The Relationship Of The Autoantibodies Against The Second Extracellular Loop Of β₃-and β₁-adrenoceptor To Heart Failure

Objective (1) The distribution of the autoantibodies against the second extracellular loop of β₃AR was investigated by ELISA using the synthetic peptide corresponding to the sequence of the second extracellular loop of β₃AR, and biological effects of the autoantibodies on the cardiomyocytes were also observed at cell level. (2) The rats without the autoantibodies against the second extracellular loop of β₃AR were chosen to establish animal model of heart failure to observe the changes of the autoantibodies by ELISA before and after the formation of this animal model. (3) In order to investigate whether the autoantibodies against the second extracellular loop of β₁-adrenoceptor can induce cardiomyocyte apoptosis in vivo or not and its possible signaling pathway, we immunized rats with synthetic peptide corresponding to the sequence of the second extracellular loop of the β₁-adrenoceptor.(4)Genetic engineering technology was adopted to produce the required peptide corresponding to the sequence of the second extracellular loop of the β₁-adrenoceptor. Materials and Methods (1) The anti-β₃AR-autoantibodies from the sera of the patients with heart failure,healthy subjects and rats with heart failure were detected by ELISA using the synthetic peptide corresponding to the sequence of the second extracellular loop (176～202 amino acid residue) of human β₃AR.(2)The IgGs from the positive sera of patients or rats with heart failure were purified by affinity chromagraphy; The effects of the purified IgGs on the contractile responses and intracellular calcium transient of adult rat isolated cardiomyocytes were observed using motion-edge detection system and double-provocation fluorescence photomultiplier system; The effects of the purified IgGs on the L -type calcium current (ICa,L)and potassium curren(tIK) of guinea pig cardiomyocytes were observed by whole cell voltage clamp techniques; The effects on the beating rate of cultured neonatal rat cardiomyocytes and the non-desensitization/desensitization phenomenon of the purified IgGs were observed. (3)Healthy male Wistar rats were subjected to heart failure by constricting the abdominal aorta, the synthetic peptide corresponding to the sequence of the second extracellular loop of the β₃AR was used as the antigen to screen the autoantibodies against the β₃AR from the sera of the rats before or after treatment using ELISA. The biological effects of the autoantibodies from the sera of rats with heart failure were investigated using the methods mentioned above.(4)95 healthy male Wistar rats without the autoantibodies against the second extracellular loop of β₁AR were used and divided into two groups randomly:control group(n=30) and β₁AR immunized group(n=65); The β₁AR immunized group was immunized with 0.4μg/g of the synthetic peptide corresponding to the sequence of the second extracellular loop of human β₁AR; The cardiac function of the two groups was evaluated periodically; Apoptosis in the cardiac tissue was assessed by using TUNEL techniques(fluorescent or enzymatic chromogenic reaction) and agarose gel electrophoresis; caspase-3,8,9 activity of the cardiac tissue were detected using caspase-3,8,9 apoptosis detection colorimetric kit. (5)Specific primers carrying the identifiable site of different endonuclease and protective bases were designed. Using rat genomic DNA as the template, the target gene fragment of the second extracellular loop of β₁-adrenoceptor was amplified by PCR technology and recombined with the expression vector (pGEX-4T-1). After PCR screening and appraising with endonuclease and sequencing, recombined vector (pGEX-4T-1-β₁AR) was transformed into E.coli BL21(DE3). The GST fusion protein was expressed by IPTG induction and identified by SDS-PAGE electrophoresis.Results (1)The positive rate and mean geometric antibody titre of the autoantibodies against the second extracellular loop of β₃AR in the sera of the patients with heart failure were obviously higher than that of homogeneous healthy subjects by ELISA using the...