Adiponectin Upregulates The Decrease Of Cardiac Autophagy Induced By β₁-adrenergic Receptor Autoantibody Thereby Improving Cardiac Dysfunction

Cardiac insufficiency has become a global public health problem,and morbidity and mortality of patients have increased year by year.Studies have reported that immune disorders are important factors in the occurrence and development of cardiac insufficiency.According to clinical data,the serum of 40%-60%cardiac insufficiency patients haveβ₁-adrenergic receptor antibody（β₁-AA）,a product of immune disorders,which could interact withβ₁-adrenergic receptor（β₁-AR）on the surface of myocardial cell membrane.It can cause sustained injury of cardiomyocytes,induce cardiomyocyte death and lead to cardiac insufficiency,but the specific mechanism is not clear.Our team’s previous studies showed thatβ₁-AA-induced decrease of cardiac autophagy flux is involved in the occurrence and development of cardiac insufficiency.Autophagy is a conservative degradation pathway in cells.The decrease of autophagy flux induced byβ₁-AA leads to the destruction of cell homeostasis and induces the death of cardiomyocytes which participate in cardiac dysfunction.Furthermore,autophagy agonist rapamycin to up regulate autophagy flux can significantly improve the cardiac insufficiency induced byβ₁-AA.Therefore,up-regulating the decrease of autophagy flux induced byβ₁-AA is very crucial to improve cardiac insufficiency.Rapamycin is a classical drug which can up-regulate autophagy by inhibiting the activity of mTOR（mammary agert of rapamycin）.But it has obvious adverse reactions,including thrombocytopenia,decrease of insulin sensitivity and so on.Therefore,the utilization of rapamycin has potential security risk.So it is urgent to find a new safe and effective drug to up-regulate autophagy flux.Adiponectin is closely related to autophagy flux.It can up-regulate autophagy flux by binding to adiponectin receptor on the surface of cell membrane and then phosphorylated AMPK（Adenosine 5‘-monophosphate（AMP）-activated protein kinase）.Adiponectin is a kind of cytokine,which is mainly secreted by adipocytes.There is also a small amount of adiponectin secreted by cardiomyocytes.It has a wide range of myocardial protective effects,including improved atherosclerosis,reduced myocardial infarction.It has been reported that adiponectin gene knockout（APN-KO）mice has a significant decrease in cardiac autophagy flux and cardiac dysfunction.So it is not clear how adiponectin affectsβ₁-AA-induced decrease in cardiac autophagy flux and cardiac dysfunction.Whether adiponectin can be used as a potential treatment and possible regulatory mechanism for up-regulating cardiac autophagy flux and improving cardiac dysfunction need further verification.In order to investigate the effect of adiponectin onβ₁-AA-induced autophagy flux decrease and cardiac dysfunction,we used the second extracellular loop ofβ₁-adrenergic receptor（β₁-AR-ECII）to active immunize APN-KO mice and C57BL/6 mice.The OD value ofβ₁-AA in mice serum was detected by ELISA to confirm whether the active immunization model was established successfully.Western blot and Real-time PCR were used to detect myocardial autophagy flux of mice,which was active immunized for 2 w and 4 w.The results showed that adiponectin deficiency aggravatedβ₁-AA-induced autophagy flux decrease.Furthermore,the cardiac function of active immunized mice for 8and 12 weeks was detected by small animal ultrasound.Subsequently the myocardial tissue of active mice immunized for 12 weeks was taken,and the degree of myocardial fibrosis was detected by Masson staining.The above results showed that adiponectin deficiency exacerbatedβ₁-AA-induced cardiac insufficiency and myocardial fibrosis.It can be seen that adiponectin deficiency aggravates the decrease of autophagy flux induced byβ₁-AA,and then exacerbates cardiac insufficiency and myocardial fibrosis.In order to investigate whether exogenous administration of adiponectin can improve the decrease of autophagy flux and cardiomyocyte death induced byβ₁-AA.In vitro,β₁-AA purified from serum of rats immunized withβ₁-AR-ECII was applied to H9c2cardiomyocytes pretreated with adiponectin.Western blot and Real-time PCR were used to detect the autophagy flux of cardiomyocytes,and CCK-8 was used to detect the survival rate of cardiomyocytes.The results showed that adiponectin improved the decrease of autophagy flux induced byβ₁-AA and then reduced the death of cardiomyocytes.Furthermore,so as to explore the possible mechanism of adiponectin up-regulating autophagy flux of cardiomyocytes,firstly we adopted Western blot to detect the level of AMPK phosphorylation.The results showed that adiponectin could improve the decrease of AMPK phosphorylation level in cardiomyocytes induced byβ₁-AA.Futher,we pretreated H9c2 cardiomyocytes with Compound C that is the AMPK phosphorylation inhibitor.The results showed that adiponectin could not improve the decrease of AMPK phosphorylation induced byβ₁-AA.Meanwhile,the results of autophagy flux detection showed that adiponectin also irreversibly reduced the mRNA expression of LC3B and Beclin1 and LC3 II protein expression induced byβ₁-AA,suggesting that the improvement of adiponectin on the reduction of autophagosome formation induced byβ₁-AA depends on AMPK pathway.However,after Compound C pretreatment,adiponectin can still improve the accumulation of P62 protein,increase the clearance of autophagosome,suggesting that adiponectin improves the decrease in clearance of autophagosome induced byβ₁-AA,which not depends on the AMPK pathway.The purpose of this study is to provide experimental basis for the prevention and treatment ofβ₁-AA positive cardiac insufficiency.Part 1 Adiponectin deficiency aggravates the decrease of myocardial autophagy flux induced byβ₁-AA involvement exacerbated cardiac insufficiencyObjective:The active immunization model was established by adiponectin knockout（APN-KO）mice and C57BL/6 mice.The changes of autophagy flux,cardiac function and myocardial fibrosis were detected.To explore the effect of adiponectin deficiency onβ₁-AA-induced the decrease of cardiac autophagy and cardiac insufficiency.Methods:（1）Identification of transgenic gene mice:genotypes of APN-KO mice were identified by agarose gel electrophoresis;（2）Experimental group:control group（C57BL/6 mice）,active immunization group（C57BL/6 mice,APN-KO mice）;（3）β₁-AR-ECII was used to immunize 6-8 w male C57BL/6 mice and APN-KO mice.Blood was collected from tail vein of immunization mice for 2 w,4 w,8 w and 12 w.The OD value ofβ₁-AA in serum was detected by ELISA;（4）Real time PCR and Western blot were used to detect the expression of autophagy related genes and proteins in the myocardium of mice immunized for 2 and 4 weeks;（5）The cardiac function of mice in 8 w and 12 w of immunization were detected by small animal ultrasound;（6）After 12 weeks of active immunization,the myocardial tissue of mice in each group was embedded in paraffin and stained with Masson to observe the degree of myocardial fibrosis.Results:（1）genotyping of adiponectin gene knockout mice:agarose gel electrophoresis was used to detect the genotype of mice and guaranteed the success of adiponectin gene knockout;（2）Establishment ofβ₁-AA active immune mouse model:with the prolongation of the immuned time,the OD value ofβ₁-AA in the serum of mice increased significantly.And there was a statistical difference at 2 w of active immunization,indicating that the active immunization mice model was established successfully;（3）Adiponectin deficiency aggravated the decrease of myocardial autophagy flux induced byβ₁-AA:compared with the control group,the myocardial autophagy flux of APN-KO mice decreased at 2 w of active immunization,while C57BL/6 mice had no significant difference.The myocardial autophagy flux of APN-KO mice was lower than that of C57BL/6 mice at 4 w of active immunization;（4）Adiponectin deficiency aggravated cardiac insufficiency induced byβ₁-AA:compared with the control group,the cardiac function of APN-KO mice decreased significantly at 8weeks of active immunization,but there was no difference in C57BL/6 mice.The cardiac function of APN-KO mice decreased further than that of C57BL/6 mice at 12 weeks of active immunization;（5）Adiponectin deficiency aggravated myocardial fibrosis induced byβ₁-AA:compared with the control group,APN-KO mice and C57BL/6 mice showed blue staining area in the cardiac tissue,and blue staining area of APN-KO mice cardiac tissue showed more than of that C57BL/6 mice at 12 weeks of active immunization.Conclusion:Adiponectin deficiency aggravates the decrease of myocardial autophagy flux induced byβ₁-AA,and then exacerbates cardiac insufficiency and fibrosis.Part 2 Adiponectin deficiency aggravates the decrease of cardiac myocyte autophagy flux induced by β₁-AA involvement decrease of cardiomyocyte deathObjective:β₁-AA,purified from the serum of rats immunized with β1-AR-ECII,was used to act on H9c2 cardiomyocytes.And we observe whether adiponectin can improve the decline of survival rate and autophagy flux of H9c2 cardiomyocytes induced by β₁-AA after adiponectin pretreatment.Methods:（1）Preparation of β₁-AA antibody: 6-8 w male SD rats were divided into control group and active immunization group according to the random number table method,6 rats in each group.Blood was collected from tail vein of rats after active immunization for 8 w.OD value of β₁-AA in serum was detected by ELISA,then blood was collected from abdominal aorta of rats,and β₁-AA in serum was purified by affinity chromatography for in vitro experiment;（2）Cell groups: control group（1 μmol/l negative Ig G treated cells for 24 h）;β₁-AA group（1 μmol/l β₁-AA treated cells for 24 h）;β₁-AA + adiponectin group（10 ng/ml adiponectin pretreated cells for 1 h and then added 1 μmol/l β₁-AA for 24 h）;adiponectin group（10ng/ml adiponectin treated cells for 25 h）;（3）CCK-8 method was used to detect the survival rate of myocardial cells;（4）The expression of autophagy related genes and proteins in cardiomyocytes were detected by Real-time PCR and Western blot.Results:（1）The autoantibodies of β1-adrenoceptor with biological activity were successfully prepared: compared with the control group,the OD value of β₁-AA in the serum of the rats active immunization for 8 weeks was significantly increased.The β₁-AA in the serum was purified by affinity chromatography for subsequent in vitro experiments;（2）Adiponectin improved the decrease of survival rate of H9c2 cardiomyocytes induced by β₁-AA: 10 ng/ml adiponectin pretreatment could significantly reverse the decrease of survival rate of H9c2 cardiomyocytes induced by β₁-AA;（3）Adiponectin improved the decrease in autophagy flux of H9c2 cardiomyocytes induced by β₁-AA.Adiponectin pretreatment improved the expression of Beclin 1,LC3 B m RNA and LC3 II protein and the accumulation of P62 protein in H9c2 cardiomyocytes induced by β₁-AA.Conclusion:Adiponectin pretreatment can significantly improve the decrease of autophagy flux induced by β₁-AA and then reduce the death of cardiomyocytes.Part 3 Adiponectin improves decreased autophagy flux induced by β₁-AA in H9c2 cardiomyocytes partially dependent on AMPK pathwayObjective:Compound C,an AMPK phosphorylation inhibitor,was used to pretreat H9c2 cardiomyocytes to explore the possible mechanism of adiponectin improving decreased autophagy flux induced by β₁-AA.Methods:（1）Cell groups: control group（1 μmol/l negative Ig G treated cells for 24 h）;β₁-AA group（1 μmol/l β₁-AA treated cells for 24 h）;β₁-AA + adiponectin group（10 ng/ml adiponectin pretreated cells for 1 h and then added 1 μmol/l β₁-AA for 24 h）;adiponectin group（10ng/ml adiponectin treated cells for 25 h）;（1）β₁-AA + adiponectin + compound C group（H9c2 cardiomyocytes were pretreated with 20 μmol/l compound C for 30 minutes and then added 10 ng/ml adiponectin for 1 hour before adding 1 μmol/l β₁-AA for 24 h）;β₁-AA+ compound C group（20 μmol/l compound C pretreated cells for 30 minutes and then added 1 μmol/l β₁-AA for 24 h）;（2）Western blot was used to detect the level of AMPK phosphorylation;（3）The expression of autophagy related genes and proteins in cardiomyocytes were detected by real time PCR and Western blot.Results:（1）Adiponectin improved AMPK phosphorylation of H9c2 cardiomyocytes induced by β₁-AA: adiponectin pretreatment significantly improved decreased P-AMPK/AMPK ratio of H9c2 cardiomyocytes induced by β₁-AA;（2）Adiponectin improved the reduction of autophagosome formation induced by β₁-AA dependent on AMPK pathway: after pretreatment with compound C,an AMPK phosphorylation inhibitor,adiponectin not improved the decrease of Beclin1 and LC3 B m RNA expression,P-AMPK/AMPK ratio and LC3 II protein induced by β₁-AA;（3）Adiponectin improved the reduction of autophagy clearance induced by β₁-AA independent of AMPK pathway: after pretreatment with compound C,an AMPK phosphorylation inhibitor,adiponectin could still improve the accumulation of P62 protein in cardiomyocytes induced by β₁-AA.Conclusion:Adiponectin improved the decrease of AMPK phosphorylation induced by β₁-AA.When compound C was used to inhibit AMPK phosphorylation,adiponectin could not improve the decrease of autophagosome formation induced by β₁-AA,but could still improve the decrease of autophagosome clearance induced by β₁-AA,suggesting that adiponectin improved decreased autophagy flux induced by β₁-AA in cardiomyocytes partially dependent on AMPK pathway.