Abstract 3027: MicroRNA-210 is Upregulated in Cardiomyocytes in Response to Hypoxia Through p53- and Akt-Dependent Pathways and Exerts Cytoprotective Effects
MicroRNAs (miRs) are important regulators of gene expression. MicroRNA-210 (miR-210) is strongly upregulated in cancer and endothelial cells in response to hypoxia. Its role in cardiomyocyte response to hypoxia, however, is not known. We assessed the hypotheses that miR-210 is regulated by p53 and Akt, and that miR-210 upregulation is cytoprotective in cardiomyocytes. Microarray data from neonatal rat cardiomyocytes (NRCM) exposed to 0.5% O2, 1.5% O2, and normoxia for 48h demonstrated miR-210 to be the most highly upregulated miR in hypoxia. This was confirmed by quantitative RT-PCR, which showed miR-210 to be increased 25-fold. Time course experiments showed that miR-210 is significantly upregulated in NRCM as soon as 8h after hypoxia exposure, and remains elevated up to 13.7-fold at 5 days after withdrawal from hypoxia (p<0.02 for both, n=3). Since p53 and Akt signalling are involved in hypoxia, we assessed the role of these molecules in the regulation of miR-210. Hypoxia-induced upregulation of miR-210 is blunted by 55±14% in Akt−/− mouse embryonic fibroblasts (MEFs) and 72±8% in p53−/− MEFs when compared to wild-type (n=6–9, p=0.05 and 0.0006, respectively). Furthermore, treatment of H9c2 cells with an Akt inhibitor prior to hypoxia supressed miR-210 induction by 62±14% (n=3, p=0.067). Since insulin activates Akt, we then tested whether insulin also induces the expression of miR-210. Treatment of H9c2 cells with 30 nM of insulin led to a 2.1-fold increase in miR-210 levels (p=0.02). Next we assessed the role of miR-210 in cytoprotection. Treatment of H9c2 cells with miR-210 adenovirus resulted in protection against hydrogen peroxide-induced cell death when compared to GFP adenovirus control (89±4.0% vs. 76±1.6% viability, respectively, as assessed by trypan blue exclusion; n=6, p=0.02). Several putative miR-210 targets were then tested by luciferase reporter assays for regulation by miR-210. These demonstrated that a novel protein, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) is a direct miR-210 target. In conclusion, miR-210 is highly upregulated in hypoxic cardiomyocytes and is regulated by the p53 and Akt pathways. MiR-210 also appears to be cytoprotective, a function that may be mediated through its regulation of AIFM3.
This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).