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A search in the GDC Data Portal revealed 304 documented somatic mutations of the KCNJ3 gene in primary tumors (out of 10.202 cases). Most affected tumor types were carcinomas from uterus, skin and lung, while breast cancer exerted the lowest number of somatic mutations. We focused our research on 15 missense mutations within the region between TM1 and TM2, comprising the pore helix and ion selectivity signature. Expression was measured by confocal laser scan microscopy of eGFP tagged GIRK1 subunits, expressed with and without GIRK4 in oocytes of Xenopus laevis. GIRK ion currents were activated via coexpressed m2Rs and measured by the Two Electrode Voltage Clamp technique. Magnitude of the total GIRK current, as well as the fraction of current inducible by the agonist, were measured. Ion selectivity was gauged by assessment of the PNa+/PK+ ratio, calculated by the GIRK current reversal potential in extracellular media at different Na+ and K+ concentrations. None of the tested mutations was able to form functional GIRK1 homooligomeric ion channels. One of the mutations, G145A, which locates directly to the ion selectivity signature, exerted an increased PNa+/PK+ ratio. Generally, the missense mutations studied can be categorized into three groups: (i) normal/reduced expression accompanied by reduced/absent function (S132Y, F136L, E139K, G145A, R149Q, R149P, G178D, S185Y, Q186R), (ii) normal/increased expression as well as increased function (E140M, A142T, M184I) and (iii) miniscule expression but increased function relative to expression levels (I151N, G158S). We conclude, that gain of function mutations, identical or similar to categories (ii) and (iii), may potentially be involved in genesis and progression of malignancies in tissues that exert a high rate of occurrence of somatic mutations of KCNJ3.

Electrophysiologists routinely use simple voltage steps to evaluate cell membrane capacitance derived from corresponding current responses. Frequently, the resting membrane voltage Vrest is employed as holding potential for the subsequent command voltage step and more or less accurate methods are utilised to analyse the transient current. Another choice as holding potential is the peak of the "quasi steady-state" current to voltage relationship, Vpeak. The aim of this study is the systematic evaluation of capacitance estimation accuracy from voltage step experiments depending on the choice of holding potential and analysis method. In this paper, a simulation approach is employed to analyse the current response of a model patch-clamp circuit. Four commonly accepted methods are implemented, utilizing different aspects of the transient current (charge, membrane time constant, and influence of the series resistance) in various combinations and with various degrees of refinement. This simulation study indicates an acceptable accuracy of the elaborated methods for capacitance estimation at holding potentials Vrest and Vpeak over a broad range of capacitance as well as series resistance values. Simple integration of the current transient provides sufficient accuracy at holding potentials, which effectively minimizes changes in resistive membrane current flow during command voltage steps (particularly around Vpeak). However, biphasic command protocols performed at Vpeak activate voltage dependent sodium channels, thereby possibly leading to the threshold voltage for an action potential. Compared to Vrest, all methods utilizing monophasic step protocols, gain additional accuracy, when applied at Vpeak as holding potential.

Lung cancer is still a leading cause of death worldwide. In recent years, knowledge has been obtained of the mechanisms modulating ion channel kinetics and thus of cell bioelectric properties, which is promising for oncological biomarkers and targets. The complex interplay of channel expression and its consequences on malignant processes, however, is still insufficiently understood. We here introduce the first approach of an in-silico whole-cell ion current model of a cancer cell, in particular of the A549 human lung adenocarcinoma, including the main functionally expressed ion channels in the plasma membrane as so far known. This hidden Markov-based model represents the electrophysiology behind proliferation of the A549 cell, describing its rhythmic oscillation of the membrane potential able to trigger the transition between cell cycle phases, and it predicts membrane potential changes over the cell cycle provoked by targeted ion channel modulation. This first A549 in-silico cell model opens up a deeper insight and understanding of possible ion channel interactions in tumor development and progression, and is a valuable tool for simulating altered ion channel function in lung cancer electrophysiology., Author summary Advances in the understanding of functional alterations at genetic, epigenetic or protein expression and the expanding knowledge in mechanisms modulating ion channel kinetics and thus the cells’ bioelectric properties have arisen as promising cancer biomarkers and oncological targets. Our hidden Markov-based in-silico cell model represents the electrophysiology behind proliferation of the A549 cell line, explaining the cell’s rhythmic oscillation from hyperpolarized to depolarized states of the membrane potential, able to trigger the transition between cell cycle phases. The model enables the prediction of membrane potential changes over the cell cycle provoked by targeted modulation of specific ion channels, leading to cell cycle promotion or interruption. We are encouraged that the availability of this first cancer cell model will provide profound insight into possible roles and interactions of ion channels in tumor development and progression, and may aid in the testing of research hypotheses in lung cancer electrophysiology.

We set out to determine the membrane potential (Vm) of the endothelial cell line EA.hy926 and its sensitivity to the antimycotic amphotericin B (AmB), a commonly used antifungal component in cell culture media. We measured the endothelial Vm under various experimental conditions by patch clamp technique and found that Vm of AmB-treated cells is (-12.1 ± 9.3) mV, while in AmB-untreated (control) cells it is (-57.1 ± 4.1) mV. In AmB-free extracellular solutions, Vm recovered toward control levels and this gain in Vm rapidly dissipated upon re-addition of AmB, demonstrating a rapid and reversible effect of AmB on endothelial Vm. The consequences of AmB dependent alterations in endothelial transmembrane potential were tested at the levels of Ca(2+) signaling, of nucleotide concentrations, and energy metabolism. In AmB-treated cells we found substantially reduced Ca(2+) entry (to about 60% of that in control cells) in response to histamine induced endoplasmic reticulum (ER) Ca(2+) depletion, and diminished the ATP-to-ADP ratio (by >30%). Our data demonstrate a marked and experimentally relevant dependence of basic functional parameters of cultured endothelial cells on the presence of the ionophoric antimycotic AmB. The profound and reversible effects of the widely used culture media component AmB need careful consideration when interpreting experimental data obtained under respective culture conditions.

Beat to beat variability of cardiac tissue or isolated cells is frequently investigated by determining time intervals from electrode measurements in order to compute scale dependent or scale independent parameters. In this study, we utilize high-speed video camera recordings to investigate the variability of intervals as well as mechanical contraction strengths and relative contraction strengths with nonlinear analyses. Additionally, the video setup allowed us simultaneous electrode registrations of extracellular potentials. Sinoatrial node tissue under control and acetylcholine treated conditions was used to perform variability analyses by computing sample entropies and Higuchi dimensions. Beat to beat interval variabilities measured by the two recording techniques correlated very well, and therefore, validated the video analyses for this purpose. Acetylcholine treatment induced a reduction of beating rate and contraction strength, but the impact on interval variability was negligible. Nevertheless, the variability analyses of contraction strengths revealed significant differences in sample entropies and Higuchi dimensions between control and acetylcholine treated tissue. Therefore, the proposed high-speed video camera technique might represent a non-invasive tool that allows long-lasting recordings for detecting variations in beating behavior over a large range of scales.

Saxagliptin treatment has been associated with increased rate of hospitalization for heart failure in type 2 diabetic patients, though the underlying mechanism(s) remain elusive. To address this, we assessed the effects of saxagliptin on human atrial trabeculae, guinea pig hearts and cardiomyocytes. We found that the primary target of saxagliptin, dipeptidyl peptidase-4, is absent in cardiomyocytes, yet saxagliptin internalized into cardiomyocytes and impaired cardiac contractility via inhibition of the Ca

to the editor: Cell membrane capacitance C m is a fundamental property of excitable cells. It scales with the cell surface area and is therefore used for cell size estimation and most important for normalizing currents and conductances. Consequently, proper knowledge of C m is also crucial for

Objective : The left human atrium plays an important role in initiation of atrial fibrillation (AF) and the hyperpolarization activated cation current ( I f) is a candidate for contributing to abnormal automaticity. However, electrophysiological data concerning I f are not available in this cardiac region and we therefore investigated I f in human left atrial tissue. Methods : Human atrial myocytes were isolated from the left atrial appendage (LAA) and the left atrial wall (LAW) obtained from patients undergoing open heart surgery. I f was measured with the whole-cell patch-clamp technique. Results : I f densities between −70 and −110 mV were found to be significantly higher in LAA than in LAW cells. Furthermore, in the group of LAA cells the half maximal activation potential ( V 1/2) was found to be less negative ( V 1/2 of −84.3±1.9 mV, n =14/9) compared to LAW cells ( V 1/2 of −97.8±2.1 mV, n =28/9). Beta-adrenergic receptor stimulation with isoproterenol (1 μM) caused an acceleration of current activation and a V 1/2 shift to more positive potentials in cells of both regions (LAA: 8.8±2.3 mV, n =6/4 and LAW: 8.9±2.6 mV, n =6/4). Simulations using a mathematical model of the human atrial myocyte demonstrated that I f was able to induce spontaneous activity in the model at a regular rhythm due to the interplay of I f, Na+/Ca2+ exchange current and Ca2+ release of the sarcoplasmic reticulum (SR). Conclusions : Our study revealed the presence of I f in left atrial myocytes and showed that I f parameters depend on atrial region. I f current densities were sufficient to convert the mathematical model of a quiescent human atrial cell into a “pacemaker cell”. These data support the hypothesis of I f as a contributor to abnormal automaticity in human atrial tissue.

Extracardiac factors of heart rate variability have commonly been investigated using linear and nonlinear methods for a long time. Recently, intracardiac mechanisms on an electrophysiological basis have been found to be also important. This work is focused on the evaluation of complex measures of temporal signals gained with microelectrode measurements of embryonic chick heart aggregates. Septic conditions were mimicked in vitro by lipopolysaccharide (LPS) administration in order to investigate the influence on beat to beat variability. Surrogate data analysis revealed high statistical significances for normalized complexity measures.

Thienopyridines (ticlopidine, clopidogrel) are frequently used drugs in antiplatelet therapy and have been shown to exert a more pronounced negative inotropic effect than thienopyrimidinones. We hypothesized that these differences are due to a differential impact of thienopyridines and thienopyrimidinones on L-type calcium current at the single-cell level. The effects of thienopyridines and thienopyrimidinones were studied on L-type calcium current and action potential parameters with the whole-cell patch-clamp technique in isolated myocytes from guinea pig ventricle and human atrial appendage. Ticlopidine showed the greatest impact on the L-type calcium current in guinea pig myocytes. It significantly reduced L-type calcium current density as well as shifted half maximal inactivation potential to more negative potentials compared to clopidogrel (at 30 μmol/L) and to all thienopyrimidinones (30 and 100 μmol/L). Clopidogrel significantly reduced the L-type calcium current density as well as shifted the half maximal inactivation potential to more negative potentials compared to all thienopyrimidinones at 100 μmol/L only. In contrast, thienopyrimidinones did not affect L-type calcium current properties. The significant different effects of thienopyridines and thienopyrimidinones could also be demonstrated in human atrial myocytes. The more pronounced negative inotropic effect of thienopyridines is well explained by our results demonstrating a differential impairment of L-type calcium current by thienopyridines and thienopyrimidinones. L-type calcium current impairment by thienopyridines may be of special relevance for patients with cardiac diseases characterized by ionic remodelling.

Thienopyridines are frequently used drugs in the management of ischemic heart diseases or thrombotic events due to its antiplatelet as well as thrombolytic properties. Recently it was shown, that these compounds exert a negative inotropic effect in the isolated guinea pig heart. Using voltage clamp technique we could demonstrate a L-type calcium current impairment possibly underlying these effects. The aim of the study is to further elucidate the effects of thienopyridines on tissue level using high resolution optical mapping in guinea pig papillary. Our experimental setup features a 16 by 16 photodiode array with individual 256 custom-built current-to-voltage amplifiers. A frequency doubled continuous wave Neodymium-Yag laser (532 nm, 2 W) is used as excitation source. The tissue is stained by incubation with the voltage sensitive dye di-4-ANEPPS. The use of different objective-magnifications (10x, 20x, 40x, 63x) allows a multi-scale based analysis with resolutions up to 15 micrometers. The setup provides a detailed view on excitation propagation. In addition, the application of external electric field pulses during the depolarization phase of the tissue sample reveals local inhomogeneities in the membrane-potential distribution at the tissue surface (and possibly 2-3 cell layers below the surface).[1] This allows us to quantify the electrical inhomogeneity of the preparation. Experiments demonstrate the importance of optical membrane potential measurements, which provide new information unattainable with other methods.[1] Windisch, H. et al. (2007): Quantification of shock-induced microscopic virtual electrodes assessed by subcellular resolution optical potential mapping in guinea pig papillary muscle. J Cardiovasc Electrophysiol 18(10), 1086-1094

In sepsis, systemic inflammatory response syndrome (SIRS), and multiorgan dysfunction syndrome (MODS), a severe prognostically relevant cardiac autonomic dysfunction exists, as manifested by a strong attenuation of sympathetically and vagally mediated heart rate variability (HRV). The mechanisms underlying this attenuation are not limited to the nervous system. They also include alterations of the cardiac pacemaker cells on a cellular level. As shown in human atrial cardiomyocytes, endotoxin interacts with cardiac hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels, which mediate the pacemaker current If and play an important role in transmitting sympathetic and vagal signals on heart rate and HRV. Moreover, endotoxin sensitizes cardiac HCN channels to sympathetic signals. These findings identify endotoxin as a pertinent modulator of the autonomic nervous regulation of heart function. In MODS, the vagal pathway of the autonomic nervous system is particularly compromised, leading to an attenuation of the cholinergic antiinflammatory reflex. An amelioration of the blunted vagal activity appears to be a promising novel therapeutic target to achieve a suppression of the inflammatory state and thereby an improvement of prognosis in MODS patients. Preliminary data revealed therapeutic benefits (increased survival rates and improvements of the depressed vagal activity) of the administration of statins, beta-blockers, and angiotensin-converting enzyme inhibitors in patients with MODS.

LPSs trigger the development of sepsis by gram-negative bacteria and cause a variety of biological effects on host cells, including alterations on ionic channels. Because heart rate variability is reduced in human sepsis and endotoxemia, we hypothesized that LPS affects the pacemaker current I(f) in human heart, which might--at least in part--explain this phenomenon. Isolated human myocytes from right atrial appendages were incubated for 6 to 10 h with LPS (1 and 10 microg/mL) and afterwards used to investigate the pacemaker current I(f). I(f) was measured with the whole-cell patch-clamp technique (at 37 degrees C). Incubation of atrial myocytes with 10 microg/mL LPS was found to significantly impair I(f) by suppressing the current at membrane potentials positive to -80 mV and slowing down current activation, but without effecting maximal current conductance. Furthermore, in incubated cells (10 microg/mL), the response of I(f) to [beta]-adrenergic stimulation (1 microM isoproterenol) was significantly larger compared with control cells (shift of half-maximal activation voltage to more positive potentials amounted to -10 and -14 mV in untreated and treated cells, respectively). Simulations using a spontaneously active sinoatrial cell model demonstrated that LPS-induced I(f) impairment reduced the responsiveness of the model cell to fluctuations of autonomic input. This study showed a direct impact of LPS on the cardiac pacemaker current I(f). The LPS-induced I(f) impairment may contribute to the clinically observed reduction in heart rate variability under septic conditions and in cardiac diseases such as heart failure, where endotoxin can be of pathophysiological relevance.

Objective : It was our aim to investigate effects of human LDL, copper-, or AAPH-oxidized over different periods of time to different degrees (ox-LDL), on viability and electrophysiological parameters of isolated ventricular myocytes of guinea pigs. Methods : Guinea pig ventricular myocytes were incubated with ox-LDL or native LDL (at 0.5 mg/ml) for 12 h, and afterwards myocyte damage, action potentials, and transmembrane ion currents were studied (at 37 °C). Results : Ox-LDL was found to induce severe myocyte damage, whereas native LDL had no effect. Myocyte damage was dependent on the content of total lipid hydroperoxides in both copper-oxidized and AAPH-oxidized LDL. Incubation with ox-LDL led to intense contractile and electrophysiological effects including prolongation of action potential duration, depolarization of resting membrane potential, spontaneous activity, generation of afterdepolarizations, and modification of transmembrane ion currents (e.g. inward rectifier, calcium, and background currents). Conclusions : Ox-LDL induced cell damage and irregular electrical activity in ventricular myocytes. These effects were dependent on the lipid hydroperoxide content of ox-LDL and were similar to oxidative stress (OS) induced by various OS-generating systems. The observed effects may play a role for functional cardiac abnormalities in patients with increased ox-LDL levels.

I(f) was shown to be present in adult human atrial and ventricular myocytes but data obtained from infant myocytes are lacking. We have studied I(f) in isolated ventricular myocytes from children undergoing surgical correction of tetralogy of Fallot (TOF; n = 5; mean age: 15.3 months). All recordings were made with the patch clamp technique in the whole cell mode at a temperature of 36-37 degrees C. A modified Tyrode solution containing 25 mM KCl was used to amplify I(f). Considering I(f) to be present when its current density at -120 mV was greater than 0.5 pA/pF, I(f) could be found in 28 out of 32 myocytes (88%). The mean current density was -2.01 +/- 0.3 pA/pF (mean +/- S.E.M.). First current activation occurred at -70 mV and I(f) could be reversibly inhibited by superfusing the myocytes with CsCl (2 mM). Half maximal activation (V(1/2)) of I(f) was at -80.3 +/- 1.0 mV (n = 28). Beta-adrenergic receptor stimulation with isoproterenol (1 microM) caused an acceleration of current activation and a shift of V(1/2) by 7.88 +/- 1.8 mV (n = 10) to less negative potentials. This study provides first evidence that the hyperpolarization-activated pacemaker current I(f) is present in infant human ventricular myocytes. Our results suggest that I(f) in ventricle of infants suffering from TOF has similar properties as I(f) in adult ventricle.

Inhomogeneities in the tissue are a determining factor for cardiac function and can be either structural or functional. Using the voltage sensitive fluorescent dye di-4-ANEPPS on incubation stained guinea pig preparations allows to map the tissue's response to externally applied electric shocks. Previous work on ventricular muscle preparations revealed a membrane potential morphology that could be relevant during defibrillation [1]. Using a two pulse stimulus protocol we are able to map the excitation response to a short triggering field pulse and to consequently map the membrane potential morphology resulting from a second field pulse, which is applied in the depolarized state and is attributed to the interaction of the electric field with the tissue. This approach is now carried over to atrial preparations. A custom developed object-positioning and image-stitching method allows for wide view analysis of the atrial tissue structure. Objective-magnifications of 10x, 20x, 40x and 63x enable a multi-scale based analysis with resolutions up to 15 micrometers. This is augmented with information from transmitted light microscopy so that it is possible to correlate the membrane potential patterns with the visible structure of the atrial tissue, providing further insight into atrial excitation and conduction.[1] Windisch,H. et al. (2007) Quantification of shock-induced microscopic virtual electrodes assessed by subcellular resolution optical potential mapping in guinea pig papillary muscle. J Cardiovasc Electrophysiol; 18(10):1086-1094