Identification of a cardiac sodium channel insensitive to synthetic modulators.

BACKGROUND: DPI 201-106 (DPI) was the first synthetic compound showing cardioselective modulation of voltage-gated sodium channels (VGSCs) resulting in a positive inotropic effect. Currently, the exact mode of action for this class of compounds is not known. METHODS: Effects of different natural and synthetic sodium channel modulators were investigated in cardiac tissue of several species with conventional electrophysiologic methods. RESULTS: In electrically driven cardiac tissues, all compounds investigated increased force of contraction (FC) and action potential duration (APD) with increasing concentrations except for DPI in cattle trabecular muscle, which demonstrated no effect. Interestingly, calculation of EC50 levels at 30% repolarization demonstrates that natural VGSC-ligands were highly potent in prolonging the APD in cattle whereas no positive trends could be obtained for DPI and SDZ 211-939 (SDZ) in cattle. CONCLUSIONS: These results demonstrate that the binding site for DPI and SDZ is distinct from sites 2 or 3 of the VGSC alpha-subunit. Moreover, this is the first time that these compounds show no effect or even shortening of APD. This finding will enable the characterization of the mode of action and probably the binding site for synthetic VGSC-modulators.

Generating and influencing Torsades de Pointes--like polymorphic ventricular tachycardia in isolated guinea pig hearts.

Torsades de Pointes (TdP) is a polymorphic ventricular arrhythmia which can degenerate into ventricular fibrillation. The most typical symptom of TdP is the ECG morphology where QRS complexes seem to rotate around the isoelectric baseline. Bradycardia and delayed repolarization are regarded as pathophysiologic predispositions. For better understanding of the pathophysiology and the evaluation of therapeutic or proarrhythmic potential of drugs, a functional experimental model is needed. In the present study, an experimental model of polymorphic tachyarrhythmias taken as TdP equivalents in isolated guinea pig hearts was developed. The hearts were perfused by the Langendorff technique. Bradycardia was induced by dissection of the sinus node, and prolongation of the QT interval by infusion of two inhibitors of the sodium channel inactivation, veratridine and DPI 201-106. TdP equivalents were triggered reproducibly by application of electrical single stimuli at the end of the T wave. Experiments with different concentrations of the channel active substances alone and in combination, with different perfusion times and mode of electrical stimulation (single pulse versus train stimulation), showed the highest incidence for TdP equivalents by means of an initial 30 min long infusion of 0.5 microM each veratridine and DPI 201-106 in combination with electrical single stimuli. After finishing the infusion with the channel active substances but still with lasting effects from them. TdP equivalents were triggered repeatedly in five of six experiments. The reasons for this increased TdP susceptibility after finishing the infusion are not known. In a separate series of six similarly arranged experiments, the incidence for TdP equivalents could be decreased from 83% to 12.5% (p < 0.001) by increasing the concentration of magnesium in the perfusate from 1.17 to 5.0 mM. With these experiments, the clinically known therapeutic effect of magnesium suppressing TdP could be demonstrated in an in vitro model for the first time. The results suggest that this model could be used as a base for further studies of clinical relevant drugs, especially antiarrhythmic agents, to obtain hints of possible risks of proarrhythmic effects or of suitability for therapeutic use at TdP attacks.

Effects of the new class III antiarrhythmic drug E-4031 on myocardial contractility and electrophysiological parameters.

The effects of the new class III antiarrhythmic agent E-4031 were investigated in different guinea pig cardiac preparations. In left atria, E-4031 (10(-8)-10(-5) M) prolonged the functional refractory period up to 45% and reduced the frequency of spontaneously beating right atria by 32%. In papillary muscles, E-4031 (3 x 10(-8)-3 x 10(-7) M) reversibly prolonged the action potential duration (APD70) of fast and slow APs by 68 and 51%, respectively. Vmax, resting potential, and AP amplitude (APA) were not altered. In isolated ventricular myocytes, E-4031 reversibly prolonged the APD90 from 275 ms (control) to 1,496 ms (10(-6) M), pD2 value 6.5. The current changes that underlie the AP-prolonging effect were also studied in ventricular myocytes: in concentrations up to 10(-5) M), E-4031 did not affect the Na+ or Ca2+ inward current but reduced the delayed rectifier (IK) tail current by 76% (10(-7) M). Contractility was enhanced by E-4031 in isolated atria by 20% (3 x 10(-7) M) and increased cell shortening in ventricular myocytes. Thus, the class III antiarrhythmic action of E-4031 is due to a selective reduction of outward currents.

Identification of cardiotonic sodium channel activators by potassium depolarization in isolated guinea-pig atria.

The inotropic actions of various drugs known to increase force of contraction in isolated mammalian cardiac muscle were investigated in electrically driven (1 Hz) guinea-pig left atria under both normal [K+]o (4.7 mM) and high [K+]o (22 mM). Under normal [K+]o a concentration-dependent increase in force of contraction could be confirmed with the beta-adrenoceptor agonist, isoprenaline, the cyclase activator, forskolin, the inhibitors of the cyclic AMP-phosphodiesterase (PDE), amrinone, IBMX, and OPC 8212, the Na+ channel activators, DPI 201-106, SDZ 210-921, veratridine, and ATX II, the Na(+)-ionophore monensin, the inhibitor of Na+/K(+)-ATPase, ouabain, and the Ca2+ channel activators, Bay K 8644, CGP 28 H 392, and SDZ 202-791. Partial depolarization of the muscle preparations by increasing [K+]o in the organ bath to 22 mM completely abolished the positive inotropic action of the Na+ channel-activating drugs. In contrast, the effects of the other compounds were still present, although changes in the maximal force development were observed. The efficacy of the PDE inhibitors amrinone and IBMX were slightly increased; the maximal effects of isoprenaline, monensin, forskolin, and OPC 8212 were unchanged; the effect of ouabain decreased to about half maximal values; while the efficacy of the Ca2+ channel activators were either unchanged (CGP 28 392) or decreased (Bay K 8644 and SDZ 202-791). The results suggest that inactivation of cardiac fast Na+ channels by partially depolarizing isolated, electrically driven atria is a suitable model to distinguish between cardiotonic agents acting through activation of Na+ channels and those with other mechanisms of action.

Evidence for different receptor sites for the novel cardiotonic S-DPI 201-106, ATX II and veratridine on the cardiac sodium channel.

DPI 201-106 (4-[3-(4-diphenylmethyl-1-piperazinyl)-2-hydroxypropoxy]-1 H-indole-2-carbonitrile, DPI) is a novel cardiotonic which prolongs the open state of the cardiac sodium channel. The interactions of the sea anemone toxin ATX II and of veratridine with the active enantiomer of (racemic) DPI, S-DPI, were investigated by making electrophysiologic measurements in guinea-pig papillary muscles. The prolongation of the action potential duration (APD) by S-DPI was potentiated in the presence of ATX II, suggesting that the two agents bound to different sites. Veratridine alone increased APD slowly over 4 h. S-DPI greatly accelerated and potentiated the effect of veratridine. Conversely, veratridine increased the efficacy of S-DPI but decreased its potency, both about 4-fold. All effects were TTX-sensitive. It is concluded that the three agents bind to different sites at the cardiac Na+ channel and that these sites are allosterically coupled.