Abstract 3026: MicroRNA Expression Profiling in the Ischemic Preconditioned Heart: Functional Role of a MiR-1/GATA-4/MiR-144, -451, and -762 Circuit
While a wealth of data has uncovered a distinctive microRNA (miRNA) expression pattern that occurs in the setting of physiological and pathological stress, relatively little is known about miRNA regulation and response to the ischemic preconditioning (IPC). In this study, we performed miRNA array analysis in the mouse heart preconditioned with six cycles of ischemia (5 min), via coronary artery occlusion, and reperfusion (5 min) in vivo. Ten miRNAs were up-regulated including miR-144, -451, -762, -294*, -142–3p,-290 –5p, -29a, -665, -32, and -183*, and 14 miRNAs were down-regulated including miR-1, -574 –5p, -675–5p, -467b*, -467e*, -466a-5p, -466b-5p, -466d-3p, -466d-5p, -466i, -208b, -297b-3p, -873, and -200a in preconditioned murine hearts, compared with the shams (n=6 hearts, fdr<0.1). The most dysregulated miRNAs (miR-1, -144, -451, and -762) were further validated by RT-PCR. To determine whether these dysregulated miRs are functionally relevant, miR mimics and inhibitors to miR-1, -144, -451, and -762 were expressed in rat H9c2 cardiomyocytes. To simulate ischemia, miR-transfected cells were serum starved for 2 h, then incubated in culture medium containing 200 μM H2O2 for 1 h. Cell viability was measured by MTS incorporation, and showed that overexpression of miR-1 enhanced cell death triggered by oxidative stress, while miR-1 inhibitor exhibited the cytoprotective effects. Conversely, overexpression of miR-144, -451, or -762 reduced cell deaths upon oxidative stress, while knock-down of miR-144, -451, or -762 showed detrimental effects. More interestingly, these 4 miRs formed a circuit through the hub of GATA-4, an important cardiac transcription factor. We confirmed that miR-1 negatively regulated GATA-4 expression, and overexpression of GATA-4 resulted in the upregulation of miR-144, -451, and -762 in H9c2 cells. Western-blots also showed that GATA-4 levels were increased by 2.3-fold in the preconditioned hearts, consistent with the downregulation of miR-1 by 1.3-fold and upregulation of miR-144 (2-fold), miR-451 (1.8-fold), and miR-762 (1.5-fold). Taken together, our data demonstrate that a circuit of miR-1/GATA-4/miR-144, -451, and -762 is involved in the regulation of cardioprotective effects by IPC.