Why Is Only Type 1 Electrocardiogram Diagnostic of Brugada Syndrome? Mechanistic Insights From Computer Modeling

Background: Three types of characteristic ST-segment elevation are associated with Brugada syndrome but only type 1 is diagnostic. Why only type 1 ECG is diagnostic remains unanswered. Methods: Computer simulations were performed in single cells, 1-dimensional cables, and 2-dimensional tissues to investigate the effects of the peak and late components of the transient outward potassium current (I to ), sodium current, and L-type calcium current (I Ca,L ) as well as other potassium currents on the genesis of ECG morphologies and phase 2 reentry (P2R). Results: Although a sufficiently large peak I to was required to result in the type 1 ECG pattern and P2R, increasing the late component of I to converted type 1 ECG to type 2 ECG and suppressed P2R. Increasing the peak I to promoted spiral wave breakup, potentiating the transition from tachycardia to fibrillation, but increasing the late I to prevented spiral wave breakup by flattening the action potential duration restitution and preventing P2R. A sufficiently large I Ca,L conductance was needed for P2R to occur, but once above the critical conductance, blocking I Ca,L promoted P2R. However, selectively blocking the window and late components of I Ca,L suppressed P2R, countering the effect of the late I to . Blocking either the peak or late components of sodium current promoted P2R, with the late sodium current blockade having the larger effect. As expected, increasing other potassium currents potentiated P2R, with ATP-sensitive potassium current exhibiting a larger effect than rapid and slow component of the delayed rectifier potassium current. Conclusions: The peak I to promotes type 1 ECG and P2R, whereas the late I to converts type 1 ECG to type 2 ECG and suppresses P2R. Blocking the peak I Ca,L and either the peak or the late sodium current promotes P2R, whereas blocking the window and late I Ca,L suppresses P2R. These results provide important insights into the mechanisms of arrhythmogenesis and potential therapeutic targets for treatment of Brugada syndrome.

Three-Week-Old Rabbit Ventricular Cardiomyocytes as a Novel System to Study Cardiac Excitation and EC Coupling

Cardiac arrhythmias significantly contribute to cardiovascular morbidity and mortality. The rabbit heart serves as an accepted model system for studying cardiac cell excitation and arrhythmogenicity. Accordingly, primary cultures of adult rabbit ventricular cardiomyocytes serve as a preferable model to study molecular mechanisms of human cardiac excitation. However, the use of adult rabbit cardiomyocytes is often regarded as excessively costly. Therefore, we developed and characterized a novel low-cost rabbit cardiomyocyte model, namely, 3-week-old ventricular cardiomyocytes (3wRbCMs). Ventricular myocytes were isolated from whole ventricles of 3-week-old New Zealand White rabbits of both sexes by standard enzymatic techniques. Using wheat germ agglutinin, we found a clear T-tubule structure in acutely isolated 3wRbCMs. Cells were adenovirally infected (multiplicity of infection of 10) to express Green Fluorescent Protein (GFP) and cultured for 48 h. The cells showed action potential duration (APD90 = 253 ± 24 ms) and calcium transients similar to adult rabbit cardiomyocytes. Freshly isolated and 48-h-old-cultured cells expressed critical ion channel proteins: calcium voltage-gated channel subunit alpha1 C (Cavα1c), sodium voltage-gated channel alpha subunit 5 (Nav1.5), potassium voltage-gated channel subfamily D member 3 (Kv4.3), and subfamily A member 4 (Kv1.4), and also subfamily H member 2 (RERG. Kv11.1), KvLQT1 (K7.1) protein and inward-rectifier potassium channel (Kir2.1). The cells displayed an appropriate electrophysiological phenotype, including fast sodium current (INa), transient outward potassium current (Ito), L-type calcium channel peak current (ICa,L), rapid and slow components of the delayed rectifier potassium current (IKr and IKs), and inward rectifier (IK1). Although expression of the channel proteins and some currents decreased during the 48 h of culturing, we conclude that 3wRbCMs are a new, low-cost alternative to the adult-rabbit-cardiomyocytes system, which allows the investigation of molecular mechanisms of cardiac excitation on morphological, biochemical, genetic, physiological, and biophysical levels.

High-Throughput Drug Screening System Based on Human Induced Pluripotent Stem Cell-Derived Atrial Myocytes ∼ A Novel Platform to Detect Cardiac Toxicity for Atrial Arrhythmias

Evaluation of proarrhythmic properties is critical for drug discovery. In particular, QT prolongation in electrocardiograms has been utilized as a surrogate marker in many evaluation systems to assess the risk of torsade de pointes and lethal ventricular arrhythmia. Recently, new evaluation systems based on human iPS cell-derived cardiomyocytes have been established. On the other hand, in clinical situations, it has been reported that the incidence of atrial arrhythmias such as atrial fibrillation has been increasing every year, with the prediction of a persistent increase in the near future. As to the increased incidence of atrial arrhythmias, in addition to the increased population of geriatric patients, a wide variety of drug treatments may be related, as an experimental method to detect drug-induced atrial arrhythmia has not been established so far. In the present study, we characterized the atrial-like cardiomyocytes derived from human induced pluripotent stem cells and examined their potential for the evaluation of drug-induced atrial arrhythmia. Atrial-like cardiomyocytes were induced by adding retinoic acid (RA) during the process of myocardial differentiation, and their characteristics were compared to those of RA-free cardiomyocytes. Using gene expression and membrane potential analysis, it was confirmed that the cells with or without RA treatment have atrial or ventricular like cardiomyocytes, respectively. Using the ultra-rapid activating delayed rectifier potassium current (IKur) channel inhibitor, which is specific to atrial cardiomyocytes, Pulse width duration (PWD) 30cF prolongation was confirmed only in atrial-like cardiomyocytes. In addition, ventricular like cardiomyocytes exhibited an early after depolarization by treatment with rapidly activating delayed rectifier potassium current (IKr) channel inhibitor, which induces ventricular arrhythmia in clinical situations. Here, we have established a high-throughput drug evaluation system using human iPS cell-derived atrial-like cardiomyocytes. Based on the obtained data, the system might be a valuable platform to detect potential risks for drug-induced atrial arrhythmias.

Investigating the Cocaine-induced Reduction of Potassium Current on the Generation of Action Potentials Using a Computational Model

Introduction: Drugs of abuse, including cocaine, affect different brain regions and lead to pathological memories. These abnormal memories may occur due to the changes in synaptic transmissions or variations in synaptic properties of neurons. It has been shown that cocaine inhibits delayed rectifying potassium currents in affected regions of the brain and can have a role in the formation of pathological memories. Purpose: This study investigates how the change in the conductance of delayed rectifying potassium channels can affect the produced action potentials using a computational model. Methods: We present a computational model with different channels and receptors, including sodium, potassium, calcium, NMDARs, and AMPARs, which can produce burst-type action potentials. In the simulations, by changing the delayed rectifying potassium conductance bifurcation diagram is calculated. Conclusion: Results show that for a specific range of potassium conductance, a chaotic regime emerges in produced action potentials. These chaotic oscillations may play a role in inducing abnormal memories.

Aminophylline affects the cardiac rat inward rectifier potassium current in a dual way

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Ministry of Education, Youth and Sports of the Czech Republic Introduction Aminophylline, a bronchodilator used in clinical practice to treat namely severe astma attacks, often induces atrial fibrillation in patients. Modifications of the inward rectifier potassium current IK1 are known to play a role in the genesis of fibrillation. Purpose We aimed to investigate the effect of aminophylline at clinically-relevant concentrations between 3 and 100 µM on IK1 in isolated rat ventricular myocytes. Methods Experiments were performed by the whole cell patch clamp technique on enzymatically isolated rat right ventricular myocytes at room temperature. IK1 was measured as the current sensitive to 100 µM Ba2+. Results We observed a dual steady-state effect of aminophylline at most of the applied concentrations. Either inhibition or activation was apparent in individual cells during application of aminophylline at a given concentration. The smaller was magnitude of the control IK1, the more likely was activation of the current in the presence of aminophylline and vice versa (tested at 10 and 30 µM). The effect was voltage-independent and fully reversible during the subsequent wash-out. The mean aminophylline effect was inhibitory at all concentrations (10, 15, 4, and 23%-inhibition at -50 mV at 3, 10, 30, and 100 µM, respectively). Using a modified version of the population model of IK1 channels that we published before, the dual effect can be explained by interaction of aminophylline with two channel populations in a different way, the first one being inhibited and the second one being activated by the drug. Considering various fractions of these two channel populations in individual cells, varying effects observed in the measured cells can be simulated. Conclusions Aminophylline at clinically-relevant concentrations affects IK1 in rat ventricular myocytes in a dual way, showing both the steady-state activation and inhibition in various cells, even at the same concentration. It may be related to a different effect of the drug on various Kir2.x subunits forming the heterotetrameric IK1 channels present at the cell membrane of a single cell.

Calcium/calmodulin-dependent protein kinase II stimulates the inward rectifier potassium current in beta-adrenergic adaptation of ventricular cardiomyocytes

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): NEW NATIONAL EXCELLENCE PROGRAM OF THE MINISTRY FOR INNOVATION AND TECHNOLOGY FROM THE SOURCE OF THE NATIONAL RESEARCH, DEVELOPMENT AND INNOVATION FUND. Introduction and purpose Acute β-adrenergic receptor (β-AR) stimulation shortens the ventricular action potential (AP). This effect is mainly regulated by the β- adrenergic stimulation of the cardiac potassium currents. Our aim was to investigate the extent of calcium/calmodulin-dependent protein kinase II (CaMKII) involvement in mediating the effect of β-AR activation on the inward rectifier potassium current – I K1 . Methods We carried out our experiments on isolated cardiomyocytes originating from canine left ventricles. The inward rectifier potassium current – I K1 was measured under a "canonical" AP under action potential voltage clamp conditions. Data were collected in four study groups [1] Control conditions (CTRL) [2] Inhibition of CaMKII with 1 µM KN-93 (KN-93) [3] Inhibition of PKA with 3 µM H-89 (H-89) [4] Acute β-adrenergic stimulation with 10 nM isoproterenol (ISO) [5] β-adrenergic stimulation with CaMKII inhibition (KN-93 + ISO) [6] β-adrenergic stimulation with PKA inhibition (H-89 + ISO) [7] β-adrenergic stimulation with inhibited PKA and CaMKII (KN-93 + H-89 + ISO) Results I K1 current amplitude did not differ among the studied groups, the total carried charge however was significantly, about 30 % larger in the ISO group compared to CTRL, and about 20 % larger compared to KN-93 + ISO. Under beta- adrenergic stimulation, I K1 starts to activate earlier during the AP plateau. I K1 density was about 3 times greater both at +20 mV and at 0 mV membrane potential under the command "canonical" AP in ISO compared to CTRL. Similarly, I K1 density was about 60 % and 90 % larger at +20 mV and at 0 mV, respectively, in KN-93 + ISO compared to KN-93. Similar results have been obtained by conventional voltage-clamp technique. Conclusion Based on the results of our researches the CaMKII activation plays an important role in β -adrenergic stimulation the I K1 potassium current.