Mitsuru Hayase

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.

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.

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.

Growth hormone interferes with the progression of myocarditis in rats.

In this study, we investigated whether recombinant human growth hormone (rhGH) influences the progression of myocarditis. We induced experimental autoimmune myocarditis in F344 rats by subcutaneous injection of cardiac myosin, and divided the rats into three groups: (1) control group, saline injection; (2) pre-treated group, subcutaneous injection of rhGH (100 mIU/rat/day for 10 days) before induction of experimental autoimmune myocarditis; and (3) post-treated group, subcutaneous injection of rhGH (100 mIU/rat/day for 10 days) after induction of experimental autoimmune myocarditis. On the 35th day after induction of experimental autoimmune myocarditis, all rats were sacrificed and the hearts were examined. The increase in body weight was smaller in the control group than the pre-treated group and the rate of heart weight/body weight was larger in the control group than in the two treated groups. Histopathologically, rats in the control group showed multifocal infiltration by inflammatory cells, mainly neutrophils, lymphocytes and macrophages, extensive fibrosis, and a higher proportion of mast cells in the inflamed region. In contrast, rats in the two treated groups showed only minor changes. We found that rhGH did not influence the distribution of lymphocytes in peripheral blood in the three groups, and that rhGH induced G1 checkpoint dysfunction, thereby arresting the cell cycle in G1 and inhibiting the proliferation of mast cells in vitro. These findings suggest a possible role for mast cells in the progression of myocarditis and the rhGH may be a candidate for use as a new tool to treat myocarditis.