The Role Of Cardiac Fibrosis Induced By β1-Adrenoceptor Autoantibody And The Underlying Mechanisms

Background Cardiac fibrosis is one of the significant causes of heart failure, arrhythmia, and cardiac sudden death. Prevention and reverse of cardiac fibrosis is the key target of clinical therapy. The overstimulation of β-adrenergic receptor plays an important role in cardiac fibrosis. However, the origination and specific mechanism are not yet clarified clearly. A lot of investigations reported that β1-adrenergic autoantibody(β1-AA for short) was found in dilated cardiomyopathy, heart failure and other cardiovascular diseases. And the positivity and titres in various cardiovascular diseases were much higher than that in healthy subjects. Then researches indicated that β1-AA have the similar effects as catecholamines. The autoantibody activated β1-adrenergic receptor on the surface of cardiomyocytes, leading to cardiomyocytes death and cardiac dysfunction. Interestingly, the downstream signalling lacks of desensitization in cardiomyocytes after being stimulated by β1-AA. Our previous study reported that β1-AA was associated with cardiac fibrosis in heart failure model. Nevertheless, whether or not β1-AA induces cardiac fibrosis directly need to be further studied. The proliferation and secretion of cardiac fibroblasts are the crucial conditions. We previously found that β1-AA promoted the proliferation and secretion of T lymphocytes via β1-adenoceptor pathway. The effects of β1-AA on the proliferation and secretion in cardiac fibroblasts have not been clear. It was reported that β1/β2-AR agonist isoprenaline promoted proliferation and secretion in cardiac fibroblasts through β2-adenoceptor. And β1-AR and β2-AR have the same Gs signalling and the switch from Gs to Gi, which suggested that β1-AR and β2-AR were involved in cardiac fibrosis. Here we attempt to observe the effect of β1-AA on cardiac fibrosis in a β1-AA passive immunization during 16 weeks. The specific mechanisms of β1-AA induced cardiac fibrosis were explored in the cellular level. Our study will provide new experiments on therapy of relative patients with cardiac disease.Methods 1. Preparation of monoclonal antibodies to β1-adrenoceptor Long 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-C)and short peptide(C-H-W-W-R-A-E-S-D-E-A-R-R) were respectively synthesized according to the amino acid sequence of the second extracellular loop of β1-adrenergicreceptor(β1-AR-ECII,197aa-223 aa,H-W-W-R-A-E-S-D-E-A-R-R-C-YN-D-P-K-C-C-D-F-V-T-N-R-A, the homology rate among human, mouse and rat is 100%) and were coupled as an antigen for immunizing mice. Hybridoma cell lines secreting β1-AR monoclonal antibody(β1-AR m Ab) were generated by hybridoma technology. β1-AR m Ab were purified from the ascites of BALB/c mice using a Mab Trap kit. The binding of β1-AR m Ab and β1-AR was detected by SA-ELISA. And the functional activity of β1-AR m Ab was tested by neonatal mouse cardiomyocytes beating frequency. 2. Animal experiments 6-8 weeks old, normal male BALB/c mices were selected and divided into three groups: 1 saline group; 2 β1-AA group; 3 isoprenaline group. β1-AA was injected intraperitoneally with a concentration of 5μg/g once every two weeks during 16 weeks to establish a β1-AA-positive-mice model. Isoprenaline was also injected intraperitoneally with a concentration of 5μg/g every day as a positive control.Echocardiography was used to measure cardiac function of each group. HE and masson trichrome staining were used to evaluate the cardiac morphology and fibrosis relatively. Real time PCR was used to detect the gene expression of collagen and Ⅰcollagen Ⅲ. Western blot was used to determine the protein expression of collagen Ⅰand collagen Ⅲ. 3. Cell experiments Neonatal rat cardiomyocytes and cardiac fibroblasts were isolated by different time of attaching. CCK8 assay and CFSE labeling were used to determine cardiac fibroblast proliferation. The interaction between β1-AA and β1-AR on the surface of primary cardiac fibroblasts was detected by immunofluorescence cellular staining, immunoprecipitation and BLItz system. CAMP production of cells was detected by [125I] c AMP RIA KIT. Western Blot was used to detect the protein expression of P38 MAPK, ERK1/2, PKA activity, etc. Cellular apoptosis was determined by Annexin Ⅴ/PI apoptosis kit. The laser scanning confocal microscope was used to observe the cellular identification and dynamic change during internalization.Results 1. β1-AA induced cardiac dysfunction, in term of decreasing in ejection fraction and fraction shortening during passive immunization. At the 16 th week of passive β1-AA immunization, there was a lot of collagen deposition in β1-AA group. Moreover, gene and protein expression of collegen Ⅰand Ⅲwere increased significantly. 2. β1-AA promoted cardiac fibroblast proliferation, which was partially reversed by β1-AR blocker metoprolol, PKA inhibitor H89 and β2-AR blocker ICI118551. These results indicated that β1-AR and β2-AR were involved in proliferation in cardiac fibroblasts. β1-AA bound to β1-AR but not to β2-AR, and subsequently activated c AMP/PKA pathway. Besides, β1-AA promoted the proliferation of cardiac fibroblasts through P38 MAPK and ERK1/2, which was reversed by P38 MAPK inhibitor SB203580 and ERK1/2 inhibitor PD98059 partially. In addition, the conditional medium from cardiac fibroblasts stimulated with β1-AA, which was neutralized by co-incubation with peptide of β1-AR ECⅡ, induced cardiomyocytes apoptosis. 3. β1-AA increased PKA activity and P38 MAPK persistently within 1 h, which suggested that β1-AA activated the downstream signaling lack of desensitization. Besides, a small amount of internalized β1-AR induced by β1-AA may be involved in the persistent activated signaling.Conclusion β1-AA promotes cardiac fibrosis directly in a β1-AA passive immunization mouse model. β1-AA binds to β1-AR but not to β2-AR, promoting proliferation and secretion of cardiac fibroblasts through β1-AR. Besides, β2-AR, P38 MAPK and ERK1/2 pathway also may be involved in the proliferation of cardiac fibroblasts. Our findings will help the understanding of pathological mechanism in heart failure induced by β1-AA, and provide the experimental evidence for therapy of relative cardiovascular diseases.