Liraglutide Relieves Myocardial Damage By Promoting Autophagy Via AMPK Signaling Pathway In Zucker Diabetic Fatty Rat

BackgroundCardiovascular complication of diabetes is a major cause of death in diabetic patients and the incidence of it is increasing gradually year by year.As an important member of cardiovascular complication of diabetes,diabetic cardiomyopathy(DCM)occurs independent of overt coronary artery disease,hypertension or other heart disease in diabetic patients.DCM is characterised by structural and functional abnormalities,such as left ventricular dysfunction and prominent interstitial fibrosis.It ultimately leads to heart failure,arrhythmia,etc.,and even death.Therefore,the effective treatment of DCM is significant to reduce the mortality of diabetic patients.Nonetheless,there is no clinically available effective treatment for the DCM at present.Liraglutide(LG),a human glucagon-like peptide 1(GLP-1)analogue,is a glucose-lowering agent that is used clinically to treat type 2 diabetic mellitus(T2DM).Clinical trials and animal experiments have shown that GLP-1 has cardiovascular protective effects,such as improving cardiac function in patients with coronary ischemia or slowing the progression of atherosclerosis.However,the effects of LG on DCM and its underlying mechanisms have not yet been absolutely elucidated.Autophagy is an intracellular catabolic pathway to maintain cellular homeostasis,which degrades denatured proteins and damaged organelles such as mitochondria.Autophagy,as a dynamic process,is known as "autophagic flux".Previous studies indicate that autophagy is involved in the development of DCM.Studies indicated that LG increased the viability of hepatocytes and pancreatic ?-cells by promoting autophagy,whereas the question of whether LG exerts cardioprotective effects in DCM by regulating autophagy has not been absolutely revealed.In addition,AMPK is a classic upstream regulator of autophagy and the loss of AMPK resulted in defective autophagy.It has been reported that LG activates AMPK in skeletal muscle cells and hepatic cells.Hence,we hypothesize that LG protects against diabetic myocardial damage by enhancing autophagic flux via the AMPK signaling pathway.Objectives1.To verify the cardioprotective effect of LG on diabetic myocardium in ZDF rat model and hyperglucose cell model.2.To explore the effect of LG on the autophagy in diabetic myocardium and its underlying molecular mechanisms.Methods1.In vivo studyZDF rats(ZDF-Leprfa)and their lean controls(ZDF-Crl)(8 weeks)were used.The ZDF-Leprfa rats were fed with a high-fat-carbohydrate chow whereas the ZDF-Crl rats were fed with a normal chow diet.The ZDF rats were randomly divided into five groups:(1)control group(Control);(2)DCM group;(3)DCM+LG group;(4)DCM+CQ group;and(5)LG+CQ group.LG was subcutaneous injected(200?g/kg)and CQ was intraperitoneal injected(25 mg/kg).LG and CQ were injected once a day for 8 weeks.At week 16,blood samples were collected for biochemical analysis such as blood glucose(Glu),cholesterol(CHOL)and triglycerides(TG).Echocardiography was taken one day before rats' sacrifices.The rats were sacrificed,and the hearts were extracted and fixed immediately.Subsequently,the TEM?IHC?Masson-trichrome staining and WB were taken.2.In vitro studyNeonatal rat cardiomyocytes(NRCs)were isolated from newborn(1-to 3-day-old)Sprague Dawley rats.The NRCs were cultured in DMEM with high glucose to mimic a hyperglycemic state in vivo and establish hyperglucose cell model.NRCs were divided into the following groups:(1)control group(Control);(2)high glucose group(HG);(3)HG+100nM LG group(HG+LG);(4)HG+25?M CQ group(HG+CQ);(5)HG+LG+CQ group.The cells were cultured in high glucose for 96 h and were administered with LG and CQ for 24h.The culture medium was changed daily.MTS was used to detect myocardial cell viability.The level of myocardial apoptosis was analyzed by TUNEL assay.Ad-mRFP-GFP-LC3 transfection was performed to observe the changes of autophagosome and autolysosome.WB was used to detect the expression of autophagy marker proteins(LC3,p62)and AMPK signal pathway related proteins(AMPK,mTOR).Results1.LG relieves myocardial cell injury in vivo and vitro.(1)LG reduced the levels of blood glucose(Glu),trilyleride(TG)and cholesterol(CHOL)in ZDF rat.The result of blood biochemical test showed that the levels of Glu,TG and CHOL were significantly decreased in the LG group comparing with the DCM group.(2)LG relieves myocardial injury in ZDF rat.The result of echocardiography showed that the left ventricular fraction shortening(LVFS)was increased in LG group comparing with DCM group.In the blood biochemical test,the levels of serum creatine kinase(CK)and serum lactate dehydrogenase(LDH)was decreased in LG group.However,the effect of LG was eliminated in the LG+CQ group(P<0.05).(3)LG mitigated high glucose-induced injury in NRCs.The result of MTS assay showed that the treatment of LG increased the cell viability.The result of TUNEL assay showed that the cell death caused by glucotoxicity was decreased in LG group.Notably,the protective effect of LG was partly blocked by CQ,an autophagy inhibitor in the above assays.2.LG alleviated myocardial fibrosis in the ZDF.Masson-trichrome staining was performed.The result showed that the collagen volume fraction(CVF)was significantly reduced in the LG group comparing with DCM group,while CVF was higher in the LG+CQ group than in the LG group.3.LG enhanced autophagy in myocardial cell.(1)LG enhanced autophagy in the diabetic myocardium of the ZDF rats.TEM showed that the number of autophagosomes was increased in the LG group comparing with DCM group.The results of the IHC analysis showed that the DCM group had a smaller LC3B ratio of IOD/Area in comparison with the LG group.The results of WB showed that the level of LC3 ?/? was decreased accompanying an increased level of p62 in the DCM group.After LG treatment,the ratio of LC3 ?/?increased and the level of p62 was down-regulated.In the above methods,the effect of LG on autophagy was almost abolished by CQ,an autophagy inhibitor.(2)LG enhanced autophagic flux in the NRCs exposed to high glucose.To further investigate the auxo-action of LG on the autophagic flux,tandem fluorescent mRFP-GFP-LC3(Ad-LC3-NRCs)was performed on the NRCs.After treatment with LG,the number of yellow puncta(autophagosomes)and red puncta(autolysosomes)increased significantly compared with the HG group.However,co-treatment with the autophagy inhibitor 3-MA decreased the number of yellow puncta and red puncta.The results of WB showed that the ratio of LC3 ?/? significantly decreased in the HG group,while the level of LC3 ?/? was up-regulated in the presence of LG compared with the HG group.This ratio was increased in the HG+LG+CQ group compared with the HG+CQ group.4.LG activated the AMPK signaling pathway in NRCs exposed to high glucose.The results of WB showed that the ratio of p-AMPK/AMPK(the phosphorylation of AMPK)was increased and the ratio of p-mTOR/mTOR(the phosphorylation of mTOR)were decreased in the LG group.Conclusions1.LG relieves diabetic myocardial injury by promoting autophagy in myocardial cell.2.AMPK signaling pathway may mediate the autophagy-promoting effect of LG in DCM.