The Role And Mechanism Research Of Melatonin In Diabetic Cardiomyopathy By Regulating Mst1

The latest report from the International Diabetes Federation?IDF?shows that globally the total number of adults with diabetes is about 425 million in 2017.It is estimated that the total number of patients with diabetes globally will reach 629 million by 2045.The prevalence of diabetes globally in 2017?age 20⁷9 years?is about 8.8%and is expected to reach 9.9%by 2045,while the prevalence of diabetes in China is 10.9%.The number of patients with diabetes in China reached 134.3 million.The number of deaths from diabetes in 2017 was about 4 million people?age 20⁷9 years?.In China there was about 840,000patients died of diabetes.Diabetes is an independent risk factor which increases the morbidity and mortality of cardiovascular diseases.Diabetes can lead to the damaged heart filling,heart systolic function drops,myocardial hypertrophy and fibrosis.We call it the diabetic cardiomyopathy.Therefore,how to prevent and delay the diabetic myocardial injury is the key problem in diabetes management.Synthesis and secretion of melatonin is mainly in the pineal gland.The previous researches showed that melatonin regulated a variety of physiological and neuroendocrine function,and the cardiovascular system.Melatonin can scavenge free radicals and has the antioxidant function.It can also regulate blood pressure and inhibit the process of the atherosclerosis.As a key component in the Hippo pathway,mammals STE20 sample kinase 1?Mst1?regulates cell apoptosis and proliferation.Mst1 exists in the heart tissue and determines the fate of the cardiomyocytes by regulating autophagy.Recent studies have shown that Mst1signaling pathway regulates many cardiovascular diseases,including aortic dissection,aortic aneurysm,atherosclerosis and myocardial infarction injury.However,whether the Mst1 signaling pathway can regulate the development of diabetic cardiomyopathy is still not clear.Therefore,this study explores the role of melatonin in diabetic cardiomyopathy by regulating Mst1 signaling pathway respectively from animal and cell level and provides new targets for the diagnosis and treatment of diabetic cardiomyopathy.?Objective?1.To investigate the effects of melatonin on cardiac function in diabetic mice by constructing the diabetic mouse model.2.Isolation and cultivation of cardiomyocytes from the neonatal mice.To observe the effects of melatonin on cardiomyocyte autophagy level under high glucose culture conditions.3.To investigate the role of Mst1 in the diabetic cardiomyopathy and confirm the role of Mst1/Beclin1 signaling pathway mediating autophagy in the diabetic cardiomyopathy.4.To confirm whether melatonin protects the heart from the diabetic cardiomyopathy by regulating the Mst1.?Methods?1.The C57 mice were randomly divided into the following four groups:wild type mice group?Con?,melatonin intervention group?Con+Mel?,diabetic group?DM?,diabetes melatonin intervention group?DM+Mel?.2.The wild type mice and Mst1^-/-mice were randomly divided into four groups:wild type mice group?WT?,Mst1 knockout mice group(Mst1^-/-),diabetic mice group?DM?,diabetes Mst1 knockout mice group(DM+Mst1^-/-).3.The wild type mice and Tg-Mst1 mice were randomly divided into four groups:wild type mice group?NTg?,Mst1 overexpression mice group?Tg-Mst1?,diabetic mice group?DM?,diabetes Mst1 overexpression mice group?DM+Tg-Mst1?.4.The wild type mice and Mst1^-/-mice were randomly divided into six groups:wild type mice group?Con?,diabetic mice group?DM?,Mst1 knockout mice group(Mst1^-/-),melatonin intervention Mst1 knockout mice group(Mst1^-/-+Mel),diabetes Mst1 knockout mice group(Mst1^-/-+DM),melatonin intervention diabetes Mst1 knockout mice group(Mst1^-/-+DM+Mel).5.The wild type mice and Tg-Mst1 mice were randomly divided into six groups:wild type mice group?Con?,diabetic mice group?DM?,Mst1 overexpression mice group?Tg-Mst1?,melatonin intervention Mst1 overexpression mice group?Tg-Mst1+Mel?,diabetes Mst1overexpression mice group?Tg-Mst1+DM?,melatonin intervention diabetes Mst1overexpression mice group?Tg-Mst1+DM+Mel?.6.Diabetes was induced by intraperitoneal?i.p.?injections of STZ?50 mg/kg/d?.The mice with diabetes were daily treated with melatonin?20 mg/kg/day?for 4 weeks.After 3 months,we examined the cardiac function of the mice using Echocardiogram.7.Cultured primary cardiomyocytes were treated with 100 uM melatonin for four hours.High glucose?33 mmol/L glucose?was used to simulate diabetes model.Cells were randomly divided into the four groups:control group?Con?;Melatonin intervention group?Con+Mel?;high glucose group?HG?;Melatonin intervention high glucose group?HG+Mel?.8.Cultured primary cardiomyocytes were transfected with Mst1 shRNA adenovirus.High glucose?33 mmol/L glucose?was used to simulate diabetes model.Cells were randomly divided into the six groups:control group?Con?;Adenovirus control group?Con+Ad-LacZ?;Mst1 knockdown group?Con+Ad-sh-Mst1?;high glucose group?HG?;Adenovirus high glucose control group?HG+Ad-LacZ?;high glucose Mst1 knockdown group?HG+Ad-sh-Mst1?.9.Cultured primary cardiomyocytes were transfected with Mst1 adenovirus.High glucose?33 mmol/L glucose?was used to simulate diabetes model.Cells were randomly divided into the six groups:control group?Con?;Adenovirus control group?Con+Ad-Control?;Mst1 overexpression group?Con+Ad-Mst1?;high glucose group?HG?;Adenovirus high glucose control group?HG+Ad-Control?;high glucose Mst1 overexpression group?HG+Ad-Mst1?.10.Cultured primary cardiomyocytes were transfected with Mst1 shRNA adenovirus,and then treated with 100 uM melatonin for four hours.High glucose?33 mmol/L glucose?was used to simulate diabetes model.Cells were randomly divided into the following four groups:Mst1 knockdown group?Ad-sh-Mst1?;Melatonin intervention Mst1 knockdown group?Ad-sh-Mst1+Mel?;high glucose Mst1 knockdown group?HG+Ad-sh-Mst1?;Melatonin intervention high glucose Mst1 knockdown group?HG+Ad-sh-Mst1+Mel?.11.Cultured primary cardiomyocytes were transfected with Mst1 adenovirus,and then treated with 100 uM melatonin for four hours.High glucose?33 mmol/L glucose?was used to simulate diabetes model.Cells were randomly divided into the following four groups:Mst1 overexpression group?Ad-Mst1?;Melatonin intervention Mst1 overexpression group?Ad-Mst1+Mel?;high glucose Mst1 overexpression group?HG+Ad-Mst1?;Melatonin intervention high glucose Mst1 overexpression group?HG+Ad-Mst1+Mel?.12.The expression level of the proteins involved in autophagy in different groups was examined by Western blotting.13.Cultured primary cardiomyocytes were transfected with GFP-LC3 adenovirus.To detect GFP-LC3 level in primary cardiomyocytes.14.To observe mitochondria and autophagosome morphology and quantity in myocardial tissue and primary cardiomyocytes using Transmission electron microscope.15.Mitochondrial membrane potential of primary cardiomyocytes in different groups was detected by JC-1 staining.?Results?1.Melatonin alleviates diabetes-induced cardiac dysfunction and increases myocardial autophagy level.2.Mst1 knockout(Mst1^-/-)promotes myocardial glucose intake,improves diabetes-induced cardiac dysfunction and increases myocardial autophagy level.3.Mst1 overexpression inhibits myocardial glucose intake,aggravates diabetes-induced cardiac dysfunction and decreases myocardial autophagy level.4.The TEM results suggest that Mst1 knockout promotes autophagosome formation in the hearts of diabetic mice.Mst1 knockdown increases autophagy level and the mitochondrial membrane potential,inhibits the apoptosis in primary cardiomyocytes under high glucose culture.5.Melatonin couldn't improve cardiac function in Mst1 knockout diabetes mice,unable to increase the autophagy level.It couldn't increase the autophagy level after down-regulating Mst1 in cardiomyocytes under high glucose culture.The results suggest that Mst1 knockout inhibits the protective function of melatonin in the diabetic cardiomyopathy.6.Melatonin could improve cardiac function and increase the autophagy level in Mst1overexpression diabetes mice.Melatonin increased the autophagy level after up-regulating Mst1 in cardiomyocytes under high glucose culture.The results suggest that melatonin protects the heart from the diabetic cardiomyopathy by inhibiting Mst1.7.Western blotting results showed that p-Mst1,Mst1 expression and the ratio of p-Mst1/Mst1 were increased in the hearts of diabetic mice,while autophagy marker protein Beclin1 and LC3-?expression were decreased.Autophagy marker proteins Beclin1 and LC3-?expression were increased significantly in the hearts of Mst1 knockout diabetic mice.Melatonin inhibited the p-Mst1 and Mst1 expression,increased autophagy marker protein Beclin1 and LC3-?expression level in the hearts of the diabetic mice.?Conclusion?Through the research of this project,we found that melatonin improved diabetes-induced cardiac dysfunction and increased cardiac autophagy level.Mst1 knockout can improve diabetes-induced cardiac dysfunction and increase cardiac autophagy level.Melatonin cannot further improve cardiac dysfunction in the Mst1 knockout diabetic mice.Therefore,melatonin protects the heart from the diabetic cardiomyopathy by regulating Mst1 signaling pathway and provides new targets for the diagnosis and treatment of the diabetic cardiomyopathy.