Autoantibodies in against human ventricular myosin in sera of patients with acute and chronic myocarditis

JACC Vol. 23, No . I January 1994 :146-53

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MYOCARDITIS

Autoantibodies Against Human Ventricular Myosin in Sera of Patients yocarditis With Acute and Chronic M

BERNWARD LAUER, MD, KLAUS PADBERG, MD, HEINZ-PETER SCHULTHEISS, MD, BODO-ECKHARD STRAUER, MD, FACC Daisseldorf. Germany

Objectives . The present study Investigated the presence of anlimyosin autoantibodies in sera of patients with myocarditis and in three control groups : healthy blood donors, patients with alcoholic cardiomyopathy and patients with other cardiac diseases . Background. An increasing body of evidence indicates that in the course of myocarditis, autoimmunologic mechanisms may play a pathogenetic role. Animal studies with Coxsackie 113 virusinduced murine myocarditis could demonstrate the appearance of circulating autoantibedies against cardiac myosin . Methods . Sera were analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blot with human left ventricular

myosin as antigen . Results. Seventeen (42%) of 40 serum samples from patients

with myocarditis showed antibody-binding against myosin, whereas only 1 (2.5%) of 39 samples from healthy blood donors and 9 (21%) of 43 samples from patients with other cardiac

Currently, most clinical cases of myocarditis in humans are suspected to be of viral origin (1,2) . Viral ribonucleic acid (RNA) has been detected in a certain percent of patients with myocarditis and dilated cardiomyopathy (3) . Although the pathogenetic mechanisms are only vaguely understood, an increasing body of evidence indicates that in the course of myocarditis, autoimmunologic mechanisms may play a pathogenetic role . Autoimmunity initiated by the viral infection may aggravate and perpetuate myocarditis and perhaps result in dilated cardiomyopathy . In humans, several autoantigens have been identified, including the adenosine nucleotide translocator (ANT), the calcium chani el, the Connexon and the beta-adrenoceptor (4-8) . In experimental studies (5,6), it was shown that the autoantibodies against adenosine nucleotide translocator influence the function of the carrier protein in vitro and impair cardiac function in vivo . This suggests a pathogenetic role of

From the Medizinische Klinik B . Abteilung fur Kardiologie . Pneumologie and Angiologie, University of Diisseldorf, Ditsseldorf, Germany . This study was supported by Grant Nr La 59911 .2 from the Deutsche Forschungsgemeinschaft, Bonn, Germany . Manuscript received June 3,1992 ; revised manuscript received August 11 . 1993, accepted August 27. 1993 . Address for correspondence : Dr . Bernward Lauer, Medizinische Klinik B, University of DUsseldorf . Moorenstrasse 5, 40225 Diisseldorf, Germany . ®1994 by the American College of Cardiology

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 could be detected. In Western blots, the aulimyosin antibodies in patients with myocarditis bound to the myosin heavy chain . Using protein-A sepharose chromatography, it could be shown that the antimyosin autoantibodies are of the immunoglobulin G (IgG) type. In ELISA, the antimyosin autoantibodies bind equally to myosin prepared from either cardiac or skeletal muscle, respectively. Conclusions. These results demonstrate the presence of autoantibodies against human ventricular myosin in patients with myocarditis . The prevalence of these autoantibodies is significantly higher in patients with myocarditis than in patients with other cardiac diseases. No organ specificity of the autoantibodies could be detected . (J Am Coll Cardiol 1994;23 :146-53)

these autoantibodies in the course of myocarditis and dilated cardiomyopathy . Animal studies (9) with Coxsackie B3 virus--induced murine myocarditis demonstrated the appearance of circulating autoantibodies against cardiac tissue . In Western blot analysis, these antibodies react with the heavy chain of

cardiac myosin (10,11) . Immunization of the animals with cardiac myosin also leads to alterations resembling virusinduced myocarditis (12) . In the present study, we show that autoantibodies against human ventricular myosin can be detected in patients with myocarditis .

Methods Study group . Serum samples from 40 patients with acute or chronic myocarditis and 94 control subjects -12 with alcoholic cardiomyopathy, 43 patients with other cardiac diseases and 39 healthy control subjects-were studied . Blood samples for immunologic studies were taken at the time of routine laboratory tests . Patients were examined with invasive and noninvasive techniques, including physical examination, routine laboratory tests, rest and exercise electrocardiography, echocardiography, right and left heart 0735-10971941$6 .00



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catheterization, coronary angiography, left ventriculography and right heart myocardial biopsy . All patients were considered to have possible myocarditis on the basis of history and clinical presentation . Endomyocardial biopsy specimens were examined histologically according to the Dallas criteria (13) and immunohistologically (14,15) . At least seven biopsy specimens from each patient were analyzed . Histologic sections were analyzed by light microscopy for evidence of myocardial necrosis, interstitial fibrosis and the presence of lymphocytic infiltrates . Because evaluation of myocardial biopsy specimens by light microscopy alone is difficult (16,17) and has a high rate of interobserver variability (18) and sampling error(19), this technique is thought to be rather insensitive in the diagnosis of myocarditis (20). We therefore also examined the biopsy specimens from our patients by immunohistologic techniques developed with various monoclonal antibodies . Antibodies directed against surface antigens of human lymphocytes (CD3, CD4, CD8) were used to detect and quantitate lymphocytic infiltrates in myocardial tissue (14,15,21-23) . Additionally, with antibodies against the major histocornpatibiiity antigens (MHC) . an increased expression of MHC I and 11 antigens could be detected . With these techniques, the diagnostic accuracy in the biopsy specimens could be increased (24) and the interobserver variability minimized (14,15) . Acute myocarditis was diagnosed when histologic sections from endomyocardial biopsy specimens revealed lymphocytic infiltrates in the region of myocardial necrosis . When lymphocytic infiltrates were seen by light microscopy in histologic sections without myocyte necrosis, the biopsy was repeated after 3 months . Ongoing myocarditis was diagnosed when the lymphocytic infiltrates had persisted or were increased at the second biopsy . When the lymphocytic infiltrates had disappeared, the patients were not classified as having myocarditis . When the immunohistologic examination revealed lymphocytic infiltrates and increased expression of MHC I and II antigens, the biopsy was also repeated after 3 months . When the lymphocytic infiltrates and increased expression of MHC I and It antigens persisted in two consecutive biopsies, a chronic inflammatory process of the myocardium was assumed . These patients were classified as having chronic myocarditis . A total of 198 consecutive patients with clinically suspected myocarditis were analyzed according to these criteria. On the basis of the Dallas criteria (13), which were meant to provide "a simple reproducible working classification" (17) rather than a definitive classification of acute myocarditis or a "conditio sine qua non" for the diagnosis of myocarditis (17), three of our patients met the criteria for acute myocarditis and five other patients for ongoing myocarditis. In 32 patients . the diagnosis of chronic myocarditis was made on the basis of immunohistologic data . The clinical data from these 40 patients are given in Table 1 . Patients were excluded if they had coronary heart disease at selective coronary angiography, systemic blood pressure

1 47

'fable 1 . Clinical Findings in 40 Patients With Myocarditis Patients (no.p P~,~i831i0n

Fatigue Pericardial effusion New rhythm disturbances Electrocardiographic changes Elevated creatine kinase level Left ventricular dysfunction

>150190 mm Hg, concomitant systemic or endocrinologic diseases known to cause left ventricular dysfunction or excessive alcohol consumption . Three groups of control subjects were enrolled in this study . Control group I comprised 43 patients with various cardiac diseases, including 31 patients with coronary artery disease, 7 with valvular heart disease and 5 with hypertensive heart disease . Control group lI comprised 12 patients with alcoholic cardiomyopathy . These patients presented

with impaired left ventricular function and had a history of excessive alcohol consumption (>I g of alcohoUkg body weight per day) . Clinical and laboratory findings as well as the histologic examination revealed no cause of impaired left ventricular function other than excessive alcohol consumption . Control group III consisted of 39 healthy persons with no history of myocardial disease . All subjects provided informed consent before participating in the study . The study protocol was approved by the ethics committee of the University of Dilsseldorf. Preparation of myosin. Myosin was prepared according to the method of Offer et al . (25), with slight modifications according to the method of L,ompre et al . (26) . Briefly, cardiac muscle specimens were obtained from the pathologic institute within 24 h after death . After homogenization, myosin was extracted in modified Guba-Straub solution (potassium chloride 0.3 mol/liter, magnesium chloride I mmollliter, sodium phosphate 150 mmollliter, sodium pyrophosphate 10 mmollliter, ethylenediaminetetraacetic acid (EDTA) 10 mmollliter, beta-mercaptoethanol 1%, pH 6 .5), centrifuged and then precipitated by lowering the ionic strength with EDTA I mmollliter, beta-mercaptoethanol 1%, pH 6 .5 . After dissociating actin with sodium pyrophosphate 100 mmollliter, adenosine 5'-triphosphate (ATP) 6 mmol/liter in EDTA 4 mmollliter, dithiothreitol 3 mmol/liter, pH 7 .0, further purification was carried out by ammonium sulfate fractionation . For immunization procedures, the myosin was further purified by ion exchange chromatography on DEAE Sephadex, using a potassium gradient from 0 to 500 mmol/ liter . The myosin solution was then dialyzed overnight against sodium chloride 500 mmollliter, tris(hydroxymethyl)aminomethane (Tris)/hydrochloride 10 mmollliter, EDTA I mmol/liter, pH 6 .5 at 4°C and stored in this solution . The purity was routinely checked by sodium dodecylsulfate gel



148

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electrophoresis . Skeletal muscle myosin was prepared using the same protocol from human iliopsoas muscle . Preparation of antihuman ventricular myosin antibodies . Antibodies against human ventricular myosin were raised in rabbits as described by Schwartz et al . (27) . One week before immunization, blood was taken from the rabbits as preimmune serum . Rabbits received an injection of the purified protein in Freund's incomplete adjuvant every 2 weeks for 2 months. Blood was drawn before each immunization procedure from the auricular artery for antibody titer monitoring . Antibody titers were maximal after five injections. The immunoglobulin G (IgG) fractions of human sera and rabbit antisera were prepared on a protein-A sepharose column (28) . Protein-A sepharose was equilibrated with phosphate-buffered saline solution with 0 .05% azide in an appropriate column . A total of 2 ml of serum was run on the column and the fraction of unbound protein was collected ("wash fraction") . The IgG bound to the column was eluted with 0 .1 mol/liter citrate, 0 .5 moUliter sodium chloride, pH 2 .9 immediately neutralized with 2 mol/liter Tris to a final pH of 7 .4, then dialyzed against phosphate-buffered saline solution overnight and concentrated by ultrafiltration . Enzyme-linked immunosorbent assay (ELISA) . The ELISA technique was performed as previously described by Lem and Hales (29) . Briefly, I Aglwell of purified myosin in phosphate-buffered saline solution was air dried on polyvinyl chloride 96 well plates . Free binding sites were blocked by incubating the plates with ELISA diluent (0 .2% Tween, 2% bovine serum albumin, 0 .4 mmol/liter Thimerosal, in phosphate-buffered saline solution) for I h, then antisera or patient sera diluted in ELISA diluent were added . After incubation at room temperature for 90 min, plates were washed three times in phosphate-buffered saline solution and 0 .2% Tween, and affinity-purified peroxidase-labeled goat antirabbit or antihuman antibody was added, respectively . After 90 min of incubation, the plates were rewashed three times and substrate (0 .4 mg/ml o-phenylenediamine, 0 .01% hydrogen peroxide in citrate buffer, pH 5 .0) was added . The reaction was stopped after 30 min with 4N sulfuric acid and the samples were read at 492 nm in an automated ELISA scanner . To test the specificity of the antibody binding in ELISA, 10 µl of serum in 400µl of phosphate-buffered saline solution was preincubated with 100 Ag of purified myosin from different human muscles (human left ventricle, human iliopsoas, human sartorius) overnight at 25°C before the ELISA procedure . Sera were also tested against myosin prepared from the different muscles . All sera were tested in duplicate . Western blot analysis. Western blot analysis was carried out as described previously . Purified myosin was loaded on a 8% acrylamide gel with a 4 .5% stacking gel (30) . After migration for 1 h at 125 V and 4 h at 250 V with weight markers as standard, the proteins were transferred to nitrocellulose sheets (31) with 195 mA for 2 h in 70% methanol in Tris/glycine buffer, pH 8 .3 . Appropriate stripes were cut out,

free binding sites blocked with phosphate-buffered saline solution, 2% bovine serum albumin overnight . The stripes were then incubated with the first antibody (rabbit antimyosin antisera or patikof sera . respectively) in phosphatebuffered saline wbnioA, d .2% Tween . After washing three times with phosphate-hv,&red saline solution, 0 .2% Tween, the second antibody was added (peroxidase-labeled antirabbit IgG or antihuman IgO, respectively) . The coloration was carried out in the dark with 0 .5% 4-chloro-naphthol, 0.1% hydrogen peroxide in methanol 15% in phosphate-buffered saline solution for 30 min . The specificity of the antibody binding to myosin was tested by preincubating 30 Al of serum in 3 ml of phosphate-buffered saline solution, Tween with 300 pg of myosin prepared from different muscles overnight at 25°C before incubation with the nitrocellulose sheets . Statistical analysis . The threshold for the detection of antimyosin autoantibodies in sera during ELISA was defined as the mean value ± 2 SD of the sera from 39 apparently healthy persons (control group 111) . The two-tailed chisquare test for independent populations was used to compare the prevalence of antimyosin antibodies in the different study groups . Statistical significance was defined as p < 0 .05 .

Results Autoantibodies against human ventricular myosin in sera from patients with myocarditis, alcoholic cardiomyopathy and other cardiac diseases. To study whether sera from patients with myocarditis contained anticardiac myosin autoantibodies, the sera were tested by ELISA with human ventricular myosin as antigen . Control values were established with sera from 39 apparently healthy persons (control group 111) . Sera were assumed positive for antimyosin antibodies if the binding in ELISA was above the mean value ± SD of the control group III . Figure f shows the extinction in ELISA of the sera from patients with myocarditis, alcoholic cardiomyopathy, other cardiac diseases and healthy blood donors . Table 2 gives the percent of antimyosin antibody-positive patients in the various groups . In 17 (42%) of 40 serum samples from patients with myocarditis, antibodies against human ventricular myosin were detected (p < 0 .05 vs . control group 111) . One of the 3 patients with acute myocarditis, 2 of the 5 patients with ongoing myocarditis and 14 of the 32 patients with immunohistologically diagnosed chronic myocarditis had antimyosin autoantibodies . There was no statistically significant difference in hemodynamic variables (cardiac index, ejection fraction, left ventricular end-diastolic pressure) between patients with myocarditis who had antimyosin antibody-positive versus antimyosin antibody-negative findings (data not shown) . None of the 12 patients with alcoholic cardiomyopathy showed antimyosin autoantibodies . In patients with other cardiac diseases, only 9 (21%) of 43 serum samples contained antibodies against human ven-



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Extinction

149

Extinction 2

I 2 4

I

I 1_

MC

Co I

CO 11

`` More after preincubation with h,_ -nan cardiac myosin

CO III

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

tricular myosin (p < 0 .05 vs . patients with myoca, difis, p < 0.05 vs . control group III) . These data demonstrate that many serum samples from patients with myocarditis contain autoantibodies against human cardiac ventricular myosin . The prevalence of antibodies against myosin is significantly higher in patients with myocarditis than in patients with other cardiac diseases .

Figure 2 . Specificity of the antibody binding for human ventricular myosin . Enzyme-linked immunosorbent assay (ELISA) was performed with serum from patients with myocarditis before and after preincubation of the serum with human ventricular myosin (see Melhnrls) . The black line indicates the mean value ± 2 SD for healthy persons (control group 111) as the threshold for the detection of antimyosin autoantibodies .

ventricular myosin before the ELISA procedure (Fig . 2). It was shown that preincubation of the sera with human ventricular myosin completely prevented the binding of the sera to human ventricular myosin . This showed that the binding in ELISA was specific for human ventricular myosin .

Table 2 shows the prevalence of antimyosin autoantibodies in sera from patients with myocarditis and from the

Localization of the antigenic determinant on the myosin molecule. The myosin molecule consists of six polypeptide

control patients at different serum dilutions . Nine (22 .5%) of the 40 patients with myocarditis showed antimyosin antibodies 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 patients

chains, two heavy chains of 200 kD (MHQ and four lighter polypeptides (two of 27 kD [MLCII and two of 10 M JMLC21) . To study which myosin subunit the antimyosin autoantibodies are directed against, the sera were tested

with other cardiac diseases, in only 3 (7%) of 43 could antimyosin antibodies be detected at a dilution of 1 :160 (p < 0.05 vs . myocarditis) ; at serum dilutions of 1 :640, 1 :2,560

against human ventricular myosin in Western blots . Polyclonal antibodies against human ventricular myosin raised in rabbits served as positive control sera . The polyclonal rabbit antihuman myosin antibodies bound against the myosin heavy chain (200 M) and against the myosin light chains

and 1 :10,240, no antibodies were demonstrated in these patients. To test the specificity of the antibody binding in ELISA for human ventricular myosin, the sera that showed antibody binding in ELISA were preincubated with human

Table 2 . Prevalence of Antimyosin Autoantibody-Positive Sera in Patients With Myocarditis and Control Groups Serum Dilution

1 :40

1 :160

1 :640

1 :2,560

1 :10 .240

Myocarditis (n = 40) Co I (n = 43) Co 11 (n = 12) CO III (n = 39)

17* (42%) 9r (21%) 0 (0%) 1 (2 .5%)

9*

4 (l07c) 0 (2 .3%) 0 (0%) 0 (0%)

3 (7 .5%) 0 (0%) 0 (0%) 0 (0%)

1 (2 .5--/,) 0 (0%) 0 (VC) 0 (0%)

(22 .5%) 3 (7.0%) 0 (0%) 0 (0%)

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

(migrating together in the 8% acrylamide gel) (Fig . 3) . Sera from patients with myocarditis that showed positive antibody binding in ELISA bound against the 200-kD protein, which is the myosin heavy chain . Control sera showed no antibody binding in Western blots . After preincubation of the sera with human ventricular myosin the autoantibody binding to the 200-0 protein was inhibited . In the region of the myosin light chains (27,000 D, 20,000 D), no autoantibody binding could be detected in the patient sera Classification of the autoantibodies against myosin . To classify the immunoglobulin type of the autoantibodies against myosin, IgG fractions from the 16 antibodycontaining human sera were prepared on a protein-A sepharose column . The various elution fractions were tested for binding to human ventricular myosin in ELISA. As shown for one serum sample in Figure 4, the IgG fraction bound strongly to myosin, whereas the wash fraction con-



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January 1994 :146-53

Extinction MHC

2

1 r

o

before incubation 1

2

3

4

5

6

~

after preincubation with cardiac skeletal myosin myosin

Extinction

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

taining other serum proteins, including other immunoglobulins, did not bind to myosin . This demonstrated that the autoantibodies against human ventricular myosin in patients with 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 with myocarditis is shown . The black line indicates the mean value ± 2 SD for healthy persons (control group 111) as the threshold for the detection of antimyosin autoantibodies . IgG fraction = ELISA was performed with the IgG fraction of the serum (see Methods) . Serum = ELISA was performed with serum from a patient with myocarditis ; "wash" fraction = ELISA was performed with the fraction of the serum that was not bound to a protein-A sepharose column (see Methods) . Extinction

Serum

" h" j fraction

2-

I

cardiac myosin

skeletal myosin

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

Specificity of the antimyosin antibodies for cardiac ventric-

ular myosin. To examine whether the antimyosin autoantibodies found in sera from patients with myocarditis are directed specifically against human ventricular myosin, antibody-positive sera were preincubated with myosin prepared from different striated muscles (human left ventricle, human skeletal muscle) . Figure 5A shows the results for seven serum samples . Preincubating the samples with any of the different myosin preparations inhibited autoantibody binding against human ventricular myosin in ELISA . Using either myosin from skeletal or cardiac muscle as antigen in ELISA showed that the sera bound equally to human ventricular myosin and skeletal myosin (Fig . 5B). This indicated that there is apparently no organ specificity of the antimyosin autobodies for human ventricular myosin .

Discussion fraction

Autoantibodies in Imyocarditis . In patients with myocarditis, several studies have demonstrated autoantibodies



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against cardiac proteins . Immunofluorescence studies (3236) showed autoantibodies against different cardiac structures (namely, membranes, mitochondria, cytoplasmic antigens) . Further studies identified the adenine nucleotide translocator of the inner mitochondrial membrane (4,37,38), calcium channel (7), Connexon (33) and cardiac betaadrenoreceptor (8) as autoantigens in myocarditis and dilated cardiomyopathy . For the adenine nucleotide translocator, a possible functional significance was shown (5,6) . The hypothesis that autoantibodies against myosin may play an important role in the pathogenesis of myocarditis is derived from animal studies . In animal models, antibodies against cardiac myosin have been demonstrated . Infection with Coxsackie-B3 virus leads to the development of myocarditis in genetically defined mice (39) . In the course of Coxsackie-B3 virus-induced murine myocarditis, heartspecific autoantibodies have been demonstrated (li) . In Western blot analysis, these antibodies bind to a 200-kD band that is thought to be the myosin heavy chain (10) . Immunization with myosin leads to histologic and immunehistologic alterations in mice hearts similar to those with Coxsackie-B3 virus infection (12) . From experimental data (40), a possible pathogenetic role of the antibodies was suspected . The present study clearly shows the existence of autoantibodies against human ventricular myosin in patients with myocarditis . These autoantibodies are directed against the myosin heavy chain . No antibody binding was seen with the myosin light chains . Recently, antibodies against myosin heavy chains were demonstrated in patients with the clinical presentation of dilated cardiomyopathy (41) . which in many cases is thought to be a sequela of myocarditis . In this study, the sensitive immunohistologic techniques for detecting lymphocytic infiltrates in myocardial biopsy specimens were not applied . Histologic and immunologic methods for the diagnosis of myocarditis . In the present study, the diagnosis of myccarditis was made using histologic (Dallas criteria [131) and mmunohistologic methods . The Dallas classification for the diagnosis of acute myocarditis was initially established by a group of outstanding pathologists . The goals of the group were to: 1) produce a morphologic definition of myocarditis, 2) develop histologic criteria for the diagnosis of myocarditis, 3) establish a simple reproducible working classification, 4) outline the problems and pitfalls in establishing the diagnosis of myocarditis, 5) assess the applicability and reproducibility of the classification system itself, and 6) make this information available to other pathologists and clinicians (13,17) . However, as these pathologists (17) have emphasized, the Dallas criteria were not meant to establish a definitive diagnosis of myocarditis and should not be misunderstood and misinterpreted as a "conditio sine qua non" of the diagnosis of myocarditis . The present study used the Dallas criteria (13) in addition to immunohistologic techniques for the detection of lymphocytic infiltrates (14,15,18-20) and an increased expression of MHC I and 11 antigens . With these techniques, the diagnos-

15 1

tic accuracy was increased (24) and the interobserver variability was minimized (14,15) . We believe that the fact that in two consecutive biopsy specimens, chronic lymphocytic infiltrates and an increased expression of MHC I and II antigens could be detected points to a chronic inflammatory process in the myocardium and therefore these patients were classified as having chronic myocarditis . In the present study, 3 patients met the histologic Dallas criteria for acute myocarditis and 5 for ongoing myocarditis ; 32 patients met the immunohistologic criteria for chronic myocarditis . In I patient with acute myocarditis, 2 patients with ongoing myocarditis and 14 patients with chronic myocarditis, antimyosin antibodies could be detected . Because of the small number of patients with acute myocarditis and ongoing myocarditis, a comparison between these groups and the chronic myocarditis group is not appropriate . Acute myocarditis can resolve spontaneously, or it may lead to a chronic inflammatory, perhaps autoimmunologically mediated, process . In the present study, only one of three patients with acute myocarditis showed antimyosin autoantibodies . Because the acute phase of the disease is of short duration, one cannot expect that IgG autoantibodies against an intracellular protein as a possible marker of an autoimmune process are always detectable at this early stage of the disease . Therefore, it is not surprising that only one of the three patients with acute myocarditis showed autoantibodies against human ventricular myosin . In the present study, we found no significant difference in hemodynamic variables between patients with myocarditis who had antimyosin antibody positive versus negative findings . At least in some patients, myocarditis is thought to be a chronic process . In the present study, the hemodynamic variables were evaluated at only one time point in this chronic process . Longitudinal studies are necessary to evaluate the relation between changes in hemodynamic variables and the persistence of antimyosin autoantibodies in patients with myocarditis . Antimyosin Ruto2atibodles in human cardiac disease . Patients with impaired ventricular function due to alcoholic cardiornyopathy did not show autoantibodies against human ventricular myosin . This is a strong indicator that the presence of antimyosin autoantibodies is linked to a chronic inflammatory, perhaps autoirnmunotogically mediated, process rather than to the disturbed left ventricular function itself. In the present study, patients with other cardiac diseases also showed autoantibodies against human ventricular myosin but to a significantly lower extent . The prevalence of antimyosin autoantibodies in patients with other cardiac diseases is similar to that in patients 1 year after cardiac surgery (42) . In the study of Caforio et al . (41), only 1 of 12 serum samples from patients with heart failure due to other cardiac diseases showed antimyosin antibodies . Their control group comprised preselected patients with no antibody binding to heart tissue in immunofluorescence studies (41) . High antimyosin autoantibody titers were more common i n p atients with myocarditis than in those with other



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cardiac diseases . This might be interpreted as indirect evidence for an autoimmunologic process in patients with myocarditis . The present study could not demonstrate any organ specificity of the antimyosin autoantibodies in patients with myocarditis . In ELISA, the antibodies bind equally to ventricular and skeletal myosin, and the binding was equally inhibited by myosin from ventricular or skeletal muscle . This is not surprising because the beta-myosin heavy chain of human cardiac muscle and the myosin heavy chain of slow skeletal muscle are products of the same gene (43), and the ventricular myosin heavy chain in humans is composed of 90% to 95% of beta-myosin heavy chain and only 5% to 10% of alpha-myosin heavy chain (44) . In mice, immunofluorescence studies (10) with cardiac specific antimyosin antibodies demonstrated a striational pattern (10) . In humans, antibodies showing a striational pattern in immunofluorescence were not organ specific and were bound equally to cardiac and skeletal muscle (45) . Because the autoantibodies demonstrated in the present study are directed against the myosin heavy chain, no specificity of the autoantibodies for either cardiac or skeletal myosin could be expected . Possible mechanisms of autolmmunity in myocarditis . The present study provides further evidence of an autoimmunologic process in human myocarditis (40) . The induction of autoantibodies in the course of myocarditis and dilated cardiomyopathy is not yet clear . One possible explanation would be "molecular mimicry ." Schwimmbeck et al . (46), using computer analysis of protein sequences, identified regions of high homology between Coxsackie B3 virus and rabbit cardiac myosin (46) . Using synthetic peptides identical to those in these regions, an immunologic crossreactivity between Coxsackie B3 virus peptides and myosin peptides was demonstrated (46) . In contrast, the antimyosin autoantibodies produced in Coxsackie B3 virus-induced myocarditis in AJJ mice did not cross-react with the virus (47) . This argues against molecular mimicry as the pathoLenetic pathway of autoimmunity in inflammatory heart disease . Another possibility is the exposition of sequestered antigens to the immune system in the course of myocarditis . Intracellular proteins such as myosin are possibly exposed to the immunogenic cells, thus perpetuating the autoimmunologic process . The pathogenetic mechanisms by which autoantibodies directed against intracellular proteins such as myosin may play a role in the autoimmunologic process remain controversial . It cannot be excluded that the antibodies may penetrate into the myocardial cells and impair cardiac function (5,6) . Conversely, assessment of whether the detection of autoantibodies against human ventricular myosin may serve as an index for the activity of the inflammatory process in the heart and be useful for monitoring the clinical course of the disease will require further study . In murine autoimmune myocarditis, the antimyosin autoantibodies do not seem to be involved in the induction of myocarditis (48) . In this context, it should be noted that scintigraphic studies (49)

with antimyosin antibodies suggest a lysis of heart cells in patients with myocarditis and dilated cardio*nyopathy . Conclusions. The present study demonstrates the presence of autoantibodies against human ventricular myosin in patients with myocarditis . The prevalence of antimyosin autoantibodies is significantly higher in patients with myocarditis than in patients with other cardiac diseases . The antimyosin autoantibodies are of the IgG type . They are directed against the myosin heavy chain and react with myosin from cardiac and skeletal muscles .

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