Positive inotropic effect of porcine left ventricular extract on canine ventricular muscle

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1 Br. J. Phrmcol. (1990), 101, zt./\f--i Mcmilln Press Ltd, 1990 Positive inotropic effect of porcine left ventriculr extrct on cnine ventriculr muscle # ts. Nvrtnm, tt. hu, # M. Agnyo, # t*d. Bose & 1 # tj.. Khtter # Deprtment of Internl Medicine, tphrmcology & Therpeutics nd *Anesthesi, University of Mnito, Winnipeg, Mnito, nd, R3E OZ2 1 We previously isolted n extrct from porcine left ventricle tht possessed digitlis-like properties such s inhiition of crdic nd kidney N+, K+-ATPse, displcement of [3H]-ouin from its inding sites nd cross rectivity with digoxin ntiodies. The extrct lso hd positive inotropic effect on the guine-pig hert. 2 In the present study the positive inotropic response of the extrct ws chrcterized in cnine right ventriculr trecule. Mximum inotropic response ( %) ws produced y 300,1 nd the hlf mximl increse occurred with 125,ul of the extrct. 3 Ougenin produced ftercontrctions in rpidly pced trecule. Equipotent nd even greter mounts of the extrct did not produce ftercontrctions. 4 The extrct incresed the mplitude of the delyed component () of iphsic contrctions produced y replcing out 92-96% of the externl with Sr. A smller increse in the size of the erly component (P1) ws lso seen. 5 The extrct decresed post-rest potentition fter rest for 30s nd 2 min. After 8 min of rest, post-rest potentition ws converted to post-rest depression. 6 The extrct (20#u1) produced decrese in the mplitude of the post-rest rpid cooling contrcture (R) t ll rest intervls. The stedy stte R, lthough greter thn tht in the control muscle, ws incresed to lesser extent thn the size of the stedy stte electriclly driven contrctions. 7 It is suggested tht the extrct from porcine left ventricle produces positive inotropic response y incresing the trns-srcolemml influx of. It lso hs dditionl effect(s) on the srcoplsmic reticulum in tht it my fcilitte the loss of from the srcoplsmic reticulum nd/or inhiit the uptke of y the orgnelle. Introduction Isoltion of digitlis-like sustnce (DLS) from iologicl mterils such s plsm, crdic tissue, plcent etc. hs een ttempted y mny lortories (Gruer et l., 1980; Kelly et l., 1985; Blzn et l., 1986, Er et l., 1986). In our lortory, we isolted crude frction from porcine left ventricle, fter extrction of lipids, chromtogrphic seprtion nd deslting (Khtter et l., 1986). The extrct hd digitlis-like properties such s inhiition of crdic nd kidney N', K+- ATPse dose-dependently, displcement of [3H]-ouin from its inding sites nd cross rectivity with ntiodies to digoxin (Khtter et l., 1986; Agnyo & Khtter 1990; Khtter et l., 1990). We lso investigted the effect of this extrct on 86R uptke, which is representtion of functionl N' pump ctivity. The extrct inhiited the uptke of 86R in dose-dependent fshion, indicting tht it inhiits the N pump. Even though mny lortories hve een le to demonstrte the presence of digitlis-like sustnce(s) either immunologiclly or iochemiclly, the functionl chrcteriztion of positive inotropism ws not done in these studies (Gruer et l., 1980; Kelly et l., 1985; Blzn et l., 1986; Er et l., 1986). The crude extrct otined in our lortory from porcine ventricle ws consistently inotropic in guine-pig isolted herts (Khtter et l., 1986). Furthermore, in our previous studies we lso excluded the possiility of ctecholmines eing responsile for the inotropic effect ecuse: () it ws not locked y 8-drenoceptor ntgonist, () the extrct did not compete for [3H]-(-)-dihydrolprenolol inding (Khtter et l., 1986) nd (c) in high performnce liquid chromtogrphy (h.p.l.c.) nlysis it did not 1 Author for correspondence t: Section of rdiology, Deprtment of Medicine, GF 330, Helth Sciences entre, 700 Willim Ave., Winnipeg, Mnito, nd R3E OZ3. hve similr peks s those of drenline, nordrenline or dopmine (Agnyo & Khtter, 1990). Essentilly the extrct otined from porcine left ventricle hd contrctile, immunologicl nd iochemicl properties like those of digitlis. The present study ws undertken to chrcterize further the positive inotropic response of the extrct on cnine ventriculr trecule, in order to investigte its possile mechnism of ction. Methods Isoltion ofdigitlis-like sustnce (DLS)-contining extrctfrom porcine left ventricle The detiled methodology for the isoltion of the crude porcine ventriculr extrct hs een descried previously (Khtter et l., 1986). A homogente of the porcine left ventricle ws otined, lipids were extrcted with methnol nd diethyl ether nd the frction sujected to column chromtogrphy (Sephdex G fine ed) nd deslting. The finl extrct from 150g porcine left ventricle ws neutrlized to ph 7.4 with 1 M Tris nd djusted to totl volume of 5 ml. Effect ofdls contining extrct on cnine right ventriculr trecule Experimentl preprtion Tissue preprtion nd tension mesurements were crried out s descried previously (Hryshko et l., 1989). Briefly, mongrel dogs of either sex (8-12kg) were nesthetized with sodium pentoritone (35mgkg-1, i.v.), herts were rpidly excised nd thin, free. running right ventriculr trecule were dissected from the wll of the hert. Isolted muscle preprtions were trnsferred to 5 ml verticl th contining oxygented (95% 2, 5% 02) Kres Henseleit (KH) solution (in mm: Nl 118,

2 INOTROPI EFFET OF PORINE LEFT VENTRIULAR EXTRAT 371 K1 4.7 l2 2.5, MgSO4 1.2, KH2PO4 1.4, NHO3 25 nd glucose 11) mintined t ph of 7.4 nd t temperture of 370. Muscles were field stimulted y pulses delivered from computer-controlled stimulus sequencer (Boyechko & Bose, 1982) with frequency of 0.5 Hz nd mplitude of % ove threshold. Muscles were stretched to n optiml length for mximl isometric force production nd isometric contrctions were recorded with force trnsducer (Grss FT-03). Periods of rest rnging from s were imposed on n otherwise constnt stimulus pttern. Dose-response of the porcine extrct ws tested y ddition of incresing mounts of the extrct to the tissue th in cumultive mnner. Rpid cooling contrcture (R) The pprtus employed to elicit rpid cooling contrcture (R) hs een descried in detil elsewhere (Hryshko et l., 1989). Briefly, the muscle chmer consists of single th nd two reservoirs contining thing medium t 370 nd -40 connected in prllel to the th. This llowed instntneous (<1 s) chnging of th solutions. Rs were elicited y cooling the preprtion from 370 to 0-20 y chnging the solution. Rs were otined in plce of norml electricl twitch (stedy stte R) or fter vrile periods of rest (post-rest R). Biphsic contrction Biphsic contrction in cnine trecule ws produced s descried y King & Bose (1983). Trecule were equilirted in KH solution y stimulting t 0.5 Hz for 1 hour. This ws followed y replcement of solution with SrKH (identicl composition to KH except tht it contined 2.5 mm Sr2+ insted of l2), which produced series of chnges in the shpe of contrctions. After 10-20min in SrKH, the two components designted s phse 1 (P1) nd phse 2 () hd pproximtely equl tensions. The iphsic contrction ws mintined y the ddition of mM 2 + to the externl medium contining 2.5 mm Sr2. Drugs Ougenin nd sotlol were otined from Sigm hemicl o. nd solutions were mde in distilled deionized wter. All chemicls for physiologicl solutions were regent grde. Sttistics Sttisticl significnce ws determined with Student's t test. The level of significnce chosen for ll experiments ws P < Dt hve een expressed s men + s.e.men. Results Positive inotropic effect of extrct The dose-response reltionship of the positive inotropic effect of the extrct ws studied y cumultive ddition. Figure 1 shows the typicl fst (l nd ) nd slow (ic nd d) recording of tension development in cnine trecule. Addition of 50pl of the extrct produced smll initil negtive inotropic effect followed y mrked positive inotropic response within few seconds (Figure ic). The mximum positive inotropic response ws seen 2-3 min fter ddition of the extrct. The log dose-response plot is shown in Figure 2 which clerly illustrtes sigmoid dose-response reltionship with the mximum increse in developed tension of %. The hlf mximl increse in developed tension (ED5o) ws produced y 125,u1 of the extrct. Under similr experimentl conditions, the dose-response reltionship of ougenin ws lso studied. Mximum inotropic effect ws produced y 10-6M ougenin. Higher concentrtions of ougenin cused ftercontrctions, increse in distolic tension nd decrese in ctive tension. The positive inotropic effect of the extrct is expressed s percentge of the mximum response to ougenin in Figure 2. A volume of 100#l of the extrct ws pproximtely equipotent to 10-6M ougenin nd u1 extrct produced 300% greter inotropy compred to tht produced y mximum concentrtion of ougenin. In ddition to the increse in developed tension, the durtion of contrction ws lso prolonged in the presence of the extrct (Figure l). The prolongtion of the totl contrction durtion ws cused y the prolonged time to pek tension s well s y slowing of relxtion. The prolongtion of contrction durtion ws dose-dependent nd the mximum prolongtion of contrction occurred with similr dose ( ,pl) to tht producing ner mximum increse in developed tension. The durtion of contrction ws unchnged in the presence of ougenin (Figure l). 9L (iii) (ii (ii 100 ms (i) d i g s Figure 1 Effect of extrct from porcine left ventricle on tension of electriclly driven trecule from cnine right ventricle. () Tension development in (i) control nd (ii) with 50pl extrct nd (iii) 350#1 extrct. () Tension development in (i) control nd (ii) ougenin 10-6M; (c) nd (d) show the time course of positive inotropy in response to the extrct (5 pl) nd ougenin (10-6M) respectively t slow recording speed.

3 372 S. NAVARATNAM et l. 0 Uo o 40 ) 0O 0) 01)._ o o 01).) o 0 ^ E._ ' m O > ) 0m *- ms cn 0 m I4- ) ooo - I DLS-contining frction (l) DLS-contininrg frction (RIl) Figure 2 () umultive log dose-effect of the extrct on the contrctile force of cnine trecule. () umultive log-dose resp( extrct s percentge of the mximum positive inotropic r ougenin (10-6M). Results re shown s men of 5 experiments; s.e.men indicted y verticl rs in (). Asence ofdigitlis-like toxicity As demonstrted in Figure 2, the mximum response ws produced y l of the extrct increse in the mount of extrct did not produce lny significnt chnge in either developed tension or restinjg tension. More importntly, there were no signs of toxicity e.g. oscilof stimu- ltory ftercontrctions, prticulrly t fster rtes ltion. Isolted trecule were treted with 3501 of extrct (Figure 3) or 5 x 10-6M ougenin (Figure 3).nd driven t fster rte (sic cycle length of 300 ms). The 1 st driven contrction ws followed y fter-contrction of thie muscles tht were treted with ougenin, which is chrc:teristic of digitlis toxicity. However, the muscles tht were treted with the extrct did not mnifest these ftercontrctions. Biphsic response The iphsic contrctions were produced y replcing 92-96% of clcium in the thing solution with strontium. These conditions produced prolonged contrction with two distinct phsic components, the erly component Pt nd the lte component. Addition of 2O-40ul of extrct in the presence of 10- M sotlol ( fl-drenoceptor ntgonist) produced sustntil increse in the mplitude of the ltter component nd smller increse in PI (Figure 4). The pooled dt from 5-6 experiments re illustrted in Figure 4. It demonstrtes tht the extrct produces significnt increse 500 in the mplitude of PI nd ut the effect on is greter thn tht on P1. Rest potentition Figure 5 shows the contrctile response of cnine isolted right ventriculr trecule to 30s, 2 nd 8 min rest periods. The control muscle (Figure 5) shows post-rest potentition fter ll three rest intervls. Addition of DLS contining extrct produced rest depression fter 8 min rest nd the mplitudes of post-rest contrction fter 30s nd 2 min were smller thn tht of control muscle. Rpid cooling contrcture (R) The typicl response of the isolted trecule to rpid cooling is illustrted in Figure 6. The control muscle shows 500 progressive decrese in the mplitude of the cooling contrcture with incresing durtion of rest prior to cooling (Figure 6). In the presence of extrct (200pl) (Figure 6), there ws onse to the decrese in the mplitude of the post-rest R t ll rest ^esponse to durtions. The stedy stte R fter the ddition of the extrct ws greter thn tht in the control muscle. However, its size when expressed s percentge of the preceding stedy stte contrction, ws decresed compred to control stedy stte R similrly expressed. The pooled dt from 4-5 experiments re depicted in Figure 7. It cn e clerly seen inotropic tht the mplitudes of rpid cooling contrcture t stedy t. Further stte nd fter periods of rest were significntly smller in the presence of the extrct s compred to control muscle. P1 d* * o 100 ontrctile force (% chnge) P1 2s Figure 3 nine right ventriculr trecule were driven rpidly (sic cycle length 30Oms). esstion of drive in the presence of () 350ju1 of extrct nd () 5 x 10-6M ougenin. Arrow with dshed line depicts ctive tension developed prior to ddition of the gent. Solid rrow shows ftercontrction in the presence of ougenin. ontrol 100 DLS frction ms Figure 4 Effect of extrct on iphsic contrction produced y sustituting 92-96% externl y Sr. () Pooled dt from 5 experiments showing the effect of 20pul of extrct on the two components (PI nd ) of the iphsic contrction. () Effect of extrct on iphsic contrction showing the predominnt effect on PI.

4 INOTROPI EFFET OF PORINE LEFT VENTRIULAR EXTRAT 373 ontrol I1g A~~~~~~~~Jn] Iy-_wiiu_ ilj I_ DLS frction gl3aj 30 s rest Figure 5 Typicl response of cnine ventriculr trecule to tretment with extrct (). 2 min rest 8 min rest vrying periods of rest in n untreted muscle () nd fter the Discussion The present study ws undertken in order to chrcterize the positive inotropic effect of DLS-contining extrct from porcine left ventricle. In greement with our previous dt (Khtter et l., 1986), the present results confirmed tht the extrct is s potent positive inotropic gent in the dog s it is on guine-pig hert. As descried erlier, the extrct is n inhiitor of the N pump nd displces [3H]-ouin from its high ffinity inding sites (Khtter et l., 1986; Agnyo & Khtter, 1990). Hence it is resonle to hypothesize tht the inotropic response my lso e produced y the inhiition of N', K+-ATPse nd susequent effect on N'-2+ exchnge nd elevtion of intrcellulr 2". In support of our hypothesis, the studies on iphsic contrction hve indicted tht the extrct primrily increses the delyed slow component (), which is cused y divlent ction entry cross srcolemm (King & Bose, 1983). The extrct lso incresed the mplitude of the erly component (lthough to smller extent thn ). This indictes n increse in the relese of ctivtor ction from the srcoplsmic reticulum. The ltter effect my in fct e secondry response to incresed srcolemml 2+ influx. rdic glycosides which cuse inotropy vi n effect on N'-2+ exchnge lso increse (King, 1982). However, unlike crdic glycosides which either decrese or do not chnge the durtion of contrction, the extrct significntly prolonged it. This ws due to the prolonged time to pek tension nd hlf mximl relxtion. The durtion of contrction is determined y the durtion of srcolemml 2+ influx, srcoplsmic reticulr 2+ relese s well s 2+ re-uptke (Lnger, 1973). Hence if the extrct increses the influx of 2+ vi voltge-gted clcium chnnels, it my prolong the durtion of the ction potentil in similr mnner to clcium chnnel gonist (Jnury et l., 1988) nd thus my lso prolong the contrction durtion. Alterntively, the extrct my increse the influx of 2+ in mnner similr to digitlis ut my lso hve n dditionl effect on the srcoplsmic reticulum tht my e responsile for the prolongtion of contrction. Aftercontrctions fter rpid pcing of the hert re chrcteristic of digitlis toxicity (Ferrier & Moe, 1973) nd re cused y the oscilltory relese of 2+ during distole from the 2+ overloded srcoplsmic reticulum (Tsien et l., 1978). The sence of digitlis-like toxicity even with the mximum dose of DLS-contining extrct would indicte tht either the incresed trns-srcolemml influx of 2+ y digitlis-like mechnism is mtched y concomitnt increse in efflux, or the extrct hs n dditionl effect which protects the crdic tissue from clcium overlod. In ny event, the sence of 2 + overlod type toxicity would suggest tht the extrct my hve etter inotropic chrcteristics thn digitlis. In the present study we lso investigted the effect of the extrct on post-rest potentition nd rpid cooling contrcture, which provide n estimte of the functionl cpcity of srcoplsmic reticulum to store nd relese 2 + (Hryshko et l., 1989). In the presence of extrct, there ws reduction in the extent of post-rest potentition fter 30s nd 2 min rest nd conversion of post-rest potentition to post-rest depression fter rest for 8 min. Much experimentl evidence suggests tht post-rest potentition is due to the redistriution of clcium within the srcoplsmic reticulum during distole (Bers, 1985; Bridge, 1986; Bers et l., 1987; Bose et l., 1988) ontrol 0.5 g DLS frction 0.5 A Stedy stte 2 min rest 8 min rest Figure 6 Typicl response of cnine trecule to rpid cooling from 370 to 0 in less thn second. The rpid cooling contrctures were otined in plce of regulrly driven et or fter 2 nd 8 min of rest period. The rrows under the individul recordings represent the time of initition of cooling. () Represents the control trcing nd () trcing fter dministrtion of extrct from porcine left ventricle.

5 374 S. NAVARATNAM et l Stedy 2 min 8 min stte rest rest Figure 7 Pooled dt from 4 experiments demonstrting the effect of extrct on rpid cooling contrcture during stedy stte nd fter 2 nd 8min of rest. *Significntly different from pproprite control vlue (P < 0.05). nd the progressive reduction in the post-rest contrction mplitude with incresing durtion of rest is due to the continuous ut slow loss of clcium from the srcoplsmic reticulum during distole (Wohlfrt & Nole 1982; Bose et l., 1988). The present oservtion of enhncement of the ltter process y the extrct, in spite of its potent positive inotropic response, indictes tht it my lso fcilitte the loss of 2+ from srcoplsmic reticulum. This ws further supported y the oservtion with rpid cooling contrcture. In the presence of the extrct there ws decrese in rpid cooling contrcture fter ll rest intervls, gin indicting tht the extrct fcilitted the loss of 2+ from srcoplsmic reticulum. In ddition, the mplitude of the stedy stte R immeditely following trin of regulr stimuli ws smller percent of the preceding stedy stte contrction compred to control stedy stte R. This would men tht the extrct my lso impir the uptke of 2+ y the srcoplsmic reticulum resulting in reltively lower content of srcoplsmic reticulr 2+ (Bouchrd et l., 1989). This is in contrst to the effect of the chnnel gonist, By K 8644 which increses the mplitude of stedy stte R even though it, like the extrct, decreses the size of the R fter vrious durtions of rest. In summry, the present study indictes tht the DLScontining extrct from porcine left ventricle produces positive inotropic response y incresing the influx of srcolemml 2. It lso hs dditionl effects on the srcoplsmic reticulum which my fcilitte the loss nd/or inhiit the uptke of 2+ nd which my lso e responsile for the prolonged contrction durtion. Electrophysiologicl studies to define the effect of the extrct on trnsmemrne ction potentil nd on whole cell ionic current(s), however, re necessry to elucidte further its mechnism of ction. This project ws supported y grnts to J..K. nd D.B. from the Medicl Reserch ouncil of nd. References AGBANYO, M. & KHATTER, J.. (1990). Purifiction nd chrcteriztion of endogenous digitlis-like sustnce (DLS) from pig hert. Reserch ommun. hem. Pthol. Phrmcol., 68, BALZAN, S., GHIONE, S., LERIO, A. & MONTALI, K. (1986). orreltion etween endogenous digoxin-like immunorectivity nd 3Houin displcement on erythrocyte memrnes in extrcts of humn plsm. lin. Biochem., 19, BERS, D.M. (1985). influx nd srcoplsmic reticulum relese in crdic muscle ctivtion during post-rest recovery. Am. J. Physiol., 248, H366-H381. BERS, D.M., BRIDGE, J.H.B. & MALEOD, K.T. (1987). The mechnism of rynodine ction in crdic muscle ssessed with clciumsensitive microelectrode nd rpid cooling contrcture. n. J. Physiol. 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