Kohei Teraoka

Dilated cardiomyopathy defines serum autoantibodies against G-protein-coupled cardiovascular receptors

In order further to identify the prevalence of anti-receptor autoantibodies in the sera of patients with dilated cardiomyopathy (DCM), we attempted to detect autoantibodies against a series of G-protein-coupled cardiovascular receptors in a well-defined population of DCM patients from Japan. Peptides corresponding to the sequences of the second extracellular loops of the human beta 1 and beta 2 adrenoceptors, alpha 1 adrenoceptors, M2 muscarinic acetylcholine receptors and angiotensin II-1 (AT1) receptors were used as antigens in an enzyme immunoassay to screen the sera from patients with DCM (n = 28). Nine sera from patients with DCM (32%) and 2 sera from healthy subjects (9%) recognized the beta 1 adrenoceptor peptide. Ten sera from patients (36%) and 3 sera from healthy subjects (13%) recognized the M2 receptor peptide. Thirty-six per cent of the patients with autoantibody against the beta 1 adrenoceptor peptide. Ten sera from patients (36%) and 3 sera from healthy subjects (13%) recognized the M2 receptor peptide. Thirty-six per cent of the patients with autoantibody against the beta 1 adrenoceptor had autoantibody against the M2 receptor. However, no significantly high frequencies of autoantibodies against the beta 2 adrenoceptor, alpha 1 adrenoceptor and AT1 receptor were found in DCM patients. Our results demonstrate that a subgroup of patients with DCM have a specific spectrum of autoantibodies which are specifically directed against the second extracellular loops of the beta 1 adrenoceptors and M2 muscarinic receptors rather than other cardiovascular receptors.

Active immunization of combined β1-adrenoceptor and M2-muscarinic receptor peptides induces cardiac hypertrophy in rabbits

BACKGROUND The high prevalence of patients with dilated cardiomyopathy (DCM) with anti-beta1-adrenoceptor and/or anti-M2-muscarinic receptor autoantibodies in their sera has been observed. However, the pathophysiological role of these autoantibodies in the development of cardiomyopathy is unknown. We previously reported an experimental model of early-stage DCM-like cardiomyopathy induced by immunizing rabbits for 1 year with synthetic peptides corresponding to the sequence of the second extracellular loop of either beta1-adrenoceptor or M2-muscarinic receptor. Because approximately half the sera of patients with DCM that recognize one of the two receptor sequences also recognize the second sequence, a model was created in rabbits simultaneously immunized with the synthetic peptides corresponding to the second extracellular loop of the beta1-adrenoceptor and M2-muscarinic receptor. METHODS AND RESULTS All rabbits (n = 8) immunized with both peptides had a high titer of both anti-beta1-adrenoceptor and anti-M2-muscarinic receptor autoantibodies in their sera, whereas none of the sera from control rabbits injected with saline (n = 9) was positive. No significant cross-reaction with peptides other than those used for immunization was found. The weight of the hearts of immunized rabbits increased significantly. The hearts of immunized rabbits showed marked concentric left ventricular hypertrophy with mild inflammatory cell infiltration. In these rabbits, mild or moderate interstitial fibrosis was also observed. In electron micrographs, immunized rabbits showed focal myofibrillar lysis, loss of myofilament, and a marked increase in the number of mitochondria and deposition of dense granules in both sarcoplasm and myofibrils. Conversely, one of the control rabbits showed scant mononuclear cell infiltration. However, in this control rabbit, no significant alteration was found by electron microscopy. CONCLUSION Our results showed the coexistence of both anti-beta1-adrenoceptor and anti-M2-muscarinic receptor autoantibodies in the sera has pathophysiological importance, shown by their ability to induce cardiac hypertrophy in rabbits.

Transfer of rabbit autoimmune cardiomyopathy into severe combined immunodeficiency mice.

Summary: Growing evidence suggests that the autoimmune mechanism plays an important role in the pathogenesis of dilated cardiomyopathy. The purpose of this study was to evaluate the effect on the cardiac structure and function by the transfer of immunoglobulin G (IgG) and/or lymphocytes from rabbits immunized with a synthetic peptide corresponding to the sequence of the second extracellular loop of &bgr;1‐adrenoceptor (&bgr; peptide) into severe combined immunodeficiency (SCID) mice. CB‐17 SCID mice were injected intraperitoneally with 2 mg of IgG and/or 1 × 107 peripheral blood lymphocytes (PBL) from either rabbits immunized with both &bgr;1 peptide and adjuvant (&bgr; group), and adjuvant or rabbits with adjuvant only (N group). Thirty‐five SCID mice were divided into seven groups: (1) N‐IgG group; (2) N‐PBL group; (3) N‐IgG+PBL group; (4) &bgr;‐IgG group; (5) &bgr;‐PBL group; (6) &bgr;‐IgG+PBL group; and (7) control group. Morphological, serological and endocrinological studies were performed 70 days after the transfer. Results showed that heart weight and heart weight/body weight ratio in the &bgr;‐IgG+PBL group tended to be increased as compared with those in other groups. All mice in the &bgr;‐IgG group, two in the &bgr;‐PBL group and four in the &bgr;‐IgG+PBL group showed high titer of rabbit anti‐ &bgr;1‐adrenoceptor antibodies. Brain natriuretic peptide in the &bgr;‐IgG+PBL group showed a significant increase as compared with those in the control group and N‐IgG+PBL. Pathohistologically, focal infiltration of inflammatory cells in the myocardium was observed in one mouse of the &bgr;‐IgG+PBL group. Rabbit CD3‐positive T‐lymphocytes in the myocardium were observed in two mice of the &bgr; group. In conclusion, transfer of IgG and PBL from rabbits immunized with &bgr;1 peptide was able to induce the early stages of myocardial damage in SCID mice. These data provide direct evidence that the autoimmune mechanism is important in the pathogenesis of dilated cardiomyopathy.

Protective effect of bisoprolol on beta-1 adrenoceptor peptide-induced autoimmune myocardial damage in rabbits.

Idiopathic dilated cardiomyopathy is a severe disease of unknown etiology. Accumulating evidence suggests that agonist-like autoantibodies against the β1 adrenoceptor in the circulation of dilated cardiomyopathy may play an important role. The aim of this study was to evaluate the effects of the selective β1-adrenoceptor blocker, bisoprolol, on β1-adrenoceptor peptide induced autoimmune myocardial damage.In the animal model of autoimmune cardiomyopathy induced by active immunization of rabbits with β1-adrenoceptor peptide, bisoprolol was given at a dose of 3 mg/day throughout the study period. Our results showed high titer of anti-β1-adrenoceptor antibody in the immunized group throughout the study but not in the group receiving only bisoprolol. Cross-reactivity ot β2 adrenoceptors was observed in some of the immunized rabbits, but disapearred almost entirely after 6 months. As compared to the β1-adrenoceptor peptide immunized group without bisoprolol treatment, bisoprolol treated β1-receptor peptide immunized group showed increase in the wall thickness and decreases in cavity dimension in anatomical measurements and only mild alterations in macro- and microscopic examinations.Thus, our study clearly demonstrated a beneficial effect of bisoprolol in rabbits who have developed autoimmune myocardial damage.ZusammenfassungDie idiopathische dilatative Kardiomyopathie ist eine schwere Erkrankung unbekannter Ätiologie. Es gibt zunehmend mehr Hinweise, dass Autoantikörpern mit agonistischer Wirkung für den β1-Rezeptor im Kreislauf bei dilatativer Kardiomyopathie eine Bedeutung zukommt. Das Ziel der Studie war, die Auswirkungen einer selektiven β1-Rezeptoren-Blockade mit Bisoprolol auf autoimmun bedingte Myokardschädigungen, die nach Verabreichung eines β1-Adrenorezeptorpeptids auftreten, zu untersuchen.Kaninchen wurden aktiv mit einem β1-Adrenorezeptorpeptid immunisiert und Bisoprolol in einer Dosierung von 3 mg/Tag verabreicht. Es wurden hohe Antikörpertiter gegen den β1-Rezeptor in der immunisierten Gruppe, nicht aber in der nur mit Bisoprolol behandelten Gruppe beobachtet. Bei einem Teil der immunisierten Tiere wurde anfänglich eine Kreuzreaktivität gegen den β2-Rezeptor beobachtet, die aber nach sechs Monaten verschwand. Im Vergleich zum immunisierten Gruppe ohne Bisoprolol-Behandlung wissen die zusätzlich mit Bisoprolol behandelten immunisierten Tiere eine Zunahme der ventrikulären Wanddicke und eine Abnahme des ventrikulären Innenvolumens, verbunden mit nur geringfügigen makroskopischen und mikroskopischen Veränderungen auf.Die Studie zeigt daher günstige Auswirkungen einer Bisoprolol-Behandlung bei Kaninchen, die autoimmun bedingte Myokardschädigungen aufweisen.

Transfer of immune components from rabbit autoimmune cardiomyopathy into severe combined immunodeficiency (SCID) mice induces cardiomyopathic changes

Background: Growing evidence suggests that autoimmune mechanism plays an important role in the pathogenesis of cardiomyopathy. The purpose of this study was to investigate whether passive transfer of IgG and/or lymphocytes from rabbits with autoimmune cardiomyopathy is able to reproduce cardiomyopathic changes in severe combined immunodeficiency (SCID) mice. Methods and results: SCID mice were injected intraperitoneally with IgG and/or peripheral blood lymphocytes (PBL) from either rabbits immunized with both β1-adrenoceptor peptide and M2-muscarinic receptor peptide (β1+M2 group) or rabbits with adjuvant (N group). Thirty five SCID mice were divided into seven groups; N-IgG, N-PBL, N-IgG & PBL, (β1+M2)-IgG, (β1+M2)-PBL, (β1+M2)-IgG & PBL and control groups. Heart weight in three (β1+M2) groups were significantly increased. All mice in three (β1+M2) groups showed high titer of rabbit anti-β1 adrenocepter autoantibodies, and 4 mice in the (β1+M2)-PBL group and 3 mice in the (β1+M2)-IgG & PBL group showed a significant increase in titer of rabbit anti-M2-muscarinic receptor autoantibodies. Focal infiltration of inflammatory cells in the myocardium was observed in the (β1+M2)-IgG & PBL group. In the (β1+M2)-PBL group and (β1+M2)-IgG & PBL group, cardiomyocyte diameters were significantly increased. Some myocytes of the (β1+M2)-IgG & PBL group exhibited intracellular edema, clumps of Z-band and increased numbers of mitochondria by using electron microscopy. Conclusion: Transfer of IgG and PBL from rabbits immunized with combined β1 and M2 peptides was able to reproduce the early stage of cardiomyopathic changes in SCID mice

Beneficial effect of muscarinic-2 antagonist on dilated cardiomyopathy induced by autoimmune mechanism against muscarinic-2 receptor.

Summary: We have previously shown that a peptide corresponding to the sequence of the second extracellular loop of the human muscarinic‐2 (M2) receptor (M2‐peptide) was able to induce an autoimmune cardiomyopathy in rabbits. In this study, we investigated the effect of M2‐antagonist (otenzepad) on M2‐peptide‐induced cardiomyopathy in rabbits. New Zealand White rabbits were divided into four groups: 1) control group, saline injection; 2) M2‐peptide group, M2‐peptide injection; 3) M2‐antagonist group, otenzepad (30 mg/day) orally and saline injection; and (4) M2‐antagonist + M2‐peptide group, otenzepad (30 mg/day) orally and M2‐peptide injection. The study duration was 1 year. Saline or peptide was injected once a month. All rabbits in both the M2‐peptide group and the M2‐antagonist + M2‐peptide group had high titers of anti‐M2‐ autoantibodies in their sera. Rabbits in the M2‐peptide group showed an increase in heart weight, wall thinning and dilatation of the right ventricle. On the contrary, rabbits in the M2‐antagonist + M2‐peptide group had normal heart weight and shape. All rabbits in the M2‐peptide group showed multifocal degeneration and necrosis of myocardial cells with moderate infiltration of inflammatory cells, while four rabbits in the M2‐antagonist + M2‐peptide group showed slight infiltration of inflammatory cells with normal myocardial cells and interstitium, and another three showed no histological changes in the hearts. In conclusion, M2‐antagonist protects the myocardium from injury induced by autoimmune mechanism against M2‐muscarinic receptor.

Beneficial effect of angiotensin-converting enzyme inhibitor on dilated cardiomyopathy induced by autoimmune mechanism against β1-adrenoceptor

We have shown that a peptide corresponding to the sequence of the second extracellular loop of the human beta1-adrenoceptor (beta1-peptide) was able to induce an autoimmune cardiomyopathy in rabbits. In this study, we examined the effect of angiotensin-converting enzyme inhibitor (ACEI) on beta1-peptide-induced cardiomyopathy. Rabbits were divided into four groups: (1) control group (n= 6) receiving saline injection; (2) beta1-peptide group (n = 8) immunized with beta1-peptide; (3) ACEI group (n = 6), lisinopril (3 mg/day) given orally and receiving saline injection; and (4) ACEI + beta1-peptide group (n = 7), lisinopril (3 mg/day) given orally and immunized with beta1-peptide. Our results showed that, after 1 year, all rabbits in the beta1-peptide group had an increase in heart weight, wall thinning and dilatations of both ventricles as compared with rabbits in the ACEI + beta1-peptide group that had normal heart weight and shape. All rabbits in the beta1-peptide group exhibited multifocal degeneration and necrosis of myocardial cells with moderate infiltration of inflammatory cells. In the ACEI + beta1-peptide group, three rabbits showed focal degeneration and necrosis of myocardial cells accompanied by mononuclear cells. The lesions in this group were apparently less marked than those in the beta1-peptide group. In conclusion, ACEI protects the myocardium from injury induced by an autoimmune mechanism against beta1-adrenoceptor.