Transintaral Heterogeneity of Nore Human Hearts

Transcription

1 JACC Vol. 23, No Trnsintrl Heterogeneity of Nore Humn Herts SCOTT L. BEAU, MD, JEFFREY E. SAFFITrZ, MD, PH Sint Louis, Missouri, FACC it, Uptke , - Objectives. The im of this study ws to determine whether regionl heterogeneity in myocrdil sympthetic neurl function mesured by the uptke of norepinephrine could ccount for the sptil heterogeneity of bet-drenergic receptor down-regultion tht occurs in the filing humn hert. Bckground. Myocrdil bet-drenergic receptor density nd function re diminished in ptients with chronic hert filure. Down-regultion occurs predominntly in the subendocrdium, suggesting tht locl rther thn systemic ltertions in sympthetic neurl function my be responsible. Although some studies hve implicted hypefunction of crdic sympthetic nerves with defective norepinephrine uptke, others suggest incresed crdic sympthetic nerve ctivity with unimpired uptke. Methods. We mesured norepinephrine uptke by incubting trnsmurl slices of the left ventricle from 19 ptients who hd chronic hert filure nd three nonfiling control herts with [3H]norepinephrine with or without desiprmine, neuronl uptke blocker. The density of uptke sites ws mesured in subepicrdil nd subendocrdil myocyte regions with light microscopic utordiogrphy. Results. Although the mount of [ -%norepinephrine uptke vried considerbly i n filing ventricles, uptke ws directly proportionl (r = 0.46, p < 0.05) to bet I -drenergic receptor density mesured in dditionl slices with rdiolignd binding ssys. In ddition, mrked hnsmurl heterogeneity in I,% norepinephrine uptke ws consistently observed in filing ventricles. Uptke in subendocrdil myocyte regions ws significntly less thn in subepicrdil regions (men I±SDJ subepicrdil/subendocrdil uptke rtio 4.7 ± 4.8, p < 0.01). The extent of trnsmurl heterogeneity in norepinephrine uptke ws similr in ptients with idiopthic nd ischemic crdlotnyopthy. In contrst, nonfiling herts exhibited more uniform trnsmurl ['H]norepinephrine uptke (subepicrdil/ subendocrdil uptke rtio 1.8 ± 1.2, p = NS). Conclusions. Specific [3H]norepinephrine ccumultion is pproximtely fivefold lower in subendocrdil regions of filing left ventricles thn in subepicrdil regions. These findings support the hypothesis tht subendocrdil defect in norepinephrine uptke my chroniclly elevte locl interstitil ctecholmine levels nd thereby down-regulte bet-drenergic receptors in sptilly heterogeneous distribution. (J Am Coll Cordil 1994, ) Myocrdil bet-drenergic receptor density nd function re diminished in ptients with chronic congestive hert filure (1-4). Severl lines of evidence suggest tht locl rther thn systemic fctors my be primrily responsible in mediting bet-receptor down-regultion in the filing hert (4-6). For exmple, decresed bet-drenergic receptor density nd drenergic neurotrnsmitter depletion hve been observed in the filing right ventricle but not in the norml left ventricle of ptients with primry pulmonry hypertension (6). Becuse bet-drenergic receptors re known to be From the Deprtments of Pthology nd Medicine, Wshington University School of Medicine, Sint Louis, Missouri. This study ws supported by Grnt HL-17646, SCOR in Coronry nd Vsculr Diseses from the Ntionl Hert, Lung, nd Blood Institute, Ntionl Institutes of Helth. Betbesd, Mrylnd. Dr. Beu ws supported by n Americn College of Crdiology Merck Adult Crdiology Reserch Fellowship Awrd, Dr. Sffitz ws supported by n Estblished Investigtor Awrd from the Americn Hert Assocition. Dlls, Texs. Mnuscript received April 12, 1993 ; revised mnuscript received August , ccepted October 14, Address for gousmildenc : Dr. Jeffrey E. Sffitz, Deprtment of Pthology, Box 8118, Wshington University School of Medicine, 660 South Euclid Avenue, Sint Louis, Missouri by the Americn College of Crdiology down-regulted in response to prolonged gonist exposure (7,8), elevtions in locl ctecholmine concentrtion re likely to be criticl determinnts of bet-drenergic receptor density nd function in the filing hert. Locl myocrdil neurotrnsmitter concentrtion is determined by severl fctors, one of the most importnt being uptke of norepinephrine into presynptic nerve terminls by the neuronl uptke mechnism (9,10). To elucidte potentil mechnisms tht might medite down-regultion of myocrdil bet-drenergic receptors, severl investigtors hve studied norepinephrine kinetics in ptients with hert filure, but the results hve been inconsistent. Swedberg et l. (11,12) reported n incresed ortocoronry sinus norepinephrine grdient in ptients with congestive hert filure. Some (13-15) hve concluded tht the cuse of incresed spillover of norepinephrine from the hert in ptients with congestive filure is incresed sympthetic neurl dischrge, wheres others (16,17) hve reported defect in ctecholtnine uptke. In previous utordiogrphic study of the distribution of bet-drenergic receptors in the filing humn hert, we (4) observed tht bet-receptor down-regultion occurs in /94/$7.00

2 580 BEAU AND SAFFITZ NOREPINEPHRINE UPTAKF IN HEART FAILURE JACC Vol. 23. No. 3 'tble 1. Clinicl Chrcteristics of Hert Trnsplnt Recipients Age (y) 45.3 ± 3.1 Rce White 16 Blck 3 Gender wle 17 Femle 2 Durtion of symptoms (mo) 32.7 ± 8.5 New York Hert Assocition functionl clss 2 III 6 IV Hemodynmic index Crdic output (liters/min) 4.93 ± 0.35 Crdic index (liters/min per m 2) Left ventriculr ejection frction (%) 22.2 IS Pulmonry cpillry wedge pressure (mm Hg) Vlues presented re men vlue t SE or number of ptients. trnsmurlly heterogeneous fshion nd is ttributble primrily to selective reduction of bet-receptors in the subendocrdium. This observtion suggests tht if locl derngements in ctecholmine function medite myocrdil bet-drenergic receptor down-regultion, then such derngements my be heterogeneously distributed. Accordingly, we developed n utordiogrphic method to chrcterize the trnsmurl distribution of norepinephrine uptke in left ventriculr slices of filing humn herts. With this pproch, we sought to determine whether potentil regionl ltertions in ctecholnine uptke might ccount for the mrked sptil heterogeneity of bet-receptor downregultion. Methods Tissue procurement. Becuse of the limited vilbility of fresh, vible humn myocrdium, preliminry experiments designed to chrcterize [ 3H]norepinephrine uptke conditions were performed using norml cnine left ventriculr myocrdium. Subsequent norepinephrine uptke studies were performed using freshly excised herts obtined from I I ptients with idiopthic dilted crdiomyopthy nd 8 ptients with ischemic crdiomyopthy who were undergoing hert trnsplnttion t Brnes Hospitl between Mrch 1991 nd October Selected clinicl fetures of these trnsplnt recipients re shown in Tble 1. Eight ptients were being treted with intrvenous ctecholmine therpy t the time of hert trnsplnttion nd ll ptients were tking other medictions including diuretic drugs, digoxin nd fterlod reducers (including ngiotensin-converting enzyme inhibitors). Freshly excised, trnsmurl smples of left ventriculr myocrdium were lso obtined from three nonfiling control herts. These smples cme from three herts not used for trnsplnttion, two from vitl orgn donors nd one from vlve homogrft donor. The donors included two motor vehicle ccident victims ( 38-yer old mn nd 3-yer old boy) with mssive closed hed injury nd 43-yer old womn with brinstem hernition fter cerebrovsculr ccident. All three subjects hd been treted with inotropic mediction within 24 h of tissue hrvest, but none hd clinicl history of congestive hert filure. Informed consent for reserch use of humn tissue ws obtined in ll cses. The study protocol ws pproved by the Wshington University Humn Studies Committee on October 28, Protocols involving niml tissue were pproved by the Wshington University Animl Studies Committee on November 26, [ 3H]Norepinephrine uptke ctivity. Uptke of [ 3 H]norepinephrine in myocrdil slices ws ssessed using procedures similr to those described by Shrm nd Bnerjee (18) nd Ling et l. (5). Thin (-0.5 mm), trnsmurl slices of fresh left ventriculr myocrdium were prepred with Stdie-Riggs microtome (Thoms Scientific) nd stored briefly in cold, oxygented, modified Krebs solution (contining in mmol/liter: sodium chloride 118, potssium chloride 4.7, clcium chloride 2.5, mgnesium chloride 0.54, sodium bicrbonte 25, sodium phosphte (monobsic) 1, nd dextrose 11, s well s 200 mg/liter of scorbic cid, 20 mg/liter of EDTA nd 20 mg/liter of prgyline). Totl ['H]norepinephrine uptke ws mesured in triplicte slices incubted t 37 C in modified Krebs solution contining 25 nmollliter of D.L-[7-3 H(N)]norepinephrine hydrochloride (8 to 15 Ci/mmol. New Englnd Nucler) in the presence or bsence of 0.3 limolaher of desiprmine, specific inhibitor of uptke function. In preliminry studies using cnine myocrdium, nonspecific uptke ws lso mesured by incubting tissue slices with [3 H]norepinephrine (without desiprmine) t 4 C. After incubtion, tissue slices were rinsed three times in ice-cold modified Krebs solution in the bsence of [3 H]norepinephrine or desiprmine nd blotted on filter pper. To determine totl specific [ 3 H]norepinephrine uptke per mg dry tissue weight, some slices incubted with ['H]norepinephrine with nd without desiprmine were desiccted overnight in n oven t 50 C, weighed, solubilized with 0.5 ml of Solvble (NEN DuPont) nd rdioctivity mesured with liquid scintilltion spectrometry. Preliminry experiments in multiple trnsmurl slices of left ventricle obtined from five cnine herts nd nlyzed in five seprte preprtions indicted tht specific uptke ws liner between 5 nd 30 min of incubtion nd represented -75% of the totl uptke whether nonspecific binding ws ssessed t 4 C or in the presence of desiprmine. In subsequent experiments using humn tissue, desiprmine ws used to ssess nonspecific binding. Rdiolignd binding ssys. The totl content of betdrenergic receptors in trnsmurl sections of left ventriculr myocrdium ws determined by Sctchrd nlysis of binding isotherms, s previously reported (19). Briefly, trnsmurl frozen sections of unfixed tissue, 12 lam thickness, were incubted under stedy stte conditions with

3 JACC Vol. 23, No. 3 Mrch 1, 1994 : BEAU AND SAFFITZ 581 selected concentrtions (1.1 to 107 pmol/liter) of (-)[ 1 '-5 1] iodocynopindolol (1,900 to 2,200 uci/mmol ; New Englnd Nucler) in the presence or bsence of 10' mol/liter of L-proprnolol. After rinsing to remove nonspecificlly bound lignd, sections were trnsferred to tubes nd rdioctivity ws quntified with gmm scintilltion spectrometry. The reltive proportions of bet,- nd bet 2 -drenergic receptors in trnsmurl sections of left ventriculr myocrdium of control nd filing herts were determined by incubting tissue sections with 25 pmol/liter iodocynopindolol in the imesencee or bsence of 10-7 molhiter CGP A, highly bet,-selective ntgonist. We (19) hve previously shown tht under these conditions, binding of iodocynopindolol to bet,-sites is inhibited by -95%, wheres binding to bet,-sites is inhibited by only -1%. Bet, -drenergic receptor density vlues in trnsmurl sections were clculted by multiplying the totl bet-receptor density, determined with binding isotherms, by the proportion of the bet, subtype, determined in sections incubted with rdiolignd nd CG020712A. The vlues were corrected to ccount for the 5% iodocynopindolol binding to bet, sites nd the 1% inhibition of iodocynopindolol binding to bet2 sites. Autordiogrphy. The distribution nd mount of [3 H]norepinephrine uptke in trnsmurl sections of filing nd control ventricles were determined with quntittive light microscopic utordiogrphy. Myocrdil slices incubted with [3 Hjnorepinephrine with or without desiprmine were frozen nd trnsmurl sections 12-Am thick were cut with cryostt microtome, mounted on geltin-coted slides nd ir dried. Geltin-coted coverslips were coted with Kodk NTB2 nucler trck emulsion (Estmn Kodk), dried t room temperture for L-3 h nd glued t one end to the slides contining the rdiolbeled myocrdil sections. The preprtions were stored in light-tight boxes t 4 C for pproximtely 6 weeks, fter which the unglued edge of ech coverslip ws gently lifted from the slides nd the emulsion ws developed with Kodk D19 developer (diluted 1 :1 with wter) for 3 min nd fixed with Kodk fixer for I min t 25 C. Tissue sections were then stined with hemtoxylin-eosin nd the coverslips were seled permnently to the slides. The tissue nd overlying utordiogrphic grins in the emulsion lyer were exmined by light microscopy nd photogrphed nd grin densities determined by counting grins per unit re of subepicrdium nd subendocrdium (defined s tissue regions included within five high power fields of the epicrdil nd endocrdil surfces, respectively, t mgnifiction of x630). Dt from individul sections were clculted s men vlues of grin density mesurements from >_10 distinct microscopic fields. Composite dt points were determined from the verge of t lest three seprte sections per ptient. [ 3 H]Norepinephrine relese experiments. Additionl tissue slices from three filing herts were incubted t 37 C for 15 min in oxygented, modified Krebs solution contining 25 nmol/liter of (3 HInorepinephrine with or without 0.3 jumol/liter of desiprmine. After three rinses in ice-cold modified Krebs solution, the tissue slices were blotted onn filter pper. Ech slice ws cut into three pproximtely equl portions, yielding subendocrdil, midmyocrdil nd subepicrdil smples. The subepicrdil nd subendocrdil smples were plced in seprte vils contining 8 ml of fresh, oxygented modified Krebs solution nd, fter incubtion t 37 C for 10 min, 80 gi of 10-2 mol/liter tyrmine ws dded to yield finl tyrmine concentrtion of 10' mol/liter, The mount of tyrmine-induced [3 H]norepinephrine relese ws mesured with liquid scintilltion spectrometry in liquots removed t selected intervls over the ensuing 15 min. Bckground relese of [3 Hjnorepinephrine ws determined by simultneously incubting dditionl epicrdil nd endocrdil smples without tyrmine. High performnce liquid chromtogrphic nlysis of tissue rdioctivity. The chemicl composition of tissue rdioctivity fter selected intervls of [3 H]norepinephrine uptke or relese ws nlyzed with liquid chromtogrphy, using sensitive electrochemicl detection method s previously described (20). Dt nlysis, Results re expressed s men vlue ± SD. Sttisticl nlyses were performed on Mcintosh l1ci computer using the Stt View 4.0 ppliction pckge. Differences in totl [ 3 Hjnorepinephrine uptke between groups were nlyzed using one-wy nlysis of vrince (ANOVA) with the Scheffd test. Anlysis of the difference in subepicrdil nd subendocrdil uptke nd relese within individul groups ws performed using the difference method nd testing the significnce of the differences between the men subendocrdil nd men subepicrdil uptke or relese. The reltion between [ 3 Hjnorepinephrine uptke nd bet,-receptor density ws nlyzed using the Fisher r to z trnsformtion of the correltion. Significnce ws defined s p < Results Totl [3 H]norepinephrine uptke in control nd filing humn myocrdium. When group dt were compred, no significnt difference ws observed in the men totl specific uptke in slices of filing nd control left ventricles (specific [3 Hjnorepinephrine uptke ± 17.0 fmol/mg dry tissue weight in nonfiling control ventricles vs ± 50.6 fmol/mg dry tissue weight in filing herts) nor ws difference in uptke observed between control ventricles nd subsets of filing ventricles ctegorized on the bsis of etiology (Fig. 1). Specific [ 3 Hjnorepinephrine uptke in ptients receiving intrvenous dobutmine therpy before hert trnsplnttion ws not significntly different from tht observed in herts of ptients not treted with dobutmine. The mount of totl specific [3 H]norepinephrine uptke vried considerbly mong smples of filing left ventriculr myocrdium, with vlues rnging from 3.8 to fmol/mg dry tissue weight. However, s shown in Figure 2, the totl mount of specific [3 HInorepinephrine uptke ws directly

4 582 BEAU AND SAFFITZ JACC Vol. 23, No. 3 Mrch I : I 10. [ F we IC +DOWT -MWT Flpre 1. Specific [ 3 fflnorepinephrine ([3H]NE) uptke in control nd filing humn left ventriculr myocrdium. Triplicte trnsmurt slices of ech ventricle were incub,, tej with 25 nmol/liter of (IHInorepinephrine in the presence or bsence of 0.3 pmouliter of desiprmine for 15 min. Specific uptke ws clculted by subtrcting nonspecific from totl uptke in ech slice. Grouped dt re expressed s men vlue ± SD for control nonfiling (NF, n = 3), filing (F. n = 19), idiopthic crdiomyopthy (IDC, n = 11) ischemic crdiomyopthy (ISC, n = 8). dobutmine-treted (+ DOBUT, n - 8) nd dobutmine-untreted (- DOBUT, n = 11) herts. proportionl to the bet 1 -drenergic receptor content of the tissue, determined in seprte trnsmurl slices with rdiolignd binding ssys (r = 0.46, p < 0.05). Tromurl dwbution of PHInorepinephrine uptke in flling nd control left ventricles. Techniclly cceptble utordiogrphic preprtions were vilble for nlysis in ll 3 nonfiling control nd It filing left ventricles. Figure 3 shows representtive utordiogrphs prepred from the YweL Reltion between specific Ninorepinephrine WHINE) uptke nd bet,-drenergic receptor density in trnsmuri slices of filing left ventricles SPECIFIC [3H]NE UPTAKE (frrl/nig dry weight) hert of ptient with idiopthic dilted crdiomyopthy. It demonstrtes tht the mount of specific I'Hlnorepinephrine uptke ws considerbly less in regions of closely pcked myocytes in the subendocrdium thn in myocyte regions of the subepicrdium. Figure 4 summrizes the utordiogrphic mesurements obtined in the subepicrdil nd subendocrdil myocyte regions of the 3 nonfiling control nd 16 filing left ventricles. Mrked trnsmurl heterogeneity in 13 Hlnorepinephrine uptke ws seen in the filing ventricles. Specific [3 H]norepinephrine uptke ws significntly lower in subendocrdil myocvte regions thn in subepicrdil myocyte regions in the hert filure group (subepicrdil :subendocrdil uptke rtio 4.7 ± 4.8, p < 0.01). The mgnitude of the subepicrdil/subendocrdil uptke rtio ws similr in both the idiopthic dilted (4.6 ± 4.5) nd ischemic (5.0 ± 5.3) crdiomyopthy subgroups. In contrst, the trnsmurl distribution of specific uptke in the nonfiling control herts ws substntilly more uniform. There ws no significnt difference between the mount of subepicrdil nd subendocrdil uptke in these herts. Spontneous nd tyrndwinduced relese of [3 H]norepi. nephrine In filing myocrdium. To determine whether the differences in mounts of subepicrdil nd subendocrdil rdioctivity mesured fter 15-min incubtion intervl could be due to regionl differences in [ 3H]norepinephrine relese, we mesured spontneous nd tyrmine-induced ['Hlnorepinephrine relese in subepicrdil nd subendocrdil smples prepred from three filing humn left ventricles. Figure 5 shows the mount of [3 H]norepinephrine relesed from subepicrdil nd subendocrdil smples incubted in the presence or bsence of tyrmine. No significnt regionl differences were observed in the mount of (3111norepinephrine relesed spontneously (without tyrmine) or in response to tyrmine fter 15 min of incubtion. FlIgh performnce liquid chromtogrphic determintion of norepinephrine metbolich To determine whether [3 HInorepinephrine ws being metbolized under the experimentl conditions employed, the chemicl composition of tissue rdioctivity in three herts ws nlyzed with high performnce liquid chromtogrphy. After incubtion of slices with [3 H]norepinephrine for 15 min, norepinephrine ws extrcted from tissue nd processed for nlysis with high performnce liquid chromtogrphy. Elunt ws collected in 1-min frctions, nd rdioctivity in ech frction mesured by liquid scintilltion spectrometry ws compred with totl rdioctivity mesured in n liquot of smple not subjected to high performnce liquid chromtogrphy. Approximtely 90% of tissue rdioctivity tht hd ccumulted fter 15 min of incubtion eluted in single frction with retention time (3 to 4 min) chrcteristic of unmetbolized norepinephrine. Similr results (91% elution in norepinephrine frction) were obtined for tissue slices from tyrmine relese experiments tht hd been incubted t 3'7*C in oxygented buffer for 45 min.

5 JACC Vol. 23, No. 3 BEAU AND SAFFITZ 413 Figure 3. Representtive utordiogrphs showing reltive grin densities in myocyte regions of filing humn left ventriculr slices incubted with 25 nmoll liter of ['H]norepinephrine in the bsence (left pnels) or presence (right pnels) of 0.3 pmol/liter of desiprmine. Top, Subepicrdil myocyte regions. Bottom, Subendocrdil myocyte regions. Mgnifiction is the sme in ll pnels. Originl mgnifiction x400 (br = 25 pm). Discussion Ptients with chronic congestive hert filure re known to hve depleted crdic norepinephrine stores (21,22) de- Figure 4. Rtios of reltive specific grin densities in subepicrdil nd subendocrdil myocyte regions in control nd filing ventricles. At lest 10 grin density mesurements were mde in ech loction in triplicte smples from ech ventricle. Grouped dt re expressed s men vlue ± SD for the rtio of subepicrdil/ subendocrdil ['H]norepinephrine ([3H]NE) uptke in control non' filing (NF, n = 3), filing (F, n = 16), idiopthic crdiomyopthy (IDC, n = 9), ischemic crdiomyopthy (ISC, n = 7), dobutminetreted (+ DOBUT, n = 7) nd dobutmine-untreted (- DOBUT, n = 9) herts *p < tp < M 0 Z Z 'U" -2 U. 0 Q = 4 Loul 6 3 CO 2 W 0 t NF F IDC Isc +DOBLFF -DOBUT spite incresed circulting ctecholmine levels (23-26). Furthermore, myocrdil interstitil concentrtions of norepinephrine pper to be incresed in ptients with hert filure (12,16). The norml hert extrcts lrge percent of perfused norepinephrine by uptke into sympthetic nerve terminls (9,10), nd bnormlities in this uptke mechnism in ptients with chronic hert filure hve been implicted s mjor cuse of ltertions in crdic norepinephrine processing (16,17). However, some investigtors (13-15) hve suggested tht primry increse in crdic sympthetic nerve ctivity is the underlying defect tht promotes derngements in crdic norepinephrine spillover in ptients with hert filure. [3 H]Norepinephrine uptke nd bet,-2drenergic receptor density. In the present study, the mount of specific ['H]norepinephrine uptke ws highly vrible mong slices prepred from nonfiling control herts nd filing herts. The totl mount of rdioctivity mesured in vitro fter incubtion of tissue slices in solution contining [3 H]norepinephrine depends on mny vribles, including reltive rtes of uptke nd relese, the sizes nd metbolic turnover rtes of endogenous norepinephrine pools nd, probbly most importnt, the reltive distribution of lrge nerves nd richly innervted vessels within individul tissue slices. Despite these technicl limittions, we did observe direct reltion between the mount of totl specific [3 H]norepinephrine ccumultion nd totl tissue bet, -drenergic : receptor density in slices of filing myocrdium. The gretest

6 584 BEAU AND SAFFITZ JACC Vol. 23, No. 3 Mrch 1, 1994: SPONTANEOUS TYRAMINE-INDUCED Figure 5. Spontneous nd tyrmine-induced ['H]norepinephrine ([SHINE) relese in filing ventricles (n = 3). Triplicte slices of myocrdium were incubted for 15 min with 25 nmolner of ['H]norepinephrinc. Subepicrdil (htched brs) nd subendocrdil (WM brs) regions were then trnsferred to fresh buffer nd incubted with or without 10' mol1liter of tyrmine for n dditionl 15 min to promote relese. Grouped dt re expressed s men vlue ± SD for [3H]norepinephrine relese s percent of totl ['H]norepinephrine ccumultion. (Totl ['Hlnorepinephrine ccumultion ws clculted by dding the totl mount of ['H]norepinephrine relesed to the mount of ['H]norepinephrine remining in the tissue t the end of the relese intervl.) mount of receptor down-regultion (ttit is, the lowest bet,-receptor density) ws ssocited with the lowest ccumultion of [3 H]norepinephrine. Sttisticl nlysis indicted tht only -21% of the vrince in the bet-receptor dt could be ttributed to differences in ['H]norepinephrine uptke. However, becuse of the difficulties involved in compring the totl mount of ['HJnorepinephrine ccumultion in whole slices of control nd filing herts, we devised novel utordiogrphic method to compre regionl ccumultion of [3 H]norepinephrine within regions of compct myocytes in trnsmurl left ventriculr slices. Autordiogrphic delinetion of regionl PHInorepinephrine uptke. Specific uptke of [3 H]norepinephrine ws mesured utordiogrphiclly under conditions in which trcer uptke ws liner, inhibitble by desiprmine nd not subject to significnt net ctbolism. This pproch ws 'csed on our previous observtions (4) tht bet-drenergic receptors re down-regulted in the filing hert in sptilly heterogeneous fshion, such tht the degree of downregultion is significntly more extensive in subendocrdil thn in subepicrdil regions. Our observtions in the present study indicte tht specific ['H]norepinephrine ccumultion ws fivefold lower in subendocrdil myocyte regions of filing left ventricles thn in subepicrdil myocyte regions. Although the trnsmurl distribution of specific [ 3H]norepinephrine uptke ws more uniform in the nonfiling control herts, the number of control herts ws smll becuse of unvoidble limittions in the vilbility of vible, fresh, norml humn myocrdium. However, the more uniform trnsmurl pttern of norepinephrine uptke is consistent with the distribution of bet-receptors in norml myocrdium, which is lso uniform (4,27). Limittions of the study. Potentil confounding vribles in our study include tretment of ptients with hert filure with intrvenous dobutmine nd digoxin, medictions known to interct with components of the drenergic nervous system. However, no differences in totl or regionl ccumultion of [3 H]norepinephrine uptke were detected in ptients with hert filure who were nd were not treted with intrvenous ctecholmines before hert trnsplnttion. Furthermore, ll control herts hd lso been exposed to intrvenous ctecholmines within 24 h of tissue hrvest. In ddition, lthough ptients with hert filure hd been receiving tretment with digoxin, digitlis glycosides hve been shown to only wekly inhibit neuronl uptke of [ 3H]norepinephrine in vitro in humn tril muscle t concentrtions fr exceeding therpeutic levels (28). Tissue edem nd fibrosis might retrd in vitro [ 3 H]norepinephrine uptke in myocrdil tissue slices from ptients with congestive hert filure. However, we selected regions free of grossly visible fibrosis for determintion of uptke cpcity, nd grin density mesurements were performed in microscopic fields composed of closely pcked myocytes tht ppered to be uninvolved by edem or fibrosis. Brber et l. (29) hve shown tht vible regions picl to myocrdil infrcts my be denervted. Regionl sympthetic denervtion my be n importnt determinnt of sptil heterogeneity in norepinephrine uptke ctivity in herts of ptients with ischemic crdiomyopthy. However, in the present studies, tissues were smpled from regions distnt from res of scrring nd not locted picl to heled infrcts. Thus, the effects of regionl sympthetic denervtion on heterogeneity in norepinephrine uptke in ischemic crdiomyopthy smples ws probbly limited. Our results do not indicte whether potentil subendocrdil defect in norepinephrine uptke in hert filure precedes nd promotes desensitiztion nd down-regultion of myocrdil bet-receptors. Perhps fctors promoting myocrdil overlod sttes or direct myocyte injury re responsible for both the decrese in neuronl uptke of norepinephrine nd the diminution of bet-receptor density nd responsiveness. Recent dvnces in the noninvsive ssessment of myocrdil drenergic function, using [1231 ] _ met-iodobenzylgunidine scintigrphy to evlute uptke- I function (30) nd positron emission tomogrphy to mesure myocrdil bet-drenergic receptor density (31), my eventully help resolve this question. The mechnism underlying the observed reduction in subendocrdil norepinephrine uptke ctivity hs not been ddressed in these experiments. Further studies to evlute the distribution of sympthetic nerve terminl density nd specific neuronl uptke binding sites seem wrrnted.

7 JACC Vol. 23, No. 3 BEAU AND SAFFITZ 585 Clinicl implictions. Our results my hve implictions regrding the pthogenesis of mlignnt ventriculr tchyrrhythmis in ptients with hert filure. Regionl heterogeneity of electrophysiologic function hs been well documented in models of hert filure (32,33). The trnsmurl grdient in norepinephrine uptke function chrcterized in our study my potentite nd mplify such electricl heterogeneity, thus incresing the likelihood of developing reentrnt circuits. Loclly elevted levels of interstitil ctecholmines my lso potentite triggered ctivity, especilly in light of evidence tht ctecholmines result in incresed delyed fterdepolriztions in certin preprtions (34). Evlution of filing herts with multichnnel electrophysiologic mpping devices might further elucidte these potentil consequences of sptilly heterogeneously ltered sympthetic ctivity. We thnk Timothy Tolley nd Suresh D. Shh for technicl ssistnce ; Louise Schoelch nd Wlter Clermont for photogrphic ssistnce ; Cindy Behrends, RN, Peggy Emmert, RN, Crol Cross, RN nd Lurie Schulte for ssistnce with humn tissue procurement ; nd Susn Johnson for preprtion of the mnuscript. References I. Bristow MR, Ginsburg R. Minobe W, et l. Decresed ctecholmine sensitivity nd A-drenergic-receptor density in filing humn herts. N Engl J Med 1982 ;307 : Bristow MR, Ginsburg R, Umns V, et l. P,- nd )3 -drenergic receptor subpopultions in nonfiling nd filing humn ventriculr myocrdium : coupling of both receptor subtypes to muscle contrction nd selective 0,-receptor down-regultion in hert filure. Circ Res 1986 ;59: Dennis AR, Mrsh JD, Quigg RJ, Gordon JB. Colucci WS. p-drenergic receptor number nd denylte cyclse function in denervted trnsplnted nd crdiomyopthic humn herts. Circultion 1989 ;79: Murphree SS, Sffitz JE. Distribution of P-drenergic receptors in filing humn myocrdium : implictions for mechnisms of down-regultion. 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