Effect And Mechanism Of Iron Overload On Ferroptosis In Cardiomyocytes Induced By GPX4 Methylation

Objective Cardiovascular disease is a serious threat to human life and health.Its incidence rate and mortality rate are increasing year by year.The death of cardiac cells is closely related to the occurrence of cardiovascular diseases such as myocardial infarction.Ferroptosis is a new form of programmed cell death recently.The current research shows that ferroptosis is closely related to various diseases,such as neurodegenerative diseases,tumor and cardiovascular diseases.Some studies suggest that iron overload of cardiomyocytes is involved in the occurrence of heart disease.It is unclear whether the mechanism of iron overload can induce ferroptosis in cardiomyocytes.Glutathione peroxidase 4(GPX4)is a key regulatory factor of glutathione dependence in ferroptosis regulation pathway.It can protect cells from accumulation of lipid peroxide by removing free radicals and participating in the hydrolysis of lipid peroxide.Glutathione deletion or inhibition of GPX4 expression can promote ferroptosis.It has been shown that GPX4 is involved in cardiovascular disease.However,the role and mechanism of GPX4 in the ferroptosis of myocardial cells is unclear.Therefore,this study aims to explore the effect of GPX4 on the ferroptosis of myocardial cells and its molecular mechanism.Method H9c2 rat cardiomyocytes were treated with 500 μmol / L ferric citrate(Fac)to induce ferroptosis;The protein expression levels of GPX4 and 12/15-LOX were detected by Western Blot;Cardiomyocyte death was detected by Propidium iodide(PI)staining;malondialdehyde(MDA)content was detected by fluorescence spectrophotometry to analyze lipid peroxidation;Overexpression or inhibition of GPX4 and other proteins were used to analyze the effects on ferroptosis and lipid peroxidation of cardiomyocytes;GPX4methylation was detected by using methylation inhibitors.Result In this study,Fac induced ferroptosis in H9c2 cells;GPX4 protein level was down regulated in H9c2 cardiomyocytes after Fac treatment.Overexpression of GPX4 inhibited ferroptosis and lipid peroxidation induced by Fac,while inhibition of endogenous GPX4 expression could promote ferroptosis and lipid peroxidation induced by Fac,indicating that GPX4 can be used as a protective gene to regulate the occurrence and development of ferroptosis in cardiomyocytes;In terms of mechanism,our study demonstrated that Fac induces lipid peroxidation and ferroptosis of cardiomyocytes via enhancing methylation of GPX4;In addition,12/15-lipoxygenase(12/15-LOX)is a downstream target of GPX4;Therefore,we also demonstrated that knockdown of 12/15-LOX can inhibit Fac-induced ferroptosis and lipid peroxidation in cardiomyocytes.Conclusion Iron overload can induce ferroptosis in cardiomyocytes by down regulating the expression of GPX4 and promoting lipid peroxidation.GPX4 plays a protective role by inhibiting ferroptosis.Further studies suggest that high iron induces DNA methylation of GPX4 gene in cardiomyocytes.And 12/15-LOX,a downstream target of GPX4,is also involved in iron overload induced ferroptosis in cardiomyocytes.This study revealed the molecular mechanism of iron overload induced ferroptosis in cardiomyocytes,and provided a new target for the prevention and treatment of cardiovascular diseases.