146

Objectives . The present study Investigated the presence ofanlimyosin autoantibodies in sera of patients with myocarditis andin three control groups: healthy blood donors, patients withalcoholic cardiomyopathy and patients with other cardiac dis-eases .

Background. An increasing body of evidence indicates that inthe course of myocarditis, autoimmunologic mechanisms may playa pathogenetic role. Animal studies with Coxsackie 113 virus-induced murine myocarditis could demonstrate the appearance ofcirculating autoantibedies against cardiac myosin .

Methods . Sera were analyzed by enzyme-linked immunosor-bent assay (ELISA) and Western blot with human left ventricularmyosin as antigen .

Results. Seventeen (42%) of 40 serum samples from patientswith myocarditis showed antibody-binding against myosin,whereas only 1 (2.5%) of 39 samples from healthy blood donorsand 9 (21%) of 43 samples from patients with other cardiac

Currently, most clinical cases of myocarditis in humans aresuspected to be of viral origin (1,2) . Viral ribonucleic acid(RNA) has been detected in a certain percent of patients withmyocarditis and dilated cardiomyopathy (3) . Although thepathogenetic mechanisms are only vaguely understood, anincreasing body of evidence indicates that in the course ofmyocarditis, autoimmunologic mechanisms may play apathogenetic role. Autoimmunity initiated by the viral infec-tion may aggravate and perpetuate myocarditis and perhapsresult in dilated cardiomyopathy .

In humans, several autoantigens have been identified,including the adenosine nucleotide translocator (ANT), thecalcium chani el, the Connexon and the beta-adrenoceptor(4-8) . In experimental studies (5,6), it was shown that theautoantibodies against adenosine nucleotide translocator in-fluence the function of the carrier protein in vitro and impaircardiac function in vivo. This suggests a pathogenetic role of

From the Medizinische Klinik B . Abteilung fur Kardiologie . Pneumologieand Angiologie, University of Diisseldorf, Ditsseldorf, Germany. This studywas supported by Grant Nr La 59911 .2 from the Deutsche Forschungsgemein-schaft, Bonn, Germany .

Manuscript received June 3,1992 ; revised manuscript received August 11 .1993, accepted August 27. 1993 .

Address for correspondence : Dr. Bernward Lauer, Medizinische KlinikB, University of DUsseldorf. Moorenstrasse 5, 40225 Diisseldorf, Germany .

®1994 by the American College of Cardiology

JACC Vol. 23, No . IJanuary 1994 :146-53

MYOCARDITIS

Autoantibodies Against Human Ventricular Myosin in Sera of PatientsWith Acute and Chronic M yocarditisBERNWARD LAUER, MD, KLAUS PADBERG, MD, HEINZ-PETER SCHULTHEISS, MD,

BODO-ECKHARD STRAUER, MD, FACC

Daisseldorf. Germany

diseases showed autoantibodies against myosin (p < 0 .05 vs.myocarditis) . In sera from patients with alcoholic cardiomyopathy(a = 12), no antibodies against human ventricular myosin couldbe detected. In Western blots, the aulimyosin antibodies inpatients with myocarditis bound to the myosin heavy chain . Usingprotein-A sepharose chromatography, it could be shown that theantimyosin autoantibodies are of the immunoglobulin G (IgG)type. In ELISA, the antimyosin autoantibodies bind equally tomyosin prepared from either cardiac or skeletal muscle, respec-tively.

Conclusions. These results demonstrate the presence of au-toantibodies against human ventricular myosin in patients withmyocarditis . The prevalence of these autoantibodies is signifi-cantly higher in patients with myocarditis than in patients withother cardiac diseases. No organ specificity of the autoantibodiescould be detected .

(J Am Coll Cardiol 1994;23:146-53)

these autoantibodies in the course of myocarditis and dilatedcardiomyopathy .

Animal studies (9) with Coxsackie B3 virus--inducedmurine myocarditis demonstrated the appearance of circu-lating autoantibodies against cardiac tissue . In Western blotanalysis, these antibodies react with the heavy chain ofcardiac myosin (10,11). Immunization of the animals withcardiac myosin also leads to alterations resembling virus-induced myocarditis (12) .

In the present study, we show that autoantibodies againsthuman ventricular myosin can be detected in patients withmyocarditis .

MethodsStudy group . Serum samples from 40 patients with acute

or chronic myocarditis and 94 control subjects-12 withalcoholic cardiomyopathy, 43 patients with other cardiacdiseases and 39 healthy control subjects-were studied .Blood samples for immunologic studies were taken at thetime of routine laboratory tests . Patients were examinedwith invasive and noninvasive techniques, including physi-cal examination, routine laboratory tests, rest and exerciseelectrocardiography, echocardiography, right and left heart

0735-10971941$6 .00

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catheterization, coronary angiography, left ventriculographyand right heart myocardial biopsy .

All patients were considered to have possible myocarditison the basis of history and clinical presentation . Endomyo-cardial biopsy specimens were examined histologically ac-cording to the Dallas criteria (13) and immunohistologically(14,15) . At least seven biopsy specimens from each patientwere analyzed . Histologic sections were analyzed by lightmicroscopy for evidence of myocardial necrosis, interstitialfibrosis and the presence of lymphocytic infiltrates . Becauseevaluation of myocardial biopsy specimens by light micros-copy alone is difficult (16,17) and has a high rate of interob-server variability (18) and sampling error(19), this techniqueis thought to be rather insensitive in the diagnosis of myo-carditis (20). We therefore also examined the biopsy speci-mens from our patients by immunohistologic techniquesdeveloped with various monoclonal antibodies . Antibodiesdirected against surface antigens of human lymphocytes(CD3, CD4, CD8) were used to detect and quantitate lym-phocytic infiltrates in myocardial tissue (14,15,21-23) . Addi-tionally, with antibodies against the major histocornpatibiiityantigens (MHC) . an increased expression of MHC I and 11antigens could be detected . With these techniques, thediagnostic accuracy in the biopsy specimens could be in-creased (24) and the interobserver variability minimized(14,15) .

Acute myocarditis was diagnosed when histologic sec-tions from endomyocardial biopsy specimens revealed lym-phocytic infiltrates in the region of myocardial necrosis .When lymphocytic infiltrates were seen by light microscopyin histologic sections without myocyte necrosis, the biopsywas repeated after 3 months . Ongoing myocarditis was

diagnosed when the lymphocytic infiltrates had persisted orwere increased at the second biopsy . When the lymphocyticinfiltrates had disappeared, the patients were not classifiedas having myocarditis . When the immunohistologic exami-nation revealed lymphocytic infiltrates and increased expres-sion of MHC I and II antigens, the biopsy was also repeatedafter 3 months . When the lymphocytic infiltrates and in-creased expression of MHC I and It antigens persisted intwo consecutive biopsies, a chronic inflammatory process ofthe myocardium was assumed . These patients were classi-fied as having chronic myocarditis .

A total of 198 consecutive patients with clinically sus-pected myocarditis were analyzed according to these crite-ria. On the basis of the Dallas criteria (13), which were meantto provide "a simple reproducible working classification"(17) rather than a definitive classification of acute myocardi-tis or a "conditio sine qua non" for the diagnosis ofmyocarditis (17), three of our patients met the criteria foracute myocarditis and five other patients for ongoing myo-carditis. In 32 patients . the diagnosis of chronic myocarditiswas made on the basis of immunohistologic data . Theclinical data from these 40 patients are given in Table 1 .

Patients were excluded if they had coronary heart diseaseat selective coronary angiography, systemic blood pressure

LAUER ET AL .

147ANTIMY®Slid AUTGANTIB0IJIES IN HUMAN MYOCARDITIS

'fable 1. Clinical Findings in 40 Patients With Myocarditis

P~,~i831i0nFatiguePericardial effusionNew rhythm disturbancesElectrocardiographic changesElevated creatine kinase levelLeft ventricular dysfunction

Patients(no.p

>150190 mm Hg, concomitant systemic or endocrinologicdiseases known to cause left ventricular dysfunction orexcessive alcohol consumption .

Three groups of control subjects were enrolled in thisstudy. Control group I comprised 43 patients with variouscardiac diseases, including 31 patients with coronary arterydisease, 7 with valvular heart disease and 5 with hyperten-sive heart disease . Control group lI comprised 12 patientswith alcoholic cardiomyopathy . These patients presentedwith impaired left ventricular function and had a history ofexcessive alcohol consumption (>I g of alcohoUkg bodyweight per day) . Clinical and laboratory findings as well asthe histologic examination revealed no cause of impaired leftventricular function other than excessive alcohol consump-tion . Control group III consisted of 39 healthy persons withno history of myocardial disease .

All subjects provided informed consent before participat-ing in the study. The study protocol was approved by theethics committee of the University of Dilsseldorf.

Preparation of myosin. Myosin was prepared accordingto the method of Offer et al . (25), with slight modificationsaccording to the method of L,ompre et al . (26) . Briefly,cardiac muscle specimens were obtained from the patho-logic institute within 24 h after death . After homogeni-zation, myosin was extracted in modified Guba-Straubsolution (potassium chloride 0.3 mol/liter, magnesium chlo-ride I mmollliter, sodium phosphate 150 mmollliter, sodiumpyrophosphate 10 mmollliter, ethylenediaminetetraaceticacid (EDTA) 10 mmollliter, beta-mercaptoethanol 1%, pH6.5), centrifuged and then precipitated by lowering the ionicstrength with EDTA I mmollliter, beta-mercaptoethanol 1%,pH 6.5. After dissociating actin with sodium pyrophosphate100 mmollliter, adenosine 5'-triphosphate (ATP) 6 mmol/literin EDTA 4 mmollliter, dithiothreitol 3 mmol/liter, pH 7 .0,further purification was carried out by ammonium sulfatefractionation . For immunization procedures, the myosin wasfurther purified by ion exchange chromatography on DEAESephadex, using a potassium gradient from 0 to 500 mmol/liter. The myosin solution was then dialyzed overnightagainst sodium chloride 500 mmollliter, tris(hydroxymethyl)-aminomethane (Tris)/hydrochloride 10 mmollliter, EDTAI mmol/liter, pH 6.5 at 4°C and stored in this solution . Thepurity was routinely checked by sodium dodecylsulfate gel

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LAUER ET AL_

electrophoresis. Skeletal muscle myosin was prepared usingthe same protocol from human iliopsoas muscle .

Preparation of antihuman ventricular myosin antibodies .Antibodies against human ventricular myosin were raised inrabbits as described by Schwartz et al . (27) . One weekbefore immunization, blood was taken from the rabbits aspreimmune serum. Rabbits received an injection of thepurified protein in Freund's incomplete adjuvant every 2weeks for 2 months. Blood was drawn before each immuni-zation procedure from the auricular artery for antibody titermonitoring . Antibody titers were maximal after five injec-tions.

The immunoglobulin G (IgG) fractions of human sera andrabbit antisera were prepared on a protein-A sepharosecolumn (28). Protein-A sepharose was equilibrated withphosphate-buffered saline solution with 0 .05% azide in anappropriate column. A total of 2 ml of serum was run on thecolumn and the fraction of unbound protein was collected("wash fraction"). The IgG bound to the column was elutedwith 0.1 mol/liter citrate, 0 .5 moUliter sodium chloride, pH2.9 immediately neutralized with 2 mol/liter Tris to a final pHof 7.4, then dialyzed against phosphate-buffered saline solu-tion overnight and concentrated by ultrafiltration .

Enzyme-linked immunosorbent assay (ELISA). TheELISA technique was performed as previously described byLem and Hales (29) . Briefly, I Aglwell of purified myosin inphosphate-buffered saline solution was air dried on polyvinylchloride 96 well plates . Free binding sites were blocked byincubating the plates with ELISA diluent (0 .2% Tween, 2%bovine serum albumin, 0 .4 mmol/liter Thimerosal, in phos-phate-buffered saline solution) for I h, then antisera orpatient sera diluted in ELISA diluent were added . Afterincubation at room temperature for 90 min, plates werewashed three times in phosphate-buffered saline solution and0.2% Tween, and affinity-purified peroxidase-labeled goatantirabbit or antihuman antibody was added, respectively .After 90 min of incubation, the plates were rewashed threetimes and substrate (0 .4 mg/ml o-phenylenediamine, 0 .01%hydrogen peroxide in citrate buffer, pH 5 .0) was added . Thereaction was stopped after 30 min with 4N sulfuric acid andthe samples were read at 492 nm in an automated ELISAscanner .

To test the specificity of the antibody binding in ELISA,10 µl of serum in 400µl of phosphate-buffered saline solutionwas preincubated with 100 Ag of purified myosin fromdifferent human muscles (human left ventricle, human ilio-psoas, human sartorius) overnight at 25°C before the ELISAprocedure. Sera were also tested against myosin preparedfrom the different muscles . All sera were tested in duplicate .

Western blot analysis. Western blot analysis was carriedout as described previously. Purified myosin was loaded ona 8% acrylamide gel with a 4 .5% stacking gel (30) . Aftermigration for 1 h at 125 V and 4 h at 250 V with weightmarkers as standard, the proteins were transferred to nitro-cellulose sheets (31) with 195 mA for 2 h in 70% methanol inTris/glycine buffer, pH 8 .3. Appropriate stripes were cut out,

free binding sites blocked with phosphate-buffered salinesolution, 2% bovine serum albumin overnight . The stripeswere then incubated with the first antibody (rabbit antimyo-sin antisera or patikof sera. respectively) in phosphate-buffered saline wbnioA, d .2% Tween. After washing threetimes with phosphate-hv,&red saline solution, 0.2% Tween,the second antibody was added (peroxidase-labeled antirab-bit IgG or antihuman IgO, respectively). The coloration wascarried out in the dark with 0 .5% 4-chloro-naphthol, 0.1%hydrogen peroxide in methanol 15% in phosphate-bufferedsaline solution for 30 min . The specificity of the antibodybinding to myosin was tested by preincubating 30 Al ofserum in 3 ml of phosphate-buffered saline solution, Tweenwith 300 pg of myosin prepared from different musclesovernight at 25°C before incubation with the nitrocellulosesheets .

Statistical analysis. The threshold for the detection ofantimyosin autoantibodies in sera during ELISA was definedas the mean value ± 2 SD of the sera from 39 apparentlyhealthy persons (control group 111) . The two-tailed chi-square test for independent populations was used to com-pare the prevalence of antimyosin antibodies in the differentstudy groups . Statistical significance was defined as p <0.05 .

ResultsAutoantibodies against human ventricular myosin in sera

from patients with myocarditis, alcoholic cardiomyopathy andother cardiac diseases. To study whether sera from patientswith myocarditis contained anticardiac myosin autoantibod-ies, the sera were tested by ELISA with human ventricularmyosin as antigen . Control values were established with serafrom 39 apparently healthy persons (control group 111) . Serawere assumed positive for antimyosin antibodies if thebinding in ELISA was above the mean value ± SD of thecontrol group III .

Figure f shows the extinction in ELISA of the sera frompatients with myocarditis, alcoholic cardiomyopathy, othercardiac diseases and healthy blood donors . Table 2 gives thepercent of antimyosin antibody-positive patients in thevarious groups . In 17 (42%) of 40 serum samples frompatients with myocarditis, antibodies against human ventric-ular myosin were detected (p < 0 .05 vs. control group 111) .One of the 3 patients with acute myocarditis, 2 of the 5patients with ongoing myocarditis and 14 of the 32 patientswith immunohistologically diagnosed chronic myocarditishad antimyosin autoantibodies . There was no statisticallysignificant difference in hemodynamic variables (cardiacindex, ejection fraction, left ventricular end-diastolic pres-sure) between patients with myocarditis who had antimyosinantibody-positive versus antimyosin antibody-negativefindings (data not shown) . None of the 12 patients withalcoholic cardiomyopathy showed antimyosin autoantibod-ies. In patients with other cardiac diseases, only 9 (21%) of43 serum samples contained antibodies against human ven-

JACC Val . 23 . No . 1January 1994 : 1 46 -53

Extinction

I2 4

I 1_ I

MC

Co I

CO 11

CO III

Figure 1 . Extinction in ELISA of the sera from patients withmyocarditis (MC), other cardiac diseases (control (Col group 1),alcoholic cardiomyopathy (control group 11) and healthy blooddonors (control group 111) with human ventricular myosin as antigen(serum dilution 1 :40) . The black line indicates the mean value ± 2SD for healthy persons (control group 111) as the threshold for thedetection of antimyosin autoantibodies .

tricular myosin (p < 0.05 vs. patients with myoca, difis, p <0.05 vs. control group III) . These data demonstrate thatmany serum samples from patients with myocarditis containautoantibodies against human cardiac ventricular myosin .The prevalence of antibodies against myosin is significantlyhigher in patients with myocarditis than in patients withother cardiac diseases .

Table 2 shows the prevalence of antimyosin autoantibod-ies in sera from patients with myocarditis and from thecontrol patients at different serum dilutions . Nine (22 .5%) ofthe 40 patients with myocarditis showed antimyosin antibod-ies at a serum dilution of 1 :160, 4 (10%) at a dilution of 1 :640,3 (15%) at 1 :2,560 and 1 (2 .5%) at 1 :10,240 . In the patientswith other cardiac diseases, in only 3 (7%) of 43 couldantimyosin antibodies be detected at a dilution of 1 :160 (p <0.05 vs. myocarditis) ; at serum dilutions of 1 :640, 1 :2,560and 1 :10,240, no antibodies were demonstrated in thesepatients. To test the specificity of the antibody binding inELISA for human ventricular myosin, the sera that showedantibody binding in ELISA were preincubated with human

Table 2 . Prevalence of Antimyosin Autoantibody-Positive Sera inPatients With Myocarditis and Control Groups

*p < 0 .05 versus control (Co) groups I (other cardiac diseases), It(alcoholic cardiomyopathy) and III (healthy blood donors) . tp < 0 .05 versusmyocarditis and control groups II and Ill .

LAUER ET AL,

149ANTIMYOSIN AUTOANTIBODIES IN HUMAN MYOCARDITIS

Extinction2

``More

afterpreincubation with h,_ -nan cardiac myosin

Figure 2 . Specificity of the antibody binding for human ventricularmyosin. Enzyme-linked immunosorbent assay (ELISA) was per-formed with serum from patients with myocarditis before and afterpreincubation of the serum with human ventricular myosin (seeMelhnrls) . The black line indicates the mean value ± 2 SD forhealthy persons (control group 111) as the threshold for the detectionof antimyosin autoantibodies .

ventricular myosin before the ELISA procedure (Fig . 2). Itwas shown that preincubation of the sera with humanventricular myosin completely prevented the binding of thesera to human ventricular myosin. This showed that thebinding in ELISA was specific for human ventricular myo-sin .

Localization of the antigenic determinant on the myosinmolecule. The myosin molecule consists of six polypeptidechains, two heavy chains of 200 kD (MHQ and four lighterpolypeptides (two of 27 kD [MLCII and two of 10 MJMLC21) . To study which myosin subunit the antimyosinautoantibodies are directed against, the sera were testedagainst human ventricular myosin in Western blots . Poly-clonal antibodies against human ventricular myosin raised inrabbits served as positive control sera . The polyclonal rabbitantihuman myosin antibodies bound against the myosinheavy chain (200 M) and against the myosin light chains(migrating together in the 8% acrylamide gel) (Fig . 3) . Serafrom patients with myocarditis that showed positive anti-body binding in ELISA bound against the 200-kD protein,which is the myosin heavy chain. Control sera showed noantibody binding in Western blots .

After preincubation of the sera with human ventricularmyosin the autoantibody binding to the 200-0 protein wasinhibited . In the region of the myosin light chains (27,000 D,20,000 D), no autoantibody binding could be detected in thepatient sera

Classification of the autoantibodies against myosin . Toclassify the immunoglobulin type of the autoantibodiesagainst myosin, IgG fractions from the 16 antibody-containing human sera were prepared on a protein-A

sepharose column. The various elution fractions were testedfor binding to human ventricular myosin in ELISA. Asshown for one serum sample in Figure 4, the IgG fractionbound strongly to myosin, whereas the wash fraction con-

SerumDilution 1 :40 1 :160 1 :640 1 :2,560 1 :10 .240

Myocarditis 17* 9* 4 3 1(n = 40) (42%) (22 .5%) (l07c) (7 .5%) (2 .5--/,)Co I 9r 3 0 0 0(n = 43) (21%) (7.0%) (2 .3%) (0%) (0%)Co 11 0 0 0 0 0(n = 12) (0%) (0%) (0%) (0%) (VC)CO III 1 0 0 0 0(n = 39) (2 .5%) (0%) (0%) (0%) (0%)

150

LAUER ET AL .ANTIMYOSIN AUTOANTIBODIES IN HUMAN MYOCARDITIS

MHC

1 2

3 4 5 6

Figure 3. Western blot analysis of antimyosin antibodies . Humanventricular myosin was subjected to polyacrylamide gel electro-phoresis, transferred to nitrocellulose sheets and incubated withdifferent sera (see Methods) . Column 1, Rabbit serum with poly-clonal antibodies against human ventricular myosin . Column 2,Human serum containing antimyosin autoantibodies . Column 3,Immunoglobulin 0 fraction prepared from human serum containingantimyosin autoantibodies . Column 4, Human control serum Inoantibody binding in enzyme-linked immunosorbent assay IELISAI) .Column 3, Immunoglobulin G fraction prepared from human controlserum (no antibody binding in ELISA) . Column 6, Molecular weight(MW) standard after polyacrylamide gel electrophoresis (CoomassieBB coloration) . MHC = myosin heavy chain ; MLC = myosin lightchain .

taining other serum proteins, including other immunoglobu-lins, did not bind to myosin . This demonstrated that theautoantibodies against human ventricular myosin in patientswith myocarditis are of the IgG type .

Figure 4. Antibody binding in enzyme-linked immunosorbent assay(ELISA) of serum, immunoglobulin G (lgG) fraction of serum and"wash fraction ." A representative example from one patient withmyocarditis is shown . The black line indicates the mean value ±2 SD for healthy persons (control group 111) as the threshold for thedetection of antimyosin autoantibodies . IgG fraction = ELISA wasperformed with the IgG fraction of the serum (see Methods) . Serum= ELISA was performed with serum from a patient with myocar-ditis ; "wash" fraction = ELISA was performed with the fraction ofthe serum that was not bound to a protein-A sepharose column (seeMethods) .

Extinction

Serum "j

h"fraction fraction

Extinction

21

Extinction

2-

cardiacmyosin

r

o

~before after preincubation with

incubation

cardiac

skeletalmyosin

myosin

JACC Vol . 23 . No . BJanuary 1994 :146-53

Iskeletalmyosin

Figure 5 . Specificity of the antimyosin autoantibodies fur myosinfrom different striated muscles . A. Sera were preincubated withhuman ventricular myosin (cardiac myosin) or myosin from humanskeletal muscle (skeletal myosin), respectively (see Methods) . B,Antibody binding in enzyme-linked immunosorbent assay (ELISA)with myosin from different striated muscles as antigen . The blackline indicates the mean value ± 2 SD for healthy persons (controlgroup 111) as the threshold for the detection of antimyosin autoanti-bodies. Cardiac myosin = myosin prepared from human left ventri-cle (see Methods) . Skeletal myosin = myosin prepared from humanskeletal muscle (see Methods) .

Specificity of the antimyosin antibodies for cardiac ventric-ular myosin. To examine whether the antimyosin autoanti-bodies found in sera from patients with myocarditis aredirected specifically against human ventricular myosin, an-tibody-positive sera were preincubated with myosin pre-pared from different striated muscles (human left ventricle,human skeletal muscle) . Figure 5A shows the results forseven serum samples . Preincubating the samples with any ofthe different myosin preparations inhibited autoantibodybinding against human ventricular myosin in ELISA . Usingeither myosin from skeletal or cardiac muscle as antigen inELISA showed that the sera bound equally to humanventricular myosin and skeletal myosin (Fig . 5B). Thisindicated that there is apparently no organ specificity of theantimyosin autobodies for human ventricular myosin .

DiscussionAutoantibodies in Imyocarditis . In patients with myocar-

ditis, several studies have demonstrated autoantibodies

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ANTIMYOSIN AUTOANTIBODIES IN HUMAN MYnCARDITIS

against cardiac proteins . Immunofluorescence studies (32-36) showed autoantibodies against different cardiac struc-tures (namely, membranes, mitochondria, cytoplasmic anti-gens). Further studies identified the adenine nucleotidetranslocator of the inner mitochondrial membrane (4,37,38),calcium channel (7), Connexon (33) and cardiac beta-adrenoreceptor (8) as autoantigens in myocarditis and di-lated cardiomyopathy . For the adenine nucleotide translo-cator, a possible functional significance was shown (5,6) .The hypothesis that autoantibodies against myosin may playan important role in the pathogenesis of myocarditis isderived from animal studies . In animal models, antibodiesagainst cardiac myosin have been demonstrated . Infectionwith Coxsackie-B3 virus leads to the development of myo-carditis in genetically defined mice (39) . In the course ofCoxsackie-B3 virus-induced murine myocarditis, heart-specific autoantibodies have been demonstrated (li) . InWestern blot analysis, these antibodies bind to a 200-kDband that is thought to be the myosin heavy chain (10) .Immunization with myosin leads to histologic and immune-histologic alterations in mice hearts similar to those withCoxsackie-B3 virus infection (12). From experimental data(40), a possible pathogenetic role of the antibodies wassuspected. The present study clearly shows the existence ofautoantibodies against human ventricular myosin in patientswith myocarditis . These autoantibodies are directed againstthe myosin heavy chain . No antibody binding was seen withthe myosin light chains . Recently, antibodies against myosinheavy chains were demonstrated in patients with the clinicalpresentation of dilated cardiomyopathy (41) . which in manycases is thought to be a sequela of myocarditis . In this study,the sensitive immunohistologic techniques for detecting lym-phocytic infiltrates in myocardial biopsy specimens were notapplied .

Histologic and immunologic methods for the diagnosis ofmyocarditis . In the present study, the diagnosis of myccar-ditis was made using histologic (Dallas criteria [131) andmmunohistologic methods . The Dallas classification for thediagnosis of acute myocarditis was initially established by agroup of outstanding pathologists . The goals of the groupwere to: 1) produce a morphologic definition of myocarditis,2) develop histologic criteria for the diagnosis of myocardi-tis, 3) establish a simple reproducible working classification,4) outline the problems and pitfalls in establishing thediagnosis of myocarditis, 5) assess the applicability andreproducibility of the classification system itself, and6) make this information available to other pathologists andclinicians (13,17) . However, as these pathologists (17) haveemphasized, the Dallas criteria were not meant to establish adefinitive diagnosis of myocarditis and should not be mis-understood and misinterpreted as a "conditio sine qua non"of the diagnosis of myocarditis .

The present study used the Dallas criteria (13) in additionto immunohistologic techniques for the detection of lympho-cytic infiltrates (14,15,18-20) and an increased expression ofMHC I and 11 antigens . With these techniques, the diagnos-

tic accuracy was increased (24) and the interobserver vari-ability was minimized (14,15) . We believe that the fact that intwo consecutive biopsy specimens, chronic lymphocyticinfiltrates and an increased expression of MHC I and IIantigens could be detected points to a chronic inflammatoryprocess in the myocardium and therefore these patients wereclassified as having chronic myocarditis . In the presentstudy, 3 patients met the histologic Dallas criteria for acutemyocarditis and 5 for ongoing myocarditis ; 32 patients metthe immunohistologic criteria for chronic myocarditis . In Ipatient with acute myocarditis, 2 patients with ongoingmyocarditis and 14 patients with chronic myocarditis, an-timyosin antibodies could be detected . Because of the smallnumber of patients with acute myocarditis and ongoingmyocarditis, a comparison between these groups and thechronic myocarditis group is not appropriate .

Acute myocarditis can resolve spontaneously, or it maylead to a chronic inflammatory, perhaps autoimmunologi-cally mediated, process . In the present study, only one ofthree patients with acute myocarditis showed antimyosinautoantibodies . Because the acute phase of the disease is ofshort duration, one cannot expect that IgG autoantibodiesagainst an intracellular protein as a possible marker of anautoimmune process are always detectable at this early stageof the disease . Therefore, it is not surprising that only one ofthe three patients with acute myocarditis showed autoanti-bodies against human ventricular myosin .

In the present study, we found no significant difference inhemodynamic variables between patients with myocarditiswho had antimyosin antibody positive versus negative find-ings. At least in some patients, myocarditis is thought to bea chronic process. In the present study, the hemodynamicvariables were evaluated at only one time point in thischronic process . Longitudinal studies are necessary to eval-uate the relation between changes in hemodynamic variablesand the persistence of antimyosin autoantibodies in patientswith myocarditis .

Antimyosin Ruto2atibodles in human cardiac disease . Pa-tients with impaired ventricular function due to alcoholiccardiornyopathy did not show autoantibodies against humanventricular myosin. This is a strong indicator that the pres-ence of antimyosin autoantibodies is linked to a chronicinflammatory, perhaps autoirnmunotogically mediated, pro-cess rather than to the disturbed left ventricular functionitself. In the present study, patients with other cardiacdiseases also showed autoantibodies against human ventric-ular myosin but to a significantly lower extent . The preva-lence of antimyosin autoantibodies in patients with othercardiac diseases is similar to that in patients 1 year aftercardiac surgery (42) . In the study of Caforio et al . (41), only1 of 12 serum samples from patients with heart failure due toother cardiac diseases showed antimyosin antibodies . Theircontrol group comprised preselected patients with no anti-body binding to heart tissue in immunofluorescence studies(41). High antimyosin autoantibody titers were more com-mon in patients with myocarditis than in those with other

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cardiac diseases. This might be interpreted as indirect evi-dence for an autoimmunologic process in patients withmyocarditis . The present study could not demonstrate anyorgan specificity of the antimyosin autoantibodies in patientswith myocarditis . In ELISA, the antibodies bind equally toventricular and skeletal myosin, and the binding was equallyinhibited by myosin from ventricular or skeletal muscle . Thisis not surprising because the beta-myosin heavy chain ofhuman cardiac muscle and the myosin heavy chain of slowskeletal muscle are products of the same gene (43), and theventricular myosin heavy chain in humans is composed of90% to 95% of beta-myosin heavy chain and only 5% to 10%of alpha-myosin heavy chain (44) . In mice, immunofluores-cence studies (10) with cardiac specific antimyosin antibod-ies demonstrated a striational pattern (10) . In humans,antibodies showing a striational pattern in immunofluores-cence were not organ specific and were bound equally tocardiac and skeletal muscle (45) . Because the autoantibodiesdemonstrated in the present study are directed against themyosin heavy chain, no specificity of the autoantibodies foreither cardiac or skeletal myosin could be expected .

Possible mechanisms of autolmmunity in myocarditis . Thepresent study provides further evidence of an autoimmuno-logic process in human myocarditis (40) . The induction ofautoantibodies in the course of myocarditis and dilatedcardiomyopathy is not yet clear . One possible explanationwould be "molecular mimicry ." Schwimmbeck et al . (46),using computer analysis of protein sequences, identifiedregions of high homology between Coxsackie B3 virus andrabbit cardiac myosin (46) . Using synthetic peptides identi-cal to those in these regions, an immunologic cross-reactivity between Coxsackie B3 virus peptides and myosinpeptides was demonstrated (46) . In contrast, the antimyosinautoantibodies produced in Coxsackie B3 virus-inducedmyocarditis in AJJ mice did not cross-react with the virus(47). This argues against molecular mimicry as the pathoLe-netic pathway of autoimmunity in inflammatory heart dis-ease . Another possibility is the exposition of sequesteredantigens to the immune system in the course of myocarditis .Intracellular proteins such as myosin are possibly exposed tothe immunogenic cells, thus perpetuating the autoimmuno-logic process .

The pathogenetic mechanisms by which autoantibodiesdirected against intracellular proteins such as myosin mayplay a role in the autoimmunologic process remain contro-versial . It cannot be excluded that the antibodies maypenetrate into the myocardial cells and impair cardiac func-tion (5,6) . Conversely, assessment of whether the detectionof autoantibodies against human ventricular myosin mayserve as an index for the activity of the inflammatory processin the heart and be useful for monitoring the clinical courseof the disease will require further study . In murine autoim-mune myocarditis, the antimyosin autoantibodies do notseem to be involved in the induction of myocarditis (48) . Inthis context, it should be noted that scintigraphic studies (49)

with antimyosin antibodies suggest a lysis of heart cells inpatients with myocarditis and dilated cardio*nyopathy .

Conclusions. The present study demonstrates the pres-ence of autoantibodies against human ventricular myosin inpatients with myocarditis . The prevalence of antimyosinautoantibodies is significantly higher in patients with myo-carditis than in patients with other cardiac diseases . Theantimyosin autoantibodies are of the IgG type . They aredirected against the myosin heavy chain and react withmyosin from cardiac and skeletal muscles .

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