Mst1 Inhibits CMECs Autophagy And Participates In The Development Of Diabetic Coronary Microvascular Dysfunction

According to the International Diabetes Federation(IDF)statistics show that globally the total number of patients with diabetes is 415 million and the number of patients with prediabetes is 318 million by 2015.With the current annual growth rate is expected,without any intervention,by 2040,the total number of patients with diabetes will reach 642 million,while the number of pre-diabetes population will reach up to 481 million people.In the past thirty years,the prevalence of diabetes in China has increased by 17 times,from the total population of the population to the growth rate of 0.67% to 11.6%.Cardiovascular(CV)complications are major public health issues which account for a substantial proportion of the morbidity and mortality in diabetic patients.Endothelium is an important component of the cardiovascular system,which affects many aspects of microvascular function,including pressure,flow rate,permeability,angiogenesis and blood rheology.Vascular endothelial dysfunction in diabetes may lead to clinical manifestations of microvascular disease.Our previous studies demonstrated that coronary microvascular dysfunction occurred in the early stages of diabetes as manifested by impaired microvascular barrier dysfunction,increased oxidative stress and apoptosis in the cardiac microvascular endothelial cells(CMECs).However,little is known about the mechanisms underlying coronary microvascular damage associated with diabetes.Thus,to better understand the pathogenesis and prevent coronary microvascular damage are central issues in the management of diabetic patients.As the barrier between blood glucose and cardiomyocytes,endothelium is thought to play a major role in the pathogenesis of diabetes-associated cardiovascular diseases(CVD).Interestingly,autophagy has recently emerged as a potential novel target for the treatment of cardiovascular diseases.In particular,Lenoir and colleagues demonstrated that endothelial cell and podocyte autophagy synergistically protected from diabetes-induced glomerulosclerosis.Nevertheless,there are currently no data on the role of CMECs autophagy in diabetes.Mst1(mammalian Ste20-like kinase 1)is a serine-threonine kinase which has been implicated in diverse biological functions,including autophagy,apoptosis and oxidative stress.Mst1 has been reported to promote cardiac dysfunction in mice subjected to myocardial infarction(MI)through inhibition of autophagy.Moreover,our previous study also demonstrated that up-regulating autophagy through Mst1 inhibition alleviates postinfarction cardiac dysfunction.In the heart,Mst1 is widely distributed in cardiomyocytes and endothelial cells.Mst1 can directly inhibit the activity of Beclin1.However,the direct role of Mst1/Beclin1 signaling in the development of coronary microvascular damage in diabetes is not fully understood.The objective of the present study was to investigate the precise involvement of autophagy and the underlying mechanisms in the pathogenesis of coronary microvascular disease in diabetes.?Objective?1.To investigate the effects of Mst1 gene knockout and Mst1 gene overexpression on cardiac function and myocardial microvascular injury in diabetic mice.2.Isolation,cultivation and identification of CMECs,using the adenoviruses harboring Mst1(Ad-Mst1)(MOI: 100),Mst1 sh RNA(Ad-sh-Mst1)(MOI: 100)transfected the CMECs.After 36 h,the CMECs were treated either in the absence(5.5 m M)or presence of high(33 m M)glucose for 48 h to observe the level of autophagy.3.To demonstrate that autophagy inhibition in CMECs plays an important role in the coronary microvascular dysfunction in diabetes and confirm that Mst1/Beclin1 signaling is involved in the development of coronary microvascular damage in diabetes?Methods?1.Mice were divided into the following groups:(i)Wide-type(WT);(ii)Mst1-/-;(iii)DM;(iv)DM + Mst1-/-;(A)Non-transgenic mice(NTg);(B)Tg-Mst1;(C)DM + NTg;(D)DM + Tg-Mst1;2.Diabetes were induced by intraperitoneal(i.p.)injections of STZ for 5 days and maintained for another 3 months as previously described.Animals with glucose levels no less than 16.6 mmol/L were classified as diabetes(DM).To examine the systolic function of the mice using Echocardiogram.3.Cardiac function was measured by an echocardiography system.4.Cardiac microvessels integrity was examined by scanning electron microscopy.5.Isolation,cultivation and identification of CMECs,using the adenoviruses harboring Mst1 sh RNA(Ad-sh-Mst1)(MOI: 100)transfected the CMECs.Then they were randomly divided into 6 groups: Con(5.5 mmol/L glucose),Con+Lac Z(5.5 mmol/L glucose),Con+Ad-sh-Mst1(5.5 mmol/L glucose),HG(33mmol/L glucose),HG+Lac Z(33mmol/L glucose)and HG+ Ad-sh-Mst1(33mmol/L glucose).6.Isolation,cultivation and identification of CMECs,using the adenoviruses harboring Mst1(Ad-Mst1)(MOI: 100)transfected the CMECs.Then they were randomly divided into 6 groups: Con(5.5 mmol/L glucose),Con+Ad-Control(5.5 mmol/L glucose),Con+Ad-Mst1(5.5 mmol/L glucose),HG(33mmol/L glucose),HG+ Ad-Control(33mmol/L glucose)and HG+ Ad-Mst1(33mmol/L glucose).7.The protein expression of Mst1 gene after adenovirus transfected the CMECs were examined by Western blot analysis.8.Fluorescence microscopic detection of GFP-LC3 was conducted according to the manufacturer's instructions.9.Fluorescence microscopic detection of aggresomes and p62 were conducted according to the manufacturer's instructions.10.Using the transmission electron microscope,the autophagosome in CMECs were observed.11.Apoptosis of CMECs was detected by TUNEL assay using a Cell Death De-tection Kit(Roche,Penzberg,Germany)according to the manufacturer's instructions.12.Mitochondrial membrane potential in CMECs were detected by JC-1 staining.13.The sirt1?Beclin1?LC3??LC3?and p62 protein levels of the CMECs were detected by Western blotting.?Results?1.Mst1 knockout preserved cardiac microvessels integrity and improves cardiac function in diabetic mice.2.In diabetic mice,Mst1 overexpression aggravated cardiac microvessels disorder and cardiac dysfunction.3.CMECs were isolated as previously described with minor modifications.Dil-acetylated low-density lipoprotein intake assay was used to identify CMECs.4.Mst1 knockout enhanced autophagy while reduces apoptosis in CMECs subjected to high glucose culture.5.Mst1 overexpression inhibited autophagy while increases apoptosis in CMECs subjected to high glucose culture.6.Ad-sh-Mst1 transfection enhanced autophagic flux as evidenced by increased LC3-II/LC3-1 ratio and decreased p62 expression in the presence of bafilomycin A1,a lysosomal inhibitor to evaluate autophagic flux.In addition,elevated LC3-II/LC3-1 ratio and less p62 expression were also demonstrated by Western blotting in the Ad-shMst1 transfected CMECs.The p-Mst1/Mst1 ratio was elevated while Beclin1 expression was decreased in the high glucose cultured CMECs,which may responsible for the development of diabetic coronary microvascular dysfunction7.Ad-Mst1 transfection inhibited autophagic flux,which was evidenced by decreased ratio of LC3-II/LC3-? and increased p62 expression after blocking lysosomal activity with bafilomycin A.Western blot analysis also revealed reduction in the ratio of LC3-II/LC3-? and accumulation of p62 in the CMECs transfected with Ad-Mst1 irrespective of normal glucose and high glucose culture.?Conclusion?Collectively,our data demonstrate that autophagy inhibition in CMECs plays an important role in the coronary microvascular dysfunction in diabetes.Beclin1 may function as downstream of Mst1 in the development of diabetic coronary microvascular dysfunction.