Abstract 3028: Cardiac-Specific Overexpression of microRNA-494 Protects Against Ischemia/Reperfusion-Induced Injury by Targeting PTEN, ROCK1 and CaMKIIΔ
We recently observed that microRNA-494 (miR-494) was downregulated in murine hearts upon ischemia/reperfusion (I/R) in vivo, consistent with other findings in human infarcted hearts. However, miR-494 was significantly upregulated in ex vivo I/R murine hearts. To further elucidate the functional role of miR-494 in both ex vivo and in vivo I/R-induced cardiac injury, we generated a mouse model with cardiac-specific overexpression of miR-494. All miR-494 transgenic (TG) mice were healthy and showed no apparent cardiac morphological or pathological abnormalities. TG hearts with 4-fold overexpression of miR-494 and wild-type hearts were then subjected to global no-flow I/R (40 min/120 min) using the Langendorff preparation. TG hearts exhibited improved recovery of contractile performance over the whole reperfusion period. This improvement was accompanied by a 2.5-fold decrease in lactate dehydrogenase released from the TG hearts. The extent of apoptotic cell death was also significantly decreased by ~70%, which was measured by TUNEL staining and DNA fragmentation. Furthermore, in vivo experiments of 30 min myocardial ischemia, via coronary artery occlusion, followed by 24 h reperfusion, showed that the infarct-to-risk region ratio was 6.1±1.4% in TG hearts (n=7) compared to 21.5±3.1% in WT hearts (n=9, p<0.001). Using Target-Scan software, we identified PTEN, ROCK1 and CaMKIIΔ, three well-known proteins that are detrimental in I/R, as important candidate targets for miR-494. To test this prediction, we examined the expression levels of PTEN, ROCK1 and CaMKIIΔ in TG and WT hearts. Indeed, the protein levels of PTEN, ROCK1 and CaMKIIΔ were decreased by 60%, 55%, and 30%, respectively, in miR-494 hearts, relative to WT hearts. Furthermore, we analyzed the effect of miR-494 on 3′UTRs’ activities of PTEN, ROCK1 and CaMKIIΔ utilizing a luciferase reporter assay. In agreement with the protein level data, the 3′UTRs’ activities of PTEN, ROCK1 and CaMKIIΔ were strongly inhibited (up to 4-fold) by miR-494. Taken together, our findings demonstrate that increased miR-494 levels in the heart protect against I/R-induced injury via antithetical regulation of PTEN, ROCK1 and CaMKIIΔ. Thus, miR-494 may constitute a new therapeutic target for ischemic heart diseases.