Cell Action Potential Duration Research Articles

Telomerase Reverse Transcriptase Regulates Intracellular Ca2+ Homeostasis and Mitochondrial Function via the p53/PGC-1α Pathway in HL-1 Cells.

Telomere shortening is strongly associated with cardiovascular aging and disease, and patients with shorter telomeres in peripheral blood leukocytes are at higher risk of cardiovascular diseases such as heart failure and atrial fibrillation (AF). Telomerase reverse transcriptase (TERT) maintains telomere length, and overexpression of TERT has been shown to reduce cardiomyocyte apoptosis and myocardial infarct size, and extend the lifespan of aged mice. However, the specific impact of TERT on the electrophysiology of cardiomyocytes remains to be elucidated. The aims of this study were to evaluate the role of TERT in Ca2+ homeostasis and mitochondrial function in atrial myocytes as well as the underlying mechanisms. TERT overexpressed and silenced HL-1 cells were constructed with lentiviruses, and the respective empty lentiviral vectors were used as negative controls. Then the patch clamp technique was used to record the electrophysiological characteristics such as cell action potential duration (APD) and L-type Ca2+ currents (ICa,L), flow cytometry was used to detect intracellular Ca2+ concentration and mitochondrial membrane potential (MMP), and the Seahorse assay was used to measure the oxygen consumption rate (OCR). TERT silencing led to intracellular Ca2+ overload, shortened APD, decreased ICa,L current density, altered Ca2+ gating mechanism, decreased MMP and OCR, and increased reactive oxygen species (ROS), whereas TERT overexpression led to the reverse effects. Additionally, TERT silencing resulted in intracellular Ca2+ overload with decreased expression of the SERCA2a, CaV1.2, and NCX1.1, whereas TERT overexpression had opposing effects. Furthermore, we discovered that TERT could regulate the expression of p53 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). The expression of PGC-1α was downregulated by the p53 agonist Tenovin-6 but upregulated by the p53 inhibitor PFTα. The effects of the PGC-1α inhibitor SR-18292 on intracellular Ca2+ and cell electrophysiology were similar to those of silencing TERT, whereas the PGC-1α agonist ZLN005 produced comparable outcomes to TERT overexpression. TERT silencing-induced Ca2+ overload and mitochondrial dysfunction may be one mechanism of age-related AF. Overexpression of TERT reduced the basis for arrhythmia formation such as AF, suggesting a favorable safety profile for TERT therapy. TERT regulated intracellular Ca2+ homeostasis and mitochondrial function through the p53/PGC-1α pathway. In addition, PGC-1α might be a novel target for AF, suggesting that intervention for AF should be not limited to abnormal cation handling.

Repolarization heterogeneity within the myocardial infarction border zone correlates with variability of myocyte remodeling

Abstract Background Myocardial infarction (MI) results in a regional scar, with a border zone (BZ) of surviving myocytes interspersed with fibrosis providing an anatomical substrate for re-entry. Heterogeneous repolarization within the BZ may add a functional component aggravating re-entrant arrhythmias. Purpose We studied BZ heterogeneity and developed novel methodology for high resolution mapping of local in vivo activation-repolarization intervals (ARI) within the BZ and for studying the relation to cellular action potential (AP) profiles of cells isolated from the BZ. Methods Anterior-septal myocardial infarction was induced in 5 domestic pigs by 120-minute occlusion of the left anterior descending artery followed by reperfusion (18.9±4.7% of the left ventricle). After 1-month, electro-anatomical mapping was performed. Contact mapping was used to define the BZ (bipolar voltage 0.5–1.5mV). A non-contact recording of a 64-electrode array was translated to 2048 non-contact electrograms distributed over the LV. The non-contact electrograms were processed to determine the ARIs using a custom-made algorithm, validated against monophasic action potential recordings. After 2–4 days recovery, single cardiomyocytes were enzymatically isolated from the anterior-septal BZs and remote regions. Cardiomyocytes were field stimulated at 1Hz at 37°C and cellular AP duration (APD) was optically recorded (fluorescent voltage-sensitive dye Di-8-Annepps). Results In vivo, regional ARIs tended to be longer in the BZs than remote. ARI heterogeneity, quantified as the standard deviation of ARIs in a neighborhood of 1cm radius, was increased in the BZ (anterior BZ: 3.4±1.0 ms, P=0.052, septal BZ: 3.6±1.7 ms, P=0.027 vs remote: 2.0±0.5 ms). Cellular APD was measured in large population samples (>100 cells per region in each pig) and was longer in BZ myocytes compared to the remote region. Cellular APD heterogeneity, measured as the standard deviation within cell population samples pooled by region per animal, was increased in the BZ (anterior BZ: 105.9±17.0 ms, P=0.0010; septal BZ: 98.1±20.8 ms, P=0.0127 vs remote: 73.9±8.6 ms). Cell APD correlated to in vivo ARI (R2=0.34, P=0.021) and cellular heterogeneity correlated strongly with in vivo heterogeneity (R2=0.67, P=0.002). Conclusion In the BZ of MI, in vivo regional heterogeneity adds a functional substrate for re-entry that may result from heterogeneous cellular remodeling and increased cell-cell APD variability. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): KU Leuven BOF-C1 “Blood pressure induced premature ventricular beats as triggers for ventricular arrhythmia in ischemic cardiomyopathy”

In silico assessment of the effects of quinidine, disopyramide and E-4031 on short QT syndrome variant 1 in the human ventricles.

AimsShort QT syndrome (SQTS) is an inherited disorder associated with abnormally abbreviated QT intervals and an increased incidence of atrial and ventricular arrhythmias. SQT1 variant (linked to the rapid delayed rectifier potassium channel current, IKr) of SQTS, results from an inactivation-attenuated, gain-of-function mutation (N588K) in the KCNH2-encoded potassium channels. Pro-arrhythmogenic effects of SQT1 have been well characterized, but less is known about the possible pharmacological antiarrhythmic treatment of SQT1. Therefore, this study aimed to assess the potential effects of E-4031, disopyramide and quinidine on SQT1 using a mathematical model of human ventricular electrophysiology.MethodsThe ten Tusscher et al. biophysically detailed model of the human ventricular action potential (AP) was modified to incorporate IKr Markov chain (MC) formulations based on experimental data of the kinetics of the N588K mutation of the KCNH2-encoded subunit of the IKr channels. The modified ventricular cell model was then integrated into one-dimensional (1D) strand, 2D regular and realistic tissues with transmural heterogeneities. The channel-blocking effect of the drugs on ion currents in healthy and SQT1 cells was modeled using half-maximal inhibitory concentration (IC50) and Hill coefficient (nH) values from literatures. Effects of drugs on cell AP duration (APD), effective refractory period (ERP) and pseudo-ECG traces were calculated. Effects of drugs on the ventricular temporal and spatial vulnerability to re-entrant excitation waves were measured. Re-entry was simulated in both 2D regular and realistic ventricular tissue.ResultsAt the single cell level, the drugs E-4031 and disopyramide had hardly noticeable effects on the ventricular cell APD at 90% repolarization (APD90), whereas quinidine caused a significant prolongation of APD90. Quinidine prolonged and decreased the maximal transmural AP heterogeneity (δV); this led to the decreased transmural heterogeneity of APD across the 1D strand. Quinidine caused QT prolongation and a decrease in the T-wave amplitude, and increased ERP and decreased temporal susceptibility of the tissue to the initiation of re-entry and increased the minimum substrate size necessary to prevent re-entry in the 2D regular model, and further terminated re-entrant waves in the 2D realistic model. Quinidine exhibited significantly better therapeutic effects on SQT1 than E-4031 and disopyramide.ConclusionsThe simulated pharmacological actions of quinidine exhibited antiarrhythmic effects on SQT1. This study substantiates a causal link between quinidine and QT interval prolongation in SQT1 and suggests that quinidine may be a potential pharmacological agent for treating SQT1 patients.

Effects of amiodarone on short QT syndrome variant 3 in human ventricles: a simulation study

BackgroundShort QT syndrome (SQTS) is a newly identified clinical disorder associated with atrial and/or ventricular arrhythmias and increased risk of sudden cardiac death (SCD). The SQTS variant 3 is linked to D172N mutation to the KCNJ2 gene that causes a gain-of-function to the inward rectifier potassium channel current (IK1), which shortens the ventricular action potential duration (APD) and effective refractory period (ERP). Pro-arrhythmogenic effects of SQTS have been characterized, but less is known about the possible pharmacological treatment of SQTS. Therefore, in this study, we used computational modeling to assess the effects of amiodarone, class III anti-arrhythmic agent, on human ventricular electrophysiology in SQT3.MethodsThe ten Tusscher et al. model for the human ventricular action potentials (APs) was modified to incorporate IK1 formulations based on experimental data of Kir2.1 channels (including WT, WT-D172N and D172N conditions). The modified cell model was then implemented to construct one-dimensional (1D) and 2D tissue models. The blocking effects of amiodarone on ionic currents were modeled using IC50 and Hill coefficient values from literatures. Effects of amiodarone on APD, ERP and pseudo-ECG traces were computed. Effects of the drug on the temporal and spatial vulnerability of ventricular tissue to genesis and maintenance of re-entry were measured, as well as on the dynamic behavior of re-entry.ResultsAmiodarone prolonged the ventricular cell APD and decreased the maximal voltage heterogeneity (δV) among three difference cells types across transmural ventricular wall, leading to a decreased transmural heterogeneity of APD along a 1D model of ventricular transmural strand. Amiodarone increased cellular ERP, prolonged QT interval and decreased the T-wave amplitude. It reduced tissue’s temporal susceptibility to the initiation of re-entry and increased the minimum substrate size necessary to sustain re-entry in the 2D tissue.ConclusionsAt the therapeutic-relevant concentration of amiodarone, the APD and ERP at the single cell level were increased significantly. The QT interval in pseudo-ECG was prolonged and the re-entry in tissue was prevented. This study provides further evidence that amiodarone may be a potential pharmacological agent for preventing arrhythmogenesis for SQT3 patients.

A cellular automaton model for electrocardiogram considering the structure of heart

The electrocardiogram (ECG) has broad applications in clinical diagnosis and prognosis of cardiovascular diseases. The accurate description for the question how the ECG come from the cardiac electrical activity is helpful for understanding the corresponding relation between the ECG waveform and cardiovascular disease. Experience is the primary method of studying the ECG, but the computer simulation method makes it more convenient to explore the effect of given factor for ECG waveform. Cellular automaton is a simple and effective computer simulation method. However, the cellular automaton model considering the main structure of the heart is not yet established. Therefore, we propose a cellular automaton model for the ECG considering the atria, the ventricle, and the ventricular septum. With this model, the conduction of the myocardial electrical activation is simulated by following the field potentials under healthy and diseased conditions, and the underlying mechanisms are analyzed. Through the computer simulations and analyses the results are obtained as follows. First, the conduction process of the electrical signal in this model is the same as that in the real heart. Second, under the healthy conditions, the behavior of the field potential appears as normal ECG, in which the P wave and the QRS wave group come from the depolarization of the atria and ventricle, respectively, on the other hand, the T wave and J wave come from the repolarization of the ventricle. The computer results support the conclusion that the J wave appears just because the existence of the notch in the epicardial transmembrane potential curve. Third, the endocardium ischemia conditions result in the T wave inversion. The mechanism is that the action potential duration of the ischemic endocardial cells is shorter than that under normal conditions, which makes larger the transmembrane potential gradient between the endocardium and the subepicardium, and then contributes a more negative value to the field potential. Fourth, the epicardium ischemia leads to the higher T wave, and this is because the shorter action potential duration of the ischemic epicardial cells brings in a larger transmembrane potential gradient between the epicardium and subepicardium, which makes the field voltage larger. Fifth, the T wave appears earlier under the through-wall ischemia. The action potential durations of cells of the endocardium, the epicardium, and the subepicardium all become shorter under the through-wall ischemia, then the repolarization processes of all of these three walls are ended earlier, which leads to the earlier T wave. The cellular automaton model proposed in this paper provides a reference for the further study of ECG.

Spatial Profiles of Electrical Mismatch Determine Vulnerability to Conduction Failure Across a Host–Donor Cell Interface

Electrophysiological mismatch between host cardiomyocytes and donor cells can directly affect the electrical safety of cardiac cell therapies; however, the ability to study host-donor interactions at the microscopic scale in situ is severely limited. We systematically explored how action potential (AP) differences between cardiomyocytes and other excitable cells modulate vulnerability to conduction failure in vitro. AP propagation was optically mapped at 75 μm resolution in micropatterned strands (n=152) in which host neonatal rat ventricular myocytes (AP duration=153.2±2.3 ms, conduction velocity=22.3±0.3 cm/s) seamlessly interfaced with genetically engineered excitable donor cells expressing inward rectifier potassium (Kir2.1) and cardiac sodium (Na(v)1.5) channels with either weak (conduction velocity=3.1±0.1 cm/s) or strong (conduction velocity=22.1±0.4 cm/s) electrical coupling. Selective prolongation of engineered donor cell AP duration (31.9-139.1 ms) by low-dose BaCl2 generated a wide range of host-donor repolarization time (RT) profiles with maximum gradients (∇RT(max)) of 5.5 to 257 ms/mm. During programmed stimulation of donor cells, the vulnerable time window for conduction block across the host-donor interface most strongly correlated with ∇RT(max). Compared with well-coupled donor cells, the interface composed of poorly coupled cells significantly shortened the RT profile width by 19.7% and increased ∇RT(max) and vulnerable time window by 22.2% and 19%, respectively. Flattening the RT profile by perfusion of 50 μmol/L BaCl2 eliminated coupling-induced differences in vulnerability to block. Our results quantify how the degree of electrical mismatch across a cardiomyocyte-donor cell interface affects vulnerability to conduction block, with important implications for the design of safe cardiac cell and gene therapies.

Selective targeting of gain-of-function KCNQ1 mutations predisposing to atrial fibrillation.

Atrial fibrillation is the most common sustained cardiac arrhythmia in adults. We hypothesized that gain-of-function KCNQ1 mutations previously associated with familial atrial fibrillation have distinct pharmacological properties that may enable targeted inhibition. Wild-type (WT) KCNQ1 or the familial atrial fibrillation mutation KCNQ1-S140G was heterologously coexpressed with KCNE1 to enable electrophysiological recording of the slow delayed rectifier current (IKs) and investigation of pharmacological effects of the IKs selective blocker HMR-1556. Coexpression of KCNQ1-S140G with KCNE1 generated potassium currents (S140G-IKs) that exhibited greater sensitivity to HMR-1556 than WT-IKs. Enhanced HMR-1556 sensitivity was also observed for another gain-of-function atrial fibrillation mutation, KCNQ1-V141M. Heteromeric expression of KCNE1 with both KCNQ1-WT and KCNQ1-S140G generated currents (HET-IKs) with gain-of-function features, including larger amplitude, a constitutively active component, hyperpolarized voltage dependence of activation, and extremely slow deactivation. A low concentration of HMR-1556, which had little effect on WT-IKs but was capable of inhibiting the mutant channel, reduced both instantaneous and steady state HET-IKs to levels that were not significantly different from WT-IKs and attenuated use-dependent accumulation of the current. In cultured adult rabbit left atrial myocytes, expression of S140G-IKs shortened action potential duration compared with WT-IKs. Application of HMR-1556 mitigated S140G-IKs-induced action potential duration shortening and did not alter action potential duration in cells expressing WT-IKs. The enhanced sensitivity of KCNQ1 gain-of-function mutations for HMR-1556 suggests the possibility of selective therapeutic targeting, and, therefore, our data illustrate a potential proof of principle for genotype-specific treatment of this heritable arrhythmia.

Increased Vulnerability of Human Ventricle to Re-entrant Excitation in hERG-linked Variant 1 Short QT Syndrome

The short QT syndrome (SQTS) is a genetically heterogeneous condition characterized by abbreviated QT intervals and an increased susceptibility to arrhythmia and sudden death. This simulation study identifies arrhythmogenic mechanisms in the rapid-delayed rectifier K+ current (IKr)-linked SQT1 variant of the SQTS. Markov chain (MC) models were found to be superior to Hodgkin-Huxley (HH) models in reproducing experimental data regarding effects of the N588K mutation on KCNH2-encoded hERG. These ionic channel models were then incorporated into human ventricular action potential (AP) models and into 1D and 2D idealised and realistic transmural ventricular tissue simulations and into a 3D anatomical model. In single cell models, the N588K mutation abbreviated ventricular cell AP duration at 90% repolarization (APD90) and decreased the maximal transmural voltage heterogeneity (δV) during APs. This resulted in decreased transmural heterogeneity of APD90 and of the effective refractory period (ERP): effects that are anticipated to be anti-arrhythmic rather than pro-arrhythmic. However, with consideration of transmural heterogeneity of IKr density in the intact tissue model based on the ten Tusscher-Noble-Noble-Panfilov ventricular model, not only did the N588K mutation lead to QT-shortening and increases in T-wave amplitude, but δV was found to be augmented in some local regions of ventricle tissue, resulting in increased tissue vulnerability for uni-directional conduction block and predisposing to formation of re-entrant excitation waves. In 2D and 3D tissue models, the N588K mutation facilitated and maintained re-entrant excitation waves due to the reduced substrate size necessary for sustaining re-entry. Thus, in SQT1 the N588K-hERG mutation facilitates initiation and maintenance of ventricular re-entry, increasing the lifespan of re-entrant spiral waves and the stability of scroll waves in 3D tissue.

Distinct regulation of cardiac If current via thyroid receptors alpha1 and beta1

Thyroid hormone (TH) markedly modulates cardiovascular function and heart rate. The pacemaker current I(f) and encoding hyperpolarization-activated cation (HCN) genes have been identified as TH targets. To analyze the specific contribution and functional significance of thyroid receptor isoforms responsible for HCN gene transactivation, we generated transgenic neonatal rat cardiomyocytes with adenovirus-mediated overexpression of the thyroid receptors alpha1 (TR alpha 1) and beta1 (TR beta 1), and analyzed native I(f) current and expression levels of the underlying molecular components HCN2 and HCN4. Initial results revealed that spontaneous beating activity was higher in TR alpha 1- and lower in TR beta 1-expressing cardiomyocytes. This was associated with accelerated depolarization velocity and abbreviated action potential duration in cells overexpressing TR alpha 1, while TR beta 1 suppressed phase 4 depolarization and prolonged action potentials. Consistently, TR alpha 1-infected myocytes exhibited larger I(f) current densities along with increased HCN2 and HCN4 mRNA and protein levels. In contrast, HCN2 gene expression was not significantly affected by TR beta 1. TR beta 1 exclusively suppressed HCN4 transcription. T3 application led to significant effects only in controls and TR alpha 1-infected cardiomyocytes; whereas, no ligand-dependent actions were observed in TR beta 1-expressing neonatal cardiomyocytes. Our results demonstrate that TR alpha 1 and TR beta 1 divergently regulate cardiac pacing activity. TH-induced positive chronotropic effects are likely to be mediated by TR alpha 1 through enhanced expression of I(f) pacemaker current and its underlying genes.

Antibodies with beta-adrenergic activity from chronic chagasic patients modulate the QT interval and M cell action potential duration

The aim of this study was to investigate whether the sera from chronic chagasic patients (CChPs) with beta-1 adrenergic activity (Ab-beta) can modulate ventricular repolarization. Beta-adrenergic activity has been described in CChP. It increases the L-type calcium current and heart rate in isolated hearts, but its effects on ventricular repolarization has not been described. In isolated rabbit hearts, under pacing condition, QT interval was measured under Ab-beta perfusion. Beta-adrenergic activity was also tested in guinea pig ventricular M cells. Furthermore, the immunoglobulin fraction (IgG-beta) of the Ab-beta was tested on Ito, ICa, and Iks currents in rat, rabbit, and guinea pig myocytes, respectively. Beta-adrenergic activity shortened the QT interval. This effect was abolished in the presence of propranolol. In addition, sera from CChP without beta-adrenergic activity (Ab-beta) did not modulate QT interval. The M cell action potential duration (APD) was reversibly shortened by Ab-beta. Atenolol inhibited this effect of Ab-beta, and Ab- did not modulate the AP of M cells. Ito was not modulated by isoproterenol nor by IgG-beta. However, IgG-beta increased ICa and IKs. The shortening of the QT interval and APD in M cells and the increase of IKs and ICa induced by IgG-beta contribute to repolarization changes that may trigger malignant ventricular arrhythmias observed in patients with chronic chagasic or idiopathic cardiomyopathy.

Hemin Enhances Cardiac Performance in Response to Pressure Overload (PO): Implications for Treatment of Heart Failure

Myocardial hypertrophy induced by PO leads to heart failure. We reported previously that hemin reduces hypertrophy and enhances cardiac performance in response to PO in rat. Here, we investigated the mechanism by which hemin enhances cardiac function in response to PO. Animals were treated two days prior- and every three days postoperatively after banding of the ascending aorta for 10 weeks. Morphometric analysis revealed that hemin prevented PO-induced hypertrophy. Pressure-volume analysis revealed enhanced cardiac performance in banded and sham animals treated with hemin. To investigate the positive inotropic effect of hemin, single cell action potential duration, fractional shortening, and calcium transients were assessed. Sham animals treated with hemin displayed increased calcium transients under normal and β-adrenergic stimulation suggesting that the positive inotropic effect of hemin is mediated via alterations in calcium release. Because hemin stimulates CO production via induction of HO-1, we tested whether the anti-hypertrophic action of hemin is mediated via cGMP, by inhibiting its production with ODQ. Treatment with ODQ prevented the anti-hypertrophic effect of hemin but not the positive inotropic effect, suggesting that the anti-hypertrophic and inotropic effects of hemin are mediated by independent signaling mechnisms. Supported by grants from CIHR and HSFO to LG Melo and CA Ward.

I Ks blockade reduces dispersion of repolarization in heart failure

The slow and rapid (I(Kr)) components of I(K) are major determinants of ventricular repolarization. Unlike I(Kr), which is homogeneously expressed across the transmural wall, I(Ks) expression is reduced in midmyocardial cells and presumably contributes significantly to transmural dispersion of repolarization. Increased dispersion of repolarization during pharmacologic blockade of I(Kr) is proarrhythmic, primarily due to relatively selective prolongation of midmyocardial cell action potential duration (APD). The mechanisms underlying proarrhythmia in heart disease associated with impaired repolarization, such as heart failure, are unknown. We hypothesize that, in contrast to I(Kr) blockade, I(Ks) blockade will have little effect on midmyocardial cells and hence decrease dispersion of repolarization in heart failure. The purpose of this study was to determine the effect of blockade of the slow component of the delayed rectifier current (I(Ks)) on arrhythmogenic dispersion of repolarization and proarrhythmia in heart failure. Optical action potentials were simultaneously recorded from 256 sites spanning the transmural wall of the arterially perfused canine wedge preparation. Hearts from dogs with heart failure induced by rapid pacing (n = 6) were compared with normals (n = 6). Baseline dispersion of repolarization, as measured from the range of transmural APD during stimulation at a cycle length of 2,000 ms, was significantly higher in heart failure (75 +/- 24 ms) compared with controls (39 +/- 21 ms, P < .04). I(Ks) blockade with 30 microM chromanol decreased dispersion of repolarization by 40% (P < .02) in heart failure, reducing it to values found in normals. Decreased dispersion of repolarization was due to a larger, relatively selective, drug-induced APD prolongation of epicardial (23%) compared with midmyocardial cells (9%, P < .02). VT could not be induced in failing hearts under conditions of I(Ks) blockade, and no proarrhythmia was observed. I(Ks) blockade significantly reduced heart failure-induced dispersion of repolarization to values seen in nonfailing hearts. By prolonging repolarization without increasing dispersion of repolarization, I(Ks) blockade may have antiarrhythmic effects without creating proarrhythmia.

Role of BK Potassium Channels Shaping Action Potentials and the Associated [Ca2+]i Oscillations in GH3 Rat Anterior Pituitary Cells

Measurements of electrical activity and intracellular Ca²⁺ levels were performed in perforated-patch clamped GH₃ cells to determine the contribution of large-conductance calcium-activated K⁺ (BK) channels to action potential repolarization and size of the associated Ca²⁺ oscillations. By examining the dependence of action potential (AP) duration on extracellular Ca²⁺ levels in the presence and the absence of the specific BK channel blocker paxilline, it is observed that plateau-like action potentials are associated to low densities of paxilline-sensitive currents. Extracellular Ca²⁺ increases or paxilline additions are not able to largely modify action potential duration in cells showing a reduced expression of BK currents. Furthermore, specific blockade of these currents with paxilline systematically elongates AP duration, but only under conditions in which short APs and/or prominent BK currents recorded under voltage-clamp mode are present in the same cells. Our data indicate that in GH₃ cells, BK channels act primarily ending the action potential and suggest that by contributing to fine-tuning cellular electrical properties and hence intracellular Ca²⁺ variations, BK channels may play an important role on time- and cell-dependent modulation of physiological outputs in adenohypophyseal cells.

MEMBRANE AND TISSUE LEVEL CONTRIBUTIONS TO PURKINJE-VENTRICULAR INTERACTIONS: A MODEL STUDY

Purkinje-to-ventricular (P-to-V) propagation and electrotonic modulation of repolarization at discrete Purkinje-ventricular junctions (PVJs) depend on differences in the ionic currents and tissue structure of the P network and the V myocardium. We used computer simulations to assess these membrane and tissue level contributions to P-V interactions. At the membrane level, we used the DiFrancesco-Noble membrane equations to model P ionic kinetics and the Luo-Rudy dynamic membrane equations to model V ionic kinetics. At the tissue level, we modeled the P network as a layer of branching cables, and we modeled a single myocardial sheet with an anisotropic layer of excitable cells. P-to-V propagation was enhanced at the tissue level when multiple wavefronts in the branching P network collided at the PVJ. At the membrane level, P-to-V propagation was enhanced by a reduced transient outward current (Ito) in the P layer. Repolarization at the PVJ was also modulated by both membrane and tissue level contributions. Under nominal conditions, action potential duration (APD) shortened in the P layer and prolonged in the V layer. However, when the V mass was reduced, both P and V cell APDs shortened during coupling with nominal Ito. Subsequent Ito inhibition restored coupling-induced prolongation of the V action potential in the reduced V mass. These results suggest that under physiologic conditions, both membrane and tissue level contributions to P-V interactions are important, while membrane level contributions become even more important under pathologies that reduce the difference in P and V tissue size, particularly in the setting of healed myocardial infarction.

Amiodarone reduces transmural heterogeneity of repolarization in the human heart

Objectives. The present work was designed to test the effects of amiodarone therapy on action potential characteristics of the three cell types observed in human left ventricular preparations.Background. The electrophysiologic basis for amiodarone’s exceptional antiarrhythmic efficacy and low proarrhythmic profile remains unclear.Methods. We used standard microelectrode techniques to investigate the effects of chronic amiodarone therapy on transmembrane activity of the three predominant cellular subtypes (epicardial, midmyocardial [M] and endocardial cells) spanning the human left ventricle in hearts explanted from normal, heart failure and amiodarone-treated heart failure patients.Results. Tissues isolated from the ventricles of heart failure patients receiving chronic amiodarone therapy displayed M cell action potential duration (404 ± 12 ms) significantly briefer (p < 0.05) than that recorded in tissues isolated from normal hearts (439 ± 22 ms) or from heart failure patients not treated with amiodarone (449 ± 18 ms). Endocardial cells from amiodarone-treated heart failure patients displayed longer (p < 0.05) action potential duration (363 ± 10 ms) than endocardial cells isolated from normal hearts (330 ± 6 ms). As a consequence, the heterogeneity of ventricular repolarization in tissues from patients treated with amiodarone was considerably smaller than in the two other groups, especially at long pacing cycle lengths.Conclusions. These findings may explain, at least in part, the reduction of ventricular repolarization dispersion and the lower incidence of torsade de pointes observed with chronic amiodarone therapy as compared with other class III agents.

Evolution and resolution of long-term cardiac memory.

Cardiac memory (CM) refers to T-wave changes induced by ventricular pacing or arrhythmia that accumulate in magnitude and duration with repeated episodes of abnormal activation. We report herein the kinetics of long-term CM and its association with the ventricular action potential. Dogs were paced from the ventricles at rates of 110 to 120 bpm for approximately 3 weeks. CM characterized by gradual sinus rhythm T vector rotation toward the paced QRS vector evolved in all dogs regardless of pacing site (left ventricular [LV] anterior apex or base, posterior LV, or right ventricular free wall). Cardiac hemodynamics and myocardial flow (microsphere studies) were unaltered by the pacing. Recovery time for the memory T wave to return to control increased with duration of the previous pacing. The protein synthesis inhibitor cycloheximide markedly (P<.05) and reproducibly attenuated evolution of CM. When pacing was performed from the atrium, CM did not occur. Standard microelectrode techniques were used to study action potential from the LV free wall of control and CM dogs. CM was associated with increased action potential duration in epicardial and endocardial but not midmyocardial cells, significantly altering the transmyocardial gradient for repolarization. CM is a dynamic process for which the final T vector is predicted by the paced QRS vector and which is associated with significant changes in epicardial and endocardial but not midmyocardial cell action potential duration, such that the transmural gradient of repolarization is altered. It is unaccompanied by evidence of altered hemodynamics or flow, requires a change in pathway of activation, and appears to require new protein synthesis.

Effects of Sodium Channel Block with Mexiletine to Reverse Action Potential Prolongation in In Vitro Models of the Long QT Syndrome

Recent clinical studies have reported a greater effectiveness of sodium channel block with mexiletine to abbreviate the QT interval in patients with the chromosome 3 variant (SCN5A, LQT3) of the long QT syndrome (LQTS) than those with the chromosome 7 form of the disease (HERG, LQT2), suggesting the possibility of gene-specific therapy for the two distinct forms of the congenital LQTS. Experimental studies using the arterially perfused left ventricular wedge preparation have confirmed these clinical observations on the QT interval but have gone on to further demonstrate a potent effect of mexiletine to reduce dispersion of repolarization and prevent torsades de pointes (TdP) in both LQT2 and LQT3 models. A differential action of sodium channel block on the three ventricular cell types is thought to mediate these actions of mexiletine. This study provides a test of this hypothesis by examining the effects of mexiletine in isolated canine ventricular epicardial, endocardial, and M region tissues under conditions that mimic the SCN5A and HERG gene defects. We used standard microelectrode techniques to record transmembrane activity from endocardial, epicardial, mid-myocardial, and transmural strips isolated from the canine left ventricle. d-Sotalol, an IKr blocker, was used to mimic the HERG defect (LQT2), and ATX-II, which increases late Na channel current, was used to mimic the SCN5A defect (LQT3). d-Sotalol (100 microM) preferentially prolonged the action potential of the mid-myocardial M cell (APD90 increased from 340 +/- 65 to 623 +/- 203 msec) as did ATX-II (10 to 20 nM; APD90 increased from 325 +/- 51 to 580 +/- 178 msec; basic cycle length = 2000 msec), thus causing a marked increase in transmural dispersion of repolarization (TDR). Mexiletine (2 to 20 microM) dose-dependently reversed the ATX-II-induced prolongation of APD90 in all three cell types. Mexiletine also reversed the d-sotalol-induced prolongation of the M cell action potential duration (APD), but had little effect on the action potential of epicardium and endocardium. Due to its preferential effect to abbreviate the action potential of M cells, mexiletine reduced the dispersion of repolarization in both models. Low concentrations of mexiletine (5 to 10 microM) totally suppressed early afterdepolarization (EAD) and EAD-induced triggered activity in both models. Our results indicate that the actions of mexiletine are both cell and model specific, but that sodium channel block with mexiletine is effective in reducing transmural differences in APD and in abolishing triggered activity induced by d-sotalol and ATX-II. The data suggest that mexiletine's actions to reduce TDR and prevent the induction of spontaneous and programmed stimulation-induced TdP in these models are due to a preferential effect of the drug to abbreviate the APD of the M cell and to suppress the development of EADs. The data provide further support for the hypothesis that block of the late sodium current may be of value in the treatment of LQT2 as well as LQT3 and perhaps other congenital and acquired (drug-induced) forms of LQTS.

Sodium channel block with mexiletine is effective in reducing dispersion of repolarization and preventing torsade des pointes in LQT2 and LQT3 models of the long-QT syndrome.

This study examines the contribution of transmural heterogeneity of transmembrane activity to phenotypic T-wave patterns and the effects of pacing and of sodium channel block under conditions mimicking HERG and SCN5A defects linked to the congenital long-QT syndrome (LQTS). A transmural ECG and transmembrane action potentials from epicardial, M, and endocardial or Purkinje cells were simultaneously recorded in an arterially perfused wedge of canine left ventricle. d-Sotalol was used to mimic LQT2, whereas ATX-II mimicked LQT3. d-Sotalol caused a preferential prolongation of the M cell action potential duration (APD90, 291+/-14 to 354+/-35 ms), giving rise to broad and sometimes low-amplitude bifurcated T waves and an increased transmural dispersion of repolarization (TDR, 51+/-15 to 72+/-17 ms). QT interval increased from 320+/-13 to 385+/-37 ms. ATX-II produced a preferential prolongation of the M cell APD90 (280+/-25 to 609+/-49 ms) and caused a marked delay in the onset of the T wave and a sharp rise in TDR (40+/-5 to 168+/-40 ms). QT-, APD90-, and dispersion-rate relations were much steeper in the ATX-II than in the d-sotalol model. Mexiletine (2 to 20 micromol/L) dose-dependently abbreviated the QT interval and APD90 of all cell types, more in the ATX-II than in the d-sotalol model, but decreased TDR equally in the two models. Mexiletine 2 to 5 micromol/L totally suppressed spontaneous torsade de pointes (TdP) and reduced the vulnerable window during which single extrastimuli could induce TdP in both models. Higher concentrations of mexiletine (10 to 20 micromol/L) totally suppressed stimulation-induced TdP. Our results suggest that although pacing and sodium channel block are very effective in abbreviating the QT interval and TDR in LQT3, these therapeutic approaches may also be valuable in reducing the incidence of arrhythmogenesis in LQT2.

Ionic mechanism responsible for prolongation of cardiac action-potential duration by berberine.

This study was designed to investigate the effects of berberine on membrane currents forming the repolarization phase of action potentials in isolated guinea pig ventricular myocytes by using the patch-clamp technique. Application of berberine (3-30 microM) to the current-clamped myocytes produced a significant prolongation of action-potential duration (APD), which was concentration dependent. However, this agent (3-30 microM) did not affect the resting potential and action-potential amplitude. The prolongation of APD caused by berberine was not attenuated by tetrodotoxin (TTX, 10 microM), and TTX (10 microM) failed to shorten APD in cells pretreated with 30 microM berberine. Under the voltage-clamp conditions, berberine (3-30 microM) inhibited the delayed rectifier K+ currents (I(K)). Under conditions in which the rapidly activating components (I(Kr)) and slowly activating component (I(Ks)) were dissected out, berberine was shown to block I(Ks) without affecting I(Kr). Application of berberine (3-30 microM) increased the Na+-Ca2+ exchange currents, which were completely abolished by 5 mM NiCl. The L-type Ca2+ currents (I(Ca)) also were increased by 3-30 microM berberine, but the threshold potential, the potential at which I(Ca) was maximal, and the apparent reversal potential remained unchanged. Berberine at either 3 or 30 microM did not affect the inward rectifier K+ currents. This study suggests that the prolongation of cardiac repolarization by berberine is mainly caused by the inhibition of I(Ks) and increase of I(Ca).

Cytosolic magnesium modulates calcium channel activity in mammalian ventricular cells

The effect of cytosolic free Mg2+ concentration on the regulation of myocardial function was studied by dialyzing isolated guinea pig ventricular myocytes with different internal Mg2+ concentrations [( Mg2+]i). We found that elevation of [Mg2+]i shortened the action potential and suppressed the Ca2+ current. Mean values recorded for action potential duration in cells dialyzed with solutions containing 0, 1.3, and 9.4 mM Mg2+ were 620 +/- 40, 400 +/- 25, and 60 +/- 10, respectively. The suppressive effect of [Mg2+]i on the action potential duration correlated significantly with the suppressive effects of [Mg2+]i on the Ca2+ current. In cells dialyzed with nominally zero Mg2+, calcium current was prominent (3.5 +/- 0.58 nA). At [Mg2+]i of 1.4 mM, calcium current was significantly smaller than in zero [Mg2+]i and was almost completely inhibited by dialysis of the cell with 9.4 mM Mg2+. The Mg2+-induced block of the Ca2+ current was due to steady-state inactivation of the high threshold calcium channel. The block was observed in the presence or absence of adenosine 3',5'-cylic monophosphate and was not reversed by elevation of external Ca2+ concentration, addition of adrenaline, or large negative potentials. These data suggest that cytosolic Mg2+ regulates Ca2+ channel activity by a novel mechanism, unrelated to its effect as a blocking particle of the open channel.