Angiotensin â…¡-induced P53-dependent Cardiac Cell Death: Its Prevention By Metallothionein In Vivo And In Vitro

Oxidative stress plays an important role in the development of diabetic complications including cardiomyopathy. So antioxidant therapies become more and more critical in the treatment of diabetic complications. Metallothioneins (MTs) are a group of intracellular metal-binding proteins characterized by a low molecular mass (6–7 kDa), high cysteine content (20 of the 61–62 amino acids). The critically biochemical feature of MT is its strict conservation of the cysteine residues that may be required for the characteristic metal binding properties of this protein. As a potent antioxidant MT has been found to play a critical role in preventing diabetic complications. Previous studies using an animal model over-expressing metallothionein specifically in the heart have shown that MT protects heart against multiple insults. In present study, we hope to establish a stable MT-IIA over- expressing cardiac cell line, and to evaluate its anti-oxidative property.It is proved that angiotensin II increased in the diabetic heart and related to the pathogenesis of diabetic cardiomyopathy. Angiotensin II could induced cardiac cell apoptosis and fibrosis induced by oxidative and nitrosative stress which was proved by our previous works showed the antioxidant protein MT can significantly prevent diabetic cardiomyopathy and suppress angiotensin II induced NADPH oxidase activation, nitrosative stress, apoptosis in the heart. Tumor suppressor p53 is normally activated by cellular stress and mediates a growth- suppressive response that involves cell cycle arrest and apoptosis. Oxidative damage to DNA was found in cardiac cells isolated from diabetic patients and rats with streptozotocin-induced diabetes mellitus. Since oxidative DNA damage is the well-known stimulator for p53 expression and function and oxidative stress also plays an important role in the development of diabetic cardiomyopathy , we assume that diabetes-derived oxidative stress cause oxidative DNA damage that turns on p53 expression to initiate cardiac cell death. To date, a few studies have explored the p53 expression in the diabetic heart; however, whether p53 expression directly involve in the induction of apoptotic cell death in the diabetic heart remains under investigated. Therefore, efforts to achieve a better understanding of the mechanisms contributing to cardiac cell death under diabetic condition are crucial to the development of new therapeutic strategies.So how about P53 ? Is MT can prevent P53 expression in the apoptosis induced by angiotensin II? These were the questions we would answered in this study.Method: 1) Rat heart-derived H9c2 cell line was stably transfected with a vector -lipofectamine 2000, in which the human MT-IIA gene was placed under the control of the constitutively activeβ-actin promoter. RT-PCR and Western-blotting proved the expression of MT and also showed its anti-oxidant property exposed to hydrogen peroxide or subjected to hypoxia/reoxygenation challenge. 2)Then we used these two cell lines and MT over-expression mouse model–MT-TG to study the effect of angiotensin II on cardiac cells and MT protective function in resistance of this attack. Firstly we tested cardiac cell apoptosis in Vivo and in Vitro by Cell Death Detection ELISA , western-blotting of caspase-3 and TUNEL staning. Then protein level of T-p53, P-p53, Bax/Bcl-2 were tested to show that p53 pathway involved in this process.Results: 1) The transfected cell line (H9c2MT7) exhibited similar growth kinetics and morphology to WT H9c2. RT-PCR and Western blotting analysis showed that H9c2MT had a remarkable increased MT protein level compared with the parent cell line H9c2. Addition of 25μM ZnSO4 had an undetectable effect on the induction of endogenous MT, but it likely stabilized the MT protein that is expressed only in H9c2MT7 cells. H9c2MT7 cells showed marked reduction in ROS production when exposed to hydrogen peroxide or subjected to hypoxia/reoxygenation challenge evaluated by dihydroethidium staining. In addition, transfection of MT showed cellular resistance to cadmium toxicity. 2) Results of Cell Death Detection ELISA , western-blotting of caspase-3 and TUNEL staning showed apoptosis obviously increasing in H9c2 cells and FVB mice heart, while in H9c2 MT7 and MT-TG mice heart there were no apparent increasing. It also showed T-p53, P-p53 and Bax/Bcl-2 were involved in these apoptosis process and MT can suppress the increasing of T-p53, P-p53 and Bax/Bcl-2.Conclusions: 1) We have successfully established a stable human MT-IIA over-expressing cardiac cell line; and this cell line showed a markedly increased oxidative protection and would be useful for dissection of the mechanisms of MT in the cardiac protection. 2)Angiotensin II can induced apoptotic cell death in heart and H9c2 cells, and this process was mainly through p53 pathway, and MT can attenuate this effect.