003 1 -3998/87/2203-0282$02.00/0 PEDIATRIC RESEARCH Vol. 22, No. 3. 1987 Copyright (cl 1987 International Pcdiatric Research Foundation. Inc Printed in L' S A.
Effect of Milrinone on Myocardial Mechanical Function and Cyclic AMP Content in the Fetal Rabbit
SHUN-ICHI OGAWA, TOSHIO NAKANISHI, KIYOMI KAMATA, AND ATSUYOSHI TAKA0 Pediatric Curdiology, Hcart Insfilure ofJapan, Tokj,o Wornrn'.~Medical College, Tokyo, Japan
ABSTRACT. The effect of milrinone on mechanical func- METHODS tion was studied in the isolated arterially perfused heart of the fetal (28th day of gestation) and newborn rabbits. The The experiments utilized the fetus at the 28th day of gestation inotropic effect of milrinone in the fetus was significantly (term 3 1 days) and 3- to 5-day-old newborn New Zealand White less than in the newborn. After milrinone infusion, myo- rabbits. After the doe was killed by a sharp blow to the head the cardial cyclic AMP levels increased significantly in the two fetuses were delivered by cesarian section and used within 1 min age groups and the fetal values before and after milrinone after delivery. The fetal and newborn rabbits were killed by a infusion were not significantly different from the newborn sharp blow to the head. The heart was then excised from the values. In our previous study the inotropic effects of dibu- chest cavity and used for mechanical function study. tyryl cyclic AMP and high extracellular calcium in the Perfusion solution. The control Krebs-Henseleit solution con- fetus were significantly less than in the newborn. These tained in mM: NaC1, 118; KCI, 6; CaClz, 1.5; glucose, 6; MgCIZ, data suggest that the diminished inotropic effect of milri- 1; NaHC03, 24; NaH2P0,, 0.436. KC1 concentration was rela- none in the fetus may be due, at least in part, to the tively high because at 6 mM KC1 arrhythmia was observed less decreased inotropism of cyclic AMP and calcium. (Pediatr frequently than at 4 mM. Changing Kf concentrations from 4 Res 22: 282-285, 1987) to 6 mM did not alter mechanical function. The control solution was equilibrated with 95% O2 and 5% COz yielding a final pH Abbreviations of 7.35-7.42. Milrinone (a gift from Sterling-Winthrop) was freshly prepared by dissolving the powder in 0.5 lactic acid IBMX, isobutylmethylxanthin N and an aliquot was added to the perfusate to achieve final DT, developed tension RT, resting tension concentrations of lo-' to 5 M. An aliquot of 0.5 N lactic acid was also added to the control perfusate to achieve the same +dT/dt max, maximum rate of tension development concentrations as the perfusate containing milrinone. IBMX %RT, half-time to relaxation (Nakarai) was suspended in the perfusate to achieve final con- centrations of 1 0-5 to 1 0-4 M. Experimenfulprrparation.Experiments were performed in the isolated, arterially perfused ventricular preparation as described Milrinone is a new analogue of amrinone and its inotropic previously (8-13). The aorta was canulated with PE-50 polyeth- effect has been evaluated mainly in the adult mammalian myo- ylene canula and then perfused with oxygenated perfusate at a cardium (1, 2). Although the precise mechanisms of the inotropic constant perfusion rate of 2.5 ml/g tissue per min using a Harvard effect remain unclear, amrinone and milrinone may inhibit pump. The base of the right and left ventricle was fixed between phosphodiesterase activity and increase myocardial cyclic AMP two Harmon forceps and the apex was attached to the Statham content (1-5). This may increase cytosolic calcium concentra- (UC 3) force transducer using a silk suture. The muscle was tions and cause a positive inotropic effect (2). Developmental stimulated at 90 bpm, and its temperature was maintained at 37 changes in the effect of amrinone and milrinone have not been k 0.5" C. In some experiments, the muscle was stimulated at 40 studied extensively. Binah ct ul. (6) showed that amrinone had a bpm and its temperature was maintained at 27" C. The following negative, rather than positive, inotropic effect in the newborn parameters of mechanical function were monitored continu- dog heart. Recently, a preliminary report of Binah et ul. (7) ously: DT, RT, +dT/dt max, and %RT. showed that milrinone induced a minimal but significant positive In the present study a whole heart was suspended at three inotropic effect in the newborn dog. The relationship between points and DT represents one of three force vectors. Because the the inotropism of these drugs and cyclic AMP metabolism in the muscle preparation was not cylindrical, parameters of mechani- fetal and newborn myocardium remains unclear. Therefore, this cal function were normalized for the wet weight rather than for study was designed to investigate the effect of milrinone on 1) the cross-sectional area. Although the papillary muscle prepara- mechanical function and 2) myocardial cyclic AMP content in tion may be more desirable for a mechanical function study, the the isolated fetal and newborn heart of the rabbit. whole heart preparation was used in the present study to perform mechanical and biochemical studies in the same tissue. It must be noted, however, that although the heart weights changed with Rece~vedDecember 1, 1986: accepted April 3. 198'7. growth, the geometry of the preparation remained similar. It Correspondence Toshio Nakanish~,M.D., Pediatric Cardiology, Heart Institute must be also mentioned that since the heart was perfused retro- of Japan, Tokyo Women's Medical College, I0 Kawadacho, Shinjuku. Tokyo, grade via the aorta, this preparation is different from the working Japan. Supported by Research Grants 61 770706 and 6 177071 1 from the Japanese heart model described by Neely et al. (20). Mlnistry of Education, Science, and Culture and a grant-in-aid from Japan Research Expcrimentulprotocol. Initially the muscles were perfused with Promotion Society for Cardlovascular Diseases. a control solution containing 1.5 mM calcium for 60 min to 28 allow fir stabili/ation ol' the mechanical f~~nction.I>ur-ing the mcnt the iriotropic ctTcct of i~igll[('a]<, and isoprotc.1-end ~rsing initial 40 mi11 01' each experiment. the length of' the muscle tlic present preparation ~icr-cidentical to the prc\ iolrsl! ~.cpc~r-tcd preparation was ad,iusted so that the tension was ecl~r:il to 00"; data (8. 17. 13). 01' the maximal tension. Alicr- this initial period. hot11 I-csting Iilii,(,i 01 ~~ie/~-itiot~c'.1.actic acid ~rsciiin tlic present stud! (I0 ' tcnsion and the length-tension relationship reniarncd unchangcti to 5 x I0 ' b1) had no signiticant ct'ii.c.ts 011 mcclianic~alI'~rricti~ri. under contl-ol contlitrons. 'I he li~llowingstudies wcrc then per- I'!l,ical data of the c\pc~-irnent\using milrinonc \lio\\n in Sornicd. I'rgur-e I. Vilrinonc rnl'usion caused \ignific:rnt incr.casc\ in I> I l:'/)czc.i (I/ .1fil/.i1ioii(,.,Alicr st:~l>ili/ationof mechanical function. in tlie ne\\born. but not in the ktus. Signilicant ilrff;'rcnce In 1) 1 the hcar-t was per-Sirscd with solutions containing I x I0 '. 5 x l>ct\\ccn the neuborn and ktus \\ere ot~,cr\cdat conccntratiori\ I0 ', and I x 10 ' and 5 x I0 " M rnilrrnonc (11 = 7 in each age ol' IO~'M and 5 x 10 ' M (f-ig. 2). .lfic~-riiilrinonc inl'ir\rori gro~rp).I'lie (lur;~tiorioSj~e~-f'~rsio~i \\,;IS 30 min at each milr-inone +d.l'/dt(ma) incrcaseri sigriiticantl> t>oth in the rie\\.bor.ri and concentr:~tion. Mechanical function reached a new stcad) state fetus but the iricren\e in tlie rct~rs\\as rnir~imal(I'ig. 3). It 5 x within I0 min alicl- 5uitching to a nc\\ lnilrinonc concentration. I0 ' M rnilrinone. +dr/dt(max) increased to 10 I -t I O"( 01' [hc and all rncas~~rcr-ncntsuerc made at 10 min. In additional cxper-imcnts, hearts were perfused uitti solutions coritaining lactic acid ol'thc same concentrations as the per-f'usatc Fetus Control M~lr~nonelo M containing milrinonc. I'hc duratron of perfusion was \irnilar- to 15 that dcscrrl~edabove. In another- \cries of cxpcr-iments. the heart was initial11 per- p::[A fUscd with a \olutron containing 5 x 10 ' M niilrrnone lijr 10 + 0 min. Aficr :I ricu8steady state was obt:~incd.tlic hc:trt Lvas ~~crl'~~seti lor with a solution containing hot11 milrinonc (5 x 10 ,' M) and 15 mM calcilrni li~r10 mrn and \vlietlicr in the heart pcrf~sed\\ it11 solution containing rnilrinonc contractile fi~rcecan be increitscti further by :III additronal inotropic agent was studied. - 1oL I:'/)c,c./ (I/ 11{2/,\'. I13MX is a potent phospliodiestcr:~~~rnhihitor (2. 3). I'hc cl'fi.ct ol' IIIMX \\a\ \t~rdicd in a srniilar fi~sliion Control Mllr~none10 M Newborn dcscrikctl ahovc (ti - 0 in each i~gcgro~~p). I':~ramctc~-sclescr-ihing rncclianical furiction wcr-e c\l~re~scda5 sr a I,cl-ccnt;ll:c of'control value5 and :I g/g tissue \vet \vcrglit :if ~.oi,io-(/ici/(.~.i./ii, . I \/IJc~oriic~rci. Afier stahili/atiori of' rncctlan- ical Ilrnctron. hear-15\\cr-c per-l'irsed mith 5olutionf contarning 5 x I0 " M milririoric or- IIIMX fi)l- I0 niin and then t'ro/cn \\it11 mct:~l clamp\ in Irclirrd riitrogcn. C'oritrol hearts \vc~-cpi~rt'useti wit11 solution5 u.11icli did rio~cont;~ir) mrlririonc or 113MX and fro/cn in liquid nrtr-ogcn. Mi~\clcswere then homogcnr/cd in ten volumes of'Of; 11-rcliloroaccticacid using a ground-gli~ssliomog- crii/cr and ccntrrl'~rgctlat 0" ( . 'I he \ul>crriatant \\ancutr-al~/cd by adding ('a('0; ( 14) and ri%~itr-il'~rgcc1again. ('ycl~c,21.11' <,on- ccntratiori in the ncutr:rli/cd super-natant \\:is riie:~~~rri~ilI>> I.;IC~IO- imrnunoassa~rncthods (I i) using cAMI' ["'I] RIA Kit (Uc1.r lingland Nuclear. I3oston. MA). 1-1g I. I y~ic,:~lc\pCrir1i~~~~t\ tli:~t \lie\\ 1111, ~,lT~~c,t01' ~iiil~~~~io~ic~or1 1) I .Y/tii~.s/ic~ii/ci~rci/i.\c\. Ilcslllt\ \\ere cxyrc\scd as mean i SI.. anti ti I 'dt I IIL, e\pcrimi.lit\ \\i'[c j~~~ili)llii~~il;it .;Yr (- I Iic 111011.01111. Statistical signrlicaricc of' the ~~illi'rc~icebctwccr~ g1.0~11) riicari\ ellCi'~of rn~li-111oncin tlii. l?tu\ (7SlIi LI:I! ol'~~~t~it~orii\\:I\ It,\\ 1/1;111 111 was dctcrmirieil using the Student'\ I tc\t (I 0). I'cr-cent changes tI1c 11c\\ t>orlI. wcrc conil,arccl using nonparamctric method5 (M:ilco\ori's rank surn test) (17). Statistical signilicaricc ol' rc\ponsc to m~lr-inonc 2nd IRMX tva\ anal>,/cd ~lsirig:I r):~i~-cd/ tesl :II~C~I-C~CYI~CC~. I~IC;ISLII-CI~~CTI~Sol'analysis 01' \arianee. 1.h~prohahilit> \\its ron- sidcred to Ijc signilica~itif' the 11 < 0.05. -Newborn ---0--- Fetus
Mcchanic:~lI'l~nctiofi data under control conditions in the Ic'tl~s were not signrlicantl!. tiilkrent fr-om tliosc in the nc\\,l?orn ( I i~hlc I). Altho~~gliI) I \\,:I\ slight11 lower than the prc\iouj data (Xi. we thought that the present prcl,ar-;~trori was acccpt:~l>lcl>cc,ausc myocar-cIi;il li~gli c1ie1g1 pIios1)11;1tc C~II~CII~I~;I~I~IISill IIIIISCIC\ which showed mcch:~nical Ilrnction similar to the present data wcrc not dcpr-c\\cd (I?). I.'ur-thcrmorc. in a prclimina~-! c\pcr-i-
* Value\ arc ~~ic;~risf SI.. I hc tiara wcrc obtained :it 37" (' 1 hcrc was I ~g.7 Illkct ot'mlli-inonc on I) I (tcmpcrat~lrc.37" C'i * sign~Iic~:~litl~ 110 signilica~itcli1Te1-cncc hcrwccn thc 78-day lbtus and ncwhorn. [[I < 0.05) ti~fkrcntfrom tlic ~aluc'11 tlic nc\\t>orl) 284 OGAWA ET AL.
Milrinone 1 0-4M Newborn /-. ----0--- Fetus
f I Newborn Fetus Fig. 4. Effect of milrinone on Y2RT (temperature 37" C). Shortening Q I o-~ 5x 1 o-~ of %RT in the fetus was not significantly different from the newborn Milrinone (M) value. Fig. 3. Effect of milrinone on +dT/dt(max) (temperature 37" C). *significantly different from the newborn value. Newborn ----Om-- Fetus control in the newborn and only to 126 + 6% of the control in the fetus (significant difference between the newborn and fetus: p < 0.001). The inotropic effect of milrinone in the muscle maintained at 27" C was not significantly different from that at 37" C (Figs. 2 and 7). Milrinone M) decreased I/?RT similarly in the newborn and fetus (Fig. 4). Since the inotropic effect of milrinone was minimal in the fetus, the inotropic effect of milrinone and high [Ca],, was studied to examine whether other inotropic agent can increase +dT/ dt(max) further in the fetus. High [Ca], (15 mM) caused addi- tional inotropic effect in the heart perfused with milrinone in the fetus (+dT/dt max = 185 f 37% of control, n = 4) and newborn (265 t- 25%, n = 4). Efect of IBMX. Whether the age-related difference in the inotropic effect of milrinone is observed in the other phospho- diesterase inhibitors was studied. After IBMX infusion, signifi- cant increases in DT and +dT/dt(max) were observed in the two age groups but the effect in the fetus was significantly less than Fig. 5. Effect of IBMX on DT (temperature 37" C). * significantly in the newborn (Figs. 5 and 6). different from the newborn value. Mvocurdiul cyclic AMY content. The control value of myocar- dial cyclic AMP content was similar in the two age groups. Milnnone and IBMX infusion increased tissue cyclic AMP levels significantly, and the value after milrinone or IBMX infusion in the fetus was similar to the value in the newborn (Table 2).
DISCUSSION This study demonstrated that in the isolated heart preparation the increase of DT and +dT/dt(max) during milrinone infusion in the fetus was less than in the newborn. This finding is in agreement with the data of Binah et a/. (7) who showed that the inotropic effect of milrinone increased with age in the dog heart. The present study also showed that the effect of milrinone on myocardial cyclic AMP levels was similar in the fetus and newborn. Therefore, it is unlikely that the decreased inotropic effect of milrinone in the fetus is explained by the response of myocardial cyclic AMP levels. Cyclic AMP, by activating protein kinase, enhances calcium release from the sarcoplasmic reticu- lum and causes an increase in intracellular calcium and a positive IBMX (M) inotropic effect. Cyclic AMP also accelerates the relaxation proc- Fig. 6. Effect of IBMX on +dT/dt(max) (temperature 37" C). * sig- ess by stimulating calcium uptake by the sarcoplasmic reticulum. nificantly different from the newborn value. Different inotropic effects of milrinone in the newborn and fetus may be due to the age-related differences in I) the process from cyclic AMP to sarcoplasmic reticulum and/or 2) the inotropic Ca sequestration by the sarcoplasmic reticulum but may be effect of calcium per se. related to the function of the elastic elements of the preparation. In the present study, enhancement of the relaxation process However, when the heart was perfused with a solution containing by milrinone, expressed as a shortening of '/2RT, was similar in ryanodine, an inhibitor of Ca uptake by the sarcoplasmic retic- the newborn and fetus. This suggests that the effect of milrinone ulum, %RT prolonged significantly (data not shown). This sug- on the sarcoplasmic reticulum is similar in the two age groups. gests that l/2RT is indeed related to the function of the sarco- One may argue that Y2RT may not be related to the function of plasmic reticulum. 0Calc~um ui lsoproterenol Alicr I13MX CACycllc AMP Aficr Ul8E IBMX Age ('ontrol (pmol/mg mill-~n~rie tissue wct wt) M~lr~none
* Values arc meall\ + SI:. Nurnhcrs in ~~arcntllcscsarc numhcrs of rncasul-crncnls. Values alicr (11-uginl'u5ion were significantl) (11 < 0.05) greater than contr-ol In all age groups. 'Thcrc was no significant dilfcrcncc hctwccn thc lktal and ncwborn values.
In the prcscnt study, the maximal inotropic clkct of both milrinonc and 113MX in the f'ctus was less than in the ncubor-n. This suggests that the inotropic clkct of phosphodicstcrase in- hibitors in general is diniinislicd in the fctal myocardiuni. We Newborn Fetus have previously studied the inotropic cffcct ofcalcit~m(15 mM). I ig. 7. ('ompar~\on ol' milrlnonc \\it11 other ~notr-ol>icagent\. 1'11~. isoprotcrcnol (I0 ' M), and dibutyr-yl cyclic AMP (I0 M) under- data of calc~~~rn.1soprotc1-~110I. iilhut)~->I c>clic ,1511' arc I'rom Ilcli.rsncc the identical cxpcrirncntal conditions used in the prcscnt stud) I4 211 eperlmcnts \\ere perli>rnlcci at 117" (' 2nd thc I~~LI\L.IL,\\;I\ (8, 13). 'l'lic inotropic cft'cct ol' dibutyl-yl cyclic ,AM['. which \timulatcd at 40 hpm, I-llc cf'lkct of all no tropic agiLnt\\ti~ci~cd in tllc passcs the cell ~ncmbrancantl acti~atcsprotein kinasc directlq l'ctus \\.ere ~lppro\lniatcl>30'; ol'thc nc\\boril \aluc\ (17). was lcss in the fetal hcar-t tlian in the newborn (I-ig. 7). This supports the liypothcsis tliat tlic diminished response to milri- none in the fetus rcsultcd from the diffi.rcncc in the process after I ~IOLI~I\ \, S~:III~L~\(;I>. jl~i:11-1 .I( . \\.:i11011 I 11 l'lS3 ('II:I~~I~~~L~II/:~~I~~II111 1lic the cyclic AMP increase. C;II~IO~OIIICcII;.cI\ of ~~lilrii~oi~e..I TIC\\ .ii1~1 l1012111 i..i~tIi:ic1~1~1\r1~11112. (111 130th milrinonc and II3MX increase the amount of' calcium ~\ol:~tcclt~\si~~,\ l'1011i \c\c~~l ,1111r11:il \l>e,c~c\I ('J~~III\J\Cl'l~~irri~~~i~ol i SO4 rcacliing the myolilarncnt and cause the inotropic cfkct. In 011s Sl l previous study. the inotropic clkct of high [('a],, in the f'ctus bas 2 Scliol/ 11 lt)S4 l~i<~ticip~c~ILI~ ,i~~dtl~e,~r 111cx'll,i111\i11\ :IL,~IIIII J \III ('011 ~':ll~liol4 .;so-.:~17 diminished (Fig. 7). 'I'hcrcfi)rc. it is likely that the dccrcased 3 I ntloh hl. \~L~~~i;~\li~l.~S 1.1ii~1 \ I')S? I'~~\I~I\cIII~II.II~~I~.~CI~~CI 01 .111111iioi1i, 111 inotropic cft'cct of milrinonc in the fi.tus may be due. at Icast in iclat~on1i1 c!i,l~i. nl~clci~t~cicII~~I.I~~O~I\III 111 1111, L.~IIIII~\~111rii.~ihi1 I~III\L.~C .I part, to the flgc-r-clatcddil1i.r-cncc in the cfl'cct ofcalciuni. l~li:~i~ii;icolI \I> I I1ci 22 I 7-<--8 ? -1'hc preclsc mechanisms ol' the diminished inotropis~ll ol' 4 111