Milan Stengl

Increased Short-Term Variability of Repolarization Predicts d-Sotalol–Induced Torsades de Pointes in Dogs

Background—Identification of patients at risk for drug-induced torsades de pointes arrhythmia (TdP) is difficult. Increased temporal lability of repolarization has been suggested as being valuable to predict proarrhythmia. The predictive value of different repolarization parameters, including beat-to-beat variability of repolarization (BVR), was compared in this serial investigation in dogs with chronic AV block. Methods and Results—In anesthetized dogs with electrically remodeled hearts, the dose-dependent difference in drug-induced TdP (d-sotalol, 2 and 4 mg/kg IV over 5 minutes, 25% and 75% TdP, respectively) could not be accounted for by prolongation of QTc (410±37 to 475±60 versus 415±47 to 484±52 ms, respectively). BVR was quantified by Poincaré plots at baseline and immediately before onset of d-sotalol–induced extrasystolic activity. TdP occurrence was associated with an increase in short-term variability (STV) of the left ventricular monophasic action potential duration (3.5±1.5 to 5.5±1.6 versus 3.0±0.7 to 8.6±3.8 ms, respectively), which was reversible when TdP was abolished by IK,ATP activation. The absence of TdP despite QTc prolongation after chronic amiodarone treatment could also be explained by an unchanged STV. In experiments with isolated ventricular myocytes, STV increased after IKr block and was highest in cells that subsequently showed early afterdepolarizations. Conclusions—Proarrhythmia is not related to differences in prolongation of repolarization but corresponds to BVR, here quantified as STV of the left ventricle. STV could be a new parameter to predict drug-induced TdP in patients.

Abrupt rate accelerations or premature beats cause life-threatening arrhythmias in mice with long-QT3 syndrome

Deletion of amino-acid residues 1505–1507 (KPQ) in the cardiac SCN5A Na+ channel causes autosomal dominant prolongation of the electrocardiographic QT interval (long-QT syndrome type 3 or LQT3). Excessive prolongation of the action potential at low heart rates predisposes individuals with LQT3 to fatal arrhythmias, typically at rest or during sleep. Here we report that mice heterozygous for a knock-in KPQ-deletion (SCN5AΔ/+) show the essential LQT3 features and spontaneously develop life-threatening polymorphous ventricular arrhythmias. Unexpectedly, sudden accelerations in heart rate or premature beats caused lengthening of the action potential with early afterdepolarization and triggered arrhythmias in Scn5aΔ/+ mice. Adrenergic agonists normalized the response to rate acceleration in vitro and suppressed arrhythmias upon premature stimulation in vivo. These results show the possible risk of sudden heart-rate accelerations. The Scn5aΔ/+ mouse with its predisposition for pacing-induced arrhythmia might be useful for the development of new treatments for the LQT3 syndrome.

Probing the Contribution of IKs to Canine Ventricular Repolarization: Key Role for β-Adrenergic Receptor Stimulation

Background—In large mammals and humans, the contribution of IKs to ventricular repolarization is still incompletely understood. Methods and Results—In vivo and cellular electrophysiological experiments were conducted to study IKs in canine ventricular repolarization. In conscious dogs, administration of the selective IKs blocker HMR 1556 (3, 10, or 30 mg/kg PO) caused substantial dose-dependent QT prolongations with broad-based T waves. In isolated ventricular myocytes under baseline conditions, however, IKs block (chromanols HMR 1556 and 293B) did not significantly prolong action potential duration (APD) at fast or slow steady-state pacing rates. This was because of the limited activation of IKs in the voltage and time domains of the AP, although at seconds-long depolarizations, the current was substantial. Isoproterenol increased and accelerated IKs activation to promote APD95 shortening. This shortening was importantly reversed by HMR 1556 and 293B. Quantitatively similar effects were obtained in ventricular-tissue preparations. Finally, when cellular repolarization was impaired by IKr block, IKs block exaggerated repolarization instability with further prolongation of APD. Conclusions—Ventricular repolarization in conscious dogs is importantly dependent on IKs. IKs function becomes prominent during &bgr;-adrenergic receptor stimulation, when it promotes AP shortening by increased activation, and during IKr block, when it limits repolarization instability by time-dependent activation. Unstimulated IKs does not contribute to cellular APD at baseline. These data highlight the importance of the synergism between an intact basal IKs and the sympathetic nervous system in vivo.

Accumulation of slowly activating delayed rectifier potassium current (IKs) in canine ventricular myocytes

In guinea‐pig ventricular myocytes, in which the deactivation of slowly activating delayed rectifier potassium current (IKs) is slow, IKs can be increased by rapid pacing as a result of incomplete deactivation and subsequent current accumulation. Whether accumulation of IKs occurs in dogs, in which the deactivation is much faster, is still unclear. In this study the conditions under which accumulation occurs in canine ventricular myocytes were studied with regard to its physiological relevance in controlling action potential duration (APD). At baseline, square pulse voltage clamp experiments revealed that the accumulation of canine IKs could occur, but only at rather short interpulse intervals (< 100 ms). With action potential (AP) clamp commands of constant duration (originally recorded at rate of 2 Hz), an accumulation was only found at interpulse intervals close to 0 ms. Transmembrane potential recordings with high‐resistance microelectrodes revealed, however, that at the fastest stimulation rates with normally captured APs (5 Hz) the interpulse interval exceeded 50 ms. This suggested that no IKs accumulation occurs, which was supported by the lack of effect of an IKs blocker, HMR 1556 (500 nM), on APD. In the presence of the β‐adrenergic receptor agonist isoproterenol (isoprenaline, 100 nM) the accumulation with AP clamp commands of constant duration was much more pronounced and a significant accumulating current was found at a relevant interpulse interval of 100 ms. HMR 1556 prolonged APD, but this lengthening was reverse rate dependent. AP clamp experiments in a physiologically relevant setting (short, high rate APs delivered at a corresponding rate) revealed a limited accumulation of IKs in the presence of isoproterenol. In conclusion, a physiologically relevant accumulation of IKs was only observed in the presence of isoproterenol. Block of IKs, however, led to a reverse rate‐dependent prolongation of APD indicating that IKs does not have a dominant role at short cycle lengths.

Extracellular divalent cations block a cation non‐selective conductance unrelated to calcium channels in rat cardiac muscle

1 The effect of removing extracellular divalent cations on resting potential (Vrest) and background conductance of rat cardiac muscle was studied. Vrest was measured with 3 M KCl‐filled microelectrodes in papillary muscles, or with a patch electrode in ventricular myocytes. Whole‐cell membrane currents were measured in myocytes using step or ramp voltage commands. 2 In both muscles and single cells, decrease or removal of Ca2+o and Mg2+o caused a nifedipine‐resistant depolarization, which was reversed upon readmission of Ca2+o or Mg2+o (half‐maximal effect at 0.8 mM Ca2+o or 3 mM Mg2+o in muscles). 3 In single myocytes, removal of Ca2+o and Mg2+o had no effect on the seal resistance in non‐ruptured cell‐attached recordings, but reversibly induced a current with a reversal potential (Vrev) of −8 ± 3.4 mV (with internal Cs+; mean. s.e.m., n= 23) during whole‐cell recordings. The current was insensitive to nifedipine (3–100 μM) or amiloride (1 mM). Vrev was insensitive to changes in the equilibrium potential for chloride ions (Ecl. 4 The current induced in the absence of extracellular divalent cations was blocked in a concentration‐dependent manner by Ca2+o. (At −80 mV, the affinity constant Kca was 60 μM with a Hill coefficient of 0.9.) KCa was voltage dependent at positive but not negative potentials. Mg2+o, Ni2+o, Sr2+o, Ba2+o, Cd2+o and Gd3+o also blocked the current. 5 In 0 mM Na+ (145 mM NMDG+), the inward component of the divalent cation‐sensitive current was decreased and Vrev shifted to more negative potentials. 6 These results suggest that a novel conductance pathway, permeable to monovalent cations but not to OF and blocked by divalent cations, exists in ventricular myocytes.

Sudden cardiac death in dogs with remodeled hearts is associated with larger beat–to–beat variability of repolarization

AbstractIncreased proarrhythmia in dogs with chronic AV block (AVB) has been explained by ventricular remodeling causing a decrease in repolarization reserve. Beat–to–beat variability of repolarization (BVR) has been suggested to reflect repolarization reserve, in which high variability represents diminished reserve and larger propensity for repolarization–dependent ventricular arrhythmia. A subset of chronic AVB dogs (10%) suffers sudden cardiac death (SCD). With the assumption that repolarization defects constitute a potentially lethal proarrhythmic substrate, we hypothesized that BVR in SCD dogs are larger than in matched control chronic AVB dogs.From a population of 200 chronic AVB dogs, initially two groups were chosen retrospectively: 8 dogs that died suddenly (SCD) and 8 control dogs. Control dogs had a longer lifespan after AVB (10 to 18 weeks) than SCD dogs (5 to 10 weeks). All dogs had undergone electrophysiological testing under anesthesia where ECG, left and right ventricular endocardial monophasic action potentials (MAP) were recorded. BVR was assessed from 30 consecutive beats, illustrated by Poincaré plots and was the only parameter discriminating between SCD and control group. All other electrophysiological parameters (RR, QT and MAP durations) were comparable for the two groups. Extending the number of animals and groups confirmed a larger BVR in the SCD group (SCD: 5.1 ± 2.7; n = 11 versus control: 2.5 ± 0.4 ms; n = 61; P < 0.05) and showed reverse–use dependence of BVR. In comparison, dogs with acute AVB had low variability (1.3 ± 0.3 ms; n = 9; P < 0.05 versus chronic AVB).Cardiac electrical remodeling after AVB is associated with an increase in beat–to–beat variability of repolarization. Chronic AVB dogs displaying further elevated variability of repolarization are prone to arrhythmia–related SCD.

Phenylephrine-induced stimulation of Na+/Ca2+ exchange in rat ventricular myocytes

OBJECTIVE The effect of an alpha-adrenergic agonist, phenylephrine, on the Na+/Ca2+ exchange current in rat ventricular myocytes was investigated. METHODS The Na+/Ca2+ exchange current was measured at room temperature in rat ventricular myocytes as the whole-cell current induced by addition of extracellular Na+ and Ca2+, while blocking Na+ current by setting the holding potential at -30 mV, K+ currents by intracellular Cs+, TEA+ and by extracellular Ba2+, Ca2+ current by nifedipine and Na+ pump current by ouabain or by 0 extracellular K+. RESULTS Under these experimental conditions, application of external Na+ and Ca2+ induced a current which was further increased by phenylephrine. Phenylephrine (80 microM) increased the current by up to 31.0 +/- 5.4% of control at all membrane potentials tested both below and above the reversal potential. The reversal potential (+21.0 +/- 3.2 mV), which corresponded with the theoretical reversal potential for the Na+/Ca2+ exchange current under our ionic conditions (+21.3 mV), was not changed by phenylephrine (+23.2 +/- 4.1 mV). Applying phenylephrine in the absence of Na+/Ca2+ exchange (0 Na+e, 0 Ca2+e) did not change the current. The effect was resistant to propranolol, a beta-adrenergic blocker, but prevented by prazosin, an alpha-receptor antagonist, by neomycin, an inhibitor of phospholipase C, and by chelerythrine, a selective inhibitor of protein kinase C. Phorbol 12-myristate 13-acetate failed to stimulate the current. The effect remained similar under conditions of high (HEPESi = 5 mM) and low (HEPESi = 0.5 mM) intracellular pH buffering. CONCLUSION Our data indicate that phenylephrine stimulates the Na+/Ca2+ exchange, both in the forward and the reverse modes, probably via a protein kinase C-dependent pathway.

Modulation of transient outward current by extracellular protons and Cd2+ in rat and human ventricular myocytes.

1 The effects of extracellular acidosis and Cd2+ on the transient outward current (Ito) have been investigated in rat and human ventricular myocytes, using the whole‐cell patch‐clamp technique. 2 In rat myocytes, exposure to acidic extracellular solution (pH 6.0) shifted both steady‐state activation and inactivation curves to more positive potentials, by 20.5 ± 2.7 mV (mean ± s.e.m.; n= 4) and 19.8 ± 1.2 mV, respectively. Cd2+ also shifted the activation and inactivation curves in a positive direction in a concentration‐dependent manner. 3 In human myocytes, the steady‐state activation and inactivation curves were located at more positive potentials. The effect of Cd2+ was similar, but acidosis had less effect than in rat myocytes (e.g. pH 6.0 shifted activation by only 7.2 ± 2.2 mV and inactivation by 13.7 ± 0.5 mV; n= 4). 4 In both species, the effect of acidosis decreased with increasing concentrations of Cd2+ and vice versa, suggesting competition between H+ and Cd2+ for a common binding site. 5 The data indicate that acidosis and divalent cations influence Ito via a similar mechanism and act competitively in both rat and human myocytes, but that human cells are less sensitive to the effects of acidosis.

Beat-to-beat variability is an independent and early indicator for d-sotalol-induced arrhythmias in vivo

Background: Beat-to-beat variability of repolarization (BVR) has been associated with sudden cardiac death in patients and with drug-induced proarrhythmia in isolated rabbit hearts. This study tried to relate repolarization parameters, including BVR. to drug induced torsades de pointes (TdP) arrhythmias in dogs. Methods: 2 and 4 “g/kg d-Sotalol (dS) were administered in a random crossover design to 8 anesthetized dogs with chronic AV block. Endocardial MAP durations (MAPD) from both ventricles (LV and RV) and surface EGG were recorded; Interventricular dispersion (LV - RV MAPD) and extrasystolic (ES) frequency were calculated. BVR was quantified by the area of the Pomcar~ plots (figure) for LV, RV MAPD and QT during 30.beat periods at baseline (BL) and in the presence of dS just prior to extrasystolic activity preceding TdP. Results: The repolarization parameters (QT, LV, RV MAPD and dispersion) could not account for the difference in TdP occurrence (218 vs. 618 after 2 and 4 mglkg dS, respectively). In contrast, the LV Potncare plot area and the frequency of multiple extrasystoles .(mES) were higher in the 4 mglkg group. BVR was present before ES occurred and independent of the other repolarization parameters. Conclusions: Where the QT-duration and the other repolarization parameters failed, beat-to-beat variability of the LV and the frequency of “ES corresponded with the dosedependent TdP induction. BVR is an Independent and early measurement that could become important in predicting drug-induced TdP.