β-Adrenergic Blocking Property of dl-Sotalol Maintains Class III Efficacy in Guinea Pig Ventricular Muscle After Isoproterenol
Background Catecholamines antagonize the efficacy of several class III antiarrhythmic agents. To determine the role of the intrinsic β-adrenergic blocking property of dl-sotalol in maintaining class III efficacy during a high-catecholamine state, we compared the electrophysiological properties of dl-sotalol with those of d-sotalol, which is devoid of significant β-adrenergic blocking effect, before and after isoproterenol infusion.
Methods and Results Action potential duration at 90% repolarization (APD90) was prolonged in isolated guinea pig papillary muscles perfused with d-sotalol and dl-sotalol 10−4 mol/L over stimulation cycle lengths from 200 to 2000 ms. The increases in APD90 for d-sotalol and dl-sotalol over control were 10.9±2.5 to 23.7±4.8 ms and 27.9±4.0 to 39.0±5.6 ms, respectively. APD90 shortened to less than control in papillary muscles treated with d-sotalol but not dl-sotalol on addition of isoproterenol 10−6 mol/L: −31.2±3.5 to −18.3±4.8 ms and 10.5±3.6 to 33.3±7.8 ms, respectively, P<.003. Single guinea pig ventricular myocytes were studied by the whole-cell patch clamp method. Time-dependent (Iout) and total (Itot) outward current in response to a 300-ms pulse to 20 mV and tail current (Itail) to −35 mV were measured after Ca2+ channel block and Na+ channel inactivation. Iout, Itail, and Itot were reduced in myocytes perfused with d-sotalol and dl-sotalol 10−4 mol/L: Iout, −36.1±4.1%, −40.5±3.3%; Itail, −59.3±4.6%, −62.2±11.1%; Itot, −27.3±4.3%, −50.0±11.8%. Iout and Itot increased to a greater degree in myocytes treated with d-sotalol than dl-sotalol on addition of isoproterenol 10−6 mol/L: Iout, 100.3±20.6%, 11.3±7.6%, P=.002; Itot, 86.8±39.2%, −41.1±20.9%, P=.01. Itail tended to increase more in myocytes treated with d-sotalol than dl-sotalol on addition of isoproterenol, but the difference was not significant (−9.1±13.5%, −28.0±9.0%).
Conclusions The β-adrenergic blocking property of dl-sotalol maintains APD prolongation and repolarizing outward current block during isoproterenol infusion in guinea pig ventricular muscle. Extrapolation of these data to a clinical setting may explain the efficacy of dl-sotalol in diminishing ventricular arrhythmia recurrence.
The drug dl-sotalol is effective therapy for ventricular tachyarrhythmias.1 As a class III antiarrhythmic agent, the mechanism of action of dl-sotalol is to block repolarizing outward current, prolonging cardiac action potential duration (APD) and refractory period and thereby reducing the likelihood of reentrant arrhythmias.2 3 4 5 6 dl-Sotalol blocks a rapid component of the delayed rectifier K+ current, IKr7 In addition, dl-sotalol has significant β-adrenergic blocking property. In contrast, d-sotalol, an equipotent IKr blocker, has minimal β-adrenergic blocking property.8 A slow component of the delayed rectifier K+ current, IKs, and a Cl− current (ICl) are upregulated by the β-adrenergic agonist isoproterenol.9 10 Upregulation of these outward currents can counteract the APD-prolonging effects of IKr blocking agents. Pretreatment with β-adrenergic blockers maintains the APD- and refractory period–prolonging effects of IKr blocking agents exposed to isoproterenol.9 This study examines the role of the intrinsic β-adrenergic blocking property of dl-sotalol in maintaining class III effectiveness after isoproterenol.
Twenty-nine guinea pigs (200 to 300 g, male) were used for this study. Animal preparation and handling were performed according to the guiding principles of the American Society of Physiology and under a protocol approved by the Animal Care Committee at Oregon Health Sciences University. Guinea pigs were anesthetized with halothane inhalation, and the hearts were removed.
Papillary Muscle Preparation
A right ventricular papillary muscle was dissected free, mounted in a muscle bath, and superfused with 1.8 mmol/L Ca2+-Tyrode’s solution (in mmol/L: NaCl 140, KCl 5.4, MgCl2 1, glucose 10, HEPES 10, adjusted with NaOH to pH 7.35) gassed with 100% O2 and maintained at a temperature of 37.0±0.2°C.
Muscle strips were attached to a force transducer, and resting length was adjusted to produce maximum isometric force. The papillary muscle was stimulated with a pulse width of 1 ms and voltage of twice threshold. Intracellular APs were recorded on an electronic strip recorder (MacLab, AD Instruments) with 20 to 30 MΩ 3 mol/L KCl-filled microelectrodes (A-M Systems, Inc). APs were recorded at stimulation cycle lengths from 200 to 2000 ms before and 30 minutes after the addition of d- or dl-sotalol 10−4 mol/L (Bristol-Myers Squibb Pharmaceuticals) and again 30 minutes after the addition of isoproterenol 10−6 mol/L (Elkins-Sinn). A dose of 10−4 mol/L sotalol was used to maximally block IKr without significant effect on other currents.4
Ventricular Myocyte Isolation
Ventricular myocytes were isolated as previously described.11 Hearts were perfused retrogradely with 100% O2/Ca2+-free Tyrode’s solution for 5 minutes. The solution was changed to one containing collagenase (type II, 275 U/mL; Worthington Biochemical Corp) and protease (type XIV, 0.8 U/mL; Sigma Chemical Co) for 7 minutes. The heart was subsequently perfused with 0.1 mmol/L Ca2+-Tyrode’s solution for 10 minutes. Ventricles were dissected, minced, and placed in 0.1 mmol/L Ca2+-Tyrode’s solution. Cells were studied within 8 hours of dissociation.
Whole-Cell Current Recording
Whole-cell currents were recorded from calcium-tolerant myocytes with patch electrodes (A-M Systems, Inc) with resistances of 2.5 to 4 MΩ when filled with (in mmol/L): potassium glutamate 80, KCl 40, NaCl 10, MgCl2 1, MgATP 5, CaCl2 1, potassium creatine phosphate 0.5, EGTA 10, and HEPES 10, adjusted with KOH to pH 7.1. Cells were voltage-clamped with an Axopatch 1B amplifier (Axon Instruments). Series resistance was 50% to 70% compensated, and the currents were filtered at 1 kHz before digitization and storage on an LSI 1173 computer (Digital Equipment Corp).
Cells were perfused at a constant rate of 1.5 mL/min with 1.8 mmol/L Ca2+-Tyrode’s solution held at 29.0±0.2°C. Ca2+ channels were blocked with 1 to 2×10−4 mol/L Cd2+, and Na+ channels were inactivated with a 1000-ms prepulse to −40 mV. Myocytes were held at their resting membrane potential of −72 to −75 mV and depolarized at 1-minute intervals to 20 mV for 300 ms, a time frame and voltage representative of the intrinsic action potential. Time-dependent outward current (Iout) was measured as the difference between final and initial current. Total outward current (Itot) equals the final current and reflects the sum of instantaneous and time-dependent outward current. Tail current (Itail) was measured at −35 mV, near the Cl− equilibrium potential. After control determination, either d-sotalol or dl-sotalol 10−4 mol/L was added to the perfusate, and data were collected for 10 minutes. Isoproterenol 10−6 mol/L was then added to the drug perfusate, and data were collected again for 10 minutes. In all experiments, steady state was achieved during the 10-minute collection times.
The time difference in APD to 90% repolarization (APD90) between control and each drug state was calculated for each pacing cycle length. Current measurements were expressed as percent change from control (100%×[drug state−control]/control), which corrected for differences in cell size. Effects on APD in papillary muscles and currents in myocytes treated with d- or dl-sotalol were compared by a two-tailed independent-samples t test. This comparison was made before and after isoproterenol infusion. Values of P<.05 were taken as statistically significant.
Results are summarized in the Table⇓.
APD90 was rate-dependent, increasing by 114.6±5.0 to 156.5±8.4 ms from the pacing cycle lengths of 200 to 2000 ms, respectively. Results are shown in Fig 1⇓. Both d- and dl-sotalol 10−4 mol/L prolonged the APD without effect on the maximal rate of rise of the AP. There was a trend for dl-sotalol to prolong APD90 to a greater extent than d-sotalol, but this was not significant. APD shortened to less than control in muscle treated with d-sotalol but not dl-sotalol on addition of isoproterenol 10−6 mol/L.
Isolated Guinea Pig Ventricular Myocytes
Iout, Itot, and Itail in response to a 300-ms pulse to 20 mV with tail to −35 mV averaged 50.6±5.5, 74.7±16.9, and 19.1±2.8 pA, respectively. Results are shown in Fig 2⇓. Iout, Itot, and Itail were reduced in myocytes perfused with d- and dl-sotalol. There was a trend for dl-sotalol to reduce Itot to a greater extent than d-sotalol, but this was not significant. Iout increased compared with control on addition of isoproterenol in myocytes treated with both d- and dl-sotalol but to a significantly greater extent in those treated with d-sotalol. Itot remained reduced below control in myocytes treated with dl-sotalol but not d-sotalol. Itail increased but not to control values with addition of isoproterenol in myocytes treated with either d-sotalol or dl-sotalol. There was a trend for Itail to increase to a greater extent with addition of isoproterenol in myocytes treated with d-sotalol compared with dl-sotalol, but the difference was not significant.
dl-Sotalol but not d-sotalol maintains the class III effects of APD prolongation and diminished outward current during isoproterenol infusion in guinea pig ventricular muscle. This differential effect is secondary to the β-adrenergic blocking property of dl-sotalol counteracting the isoproterenol-induced increase in total outward current. In myocytes treated with d-sotalol, an instantaneous current, consistent with ICl, and an increased time-dependent current, consistent with an increase in IKs, occurred with isoproterenol infusion. These isoproterenol-induced currents increased total outward current, overwhelming the effect of IKr block by d-sotalol. Catecholamines antagonize the class III efficacy of several antiarrhythmic agents known to block IKr in humans, consistent with our findings for d-sotalol in guinea pig heart.12 13 14 15 Ionic currents responsible for repolarization in human heart have not been well characterized, and therefore, the mechanism by which catecholamines antagonize class III antiarrhythmic efficacy may be different from that in guinea pig heart.
With isoproterenol challenge, Itail did not increase to control value with either d-sotalol or dl-sotalol, and the difference between drugs was not significant. This is probably due to IKr inward rectification,7 which increases the contribution of IKr to Itail compared with Iout.
d-Sotalol and other pure IKr blockers, despite potent class III effects, may be limited in their efficacy by high-catecholamine states. The clinical efficacy of dl-sotalol may be related to the combination of class III effects, which prolong APD and thereby reduce the likelihood of reentrant arrhythmias, and β-adrenergic blocking property, which maintains class III efficacy despite high-catecholamine states.
This research was supported in part by an American Heart Association, Oregon Affiliate, grant (Dr Groh) and by National Institutes of Health grant HL-48286 (Dr Maylie).
Presented in part at the Annual Meeting of the North American Society of Pacing and Electrophysiology, Nashville, Tenn, May 11-14, 1994.
- Received September 26, 1994.
- Accepted November 25, 1994.
- Copyright © 1995 by American Heart Association
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