade, it is likely that anandamide has actions which are independent of CB1 and CB2 receptors.

Actions of the NAADP Antagonist Trans-Ned19 in Cardiac Ventricular Myocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a substance that promotes calcium release from acidic intracellular calcium stores and exerts effects on calcium transients and contractions in guinea-pig ventricular myocytes. A structural analogue of NAADP, termed NED19, acts as an antagonist of NAADP in cell homogenates from sea-urchin eggs. The aim of the present study was to investigate actions of NED19 (using the trans optical isomer) on ventricular myocytes isolated from guinea-pig hearts. Trans-NED19 (1 μM, 5 min exposure) was without significant effect on L-type calcium currents over the range −30 to +60 mV (switched voltage-clamp, step depolarizations from −40 mV). The acetoxymethylester of NAADP (NAADP-AM) was applied to ventricular myocytes with the aim of allowing permeation of NAADP-AM across the cell membrane followed by accumulation of cytosolic NAADP after de-esterification of NAADP-AM by intracellular esterases. NAADP-AM (60 nM) increased myocyte contraction by approximately 40% (n=10, field stimulation at 1 Hz). This effect of NAADP-AM was no longer observed in myocytes exposed to trans-NED19 (1 μM), consistent with an antagonist effect of trans-NED19 preventing the effects of cytosolic NAADP. Exposure of myocytes to trans-NED19 (1 μM) alone significantly reduced contraction by 17 +/- 4%, a reduction which is similar to the 20 +/- 4% previously found when myocytes were exposed to bafilomycin A1 (1 μM, presumed to act on intracellular acidic stores). Calcium transients measured using Fluo-5F were also reduced by trans-NED19 (1 μM) and these effects were inhibited by pre-exposure of the myocytes to bafilomycin A1. These observations are consistent with effects of NAADP on myocyte contraction under these conditions, that can be prevented either by interference with the function of acidic stores by bafilomycin A1 or by antagonism of the effects of NAADP by trans-NED19.

Modeling the Mechanisms of Calcium-Mediated Cardiac Arrhythmias

Dysfunction of normal calcium dynamics has been implicated in the generation of cardiac arrhythmias. It is thought that spontaneous calcium release events in the myocyte lead to the formation of intracellular calcium waves. These calcium release events occur through opening of the ryanodine receptors (RyRs) in the sarcoplasmic reticulum. In order for this to lead to an arrhythmia, these waves need to depolarize the cardiac myocyte in events know as early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs). These aberrant depolarizations must spread to adjacent cells in a propagating wave of depolarization to disrupt the normal pattern of electrical excitation of the heart. Computational compartmental ventricular myocyte models have shown that EADs and DADS can be generated by certain conditions consistent with experiments. We have developed a spatio-temporal computational model of a chain of cardiac myocytes based on the Jafri-Rice-Winslow model of the guinea pig ventricular myocyte. The model includes spatial resolution of the individual myocyte as well as a network of myocytes, calcium dynamics, and the sarcolemmal electrical activity. We use the model to explore how factors such as calcium overload, RyR calcium sensitivity, and other factors affect the generation of calcium waves. Furthermore, we also explore under what conditions the calcium wave can depolarize the myocyte and induce a propagating wave of depolarization to adjacent myocytes. These insights can be used to better understand the basic mechanisms of calcium-entrained cardiac arrhythmias.

Modeling HERG Gating Transitions

HERG (human ether-à-go-go related gene, Kv11.1, KCNH2) is a voltage-gated potassium channel with unique gating characteristics. HERG has fast voltage-dependent inactivation, relatively slow deactivation, and fast recovery from inactivation. This combination of gating kinetics makes study of HERG difficult without using mathematical models. Several HERG models have been developed, with fundamentally different organization and properties. We programmed five distinct HERG models and tested their behavior under voltage-clamp and guinea-pig ventricular myocyte action potential clamp. Four models used Markov formalisms, and one used Hodgkin-Huxley formalism. HERG behavior cannot be replicated using a Hodgkin-Huxley formalism. The Markov Models had 2 or 3 closed states, 1 open state, and 1 inactivated state. A voltage-independent activation step is required in order to replicate the experimentally observed voltage-independent rate limiting step of activation. A fundamental difference between models is the presence or absence of a transition directly from the closed state to the inactivated state. Two of the Markov models had transitions directly from the closed state to the inactivated state. Our analysis demonstrates that the only models which effectively reproduce HERG experimental data require that the closed-inactivated transitions are absent or are effectively zero compared to the closed to open transitions, rendering the closed-inactivation transition superfluous. Furthermore, the models make significantly different predictions about the behavior of the HERG during action potentials and premature stimuli. Our simulations indicate there is no direct transition between the pre-activated closed state and the inactivated state, although inactivation from a “flicker” open state is possible. The use of a model with a direct transition between closed and inactivated states with rates that are effectively zero is potentially misleading in understanding HERG gating.

Normalizing Action-Potential Morphology in Long-Term Cultures of Adult Guinea-Pig Ventricular Cardiomyocytes by Ca VBETA Expression

Adult guinea-pig ventricular myocytes (aGPVMs) exhibit enduring action-potential plateaus similar to those in humans, making this system advantageous for investigating human phase 2 phenomena. Long-term cultures of aGPVMs (Canadian J Physiol Pharmacol7:740) further offer the promise of genetic manipulation by somatic gene transfer, but have been infrequently exploited. Here, we confirm that aGPVM cultures form monolayers featuring organized myofibrils and robust contraction. Beyond this, we now demonstrate that point pacing of di-4-ANEPPS stained monolayers enable optical measurements of action potentials. At 2-Hz pacing, action potentials indeed lasted ∼250 ms, but displayed attenuated plateau phases (a, gray trace). To normalize phase 2, and to demonstrate genetic manipulability, we virally expressed Ca2+ channel CaVβ subunits that enhance L-type channels in heterologous systems. CaVβ expression in fact restored a full-bore plateau phase, as shown by the black trace in a. To confirm a role of Ca2+ current, we obtained single-channel recordings of native L-type channels. CaVβ subunits strikingly increased open probability, while decreasing inactivation (b, control; c, CaVβ expression), thus explaining the normalization of action-potential morphology. Overall, long-term cultured aGPVMs offer a powerful model system for electrophysiological exploration.

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.