- Email: [email protected]
Effect of sotalol on transmembrane ionic currents responsible for repolarization in cardiac ventricular myocytes from rabbit and guinea pig
Life Sciences, Vol. 49, pp. PL-7 - PL-12 Printed in the U.S.A.
Pergamon Press
PHARMACOLOGY LETTERS Accelerated hmW&?atiOn EFFECT OF SOTALOL ON TRANSMEMBRANE IONIC CURRENTS RESPONSIBLE FOR REPOLARIZATION IN CARDIAC VENTRICULAR MYOCYTES FROM RABBIT AND GUINEA PIG Andras Varro (1,2), Peter P. Nan&i (3) and David A. Lathrop (1,4) Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA (1); Szent-Gyijr ’ Albert Medical University, De artment of Pharmacology, Szeged D6m Ter 12, H- r 701 HUNGARY (2); University Medical School of Debrecen Department of Physiology Debrecen P.O. BOX22, H-4012 HUNGARY (3); Departme& of Physiology, Institute oiMedical Biology, University of Tromse, N-9000 Tromse, NORWAY (4) (Submitted April 22, 1991; accepted May 3, 1991; received in final form May 7, 1991)
Abstract. The effects of sotalol, a 6-adrenoceptor blocker and class III antiarrhythmic agent, on transmembraneionic currents were examined in single rabbit and guinea pig ventricular myocytes using whole-cell volta e-clamp techniques. In neither of these species did 60 pM sotalol appreciably effect the inward recta Fier,.the transient outward or the inward calcium currents. In addition, sotal did not elicit a slowly inactivating componentof the sodium current as did 1 pg/ml veratrine. In guinea pig ventricular myocytes, sotalol also significantly depressed the outward delayed rectifier current. An outward delayed rectifier current was not observed in rabbit ventricular myocytes examined at room temperature; and, under these conditions sotalol did not lengthen action potential duration. Sotal induced lengthemng of cardiac action potential duration can, therefore, be explained by depression the outward delayed rectifier current.
Introduction Many drugs that lengthen myocardial action otential duration (APD) have been demonstrated to be efficacious in the treatment of car % ac arrhythmias (1).Such drugs are classified as class III antiarrhythmic agents (2). Sotalol, a 6-adrenoceptor blocker that also increases cardiac APD (3,4), has been demonstrated to be beneficial in the treatment of cardiac rhythm disturbances ($6). Despite this recognized utility as a class III antiarrhythmic agent, few studies have described the specific effects of sotalol on cardiac ionic currents (7,8,9!; and, the effects described in these studies have been contradictory and confusin Carmehet (7) re rted that, in sheep and rabbit cardiac Purkinje fibers, sotalol primarily bloc% ed the outward dep”ayed rectifier current (Ix) while also producing moderate block of the inward rectifier current (I,). In contrast, Berger et al. (8) reported that, in sheep cardiac Purkinje fibers, concentrations of sotalol as high as 1000 pM did not affect I,. Instead, these authors (8) reported that the rimaryeffects of sotalol were block of the transient outwardcurrent (I,,) and I,. More recently, ! anguinetti and Jurkiewicz (9) have reported that, in guinea pig ventncuk myocytes, ~0~0~s primary effect is to selectively block a fast component of Ix. Still other authors have inferred that because many currents are known to be important in the control of action potential duration, sotalol may also affect the inward calcium current (10) and possibly a sodium ‘window” current (11).The experiments described here were conducted with the hope that the information obtained might help to better elucidate the mechanism by which sotalol increases cardiac action potential duration.
Correspondti Author: David A, Lathrop, Ph.D., Department of Physiology, Institute of Medical Biology, University of Tromse, N-9000 Tromse, Norway
0024-3205/91 $3.00 +.oo l-nr..7ri"h+ I,..\ ,001 Dnrr,r.",... D.-^.-.^ -7-
PL-8
Sotalol
on Membrane
Currents
Vol.
49,
No.
4,
1991
Single right ventricular myocytes were obtained by enzymatic dissociation (12). Brietly, rabbits (weighing 2 - 3 kg) and (weighing 300 - 600 g) of either sex were anmsthetized ? i.v. with sodium pentobarbitone (50 , i.v. and i.p.) following he arinization (400 i and i.p. ). The chest was was quickly remove B and mounted in a ?&l m ed rfusion apparatus. Blood was flushed ii-om the coronary vasculature during a erfusion with bicarbonate buffered solution (BBP). The BBP erfusate NaCl, 121.0; KCl, 5.0; CaCl,, 1.0; Na-acetate, 2.8; M l,, 1.0; RraHCOs, 24.0; Na&IPG,, 1.0; and glucose 5.5. This brief period of perfusion was fo $:owed by erfusion riod of a proximate P 5 min. with nominally Ca’+-free BBP containing 35 mM KC1 for a After this period, the BBP was switched to one containing O.O!?&M CaCf, 35 0 mM I&l 20.0 mM taurine, as well as 0.1 bovine albumin and 0.25 m ml collagenase (Sigma, St. Louis, MO, USA; type I) for an a(Bfl ditional40 - 60 min. All pe lYusates were warmed to 37 ‘C and continuously assed with 95% 0, and 5% CO, while perfusion pressure was maintained at 40 cm/l&O and p& was adjusted to 7.4. Following perfusion with the colla nase containing solution, the right ventricular free wall was removed diced into small ch uIZes and placed into KB medium (13). The corn sition of the KB medium utilized was (in mM): KC1 85 0. &HPG 3 0. MgC1, 5 6 N&G 5 0. Pyruvic acid, 5.0; beta-hydroxy butyric acid, 5.6; crt&ne, 5.&?a&ie 20.6; NaCl, 4.0; C&: 0.5; EGTA, 1.0 and lucose, 20.0 with the addition of polyvinyl-pyrrolidone, 50 g/l. The H of this solution was a4 ‘usted to 7.2 by addition of KOH. The tissue was eqmhbrated in I&for 60 min and isolated myocytes were obtained by gentle agitation of the minced tissue. The cell suspension thus obtained was diluted (1:lO) in KB medium and the resulting mixture was stored at 4 ‘C for u to 36 hr. One drop of cell suspension was laced in a trans arent recording chamber moun tedpon the stage of an inverted microscope (h aphot TMD, 8 Ron Co. Tokyo, Japan) and the individual myocytes were allowed to settle on the bottom of the recording chamber for at least 10 min prior to making electrical measurements. HEPES buffered Tyrode solution (HBT) was utilized as the normal au rfusate. This solution contained, in mM, NaCl, 137.0; KCl, 5.4; Na&IPG,, 0.35; K&PO,, 0.3; r aCl,, B,O;_MgCl,,1.0; HEPES, 20.0; glucose, 20.0 at a pH of 7.4. Superfusion was maintained by gravuhty flow. Suction electrodes having tip diameters less than 3 pm were fabricated from glass capillaries (Kimax-51, Kimble products USA). After fire olishing, the electrodes had resistances in the range of 2 - 4 Ma when filled with the standar B filling solution. The standard filli solution consisted of (in mM): KC1 140. Na&TP 5. MgCl,, 1; EGTA, 1; CaCl,, 0.5; pH 7.2.n&ctrode potentials were adjusted tdzero’immedia&y before formation of seals between the electrode tip and membrane. After establishing a high resistance (l- 2 GQ) seal, the cell membrane beneath the electrode tip was disrupted by further suction or by application of 1.5 V pulses 1 - 5 ms in duration. Input resistance and cell capacitance were measured during application of a 10 mV depolarizing pulse from a holding potential of -80 mV. Membrane action potentials were elicited by applying depolarizing constant current pulses that were 1.5 - ‘2 nA in amplitude and 6 to 12 ms in duration. All measurements were obtained at room temperature using an Axopatch 1B patch-clamp amplifier (Axon Instruments, Burlingame, CA, USA) in either voltage- or current-clam mode. Analog outputs from this amplifier were digitized under software control (Clampex 5. B3; Axon Instruments) using an analogue-to-digital converter (Labmaster, Cleveland, OH, USA, model TL-1) having a maximal cycle length of 125 KHz and installed on an 8 MHz IBM-AT computer. Digitized recordings thus obtained were analyzed with the aid of software developed by Axon Instruments (Clampan 5.03 and Clam fit 5.03). In addition, Student’s t-tests for either aired or unpaired data were performed to Betermine the si ‘ficance level of the changes pro a uced by the various maneuvers employed. Probability (p) V~S of less than 0.05 were considered significant.
The effect of sotalol on I,, in rabbit ventricular myocytes was evaluated from a holding potential of -50 mV in order to inactivate I,. In these experiments, the bathing solution also Under these conditions, the quasi steady-state contained 0.25 mM CdCl, to block I,. current-voltages relation established at the end of 400 ms command pulses applied every 3 8 was not effected by 60 pM sotalol (Fig. 1, upper panels)
Vol.
Sotalol
49, No. 4, 1991
~Qrominent
I,_was observed at rtentials,
PL-9
on Membrane Currents
sitive to -20 mV in 6 of the 10 rabbit
WXI~~C ar myocytes m wbxh &, was b ocked by dC1,. In these myocytes, sotalol(60 @‘I) did not affect the amplitude of I, (Fi . 1, lower panels) which was measured as the difference
between the peak outward current o%served within 15 ms after the start of the voltage pulse and the current recorded at the end of the command pulse (14). In addition, 60 @ sotal did not significantly change the inactivation time constant ( q_, ) for I, evoked during command pulses to +50 mV (61.7 + 12.7 ms before uersus 51.8 * 8.1 ms after sotalol; n = 6). A) Steady-state IV relation
4.0 .“.V
-1
l
-
A ------
CmaROL SOTALOL
Sotalol
-1.4 -1.8 iooms
B) Transient outwardcurrent
SPA
;
0’ 0
20
40
v01mgc (mV)
Fipure 1. A) Effect of 60 fl sotalol on the quasi steady-state current-voltage relation established in 10 rabbit ventricular myocytes. Holding potential = -50 mV, command pulse duration = 400 ms, command pulse frequency = 0.33 Hz. Panels to the right are of representative recordings to illustrate the effect of 60 @I sotalol when clamped to test potentials of -100, -80, -40,O and +40 mV, before (top) and after a plication of 60 pM sotalol (bottom). B) Pe 9 transient outward current before ( l ) and after ( A ) the application of 60 pM sotalol in rabbit myocytes (n = 6). Analog recordin s to the right were obtained before and after administration of 60 fl sotalol. I! olding potential = -50 mV, command potential = +50 mV, command pulse duration = 400 1119, command pulse fixquency = 0.33 Hz Inward Ca*+currents were assessed in 5 rabbit ventricular myocytes not exposed to CdCl, and not having a rominent I,. This current was measured before and after e osure to sotalol (60 a) as the d& erence between the peak inward current activated within “R t e first I5 ills of the command pulse and the current at the end of a 400 ms long comman d pulse (Fig. 2). As illustrated, sotalol effM.ed neither the amplitude nor inactivation kinetics of b, in these preparations. Before sotalol, T,, for I,-, evoked by a command pulse to 0 mV was 40.4 * 4.2 ms, and following sotalol administration it was 45.5 & 1.9 ms (NS; n = 5). Because a sodium “window” current, or slowly inactivating component of the fast Na+ current (I,), has been suggested to be important in control of APD (15) and because of the similarit of the electromechanical effects of sotalol to those produced by veratrine (16), we previou&y srulated upon ther ssibility of sotalol having direct effects on cardiac Na+ channels (11. TO test this possib’ ity, we e xamined I, in rabbit ventricular myocytes clamped to a holding potential of -90 mV. To avoid contamination of IN.by other inward currents, Ic.
PL-10
Sotalol
on Membrane
Currents
Vol.
49,
No.
4,
1991
was blocked in this series of experiments by addition of 0.25 mM Cd&. Command tentials between 40 and +40 mV were applied and the large inward currents thus evoke Z”decayed within 10 ms and were blocked by 40 J.&Itetrodotoxin (TTX). Following exposure to 1 &ml veratrine, a second slowly inactivating, TTX sensitive, corn nent to this inward current was expressed (Fig. 2e). In contrast, 60 fl sotalol failed to pro arent effect on the 1, (Fig 2d). Similar results were obtained in 6 additional rabbit ventric A) Inwardcalciumcurrent 0.2 -60
l -
CONTROL
A ----
SOTALOL
D)
(nA) ,
SOTALOL
(A) Effect of 60 fl sotalol on cal ard currents in rabbit ventricular myocytes (n = 5). Analog recordings before (B) and after (C) administration of 60 fl sotalol are illustrated at the right of the figure. Holding otential = -50 mV, test potential = 0 mV, test pulse duration = 400 ms, test pulse ip requency = 0.33 Hz Comparison of the effects of 60 p.M sotalol (D) and 1 &ml veratrine (E) on sodium current inactivation in rabbit ventricular myocytes. The veratrine effect was completely blocked by addition of 10 p.M TTX. Holding potential = -90 mV, test otential = -20 mV, test ulse duration = 400 ms, and test pulse fre uency = 0.33 R z. In panels D and E tRe bathing solution contained 0.25 mM Cd E1,. In rabbit ventricular myocytes examined at room temperature, Ix has been reported to be either very small or absent (12,17). We also could not detect an Ix in the rabbit myocytes that we examined at room temperature while using 3 s long depolarizing command pulses applied from a holdin potential of -50 mV. In rabbit, therefore, sotalol could not be demonstrated to affect lgx. In guinea pig ventricular myocytes, however, we routinely observed a large Ix at room temperature when I,, was abolished by 0.25 mM CdCl,. In our experiments utilizing guinea pig myocytes, membrane potential was held at -40 mV and 5 s long depolarizin command pulses were applied every 12 s. The amplitudes of the tail currents thus evoke d upon return to the holding otential were determined. Under these conditions, 60 fl sotalol markedly reduced the amp Petude of the outward current developed during the command pulse while the amplitudes of the outward tail currents were also decreased (Fig. 3) The effects of sotalol on action potential characteristics were examined at room temperature in both rabbit and guinea ig ventricular myocytes continuously paced at 0.5 Hz. Figure 4 illustrates representative fin d!mgs. Sotalol(60 p.M) had no effect on either diastolic potential or action potential amplitude (APA) in either rabbit or guinea pig ventricular myocytes. In 6 rabbit ventricular myocytes, therefore, diastolic potential was -72.3 f 1.2 mV before and -72.6 f 1.6 mV following 60 p.M sotalol while APA in these preparations was 116.0 ? 3.1 mV before and 114.7 -+3.3 mV after sotalol. Similarly, in 6 guinea ig myocytes, diastolic potential and APA were -72.2 + 1.0 and 115.3 + 3.5 mV before and -73. f f 1.3 and 117.3 rt:1.8 mV after 60 pM sotalol. In addition, in rabbit ventricular myocytes, 60 p.M sotalol did not change APD measured at either 50% or 90% of repolarization (APD, and AF’Dw, respectively) so that before and after sotalol APD,, and APD, were 495.7 + 52.3 and 535.3 + 56.4 ms, and 486.3 + 55.8 and 519.3 + 60.4 ms, respectively. This sotalol concentration (60 a), however, did significantly increase both APD, and APD, in guinea pig myocytes from 449.0 f 43.2 and 474.7 f 43.5 ms to 513.0 f 56.2 (p < 0.01) and 544.3 + 57.3 ms (p < 0.05).
1667 't 'ON ‘6'7 “COA
PL-12
Sotalol
on Membrane Currents
Vol.
49,
No. 4,
1991
on IKwas not examin ed in the present study, it should be noted that at 32 ‘C sotalol has been demonstrated to increase rabbit ventricular muscle APD in concentrations in the range of those utilized (20). effect of temperature
Bergeret al. (8) reported that involtage-clamped sheep Purkinje strands &is not affected by sotalol while I, is markedly decreased. Our results from guinea ig ventricular myocytes indicate the opposite-- I, is not effected while IK is substantially Elocked by sotalol. The explanation for this discrepancy inresultsis not clear. Although, sgecies+epndent differen!es could be involved, such an e lanation would ap ar unlikely as amehet s (7) demonstration of a sotalol-induced block of“pKwas obtained in s?Zeep Purkinje fibre preparations.
Additional effects of sotalol certainly cannot be ruled out and the specific effects of sotalol on Iz channels remains to be carefully worked out. Sanguinetti & Jurkiewicz (9) have, for example, recently demonstrated that, in guinea pig ventncular myocytes examined at 35 ‘C, blocked a rapidly activating component of I,. At 22 ‘C, however, these authors (9 component of IKwas either absent or its activation so slow that it could not be from Iz’s other component. The results of the resent stud reported by Carmeliet (7) and by 2 anguinetti & Jurkiewicz (9), that sotalol induced increases in action potential duration are most likely due to block of I* Acknowledeements The aotalol utilized in this study was obtained as a gift from Bristol-Myers/Squibb Pharmaceuticals, Wallingford, CT, USA. The study was funded by a grant to David A. Lathrop from the FzTd,Heart, Lung and Blood Institute of the United States National Institutes of Health CR01 References M.R. ROSEN and A.L. WIT, Am Heart J l& 829-841(1983)
E.M. VAUGHANWILLIAMS, Pharmacol Ther B 1115-121(1975) H.C. STRAUSS,J.T. BIGGERand B.F. HOFFMAN, Circ Res p 661-678 (1970)
B.N. SINGH and E.M. VAUGHANWILLIAMS, Br J Pharmacol p 675-687 (1970) pi9y44yBouL, G. ATALLAH,G. KIRKORIAN, M. LAMAUD, and P. MoLEUR, Am Heart J l!X 888-895 B.N. SINGH, Am J Cardiol & 84A-88A (1990) E. CARMELIET,J Pharmacol Exp Ther m 657-62 (1985) F. BERGER,U. BORCHARDand D. HAFNER,Naunyn-Schmiedeberg’s Arch Pharmacol XQ 696-704 (1989) M.C. SANGIJINEITIand N.K. JURKIEWICZ,J Gen Physiol $)$j195-215 (1990) D.S. ECHT, L.E. BERTE,W.T. CLUSJN,R.G. SAMIJEISSON,D.C. HARRISON,and J.W. MASON, Am
J Cardiol IiQ1082-1086 (1982)
D.A. LATHROP,Can J Physiol Pharmacol $Q 1506-1512 (1985) M. HIRAOKAand S. KAWANO J Ph siol QQ 187-212 (1989) G. ISENBERGand U. KL&XI&R Pkigers Arch s 6-18 (1982) M. HIRAOKAand S. KAWANO,Circ Res 6Q 14-26 (1987) D. ATIWELL, I. COHEN, D. ELSNER,M. OHBA, and C. OJEDA, Eur J Physiol m 137-142 (1979) D.A. LATHROPand A. VARR~, Can J Physiol Pharmacol 611463-1467 (1989) W.R. GILES and Y. IMAJZUMI,J Physiol $&j 123-145 (1988) P.J. NEUVONEN, E. ELONEN, A TANSKANEN, and J. TOHIJMILEHTO,Eur J Clin Pharmacol 24 85-89 (1981) P. SOMANI and D.L. WATSON, J Pharmacol Exp Therm 317-352 (1968) S.M. COBBE, B.S., MANLEY, and D. ALRXOPOULOS,Cardiovasc Res u 668-673 (1985)
Pergamon Press
PHARMACOLOGY LETTERS Accelerated hmW&?atiOn EFFECT OF SOTALOL ON TRANSMEMBRANE IONIC CURRENTS RESPONSIBLE FOR REPOLARIZATION IN CARDIAC VENTRICULAR MYOCYTES FROM RABBIT AND GUINEA PIG Andras Varro (1,2), Peter P. Nan&i (3) and David A. Lathrop (1,4) Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA (1); Szent-Gyijr ’ Albert Medical University, De artment of Pharmacology, Szeged D6m Ter 12, H- r 701 HUNGARY (2); University Medical School of Debrecen Department of Physiology Debrecen P.O. BOX22, H-4012 HUNGARY (3); Departme& of Physiology, Institute oiMedical Biology, University of Tromse, N-9000 Tromse, NORWAY (4) (Submitted April 22, 1991; accepted May 3, 1991; received in final form May 7, 1991)
Abstract. The effects of sotalol, a 6-adrenoceptor blocker and class III antiarrhythmic agent, on transmembraneionic currents were examined in single rabbit and guinea pig ventricular myocytes using whole-cell volta e-clamp techniques. In neither of these species did 60 pM sotalol appreciably effect the inward recta Fier,.the transient outward or the inward calcium currents. In addition, sotal did not elicit a slowly inactivating componentof the sodium current as did 1 pg/ml veratrine. In guinea pig ventricular myocytes, sotalol also significantly depressed the outward delayed rectifier current. An outward delayed rectifier current was not observed in rabbit ventricular myocytes examined at room temperature; and, under these conditions sotalol did not lengthen action potential duration. Sotal induced lengthemng of cardiac action potential duration can, therefore, be explained by depression the outward delayed rectifier current.
Introduction Many drugs that lengthen myocardial action otential duration (APD) have been demonstrated to be efficacious in the treatment of car % ac arrhythmias (1).Such drugs are classified as class III antiarrhythmic agents (2). Sotalol, a 6-adrenoceptor blocker that also increases cardiac APD (3,4), has been demonstrated to be beneficial in the treatment of cardiac rhythm disturbances ($6). Despite this recognized utility as a class III antiarrhythmic agent, few studies have described the specific effects of sotalol on cardiac ionic currents (7,8,9!; and, the effects described in these studies have been contradictory and confusin Carmehet (7) re rted that, in sheep and rabbit cardiac Purkinje fibers, sotalol primarily bloc% ed the outward dep”ayed rectifier current (Ix) while also producing moderate block of the inward rectifier current (I,). In contrast, Berger et al. (8) reported that, in sheep cardiac Purkinje fibers, concentrations of sotalol as high as 1000 pM did not affect I,. Instead, these authors (8) reported that the rimaryeffects of sotalol were block of the transient outwardcurrent (I,,) and I,. More recently, ! anguinetti and Jurkiewicz (9) have reported that, in guinea pig ventncuk myocytes, ~0~0~s primary effect is to selectively block a fast component of Ix. Still other authors have inferred that because many currents are known to be important in the control of action potential duration, sotalol may also affect the inward calcium current (10) and possibly a sodium ‘window” current (11).The experiments described here were conducted with the hope that the information obtained might help to better elucidate the mechanism by which sotalol increases cardiac action potential duration.
Correspondti Author: David A, Lathrop, Ph.D., Department of Physiology, Institute of Medical Biology, University of Tromse, N-9000 Tromse, Norway
0024-3205/91 $3.00 +.oo l-nr..7ri"h+ I,..\ ,001 Dnrr,r.",... D.-^.-.^ -7-
PL-8
Sotalol
on Membrane
Currents
Vol.
49,
No.
4,
1991
Single right ventricular myocytes were obtained by enzymatic dissociation (12). Brietly, rabbits (weighing 2 - 3 kg) and (weighing 300 - 600 g) of either sex were anmsthetized ? i.v. with sodium pentobarbitone (50 , i.v. and i.p.) following he arinization (400 i and i.p. ). The chest was was quickly remove B and mounted in a ?&l m ed rfusion apparatus. Blood was flushed ii-om the coronary vasculature during a erfusion with bicarbonate buffered solution (BBP). The BBP erfusate NaCl, 121.0; KCl, 5.0; CaCl,, 1.0; Na-acetate, 2.8; M l,, 1.0; RraHCOs, 24.0; Na&IPG,, 1.0; and glucose 5.5. This brief period of perfusion was fo $:owed by erfusion riod of a proximate P 5 min. with nominally Ca’+-free BBP containing 35 mM KC1 for a After this period, the BBP was switched to one containing O.O!?&M CaCf, 35 0 mM I&l 20.0 mM taurine, as well as 0.1 bovine albumin and 0.25 m ml collagenase (Sigma, St. Louis, MO, USA; type I) for an a(Bfl ditional40 - 60 min. All pe lYusates were warmed to 37 ‘C and continuously assed with 95% 0, and 5% CO, while perfusion pressure was maintained at 40 cm/l&O and p& was adjusted to 7.4. Following perfusion with the colla nase containing solution, the right ventricular free wall was removed diced into small ch uIZes and placed into KB medium (13). The corn sition of the KB medium utilized was (in mM): KC1 85 0. &HPG 3 0. MgC1, 5 6 N&G 5 0. Pyruvic acid, 5.0; beta-hydroxy butyric acid, 5.6; crt&ne, 5.&?a&ie 20.6; NaCl, 4.0; C&: 0.5; EGTA, 1.0 and lucose, 20.0 with the addition of polyvinyl-pyrrolidone, 50 g/l. The H of this solution was a4 ‘usted to 7.2 by addition of KOH. The tissue was eqmhbrated in I&for 60 min and isolated myocytes were obtained by gentle agitation of the minced tissue. The cell suspension thus obtained was diluted (1:lO) in KB medium and the resulting mixture was stored at 4 ‘C for u to 36 hr. One drop of cell suspension was laced in a trans arent recording chamber moun tedpon the stage of an inverted microscope (h aphot TMD, 8 Ron Co. Tokyo, Japan) and the individual myocytes were allowed to settle on the bottom of the recording chamber for at least 10 min prior to making electrical measurements. HEPES buffered Tyrode solution (HBT) was utilized as the normal au rfusate. This solution contained, in mM, NaCl, 137.0; KCl, 5.4; Na&IPG,, 0.35; K&PO,, 0.3; r aCl,, B,O;_MgCl,,1.0; HEPES, 20.0; glucose, 20.0 at a pH of 7.4. Superfusion was maintained by gravuhty flow. Suction electrodes having tip diameters less than 3 pm were fabricated from glass capillaries (Kimax-51, Kimble products USA). After fire olishing, the electrodes had resistances in the range of 2 - 4 Ma when filled with the standar B filling solution. The standard filli solution consisted of (in mM): KC1 140. Na&TP 5. MgCl,, 1; EGTA, 1; CaCl,, 0.5; pH 7.2.n&ctrode potentials were adjusted tdzero’immedia&y before formation of seals between the electrode tip and membrane. After establishing a high resistance (l- 2 GQ) seal, the cell membrane beneath the electrode tip was disrupted by further suction or by application of 1.5 V pulses 1 - 5 ms in duration. Input resistance and cell capacitance were measured during application of a 10 mV depolarizing pulse from a holding potential of -80 mV. Membrane action potentials were elicited by applying depolarizing constant current pulses that were 1.5 - ‘2 nA in amplitude and 6 to 12 ms in duration. All measurements were obtained at room temperature using an Axopatch 1B patch-clamp amplifier (Axon Instruments, Burlingame, CA, USA) in either voltage- or current-clam mode. Analog outputs from this amplifier were digitized under software control (Clampex 5. B3; Axon Instruments) using an analogue-to-digital converter (Labmaster, Cleveland, OH, USA, model TL-1) having a maximal cycle length of 125 KHz and installed on an 8 MHz IBM-AT computer. Digitized recordings thus obtained were analyzed with the aid of software developed by Axon Instruments (Clampan 5.03 and Clam fit 5.03). In addition, Student’s t-tests for either aired or unpaired data were performed to Betermine the si ‘ficance level of the changes pro a uced by the various maneuvers employed. Probability (p) V~S of less than 0.05 were considered significant.
The effect of sotalol on I,, in rabbit ventricular myocytes was evaluated from a holding potential of -50 mV in order to inactivate I,. In these experiments, the bathing solution also Under these conditions, the quasi steady-state contained 0.25 mM CdCl, to block I,. current-voltages relation established at the end of 400 ms command pulses applied every 3 8 was not effected by 60 pM sotalol (Fig. 1, upper panels)
Vol.
Sotalol
49, No. 4, 1991
~Qrominent
I,_was observed at rtentials,
PL-9
on Membrane Currents
sitive to -20 mV in 6 of the 10 rabbit
WXI~~C ar myocytes m wbxh &, was b ocked by dC1,. In these myocytes, sotalol(60 @‘I) did not affect the amplitude of I, (Fi . 1, lower panels) which was measured as the difference
between the peak outward current o%served within 15 ms after the start of the voltage pulse and the current recorded at the end of the command pulse (14). In addition, 60 @ sotal did not significantly change the inactivation time constant ( q_, ) for I, evoked during command pulses to +50 mV (61.7 + 12.7 ms before uersus 51.8 * 8.1 ms after sotalol; n = 6). A) Steady-state IV relation
4.0 .“.V
-1
l
-
A ------
CmaROL SOTALOL
Sotalol
-1.4 -1.8 iooms
B) Transient outwardcurrent
SPA
;
0’ 0
20
40
v01mgc (mV)
Fipure 1. A) Effect of 60 fl sotalol on the quasi steady-state current-voltage relation established in 10 rabbit ventricular myocytes. Holding potential = -50 mV, command pulse duration = 400 ms, command pulse frequency = 0.33 Hz. Panels to the right are of representative recordings to illustrate the effect of 60 @I sotalol when clamped to test potentials of -100, -80, -40,O and +40 mV, before (top) and after a plication of 60 pM sotalol (bottom). B) Pe 9 transient outward current before ( l ) and after ( A ) the application of 60 pM sotalol in rabbit myocytes (n = 6). Analog recordin s to the right were obtained before and after administration of 60 fl sotalol. I! olding potential = -50 mV, command potential = +50 mV, command pulse duration = 400 1119, command pulse fixquency = 0.33 Hz Inward Ca*+currents were assessed in 5 rabbit ventricular myocytes not exposed to CdCl, and not having a rominent I,. This current was measured before and after e osure to sotalol (60 a) as the d& erence between the peak inward current activated within “R t e first I5 ills of the command pulse and the current at the end of a 400 ms long comman d pulse (Fig. 2). As illustrated, sotalol effM.ed neither the amplitude nor inactivation kinetics of b, in these preparations. Before sotalol, T,, for I,-, evoked by a command pulse to 0 mV was 40.4 * 4.2 ms, and following sotalol administration it was 45.5 & 1.9 ms (NS; n = 5). Because a sodium “window” current, or slowly inactivating component of the fast Na+ current (I,), has been suggested to be important in control of APD (15) and because of the similarit of the electromechanical effects of sotalol to those produced by veratrine (16), we previou&y srulated upon ther ssibility of sotalol having direct effects on cardiac Na+ channels (11. TO test this possib’ ity, we e xamined I, in rabbit ventricular myocytes clamped to a holding potential of -90 mV. To avoid contamination of IN.by other inward currents, Ic.
PL-10
Sotalol
on Membrane
Currents
Vol.
49,
No.
4,
1991
was blocked in this series of experiments by addition of 0.25 mM Cd&. Command tentials between 40 and +40 mV were applied and the large inward currents thus evoke Z”decayed within 10 ms and were blocked by 40 J.&Itetrodotoxin (TTX). Following exposure to 1 &ml veratrine, a second slowly inactivating, TTX sensitive, corn nent to this inward current was expressed (Fig. 2e). In contrast, 60 fl sotalol failed to pro arent effect on the 1, (Fig 2d). Similar results were obtained in 6 additional rabbit ventric A) Inwardcalciumcurrent 0.2 -60
l -
CONTROL
A ----
SOTALOL
D)
(nA) ,
SOTALOL
(A) Effect of 60 fl sotalol on cal ard currents in rabbit ventricular myocytes (n = 5). Analog recordings before (B) and after (C) administration of 60 fl sotalol are illustrated at the right of the figure. Holding otential = -50 mV, test potential = 0 mV, test pulse duration = 400 ms, test pulse ip requency = 0.33 Hz Comparison of the effects of 60 p.M sotalol (D) and 1 &ml veratrine (E) on sodium current inactivation in rabbit ventricular myocytes. The veratrine effect was completely blocked by addition of 10 p.M TTX. Holding potential = -90 mV, test otential = -20 mV, test ulse duration = 400 ms, and test pulse fre uency = 0.33 R z. In panels D and E tRe bathing solution contained 0.25 mM Cd E1,. In rabbit ventricular myocytes examined at room temperature, Ix has been reported to be either very small or absent (12,17). We also could not detect an Ix in the rabbit myocytes that we examined at room temperature while using 3 s long depolarizing command pulses applied from a holdin potential of -50 mV. In rabbit, therefore, sotalol could not be demonstrated to affect lgx. In guinea pig ventricular myocytes, however, we routinely observed a large Ix at room temperature when I,, was abolished by 0.25 mM CdCl,. In our experiments utilizing guinea pig myocytes, membrane potential was held at -40 mV and 5 s long depolarizin command pulses were applied every 12 s. The amplitudes of the tail currents thus evoke d upon return to the holding otential were determined. Under these conditions, 60 fl sotalol markedly reduced the amp Petude of the outward current developed during the command pulse while the amplitudes of the outward tail currents were also decreased (Fig. 3) The effects of sotalol on action potential characteristics were examined at room temperature in both rabbit and guinea ig ventricular myocytes continuously paced at 0.5 Hz. Figure 4 illustrates representative fin d!mgs. Sotalol(60 p.M) had no effect on either diastolic potential or action potential amplitude (APA) in either rabbit or guinea pig ventricular myocytes. In 6 rabbit ventricular myocytes, therefore, diastolic potential was -72.3 f 1.2 mV before and -72.6 f 1.6 mV following 60 p.M sotalol while APA in these preparations was 116.0 ? 3.1 mV before and 114.7 -+3.3 mV after sotalol. Similarly, in 6 guinea ig myocytes, diastolic potential and APA were -72.2 + 1.0 and 115.3 + 3.5 mV before and -73. f f 1.3 and 117.3 rt:1.8 mV after 60 pM sotalol. In addition, in rabbit ventricular myocytes, 60 p.M sotalol did not change APD measured at either 50% or 90% of repolarization (APD, and AF’Dw, respectively) so that before and after sotalol APD,, and APD, were 495.7 + 52.3 and 535.3 + 56.4 ms, and 486.3 + 55.8 and 519.3 + 60.4 ms, respectively. This sotalol concentration (60 a), however, did significantly increase both APD, and APD, in guinea pig myocytes from 449.0 f 43.2 and 474.7 f 43.5 ms to 513.0 f 56.2 (p < 0.01) and 544.3 + 57.3 ms (p < 0.05).
1667 't 'ON ‘6'7 “COA
PL-12
Sotalol
on Membrane Currents
Vol.
49,
No. 4,
1991
on IKwas not examin ed in the present study, it should be noted that at 32 ‘C sotalol has been demonstrated to increase rabbit ventricular muscle APD in concentrations in the range of those utilized (20). effect of temperature
Bergeret al. (8) reported that involtage-clamped sheep Purkinje strands &is not affected by sotalol while I, is markedly decreased. Our results from guinea ig ventricular myocytes indicate the opposite-- I, is not effected while IK is substantially Elocked by sotalol. The explanation for this discrepancy inresultsis not clear. Although, sgecies+epndent differen!es could be involved, such an e lanation would ap ar unlikely as amehet s (7) demonstration of a sotalol-induced block of“pKwas obtained in s?Zeep Purkinje fibre preparations.
Additional effects of sotalol certainly cannot be ruled out and the specific effects of sotalol on Iz channels remains to be carefully worked out. Sanguinetti & Jurkiewicz (9) have, for example, recently demonstrated that, in guinea pig ventncular myocytes examined at 35 ‘C, blocked a rapidly activating component of I,. At 22 ‘C, however, these authors (9 component of IKwas either absent or its activation so slow that it could not be from Iz’s other component. The results of the resent stud reported by Carmeliet (7) and by 2 anguinetti & Jurkiewicz (9), that sotalol induced increases in action potential duration are most likely due to block of I* Acknowledeements The aotalol utilized in this study was obtained as a gift from Bristol-Myers/Squibb Pharmaceuticals, Wallingford, CT, USA. The study was funded by a grant to David A. Lathrop from the FzTd,Heart, Lung and Blood Institute of the United States National Institutes of Health CR01 References M.R. ROSEN and A.L. WIT, Am Heart J l& 829-841(1983)
E.M. VAUGHANWILLIAMS, Pharmacol Ther B 1115-121(1975) H.C. STRAUSS,J.T. BIGGERand B.F. HOFFMAN, Circ Res p 661-678 (1970)
B.N. SINGH and E.M. VAUGHANWILLIAMS, Br J Pharmacol p 675-687 (1970) pi9y44yBouL, G. ATALLAH,G. KIRKORIAN, M. LAMAUD, and P. MoLEUR, Am Heart J l!X 888-895 B.N. SINGH, Am J Cardiol & 84A-88A (1990) E. CARMELIET,J Pharmacol Exp Ther m 657-62 (1985) F. BERGER,U. BORCHARDand D. HAFNER,Naunyn-Schmiedeberg’s Arch Pharmacol XQ 696-704 (1989) M.C. SANGIJINEITIand N.K. JURKIEWICZ,J Gen Physiol $)$j195-215 (1990) D.S. ECHT, L.E. BERTE,W.T. CLUSJN,R.G. SAMIJEISSON,D.C. HARRISON,and J.W. MASON, Am
J Cardiol IiQ1082-1086 (1982)
D.A. LATHROP,Can J Physiol Pharmacol $Q 1506-1512 (1985) M. HIRAOKAand S. KAWANO J Ph siol QQ 187-212 (1989) G. ISENBERGand U. KL&XI&R Pkigers Arch s 6-18 (1982) M. HIRAOKAand S. KAWANO,Circ Res 6Q 14-26 (1987) D. ATIWELL, I. COHEN, D. ELSNER,M. OHBA, and C. OJEDA, Eur J Physiol m 137-142 (1979) D.A. LATHROPand A. VARR~, Can J Physiol Pharmacol 611463-1467 (1989) W.R. GILES and Y. IMAJZUMI,J Physiol $&j 123-145 (1988) P.J. NEUVONEN, E. ELONEN, A TANSKANEN, and J. TOHIJMILEHTO,Eur J Clin Pharmacol 24 85-89 (1981) P. SOMANI and D.L. WATSON, J Pharmacol Exp Therm 317-352 (1968) S.M. COBBE, B.S., MANLEY, and D. ALRXOPOULOS,Cardiovasc Res u 668-673 (1985)