Brigitte Pelzmann

The pacemaker current If in single human atrial myocytes and the effect of β-adrenoceptor and A1-adenosine receptor stimulation

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.

L-type calcium current in human ventricular myocytes at a physiological temperature from children with tetralogy of Fallot

OBJECTIVE The aim was to investigate the electrophysiological properties of the L-type calcium current (ICa,L) in ventricular myocytes at a physiological temperature (36-37 degrees C) isolated from children undergoing surgical repair of tetralogy of Fallot. METHODS ICa,L was recorded with the patch-clamp technique in the single electrode whose-cell mode at a physiological calcium concentration (1.8 mmol/l) at 36-37 degrees C. RESULTS Under these conditions, maximum current density averaged -5.80 +/- 0.45 pA/pF. ICa,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 ICa,L was 5.23 +/- 0.46 ms. Membrane potentials for half-maximal steady-state activation and inactivation of ICa,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. ICa,L did not completely inactivate and showed a big window current between -45 and +40 mV. The inactivation of ICa,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 ICa,L was biphasic with a fast time constant of 60.7 ms and a slow time constant of 619 ms. beta-Adrenergic stimulation with isoprenaline (1 mumol/l) increased the ICa,L density from -5.71 +/- 1.55 to -13.8 +/- 1.96 pA/pF (142%; P < 0.05) at +10 mV. CONCLUSIONS The present study demonstrates that most of the electrophysiological properties of ICa,L in ventricular myocytes isolated from children with tetralogy of Fallot resemble those of adult ventricular cells. The existence of a big calcium window current could be involved in the occurrence of early afterdepolarizations which could lead to the high incidence of arrhythmias after surgical repair of tetralogy of Fallot.

Effects of the class III antiarrhythmic drug ambasilide on outward currents in human atrial myocytes

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 Tso 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 μmol/l and 45.7 μmol/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.

Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics

Nitric oxide () is a free radical with a wide range of biological effects, but practically impossible to visualize in single cells. Here we report the development of novel multicoloured fluorescent quenching-based probes by fusing a bacteria-derived -binding domain close to distinct fluorescent protein variants. These genetically encoded probes, referred to as geNOps, provide a selective, specific and real-time read-out of cellular dynamics and, hence, open a new era of bioimaging. The combination of geNOps with a Ca2+ sensor allowed us to visualize and Ca2+ signals simultaneously in single endothelial cells. Moreover, targeting of the probes was used to detect signals within mitochondria. The geNOps are useful new tools to further investigate and understand the complex patterns of signalling on the single (sub)cellular level.

The sulphonylurea glibenclamide inhibits voltage dependent potassium currents in human atrial and ventricular myocytes

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<0.001) and 101±20 ms (P<0.05) under glibenclamide (100 μM). Our data suggest, that both repolarizing currents in human atrial myocytes, the transient outward current (Ito1) and the ultrarapid delayed rectifier current (IKur) were inhibited by glibenclamide. In human ventricular myocytes glibenclamide inhibited Ito1 without affecting the late current. Our data suggest that glibenclamide inhibits human voltage dependent cardiac potassium currents at concentrations above 10 μM.

NADH supplementation decreases pinacidil-primed IK(ATP) in ventricular cardiomyocytes by increasing intracellular ATP

The aim of this study was to investigate the effect of nicotinamide‐adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea‐pig ventricular cardiomyocytes. The pinacidil‐primed ATP‐dependent potassium current IK(ATP) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured. Membrane currents were studied using the patch‐clamp technique in the whole‐cell recording mode at 36–37°C. Adenine nucleotides were determined by HPLC. Under physiological conditions (4.3 mM ATP in the pipette solution, ATPi) IK(ATP) did not contribute to basal electrical activity. The ATP‐dependent potassium (K(ATP)) channel opener pinacidil activated IK(ATP) dependent on [ATP]i showing a significantly more pronounced activation at lower (1 mM) [ATP]i. Supplementation of cardiomyocytes with 300 μg ml−1 NADH (4–6 h) resulted in a significantly reduced IK(ATP) activation by pinacidil compared to control cells. The current density was 13.8±3.78 (n=6) versus 28.9±3.38 pA pF−1 (n=19; P<0.05). Equimolar amounts of the related compounds nicotinamide and NAD+ did not achieve a similar effect like NADH. Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6±1.88 nmol mg−1 protein versus control: 35.4±2.57 nmol mg−1 protein, P<0.000005). These data show that supplementation of guinea‐pig ventricular cardiomyocytes with NADH results in a decreased activation of IK(ATP) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP.

Adenosine inhibits the L-type calcium current in human atrial myocytes

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.

If blocking potency of ivabradine is preserved under elevated endotoxin levels in human atrial myocytes

Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS–HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial.

Estimation of outward currents in isolated human atrial myocytes using inactivation time course analysis

Abstract 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°C. 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).

Repolarizing currents in ventricular myocytes from young patients with tetralogy of Fallot

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 degrees 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 Ba(2+)-sensitive inward currents similar to the inward rectifier current (IKl) could be observed. The current density at -120 mV was -22.8 +/- 2.47 pA/pF (n = 14). A transient outward current (Itol) 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 Itol 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 IKr). 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 Itol. The observed EADs may have clinical implications.