Effects of Inotropic and Arrhythmogenie Digoxin Doses and of Digoxin-Specific Antibody on Myocardial Monovalent Cation Transport in the Dog

Transcription

1 EFFECTS OF DIGOXIN AND ANTIBODY ON CATION TRANSPORT/Hugen et al. 23 Schmier J, van Ackern K, Bruckner UB, Hakimi B, Heger W, Sims J: Investigatins n the develpment f cllaterals, crnary flw, tachyphylaxis and steal phenmenn in dgs after applicatin f Adalat. In Secnd Internatinal Nifedipine "Adalat" Sympsium, edited by W Lchner, W Braasch, and G Krneberg. New Yrk, Springer-Verlag, 1975, pp Selwyn AP, Jnes T, Turner JH, Clark J, Lavender P: Cntinuus assessment f reginal mycardial perfusin in dgs using kryptn-81m. Circ Res 42: , 1978 Shine JJ, Fgelman AM, Kattus AA, Buckberg GD, TUlisch JH: Pathphysilgy f mycardial infarctin. Ann Intern Med 87: 75-85, 1977 Turner JH, Selwyn AP, Jnes T, Evans TR, Raphael MJ, Lavender JP: Cntinuus imaging f reginal mycardial bld flw in dgs using kryptn-81m. Cardivasc Res 10: , 1976 Vater W: Mycardial xygen cnsumptin under the influence f nifedipine (Adalat) in the anesthetized dgs. In Secnd Internatinal Nifedipine "Adalat" Sympsium, edited by W Lchner, W Braasch and G Krneberg. New Yrk, Springer- Verlag, 1975, pp Effects f Intrpic and Arrhythmgenie Digxin Dses and f Digxin-Specific Antibdy n Mycardial Mnvalent Catin Transprt in the Dg THOMAS J. HOUGEN, BRIAN L. LLOYD, AND THOMAS W. SMITH SUMMARY The effects f digxin n mnvalent catin active transprt were determined in cardiac tissue btained frm dgs given intrpic, txic, r lethal dses f digxin. In hemdynamically mnitred dgs, active uptake f the K + analgue Rb + was determined in vitr in a cntrl mycardial bipsy, and then in serial bipsies frm the same dg after the infusin f [ 3 H]digxin in dses sufficient t cause a sustained psitive intrpic effect in the absence f txicity, and finally after additinal dses t induce vert txicity. Nntxic digxin dses prducing a mean increase f 20% in left ventricular (LV) dp/dt significantly reduced Rb + active transprt by 25% belw cntrl values. At the nset f digxin-induced arrhythmias, maximal LV dp/dt was 53% abve cntrl whereas active Rb + transprt was reduced by 60% belw baseline values (P < 0.001). Cntrl dgs given vehicle alne shwed n significant change in cntractility r in mnvalent catin active transprt. In anther grup f dgs given a lethal dse f digxin, Rb + active transprt was reduced 59% belw cntrl levels at the nset f vert txicity and was further reduced 80% belw cntrl at the time f nset f a fatal rhythm disturbance. When dgs were given high affinity digxin-specific IgG r Fab fragments at the nset f vert txicity, txicity was rapidly reversed, and mnvalent catin active transprt increased t 51% f cntrl at the time f restratin f sinus rhythm. Twenty-fur hurs after antibdy reversal f arrhythmias, mnvalent catin transprt values apprximated nrmal cntrl levels. These data prvide quantitative estimates f the extent f inhibitin f mnvalent catin transprt by digxin at intrpic, txic, and lethal endpints. Similar degrees f transprt inhibitin were present at the time f nset f digxin-induced arrhythmias and at the time f arrhythmia reversal by digxin-specific antibdies. Circ Res 44: 23-31, 1979 SCHATZMANN in 1953 first demnstrated that cardiac glycsides inhibit the active transprt f mnvalent catins acrss cell membranes. In the decades since this bservatin, numerus studies Frm the Cardivascular Divisin, Peter Bent Bngham Hspital, the Department f Cardilgy, Children's Hspital Medical Center, and the Departments f Medicine and Pediatrics, Harvard Medical Schl. Bstn, Massachusetts. Supprted by Natinal Institutes f Health Grants HL and HL , and by the King Trust Fund Dr. Hugen was a Charles A. Janeway Schlar. Dr. Llyd was an Overseas Fellw f the Natinal Heart Fundatin f Australia. Address fr reprints' Dr. Thmas W. Smith, Cardivascular Divisin, Peter Bent Bngham Hspital, 721 Huntingln Avenue, Bstn, Massachusetts Received April 12, 1978; accepted fr publicatin August 23, 1978 have shwn changes in ptassium (and less cmmnly in sdium) cncentratins in cardiac tissue expsed t cardiac glycsides (Grupp and Charles, 1964; Miiller 1965; Akera and Brdy, 1978; Langer, 1977; Ettinger et al., 1977; Ku et al., 1977), presumably brught abut by the inhibitry effect f cardiac glycsides n mnvalent catin transprt. Ten years after Schatzmann's bservatin, Repke (1963) advanced the hypthesis that cardiac glycside inhibitin f the mnvalent catin transprt enzyme cmplex sdium- and ptassium-activated adensine triphsphatase (Na +,K + -ATPase) is respnsible fr the therapeutic and txic effects f digitalis. Hwever, the relatinship f cardiac gly-

2 24 CIRCULATION RESEARCH VOL. 44, N. 1, JANUARY 1979 cside inhibitin f Na +,K + -ATPase and f mnvalent catin active transprt t changes in mycardial cntractility remains unsettled (Akera and Brdy, 1978; Akera et al., 1970; Besch et al, 1970; Chen et al., 1976; Rhee et al., 1976; Schwartz et al., 1975; Hugen and Smith, 1978). Althugh substantial circumstantial evidence suggesting a causal relatinship exists, direct prf f the validity f the hypthesis has nt been frthcming (fr reviews, see Schwartz et al., 1975, and Akera and Brdy, 1978). Whereas inhibitin f mycardial mnvalent catin transprt by subtxic but psitively intrpic dses f uabain in intact dgs has been demnstrated recently (Hugen and Smith, 1978), the quantitative relatinship between induced arrhythmias (txic r fatal) and mycardial mnvalent catin transprt has nt been established in an intact animal mdel. In additin, it is nt knwn whether r t what extent transprt functin is restred when txic manifestatins are reversed by digxin-specific antibdy treatment. Cardiac glycside-specific antibdy preparatins are able t reverse many f the actins f cardiac glycsides. Previus studies have shwn reversal f digitalis-induced bichemical, electrphysilgical, and hemdynamic alteratins (see review by Smith et al., 1977). Hwever, the effect f digxin-specific antibdy n mycardial mnvalent catin transprt has nt been studied. In particular, its effect has nt been investigated in an intact animal mdel f digitalis txicity. T examine mycardial mnvalent catin active transprt during digxin-induced intrpy and txicity, we studied the active uptake f llie K + analgue Rb + in serial left ventricular bipsies frm dgs given intrpic and txic dses f digxin. Additinally, t test the hypthesis that digxininduced arrhythmias wuld be assciated with a certain critical level f inhibitin f mycardial mnvalent catin transprt, and that this inhibitin wuld be reversed (tgether with the rhythm disturbance) by digxin-specific antibdies r their Fab fragments, we studied the interrelatinships amng mycardial active transprt f Rb + and the develpment and subsequent reversal by specific antibdy f digxin-induced cardiac arrhythmias. The present studies, therefre, had three bjectives: t determine whether mycardial transprt inhibitin culd be dcumented at subtxic digxin dses in the intact dg; t quantitate mycardial mnvalent catin transprt inhibitin sn after the nset f intrpy, at the nset f vert txicity, and at the nset f lethal arrhythmia; and t determine whether and t what extent mnvalent catin transprt inhibitin is reversed when electrphysilgical txicity is reversed by digxin-specific IgG r Fab fragments. Methds Animal Instrumentatin Twelve healthy adult mngrel dgs (frm 13.6 t 33 kg, mean 21.8 kg) f either sex were anesthetized with intravenus pentbarbital (30 mg/kg), intubated, and ventilated with 100% xygen using a Harvard respiratr. The chest f each dg was pened by a midline sterntmy. Instrumentatin was intrduced as described previusly (Hugen and Smith, 1978) t measure left ventricular (LV) pressure and maximum rate f change in LV pressure (dp/dt), t maintain cnstant heart rate by atrial pacing, t recrd intra-arterial pressure, and t mnitr the electrcardigram. A femral artery cannula was placed t btain bld samples fr measurement f ph, P02, PCO2, hematcrit, ptassium cncentratin, and plasma digxin cncentratin as indicated in experimental prtcls. Anther 33 healthy adult mngrel dgs f either sex (frm 11.4 t 24.1 kg, mean 19.5 kg) were anesthetized with intravenus pentbarbital (30 mg/kg), intubated with a cuffed endtracheal tube, and ventilated with 50% xygen using a Harvard respiratr, with rate and tidal vlume adjusted t maintain nrmal levels f ph, P 2) and Pc 2. Standard limb leads were attached and the electrcardigram cntinuusly mnitred. One plyethylene catheter was inserted int a frelimb vein fr administratin f drugs and anther placed in a femral vein fr bld sampling. Mycardial Bipsy Technique After intrductin f the apprpriate instrumentatin, and when the dgs were hemdynamically stable (usually within 30 minutes), transmural left ventricular bipsies were btained using a 4-mm inside diameter crkbrer attacked t a handheld pwer drill as previusly described (Hugen and Smith, 1978). In ther experiments, the bipsies were btained at designated times by rapidly perfrming a left lateral thractmy, pening the pericardium, and drilling several bipsies frm the left ventricular free wall f the beating heart. The elapsed time frm skin incisin t bipsy did nt exceed 20 secnds. Bipsy tissue was rinsed briefly in incubatin medium and then sliced lengthwise int strips less than 1 mm 2 in crss-sectinal area weighing between 3 and 15 mg each. Rubidium Transprt Measurements The techniques used t measure mycardial uptake f Rb + using M Rb + as tracer have been described in detail recently (Hugen and Smith, 1978). Briefly, the samples f mycardium were incubated fr 5 minutes at 30 C in medium cntaining, in mm cncentratins: KC1, 4.0; NaCl, 120; NaHCO 3, 24; MgCl 2, 2.0; CaCl 2, 2.5; glucse, 5.6; Na phsphate buffer, 1.1, adjusted t ph 7.4, and equilibrated with 95% O 2, 5% CO 2. After this equilibratin perid, samples were transferred t flasks cntaining the same medium except that the KC1 cncentratin was 2.0 mm, and ^RbCl (New England Nuclear) was added tgether with unlabeled RbCl t a final cncentratin f 0.1 mm. Samples were incubated fr 15 minutes at 30 C in this uptake medium in the presence r absence f 1 mm uabain. Samples were then rinsed, cunted, and weighed as previ-

3 EFFECTS OF DIGOXIN AND ANTIBODY ON CATION TRANSPORT///u#en et al. 25 usly described (Hugen and Smith, 1978). Active transprt f Rb + in nml/mg wet weight/15 min was calculated as the difference between uptake in bth the presence and absence f 1 HIM uabain. T validate and characterize further the previusly reprted methd f Rb + uptake measurement, we cmpared the relative uptake f K + t Rb + in the same mycardial tissue sample using "^Rb* and 42 K + as tracers. After a 15-minute equilibratin perid in physilgic medium, samples were transferred t medium cntaining 42 K + (specific activity 10 6 dpm//xml K +, r 2 X 10 6 dpm/ml f medium) tgether with the usual K + cncentratin f 2.0 mm used in all media. ^Rb* was present at a specific activity f 10 7 dpm//ltml (10 6 dpm/ml incubatin medium), tgether with 0.1 mm unlabeled Rb +. Measurements were made bth in the presence and in the absence f 1 mm uabain t allw estimatin f passive as well as active uptakes. Samples were remved after 15 minutes, rinsed, cunted t determine ttal 42 K + plus ^Rb" 1 " uptake, and weighed. T determine ^Rb cunts in the absence f any appreciable cntributin frm 42 K (radiactive tj, 12.4 hurs), mycardial samples were then returned t their cunting vials, stred at 4 C fr at least 7 days, and then recunted. Uptake values fr ^Rb" 1 " and 42 K + were then calculated after crrectin fr radiactive decay. The respnse f the bipsy preparatin t graded in vitr digxin cncentratin was determined t assess the sensitivity and precisin f the methd. Three healthy dgs were anesthetized with pentbarbital, intubated, and ventilated as described abve. Thirty minutes later the chest was rapidly pened and multiple LV bipsies were quickly btained frm the beating heart. Samples prepared as described abve were incubated fr 15 minutes at 30 C in physilgic medium as previusly described (Hugen and Smith, 1978) in the presence f digxin in cncentratins ranging frm zer t 10~ s M. They were then transferred t flasks cntaining the identical medium and digxin cncentratin except that ^Rb" 1 " was present fr an additinal 15-minute incubatin perid. Paired slices were identically incubated in the presence f 1 mm uabain. Passive uptake was defined as that uptake measured in the presence f 1 mm uabain; active transprt was defined as the difference between Rb + taken up in the presence and absence f 1 mm uabain. Plasma and Mycardial Digxin Determinatins Five-milliliter samples f heparinized arterial bld were withdrawn prir t and 5, 10, 15, and 30 minutes after infusin f digxin (Lanxin, Burrughs Wellcme) t which had been added suitable amunts f randmly labeled [ 3 H]digxin (New England Nuclear) fr measurement f plasma digxin cncentratins. Bld samples were als taken at the time f bipsies btained sn after the develpment f intrpy and at the nset f vert txicity. Plasma and mycardial digxin cntents were measured by quantitatin f tritium cunts as described elsewhere (Hugen and Smith, 1978). Quenching crrectin was by the use f internal standards fr bth plasma and mycardial samples. Samples were cunted until stable cunt rates were btained, using a Packard mdel 3300 scintillatin cunter with apprpriate windw settings and crrectin fr m Kh + spill int the 3 H channel. Antibdy Prductin and Characterizatin Sheep were immunized with digxin-serum albumin cnjugates as previusly described (Smith et al., 1970; Curd et al., 1971). Bld was cllected by venipuncture, and plasma separated by centrifugatin. IgG was prepared frm crude antiserum by ammnium sulfate precipitatin (Curd et al., 1971). Fab fragments were prepared by papain digestin as reprted elsewhere (Curd et al., 1971; Nisnff, 1964). Fab and IgG preparatins were passed thrugh a sterile 0.22-/um Millipre filter (Millipre Crp.) and stred at 20 C. Cncentratins f specific antibdy were determined by [ 3 H]digxin binding studies as previusly described (Smith et al., 1970). Preparatins used in the present study had average intrinsic affinity cnstants f 5 X 10 s M" 1 t 1.5 X M" 1. Nnspecific IgG and Fab were identically prepared frm sheep nt specifically immunized. Ttal prtein cncentratins f antibdy preparatins were determined by the methd f Lwry et al. (1951). Prtcls Measurement f Mnvalent Catin Transprt after Infusin f Intrpic and Txic Dses f Digxin After a cntrl bipsy was btained and hemdynamic cnditins were stable, [ 3 H]digxin in vehicle (12% ethanl in saline) was administered as an intravenus injectin f 0.05 mg/kg t six penchest dgs. After the develpment f a sustained psitive intrpic respnse, a secnd LV bipsy was btained. Sequential dses f digxin (0.05 mg/kg) were then given intravenusly at 30-minute intervals until the emergence f vert digitalis txicity, defined as the electrcardigraphic appearance f atriventricular junctinal tachycardia r ventricular tachycardia. A final LV bipsy was btained at the nset f txicity. Cntrl dgs were given similar dses f vehicle alne and were bipsied at cmparable times. Measurement f Mnvalent Catin Transprt at the Onset f Txic and Lethal Arrhythmias and after Antibdy Reversal f Arrhythmias Digxin, 0.3 mg/kg, r an equivalent vlume f vehicle (40% prpylene glycl, 10% ethanl in saline), was administered intravenusly ver 1 minute t 33 pentbarbital-anesthetized clsed-chest dgs. Sinus rhythm was present in all animals prir t digxin administratin. The dgs were divided int five grups, as fllws: A. Cntrl Grup (n = 9). Vehicle alne was

4 26 CIRCULATION RESEARCH VOL. 44, N. 1, JANUARY 1979 given t these dgs and mycardial bipsies were btained at intervals frm 15 t 100 minutes later (average, 53 minutes). A wide range f bipsy times was selected t determine whether the interval between vehicle infusin and bipsy affected Rb + transprt. B. Txicity Onset Grup (n = 5). These dgs received digxin, 0.3 mg/kg, and cardiac rhythm was mnitred until develpment f txicity (defined abve). The chest was then rapidly pened and left ventricular bipsies btained. C. Lethal Txicity Grup (n = 5). The dgs in this grup were given digxin, 0.3 mg/kg. At the nset f txicity, nnspecific antibdy was infused. The txic arrhythmia prgressed t a fatal arrhythmia in all instances (ventricular fibrillatin in fur dgs and asystle in ne), at which time the left ventricle was immediately bipsied. D. Antibdy Reversal Grup (n = 5). After administratin f digxin, 0.3 mg/kg, ventricular tachycardia ccurred in all dgs, at which pint digxin-specific antibdy (IgG) was administered intravenusly ver 3 minutes in a dse equimlar with that f administered digxin. A further antibdy infusin equal t ne-third f the amunt f antibdy initially given was then infused ver 30 minutes with a cnstant infusin pump. The electrcardigram was cntinuusly mnitred. After restratin f 30 secnds f cntinuus sinus rhythm withut ectpic beats, the chest was quickly pened and the left ventricle bipsied. E. Twenty-fur Hur Pstreversal Grup (n = 9). These dgs were treated identically t thse in the antibdy reversal grup (D) except that five received digxin-specific IgG and fur were given an equivalent number f binding sites as digxinspecific Fab fragments at the nset f txicity. After the return f nrmal sinus rhythm, the dgs were allwed t recver frm anesthesia. Twenty-fur hurs later, they were again anesthetized with intravenus pentbarbital (30 mg/kg), intubated, and ventilated as described abve. An electrcardigram was recrded. The chest was then pened and the left ventricle was bipsied. Electrically Induced Ventricular Fibrillatin T assess the pssible effect f ventricular fibrillatin n mycardial mnvalent catin transprt, ventricular fibrillatin was induced by electrical stimulatin in three f the nine dgs in the cntrl grup. The right external jugular vein was cannulated, and a 6F biplar catheter (Elecath 6F, Electr-catheter Crp.) was passed int the right ventricle. Sustained ventricular fibrillatin was induced by delivering a brief (3-secnd) train f 60 Hz alternating current impulses f 1 vlt RMS emplying a standard labratry fibrillatr (Mansfield, 1962). After an interval f ventricular fibrillatin f abut 15 secnds, equal t that in grup C (lethal txicity grup) dgs, the chest was rapidly pened and Rb + active transprt measured in left ventricular bipsy tissue as described abve. Data Analysis Data frm the pen-chest dgs were analyzed by paired -test, each dg serving as its wn cntrl. Data frm the clsed-chest grups were analyzed by unpaired t-test. Differences in values were cnsidered significant when P values were less than Results Validatin f Rb + Uptake Studies Cmparisn f the simultaneus uptake f K + and Rb + by mycardial slices in the presence and absence f uabain is summarized in Table 1. The rati f K + t Rb + uptake was 19.5 ± 0.4 (SE) in the absence f uabain, essentially identical t the 20:1 rati f K + t Rb + present in the medium. This rati did nt vary with the 10~ 3 M uabain, a secnd set f slices yielding a mean rati f 20.3 ± 0.4 in its presence. Thus, bth active and passive uptake f Rb + are interchangeable with K + under the cnditins f these experiments. The in vitr cncentratin-effect curve fr digxin inhibitin f Rb + transprt in mycardial samples is shwn in Figure 1. Cncentratins f digxin greater than 10" M culd nt be disslved reliably, and uabain was substituted fr the 10" 3 M cncentratin pint. As shwn, digxin at 10~ 8 M caused a 21 ± 8% (P < 0.05) reductin in Rb + active uptake cmpared t active uptake in the presence f vehicle alne. Half-maximal inhibitin ccurred at 8 X 10" 7 M digxin. Previus wrk frm this labratry has shwn that active uptake f Rb + by these mycardial samples is linear fr at least 30 minutes, under the incubatin cnditins used in the present set f experiments (Hugen and Smith, 1978). Plasma and Mycardial [ 3 H]Digxin Cncentratins Plasma and mycardial digxin cncentratins are shwn in Figure 2 fr the pen-chest dgs receiving an initial psitive intrpic but subtxic dse, fllwed by additinal dses f digxin t nset f vert txicity. The mean plasma digxin TABLE 1 Cmparisn f Simultaneus Rb + and K* Uptake by Canine Mycardial Bipsy Slices nml Rb + /mg/15 nml KVmg/15 K*.Rb*upmin' min* take rati Ouabain absent ± ± ± 0.4 (i = 9) d = 9) Ouabain pres ± ± ± 0.4 ent (n=8) d-8) (KT 1 M) ' Values expressed as mean ± SE f uptake in nml/mg wet weight/15

5 EFFECTS OF DIGOXIN AND ANTIBODY ON CATION TRANSPORT/tfu^erc et al. 27 cr O I i B0 60 e I 00 INOTROPIC TOXIC ',i:00 I.OOC z h- m x z 3? ce UJ < rr I I 0 10"* 10'* ICT T 10'* 10"' I0" 4 IO" S DIGOXIN CONCENTRATION (M) FIGURE 1 In vitr digxin cncentratin-effect curve fr inhibitin f Rb+ active transprt. Left ventricular bipsy tissue btained frm threepentbarbital-anesthetized dgs was preincubated fr 15 minutes at 30 C in the presence f the digxin cncentratins shwn (hrizntal axis). Ouabain was substituted fr the 10 ~:i M cncentratin pint. The tissues were incubated an additinal 15 minutes in the presence f 8 6Rb +. Cmplete inhibitin f active transprt was defined as that bserved in the presence f 10~ :> M uabain. Statistically significant (P < 0.01) transprt inhibitin was bserved at cncentratins f 10~ 8 M digxin r greater. Half maximal inhibitin ccurred at 8 X W~' M. Numbers in parentheses indicate numbers f individual tissue samples assayed; brackets indicate ne SK abve and belw the mean. cncentratin at the time f LV bipsy during intrpy was 27 ± 3 ng/ml (3.5 x 10" 8 M) and at the nset f arrhythmia 113 ± 6 ng/ml (1.5 x 10" 7 M), an increase f 4-fld. The mean left ventricular cncentratin f digxin in the specimen btained sn after the nset f intrpy was 272 ± 33 ng/g wet weight. At txicity, the mycardial cntent increased t 873 ± 79 ng/g wet weight. The larger rati f mycardial t plasma digxin cntent at txicity wuld be expected n the basis f the later sampling time. Effects f Intrpic and Txic Dses f Digxin As summarized in Table 2, there was n significant difference in mean bdy weight, hematcrit, serum ptassium cncentratin, arterial bld P 2, PCO 2, r ph between cntrl dgs and thse given PLASMA 20- -II- MYOCARDIUM 200 FIGURE 2 Plasma (vertical axis t left) and mycardial (vertical axis t right) digxin cntent after intrpic (0.05 mg/kg) and txic (mean 0.17 mg/kg) dses ffhj digxin iv. Plasma digxin cncentratins at nset f sustained intrpy (pen bar) and txicity (crsshatched bar) represent the mean ± SE fr six dgs. Mycardial digxin cntent (ng/g wet weight) in crrespnding bipsy samples is als shwn. TABLE 2 Initial Measurements frm Cntrl and Digxin-Treated Dgs digxin. Mean arterial pressure and heart rate did nt vary significantly frm the initial values at any pint during the curse f the experiments prir t nset f digxin-induced arrhythmias. The initial digxin dse f 0.05 mg/kg was selected t prduce a demnstrable psitive intrpic effect withut txicity. Fllwing this dse, n elec- Weight (kg) Hematcrit (%) Serum ptassium (meq/liter) Arterial ph Arterial P 2 (mm Hg) Arterial Pc 2 (mmmhg) Each value is the mean ± SE. Cntrl grup (n = 6) 19.5 ± ± Digxintreated grup in = ± ± ± ± ± ± 3 cr

6 28 CIRCULATION RESEARCH VOL. 44, N. 1, JANUARY 1979 trcardigraphic evidence f digxin txicity appeared, cnsistent with ur wn previus experience (Beller and Smith, 1975; Beller et al., 1975) and that f thers (Gldman et al., 1973). At the time f bipsy, shrtly after the appearance f sustained psitive intrpy, a mean f 26 ± 3 minutes after digxin administratin, maximal LV dp/dt had increased 20 ± 3% abve baseline values (P < 0.001, paired -test) as shwn in Figure 3. Additinal digxin dses (t an average ttal f 0.17 mg/kg, iv) were given t the nset f arrhythmia, manifest as ventricular tachycardia in five dgs and atriventricular dissciatin in ne. Immediately prir t the nset f arrhythmia, maximal LV dp/dt increased 53 ± 7% abve baseline values (P < 0.001, paired t-test), an average f 88 ± 8 minutes after the initial dse f digxin was infused. In cntrast, cntrl dgs given vehicle alne shwed unchanged cntractility at cmparable times (Fig. 3). Effects f Digxin n Mycardial Active Transprt f Rb + at Onset f Intrpy and Txicity Active transprt f Rb + int mycardial samples btained sn after the nset f digxin-induced intrpy was significantly reduced by 25 ± 5% (P < 0.01) belw cntrl values (Fig. 4). At the nset f digxin-induced rhythm disturbances, mycardial active uptake f Rb + was markedly reduced by 60 ± 4% (P < 0.001) belw baseline values. In cntrast, dgs receiving vehicle (12% ethanl in saline) shwed unchanged Rb% active transprt cmpared t baseline values when measured at cmparable times (Fig. 4). These data demnstrate significant inhibitin f mnvalent catin active transprt by psitively intrpic but nntxic dses f digxin. Onset f arrhythmia was assciated with markedly reduced active transprt t 60% belw cntrl levels. Time Curse f Digxin Txicity and Reversal with Antibdy There were n significant differences in mean weight, serum ptassium, hematcrit, r initial arterial P 2, Pc 2, and ph amng the five grups f dgs used in these experiments. Figure 5 summarizes the time curse f the develpment f txic arrhythmias after digxin administratin in these five grups f dgs. The time f nset f txicity was similar amng the varius digxin-treated grups, with each develping vert txicity at mean times f minutes after digxin infusin. Ventricular tachycardia was the arrhythmia in all grup B dgs. The digxin-induced rhythm disturbance prgressed t ventricular fibrillatin in fur dgs and ventricular asystle in ne dg in the lethal txicity grup'(c), a mean f 54 ± 15 minutes after digxin treatment. Overt txicity develped in the dgs subsequently underging antibdy reversal (grup D), an average f 17 ± 4 minutes after digxin administratin. Ventricular tachycardia de- M X E E 0. > 3,000 4,000 3,000 2,000 1, ,000 3,000 4,000 3,000 2,000 1,000 CONTROL. GROUP DIGOXIN-TREATED GROUP I BASELINE INOTROPY TOXICITY FIGURE 3 Effects fsubtxic and arrhythmgenic dses f digxin n left ventricular cntractility. Six dgs were given digxin, 0.05 mg/kg, iv. After the develpment f sustained intrpy (26 ± 3 minutes later), additinal dses f digxin (average ttal f 0.17 mg/kg) were infused until txicity develped. A cntrl grup (n = 6) with similar instrumentatin was infused with cmparable amunts f vehicle. With each dg as its wn cntrl, dp/dt increased 20 ± 3% (P < 0.001) at intrpy and 53 ± 7% (P < 0.001) immediately prir t txicity. Dgs given vehicle shwed unchanged cntractility at cmparable times. Shrt hrizntal bars represent the mean value frm six dgs. velped in all dgs in this grup, at which pint digxin-specific antibdy was infused. Sinus rhythm returned in all instances, a mean interval f 74 ± 9 minutes after antibdy administratin. Txic arrhythmias (ventricular tachycardia in all cases) develped in grup E (24-hur pstreversal grup) 14 ± 2 minutes after digxin administratin. Antibdy in the frm f digxin-specific IgG (n = 5) r Fab fragments (n = 4) was then infused. Sinus rhythm returned in all dgs, a mean f 69 ± 15 minutes after antibdy infusin. All remained healthy and in sinus rhythm fr the ensuing 24 hurs. As shwn in Figure 5, the time curse f antibdy reversal f txicity in grups D and E was similar. Mnvalent Catin Active Transprt at Onset and Reversal f Digxin-induced Arrhythmias Active transprt f the K + analgue Rb + in mycardial bipsy samples frm the five grups f dgs is summarized in Figure 6. Active Rb + uptake in

7 EFFECTS OF DIGOXIN AND ANTIBODY ON CATION TRANSPORT/Hu^erc et al. 29 O.I8O CONTROL GROUP I RHYTHM I SINUS TOXIC I FATAL T Bx OIGOXIN-TREATED GROUP (5) r Q. z u BASELINE INOTROPY TOXICITY FIGURE 4 Effect f digxin n mycardial active transprt f Rb +. Left ventricular bipsies after psitive intrpic dses (0.05 mg/kg) and at txicity (0.17 mg/kg, iv) were incubated at 30 C and Rb + active uptake was determined as described in the text. Cntrl dgs given vehicle alne were bipsied at cmparable times. Shrt hrizntal bars represent mean values. bipsies btained frm cntrl (grup A) dgs was ± nml Rb + /mg wet weight tissue/15 min. Rb + transprt was nt influenced by the interval between cntrl vehicle infusin and the time f bipsy. Three electrically fibrillated dgs had Rb + transprt values similar t ther animals in the cntrl grup. Thus, ventricular fibrillatin per se did nt alter Rb + active transprt measurements. Onset f digxin-txic arrhythmias (grup B) was assciated with a marked reductin in active transprt t ± nml Rb + /mg wet wt/15 min, 59 ± 4% belw the cntrl value (P < 0.001). Left ventricular bipsies btained at the time f fatal arrhythmia (grup C) indicated a further decrease f active transprt t ± nml Rb + /mg wet wt/15 min. This represented an 80 ± 5% decrease f active transprt cmpared t the cntrl value (P < 0.001). Thse dgs given digxin-specific antibdy and bipsied immediately after restratin f sinus rhythm (grup D) had partial recvery f active transprt t a level that was significantly greater than that bserved at the nset f vert txicity (0.060 ± cmpared t ± 0.004, P < 0.05), but still substantially belw cntrl active transprt values. CONTROL TOXICITY LETHAL ANTBOOY 24 H ONSET TOXICITY REVERSAL AFTER REVERSAL A C D E OftOUT FIGURE 5 Time curse f digxin-induced txic and lethal arrhythmias, and f reversal f txicity with digxin-specific antibdy. Cntrl dgs (grup A) given vehicle remained in sinus rhythm while grups B, C, D, and E received digxin, 0.3 mg/kg intravenusly, and develped txic and lethal rhythm disturbances at the times indicated. After the nset f arrhythmia, grups D and E received digxin-specific antibdy intravenusly at the nset f txicity (shwn by arrws). Grup E was allwed t recver and was bipsied the fllwing day, indicated by *. Vertical lines indicate ± 1 SE; numbers f dgs are indicated in parentheses. Bx, bipsy time. Measurement f Rb + active transprt in nine 24- hur survivrs in grup E (five IgG, fur Fab) demnstrated further recvery f pump activity t ± nml Rb + /mg wet wt/15 min, nt significantly different frm cntrl (Fig. 6). N difference was evident in mean Rb + transprt between IgG- and Fab-treated animals (IgG, cmpared with Fab, nml Rb + /mg wet wt/15 min) if J 0M - (S) <*) (S) <*) CONTROL TOXIdTV ONSET LETHAL ANTIBODY M H TOXICITY REVERSAL AFTER REVERSAL A B C D E OROUP FIGURE 6 Digxin-induced inhibitin f Rb + active transprt and reversal f inhibitin by digxin-specific antibdy. Cntrl dgs received vehicle; ther grups were given digxin 0.3 mg/kg. Tw grups (crsshatched) were treated with digxin-specific antibdy at nset f txicity. Rb + active transprt was measured in vitr in mycardial bipsy samples as described in the text. Vertical lines represent ± 1 SE; numbers f dgs are indicated in parentheses.

8 30 CIRCULATION RESEARCH VOL. 44, N. 1, JANUARY 1979 These data demnstrate that, in intact dgs given a single lethal dse f digxin, digxin-specific antibdy reversed advanced txic arrhythmias, and sinus rhythm was reestablished in all instances. Mnvalent catin active transprt returned tward nrmal at the time f reversal f txicity and was nt significantly different frm nrmal cntrl values 24 hurs after reversal. Discussin These experiments demnstrate that significant inhibitin f active transprt f the K + analgue Rb + is present at the time psitive intrpy prduced by a nntxic dse f digxin is first clearly evident in intact dgs. The impact f animal-tanimal bilgical variatin n data analysis was minimized by using each dg as its wn cntrl. We als regard as imprtant the assessment f transprt functin in intact cells by a methd sensitive t lw (1CT 8 M) cncentratins f digxin similar in magnitude t thse present in plasma f treated patients. Althugh these data d nt prve a causal relatinship between digxin-induced transprt inhibitin and psitive intrpy, they d demnstrate that a necessary (but nt sufficient) cnditin is met with regard t the Na +,K + -ATPase inhibitin hypthesis f cardiac glycside intrpic actin. This relatinship has been discussed in detail recently by a number f authrs (Akera and Brdy, 1978; Langer, 1977; Ettinger, 1977; Schwartz et al., 1975; Hugen and Smith, 1978) and will nt be cnsidered further here. Our results further indicate that the prgressin frm psitive intrpy alne t arrhythmic manifestatins f digxin txicity t fatality is assciated with marked and prgressive decreases in mycardial mnvalent catin active transprt (Figs. 5 and 6). These data are cnsistent with the recent findings f Ettinger and assciates (1977), wh detected significant decreases in mycardial K + and increases in Na + cntent in areas f dg hearts frm which acetylstrphanthidin-induced arrhythmias appeared t riginate. Althugh the effects f txic dses f digxin n mycardial mnvalent catin active transprt in intact dgs have nt been investigated previusly, the relatinship between the arrhythmic manifestatin f digitalis txicity and Na +,K + -ATPase inhibitin has been explred. Besch et al. (1970) fund 59% inhibitin f enzyme activity at the time f nset f ventricular ectpic activity in respnse t uabain infusin, while Akera et al. (1970) fund 47% inhibitin f Na +,K + -ATPase at a similar endpint. Dutta et al. (1970) demnstrated 54% and 75% inhibitin f mycardial Na +,K + -ATPase in dgs given uabain in arrhythmgenic and lethal dses, respectively. Zavecz and Dutta (1977) studied the effect f a cnstant infusin f actdigin, a shrt-acting semisynthetic glycside, n canine mycardial Na +,K + -ATPase at the nset f txicity and after return f sinus rhythm fllwing cessatin f drug infusin. These wrkers shwed that at the nset f arrhythmia, Na +,K + -ATPase was reduced by 55% but returned t cntrl values at the time f recvery t nrmal sinus rhythm. Akera et al. (1977) fund canine mycardial Na +,K + -ATPase t be reduced 73% belw cntrl levels by chrnic administratin f txic dses f digxin. Thus, enzyme inhibitin at txicity and lethal arrhythmia bserved in these prir studies are similar t the degree f mnvalent catin active transprt inhibitin reprted here. We fund that, at the nset f txicity, mnvalent catin active transprt was 59% belw cntrl in ne grup and 60% in anther; at the appearance f fatal arrhythmia, 80% inhibitin f transprt was present. The transprt activities measured in the present study represent pump activity in mycardium rather than Purkinje netwrk r ther parts f the specialized cardiac cnductin system. Althugh ne must be cautius in inferring that parallel declines in pump activity are present in mycardial cells and specialized cnductin tissue, the similarity f mnvalent catin active transprt at nset f arrhythmia and at the time f arrhythmia reversal suggests that there may be a critical level f pump activity necessary t maintain electrphysilgical stability. It is f interest that variatin in time t reach txicity did nt appear t alter the degree f catin transprt inhibitin measured at the nset f txicity, whether assessed in pen- r clsed-chest dgs. In dgs first receiving an intrpic dse fllwed by txic dses f digxin (ttal, 0.17 mg/kg), txicity develped 88 ± 8 minutes after the initial digxin was infused. In dgs given a single, rapidly fatal 0.3 mg/kg dse f digxin, ventricular tachycardia appeared after 19 ± 3 minutes. Hwever, at txicity, Rb + active transprt was reduced by 60 ± 4% belw cntrl in the first grup and 59 ± 4% in the latter grup f dgs. Als f interest in these experiments was the fact that the prgressive electrcardigraphic changes f digxin txicity fllwed a reversed pattern after administratin f digxin-specific antibdy. In mst instances, the large dse f digxin given in these studies prduced premature atriventricular junctinal and/r ventricular beats at an increasing rate until a sustained rapid junctinal r ventricular tachycardia emerged. The rhythm sequence cming ut f txicity generally retraced the sequence f arrhythmias during develpment f txicity. In parallel with this symmetry f rhythm disturbances was the degree f mnvalent catin inhibitin at nset (59-60%) and at reversal (49%) f rhythm disturbances. The delay between antibdy infusin and reversal f arrhythmia presumably is due t factrs including the time necessary fr antibdy t bind free digxin in the plasma and interstitial space, t shift the equilibrium in favr f dissciatin f digxin frm cardiac receptr sites, and t allw crrectin f cellular electrphysilgical abnrmalities.

9 EFFECTS OF DIGOXIN AND ANTIBODY ON CATION TRANSPORT/Hugen et al. 31 We cnclude that these results are cnsistent with (but d nt prve) the hypthesis that binding t and inhibitin f mycardial Na +,K + -ATPase are related t bth intrpic and txic effects f digxin. Digxin-induced arrhythmias develp in assciatin with reductin in mycardial mnvalent catin active transprt t abut 40% f levels bserved in nrmal cntrl mycardium, and lethal arrhythmias are assciated with reductin t 20% f nrmal. The reversal f advanced digxin txicity by digxin-specific antibdies r Fab fragments is assciated with the restratin f mycardial mnvalent catin transprt functin. Acknwledgments We wish t thank Thmas Manning and Edward Wdfrd fr their technical assistance, and Lucille Kelly fr typing the manuscript. References Akera T, Brdy TM: The rle f Na +,K + -ATPase in the intrpic actin f digitalis. Pharmacl Rev 29: , 1978 Akera T, Ku DD, Brdy TM: Lack f effect n brain stem and cerebral crtex Na +,K + -ATPase during heart blck prduced by chrnic digxin treatment. Eur J Pharmacl 45: , 1977 Akera T, Larsen FS, Brdy TM: Crrelatin f cardiac sdiumand ptassium-activated adensine triphsphatase activity with uabain-induced intrpic stimulatin. J Pharmacl Exp Ther 173: , 1970 Beller GA, Giamber SR, Saltz SB, Smith TW: Cardiac and respiratry effects f digitalis during chrnic hypxia in intact cnscius dgs. Am J Physil 229: , 1975 Beller GA, Smith TW: Digitalis txicity during acute hypxia in intact, cnscius dgs. J Pharmacl Exp Ther 193: , 1975 Besch HR Jr, Allen JC, Glick G, Schwartz A: Crrelatin between the intrpic actin f uabain and its effects n subcellular enzyme systems frm canine mycardium. J Pharmacl Exp Ther 171: 1-12, 1970 Chen I, Daut J, Nble D: An analysis f the actins f lw cncentratins f uabain n membrane currents in Purkinje fibers. J Physil (Lnd) 260: , 1976 Curd J, Smith TW, Jatn J-C, Haber E: The islatin f digxinspecific antibdy and its use in reversing the effects f digxin. Prc Natl Acad Sci USA 68: , 1971 Dutta S, Rhee HM, Marks BH: H 3 -uabain accumulatin and Na + -K + ATPase activities in relatin t therapeutic, txic and lethal dses f uabain in dgs. In Recent Advances n Cardiac Structure and Metablism (Mycardial Bilgy), vl 4, edited by NS Dhalla. Baltimre, University Park Press, 1974, pp Ettinger PO, Calabr J, Regan TJ, Oldewurtel HA: Origin f acetyl strphanthidin-induced ventricular arrhythmias. J Clin Invest 59: , 1977 Gldman RH, Cltart DJ, Friedman JP, Nla GT, Berke DK, Schweizer E, Harrisn DC: The intrpic effects f digxin in hyperkalemia. Relatin t (Na +,K + )-ATPase inhibitin in the intact animal. Circulatin 48: , 1973 Grupp G, Charles A: Effect f uabain and 3-acetyl strphanthidin n ptassium exchange in the dg heart in situ. J Pharmacl Exp Ther 143: , 1964 Hugen TJ, Smith TW: Inhibitin f mycardial mnvalent catin active transprt by subtxic dses f uabain in the dg. Circ Res 42: , 1978 Ku DD, Akera T, Brdy TM, Weaver LC: Chrnic digxin treatment n canine mycardial Na +,K + -ATPase. Naunyn Schmiedebergs Arch Pharmacl 301: 39-47, 1977 Langer GA: Relatinship between mycardial cntractility and the effects f digitalis n inic exchange. Fed Prc 36: , 1977 Lwry VH, Rsebrugh NS, Farr AL, Randell RJ: Prtein measurements with the Flin phenl reagent. J Bil Chem 193: , 1951 Mansfield PB: An apparatus fr elective fibrillatry cardiac arrest in experimental and clinical cardipulmnary bypass peratins. J Thrac Cardivasc Surg 43: , 1962 Miiller P: Ouabain effects n cardiac cntractin, actin ptential and cellular ptassium. Circ Res 17: 46-56, 1965 Nisnff A: Enzymatic digestin f rabbit gamma glbulin and antibdy and chrmatgraphy f digestin prducts. Methds Med Res 10: , 1964 Repke K: Influence f cardiactive principles n pump ATPase. In Prceedings f the First Internatinal Pharmaclgical Meeting, Stckhlm, vl III. New Aspects f Cardiac Glycsides, edited by W Wilbrandt, P Lindgren. Oxfrd, Pergamn Press, 1963, pp Rhee HM, Dutta S, Marks BH: Cardiac NaK-ATPase activity during psitive intrpic and txic actins f uabain. Eur J Pharmacl 37: , 1976 Schatzmann HJ: Herzglykside als Hemmstffe fiir den aktiven Kalium und Natriumtransprt durch die Erythrcytenmembran. Helv Physil Pharmacl Acta 11: , 1953 Schwartz A, Lindenmayer GE, Allen JC: The sdium-ptassium adensine triphsphatase: Pharmaclgical, physilgical and bichemical aspects. Pharmacl Rev 27: 3-134, 1975 Smith TW, Butler VP Jr, Haber E: Characteristics f antibdies f high affinity and specificity fr the digitalis glycside digxin. Bichemistry 9: , 1970 Smith TW, Butler VP Jr, Haber E: Cardiac glycside-specific antibdies in the treatment f digitalis intxicatin. In Antibdies in Human Diagnsis and Therapy, edited by R Krause, E Haber. New Yrk, Raven Press, 1977, pp Zavecz JH, Dutta S: The relatinship between Na +,K + -ATPase inhibitin and cardiac glycside-induced arrhythmia in dgs. Naunyn Schmiedebergs Arch Pharmacl 297: 91-98, 1977