Bernd Koidl

Impaired regulation of cardiac function in sepsis, SIRS, and MODS.

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.

Di-4-ANEPPS causes photodynamic damage to isolated cardiomyocytes

Action potential recordings from isolated guinea pig ventricular cells in the whole-cell recording mode were used to study the toxic and photodynamic properties of the voltage-sensitive fluorescent dye di-4-ANEPPS. Staining of the cardiomyocytes with di-4-ANEPPS (30 or 60 μM; 10 min) did not alter the action potential shape. When the stained cells were illuminated (1W/cm2) severe effects on the action potential were observed. There was a prolongation of the action potential duration, occurrence of early afterdepolarizations, reduction of the membrane resting potential and eventually inexcitability. Addition of the antioxidant catalase (100 IU/ml) to the extracellular solution delayed the onset of these effects, suggesting that reactive-oxygen-intermediates take part in di-4-ANEPPS induced photodynamic damage. Since di-4-ANEPPS is a very important tool for optical membrane potential recordings in heart tissue and single cardiomyocytes catalase might be useful in suppressing photodynamic damage during optical potential recordings.

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.

Selectivity of different calcium antagonists on T- and L-type calcium currents in guinea-pig ventricular myocytes.

Both L- and T-type calcium channels are present in the heart. In cardiac myocytes L-type calcium channels are blocked by the classical calcium channel blockers, while T-type calcium channels are thought to be insensitive to these drugs and to be selectively blocked by mibefradil. We aimed to compare the T/L calcium channel blocking selectivity of several calcium channel blockers by evaluating their effects on both components evoked in the same cell from a holding potential corresponding to the normal physiological value (-90mV). Currents were recorded in single patch-clamped guinea-pig ventricular myocytes, superfused with a Na(+)- and K(+)-free solution to abolish overlapping currents. Two dihydropyridines (amlodipine and lacidipine), verapamil diltiazem and mibefradil were tested; for each compound concentrations equieffective on L-type Ca(2+) current were used. All calcium channel blockers, at concentrations blocking less than 30% of L-type Ca(2+) current, inhibited a significant amount of T-type Ca(2+) current, varying from 0.8% (diltiazem) to 28% (mibefradil). We calculated for each compound the T/L ratio. As expected, mibefradil showed the highest T selectivity; lacidipine and diltiazem resulted to be L selective. Verapamil and amlodipine were not selective. Thus, the calcium channel blockers can be differentiated on the basis of their T/L selectivity.

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.

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.

A novel benzothiazine Ca2+ channel antagonist, semotiadil, inhibits cardiac L-type Ca2+ currents

The influence of semotiadil fumarate, a novel vasoselective Ca2+ channel antagonist with a benzothiazine skeleton, was measured on the high-threshold Ca2+ current ICa,L in guinea-pig ventricular myocytes prepared by coronary perfusion with collagenase solution. Patch- and voltage-clamp methods were used to measure ICa,L. Diltiazem, nifedipine and amlodipine were studied for comparison. Samotiadil could be shown to inhibit ICa,L in a dose-dependent manner in concentrations similar to those of diltiazem but was less effective than amlodipine and nifedipine. The IC50 for nifedipine and amlodipine was in the range between 0.1 and 1 microM and that of semotiadil and diltiazem was between 10 and 100 microM. Recovery from inactivation of ICa,L in the control and under the influence of nifedipine 0.01 microM) and amlodipine (0.1 microM) was complete alter I. Semotiadil (0.1 microM) and diltiazem (1 microM) prolonged the time to full recovery to 20 s. This significant delay in the recovery of ICa,L produced by semotiadil indicates a mode of action similar to that of the verapamil type of Ca2+ channel antagonists and masses a clear distinction between it and the dihydropyridines, which have no effect on the recovery process. The rate dependence of the effect in combination with a distinct influence of the holding potential underlines the use dependence of the mechanism underlying the effect of semotiadil. The well-known high vasoselectivity of semotiadil in combination with a relatively low Ca2+ channel antagonistic influence on the heart makes semotiadil an interesting candidate for the treatment of coronary heart diseases.

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.