Therapeutic Approaches of Ryanodine Receptor-Associated Heart Diseases.

Cardiac diseases are the leading causes of death, with a growing number of cases worldwide, posing a challenge for both healthcare and research. Therefore, the most relevant aim of cardiac research is to unravel the molecular pathomechanisms and identify new therapeutic targets. Cardiac ryanodine receptor (RyR2), the Ca2+ release channel of the sarcoplasmic reticulum, is believed to be a good therapeutic target in a group of certain heart diseases, collectively called cardiac ryanopathies. Ryanopathies are associated with the impaired function of the RyR, leading to heart diseases such as congestive heart failure (CHF), catecholaminergic polymorphic ventricular tachycardia (CPVT), arrhythmogenic right ventricular dysplasia type 2 (ARVD2), and calcium release deficiency syndrome (CRDS). The aim of the current review is to provide a short insight into the pathological mechanisms of ryanopathies and discuss the pharmacological approaches targeting RyR2.

Ion current profiles in canine ventricular myocytes obtained by the "onion peeling" technique.

The profiles of ion currents during the cardiac action potential can be visualized by the action potential voltage clamp technique. To obtain multiple ion current data from the same cell, the "onion peeling" technique, based on sequential pharmacological dissection of ion currents, has to be applied. Combination of the two methods allows recording of several ion current profiles from the same myocyte under largely physiological conditions. Using this approach, we have studied the densities and integrals of the major cardiac inward (ICa, INCX, INa-late) and outward (IKr, IKs, IK1) currents in canine ventricular cells and studied the correlation between them. For this purpose, canine ventricular cardiomyocytes were chosen because their electrophysiological properties are similar to those of human ones. Significant positive correlation was observed between the density and integral of ICa and IKr, and positive correlation was found also between the integral of ICa and INCX. No further correlations were detected. The Ca2+-sensitivity of K+ currents was studied by comparing their parameters in the case of normal calcium homeostasis and following blockade of ICa. Out of the three K+ currents studied, only IKs was Ca2+-sensitive. The density and integral of IKs was significantly greater, while its time-to-peak value was shorter at normal Ca2+ cycling than following ICa blockade. No differences were detected for IKr or IK1 in this regard. Present results indicate that the positive correlation between ICa and IKr prominently contribute to the balance between inward and outward fluxes during the action potential plateau in canine myocytes. The results also suggest that the profiles of cardiac ion currents have to be studied under physiological conditions, since their behavior may strongly be influenced by the intracellular Ca2+ homeostasis and the applied membrane potential protocol.

Complex electrophysiological remodeling in postinfarction ischemic heart failure.

Heart failure (HF) following myocardial infarction (MI) is associated with high incidence of cardiac arrhythmias. Development of therapeutic strategy requires detailed understanding of electrophysiological remodeling. However, changes of ionic currents in ischemic HF remain incompletely understood, especially in translational large-animal models. Here, we systematically measure the major ionic currents in ventricular myocytes from the infarct border and remote zones in a porcine model of post-MI HF. We recorded eight ionic currents during the cell's action potential (AP) under physiologically relevant conditions using selfAP-clamp sequential dissection. Compared with healthy controls, HF-remote zone myocytes exhibited increased late Na+ current, Ca2+-activated K+ current, Ca2+-activated Cl- current, decreased rapid delayed rectifier K+ current, and altered Na+/Ca2+ exchange current profile. In HF-border zone myocytes, the above changes also occurred but with additional decrease of L-type Ca2+ current, decrease of inward rectifier K+ current, and Ca2+ release-dependent delayed after-depolarizations. Our data reveal that the changes in any individual current are relatively small, but the integrated impacts shift the balance between the inward and outward currents to shorten AP in the border zone but prolong AP in the remote zone. This differential remodeling in post-MI HF increases the inhomogeneity of AP repolarization, which may enhance the arrhythmogenic substrate. Our comprehensive findings provide a mechanistic framework for understanding why single-channel blockers may fail to suppress arrhythmias, and highlight the need to consider the rich tableau and integration of many ionic currents in designing therapeutic strategies for treating arrhythmias in HF.

Electrophysiological Effects of the Transient Receptor Potential Melastatin 4 Channel Inhibitor (4-Chloro-2-(2-chlorophenoxy)acetamido) Benzoic Acid (CBA) in Canine Left Ventricular Cardiomyocytes.

Transient receptor potential melastatin 4 (TRPM4) plays an important role in many tissues, including pacemaker and conductive tissues of the heart, but much less is known about its electrophysiological role in ventricular myocytes. Our earlier results showed the lack of selectivity of 9-phenanthrol, so CBA ((4-chloro-2-(2-chlorophenoxy)acetamido) benzoic acid) was chosen as a new, potentially selective inhibitor. Goal: Our aim was to elucidate the effect and selectivity of CBA in canine left ventricular cardiomyocytes and to study the expression of TRPM4 in the canine heart. Experiments were carried out in enzymatically isolated canine left ventricular cardiomyocytes. Ionic currents were recorded with an action potential (AP) voltage-clamp technique in whole-cell configuration at 37 °C. An amount of 10 mM BAPTA was used in the pipette solution to exclude the potential activation of TRPM4 channels. AP was recorded with conventional sharp microelectrodes. CBA was used in 10 µM concentrations. Expression of TRPM4 protein in the heart was studied by Western blot. TRPM4 protein was expressed in the wall of all four chambers of the canine heart as well as in samples prepared from isolated left ventricular cells. CBA induced an approximately 9% reduction in AP duration measured at 75% and 90% of repolarization and decreased the short-term variability of APD90. Moreover, AP amplitude was increased and the maximal rates of phase 0 and 1 were reduced by the drug. In AP clamp measurements, CBA-sensitive current contained a short, early outward and mainly a long, inward current. Transient outward potassium current (Ito) and late sodium current (INa,L) were reduced by approximately 20% and 47%, respectively, in the presence of CBA, while L-type calcium and inward rectifier potassium currents were not affected. These effects of CBA were largely reversible upon washout. Based on our results, the CBA induced reduction of phase-1 slope and the slight increase of AP amplitude could have been due to the inhibition of Ito. The tendency for AP shortening can be explained by the inhibition of inward currents seen in AP-clamp recordings during the plateau phase. This inward current reduced by CBA is possibly INa,L, therefore, CBA is not entirely selective for TRPM4 channels. As a consequence, similarly to 9-phenanthrol, it cannot be used to test the contribution of TRPM4 channels to cardiac electrophysiology in ventricular cells, or at least caution must be applied.

Late sodium current in human, canine and guinea pig ventricular myocardium.

Although late sodium current (INa-late) has long been known to contribute to plateau formation of mammalian cardiac action potentials, lately it was considered as possible target for antiarrhythmic drugs. However, many aspects of this current are still poorly understood. The present work was designed to study the true profile of INa-late in canine and guinea pig ventricular cells and compare them to INa-late recorded in undiseased human hearts. INa-late was defined as a tetrodotoxin-sensitive current, recorded under action potential voltage clamp conditions using either canonic- or self-action potentials as command signals. Under action potential voltage clamp conditions the amplitude of canine and human INa-late monotonically decreased during the plateau (decrescendo-profile), in contrast to guinea pig, where its amplitude increased during the plateau (crescendo profile). The decrescendo-profile of canine INa-late could not be converted to a crescendo-morphology by application of ramp-like command voltages or command action potentials recorded from guinea pig cells. Conventional voltage clamp experiments revealed that the crescendo INa-late profile in guinea pig was due to the slower decay of INa-late in this species. When action potentials were recorded from multicellular ventricular preparations with sharp microelectrode, action potentials were shortened by tetrodotoxin, which effect was the largest in human, while smaller in canine, and the smallest in guinea pig preparations. It is concluded that important interspecies differences exist in the behavior of INa-late. At present canine myocytes seem to represent the best model of human ventricular cells regarding the properties of INa-late. These results should be taken into account when pharmacological studies with INa-late are interpreted and extrapolated to human. Accordingly, canine ventricular tissues or myocytes are suggested for pharmacological studies with INa-late inhibitors or modifiers. Incorporation of present data to human action potential models may yield a better understanding of the role of INa-late in action potential morphology, arrhythmogenesis, and intracellular calcium dynamics.

Mechano-electric and mechano-chemo-transduction in cardiomyocytes.

Cardiac excitation-contraction (E-C) coupling is influenced by (at least) three dynamic systems that couple and feedback to one another (see Abstract Figure). Here we review the mechanical effects on cardiomyocytes that include mechano-electro-transduction (commonly referred to as mechano-electric coupling, MEC) and mechano-chemo-transduction (MCT) mechanisms at cell and molecular levels which couple to Ca2+ -electro and E-C coupling reviewed elsewhere. These feedback loops from muscle contraction and mechano-transduction to the Ca2+ homeodynamics and to the electrical excitation are essential for understanding the E-C coupling dynamic system and arrhythmogenesis in mechanically loaded hearts. This white paper comprises two parts, each reflecting key aspects from the 2018 UC Davis symposium: MEC (how mechanical load influences electrical dynamics) and MCT (how mechanical load alters cell signalling and Ca2+ dynamics). Of course, such separation is artificial since Ca2+ dynamics profoundly affect ion channels and electrogenic transporters and vice versa. In time, these dynamic systems and their interactions must become fully integrated, and that should be a goal for a comprehensive understanding of how mechanical load influences cell signalling, Ca2+ homeodynamics and electrical dynamics. In this white paper we emphasize current understanding, consensus, controversies and the pressing issues for future investigations. Space constraints make it impossible to cover all relevant articles in the field, so we will focus on the topics discussed at the symposium.

Altered K+ current profiles underlie cardiac action potential shortening in hyperkalemia and ß-adrenergic stimulation.

Hyperkalemia is known to develop in various conditions including vigorous physical exercise. In the heart, hyperkalemia is associated with action potential (AP) shortening that was attributed to altered gating of K+ channels. However, it remains unknown how hyperkalemia changes the profiles of each K+ current under a cardiac AP. Therefore, we recorded the major K+ currents (inward rectifier K+ current, IK1; rapid and slow delayed rectifier K+ currents, IKr and IKs, respectively) using AP-clamp in rabbit ventricular myocytes. As K+ may accumulate at rapid heart rates during sympathetic stimulation, we also examined the effect of isoproterenol on these K+ currents. We found that IK1 was significantly increased in hyperkalemia, whereas the reduction of driving force for K+ efflux dominated over the altered channel gating in case of IKr and IKs. Overall, the markedly increased IK1 in hyperkalemia overcame the relative decreases of IKr and IKs during AP, resulting in an increased net repolarizing current during AP phase 3. ß-Adrenergic stimulation of IKs also provided further repolarizing power during sympathetic activation, although hyperkalemia limited IKs upregulation. These results indicate that facilitation of IK1 in hyperkalemia and ß-adrenergic stimulation of IKs represent important compensatory mechanisms against AP prolongation and arrhythmia susceptibility.

ß-adrenergic regulation of late Na+ current during cardiac action potential is mediated by both PKA and CaMKII.

Late Na+ current (INaL) significantly contributes to shaping cardiac action potentials (APs) and increased INaL is associated with cardiac arrhythmias. ß-adrenergic receptor (ßAR) stimulation and its downstream signaling via protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways are known to regulate INaL. However, it remains unclear how each of these pathways regulates INaL during the AP under physiological conditions. Here we performed AP-clamp experiments in rabbit ventricular myocytes to delineate the impact of each signaling pathway on INaL at different AP phases to understand the arrhythmogenic potential. During the physiological AP (2 Hz, 37 °C) we found that INaL had a basal level current independent of PKA, but partially dependent on CaMKII. ßAR activation (10 nM isoproterenol, ISO) further enhanced INaL via both PKA and CaMKII pathways. However, PKA predominantly increased INaL early during the AP plateau, whereas CaMKII mainly increased INaL later in the plateau and during rapid repolarization. We also tested the role of key signaling pathways through exchange protein activated by cAMP (Epac), nitric oxide synthase (NOS) and reactive oxygen species (ROS). Direct Epac stimulation enhanced INaL similar to the ßAR-induced CaMKII effect, while NOS inhibition prevented the ßAR-induced CaMKII-dependent INaL enhancement. ROS generated by NADPH oxidase 2 (NOX2) also contributed to the ISO-induced INaL activation early in the AP. Taken together, our data reveal differential modulations of INaL by PKA and CaMKII signaling pathways at different AP phases. This nuanced and comprehensive view on the changes in INaL during AP deepens our understanding of the important role of INaL in reshaping the cardiac AP and arrhythmogenic potential under elevated sympathetic stimulation, which is relevant for designing therapeutic treatment of arrhythmias under pathological conditions.

Transient receptor potential melastatin 4 channel inhibitor 9-phenanthrol inhibits K+ but not Ca2+ currents in canine ventricular myocytes.

The role of transient receptor potential melastatin 4 (TRPM4) channels has been frequently tested using their inhibitor 9-phenanthrol in various cardiac preparations; however, the selectivity of the compound is uncertain. Therefore, in the present study, the concentration-dependent effects of 9-phenanthrol on major ionic currents were studied in canine isolated ventricular cells using whole-cell configuration of the patch-clamp technique and 10 mM BAPTA-containing pipette solution to prevent the Ca2+-dependent activation of TRPM4 channels. Transient outward (Ito1), rapid delayed rectifier (IKr), and inward rectifier (IK1) K+ currents were suppressed by 10 and 30 µM 9-phenanthrol with the blocking potency for IK1 < IKr < Ito1 and partial reversibility. L-type Ca2+ current was not affected up to the concentration of 30 µM. In addition, a steady outward current was detected at voltages positive to -40 mV in 9-phenanthrol, which was larger at more positive voltages and larger 9-phenanthrol concentrations. Action potentials were recorded using microelectrodes. Maximal rate of depolarization, phase-1 repolarization, and terminal repolarization were decreased and the plateau potential was depressed by 9-phenanthrol (3-30 µM), congruently with the observed alterations of ionic currents. Significant action potential prolongation was observed by 9-phenanthrol in the majority of the studied cells, but only at 30 µM concentration. In conclusion, 9-phenanthrol is not selective to TRPM4 channels in canine ventricular myocardium; therefore, its application as a TRPM4 blocker can be appropriate only in expression systems but not in native cardiac cells.

Ca2+-activated Cl- current is antiarrhythmic by reducing both spatial and temporal heterogeneity of cardiac repolarization.

The role of Ca2+-activated Cl- current (ICl(Ca)) in cardiac arrhythmias is still controversial. It can generate delayed afterdepolarizations in Ca2+-overloaded cells while in other studies incidence of early afterdepolarization (EAD) was reduced by ICl(Ca). Therefore our goal was to examine the role of ICl(Ca) in spatial and temporal heterogeneity of cardiac repolarization and EAD formation. Experiments were performed on isolated canine cardiomyocytes originating from various regions of the left ventricle; subepicardial, midmyocardial and subendocardial cells, as well as apical and basal cells of the midmyocardium. ICl(Ca) was blocked by 0.5mmol/L 9-anthracene carboxylic acid (9-AC). Action potential (AP) changes were tested with sharp microelectrode recording. Whole-cell 9-AC-sensitive current was measured with either square pulse voltage-clamp or AP voltage-clamp (APVC). Protein expression of TMEM16A and Bestrophin-3, ion channel proteins mediating ICl(Ca), was detected by Western blot. 9-AC reduced phase-1 repolarization in every tested cell. 9-AC also increased AP duration in a reverse rate-dependent manner in all cell types except for subepicardial cells. Neither ICl(Ca) density recorded with square pulses nor the normalized expressions of TMEM16A and Bestrophin-3 proteins differed significantly among the examined groups of cells. The early outward component of ICl(Ca) was significantly larger in subepicardial than in subendocardial cells in APVC setting. Applying a typical subepicardial AP as a command pulse resulted in a significantly larger early outward component in both subepicardial and subendocardial cells, compared to experiments when a typical subendocardial AP was applied. Inhibiting ICl(Ca) by 9-AC generated EADs at low stimulation rates and their incidence increased upon beta-adrenergic stimulation. 9-AC increased the short-term variability of repolarization also. We suggest a protective role for ICl(Ca) against risk of arrhythmias by reducing spatial and temporal heterogeneity of cardiac repolarization and EAD formation.

Potassium currents in the heart: functional roles in repolarization, arrhythmia and therapeutics.

This is the second of the two White Papers from the fourth UC Davis Cardiovascular Symposium Systems Approach to Understanding Cardiac Excitation-Contraction Coupling and Arrhythmias (3-4 March 2016), a biennial event that brings together leading experts in different fields of cardiovascular research. The theme of the 2016 symposium was 'K+ channels and regulation', and the objectives of the conference were severalfold: (1) to identify current knowledge gaps; (2) to understand what may go wrong in the diseased heart and why; (3) to identify possible novel therapeutic targets; and (4) to further the development of systems biology approaches to decipher the molecular mechanisms and treatment of cardiac arrhythmias. The sessions of the Symposium focusing on the functional roles of the cardiac K+ channel in health and disease, as well as K+ channels as therapeutic targets, were contributed by Ye Chen-Izu, Gideon Koren, James Weiss, David Paterson, David Christini, Dobromir Dobrev, Jordi Heijman, Thomas O'Hara, Crystal Ripplinger, Zhilin Qu, Jamie Vandenberg, Colleen Clancy, Isabelle Deschenes, Leighton Izu, Tamas Banyasz, Andras Varro, Heike Wulff, Eleonora Grandi, Michael Sanguinetti, Donald Bers, Jeanne Nerbonne and Nipavan Chiamvimonvat as speakers and panel discussants. This article summarizes state-of-the-art knowledge and controversies on the functional roles of cardiac K+ channels in normal and diseased heart. We endeavour to integrate current knowledge at multiple scales, from the single cell to the whole organ levels, and from both experimental and computational studies.

Electrophysiological Determination of Submembrane Na(+) Concentration in Cardiac Myocytes.

In the heart, Na(+) is a key modulator of the action potential, Ca(2+) homeostasis, energetics, and contractility. Because Na(+) currents and cotransport fluxes depend on the Na(+) concentration in the submembrane region, it is necessary to accurately estimate the submembrane Na(+) concentration ([Na(+)]sm). Current methods using Na(+)-sensitive fluorescent indicators or Na(+) -sensitive electrodes cannot measure [Na(+)]sm. However, electrophysiology methods are ideal for measuring [Na(+)]sm. In this article, we develop patch-clamp protocols and experimental conditions to determine the upper bound of [Na(+)]sm at the peak of action potential and its lower bound at the resting state. During the cardiac cycle, the value of [Na(+)]sm is constrained within these bounds. We conducted experiments in rabbit ventricular myocytes at body temperature and found that 1) at a low pacing frequency of 0.5 Hz, the upper and lower bounds converge at 9 mM, constraining the [Na(+)]sm value to ∼9 mM; 2) at 2 Hz pacing frequency, [Na(+)]sm is bounded between 9 mM at resting state and 11.5 mM; and 3) the cells can maintain [Na(+)]sm to the above values, despite changes in the pipette Na(+) concentration, showing autoregulation of Na(+) in beating cardiomyocytes.

Sarcolemmal Ca(2+)-entry through L-type Ca(2+) channels controls the profile of Ca(2+)-activated Cl(-) current in canine ventricular myocytes.

Ca(2+)-activated Cl(-) current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca(2+) cycling and AP voltage-clamp) as well as in conditions designed to change [Ca(2+)]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied. The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca(2+)]i was monitored using Fura-2-AM. Setting [Ca(2+)]i to the systolic level measured in the bulk cytoplasm (1.1µM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca(2+)-entry through L-type Ca(2+) channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of ICl(Ca) in canine ventricular cells requires Ca(2+)-entry through neighboring L-type Ca(2+) channels and is only augmented by SR Ca(2+)-release. Substantial activation of ICl(Ca) requires high Ca(2+) concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

Concept of relative variability of cardiac action potential duration and its test under various experimental conditions.

Beat-to-beat variability of action potential duration (short-term variability, SV) is an intrinsic property of mammalian myocardium. Since the majority of agents and interventions affecting SV may modify also action potential duration (APD), we propose here the concept of relative SV (RSV), where changes in SV are normalized to changes in APD and these data are compared to the control SV-APD relationship obtained by lengthening or shortening of action potentials by inward and outward current injections. Based on this concept the influence of the several experimental conditions like stimulation frequency, temperature, pH, redox-state and osmolarity were examined on RSV in canine ventricular myocytes using sharp microelectrodes. RSV was increased by high stimulation frequency (cycle lengths <0.7 s), high temperature (above 37ºC), oxidative agents (H2O2), while it was decreased by reductive environment. RSV was not affected by changes in pH (within the range of 6.4-8.4) and osmolarity of the solution (between 250-350 mOsm). The results indicate that changes in beat-to-beat variability of APD must be evaluated exclusively in terms of RSV; furthermore, some experimental conditions, including the stimulation frequency, redox-state and temperature have to be controlled strictly when analyzing alterations in the short-term variability of APD.

KN-93 inhibits IKr in mammalian cardiomyocytes.

Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 is widely used in multiple fields of cardiac research especially for studying the mechanisms of cardiomyopathy and cardiac arrhythmias. Whereas KN-93 is a potent inhibitor of CaMKII, several off-target effects have also been found in expression cell systems and smooth muscle cells, but there is no information on the KN93 side effects in mammalian ventricular myocytes. In this study we explore the effect of KN-93 on the rapid component of delayed rectifier potassium current (IKr) in the ventricular myocytes from rabbit and guinea pig hearts. Our data indicate that KN-93 exerts direct inhibitory effect on IKr that is not mediated via CaMKII. This off-target effect of KN93 should be taken into account when interpreting the data from using KN93 to investigate the role of CaMKII in cardiac function.

Contribution of ion currents to beat-to-beat variability of action potential duration in canine ventricular myocytes.

Although beat-to-beat variability (short-term variability, SV) of action potential duration (APD) is considered as a predictor of imminent cardiac arrhythmias, the underlying mechanisms are still not clear. In the present study, therefore, we aimed to determine the role of the major cardiac ion currents, APD, stimulation frequency, and changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) on the magnitude of SV. Action potentials were recorded from isolated canine ventricular cardiomyocytes using conventional microelectrode techniques. SV was an exponential function of APD, when APD was modified by current injections. Drug effects were characterized as relative SV changes by comparing the drug-induced changes in SV to those in APD according to the exponential function obtained with current pulses. Relative SV was increased by dofetilide, HMR 1556, nisoldipine, and veratridine, while it was reduced by BAY K8644, tetrodotoxin, lidocaine, and isoproterenol. Relative SV was also increased by increasing the stimulation frequency and [Ca(2+)]i. In summary, relative SV is decreased by ion currents involved in the negative feedback regulation of APD (I Ca, I Ks, and I Kr), while it is increased by I Na and I to. We conclude that drug-induced effects on SV should be evaluated in relation with the concomitant changes in APD. Since relative SV was decreased by ion currents playing critical role in the negative feedback regulation of APD, blockade of these currents, or the beta-adrenergic pathway, may carry also some additional proarrhythmic risk in addition to their well-known antiarrhythmic action.

Cytosolic calcium changes affect the incidence of early afterdepolarizations in canine ventricular myocytes.

This study was designed to investigate the influence of cytosolic Ca(2+) levels ([Ca(2+)]i) on action potential duration (APD) and on the incidence of early afterdepolarizations (EADs) in canine ventricular cardiomyocytes. Action potentials (AP) of isolated cells were recorded using conventional sharp microelectrodes, and the concomitant [Ca(2+)]i was monitored with the fluorescent dye Fura-2. EADs were evoked at a 0.2 Hz pacing rate by inhibiting the rapid delayed rectifier K(+) current with dofetilide, by activating the late sodium current with veratridine, or by activating the L-type calcium current with BAY K8644. These interventions progressively prolonged the AP and resulted in initiation of EADs. Reducing [Ca(2+)]i by application of the cell-permeant Ca(2+) chelator BAPTA-AM lengthened the AP at 1.0 Hz if it was applied alone, in the presence of veratridine, or in the presence of BAY K8644. However, BAPTA-AM shortened the AP if the cells were pretreated with dofetilide. The incidence of the evoked EADs was strongly reduced by BAPTA-AM in dofetilide, moderately reduced in veratridine, whereas EAD incidence was increased by BAPTA-AM in the presence of BAY K8644. Based on these experimental data, changes in [Ca(2+)]i have marked effects on APD as well as on the incidence of EADs; however, the underlying mechanisms may be different, depending on the mechanism of EAD generation. As a consequence, reduction of [Ca(2+)]i may eliminate EADs under some, but not all, experimental conditions.

Oxidative shift in tissue redox potential increases beat-to-beat variability of action potential duration.

Profound changes in tissue redox potential occur in the heart under conditions of oxidative stress frequently associated with cardiac arrhythmias. Since beat-to-beat variability (short term variability, SV) of action potential duration (APD) is a good indicator of arrhythmia incidence, the aim of this work was to study the influence of redox changes on SV in isolated canine ventricular cardiomyocytes using a conventional microelectrode technique. The redox potential was shifted toward a reduced state using a reductive cocktail (containing dithiothreitol, glutathione, and ascorbic acid) while oxidative changes were initiated by superfusion with H2O2. Redox effects were evaluated as changes in "relative SV" determined by comparing SV changes with the concomitant APD changes. Exposure of myocytes to the reductive cocktail decreased SV significantly without any detectable effect on APD. Application of H2O2 increased both SV and APD, but the enhancement of SV was the greater, so relative SV increased. Longer exposure to H2O2 resulted in the development of early afterdepolarizations accompanied by tremendously increased SV. Pretreatment with the reductive cocktail prevented both elevation in relative SV and the development of afterdepolarizations. The results suggest that the increased beat-to-beat variability during an oxidative stress contributes to the generation of cardiac arrhythmias.

Beta-adrenergic stimulation reverses the I Kr-I Ks dominant pattern during cardiac action potential.

ß-Adrenergic stimulation differentially modulates different K(+) channels and thus fine-tunes cardiac action potential (AP) repolarization. However, it remains unclear how the proportion of I Ks, I Kr, and I K1 currents in the same cell would be altered by ß-adrenergic stimulation, which would change the relative contribution of individual K(+) current to the total repolarization reserve. In this study, we used an innovative AP-clamp sequential dissection technique to directly record the dynamic I Ks, I Kr, and I K1 currents during the AP in guinea pig ventricular myocytes under physiologically relevant conditions. Our data provide quantitative measures of the magnitude and time course of I Ks, I Kr, and I K1 currents in the same cell under its own steady-state AP, in a physiological milieu, and with preserved Ca(2+) homeostasis. We found that isoproterenol treatment significantly enhanced I Ks, moderately increased I K1, but slightly decreased I Kr in a dose-dependent manner. The dominance pattern of the K(+) currents was I Kr > I K1 > I Ks at the control condition, but reversed to I Kr < I K1 < I Ks following ß-adrenergic stimulation. We systematically determined the changes in the relative contribution of I Ks, I Kr, and I K1 to cardiac repolarization during AP at different adrenergic states. In conclusion, the ß-adrenergic stimulation fine-tunes the cardiac AP morphology by shifting the power of different K(+) currents in a dose-dependent manner. This knowledge is important for designing antiarrhythmic drug strategies to treat hearts exposed to various sympathetic tones.