367

Antibodies to Beta-Adrenergic Receptors DisclosingAgonist-Like Properties in Idiopathic DilatedCardiomyopathy and Chagas' Heart Disease

MAURICIO B. ROSENBAUM, M.D., PABLO A. CHIALE, M.D.,DALIA SCHEJTMAN, M.D., MARIANO LEVIN, PH.D.,

and MARCELO V. ELIZARI, M.D.

From the Service of Cardiology, Ramos Mejia Hospital, and the Instituto de Investigacionesen Ingenieria Genetica y Biologia Molecular (INGEBI), Buenos Aires, Argentina

Antibodies to Beta-Adrenergic Receptors. Recent studies confirm the existence of anti-bodies (Abs) to j0-adrenoceptors in patients with idiopathic dilated cardiomyopathy and Cha-gas' heart disease. These Abs can be shown to exert both stimulatory and inhibitory effects,which may play a role in the development of the cardiac abnormalities known to occur in thesediseases, including advanced heart failure. The hypothesis is advanced that Chagas' heart dis-ease and some forms of idiopathic dilated cardiomyopathy may represent, at least partially,a form of "adrenergic cardiomyopathy." (J Cardiovasc Electrophysiol, Vol. 5, pp. 367-375,April 1994)

sympathetic nervous system, congestive heart failure, adrenergic cardiomyopathy

Introduction

Analogous or equivalent antibodies (Abs) thatreact with cardiac tissues occur in variable num-bers of patients with idiopathic dilated cardiomy-opathy'-'* and Chagas' heart disease,''* suggestingan immunological link between these two diseases.Common targets for these Abs include sar-colemma,'-^"' myosin,39.'7 actin,^-' laminin,'''^'-tand tubulin.*' However, no pathogenic role hasbeen demonstrated for any of these Abs, their bio-logical significance is unclear, the diagnostic valueis limited, and identification of the antigenic deter-minants at the molecular level is lacking. Abs thatpresently look to be more promising candidates toserve as pathogenic or diagnostic markers are theAbs to: tbe ADP/ATP carrier protein of the inner

This work was supported in part by the UNDPAVorld BankAVHOSpecial Programme for Research and Training in Tropieal Dis-eases (Grant 900253) and the Fundacion de Investigaciones Cardi-ologicas Einthoven.

Address for correspondence: Mauricio B. Rosenbaum, M.D., Ser-vice of Cardiology, Ramos Mejia Hospital, Urquiza 609, FirstFloor, 1221 Buenos Aires, Argentina. Fax: 54-1-326-0752.

Manuscript received 13 April 1993; Accepted for publication 12November 1993.

mitochondrial membrane'*-2o in idiopathic dilatedcardiomyopathy; tbe R-13 synthetic peptide orautoepitope of Trypanosoma cruzi and human ribo-some P protein in Chagas' heart disease2i22; andthe /3-adrenergic receptors in both.23-27 Only thelast is discussed in tbis article.

Tbere is a group of autoimmune diseases inwhicb Abs directed against hormonal receptorsplay a major role. Myastbenia gravis, '* Graves'disease,29 and insulin-resistant diabetes^" are well-documented examples. It is remarkable that in sucbconditions the Abs, ratber tban causing directcell damage, exert functional effects tbat are path-ogenic by interacting as agonists or antagonistswitb the respective receptors. There is some evi-dence23-27 that the Abs to /3-adrenergic receptorsoccurring in patients with idiopatbic dilated car-diomyopathy and Cbagas' heart disease may alsoexert stimulatory and inhibitory actions that maybe pathogenic. Chagas' beart disease is related toan infection by T. cruzi.^^ In contrast, the etiol-ogy of idiopathic dilated cardiomyopatby isunknown, although many cases can be traced backto an initial viral infection.232-38 xhat both sbouldresult in Abs sharing tbe same or closely relatedepitope within the primary structure of tbe /3-recep-

368 Journal of Cardiovascular Electrophysiology Vol. 5, No. 4, April 1994

tors (see below) is a biological mystery. In thisarticle we discuss principally the potentiallypathogenic role of these Abs in Chagas' diseaseand idiopathic dilated cardiomyopathy.

Abs to /3-Adrenergic Receptors in IdiopathicDilated Cardiomyopathy and

Chagas' Heart Disease

Synthetic peptides derived from the predictedsequences of the human ;0,-(amino acids 183-208)and ^2-(aniino acids 172-197) adrenergic recep-tors have been used as antigens to screen for Abs. ^Abs directed against the /3|-peptide were shownto occur in 13 of 42 (31%) patients with idiopathicdilated cardiomyopathy, in 4 of 34 (12%) healthyblood donors, and in none of 17 patients withischemic cardiomyopathy. The affinity-purified Abswere shown to recognize the /3,-receptor proteinby immunoblot, and to bind in situ to human myocar-dial tissue. The occurrence of Abs against the yS,-adrenoceptors in cases of idiopathic dilated car-diomyopathy had previously been suggested,2''25,39-4ialthough based on less direct screening methods,and in a series of studies dating back to 1976,Sterin-Borda et al.23''2-44 j ad reported the presenceof Abs to the ;5-adrenergic receptors (both /3[and /?2) in the sera and IgG from patients withChagas' disease.

Table 1 shows preliminary results from an ongo-ing study (in cooperation with Dr. Johan Hoebekefrom Tours, France, and Dr. Ivonne Magnussonfrom Goteborg, Sweden), in which sera from

TABLE 1Prevalence of Abs to /?,- and yJj-Peptides in Patients withChagas' Disease, Idiopathic Dilated Cardiomyopathy, and

Idiopathic Arrhythmias and Conduction Disturbances

Patient Group(n)

Chagas' diseasen = 67

Idiopathic dilatedcardiomyopathyn = 29

Idiopathicarrhythmias andconductiondisturbancesn = 57

Healthy controlsn = 68

Cardiopathiccontrols n = 32

PrPeptlde

14(20.9%)

7(24.1%)

10(17.5%)

5(7.3%)

1• (3.1%)

Pi-Peptide

7(10.4%)

0

2(3.5%)

3(4.4%)

0

Pr+Pi-Peptides

10(14.9%)

2(6.9%)

6(10.5%)

3(4.4%)

1(3.1%)

TotalPrevalence

31(46.3%)

9(31%)

18(31.5%)

11(16.2%)

2(6.2%)

Abs = antibodies. Cardiomyopathic controls included:coronary artery disease, 4 cases; hypertrophic cardio-myopathy, 5 cases; valvular heart disease, 2 cases; hyper-tensive heart disease, 1 case.

patients with various clinical forms of idiopathicdilated cardiomyopathy, Chagas' disease, and idio-pathic cardiac arrhythmias and conduction distur-bances (without any other clinical evidence of car-diac involvement) are being tested for Abs to the/91-peptides and /Sj-peptides. It should be notedthat half the cases of idiopathic dilated cardiomy-opathy were mildly dilated forms '*' or in the earlystages."*'*'' These results confirm that Abs to /?,-adrenoceptors and /Sj-^dr^noreceptors aresignificantly prevalent in patients with idiopathicdilated cardiomyopathy and Chagas' disease. Itis remarkable that patients with idiopathic atrialand/or ventricular arrhythmias and/or intraven-tricuiar or atrioventricular block exhibited a sim-ilar prevalence of Abs to that occurring in idio-pathic dilated cardiomyopathy. This suggests thatsome of these cardiac abnormalities may bepathogenically related to, or eventually be a har-binger of, the dilated cardiomyopathy.

Functional Properties of the Abs Interactingwith /9-Adrenergic Receptors

Stimulatory as well as inhibitory adrenergiceffects can be shown to occur, depending on theexperimental setup. When spontaneously beatingcardiomyocytes (cultured neonatal rat heart cells)were incubated with a low concentration, of affinity-purified anti-/3| Abs obtained from rabbits immu-nized with the /0|-pepdde2'''*^ or from patients withidiopathic dilated cardiomyopathy,"^ a significantand rapid dose-dependent increase in beating ratewas observed. This stimulatioh was significant at0.05 nM and reached a maximum of 80% of themaximal isoproterenol stimulation at 0.25 nM. Theeffect was blocked by the selective /S,-blockingdrug bisoprolol, but not by the selective /Jj-block-ing drug ICI118,151, and was inhibited by prein-cubation of the Abs with the /?,-peptide. Nostudies on the inotropic effects of Abs from patientswith idiopathic dilated cardiomyopathy have beenreported, but both chronotropic and inotropicresponses were shown to occur when testingchagasic sera or IgG. In isolated rat atrial prepa-rations, chagasic sera caused a significant sinoa-trial acceleration,''2 as well as a positive inotropiceffect^ measured as an IgG concentration-depen-dent increase in contracdle tension. Furthermore,when guinea pig heart membranes were incubatedwith increasing concentrations of chagasic IgG,adenylate cyclase activity increased in a dose-dependent manner* ; and when chagasic IgG wasincubated with either the rat atrial preparation"^ or

Ro.ienbaum, et al. Antibodies to Beta-Adrenergic Receptors 369

rat heart cell homogenates, ^ there was a significantincrease of cAMP intracellular levels, which peakedat 2 minutes and was similar to the one inducedby isoproterenol."*' A similar response was seenwhen chagasic IgG was incubated with spleen cellsrich in /32-adrenoceptors.23 Collectively, these datasuggest that the Abs against /3-receptors occurringin patients with idiopathic dilated cardiomyopathyand Chagas' disease fulfill criteria'" for admissioninto the family of /3-receptor agonists or agonist-like substances. Binding to the receptor is rapidand of relatively high affinity; occupancy of thereceptor leads to the generation of intracellular sec-ond messenger and culminates in a physiologicalresponse. There are, however, differences from theeffects of neurohormonal agonists, as discussedbelow.

Adrenergic stimulation via the /^-receptors issubject to regulation by a process termed desen-sitization.5"" Occupancy of/3-adrenergic receptorby agonist triggers a variety of events, both rapidand delayed.'' Initially, there is a functional uncou-pling of ^-receptors from adenylate cyclase.'^ssThis stage occurs in seconds or minutes and is notassociated with a change in the total number of /3-receptors. More prolonged agonist exposure (hoursor days) leads to an absolute decrease in the num-ber of /3-adrenoceptors, frequently referred to asdownregulation,52-53 and is likewise associated withsubsensidvity to /5-adrenoceptor stimulated effects.In light of these concepts, the question iswhether the Abs against /3-receptors may, like other/5-adrenergic agonists, result in uncoupling anddownreguladon. The increase and rapid fall in intra-cellular levels of cAMP induced by chagasic IgG^suggested that desensidzation starts very early, butthis is not supported by recent studies in whichthe increase in beating frequency induced by and-/5i Abs,(from padents with dilated cardiomyopa-thy), unlike the one caused by isoproterenol,remained unchanged over a period of 6 hours."*Furthermore, this sustained chronotropic effect wasnot reversed by washing and subsequent incuba-tion for some hours in fresh culture medium nolonger containing the Abs, although it was rapidlyabolished by the specific /(3|-blocking agent biso-prolol. These new and surprising observadons ledto the idea that the conformadonal change inducedby the Abs, while causing a similar chronotropiceffect, differs from the one induced by neurohor-monal agonists in that it does not trigger the /?-adrenergic protein kinase that is responsible forthe phosphorylation of the agonist-occupied recep-tor and its subsequent rapid uncoupling.''* This lack

of short-term desensidzation may reinforce andsustain the adrenergic sdmulation, with obviousphysiological and pathogenic connotadons.

It has been suggested that the adrenergic stim-uladon induced by the Abs might also prevent theoccurrence of downreguladon."*" However, whenand-/0| Abs,2627 or sera and/or IgG from padentswith dilated cardiomyopathy^"* and Chagas' dis-ease,23.'t3.44 were incubated with cell membranesrich in ;5|-receptors23-27 using ^H-dihydroalprenololor '^'iodocyanopindolol as ligands, there was asignificant decrease in the number of radioligandbinding sites without change in the dissociationconstants. This was associated with a significantinhibition of isoproterenol-stimulated adenylatecyclase acdvity.23.54.55 Y^Q^ \^ Q^^ ^Q reconcile thisinhibitory effect apparendy resulting from down-regulation'5 with the sustained adrenergic sdmu-ladon induced by the Abs on cardiac myocytes?When membranes of C6 glioma cells rich in (3^-receptors were incubated with the Abs^'' in thepresence of the nonhydrolyzable GTP analog 5'-guanylyl imidodiphosphate (which completelyuncouples the receptor from the GTP regulatoryprotein GJ, the inhibidon of the ligand bindingwas unaffected, suggesting that G plays no essen-tial role in this process. This was confirmed inanother experiments^ using inner membranes ofEscherichia coli expressing human /?,-adrenergicreceptors but without acdvadon of the adenylatecyclase. The inhibidon of the ligand binding againwas unchanged. This is consistent with other stud-ies suggesdng that "there appears to be both cAMP-dependent and cAMP-independent mechanismsthat lead to downreguladon."'053 j ^ jg t^en possi-ble that the inhibidon of ligand binding caused bythe Abs may be, unlike the one induced by neu-rohormonal agonists, cAMP-independent. Furdierevidence that the downreguladon caused by theAbs is mechanistically different from the oneinduced by neurohormonal agonists has beenreported.'' The available evidence seems to sug-gest that the Abs against /3-receptors occurring inpadents with idiopathic dilated cardiomyopathy(and Chagas' heart disease) may cause a form ofdownreguladon that may eventually result in rel-evant subsensidvity of ;5-adrenergic acdvity, andpardcipate in the documented decline of/3-adreno-ceptors in the failing myocardium of such padents.

The Problem of Heart Failure

Idiopathic dilated cardiomyopathy and Cha-gas' heart disease often result in a globally dilated

370 Journal of Cardiovascular Electrophysiology Vol. 5, No. 4, April 1994

heart and congestive heart failure. Heart failureis known to activate "homeostatically" the sym-pathetic nervous system and to.be associated withhigh levels of circulating catecholamines.^* Thisin turn prompts the occurrence of downregula-tion of the /3-adrenoceptors, associated with adiminished contractile response to /3-adrenergicagonists."-^' More specifically, there is a selec-tive downregulation of the /0,-receptors, whereasthe ;02"rsceptors are only marginally or not atall affected.fio However, other studies^'«' suggestthat the inhibition of sympathetic responsesmay be associated with a more global abnormalityof the entire /S-adrenergic receptor system. Thissystem consists of three functionally distinct,membrane-bound proteins: (a) the receptor itself;(b) various signal-transducing and regulatory pro-teins termed "G proteins," which may either stim-ulate Gs or inhibit Q; and (c) the catalytic adeny-late cyclase subunit, which synthesizes theintracellular second messenger cAMR'^-^^^'^^Studies on membranes prepared from failinghuman ventricular myocardium show that, in addi-tion to downregulation, there is an approximate30% increase in Gj activity.*'" Agonist occu-pancy of receptors coupled to Gj may cause inhi-bition of contraction, and also result in what hasbeen referred to as "uncoupling of /02-receptors,"implying a diminished functional activity with-out any loss in the number of /Sj-receptors.'^^All these changes have the potential for thegeneration of inhibitory effects on the responsesto adrenergic stimulation, and are probably causedby chronic exposure to increased amounts of cat-echolamines.*^

Are these changes similar in aU cases of humanheart failure? They probably are, but differencesare likely to occur, depending on the degree ofheart failure" and, particularly, on the underlyingpathophysiology. For example, when patients withidiopathic dilated cardiomyopathy and Chagas'heart disease reach the stage of congestive heartfailure, they may have, in addition to increasedlevels of catecholamines of neurohormonal ori-gin, a second population of /3-adrenergic agonistsconsisting of circulating Abs. Additive effects ofthese two different populations of agonists actingon cardiac /S-adrenoceptors would be expected toresult in an "enhancement of downregulation."However, the most relevant studies on the changesinduced by heart failure on the /3-adrenergic sys-tem relied heavily on very advanced cases(explanted hearts) of idiopathic dilated car-diomyopathy, and failed to discern how many of

the changes were solely related to the failing heartor to the cardiomyopathy itself. An answer to thisquestion would require comparative studies withcases of heart failure other than dilated car-diomyopathy, under rigorously similar conditions(same degree of heart failure, same levels of cir-culating catecholamines, same technical proce-dures, etc.), but such studies are practically nonex-istent. In one study** in which the effects of heartfailure on the /3-adrenergic system were compa-rably assessed in explanted hearts from patientswith idiopathic dilated cardiomyopathy andischemic cardiomyopathy, the "idiopathic" ven-tricles exhibited a greater degree of total /3-recep-tor or /3,-receptor downregulation than the"ischemic" ventricles. Although the authors attrib-uted this difference to a lesser degree of adren-ergic activation in the ischemic hearts related tothe sympathetic denervation that may result frommyocardial infarction, it seems more plausible tospeculate that there is a greater amount of adren-ergic drive in the idiopathic hearts, due to the ago-nist additive effect of the anti-/0, receptor Abs.This should result in an "absolute" enhancementof downregulation in the latter, and would cer-tainly explain why treatment with /3-blocking drugsmay be beneficial in idiopathic dilated cardiomy-opathy and much less or not at all in ischemiccardiomyopathy ''*^"*' or other causes of heart fail-ure. However, in contrast with the enhanced down-regulation in the idiopathic ventricles, subsensi-tivity of /3-receptors to agonist-mediatedstimulation was found to be greater in the ischemicventricles,** indicating a functional uncouplingthat was related to an abnormality in G proteinactivation of unknown mechanism. Furthermore,in a previous comparative (but less extensive)study of idiopathic dilated cardiomyopathy andischemic cardiomyopathy,*^ different and evenopposite results were obtained: for a similar degreeof downregulation, subsensitivity was greater inthe idiopathic than in the ischemic ventricles. Fur-ther studies are needed to define more preciselythe nature of the differences between idiopathicdilated cardiomyopathy and other etiologicallydiverse forms of heart failure and their effectson the /3-adrenergic receptor system. It will be ofgreat interest to examine: (a) whether the differ-ences correlate with the presence of anti-/3| andanti-/52 Abs in the patients with idiopathic dilatedcardiomyopathy (and Chagas' heart disease); and(b) whether the differences also occur during ear-lier stages of these diseases, prior to the onset ofheart failure.

Rosenbaum, et al. Antibodies to Beta-Adrenergic Receptors 371

The Adrenergic Cardiomyopathies

If idiopathic dilated cardiomyopathy and Cha-gas' heart disease are characterized hy an increasedambunt of adrenergic activation mediated hy Ahs,this could result in cardiotoxic effects similar tothose known to he caused hy catecholamines.''""Patients with pheochromocytomas have heen shownto exhibit a wide spectrum of cardiac ahnormali-des, from cases that look like an acute myocardialinfarction™ to those presenting as a chronic dilatedcardiomyopathy.^o''*-'* An infarct-like patternwas the rule when large doses of isoproterenolwete given to rats,''''^'''''' and was similar to theso-called fulminant*" or catastrophic" forms ofacute chagasic or viral myocarditis.*' In one study,*"cdrdiac changes similar to those found inhuiirian Chagas' disease" were reported, includ-ing the very characteristic apical aneurysm. Whenlower doses were used.^^'" the main changes con-sisted of disseminated microfocal lesions associ-ated with a mononuclear cell infiltrate, whichresembled the histologic pattem seen in most casesof Chagas' heart disease^' and in some cases ofidiopathic dilated cardiomyopathy. 3.84 ^ remark-ahle feature of the lesions caused hy catecholaminesis that their distribution is far from homoge-nedus. In all studies, involvement of the left ven-tricle was more severe,'"*''^''''''' and this was espe-cially apparent in the subendocardial layers andleft ventricular apex. Another striking feature isthat the necrotic or degenerative lesions are accom-panied by a mononuclear cell infiltrate, rather thanthe usual polymorphonuclear leukocytic responseseen in acute myocardial necrosis.'"'-^ ''''' ^ Com-parable lesions were noted to occur following the"chronic" administration of isoproterenol to Wis-tar rats,*^ and when the heart tissue was utilizedfor biochemical and physiological studies, the fol-lowing changes were observed: (a) a 34% decreasein the density of /0-receptors; (b) a large reductionin the basal and maximal activities of isoproterenol-stimulated adenyl cyclase; (c) a reduction in theintracellular cAMP concentration, both basal andisoproterenol stimulated; and (d) a large reductionin maximal tension development when ventricu-lar strips were stimulated with isoproterenol. Theseresults are similar to those obtained from the hearttissues of patients with idiopathic dilated car-diomyopathy,*'so and are consistent with the invitro effects of the anti-/0-receptor Abs occurringin patients with dilated cardiomyopathy and Cha-gas' disease. In view of these many similarities,we postulate that these diseases may be the expres-

sion of an "adrenergic cardiomyopathy," mediatedby the chronic stimulating effects of the Abs. Fur-ther studies will be needed to test this hypothe-sis, and to demonstrate whether these Ahs are reallypathogenic or merely a coincidental association.Our hypothesis does not exclude other mechanismsthat may play a role in the natural course ofthese diseases (for review, see references 2, 32,33, 86, and 87), the discussion of which is beyondthe scope of this article.

Regional Myocardial Injury in the AdrenergicCardiomyopathies

Many cases of Chagas' heart disease and idio-pathic dilated cardiomyopathy show a selective orpredominant involvement of distinct parts of theheart. The infarct-like pattem seen in chronic oracute cases of both diseases, and the apical ven-tricular aneurysm of Chagas' disease, are con-spicuous manifestations of a regional involvement.We have recently identified a clinical form of Cha-gas' disease in which there is exclusive or pre-vailing involvement of the conducting tissues ofthe heart. There is also an amazing "predilec-tion" of Chagas' disease for the right bundle branch,in contrast to the preference of idiopathic dilatedcardiomyopathy for the left bundle branch, whileinvolvement of the sinoatrial node is frequent inthe former but not in the latter. Awareness of thesefeatures now brings into focus the existence ofregional differences in the lesions caused hy cat-echolamines. It is well known that sympathetic ter-minals are distributed nonuniformly across themyocardium,** and this seems also to correspondto an inhomogeneous distribution of /0-adrener-gic receptors.*' This uneven distribution appearsto involve both ;S,-receptors and /?2-receptors.*^-"In a recent study,*^ the density of /0-adrenergicreceptors was found to be higher in the subendo-cardium than in the subepicardium from normalhuman ventricles. This may explain the more severelesions caused by catecholamines in the suben-docardium, as previously discussed. In contrast,the /3-receptor density was lower in the subendo-cardium than in the subepicardium of tissue slicesfrom the hearts of patients with severe heart fail-ure, 2 suggesting a greater degree of downregula-tion in the former than in the latter, also compat-ible with the greater effects of catecholamines atthe subendocardial level. Further studies will beneeded to determine the comparative density of/01-receptors and /Jj-receptors in all four chambersof the heart, in the subendocardium versus subepi-

372 Journal of Cardiovascular Electrophysiology Vol. 5, No. 4, April 1994

cardium, in the apex as compared to the base ofthe heart, and in the ventricular conduction sys-tem and in the sinoatrial region. This is likely toimprove our understanding of some of the puz-zling regional differences that occur in the adren-ergic cardiomyopathies.

Summary and Conclusions

Abs against peptides derived from the human/S,-adrenergic and/or /Jj-adrenergic receptors havebeen shown to occur in 35% of patients with idio-pathic dilated cardiomyopathy, 60% of chagasicpatients, and 12% of control cases. The same Absoccur in 32.5% of patients with idiopathic cardiacarrhythmias and conduction disturbances in theabsence of structural heart disease, suggesting thatsome of these so-far enigmatic abnormalities maybe pathogenically linked to idiopathic dilated car-diomyopathy, and eventually may serve to identifyearly stages and/or patients at risk of developingthe dilated cardiomyopathy. Several studies showthat these Abs can activate the /3-adrenoceptors, butin contrast to neurohormonal agonists, this acti-vation fails to be followed by short-term desensi-tization. However, a long-term desensitization doesoccur as a form of downregulation, which is alsomechanistically different from the one induced byneurohormonal agonists. This may explain whydownregulation of /3-adrenoceptors is enhanced inthe failing heart from patients with idiopathic dilatedcardiomyopathy as compared with ischemic car-diomyopathy, and also why treatment with /3-block-ing drugs may be beneficial in the former but notin the latter. The sustained /3-adrenergic activationmediated by the Abs may also cause direct dele-terious effects and play a role in the occurrence ofmyocardial lesions. These lesions and concomi-tant biochemical changes are similar to those knownto be induced by catecholamines, supporting thehypothesis that Chagas' heart disease and someforms of idiopathic dilated cardiomyopathy maybe the expression of an Ab-mediated adrenergiccardiomyopathy.

Acknowledgment: The authors are indebted to Mrs. CeciliaMcKeon for her secretarial work.

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