The yield of a comprehensive investigation protocol for the diagnosis of true idiopathic ventricular fibrillation in a real-life clinical setting.

Traditionally, aborted cardiac arrest (ACA) due to documented ventricular fibrillation (VF) in the absence of structural heart disease has been termed idiopathic VF. By careful evaluation, a specific etiology can be found in a substantial proportion of patients. The aim of this survey was to assess the yield of an advanced diagnostic work-up to reveal a causative etiology in a real-life clinical setting. Patients from the University Hospital Brno's ACA database were analyzed (514 patients in total). Forty-six patients (31 males) fulfilled the inclusion criteria, which were: (1) absence of structural pathology on echocardiography; (2) absence of coronary artery disease; and (3) absence of reversible cause of ACA. The diagnostic work-up consisted in cardiac magnetic resonance imaging, stress testing, sodium channel blocker challenge, and genetic testing according to the availability of the method and patient compliance. A specific disease was found in 17 individuals (37.0%), although at least one diagnostic step was refused by 13 patients (28.3%). True idiopathic VF was confirmed in 7 patients (15.2%), for whom the entire diagnostic work-up did not reveal any specific pathology. Our real-life survey shows that, even with an incomplete diagnostic work-up (due to the unavailability of a particular method or variable patient compliance), a specific diagnosis can be identified in more than one third of the cases of "idiopathic" VF, which can thus enable targeted treatment and family screening.

Sildenafil affects the human Kir2.1 and Kir2.2 channels at clinically relevant concentrations: Inhibition potentiated by low Ba2.

Sildenafil (Viagra), the first approved and widely used oral drug for the treatment of erectile dysfunction, was occasionally associated with life-threatening ventricular arrhythmias in patients. Since inward rectifier potassium current (I K1) may considerably contribute to this arrhythmogenesis, we investigated the effect of sildenafil on the human Kir2.1 and Kir2.2, the prevailing subunits forming the ventricular I K1 channels. Experiments were performed by the whole-cell patch clamp technique at 37°C using Chinese hamster ovary cells transiently expressing the human Kir2.1 and Kir2.2 channels. Changes of both the inward and outward current components (at -110 and -50 mV, respectively) were tested to be able to consider the physiological relevance of the sildenafil effect (changes at -110 and -50 mV did not significantly differ, results at -50 mV are listed below). A significant Kir2.1 inhibition was observed at all applied sildenafil concentrations (16.1% ± 3.7%, 20.0% ± 2.6%, and 15.0% ± 3.0% at 0.1, 1, and 10 µM, respectively). The inhibitory effect of 0.1 µM sildenafil was potentiated by the presence of a low concentration of Ba2+ (0.1 µM) which induced only a slight Kir2.1 inhibition by 5.95% ± 0.75% alone (the combined effect was 35.5% ± 3.4%). The subtherapeutic and therapeutic sildenafil concentrations (0.1 and 1 µM) caused a dual effect on Kir2.2 channels whereas a significant Kir2.2 activation was observed at the supratherapeutic sildenafil concentration (10 µM: 34.1% ± 5.6%). All effects were fully reversible. This is the first study demonstrating that sildenafil at clinically relevant concentrations inhibits both the inward and outward current components of the main human ventricular I K1 subunit Kir2.1. This inhibitory effect was significantly potentiated by a low concentration of environmental contaminant Ba2+ in agreement with recently reported data on rat ventricular I K1 which additionally showed a significant repolarization delay. Considering the similar subunit composition of the human and rat ventricular I K1 channels, the observed effects might contribute to sildenafil-associated arrhythmogenesis in clinical practice.

A new approach to the determination of tubular membrane capacitance: passive membrane electrical properties under reduced electrical conductivity of the extracellular solution.

The transverse-axial tubular system (tubular system) of cardiomyocytes plays a key role in excitation-contraction coupling. To determine the area of the tubular membrane in relation to the area of the surface membrane, indirect measurements through the determination of membrane capacitances are currently used in addition to microscopic methods. Unlike existing electrophysiological methods based on an irreversible procedure (osmotic shock), the proposed new approach uses a reversible short-term intermittent increase in the electrical resistance of the extracellular medium. The resulting increase in the lumen resistance of the tubular system makes it possible to determine separate capacitances of the tubular and surface membranes. Based on the analysis of the time course of the capacitive current, computational relations were derived to quantify the elements of the electrical equivalent circuit of the measured cardiomyocyte including both capacitances. The exposition to isotonic low-conductivity sucrose solution is reversible which is the main advantage of the proposed approach allowing repetitive measurements on the same cell under control and sucrose solutions. Experiments on rat ventricular cardiomyocytes (n = 20) resulted in the surface and tubular capacitance values implying the fraction of tubular capacitance/area of 0.327 ± 0.018. We conclude that the newly proposed method provides results comparable to the data obtained by the currently used detubulation method and, in addition, by being reversible, allows repeated evaluation of surface and tubular membrane parameters on the same cell.

Fraction of the T-Tubular Membrane as an Important Parameter in Cardiac Cellular Electrophysiology: A New Way of Estimation.

The transverse-axial tubular system (t-tubules) plays an essential role in excitation-contraction coupling in cardiomyocytes. Its remodelling is associated with various cardiac diseases. Numerous attempts were made to analyse characteristics essential for proper understanding of the t-tubules and their impact on cardiac cell function in health and disease. The currently available methodical approaches related to the fraction of the t-tubular membrane area produce diverse data. The widely used detubulation techniques cause irreversible cell impairment, thus, distinct cell samples have to be used for estimation of t-tubular parameters in untreated and detubulated cells. Our proposed alternative method is reversible and allows repetitive estimation of the fraction of t-tubular membrane (f t) in cardiomyocytes using short-term perfusion of the measured cell with a low-conductive isotonic sucrose solution. It results in a substantial increase in the electrical resistance of t-tubular lumen, thus, electrically separating the surface and t-tubular membranes. Using the whole-cell patch-clamp measurement and the new approach in enzymatically isolated rat atrial and ventricular myocytes, a set of data was measured and evaluated. The analysis of the electrical equivalent circuit resulted in the establishment of criteria for excluding measurements in which perfusion with a low conductivity solution did not affect the entire cell surface. As expected, the final average f t in ventricular myocytes (0.337 ± 0.017) was significantly higher than that in atrial myocytes (0.144 ± 0.015). The parameter f t could be estimated repetitively in a particular cell (0.345 ± 0.021 and 0.347 ± 0.023 in ventricular myocytes during the first and second sucrose perfusion, respectively). The new method is fast, simple, and leaves the measured cell intact. It can be applied in the course of experiments for which it is useful to estimate both the surface and t-tubular capacitance/area in a particular cell.

Combination of Sildenafil and Ba2+ at a Low Concentration Show a Significant Synergistic Inhibition of Inward Rectifier Potassium Current Resulting in Action Potential Prolongation.

Sildenafil (Viagra) is a vasodilator mainly used in the treatment of erectile dysfunction. Atrial or ventricular fibrillation may rarely occur as a side effect during sildenafil therapy. Although changes in inward rectifier potassium currents including I K1 are known to contribute to the pathogenesis of fibrillation, the effect of sildenafil on I K1 has not been studied. In experiments, Ba2+ is used as a specific inhibitor of I K1 at high concentrations (usually 100 µM). Being an environmental contaminant, it is also present in the human body; Ba2+ plasmatic concentrations up to 1.5 µM are usually reported in the general population. This study was primarily aimed to investigate changes of I K1 induced by sildenafil in a wide range of concentrations (0.1-100 µM). Additionally, the effect of combination of sildenafil and Ba2+ at selected clinically-relevant concentrations was tested, at 0.1 µM both on I K1 and on the action potential duration (APD). Experiments were performed by the whole-cell patch-clamp technique on enzymatically isolated rat ventricular cardiomyocytes, mostly at 23°C with the exception of APD measurements which were performed at 37°C as well. Sildenafil caused a significant, reversible, and concentration-dependent inhibition of I K1 that did not differ at -50 and -110 mV. Simultaneous application of sildenafil and Ba2+ at 0.1 µM revealed a massive inhibition of both inward and outward components of I K1 (this synergy was missing at other tested combinations). The combined effect at 0.1 µM (45.7 ± 5.7 and 43.0 ± 6.9% inhibition at -50 and -110 mV, respectively) was significantly higher than a simple sum of almost negligible effects of the individual substances and it led to a significant prolongation of APD at both 23 and 37°C. To our knowledge, similar potentiation of the drug-channel interaction has not been described. The observed massive inhibition of I K1 induced by a combined action of the vasodilator sildenafil and environmental contaminant Ba2+ at a low concentration and resulting in a significant APD prolongation may contribute to the genesis of arrhythmias observed in some patients treated with sildenafil.

Aminophylline at clinically relevant concentrations affects inward rectifier potassium current in a dual way.

Bronchodilator aminophylline may induce atrial or less often ventricular arrhythmias. The mechanism of this proarrhythmic side effect has not been fully explained. Modifications of inward rectifier potassium (Kir) currents including IK1 are known to play an important role in arrhythmogenesis; however, no data on the aminophylline effect on these currents have been published. Hence, we tested the effect of aminophylline (3-100 µM) on IK1 in enzymatically isolated rat ventricular myocytes using the whole-cell patch-clamp technique. A dual steady-state effect of aminophylline was observed; either inhibition or activation was apparent in individual cells during the application of aminophylline at a given concentration. The smaller the magnitude of the control IK1, the more likely the activation of the current by aminophylline and vice versa. The effect was reversible; the relative changes at -50 and -110 mV did not differ. Using IK1 channel population model, the dual effect was explained by the interaction of aminophylline with two different channel populations, the first one being inhibited and the second one being activated. Considering various fractions of these two channel populations in individual cells, varying effects observed in the measured cells could be simulated. We propose that the dual aminophylline effect may be related to the direct and indirect effect of the drug on various Kir2.x subunits forming the homo- and heterotetrameric IK1 channels in a single cell. The observed IK1 changes induced by clinically relevant concentrations of aminophylline might contribute to arrhythmogenesis related to the use of this bronchodilator in clinical medicine.

Divergent estimates of the ratio between Na+-Ca2+ current densities in t-tubular and surface membranes of rat ventricular cardiomyocytes.

The ratio between Na+-Ca2+ exchange current densities in t-tubular and surface membranes of rat ventricular cardiomyocytes (JNaCa-ratio) estimated from electrophysiological data published to date yields strikingly different values between 1.7 and nearly 40. Possible reasons for such divergence were analysed by Monte Carlo simulations assuming both normal and log-normal distribution of the measured data. The confidence intervals CI95 of the mean JNaCa-ratios computed from the reported data showed an overlap of values between 1 and 3, and between 0.3 and 4.3 in the case of normal and log-normal distribution, respectively. Further analyses revealed that the published high values likely result from a large scatter of data due to transmural differences in JNaCa, dispersion of cell membrane capacitances and variability in incomplete detubulation. Taking into account the asymmetric distribution of the measured data, the reduction of mean current densities after detubulation and the substantially smaller CI95 of lower values of the mean JNaCa-ratio, the values between 1.6 and 3.2 may be considered as the most accurate estimates. This implies that 40 to 60% of Na+-Ca2+ exchanger is located at the t-tubular membrane of adult rat ventricular cardiomyocytes.

ATP-sensitive potassium channels: key players in pathophysiology of many diseases.

ATP-sensitive potassium channels have been an intensively studied type of protein complexes incorporated in the cell membrane for several decades. Their unique function makes them special, as they create a connection between the metabolic state and membrane voltage of the cell. This position of a bridge involved in many cellular cascades allow them to participate in various processes at often surprising positions in nearly all organ systems of the body, from the pancreas, heart muscle or retina, to the central nervous system. This review summarizes the most important roles of ATP-sensitive potassium channels focusing on their possible clinical use within particular organ systems.

Long-QT founder variant T309I-Kv7.1 with dominant negative pattern may predispose delayed afterdepolarizations under ß-adrenergic stimulation.

The variant c.926C > T (p.T309I) in KCNQ1 gene was identified in 10 putatively unrelated Czech families with long QT syndrome (LQTS). Mutation carriers (24 heterozygous individuals) were more symptomatic compared to their non-affected relatives (17 individuals). The carriers showed a mild LQTS phenotype including a longer QTc interval at rest (466 ± 24 ms vs. 418 ± 20 ms) and after exercise (508 ± 32 ms vs. 417 ± 24 ms), 4 syncopes and 2 aborted cardiac arrests. The same haplotype associated with the c.926C > T variant was identified in all probands. Using the whole cell patch clamp technique and confocal microscopy, a complete loss of channel function was revealed in the homozygous setting, caused by an impaired channel trafficking. Dominant negativity with preserved reactivity to ß-adrenergic stimulation was apparent in the heterozygous setting. In simulations on a human ventricular cell model, the dysfunction resulted in delayed afterdepolarizations (DADs) and premature action potentials under ß-adrenergic stimulation that could be prevented by a slight inhibition of calcium current. We conclude that the KCNQ1 variant c.926C > T is the first identified LQTS-related founder mutation in Central Europe. The dominant negative channel dysfunction may lead to DADs under ß-adrenergic stimulation. Inhibition of calcium current could be possible therapeutic strategy in LQTS1 patients refractory to ß-blocker therapy.

Aminophylline Induces Two Types of Arrhythmic Events in Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiac side effects of some pulmonary drugs are observed in clinical practice. Aminophylline, a methylxanthine bronchodilator with documented proarrhythmic action, may serve as an example. Data on the action of aminophylline on cardiac cell electrophysiology and contractility are not available. Hence, this study was focused on the analysis of changes in the beat rate and contraction force of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and HL-1 cardiomyocytes in the presence of increasing concentrations of aminophylline (10 µM-10 mM in hPSC-CM and 8-512 µM in HL-1 cardiomyocytes). Basic biomedical parameters, namely, the beat rate (BR) and contraction force, were assessed in hPSC-CMs using an atomic force microscope (AFM). The beat rate changes under aminophylline were also examined on the HL-1 cardiac muscle cell line via a multielectrode array (MEA). Additionally, calcium imaging was used to evaluate the effect of aminophylline on intracellular Ca2+ dynamics in HL-1 cardiomyocytes. The BR was significantly increased after the application of aminophylline both in hPSC-CMs (with 10 mM aminophylline) and in HL-1 cardiomyocytes (with 256 and 512 µM aminophylline) in comparison with controls. A significant increase in the contraction force was also observed in hPSC-CMs with 10 µM aminophylline (a similar trend was visible at higher concentrations as well). We demonstrated that all aminophylline concentrations significantly increased the frequency of rhythm irregularities (extreme interbeat intervals) both in hPSC-CMs and HL-1 cells. The occurrence of the calcium sparks in HL-1 cardiomyocytes was significantly increased with the presence of 512 µM aminophylline. We conclude that the observed aberrant cardiomyocyte response to aminophylline suggests an arrhythmogenic potential of the drug. The acquired data represent a missing link between the arrhythmic events related to the aminophylline/theophylline treatment in clinical practice and describe cellular mechanisms of methylxanthine arrhythmogenesis. An AFM combined with hPSC-CMs may serve as a robust platform for direct drug effect screening.

Acute effects of alcohol on cardiac electrophysiology and arrhythmogenesis: Insights from multiscale in silico analyses.

Acute excessive ethyl alcohol (ethanol) consumption alters cardiac electrophysiology and can evoke cardiac arrhythmias, e.g., in 'holiday heart syndrome'. Ethanol acutely modulates numerous targets in cardiomyocytes, including ion channels, Ca2+-handling proteins and gap junctions. However, the mechanisms underlying ethanol-induced arrhythmogenesis remain incompletely understood and difficult to study experimentally due to the multiple electrophysiological targets involved and their potential interactions with preexisting electrophysiological or structural substrates. Here, we employed cellular- and tissue-level in-silico analyses to characterize the acute effects of ethanol on cardiac electrophysiology and arrhythmogenesis. Acute electrophysiological effects of ethanol were incorporated into human atrial and ventricular cardiomyocyte computer models: reduced INa, ICa,L, Ito, IKr and IKur, dual effects on IK1 and IK,ACh (inhibition at low and augmentation at high concentrations), and increased INCX and SR Ca2+ leak. Multiscale simulations in the absence or presence of preexistent atrial fibrillation or heart-failure-related remodeling demonstrated that low ethanol concentrations prolonged atrial action-potential duration (APD) without effects on ventricular APD. Conversely, high ethanol concentrations abbreviated atrial APD and prolonged ventricular APD. High ethanol concentrations promoted reentry in tissue simulations, but the extent of reentry promotion was dependent on the presence of altered intercellular coupling, and the degree, type, and pattern of fibrosis. Taken together, these data provide novel mechanistic insight into the potential proarrhythmic interactions between a preexisting substrate and acute changes in cardiac electrophysiology. In particular, acute ethanol exposure has concentration-dependent electrophysiological effects that differ between atria and ventricles, and between healthy and diseased hearts. Low concentrations of ethanol can have anti-fibrillatory effects in atria, whereas high concentrations promote the inducibility and maintenance of reentrant atrial and ventricular arrhythmias, supporting a role for limiting alcohol intake as part of cardiac arrhythmia management.

Distribution of data in cellular electrophysiology: Is it always normal?

The distribution of data presented in many electrophysiological studies is presumed to be normal without any convincing evidence. To test this presumption, the cell membrane capacitance and magnitude of inward rectifier potassium currents were recorded by the whole-cell patch clamp technique in rat atrial myocytes. Statistical analysis of the data showed that these variables were not distributed normally. Instead, a positively skewed distribution appeared to be a better approximation of the real data distribution. Consequently, the arithmetic mean, used inappropriately in such data, may substantially overestimate the true mean value characterizing the central tendency of the data. Moreover, a large standard deviation describing the variance of positively skewed data allowed 95% confidence interval to include unrealistic negative values. We therefore conclude that the normality of the electrophysiological data should be tested in every experiment and, if rejected, the positively skewed data should be more accurately characterized by the median and interpercentile range or, if justified (namely in the case of log-normal and gamma data distribution), by the geometric mean and the geometric standard deviation.

Current density as routine parameter for description of ionic membrane current: is it always the best option?

The current density (J) is a parameter routinely used to characterize individual ionic membrane currents. Its evaluation is based on the presumption that the magnitude of whole-cell ionic membrane current (I) is directly proportional to the cell membrane capacitance (C), i.e. I positively and strongly correlates with C and the regression line describing I-C relation intersects the y-axis close to the origin of coordinates. We aimed to prove the presumption in several examples and find whether the conversion of I to J could be always beneficial. I-C relation was analysed in several potassium currents, measured in rat atrial myocytes (in inward rectifier currents, IK1, and both the constitutively active and acetylcholine-induced components of acetylcholine-sensitive current, IK(Ach)CONST and IK(Ach)ACH), and in rat ventricular myocytes (transient outward current Ito). I-C correlation was estimated by the Pearson coefficient (r). A coefficient (k) was newly suggested describing deviation of the regression intercept from zero in currents with considerable r value. Based on mathematical simulations, I was satisfactorily proportional to C when r ≥ 0.6 and k ≤ 0.2 which was fulfilled in IK1 and IK(Ach)ACH (r = 0.84, k = 0.20, and r = 0.61, k = 0.06, respectively). I-C correlation was significantly positive, but weak in IK(Ach)CONST (r = 0.42), and virtually missing in Ito (r = 0.04). The impaired I-C proportionality in IK(Ach)CONST and Ito likely reflects heterogeneity of the channel expression. We conclude that the conversion of I to J should be avoided when I-C proportionality is absent. Otherwise, serious misinterpretation of data may arise.

The intriguing effect of ethanol and nicotine on acetylcholine-sensitive potassium current IKAch: Insight from a quantitative model.

Recent experimental work has revealed unusual features of the effect of certain drugs on cardiac inwardly rectifying potassium currents, including the constitutively active and acetylcholine-induced components of acetylcholine-sensitive current (IKAch). These unusual features have included alternating susceptibility of the current components to activation and inhibition induced by ethanol or nicotine applied at various concentrations, and significant correlation between the drug effect and the current magnitude measured under drug-free conditions. To explain these complex drug effects, we have developed a new type of quantitative model to offer a possible interpretation of the effect of ethanol and nicotine on the IKAch channels. The model is based on a description of IKAch as a sum of particular currents related to the populations of channels formed by identical assemblies of different α-subunits. Assuming two different channel populations in agreement with the two reported functional IKAch-channels (GIRK1/4 and GIRK4), the model was able to simulate all the above-mentioned characteristic features of drug-channel interactions and also the dispersion of the current measured in different cells. The formulation of our model equations allows the model to be incorporated easily into the existing integrative models of electrical activity of cardiac cells involving quantitative description of IKAch. We suppose that the model could also help make sense of certain observations related to the channels that do not show inward rectification. This new ionic channel model, based on a concept we call population type, may allow for the interpretation of complex interactions of drugs with ionic channels of various types, which cannot be done using the ionic channel models available so far.

Impaired Adrenergic/Protein Kinase A Response of Slow Delayed Rectifier Potassium Channels as a Long QT Syndrome Motif: Importance and Unknowns.

The slow delayed rectifier potassium current (IKs) significantly contributes to cardiac repolarization under specific conditions, particularly at stimulation by the protein kinase A (PKA) during increased sympathetic tone. Impaired PKA-mediated stimulation of IKs channels may considerably aggravate dysfunction of the channels induced by mutations in the KCNQ1 gene that encodes the structure of the α-subunit of IKs channels. These mutations are associated with several subtypes of inherited arrhythmias, mainly long QT syndrome type 1, less commonly short QT syndrome type 2, and atrial fibrillation. The impaired PKA reactivity of IKs channels may significantly increase the risk of arrhythmia in these patients. Unfortunately, only approximately 2.7% of the KCNQ1 variants identified as putatively clinically significant have been studied with respect to this problem. This review summarizes the current knowledge in the field to stress the importance of the PKA-mediated regulation of IKs channels, and to appeal for further analysis of this regulation in KCNQ1 mutations associated with inherited arrhythmogenic syndromes. On the basis of the facts summarized in our review, we suggest several new regions of the α-subunit of the IKs channels as potential contributors to PKA stimulation, namely the S4 and S5 segments, and the S2-S3 and S4-S5 linkers. Deeper knowledge of mechanisms of the impaired PKA response in mutated IKs channels may help to better understand this regulation, and may improve risk stratification and management of patients suffering from related pathologies.

Inward rectifying potassium currents resolved into components: modeling of complex drug actions.

Inward rectifier potassium currents (I Kir,x) belong to prominent ionic currents affecting both resting membrane voltage and action potential repolarization in cardiomyocytes. In existing integrative models of electrical activity of cardiac cells, they have been described as single current components. The proposed quantitative model complies with findings indicating that these channels are formed by various homomeric or heteromeric assemblies of channel subunits with specific functional properties. Each I Kir,x may be expressed as a total of independent currents via individual populations of identical channels, i.e., channels formed by the same combination of their subunits. Solution of the model equations simulated well recently observed unique manifestations of dual ethanol effect in rat ventricular and atrial cells. The model reflects reported occurrence of at least two binding sites for ethanol within I Kir,x channels related to slow allosteric conformation changes governing channel conductance and inducing current activation or inhibition. Our new model may considerably improve the existing models of cardiac cells by including the model equations proposed here in the particular case of the voltage-independent drug-channel interaction. Such improved integrative models may provide more precise and, thus, more physiologically relevant results.

[Functional impact of hERG: from physiological role to target of anticancer therapy]. / Funkcní význam hERG: od fyziologické role po cíl protinádorové terapie.

The human ether-à-go-go related gene (hERG; officially designated as KCNH2) encodes the structure of protein forming α-subunit of voltage-gated ion channel which conducts the rapid component of delayed rectifier K+ current (IKr). This current plays an important role namely in the cardiac repolarization. Mutations in hERG result in inherited arrhythmogenic syndromes characterized by a lenghtening or shortening of QT interval on the electrocardiogram and by an increased occurrence of life-threatening arrhythmias. This review also introduces hERG channels as a part of regulatory mechanisms of the smooth muscle contractility, neuronal activity, release of several hormones, and of proliferation and apoptosis of cancer cells. There are also mentioned some of the diseases arising from hERG channel dysfunction, and some possibilities of use of hERG gene/channel as a diagnostic marker and potential therapeutic target in various diseases, namely in cancer.Key words: cancer - epilepsy - hERG - KCNH2 - K+ channel - LQTS - membrane potential - muscle contraction - proliferation - schizophrenia.