The New Mechanism Of Diabetic Cardiomyopathy: Pink1/Parkin Pathway-induced Mitophagy

Recently, with the change of life style and food habits, the prevalence of diabetes mellitus is growing rapidly. The number about the new patients with diabetes is above 7 million per year. It is estimated that globally the number of adults affected with diabetes will increase from 135 million in 1995 to 300 million by 2025. At present, China has more than 94 million diabetic patients and has the most diabetic patients in the world. Meanwhile, The dysfunction from cardiovascular system is one of the most one of the diabetes-inducd damage of the patients with diabetes mellitus, about 75% of which exist risk of cardiovascular system diseases. so, Cardiovascular complications are the leading cause of diabetes-related morbidity and mortality. Now, although there are a variety of Strategies for the prevention and treatment of diabetic myocardial damage, the morbidity and mortality of diabetes-induced cardiovascular complications don’t sharply decreased. In the future, the prevention and control task is very high.Diabetic cardiomyopathy(DCM)has been defined as ventricular dysfunction that occurs independently of coronary artery disease and hypertension.The early characteristic of DCM is that the left ventricular stiffness, diastolic function and decreased complianced. Eventually, DCM is to become progressively to the congestive heart failure. Although chronic hyperglycemia plays an important role in the pathogenesis of diabetic complications, the meschanism of DCM remains unclear. It has an important theoretical and clinical value to make clear the key mechanisms for improving the outcome of patients with DCM.It has been confirmed that the mitochondrial dysfunction is one of the physiological mechanism of DCM. So, how to clean out effectively the damaged mitochondrial has been a hotspot maker in the DCM research in recent years. Pink1/Pakrin induced-mitophagy can selectively clear the damaged mitochondrial in cardiomyocyte, which has an important role in maintaining myocardial cell homeostasis and improving heart function. The role of Pink1/Parkin regulated mitophagy in DCM remains unclear. According to the current research progress, the aims in this study are as follows: ① The change of Pink1/Parkin induce mitophagy in diabetic cardiomyopathy. ② Wether the change of Pink1/Parkin pathway or not is the cause of DCM. ③ Wether the Pink1/Parkin pathway or not is involved in the progress of DCM through mitophagy.【Objective】 1. To establish diabetic cardiomyopathy model and investigate that the change of Pink1/Parkin pathway induce-mitophagy in diabetic cardiomyopathy. 2. To establish the DCM model in Parkin-/- mouse and primary cardiomyocytes and in vivo and vitro confirm that wether the changes of Pink1/Parkin pathway or not is the cause of DCM. 3. Using the autophagy inhibitor 3-MA, To clarify that wether the changes of Pink1/Parkin pathway or not is involved in the progress of DCM though mitophagy and confirm that Pink1/Parkin induced-mitophagy is one of the new mechanisms of DCM.【Methods】 1. The forty mice were randomly divided into two groups, which was: Controlgroup(Control) and diabetic cardiomyopathy group(DM). 2. Twenty C57 male mice in DM received the streptozotocin by intraperitoneal injection(STZ, 50 mg/kg). The progress lasted 5 days. The fasting blood glucose > 11.1 mmol/L was considered as the successful model. In the 8th week of diabetic model, diabetic cardiomyopathy modle was established. 3. To examine the systolic function of the mice using Echocardiogram. 4. To observe the pathological changes of the mice cardiac tissue using HE and Masson. 5. Using immunofluorescent labeling with TUNEL, cardiomyocyte apoptosis in mice cardiac tissue was observed with a fluorescence microscopy. 6. Using the transmission electron microscope, the mitochondrial morphology and the autophagosome in cardiac tissue were observed. 7. The Pink1、Parkin、Mfn1、Mfn2、Nix、Beclin1、P62、LC3Ⅱand LC3Ⅰprotein levels of cardiac tissue were detected by Western blotting. 8. The C57 mice and the Parkin-/- mice were randomly divided into 4 groups respectively, which was: Control group(WT), Control+STZ group(WT-STZ), Parkin-/-group(Parkin) and Parkin-/-+STZ goup(Parkin-STZ). In the 8th week of diabetic mice, the systolic function was detected using Echocardiogram. 9. Cultivated the primary cardiomyocytes from neonatal rats were divided into the following groups: Con+Lac Z(5.5 mmol/L glucose), Con+Parkin(5.5 mmol/L glucose), Con+Parkin+3-MA(5.5 mmol/L glucose), HG+Lac Z(33mmol/L glucose), HG+Parkin(33mmol/L glucose) and HG+Parkin+3-MA(33mmol/L glucose). 10. Using immunofluorescent labeling with TUNEL, the cultured cardiomyocyte apoptosis was observed with a fluorescence microscopy. 11. Using the transmission electron microscope, the mitochondrial morphology and the number of autophagosome in cardiomyocyte were observed. 12. The GFP-LC3 level in primary cardiomyocytes was detected with a fluorescence microscopy 13. The Pink1、Parkin、Mfn1、Mfn2、Nix、Beclin1、P62、LC3Ⅱand LC3Ⅰprotein levels in primary cardiomyocytes were detected by Western blotting.【Results】 1. The cardiac systolic function decreased signifcantly and cell apoptosis in cardiac tissues increased in diabetes group, compared with non diabetes. The myocardial fiber from DM guoup was thicker than that from control group, some of which has been fractured and were arranged in disorder. The fibrillation in cardiac tissues in both of control group and DM group were not obvious. The transmission electron microscope observation indicated the damaged mitochondrial morphology and decreased mitochondria autophagosome in DM group. 2. Compared with that of control group, the expression of Parkin、Mfn2、Nix and Beclin1 in DM group decreased significantly, in which the protein levels of Mfn1 and Opa1 significantly increased. There was no difference about the expression of Pink1 and Drp1 in the both group. Western-blot showed that autophagy in diabetic mice heart decreased sharply,compared with that in non diabetes. 3. Diabetes mellitus triggered the contractile dysfunction as evidenced by the echocardiogram,the effects of which were accentuated by Parkin-/-. 4. Cultured cardiac myocytes were transfected with parkin overexpression lentivirus. High glucose-induced cells death and apoptosis were also reversed by parkin overexpression. High glucose-induced mitochondria ultrastructure impairment in cardiomyocytes was also offset partially by Parkin overexpression. Given the autophagy inhibitor-3-methyladenine(3-MA), the effect of the protection and the enhancing mitophagy in cardiomyocytes by parkin overexpression disappeared. 5. The western-blot analysis and transfecting GFP-LC3 revealed the same result that high glucose challenge dampened autophagy in myocytes as evidenced by enhanced p62 levels, decreased levels of LC3 B positive puncta and protein level, the effect of which was alleviated by parkin overexpression. 6. The expression of Mfn2、Nix and beclin1, which were related with autophagy, decreased in High glucose condition, the effect of which was alleviated by parkin overexpression. The expression of Mfn1 and Opa1 increased significantly in High glucose condition, parkin overexpression decreased the Mfn1 protein level, without the effect of the Opa1 level. Level of Pink1 was unchanged in all groups.【Conclusion】 Our results suggested that the inhibition of Pink1/Parkin pathway inducedmitophagy played a vital role in the myocardial damage from diabetes, which was probably one of the mechanisms of DCM.