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Excessive expression of subunit 1 of GIRK1 in ER+ breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10AGIRK1 with the control (MCF10AeGFP). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.

Many cardiac insults causing atrial remodeling are linked to either stretch or tachycardia, but a comparative characterization of their effects on early remodeling events in human myocardium is lacking. Here, we applied isometric stretch or sustained tachycardia at 2.5 Hz in human atrial trabeculae for 6 h followed by microarray gene expression profiling. Among largely independent expression patterns, we found a small common fraction with the microRNA miR-1183 as the highest up-regulated transcript (up to 4-fold). Both, acute stretch and tachycardia induced down-regulation of the predicted miR-1183 target genes ADAM20 and PLA2G7. Furthermore, miR-1183 was also significantly up-regulated in chronically remodeled atrial samples from patients with persistent atrial fibrillation (3-fold up-regulation versus sinus rhythm samples), and in ventricular myocardium from dilative cardiomyopathy hearts (2-fold up-regulation) as compared to non-failing controls. In sum, although stretch and tachycardia show distinct transcriptomic signatures in human atrial myocardium, both cardiac insults consistently regulate the expression of miR-1183 and its downstream targets in acute and chronic remodeling. Thus, elevated expression of miR-1183 might serve as a tissue biomarker for atrial remodeling and might be of potential functional significance in cardiac disease.

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.

Glucagon-like peptide 1 (GLP-1) receptor agonists increase the intracellular calcium levels of atrial cardiomyocytes in a protein kinase A dependent manner. This elicits a positive inotropic effect. Furthermore, the translocation of GLUT1 is promoted. The relevance of the latter process is unclear, therefore we assessed the influence of energy substrates. Muscle strips (trabeculae; n = 86) were isolated from human right atrial appendages obtained from patients undergoing heart surgery (n = 34), mounted on hooks, electrically stimulated (1 Hz) and treated with a single dose of exenatide (15 nM), GLP-1(7–36) amide (180 nM), GLP-1(9–36) amide (200 nM), isoproterenol (100 nM), or increasing concentrations of calcium (4.0 and 7.2 mM). Either 11.2 mM glucose or 22.4 mM pyruvate served as the energy substrate. Developed force, diastolic tension and relaxation parameters were recorded and analyzed. Administration of exenatide and GLP-1(7–36) amide, but not GLP-1(9–36) amide, led to a transient positive inotropic effect in the presence of pyruvate and glucose. This effect tended to be more pronounced in glucose-treated muscle strips at maximal developed force and steady state conditions. Both isoproterenol and calcium exerted a strong positive inotropic effect with no difference regarding the energy substrate. In conclusion, the positive inotropic effect of GLP-1 receptor agonists is more pronounced in glucose enriched Tyrode’s solution, which might be linked to the previously reported translocation of GLUT1.

Padagogische Hochschulen sind im Kreis der tertiaren bzw. postsekundaren Bildungseinrichtungen Osterreichs die jungsten Institutionen. Mit dem Hochschulgesetz 2005 erhielten neben den offentlichen Universitaten, Fachhochschulen und privaten Universitaten auch Padagogische Hochschulen den Hochschulstatus, sodass 2007 aus drei Vorgangerinstitutionen – den Padagogischen Akademien, den Padagogischen Instituten und den Berufsbildenden Akademien – neun offentliche Padagogische Hochschulen und daruber hinaus funf private Padagogische Hochschulen entstanden. Letztere befinden sich meist in kirchlicher Tragerschaft.

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.

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

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes.

We recorded transmembrane potential in whole cell recording mode from small clusters (2–4 cells) of spontaneously beating 7-day embryonic chick ventricular cells after 1–3 days in culture and investigated effects of the blockers D-600, diltiazem, almokalant, and Ba2+. Electrical activity in small clusters is very different from that in reaggregates of several hundred embryonic chick ventricular cells, e.g., TTX-sensitive fast upstrokes in reaggregates vs. TTX-insensitive slow upstrokes in small clusters (maximum upstroke velocity ∼100 V/s vs. ∼10 V/s). On the basis of our voltage- and current-clamp results and data from the literature, we formulated a Hodgkin-Huxley-type ionic model for the electrical activity in these small clusters. The model contains a Ca2+current ( ICa), three K+currents ( IKs, IKr, and IK1), a background current, and a seal-leak current. ICagenerates the slow upstroke, whereas IKs, IKr, and IK1contribute to repolarization. All the currents contribute to spontaneous diastolic depolarization, e.g., removal of the seal-leak current increases the interbeat interval from 392 to 535 ms. The model replicates the spontaneous activity in the clusters as well as the experimental results of application of blockers. Bifurcation analysis and simulations with the model predict that annihilation and single-pulse triggering should occur with partial block of ICa. Embryonic chick ventricular cells have been used as an experimental model to investigate various aspects of spontaneous beating of cardiac cells, e.g., mutual synchronization, regularity of beating, and spontaneous initiation and termination of reentrant rhythms; our model allows investigation of these topics through numerical simulation.

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.

OBJECTIVES: Currently available cardioplegic solutions provide excellent protection in patients with normal surgical risk; in high-risk patients, however, such as in emergency coronary artery bypass surgery, there is still room for improvement. As most of the cardioplegic solutions primarily protect myocytes, the addition of substances for protection of the endothelium might improve their protective potential. The nitric oxide donor, S-nitroso human serum albumin (S-NO-HSA), which has been shown to prevent endothelial nitric oxide synthase uncoupling, was added to the newly developed histidine-tryptophan-ketoglutarat (HTK-N) cardioplegia in an isolated heart perfusion system after subjecting rats to acute myocardial infarction (MI) and reperfusion. METHODS: In male Sprague-Dawley rats, acute MI was induced by ligation for 1 h of the anterior descending coronary artery. After 2 h of in vivo reperfusion hearts were evaluated on an isolated erythrocyte-perfused working heart model. Cold ischaemia (4°C) for 60 min was followed by 45 min of reperfusion. Cardiac arrest was induced either with HTK (n= 10), HTK-N (n= 10) or HTK-N + S-NO-HSA (n= 10). In one group (HTK-N + S-NO-HSA plus in vivo S-NO-HSA; n= 9) an additional in vivo infusion of S-NO-HSAwas performed. RESULTS: Post-ischaemic recovery of cardiac output (HTK: 77 ± 4%, HTK-N: 86 ± 7%, HTK-N + S-NO-HSA: 101 ± 5%, in vivo S-NO-HSA: 93 ± 8%), external heart work (HTK: 79 ± 5%, HTK-N: 83 ± 3%, HTK-N + S-NO-HSA: 101 ± 8%, in vivo S-NO-HSA: 109 ± 13%), coronary flow (HTK: 77 ± 4%, HTK-N: 94 ± 6%, HTK-N + S-NO-HSA: 118 ± 15%, in vivo S-NO-HSA: 113 ± 3.17%) [HTK-N + S-NO-HSA vs HTK P< 0.001; HTK-N + S-NO-HSA vs HTK-N P< 0.05] and left atrial diastolic pressure (HTK: 122 ± 31%, HTK-N: 159 ± 43%, HTK-N + S-NO-HSA: 88 ± 30, in vivo S-NO-HSA: 62 ± 10%) [HTK-N + S-NO-HSA vs HTK P< 0.05; in vivo S-NO-HSA vs HTK-N P< 0.05] were significantly improved in both S-NO-HSA-treated groups compared with HTK and HTK-N, respectively. This was accompanied by better preservation of highenergy phosphates (adenosine triphosphate; energy charge) and ultrastructural integrity on transmission electron microscopy. However, no additional benefi to fin vivo S-NO-HSA infusion was observed. CONCLUSIONS: Addition of the NO donor, S-NO-HSA refines the concept of HTK-N cardioplegia in improving post-ischaemic myocardial perfusion. HTK-N with S-NO-HSA is a possible therapeutic option for patients who have to be operated on for acute MI.

It was the aim of our study to investigate the effects of the sulphonylurea glibenclamide on voltage dependent potassium currents in human atrial myocytes. The drug blocked a fraction of the quasi steady state current (ramp response) which was activated positive to −20 mV, was sensitive to 4-aminopyridine (500 μM) and was different from the ATP dependent potassium current IK(ATP). Glibenclamide dose dependently inhibited both, the peak as well as the late current elicited by step depolarization positive to −20 mV. The IC50 for reduction in charge area of total outward current was 76 μM. The double-exponential inactivation time-course of the total outward current was accelerated in the presence of glibenclamide with a τfast of 12.7±1.5 ms and a τslow of 213±25 ms in control and 5.8±1.9 ms (P

Objective: It was the aim of our study to describe repolarizing currents in ventricular myocytes isolated from children with tetralogy of Fallot. This is the first report on outward currents in ventricular myocytes from children. Methods: Ventricular myocytes were isolated from tissue samples of the outflow tract of the right ventricle which were obtained during corrective surgery of tetralogy of Fallot. Action potentials and whole-cell currents were recorded with the patch clamp technique at a temperature of 36–37°C. Results: The mean resting potential was −71.7±1.92 mV, action potential amplitude was 110±2.96 mV and action potential duration at 90% repolarization was 794±99.5 ms ( n =12). In four out of 12 myocytes early afterdepolarizations (EADs) were observed. Upon hyperpolarization Ba2+-sensitive inward currents similar to the inward rectifier current ( I K1) could be observed. The current density at −120 mV was −22.8±2.47 pA/pF ( n =14). A transient outward current ( I to1) could be recorded in all myocytes studied, the current density varied from 0.3 to 8.6 pA/pF with a mean of 3.77±0.47 pA/pF at +40 mV ( n =38). Recovery of I to1 from inactivation was fast (70% recovery within 100 ms), rate-dependent reduction amounted to 38.2% at 4 Hz. A delayed rectifier current was seen in only two out of 38 myocytes (rapid component I Kr). Conclusions: The electrophysiological characteristics of right ventricular myocytes isolated from children with tetralogy of Fallot resemble in most cases subendocardial myocytes from adults. The most prominent difference is a fast recovery from inactivation as well as a small rate dependent reduction of I to1. The observed EADs may have clinical implications.

The aim was to investigate outward currents in single, isolated, human, atrial myocytes and to determine the relative contribution of individual current components to the total outward current. Currents were recorded using the whole-cell patch-clamp technique at 36-37 degreesC. Individual outward current components were estimated from recordings of total outward current using a mathematical procedure based on the inactivation time course of the respective currents. This method allows estimation of outward currents without the use of drugs or conditioning voltage-clamp protocols to suppress individual current components. A rapidly activating and partially inactivating total outward current was recorded when myocytes were voltage clamped at potentials positive to -20 mV (peak current density 24. 0+/-0.97 pA/pF at +40 mV; n=107 cells, 33 patients). This total outward current comprised three overlapping currents: a rapidly inactivating, transient, outward current (Ito1) a slowly and partially inactivating current (ultrarapid delayed rectifier, IKur) and a third current component which most probably reflects a non selective cation current (not characterized). The average current densities at +40 mV were 8.92+/-0.44 pA/pF for Ito1 and 15.1+/-0.72 pA/pF for IKur (n=107 cells). Recovery from inactivation was bi-exponential for both currents and was faster for Ito1. A slowly activating delayed rectifier current (IK) was not found. The current densities of peak Ito1 and IKur varied strongly between individual myocytes, even in those from the same patient. The ratio IKur/Ito1 was 0.5-6.9 with a mean of 1.98+/-0.11 (n=107 cells), suggesting that IKur is the main repolarizing current. The amplitudes of the total outward current, Ito1 and IKur, and the ratio of the latter two were independent of patient age (16-87 years).

1 We used single human atrial myocytes to study If occurrence, properties and pharmacological modulation. Cells were obtained by chunk enzymatic digestion from samples of right atrial appendages of patients undergoing corrective cardiac surgery. 2 Patch-clamped cells in the whole-cell configuration were superfused with a modified Tyrode solution to reduce contamination by interfering currents and to amplify If. The average cell membrane capacitance was 85.06±2.41 pF (n=531). Data were consistent with the geometrical dimensions of the cells (length 94.2±1.89 μm, width 17.9±0.42 μm, n=126). 3 When hyperpolarizing to −120 mV from a holding potential of −40 mV, 252 of 306 tested cells (82%) expressed a hyperpolarization-activated inward current (If density =3.77±0.25 pA pF−1); the current was considered to be present in a given cell if its density at −120 mV was larger than 0.5 pA pF−1. 4 Current activation was sigmoidal and fitted a Boltzmann model; the average activation curve (n=25) showed a maximum current amplitude of 205.97±19.94 pA, corresponding to 3.87±0.63 pA pF−1, voltage of half-maximal activation (V1/2) at −86.68±2.19 mV and a slope of −11.39±0.69 mV. The reversal potential of If measured by tail-current analysis was −13.07±1.92 mV (n=6). The addition of CsCl (5 mM) fully and reversibly blocked the current. 5 In the presence of the β-adrenoceptor agonist isoprenaline (Iso, 1 μM), V1/2 was significantly shifted toward less negative potentials by 6.06±1.96 mV (n=16, P=0.0039). The selective A1-adenosine receptor agonist cyclopentyladenosine (CPA, 1 μM) caused a statistically significant shift of V1/2 toward more negative potentials with respect to the control curve, both in the absence (−7.37±1.83 mV, P=0.0005, n=11) and in the presence of 1 μM Iso (−4.97±1.78, P=0.031, n=6). 6 These results demonstrate that a current with the properties of If described in cardiac primary and secondary pacemakers occurs in the majority of human atrial cells. While the pathophysiological relevance of If in human atrial tissue remains to be defined, our data clearly show that it is modulated through stimulation of β-adrenoceptors and A1-adenosine receptors. British Journal of Pharmacology (1997) 122, 963–969; doi:10.1038/sj.bjp.0701473

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

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

The aim of this study was to investigate the effects of the potassium channel openers (PCOs) cromakalim and pinacidil on the ATP-dependent potassium current I K(ATP) in human atrial myocytes. Cells were isolated from the right atrial appendage obtained during cardiac surgery. Membrane currents were studied with the patch-clamp technique in the whole-cell recording mode at 36°–37°C. Under physiological conditions (4.3 mmol/l ATP in the pipette solution, ATPi) I K(ATP) did not contribute to basal electrical activity. When ATPi was omitted from the pipette solution I K(ATP) activated with a time lag of 4.92±0.92 min (n=6) and was completely inhibited by glibenclamide. Using 4.3 mmol/l ATPi I K(ATP) at +30 mV was increased by 2.04±0.51, 7.24±1.65 and 13.22±3.71 pA/pF (n=7) with 10, 30 and 100 µmol/l cromakalim, respectively, and by 3.24±0.98 (n=6), 4.07±0.48 (n=10) and 3.46±1.23 pA/pF (n=6) with 10, 30 and 100 µmol/l pinacidil, respectively. Control current density was 5.39±0.47 pA/pF (n=39). Using 1 mmol/l ATPi I K(ATP) showed a more pronounced activation (4.81±3.28, n=6; 9.78±2.60, n=7; and 15.1±4.18 pA/pF, n=6; with 10, 30 and 100 µmol/l pinacidil, respectively). I K(ATP) activated by both compounds could be effectively inhibited by glibenclamide. Repetitive exposure to pinacidil (30 µmol/l at 4.3 mmol/l ATPi) caused a potentiation of I K(ATP). Current density at +30 mV was increased by 87% during the first and by 401% during the second pinacidil application (n=5). The data presented in this paper provide new information about electrophysiological characteristics of human atrial I K(ATP) and its modulation by the PCOs cromakalim and pinacidil and suggest species-dependent differences.

Objective: The aim was to investigate the electrophysiological properties of the L-type calcium current ( I Ca,L) in ventricular myocytes at a physiological temperature (36–37°C) isolated from children undergoing surgical repair of tetralogy of Fallot. Methods: I Ca,L was recorded with the patch-clamp technique in the single electrode whole-cell mode at a physiological calcium concentration (1.8 mmol/l) at 36–37°C. Results: Under these conditions, maximum current density averaged −5.80±0.45 pA/pF. I Ca,L showed a bell-shaped current–voltage relationship: the current activated at −37.7±1.36 mV, peaked at +9.41±1.60 mV and reversed at +57.7±2.12 mV ( n =17). At +10 mV, time to peak of I Ca,L was 5.23±0.46 ms. Membrane potentials for half-maximal steady-state activation and inactivation of I Ca,L were −6.02 and −20.4 mV, respectively, the slope factors were 7.16 mV for steady-state activation and 6.49 mV for steady-state inactivation. I Ca,L did not completely inactivate and showed a big window current between −45 and +40 mV. The inactivation of I Ca,L showed a biexponential time course with a fast time constant ranging from 9.11 to 12.9 ms and a slow time constant ranging from 60.9 to 220 ms between −30 and +30 mV. Only the slow time constant showed a pronounced voltage dependency. The recovery from inactivation of I Ca,L was biphasic with a fast time constant of 60.7 ms and a slow time constant of 619 ms. β-Adrenergic stimulation with isoprenaline (1 μmol/l) increased the I Ca,L density from −5.71±1.55 to −13.8±1.96 pA/pF (142%; P

We have studied the inhibitory influence of the class III antiarrhythmic drug ambasilide (LU 47110) on the transient outward current Ito1 and the sustained current Iso following inactivation of Ito1, in human atrial myocytes. The two currents are separated by a mathematical procedure based on the amplitudes and time constants of the biexponential inactivation of the total outward current. The frequency dependence, the recovery from inactivation and the kinetics of activation and inactivation are described. Ambasilide reversibly and concentration dependently inhibited Ito1, Iso and the sodium current INa. Concentration required for half maximal inhibition (IC50) for the effects on Ito1 and Iso were 23.3 mumol/l and 45.7 mumol/l respectively, concentrations shown by others to be effective in terminating and preventing fibrillation in a dog atrial arrhythmia model. Ambasilide not only reduced the amplitude of Ito1 and Iso but also accelerated the time course of inactivation from 14.22 to 6.69 ms and from 202.3 to 87.9 ms respectively. The amplitude of Ito1 showed only a small dependence on stimulation frequency characteristic for human atrial myocytes, whereas Iso was reduced significantly at higher stimulation frequencies. Ambasilide did not change these relationships (0.1-4 Hz) and therefore did not show the reverse use-dependence known from other class III antiarrhythmic agents and which is an important property for a prospective antiarrhythmic drug. The lack of an effect of ambasilide on both steady-state activation and inactivation of Ito1, and the time constant of recovery from inactivation, suggests that ambasilide acts by changing conductance rather than by influencing the gating mechanism. The described characteristics make ambasilide an interesting substance in the group of class III antiarrhythmic drugs.

The effects of adenosine on the L-type Ca2+ current (Ica) were studied in human atrial myocytes using the whole-cell voltage clamp technique. Ica was recorded under physiological calcium concentrations (1.8 mmol/l) at 37 degrees C. Under these conditions the current density of basal Ica averaged 4.0 pA/pF. Isoprenaline (1 mumol/l) increased basal Ica to 249.7%. Adenosine (100 mumol/l) in the presence of isoprenaline (1 mumol/l) decreased Ica from the level obtained with isoprenaline to 87.5% of basal Ica. Adenosine (0.1 to 100 mumol/l) also reduced basal Ica, maximally to 64.5% of control. Activation and inactivation parameters of basal Ica were not significantly different between adenosine (100 mumol/l) and control recordings. Our results show that adenosine affects both basal and isoprenaline stimulated Ica in human atrial myocytes. Although a considerable decrease of basal Ica was seen, we conclude that the action of adenosine on L-type Ca2+ current in human atrial myocytes is mainly antiadrenergic. Both effects may contribute to the antiarrhythmic properties of adenosine.