Epac1 Is Involved In Decreased Autophagy In Cardiomyocyte Induced By β₁-Adrenergic Receptor Autoantibody And Its Receptor Mechanism

Morbidity and mortality of heart dysfunction are increasingly in worldwide.Cardiomyocytes are terminally differentiated cells with limited regenerative capacity.A large number of cardiomyocytes loss could impaire cardiac function,which leads to heart dysfunction.A study has shown that autophagy-associated cell death,is the most common types of cell death in heart dysfunction.However,the mechanism of decreased autophagy in heart dysfunction is not well understood.A large number of previous studies have found that the positive rate of β₁-adrenergic receptor autoantibody（ β₁-AA）in the serum of cardiovascular diseases patients is higher than that of normal people.Ourprevious study found that decreased cardiomyocyte autophagy induced by β₁-AA,which was involved in β₁-AA-induced cardiomyocyte death and even heart dysfunction.Further studies have found that β₁-AA can reduce the level of cardiomyocyte autophagy by activating β₁-AR-cAMP and then activating protein kinase A（PKA）.However,decreased cardiomyocytes autophagy induced by β₁-AA cannot completely reversed by inhibitor of PKA.It is suggested that there are other factors contributed to decreased cardiomyocyte autophagy induced by β₁-AA.,Epac1 is cAMP another downstream signaling pathway.Whether Epac1 is involved in decreased autophagy induced by β₁-AA and its related mechanisms are still unclear.Therefore,in this study,8-week-old male SD rats were actively immunized with the β₁-AR-ECII antigen peptide for 2 w,4 w,6 w,8 w.The OD value of β₁-AA in the serum was detected by SA-ELISA to identify whether the rat models were established successfully.The β₁-AA in rat serum was purified by affinity chromatography for vitro experiments.In this research,cAMP content in myocardial tissue was detected by ELISA;PKA activation was tested by Western Blot;the protein and mRNA expression levels of autophagy-related genes LC3,Beclin1 and P62 were detected by Western blot and RT-PCR.H9c2 myocardial cells were treated with β₁-AA purified by affinity chromatography,then the expression of cAMP,p-PKA and were observed.Autophagy were further examined after pretreated with the PKA inhibitor H89.It was found that long-term stimulation of β₁-AA caused cAMP,p-PKA in myocardial tissue expression increased significantly and the level of autophagy decreased obviously.Inhibition of PKA could outstandingly rescue the decreased cardiomyocyte autophagy induced by β₁-AA.However,decreased autophagy induced by β₁-AA could only be recovered partially by inhibitor PKA.Therefore,the expression of Epac1 was detected in vivo and vitro by Western blot furtherly.The results showed that the expression of Epac1 induced by β₁-AA was increased.Cardiomyocyte autophagy was observed after pretreated with Epac1 inhibitor ESI-09 in H9c2 cardiomyocytes cells.It was found that inhibition of Epac1 obviously improved the level of autophagy induced by β₁-AA.Next,we focused on the possible mechanism that increased expression of Epac1 in cardiomyocytes induced by β₁-AA. β₁-AA,an autoantibody of the second extracellular loop of β₁-AR,could specifically activate β₁-AR to exert effects.Long-term β₁-AR stimulation can cause left ventricular dilatation and heart dysfunction.Other studies have indicated that β₁-AR and β₂-AR can form heterodimers and enhance the biological effects of β₁-AR.Our team has previously found that β₂-AR does not directly bind to β₁-AA,but can enhance the proliferation of cardiac fibroblasts induced by β₁-AA bound to β₁-AR.Therefore,we speculate that both β₁-AR and β₂-AR may be involved in increasing the expression of Epac1 toinhibitor autophagy in cardiomyocytes induced by β₁-AA.To investigate the role of β₁-AR and β₂-AR in increased Epac1 expression,we selected β₁-AR KO mice and β₂-AR KO mice,meanwhile established a model of active immunization with β₁-AR-ECII antigen peptides.Then the expression of Epac1 and the levelof autophagy in myocardium were detected.The results suggested that both β₁-AR and β₁-AR were involved in the enhanced Epac1 expression anddecreased autophagy in myocardial tissue induced by β₁-AA.Further,we try to investigate how β₂-AR affects the increased Epac1 expression induced by β₁-AA.It is well known that,unlike β₁-AR which only couples Gs protein, β₂-AR can couple both Gs and Gi proteins.Studies have shown that overexpression of β₂-AR can significantly inhibit the functional damage of β₁-AR. β₂-AR inverse agonist ICI118551 could biased activate the Gi signaling pathway to alleviate this damage.The results suggested that the β₂-AR/Gi signaling pathway played an important role in function of β₁-AR.In order to explore the role of the β₂-AR/Gi signaling pathway enhanced the expression of Epac1 induced by β₁-AA in cardiomyocytes.We selected ICI 118551 to pre-treat H9c2 cardiomyocyte cell before interfered with β₁-AA.The results demonstrated that ICI 118551 significantly reversed the increased Epac1 expression,decreased autophagy and reduced cell viability in cardiomyocytes induced by β₁-AA.The results suggested that β₁-AA could up-regulate the expression of Epac1 in cardiomyocytes by inhibiting the β₂-AR/Gi signaling pathway.Part I Epac1 is involved in decreased cardiomyocyte autophagy induced by β₁-AAObejective:In this study,the β₁-AR-ECII antigen peptide was used to actively immunize SD rats to establish a model.Meanwhile,serum-purified β₁-AA was applied to H9c2 myocardial cells to investigate whether Epac1 was involved in decreased cardiomyocyte autophagy induced by β₁-AA.Method:（1）The second extracellular loop of β₁-AR,an antigen peptide,was used to immunize8-week-old rat,and the β₁-AA in serum was detected by ELISA at 2 w,4 w,6 w,8 w,respectively.The OD value identify whether the model is successfully established;（2）Purifying β₁-AA in serum by affinity chromatography and its concentration was tested by BCA method,which was used to treat cell experiments;（3）ELISA was used to detect the changes of cAMP concentration treated with β₁-AA for 2w,4 w,8 w in cardiomyocytes and 12 h,24 h and 36 h in H9c2 myocardial cells;ELISA was used to detect the changes of cAMP concentration changes in heart tissue of the at 2 w,4 w,8 w,and the changes of cAMP concentration in H9c2 myocardial cells to treated with β₁-AA at 12 h,24 h and 36 h.（4）Western blot was used to examine the expression of p-PKA and Epac1 protein in different time points of β₁-AA stimulation.The expression of autophagy-related proteins and genes were detected by Western blot and RT-PCR.Western blot was used to examine the expression of p-PKA and Epac1 protein in β₁-AA stimulated myocardial tissue at different times,The expression of autophagy-related proteins and genes were detected by Western blot and RT-PCR.Results:（1）The OD value of β₁-AA was significantly increased in the serum of actively immunized rats with β₁-AR-ECII antigen peptide with the prolongation of immunization time.（2）β₁-AA can cause accumulation of cAMP and increased PKA activity in myocardial tissue and H9c2 cardiomyocytes.（3）β₁-AA induced a significant decreased autophagy in myocardial tissue and H9c2 cells.（4）Inhibition of PKA can partially reverse the extent of decreased levels of myocardial autophagy induced by β₁-AA.（5）Epac1 is involved in declined myocardial autophagy induced by β₁-AA.（6）Epac1 may promot IP3 expression which participate in reduced autophagy induced by β₁-AA.Conclusion: β₁-AA can induce the decrease of myocardial autophagy level by cAMP-PKA/Epac1signaling pathway,and Epac1 can promote the expression of IP3 to inhibit the level of myocardial autophagy.Part II β1-AR and β2-AR are involved in increased Epac1 expression induced by β₁-AA in cardiomyocytesObjective:In this study,the β₁-AR-ECII antigen peptide was used to actively immunize C57BL/6 mice, β₁-AR KO mice and β₂-AR KO mice to establish model,and β₁-AA-stimulated H9c2 cardiomyocytes was used to study the receptor mechanism of β₁-AA promoting the increase of Epac1 expression in cardiomyocytes.Method:（1）Detecting genotypic of knockout mice by agarose gel electrophoresis;（2）Using the second extracellular loop of β₁-AR as the antigen peptide to active immunization C57BL/6 mice, β₁-AR KO mice and β₂-AR KO mice for 4 weeks,respectively.The OD value of β₁-AA in serum was detected by ELISA to detect whether the model was successfully established;（3）The expression of Epac1 and in autophagy-related protein and gene myocardial tissues in β₁-AR KO mice, β₂-AR KO rats was detected by Western blot and RT-PCR.Epac1,autophagy-related protein and gene expression of H9c2 cardiomyocytes pretreated with atenolol and ICI 118551 before stimulation with β₁-AA were detected by Western blot and RT-PCR;（1）Immunohistochemistry was used to detect the in situ expression of Epac1 in myocardial tissue;（2）CCK-8 was used to test survival rate of H9c2 cardiomyocytes induced by β₁-AA after atenolol and ICI 118551 preconditioning.Results:（1）Genotypic identification of β₁-AR KO mice and β₁-AR KO mice.（2）The mice actively immunized with β₁-AR-ECII were established.（3）β₁-AR contributed to increased Epac1and decreased autophagy with the existence of β₁-AA in myocardial tissue.（4）β₁-AR was involved in increased Epac1,decreased autophagy levels,and reduced survival induced by β₁-AA in H9c2 cardiomyocytes.（5）β₂-AR took part in the increase in Epac1 and the decrease in autophagic induced by β₁-AA in myocardial tissue.（6）β₂-AR/Gi signaling pathway antagonizes increased Epac1,decreased autophag level and r educed survival rate of H9c2 cardiomyocytes induced by β₁-AA.Conclusion:Both β₁-AR and β₂-AR are involved in the increase of Epac1 expression in myocardial induced by β₁-AA,which in turn induces decreased myocardial autophagy and declined survival rate.