Role And Molecular Mechanism Of Mfn2-mediated Mitochondrial Dynamics Dysfunction In Diabetic Cardiomyopathy

According to the World Health Organization and the International Diabetes Federation,the number of patients with diabetes worldwide has reached 66 million in 2011.It is estimated that there will be 500 million people with diabetes and pre-diabetes worldwide by 2030.Diabetes has become a leading cause of threats to human health.Cardiovascular complications of diabetes are the leading cause of death and disability in diabetes.Diabetic cardiomyopathy is defined as heart failure syndrome which occurs in patients with diabetes,mainly characterized by left ventricular hypertrophy and left ventricular dysfunction independent of the occurrence of vascular lesions such as coronary heart disease and atherosclerosis.Exploring the pathogenesis of diabetic cardiomyopathy and finding its potential therapeutic targets is an important issue needs to be solved urgently.As an energy factory inside the cell,mitochondria are widely involved in various physiological processes such as ATP synthesis and oxidative stress,which are important for maintaining cell survival and homeostasis.While exerting various physiological functions,mitochondria adjust their own morphology to adapt to different cell states through continuous fusion and fission,which is defined as mitochondrial dynamics.It is generally believed that excessively dividing mitochondria will cause adverse effect to cell,as excessive mitochondrial fission will decrease mitochondrial membrane potential,reduce ATP production and increase oxidative stress.Moderate promotion of mitochondrial fusion enhances mitochondrial function.A study published in Circulation demonstrated that there is significant mitochondrial excessive fission in myocardial tissue of diabetic patients,suggesting that mitochondrial dynamics may be involved in the development of diabetic cardiomyopathy.This study intends to demonstrate whether mitochondrial dynamics is involved in the pathogenesis of diabetic cardiomyopathy and its underlying molecular mechanisms.Objectives: 1.Explore whether mitochondrial dynamics is involved in the pathogenesis of type 2 diabetic cardiomyopathy.2.Explain the underlying molecular mechanism of mitochondrial dynamics in the development of diabetic cardiomyopathy.3.Explore the upstream molecular regulation mechanism regulating mitochondrial dynamics in diabetic myocardium Methods: Part I: Mfn2 mediated mitochondrial fission contributed to the pathogenesis of diabetic cardiomyopathyPurchase 8-weeks-old type 2 diabetes model mouse db/db mice and their strict control mice db/+,normal feeding,detect the mice's blood sugar,blood lipids,food intake and body weight changes every two weeks.Echocardiographic was used to determine cardiac function in mice;transmission electron microscopy was used to observe myocardial mitochondrial morphology in mice.Myocardial mitochondrial dynamics related protein expression(Drp1,Fis1,Mfn1,Mfn2,Opa1)were analyzed by Western Blotting.Part II: Mitochondrial fission caused by down-regulation of Mfn2 promotes the development of diabetic cardiomyopathyMfn2 overexpressing adenovirus was constructed,and control adenovirus(Ad-EV)and Mfn2 overexpressing adenovirus(Ad-Mfn2)were injected into control group(db/+)and model mice(db/db)at 12 weeks of age,Western Blotting and immunohistochemistry were used to detect the transfection efficiency of adenovirus after 1 week and 4 weeks of myocardial injection.After 4 weeks of injection,cardiac function was monitored by echocardiography and myocardial mitochondrial morphological changes was observed by transmission electron microscopy.Gross morphological changes of the heart were observed by HE staining;myocardial fibrosis was observed by Masson staining;myocardial hypertrophy related indexes were observed by WGA staining;myocardial apoptosis was detected by TUNEL staining;and myocardial oxidative stress related indicators were determined.Part III: In-vitro primary cardiomyocytes were used to further explore the role of Mfn2 in diabetic cardiomyopathyRat primary cardiomyocytes were isolated,cultured with high glucose and high fat(25 mM glucose + 500 ?M sodium palmitate),and transfected with adenovirus to overexpress and knock down Mfn2,respectively.Mito-Tracker Red staining was used to observe mitochondrial morphology of primary cardiomyocytes.Mitochondrial membrane potential changes were detected by JC-1 staining;mitochondrial cytochrome C release was detected by Western Blotting;myocardial apoptosis was detected by flow cytometry;cellular mitochondrial ROS content was detected by cellular ROS staining and Mito-SOX staining;Seahorse were used to detect mitochondrial function related indicators.Part IV: Exploring the upstream regulation mechanism of Mfn2 in diabetic heartGEO public database was used to analyze the potential transcription and expression of the potential molecule in diabetic db/db mice was detected by Western Blotting,which was consistent with the expression of Mfn2.The molecule was overexpressed in the primary cardiomyocytes to determine the effect of the molecule on Mfn2 expression and mitochondrial dynamics.ChIP and dual luciferase reporter assays were used to further identify possible binding sites for this molecule in the Mfn2 promoter region.Results: 1.Compared with control db/+ mice,type 2 diabetes db/db mice showed significant weight gain.Blood glucose levels and blood lipids of db/db mice were significantly higher than control mice.From 12 weeks of age,the left ventricular ejection fraction and left ventricular short axis shortening rate of db/db mice decreased significantly.At 16 weeks of age,the cardiac function of db/db mice was further reduced.These data indicate that type 2 diabetic db/db mice develop symptoms of diabetic cardiomyopathy starting at 12 weeks of age.2.With the decrease of heart function,myocardial mitochondria of diabetic mice showed excessive fission from 12 weeks of age compared with the same age control db/+ mice.The individual mitochondria in the tissue is smaller and the number of mitochondria per unit area is greater.Western Blotting analysis showed that the expression of fusion-related proteins Mfn1,Opa1 and fission-related proteins Drp1 and Fis1 were not significantly different between diabetic myocardium and control myocardium.Only fusion-related protein Mfn2 was significantly reduced in diabetic myocardium of db/db mice.3.After 4 weeks of injection of Mfn2 overexpressing adenovirus,Western Blotting and immunohistochemical staining indicated that compared with the empty-borne virus(Ad-EV)injection group,the expression of Mfn2 in Ad-Mfn2 mouse myocardium was significantly increased,which was about 1.8 times that of the control group.Electron microscopy showed that the excessive fission of myocardial mitochondria was significantly inhibited by the increase of Mfn2 expression.Echocardiographic results showed that myocardial-specific overexpression of Mfn2 adenovirus effectively improved cardiac function in diabetic mice.HE staining,WGA staining,Masson staining and TUNEL staining all indicated that myocardial specific Mfn2 overexpression improved the pathophysiological indexes of diabetic cardiomyopathy.4.The results of cell experiments showed that compared with normal cultured primary cardiomyocytes,HG/HF cultured cardiomyocytes showed obvious mitochondrial excessive fission,accompanied by significantly down-regulated Mfn2 expression.After transfection of primary cardiomyocytes with Mfn2 knockdown or overexpressing adenovirus for 48 hours,Western Blotting results showed that the virus transfection efficiency was in line with expectations.Mito-tracker staining showed that Mfn2 overexpression significantly inhibited mitochondrial fission induced by HG/HF,while knockdown of Mfn2 in normal cultured cardiomyocytes directly induced myocardial mitochondrial fission.JC-1 detection and Western Blotting results showed that Mfn2 overexpression inhibited the loss of myocardial mitochondrial membrane potential caused by HG/HF,and further inhibited cytochrome C leakage into cytoplasm.Normal cell knockdown of Mfn2 directly induces a decrease in mitochondrial membrane potential.Flow cytometry and Western Blotting showed that overexpression of Mfn2 inhibited apoptosis induced by HG/HF,while normal cells knockdown of Mfn2 induced apoptosis directly.5.Cellular ROS staining and mitochondrial mito-SOX staining suggest that Mfn2 overexpression significantly inhibits oxidative stress induced by HG/HF,while normal cell knockdown Mfn2 can directly induce increased oxidative stress.The results of Seahorse cell energy metabolism test showed that mitochondrial oxidative respiration was significantly damaged in HG/HF cultured cardiomyocytes and Mfn2 overexpression reversed the mitochondrial function damage.Normal cardiomyocytes can directly induce loss of mitochondrial function after knocking down Mfn2.6.Analysis of GEO public database data showed that PPAR? showed strong correlation with Mfn2,suggesting that PPAR? is likely to be a transcription factor of Mfn2.Overexpression of PPAR? in primary cardiomyocytes significantly enhanced Mfn2 expression and inhibited mitochondrial fission induced by high glucose and high fat.Chromatin immunoprecipitation experiments showed that PPAR? binds to the Mfn2 promoter region.Dual luciferase reporter assay further confirmed that PPAR? can directly bind to the Mfn2 promoter region "GACTGGGGACGGGGTAA" sequence to promote Mfn2 transcription.Conclusion 1.Mitochondrial excessive fission caused by Mfn2 downregulation is involved in the pathogenesis of diabetic cardiomyopathy.2.Mfn2-mediated mitochondrial fusion improved cardiac function in type 2 diabetic mice by improving mitochondrial function,decreasing apoptosis and reducing oxidative stress.3.Decreased expression of PPAR? in diabetic myocardium is the main reason for the down-regulation of Mfn2 expression.PPAR? directly binds to the Mfn2 promoter region within ?GACTGGGGACGGGGTAA? to promote Mfn2 transcription.