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1979

Laboratory Investigation

Specific lK, Blockade: A NewAntiarrhythmic Mechanism?

Effect of RP58866 on Ventricular Arrhythmiasin Rat, Rabbit, and Primate

Sian A. Rees, BSc, and Michael J. Curtis, PhD

Background. The effectiveness of blockade of the inwardly rectifying K' current (IK1) in prevention ofarrhythmias is unknown. We have examined the antiarrhythmic potential of a new selective IKI blocker,RP58866, in rat, rabbit, and primate (marmoset) isolated hearts in the settings of acute ischemia andreperfusion.Methods and Results. In concentration-response studies (n=12 per group), the drug reduced ischemia-

induced ventricular fibrillation (VF) in rat from control incidence of 100 to 50%o, 17% (p<0.05), and 0%7(p<0.05) at 1, 3, and 10 gmol/L, respectively. RP58866 produced significant bradycardia at the 3- and10-,gmol/L concentrations and significant QT interval widening at all three concentrations (p<0.05).When rat hearts (n=12 per group) were paced (5 Hz) via the left atrium to prevent bradycardia, theantiarrhythmic effects of 10-,umol/L RP58866 were unmodified (ischemia-induced VF incidence wasreduced by drug from 83% in control hearts to 8%; p<0.05). Similarly, pacing did not prevent the drug'sQT-widening activity at 90% repolarization (QT90 was 64±3 msec in control hearts versus 128±17 msecin the presence of 10 ,umol/L of drug after 10 minutes of ischemia; p<O.05). These values are similar toequivalent values in unpaced hearts (65±3 msec in control hearts versus 159±15 msec with 10 ,umol/L ofdrug; p<O.05). In separate groups of rat hearts (n=10 per group) subjected to 10 minutes of ischemia,reperfusion-induced VF incidence was reduced from 90%,o in control hearts to 10%o (p<O0.05), 0%o (p<O0.05),and 0%o (p<0.05) by 1-, 3-, and 10-,umol/L RP58866. To examine whether drug actions were species-specific, we performed further studies in rabbit and primate using the middle concentration of RP58866(3 ,umol/L). Ischemia-induced VF incidence was too low in these species to assess the effects of the drug.However, RP58866 widened QT interval (p<0.05), slowed heart rate (p<0.05), and reduced the incidenceof reperfusion-induced VF from 67% to 8% (p<0.05) in rabbit. Furthermore, in the more clinicallyrelevant primate species (marmoset; n=9-12 per group), RP58866 (3 ,umol/L) abolished ischemia-induced VT (36% incidence in control hearts; p<0.05) and significantly reduced the incidence ofischemia-induced ventricular premature beats from 91% to 33% (p<0.05). The drug was also effectiveagainst reperfusion VF in primates (incidence reduced from 64% in control hearts to 11%;p<0.05). As inrat and rabbit, RP58866 significantly widened QT interval in primate and caused bradycardia before andduring ischemia. RP58866 had no significant influence on coronary flow in any species. Finally, in furtherstudies on rat, QT widening by RP58866 was found to persist relatively unmodified in nonischemic heartsperfused with solution containing K' elevated to 8 mmol/L to mimic the early ischemic milieu.

Conclusions. RP58866, a selective IKI blocker, is a potent and efficacious new antiarrhythmic drug inischemia and reperfusion in rat, rabbit, and primate. When tested in rat, pharmacological activity wasundiminished by cardiac pacing or elevation of extracellular K'. (Circulation 1993;87:1979-1989)KEY WORDs * ventricular fibrillation * myocardial ischemia * reperfusion * primates

P otassium (K') channel blockers and their use asantiarrhythmic agents have received much at-tention recently. Such drugs are generally des-

ignated as class III antiarrhythmics, because they pro-

long action potential duration (APD) and/or QTinterval in the absence of significant effects on conduc-tion velocity.' However, until recently, all the available

From the Cardiovascular Research Laboratories, Departmentof Pharmacology, Division of Biomedical Sciences, King's College,University of London.

Supported in part by the British Pharmacological Society(S.A.R. is supported by the AJ. Clark Award). Additional supportwas kindly provided by The Royal Society (grant referenceT/2.BdeV/SG) and equipment by Unilever plc (UK).

Presented in part at the 64th Scientific Sessions of the AmericanHeart Association, Anaheim, Calif., November 11-14, 1991; the65th Scientific Sessions of the American Heart Association, New

Orleans, La., November 16-19, 1992; the British PharmacologicalSociety Summer Meeting, Dublin, Ireland, July 1992; and the 13thMeeting of the European Section of the International Society forHeart Research, Heidelberg, Germany, September 1992.

Supported by the A.J. Clark Award from the British Pharma-cological Society (S.A.R.).Address for reprints: Michael J. Curtis, PhD, Cardiovascular

Research Laboratories, Pharmacology Group, Division of Bio-medical Sciences, King's College, University of London, ManresaRoad, London SW3 6LX, UK.

Received August 19, 1992; revision accepted February 3, 1993.

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1980 Circulation Vol 87, No 6 June 1993

FIGURE 1. Diagram showing structure of RP58866.

K' channel blockers have been relatively nonselective,blocking more than one K' channel, other channels, andreceptors as well. Recently, new agents have beendeveloped that not only affect APD selectively but affectparticular cardiac repolarizing K' currents selectively.These include tedisamil, which blocks the transientoutward current (Io2); glibenclamide, which blocks theATP-dependent K' channel [IK(ATP)] and prolongs APDselectively during ischemia3; UK66,914, which blocksthe delayed rectifying K' current (IK)4,5; and RP58866,an inwardly rectifying K' current (IK1) blocker.6-8 Theseagents make it possible to examine the value of selectiveblockade of an individual K' current as a mechanism forsuppression of arrhythmias.

Until recently, the majority of work on class III agentshas focused on IK blockers. Hitherto, it has provendifficult to unequivocally link blockade of a particularchannel to arrhythmia suppression, since one cannotmeasure channel block and arrhythmia suppressioncontiguously. We have shown that UK66,914, a new andhighly selective IK blocker, widens QT interval andreduces the incidence of reperfusion-induced arrhyth-mias by more than 50% in rabbit, a species possessingfunctional cardiac IK,9 whereas in rat, a species deficientin IK,'0 the drug is devoid of activity." This shows thatIK block is a specific mechanism for suppression ofventricular fibrillation (VF) during reperfusion. Thereis currently much interest in blockade of IK(ATP) and itseffectiveness as an antiarrhythmic mechanism, but re-sults are in conflict with some studies that show IK(ATP)blockers to be beneficial3"12 and others that find themineffective.'3"4 Block of I, has been examined by Tsu-chihashi and Curtis using tedisamil.'5 Although tedis-amil had no effect on the incidence of ischemia- orreperfusion-induced VF in rat in vitro, it decreased theduration of VF once it had started. In rat in vivo,tedisamil reduces ischemia-induced VF incidence,'6 al-though this occurs only at high doses beyond the rangeover which the drug is selective for IJ,.2

Until recently, there has been very little informationon the utility of IK1 blockade as an antiarrhythmicmechanism. This is primarily a consequence of the lackof selective agents available to probe this channel. Inthe present study, our aim was to investigate the effectOf IK1 blockade on arrhythmias and QT interval usingthe novel IK1 blocker RP58866 (Figure 1). This agent hasbeen characterized pharmacologically. At concentra-tions up to 30 ,tmol/L, RP58866 is a highly selectiveblocker of IK1 and has no effect on inward currents (INaand Ica) or other potassium currents [IK and IK(ATP)I; itprolongs APD, QT interval, and atrial, nodal, andventricular refractory periods with no effects on con-duction velocity or excitability, and it has no effect onresting membrane potential.6-8"17 Thus, at concentra-tions up to 30 umol/L, RP58866 is a highly selective tool

for probing the utility of lK, blockade as an antiarrhyth-mic mechanism.We investigated the effects of RP58866 in three

species: rat, rabbit, and marmoset. Rat was used as afirst-line bioassay. Since rat ventricle possesses no func-tional IK'0 18 and since neither lK blockers'9 nor selectiveIto blockers'520 possess antifibrillatory activity in theperfused rat heart, the preparation is particularly suitedto detecting potentially antiarrhythmic effects of IKiblockade because, although RP58866 (<30 ttmol/L)does not block IK or l,,6,7 use of rat provides additionalreason for excluding IK or It,, block as mechanismspotentially contributing to any antifibrillatory effects ofthe drug. Rabbit was used to confirm actions on reper-fusion arrhythmias in a second species. Marmoset wasused to determine whether the drug at a concentrationeffective in rat could reduce ischemia- and reperfusion-induced arrhythmias in primate hearts.Our preliminary findings have been presented in part

to the American Heart Association,21,22 the BritishPharmacological Society,23 and the International Soci-ety for Heart Research.24

MethodsThe perfusion technique, methods for induction of

ischemia and reperfusion, methods for verification andquantification of occluded-zone size, and the techniquesfor recording, quantifying, and analyzing data have allbeen described previously.25-27 All experiments wereperformed in accordance with the American Physiolog-ical Society guidelines and the United Kingdom HomeOffice "Guide on the Operation of the Animals (Scien-tific Procedures) Act 1986."

Animals and General Experimental MethodsRats (male Wistar; Bantin and Kingman, UK; 250-

300 g) were anesthetized with pentobarbitone (60mg- kg-1 i.p.). Hearts were excised 30 seconds after anintravenous injection of 250 units sodium heparin andplaced into ice-cold perfusion solution containing (inmmol/L): NaCl 118.5, NaHCO3 25.0, MgSO4 1.2,NaH2PO4 1.2, CaCl2 1.4, KCl 3.0, and glucose 11.1.Hearts were perfused according to the Langendorffmethod,28 with perfusion solution delivered at 37°C andpH 7.4. All solutions were filtered (5-,um pore size)before use. Perfusion pressure was constant and equiv-alent to 100 cm H20. A unipolar electrogram (ECG)was recorded by implanting a stainless steel wire elec-trode into the center of the region to become ischemic,with a second electrode connected to the aorta. ECGconfiguration, including QT interval at 100% and 90%repolarization, was evaluated as described previously.27A traction-type coronary occluder consisting of a silksuture (Mersilk, 4-0) threaded through a polyethyleneguide was used for coronary occlusion. The suture waspositioned loosely around the left main coronary arterybeneath the left atrial appendage. Regional ischemiawas induced by tightening the occluder and reperfusionby releasing it.To exclude the possibility that any beneficial effects

seen in rat studies are the result of the unusual cardiacelectrophysiology of the rat,1529 we repeated essentialparts of the studies in rabbit, which has markedlydifferent cardiac action potential morphology, heartrate, and intracellular cation content.30


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Rees and Curtis Antiarrhythmic Action of RP58866 1981

Rabbits (male New Zealand White; Froxfield, UK;0.9-1.2 kg; n= 12 per group) were anesthetized bymeans of pentobarbitone (60 mg * kg` i.v.) with sodiumheparin (250 units i.v.). Perfusion was as described forthe rat except that the Ca21 concentration of theperfusion solution was raised to 2.8 mmol/L to raisesusceptibility to arrhythmias, as described previously.3'The ECG was recorded in the same way as for ratstudies.To examine the actions of RP58866 in an animal

species presumed to be more closely allied to human,studies were performed in primate. Marmosets (maleCallithrix jaccus; 300-350 g; n=9-12 per group) wereanesthetized with Saffan (a mix of 0.9% a-vaxilone plus0.3% a-dolone, of which we gave 2 mL/kg i.m.). Theheart was arrested and exsanguinated in vivo by inser-tion of a 23-gauge needle into the left ventricle forinfusion of ice-cold Krebs' solution (constituents asdescribed above for rat studies). This was done primar-ily because the animals were used jointly in separatestudies requiring in vivo exsanguination of other organs(e.g., brain). Cardiac excision and LangendorfF perfu-sion were performed as described above for rat andrabbit.

Experimental ProtocolsIschemia-induced arrhythmias in unpaced rat hearts.

The first protocol was designed to allow identification ofantiarrhythmic activity of RP58866 and used the iso-lated rat heart model. Hearts (n=12 per group) wereperfused for an initial 5 minutes with salt solution(constituents described above), then solution wasswitched in a randomized, blinded fashion to one of foursolutions modified by addition of stock solutions tocontain 0-, 1-, 3-, or 10-,mol/L RP58866. The0-,mol/L stock solution was vehicle (deionized water).After a further 5 minutes of perfusion, the left coronaryartery was occluded. After 30 minutes of ischemia, theoccluder was released to achieve reperfusion.

Reperfusion-induced arrhythmias in unpaced rat hearts.Owing to the expected high incidence of sustained VFin the control group during 30 minutes of ischemia,19,20a separate study was performed to investigate the effectof the drug on reperfusion-induced arrhythmias. Hearts(n= 10 per group) were perfused, randomized to one offour solutions (0-, 1-, 3-, or 10-,umol/L RP58866), andsubjected to regional ischemia as above. In these heartshowever, the occluder was released to achieve reperfu-sion after a shorter duration of ischemia (10 minutes).This duration of ischemia was used because, although itis associated with a high susceptibility to reperfusion-induced VF, it is sufficiently brief to preclude a highincidence of sustained ischemia-induced VF,25 whichwould otherwise interfere with assessment of reperfu-sion-induced arrhythmias in this model.Arrhythmias in paced rat hearts. In previous studies we

found that tacrine, a nonselective IK1 blocker,32 pro-duces sinus bradycardia,33,34 so we anticipated the pos-sibility of bradycardia with RP58866. To examine therole of any bradycardia in modulating the antiarrhyth-mic actions of RP58866, we performed additional stud-ies in paced rat hearts (n= 12 per group). Methods wereas for unpaced hearts (above) except that RP58866 wasinvestigated at the highest concentration only (10 ,umol/L). Hearts were paced with a Harvard Student Stimu-

lator using stainless steel electrodes impaled in the leftatrium (twice threshold voltage; pulse width, 0.05 sec-ond). The stimulation frequency chosen (5 Hz) pro-duces a heart rate slightly above that expected inunpaced control hearts after 10 minutes of regionalischemia. QT was measured at 90% repolarization(QTgo) in these experiments, since the end of repolar-ization was obscured, in some hearts, by the stimulusartifact, precluding accurate assessment of QT at 100%repolarization.Potassium dependence ofRP58866's effects on QT and

RR in rat. Many class III agents lose their pharmacolog-ical activity in depolarized tissue and high-K' environ-ments.35-37 In previous studies, using the present ratmodel, we found that effects of certain potassiumchannel blocker drugs on QT interval are attenuated byextracellular K' elevation.20 Since ischemia elevatesextracellular K' concentration, exacerbation or diminu-tion of QT widening by perfusion with raised-K' solu-tion can give an indication of whether this importantcomponent of the ischemic milieu is likely to promote orantagonize effects of the drug. Thus, rat hearts (n=10per group) were perfused with each of four modifiedsolutions (8-mmol/L K', 3-mmol/L K', 3-mmol/L K'plus 3-,umol/L RP58866, and 8-mmol/L K' plus3-,umol/L RP58866) for sequential 10-minute periods.During each period, three recordings were taken (after1, 5, and 9 minutes of perfusion) of QT, RR, and PRintervals. PR was corrected for changes in RR (PRc)because (unlike QT) it is rate-dependent in rat.38

Ischemia- and reperfusion-induced arrhythmias in rab-bit hearts. Hearts (n= 12 per group) were subjected to 30minutes of ischemia as for rat studies. We were able touse the same rabbits for study of ischemia- and reper-fusion-induced arrhythmias, because isolated heartsfrom this species have a much lower susceptibility toischemia-induced sustained VF than rat.39 To limitanimal use, we examined only two groups, control heartsand 3-,umol/L RP58866.

Ischemia- and reperfusion-induced arrhythmias in mar-moset hearts. There are no published data on ischemiaor reperfusion arrhythmias in marmoset. In the marmo-set studies, we followed a protocol similar to that for ratand used similar perfusion solution. Hearts were sub-jected to 30 minutes of ischemia by ligation of the leftanterior descending coronary artery close to its origin ata position approximately equivalent to that for rat andrabbit studies. Two groups were studied, control hearts(n=12) and 3-,umol/L RP58866 (n=9).

Measurement of Occluded-Zone SizeTwo independent methods were used to verify occlu-

sion and to delineate the occluded zone from unin-volved tissue. First, coronary flow was measured bytimed collection of coronary effluent in a graduatedcylinder; occlusion was verified by comparing flow at 1minute before occlusion with flow at 1 minute afterocclusion and was quantified in terms of the percentagereduction in flow. Second, at the end of 5 minutes ofreperfusion, readmission of flow was verified and thesize of the formerly occluded zone was quantified by thedisulfine blue dye exclusion method.25 Occluded-zonesize was quantified as a percentage of total ventricularweight. Values of coronary flow in the uninvolved tissueand the reperfused zone were calculated from the total


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coronary flow and the weights of the occluded zone andthe uninvolved zone, as described previously.26 Weexpected occluded-zone sizes of approximately 40%total ventricular weight in rat and rabbit." The defi-ciency of coronary collaterals in rat and rabbit40 meansthat occluded zone determined by dye is equivalent tozone by flow reduction.26 In marmoset (for which nodata exist), we aimed for an occluded-zone size equiv-alent to that in the other species by placing the coronaryoccluder in an equivalent location. Collateral flow inmarmoset was determined by comparing estimates ofoccluded-zone size measured by flow reduction and dyemethods, since dye exclusion delineates the zone ofunderperfusion, whereas flow reduction defines theextent of global reduction. Collateral flow was calcu-lated as milliliters per minute per gram of underper-fused myocardium and was expressed as percent of flowin the uninvolved zone (delineated by dye).

Exclusion CriteriaAny rat heart with a sinus rate of <250 beats per

minute or a coronary flow of > 18 mL/min or <8mL/min at 5 minutes before the onset of ischemia (andbefore randomization) was excluded. Likewise, any rab-bit with a sinus rate of <200 beats per minute or acoronary flow of >80 mL/min or <35 mL/min wasexcluded.3' Exclusion criteria for marmoset could not bedefined before the study because no data base exists.Therefore, we applied le Chatlier's principle and ex-cluded any heart for which heart rate or coronary flowvalues lay more than 2 SD from the group mean. For allthree species, any heart not in sinus rhythm during the2 seconds before reperfusion was excluded from thereperfusion sample.

Arrhythmia Diagnosis and ECG AnalysisA digital storage-type oscilloscope (model DS0400,

Gould) and a Gould chart recorder (model RS3200)were used in the identification and analysis of wave-forms and diagnosis of arrhythmias. Arrhythmias weredefined according to the Lambeth Conventions4' withslight modification.'5 Ventricular premature beats(VPB) were defined as discrete and identifiable prema-ture QRS complexes; a run of four or more VPBs wasdefined as ventricular tachycardia (VT). VF was definedas a signal from which individual QRS deflections varyin amplitude and coupling interval on a cycle-to-cyclebasis.Very often, one type of arrhythmia may convert into

another type without an intervening period of sinusrhythm; this has been amply illustrated (for one of themodels used in the present study) by anecdote inpublished papers.26 When this conversion occurs, eachtype of arrhythmia is recorded as having been presentduring the period of assessment. During ischemia, VFgenerally initiates directly from sinus rhythm, whereasduring reperfusion, VF generally begins after a run ofVT of increasing complexity and irregularity.26 Use offast chart speed recording or fast sweep speed oscillo-scope monitoring in conjunction with strict adherence tothe Lambeth Conventions41 with appropriate modifica-tions'5 is necessary to permit objective assessment of theclassification, time of onset, and duration of ventriculartachyarrhythmias. Furthermore, blinded evaluation of

0 % Repolarzation

l9 palat190 % RepolarizationV----- 100 % Repolarization

QT90

QT100FIGURE 2. Diagram showing measurement of QT90 andQT10, (in milliseconds) in a typical nonischemic electrogram.

From the ECG, the incidence and the time to onset ofarrhythmias, the RR interval, and the QT interval mea-

sured at the point of 100% repolarization (or at 90%repolarization in paced heart studies) with on-screen

cursors were obtained (see Figure 2). In isolated rat heart,QT is rate independent, but in rabbit, QT appears to varywith heart rate. For this reason, we have presented rat QTdata uncorrected (QT), but have corrected rabbit datausing Zbinden et al's42 formula (QTz=QT/RR). Appro-priate QT correction for marmoset is unknown. For thisreason, we have expressed QT uncorrected (QT), cor-

rected by Zbinden's formula (QTz), and corrected byBazett's43 formula (QTc=QTIN/Rii).Drugs and Materials

RP58866 (batch number PHI 2050) was a gift fromRhone-Poulenc Rorer (France). It was stored as a

10-3-mol/L stock solution in deionized water. All saltswere reagent grade chemicals from Sigma Chemical Co.Water for preparing perfusion solution was suppliedwith a reverse osmosis system (Milli-RO 10 and Milli-Q50, Millipore Ltd).

StatisticsMeasurement of all variables was performed in a

blinded manner, permitting use of sampling-based sta-tistics. Gaussian-distributed variables were expressed asmean±SEM and were subjected to ANOVA. In rat, iftreatment constituted a significant source of variance,each group was compared with the control by Dunnett'stest. In rabbit and marmoset studies in which only onedrug concentration was used, Gaussian-distributed datawere evaluated by unpaired t test. In high-K' perfusionstudies, ECG intervals were evaluated by paired analy-sis. The time to onset of first arrhythmia was log10-transformed to generate a Gaussian-distributed variableas described previously.25 Binomially distributed vari-ables were compared by x2 test with Yates's correctionwhere appropriate." A value of p<0.05 was taken asindicative of a statistically significant difference betweenvalues.

ResultsIschemia and Reperfusion Studies in RatRP58866 significantly reduced the incidence of is-

chemia-induced arrhythmias in a concentration-depen-dent manner. At the highest drug concentration, VT

all variables is mandatory. andW were completely abolished (p< 0.05), and VPB


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VF VT VPB VF VT VPB VF VT VPB VF VT VPB

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RP-58866 (EiM)

FIGURE 3. Bar graphs showing effect of 0-, 1-, 3-, and10-,tmolIL RP58866 on group incidences (%) of ischemia-induced ventricularfibrillation (VF) (solid bars), ventriculartachycardia (VT) (hatched bars), and ventricular prematurebeats (VPB) (open bars) in rat (n=12 hearts per group).*p<0.05 vs. control hearts.

incidence was significantly reduced (Figure 3). In the10-ttmol/L group, four of 12 hearts were entirely free ofarrhythmias during 30 minutes of ischemia. The onset ofischemia-induced VF was slightly delayed from3.1±0.03 log1o seconds in control hearts to 3.2±0.02log10 seconds in 1-gmol/L RP58866-treated hearts(p<0.05) (only two hearts of the 24 treated with the two

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FIGURE 5. Bar graphs showing effect of 0-, 1-, 3-, and10-1£molIL RP58866 on group incidences (%) ofreperfusion-induced ventricularfibrillation (VF) (solid bars), ventriculartachycardia (VT) (hatched bars), and ventricular prematurebeats (VPB) (open bars) in rat (n=10 hearts per group).*p<0.05 vs. controls.

higher concentrations of drug developed VF, so onsettimes could not be assessed in these groups). Whenischemia-induced arrhythmias were analyzed in relationto their time course of occurrence, it was found thatRP58866 reduced the occurrence of VPB, VT, and VFto a similar degree in each successive 5-minute interval,although the effects were statistically significant only

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FIGURE4. BargraphsshowingeffectofO-,umol/L (panelA), 1-,molIL (panelB), 3-,pmol/L (panel C), and lO-,mol/L (panel

D) RP58866 on the time course of occurrence of ischemia-induced ventricular fibrillation (solid bars), ventricular tachycardia(hatched bars), and ventricular premature beats (open bars) in rat (n=12 hearts per group).

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TABLE 1. Effect of RP58866 on QT Interval and RR Interval in Rat

QT (msec) RR (msec)

Time Control hearts 1.0 ,.mol/L 3.0 jumol/L 10.0 /umol/L Control hearts 1.0 1umol/L 3.0 ,umol/L 10.0 ,umol/L-10 91±8 83±3 82±2 84±3 206±9 202±6 210±8 228±10-1 109±11 153±10* 154±17* 213±24* 229±12 249±9 286±17* 338±33*+10 164±16 185±16 221±15* 298±33* 284±13 310±10 364±18* 446+39*

Time refers to minutes before (-) and after (+) the onset of ischemia.*p<0.05 vs. time-matched control hearts.

when control incidences were sufficiently high to permit"detection" of the effects (Figure 4).RP58866 was also concentration-dependently effec-

tive against reperfusion-induced arrhythmias in rat (Fig-ure 5). The 3-,umol/L concentration abolished VF, andthe 10-,umol/L concentration abolished VF and VT andreduced VPB incidence (p<0.05). RP58866 signifi-cantly widened QT and RR intervals (Table 1) beforeand during ischemia.RP58866 (examined at the highest concentration

only) retained its antiarrhythmic actions on ischemia-and reperfusion-induced arrhythmias when hearts werepaced at a frequency of 5 Hz (Figure 6). In paced hearts,the onset of the first ischemia-induced arrhythmia wassignificantly delayed by the drug from 2.70±0.02 to

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FIGURE 6. Bar graphs. PanelA shows the effect ofRP58866on incidences (%) ofischemia-induced ventricularfibrillation(VF), ventricular tachycardia (VT), and ventricular prema-ture beats (VPB) in rat heartspaced at 5Hz (n=12 heartspergroup). Control hearts are represented by solid bars and10-,umol/L RP58866-treated hearts by open bars. *p<0.05vs. control hearts (0 ,umolIL). The drug retained its ability to

significantly reduce the incidence of ischemia-induced VF,VT, and VPB when hearts were paced. Panel B shows theeffect of RP58866 on incidences (%) of reperfusion-inducedVF, VT, and VPB in rat heartspaced at 5 Hz (n= 7-12 heartsper group). Control hearts are represented by solid bars and10-,umolIL RP58866-treated hearts by open bars. *p<0.05vs. control hearts (0 ,umol/L). The drug retained its ability tosignificantly reduce the incidence of reperfusion-induced VF,VT, and VPB when hearts were paced.

3.0+0.04 logl0 seconds. The drug also retained its abilityto widen QT interval when hearts were paced (Table 2).RP58866 had no effect on coronary flow or occluded-

zone size. Coronary flow 1 minute before the onset ofischemia was 12±0.7, 10±0.8, 12±0.6, and 12± 1.1mL- min-l g-1 in control, 1-,mol/L, 3-,umol/L, and10-,umol/L groups, respectively (p=NS), and recovery

of coronary flow on reperfusion was 9+1.5, 12+2.0,11±1.1, and 9+1.2 mL min-l g- 1 of reperfused tissue,respectively (p=NS). Occluded-zone sizes were similarin each group, values being 41±3%, 40±3%, 40±2%,and 42±2% of total ventricular weight with increasingconcentrations of drug (p=NS). Thus, antiarrhythmiceffects were not secondary to changes in flow or changesin occluded-zone size.

Potassium Dependence of RP58866's Effects on QT,RR, and PR Intervals in RatThe effects of elevation of extracellular K' on the

ability of RP58866 to widen QT interval were examinedin the absence of ischemia in separate groups of rathearts (Figure 7). Switching from 8-mmol/L K' to3-mmol/L K' perfusion solution had no significanteffect on QT (93±5 versus 99±8 msec). In 3-mmol/L K'solution, 3-ttmol/L RP58866 widened QT within 1minute to 131±12 msec (p<0.05) and continued towiden it to 156±11 msec (p<0.05) after 9 minutes.When K' was increased to 8 mmol/L in the presence ofdrug, there was a significant QT shortening (p<0.05) to133-12 msec, although by 9 minutes after the change,QT had lengthened to 141 ± 10 msec.

Switching from high to low K' in the absence of drugwidened RR from 206±9 to 221±7 msec (p<0.05). Onintroduction of RP58866, RR was further widened to264±11 msec (p<0.05). When K' was increased to 8mmol/L in the presence of drug, no additional brady-cardia was seen.RP58866 had no effect on PR interval corrected for

heart rate (PRJ). PR, was significantly shortened, from47±1 to 40±2 msec, after switching from 8 mmol/L to 3

TABLE 2. Effect of RP58866 on QT Interval Measured at 90%oRepolarization in Paced and Unpaced Rat Hearts

QTgo (msec) in paced QTgo (msec) in unpacedhearts hearts

Time Control hearts RP58866 Control hearts RP58866-10 54+3 56±4 47+2 50±2-1 54±3 70±5* 48±2 87±6*+1 49±5 104±14* 53±3 120±15*+10 64±3 128±17* 65±3 159±15*

QT90, QT interval measured at 90% repolarization. Time refersto minutes before (-) and after (+) the onset of ischemia.

*p<0.05 vs. time-matched control hearts.

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600 5 10 15 20 25 30 35 40

Time (min)

FIGURE 7. Graph showing effect ofRP58866 on QTinterval(msec) in rat (n=10 per group) before and at various timesafter the onset of ischemia. Values are mean±SEM forperfusion with solution containing 8-mmolIL K' (0-10 min-utes), 3-mmolIL K' (10-20 minutes), 3-mmolIL K' plus3-pumolIL RP58866 (20-30 minutes), and 8-mmolIL K+ plus3-limolIL RP58866 (30-40 minutes). Thepresence ofdrug isindicated by the hatched background shading. Switchingfrom8-mmolIL K' to 3-mmolIL K' solution had no significanteffect on QT. In 3-mmolIL K' solution, 3-iumol/L RP58866widened QT, and when K' was raised to 8 mmolIL in thepresence of drug, there was a significant QT shortening.

mmol/L of K' in the absence of drug. Introduction ofRP58866 had no effect on PR, (39±3 msec), and raisingK' in the presence of drug caused PR, to lengthen backto 50±4 msec (p<0.05).

Effects ofRP58866 in RabbitConfirmatory studies were undertaken in rabbit, us-

ing a concentration of RP58866 (3 limol/L) found to beeffective in rat. Effects in rabbit were essentially similarto effects in rat. Control incidence of arrhythmias is verylow in rabbit isolated heart during ischemia (Figure8A), which precludes assessment of any antiarrhythmicactivity a drug might possess in this setting, as demon-strated previously.39 This does, however, make the rab-bit an ideal species in which to detect any drug-inducedproarrhythmic events. Figure 6B shows that RP58866had no proarrhythmic activity.

Reperfusion-induced VF occurred with a high inci-dence (eight of 12) in control rabbit hearts (as expect-ed39), permitting analysis of the antiarrhythmic effectsof RP58866. RP58866 significantly reduced the inci-dence of the most severe reperfusion-induced arrhyth-mias (VF and VT) but had no effect on VPB incidence(Figure 8B).As in rat, RP58866 elicited significant bradycardia

and caused significant widening of QT interval (correct-ed for alterations in rate; Table 3) but was devoid ofeffects on coronary flow (Table 3) in rabbit. Occluded-zone sizes were slightly higher than values in rat at48±1% and 46±3% of ventricular weight in control anddrug-treated hearts, respectively (p=NS).

Effects of RP58866 in PrimateRP58866 was examined in marmoset at the same

concentration (3 ,umol/L) as studied in rabbit. Theactions were essentially similar in rat and rabbit. Theslightly higher control incidence of VT and VPB duringischemia gave scope for assessment of antiarrhythmicactivity in this setting. Both VT and VPB incidences

A.

100-

80 -

_c 60-0

Cc 40--

20-

W VT VPB

Arrhythmia type

B.

000*0c

100 -

80

60-

40

20- *

VF _| F VT | | VPB

Arrhythmia type

FIGURE 8. Bargraphs. PanelA shows the effect ofRP58866on incidences (%) ofischemia-induced ventricularfibrillation(VF), ventricular tachycardia (VT), and ventricular prema-ture beats (VPB) in perfused rabbit hearts (n=12 hearts pergroup). Control hearts are represented by solid bars and3-,umolIL RP58866 -treated hearts by open bars. The inci-dence ofischemia-induced arrhythmias was too low to permitassessment of the drug's antiarrhythmic activity; however, noproarrhythmia was detected. Panel B shows the effect ofRP58866 on incidences (%) of reperfusion-induced VF, VT,and VPB in perfused rabbit hearts (n=12 hearts per group).Control hearts are represented by solid bars and 3-,umol/LRP58866-treated hearts by open bars. *p<0.05 vs. controlhearts (0 ,umolIL). The drug significantly reduced the inci-dence of reperfusion-induced VF and VT, confirming resultsin rat.

were significantly reduced by drug (from 36% to 0% andfrom 91% to 33%, respectively; p<0.05). VF incidencewas very low in marmoset during ischemia, but therewas no proarrhythmia (Figure 7A). During reperfusion,RP58866 significantly reduced VF incidence, from 64%to 11%, but had no significant effects on either VT orVPB incidence (Figure 9B).RP58866 elicited bradycardia and QT widening in

marmoset (Tables 4 and 5). Changes were similar tothose seen in rat and rabbit. QT was expressed in threedifferent ways, since appropriate heart rate correction isnot known for marmoset. QT uncorrected for heart ratewas widened in marmoset to an extent similar to that inrat. For example, values 10 minutes after the onset ofischemia were increased by 70% in marmoset and 82%in rat by 3 umol/L of drug (compared with time-matched control values). Likewise, QTz was widened toa similar extent in marmoset and rabbit 10 minutes afterischemia onset (by 18% and 16%, respectively). Thisindicates that when appropriate (species-dependent)correction for heart rate is applied, RP58866 affects QTsimilarly in different species.


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TABLE 3. Effect of RP58866 on QTz Interval, RR Interval, and Coronary Flow in Rabbit

QTz RR (msec) Coronary flow (mL min- 1 . g 1)

Time Control hearts 3 ,umol/L Control hearts 3 gmol/L Control hearts 3 gmol/L-10 0.62±0.02 0.61±0.01 260±8 253±5 15±1.1 16±0.9-1 0.62±0.02 0.73±0.02* 294+6 334±10* 13±1.0 13±0.8+10 0.61±0.02 0.71±0.02* 336±17 383±12* 6±0.6 6±0.6

QTz, QT interval corrected by Zbinden's formula.42 Time refers to minutes before (-) and after (+) the onset of ischemia.*p<0.05 vs. time-matched control hearts.

RP58866 had no effect on flow before or duringischemia (Table 5) or on recovery of flow on reperfusion(13+ 1 mL - mind` g`1 of reperfused tissue versus 15+1mL* min 1. g` in control and drug-treated hearts, re-spectively; p=NS). Occluded-zone sizes were similar incontrol and drug groups whether measured by flow-reduction or dye-exclusion methods (in control group,45±1% by dye method and 45±3% by flow reductionversus in drug-treated hearts, 47±3% by dye method

00(D.5c

0

0

A.

100-

80 -

60 -

40- L20

V | | VT | | VPB |Arrhythmia type

B.

100 -

80 -

60

40- ul20-

| vi| | VPB |

Anhythmia type

FIGURE 9. Bargraphs. PanelA shows the effect ofRP58866on incidences (%) ofischemia-induced ventricularfibrillation(VF), ventricular tachycardia (VT), and ventricular prema-ture beats (VPB) inperfused marmoset hearts. Control hearts(n= 12) are represented by solid bars and 3-,umol/LRP58866-treated hearts (n=9) by open bars. The drugsignificantly reduced the incidence of ischemia-induced VTand VPB. The incidence of VF was too low to permitassessment ofantifibrillatory activity. PanelB shows the effectof RP58866 on incidences (%) of reperfusion-induced VF,VT, and VPB in perfused marmoset hearts. Control hearts(n=12) are represented by solid bars and 3-pimolILRP58866-treated hearts (n=9) by open bars. *p<0.05 vs.

control hearts (0 gmol/L). The drug significantly reduced theincidence of reperfusion-induced VF, but had no significanteffect on VT or VPB.

and 46±3% by flow-reduction measurements; p=NS).The consistency between flow and dye measures ofoccluded-zone size, reminiscent of equivalent data forrat,26 indicates an absence of functional coronary col-lateral vessels in this species, as is the case for rat andrabbit.40 The similarity between control and druggroups for each of these estimates indicates an inabilityof RP58866 to increase collateral flow in this species.Findings were similar for rat and rabbit.

DiscussionClass III antiarrhythmic drugs represent a potential

alternative to class I drugs in ischemic heart disease,given the problems associated with the latter45 and inview of the antiarrhythmic efficacy of agents such asD-sotalol.46-48 However, class III agents may have aninherent disadvantage in that their actions are com-monly diminished with the short coupling intervalstypical of ventricular tachyarrhythmias.49 This loss ofactivity at shorter cycle lengths is least prominent withamiodarone but appears to be a common feature ofmost class III agents,'0505' and it has been argued thattherapeutic efficacy may be jeopardized as a result ofit.52 The reason for this is not clear. However, it may notbe a result of reverse use-dependence, as purported byHondeghem and Snyders,52 since others have reportedconventional use-dependence with class III agents.49,53

Class III agents are not a homogeneous class, how-ever. Subclassification is possible in terms of the partic-ular K' channel blocked by the agent, according to thesuggestion of the Sicilian gambit54 as discussed in moredetail recently by Rosen et a155 and Colatsky.56 Mostclass III agents block IK.49 However, some investiga-tional agents, such as tacrine, also block 'K1.32 The lattermay represent a viable alternative to 1K blockers. How-ever, their effectiveness as antiarrhythmics is notestablished.The present study is the first to examine the potential

of a new and highly selective lKi blocker in prophylaxisof ischemia- and reperfusion-induced arrhythmias. Weobserved consistent antiarrhythmic effects in all threespecies studied (rat, rabbit, and marmoset), with effectsbeing concentration-dependent where studied (in rat).Effects were accompanied by equivalent QT wideningand bradycardia in each species.The first question to consider is whether these effects

were direct or secondary to "anti-ischemic" actions. Anindirect antiarrhythmogenic action (e.g., via some anti-ischemic property) can be ruled out. The reasons for thisare as follows. First, anti-ischemic interventions delay theonset of ischemia-induced and reperfusion-induced VFsusceptibility (shown explicitly in the rat model57); theydo not suppress arrhythmias throughout the time courseof ischemia, in contrast to the findings with RP58866 in


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TABLE 4. Effect of RP58866 on QT Interval Corrected and Uncorrected in Primate

QT QTz QT.

Time Control hearts RP58866 Control hearts RP58866 Control hearts RP58866

-10 137+3 148+6 0.51±0.02 0.53±0.03 8±0.2 8±0.4-1 151±6 231±14* 0.58±0.03 0.64±0.05 9±0.4 12±0.7*+10 159±10 270±18* 0.56±0.02 0.66±0.04* 9±0.5 13±0.6*

QT represents uncorrected QT interval; QTz represents QT corrected for changes in heart rate by Zbinden's formula (see text); QT,represents QT corrected by Bazett's formula (see text). Time refers to minutes before (-) and after (+) the onset of ischemia.


the rat (Figure 4). Second, pacing rat hearts failed toreverse the protective effects of the drug, as would beexpected if anti-ischemic actions contributed to the anti-arrhythmic effects. Therefore, the drug can be presumedto have affected arrhythmias as a consequence of a directeffect on cardiac electrophysiology.We used the rat to examine the role of ventricular IKI

block versus other cellular mechanisms in mediating theantiarrhythmic effects. Since previous work has shownthat UK66,914, a selective 1K blocker,5 has no effect onheart rate, QT interval, or arrhythmias in rat'9; sinceadult rat ventricle is deficient in functional IK10'18; andsince RP58866 does not block 'K over the concentrationrange used in the present study,6-8 then 'K block can beexcluded as a mechanism contributing to the drug'santiarrhythmic effects. Also, since previous work hasshown that I,. block with tedisamil is ineffective inpreventing VF in the isolated rat heart model'5 andsince RP58866 does not alter the action potential shapein the "notch" region where I, is active,7,8 then I,. blockwould not appear to be sufficient to explain any antiar-rhythmic effect of the drug in rat. In addition, RP58866has no effect on INa, IK(ATP), or T or L calcium currents,67so block of these currents can be excluded as mecha-nisms contributing to the drug's effects on arrhythmias.However, RP58866 does have established effects on IK1.These effects lead to APD widening at voltages negativeto 1 mV.8 Furthermore, in contrast to UK66,914,19RP58866 widens QT and prevents VF in rabbit and inrat (UK66,914 is effective in rabbit but has no pharma-cological activity in rat). On the basis of these data, 1K1blockade appears to be a mechanism sufficient to ac-count for the antiarrhythmic effects observed in rat inthe present study.An indication that the drug may be a specific IKI

blocker but not necessarily a completely selectiveblocker is that it caused pronounced bradycardia in allthree species tested. This is puzzling, because althoughthe periphery of the sinoatrial node has been shown topossess 1K1 ,58 the center of the node, which is responsi-

TABLE 5. Effect of RP58866 on RR Interval and Coronary Flowin Marmoset

Coronary flowRR (msec) (mL * min-l g-')

Time Control hearts RP58866 Control hearts RP58866

-10 271+9 286+14 12+0.6 13±0.7-1 265±11 373±28* 11±0.6 13±0.9+10 270±18 418±35* 6±0.4 6+0.4

Time refers to minutes before (-) and after (+) the onset ofischemia.


ble for determining heart rate, is believed to be devoidof this current. For this reason, effects on currents yetuntested, such as the hyperpolarization-activated in-ward current (If), which appears to be important indetermining pacemaker rate, and subtypes of , ('toland Ito259) cannot be ruled out completely. However, theimportant issue in relation to the present study iswhether any such effects contributed to the suppressionof arrhythmias produced by the drug via the resultantreduction in heart rate. Additional studies indicatedthat drug-induced bradycardia was not responsible forthe antiarrhythmic effects seen, since cardiac pacing viathe left atrium failed to diminish the drug's ability toreduce and abolish ischemia- and reperfusion-inducedVF. Concomitant with this, QT interval remained wid-ened by the drug in paced hearts. These findings suggestthat the drug is protective primarily via its direct actionsin the ventricles (cellular mechanism discussed above)independent of any additional benefit associated withthe sinus bradycardia it elicits.

Ischemia elevates extracellular K' concentration,60and the associated diastolic depolarization may dimin-ish the pharmacological activity of K' channel blockingdrugs.49 In the present study, we observed only slightattenuation of the QT-widening effects of RP58866 inhearts perfused with 8- versus 3-mmol/L K'. Thisindicates that RP58866 loses little of its pharmacologi-cal activity in a high-K' environment equivalent to thatof acute ischemia. This contrasts markedly with otherclass III agents, such as D-sotalol.36 If the antiarrhythmicactivity of class III agents is mediated primarily viaactions in the involved zone rather than in the adjacentuninvolved zone, then this action of RP58866 wouldappear to lend a potential advantage over other class IIIdrugs.

In view of the caution expressed over data derivedfrom rat studies,29 we sought to establish that RP58866was effective in additional species. Studies in rabbitrevealed good protection against reperfusion-inducedarrhythmias accompanied by QT widening and brady-cardia, findings equivalent to those in rat.We could not examine antiarrhythmic effects against

ischemia-induced arrhythmias in rabbit owing to the lowincidence of such arrhythmias in control hearts, as wehave found in previous studies in this species.39 How-ever, this allowed us to examine possible proarrhythmiceffects of the drug. RP58866 was devoid of such activityduring ischemia and reperfusion. Carlsson et a16' haveshown that class III agents can induce torsade depointes in rabbit when administered in conjunction withan a1-agonist but in the absence of ischemia and reper-fusion. It would appear from the present and previousstudies31 that potassium channel blocking drugs such as


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RP58866 do not provoke torsade de pointes in rabbithearts in the absence of "priming" with an a1-agonist.The present study demonstrated, for the first time,

pharmacological prevention of both ischemia- andreperfusion-induced arrhythmias in isolated perfusedmarmoset hearts. This model has not previously beenused for such studies because of high costs. We wereable to perform the present studies because a limitednumber of hearts were made available to us by col-leagues in our department. The lack of data base withthis model is a disadvantage. However, the model wasused under the assumption that because it is primate, itis likely to be more "relevant" to humans. The lack ofdata base, however, does mean that we cannot saywhether RP58866 is more or less effective than otherdrugs in the model. The important point from theprimate studies is that the drug was effective in themodel, reducing arrhythmias and widening QT intervalin a manner equivalent to that seen in rat and rabbit.The model itself appears to be more similar to therabbit than the rat model in terms of heart rate, QTinterval, heart size, and susceptibility to ischemia- andreperfusion-induced arrhythmias. Occluded-zone sizeswere similar in control and drug groups, whether mea-sured by dye or flow reduction (as was the case for theother two species used). The latter indicates that themarmoset heart, like that of the rat and rabbit, isdeficient in functional coronary collaterals.

Finally, although we have established that IK, block-ade is a mechanism sufficient to account for the effectsof RP58866 on VF during ischemia and reperfusion bylKi blockade, we have not addressed the mechanism bywhich lK, blockade leads to VF suppression at thesyncytial level (e.g., reentry, abnormal automaticity,flow of injury current). This is a difficult question toaddress. During a 30-minute period of ischemia in theisolated rat heart (the model used most intensively inthe present studies), initiation of VF appears to occurvia flow of injury current between ischemic and non-ischemic tissue.62 IKi blockade by RP58866 may becapable of preventing this from occurring either byreducing diastolic injury potential (by causing partialdepolarization of nonischemic tissue) or by reducingsystolic injury potential (by altering the shape of theterminal phase of the action potential). Ischemia-in-duced VPB and VT and reperfusion-induced VF ap-pear to be initiated by mechanisms other than injurycurrent flow,62 but as yet there is no proof of which ofthe possible mechanisms is most important.63 However,ischemia-induced VF may be initiated by a mechanismdifferent from that for VT and VPBs2662; moreover,modulation of VPBs preferentially at later (>20 min-utes) versus earlier stages of ischemia may indicate thatarrhythmogenic mechanisms may vary during a 30-minute period of ischemia as well as between differentarrhythmia subtypes.62 Thus, the present observationthat RP58866 appears to suppress ischemia-inducedVF, VT, and VPBs and to do so equally well at differentstages of a 30-minute period of ischemia indicates thatit may be inappropriate to view the drug as having aselective action on any specific arrhythmogenic mecha-nism (e.g., flow of injury current versus reentry). In-stead, the drug, via IK1 blockade, may have a broaderspectrum of action on so-called syncytial arrhyth-mogenic mechanisms.

In conclusion, we have demonstrated the first obser-vation of antiarrhythmic effects of a selective lKi block-ing drug in the setting of acute ischemia and reperfusionin three species, including a primate. lK, blockade byRP58866, producing a widening of QT interval (which,unusually for a K' channel blocker, appears to persistwith heart rate maintained by pacing and in tissueexposed to elevated K' concentration), may represent auseful new antiarrhythmic mechanism. Other as yetundiscovered pharmacological actions may contributeto the antiarrhythmic effects and the QT wideningobserved, although their invocation is not necessary.

AcknowledgmentsWe would like to thank Dr. Peter Collins, without whom the

primate studies would not have been possible. We would alsolike to thank Dr. Icilio Cavero for the gift of RP58866.

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S A Rees and M J Curtisarrhythmias in rat, rabbit, and primate.

Specific IK1 blockade: a new antiarrhythmic mechanism? Effect of RP58866 on ventricular

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