Uric acid promotes cardiomyocyte apoptosis through regulating microRNA-21/PTEN/AKT/eNOS axis in myocardial ischemia/reperfusion injury

Myocardial ischemic/reperfusion injury (MI/RI) results from severe impairment of coronary blood supply and leads to irreversible cell death, with limited therapeutic possibilities. Uric acid (UA) is a purine nucleotide metabolite that is synthesized in the liver from xanthine via xanthine oxidase and affects various vasoactive mediators. The aim of this study was to investigate the effect of UA on cardiomyocyte apoptosis and its molecular mechanism in MI/RI. H9c2 cells were treated with UA after which cell viability, apoptosis, and caspase activation were evaluated by CCK-8, flow cytometry and Western Blotting. Meanwhile, eNOS expression and NO content were measured using the fluorescent indicator DAF-FM diacetate and Cayman's NOS activity assay kit. Then, the roles of UA on miR-21 expression were investigated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Further, the potential target genes of miR-21 were predicted by bioinformatics analyses and confirmed by dual-luciferase reporter assay. Finally, H9c2 cells were transfected with miR-21 inhibitor or mimic to confirm the role of miR-21 on the protein expression of PTEN/AKT/eNOS pathway and UA-induced apoptosis. Our study showed that UA inhibited H9c2 cells growth in a concentration-and time-dependent manner. UA at 15 mg/dl effectively promoted apoptotic cell death, increased the activities of caspase-3 and PARP, which was along with dysregulation of Bcl-2 and Bax protein in activated apoptotic pathway. In addition, UA caused a dose-dependent decrease in the NO production through inhibiting eNOS phosphorylation in cultured H9c2 stimulated by Bradykinin. We further demonstrated that UA inhibited miR-21 expression in a concentration-and time-dependent manner and PTEN was identified as a target of miR-21. Knockdown of miR-21 decreased eNOS and AKT phosphorylation and miR-21 overexpression could alleviate the cell apoptosis induced by UA in H9c2 cells. Collectively, the findings that UA inhibits cardiomyocyte growth, call for careful reconsideration of the role of UA in MI/RI. Our data also show that UA increases cardiomyocyte apoptosis through regulating miR-21/PTEN/AKT/eNOS axis, indicating that interruption of miR-21/PI3K/Akt/eNOS may represent a novel therapeutic strategy in MI/RI.