The Role And Application Of RhoA/ROCK Signaling Pathway In The Pathogenesis Of Diabetes Cardiomyopathy

Objective:This study aims to determine whether Rho A/ROCK signaling pathway is involved in myocardial damage in type 2 diabetes mellitus（T2DM）through regulating myocardial autophagy,and what is the possible molecular mechanisms,attempting to provide new ideas and targets for the prevention and treatment of diabetic cardiomyopathy.Methods:In this study,T2DM rat models were established by streptozotocin combined with a high-fat diet,and the molecular biological techniques（real time PCR and western blotting）,the histological analysis（tissue electron microscopy,HE and Masson staining）and animal ultrasound techniques were used for evaluating the rat models.The T2DM rat models were intervened by inhibitors or agonists,fasudil 10mg/kg/d,SP60012515mg/kg/d,Rapamycin 1mg/kg/d,3-MA 15 mg/kg/d,intraperitoneally for 4weeks.The improvements of cardiac structure and function were assessed by molecular biological techniques,histological analysis and ultrasound techniques.Primary neonatal rat cardiomyocytes were cultured in high glucose（30m M）and high insulin(10^-7M)medium to establish high glucose and high insulin cardiomyocyte model.The apoptotic proteins and autophagy function of cardiomyocytes were detected by real time PCR and western blotting methods,and the molecular mechanisms was analyzed.Results:1.The evaluations of diabetic cardiomyopathy rat models:After a single intraperitoneal injection of streptozotocin combined with high-fat feeding for34W,the blood glucose of Wistar rats remained at a high level.Histological analysis showed that the content of myocardial collagen and the cross-sectional area of myocardial cells were significantly increased in diabetic rats;functional examination showed that the EF and FS of left ventricle and the E/A value of mitral valve were significantly lower,indicating the seriously damaged cardiac systolic and diastolic functions.The abnormalities of cardiac structure and function demonstrated success in diabetic cardiomyopathy rat models.2.Rho A/ROCK signaling pathway is involved in myocardial damage induced by diabetic cardiomyopathy:The results of immunohistochemistry and western blotting showed that the expressions of LC3B,Atg5 and Casp3 in diabetic rats were up-regulated,and Rho A/ROCK signaling pathway was activated,according to significant increase of the key proteins ROCK1 and ROCK2.3.Rho A/ROCK signaling pathway participates in myocardial damage induced by diabetic cardiomyopathy via regulating autophagy:Fasudil inhibited the expression of ROCK1 and ROCK2,LC3B,Atg5 and Casp3 in primary neonatal rat cardiomyocytes stimulated by high glucose and high insulin.High glucose and high insulin significantly up-regulated the protein levels of JNK and Beclin1 in cardiomyocytes,which could be inhibited by JNK inhibitors.4.Inhibition of Rho A/ROCK signaling pathway improves myocardial damage in diabetic cardiomyopathy rats:Fasudil significantly inhibited Rho A/ROCK signaling pathway by down-regulated ROCK1 and ROCK2 in myocardial tissue,and decreased the content of myocardial collagen and cross-sectional area of myocardial cells,and improved cardiac dysfunction.Moreover,inhibited Rho A/ROCK signaling pathway by fasudil reduced the autophogosome in myocardial tissue,and down-regulated the protein expressions of LC3B,Atg5.Blocking the downstream signal molecule JNK of Rho A/ROCK signaling pathway also improved the myocardial autophagy by reducing the expression of Beclin1.Conclusions:1.Autophagy dysfunction is involved in myocardial damage of diabetic cardiomyopathy induced by T2DM.2.Rho A/ROCK signaling pathway participates in myocardial damage of diabetic cardiomyopathy by regulating myocardial autophagy via ROCK/JNK/Beclin1 axis.