Molecular Mechanism Of Up-regulation Of HMGB1 By Cardiomyocyte Under Diabetic Condition

Background and objectiveDiabetes Mellitus(DM)is one of the most common metabolic disorders and is a very common in clinical setting.Patients with DM are secondary to insulin insufficiency or decreased sensitivity of the body system to insulin.Diabetes is a serious and increasing global health burden worldwide.It now becomes an important contributing factor for the high incidence of cardiovascular disease.It is expected that the number of adult diabetic patients would reach 300 million by 2025.Diabetic cardiomyopathy(DCM)is a complication of patients with diabetes.Hyperglycemia of patients with DM plays a key role in the induction of DCM.But the molecular mechanisms of the development of myocardial dysfunction in DM remain unclear.Hallmark features of the diabetic cardiomyopathy are myocardial hypertrophy and fibrosis.Ventricular diastolic dysfunction has been described as an early functional alteration in the patients with DM followed by ventricular systolic dysfunction.Diabetes is a pro-inflammatory state and many groups have reported an increase in tissue concentrations of cytokines in various mouse models of diabetes suggesting an important contribution of inflammation to the development of diabetic myocardial dysfunction.Many of the molecular mechanisms that increase fibrosis and myocardial inflammation can activate pro-apoptotic signalling pathways or activate necrosis signaling.Underlying mechanisms for increased rates of apoptosis include increased production of reactive oxygen species(ROS),increased circulating inflammatory cytokines and chemokines,caspase activation,Fas-receptor-dependent and mitochondrion-dependent apoptosis,ER stress,increased activation of the TGF-? signalling pathway,increased local activation of the renin-angiotensin-aldosterone system.A study from our group indicated that cardiomyocytes increase the expression of the high-mobility group box1(HMGB1)protein,which mediates cardiomyocyte-fibroblast communication,thus resulting in increased collagen production and myocardial fibrosis.The aim of present study is to explore the intracellular signaling pathway involved in the regulation of HMGB1 expression by the cardiomyocytes under diabetic condition.MethodsThe research is divided into two parts:Part I:Cardiomyocytes were isolated from neonatal rat heart and cell model was established by using high glucose(HG,30mM)in the experiments.Mannitol(Mtl,5.5mM HG+24.5mM Mtl)was served as osmolarity controls.Proteins were extracted from cardiomyocytes.The protein expression and phosphorylation of PI3K?,Akt,HMGB1 was analyzed by Western blot in the study.Released HMGB1 was analyzed with enzyme-linked immunosorbent assay(ELISA).Part ?:PI3K? CRISPR/Cas9 knockout plasmid and AS 605240were used to interfere the PI3K?.Akt inhibitior A6730 and Akt siRNA were used to interfere Akt.Cardiomyocyte oxidative stress was determined with a DCFDA fluorescence probe.Treatment of cardiomyocytes with an antioxidant(Mitotempo)to determine the relationship of activation of PI3K?/Akt and ROS.Results and Conclusions:1.Condition of cardiomyocytes with HG activates PI3K? and Akt,and up-regulates cardiomyocyte HMGB1 expression.2.HG-induced HMGB1 expression in cardiomyocytes is regulated by the PI3K?/Akt intracellular signaling pathway.3.Condition of cardiomyocytes with HG results cardiomyocyte oxidative stress,and subsequent activation of PI3K?/Akt pathway and HMGB1 production.In conclusion,under diabetic condition,cardiomyocytes incur an oxidative stress which further activates the PI3K?/Akt-signaling pathway leading to increase in HMGB1 production.