Helmut A. Tritthart

A mechanistic interpretation of the action of toxin II from Anemonia sulcata on the cardiac sodium channel

Cardiac sodium channels, modified by Anemonia sulcata toxin II, have been analyzed by the patch-clamp method. The open state of the modified sodium channels proved to be prolonged highly significantly and reopening from a closed state denoted c*-state frequently occurred, interrupted by silent periods, denoted i*-state. Activation from the c*-state was apparently not affected by toxin action, whereas activation from the i*-state was markedly prolonged. Upon higher depolarizations toxin-induced sodium channels disappeared and this behaviour has been attributed to dissociation of the toxin from the channel by use of a special pulse-protocol. The onset of the toxin effect on the action potential proved to depend on stimulation, and it is concluded that the toxin binds preferentially to the open (o)-state. Taking together the results, a kinetic scheme is suggested for action of the toxin on the cardiac sodium channel.

Assessment of the conduction of the cardiac impulse by a new epicardiac surface and stimulation technique (SST-ECG) in Langendorff perfused mammalian hearts.

The assessment of the effects on the conduction of the cardiac impulse and of refractoriness simultaneously from multiple cardiac structures is important to evaluate the mode of action of new compounds, as well as to investigate undesired cardiac side effects. The measurements of intracardiac electrical activities of special structures have required catheter methods or averaging techniques to produce clear deflections of low-level potential wave forms. We have developed an epicardial surface electrocardiogram (S-ECG) recording technique to detect continuously sinus node and His-bundle activities in spontaneously beating Langendorff perfused heart preparations. A bipolar surface stimulation technique electrocardiogram (ST-ECG) could be combined with this S-ECG method to assess the effect of pacing at maximal rate on the refractoriness of the sino-atrial, AV-nodal, and His-bundle conduction, as well as that of the atrial and ventricular myocardium. Epicardial surface electrodes were also used for vector analysis of the signals, which include those of the sinus node and His-bundle potentials, in addition to the usual atrial and ventricular loops. The present results demonstrate that this new epicardial surface ECG recording technique, combined with the described SST-ECG, provided a way to improve or replace more elaborate intracardiac techniques used in isolated hearts or in animal experiments.

Isoproterenol, norepinephrine and phosphodiesterase inhibitors are blockers of the depressed fast Na+-system in ventricular muscle fibers

It is generally agreed that β-adrenergic transmitters do not influence the maximum rate of rise of action potentials of cardiac fibers with an intact fast Na+-system. Inhibitory effects of epinephrine have only been reported for a Mg-activated fast current [9]. Fibers with a depressed fast Na+-system have been studied under various experimental conditions, some of them mimicking infarct-like environments [1,5]. In some respects the depressed fast sodium-system reacts differently, e.g. recovery from inactivation [3], Ca2+-effects [10], TTX-influences [6] and susceptibility to antiarrhythmic drug influence [4]. We have studied the influence of isoproterenol, norepinephrine and of phosphodiesterase inhibitors on the depressed fast Na+-system in guinea-pig papillary muscles and gained results which are completely different to those in fibers with intact fast Na+-system.

Calcium ion effects on the rising phases of action potentials obtained from guinea-pig papillary muscles at different potassium concentrations.

Abstract In guinea-pig papillary muscles, action potentials, rates of rise ( v max and conduction velocities were measured at various potassium concentrations. An increase in potassium reduced v max in an S-shaped relation to the membrane potential but conduction velocity was influenced differently. At 18 to 20 m m K 0 + , the rising phase often included a remainder of the fast depolarization and v max showed two peaks; the first peak could be abolished by TTX (10 −6 m ), the second peak by D600 (1 μg/ml, 1 Hz). The v max (second peak) of the rising phase of the slow responses as well as overshoot and amplitude was clearly correlated ( r = 0.99) to the log of Ca 0 2+ . The increase in Ca 0 2+ concentration caused an elevation of both overshoot and amplitude of 34.22 mV per decade. In contrast, the v max of the first peak was correlated to the linear Ca 0 2+ concentration. At K 0 + 20 m m increase of Ca 0 2+ led to a marked increase of the first and Na + -dependent peak of v . This v max was increased 4.23 times by a Ca 0 2+ increase from 2.5 to 10 m m . Accordingly, impaired conduction due to 20 m m K 0 + was almost normalized by 10 m m Ca 0 2+ . A Ca 0 2+ -increase (2.5 to 10 m m ) shifted the curve of Na + -inactivation by 4.26 mV in the depolarization direction and reduced v max at normal membrane potential. The results confirm the dependence of the rising phase and the overshoot of slow responses to the log Ca 0 2+ but indicate strong and simultaneous Ca 0 2+ -effects on recovery of the Na + -system which may add to or even dominate the overall changes in v max of slow responses.

Effect of dequalinium on K1735-M2 melanoma cell growth, directional migration and invasion in vitro.

Cationic lipophilic compounds have an antiproliferative effect on certain tumour systems in vitro and in vivo. We have investigated whether the cationic lipophilic compound dequalinium affects not only proliferation but also motility and invasion of the highly metastatic and highly invasive melanoma cell line K1735-M2. Proliferation was assessed in monolayer cultures and in multicellular spheroids, motility was estimated in the assay of directional migration, and invasiveness was tested through confrontation cultures of tumour multicellular spheroids with embryonic chick heart tissue evaluated by computerized image analysis. 2 mumol/l dequalinium impaired melanoma cell proliferation, reduced directional migration and significantly blocked invasion in vitro. On the ultrastructural level, dequalinium caused obvious changes in mitochondria of both melanoma and embryonic chick heart cells. The mechanisms of the antiproliferative, antimigrating and antiinvasive effects remain to be determined. Inhibition of protein kinase C, calmodulin antagonism, DNA intercalation and/or direct effects on mitochondrial functions may be considered.

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.

Modulation of cardiac impulse generation and conduction by nifedipine and verapamil analyzed by a refined surface ECG technique in Langendorff perfused guinea pig hearts

SummaryUsing a modified Langendorff system, a special ECG recording technique and appropriate placement of two silver wire electrodes, early atrial and His bundle activity can be detected continuously from the surface of intact and spontaneously beating guinea pig hearts. This new method was applied to measure the direct and inhibitory effects of nifedipine and verapamil on impulse generation and conduction in isolated and perfused guinea pig hearts. Depression of sinoatrial conduction was the most prominent effect of nifedipine. In all concentrations applied (10−7 M, 10−6 M, 10−5 M) nifedipine predominantly led to sinoatrial blocks of different degrees. Heart rate decreased slightly in a dosedependent manner. PQ and HV duration remained essentially constant. In the highest concentration of nifedipine (10−5 M), sinus node activity was so depressed that AV dissociation or ventricular rhythm developed. Only in one out of eight experiments with cumulative increase of nifedipine concentrations to 10−5 M was the AV node affected by nifedipine and a second-degree AV block developed (10−6 M).Verapamils inhibitory effects on the rate of impulse initiation in the sinus node were more pronounced than those of nifedipine, but the inhibition of sinoatrial conduction by verapamil was less marked. At 10−6 M verapamil, the incidence of sinoatrial blocks and of ventricular rhythm was similar to the incidence of first degree AV blocks. PQ time (+14%) but also HV time (+12%) were prolonged under the influence of this concentration of verapamil. At the highest concentration of verapamil (10−5 M) applied for 10 min, ventricular rhythm developed in five out of eight experiments, as well as one second and two third-degree AV blocks.The results confirm that the simultaneous measurement of sinus node activity of sinoatrial and atrioventricular conduction and of HV duration is feasible with this ECG technique, to evaluate the inhibitory effects of Ca-antagonists on sinus and AV node activity in the intact heart.

CP-96,345, a non-peptide antagonist of substance P. III. Cardiovascular effects in mammals unrelated to actions on substance P receptors.

SummaryThe cardiovascular effects of CP-96,345, a non-peptide antagonist of substance P, were analyzed in vivo and in vitro. In the anaesthetized rat, the i.v. injection of 3 µmolkg−1 of CP-96,345 induced a fall in mean arterial blood pressure and a reduction in heart rate. Similar effects were obtained with the enantiomer CP-96,344 ((2R,3R)-cis-isomer of CP-96,345) which does not interact with substance P receptors. Both enantiomers, at a concentration of 10 gM, decreased the beating frequency of the isolated atria and of the isolated perfused heart of the guinea-pig to a similar extent, and caused transient coronary dilatation. CP-96,345 (10 µM) decreased the spontaneous sinus rate, prolonged the atrioventricularnodal conduction interval and the His-bundle conduction interval of the perfused guinea-pig heart. The intraventricular spread of conduction was markedly inhibited. During programmed stimulation 10 min after the beginning of the drug application, the effective refractory periods evaluated by stimulation with premature beats, as well as rate dependent effective refractory periods, of the atrioventricular node, of the atrial and of the ventricular myocardium, were prolonged. Sinus node recovery time was also prolonged. It was concluded that these cardiac effects of CP-96,345 were not caused by an action of the compound on substance P receptors.

Continuous ECG measurements of intracardiac activity from the surface of Langendorff-perfused guinea pig hearts

SummaryThe present paper describes a method by which it is possible to continuously detect early atrial and His-bundle activity from the surface of intact Langendorff-perfused guinea-pig hearts, by appropriate placement of two electrodes and the use of a custom designed instrumentation-amplifier. In some experiments the surface ECG recordings were compared with intracardiac ECG recordings. No difference in ECG durations could be observed between intracardiac and extracardiac measurements. In further experiments changes in ECG durations and heart rate were measured for 2 h. After 30 min equilibration time, no changes in heart rate and conduction time could be observed. In order to locate the best surface electrode positions to detect His-bundle activity, vector ECG recordings were taken at high gain. This vector ECG signal contained a His-loop which was split into a larger and a smaller part. The main and initial vector was directed to the left and the smaller to the right ventricle. The best recordings of the His-bundle activity could be observed when the electrodes were positioned as follows; one in a posterior position, near the valve plane and the other one in the opposite position near the initial part of the anterior interventricular artery and in the direction of the large His-loop.We conclude that the ECG surface recordings are a valuable tool for measuring impulse propagation through various segments of the cardiac conduction system in preparations of guinea-pig hearts, perfused by the Langendorff method.

Stereoselective electrophysiological effects of propafenone in Langendorff perfused guinea pig hearts.

SummaryThe present study was focused on the stereoselective electrophysiological effects of (R)-and (S)-propafenone·HCl evaluated in isolated Langendorff perfused guinea pig hearts. Conduction intervals were measured using an ECG-recording method of high resolution. Refractory periods of the different parts of the myocardium were determined by stimulation with premature stimuli, as well as by stimulation with increasing pacing rate (rate-dependent/refractory periods). Drug concentrations of 0.1, 1 and 3 μM were tested. Both compounds induced a dose-dependent increase in AV-nodal, His-bundle, and intraventricular conduction time which reached significance (p<0.01) following 3 μM of either compound. Sinus rate was also dose-dependently and significantly reduced.(R)- and (S)-propafenone·HCl induced a marked prolongation of the rate-dependent refractory period of sino-atrial (by 140±22%, p<0.01 and by 141±14%, p<0.01, respectively) and AV-nodal (by 34±22%, p<0.01 and by 42±15%, p<0.01, respectively) conduction and of the atrial (by 182±21%, p<0.01 and by 195±15%, p<0.01, respectively) and ventricular (by 93±16%, p<0.01 and by 88±16%, p<0.01, respectively) myocardium. The effective refractory periods evaluated by stimulation with premature stimuli were also significantly prolonged under the influence of (R)- and (S)-propafenone·HCl, except the ventricular myocardial refractoriness by (R)-propafenone·HCl (increase to 114±23%, n.s.). Both compounds showed a strong rate-dependence of their effects and, thus, the refractory periods evaluated by stimulation with increasing pacing rate were significantly more prolonged than the refractory periods evaluated by stimulation with premature stimuli. The main difference between the effects of (R)- and (S)-propafenone·HCl on the cardiac electrical activity is the lack of effect of (R)-propafenone·HCl on the ventricular myocardial refratoriness evaluated by stimulation with premature stimuli.