Evidence for Reduction of Norepinephrine Uptake Sites in the Failing Human Heart

Transcription

1 146 JACC Vl. 25, N. 1 HEART FAILURE Evidence fr Reductin f Nrepinephrine Uptake Sites in the Failing Human Heart MICHAEL BOHM, MD, KARL LA ROSI~E, MD, ROBERT H. G. SCHWINGER, MD, ERLAND ERDMANN, MD, FACC Clgne, Germany Objectives. This study investigated the rle f neurnal uptake f nrepinephrine (uptake-l) in human heart failure as a lcal factr fr altering cncentratins f nrepinephrine at the cardiac mycyte membranes. Backgrund. Several beta-adrenergic neureffectr defects ccur in heart failure. Whether an alteratin in nrepinephrine uptake-1 ccurs is still unreslved. Methds. The rle f nrepinephrine uptake-1 was studied in electrically stimulated (1 Hz, 37 C) human ventricular cardiac preparatins and islated mycardial membranes. Results. The effectiveness f nrepinephrine in increasing the frce f cntractin was decreased in relatin t the degree f heart failure. In cntrast, the ptency f nrepinephrine was increased in failing hearts (New Yrk Heart Assciatin functinal class IV) in relatin t the cncentratins prducing 50% f the maximal effect (ECs). The ECs values fr isprterenl, which is nt a substrate fr nrepinephrine uptake-l, were reduced in mycardium in functinal classes II t III and IV cmpared with thse in nnfailing mycardium. The uptake inhibitrs ccaine and desipramine (3/~ml/liter) ptentiated the psitive intrpic effects f nrepinephrine in nnfailing mycardium (p < 0.05) but nt in functinal class IV mycardium. Radiligand binding experiments using the uptake inhibitr laydrgen-3 mazindl revealed a significant decrease by -30% in nrepinephrine uptake-1 carrier density in functinal classes II t III and IV mycardium versus nnfailing mycardium (p < 0.05). Cnclusins. In human heart failure, there is a presynaptic defect in the sympathetic nervus system, leading t reduced uptake-1 activity. This defect in the failing heart can be mimicked by the effects f uptake blcking agents, such as ccaine and desipramine, in the nnfailing heart nly. Cmprmised nrepinephrine uptake-1 in functinal class IV cannt be further increased by ccaine and desipramine. The pathphysilgic cnsequences culd be an increased synaptic cncentratin f nrepinephrine predispsing t adenylyl cyclase desensitizatin. (J Am Cil Cardil 1995;25:146-53) In failing human mycardium, the blunted cardiac respnsiveness t beta-adrenergic stimulatin is due t a reductin in betal-adrenceptr density (1,2) and an increase in Gi-alpha prteins (3-5), which culd be induced by an increased level f circulating catechlamines (6-8), an increase in neurnal release f nrepinephrine (9) r an impairment f neurnal uptake f nrepinephrine, r a cmbinatin f these mechanisms. Cardiac neurnal uptake is the predminant mechanism fr terminating the actin f nrepinephrine n betaadrenceptrs when catechlamines are released by sympathetic nerve terminals (10). In patients with heart failure, data arguing against (11,12) r in favr (13) f reduced mycardial nrepinephrine uptake exist. Hwever, direct experimental evidence in islated failing human mycardium in favr f reduced nrepinephrine uptake as well as its ptential functinal cnsequences is limited. The purpse f this study was t Frm the Klinik III f/jr Innere Medizin der Universit/it zu K6ln, Clgne, Germany. Dr. B6hm was supprted by the Deutsche Frschungsgemeinschaft, Bnn, Germany. Manuscript received May 10, 1994; revised manuscript received July 15, 1994, accepted August 3, Address fr crresnndence: Dr. Michael B6hm, Klinik III fiir Innere Medizin der Universit~it zu K61n, Jseph Stelzmann Strasse 9, D Clgne, Germany. use functinal and bichemical techniques t investigate the rle f neurnal uptake f nrepinephrine (uptake-i) in human heart failure as a lcal factr fr altering cncentratins f nrepinephrine at the cardiac mycyte membranes. Studies were perfrmed in electrically stimulated human ventricular cardiac preparatins and islated mycardial membranes. Tissue was btained frm patients wh underwent mitral valve replacement r heart transplantatin. Fr cmparisn, tissue frm nnfailing dnr hearts was studied. Methds Mycardial tissue. Mycardium frm terminally failing human hearts was btained frm patients after cardieetmy during cardiac transplantatin. The patients had dilated r ischemic cardimypathy and were in New Yrk Heart Assciatin functinal class IV n the basis f clinical symptms and signs as judged by the attending cardilgist shrtly befre peratin. During mitral valve replacement, tissue frm mderately failing hearts was btained. All patients gave written infrmed cnsent befre peratin. Clinical details f the patients included in the study are shwn in Table 1. Flunitrazepam and pancurnium brmide with isflurane were used fr general anesthesia. Cardiac surgery was perfrmed during 1995 by the American Cllege f Cardilgy /95/$ (94)00353-R

2 JACC Vl. 25, N. 1 BOHM ET AL. 147 NOREPINEPHRINE UPTAKE IN HEART FAILURE Table 1. Clinical Characteristics Obtained at Preperative Cardiac Catheterizatin fr Patients Prviding Study Mycardium Samples LVEDP LVEDV EF CI Gender/Age (yr) NYHA Diagnsis (mm Hg) (ml) (%) (liters/min m 2) Mean _+SEM Mean _+SEM Mitral Valve Replacement F/57 II-III MR F/67 li-iii MR F/46 I1-Ili MR > MS M/44 II-III MS > MR M/46 II-III MR M/59 II-III MR F/65 II-II1 MS F/68 II-III MS II-III _+1.6 _+39.1 _+3.7 _+0.2 Cardiac Transplantatin I71/55 IV DCM M/50 IV DCM M/47 IV DCM M/42 IV DCM M/51 IV DCM M/49 IV DCM M/39 IV ICM M/63 IV ICM.... M/48 IV ICM _+2.2 _+2.6 _+53.5 _+4.4 _+0.2 CI = cardiac index; DCM = dilated cardimypathy; EF - ejectin fractin; F - female; ICM = ischemic cardimypathy; LVEDP = left ventricular end-diastlic pressure; LVEDV - left ventricular end diastlic vlume; M - male; MR = predminant mitral regurgitatin; MS = predminant mitral stensis; NYHA = New Yrk Heart Assciatin functinal class (II t III - mderately failing mycardium, IV = severely failing mycardium); -- = data nt available. cardipulmnary bypass with cardiplegic arrest during hypthermia. The cardiplegic slutin (mdified Bretschneidcr slutin) cntained (in mml/liter) NaCI 15, KC1 10, MgC12 4, histidine 180, tryptphan 2, mannitl 30 and ptassium dihydrgen xglutarate 1. Nnfailing mycardium was btained frm five dnrs wh were declared brain dead as a result f traumatic injury. These hearts culd nt be transplanted fr technical reasns. Echcardigraphic evaluatin shwed nrmal left ventricular functin. Invasive hemdynamic measurements were nt perfrmed. Nne f the dnrs f the nnfailing hearts received sustained catechlamine treatment. Hwever, blus injectins f catechlamines were dcumented in three patients during resuscitatin after acute treatment f trauma. Thus, these hearts cannt be termed nrmal but nly nnfailing. The reasns fr nt transplanting the hearts were fever, pericardial effusin r evidence fr crnary heart disease. The use f human cardiac tissues was apprved by the ethical cmmittees f the Universities f Munich and Clgne. Islated cardiac preparatin and measurement f frce f cntractin. Immediately after excisin, papillary muscle strips were placed in ice-cld preaerated!.tyrde slutin and delivered t the labratry within 10 min. The experiments were perfrmed n islated electrically driven muscle preparatins. Muscle strips f unifrm size with muscle fibers running apprximately parallel t the length f the strips were dissected under micrscpic cntrl using scissrs in aerated mdified Tyrde slutin (see cmpsitin later). Cnnective tissue was carefully trimmed away. The muscles were suspended in an rgan bath (75 ml) maintained at 37 C and cntaining a mdified Tyrde slutin f the fllwing cmpsitin (in retl/liter): NaC , KC1 5.4, MgC , CaC12 1.8, NaHCO , NaH2PO , glucse 5.0, ascrbic acid 0.28, ethylenediaminetetraacetic acid (EDTA) The bathing slutin was cntinuusly aerated with 95% xygen and 5% carbn dixide. The muscles were stimulated by tw platinum electrdes using field stimulatin frm a Grass $88 stimulatr (frequency 1 Hz, impulse duratin 5 ms, intensity 10% t 20% greater than threshld). Each muscle was stretched t the length at which frce f cntractin was maximal. The frce at rest was kept cnstant thrughut the experiment. The resultant tensin was measured ismetrically with an inductive frce transducer (W. Fleck, Mainz, Germany) attached t a Guld recrder. Preparatins were allwed t equilibrate fr at least 90 min, with the bathing slutin changed nce after -45 min. Experimental details have been described elsewhere (3). Membrane preparatin and radiligand binding experiments. Left ventricular mycardial tissue was chilled in 30 ml f ice-cld hmgenizatin buffer (20 mml/liter f tris(hydrxymethyl)aminmethane [Tris] hydrchlride, 4 mml/liter f EDTA and 1 mml/liter f dithitheitl, ph 7.4). Cnnective tissue was trimmed away; mycardial tissue was minced

3 148 BI3HM ET AL. JACC Vl. 25, N. 1 NOREPINEPHRINE UPTAKE IN HEART FAILURE with scissrs; and membranes were hmgenized with a mtrdriven glass-tefln hmgenizer fr 1 min. The membrane preparatin was then hmgenized by hand with a glass-glass hmgenizer. The hmgenate was spun at 484 g (Beckman JA 20 rtr) fr 15 min. The supernatant was filtered thrugh fur layers f gauze, diluted with an equal vlume f ptassium chlride (1 ml/liter) and stred n ice fr 10 min. This suspensin was centrifuged at 100,000 g fr 30 min. The pellet was resuspended in 50 vlumes f incubatin buffer (50 mml/liter f Tris He1, 120 mml/liter f NaC1, 5 mml/liter f KC1, ph 7.4) and hmgenized fr 1 rain with a glass-glass hmgenizer. This suspensin was recentrifuged at 100,000 g fr 45 min. The final pellet was resuspended in incubatin buffer and stred at -80 C. Strage did nt alter the results. Prtein was measured accrding t Lwry et al. (14) using bvine serum as standard. The assays were perfrmed in a ttal vlume f 250/zl. The incubatin was carried ut at 22 C fr 60 min. These cnditins allwed cmplete equilibratin f the receptr with the radiligand. The reactin was terminated by rapid vacuum filtratin thrugh Whatman GF/C filters that were presaked with 0.1% 3-([3-chelamidpryl]- dimethylamni)-l-prpansulfate (CHAPS) t reduce nnspecific binding. The filters were washed immediately three times with 6 ml f ice-cld incubatin buffer t remve any unbund radiligand. All experiments were perfrmed in triplicate. Radiactivity was determined in an LKB cunter (Pharmacia LKB, Freiburg, Germany). Mycardial nrepinephrine uptake carrier sites were studied using hydrgen-3 (H-3) mazindl (specific activity 24.7 Ci/mml) as radilabeled ligand (1 t 50 nml/liter). Specific binding f H-3 mazindl was defined in the presence f desipramine (100/sml/liter). Materials. (S)-(-)-Nrepinephrine was frm Merck- Schuchardt (Hhenbrunn, Germany). Atrpine sulfate and desipramine were purchased frm Sigma Chemie GmbH (Deisenhfen, Germany). Ccaine slutin was prepared in the pharmacy f the Klinikum GrBhadern (University f Munich). Hydrgen-3 mazindl (specific activity 24.7 Ci/ mml) was frm New England Nuclear-DuPnt GmbH (Dreieich, Germany). All ther cmpunds used were f analytic grade r the best grade cmmercially available. Only deinized and twice-distilled water was used thrughut. Statistical evaluatin. All data are mean value _SEM. Statistical significance was estimated with the Student t test fr unpaired bservatins and analysis f variance. The Scheff6 prcedure fr multiple cmparisns was applied. A p value <0.05 was cnsidered significant. The drug cncentratins prducing 50% f the maximal effect (ECs0) were graphically determined fr each individual experiment. Results Effects f nrepinephrine and isprterenl. Figure 1 shws the psitive intrpic effects f nrepinephrine in islated electrically driven papillary muscle strips frm mderately (functinal classes II t III) and severely (functinal Z E *d e- ID Q e- 0 - m /! r-.9 ~'100 ~ ~ 0 NF (n=5) ~ f I'~ NYHA,l-Ill (n=6) / I / i i i i i i i i, Cncentratin f nredinephrine OJml/I)., NF (n=5) 8 ~ NYHAII-III{n=6),//'/ ~ 50 ~E ='6 Q t/ i, i i i Cncentratin f nrepinephrine (/Jml/I) Figure l. Cncentratin-respnse curves fr the effect f nrepinephrine (0.01 t 100 ~ml/liter) n ismetric frce f cntractin in islated electrically driven papillary muscle strips frm nnfailing dnr hearts (NF) and frm patients underging mitral valve replacement (New Yrk Heart Assciatin [NYHA] functinal classes II t III) r cardiac transplantatin (functinal class IV). class IV) failing as well as nnfailing mycardium. Nrepinephrine cncentratin dependently increased frce f cntractin in bth grups. As expected, the psitive intrpic effect f nrepinephrine was reduced in papillary muscle strips frm functinal classes II t III and IV cmpared with that in nnfailing mycardium. T evaluate the ptency f the betaadrenceptr agnist, cncentratin-respnse curves fr nrepinephrine are shwn in percent f the maximal effect in each preparatin. Unexpectedly, the ptency f nrepinephrine t increase frce f cntractin was increased in functinal classes II t III and IV mycardium cmpared with nnfailing mycardium (Fig. 1, bttm). Crrespndingly, the EC5 values were significantly lwer in failing than in nnfailing mycardium (Table 2). The increased ptency f nrepinephrine despite reduced maximal effects culd either reflect mre efficient cupling f the remaining beta-adrenceptrs r a reduced inactivatin f nrepinephrine by uptake-1 in failing mycardium. T address the latter, we cmpared the effects f nrepinephrine with thse f isprterenl, which is nt a substrate fr uptake-1 (15,16), under same experimental cn-

4 JACC Vl. 25, N. 1 BOHM ET AL. 149 NOREPINEPHRINE UPTAKE IN HEART FAILURE Table 2. ECs0 Values fr Effect f Isprterenl and Nrepinephrine Alne r With Ccaine r Desipramine ECs Values (/zml/liter) Study Cnditins NF NYHA II-III NYHA IV Isprterenl ( ) ( )* 0.06 ( )* NE 1.7 ( ) 0.50 ( )* 0.59( )* NE + ccaine 0.58 ( ) ( ) 1.1 ( ) NE + desipramine 0.51 ( )~ 0.27 ( ) 0.85 ( ) *p < 0.05 versus nnfailing rnycardium (NF). "~p < 0.05 versus nrepinephrine (NE). ECs values = cncentratin prducing half-maximal effects (range); NYHA = New Yrk Heart Assciatin functinal class (see Table 1). ditins. The results are summarized in Figure 2. As with nrepinephrine, the maximal effects f isprterenl were reduced in relatin t the severity f heart failure (Fig. 2, tp). As shwn in Figure 2, bttm, the effects f isprterenl were less ptent in failing than in nnfailing mycardium, als in relatin t the stage f the disease. Cnsistently, the ECs values were significantly higher in functinal classes II t III and IV mycardium than in nnfailing mycardium. In summary, ppsing results n the ptency t increase frce f Figure 2. Cncentratin-respnse curves fr the effect f isprterenl (isprenaline) ( t 1 #ml/liter) n ismetric frce f cntractin in islated electrically driven papillary muscle strips frm nnfailing dnr hearts (NF) and frm patients underging mitral valve replacement (New Yrk Heart Assciatin [NYHA] functinal classes II t III) r cardiac transplantatin (functinal class IV), Z 24,9._c 03 ~2 0 e NF (n=9) / NYHA I1-111 (n=12) f I NYHA IV (n=9) / i Cncentratin f isprenaline (/Jml/I) / cntractin were btained with isprterenl, which is nt a substrate fr uptake-1 (15,16), and the physilgically ccurring beta-adrenceptr agnist nrepinephrine, which is primarily inactivated by uptake-l, uptake int presynaptic stres (17). T further substantiate the evidence fr a reduced uptake-1 in failing mycardium, experiments with the uptake-1 inhibitrs desipramine and ccaine were perfrmed. Sme difference in the effect f these uptake-1 inhibitrs n the cncentratin-respnse curve f nrepinephrine in the different grups culd be expected if there was an alteratin in nrepinephrine carrier. Figure 3 shws cncentratin-respnse curves fr nrepinephrine alne and in the presence f desipramine (3 /zml/liter) r ccaine (3 /~ml/liter) in cardiac preparatins frm nnfailing mycardium. At 3/~ml/liter f desipramine r ccaine, frce f cntractin was reduced by -15% (nt shwn). Higher cncentratins were nt used because f the negative intrpic effects f the uptake-1 inhibitrs. Ccaine and desipramine shifted the cncentratinrespnse curve f nrcpinephrine t the left in nnfailing mycardium. The EC50 values were significantly reduced cmpared with thse fr nrepinephrine alne (Table 2). Figure 4 summarizes the data in functinal class IV mycardium. Neither desipramine nr ccaine affected the cncentratin- Figure 3. Cncentratin-respnse curves fr the effects f nrepinephrine (0.01 t 100/~ml/liter) alne and in the presence f ccaine (3 /~ml/liter) r desipramine (3 /zml/liter) n ismetric frce f cntractin in islated electrically driven papillary muscle strips frm nnfailin dnr hearts. 0 IO0 0 NF (n=9) t f ~ / / / - NYHA I,-II, (n=12) f / / "222/ t Nnfailing mycardium 50 ~~.E E /// ' 0.01 ' 0.1 i Cncentratin f isdrenaline (/Jml/I) ~ ~ s._~ E e- // / O Nrepinephrine (NE), (n=5) /.~ y ' / 7" NE in the presence f desipramine (3 uml/i), (n=4) ~" J NE in the presence f ccaine (3 ~mvi), (n=5) i i i,, Cncentratin f nrepinephrine ~ml/i)

5 150 BOHM ET AL. JACC Vl. 25, N. 1 NOREPINEPHRINE UPTAKE IN HEART FAILURE t t II1 A t m 900 c ~3 "0~,,~/'/ O Nrepinephrine <NE'), (n:6>._~ E ~ /'.L,/ NE in the presence f ~~~ /.~T I desiprarnine (3 uml/i), (n=4) NE in the presence f ccaine (3/zml/l), (n=5), Cncentratin f nrepinelhrine (pml/i) Figure 4. Cncentratin-respnse curves fr the effects f nrepinephrine (0.01 t 100/zml/liter) alne and in the presence f ccaine (3 /sml/liter) r desipramine (3 /~ml/liter) n ismetric frce f cntractin in islated electrically driven papillary muscle strips frm patients underging heart transplantatin (New Yrk Heart Assciatin [NYHA] functinal class IV). respnse curves in functinal class IV mycardium. The ECs values f nrepinephrine alne r in the presence f ccaine r desipramine in functinal class IV mycardium were nt significantly different frm thse in nnfailing mycardium in the presence f uptake-1 inhibitrs. Thus, uptake-1 inhibitin in nnfailing mycardium prduced a situatin similar t that in failing mycardium under cntrl cnditins. Nrepinephrine uptake carrier sites. T quantitate nrepinephrine uptake carrier sites directly, radiligand binding experiments were perfrmed in functinal classes II t III and IV mycardium and in nnfailing mycardium. Hydrgen-3 mazindl binding experiments were perfrmed as described elsewhere (18). Hydrgen-3 mazindl binding was strngly NaC1 dependent. Maximal specific binding was btained in the presence f 120 mml/liter f NaCI (nt shwn). Radiligand saturatin binding experiments (in the presence f 120 mml/ liter f NaC1) revealed cncentratin-dependent binding f the radiligand. Scatchard (19) analysis f the binding data shwed mnphasic binding with a specific binding f -30% at the dissciatin cnstant (K) (nt shwn). The density f nrepinephrine uptake carrier sites btained with these techniques is shwn in Figure 5. There was a reductin by -30% in uptake sites in functinal classes II t III and IV mycardium cmpared with nnfailing mycardium (p < 0.05). The abslute numbers and K values are summarized in Table 3. Discussin It is widely accepted that there is a general activatin f the sympathetic nervus system in heart failure (6-8,20), which results in increased circulating nrepinephrine levels at rest and during exercise (6-8) and crrelates with the prgnsis f patients with heart failure (6,8). In additin, Swedberg et al. (9) bserved an increased release f nrepinephrine frm the mycardium f patients with heart failure cmpared with 0 E 0 * p<o.05 Figure 5. Density f nrepinephrine uptake carrier sites in cardiac membranes frm the human left ventricle frm nnfailing (NF) dnr hearts and frm patients underging mitral valve replacement (New Yrk Heart Assciatin [NYHA] functinal classes II t III) and heart transplantatin (functinal class IV). Densities were investigated with radiligand saturatin experiments. See Methds. cntrl subjects. The latter finding culd result frm increased activity f mycardial sympathetic neurns r impaired uptake f nrepinephrine frm the synaptic cleft, r bth. Further evidence fr lcal alteratin f mycardial nrepinephrine (i.e., uptake, release and synthesis) has been reprted. There are reduced stres f mycardial nrepinephrine (18,21,22) and a reduced mycardial turnver, including defects in catechlamine synthesis (23,24) as well as reduced effects f the indirectly acting sympathmimetic drugs tyramine (25) and dpexamine (26). Cntrversy exists abut the invlvement f nrepinephrine uptake site mechanisms in heart failure. Rse et al. (13) reprted a reductin in the rate f nrepinephrine uptake and f nrepinephrine release with a multiple indicatr technique and reprted a reductin in nrepinephrine release frm the mycardium f patients with dilated cardimypathy. Hwever, Hasking et al. (12) prvided evidence fr unchanged nrepinephrine uptake as judged frm the kinetics f tritiated nrepinephrine cnstantly applied t the mycardium f patients with heart failure. In agreement with the latter reprt, Meredith et al. (11) reprted data n the cardiac utflw f the nrepinephrine precursr dihydrxyphenylalanine, the intraneural metablite f nrepinephrine dihydrxyphenylglycl and f tritium-labeled nrepinephrine. The latter reprt was in Table 3. Binding Characteristics f Hydrgen-3 Mazindl t Human Left Ventricular Membrane Preparatins Bmux (fml H-3 mazindl bund/mg prtein) K D (nml/liter) NF (n = 5) 1,102 _ ( ) NYHA li-iii (n = 5) 724 _+ 77* 36.4 ( ) NYHA IV (n = 9) 809 ± 52* 28.4 ( ) *p < 0.05 versus nnfailing mycardium (NF). Data presented are mean value +_ SEM r value (range). Bm~, = maximal number f binding sites; KD - dissciatin cnstant; NYHA = New Yrk Heart Assciatin functinal class (see Table I).

6 JACC Vl. 25, N. 1 BOHM ET AL. 151 NOREPINEPHRINE UPTAKE IN HEART FAILURE favr f an increased neurnal firing rate f sympathetic nerves in the presence f unchanged neurnal uptake. The discrepancies amng available studies are apparently influenced by the different techniques used and the prblem f differentiating between sympathetic activity and catechlamine uptake-1 in viv. Cardiac uptake-1 and cntractility. T vercme the prblems inherent in in viv studies, we investigated the effects f nrepinephrine cmpared with isprterenl, which is nt a substrate f uptake-l, uptake int presynaptic stres (15,16) in islated human mycardium in vitr. The efficiency f bth beta-adrenceptr agnists in increasing frce f cntractin was reduced in relatin t severity f heart failure. These findings were reprted earlier by several grups (1-3,27) and might be related t pstsynaptic alteratins in beta-adrenergic signal transductin invlving beta-adrenceptr dwnregulatin r uncupling (1-3,28) and increased levels f mycardial Gi-alpha (4-6). Hwever, the ppsite results were btained with the tw beta-adrenergic agnists when their ptency was cmpared. As expected, and as previusly reprted (1), the ptency f isprterenl was bserved t be reduced in failing mycardium. This culd be the result f beta-adrenceptr uncupling and dwn-regulatin (1,2,27,28). Hwever, the ptency f nrepinephrine t increase frce f cntractin was increased in failing human mycardium. This finding is cmpatible with the cncept that exgenusly applied nrepinephrine is nt inactivated because f a reduced capacity fr nrepinephrine uptake. If this were the case, ne wuld expect that the cncentratin-respnse curve f nrepinephrine wuld be affected differently by nrepinephrine uptake-1 inhibitrs, such as desipramine r ccaine. Indeed, ccaine and desipramine shifted the cncentratinrespnse curves f nrepinephrine t the left in nnfailing mycardium but did nt affect the effect f nrepinephrine in failing human mycardium. Thus, anther discrepancy exists between the in viv data reprted by Meredith et al. (11) and the in vitr data n islated mycardium reprted in the present study, sme f which culd be due t the fact that Meredith et al. (11) studied in viv nrepinephrine uptake in patients with mderate heart failure. Thus, nrepinephrine uptake in the whle heart, including the vasculature and the arteries, is determined by using the techniques f Meredith et al. (11). The experiments perfrmed in the present study exclusively address alteratins in nrepinepbrine uptake in left ventricular mycardium. Cnclusins drawn frm these data cannt be uncritically extraplated t ther parts f the heart, such as the arteries r vasculature. Therefre, the different results indirectly indicate evidence f differences in nrepinephrine uptake r even sympathetic innervatin f different parts r cmpnents f the failing human heart. Finally, Meredith et al. (11) studied patients with hypertensin r ischemic heart disease, r bth, at the stage f mderate heart failure. In the present study, we evaluated mycardium frm patients with terminal heart failure due t ischemic dilated cardimypathy as well as mycardial tissue frm patients with mitral valve replacement. Therefre, the underlying cardiac disease culd als have affected these differences. Nrepinephrine uptake carrier sites in islated mycardial membranes. T substantiate functinal findings with a bichemical technique, we labeled nrepinephrine uptake-1 carrier sites with the radiligand H-3 mazindl. This cmpund has been reprted t label catechlamine uptake carrier sites in the brain (18). In the present study, we bserved a significant reductin by -40% in H-3 mazindl binding sites in left ventricular particulate fractins frm failing hearts cmpared with fractins frm nnfailing hearts. In right heart failure in dgs caused by tricuspid valve avulsin, Liang et al. (29) reprted a significant reductin by -50% f H-3 mazindl binding sites in the right but nt left ventricle. The reductin f binding sites clsely was crrelated with the depressed nrepinephrine uptake activity in the same hearts. These investigatrs cncluded that lcal mechanisms culd be related t the impairment f nrepinephrine uptake cnfined t the right ventricle in their mdel. Nrepinephrine uptake inhibitrs failed t influence the cncentratin-respnse curve fr nrepinephrine, althugh the number f H-3 mazindl binding sites was reduced by 30%. Thus, a large prtin f uptake-1 carriers in the present study f human heart might nt participate in active nrepinephrine inactivatin. Study limitatins, A ptential limitatin f the radiligand binding experiments is the use f a high cncentratin f desipramine (100/zml/liter) t determine nnspecific binding in experiments characterizing nrepinephrine uptake carrier sites in human ventricular tissue. Thus it is pssible that desipramine displaces H-3 mazindl binding frm nnspecific binding sites. Hwever, we culd nt reprduce the data f Liang et al. (29), wh reprted numbers f H-3 mazindl binding sites similar t thse herein by using 0.3 ~ml/liter f desipramine. Thus, H-3 mazindl binding t human mycardial membranes is mre similar t binding sites in striatal membranes, where -30 t 100 /~ml/liter f desipramine is necessary t displace H-3 mazindl binding (18). Such difficulties in quantifying nrepinephrine uptake carrier sites by binding experiments emphasize the necessity f applying functinal as well as bichemical methds t investigate reduced nrepinephrine uptake-1 in failing mycardium. Evidence fr a reductin in nrepinephrine uptake effects has been bserved in the transplanted human heart. After transplantatin, degeneratin f the pstganglinic ventricular nerve terminal has been bserved in histlgic (30) and immunhistlgic (31) investigatins. Cnsistently, as in the failing human heart (21,22), nrepinephrine depletin ccurs in the transplanted heart (26,32). Failing mycardium is als similar t transplanted heart mycardium with regard t uptake mechanisms. In heart transplant recipients the effect f epinephrine n mycardial frce f cntractin was bserved t increase in viv, whereas the effect f isprterenl was unchanged (33). Desipramine treatment in healthy vlunteers enhanced the effect f epinephrine but was ineffective in heart transplant recipients (33). T ur knwledge these are the first

7 152 BOHM ET AL. JACC Vl. 25, N. 1 NOREPINEPHRINE UPTAKE IN HEART FAILURE Nnfailing mycardium ~ t i c clelt l cardiac muscle cell Failing mycardium /f~'~'l ATP camp,,- nrepinephrine nrepinephdneuptake carder site t reduce beta-adrenceptr numbers in dgs with an intact mycardial nrepinephrine uptake mechanism (34), but betaadrenceptr dwn-regulatin was bserved in dgs after cardiac denervatin (34), a cnditin that is knwn t impair mycardial nrepinephrine uptake (35). Thus, defective nrepinephrine uptake int presynaptic stres is likely t cntribute t adenylyl cyclase desensitizatin, which is regarded as an imprtant mechanism f mycardial dysfunctin in heart failure (Fig. 6). Cnclusins. Functinal and bichemical evidence indicate that nrepinephrine uptake is impaired in failing human hearts. This defect is likely t alter the prfile f the hemdynamic respnse t nrepinephrine in viv and might play a pathphysilgically imprtant rle in generating pstsynaptic alteratins, such as beta-adrenceptr dwn-regulatin and increased Gi-alpha levels, leading t adenylyl cyclase desensitizatin. % synaptic deft is t~* p-ar [lr] IN. ~,-.~ cardiac muscle cell / ~ ATP ~ Figure 6. Scheme f functinal defects in the failing human mycardium. Dwn-regulatin f cardiac beta-adrenceptrs (/3-AR) and an increase in inhibitry guanine binding prteins (Gcalpha) lead t a reduced frmatin f the intracellular secnd messenger cyclic adensine mnphsphate (camp). In additin t these pstsynaptic changes, there culd be a presynaptic defect in the sympathetic nerve system cnsisting f a reduced density f nrepinephrine uptake carrier sites. The cnsequence culd be an increase in the cncentratin f nrepinephrine in the synaptic cleft. AC = adenylyl cyclase; ATP = adensine triphsphate; M-Ch = m-chlinceptrs; /i (Ors) : Gi (G~)-prtein subunit alpha; /3 (7) = G-prtein subunit beta (gamma). findings t demnstrate the similarity f the failing and the transplanted human heart. Clinical implicatins. In patients with heart failure as well as in heart transplant recipients, there will be a higher rati f intrpic t vascnstrictr effects (i.e., a higher rati f betaadrenergic t alpha-adrenergic stimulatin). These findings indicate sme imprtant cnsequences in the initiatin f pstsynaptic adenylyl cyclase desensitizatin f the failing heart. A defective nrepinephrine uptake culd cntribute t high cncentratins f nrepinephrine in the synaptic cleft. In additin, elevated plasma nrepinephrine cncentratins cntribute t beta-adrenceptr dwn-regulatin even in situatins in which the release f nrepinephrine frm the sympathetic cardiac neurns is lw because cardiac nrepinephrine is nt inactivated by uptake-1. 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