| Objective: In recent years, it has been demonstrated that the autoantibodies against β1-adrenoceptor can be detected in the sera of the patients with dilated cardiomyopathy and other heart diseases. The autoantibodies are characterized by their "agonist-like" activity on cardiac β1-adrenoceptor without desensitization. The previous research showed that the antibodies were able to induce cardiomyocytes pathological injuries and cardiac dysfunctional during their long-term action on the heart. However, the mechanisms responsible for the antibody-induced injuries are unclear. Therefore, in this study, in an attempt to clarify whether apoptosis are involved in the injury process, we observed the biological and apoptotic effects of the antibodies on cultured nenatal rat cardiomyocytes.Materials and Methods: Wistar rats were immunized with the peptides corresponding to the second extracellular loop of the β1-adrenoceptor to induce the production of anti-β1-adrenoceptor autoantibodies in the sera. The control group received saline instead of the antigen peptides according to the same procedure as the immunized group. Anti-β1-adrenoceptor autoantibodies were detected using SA-ELISA assay. IgG in the sera fromthe two groups were purified with saltingout and DEAE-cellulose ion exchange chromatography technique. The effects of IgG from each group on the beating rate of cultured neonatal rat cardiomyocyte were observed Furthermore, after incubation for 48h, the formation of DNA ladder was detected using agarose gel electrophoresis, and the number of apoptotic cell were measured with flow cytometry. To analyse the mechanism of antibody-induced apoptosis, we also observed the effects of anti-p,-adrenoceptor monoclonal antibodies and isoproterenol (Iso) on the cultured neonatal rat cardiomyocytes as comparison. Results:1. The geometric mean of the antibody titers of immunized rats was less than 1:10 at the end of 2 weeks after prior immunization, while the geometric mean rised to 1:142.5±5.96 at the end of 2 weeks after a booster immunization. These demonstrated that the corresponding autoantibodies could be detected in the sera of animals after i.h. injection of antigen peptides. The amount of produced antibody increased with time. No immune response was found in the sera of control group. IgG from control group were regarded as negative control in the following experiments.2. The IgG derived from positive sera, the monoclonal antibody or Iso were then added to the incubation medium of cultured beating neonatal rat cardiomyocytes, they all increased the heart beating rate (beats/min), showing an "agonist-like" activity. IgG from positive sera at 0.3μ.M, 0.6μM and 1.2μM increased the beating rate by 17.2±7.05, 22.6±6.27 and 30.4±11.95 respectively, which were significant as compared with theabove-mentioned increasing beating rate of negative control group. Monoclonal antibodies at 10nM, 50nM and 100nM increased it by 20.4±6.62, 24.8±8.61and 35.6±13.61; Iso at 10μM increased by 51.0±19.42. These were also significant as compared with the pretreatment beating rate.3. Agorose gel electorphoresis showed a typical ladder formation characteristic of apoptosis when cardiac myocytes were exposed to IgG (1.2μ) from positive sera, monoclonal antibodies (100nM) or Iso (10μM) for 48h.4. Flow cytometry also demonstrated that IgG from positive sera and monoclonal antibodies increased the number of apoptotic myocytes at 48h. The former at 0.3μM, 0.6μM and 1.2μM increased the apoptotic percentage (%) by 11.48±2.76, 14.10±2.65 and 21.57±4.22, respectively; the latter at 10nM, 50nM and 100nM increased it by 14.32±2.71, 17.61±2.25 and 30.39±1.81, respectively. These effects were also highly significant as compared with control.Conclusions:1. The high level autoantibodies against b1-adrenoceptor occured in the sera of rats immunized with the peptides corresponding to the second extracellular loop of the cardiac b1-adrenoc...
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