During the past decade, convincing evidence has suggested that autoimmunity plays an important role in the pathology of dilated cardiomyopathy (DCM) (1-10). We have previously demonstrated the presence of circulating autoantibodies against the human β1-adrenoceptor in approximately 32% of patients with DCM (11). Moreover, these autoantibodies were functionally active both in vitro (3,10,12,13) and in vivo (14) in the cardiovascular system. The immunization of rabbits with receptor peptide was able to induce cardiomyopathy (14).
Angiotensin-converting enzyme inhibitor (ACEI) has been shown to improve the quality of life and prognosis in patients with chronic heart failure (15,16). Moreover, ACEI was able to induce regression in myocardial hypertrophy and cardiac interstitial fibrosis in spontaneously hypertensive rats with left ventricular hypertrophy (17-19). It is our hypothesis that ACEI can exert a protective effect on cardiomyopathic damage induced by β1-receptor autoimmunity.
In this study, we investigate the effect of ACEI, lisinopril, on cardiomyopathic changes induced by immunizing rabbits with β1-peptide.
MATERIALS AND METHODS
Peptide corresponding to the second extracellular loop of human β1-adrenoceptor (β1-peptide), H-W-W-R-A-E-S-D-E-A-R-R-C-Y-N-D-P-K-C-C-D-F-V-T-N-R, was used, and it was synthesized commercially by TORAY Research Center, Inc. (Tokyo, Japan).
A total of 40 rabbits was divided into four groups with 10 animals in each: the control group, the β1-peptide group, the ACEI group and the ACEI + β1-peptide group. All rabbits in the β1-peptide group and the ACEI + β1-peptide group were immunized subcutaneously with β1-peptide (1 mg peptide + 1 ml complete Freund's adjuvant per animal), whereas in the control group and the ACEI group only saline and Freund's adjuvant of the same volume as in the β1-peptide group and ACEI + β1-peptide group were injected. The experiment was carried out during 1 year using the same procedure as that described in our recent publication (14). Both the ACEI and the ACEI + β1-peptide groups were treated with 3 mg lisinopril, which was mixed with the drinking water each day. All rabbits were bled monthly, and vital organs were harvested for later analysis at the end of the experiment. Animal models and experimental procedures were approved by the Animal Ethics Committee, Kanazawa Medical University, Japan.
Plasma concentration of lisinopril was measured by high-performance liquid chromatographic methods.
Enzyme-linked immunosorbent assay
The enzyme-linked immunosorbent assay (ELISA) experiments were performed as described in our previously published work (14).
Cardiac anatomic measurements, light microscopy and electron microscopy
A detailed description of these methods has been provided in our previously published work (14).
All values are expressed as the mean ± SD. The statistics were calculated using StatView 5.0 (SAS Institute Inc., North Carolina, U.S.A.).
Two rabbits in the β1-peptide group, four in the ACEI group, three in the ACEI + β1-peptide group and four rabbits in the control group died within 4 months as a result of spinal injury. Two rabbits in the β1-peptide group died suddenly after 5 and 9 months, respectively. Accordingly, we studied six rabbits in the control group, eight in the β1-peptide group, six in the ACEI group and seven rabbits in the ACEI + β1-peptide group, except for the rabbits that died due to spinal injury.
Plasma concentration of lisinopril
All rabbits in the ACEI group and the ACEI + β1-peptide group showed satisfying plasma concentration of lisinopril throughout the experiment (672 ± 209.8 ng/ml in the ACEI group, 462 ± 173.9 ng/ml in the ACEI + β1-peptide group). There was no difference between the two groups.
Enzyme linked immunosorbent assay
Both the β1-peptide group and the ACEI + β1-peptide group showed increasing titer of anti-β1-adrenoceptor antibody throughout the study. The main immunoglobulin (Ig) isotype in most antisera was IgG. This trend was not seen in the control or ACEI groups, which maintained insignificant levels of antibody throughout the experiment (Fig. 1).
Body weight and heart weight
There was a significant difference among the β1-peptide, ACEI and ACEI + β1-peptide groups in terms of final body weight. The heart weight and heart : body weight ratio in the β1-peptide group were significantly increased as compared with those in the control group. On the other hand, there were no differences between the ACEI group and ACEI + β1-peptide group in terms of final body weight, heart weight and heart : body weight ratio (Table 1).
In the β1-peptide group, the hearts showed mild to moderate dilatation with wall thinning in both ventricles. These changes were more prominent in the right ventricle. The hearts in the control group, the ACEI group and ACEI + β1-peptide group showed normal chamber size and wall thickness. In all groups, there was neither appreciable fluid in the pericardial, pleural and peritoneal cavities nor obvious changes in the lungs, liver, kidneys and spleen.
Cardiac anatomic measurements
The cavity dimensions of the right and left ventricles were enlarged, and the wall thickness of the right and left ventricles was decreased in the β1-peptide group as compared with that in the control group. However, there were no differences between the ACEI group and ACEI + β1-peptide group in terms of anatomical measurements (Table 2).
Light microscopic findings
In all rabbits of the β1-peptide group, the hearts showed significant alterations, as shown by multifocal degeneration and necrosis of myocardial cells together with infiltration of inflammatory cells (Fig. 2a). These inflammatory cells were mainly mononuclear cells (lymphocytes and macrophages). These changes were present in the walls of both ventricles and interventricular septum, but the right ventricle was more commonly affected than the left. Subendocardial mild to moderate fibrosis was occasionally found (Fig. 2b). In the ACEI + β1-peptide group, four of seven rabbits showed no histological changes in the hearts (Fig. 2c). The other three exhibited focal degeneration and necrosis of myocardial cells accompanied by mononuclear cells such as lymphocytes and macrophages (Fig. 2d). The lesions in this group were less marked than those found in the β1-peptide group. In the ACEI group, three rabbits showed no histological changes in the hearts, but the other three rabbits showed an occasional focus of scant mononuclear cell infiltration with normal myocardial cells (Figs 2e and f). In the control group, five rabbits were found to have no histological change in the heart (Fig. 2g), whereas one rabbit had slight degenerative changes accompanied by scant mononuclear cell infiltration in the heart.
Other organs such as lungs, liver, kidneys and spleen were also examined by light microscopy. In all rabbits of the β1-peptide group, there was mild congestion in the lungs and liver, but no change in kidneys and spleen. The other three groups showed no obvious change in lungs, liver, kidneys or spleen.
Electron microscopic findings
In the β1-peptide group, the hearts exhibited significant alterations as shown by focal myofibrillar lysis, loss of the normal myofilament banding pattern with hypercontraction bands, increase in the number of mitochondria, and deposition of dense granules in both the sarcoplasm and myofibrils. The contour of myocytes was irregular, and plasma membranes were discontinuous in some cells. All altered myocytes were fairly widely distributed throughout the myocardium. The interstitium showed edema, deposits of flocculent serum proteins, occasional activated fibroblasts and increased amounts of collagen fibers. Lymphocytes and macrophages were also found. In the ACEI + β1-peptide group, ACEI group and control group, no obvious alterations were observed in spite of their slight to mild light microscopic abnormalities.
Since the epitopic targets of the autoantibodies against the β1-adrenoceptor was defined in DCM (3,9-13), the opportunity to define the importance of this autoimmune component in the development of DCM was examined by setting up an experimental model of active immunization according to one of the criteria advanced by Rose and Bona (20). In 1997, we established an experimental model in rabbits by immunization for 1 year with a synthetic peptide corresponding to the sequence of the second extracellular loop of β1-adrenoceptor to resemble the early stage of cardiomyopathy (14).
We used the same animal model, as already described, in order to evaluate the effects of ACEI, lisinopril, in rabbits. As was shown in the results section, immunization is successful since only the immunized animals produced antibodies with increasing titers until the second experimental month, after which they retained constantly high levels throughout the study period. The DCM-like changes seen in rabbits of the β1-peptide group were moderate, with increased heart weight, dilated ventricles and marked wall thinning. The unimmunized groups (control and ACEI groups) were, as expected, almost unaffected, showing only very mild microscopic signs of DCM in rare cases. What is more interesting is that the rabbits in the ACEI + β1-peptide group were significantly protected from developing DCM by the ACEI treatment; only a few of them had pathological alterations, which in all cases were apparently much milder than those of the β1-peptide group. Thus, this study demonstrated unambiguously that blocking of the renin-angiotensin system (RAS) exerts a beneficial effect in rabbits that have developed DCM on an autoimmune basis.
In the hearts of rabbits immunized with β1-peptide, rabbit IgG was localized on the sarcolemma of cardiomyocytes. CD4 and CD8 T cells and macrophages were recognized in the myocardium (21). Moreover, both major histocompatibility complex class I and class II were expressed in the myocardium (21). Based on these immunohistological findings, the following mechanism of myocardial injury induced by β1-peptide may be proposed: (1) antibody-dependent cell-mediated cytotoxicity; (2) T-cell-mediated cytotoxicity; and (3) antibody-mediated cellular dysfunction.
The anti-β1-adrenoceptor antibodies from immunized rabbits have been shown to display agonist-like effect against β1-adrenoceptor (unpublished data). In addition, the β-adrenoceptor blocking agent, bisoprolol, completely protected the myocardium from injury induced by the autoimmune mechanism against β1-adrenoceptor (22). Based on these findings, we speculated that myocardial injury in the β1-peptide group might be induced by antibody-mediated cellular dysfunction, i.e chronic stimulation by autoantibodies against β1-adrenoceptor might result in considerable myocardial hypertrophy, necrosis and fibrosis (23).
In the present state of our knowledge, it is impossible to describe the exact mechanism for myocardial protection by ACEI, lisinopril. ACEI may exert its immunomodulatory effect through the stimulation of T lymphocytes and suppression of interleukin-12 (24,25). It is also highly possible that the inhibition of the RAS by ACEI may result in protective effects on the cardiovascular alterations induced by antibodies. As we know, systemic and/or local activation of the RAS is seen in chronic heart failure due to cardiomyopathy (26-28). Angiotensin II plays a major role in modulating extracellular fluid volume and systemic vascular resistance. It is now known that angiotensin II is also involved in cell growth and differentiation, and that it plays a role in ventricular hypertrophy and fibrosis, vascular media hypertrophy, and structural alterations of the heart (29,30). Moreover, the RAS and adrenergic nervous system are cross-regulated by interactive compensatory mechanisms (31). In chronic heart failure, increased adrenergic activity is also seen (32). Norepinephrine also has effects on the growth of cardiovascular tissue and may thereby play an important role in the remodeling of the heart (22,23). The inhibition of RAS by ACEI would attenuate the activity of the adrenergic nervous system and result in cardiac protection in rabbits immunized with β1-peptide.
As a future perspective, it would of course be of interest to set up controlled clinical studies to investigate whether the protective effect of ACEI observed in animals also applies to human DCM patients on an autoimmune basis.
Acknowledgement: This work is supported by Kanazawa Medical University Project Research Grant (P99-6).
1. Schultheiss HP, Bolte HD. Immunological analysis of autoantibodies against the adenine nucleotide translocator in dilated cardiomyopathy
. J Mol Cell Cardiol
2. Wolff PG, Kuhl U, Schultheiss HP. Laminin distribution and autoantibodies to laminin in dilated cardiomyopathy
and myocarditis. Am Heart J
3. Magnusson Y, Marullo S, Hoyer S, et al. Mapping of a functional autoimmune epitope on the β1-adrenergic receptor in patients with idiopathic dilated cardiomyopathy
. J Clin Invest
4. Neumann DA, Burek CL, Baughman KL, Rose NR, Herskowitz A. Circulating heart-reactive antibodies in patients with myocarditis or cardiomyopathy
. J Am Coll Cardiol
5. Caforio AL, Bonifacio E, Stewart JT, et al. Novel organ-specific circulating cardiac autoantibodies in dilated cardiomyopathy
. J Am Coll Cardiol
6. Fu MLX, Magnusson Y, Bergh CH, et al. Localization of a functional autoimmune epitope on the muscarinic acetylcholine receptor-2 in patients with idiopathic dilated cardiomyopathy
. J Clin Invest
7. Latif N, Baker CS, Dunn MJ, Rose ML, Brady P, Yacoub MH. Frequency and specificity of anti-heart antibodies in patients with dilated cardiomyopathy
detected using SDS-PAGE and Western blotting. J Am Coll Cardiol
8. Lauer B, Padberg K, Schultheiss HP, Strauer BE. Autoantibodies against human ventricular myosin in sera of patients with acute and chronic myocarditis. J Am Coll Cardiol
9. Fu MLX, Hoebeke J, Matsui S, et al. Autoantibodies against cardiac G-protein-coupled receptors define different populations with cardiomyopathies but not with hypertension. Clin Immunol Immunopathol
10. Magnusson Y, Wullkat G, Waagstein F. Autoimmunity
in idiopathic dilated cardiomyopathy
. Characterization of antibodies against the β1-adrenoceptor
with positive chronotropic effect. Circulation
11. Matsui S, Fu MLX, Shimizu M, et al. Dilated cardiomyopathy
defines serum autoantibodies against G-protein-coupled cardiovascular receptors. Autoimmunity
12. Magnusson Y, Wallukat G, Guillet JG, Hjalmarson Å, Hoebeke J. Functional analysis of rabbit anti-peptide antibodies which mimic autoantibodies against the β1-adrenergic receptor in patients with idiopathic dilated cardiomyopathy
. J Autoimmun
13. Wallukat G, Wollenberger A, Morwinski R, Pitschner HF. Anti-β1-adrenoceptor
autoantibodies with chronotropic activity from the serum of patients with dilated cardiomyopathy
: mapping of epitopes in the first and second extracellular loops. J Mol Cell Cardiol
14. Matsui S, Fu MLX, Katsuda S, et al. Peptides derived from cardiovascular G-protein-coupled receptors induce morphological cardiomyopathic changes in immunized rabbits. J Mol Cell Cardiol
15. The CONSENSUS Trial Group. Effect of enalapril on mortality in severe congestive heart failure: results of the cooperative North Scandinavian enarapril survival study (CONSENSUS). N Engl J Med
16. Pfeffer MA, Braunwald E, Moye LA, et al. Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med
17. Brilla CG, Matsubara L, Weber KT. Advanced hypertensive heart disease in spontaneously hypertensive rats. Lisinopril-mediated regression of myocardial fibrosis. Hypertension
18. Brooks WW, Bing OH, Robinson KG, Slawsky MT, Chaletsky DM, Conrad CH. Effect of angiotensin-converting enzyme inhibition on myocardial fibrosis and function in hypertrophied and failing myocardium from the spontaneously hypertensive rat. Circulation
19. Susic D, Varagic J, Frohlich ED. Pharmacologic agents on cardiovascular mass, coronary dynamics and collagen in aged spontaneously hypertensive rats. J Hypertens
20. Rose NR, Bona C. Defining criteria for autoimmune diseases. Immunol Today
21. Hayase M, Matsui S, Katsuda S, et al. Immune mechanism of myocardial injury due to anti-β1-adrenoceptor
autoantibody: immunohistological study of myocardial disorders. J Cardiovasc Pharmacol
22. Matsui S, Fu LXM. The protective effect of bisoprolol on beta 1 receptor autoimmunity
in the rabbits. Herz
2000 (in press).
23. Engelhardt S, Hein L, Wiesmann F, Lohse MJ. Progressive hypertrophy and heart failure in β1-adrenergic receptor transgenic mice. Proc Natl Acad Sci USA
24. Tarkowski A, Carlsten H, Herlitz H, Westberg G. Differential effects of captopril and enalapril, two angiotensin converting enzyme inhibitors, on immune reactivity in experimental lupus disease. Agents Actions
25. Constantinescu CS, Goodman DB, Ventura ES. Captopril and lisinopril suppress production of interleukin-12 by human peripheral blood mononuclear cells. Immunol Lett
26. Pinto YM, Buikema H, van Gilst WH, Lie KI. Activated tissue renin-angiotensin systems add to the progression of heart failure. Basic Res Cardiol
27. Nicholls DP, Onuoha GN, McDowell G, et al. Neuroendocrine changes in chronic cardiac failure. Basic Res Cardiol
28. Pathak SK, Kukreja RC, Hess M. Molecular pathology of dilated cardiomyopathies. Curr Problems Cardiol
29. Dzau VJ. Implications of local angiotensin production in cardiovascular physiology and pharmacology. Am J Cardiol
30. Griffin SA, Brown WCB, MacPherson F, et al. Angiotensin II causes vascular hypertrophy in part by a non-presser mechanism. Hypertension
31. Cody RJ. The sympathetic nervous system and the renin-angiotensin-aldosteron system in cardiovascular disease. Am J Cardiol
32. Benedict CR, Weiner DH, Johnstone DE, et al. Comparative neurohumoral responses in patients with preserved and impaired left ventricular ejection fraction: results of the Studies of Left Ventricular Dysfunction (SOLVED) registry. J Am Coll Cardiol
The symposium and the publication of this supplement were supported by an educational grant from Novartis Pharma K.K. Tokyo, Japan.