Antiarrhythmic actions of verapamil against ischaemic arrhythmias in the rat

Br. J. Pharma. (1984), 83,373-385 Antiarrhythmi ations of verapamil against ishaemi arrhythmias in the rat M.J. Curtis, B.A. MaLeod & M.J.A. Walker Department of Pharmaology & Therapeutis, Faulty of Mediine, The University of British Columbia, 2176 Health Sienes Mall, Vanouver, B.C., V6T 1W5, Canada 1 The ations of intravenous verapamil against arrhythmias indued by olusion of a oronary artery were investigated in onsious rats. 2 Verapamil (2-2mgkg-1, i.v. given pre-olusion) dose-dependently redued arrhythmias in rats with either large or small oluded zones at an ED5 of 6 mg kg-'. This dose was effetive when given immediately post-olusion. 3 Severe arrhythmias, as opposed to PVC, were preferentially redued. 4 In onsious, and pentobarbitone-anaesthetized rats, verapamil (6 mg kg-) had different effets on eletrially-indued arrhythmias, and the ECG, from an equi-effetive anti-arrhythmi dose of quinidine (2mgkg-1, i.v.). Quinidine dereased following frequeny, but inreased threshold urrent and pulse width, whereas verapamil did not. Both drugs inreased P-R interval, but only quinidine inreased QRS and Q-T intervals. 5 Thirty minutes post-olusion, the verapamil ontent of tissue and blood was determined after a 6 mg kg-1 dose given pre- or post-olusion. Measurable levels of verapamil were found in both normal and ishaemi myoardium. Plasma and plasma water onentrations were 3.6 +.8 ltmol - 1 and.6±.1 Mmoll-1 (x±s.e.mean), respetively following post-olusion administration vs. 2.7 ± 1.2 and.24 ±.4 for pre-olusion administration. 6 Plasma water onentrations were lose to IC5 values for inhibition of ontratility in rat atria and ventriles. Similar onentrations depressed slow ation potentials indued in rat ventriles by raised K+ 7 We suggest that the ability of verapamil to prevent severe ventriular arrhythmias following myoardial ishaemia in the onsious rat is largely due to the alium antagonist effets of the drug. Introdution A variety of drugs have been shown to redue experimental arrhythmias due to irreversible myoardial ishaemia. Unfortunately in many experiments only single doses were studied by methods of unertain preision and auray. Our method of oluding a oronary artery in onsious rats allows responses to ishaemia and infartion to be assessed with preision and auray. The model has been used previously to assess established antiarrhythmis (Johnston et al., 1983a), prostaylin and aspirin (Johnston et al., 1983b), halothane (MaLeod et al., 1983) and similar anaesthetis (Jang et al., 1983). Arrhythmias in this model were not redued by P-blokade or sympathetomy (Botting et al., 1983). We have now investigated the antiarrhythmi ations of verapamil in this model in onjuntion with studies into other ations of verapamil on rat myoardium. Verapamil is well-established as a treatment for supra-ventriular antiarrhythmias (Surawiz, 1982). Antiarrhythmi ativity has also been demonstrated in a variety of experimental preparations. Verapamil (.1 to.5 mgkg-1) has antiarrhythmi ations against arrhythmias indued by oronary olusion in dogs (Brooks et al., 198; Temesy et al., 1982; Thale et al., 1982). Verapamil has also been reported to be antiarrhythmi following oronary artery olusion in the autely prepared anaesthetized rat (Bemauer, 1982; Mertz & Kaplan, 1982). Attempts were not made to determine the mehanism or site of ation of verapamil. Unfortunately in these, and other studies, a range of doses of verapamil was not studied. The doses whih were studied appeared to orrespond with those used linially for supraventriular arrhythmias. Bolus injetions of verapamil (.1 mg kg-1 ) may transiently raise plasma onentrations suffiiently to redue alium urrents in the The Mamillan Press Ltd 1984

374 M.J. CURTIS etal. atrioventriular node, and so abruptly terminate supra-ventriular tahyarrhythmias, but there is no reason to assume that suh a bolus will ontinuously suppress ishaemia-indued ventriular arrhythmias. In order to redue ishaemia-indued ventriular arrhythmias it is possible that verapamil onentrations exeeding those of benefit in supraventriular arrhythmias must be maintained. To investigate verapamil's possible antiarrhythmi ations against ishaemi-indued ventriular arrhythmias we performed a dose-response study of verapamil's antiarrhythmi ation in onsious rats. We subsequently analysed further the ation of verapamil (at an antiarrhythmi ED5) by pharmaologial and pharmaokineti studies. Methods Olusion in onsious rats Our method of produing oronary olusion in onsious rats has been used in a number of studies (see above for referenes). The model's preision and auray, have been desribed in detail (Johnston et al., 1981; 1983a). In brief, a oronary artery oluder, aorti and venous annulae, and permanent ECG leads were surgially implanted under halothane anaesthesia seven days before testing. The olusion site was varied to produe either large (LOZ), or small oluded zones (SOZ). Zone size was pre-determined by hoosing a high or low olusion site on the LAD oronary tree. On the day of study the animal was plaed in its home age with leads and annulae onneted. After 3 min, appropriate ontrol or verapamil solutions were given by intravenous infusion over 1 min in a total volume of 1 ml or less. Thereafter, a further 1 min elapsed before olusion. One group of SOZ rats reeived verapamil 6 mg kg- infusions, beginning immediately post-olusion, and ontinuing for 15 min. Infusions were temporarily stopped if diastoli blood pressure fell below 6 mmhg. Different oluded zone size groups were used sine previous studies (Johnston et al., 1983a) showed that arrhythmia sores following olusion are a square root funtion of the oluded zone volume (i.e., a linear funtion of the interfae area). Furthermore, non-arrhythmi mortality is low in SOZ rats. Treatments, proedures and reord analyses were performed blind and randomized. Antiarrhythmi studies Ishaemi arrhythmias Four doses of verapamil (.2, 2, 6 and 2 mg kg-') were tested in LOZ (greater than 3% of total ventriular weight) rats. Two, 6 and 2 mg kg- were also tested in SOZ (less than 3% of total ventriular weight) rats. Five ontrol groups (2 of SOZ, and 3 of LOZ) were used. All groups, treated and ontrol, ontained nine rats. After olusion rats were ontinuously monitored for 4 h, or until death. Survivors for 24 h were monitored for another 3 min before they were killed and their hearts exised. Perfusion of the heart with dye was used to determine the oluded zone (OZ) followed by tetrazolium for determination of the infart zone (IZ) (Johnston et al., 1983a). In animals dying before 24 h only OZ was determined. Arrhythmias, arrhythmia sore, oluded and infarted zones, blood pressure, heart rate, ECG and mortality were reorded. Statistial analyses was applied as previously desribed (Johnston et al., 1981; 1983a). Eletrially-indued arrhythmias Verapamil and quinidine were tested against eletrially-indued arrhythmias in onsious and anaesthetized rats by methods similar to those of Martinez & Crampton (1981) and Marshall et al. (1983). Stainless steel eletrodes were permanently implanted,.3 m apart, into the left ventrile, and their free ends exteriorised in the nek region. Stimulation parameters for fibrillo-flutter were measured in hronially prepared onsious, and autely prepared pentobarbitone (5 mg kg-' i.p.) anaesthetized rats. The threshold urrent (at 5 Hz and.8 ms) induing a preipitous fall in blood pressure was determined together with the threshold pulse width (at 5 Hz and 2 x threshold urrent) and maximum following frequeny (at.8 ms, 2 x threshold urrent). The latter was revealed as an abrupt large inrease in pulse pressure as the heart failed to follow, on a 1: 1 basis, a steadily inreasing frequeny of stimulation from 5 to 3 Hz. ECG and blood pressure reords showed that, as the frequeny of stimulation inreased, the heart was eventually 'aptured' with resulting hanges in the axis of the ECG. This ECG hange was maintained as the frequeny inreased while pulse and mean blood pressures fell. Eventually the heart failed to follow stimulation on a 1: 1 basis and, as a result of the dropped beat, the heart was able to fill during diastole resulting in a large inrease in pulse pressure for the next beat. The ECG showed the missing beat, and sometimes a hange in omplex shape. As this first missed beat produed suh an easily disernible hange in blood pressure it was used as the learest end point. The mean of three determinations were obtained 5 min before, and 5, 15,45 and 6 min after drug administration. Anaesthetized rats reeived only one drug with no saline ontrol, while eah onsious animal, ating as its own ontrol, reeived eah drug, and saline, with 3 days between tests.

ANTIARRHYTHMIC ACTIONS OF VERAPAMIL IN ISCHAEMIA 375 Verapamil (6 mg kg- l ) was ompared with a dose of quinidine (2mgkg-1) previously shown to redue ishaemia-indued arrhythmias by 5% in our model (Johnston et al., 1983a). Quinidine was given by slow injetion over 5-1 min whereas verapamil was infused slowly to avoid hypotension. Verapamil infusions took 4 min in anaesthetized rats but only 5 min in onsious rats. From ECG reords obtained on a Grass polygraph (Model 79B pre-amplifier type 7P1A, band width.8-5hz), P-P, P-R, QRS and Q-T intervals were measured. The first two variables were onventionally measured whereas QRS was measured from Q to the isoeletri point on the RS wave. The T wave is not easily seen in the rat ECG and so a standardized postion on the S-T omplex was taken as a measure of the position of T. ECG reords were analysed in a blind manner from reords obtained at 1 mmshart speed. Tissue and Blood levels of verapamil ty minutes after olusion, whih is after the major period for arrhythmias, animals were killed and heart and blood olleted. On the basis of visual inspetion hearts were divided into ishaemi and nonishaemi tissue. Small samples were also taken from the entre of the ishaemi zone. Verapamil onentrations were determined in myoardial tissue, in blood, in plasma, and in plasma ultrafiltrates (plasma entrifuged with CF 25 Centriflo Amion membranes at 21 g) by modifiations of the published tehniques for extration of blood (Cole et al., 1981) and tissue (MAllister & Howell, 1976) with subsequent detetion by h.p.l.. (Kuwada et al., 1981). In vitro studies on ontratility and intraellularpotentials These were designed to determine the myoardial effets of a onentration range of verapamil ommensurate with those found in vivo. Right and left atria were disseted from hearts of 3 g Sprague- Dawley rats and suspended in Krebs-Henseleit solution at 35 C as previously desribed (Au et al., 198). Initial umulative alium onentrationresponse urves were obtained (for inotropism) and Ea) E Tissue and blood levels of verapamil were determined 3 min post-olusion after 6 mg kg- li.v. given either pre- or post-olusion in SOZ rats. Thira - 3 min post-olusion I r- 8 - b - 4 h post-olusion E 4-, -3 E 2o 6 1 41- -o._r 2 2 6 I2 2 2 6 2.2 2 6 2 mg kg-' iv. Figure 1 The effet of various doses of verapamil on mean arrhythmia sores in rats with large or small oluded zones. Various doses of verapamil were given i.v. pre-olusion to rats with large (-) or small (A) oluded zones. Symbol (A) indiates 6 mgkg- verapamil given post-olusion. The % redution in arrhythmia sore from the appropriate mean ontrol value (LOZ or SOZ) is given on the ordinate and loglo dose in the absissa sale. Arrhythmia sores (see Methods) were measured for the -3min (a) and -4h (b) post-olusion periods. Altogether 13 groups of rats (9 rats per group) were studied and values for the 2 SOZ and 3 LOZ groups of ontrols were aumulated. The rosses indiate estimates of ED5 values.

376 M.J. CURTIS etal. then, in the presene of the determined EC5 onentration of alium, umulative negative inotropism urves were obtained for verapamil. Calium and verapamil dose-response urves were also determined for perfused rat ventriles stimulated at 18 or 4 beats min-'. Developed pressure within ventriles was measured with intraventriular balloons at a diastoli pressure of 15 mmhg. The method of Inoue et al. (1979) was used to asertain the effet of verapamil on slow alium potentials generated in the guinea-pig papillary, and ventrile musle, by exposure to 2 mm potassium and 1-6 M isoprenaline. 1 a 81oo Results Dose-response data for antiarrhythmi effiay ofverapamil Verapamil.2-2mg kg-l i.v. produed a dosedependent redution in the arrhythmias indued by oronary olusion as judged by redutions in arrhythmia sore from ontrol values (Figure la and b). The arrhythmia sore summarises the arrhythmi history following oronary olusion. Twenty mgkg-' was almost 1% effetive in suppressing olusion-indued arrhythmias in both the - 3 min b 1) a-) -o Cf-) E._ ~ C.) -1 61-4 F 2 F 1 8 1- - 3 min post-olusion A d r 9', I I 2 6 2 6-4 h post-olusion - ~~A. C-) ;r_ U) 6 1 4 F 2 _ + A+ A - A + / OL t9 1 I 2 6 2 6 2 6 2 6 mg kg-' iv. FIgure 2 The effet of various doses of verapamil on the inidene of ventriular flutter (a and b) and fibrillation ( and d) in rats with large or small oluded zones. In (a) and (b) the inidene of ventriular flutter in groups of LOZ or SOZ rats given 2, 6, or 2 mg kg-1 verapamil pre-oulsion is expressed as the perentage redution from ontrol inidene (LOZ or SOZ) -3 min (a) or -4 h (b) after olusion. Data from the.2mg kg-1 LOZ group is exluded from the figure. The symbol () indiates LOZ rats and (A) indiates SOZ rats. In () and (d) the inidenes of ventriular fibrillation are shown: -3 min () and -4 h post-olusion (d). The open symbol refers to SOZ rats given 6 mg kg-1 post-olusion. Crosses indiate estimates of ED5 values. The ontrol inidenes of ventriular flutter for SOZ were 67% (-3 min) and 78% (-4 h). For ventriular fibrillation the inidenes were 56% and 89% respetively. For LOZ the orresponding figures were 74% and 82% for tahyardia and 74% and 89% for fibrillation.

ANTIARRHYTHMIC ACTIONS OF VERAPAMIL IN ISCHAEMIA 377 and -4h post-olusion arrhythmi periods (see also Johnston etal., 1981; 1983a). The ED5 (i.e., the dose produing a 5% redution in arrhythmia sore from ontrol) was approximately 6 mg kg-1, in both SOZ and LOZ rats. In the LOZ groups, animals were lost from the study beause of non-arrhythmi deaths. These deaths ourred at 3, 3 and 18 min post-olusion in rats given 2 mg kg- I and 16,21,25, 28, 31 and 34min post-olusion with 2mg kg-. Thus most deaths ourred after the first major arrhythmi period (4-15 min post-olusion) in the -3 min post-olusion observation period. Arrhythmias were suppressed by verapamil in both SOZ and LOZ rats, despite differenes in ontrol arrhythmia sores for the two groups. Control arrhythmia sores in SOZ rats were 3.3 +.5 (x±s.e.mean) and 4.2±.5 for the -3min and -4 h periods, respetively, versus 4.8 ±.5 and 5.3 +.4 in LOZ. Verapamil (6 mg kg-') given postolusion to SOZ rats was just as effetive as when given pre-olusion. Previous studies (Johnston et al., 1983a) showed the relationship between arrhythmia sore and oluded zone size to be suh that arrhythmia sore Table 1 The effet of various doses of verapamil on the number of premature ventriular ontrations (PVC) (as logio) indued by olusion Treatment logjo PVC -3min post-olusion Large oluded zones (LOZ) Control (n= 27) 1.4±.1 2mgkg-' (n=9) 1.5±.2 6mgkg- I(n=9) 1.2+.3 2 mg kg- (n = 9) 1.2+.2 Small oluded zones (SOZ) Control (n= 18) 1.5 +.1 2mgkg-' (n=9) 1.+.2 6mgkg-' (n= 9) 1.3 ±.2 6 mg kg-' Post (n = 9) 1.3 ±.2 2mgkg-' (n=9).4+.1* logio PVC -4 h post-olusion 1.7 +.4 1.9+.4 1.7 +.4 1.3+.2 2.4 +.2 2.3 +.3 2.4 +.3 2.4+.3 1.1 +.3* Values are given as the mean (± s.e.mean) of log,o PVC in the -3min and -4h post-olusion periods. Statistial signifiane for differenes from ontrol is indiated by * for P <.5. Control groups for SOZ (2) and LOZ (3) were amalgamated. Deaths limited PVC ourrene in LOZ rats. All doses were given pre-olusion exept for 6 mg kg-' Post where the drug was given postolusion. (AS) = a + bi, where a = interept, b = slope and I = estimated area of interfae between ishaemi and normal tissue. Control rats in this study obeyed the same relationship with oeffiients of a = m2 and b = 6.5 AS/m2 for an assumed average ventrile wall thikness of.25 m and the radius of a 5% OZ to be.65 m. Data for verapamil also obeyed the same linear relationship with a ommon slope and variable extrapolated interepts (a) of.2 m2 for 2 mg kg- I verapamil,.3 m2 for 6 mg kg- I verapamil and.6 m2 for 2 mg kg- I verapamil. The antiarrhythmi effetiveness of verapamil was also seen as a redued inidene of ventriular flutter (a,b) and fibrillation (,d) (Figure 2). Verapamil was most effetive in reduing the inidene of these severe arrhythmias in SOZ rats. Twenty mg kg-i verapamil prevented almost all severe arrhythmias, whether flutter (a,b) or fibrillation (,d), espeially in SOZ versus LOZ rats. The 6 mg kg-l post-olusion dose in SOZ rats was less effetive against severe arrhythmias than the pre-olusion dose. The group inidene of fatal irreversible ventriular fibrillation was also redued by verapamil (see Table 2). While verapamil markedly redued arrhythmia and the inidene of ventriular flutter and sore, fibrillation, it had a lesser effet on the number of premature ventriular ontrations (PVC) (Table 1). Even at the highest dose, verapamil did not markedly redue the number of PVCs in the large infart group (25% redution only). The only dose whih redued the number of PVCs signifiantly was 2 mg kg-l in SOZ rats. Effets of verapamil on infarted zone size and mortality None of the verapamil treatments hanged the size of the oluded or infarted zones (Table 2) in either SOZ or LOZ rats although the drug produed a dose-dependent inrease in non-arrhythmi deaths in LOZ animals (e.g., 6/9 rats at 2mg kg-1) while reduing the number of deaths due to irreversible ventriular fibrillation from 1/27 in ontrols to /9. Non-arrhythmi deaths were assoiated with profound falls in blood pressure and were onsidered to be due to ardia output failure. Non-arrhythmi mortality in SOZ rats was not inreased by verapamil although the ourrene of irreversible ventriular fibrillation was redued (by 1% for 6 and 2mgkg-'). Effets of verapamil on the post-olusion ECG The hanges in the S-T segment expressed as dstr and R wave size following olusion were not influened in a preditable manner by verapamil treatment (Table 2).

378 M.J. CURTIS etal. Table 2 The effet of various doses of verapamil on oluded and infart zone sizes, on mortality and on ECG hanges indued by olusion Dose Control 2mgkg-1 6mgkg-1 2 mg kg-1 Control 2mgkg-1 6mgkg-1 6 mg kg-' Post 2 mg kg-' n 27 99 9 n 18 99 9 9 Oluded zone Infart zone (as % ventriular weight) 4±1 4±3 38+2 4+2 22+1 23+1 23+1 22+3 21±1 Mortality postolusion as number in group -4h -24h Large oluded zone (LOZ) NA A 25±4 3 1 32 3 3 26 2 2 31 6 Small oluded zone (SOZ) NA A 16±1 1 5 16+2 1 1 17+1 1 13+3 1 1 14+1 1 16 76 6 9 2 2 3 1 dstr.29 +.9.29 +.3.34 ±.5.22 ±.2 (n= 4-22).23 ±.3.25 ±.4.33 +.9.19+.6.21 ±.4 (n= 6-13) ECG (1 h) R.63 ±.7.52±.5.74 ±.5.64 ±.9.64+.6.63 ±.8.59±.13.66 ±.8.34+.11 Control groups for LOZ (3) and SOZ (2) were amalgamated to give a single ontrol group for eah oluded zone size (large or small) beause analysis showed no differenes between groups within the LOZ or SOZ groupings. Oluded and infarted zones were estimated as indiated in Methods. Non-arrhythmi mortality (NA), in the - 4 h period, is given as the number of dead animals in the group (n originally = 9 exept for ontrol LOZ where n = 27 and ontrol SOZ where n = 18). Arrhythmi deaths by 4 h post-ligation are indiated by A. Total mortality by 24 h (arrhythmi and non-arrhythmi) is given in the third olumn. ECGs at 1 h post-olusion were analysed as desribed in Methods for S-T segment elevation and inrease in R-wave size (R). The S-T segment elevations were orreted for R-wave size (dstr). All doses were given pre-olusion, exept for 6 mg kg-1 Post where drug treatment was started immediately post-olusion. Values are i±s.e.mean exept for mortality figures. The s.e.mean is not given where n < 4. Effets of verapamil on heart rate and blood pressure In onsious rats verapamil infusion sometimes redued diastoli blood pressure to 55-6 mmhg, but despite this, animals remained onsious although relatively immobile with their heads held down. At this blood pressure infusions were stopped, but they ould be reommened within 5 min as blood pressure reovered quite rapidly. In both SOZ and LOZ rats, 1 min pre-olusion, verapamil produed a dose-dependent fall in blood pressure (Figure 3a) with an ED25 of 8 mg kg- and 1 an ED5 of 3 mg kg-'. After 1 h of olusion (Figure 3b), blood pressure was still redued by verapamil in a doserelated manner, partiularly in LOZ rats. Verapamil also aused a dose-related bradyardia prior to olusion (Figure 3) but this relationship was lost 1 h after olusion (Figure 3d). The ED5 for heart rate redution in the pre-olusion period was approximately 3mg kg-'. A slight tahyardia was seen with the lowest dose of verapamil. The ratios of ED5 values were estimated to be at least 5 for vasodepressor to antiarrhythmi effet, and 5 for heart rate to antiarrhythmi effet. Effets of verapamil and quinidine on eletriallyindued arrhythmias As verapamil at high onentrations is known to have quinidine-like ations on the rise rate of myoardial intraellular potentials in vitro (Bayer et al., 1975), we determined whether 6 mg kg-1 verapamil (i.e., the ED5 against ishaemia-indued arrhythmias) had quinidine-like effets on eletrially-indued arrhythmias, and on the ECG. Rise rate depression should redue sensitivity to eletrially-indued arrhythmias and widen the QRS interval. Quinidine also widens the Q-T interval and lowers following frequeny by virtue of prolonging ation potential duration. We determined whether verapamil at 6mg kg- had the 1 same ations as 2mg kgquinidine (Figure 4). Pre-drug values for the stimulation thresholds (Table 3a) varied signifiantly between onsious

ANTIARRHYTHMIC ACTIONS OF VERAPAMIL IN ISCHAEMIA 379 failed to show quinidine-like effets on thresholds or following frequeny. In the rat ECG before drug treatment (Table 3a), the QRS and Q-T intervals were longer in anaesthetized than in onsious rats. These differenes were probably due to differenes in heart rate (Table 3b) between the two groups. Drug-indued hanges (Figure 4b) were potentiated in anaesthetized animals and suh potentiation was greatest for quinidine. Both quinidine and verapamil prolonged P-R intervals. Quinidine failed to give an atropinelike shortening. Only quinidine produed the large inrease in Q-T interval (unorreted for rate) exa 1I b a) 8 - U, Ue a) aq 61 C) ) E ~ -o 4 21_ ' ioor 8 A7 I I I 1 2 6 2 6 1 min pre-olusion d _ A 2 6 2 I6 2 6 2 6 1 h post-olusion a) 6 o 4 I 2- ' A: / + 2 6 2 6 2 6 2 6 mg kg-' i.v. Figure 3 The effet of verapamil on blood pressure (a and b) and heart rate ( and d) before (a and ), and 1 h after olusion (b and d). Three different doses of verapamil were given to groups of rats with large () or small (A) oluded zones. Blood pressure and heart rate data 1 min before olusion were used to express the effets of verapamil as perentage redution in blood pressure (a), and heart rate () from ontrol group value. Additionally, 1 h post-olusion values are given in (b) and (d). The dotted lines indiate extrapolations neessary to give estimates of ED5 values (indiated by rosses). Control values in SOZ rats for mean blood pressure were 122 ± 3 mmhg at - 1 min and 113 ± 4 mmhg at 1 h while values for heart rate were 4 ± 9 and 36 ± 15 beats min-i respetively. Values for LOZ rats were 113 ± 3 mmhg at -1 min, 12 + 3 mmhg at 1 h, 45 + 1 beats min-1 and 3 8 ± 2 beats min - l. and anaesthetized rats. Threshold pulse width was longer in onsious versus anaesthetized ontrols. In onsious rats, threshold urrent ould not be determined due to limitations in our stimulator and so threshold voltage was used instead. Quinidine produed the expeted inreases in threshold urrent, and pulse width, with a redution in following frequeny (Figure 4a); hanges were most marked in anaesthetized rats. Verapamil had different effets; it inreased following frequeny without altering threshold urrent or pulse width; again hanges were greatest in anaesthetized rats. Thus verapamil had a different spetrum of ativity from quinidine and

38 M.J. CURTIS etal. 1oo Change in maximum 8 1 following frequeny 6-4 - Change in threshold urrent Change in threshold pulse width FIgure 4 - J ).E QE 2 - -2 - -4-1 8 6 4 2-2 4 2 - - -2 - -4 - b I I I 1 5 3 6 Change in P-R interval 3 6 Change in mean blood pressure.... a..o*-- *--- *-- ^...'.-. - A. r I 15 3 6 Change in QRS width I"R -MF" 1 5 3 6 Change in heart rate i: F::::2 ::::i::::::a, -- 2-t S.2::::a... I I 1 5 3 6 Change in Q-T width 15 3 6 5 3 6 6-1. 3 I 6 1 5 3 6 15 3 6 - Time (min) The effet of verapamil and quinidine treatment on stimulation variables, ECG, blood pressure and heart rate in anaesthetized and onsious rats. Rats were treated with verapamil 6 mg kg-' (triangles) or quinidine 2 mg kg-1 (irles) aording to the shedules given in Methods. Injetions were ompleted at time zero and hanges in (a) the stimulation variables (following frequeny, threshold urrent and pulse width), (b) ECG (P-R, QRS and Q-T intervals) and () blood pressure and heart rate are expressed as perentage hange from pre-drug values for anaesthetized animals (filled symbols), and from saline ontrol values for onsious rats (open symbols). Control values are given in Table 4. peted of a drug that prolongs ation potentials. Quinidine, presumeably via depression of ation potential rise rate, also widened the QRS in both anaesthetized and onsious rats (Figure 3a) whereas verapamil did not. As was the ase with eletriallyindued arrhythmias, verapamil failed to show quinidine-like effets on the ECG. Blood pressure and heart rate varied between onsious and anaesthetized rats (Table 3b). Marked hypertension ourred in anaesthetized animals. Both variables were lowered by verapamil and quinidine (Figure 4) to approximately equal degrees in anaesthetized rats. In onsious rats, quinidine was not as effetive as verapamil in lowering blood pressure and neither drug redued heart rate. As mentioned above, marked differenes between onsious and anaesthetized animals were seen in those responses hanged by verapamil, and/or quinidine (Figure 4). Pentobarbitone appeared to both potentiate and prolong the ation of both drugs. Interpolation in Figure 4, gave pharmaologial halflives of 3-4 min for quinidine's effets on ECG and stimulation variables. Data for verapamil ould not be so readily interpolated. Thus verapamil (6 mg kg- 1, i.v.) and quinidine (2 mg kg-1, i.v.) had different effets both on the ECG and on sensitivity to eletrial indution of arrhythmias.

ANTIARRHYTHMIC ACTIONS OF VERAPAMIL IN ISCHAEMIA 381 Table 3 Eletrially-indued ventriular flutter variables, blood pressure and heart rate in anaesthetised and onsious rats prior to drug treatment A. Stimulation harateristis Thresholds for: ECG (ms) Maximum follow. freq. Current Pulse width Group (Hz) (HA) (ins) P-R QRS Q-T Pentobarbitone-anaesthetized rats Pre-verapamil 17+ 1 219+ 15.18+.1 46+2 25+ 1 79+5 Pre-quinidine 2+ 2 29+1.17 +.1 46+ 2 24+ 2 79 + 5 Consious rats Volts Pre-verapamil 17+1 2.6+.4.21+.2* 45+1 21+1* 6+3* Pre-quinidine 18+ 2 2.4±.3.26 +.4* 46± 1 22+1* 64+4* B. Blood pressure and heart rates Group Blood pressure (mmhg) Heart rate Pentobarbitone-anaesthetized Rats Pre-verapamil 16 + 6/137 + 5 (sys/dias) 384 + 1 Pre-quinidine 16 + 7/13 ± 9 (sys/dias) 392 + 15 Consious rats Pre-verapamil Pre-quinidine 113 + 2* (mean pressure) 122 + 3* (mean pressure) 413 + 14 416 ± 17 Values are given as x±s.e.mean for 5-6 rats; they were measured prior to admiistration of the drug indiated. The threshold stimulation variables were determined by means of silver eletrodes inserted in the ventriles, and using the tehnique desribed in Methods. The ECG readings were from traes reorded after testing stimulation harateristis. Intervals were measured as indiated in Methods. * indiates P <.5, for differene between onsious and anaesthetized rats. Blood and tissue levels of verapamil in oronary oluded rats in the oluded tissue were, even when given post- olusion, lose to onentrations found in normal tissue. Oluded tissue ontained 86% of the ver- The results of pharmaokineti studies in rats are apamil ontent of normal. Although a degree of summarised in Figure 5. Conentrations of verapamil ross-ontamination ould be expeted beause of Table 4 In vitro sensitivity of atrial and ventriular tissue to alium and verapamil and effet of verapamil on slow alium potentials Sensitivity to alium Sensitivity to verapamil Tissue ED5 (mm) Slope ED5 (LM) Slope (A) Inotropi responses Right atria 2.7 +.5 2.6 +.4.35 ±.1 1.3 +.3 Leftatria(18bmin 1) 2.7+.4 2.5+.2.4±.5.8+.1 (4bmin- 1).19 +.11 Ventriles(18bmin- 1).5+.1 1.2+.2.14±.8 1.+.1 (4 b min- 1).6 +.3 (B) Slow aldum potentials (Effetive verapamil onentration range for inhibition) Rat ventrile musle.1 to 1 MM Guinea pig papillary musle.1 to 1 tlm The values in (A) are x±s.e.mean (n=5-6) alulated from umulative dose-response urves for alium and verapamil obtained as explained in Methods. The slope value is the Hill oeffiient for the urves. Values in (B) are a range of effetive onentrations obtained for 4-6 preparation with one ell per preparation examined. The height of the slow response was depressed by 2-8%.

382 M.J. CURTIS etal. 3 l 25 Oluded zone Normal zone 5- Whole Plasma Ultrablood filtrate g 2-- a1, 4-.3 5- -- E 15- I 3-2- -a FIgure 5 Verapamil ontent of heart tissue and blood matries 3 min after olusion and after 6 mg kg-' given i.v. pre- or post-olusion. Values are given as x±s.e.mean for ardia tissue (normal or oluded zone), and for whole blood, plasma and plasma ultrafiltrate (n = 5-6). Values for small samples from the entre of the oluded zone are indiated by the symbol (U). Values obtained when verapamil was given pre-ligation are shown in open olumns whereas values from post-olusion treatment are shown in filled olumns. - imperfetions in the method of separating ishaemi from normal tissue, it appears that verapamil distributes into the oluded zone. Surprisingly, samples taken from the entre of the zone had the highest verapamil ontents. This was partiularly so when verapamil was given pre-olusion. Despite the large dose, the plasma onentration of verapamil 3 min post-olusion was below 1-5 M. Verapamil was plasma-protein bound. Binding was 82 ± 4 and 84 ± 4% for the two groups. The mean plasma water verapamil onentrations were.6 ±.1,sM for the post group vs..2 ±.4 tlm for the pre values whih orresponded with in vitro onentrations supressing ontratility (Table 4). Verapamil metabolites In animals given 6 mg kg-l verapamil pre-olusion, a number of metabolites were found. The two major metabolites (as verapamil equivalents) aumulated in the ishaemi tissue to 9.7±3.1pmolkg-1 (x±s.e.mean) and 5.4±1.3 respetively versus 1.5±.7 and 1.2±.2[imolkg-1 in normal tissue. These metabolites were not plasma-protein bound; plasma onentrations were.53 ±.9 and.28 +.4 RiM. Effets of verapamil on myoardial tissue Both atrial and ventriular tissue showed doserelated positive inotropi responses to low onentrations of extraellular alium. In atrial tissues the alium EC5 (Table 4) was lose to the onentration (2.5 mm) in our Krebs-Henseleit solution whereas, in ventriles, the EC5 was less. EC5 estimations for verapamil's suppression of ontratility (Table 4) were made in the presene of the appropriate alium EC5s Verapamil was equi-effetive in paed and unpaed atrial tissue and more ative in ventriular versus atrial tissue, despite the differenes (slope and EC5 values) in alium-response urves for the two tissues. EC5 values for verapamil were lower at higher stimulation frequenies exept between 4 vs. 6 beats per min. In eletrophysiologial experiments, verapamil was found to inhibit slow alium potentials (indued by 15 mm K+ and isoprenaline) at onentrations similar to those inhibiting ontratility (Table 4a vs. 4b). Disussion The aim of this study was to demonstrate antiarrhythmi ations for verapamil against ishaemia-indued arrhythmias, and to determine the various fators involved. The results demonstrated unambiguously that verapamil is antiarrhythmi in the rat whether given pre- or post-olusion. The rat is now frequently used for ishaemia and infartion studies (Au et al., 1979; Clark et al., 198; Marshall et al., 1981). In onsious rats we demonstrated antiarrhythmi ativity for a number of drugs (MaLeod et al., 1983; Jang et al., 1983; Johnston et al., 1983b) inluding quinidine (Johnston et al., 1983a). Despite a small heart size and differenes in ardia physiology, the rat is sensitive to antiarrhythmis and is suitable for the qualitative and quantitative evaluation of drugs

ANTIARRHYTHMIC ACTIONS OF VERAPAMIL IN ISCHAEMIA 383 ative against ishaemi-arrhythmias. Comparison between anaesthetized ishaemi rats, pigs and dogs showed no fundamental differenes between the three speies (Bergey et al., 1982). In anaesthetized rats, Kane et al. (1981) ould not show antiarrhythmi ativity for.1 mg kg- I verapamil but were able to show it for niludipine, nisoldipine and nifedipine (Fagbemi & Parratt, 1981). In a similar study.2 mg kg-1 verapamil was moderately antiarrhythmi (Mertz & Kaplan, 1982). Bernauer (1982) found verapamil to be antiarrhythmi at 1 and 2 mg kg-l while Muller & Wilsmann (1982) showed minimal ativity for.5 mg kg-l verapamil; it was less effetive than 5 mg kg-1 quinidine. The authors stressed the antiarrhythmi importane of sodium hannel blokade. The above rat studies used low doses of verapamil ompared with those used in the present study. This may aount for equivoal evidene of verapamil's effetiveness. Previous studies used autely prepared anaesthetized animals in whih high doses of verapamil are poorly tolerated. We found anaesthetized rats to be very sensitive to the adverse ardiovasular effets of verapamil and quinidine. The resistane of onsious animals possibly reflets a superior ardiovasular status, inreased drug metabolism, and/or intat reflexes. In dogs with myoardial ishaemia, verapamil has been shown to be antiarrhythmi in the limited dose range tested (see refs. in Introdution). Unfortunately, suh studies laked ontrol fo; oluded zone size despite its importane in arrhythmogenesis (Austin et al., 1982). Our rat model allows dose-response studies, with ontrol for oluded zone size, while monitoring other responses to oronary olusion. We have shown that verapamil redues arrhythmia sore in a dose-related manner as well as reduing the inidene of major arrhythmias (ventriular flutter and fibrillation). PVCs were minimally redued whih perhaps reflets the different arrhythmogeni mehanisms for PVC, and/or the frequenydependent nature of verapamil's ation (Sanguinetti & West, 1982). While verapamil was antiarrhythmi against severe arrhythmias, and thus prevented deaths due to irreversible ventriular fibrillation, it also aused deaths by non-arrhythmi mehanisms. Doses preventing arrhythmias produed vasodilatation and ardia depression suffiient, when oupled with ishaemia, to ause death due to redued ardia output. However, if the OZ size were limited, as in the SOZ animals, suh deaths did not our. With the effiay of verapamil established, we investigated various fators involved. Above 1-5 M in vitro, verapamil has some quinidine-like ations (Bayer et al., 1975) but suh a onentration is 1 x the ED5 for inhibiting myoardial ontratility (this study), or slow alium urrents (Sanguinetti & West, 1982; Mobyvda & Sperelakis, 1983). Verapamil, at 1-7 M, is antiarrhythmi in perfused rat hearts with oronary olusion (Woodward, 1981). Plasma water onentrations of verapamil (.6 ±.1 tim post-olusion and.24 ±.4 fim preolusion) 3 min after 6 mg kg- i.v. would indiate that only alium antagonism was present in the rats used in this study. Verapamil's pharmaokinetis in rats indiates a very short half-life. In dogs, verapamil has a 2 min t1/2 alpha phase and a 2 h t1/2 3-phase (Hashimoto et al., 1982) suh that.3 mg kg-1 gives a maximum beta phase plasma onentration of.3 pmol 1-. Extrapolation of our 6mg kg- data suggests plasma onentrations at 3 min postolusion of.8timoll-1 for.2mgkg-1, up to.8 timol l-i for 2 mg kg-'). If inhibition of ontratility (Table 4) involves inhibition of the same alium urrents as those involved in ishaemi arrhythmogenesis in the rat then verapamil's antiarrhythmi ation may be due to alium antagonism. The other evidene that supports this view is that verapamil did not have quinidine-like ations on eletrial arrhythmias and ECG. Quinidine raised thresholds and lowered following frequeny, whereas verapamil did not. Only quinidine widened QRS width and Q-T intervals. This latter data was similar to that of Tisne-Versailles et al. (1982). Muller & Wilsmann (1982) showed verapamil is 1-4 x more potent than quinidine in effets on blood pressure, heart rate, and PR interval but equipotent in widening the QRS width. QRS widening was not signifiant with 1mgkg-1 verapamil in anaesthetized rats. Pharmaokineti evidene further suggested verapamil was antiarrhythmi by virtue of alium antagonism. In order to reveal antiarrhythmi ations of verapamil high doses had to be given. These lowered blood pressure, redued heart rate, and, in LOZ rats, inreased nonarrhythmi mortality. However, the ED5 values (Figure 2) for suh effets were greater than the antiarrhythmi ED5. In view of high onentrations of metabolites found in the oluded zone, and the possibly greater antiarrhythmi effiay of pre-olusion administration, the possibility exists that a verapamil metabolite(s) was responsible for observed antiarrhythmi ations. Three ardia loi for verapamil's antiarrhythmi ations are obvious; the ishaemi zone, the interfae zone and normal tissue. Extra-ardia loi are possible although blood pressure lowering alone does not aount for verapamil's antiarrhythmi ations sine other vasodepressors are not antiarrhythmi (Au et al.,1983; Jang et al., 1983). Furthermore, verapamil's vasodepressor dose-response urve was different from that for antiarrhythmi ation. Pharmaokineti data did not exlude the oluded

384 M.J. CURTIS etal. zone sine both verapamil and metabolites were found there. The high onentrations found annot readily be explained although the rat heart has small (1,iM) ollaterals whih may deliver verapamil. Alternatively, venous drainage may result in verapamil entering the zone retrogradely. Whatever the ause, verapamil, and partiularly its metabolites, are sequestered in both normal and ishaemi myoardium but it is unlikely that all this verapamil is available for effet. Possibly verapamil binds in a non-speifi manner to myoardium. With regard to the interfae zone being a possible lous of ation, our model of arrhythmogenesis (Johnston et al., 1983) of A = a + bi, indiates that verapamil inreased a. In effet this redues the effetiveness of I sine I (i.e. OZ or IZ) did not hange (Table 2). It is possible that verapamil (or metabolites) redues the interfae arrhythmogeniity or redues eletrophysiologial disparity between ishaemi and normal tissue, whih amounts to the same thing. In dogs, verapamil redues disparity between ondution times in normal and ishaemi tissue (Nakaya et al., 198; Moses, 1981; Fondaaro et al., 1978) and normalizes eletrophysiologial hanges indued by exposure to aidi, hyperkalaemi, and hypoxi onditions (Kimura et al., 1982). Finally, if oluded or interfae zones are the site of ation, and alium blokade the mehanism, our study suggests a design speifiation for a more effetive anti-fibrillatory drug. A myoardial alium antagonist, without vasular effets, would be a partiular effetive antiarrhythmi if it preferentially aumulated at, or beame ativated by, the aid ph enountered in ishaemia. A frequeny-dependent ation would seletively redue high frequeny arrhythmias suh as ventriular flutter and fibrillation. Expert tehnial assistane was given throughout by Kathleen Johnston. T. Yuswak is thanked for his help with some of the experiments. The expert tehnial guidane of Dr R. Wall was used for verapamil determinations and G.A. 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